Compositions and methods based on fluorophore diffusion

Abstract

This disclosure provides a method for detecting an analyte in a sample, wherein the sample is introduced into an analytical chamber along with emulsion droplets or gel beads. In another aspect, this disclosure provides designs for formulating emulsion droplets or gel beads such that they can be used for the large-scale parallel detection of analytes. Formulations containing specific combinations of fluorescent particles allow for the optical determination of the identity of each fluorescent particle. These combinations are based on the particle fluorescence emission wavelength, fluorescence excitation wavelength, and particle count.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 62 / 935,766, filed November 15, 2019; U.S. Provisional Patent Application Serial No. 63 / 022,324, filed May 8, 2020; and U.S. Provisional Patent Application Serial No. 63 / 052,310, filed July 15, 2020, all of which are incorporated herein by reference in their entirety for all purposes. Technical Field

[0003] This disclosure provides methods for detecting analytes and their binding interactions, and the design of formulations for said methods (e.g., formulations comprising emulsion droplets and / or gel beads). Background Technology

[0004] Samples of complex biological media (e.g., blood) contain specific components that can be used to diagnose diseases. Despite the presence of similar components at greater concentrations, there is a need to reliably measure the concentration of these specific components. These specific components, acting as "analytes," can include proteins, small molecules, or nucleic acids. Measurement is typically achieved by using a complementary component that binds to the analyte. This complementary component is called an "analyte binding reagent." An example of an analyte and analyte binding reagent pair is an antigen and an antibody.

[0005] Methods for measuring the concentration of analytes of specific components are known. However, some of these analytes, at concentrations currently undetectable, may be biologically significant, requiring new techniques with high inherent sensitivity and specificity. Furthermore, the complexity of culture media increases the risk of false positives and false negatives, which can be mitigated by new techniques for disease biomarker diagnosis. In addition, the discovery of novel disease biomarkers and new drug therapies are areas of research that will benefit from improved methods. This disclosure addresses these and other needs. Summary of the Invention

[0006] This disclosure provides a method for detecting an analyte in a sample, wherein the sample is introduced into an analytical chamber along with an emulsion droplet. The droplet contains fluorescent particles having a surface-binding reagent capable of binding with the analyte. The droplet is demulsified within the analytical chamber, and then the reagent binds to the analyte. The diffusivity of the fluorescent particles is reduced by binding. Fluorescent excitation light with a controlled phase or spatial pattern passes through the analytical chamber, and fluorescent emission light exits the analytical chamber. The fluorescent emission light is imaged. As the fluorescent particles diffuse into and out of a high-electric-field region, the fluorescence changes. The change in fluorescence is a function of the diffusivity of the fluorescent particles and thus provides information about the presence of the analyte. Conversely, when the analyte is explicitly provided, these methods can also be used to determine whether binding occurs in a population of different candidate reagents.

[0007] A key feature of this disclosure is that it allows the use of large-scale reagent libraries derived from simple vials, where the mapping of reagents to fluorescence emission behavior is achieved through diffusion-expanded constellation-like population imaging of fluorescent particles. In another aspect, this disclosure provides designs for formulating emulsion droplets or gel beads that can be used to detect analytes in a large-scale parallel manner. Formulations containing specific combinations of fluorescent particles allow for the optical determination of the identity of each fluorescent particle. These combinations are based on the particle fluorescence emission wavelength, fluorescence excitation wavelength, and particle count.

[0008] In some embodiments, this document discloses a method for analyzing an analyte, comprising: contacting (i) a first composition with (ii) a second composition, the first composition comprising a liquid phase and a sample in the liquid phase, the second composition comprising a liquid matrix and a formulation encapsulated in the liquid matrix, wherein the formulation comprises a plurality of fluorescent constructs and one or more analyte-interacting reagents attached to the fluorescent constructs; and combining the liquid phase and the formulation such that the analyte in the sample interacts with the one or more of the analyte-interacting reagents attached to the fluorescent constructs to generate a detectable signal, wherein the detectable signal is analyzed to detect the presence or absence, amount or concentration, and / or activity of the analyte in the sample. In some embodiments, the analyte-interacting reagent is an analyte-binding reagent.

[0009] In any of the foregoing embodiments, during the merging step, the liquid phase and the formulation can be fused into a single fluid. In any of the foregoing embodiments, the analyte in the sample can specifically bind to the analyte-interacting reagent.

[0010] In any of the foregoing embodiments, the analyte in the sample and the analyte-interacting reagent can participate in a reaction, such as an enzymatic reaction catalyzed by the analyte, the analyte-interacting reagent, and / or the reagent in the sample and / or the formulation.

[0011] In any of the foregoing embodiments, the plurality of fluorescent constructs may comprise one or more fluorescent particles, one or more fluorescent small molecules, one or more fluorescent peptides or proteins, one or more fluorescent dyes, or any combination thereof.

[0012] In any of the foregoing embodiments, the formulation may comprise one or more polymer particles, each of which comprises one or more fluorescent particles, one or more fluorescent small molecules, one or more fluorescent peptides or proteins, one or more fluorescent dyes, or any combination thereof.

[0013] In any of the foregoing embodiments, the plurality of fluorescent constructs may include one or more fluorescent semiconductor nanoparticles. In any of the foregoing embodiments, the plurality of fluorescent constructs may include one or more quantum dots.

[0014] In any of the foregoing embodiments, the analyte interacting reagent may be directly attached to one or more of the fluorescent constructs.

[0015] In any of the foregoing embodiments, the analyte-interacting reagent may be indirectly attached to one or more of the fluorescent constructs, for example, via a connector or a common binding partner.

[0016] In any of the foregoing embodiments, the analyte interacting reagent may be covalently or non-covalently attached to one or more of the fluorescent constructs.

[0017] In any of the foregoing embodiments, each of the one or more fluorescent constructs may have one or more analyte interacting reagents to which it is attached, or only a subset of the one or more fluorescent constructs may have one or more analyte interacting reagents to which it is attached.

[0018] In any of the foregoing embodiments, the second composition may comprise, for example, an emulsion of a liquid matrix and a formulation, or the second composition may comprise, or a composition other than an emulsion.

[0019] In any of the foregoing embodiments, the formulation may comprise aqueous droplets or aqueous droplets.

[0020] In any of the foregoing embodiments, the formulation may comprise or be, for example, a gel in the form of gel beads. In some embodiments, the gel comprises a plurality of fluorescent constructs and is capable of releasing the plurality of fluorescent constructs, for example, upon melting or otherwise disintegrating the gel (e.g., depolymerization of the gel by means of enzymatic digestion).

[0021] In any of the foregoing embodiments, the liquid phase may be water, and the formulation may be soluble in water and / or miscible with water, or the liquid matrix may be immiscible or substantially immiscible with water.

[0022] In any of the foregoing embodiments, the liquid matrix may comprise lipids, oils, hydrocarbon fluids, fluorocarbon fluids, chlorocarbon fluids, bromocarbon fluids, iodocarbon fluids, organosilicon fluids, or mixtures thereof.

[0023] In any of the foregoing embodiments, the liquid matrix may be selected from the group consisting of lipids, oils, hydrocarbon fluids, fluorocarbon fluids, chlorinated hydrocarbon fluids, brominated hydrocarbon fluids, iodinated hydrocarbon fluids, silicone fluids, and mixtures thereof.

[0024] In any of the foregoing embodiments, the combination of the liquid phase and the formulation can be performed in an analytical laboratory.

[0025] In any of the foregoing embodiments, the merging step may include merging adjacent streams of the first composition and the formulation, respectively.

[0026] In any of the foregoing embodiments, the merging step may include merging the cross flows of the first composition and the formulation, respectively.

[0027] In any of the foregoing embodiments, the merging step may include merging the vertical flows of the first composition and the formulation, respectively.

[0028] In any of the foregoing embodiments, the merging step may include merging the inclined streams of the first composition and the formulation, respectively.

[0029] In any of the foregoing embodiments, the merging step may include merging the relative flows of the first composition and the formulation, respectively.

[0030] In any of the foregoing embodiments, the merging step may include merging the concentric flows of the first composition and the formulation, respectively.

[0031] In any of the foregoing embodiments, the first composition and / or formulation may contain one or more demulsifiers.

[0032] In any of the foregoing embodiments, the first composition and / or formulation may contain one or more gelling agents.

[0033] In any of the foregoing embodiments, the first composition and / or formulation may contain one or more viscosity enhancers.

[0034] In any of the foregoing embodiments, the first composition may be a first emulsion comprising the liquid phase as emulsified droplets in a first liquid matrix, and the liquid matrix of the second composition may be a second liquid matrix.

[0035] In any of the foregoing embodiments, the first liquid matrix and the second liquid matrix may be the same or different.

[0036] In any of the foregoing embodiments, the first liquid matrix and the second liquid matrix may be independently selected from the group consisting of lipids, oils, hydrocarbon fluids, fluorocarbon fluids, chlorinated hydrocarbon fluids, bromine hydrocarbon fluids, iodine hydrocarbon fluids, organosilicon fluids, and any suitable combination thereof.

[0037] In any of the foregoing embodiments, the merging step may include applying an electric field to the first composition and / or the second composition.

[0038] In any of the foregoing embodiments, the first composition and the second composition may each comprise surfactants with opposite charges, and the merging step may include bringing the surfactants with opposite charges into contact with each other.

[0039] In any of the foregoing embodiments, the merging step may include applying a demulsifier to the first composition and / or the second composition, or the merging step may not include applying a demulsifier.

[0040] In any of the foregoing embodiments, the merging step may include applying heat to the first composition and / or the second composition.

[0041] In any of the foregoing embodiments, the merging step may include applying a gel depolymerizing agent to the first composition and / or the second composition.

[0042] In any of the foregoing embodiments, the detectable signal may be a fluorescence signal.

[0043] In any of the foregoing embodiments, the detectable signal can be induced by excitation light, which includes, is substantially composed of or is composed of unpolarized light.

[0044] In any of the foregoing embodiments, the detectable signal can be induced by excitation light, which includes, is substantially composed of, or is composed of linearly polarized light.

[0045] In any of the foregoing embodiments, the detectable signal can be induced by excitation light, which includes, is substantially composed of or is composed of circularly polarized light.

[0046] In any of the foregoing embodiments, the detectable signal can be induced by excitation light, which includes, is substantially composed of, or is composed of elliptically polarized light and / or cocycloidically polarized light.

[0047] In any of the foregoing embodiments, the detectable signal can be induced by excitation light, which includes, is substantially composed of, or is composed of light of a single wavelength.

[0048] In any of the foregoing embodiments, the detectable signal can be induced by excitation light, which includes, is substantially composed of or is composed of polychromatic light.

[0049] In any of the foregoing embodiments, the detectable signal can be induced by excitation light, which includes, is substantially composed of or consists of incoherent light.

[0050] In any of the foregoing embodiments, the detectable signal can be induced by excitation light, which includes, is substantially composed of or is composed of coherent light.

[0051] In any of the foregoing embodiments, the detectable signal can be induced by excitation light, which includes, is substantially composed of, or is composed of continuous light.

[0052] In any of the foregoing embodiments, the detectable signal can be induced by excitation light, which includes, is substantially composed of, or is composed of pulsed light.

[0053] In any of the foregoing embodiments, the detectable signal can be induced by excitation light, which includes, is substantially composed of, or is composed of light applied at a single incident angle.

[0054] In any of the foregoing embodiments, the detectable signal can be induced by excitation light, which includes, is substantially composed of, or is composed of light applied at a set of incident angles.

[0055] In any of the foregoing embodiments, the method may include applying an external electric field to the merged composition, wherein the external electric field is sufficient to induce electrophoretic movement of fluorescent constructs within the merged composition. In some embodiments, the external electric field includes, consists substantially of, or consists of: (i) a constant electric field applied in a constant direction; (ii) a pulsed electric field applied in a constant direction; and / or (iii) an oscillating electric field applied in a constant direction.

[0056] In any of the foregoing embodiments, the external electric field may include, consist substantially of, or consist of: (i) a constant electric field switching between multiple directions; (ii) a pulsed electric field switching between multiple directions; and / or (iii) an oscillating electric field switching between multiple directions.

[0057] In any of the foregoing embodiments, the method may include applying a force to the combined composition to reduce or eliminate nonspecific interactions between the analyte molecules and the fluorescent construct.

[0058] In any of the foregoing embodiments, the method may include applying sound waves to the combined composition, wherein the sound waves optionally include high-frequency sound waves, such as ultrasound.

[0059] In any of the foregoing embodiments, the method may include applying fluorescence excitation light to the combined composition. In some embodiments, the fluorescence excitation light is the fluorescence excitation light used in confocal microscopy, structured illumination microscopy (SIM), stochastic optical reconstruction microscopy (STORM), point-scan two-photon microscopy, scan line angle projection microscopy (SLAPMi), ghosting imaging (GI), and / or sparse constrained ghosting imaging (GISC).

[0060] In any of the foregoing embodiments, the method may include allowing the fluorescent emitted light to be refracted or internally reflected by droplets having a refractive index different from that of the matrix, thereby generating an image pattern that can be deconvolved to identify the location of the fluorophore.

[0061] In some aspects, this document discloses a method for analyzing an analyte, comprising: contacting (i) a first composition with (ii) a second composition, the first composition comprising a liquid phase and a sample in the liquid phase, the second composition comprising a liquid matrix and a formulation encapsulated in the liquid matrix, wherein the formulation comprises a plurality of fluorescent constructs and one or more analyte-interacting reagents attached to the fluorescent constructs; combining the liquid phase and the formulation in an analytical chamber such that the analyte in the sample interacts with the one or more of the analyte-interacting reagents attached to the fluorescent constructs; directing excitation light through the analytical chamber to induce fluorescence in the fluorescent constructs, wherein the fluorescence depends on the position of the fluorescent construct within a local phase angle of the excitation light; detecting and / or measuring fluorescence emission as the fluorescent constructs in each formulation diffuse in the combined composition; determining the identity of each analyte-interacting reagent present in each formulation based on a pattern of fluorescence emission wavelengths during diffusion, wherein variations in the random behavior of fluorescence emission provide an indication of the presence or absence, amount, and / or activity of the analyte in the sample. In some embodiments, the excitation light has a controlled phase and wavelength. In any of the foregoing embodiments, the excitation light may be or may include a standing wave.

[0062] In any of the foregoing embodiments, the excitation light may be coherent, and the phase of the coherent excitation light is controlled such that the peaks and troughs of the excitation light move through the fluorescent construct, sufficient to cause corresponding oscillations in the fluorescence of the fluorescent construct.

[0063] In any of the foregoing embodiments, the excitation light may be elliptically polarized coherent excitation light, and the ellipticity of the excitation light may be controlled such that the peaks and troughs of the excitation light move through the fluorescent construct, sufficient to cause corresponding oscillations in the fluorescence of the fluorescent construct.

[0064] In any of the foregoing embodiments, the excitation light may have a controlled spatial pattern and / or a controlled wavelength. In some embodiments, the excitation light is applied as a patterned spatial array across the entire analytical chamber, such that the light intensity varies for different points within the analytical chamber, sufficient to cause a fluorescence change in the fluorescent construct as it moves between these different points within the analytical chamber.

[0065] In any of the foregoing embodiments, the patterned spatial array of excitation light can be moved throughout the analysis chamber, such that the light intensity varies with time and for different points within the analysis chamber, sufficient to cause fluorescence changes in the fluorescent construct.

[0066] In any of the foregoing embodiments, detecting and / or measuring fluorescence emission may include detecting and / or measuring the magnitude of fluorescence emission.

[0067] In any of the foregoing embodiments, a pattern of the fluorescence emission wavelengths during diffusion can be determined near the location where each formulation has been incorporated with the liquid phase.

[0068] In any of the foregoing embodiments, during the merging step, the liquid phase and the formulation can be fused into a single fluid.

[0069] In any of the foregoing embodiments, the analyte in the sample can specifically bind to the analyte-interacting reagent.

[0070] In any of the foregoing embodiments, the analyte in the sample and the analyte-interacting reagent can participate in a reaction, such as an enzymatic reaction catalyzed by the analyte, the analyte-interacting reagent, and / or the reagent in the sample and / or the formulation.

[0071] In any of the foregoing embodiments, the plurality of fluorescent constructs may comprise one or more fluorescent particles, one or more fluorescent small molecules, one or more fluorescent peptides or proteins, one or more fluorescent dyes, or any combination thereof.

[0072] In any of the foregoing embodiments, the formulation may comprise one or more polymer particles, each of which comprises one or more fluorescent particles, one or more fluorescent small molecules, one or more fluorescent peptides or proteins, one or more fluorescent dyes, or any combination thereof.

[0073] In any of the foregoing embodiments, the plurality of fluorescent constructs may include one or more fluorescent semiconductor nanoparticles.

[0074] In any of the foregoing embodiments, the plurality of fluorescent constructs may contain one or more quantum dots.

[0075] In any of the foregoing embodiments, the analyte interacting reagent may be directly attached to one or more of the fluorescent constructs.

[0076] In any of the foregoing embodiments, the analyte-interacting reagent may be indirectly attached to one or more of the fluorescent constructs, for example, via a connector or a common binding partner.

[0077] In any of the foregoing embodiments, the analyte interacting reagent may be non-covalently attached to one or more of the fluorescent constructs.

[0078] In any of the foregoing embodiments, the analyte interacting reagent may be covalently attached to one or more of the fluorescent constructs.

[0079] In any of the foregoing embodiments, each of the one or more of the fluorescent constructs may have one or more analyte interaction reagents to which it is attached.

[0080] In any of the foregoing embodiments, a subset of one or more of the fluorescent constructs may have one or more analyte interaction reagents to which they are attached.

[0081] In any of the foregoing embodiments, the second composition may comprise, for example, an emulsion of a liquid matrix and a formulation.

[0082] In any of the foregoing embodiments, the formulation may comprise aqueous droplets or aqueous droplets.

[0083] In any of the foregoing embodiments, the second composition may comprise or comprise a composition other than an emulsion.

[0084] In any of the foregoing embodiments, the formulation may comprise or be, for example, a gel in the form of gel beads. In some embodiments, the gel comprises a plurality of fluorescent constructs and is capable of releasing the plurality of fluorescent constructs, for example, upon melting or otherwise disintegrating the gel (e.g., depolymerization of the gel by means of enzymatic digestion).

[0085] In any of the foregoing embodiments, the liquid phase may be water, and the formulation may be soluble in water and / or miscible with water.

[0086] In any of the foregoing embodiments, the liquid matrix may be substantially immiscible with water.

[0087] In any of the foregoing embodiments, the liquid matrix may comprise lipids, oils, hydrocarbon fluids, fluorocarbon fluids, chlorinated hydrocarbon fluids, brominated hydrocarbon fluids, iodinated hydrocarbon fluids, silicone fluids, or mixtures thereof.

[0088] In any of the foregoing embodiments, the liquid matrix may be selected from the group consisting of lipids, oils, hydrocarbon fluids, fluorocarbon fluids, chlorinated hydrocarbon fluids, brominated hydrocarbon fluids, iodinated hydrocarbon fluids, silicone fluids, and mixtures thereof.

[0089] In any of the foregoing embodiments, the combination of the liquid phase and the formulation can be performed in an analytical laboratory.

[0090] In any of the foregoing embodiments, the merging step may include merging adjacent streams of the first composition and the formulation, respectively.

[0091] In any of the foregoing embodiments, the merging step may include merging the cross flows of the first composition and the formulation, respectively.

[0092] In any of the foregoing embodiments, the merging step may include merging the vertical flows of the first composition and the formulation, respectively.

[0093] In any of the foregoing embodiments, the merging step may include merging the inclined streams of the first composition and the formulation, respectively.

[0094] In any of the foregoing embodiments, the merging step may include merging the relative flows of the first composition and the formulation, respectively.

[0095] In any of the foregoing embodiments, the merging step may include merging the concentric flows of the first composition and the formulation, respectively.

[0096] In any of the foregoing embodiments, the first composition and / or formulation may contain one or more demulsifiers.

[0097] In any of the foregoing embodiments, the first composition and / or formulation may contain one or more gelling agents.

[0098] In any of the foregoing embodiments, the first composition and / or formulation may contain one or more viscosity enhancers.

[0099] In any of the foregoing embodiments, the first composition may be a first emulsion comprising the liquid phase as emulsified droplets in a first liquid matrix, and the liquid matrix of the second composition may be a second liquid matrix.

[0100] In any of the foregoing embodiments, the first liquid matrix and the second liquid matrix may be the same or different.

[0101] In any of the foregoing embodiments, the first liquid matrix and the second liquid matrix may be independently selected from the group consisting of lipids, oils, hydrocarbon fluids, fluorocarbon fluids, chlorinated hydrocarbon fluids, bromine hydrocarbon fluids, iodine hydrocarbon fluids, organosilicon fluids, and any suitable combination thereof.

[0102] In any of the foregoing embodiments, the merging step may include applying an electric field to the first composition and / or the second composition.

[0103] In any of the foregoing embodiments, the first composition and the second composition may each comprise surfactants with opposite charges, and the merging step includes contacting the surfactants with opposite charges with each other.

[0104] In any of the foregoing embodiments, the merging step may include applying a demulsifier to the first composition and / or the second composition.

[0105] In any of the foregoing embodiments, the merging step may not require the application of a demulsifier.

[0106] In any of the foregoing embodiments, the merging step may include applying heat to the first composition and / or the second composition.

[0107] In any of the foregoing embodiments, the merging step may include applying a gel depolymerizing agent to the first composition and / or the second composition.

[0108] In any of the foregoing embodiments, the detectable signal may be or includes a fluorescence signal.

[0109] In any of the foregoing embodiments, the detectable signal can be induced by excitation light, which includes, is substantially composed of or is composed of unpolarized light.

[0110] In any of the foregoing embodiments, the detectable signal can be induced by excitation light, which includes, is substantially composed of, or is composed of linearly polarized light.

[0111] In any of the foregoing embodiments, the detectable signal can be induced by excitation light, which includes, is substantially composed of or is composed of circularly polarized light.

[0112] In any of the foregoing embodiments, the detectable signal can be induced by excitation light, which includes, is substantially composed of, or is composed of elliptically polarized light and / or cocycloidically polarized light.

[0113] In any of the foregoing embodiments, the detectable signal can be induced by excitation light, which includes, is substantially composed of, or is composed of light of a single wavelength.

[0114] In any of the foregoing embodiments, the detectable signal can be induced by excitation light, which includes, is substantially composed of or is composed of polychromatic light.

[0115] In any of the foregoing embodiments, the detectable signal can be induced by excitation light, which includes, is substantially composed of or consists of incoherent light.

[0116] In any of the foregoing embodiments, the detectable signal can be induced by excitation light, which includes, is substantially composed of or is composed of coherent light.

[0117] In any of the foregoing embodiments, the detectable signal can be induced by excitation light, which includes, is substantially composed of, or is composed of continuous light.

[0118] In any of the foregoing embodiments, the detectable signal can be induced by excitation light, which includes, is substantially composed of, or is composed of pulsed light.

[0119] In any of the foregoing embodiments, the detectable signal can be induced by excitation light, which includes, is substantially composed of, or is composed of light applied at a single incident angle.

[0120] In any of the foregoing embodiments, the detectable signal can be induced by excitation light, which includes, is substantially composed of, or is composed of light applied at a set of incident angles.

[0121] In any of the foregoing embodiments, the method may include applying an external electric field to the merged composition, wherein the external electric field is sufficient to induce electrophoretic movement of fluorescent constructs within the merged composition. In some embodiments, the external electric field includes, constitutes, or is constituted by a constant electric field applied in a constant direction.

[0122] In any of the foregoing embodiments, the external electric field may include, consist of, or be composed of a pulsed electric field applied in a constant direction.

[0123] In any of the foregoing embodiments, the external electric field may include, consist substantially of, or consist of: (i) an oscillating electric field applied in a constant direction; (ii) a constant electric field switching between multiple directions; (iii) a pulsed electric field switching between multiple directions; and / or (iv) an oscillating electric field switching between multiple directions.

[0124] In any of the foregoing embodiments, the analyte may comprise a protein moiety, and the analyte-interacting agent may comprise a protein binding agent, such as an antibody.

[0125] In any of the foregoing embodiments, the analyte may comprise a polynucleotide sequence, and the analyte interacting agent may comprise a sequence capable of hybridizing with the polynucleotide sequence, such as a sequence complementary to the polynucleotide sequence. In some embodiments, the method further includes, for example, using a temperature control device of the analysis chamber and / or using targeted local heating of quantum dots with focused light to unwind and / or anneal the hybridized sequence.

[0126] In any of the foregoing embodiments, the method may include moving an image of each fluorescent construct across the entire surface of the optical detector using a nutation transparency window and / or a rotating prism.

[0127] In some aspects, this document discloses a method for preparing a group of aqueous droplets, comprising preparing each aqueous droplet suspended in a water-immiscible liquid matrix to contain a plurality of fluorescent constructs having combinations of fluorescent emission colors, the fluorescent constructs having a certain count for each fluorescent emission color, and each fluorescent construct having zero, one, or more reagents attached thereto, wherein the identity of said reagent is specific to the color of the fluorescent construct to which it is attached and / or specific to the combination of color and count of the fluorescent constructs within each aqueous droplet. In some embodiments, the method further includes collecting the group of aqueous droplets into a container.

[0128] In any of the foregoing embodiments, the liquid matrix may be immiscible with water.

[0129] In any of the foregoing embodiments, the liquid matrix may be selected from the group consisting of lipids, oils, hydrocarbon fluids, fluorocarbon fluids, chlorinated hydrocarbon fluids, brominated hydrocarbon fluids, iodinated hydrocarbon fluids, silicone fluids, and any suitable combination or mixture thereof.

[0130] In any of the foregoing embodiments, the liquid matrix can be formulated into a gel.

[0131] In any of the foregoing embodiments, the liquid matrix can be vitrified at low temperatures.

[0132] In any of the foregoing embodiments, the liquid matrix can be cured at low temperatures.

[0133] In any of the foregoing embodiments, the liquid matrix may have a refractive index that matches that of the aqueous droplets suspended or encapsulated in the liquid matrix.

[0134] In any of the foregoing embodiments, the liquid matrix may have a higher or lower refractive index compared to the refractive index of the aqueous droplets suspended or encapsulated in the liquid matrix.

[0135] In any of the foregoing embodiments, the liquid matrix may be non-Newtonian.

[0136] In any of the foregoing embodiments, the liquid matrix may be shear-thinned.

[0137] In any of the foregoing embodiments, the liquid matrix may be shear-thickened.

[0138] In any of the foregoing embodiments, the liquid matrix may have a high viscosity.

[0139] In any of the foregoing embodiments, the liquid matrix may have low viscosity.

[0140] In any of the foregoing embodiments, the liquid matrix may have a density that matches that of the aqueous droplets suspended or encapsulated in the liquid matrix.

[0141] In any of the foregoing embodiments, the liquid matrix may have a smaller density compared to the density of aqueous droplets suspended or encapsulated in the liquid matrix.

[0142] In any of the foregoing embodiments, the liquid matrix may have a higher density compared to aqueous droplets suspended or encapsulated in the liquid matrix.

[0143] In any of the foregoing embodiments, these fluorescent constructs may be selected from the group consisting of: quantum dots; fluorescent proteins; fluorescent molecules; polymer particles containing quantum dots, fluorescent proteins, or fluorescent molecules; fluorescent particles each connected to another fluorescent particle via a chemical bonding system; and fluorescent particles each connected to a magnetic particle via a chemical bonding system.

[0144] In any of the foregoing embodiments, the fluorescent construct may include one or more analyte binding agents that are bound to its surface.

[0145] In any of the foregoing embodiments, the aqueous droplet may contain one or more magnetic particles.

[0146] In any of the foregoing embodiments, aqueous droplets can be stabilized using one or more surfactants.

[0147] In any of the foregoing embodiments, the fluorescent construct may be connected to fluorescent particles and / or magnetic particles via a connector.

[0148] In any of the foregoing embodiments, the aqueous droplet assembly may contain different combinations of fluorescent construct emission colors and counts.

[0149] In some embodiments, this document discloses a method for formulating a set of gel beads, comprising each gel bead formulated in a gel matrix removable by physical or chemical means, wherein each gel bead contains a plurality of fluorescent constructs having combinations of fluorescent emission colors, the fluorescent constructs having a certain count for each fluorescent emission color, and each fluorescent construct having zero, one, or more reagents attached thereto, wherein the identity of said reagent is specific to the color of the fluorescent construct to which it is attached and / or specific to the combination of color and count of the fluorescent constructs within each gel bead. In some embodiments, the method further includes collecting the set of gel beads into a container.

[0150] In any of the foregoing embodiments, these fluorescent constructs may be selected from the group consisting of: quantum dots; fluorescent proteins; fluorescent molecules; polymer particles containing quantum dots, fluorescent proteins, or fluorescent molecules; fluorescent particles each connected to another fluorescent particle via a chemical bonding system; and fluorescent particles each connected to a magnetic particle via a chemical bonding system.

[0151] In any of the foregoing embodiments, the fluorescent construct may include one or more analyte binding agents that are bound to its surface.

[0152] In any of the foregoing embodiments, the gel beads may contain one or more magnetic particles.

[0153] In any of the foregoing embodiments, the fluorescent construct may be connected to fluorescent particles and / or magnetic particles via a connector.

[0154] In any of the foregoing embodiments, the gel bead assembly may contain different combinations of fluorescent construct emission colors and counts.

[0155] In any of the foregoing embodiments, the method may include melting the gel at an elevated temperature.

[0156] In any of the foregoing embodiments, the method may include depolymerizing the gel using one or more enzymes.

[0157] In any of the foregoing embodiments, the method may include depolymerizing the gel using one or more chemical agents.

[0158] In any of the foregoing embodiments, the method may include depolymerizing the gel by light.

[0159] In any of the foregoing embodiments, the gel bead assembly may contain different combinations of fluorescent construct emission colors and counts.

[0160] In any of the foregoing embodiments, the method may include applying light to a composition comprising a gel-forming monomer, a photocatalyst, and a plurality of fluorescent constructs to selectively catalyze the polymerization of the gel-forming monomer to form gel beads comprising a subset of the plurality of fluorescent constructs.

[0161] In some aspects, this document discloses a composition comprising the set of aqueous droplets and / or gel beads formulated by the method described in any of the foregoing embodiments.

[0162] In any of the foregoing embodiments, each of the aqueous droplets may optionally contain an antifreeze, such as ethylene glycol or glycerin, to allow for frozen storage, for example.

[0163] In some embodiments, this document discloses a method for analyzing an analyte, comprising: (i) contacting a first composition with (ii) a second composition, the first composition comprising a first liquid matrix and a sample encapsulated in the first liquid matrix, the second composition comprising a second liquid matrix and a formulation encapsulated in the second liquid matrix, wherein the formulation comprises a plurality of fluorescent constructs and one or more analyte-interacting reagents attached to the fluorescent constructs; and combining the sample with the formulation such that the analyte in the sample interacts with the one or more of the analyte-interacting reagents attached to the fluorescent constructs to generate a detectable signal, wherein the analyte is optionally located at a boundary between the first liquid matrix and the sample, wherein the detectable signal is analyzed to analyze the presence or absence, amount or concentration, and / or activity of the analyte in the sample. In some embodiments, the first liquid matrix and the sample have the same or substantially the same refractive index.

[0164] In any of the foregoing embodiments, the first liquid matrix and the second liquid matrix may be the same or different. In any of the foregoing embodiments, the sample may be a solution, and the analyte may be located at the boundary between the first liquid matrix and the sample. In any of the foregoing embodiments, after the sample is combined with the formulation, the sample and the formulation may remain encapsulated in the first liquid matrix and / or the second liquid matrix.

[0165] In any of the foregoing embodiments, the method may further include applying excitation light to the plurality of fluorescent constructs, and the detectable signal comprising fluorescence emission from the plurality of fluorescent constructs, wherein the excitation light comprises simple ultraviolet light or pulsed ultraviolet light. In any of the foregoing embodiments, the method may further include analyzing the diffuseness of the plurality of fluorescent constructs, the analysis comprising analyzing the detectable signal. In any of the foregoing embodiments, the method may further include analyzing the change in diffuseness of the plurality of fluorescent constructs with increasing and / or decreasing temperature. In any of the foregoing embodiments, the change in diffuseness may be analyzed to provide a melting temperature indicative of the interaction between the analyte and the analyte-interacting reagent.

[0166] In any of the foregoing embodiments, the first composition may comprise a first oil and an aqueous sample encapsulated in the first oil, and the second composition may comprise a second oil and an aqueous formulation encapsulated in the second oil. In any of the foregoing embodiments, the aqueous formulation may comprise a plurality of quantum dots and one or more analyte binding agents attached to the quantum dots. In any of the foregoing embodiments, the aqueous sample and the aqueous formulation may form a combined aqueous composition retained encapsulated in an oil, and the combined aqueous composition and its encapsulating oil may have the same or substantially the same refractive index.

[0167] In any of the foregoing embodiments, the method may further include detecting the change in the diffusivity of a plurality of quantum dots encapsulated in oil as the temperature increases and / or decreases.

[0168] In some embodiments, this document discloses a method for analyzing cells, comprising: contacting (i) a first composition with (ii) a second composition, the first composition comprising a first liquid matrix and a sample encapsulated in the first liquid matrix, wherein the sample comprises single cells; the second composition comprising a second liquid matrix and a formulation encapsulated in the second liquid matrix, wherein the formulation comprises a plurality of fluorescent constructs and one or more analyte-interacting reagents attached to the fluorescent constructs, wherein: (a) the single cells are lysed in the sample to release one or more cellular components or (b) the single cells are not lysed in the sample, and optionally a cell lysis agent is provided in the second composition; and combining the sample with the formulation such that the cellular components interact with the one or more analyte-interacting reagents attached to the fluorescent constructs to generate a detectable signal, wherein the detectable signal is analyzed to analyze the presence or absence, amount or concentration, and / or activity of the cellular components in the single cells. In some implementations, the method includes analyzing multiple single cells from a cell population and comparing the presence or absence, amount or concentration and / or activity of the cellular components in a first single cell with the presence or absence, amount or concentration and / or activity of the cellular components in a second single cell to infer cellular heterogeneity in the cell population.

[0169] In some embodiments, this document discloses a method for analyzing an analyte, comprising: contacting a first emulsion and a second emulsion with a sample, wherein: the first emulsion comprises a first formulation encapsulated in a first liquid matrix, wherein the first formulation comprises a first plurality of fluorescent constructs and a first reagent attached to one or more of the fluorescent constructs, and a first free agent; the second emulsion comprises a second formulation encapsulated in a second liquid matrix, wherein the second formulation comprises a second plurality of fluorescent constructs and a second reagent attached to one or more of the fluorescent constructs, and a second free agent; and the sample comprises an analyte, wherein the first reagent and the second reagent are capable of binding to the analyte; and the first emulsion is then... The emulsion and the second emulsion are demulsified to allow the first plurality of fluorescent constructs and the second plurality of fluorescent constructs, as well as the first free agent and the second free agent, to diffuse in the sample. This allows at least one of the first plurality of fluorescent constructs and / or at least one of the second plurality of fluorescent constructs to interact with the analyte in the presence of the first free agent and the second free agent to generate a detectable signal. The detectable signal is analyzed to determine the presence or absence, amount or concentration, and / or activity of the analyte in the sample, and / or a first relationship between the first reagent and the second free agent and the analyte, and / or a second relationship between the second reagent and the first free agent and the analyte. In some embodiments, the first relationship is a synergistic binding effect between the first reagent and the second free agent and the analyte, and / or the second relationship is a synergistic binding effect between the second reagent and the first free agent and the analyte. In any of the foregoing embodiments, the first relationship may be an allosteric binding effect between the first reagent and the second free agent and the analyte, and / or the second relationship may be an allosteric binding effect between the second reagent and the first free agent and the analyte.

[0170] In some embodiments, this document discloses a method for labeling interactions with proteins, comprising: contacting (i) a first composition with (ii) a second composition, the first composition comprising a first liquid matrix and a sample encapsulated in the first liquid matrix, wherein the sample comprises a protein; the second composition comprising a second liquid matrix and a formulation encapsulated in the second liquid matrix, wherein the formulation comprises a plurality of fluorescent constructs and one or more analyte-interacting reagents attached to the fluorescent constructs; and combining the sample with the formulation such that the protein in the sample interacts with the one or more of the analyte-interacting reagents attached to the fluorescent constructs to generate a detectable signal, wherein the detectable signal is analyzed to analyze the interaction between the analyte-interacting reagent and the protein. In some embodiments, the analyte-interacting reagent comprises a flexible connector to reduce or avoid steric hindrance in interaction with the protein, optionally wherein the analyte-interacting reagent comprises one or more dendritic polymers. In any of the foregoing embodiments, the protein in the sample may interact with the analyte-interacting reagent in the presence of a weak gelling agent for controlling convection and / or slowing diffusion. In any of the foregoing embodiments, the method may be used for protein surface labeling.

[0171] In some embodiments, this document discloses a method for generating a population of emulsion droplets, comprising: mixing a first population of emulsion droplets, each containing one or more first fluorescent constructs, a second population of emulsion droplets, and a third population of emulsion droplets, in any suitable order, in a chamber containing a liquid matrix immiscible with first, second, and third emulsion droplets; merging the first and second populations of emulsion droplets with the third population of emulsion droplets to form merged emulsion droplets in the liquid matrix in the chamber, wherein the first and second fluorescent constructs are present in the merged emulsion droplets in a defined ratio; and dividing the merged emulsion droplets into a fourth population of emulsion droplets, wherein at least 90% of the fourth population of the fourth population contains the first and second fluorescent constructs in a defined ratio, thereby generating a fourth population of emulsion droplets. In some embodiments, the first emulsion droplet, the second emulsion droplet, and the third emulsion droplet are aqueous droplets, wherein the aqueous droplets optionally contain an antifreeze agent, such as ethylene glycol or glycerin.

[0172] In any of the foregoing embodiments, the first fluorescent construct and / or the second fluorescent construct may comprise quantum dots. In any of the foregoing embodiments, the method may further include the step of mixing the contents of the merged emulsion droplets. In any of the foregoing embodiments, the first fluorescent construct and the second fluorescent construct may be uniformly distributed within the merged emulsion droplets. In any of the foregoing embodiments, the merging step may include demulsifying the first emulsion droplet, the second emulsion droplet, and the third emulsion droplet.

[0173] In any of the foregoing embodiments, at least 95% of the fourth emulsion droplets in the population may contain the first fluorescent construct and the second fluorescent construct at the defined ratio. In any of the foregoing embodiments, at least 99% of the fourth emulsion droplets in the population may contain the first fluorescent construct and the second fluorescent construct at the defined ratio. In any of the foregoing embodiments, each of the fourth emulsion droplets in the population may contain the first fluorescent construct and the second fluorescent construct at the defined ratio.

[0174] In some embodiments, this document discloses a method for analyzing an analyte, comprising: contacting a first emulsion droplet, a second emulsion droplet, and a third emulsion droplet, wherein: the first emulsion droplet comprises a first formulation encapsulated in a first liquid matrix, wherein the first formulation comprises a first plurality of fluorescent constructs and a reagent attached to one or more of the fluorescent constructs; the second emulsion droplet comprises a second formulation encapsulated in a second liquid matrix, wherein the second formulation comprises a second plurality of fluorescent constructs and a free agent S; and the third emulsion droplet comprises a third formulation encapsulated in a third liquid matrix, wherein the third formulation comprises a free agent R; and fusing the first emulsion droplet, the second emulsion droplet, and the third emulsion droplet to allow the first plurality of fluorescent constructs and the second plurality of fluorescent constructs and the free agents S and R to diffuse in the fused emulsion droplet, wherein the reagent and the free agent R interact in the presence of the free agent S to generate a detectable signal, wherein the detectable signal indicates an interaction between the reagent, the free agent R, and the free agent S. In some embodiments, the first liquid matrix, the second liquid matrix, and / or the third liquid matrix are identical. In any of the foregoing embodiments, the first formulation, the second formulation, and the third formulation may be aqueous, the first plurality of fluorescent constructs and the second plurality of fluorescent constructs may be quantum dots, and the second plurality of fluorescent constructs may not interact with the free agent S or the free agent R. In any of the foregoing embodiments, the free agent R may be a acceptor of the reagent, and the free agent S may be a cooperating molecule that binds between R and the acceptor. In any of the foregoing embodiments, the fusion may be controllable, for example, by a laser pulse.

[0175] In some embodiments, this document discloses compositions comprising a first emulsion droplet, a second emulsion droplet, and a third emulsion droplet, wherein: the first emulsion droplet comprises a first formulation encapsulated in a first liquid matrix, wherein the first formulation comprises a first plurality of fluorescent constructs and a reagent attached to one or more of the fluorescent constructs; the second emulsion droplet comprises a second formulation encapsulated in a second liquid matrix, wherein the second formulation comprises a second plurality of fluorescent constructs and a free agent S; and the third emulsion droplet comprises a third formulation encapsulated in a third liquid matrix, wherein the third formulation comprises a free agent R; wherein the reagent is capable of interacting with the free agent R in the presence of the free agent S to generate a detectable signal indicating the interaction between the reagent, the free agent R, and the free agent S.

[0176] In any of the foregoing embodiments, the second plurality of fluorescent constructs may be quantum dots each having a particularly thick or thin shell, which reduces or enhances their diffusion rate sufficiently to allow them to be distinguished from quantum dots of the same color but with reagents attached thereto.

[0177] In some embodiments, this document discloses a method for analyzing an analyte, comprising: contacting a first emulsion droplet, a second emulsion droplet, and a third emulsion droplet, wherein: the first emulsion droplet comprises a first formulation encapsulated in a first liquid matrix, wherein the first formulation comprises a first plurality of fluorescent constructs and a reagent attached to one or more of the fluorescent constructs; the second emulsion droplet comprises a second formulation encapsulated in a second liquid matrix, wherein the second formulation comprises one or more gel beads encapsulating a second plurality of fluorescent constructs, and a free agent S; and the third emulsion droplet comprises a third emulsion... A third formulation in a matrix, wherein the third formulation comprises a free agent R; and the first emulsion droplet, the second emulsion droplet, and the third emulsion droplet are fused to allow the first plurality of fluorescent constructs, the one or more gel beads, and the free agents S and R to diffuse within the fused emulsion droplet, wherein the second plurality of fluorescent constructs in each gel bead do not diffuse outside the gel bead, wherein the reagent interacts with the free agent R in the presence of the free agent S to generate a detectable signal, and wherein the detectable signal indicates the interaction between the reagent, the free agent R, and the free agent S.

[0178] In some embodiments, this document discloses compositions comprising a first emulsion droplet, a second emulsion droplet, and a third emulsion droplet, wherein: the first emulsion droplet comprises a first formulation encapsulated in a first liquid matrix, wherein the first formulation comprises a first plurality of fluorescent constructs and a reagent attached to one or more of the fluorescent constructs; the second emulsion droplet comprises a second formulation encapsulated in a second liquid matrix, wherein the second formulation comprises one or more gel beads encapsulating a second plurality of fluorescent constructs, and a free agent S; and the third emulsion droplet comprises a third formulation encapsulated in a third liquid matrix, wherein the third formulation comprises a free agent R, wherein the second plurality of fluorescent constructs in each gel bead do not diffuse to the exterior of the gel bead, wherein the reagent is capable of interacting with the free agent R in the presence of the free agent S to generate a detectable signal indicating the interaction between the reagent, the free agent R, and the free agent S.

[0179] In any of the foregoing embodiments, the contacting step may further include contacting the first emulsion droplet, the second emulsion droplet, and / or the third emulsion droplet with a fourth emulsion droplet, wherein the fourth emulsion droplet comprises a fourth formulation encapsulated in a fourth liquid matrix, wherein the fourth formulation comprises a third plurality of fluorescent constructs and one or more gel beads encapsulating the fourth plurality of fluorescent constructs.

[0180] In any of the foregoing embodiments, the contacting step may further include contacting the first emulsion droplet, the second emulsion droplet, the third emulsion droplet and / or the fourth emulsion droplet with a fifth emulsion droplet, wherein the fifth emulsion droplet comprises a fifth formulation encapsulated in a fifth liquid matrix, wherein the fifth formulation comprises a fifth plurality of fluorescent constructs.

[0181] In any of the foregoing embodiments, the first liquid matrix, the second liquid matrix, the third liquid matrix, the fourth liquid matrix, and / or the fifth liquid matrix may be the same. In any of the foregoing embodiments, the first formulation, the second formulation, the third formulation, the fourth formulation, and / or the fifth formulation may be aqueous, the first plurality of fluorescent constructs, the second plurality of fluorescent constructs, the third plurality of fluorescent constructs, the fourth plurality of fluorescent constructs, and / or the fifth plurality of fluorescent constructs may be quantum dots, and the gel beads may not interact with the free agent S or the free agent R. In any of the foregoing embodiments, the free agent R may be a acceptor of the reagent, and the free agent S may be a cooperating molecule for binding between R and the acceptor. In any of the foregoing embodiments, the fusion may be controllable, for example, by laser pulses. Attached Figure Description

[0182] To better understand this disclosure, methods according to this disclosure will now be described by way of example only, with reference to the accompanying drawings.

[0183] Figure 1 This is a schematic diagram of an exemplary analytical chamber, in which an aqueous sample flows through the analytical chamber and fluorescent excitation light passes through the analytical chamber.

[0184] Figure 2 This is a schematic diagram of an exemplary flat optical chamber with a sample inlet and a sample outlet. A sample can be added as a large droplet in an oil matrix through the sample inlet, wherein the large droplet is compressed into a pancake shape within the chamber. The pancake-shaped droplet, together with the oil matrix, can occupy all or part of the chamber cavity. A reagent emulsion can be injected into the chamber through one or more holes at the bottom of the chamber. Fluorescent excitation light passes through the chamber from the bottom, and visible light from the fluorescence emission is detected from above the chamber.

[0185] Figure 3 This is a schematic diagram of an exemplary analytical chamber in which a set of aqueous samples passes through the chamber, and fluorescent excitation light passes through the chamber.

[0186] Figure 4 This is an exemplary schematic diagram of emulsion droplets being swept across an analytical chamber and then demulsified into a constellation of fluorescent particles.

[0187] Figure 5 This is a schematic diagram of a representative camera image from the analysis chamber, showing two different emulsion droplets being swept across the chamber and then demulsified into a constellation of fluorescent particles.

[0188] Figure 6 This is a schematic side view of an exemplary optical chamber, showing emulsion droplets being swept across the chamber and then demulsified to fuse with larger sample droplets (e.g., as in combination). Figure 2 The quantum dots from each emulsion droplet form an expanded constellation of fluorescent particles within the larger sample droplet. For example, during expansion, protein H on the blue quantum dots from the emulsion droplet binds to the analyte in the larger sample droplet. Weak pulses of the electric field can be used to demulsify the droplets, allowing the quantum dots to diffuse into the larger sample droplet. The quantum dots from each emulsion droplet form an expanded constellation of quantum dots, and laminar distribution can be mitigated by introducing static friction through the addition of a weak gelling agent.

[0189] Figure 7This is a schematic top view of an exemplary optical chamber, showing two types of emulsion droplets (Type A with 3 red, 1 green, and 4 blue droplets, and Type B with 4 red, 1 yellow, and 2 blue droplets) swept through the chamber and then demulsified to fuse with the sample, each expanding into a constellation-like population of fluorescent particles in the sample. Blue quantum dots from Type A emulsion droplets are coated with protein H, while blue quantum dots from Type B emulsion droplets are coated with protein C. Analytes in the sample bind to protein H but not to protein C. During expansion, analytes bind to the blue quantum dots from Type A emulsion droplets but not to the blue quantum dots from Type B emulsion droplets.

[0190] Figure 8 This is a schematic diagram of an exemplary chamber with multiple channels for multi-channel analysis, for example, for synergistic small molecule assemblages. The emulsion droplets can be injected into individual channels at a controlled rhythm.

[0191] Figure 9 This is an exemplary schematic diagram of emulsion droplets being swept across an analytical chamber and then demulsified into a constellation of fluorescent particles, wherein the sample itself is in the form of large droplets, and the analyte (e.g., a surfactant analyte) is concentrated at the interface between the aqueous sample droplets and a water-immiscible matrix.

[0192] Figure 10 This is a schematic side view of an exemplary optical chamber, showing emulsion droplets being swept across the chamber and then demulsified to fuse with larger sample droplets (e.g., as in combination). Figure 2 The quantum dots (as described above) expand into a constellation-like population of fluorescent particles within the large sample droplets. For example, during expansion, protein H on the blue quantum dots from the emulsion droplets binds to surfactant analytes in the sample, where the surfactant analytes are concentrated at the interface between the aqueous sample droplets and the water-immiscible matrix. Weak pulses of the electric field can be used to demulsify the droplets, allowing the quantum dots to diffuse into the large sample droplets. The quantum dots from each emulsion droplet form an expanded constellation-like population of quantum dots, and laminar distribution can be mitigated by introducing static friction through the addition of a weak gelling agent.

[0193] Figure 11 This is a schematic diagram of an exemplary optical system for measuring fluorescence emission light from an exemplary analysis chamber.

[0194] Figure 12This is a schematic diagram of an exemplary optical system for measuring fluorescence emission light from an exemplary analytical chamber, where SLAPMi image data is collected by a camera and the Brownian motion of each quantum dot in each constellation group is tracked over time. An exemplary diagram of a droplet constellation group is shown, and many simultaneous droplet constellations exist in the camera image. In the exemplary diagram, binding events have occurred for the blue quantum dots at 13, 16, 23, and 28 s, and measurements of kinetics or concentrations are given, while no binding events are shown for the red or green quantum dots. This is because these blue quantum dots bind with 4 blue + 3 red + 1 green (… Figure 7 The correlation between the droplets (type A emulsion droplets in the sample) indicates that these blue quantum dots are coated with protein H, and therefore bind between protein H and the analyte in the sample.

[0195] Figure 13 This is an example of a nutation disk and a schematic diagram of the camera image expected in the absence and presence of a nutation disk.

[0196] Figure 14 This is a schematic diagram of exemplary coherent fluorescence excitation light passing through the analysis chamber.

[0197] Figure 15 This is a schematic diagram of an exemplary spatially patterned fluorescence excitation light passing through the analysis chamber.

[0198] Figure 16 This is a schematic diagram of an exemplary vial containing five different aqueous droplet formulations.

[0199] Figure 17 This is a schematic diagram of an exemplary vial containing aqueous droplets of a reagent emulsion in an oil matrix, wherein each droplet contains a specific combination of quantum dot colors in a specific proportion.

[0200] Figure 18 Exemplary quantum dots that can be functionalized, for example, with antibodies, are shown, as well as an exemplary method for generating emulsion droplets containing quantum dots.

[0201] Figure 19 Exemplary quantum dots are shown attached to a dendritic polymer.

[0202] Figure 20 An exemplary method for the synthesis of reagent emulsions is shown, which utilizes binary emulsion fusion for a customized emulsion droplet composition.

[0203] Figure 21 An exemplary method for batch preparation of reagent emulsions is shown, using the exemplary chamber disclosed herein as the preparation chamber.

[0204] Figure 22An exemplary method for detecting surfactant analytes is shown, wherein an emulsion droplet is introduced into a chamber to contact a sample droplet before using an electric field pulse to fuse the droplet.

[0205] Figure 23 An exemplary method for detecting surfactant analytes is shown, wherein an emulsion droplet and a sample droplet are fused together by electrostatic attraction.

[0206] Figure 24 An exemplary method is shown to correlate the measured random diffusion behavior of quantum dots with the concentration of a compound, which includes molecules in a sample and in adjacent droplets that have expanded into overlapping constellation-like populations.

[0207] Figure 25 Figure A in the figure shows the results of an exemplary fluorescence polarization assay (FPA), in which unbound quantum dots rotate rapidly, resulting in fluorescence emission with randomized polarization (e.g., dissociation after association), while bound quantum dots rotate more slowly, resulting in fluorescence emission with retained polarization (e.g., association after association).

[0208] Figure 25 Figure B in the diagram shows the results of an exemplary thermal displacement measurement, where the temperature of the optical chamber can be gradually increased as quantum dots attached to an analyte (e.g., a protein) are observed. The diffusivity increases smoothly with increasing temperature. Once the analyte begins to melt, the smooth rise shows a decrease at a specific temperature. The presence of compounds in the sample can increase the melting temperature, indicating the binding of one or more compounds to the quantum dots.

[0209] Figure 25 Figure C in the diagram shows the results of an exemplary fluorescence quenching assay. Compounds containing heavy atoms bind to quantum dots, causing the quantum dots to quench fluorescence.

[0210] Figure 26 The exemplary methods disclosed herein are compared with existing technologies.

[0211] Figure 27 An exemplary method for labeling emulsion droplets using free quantum dots is shown.

[0212] Figure 28 An exemplary method for labeling emulsion droplets using free quantum dots and microbeads (e.g., gel beads) is shown.

[0213] Figure 29 The simulation shows a projected image of 90 quantum dots, with nine constellation-shaped clusters of ten quantum dots slowly spreading outwards. The binding interaction between the quantum dots and phosphodiesterase was simulated.

[0214] Figure 30 Shown in Figure 29Exemplary molecular association and / or dissociation events during the simulation are shown.

[0215] Figure 31 This is a schematic diagram of an exemplary optical system for measuring fluorescence emission light from an exemplary analytical chamber. A rotating prism (e.g., as in a Risley prism scanner) can be used instead of a diffraction grating (e.g., as in...). Figure 12 (as shown in the image).

[0216] Figure 32 An exemplary camera image using a Risley prism scanner is shown. Detailed Implementation

[0217] The selected embodiments of this disclosure will be outlined separately, and methods according to various embodiments of this disclosure will then be described. The embodiments and examples are intended to illustrate this disclosure and should not be construed as limiting it.

[0218] All publications mentioned in this application, including patent documents, scientific articles, and databases, are incorporated in their entirety by reference for all purposes, to the extent that each individual publication is individually incorporated by reference. Where a definition presented herein contradicts or is inconsistent with a definition presented in a patent, application, published application, or other publication incorporated herein by reference, the definition presented herein shall prevail over the definition presented herein by reference.

[0219] In some implementations, this document provides methods suitable for disease biomarker diagnosis, disease biomarker discovery, drug therapy discovery, and / or drug therapy evaluation. In one aspect, this document provides methods for detecting analytes that interact with a binding reagent.

[0220] In some embodiments, the assays, compositions, and methods disclosed herein include the use of heterogeneous assays or homogeneous assays, or a combination of both, in different steps. Heterogeneous assays utilize a stationary surface of the analyte-binding reagent exposed to a solubilized sample, while homogeneous assays utilize a fully solubilized analyte-binding reagent and sample.

[0221] Without departing from the true scope and spirit of this disclosure, aspects of the following exemplary determinations may be included in or excluded from the currently disclosed assay compositions and methods. Such embodiments having aspects of the following exemplary determinations, whether included or excluded, are intended to fall within the scope of this disclosure.

[0222] One exemplary assay involves the use of an electrode in which an analyte specifically binds to an electrode surface that has been derivatized with a reagent that binds to the analyte contained in the sample. This binding interaction alters the electron transport behavior of the electrode in a way that allows for the determination of the presence or concentration of the analyte.

[0223] A second exemplary assay is enzyme-linked immunosorbent assay (ELISA). ELISA uses a stationary surface coated with a series of different analyte binding reagents, which is typically exposed to a labeled sample for 30 to 120 minutes, rinsed, and then scanned for bound labels. These labels can be fluorescent, radioactive, magnetic, or have some other detectable property. For fluorescent markers, rinsing can be avoided by making the stationary surface transparent and using an evanescent field, for example, as described in patent application US2015 / 0010903A1.

[0224] A third exemplary assay is surface plasmon resonance (SPR), such as that used by Biacore. SPR uses a stationary surface coated with a series of different analyte-binding reagents, which is exposed to the sample for several minutes, and then the surface plasmon resonance is scanned. Total internal reflection from the array produces a quantitative change in the reflection angle, for example, as described in U.S. Patent Nos. 7,3732,55, 7,262,866, 7,081,958, 7,012,694, 6,999,175, 6,775,003, 6,714,303, 6,589,798, 6,493,097, 6,127,183, 5,965,456, and 5,641,640.

[0225] A fourth exemplary assay is a homogeneous assay. These include agglutination assays and fluorescence resonance energy transfer (FRET) assays. Additionally, differential scanning fluorometry (DSF) exists, for example, as used by ThermaFluor. DSF uses a hydrophobic fluorescent dye embedded in the hydrophobic center of a protein, which is exposed to an aqueous solvent as the temperature rises and the protein molecule melts. This exposure prevents the fluorescent dye from being quenched. The reagents binding to the protein affect the melting point and thus the temperature at which fluorescence is not quenched. This provides an analytical signal for binding interactions, for example, as described in U.S. Patent Nos. 6,214,293, 6,036,920, and 6,020,141.

[0226] The fifth exemplary assay involves the use of particles that are both fluorescent and magnetic and can bind to an analyte, such as those described in U.S. Patent No. 9,658,219, wherein the particles are allowed to bind to the analyte, the particles are magnetically fixed to a surface containing an evanescent wave, and detected by optical attenuation or fluorescence.

[0227] A sixth exemplary determination involves the use of derived fluorescent quantum dots that can bind to a specific analyte, such as those described in U.S. Patent No. 9,664,667, wherein the quantum dots are allowed to bind to the analyte, the quantum dots are placed near a surface containing an evanescent wave, and detected individually by optical measurements. Brownian motion of each quantum dot causes it to randomly enter and exit the evanescent field at a certain diffusion rate. This motion produces a corresponding change in the fluorescence intensity of the particle. Optical tracking provides a measurement of the diffusion rate of the quantum dots. The diffusion rate is influenced by binding interactions, which tend to increase the effective size of the quantum dots and decrease their diffusion rate. The address space of the combination is limited by a finite number of different colors of the quantum dots. A larger address space can be obtained by using a heterogeneous structure composed of different quantum dots. Such heterogeneous structures are difficult to synthesize with uniform compositions and diffusion rates, for example, as described by Yu, Wan, et al. (2009). Fluorescent bands broaden, fluorescence quantum yield decreases, and the larger size reduces the sensitivity of binding interaction measurements. Aside from using such heterogeneous structures, descriptions of how to expand the address space are not readily apparent. Furthermore, descriptions of how to measure the transition in diffusion rate during binding interactions are not readily apparent. Finally, descriptions of how to measure the association and dissociation constants of binding interactions are not readily apparent.

[0228] The seventh exemplary determination involves using an aqueous emulsion to contain a sample and deliver it to a measuring device, for example, as described in Japanese Science and Technology Agency Patent Publication WO2002 / 068104 (including U.S. Patent Nos. 7,268,167, 7,772,287, 7,717,615 and 7,375,140) and Patent Publication WO2005 / 089921 (including U.S. Patent No. 8,741,192).

[0229] The eighth exemplary assay involves using an aqueous emulsion to measure the effectiveness of antibiotics and synergistic compounds against a variety of bacteria, for example, as described by Kulesa, Kehe, et al. (2018). Random pairs of droplets are contained in a chamber, fused using an electric field stimulation, and the behavior of the individual resulting fused droplets with captured bacteria is then observed over time. The droplets are labeled with a set of quantum dots, which allows identification of the antibiotic, compound, and bacteria present in each droplet. This would require the use of a fixed array of chambers, each containing random pairs of droplets, to achieve binary pairing of droplets. Instead, this disclosure avoids the requirement of a fixed array and instead uses binary interactions without any pairs contained. This allows for simpler analytical chambers with random arrays of droplets, for example, as described in the embodiments provided herein.

[0230] The ninth exemplary determination involves the use of confocal microscopy. This method uses point illumination and a pinhole in the optical conjugate plane. This eliminates defocused light, providing higher resolution and greater accuracy when determining the location of small objects.

[0231] The tenth exemplary determination involves the use of structured illumination microscopy (SIM). This method uses interference patterns to generate moiré patterns. When determining the location of small objects, the modulated image is mathematically deconvolved to produce higher resolution and greater accuracy.

[0232] The eleventh exemplary determination involves the use of stochastic optical reconstruction microscopy (STORM). This method uses very low-intensity illumination to randomly activate fluorophores, thereby allowing for the temporary separation of individual fluorophores. When determining the location of small objects, a set of images is combined to produce higher resolution and greater accuracy.

[0233] The twelfth exemplary determination involves the use of point-scanning two-photon microscopy. This method scans short pulses of intense light across the entire sample, where the wavelength of the light is longer than that required to excite the fluorophore. At high intensity, two photons can simultaneously enter the fluorophore, doubling the effective energy and causing the fluorophore to fluoresce. This method enables high-resolution imaging of scattering samples.

[0234] The thirteenth exemplary determination involves the use of scan line angle projection microscopy (SLAPMi). This is an improvement on point-scanning two-photon microscopy, in which the excited focal lines are applied at multiple angles, similar to computed tomography methods. This method allows for very rapid image generation while maintaining high resolution within the scattering sample, as described, for example, by Kazemipour, Novak, et al. and WO2018208687A1.

[0235] The fourteenth exemplary assay involves competitive protein binding using a fluorescence quencher. A set of quantum dots is generated in which bromophenol blue is bound to a surface, with each color of quantum dot exhibiting a different binding strength to bromophenol blue. The bromine atoms of the bromophenol blue produce a heavy atom effect that quenches the fluorescence of the associated quantum dots. The addition of a sample protein will competitively adsorb bromophenol blue molecules from a subset of the quantum dots. The free quantum dots are then made to have enhanced fluorescence. Linear discriminant analysis is then used to determine the binding strength of the sample protein, for example, as described by Xu, Zhang, et al. (2016). Graphene can also be used for fluorescence quenching, for example, as described by Chou, De, et al. (2012).

[0236] Methods exist that use sample oligonucleotides already labeled with molecular fluorophores. In this disclosure, a fluorophore is not required because diffusion rate is the analytical signal. However, sample oligonucleotides can be labeled with heavy atoms (such as bromine) instead of fluorophores. If the label is attached to the proximal end of the oligonucleotide, it can partially quench the quantum dot fluorescence. This quenching will provide additional analytical data.

[0237] The fifteenth exemplary assay involves the use of phage display. A mixed population of phages with random mutations and externally expressed random proteins is generated. This mixture is applied to a plate having an array of sample proteins attached thereto. Some phages will attach to the sample proteins. Excess phages are washed away, and the bound residue is sequenced for DNA. The DNA sequence encodes the protein that will bind to the sample protein.

[0238] The sixteenth exemplary assay is the Suspension Array Technique (SAT). A mixed population of microsphere beads is generated, each containing a combination of quantum dot emission colors at a specific intensity ratio, and coated with a specific reagent (e.g., an antigen or oligonucleotide). When fluorescent excitation light is applied, the pattern of fluorescence emission colors and amplitudes provides identification of each bead, thereby providing identification of its reagent coating. Upon incubation with a sample, specific sample molecules bind to the specific bead-binding reagent; subsequent washing and incubation with fluorescent molecules create molecular interlayers, and the bead population is characterized by flow cytometry, which identifies the bead-binding reagent via quantum dot fluorescence and the occurrence of binding via fluorescent molecule fluorescence. This method is commercially used by Bio-Rad and other companies and is known as the Luminex xMAP and Bio-Plex technologies.

[0239] In one aspect, this document provides a method for detecting an analyte interacting with an analyte binding agent, comprising: (a) providing a first liquid, wherein the first liquid comprises a portion of a sample, for example, a biological sample may be mixed with and / or diluted with a buffer to form the first liquid; and (b) providing an emulsion formed between (i) a liquid droplet comprising a second liquid and (ii) a liquid matrix, wherein the liquid droplet comprises a plurality of fluorescent constructs having an analyte binding agent attached to all or a group of fluorescent constructs. In some embodiments, both the first liquid and the second liquid are aqueous, and the liquid droplet is an aqueous droplet. In some embodiments, the method further comprises contacting the first liquid with the aqueous droplet to form a combination of the first liquid and the second liquid under conditions that allow the first liquid and the second liquid to merge into a single fluid. In some embodiments, the method further comprises detecting a signal generated by the binding interaction of the analyte with the analyte binding agent, the signal providing an indication of the presence or absence, amount or concentration, and / or characteristics or activity (e.g., binding activity) of the analyte. In any of the foregoing embodiments, the emulsion comprises a plurality of liquid droplets in a liquid matrix.

[0240] Methods for forming mixed droplets and generating and manipulating microfluidic droplets are known, see, for example, US 9,364,803 and US2019 / 0060861, and can be used in conjunction with one or more embodiments disclosed herein.

[0241] Methods for controlled fusion and sample analysis of emulsion droplets are described in the following documents: U.S. Provisional Application No. 62 / 768,743, filed November 16, 2018, entitled “Methods for the Detection of Analyte Concentrations and Binding Interactions”; U.S. Provisional Application No. 62 / 768,754, filed November 16, 2018, entitled “Methods for Controlled Merging of Emulsion Droplets”; and U.S. Serial No. 16 / 685,376, filed November 15, 2019, entitled “Compositions and Methods for Controllably Merging Emulsion Droplets and Sample Analysis”, the contents of which are incorporated herein by reference in their entirety for all purposes. Figure 20An exemplary method for synthesizing reagent emulsions is illustrated, which utilizes binary emulsion fusion for a customized emulsion droplet composition. Each initial droplet may contain a specific reagent or may contain a single cell, such as a cell that has been lysed or is to be lysed. In some embodiments, binary emulsion droplets are provided and contain two different dyes (e.g., fluorescent dyes).

[0242] In some embodiments, methods including controlled fusion of emulsion droplets are used to generate emulsion droplets containing various combinations of quantum dots, fluorescent dyes, and / or reagents. In some embodiments, different electrostatic charges and / or different surfactants are used to assemble binary emulsion droplets. In some embodiments, the method includes contacting (i) a first emulsion containing a first aqueous droplet in a first liquid matrix with (ii) a second emulsion containing a second aqueous droplet in a second liquid matrix under conditions allowing the first aqueous droplet and the second aqueous droplet to fuse to form fused droplets, wherein the fusion is controlled and provided by: (i) the first aqueous droplet containing a first redox substance and a first liquid matrix containing a first electrolyte, and the second aqueous droplet containing a second redox substance and a second liquid matrix containing a second electrolyte, wherein the first aqueous droplet and the second aqueous droplet are each in contact with an electrode, thereby causing the... (ii) charge transfer between the aqueous droplet and the electrode; (iii) a first emulsion stabilized with a first charged surfactant and a second emulsion stabilized with a second charged surfactant having an opposite charge to the first charged surfactant; (iv) contacting the first emulsion with a positive electrode sufficient to induce electrostatic charging of the first aqueous droplet and contacting the second emulsion with a negative electrode sufficient to induce electrostatic charging of the second aqueous droplet; and / or (iv) the first aqueous droplet containing a first magnetic particle and the second aqueous droplet containing a second magnetic particle, wherein an external magnetic field is applied to generate an attractive force between the first magnetic particle and the second magnetic particle.

[0243] In some embodiments, this document provides a method for electrostatically charging droplets, for example, thereby generating negatively or positively charged droplets for fusion with droplets of opposite charge, such as... Figure 20As shown. In some embodiments, a tube or channel with an inner diameter or size approximately the same as that of the droplet is used. For example, the emulsion can flow through the tube or channel. In some embodiments, the tube or channel is (or contains) a charged hydrophobic conductor, and charge can be transferred to the passing droplet without damaging it. For two such channels (with opposite charges), the emulsion droplet flow is immediately directed into a non-conductive Y-shaped device, causing the oppositely charged droplets to coalesce into binary pairs. Electrostatic adsorption to the conductor may occur because the ionic attraction is greater than the electrostatic repulsion. In some embodiments, a gelling agent is added to the oil matrix, and the two oppositely charged channels are cooled to gel the oil matrix, thereby helping to push the droplets away from the conductive electrode, followed by heating under the Y-shape. The oil can be gelled by using reagents (e.g., polyisobutylene and polydimethylsiloxane).

[0244] This document also provides a method for generating emulsion droplet populations, comprising: in a chamber containing a liquid matrix immiscible with the first emulsion droplet, the second emulsion droplet, and the third emulsion droplet, respectively, a first emulsion droplet population comprising one or more first fluorescent constructs, a second emulsion droplet population comprising one or more second fluorescent constructs, and a third emulsion droplet (e.g., as shown in the image) Figure 21 As shown, large droplets containing reagent R are mixed in any suitable order; the first and second emulsion droplet groups are combined with the third emulsion droplet to form combined emulsion droplets in the liquid matrix in the chamber, wherein the first and second fluorescent constructs are present in the combined emulsion droplets in a defined ratio; the combined emulsion droplets are divided into a fourth emulsion droplet group, wherein at least 90% of the fourth emulsion droplets in the group contain the first and second fluorescent constructs in a defined ratio, thereby generating a fourth emulsion droplet group. A weak electrical pulse can be used to break up the emulsion in the chamber and combine the first, second, and third emulsion droplets. The contents of the combined emulsion droplets can be mixed such that the first and / or second fluorescent constructs, for example, quantum dots, can be uniformly distributed in the combined emulsion droplets. Statistically, virtually all fourth emulsion droplets generated from the merged emulsion droplets contain, in a defined ratio, both a first fluorescent construct and a second fluorescent construct. In some embodiments, at least 99% of the fourth emulsion droplets in the population contain, in the defined ratio, both the first and second fluorescent constructs.

[0245] In some embodiments, this document provides a method for detecting an analyte interacting with an analyte binding agent, comprising contacting (i) a first liquid with (ii) an emulsion, the first liquid comprising a portion of a sample; the emulsion being formed between a liquid droplet comprising a second liquid and a liquid matrix, wherein the liquid droplet comprises a plurality of fluorescent constructs having an analyte binding agent attached to all or a set of fluorescent constructs. In some embodiments, both the first liquid and the second liquid are aqueous, and the liquid droplet is an aqueous droplet. In some embodiments, the method further comprises forming a combination of the first liquid and the second liquid under conditions that allow the first liquid and the second liquid to fuse into a single fluid. For example, the method may include breaking the emulsion to allow the liquid droplets to fuse into the first liquid. In some embodiments, the emulsion comprises a plurality of liquid droplets suspended or encapsulated in the liquid matrix. In some embodiments, the method further comprises detecting a signal generated by the binding interaction of the analyte with the analyte binding agent, the signal providing an indication of the presence or absence, amount or concentration, and / or properties or activity (e.g., binding activity) of the analyte.

[0246] In some embodiments, this document provides a method for detecting an analyte interacting with an analyte-binding reagent, the method comprising: (a) providing a first liquid containing the analyte; (b) providing an emulsion formed between (i) a reagent droplet containing a second liquid and (ii) a water-immiscible host matrix, wherein the reagent droplet contains a plurality of fluorescent constructs having an analyte-binding reagent attached to all or a group of the fluorescent constructs; (c) mixing the first liquid and the reagent droplet together in an analytical chamber, allowing the first liquid and the reagent droplet to fuse into a single fluid; (d) after said mixing, guiding excitation light having a controlled phase through the analytical chamber sufficient to induce fluorescence in one or more of the fluorescent constructs, wherein said fluorescence depends on the position of the fluorescent construct within a local phase angle of said excitation light; and (e) after said mixing step, detecting fluorescence emission and / or measuring a magnitude of said fluorescence emission. In some embodiments, the method further includes identifying each analyte-binding reagent present in each reagent droplet based on, for example, a pattern of fluorescence emission wavelengths near the location where each droplet fuses with the first liquid. In some implementations, variations in the random behavior of fluorescence emission values ​​provide an indication of the presence or absence, amount or concentration, and / or properties or activity (e.g., binding activity) of an analyte.

[0247] In some embodiments, this document provides a method for detecting an analyte interacting with an analyte-binding reagent, comprising contacting a first liquid with an emulsion in an analytical chamber, the first liquid containing the analyte, the emulsion being formed between (i) reagent droplets containing a second liquid and (ii) a water-immiscible host matrix, wherein the reagent droplets contain a plurality of fluorescent constructs having an analyte-binding reagent attached to all or a group of fluorescent constructs. In some embodiments, the method further includes allowing the first liquid and the reagent droplets to fuse into a single fluid. For example, the method may include breaking the emulsion to allow the reagent droplets to fuse into the first liquid. In some embodiments, the method further includes guiding excitation light having a controlled phase through the analytical chamber after the mixing, sufficient to induce fluorescence in one or more of the fluorescent constructs, wherein the fluorescence depends on the position of the fluorescent construct within a local phase angle of the excitation light. In some embodiments, the method further includes detecting fluorescence emission and / or measuring a magnitude of fluorescence emission after the mixing step. In some embodiments, the method further includes identifying each analyte-binding reagent present in each reagent droplet based on, for example, a pattern of fluorescence emission wavelengths near the location where each droplet fuses with the first liquid. In some implementations, variations in the random behavior of fluorescence emission values ​​provide an indication of the presence or absence, amount or concentration, and / or properties or activity (e.g., binding activity) of an analyte.

[0248] In some embodiments, this document provides a method for detecting the interaction between an analyte and an analyte-binding reagent in a sample, wherein the method comprises: (a) providing a first liquid containing the analyte; (b) providing an emulsion formed between (i) a reagent droplet containing a second liquid and (ii) a water-immiscible host matrix, wherein the reagent droplet contains a plurality of fluorescent constructs having an analyte-binding reagent attached to all or a group of fluorescent constructs; (c) mixing the first liquid and the reagent droplet together in an analytical chamber, allowing the first liquid and the reagent droplet to fuse into a single fluid; (d) after said mixing, guiding a controlled spatial pattern of excitation light through the analytical chamber sufficient to induce fluorescence in one or more of the fluorescent constructs, wherein said fluorescence depends on the position of the fluorescent construct within the local spatial pattern of said excitation light; and (e) after said mixing step, detecting fluorescence emission and / or measuring a magnitude of fluorescence emission. In some embodiments, the method further includes determining the identity of each analyte-binding reagent present in each reagent droplet based on, for example, a pattern of fluorescence emission wavelengths near the location where each droplet fuses with the first liquid. In some implementations, variations in the random behavior of fluorescence emission values ​​provide an indication of the presence or absence, amount or concentration, and / or properties or activity (e.g., binding activity) of an analyte.

[0249] In some embodiments, this document provides a method for detecting an analyte interacting with an analyte-binding reagent, comprising contacting a first liquid with an emulsion in an analytical chamber, the first liquid containing the analyte, the emulsion being formed between (i) reagent droplets containing a second liquid and (ii) a water-immiscible host matrix, wherein the reagent droplets contain a plurality of fluorescent constructs having an analyte-binding reagent attached to all or a group of fluorescent constructs. In some embodiments, the method further includes allowing the first liquid and the reagent droplets to fuse into a single fluid. For example, the method may include breaking the emulsion to allow the reagent droplets to fuse into the first liquid. In some embodiments, the method further includes guiding a controlled spatial pattern of excitation light through the analytical chamber after the mixing, sufficient to induce fluorescence in one or more of the fluorescent constructs, wherein the fluorescence depends on the position of the fluorescent construct within the local spatial pattern of the excitation light. In some embodiments, the method further includes detecting fluorescence emission and / or measuring the magnitude of fluorescence emission after the mixing step. In some embodiments, the method further includes determining the identity of each analyte-binding reagent present in each reagent droplet based on, for example, a pattern of fluorescence emission wavelengths near the location where each droplet fuses with the first liquid. In some implementations, variations in the random behavior of fluorescence emission values ​​provide an indication of the presence or absence, amount or concentration, and / or properties or activity (e.g., binding activity) of an analyte.

[0250] In some aspects, the methods disclosed herein use a sample liquid containing an emulsion mixture of one or more analytes and aqueous reagent droplets. For example, each aqueous reagent droplet may contain one or more analyte-binding reagents. In some aspects, the sample liquid contains one or more analytes to be analyzed by the methods disclosed herein. The sample liquid may be in the form of emulsified droplets. In some aspects, each reagent droplet contains one or more fluorescent constructs, such as fluorophores or particles containing fluorophores. In some embodiments, one or more of the fluorescent constructs contain reagents capable of binding to one or more analytes. In some embodiments, the reagent is attached to the surface of one or more fluorescent constructs. For example, the reagent may be covalently or non-covalently attached to the surface, and the reagent may be directly attached to the surface or indirectly attached to the surface, for example, via a connector.

[0251] In some aspects, after the sample liquid and the reagent droplets are brought into contact within the analytical chamber, the sample liquid and the liquid within the reagent droplets can be combined, for example, fused into a homogeneous liquid. In some embodiments, during liquid merging, fluorescent constructs containing one or more analyte-interacting (e.g., analyte-binding) reagents are allowed to interact with one or more analytes; for example, binding interactions may occur. In some aspects, excitation light is applied to irradiate the liquid mixture, producing fluorescence emission, and the fluorescence emission of each fluorescent construct is continuously monitored and tracked. In some aspects, fluorescence emission is continuously monitored and tracked for all fluorescent constructs from the same reagent droplet.

[0252] In some embodiments, the phase or spatial pattern of the excitation light is controlled, such that the electric and magnetic field vectors of the excitation light are non-uniform throughout the analytical chamber. As the fluorescent construct (e.g., fluorescent particles, such as quantum dots) moves with Brownian motion in the mixed liquid, its fluorescence will vary with the magnitudes of the local electric and magnetic field vectors. In some aspects, the movement is influenced by binding interactions with the analyte, and thus affects the random behavior of the magnitude of fluorescence emission. In some aspects, the shift in the random behavior of fluorescence emission magnitude after mixing constitutes a measurement of the analyte, such as its presence or absence, amount or concentration, and / or properties or activity (e.g., binding activity).

[0253] In some embodiments, the identity of the reagent is determined by a pattern of fluorescence wavelengths associated with each droplet. In some embodiments, after mixing, the fluorescent constructs within the droplets begin to diffuse outward through the sample liquid. In some aspects, this outward diffusion forms a slowly expanding constellation-like population of localized fluorescent constructs, such as quantum dots. In some aspects, the expanding constellation-like population of fluorescent constructs unfolds and allows for the detection of individual fluorescent constructs. In some aspects, the population of fluorescent constructs within the droplet is large enough to allow for the unique identification of individual droplets and therefore their associated one or more reagents. In some aspects, the population of fluorescent particles within the droplet allows for the individual differentiation of fluorescent constructs by optical systems or detection algorithms.

[0254] In some embodiments, methods for analyzing analytes are disclosed herein, such as for detecting the presence or absence, amount, and / or activity of an analyte in a sample. In some embodiments, the method includes contacting (i) a first composition with (ii) a second composition, the first composition comprising a liquid phase and a sample in the liquid phase, the second composition comprising a liquid matrix and a formulation encapsulated in the liquid matrix, wherein the formulation comprises a plurality of fluorescent constructs and one or more analyte-interacting reagents attached to the fluorescent constructs. In some embodiments, the method further includes, for example, combining the liquid phase and the formulation under conditions that allow the liquid phase and the formulation to fuse into a single fluid. In some embodiments, the method further includes allowing the analyte in the sample to interact with one or more of the analyte-interacting reagents attached to the fluorescent constructs. In some embodiments, the interaction between the analyte in the sample and the analyte-interacting reagent generates a detectable signal. In some embodiments, the detectable signal is analyzed, for example, for detecting the presence or absence, amount, and / or activity of an analyte in the sample.

[0255] The section headings used in this article are for organizational purposes only and are not intended to limit the topics described.

[0256] I. Reagent diffusion-based sample analysis methods

[0257] In some embodiments, the aqueous sample to be analyzed is contacted with an emulsion, wherein the emulsion is formed from droplets of water containing fluorescent particles that can bind to one or more analytes in the sample. In some embodiments, the contact is performed in an analytical chamber. Inside the analytical chamber, the emulsion droplets and the aqueous sample are fused, allowing the fluorescent particles to bind to one or more analytes in the sample. In some embodiments, fluorescent excitation light is applied to the fused mixture. The characteristics of the fluorescence emission are measured, for example, to provide readings of the concentration and / or binding interactions of one or more analytes, which can provide useful information about biological samples, such as liquid biopsy samples used for cancer detection in patients.

[0258] In some implementations, an aqueous sample is mixed with multiple reagent droplets in an analytical chamber. The aqueous sample may contain an analyte to be analyzed. Each reagent droplet may contain one or more fluorescent particles, at least one of which is attached to a reagent capable of interacting with one or more analytes in the sample. For example, each of the one or more fluorescent particles may have a surface coated with one or more analyte-binding reagents.

[0259] In some embodiments, the aqueous sample may exist in the form of sample droplets in an immiscible matrix. In some embodiments, the surface of the sample droplets contains or is treated with a surfactant. In some embodiments, the reagent droplets are emulsions or gel beads contained within a water-immiscible host matrix, or within an aqueous matrix. In some embodiments, the surface of the reagent droplets may be stabilized with a surfactant. When surfactants are used for both the aqueous sample and the reagent droplets, in some embodiments, the two surfactants may carry the same charge, opposite charges, or no charge.

[0260] In some embodiments, the surfactant is or comprises an amphiphilic molecule containing a polar hydrophilic group and a nonpolar hydrophobic group. The polar hydrophilic group may be positively charged by the cationic portion, negatively charged by the anionic portion, and be nonionic or amphoteric. Examples of cationic surfactants include benzalkonium chloride, benzyl chloride, cetrimonium bromide, stearic acid chloride, tetramethylammonium hydroxide, and thonzonium bromide. Examples of anionic surfactants include sodium alkyl sulfate, alkylbenzene sulfonates, chlorothioesters, perfluoroalkyl sulfonic acids, phospholipids, and thioesters. Examples of nonionic surfactants include alkyl polyglycosides, alkyl glucosides, cetyl alcohol, glyceryl monostearate, maltodextrin, nonyl alcohol ether, polysorbate, and sorbitan stearate. Examples of amphoteric surfactants include iminopropionate, iminoacetate, lauryl betaine, betaine citrate, sodium hydroxymethylglycinate, sodium lauryl amphoteric acetate, and (carboxymethyl)dihydroxymethylammonium hydroxide. Amphoteric surfactants are also known as zwitterionic surfactants.

[0261] In some embodiments, the emulsions are stabilized by one or more surfactants because their droplets have similar surface charges that repel other droplets, thereby minimizing droplet aggregation. In some embodiments, the surfactants self-assemble onto the surface of each droplet such that the polar hydrophilic groups of the surfactant molecules face the droplet, and the nonpolar hydrophobic groups of the surfactant molecules are away from the reagent droplet.

[0262] In some embodiments, the surface of the aqueous sample or reagent droplet is saturated with proteins that may exist as membrane proteins within the cell. These proteins have hydrophobic components that extend into the phospholipid bilayer of the cell membrane. In some embodiments, these proteins are considered surfactants.

[0263] In some embodiments, reagent droplets of the emulsion are suspended in a host matrix. In some embodiments, the host matrix of each emulsion is a water-immiscible liquid or liquid crystal. Examples of host matrices are lipids, oils, hydrocarbon fluids, fluorocarbon fluids, chlorinated hydrocarbon fluids, brominated hydrocarbon fluids, iodinated hydrocarbon fluids, silicone fluids, and mixtures thereof. Commercially available fluorocarbon fluids include Fluorinert FC-40, Fluorinert FC-43, Fluorinert FC-70, Fluorinert FC-75, Fluorinert FC-3283, and perfluoronaphthenes. In some embodiments, the host matrix is ​​selected such that its refractive index is close to that of the aqueous sample, thereby minimizing optical distortion in the analytical chamber. In some embodiments, the host matrix is ​​selected such that it forms a gel or vitrifies at low temperatures to enhance long-term storage stability.

[0264] In some embodiments, the density of the reagent droplets relative to their host matrix imposes gravitational-driven motion on the droplets, e.g., causing them to float upwards or sink downwards, which tends to compact the droplets together and reduce emulsion stability. In some embodiments, gravitational-driven movement is mitigated by Brownian motion, thereby improving emulsion stability. In some embodiments, the methods disclosed herein include promoting Brownian motion of the reagent droplets to mitigate gravitational-driven motion of the droplets.

[0265] In some embodiments, emulsions are formed using known methods by combining a slowly flowing aqueous composition (e.g., an aqueous medium) with a rapidly flowing water-immiscible composition (e.g., a water-immiscible medium) within a flow cell. In some embodiments, the method produces aqueous droplets of highly uniform size within a water-immiscible host matrix. Secondary emulsions can also be formed consisting of water-immiscible droplets within the aqueous droplets, the water-immiscible droplets themselves being within a water-immiscible host matrix. Such emulsion methods are commercially performed, for example by the Japan Science and Technology Agency. The production of equivalent gel beads is commercially performed, for example by xTAG and MagPlex microspheres manufactured by Luminex Corporation.

[0266] In some embodiments, the aqueous composition contains multiple fluorescent particles, for example, the resulting aqueous droplets contain fluorescent particles. In some embodiments, the concentration of fluorescent particles in the aqueous composition is set such that a statistically significant population of fluorescent particles exists in each aqueous droplet, wherein the number of fluorescent particles is approximately known. In some embodiments, fluorescence-based classification of the resulting droplets can be performed to produce a population of droplets containing a more controlled population of fluorescent particles. Water-immiscible droplets within the secondary emulsion may also contain fluorescent particles.

[0267] In some embodiments, the aqueous composition contains a mixture of fluorescent particles having a known set of fluorescence emission spectra (e.g., colors), each fluorescent particle having a known concentration in the aqueous composition. The resulting droplets can be collected into the resulting emulsion.

[0268] In some embodiments, the aqueous composition contains fluorescent particles having a known concentration of a known single fluorescence emission spectrum (e.g., color). In some embodiments, a plurality of such aqueous compositions are used to produce individual emulsions. Two or more individual emulsions can then be fused, for example, individual droplets from each individual emulsion can be fused into a larger droplet. The larger droplet can be collected into the resulting emulsion.

[0269] In some embodiments, the population of fluorescent constructs (e.g., particles) in each droplet of the resulting emulsion comprises a combination of different emission wavelengths with different fluorescent construct counts. For example, droplets can be fabricated each having approximately 6 fluorescent particles emitting red light, 9 fluorescent particles emitting green light, and 3 fluorescent particles emitting blue light. In some embodiments, the different particle populations uniquely label the droplets, and the combination of different emission wavelengths and different fluorescent construct counts collectively provides a large address space.

[0270] In some implementations, the population of fluorescent constructs (e.g., particles) in each droplet is large enough to ensure that the number of fluorescent particles in each droplet is statistically consistent during manufacturing, yet small enough to allow an optical detection system to distinguish individual fluorescent constructs (e.g., particles). Examples of fluorescent constructs (e.g., particles) include quantum dots, carbon dots, green fluorescent protein (GFP) molecules, luciferin molecules, and the commercial dyes Alexa Fluor 405 and Alexa Fluor 647.

[0271] In some embodiments, the fluorescent construct is a quantum dot, which typically has a semiconductor core surrounded by a protective layer and is generally about 5 nm in diameter. In some embodiments, a reagent layer that can bind to the analyte is fixed to the outer surface of one or more quantum dots. Typical protein analytes range in diameter from 10 nm to 50 nm. Therefore, the attachment of a protein analyte to a fluorescent construct (e.g., a particle such as a quantum dot) typically produces large changes in the hydrodynamic properties of the fluorescent construct.

[0272] In some embodiments, fluorescent particles with a bola structure are synthesized and used herein. These fluorescent particles typically comprise two fluorescent particles connected by a flexible connector (e.g., a homogeneous bola) or by a single fluorescent particle, and a magnetic particle connected by a flexible connector (e.g., a heterogeneous bola). An example of using such a bola structure is for fluorescence resonance energy transfer (FRET) analysis. In some embodiments, a flexible connector is used, and said flexible connector exhibits binding interactions with nucleic acids in aqueous samples, wherein the binding interactions affect the flexibility of the connector and thus affect the mobility of the fluorescent particles. In some embodiments, a magnetic field with a heterogeneous bola is used to control the movement of the fluorescent particles within the heterogeneous bola, for example by dragging the fluorescent particles in circular motion within and outside the path of the fluorescent excitation light. Other types of fluorescent constructs, such as fluorescent proteins, fluorescent molecules, and polymer spheres containing quantum dots, fluorescent proteins, fluorescent molecules, and combinations thereof, can be used in the methods or compositions disclosed herein.

[0273] In general, quantum dots have a fairly broad excitation spectrum, meaning that a single excitation light can be used to excite a wide variety of quantum dots, resulting in fluorescence of different colors. Typically, ultraviolet light is used for quantum dot excitation. Unbound by any particular theory, the excitation of fluorescent constructs can be generated by the electric field vector of the excitation light.

[0274] Figure 18 Exemplary quantum dots that can be functionalized, for example, with antibodies are shown. An aqueous solution sample containing quantum dots can be passed through a channel in which a water-immiscible matrix (e.g., oil) flow intercepts the aqueous solution flow to produce emulsion droplets containing quantum dots. Dendritic polymer quantum dots are known, and dendritic polymers (e.g., PAMAM dendritic polymers) can be linked to functionalized quantum dots, such as... Figure 19 As shown. Methods for synthesizing PAMAM dendritic polymers are known. See, for example, Hong et al., Langmuir 2005, 21, 4257-4261.

[0275] If near-infrared two-photon fluorescence excitation is used, and if the sample contains monomers and photocatalysts, the application of focused ultraviolet light can be used to form polymeric gel beads within the optical chamber, thereby capturing specific quantum dots for subsequent collection. SLAPMi technology uses near-infrared two-photon fluorescence excitation and can significantly reduce image scattering in complex biological samples. Two-photon excitation also provides better particle localization than standard excitation with ultraviolet light and eliminates background fluorescence. Furthermore, the optical technique can collect data at 1000 samples / second.

[0276] Quantum dots have amine or carboxyl groups on their surface, which can be used to attach proteins or oligonucleotides.

[0277] Because quantum dots are much denser than water, a brief application of ultrasound will cause the water around each quantum dot to oscillate, which can loosen non-specifically bound materials but not the target.

[0278] In some respects, using quantum dots instead of gel beads offers several advantages. For example, only one molecule needs to be attached to a quantum dot to detect its diffusion properties, whereas gel beads require many analytes bound to their surface. This makes the method faster and more sensitive. Furthermore, using multiple quantum dots of each color implies multiple measurements. As shown in the Brownian motion diagram, binding occurs several times, and together they provide a clearer measurement of the binding kinetics. Using SLAPMi optics, binding can be measured at 1000 Hz. Finally, the analytes do not need to be labeled, as the diffusion behavior of the quantum dots is the analytical signal.

[0279] For weakly bound analytes, the Brownian motion graph may also reveal accidental dissociation events, indicated by a sudden increase in the diffusion rate of a particular quantum dot. This is a fast, real-time measure of the binding interaction.

[0280] When irradiated by a fluorescent excitation source, quantum dots can exhibit random flickering of fluorescence emission (intermittent photoluminescence). This phenomenon has been extensively studied, and existing literature suggests that it may be due to localized electric fields caused by high excitation power, nonradiative Auger recombination, and / or surface trap-induced recombination. In some embodiments, this flickering is reduced by modifying the chemical properties of the quantum dot surface, for example, the work of Thomas, Ghimire et al. (2018). In some embodiments, this flickering is reduced by using gradient alloy quantum dots, for example, the work of Zhang, Wang, et al. (2019). In some embodiments, this flickering is reduced by using a thick shell, for example, the work of Reid, McBride, et al. (2018).

[0281] In some implementations, fluorescence quenching assays are used to detect the random constellation-like population diffusion disclosed herein. Compounds containing heavy atoms bound to the quantum dots may cause quenching of the quantum dot fluorescence. Inherent quantum dot scintillation may interfere with fluorescence quenching analysis, and as disclosed herein, several techniques can be used to limit inherent quantum dot scintillation.

[0282] Because multiple quantum dots can be used within each droplet or gel bead, it is easy to exclude those that flicker from the data analysis and use only the remaining quantum dots that do not flicker during the measurement.

[0283] Typically, the emission spectrum of quantum dots is relatively narrow and depends on the semiconductor composition and the physical size of the quantum dots. At room temperature, in the visible light range, a single quantum dot typically has a fluorescence emission spectral band (e.g., color) that is about 10 to 15 nm wide.

[0284] Quantum dots with a combined color width of 30 to 50 nm are commercially available. In these examples, the 30 to 50 nm width of each fluorescence emission band (e.g., color) means that when using quantum dot particles, only about nine emission bands can be spectrally distinguished. Therefore, for a collection of fused droplets each containing only one fluorescent particle, only nine combinations of samples and reagents in the collection can be simultaneously and definitively identified. In these examples, the fluorescent particle serves as a marker in an address space with only nine combinations.

[0285] In some embodiments, this disclosure includes fused droplets containing multiple quantum dots of various emission colors in various counts, and with an address space much larger than that of droplets each containing only one fluorescent particle. For example, the fused droplets may contain six reagent-labeled red quantum dots, nine unlabeled green quantum dots, and three unlabeled blue quantum dots.

[0286] In some implementations, for nine possible colors and three possible values ​​for each color, the address space becomes r(n! / (r!(n–r)!), a sum of r = 1 to n, where n = 9 × 3 = 27. This gives a total of 2,359,296 combinations.

[0287]

[0288] If more than one reagent label is used, the quantity may be larger.

[0289] The address space can be further increased by using fluorescent particles with different fluorescence lifetimes and by using an optical detection system capable of measuring fluorescence lifetimes. The address space can be further increased by using chemicals on the fluorescent particles that cause fluorescence quenching during binding interactions. The address space can also be further increased by using chemicals (other than reagent molecules) that cause absorption at some wavelengths of the fluorescence excitation light on the fluorescent particles. Such chemicals reduce fluorescence if the fluorescence excitation light is confined to such wavelengths. These chemicals can be dyes with bandpass, bandstop, notch, high-pass, or low-pass absorption spectra. By using fluorescence excitation light with controlled wavelengths, fluorescent particles with the same emission wavelength but different dye coatings can be distinguished. For example, when the fluorescence excitation light is switched between 350 nm and 450 nm, a red quantum dot with a 350 nm absorbing dye coating and a second red quantum dot with a 450 nm absorbing dye coating can be distinguished based on fluorescence intensity.

[0290] In some embodiments, the mixing of an emulsion of aqueous sample with reagent droplets can be achieved by a stream flowing into the thin-layer analysis chamber. In some embodiments, the analysis chamber is sized such that its internal volume is much smaller than its length and width, exhibiting a sheet-like shape with a top and bottom surface and thin edges. In some embodiments, the walls of the analysis chamber are treated to be hydrophobic. In some embodiments, the walls of the analysis chamber comprise a chemically bonded surface or a thin coating of oil or other water-immiscible liquid (e.g., an emulsion matrix).

[0291] In some implementations, the aqueous sample flows into one end of the analysis chamber, and as it passes through the chamber and flows out from the opposite end, it spreads into a thin layer. For example, as... Figure 1 As shown, aqueous sample 3 flows into analysis chamber 1, diffuses into a flat area, and then exits through outlet 4. Fluorescent excitation light 2 from one side of the analysis chamber passes through the analysis chamber, and fluorescence emission 5 exits the analysis chamber on the other side. Emulsion droplets containing quantum dots can be introduced into the chamber, for example, as... Figure 2 As shown in the image.

[0292] In some implementations, a set of aqueous samples flows into multiple inlets of the analytical chamber. For example, such as Figure 3 As shown, a group of aqueous samples 3 flows into the analysis chamber 1, through a thin-layer region, and then exits at the outlet 4. Fluorescent excitation light 2 from one side of the analysis chamber passes through it, and fluorescent emission 5 exits on the other side. The analysis chamber may include multiple separate chambers or channels, each with its own... Figure 3 The text shows separate entrances and separate exits.

[0293] In some embodiments, an aqueous sample is introduced into the analytical chamber before the emulsion or gel beads are introduced. In some embodiments, an aqueous sample is introduced into the analytical chamber after the emulsion or gel beads are introduced. In some embodiments, an aqueous sample and an emulsion or gel beads are introduced into the analytical chamber simultaneously. In some embodiments, when the emulsion or gel beads are introduced into the chamber, the aqueous sample flows through the analytical chamber, for example, entering the chamber through an inlet and exiting the chamber through an outlet. In some embodiments, the emulsion droplets or gel beads are spread or otherwise distributed across the entire inner surface of the analytical chamber to facilitate subsequent analysis based on the diffusion of fluorophores from the emulsion droplets or gel beads; for example, the emulsion or gel beads may be swept across by the aqueous sample in one or more directions. In some embodiments, an external force may be applied to the chamber during or after the introduction of the emulsion droplets or gel beads to distribute the emulsion droplets or gel beads on the inner surface of the chamber. In some embodiments, gravity may be used to distribute the emulsion droplets or gel beads. For example, the analytical chamber may be shaken, rotated, tilted, or otherwise moved or agitated.

[0294] In some embodiments, a water-immiscible matrix (e.g., fluorocarbon oil) is pumped into the inlet of the analytical chamber, causing the aqueous sample to be propelled through the chamber and exit from the distal end. In some embodiments, a weak gelling agent is added to the aqueous sample to minimize laminar flow through the analytical chamber. In some embodiments, reagent emulsion droplets or gel beads are swept along with the sample flow by gravity and the aid of a weak gelling agent.

[0295] The analytical chamber may include a substantially flat, rigid substrate on which emulsion droplets or gel beads are situated or assembled. In some embodiments, the substrate is transparent to radiation at excitation and emission wavelengths used to excite and detect various labels (e.g., fluorescent dyes, quantum dots, plasmon resonance particles, nanoclusters, or any suitable combination thereof), for example, between approximately 400-900 nm. Materials (e.g., glass, plastic, quartz, etc.) are suitable. Emulsion droplets or gel beads can be adhered to the substrate, and can optionally be adhered using any of a variety of methods. The substrate may be coated with or without a substance that enhances adhesion or bonding, such as silanes, polylysine, etc. The substrate may have pores or depressions to accommodate the emulsion droplets or gel beads before they are incorporated into the sample to be analyzed. In various embodiments, raised barriers or masks may be used for this purpose.

[0296] In some implementations, as the aqueous sample passes through the analysis chamber, the emulsion flows into an orifice on one side of the analysis chamber. For example, as... Figure 4As shown, when the aqueous sample 3 flows through the analysis chamber 1, an emulsion or gel bead 5 flows into the analysis chamber. Each emulsion droplet or gel bead forms a protrusion 6, sweeping downwards along the length of the analysis chamber 1 and being trapped under a sheet of aqueous sample 3 (much like a small ball under a carpet), pressing the emulsion droplet very close to the aqueous sample. The emulsion droplet (e.g., protrusion 6) can be an aqueous droplet separated from the aqueous sample by a thin layer of a water-immiscible matrix (e.g., oil). At this stage, fluorescent particles (and any associated analyte interacting reagents) are trapped within the emulsion droplet and do not contact the aqueous sample. The swept protrusion 7 demulsifies into small constellation-like groups 8 of fluorescent particles. This constellation-like group diffuses outwards into larger constellation-like groups 9. Some fluorescent particles may combine with analyte molecules 4 in the aqueous sample, affecting the diffusion rate of the fluorescent particles. During this process, fluorescent excitation light 2 is guided through the analysis chamber 1.

[0297] In some embodiments, the application of a transient strong electric field is used to stimulate demulsification, wherein the emulsion droplets are fused with the aqueous sample, allowing the fluorescent particles of the reagent to interact with the sample analyte. Other methods can also be used to induce demulsification, such as by using surfactants or demulsifiers with opposite charges added to the sample or a water-immiscible host matrix. In some embodiments, the application of the electric field is used to induce electrophoretic motion of the components of the fused aqueous sample and the emulsion droplets.

[0298] In some embodiments, multiple pores containing various different emulsions are used. In other embodiments, the multiple pores are spaced sufficiently so that the emulsion droplets do not interact.

[0299] In some implementations, the analytical chamber is shaped such that the aqueous sample stream forms a long, meandering path through it. This provides an extended duration for the fluorescent particles to be observed. Different emulsions can be added at different times, allowing the emulsion droplets to be spaced apart.

[0300] In some implementations, the analytical chamber includes multiple inlets for simultaneously flowing multiple aqueous samples. These flows can be separated by barriers. Emulsion droplets can flow into the junction between two aqueous sample inlets.

[0301] In some embodiments, the emulsion droplets may contain a gelled fluid. The gelled fluid may be melted or enzymatically depolymerized upon insertion into the analytical chamber. In some embodiments, gel beads containing fluorescent particles may be used instead of emulsion droplets. In some embodiments, the fluorescent particles are released after gel melting or enzymatic depolymerization. Alternatively, the emulsion droplets and their water-immiscible host matrix may be replaced by gel beads suspended in an aqueous matrix, wherein the fluorescent particles are released after gel melting or enzymatic depolymerization of the gel.

[0302] In some implementations, the aqueous sample exists as sample droplets in an immiscible matrix, for example, as Figure 6 or Figure 9 As shown. Some analytes can concentrate at the surface interface between the sample droplet and the water-immiscible matrix. In this case, the diffusion of fluorescent particles may be largely confined to the two-dimensional plane of the surface interface. In some embodiments, the change in the diffusion behavior of fluorescent particles from three-dimensional motion to two-dimensional motion indicates the binding between the fluorescent particles and the analyte. Examples of such analytes include cell membrane proteins.

[0303] Figure 9 The illustration shows, for example, that when an aqueous sample 3 is present in the analysis chamber, emulsion droplets or gel beads 5 can be allowed to flow into the analysis chamber 1. Each emulsion droplet or gel bead forms a protrusion 6, and these protrusions can be swept downwards along the length of the analysis chamber or otherwise spread out on the surface of the chamber. The emulsion droplets or gel beads can be trapped under a sheet of aqueous sample 3, much like small balls under a carpet. In some embodiments, the emulsion droplets or gel beads are pressed very close to the aqueous sample. The emulsion droplets (e.g., protrusions 6) can be aqueous droplets separated from the aqueous sample by a thin layer of a water-immiscible matrix (e.g., oil). At this stage, fluorescent particles (and any associated analyte-interacting reagents) are trapped within the emulsion droplets and do not contact the aqueous sample. Then, for example, upon the application of an electrical pulse, the swept protrusions 7 can be demulsified, causing the thin gaps in the water-immiscible matrix to break, thereby allowing each aqueous droplet to fuse into the aqueous sample. The fluorescent particles (and any associated analyte interacting reagents) within each aqueous droplet are no longer confined to a water-immiscible matrix and can diffuse throughout the aqueous sample to form small constellation-like populations 8. These constellation-like populations diffuse outwards into larger constellation-like populations 9. Some fluorescent particles can combine with analyte molecules 4 in the aqueous sample, affecting the diffusion rate of the fluorescent particles. For example... Figure 4 As shown, analyte molecules 4 can be concentrated at the interface between the aqueous sample 3 and the water-immiscible matrix. In at least a portion of this process, fluorescent excitation light 2 is guided through the analytical chamber 1. For example, fluorescent excitation light can be used during the expansion of a small constellation 8 into a larger constellation 9, wherein all or a subset of the fluorescent particles of one or more emulsion droplets are captured, and their fluorescence emission characteristics are recorded and / or analyzed.

[0304] In some implementations, the analytical chamber is constructed such that fluorescent excitation light can be applied to the fused aqueous sample and emulsion droplets. The applied fluorescent excitation light enters the surface of the analytical chamber, passes through the fused aqueous sample and emulsion droplets, and exits from other surfaces.

[0305] In some implementations, the surface within the analysis chamber can be textured to alter the flow of fluid through the chamber or to change the movement of droplets within it. For example, grooves can be created on the surface to ensure that droplets move along a specific path.

[0306] In some embodiments, the flow rate of the aqueous sample through the analysis chamber is constant. In other embodiments, the flow rate of the aqueous sample through the analysis chamber varies over time. For example, the flow rate of the aqueous sample through the analysis chamber may be pulsed.

[0307] In some implementations, acoustic waves, such as high-frequency sound waves (e.g., ultrasound), are applied to the analytical chamber. For example, acoustic standing waves can be used to induce controlled oscillations (or cavitation) in the aqueous sample and its contained fluorescent particles, thereby altering the diffusion properties of the fluorescent particles. This can be particularly noticeable if the density of the fluorescent particles differs significantly from that of the aqueous sample. By rapidly moving the environment around the fluorescent particles, it is possible to distinguish weakly bound components that are nonspecifically adsorbed onto the fluorescent particles. In some aspects, the method involves applying acoustic waves across the entire width of the analytical chamber, thereby imparting a degree of non-random motion to the quantum dots, which helps distinguish between random diffusion rate fluctuations and rapidly alternating association and dissociation behavior.

[0308] In some embodiments, quantum dots have a significantly higher density than water; for example, quantum dots may contain PbS or CdS. In some embodiments, the quantum dots are subjected to ultrasonic treatment (e.g., ultrasonic treatment) to cause them to vibrate more than Brownian motion, and this vibration can remove non-specifically bound materials. Increasing the ultrasonic power can indicate the strength of the bond with the quantum dots.

[0309] In some implementations, a magnetic field is applied to the analytical chamber. For example, reagent droplets manufactured to contain magnetic particles can be drawn in a controlled direction by the applied magnetic field.

[0310] In some implementations, the fluorescent particles may bind weakly to a quencher that can competitively adsorb onto sample proteins, resulting in increased fluorescence of the fluorescent particles.

[0311] In some embodiments, the fluorescence excitation light is unpolarized. In some embodiments, the fluorescence excitation light is linearly, circularly, elliptically, or cocycloidically polarized. The propagation mode through the analysis chamber can be transverse electromagnetic (TEM), transverse electric (TE), transverse magnetic (TM), or a mixture thereof.

[0312] In some embodiments, the fluorescence excitation light is coherent. In other embodiments, the fluorescence excitation light is continuous or pulsed. The fluorescence excitation light can be applied to the analytical chamber at various incident angles. In some embodiments, the fluorescence excitation light includes, is substantially composed of, or is composed of infrared light. In some embodiments, the use of infrared light means that optical scattering is reduced or minimized, thereby allowing for precise quantum dot localization.

[0313] In some embodiments, the fluorescent excitation light is spatially uniform. In other embodiments, the fluorescent excitation light is spatially patterned. The fluorescent excitation light may consist of a single frequency, multiple frequencies, or serve as a frequency comb. Optical frequency combs can be used to provide equidistant frequency markings in infrared, visible, and ultraviolet light, and can correlate unknown optical frequencies with radio or microwave frequency references. Methods for generating frequency combs are known in the art, for example, as disclosed in U.S. Patent No. 7,982,944.

[0314] In some embodiments, fluorescence excitation light is applied in a manner similar to confocal microscopy. In some embodiments, fluorescence excitation light is applied in a manner similar to structured illumination microscopy (SIM). In some embodiments, fluorescence excitation light is applied in a manner similar to stochastic optical reconstruction microscopy (STORM). In some embodiments, fluorescence excitation light is applied in a manner similar to point-scan two-photon microscopy. In some embodiments, fluorescence excitation light is applied in a manner similar to scan line angle projection microscopy (SLAPMi). In some embodiments, fluorescence excitation light is applied in a manner similar to ghost imaging (GI) or sparse constraint ghost imaging (GISC). In some embodiments, using fluorescence excitation light according to the above techniques facilitates rapid and precise localization of fluorescent particles within a scattering medium such as a biological sample.

[0315] In some implementations, the fluorescent emitted light is refracted or internally reflected by droplets having a refractive index different from that of the matrix, thereby producing an image pattern that can be deconvolved to identify the location of the fluorophore.

[0316] In some embodiments, the aqueous sample is transparent to the fluorescent excitation light. In some embodiments, the excitation light causes the fluorescent particles to fluoresce and emit fluorescence in all directions. In some embodiments, stray excitation light is removed by using a filter that absorbs the frequency of the excitation light, or by using a reflective surface that preferentially reflects the frequency of the excitation light.

[0317] In some implementations, the analytical chamber is constructed such that fluorescence emission can be directed to an optical system. For example, as... Figure 11As shown, analysis chamber 1 contains fluorescent particles within the aqueous sample 3. Fluorescent excitation light 2 is guided through the analysis chamber, causing the fluorescent particles to emit fluorescent light. Lens 4 focuses the fluorescent emission light from the fluorescent particles onto a diffraction grating 5. Another lens 6 focuses the light from the diffraction grating 5 onto a camera 7. The use of a diffraction grating is optional.

[0318] In some implementations, when the emulsion droplets first fuse with the aqueous sample, their fluorescent particles approach each other. If the emulsion droplets contain, for example, approximately six red-emitting fluorescent particles, nine green-emitting fluorescent particles, and three blue-emitting fluorescent particles, then the camera can observe bright red spots, very bright green spots, and dim blue spots along lines generated by a diffraction grating. After applying a transient strong electric field to stimulate emulsion demulsification, the fluorescent particles can be released from the emulsion droplets and can move randomly via Brownian motion, for example, when the aqueous sample is not flowing. As individual fluorescent particles diffuse outward in the aqueous sample, they eventually travel a distance sufficient for the camera to resolve them, thus allowing the tracking of individual intensities of the fluorescent particles. In some implementations, as diffusion continues, the movement of the fluorescent particles and the fluorescence intensity are tracked over time, allowing the camera to observe an expanding constellation-like population.

[0319] In some implementations, the spectral composition and relative color intensity of the constellation-like population provide a means for identifying the original emulsion droplets and thus the one or more analyte binding reagents contained therein.

[0320] In some embodiments, a large number of different emulsion droplets are fused into an aqueous sample, where each droplet can be identified by a camera. For example, the emulsion droplet source can be a vial containing a library of emulsion droplets, where each droplet has a unique combination of fluorescence emission spectral composition and relative color intensity of one or more specific analyte-binding agents it contains. In some embodiments, these droplets are randomly fused into the aqueous sample, and the diffuse constellation-like population of each droplet is observed, providing identification of one or more analyte-binding agents present in the droplets. Figure 5 The image shows a representative set of camera images. For example, the analytical chamber contains a flowing aqueous sample 2 carrying two different emulsion droplets 3. As the two emulsion droplets are carried by the aqueous sample, they are demulsified to form two dense constellation-like groups 4 of fluorescent particles. The fluorescent particles slowly diffuse outward, expanding the two constellation-like groups to form an extended constellation-like group 5. Molecules of analyte 8 in the sample bind to the fluorescent particles 6. These constellation-like groups continue to expand, eventually overlapping each other in 7. Continuous optical tracking of each fluorescent particle allows identification of which emulsion droplet it originated from, and thus the identity of its analyte binding reagent.

[0321] In some implementations, the excitation light is spatially uniform, and the random diffusion of fluorescent particles results in a corresponding random variation in the fluorescence emission position of each fluorescent particle.

[0322] In some implementations, the excitation light is structured in a spatial pattern, and the random diffusion of fluorescent particles into and out of regions with high excitation light intensity results in a corresponding random variation in the fluorescence emission of each fluorescent particle.

[0323] In some embodiments, the fluorescent particles chemically associate with the analyte and, optionally, are covalently bonded. In some embodiments, the random motion of the fluorescent particles is influenced by binding interactions with the analyte. For example, binding interactions can increase hydrodynamic drag on the fluorescent particles.

[0324] In some embodiments, changes in the random motion of fluorescent particles caused by binding interactions lead to changes in the random behavior of fluorescence emission position and / or magnitude. In some embodiments, the degree of random behavior of fluorescence emission position and / or magnitude constitutes an optical detection signal for the binding interaction between the analyte and the reagent after each droplet demulsification (or gel depolymerization) event. In some embodiments, when fluorescent particles are not bound, the degree of random behavior among all fluorescent particles having a given emission color is compared to establish a relative baseline of random behavior.

[0325] In some implementations, the attachment of fluorescent particles to one side of the analyte creates an asymmetric geometry, thereby influencing random motion. In some implementations, statistically, the motion is not purely random but is constrained by the asymmetric geometry. In addition to the magnitude of Brownian motion, various statistical methods can be used to distinguish the random motion of bound fluorescent particles from the random motion of unbound fluorescent particles with symmetrical geometry.

[0326] In some embodiments, the presence of an analyte can be determined by the presence of random behavioral changes in fluorescence emission values. In some embodiments, the concentration of the analyte in the sample can be determined by the time required for a random behavioral change in fluorescence emission values. In some embodiments, the concentration of the analyte in the sample can be determined by the proportion of fluorescent particles exhibiting random behavioral changes in fluorescence emission values. This approach is typically suitable when the analyte in the sample is at a sufficiently low concentration and not all fluorescent particles bind to analyte molecules.

[0327] In some implementations, the concentration of the analyte is determined by the saturation of the binding sites on the fluorescent particles, which can be determined by comparing a measured change in the random behavior of the fluorescence emission value with an expected change in the random behavior of the fluorescence emission value.

[0328] In some implementations, the association and dissociation constants of the binding interaction between the analyte and the reagent are determined by varying the concentration of the aqueous sample, for example, by diluting the initial sample to provide diluted samples of various concentrations. For example, for a series of measurements in which the analyte concentration decreases continuously, the binding interaction (as shown by random motion) decreases continuously. Since the binding interaction at equilibrium depends on the concentration, a graph of the binding interaction versus concentration produces the association and dissociation constants.

[0329] In some embodiments, the association and dissociation constants of the binding interaction between the analyte and the reagent are determined by measuring the change in fluorescence emission values ​​over time according to random behavior. For example, methods are disclosed herein that include allowing association to occur for a period of time, followed by dissociation for a period of time, and then re-association. In some embodiments, the random behavior of fluorescence emission values ​​over time during association and dissociation indicates analyte concentration, binding strength, and / or binding kinetics.

[0330] In some implementations, the expanded constellation-like population extends sufficiently to overlap with other constellation-like populations in the analysis chamber. After this stage, the unique identification of each fluorescent particle becomes more complex, but can be managed by tracking fluorescent particles over time and / or applying statistical techniques.

[0331] For example, as constellation groups expand, each fluorescent particle will be identified by its constellation group. Eventually, an identified fluorescent particle may randomly overlap with an identified fluorescent particle of the same color from another constellation group and then continue to diffuse from the overlap. Statistical techniques can be used to identify which particle comes from which constellation group, and thus which initial emulsion droplet. For each of the two types of fluorescent particles, there is a 50% probability that its observed diffusing behavior is due to one analyte binding agent and another 50% probability that it is due to the other analyte binding agent. However, since there are multiple particles of each type, the diffusing behavior of a vaguely identified fluorescent particle can be compared with other particles of that type, and probabilities can be assigned to possible identities.

[0332] Compositions and methods for tracking single particles are known. See, for example, Zhang et al., “QuantumDot Based Biotracking and Biodetection,” Anal. Chem. 2019, 91, 532-547. In these methods, fluorophores (e.g., quantum dots, fluorescent proteins, or gold nanodots) are attached to molecules, and then the fluorophores are randomly dragged by their molecules on or within the cell membrane to facilitate the observation and study of cellular transport processes. Researchers have found that quantum dots and gold nanodots exhibit the expected spatial and inertial effects compared to molecular fluorophores.

[0333] In some respects, the compositions and methods disclosed herein differ from known single-particle tracking techniques. For example, as disclosed herein, the diffusion rate of quantum dots is used to measure the presence and / or strength of binding interactions, and the identity of quantum dots is derived from their association with other quantum dots within their emulsion droplets and extended constellation-like populations.

[0334] In some implementations, a degree of randomness is provided during the fabrication of aqueous droplets in relation to a specific count of fluorescent particles. For example, when attempting to fabricate aqueous droplets with 6 red-emitting fluorescent particles, 9 green-emitting fluorescent particles, and 3 blue-emitting fluorescent particles, some droplets with 5 red-emitting, 9 green-emitting, and 4 blue-emitting fluorescent particles may also exist. As long as the identity of these aqueous droplets is unambiguous, they can still be used by the analysis algorithm. If their identity is undefined, then optical data from the fluorescent particles associated with those aqueous droplets can be excluded from the analysis.

[0335] In some implementations, a weak gelling agent is added to the aqueous sample to minimize laminar flow through the analytical chamber. Laminar flow typically tends to spread out constellations of fluorescent particles. In cases where multiple aqueous samples flow into the analytical chamber via multiple inlets, the gelling agent is used to suppress convective mixing of the various aqueous samples.

[0336] In some implementations, a viscosity enhancer is added to the aqueous sample to slow the diffusion of fluorescent particles in the analytical chamber. Examples of viscosity enhancers include glycerol, polyethylene glycol, and polyvinyl alcohol.

[0337] In some implementations, non-Newtonian agents are added to the aqueous sample to further alter the diffusion of fluorescent particles in the analytical chamber. These include shear thinners and / or shear thickeners.

[0338] In some embodiments, an electric field is applied to the analytical chamber. The charged components of the fused aqueous sample (e.g., fluorescent particles and protein molecules) can then be subjected to forces that result in deterministic motion (as opposed to nondeterministic random motion). This is called electrophoretic motion. If the electric field is oscillatory, then these charged components will also oscillate. In some embodiments, this oscillation is useful for identifying nonspecific binding interactions. In some embodiments, nonspecific binding is weak while analyte binding is strong, and the oscillation of the fluorescent particles can temporarily shake off the weakly bound components. This can be observed in the random behavior of fluorescence emission values.

[0339] In some embodiments, the instantaneous strong electric field used to stimulate demulsification causes a momentary shift in the position of the fluorescent particles due to electrostatic interactions. In some embodiments, the signal can be used for analysis because it depends on the net charge of the fluorescent particles.

[0340] Each camera image is a collection of light arriving within the image exposure time. In some implementations, if the diffusion motion of fluorescent particles is slower than the exposure time, then the fluorescent emission light forms a set of small focal points in each camera image, and the movement of these points across a series of camera images provides information about the diffusion motion of the fluorescent particles.

[0341] If the diffusion of fluorescent particles is faster than the exposure time, the emitted fluorescent light forms a set of large, blurred spots in each camera image; during exposure, the small focal point of each fluorescent particle follows a random path that blurs into large spots, making analysis through a series of camera images difficult. In some implementations, a nutation disk is used to capture even faster motion. For example, as... Figure 13 As shown, fluorescent emission light 4 is passed through a nutation disk 1 with a vertical axis 2. The vertical axis 2 rotates about a second axis 3, and the emitted light 5 is given a translation. Instead of each fluorescent particle generating a point on the camera's focal plane, the point must be optically tracked over a set of exposures, during which the point is rapidly moved in a circular (or other pattern) manner by the nutation disk. Instead of forming a set of large, blurred spots 6 in the camera image, the random motion of these points is stretched into an approximately circular shape 7 in the camera image, where deviations from the circle indicate random motion occurring during the exposure. In this way, more pixels are used to measure the fluorescent emission light from each fluorescent particle, thus allowing for higher temporal resolution. In some embodiments, a more sensitive camera is used to compensate for the reduced photon rate / pixel rate due to the use of the nutation disk.

[0342] In some implementations, the methods disclosed herein include implementing active system feedback, wherein a population of aqueous droplets inserted into the analysis chamber is altered based on measurements obtained from earlier aqueous droplets. For example, an initial population of aqueous droplets is determined to indicate the binding of the analyte to a class of reagents, and a second population of aqueous droplets is inserted into the analysis chamber to probe the details of that class of reagents.

[0343] II. Methods for optical analysis including reagent diffusion.

[0344] Various methods can be used for the optical analysis of the diffusion of fluorescent constructs, which may or may not bind to analytes in the sample.

[0345] In some embodiments, the optical methods disclosed herein include fluorescence excitation methods, which include methods using ultraviolet (UV) light to provide simple UV irradiation, structured UV irradiation, standing waves, and / or pulsed / polarized light. In some embodiments, the optical methods disclosed herein include near-IR two-photon fluorescence excitation. For example, high-intensity lasers with short pulses can be used to provide better precision and / or less background fluorescence in scattering samples.

[0346] In some embodiments, the optical methods disclosed herein include fluorescence imaging methods. In some cases, a simple CCD with a nutation disk is used, wherein point sources (e.g., quantum dots) are distributed in a circular pattern to achieve higher velocities, for example, as... Figure 13 In some embodiments, the imaging methods disclosed herein include scan line angle projection microscopy (SLAPMi), for example, as disclosed in Kazemipour et al., “Kilohertz frame-rate two-photon tomography,” Nature Methods 16(8), 778-786 (2019). In some embodiments, the imaging methods disclosed herein include ghost imaging, for example, as disclosed in Li et al., “Single-frame wide-field nanoscopy based on ghost imaging via sparsity constraints,” Optica 6(12), 1515-1523 (2019). In some embodiments, the optical methods disclosed herein include optical position tracking.

[0347] Any suitable combination of the excitation, imaging, and / or optical position tracking methods disclosed herein can be used. Switching between two or more optical techniques during optical measurements to obtain the data required by the operator may be advantageous.

[0348] In one aspect, this document discloses a method comprising contacting an aqueous sample to be analyzed with an emulsion, wherein the emulsion comprises droplets of water containing a fluorescent construct (e.g., fluorescent particles) capable of binding to one or more analytes in the aqueous sample. In some embodiments, the contact is performed in an analytical chamber configured for optical analysis of the fluorescent construct, including diffusion of the fluorescent construct before and / or after the droplets fuse with the aqueous sample. In some embodiments, the analytical chamber is configured to allow the emulsion droplets to fuse with the aqueous sample. In some embodiments, fluorescence excitation light is applied at a controlled phase angle. In some embodiments, the fluorescence is characterized to provide concentration and binding interaction data for various analytes, which can provide useful information about the biological sample.

[0349] In some embodiments, the fluorescence excitation light is or includes a standing wave, wherein the peaks and troughs of the light wave form a series of parallel planes with equal electric field values, spaced one wavelength apart. In some embodiments, the fluorescence excitation light is vertically guided through an analytical chamber, and the analytical chamber contains multiple parallel planes with equal electric field values. For example, as... Figure 14 As shown, coherent fluorescence excitation light 2 is guided through analysis chamber 1 to generate a set of planes 4 with the maximum electric field value, because each photon wave 3 is in phase with all other photon waves.

[0350] The standing wave of the fluorescent excitation light can be generated within the resonant cavity by coherent light from a laser. Alternatively, a single electric field plane can be generated by an evanescent wave on the surface of the analysis chamber. If the analysis chamber 1 contains fluorescent particles, those particles 6 located at or near the parallel plane will emit maximum fluorescence, while those particles 5 between the planes (at the nodes) will emit minimum fluorescence. Unbound by any particular theory, the fluorescence intensity of a fluorescent particle depends on the magnitude of its local electric field. In some embodiments, the fluorescent particles diffuse randomly over time, moving in and out of the parallel plane. As they do so, their fluorescence intensity exhibits a corresponding change. Therefore, the fluorescence intensity of each fluorescent particle provides an optical detection signal characterizing the diffusion behavior of the fluorescent particles.

[0351] In some embodiments, the fluorescence color of each particle, together with the fluorescence colors of its neighboring particles, is used to identify the analyte binding reagent on the surface of each fluorescent particle. In some embodiments, fluorescent particles and their neighboring fluorescent particles are traced back to the same emulsion droplet, which can be uniquely identified among multiple emulsion droplets, and the identity of the emulsion droplet indicates the identity of the analyte binding reagent associated with each fluorescent particle.

[0352] In some embodiments, when the analyte binding agent binds to the analyte in the sample, the fluorescent particles exhibit altered diffusion characteristics that affect the optical detection signal. In some embodiments, monitoring the fluorescence intensity of each fluorescent particle over time provides a measurement of the binding interactions that occur.

[0353] In some implementations, the phase angle of the coherent fluorescence excitation light shifts during measurement, causing the parallel plane to move through the analytical chamber. In this case, in addition to the fluorescent particles moving through the parallel plane, the parallel plane also moves through the fluorescent particles. This shift in the phase angle of the coherent fluorescence excitation light can be achieved through one or more mechanisms. For example, by physically altering the distance between the fluorescence excitation source and the analytical chamber, using interferometry, changing the composition of the medium separating the fluorescence excitation source and the analytical chamber, using elliptically polarized fluorescence excitation light, or by mixing two or more wavelengths of coherent fluorescence excitation light to form a beat frequency.

[0354] In some implementations, controlled shifting of the phase angle provides measurement advantages. For example, it can provide increased measurement speed for rapid detection. It can generate oscillations of known frequencies of fluorescence intensity, which can be used to distinguish the fluorescence background of a sample.

[0355] In another aspect, this document discloses a method comprising contacting an aqueous sample to be analyzed with an emulsion, wherein the emulsion comprises droplets of water containing a fluorescent construct (e.g., fluorescent particles) capable of binding to one or more analytes in the aqueous sample. In some embodiments, the contact is performed in an analytical chamber configured for optical analysis of the fluorescent construct, including diffusion of the fluorescent construct before and / or after the droplets fuse with the aqueous sample. In some embodiments, the analytical chamber is configured to allow the emulsion droplets to fuse with the aqueous sample. In some embodiments, fluorescence excitation light is applied in a controlled spatial pattern. In some embodiments, the fluorescence is characterized to provide concentration and binding interaction data for various analytes, which can provide useful information about the biological sample.

[0356] In some implementations, the fluorescent excitation light forms a spatial pattern, wherein the light waves form a series of regions with equal electric field values. If this fluorescent excitation light is vertically guided through an analysis chamber, the analysis chamber will contain a pattern of these equal electric field values. For example, as... Figure 9 As shown, spatially patterned fluorescent excitation light 2 is guided through analysis chamber 1 to generate a set of regions 4 with maximum electric field values. Unbound by any particular theory, the fluorescence intensity of the fluorescent particles depends on the magnitude of their local electric field. In analysis chamber 1 containing fluorescent particles, fluorescent particles 6 located in or near region 4 will fluoresce, while those particles 5 between regions will not fluoresce.

[0357] Spatially patterned fluorescence excitation light can be generated using structured light, where the pattern is projected through an analysis chamber. The pattern can be generated by a mask, microelectromechanical systems (MEMS), or digital micromirror devices (DMDs). The pattern can consist of a grid, a set of lines, a set of dots, or any geometric array that can be used to obtain optical measurements.

[0358] Spatially patterned fluorescent excitation light can be rotated by a rotating polarizer to produce rotated polarized light. Therefore, the edges of the pattern will change the magnitude of the electric field vector, and the fluorescence emission intensity of the fluorescent particles at the edges will also change accordingly.

[0359] In some implementations, fluorescent particles diffuse randomly over time, moving in and out of the region. As they do so, their fluorescence intensity exhibits corresponding changes. Therefore, the fluorescence intensity of each fluorescent particle provides an optical detection signal characterizing the diffusion behavior of the fluorescent particles. The fluorescence color of each particle, along with the fluorescence colors of its neighboring particles, can be used to identify analyte-binding agents on the surface of each fluorescent particle. If an analyte-binding agent binds to an analyte in the sample, the fluorescent particle will exhibit altered diffusion characteristics, thus affecting the optical detection signal. Therefore, monitoring the fluorescence intensity of each fluorescent particle over time provides a measurement of the binding interactions that have occurred.

[0360] In some implementations, the region of fluorescence excitation light is moved during measurement, causing the region to move through the analytical chamber. In this case, in addition to the fluorescent particles moving through the region, the region also moves past the fluorescent particles. This movement of the region can be achieved through one or more mechanisms, such as mechanical control of the mask position or electronic control of the MEMS or DMD. Another example is using a rotating reflector or refractor to rapidly scan points across the entire region, as used in confocal microscopy. Yet another example is using a rotating reflector with a refractor to change the angle of the fluorescence excitation light passing through the analytical chamber.

[0361] In some implementations, specific fluorescent particles are actively tracked via electronic control of a MEMS or DMD. The optical determination of the fluorescent particle's location can be used to ensure that only the area at or around the fluorescent particle is illuminated. This allows for higher illumination intensities, but higher intensities can overheat the analysis chamber.

[0362] In some implementations, specific fluorescent particles are actively controlled by optical tweezers. This can be achieved by focusing a laser beam so that its focal point is located at or near the fluorescent particle. If the fluorescent particle is a dielectric, it will be attracted to the focal point of the laser beam, where the electric field is strongest. Furthermore, the photon momentum involved in the excitation and fluorescence processes will be transferred to the fluorescent particle, thus affecting its motion.

[0363] Optical techniques commonly used in microscopy can be used to improve information about the position of each fluorescent particle. For example, confocal microscopy, structured illumination microscopy, and stochastic optical reconstruction microscopy are techniques for improving image resolution.

[0364] In some implementations, controlled movement of the region provides measurement advantages. For example, it can provide increased measurement speed useful for detection. Additionally, it can generate oscillations of known frequencies of fluorescence intensity, which can be used to distinguish the fluorescence background of the sample.

[0365] III. Compositions and formulations.

[0366] In specific embodiments, this document provides compositions and formulations of emulsion droplets and / or gel beads. For example, emulsions of aqueous droplets in a water-immiscible matrix are formulated with different compositions. The aqueous droplets may contain quantum dots or other fluorescent particles, magnetic particles, dissolved electrolytes or other small molecules, proteins or other macromolecules, and surfactants. In other examples, gel beads in an aqueous matrix are formulated with different compositions. The gel beads may contain quantum dots or other fluorescent particles, magnetic particles, and proteins or other macromolecules. The gels forming the beads can be designed to be removed by melting or enzymatic depolymerization.

[0367] In some embodiments, a group of monochromatic quantum dots is chemically treated such that the surface of the quantum dots is coated (derived) with a reagent (e.g., a specific protein). This process is repeated with other groups of monochromatic quantum dots and other proteins, producing a large number of quantum dot groups. For example, for multiple quantum dot colors, there might be vials of 100 red-light-emitting quantum dots (each coated with a unique protein), plus vials of 100 yellow-light-emitting quantum dots (each coated with a unique protein), and so on. In some embodiments, this can be performed within a microfluidic device.

[0368] In some embodiments, equal samples of specific vials containing red quantum dots are mixed with equal samples of specific vials containing yellow quantum dots of different sizes to produce an aqueous mixture of red and yellow quantum dots in a known ratio, each color type having a known protein coating on its surface. In some embodiments, this can be performed within a microfluidic device.

[0369] In some embodiments, an emulsion is formed using known methods by combining a slowly flowing aqueous mixture with a rapidly flowing water-immiscible composition (e.g., a water-immiscible medium) within a flow cell. In some embodiments, the method produces aqueous droplets of highly uniform size within a water-immiscible bulk matrix.

[0370] In some embodiments, the resulting emulsion contains droplets of quantum dots emitting a first color (e.g., red) and quantum dots emitting a second color (e.g., yellow) in a known ratio, each color type having a known protein coating on its surface. An exemplary emulsion droplet composition is shown in... Figure 16 and Figure 17 middle.

[0371] In some embodiments, an emulsion containing quantum dots emitting different colors at known ratios is prepared using the preparation chamber disclosed herein, for example, such as... Figure 21As shown and described in Example 13. In some embodiments, separate populations of emulsion droplets with defined quantum dot contents are generated in parallel and can be merged at any suitable ratio, for example, for the analytical chamber disclosed herein.

[0372] In some embodiments, gel beads are formed by adding gel beads to an aqueous mixture, followed by the addition of an organic solvent to cause the gel beads to swell. This allows quantum dots to migrate into the gel beads. Subsequently, the organic solvent is removed to reduce swelling and trap the quantum dots within the gel beads.

[0373] In some embodiments, the resulting gel beads have a known ratio of quantum dots emitting a first color (e.g., red) and quantum dots emitting a second color (e.g., yellow), each color type having a known protein coating on its surface.

[0374] Methods for using aqueous emulsions to deliver droplets containing an aqueous sample to a measuring device are known in the art and are applicable to this disclosure. Exemplary methods for using emulsions are described in WO2002 / 068104, US 7,268,167, US 7,772,287, US 7,717,615, and US 7,375,140, ​​WO2005 / 089921, and US 8,741,192, all of which are incorporated herein by reference in their entirety for all purposes. Exemplary methods for using gel beads are described in US 7645868, US 8624014, US7718262, US 8283037, US 8568881, US 8968874, US 9376613, and their foreign corresponding patents, all of which are incorporated herein by reference in their entirety for all purposes. Some technical methods relate to nonspecific compositions of emulsions or gel beads. For example, in these methods, nonspecific emulsions or gel beads cannot be distinguished from each other by, for example, uniquely identifiable address labels or barcodes. Therefore, there is a need for formulations that provide novel functionalities not found in generally nonspecific formulations.

[0375] In some implementations, the designs described herein are applicable to specific formulations containing aqueous droplets (and gel beads) of reagents identifiable within a large address space, wherein said reagents can be used to detect analytes, discover disease biomarkers, discover drug therapies, and evaluate drug therapies in disease biomarker diagnosis. The address space is effectively increased by combining the different fluorescence emission colors of fluorescent particles with the different counts of these particles. Attached to the fluorescent particles is the reagent for detecting the analyte. Continuous optical tracking of individual fluorescent particles allows the behavior of the fluorescent particles to be mapped to their identification.

[0376] A large number of different aqueous droplets (or gel beads) can be contained in a single vial and used directly in analytical instruments. The large address space allows for precise labeling, even when different reagents are present on fluorescent particles of the same color. Reagent libraries within the vials can be manufactured, stored, and dispensed as needed.

[0377] For example, within a small vial of aqueous droplets, a subgroup X of the aqueous droplets may contain approximately 6 red fluorescent particles (coated with reagent A), 9 green fluorescent particles (coated with reagent B), and 3 blue fluorescent particles (coated with reagent C).

[0378] Another subgroup Y of aqueous droplets may contain approximately 6 red fluorescent particles (coated with reagent D), 12 yellow fluorescent particles (coated with reagent E), and 3 blue fluorescent particles (coated with reagent F).

[0379] Another subgroup Z of aqueous droplets may contain approximately 6 red fluorescent particles (coated with reagent G), 9 green fluorescent particles (coated with reagent H), and 6 blue fluorescent particles (coated with reagent I).

[0380] Ultimately, vials of aqueous droplets can contain many of these subgroups. The combination of fluorescence emission color and counting provides a large address space, allowing reagents to uniquely identify their fluorescent particles.

[0381] During use, the aqueous droplets are demulsified, releasing internal fluorescent particles that interact with any analytes that may be present in their vicinity.

[0382] The identity of each reagent can be maintained by optically tracking all fluorescent particles during the use of the aqueous droplet vial. In this example, the aqueous droplet vial contains droplets with red fluorescent particles attached to reagent A, reagent D, and reagent G. By associating each fluorescent particle with its original droplet, a clear labeling of the red fluorescent particles to the specific reagent (A, D, or G) attached to the original droplet is achieved, the original droplet having a unique combination of fluorescence emission color and fluorescent particle count. Continuous tracking from the moment of demulsification enables this association.

[0383] During use, the optical behavior of each fluorescent particle provides a signal relevant to the detection of the analyte.

[0384] By detecting optical trajectories and optical behavior together, measuring signals and the identification of reagents associated with those signals, the sample in the test can be characterized.

[0385] These compositions and their formulations have broad applicability to the diagnosis of disease biomarkers in complex biological media. Furthermore, such designs can also be used for disease biomarker discovery, drug discovery, and drug evaluation, where the samples are known and relatively uncomplicated. A significant advantage of these designs over existing designs is that the designs disclosed herein can be readily scaled to arbitrarily large parallelizations without being limited to fixed plate arrays. These designs are also unaffected by pH or other solution properties, can be used with opaque samples, and can utilize very small sample volumes with minimal transport loss.

[0386] The designs disclosed herein can be used in methods for detecting the presence, absence, or extent of binding interactions between an analyte and an analyte-binding reagent, and for determining the presence, absence, or amount (e.g., concentration) of an analyte in a sample.

[0387] These designs are useful in methods for discovering and characterizing binding interactions between known analytes and their binding agents, and in methods for diagnosing the presence of analytes in biological samples. Current methods for performing these tasks suffer from several limitations, such as limitations in analysis speed, array size, requirements for large sample volumes, and analyte handling losses. The designs described herein allow for methods that overcome many of these limitations and provide novel approaches to detecting intermolecular interactions and measuring a wide range of analytes, including those with clinical significance and / or diagnostic relevance. Using an emulsion instead of a fixed array plate allows for the formation of arrays of arbitrary sizes, allows for small sample volumes, and avoids sample contact with the channel surfaces that could adsorb analytes. This use occurs over a short timeframe, during which the binding interaction process can be monitored.

[0388] One specific area where these designs are useful is biotechnology, for measuring protein-protein interactions. Every biological system uses an enormous number of proteins that interact within a complex network that depends on many factors. Disease distorts this network, adding or removing components and interaction pathways. Understanding these systems allows for early diagnosis of diseases in subjects (e.g., human patients) and methods for chemically repairing these systems through drug therapy. The most numerous and stable proteins in these systems have been partially studied, but further research is needed. Adding new and more powerful tools to the full skill set of medical researchers, such as those using the designs described in this paper, will deepen our understanding of protein networks and allow for the development of new diagnostics and new drug therapies.

[0389] IV. How to use.

[0390] One specific area where these methods are useful is in biotechnology for measuring protein interactions. Every biological system uses an enormous number of proteins that interact within a complex network that depends on many factors. Disease distorts this network, adding or removing components and interaction pathways. Understanding these systems allows for early diagnosis of diseases and methods for chemically repairing these systems through drug therapy. The most numerous and stable proteins in these systems have been partially studied, but further research is needed. Adding new and more powerful tools (such as those described in this paper) to the full skill set of medical researchers will deepen our understanding of protein networks and allow for the development of new diagnostics and new drug therapies.

[0391] The methods disclosed herein are broadly applicable to the diagnosis of disease biomarkers in complex biological media. Furthermore, such methods can also be used for disease biomarker discovery, drug discovery, and drug evaluation, where the samples are known and relatively uncomplicated. A significant advantage of these methods over existing approaches is that they can be easily scaled to arbitrarily large parallelizations without being limited to fixed plate arrays. These methods are also unaffected by pH or other solution properties, can be used with opaque samples, and can utilize very small sample volumes with minimal transport loss.

[0392] The methods disclosed herein can be used to detect the presence, absence, or extent of binding interactions between an analyte and an analyte binding reagent, and to determine the presence, absence, or amount (e.g., concentration) of an analyte in a sample.

[0393] These methods are useful for discovering and characterizing binding interactions between known analytes and their binding agents, and for diagnosing the presence of analytes in biological samples. Current methods for performing these tasks suffer from several limitations, such as limitations in analysis speed, array size, requirements for large sample volumes, and analyte processing losses. The method described in this paper addresses many of these limitations and provides a novel approach for detecting intermolecular interactions and measuring a wide range of analytes, including those with clinical significance and / or diagnostic relevance. Using an emulsion instead of a fixed array plate allows for the formation of arrays of arbitrary sizes, permits small sample volumes, and avoids sample contact with the channel surfaces that could adsorb analytes. This mixing occurs over a short timeframe, during which the binding interaction process can be monitored.

[0394] In some embodiments, the methods and compositions disclosed herein can be used to study synergistic effects between two or more agents. In some aspects, methods and compositions are provided to study druggable targets. Most protein-protein interactions are currently considered undrugable, including cancer proteins (such as KRas) and protein degradation products (such as E3 ubiquitin ligases). Such protein-protein interactions can be measured using the currently disclosed compositions and methods, for example, on a wide, flat surface, by providing an intervening molecule (such as a small molecule or antibody) that reduces / disrupts protein-protein interactions in some variants and / or promotes / enhances them in others. In some aspects, methods and compositions are provided to study allosteric interactions, for example, where protein-protein interactions are disrupted by small effector molecules at distal sites. In some aspects, methods and compositions are provided for single-cell analysis. For example, a single lysed cell can be provided for each droplet, where binding with all cellular components present in the droplet provides a more realistic measurement of interactions. Furthermore, by observing individual single cells within a population, a measure of cell population heterogeneity can be provided.

[0395] In some embodiments, the methods and compositions disclosed herein can be used for proteomic analysis based on protein labeling and / or activity. In some aspects, probe kits for proteomic analysis based on protein labeling and / or activity are provided herein, comprising dendritic polymer-coated quantum dots (QDs). In some embodiments, the QD-tethered probe comprises a flexible portion capable of interacting with motifs of various geometries in the protein, such as binding / reaction pockets and / or cavities. In some embodiments, the methods and compositions disclosed herein are used independently of mass spectrometry. In some embodiments, artificial intelligence can be used to infer the possible shapes of the protein and suggest potential probe designs.

[0396] In some embodiments, the methods and compositions disclosed herein can be used to study weak interactions. For example, small molecules near proteins can rapidly jump between associated and dissociated states with proteins, and these transitional interactions can be studied using the methods and compositions disclosed herein.

[0397] V. Definition

[0398] Unless otherwise defined, all technical terms, symbols, and other technical and scientific terms or terminology used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, for clarity and / or ease of reference, terms are defined herein with their commonly understood meanings, and the inclusion of such definitions herein should not necessarily be construed as indicating a material difference from the meanings commonly understood in the art. All patents, applications, published applications, and other publications mentioned herein are incorporated herein by reference in their entirety. If a definition set forth in this section is contrary to or otherwise inconsistent with a definition set forth in a patent, application, or other publication incorporated herein by reference, the definition set forth in this section shall prevail over the definition incorporated herein by reference.

[0399] Unless the context clearly indicates otherwise, as used herein, the singular forms “a” and “described” include multiple indicators. For example, “a” means “at least one” or “one or more”. It should be understood that aspects and variations described herein include “consisting of aspects and variations” and / or “substantially consisting of aspects and variations”.

[0400] Throughout this disclosure, all aspects of the claimed subject matter are presented in scope. It should be understood that this scope-based description is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Therefore, the scope description should be considered to include all possible sub-scopes precisely disclosed, as well as individual numerical values ​​within that scope. For example, where a numerical range is provided, it should be understood that every intermediate value between the upper and lower limits of that range, and any other specified value or intermediate value within that range, is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may be independently included in the smaller range and are also encompassed within the claimed subject matter, subject to any specifically excluded limits within the range. Where a specified scope includes one or both limits, the scope excluding one or both of those included limits is also included within the invention. This applies regardless of the width of the scope.

[0401] As used herein, the term "about" refers to a typical range of error for readily known individual values. References to "about" a value or parameter herein include (and describe) embodiments of variations of said value or parameter itself. For example, a description of "about X" includes a description of "X". In some embodiments, "about X" refers to a value of ±25%, ±10%, ±5%, ±2%, ±1%, ±0.1%, or ±0.01% of X.

[0402] As used herein, a composition refers to any mixture of two or more products, substances, or compounds (including cells). It can be a solution, suspension, liquid, powder, paste, aqueous solution, non-aqueous solution, or any combination thereof.

[0403] As used in this article, “subject” can include mammals, such as humans or other animals, and is typically a human.

[0404] In this application, the term "analyte" can include a specific component of a complex biological media (such as blood) and can consist of proteins, small molecules, or nucleic acids. The term "analyte" can also refer to a type of molecule to be measured in terms of concentration, presence, size, or binding interaction characteristics. The term "analyte" can also refer to bacteria or viruses. The term "analyte" can also refer to a type of molecule that binds to the interface between an aqueous droplet and a water-immiscible host matrix, or between an aqueous droplet and a secondary emulsion containing a water-immiscible droplet. The term "analyte" can also refer to oligonucleotides or cells, such as blood cells or bacteria, which can be detected by imaging methods.

[0405] In this application, the term "analyte binding reagent" may include components that bind to or interact with an analyte, typically exhibiting high selectivity and high affinity.

[0406] In this application, the term "particle" refers to a collection of atoms, such as fluorescent or magnetic, or both. Typically, a particle is a substantially insoluble solid in the context in which it is described.

[0407] In this application, “flocculation” involving droplets refers to the adhesion of more than two types of droplets together, typically forming a large amorphous substance.

[0408] In this application, the term "electric field vector" may include the electric field component of an evanescent wave near the refractive index transition.

[0409] In this application, the term "random" can include nondeterministic behavior as well as nondeterministic behavior that contains a degree of deterministic behavior.

[0410] In this application, the term "binding interaction" can include a combination of analytes and reagents having specific association and dissociation constants under specific conditions. It can also refer to the interaction between a reagent and bacteria or viruses. The occurrence of binding interactions can be observed by detecting the various signals described herein.

[0411] As used herein, “signal” or “measurement signal” can include any detectable emission or observable change. Examples include fluorescence emission, color changes, or changes in size or appearance. In each case, the signal can be detected as described herein or using techniques and devices known in the art (e.g., CCD, CMOS, camera, etc.).

[0412] In this application, the term "electric field vector" may include the electric field component of the photons of the excitation light.

[0413] In this application, the term "magnetic field vector" may include the magnetic field component of the photons that excite the light.

[0414] As used herein, the terms "fluorescent dye" and "fluorophore" can include portions that absorb light energy defining an excitation wavelength and emit light energy at different wavelengths. In some examples, combinations of fluorophores can be used according to a labeling scheme known as "combinatorial multicolor coding," which is described in U.S. Patent No. 6,632,609 and Speicher et al., Nature Genetics, 12:368-375, 1996.

[0415] As used herein, "spectrally distinguishable" means that, under operating conditions, markers (e.g., fluorophores such as quantum dots) can be distinguished based on their spectral characteristics, particularly the wavelengths of fluorescence emission. For example, the identity of one or more fluorophores may be associated with different wavelengths of maximum light emission intensity, or possibly with the intensity ratio of different wavelengths. One or more spectral characteristics of markers used for detection and identification are referred to herein as "color". It should be understood that markers are typically identified based on specific spectral characteristics.

[0416] In this application, the description of the fluorescence mechanism by the electric field vector of a photon wave is not exclusive. Other mechanisms by which photon waves can induce fluorescence in fluorescent particles are included.

[0417] In this application, the term "rotated polarization" can include light waves whose polarization plane rotates about the transmission axis. For example, rotated polarized light can be generated by a rotating polarizer.

[0418] In this application, the term "random" can include nondeterministic behavior as well as nondeterministic behavior that contains a degree of deterministic behavior.

[0419] In this application, the term "nutation" can include the movement of an optical element that changes angle or deflects light rays by an amount proportional to the movement. For example, a transparent disk having a vertical axis rotating about a second axis (sweeping out a conical shape) exhibits nutation. Light rays passing through the disk parallel to the second axis will be deflected proportionally to the nutation.

[0420] In this application, the term "diffusion" may include the diffusion properties of one or more specific particles.

[0421] In this application, the terms "fluorocarbon fluid," "chlorinated carbon fluid," "bromine carbon fluid," and "iodine carbon fluid" can include liquid halogenated carbon compounds and solid halogenated carbon compounds that can form fluids or solutions.

[0422] The embodiments listed below represent selected aspects of this disclosure. While these are generally described as 'including' specific features and / or steps, this disclosure also includes corresponding embodiments 'consistently composed of specific features and / or steps' and 'composed of specific features and / or steps'.

[0423] VI. Exemplary Implementation Scheme

[0424] The provided implementation scheme is as follows:

[0425] 1. A method for detecting the presence of an analyte in a sample, comprising:

[0426] a. Provide a first liquid, wherein the first liquid comprises a portion of the sample;

[0427] b. An emulsion of a second liquid comprising aqueous droplets in a liquid matrix, wherein the aqueous droplets comprise an array of fluorescent particles having an analyte binding agent attached to a set of their surfaces;

[0428] c. Contact the first liquid with the aqueous droplets to form a combination of the first liquid and the second liquid, under conditions that allow the first liquid and the second liquid to merge into a single fluid; and

[0429] Examine the signal generated by the binding interaction between the analyte and the analyte binding reagent.

[0430] 2. The method of embodiment 1, wherein the aqueous droplet comprises a fluorescent label or fluorescent particles. In some such embodiments, one or more aqueous droplets comprise fluorescent particles, such as quantum dots, fluorescent proteins, or fluorescent molecules; or polymer particles containing quantum dots, fluorescent proteins, or fluorescent molecules, and the fluorescent particles are thus present in the aqueous droplet. In some such embodiments, the analyte binding agent associates with or binds to the quantum dots.

[0431] 3. The method according to any one of embodiments 1-2, wherein the aqueous droplets are gel beads.

[0432] 4. The method according to any one of embodiments 1-3, wherein the aqueous droplets are not emulsions.

[0433] 5. The method according to any one of embodiments 1-4, wherein the liquid matrix is ​​immiscible with water. The first liquid matrix may be selected from lipids, oils, hydrocarbon fluids, fluorocarbon fluids, chlorinated hydrocarbon fluids, brominated hydrocarbon fluids, iodinated hydrocarbon fluids, silicone fluids, and mixtures thereof.

[0434] 6. The method of any one of embodiments 1-5, wherein the mixing of the first liquid and the aqueous droplets is carried out in an analytical chamber.

[0435] 7. The method of any one of embodiments 1-6, wherein contacting the first liquid with the aqueous droplets comprises mixing the following:

[0436] a. Adjacent flows of the first liquid and the aqueous droplets; or

[0437] b. The lateral flow of the first liquid and the aqueous droplets; or

[0438] c. The vertical flow of the first liquid and the aqueous droplets; or

[0439] d. An oblique flow of the first liquid and the aqueous droplets; or

[0440] e. The relative flow between the first liquid and the aqueous droplets; or

[0441] f. The concentric flow of the first liquid and the aqueous droplets.

[0442] 8. The method of any one of embodiments 1-7, wherein contacting the first liquid with the aqueous droplets comprises mixing the first liquid and a stream of the aqueous droplets comprising at least one demulsifier.

[0443] 9. The method of any one of embodiments 1-8, wherein contacting the first liquid with the aqueous droplets comprises mixing the first liquid and a stream of the aqueous droplets comprising at least one surfactant.

[0444] 10. The method of any one of embodiments 1-9, wherein contacting the first liquid with the aqueous droplets comprises mixing the first liquid and a stream of the aqueous droplets comprising at least one gelling agent.

[0445] 11. The method of any one of embodiments 1-10, wherein contacting the first liquid with the aqueous droplets comprises mixing the first liquid and a stream of the aqueous droplets comprising at least one viscosity enhancer.

[0446] 12. The method of any one of embodiments 1-11, wherein the first liquid may exist as a first emulsion in the form of emulsified droplets within a liquid matrix. The liquid matrix may be selected from lipids, oils, hydrocarbon fluids, fluorocarbon fluids, chlorinated hydrocarbon fluids, brominated hydrocarbon fluids, iodinated hydrocarbon fluids, silicone fluids, and mixtures thereof.

[0447] 13. The method of any one of embodiments 1-12, wherein contacting the first liquid with the aqueous droplets comprises fusing the aqueous phase into a fused liquid in such a manner as follows:

[0448] a. Apply an electric field; or

[0449] b. Use surfactants with opposite charges on the first liquid and the second emulsion; or

[0450] c. Use a demulsifier; or

[0451] d. Do not use demulsifying agents; or

[0452] e. Apply heat; or

[0453] f. Apply a gel depolymerizing agent.

[0454] 14. The method of any one of embodiments 1-13, wherein the signal generated in the fused liquid by the binding of the analyte with the analyte binding reagent is a fluorescent signal.

[0455] 15. The method of any one of embodiments 1-14, wherein the signal is induced by excitation light, the excitation light having at least one characteristic selected from the group consisting of:

[0456] a. Non-polarized;

[0457] b. Linearly polarized;

[0458] c. Circularly polarized;

[0459] d. Elliptically polarized;

[0460] e. Paracycloid polarization;

[0461] f. Single wavelength;

[0462] g. Multiple wavelengths;

[0463] h. Incoherent;

[0464] i. Relevant;

[0465] j. continuous;

[0466] k. Pulsating;

[0467] l. Applied at a single angle of incidence; and

[0468] m. applied with a set of incident angles.

[0469] 16. The method of any one of embodiments 1-15, wherein an external electric field is applied to the fused liquid in a manner selected from the group consisting of:

[0470] a. A constant electric field in a constant direction;

[0471] b. A pulsed electric field in a constant direction;

[0472] c. Oscillating electric field in a constant direction;

[0473] d. A constant electric field that switches between multiple directions;

[0474] e. A pulsed electric field switching between multiple directions; and

[0475] f. Oscillating electric field switching between multiple directions,

[0476] This is sufficient to induce electrophoretic motion of particles within the fused liquid.

[0477] 17. A method for detecting an analyte that interacts with a binding reagent, comprising:

[0478] a. Provide a first liquid containing the analyte;

[0479] b. Providing an emulsion of a second liquid, the emulsion of the second liquid comprising aqueous reagent droplets within a water-immiscible host matrix, wherein the reagent droplets contain an aggregate of fluorescent particles having an analyte binding reagent attached to a set of their surfaces;

[0480] c. In the analysis chamber, the first liquid and the reagent droplets are mixed together, allowing the first liquid and the reagent droplets to merge into a single fluid;

[0481] d. After the mixing, an excitation light with a controlled phase and wavelength is guided through the analysis chamber, sufficient to induce fluorescence in the fluorescent particles, wherein the fluorescence depends on the position of the fluorescent particles within a local phase angle of the excitation light;

[0482] e. After the mixing process, detect and / or measure the amount of fluorescence emission;

[0483] f. Based on the pattern of fluorescence emission wavelengths near the point of fusion of each droplet with the first liquid, determine the identity of each analyte binding reagent present within each reagent droplet; and

[0484] Variations in the random behavior of fluorescence emission values ​​provide an indication of the presence, concentration, and / or binding interactions of the analyte.

[0485] 18. The method of embodiment 17, wherein the aqueous droplet comprises a fluorescent label or fluorescent particles. In some such embodiments, one or more aqueous droplets comprise fluorescent particles, such as quantum dots, fluorescent proteins, or fluorescent molecules; or polymer particles containing quantum dots, fluorescent proteins, or fluorescent molecules, and the fluorescent particles are thus present in the aqueous droplet. In some such embodiments, the analyte binding agent associates with or binds to the quantum dots.

[0486] 19. The method of any one of embodiments 17-18, wherein the aqueous droplets are gel beads.

[0487] 20. The method of any one of embodiments 17-19, wherein the aqueous droplets are not emulsions.

[0488] 21. The method of any one of embodiments 17-20, wherein the liquid matrix is ​​immiscible with water. The first liquid matrix may be selected from lipids, oils, hydrocarbon fluids, fluorocarbon fluids, chlorinated hydrocarbon fluids, brominated hydrocarbon fluids, iodinated hydrocarbon fluids, silicone fluids, and mixtures thereof.

[0489] 22. The method of any one of embodiments 17-21, wherein the mixing of the first liquid and the aqueous droplets is carried out in an analytical chamber.

[0490] 23. The method of any one of embodiments 17-22, wherein contacting the first liquid with the aqueous droplets comprises mixing the following:

[0491] a. Adjacent flows of the first liquid and the aqueous droplets; or

[0492] b. The lateral flow of the first liquid and the aqueous droplets; or

[0493] c. The vertical flow of the first liquid and the aqueous droplets; or

[0494] d. An oblique flow of the first liquid and the aqueous droplets; or

[0495] e. The relative flow between the first liquid and the aqueous droplets; or

[0496] f. The concentric flow of the first liquid and the aqueous droplets.

[0497] 24. The method of any one of embodiments 17-23, wherein contacting the first liquid with the aqueous droplets comprises mixing the first liquid and a stream of the aqueous droplets comprising at least one demulsifier.

[0498] 25. The method of any one of embodiments 17-24, wherein contacting the first liquid with the aqueous droplets comprises mixing the first liquid and a stream of the aqueous droplets comprising at least one surfactant.

[0499] 26. The method of any one of embodiments 17-25, wherein contacting the first liquid with the aqueous droplets comprises mixing the first liquid and a stream of the aqueous droplets comprising at least one gelling agent.

[0500] 27. The method of any one of embodiments 17-26, wherein contacting the first liquid with the aqueous droplets comprises mixing the first liquid and a stream of the aqueous droplets comprising at least one viscosity enhancer.

[0501] 28. The method of any one of embodiments 17-27, wherein the first liquid may exist as a first emulsion in the form of emulsified droplets within a liquid matrix. The liquid matrix may be selected from lipids, oils, hydrocarbon fluids, and fluorocarbon fluids, chlorinated hydrocarbon fluids, brominated hydrocarbon fluids, iodinated hydrocarbon fluids, silicone fluids, and mixtures thereof.

[0502] 29. The method of any one of embodiments 17-28, wherein contacting the first liquid with the aqueous droplets comprises fusing the aqueous phase into a fused liquid in such a manner as:

[0503] a. Apply an electric field; or

[0504] b. Use surfactants with opposite charges on the first liquid and the second emulsion; or

[0505] c. Use a demulsifier; or

[0506] d. Do not use demulsifying agents; or

[0507] e. Apply heat; or

[0508] f. Apply a gel depolymerizing agent.

[0509] 30. The method of any one of embodiments 17-29, wherein the signal generated in the fused liquid by the binding of the analyte with the analyte binding agent is a fluorescent signal.

[0510] 31. The method of any one of embodiments 17-30, wherein the signal is induced by excitation light, the excitation light having at least one characteristic selected from the group consisting of:

[0511] a. Non-polarized;

[0512] b. Linearly polarized;

[0513] c. Circularly polarized;

[0514] d. Elliptically polarized;

[0515] e. Paracycloid polarization;

[0516] f. Single wavelength;

[0517] g. Multiple wavelengths;

[0518] h. Incoherent;

[0519] i. Relevant;

[0520] j. continuous;

[0521] k. Pulsating;

[0522] l. Applied at a single angle of incidence; and

[0523] m. applied with a set of incident angles.

[0524] 32. The method of any one of embodiments 17-31, wherein the phase of the coherent excitation light is controlled such that the peaks and troughs of the light wave move past the fluorescent particles sufficiently to cause corresponding oscillations in the fluorescence of the fluorescent particles.

[0525] 33. The method of any one of embodiments 17-32, wherein the ellipticity of the ellipticized coherent excitation light is controlled such that the peaks and troughs of the light wave move past the fluorescent particles sufficiently to cause corresponding oscillations in the fluorescence of the fluorescent particles.

[0526] 34. The method of any one of embodiments 17-33, wherein a nutation transparent window and / or a rotating prism moves an image of each fluorescent particle across the entire surface of the optical detector.

[0527] 35. The method of any one of embodiments 17-34, wherein an external electric field is applied to the fused liquid in a manner selected from the group consisting of:

[0528] a. A constant electric field in a constant direction;

[0529] b. A pulsed electric field in a constant direction;

[0530] c. Oscillating electric field in a constant direction;

[0531] d. A constant electric field that switches between multiple directions;

[0532] e. A pulsed electric field switching between multiple directions; and

[0533] f. Oscillating electric field switching between multiple directions,

[0534] This is sufficient to induce electrophoretic motion of particles within the fused liquid.

[0535] 36. A method for detecting the interaction between an analyte and an analyte binding reagent in a sample, wherein the method comprises:

[0536] a. Provide a first liquid containing the analyte;

[0537] b. Providing an emulsion of a second liquid, the emulsion of the second liquid comprising aqueous reagent droplets within a water-immiscible host matrix, wherein the reagent droplets contain an aggregate of fluorescent particles having an analyte binding reagent attached to a set of their surfaces;

[0538] c. In the analysis chamber, the first liquid and the reagent droplets are mixed together, allowing the first liquid and the reagent droplets to merge into a single fluid;

[0539] d. After the mixing, an excitation light having a controlled spatial pattern and wavelength is guided through the analysis chamber, sufficient to induce fluorescence in the fluorescent particles, wherein the fluorescence depends on the position of the fluorescent particles within the local spatial pattern of the excitation light;

[0540] e. After the mixing process, detect and / or measure the amount of fluorescence emission;

[0541] f. Based on the pattern of fluorescence emission wavelengths near the point of fusion of each droplet with the first liquid, determine the identity of each analyte binding reagent present within each reagent droplet; and;

[0542] Variations in the random behavior of fluorescence emission values ​​provide an indication of the presence, concentration, and / or binding interactions of the analyte.

[0543] 37. The method of embodiment 36, wherein the aqueous droplet comprises a fluorescent label or fluorescent particles. In some such embodiments, one or more aqueous droplets comprise fluorescent particles, such as quantum dots, fluorescent proteins, or fluorescent molecules; or polymer particles containing quantum dots, fluorescent proteins, or fluorescent molecules, and the fluorescent particles are thus present in the aqueous droplet. In some such embodiments, the analyte binding agent associates with or binds to the quantum dots.

[0544] 38. The method of any one of embodiments 36-37, wherein the aqueous droplets are gel beads.

[0545] 39. The method of any one of embodiments 36-38, wherein the aqueous droplets are not emulsions.

[0546] 40. The method of any one of embodiments 36-39, wherein the liquid matrix is ​​immiscible with water. The first liquid matrix may be selected from lipids, oils, hydrocarbon fluids, fluorocarbon fluids, chlorinated hydrocarbon fluids, brominated hydrocarbon fluids, iodinated hydrocarbon fluids, silicone fluids, and mixtures thereof.

[0547] 41. The method of any one of embodiments 36-40, wherein the mixing of the first liquid and the aqueous droplets is carried out in an analytical chamber.

[0548] 42. The method of any one of embodiments 36-41, wherein contacting the first liquid with the aqueous droplets comprises mixing the following:

[0549] a. Adjacent flows of the first liquid and the aqueous droplets; or

[0550] b. The lateral flow of the first liquid and the aqueous droplets; or

[0551] c. The vertical flow of the first liquid and the aqueous droplets; or

[0552] d. An oblique flow of the first liquid and the aqueous droplets; or

[0553] e. The relative flow between the first liquid and the aqueous droplets; or

[0554] f. The concentric flow of the first liquid and the aqueous droplets.

[0555] 43. The method of any one of embodiments 36-42, wherein contacting the first liquid with the aqueous droplets comprises mixing the first liquid and a stream of the aqueous droplets comprising at least one demulsifier.

[0556] 44. The method of any one of embodiments 36-43, wherein contacting the first liquid with the aqueous droplets comprises mixing the first liquid and a stream of the aqueous droplets comprising at least one surfactant.

[0557] 45. The method of any one of embodiments 36-44, wherein contacting the first liquid with the aqueous droplets comprises mixing the first liquid and a stream of the aqueous droplets comprising at least one gelling agent.

[0558] 46. ​​The method of any one of embodiments 36-45, wherein contacting the first liquid with the aqueous droplets comprises mixing the first liquid and a stream of the aqueous droplets comprising at least one viscosity enhancer.

[0559] 47. The method of any one of embodiments 36-46, wherein the first liquid may be present as a first emulsion in the form of emulsified droplets within a liquid matrix. The liquid matrix may be selected from lipids, oils, hydrocarbon fluids, fluorocarbon fluids, chlorinated hydrocarbon fluids, brominated hydrocarbon fluids, iodinated hydrocarbon fluids, silicone fluids, and mixtures thereof.

[0560] 48. The method of any one of embodiments 36-47, wherein contacting the first liquid with the aqueous droplets comprises fusing the aqueous phase into a fused liquid in such a manner as:

[0561] a. Apply an electric field; or

[0562] b. Use surfactants with opposite charges on the first liquid and the second emulsion; or

[0563] c. Use a demulsifier; or

[0564] d. Do not use demulsifying agents; or

[0565] e. Apply heat; or

[0566] f. Apply a gel depolymerizing agent.

[0567] 49. The method of any one of embodiments 36-48, wherein the signal generated in the fused liquid by the binding of the analyte with the analyte binding reagent is a fluorescent signal.

[0568] 50. The method of any one of embodiments 36-49, wherein the signal is induced by excitation light, the excitation light having at least one characteristic selected from the group consisting of:

[0569] a. Non-polarized;

[0570] b. Linearly polarized;

[0571] c. Circularly polarized;

[0572] d. Elliptically polarized;

[0573] e. Paracycloid polarization;

[0574] f. Single wavelength;

[0575] g. Multiple wavelengths;

[0576] h. Incoherent;

[0577] i. Relevant;

[0578] j. continuous;

[0579] k. Pulsating;

[0580] l. Applied at a single angle of incidence; and

[0581] m. applied with a set of incident angles.

[0582] 51. The method of any one of embodiments 36-50, wherein the excitation light is applied as a patterned spatial array over the entire analytical chamber such that the light intensity varies at different points within the analytical chamber, sufficient to cause a fluorescence change in the fluorescent particles as they move.

[0583] 52. The method of any one of embodiments 36-51, wherein the patterned spatial array of excitation light is moved over the entire analysis chamber such that the light intensity varies at different points and times within the analysis chamber, sufficient to cause fluorescence changes in the fluorescent particles.

[0584] 53. The method of any one of embodiments 36-52, wherein a nutation transparent window and / or a rotating prism moves an image of each fluorescent particle on the surface of the optical detector.

[0585] 54. The method of any one of embodiments 36-53, wherein an external electric field is applied to the fused liquid in a manner selected from the group consisting of:

[0586] a. A constant electric field in a constant direction;

[0587] b. A pulsed electric field in a constant direction;

[0588] c. Oscillating electric field in a constant direction;

[0589] d. A constant electric field that switches between multiple directions;

[0590] e. A pulsed electric field switching between multiple directions; and

[0591] f. Oscillating electric field switching between multiple directions,

[0592] This is sufficient to induce electrophoretic motion of particles within the fused liquid.

[0593] 55. A design for preparing a set of aqueous droplets, comprising:

[0594] a. Providing a water-immiscible liquid matrix for suspending the aqueous droplets;

[0595] b. Formulate each aqueous droplet to contain fluorescent particles with a specific combination of fluorescent emission colors;

[0596] c. Formulate each aqueous droplet to contain a specific count of fluorescent particles with each fluorescent emission color;

[0597] d. Each fluorescent particle is formulated with zero, one, or more reagents attached thereto, wherein the identity of said reagent is specific to the color of its particle and specific to the combination of color and count within an aqueous droplet containing it; and

[0598] Aqueous droplets are collected in containers for storage.

[0599] 56. The method of embodiment 55, wherein the liquid matrix is ​​immiscible with water. The first liquid matrix may be selected from lipids, oils, hydrocarbon fluids, fluorocarbon fluids, chlorinated hydrocarbon fluids, brominated hydrocarbon fluids, iodinated hydrocarbon fluids, silicone fluids, and mixtures thereof.

[0600] 57. The method of any one of embodiments 55-56, wherein the liquid matrix is ​​formulated to have characteristics selected from the group consisting of:

[0601] a. gel;

[0602] b. Vitrification at low temperatures;

[0603] c. Curing at low temperature;

[0604] d. The refractive index matches the refractive index of the aqueous droplet to which it is suspended;

[0605] e. Its refractive index is higher than that of the aqueous droplets to which it is suspended;

[0606] f. Its refractive index is lower than that of the aqueous droplets to which it is suspended;

[0607] g. Non-Newtonian;

[0608] h. Shear thinning;

[0609] i. Shear-thickening;

[0610] j. Increased viscosity;

[0611] k. Reduced viscosity;

[0612] l. Its density matches the density of the aqueous droplets in which it is suspended;

[0613] m. Its density is less than the density of the aqueous droplets in which it is suspended;

[0614] n. A substance with a density greater than that of the aqueous droplets in which it is suspended.

[0615] 58. The method of any one of embodiments 55-57, wherein the fluorescent particles may be selected from the group consisting of:

[0616] a.Quantum dots;

[0617] b. Fluorescent proteins;

[0618] c. Fluorescent molecules;

[0619] d. Polymer particles containing quantum dots, fluorescent proteins, or fluorescent molecules;

[0620] e. Fluorescent particles, each connected to another fluorescent particle via a chemical bonding system;

[0621] f. Fluorescent particles, each connected to a magnetic particle via a chemical bonding system.

[0622] 59. The method of any one of embodiments 55-58, wherein the fluorescent particles can be formulated to have an analyte binding reagent bound to their surface.

[0623] 60. The method of any one of embodiments 55-59, wherein the aqueous droplets may be formulated to contain one or more magnetic particles.

[0624] 61. The method of any one of embodiments 55-60, wherein the aqueous droplets are stabilized with one or more surfactants.

[0625] 62. The method as described in any one of embodiments 55-61, wherein the fluorescent particles can be formulated to be connected to another fluorescent particle or magnetic particle via a connector.

[0626] 63. The method of any one of embodiments 55-62, wherein the set of aqueous droplets contains different combinations of fluorescent particle emission colors and fluorescent particle counts.

[0627] 64. A design for formulating a set of gel beads, comprising:

[0628] a. Provides an aqueous matrix for the suspension gel beads;

[0629] b. Provide a gel matrix that can be removed by physical or chemical methods;

[0630] c. Formulate each gel bead to contain fluorescent particles with a specific combination of fluorescent emission colors;

[0631] d. Formulate each gel bead to contain a specific count of fluorescent particles with each fluorescence emission color;

[0632] e. Each fluorescent particle is formulated with zero, one, or more reagents attached thereto, wherein the identity of said reagent is specific to the color of its particle and specific to the combination of color and count within the gel beads containing it; and

[0633] The gel beads are collected into a container for storage.

[0634] 65. The method of embodiment 64, wherein the liquid matrix is ​​immiscible with water. The first liquid matrix may be selected from lipids, oils, hydrocarbon fluids, fluorocarbon fluids, chlorinated hydrocarbon fluids, brominated hydrocarbon fluids, iodinated hydrocarbon fluids, silicone fluids, and mixtures thereof.

[0635] 66. The method of any one of embodiments 64-65, wherein the liquid matrix is ​​formulated to have characteristics selected from the group consisting of:

[0636] a. gel;

[0637] b. Vitrification at low temperatures;

[0638] c. Curing at low temperature;

[0639] d. The refractive index matches the refractive index of the aqueous droplet to which it is suspended;

[0640] e. Its refractive index is higher than that of the aqueous droplets to which it is suspended;

[0641] f. Its refractive index is lower than that of the aqueous droplets to which it is suspended;

[0642] g. Non-Newtonian;

[0643] h. Shear thinning;

[0644] i. Shear-thickening;

[0645] j. Increased viscosity;

[0646] k. Reduced viscosity;

[0647] l. Its density matches the density of the aqueous droplets in which it is suspended;

[0648] m. Its density is less than the density of the aqueous droplets in which it is suspended;

[0649] n. A substance with a density greater than that of the aqueous droplets in which it is suspended.

[0650] 67. The method of any one of embodiments 64-66, wherein the fluorescent particles may be selected from the group consisting of:

[0651] a.Quantum dots;

[0652] b. Fluorescent proteins;

[0653] c. Fluorescent molecules;

[0654] d. Polymer particles containing quantum dots, fluorescent proteins, or fluorescent molecules.

[0655] 68. The method of any one of embodiments 64-67, wherein the fluorescent particles can be formulated to have an analyte binding reagent bound to their surface.

[0656] 69. The method of any one of embodiments 64-68, wherein the gel beads may be formulated to contain one or more magnetic particles.

[0657] 70. The method of any one of embodiments 64-69, wherein fluorescent particles can be formulated to be connected to another fluorescent particle or magnetic particle via a connector.

[0658] 71. The method of any one of embodiments 64-70, wherein the gel beads are formulated to have characteristics selected from the group consisting of:

[0659] a. Melt the gel at an elevated temperature;

[0660] b. Depolymerizing the gel using enzymes;

[0661] c. Depolymerizing the gel using chemical agents;

[0662] d. Depolymerize the gel by light.

[0663] 72. The method of any one of embodiments 64-71, wherein the set of gel beads contains different combinations of fluorescent particle emission colors and fluorescent particle counts.

[0664] VII. References

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[0714] VIII. Examples

[0715] The following examples are for illustrative purposes only and are not intended to limit the scope of this disclosure.

[0716] Example 1: Methods and compositions for the detection and / or analysis of disease biomarkers.

[0717] Select a small vial of reagent emulsion from the cold storage and install it in a benchtop instrument. This reagent emulsion contains various types of aqueous droplets in fluorocarbon oils. One type

[0718] Subpopulation X contains approximately 6 red-emitting quantum dots (coated with antigen A), 9 green-emitting quantum dots (coated with antigen B), and 3 blue-emitting quantum dots (coated with antigen C). Another type (subpopulation Y) contains approximately 6 red-emitting quantum dots (coated with antigen D), 12 yellow-emitting quantum dots (coated with antigen E), and 3 blue-emitting quantum dots (coated with antigen F). Yet another type (subpopulation Z) contains approximately 6 red-emitting quantum dots (coated with antigen G), 9 green-emitting quantum dots (coated with antigen H), and 6 blue-emitting quantum dots (coated with antigen I). The vials may contain thousands of these unique types, forming an antigen library that can bind to a wide variety of disease biomarkers.

[0719] A blood plasma sample is mixed with a weak gelling agent and added to a benchtop instrument. This blood plasma is to be tested for the presence of antibody A, which binds to antigen A. Antibody A is a naturally occurring disease marker.

[0720] The benchtop instrument forms large droplets of blood plasma in a fluorocarbon oil and pumps these droplets to the inlet end of a flat, wide analytical chamber. Simultaneously, a reagent emulsion is pumped through a series of small orifices near the inlet end to the flat side of the analytical chamber. The reagent emulsion droplets are trapped beneath the large blood plasma droplets, much like small balls under a carpet. Pumping the fluorocarbon oil to the inlet end of the analytical chamber causes the blood plasma sample droplets to be propelled through the chamber and discharged from the distal end. With the aid of gravity and a weak gelling agent, the reagent emulsion droplets are swept away.

[0721] A brief pulse of the electric field causes the thin gap between the fluorocarbon oil (the reagent emulsion droplets and the blood plasma) to break, thus demulsifying the emulsion. The quantum dots within each droplet can now diffuse freely into the blood plasma.

[0722] Quantum dots, as tightly packed clusters, begin their diffusion journey, confined together within the droplet. Slowly, the quantum dots begin to diffuse outward, forming an ever-expanding constellation-like group.

[0723] Ultimately, the red quantum dots from the X-type droplets encountered antibody A molecules in the blood plasma. A binding interaction occurred, forming a structure of the sum of its dimensions. Since antibodies and quantum dots have the same size scale, within the 10 nanometer diameter range, the binding typically doubles the size and more than halves the diffusivity. Other quantum dots do not bind strongly to any present molecules, so their diffusivity remains constant. Weak binding of quantum dots to nonspecifically adsorbed molecules tends to reduce the diffusivity of the quantum dots. To eliminate or reduce nonspecific binding, high-frequency acoustic waves are then applied, causing vibration or cavitation of the solution environment around each quantum dot (because quantum dots have a significantly higher density than water). The X-type–antibody A structure remains intact, but the weakly bound nonspecifically adsorbed structures are shaken apart, restoring the original diffusivity of the quantum dots. Only the X-type–antibody A structure exhibits half the diffusivity.

[0724] During demulsification and quantum dot diffusion, intense pulses of infrared light are projected through the analysis chamber as a set of focal lines, similar to those used in scan-line angle projection microscopy. Two-photon excited fluorescence emission is collected by a camera, and tomographic algorithms are applied to the optical data to reconstruct the movement of the quantum dots. The use of infrared light means that optical scattering (due to its large component in blood plasma) is reduced or minimized, allowing for precise quantum dot localization.

[0725] Data regarding the movement of each quantum dot were analyzed. Each quantum dot exhibited random motion in the x, y, and z directions over time, with the magnitude of the motion depending on the diffusion rate. Some quantum dots were observed to have initially high diffusion rates, but subsequently gradually decreased. Other quantum dots maintained high diffusion rates. In this embodiment, only those red quantum dots tracked by the X-shaped droplet (identified by the unique characteristics of their 6 red, 9 green, and 3 blue quantum dots) showed a step change in diffusion rate, indicating the presence of antibody A in the blood plasma sample.

[0726] In some reference methods for measuring such binding interactions, the binding of antibody A to antigen A can take tens of minutes. This is due to several factors: 1) antigen A is attached to a fixed surface, so only antibody A can freely diffuse toward antigen A; 2) many antibody A molecules must bind to a set of attached antigen A molecules to provide a signal; and 3) some interactions may have slow binding kinetics. Slow binding kinetics may be caused by complex fitting that is impossible for the position and orientation of randomly moving molecules. In this embodiment disclosed herein, both antibody A and antigen A can diffuse toward each other, one antibody A attached to a red quantum dot (coated with antigen A) is sufficient to provide a signal, and the presence of six individual red quantum dots increases the likelihood of binding interactions with antibody A molecules in the sample. Therefore, instead of requiring tens of minutes of incubation time to allow binding interactions to occur, the incubation time using the method disclosed herein is significantly reduced.

[0727] The commercial method of the levitation array technology in Luminex xMAP (Bio-Rad) uses beads, each with a pattern of fluorescent emission color and amplitude, to provide identification of each bead, thereby providing identification of the reagent coating on each bead. In this patent application, droplets with patterns of fluorescent emission color and amplitude are used to provide identification of each droplet. However, there are fundamental differences: 1) the droplets in this embodiment are not coated with reagent, while the quantum dots of the droplets are coated with reagent; 2) the droplets are not identified when intact, but rather by tracking their component quantum dots over time; 3) the need for a washing step and the formation of molecular interlayers is eliminated in this embodiment; and 4) this embodiment does not require the use of fluorescent ligands to determine the occurrence of binding. Therefore, the compositions and methods disclosed in this embodiment, such as those based on random constellation-like population diffusion, offer many advantages over levitation array technology, such as shorter incubation times, no need for washing, no need for molecular interlayer formation, and no need for fluorescent ligands representing additional cost, such as... Figure 26 As shown.

[0728] Example 2: Methods and compositions for the detection and / or analysis of multiple biomarkers.

[0729] Essentially as described in Example 1, the composition and method are also used in multiplex analyses that include high-throughput biomarker detection and / or analysis. Instead of detecting only antibody A in blood plasma, the absence / presence, amount, or activity of antibodies A through I, which bind to antigen A through antigen I, are tested in the sample. Each of antibody A through antibody I is a biomarker for one or more diseases or conditions.

[0730] Specific binding between antigens and antibodies results in approximately a 50% reduction in diffusion rate. Because the movement of each quantum dot is tracked and analyzed, quantum dots that form part of a specific antigen / antibody complex exhibit an initial high diffusion rate (before binding), then gradually decrease after binding, and remain at a low diffusion rate even after reducing or eliminating nonspecific adsorption. Quantum dots that do not bind to antibodies maintain a high diffusion rate. The diffusion rate of quantum dots that only nonspecifically bind antibodies may decrease, but recovers to a high diffusion rate after reducing or eliminating nonspecific adsorption.

[0731] In this embodiment, multiple detection and analysis can be performed as long as some quantum dots can be traced back to one or more droplets from which they originated. For example, observation of a step change in the diffusion rate of red quantum dots traced back to type X droplets (identified by the unique characteristics of its 6 red, 9 green, and 3 blue quantum dots) indicates the presence of antibody A in a blood plasma sample, while observation of the constant high diffusion rate of green quantum dots traced back to type X droplets indicates the presence of antibody B in the sample. Similarly, observation of a step change in the diffusion rate of red quantum dots traced back to type Y droplets (identified by the unique characteristics of its 6 red, 12 yellow, and 3 blue quantum dots) indicates the presence of antibody D in a blood plasma sample, while observation of the constant high diffusion rate of red quantum dots traced back to type Z droplets (identified by the unique characteristics of its 6 red, 9 green, and 6 blue quantum dots) indicates the absence of antibody G in the sample. The amount or concentration of each analyte antibody can also be quantified.

[0732] Example 3: Compositions and methods for drug discovery.

[0733] Select a vial of reagent emulsion from a cold storage facility and mount it in a benchtop instrument. This reagent emulsion contains various types of aqueous droplets in a fluorocarbon oil. One type (referred to as subpopulation X) contains approximately 6 red-emitting quantum dots (coated with ligand A), 9 green-emitting quantum dots (coated with ligand B), and 3 blue-emitting quantum dots (coated with ligand C). Another type (referred to as subpopulation Y) contains approximately 6 red-emitting quantum dots (coated with ligand D), 12 yellow-emitting quantum dots (coated with ligand E), and 3 blue-emitting quantum dots (coated with ligand F). Yet another type (referred to as subpopulation Z) contains approximately 6 red-emitting quantum dots (coated with ligand G), 9 green-emitting quantum dots (coated with ligand H), and 6 blue-emitting quantum dots (coated with ligand I). Vials can contain thousands of such unique types, forming a screening library of candidate drugs.

[0734] The drug target (receptor R) solution sample is mixed with a weak gelling agent and added to a benchtop instrument. The drug target is then tested against a large screening library of candidate drugs, for example, ligands A through I, to screen for ligands that specifically target receptor R.

[0735] A benchtop instrument forms large droplets of a drug target solution in a fluorocarbon oil and pumps these droplets to the inlet end of a flat, wide analytical chamber. Simultaneously, a reagent emulsion is pumped through a series of small orifices near the inlet end to the flat side of the analytical chamber. The reagent emulsion droplets are trapped beneath the large droplets of drug target solution, much like small balls under a carpet. Pumping the fluorocarbon oil to the inlet end of the analytical chamber causes the drug target solution droplets to be propelled through the chamber and discharged from the distal end. With the aid of gravity and a weak gelling agent, the reagent emulsion droplets are swept away.

[0736] A brief pulse of the electric field causes the thin gap between the fluorocarbon oil (the reagent emulsion droplets and the drug target solution) to break, thus demulsifying the emulsion. Quantum dots in each droplet can now diffuse freely into the drug target solution.

[0737] Quantum dots, as tightly packed clusters, begin their diffusion journey, confined together within the droplet. Slowly, the quantum dots begin to diffuse outward, forming an ever-expanding constellation-like group.

[0738] Ultimately, specific quantum dots encounter drug target molecules in the drug target solution and undergo binding interactions, forming a structure of the sum of their sizes. If the drug target and the quantum dots have the same size scale, within the 10 nanometer diameter range, the binding interaction will double the size and more than halve the diffusivity. Other quantum dots will have a constant diffusivity.

[0739] During demulsification and quantum dot diffusion, a pattern of ultraviolet light is projected as a series of closely spaced parallel beams through the analysis chamber. Fluorescent emission light is collected by a camera. As the quantum dots diffuse randomly in and out of the parallel beams, their fluorescence fluctuates accordingly.

[0740] Data on fluorescence intensity for each quantum dot were analyzed. Each quantum dot exhibited random motion in the x, y, and z directions over time, with the magnitude of motion in the x-direction (perpendicular to the parallel beam) depending on the diffusivity. Some quantum dots were observed to have initially high diffusivity, but subsequently gradually decreased diffusivity. Other quantum dots maintained high diffusivity. Tracking the quantum dots with step changes over past time revealed that, for example, they were all blue quantum dots originating from Y-shaped droplets (identified by the unique characteristics of their 6 red, 12 yellow, and 3 blue quantum dots). This suggests that ligand F has a binding interaction with the drug target, and that ligands targeting receptor R may be good candidates for detailed investigation.

[0741] Example 4: Extraction of a composition and method for quenching removal.

[0742] A vial of quantum dots was synthesized and mounted in a benchtop instrument. These quantum dots were coated with bromophenol blue. Red-emitting quantum dots were coated with bromophenol blue containing weak bonds, yellow-emitting quantum dots with medium-strength bonds, and blue-emitting quantum dots with strong bonds. The vial contained millions of such quantum dots. The presence of bromophenol blue resulted in a heavy atom effect, which reduced the fluorescence efficiency of the quantum dots.

[0743] The solution of the candidate drug (from the screening library) in free solution was also added to the benchtop instrument.

[0744] The benchtop instrument pumps the candidate drug to the inlet of a flat, wide analytical chamber. Simultaneously, quantum dots are pumped through a series of small orifices near the inlet to the flat side of the analytical chamber. Water is pumped to the inlet of the analytical chamber, causing the candidate drug and quantum dots to be propelled through the chamber and discharged from the distal end.

[0745] Quantum dots diffuse throughout the candidate drug solution.

[0746] Ultimately, specific quantum dots encounter candidate drug molecules and undergo binding interactions, extracting or replacing bromophenol blue from the quantum dots. This prevents the quantum dots from quenching and allows them to emit brighter fluorescence.

[0747] Data on fluorescence intensity for each quantum dot were analyzed. Each quantum dot exhibited fluorescence over time. Some quantum dots were observed to initially exhibit low fluorescence but then gradually reach higher fluorescence levels. Other quantum dots remained exactly at low fluorescence. This step change indicates that the candidate drug molecule has successfully interacted with the specific quantum dot.

[0748] In this embodiment, if the candidate drug has caused increased fluorescence in red- and yellow-emitting quantum dots, but not in blue-emitting quantum dots, then this indicates that the candidate drug can replace weakly and moderately bonded bromophenol blue, but not strongly bonded bromophenol blue. This provides information about the binding strength between the candidate drug and the surface chemistry of the quantum dots initially used to bind bromophenol blue.

[0749] Example 5: Quencher-labeled oligonucleotides.

[0750] Select a vial of reagent emulsion from the cold storage and mount it in a benchtop instrument. This reagent emulsion contains various types of aqueous droplets in a fluorocarbon oil. One type (referred to as subpopulation X) contains approximately 6 red-emitting quantum dots (coated with oligonucleotide A), 9 green-emitting quantum dots (coated with oligonucleotide B), and 3 blue-emitting quantum dots (coated with oligonucleotide C). Another type (referred to as subpopulation Y) contains approximately 6 red-emitting quantum dots (coated with oligonucleotide D), 12 yellow-emitting quantum dots (coated with oligonucleotide E), and 3 blue-emitting quantum dots (coated with oligonucleotide F). Yet another type (referred to as subpopulation Z) contains approximately 6 red-emitting quantum dots (coated with oligonucleotide G), 9 green-emitting quantum dots (coated with oligonucleotide H), and 6 blue-emitting quantum dots (coated with oligonucleotide I). The vial can contain thousands of such unique types, forming an oligonucleotide library that can hybridize with various oligonucleotides present in the sample.

[0751] DNA fragments are extracted from a collection of cells to produce a sample containing oligonucleotides. These oligonucleotides are labeled with a fluorescent quenching reagent (such as a reagent containing heavy atoms), and the sample is added to a benchtop instrument. The presence of oligonucleotide A* in the sample, which can hybridize with oligonucleotide A, is tested; for example, A* could be a complementary sequence to oligonucleotide A.

[0752] The benchtop instrument pumps the sample to the inlet of a flat, wide analytical chamber. Simultaneously, a reagent emulsion is pumped through a series of small orifices near the inlet to the flat side of the analytical chamber. The reagent emulsion droplets float on top of the sample. Pumping water to the inlet of the analytical chamber causes the sample to be propelled through the chamber and discharged from the distal end. With the aid of gravity, the reagent emulsion droplets are swept away.

[0753] A brief pulse of the electric field causes the emulsion to break down. The quantum dots in each droplet can now diffuse freely into the sample.

[0754] Quantum dots, as tightly packed clusters, begin their diffusion journey, confined together within the droplet. Slowly, the quantum dots begin to diffuse outward, forming an ever-expanding constellation-like group.

[0755] Ultimately, the red quantum dot from the X-shaped droplet encountered the oligonucleotide A* molecule in the sample. Hybridization occurred between oligonucleotide A and oligonucleotide A*, forming a structure with a quencher linked to the quantum dot. In the case of double-stranded oligonucleotides, unwinding and annealing of the oligonucleotides can be optionally used.

[0756] The heating and cooling required for unwinding and annealing can be achieved by controlling the temperature of the analysis chamber or by using focused light to target and locally heat specific quantum dots.

[0757] During demulsification and quantum dot diffusion, ultraviolet light is projected through the analysis chamber, and the fluorescence emission light is collected and applied using 2D spectroscopy.

[0758] Data regarding the position and fluorescence emission color of each quantum dot were analyzed. Some quantum dots exhibited reduced fluorescence intensity due to the attached quencher. In this embodiment, only those red quantum dots tracked by the X-shaped droplet (identified by the unique characteristics of their 6 red, 9 green, and 3 blue quantum dots) showed reduced fluorescence intensity, indicating the presence of oligonucleotide A*.

[0759] Increasing the temperature causes melting, providing additional information about the degree of complementarity between oligonucleotide A* and oligonucleotide A, as the melting temperature is indicated by the recovery of fluorescence intensity.

[0760] The preferred quencher is located at the end of the oligonucleotide in the sample, closest to the quantum dot.

[0761] Optionally, the sample may contain monomers with photocatalysts. Applying focused light of an appropriate wavelength to specific quantum dots can lead to the formation of gel beads containing quantum dots. The resulting gel beads can then be separated using standard methods for gel bead management, such as filtration.

[0762] Example 6: Magnetically labeled oligonucleotides.

[0763] Select a vial of reagent emulsion from the cold storage and mount it in a benchtop instrument. This reagent emulsion contains various types of aqueous droplets in a fluorocarbon oil. One type (referred to as subpopulation X) contains approximately 6 red-emitting quantum dots (coated with oligonucleotide A), 9 green-emitting quantum dots (coated with oligonucleotide B), and 3 blue-emitting quantum dots (coated with oligonucleotide C). Another type (referred to as subpopulation Y) contains approximately 6 red-emitting quantum dots (coated with oligonucleotide D), 12 yellow-emitting quantum dots (coated with oligonucleotide E), and 3 blue-emitting quantum dots (coated with oligonucleotide F). Yet another type (referred to as subpopulation Z) contains approximately 6 red-emitting quantum dots (coated with oligonucleotide G), 9 green-emitting quantum dots (coated with oligonucleotide H), and 6 blue-emitting quantum dots (coated with oligonucleotide I). The vial can contain thousands of such unique types, forming an oligonucleotide library that can hybridize with various oligonucleotides present in the sample.

[0764] DNA fragments are extracted from a collection of cells to produce a sample containing oligonucleotides. These oligonucleotides are labeled with magnetic particles, and the sample is then fed into a benchtop instrument. The presence of oligonucleotide A* in the sample, which can hybridize with oligonucleotide A, is tested; for example, A* could be a complementary sequence to oligonucleotide A.

[0765] The benchtop instrument pumps the sample to the inlet of a flat, wide analytical chamber. Simultaneously, a reagent emulsion is pumped through a series of small orifices near the inlet to the flat side of the analytical chamber. The reagent emulsion droplets float on top of the sample. Pumping water to the inlet of the analytical chamber causes the sample to be propelled through the chamber and discharged from the distal end. With the aid of gravity, the reagent emulsion droplets are swept away.

[0766] A brief pulse of the electric field causes the emulsion to break down. The quantum dots in each droplet can now diffuse freely into the sample.

[0767] Quantum dots, as tightly packed clusters, begin their diffusion journey, confined together within the droplet. Slowly, the quantum dots begin to diffuse outward, forming an ever-expanding constellation-like group.

[0768] Ultimately, the red quantum dot from the X-shaped droplet encountered the oligonucleotide A* molecule in the sample. Hybridization occurred between oligonucleotide A and oligonucleotide A*, forming a structure with magnetic particles connected to the quantum dot. In the case of double-stranded oligonucleotides, unwinding and annealing of the oligonucleotides can be optionally used.

[0769] During demulsification and quantum dot diffusion, a standing wave of ultraviolet light is projected through the analysis chamber as a set of closely spaced electric field amplitudes in a parallel plane. Fluorescent emission light is collected by a camera. As the quantum dots diffuse randomly in and out of the parallel plane, their fluorescence fluctuates accordingly.

[0770] An oscillating magnetic field is applied through the analysis chamber. Quantum dots with interconnected magnetic particles are drawn in a non-random path by the standing wave of ultraviolet light.

[0771] Data on fluorescence intensity for each quantum dot were analyzed. Each quantum dot exhibited random motion in the x, y, and z directions over time, with the magnitude of motion in the z-direction (perpendicular to the parallel plane) depending on the diffusivity and being magnetically attracted. Some quantum dots were observed to have initial random fluorescence intensity but subsequently exhibit additional non-random oscillations in fluorescence intensity. Other quantum dots maintained random diffusivity. Tracking the quantum dots that showed step changes over past time revealed, for example, that they were all red quantum dots originating from an X-shaped droplet (identified by the unique characteristics of their 6 red, 9 green, and 3 blue quantum dots). This indicates that oligonucleotide A* was present in the initial DNA sample.

[0772] The components of the aforementioned five embodiments can be used in combination depending on the measurement results.

[0773] Example 7: Method and composition for measuring thermal displacement.

[0774] An emulsion stream is prepared and injected into a benchtop instrument. The emulsion contains various types of aqueous droplets in a fluorocarbon oil having the same refractive index as the aqueous droplets. One type (referred to as subpopulation X) contains approximately 6 red-emitting quantum dots (coated with drug target receptor A), 9 green-emitting quantum dots (uncoated), 3 blue-emitting quantum dots (uncoated), and ligand U in free solution. Another type (referred to as subpopulation Y) contains approximately 6 red-emitting quantum dots (coated with receptor A), 12 yellow-emitting quantum dots (uncoated), 3 blue-emitting quantum dots (uncoated), and ligand V in free solution. Yet another type (referred to as subpopulation Z) contains approximately 6 red-emitting quantum dots (coated with receptor A), 9 green-emitting quantum dots (uncoated), 6 blue-emitting quantum dots (uncoated), and no ligands in free solution. Vials can contain thousands of such unique types, forming, for example, screening libraries for candidate drugs.

[0775] The benchtop instrument pumps the emulsion to the inlet of a flat, wide analytical chamber. Quantum dots within each aqueous droplet diffuse randomly within the droplet's volume. In some droplets, ligands in free solution can bind to acceptor A. In some cases, the binding of small ligands has a negligible effect on the diffusion properties of the attached quantum dots.

[0776] During quantum dot diffusion, ultraviolet light (e.g., pulses of polarized ultraviolet light) is projected through the analytical chamber. In some cases, an oil with the same refractive index as the aqueous droplet (RI = 1.33) is used (e.g., perfluoronaphthalene, RI = 1.31). Since the refractive indices of the fluorocarbon oil and the aqueous droplet are the same or substantially the same, there is no or almost no boundary refraction.

[0777] Fluorescent emission is collected by a camera (or a set of cameras) as a collection of fluorescent dot sources originating from quantum dots. As the fluorescent dot sources diffuse randomly within each droplet, the camera records the positions of these sources (and thus the positions of the quantum dots). The aqueous droplets are large enough to sufficiently disperse the quantum dots for individual detection. Typically, the separation distance in the focal plane is at least 500 nm. Furthermore, the camera records the timing and polarization of the fluorescent dot sources. Techniques such as time-resolved fluorescence (TRF) are used to collect fluorescence only from a brief time window after excitation to provide differentiation from background fluorescence against the sample matrix. Techniques such as fluorescence polarization assays (FPA), such as fluorescence polarization immunoassay (FPIA), can be used to collect polarization data to provide information about the rotational speed of the quantum dots. Fast-spinning (unbound) quantum dots emit fluorescence with random polarization, while slow-spinning (bound) quantum dots emit fluorescence with a degree of excitation polarization. For example, as... Figure 25As shown in Figure A, pulsed polarized fluorescence excitation is used to distinguish between fast-spinning (unbound) quantum dots and slow-spinning (bound) quantum dots. Unbound quantum dots spin rapidly, resulting in fluorescence emission with random polarization (e.g., dissociation after association), while bound quantum dots spin more slowly, resulting in fluorescence emission with retained polarization (e.g., association after association).

[0778] During quantum dot diffusion, the temperature in the analytical chamber rises slowly. The diffusion rate and rotation of the quantum dots increase proportionally with temperature. Above a certain temperature, acceptor A begins to melt (unfold). This has a significant effect on the diffusion properties of the attached red quantum dots. The binding of ligands to acceptor A in free solution helps to hold acceptor A together and increases the melting temperature. Typical thermal shift assays (TSA) require an additional component, a quenchable dye. Measuring diffusion (rather than fluorescence intensity) avoids the need for a quenchable dye.

[0779] Data regarding the fluorescence position and polarization of each quantum dot were analyzed. Each quantum dot exhibits random motion in the x, y, and z directions over time, and the magnitudes of the optically detected motion in the x and y directions depend on the diffusivity. Similarly, each quantum dot exhibits random rotation over time, and the rotational speed depends on the diffusivity. With increasing temperature, the diffusivity of each quantum dot increases smoothly with an ascending slope. However, as acceptor A begins to melt, the smoothly ascending slope indicates a decrease in diffusivity, marking the melting temperature, for example, as... Figure 25 Figure B in the diagram shows the relationship between the diffusion rate and temperature. Tracking the diffusion rate relative to temperature is used to detect and / or analyze, for example, one or more binding interactions between ligands and receptors. In one example, all the red quantum dots within the Y-type droplet (identified by its unique characteristics of 6 red, 12 yellow, and 3 blue quantum dots) have a melting temperature of 83.3 °C, and all the red quantum dots within the Z-type droplet (identified by its unique characteristics of 6 red, 9 green, and 6 blue quantum dots) have a melting temperature of 79.8 °C. The 3.5 °C temperature difference indicates that ligand V (in the free solution within the Y-type droplet) has a binding interaction with the drug target receptor A (on the red glowing quantum dots), while the Z-type droplet, which does not contain the ligand in the free solution, serves as a control, thus identifying ligand V as a good candidate for detailed studies. In another example, the red quantum dots within the X-type droplet (identified by its unique characteristics of 6 red, 9 green, and 3 blue quantum dots) have a melting temperature T0. m1 The red quantum dots within the Y-shaped droplet (identified by its unique combination of 6 red, 12 yellow, and 3 blue quantum dots) possess a melting temperature T. m2 Furthermore, the red quantum dots within the Z-shaped droplet (identified by the unique characteristics of its 6 red, 9 green, and 6 blue quantum dots) possess a melting temperature T. m3 The temperature difference Tm1 -T m3 and T m2 -T m3 A comparison was made to determine whether ligand U (in a free solution within an X-shaped droplet) had a stronger or weaker binding interaction with the drug target receptor A compared to ligand V (in a free solution within a Y-shaped droplet).

[0780] Example 8: Methods and compositions for single-cell detection.

[0781] An emulsion stream was prepared and injected into a benchtop instrument. The emulsion contained various types of aqueous droplets in a fluorocarbon oil having the same refractive index as the aqueous droplets. One type (referred to as subpopulation X) contained approximately 6 red-emitting quantum dots (coated with antibody A), 9 green-emitting quantum dots (coated with antibody B), 3 blue-emitting quantum dots (coated with antibody C), a single cell, and a lyase. Another type (referred to as subpopulation Y) contained approximately 6 red-emitting quantum dots (coated with antibody D), 12 yellow-emitting quantum dots (coated with antibody E), 3 blue-emitting quantum dots (coated with antibody F), a single cell, and a lyase. Yet another type (referred to as subpopulation Z) contained approximately 6 red-emitting quantum dots (coated with antibody G), 9 green-emitting quantum dots (coated with antibody H), 6 blue-emitting quantum dots (coated with antibody I), a single cell, and a lyase. For any subpopulation X, Y, or Z droplet, the droplet could be fabricated to contain a single cell, and then the lyase could be added to the droplet. Alternatively, droplets can be manufactured to contain lysin, and then individual cells can be added to the droplets. Vials can contain thousands of such unique types and form libraries, such as diagnostic reagents.

[0782] The benchtop instrument pumps the emulsion to the inlet of a flat, wide analytical chamber. Quantum dots within each aqueous droplet diffuse randomly within the droplet's volume.

[0783] Lysins rupture the cells within each droplet, for example, upon cell / lyase contact, to release one or more cellular components. Specific quantum dots encounter cellular components and bind to them, forming structures of the sum of their sizes. For cellular components of the same size scale as the quantum dots, within a 10-nanometer diameter range, the binding interaction doubles the size and more than halves the diffusivity, while quantum dots without bound cellular components exhibit a constant diffusivity.

[0784] During the quantum dot diffusion process, ultraviolet light is projected through the analysis chamber. In some cases, an oil with the same refractive index as the aqueous droplet (RI = 1.33) is used (e.g., perfluoronaphthalene, RI = 1.31). Since the refractive indices of the fluorocarbon oil and the aqueous droplet are the same or substantially the same, there is no or almost no boundary refraction.

[0785] Fluorescent emission light is collected by a camera as a collection of fluorescent dot sources originating from the quantum dots. As these fluorescent dot sources diffuse randomly within each droplet, the camera records their positions (and thus the positions of the quantum dots). The aqueous droplets are large enough to sufficiently disperse the quantum dots for individual detection. Typically, a separation distance of at least 500 nm is required in the focal plane. In some cases, this is achieved by flattening the droplets into a pancake shape within the analysis chamber.

[0786] Data regarding the fluorescence position of each quantum dot were analyzed. Each quantum dot exhibited random motion in the x, y, and z directions over time, and the magnitude of the motion detected optically in the x and y directions depended on the diffusivity. Some quantum dots were observed to have initially high diffusivity, but subsequently gradually decreased diffusivity. Other quantum dots maintained high diffusivity. Quantum dots that tracked step changes over past time were used to detect and / or analyze, for example, one or more binding interactions between antibodies and cellular components. In one example, the blue quantum dots within a Y-shaped droplet (identified by the unique characteristics of its 6 red, 12 yellow, and 3 blue quantum dots) had initially high diffusivity, but subsequently gradually decreased diffusivity, indicating that antibody F on the blue quantum dots had binding interactions with one or more components of a specific cell lysed within the Y-shaped droplet. In some cases, each droplet of the Y-shaped microdroplet encapsulated a single cell of a cell population, and comparing the behavior of all blue quantum dots within the Y-shaped droplet group provided information about the heterogeneity of the cell population.

[0787] Example 9: Methods and compositions for surfactant analytes.

[0788] An emulsion stream was prepared and injected into a benchtop instrument. The emulsion contained various types of aqueous droplets in a fluorocarbon oil, the fluorocarbon oil having the same refractive index as the aqueous droplets. One type (referred to as subpopulation X) contained approximately 6 red-emitting quantum dots (coated with drug target receptor A), 9 green-emitting quantum dots (coated with drug target receptor B), 3 blue-emitting quantum dots (coated with drug target receptor C), and surfactant ligand U adsorbed on the droplet wall. Another type (referred to as subpopulation Y) contained approximately 6 red-emitting quantum dots (coated with receptor A), 12 yellow-emitting quantum dots (coated with receptor D), 3 blue-emitting quantum dots (coated with receptor C), and surfactant ligand V adsorbed on the droplet wall. Yet another type (referred to as subpopulation Z) contained approximately 6 red-emitting quantum dots (coated with receptor A), 9 green-emitting quantum dots (coated with receptor B), 6 blue-emitting quantum dots (coated with receptor C), and no ligands. The vials can contain thousands of such unique types and form, for example, screening libraries for candidate drugs.

[0789] The benchtop instrument pumps the emulsion to the inlet of a flat, wide analytical chamber. Quantum dots within each aqueous droplet diffuse randomly within the droplet's volume. Specific quantum dots encounter surfactant ligand molecules on the droplet wall, and the ligands bind to available acceptors. The negligible mobility of the ligands perpendicular to the wall significantly influences the diffusion properties of the attached quantum dots.

[0790] For example, Figure 22 A narrow channel in an optical chamber, such as above an opening, is shown to capture sample droplets. One or more emulsion droplets can be introduced through the opening to contact the sample droplets. A weak pulse of electric field is applied to fuse the emulsion droplets with the small sample droplets, allowing quantum dots to diffuse into the sample droplets. Oils with the same refractive index as the aqueous sample can be used; for example, perfluoronaphthene (RI = 1.31) and mineral oil can be used. The quantum dots diffuse onto the walls of the sample droplets and adhere to the surfactant analytes on the droplet walls, and the binding event is detected.

[0791] Figure 23 This study demonstrates the introduction of emulsion droplets into an oil matrix via an opening, and the fusion of the emulsion droplets and sample droplets by electrostatic attraction, thereby allowing quantum dots to diffuse into the sample droplets. The quantum dots diffuse onto the walls of the sample droplets and adhere to surfactant analytes on the droplet walls, and binding events are detected.

[0792] During the quantum dot diffusion process, ultraviolet light is projected through the analysis chamber. In some cases, an oil with the same refractive index as the aqueous droplet (RI = 1.33) is used (e.g., perfluoronaphthalene, RI = 1.31). Since the refractive indices of the fluorocarbon oil and the aqueous droplet are the same or substantially the same, there is no or almost no boundary refraction.

[0793] Fluorescent emission light is collected by a camera as a collection of fluorescent dot sources originating from quantum dots. As these fluorescent dot sources diffuse randomly within each droplet, the camera records their positions (and thus the positions of the quantum dots). In some cases, the aqueous droplets are large enough to sufficiently disperse the quantum dots for individual detection. Typically, the separation distance in the focal plane is at least 500 nm, and this can be achieved by flattening the droplets into a pancake shape within the analysis chamber.

[0794] Data regarding the fluorescence position of each quantum dot were analyzed. Each quantum dot exhibited random motion in the x, y, and z directions over time, and the magnitude of the motion detected optically in the x and y directions depended on the diffusivity. Some quantum dots were observed to have initially high diffusivity, but subsequently gradually decreased diffusivity. Other quantum dots maintained high diffusivity. Quantum dots that tracked step changes over past time were used to detect and / or analyze, for example, one or more binding interactions between receptors and surfactant ligands. In one example, a blue quantum dot derived from a Y-type droplet (identified by its unique characteristics of 6 red, 12 yellow, and 3 blue quantum dots) had an initially high diffusivity, but subsequently gradually decreased diffusivity (e.g., while X-type or Z-type blue quantum dots did not exhibit a gradual decrease to a lower diffusivity), indicating that receptor C has a binding interaction with surfactant ligand V, and that ligand V is a good candidate for detailed studies.

[0795] Example 10: Compositions and methods for the discovery of synergistic or allotropic drugs.

[0796] Select a small vial of reagent emulsion from the cold storage and install it in a benchtop instrument. This emulsion contains various types of aqueous droplets in a fluorocarbon oil. One type (referred to as subpopulation X) contains approximately 6 red-emitting quantum dots (coated with antigen A), 9 green-emitting quantum dots (coated with antigen B), 3 blue-emitting quantum dots (coated with antigen C), and ligand U in free solution. Another type (referred to as subpopulation Y) contains approximately 6 red-emitting quantum dots (coated with antigen D), 12 yellow-emitting quantum dots (coated with antigen E), 3 blue-emitting quantum dots (coated with antigen F), and ligand V in free solution. Yet another type (referred to as subpopulation Z) contains approximately 6 red-emitting quantum dots (coated with antigen G), 9 green-emitting quantum dots (coated with antigen H), 6 blue-emitting quantum dots (coated with antigen I), and ligand W in free solution. Antigens A, B, C, D, E, F, G, H, and I are candidates that specifically bind to antibody K. Vials can contain thousands of these unique types, forming screening libraries for, for example, candidate drugs.

[0797] Add the antibody K solution sample (optionally mixed with a weak gelling agent) to the benchtop instrument. For a large screening library of candidate drug combinations, such as antigens A to I and ligands V to W, test the synergistic effect or allotropy of the antibody to screen for combinations that bind to antibody K.

[0798] A benchtop instrument forms large droplets of antibody K solution in a fluorocarbon oil and pumps these droplets to the inlet end of a flat, wide analytical chamber. Simultaneously, an emulsion is pumped through a series of small orifices near the inlet end to the flat side of the analytical chamber. The emulsion droplets are trapped beneath the large droplets of antibody K solution, much like small balls under a carpet. Pumping the fluorocarbon oil to the inlet end of the analytical chamber causes the antibody K solution droplets to be propelled through the chamber and discharged from the distal end. With the aid of gravity and a weak gelling agent, the emulsion droplets are swept away.

[0799] A brief pulse of the electric field causes the thin gap between the fluorocarbon oil (the emulsion droplet and the antibody K solution) to break, thus demulsifying the emulsion droplet. The quantum dots in each droplet can now diffuse freely into the antibody K solution.

[0800] Quantum dots begin their diffusion journey as tight clusters, confined together within the droplet. Slowly, the quantum dots begin to diffuse outward, forming ever-expanding constellation-like groups. Furthermore, various ligands also expand outward, with their concentrations decreasing and overlapping. This results in each quantum dot experiencing a unique combination of ligands in its immediate environment.

[0801] Specific quantum dots encounter antibody K molecules and activated ligands in drug target solutions, and binding interactions occur, forming structures of the sum-size. For drug targets with the same size scale as quantum dots, within the 10-nanometer diameter range, binding interactions double the size and more than halve the diffusion rate, while quantum dots that actually undergo binding interactions have a constant diffusion rate.

[0802] During demulsification and quantum dot diffusion, ultraviolet light is projected through the analysis chamber. Fluorescent emission light is collected by a camera as a collection of fluorescent dot sources originating from the quantum dots. As the fluorescent dot sources diffuse randomly, the camera records the positions of these fluorescent dot sources (and thus the positions of the quantum dots).

[0803] Data on the fluorescence position of each quantum dot were analyzed. Each quantum dot exhibited random motion in the x, y, and z directions over time, with the magnitude of motion in the x and y directions depending on the diffusivity. Some quantum dots maintained high diffusivity. Other quantum dots were observed to have initially high diffusivity but subsequently gradually decreased diffusivity. Tracking quantum dots exhibiting step changes over past time, it was found that, for example, the blue quantum dots showing a step decrease in diffusivity originated from Y-type droplets (identified by the unique characteristics of their 6 red, 12 yellow, and 3 blue quantum dots) and were close to a constellation-like population of Z-type droplets. This suggests that antigen F (from the Y-type droplet population) has a binding interaction with antibody K in the presence of ligand W (from the Z-type droplet population), and that pairing antigen F with ligand W could be a good candidate for detailed study of co-orthogonal or allosteric interactions with antibody K. Since the ligands have known compositions, their diffusivity was directly calculated, thus generating a plot of their expected concentrations over time within the analytical chamber. In some examples, a weak gelling agent was added to minimize convection and diffusion rates.

[0804] For example, such as Figure 24 As shown, cooperative molecular combinations can be determined. The diffusion coefficient is known, so the measured random diffusion behavior of the green quantum dots (indicated by arrows) can be correlated with the concentration of the compound, including molecules that have expanded into overlapping constellation-like groups in the sample and adjacent droplets.

[0805] Example 11: Compositions and methods for protein labeling.

[0806] Select a small vial of reagent emulsion from the cold storage and install it in a benchtop instrument. This emulsion contains various types of aqueous droplets in a fluorocarbon oil. One type (referred to as subgroup X) contains approximately 6 reddish quantum dots (dendritic polymers with ligand A covering the tip), 9 greenish quantum dots (dendritic polymers with ligand B covering the tip), and 3 blueish quantum dots (dendritic polymers with ligand C covering the tip). Another type (referred to as subgroup Y) contains approximately 6 reddish quantum dots (dendritic polymers with ligand D covering the tip), 12 yellowish quantum dots (dendritic polymers with ligand E covering the tip), and 3 blueish quantum dots (dendritic polymers with ligand F covering the tip). Another type (called subpopulation Z) contains approximately 6 red quantum dots (dendritic polymers with ligand G covering the tips), 9 green quantum dots (dendritic polymers with ligand H covering the tips), and 6 blue quantum dots (dendritic polymers with ligand I covering the tips). Vials can contain thousands of these unique types, forming a screening library of protein probes.

[0807] The use of dendritic polymers is useful for reducing or avoiding steric hindrance, but it is not strictly necessary, as other components (such as flexible joints) can be used.

[0808] A protein K solution sample (optionally mixed with a weak gelling agent to control convection or slow diffusion) is added to the benchtop instrument. The interaction between the protein and ligands (e.g., ligands A through I) is tested to characterize the surface of protein K.

[0809] A benchtop instrument forms large droplets of protein K solution in a fluorocarbon oil and pumps these droplets to the inlet end of a flat, wide analytical chamber. Simultaneously, a reagent emulsion is pumped through a series of small orifices near the inlet end to the flat side of the analytical chamber. The emulsion droplets are trapped beneath the large droplets of protein K solution, much like small balls under a carpet. Pumping the fluorocarbon oil to the inlet end of the analytical chamber causes the protein K solution droplets to be propelled through the chamber and discharged from the distal end. With the aid of gravity and a weak gelling agent, the emulsion droplets are swept away.

[0810] A brief pulse of the electric field causes the thin gap between the fluorocarbon oil (the emulsion droplet and the protein K solution) to break, thus demulsifying the emulsion droplet. The quantum dots in each droplet can now diffuse freely into the protein K solution.

[0811] Quantum dots begin their diffusion journey as tight clusters, confined together within a droplet. Slowly, they begin to diffuse outward, forming ever-expanding constellation-like groups. Eventually, specific quantum dots encounter protein K molecules in the protein K solution and undergo binding interactions, forming structures of the same size. For example, if the protein K and quantum dots are of the same size scale, within the 10-nanometer diameter range, the binding interactions will double the size and more than halve the diffusivity. Quantum dots that do not undergo binding interactions have a constant diffusivity.

[0812] During demulsification and quantum dot diffusion, ultraviolet light is projected through the analysis chamber. Fluorescent emission light is collected by a camera as a collection of fluorescent dot sources originating from the quantum dots. As the fluorescent dot sources diffuse randomly, the camera records the positions of these fluorescent dot sources (and thus the positions of the quantum dots).

[0813] Data regarding the fluorescence position of each quantum dot were analyzed. Each quantum dot exhibits random motion in the x, y, and z directions over time, and the magnitude of the motion detected optically in the x and y directions depends on the diffusivity. Some quantum dots maintain high diffusivity. Other quantum dots were observed to have initially high diffusivity but subsequently gradually decrease it. Quantum dots exhibiting step changes in diffusivity over past time were tracked, revealing that, for example, the blue quantum dot exhibiting a step decrease in diffusivity originated from a Y-shaped droplet (identified by the unique characteristics of its 6 red, 12 yellow, and 3 blue quantum dots). This indicates that ligand F has a binding interaction with protein K. Because the ligand is located at the top of the dendritic polymer, it has fundamental freedom to probe the surface features of protein K without steric hindrance. Strong (persistent) and weak (transient) interactions can be detected and characterized by the timing of diffusivity step changes. Information about which ligands interact and do not interact is combined with the known structure of the ligands to assemble a map of the protein K surface.

[0814] Example 12: Method and composition for the determination of large droplets.

[0815] An emulsion stream was prepared and injected into a benchtop instrument. The emulsion contained various types of aqueous droplets in a fluorocarbon oil, the fluorocarbon oil having the same refractive index as the aqueous droplets. One type (referred to as subpopulation X) contained approximately 6 red-emitting quantum dots (coated with drug target receptor A), 9 green-emitting quantum dots (coated with drug target receptor B), 3 blue-emitting quantum dots (coated with drug target receptor C), and ligand U in free solution. Another type (referred to as subpopulation Y) contained approximately 6 red-emitting quantum dots (coated with drug target receptor D), 12 yellow-emitting quantum dots (coated with drug target receptor E), 3 blue-emitting quantum dots (coated with drug target receptor F), and ligand V in free solution. Yet another type (referred to as subpopulation Z) contained approximately 6 red-emitting quantum dots (coated with drug target receptor G), 9 green-emitting quantum dots (coated with drug target receptor H), 6 blue-emitting quantum dots (coated with drug target receptor I), and ligand W in free solution. The vials can contain thousands of such unique types, forming a screening library of candidate drugs.

[0816] The benchtop instrument pumps the emulsion to the inlet of a flat, wide analytical chamber. Quantum dots within each aqueous droplet diffuse randomly within the droplet's volume. Eventually, specific quantum dots encounter ligands and undergo binding interactions, forming structures of the sum of their sizes. If the ligands and quantum dots have the same size scale, within the 10-nanometer diameter range, the binding interactions will double the size and more than halve the diffusion rate, while quantum dots that do not undergo binding interactions have a constant diffusion rate.

[0817] During the quantum dot diffusion process, ultraviolet light is projected through the analysis chamber. Since the refractive indices of the fluorocarbon oil and the aqueous droplets are the same or substantially the same, there is no or almost no boundary refraction. Fluorescent emission light is collected by a camera as a collection of fluorescent dot sources originating from the quantum dots. As the fluorescent dot sources diffuse randomly within each droplet, the camera records the positions of these sources (and thus the positions of the quantum dots). In some cases, the aqueous droplets are large enough to sufficiently disperse the quantum dots for individual detection. Typically, the separation distance in the focal plane is at least 500 nm. This can be achieved by flattening the droplets into a pancake shape within the analysis chamber.

[0818] Data regarding the fluorescence position of each quantum dot were analyzed. Each quantum dot exhibits random motion in the x, y, and z directions over time, and the magnitude of the motion optically detected in the x and y directions depends on the diffusivity. Some quantum dots maintain high diffusivity. Other quantum dots were observed to have initially high diffusivity but subsequently gradually decrease it. Quantum dots exhibiting step changes in diffusivity over past time were tracked, and it was found that, for example, the blue quantum dot exhibiting a step decrease in diffusivity originated from a Y-shaped droplet (identified by the unique characteristics of its 6 red, 12 yellow, and 3 blue quantum dots). This suggests that ligand V has a binding interaction with acceptor F and may be a good candidate for detailed investigation.

[0819] Example 13: Compositions and methods for droplet preparation.

[0820] A method for producing a controlled emulsion composition is provided. A preparation chamber and reagents associated with the preparation of the controlled composition of the emulsion are also provided. For example, such as... Figure 21 As shown, an aqueous solution sample containing a specific type of quantum dots can be passed through a channel in which a water-immiscible matrix (e.g., oil) flow interrupts the aqueous solution flow to generate emulsion droplets containing the specific type of quantum dots, for example, emulsion droplets each containing one red quantum dot. Standard droplet generation devices, such as flow focusing devices, can be used. Other emulsion droplets can be generated in a similar manner, preferably in parallel, individually, for example, to provide groups of emulsion droplets each containing two blue quantum dots and groups of emulsion droplets each containing three violet quantum dots. These reagent emulsions can be stored before use.

[0821] Select a set of vials of reagent emulsions from the cold storage and install them in a benchtop instrument. These emulsions contain various types of aqueous droplets in fluorocarbon oils. One type (referred to as vial X) contains droplets with reddish quantum dots (coated with antibody A). Another type (referred to as vial Y) contains droplets with blued quantum dots (coated with antibody B). Yet another type (referred to as vial Z) contains droplets with violetd quantum dots (coated with antibody C).

[0822] Add the sample of reagent R in free solution to the benchtop instrument.

[0823] A benchtop instrument forms large droplets of reagent R solution in a fluorocarbon oil and pumps these droplets to the inlet end of a flat, wide preparation chamber (similar to the analysis chamber in other examples). Simultaneously, each emulsion is pumped through a series of small orifices near the inlet end to the flat side of the preparation chamber. Emulsion droplets are trapped beneath the large droplets of reagent R solution, much like small balls under a carpet. Note that emulsion droplets can be of any size and are not necessarily beneath the large droplets; emulsion droplets may be adjacent to, but not beneath, the large droplets. Pumping the fluorocarbon oil to the inlet end of the preparation chamber causes droplets of reagent R solution to be propelled through the preparation chamber and discharged from the distal end. With the aid of gravity and grooves on the surface of the preparation chamber, the emulsion droplets are swept away, limiting their migration and causing them to remain beneath the large droplets.

[0824] The flow rate of each emulsion into the preparation chamber is controlled to provide a controlled total number of red, blue, and violet quantum dots under large droplets.

[0825] A brief pulse of the electric field causes the thin gap between the fluorocarbon oil (the emulsion droplet and reagent R solution) to break, resulting in emulsion demulsification. Quantum dots in each droplet can now freely diffuse into the reagent R solution. Large droplets containing quantum dots now exhibit a controlled composition of red (antibody A), blue (antibody B), and violet (antibody C) quantum dots, as well as reagent R, although not in a homogeneous mixture.

[0826] Large droplets are expelled from the far end of the preparation chamber. The contents of the large droplets are mixed using standard microfluidic methods, such as via a meandering or turbulent path.

[0827] Large droplets are introduced into a standard droplet generating apparatus, such as a flow focusing apparatus. This forms a large population of smaller droplets from the larger droplets, all having the same composition, for example, where each smaller droplet contains one red quantum dot, two blue quantum dots, and three violet quantum dots. These smaller droplets are collected and stored in vials for later use, for example, in an analytical chamber as described in other embodiments. Other emulsion droplets can be generated separately, preferably in parallel, using the preparation chamber described herein, in a similar manner, for example, to provide a population of emulsion droplets each containing one yellow quantum dot, one green quantum dot, one dark blue quantum dot, and three red quantum dots; and a population of emulsion droplets each containing one green quantum dot, one orange quantum dot, two yellow quantum dots, and two dark blue quantum dots. These populations can be stored and used separately in the analytical chamber, or they can be mixed in defined proportions for storage and / or use, as described in other embodiments.

[0828] Depending on the need to generate a group of small droplets, the preparation chamber can be designed to be parallel to or sequential with other preparation chambers.

[0829] Example 14: Compositions and methods for drug discovery using emulsified targets.

[0830] Essentially as described in Example 3, the composition and method are also used with emulsified drug targets.

[0831] Select a small bottle of reagent emulsion from the cold storage and install it in the benchtop instrument. This reagent emulsion contains various types of aqueous droplets in a fluorocarbon oil, optionally containing additives to adjust the refractive index for a close match to the aqueous droplets. For example... Figure 27 As shown, one type (referred to as subpopulation X) contains approximately one red quantum dot (coated with ligand A), one orange quantum dot (coated with ligand B), and six blue quantum dots (coated with ligand C). Another type (referred to as subpopulation Y) contains approximately four red quantum dots (coated with ligand D), two orange quantum dots (coated with ligand E), and two blue quantum dots (coated with ligand F). Yet another type (referred to as subpopulation Z) contains approximately five red quantum dots (coated with ligand G), one orange quantum dot (coated with ligand H), and two blue quantum dots (coated with ligand I). Vials can contain thousands of such unique types, forming a screening library of candidate drugs. The aqueous droplets may optionally contain an antifreeze agent, such as ethylene glycol or glycerol, to allow for cryogenic storage.

[0832] An emulsion of the drug target (receptor R) solution is also added to the benchtop instrument. This target emulsion contains aqueous droplets in a fluorocarbon oil, which optionally contains additives to adjust the refractive index for close matching of the aqueous droplets. The drug target is tested against a large screening library of candidate drugs, for example, ligands A through I, to screen for ligands that specifically target receptor R.

[0833] Select a small bottle of synergistic emulsion from the cold storage and optionally add it to the benchtop instrument. This synergistic emulsion contains various types of aqueous droplets in a fluorocarbon oil, which optionally contains additives (e.g., carbobromine or carboiodine compounds) to adjust the refractive index for close matching of the aqueous droplets. Figure 27As shown, one type (referred to as subgroup χ) contains approximately 1 yellow quantum dot, 4 green quantum dots, and 2 violet quantum dots, along with a cooperating candidate α. Another type (referred to as subgroup ψ) contains approximately 5 green quantum dots, 2 violet quantum dots, and a cooperating candidate β. Yet another type (referred to as subgroup Ω) contains approximately 3 green quantum dots, 4 violet quantum dots, and a cooperating candidate γ. Vials can contain thousands of such unique types, forming a screening library of cooperating candidates. Aqueous droplets may optionally contain antifreeze, such as ethylene glycol or glycerol, to allow for cryogenic storage. Because these quantum dots will not be used for binding interactions, they can be formulated with shells that are particularly thick or thin, reducing or enhancing their diffusivity sufficiently to allow differentiation of quantum dots coated with ligands of the same color. This allows for a significant increase in address space. For example, a red quantum dot coated with ligand A can be optically distinguished from a very thick, shelled red quantum dot without coating.

[0834] The benchtop instrument pumps droplets from two or three emulsion sources to the inlet of a flat, wide analytical chamber. The emulsion droplets are randomly dispersed within a flat flocculation array. Pumping a fluorocarbon oil to the inlet of the analytical chamber causes the emulsion droplets to be propelled through the chamber and discharged from the distal end.

[0835] The droplets are imaged to determine the chemical composition of each droplet based on the quantum dot composition. Calculations are performed to determine which contacting droplets would provide the most useful information if they were to fuse.

[0836] As described by Hayat et al., Biosensors 2019, 9, 129, a brief pulse of focused laser between two contacting droplets causes the thin gaps in the fluorocarbon oil to break, fusing the droplet pair into a larger droplet. The contents of each source droplet can now be freely mixed within the resulting larger droplet. The composition of the fluorocarbon oil, surfactant, or aqueous droplet can be tailored to enhance the effectiveness of laser pulse fusion. The laser can be structured or pulsed to improve the effectiveness of laser pulse fusion. The thickness of the analytical chamber can be circulated to redistribute the droplets for optimal contact.

[0837] For example, if X-type droplets, χ-type droplets, and target droplets are fused, a larger droplet will be produced containing approximately one red quantum dot (coated with ligand A), one orange quantum dot (coated with ligand B), six blue quantum dots (coated with ligand C), one yellow quantum dot, four green quantum dots, two purple quantum dots, cooperating candidate α, and acceptor R.

[0838] The quantum dots within the resulting larger droplets diffuse freely within their volume.

[0839] Ultimately, specific quantum dots, in the presence of synergistic candidates, encounter drug target molecules and undergo binding interactions, forming a structure of the sum-size. If the drug target and quantum dots have the same size scale, within the 10-nanometer diameter range, the binding interaction will double the size and more than halve the diffusivity. Other quantum dots will have a constant diffusivity.

[0840] During quantum dot diffusion, structured near-infrared light is projected through the analysis chamber. Fluorescent emission light generated by two-photon excitation is collected by a camera. As the quantum dots randomly diffuse into the resulting larger droplets, their fluorescence positions change accordingly.

[0841] Data regarding the fluorescence position of each quantum dot were analyzed. Each quantum dot exhibited random motion in the x, y, and z directions over time, and the magnitude of the motion detected optically in the x and y directions depended on the diffusivity. Some quantum dots maintained high diffusivity. Other quantum dots were observed to have initially high diffusivity but subsequently gradually decreased diffusivity. Quantum dots exhibiting step changes in diffusivity over past time were tracked, and it was found that, for example, the blue quantum dot exhibiting a step decrease in diffusivity originated from an X-type droplet (identified by the unique features of its 1 red, 1 orange, and 6 blue quantum dots) and fused with a χ-type droplet (identified by the unique features of its 1 yellow, 4 green, and 2 purple quantum dots). This suggests that ligand C has a binding interaction with acceptor R in the presence of the cooperating candidate α, and is likely a good candidate for detailed studies.

[0842] Example 15: Compositions and methods for drug discovery using gel beads.

[0843] Essentially as described in Example 14, the composition and method are also used with a mixture of free quantum dots and gel beads containing quantum dots. The use of a mixture of free quantum dots (e.g., with a ligand coating) and gel beads (containing quantum dots solely for droplet recognition) allows for a very large address space.

[0844] Select a small bottle of reagent emulsion from the cold storage and install it in the benchtop instrument. This reagent emulsion contains various types of aqueous droplets in fluorocarbon oils. For example... Figure 28As shown, one type (referred to as subpopulation X) contains approximately one red quantum dot (coated with ligand A), one orange quantum dot (coated with ligand B), and six blue quantum dots (coated with ligand C). Another type (referred to as subpopulation Y) contains approximately four red quantum dots (coated with ligand D), two orange quantum dots (coated with ligand E), and two blue quantum dots (coated with ligand F). Yet another type (referred to as subpopulation Z) contains approximately two red quantum dots (coated with ligand G); one orange quantum dot (coated with ligand H); one blue quantum dot (coated with ligand I); and gel beads containing 60% yellow quantum dots, 20% blue quantum dots, and 20% purple quantum dots. Vials can contain thousands of these unique types, forming a screening library of candidate drugs.

[0845] These gel beads are commercially available and, in some cases, are formed by chemically expanding empty gel beads, soaking them in the presence of free quantum dots, and then chemically de-expanding the gel beads to fix the quantum dots therein.

[0846] The quantum dots within the gel beads do not diffuse randomly among themselves and are therefore optically distinguishable from free quantum dots. Multiple types of gel beads can be used in the same droplet, which also contains free quantum dots with ligands coated on their surfaces, providing a vast address space.

[0847] An emulsion of the drug target (receptor R) solution was also added to the benchtop instrument. This target emulsion contained aqueous droplets in a fluorocarbon oil. The drug target was tested against a large screening library of candidate drugs, for example, ligands A through I, to screen for ligands that specifically target receptor R.

[0848] Select a small bottle of synergistic emulsion from the cold storage and optionally add it to the benchtop instrument. This synergistic emulsion contains various types of aqueous droplets in fluorocarbon oils. For example... Figure 28 As shown, one type (referred to as subpopulation χ) contains gel beads with 60% green quantum dots, 20% yellow quantum dots, and 20% purple quantum dots, along with synergistic candidate α. Another type (referred to as subpopulation ψ) contains gel beads with 60% yellow quantum dots, 20% purple quantum dots, and 20% green quantum dots, along with synergistic candidate β. Yet another type (referred to as subpopulation Ω) contains gel beads with 60% yellow quantum dots, 20% purple quantum dots, and 20% blue quantum dots; and gel beads with 50% red quantum dots, 25% purple quantum dots, and 25% yellow quantum dots, along with synergistic candidate γ. Vials can contain thousands of these unique types, forming a screening library of synergistic candidates.

[0849] The benchtop instrument pump...

Claims

1. A method for analyzing an analyte, comprising: Contact (i) a first composition with (ii) a second composition, the first composition comprising a liquid phase and a sample in the liquid phase, the second composition comprising a liquid matrix and a formulation encapsulated in the liquid matrix, wherein the formulation comprises a plurality of fluorescent constructs and one or more analyte interaction reagents attached to the fluorescent constructs; as well as The liquid phase and the liquid matrix are combined such that the analytes in the sample interact with one or more of the analyte-interacting reagents attached to the fluorescent construct to generate a detectable signal. The detectable signal is analyzed using optical position tracking, and the analysis shows the presence or absence, amount or concentration and / or activity of the analyte in the sample, and the analyte in the sample is capable of specifically binding to the analyte-interacting reagent.

2. The method of claim 1, wherein the analyte interaction reagent is an analyte binding reagent.

3. The method of claim 1, wherein during the merging process, the liquid phase and the liquid matrix are fused into a single fluid.

4. The method of claim 1, wherein the analyte in the sample and the analyte-interacting reagent are capable of participating in the reaction.

5. The method of claim 1, wherein the plurality of fluorescent constructs comprises one or more fluorescent particles, one or more fluorescent small molecules, one or more fluorescent peptides or proteins, one or more fluorescent dyes, or any combination thereof.

6. The method of claim 1, wherein the formulation comprises one or more polymer particles, each of the one or more polymer particles comprising one or more fluorescent particles, one or more fluorescent small molecules, one or more fluorescent peptides or proteins, one or more fluorescent dyes or any combination thereof.

7. The method of claim 1, wherein the plurality of fluorescent constructs comprises one or more fluorescent semiconductor nanoparticles.

8. The method of claim 1, wherein the plurality of fluorescent constructs comprises one or more quantum dots.

9. The method of claim 1, wherein the analyte interacting agent is directly attached to one or more of the fluorescent constructs.

10. The method of claim 1, wherein the analyte interacting reagent is indirectly attached to one or more of the fluorescent constructs via a connector or a common binding partner.

11. The method of claim 1, wherein the analyte interacting reagent is covalently or non-covalently attached to one or more of the fluorescent construct.

12. The method of claim 1, wherein each of the one or more fluorescent constructs has one or more analyte interacting reagents to which it is attached, or only a subset of the one or more fluorescent constructs has one or more analyte interacting reagents to which it is attached.

13. The method of claim 1, wherein the second composition comprises or is an emulsion of the liquid matrix and the liquid matrix.

14. The method of claim 1, wherein the formulation comprises aqueous droplets or aqueous droplets.

15. The method of claim 14, wherein the second composition does not contain an emulsion or is not an emulsion.

16. The method of claim 1, wherein the formulation comprises a gel.

17. The method of claim 16, wherein the gel comprises the plurality of fluorescent constructs and is capable of releasing the plurality of fluorescent constructs.

18. The method of claim 1, wherein the liquid phase is water, and the formulation is soluble in water and / or miscible with water.

19. The method of claim 1, wherein the liquid matrix is ​​immiscible or substantially immiscible with water.

20. The method of claim 1, wherein the liquid matrix comprises lipids, oils, hydrocarbon fluids, fluorocarbon fluids, chlorinated hydrocarbon fluids, brominated hydrocarbon fluids, iodinated hydrocarbon fluids, organosilicon fluids, or mixtures thereof.

21. The method of claim 1, wherein the liquid matrix is ​​selected from the group consisting of lipids, oils, hydrocarbon fluids, fluorocarbon fluids, chlorinated hydrocarbon fluids, brominated hydrocarbon fluids, iodinated hydrocarbon fluids, organosilicon fluids, and mixtures thereof.

22. The method of claim 1, wherein the merging of the liquid phase and the liquid matrix is ​​performed in an analytical chamber.

23. The method of claim 1, wherein the merging comprises merging adjacent streams of the first composition and the formulation, respectively.

24. The method of claim 1, wherein the merging comprises merging the cross flows of the first composition and the formulation, respectively.

25. The method of claim 1, wherein the merging comprises merging the vertical flows of the first composition and the formulation, respectively.

26. The method of claim 1, wherein the merging comprises merging the inclined streams of the first composition and the formulation, respectively.

27. The method of claim 1, wherein the merging comprises merging the relative flows of the first composition and the formulation, respectively.

28. The method of claim 1, wherein the merging comprises merging concentric flows of the first composition and the formulation, respectively.

29. The method of claim 1, wherein the first composition and / or the formulation comprises one or more demulsifiers.

30. The method of claim 1, wherein the first composition and / or the formulation comprises one or more gelling agents.

31. The method of claim 1, wherein the first composition and / or the formulation comprises one or more viscosity enhancers.

32. The method of claim 1, wherein the first composition is a first emulsion comprising the liquid phase as emulsified droplets in a first liquid matrix, and the liquid matrix of the second composition is a second liquid matrix.

33. The method of claim 32, wherein the first liquid matrix and the second liquid matrix are the same.

34. The method of claim 32, wherein the first liquid matrix and the second liquid matrix are different.

35. The method of claim 32, wherein the first liquid matrix and the second liquid matrix are independently selected from the group consisting of lipids, oils, hydrocarbon fluids, fluorocarbon fluids, chlorinated hydrocarbon fluids, brominated hydrocarbon fluids, iodinated hydrocarbon fluids, silicone fluids, and any suitable combination thereof.

36. The method of claim 1, wherein the merging comprises applying an electric field to the first composition and / or the second composition.

37. The method of claim 1, wherein the first composition and the second composition each comprise surfactants with opposite charges, and the merging comprises contacting the surfactants with opposite charges with each other.

38. The method of claim 1, wherein the merging comprises applying a demulsifier to the first composition and / or the second composition.

39. The method of claim 1, wherein the merging does not include applying a demulsifier.

40. The method of claim 1, wherein the merging comprises applying heat to the first composition and / or the second composition.

41. The method of claim 1, wherein the merging comprises applying a gel depolymerizing agent to the first composition and / or the second composition.

42. The method of claim 1, wherein the detectable signal is a fluorescence signal.

43. The method of claim 1, wherein the detectable signal is induced by excitation light, the excitation light comprising, substantially comprising, or comprising unpolarized light.

44. The method of claim 1, wherein the detectable signal is induced by excitation light, the excitation light comprising, substantially comprising, or comprising linearly polarized light.

45. The method of claim 1, wherein the detectable signal is induced by excitation light, the excitation light comprising, substantially comprising, or comprising circularly polarized light.

46. ​​The method of claim 1, wherein the detectable signal is induced by excitation light, the excitation light comprising, substantially comprising, or comprised of elliptically polarized light and / or cocycloidically polarized light.

47. The method of claim 1, wherein the detectable signal is induced by excitation light, the excitation light comprising, substantially comprising, or comprising of light of a single wavelength.

48. The method of claim 1, wherein the detectable signal is induced by excitation light, the excitation light comprising, substantially comprising, or comprising polychromatic light.

49. The method of claim 1, wherein the detectable signal is induced by excitation light, the excitation light comprising, substantially comprising, or comprising incoherent light.

50. The method of claim 1, wherein the detectable signal is induced by excitation light, the excitation light comprising, substantially comprising, or comprising coherent light.

51. The method of claim 1, wherein the detectable signal is induced by excitation light, the excitation light comprising, substantially comprising, or comprising of continuous light.

52. The method of claim 1, wherein the detectable signal is induced by excitation light, the excitation light comprising, substantially comprising, or comprising of pulsed light.

53. The method of claim 1, wherein the detectable signal is induced by excitation light, the excitation light comprising, substantially comprising, or comprised of light applied at a single incident angle.

54. The method of claim 1, wherein the detectable signal is induced by excitation light, the excitation light comprising, substantially comprising, or comprised of light applied at a set of incident angles.

55. The method of claim 1, further comprising applying an external electric field to the combined composition, wherein the external electric field is sufficient to induce electrophoretic movement of the fluorescent construct within the combined composition.

56. The method of claim 55, wherein the external electric field comprises, consists essentially of, or consists of: (i) A constant electric field applied in a constant direction; (ii) A pulsed electric field applied in a constant direction; And / or (iii) an oscillating electric field applied in a constant direction.

57. The method of claim 55, wherein the external electric field comprises, substantially consists of, or consists of the following: (i) A constant electric field that switches between multiple directions; (ii) Pulsed electric fields switching between multiple directions; And / or (iii) an oscillating electric field that switches between multiple directions.

58. The method of claim 1, further comprising applying a force to the combined composition to reduce or eliminate nonspecific interactions between the analyte molecule and the fluorescent construct.

59. The method of claim 1, further comprising applying acoustic waves to the combined composition.

60. The method of claim 1, further comprising applying fluorescent excitation light to the combined composition.

61. The method of claim 60, wherein the fluorescence excitation light is a fluorescence excitation light used in the following: confocal microscopy, structured illumination microscopy (SIM), stochastic optical reconstruction microscopy (STORM), point scan two-photon microscopy, scan line angle projection microscopy (SLAPMi), ghosting imaging (GI) and / or sparse constrained ghosting imaging (GISC).

62. A method for analyzing an analyte, comprising: Contact (i) a first composition with (ii) a second composition, the first composition comprising a liquid phase and a sample in the liquid phase, the second composition comprising a liquid matrix and a formulation encapsulated in the liquid matrix, wherein the formulation comprises a plurality of fluorescent constructs and one or more analyte interaction reagents attached to the fluorescent constructs; In the analytical chamber, the liquid phase and the liquid matrix are combined such that the analytes in the sample interact with one or more of the analyte-interacting reagents attached to the fluorescent construct; The excitation light is guided through the analysis chamber to generate a detectable signal; When the fluorescent construct in each formulation diffuses in the combined composition, fluorescence emission is detected and / or measured; The identity of each analyte interacting agent present in each formulation is determined based on the pattern of fluorescence emission wavelengths during diffusion. The random behavior of the fluorescence emission position and / or fluorescence emission magnitude provides an indication of the presence or absence, amount and / or activity of the analyte in the sample, wherein the analyte in the sample is capable of specifically binding to the analyte-interacting reagent.

63. The method of claim 62, wherein the excitation light has a controlled phase and wavelength.

64. The method of claim 62, wherein the excitation light is a standing wave.

65. The method of claim 62, wherein the excitation light is a coherent excitation light, and the phase of the coherent excitation light is controlled such that the peaks and troughs of the excitation light move through the fluorescent construct sufficiently to cause corresponding oscillations in the fluorescence of the fluorescent construct.

66. The method of claim 62, wherein the excitation light is an elliptically polarized coherent excitation light, and the ellipticity of the excitation light is controlled such that the peaks and troughs of the excitation light move through the fluorescent construct sufficiently to cause corresponding oscillations in the fluorescence of the fluorescent construct.

67. The method of claim 62, wherein the excitation light has a controlled spatial pattern and wavelength.

68. The method of claim 67, wherein the excitation light is applied as a patterned spatial array over the entire analytical chamber such that the intensity of the excitation light varies at different points within the analytical chamber, sufficient to cause a fluorescence change in the fluorescent construct as it moves between the different points within the analytical chamber.

69. The method of claim 67, wherein the patterned spatial array of excitation light is moved throughout the analysis chamber such that the light intensity varies with time and for different points within the analysis chamber, sufficient to cause a fluorescence change in the fluorescent construct.

70. The method of claim 62, wherein the detection and / or measurement of fluorescence emission includes detecting and / or measuring the magnitude of fluorescence emission.

71. The method of claim 62, wherein the pattern of the fluorescence emission wavelength during diffusion is determined near the location where each formulation has been incorporated with the liquid phase.

72. The method of claim 62, wherein during the merging process, the liquid phase and the liquid matrix are fused into a single fluid.

73. The method of claim 62, wherein the analyte in the sample and the analyte-interacting reagent are capable of participating in the reaction.

74. The method of claim 62, wherein the plurality of fluorescent constructs comprises one or more fluorescent particles, one or more fluorescent small molecules, one or more fluorescent peptides or proteins, one or more fluorescent dyes, or any combination thereof.

75. The method of claim 62, wherein the formulation comprises one or more polymer particles, each of the one or more polymer particles comprising one or more fluorescent particles, one or more fluorescent small molecules, one or more fluorescent peptides or proteins, one or more fluorescent dyes or any combination thereof.

76. The method of claim 62, wherein the plurality of fluorescent constructs comprises one or more fluorescent semiconductor nanoparticles.

77. The method of claim 62, wherein the plurality of fluorescent constructs comprises one or more quantum dots.

78. The method of claim 62, wherein the analyte interacting agent is directly attached to one or more of the fluorescent constructs.

79. The method of claim 62, wherein the analyte interacting reagent is indirectly attached to one or more of the fluorescent construct.

80. The method of claim 62, wherein the analyte interacting reagent is non-covalently attached to one or more of the fluorescent construct.

81. The method of claim 62, wherein the analyte interacting reagent is covalently attached to one or more of the fluorescent construct.

82. The method of claim 62, wherein each of the one or more fluorescent constructs has one or more analyte interacting reagents to which it is attached.

83. The method of claim 62, wherein a subset of one or more of the fluorescent constructs has one or more analyte interacting reagents to which they are attached.

84. The method of claim 62, wherein the second composition comprises the liquid matrix and an emulsion of the formulation.

85. The method of claim 62, wherein the formulation comprises aqueous droplets, wherein the aqueous droplets optionally comprise an antifreeze agent, such as ethylene glycol or glycerin.

86. The method of claim 62, wherein the second composition does not contain an emulsion or is not an emulsion.

87. The method of claim 62, wherein the formulation comprises a gel.

88. The method of claim 87, wherein the gel comprises the plurality of fluorescent constructs and is capable of releasing the plurality of fluorescent constructs.

89. The method of claim 62, wherein the liquid phase is water, and the formulation is soluble in water and / or miscible with water.

90. The method of claim 62, wherein the liquid matrix is ​​immiscible or substantially immiscible with water.

91. The method of claim 62, wherein the liquid matrix comprises lipids, oils, hydrocarbon fluids, fluorocarbon fluids, chlorinated hydrocarbon fluids, brominated hydrocarbon fluids, iodinated hydrocarbon fluids, silicone fluids, or mixtures thereof.

92. The method of claim 62, wherein the liquid matrix is ​​selected from the group consisting of lipids, oils, hydrocarbon fluids, fluorocarbon fluids, chlorinated hydrocarbon fluids, brominated hydrocarbon fluids, iodinated hydrocarbon fluids, organosilicon fluids, and mixtures thereof.

93. The method of claim 62, wherein the merging of the liquid phase and the liquid matrix is ​​performed in an analytical chamber.

94. The method of claim 62, wherein the merging comprises merging adjacent streams of the first composition and the formulation, respectively.

95. The method of claim 62, wherein the merging comprises merging the cross flows of the first composition and the formulation, respectively.

96. The method of claim 62, wherein the merging comprises merging the vertical flows of the first composition and the formulation, respectively.

97. The method of claim 62, wherein the merging comprises merging the inclined streams of the first composition and the formulation, respectively.

98. The method of claim 62, wherein the merging comprises merging the relative flows of the first composition and the formulation, respectively.

99. The method of claim 62, wherein the merging comprises merging concentric flows of the first composition and the formulation, respectively.

100. The method of claim 62, wherein the first composition and / or the formulation comprises one or more demulsifiers.

101. The method of claim 62, wherein the first composition and / or the formulation comprises one or more gelling agents.

102. The method of claim 62, wherein the first composition and / or the formulation comprises one or more viscosity enhancers.

103. The method of claim 62, wherein the first composition is a first emulsion comprising the liquid phase as emulsified droplets within a first liquid matrix, and the liquid matrix of the second composition is a second liquid matrix.

104. The method of claim 103, wherein the first liquid matrix and the second liquid matrix are the same.

105. The method of claim 103, wherein the first liquid matrix and the second liquid matrix are different.

106. The method of claim 103, wherein the first liquid matrix and the second liquid matrix are independently selected from the group consisting of lipids, oils, hydrocarbon fluids, fluorocarbon fluids, chlorinated hydrocarbon fluids, brominated hydrocarbon fluids, iodinated hydrocarbon fluids, silicone fluids, and any suitable combination thereof.

107. The method of claim 62, wherein the merging comprises applying an electric field to the first composition and / or the second composition.

108. The method of claim 62, wherein the first composition and the second composition each comprise surfactants with opposite charges, and the merging comprises contacting the surfactants with opposite charges with each other.

109. The method of claim 62, wherein the merging comprises applying a demulsifier to the first composition and / or the second composition.

110. The method of claim 62, wherein the merging does not include applying a demulsifier.

111. The method of claim 62, wherein the merging comprises applying heat to the first composition and / or the second composition.

112. The method of claim 62, wherein the merging comprises applying a gel depolymerizing agent to the first composition and / or the second composition.

113. The method of claim 62, wherein the detectable signal is a fluorescence signal.

114. The method of claim 62, wherein the detectable signal is induced by excitation light, the excitation light comprising, substantially comprising, or comprising unpolarized light.

115. The method of claim 62, wherein the detectable signal is induced by excitation light, the excitation light comprising, substantially comprising, or comprising linearly polarized light.

116. The method of claim 62, wherein the detectable signal is induced by excitation light, the excitation light comprising, substantially comprising, or comprising circularly polarized light.

117. The method of claim 62, wherein the detectable signal is induced by excitation light, the excitation light comprising, substantially comprising, or comprised of elliptically polarized light and / or cocycloidically polarized light.

118. The method of claim 62, wherein the detectable signal is induced by excitation light, the excitation light comprising, substantially comprising, or comprising of light of a single wavelength.

119. The method of claim 62, wherein the detectable signal is induced by excitation light, the excitation light comprising, substantially comprising, or comprising polychromatic light.

120. The method of claim 62, wherein the detectable signal is induced by excitation light, the excitation light comprising, substantially comprising, or comprising incoherent light.

121. The method of claim 62, wherein the detectable signal is induced by excitation light, the excitation light comprising, substantially comprising, or comprising coherent light.

122. The method of claim 62, wherein the detectable signal is induced by excitation light, the excitation light comprising, substantially comprising, or comprising of continuous light.

123. The method of claim 62, wherein the detectable signal is induced by excitation light, the excitation light comprising, substantially comprising, or comprising of pulsed light.

124. The method of claim 62, wherein the detectable signal is induced by excitation light, the excitation light comprising, substantially comprising, or comprising light applied at a single incident angle.

125. The method of claim 62, wherein the detectable signal is induced by excitation light, the excitation light comprising, substantially comprising, or comprising of light applied at a set of incident angles.

126. The method of claim 62, further comprising applying an external electric field to the combined composition, wherein the external electric field is sufficient to induce electrophoretic movement of the fluorescent construct within the combined composition.

127. The method of claim 126, wherein the external electric field comprises, is substantially composed of, or is composed of a constant electric field applied in a constant direction.

128. The method of claim 126, wherein the external electric field comprises, is substantially composed of, or is composed of a pulsed electric field applied in a constant direction.

129. The method of claim 126, wherein the external electric field comprises, substantially consists of, or consists of the following: (i) An oscillating electric field applied in a constant direction; (ii) A constant electric field that switches between multiple directions; (iii) Pulsed electric fields switching between multiple directions; And / or (iv) an oscillating electric field that switches between multiple directions.

130. The method of claim 1, wherein the analyte comprises a protein moiety, and the analyte interacting agent comprises a protein binding agent.

131. The method of claim 1, wherein the analyte comprises a polynucleotide sequence, and the analyte interacting reagent comprises a sequence capable of hybridizing with the polynucleotide sequence.

132. The method of claim 131, further comprising using a temperature control device for the analysis chamber and / or using targeted local heating of quantum dots with focused light to unwind and / or anneal the hybridized sequences.

133. The method of claim 1, further comprising moving an image of each fluorescent construct across the entire surface of the optical detector using a nutation transparency window and / or a rotating prism.

134. A method for preparing a set of aqueous droplets, comprising contacting (i) a first composition with (ii) a second composition, the first composition comprising a liquid phase and a sample in the liquid phase, the second composition comprising a liquid matrix and a formulation encapsulated in the liquid matrix, wherein the formulation comprises a plurality of fluorescent constructs and one or more analyte interaction reagents attached to the fluorescent constructs; The liquid phase and the formulation are combined such that the analytes in the sample interact with one or more of the analyte-interacting reagents attached to the fluorescent constructs to generate a detectable signal; then, each aqueous droplet is prepared wherein multiple fluorescent constructs contain combinations of fluorescent emission colors, each fluorescent emission color having a certain count, and each fluorescent construct has zero, one or more analyte-interacting reagents attached to it. The identity of the analyte-interacting reagent is specific to the color of the fluorescent construct it is attached to and / or specific to the combination of color and count of the fluorescent construct within each aqueous droplet. The detectable signal is analyzed using optical position tracking, and the analysis shows the presence or absence, amount or concentration, and / or activity of the analyte in the sample. The analyte in the sample can specifically bind to the analyte interaction reagent.

135. The method of claim 134, further comprising collecting the aqueous droplet assembly into a container.

136. The method of claim 134, wherein the liquid matrix is ​​immiscible with water.

137. The method of claim 134, wherein the liquid matrix is ​​selected from the group consisting of lipids, oils, hydrocarbon fluids, fluorocarbon fluids, chlorinated hydrocarbon fluids, brominated hydrocarbon fluids, iodinated hydrocarbon fluids, organosilicon fluids, and any suitable combination or mixture thereof.

138. The method of claim 134, wherein the liquid matrix is ​​formulated into a gel.

139. The method of claim 134, wherein the liquid matrix is ​​vitrified at a low temperature.

140. The method of claim 134, wherein the liquid matrix is ​​solidified at a low temperature.

141. The method of claim 134, wherein the liquid matrix has a refractive index that matches the refractive index of the aqueous droplets suspended in the liquid matrix; or wherein the liquid matrix has a higher refractive index than the refractive index of the aqueous droplets suspended in the liquid matrix; or wherein the liquid matrix has a lower refractive index than the refractive index of the aqueous droplets suspended in the liquid matrix.

142. The method of claim 134, wherein the liquid matrix is ​​non-Newtonian.

143. The method of claim 134, wherein the liquid matrix is ​​shear-thinned.

144. The method of claim 134, wherein the liquid matrix is ​​shear-thickened.

145. The method of claim 134, wherein the liquid matrix has a high viscosity.

146. The method of claim 134, wherein the liquid matrix has a low viscosity.

147. The method of claim 134, wherein the liquid matrix has a density matching that of the aqueous droplets suspended in the liquid matrix.

148. The method of claim 134, wherein the liquid matrix has a smaller density than the density of aqueous droplets suspended in the liquid matrix.

149. The method of claim 134, wherein the liquid matrix has a density greater than that of the aqueous droplets suspended in the liquid matrix.

150. The method of claim 134, wherein the fluorescent construct is selected from the group consisting of: quantum dots; fluorescent proteins; fluorescent molecules; polymer particles containing quantum dots, fluorescent proteins, or fluorescent molecules; fluorescent particles each connected to another fluorescent particle via a chemical bonding system; and fluorescent particles each connected to a magnetic particle via a chemical bonding system.

151. The method of claim 134, wherein the fluorescent construct comprises one or more analyte binding agents bound to its surface.

152. The method of claim 134, wherein the aqueous droplet contains one or more magnetic particles.

153. The method of claim 134, wherein the aqueous droplets are stabilized with one or more surfactants.

154. The method of claim 134, wherein the fluorescent construct is connected to fluorescent particles and / or magnetic particles via a connector.

155. The method of claim 134, wherein the aqueous droplet assembly contains different combinations of fluorescent construct emission colors and counts.

156. A method for formulating a set of gel beads, comprising contacting (i) a first composition with (ii) a second composition, the first composition comprising a liquid phase and a sample in the liquid phase, the second composition comprising a liquid matrix and a formulation encapsulated in the liquid matrix, wherein the formulation comprises a plurality of fluorescent constructs and one or more analyte interaction reagents attached to the fluorescent constructs; The liquid phase and the formulation are combined such that the analyte in the sample interacts with one or more of the analyte-interacting reagents attached to the fluorescent construct to generate a detectable signal; then... Each gel bead is formulated in a gel matrix that can be removed by physical or chemical methods, wherein each gel bead contains multiple fluorescent constructs having combinations of fluorescent emission colors, each fluorescent emission color having a certain count, and each fluorescent construct having zero, one, or more reagents attached to it. The identity of the analyte-interacting reagent is specific to the color of the fluorescent construct it is attached to and / or specific to the combination of color and count of the fluorescent construct within each gel bead. The detectable signal is analyzed using optical position tracking, and the analysis shows the presence or absence, amount or concentration, and / or activity of the analyte in the sample. The analyte in the sample can specifically bind to the analyte interaction reagent.

157. The method of claim 156, further comprising collecting the gel bead assembly into a container.

158. The method of claim 156, wherein the fluorescent construct is selected from the group consisting of: quantum dots; fluorescent proteins; fluorescent molecules; polymer particles containing quantum dots, fluorescent proteins, or fluorescent molecules; fluorescent particles each connected to another fluorescent particle via a chemical bonding system; and fluorescent particles each connected to a magnetic particle via a chemical bonding system.

159. The method of claim 156, wherein the fluorescent construct comprises one or more analyte binding agents bound to its surface.

160. The method of claim 156, wherein the gel beads contain one or more magnetic particles.

161. The method of claim 156, wherein the fluorescent construct is connected to fluorescent particles and / or magnetic particles via a connector.

162. The method of claim 156, wherein the gel bead assembly contains different combinations of fluorescent construct emission colors and counts.

163. The method of claim 156, wherein the gel is melted at an elevated temperature.

164. The method of claim 156, further comprising depolymerizing the gel using one or more enzymes.

165. The method of claim 156, further comprising depolymerizing the gel using one or more chemical agents.

166. The method of claim 156, further comprising depolymerizing the gel by light.

167. The method of claim 156, wherein the gel bead assembly contains different combinations of fluorescent construct emission colors and counts.

168. The method of claim 156, the method comprising applying light to a composition comprising a gel-forming monomer, a photocatalyst, and a plurality of fluorescent constructs to selectively catalyze the polymerization of the gel-forming monomer to form gel beads comprising a subset of the plurality of fluorescent constructs.

169. A composition comprising the set of aqueous droplets and / or gel beads formulated as described in claim 134.

170. A method for analyzing an analyte, comprising: Contact (i) a first composition with (ii) a second composition, the first composition comprising a first liquid matrix and a sample encapsulated in the first liquid matrix, the second composition comprising a second liquid matrix and a formulation encapsulated in the second liquid matrix, wherein the formulation comprises a plurality of fluorescent constructs and one or more analyte interaction reagents attached to the fluorescent constructs; as well as The first liquid matrix and the second liquid matrix are combined such that the analytes in the sample interact with one or more of the analyte-interacting reagents attached to the fluorescent construct to generate a detectable signal. The detectable signal is analyzed using optical position tracking, and the analysis shows the presence or absence, amount or concentration, and / or activity of the analyte in the sample. The analyte in the sample can specifically bind to the analyte interaction reagent.

171. The method of claim 170, wherein the analyte is located at the boundary between the first liquid matrix and the sample.

172. The method of claim 170, wherein the first liquid matrix and the sample have the same or substantially the same refractive index.

173. The method of claim 170, wherein the first liquid matrix and the second liquid matrix are the same.

174. The method of claim 170, wherein the first liquid matrix and the second liquid matrix are different.

175. The method of claim 170, wherein the sample is a solution and the analyte is located at the boundary between the first liquid matrix and the sample.

176. The method of claim 170, wherein after combining the sample with the formulation, the sample and the formulation remain encapsulated in the first liquid matrix and / or the second liquid matrix.

177. The method of claim 170, further comprising applying excitation light to the plurality of fluorescent constructs, and the detectable signal comprising fluorescence emission from the plurality of fluorescent constructs, wherein the excitation light comprises simple ultraviolet light or pulsed ultraviolet light.

178. The method of claim 170, further comprising analyzing the diffusion of the plurality of fluorescent constructs, the analysis including analyzing the detectable signal.

179. The method of claim 170, further comprising analyzing the change in the diffuseness of the plurality of fluorescent constructs with increasing and / or decreasing temperature.

180. The method of claim 179, wherein the change in diffusion is analyzed to provide a melting temperature indicating the interaction between the analyte and the analyte-interacting reagent.

181. The method of claim 170, wherein the first composition comprises a first oil and an aqueous sample encapsulated in the first oil, and the second composition comprises a second oil and an aqueous formulation encapsulated in the second oil.

182. The method of claim 181, wherein the aqueous formulation comprises a plurality of quantum dots and an analyte binding agent attached to one or more of the quantum dots.

183. The method of claim 182, wherein the aqueous sample and the aqueous formulation form a combined aqueous composition retained in an oil, and the combined aqueous composition and its encapsulating oil have the same or substantially the same refractive index.

184. The method of claim 183, further comprising detecting changes in the diffusivity of the plurality of quantum dots encapsulated in the oil as temperature increases and / or temperature decreases.

185. A method for analyzing cells, comprising: Contact (i) a first composition with (ii) a second composition, the first composition comprising a first liquid matrix and a sample encapsulated in the first liquid matrix, wherein the sample comprises single cells, and the second composition comprising a second liquid matrix and a formulation encapsulated in the second liquid matrix, wherein the formulation comprises a plurality of fluorescent constructs and one or more analyte-interacting reagents attached to the fluorescent constructs, wherein: (a) the single cells are lysed in the sample to release one or more cellular components or (b) the single cells are not lysed in the sample; and The first liquid matrix and the second liquid matrix are combined such that the cellular components interact with one or more of the analyte-interacting reagents attached to the fluorescent construct to generate a detectable signal. The detectable signal is analyzed using optical position tracking, and the analysis shows the presence or absence, amount or concentration, and / or activity of the analyte in the sample. The analyte in the sample can specifically bind to the analyte interaction reagent.

186. The method of claim 185, wherein the second composition optionally includes a cell lysis agent.

187. The method of claim 186, wherein the method comprises analyzing a plurality of single cells from a cell population and comparing the presence or absence, amount or concentration and / or activity of the cellular components in a first single cell with the presence or absence, amount or concentration and / or activity of the cellular components in a second single cell to infer cellular heterogeneity in the cell population.

188. A method for analyzing an analyte, comprising: The first emulsion and the second emulsion are brought into contact with the sample, wherein: The first emulsion comprises a first formulation encapsulated in a first liquid matrix, wherein the first formulation comprises a first plurality of fluorescent constructs and a first reagent attached to one or more of the fluorescent constructs, and a first free agent; The second emulsion comprises a second formulation encapsulated in a second liquid matrix, wherein the second formulation comprises a second plurality of fluorescent constructs and a second reagent attached to one or more of the fluorescent constructs, and a second free agent; and The sample contains an analyte, wherein the first reagent and the second reagent are capable of binding to the analyte; and The first and second emulsions are demulsified to allow the first and second plurality of fluorescent constructs, as well as the first and second free agents, to diffuse in the sample, wherein at least one of the first plurality of fluorescent constructs and / or at least one of the second plurality of fluorescent constructs is allowed to interact with the analyte in the presence of the first and second free agents to generate a detectable signal. The detectable signal is analyzed using optical position tracking, wherein the analysis reveals the presence or absence, amount or concentration and / or activity of the analyte in the sample, and / or a first relationship between the first reagent and the second free agent and the analyte, and / or a second relationship between the second reagent and the first free agent and the analyte. The analyte in the sample can specifically bind to the analyte interaction reagent.

189. The method of claim 188, wherein the first relationship is a synergistic binding effect between the first reagent and the second free agent with the analyte, and / or the second relationship is a synergistic binding effect between the second reagent and the first free agent with the analyte.

190. The method of claim 188, wherein the first relationship is an allosteric binding effect between the first reagent and the second free agent and the analyte, and / or the second relationship is an allosteric binding effect between the second reagent and the first free agent and the analyte.

191. A method for labeling protein-protein interactions, comprising: Contact (i) a first composition with (ii) a second composition, the first composition comprising a first liquid matrix and a sample encapsulated in the first liquid matrix, wherein the sample comprises a protein, and the second composition comprising a second liquid matrix and a formulation encapsulated in the second liquid matrix, wherein the formulation comprises a plurality of fluorescent constructs and one or more analyte interaction reagents attached to the fluorescent constructs; as well as The first liquid matrix and the second liquid matrix are combined such that the protein in the sample interacts with one or more of the analyte-interacting reagents attached to the fluorescent construct to generate a detectable signal. The detectable signal is analyzed using optical position tracking, and the analysis reveals the interaction between the analyte-interacting reagent and the protein. The analyte in the sample can specifically bind to the analyte interaction reagent.

192. The method of claim 191, wherein the analyte interaction reagent comprises a flexible connector to reduce or avoid steric hindrance in interacting with the protein.

193. The method of claim 191, wherein the protein in the sample interacts with an analyte interaction reagent in the presence of a weak gelling agent for controlling convection and / or slowing diffusion.

194. The method of claim 191, wherein the method is used for protein surface labeling.

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