Method for detecting analytes

Abstract

The present invention provides a method for detecting an analyte in a sample, the method comprising the following steps: (i) providing a mixture comprising a sample and a reporter reagent to a device, wherein the device comprises a substrate having an optical component attached to the surface of the substrate and a binding component; (ii) binding a portion of the reporter reagent to the surface of the substrate in proportion to the concentration of the analyte by the binding component; (iii) irradiating the device with electromagnetic radiation for absorption by a photosensitive agent of the reporter reagent, such that the photosensitive agent of the bound portion of the reporter reagent interacts with the optical component to change the optical component from a first optical state to a second optical state, thereby forming a set of local regions of the optical component having the second optical state on the substrate; and (iv) detecting the set of local regions having the second optical state on the substrate.

Description

Technology Field

[0001] The present invention relates to a method for detecting an analyte, in particular to a method for detecting individual binding events resulting from the presence of an analyte in a sample. Background Technology

[0002] There are many techniques available to measure biologically relevant parameters in human samples, such as blood, plasma, serum, and tissues. A common method involves using a capture reagent that binds to the target of interest and a reporter reagent that is labeled. The capture reagent can be bound to a solid phase, such as a microtitre plate, beads, or a membrane. When the sample is incubated with the capture reagent, the analyte binds to the capture reagent. The reporter reagent also binds to the analyte. Subsequently, the excess reporter can be removed (by washing), and the amount of reporter reagent can be measured to determine the amount of the analyte present in the sample. There are various variations in how this type of binding analysis can be performed. For example, the analyte may bind to the capture reagent first, and then the reporter may be added in a separate step, or the analyte may bind to the reporter first and then to the capture reagent.

[0003] There is a wide range of reagents that can be used as capture and reporter in this type of binding assay, including nucleic acids, carbohydrates, antigens, peptides, proteins, and antibodies. There is also a broad range of target analytes, including peptides, proteins, antibodies, nucleic acids, cells, carbohydrates, small molecules, therapeutic agents, abused drugs, steroids, hormones, lipids, and the like.

[0004] Analysis using antibodies is generally referred to as immunoassay. Immunoassays can take various forms. For example, when a capture antibody is used to capture an analyte and a reporter antibody is used to generate a measurable signal, this is generally called a sandwich immunoassay. An alternative form is known where a binder is attached to a solid phase and the target analyte competes with a labeled reagent in solution, which also binds to the binder. In the absence of the analyte, a high signal is obtained because a high level of the labeled reagent binds. In the presence of the analyte, part of the binding site is blocked, causing the labeled reagent to bind less and the signal to be reduced. Such analyses are generally known as inhibition or competition analyses. Several types of competition analyses are known. For example, an antibody can be bound to a solid phase, and a labeled analyte (or an analyte analog) can compete for the antibody's binding site. Alternatively, an analyte analog can be immobilized, and the labeled antibody can bind to this surface. If the analyte is present in the sample, it will bind to the antibody in solution, preventing binding to the surface and reducing the signal.

[0005] There are various analytical formats and different types of labels that can be used. For example, analyses can be heterogeneous in that excess labels are removed before the measurement is performed, for instance, using a washing step. Removal of excess labels may also be achieved by allowing the sample and reporter to flow through a capture zone. This approach is used, for instance, in immunochromatography or lateral flow strips used in rapid tests for infectious diseases and pregnancy. Alternatively, homogeneous assays are known, in which excess reporters are not removed. Homogeneous assays tend to rely on the proximity of the capture zone and reporter to generate some form of signal. One example of a homogeneous assay is an agglutination assay, where particles bind together in solution. Aggregated particles cause light scattering, which can be measured by turbidimetry or nephelometry. An additional example of a homogeneous assay using particles is the luminescent oxygen channeling immunoassay (LOCI), which is described in more detail below.

[0006] Another example of homogeneous analysis is fluorescence resonance energy transfer (FRET), where the capture and reporter reagents are donor and acceptor fluorophores, respectively, and the excitation of the donor transfers energy to the acceptor and then emits light.

[0007] One homogeneous analytical format that functions in whole blood without removing cellular material is the pyro-optical immunoassay. Capture antibodies are coated onto a pyroelectric polyvinylidene (PVDF) sensor, and carbon particles are used as reporters. When light is shone on the sample, a signal is generated, causing the particles to heat up locally. The particles bound to the sensor transfer energy to the pyroelectric sensor, inducing thermal stress that is detected as an electrical signal. The more carbon bonds there are, the stronger the signal.

[0008] Labels attached to the reporter binder can be light-absorbing agents, such as dyes, gold particles, or stained latex microspheres. In principle, larger particles absorb more light and can generate more signals. However, as described in more detail below, there are size limitations that make microparticle labels impractical for use in analysis. Luminescent labels, such as fluorescent, chemiluminescent, bioluminescent, and electrochemiluminescent labels, are also known. Luminescent labels have also been encapsulated into particles for specific types of analysis. Signal amplification may be performed using enzymatic or catalytic reactions. Enzymes can be used to convert the substrate from a leuco dye into a colored form, or into a fluorescent or luminescent form. Since it is common to remove excess enzymes using a washing step before adding the substrate, the signal is generated only by the enzyme specifically bound to the analyte.

[0009] Label-free immunoassays are also known, such as those using surface plasmon resonance as a signal transduction method. However, label-free assays tend to lack the sensitivity of assays that use labels to enhance the signal.

[0010] Further information on the field of immunoassay can be found in "The Immunoassay Handbook: 4th Edition: Theory and Applications of Ligand Binding, ELISA and Related Techniques", Ed. D. Wild, Elsevier Science, 2013.

[0011] All binding analyses, including immunoassays, have limitations in terms of the minimum and maximum concentrations of analytes that can be reliably measured.

[0012] The signal maximum is generally limited by factors such as the total amount of capture antibody capable of binding to the analyte and the total amount of reporter antibody to generate the signal. If the capture antibody is immobilized on a solid phase, the surface area of ​​the solid phase can limit the upper limit of detection. Additionally, some signal transduction techniques may exhibit a saturation tendency similar to colorimetric methods, depending on the path length required for light to pass through the sample. Luminescence methods are less susceptible to saturation because the detector gain can be attenuated to handle higher levels of light emission. In heterogeneous assays, when all antibody binding sites are filled with the analyte, the signal maximum is reached and the system becomes saturated. Excess analyte is typically removed during a washing step before adding the reporter. Homogeneous assays can also suffer from an effect known as a high-dose hook, where the analyte concentration is greater than the effective concentration of the capture and / or reporter antibodies. In this case, at very high concentrations, all binding sites of the capture and reporter may be blocked, reducing the analytical signal and potentially leading to erroneous results.

[0013] Low levels of detection are determined by various factors. Generally, all analyses will be influenced by properties such as the quality of the antibodies used (affinity and specificity) and the cross-reactivity between the antibody and the associated analyte. The lower limit of detection also depends on factors affecting the signal-to-noise ratio of the analysis setup and system design. For example, in a standard enzyme-linked immunosorbent assay (ELISA), a capture antibody is coated onto the surface of a 96-well microtiter plate, and then the sample is incubated in the wells to capture the analyte. After washing the wells, an excess of reporter is added to bind to the captured analyte. Subsequently, the excess reporter is washed away, and a substrate capable of reacting with the enzyme is added to convert it into an active form. For example, colorless leuco dyes such as 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) can be converted into a green form by oxidizing horseradish peroxidase in the presence of hydrogen peroxide. When the analyte is present in very small amounts (e.g., less than 1 picomole), only a very small amount of the enzyme bound to the well surface will be present. ABTS reacts with the enzyme to produce the green form, then diffuses into most of the fluid, creating a solution so dilute that it is indistinguishable from the background signal. The auto-conversion of the substrate may also produce colors that interfere with the measurement. Similarly, other detection methods, such as fluorescence, may face difficulties due to interfering factors and autofluorescence from components in the sample or reaction well.

[0014] Another confounding factor in immunoassays may be the non-specific binding of reporter reagents to capture surfaces. For example, in the ELISA assay described above, microtitre wells are coated with a protein layer, some of which may be denatured during the coating process. It is not uncommon for a reporter to bind to the capture surface area during the assay. If the substrate is inverted to allow this reporter to contribute to the overall signal, it becomes impossible to distinguish between the signal from the specifically bound reporter and the signal from the non-specifically bound reporter. Non-specific binding can also be facilitated by many components present in the original sample that bind to the capture surface during initial incubation, modifying the surface properties of the capture layer and creating a surface capable of binding the reporter. Minimizing non-specific binding of the reporter involves carefully optimizing all reagents and reaction conditions used during the assay, including antibodies, detergents, temperature, and ionic strength.

[0015] Generally, the detection limit of conventional immunoassays is approximately 0.1 picomolar to 1 nanomolar, depending on the assay method. Developing assays with very low detection limits using conventional approaches often requires significant optimization, along with rigorous washing steps to reduce non-specific binding and maximize signal-to-noise ratios. Additionally, the capture surface is often small relative to the sample volume to ensure that the signal is sufficiently high compared to the background.

[0016] One approach used to bypass signal-to-noise issues and improve detection limits is to measure individual coupled events and count these coupled events as "on" or "off" events if the measurement exceeds a localized threshold. In this way, much background noise can be eliminated. This can be inferred from the digitization of audio or communication signals. Such digital analysis has been shown to reach detection limits that were previously unattainable using conventional analog methods. For example, low femtomolar (10 -15 mol / L) and even atmolar (10 -18 Detection limits in the range of mol / L have been reported. For example, "Evaluation of highly sensitive immunoassay technologies for quantitative measurements of sub-pg / mL levels of cytokines in human serum", Yeung et al , Journal of Immunological Methods, 2016, 437 , 53 and "Digital Detection of Biomarkers Assisted by Nanoparticles:Application to Diagnostics", Cretich et al , Trends in Biotechnology, 2015, 33 Refer to , 343.

[0017] Most labels / reporters used in analysis (e.g., fluorescent dyes, dyes, etc.) cannot be viewed individually using a wide-field microscope even at high magnification because their size is below the diffraction limit of visible light. Therefore, the presence of these labels can only be measured as a bulk phenomenon, rather than by counting each label individually. In contrast, particulate labels, such as latex particles, can theoretically be visualized with a wide-field optical microscope if they exceed a certain size. Depending on the optical setup, numerical aperture, and microscope type, visualization of particles can begin when their diameter exceeds several hundred microns.

[0018] However, using particles of this size as labels to monitor individual binding events (e.g., antibody-antigen interactions) on capture surfaces becomes impractical for several reasons. For instance, particles of this size diffuse very slowly compared to other types of labels, impairing reaction rates on planar surfaces. Additionally, they begin to exhibit macroscopic buoyancy effects; if the particle density differs significantly from that of the medium containing them, they may settle or float, which can cause problems with the assay format. Particles of this size also tend to bind non-specifically to surfaces, resulting in high background that is difficult to remove. Finally, a washing step is required to remove excess particles. However, large particles begin to experience shear effects in the presence of fluid flow, and as the shear force applied to the particles exceeds the bursting strength of antibody-antigen interactions (approx. 60–250 pN), the particles are washed away ("Rapid Femtomolar Bioassays in Complex Matrices Combining Microfluidics and Magnetoelectronics", Mulvaney et al, Biosensors and Bioelectronics, 2007, 23 , see 191).

[0019] Examples of digital analysis include SIMOA (Quanterix Single Molecule Array) systems and SMC (Singulex Single Molecule Counting) systems.

[0020] The Quanterix SIMOA system captures analytes from solution using antibody-coated paramagnetic beads. Subsequently, the magnetic beads are washed, and an enzyme-labeled reporter antibody is added. The number of beads is sufficient to minimize the probability of having more than one analyte and reporter per bead. After washing the beads again, they are loaded into an array of microwells capable of holding only one bead per well. The volume of the microwells is on the femtoliter scale. When the enzyme is attached to a bead, the fluorescent substrate in the well is flipped. The small dimensions of the wells prevent excessive diffusion of the fluorescent product. Subsequently, if the fluorescence exceeds a threshold value, each well is counted as an "on" or "off" event.

[0021] The SMC system is used in the Singulex Clarity instrument and Merck Millipore’s Erenna and SMCxPRO systems. The basic measurement technique is the same for all three systems. Magnetic beads coated with capture antibodies are used to capture the target analyte in the sandwich assay. Fluorescently labeled reporter antibodies also bind to the beads in the presence of the analyte. The beads are pulled by magnets, and the excess fluorescently tagged reporters are washed away. Subsequently, an elution buffer is added to induce the dissociation of the sandwich complex, and the samples are transferred to a measurement vessel. Then, the presence of the fluorescent tag is measured using a confocal fluorescent microscope, which sequentially irradiates small samples to confirm the presence of the fluorescent tag. If the signal for each individual measurement is higher than a threshold, that measurement is counted as an "on" event.

[0022] Several independent academic reviews on high-sensitivity immunoassays have emphasized that digital approaches to immunoassays can improve detection limits to unprecedented levels (see Yeung and Cretich above).

[0023] The detection limits of Quanterix and Singulex systems depend on the volume of the sample used for analysis. For a 10-microliter serum or plasma sample, the theoretical limit would be the detection of a single-binding event corresponding to one molecule. However, in terms of molar concentration, this corresponds to 100,000 molecules per liter of sample, or 0.16 × 10⁻⁶ molecules per liter. -18 It corresponds to a mole (0.16 atmoles).

[0024] However, the Quanterix and Singulex systems described above are complex and cumbersome, each requiring multiple washing and transfer steps. Furthermore, analysis can only be performed on samples that do not contain cellular material, and the systems require expensive instruments to achieve the performance they provide. Therefore, there remains a need for simpler and more cost-effective high-sensitivity systems. means of solving the problem

[0025] Accordingly, the present invention provides a method for detecting an analyte in a sample, said method comprising the following steps:

[0026] (i) a step of providing a mixture comprising a sample and a reporter reagent to a device, wherein the device comprises a substrate having an optical component and a binding component attached to the surface of the substrate;

[0027] (ii) a step of binding a portion of the reporter reagent to the surface of the substrate in proportion to the concentration of the analyte by means of the binding component;

[0028] (iii) irradiating the device with electromagnetic radiation for absorption by the photosensitive agent of the reporter reagent, so that the photosensitive agent of the combined reporter reagent portion interacts with the optical component to change the optical component from a first optical state to a second optical state, thereby forming a set of local regions of the optical component having the second optical state on the substrate; and

[0029] (iv) a step of detecting a set of local regions having a second optical state on the above-mentioned material;

[0030] Accordingly, the present invention provides a method for detecting an analyte in a sample in which only a reporter reagent located near the surface of the substrate generates a signal (a local region of the optical component in a second optical state) and a signal (a set of local regions of the optical component in a second optical state) is detected. Accordingly, the method of the present invention simplifies the digital detection of an analyte. The method of the present invention facilitates homogeneous analysis of a sample range including a sample containing cellular material. Brief explanation of the drawing

[0031] The present invention will now be described with reference to the drawings, wherein: FIG. 1 illustrates various components that can be used in the method of the present invention; FIG. 2 illustrates a device in which a reporter reagent is bound to the surface of a substrate before investigation; FIG. 3 illustrates the apparatus of FIG. 2 being investigated; FIG. 4 illustrates the apparatus of FIG. 3 after investigation; FIG. 5 illustrates a device in which a whole blood sample and reporter reagent are present before investigation; FIG. 6 illustrates the apparatus of FIG. 5 after investigation; FIG. 7 illustrates an apparatus used in the method of the present invention, in which a reporter reagent is bound to the surface of a substrate in proportion to the concentration of the analyte by a binding component prior to irradiation; FIG. 8 illustrates the apparatus of FIG. 7 after investigation; FIG. 9 illustrates an optical setup for detection; FIG. 10 illustrates a substrate and a well generated for the method of the present invention; FIG. 11 illustrates a sample chamber created using the substrate and well of FIG. 10; FIG. 12 illustrates the detection results of Example 5; FIG. 13 illustrates the detection results of Example 6; Figure 14 illustrates the detection results of Example 7. Specific details for implementing the invention

[0032] The method of the present invention is used to detect an analyte in a sample (this may be achieved through the detection of a complex or derivative of the analyte).

[0033] The components of FIG. 1 are as follows: analyte (1); photosensitizer (2); streptavidin-coated latex particle infused with photosensitiser (3); antibody-coated latex particle infused with photosensitiser (4); antibody (5); photosensitizer-labeled antibody (6); fluorescent dye (7); non-fluorescent dye (8); polymerized streptavidin (9); fluorescent polystreptavidin dye conjugate (10); non-fluorescent polystreptavidin dye conjugate (11); biotin-BSA conjugate (12); red blood cell (13); Aminodextran-biotin-dye conjugate in a fluorescent state (14); and an aminodextran-biotin-dye conjugate in a non-fluorescent state (15).

[0034] Step (i) of the method of the present invention comprises providing a mixture comprising a sample and a reporter reagent to a device, wherein the device comprises a substrate (17) having an optical component and a binding component attached to the surface of a substrate (17). The sample and the reporter reagent may be mixed in advance before adding the mixture to the device, or the sample and the reporter reagent may be added sequentially to the device to form the mixture. The mixture may also include additional reagents, but preferably the mixture consists of a sample and a reporter reagent.

[0035] To explain the principle underlying the present invention, FIG. 2 illustrates an apparatus in which a reporter reagent is bound to the surface of a substrate prior to irradiation. The apparatus comprises a substrate (17) and a sample chamber (16) for holding a sample containing an analyte dissolved or suspended therein. The substrate may be any substrate that allows detection of a set of local regions having a second optical state on the substrate. Preferably, the substrate is a transparent substrate, and more preferably, the substrate is glass.

[0036] The substrate (17) has an aminodextran-biotin-dye conjugate (14) attached to the surface of the substrate (17) via a biotin-BSA conjugate (12) and polymerized streptavidin (9). The dye acts as an optical component, and the biotin acts as a binding component. The biotin-BSA conjugate (12) and polymerized streptavidin (9) are inert macromolecules that facilitate the attachment of the optical and binding components to the surface of the substrate (17). This approach is used when the optical component is water-soluble, as it needs to be fixed to the surface of the substrate (17) to immobilize the optical component.

[0037] Although the optical and bonding components are illustrated in this manner, any technique for maintaining the optical and bonding components in close proximity to the surface of the substrate (17) may be applied. For example, the optical and bonding components may be bonding components attached to separate reagents and optical components.

[0038] The optical component may also be encapsulated within a polymer layer coated on the surface of the substrate (17) and the surface of the bonding component attached to the polymer layer. The polymer may be silicone, polystyrene or polyisobutylene, or any other suitable polymer plastic that can be used to encapsulate the optical component. This approach may be used when the optical component is water-insoluble.

[0039] Alternatively, the gel / hydrogel layer may be impregnated with an optical component, and the gel / hydrogel layer may be coated on the surface of the substrate (17) and a binding component attached to the gel / hydrogel layer.

[0040] Step (ii) of the method of the present invention comprises causing a portion of the reporter reagent to bind to the surface of the substrate in proportion to the concentration of the analyte by means of a binding component. This can be achieved by leaving the device undisturbed for a certain period of time, for example, 10 minutes.

[0041] In FIG. 2, the photosensitized streptavidin-coated latex particles (3) are bonded to the surface of the substrate (17) by biotin on the aminodextran-biotin-dye conjugate (14). The photosensitized streptavidin-coated latex particles (3) act as a reporter reagent.

[0042] Although the reporter reagent is depicted as being bound to the surface of the substrate (17) in this manner, when an analyte is present, the reporter reagent is bound to the surface of the substrate in proportion to the concentration of the analyte. For example, when the binding component and the reporter reagent are antibodies and the analyte is an antigen, the reporter reagent binds to the binding component through the analyte to form a so-called "sandwich" complex as discussed below with reference to FIGS. 7 and 8.

[0043] All steps up to this point were performed in the absence of light. Step (iii) of the method of the present invention comprises irradiating the device with electromagnetic radiation for absorption by the photosensitive agent of the reporter reagent so that the photosensitive agent of the combined reporter reagent part interacts with the optical component to change the optical component from a first optical state to a second optical state, thereby forming a set of local regions of the optical component having a second optical state on the substrate (17).

[0044] FIG. 3 illustrates the apparatus of FIG. 2 irradiating electromagnetic radiation (typically referred to as "light"), preferably visible light. The light source may be, for example, an LED (18). The light source illuminates the sample chamber (16) with light of a suitable wavelength to excite the photosensitive material (2). The wavelength varies depending on the photosensitive material, but a preferred wavelength is 680 nm. The apparatus is generally irradiated for at least 30 seconds. Preferably, the apparatus is exposed to the electromagnetic radiation source for at least 1 second, more preferably at least 2 seconds, more preferably at least 5 seconds, more preferably at least 20 seconds, and most preferably at least 30 seconds. This ensures that there is an irreversible optical change on the substrate surface, so that long-lasting and transient binding events can be distinguished.

[0045] FIG. 4 illustrates the apparatus of FIG. 3 after irradiation. The photosensitive agent (2) interacts with the dye optical component of the aminodextran-biotin-dye conjugate (14) to change the dye from a fluorescent state to a non-fluorescent state. The aminodextran-biotin-dye conjugate (14) in the fluorescent state becomes the aminodextran-biotin-dye conjugate (15) in the non-fluorescent state. Only the dye very close to the photosensitive agent (2) changes from the first optical state to the second optical state.

[0046] The dye is indicated to change from a fluorescent state to a non-fluorescent state, while other optical components undergo alternative changes. In one embodiment, when in the first optical state, the optical component absorbs light, preferably visible light, at one or more wavelengths, and when in the second optical state, the optical component absorbs light, preferably visible light, at one or more different wavelengths. In this embodiment, the optical component undergoes a color change.

[0047] delete

[0048] Alternative first and second optical states may include changes in polarization, fluorescence lifetime, refractive index, light scattering (including Raman scattering), and other optical effects.

[0049] FIG. 5 shows a device in which a whole blood sample and a reporter reagent are present before investigation. The whole blood sample also contains additional components such as red blood cells (13). Preferably, the substrate (17) forms the upper part of the sample chamber (16) so that red blood cells (13) are deposited from the substrate (17).

[0050] A portion of the reporter reagent is bound to the surface of the substrate (17) by a binding component. Thus, the sample contains the bound reporter reagent free in the solution and the unbound reporter reagent. The depth of the sample chamber (16) is designed to minimize the diffusion path length of the reporter reagent and to allow equilibrium to be achieved quickly. Generally, the depth of the sample chamber is 50 to 200 μm.

[0051] Afterward, the device is irradiated as described above, and FIG. 6 shows the device of FIG. 5 after irradiation. Any photosensitive material near the optical component interacts to cause the optical component to change from a first optical state to a second optical state. In this way, the photosensitive material of the combined reporter reagent portion interacts with the optical component to cause the optical component to change from a first optical state to a second optical state, thereby forming a set of local regions of the optical component having a second optical state on the substrate (17).

[0052] FIG. 7 illustrates an apparatus used in the method of the present invention, in which a reporter reagent is bound to the surface of a substrate in proportion to the concentration of the analyte by a binding component prior to investigation.

[0053] In a typical sandwich immunoassay using the method of the present invention, the substrate (17) has a polystreptavidin-dye conjugate (11) attached to the surface of the substrate (17) via a biotin-BSA conjugate (12), and an antibody (5) attached to the polystreptavidin-dye conjugate (11). The dye acts as an optical component, and the antibody acts as a binding component. Although the optical and binding components are illustrated in this manner, any technique for keeping the optical and binding components close to the surface of the substrate (17), such as the technique described above, may be applied.

[0054] In the method of the present invention, the sample chamber (16) is filled with a sample containing an analyte (1). A reporter reagent, such as a photosensitive labeled antibody (6), is also added to the sample chamber (16). When using a whole blood sample, the sample may contain additional components such as red blood cells (13).

[0055] Afterward, equilibrium is achieved. The photosensitive antibody (6) is bound to the surface of the substrate (17) by the antibody (5) in proportion to the concentration of the analyte (1). The photosensitive antibody (6) acts as a reporter reagent. With an excess of the photosensitive antibody (6), all the analyte (1) forms a sandwich complex. Thus, a portion of the reporter reagent is bound to the surface of the substrate (17) in proportion to the concentration of the analyte (1) by the binding component. Thus, the sample contains the bound reporter reagent free in the solution and the unbound reporter reagent.

[0056] FIG. 8 illustrates the apparatus of FIG. 7 after irradiation. The photosensitive agent (2) interacts with the dye optical component of the polystreptavidin-dye conjugate (11) to change the dye from a non-fluorescent state to a fluorescent state. The polystreptavidin-dye conjugate (11) in the non-fluorescent state becomes a polystreptavidin-dye conjugate (10) in the fluorescent state. Only the dye very close to the photosensitive agent (2) changes from the first optical state to the second optical state.

[0057] The reporter reagent must be permanently bound to the surface of the substrate (17) for the entire irradiation period to completely switch the first optical state to the second optical state. Any reporter reagent not bound to the solution prevents the optical component from changing from the first optical state to the second optical state.

[0058] This offers a significant advantage over other digital analysis methods in that a washing step is not required. In this way, the method of the present invention is a homogeneous analysis. In conventional analysis, because unbound reporter reagents interfere with the signal generated by bound reporter reagents, unbound reporter reagents must be separated from bound reporter reagents before any measurement is performed. However, due to localized surface changes provided by the present invention, bound reporter reagents and unbound reporter reagents can be distinguished. In fact, the ability to distinguish between reporter reagents (i.e., bound) in close proximity to the surface of the substrate (17) and reporter reagents in the bulk solution (i.e., unbound) is a particular advantage of the present invention. Preferably, steps (i) through (iii) are performed in the absence of a washing step, that is, the method is performed without removing the sample from the substrate in steps (i), (ii), and (iii).

[0059] The photosensitizer in the bound reporter reagent portion interacts directly with the optical component to cause a change (e.g., the photosensitizer is excited and directly transfers this energy to the optical component), or interacts indirectly with the optical component to cause a change through an additional reagent (e.g., the photosensitizer is excited, transfers this energy to an additional component, and then transfers this energy to the optical component).

[0060] In a preferred embodiment, absorption by the photosensitizer is intended to interact with a pre-activator reagent present in the mixture to produce an activator reagent, said activator reagent is intended to interact with an optical component to cause a change from a first optical state to a second optical state.

[0061] The pre-activator reagent may be present in the sample, or the pre-activator reagent may be added as an additional reagent to a mixture of the sample and the reporter reagent. In a preferred embodiment, the pre-activator reagent is triplet oxygen. In another preferred embodiment, the activator reagent is a reactive oxygen species (ROS). Preferably, the ROS is selected from hydroxyl radicals, superoxide, peroxide, organic peroxide, peroxynitrite, singlet oxygen, and mixtures thereof. More preferably, the activator reagent is singlet oxygen. Singlet oxygen is a preferred activator reagent because it has a short half-life and a finite diffusion path length (up to 200 nm under aqueous conditions).

[0062] Although not bound by theory, it is believed that the photosensitive agent absorbs light to generate an excited state capable of undergoing intersystem switching (ISC) with oxygen present in the sample and in proximity to the reporter reagent, thereby producing singlet oxygen. Subsequently, the singlet oxygen continues to interact with the optical component as discussed below. In a particularly preferred embodiment, the pre-activator reagent is triplet oxygen, and the activator reagent is singlet oxygen.

[0063] Singlet oxygen has previously been used in the immunoassay of Luminescent Oxygen Channeling Immunoassay (LOCI). LOCI immunoassay is a homogeneous, non-digital assay using donor and recipient beads. The donor bead generates singlet oxygen upon illumination at 680 nm, and the recipient bead generates a chemiluminescent signal when activated by singlet oxygen. The binding of the donor to the recipient bead is facilitated by antibody-antigen binding. The reaction mixture is typically illuminated for 0.5 to 1.0 seconds, after which the luminescence signal is measured for 0.5 to 1.0 seconds. Crucially, the measurement occurs in the presence of all unbound donor and recipient beads. Spatial separation of the beads minimizes background signals; however, due to the short measurement time, LOCI assay cannot distinguish between long-lived binding events and transient binding events. This assay can achieve a detection limit of approximately 1–5 pg / mL for the most sensitive assays, e.g., Interleukin 6 (IL-6) or Thyroid-Stimulating Hormone (TSH). The method of the present invention further minimizes background signals and must remain present during the illumination period because the "donor" particle, which is the reporter reagent, must be in close proximity to the surface of the substrate (17) rather than the particle in the solution for the signal to be detected. Therefore, the method of the present invention is more sensitive than LOCI analysis and can detect analytes at lower concentrations.

[0064] In a preferred embodiment, the optical component is a dye. Preferably, the optical component is selected from one of the following dyes and mixtures thereof:

[0065]

[0066] Dyes (1)-(5) are known and are used as cellular probes to monitor ROS formation in cells under oxidative stress. However, these dyes are not known to be used for standard or digital immunoassays. Dyes (6) are common fluorescent dyes. There are various other commercially available fluorescent dyes suitable for use in the present invention, including Alexafluor dye, Bodipy dye, Rhodamine dye, Texas red, Oregon green, Cascade yellow, Pacific blue, etc. For additional examples, see The Molecular Probes Handbook, Thermo Fisher Scientific.

[0067] SOSG (Dye singlet oxygen sensor green) (1) reacts with singlet oxygen to change from a weakly fluorescent form to a highly fluorescent form. Singlet oxygen reacts with an anthracenyl group to form endoperoxide. In a preferred embodiment, the optical component is SOSG.

[0068] Boron-dipyromethen dye 581 / 591 (BODIPY 581 / 591 )(2) reacts with reactive oxygen species such as singlet oxygen and hydroxyl radicals, causing a hypochromic shift at the maximum fluorescence excitation / emission.

[0069] The dyes (3)-(5) share the same core structure and react with a general oxidizing agent containing singlet oxygen to be converted into a fluorescent form. Thus, these dyes can be converted from a leuco state to a fluorescent state.

[0070] It has been found that the dye (6) reacts with singlet oxygen to convert from a fluorescent form to a non-fluorescent form. It has been discovered that by using fluorescein, it is possible to completely remove fluorescence near the photosensitive material, thereby creating a dark, non-fluorescent region on the fluorescent substrate. Thus, in a preferred embodiment, the optical component is fluorescein. More preferably, the optical component is fluorescein, the first optical state is fluorescent, and the second optical state is non-fluorescent. Most preferably, the optical component is fluorescein, the first optical state is fluorescent, the second optical state is non-fluorescent, and the set of local regions is a dark, non-fluorescent region on the substrate.

[0071] Preferably, the optical component is fluorescent when in one of the first and second optical states, and is non-fluorescent when in the other of the first and second optical states. In one embodiment, the optical component is non-fluorescent when in the first optical state, and the optical component is fluorescent when in the second optical state. However, more preferably, the optical component is fluorescent when in the first optical state, and the optical component is non-fluorescent when in the second optical state.

[0072] In another preferred embodiment, when in a first optical state, the optical component emits fluorescence at one or more wavelengths, and when in a second optical state, the optical component emits fluorescence at one or more different wavelengths. In this embodiment, the optical component shifts its fluorescence excitation / emission maximum.

[0073] Preferably, the change from the first optical state to the second optical state is irreversible. This allows for subsequent scanning of the substrate to identify the region where the change in optical state occurred.

[0074] Step (iv) of the method of the present invention comprises detecting a set of local regions having a second optical state on a substrate.

[0075] An optical component having a second optical state forms a set of local regions on a substrate. Advantageously, the local regions having the second optical state can be counted as individual combined events. Thus, the method of the present invention is suitable for performing digital analysis. However, a large change may be detected if there are many combined events such that most of the optical components are in the second optical state.

[0076] In a preferred embodiment, a set of local regions having a second optical state on the substrate is detected by counting local regions within the set of local regions having a second optical state on the substrate or by measuring the set of local regions having a second optical state on the substrate as a bulk property. More preferably, a set of local regions having a second optical state on the substrate is detected by counting local regions within the set of local regions having a second optical state on the substrate.

[0077] A local region having a second optical state may need to be above a threshold corresponding to the background signal depending on the first and second optical states. For example, the sample may have some background autofluorescence, but if this does not exceed the threshold, it should not affect the signal due to the digital nature of the analysis. Background autochemiluminescence tends not to occur.

[0078] Additionally, the surface of the substrate may be scanned to provide a 2D image of the surface before irradiation with a photosensitive agent, and then scanned after irradiation; to reduce interference from artifacts, contaminants, and any autofluorescence on the sample itself or on the surface, the background is subtracted from the background of the pre-illumination image in the post-illumination image. Accordingly, in a preferred embodiment, the method of the present invention further comprises the step of subtracting any component having a second optical state on the substrate detected before irradiating the device with electromagnetic radiation from a set of local regions having a second optical state on the substrate detected in step (iv). In this way, the background-subtracted image will display only changes in the optical properties of the substrate (e.g., changes in fluorescence intensity).

[0079] A set of local regions having a second optical state on the substrate is generally individual regions on the substrate. However, some local regions may be excluded from detection due to their shape. Local regions corresponding to individual binding events tend to be uniform and circular, but some local regions may have irregular shapes corresponding to artifacts. Additionally, some local regions may be larger than other regions where particles are clustered together. Therefore, in a preferred embodiment, only uniform circular local regions having a second optical state on the substrate are detected.

[0080] A set of local regions on the substrate can be detected using simple optical means. In a preferred embodiment, a set of local regions having a second optical state on the substrate is detected using an optical microscope. An optical setup suitable for detection is shown in FIG. 9. More preferably, a set of local regions having a second optical state on the substrate is detected using a wide-field microscope. A wide-field microscope is the simplest form of microscope in which the entire sample is simultaneously illuminated and imaged, compared to more complex techniques such as a confocal microscope, in which only one single focal spot is illuminated and recorded at a time. The advantage of a confocal microscope is that it can eliminate out-of-focus haze and capture image stacks through the depth of the sample, thereby increasing contrast. Super-resolution microscopy techniques such as photo-activated localization microscopes (PALM or FPALM) and stochastic optical reconstruction microscopes (STORM) are also known. These methods add complexity and cost compared to simple wide-field methods.

[0081] When using dyes (1) and (3)-(5), a fluorescence hotspot (or increased fluorescence in the case of many binding events) can be observed on the substrate surface where the optical component is switched to a second optical state. To detect this fluorescence, the substrate can be illuminated at the excitation wavelength of the dye, and the surface of the substrate can be scanned for luminescence.

[0082] When using dye (2), a decrease in fluorescence emission at 610 nm or an increase in fluorescence emission at 515 nm can be measured, or both wavelengths can be monitored.

[0084] When using the dye (6), a dark spot (or, if there are many coupling events, a dark image) can be observed on the surface of the substrate where the optical component has been converted to a second optical state. To detect such dark spots or images, a wide-field fluorescence microscope equipped with a light source (e.g., an LED) and a photodetector (e.g., a camera such as a photomultiplier tube or CCD) can be used, using excitation and emission filters suitable for fluorescein detection (e.g., excitation at 490 nm and emission at 520 nm).

[0085] The photosensitive material is in close proximity to the substrate when the binding event occurs. That is, the photosensitive material is sufficiently close to the surface of the substrate to interact with the optical component and convert it from a first optical state to a second optical state upon irradiation by the device. However, the actual distance between the photosensitive material and the substrate surface will vary depending on the size and properties of the photosensitive material, the size and properties of the binding component, the reporter reagent and analyte, and the characteristics of the sample medium.

[0086] The binding component has a binding site capable of binding a reporter reagent in proportion to the concentration of the analyte in the sample. Proportionality is important for the function of the analysis, as binding must depend on the analyte concentration to determine any meaningful measurement of the analyte concentration. Depending on the type of analysis performed, binding may be directly or indirectly proportional to the analyte concentration. In the case of non-competitive analyses, such as immunoassays, binding is directly proportional to the analyte concentration, whereas in the case of competitive analyses, binding is indirectly proportional to the analyte concentration.

[0087] A specific type of competitive assay is provided in which an antibody against the analyte is immobilized on a substrate, and a labeled analog of the analyte is introduced into the sample. Subsequently, the analyte and its labeled analog compete for the antibody on the surface. In the absence of the analyte, the labeled analog will bind at the maximum possible rate. However, in the presence of the analyte, the antibody on the substrate is filled with the analyte, and the binding rate of the analog is reduced.

[0088] The binding component may be adapted to bind to the analyte, or a complex or derivative of the analyte, in which case the reporter reagent will bind to the binding component in the presence of the analyte, or a complex or derivative of the analyte. In this case, the binding component has a binding site capable of binding to the reporter reagent in the presence of the analyte, or a complex or derivative of the analyte. However, binding is still proportional to the concentration of the analyte.

[0089] Alternatively, the binding component itself may be an analog of the analyte, and the reporter reagent binds directly to the binding component (it is an analog because it binds to the surface of the substrate via covalent or non-covalent interactions). In this case, the binding component will compete for the binding of the reporter reagent with the unbound analyte, or the unbound complex or derivative of the analyte. Therefore, the binding component will simply be able to bind to the reporter reagent.

[0090] By determining the degree of binding of the reporter reagent to the binding component (directly or mediated by an analyte / analyte complex or derivative), the concentration of the analyte in the sample can be measured.

[0091] The analysis also requires the presence of a reporter reagent. The reporter reagent contains a photosensitizer. The photosensitizer absorbs electromagnetic waves and can interact with the optical component. This is an interaction that causes the optical component to change from a first optical state to a second optical state.

[0092] Therefore, photosensitizers can be composed of any material capable of interacting with electromagnetic radiation in this manner. Suitable photosensitizers are known in photodynamic therapy (PDT) as photodynamic therapy (PDT) reagents. PDT reagents are used in cancer treatment and dermatology to destroy cells upon illumination (Shafirstein et al, Cancers, 2017, 9, 12; Wan and Lin, Clinical, Cosmetic and Investigational Dermatology, 2014, 7, 145).

[0093] Upon irradiation with electromagnetic radiation, PDT reagents are promoted to an excited triplet state. This excited triplet state can interact directly with cellular components, known as the Type I process, or with oxygen, known as the Type II process. Both Type I and Type II processes can lead to the formation of ROS. In the Type II process, the major product is singlet oxygen via cross-system mechanisms.

[0094] Singlet oxygen is a highly reactive excited state of oxygen. It can undergo various reactions before decaying, including Diels-Alder type reactions and ene reactions. Additionally, it undergoes common oxidation reactions with sulfur and nitrogen-containing compounds. The unrestrained reactivity of singlet oxygen is one of the reasons it is used in photodynamic therapy.

[0095] A wide range of photosensitizing compounds are known, including porphyrins, chlorines (e.g., pyropheophorbide-a), phthalocyanines, and other polyaromatic species (see, e.g., Antibody-Directed Phototherapy, Pye et al, Antibodies, 2013, 2, 270).

[0096] In one embodiment, the photosensitizer is selected from porphyrins, chlorines, phthalocyanines, and other polyaromatic species. In a preferred embodiment, the photosensitizer is pyropheophorbide-a (PPa). PPa has the following structure:

[0097]

[0098] Piropheophorbide-a (PPa)

[0099] Other photosensitizers that promote reactions without generating ROS may be used. These include Ru(II)-tris(2,2'-bipyridyl) dichloride, which catalyzes the reduction of dichlorodiphenyltrichloroethane in the presence of a reducing agent; and benzophenone / eosin, which catalyzes the cis-trans isomerization of stilbenes and chlorophyll and catalyzes the reaction of carbon dioxide and water to produce carbohydrates. Photosensitizers are known to be able to catalyze polymerization reactions, which can also alter the optical properties of the substrate. For example, Dyes as Photoinitiators or Photosensitizers of Polymerization Reactions, Fouassier et al , Materials, 2010, 3 Refer to , 5130.

[0100] The characteristics of the binding component and the reporter reagent vary depending on the characteristics of the analyte, but are preferably antibodies. The method of the present invention is particularly applicable to immunoassays. In a particularly preferred embodiment, the binding component is an antibody produced against the analyte or a complex or derivative of the analyte, and the reporter reagent comprises an antibody produced against the analyte or a complex or derivative of the analyte. In principle, a single molecule may be used for each reagent, but in practice, the binding component and the reporter reagent are a group of molecules. The term "antibody" preferably includes Fab fragments, single-chain variable fragments (scFv), and recombinant binding fragments within its scope.

[0101] As an alternative to antibody-antigen reactions, the binding component, reporter reagent, and analyte may be first and second nucleic acids in which the first and second nucleic acids are complementary, or a reagent containing avidin or a derivative thereof and an analyte containing biotin or a derivative thereof, and vice versa. The binding component and reporter reagent may also be aptamers. This system is not limited to biological analysis and may be applied, for example, to the detection of heavy metals in water. The system also does not need to be limited to liquids, and any fluid system, such as for the detection of enzymes, cells, and viruses in the air, may be used.

[0102] The maximum observable signal is the maximum signal achievable when monitoring the photosensitizer bound to the surface. The binding of particles to the substrate is governed by the diffusion rates of the analyte and reporter reagents, which are ultimately heavily influenced by the hydrodynamic radii of these components and the viscosity / temperature of the sample.

[0103] The apparatus used in the method of the present invention may further include a control device that compensates for the natural variability of the components of the measurement system, the variability of the sample being measured, and the variability of environmental conditions during measurement. This can be achieved by exposing the sample to a reagent on the surface of the substrate. Different reagents are generally located in different areas of the surface of the substrate, and these areas are coated with different reagents. These controls are defined as "negative" and "positive" controls in the sense that the negative control should approximate the signal expected when the analyte is absent, and the positive control should approximate the signal expected when the analyte saturates the system.

[0104] To achieve detection with these control groups, the device of the present invention preferably comprises a binding component, a negative control reagent, and a positive control reagent, each of which is attached to the surface of a substrate as described above.

[0105] The binding components are as described above.

[0106] The negative control reagent has a lower affinity for the reporter reagent under analytical conditions than the binding component. Therefore, the negative control reagent provides a negative control. It is important to consider affinity under analytical conditions. This is because, in the case of non-competitive analysis, the affinity of the binding component for the reporter reagent is modulated by the presence of the analyte or the presence of an analyte complex or derivative. Thus, in the absence of the analyte or its complex or derivative, neither the binding component nor the negative control reagent has any affinity for the reporter reagent. However, in the presence of the analyte or its complex or derivative, the negative control reagent has a lower affinity for the reporter reagent than the binding component.

[0107] Additionally, in the mode in which the binding component binds to the analyte, or the complex or derivative of the analyte, the negative control reagent preferably has a lower affinity for the analyte or, where applicable, the complex or derivative of the analyte than the binding component. The negative control reagent is preferably a protein, more preferably an antibody. The negative control reagent generally has chemical and physical properties similar to the binding component, but under analytical conditions, provides little or no affinity for the reporter reagent. In a particularly preferred mode, the negative control reagent essentially has no affinity for the reporter reagent under analytical conditions. Preferably, the negative control reagent essentially does not provide affinity for the analyte or the complex or derivative of the analyte. That is, the binding of the reporter reagent, or, where applicable, the analyte or the complex or derivative of the analyte, to the negative control reagent is non-specific. In this way, the negative control reagent can compensate for the non-specific binding of the reporter reagent to the binding component.

[0108] A positive control reagent binds to a reporter reagent and has an affinity for the reporter reagent, providing a positive control because the concentration of the analyte sample or, when used, the concentration of the analyte complex or derivative is less affected than that of the binding component. Preferably, the positive control reagent has an affinity for the reporter reagent that is essentially independent of the concentration of the analyte or the concentration of the analyte complex or derivative. More preferably, the positive control reagent has a higher affinity for the reporter reagent than the binding component under analytical conditions. In this way, the positive control reagent measures the maximum signal expected in the system.

[0109] To increase the dynamic range of the analysis performed according to the present invention and to improve precision, it is desirable to have the binding component at multiple locations on the substrate. These locations can be adjusted to different sensitivities by varying the concentration of the binding component at each location. Each location may also have its own negative and positive control reagents that serve as controls for various dynamic ranges. This is particularly applicable to competitive analyses that are sensitive to the concentration of each individual component constituting the system.

[0110] The above description allows the analysis to reach equilibrium before activating the photosensitizer, but it is also possible to monitor the kinetics of binding events by illuminating the photosensitizer for distinct periods over time and monitoring binding events occurring during the reaction process before reaching equilibrium.

[0111] The analyte can be a macromolecule or a small molecule. Macromolecules are generally proteins, such as protein-based hormones, and may also be parts of larger particles, such as viruses, bacteria, cells (e.g., red blood cells), or prions. Small molecules can be drugs.

[0112] The term “small molecule” as used herein is a term of the art used to distinguish it from macromolecules such as proteins and nucleic acids. Small molecules are often referred to as “haptens” in the field of immunoassay and are small molecules capable of inducing an immune response when attached to large carrier molecules, such as proteins, and include molecules such as hormones and synthetic drugs. These types of small molecules will generally have a molecular weight of 2,000 or less, often 1,000 or less, and even 500 or less. Although the analyte may undergo chemical reactions or initial complex formation events before binding to the binding component, the binding component may be tuned to bind to the analyte itself. For example, the analyte may be protonated / deprotonated at the pH of the analysis conditions. Thus, the analyte bound to the binding component may be the analyte itself or a derivative of the analyte; both are included within the scope of the present invention.

[0113] In a preferred embodiment, the present invention may be used to detect the presence of multiple analytes simultaneously in the same sample. Different binding components may be used at different locations on the substrate for the measurement of each analyte. Sandwich and competitive analyses may be performed in parallel, and the analyses may use negative and positive controls as described above, or there may be a separate control for each analyte being measured.

[0114] The sample suspected of containing the analyte of interest will generally be a fluid sample, e.g., a liquid sample, and generally a biological sample, e.g., a biological sample such as blood, plasma, saliva, serum, tracheal fluid, cerebrospinal fluid, or urine. The sample may contain suspended particles and may be whole blood. In a preferred embodiment, the sample is untreated, and more preferably, an untreated fluid. Untreated means that the sample / fluid is not pretreated by filtration, dilution, or any other pretreatment step before being mixed with the reporter reagent and other analyte components. An advantage of the method of the present invention is that analysis can be performed on a sample containing suspended particles without excessively affecting the analysis results.

[0115] In a preferred embodiment, the sample is whole blood. It is not surprising that the components of whole blood do not interfere with the detection method of the present invention. It is common practice to remove red blood cells from blood to measure the fluorescence of blood plasma or serum components due to unpredictable light scattering by cellular components that differ from sample to sample. However, in the method of the present invention, since the fluorescence will be measured on a substrate and individual binding events can be measured, the measurement can take place in whole blood.

[0116] The sample is generally in the order of microliters (e.g., 1-100 μL, preferably 1-10 μL). To hold the fluid sample, the substrate is preferably positioned in a sample chamber having one or more side walls, an upper surface, and a lower surface. Accordingly, the apparatus used in the method of the present invention preferably further comprises a chamber for holding a sample containing an analyte in contact with the substrate.

[0117] A potential additional source of background interference is the deposition of suspended particles on the substrate surface, including the sample's reporter reagent and cellular components. Such sources of interference can be reduced by positioning the substrate over the bulk solution, for example, on the upper surface of the reaction chamber. Thus, even if deposition occurs, it does not interfere with the substrate. Preferably, the substrate forms the upper surface as illustrated in the drawings. Clearly, the optical and binding components will be located on the inner surface of the chamber to allow contact with the sample. Such variations and other variations are included within the scope of the present invention.

[0118] The sample can be simply maintained, for example, inside a capillary channel by surface tension.

[0119] The reporter reagent and optionally one or more additional reagents are preferably stored in a chamber integrated into the device used in the method of the present invention.

[0120] The method of the present invention is particularly useful in point-of-care (POC) testing. POC testing is defined as a diagnostic test, or bedside test, at or near the point of treatment. POC testing enables convenient and rapid testing, thereby improving decision-making and classification while allowing for better allocation of hospital resources, such as accident and emergency care and beds. This contrasts with conventional testing, where samples are collected at the point of care and sent to a laboratory for testing. With such tests, it often takes hours or days to obtain results, during which time patients must continue to be managed without the desired information. POC testing often utilizes test kits accompanied by portable devices.

[0121] The method of the present invention is particularly useful for monitoring the concentration or presence / absence of an analyte that is generally present in very low abundances. Potential applications include the measurement of biomarkers for heart disease (e.g., high-sensitivity troponin), infectious disease (e.g., hepatitis C core antigen), aging / dementia (e.g., amyloid beta and tau, which are Alzheimer's disease markers), cytokines, and oncology (e.g., circulating tumor markers).

[0122] The present invention also provides an apparatus for detecting an analyte in a sample, the apparatus comprising a substrate having an optical component and a binding component attached to the surface of the substrate, wherein the optical component can change from a first optical state to a second optical state in response to interaction with an irradiated photosensitive agent of a reporter reagent bound to the surface of the substrate in proportion to the concentration of the analyte in the sample, thereby forming a set of local regions of the optical component having the second optical state on the substrate.

[0123] The features of the device are as described above for the device used in the method of the present invention.

[0124] In a preferred embodiment, the device further includes a chamber for holding a mixture of the sample and the reporter reagent.

[0125] The device of the present invention may include a radiation source configured to generate electromagnetic radiation and a detector capable of precisely determining the position of a photosensitive material on a substrate by detecting an optical component in a second optical state.

[0126] The device of the present invention may take the form of a cartridge used with a separate reader. The reader may integrate a radiation source and a detector. The reader is preferably a portable reader. Preferably, the device comprises a cartridge, the substrate is contained within the cartridge, and the device further comprises a detector for detecting a set of local regions having a second optical state on the substrate. The present invention may also provide a cartridge comprising a substrate and optical and coupling components as defined herein. The cartridge is preferably a disposable cartridge.

[0127] The present invention also provides a system for detecting an analyte in a sample, wherein the system comprises: an apparatus of the present invention; and a reporter reagent for forming a mixture comprising a sample, wherein the reporter reagent comprises a photosensitive agent, and the photosensitive agent absorbs electromagnetic radiation and interacts with an optical component to change the optical component from a first optical state to a second optical state.

[0128] Preferably, the photosensitive agent can absorb electromagnetic radiation and interact with a pre-activator reagent present in the mixture to produce an activator reagent, and the activator reagent can interact with the optical component to change the optical component from a first optical state to a second optical state.

[0129] In a preferred embodiment, the system of the present invention is essentially composed of the features described above. "Essentially" means that no other functions are required to perform the analysis.

[0130] The present invention will now be described with reference to the following examples, which are not intended to limit the invention.

[0131] Examples

[0132] Biotinized BSA was prepared according to techniques known in the art. Alphascreen donor beads containing encapsulated singlet oxygen photosensitizers were supplied by Perkin Elmer.

[0133] Example 1

[0134] 70 kD aminodextran-biotin-fluorescein conjugate

[0135] 10 mg of aminodextran (70 kD) was weighed and placed in a glass vial, and diluted to 2 mg / mL in 100 mM potassium phosphate buffer. 250 μL of this solution was dispensed into a 2 mL cryotube. A 4 mg / mL biotin-N-hydroxysuccinimide (NHS) ester solution was prepared in DMSO, and 16.2 μL was added to the cryotube. The sample was mixed on a roller at 20 °C for 30 minutes, while a 20 mg / mL fluorescein-NHS ester solution was prepared in DMSO.

[0136] After reacting with biotin-NHS ester for 30 minutes, 8.5 μL of a 20 mg / mL fluorescein-NHS ester solution was added, and the mixture was mixed on a roller at 20 °C for an additional 60 minutes. Subsequently, 11.3 μL of a 10 mg / mL glycine solution was added to quench the reaction, and the mixture was mixed on a roller at 20 °C for 20 minutes. The sample was desalted with 50 mM phosphate buffer using a Sephadex G-25 PD10 column. Absorbance was measured at 280 nm and 495 nm using a Nanodrop2000 spectrophotometer to determine concentration and fluorescein incorporation. The sample was then filtered using a Minisart (Sartorius) filter with a 0.2 μm filtration rate. The calculated biotin and fluorescein incorporation values ​​were 1.2 and 4.3, respectively.

[0137] Example 2

[0138] Polymerized streptavidin (polystreptavidin)

[0139] Polymerized streptavidin was prepared according to techniques known in the art. In summary, a fraction of streptavidin (1 mg / mL) was activated with 9.7 molar equivalents of SMCC (N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) for 1 hour, the reaction mixture was quenched with glycine, and the product was desalted on a PD10 size-exclusion column. At the same time, a separate fraction of streptavidin was reacted with 9.7 molar equivalents of SATA (N-succinimidyl-s-acetyl-thioacetate) for 1 hour, then desalted with hydroxylamine and purified on a PD10 size-exclusion column.

[0140] SATA-streptavidin and SMCC-streptavidin were combined in a 3:2 molar ratio for 30 minutes, after which NEM (N-ethyl maleimide) was added to quench the reaction mixture. Subsequently, the product was purified on a G25 Sephadex column to obtain polymerized streptavidin. The concentration of the product was measured by UV-vis spectroscopy and adjusted to 1.0 mg / mL. No further characterization was performed.

[0141] Example 3

[0142] Preparation of substrate surface 1

[0143] A 1cm x 1cm square piece with a diameter of 6 mm and a thickness of 200 μm was attached to a 22mm x 22mm glass cover slip (20) (brand, catalog number 4700 55) to create a shallow well (21).

[0145] Then, 30 μL of biotinylated BSA (10 μg / mL in 40 mM phosphate buffer) was added to this well and incubated for 2 hours, after which it was washed with wash buffer (40 mM phosphate, 2% sucrose, 0.9% NaCl, 0.03% BSA). Subsequently, 30 μL of polystreptavidin (10 μg / mL in 40 mM phosphate buffer) was added to the well and incubated for 60 minutes, after which it was washed three times with wash buffer. Then, 30 μL of 70 kD aminodextran-biotin-fluorescein conjugate (10 μg / mL in 40 mM phosphate buffer) was incubated for 30 minutes, after which it was washed three times with wash buffer and air-dried at room temperature.

[0146] Example 4

[0147] Binding of streptavidin beads on the surface

[0148] Streptavidin-coated Alphascreen donor beads (Perkin Elmer catalog number 6760002S, 5 mg / mL) were diluted 1 / 1000 with 40 mM phosphate buffer, 20 μL of this solution was added to the substrate of Example 3, incubated for 2 hours, washed three times with wash buffer, and dried. This was performed under low light conditions using a green filter over a light fixture. The streptavidin-coated beads bind to the free biotin groups of the aminodextran-biotin-fluorescein conjugate as shown in Figure 2.

[0149] Example 5

[0150] Generation of non-fluorescent spots in the fluorescent layer

[0151] 20 μL of H2O was added to the substrate, and the entire surface was illuminated using a red LED at 680 nm to provide a total light output of 15 mW, which is approximately 0.5 mW / mm² on the surface as shown in Fig. 3. 2It corresponds to the luminous flux. After 5 minutes of illumination, the liquid was removed and the surface was dried. Subsequently, the substrate was inverted and attached to the top of a glass slide as shown in Fig. 11. It was then imaged using a Leica DMR wide-field fluorescence microscope with a 100x oil immersion lens and a fluorescein set, using a CoolLED pE-300 light source and a Leica DFC9000GT digital camera (2048 x 2048 pixels). Fig. 9 shows the optical setup.

[0152] FIG. 4 shows how a dye molecule of aminodextran very close to a bound bead is converted from a fluorescent form (14) to a non-fluorescent form (15) due to the flux of singlet oxygen generated by bead 3 when illuminated at 680 nm as described above.

[0153] Figure 12 is a partial image of the substrate surface taken with a microscope camera. Areas with distinct dark spots where beads are bonded can be observed. There are larger spots where beads are clustered. However, smaller, uniform spots can be clearly distinguished as individual bonding events because the beads remain near the surface during a 5-minute illumination period.

[0154] Example 6

[0155] A substrate surface was prepared as described in Examples 3 and 4 above, and beads were bonded to the surface. Subsequently, 20 μL of human plasma was added to the surface, and bead excitation and imaging were performed as described in Example 5 above. Microscope camera images of a portion of the substrate surface are shown in FIG. 13. Discontinuous dark regions are observed on the substrate surface corresponding to the individually bonded beads, which indicates that the components of human plasma do not prevent or extinguish the singlet oxygen reaction with the active dye during the illumination phase.

[0156] Example 7

[0157] A substrate surface was prepared as described in Examples 3 and 4 above, and beads were bonded to the surface. Subsequently, 20 μL of fresh human blood was added to the surface, and bead excitation and imaging were performed as described in Example 5 above. Microscope camera images of a portion of the substrate surface are shown in FIG. 14. Discontinuous dark regions were observed on the substrate surface corresponding to the individually bonded beads, which indicates that the cellular components of human blood do not prevent or extinguish the singlet oxygen reaction with the active dye during the illumination phase.

[0158] Example 8

[0159] Polystreptavidin - SOSG (singlet oxygen sensor green) conjugate

[0160] Six vials of SOSG (singlet oxygen sensor green) (Thermo Fisher, catalog number S36002, 100 μg per vial) were removed from the freezer and thawed at room temperature for 30 minutes. Subsequently, they were reconstituted in methanol and pooled to yield a total volume of 200 μL. The SOSG was activated by adding 19.1 μL of N-hydroxysuccinimide (6 mg / mL in 25 mM MES buffer), followed by the addition of 31.9 μL of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (6 mg / mL in 25 mM MES buffer), and mixing the solution on a roller at 20 °C for 15 minutes. 1 mL of 1 mg / mL polystreptavidin solution (in 100 mM phosphate buffer) was dispensed into a freezing tube.

[0161] After 15 minutes of incubation, 90.7 μL of activated SOSG was added, and the sample was mixed on a roller at 20 °C for approximately 65 hours. Subsequently, the sample was desalted with 50 mM phosphate buffer using a Sephadex G-25 PD10 column. Absorbance was measured at 280 nm and 520 nm using a Nanodrop2000 spectrophotometer to determine concentration and SOSG incorporation. After adding 25 μL of Proclin 950 to the sample, it was filtered to 0.2 μm using a Minisart filter. SOSG incorporation was calculated as 1.8 moles per mole of streptavidin monomer.

[0162] Example 11

[0163] Preparation of substrate surface 2

[0164] A 1 cm x 1 cm square piece of pressure-sensitive adhesive (19) with a diameter of 6 mm and a thickness of 200 μm was attached to a 22 mm x 22 mm glass cover slip (20) (brand GMBH, catalog number 4700 55) to create a shallow well (21) as shown in FIG. 10.

[0165] Then, 30 μL of biotinylated BSA (10 μg / mL in 40 mM phosphate buffer) was added to this well and incubated for 2 hours, after which it was washed with wash buffer (40 mM phosphate, 2% sucrose, 0.9% NaCl, 0.03% BSA). Then, 30 μL of polystreptavidin-SOSG conjugate (10 μg / mL in 40 mM phosphate buffer) was added to the well and incubated for 60 minutes, after which it was washed three times with wash buffer. Subsequently, 30 μL of biotinylated anti-TSH antibody 5409 (which recognizes the conjugation between the alpha and beta subunits of TSH with a dissociation constant of 50 pM) (2 μg / mL in 40 mM phosphate buffer) was added and incubated for 30 minutes, after which it was washed three times with wash buffer and air-dried at room temperature.

[0166] Example 12

[0167] Preparation of Piropheofovid-α Conjugate of Anti-TSH Antibody 5407

[0168] Antibody 5407 (recognizing the beta subunit of TSH with a dissociation constant of 180 pM) was provided by Medix Biochemica, and pyropheophorbide-a (PPa) was provided by Frontier Scientific (Logan, Utah). PPa is converted to succinimidyl ester, and "Targeted photodynamic therapy with multiply-loaded recombinant antibody fragments, Bhatti et al , Int. J. Cancer, 2008, 122 It was bound to the antibody using the technique described in , 1155." PPa molar incorporation was calculated as 3.5 per mole of antibody.

[0169] Example 13

[0170] Creation of discontinuous fluorescent spots

[0171] After inverting the substrate surface (17) and removing the adhesive release liner, the substrate was attached to an acrylic sheet (22) having two small holes (23) on both sides of the substrate well as shown in the profile of FIG. 11.

[0172] A reaction mixture was prepared containing TSH-spiked TSH-free whole blood with a 5407-PPa antibody conjugate (10 ng / mL) and a series of dilutions at concentration ranges (1 nM, 100 pM, 10 pM, 1 pM, 100 fM, 10 fM). The mixture (about 6 μL) was transferred to the substrate with a pipette through one of the holes (23), and then the holes (23) were sealed with grease.

[0173] After incubating the chamber in the dark for 10 minutes, it was illuminated at 680 nm using a red LED with a total light output of 15 mW. After 2 minutes of illumination, the substrate surface was imaged using the optical setup as described in Fig. 9 and above. A discrete fluorescence region can be observed where PPa was bound to the substrate surface for the entire 2 minutes. TSH can be quantified as the background signal above at an estimated concentration of 50 femtomolar (3 times the standard deviation of the spot count for zero analyte samples).

Claims

Claim 1 A method for detecting an analyte in a sample, wherein the method comprises: (i) providing a mixture comprising a sample and a reporter reagent to a device, wherein the device comprises a substrate having an optical component and a binding component attached to the surface of the substrate; (ii) binding a portion of the reporter reagent to the surface of the substrate in proportion to the concentration of the analyte by means of the binding component; (iii) irradiating the device with electromagnetic radiation for absorption by a photosensitive agent of the reporter reagent, such that the photosensitive agent of the bound portion of the reporter reagent interacts with the optical component to change the optical component from a first optical state to a second optical state, thereby forming a set of local regions of the optical component having the second optical state on the substrate, wherein the optical component is fluorescent when in the first optical state and is non-fluorescent when in the second optical state, and the change from the first optical state to the second optical state is irreversible. and (iv) a step of detecting a set of local regions having a second optical state on the above-mentioned substrate; wherein the local regions having the second optical state are counted as individual combined events. Claim 2 A detection method according to claim 1, wherein absorption by the photosensitive agent is intended to interact with a pre-activator reagent present in the mixture to produce an activator reagent, and the activator reagent is intended to interact with an optical component to cause a change from a first optical state to a second optical state. Claim 3 A detection method according to paragraph 2, wherein the activator reagent is a reactive oxygen species. Claim 4 A detection method according to paragraph 3, wherein the reactive oxygen species is singlet oxygen. Claim 5 A detection method according to any one of claims 1 to 4, wherein a set of local regions having a second optical state on the substrate is detected using an optical microscope. Claim 6 A detection method according to claim 1, wherein the optical component absorbs light of one or more wavelengths when in a first optical state, and the optical component absorbs light of one or more different wavelengths when in a second optical state. Claim 7 delete Claim 8 delete Claim 9 delete Claim 10 A detection method according to claim 1, wherein steps (i) to (iii) are performed without a washing step. Claim 11 A detection method according to claim 1, wherein the sample is untreated. Claim 12 A detection method according to claim 1, wherein the device is irradiated with electromagnetic radiation for at least 1 second. Claim 13 An apparatus for detecting an analyte in a sample, the apparatus comprises a substrate having an optical component and a binding component attached to the surface of the substrate, wherein the optical component changes from a first optical state to a second optical state in response to interaction with a photosensitive agent irradiated by a reporter reagent bound to the surface of the substrate in proportion to the concentration of the analyte in the sample, thereby forming a set of local regions of the optical component having the second optical state on the substrate, wherein the optical component is fluorescent when in the first optical state and the optical component is non-fluorescent when in the second optical state, the change from the first optical state to the second optical state is irreversible, and the local regions having the second optical state are counted as individual binding events. Claim 14 A device according to claim 13, wherein the device comprises a cartridge, the substrate is within the cartridge, and the device further comprises a detector for detecting a set of local regions having a second optical state on the substrate. Claim 15 A system for detecting an analyte in a sample, wherein the system comprises: an apparatus described in claim 13 or 14; and a reporter reagent for forming a mixture comprising a sample, wherein the reporter reagent comprises a photosensitive agent, and the photosensitive agent absorbs electromagnetic radiation and interacts with an optical component to change the optical component from a first optical state to a second optical state.

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