Methods for detecting analytes

Abstract

The present invention provides a method for detecting two or more analytes in a sample, comprising steps (i) to (v). Accordingly, the present invention provides a method for detecting two or more analytes in a sample, wherein only a reporter reagent near the surface of the substrate generates a signal, and this signal is a local region of an optical component in a second optical state. What is detected is a set of local regions of an optical component in a second optical state.

Description

Technical Field

[0001] The present invention relates to a method for detecting an analyte, particularly a method for detecting two or more analytes in a sample.

Background Art

[0002] There are many techniques available for the measurement of biologically relevant parameters in human samples such as blood, plasma, serum, tissue, etc. A common method is to use a capture reagent that binds to the target of interest and a reporter reagent that has some kind of label. The capture reagent can be bound to a solid phase such as a microtiter 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. Then, the excess reporter can be removed (by washing), and by measuring the amount of the reporter reagent, the amount of the analyte present in the sample can be measured. There are many different variations on how these types of binding assays can be performed. For example, the analyte may first bind to the capture reagent, and then the reporter may be added in a separate step, or the analyte may first bind to the reporter and then bind to the capture reagent.

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

[0004] Assays that use antibodies are generally called immunoassays. Immunoassays can take many forms. For example, when a capture antibody is used to capture the analyte and a reporter antibody is used to generate a measurable signal, this is generally called a sandwich immunoassay. Alternative forms are also known in which the binder is immobilized on a solid phase and the target analyte is in solution, competing with a labeling reagent that also binds to the binder. If the analyte is not present, a high level of labeling reagent binds, resulting in a high signal. If the analyte is present, part of the binding site is blocked, resulting in a small amount of labeling reagent binding and a reduced signal. These assays are generally known as inhibitory assays or competitive assays. Several types of competitive assays are known. For example, the antibody may be bound to a solid phase, and the labeling analyte (or an analogue of the analyte) can compete for the binding site on the antibody. Alternatively, the analogue of the analyte may be immobilized, and the labeling antibody can be bound to this surface. If the analyte is present in the sample, this analyte binds to the antibody in solution, preventing binding to the surface and reducing the signal.

[0005] Many forms of assays exist, and a large number of different types of labels are available. Assays can be heterogeneous, in which case excess labeling is removed before measurement, for example, by using a washing step. Removal of excess labeling can also be achieved by flowing the sample and reporter beyond the capture area. This approach is used, for example, in immunochromatography or lateral flow strips used in rapid tests such as infectious disease tests and pregnancy tests. Alternatively, homogeneous assays are known in which excess reporter is not removed. Homogeneous assays tend to rely on the proximity of the capture and reporter to produce some form of signal. An example of a homogeneous assay is the agglutination assay in which particles bind together in solution. The aggregated particles cause light scattering, which can be measured by turbi geometry or nephelometry. A further 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 a homogeneous assay is fluorescence resonance energy transfer (FRET). In this assay, the capture reagent and reporter reagent are a donor fluorophore and an acceptor fluorophore, respectively. Excitation of the donor leads to energy transfer to the acceptor, followed by luminescence.

[0007] One type of homogeneous assay that functions in whole blood without removing cellular material is the pyro-optical immunoassay. The capture antibody is coated onto a pyroelectric polyvinylidene PVDF sensor, and carbon particles are used as reporters. The signal is generated by irradiating the sample with light, causing localized heating of the particles. Those bound to the sensor transfer energy to the pyroelectric sensor, generating thermal stress that is detected as an electrical signal. The more carbon particles bound, the stronger the signal.

[0008] The label immobilized on the reporter binder can be a light-absorbing substance such as a dye, gold particles, or stained latex microparticles. Larger particles can, in principle, absorb more light and generate a stronger signal. However, as will be discussed in more detail below, particle labeling has size limitations and is not practical for use in assays. Luminescent labels, such as fluorescent, chemiluminescent, bioluminescent, and electrochemiluminescent labels, are also known. Luminescent labels are also encapsulated within particles in certain types of assays. Signal amplification may also be performed using enzymatic or catalytic reactions. Enzymes may be used to convert the substrate from a leuco dye to a colored form, or to a fluorescent or luminescent form. Excess enzyme is typically removed using a washing step before the substrate is added, so that the signal is generated only by enzymes specifically bound to the analyte.

[0009] Immunoassays that do not use labels, such as those employing surface plasmon resonance, are also known as signal transduction methods. However, label-free assays tend to lack the sensitivity of assays that use labels to enhance the signal.

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

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

[0012] The maximum signal is generally limited by factors such as the total amount of capture antibody available to bind to the analyte and the total amount of reporter antibody generating the signal. If the capture antibody is immobilized on a solid phase, the upper limit of detection may be limited by the surface area of ​​the solid phase. Furthermore, some signal conversion techniques, such as colorimetric methods, tend to saturate depending on the optical path length required for light to pass through the sample. Luminescence methods tend to be less prone to saturation because the detector amplification can be attenuated to cope with higher levels of emission. In heterogeneous assays, the system reaches its maximum signal and saturates when all antibody binding sites are filled with analyte. Excess analyte is usually removed in a washing step before the reporter is added. Homogeneous assays can also be affected by what is known as high-dose hooking, where the concentration of the analyte is higher than the effective concentration of the capture antibody and / or reporter antibody. In this case, at extremely high concentrations, all binding sites on the capture and reporter may be blocked, leading to erroneous results by reducing the assay signal.

[0013] Low levels of detection are governed by various factors. Generally, all assays are influenced by attributes such as the quality (affinity and specificity) of the antibody used, as well as the cross-reactivity between the antibody and the analyte in question. The lower limit of detection also depends on factors affecting the signal-to-noise ratio of the assay setup and system design. For example, in a standard enzyme-linked immunosorbent assay (ELISA), the capture antibody is coated onto the surface of a 96-well microtiter plate, and then the sample is incubated in the wells to obtain the captured analyte. The wells are washed, and then an excess amount of reporter is added and bound to the captured analyte. The excess reporter is then washed away, and a substrate that can react with the enzyme is added and converted to the active form. For example, a colorless leuco dye such as 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) can be converted to a green oxidized form by horseradish peroxidase in the presence of hydrogen peroxide. When the analyte is present in very small amounts (e.g., less than 1 picomole), the amount of enzyme that binds to the well surface will be extremely small. ABTS reacts with the enzyme to produce a green form, which then diffuses into the majority of the fluid, creating a solution so dilute that it cannot be distinguished from the background signal. Autoconversion of the substrate can also produce a color that interferes with the measurement. Similarly, other detection methods, such as fluorescence, can also be affected by the autofluorescence of interfering factors and components in the sample or reaction well.

[0014] Another confounding factor in immunoassays can be the nonspecific binding of the reporter reagent to the capture surface. For example, in the ELISA assay described above, the microtiter wells are coated with a layer of protein, some of which may denature during the coating process. It is not uncommon for the reporter to bind to a region of the capture surface during the assay. If this reporter causes substrate turnover and contributes to the overall signal, it becomes impossible to distinguish between a signal from a specifically bound reporter and a signal from a nonspecificly bound reporter. Nonspecific binding can also be facilitated by many components present in the original sample, which bind to the capture surface during the initial incubation, modifying the surface properties of the capture layer to create a surface that can bind to the reporter. Minimizing nonspecific reporter binding involves careful optimization of all reagents and reaction conditions used during the assay, including antibodies, surfactants, temperature, and ionic strength.

[0015] Generally, the detection limits of conventional immunoassays range from approximately 0.1 picomoles to approximately 1 nanomolar, depending on the assay method. Developing assays with extremely low detection limits using conventional approaches often requires significant optimization, including stringent washing steps to reduce nonspecific binding and maximize the signal-to-noise ratio. Furthermore, the capture surface is often small relative to the sample volume to ensure that the signal is sufficiently high against the background.

[0016] One approach used to avoid problems associated with the signal-to-noise ratio 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 local threshold. In this way, much of the background noise can be eliminated. Similarities can be demonstrated digitally with acoustic or communication signals. These digital assays have been shown to reach detection limits that could not be achieved using conventional similar methods in the past. For example, low femtomole (10-15 mol / L), and even atomole (10 -18 Detection limits in the range of mol / L have been reported. See, 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, 343.

[0017] The vast majority of labels / reporters used in assays (e.g., fluorophores, dyes, etc.) are so small that they cannot be individually visualized using a wide-field microscope, even at high magnification. Therefore, the presence of these labels can only be measured as a bulk phenomenon, not by counting each label. In contrast, particulate labels such as latex particles can theoretically be visualized by a wide-field optical microscope if they exceed a certain size. Depending on the optical setup and the numerical aperture and type of microscope, particle visualization may begin when the diameter exceeds several hundred nanometers.

[0018] However, using particles of this size as labels to monitor individual binding events (e.g., antibody-antigen interactions) on a capture surface is impractical for several reasons. For example, particles of this size diffuse very slowly compared to other types of labels, impairing reaction rates on planar surfaces. They also begin to exhibit macroscopic buoyancy effects, precipitating or floating if the particle density differs significantly from the density of the medium they contain, potentially causing problems with the assay. Particles of this size are particularly prone to non-specific binding to surfaces, causing high background noise that is difficult to remove. Ultimately, excess particles must be removed, necessitating a washing step. However, larger particles begin to experience shearing effects in the presence of fluid flow, and if the shearing force on the particle becomes greater than the breaking strength of the antibody-antigen interaction (approximately 60 pN to 250 pN), the particle will be washed away (see "Rapid Femtomolar Bioassays in Complex Matrices Combining Microfluidics and Magnetoelectronics," Mulvaney et al., Biosensors and Bioelectronics, 2007, 23, 191).

[0019] Examples of digital assays include the Quanterix Single Molecule Array (SIMOA) system and the Merck Millipore Single Molecule Counting (SMC) system.

[0020] The Quanterix SIMOA system captures analytes from solution using paramagnetic beads coated with antibodies. The magnetic beads are then washed, and an enzyme-labeled reporter antibody is added. The number of beads is sufficient to minimize the probability of having multiple analytes and reporters per bead. The beads are washed again and then loaded into an array of microwells, each capable of holding only one bead. The microwell volume is on a femtoliter scale. If the beads contain the enzyme to which they are bound, the fluorescent substrate in the well is turned over. Smaller wells prevent excessive diffusion of the fluorescent product. Each well is then counted as an "on" or "off" event depending on whether the fluorescence exceeds a threshold.

[0021] The SMC system is used in the Merck Millipore Erenna and SMCxPRO systems. The basic measurement technique is the same in both systems. Magnetic beads coated with capture antibody are used to capture the target analyte in the sandwich assay. A fluorescently labeled reporter antibody also binds to the beads in the presence of the analyte. The beads are pulled down by a magnet, and any excess fluorescently tagged reporter is washed away. Elution buffer is then added to dissociate the sandwich complex, which is then transferred to the measurement vessel. The presence of the fluorescent tag is then measured using a confocal fluorescence microscope, which sequentially examines small amounts of sample to determine whether or not the fluorescent tag is present. If the signal for each individual measurement exceeds the threshold, this is counted as an "on" event in that measurement.

[0022] Several independent academic reviews of high-sensitivity immunoassays highlight that digital approaches to immunoassays enable unexpected improvements in detection limits (see Yeung and Cretich cited above).

[0023] The detection limits of the Quanterix and Merck Millipore systems depend on the volume of the sample used in the assay. For a 10-microliter serum or plasma sample, the theoretical limit is the detection of a single binding event corresponding to one molecule. However, in terms of molar concentration, this is 100,000 molecules per liter of sample, or 0.16 × 10⁶ molecules per liter. -18 This corresponds to a mole (0.16 atmole).

[0024] However, the Quanterix and Merck Millipore systems mentioned above are complex and cumbersome, requiring numerous washing and transfer steps. Furthermore, these assays can only be performed on samples that do not contain cell material, and the systems require expensive equipment to achieve the performance they offer. Therefore, there is still a need for simpler, more cost-effective, and highly sensitive systems.

[0025] Furthermore, the Quanterix and Merck Millipore systems measure one analyte per sample. Measuring multiple analytes within the same sample in a homogeneous assay format is typically achieved through one of two approaches. The first approach uses separate physical regions containing a separate reporter reagent for each analyte and a binding component (capture reagent) that binds to each reporter reagent in the presence of the analyte. Thus, this approach achieves multiple measurements through spatial separation. However, if there are five analytes, five reporting reagents, and five binding components (capture reagents), 80% of the reporter reagents in each measurement region could become undesirable reporter reagents that interfere with the measurement. The second approach uses optical labeling that can be visualized at individual wavelengths. However, this approach requires the use of electromagnetic radiation at different wavelengths, which necessitates multiple light sources and / or complex optical filters, diffraction gratings, etc.

[0026] International Publication No. 2020 / 260865 pamphlet describes a digital assay method that uses a photosensitizer reagent to convert an optical component from a first optical state to a second optical state. However, there is still a need to optimize this system to simultaneously measure (multiplex) multiple analytes without increasing the complexity or interference between each measurement.

Summary of the Invention

[0027] Therefore, the present invention is a method for detecting two or more analytes in a sample, comprising: (i) providing to a device a mixture comprising a sample and two or more reporter reagents, wherein each of the two or more reporter reagents comprises a photosensitizer, and the device comprises a substrate having an optical component and two or more binding components, and the optical component and the two or more binding components are bound to the surface of the substrate; (ii) enabling a portion of each of the two or more reporter reagents to bind to the surface of the substrate in proportion to the concentration of the corresponding analyte by the corresponding binding component; (iii) irradiating the device with electromagnetic radiation of one wavelength for absorption by at least one of the photosensitizers of the two or more reporter reagents, such that at least one of the photosensitizers of the two or more reporter reagents bound to the surface of the substrate absorbs the electromagnetic radiation and interacts with the optical component to change the optical component from a first optical state to a second optical state, thereby forming at least one set of local regions of the optical component having the second optical state on the substrate; (iv) Optionally, detecting at least one set of local regions of at least one optical component having a second optical state on the substrate formed in step (iii), and repeating step (iii) with electromagnetic radiation of the same or different wavelengths for absorption by at least one of the photosensitizers of two or more reporter reagents, whereby at least one of the photosensitizers of two or more reporter reagents bound to the surface of the substrate absorbs the electromagnetic radiation and interacts with the optical component to change the optical component from the first optical state to the second optical state, thereby forming at least one set of local regions of the optical component having the second optical state on the substrate; and (v) When step (iv) is carried out, detecting at least one set of local regions of the optical component having the second optical state on the substrate formed in step (iv), or when step (iv) is not carried out, detecting two or more sets of local regions of the optical component having the second optical state on the substrate formed in step (iii) comprising providing a method wherein the two or more sets of local regions are distinguishable from each other by the formation rate of the set of local regions, the formation order of the set of local regions, or the size of the local regions optionally in combination with the formation rate or the formation order of the set of local regions.

[0028] Thus, the present invention provides a method for detecting two or more analytes in a sample, wherein only the reporter reagents proximal to the surface of the substrate provide a signal, and this signal is a local region of an optical component in the second optical state. What is detected is a set of local regions of the optical component in the second optical state. Thus, the present invention simplifies the digital detection of two or more analytes and facilitates homogeneous assays for a range of samples, including those containing cellular material.

[0029] The fact that there are two or more sets of local regions that are distinguishable from each other, depending on the formation rate of the sets of local regions, the formation order of the sets of local regions, or the size of the local regions in an optional combination of the formation rate or the formation order of the sets of local regions, provides a simple method for detecting and identifying two or more analytes in a sample.

[0030] Unlike typical methods for measuring two or more analytes in a sample using homogeneous assays, the method of the present invention does not require the separation of two or more binding components on the surface of the substrate, nor does it require the use of electromagnetic radiation of different wavelengths to detect two or more analytes in the sample.

[0031] However, when two or more binding components on the surface of a substrate are separated into different regions, specific binding of a reporter reagent that is expected to bind and nonspecific binding of an undesirable reporter reagent can be distinguished because two or more sets of local regions are distinguished from each other by the rate of formation of the local region sets, the order in which the local region sets are formed, or the size of the local region sets, which is a combination of the rate of formation of the local region sets or the order in which the local region sets are formed and a combination of both. Compared to a multiplex assay in which all different reporter reagents are added to a single sample and binding is identified only by changing the position of the binding components, the reduction of this interference and the improvement of accuracy in the method of the present invention are valuable advantages.

[0032] Therefore, the present invention provides an improved method for detecting two or more analytes in a sample.

[0033] The present invention will be described below with reference to the drawings. [Brief explanation of the drawing]

[0034] [Figure 1] Figure 1 shows various components that may be used in the method of the present invention. [Figure 2]Figure 2 shows a device in which two reporter reagents of different sizes are bonded to the surface of a substrate before irradiation. [Figure 3] Figure 3 shows the device shown in Figure 2 being irradiated. [Figure 4] Figure 4 shows the device from Figure 3 after irradiation. [Figure 5] Figure 5 shows a representative example of two sets of local regions having a second optical state on a substrate, where the two sets of local regions are distinguishable from each other by the size of the local regions that are visualized as faded dark regions of different sizes in the fluorescent layer. [Figure 6] Figure 6 shows an optical setup for detection that allows two electromagnetic radiation sources to be focused through an objective lens. [Figure 7] Figure 7 shows a device in which two reporter reagents containing different photosensitizers are bonded to the surface of a substrate before irradiation. [Figure 8] Figure 8 shows the device from Figure 7 being irradiated with wavelength a. [Figure 9] Figure 9 shows the device from Figure 8 after irradiation with wavelength a. [Figure 10] Figure 10 shows the device from Figure 9 being irradiated with wavelength b. [Figure 11] Figure 11 shows the device from Figure 10 after irradiation with wavelength b. [Figure 12] Figure 12 shows the substrate and wells prepared for the method of the present invention. [Figure 13] Figure 13 shows a sample chamber prepared using the substrate and wells shown in Figure 12. [Figure 14] Figure 14 shows a device in which two types of binding components are bound to separate regions of the substrate (binding component 2a in well 1 and binding component 2b in well 2), and two reporter reagents of different sizes are bound to the surface of the substrate before irradiation. [Figure 15]Figure 15 shows the device shown in Figure 14 being irradiated. [Figure 16] Figure 16 shows the device from Figure 15 after irradiation. [Figure 17] Figure 17 shows a device in which each of the two binding components is bound to separate regions of the substrate (binding component 2a in well 1 and binding component 2b in well 2), and two reporter reagents containing different photosensitizers are bound to the surface of the substrate before irradiation. [Figure 18] Figure 18 shows the device shown in Figure 17 being irradiated. [Figure 19] Figure 19 shows the device from Figure 18 after irradiation. [Modes for carrying out the invention]

[0035] The method of the present invention is used to detect two or more analytes in a sample (which may be via the detection of a complex or derivative of the analytes). The two or more analytes are different types of analytes and form two or more sets of local regions having a second optical state on the substrate. Therefore, the method of the present invention is used to detect multiple analytes simultaneously (multiplexing). There is no theoretical upper limit to the number of analytes that can be detected, but the number of detectable analytes may be limited by the practical need to reliably distinguish between different sets of local regions.

[0036] In a preferred embodiment, the method of the present invention is a method for detecting 2 to 10 analytes in a sample, more preferably 2 to 8 analytes in a sample, even more preferably 2 to 6 analytes in a sample, and most preferably 2 to 4 analytes in a sample.

[0037] The components in Figure 1 are: photosensitizers 1a and 1b stimulated by radiation of different wavelengths; antibodies 2a and 2b that bind to different analytes; antibody-coated latex particles 3 injected with photosensitizer 1a (hereinafter also called photosensitizer-labeled antibody 3); antibody-coated latex particles 4 injected with photosensitizer 1a (hereinafter also called photosensitizer-labeled antibody 4), which are of a different size from particle 3; and antibody-coated latex particles 5 injected with photosensitizer 1b (hereinafter also called photo These are: sensitizer-labeled antibody 5 (also called); protein analytes 6a and 6b; fluorescent optical component 7; non-fluorescent optical component 8; streptavidin 9, streptavidin 10 labeled with the fluorescent optical component (hereinafter also called streptavidin-dye conjugate 10); streptavidin 11 labeled with the non-fluorescent optical component; and biotinylated BSA 12 (hereinafter also called BSA-biotin conjugate 12).

[0038] Step (i) of the method of the present invention is a step of providing a device with a sample and a mixture comprising two or more reporter reagents, each of which comprises a photosensitizer, and the device comprises a substrate 14 having an optical component and two or more binding components, wherein the optical component and the two or more binding components are bonded to the surface of the substrate 14.

[0039] The sample and two or more reporter reagents may be pre-mixed before adding the mixture to the device, or the sample and two or more reporter reagents may be added sequentially to the device to form the mixture. The mixture typically contains additional reagents, such as buffers, surfactants, and other additives.

[0040] Each of the two or more reporter reagents corresponds to a different analyte and binding component. For example, the first reporter reagent corresponds to the first analyte and the first binding component, the second reporter reagent corresponds to the second analyte and the second binding component, and so on.

[0041] To illustrate the fundamental principle of the present invention, Figure 2 shows a device in which two reporter reagents are bonded to the surface of a substrate before irradiation. The device comprises a substrate 14 and a sample chamber 13 for holding a sample containing protein analytes 6a and 6b dissolved or suspended within it. The substrate can be any substrate that allows detection of two or more sets of local regions having a second optical state on the substrate. Preferably, the substrate is planar. Preferably, the substrate is transparent, and more preferably, the substrate is glass or plastic.

[0042] The substrate 14 has antibodies 2a and 2b bound to a streptavidin-dye conjugate 10 which is bound to the surface of the substrate 14 via a BSA-biotin conjugate 12. The dye acts as an optical component, and the antibodies act as binding components. The BSA-biotin conjugate 12 is an inert polymer that facilitates the binding of the optical and binding components to the surface of the substrate 14.

[0043] Although the optical and binding components are shown in this manner, any technique can be applied to retain the optical and binding components proximal to the surface of the substrate 14. For example, the optical and binding components may be a single reagent. The optical component may also be encapsulated within a polymer layer coated on the surface of the substrate 14, and the binding component is bonded to that 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.

[0044] Alternatively, the gel layer, for example, the hydrogel layer, may be impregnated with optical components, the gel / hydrogel layer may be coated on the surface of the substrate 14, and the binding components may adhere to the gel / hydrogel layer.

[0045] Step (ii) of the method of the present invention includes binding a portion of each of two or more reporter reagents to the surface of a substrate in proportion to the concentration of the corresponding analyte using a corresponding binding component. As described above, each of the two or more reporter reagents corresponds to a different analyte and binding component. For example, the first reporter reagent corresponds to a first analyte and a first binding component, the second reporter reagent corresponds to a second analyte and a second binding component, and so on. Therefore, the corresponding analyte means an analyte specific to the reporter reagent, and the corresponding binding component means a binding component specific to the reporter reagent.

[0046] Step (ii) can be achieved by leaving the device undisturbed for a certain period of time, for example, 10 minutes.

[0047] In Figure 2, photosensitizer-labeled antibody 3 is bound to the substrate surface by antibody 2a, and photosensitizer-labeled antibody 4 is bound to the substrate surface by antibody 2b. Photosensitizer-labeled antibody 3 and photosensitizer-labeled antibody 4 act as reporter reagents.

[0048] Two or more reporter reagents bind to the surface of the substrate in proportion to the concentration of the corresponding analytes. For example, if two or more binding components and two or more reporter reagents are antibodies, and two or more analytes are antigens, each of the two or more reporter reagents binds to the corresponding binding component via the corresponding analyte, forming a so-called "sandwich" complex. In Figure 2, photosensitizer-labeled antibody 3 binds to antibody 2a via protein analyte 6a. Similarly, photosensitizer-labeled antibody 4 binds to antibody 2b via protein analyte 6b. Other binding events, such as antibody-hapten binding or nucleic acid binding, are also possible.

[0049] All steps up to this point are performed in the absence of light. Step (iii) of the method of the present invention includes irradiating the device with electromagnetic radiation of one wavelength to absorb a photosensitizer by at least one of two or more reporter reagents, so that at least one of the two or more reporter reagent photosensitizers bonded to the surface of the substrate absorbs the electromagnetic radiation and interacts with the optical components to change the optical components from a first optical state to a second optical state, thereby forming a local region of at least one set of optical components having the second optical state on the substrate 14.

[0050] Figure 3 shows the device of Figure 2, which is irradiated with electromagnetic radiation, preferably visible light. The light source may be, for example, an LED 15. The light source irradiates the sample chamber 13 with light of an appropriate wavelength, exciting the photosensitizer 1a of the photosensitizer-labeled antibody 3 and the photosensitizer-labeled antibody 4.

[0051] The wavelength varies depending on the photosensitizer. The device is typically irradiated for 10 seconds. Preferably, the device is irradiated with electromagnetic radiation for 1 to 30 seconds, more preferably 2 to 20 seconds, even more preferably 5 to 15 seconds, and most preferably 10 seconds. This ensures that there is an irreversible optical change on the surface of the substrate, allowing for the distinction between permanent and transient bonding events.

[0052] Figure 4 shows the device from Figure 3 after irradiation. The photosensitizer 1a of photosensitizer-labeled antibody 3 and photosensitizer-labeled antibody 4 interact with the dye optical component of the streptavidin-dye conjugate 10, changing the dye from a fluorescent state to a non-fluorescent state. The fluorescent streptavidin-dye conjugate 10 becomes the non-fluorescent streptavidin-dye conjugate 11. Only the dye adjacent to the photosensitizer 1a changes from the first optical state to the second optical state.

[0053] Alternative first and second optical states may include changes in optical polarization, fluorescence lifetime, refractive index, light scattering (including Raman scattering), phosphorescence, and other optical effects.

[0054] Figures 2 to 4 show one of each of the two reporter reagents bound to the surface of the substrate 14. In reality, however, numerous identical reporter reagents are bound to the surface of the substrate 14, generating multiple local regions having a second optical state on the substrate within a set of local regions having a second optical state on the substrate. When using the components of Figure 1, these local regions having a second optical state on the substrate can be visualized as separate regions having a non-fluorescent streptavidin-dye conjugate 11.

[0055] Such individual regions can be seen in Figure 5, which shows representative examples of two sets of local regions having a second optical state on the substrate. These two sets of local regions are distinguishable from each other by their size and can be visualized as faded dark regions of different sizes in the fluorescent layer. There is a clear difference in size between the sets of local regions, with one set of local regions being visualized as a large dark region and the other as a small dark region. The white areas are artifacts and should be ignored.

[0056] Furthermore, the method of the present invention detects two or more analytes using two or more reporter reagents. The two or more reporter reagents are different types of reporter reagents for forming two or more sets of local regions having a second optical state on a substrate, which are distinguishable from each other by the rate of formation of the sets of local regions, the order in which the sets of local regions are formed, or the size of the local regions in an optional combination of the rate of formation of the sets of local regions or the order in which the sets of local regions are formed.

[0057] Furthermore, excess particles that are not bound to the surface may be suspended in the sample. If the sample is whole blood, the red blood cells are suspended in the culture medium. Preferably, the substrate 14 forms the top of the sample chamber 13, allowing the red blood cells to settle from the substrate 14.

[0058] A portion of each of the two or more reporter reagents binds to the surface of the substrate 14 by its corresponding binding component. Therefore, the sample contains both bound reporter reagents and unbound reporter reagents free in the solution. The depth of the sample chamber 13 is designed to minimize the length of the diffusion pathways for the two or more reporter reagents, allowing for rapid binding. Typically, the depth of the sample chamber is 50–200 μm.

[0059] In the method of the present invention, the sample chamber 13 is filled with a sample containing two or more analytes. Two or more reporter reagents, such as photosensitizer-labeled antibody 3 and photosensitizer-labeled antibody 4, are also added to the sample chamber 13. If a whole blood sample is used, the sample may also contain additional components such as red blood cells.

[0060] After a certain period of time, portions of each of the two or more reporter reagents, such as photosensitizer-labeled antibody 3 and photosensitizer-labeled antibody 4, bind to the surface of the substrate 14 by corresponding binding components, such as antibody 2a and antibody 2b, in proportion to the concentration of the corresponding analytes, such as protein analytes 6a and protein analytes 6b. An excess of reporter reagent is present, resulting in a significant proportion of each of the two or more analytes forming a sandwich complex. Thus, a proportion of each of the two or more reporter reagents binds to the surface of the substrate 14 by corresponding binding components, in proportion to the concentration of the corresponding analyte. Therefore, the sample contains both bound reporter reagents and unbound reporter reagents free in the solution.

[0061] After irradiation, the excited photosensitizer interacts with the optical components, changing them from a first optical state to a second optical state. For example, streptavidin 10 labeled with an optical component in a fluorescent state becomes streptavidin 11 labeled with an optical component in a non-fluorescent state. Only the optical components adjacent to the excited photosensitizer change from the first optical state to the second optical state.

[0062] The photosensitizer is in close proximity to the substrate when a binding event occurs. That is, the photosensitizer is in close proximity to the substrate surface and interacts with the optical components upon irradiation of the device, converting them from a first optical state to a second optical state. However, the actual distance between the photosensitizer and the substrate surface varies depending on several variability factors, including the size and properties of the photosensitizer, the binding components, the size and properties of the reporter reagent and analyte, and the properties of the sample medium.

[0063] Any excited photosensitizer located proximal to the optical component interacts with it, changing the optical component from a first optical state to a second optical state. In this way, each excited photosensitizer of two or more reporter reagents bound to the surface of the substrate interacts with the optical component, changing it from a first optical state to a second optical state, thereby forming two or more sets of local regions having the second optical state on the substrate 14.

[0064] The reporter reagent containing the excited photosensitizer must remain permanently bound to the surface of the substrate 14 for the entire duration of irradiation in order to achieve a complete conversion from the first optical state to the second optical state. If the reporter reagent containing the excited photosensitizer is only temporarily bound to the surface for a short period of the irradiation duration, an incomplete conversion to the second optical state will occur, which can be detected by an algorithm used to measure the size, shape, and intensity of individual regions. Any unbound reporter reagent in solution will not significantly change the optical components from the first optical state to the second optical state.

[0065] This offers a significant advantage over other digital assay methods in that it eliminates the need for a washing step. Thus, the method of the present invention is a homogeneous assay. In conventional assays, unbound reporter reagents interfere with the signal produced by bound reporter reagents, so unbound reporter reagents must be separated from bound reporter reagents before any measurement. However, the localized surface changes provided by the present invention allow for the distinction between bound and unbound reporter reagents. In fact, the ability to distinguish between reporter reagents adjacent to the surface of the substrate 14 (i.e., bound) and reporter reagents in the bulk solution (i.e., unbound) is a particular advantage of the present invention. Preferably, steps (i) to (iii) are carried out in the absence of a washing step, i.e., the method is carried out without removing the sample from the substrate in steps (i), (ii), and (iii).

[0066] Step (iv) of the method of the present invention optionally includes detecting a local region of at least one set of optical components having a second optical state on a substrate formed in step (iii), and repeating step (iii) with electromagnetic radiation of the same or different wavelengths to be absorbed by at least one of two or more reporter reagent photosensitizers, wherein at least one of the two or more reporter reagent photosensitizers bonded to the surface of the substrate absorbs the electromagnetic radiation and interacts with the optical components to change the optical components from a first optical state to a second optical state, thereby forming a local region of at least one set of optical components having a second optical state on the substrate.

[0067] Step (iv) is optional, and two or more sets of local regions having a second optical state are sequentially formed on the substrate by two or more irradiation steps, and detection is required after each irradiation step.

[0068] If step (iv) is performed, detection of at least one set of local regions of optical components having a second optical state on the substrate formed in step (iii) is preferably performed by image analysis software capable of distinguishing between actual bonding events and transient bonding events and surface artifacts by surface intensity and morphology analysis. Detection may be performed in another step after the optical components have been converted from the first optical state to the second optical state. Alternatively, detection may be performed during the process of conversion to the second optical state.

[0069] Any method can be used to successfully identify individual coupling events. For example, an initial image of the surface can be obtained by first using the excitation wavelength of the optical component, then irradiating with electromagnetic radiation to excite the photosensitizer, and finally irradiating with the excitation wavelength of the optical component.

[0070] Alternatively, it is possible to capture a series of images (or video files) of the surface while irradiating it with electromagnetic radiation that simultaneously excites both the optical components and the photosensitizer. Any combination of optical filters, dichroic mirrors, and illumination sequences is permitted to enable the identification of coupled events.

[0071] The second optical state forms a set of local regions on the substrate. Advantageously, local regions having the second optical state can be counted as individual binding events. Therefore, the method of the present invention is suitable for performing digital assays. However, if there are many binding events in which the majority of the optical components are in the second optical state, bulk changes may be detected.

[0072] In a preferred embodiment, at least one set of local regions having a second optical state on the substrate is detected by counting the local regions within each set of local regions having a second optical state on the substrate, or by measuring at least one set of local regions having a second optical state on the substrate as a bulk characteristic. More preferably, at least one set of local regions having a second optical state on the substrate is detected by counting the local regions within each set of local regions having a second optical state on the substrate.

[0073] A localized region with a second optical state may need to be above or below a threshold corresponding to the background signal, depending on the first and second optical states. For example, if the first optical state is non-fluorescent and the second optical state is fluorescent, the fluorescence level of the second optical state may need to exceed a threshold before detection. This can eliminate any interference from the sample's autofluorescence. Alternatively, if the first optical state is fluorescent and the second optical state is non-fluorescent, the fluorescence level of the second optical state may need to be below a threshold before detection.

[0074] Local regions on the substrate having a second optical state are typically individual regions on the substrate. However, some local regions may be excluded from detection due to their morphology. Local regions corresponding to individual binding events tend to be uniform and circular, but some local regions may be irregular in shape and may represent artifacts. Furthermore, some local regions may be larger than others if particles are aggregated together, or smaller if only transient binding events occur. Therefore, in a preferred embodiment, only uniform and circular local regions on the substrate having a second optical state are detected.

[0075] At least one set of localized regions on a substrate can be detected using simple optical means. In a preferred embodiment, at least one set of localized regions having a second optical state on the substrate is detected using an optical microscope. A suitable optical setup for detection is shown in Figure 6. More preferably, at least one set of localized regions having a second optical state on the substrate is detected using wide-field microscopy. Wide-field microscopy is the simplest form of microscopy, which allows the entire sample to be illuminated and imaged simultaneously, compared to more complex techniques such as confocal microscopy, where only a single focal point is illuminated and recorded at a time. The advantages of confocal microscopy are increased contrast by eliminating out-of-focus haze and the ability to obtain image stacking through the depths of the sample. Super-resolution microscopy techniques, such as photoactivated localization microscopy (PALM or FPALM) and stochastic optical reconstruction microscopy (STORM), are also known. These methods are more complex and costly than simple wide-field methods.

[0076] To detect at least one set of localized regions on a substrate, a wide-field fluorescence microscope equipped with a light source (e.g., an LED) and a photodetector (e.g., a camera such as a CCD) can be used with excitation and emission filters suitable for detecting specific optical components.

[0077] If step (iv) is carried out, step (iii) is repeated in the same manner as the irradiation step (iii), except that electromagnetic radiation of the same or different wavelengths is used. A preferred embodiment of step (iii) also applies to the irradiation in step (iv).

[0078] In a preferred embodiment, an optional step (iv) of the method of the present invention includes repeating step (iii) using electromagnetic radiation of the same wavelength. This provides a simpler method for detecting and identifying two or more analytes in a sample than the common method of measuring two or more analytes in a sample in a homogeneous assay format using electromagnetic radiation of different wavelengths. This method can be used when two or more reporter reagents differ from each other in size, gas permeability, photosensitizer reactivity, amount of photosensitizer, or a combination thereof. In this case, the electromagnetic radiation used in step (iii) and the optional step (iv) corresponds to the excitation wavelengths of the photosensitizers of the two or more reporter reagents. In this embodiment, two or more sets of local regions are distinguishable from each other by the rate of formation of the set of local regions, or by the size of the local regions in combination with the rate of formation of the set of local regions.

[0079] Preferably, an optional step (iv) of the method of the present invention includes repeating step (iii) with electromagnetic radiation of the same wavelength for a longer duration and / or at a higher intensity. This subsequently forms a set of local regions that are formed at a slower rate.

[0080] In an alternative preferred embodiment, an optional step (iv) of the method of the present invention comprises repeating step (iii) using electromagnetic radiation of different wavelengths. This method is used when two or more reporter reagents are optionally different from each other by the excitation wavelength of the photosensitizer in combination with their size. In this case, the electromagnetic radiation used in step (iii) and the optional step (iv) corresponds to the excitation wavelengths of the photosensitizers of the two or more reporter reagents. In this embodiment, the local regions of two or more sets are distinguishable from each other by the formation order of the sets of local regions, or by the size of the local regions in combination with the formation order of the sets of local regions.

[0081] Step (v) of the method of the present invention includes, if step (iv) is performed, detecting at least one set of local regions having a second optical state on the substrate formed in step (iv), or, if step (iv) is not performed, detecting two or more sets of local regions having a second optical state on the substrate formed in step (iii), wherein the two or more sets of local regions are distinguishable from each other by the formation rate of the set of local regions, the formation order of the set of local regions, or the size of the local regions in an optional combination of the formation rate of the set of local regions or the formation order of the set of local regions.

[0082] Therefore, if step (iv) is not performed, step (iii) irradiates the device with electromagnetic radiation of one wavelength for absorption by the respective photosensitizers of two or more reporter reagents, and as a result, the respective photosensitizers of the two or more reporter reagents bound to the surface of the substrate absorb the electromagnetic radiation and interact with the optical components, changing the optical components from a first optical state to a second optical state, thereby forming two or more sets of local regions on the substrate having the second optical state. In this example, the set of local regions differs in size.

[0083] Step (v) is carried out in a manner similar to the detection in the optional step (iv). Preferred embodiments of detection in the optional step (iv) are also applicable to step (v).

[0084] In the method of the present invention, two or more analytes are detected in the sample, and as a result, two or more sets of local regions having a second optical state are formed on the substrate. The two or more sets of local regions can be distinguished from each other by the formation rate of the sets of local regions, the formation order of the sets of local regions, or the size of the local regions, which is optionally combined with the formation rate or formation order of the sets of local regions.

[0085] In other words, two sets of local regions can be distinguished from each other by the rate of formation of the sets of local regions, the order in which the sets of local regions are formed, or the size of the local regions. Furthermore, if two sets of local regions can be distinguished from each other by the size of the local regions, then the two sets of local regions can optionally be further distinguished from each other by the rate of formation of the sets of local regions or the order in which the sets of local regions are formed. That is, two sets of local regions can be distinguished from each other by the size of the local regions and the rate of formation of the sets of local regions. Or, two sets of local regions can be distinguished from each other by the size of the local regions and the order in which the sets of local regions are formed.

[0086] The difference between formation rate and formation order lies in the fact that when two sets of local regions can be distinguished from each other by their formation rate, using excitation at the same wavelength will always result in the formation of the two sets of local regions in the same order. On the other hand, when two sets of local regions can be distinguished from each other by their formation order, the formation order of the two sets of local regions can be switched by changing the order in which two different excitation wavelengths are used.

[0087] The formation rate of a set of local regions refers to the length of time it takes for a set of local regions to form to a predetermined size using electromagnetic radiation of a certain wavelength.

[0088] In a preferred embodiment, at least two of the two or more sets of local regions can be distinguished from one another by the rate at which the sets of local regions are formed. Preferably, two or more sets of local regions can be distinguished from one another by the rate at which the sets of local regions are formed. This is a clear difference, and the concentration of one analyte can be calculated by counting the number of local regions in the set of local regions after a first irradiation step, and the concentration of another analyte can be calculated by counting the number of local regions in the set of local regions after a second irradiation step using the same wavelength. This provides a simple method for detecting and distinguishing two or more analytes in a sample.

[0089] In contrast to typical methods for measuring two or more analytes in a sample using homogeneous assays, the method of the present invention therefore does not require the use of electromagnetic radiation of different wavelengths to detect two or more analytes in a sample. This further simplifies the detection and identification of two or more analytes in a sample.

[0090] The formation sequence of local region sets means that a set of local regions is formed sequentially in response to irradiating the device with different excitation wavelengths of the photosensitizer.

[0091] In a preferred embodiment, at least two of the two or more sets of local regions can be distinguished from one another by the order in which the sets of local regions were formed. Preferably, the two or more sets of local regions can be distinguished from one another by the order in which the sets of local regions were formed. Again, and this is also a clear difference, the concentration of one analyte can be calculated by counting the number of local regions in the set of local regions formed after the first irradiation step, and the concentration of another analyte can be calculated by counting the total number of local regions in the set of local regions formed after the second irradiation step using a different wavelength and subtracting the number of local regions in the set of local regions formed after the first irradiation step. This provides a simple method for detecting and distinguishing two or more analytes in a sample.

[0092] The size of the local region means the size of the local region at a given time, or the maximum size that can be achieved, preferably the maximum size that can be achieved.

[0093] In a set of local regions of an optical component having a second optical state on the substrate, each local region is the same size, and a local region in one set of local regions should be significantly larger or smaller than a local region in another set of local regions. Preferably, in a set of local regions of an optical component having a second optical state on the substrate, each local region is the same size.

[0094] As described above, the local regions corresponding to individual binding events tend to be uniform and circular. Therefore, two or more sets of local regions can preferably be distinguished from each other by the diameter of the local regions.

[0095] In a preferred embodiment, at least two of the two or more sets of local regions can be distinguished from each other by the size of the local regions. Preferably, two or more sets of local regions can be distinguished from each other by the size of the local regions. Again, and this is also a clear difference, the concentrations of two or more analytes can be easily calculated by counting the number of local regions in each set of local regions. This provides a simple method for detecting and identifying two or more analytes in a sample.

[0096] In another preferred embodiment, two or more sets of local regions are preferably distinguishable from one another by the size of the local regions and the rate at which the sets of local regions are formed.

[0097] Alternatively, two or more sets of local regions are preferably distinguishable from each other by the size of the local regions and the order in which the sets of local regions are formed.

[0098] By using two or more different reporter reagents, two or more sets of local regions can be distinguished from each other by the formation rate of the local region set, the formation order of the local region set, or the size of the local region set, which is an optional combination of the formation rate or formation order of the local region set.

[0099] Two or more reporter reagents have different physicochemical properties. There are many physicochemical properties that can be modified so that two or more sets of local regions are distinguishable from each other by the rate of formation of the sets of local regions, the order in which the sets of local regions are formed, or the size of the sets of local regions, which is optionally combined with the rate of formation of the sets of local regions or the order in which the sets of local regions are formed. These physicochemical properties include size, material properties (e.g., gas permeability), type of photosensitizer (e.g., wavelengths that can be activated, reactivity of the photosensitizer), and / or amount of photosensitizer.

[0100] In a preferred embodiment, two or more reporter reagents differ from each other in terms of size, gas permeability, excitation wavelength of the photosensitizer, reactivity of the photosensitizer, amount of the photosensitizer, or a combination thereof. More preferably, two or more reporter reagents differ from each other in terms of size, gas permeability, excitation wavelength of the photosensitizer, amount of the photosensitizer, or a combination thereof. Most preferably, two or more reporter reagents differ from each other in size and / or excitation wavelength of the photosensitizer.

[0101] Preferably, at least two of the two or more reporter reagents are of different sizes. When the effect of the photosensitizer on the optical component is limited to a certain distance from the reporter reagent, a larger reporter reagent can interact with the optical component at a greater distance from the center of the reporter reagent. Furthermore, a larger size allows for the association of a larger amount of photosensitizer with the reporter reagent, increasing the activity of the reporter reagent. This increased activity facilitates the interaction between the photosensitizer and the optical component of the reporter reagent, allowing for the more rapid formation of a set of local regions, which are also larger in size. Therefore, preferably, at least two of the two or more sets of local regions are distinguishable from each other by the rate of formation of the set of local regions and the size of the local regions when selectively combined. Preferably, the two or more reporter reagents are of different sizes.

[0102] Preferably, at least two of the two or more reporter reagents differ from each other in terms of gas permeability. For example, higher gas permeability promotes faster interaction between the photosensitizer and optical component of the reporter reagent, leading to faster formation of sets of local regions and / or sets of local regions having larger sizes. Thus, at least two of the two or more sets of local regions are distinguishable from each other by the rate of formation of the sets of local regions, or by the size of the local regions, optionally combined with the rate of formation of the sets of local regions. Preferably, the two or more reporter reagents differ from each other in terms of gas permeability.

[0103] Preferably, at least two of the two or more reporter reagents differ from each other depending on the excitation wavelength of the photosensitizer. Therefore, at least two of the two or more sets of local regions are distinguishable from each other by the formation order of the sets of local regions. Preferably, the two or more reporter reagents differ from each other depending on the excitation wavelength of the photosensitizer.

[0104] This can be illustrated in Figures 7 to 11. Figure 7 shows a device in which two reporter reagents containing photosensitizers stimulated by radiation of different wavelengths are bound to the surface of a substrate before irradiation. Photosensitizer-labeled antibody 3 is bound to the surface of the substrate by antibody 2a, and photosensitizer-labeled antibody 5 is bound to the surface of the substrate by antibody 2b. Photosensitizer-labeled antibody 3 and photosensitizer-labeled antibody 5 act as reporter reagents.

[0105] Figure 8 shows the device of Figure 7 being irradiated with electromagnetic radiation of a single wavelength, for example, 680 nm. The light source may be, for example, an LED 15a. The light source irradiates the sample chamber 13 with light of an appropriate wavelength, exciting the photosensitizer 1a of the photosensitizer-labeled antibody 3.

[0106] Figure 9 shows the device from Figure 8 after irradiation. The photosensitizer 1a of the photosensitizer-labeled antibody 3 interacts with the dye optical component of the streptavidin-dye conjugate 10, changing the dye from a fluorescent state to a non-fluorescent state. The streptavidin-dye conjugate 10 in the fluorescent state becomes the streptavidin-dye conjugate 11 in the non-fluorescent state. Only the dye adjacent to the photosensitizer 1a changes from the first optical state to the second optical state, forming a set of local regions of optical components having the second optical state on the substrate.

[0107] Next, this set of local regions can be detected before irradiating the device with electromagnetic radiation of different wavelengths for absorption by photosensitizer 1b of the photosensitizer-labeled antibody 5.

[0108] Figure 10 shows the device of Figure 9 being irradiated with electromagnetic radiation of a different wavelength, for example, 350 nm. The light source may be, for example, LED 15b. The light source irradiates the sample chamber 13 with light of the appropriate wavelength, exciting the photosensitizer 1b of the photosensitizer-labeled antibody 5.

[0109] Figure 11 shows the device from Figure 10 after irradiation. The photosensitizer 1b of the photosensitizer-labeled antibody 5 interacts with the dye optical component of the streptavidin-dye conjugate 10, changing the dye from a fluorescent state to a non-fluorescent state. The streptavidin-dye conjugate 10 in the fluorescent state becomes the streptavidin-dye conjugate 11 in the non-fluorescent state. Only the dye adjacent to the photosensitizer 1b changes from the first optical state to the second optical state, forming an additional set of local regions of the optical component having the second optical state on the substrate.

[0110] Subsequently, an additional set of these local regions can be detected. Thus, the two sets of local regions are distinguishable from each other by the order in which the sets of local regions were formed.

[0111] Preferably, at least two of the two or more reporter reagents differ from each other due to the reactivity of their photosensitizers. For example, the photosensitizer of one reporter reagent may absorb more electromagnetic radiation and / or interact more strongly with the optical components than the photosensitizer of another reporter reagent, potentially leading to faster formation of sets of local regions and / or sets of local regions having larger sizes. Thus, at least two of the two or more sets of local regions are distinguishable from each other by the rate of formation of the sets of local regions, or by the size of the local regions in an optionally selective combination of the rate of formation of the sets of local regions. Preferably, the two or more reporter reagents differ from each other due to the reactivity of their photosensitizers.

[0112] Preferably, at least two of the two or more reporter reagents differ from each other in terms of the amount of photosensitizer. For example, a reporter reagent containing a larger amount of photosensitizer absorbs more electromagnetic radiation and interacts more with the optical component than another reporter reagent containing a smaller amount of photosensitizer, resulting in the faster formation of a set of local regions and / or a set of local regions having larger local regions. Thus, at least two of the two or more sets of local regions are distinguishable from each other by the rate of formation of the set of local regions, or by the size of the local regions in an optional combination of the rate of formation of the set of local regions. Preferably, the two or more reporter reagents differ from each other in terms of the amount of photosensitizer.

[0113] Two or more reporter reagents may differ from each other by one or more physicochemical properties. For example, four analytes in a sample may be detected using two reporter reagents of different sizes and two reporter reagents with different photosensitizer excitation wavelengths, and the four sets of local regions formed may be distinguishable from each other by the size of the local regions and the order in which the sets of local regions are formed.

[0114] In a preferred embodiment, the method of the present invention is (i) A step of providing a device with a mixture comprising a sample and two or more reporter reagents, wherein each of the two or more reporter reagents comprises a photosensitizer, and the device comprises a substrate having an optical component and two or more binding components, wherein the optical component and the two or more binding components are bonded to the surface of the substrate; (ii) A step that enables a portion of each of two or more reporter reagents to bind to the surface of a substrate in proportion to the concentration of the corresponding analyte by the corresponding binding component; (iii) Irradiating the device with electromagnetic radiation of one wavelength for absorption by the photosensitizers of two or more reporter reagents, so that the photosensitizers of the two or more reporter reagents bonded to the surface of the substrate absorb the electromagnetic radiation and interact with the optical components, causing the optical components to change from a first optical state to a second optical state, thereby forming two or more local regions of optical components having the second optical state on the substrate; and (iv) A step of detecting two or more localized regions having a second optical state on the substrate. Includes, Two or more sets of local regions are distinguishable from each other by the size of the local region.

[0115] In this embodiment, two or more reporter reagents are preferably different from each other in terms of size, gas permeability, photosensitizer reactivity, amount of photosensitizer, or a combination thereof.

[0116] In another preferred embodiment, the method of the present invention is (i) A step of providing a device with a mixture comprising a sample and two or more reporter reagents, wherein each of the two or more reporter reagents comprises a photosensitizer, and the device comprises a substrate having an optical component and two or more binding components, wherein the optical component and the two or more binding components are bonded to the surface of the substrate; (ii) A step that enables a portion of each of two or more reporter reagents to bind to the surface of a substrate in proportion to the concentration of the corresponding analyte by the corresponding binding component; (iii) Irradiating the device with electromagnetic radiation of one wavelength for absorption by at least one of two or more reporter reagent photosensitizers, so that at least one of the two or more reporter reagent photosensitizers bonded to the surface of the substrate absorbs the electromagnetic radiation and interacts with the optical components to change the optical components from a first optical state to a second optical state, thereby forming a local region of at least one set of optical components having the second optical state on the substrate; (iv) detecting at least one set of local regions of optical components having a second optical state on the substrate formed in step (iii), repeating step (iii) with electromagnetic radiation of the same or different wavelengths for absorption by at least one of two or more reporter reagent photosensitizers, so that at least one of the two or more reporter reagent photosensitizers bonded to the surface of the substrate absorbs the electromagnetic radiation and interacts with the optical components, changing the optical components from a first optical state to a second optical state, thereby forming at least one set of local regions of optical components having a second optical state on the substrate; and (v) A step of detecting a local region of at least one set of optical components having a second optical state on the substrate formed in step (iv). Includes, Two or more sets of local regions can be distinguished from each other by the formation rate of the sets of local regions, the formation order of the sets of local regions, or the size of the local regions in combination with the formation rate or formation order of the sets of local regions.

[0117] In this embodiment, two or more reporter reagents are preferably different from each other in terms of size, gas permeability, excitation wavelength of the photosensitizer, reactivity of the photosensitizer, amount of the photosensitizer, or a combination thereof.

[0118] In a preferred embodiment, the method of the present invention is (i) A step of providing a device with a mixture comprising a sample and two or more reporter reagents, wherein each of the two or more reporter reagents comprises a photosensitizer, and the device comprises a substrate having an optical component and two or more binding components, wherein the optical component and the two or more binding components are bonded to the surface of the substrate; (ii) A step that enables a portion of each of two or more reporter reagents to bind to the surface of a substrate in proportion to the concentration of the corresponding analyte by the corresponding binding component; (iii) Irradiating the device with electromagnetic radiation of one wavelength for absorption by at least one of two or more reporter reagent photosensitizers, so that at least one of the two or more reporter reagent photosensitizers bonded to the surface of the substrate absorbs the electromagnetic radiation and interacts with the optical components to change the optical components from a first optical state to a second optical state, thereby forming a local region of at least one set of optical components having the second optical state on the substrate; (iv) detecting at least one set of local regions of optical components having a second optical state on the substrate formed in step (iii), repeating step (iii) with electromagnetic radiation of the same wavelength for absorption by at least one of two or more reporter reagent photosensitizers, so that at least one of the two or more reporter reagent photosensitizers bound to the surface of the substrate absorbs the electromagnetic radiation and interacts with the optical components to change the optical components from a first optical state to a second optical state, thereby forming at least one set of local regions of optical components having a second optical state on the substrate; and (v) A step of detecting a local region of at least one set of optical components having a second optical state on the substrate formed in step (iv). Includes, Two or more sets of local regions can be distinguished from one another by the formation rate of the set of local regions, or by the size of the local regions in combination with the formation rate of the set of local regions.

[0119] In this embodiment, two or more reporter reagents are preferably different from each other in terms of size, gas permeability, photosensitizer reactivity, amount of photosensitizer, or a combination thereof.

[0120] In another preferred embodiment, the method of the present invention is (i) A step of providing a device with a mixture comprising a sample and two or more reporter reagents, wherein each of the two or more reporter reagents comprises a photosensitizer, and the device comprises a substrate having an optical component and two or more binding components, wherein the optical component and the two or more binding components are bonded to the surface of the substrate; (ii) A step that enables a portion of each of two or more reporter reagents to bind to the surface of a substrate in proportion to the concentration of the corresponding analyte by the corresponding binding component; (iii) Irradiating the device with electromagnetic radiation of one wavelength for absorption by at least one of two or more reporter reagent photosensitizers, thereby causing at least one of the two or more reporter reagent photosensitizers bonded to the surface of the substrate to absorb the electromagnetic radiation and interact with the optical components to change the optical components from a first optical state to a second optical state, thereby forming a local region of at least one set of optical components having the second optical state on the substrate; (iv) detecting at least one set of local regions of optical components having a second optical state on the substrate formed in step (iii), repeating step (iii) with electromagnetic radiation of different wavelengths for absorption by at least one of two or more reporter reagent photosensitizers, so that at least one of the two or more reporter reagent photosensitizers bound to the surface of the substrate absorbs the electromagnetic radiation and interacts with the optical components to change the optical components from a first optical state to a second optical state, thereby forming at least one set of local regions of optical components having a second optical state on the substrate; and (v) A step of detecting a local region of at least one set of optical components having a second optical state on the substrate formed in step (iv). Includes, Two or more sets of local regions are distinguishable from each other by the formation order of the sets of local regions, or by the size of the local regions in combination with the formation order of the sets of local regions.

[0121] In this embodiment, two or more reporter reagents are preferably different from each other by their size and the excitation wavelength of the photosensitizer, which can be optionally selected.

[0122] In a preferred embodiment, two or more binding components are bound to the same region on the surface of the substrate. The two or more analytes are identified by the rate of formation of sets of local regions, the order of formation of sets of local regions, or the size of local regions in a choice of combinations of the rate of formation of sets of local regions or the order of formation of sets of local regions. Thus, in contrast to typical methods for measuring two or more analytes in a sample in a homogeneous assay format, it is not necessary to separate the two or more binding components on the surface of the substrate. Furthermore, since the method of the present invention uses a single substrate, a small sample volume is required in multiplexed assays. This provides a simple method for detecting and identifying two or more analytes in a sample.

[0123] In an alternative, preferred embodiment, two or more binding components are bound to separate regions on the surface of the substrate. Each of these separate regions contains only a specific type of binding component. Thus, only one reporter reagent binds to each separate region in the presence of the corresponding analyte, and therefore facilitates the binding of a specific reporter reagent to a specific binding component. When these regions are investigated individually, in principle, it is not necessary to distinguish between sets of local regions. The concentration of a particular analyte may be calculated from the set of local regions formed on the separate regions. However, the fact that there is a distinguishable set of local regions means that specific binding of the reporter reagent that is expected to bind and nonspecific binding of the reporter reagent that is undesirable can be distinguished. Nonspecific binding of the reporter reagent is common to all immunoassays and is often corrected by performing control measurements to monitor the level of nonspecific binding, as will be described in more detail below. In this particular embodiment, as shown in Examples 3 and 4, the separate regions can act as negative controls for each other. These negative controls can correct for variability between different samples. This reduction in interference and improvement in accuracy represent a significant advantage over multiplexed assays that identify binding only by adding all the various reporter reagents to a single sample and varying the binding component depending on the location.

[0124] Each photosensitizer of two or more reporter reagents bonded to the surface of a substrate can either directly interact with the optical component to induce a change (e.g., the photosensitizer is excited and directly transfers this energy to the optical component), or it can indirectly interact with the optical component to induce a change via an additional reagent (e.g., the photosensitizer is excited, transfers this energy to an additional component, and then transfers this energy back to the optical component).

[0125] In a preferred embodiment, each photosensitizer of two or more reporter reagents absorbs electromagnetic radiation and interacts with a preactivator reagent present in the mixture to generate an activator reagent. The activator reagent then interacts with the optical component, changing it from a first optical state to a second optical state. Thus, two or more reporter reagents can generate an activator reagent from a preactivator reagent present in the mixture when absorbing electromagnetic radiation, and the optical component can be changed from a first optical state to a second optical state through interaction with the activator reagent.

[0126] The preactivator reagent may be present in the sample, or it may be added as an additional reagent to a mixture of the sample and two or more reporter reagents. In a preferred embodiment, the preactivator reagent is ground-state triplet oxygen. In another preferred embodiment, the activator reagent is a reactive oxygen species (ROS). Preferably, the ROS is selected from hydroxyl radicals, superoxides, peroxides, organic peroxides, peroxynitrites, singlet oxygen, and mixtures thereof. More preferably, the ROS is singlet oxygen. Singlet oxygen is a preferred activator reagent because it has a short half-life and a limited diffusion path length (typically less than 1 micron under aqueous conditions).

[0127] The photosensitizer absorbs light and enters an excited state, which is thought to undergo intersystem cross-reaction (ISC) with oxygen present in the sample and in close proximity to the reporter reagent, potentially generating singlet oxygen. Subsequently, the singlet oxygen interacts with the optical components, as described below. In a particularly preferred embodiment, the preactivator reagent is ground-state triplet oxygen, and the activator reagent is singlet oxygen.

[0128] Singlet oxygen has been used in immunoassays, such as the luminescent oxygen channeling immunoassay (LOCI). The LOCI immunoassay is a homogeneous, non-digital assay that uses donor beads and acceptor beads to measure bulk phenomena. Donor beads generate singlet oxygen upon irradiation at 680 nm, and acceptor beads produce a chemiluminescent signal when activated by singlet oxygen. Binding of donor beads to acceptor beads is facilitated by antibody-antigen binding. The reaction mixture is typically irradiated for 0.5 to 1.0 seconds, and then the luminescence signal is measured for 0.5 to 1.0 seconds. Importantly, the measurement is performed in the presence of all unbound donor and acceptor beads. Spatial separation of the beads minimizes background signal, but due to the short measurement time, the LOCI assay cannot distinguish between persistent and transient binding events. This assay can achieve a detection limit of approximately 1 to 5 pg / mL in its most sensitive assays, such as those for interleukin-6 (IL-6) or thyroid-stimulating hormone (TSH). The method of the present invention further minimizes background signal because, for a signal to be detected, the "donor" particle, the reporter reagent, must be in close proximity to the surface of the substrate 11, rather than to particles in solution, and must be present for the duration of the irradiation to generate a signal higher (or lower) than the threshold criteria used in the image analysis software. Therefore, the method of the present invention is more sensitive than the LOCI assay and can detect lower concentrations of analytes.

[0129] In a preferred embodiment, the optical component is a dye. Preferably, the optical component is selected from any of the following fluorescent dyes and mixtures thereof:

[0130] [ka]

[0131] Dyes (1), (2), and (3) are common organic fluorophores that can attach to proteins and other macromolecules via attached N-hydroxysuccinimide groups, which react with amine groups to form covalent amide bonds. These dyes, along with a wide range of other fluorophores, may be irreversibly converted to a non-fluorescent state by the methods of the present invention.

[0132] Preferably, the optical component is fluorescent when in either the first or second optical state, and non-fluorescent when in the other of the two optical states. In one embodiment, the optical component in the first optical state is fluorescent and the optical component in the second optical state is non-fluorescent, or the optical component in the first optical state is non-fluorescent and the optical component in the second optical state is fluorescent. However, more preferably, the optical component in the first optical state is fluorescent and the optical component in the second optical state is non-fluorescent.

[0133] Preferably, the change from the first optical state to the second optical state is irreversible. This allows the substrate to be subsequently scanned to identify the region where the change in optical state has occurred.

[0134] The assay requires the presence of two or more binding components, and the number of these binding components corresponds to the number of analytes being detected.

[0135] Two or more binding components have binding sites that allow them to bind the corresponding reporter reagent in proportion to the concentration of the corresponding analyte in the sample. This proportionality is important for the function of the assay, as binding must depend on the concentration of the analyte for any meaningful measurement of the concentration of the analyte to be determined. Depending on the type of assay being performed, binding may be directly proportional to or inversely proportional to the concentration of the analyte. In non-competitive assays, such as immunoassays, binding is directly proportional to the concentration of the analyte, while in competitive assays, binding is inversely proportional to the concentration of the analyte.

[0136] A specific type of competitive assay is presented in which an antibody against an analyte is immobilized on a substrate, and a labeled analog of the analyte is introduced into the sample. The analyte and its labeled analog then "compete" for the antibody on the surface. In the absence of the analyte, the labeled analog binds at the maximum possible rate. However, in the presence of the analyte, the antibody on the substrate becomes saturated with the analyte, and the binding rate of the analog decreases.

[0137] Two or more binding components can be adapted to bind to the corresponding analyte, or a complex or derivative of the corresponding analyte, in which case the two or more reporter reagents bind to the corresponding binding components in the presence of the corresponding analyte, or a complex or derivative of the corresponding analyte. In this case, the two or more binding components have binding sites that are capable of binding to the corresponding reporter reagent in the presence of the corresponding analyte or a complex or derivative of the corresponding analyte. However, the binding is still proportional to the concentration of the corresponding analyte.

[0138] Alternatively, the two or more binding components themselves may be analogs of the corresponding analytes, and the corresponding reporter reagent binds directly to the two or more binding components (these are analogs because they are bound to the surface of the substrate by either covalent or non-covalent interactions). In this case, the two or more binding components compete with the unbound analyte, or a complex or derivative of the unbound analyte, for binding to the corresponding reporter reagent. Therefore, the two or more binding components can easily bind to the corresponding reporter reagent.

[0139] By determining the degree of binding (either directly or mediated by the corresponding analyte / or a complex or derivative of the corresponding analyte) of two or more reporter reagents to their corresponding binding components, a measurement of the concentration of the corresponding analyte in the sample is obtained. It is customary for the system to be pre-calibrated at the time of manufacture and to generate a calibration curve used to convert the instrument signal to the concentration of the analyte measured in the sample.

[0140] The assay also requires the presence of two or more reporter reagents, and the number of reporter reagents corresponds to the number of analytes to be detected.

[0141] Each of the two or more reporter reagents contains a photosensitizer. The photosensitizer absorbs electromagnetic radiation, enabling it to interact with the optical component. This interaction changes the optical component from a first optical state to a second optical state.

[0142] Therefore, photosensitizers can be composed of any material capable of interacting with electromagnetic radiation in this manner. Suitable photosensitizers are known as PDT reagents in photodynamic therapy (PDT). PDT reagents are used in cancer therapy and dermatology to destroy cells upon irradiation (Shafirstein et al., Cancers, 2017, 9, 12; Wan and Lin, Clinical, Cosmetic and Investigational Dermatology, 2014, 7, 145).

[0143] Upon irradiation with electromagnetic radiation, the PDT reagent is promoted to an excited triplet state. This excited triplet state can directly interact with cellular components, which is called the type I process, or it can interact with oxygen, which is called the type II process. Both type I and type II processes can lead to the formation of ROS. In the type II process, the main product is singlet oxygen via an intersystem cross-reaction mechanism.

[0144] Singlet oxygen is a highly reactive excited state of oxygen. Before decaying, it can undergo numerous reactions, including the Diels-Alder reaction and the Ehn reaction. It also undergoes common oxidation reactions with sulfur and nitrogen-containing compounds. This indiscriminate reactivity of singlet oxygen is one reason it is used in photodynamic therapy.

[0145] A wide range of photosensitizer compounds are known, including porphyrins, chlorins (e.g., pyropheophorbid-a), phthalocyanines, and other polycyclic aromatic species (see, e.g., Antibody-Directed Phototherapy, Pye et al., Antibodies, 2013, 2, 270).

[0146] In one embodiment, the photosensitizer is selected from porphyrins, chlorins, phthalocyanines, and other polycyclic aromatic species. In a preferred embodiment, the photosensitizer is, for example, a silicon phthalocyanine derivative as shown below:

[0147] [ka]

[0148] The properties of the binding components and reporter reagents depend on the properties of the analyte, but they preferably include antibodies. The method of the present invention is particularly applicable in immunoassays. In a particularly preferred embodiment, two or more binding components are antibodies produced against the corresponding analyte or a complex or derivative of the corresponding analyte, and two or more reporter reagents include antibodies produced against the corresponding analyte or a complex or derivative of the corresponding analyte. In principle, a single molecule may be used for each reagent, but in practice, the binding components and reporter reagents are a collection of molecules. The term "antibody" preferably includes Fab fragments, single-chain variable fragments (scFv), and recombinant binding fragments within that range.

[0149] As an alternative to antibody-antigen reactions, the binding component, reporter reagent, and analyte may be a first nucleic acid and a second nucleic acid 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, or vice versa. The binding component and reporter reagent may also be aptamers. This system is not limited to biological assays and can also be applied, for example, to the detection of heavy metals in water. This system is also not limited to liquids and any fluid system may be used, for example, to detect enzymes, cells, and viruses in air.

[0150] The photosensitizer may be located inside or outside each of two or more reporter reagents. Preferably, each of the two or more reporter reagents contains polymer particles, and the photosensitizer is encapsulated within the polymer particles. Alternatively, each of the two or more reporter reagents further contains polymer particles, and the photosensitizer is coated onto the surface of the polymer particles. Suitable polymer particles generally include latex particles made from polystyrene or polystyrene copolymers. These polymer particles may contain functional groups on their surface, such as carboxyl groups, which can be used to form covalent bonds. Such polymer particles swell in a nonpolar solvent, allowing for the injection / encapsulation of hydrophobic organic molecules.

[0151] More preferably, each of the two or more reporter reagents further comprises polymer particles and one or more binding domains, the photosensitizer is encapsulated within the polymer particles and the one or more binding domains are coated onto the polymer particles. Alternatively, each of the two or more reporter reagents further comprises polymer particles and one or more binding domains, the photosensitizer and the one or more binding domains are co-coated onto the polymer particles. The binding domains may be antibodies or nucleic acids, etc., as described above, depending on the properties of the analyte. However, preferably, the binding domains are antibodies.

[0152] The maximum observable signal is the maximum signal that can be achieved when monitoring the photosensitizer bound to the surface. The binding of particles to the substrate is determined by the diffusion rates of the analyte and reporter reagent, which is then primarily determined by the hydrodynamic radius of these components and the viscosity / temperature of the sample.

[0153] The device used in the method of the present invention may further include controls to compensate for natural variations in the components of the measurement system, variations in the sample being measured, and variations in environmental conditions during measurement. This can be achieved by exposing the sample to reagents on the surface of a substrate. Different reagents are typically placed in different areas on the surface of the substrate, and these areas are coated with different reagents. These controls are defined as “negative” controls and “positive” controls in the sense that a negative control should approximate the signal expected in the absence of the analyte, and a positive control should approximate the signal expected when the analyte saturates the system.

[0154] To achieve detection using these controls, the device of the present invention preferably includes two or more binding components together with negative and positive control reagents for those two or more binding components, each of which is bound to the surface of the substrate as described above. The negative and positive control reagents may be a single negative and a single positive control reagent for all of the two or more binding components, or each of the two or more binding components may have its own negative and positive control reagents.

[0155] However, if each of the two or more binding components is bound to a separate region on the surface of the substrate, a negative control reagent may not be necessary, as the separate regions can act as negative controls for each other, as described above and in Examples 3 and 4. Therefore, the device of the present invention preferably contains two or more binding components together with a positive control reagent, each of which is bound to the surface of the substrate as described above. The positive control reagent may be a single positive control reagent for all of the two or more binding components, or each of the two or more binding components may have its own positive control reagent.

[0156] The two or more binding components are as described above.

[0157] Under assay conditions, the negative control reagent has a lower affinity for two or more reporter reagents than the corresponding binding component. Therefore, the negative control reagent provides a negative control. It is important to consider affinity under assay conditions because, in non-competitive assays, the affinity of two or more binding components for the corresponding reporter reagent is mediated by the presence of the corresponding analyte or a complex or derivative of the corresponding analyte. Therefore, in the absence of the corresponding analyte or a complex or derivative of the corresponding analyte, neither the two or more binding components nor the negative control reagent have affinity for the corresponding reporter reagent. However, in the presence of the corresponding analyte or a complex or derivative of the corresponding analyte, the negative control reagent has a lower affinity for two or more reporter reagents than the corresponding binding component.

[0158] Furthermore, in embodiments in which two or more binding components bind to the corresponding analyte or a complex or derivative of the corresponding analyte, the negative control reagent preferably has a lower affinity for the corresponding analyte, or, if used, the complex or derivative of the corresponding analyte, than the two or more binding components. The negative control reagent is preferably a protein, and more preferably an antibody. The negative control reagent typically has similar chemical and physical properties to the two or more binding components, but provides little or no affinity to the corresponding reporter under the conditions of the assay. In a particularly preferred embodiment, the negative control reagent has essentially no affinity for the two or more reporter reagents under the conditions of the assay. Preferably, the negative control reagent provides essentially no affinity for the corresponding analyte or a complex or derivative of the corresponding analyte. That is, the binding of the two or more reporter reagents, or, where applicable, the corresponding analyte or complex or derivative of the corresponding analyte, to the negative control reagent is nonspecific. Thus, the negative control reagent can compensate for the nonspecific binding of the two or more reporter reagents to the corresponding binding components. In a particular embodiment, a software algorithm uses data from the negative control region as part of a calculation to obtain the concentration of the analyte.

[0159] A positive control reagent binds to two or more reporter reagents and has affinity for two or more reporter reagents whose concentration in the sample is less affected by the corresponding analyte, or, if used, a complex or derivative of the corresponding analyte, than that of the corresponding binding component, thus providing a positive control. Preferably, the positive control reagent has affinity for two or more reporter reagents that are essentially independent of the concentration of the corresponding analyte or a complex or derivative of the corresponding analyte. More preferably, under the conditions of the assay, the positive control reagent has higher affinity for two or more reporter reagents than for the corresponding binding component. In this way, the positive control reagent measures the maximum expected signal in the system. If the binding of the positive control reagent to two or more reporter reagents is mediated by a mechanism in which the positive control reagent recognizes all different types of reporter reagents, it is possible to measure the binding of all two or more reporter reagents in a single positive control region, and furthermore, it is possible to distinguish them from one another (i.e., it is possible to have a range of positive control measurement).

[0160] In both negative and positive control measurements, the software algorithm can detect abnormal binding patterns that may result in error messages and terminate the measurement process.

[0161] The above explanation allows the assay to be incubated for a certain period of time before activating the photosensitizer, but the dynamics of the binding event can be monitored by irradiating the photosensitizer at discontinuous intervals over time and monitoring the binding event that occurs throughout the reaction.

[0162] The present invention is used to simultaneously detect the presence of multiple analytes in the same sample. Sandwich assays and competitive assays may be performed in parallel, and these assays may use the same negative and positive controls as described above, or separate controls may exist for each analyte to be measured.

[0163] Two or more analytes are distinct from one another. The two or more analytes may be macromolecules and / or small molecules. Macromolecules are typically proteins, such as protein-based hormones, which may also be part of larger particles such as viruses, bacteria, cells (e.g., red blood cells), or prions. Small molecules may be drugs.

[0164] As used herein, the term “small molecule” is a term specific to this art and is used to distinguish it from macromolecules such as proteins and nucleic acids. Small molecules are often called “haptens” in the field of immunoassays and are small molecules that can induce an immune response when attached to larger carrier molecules such as proteins, and are also found in molecules such as hormones and synthetic drugs. This type of small molecule typically has a molecular weight of 2,000 or less, often 1,000 or less, and even 500 or less. Two or more binding components may be adapted to bind to the corresponding analyte itself, although the corresponding analyte may undergo a chemical reaction or an initial complexation event before binding to the two or more binding components. For example, two or more analytes may be protonated / deprotonated at the pH of the assay conditions. Thus, the two or more analytes bound to the corresponding binding components may be the analyte itself or derivatives of the analyte, both of which are within the scope of the present invention.

[0165] In addition to detecting different analytes, two or more analytes may also include different forms of the same analyte, depending on the specificity of the antibody. For example, proteins with post-translational modifications may contain different phosphorylation or glycation sites, and multiplexing can be used to detect different fragments of the target protein.

[0166] More than one reporter reagent can also be selected based on the concentrations of more than one analyte. For example, for low-abundance analytes, a small, high-affinity reporter reagent should be used to maximize the sensitivity of the assay. Under these conditions, the reporter reagent captures more of the analyte and diffuses rapidly onto the substrate surface, thereby maximizing the binding event and improving the signal-to-noise ratio. Conversely, for high-abundance analytes, a large, low-affinity reporter reagent should be used to prevent saturation. Larger reporter reagents have a larger surface area, resulting in slower diffusion onto the substrate surface and lower sensitivity, but allowing for measurement up to higher concentrations.

[0167] Samples suspected of containing the target analyte are generally fluid samples, such as liquid samples, and typically biological samples such as body fluids, e.g., blood, plasma, saliva, serum, intraocular fluid, cerebrospinal fluid, or urine. Samples may contain suspended particles or may be whole blood. In preferred embodiments, the sample is untreated, and more preferably, untreated fluid. Untreated means that the sample / fluid has not been pretreated by filtration, dilution, or other pretreatment steps before being mixed with the reporter reagent and other assay components. An advantage of the method of the present invention is that the assay can be performed on samples containing suspended particles without excessively affecting the assay results.

[0168] In a preferred embodiment, the sample is whole blood. It is surprising that the components of whole blood do not interfere with the detection method of the present invention. To measure the fluorescence of plasma or serum components of blood, it is common to remove red blood cells from the blood due to unpredictable scattering of light by different cellular components in each sample. However, in the method of the present invention, since fluorescence can be measured on a substrate using an imaging system with a shallow depth of field, and since individual binding events can be measured, the measurement can be performed in whole blood.

[0169] The sample is typically on the order of microliters (e.g., 1 to 100 μL, preferably 1 to 10 μL). To hold the fluid sample, the substrate is preferably placed in a sample chamber having one or more side walls, a top surface, and a bottom surface. Thus, the device used in the method of the present invention preferably further includes a chamber for holding the sample containing the analyte in contact with the substrate.

[0170] A potential further source of background interference is the sedimentation of suspended particles, including the reporter reagent and cellular components of the sample, onto the surface of the substrate. This interference source can be mitigated by placing the substrate on top of the bulk solution, for example, on the top surface of the reaction chamber. Therefore, any sedimentation that occurs will not interfere with the substrate. Preferably, the substrate forms a top surface as shown in the figure. Preferably, the substrate is substantially planar. More preferably, the substrate is planar. "Substantially planar" means that the substrate deviates from planarity only to the extent that it maintains its function in the invention, for example, so that the entire substrate is maintained within a single focal range during imaging. Obviously, the optical components and two or more binding components are located on the inner surface of the chamber to allow contact with the sample. This modification and other modifications are included within the scope of the invention.

[0171] The sample can be easily held, for example, by the surface tension inside the capillary channel.

[0172] Two or more reporter reagents and optionally one or more additional reagents are preferably stored in a chamber incorporated into the device used in the method of the present invention.

[0173] The method of the present invention is particularly useful for point-of-care (POC) testing. POC testing is defined as diagnostic testing performed in or near a clinical setting, i.e., bedside testing. POC testing enables convenient and rapid testing, allowing for improved decision-making and triage, and enabling better allocation of accident and emergency medical care, as well as hospital resources such as beds. This is in contrast to conventional testing, where samples are collected in the clinical setting and then sent to a laboratory for testing. Such testing often takes several hours or even days to obtain results, during which time patients must continue treatment without the desired information. POC testing often uses a test kit in combination with portable equipment.

[0174] The method of the present invention is particularly useful for monitoring the concentration or presence / absence of analytes, which are typically present in very low amounts. Potential applications include the measurement of biomarkers in cardiovascular disease (e.g., highly sensitive troponin), infectious diseases (e.g., hepatitis C core antigen), aging / dementia (e.g., Alzheimer's disease markers amyloid-beta and phosphorylated tau), cytokines, and oncology (e.g., circulating tumor markers).

[0175] The present invention also provides a device for detecting two or more analytes in a sample, wherein the device comprises a substrate having an optical component and two or more binding components, the optical component and two or more binding components adhering to the surface of the substrate, the optical component changing from a first optical state to a second optical state in response to interaction with a photosensitizer irradiated with two or more reporter reagents bound to the surface of the substrate in proportion to the concentration of the corresponding analytes in the sample, thereby forming two or more sets of local regions having the second optical state on the substrate, and the two or more sets of local regions being distinguishable from each other by the formation rate of the sets of local regions, the formation order of the sets of local regions, or the size of the local regions in an optionally selected combination of the formation rate of the sets of local regions or the formation order of the sets of local regions.

[0176] The features of this device are as described above with respect to the device used in the method of the present invention.

[0177] In a preferred embodiment, the device further includes a chamber for holding a mixture of the sample and two or more reporter reagents.

[0178] The device may include one or more radiation sources adapted to generate electromagnetic radiation, and a detector adapted to detect a second optical state, thereby enabling precise determination of the position of the photosensitizer relative to the substrate.

[0179] The device may take the form of a cartridge used with another reader. The reader may incorporate a radiation source and a detector. The reader is preferably a portable reader. Preferably, the device includes a cartridge in which a substrate is located, and the device further includes 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 containing a substrate and optical and bonding components as defined herein. The cartridge is preferably a disposable cartridge.

[0180] The present invention also provides a system for detecting two or more analytes in a sample, comprising the above-described device and two or more reporter reagents for forming a mixture containing the sample, each of the two or more reporter reagents comprising a photosensitizer, the photosensitizer absorbing electromagnetic radiation and interacting with an optical component, thereby changing the optical component from a first optical state to a second optical state.

[0181] Preferably, the photosensitizer can absorb electromagnetic radiation and interact with a preactivator reagent present in the mixture to generate an activator reagent, which in turn can interact with the optical component to change it from a first optical state to a second optical state.

[0182] In a preferred embodiment, the system of the present invention is essentially comprised of the above-described features. "Essentially" means that no other features are required to perform the assay.

[0183] The present invention will be described with reference to the following examples, but these examples are not intended to be limiting. These examples demonstrate that two or more reporter reagents bind to corresponding binding components via corresponding analytes. However, it should be understood that various assay forms are conceivable in which the binding of two or more reporter reagents to corresponding binding components is promoted or inhibited by the presence of corresponding analytes in solution. These examples are provided to demonstrate a mode of transformation of an optical component from a first optical state to a second optical state. [Examples]

[0184] Example 1

[0185] The measurement is performed in a single reaction well. The reaction well shown in Figure 12 can be prepared by taking a 20x20 mm piece of 175 micron thick polymethyl methacrylate (PMMA), attaching a 1 cm x 1 cm square piece of 100 μm thick pressure-sensitive adhesive (PSA) 16 with a 6 mm diameter hole to a cover glass 17, and creating a shallow well 18.

[0186] The surface is first coated with biotinylated BSA and then with Cy2-labeled streptavidin using methods known in the art. The reaction well is then simultaneously coated with two biotinylated capture antibodies, one against analyte A and the other against analyte B. The reaction chamber can then be prepared by removing the release liner from the PSA, inverting the substrate, and attaching it to a PMMA sheet 19 with two small holes 20 drilled into it, as shown in the profile of Figure 13.

[0187] The sample is mixed with two reporter reagents. The reporter reagent for analyte A contains 50 nm beads coated with an antibody against analyte A (photosensitizer-labeled antibody 3). The reporter reagent for analyte B contains 250 nm beads coated with an antibody against analyte B (photosensitizer-labeled antibody 4). Both reporter reagents are injected with the same photosensitizer stimulated at the same wavelength (680 nm). Photosensitizer-labeled antibody 3 generates a spot with a diameter of 400 nm, and photosensitizer-labeled antibody 4 generates a spot with a diameter of 1000 nm.

[0188] This mixture is added to the reaction well and incubated (to enable binding) to bind photosensitizer-labeled antibody 3 to the substrate surface by antibody 2a and photosensitizer-labeled antibody 4 to the substrate surface by antibody 2b, as shown in Figure 2. The sensor is then irradiated with 680 nm light to simultaneously activate both reporter reagents, as shown in Figure 3. Rows of large and small spots are formed on the surface, as shown in Figures 4 and 5. The surface is then imaged by 490 nm fluorescence excitation. The concentration of analyte A is calculated from the number of small spots, and the concentration of analyte B is calculated from the number of large spots.

[0189] Example 2

[0190] The measurement will be performed in one reaction well. The reaction well will be prepared in the same manner as in Example 1.

[0191] The sample is mixed with two types of reporter reagents. The reporter reagent for analyte A contains 50 nm beads coated with an antibody against analyte A (photosensitizer-labeled antibody 3). The reporter reagent for analyte B contains 50 nm beads coated with an antibody against analyte B (photosensitizer-labeled antibody 5). Photosensitizer-labeled antibody 3 is stimulated at 680 nm, and photosensitizer-labeled antibody 5 is stimulated at 350 nm.

[0192] This mixture is added to the reaction well and incubated to conjugate photosensitizer-labeled antibody 3 to the substrate surface by antibody 2a and photosensitizer-labeled antibody 5 to the substrate surface by antibody 2b (to enable conjugation), as shown in Figure 7. First, the sensor is irradiated with 680 nm light to generate spots as shown in Figures 8 and 9. These are then imaged and counted by 490 nm fluorescence excitation. Subsequently, the sensor is irradiated with 350 nm light to generate additional spots as shown in Figures 10 and 11. These additional spots are then counted by 490 nm fluorescence excitation.

[0193] The concentration of analyte A is calculated from the first series of spots formed, and the concentration of analyte B is calculated from the second series of spots formed (by subtracting the number of spots in the first series from the total number of spots).

[0194] Example 3

[0195] The measurements will be carried out in two reaction wells. The reaction wells will be prepared in the same manner as in Example 1.

[0196] Reaction well 1 is coated with an antibody against analyte A, and reaction well 2 is coated with an antibody against analyte B.

[0197] The sample is mixed simultaneously with two reporter reagents. The reporter reagent for analyte A contains 50 nm beads coated with an antibody against analyte A (photosensitizer-labeled antibody 3). The reporter reagent for analyte B contains 250 nm beads coated with an antibody against analyte B (photosensitizer-labeled antibody 4). Both reporter reagents are stimulated at the same wavelength (680 nm). Photosensitizer-labeled antibody 3 produces a spot with a diameter of 400 nm, and photosensitizer-labeled antibody 4 produces a spot with a diameter of 1000 nm.

[0198] This mixture is added to each well and incubated for 10 minutes to conjugate photosensitizer-labeled antibody 3 to the surface of the substrate in well 1 by antibody 2a, and photosensitizer-labeled antibody 4 to the surface of the substrate in well 2 by antibody 2b, as shown in Figure 14. After incubation (to allow conjugation in both wells), each well is stimulated with 680 nm light to generate dark spots in each well, as shown in Figures 15 and 16. These are imaged and counted by 490 nm fluorescence excitation.

[0199] In well 1, the concentration of analyte A is calculated by counting the number of small spots. Any large spots generated by nonspecific binding of reporter B are ignored. Similarly, in well 2, the concentration of analyte B is calculated by counting the number of large spots. Any small spots generated by nonspecific binding of reporter A are ignored.

[0200] Wells 1 and 2 act as negative controls for each other. In this regard, the large spots produced by nonspecific binding of reporter B in well 1 act as a negative control for the large spots produced by specific binding of reporter B in well 2. Subtracting the number of any large spots produced by nonspecific binding of reporter B in well 1 from the number of large spots produced by specific binding of reporter B in well 2 provides a more accurate calculation of the concentration of analyte B.

[0201] Similarly, any small spots generated by nonspecific binding of reporter A in well 2 act as a negative control to the small spots generated by specific binding of reporter A in well 1. Subtracting the number of any small spots generated by nonspecific binding of A in well 2 from the number of small spots generated by specific binding of reporter A in well 1 provides a more accurate calculation of the concentration of analyte A.

[0202] Furthermore, if nonspecific binding exceeds a threshold in any well, the system can inform the user that there may be an abnormality in the measurement.

[0203] Example 4

[0204] The measurements will be carried out in two reaction wells. The reaction wells will be prepared in the same manner as in Example 1.

[0205] Reaction well 1 is coated with an antibody against analyte A, and reaction well 2 is coated with an antibody against analyte B.

[0206] The sample is mixed simultaneously with two reporter reagents. The reporter reagent for analyte A contains 50 nm beads coated with an antibody against analyte A (photosensitizer-labeled antibody 3). The reporter reagent for analyte B contains 50 nm beads coated with an antibody against analyte B (photosensitizer-labeled antibody 5). Photosensitizer-labeled antibody 3 is stimulated at 680 nm, and photosensitizer-labeled antibody 5 is stimulated at 350 nm.

[0207] This mixture is added to each well and incubated for 10 minutes to conjugate photosensitizer-labeled antibody 3 to the surface of the substrate in well 1 by antibody 2a, and photosensitizer-labeled antibody 5 to the surface of the substrate in well 2 by antibody 2b, as shown in Figure 17. After incubation (to allow binding in both wells), each well is stimulated with 680 nm light to generate dark spots, mainly in well 1. Each well is then imaged and the number of spots generated in well 1 is counted by 490 nm fluorescence excitation. Subsequently, each well is stimulated with 350 nm light to generate dark spots, mainly in well 2. The number of spots generated in well 2 is then counted by 490 nm fluorescence excitation. Figure 18 shows well 1 stimulated with 680 nm light and well 2 stimulated with 350 nm light, and Figure 19 shows the dark spots formed in both well 1 and well 2 after the illumination sequence. For brevity, Figures 18 and 19 show only the formation of specific dark spots due to specific binding. These do not contain any dark spots formed by nonspecific binding.

[0208] In well 1, the concentration of analyte A is calculated using the number of spots, and similarly, the concentration of analyte B is calculated using the number of spots in well 2.

[0209] Wells 1 and 2 act as negative controls for each other. In this regard, the spots produced by nonspecific binding of reporter B in well 1 act as a negative control for the spots produced by specific binding of reporter B in well 2. Subtracting the number of any spots produced by nonspecific binding of reporter B in well 1 from the number of spots produced by specific binding of reporter B in well 2 provides a more accurate calculation of the concentration of analyte B.

[0210] Similarly, any spots generated by nonspecific binding of reporter A in well 2 act as a negative control for the spots generated by specific binding of reporter A in well 1. Subtracting the number of spots generated by nonspecific binding of reporter A in well 2 from the number of spots generated by specific binding of reporter A in well 1 provides a more accurate calculation of the concentration of analyte A.

Claims

1. A method for detecting two or more analytes in a sample, (i) A step of providing a sample and a mixture comprising two or more reporter reagents to a device, wherein each of the two or more reporter reagents comprises a photosensitizer, and the device comprises a substrate having an optical component and two or more binding components, wherein the optical component and the two or more binding components are bonded to the surface of the substrate; (ii) A step that enables a portion of each of two or more reporter reagents to bind to the surface of a substrate in proportion to the concentration of the corresponding analyte by the corresponding binding component; (iii) Irradiating the device with electromagnetic radiation of one wavelength for absorption by at least one of two or more reporter reagent photosensitizers, so that at least one of the two or more reporter reagent photosensitizers bonded to the surface of the substrate absorbs the electromagnetic radiation and interacts with the optical components to change the optical components from a first optical state to a second optical state, thereby forming a local region of at least one set of optical components having the second optical state on the substrate; (iv) Optionally, detect at least one set of local regions of optical components having a second optical state on the substrate formed in step (iii), repeat step (iii) with electromagnetic radiation of the same or different wavelengths for absorption by at least one of two or more reporter reagent photosensitizers, thereby causing at least one of the two or more reporter reagent photosensitizers bound to the surface of the substrate to absorb the electromagnetic radiation and interact with the optical components to change the optical components from a first optical state to a second optical state, thereby forming at least one set of local regions of optical components having a second optical state on the substrate; (v) If step (iv) is performed, a step of detecting at least one set of local regions having a second optical state on the substrate formed in step (iv), or if step (iv) is not performed, a step of detecting two or more sets of local regions having a second optical state on the substrate formed in step (iii). Includes, A method wherein two or more sets of local regions are distinguishable from one another by the formation rate of the sets of local regions, the formation order of the sets of local regions, or the size of the local regions in an arbitrary combination of the formation rate of the sets of local regions or the formation order of the sets of local regions.

2. The method according to claim 1, wherein two or more reporter reagents differ from each other in terms of size, gas permeability, excitation wavelength of the photosensitizer, reactivity of the photosensitizer, amount of the photosensitizer, or a combination thereof.

3. The method described above is (i) A step of providing a device with a mixture containing a sample and two or more reporter reagents, wherein each of the two or more reporter reagents contains a photosensitizer, and the device contains a substrate having an optical component and two or more binding components, the optical component and the two or more binding components being bonded to the surface of the substrate; (ii) A step that enables a portion of each of two or more reporter reagents to bind to the surface of a substrate in proportion to the concentration of the corresponding analyte by the corresponding binding component; (iii) Irradiating the device with electromagnetic radiation of one wavelength for absorption by the photosensitizers of two or more reporter reagents, so that the photosensitizers of the two or more reporter reagents bonded to the surface of the substrate absorb the electromagnetic radiation and interact with the optical components to change the optical components from a first optical state to a second optical state, thereby forming two or more local regions of optical components having a second optical state on the substrate; and (iv) A step of detecting two or more sets of local regions having a second optical state on the substrate. Includes, The method according to claim 1, wherein the two or more sets of local regions are distinguishable from each other by the size of the local regions.

4. The method according to claim 3, wherein two or more reporter reagents differ from each other in terms of size, gas permeability, reactivity of the photosensitizer, amount of the photosensitizer, or a combination thereof.

5. The method described above is (i) A step of providing a sample and a mixture comprising two or more reporter reagents to a device, wherein each of the two or more reporter reagents comprises a photosensitizer, and the device comprises a substrate having an optical component and two or more binding components, wherein the optical component and the two or more binding components are bonded to the surface of the substrate; (ii) A step that enables a portion of each of two or more reporter reagents to bind to the surface of a substrate in proportion to the concentration of the corresponding analyte by the corresponding binding component; (iii) Irradiating the device with electromagnetic radiation of one wavelength for absorption by at least one of two or more reporter reagent photosensitizers, so that at least one of the two or more reporter reagent photosensitizers bonded to the surface of the substrate absorbs the electromagnetic radiation and interacts with the optical components to change the optical components from a first optical state to a second optical state, thereby forming at least one local region of the optical components having the second optical state on at least the substrate; (iv) detecting at least one set of local regions of an optical component having a second optical state on the substrate formed in step (iii), repeating step (iii) with electromagnetic radiation of the same or different wavelengths for absorption by at least one of two or more reporter reagent photosensitizers, so that at least one of the two or more reporter reagent photosensitizers bonded to the surface of the substrate absorbs the electromagnetic radiation and interacts with the optical component to change the optical component from a first optical state to a second optical state, thereby forming at least one set of local regions of an optical component having a second optical state on the substrate; and (v) A step of detecting a local region of at least one set of optical components having a second optical state on the substrate formed in step (iv). Includes, The method according to claim 1 or 2, wherein the two or more sets of local regions are distinguishable from each other by the formation rate of the sets of local regions, the formation order of the sets of local regions, or the size of the local regions in combination with the formation rate of the sets of local regions or the formation order of the sets of local regions.

6. The method according to any one of claims 1 to 5, wherein each of two or more binding components is bound to a separate region on the surface of the substrate.

7. The method according to any one of claims 1 to 6, wherein each of the two or more reporter reagents further contains polymer particles, and a photosensitizer is encapsulated within the polymer particles.

8. The method according to any one of claims 1 to 7, wherein each of the two or more reporter reagents, the photosensitizer, absorbs electromagnetic radiation and interacts with a preactivator reagent present in the mixture to generate an activator reagent that interacts with the optical component, thereby changing the optical component from the first optical state to the second optical state.

9. The method according to claim 8, wherein the activator reagent is a reactive oxygen species.

10. The method according to claim 9, wherein the reactive oxygen species is singlet oxygen.

11. The method according to any one of claims 1 to 10, wherein the first optical state is fluorescent and the second optical state is non-fluorescent.

12. The method according to any one of claims 1 to 11, wherein the change from the first optical state to the second optical state is irreversible.

13. The method according to any one of claims 1 to 12, wherein steps (i) to (iii) are performed in the absence of a cleaning step.

14. The method according to any one of claims 1 to 13, wherein the sample is untreated.

15. The method according to any one of claims 1 to 14, wherein the device is irradiated with electromagnetic radiation for a longer period than one second.

Images