Methods for detecting analytes

Abstract

The present invention relates to a method for detecting an analyte, comprising steps (i) to (v), which is suitable for detecting an analyte in a sample over a wide range of concentrations. Accordingly, the present invention provides a method for detecting an analyte 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 expanding the range of detectable concentrations of an analyte.

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 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 reporters, 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, Quanterix and Merck Millipore systems become saturated at high concentrations of the analyte, making it impossible to measure such high concentrations without diluting the sample.

[0026] Generally, it is difficult for an assay to achieve both an extremely low detection limit and a wide dynamic range. This tendency is particularly pronounced in homogeneous assays that do not use a washing step. The dynamic range is mainly influenced by the total amount of capture reagent and reporter reagent, which, depending on the assay format, is often related to the surface area containing the capture reagent and / or reporter reagent. Using a large amount of reporter reagent over a large surface area generally increases nonspecific binding and background, negatively impacting the detection limit.

[0027] 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 extremely low and extremely high concentrations of analytes on the same sample without splitting or diluting the sample.

Summary of the Invention

[0028] Therefore, the present invention is a method for detecting an analyte in a sample suitable for detecting over a wide range of concentrations, (i) providing a sample, a first reporter reagent, and a second reporter reagent to a device, wherein each of the first and second reporter reagents contains a photosensitizer, each of the first and second reporter reagents can individually bind to an analyte, the device includes a substrate having an optical component and a binding component, and the optical component and the binding component are bound to the surface of the substrate; (ii) by mixing the sample with the first and second reporter reagents, (ii)(a) when the concentration of the analyte in the sample is lower than the concentration of the first reporter reagent, a portion of the first reporter reagent binds to the analyte and the substrate surface in proportion to the concentration of the analyte, and a portion of the second reporter reagent binds to the substrate surface independently of the concentration of the analyte, or (ii)(b) when the concentration of the analyte in the sample is higher than the concentration of the first reporter reagent, the first reporter reagent binds to the analyte and the substrate surface in proportion to the concentration of the analyte, and a portion of the second reporter reagent binds to the remaining analyte that is not bound to the first reporter reagent and the substrate surface in proportion to the concentration of the remaining analyte that is not bound to the first reporter reagent, wherein the binding to the substrate surface is by the binding component; (iii) irradiating the device with electromagnetic radiation at one wavelength to cause at least the photosensitizer of the first reporter reagent bound to the substrate surface to absorb the electromagnetic radiation and interact 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 in the optical component having the second optical state on the substrate; (iv) detecting, if present, the one set of local regions in the optical component having the second optical state formed on the substrate by the first reporter reagent in step (iii), and repeating step (iii) using electromagnetic radiation of the same or different wavelength absorbed by the photosensitizer of the second reporter reagent, such that the photosensitizer of the second reporter reagent bound to the substrate surface 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 one set of local regions in the optical component having the second optical state on the substrate; and (v) detecting, when step (iv) is carried out, the one set of local regions in the optical component having the second optical state formed on the substrate by the second reporter reagent in step (iv), or, when step (iv) is not carried out, detecting the two sets of local regions in the optical component having the second optical state formed on the substrate by the first and second reporter reagents in step (iii), wherein the two sets of local regions are distinguishable from each other by the formation rate of a plurality of sets of local regions, the formation order of a plurality of sets of local regions, or, optionally, the size of the local regions in combination with the formation rate of a plurality of sets of local regions or the formation order of a plurality of sets of local regions, A method comprising the above steps is provided.

[0029] Accordingly, the present invention provides a method for detecting an analyte in a sample, wherein only a reporter reagent near the surface of the substrate generates a signal, and that signal is a local region of an optical component in a second optical state. The target to be detected is a set of local regions of an optical component in a second optical state. Thus, the present invention simplifies the digital detection of analytes and assists homogeneous assays in a wide range of samples, including those containing cellular material.

[0030] Step (ii) of the method of the present invention means that when the concentration of the analyte is low, the first reporter reagent preferentially binds to the analyte and the second reporter reagent binds nonspecifically to the substrate surface, whereas when the concentration of the analyte is high, the first reporter reagent binds to the analyte and a portion of the second reporter reagent binds to the remaining analyte.

[0031] The fact that the first and second reporter reagents each form one set of local regions, and that the two sets of local regions are distinguishable from each other, means that, at low concentrations of the analyte, the nonspecific binding of the second reporter reagent to the substrate surface is distinguishable from the specific binding of the first reporter reagent to the substrate surface and can be ignored. This reduces interference when detecting analytes present at low concentrations in the sample, improving the accuracy and detection limit of the assay.

[0032] The fact that a first reporter reagent and a second reporter reagent are used, that is, that each of the first and second reporter reagents can be individually bound to the analyte, means that high concentrations of the analyte can also be detected without compromising the detection limit.

[0033] Therefore, the method of the present invention expands the dynamic range of the assay, making it possible to detect the analyte at both extremely low and extremely high concentrations. This is particularly difficult to achieve in homogeneous assays that do not have multiple washing steps to remove nonspecific binding.

[0034] Therefore, the present invention provides an improved method for detecting analytes in a sample.

[0035] The present invention will be described below with reference to the drawings. [Brief explanation of the drawing]

[0036] [Figure 1] Figure 1 shows various components that can be used in the method of the present invention. [Figure 2] Figure 2 shows a device in which first and second reporter reagents of different sizes are bound to the substrate surface before irradiation. [Figure 3] Figure 3 shows the device from 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 the substrate. In this case, the two sets of local regions are distinguishable from each other by the size of the local regions, which are visualized as bleached, dark regions of different sizes in the fluorescent layer. [Figure 6] Figure 6 shows an optical configuration for detection that allows two electromagnetic radiation sources to be focused through an objective lens. [Figure 7] Figure 7 shows a device in which first and second reporter reagents containing different photosensitizers are bound to the substrate surface before irradiation. [Figure 8] Figure 8 shows the device from Figure 7 being irradiated at 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. [Modes for carrying out the invention]

[0037] The method of the present invention is used to detect analytes in a sample (sometimes through the detection of analyte complexes or derivatives). The method of the present invention is suitable for detecting analytes over a wide range of concentrations. The concentration of the analyte can be measured over a wide range of six orders of magnitude or more.

[0038] The components of Figure 1 are as follows: photosensitizers 1a and 1b, excited by radiation of different wavelengths; antibody 2; antibody-coated latex particles 3 (hereinafter also referred to as photosensitizer-labeled antibody 3) injected with photosensitizer 1a; antibody-coated latex particles 4 (hereinafter also referred to as photosensitizer-labeled antibody 4) which are of a different size from particle 3 but similarly injected with photosensitizer 1a; antibody-coated latex particles 5 (hereinafter also referred to as photosensitizer-labeled antibody 5) injected with photosensitizer 1b; protein analyte 6; fluorescent optical component 7; non-fluorescent optical component 8; streptavidin 9; streptavidin 10 (hereinafter also referred to as streptavidin-dye conjugate 10) labeled with the fluorescent optical component; streptavidin 11 (hereinafter also referred to as streptavidin-dye conjugate 12) labeled with the non-fluorescent optical component; and biotinylated BSA 12 (hereinafter also referred to as BSA-biotin conjugate 12).

[0039] Step (i) of the method of the present invention includes providing a sample, a first reporter reagent, and a second reporter reagent to a device, wherein each of the first and second reporter reagents comprises a photosensitizer, each of the first and second reporter reagents can be individually bound to an analyte, and the device comprises a substrate having an optical component and a binding component, the optical component and the binding component being bound to the surface of the substrate.

[0040] The sample and the first and second reporter reagents may be mixed according to step (ii) of the method of the present invention to form a mixture containing the sample and the first and second reporter reagents, after which the mixture may be added to the device, or the sample and the first and second reporter reagents may be added sequentially to the device and mixed according to step (ii) of the method of the present invention. Alternatively, the first and second reporter reagents may be supplied to the device and stored therein, after which the sample may be added and mixed according to step (ii) of the method of the present invention.

[0041] Step (ii) of the method of the present invention includes a step of mixing a sample with first and second reporter reagents to enable either (ii)(a) when the concentration of the analyte in the sample is less than the concentration of the first reporter reagent, a portion of the first reporter reagent binds to the analyte and the substrate surface in proportion to the concentration of the analyte, and a portion of the second reporter reagent binds to the substrate surface regardless of the concentration of the analyte, or (ii)(b) when the concentration of the analyte in the sample is greater than the concentration of the first reporter reagent, the first reporter reagent binds to the analyte and the substrate surface in proportion to the concentration of the analyte, and a portion of the second reporter reagent binds to the remaining analyte and the substrate surface that are not bound to the first reporter reagent in proportion to the concentration of the remaining analyte that are not bound to the first reporter reagent, wherein the binding to the substrate surface is due to the binding component.

[0042] In step (ii), the sample is mixed with the first and second reporter reagents. Thus, the sample and the first and second reporter reagents form a mixture containing the sample and the first and second reporter reagents. The mixture typically contains additional reagents such as buffers, surfactants, and other additives.

[0043] The sample may be mixed simultaneously with the first and second reporter reagents, or the sample may be mixed sequentially with the first and second reporter reagents.

[0044] In a preferred embodiment, the first reporter reagent is added to the sample before the second reporter reagent. This ensures that the analyte in the sample binds to the first reporter reagent before the second reporter reagent is introduced, thereby extending the dynamic range of the assay. If the first reporter reagent exhibits high affinity for the analyte with a slow dissociation rate (e.g., half-life of more than 60 minutes), the dissociation of the analyte from the first reporter reagent within the assay timeframe will be negligible, regardless of the affinity of the second reporter reagent for the analyte. This ensures that the first reporter reagent remains bound to the analyte.

[0045] Adding the first reporter reagent to the sample before the second reporter reagent is easily achievable in fluid devices where the analyte moves along a channel and sequentially merges with the reagents under either capillary flow or pump-driven conditions.

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

[0047] If the concentration of the analyte in the sample is lower than the concentration of the first reporter reagent (i.e., the concentration of the analyte is low), step (ii)(a) is applied, in which a portion of the first reporter reagent binds to the analyte and the substrate surface in proportion to the concentration of the analyte (specific binding of the first reporter reagent), and a portion of the second reporter reagent binds to the substrate surface regardless of the concentration of the analyte (nonspecific binding of the second reporter reagent). Here, binding to the substrate surface is due to the binding component. In this case, the sample contains the bound first and second reporter reagents and the unbound first and second reporter reagents free in the solution. Since the binding is in equilibrium, a small amount of free analyte may be present in the solution. The portion of the second reporter reagent bound to the substrate surface regardless of the concentration of the analyte is only a small portion of the second reporter reagent and is nonspecific binding.

[0048] If the concentration of the analyte in the sample exceeds the concentration of the first reporter reagent (i.e., the concentration of the analyte is high), step (ii)(b) is applied, and the first reporter reagent binds to the analyte and the substrate surface in proportion to the concentration of the analyte, and a portion of the second reporter reagent binds to the remaining analyte and the substrate surface in proportion to the concentration of the remaining analyte not bound to the first reporter reagent (specific binding of the first and second reporter reagents). Here, binding to the substrate surface is due to the binding component. In this case, the sample contains the bound first and second reporter reagents and the unbound second reporter reagent free in the solution. Since the binding is in equilibrium, a small amount of analyte and unbound first reporter reagent may be present in the solution in a free state. Also, a small amount of the second reporter reagent may be bound to the substrate surface regardless of the concentration of the analyte (nonspecific binding of the second reporter reagent).

[0049] The fact that each of the first and second reporter reagents forms one set of local regions, and that the two sets of local regions are distinguishable from each other, means that, at low concentrations of the analyte, the nonspecific binding of the second reporter reagent is distinguishable from the specific binding of the first reporter reagent. This nonspecific binding can be ignored when calculating the concentration of the analyte in the sample. This reduces interference when detecting analytes present at low concentrations in the sample, improving the accuracy of the assay and lowering the detection limit.

[0050] The fact that a first reporter reagent and a second reporter reagent are used, that is, that each of the first and second reporter reagents can bind to the analyte individually, means that even after the first reporter reagent is saturated with the analyte, high concentrations of the analyte can still be detected. The fact that each of the first and second reporter reagents can bind to the analyte individually means that once a single molecule of the analyte binds to a binding domain on the reporter reagent, it will not additionally bind to another binding domain on the same or a different reporter reagent.

[0051] Therefore, the method of the present invention expands the dynamic range of the assay, making it possible to detect the analyte at both extremely low and extremely high concentrations. This is particularly difficult to achieve with homogeneous assays.

[0052] The application of step (ii)(a) or step (ii)(b) depends on the concentration of the first reporter reagent. Preferably, the concentration of the first reporter reagent is set relatively low to minimize nonspecific binding of the first reporter reagent. To broaden the assay range and prevent saturation of both reporter reagents, the second reporter reagent is generally concentrated at a higher concentration than the first reporter reagent.

[0053] For the purpose of illustrating the principle underlying the present invention, Figure 2 shows a device in which the first and second reporter reagents are bound to the surface of a substrate before irradiation. The device comprises a substrate 14 and a sample chamber 13 for holding a sample in which the protein analyte 6 is dissolved or suspended. The substrate may be any substrate that allows detection of two 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.

[0054] The substrate 14 has an antibody 2 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 functions as an optical component, and the antibody functions as a binding component. The BSA-biotin conjugate 12 is an inert polymer that assists in the binding of the optical component and the binding component to the surface of the substrate 14.

[0055] Although the optical and binding components are shown in this manner, any technique can be applied to retain the optical and binding components near the surface of the substrate 14. For example, the optical and binding components may be a single reagent. The optical components may also be encapsulated within a polymer layer coated on the surface of the substrate 14, and the binding components may be bonded to the polymer layer. The polymer may be silicone, polystyrene, polyisobutylene, or other suitable polymer plastics that can be used to encapsulate the optical components.

[0056] Alternatively, an optical component may be impregnated into a gel layer, such as a hydrogel layer, and the gel / hydrogel layer may be coated onto the surface of the substrate 14, thereby bonding the binding component to the gel / hydrogel layer.

[0057] In Figure 2, photosensitizer-labeled antibodies 3 and 4 are bound to the surface of the substrate by antibody 2. Photosensitizer-labeled antibodies 3 and 4 act as reporter reagents.

[0058] At least a portion of the first reporter reagent binds to the surface of the analyte and substrate via a binding component in proportion to the concentration of the analyte. When the concentration of the analyte is high, the first reporter reagent binds to the surface of the analyte and substrate via a binding component in proportion to the concentration of the analyte, and a portion of the second reporter reagent binds to the surface of the remaining analyte and substrate not bound to the first reporter reagent via a binding component in proportion to the concentration of the remaining analyte not bound to the first reporter reagent. For example, if the binding component and the first and second reporter reagents are antibodies and the analyte is an antigen, the reporter reagents bind to the binding component via the analyte, forming a so-called "sandwich" complex. In Figure 2, photosensitizer-labeled antibodies 3 and 4 bind to antibody 2 via protein analyte 6. Other binding events, such as antibody-hapten binding or nucleic acid binding, are also possible.

[0059] 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 at one wavelength so that at least the photosensitizer of the first reporter reagent bound to the surface of the substrate absorbs 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 14.

[0060] Figure 3 shows the device of Figure 2 being 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 a wavelength suitable for exciting the photosensitizer 1 of the photosensitizer-labeled antibodies 3 and 4.

[0061] The wavelength varies depending on the photosensitizer. Typically, the device is irradiated for at least 10 seconds. Preferably, the device is irradiated with electromagnetic radiation for 1 to 30 seconds, more preferably 2 to 20 seconds, more preferably 5 to 15 seconds, and most preferably 10 seconds. This ensures that irreversible optical changes are present on the surface of the substrate, allowing for the distinction between long-lived and transient bonding events.

[0062] Figure 4 shows the device from Figure 3 after irradiation. The photosensitizers 1, labeled antibodies 3 and 4, interact with the optical components 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 1 changes from the first optical state to the second optical state.

[0063] Other first and second optical states include changes in light polarization, fluorescence lifetime, refractive index, light scattering (including Raman scattering), phosphorescence, and other optical effects.

[0064] Figures 2 to 4 show how one of the first and second reporter reagents are bound to the surface of the substrate 14. However, in reality, multiple instances of the same reporter reagent are bound to the surface of the substrate 14, forming multiple local regions having a second optical state on the substrate in a set of multiple local regions having a second optical state on the substrate. Using the components in Figure 1, these local regions having a second optical state on the substrate are visualized as separate regions from the non-fluorescent streptavidin-dye conjugate 11.

[0065] Such distinct regions can be seen in Figure 5, which shows a representative example of two sets of local regions having a second optical state on the substrate. In this case, the two sets of local regions can be distinguished from each other by the size of the local regions, which are visualized in the fluorescent layer as bleached dark regions of different sizes. A clear size difference exists between the sets of local regions, with one set being visualized as a large dark region and the other as a small dark region. White regions are artifacts and should be ignored.

[0066] Furthermore, the method of the present invention uses a first reporter reagent and a second reporter reagent. The first and second reporter reagents are different types of reporter reagents that form two sets of local regions having a second optical state on a substrate. These two sets of local regions are distinguishable from each other by the formation rate of multiple sets of local regions, the formation order of multiple sets of local regions, or, optionally, by the size of the local regions in combination with the formation rate or formation order of multiple sets of local regions.

[0067] In addition, excess particles that are not bound to the surface may be suspended in the sample. If the sample is whole blood, red blood cells will be suspended in the culture medium. Preferably, the substrate 14 forms the top of the sample chamber 13 so that the red blood cells can be settled and removed from the substrate 14.

[0068] The depth of the sample chamber 13 is designed to minimize the diffusion path length of the first and second reporter reagents and to allow for rapid binding of the first and second reporter reagents. Typically, the depth of the sample chamber is 50–200 μm.

[0069] In the method of the present invention, the sample chamber 13 is filled with a sample containing the analyte. First and second reporter reagents, such as photosensitizer-labeled antibodies 3 and 4, are also added to the sample chamber 13. In a preferred embodiment, the first reporter reagent is added to the sample before the second reporter reagent. If a whole blood sample is used, the sample may contain additional components such as red blood cells.

[0070] After a certain period, at least a portion of the first reporter reagent, such as the photosensitizer-labeled antibody 3, binds to the surface of the analyte 6 and the substrate 14 in proportion to the concentration of the analyte 6, by a binding component such as antibody 2. If the concentration of the analyte is high, the first reporter reagent binds to the surface of the analyte and the substrate in proportion to the concentration of the analyte, by a binding component such as antibody 2, and a portion of the second reporter reagent, such as the photosensitizer-labeled antibody 3, binds to the surface of the remaining analyte and the substrate 14 that are not bound to the first reporter reagent, in proportion to the concentration of the remaining analyte that is not bound to the first reporter reagent 6, by a binding component such as antibody 2.

[0071] By including a sufficient amount of the first reporter reagent and an excess amount of the second reporter reagent in the target analyte, the maximum amount of analyte forms a sandwich complex. To maximize sensitivity (by reducing nonspecific binding of the first reporter reagent to the substrate surface), it is preferable to use a relatively small amount of the first reporter reagent, and to maximize the dynamic range (by preventing saturation of the second reporter reagent), it is preferable to use a relatively large amount of the second reporter reagent. In practice, the actual amount of each reporter reagent is assay-specific and depends on the preferred measurement range.

[0072] 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 a fluorescent optical component becomes streptavidin 11 labeled with a non-fluorescent optical component. Only the optical components adjacent to the excited photosensitizer change from the first optical state to the second optical state.

[0073] When a binding event occurs, the photosensitizer is located near the substrate. That is, the photosensitizer is close enough to the substrate surface to interact with the optical component upon irradiation of the device, converting the optical component from a first optical state to a second optical state. However, the actual distance between the photosensitizer and the substrate surface depends on many variables, including the size and properties of the photosensitizer, the size and properties of the binding component, the first and second reporter reagents and analytes, and the properties of the sample medium.

[0074] 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. As a result, at least one excited photosensitizer of the first reporter reagent bound to the substrate surface interacts with the optical component, changing it from a first optical state to a second optical state, thereby forming at least one set of local regions having the second optical state on the substrate 14.

[0075] The first and second reporter reagents, containing the excited photosensitizer, must be permanently bound to the surface of the substrate 14 throughout the entire irradiation period to achieve a complete conversion from the first optical state to the second optical state. If the first and second reporter reagents, containing the excited photosensitizer, are only transiently bound to the surface for part of the irradiation period, the conversion to the second optical state is incomplete, which can be detected by algorithms used to measure the size, shape, and intensity of separate regions. Unbound first and second reporter reagents in solution do not significantly alter the optical components from the first optical state to the second optical state.

[0076] This offers a significant advantage compared to 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, the unbound reporter reagent interferes with the signal produced by the bound reporter reagent, so it is necessary to separate the unbound reporter reagent from the bound reporter reagent before measurement. However, the localized surface changes provided by the present invention allow for the distinction between the bound and unbound reporter reagents. In fact, the ability to distinguish between the reporter reagent in close proximity to the surface of the substrate 14 (i.e., bound) and the reporter reagent in the bulk solution (i.e., unbound) is a special advantage of the present invention. Preferably, steps (i) to (iii) are carried out without a washing step. That is, the method of the present invention is carried out in steps (i), (ii), and (iii) without removing the sample from the substrate.

[0077] Step (iv) of the method of the present invention includes optionally detecting a set of local regions of an optical component having a second optical state, which was formed on the substrate by the first reporter reagent in step (iii), and repeating step (iii) using electromagnetic radiation of the same or different wavelengths absorbed by the photosensitizer of the second reporter reagent, such that a photosensitizer of the second reporter reagent bonded to the surface of the substrate absorbs 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 a set of local regions of an optical component having a second optical state on the substrate.

[0078] Step (iv) is optional and is performed when detection is required after each irradiation step, in which two sets of local regions having a second optical state are sequentially formed on the substrate by two irradiation steps.

[0079] When performing step (iv), the detection of a set of local regions in the optical component having a second optical state formed on the substrate in step (iii) is preferably performed by image analysis software that can distinguish between actual bonding events and transient bonding events and surface artifacts by analyzing surface intensity and morphology. The detection may be performed in a separate step after the optical component has been converted from the first optical state to the second optical state. Alternatively, the detection may be performed during the conversion to the second optical state.

[0080] Any method can be used to successfully identify individual coupling events. For example, an initial image of the surface can be obtained using a final step of irradiation with the excitation wavelength of the optical component, followed by irradiation with electromagnetic radiation to excite the photosensitizer, and then irradiation with the excitation wavelength of the optical component.

[0081] Alternatively, it may be possible to acquire 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.

[0082] The second optical state forms a series of local regions on the substrate. Advantageously, these local regions with 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 numerous binding events exist and the majority of the optical components are in the second optical state, bulk changes may be detected.

[0083] In a preferred embodiment, a set of local regions having a second optical state on the substrate is detected by counting local regions within a set of local regions having a second optical state on the substrate, or by measuring a set of local regions having a second optical state on the substrate as a bulk characteristic. More preferably, a set of local regions having a second optical state on the substrate is detected by counting local regions within a set of local regions having a second optical state on the substrate.

[0084] A local 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 be above a threshold before detection. This eliminates 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.

[0085] Local regions on the substrate that have a second optical state are typically discrete 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 uniformly circular, but some local regions may have a non-uniform shape, which corresponds to artifacts. Furthermore, some local regions may be larger than others when particles are aggregated together, or smaller when only transient binding events are occurring. Therefore, in a preferred embodiment, only uniformly circular local regions on the substrate that have a second optical state are detected.

[0086] A set of localized regions on a substrate can be detected using simple optical means. In a preferred embodiment, a set of localized regions having a second optical state on the substrate is detected using an optical microscope. A suitable optical configuration for detection is shown in Figure 6. More preferably, a set of localized regions having a second optical state on the substrate is detected using a wide-field microscope. A wide-field microscope is the simplest form of microscope that simultaneously illuminates and images the entire sample, compared to more complex techniques such as confocal microscopy, which illuminates and records only one focal point at a time. The advantages of confocal microscopy are improved contrast due to the removal of out-of-focus haze and the ability to obtain a stack of images across the entire depth 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 expensive than simple wide-field methods.

[0087] To detect a 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.

[0088] When step (iv) is performed, 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).

[0089] 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 method may be used when the first and second 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 wavelength of the photosensitizers of the first and second reporter reagents. In this embodiment, the two sets of local regions are distinguishable from each other by the formation rate of multiple sets of local regions, or by the size of the local regions in combination with the formation rate of multiple sets of local regions.

[0090] Preferably, any step (iv) of the method of the present invention includes repeating step (iii) for a longer period and / or at a higher intensity using electromagnetic radiation of the same wavelength. This results in the subsequent formation of multiple sets of local regions with slower formation rates.

[0091] In another preferred embodiment, an optional step (iv) of the method of the present invention includes repeating step (iii) using electromagnetic radiation of different wavelengths. In this method, the first and second reporter reagents may be used differently from each other depending on the excitation wavelength of the photosensitizer, and optionally in combination with size. In this case, the electromagnetic radiation used in step (iii) and the optional step (iv) corresponds to the excitation wavelength of the photosensitizer of the first and second reporter reagents. In this embodiment, two or more sets of local regions are distinguishable from each other by the formation sequence of the sets of local regions, or by the size of the local regions in combination with the formation sequence of the sets of local regions.

[0092] Step (v) of the method of the present invention includes, if step (iv) is performed, detecting one set of local regions in an optical component having a second optical state formed on the substrate by a second reporter reagent in step (iv), or, if step (iv) is not performed, detecting two sets of local regions having a second optical state formed on the substrate by the first and second reporter reagents in step (iii). Here, the two sets of local regions are distinguishable from each other by the formation rate of multiple sets of local regions, the formation order of multiple sets of local regions, or, optionally, the size of the local regions in combination with the formation rate of multiple sets of local regions or the formation order of multiple sets of local regions.

[0093] Therefore, if step (iv) is not performed, step (iii) is to irradiate the device with electromagnetic radiation at one wavelength so that each of the photosensitizers of the first and second reporter reagents bound to the substrate surface absorbs electromagnetic radiation and interacts with the optical component, changing the optical component from a first optical state to a second optical state, thereby forming two sets of local regions on the substrate in the optical component having the second optical state. In this case, the sets of local regions are of different sizes.

[0094] Step (v) is carried out in the same manner as the detection in any step (iv), except that if step (iv) is not performed, more than one set of local regions having a second optical state formed on the substrate by the first and second reporter reagents in step (iii) are detected. Preferred embodiments of the detection in any step (iv) also apply to step (v).

[0095] In the method of the present invention, two sets of local regions having a second optical state are detected on the substrate. The two sets of local regions can be distinguished from each other by the formation rate of multiple sets of local regions, the formation order of multiple sets of local regions, or, optionally, by the size of the local regions in combination with the formation rate or formation order of multiple sets of local regions.

[0096] In other words, two sets of local regions can be distinguished from each other by the formation rate of multiple sets of local regions, the formation order of multiple sets of local regions, or the size of the local regions. Furthermore, if two sets of local regions are distinguishable from each other by their size, they may also be distinguishable from each other by the formation rate of multiple sets of local regions or the formation order of multiple sets of local regions. That is, two sets of local regions may be distinguishable from each other by their size and the formation rate of multiple sets of local regions. Alternatively, two sets of local regions may be distinguishable from each other by their size and the formation order of multiple sets of local regions.

[0097] The difference between formation rate and formation order is that when two sets of local regions are distinguishable by their formation rate, they are always formed in the same order when using excitation of the same wavelength. However, when two sets of local regions are distinguishable by their formation order, the formation order of the two sets of local regions can be switched by changing the order in which the two excitation wavelengths are used.

[0098] The formation rate of multiple sets 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 single wavelength.

[0099] In a preferred embodiment, two sets of local regions are distinguishable from each other by the formation rate of multiple sets of local regions. This is a clear difference; for low concentrations of analytes, if the number of local regions in the first set of local regions falls below a threshold, the number of local regions in the first set of local regions after a short first irradiation step is counted, while the number of local regions in the second set of local regions after a second irradiation step using the same wavelength for a longer period and / or higher intensity is not counted. Since the second reporter reagent does not generate local regions during the first irradiation period, there is no interference from the second reporter reagent to measurements using the first reporter reagent. Therefore, the nonspecific binding of the second reporter reagent does not hinder the detection of low concentrations of analytes, improving the accuracy and detection limit of the assay.

[0100] In the case of high-concentration analytes, if the number of local regions in the first set of local regions exceeds the threshold, the number of local regions in both the first and second sets of local regions after the first and second irradiation steps are counted. Therefore, both low and high-concentration analytes can be detected, and the dynamic range of the assay is extended.

[0101] The formation sequence of multiple sets of local regions means that multiple sets of local regions are formed sequentially by irradiating the device with different excitation wavelengths of the photosensitizer.

[0102] In a preferred embodiment, the two sets of local regions are distinguishable from each other by the formation sequence of the multiple sets of local regions. In this case as well, this is a clear difference; if the number of local regions in the first set of local regions is below a threshold for low concentrations of analytes, the number of local regions in the first set of local regions after the first irradiation step is counted, while the number of local regions in the second set of local regions after a second irradiation step using a different wavelength is not counted. Since the second reporter reagent does not generate local regions during the first irradiation step, there is no interference from the second reporter reagent to measurements using the first reporter reagent. Therefore, the nonspecific binding of the second reporter reagent does not hinder the detection of low concentrations of analytes, improving the accuracy and detection limit of the assay.

[0103] For high concentrations of analytes, if the number of local regions in the first set of local regions exceeds a threshold, the number of local regions in both the first and second sets of local regions after the first and second irradiation steps using different wavelengths are counted. Therefore, both low and high concentrations of analytes can be detected, extending the dynamic range of the assay.

[0104] The size of the local region refers to the size of the local region at a given time point in time, or the maximum size that can be achieved, and preferably refers to the maximum size that can be achieved.

[0105] The size of each local region in a set of local regions of an optical component having a second optical state on the substrate should be the same, and the local region in one set should be significantly larger or significantly smaller than the local region in another set. Preferably, the size of each local region in a set of local regions of an optical component having a second optical state on the substrate is the same.

[0106] As described above, the local regions corresponding to individual binding events tend to be uniform and circular. Therefore, two sets of local regions are preferably distinguishable from each other by the diameter of the local regions.

[0107] In a preferred embodiment, the two sets of local regions are distinguishable from each other by the size of the local regions. In this case as well, this is a clear distinction, and if the number of local regions in the first set of local regions falls below a threshold at low concentrations of the analyte, the number of local regions in the first set of local regions is counted, while the number of local regions in the second set of local regions is not counted. Therefore, nonspecific binding of the second reporter reagent does not interfere with the detection of low concentrations of the analyte, improving the accuracy and detection limit of the assay.

[0108] In the case of a high-concentration analyte, if the number of local regions in the first set of local regions exceeds the threshold, the number of local regions in both the first and second sets of local regions are counted. Therefore, both low and high concentrations of analytes can be detected, and the dynamic range of the assay is extended.

[0109] 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 formation rate of the multiple sets of local regions.

[0110] Alternatively, two or more sets of local regions are preferably distinguishable from one another by the size of the local regions and the formation order of the multiple sets of local regions.

[0111] By using the first and second reporter reagents, the two sets of local regions can be distinguished from each other by the formation rate of multiple sets of local regions, the formation sequence of multiple sets of local regions, or, in some cases, by the size of the local regions in combination with the formation rate or formation sequence of multiple sets of local regions.

[0112] The first and second reporter reagents have different physicochemical properties. The number of physicochemical properties can vary, allowing the two sets of local regions to be distinguished from each other by the formation rate of multiple sets of local regions, the formation order of multiple sets of local regions, or, in some cases, by the size of the local regions in combination with the formation rate or formation order of multiple sets of local regions. These physicochemical properties include size, material properties (e.g., gas permeability), type of photosensitizer (e.g., activatable wavelength, reactivity of the photosensitizer), and / or amount of photosensitizer.

[0113] In a preferred embodiment, the first and second reporter reagents differ from each other in size, gas permeability, excitation wavelength of the photosensitizer, reactivity of the photosensitizer, amount of the photosensitizer, or a combination thereof. More preferably, the first and second reporter reagents differ from each other in size, gas permeability, excitation wavelength of the photosensitizer, amount of the photosensitizer, or a combination thereof. Most preferably, the first and second reporter reagents differ from each other in size and / or excitation wavelength of the photosensitizer.

[0114] Preferably, the first and second reporter reagents differ from each other in size. If the effect of the photosensitizer on the optical component is limited to a finite 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, the larger size allows more photosensitizer to bind to the reporter reagent, increasing the activity of the reporter reagent. This increased activity promotes the interaction between the photosensitizer of the reporter reagent and the optical component, potentially leading to the formation of larger local regions more rapidly. In this way, the two sets of local regions are preferably distinguishable from each other by the size of the local regions, and possibly in combination with the formation rate of multiple sets of local regions.

[0115] In a preferred embodiment, the first and second reporter reagents are of different sizes, with the first reporter reagent being smaller than the second. In this case, the first reporter reagent diffuses to the substrate surface more rapidly than the second reporter reagent, so that, for any predetermined incubation period, at low concentrations of analytes, more of the first reporter reagent can bind to the substrate surface than the second reporter reagent, proportional to the concentration of the analyte. Therefore, the first reporter reagent forms more local regions on the substrate in the optical component having a second optical state than the second reporter reagent. These local regions are smaller in the case of the first reporter reagent than in the case of the second reporter reagent. At high concentrations of analytes, the first reporter reagent binds to the substrate surface proportionally to the analyte concentration, and a portion of the second reporter reagent binds to the substrate surface proportionally to the concentration of the remaining analyte. The binding of the first and second reporter reagents is distinguishable by the size of the local regions, thus extending the dynamic range of the assay. Furthermore, if the second reporter reagent is larger than the first reporter reagent, the second reporter reagent can have more binding domains, thereby preventing saturation.

[0116] Preferably, the first and second reporter reagents differ from each other in size, affinity to the analyte, and dissociation rate. In this case, the first reporter reagent is smaller, has a higher affinity to the analyte, and a slower dissociation rate than the second reporter reagent. When considering affinity, it is important to consider not only the overall affinity constant but also the dissociation rate (off-rate). If the first reporter reagent has high affinity and a slow dissociation rate (relative to the assay period), and the second reporter reagent has low affinity and a fast dissociation rate (relative to the assay period), then as the assay time progresses, most of the analyte will bind to the first reporter reagent. Furthermore, as mentioned above, the first reporter reagent diffuses to the substrate surface more rapidly than the second reporter reagent. Due to these factors, at low concentrations of analyte, more of the first reporter reagent can bind to the substrate surface than the second reporter reagent, proportional to the analyte concentration. Therefore, the first reporter reagent forms more local areas on the substrate in the optical component having a second optical state than the second reporter reagent. This localized area is smaller with the first reporter reagent than with the second reporter reagent. At high analyte concentrations, the first reporter reagent binds to the substrate surface in proportion to the analyte concentration, while a portion of the second reporter reagent binds to the substrate surface in proportion to the remaining analyte concentration. Since the binding of the first and second reporter reagents is distinguishable by the size of the localized area, the dynamic range of the assay is extended.

[0117] In a particularly preferred embodiment, the first reporter reagent is added to the sample before the second reporter reagent, and the amount of the first reporter reagent is smaller than that of the second reporter reagent. Combining these two approaches ensures that low concentrations of analytes bind only to the first reporter reagent, thereby improving the dynamic range of the assay.

[0118] Preferably, the first and second 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, thereby accelerating the formation of a set of local regions and / or forming a set of local regions having larger local regions. In this way, the two sets of local regions are distinguishable from each other by the formation rate of multiple sets of local regions, or by the size of the local regions, optionally combined with the formation rate of multiple sets of local regions.

[0119] Preferably, the first and second reporter reagents differ from each other depending on the excitation wavelength of the photosensitizer. In this way, the two sets of local regions are distinguishable from each other by the formation sequence of the multiple sets of local regions.

[0120] This is shown in Figures 7 to 11. Figure 7 shows a device in which first and second reporter reagents containing photosensitizers stimulated by different radiation wavelengths are bound to the substrate surface before irradiation. Photosensitizer-labeled antibody 3 is bound to the substrate surface by antibody 2, and photosensitizer-labeled antibody 5 is bound to the substrate surface by antibody 2. Photosensitizer-labeled antibodies 3 and 5 act as reporter reagents.

[0121] 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, LED 15a. The light source irradiates the sample chamber 13 with light of a wavelength appropriate for exciting the photosensitizer 1a of the photosensitizer-labeled antibody 3.

[0122] 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 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, forming a set of local regions of optical components having the second optical state on the substrate.

[0123] This set of localized regions can then be detected before irradiating the device with electromagnetic radiation of different wavelengths, which will subsequently be absorbed by the photosensitizer 1b of the photosensitizer-labeled antibody 5.

[0124] Figure 10 shows the device of Figure 9 being irradiated with electromagnetic radiation of different wavelengths, for example, 350 nm. The light source may be, for example, LED 15b. The light source irradiates the sample chamber 13 with light of a wavelength appropriate for exciting the photosensitizer 1b of the photosensitizer-labeled antibody 5.

[0125] 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 fluorescent streptavidin-dye conjugate 10 becomes the non-fluorescent streptavidin-dye conjugate 11. Only the dye adjacent to the photosensitizer 1b changes from the first optical state to the second optical state, forming a further set of local regions of optical components having the second optical state on the substrate.

[0126] This additional set of local regions then becomes detectable. In this way, the two sets of local regions are distinguishable from each other by the formation order of the multiple sets of local regions.

[0127] Preferably, the first and second reporter reagents differ from each other in terms of the reactivity of their photosensitizers. For example, the photosensitizer of one reporter reagent absorbs more electromagnetic radiation and / or interacts more strongly with the optical component than the photosensitizer of the other reporter reagent, thereby allowing for the formation of a set of local regions more rapidly and / or the formation of a set of local regions having larger local regions. In this way, the two sets of local regions are distinguishable from each other by the formation rate of multiple sets of local regions, or by the size of the local regions, which may be a combination of the formation rate of multiple sets of local regions.

[0128] Preferably, the first and second reporter reagents differ from each other in terms of the amount of photosensitizer. For example, a reporter reagent containing more photosensitizer absorbs more electromagnetic radiation and / or interacts more strongly with the optical component than another reporter reagent containing less photosensitizer, thereby causing the formation of a set of local regions to occur more rapidly and / or forming a set of local regions with larger local regions. In this way, the two sets of local regions are distinguishable from each other by the formation rate of the multiple sets of local regions, or by the size of the local regions, which may be a combination of the formation rate of the multiple sets of local regions.

[0129] The first and second reporter reagents may differ from each other in one or more physicochemical properties.

[0130] In a preferred embodiment, the method of the present invention is (i) A step of providing a sample, a first reporter reagent, and a second reporter reagent to a device, wherein each of the first and second reporter reagents contains a photosensitizer, each of the first and second reporter reagents can be individually bound to an analyte, and the device includes a substrate having an optical component and a binding component, the optical component and the binding component being bound to the surface of the substrate; (ii) A step that enables either (ii)(a) when the concentration of the analyte in the sample is less than the concentration of the first reporter reagent, a portion of the first reporter reagent binds to the analyte and the substrate surface in proportion to the concentration of the analyte, and a portion of the second reporter reagent binds to the substrate surface regardless of the concentration of the analyte, or (ii)(b) when the concentration of the analyte in the sample is greater than the concentration of the first reporter reagent, the first reporter reagent binds to the analyte and the substrate surface in proportion to the concentration of the analyte, and a portion of the second reporter reagent binds to the remaining analyte and the substrate surface that are not bound to the first reporter reagent in proportion to the concentration of the remaining analyte that are not bound to the first reporter reagent, wherein the binding to the substrate surface is due to a binding component; (iii) Irradiating the device with electromagnetic radiation at one wavelength so that each of the photosensitizers of the first and second reporter reagents bonded to the surface of the substrate absorbs 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 two sets of local regions on the substrate in which the optical component has a second optical state; and (iv) A step of detecting two sets of local regions on a substrate having a second optical state, Includes, The two sets of local regions are distinguishable from each other by the size of the local region.

[0131] In this embodiment, the first and second reporter reagents are preferably different from each other in terms of size, gas permeability, photosensitizer reactivity, amount of photosensitizer, or a combination thereof.

[0132] In a preferred embodiment, the method of the present invention is (i) A step of providing a sample, a first reporter reagent, and a second reporter reagent to a device, wherein each of the first and second reporter reagents contains a photosensitizer, each of the first and second reporter reagents can be individually bound to an analyte, and the device includes a substrate having an optical component and a binding component, the optical component and the binding component being bound to the surface of the substrate; (ii) A step that enables either (ii)(a) when the concentration of the analyte in the sample is less than the concentration of the first reporter reagent, a portion of the first reporter reagent binds to the analyte and the substrate surface in proportion to the concentration of the analyte, and a portion of the second reporter reagent binds to the substrate surface regardless of the concentration of the analyte, or (ii)(b) when the concentration of the analyte in the sample is greater than the concentration of the first reporter reagent, the first reporter reagent binds to the analyte and the substrate surface in proportion to the concentration of the analyte, and a portion of the second reporter reagent binds to the remaining analyte and the substrate surface that are not bound to the first reporter reagent in proportion to the concentration of the remaining analyte that are not bound to the first reporter reagent, wherein the binding to the substrate surface is due to a binding component; (iii) Irradiating the device with electromagnetic radiation at one wavelength so that the photosensitizer of the first reporter reagent bonded to the surface of the substrate absorbs 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 a set of local regions on the substrate in which the optical component has the second optical state; (iv) A set of local regions of optical components having a second optical state, formed on the substrate by the first reporter reagent in step (iii), is detected, and step (iii) is repeated using electromagnetic radiation of the same or different wavelengths absorbed by the photosensitizer of the second reporter reagent; and the photosensitizer of the second reporter reagent, bound to the surface of the substrate, absorbs 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 set of local regions of optical components having a second optical state on the substrate; and (v) A step of detecting a set of local regions in an optical component having a second optical state, which is formed on the substrate by a second reporter reagent in step (iv), Includes, The two sets of local regions can be distinguished from each other by the formation rate of the multiple sets of local regions, the formation order of the multiple sets of local regions, and, if applicable, by the size of the local regions.

[0133] 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.

[0134] In a preferred embodiment, the method of the present invention is (i) A step of providing a sample, a first reporter reagent, and a second reporter reagent to a device, wherein each of the first and second reporter reagents contains a photosensitizer, each of the first and second reporter reagents can be individually bound to an analyte, and the device includes a substrate having an optical component and a binding component, the optical component and the binding component being bound to the surface of the substrate; (ii) A step that enables either (ii)(a) when the concentration of the analyte in the sample is less than the concentration of the first reporter reagent, a portion of the first reporter reagent binds to the analyte and the substrate surface in proportion to the concentration of the analyte, and a portion of the second reporter reagent binds to the substrate surface regardless of the concentration of the analyte, or (ii)(b) when the concentration of the analyte in the sample is greater than the concentration of the first reporter reagent, the first reporter reagent binds to the analyte and the substrate surface in proportion to the concentration of the analyte, and a portion of the second reporter reagent binds to the remaining analyte and the substrate surface that are not bound to the first reporter reagent in proportion to the concentration of the remaining analyte that are not bound to the first reporter reagent, wherein the binding to the substrate surface is due to a binding component; (iii) Irradiating the device with electromagnetic radiation at one wavelength so that the photosensitizer of the first reporter reagent bonded to the surface of the substrate absorbs 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 a set of local regions on the substrate in which the optical component has the second optical state; (iv) A set of local regions of optical components having a second optical state, formed on the substrate by the first reporter reagent in step (iii), is detected, and step (iii) is repeated using electromagnetic radiation of the same wavelength absorbed by the photosensitizer of the second reporter reagent; and the photosensitizer of the second reporter reagent, bound to the surface of the substrate, absorbs 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 set of local regions of optical components having a second optical state on the substrate; and (v) A step of detecting a set of local regions in an optical component having a second optical state, which is formed on the substrate by a second reporter reagent in step (iv), Includes, The two sets of local regions are distinguishable from each other by their formation rate and, if applicable, by the size of the local regions.

[0135] In this embodiment, two or more reporter reagents are preferably different from each other by size, gas permeability, reactivity of the photosensitizer, amount of the photosensitizer, or a combination thereof.

[0136] In another preferred embodiment, the method of the present invention is (i) A step of providing a sample, a first reporter reagent, and a second reporter reagent to a device, wherein each of the first and second reporter reagents contains a photosensitizer, each of the first and second reporter reagents can be individually bound to an analyte, and the device includes a substrate having an optical component and a binding component, the optical component and the binding component being bound to the surface of the substrate; (ii) A step that enables either (ii)(a) when the concentration of the analyte in the sample is less than the concentration of the first reporter reagent, a portion of the first reporter reagent binds to the analyte and the substrate surface in proportion to the concentration of the analyte, and a portion of the second reporter reagent binds to the substrate surface regardless of the concentration of the analyte, or (ii)(b) when the concentration of the analyte in the sample is greater than the concentration of the first reporter reagent, the first reporter reagent binds to the analyte and the substrate surface in proportion to the concentration of the analyte, and a portion of the second reporter reagent binds to the remaining analyte and the substrate surface that are not bound to the first reporter reagent in proportion to the concentration of the remaining analyte that are not bound to the first reporter reagent, wherein the binding to the substrate surface is due to a binding component; (iii) Irradiating the device with electromagnetic radiation at one wavelength so that the photosensitizer of the first reporter reagent bonded to the surface of the substrate absorbs 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 a set of local regions on the substrate in which the optical component has the second optical state; (iv) A set of local regions of optical components having a second optical state, formed on the substrate by the first reporter reagent in step (iii), is detected, and step (iii) is repeated using electromagnetic radiation of different wavelengths absorbed by the photosensitizer of the second reporter reagent; and the photosensitizer of the second reporter reagent, bound to the surface of the substrate, absorbs 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 set of local regions of optical components having a second optical state on the substrate; and (v) A step of detecting a set of local regions in an optical component having a second optical state, which is formed on the substrate by a second reporter reagent in step (iv), Includes, The two sets of local regions can be identified from each other by the formation order of the multiple sets of local regions, and, if applicable, by the size of the local regions.

[0137] In this embodiment, two or more reporter reagents are preferably different from each other, depending on the excitation wavelength of the photosensitizer, and optionally in combination with size.

[0138] The photosensitizers of the first and second reporter reagents bound to the surface of the 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 they can indirectly interact with the optical component through an additional reagent to induce a change (e.g., the photosensitizer is excited, transfers this energy to the additional component, and then transfers this energy to the optical component).

[0139] In a preferred embodiment, the photosensitizers of the first and second reporter reagents absorb electromagnetic radiation and interact with the preactivator reagent present in the mixture of the sample and the first and second reporter reagents to generate an activator reagent that interacts with the optical component, thereby changing the optical component from a first optical state to a second optical state. Thus, the first and second reporter reagents can generate an activator reagent from the preactivator reagent present in the mixture by absorbing electromagnetic radiation, and the optical component can change from a first optical state to a second optical state by interacting with the activator reagent.

[0140] The preactivator reagent may be present in the sample, or it may be added as an additional reagent to the mixture of the sample and the first and second 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).

[0141] The photosensitizer is thought to absorb light to generate an excited state, which then undergoes intersystem cross-reaction (ISC) with oxygen present in the sample and near the reporter reagent to produce singlet oxygen. Subsequently, as described below, the singlet oxygen interacts with the optical components. In a particularly preferred embodiment, the preactivator reagent is ground-state triplet oxygen, and the activator reagent is singlet oxygen.

[0142] Traditionally, singlet oxygen has been used in immunoassays such as the luminescent oxygen channeling immunoassay (LOCI). The LOCI immunoassay is a homogeneous, non-digital assay using donor beads and acceptor beads to measure bulk phenomena. Donor beads generate singlet oxygen when irradiated at 680 nm, and acceptor beads produce a chemiluminescent signal when activated by singlet oxygen. Binding between donor and acceptor beads is facilitated by antibody-antigen binding. The reaction mixture is typically irradiated for 0.5–1.0 seconds, and then the luminescence signal is measured for 0.5–1.0 seconds. Importantly, the measurement is performed in the presence of all unbound donor and acceptor beads. Spatially separating the beads minimizes background signaling. However, due to the short measurement time in the LOCI assay, it is not possible to distinguish between long-term and transient binding events. The assay can achieve a detection limit of approximately 1–5 pg / mL for the most sensitive assays, such as interleukin-6 (IL-6) or thyroid-stimulating hormone (TSH). The reporter reagent, which is a "donor" particle, needs to be in close proximity to the surface of the substrate 11 rather than to particles in solution for the signal to be detected, and must be present during the illumination period to generate a signal higher (or lower) than the threshold used by the image analysis software. Therefore, the method of the present invention further minimizes background signaling. Consequently, the method of the present invention is more sensitive than the LOCI assay and can detect low concentrations of analytes.

[0143] 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.

[0144] [ka]

[0145] Dyes (1), (2), and (3) are common organic fluorophores that can bind to proteins and other macromolecules via N-hydroxysuccinimide groups that react with amine groups to form covalent amide bonds. These dyes, along with a wide range of other fluorophores, can be irreversibly converted to a non-fluorescent state by the methods of the present invention.

[0146] Preferably, the optical component is fluorescent when in one of the first and second optical states, and non-fluorescent when in the other of the first and second 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 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.

[0147] Preferably, the change from the first optical state to the second optical state is irreversible. This allows for subsequent scanning of the substrate and identification of the region where the change in optical state has occurred.

[0148] The assay also requires the presence of a binding component.

[0149] The binding component has a binding site that can bind at least one first reporter reagent in proportion to the concentration of the analyte in the sample. Proportionality is important for the functionality of the assay because binding must depend on the concentration of the analyte in order to measure the concentration of the analyte as a meaningful measurement. Depending on the type of assay performed, binding may be directly or indirectly 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 indirectly proportional to the concentration of the analyte.

[0150] In a specific form of competitive assay, an analogue of the analyte is immobilized on a substrate, and each of the first and second reporter reagents is presented in a form containing an antibody that targets the analyte. When the analyte is absent, the first and second reporter reagents bind to the substrate surface. In a competitive assay, the first reporter reagent is added to the sample before the second reporter reagent, or the first reporter reagent has a higher affinity for the analyte than the second reporter reagent. At low concentrations of the analyte, binding of the first reporter reagent to the substrate surface is inhibited, and as the analyte concentration increases, binding of the first reporter reagent to the substrate surface decreases. At high concentrations of the analyte, binding of both the first and second reporter reagents to the substrate surface is inhibited, and further, as the analyte concentration increases, binding of the first and second reporter reagents to the substrate surface decreases.

[0151] The binding component can be adapted to bind to the analyte, or a complex or derivative of the analyte, in which case the first and second reporter reagents bind to the binding component in the presence of the analyte, or a complex or derivative of the analyte. In this case, the binding component has binding sites that can bind to the first and second reporter reagents in the presence of the analyte, or a complex or derivative of the analyte. However, the binding is still proportional to the concentration of the analyte.

[0152] Alternatively, the binding component itself may be an analog of the analyte, and the first and second reporter reagents bind directly to the binding component (they are analogs because they are bound to the surface of the substrate via either covalent or non-covalent interactions). In this case, the binding component competes with the unbound analyte, or a complex or derivative of the unbound analyte, for binding to the first and second reporter reagents. Therefore, the binding component can simply bind to the first and second reporter reagents.

[0153] The concentration of an analyte in a sample can be measured by measuring the degree to which the first and second reporter reagents bind to the binding component (either directly or mediated by an analyte / analyte complex or derivative). The system is typically pre-calibrated at the time of manufacture, and a calibration curve is generated that is used to convert the instrument signal to the analyte concentration measured in the sample.

[0154] The assay also requires the presence of a first reporter reagent and a second reporter reagent. The first and second reporter reagents each contain a photosensitizer. The photosensitizer can absorb electromagnetic radiation and interact with the optical component. This interaction causes the optical component to change from a first optical state to a second optical state.

[0155] Therefore, photosensitizers can be composed of any material capable of interacting with electromagnetic radiation in this manner. Suitable photosensitizers are known as PDT reagents from photodynamic therapy (PDT). PDT reagents are used in cancer treatment 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).

[0156] When irradiated with electromagnetic radiation, the PDT reagent is promoted to an excited triplet state. This excited triplet state can either directly interact with cellular components in a so-called type I process, or interact with oxygen in a so-called 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.

[0157] Singlet oxygen is a highly reactive excited state of oxygen. Before decaying, it can undergo a variety of reactions, including Diels-Alder reactions and EEN reactions. It also undergoes common oxidation reactions with sulfur-containing and nitrogen-containing compounds. The indiscriminate reactivity of singlet oxygen is one reason why it is used in photodynamic therapy.

[0158] A wide range of photosensitizer compounds are known, including porphyrins, chlorins (e.g., pyropheoforbid-a), phthalocyanines, and other polycyclic aromatic species (see, e.g., Antibody-Directed Phototherapy, Pye et al., Antibodies, 2013, 2, 270).

[0159] In one embodiment, the photosensitizer is selected from porphyrins, chlorins, phthalocyanines, and other polycyclic aromatic species. In a preferred embodiment, the photosensitizer is a silicon phthalocyanine derivative, such as the examples shown below.

[0160] [ka]

[0161] The properties of the binding component and the first and second reporter reagents depend on the properties of the analyte, but they preferably contain antibodies. The method of the present invention is particularly applicable to immunoassays. In a particularly preferred embodiment, the binding component is an antibody produced against the analyte or a complex or derivative of the analyte, and the first and second reporter reagents contain antibodies produced against the analyte or a complex or derivative of the analyte. In principle, a single molecule can be used for each reagent, but in practice, the binding component and the first and second reporter reagents are aggregates of molecules. The term "antibody" preferably includes, within its scope, Fab fragments, single-chain variable fragments (scFv), and recombinant binding fragments.

[0162] As an alternative to antibody-antigen reactions, the binding component, first and second reporter reagents, and analytes may be first and second nucleic acids in which the first and second nucleic acids are complementary, or reagents containing avidin or a derivative thereof, and analytes containing biotin or a derivative thereof, or vice versa. The binding component and the first and second reporter reagents 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. Furthermore, the system is not necessarily limited to liquids and can be used to detect any fluid system, such as enzymes, cells, and viruses in the air.

[0163] The photosensitizer may be present inside or outside the first and second reporter reagents, respectively. Preferably, each of the first and second reporter reagents contains polymer particles, and the photosensitizer is encapsulated within the polymer particles. Alternatively, each of the first and second reporter reagents further contains polymer particles, and the photosensitizer is coated onto 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.

[0164] More preferably, each of the first and second reporter reagents further comprises polymer particles and one or more binding domains, where the photosensitizer is encapsulated within the polymer particles and one or more binding domains are coated onto the polymer particles. Alternatively, each of the first and second reporter reagents further comprises polymer particles and one or more binding domains, where the photosensitizer and one or more binding domains are coated onto the polymer particles. The binding domains may be antibodies or nucleic acids, etc., depending on the properties of the analyte, as described above. However, it is preferable that the binding domains are antibodies.

[0165] The observable maximum signal is the maximum signal achievable when monitoring the photosensitizer binding to the surface. Particle binding to the substrate is governed by the diffusion rates of the analyte and the first and second reporter reagents, and further, primarily by the hydrodynamic radii of these components and the viscosity / temperature of the sample.

[0166] 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 of the substrate surface, and these areas are coated with different reagents. These controls are defined as “negative” controls and “positive” controls, meaning that the negative control should approximate the signal expected in the absence of the analyte, and the positive control should approximate the signal expected when the analyte saturates the system.

[0167] To achieve detection using these controls, the device of the present invention preferably comprises a binding component, a negative control reagent, and a positive control reagent. These are each bound to the substrate surface as described above.

[0168] The binding components are as described above.

[0169] Under assay conditions, the negative control reagent has lower affinity for the first and second reporter reagents than the binding component. Therefore, the negative control reagent functions as a negative control. It is important to consider affinity under assay conditions. This is because, in non-competitive assays, the affinity of the binding component to the reporter reagent is mediated by the presence of the analyte or a complex or derivative of the analyte. Therefore, in the absence of the analyte or a complex or derivative of the analyte, neither the binding component nor the negative control reagent has affinity for the reporter reagent. However, in the presence of the analyte or a complex or derivative of the analyte, the negative control reagent has lower affinity for the reporter reagent than the binding component.

[0170] Furthermore, in embodiments where the binding component binds to the analyte or a complex or derivative of the analyte, the negative control reagent preferably has lower affinity for the analyte, or, if used, the complex or derivative of the analyte, than the binding component. 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 binding component but shows little or no affinity for the reporter reagent under assay conditions. In a particularly preferred embodiment, the negative control reagent has substantially no affinity for the reporter reagent under assay conditions. Preferably, the negative control reagent shows substantially no affinity for the analyte or a complex or derivative of the analyte. That is, the binding of the reporter reagent, or, where applicable, the analyte or a complex or derivative of the analyte, to the negative control reagent is nonspecific. In this way, the negative control reagent can compensate for the nonspecific binding of the reporter reagent to the binding component. In a particular embodiment, a software algorithm uses data from the negative control region as part of a calculation to obtain the analyte concentration.

[0171] The positive control reagent binds to the first and second reporter reagents and has an affinity for the first and second reporter reagents that is less affected by the concentration of the analyte sample, or, if used, the analyte complex or derivative in the sample, compared to the binding component, and therefore functions as a positive control. Preferably, the positive control reagent has an affinity for the first and second reporter reagents that is substantially independent of the concentration of the analyte or the analyte complex or derivative. More preferably, the positive control reagent has a higher affinity for the first and second reporter reagents than the binding component under assay conditions. In this way, the positive control reagent measures the maximum signal expected in the system. In one embodiment, the positive control reagent may bind to only the first reporter reagent, or only the second reporter reagent, or it may bind to both reporter reagents.

[0172] For both negative and positive control measurements, the software algorithm can detect abnormal coupling patterns that could cause error messages and interrupt the measurement process.

[0173] While the above explanation allows for incubation of the assay for a certain period before activating the photosensitizer, it is also possible to monitor the dynamics of the binding event by irradiating the photosensitizer over discontinuous time intervals and monitoring the occurrence of the binding event during the reaction process.

[0174] The analytes may be macromolecules or small molecules. Macromolecules are typically proteins, such as protein-based hormones, and may also be parts of larger particles such as viruses, bacteria, cells (e.g., red blood cells), or prions. Small molecules may be drugs.

[0175] As used herein, the term “small molecule” is a term specific to the art and is used to distinguish it from macromolecules such as proteins and nucleic acids. Small molecules, often called “haptens” in the field of immunoassays, are small molecules that can induce an immune response when bound to larger carrier molecules such as proteins, and include 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. The binding component may be adapted to bind to the analyte itself, although the analyte may undergo a chemical reaction or an initial complex formation event before binding to the binding component. For example, the analyte may be protonated / deprotonated at the pH of the assay conditions. Thus, the analyte that binds to the binding component may be the analyte itself or a derivative of the analyte, both of which are encompassed within the scope of the present invention.

[0176] In a preferred embodiment, the present invention can be used to simultaneously detect the presence of multiple analytes in the same sample. Different binding components can be used at different positions on the substrate for the measurement of each analyte. Sandwich assays and competitive assays can be performed in parallel. The assays may use the same negative and positive controls as described above, or separate controls may be used for each analyte being measured.

[0177] A sample suspected of containing the analyte of interest is generally a fluid sample, such as a liquid sample, and is typically a biological sample such as a body fluid, e.g., blood, plasma, saliva, serum, intraocular fluid, cerebrospinal fluid, or urine. The sample may contain suspended particles or may be whole blood. In a preferred embodiment, the sample is untreated, and more preferably, an 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 elements. The 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.

[0178] 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. Because different cellular components in each sample cause unpredictable light scattering, it is common practice to remove red blood cells from blood in order to measure the fluorescence of plasma or serum components of blood. However, in the method of the present invention, since fluorescence is measured on a substrate using an imaging system with a shallow depth of field, and individual binding events can be measured, the measurement can be performed on whole blood.

[0179] The sample is typically in microliter units (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. Therefore, the device used in the method of the present invention preferably further comprises a chamber for holding the sample containing the analyte in contact with the substrate.

[0180] A further potential source of background interference is the sedimentation of suspended particles, including first and reporter reagents and cellular components of the sample, onto the substrate surface. This interference source can be reduced by placing the substrate above the bulk solution, for example, on the top surface of the reaction chamber. Thus, even if sedimentation occurs, it 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 within a single focal range during imaging. Clearly, the optical and binding components are on the inner surface of the chamber and are capable of contact with the sample. This and other modifications are included within the scope of the invention.

[0181] The sample can be held, for example, simply by the surface tension inside the capillary channel.

[0182] The first and second 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.

[0183] The method of the present invention is particularly useful in point-of-care (POC) testing. POC testing is defined as a diagnostic test performed in or near a clinical setting, i.e., a bedside test. POC testing enables convenient and rapid testing, improving decision-making and triage, while also allowing for more appropriate allocation of hospital resources such as accident and emergency treatment and hospital beds. This is in contrast to conventional testing, where samples are collected at the clinical setting and then sent to a laboratory for testing. Such tests often take several hours to several days to yield results, during which time treatment must continue without the necessary information. In POC testing, test kits are often used in combination with portable equipment.

[0184] The method of the present invention is particularly useful for monitoring the concentration or presence / absence of analytes that are typically present in very small amounts. Potential applications include biomarker measurement in cardiac disease (e.g., highly sensitive troponin), infectious diseases (e.g., hepatitis C core antigen), aging / dementia (e.g., amyloid-beta and phosphorylated τ, which are Alzheimer's disease markers), cytokines, and oncology (e.g., circulating tumor markers).

[0185] The present invention also provides an analyte detection device suitable for detecting analytes in a sample over a wide range of concentrations. The device comprises: a substrate having an optical component and a binding component, wherein the optical component and the binding component are bound to the surface of the substrate, and the optical component can change from a first optical state to a second optical state in response to interaction between a first reporter reagent and a second reporter reagent irradiated with a photosensitizer bound to the surface of the substrate; wherein (ii)(a) when the concentration of the analyte in the sample is less than the concentration of the first reporter reagent, a portion of the first reporter reagent binds to the analyte and the substrate surface in proportion to the concentration of the analyte, and a portion of the second reporter reagent binds to the substrate surface regardless of the concentration of the analyte, or (ii)(b) when the concentration of the analyte in the sample is greater than the concentration of the first reporter reagent Either a first reporter reagent binds to the analyte and the substrate surface in proportion to the concentration of the analyte, or a portion of the second reporter reagent binds to the remaining analyte and the substrate surface in proportion to the concentration of the remaining analyte not bound to the first reporter reagent; here, the binding to the substrate surface is due to the binding component, thereby forming two sets of local regions having a second optical state on the substrate; here, the two sets of local regions are distinguishable from each other by the formation rate of multiple sets of local regions, the formation order of multiple sets of local regions, or the size of the local regions in combination with the formation rate or formation order of multiple sets of local regions.

[0186] The characteristics of the device are as described above for the device used in the method of the present invention.

[0187] In a preferred embodiment, the device further includes a chamber for holding a sample and first and second reporter reagents.

[0188] The device may include one or more radiation sources tuned to generate electromagnetic radiation corresponding to the excitation wavelengths of the photosensitizers of first and second reporter reagents, and a detector tuned to detect a second optical state, thereby precisely determining the position of the photosensitizer relative to the substrate.

[0189] The device may be in the form of a cartridge used with a separate reader. The reader may incorporate a radiation source and a detector. The reader is preferably a portable reader. Preferably, the device includes a cartridge, the substrate is located within the cartridge, and the device further includes a detector for detecting two sets of local regions having a second optical state on the substrate. The present invention may also provide a cartridge comprising a substrate as defined herein, as well as optical and bonding components. The cartridge is preferably a disposable cartridge.

[0190] The present invention can also provide an analytic detection system suitable for detecting analytics in a sample over a wide range of concentrations, comprising the above-described device, as well as first and second reporter reagents for mixing with the sample, each of which comprises a photosensitizer that absorbs electromagnetic radiation and interacts with the optical component to change the optical component from a first optical state to a second optical state.

[0191] Preferably, the photosensitizer absorbs electromagnetic radiation and interacts with a preactivator reagent present in the mixture of the sample and the first and second reporter reagents to generate an activator reagent, which in turn interacts with the optical component to change it from a first optical state to a second optical state.

[0192] In a preferred embodiment, the system of the present invention comprises substantially the above features. "Substantially" means that no other features are required to perform the assay.

[0193] 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 the first and second reporter reagents bind to the conjugated component via the analyte. However, it should be understood that a variety of assay forms are conceivable. These examples are provided to demonstrate the mode of transformation of the optical component from a first optical state to a second optical state. [Examples]

[0194] Example 1

[0195] The reaction well shown in Figure 12 can be formed by cutting a 175 micron thick polymethyl methacrylate (PMMA) sheet to a size of 20 × 20 mm, and attaching a 1 cm × 1 cm square piece of 100 μm thick pressure-sensitive adhesive (PSA) 16 with a 6 mm diameter hole to it, along with a coverslip 17, to create a shallow well 18.

[0196] The surface is first coated with biotinylated BSA using a known method, and then coated with Cy2-labeled streptavidin. After coating the wells with biotinylated capture antibody (binding component), the release liner is removed from the PSA, the substrate is turned over, and the reaction chamber can be prepared by attaching it to an acrylic sheet 19 with two small holes 20 drilled into it, as shown in the side view of Figure 13.

[0197] The sample is first mixed with small reporter beads (50 nm in diameter) bound with a high-affinity antibody for 1 minute, followed by the addition of larger reporter beads (250 nm) coated with the same antibody. The concentration (particles / mL) of the larger beads is five times that of the smaller beads. The same photosensitizer is injected into the two different reporter reagents. The sample is then incubated in a reaction well with a fluorescent sensor surface containing the capture antibody. After 8 minutes of incubation, the sensor is irradiated with red light (680 nm) for 10 seconds, and an image of the fluorescent surface is taken using a 480 nm excited objective lens / camera.

[0198] On the surface of the fluorescence sensor, individual bleached spots are formed by the binding of beads due to the presence of analytes in the sample. Spots formed by two different types of bead clusters can be clearly distinguished by their size. 50 nm beads form spots with a diameter of 400 nm, and 250 nm beads form spots with a diameter of 1000 nm.

[0199] This instrument incorporates an algorithm for classifying and counting spots on the surface. If the number of small spots is relatively small, larger spots are excluded, and the signal is calculated only from the small spots. Nonspecific binding of large beads does not affect the calculation of the analyte concentration. If the number of small spots exceeds a threshold, the larger spots are also counted and used in the algorithm to calculate the analyte concentration.

[0200] Example 2

[0201] The assay is carried out in the same manner as in Example 1, except that the first and second reporter reagents are the same size and are activated using different excitation wavelengths. The first reporter reagent is excited at a wavelength of 680 nm, and the second reporter reagent is excited at a wavelength of 350 nm.

[0202] First, mix the sample with the first reporter reagent as described above. Add the second reporter reagent in a 5-fold excess. After incubation, irradiate the sensor with a wavelength of 680 nm to count the spots, and then irradiate with a wavelength of 350 nm to count further spots.

Claims

1. A method for detecting an analyte, which is suitable for detecting the analyte in a sample over a wide range of concentrations, (i) A step of providing the sample, a first reporter reagent, and a second reporter reagent to a device, wherein each of the first and second reporter reagents comprises a photosensitizer, each of the first and second reporter reagents can individually bind to the analyte, and the device comprises a substrate having an optical component and a binding component, wherein the optical component and the binding component are bound to the surface of the substrate; (ii) A step that enables either (ii)(a) when the concentration of the analyte in the sample is less than the concentration of the first reporter reagent, a portion of the first reporter reagent binds to the analyte and the substrate surface in proportion to the concentration of the analyte, and a portion of the second reporter reagent binds to the substrate surface regardless of the concentration of the analyte, or (ii)(b) when the concentration of the analyte in the sample is greater than the concentration of the first reporter reagent, the first reporter reagent binds to the analyte and the substrate surface in proportion to the concentration of the analyte, and a portion of the second reporter reagent binds to the remaining analyte and the substrate surface that are not bound to the first reporter reagent in proportion to the concentration of the remaining analyte that are not bound to the first reporter reagent, wherein the binding to the substrate surface is due to the binding component; (iii) Irradiating the device with electromagnetic radiation at one wavelength so that at least the photosensitizer of the first reporter reagent bonded to the surface of the substrate absorbs 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) The photosensitizer of the second reporter reagent bonded to the surface of the substrate absorbs 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 a set of local regions of the optical component having the second optical state on the substrate, the set of local regions of the optical component having the second optical state formed on the substrate by the first reporter reagent in step (iii), the step is optionally detected, and step (iii) is repeated using electromagnetic radiation of the same or different wavelengths absorbed by the photosensitizer of the second reporter reagent; and (v) If step (iv) is performed, in step (iv) one set of local regions of the optical component having the second optical state formed on the substrate by the second reporter reagent, or if step (iv) is not performed, in step (iii) two sets of local regions having the second optical state formed on the substrate by the first and second reporter reagents, The two sets of local regions are, in some cases, distinguishable from one another by the formation speed of the multiple sets of local regions, the formation order of the multiple sets of local regions, or, in some cases, by the size of the local regions in combination with the formation speed of the multiple sets of local regions or the formation order of the multiple sets of local regions, in a process, Methods that include...

2. The method according to claim 1, wherein the first reporter reagent is added to the sample before the second reporter reagent.

3. The method according to claim 1 or 2, wherein the first and second 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.

4. The method according to claim 3, wherein the first and second reporter reagents are of different sizes, and the first reporter reagent is smaller than the second reporter reagent.

5. The method described above is (i) A step of providing the sample, a first reporter reagent, and a second reporter reagent to a device, wherein each of the first and second reporter reagents comprises a photosensitizer, each of the first and second reporter reagents can individually bind to the analyte, and the device comprises a substrate having an optical component and a binding component, wherein the optical component and the binding component are bound to the surface of the substrate; (ii) A step that enables either (ii)(a) when the concentration of the analyte in the sample is less than the concentration of the first reporter reagent, a portion of the first reporter reagent binds to the analyte and the substrate surface in proportion to the concentration of the analyte, and a portion of the second reporter reagent binds to the substrate surface regardless of the concentration of the analyte, or (ii)(b) when the concentration of the analyte in the sample is greater than the concentration of the first reporter reagent, the first reporter reagent binds to the analyte and the substrate surface in proportion to the concentration of the analyte, and a portion of the second reporter reagent binds to the remaining analyte and the substrate surface that are not bound to the first reporter reagent in proportion to the concentration of the remaining analyte that are not bound to the first reporter reagent, wherein the binding to the substrate surface is due to the binding component; (iii) Irradiating the device with electromagnetic radiation at one wavelength so that each of the photosensitizers of the first and second reporter reagents bonded to the surface of the substrate absorbs 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 two sets of local regions of the optical component having the second optical state on the substrate; and (iv) A step of detecting the two sets of local regions having the second optical state on the substrate, Includes, The two sets of local regions are distinguishable from each other by the size of the local region. The method according to claim 1, 2, or 4.

6. The method described above is (i) A step of providing the sample, a first reporter reagent, and a second reporter reagent to a device, wherein each of the first and second reporter reagents comprises a photosensitizer, each of the first and second reporter reagents can individually bind to the analyte, and the device comprises a substrate having an optical component and a binding component, wherein the optical component and the binding component are bound to the surface of the substrate; (ii) A step that enables either (ii)(a) when the concentration of the analyte in the sample is less than the concentration of the first reporter reagent, a portion of the first reporter reagent binds to the analyte and the substrate surface in proportion to the concentration of the analyte, and a portion of the second reporter reagent binds to the substrate surface regardless of the concentration of the analyte, or (ii)(b) when the concentration of the analyte in the sample is greater than the concentration of the first reporter reagent, the first reporter reagent binds to the analyte and the substrate surface in proportion to the concentration of the analyte, and a portion of the second reporter reagent binds to the remaining analyte not bound to the first reporter reagent and the substrate surface in proportion to the concentration of the remaining analyte not bound to the first reporter reagent, wherein the binding to the substrate surface is due to the binding component; (iii) Irradiating the device with electromagnetic radiation at one wavelength so that the photosensitizer of the first reporter reagent bonded to the surface of the substrate absorbs 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 a set of local regions of the optical component having the second optical state on the substrate; (iv) A step in which the photosensitizer of the second reporter reagent bonded to the surface of the substrate absorbs 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 a set of local regions of the optical component having the second optical state on the substrate, the set of local regions of the optical component having the second optical state formed on the substrate by the first reporter reagent in step (iii), and the step in (iii) is repeated using electromagnetic radiation of the same or different wavelengths absorbed by the photosensitizer of the second reporter reagent; and (v) A step of detecting the set of local regions of the optical component having the second optical state, which is formed on the substrate by the second reporter reagent in step (iv), Includes, The two sets of local regions are distinguishable from each other by the formation rate of the multiple sets of local regions, the formation order of the multiple sets of local regions, or the size of the local regions when combined with the formation rate of the multiple sets of local regions or the formation order of the multiple sets of local regions. The method according to any one of claims 1 to 4.

7. The method according to any one of claims 1 to 6, wherein each of the first and second reporter reagents further comprises polymer particles, and the photosensitizer is encapsulated within the polymer particles.

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

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