Method for detecting analytes

Abstract

The present invention provides a method for detecting an analyte in a sample and an apparatus for carrying out said method. The method of the present invention comprises the steps of: (i) providing a mixture comprising a sample, a reporter reagent and a pre-activation reagent to a device, the device comprising a substrate having an optical element and a binding element, the optical element and the binding element being bound to a surface of the substrate, the reporter reagent being capable of generating reactive oxygen species from the pre-activation reagent upon absorption of electromagnetic radiation, and the optical element being capable of changing from a first optical state to a second optical state upon reaction with the reactive oxygen species; (ii) allowing a portion of the reporter reagent to bind to the surface of the substrate in proportion to the concentration of the analyte by the binding member; (iii) irradiating the device with electromagnetic radiation for absorption by the reporter reagent, thereby forming a set of localized regions of optical elements having the second optical state on the substrate, wherein prior to irradiation, the substrate is contacted with a deuterium-enriched liquid and / or the substrate has a deuterium-enriched layer on its surface; (iv) detecting a set of localized regions on the substrate having the second optical state.

Description

Technical Field

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

Background Art

[0002] Many techniques are available for measuring 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 a target of interest and a reporter reagent having a certain 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 is removed (by washing) and the amount of the reporter reagent is measured to obtain a measurement of the amount of the analyte present in the sample. There are various variations on how these types of binding assays can be performed. For example, the analyte can first be bound to the capture reagent and then the reporter can be added in a separate step, or the analyte can first be bound to the reporter and then to the capture reagent.

[0003] In this type of binding assay, a wide variety of reagents can be used as capture and reporter, including nucleic acids, carbohydrates, antigens, peptides, proteins and antibodies, etc. Also, the target analytes are diverse and include peptides, proteins, antibodies, nucleic acids, cells, carbohydrates, small molecules, therapeutic agents, abused drugs, steroids, hormones, lipids, etc.

[0004] Assays using antibodies are generally called immunoassays. There are various forms of immunoassays. For example, when a capture antibody is used to capture an analyte and a reporter antibody is used to generate a measurable signal, this is generally called a sandwich immunoassay. Alternative forms are known where a binding reagent is attached to a solid phase, and in solution, the target analyte competes with a labeling reagent that also binds to the binding reagent. If the analyte is absent, a high level of labeling reagent binds, resulting in a high signal. If the analyte is present, part of the binding site is blocked, reducing the amount of labeling reagent that binds and thus reducing the signal. These assays are generally known as inhibitory assays or competitive assays. Several types of competitive assays are known. For example, an antibody can be bound to a solid phase, and a labeled analyte (or an analogue of the analyte) can compete for binding sites on the antibody. Alternatively, an analogue of the analyte can be immobilized, and a labeled antibody can be bound to this surface. If the analyte is present in the sample, the analyte binds to the antibody in solution, preventing binding to the surface and reducing the signal.

[0005] Many forms of assays exist, and a wide variety of labels can be used. For example, an assay can be heterogeneous in that excess labeling is removed before measurement by using a washing step. Excess labeling can also be removed by flowing the sample and reporter through 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 capture and reporter being in close proximity to generate some kind of signal. An example of a homogeneous assay is an agglutination assay in which particles bind in solution. The aggregated particles cause light scattering, which can be measured by turbidimetry or nephelometry. A further example of a homogeneous assay using particles is the luminescent oxygen channeling immunoassay (LOCI), which will be described in more detail below.

[0006] Another example of a homogeneous assay is fluorescence resonance energy transfer (FRET), where the capture reagent and reporter reagent are the donor and acceptor fluorophores, respectively. Excitation of the donor leads to energy transfer to the acceptor, followed by emission.

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

[0008] The label that binds to the reporter-binding reagent can be a light-absorbing substance such as a dye, gold particles, or stained latex microspheres. In principle, the larger the particle, the more light it can absorb and the more signal it can generate. However, as detailed below, particle labels have size limitations and are not practical for use in assays. Luminescent labels such as fluorescent labels, chemiluminescent labels, bioluminescent labels, and electrochemiluminescent labels are also known. Luminescent labels are encapsulated in particles for specific types of assays. Signal amplification can also be performed using enzymatic or catalytic reactions. Enzymes can be used to convert the substrate from a leuco dye to a colored form, or to a fluorescent or luminescent form. It is common practice to remove excess enzyme using a washing step before adding the substrate, thereby ensuring 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 as a signal transduction method, are also known. 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," Ed. 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, can be prone to saturation depending on the path length that light must travel through the sample. Luminescence methods have a reduced tendency to saturate because the detector gain can be attenuated to cope with higher levels of emission. In heterogeneous assays, the maximum signal is reached and the system saturates when all antibody binding sites are filled with analyte. Excess analyte is usually removed in a washing step before adding the reporter. In homogeneous assays, the concentration of analyte can be higher than the effective concentration of the capture antibody and / or reporter antibody, a phenomenon known as high-dose hooking. In this case, at very high concentrations, all binding sites on the capture and reporter may be blocked, reducing the assay signal and potentially leading to erroneous results.

[0013] Low detection levels are affected by various factors. Generally, all assays are affected by the quality of the antibody used (affinity and specificity) and its characteristics, such as cross-reactivity between the antibody and the analyte. The lower limit of detection also depends on the assay settings and factors that affect the signal-to-noise ratio of the 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 capture the analyte. The wells are washed, and then an excess of reporter is added to bind to the captured analyte. The excess reporter is then washed away, and a substrate that can react with the enzyme is added to convert it to its 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. If the amount of analyte is very small (e.g., less than 1 picomole), only a very small amount of enzyme will be present on the surface of the wells. ABTS reacts with the enzyme to produce a green morphology, which then diffuses into the majority of the liquid, creating a solution so dilute that it is indistinguishable from the background signal. Automated substrate conversion may also produce colors that interfere with the measurement. Similarly, other detection methods, such as fluorescence, can be affected by interfering factors or autofluorescence from elements 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 be denatured 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 turns over the substrate and contributes to the overall signal, it becomes impossible to distinguish between a signal caused by a specifically bound reporter and one caused by a nonspecifically bound reporter. Nonspecific binding can also be promoted by many elements present in the original sample, which can bind to the capture surface during the initial incubation and modify the surface properties of the capture layer to create a surface that can bind to the reporter. To minimize nonspecific reporter binding, all reagents and reaction conditions used in the assay, including antibodies, surfactants, temperature, and ionic strength, must be carefully optimized.

[0015] Generally, the detection limit of conventional immunoassays is approximately 0.1 picomoles to 1 nanomoles, depending on the assay method. Developing assays with very low detection limits using conventional approaches often requires extensive optimization, including rigorous 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 relative to the background.

[0016] One approach used to avoid problems related to the signal-to-noise ratio and improve the detection limit is to measure individual coupled events and count these coupled events as "on" or "off" events if the measured value exceeds a local threshold. In this way, much of the background noise can be removed. This can be analogous to the digitization of audio and communication signals. These digital assays have been shown to reach detection limits that were previously unattainable using conventional analog methods. 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," Trends in Biotechnology, 2015, 33, 343.

[0017] Most labels / reporters (fluorescent dyes, pigments, etc.) used in assays are too small to be individually observed even at high magnification using a wide-field microscope. Therefore, the presence of these labels can only be measured as a bulk phenomenon, not by counting individual labels. In contrast, particle labels such as latex particles can theoretically be visualized with a wide-field optical microscope if they exceed a certain size. Depending on the optical setup, if the particle diameter is several hundred nanometers or more, visualization of the particles can be initiated, depending on the numerical aperture and the type of microscope.

[0018] However, using particles of this size as labels to monitor individual binding events (such as antibody-antigen interactions) on the capture surface is impractical for several reasons. For example, particles of this size diffuse very slowly compared to other types of labels, impairing the reaction rate on a planar surface. Also, particles of this size begin to exhibit macroscopic buoyancy effects, and will settle or float if the density of the particles differs significantly from that of the medium containing them, which can cause problems with the assay format. Particles of this size tend to bind nonspecifically to surfaces, causing high background noise that is difficult to remove. Finally, excess particles need to be removed, requiring a washing step. However, larger particles begin to experience shear effects when liquid flow is present, and the shear force acting on the particles becomes greater than the breaking strength of the antibody-antigen interaction (approximately 60-250 pN), causing the particles to 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 amount of beads is sufficient to minimize the possibility of each bead containing multiple analytes and reporters. The beads are washed again and then packed into an array of microwells, each capable of holding only one bead. The volume of the microwells is on a femtoliter scale. If the enzyme is bound to the beads, the fluorescent substrate in the well changes. The small size of the wells prevents excessive diffusion of the fluorescent product. Each well is counted as an "on" or "off" event if the fluorescence exceeds a threshold.

[0021] The SMC system is used in Merck Millipore's Erenna and SMCxPRO systems. The basic measurement technique is the same in both systems. Magnetic beads coated with capture antibodies 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 induce dissociation of the sandwich complex, and it is then transferred to the measurement vessel. The presence of the fluorescent tag is then measured using a confocal fluorescence microscope, and small amounts of sample are examined sequentially to determine whether the fluorescent tag is present. If the signal of an individual measurement exceeds the threshold, it is counted as an "on" event for that measurement.

[0022] Several independent academic reviews on highly sensitive immunoassays have highlighted that digital approaches to immunoassays enable unprecedented improvements in detection limits (see Yeung and Cretich, references mentioned above).

[0023] The detection limits of the Quanterix and Merck Millipore systems vary depending on the amount of sample used in the assay. In the case of a 10 microliter serum or plasma sample, the theoretical limit is the detection of a single binding event corresponding to 1 molecule. However, in terms of molar concentration, this corresponds to 100,000 molecules per liter of sample, or 0.16×10 -18 mol (0.16 attomol) per liter. Summary of the Invention Problems to be Solved by the Invention

[0024] However, the above-mentioned Quanterix and Merck Millipore systems are complex and difficult to handle, each requiring many washing steps and transfer steps. Furthermore, the assay can only be performed on samples that do not contain cellular material, and expensive equipment is required to achieve the performance provided by the system. Therefore, a simpler and more cost-effective high-sensitivity system is still needed. Means for Solving the Problems

[0025] Therefore, the present invention provides a method for detecting an analyte in a sample, the method comprising the following steps: (i) providing a mixture comprising a sample, a reporter reagent, and a pre-activation reagent to an apparatus, the apparatus comprising a substrate having an optical element and a binding element, the optical element and the binding element being bound to the surface of the substrate, the reporter reagent being capable of generating reactive oxygen species from the pre-activation reagent by absorption of electromagnetic radiation, and the optical element being capable of changing from a first optical state to a second optical state by reaction with the reactive oxygen species; (ii) enabling a portion of the reporter reagent to bind to the surface of the substrate in proportion to the concentration of the analyte by the binding element; (iii) irradiating the device with electromagnetic radiation for absorption by the reporter reagent, thereby forming a set of local regions of the optical element having the second optical state on the substrate, wherein the substrate is contacted with a deuterium-enriched liquid before irradiation and / or the substrate has a deuterium-enriched layer on its surface; and (iv) detecting the set of local regions having the second optical state on the substrate.

[0026] Thus, the present invention provides a method for detecting an analyte in a sample, wherein only the reporter reagent near the surface of the substrate produces a signal, and the signal is a local region of an optical element in the second optical state. What is detected is a set of local regions of the optical element in the second optical state. The signal is generated by the reaction of reactive oxygen species with the optical element, and a richer deuterium environment provides a larger signal that is easier to image. Thus, the present invention simplifies the digital detection of analytes and facilitates homogeneous assays for various samples, including samples containing cellular material.

Advantages of the Invention

[0027] The present invention is used for detecting an analyte in a sample (which may also be through the detection of a complex or derivative of the analyte).

Brief Description of the Drawings

[0028] Hereinafter, the present invention will be described with reference to the drawings. [Figure 1] FIG. 1 shows various elements that can be used in the present invention. [Figure 2] FIG. 2 shows a device in which a reporter reagent binds to the surface of a substrate through the formation of a sandwich with an analyte. [Figure 3] FIG. 3 shows the device during irradiation. [Figure 4] FIG. 4 shows the device after irradiation in a non-deuterium-rich environment. [Figure 5] FIG. 5 shows the device after irradiation in a deuterium-rich environment. [Figure 6]Figure 6 shows a homogeneous assay format in which excess labeled reporter and cellular elements are present in the solution during the assay period. [Figure 7] Figure 7 shows the optical configuration for detection. [Figure 8] Figure 8 shows the substrate and wells prepared for the present invention. [Figure 9] Figure 9 shows a sample chamber prepared using the substrate and wells shown in Figure 8. [Figure 10] Figure 10 shows the detection results for Example 4. [Figure 11] Figure 11 shows the detection results for Example 5. [Modes for carrying out the invention]

[0029] The components of Figure 1 are: analyte 1; photosensitizer 2; capture antibody 3; reporter antibody complex of latex particles injected with photosensitizer 4; fluorescent dye 5; bleached fluorescent dye 6; fluorescently labeled streptavidin 7; fluorescently labeled streptavidin (bleached) 8; biotin-labeled BSA 9 and red blood cells 10.

[0030] Step (i) of the method of the present invention comprises providing a mixture comprising a sample, a reporter reagent, and a pre-activating reagent to an apparatus, the apparatus comprising a substrate 12 having optical elements and binding elements, the optical elements and binding elements being bonded to the surface of the substrate 12. The sample, reporter, and pre-activating reagent may be pre-mixed before adding the mixture to the apparatus; or the sample, reporter reagent, and pre-activating reagent may be continuously added to the apparatus to form the mixture. The mixture may contain additional reagents, but preferably the mixture consists of a sample, a reporter reagent, and a pre-activating reagent.

[0031] For the purpose of explaining the underlying principle of the present invention, Figure 2 shows an apparatus in which a reporter reagent is bonded to the surface of a substrate via analyte 1 before irradiation. The apparatus comprises a substrate 12 and a sample chamber 24 for holding a sample containing a dissolved or suspended analyte. The substrate may be any substrate that allows detection of a set of local regions having a second optical state on the substrate. Preferably, the substrate is a transparent substrate, and more preferably, the substrate is glass. The substrate 12 is typically planar.

[0032] Substrate 12 has a capture antibody 3 bound to its surface via fluorescently labeled streptavidin 7 and biotin-labeled BSA 9. The fluorescent dye functions as an optical element, and the capture antibody 3 functions as a binding element. Biotin-labeled BSA and streptavidin are inert polymers that facilitate the binding of the optical and binding elements to the surface of substrate 12. This approach is used when the optical elements are water-soluble, as the optical elements need to be immobilized on the surface of substrate 12 in order to fix the substrate 12.

[0033] Although the optical elements and binding elements are shown in this manner, any technique for holding the optical elements and binding elements near the surface of the substrate 12 is applicable. For example, the optical elements and binding elements may be separate reagents, or the binding elements and optical elements may be bonded together.

[0034] The optical elements may also be encapsulated within a polymer layer coated on the surface of the substrate 12, and the binding elements 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 elements. This approach can be used when the optical elements are water-insoluble.

[0035] Alternatively, an optical element may be impregnated into a layer of gel, such as a hydrogel, the gel / hydrogel layer may be coated onto the surface of the substrate 12, and the binding element may be bonded to the gel / hydrogel layer.

[0036] Step (ii) of the method of the present invention includes binding a portion of the reporter reagent to the surface of the substrate in proportion to the concentration of the analyte using a binding element. This step can be achieved by leaving the apparatus in place for a certain period of time, for example, 10 minutes.

[0037] In Figure 2, the captured antibody 3 is bound to the surface of the substrate 12 via streptavidin labeled with the fluorescent dye 7 and BSA9 labeled with biotin. The antibody complex 4 of the photosensitizer-impregnated latex particle functions as a reporter reagent.

[0038] The reporter reagent is shown to be bound to the surface of the substrate 12 via analyte 1. Thus, in the presence of the analyte, the reporter reagent binds to the surface of the substrate in a form called a “sandwich” complex, as described below with reference to Figure 6, in proportion to the concentration of the analyte.

[0039] All steps up to this point are carried out in the absence of light. Step (iii) of the method of the present invention includes irradiating the apparatus with electromagnetic radiation for absorption by a reporter reagent, thereby forming a set of local regions of optical elements having a second optical state on the substrate 12. Step (iii) of the method of the present invention further includes providing a deuterium-rich environment, by contacting the substrate with a deuterium concentrate before irradiation and / or the substrate having a deuterium-concentrated layer on its surface.

[0040] Accordingly, the present invention provides a method in which an apparatus is irradiated with electromagnetic radiation, and this radiation is absorbed by a photosensitizer in a reporter reagent. The reporter reagent then generates reactive oxygen species from a pre-activating reagent, which react with the optical elements, changing from a first optical state to a second optical state, thereby forming a set of local regions of the optical elements having the second optical state on the substrate 12.

[0041] Figure 3 shows the apparatus of Figure 2 being irradiated by light source 13 with electromagnetic radiation (commonly referred to as "light"), preferably visible light. The light source irradiates the sample chamber 24 with light of an appropriate wavelength to excite the photosensitizer injected into the latex particles 4. The wavelength depends on the photosensitizer, but a preferred wavelength is 680 nm. Typically, the apparatus is irradiated for at least 30 seconds. Preferably, the apparatus is irradiated with electromagnetic radiation for more than 1 second, more preferably at least 2 seconds, more preferably at least 5 seconds, more preferably at least 10 seconds, more preferably at least 15 seconds, more preferably at least 20 seconds, more preferably at least 25 seconds, and most preferably at least 30 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.

[0042] Figure 4 shows the apparatus of Figure 3 after irradiation, where the substrate was not in contact with the deuterium concentrate before irradiation. Reactive oxygen species generated by the photosensitizer in latex particle 4 react with the dye optical elements of streptavidin 7 labeled with a fluorescent dye, changing the dye from a fluorescent state to a non-fluorescent state. Streptavidin 7 labeled with a fluorescent dye in the fluorescent state becomes streptavidin 8 labeled with a fluorescent dye in the non-fluorescent state. Only the dye in close proximity to the reporter reagent changes from the first optical state to the second optical state.

[0043] Figure 5 shows the apparatus from Figure 3 after irradiation, where the substrate was in contact with deuterium concentrate before irradiation. Reactive oxygen species generated by the photosensitizer in latex particle 3 react with the dye optical elements of streptavidin 7 labeled with a fluorescent dye, changing the dye from a fluorescent state to a non-fluorescent state. Streptavidin 7 labeled with a fluorescent dye in the fluorescent state becomes streptavidin 8 labeled with a fluorescent dye in the non-fluorescent state. Compared to Figure 4, the dye located farther away from the reporter reagent changes from the first optical state to the second optical state.

[0044] While it has been shown that the dye changes from a fluorescent state to a non-fluorescent state, other optical elements undergo different changes. In one embodiment, the dye changes from a non-fluorescent state to a fluorescent state. In another embodiment, the optical element in the first optical state absorbs light of one or more first wavelengths, and the optical element in the second optical state absorbs light of one or more second wavelengths, where the first and second wavelengths are different. The light is preferably visible light. The first and second wavelengths are different. In this embodiment, the optical element undergoes a change in color.

[0045] Alternative first and second optical states include changes in optical polarization, fluorescence lifetime, refractive index, light scattering (including Raman scattering), and other optical effects.

[0046] Figure 6 shows the apparatus with a whole blood sample and excess reporter reagent present after irradiation. The whole blood sample also contains additional components such as red blood cells 10. Preferably, the substrate 12 forms the upper part of the sample chamber 24, allowing the red blood cells 10 to settle from the substrate 12.

[0047] A portion of the reporter reagent is bound to the surface of the substrate 12 by a sandwich complex of analyte 1 and capture antibody 3. Therefore, the sample contains both bound and unbound reporter reagent in solution. The depth of the sample chamber 24 is designed to minimize the diffusion path length of the reporter reagent and to ensure rapid equilibrium. Typically, the depth of the sample chamber is 50–200 μm.

[0048] In a typical sandwich immunoassay using the present invention, the substrate 12 has a capture antibody 3 bound to its surface via a fluorescently labeled streptavidin 7 and a biotin-labeled BSA 9. The dye functions as an optical element, and the antibody functions as a binding element. Although the optical and binding elements are shown in this manner, any technique for holding the optical and binding elements in close proximity to the surface of the substrate 12 is applicable, as described above.

[0049] In this invention, the sample chamber 24 is filled with a sample containing the analyte 1. A reporter reagent, such as an antibody complex 4 of latex particles injected with a photosensitizer, is also added to the sample chamber 24. When a whole blood sample is used, the sample may contain additional components such as red blood cells 10. Whole blood and plasma samples naturally contain dissolved oxygen, which functions as a pre-activating reagent.

[0050] Next, equilibrium is achieved. The antibody complex 4 of the photosensitizer-injected latex particles binds to the surface of the substrate 12 by the capture antibody 3 in proportion to the concentration of analyte 1. The antibody complex 4 of the photosensitizer-injected latex particles functions as a reporter reagent. Because there is an excess of the antibody complex 4 of the photosensitizer-injected latex particles, all of the analyte 1 forms a sandwich complex. Thus, a portion of the reporter reagent binds to the surface of the substrate 12 by the binding element in proportion to the concentration of analyte 1. Therefore, the sample contains bound reporter reagent and unbound reporter reagent released into the solution.

[0051] The reporter reagent must permanently bind to the surface of the substrate 12 throughout the entire irradiation period in order to achieve a complete conversion from the first optical state to the second optical state. A reporter reagent that does not permanently bind to the surface of the substrate 12 throughout the entire irradiation period will cause a reduction in the change of the optical element from the first optical state to the second optical state, resulting in a weaker signal. An unbound reporter reagent in solution will not change the optical element from the first optical state to the second optical state.

[0052] This offers a significant advantage over other digital assay methods in that it eliminates the need for a washing step. Thus, the present invention can be performed as 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 make it possible to distinguish 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 12 (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) and (ii) are performed without a washing step, i.e., the method of the present invention is performed without removing the sample from the substrate in steps (i) and (ii).

[0053] The photosensitizer in the bound reporter reagent portion interacts indirectly with the optical element, causing a change via the additional reagent; that is, the photosensitizer is excited and transfers this energy to the additional element, which then transfers this energy to the optical element.

[0054] In a preferred embodiment, absorption by the photosensitizer is for interaction with a pre-activating reagent present in the mixture to generate reactive oxygen species, the activating reagent reacting with the optical element to change the optical element from a first optical state to a second optical state.

[0055] The pre-activating reagent may be present in the sample, or it may be added as an additional reagent to the mixture of the sample and reporter reagent. The pre-activating reagent may be ground state oxygen, also known as triplet oxygen. The pre-activating reagent may be any reagent that can interact with the photosensitizer after irradiation with electromagnetic radiation to produce a reactive oxygen species. In a preferred embodiment, the pre-activating reagent is triplet oxygen. In another preferred embodiment, the activating reagent is a reactive oxygen species. Preferably, the reactive oxygen species is selected from hydroxyl radicals, superoxides, peroxides, organic peroxides, peroxynitrites, singlet oxygen, and mixtures thereof. More preferably, the reactive oxygen species is singlet oxygen.

[0056] 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 continues to react with the optical elements. In a particularly preferred embodiment, the pre-activating reagent is triplet oxygen, and the activating reagent is singlet oxygen.

[0057] Traditionally, singlet oxygen has been used in immunoassays of the luminescent oxygen channeling immunoassay (LOCI). The LOCI immunoassay is a homogeneous, non-digital assay using donor beads and acceptor beads. Donor beads generate singlet oxygen upon irradiation 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. Because the beads are spatially separated, background signaling is minimized; however, due to the short measurement time in the LOCI assay, it is not possible to distinguish between long-term binding events and transient binding events. Unbound beads in close proximity to each other can also generate unwanted background signaling. The LOCI assay, for example, can achieve a detection limit of approximately 1–5 pg / mL in its most sensitive assay for interleukin-6 (IL-6) and thyroid-stimulating hormone (TSH). In this invention, in order to detect the signal, the "donor" particle, the reporter reagent, must be in close proximity to the surface of the substrate 12, rather than to particles in solution, and must remain there throughout the irradiation period. Therefore, this invention is more sensitive than the LOCI assay and can detect analytes at lower concentrations.

[0058] In one embodiment, the substrate is contacted with a deuterium concentrate before irradiation. Any method of providing the deuterium concentrate may be suitable for use in the present invention. The deuterium concentrate may be provided by contacting the sample with the deuterium concentrate before analysis. For example, the sample may be diluted or washed with a deuterium-rich liquid before irradiation. The deuterium concentrate preferably contains deuterium oxide and may be a deuterium concentration buffer. Using deuterium oxide is not prohibitively expensive.

[0059] In another embodiment, the substrate has a deuterium-concentrated layer on its surface. Any method of providing a deuterium-concentrated layer on the surface of the substrate may be suitable for use in the present invention. This layer may be deuterium-concentrated by proton exchange with a deuterated solution, such as deuterium oxide, or the layer may be synthesized using a deuterium-rich starting material.

[0060] The deuterium-concentrated layer may contain deuterium-concentrated polymers such as deuterium-concentrated proteins or deuterium-concentrated polysaccharides. The deuterium-concentrated polymer may be in the form of a deuterium-concentrated gel, particularly a hydrogel. Preferably, the deuterium-concentrated layer contains a deuterium-concentrated protein layer or a deuterium-concentrated polysaccharide layer.

[0061] This layer can be prepared by hydrogen-deuterium exchange of a deuterium-poor starting material. Alternatively, deuterium-concentrated polysaccharides or deuterium-concentrated proteins can be prepared by biosynthesis. In another embodiment, the deuterium-concentrated layer may include a deuterium-concentrated gel, which is synthesized using a deuterium-rich starting material. In yet another embodiment, the deuterium-concentrated layer may include a hydrogel, and the solid and / or liquid phases may be deuterium-concentrated. The deuterium-concentrated solid phase may be prepared from a deuterium-rich starting material, or the liquid phase may include a deuterium concentrate. Preferably, the deuterium concentrate is also deuterium oxide.

[0062] In a further embodiment, the substrate is brought into contact with a deuterium concentrate before irradiation, and the substrate has a deuterium concentrate layer on its surface.

[0063] Deuterium exchange is a well-known chemical reaction in which a covalently bonded hydrogen atom is replaced by a deuterium atom. This is most easily applied to exchangeable protons. Exchangeable hydrogen atoms are usually bonded to hydroxyl or amino groups, but thiols can also be targets for deuterium exchange. These are typically introduced using D2O. Non-exchangeable hydrogen atoms within molecules can also be replaced with deuterium, but this requires a deuterated starting material.

[0064] The surface of the substrate is typically deuterized by using a deuterated polymer. Deuterium exchange can be used for proteins and polysaccharides, particularly those containing amino or hydroxyl groups. However, since these exchangeable deuterium atoms may be lost to the bulk solution when the substrate comes into contact with the sample, it is preferable to use pre-deuterated starting materials for the substrate elements.

[0065] When deuterium concentration is performed using a sample processing or washing step, deuterium exchange is a preferred approach.

[0066] Reactive oxygen species, once generated, have a maximum distance they can travel from their source. Therefore, the reaction between the reactive oxygen species and the optical element attached to the surface of the substrate forms a localized region centered on the reporter reagent.

[0067] The concentration of reactive oxygen species as a function of distance from the reporter molecule is thought to be governed by three factors: radial diffusion, the lifetime of the reactive oxygen species, and the bimolecular reaction rate of the reactive oxygen species.

[0068] The bimolecular reaction of reactive oxygen species can be productive in that the reactive oxygen species react with molecules of the optical element, changing the optical element from a first optical state to a second optical state.

[0069] Bimolecular reactions of reactive oxygen species can be unproductive in that the reactive oxygen species reacts with molecules that are not optical elements, resulting in the quenching of the reactive oxygen species. An example of an unproductive reaction is the reaction of a reactive oxygen species with a reactive hydrogen atom, such as the hydrogen atom of an NH or OH functional group. Reactive oxygen species such as singlet oxygen are also known to form adducts with aromatic groups such as histidine residues in proteins.

[0070] It was found that reducing the effective concentration of active hydrogen atoms around the reporter reagent reduced the ratio of unproductive reactions to productive reactions of reactive oxygen species. In this way, more optical element molecules within the region of the bound reporter reagent changed from the first optical state to the second optical state, resulting in greater signal contrast. Furthermore, the time required to convert the optical elements from the first optical state to the second optical state was reduced.

[0071] Reducing the effective concentration of active hydrogen atoms around the reporter reagent causes reactive oxygen species to move further away from the reporter reagent, resulting in a larger local area of ​​the optical element in the second optical state. This larger local area can be easily visualized using simple optical methods.

[0072] This invention provides a method for reducing the effective concentration of reactive hydrogen atoms around a reporter reagent by using a deuterium-enriched analog of a mixture or a deuterium-enriched analog of a component of an apparatus. It is believed that the reaction rate with reactive oxygen species decreases due to the dynamic isotope effect when hydrogen is replaced with deuterium.

[0073] Additional approaches to reduce the effective concentration of reactive hydrogen atoms around the reporter reagent are suitable for carrying out the present invention, such as using materials with low reactive hydrogen content, for example, by using an inert polymer instead of a layer formed of polysaccharides or proteins to prepare the components of the apparatus.

[0074] Other approaches to reduce the non-productive reactions of reactive oxygen species may also be included in the methods and apparatus of the present invention.

[0075] Therefore, the present invention provides a deuterium-rich environment in which reactive oxygen species are generated. In this way, the reactive oxygen species diffuse more from the reporter reagent than in a deuterium-poor environment, thus generating a larger signal. In one embodiment, a larger signal is generated by the reporter reagent. In another embodiment, a signal of the same size is generated using smaller reporter reagent particles. Furthermore, it is also possible to generate a signal of a specific size more quickly or to stimulate the photosensitizer using lower intensity electromagnetic radiation.

[0076] The advantage of generating a large signal is that it is easier to detect than a small signal. In a standard optical microscope, the lens selection (including the numerical aperture and magnification) and the measurement wavelength determine the system's resolution and depth of field. To measure dark spots, it is preferable to use the lowest possible magnification lens. Low-magnification lenses have a deep depth of field and a wide field of view. This makes it easier to focus the lens on a surface and allows for the visualization of a larger surface area without moving the lens or substrate relative to each other. This reduces the complexity and cost of the equipment.

[0077] While the sample and reporter may be mixed before being provided to the instrument, incubation and binding are typically performed with small amounts without mixing. Therefore, it is advantageous for the reporter reagent to contain smaller particles, as smaller particles diffuse more rapidly onto the substrate surface. Furthermore, smaller particles have less steric constraint and greater rotational energy, resulting in a higher binding rate upon diffusion onto the substrate surface. A higher binding rate allows for assays to be performed within shorter timeframes, which is particularly beneficial for point-of-care applications. Moreover, a higher binding rate enables more sensitive assays.

[0078] Therefore, the method of the present invention facilitates the detection of the signal formed in step (iii) of the present invention.

[0079] In a preferred embodiment, the optical element is a dye. Preferably, the optical element is selected from one of the following dyes and mixtures thereof: [ka]

[0080] Dyes (1) to (5) are known and used as cell probes to monitor the formation of reactive oxygen species within cells under oxidative stress. However, these dyes are not known to be used in standard or digital immunoassays. Dyes (6) are common fluorescent dyes. Dyes (7) and (8) are known to react specifically with singlet oxygen, but their use in immunoassays is unknown. A wide range of other commercially available fluorescent dyes suitable for use in this invention exist, including Alexafluor dyes, BODIPY dyes, Rhodamine dyes, Texas Red, Oregon Green, Cascade Yellow, Pacific Blue, etc. For further examples, see The Molecular Probes Handbook by Thermo Fisher Scientific.

[0081] The dye singlet oxygen sensor green (SOSG) (1) reacts with singlet oxygen to convert from a weak fluorescence form to a strong fluorescence form. Singlet oxygen reacts with anthracenyl groups to form endoperoxides. In a preferred embodiment, the optical element is SOSG.

[0082] Boron dipyromethene pigment 581 / 591 (BODIPY 581 / 591 (2) reacts with reactive oxygen species such as singlet oxygen and hydroxyl radicals, causing a shallow color shift in the maximum fluorescence excitation / emission value.

[0083] Dyes (3) to (5) share the same core structure and react with common oxidizing agents containing singlet oxygen to be converted into a fluorescent form. Therefore, these dyes can be converted from a leuco state to a fluorescent state.

[0084] It has been found that dye (6) reacts with singlet oxygen to convert from a fluorescent form to a non-fluorescent form. Surprisingly, it was found that fluorescein can completely remove fluorescence near the photosensitizer, creating a dark non-fluorescent region on the fluorescent substrate. Dyes (7) and (8) fluoresce under UV light and react with singlet oxygen to produce endoperoxides, generating non-fluorescent products.

[0085] Preferably, the optical element 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 element is non-fluorescent when in the first optical state, and fluorescent when in the second optical state. However, more preferably, the optical element is fluorescent when in the first optical state, and non-fluorescent when in the second optical state.

[0086] In another preferred embodiment, when in a first optical state, the optical element fluoresces at one or more wavelengths, and when in a second optical state, the optical element fluoresces at one or more other wavelengths. In this embodiment, the optical element shifts its fluorescence excitation / emission maximum value.

[0087] 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.

[0088] Step (iv) of the method of the present invention includes detecting a set of local regions having a second optical state on a substrate.

[0089] Optical elements having a second optical state form a set of local regions on the substrate. Advantageously, these local regions having a second optical state can be counted as individual binding events. Therefore, the present invention is suitable for performing digital assays. However, if numerous binding events exist and the majority of the optical elements are in the second optical state, bulk changes can be detected.

[0090] In a preferred embodiment, a set of local regions having a second optical state on the substrate is detected by counting local regions within the set of local regions having a second optical state on the substrate, or by measuring the set of local regions having a second optical state. It is also detected by measuring the 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 the set of local regions having a second optical state on the substrate.

[0091] Local regions with a second optical state may need to exceed a threshold corresponding to either a background signal or a transient binding event, depending on the first and second optical states. For example, a sample may contain background autofluorescence, but if this does not exceed a threshold, the signal will not be affected due to the digital nature of the assay. Computer software can be used to detect the presence of local regions and distinguish between local regions that exceed the threshold and those that fall below the threshold.

[0092] Furthermore, the surface of the substrate is scanned before irradiation with the photosensitizer to obtain a 2D image of the surface, and scanned again after irradiation, so that the pre-irradiation image is reduced from the post-irradiation image by subtracting the background, artifacts, contaminants, and autofluorescence of the surface or the sample itself.Therefore, in a preferred embodiment, the method of the present invention further includes the step of subtracting any elements having a second optical state on the substrate detected before irradiating the apparatus with electromagnetic radiation from the set of local regions having a second optical state on the substrate detected in step (iv).In this way, the image with the background subtracted displays only the changes in the optical properties of the substrate (such as changes in fluorescence intensity).

[0093] The set of local regions having a second optical state on the substrate is typically discrete regions on the substrate. However, some local regions may be excluded from detection due to their morphology. While local regions corresponding to individual coupling events tend to be uniformly circular, some local regions may have non-uniform shapes corresponding to artifacts. Furthermore, some local regions may be larger than other local regions where particles have aggregated. Therefore, in a preferred embodiment, only uniformly circular local regions having a second optical state on the substrate are detected.

[0094] A set of localized regions on a substrate can be detected using simple optical means. A suitable optical configuration for detection is shown in Figure 7. The components of Figure 7 are: objective lens 14; dichroic mirror 15; radiation filter 16; photodetector / camera 17; microprocessor 18; data output 19; excitation filter 20; and light source (LED / laser) 21. In a preferred embodiment, a set of localized regions on the substrate having a second optical state is detected using an optical microscope. More preferably, a set of localized regions on the substrate having a second optical state 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 microscopes that illuminate and record only one focal point at a time. The advantages of confocal microscopes 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 costly compared to simple wide-field methods.

[0095] When dyes (1) and (3) to (5) are used, a localized fluorescent region (or increased fluorescence if there are many binding events) is observed on the surface of the substrate where the optical elements are converted to a second optical state. To detect this fluorescence, the substrate can be illuminated with the excitation wavelength of the dye, and the surface of the substrate can be scanned to induce emission.

[0096] When dye (2) is used, a decrease in fluorescence emission at 610 nm or an increase in fluorescence emission at 515 nm is measured, or both wavelengths are monitored.

[0097] When dyes (6) to (8) are used, dark localized regions (or dark images if there are many binding events) are observed on the surface of the substrate where the optical elements are converted to a second optical state. To detect these dark spots or images, a wide-field fluorescence microscope equipped with a light source (e.g., an LED) and a photodetector (a camera such as a photomultiplier tube or CCD) can be used, with excitation and emission filters suitable for detecting each fluorescent dye molecule (for example, fluorescein can be detected with excitation at 490 nm and emission at 520 nm).

[0098] When a binding event occurs, the photosensitizer is located in close proximity to the substrate. That is, the photosensitizer is located in close proximity to the surface of the substrate and interacts with the optical element, converting the optical element from a first optical state to a second optical state when the device is irradiated. However, the actual distance between the photosensitizer and the surface of the substrate depends on many variables, such as the size and properties of the photosensitizer, the size and properties of the binding element, the reporter reagent and analyte, and the properties of the sample medium.

[0099] The binding element has a binding site that can bind the 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 in 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 immunoassay assays, binding is directly proportional to the concentration of the analyte, while in competitive assays, binding is indirectly proportional to the concentration of the analyte.

[0100] One specific type of competitive assay involves immobilizing an antibody against the analyte on a substrate and introducing a labeled analog of the analyte into the sample. The analyte and its labeled analog "compete" for the antibody on the surface. In the absence of the analyte, the labeled analog binds at the maximum possible rate. However, in the presence of the analyte, the analyte concentrates on the antibody on the substrate, reducing the binding rate of the analog.

[0101] The binding element may be adapted to bind to the analyte, or a complex or derivative of the analyte, in which case the reporter reagent binds to the binding element in the presence of the analyte, or a complex or derivative of the analyte. In this case, the binding element has a binding site that can bind to the reporter reagent 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.

[0102] Alternatively, the binding element itself may be an analog of the analyte, and the reporter reagent binds directly to the binding element (it is an analog because it is bound to the surface of the substrate via either covalent or non-covalent interactions). In this case, the binding element competes with the unbound analyte, or a complex or derivative of the unbound analyte, for binding of the reporter reagent. Therefore, the binding element can simply bind to the reporter reagent.

[0103] The concentration of the analyte in the sample is measured by determining the degree of binding (directly or mediated by the analyte / complex or derivative of the analyte) to the binding element of the reporter reagent.

[0104] The assay also requires the presence of a reporter reagent. The reporter reagent of the present invention contains a photosensitizer. Upon absorbing electromagnetic radiation, the photosensitizer reacts with a pre-activating reagent to generate reactive oxygen species. These reactive oxygen species continue to interact with the optical element. This interaction causes the optical element to change from a first optical state to a second optical state.

[0105] Therefore, photosensitizers can be composed of any material capable of interacting with electromagnetic radiation in this manner. Suitable photosensitizers are known in photodynamic therapy (PDT) as PDT reagents. 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).

[0106] 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 reactive oxygen species. In the type II process, the main product is singlet oxygen via an intersystem cross-reaction mechanism.

[0107] 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.

[0108] A wide range of photosensitizer compounds are known, including porphyrins, chlorins (e.g., pyrofeoforbid-a), phthalocyanines, and other polycyclic aromatic species (see, e.g., Antibody-Directed Phototherapy, Pye et al., Antibodies, 2013, 2, 270).

[0109] In one embodiment, the photosensitizer is selected from porphyrins, chlorins, phthalocyanines, and other polycyclic aromatic species. The photosensitizer can be directly bound to the reporter reagent via covalent bonds. Alternatively, multiple photosensitizers can be encapsulated within particles, such as polystyrene latex particles, and antibodies can be bound to these particles. The advantage of using particles is that more photosensitizer can be bound to the reporter reagent. Furthermore, many photosensitizers are polyaromatic species with low solubility in aqueous media. By encapsulating them in colloidal particles, solubility problems can be avoided. Polystyrene latex particles are available in a wide range of sizes, with diameters of 10 nanometers or more.

[0110] The properties of the binding element and reporter reagent depend on the properties of the analyte, but they are preferably antibodies. The present invention is particularly applicable to immunoassays. In a particularly preferred embodiment, the binding element is an antibody produced against the analyte or a complex or derivative of the analyte, and the reporter reagent contains an antibody 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 element and reporter reagent are aggregates of molecules. The term "antibody" preferably encompasses Fab fragments, single-chain variable fragments (scFv), and recombinant binding fragments.

[0111] As an alternative to antibody-antigen reactions, the binding element, reporter reagent, and analyte may be first and second nucleic acids, which may be complementary, or the reagent may contain avidin or a derivative thereof, and the analyte may contain biotin or biotin, or vice versa. The binding element and reporter reagent may also be aptamers. This system is not limited to biological assays and can 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 in any liquid system, such as the detection of enzymes, cells, and viruses in air.

[0112] The maximum observable signal is the maximum signal that can be achieved when monitoring the photosensitizer binding to the surface. Particle binding to the substrate is governed by the diffusion rates of the analyte and reporter reagent, and further, primarily by the hydrodynamic radii of these elements and the viscosity / temperature of the sample.

[0113] The apparatus used in 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 is achieved by exposing the sample to reagents on the surface of a substrate. Different reagents are typically placed in different areas on the surface of the substrate, and these areas are coated with different reagents. These controls are defined as “negative” controls and “positive” controls, 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.

[0114] To achieve detection using these controls, the apparatus of the present invention preferably comprises a binding element, a negative control reagent, and a positive control reagent, which are each attached to the surface of the substrate as described above.

[0115] The connecting elements are as described above.

[0116] The negative control reagent has a lower affinity for the reporter reagent than the binding element under assay conditions. Therefore, the negative control reagent provides a negative control. It is important to consider affinity under assay conditions. This is because, in non-competitive assays, the affinity of the binding element 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 element 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 a lower affinity for the reporter reagent than the binding element.

[0117] Furthermore, in embodiments where the binding element binds to the analyte or a complex or derivative of the analyte, it is preferable that the negative control reagent has a lower affinity for the analyte, or, if used, a complex or derivative of the analyte, than the binding element. 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 element, 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, if 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 element. Furthermore, if the binding of the reporter to the negative control exceeds a threshold, an error code may be triggered, and the measurement may be terminated.

[0118] The positive control reagent binds to the reporter reagent and has an affinity for the reporter reagent that is less affected by the concentration of the analyte in the sample, or the analyte complex or derivative if used, compared to the binding element, thus providing a positive control. Preferably, the positive control reagent has an affinity for the reporter reagent 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 reporter reagent than the binding element under assay conditions. In this way, the positive control reagent measures the maximum signal expected in the system.

[0119] To increase the dynamic range of the assay performed according to the present invention and simultaneously improve its accuracy, it is preferable to have the binding element at multiple positions on the substrate. These positions can be adjusted to different sensitivities by varying the concentration of the binding element at each position. Each position may have its own negative control reagents and positive control reagents that function as controls for various dynamic ranges. This is particularly applicable to competitive assays that are particularly sensitive to the concentrations of individual elements constituting the system.

[0120] In the above explanation, the assay is designed to reach equilibrium before the photosensitizer is activated. However, the dynamics of the binding event can also be monitored by irradiating the photosensitizer at discontinuous intervals over time and monitoring the binding event occurring during the reaction before equilibrium is reached.

[0121] 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.

[0122] 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 element 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 element. For example, the analyte may be protonated / deprotonated at the pH of the assay conditions. Thus, the analyte that binds to the binding element may be the analyte itself or a derivative of the analyte; both are encompassed within the scope of the present invention.

[0123] In a preferred embodiment, the present invention can be used to simultaneously detect the presence of multiple analytes in the same sample. Different binding elements 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, and the assays may use the same negative and positive controls as described above, or separate controls may be used for each analyte being measured.

[0124] 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 liquid. Untreated means that the sample / liquid has not been pretreated by filtration, dilution, or other pretreatment steps before being mixed with the reporter reagent and other assay elements. However, treating the sample to provide a deuterium-concentrated mixture, e.g., dilution with a deuterium concentrate, or incubation with a deuterium concentrate is within the scope of the present invention. An advantage of the present invention is that the assay can be performed on samples containing suspended particles without excessively affecting the assay results. Preferably, the sample is untreated except for deuterium concentration.

[0125] 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. Due to unpredictable light scattering by different cellular components in each sample, it is common practice to remove red blood cells from blood to measure the fluorescence of plasma or serum components of blood. However, in the present invention, since fluorescence is measured on the substrate and individual binding events can be measured, the measurement can be performed with whole blood.

[0126] The sample is typically on the order of microliters (e.g., 1 to 100 μL, preferably 1 to 10 μL). To hold the liquid 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 apparatus used in the present invention preferably further comprises a chamber for holding the sample containing the analyte in contact with the substrate.

[0127] A potential additional source of background interference is the settling of suspended particles, including 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. Therefore, even if settling 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 remains within a single focal range or field of view during imaging. Obviously, the optical and coupling elements are located 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.

[0128] The sample can be held simply by the surface tension inside the capillary channel, for example.

[0129] The reporter reagent and optionally one or more additional reagents are preferably stored in a chamber incorporated into the apparatus used in the present invention.

[0130] This invention is particularly useful in point-of-care (POC) testing. POC testing is defined as a diagnostic test performed at or near the point of care, 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 point of care 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. POC testing often uses a combination of test kits and portable devices.

[0131] 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 biomarkers to be measured 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, Alzheimer's disease markers, nerve filament light chains), cytokines, and oncology (e.g., circulating tumor markers).

[0132] The present invention also provides an apparatus for detecting an analyte in a sample, the apparatus comprising a substrate having optical elements and bonding elements, the optical elements and bonding elements being bonded to the surface of the substrate, a photosensitizer capable of generating reactive oxygen species from a pre-activated reagent by absorption of electromagnetic radiation, the optical elements capable of changing from a first optical state to a second optical state by reaction with the reactive oxygen species, and the substrate having a deuterium-enriched layer on its surface.

[0133] The characteristics of the apparatus are as described above for the apparatus used in the present invention.

[0134] In a preferred embodiment, the apparatus further includes a chamber for holding a mixture of the sample and the reporter reagent.

[0135] The apparatus of the present invention may include a radiation source adapted to generate electromagnetic radiation, and a detector adapted to detect the optical elements of a second optical state, thereby enabling precise determination of the position of the photosensitizer relative to the substrate.

[0136] The apparatus of the present invention 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 apparatus includes a cartridge, the substrate is located within the cartridge, and the apparatus further includes a detector for detecting a set of local regions having a second optical state on the substrate. The present invention may also provide a cartridge including a substrate, optical elements and coupling elements as defined herein. The cartridge is preferably a disposable cartridge.

[0137] The present invention also relates to a system for detecting analytes in a sample, comprising: the apparatus of the present invention; and a reporter reagent and a pre-activating reagent for forming a mixture containing the sample, wherein the reporter reagent can generate reactive oxygen species from the pre-activating reagent by absorption of electromagnetic radiation, and an optical element can change from a first optical state to a second optical state by reaction with the reactive oxygen species, and the substrate can be brought into contact with a deuterium concentrate before irradiation.

[0138] Preferably, the photosensitizer of the reporter reagent can absorb electromagnetic radiation and interact with the pre-activating reagent present in the mixture to generate reactive oxygen species, which react with the optical element to change the optical element from a first optical state to a second optical state.

[0139] 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. [Examples]

[0140] The present invention will be described with reference to the following examples, but these are not limiting.

[0141] material Biotinylated bovine serum albumin (BSA) and fluorescein-labeled streptavidin were prepared according to techniques known in the art. These materials are also commercially available. Anti-TSH antibody 5407 (Medix Biochemica) was conjugated to aldehyde-coated Alphascreen donor beads (Perkin Elmer catalog no. 6762013) using a protocol available from Perkin Elmer. These protocols involved incubating the beads and antibody overnight in the presence of cyanoboron hydride, followed by the addition of (carboxymethoxy)amine to halt the reaction. Excess antibody was removed by centrifugation and washing.

[0142] Example 1 Silane glass cover slip A fresh piranha solution was prepared by adding 50 mL of 30% hydrogen peroxide solution to 150 mL of concentrated sulfuric acid. Next, 22 mm x 22 mm coverslips 23 (Brand, catalog no. 470055) were placed on a stainless steel rack and immersed in the piranha solution for 30 minutes, followed by rinsing three times with deionized water, and then three times with isopropanol. The coverslips were then immersed for 2 minutes in a fresh % dichloro(methyl)phenylsilane (Merck, catalog no. 440116) isopropanol solution. They were then washed three times with isopropanol and dried in a 100°C oven for 1 hour.

[0143] Example 2 Preparation of the substrate surface A 1cm x 1cm square piece of 200μm thick pressure-sensitive adhesive (PSA) 22, from which a 6mm diameter hole had been cut out, was attached to the silane-treated coverslip 23 described in Example 1 to create a shallow well 24 as shown in Figure 8. Next, 50μL of biotinylated BSA (10μg / mL in 40mM phosphate buffer) was added to the well and incubated for 2 hours, after which it was washed with washing buffer (40mM phosphate, 2% sucrose, 0.9% NaCl, 0.03% BSA). Then, 30μL of fluorescein-labeled streptavidin (10μg / mL in 40mM phosphate, 2% sucrose, 0.9% NaCl, 0.03% BSA) was added to the well and incubated for 60 minutes, after which it was washed three times with washing buffer. Next, 30 μL of biotinylated 5409 anti-TSH antibody (20 μg / mL in 40 mM phosphate buffer, 2% sucrose, 0.9% NaCl, and 0.03% BSA) was incubated for 30 minutes, washed three times with wash buffer, and then dried in air at room temperature.

[0144] Next, the release liner was removed from the PSA, the substrate was turned over, and mounted onto an acrylic sheet 25 with two small holes 26 drilled to form a reaction chamber, as shown in the profile of Figure 9.

[0145] Example 3 Binding to the surface of photosensitizer beads Alphascreen donor beads (0.5% solids) of 200 nm coated with anti-TSH antibody were diluted 1 / 500 in a TSH (100 μg / mL) solution in phosphate buffer containing 0.5% BSA and 0.05% Tween-20. 8 μL of this mixture was pipetted into a reaction chamber through one well and incubated in the dark for 30 minutes. After 30 minutes, the chamber was washed three times with phosphate buffer.

[0146] Example 4 Generation of bleached areas in a non-deuterated environment The reaction chamber of Example 3 was illuminated for 1 minute using a narrow-cone angle 680 nm LED with a total light output of 15 mW, focused on the surface of the coverslip. The surface was then imaged using a Euromex iScope epifluorescence microscope equipped with a mercury discharge bulb and a GXCam HiChrome MET camera (1080 × 1920 pixels) with a fluorescein excitation / emission filter set mounted on a 60X air lens with a numerical aperture of 0.85. The optical configuration is shown in Figure 7. A representative image of a portion of the surface is shown in Figure 10. Each dark spot represents the location where a binding event occurred on the surface. The approximate diameter of the spots in this image is 0.4 μm.

[0147] Example 5 Formation of bleached regions in a deuterized environment The second reaction chamber was prepared in the same manner as in Example 3, except that the three final washes were performed in a deuterated phosphate buffer prepared by dissolving phosphate buffer tablets in deuterium oxide. The surface was imaged in the same manner as in Example 4. A representative image of the surface is shown in Figure 11. In this example, the dark spots had a diameter of approximately twice that of Example 4 and an average size of 0.8 μm.

Claims

1. A method for detecting an analyte in a sample, comprising the following steps: (i) A step of providing a mixture comprising a sample, a reporter reagent, and a pre-activating reagent to an apparatus, wherein the apparatus comprises a substrate having optical elements and bonding elements, the optical elements and bonding elements being bonded to the surface of the substrate, the reporter reagent being capable of generating reactive oxygen species from the pre-activating reagent by absorption of electromagnetic radiation, the optical elements being capable of changing from a first optical state to a second optical state by reaction with the reactive oxygen species, the reporter reagent comprising a photosensitizer, and the pre-activating reagent comprising triplet oxygen; (ii) A step of enabling the binding element to bind a portion of the reporter reagent to the surface of the substrate in proportion to the concentration of the analyte; (iii) A step of irradiating the apparatus with electromagnetic radiation for absorption by the reporter reagent, thereby forming a set of local regions of optical elements having the second optical state on the substrate, wherein the substrate is brought into contact with a deuterium concentrate and / or a deuterium concentrate layer is provided on the surface of the substrate before irradiation; and (iv) A step of detecting a set of local regions having the second optical state on the substrate. A method that includes this.

2. The method according to claim 1, wherein the substrate is brought into contact with a deuterium concentrate, and the contact is performed by diluting the sample before binding a portion of the reporter reagent, or by washing the substrate with the deuterium concentrate after binding a portion of the reporter reagent.

3. The method according to claim 2, wherein the deuterium concentrate contains deuterium oxide.

4. The method according to claim 1, wherein the substrate has a deuterium-concentrated layer on its surface, and the deuterium-concentrated layer contains a deuterium-concentrated polymer.

5. The method according to any one of claims 1 to 4, wherein the reactive oxygen species is singlet oxygen.

6. The method according to any one of claims 1 to 5, wherein a set of local regions having the second optical state on the substrate is detected using an optical microscope.

7. The method according to any one of claims 1 to 6, wherein the optical element in the first optical state absorbs light of one or more first wavelengths, and the optical element in the second optical state absorbs light of one or more second wavelengths, and the first and second wavelengths are different.

8. The method according to any one of claims 1 to 6, wherein the optical element of the first optical state is fluorescent and the optical element of the second optical state is nonfluorescent, or the optical element of the first optical state is nonfluorescent and the optical element of the second optical state is fluorescent.

9. The method according to any one of claims 1 to 8, wherein the change from the first optical state to the second optical state is irreversible.

10. The method according to any one of claims 1 to 9, wherein steps (i) and (ii) are performed in the absence of a cleaning step.

11. The method according to any one of claims 1 to 10, wherein the sample is untreated except for deuterium enrichment.

12. The method according to any one of claims 1 to 11, wherein the apparatus is irradiated with electromagnetic radiation for more than one second.

13. A device for detecting analytes in a sample, The apparatus comprises a substrate having an optical element and a bonding element, wherein the optical element and the bonding element are bonded to the surface of the substrate, the bonding element can bind to a reporter reagent in proportion to the concentration of an analyte in a sample, the reporter reagent contains a photosensitizer that can generate reactive oxygen species from a pre-activating reagent by absorption of electromagnetic radiation, the optical element can change from a first optical state to a second optical state by reaction with the reactive oxygen species, the substrate has a deuterium-concentrated layer on its surface, and the pre-activating reagent contains triplet oxygen.

14. The apparatus according to claim 13, further comprising a cartridge, wherein the substrate is present within the cartridge, and a detector for detecting a set of local regions having a second optical state on the substrate.

15. A system for detecting analytes in a sample, The apparatus according to claim 13 or 14, and Reporter reagent and pre-activating reagent for forming a mixture containing the aforementioned sample The system comprising, wherein the reporter reagent comprises a photosensitizer capable of generating reactive oxygen species from the pre-activating reagent by absorption of electromagnetic radiation.

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