A method for detecting trace amounts of analyte particles in a fluid sample.

Abstract

The present invention is characterized by comprising the steps of: flowing a fluid sample containing an analyte onto a substrate on which a microchamber array is formed, the first capture substance which specifically binds to the analyte fixed on its surface; the analyte binding to the first capture substance in each microchamber of the microchamber array; reacting the analyte with the second capture substance by allowing a second capture substance which specifically binds to the analyte and binds to a signal generating substance to flow onto the substrate; allowing the signal generating substance to flow onto the substrate to bind with the second capture substance; flowing a substrate solution which reacts with the signal generating substance to generate a fluorescent signal onto the substrate; allowing a hydrophobic solvent to flow onto the substrate so that when the signal generating substance and the substrate solution react inside the microchamber, the substrate solution outside the microchamber is removed; and counting and detecting the number of microchambers where the fluorescent signal was generated.

Description

Technical Field

[0001] The present invention relates to a method for detecting particles of an analyte substance, and more particularly, to a method for detecting particles of an analyte substance present in trace amounts in a fluid sample, which can more easily calculate the concentration of molecules or particles present in trace amounts in the fluid sample.

Background Art

[0002] In fluid samples, many state-of-the-art techniques for quantifying low-concentration molecules use an amplification method that increases the number of reporter molecules so as to provide a measurable signal. For example, known methods include enzyme-linked immunosorbent assay (ELISA) for amplifying antibody-based signals. There is also an immuno-PCR technique that combines gene amplification technology and antigen-antibody reaction-based immunoassay technology. The immuno-PCR method can detect ultra-trace amounts of proteins, but it must undergo a complex assay process and may generate false positive signals.

[0003] Korean Patent Publication (KR10-2011-0024846) relates to a "quantitative analysis apparatus and method for biomolecules using magnetic nanoparticles", and includes a flux concentrator that is magnetized by an external magnetic field and concentrates magnetic flux in a gap region, a microchannel for injecting a sample solution into the gap region, and a sensor unit including a magnetic sensor formed on a substrate in the gap region between the flux concentrators for detecting a magnetic field change in the gap region due to magnetic nanoparticles, thereby disclosing a configuration for improving the accuracy of quantitative analysis of biomolecules.

[0004] However, conventional techniques utilize the fact that magnetic nanoparticles induce changes in the magnetic field in the gap region, which causes a change in the output voltage of the GMR sensor. The concentration of magnetic nanoparticles is then quantitatively analyzed through the range of change in the output voltage, and the target molecule is quantitatively analyzed. However, when the target molecule is present in only trace amounts in the fluid sample, the range of change in the output voltage is too small to measure, so there is currently a need to increase the sensitivity of the quantitative analysis of the target molecule. [Overview of the project] [Problems that the invention aims to solve]

[0005] Therefore, the problem that the present invention aims to solve is to provide a method for detecting particles of an analyte that can more easily calculate the concentration of molecules or particles present in trace amounts within a fluid sample. [Means for solving the problem]

[0006] To achieve the above objectives, the present invention provides a method for detecting an analyte present in trace amounts in a fluid sample, comprising the steps of: flowing a fluid sample containing an analyte onto a substrate on which a microchamber array is formed, the microchamber array having a first capture substance fixed on its surface that specifically binds to the analyte; allowing the analyte to bind to the first capture substance in each microchamber of the microchamber array; allowing a second capture substance, which specifically binds to the analyte and binds to a signal-generating substance, to flow onto the substrate, thereby causing the analyte to react with the second capture substance; allowing the signal-generating substance to flow onto the substrate, thereby causing it to bind with the second capture substance; flowing a substrate solution that reacts with the signal-generating substance to generate a fluorescent signal onto the substrate; allowing a hydrophobic solvent to flow onto the substrate, thereby removing the substrate solution outside the microchamber once the signal-generating substance and substrate solution have reacted inside the microchambers; and counting and detecting the number of microchambers where the fluorescent signal has been generated.

[0007] According to one embodiment of the present invention, it is preferable that the inside of the microchamber is hydrophilic with the first captured substance immobilized thereon, and the outside of the microchamber is hydrophobic.

[0008] Furthermore, by calculating a first count value obtained by counting the microchambers and a second count value obtained by recognizing and counting the microchambers where the signal-generating substance and the substrate solution were mixed and a signal was generated, and by calculating the ratio of the second count value to the first count value, the amount of the analyte in the fluid sample can be estimated.

[0009] According to another embodiment of the present invention, it is preferable to generate a calibration curve showing the concentration of a fluid sample with a known concentration relative to a standard substance, and then use the generated calibration curve to correct the concentration calculation of the analyte.

[0010] According to yet another embodiment of the present invention, the amount of analyte in a fluid sample can be estimated by calculating a first count value obtained by counting the microchambers and a second count value obtained by photographing the microchamber region, dividing the microchambers that generate the fluorescence signal by image processing, and counting their number. [Effects of the Invention]

[0011] According to the present invention, the concentration of molecules or particles present in trace amounts within a fluid sample can be calculated more easily.

[0012] Furthermore, according to the present invention, the inside of the microchamber is coated with a hydrophilic solvent and the outside of the microchamber is coated with a hydrophobic solvent. When the hydrophobic solvent is flowed onto the substrate, the substrate solution outside the microchamber can be removed, and the microchamber from which the fluorescence signal is generated can be easily detected. [Brief explanation of the drawing]

[0013] [Figure 1]This is a diagram showing the configuration of an apparatus for detecting particles of an analyte present in trace amounts within a fluid sample according to the first embodiment of the present invention. [Figure 2] This figure shows the analysis chip 300 according to the first embodiment of the present invention in more detail. [Figure 3] This diagram conceptually illustrates the process of detecting an analyte according to the first embodiment of the present invention. [Figure 4] This is a flowchart of a method for detecting trace amounts of analyte particles in a fluid sample according to the first embodiment of the present invention. [Figure 5] This is a diagram showing the configuration of an apparatus for detecting particles of an analyte present in trace amounts within a fluid sample, according to a second embodiment of the present invention. [Figure 6] This figure shows in more detail an analysis chip 301 according to a second embodiment of the present invention. [Figure 7] This figure shows the binding configuration of fine particles, a first capture substance, an analyte, and a signal generating substance according to a second embodiment of the present invention. [Figure 8] This figure shows in detail the state in which fine particles are captured in a microchamber placed in the detection space according to the second embodiment of the present invention. [Figure 9] This diagram conceptually illustrates the process of detecting an analyte according to a second embodiment of the present invention. [Figure 10] This figure shows the pretreatment process for a method for detecting particles of an analyte according to a second embodiment of the present invention. [Figure 11] This figure shows the pretreatment process for a method for detecting particles of an analyte according to a second embodiment of the present invention. [Figure 12] This is a flowchart of a method for detecting trace amounts of analyte particles in a fluid sample according to a second embodiment of the present invention. [Figure 13] This is a diagram showing the configuration of an apparatus for detecting particles of an analyte present in trace amounts within a fluid sample, according to a third embodiment of the present invention. [Figure 14] This figure shows in more detail an analysis chip 302 according to a third embodiment of the present invention. [Figure 15] FIG. is a diagram showing in more detail the microwell microparticles according to the third embodiment of the present invention. [Figure 16] FIG. is a diagram conceptually showing the process of detecting an analyte according to the third embodiment of the present invention. [Figure 17] FIG. is a flowchart of a method for detecting particles of an analyte present in trace amounts in a fluid sample according to the third embodiment of the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

[0014] The present invention is a method for detecting particles of an analyte present in trace amounts in a fluid sample, comprising flowing a fluid sample containing the analyte onto a substrate on which a microchamber array is formed, in which a first capture substance that specifically binds to the analyte is immobilized on the surface; binding the analyte to the first capture substance in each microchamber of the microchamber array; flowing a second capture substance that specifically binds to the analyte and binds to a signal generating substance onto the substrate to react the analyte with the second capture substance; flowing the signal generating substance onto the substrate to bind it to the second capture substance; flowing a substrate solution that reacts with the signal generating substance to generate a fluorescence signal onto the substrate; flowing a hydrophobic solvent onto the substrate to remove the substrate solution outside the microchamber when the signal generating substance and the substrate solution react inside the microchamber; and counting and detecting the number of microchambers in which the fluorescence signal is generated.

EXAMPLE

[0015] Hereinafter, preferred embodiments that can be easily implemented by those having ordinary knowledge in the technical field to which the present invention pertains will be described in detail with reference to the accompanying drawings. However, it is self-evident to those having ordinary knowledge in the art that these embodiments are for more specifically explaining the present invention and that the scope of the present invention is not limited thereby.

[0016] The configuration of the present invention, which clarifies the solution to the problems that the present invention aims to solve, will be described in detail with reference to the accompanying drawings based on preferred embodiments of the present invention. When assigning reference numerals to the components in the drawings, the same component will be given the same reference numeral even if it is in another drawing, and it will be made clear in advance that components from other drawings may be referenced as necessary when describing the drawing. Furthermore, when describing the operating principle of a preferred embodiment of the present invention in detail, if it is determined that a specific description of a known function or configuration related to the present invention, or other miscellaneous matters, may unnecessarily obscure the gist of the present invention, such a detailed description will be omitted.

[0017] In this specification, when a part is described as being "combined" with another part, this includes not only cases where they are "directly connected" but also cases where they are "indirectly connected" with other elements in between. In this specification, the singular form includes the plural form unless otherwise specified in the text. As used in this specification, "comprises" or "comprising" does not preclude the presence or addition of one or more other components, stages, operations, or elements other than those mentioned.

[0018] Figure 1 is a diagram showing the configuration of an apparatus for detecting particles of an analyte present in trace amounts within a fluid sample according to the first embodiment of the present invention.

[0019] Figure 1(a) is a plan view of the apparatus for detecting particles of an analyte according to the first embodiment of the present invention, and Figure 1(b) is a cross-sectional view of the apparatus for detecting particles of an analyte according to the first embodiment of the present invention.

[0020] Referring to Figure 1, the apparatus for detecting particles of an analyte present in trace amounts in a fluid sample according to the first embodiment of the present invention is configured to include a cartridge 100.

[0021] The cartridge 100 comprises a fluid sample chamber 200, a waste chamber 210, a washing solution chamber 230, a second captured substance chamber 240, a signal generating substance chamber 250, a substrate solution chamber 260, and an analysis tip 300.

[0022] The upper surface of the cartridge 100 is preferably sealed with an airtight film using heat fusion, UV bonding, ultrasonic bonding, or tape, in order to prevent the multiple chambers and analysis tips 300 from being contaminated by each other or by external substances.

[0023] The fluid sample chamber 200 is configured to contain a fixed amount of fluid sample. Such a fluid sample chamber 200 can be configured to have a volume in the range of 50 μL to 10 mL. The fluid sample includes body fluids such as blood, plasma, serum, and cerebrospinal fluid.

[0024] The waste chamber 210 is configured to provide a containment space for waste generated when the fluid sample, washing solution, second capture substance, signal generating substance, and substrate solution introduced into the analysis tip 300 are discharged. Such a waste chamber 210 can be configured to have a capacity of 1 to 15 mL. In Figure 1, the waste chamber 210 is contained within the cartridge 100, but the waste chamber 210 may also be provided in a separate space outside the cartridge 100.

[0025] The washing solution chamber 230 is configured to contain a washing solution for washing the detection space 330, which contains the microchamber 30 of the analysis tip 300. The washing solution may be a solution such as PBS (Phosphate buffered saline), TBS (Tris-buffered saline), PBS-T, TBS-T, or ultrapure water.

[0026] In one embodiment, the cleaning solution in the cleaning solution chamber 230 may be injected into the inlet 310 to clean the fluid sample, second capture substance, signal generating substance, and substrate solution remaining in the detection space 330 after the reaction.

[0027] The second capture substance chamber 240 is configured to contain a second capture substance that specifically binds to the analyte and to the signal-generating substance. The second capture substance has the property of specifically binding to both the analyte and the signal-generating substance, and the signal-generating substance binds to the second capture substance which is specifically bound to the analyte, playing a role in generating a fluorescence signal proportional to the amount of analyte. For example, the second capture substance may be a secondary antibody and can be provided in a form pre-bound to the signal-generating substance.

[0028] Since the second capture substance binds to the analyte and the first capture substance, which is fixed in multiple microchambers 30, also binds to the analyte, it is preferable that the first capture substance and the second capture substance bind to different parts of the analyte.

[0029] The signal-generating substance chamber 250 is configured to contain a signal-generating substance that specifically binds to the second capture substance bound to the analyte. The signal-generating substance may be, for example, an enzyme.

[0030] The signal-generating substance is, for example, a hue source, a fluorescent or chemiluminescent enzyme precursor, which, upon contact with a substrate solution, is converted into a detectable indicator.

[0031] The signal-generating substance and the second capture substance may be housed together in either the second capture substance chamber 240 or the signal-generating substance chamber 250.

[0032] The signal-generating substance reacts with the substrate solution to generate a detectable indicator such as a fluorescent signal, and the enzyme component may include β-galactosidase, horseradish peroxidase, or alkaline phosphatase.

[0033] The substrate solution chamber 260 is configured to contain a substrate solution that reacts with the signal-generating substance to generate a signal. The substrate solution reacts with the signal-generating substance to generate a detectable indicator such as a fluorescent signal, and may contain resorufin β-D-galactopyranoside or the like.

[0034] The analysis chip 300 comprises an injection port 310 and a detection space 330.

[0035] The injection port 310 is into which a fluid sample, washing solution, second capture substance, signal generating substance, substrate solution, or a mixture thereof is injected.

[0036] The injection port 310 may further include a filter to separate and supply plasma from whole blood. The filter will filter out blood cells in the whole blood and allow the fluid sample containing plasma components to pass through. The filter can be formed with pores of various sizes and materials to filter out living cells, inorganic particles, or organic particles. The filter in this embodiment preferably has pores that can filter out cells or foreign matter larger than the analyte, and is preferably formed with pores that can filter out blood cells of approximately 5-10 μm in size. The filter can also be formed from a biologically inert material so that it can be applied to biological samples.

[0037] During the process of supplying the fluid sample to the injection port 310, the filter output surface can be pre-moistened by immersing it in buffer solution before filter filtration. Therefore, the whole blood plasma supplied to the filter input surface can pass through the filter pores without surface resistance and be smoothly filtered to the filter output surface.

[0038] Preferably, the detection space 330 has multiple microchambers 30 arranged within it, with multiple first capture substances fixed inside each microchamber.

[0039] The microchambers 30 are arranged on one surface of the detection space 330 of the analysis tip 300 at regular intervals from each other, and each microchamber is of the same size. The number of microchambers is preferably 10,000 or more, and this number can vary depending on the concentration range of the analyte to be measured. The inner surface of each microchamber 30 is immobilized with a first capture substance that specifically binds to the analyte. Furthermore, to prevent nonspecific reactions, the remaining surface without the first capture substance is preferably coated with a blocking substance such as BSA (bovine serum albumin).

[0040] When a fluid sample is injected through the injection port 310, the first capture substance fixed in the internal space of the microchamber 30 within the detection space 330 combines with the analyte contained in the fluid sample.

[0041] Subsequently, when the cleaning solution is injected through the inlet 310, the fluid sample remaining after the first captured substance and the analyte can be removed and cleaned.

[0042] On the other hand, when the second capture substance contained in the second capture substance chamber 240, which binds to the analyte and signal generating substance, is injected through the injection port 310, the analyte bound to the first capture substance will bind to the second capture substance, and thereafter, the remaining second capture substance can be removed and washed away through the washing solution injected through the injection port 310.

[0043] Next, when the signal-generating substance is injected through the injection port 310, the signal-generating substance and the second capture substance combine.

[0044] Finally, the substrate solution is injected through the injection port 310 to allow the signal-generating substance and the substrate solution to react, thereby generating a fluorescent signal. A hydrophobic solvent is then allowed to flow through the injection port 310 to remove any remaining substrate solution that has reacted with the signal-generating substance. The hydrophobic solvent is preferably an oil or a gas.

[0045] Figure 2 is a diagram showing in more detail the analysis chip 300 according to the first embodiment of the present invention.

[0046] The fluid sample, washing solution, second capture substance, signal generating substance, substrate solution, or a mixture thereof are sequentially injected through the injection port 310. The second capture substance is a capture substance for binding the analyte to the signal generating substance, which generates a detectable indicator such as a fluorescent signal, and can be provided bound to the signal generating substance, which simplifies the procedure compared to when the second capture substance and the signal generating substance are provided separately.

[0047] When the fluid sample, washing solution, second capture substance, signal generating substance, and substrate solution or a mixture thereof are injected into the injection port 310, they diffuse and flow into the detection space 330 and then flow through the microchamber 30.

[0048] Preferably, a first capture substance that binds to the analyte contained in the fluid sample is immobilized in the microchamber 30.

[0049] Figure 3 conceptually illustrates the process of detecting the target substance according to the first embodiment of the present invention.

[0050] Figure 3(a) shows that a microchamber 30 is formed in a recessed shape at the bottom of the substrate, and the first captured substance 40 is fixed in the microchamber 30. The size of the microchamber 30 is 0.5 to 15 μm, which is the minimum size that allows for optical measurement.

[0051] The microchambers 30 are arranged along the bottom surface of the detection space 330 at intervals of 0.5 μm or more from each other, and can be designed so that at least 10,000 microchambers are arranged in a uniform distribution.

[0052] The microchamber 30 is formed as a groove-shaped recess on the bottom surface of the detection space 330, and the first capture substance 40 is fixed therein to specifically capture the analyte 50.

[0053] In one embodiment of the present invention, the microchamber 30 may be formed perpendicular to the fluid flow direction from the bottom surface of the detection space 330. This ensures that the microchamber 30 and the fluid flow are perpendicular to each other. Consequently, particles inserted into the microchamber 30 cannot easily escape from the microchamber 30 along the fluid flow.

[0054] In this way, the first captured substance 40 is stably inserted and fixed within the microchamber 30, and can maintain a uniform arrangement on the detection space 330 regardless of the fluid flow.

[0055] When the first capture substance is antibody particles, the size of the antibodies is generally 10 to 50 nm, so it is preferable that a large number of antibodies are immobilized in the microchamber 30.

[0056] Figure 3(b) shows that as the fluid sample flows over the microchamber 30, the analyte 50 is captured by the first capture substance 40. Subsequently, it is preferable that the remaining fluid sample containing uncaptured analyte be washed with a washing solution.

[0057] As can be seen from Figure 3(b), since a large number of first capture substances are fixed in the microchamber 30, the number of analytes 50 fixed to the first capture substances in one microchamber 30 depends on the number of analytes and their material properties. In this case, one analyte may be equally captured in each microchamber, or the number of analytes captured may vary from microchamber to microchamber. For example, if the number of analytes is less than 60% of the number of microchambers, there is a high probability that one analyte will be captured in each microchamber. However, if the number of analytes is 60% or more of the number of microchambers, the number of analytes captured in each microchamber may vary from microchamber to microchamber. Therefore, if the number of analytes captured in each microchamber is the same, it is preferable to calculate the number of microchambers that generate a signal from a detectable indicator such as a fluorescent signal to infer the number of analytes. If the number of analytes captured in each microchamber differs, it is preferable to calculate not only the number of microchambers that generate a signal from a detectable indicator such as a fluorescent signal, but also the intensity of the signal from the detectable indicator such as a fluorescent signal generated in the microchamber to infer the number of analytes.

[0058] Any remaining substances other than the analyte inserted, fixed, or captured in the microchamber 30 by the washing solution are removed from the detection space 330. During the washing process, the substances inserted, fixed, or captured in the microchamber are fixed in the microchamber 30, which is formed to be deep perpendicular to the flow direction of the washing solution, and therefore remain fixed without being dislodged from the microchamber 30 by the flow of the washing solution. As a result, only the remaining substances that have not combined with the first captured substances inserted and fixed in the microchamber 30 within the detection space 330 and are not fixed are removed from the detection space 330 along with the flow of the washing solution.

[0059] Figure 3(c) shows the state in which the analyte 50 and the second captured substance 60 combine and react when the second captured substance 60 is flowed over the microchamber 30. After that, it is preferable that the remaining second captured substance 60 is washed away with a washing solution.

[0060] Figure 3(d) shows that when the signal-generating substance 70 is flowed over the microchamber 30, the signal-generating substance 70 and the second capture substance 60 combine and react. After that, it is preferable that the remaining signal-generating substance 70 is washed away with a washing solution.

[0061] Figure 3(e) shows that when the substrate solution is flowed over the microchamber 30, the signal-generating substance 70 reacts with the substrate solution to generate a signal of a detectable indicator, such as a fluorescent signal. After that, it is preferable to wash away the remaining substrate solution with a washing solution.

[0062] In Figure 3(b), since the number of analyte substances 50 fixed to the first captured substance differs within each microchamber 30, the number of signal-generating substances 70 that produce a detectable indicator signal such as a fluorescent signal also differs for each microchamber.

[0063] Subsequently, a hydrophobic solvent is flowed through the substrate to remove the substrate solution outside the multiple microchambers 30, and the number of microchambers where the fluorescence signal was generated is counted and detected. By coating the inside of the multiple microchambers 30 with a hydrophilic solvent and the outside of the multiple microchambers 30 with a hydrophobic solvent, and flowing the hydrophobic solvent through the substrate, it becomes possible to remove the substrate solution outside the multiple microchambers 30.

[0064] To more accurately detect the analyte contained in a fluid sample, it is preferable to associate the number of analyte particles with the brightness of the signal from a detectable indicator such as a fluorescent signal, and to count the number of analyte particles by considering the brightness of the signal from the detectable indicator such as a fluorescent signal in each microchamber.

[0065] Figure 4 is a flowchart of a method for detecting trace amounts of analyte particles in a fluid sample according to the first embodiment of the present invention.

[0066] In step 400, a fluid sample containing the analyte is flowed through a substrate on which a microchamber array is formed, on which a first capture substance that specifically binds to the analyte is fixed to the surface.

[0067] In step 410, the analyte binds to the first captured substance in each microchamber of the microchamber array 30.

[0068] In step 420, the fluid sample remaining after the binding of the first capture substance and the analyte is removed and washed.

[0069] In step 430, the analyte and the second capture substance are reacted by allowing a second capture substance, which specifically binds to the analyte and binds to the signal generating substance, to flow onto the substrate.

[0070] In step 440, the analyte and the second capture substance are reacted, and any remaining second capture substance is removed and washed.

[0071] In step 450, the signal generating substance is made to flow onto the substrate and combine with the second capture substance.

[0072] In step 460, a substrate solution that reacts with the signal-generating substance to generate a signal of a detectable indicator such as a fluorescent signal is poured onto the substrate.

[0073] In step 470, a hydrophobic solvent is flowed through the substrate to remove the substrate solution outside the microchamber while the signal generating substance and substrate solution remain bound inside the microchamber.

[0074] Preferably, the inside of the microchamber is hydrophilic, and the outside of the microchamber is hydrophobic.

[0075] In step 480, the number of microchambers that have generated a signal from a detectable indicator such as the fluorescent signal is counted and detected.

[0076] Figure 5 is a diagram showing the configuration of an apparatus for detecting particles of an analyte present in trace amounts within a fluid sample, according to a second embodiment of the present invention.

[0077] Figure 5(a) is a plan view of the apparatus for detecting particles of an analyte according to a second embodiment of the present invention, and Figure 5(b) is a cross-sectional view of the apparatus for detecting particles of an analyte according to a second embodiment of the present invention.

[0078] Referring to Figure 5, the apparatus for detecting particles of an analyte present in trace amounts in a fluid sample according to the second embodiment of the present invention comprises a magnetic device 150 and a cartridge 101.

[0079] The magnetic device 150 moves in and out of the reaction chamber 205, thereby causing separation of magnetic particles, which are an embodiment of the fine particles in the reaction chamber 205, from the residual solution. After the residual solution is removed, the cleaning solution contained in the cleaning solution chamber 231 can be injected into the reaction chamber 205 to clean the walls of the reaction chamber 205 where the magnetic particles are located.

[0080] Cartridge 101 comprises a fluid sample chamber 201, a reaction chamber 205, a waste chamber 211, a fine particle chamber 221, a washing solution chamber 231, a second captured substance chamber 241, a signal generating substance chamber 251, a substrate solution chamber 261, and an analysis tip 301.

[0081] The upper surface of the cartridge 101 is preferably sealed with an airtight film using heat fusion, UV bonding, ultrasonic bonding, or tape, in order to prevent the multiple chambers and analysis tips 301 from being contaminated by each other or by external substances.

[0082] The fluid sample chamber 201 is configured to contain a fixed amount of fluid sample. Such a fluid sample chamber 201 can be configured to have a volume in the range of 50 μL to 10 mL. The fluid sample includes body fluids such as blood, plasma, serum, and cerebrospinal fluid.

[0083] The reaction chamber 205 is configured to contain and mix a fixed amount of fluid sample and fine particles to which the first capture substance is immobilized. Such a reaction chamber 205 can be configured to have a volume in the range of 50 μL to 15 mL.

[0084] The waste chamber 211 is configured to provide a containment space for waste generated when fluid samples, fine particles, washing solution, second capture material, signal generating material, and substrate solution are discharged from the analysis tip 301. Such a waste chamber 211 may be configured to have a capacity of 1 to 30 mL. The waste chamber 211 in Figure 5 is contained within the cartridge 101, but the waste chamber 211 may be provided in a separate space outside the cartridge.

[0085] The fine particle chamber 221 is configured to contain fine particles on which the first capture substance is immobilized, and the fine particles are injected into the reaction chamber 205. The fine particles may be polymers or magnetic particles. The magnetic particles preferably have a size of 500 nm to 10 μm in diameter and contain a magnetic substance such as iron oxide. The fine particles may be spherical magnetic particles or donut-shaped microchamber particles.

[0086] The fine particles contained in the fine particle chamber 221 preferably have a first capture substance bound to their surface, which binds to the analyte. The first capture substance may include antibodies, nucleic acid molecules, peptides, and the like.

[0087] The washing solution chamber 231 is configured to contain a washing solution for washing the walls of the reaction chamber 205 or for washing the detection space 331 containing the microchamber 31 of the analysis tip 301. The washing solution may be a solution such as PBS (Phosphate buffered saline), TBS (Tris-buffered saline), PBS-T, TBS-T, or ultrapure water.

[0088] In one embodiment, the cleaning solution in the cleaning solution chamber 231 may be injected into the inlet 311 to clean the fluid sample, fine particles, second capture substance, signal generating substance, and substrate solution remaining in the detection space 331 after the reaction.

[0089] In another embodiment, the magnetic device 150 may be placed close to the reaction chamber 205, and once separation occurs between the magnetic particles, which are an embodiment of the fine particles in the reaction chamber 205, and the residual solution, the residual solution may be removed, and then the cleaning solution contained in the cleaning solution chamber 231 may be injected into the reaction chamber 205 to clean the wall surface of the reaction chamber 205 where the magnetic particles were located.

[0090] The second capture substance chamber 241 is configured to contain a second capture substance that specifically binds to the analyte and to the signal-generating substance. The second capture substance has the property of specifically binding to both the analyte and the signal-generating substance, and the signal-generating substance binds to the second capture substance which is specifically bound to the analyte, playing a role in generating a fluorescence signal proportional to the amount of analyte. For example, the capture substance may be a secondary antibody and can be provided in a form that is pre-bound to the signal-generating substance.

[0091] Since the second capture substance binds to the analyte and the first capture substance, which is fixed to the fine particles, also binds to the analyte, it is preferable that the first and second capture substances bind to different parts of the analyte.

[0092] The signal-generating substance chamber 251 is configured to contain a signal-generating substance that specifically binds to the second capture substance bound to the analyte. The signal-generating substance may be, for example, an enzyme.

[0093] The signal-generating substance is, for example, a hue source, a fluorescent or chemiluminescent enzyme precursor, which, upon contact with a substrate solution, is converted into a detectable indicator.

[0094] The signal-generating substance and the second capture substance may be housed together in either the second capture substance chamber 241 or the signal-generating substance chamber 251.

[0095] The signal-generating substance reacts with the substrate solution to generate a detectable indicator such as a fluorescent signal, and the enzyme component may include β-galactosidase, horseradish peroxidase, or alkaline phosphatase.

[0096] The substrate solution chamber 261 is configured to contain a substrate solution that reacts with the signal-generating substance to generate a signal. The substrate solution reacts with the signal-generating substance to generate a detectable indicator such as a fluorescent signal, and may contain resorufin β-D-galactopyranoside or the like.

[0097] The analysis chip 301 comprises an injection port 311 and a detection space 331.

[0098] In reaction chamber 205, a certain amount of fluid sample and fine particles to which the first capture substance is immobilized are contained and mixed, so the analyte is bound to the first capture substance immobilized on the fine particles.

[0099] The injection port 311 is into which a fluid sample, fine particles, washing solution, second capture substance, signal generating substance, and substrate solution or mixed solution are injected.

[0100] The injection port 311 may further include a filter to separate and supply particles larger than the fine particles from a mixed solution of fine particles and a fluid sample. The filter may be formed of pores of various sizes and materials to filter out living cells, inorganic particles, or organic particles, etc. The filter in this second embodiment is preferably formed of pores that can filter out particles larger than the fine particles or foreign matter. The filter may also be formed of a biologically inert material so that it can be applied to biological samples.

[0101] During the process of supplying the fluid sample to the injection port 311, the filter output surface can be pre-moistened by immersing it in buffer solution before filter filtration. Therefore, the mixed solution of fine particles and the fluid sample supplied to the filter input surface can pass through the filter pores without surface resistance and be smoothly filtered to the filter output surface.

[0102] The detection space 331 has multiple microchambers 31 arranged within it. When a mixed solution of fine particles and a fluid sample is injected through the injection port 311, the fine particles are trapped in the internal space of the microchambers 31 within the detection space 331. By placing a magnetic material 15 below the microchambers 31, if the fine particles in the internal space of the microchambers 31 are magnetic particles, they can be easily trapped.

[0103] The fine particles captured in the microchamber 31 include both fine particles of the analyte captured by the first captured substance and fine particles that were not captured. The size of the microchamber 31 is preferably larger than the diameter of the fine particles and less than twice the diameter of the fine particles.

[0104] Subsequently, when the cleaning solution is injected through the inlet 311, the fluid sample remaining after the first captured substance and the analyte can be removed and cleaned.

[0105] On the other hand, when the second capture substance contained in the second capture substance chamber 241, which binds to the analyte and signal generating substance, is injected through the injection port 311, the analyte bound to the first capture substance will bind to the second capture substance, and thereafter, the remaining second capture substance can be removed and washed away with the washing solution.

[0106] Next, when the signal-generating substance is injected through the injection port 311, the signal-generating substance and the second capture substance combine.

[0107] Finally, the substrate solution is injected through the injection port 311 to react with the signal-generating substance, thereby generating a signal of a detectable indicator such as a fluorescent signal. A hydrophobic solvent is then allowed to flow through the injection port 311 to remove any remaining substrate solution that has reacted with the signal-generating substance. The hydrophobic solvent is preferably an oil or a gas.

[0108] Figure 6 is a diagram showing in more detail an analysis chip 301 according to a second embodiment of the present invention.

[0109] Through the injection port 311, a mixed solution of the fluid sample and fine particles, a washing solution, a second capture substance, a signal generating substance, and a substrate solution or a mixed solution thereof are sequentially injected. The second capture substance is a capture substance for binding the analyte to a signal generating substance that generates a detectable indicator such as a fluorescent signal, and can be provided bound to the signal generating substance, which simplifies the procedure compared to when the second capture substance and the signal generating substance are provided separately.

[0110] When the mixed solution of the fluid sample and fine particles, the washing solution, the second capture substance, the signal generating substance, and the substrate solution or a mixture thereof are injected into the injection port 311, they diffuse and flow into the detection space 331 and then flow through the microchamber 31.

[0111] The size of the microchamber 310 is preferably larger than the diameter of the fine particles and less than twice the diameter of the fine particles.

[0112] Figure 7 shows the binding configuration of fine particles, first capture substance, analyte, and signal generating substance according to a second embodiment of the present invention.

[0113] Referring to Figure 7(a), it is shown that the first capture substance 41 is immobilized on the fine particle 1. Referring to Figure 7(b), it is shown that the analyte 51 is bound to the first capture substance 41, the second capture substance 61 is bound to the analyte 51, and the signal generating substance 71 is bound to the second capture substance 61. When the substrate solution reacts with the signal generating substance 71, a detectable indicator signal, such as a fluorescent signal, is generated.

[0114] Figure 8 is a detailed diagram showing the state in which fine particles are captured in a microchamber placed in the detection space according to the second embodiment of the present invention.

[0115] Referring to Figure 8, the magnetic material 15 is placed below the internal space of the microchamber 31 to form a magnetic field, and the figure shows a state in which magnetic particles are captured as an example of fine particles 1 in the mixed solution.

[0116] Figure 9 is a conceptual diagram illustrating the process of detecting the target substance according to the second embodiment of the present invention.

[0117] Figure 9(a) shows the state in which magnetic particles 1, to which the first capture substance 41 is immobilized, are mixed with a fluid sample in the reaction chamber 205 and injected into the injection port 311, and the magnetic particles are captured by the magnetic material 15 in each microchamber 31. The first capture substance 41 is immobilized on the magnetic particles 1.

[0118] As the mixed solution exiting from the injection port 311 flows through multiple microchambers 31, magnetic particles are captured in each containment by a magnetic material placed beneath the multiple microchambers 31.

[0119] The size of the microchamber 31 is preferably larger than the diameter of the magnetic particles 1 and smaller than twice the diameter of the magnetic particles. Therefore, the accommodation portion, which is the internal space of the microchamber 31, is larger than the size of the magnetic particles 1 and has a size such that one or fewer magnetic particles can enter.

[0120] The microchambers 31 can be arranged in an array at intervals on the bottom surface along the detection space 331 and designed to be arranged in a uniform distribution.

[0121] In one embodiment of the present invention, the depth h of the microchamber 31 may be such that d < h < 2d. Here, h is the depth of the microchamber 31, and d is the diameter of the fine particles. The depth h of the microchamber 31 is the distance from the bottom surface of the microchamber 31 to the tip of the upper inner part of the microchamber 31. Also, the width of the microchamber 31 may be such that d < w < 2d. Here, w is the width of the microchamber 30, and d is the diameter of the fine particles.

[0122] When the depth h of the microchamber 31 is smaller than the diameter d of the fine particles, the fixation of the fine particles is unstable, and there is a problem that the fine particles may escape from the microchamber 31 due to the flow of the fluid sample flowing along the detection space 331. When the depth h of the microchamber 31 exceeds twice the diameter d of the fine particles, there is a problem that the probability of two or more fine particles being captured in one chamber occurs, resulting in a decrease in analysis accuracy, and a problem that it takes time for the cleaning liquid for cleaning the fine particles and the detection space 331 to diffuse.

[0123] Also, in one embodiment of the present invention, the microchamber 31 may be formed on the bottom surface of the detection space 331 in a direction perpendicular to the flow direction of the fluid. Thereby, the microchamber 31 and the fluid flow are arranged at right angles. Therefore, the fine particles inserted into the microchamber 31 cannot easily escape from the microchamber 30 along the fluid flow.

[0124] In this way, the fine particles are stably inserted into the microchamber 31 and can maintain a uniform arrangement on the detection space 331 regardless of the fluid flow.

[0125] When the cleaning solution is injected from the injection port 311, the fluid sample containing uncaptured analyte can be removed and cleaned after the first captured substance 41 and the analyte 51 have combined. The magnetic particles 1 are not cleaned because they are fixed to each containment part of the microchamber 31 by the magnetic material.

[0126] Any remaining substances other than the magnetic particles inserted, fixed, or captured in the microchamber 31 by the washing solution are removed from the detection space 331. During the washing process, the substances inserted, fixed, or captured in the microchamber are inserted into the microchamber 31, which is formed to be deep perpendicular to the flow direction of the washing solution, and can therefore remain fixed without being dislodged from the microchamber 31 by the flow of the washing solution. As a result, only the remaining substances that were inserted into the microchamber 31 but did not bind and become fixed with the analyte in the detection space 331 are removed by flowing out of the detection space 331 along with the flow of the washing solution.

[0127] As can be seen from Figure 9(b), when the second capture substance 61, which is contained in the second capture substance chamber 241 that binds to the analyte and signal generating substance, is injected through the injection port 311, the analyte 51 bound to the first capture substance 41 will bind to the second capture substance 61, and then the remaining second capture substance 61 can be removed and washed away with the washing solution.

[0128] In Figure 9(c), when the signal-generating substance 71 is injected through the injection port 311, the signal-generating substance 71 and the second capture substance 61 bind together. After that, it is preferable that the remaining signal-generating substance 71 is washed away with a cleaning solution.

[0129] In Figure 9(d), when the substrate solution is injected through the injection port 311, the signal generating substance 71 reacts with the substrate solution to generate a signal of a detectable indicator such as a fluorescent signal. A hydrophobic solvent is then allowed to flow through the injection port 311 to remove any substrate solution that has reacted with the signal generating substance 71.

[0130] By allowing a hydrophobic solvent to flow across the substrate, the substrate solution outside the multiple microchambers 31 is removed, and the number of microchambers from which a detectable indicator signal, such as a fluorescent signal, is generated is counted for detection. When the inside of the multiple microchambers 31 is coated with a hydrophilic solvent and the outside of the multiple microchambers 30 is coated with a hydrophobic solvent, and the hydrophobic solvent is allowed to flow across the substrate, the substrate solution outside the multiple microchambers 31 can be easily removed.

[0131] To more accurately detect analyte substances contained in a fluid sample, the number of analyte particles can be associated with the brightness of a detectable indicator signal, such as a fluorescent signal. By considering the brightness of the fluorescent signal signal in each microchamber, the number of analyte particles can be counted.

[0132] Referring again to Figure 9(d), it can be confirmed that the number of signal-generating substances that produce detectable indicator signals such as fluorescent signals differs in each microchamber, and this results in differences in the brightness of the fluorescent signals.

[0133] Therefore, if the number of signal-generating substances captured in each microchamber is the same, it is preferable to calculate the number of microchambers that generate fluorescence signals and infer the number of analytes. If the number of signal-generating substances captured in each microchamber is different, it is preferable to calculate not only the number of microchambers that generate fluorescence signals, but also the intensity of the fluorescence signals generated in the microchambers, and infer the number of analytes.

[0134] According to another embodiment of the present invention, a first capture substance immobilized on fine particles is reacted with the analyte in a fluid sample within a reaction chamber 200, and after bringing a magnetic device close to the reaction chamber 200 to separate the fine particles from the fluid sample, the fluid sample that does not bind to the fine particles can be removed and washed.

[0135] Subsequently, a second capture substance that specifically binds to the analyte bound to the first capture substance is injected into the reaction chamber 200 and reacted, and a signal-generating substance that specifically binds to the second capture substance is injected into the reaction chamber 200 and reacted.

[0136] Next, a substrate solution that reacts with the signal-generating substance to generate a signal is injected into the reaction chamber 205 and mixed, and the magnetic particles in the mixed substrate solution are captured in the microchamber 31 in the detection space 331 using the magnetic material 15.

[0137] Finally, it is preferable that the particle detection device for the substance to be analyzed detects the number of microchambers 31 that generate a signal by mixing the signal-generating substance with the substrate solution and counting the number of microchambers 31 that generate a signal.

[0138] Figures 10 and 11 show the pretreatment process for a method for detecting particles of an analyte according to a second embodiment of the present invention.

[0139] First, as shown in Figure 10(a), the magnetic device drive unit 160 can move the magnetic device 150 to bring it into contact with the reaction chamber 205 for 20 minutes. The reaction chamber 205 contains a mixed solution S, which is a mixture of a certain amount of fluid sample and magnetic particles to which the first capture substance is fixed.

[0140] As a result, as shown in Figure 10(b), separation of the magnetic particles S1 and the residual solution W can occur in the reaction chamber 205.

[0141] Next, as shown in Figure 11(a), the residual solution W is removed from the reaction chamber 205, and the cleaning solution contained in the cleaning solution chamber 231 is injected into the reaction chamber 205 to clean the wall surface of the reaction chamber 205 where the magnetic particles S1 were located. After that, the magnetic device drive unit 160 moves the magnetic device 150 again, separating the magnetic device 150 from the reaction chamber 205.

[0142] Subsequently, as shown in Figure 11(a), magnetic particles S1 remain in the reaction chamber 205, and as shown in Figure 11(b), it is preferable to generate a mixed solution S2 by adding another buffer solution for injection into the inlet 311.

[0143] Figure 12 is a flowchart of a method for detecting trace amounts of analyte particles in a fluid sample according to a second embodiment of the present invention.

[0144] In step 1200, the first capture substance is mixed with the fine particles so that the first capture substance is immobilized on the fine particles. The first capture substance may include antibodies, nucleic acid molecules, peptides, etc. The fine particles are preferably magnetic particles that are magnetized.

[0145] The magnetic particles have a diameter of 500 nm to 10 μm and preferably contain a magnetic substance such as iron oxide. According to one embodiment of the present invention, the mixing may be carried out by methods such as vibration, ultrasound, or pipetting.

[0146] In step 1210, the first captured substance immobilized on the fine particles reacts with the analyte in the fluid sample within the reaction chamber 200.

[0147] The fluid sample includes bodily fluids such as blood, plasma, serum, and cerebrospinal fluid.

[0148] In step 1220, the magnetic device is brought close to the reaction chamber 200 to separate the fine particles from the fluid sample, and the fluid sample that does not bind to the fine particles is removed and washed.

[0149] After removing the fluid sample that does not bind with the fine particles, a cleaning solution is injected and mixed with the fine particles. Then, the magnetic material is brought close to the reaction chamber 200 to separate the magnetic particles from the cleaning solution, and the cleaning solution is removed, thereby cleaning the fine particles.

[0150] In step 1230, a second capture substance that specifically binds to the analyte bound to the first capture substance is injected into the reaction chamber 200 and reacted. The second capture substance that does not bind to the analyte is removed and washed.

[0151] Since the second capture substance binds to the analyte and the first capture substance, which is fixed to the fine particles, also binds to the analyte, it is preferable that the first and second capture substances bind to different parts of the analyte.

[0152] In step 1240, a signal-generating substance that specifically binds to the second capture substance is injected into the reaction chamber 200 and reacted. Signal-generating substances that do not bind to the second capture substance are removed and washed. The signal-generating substance may be, for example, an enzyme.

[0153] The signal-generating substance is, for example, a hue source, a fluorescent or chemiluminescent enzyme precursor, which, upon contact with a substrate solution, is converted into a detectable indicator.

[0154] The signal-generating substance reacts with the substrate solution to generate a signal of a detectable indicator, such as a fluorescent signal, and the enzyme component may include β-galactosidase, horseradish peroxidase, or alkaline phosphatase.

[0155] In steps 1230 and 1240, the particle detection device for the analyte washes the fine particles in the same manner as in step 1220.

[0156] In step 1250, the particle detection device for the analyte injects and mixes a substrate solution that reacts with the signal-generating substance to generate a signal into the reaction chamber 205. It is preferable to dry the fine particles remaining in the reaction chamber 205 before mixing them with the substrate solution.

[0157] The substrate solution reacts with the signal-generating substance to generate a detectable indicator signal such as a fluorescent signal, and may contain resorufin β-D-galactopyranoside or the like.

[0158] In step 1260, the particle detection device for the substance to be analyzed injects the mixed substrate solution into the inlet 301 and uses the magnetic material 15 to capture the magnetic particles in the mixed substrate solution into the microchamber 31 in the detection space 331.

[0159] To separate the substances in the microchamber 31 located in the detection space 331 of the analysis tip 301 from each other, a fluid that is immiscible with the substrate solution can be injected, preferably containing oil or air. The microchamber is preferably larger than the size of the fine particles and large enough to contain one or fewer magnetic particles.

[0160] According to another embodiment of the present invention, instead of reacting the fine particles with the second capture substance in the reaction chamber 200, the first capture substance immobilized on the fine particles may bind to the analyte, and then the fine particles may be injected into the injection port 311 so that the fine particles are captured in each microchamber 31.

[0161] Subsequently, when a second capture substance 61, which binds to the analyte and the signal generating substance, is injected through the injection port 311, the analyte 51 bound to the first capture substance 41 will bind to the second capture substance 61. In the next step, when a signal generating substance 71 is injected through the injection port 311, the signal generating substance 71 will bind to the second capture substance 61.

[0162] Furthermore, when the substrate solution is injected through the injection port 311, the signal generating substance 71 reacts with the substrate solution to generate a signal of a detectable indicator such as a fluorescent signal. By allowing a hydrophobic solvent to flow through the injection port 311, the substrate solution that remains after reacting with the signal generating substance 71 can be removed.

[0163] In step 1270, the particle detection device for the substance to be analyzed mixes the signal generating substance with the substrate solution and counts the number of microchambers 31 that generate a signal.

[0164] As one embodiment, a first count value is calculated by recognizing and counting a microchamber containing the magnetic particles, and a second count value is calculated by recognizing and counting a microchamber from which a signal was generated by mixing the signal generating substance and the substrate solution.

[0165] By calculating the ratio of the second count value to the first count value, the amount of the analyte in the fluid sample can be estimated.

[0166] In another embodiment, a region containing many of the microchambers can be photographed using a microscope and a digital camera, a first count value can be calculated by classifying the microchambers containing the magnetic particles using image processing and counting their number, and a second count value can be calculated by classifying the microchambers that generate a signal of a detectable indicator such as a fluorescent signal using image processing and counting their number.

[0167] As one embodiment of estimating the amount of particles of the analyte, the ratio of the number of microchambers that generate the fluorescence signal to the number of microchambers containing the magnetic particles can be calculated and estimated. The larger the amount of the analyte in the fluid sample, the higher the ratio, and the smaller the amount of the analyte, the lower the ratio.

[0168] As another embodiment for estimating the amount of particles of the analyte, the cartridge contains three or more standard substances whose concentrations are known in advance. After the particle detection device for the analyte according to one embodiment of the present invention generates a calibration curve showing the concentrations relative to the standard substances, the generated calibration curve can be used to correct the concentration calculation of the analyte.

[0169] Figure 13 is a diagram showing the configuration of an apparatus for detecting trace amounts of analyte particles present in a fluid sample according to a third embodiment of the present invention.

[0170] Figure 13(a) is a plan view of the apparatus for detecting particles of an analyte according to the third embodiment of the present invention, and Figure 13(b) is a cross-sectional view of the apparatus for detecting particles of an analyte according to the third embodiment of the present invention.

[0171] Referring to Figure 13, the apparatus for detecting trace amounts of analyte particles present in a fluid sample according to the third embodiment of the present invention comprises a magnetic device 152 and a cartridge 102.

[0172] The magnetic device 152 moves closer to or away from the reaction chamber 206, thereby causing separation between the microwell particles 4 and the residual solution in the reaction chamber 206. After removing the residual solution, the cleaning solution contained in the cleaning solution chamber 232 can be injected into the reaction chamber 206 to clean the wall surface of the reaction chamber 206 where the microwell particles 4 are located. Preferably, a magnetic material is formed at the bottom of the microwell particles 4.

[0173] Cartridge 102 comprises a fluid sample chamber 202, a reaction chamber 206, a waste chamber 212, a microwell fine particle chamber 222, a washing solution chamber 232, a second captured substance chamber 242, a signal generating substance chamber 252, a substrate solution chamber 262, and an analysis tip 302.

[0174] The cartridge 102 is preferably sealed with an airtight film using heat fusion, UV bonding, ultrasonic bonding, or tape, in order to prevent contamination of the multiple chambers and analysis tip 302.

[0175] The fluid sample chamber 202 is configured to contain a fixed amount of fluid sample. Such a fluid sample chamber 202 can be configured to have a volume in the range of 50 μL to 10 mL. The fluid sample includes body fluids such as blood, plasma, serum, and cerebrospinal fluid.

[0176] The reaction chamber 206 is configured to contain and mix a fixed amount of fluid sample with microwell fine particles on which the first capture substance is immobilized. Such a reaction chamber 206 can be configured to have a volume in the range of 50 μL to 15 mL.

[0177] The waste chamber 212 is configured to provide a containment space for waste generated when fluid samples, microwell particles, washing solution, second capture material, signal generating material, and substrate solution are discharged from the analysis tip 302. Such a waste chamber 212 can be configured to have a capacity of 1 to 15 mL. In Figure 13, the waste chamber 212 is contained within the cartridge 102, but the waste chamber 212 may also be provided in a separate space outside the cartridge.

[0178] The microwell fine particle chamber 222 is configured to contain microwell fine particles on which the first capture substance is immobilized, and the microwell fine particles are injected into the reaction chamber 206.

[0179] The microwell particles contained in the microwell microparticle chamber 222 preferably have a first capture substance bound to their surface, which binds to the analyte. The first capture substance may include antibodies, nucleic acid molecules, peptides, and the like.

[0180] The cleaning solution chamber 232 is configured to contain a cleaning solution for cleaning the walls of the reaction chamber 206 or the detection space 332 of the analysis tip 302. The cleaning solution may be a solution such as PBS (phosphate buffered saline), TBS (tris-buffered saline), PBS-T, TBS-T, or ultrapure water.

[0181] In one embodiment, the cleaning solution in the cleaning solution chamber 232 is injected into the inlet 312 to clean the fluid sample, fine particles, second capture substance, signal generating substance, and substrate solution remaining in the detection space 332 after the reaction.

[0182] In another embodiment, the magnetic device 152 is positioned close to the reaction chamber 206. When separation occurs between the microwell particles and the residual solution in the reaction chamber 206, the residual solution is removed, and then the cleaning solution contained in the cleaning solution chamber 232 is injected into the reaction chamber 206 to clean the wall surface of the reaction chamber 205 where the microwell particles are located. In this case, it is preferable that a magnetic material is formed beneath the microwell particles.

[0183] The second capture substance chamber 242 is configured to contain a second capture substance that specifically binds to the analyte and to the signal-generating substance. The second capture substance has the property of specifically binding to both the analyte and the signal-generating substance, and the signal-generating substance binds to the second capture substance which is specifically bound to the analyte, playing a role in generating a fluorescence signal proportional to the amount of analyte. For example, the second capture substance may be a secondary antibody and can be provided in a form that is pre-bound to the signal-generating substance.

[0184] Since the second capture substance binds to the analyte and the first capture substance, which is fixed to the microwell fine particles, also binds to the analyte, it is preferable that the first and second capture substances bind to different parts of the analyte.

[0185] The signal-generating substance chamber 252 is configured to contain a signal-generating substance that specifically binds to the second capture substance bound to the analyte. The signal-generating substance may be, for example, an enzyme.

[0186] The signal-generating substance is, for example, a hue source, a fluorescent or chemiluminescent enzyme precursor, which, upon contact with a substrate solution, is converted into a detectable indicator.

[0187] The signal-generating substance and the second capture substance may be housed together in either the second capture substance chamber 242 or the signal-generating substance chamber 252.

[0188] The signal-generating substance reacts with the substrate solution to generate a detectable indicator such as a fluorescent signal, and the enzyme component may include β-galactosidase, horseradish peroxidase, or alkaline phosphatase.

[0189] The substrate solution chamber 262 is configured to contain a substrate solution that reacts with the signal-generating substance to generate a signal. The substrate solution reacts with the signal-generating substance to generate a detectable indicator such as a fluorescent signal, and may contain resorufin β-D-galactopyranoside or the like.

[0190] The analysis chip 302 comprises an injection port 312 and a detection space 332.

[0191] In the reaction chamber 206, a fixed amount of fluid sample and microwell fine particles on which the first capture substance is immobilized are contained and mixed, so the analyte becomes bound to the first capture substance immobilized on the microwell fine particles.

[0192] The injection port 312 is into which a fluid sample, microwell fine particles, washing solution, second capture substance, signal generating substance, and substrate solution or mixed solution are injected.

[0193] The injection port 312 may further include a filter to separate and supply particles larger than the microwell microparticles from a mixed solution of microwell microparticles and a fluid sample. The filter may be formed of pores of various sizes and materials so as to be able to filter out living cells, inorganic particles, or organic particles. In this third embodiment, the filter is preferably formed of pores that can filter out particles larger than the microwell microparticles. The filter may also be formed of a biologically inert material so as to be applicable to biological samples.

[0194] During the process of supplying the fluid sample to the injection port 312, the filter output surface can be pre-moistened by immersing it in buffer solution before filter filtration. Therefore, the mixed solution of microwell fine particles and the fluid sample supplied to the filter input surface can pass through the filter pores without surface resistance and be smoothly filtered to the filter output surface.

[0195] When a mixed solution of microwell fine particles and a fluid sample is injected into the detection space 332 through the injection port 312, the microwell fine particles are arranged in the internal space of the detection space 332. By placing the magnetic material 16 beneath the microwell fine particles 4, they can be easily fixed in the internal space of the detection space 332.

[0196] Subsequently, when the cleaning solution is injected through the injection port 312, the microwell fine particles that are not fixed in the internal space of the detection space 332, the first captured substance that is not fixed to the microwell fine particles 4, and the fluid sample that is not bound to the first captured substance can be removed and cleaned.

[0197] On the other hand, when the second capture substance contained in the second capture substance chamber 242, which binds to the analyte and signal generating substance, is injected through the injection port 312, the analyte bound to the first capture substance will bind to the second capture substance, and thereafter, the remaining second capture substance can be removed and washed away with the washing solution.

[0198] Next, when the signal-generating substance is injected through the injection port 312, the signal-generating substance and the second capture substance combine.

[0199] Finally, the substrate solution is injected through the injection port 312, and the signal generating substance reacts with the substrate solution to generate a signal of a detectable indicator, such as a fluorescent signal. A hydrophobic solvent is then allowed to flow through the injection port 312 to remove any substrate solution remaining after reacting with the signal generating substance. The hydrophobic solvent is preferably an oil or a gas.

[0200] Figure 14 is a diagram showing in more detail an analysis chip 302 according to a third embodiment of the present invention.

[0201] Through the injection port 312, a mixed solution of the fluid sample and microwell fine particles, a washing solution, a second capture substance, a signal generating substance, and a substrate solution or a mixture thereof are sequentially injected. The second capture substance is a capture substance for detecting the signal of a detectable indicator such as a fluorescent signal, and can be provided bound to the signal generating substance, which simplifies the procedure compared to when the second capture substance and the signal generating substance are provided separately.

[0202] When the mixed solution of the fluid sample and microwell fine particles 4, the washing solution, the second capture substance, the signal generating substance, and the substrate solution or a mixture thereof are injected into the injection port 312, they diffuse and flow into the detection space 332, forming a filler 5 at one end of the detection space 332, causing the microwell fine particles 4 to accumulate within the detection space 332.

[0203] As shown in Figure 14, the microwell particles 4 are collected in the detection space 302, where a mixture of microwell particles containing the first capture substance fixed in the internal space of the microwell particles 4 and the analyte in the fluid sample, and microwell particles that are not bound, are present.

[0204] Figure 15 is a diagram showing in more detail the microwell fine particles according to the third embodiment of the present invention.

[0205] Referring to Figure 15, the microwell fine particles 4 have a hexagonal top surface, and the containment portion 6, which is the internal space of the microwell fine particles 4, may have one side open, or it may have a donut shape with both sides open and penetrating.

[0206] The microwell fine particle containment section 6 may be a cylindrical shape with one side closed (Figure 15(b)) or a cylindrical shape with both sides open (Figure 15(c)).

[0207] On the other hand, the first capture substance is fixed in the microwell fine particle containment section 6 and can bind to the analyte.

[0208] The containment section 6, which is the internal space of the microwell fine particles 4, and the first captured substance are coated with a hydrophilic solvent, while the external space 7 of the microwell fine particles 4 is coated with a hydrophobic solvent. When the hydrophobic solvent is flowed onto the substrate, the substrate solution in the external space 7 of the microwell fine particles 4 can be removed.

[0209] Comparing Figure 7 and Figure 15, the difference is that in Figure 15, the first captured substance is fixed in the internal space of the microwell fine particle 4, whereas in Figure 7, the first captured substance is fixed to the fine particle.

[0210] Figure 16 is a conceptual diagram illustrating the process of detecting the target substance according to the third embodiment of the present invention.

[0211] Through the injection port 312, a mixture of the fluid sample and microwell fine particles 4, a washing solution, a second capture substance, a signal generating substance, and a substrate solution or a mixture thereof are sequentially injected.

[0212] Figure 16(a) shows the state in which the microwell fine particles 4, to which the first capture substance 42 is immobilized, are injected into the injection port 312 after being mixed and reacted with the fluid sample in the reaction chamber 206, and each microwell fine particle 4 is positioned and fixed by the magnetic material 16. The first capture substance 42 is immobilized on the microwell fine particles 4, and the analyte 52 is specifically captured.

[0213] The mixture of the fluid sample and the microwell fine particles 4 can be mixed in the reaction chamber 206. The internal space of the microwell fine particles 4 is immobilized with a first capture substance, and the analyte in the fluid sample binds to the first capture substance.

[0214] When the mixture of the fluid sample and the microwell fine particles 4 is injected into the injection port 312, it diffuses and flows into the detection space 332. The mixture of the fluid sample and the microwell fine particles 4 that has passed through the detection space 332 is blocked by a plurality of fillers 5 formed at the opposite end of the injection port 312 and collects in the detection space 332.

[0215] Subsequently, when the cleaning solution is injected through the inlet 312, the fluid sample containing the analyte, or the analyte not captured by the first capture substance, can be cleaned by removing the bond between the first capture substance and the analyte.

[0216] As can be seen from Figure 16(b), when the second capture substance 62, which is contained in the second capture substance chamber 242 that binds to the analyte and signal generating substance, is injected through the injection port 312, the analyte 52 bound to the first capture substance 42 will bind to the second capture substance 62, and thereafter the remaining second capture substance 62 can be removed and washed through the washing solution.

[0217] In Figure 16(c), when the signal-generating substance 72 is injected through the injection port 312, the signal-generating substance 72 and the second capture substance 62 bind together. Subsequently, it is preferable that the remaining signal-generating substance 72 is washed away with a cleaning solution.

[0218] In Figure 16(d), when the substrate solution is injected through the injection port 312, the signal generating substance 72 reacts with the substrate solution to generate a detectable indicator signal such as a fluorescent signal. A hydrophobic solvent is then allowed to flow through the injection port 312 to remove any substrate solution that has reacted with the signal generating substance 72.

[0219] By allowing a hydrophobic solvent to flow across the substrate, the substrate solution outside the microwell particles 4 is removed, and the number of microwell particles 4 that have generated a detectable indicator signal, such as a fluorescent signal, is counted for detection. The inside 6 of the microwell particles 4 is coated with a hydrophilic solvent, and the outside 7 of the microwell particles 4 is coated with a hydrophobic solvent. When the hydrophobic solvent flows across the substrate, the substrate solution outside 7 of the microwell particles 4 can be easily removed.

[0220] To more accurately detect analyte contained in a fluid sample, the number of analyte particles can be counted by correlating the brightness of the fluorescence signal of a detectable indicator, such as a fluorescence signal, with the brightness of the fluorescence signal of each microwell fine particle 4.

[0221] Referring again to Figure 16(d), it can be confirmed that the number of signal-generating substances that produce detectable indicator signals such as fluorescent signals differs for each microwell fine particle 4, and that the brightness of the fluorescent signal varies as a result.

[0222] Figure 17 is a flowchart of a method for detecting trace amounts of analyte particles in a fluid sample according to a third embodiment of the present invention.

[0223] In step 1700, the fluid sample is mixed in the reaction chamber 206 with microwell fine particles 4 on which a first capture substance that specifically binds to the analyte is immobilized.

[0224] The mixed fluid sample and microwell fine particles 4 are injected into the injection port 312, and the first capture substance 42 is fixed in the internal space 6 of the microwell fine particles 4.

[0225] In step 1710, a mixed solution of microwell fine particles 4 and the fluid sample is flowed into the detection space 332. The microwell fine particles 4 flow through the detection space 332 with the analyte 52 in the fluid sample bound to the first captured substance 42.

[0226] In step 1720, after the first captured substance 42 and the analyte 52 have combined, the remaining fluid sample is removed and washed.

[0227] In step 1730, the analyte 52 and the second capture substance 62, which specifically binds to the analyte 52 and the signal generating substance 72, are allowed to flow into the microwell fine particles 4, thereby causing the analyte 52 and the second capture substance 62 to react.

[0228] In step 1740, the analyte 52 is reacted with the second capture substance 62, and the remaining second capture substance 62 is removed and washed.

[0229] In step 1750, the signal-generating substance 72 is made to flow into the microwell fine particles 4 and bind with the second capture substance 62.

[0230] In step 1760, a substrate solution that reacts with the signal-generating substance 72 to generate a detectable indicator signal such as a fluorescent signal is flowed through the microwell fine particles 4.

[0231] In step 1770, a hydrophobic solvent is flowed through the microwell microparticles 4, so that the substrate solution outside the microwell microparticles 4 is removed while the signal generating substance 72 and the substrate solution remain bound inside the microwell microparticles 4.

[0232] It is preferable that the interior 6 of the microwell fine particles 4 is hydrophilic, and the exterior 7 of the microwell fine particles 4 is hydrophobic.

[0233] In step 1780, the number of microwell fine particles 4 that have generated a signal from a detectable indicator such as the fluorescent signal is counted and detected.

[0234] As one embodiment, a first count value is calculated by recognizing and counting the microwell fine particles 4, and a second count value is calculated by recognizing and counting the microwell fine particles 4 that generate a signal when the signal-generating substance and the substrate solution are mixed.

[0235] By calculating the ratio of the second count value to the first count value, the amount of the analyte in the fluid sample can be estimated.

[0236] In another embodiment, a region containing the microwell fine particles 4 can be photographed using a microscope and a digital camera, a first count value can be calculated by classifying the microwell fine particles 4 by image processing and counting their number, and a second count value can be calculated by classifying the microwell fine particles 4 that generate the fluorescence signal by image processing and counting their number.

[0237] As one embodiment of estimating the amount of particles of the analyte, the ratio of the number of microchambers 4 that generate a signal from a detectable indicator such as a fluorescent signal to the total number of microwell fine particles 4 can be calculated and estimated. The larger the amount of the analyte in the fluid sample, the higher the ratio, and the smaller the amount of the analyte, the lower the ratio.

[0238] As another embodiment for estimating the amount of particles of the analyte, the cartridge contains three or more standard substances whose concentrations are known in advance. After the particle detection device for the analyte according to one embodiment of the present invention generates a calibration curve showing the concentrations of the standard substances, the generated calibration curve can be used to correct the concentration calculation of the analyte.

[0239] Figure 18 shows a microchamber containing fine particles and a microchamber generating a fluorescence signal according to a second embodiment of the present invention.

[0240] Microchambers containing fine particles that are not bound to the analyte are represented as dark cells, while microchambers that generate a fluorescence signal are represented as bright cells.

[0241] To calculate the ratio of the number of microchambers containing fine particles that do not contain the analyte to the number of microchambers that generate the fluorescence signal, instead of counting the number of cells, the area of ​​the bright cell region can be calculated after collecting the bright cells, or the area of ​​the dark cell region can be calculated after collecting the dark cells. By doing so, the ratio of the second count value to the first count value can be calculated, and the amount of the analyte in the fluid sample can be estimated.

[0242] When the first capture substance is an antibody-coated fine bead (a small, bead-like protein), and this fine particle is the fine particle according to the second embodiment of the present invention, the first capture substance captures the analyte, and the beads that generate a signal due to the second capture substance bound to the desired protein (analyte) from the serum or plasma sample, and the enzyme (signal-generating substance) bound to the second capture substance, can be called "on" beads.

[0243] When determining the concentration of the analyte according to the second embodiment of the present invention, it is preferable to determine the enzyme concentration (AEB) using a digital and analog analysis system, and the concentration is determined by the number of "on" beads (activated protein).

[0244] In serum or plasma samples, the concentration of a desired protein (analyte) can be determined when the ratio of enzyme (signal generator) to beads (fine particles) is low, or when there are no beads that generate a signal due to the enzyme, by using the Poisson distribution to convert the ratio of active beads to the enzyme concentration (AEB).

[0245] The Poisson distribution can explain the possibility of multiple events occurring, given the average number of events. If the expected average number of occurrences is μ, the probability of the exact ν occurring can be expressed as shown in Equation 1 below (where ν is a non-negative integer, ν = 0, 1, 2, 3, ---).

[0246]

number

[0247] μ is equal to the proportion of proteins bound to the beads, and ν is equal to the number of enzyme-labeled proteins carried by each subgroup of beads (i.e., 0, 1, 2, 3, etc.).

[0248] According to a second embodiment of the present invention, the same μ value as the enzyme concentration (AEB) can be determined, and this can be used as a quantitative parameter to determine the protein concentration.

[0249] Beads associated with one enzyme are indistinguishable from those associated with two enzymes, indicating that the bead population associated with one enzyme has a distribution of up to seven times the activation stage. Due to this broad distribution, when ν=0, some "off" beads (inactivated proteins) can be identified (Pμ(0)).

[0250] Using equation 1 to determine Pμ(0), and using the fact that the proportion of "off" beads is equal to the proportion of "on" beads minus 1, f on (Percentage or % activity of "on" beads) from AEB digital It is possible to determine (AEB determined digitally).

[0251]

number

[0252] The aforementioned formula 2 allows for the accurate measurement of protein (analyte) concentration in a plasma sample.

[0253] While concentration quantification using the Poisson distribution works well when the activation bead content is less than 70%, it has limitations: when the proportion of activation beads exceeds approximately 70%, the change in AEB with respect to concentration decreases, leading to increased inaccuracy in concentration measurement.

[0254] To overcome this, AEB uses the average fluorescence intensity value of the activated beads.

number

number

[0255]

number

[0256]

number

[0257]

number

[0258] In this case, it is preferable to select an array of "on" beads < 0.1 that satisfies the criteria of Formula 4.

[0259] Therefore, the enzyme dose (AEB) can be configured for both the digital and analog ranges.

[0260] Figure 19 is a graph showing the relationship between the concentration (pg / mL) and signal (au) of phosphorylated tau-181.

[0261] Phosphorylated tau-181 protein is a protein used in the diagnosis of Alzheimer's disease and is the protein corresponding to the analyte of this invention.

[0262] After the second capture substance binds to the phosphorylated tau-181 protein, and the enzyme (signal generator) binds to the second capture substance, a signal is generated by the substrate solution. Figure 19 shows the relationship between the concentration of phosphorylated tau-181 protein and the signal generation intensity.

[0263] Referring to Figure 19, it can be confirmed that the concentration of phosphorylated tau-181 protein is proportional to the signal generation intensity.

[0264] As described above, the present invention has been explained with specific details such as concrete components and limited embodiments and drawings, but these are provided to aid in a more overall understanding of the present invention, and the present invention is not limited to the above embodiments, and various modifications and variations can be made from these descriptions by those who have ordinary skill in the art to which the present invention belongs.

[0265] Therefore, the concept of the present invention should not be limited to the embodiments described, and all modifications equivalent to or comparable to the claims described below, as well as the claims described below, can be said to fall within the scope of the concept of the present invention.

Claims

1. A step of flowing a fluid sample containing the analyte onto a substrate on which a microchamber array is formed, the first capture substance which specifically binds to the analyte on its surface; A step in which the analyte binds to the first captured substance in each microchamber of the microchamber array; A step of reacting the analyte with the second capture substance by allowing a second capture substance, which specifically binds to the analyte and binds to the signal generating substance, to flow onto the substrate; A step of causing the signal-generating substance to flow onto the substrate and combine it with the second capture substance; A step of pouring a substrate solution that reacts with the signal-generating substance to generate a fluorescent signal onto the substrate; A step of removing the substrate solution outside the microchamber after a hydrophobic solvent has been flowed through the substrate and the signal generating substance and substrate solution have reacted inside the microchamber; and Steps to count and detect the number of microchambers from which the aforementioned fluorescence signal was generated. A method for detecting trace amounts of an analyte in a fluid sample, including [specific examples of analytes].

2. A method for detecting an analyte present in trace amounts in a fluid sample according to claim 1, characterized in that the inside of the microchamber is hydrophilic with the first captured substance immobilized thereon, and the outside of the microchamber is hydrophobic.

3. A method for detecting particles of an analyte present in trace amounts in a fluid sample according to claim 1, characterized by calculating a first count value obtained by counting the microchambers and a second count value obtained by recognizing and counting the microchambers that generated a signal due to the mixing of the signal-generating substance and the substrate solution, and estimating the amount of the analyte in the fluid sample by calculating the ratio of the second count value to the first count value.

4. A method for detecting trace amounts of particles of an analyte present in a fluid sample according to claim 1, characterized by generating a calibration curve showing the concentration of a fluid sample with a known concentration relative to a standard substance, and then correcting the concentration calculation of the analyte using the generated calibration curve.

5. A method for detecting particles of an analyte present in trace amounts in a fluid sample, according to claim 1, characterized by calculating a first count value obtained by counting the microchambers and a second count value obtained by photographing the microchamber region, classifying the microchambers that generate the fluorescence signal by image processing, and counting the number of such microchambers, thereby estimating the amount of the analyte in the fluid sample.

Images