Concentration method and device for rapidly detecting target to be detected in biological sample

Abstract

The invention discloses a method and a device for sample treatment in rapid detection, and particularly discloses a method for concentrating and purifying a to-be-detected target in a biological sample, which comprises the following steps: mixing and incubating the biological sample and nanoparticles, and capturing and combining the to-be-detected target in the sample by a surface antibody of the nanoparticles to form a target-nanoparticle compound; separating and recovering the target-nanoparticle complex from the sample using a microporous filtration membrane; eluting and dissociating the target to be detected from the target-nanoparticle compound by using the eluent, and recovering a concentrated solution of the target to be detected. The invention also discloses a rapid detection method of the target to be detected, a concentration device of the target to be detected in the biological sample and a method for concentrating the target to be detected by using the concentration device. By adopting the sample treatment device and the sample treatment method, the sensitivity and the specificity of immunochromatography detection can be improved, and the sample treatment device and the sample treatment method can be suitable for detection of large-volume samples and complex biological samples.

Description

Technical Field

[0001] The present invention relates to the technical field of rapid diagnostic testing, and in particular to a method and device for processing samples in rapid testing. Background Art

[0002] Rapid Diagnostic Test (RDT) can provide disease detection results in a short time. This type of test is usually simple to operate and has clear results. It has been widely used in the rapid screening of infectious diseases. Simple portable devices are very suitable for use in resource-limited environments, and can also play a role in emergency medical scenarios and home self-testing. This test can be performed by untrained personnel, does not rely on professional laboratory equipment, and can quickly provide immediate diagnostic feedback, usually within 10 minutes to 1 hour. The conventional RDT operation process is to collect biological samples from patients and apply the biological samples to immunochromatographic test strips. After waiting for 10 to 30 minutes, the test strip will display a band of a specific color, indicating whether there is a disease-related target in the sample.

[0003] However, existing rapid diagnostic tests mainly rely on immunochromatographic test strips, which are insufficient in sensitivity and specificity. In actual applications, the concentration of target molecules in samples is often very low, and even the inability to collect sufficient concentrations of targets leads to failure in the detection of low-abundance targets. There are generally problems of insufficient sensitivity and high false negative rates. In addition, due to the small sample volume of the test strip test and the insufficient target content and purity in the sample, the detection capacity is limited and the application scenarios are also restricted.

[0004] In order to solve these problems, one solution is to concentrate the large volume sample and then apply the concentrated small volume sample to the immunochromatographic test strip test. Therefore, it is particularly important to explore new sample enrichment and concentration processing technologies. At the same time, the portable sample processing device has significant advantages in the application of rapid diagnostic tests (RDT). The device is miniaturized and low-cost, easy to operate and maintain, and reduces the risk of cross-contamination during sample processing. It is suitable for application in medical environments with limited resources and home self-testing environments.

[0005] Ultrafiltration centrifuge tubes are commonly used in laboratories for sample concentration and processing. They use centrifugal force to separate different molecules or particles in the liquid through ultrafiltration membranes with specific nanopore sizes. Small molecules or solvents can be filtered out through the ultrafiltration membrane, while macromolecules, particles or microorganisms are retained on the ultrafiltration membrane to achieve sample concentration, separation or purification. However, the above methods require high-speed centrifugation operations in large centrifuges and are carried out under specific laboratory equipment. They are not suitable for rapid testing in emergency medical environments and resource-limited environments. At the same time, for complex biological samples, such as blood and urine, there are a large number of impurities such as macromolecular proteins and particles that are simultaneously concentrated and enriched in ultrafiltration centrifuge tubes, which can easily increase the problem of false positives in detection.

[0006] The U.S. patent with publication number US10837962B2 discloses a method and related device for rapid detection of target biological molecules with enhanced sensitivity. The molecules to be tested are captured and enriched using ion exchange chromatography media, and the salt concentration of the eluent is greater than 150 mM. However, complex biological samples containing a large amount of foreign proteins will greatly interfere with the adsorption effect of the ion exchange medium on the molecules to be tested, and the complex matrix interference of the biological sample will be enriched by the ion exchange medium, affecting the specificity of downstream detection. The capture of the nanoparticle surface antibody used in this application has better affinity and specificity than the ion exchange medium, which can improve the detection specificity of the downstream immunochromatographic test strips. The adsorption effect of the ion exchange medium is also easily affected by the pH of the sample, especially biological samples such as urine, where the pH difference between different samples is very large.

[0007] The magnetic separation method uses magnetic particles to capture the molecules to be tested in biological samples, concentrates and purifies the molecules to be tested by magnetic separation of magnetic particles, uses a dissociation solution to dissociate the molecules to be tested from the magnetic particle complex, and then uses magnetic adsorption to remove the magnetic particles and recover the concentrated solution of the molecules to be tested. For example, the Chinese patent application with publication number CN114341640A discloses an immunochromatography method, which uses magnetic particles to capture the molecules to be tested in biological samples. However, when the sample volume exceeds 10mL, the traditional magnetic particle enrichment and concentration method becomes unsuitable because it cannot separate large-volume samples by magnetic force, resulting in the problem of being unable to enrich large-volume samples.

[0008] Researchers in this field used microporous filtration membranes to enrich tumor cells in the previous literature "Methods for Enriching Tumor Cells with Microporous Filter Membranes of Different Pore Sizes". A polycarbonate filter membrane with a pore size of 3 μm was used to enrich tumor cells. This method mainly relies on the size of the cells to achieve enrichment. However, the size of a large number of antigen targets is much smaller than the cell size and cannot be directly filtered and enriched using conventional microporous filter membranes (>0.22 μm). In most cases, the ultrafiltration centrifuge tubes using microporous ultrafiltration membranes mentioned above can be used for the enrichment and concentration of small molecules, but cannot be used for the concentration of complex matrix samples and require large centrifuge equipment. Summary of the invention

[0009] The purpose of the present invention is to provide a method and device for sample processing in rapid detection, which involves the concentration of the target to be detected in the rapid detection, using nanoparticles to specifically capture, separate and concentrate the target to be detected in the biological sample, separating and recovering the nanoparticles by means of a microporous filtration membrane, and eluting and concentrating the target to be detected from the nanoparticles so that it can be used for immunochromatographic detection, thereby effectively improving the detection sensitivity and specificity.

[0010] The objective of the present invention is achieved through the following technical solutions: In a first aspect, the present invention provides a method for concentrating and purifying a target to be detected in a biological sample, comprising the following steps: S1. Mix and incubate the biological sample with the nanoparticles, and the target to be detected in the sample is captured and bound by the antibody coupled to the surface of the nanoparticles to form a target-nanoparticle complex; S2, separating and recovering the target-nanoparticle complex from the sample using a microporous filtration membrane; S3. Using an eluent, the target to be detected is eluted and dissociated from the target-nanoparticle complex, and a concentrated solution of the target to be detected is recovered.

[0011] As some specific embodiments of the present invention, in step S1, the biological sample includes at least one of urine, sputum, blood, cell culture fluid, and pleural effusion.

[0012] As some specific embodiments of the present invention, in step S1, the nanoparticles include at least one of polystyrene nanoparticles, silica particles, and silica magnetic particles.

[0013] As some specific embodiments of the present invention, in step S1, the incubation temperature is 4-55° C. and the incubation time is 5-60 min.

[0014] As some specific embodiments of the present invention, in step S1, any antibody is coupled to the surface of the nanoparticle by covalent binding, and the coupling method includes EDC / NHS coupling method.

[0015] As some specific embodiments of the present invention, in step S2, the microporous filtration membrane includes at least one of polyvinylidene fluoride, polyethersulfone, polytetrafluoroethylene, and nylon.

[0016] As some specific embodiments of the present invention, the particle size of the nanoparticles is 400 nm-3 μm; The pore size of the microporous filtration membrane is 200 nm-1 μm; The pore size of the microporous filtration membrane is smaller than the particle size of the nanoparticles.

[0017] As some specific embodiments of the present invention, in step S3, the eluent contains NaOH or H2SO4, and a surfactant, and the surfactant includes at least one of S9, NP-40, and Tween-20.

[0018] Furthermore, the concentration of NaOH or H2SO4 is 5-100 mM.

[0019] As some specific embodiments of the present invention, the volume ratio of the eluent to the biological sample is 1:10-1:100.

[0020] In a second aspect, the present invention provides a method for rapid detection of a target to be detected, comprising dripping a concentrated solution of the target to be detected obtained by concentrating and purifying the target by any of the methods described above onto an immunochromatographic test strip for detection.

[0021] As some specific embodiments of the present invention, the immunochromatographic test strip comprises a filter pad, and the filter pad contains 100-700 mM Tris.

[0022] As some specific embodiments of the present invention, the labeling particles of the immunochromatographic test strip include at least one of colloidal gold, latex spheres, fluorescent nanoparticles, and quantum dots.

[0023] In a third aspect, the present invention provides a device for concentrating a target to be detected, which is used in any of the above methods for concentrating and purifying a target to be detected in a biological sample, comprising: A mixing unit, a filtration unit and an elution unit; The mixing unit comprises a mixing container, wherein the mixing container is filled with nanoparticles with surface-coupled antibodies and an insertable extruder, and the insertable extruder can reciprocate along the inner wall of the mixing container in the mixing container; The filtration unit includes a microporous filtration membrane; the filtration unit can be connected to the mixing unit; The elution unit comprises an elution container, the outer shell of the elution container is an extrudable and resilient structure, the elution container is filled with an eluent, and the elution container is connected to the filtering unit.

[0024] As some specific embodiments of the present invention, a sealing ring is installed at the bottom of the insertable extruder, and the sealing ring is in close contact with the inner wall of the mixing container and reciprocates along the inner wall of the mixing container with the insertable extruder.

[0025] As some specific embodiments of the present invention, an upper opening is provided on the top of the mixing container, and the insertable extruder enters the interior of the mixing container through the upper opening.

[0026] As some specific embodiments of the present invention, the filtration unit includes a filter, and the microporous filtration membrane is fixed inside the filter by upper and lower fixers; the filter is provided with an inlet and an outlet at the upper and lower ends respectively, and the inlet can be interconnected with the bottom opening of the mixing container.

[0027] As some specific embodiments of the present invention, the elution container has an inverted funnel-shaped upper portion, an arc-shaped bottom cross-section, an elution port at the top, and the elution port is sealed and connected to the sample outlet of the filtration unit.

[0028] As some specific embodiments of the present invention, the concentration device is used to concentrate the target to be detected in the biological sample, comprising the following steps: A1. Pull the insertable squeezer to suck a large volume of biological sample into the mixing container, and the target to be detected in the biological sample is combined with the nanoparticles with antibodies coupled to the surface of the mixing container to form a target-nanoparticle complex; A2, connecting the filtration unit and the mixing unit, pressing the insertable squeezer, the target-nanoparticle complex enters the filtration unit and is intercepted by the microporous filtration membrane, and the discarded biological sample is discharged from the filtration unit; A3. Connect the elution unit and the filtration unit, repeatedly squeeze the elution container, and the eluent enters the filtration unit from the elution container to elute the target on the microporous filtration membrane from the complex with the nanoparticles, and enters the elution container through the microporous filtration membrane along with the eluent.

[0029] The concentrator of the present invention has a simple structure, is easy to operate, is economical and practical, and is portable, and is suitable for rapid diagnosis and testing in resource-limited environments and home self-testing environments.

[0030] Compared with the prior art, the present invention has the following beneficial effects: (1) The method for concentrating and purifying the target to be detected in the biological sample provided by the present invention can enrich and concentrate the target to be detected with low content in the sample, thereby improving the sensitivity of immunochromatographic detection; (2) Since there are various impurities in complex biological samples, they are easy to interfere with immunochromatographic detection, resulting in false positives and poor detection specificity. However, the present invention captures the target to be detected by coupling antibodies to the surface of nanoparticles, which can specifically bind to the target and purify the sample matrix, thereby solving the false positive problem of immunochromatographic detection and improving detection specificity. (3) In this concentration and purification method, the target to be detected is eluted from the nanoparticles, retaining all the antibody binding sites of the antigen target, which can better ensure the binding of the antigen with the antibody on the downstream immunochromatographic test strip. At the same time, the surface antibody of the nanoparticle can be the same as any antibody on the immunochromatographic test strip; (4) The miniaturized design of the sample pretreatment device makes it lightweight, easy to carry and store, and very suitable for use in resource-limited environments, such as remote areas or home self-testing environments, which improves the flexibility and accessibility of testing; the operation is simple and fast, reducing the reliance on professional skills, and effectively cooperating with the simplicity and rapidity of the immunochromatographic test strips to speed up the testing process. It does not rely on complex laboratory equipment and power supply, has strong adaptability, and can provide basic medical testing services for resource-limited environments and home self-testing environments; (5) The rapid detection device of the present invention is a disposable device that is easy to operate, has low cost, avoids cross contamination between samples, and is suitable for home self-testing environments. Compared with the magnetic separation device, the present invention uses a microporous filtration membrane as the core component of the device, which is low cost, high economic benefit, reduces resource waste, and reduces cross contamination during testing; (6) The present invention can efficiently concentrate the target to be detected in a large volume sample, solving the limitations and challenges faced by the magnetic bead enrichment method when the sample volume exceeds 10 mL, thereby improving the detection sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings: Figure 1 A schematic diagram of the structure of a device for concentrating targets to be detected in biological samples; Figure 2 Schematic diagram of aspirating biological samples for mixing vessels; Figure 3 Schematic diagram of the mixing of biological samples and nanoparticles; Figure 4 Schematic diagram of interception and separation of target-nanoparticle complexes by microfiltration membrane; Figure 5 A schematic diagram of the elution container and the filtration unit being sealed and connected; Figure 6 Schematic diagram of target recovery on the complex by dissociation of the eluent.

[0032] In the figure, 100, a device for concentrating the target to be detected in a biological sample; 120, a mixing unit; 121, a mixing container; 122, an insertable squeezer; 123, a sealing ring; 124, a bottom opening of the mixing container; 125, an upper opening of the mixing container; 200, a filtering unit; 210, a holder; 220, a microporous filter membrane; 230, a filter; 231, an injection port; 240, an outlet port; 310, nanoparticles; 311, a target-nanoparticle complex; 400, an elution unit; 410, an elution container; 420, an elution port; 430, an eluent; 500, a target to be detected. DETAILED DESCRIPTION

[0033] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several variations and improvements may be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0034] Example 1 This embodiment provides a concentration device for a target to be detected and a method for concentrating and purifying the target to be detected using the concentration device. Figure 1-6 As shown, the concentration device 100 includes a mixing unit 120 , a filtering unit 200 , and an elution unit 400 .

[0035] 1. The mixing unit 120 includes a mixing container 121, wherein the mixing container 121 contains nanoparticles 310 containing surface antibodies, and an insertable extruder 122 with a sealing ring 123 installed at the bottom enters the mixing container 121 through the upper end opening 125 of the mixing container 121, and can reciprocate along the inner wall of the mixing container 121, and the sealing ring 123 moves along the inner wall of the mixing container 121 in close contact with the inner wall of the mixing container 121, and the nanoparticles 310 are located in the cavity formed by the sealing ring 123 and the bottom end of the mixing container 121 (the nanoparticles 310 are located on the inner wall of the mixing container 121). The bottom end of the mixing container 121 is provided with a bottom end opening 124.

[0036] like Figure 2 As shown, the insertable squeezer 122 is pulled upward to suck a large volume of biological sample into the mixing container 121 from the bottom opening 124, so that the nanoparticles 310 in the mixing container 121 are mixed with the biological sample, and the target 500 to be detected in the biological sample is combined with the nanoparticles 310 to form a target-nanoparticle complex 311 ( Figure 3 ).

[0037] 2. The filtration unit 200 includes a filter 230, in which a microporous filter membrane 220 is provided. The microporous filter membrane 220 is fixed inside the filter 230 by means of fixtures 210 located at the upper and lower sides thereof; an inlet 231 and an outlet 240 are provided at the upper and lower ends of the filter 230, respectively, and the inlet 231 can be communicated with the bottom opening 124 of the mixing container 121.

[0038] like Figure 3 As shown, the filter 230 is installed at the lower part of the mixing container 121, the sample inlet 231 of the filter 230 is connected to the bottom opening 124 of the mixing container 121, and the insertable squeezer 122 is quickly moved from the upper opening 125 to the bottom opening 124 of the mixing container 120. The target-nanoparticle complex 311 in the mixing container 120 enters the filter 230 through the bottom opening 124 of the mixing container 120 and the sample inlet 231 of the filter 230 in turn, and is intercepted by the microporous filter membrane 220. The discarded biological sample is discharged from the sample outlet 240 of the filter 230 through the microporous filter membrane 220. After the sample is discharged, Figure 4 As shown, the target-nanoparticle complex is located between the microporous filtration membrane 220 and the holder 210 above it.

[0039] 3. The elution unit 400 includes an elution container 410, the shell of which is an extrudable and resilient structure, the upper part of which is in the shape of an inverted funnel, the bottom cross-section is arc-shaped, and an elution port 420 is provided at the top. The elution port 420 is sealed and connected to the sample outlet 240 of the filter 230; the elution container 410 contains an eluent 430.

[0040] like Figure 5 As shown, the elution container 410 is installed at the lower part of the filter 230, and the elution port 420 of the elution container 410 is connected to the sample outlet 240 of the filter 230. By squeezing the outer shell of the elution container 410, an external force is applied to the elution container 410. Under the action of the external force, the eluent 430 inside is transported into the filter 230 through the liquid channel formed by the top elution port 420 and the sample outlet 240 of the filter 230, and contacts the target-nanoparticle complex on the microporous filter membrane 220, and elutes the target from the complex. Due to the sealing air pressure formed in the device, the outer shell of the elution container 410 gradually rebounds, and the eluent in the filter 230 flows back to the elution container 410 through the liquid channel. The target separated from the nanoparticles enters the elution container 410 through the micropores on the microporous filter membrane 220 along with the eluent. The outer shell of the elution container 410 is squeezed repeatedly until the target on the target-nanoparticle complex is eluted, as shown in FIG. Figure 6 shown.

[0041] The steps for concentrating and enriching the target to be detected using the above-mentioned concentrating device are as follows: (1) Pulling the insertable squeezer to suck a large volume of biological sample into the mixing container through the bottom opening of the mixing container, so that the nanoparticles containing the surface antigen are mixed with the biological sample, and the target to be detected in the biological sample is combined with the nanoparticles to form a target-nanoparticle complex; (2) connecting the bottom opening of the mixing container to the sample inlet of the filter, pressing the insertable squeezer to rapidly move it from the top opening of the mixing container to the bottom opening, so that the target-nanoparticle complex in the mixing container enters the filter and is intercepted by the microporous filter membrane, and the discarded biological sample is discharged from the sample outlet of the filter through the microporous filter membrane; (3) Connect the elution port of the elution container to the sample outlet of the filter, repeatedly squeeze the outer shell of the elution container, and the eluent enters the filter and contacts the target-nanoparticle complex on the microporous filter membrane, dissociating and eluting the target from the complex with the nanoparticles and returning to the elution container with the eluent.

[0042] Example 2 This embodiment uses a concentration device for the target to be detected in the biological sample to concentrate and purify the LAM antigen in the biological sample, and then uses an immunochromatographic test strip to quickly detect it.

[0043] 1. Device for concentrating the target to be detected (1) Preparation of surface antibody-coupled polystyrene nanoparticles 1 mg of polystyrene nanoparticles (PS-COOH, particle size: 500 nm, manufactured by Maijin Bio) were activated with EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) and Sulfo-NHS (N-hydroxysulfosuccinimide), and mouse anti-LAM antibody 1# (0.15 mg / mL, purchased from Shanghai Shuoying Technology Co., Ltd.) was added for 3 h to couple the antibody to the surface of the polystyrene nanoparticles. Then, 20 μL of 10% (w / v) BSA was added for blocking for 2 h to obtain polystyrene nanoparticles coupled with antibodies. After washing the polystyrene nanoparticles 3 times with preservation solution (10 mM PBST, 1% (w / v) BSA, 0.05% (w / v) Tween-20), 100 μL of preservation solution was added to preserve the antibody-coupled polystyrene nanoparticles (10 mg / mL).

[0044] Prepare nanoparticle lyophilized solution (10 mM PBS, 5% (w / v) sucrose, 5% (w / v) dextran sulfate, 2.5% (w / v) trehalose), and dilute the antibody-coupled polystyrene nanoparticles 10 times with the lyophilized solution. Take 20 μL of the diluent and add it to the mixing container of the biological sample pretreatment device, then add 7.5 μL of 10% (w / w) NP-40, and freeze-dry the antibody-coupled nanoparticles using a freeze-drying instrument.

[0045] (2) The mixing container is made of plastic, and the sealing ring that can be inserted into the bottom of the extruder is made of rubber, which forms an airtight seal with the inner wall of the mixing container.

[0046] The microporous filter membrane in the filter unit is made of polyethersulfone with a pore size of 220 nm.

[0047] The eluent container is filled with an eluent, which is 150 μL of an aqueous solution containing 50 mM NaOH and 0.1% (w / v) S9.

[0048] 2. Immunochromatographic test strips (1) Preparation of immunocolloidal gold Take 10 mL of 0.01% (w / v) colloidal gold solution and adjust the pH to 7.0 with 0.2 M K2CO3. Then add 100 µg rabbit anti-LAM antibody 2# (purchased from Medix Biochemica) dropwise with stirring. After labeling for 30 min, add 400 μL 10% (w / v) BSA for blocking for 30 min to obtain immunocolloidal gold. Centrifuge at 8000 g for 10 min, remove the supernatant, and add 400 μL preservation solution (50 mMTris, 1% (w / v) sucrose, 0.5% (w / v) Tween-20, 0.5% (w / v) BSA, 0.02% (w / v) NaN3, pH=8.5) for storage.

[0049] (2) Preparation of immunochromatographic test strips Preparation of filter pad: Spread the prepared filter solution (360 mM Tris, 0.5% (w / v) Tween-20, 2 mM EDTA, 0.5% (w / v) PVP40000, 2% (w / v) BSA, 0.1% (w / v) Casein-Na, 0.02% (w / v) NaN3) on a glass fiber membrane (16 mm*30 cm) (2.7 mL / strip) and dry at 37°C overnight; Preparation of conjugate pad: Spread the prepared immunocolloidal gold on glass fiber membrane 8964 (5 mm*30 cm) (0.6 mL / strip) and vacuum dry; Preparation of test line and quality control line: Mouse anti-LAM antibody 1# and goat anti-rabbit IgG (purchased from Shanghai Aixin Biotechnology Co., Ltd.) were coated on fixed positions of nitrocellulose membrane as test line and quality control line, respectively, and dried at 45°C overnight; Assembly of colloidal gold chromatography test strips: Assemble the nitrocellulose membrane, absorbent pad, binding pad and filter pad on the bottom plate in sequence and cut into strips (3.75 mm) to obtain LAM colloidal gold chromatography test strips.

[0050] 3. The target concentration device concentrates the LAM antigen in the biological sample: (1) 1.5 mL of urine sample containing LAM antigen was drawn into the mixing container, mixed and incubated at room temperature for 30 min to allow the polystyrene nanoparticles coupled with mouse anti-LAM antibody 1# to fully capture the LAM antigen and form a LAM-nanoparticle complex; (2) Pressing the insertable squeezer causes the LAM-nanoparticle complex to enter the filter and be intercepted by the microporous filter membrane, and the discarded biological sample is discharged from the filter; (3) Connecting the device containing the eluent to the elution container, repeatedly squeezing the elution container, the eluent contacts the LAM-nanoparticle complex on the microporous filtration membrane, and the LAM separates from the nanoparticles and enters the eluent, which is then recovered into the eluent container to obtain a LAM concentrate.

[0051] 4. Detection of LAM antigen using colloidal gold chromatography test strips The recovered 80 μL LAM concentrate was added dropwise to the filter pad of the colloidal gold chromatography test strip, and flowed to the conjugate pad by capillary action. After being captured by the immunocolloidal gold on the conjugate pad to form a colloidal gold complex, it flowed to the nitrocellulose membrane. The colloidal gold complex was captured on the test line, and the colloidal gold particles that did not form a complex were captured on the quality control line. Wait for 15 minutes and compare the color intensity value of the test line with the color comparison card.

[0052] Example 3 The immune colloidal gold on the immunochromatographic test strip conjugate pad of Example 2 is replaced with immune red latex balls. The specific preparation method of immune red latex balls is as follows: Take 200 μg of red latex balls (manufactured by Suzhou Weidu), add NHS and EDC in the ratio of microspheres: NHS: EDC = 5:2:1, shake on a 30℃ shaker for 20 min to activate the red latex balls, then add 500 μL of coupling buffer (10 mM boric acid buffer, pH 8.0) and 20 μg of rabbit anti-LAM antibody 2#, shake on a 30℃ shaker for 2 h to couple the antibody to the red latex balls, then add 50 μL of 10% (w / v) BSA and block at room temperature for 2 h to obtain immune red latex balls. Add 500 μL of preservation solution (50mM Tris, 1% (w / v) sucrose, 0.5% (w / v) Tween-20, 0.5% (w / v) BSA, 0.02% (w / v) NaN3, pH=8.5), sonicate for 1 min, and store at 2-8℃.

[0053] The prepared immune red latex spheres were spread on a glass fiber membrane 8964 (5 mm*30 cm) (0.6 mL / strip) and vacuum dried to obtain a conjugate pad.

[0054] The remaining steps are carried out according to Example 2.

[0055] Example 4 The difference from Example 3 is that the biological sample type is pleural effusion.

[0056] Example 5 The difference from Example 2 is that the volume of the urine biological sample is 10 mL, the volume of the nanoparticle dilution in the mixing container is 60 μL, and the remaining steps are carried out according to Example 2.

[0057] Example 6 The difference from Example 3 is that the eluent is 150 μL of an eluent containing 25 mM H2SO4 and 0.1% (w / v) S9, and the remaining steps are carried out according to Example 3.

[0058] Comparative Example 1 The target to be detected in the biological sample is not concentrated and purified by the concentration device of the present invention. 80 μL of the urine biological sample containing LAM antigen is directly dripped onto the filter pad of the colloidal gold chromatography test strip, flows to the binding pad by capillary action, is captured by the immune colloidal gold particles on the binding pad to form a colloidal gold complex, and then flows to the nitrocellulose membrane. The colloidal gold complex is captured on the detection line, and the colloidal gold particles that have not formed a complex are captured on the quality control line. Wait for 15 minutes, and compare the color intensity value of the detection line with the color card.

[0059] Comparative Example 2 The difference from Comparative Example 1 is that a red latex ball chromatography test strip is used instead of a colloidal gold chromatography test strip, that is, the immune colloidal gold on the binding pad is replaced with immune red latex balls, and the preparation method of immune red latex microspheres is shown in Example 3. The remaining steps are carried out according to Comparative Example 1.

[0060] Comparative Example 3 The difference from Comparative Example 2 is that the biological sample type is pleural effusion instead of urine, and the remaining steps are carried out according to Comparative Example 2.

[0061] Comparative Example 4 This comparative example uses a magnetic separation method to concentrate the target to be detected in the biological sample.

[0062] 1. Preparation of magnetic microspheres containing surface antibodies 1 mg of magnetic microspheres (SPM-COOH, particle size: 400 nm, manufactured by Nanobrick) were activated with EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) and Sulfo-NHS (N-hydroxysulfosuccinimide), and mouse anti-LAM antibody 1# (0.15 mg / mL) was added for 3 h to couple the antibody to the surface of the magnetic microspheres. Then, 20 μL of 10% (w / v) BSA was added for blocking for 2 h to obtain antibody-coupled magnetic microspheres. After washing the magnetic microspheres three times with storage solution (10 mM PBST, 1% (w / v) BSA, 0.05% (w / v) Tween-20), 100 μL of storage solution was added to preserve the antibody-coupled magnetic microspheres (10 mg / mL).

[0063] 2. Magnetic separation method for biological samples (1) The prepared antibody-coupled magnetic spheres were diluted 10-fold with lyophilized solution (10 mM PBS, 5% (w / v) sucrose, 5% (w / v) dextran sulfate, 2.5% (w / v) trehalose). 60 μL of the magnetic sphere dilution was added to a 15 mL centrifuge tube, and 10 mL of urine sample containing LAM antigen was added, and mixed and incubated at room temperature for 30 min.

[0064] (2) The magnetic separation rack adsorbs the magnetic balls for 5 min, and then aspirates and discards the discarded urine supernatant sample.

[0065] (3) Add 150 μL of elution buffer (50 mM NaOH, 0.1% (w / v) S9) and mix well with the magnetic balls.

[0066] (4) The magnetic separation rack was used to adsorb the magnetic balls for 2 min to recover the LAM antigen concentrate.

[0067] 3. Colloidal gold chromatography test strip detection The recovered 80 μL LAM concentrate was added dropwise to the filter pad of the colloidal gold chromatography test strip, and flowed to the conjugate pad by capillary action. After being captured by the colloidal gold particles modified with rabbit anti-LAM antibody 2# on the conjugate pad to form a colloidal gold complex, it flowed to the nitrocellulose membrane. The colloidal gold complex was captured on the test line, and the colloidal gold particles that did not form a complex were captured on the quality control line. Wait for 15 minutes and compare the color intensity value of the test line with the color comparison card.

[0068] Effect Example 1. The methods of Example 2 and Comparative Example 1 were used to detect LAM biological samples of different concentrations. The detection results are shown in Table 1 below.

[0069] Table 1 Comparison of the test results of colloidal gold chromatography test strips before and after LAM biological sample treatment

[0070] According to the test results in Table 1, for the detection of urine samples with the same LAM concentration, the signal value of Example 2 is significantly stronger than that of Comparative Example 1 by 1-2 points. When the concentration is low, Comparative Example 1 will have a false negative result, but Example 2 can accurately detect. The detection sensitivity of Comparative Example 2 is 100 pg / mL, while the detection sensitivity of Example 2 is 20 pg / mL. The sample pretreatment method of the present invention is used to concentrate and purify the target to be detected in the biological sample, and the detection sensitivity is significantly improved by 5 times.

[0071] 2. The methods of Examples 3 and 4 and Comparative Examples 2 and 3 were used to detect LAM biological samples of different concentrations. The detection results are shown in Table 2 below.

[0072] Table 2 Comparison of latex ball chromatography test strips before and after LAM biological sample treatment

[0073] According to the test results in Table 2, in Comparative Examples 2 and 3, when the target is not concentrated and purified, the red latex test strips are used to detect urine samples and pleural effusion samples, and there are false positive problems. In Example 3 and Example 4, the same samples are detected after the target to be tested is concentrated using the method of the present invention, and there is no false positive problem. In particular, pleural effusion samples are typical complex matrix samples with interference from a large number of impurity proteins. In Comparative Example 3, there are extremely strong false positive signals in the detection of red latex ball chromatography test strips, and the detection results of LAM pleural effusion samples of different concentrations are the same. Example 4 solves this false positive problem using the method of the present invention.

[0074] In addition, for urine sample detection with the same LAM concentration, the signal value of Example 3 is significantly stronger than that of Comparative Example 2 by 1-2 points. The sample pretreatment method of the present invention can solve the false positive problem of complex biological sample detection, and the detection sensitivity is significantly improved.

[0075] 3. The methods of Example 5 and Comparative Example 4 were used to detect LAM biological samples of different concentrations. The results are shown in Table 3 below.

[0076] Table 3 Comparison of sample processing device and magnetic separation method for 10mL sample detection

[0077] According to the test results in Table 3, for a large volume of 10 mL urine sample, the detection signal of Example 5 is significantly higher than that of Comparative Example 4. The detection sensitivity of Comparative Example 4 is 20 pg / mL, while the detection sensitivity of Example 5 is 5 pg / mL. The sensitivity of the microporous filtration membrane sample processing device of the present invention is significantly improved by 4 times compared with the magnetic ball separation and concentration method. The problem of slow magnetic response and low recovery rate of magnetic balls in large volume samples is solved, and the efficiency of sample concentration is improved.

[0078] 4. The method of Example 6 was used to detect LAM biological samples of different concentrations. The results are shown in Table 4 below.

[0079] Table 4 Detection effect of eluent component H2SO4

[0080] According to the detection results in Table 4, the eluent component is H2SO4. The target to be detected in the biological sample is concentrated and purified by the sample pretreatment method of the present invention, and the detection sensitivity reaches 20 pg / mL, which is consistent with the detection effect of the eluent component of NaOH in Example 2.

[0081] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A method for concentrating and purifying a target to be detected in a biological sample, characterized in that: The steps include: S1. Mix and incubate the biological sample with the nanoparticles, and the target to be detected in the sample is captured and bound by the antibody coupled to the surface of the nanoparticles to form a target-nanoparticle complex; S2, separating and recovering the target-nanoparticle complex from the sample using a microporous filtration membrane; S3. Using an eluent, the target to be detected is eluted and dissociated from the target-nanoparticle complex, and a concentrated solution of the target to be detected is recovered.

2. The concentration and purification method according to claim 1, characterized in that: In step S1, the biological sample includes at least one of urine, sputum, blood, cell culture fluid, and pleural effusion.

3. The concentration and purification method according to claim 1, characterized in that: In step S1, the nanoparticles include at least one of polystyrene particles, silica particles, and silica magnetic particles; And / or, in step S2, the microporous filtration membrane includes at least one of polyvinylidene fluoride, polyethersulfone, polytetrafluoroethylene, and nylon.

4. The concentration and purification method according to claim 3, characterized in that: The particle size of the nanoparticles is 400nm-3 μm; The pore size of the microporous filtration membrane is 200 nm-1 μm; The pore size of the microporous filtration membrane is smaller than the particle size of the nanoparticles.

5. The concentration and purification method according to claim 1, characterized in that: In step S3, the eluent contains NaOH or H2SO4, and a surfactant, and the surfactant includes at least one of S9, NP-40, and Tween-20.

6. A method for rapid detection of a target to be detected, characterized in that: The method comprises dropping a concentrated solution of the target to be detected obtained by concentrating and purifying according to the method of any one of claims 1 to 5 onto an immunochromatographic test strip for detection.

7. The rapid detection method according to claim 6, characterized in that: The labeling particles of the immunochromatographic test strip include at least one of colloidal gold, latex balls, fluorescent nanoparticles, and quantum dots.

8. A device for concentrating a target to be detected, used in the method for concentrating and purifying a target to be detected in a biological sample as claimed in any one of claims 1 to 5, characterized in that: include: A mixing unit, a filtration unit and an elution unit; The mixing unit comprises a mixing container, wherein the mixing container is filled with nanoparticles with surface-coupled antibodies and an insertable extruder, and the insertable extruder can reciprocate along the inner wall of the mixing container in the mixing container; The filtration unit includes a microporous filtration membrane; the filtration unit can be connected to the mixing unit; The elution unit comprises an elution container, the outer shell of the elution container is an extrudable and resilient structure, the elution container is filled with an eluent, and the elution container is connected to the filtering unit.

9. The concentrating device according to claim 8, characterized in that: A sealing ring is installed at the bottom of the insertable extruder, and the sealing ring is in close contact with the inner wall of the mixing container and reciprocates along the inner wall of the mixing container with the insertable extruder; The filtration unit comprises a filter, and the microporous filtration membrane is fixed inside the filter by means of upper and lower fixing devices; the filter is provided with an inlet and an outlet at the upper and lower ends respectively, and the inlet can be communicated with the bottom opening of the mixing container; The upper part of the elution container is in the shape of an inverted funnel, the cross section of the bottom is arc-shaped, and the top is provided with an elution port, which is sealed and connected with the sample outlet of the filtration unit.

10. The concentrating device according to claim 8 or 9, characterized in that: The concentration device is used to concentrate the target to be detected in the biological sample, including the following steps: A1. Pull the insertable squeezer to suck a large volume of biological sample into the mixing container, and the target to be detected in the biological sample is combined with the nanoparticles with antibodies coupled to the surface of the mixing container to form a target-nanoparticle complex; A2, connecting the filtration unit and the mixing unit, pressing the insertable squeezer, the target-nanoparticle complex enters the filtration unit and is intercepted by the microporous filtration membrane, and the discarded biological sample is discharged from the filtration unit; A3. Connect the elution unit and the filtration unit, repeatedly squeeze the elution container, and the eluent enters the filtration unit from the elution container to elute the target on the microporous filtration membrane from the complex with the nanoparticles, and enters the elution container through the microporous filtration membrane along with the eluent.

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