Immunodetection device as well as preparation method and application thereof

Abstract

The invention provides an immunodetection device as well as a preparation method and application thereof, and belongs to the technical field of biological detection. The immunodetection device comprises a test strip and a small in-situ pump, and a driving part in the small in-situ pump is connected with an integrated circuit board or a chip outside the small in-situ pump through a wire and used for controlling the small in-situ pump to be opened or closed; a liquid outlet of the small in-situ pump is tightly attached to the upper part of the conjugate pad of the test strip or the upper part of the sample pad or the upper part of the middle part of the conjugate pad and the sample pad. The prepared immunodetection device is based on competitive or sandwich immunoassay, a sample is diluted on a test paper strip by a small in-situ pump, and the detection range of the test paper is adjusted according to the concentration of the sample. The immune device provided by the invention realizes rapid and accurate detection of biomarkers in a high-concentration range, can be directly used for quantitative detection of clinical samples, and is beneficial to health monitoring of nephropathy patients in hospitals or families.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and in particular relates to an immune detection device and a preparation method and application thereof. Background Art

[0002] Albumin is one of the most abundant and important proteins in the human circulatory system. As a family of globular proteins, albumin can bind and transport a variety of endogenous and exogenous compounds, and it plays an important role in human metabolism. For healthy kidneys, albumin in the blood does not enter the urine, but if the kidneys are damaged, albumin will enter the urine. Therefore, albumin in urine is an important biomarker for kidney disease. The albumin concentration in the urine of healthy people is generally less than 30 mg / L, while the protein concentration in the urine of patients is higher than 150 mg / L. Monitoring albumin in urine is of great significance for monitoring the progression of the disease in patients with chronic kidney disease. At present, the traditional methods for detecting and quantifying albumin are classic colorimetric methods, such as sulfosalicylate test, Coomassie brilliant blue (Bradford) test, bromophenol blue test, and biuret reagent test, however, these methods are inaccurate and have no selectivity for albumin in the mixture. More accurate instrumental techniques include visible absorption spectroscopy, electrochemical detection, fluorescent immunoassay, enzyme-linked immunosorbent assay (ELISA), electrical or optical biosensors, and high-performance liquid chromatography (HPLC), however these methods are often expensive, cumbersome, and time-consuming.

[0003] Lateral flow immunoassay (LFIA) has been widely used to detect diseases, pathogens, chemicals, toxins, and water pollutants. In recent years, it has been considered as one of the most promising point-of-care testing (POCT) tools in the field of bioanalysis because of its obvious advantages such as simple operation, rapid analysis, low cost, portable equipment, and user-friendliness. Ki et al. used colorimetric signals to simultaneously measure total human serum albumin and glycated albumin concentrations with detection ranges of 1 ng / mL to 1 mg / mL and 0.5 μg / mL to 3.6 mg / mL, respectively. Zangheri developed a chemiluminescence-based lateral flow immunoassay with an integrated amorphous silicon (a-Si:H) photosensor array for the quantitative detection of albumin in urine with a detection limit of 2.5 mg / L. Yasukawa developed a dual electrochemical sensor based on a test strip for the determination of albumin and creatinine, with an albumin detection range of 18.75 to 150 μg / mL. However, these detection ranges or detection limits are not compatible with the concentration ranges of healthy people (albumin below 30 mg / L) and kidney patients (albumin above 150 mg / L). The albumin concentration in patients with kidney disease is often much higher, which is a great challenge for LFIA detection. Excessively high albumin concentrations limit rapid detection methods, which makes it impossible to distinguish different concentrations in LFIA. Urine samples need to be diluted in advance, but this process reduces the ease of use of lateral flow immunoassays. Therefore, the development of a lateral flow device that can detect high-concentration albumin with a wide detection range and high accuracy is a technical problem that needs to be solved urgently at this stage. Summary of the invention

[0004] In view of this, the object of the present invention is to provide an immunoassay device and a preparation method and application thereof, wherein the immunoassay device can directly perform rapid and accurate quantitative detection of albumin, a kidney disease marker, within a high concentration range.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides an immunoassay device, comprising a test strip and a small in-situ pump, wherein the liquid outlet of the small in-situ pump is closely attached to the conjugate pad or the sample pad of the test strip or the middle part between the conjugate pad and the sample pad.

[0007] Preferably, the small in-situ pump comprises a driving component, a liquid inlet, a liquid outlet, and a solution storage chamber; the electrode layer in the driving component is connected to an integrated circuit board or chip outside the small in-situ pump through a wire.

[0008] Preferably, the types of the small in-situ pump include electrochemical pumps, electroosmotic pumps, and ultrasonic pumps.

[0009] Preferably, the conjugate pad of the test strip is coated with an antibody-bound optical marker, and the optical marker includes fluorescent nanoparticles, colloidal gold, and an enzyme that catalyzes chemiluminescence.

[0010] Preferably, the immunoassay method includes a competitive reaction method or a sandwich immunoreaction method.

[0011] Preferably, in the competitive reaction method, the nitrocellulose membrane of the test strip is coated with a detection line and a quality control line, the detection line is coated with a biomarker of the type to be detected, and the quality control line is coated with IgG; in the sandwich immunoreaction method, the nitrocellulose membrane of the test strip is coated with a detection line and a quality control line, the detection line is coated with an antibody of the biomarker of the type to be detected, and the quality control line is coated with IgG.

[0012] The present invention provides a method for preparing the immunoassay device, comprising the following steps:

[0013] The electrode layer contained in the driving component of the small in-situ pump is connected to the integrated circuit board or chip outside the small in-situ pump through a wire to control the small in-situ pump to be turned on or off; the integrated circuit board or chip can be attached to the PVC backboard card on the other side of the test strip; the nitrocellulose membrane is fixed on the upper surface of the PVC backboard card, the absorption pad is overlapped to the end part of the PVC backboard card, and the sample pad and the conjugate pad are overlapped to the starting part of the PVC backboard card in sequence to obtain the test strip; the liquid outlet of the small in-situ pump is closely attached to the conjugate pad or the sample pad of the test strip or the middle part between the conjugate pad and the sample pad.

[0014] Preferably, the immunoassay device can be placed in a 3D printed housing of a corresponding style; the 3D printed housing is prepared by 3D printing or one-piece injection molding.

[0015] The present invention provides an application of an immunoassay device, comprising the following steps: adding a sample to a small in-situ pump of the immunoassay device, diluting the sample with a diluent in the small in-situ pump, starting the small in-situ pump to release a mixture of the sample and the diluent, the mixture flowing to a conjugate pad and a nitrocellulose membrane in sequence, and observing or measuring the fluorescence color or signal intensity on a detection line and a quality control line in the immunoassay device; or adding a sample to a sample pad of the immunoassay device, starting the small in-situ pump to release the diluent, the sample and the diluent being mixed on a test strip, and then flowing to a conjugate pad and a nitrocellulose membrane in sequence, and observing or measuring the color or intensity of light signals on the detection line and the quality control line in the immunoassay device.

[0016] Preferably, the diluent dilutes the sample by a multiple of 1 / 10 to 1 / 1000000.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention uses a small in-situ pump for the first time to perform one-step fluorescent lateral flow quantitative analysis of human urine, and combines LFIA and a small in-situ pump to establish a quantitative immunoassay device for detecting albumin concentration in urine. When a urine sample is added to the present invention, the small in-situ pump first automatically releases a buffer solution for dilution. The diluted sample is then tested by the LFIA test paper, and finally the fluorescence detection result is measured for quantification.

[0019] The immunoassay device of the present invention realizes a one-step rapid and accurate detection of albumin, a kidney disease marker, within a high concentration range, and can be directly used for the quantification of albumin in the urine of clinical kidney disease patients, which helps hospitals or families monitor the health of kidney disease patients, and also provides a comprehensive solution for the detection of high-concentration markers in body fluids. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of the connection between the small in situ pump and the test strip in the immunoassay device.

[0021] Figure 2 3D shell diagram.

[0022] Figure 3 Schematic diagram of the detection principle of the immune device.

[0023] Figure 4 Schematic diagram of the ultrasonic pump above the sample pad of the test strip (the upper picture is the front view, and the lower picture is the test picture).

[0024] Figure 5 Schematic diagram of the ultrasonic pump located above the middle position between the sample pad and the conjugate pad of the test strip (the upper picture is the front view, and the lower picture is the test picture).

[0025] Figure 6 Schematic diagram of the ultrasonic pump above the conjugate pad of the test strip (the upper picture is the front view, and the lower picture is the test picture).

[0026] Figure 7 Schematic diagram of the electrochemical pump above the sample pad of the test strip (the upper picture is the front view, and the lower picture is the test picture).

[0027] Figure 8 Schematic diagram of the electrochemical pump located above the middle position between the sample pad and the conjugate pad of the test strip (the upper picture is the front view, and the lower picture is the test picture).

[0028] Fig. 9 Schematic diagram of the electrochemical pump above the conjugate pad of the test strip (the upper picture is the front view, and the lower picture is the test picture).

[0029] Fig.10 Schematic diagram of the electroosmotic pump above the sample pad of the test strip (the upper picture is the front view, and the lower picture is the test picture).

[0030] Fig.11 Schematic diagram of the electroosmotic pump located above the middle position between the sample pad and the conjugate pad of the test strip (the upper picture is the front view, and the lower picture is the test picture).

[0031] Fig.12 Schematic diagram of the electroosmotic pump above the conjugate pad of the test strip (the upper picture is the front view, and the lower picture is the test picture).

[0032] Fig.13 Results of screening of the amount of antibodies conjugated to fluorescent nanoparticles.

[0033] Fig.14 Results of the dosage screening of antibody-conjugated fluorescent nanoparticles.

[0034] Fig.15 Screening results for albumin concentration on the test line.

[0035] Fig.16 Screening results for sample addition locations.

[0036] Fig.17 Screening results for sample dilution type.

[0037] Fig.18 Screening results of sample albumin concentration dilutions.

[0038] Fig.19 Screening results of a small in situ pump in the test strip position.

[0039] Fig. 20 Filter results for Small In Situ Pumps by type.

[0040] Fig.21 Results of the screening of the addition positions of antibody-conjugated fluorescent nanoparticles.

[0041] Fig. 22 Results of the effect of the presence or absence of a small in-situ pump on the detection performance of the immunoassay device.

[0042] Fig.23 Results showing the effect of albumin concentration in the sample on the flow rate of a small in situ pump.

[0043] Fig.24 Albumin samples were diluted to 1 / 1000 for the calibration curve.

[0044] Fig.25 Albumin samples were diluted to 1 / 10000 for the calibration curve.

[0045] Fig.26 Screening results of immune device testing time.

[0046] Fig. 27 Results of specific determination of immune devices.

[0047] Fig.28 Results of stability assays of immunological devices.

[0048] Fig.29 Quantitative analysis results of clinical samples.

[0049] Fig.30 Data and errors between clinical sample concentrations determined by immunoassay device and hospital clinical sample concentrations.

[0050] In the figure, 1: small in-situ pump, 2: integrated circuit board or chip, 3: wire, 4: liquid inlet, 5: sample pad, 6: conjugate pad, 7: nitrocellulose membrane, 8: T detection line, 9: C quality control line, 10: absorption pad, 11: PVC backboard card, 12: liquid outlet, 13: driving component, 14: solution storage chamber, 15: shell area where the small in-situ pump is located, 16: shell area where the test strip is located, 17: visual area. DETAILED DESCRIPTION

[0051] The present invention provides an immunoassay device, comprising a test strip and a small in-situ pump, wherein the small in-situ pump comprises a driving component, a liquid inlet, a liquid outlet, and a solution storage chamber; the electrode layer in the driving component is connected to an integrated circuit board or a chip outside the small in-situ pump through a wire, and is used to control the opening or closing of the small in-situ pump; the liquid outlet of the small in-situ pump is closely attached to the conjugate pad of the test strip or the sample pad or the middle part between the conjugate pad and the sample pad. The immunoassay device of the present invention can be placed in a 3D printed housing of a corresponding style.

[0052] In the present invention, the small in-situ pump includes an ultrasonic pump, an electrochemical pump and an electroosmotic pump. As an implementation method, the ultrasonic pump source ( Figure 4-6 1) The drug injection pump described in the patent number CN202211685429.X applied by the applicant of the present invention is cited, wherein the film 30 and the piezoelectric ring 50 in the drug injection pump described in the source patent are the driving components described in the present invention. As another possible implementation method, the electrochemical pump ( Figure 7-9 1) The source cited patent number CN202210724624.2 applied by the applicant is described in the drug injection, the drug storage component of the source cited patent can be replaced with a cylindrical shape with a liquid outlet at the lower end, and the driving cavity in the driving component can be replaced with a hemispherical cavity. As an implementation method, the electroosmotic pump ( Figure 10-12 The driving component in 1) is sourced from the electroosmotic pump described in patent number ZL202210393564.0 applied for by the present applicant. Except for the driving component (source ZL202210393564.0), the other structures of the electroosmotic pump described in the present invention are consistent with the ultrasonic pump described in the present invention.

[0053] In the present invention, the small in-situ pump is used to store diluent in advance for sample dilution. The electrode layer of the driving component in the small in-situ pump of the present invention is connected to the integrated circuit board or chip outside the small in-situ pump through a wire, and the integrated circuit board or chip is controlled by wire or wireless control to control the opening or closing of the small in-situ pump. The integrated circuit board or chip of the present invention is integrated under the test strip and integrated with the immune device, and can also be used as an external small handheld instrument.

[0054] In the present invention, the test strip includes a sample pad, a conjugate pad, a nitrocellulose (NC) membrane, an absorption pad and a PVC backboard card. The sample pad, conjugate pad, NC membrane and absorbent pad of the present invention are overlapped and pasted on the bottom plate in sequence according to the chromatography direction. The NC membrane of the present invention is connected to the middle part of the PVC backboard card, the absorption pad is overlapped to the end part of the PVC backboard card, and the sample pad and the conjugate pad are overlapped to the starting part of the PVC backboard card. The two ends of each part overlap by 1.5 to 2.5 mm to ensure that the antibody or antigen can continue to flow from the sample pad to the absorption pad under capillary action. The sample pad and absorption pad of the present invention can be purchased from commercial channels.

[0055] In the present invention, the conjugate pad of the test strip is coated with an antibody-bound optical marker, and the optical marker includes fluorescent nanoparticles, colloidal gold, and an enzyme that catalyzes chemiluminescence. The antibody-bound optical marker of the present invention is preferably antibody-bound fluorescent nanoparticles, and the amount of the antibody-bound fluorescent nanoparticles is 1 to 5 μL, preferably 1.5 to 4.5 μL; the antibody-bound fluorescent nanoparticles are albumin antibody-bound fluorescent nanoparticles, and the amount of albumin antibody added in the preparation of the albumin antibody-bound fluorescent nanoparticles is 4 to 15 μg, preferably 5 to 10 μg. The antibody-bound fluorescent nanoparticles of the present invention are fluorescent nanoparticles that are bound to antibodies through the EDC-NHS chemical bond method.

[0056] In the present invention, the immunoassay method includes a competitive reaction method or a sandwich immunoassay. In the competitive reaction method of the present invention, the NC membrane of the test strip is coated with a detection line (T line) and a quality control line (C line), the detection line is coated with a biomarker of the type to be tested, and the quality control line is coated with IgG, preferably the detection line is coated with albumin, and the quality control line is coated with goat anti-mouse IgG. The albumin concentration of the present invention is 0.5-1.8 mg / mL, preferably 1.5 mg / mL; the goat anti-mouse IgG concentration is 0.5-1.8 mg / mL, preferably 1 mg / mL. The albumin and goat anti-mouse IgG of the present invention are respectively drawn on the detection line and the quality control line of the NC membrane at a spray speed of 0.5-1.5 μL / cm. The detection line and the quality control line of the present invention are 2-4 mm apart. The present invention dries the NC membrane coated with albumin and goat anti-mouse IgG at room temperature for 0.8 to 1.5 hours, then cuts it into membrane strips with a width of 4 to 6 mm, and stores it in a sealed bag containing a desiccant for standby use.

[0057] In the present invention, in the sandwich immunoreaction method, the NC membrane of the test strip is coated with a detection line and a quality control line, the detection line is coated with an antibody of the biomarker to be detected, and the quality control line is coated with IgG. The antibody of the biomarker to be detected in the present invention is preferably an albumin antibody, and the IgG is preferably goat anti-mouse IgG.

[0058] The present invention provides a method for preparing an immunoassay device, comprising the following steps:

[0059] The electrode layer contained in the driving component of the small in-situ pump is connected to the integrated circuit board or chip outside the small in-situ pump through a wire to control the opening or closing of the small in-situ pump, and the integrated circuit board or chip is attached to the PVC backboard card on the other side of the test strip; the NC membrane is fixed on the upper surface of the PVC backboard card, the absorption pad is overlapped to the end part of the PVC backboard card, and the sample pad and the conjugate pad are overlapped to the starting part of the PVC backboard card in turn to obtain a test strip; the liquid outlet of the small in-situ pump is placed tightly above the conjugate pad of the test strip or above the sample pad or above the middle part between the conjugate pad and the sample pad. The present invention places the immunoassay device prepared in the above steps in a 3D printed shell of the corresponding style. The present invention prepares the 3D printed shell by 3D printing or one-piece injection molding. The integrated circuit board or chip described in the present invention can be adhered to the PVC base plate on the opposite side of the starting end of the test strip and the sample pad, such as Figure 1 The 3D printed housing of the present invention is as shown. Figure 2As shown, the housing shown in the figure is the housing used after the small in-situ pump is placed above the sample pad for assembly. The present invention can also obtain a 3D printed housing of a corresponding style through 3D printing or injection molding according to the style after the small in-situ pump is placed above the sample pad and the conjugate pad or the style after the small in-situ pump is placed above the conjugate pad. The integrated circuit board or chip of the present invention can control the opening or closing of the small in-situ pump through a wired or wireless receiver.

[0060] The present invention provides an application of an immunoassay device, comprising the following steps: adding a sample to a small in-situ pump of the immunoassay device, diluting the sample with a diluent in the small in-situ pump, starting the small in-situ pump to release a mixture of the sample and the diluent, the mixture flows to the conjugate pad and the NC membrane in turn, and observing or measuring the fluorescence color or signal intensity (including particle color, fluorescence, and chemiluminescence) on the detection line and the quality control line in the immunoassay device; or adding a sample to the sample pad of the immunoassay device, starting the small in-situ pump to release the diluent, the sample and the diluent are mixed on the test strip, and then flow to the conjugate pad and the NC membrane in turn, and observing or measuring the fluorescence color or signal intensity (including particle color, fluorescence, and chemiluminescence) on the detection line and the quality control line in the immunoassay device. The diluent of the present invention is a PBS buffer (10mM, pH 7.4), which can be prepared according to conventional methods or purchased from commercial channels. The diluent of the present invention dilutes the sample at a multiple of 1 / 1000 to 1 / 10000.

[0061] In the present invention, the principle of detecting albumin in urine using an immunoassay device (such as Figure 3 As shown): a sample containing albumin is placed on a small in-situ pump or sample pad of the immunoassay device. When it flows to the conjugate pad, the antibody-bound fluorescent nanoparticles bind to the albumin in the sample and continue to flow along the NC membrane; the albumin on the test line of the NC membrane cannot bind to the albumin-antibody-bound fluorescent nanoparticles because the binding sites are occupied; the antibodies on the quality control line of the NC membrane bind to the albumin-antibody-bound fluorescent nanoparticles; using 365nm ultraviolet light as the excitation light, the fluorescent nanoparticles on the quality control line emit red fluorescence, while there is no fluorescent signal on the test line; when there is no albumin in the sample, the fluorescent nanoparticles on both the test line and the quality control line emit red fluorescence.

[0062] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0063] Example 1 Preparation of Immunoassay Device

[0064] 1. Preparation of antibody-bound fluorescent nanoparticles: Fluorescent nanoparticles are bound to antibodies via the EDC-NHS chemical bonding method.

[0065] First, 1 mL of activation buffer (20 mM MES, pH 6.5) was added to 200 μL of fluorescent nanoparticle solution to activate the carboxyl groups on the surface of the nanoparticles. The resulting solution was mixed and centrifuged at 13,000 rpm / min for 20 min, the supernatant was removed, and the nanoparticles were resuspended in 200 μL of activation buffer. Then, 5 mg of freshly prepared EDC (1% EDC in activation buffer) was added to the solution, mixed and reacted for 5 min.

[0066] Then add 15 mg of NHS (5% dissolved in activation buffer), mix and react for 10 minutes. The carboxyl groups on the surface of the fluorescent nanoparticles are activated. Next, wash three times with activation buffer, centrifuge at 13,000r / min for 20 minutes, separate the supernatant after centrifugation, and resuspend the nanoparticles in 400μL of coupling buffer (20mM MES, pH 8.5). Add 5μg of antibody to the solution, mix thoroughly on the vortex suspension, and react at room temperature for 2h. Then add 100μL of blocking buffer (10% BSA water) to the binding solution, mix thoroughly, and react at room temperature for 30 minutes, so that other sites of the fluorescent nanoparticles can be blocked to avoid nonspecific reactions. Finally, the mixed solution was centrifuged at 13,000r / min for 10 minutes, the supernatant was separated, and washed three times with coupling buffer. The nanoparticle-bound antibodies were suspended in 200 μL of storage solution (1% BSA, 0.05% Tween-20, 0.05% sucrose, 20 mM BBS buffer, pH 8.5) and stored in a refrigerator at 4°C.

[0067] 2. Preparation of immunoassay device

[0068] (1) Assembly of test strips

[0069] Materials: One sample pad (5×13.5mm 2 ), a conjugate pad (5×6.5mm 2 ), an NC film (5×25mm 2 ), an absorbent pad (5×15mm 2 ), and a PVC backplane card.

[0070] Preparation of the conjugate pad: drop 2.5 μL of the antibody-conjugated fluorescent nanoparticles prepared in step 1 onto the conjugate pad and dry for later use.

[0071] Preparation of NC membrane: stick the NC membrane on the middle position of the PVC backboard card; then, 1.5 mg / mL albumin and 1 mg / mL goat anti-mouse IgG were respectively drawn on the NC membrane through the XYZ platform dispenser at a spray speed of 1 μL / cm. The line with fixed albumin was the detection line, and the line with fixed goat anti-mouse IgG was the quality control line. The two lines were 3 mm apart; then, the fixed NC membrane was dried at room temperature for 1 h, cut into 5 mm wide strips, and stored in a sealed bag containing desiccant for later use.

[0072] Assembly of the test strip: Take the prepared PVC backing card connected to the NC membrane, connect the absorption pad to the end of the PVC backing card, and connect the sample pad and conjugate pad to the beginning of the backing card in sequence. The two ends of each part overlap by 2mm to ensure that the antibody can continue to flow from the sample pad to the absorption pad under capillary action.

[0073] (2) Assembly of immunoassay device

[0074] The edges of the film and the piezoelectric ring on opposite sides of the ultrasonic pump (the drug injection pump described in the source patent number CN115887815A) are connected to the integrated circuit board or chip outside the ultrasonic pump through wires to control the ultrasonic pump to be turned on or off;

[0075] Attach the integrated circuit board or chip to the PVC backing card on the starting end of the test strip opposite to the sample pad;

[0076] Place the outlet of the ultrasonic pump close to the sample pad of the test strip (such as Figure 4 ), or above the middle of the sample pad and the conjugate pad (as shown in Figure 5 ), or above the conjugate pad (as shown Figure 6 shown);

[0077] According to the above three assembled immunoassay device styles, a 3D printed housing is prepared by 3D printing or one-piece injection molding;

[0078] Place the immunoassay device assembled in the above steps into the 3D printed housing.

[0079] Example 2 Preparation of Immunization Device

[0080] The difference from Example 1 is that the ultrasonic pump is replaced by an electrochemical pump, and the liquid outlet of the electrochemical pump is closely attached to the sample pad of the test strip (such as Figure 7 ), or above the middle of the sample pad and the conjugate pad (as shown in Figure 8 ), or above the conjugate pad (as shown Fig. 9 The remaining steps are the same as those in Example 1.

[0081] Among them, the electrochemical pump source patent number is the drug injection pump described in CN202210724624.2, wherein the drug storage component described in the source patent is cylindrical and has a liquid outlet at the lower end, as in the present invention Figure 7-9 As shown in 14, the driving cavity in the driving component is a hemispherical cavity, as shown in the present invention. Figure 7-9 As shown in Figure 13.

[0082] Example 3 Preparation of Immunization Device

[0083] The difference from Example 1 is that the ultrasonic pump is replaced by an electroosmotic pump, and the liquid outlet of the electroosmotic pump is close to the sample pad of the test strip (such as Fig.10 ), or above the middle of the sample pad and the conjugate pad (as shown in Fig.11 ), or above the conjugate pad (as shown Fig.12 The remaining steps are the same as those in Example 1.

[0084] Wherein, the electroosmotic pump ( Figure 10-12 The driving component in 1) is derived from the electroosmotic pump described in patent number ZL202210393564.0 applied for by the present applicant. Except for the driving component (derived from the above patent), the other structures of the electroosmotic pump described in the present invention are consistent with the ultrasonic pump described in the present invention.

[0085] Example 4 Detection method of immune device

[0086] The sample was added in a volume of 0.1 μL to the small in-situ pump of the immunoassay device, which contained 999.9 μL of PBS buffer (10 mM, pH 7.4) for dilution. The small in-situ pump was electrically operated to release the mixture of the sample and PBS buffer, which flowed to the conjugate pad and NC membrane in turn. After 5 minutes, the immunoassay analyzer observed the fluorescence signal on the detection line under 365 nm light, which excited the beads to produce a fluorescence signal. The immunoassay analyzer measured the fluorescence signal intensity of the detection line.

[0087] Example 5 Detection Method of Immune Device

[0088] The difference from Example 4 is that the sample is dripped onto the sample pad in an amount of 0.1 μL, the small in-situ pump contains 999.9 μL of PBS buffer, the small in-situ pump is electrically operated to release the PBS buffer, and the sample and the PBS buffer are mixed on the test strip and flow to the conjugate pad and the NC membrane in sequence. The remaining steps are the same as in Example 4.

[0089] Example 6 Preparation and Detection Optimization of Immunoassay Device

[0090] 1. Screening of antibodies conjugated with fluorescent nanoparticles

[0091] The amount of antibody conjugated with the fluorescent nanoparticles was set to 2.5 μg, 5 μg and 10 μg, and the fluorescent nanoparticles conjugated with the antibody of the above three amounts were prepared by the method for preparing fluorescent nanoparticles conjugated with antibodies in step 1 of Example 1, wherein the antibody is an albumin antibody. The immune device was prepared by the method for preparing the immune device in step 2 of Example 1, such as Figure 5 shown.

[0092] The albumin concentrations in the samples were set to 0, 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 5 mg / mL, and 10 mg / mL, respectively. The samples with different albumin concentrations were measured using the detection method in Example 4, and the fluorescence signal intensity of the detection line was measured by the immunoassay analyzer. The results are shown in FIG. Fig.13 shown.

[0093] like Fig.13 It was shown that different amounts of antibodies (2.5 μg, 5 μg, and 10 μg) conjugated to fluorescent nanoparticles had an effect on the color intensity and sensitivity of LFIA. 5 μg and 10 μg of antibodies conjugated to fluorescent nanoparticles had better detection intensity and sensitivity. The test results did not differ much between 5 μg and 10 μg of antibodies, which may be due to the saturation of antibodies on the surface of fluorescent nanoparticles. Therefore, 5 μg of antibodies were selected to conjugate to nanoparticles to prepare antibody-bound fluorescent nanoparticles for the preparation of immunoassay devices.

[0094] 2. Screening of dosage of antibody-bound fluorescent nanoparticles

[0095] The antibody-bound fluorescent nanoparticles were prepared according to step 1 of Example 1, and the antibody-bound fluorescent nanoparticles were set to 1 μL, 2.5 μL, and 5 μL, wherein the antibody was an albumin antibody, and the immune device was prepared according to the method for preparing the immune device in step 2 of Example 1, such as Figure 5 shown.

[0096] The albumin concentrations in the samples were set to 0, 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 5 mg / mL, and 10 mg / mL, respectively. The samples with different albumin concentrations were measured using the detection method in Example 4, and the fluorescence signal intensity of the detection line was measured by the immunoassay analyzer. The results are shown in Fig.14 .

[0097] like Fig.14 As shown, 1 μL of antibody-bound fluorescent nanoparticles resulted in a weakened signal intensity, while 2.5 μL and 5 μL of antibody-bound fluorescent nanoparticles had better fluorescence intensity and sensitivity. At the same time, compared with 2.5 μL of antibody-bound fluorescent nanoparticles, the signal intensity of the immunodevice test results prepared by 5 μL of antibody-bound fluorescent nanoparticles was not much improved, and 2.5 μL of fluorescent nanoparticles were selected to be coated in the conjugate pad to prepare the immunodevice.

[0098] 3. Screening of capture antibody (albumin antibody) concentration on the test line

[0099] Antibody-bound fluorescent nanoparticles were prepared according to step 1 of Example 1, wherein the antibody was an albumin antibody. The albumin antibody concentration on the test line was set to 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, and 2 mg / mL, and an immune device was prepared according to the method for preparing an immune device according to step 2 of Example 1, such as Figure 5 shown.

[0100] The albumin concentrations in the samples were set to 0, 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 5 mg / mL, and 10 mg / mL, respectively. The samples with different albumin concentrations were measured using the detection method in Example 4, and the fluorescence signal intensity of the detection line was measured by the immunoassay analyzer. The results are shown in Fig.15 .

[0101] like Fig.15 As shown, as the albumin antibody concentration of the test line increases, the signal intensity increases. When the albumin antibody concentration of the test line exceeds 2 mg / mL, a false positive test result occurs, which is an erroneous signal, so it is not displayed. Therefore, it is most appropriate to select 1.5 mg / mL as the albumin concentration of the test line.

[0102] 4. Screening of sample addition location

[0103] The fluorescent nanoparticles bound to antibodies were prepared according to step 1 of Example 1, wherein the antibody was an albumin antibody. The immune device was prepared according to the method for preparing the immune device according to step 2 of Example 1, such as Figure 5 shown.

[0104] The albumin concentrations in the samples were set to 0, 0.2 μg / mL, 0.4 μg / mL, 0.6 μg / mL, 0.8 μg / mL, and 1 μg / mL, respectively. The detection methods of Example 4 and Example 5 were used to measure samples with different albumin concentrations, and the immunoassay analyzer measured the fluorescence signal intensity of the detection line. Fig.16 .

[0105] like Fig.16 The results showed no difference when the sample was added to the small in-situ pump or on the sample pad. Since the amount of sample added was small, it was diluted quickly whether added to the sample pad or the pump. There was no significant difference in the test results.

[0106] 5. Screening of sample dilution types

[0107] The fluorescent nanoparticles bound to antibodies were prepared according to step 1 of Example 1, wherein the antibody was an albumin antibody. The immune device was prepared according to the method for preparing the immune device according to step 2 of Example 1, such as Figure 5 shown.

[0108] The albumin concentrations in the samples were set to 0, 0.2 μg / mL, 0.4 μg / mL, 0.6 μg / mL, 0.8 μg / mL, and 1 μg / mL, respectively. The samples with different albumin concentrations were diluted in a small in-situ pump or in an external PBS buffer. The samples with different albumin concentrations were measured using the detection method of Example 4, and the fluorescence signal intensity of the detection line was measured by an immunoassay analyzer. The results are shown in Fig.17 .

[0109] like Fig.17 It shows that whether the sample is diluted in the small in-situ pump or in the external PBS buffer, there is a high consistency. This shows that the small in-situ pump has a good dilution effect, which can simplify the dilution steps and achieve one-step detection.

[0110] 6. Screening of sample albumin concentration dilution

[0111] The fluorescent nanoparticles bound to antibodies were prepared according to step 1 of Example 1, wherein the antibody was an albumin antibody. The immune device was prepared according to the method for preparing the immune device according to step 2 of Example 1, such as Figure 5 shown.

[0112] The albumin concentrations in the samples were set to 0, 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 5 mg / mL, and 10 mg / mL, respectively. Different amounts of PBS buffer were stored in a small in-situ pump to dilute the sample albumin concentrations to 1 / 100, 1 / 1000, and 1 / 10000 of the original concentrations. The detection method of Example 4 was used to measure samples with different albumin concentrations, and the immunoassay analyzer measured the fluorescence signal intensity of the detection line. The results are shown in Fig.18 .

[0113] like Fig.18 As shown in the figure, when the albumin concentration is diluted to 1 / 1000 of the original concentration, the test results can hardly distinguish albumin with a concentration of more than 1 mg / mL. When the albumin concentration is diluted to 1 / 10000, the signal intensity is the strongest and the linear range of the test results is the widest. When the albumin concentration is diluted to 1 / 100, the test results cannot distinguish albumin of different concentrations. In order to obtain a stronger signal, the albumin concentration is diluted to 1 / 10000 of the original concentration.

[0114] 7. Screening of small in-situ pumps at the test strip position

[0115] The albumin concentrations in the samples were set to 0, 0.2 μg / mL, 0.4 μg / mL, 0.6 μg / mL, 0.8 μg / mL, and 1 μg / mL, respectively. The immune device was prepared using Example 1. Figure 4The apparatus shown in Figure 6 was used to measure samples with different albumin concentrations using the detection method of Example 4, and the immunoassay analyzer was used to measure the fluorescence signal intensity of the detection line. Fig.19 .

[0116] like Fig.19 The results showed that the location of the small in-situ pump on the test strip had no effect on the test results. Since the pumps were not far apart, they did not affect the dilution and mixing of the liquid, resulting in similar test results.

[0117] 8. Selection of small in-situ pump types

[0118] The immunological device prepared in Example 1 is used, and the small in-situ pump is set as an ultrasonic pump or an electroosmotic pump, such as Figure 5 and Fig.11 The device shown.

[0119] The albumin concentrations in the samples were set to 0, 0.2 μg / mL, 0.4 μg / mL, 0.6 μg / mL, 0.8 μg / mL, and 1 μg / mL, respectively. The samples with different albumin concentrations were measured using the detection method of Example 4, and the fluorescence signal intensity of the detection line was measured by the immunoassay analyzer. The results are shown in Fig. 20 .

[0120] like Fig. 20 The results show that the small in-situ pump type has no effect on the test results. Since the pumps are located not far apart, they do not affect the dilution and mixing of the liquids, resulting in similar test results.

[0121] 9. Screening of the location of adding antibody-bound fluorescent nanoparticles

[0122] The immune device prepared in Example 1 is prepared by placing the antibody-bound fluorescent nanoparticles in a small in-situ pump according to the preparation method of Example 1, such as Figure 5 The device shown.

[0123] The albumin concentrations in the samples were set to 0, 0.2 μg / mL, 0.4 μg / mL, 0.6 μg / mL, 0.8 μg / mL, and 1 μg / mL, respectively. The samples with different albumin concentrations were measured using the detection method of Example 4, and the fluorescence signal intensity of the detection line was measured by the immunoassay analyzer. The results are shown in Fig.21 .

[0124] like Fig.21 It was shown that both methods can achieve the detection of albumin, but the method of antibody-conjugated fluorescent nanoparticles on the conjugated pad makes the detection results more accurate and the linear range of detection wider. The reason is that the flow of antibody-conjugated fluorescent nanoparticles is affected by the small in-situ pump, and some antibody-conjugated fluorescent nanoparticles remain in the small in-situ pump, resulting in a weak signal intensity.

[0125] Example 7 Performance Testing of Immunization Device

[0126] 1. The influence of the presence or absence of a small in-situ pump on the detection performance of the immune device

[0127] Antibody-bound fluorescent nanoparticles were prepared according to step 1 of Example 1, wherein the antibody was an albumin antibody. Immunoassay devices with and without a small in-situ pump were prepared according to the preparation method of the immunoassay device in step 2 of Example 1. The immunoassay device with a small in-situ pump was Figure 5 shown.

[0128] The sample with an albumin concentration of 0 to 10 mg / mL was measured by the detection method of Example 4, and the fluorescence signal intensity of the detection line was measured by the immunoassay analyzer. Fig. 22 .

[0129] like Fig. 22 It shows that when the albumin concentration of the sample is higher than 1 mg / mL, the fluorescence intensity on the test line of the immunoassay without a small in-situ pump does not change much, and quantitative detection cannot be achieved. Albumin samples of different concentrations (0-10 mg / mL) can be distinguished by the immunoassay with a small in-situ pump. This shows that the addition of a small in-situ pump in the immunoassay can effectively increase the detection range.

[0130] 2. Effect of albumin concentration in the sample on the flow rate of the small in-situ pump

[0131] The sample albumin concentration was set to 0, 10 μg / ml, 100 μg / ml, 500 μg / ml, 1 mg / ml, 5 mg / ml, and 10 mg / ml. The immunoassay device prepared in Example 1 was used. Figure 5 As shown, samples with different albumin concentrations were measured using the detection method of Example 4, the time required for the solution in the small in-situ pump to be released was recorded, and the flow rate corresponding to each albumin sample was calculated. Fig.23 .

[0132] like Fig.23 It is shown that the flow rate of the small in-situ pump varies with the albumin concentration in the sample. As the albumin concentration increases, the flow rate decreases. When the albumin concentration in the small in-situ pump is 0.1 mg / ml, the flow rate of the small in-situ pump can reach 100 μL / min. When the albumin concentration is 10 mg / mL, the flow rate drops to 10 μL / min. In the test, 999.9 μL of buffer is added to the small in-situ pump, and then 0.1 μL of sample is dripped into the pump. Since the concentration is diluted to 1 / 10000, detection can be achieved at a high flow rate.

[0133] 3. Testing of calibration curves of albumin samples diluted to 1 / 1000 and 1 / 10000

[0134] The immune device prepared in Example 1 is used, Figure 5 The device shown. The detection method in Example 4 was used, wherein the small in-situ pump was filled with 99.9 μL PBS buffer or 999.9 μL PBS buffer, and the sample was dripped into the small in-situ pump in an amount of 0.1 μL to detect samples with protein concentrations of 0 to 10 mg / mL or 0 to 100 mg / mL, respectively. The immunoassay analyzer measured the fluorescence intensity of the detection line. The results are shown in Figure 24-25 .

[0135] like Fig.24 It was shown that when albumin was diluted to 1 / 1000, the linear range of the calibration curve was 0-10 mg / mL, the linear equation of the added albumin sample was y=-1350x+13481 (x is the albumin concentration, y is the test value T)), and the coefficient was 0.97.

[0136] like Fig.25 It shows that when the albumin sample is diluted to 1 / 10000, the linear range of 0 to 100 mg / mL can be achieved. The linear equation for albumin detection is y=-98.51x+10840 (x is the albumin concentration, y is the detection value T), the coefficient is 0.90, and the minimum detection limit after dilution is 0.1 ng / mL.

[0137] 4. Determination of the detection time of immune devices

[0138] The immune device prepared in Example 1 is used, Figure 5 As shown, the sample with a protein concentration of 50 ng / mL was detected by the detection method in Example 4. The immunoassay analyzer measured the fluorescence intensity of 0 to 300 s on the detection line. Fig.26 .

[0139] like Fig.26 As shown, when the sample was added to the immunodevice, the detection signal increased to a saturated state within the first 150 seconds, and after a reaction time of 150 seconds, the signal intensity no longer changed, indicating that the immunodevice can complete the detection of albumin in the sample within 3 minutes.

[0140] 5. Specificity determination of immune devices

[0141] The immune device prepared in Example 1 is used, Figure 5 As shown, samples containing ALB, NGAL, Cysc, Kim-1 and IgG were detected by the detection method in Example 4, and the concentration of each sample was 1 μg / mL. The immunoassay analyzer measured the fluorescence intensity on the detection line. The results are shown in Fig. 27 .

[0142] The cross-reactivity of the LFIA was tested by adding different analytes (ALB, NGAL, Cysc, Kim-1, and IgG) to the test strip. If analytes other than albumin react with the antibody on the conjugate pad, there will be no signal in the test line, and vice versa. Fig. 27 As shown, only when albumin was added, the test line had a weak signal, while for other analytes, the fluorescence signal was consistent and strong, indicating that the immunoassay device had good specificity for the detection of albumin.

[0143] 6. Stability of the immune system

[0144] The immune device prepared in Example 1 is used, Figure 5 As shown, the immunoassay device was stored in a refrigerator at 4°C and the changes in signal intensity were observed within 30 days.

[0145] The sample with albumin concentration of 100 ng / mL was tested by the detection method in Example 4. The fluorescence intensity on the detection line at different days was measured by the immunoassay analyzer. The results are shown in Fig.28 .

[0146] like Fig.28 As shown in the figure, during this period, the fluorescence signal decreased by 16.67%, but still maintained a strong signal intensity. The results show that the test strip has good stability within 30 days.

[0147] Example 8 Quantitative Analysis of Clinical Samples by Immunoassay Device

[0148] The research ethics committee of Peking University First Hospital approved the protocol of this clinical study (approval number: 2022-714)

[0149] 1. Obtain clinical standard curve

[0150] The immune device prepared in Example 1 is used, Figure 5 The immunoassay device shown was used to test 14 clinical urine albumin samples of different concentrations using the detection method of Example 4. The immunoassay analyzer measured the fluorescence intensity on the detection line. The results are shown in Fig.29 .

[0151] like Fig.29 As shown, the data points are the test results of clinical samples using an immunoassay analyzer, and the black line is the fitted standard curve obtained from clinical samples. The immunoassay device can successfully detect high-concentration clinical samples exceeding 1 mg / mL. The linear equation for albumin detection is y=-1133.9x+10076.4, with a determination coefficient of 0.967. The test results have a good linear range, the clinical result range is 0.1-10 mg / mL, and the detection limit after dilution is 0.1 ng / mL.

[0152] 2. Testing of 14 clinical samples of unknown concentration

[0153] The immune device prepared in Example 1 is used, Figure 5 The device shown in the figure is used to test 14 clinical samples of unknown concentration using the detection method of Example 4. The immunoanalyzer measures the fluorescence intensity on the detection line. The measured fluorescence intensity is substituted into the standard curve equation of step 1 to obtain the concentration of each clinical sample of unknown concentration. At the same time, the immunoturbidimetry method (Beckman Coulter, AU5800) used by Peking University First Hospital is used to measure the above 14 clinical samples of unknown concentration to obtain the corresponding hospital clinical sample concentrations.

[0154] The absolute error was obtained by comparing the clinical sample concentration determined by the immunoassay device with the hospital clinical sample concentration. The results are shown in Fig.30 and Table 1.

[0155] Table 1 Comparison of clinical sample concentrations measured by immunoassay device and hospital clinical sample concentrations

[0156]

[0157]

[0158] like Fig.30 The results in Table 1 show that the absolute error of the fluorescence intensity of the clinical sample concentration measured by the immunoassay device and the hospital clinical sample concentration is within 500, and the relative error is within 20%. This shows that the test results and clinical results are within the confidence range, and the albumin concentration in the urine of different patients is successfully measured. Moreover, the test results have good reliability and repeatability. Therefore, the immunoassay device designed by the present invention can successfully achieve the dilution and quantitative detection of albumin in urine to detect and diagnose kidney diseases, and can be used as an auxiliary tool for clinical testing.

[0159] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An immune detection device, characterized in that: The invention comprises a test strip and a small in-situ pump, wherein the liquid outlet of the small in-situ pump is closely attached to the upper part of the conjugate pad or the upper part of the sample pad or the upper part of the middle part between the conjugate pad and the sample pad of the test strip.

2. The immunoassay device according to claim 1, characterized in that: The small in-situ pump comprises a driving component, a liquid inlet, a liquid outlet, and a solution storage chamber; the electrode layer in the driving component is connected to an integrated circuit board or a chip outside the small in-situ pump through a wire.

3. The immune detection device according to claim 1, characterized in that: The types of the small in-situ pumps include electrochemical pumps, electroosmotic pumps, and ultrasonic pumps.

4. The immunoassay device according to claim 1, characterized in that: The conjugate pad of the test strip is coated with an optical marker combined with an antibody, and the optical marker includes fluorescent nanoparticles, colloidal gold, and an enzyme that catalyzes chemiluminescence.

5. The immunoassay device according to claim 4, characterized in that: The immunoassay method includes a competitive reaction method or a sandwich immunoassay method.

6. The immunoassay device according to claim 5, characterized in that: In the competitive reaction method, the nitrocellulose membrane of the test strip is coated with a detection line and a quality control line, the detection line is coated with a biomarker of the type to be tested, and the quality control line is coated with IgG; In the sandwich immunoreaction method, the nitrocellulose membrane of the test strip is coated with a detection line and a quality control line, the detection line is coated with antibodies of the biomarker to be detected, and the quality control line is coated with IgG.

7. The method for preparing the immunoassay device according to any one of claims 1 to 6, characterized in that: The steps include: The electrode layer contained in the driving component of the small in-situ pump is connected to the integrated circuit board or chip outside the small in-situ pump through a wire to control the opening or closing of the small in-situ pump; the integrated circuit board or chip can be attached to the PVC backboard card on the other side of the test strip; Fix the nitrocellulose membrane on the upper surface of the PVC backboard card, overlap the absorption pad on the end part of the PVC backboard card, and overlap the sample pad and the conjugate pad on the beginning part of the PVC backboard card in sequence to obtain a test strip; Place the liquid outlet of the small in-situ pump close to the conjugate pad or the sample pad of the test strip or the middle part between the conjugate pad and the sample pad.

8. The method for preparing the immunoassay device according to claim 7, characterized in that: The immune detection device can be placed in a 3D printed housing of a corresponding style; The 3D printed housing is prepared by 3D printing or one-piece injection molding.

9. An application of an immunoassay device, characterized in that: The method comprises the following steps: adding a sample to a small in-situ pump of the immunoassay device as claimed in any one of claims 1 to 6, diluting the sample with a diluent in the small in-situ pump, starting the small in-situ pump to release a mixture of the sample and the diluent, wherein the mixture flows to a conjugate pad and a nitrocellulose membrane in sequence, and observing or measuring the color or intensity of light signals on a detection line and a quality control line in the immunoassay device; or adding a sample to a sample pad of the immunoassay device as claimed in any one of claims 1 to 6, starting the small in-situ pump to release the diluent, wherein the sample and the diluent are mixed on a test strip and then flow to a conjugate pad and a nitrocellulose membrane in sequence, and observing or measuring the fluorescence color or signal intensity on a detection line and a quality control line in the immunoassay device.

10. The use according to claim 9, characterized in that: The diluent dilutes the sample at a multiple of 1 / 10 to 1 / 1000000.

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