Lateral flow immunoassay device for albumin detection

Abstract

The invention belongs to the technical field of albumin analysis, and particularly relates to a lateral flow immunoassay device for albumin detection. According to the technical scheme, a transparent glass substrate is adopted to replace an existing plastic back plate, and silanization modification is carried out on partial area of the transparent glass substrate so as to fix an antibody; the lateral flow immunoassay device for albumin detection, which is prepared by replacing an antibody fixed on a nitrocellulose membrane, can solve the technical problems recorded in the prior art in a lateral chromatography immunoassay method, and has the advantages of improving the detection concentration range and detection limit of a low-concentration albumin sample, improving the detection sensitivity, reducing the use amount of colloidal gold, reducing the cost and the like. The technical effect of biological detection of albumin detection is enhanced.

Description

Technical Field

[0001] The invention belongs to the technical field of albumin analysis, and in particular relates to a lateral flow immunoassay device for albumin detection. Background Art

[0002] Current albumin analysis tools have been developed for decades, and many methods can be applied to analysis, such as enzyme-linked immunosorbent assay (ELISA), lateral flow immunoassay (LFIA), chemiluminescent enzyme immunoassay (CLEIA), surface plasmon resonance (SPR), time-resolved fluorescence, electrochemistry, capillary electrophoresis and high-performance liquid chromatography, as well as microfluidics. Among these detection methods, LFIA is considered to be one of the most promising tools for preclinical diagnosis due to its convenient operation process, low cost and rapid response. Lateral flow detection technology is not only one of the important research tools in the field of scientific research, but is also often used in our daily lives. For example, the most common early pregnancy test strips, blood glucose monitoring test strips and infectious disease rapid diagnostic kits, such as the new crown antigen and antibody rapid detection kits, are all based on the principle of lateral flow detection technology.

[0003] Lateral Flow Immunoassay (LFIA) is a simple and rapid immunological detection technology that appeared in the early 1980s and is based on the combination of immunoassay and chromatographic technology.

[0004] The principle of lateral flow immunoassay technology is to fix specific antigens or antibodies in the form of strips on a nitrocellulose membrane (NC membrane), and adsorb another labeled antigen or antibody on the marker pad that has been labeled with the antigen or antibody. When the sample to be tested is added to the sample pad at one end of the test strip, it moves forward through capillary action, dissolves the labeled antigen or antibody on the marker pad and reacts with each other, and then moves to the detection zone where the antigen or antibody color marker is fixed. The complex after the reaction of the test object and the marker specifically binds to it and is retained, and aggregates on the detection zone, and intuitive results are obtained through visually observable color markers. Antibody immobilization on the detection device is a key step in albumin detection, which can determine the detection concentration range, detection limit, detection rate and sensitivity of the sample.

[0005] However, traditional paper-based LFIA methods usually have the following problems:

[0006] 1) The capture antibody is fixed on the nitrocellulose membrane, and the nitrocellulose membrane is fixed on the white base plate. However, the nitrocellulose membrane and the base plate are not transparent, which is not conducive to the detection of optical signals.

[0007] 2) Colloidal gold is a representative of colored lateral flow markers. Colloidal gold is a nanoparticle with a surface double-layer structure formed by chloroauric acid in the presence of a reducing agent. It is simple to prepare, has stable performance, and has good biocompatibility. It can bind to proteins, nucleic acids, etc. through non-covalent bonds. The labeling process is simple and rapid, which has attracted widespread attention and application in the field of rapid detection. However, its sensitivity is still low for the detection of low-concentration samples, and the cost of preparing raw materials is relatively high.

[0008] 3) In LFIA, the sample flows through the membrane driven by capillary forces. The pore size of the absorbent material / membrane and the sample viscosity are two parameters that have a direct impact on the flow rate of the sample through the system; as for the flow speed / rate, it is a key factor affecting the detection efficiency; in the case of a large influenza epidemic, improving the detection efficiency is of great significance.

[0009] At present, due to the above-mentioned technical problems, most LFIAs can only be used for qualitative diagnosis. Improving the sensitivity of LFIA technology and performing quantitative analysis of the detected objects are the future development trends. Summary of the invention

[0010] The present invention aims at the technical problems 1) to 3) described above and provides a lateral flow immunoassay device for albumin detection.

[0011] The technical solution adopted to solve the technical problem of the present invention is:

[0012] A lateral flow immunoassay device for albumin detection, comprising one or more strips formed by sequentially connecting the following components:

[0013] a) a sample pad for receiving a sample to be tested;

[0014] b) a binding pad for temporarily storing gold labels;

[0015] c) a detection area for fixing antibodies, a nitrocellulose membrane is arranged above the detection area, and two ends of the nitrocellulose membrane overlap with the binding pad and the absorption pad respectively, and the overlapping length is 1-5 mm;

[0016] d) absorbent pads for increasing capillarity;

[0017] And it is characterized in that it also includes e) a back plate for supporting the above-mentioned components, the back plate is selected from transparent glass, and there is a detection area on the upper surface of the transparent glass that can provide fixed antibodies and colloidal gold markers, and the transparent glass is pre-treated by a silanization method.

[0018] Optionally, the sample pad mainly plays the role of loading samples, and the material can be selected as polyester fiber membrane or glass fiber membrane.

[0019] Optionally, the binding pad for temporarily storing the gold label is generally made of glass fiber, non-woven fabric or polyester fiber with good water absorption and uniformity. According to different usage requirements, the material, thickness, release rate and pre-immersion treatment of the binding pad are adjusted to ensure the binding stability of the conjugate and its continuous and uniform migration to the area for fixing the antibody.

[0020] Optionally, the size of the detection area is 1-5 mm in width and 2-30 mm in length; more preferably, the size of the detection area is 1×30 mm.

[0021] Optionally, the adsorption pad for increasing the capillary phenomenon is mainly used for absorbing waste liquid, and is generally made of absorbent paper with stable performance, large water absorption capacity and good water absorption performance.

[0022] Optionally, the silanization agent is 3-aminopropyltriethoxysilane.

[0023] Optionally, the transparent glass is subjected to silanization by: the glass substrate is first ultrasonically treated in water, and the ultrasonic treatment conditions are 100-150W, 40KHz, and 10-30 minutes. After rinsing with ultrapure water and drying in nitrogen, the glass substrate is placed in a UV ozone cleaner or oxygen plasma for 10-30 minutes to remove organic pollutants. Then the glass substrate is immersed in a newly prepared silane coupling agent solution, which is an anhydrous toluene solvent with a silane coupling agent accounting for 1-10% by volume, and is immersed at 90-120°C for 1-2 hours. Subsequently, the glass slide is thoroughly cleaned with an ethanol solution and dried at room temperature before use.

[0024] Optionally, the transparent glass is subjected to silanization by: the glass substrate is first ultrasonically treated in water, and the ultrasonic treatment conditions are 120W, 40KHz, and 10 minutes. After rinsing with ultrapure water and drying in nitrogen, the glass substrate is placed in a UV ozone cleaning machine for 20 minutes to remove organic pollutants. The glass substrate is then immersed in a newly prepared silane coupling agent solution, which is an anhydrous toluene solvent with a silane coupling agent accounting for 5% by volume, and immersed at 110°C for 2 hours. Subsequently, the glass slide is thoroughly cleaned with an ethanol solution and dried at room temperature before use.

[0025] Optionally, the lateral flow immunoassay device for albumin detection can also be used in clinical medical disease detection, biomedicine, environmental detection, food hygiene and other detection fields.

[0026] The technical effects achieved by the present invention are specifically as follows:

[0027] 1) The present invention discloses a lateral flow immunoassay device for albumin detection and its use. The inventors surprisingly found that in the traditional paper-based LFIA detection, the detection antibody layer is sprayed on the nitrocellulose membrane. Due to the three-dimensional structure of the membrane, the colloidal gold nanoparticles are unevenly distributed. This defect can be solved in the glass-based LFIA because the immobilization occurs on a two-dimensional plane and the antibody-bound liquid is evenly distributed on the glass surface, thereby reducing the distribution of colloidal gold per unit area, reducing the detection concentration and detection limit, and the detection limit of 10 ng / mL can complete the sample detection; at the same time, the cost of the lateral flow immunoassay device for albumin detection can also be reduced.

[0028] 2) In traditional paper-based LFIA detection, analytes move within the nitrocellulose membrane to reach the detection line, which limits their movement speed and the time of the entire process. However, based on the improvement of the technical solution of the present invention, in glass-based LFIA, analytes can pass through the nitrocellulose membrane as well as the overlapping space between the glass and nitrocellulose membrane, as shown in the attached manual. Figure 1 The gaps in the network, where restrictions on movement are greatly reduced, can improve detection efficiency. The huge improvement in detection efficiency brings huge advantages to future commercial applications. It is beneficial in pandemic situations where efficient detection is required for preliminary screening of diseases. For example, the shortening of nucleic acid detection time for epidemics is beneficial to urban management.

[0029] Therefore, the technical solution of the present invention is to replace the existing plastic polyvinyl chloride (PVC) bottom plate with a transparent glass substrate, and to perform silanization modification on a part of the transparent glass substrate for fixing antibodies, and to replace the detection device prepared by fixing antibodies on the nitrocellulose membrane in the lateral flow immunoassay method, which can solve the technical problems recorded in the prior art, and has the technical effects of improving the detection concentration range and detection limit of low-concentration albumin samples, improving detection sensitivity, improving detection efficiency, reducing the amount of colloidal gold, and enhancing albumin biological detection. Compared with the prior art, the lateral flow chromatography detection device of the present invention has achieved corresponding unexpected technical effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0031] Figure 1 It is a schematic diagram of the structure of the lateral flow immunoassay device in the present invention;

[0032] Figure 2 It is a schematic diagram of the structure of a lateral flow immunoassay device in the prior art;

[0033] Figure 3 It is a schematic diagram of the signal display of the lateral flow immunoassay device of the present invention;

[0034] Figure 4 Glass-based LFIA test results; (a) is a photographic image, (b) is an optical microscope image (the upper line is the test line, the lower line is the control line, and the concentrations of the sample albumin solutions are from left to right: 0 ng / mL, 1 ng / mL, 2 ng / mL, 4 ng / mL, 6 ng / mL, 8 ng / mL, 10 ng / mL, 100 ng / mL, 200 ng / mL, 800 ng / mL, 10 μg / mL, 40 μg / mL, 80 μg / mL, 100 μg / mL);

[0035] Figure 5 is a calibration curve for lateral flow immunoassay of albumin in the present invention;

[0036] Figure 6 It is the detection time of different concentrations of albumin in the lateral flow immunoassay of the present invention;

[0037] Figure 7 Comparison of the specificity of lateral flow immunoassay for albumin in the present invention (comparison with different proteins NGAL, HCG, CTNI, Kim);

[0038] Figure 8 The temporal stability of the lateral flow immunoassay for albumin in the present invention;

[0039] Fig. 9 It is a comparison curve between the albumin in the clinical patient sample detected by lateral flow immunoassay in the present invention and the clinical detection result in the hospital;

[0040] Fig.10 This is a photo of a test paper for lateral flow immunoassay of albumin in clinical patient samples in the present invention;

[0041] Fig.11 Comparison data between the albumin in the lateral flow immunoassay of the present invention and the clinical test results in the hospital;

[0042] Fig.12This is a picture of the traditional paper-based LFIA test (sample albumin solution concentration from left to right: 10ng / mL, 100ng / mL, 200ng / mL, 600ng / mL, 1μg / mL, 10μg / mL, 20μg / mL, 40μg / mL, 60μg / mL, 80μg / mL, 100μg / mL, 200μg / mL, 400μg / mL, 600μg / mL, 800μg / mL, 1mg / mL);

[0043] Fig.13 This is the traditional paper-based LFIA test curve;

[0044] Fig.14 It is the detection time of traditional test strips for different concentrations of albumin;

[0045] Fig.15 Gold particle distribution diagram, where (a) is the distribution diagram of gold particles on the paper substrate, and (b) is the distribution diagram of gold particles on the glass substrate.

[0046] Wherein, the accompanying drawings are marked as follows: 1-sample pad, 2-conjugate pad, 3-nitrocellulose membrane, 4-absorption pad, 5-test line, 6-control line, 7-transparent glass, 8-sample pad, 9-conjugate pad, 10-absorption pad, 11-PVC bottom plate, 12-test line, 13-control line, 14-nitrocellulose membrane. DETAILED DESCRIPTION

[0047] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0048] Embodiment 1:

[0049] This embodiment provides a lateral flow immunoassay device for albumin detection. Figure 1As shown, by installing the sample pad, conjugate pad, nitrocellulose membrane and absorption pad on a back plate (1×60 mm) for supporting the above components, there is a detection area for fixing antibodies on the back plate, the back plate is selected from transparent glass, and the transparent glass is obtained by silanization. The detection area is set in the center of the back plate, and a nitrocellulose membrane is set above the detection area. The two ends of the nitrocellulose membrane overlap with the conjugate pad and the absorption pad respectively, and the overlapping length is 2 mm. There is a gap between the nitrocellulose membrane and the transparent glass back plate, and there is a detection line on the transparent glass. Albumin (0.8 mg / mL) is distributed on the glass surface test line at a volume ratio of 2 μL / cm, and goat anti-mouse IgG (1 mg / mL) is selected as the control line capture antibody and distributed on the glass surface at a volume of 2 μL / cm, 4 mm away from the test line. When using a self-designed 3D printed clip to connect it to the albumin for testing, the albumin solution is gently dripped on the sample pad, and the test stroke is dried at room temperature for 30 minutes. As shown Figure 3 As shown in the figure, based on the results of the competitive reaction from the test line and the control line, a strong red color appears on the T line (test line), which indicates a negative result, indicating that there is no albumin analyte in the test fluid. A light red color appears on the T line (test line), which indicates a positive result, indicating that there is albumin analyte in the test fluid.

[0050] The specific modification method of the detection area is as follows: First, the glass substrate is placed in water for ultrasonic treatment (120W, 40KHz) for 10 minutes. After rinsing with ultrapure water and drying in nitrogen, the glass substrate is placed in a UV ozone cleaning machine for 20 minutes to remove organic pollutants. The glass substrate is then immersed in a newly prepared 3-aminopropyltriethoxysilane solution (5% by volume in anhydrous toluene) and immersed at 110°C for 2 hours. Subsequently, the glass substrate is thoroughly cleaned with an ethanol solution and dried at room temperature before use.

[0051] Comparative Example 1

[0052] This embodiment provides a lateral flow immunoassay device for albumin detection. Figure 2As shown, the LFIA test strip was assembled by mounting the sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad on a PVC base plate (1×60 mm). The nitrocellulose membrane was placed in the center of the PVC base plate and carefully covered with the immobilized antibodies on the PVC base plate. The conjugate pad containing colloidal gold and conjugated anti-albumin IgG was attached to the nitrocellulose membrane with a 2 mm overlap. Similarly, the absorbent pad was attached to the nitrocellulose membrane on the other side with a 2 mm overlap, and the sample pad was placed on the conjugate pad. Albumin (0.8 mg / mL) was immobilized to the glass substrate surface with a volume dispense of 2 μL / cm. Goat anti-mouse IgG (1 mg / mL) was selected as the control line capture antibody and dispensed to the glass surface with a volume of 2 μL / cm, 4 mm away from the test line. After assembling the test strip, it was attached to the protein chip using a self-designed 3D printed clip. When performing the LFIA test, the albumin solution was gently dropped on the sample pad and the test stroke was dried at room temperature for 30 minutes. The results seen on the test and control lines can then be viewed and analyzed through the glass.

[0053] The test devices of Example 1 of the present invention and Comparative Example 1 were tested in terms of detection limit, detection concentration, response time, colloidal gold distribution, etc., and the specific effects were compared as follows:

[0054] 1) Comparison of sample detection limits

[0055] like Figure 4 As shown in the figure, the performance of glass-based LFIA and paper-based LFIA was compared by analyzing a series of albumin solutions with different concentrations. A competitive detection method was used in this process, and the results are explained in Figure 4 The color of the test line in the figure decreases as the albumin concentration in the sample solution increases. The fundamental reason for this phenomenon is the principle of competitive detection. When there are fewer available sites on the colloidal gold and anti-albumin antibody conjugate, the test line albumin captures less colloidal gold. This occurs when a large amount of sample albumin occupies the vacant sites on the conjugate. Figure 4 (a) and Figure 4 As shown in (b), in the glass-based LFIA test, when the albumin concentration increases, the color intensity of the test line becomes lighter and disappears in a 100 μg / mL albumin solution. Figure 5 This is the calibration curve for lateral flow immunoassay of albumin, with a detection limit of 10 ng / mL. The calibration curve signal comes from ImageJ. The darker the color, the smaller the ImageJ signal (black signal is set to 0, white signal is set to 255). Fig.13 As shown in Figure 2, the detection limit of the paper-based LFIA was found to be 100 ng / mL, which is 1000% higher than that of the glass-based LFIA. Therefore, the glass-based LFIA showed a lower detection limit in the experiment and has an advantage in real-time detection.

[0056] 2) Comparison of sample detection concentration

[0057] The data of paper-based LFIA is as follows Fig.13 As shown in the figure, the detection signal increases sharply in the concentration range of 100 ng / mL to 100 μg / mL. This huge jump in the concentration level from 1 μg / mL to 10 μg / mL reduces its detection range and it is impossible to establish a regression equation in the entire analytical range. In addition, the paper-based LFIA shows convincing detection results when the albumin concentration is 100 ng / mL to 100 μg / mL. However, when the albumin concentration is below its detection limit of 100 ng / mL, the paper-based LFIA cannot produce concentration changes that distinguish the albumin solution. The glass-based LFIA solves the above problems. It increases the detection sensitivity to the limit of 10 ng / mL and expands the detection range to 10 ng / mL to 100 μg / mL, which reduces the detection limit concentration by 10 times. In summary, the glass-based LFIA has advantages in both detection range and limit, which is very promising in actual product applications.

[0058] 3) Comparison of sample response time

[0059] Another advantage of glass-based LFIA testing is its high detection efficiency. In traditional paper-based LFIA, analytes move within the nitrocellulose membrane to reach the detection line, which limits their movement speed and the time of the entire process. However, in glass-based LFIA, analytes can pass through the nitrocellulose membrane as well as the cross-space between the glass and nitrocellulose membrane, where the restriction of movement is greatly reduced. Figure 6 and Fig.14 The detailed flow times of the two methods at different albumin concentrations are listed. The average detection time of the glass-based method was 147.8 seconds, while that of the paper-based method was 206.6 seconds, increasing the detection efficiency by about 28.5%. The huge improvement in detection efficiency brings great potential for future commercial applications, which is advantageous in pandemic situations where efficient detection is needed for preliminary screening of diseases.

[0060] 4) Selectivity and stability of LFIA

[0061] The cross-reaction of LFIA was tested by dropping different analytes NGAL, HCG, CTNI and Kim on the test strip. Observe whether there is a signal on the T line and C line to determine whether there is a non-specific reaction. When NGAL, HCG, CTNI and Kim were added to LFIA, there was no test signal, indicating that LFIA has good specificity. The results are shown in Figure 8 shown.

[0062] In terms of stability, the glass-LFIA test strips were stored in a dark, dry environment at room temperature to observe whether the signal intensity changed after 30 days. Fig. 9 As shown, the signal remained unchanged over 30 days, indicating that the glass-LFIA test strip has good selectivity and stability.

[0063] 5) Distribution of gold nanoparticles

[0064] like Fig.15 (a) As shown on the left, in the electron microscopy image results, it can be seen that the shape of the fiber is uneven, with concave and convex areas in its cross section, with different potentials to attach gold nanoparticles. Fig.15 (a) right shows the distribution pattern of gold nanoparticles in the paper-based LFIA, where the colloidal gold nanoparticles are mainly located in the convex part of the cross section. Fig.15 (b) As shown on the right, gold nanoparticles located in the glass slide produce a uniform distribution pattern. The plate-like distribution of antibody-gold conjugates is also advantageous during antigen detection because each antibody has a similar probability of binding to the target protein. Therefore, the transition of immobilizing the detection antibody from the nitrocellulose membrane to the glass surface improves the distribution of the nanoparticles, reduces the amount of gold nanoparticles used, and reduces costs.

[0065] It is to be understood that the above embodiments are merely exemplary embodiments used to illustrate the principles of the present invention, but the present invention is not limited thereto. For those of ordinary skill in the art, various modifications and improvements can be made without departing from the spirit and essence of the present invention, and these modifications and improvements are also considered to be within the scope of protection of the present invention.

Claims

1. A lateral flow immunoassay device for albumin detection, comprising one or more strips formed by sequentially connecting the following components: a) a sample pad for receiving a sample to be tested; b) a binding pad for temporarily storing gold labels; c) a detection area for fixing antibodies, a nitrocellulose membrane is arranged above the detection area, and two ends of the nitrocellulose membrane overlap with the binding pad and the absorption pad respectively, and the overlapping length is 1-5 mm; d) absorbent pads for increasing capillarity; and characterized in that, It also includes e) a back plate for supporting the above components, the back plate is selected from transparent glass, the upper surface of the transparent glass has a detection area that can provide fixed antibodies and colloidal gold markers, and the transparent glass is pre-treated by a silanization method.

2. A lateral flow immunoassay device for albumin detection according to claim 1, characterized in that: The sample pad is a polyester fiber membrane or a glass fiber membrane.

3. A lateral flow immunoassay device for albumin detection according to claim 1, characterized in that: The binding pad for temporarily storing the gold standard is made of a free combination of glass fiber, non-woven fabric or polyester fiber.

4. A lateral flow immunoassay device for albumin detection according to claim 1, characterized in that: The dimensions of the detection area are 1-5 mm in width and 2-30 mm in length.

5. A lateral flow immunoassay device for albumin detection according to claim 1, characterized in that: The absorbent pad for increasing the capillary phenomenon is mainly used for absorbing waste liquid and is made of absorbent paper with stable performance, large water absorption capacity and good water absorption performance.

6. A lateral flow immunoassay device for albumin detection according to claim 1, characterized in that: The reagent used for the silanization is 3-aminopropyltriethoxysilane.

7. A lateral flow immunoassay device for albumin detection according to claim 1, characterized in that: The method for silanization of the transparent glass is as follows: the glass substrate is firstly subjected to ultrasonic treatment in water, the ultrasonic conditions being 100-150W, 40KHz, and 10-30 minutes; after being rinsed with ultrapure water and dried in nitrogen, the glass substrate is placed in an ultraviolet ozone cleaning machine or oxygen plasma for treatment for 10-30 minutes to remove organic pollutants; then the glass substrate is immersed in a newly prepared silane coupling agent solution, the silane coupling agent solution being an anhydrous toluene solvent in which the silane coupling agent accounts for 1-10% by volume, and immersed at 90-120° C. for 1-2 hours; Subsequently, the glass slides were thoroughly cleaned with an ethanol solution and dried at room temperature before use.

8. A lateral flow immunoassay device for albumin detection according to claim 7, characterized in that: The method for silanization of the transparent glass is as follows: the glass substrate is firstly ultrasonically treated in water, and the ultrasonic conditions are 120W, 40KHz, and 10 minutes; after being rinsed with ultrapure water and dried in nitrogen, the glass substrate is placed in an ultraviolet ozone cleaning machine for 20 minutes to remove organic pollutants; then the glass substrate is immersed in a newly prepared silane coupling agent solution, which is an anhydrous toluene solvent with a silane coupling agent accounting for 5% by volume, and is immersed at 110°C for 2 hours; subsequently, the glass slide is thoroughly cleaned with an ethanol solution and dried at room temperature before use.

9. A lateral flow immunoassay device for albumin detection according to any one of claims 1 to 8, characterized in that: Compared with the paper-based detection limit concentration of 100 ng / mL, the detection limit concentration was reduced to 10 ng / mL, and the detection time was increased by 28.5%.

10. A lateral flow immunoassay device for albumin detection according to any one of claims 1 to 8, characterized in that: It can also be used in clinical medical disease detection, biomedicine, environmental testing, food hygiene and other testing fields.

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