Chemiluminescence lateral flow immunoassay method and application

Abstract

The application discloses a chemiluminescence lateral flow immunoassay method and application, and belongs to the technical field of detection. Paraffin liquid is coated on T lines and C lines of a nitrocellulose membrane, and the volume of the paraffin liquid drop is controlled, so that the transparency of the nitrocellulose membrane can be adjusted in the range of 12.47% to 78.53%. The chemiluminescence lateral flow immunoassay method and application based on the paraffin liquid modified nitrocellulose membrane can be applied to detection of a typical acute myocardial infarction biomarker, i.e. cardiac troponin I (cTnI), and the lower limit of detection can reach 0.063 ng / mL, and the method has high sensitivity and reliability. Moreover, the method can effectively distinguish clinical samples with cTnI concentrations in the range of 1 to 5.1 ng / mL, and has high diagnostic accuracy.

Description

Technical Field

[0001] This invention relates to the field of detection technology, and in particular to a chemiluminescent lateral flow immunoassay method and its application. Background Technology

[0002] Lateral flow detection (LFA) has long been a commonly used method for the rapid detection of chemical substances or proteins in a wide range of fields, including human medical health, food quality, animal health, and environmental monitoring, due to its advantages such as ease of production, low cost, simple operation, and short response time.

[0003] However, lateral flow detection methods still require higher sensitivity for many biomarkers. To improve the performance of lateral flow detection methods, many researchers have focused on studying novel conjugates by modifying antigen or antibody proteins on nanoscale crystals. These include not only traditional gold nanoparticles (AuNPs) but also magnetic beads, carbon-based materials, or quantum dots. However, the synthesis of many novel nanoscale materials requires complex equipment and a series of chemicals; in addition, some products have uneven particle size distribution and severe aggregation, which keeps much of the research at the laboratory stage.

[0004] In recent years, the application of chemiluminescence (CL), with its high signal-to-noise ratio, in lateral flow detection systems has attracted considerable attention. In chemiluminescence-lateral flow detection, nanoparticles generate a stronger light signal in the dark, rather than the color of the conjugated nanoparticles embedded in the nitrocellulose membrane can be directly read by the naked eye. For example, Chen et al. reported a comparison of signal readout between conventional lateral flow detection and chemiluminescence-lateral flow detection; when detecting procalcitonin, the signal readout level of chemiluminescence-lateral flow detection was 0.1 pg / mL, while that of conventional lateral flow detection was 10 pg / mL.

[0005] In addition, many reports have modified the structure of nitrocellulose membranes (NC membranes), with some results showing that the slower flow rate of protein-modified nanoparticles from the conjugated pad to the detection line allows for a more complete biorecognition process, thereby improving the performance of lateral flow detection methods. For example, Rivas et al. reported that nitrocellulose membranes printed with wax column patterns improved the detection limit by approximately 3 times compared to unmodified membranes by providing delay and pseudo-turbulence during microcapillary flow.

[0006] In fact, nitrocellulose membranes are white, and the conjugates within the nitrocellulose membrane may not be able to display most of their light or color to the detector or observer, thus affecting the light signal when used in lateral flow detection or chemiluminescence-lateral flow detection methods.

[0007] Many filter papers with an initial visible color have been reported to exhibit high transparency. For example, Liu et al. reported that filling the pores of porous polyvinylidene fluoride (PVDF) membranes with ionic liquids increased the transmittance of the product membrane in the visible light range from 0% to 94.8%. However, most transparent papers composed of nanoscale particles or fibers have a high density and lack a sufficient number of microchannels to meet the requirements of popular side-flow detection methods based on the flow of analytical solutions. Summary of the Invention

[0008] The purpose of this invention is to provide a chemiluminescent lateral flow immunoassay method and its application, in order to solve the problems existing in current chemiluminescent lateral flow immunoassay methods.

[0009] To achieve the above objectives, the present invention provides a chemiluminescent lateral flow immunoassay method, which is based on a paraffin-modified nitrocellulose membrane for chemiluminescent lateral flow detection. The preparation of the paraffin-modified nitrocellulose membrane includes coating the test line and control line areas of the nitrocellulose membrane with paraffin-modified material.

[0010] Preferably, the adjustable transparency of the nitrocellulose membrane is achieved by adjusting the coating volume of paraffin liquid on the nitrocellulose membrane, which ranges from 12.47% to 78.53%.

[0011] Preferably, the coating volume of the paraffin liquid is 0.1~6μL.

[0012] Preferably, the light attenuation constant of the chemiluminescence side-flow detection method is reduced to -1.35 after modification with paraffin liquid.

[0013] Secondly, the present invention provides an application of the above-mentioned chemiluminescent lateral flow immunoassay method in the detection of cardiac troponin I.

[0014] Preferably, when detecting 20 ng / mL cardiac troponin I, the light attenuation constant is -1.35.

[0015] Thirdly, the present invention provides an application of the above-described chemiluminescent lateral flow immunoassay method in the diagnosis of myocardial injury for non-medical purposes.

[0016] Preferably, the chemiluminescent lateral flow immunoassay method can effectively distinguish samples with cardiac troponin I concentrations between 1 and 5.1 ng / mL.

[0017] Fourthly, this invention provides an application of the above-mentioned chemiluminescent lateral flow immunoassay method in a paper-based biosensor, wherein the preparation method of the paper-based biosensor includes: S1. Au-HRP-Ab or Au-Ab conjugates are prepared by physical adsorption method; S2. Adhere the sample pad, conjugate pad, paraffin-modified nitrocellulose membrane, and absorbent pad in sequence to assemble the test strip; S3. Drop Au-HRP-Ab or Au-Ab conjugate onto the conjugate pad and allow it to dry. S4. Spray cTnI capture antibody onto the T line of the nitrocellulose membrane and anti-species secondary antibody onto the C line. S5. Use 3D printed rectangular blocks to determine the coating positions of paraffin liquid on the T-line and C-line of the nitrocellulose membrane and then coat the membrane with paraffin liquid for modification.

[0018] Therefore, the chemiluminescent lateral flow immunoassay method and its application of the present invention have the following beneficial effects: (1) Coating the test (T) line and control (C) line areas of the nitrocellulose membrane with paraffin liquid can enhance the light signal intensity without causing structural degradation of the nitrocellulose membrane; (2) Paraffin coating can reduce light scattering between pores of nitrocellulose membrane. By adjusting the coating volume of paraffin on nitrocellulose membrane, the nitrocellulose membrane can achieve adjustable transparency in the range of 12.47% to 78.53%. (3) Coating the test (T) line and control (C) line areas of the nitrocellulose membrane with paraffin liquid can optimize the transmittance of the light signal and reduce the light attenuation constant. The paraffin liquid treatment reduces the light attenuation constant of CL-LFA to -1.35, which is more conducive to signal detection than the untreated NC membrane (light attenuation constant is -1.72). (4) The detection method of the present invention can be used to detect cardiac troponin I (cTnI). The test shows that its detection limit can reach 0.063 ng / mL, indicating that the detection method of the present invention has high sensitivity and reliability. (5) The detection method of the present invention can effectively distinguish clinical samples with cTnI concentrations of 1-5.1 ng / mL, thereby improving the diagnostic accuracy of myocardial injury.

[0019] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0020] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 A schematic diagram of the preparation process of a transparent nitrocellulose membrane; Figure 2A schematic diagram illustrating the process of applying a paraffin-modified nitrocellulose membrane to the detection of cTnI chemiluminescent conjugates; part a shows the preparation process of the chemiluminescent conjugate (AuNP-Ab-HRP / BSA); part b shows the paraffin-modified nitrocellulose membrane and detection device after adding the analytical solution; part c shows the chemiluminescence enhancement effect after adding paraffin solution at the T line. Figure 3 The UV-Vis spectra of AuNPs and AuNP-Ab-HRP are shown. Figure 4 DLS spectra of AuNPs and AuNP-Ab-HRP; Figure 5 TEM images of AuNPs (1) and HRP-Ab modified AuNPs (2); Figure 6 Images of nitrocellulose membranes modified with paraffin liquid of different volumes; Figure 7 The images are scanning electron microscope (SEM) images of nitrocellulose membranes before and after modification with 6 μL of paraffin solution; (1) is a scanning electron microscope image of the plane of the nitrocellulose membrane before modification with paraffin solution; (2) is a scanning electron microscope image of the side of the nitrocellulose membrane before modification with paraffin solution; (3) is a scanning electron microscope image of the plane of the nitrocellulose membrane after modification with paraffin solution; and (4) is a scanning electron microscope image of the side of the nitrocellulose membrane after modification with paraffin solution. Figure 8 Comparison of the flowability of nitrocellulose membranes modified with different volumes of paraffin liquid; Figure 9 XPS spectra of nitrocellulose membranes before and after modification with paraffin liquid of different volumes; Figure 10 The UV-Vis spectra of nitrocellulose membranes before and after modification with paraffin liquid of different volumes; Figure 11 Fourier transform infrared spectra of nitrocellulose membranes before and after modification with paraffin liquid of different volumes; Figure 12 Comparison of blue light signals of nitrocellulose membranes before and after modification with different volumes of paraffin solution at 20 ng / mL cTnI; Figure 13 The CL intensity decay of the T line of nitrocellulose membranes before and after modification with different volumes of paraffin solution at 20 ng / mL cTnI was observed within 120 seconds. Figure 14 The CL intensity decay of the C-line of nitrocellulose membranes before and after modification with different volumes of paraffin solution at 20 ng / mL cTnI was measured within 120 seconds. Figure 15 To detect 0.5 ng / mL cTnI, CL images of NC membranes were obtained by processing different volumes of liquid. Figure 16 To detect the CL intensity decay of the T line within 120 seconds at 0.5 ng / mL cTnI; Figure 17 To detect the CL intensity decay of the C-line within 120 seconds at 0.5 ng / mL cTnI; Figure 18 The colorimetric results of paraffin-modified nitrocellulose membrane CL-LFA in detecting different concentrations of cTnI are shown. The star symbol represents the detection limit at different detection times. Figure 19 The colorimetric measurements were performed on the T-line and C-line regions modified with 0.1 μL of paraffin solution on nitrocellulose strips. Figure 20 Optical images of the T-line and C-line after processing when detecting different concentrations of cTnI antigen in CL-LFA assay; Figure 21 The measured color intensity of the T-line and C-line regions modified with 0.1 μL of paraffin solution on the NC strip during CL-LFA testing; Figure 22 For without Figure 18 Color intensity distribution of background intensity detected by CL-LFA; Figure 23 For without Figure 20 Color intensity distribution of background intensity detected by CL-LFA; Figure 24 Calibration curves were generated for detecting CL intensity at different cTnI concentrations ranging from 0.038 to 10 ng / mL using different detection times (t=0 s, 30 s, and 60 s). Figure 25 The steps and schematic diagram for detecting cTnI in real human serum samples using the CL-LFA method based on transparent paper; Figure 26 To detect the CL phenomenon and results on the test strip when different serum samples are tested. Detailed Implementation

[0022] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0023] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0024] The instruments, equipment, reagents, and materials used in the examples were all obtained commercially.

[0025] Example 1 like Figure 1 As shown, nitrocellulose membranes were immersed in a typical transparent solution—paraffin solution—to obtain optically transparent nitrocellulose membranes.

[0026] Example 2 Figure 2 The preparation process of a chemiluminescent conjugate (AuNP-Ab-HRP / BSA) for high-sensitivity detection of cardiac troponin I (cTnI) is shown. The antibody is conjugated with horseradish peroxidase (HRP) through avertin-biotin interaction, and the resulting antibody-HPR is absorbed onto AuNPs.

[0027] The specific preparation process is as follows: Figure 2 As shown in part a, it includes: (1) Preparation of AuNP-Ab-HRP conjugate: Add 8 μL of 0.1M sodium carbonate solution to 0.3 mL of AuNP solution, then add 5 μL of 1 mg / mL SA-HRP and 10 μL of 1 mg / mL biotinylated cTnI detection antibody. Incubate the mixture on a horizontal shaker for about 60 minutes to obtain the final product.

[0028] like Figure 3 The image shows the UV-Vis spectra of AuNPs and AuNP-Ab-HRP. The wavelength shift from ~534 nm to ~541 nm indicates that the surface modification was successful.

[0029] like Figure 4 The image shows the DLS spectra of AuNPs and AuNP-Ab-HRP, revealing their particle distribution. The intensity increased from ~50 nm to ~200 nm before and after modification, indicating successful absorption of the detection antibody protein on the AuNP surface. However, this does not necessarily imply significant contact between AuNPs. Notably, the Ab-HRP-modified gold aggregates still exhibit a noticeable peak at ~20 nm.

[0030] like Figure 5 The image shows TEM images of AuNPs (1) and HRP-Ab modified AuNPs (2). It can be seen from the image that the average crystal size of the particles is ~20nm and there is no serious aggregation phenomenon.

[0031] (2) Preparation of AuNP-Ab-HRP / BSA conjugate: 20 μL of 100 mg / mL BSA solution was added to the AuNP-Ab-HRP conjugate and mixed well. The mixture was incubated on a horizontal shaker for 30 minutes. Finally, the mixture was centrifuged at 10,000 rpm for 8 minutes, the supernatant was removed, and 50 μL of recovery solution (TBS: 1% BSA, 5% sucrose, and 0.1% Tween 20) was added to redisperse the mixture. The prepared AuNP-Ab-HRP / BSA conjugate solution was stored at 4°C for further use.

[0032] Figure 2 As shown in Figure b, after the addition of the analytical solution, a sandwich immunoassay structure was formed on the detection line (T line), and the remaining conjugate was captured on the control line (C line).

[0033] After 30 minutes of sufficient biological reaction on the LFA test strip, paraffin solution was dropped into the T and C line regions to induce transparency, and then CL matrix solution was applied to the nitrocellulose membrane.

[0034] like Figure 2 As shown in Figure b, the detection device consists of a 3D-printed chamber containing an LFA that captures microdroplets via a Bluetooth-controlled switch. Two biosensing modes are demonstrated: colorimetric mode and chemiluminescence mode.

[0035] Figure 2 As shown in Figure c, the addition of a chemiluminescent matrix solution resulted in stronger chemiluminescent signals in the paraffin-modified T-line and C-line regions.

[0036] Example 3 Using a pipette, apply 0, 0.1 μL, 0.3 μL, 0.6 μL, 0.9 μL, 1.2 μL, 1.5 μL, 3 μL, and 6 μL of paraffin solution to a porous nitrocellulose membrane (1.5 mm × 2.5 mm × 200 μm).

[0037] like Figure 6 The images shown are of nitrocellulose membranes modified with paraffin liquid of different volumes. These images were obtained by dropping paraffin liquid onto porous nitrocellulose membranes with a pore size of 15 μm. The initial porous nitrocellulose membranes are typically white; as the volume of paraffin liquid increases, obvious diffusion and a gradual expansion of the transparent area can be observed.

[0038] Then, the nitrocellulose membranes before and after modification with 6 μL of paraffin solution were characterized using scanning electron microscopy, such as... Figure 7 As shown.

[0039] Figure 7Images (1) and (2) show the porous structure composed of nitrocellulose microfibers and particles from the vertical and lateral perspectives, respectively. After modification with paraffin liquid, it can be observed that many microchannels and pores have been filled, and due to the solidification of the paraffin liquid, the number of pores is also very small.

[0040] Figure 7 The illustrations in (3) and (4) show the contact angle test results of the nitrocellulose membrane surface before and after modification with 6 μL of paraffin solution. They show that at 13 seconds, the latter has poorer hydrophilicity than the former, with a contact angle of 72.75°.

[0041] Example 4 To test the hydrophilicity of nitrocellulose membranes before and after modification with different volumes of paraffin solution (0, 0.1 μL, 0.3 μL, 0.6 μL, 0.9 μL, 1.2 μL, 1.5 μL, 3 μL, and 6 μL), 1 μL of gold nanoparticle colloidal solution was dropped onto a lateral flow test strip coated with different volumes of paraffin solution, followed by 15 μL of PBS solution, for flowability testing. A stopwatch was started simultaneously with the addition of the PBS solution, and each volume of sample was tested three times.

[0042] The impregnation locations of the paraffin liquid at the T and C lines were determined by 3D-printed rectangular blocks with a hole (diameter: 0.8 mm; depth: 0.5 mm) to hold the transparent paraffin liquid. The block structure was designed by Autodesk Inventor. The 3D-printed blocks measured 1.5 mm × 2.5 mm × 0.5 mm, and each UV-irradiated sheet was 0.04 mm thick.

[0043] Liquidity test results as follows Figure 8 As shown, the average flow times of nitrocellulose membranes modified with 0, 0.1, 0.3, 0.6, 0.9, 1.2 and 1.5 μL of paraffin solution were 66.33, 135, 175, 200.33, 203.33, 267 and 308 seconds, respectively, which again confirms that the addition of paraffin solution has a significant impact on the hydrophilicity of nitrocellulose membranes.

[0044] like Figure 9 The figure shows the XPS spectra of C1s of nitrocellulose membranes before and after modification with paraffin solution of different volumes. As the volume of paraffin solution increases from 0 μL to 0.6 μL, the peak region of C1s gradually shifts from ~284.66 eV to ~284.2 eV. This may be due to the gradual replacement of the C-C bonds on the surface of the nitrocellulose membrane with the CH bonds (-CnH) in the paraffin solution. 2n+2The mixture covers the surface. C1s calibration was not used here; instead, raw data obtained using XPS technology are presented. No significant change was observed when the volume of the paraffin solution increased from 0.6 μL to 6 μL, indicating that a thick layer of paraffin solution exists on the NC surface, thus preventing X-ray photoelectrons from penetrating the paraffin solution to reach the NC surface.

[0045] like Figure 10 The image shows the UV-Vis spectra of nitrocellulose membranes before and after modification with different volumes of paraffin solution. The average transmittances of the nitrocellulose membranes modified with 0, 0.1, 0.3, 0.6, 0.9, 1.2, 1.5, and 6 μL of paraffin solution in the 360-760 nm range were 12.47%, 21.86%, 25.53%, 28.84%, 35.4%, 40.15%, 65.23%, and 78.53%, respectively, indicating that the more paraffin solution added, the higher the transmittance of the film product.

[0046] Figure 11 The Fourier transform infrared spectra of nitrocellulose membranes before and after modification with different volumes of paraffin solution are shown. The initial nitrocellulose membranes were observed at ~3505.28, ~2916.16, ~1636.58, ~1274.21, ~1053.29, and ~825.59 cm⁻¹. -1 Peaks appeared at the wavelengths corresponding to OH extension, CH extension, NO2 asymmetric extension, CO extension, and O-NO2 extension, respectively. All modified products showed peaks at ~2920, ~1459, and ~1376 cm⁻¹. -1 Peaks appeared at all wavelengths, corresponding to aliphatic, CH bending vibration and -CH3 in-plane deformation, respectively, which further proves the existence of paraffin liquid covering the NC surface.

[0047] Example 4 As described in Example 3, adding paraffin solution can make the nitrocellulose membrane completely or partially transparent, but a completely transparent nitrocellulose membrane has poor hydrophilicity, which is not conducive to rapid detection and immersion of the CL substrate solution. In this example, 0.1 or 0.3 μL of paraffin solution was used for further testing.

[0048] (1) Testing was performed using a high cTnI concentration (20 ng / mL). like Figure 12 As shown, direct observation of nitrocellulose membranes before and after paraffin liquid modification reveals a strong blue light signal even after a 120-second exposure time.

[0049] like Figure 13As shown, before the addition of paraffin solution, the T-line in the CL-LFA test exhibited an approximately linear decay of light intensity from 237.57 to 151.08 over a time period of 0 to 120 seconds, with a k-slope of -0.74. The addition of 0.1 μL of paraffin solution reduced the rate of light intensity decay; over the time period of 0 to 120 seconds, the light intensity decreased from 235.09 to 168.59, with a k-slope of -0.58. The light intensity range of the paraffin-modified sample was reduced from 234.97 to 133.92, with a k-slope of -0.86.

[0050] After applying 0.1 μL of paraffin solution to the T-line and C-line regions, the light attenuation constant was -1.35 when detecting cardiac troponin I (cTnI) (a key biomarker of myocardial injury) at 20 ng / mL, which is lower than the light attenuation constant of the NC membrane (-1.72).

[0051] like Figure 14 As shown, the C-line intensity exhibits a stable pattern, remaining almost constant at a value of approximately 250, demonstrating the effectiveness of the CL-LFA test.

[0052] (2) In order to test whether the system can test lower concentrations of cTnI, a cTnI concentration of 0.5 ng / mL was selected for a similar experiment.

[0053] like Figure 15 As shown, direct observation of the nitrocellulose membrane before and after paraffin liquid modification revealed a clear blue light signal even after 60 seconds.

[0054] like Figure 16 As shown, before the addition of paraffin solution, the T-line in the CL-LFA test exhibited an approximately linear decay of light intensity from 95.87 to 0 during the 0-60 second time period, with a k-slope of -1.72. The T-line region modified with 0.1 μL of paraffin solution not only reduced the light intensity decay rate from 93.92 to 10.66 (k-slope of -1.72) during the 0-60 second time period, but also achieved a complete improvement in light intensity. Unfortunately, the light intensity of the 0.3 μL paraffin-modified sample decreased from 85.28 to ~0, with a k-slope of -1.59.

[0055] like Figure 17 As shown, similar to the T line, the C line intensity exhibits a stable pattern, remaining almost constant at a value of ~250, which also demonstrates the effectiveness of the CL-LFA test.

[0056] Adding 0.1 μL of paraffin liquid to the nitrocellulose membrane resulted in slower light intensity decay and a stronger light signal. This is because some transparent areas can generate more light, while adding more 0.3 μL of paraffin liquid provides more barriers, making it more difficult for the CL substrate to come into contact with the conjugate.

[0057] Example 5 Based on the results of testing different cTnI antigen solution concentrations after modification with 0.1 μL paraffin solution, the optimal performance of CL-LFA was determined. cTnI antigen solutions with concentration gradients (0, 0.039, 0.078, 0.156, 0.312, 0.625, 1.25, 2.5, 5, 10 and 20 ng / mL) were designed for detection experiments.

[0058] Figure 18 Optical images of the processed T-line used in LFA assays to detect gradient concentrations of cTnI antigen are shown. In visual observation mode, the LOD can reach 1.25 ng / mL; no obvious positive signal is observed within the cTnI antigen concentration range of 0-0.6 ng / mL.

[0059] Figure 19 The image shows the measured colorimetric values ​​of the T and C line regions modified with 0.1 μL of paraffin solution on the NC strip during LFA detection. When the cTnI antigen concentration is 20 ng / mL, two distinct troughs appear on the red line, corresponding to the valid T and C lines; when the cTnI antigen concentration is 0, only one distinct trough appears on the blue line, corresponding to the valid C line.

[0060] Figure 20 Optical images of the treated T-line and C-line are shown in the CL-LFA assay for detecting different concentrations of cTnI antigen. The detection limits were 0.039 ng / mL when the illumination time was stopped at 0 seconds and 30 seconds, respectively; and 0.078 ng / mL when the illumination time was stopped at 60 seconds.

[0061] Figure 21 The measured color intensities of the T and C lines modified with 0.1 μL of paraffin solution on the NC strip are shown in the CL-LFA assay. Similarly, when the cTnI antigen concentration is 20 ng / mL, the red line shows two distinct peaks, corresponding to the effective T and C lines; when the cTnI antigen concentration is 0, the blue line shows only one distinct peak, corresponding to the effective C line. Clearly, the T and C lines modified with 0.1 μL of paraffin solution exhibit better performance in CL-LFA than in LFA.

[0062] Figure 22 The display shows the color intensity distribution detected by LFA, without... Figure 18 The background intensity is shown. When the cTnI antigen concentration is higher than LOD (1.25 ng / mL), the color intensity increases with the increase of cTnI antigen concentration; however, when the cTnI antigen concentration is lower than LOD, the detection resolution is not ideal and fluctuates between ~8.

[0063] Figure 23 Showing without Figure 20 The background intensity is shown in the color intensity distribution of CL-LFA detection. When the light exposure was stopped at 0 and 30 seconds, the CL intensity of the detected cTnI antigen was in the concentration range of 0.039-5 ng / mL, and the color intensity increased linearly with the increase of cTnI antigen concentration; however, when the cTnI antigen concentration was below 0.038 ng / mL, the detection resolution failed. When the light exposure was stopped for 60 seconds, the CL intensity of the detected cTnI antigen was in the concentration range of 0.075-5 ng / mL; however, when the cTnI antigen concentration was below 0.075 ng / mL, the detection resolution became unsatisfactory.

[0064] Figure 24 The fitted curves for CL intensity (excluding background intensity) in the concentration range of 0-2.5 ng / mL are shown, with the fitted expression being y = 282.78 - 178.47 * exp(-0.6x), R0. 2 =0.994.

[0065] Example 6 Human serum clinical testing (1) In order to test the selectivity of LFA based on Au-HRP-Ab for detecting cTnI, different antigens were detected using transparent nitrocellulose membrane strips: cTnI, MYO, creatine kinase MB (CK-MB), human serum albumin (HSA), human chorionic gonadotropin (HCG) and immunoglobulin G (IgG).

[0066] The concentration of each protein was 100 ng / mL, and the detection steps included: Add 1 μL of Au-HRP-Ab solution to a transparent nitrocellulose membrane strip, wait for the strip to dry, then add 15 μL of the above protein in PBS solution to the strip. After about 10 minutes, add 30 μL of PBS to rinse the strip, and wait for it to dry and develop color.

[0067] The results showed that the CL intensity and color intensity of the first four biomarkers were limited, but the intensity of cTnI was clearer in LFA and CL-LFA modes.

[0068] (2) Detection of cTnI in real human serum on paraffin-modified nitrocellulose test strips.

[0069] 10 μL of the conjugate solution was mixed with human serum, and then the mixture was added to a clear nitrocellulose membrane strip. After the same washing procedure, the light signal on the test strip was obtained, and the results were compared with the immunoturbidimetric assay (Beckman Coulter, AU5800) used in the hospital.

[0070] Figure 25A schematic diagram of the actual clinical test is shown. Human serum samples numbered 1-9 were obtained from patients, containing different concentrations of cTnI (1, 3.1, 3.4, 409.7, 816.2, 905.3, 3154.1, 5102.4, and 4242.2 pg / mL), and were thoroughly mixed with the conjugate before CL-LFA testing.

[0071] Figure 26 Images from a CL-LFA camera show T-line and C-line detection after paraffin solution treatment, used to detect cTnI antigen in real clinical human serum. Generally, the intensity of the T-line increases with increasing cTnI concentration, while the intensity of the C-line remains at a relatively constant level.

[0072] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A chemiluminescent lateral flow immunoassay method, characterized in that, Chemiluminescent side-flow detection is based on paraffin-modified nitrocellulose membrane. The preparation of the paraffin-modified nitrocellulose membrane includes coating the test line and control line areas of the nitrocellulose membrane with paraffin-modified material.

2. The chemiluminescent lateral flow immunoassay method according to claim 1, characterized in that: Adjustable transparency of nitrocellulose membranes, ranging from 12.47% to 78.53%, is achieved by adjusting the coating volume of paraffin liquid on the nitrocellulose membrane.

3. The chemiluminescent lateral flow immunoassay method according to claim 2, characterized in that: The coating volume of paraffin liquid is 0.1~6μL.

4. The chemiluminescent lateral flow immunoassay method according to claim 1, characterized in that: After modification with paraffin liquid, the light attenuation constant of the chemiluminescent lateral flow immunoassay method was reduced to -1.

35.

5. The application of the chemiluminescent lateral flow detection method as described in any one of claims 1 to 4 in the detection of cardiac troponin I.

6. The application according to claim 5, characterized in that: When detecting cardiac troponin I at 20 ng / mL, the light attenuation constant was -1.

35.

7. The application of the chemiluminescent lateral flow immunoassay method as described in any one of claims 1 to 4 in the diagnosis of myocardial injury for non-medical purposes.

8. The application according to claim 7, characterized in that: Chemiluminescent lateral flow immunoassay can effectively distinguish samples with cardiac troponin I concentrations ranging from 1 to 5.1 ng / mL.

9. The application of the chemiluminescent lateral flow immunoassay method as described in any one of claims 1 to 4 in a paper-based biosensor, characterized in that, Methods for preparing paper-based biosensors include: S1. Au-HRP-Ab or Au-Ab conjugates are prepared by physical adsorption method; S2. Adhere the sample pad, conjugate pad, paraffin-modified nitrocellulose membrane, and absorbent pad in sequence to assemble the test strip; S3. Drop Au-HRP-Ab or Au-Ab conjugate onto the conjugate pad and allow it to dry. S4. Spray cTnI capture antibody onto the T line of the nitrocellulose membrane and anti-species secondary antibody onto the C line. S5. Use 3D printed rectangular blocks to determine the coating positions of paraffin liquid on the T-line and C-line of the nitrocellulose membrane and then coat the membrane with paraffin liquid for modification.

Images