Zwitterionic polymer modified lateral flow chromatography sensor and application thereof in detection of microRNA in milk

Abstract

The invention relates to a zwitterionic polymer modified lateral flow chromatography sensor and application thereof in detection of microRNA in milk, and belongs to the field of biological detection. The zwitterionic polymer can resist interference of protein, fat and other interferents in milk on detection, and a tedious pretreatment process is avoided. A signal element is Au (at) Pt, and the microRNA in milk can be subjected to photo-thermal qualitative / quantitative detection, so that the problem that the traditional colorimetric detection result is easily interfered by the color of a sample matrix is solved.

Description

Technical Field

[0001] The invention belongs to the field of biological detection, and in particular relates to a zwitterionic polymer modified lateral flow chromatography sensor and an application thereof in detecting microRNA in milk. Background Art

[0002] MicroRNA (microRNA) is a class of short non-coding RNAs of approximately 19 to 25 nucleotides that play an important role in every biological process, including cell proliferation, differentiation, apoptosis, and other physiological or pathological processes. MicroRNA in milk has the potential to serve as a biomarker for bovine mastitis. Mastitis is an inflammatory disease, and early detection and treatment can help early recovery without significantly reducing reproductive performance and help prevent transmission to other cows.

[0003] Traditional detection methods for microRNA include Northern blot hybridization, microarray, and real-time quantitative PCR (qRT-PCR). Northern blot hybridization is considered the gold standard for detecting microRNA due to its high sensitivity, but it is time-consuming and requires a large amount of sample. Microarray has the ability of high throughput and multiplexing, but its sensitivity is low and it is also time-consuming; qRT-PCR has the advantages of strong specificity and high sensitivity, but due to the short sequence of microRNA, tailing or stem-loop methods are often required, which limits its application. Lateral flow chromatography sensors have the advantages of simple operation, short detection time, portability and low cost, and are suitable for detection on site and in resource-scarce or remote areas. Summary of the invention

[0004] The present invention aims to provide a method for detecting microRNA in milk which does not require pre-treatment and can resist the influence of the color of the milk matrix itself on the detection result.

[0005] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention is: The first aspect of the present invention is to provide a method for modifying a lateral flow chromatography sensor sample pad, the method comprising the following steps: 1) Dissolve dopamine and zwitterion monomers in Tris buffer, with the mass ratio of dopamine to zwitterion being 1:7-9; 2) Add CuSO4 and H2O2 in sequence; 3) Pre-wet the sample pad with ethanol, then place the sample pad in the solution formed in step 2) and soak and shake at 30-40 degrees Celsius for 3-5 hours; 4) Wash with deionized water and dry.

[0006] Furthermore, in step 1), the concentration of dopamine and zwitterion added is 1-3 mg / mL, and the concentration of zwitterion monomer is 8-24 mg / mL; preferably, the concentration of dopamine added is 2 mg / mL, and the concentration of zwitterion monomer added is 16 mg / mL.

[0007] Furthermore, in step 2), the concentrations of CuSO4 and H2O2 added are 4-6 mM and 18-21 mM, respectively; preferably, the concentrations of CuSO4 and H2O2 are 5 mM and 19.6 mM, respectively.

[0008] The second aspect of the present invention provides a modified lateral flow chromatography sensor sample pad prepared by the method described in the first aspect.

[0009] The third aspect of the present invention provides use of the sample pad described in the second aspect in detecting target nucleic acid in a sample. Preferably, the application is a non-diagnostic application.

[0010] Furthermore, the zwitterionic polymer modified lateral flow chromatography sensor uses Au@Pt as a signal element and uses a photothermal method to detect the target nucleic acid sequence; Furthermore, the sample is milk, and the target nucleic acid is microRNAs.

[0011] Furthermore, it is characterized in that the diameter of the gold nanoparticles of the signal element is 10-15 nm, and the diameter of the Au@Pt particles is 30-50 nm.

[0012] Furthermore, the steps of the application are: 1) preparing a lateral flow chromatography sensor by a conventional method in the art using the modified sample pad; 2) Mix the sample to be tested, running buffer and system buffer, add target standards of different concentrations, and immerse one end of the lateral flow chromatography sensor sample pad in the mixed solution; 3) Add gold nanoparticles coupled with capture probes and 2% BSA onto the conjugate pad, and drive the solution upward by capillary force; 4) Let it stand and record the temperature of the T line with a handheld thermal imager. Then, use a laser transmitter to irradiate the T line at a wavelength of 808 nm for 4 minutes and then record the temperature of the T line with a handheld thermal imager again. The difference between the two temperatures is used as a quantitative signal.

[0013] Furthermore, the lateral flow chromatography sensor comprises a sample pad, a conjugate pad, a water absorbent pad, an NC membrane and a back plate; preferably, the preparation process of the lateral flow chromatography sensor is: a. The biotin-labeled T-line nucleic acid sequence and the C-line nucleic acid sequence were diluted with PBS buffer and mixed with an equal amount of streptavidin and incubated overnight at room temperature to prepare a T-line mixture and a C-line mixture; b. Load the T-line mixed liquid into the nozzle 1 of the three-dimensional spray point platform and fix it at a position away from the lower edge of the NC film. Load the C-line mixed liquid into the nozzle 2 of the three-dimensional spray point platform and also fix it at a position away from the lower edge of the NC film. Make the distance between the T-line nozzle and the C-line nozzle 0.4~0.7 cm, with the C-line closer to the lower edge. Glue the NC film to the PVC bottom plate, spray the T-line and C-line mixed liquid evenly on the NC film, dry the sprayed NC film, and stick the absorbent pad close to the upper edge of the back plate; c. Stick the bonding pad to 0.5~1 cm from the bottom edge of the back plate; d. Stick the sample pad to the backing plate close to the lower edge of the backing plate, leaving part of the binding pad exposed; e. Use a programmable cutting machine to cut the pasted LFB strips into 3~4 mm wide sensors and store them at room temperature for future use.

[0014] Furthermore, the capture probe sequence is SH-AAATGGGGTATTT; the sequence of the detection line (T line) marker is AAACTGACAGGG-Biotin; the control line (C line) is Biotin-AAAAAATACCCCA; the system buffer is 4×SSC, 10 mM Tris-Hac, 0.002% TritionX-100, 0.1% Tween20, and the MgCl2 concentration is 0.8 mol / L; The running buffer is a conventional antigen detection buffer in the art, preferably the buffer in the Oriental Gene Antigen Rapid Detection Reagent.

[0015] The beneficial effects achieved by the present invention are: 1) The introduction of copper ions forms a metal phenol network with the catechol group, further improving the stability of the coating. In addition to being an anchoring agent for SBMA, PDA can also be used as an initiator to initiate the polymerization of SBMA monomers, improving the anti-interference ability of the coating; 2) The quantitative calibration curve of the zwitterionic polymer modified lateral flow chromatography sensor showed a good linear relationship between 0.18 nM and 1 nM. The minimum detection limit of this detection method in milk samples was 0.107 nM. The final recovery rate was between 99% and 114%, which proved that this detection method had good accuracy.

[0016] 3) The detection performance of the modified lateral flow chromatography sensor did not decrease significantly within 14 days, indicating that its detection performance was good. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Characterization diagram of zwitterion modified lateral flow chromatography sensor; A: Synthesis schematic diagram; B: SEM image of modified lateral flow chromatography sensor.

[0018] Figure 2 This is the anti-interference verification diagram of the zwitterion-modified lateral flow chromatography sensor; A: anti-BSA protein adsorption simulation curve; B: anti-fluorescent protein adsorption diagram.

[0019] Figure 3 Characterization diagram of Au@Pt-DNA material; A: Synthesis schematic diagram; B: Au@Pt TEM image; C: Au@Pt-DNA TEM image.

[0020] Figure 4 These are the optimization diagrams of the detection system. A: Optimization diagram of probe nucleic acid dosage; B: Optimization diagram of buffer salt concentration; C: Optimization diagram of cation types; D: Optimization diagram of anion types.

[0021] Figure 5 Qualitative / quantitative detection diagram of the modified lateral flow chromatography sensor; A: qualitative detection diagram; B: quantitative detection linear fitting diagram.

[0022] Figure 6 The specificity diagram (A) and stability diagram (B) of modified lateral flow chromatography. DETAILED DESCRIPTION

[0023] The following is a further description of the concept of the present invention and the technical effects produced in conjunction with specific embodiments, so as to fully understand the purpose, features and effects of the present invention. The methods are conventional methods unless otherwise specified. The materials can be obtained from public commercial channels unless otherwise specified. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations of the present invention. It should be noted that the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

[0024] Example 1 Lateral Flow Chromatography Sensor Modification The experimental process is as follows Figure 1As shown in A, the principle is to modify the sample pad by using CuSO4 and H2O2 to generate reactive oxygen species to accelerate the deposition of PDA / SBMA anti-interference coating. Theoretically, in an alkaline environment, dopamine is first oxidized to dopamine quinone, and then 5,6-dihydroxyindole is formed through intramolecular cyclization. Next, part of 5,6-dihydroxyindole will be further oxidized to 5,6-indolequinone. Unoxidized 5,6-dihydroxyindole and 5,6-indolequinone will be converted into semiquinone free radicals through a single electron exchange reaction, but the free radicals in this state are unstable, so relatively stable free radicals will be formed through electronic resonance. These free radicals will promote DA to become PDA through coupling reaction. However, without additional assistance, this process is very slow. CuSO4 and H2O2 will produce O2 ·— , OH2 · and OH · Active oxygen species greatly accelerate the oxidation and polymerization of dopamine, which is more conducive to the co-deposition of SBMA and PDA. This co-deposition is different from other non-covalent interactions. There is a covalent interaction between PDA and SBMA, which is more conducive to improving the uniformity and stability of the zwitterionic polymer anti-interference layer coating. The introduction of copper ions forms a metal phenol network with catechol groups, further improving the stability of the coating. In addition, PDA can be used as an anchor for SBMA and an initiator for initiating the polymerization of SBMA monomers, improving the anti-interference ability of the coating.

[0025] According to the above experimental ideas, the following experiments are involved: 1. CuSO4 / H2O2-assisted dopamine / zwitterion-modified lateral flow chromatography sensor; 1) Dissolve dopamine and zwitterion monomers in 10 mL of Tris buffer (50 mM, pH 8.5), control the dopamine concentration to 2 mg / mL, and the zwitterion concentration to 16 mg / mL (the mass ratio of dopamine to zwitterion sulfobetaine methacrylate is 1:8); 2) CuSO4 (5 mM) and H2O2 (19.6 mM) were added sequentially; 3) Pre-wet the sample pad with ethanol, then place the sample pad in the solution formed in step 2) and soak and shake at 35 degrees Celsius for 4 hours; 4) Then wash with deionized water and dry at 50 °C for 1 h.

[0026] 2. SEM electron microscope observation sample pad modification: After drying, cut the sample into a suitable size and paste it on the sample stage with conductive glue, and remove surface impurities by air sweeping. Because the conductivity of the film is poor, it needs to be sprayed with gold to enhance the conductivity for easy detection. The test scanning voltage is 3 kV and the working distance is 3.9-4.7 mm. Electron microscopy results Figure 1 As shown in Figure B: the sample pad modified with dopamine / SBMA is black, and the unmodified sample pad is white. Both surfaces are flat and burr-free, indicating that the modification does not change the fiber itself. In the SEM electron microscope image, it can be observed that the interface of the unmodified sample pad is smooth, without obvious granularity, and there is no cross-linking between the filamentous fibers, while the interface of the dopamine and zwitterionic polymer modified sample pad is rough and granular, indicating that the coating is successfully applied. The introduction of the coating makes the network structure of the sample pad more compact, enhances the steric hindrance effect, and improves the anti-interference ability of large molecular interferences.

[0027] 4. Quantitative test of adsorbed protein on zwitterionic polymer modified and unmodified sample pads using BCA method: In order to further verify the universal anti-interference ability of the anti-interference coating, the BSA protein adsorption at the interface of the modified / unmodified sample pad was detected using the BCA protein detection kit.

[0028] 1) Fit the standard curve with absorbance as the ordinate and BSA concentration as the abscissa; 2) Then, the modified and unmodified sample pads were immersed in 5 mg / ml BSA solution at the same time, shaken at 60 rpm for 3 h, and unbound proteins were washed with PBS buffer; 3) Immerse the modified / unmodified sample pad in 1% SDS solution, shake at 60 rpm for 2 hours, and measure the absorbance of the solution at 562 nm using an ELISA reader. Substitute the average of the three absorbance measurements into the standard curve to obtain the BSA adsorption concentration. The protein adsorption reduction rate is calculated as y=1-unmodified adsorption concentration / modified adsorption concentration×100%.

[0029] 4) The zwitterionic polymer-modified and unmodified sample pads were immersed in 10 ng / ml green fluorescent protein, shaken at 60 rpm for 3 h, and then the unbound proteins were washed away with PBS buffer and observed under a fluorescence microscope.

[0030] Figure 2 As shown in A, a linear fit was performed with protein concentration as the horizontal axis and absorbance as the vertical axis. The linear equation was y=0.62327x+0.08402, R 2=0.99936, indicating that the equation has a good linear relationship. After testing, the average value of the three tests of absorbance measured by unmodified / modified was substituted into the linear equation, and the adsorbed protein concentrations of 0.691% and 0.037% were obtained respectively. The protein adsorption was calculated to be 94.7% lower than that of unmodified. This shows that the PDA / SBMA anti-interference coating has excellent anti-interference ability and can be successfully applied to the modification of lateral flow chromatography sensors.

[0031] Figure 2 As shown in Figure B and Table 1, under the interference of high and low concentrations of fluorescent protein, the anti-interference coating has good anti-interference ability, and the fluorescence intensity is significantly reduced compared with the unmodified sample pad. Under the interference of high concentration of fluorescent protein, the fluorescence of the unmodified sample pad is significantly improved compared with the interference of low concentration of fluorescent protein, but the interface fluorescence intensity of the sample pad modified with the anti-interference coating is only slightly increased, which proves that the anti-interference coating has good anti-interference ability under the interference of low and high concentration of protein. Compared with the unmodified sample pad, the adsorption of low concentration of fluorescent protein decreased by 48.9%, and the adsorption of high concentration of fluorescent protein decreased by 53.7%.

[0032] Table 1 Effect of zwitterion-modified sample pad on protein adsorption

[0033] Example 2 Preparation of gold nanoparticles and coupling with nucleic acid probes 1) AuNPs (500 μL) were mixed with 200 μL 2% weight PVP, 40 uL 100 mg / mL ascorbic acid, and 24.4, 36.6, and 48.8 μL 100 mM H2PtCl6, and the mixture was incubated and heated at 70°C for 30 minutes. After the temperature dropped to room temperature, it was washed three times with ethanol and water alternately, and stored for a long time in a dark place at 4°C ( Figure 3 Middle A).

[0034] 2) Parameters for detecting gold nanoparticles: The solution from step 1) was dripped onto a copper grid and the solvent was removed by vacuum drying. Then, the sample was loaded into the TEM, and the appropriate accelerating voltage and electron beam focus were selected to observe the morphology, size, and dispersion of the nanoparticles using bright field and dark field modes.

[0035] Figure 3 As shown in B, the synthesized Au@Pt nanoparticles have a diameter of about 35 nm, forming a typical spherical core-shell structure. The darker inner core is the colloidal gold nanoparticle, and the interface is loaded with a layer of Pt shell, which is relatively transparent in color. Figure 3As shown in Figure C, the interface morphology of gold nanoparticles has not changed significantly compared with Au@Pt, and still maintains a spherical core-shell structure, indicating that coupling with nucleic acids will not affect its application in lateral flow chromatography sensors.

[0036] 3) Coupling of gold nanoparticles and nucleic acid capture probes The method of using frozen coupled nucleic acid: take 100 μL of the mixture solution obtained above and mix it evenly with 0.5, 1, 1.5, 2, and 2.5 uL of 100 μM concentration DNA capture probe, freeze it at -20 ℃ for half an hour, thaw it at room temperature, wash it three times with deionized water, and store it for a long time in a dark place at 4 ℃.

[0037] like Figure 4 As shown, Figure 4 A: Using the frozen nucleic acid coupling method, in a 100 μL Au@Pt system environment, adding 2 uL of 100 μM nucleic acid capture probe has the best detection performance. When it is less than 2 μL, the capture probes on the nanoparticle interface are not fully coupled, affecting the capture efficiency of the target. When it is higher than 2 μL, steric hindrance may be generated between the excess capture probes, affecting the anchoring of the capture probes on the nanoparticle interface.

[0038] Figure 4 B in the figure: MgCl2 is used as a representative salt to optimize the salt concentration in the detection system. When the salt concentration in the system is 0.8 mol / L, the detection performance of the flow chromatography sensor is optimal. As the salt concentration increases, the detection performance decreases. This may be because the excessively high salt concentration causes the negative charge at the colloidal gold interface to be neutralized, forming an electronic double layer, which hinders the aggregation and color development of gold-platinum nanoparticles.

[0039] Figure 4 C: Keep the cation concentration at 0.8 mol / L and compare Na + , K + , Ba 2+ Mg 2+ Effects of four cations on the detection system. Divalent cations improve the detection performance more than monovalent cations, and Mg 2+ The difference between Mg and Mg is significantly higher than that of the other three cations, which may be because monovalent cations are more likely to form outer layer interactions with nucleic acids and have poor binding stability, while Mg 2+ There is a stronger electrostatic interaction, and it is beneficial for the bases to form hydrogen bonds with the phosphate groups on the main chain, which greatly improves the hybridization rate and hybridization rate between nucleic acids.

[0040] Figure 4 D: in CH3COO - , Cl -、NO 2- 、SO4 2- Among the four anions, Cl - ions have the best effect on improving detection ability, which may be because the electrostatic repulsion between monovalent anions and the phosphate groups in the nucleic acid backbone is smaller and does not cause changes in the pH of the system, while CH3COO - and NO 2- It is a weak acid radical, which is easily hydrolyzed, causing the pH in the system to decrease, resulting in a decrease in detection performance.

[0041] Example 3 Comparison of the use of zwitterionic polymer modified / unmodified lateral flow chromatography sensors by the spike method 1) Preparation of lateral flow chromatography sensor: It mainly consists of five parts: a sample pad modified by the method described in Example 1, a binding pad, a water-absorbing pad, an NC membrane and a backing plate. The specific operation steps are: a. Dilute the biotin-labeled T-line nucleic acid sequence and C-line nucleic acid sequence to 100 μM with PBS buffer, mix with an equal amount of streptavidin, and incubate overnight at room temperature to prepare a T-line mixed solution and a C-line mixed solution; b. Load the T-line mixed liquid into the nozzle 1 of the three-dimensional spray platform and fix it at a position 1.1 cm away from the lower edge of the NC membrane. Load the C-line mixed liquid into the nozzle 2 of the three-dimensional spray platform and fix it at a position 1.6 cm away from the lower edge of the NC membrane. Make sure that the distance between the T-line nozzle and the C-line nozzle is 0.5 cm. Glue the NC membrane to the PVC base plate, set the scribing speed to 1.0 µL / cm, spray the T-line and C-line mixed liquid evenly on the NC membrane, dry the sprayed NC membrane at 37℃ for 3 h, and then stick the absorbent pad close to the upper edge of the backing plate. c. Stick the bonding pad to about 0.7 cm from the bottom edge of the backing plate; d. Stick the sample pad to the backing plate close to the lower edge of the backing plate, leaving part of the binding pad exposed; e. Use a programmable strip cutter to cut the pasted LFB strips into 3.5 mm wide sensors and store them at room temperature for future use.

[0042] 2) Mix 20 μL of commercial milk, 20 μL of running buffer (the buffer used in this example is the buffer in the Oriental Gene Antigen Rapid Detection Reagent) and 60 μL of the system buffer with the best performance (4×SSC, 10 mM Tris-Hac, 0.002% TritionX-100, 0.1% Tween20, 0.2mol / L MgCl2 used in this example), add target standards of different concentrations, and immerse one end of the lateral flow chromatography sensor sample pad in the mixed solution.

[0043] 3) 4 μL of the gold nanoparticles coupled with the capture probe prepared in Example 2 and 0.5 uL of 2% BSA were dripped onto the conjugate pad, and the solution was driven upward by capillary force. After 5 minutes of rest, the temperature of the T line was recorded with a handheld thermal imager, and then the T line was irradiated with a laser emitter at a wavelength of 808 nm for 4 minutes and then the temperature of the T line was recorded with a handheld thermal imager again, and the difference between the two temperatures was used as a quantitative signal.

[0044] The results are as follows Figure 5 As shown, Figure 5 A: The detection performance of zwitterionic polymer modified / unmodified lateral flow chromatography sensors using Au@Pt as signal expression nanoparticles in milk was compared by the spike method. It was found that the qualitative limit of the zwitterionic polymer modified lateral flow chromatography sensor was 0.18 nM, and the qualitative limit of the unmodified lateral flow chromatography sensor was 0.5 pM, with a qualitative sensitivity of 2.78 times. This proves that the zwitterionic polymer coating has a significant effect on resisting the interference of complex matrices in milk, and when Au@Pt is used as a signal expression nanoparticle, its qualitative limit is lower than that of colloidal gold, and the black color is easier to be recognized by the naked eye.

[0045] Figure 5 As shown in B, a handheld thermal imager was used to take photos and record them before and after irradiation at 808 nm for 4 minutes, and the temperature difference was used as the signal intensity as the ordinate, and the corresponding target concentration was used as the abscissa to draw a calibration curve. It can be seen that the detection quantitative calibration curve of the zwitterionic polymer modified lateral flow chromatography sensor between 0.18 nM and 1 nM showed a good linear relationship, and its linear equation was y=14.23054x-0.01281, and the relative relationship R 2 =0.9801, based on the calculation formula of "3×standard deviation / slope of linear regression equation", the minimum detection limit of quantitative detection of this detection method in milk samples is 0.107 nM. In addition, the calibration curve is drawn between 0.18 nM and 1 nM with the peak area of ​​image J quantification as the ordinate and the corresponding target concentration as the abscissa, but the calibration curve made in this way has no linear relationship and cannot meet the needs of predicting the target concentration. The spike recovery experiment was performed on samples with target concentrations of 1 nM, 0.7 nM and 0.5 nM, and the final recovery rate was between 99% and 114%, proving that this detection method has good accuracy.

[0046] Example 4 Detection of miRNAs in milk using a zwitterionic polymer-modified lateral flow chromatography sensor 1) preparing a lateral flow chromatography sensor for detection as described in step 1) of Example 3; 2) Mix 20 μL of milk, 20 μL of running buffer (same as in Example 3) and 60 μL of system buffer (same as in Example 3), add target standards of different concentrations, and immerse one end of the lateral flow chromatography sensor sample pad in the mixed solution. 3) 4 μL of the gold nanoparticles coupled with the capture probe prepared in Example 2 and 0.5 μL of 2% BSA were dropped onto the conjugate pad, and the solution was driven to flow upward by capillary force.

[0047] 4) After 5 minutes of rest, the temperature of the T line was recorded with a handheld thermal imager. The T line was irradiated with a laser transmitter at a wavelength of 808 nm for 4 minutes and the temperature of the T line was recorded with a handheld thermal imager again. The difference between the two temperatures was used as a quantitative signal.

[0048] 5) Method-specific detection Mix 20 μL of commercial milk, 20 μL of running buffer and 60 μL of the system buffer with the best performance, add equal amounts of target standards of different sequences, immerse one end of the lateral flow chromatography sensor sample pad in the mixed solution, drop 4 μL of the gold nanoparticles coupled with the capture probe prepared in Example 2 on the binding pad, and drive the solution upward by capillary force. After standing for 5 minutes, observe the experimental results. If black stripes appear on both the T line and the C line, the experimental result is positive; if only the C line appears red stripes, the experimental result is negative. Use a handheld thermal imager to record the temperature of the T line, then use a laser transmitter to irradiate the T line at a wavelength of 808 nm for 4 minutes, and then use a handheld thermal imager to record the temperature of the T line, and use the difference between the two temperatures as a quantitative signal.

[0049] Figure 6 Figure A shows that under the optimal experimental conditions, the zwitterionic polymer-modified lateral flow chromatography sensor was used to detect five types of microRNAs related to verification in milk: Bta-miRNA-223, Bta-miRNA-21, Bta-miRNA-155, Bta-miRNA-383, and Bta-miRNA-183. The results are shown in Figure 6. When detecting the target Bta-miRNA-223, its signal intensity was significantly higher than that of the other four groups, and no false positives were observed by naked eye, proving that the detection method has good specificity.

[0050] 6) Stability test The prepared lateral flow chromatography sensor was stored in a vacuum bag and taken out for spiked testing at 0, 3, 7, and 14 days, respectively, to verify its stability with the test performance as the standard. The temperature of the T line was recorded with a handheld thermal imager, and then the T line was irradiated with a laser transmitter at a wavelength of 808 nm for 4 minutes and then recorded with a handheld thermal imager, and the difference between the two temperatures was used as a quantitative signal.

[0051] Figure 6 As shown in Figure B, the detection performance of the lateral flow chromatography sensor in milk is used as a measure to test its stability, and the samples are tested at 0, 3, 7, and 14 days. Its detection performance did not decrease significantly within 14 days, indicating that its detection performance is good.

[0052] The embodiments described above are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work shall all fall within the scope of protection of the present invention.

Claims

1. A method for modifying a lateral flow chromatography sensor sample pad, characterized in that: The method comprises the following steps: 1) Dissolve dopamine and zwitterion monomers in Tris buffer, with the mass ratio of dopamine to zwitterion being 1:7-9; 2) Add CuSO4 and H2O2 in sequence; 3) Pre-wet the sample pad with ethanol, then place the sample pad in the solution formed in step 2) and soak and shake at 30-40 degrees Celsius for 3-5 hours; 4) Wash with deionized water and dry.

2. The method according to claim 1, characterized in that In step 1), the added concentration of dopamine and zwitterion is 1-3 mg / mL, and the concentration of zwitterion monomer is 8-24 mg / mL.

3. The method according to claim 1, characterized in that: Step 2) Add CuSO4 and H2O2 at concentrations of 4~6mM and 18~21mM.

4. The modified lateral flow chromatography sensor sample pad prepared by the method according to claim 1.

5. An application of the sample pad according to claim 4 in detecting target nucleic acid in a sample, characterized in that: The sample pad is used to prepare a lateral flow chromatography sensor, and the application is a non-diagnostic application.

6. The use according to claim 5, characterized in that: The lateral flow chromatography sensor uses Au@Pt as a signal element and adopts a photothermal method to detect a target nucleic acid sequence, wherein Au@Pt is coupled to a capture probe of the target nucleic acid.

7. The use according to claim 5 or 6, characterized in that: The sample is milk, and the target nucleic acid is microRNAs.

8. The use according to claim 6, characterized in that: The diameter of the gold nanoparticles of the signal element is 10-15 nm, and the diameter of the Au@Pt particles is 30-50 nm.

9. The use according to claim 8, characterized in that: The steps of the application are: 1) preparing a sample pad to obtain a lateral flow chromatography sensor, wherein a biotin-coupled T line sequence and a C line sequence are respectively labeled in the sensor; 2) Mix the sample to be tested, running buffer and system buffer, add target standards of different concentrations, and immerse one end of the lateral flow chromatography sensor sample pad in the mixed solution; 3) Add Au@Pt coupled with capture probe and 2% BSA onto the conjugate pad, and drive the solution upward by capillary force; 4) Let it stand, record the temperature of the T line with a handheld thermal imager, then use a laser transmitter to irradiate the T line at a wavelength of 808 nm for 3 to 5 minutes, then record the temperature of the T line with a handheld thermal imager again, and use the difference between the two temperatures as a quantitative signal.

10. The use according to claim 9, characterized in that: The capture probe sequence is SH-AAATGGGGTATTT; the T line sequence is AAACTGACAGGG-Biotin; the C line sequence is Biotin-AAAAAATACCCCA; the system buffer is 4×SSC, 10 mM Tris-Hac, 0.002% TritionX-100, 0.1% Tween20, and the MgCl2 concentration is 0.8 mol / L.

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