A lateral flow chromatographic analysis method and processing technology based on substrate delayed release automatic signal amplification

Abstract

The application discloses a kind of based on substrate delayed-release automatic signal amplification lateral flow chromatography analysis method and processing technology, belong to biosensor technical field, including, the sample to be measured is mixed with running buffer, and prepared mixed solution;70-150 μL mixed solution is dropped into SGF-LFA test paper sample pad, and the detection result is judged after 5-10 minutes;The SGF-LFA test paper includes PVC bottom plate, sample pad, gold mark pad, NC membrane and absorbent pad and substrate slow-release device SGF;NC membrane includes SA-Bio-DNAT, SA-Bio-DNAC and signal probe Au@Pt@PolyA-SDNA on it.The SGF-LFA of the application reaches higher naked-eye detection sensitivity for the detection of H1N1, and the sensitivity of traditional Au@PtNPs-based LFA colorimetric detection is increased by 25 times respectively, and is equivalent to the colorimetric sensitivity of Au@PtNPs catalyzed LFA based on manual drop color developing substrate, and can be used for naked-eye detection to large quantities of samples.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biosensors, and particularly relates to a lateral flow chromatographic analysis method and processing technology based on automatic signal amplification of substrate delayed release. BACKGROUND

[0002] Lateral flow assay (LFA) is the most commonly used POCT detection technology, which is widely used in food quality supervision, environmental monitoring and disease screening fields. Gold nanoparticle-based lateral flow assay (AuNP-LFA) has become one of the most commonly used rapid detection methods in food safety, clinical diagnosis and environmental monitoring due to its fast analysis speed, simple operation and low cost. Compared with laboratory testing technology, AuNP-LFA has certain limitations: low sensitivity, which greatly hinders the application of AuNP-LFA in rapid detection of trace targets.

[0003] In order to realize high sensitivity of LFA, some signal amplification strategies, such as nano-enzyme catalysis (NC), nanoparticle aggregation (NA), metal in-situ growth (MISG), and branched DNA technology (bDNA), are introduced into LFA. Through nano-enzyme catalysis of substrate color development, the sensitivity of LFA can be improved by tens or even hundreds of times. However, these methods require additional operation steps, such as preparation of substrate and manual addition of substrate for catalysis, which increases the complexity of experimental operation.

[0004] In the MISG strategy, Au 3+ , Cu 2+ or Ag + metal ions are reduced to metal crystals by reducing agents such as hydroxylamine (HA), ascorbic acid and p-benzoquinol, and deposited on the surface of AuNPs, thereby expanding the size of AuNPs, which leads to significant enhancement of colorimetric signal in the detection area, but at the same time, unnecessary self-nucleation and metal ion growth occur, which usually leads to a higher background signal and may cause false positive results. Branched DNA is an artificially synthesized branched DNA that can be combined with various enzymes to amplify the captured target signal for detection, however, it is usually carried out in a microplate.

[0005] In recent years, some colorimetric detection methods combining traditional LFA characteristics with one-step catalysis have attracted the interest of researchers. A delayed and non-delayed channel was designed on a nitrocellulose membrane using sequential wax dyeing technology for transporting gold enhancement solution in immunoassay, which simplified the multi-step operation of gold enhancement to one step; only one injection was needed to achieve signal amplification. There is also the use of two chemical release fibers embedded with color developing substrate and hydrogen peroxide respectively, under the permeation of sample solution, it slowly exudes and flows to the detection line, which takes a certain amount of time, which will produce a delay time between immunoassay and substrate catalysis, thereby realizing an automatic catalysis process. SUMMARY

[0006] This section is intended to summarize some aspects of the embodiments of the present application and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section as well as in the abstract and the title of the application in order to avoid obscuring the summary of the disclosure of the present application, and such simplifications or omissions are not intended to limit the scope of the present application in any manner.

[0007] In view of the above and / or problems existing in the prior art, the present application is proposed.

[0008] Therefore, the purpose of the present application is to overcome the deficiencies in the prior art, and to provide a lateral flow chromatographic analysis method based on substrate delayed release automatic signal amplification.

[0009] To solve the above technical problems, the present application provides the following technical scheme: a lateral flow chromatographic analysis method based on substrate delayed release automatic signal amplification, comprising,

[0010] Mixing the sample to be tested with a running buffer to prepare a mixed solution;

[0011] Drop 70-150 μL of the mixed solution on the sample pad of the SGF-LFA test paper, and read the detection result after 5-10 minutes;

[0012] The running buffer comprises 4xSSC, 0.5% Tween-20, and pH 7.4.

[0013] The SGF-LFA test paper comprises a PVC base plate, a sample pad, a gold label pad, an NC membrane, and an absorbent pad and a substrate slow-release device SGF.

[0014] The NC membrane comprises SA-Bio-DNAT, SA-Bio-DNAC, and a signal probe Au@Pt@PolyA-SDNA.

[0015] As a preferred scheme of the method of the present application, in the SGF-LFA test paper, the SA-Bio-DNAT and the SA-Bio-DNAC are fixed on the nitrocellulose membrane by non-covalent interaction.

[0016] As a preferred scheme of the method of the present application, in the PVC base plate, the sample pad, the gold label pad, the NC membrane, and the absorbent pad are sequentially pasted, and then the SGF is fixed on the sample pad.

[0017] The NC membrane is sprayed with SA-Bio-DNAT on the test line and with SA-Bio-DNAC on the control line, and is dried at 37-40℃, and the distance between the test line and the control line is 4-8 mm.

[0018] As a preferred scheme of the method of the present application, wherein: the preparation method of the SGF-LFA test paper comprises,

[0019] Preparation of substrate slow-release device SGF: mix a solution of 0-10% PVA with a DAB solution to obtain a color developing solution, cut a glass fiber membrane GF into a long strip with a width of 2-5 mm, and immerse it in the color developing solution for 5 min, and then dry it in a 37°C drying oven for 1 h;

[0020] Preparation of signal probe Au@Pt@PolyA-SDNA: Au@Pt NPs with a diameter of 23 nm are synthesized by a seed-mediated method, and a traditional salt aging method is used to construct the Au@Pt@PolyA-SDNA nanoprobes;

[0021] Assembly of the test paper: sequentially attach the sample pad, gold-labeled pad, NC membrane, and absorbent pad on the PVC adhesive back, with an overlap of 2 mm each, immerse the SGF in a 60%-100% sucrose solution, immediately take it out, and attach it to the sample pad, and dry it in a 40°C oven for 1 h;

[0022] Spray SA-Bio-DNAT on the T line of the NC membrane and spray SA-Bio-DNAC on the C line using a three-dimensional spray point platform, and dry them at 30-37°C for 1-2 h, and after drying, use a cutting machine to cut the assembled test paper strip into a long strip with a width of 2-4 mm, and store it in a self-sealing bag.

[0023] The method as described above, characterized in that: the preparation method of the Au@Pt NPs comprises,

[0024] Add 100 mL of 0.01% HAuCl4 to a 250 mL conical flask, heat and stir until the solution boils, and maintain for 1-2 min;

[0025] Subsequently, quickly add 2 mL of 1% trisodium citrate solution, continue to heat and stir, and the color of the solution gradually changes from light yellow to deep purple and finally to wine red to obtain an AuNPs solution;

[0026] The obtained AuNPs solution is used as a seed solution, 1 mL of 0.1 mol / L ascorbic acid is added to the AuNPs seed solution, then 1.5 mL of 0.01 g / mL H2PtCl6 is added for mixing, and the obtained mixed solution is heated at 90°C for 30 min to obtain Au@Pt NPs;

[0027] Cool the prepared Au@Pt NPs solution to room temperature, and store it at 4°C for standby use.

[0028] As a preferred scheme of the method, the Au@Pt@PolyA-SDNA nanoprobe is prepared by the method comprising the following steps:

[0029] 5 μL-15 μL of 100 μmol / L PolyA-SDNA is added into 1 mL of 10 nmol / L Au@PtNPs and mixed uniformly;

[0030] Then 20 μL of 500 mmol / L citric acid buffer solution with pH 3 is added, and after uniform mixing, the mixture is incubated at room temperature for 30 min-60 min;

[0031] After the incubation is completed, 60 μL of 500 mmol / L HEPES buffer solution with pH 7.6 is added to adjust the pH of the Au@PtNPs solution to neutral, and then the mixture is incubated at room temperature for 2 h-4 h;

[0032] Then the mixture is centrifuged at 10000 r / min for 20 min, the precipitate is resuspended in a resuspension solution, centrifuged at 10000 r / min for 20 min, and the unreacted nucleic acid is removed by centrifugation for three times. Finally, 400 μL of resuspension solution is added, and the mixture is stored at 4°C for standby.

[0033] As a preferred scheme of the method, the resuspension solution formula is 20 mmol / L Na3PO4, 5% BSA, 10% sucrose, and 0.25% Tween-20.

[0034] As a preferred scheme of the method, the SA-Bio-DNAT and the SA-Bio-DNAC are prepared by the method comprising the following steps:

[0035] SA and DNAT and DNAC are respectively incubated at 4°C and 400 rpm for 2 h in an incubator according to a molar ratio of 1:1-1:12;

[0036] Then the mixture is transferred to a MWCO 30 kDa ultrafiltration tube, centrifuged at 6000 r / min for 20 min, and the unbound Bio-DNAT and Bio-DNAC are washed by centrifugation with 400 μL of 0.01M PBS with pH 7.2 twice;

[0037] The conjugate is resuspended in 10 mmol / L PBS to make the total volume of the conjugate 300 μL, and the mixture is stored in a 4°C refrigerator for standby

[0038] As a preferred scheme of the method, the sample to be detected comprises an H1N1 sample.

[0039] As a preferred scheme of the method, the concentration of the H1N1 sample is 0.02-50 nM.

[0040] The present application has the following advantages:

[0041] (1) The present application uses PVA solution to fix DAB in the glass fiber membrane, only needs to be mixed simply, prepares the color developing substrate solution, and immerses the glass fiber membrane in the color developing solution, and after drying treatment, the substrate automatic release device SGF is obtained, which is simple, economical, and has good compatibility with the existing test paper processing technology.

[0042] (2) The present application further develops an automatic signal amplification lateral flow chromatography test paper SGF-LFA based on substrate delayed release based on the above work, PVA and sucrose form two barriers, which are continuously dissolved with the flow of the Yangping solution, which forms a time interval between the probe capture and nanoscale enzyme catalysis; when the color developing substrate flows through the detection area, strong color response will be generated under the catalysis of nanoscale enzyme; therefore, the SGF-LFA test paper has high sensitivity, rapidness, and excellent selectivity and repeatability, and therefore has high potential for application in H1N1 detection.

[0043] (3) The SGF-LFA in the present application has high naked eye detection sensitivity for H1N1 detection, which is 25 times higher than the sensitivity of the traditional Au@PtNPs-based LFA colorimetric detection, and is equivalent to the colorimetric sensitivity of the Au@PtNPs catalyzed LFA based on manual drop of color developing substrate, and can be used for naked eye detection of a large number of samples. BRIEF DESCRIPTION OF DRAWINGS

[0044] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor. Among them,

[0045] Figure 1 It is a schematic diagram for detecting H1N1 by SGF-LFA of the present application.

[0046] Figure 2 It is an Au@PtNPs characterization image of the present application, wherein (A) is a TEM image of Au@PtNPs; (B) is an ultraviolet-visible absorption spectrum of Au@PtNPs.

[0047] Figure 3 It is a SEM image of SGF of the present application, wherein (A) is a SEM image of glass fiber; (B) is a SEM image of SGF.

[0048] Figure 4The picture for verifying the feasibility of the SGF-LFA test paper of the application.

[0049] Figure 5 The optimization experiment of the SGF of the application, wherein (A) is PVA concentration optimization; (B) is sucrose concentration optimization.

[0050] Figure 6 The optimization of the SGF-LFA recognition element of the application, wherein (A) is the molar ratio of SA and Bio-DNA T ; (B) is the optimization of the volume of Au@Pt@PolyA-SDNA probe.

[0051] Figure 7 The determination of the detection sensitivity of the SGF-LFA test paper, wherein (A) is the visual detection range of H1N1 based on the Au@Pt nanocatalytic, manual dropwise addition of color developing substrate mode; (B) is the visual detection range of the SGF-LFA test paper of the application.

[0052] Figure 8 (A) is the specificity of the SGF-LFA test paper of the application; (B) is the repeatability of the SGF-LFA test paper of the application. DETAILED DESCRIPTION

[0053] In order to make the above objectives, features and advantages of the application more obvious and easy to understand, the specific embodiments of the application will be described in detail below with reference to the description of the application.

[0054] In the following description, a lot of specific details are set forth in order to provide a thorough understanding of the application, but the application can also be implemented in other ways different from those described herein, and those skilled in the art can make similar generalizations without departing from the connotation of the application, therefore the application is not limited by the specific embodiments disclosed below.

[0055] Secondly, the "one embodiment" or "embodiment" referred to herein means that the specific features, structures or characteristics can be included in at least one implementation of the application. "In one embodiment" appearing in different places in the specification does not mean the same embodiment, nor is it an independent or alternative embodiment that excludes other embodiments.

[0056] The preparation method of SA-Bio-DNAT and SA-Bio-DNAC in the application:

[0057] SA and DNA T (biotinylated single-stranded nucleic acid, both purchased from Sangon), DNA C(Biotinylated single-stranded nucleic acid, purchased from Sangon), according to the molar ratio of 1:1-1:12, incubate at 4℃, 400 rpm for 2h in the incubator;

[0058] Then the mixture was transferred to the MWCO 30 kDa ultrafiltration tube, put into the centrifuge 6000r / min centrifugation 20min, for washing unbound Bio-DNAT, Bio-DNAC with 400μL, 0.01M, PH7.2 PBS centrifugation, repeated twice;

[0059] The conjugate was resuspended in 10mmol / L PBS to make the total volume of the conjugate 300μL, and stored in the refrigerator at 4℃ for standby. Example 1

[0060] This example provides the preparation and characterization of Au@PtNPs, Au@Pt@PolyA-SDNA and SGF, the main steps are as follows:

[0061] 1. Preparation of AuNPs:

[0062] Gold nanoparticles were synthesized by seed growth method, the specific steps are as follows:

[0063] Sodium citrate solution (2.2mM, 75mL) was injected into the conical flask, heated for 10 minutes;

[0064] Then add 0.8mL HAuCl4 (25 mM), reaction for 8 minutes;

[0065] Observe the color change from yellow to deep pink, prepare gold seed immediately cooled to 90℃;

[0066] Then, under constant temperature of 90℃, add 0.8mL sodium citrate solution (60mM), after 2 minutes, add 0.8mL HAuCl4 (25mM), reaction for 30 min to obtain 23nm AuNPs;

[0067] 2. Preparation of Au@PtNPs:

[0068] The obtained AuNPs solution was used as seed solution,

[0069] 1 mL ascorbic acid (0.1 mol / L) was added to 100 mL AuNPs seed solution, then 1.5 mL H2PtCl6 (0.01 g / mL) was added for mixing.

[0070] The above mixed solution was heated at 90℃ for 30 min to obtain Au@PtNPs.

[0071] The prepared Au@PtNPs solution was cooled to room temperature and stored at 4℃ for standby.

[0072] TEM images of Au@Pt NPs are shown in FIG. 1. Figure 2 As shown in FIG. 1, the Au@Pt NPs with a thorn-like shape were synthesized.

[0073] 3. Preparation of signal probe Au@Pt@PolyA-SDNA

[0074] 5 μL-15 μL (100 μmol / L) of PolyA-SDNA (purchased from Sangon) was added to 1 mL of 4-fold concentrated Au@Pt NPs (10 nmol / L) and mixed uniformly;

[0075] Then 20 μL (500 mmol / L, pH 3) of citric acid buffer was added;

[0076] After mixing uniformly, incubation was performed at room temperature for 30 min-60 min;

[0077] After completion of the incubation, 60 μL (pH 7.6) of 500 mmol / L HEPES buffer was added to adjust the pH of the Au@Pt NPs solution to neutral, and then incubation was performed at room temperature for 2 h-4 h;

[0078] Then centrifugation was performed at 10000 r / min for 20 min, and the precipitate was resuspended in a resuspension solution (20 mmol / L Na3PO4, 5% BSA, 10% sucrose, 0.25% Tween-20);

[0079] Centrifugation was performed at 10000 r / min for 20 min, and the unreacted nucleic acid was removed by centrifugation for three times, and finally 400 μL of resuspension solution was added and stored at 4°C for standby.

[0080] 4. Preparation of SGF

[0081] A solution of 5% PVA was mixed with a DAB solution (3,3'-diaminobenzidine DAB was dissolved in DMSO to obtain a DAB solution, and the concentration was 40 mM) to obtain a color developing solution, and the volume ratio of the PVA solution to the DAB solution was 1:1;

[0082] The glass fiber membrane (GF) was cut into a long strip with a width of 2-5 mm and immersed in the color developing solution for 5 min;

[0083] Then, drying was performed in a 37°C drying oven for 1 h.

[0084] The SEM images of the SGF before and after preparation are shown in FIG. 2. Figure 3 As can be seen from the SEM images, the membrane without embedding the substrate has a larger pore size and a higher porosity, and the membrane after the substrate immersion treatment has a significantly smaller pore size, which has a certain interception effect on the fluid.

[0085] 5. SGF-LFA test paper assembly

[0086] The sample pad, gold label pad, NC membrane and absorbent pad were sequentially attached to the PVC adhesive backing, each with an overlap of 2 mm. The SGF was immersed in a 100% sucrose solution and immediately removed and attached to the sample pad. The assembly was dried in an oven at 40°C for 1 h. Example 2

[0087] Method feasibility verification:

[0088] A methylene blue indicator was used instead of DAB to prepare a color developing substrate, which was embedded in a glass fiber paper for simulation experiments.

[0089] Release process simulation of the color developing substrate: The glass fiber membrane was immersed in a mixture containing 10 mM methylene blue and 2.5% PVA for 5 min, then removed and dried at 37°C for 1 h, fixed on the sample pad, and dried in an oven for 1 h. 150 μL of sample solution was loaded onto the sample pad.

[0090] After running the test paper strip, the movement trajectory of the indicator on the test paper strip could be clearly seen.

[0091] As shown in Figure 4 , with the flow of the running buffer, the indicator slowly flows forward with the dissolution of PVA and sucrose crystals, and within 8 min, the indicator does not reach the detection line. This shows that the SGF can act as a barrier to provide a time interval between dye delivery and probe capture. Around 15 min, the indicator reaches the absorbent pad, and within this time, the detection process is basically complete. This simulation experiment provides a feasibility basis for the delayed release of the substrate based on the membrane. Example 3

[0092] Preparation of a lateral flow chromatography test paper based on automatic signal amplification of delayed release of the substrate:

[0093] 1. Preparation of the test paper

[0094] The SGF-LFA test paper includes a PVC base plate, a sample pad, a gold label pad, an NC membrane and an absorbent pad, as well as a substrate release device SGF.

[0095] The length of the overlap between the sample pad and the PVC adhesive backing is 2 mm, and the sample pad is placed above the PVC adhesive backing. The length of the overlap between the sample pad, the gold label pad and the NC membrane is 2 mm, and the sample pad membrane is placed above the gold label pad, and the gold label pad is placed above the NC membrane. The length of the overlap between the NC membrane and the absorbent pad is 2 mm, and the absorbent pad is placed above the NC membrane. The SGF was immersed in a 100% sucrose solution and immediately removed and attached to the sample pad. The assembly was dried in an oven at 40°C for 1 h.

[0096] The SA-Bio-DNA coupled at a molar ratio of 1:8 was sprayed on the test line (T line) of the NC membrane using a three-dimensional spray point platform T The SA-Bio-DNA coupled at a molar ratio of 1:8 was sprayed on the control line (C line) C , and dried at 30-37℃ for 2h. The distance between the test line and the control line was 4mm, and the sample was dried in an oven at 37℃ for 4h.

[0097] Then the assembled test strip was cut into 4mm wide strips using a cutting machine and stored in a self-sealing bag.

[0098] 2. Establishment of the detection method based on the SGF-LFA test strip

[0099] 150μL of different concentrations of H1N1 (0, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 5, 20, 50nM) were added to the sample pad, and the results were detected after 15 minutes using a portable reader.

[0100] Among them, the H1N1 was diluted into different concentration standard solutions (0, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 5, 20, 50nM) using a running buffer solution (600mM NaCl, 80mM sodium citrate, 0.5% Tween 20).

[0101] If H1N1 exists in the sample, a segment of the H1N1 nucleic acid chain hybridizes with the detection probe to form Au@Pt@PolyA-SDNA-H1N1, which is captured by the capture nucleic acid Bio-DNA T on the detection line to form Au@Pt@PolyA-SDNA-H1N1-DNAT, thereby depositing on the T line to produce color. If H1N1 does not exist, the detection probe will not be captured by the capture probe on the T line, and the T line will not change color.

[0102] Regardless of the presence or absence of H1N1, the detection probe can hybridize with the Bio-DNA C on the control line to form Au@Pt@PolyA-SDNA-DNAC, thereby accumulating color changes. After the capture process is complete, the color developing substrate flows forward with the dissolution of PVA and sucrose, is catalyzed by the Au@PtNPs probe on the T line and the C line, produces strong brown color, and realizes signal amplification.

[0103] The schematic diagram of detecting H1N1 by SGF-LFA is shown in Figure 1 . Example 4

[0104] Optimization of the SGF-LFA detection method:

[0105] 1. Optimization of SGF:

[0106] (1) Optimization of PVA concentration

[0107] Under the condition of controlling other conditions unchanged (SA and Bio-DNA T , SA and Bio-DNA C molar ratio 1:8, GF width 4mm), PVA and DAB were mixed, and after mixing, the PVA concentration in the solution was 0, 2.5, 5, 10% respectively to prepare SGF; the SGF-LFA program was run, and the C-line signal intensity was detected in the portable reader.

[0108] The results are shown in Figure 5 A, when the PVA content is 2.5%, the signal intensity reaches the maximum.

[0109] (2) Optimization of sucrose concentration

[0110] Under the condition of controlling other conditions unchanged (SA and Bio-DNA T , SA and Bio-DNA C molar ratio 1:8, SGF width 4mm, PVA concentration 2.5%), different concentrations of sucrose solution (70, 80, 90, 100%) were used to fix the SGF; the SGF-LFA test paper was run, and the C-line signal intensity was detected in the portable reader.

[0111] The results are shown in Figure 5 B, 100% sucrose concentration better controls the release of the substrate and reduces background interference.

[0112] 2, Optimization of SGF-LFA recognition elements

[0113] (1) SA and Bio-DNA T molar ratio

[0114] Under the condition of controlling other conditions unchanged (PVA concentration 2.5%, sucrose concentration 100%), SA and Bio-DNA T were incubated according to different molar ratios (1:1, 1:4, 1:8, 1:12), and the T-line signal intensity was detected by the portable reader to judge the signal strength, and the results are shown in Figure 6 A, the best molar ratio is 1:8.

[0115] (2) Volume of signal probe Au@Pt@PolyA-SDNA

[0116] Under the condition of controlling other conditions unchanged (PVA concentration 2.5%, sucrose concentration 100%, SA and Bio-DNA TThe volume of Au@Pt@PolyA-SDNA probe was changed (0.6, 0.8, 1, 1.2, 1.4 μL), and the signal intensity of T line was detected by using a portable reader to determine the signal intensity. The results are shown in Figure 6 B, and the optimal probe addition amount is 1 μL. Example 5

[0117] Detection of H1N1:

[0118] 1. Determination of the detection sensitivity of H1N1 SGF-LFA test paper:

[0119] Under the optimal detection conditions (PVA concentration of 2.5%, sucrose concentration of 100%, SA and Bio-DNA T The visual detection limit of the traditional Au@Pt NPs nanocatalytic LFA for H1N1 is 0.5 nM at 0-50 nM, and the analysis performance of the manual substrate drop catalytic mode and our SGF-LFA test paper is compared.

[0120] As shown in Figure 7 With the decrease of the concentration of H1N1, the detection probe accumulated on the detection line gradually decreases, and the color signal gradually weakens. When the concentration of H1N1 is 0.02 nM, the color of the detection line is almost invisible. For the mode of manual drop of substrate to realize signal amplification and our automatic delivery of substrate detection mode, the T line signal intensity and the concentration of H1N1 have a good linear relationship between 0.02-50 nM, and the detection performance of H1N1 in the two catalytic modes is almost equivalent. After catalysis, the detection sensitivity of H1N1 is improved by 25 times compared with the results before catalysis.

[0121] 2. Selectivity and repeatability analysis:

[0122] Typical reverse transcription nucleic acids of influenza viruses, including H3N2, H9N2 and H5N1, were selected for the experiment to verify the selectivity of SGF-LFA. The single sample, mixed sample and blank sample of the above nucleic acid chains were detected by SGF-LFA test paper, as shown in Figure 8 A, only when the target H1N1 exists, the T line of the test strip will have a strong color signal, and the test strips of H3N2, H9N2, H5N1 and blank control only have weak signal reaction. This result shows that the automatic signal amplification lateral flow chromatographic sensor based on substrate delayed release constructed in this experiment has good selectivity.

[0123] The repeatability of the test paper is crucial for accurate detection of target objects, as shown in Figure 8As shown in B, 14 groups of parallel H1N1 were detected, and the results showed that the signal intensity difference between the 14 test strips was not large, which also proved that the sensor had good stability and provided credibility for the accuracy of the target detection. Example 6

[0124] Detection of H1N1 in serum samples:

[0125] The serum matrix was diluted 10 times with running buffer, and H1N1 was diluted to 0, 1, 5, 20 nM with the diluted serum, and tested by SGF-LFA test paper, and the concentration of H1N1 after spiking was obtained according to the standard curve in Example 5, and the recovery rate after spiking was calculated:

[0126] As shown in Table 1, in the detection mode of SGF-LFA test paper, the recovery rate of H1N1 was between 96% and 102.7%, and the relative standard deviation was not more than 6.3%, the concentration of H1N1 detected in the serum was equivalent to the amount added, which indicated that the sensor had good accuracy and stability in detecting H1N1 in actual samples. Therefore, the SGF-LFA test paper provided by the application can be used for rapid detection of H1N1.

[0127] Table 1 Recovery experiment of H1N1 in serum (n=3)

[0128]

[0129] Note: a not detected, each data represents the average of three replicates

[0130] It can be seen that the nano sensor constructed by the application has good anti-interference performance and good specific response to H1N1.

[0131] The application selects a commercial glass fiber membrane (GF) to encapsulate the chromogenic substrate (SGF), and integrates it with LFA, which can control the release of the substrate in LFA. The parameters of the base film and the detection system are optimized, a SGF-based autocatalytic LFA (SGF-LFA) is developed, and it is applied to detect the reverse transcription nucleic acid H1N1 of influenza A virus.

[0132] It should be noted that the above examples are only used to illustrate the technical solutions of the application and not to limit it. Although the application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the application can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the application, which should be covered in the scope of the application.

Claims

1. A sideflow tomography method based on substrate delayed release and automatic signal amplification, characterized in that: include, The sample to be tested is mixed with the running buffer to prepare a mixed solution; Add 70-150 μL of the mixed solution to the sample pad of the SGF-LFA test strip, and read the test result after 5-10 minutes. The running buffer comprises 4×SSC, 0.5% Tween-20, and pH 7.

4. The SGF-LFA test strip includes a PVC base plate, a sample pad, a gold label pad, an NC membrane, an absorbent pad, and a substrate slow-release device SGF. The preparation method of the SGF-LFA test paper includes preparing the substrate slow-release device SGF: mixing a 0-10% polyvinyl alcohol (PVA) solution with a DAB solution to obtain a colorimetric solution, cutting the glass fiber membrane GF into strips 2-5 mm wide, immersing them in the colorimetric solution for 5 min, and then drying them in a 37°C drying oven for 1 h. Preparation of signal probe Au@Pt@PolyA-SDNA: Au@PtNPs with a diameter of 23 nm were synthesized by seed-mediated method, and Au@Pt@PolyA-SDNA nanoprobes were constructed using the traditional salt aging method; Assemble the test strip: Place the sample pad, gold label pad, NC membrane and absorbent pad on the PVC backing in sequence, overlapping each one by 2 mm. Immerse the SGF in a 60%-100% sucrose solution, remove it immediately, and stick it on the sample pad. Dry in an oven at 40℃ for 1 hour. SA-Bio-DNAT was sprayed onto the T line of the NC membrane using a three-dimensional spraying platform, and SA-Bio-DNAC was sprayed onto the C line. The membrane was then dried at 30-37°C for 1-2 hours. After drying, the assembled test strips were cut into strips 2-4 mm wide using a cutting machine, placed in a resealable bag, and sealed for storage.

2. The method as described in claim 1, characterized in that: In the SGF-LFA test paper, SA-Bio-DNAT and SA-Bio-DNAC are immobilized on the nitrocellulose membrane through non-covalent interactions.

3. The method as described in claim 1 or 2, characterized in that: The sample pad, gold label pad, NC film and absorbent pad are sequentially glued onto the PVC base plate, and then the SGF is fixed on the sample pad. SA-Bio-DNAT is sprayed on the test line of the NC membrane, and SA-Bio-DNAC is sprayed on the control line. The membrane is then dried at 37-40°C, and the distance between the test line and the control line is 4-8 mm.

4. The method as described in claim 1, characterized in that: The Au@PtNPs are prepared by the following methods: Add 100 mL of 0.01% HAuCl4 to a 250 mL Erlenmeyer flask, heat and stir until the solution boils violently, and maintain for 1-2 minutes; Subsequently, 2 mL of 1% trisodium citrate solution was quickly added, and the mixture was heated and stirred continuously. The color of the solution gradually changed from light yellow to dark purple and finally to wine red, thus obtaining the AuNPs solution. The obtained AuNPs solution was used as the seed solution. 1 mL of 0.1 mol / L ascorbic acid was added to the AuNPs seed solution, and then 1.5 mL of 0.01 g / mL H2PtCl6 was added and mixed. The resulting mixture was heated at 90℃ for 30 min to obtain Au@PtNPs. The prepared Au@PtNPs solution was cooled to room temperature and stored at 4°C for later use.

5. The method as described in claim 1, characterized in that: The Au@Pt@PolyA-SDNA nanoprobe is prepared by the following method: Add 5 μL-15 μL of 100 μmol / L PolyA-SDNA to 1 mL of 10 nmol / L Au@PtNPs and mix thoroughly. Then add 20 μL of 500 mmol / L, pH 3 citrate buffer, mix well, and incubate at room temperature for 30-60 min. After incubation, add 60 μL of 500 mmol / L HEPES buffer (pH 7.6) to adjust the pH of the Au@PtNPs solution to neutral, and then incubate at room temperature for 2-4 hours. After centrifugation at 10000 rpm for 20 min, the precipitate was resuspended in resuspending solution and centrifuged again at 10000 rpm for 20 min. This process was repeated three times to remove unreacted nucleic acids. Finally, 400 μL of resuspending solution was added and the mixture was stored at 4°C for later use.

6. The method as described in claim 5, characterized in that: The resuspension formulation is 20 mmol / L Na3PO4, 5% BSA, 10% sucrose, and 0.25% Tween-20.

7. The method as described in claim 1, characterized in that: The preparation methods of SA-Bio-DNAT and SA-Bio-DNAC include, SA and DNA respectively T DNA C Incubate at 4°C and 400 rpm for 2 hours in an incubator at a molar ratio of 1:1 to 1:

12. The mixture was then transferred to an MWCO 30 kDa ultrafiltration tube and centrifuged at 6000 rpm for 20 min. To wash away unbound Bio-DNAT and Bio-DNAC, the mixture was centrifuged twice with 400 µL of 0.01 M PBS at pH 7.

2. The conjugate was resuspended in 10 mmol / L PBS to make a total volume of 300 µL and stored at 4 °C for later use.

8. The method according to any one of claims 1, 2, 4 to 7, characterized in that: The samples to be tested include H1N1 samples.

9. The method as described in claim 8, characterized in that: The concentration of the H1N1 sample was 0.02~50 nM.

Images