A dual-function immunochromatographic test strip and its application

Abstract

The invention discloses a dual-function immunochromatographic test strip and its application, which relates to the fields of medical inspection and food safety detection. The test strip includes a GO@PBNP labeled antibody complex, and its preparation method includes: S1, uniformly mixing a monolayer graphene oxide solution with polyvinyl pyrrolidone, and uniformly dispersing it in hydrochloric acid together with a potassium ferrocyanide solution to obtain a dispersion; S2, adding polyvinyl pyrrolidone to the dispersion, reacting to grow Prussian blue particles under heating conditions, centrifuging, washing, and redissolving to obtain GO@PBNP; S3, adding the antibody to be labeled to GO@PBNP, adding a blocking agent after sufficient mixing, centrifuging, washing, and redissolving to obtain. The beneficial effect of the present invention is to use graphene oxide as a carrier, in situ grow Prussian blue nanoparticles to synthesize GO@PBNP, and use it as a beacon carrier to prepare an immunochromatographic test strip with colorimetric and photothermal dual-function signals, and the detection sensitivity is high.

Description

Technical Field

[0001] The invention belongs to the field of medical inspection and food safety detection, and specifically relates to a dual-function immunochromatographic test strip and an application thereof. Background Art

[0002] Immunochromatography technology has the advantages of fast detection speed, good specificity, simple operation and low cost. It is currently the most mature on-site rapid detection technology. It has developed rapidly in recent years and is widely used in clinical examinations, food safety, medical testing and environmental pollutant monitoring. The development of immunochromatography technology can be divided into three categories: one is to use signal amplification systems (nanozymes, streptavidin-avidin systems) to improve detection sensitivity; the second is to develop new markers (such as nanomaterials) to increase signal output values. The third is to use immunomagnetic separation and enrichment technology to separate and concentrate the target to solve the matrix interference of complex samples.

[0003] However, the colloidal gold immunochromatographic test strips commonly used at present have low sensitivity, are easily interfered by the matrix, and have certain limitations. Summary of the invention

[0004] The technical problem to be solved by the present invention is that the existing colloidal gold immunochromatographic test strips have insufficient detection sensitivity and slow detection speed. The present invention provides a dual-function immunochromatographic test strip prepared by using graphene oxide in-situ grown Prussian blue nanomaterials as beacon carriers and its application. The GO@PBNP is prepared based on the self-assembly method of polyvinyl pyrrolidone. The prepared GO@PBNP has good dispersibility, good biocompatibility, good colorimetric performance, and superior photothermal performance. It can be used as a new colorimetric / photothermal signal label in dual-function immunochromatographic test strips. Compared with colloidal gold test strips, dual-function immunochromatographic test strips prepared by using graphene oxide in-situ grown Prussian blue nanomaterials as beacon carriers have the advantages of high sensitivity and short detection time.

[0005] The technical solution of the present invention is as follows:

[0006] The first aspect of the present invention provides a dual-function immunochromatographic test strip, the dual-function immunochromatographic test strip comprising a GO@PBNP labeled antibody complex;

[0007] The method for preparing the GO@PBNP labeled antibody complex comprises the following steps:

[0008] S1, uniformly mixing the monolayer graphene oxide solution and polyvinyl pyrrolidone, and uniformly dispersing the mixture in hydrochloric acid together with the potassium ferrocyanide solution to obtain a dispersion;

[0009] S2, continue to add polyvinyl pyrrolidone to the dispersion and mix evenly, react and grow Prussian blue particles under heating conditions, centrifuge to obtain a precipitate, wash it, and then redissolve it in pure water to obtain GO@PBNP;

[0010] S3, adding the antibody to be labeled to the GO@PBNP, adding a blocking agent after sufficient mixing, centrifuging to obtain a precipitate after the reaction, and redissolving the obtained precipitate to prepare a GO@PBNP labeled antibody complex.

[0011] Optionally, in step S1, the method for preparing the single-layer graphene oxide solution comprises the following steps:

[0012] The monolayer graphene oxide dispersion is ultrasonically treated, centrifuged, and the precipitate is collected and resuspended in water to obtain a monolayer graphene oxide solution;

[0013] The thickness of the single-layer graphene oxide in the single-layer graphene oxide dispersion is 0.8 nm to 1.2 nm; the flake diameter is less than 500 nm; the power of the ultrasonic treatment is 500 W to 1000 W; the ultrasonic time is 10 min to 20 min; and the centrifugal speed is 8000 r / min to 12000 r / min.

[0014] Optionally, in step S1, the concentration of the monolayer graphene oxide solution is 1.5 mg / mL~2.5 mg / mL, the molecular weight of polyvinyl pyrrolidone is 10000, the concentration of polyvinyl pyrrolidone is 8 mg / mL~12 mg / mL, the concentration of hydrochloric acid is 0.008 M~0.012 M, the concentration of potassium ferrocyanide solution is 8 mg / mL~12 mg / mL, and the added volume ratio of the monolayer graphene oxide solution, polyvinyl pyrrolidone, hydrochloric acid and potassium ferrocyanide solution is 4~6:12~18:25~35:5~7.

[0015] Optionally, in step S2, the molecular weight of polyvinyl pyrrolidone is 40,000, the concentration of polyvinyl pyrrolidone is 0.15 g / mL~0.25 g / mL, the added volume of polyvinyl pyrrolidone is 1.5 times~2.5 times the volume of the monolayer graphene oxide solution, the heating temperature is 70°C~90°C, and the heating time is 18 h~22 h; the centrifugal conditions include: the rotation speed is 3000r / min~12000 r / min, and the time is 10 min~30 min.

[0016] Optionally, step S3 includes the following specific steps:

[0017] The graphene in situ grown Prussian blue nanomaterial was ultrasonically treated for 4 min to 6 min, a 0.015 M to 0.025 M borate buffer with a pH of 5.0 to 9.0 was added, and then the antibody to be labeled was added, and a blocking agent was added after sufficient mixing, and the mixture was blocked at room temperature for 0.5 h to 1.5 h. After the reaction, the precipitate was centrifuged and the obtained precipitate was redissolved with a 0.008 M to 0.012 M phosphate buffer with a pH of 7.4 to obtain a GO@PBNP labeled antibody complex;

[0018] Among them, the concentration of the antibody to be labeled is 10 μg / mL~100μg / mL, the concentration of the blocking agent is 1%~10%, and the blocking agent is any one or more of bovine serum albumin, casein, ovalbumin, and skim milk; the centrifugal speed is 8000 r / min~10000 r / min, and the time is 10 min~20 min; the volume of the solution after reconstitution is 1 / 20~1 / 5 of the initial volume.

[0019] Optionally, the antibody to be labeled includes a monoclonal antibody, a polyclonal antibody, a nanobody, or a phage-expressed antibody.

[0020] Optionally, the dual-function immunochromatographic test strip comprises:

[0021] Base plate;

[0022] A nitrocellulose membrane is arranged on the bottom plate, and the nitrocellulose membrane has detection lines and quality control lines arranged at intervals;

[0023] A glass fiber mat is arranged at one end of the nitrocellulose membrane having a detection line, one end of the glass fiber mat is arranged on the bottom plate, and the other end is arranged on the nitrocellulose membrane, and the GO@PBNP labeled antibody complex is arranged on the glass fiber mat;

[0024] Absorbent paper is arranged at one end of the nitrocellulose membrane having a quality control line, one end of the absorbent paper is arranged on the bottom plate, and the other end is arranged on the nitrocellulose membrane;

[0025] A sample pad has one end arranged on the bottom plate and the other end arranged on the glass fiber pad.

[0026] Optionally, the method of disposing the GO@PBNP labeled antibody complex on the glass fiber mat comprises:

[0027] The GO@PBNP labeled antibody complex was sprayed onto the glass fiber mat at a volume of 7 μL / cm~9 μL / cm and vacuum dried for 1.5 h~2.5 h.

[0028] Optionally, the test line is coated with an artificially coupled antigen of the object to be tested or an antibody to be tested, and the quality control line is coated with a goat anti-mouse antibody;

[0029] The method for forming a detection line and a quality control line on the nitrocellulose membrane comprises the following steps:

[0030] Use 0.01 M to 0.5 M, pH 6.0 to 8.0 PBS solution to adjust the concentration of the coated object artificially coupled antigen or the object antibody and the goat anti-mouse antibody to 0.01 mg / mL to 10.0 mg / mL respectively; wherein the object antibody includes monoclonal antibody, polyclonal antibody, nano antibody, phage expressed antibody;

[0031] The artificially coupled antigen or antibody to be detected is sprayed on the upper part of the nitrocellulose membrane as a detection line, and the goat anti-mouse antibody is sprayed on the lower part of the nitrocellulose membrane as a quality control line; wherein the spraying amount of the artificially coupled antigen or antibody to be detected and the goat anti-mouse antibody is 0.25 μL / cm~0.74 μL / cm;

[0032] The nitrocellulose membrane sprayed with the test line and the quality control line is dried.

[0033] The second aspect of the present invention provides an application of the dual-function immunochromatographic test strip, wherein the dual-function immunochromatographic test strip is used to detect pesticides or pathogenic bacteria, and the detection method comprises the following steps:

[0034] The sample to be tested is added to the dual-function immunochromatographic test strip. The reaction time is 3 minutes to 15 minutes. The concentration of the sample to be tested is calculated by reading the grayscale of the test strip and the imaging data of the infrared imager to achieve quantitative detection, or the qualitative judgment of the sample to be tested can be achieved by visually observing the detection line and quality control line on the dual-function immunochromatographic test strip for the presence of colored bands.

[0035] The present invention has at least one of the following beneficial effects:

[0036] The present invention uses graphene oxide as a carrier, in situ grows Prussian blue nanoparticles to synthesize a new type of nanomaterial (GO@PBNP), and uses GO@PBNP as a beacon carrier to prepare a dual-function immunochromatographic test strip; compared with traditional colloidal gold test strips, the dual-function immunochromatographic test strip prepared by the present invention has colorimetric and photothermal dual-function signals, and the signal is strong. The prepared dual-function immunochromatographic test strip has high detection sensitivity and can realize rapid quantitative or qualitative detection of various analytes. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1This is a schematic diagram of the structure of a dual-function immunochromatographic test strip, wherein: 1 is a sample pad, 2 is a glass fiber pad, 3 is a nitrocellulose membrane, 4 is a detection line, 5 is a quality control line, 6 is a bottom plate, and 7 is absorbent paper.

[0038] Figure 2 Shown are the UV absorption spectra of GO and GO@PBNP.

[0039] Figure 3 Shown are the particle sizes of GO, GO@PBNP, and GO@PBNP-labeled clenbuterol antibody complex (GO@PBNP@mAb).

[0040] Figure 4 Shown is the transmission electron microscopy image of GO@PBNP.

[0041] Figure 5 Shown is a colorimetric picture of a dual-functional immunochromatographic test strip with GO@PBNP as a beacon carrier for detecting clenbuterol.

[0042] Figure 6 Shown is a photothermal image of a dual-functional immunochromatographic test strip using GO@PBNP as a beacon carrier to detect clenbuterol.

[0043] Figure 7 Shown is a colorimetric image of the traditional colloidal gold test strip for detecting clenbuterol.

[0044] Figure 8 Shown is the actual colorimetric picture of the dual-functional immunochromatographic test strip using GO@PBNP as the beacon carrier to detect carbofuran.

[0045] Fig. 9 Shown is a photothermal image of a dual-functional immunochromatographic test strip using GO@PBNP as a beacon carrier to detect carbofuran.

[0046] Fig.10 Shown is a colorimetric picture of the dual-functional immunochromatographic test strip with GO@PBNP as the beacon carrier for detecting Pseudomonas aeruginosa.

[0047] Fig.11 Shown is a photothermal image of a dual-functional immunochromatographic test strip with GO@PBNP as the beacon carrier for detecting Pseudomonas aeruginosa. DETAILED DESCRIPTION

[0048] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0049] An embodiment of the present invention provides a method for preparing a nanomaterial based on graphene oxide in situ grown Prussian blue (GO@PBNP), comprising the following steps: pre-treating a single layer of graphene oxide and uniformly mixing it with polyvinyl pyrrolidone, dispersing it in hydrochloric acid together with potassium ferrocyanide and uniformly stirring it, vigorously stirring the polyvinyl pyrrolidone and then reacting to grow Prussian blue particles under heating conditions, centrifuging to obtain a precipitate, washing it, and then redissolving it in pure water to obtain the graphene in situ grown Prussian blue nanomaterial.

[0050] In the above preparation method, the final concentrations of monolayer graphene oxide and potassium ferrocyanide after being dissolved in the solvent are 2 mg / mL and 1.65 mg / mL, respectively. The pretreatment of monolayer graphene oxide is carried out according to the following steps: the monolayer graphene oxide dispersion is ultrasonically treated, centrifuged, and the precipitate is collected and resuspended in water to obtain pretreated monolayer graphene oxide; the thickness of the monolayer graphene oxide in the monolayer graphene oxide dispersion is 0.8~1.2 nm; the sheet diameter is less than 500 nm; the power of the ultrasonic treatment is 500~1000 W; the ultrasonic time is 15 min; the speed of the centrifugation is 10000 r / min. The molecular weight of the polyvinyl pyrrolidone used for the first time is 10000, and the molecular weight of the polyvinyl pyrrolidone used for the second time is 40000. The conditions of the centrifugation include: the speed is 3000~12000 r / min, and the time is 10~30 min. The heating temperature is 80 ℃ and the time is 20 h.

[0051] Based on the above-prepared graphene oxide in-situ grown Prussian blue nanomaterial, another embodiment of the present invention further provides a dual-function immunochromatographic test strip, which includes a glass fiber mat sprayed with a GO@PBNP labeled antibody complex.

[0052] The preparation steps of the glass fiber mat sprayed with the GO@PBNP labeled antibody complex are as follows: adding the antibody to be labeled to the prepared GO@PBNP, adding the blocking agent after sufficient mixing, reacting at room temperature, centrifuging to obtain the precipitate, re-dissolving the obtained precipitate to prepare the GO@PBNP labeled antibody complex; spraying the prepared GO@PBNP labeled antibody complex onto the glass fiber mat at a volume of 8 μL / cm, vacuum drying for 2 h, and storing it in a drying cabinet for later use.

[0053] In some embodiments, the GO@PBNP is ultrasonicated for 5 min, and then the concentration is adjusted with a 0.02 M borate buffer with a pH of 5.0 to 9.0. The antibody labeling concentration is 10 μg / mL to 100 μg / mL, and the concentration of the blocking agent after the blocking agent is added is 1% to 10%, and the blocking agent is any one of bovine serum albumin, casein, ovalbumin, and skim milk.

[0054] In some embodiments, the antibody to be labeled in the glass fiber mat of the GO@PBNP labeled antibody complex comprises a monoclonal antibody, a polyclonal antibody, a nanobody or a phage-expressed antibody.

[0055] In some embodiments, the room temperature blocking time is 1 h, the centrifugal speed is 8000-10000 r / min, and the time is 10-20 min.

[0056] In some embodiments, the precipitate after centrifugation is re-dissolved with 0.01M pH 7.4 phosphate buffer (PBS) to 1 / 10 of the initial volume to obtain a GO@PBNP labeled antibody complex, which is then stored at 4°C for later use.

[0057] In some embodiments, the bifunctional immunochromatographic test strip comprises a bottom plate, a sample pad, a glass fiber pad sprayed with a GO@PBNP labeled antibody complex, a nitrocellulose membrane and absorbent paper sprayed with an artificial antigen coupled to the test object as a detection line and a sheep anti-mouse antibody as a quality control line.

[0058] The nitrocellulose membrane of the present invention is coated with an artificially coupled antigen or antibody of the object to be detected as a detection line, and is coated with a goat anti-mouse antibody (secondary antibody) as a quality control line; wherein the preparation method of the nitrocellulose membrane comprises the following steps:

[0059] (1) Use 0.01-0.5 M PBS (pH 6.0-8.0) solution to adjust the concentration of the coated object, the analyte artificial conjugated antigen or the analyte antibody, and the goat anti-mouse antibody to 0.01-10.0 mg / mL respectively;

[0060] (2) Spray the artificially conjugated antigen or antibody of the analyte after adjusting the concentration onto the upper part of the nitrocellulose membrane as the test line, and spray the goat anti-mouse antibody onto the lower part of the nitrocellulose membrane as the quality control line. The test line and the quality control line are separated by a certain distance, and the spray volume of both is 0.25-0.74 μL / cm;

[0061] (3) Dry the nitrocellulose membrane sprayed with the test line and quality control line at 37°C overnight and store it in a dry environment at room temperature for future use.

[0062] In some embodiments, the assembly of a bifunctional immunochromatographic test strip prepared with GO@PBNP as a beacon carrier is performed by overlapping and pasting the following materials on the bottom plate: a sample pad, a glass fiber pad sprayed with a GO@PBNP-labeled antibody complex, a nitrocellulose membrane and absorbent paper sprayed with an artificial antigen or antibody coupled to the test object as a detection line and a sheep anti-mouse antibody as a quality control line, thereby assembling the bifunctional immunochromatographic test strip prepared with GO@PBNP as a beacon carrier of the present invention.

[0063] Another embodiment of the present invention provides a detection process of a dual-function immunochromatographic test strip prepared using GO@PBNP as a beacon carrier, comprising:

[0064] The sample to be tested is added to the bifunctional immunochromatographic test strip prepared with GO@PBNP as the beacon carrier. The reaction time is 3 to 15 minutes. The concentration of the test sample is calculated by reading the grayscale of the test strip and the imaging data of the infrared imager to achieve quantitative detection, or the qualitative judgment of the test sample can be achieved by observing the test line and the quality control line with the naked eye to see if there are colored bands.

[0065] Graphene oxide has a two-dimensional crystal structure, rich functional groups on the surface, good water solubility and biocompatibility. It can be used as an effective carrier to combine with other nanomaterials to form a composite structure with improved performance. It is precisely because the preparation of graphene oxide is relatively simple, the output is large, the price is low, the raw material graphite for synthesis is also abundant, and it can be combined with metals, inorganic non-metals, polymers and other materials to synthesize composite materials with better performance. Therefore, graphene oxide has become one of the nanomaterials with the most extensive application prospects. Previous studies have reported that graphene oxide materials are very suitable for the construction of biosensors due to their outstanding electronic properties such as photoluminescence quenching ability, good electrical conductivity and huge surface area. Prussian blue nanoparticles are a metal-organic framework coordinated by trivalent iron and ferrocyanide. They have a cubic structure, strong absorption in the near-infrared (NIR) region (700~900 nm), and stable photothermal conversion performance. The synthesis of Prussian blue nanoparticles is easy to control. By regulating the precursor substances and controlling the reaction conditions, PBNPs of different target sizes can be synthesized.

[0066] Based on the above situation, the inventors used graphene oxide as a carrier to in situ grow Prussian blue nanoparticles to synthesize a new type of nanomaterial (GO@PBNP), which is applied to the fields of medical testing and rapid food safety detection and improves the stability and detection sensitivity of the test strip. The dual-function immunochromatographic test strip prepared with graphene oxide in situ grown Prussian blue nanomaterial as a beacon carrier has higher sensitivity than the traditional colloidal gold test strip; compared with the traditional nanoprobe, it has a larger surface area, better dispersibility and stability, and higher signal value, thereby greatly improving the detection sensitivity of the test strip. The dual-function immunochromatographic test strip made of a bottom plate, a sample pad, a glass fiber pad sprayed with a GO@PBNP labeled antibody complex, a nitrocellulose membrane sprayed with a detection line and a quality control line, and a water-absorbing paper can be used to quantitatively detect the concentration of the analyte in the sample. The dual-function immunochromatographic test strip based on graphene oxide in situ grown Prussian blue nanomaterial has the characteristics of high stability, high sensitivity, and short detection time. At present, there is no report on the application of GO@PBNP with dual colorimetric and photothermal functional signals as a nanoprobe in immunochromatography.

[0067] The present invention is further described in detail below with specific examples, but the present invention is not limited to the following specific examples.

[0068] Example 1: Preparation of GO@PBNP

[0069] 1. Solution preparation: 0.01 M hydrochloric acid;

[0070] 2. Pretreatment of monolayer graphene oxide (GO): 5 mL GO (2 mg / mL) was thoroughly mixed with 5 mL deionized water, ultrasonicated for 10 min, centrifuged at 10000 r / min for 10 min, discarded the supernatant, and resuspended in 5 mL deionized water.

[0071] 3. Reaction: Dissolve 5 mL of pretreated GO (2 mg / mL) in 15 mL of 10 mg / mL polyvinyl pyrrolidone (PVP, Mw=10000) solution and stir evenly at room temperature for 24 h. Centrifuge at 12000 r / min for 20 min to remove unbound PVP, resuspend in 30 mL of 0.01 M hydrochloric acid, add 6 mL of 10 mg / mL potassium ferrocyanide solution, and stir evenly for 12 h. Under vigorous stirring, add 10 mL of 0.2 g / mL polyvinyl pyrrolidone (PVP, Mw=40000) solution and react at 80 ℃ for 15 h.

[0072] 4. Washing: After the reaction is completed, centrifuge (speed is 12000 r / min, time is 15 min) to take the precipitate, wash the precipitate and then dissolve it in deionized water to obtain GO@PBNP, which is then stored at 4 ℃.

[0073] Figure 2 Shown are the UV absorption spectra of GO and GO@PBNP; Figure 4 Shown are transmission electron microscopy images of GO@PBNP, which respectively demonstrate the ultraviolet absorption characteristics and morphological characteristics of GO@PBNP.

[0074] Example 2: Preparation of a dual-function immunochromatographic test strip for detecting clenbuterol using GO@PBNP as a beacon carrier

[0075] 1. Preparation process of dual-function immunochromatographic test strips

[0076] The dual-function immunochromatographic test strip comprises a PVC base plate, and a sample pad, a glass fiber pad, a nitrocellulose membrane and absorbent paper which are overlapped and pasted on the base plate at one time.

[0077] 1. Preparation of nitrocellulose membrane:

[0078] Clenbuterol whole antigen and goat anti-mouse antibody were coated on nitrocellulose membrane: the concentration of clenbuterol whole antigen was diluted to 1 mg / mL with 0.01 M PBS, pH 7.4, and the resulting solution was sprayed on the membrane as the test line; the concentration of goat anti-mouse antibody was diluted to 0.5 mg / mL, and the resulting solution was sprayed on the membrane as the quality control line. The spray volume of both lines was 0.74 μL / cm, the detection line was 10 mm away from the top edge of the membrane, and the interval between the two lines was 5 mm. The membrane was dried at 37°C for 12 h and stored in a drying cabinet for future use.

[0079] 2. Preparation of glass fiber mat sprayed with GO@PBNP labeled antibody complex:

[0080] Take 50 μL of GO@PBNP prepared in Example 1 and ultrasonicate for 5 minutes, mix with 450 μL of 0.02 M borate buffer at pH 8.0, shake and mix, add 2.5 μg of clenbuterol monoclonal antibody, mix thoroughly, shake for 3 h, add 50 μL of bovine serum albumin with a final concentration of 10%, block at room temperature for 1 h, centrifuge at 8000 r / min for 20 min, and re-dissolve the precipitate with 0.01M pH 7.4 phosphate buffer (PBS) to 1 / 10 of the initial volume. Spray on the glass fiber mat at a volume of 8 μL / cm, vacuum dry for 2 h, and store in a drying cabinet for later use.

[0081] Figure 3Shown are the particle sizes of GO, GO@PBNP, and GO@PBNP labeled clenbuterol antibody complex (GO@PBNP@mAb); it shows that the particle sizes of the three gradually increase.

[0082] 3. Assemble the test strips:

[0083] ⑴ The sample pad size is 1×30 cm;

[0084] ⑵ Glass fiber mat sprayed with GO@PBNP labeled antibody complex, with specifications of 0.8×30 cm;

[0085] ⑶ Nitrocellulose membrane sprayed with test lines and quality control lines, with specifications of 2.5×30 cm;

[0086] (4) Absorbent paper, size: 1.2×30 cm;

[0087] ⑸ PVC base plate, specifications are 5.5×30 cm.

[0088] like Figure 1 As shown in the figure, the composition of the dual-function immunochromatographic test strip is as follows: on the PVC base plate, a nitrocellulose membrane sprayed with a detection line and a quality control line, a glass fiber pad sprayed with a GO@PBNP labeled antibody complex, a sample pad and absorbent paper are sequentially overlapped and pasted. After pasting, it is cut into 4×55 mm test strips by a strip cutter, loaded into a plastic card shell, compressed and loaded into an aluminum foil bag, and after adding a desiccant, it is sealed and stored. The shelf life at room temperature is 12 months.

[0089] 2. Establishment of the standard curve of clenbuterol dual-function immunochromatographic test strip based on GO@PBNP as beacon carrier:

[0090] Prepare the standard curve of the test strips: spike clenbuterol into the negative matrix so that its concentrations in the negative matrix are: 0, 0.001, 0.01, 0.1, 1, 10, 100 ng / mL.

[0091] The clenbuterol content in the above samples was detected using the above-assembled dual-function immunochromatographic test strips, and the detection method was as follows:

[0092] The above samples were added to the sample pads respectively, and the reaction time was 10 minutes.

[0093] As a control, the above samples were added to traditional colloidal gold test strips, and the reaction time was 10 minutes.

[0094] Figure 5 , Figure 6 The figure shows a physical picture of the GO@PBNP dual-function immunochromatographic test strip of the present invention being applied to the detection of clenbuterol; Figure 5 , Figure 6 The GO@PBNP dual-function immunochromatographic test strip of the present invention was used to detect different concentrations of clenbuterol, and the detection line of the test strip showed different colorimetric signals and photothermal signals. As the concentration of clenbuterol increased, the colorimetric signal gradually weakened, and it was visible to the naked eye that there was an obvious inhibitory effect at 0.1 ng / mL, and the line was completely eliminated at 1 ng / mL. The photothermal signal can effectively improve the sensitivity of the detection. Figure 7 The traditional colloidal gold test strips were used to detect different concentrations of clenbuterol. Under naked eye conditions, a significant inhibitory effect was observed at 1 ng / mL, and the line completely disappeared at 5 ng / mL. This indicates that compared with traditional colloidal gold test strips, the GO@PBNP-based dual-function immunochromatographic test strips are effective and have superior performance for detecting clenbuterol.

[0095] Example 3: Preparation of a dual-function immunochromatographic test strip for detecting carbofuran using GO@PBNP as a beacon carrier

[0096] 1. Preparation process of dual-function immunochromatographic test strips

[0097] The dual-function immunochromatographic test strip comprises a PVC base plate, and a sample pad, a glass fiber pad, a nitrocellulose membrane and absorbent paper which are overlapped and pasted on the base plate at one time.

[0098] 1. Preparation of nitrocellulose membrane;

[0099] The whole carbofuran antigen and goat anti-mouse antibody were coated on the nitrocellulose membrane: the concentration of the whole carbofuran antigen was diluted to 1 mg / mL with 0.01 M PBS, pH 7.4, and the resulting solution was sprayed on the membrane as the test line; the concentration of the goat anti-mouse antibody was diluted to 0.5 mg / mL, and the resulting solution was sprayed on the membrane as the quality control line. The spray volume of the two lines was 0.74 μL / cm, the detection line was 10 mm away from the top edge of the membrane, and the interval between the two lines was 5 mm. The membrane was dried at 37°C for 12 h and stored in a drying cabinet for future use.

[0100] 2. Preparation of glass fiber mat sprayed with GO@PBNP labeled antibody complex:

[0101] Take 50 μL of GO@PBNP prepared in Example 1 and ultrasonicate for 5 minutes, mix with 450 μL of 0.02 M borate buffer at pH 6.0, oscillate and mix, add 2.5 μg of carbofuran monoclonal antibody, mix thoroughly, shake for 3 h, add 50 μL of bovine serum albumin with a final concentration of 10%, block at room temperature for 1 h, centrifuge at 8000 r / min for 20 min, and re-dissolve the precipitate with 0.01M phosphate buffer (PBS) at pH 7.4 to 1 / 10 of the initial volume. Spray on the glass fiber mat at a volume of 8 μL / cm, vacuum dry for 2 h, and store in a drying cabinet for later use.

[0102] 3. Assemble the test strips:

[0103] ⑴ The sample pad size is 1×30 cm;

[0104] ⑵ Glass fiber mat sprayed with GO@PBNP labeled antibody complex, with specifications of 0.8×30 cm;

[0105] ⑶ Nitrocellulose membrane sprayed with test lines and quality control lines, with specifications of 2.5×30 cm;

[0106] (4) Absorbent paper, size: 1.2×30 cm;

[0107] ⑸ PVC base plate, specifications are 5.5×30 cm.

[0108] Paste the above materials in sequence according to the positions of the components in the test strip structure diagram. After assembly, use a cutter to cut into 4×55 mm test strips, put them into a plastic card shell, press them tightly and put them into an aluminum foil bag. After adding desiccant, seal and store. The shelf life at room temperature is 12 months.

[0109] 2. Establishment of the standard curve of the carbofuran dual-function immunochromatographic test strip based on GO@PBNP as the beacon carrier:

[0110] Prepare a standard curve for the test strips: add carbofuran to the negative matrix so that its concentrations in the negative matrix are: 0, 0.01, 0.1, 1, 5, 10, 50, 100, 500 ng / mL.

[0111] The content of carbofuran in the above samples was detected using the above-assembled dual-function immunochromatographic test strips, and the detection method was as follows:

[0112] The above samples were added to the sample pads respectively, and the reaction time was 10 minutes.

[0113] Figure 8 , Fig. 9The figure shows the physical picture of the GO@PBNP dual-function immunochromatographic test strip of the present invention applied to the detection of carbofuran; it shows that the GO@PBNP dual-function immunochromatographic test strip of the present invention detects carbofuran of different concentrations, and the detection line of the test strip presents different colorimetric and photothermal signals. As the concentration of carbofuran increases, the colorimetric and photothermal signals gradually weaken. It can be seen with the naked eye that there is an obvious inhibitory effect at 1 ng / mL, and the line has completely disappeared at 50 ng / mL, indicating that the dual-function immunochromatographic test strip based on GO@PBNP is effective in detecting carbofuran.

[0114] Example 4: Preparation of a double-antibody sandwich bifunctional immunochromatographic test strip using GO@PBNP as a beacon carrier to detect Pseudomonas aeruginosa

[0115] 1. Preparation process of dual-function immunochromatographic test strips

[0116] 1. Preparation of test line and quality control line on nitrocellulose membrane:

[0117] Pseudomonas aeruginosa monoclonal antibody and goat anti-mouse antibody were coated on nitrocellulose membrane: the concentration of Pseudomonas aeruginosa monoclonal antibody was diluted to 2 mg / mL with 0.01 M PBS, pH 7.5, and the resulting solution was sprayed on the membrane as the test line; the concentration of goat anti-mouse antibody was diluted to 0.5 mg / mL, and the resulting solution was sprayed on the membrane as the quality control line. The spray volume of both lines was 0.74 μL / cm, the detection line was 10 mm away from the top edge of the membrane, and the interval between the two lines was 5 mm. Dry at 37 °C for 12 h and store in a drying cabinet for later use.

[0118] 2. Preparation of glass fiber mat sprayed with GO@PBNP labeled antibody complex:

[0119] Take 50 μL of GO@PBNP prepared in Example 1 and ultrasonically mix for 5 minutes with 450 μL of 0.02 M borate buffer at pH 8.0, oscillate and mix, add 10 μg of Pseudomonas aeruginosa polyclonal antibody, mix thoroughly, shake for 2 h, add 50 μL of casein with a final concentration of 1%, block at room temperature for 1.5 h, centrifuge at 8000 r / min for 20 min, and re-dissolve the precipitate with 0.01M phosphate buffer (PBS) at pH 7.4 to 1 / 10 of the initial volume. Spray it onto the glass fiber mat at a volume of 8 μL / cm, vacuum dry for 2 h, and store it in a drying cabinet for later use.

[0120] 3. Assemble the test strips:

[0121] ⑴ The specifications of filter paper and sample pad are 1×30 cm;

[0122] ⑵ Glass fiber mat sprayed with GO@PBNP labeled antibody complex, with specifications of 0.8×30 cm;

[0123] ⑶ Nitrocellulose membrane sprayed with test lines and quality control lines, with specifications of 2.5×30 cm;

[0124] (4) Absorbent paper, size: 1.2×30 cm;

[0125] ⑸ PVC base plate, specifications are 5.5×30 cm.

[0126] Paste the above materials in sequence according to the positions of the components in the test strip structure diagram. After assembly, use a cutter to cut into 4×55 mm test strips, put them into a plastic card shell, press them tightly and put them into an aluminum foil bag. After adding desiccant, seal and store. The shelf life at room temperature is 12 months.

[0127] 2. Establishment of the standard curve of Pseudomonas aeruginosa dual-function immunochromatographic test strip based on GO@PBNP as beacon carrier:

[0128] Prepare the standard curve of the test strip: add carbofuran to the negative matrix to make the concentrations in the negative matrix: 0, 10 3 , 2×10 3 , 5×10 3 , 10 4 , 2×10 4 , 5×10 4 , 10 5 , 2×10 5 , 5×10 5 CFU / mL.

[0129] The content of Pseudomonas aeruginosa in the above samples was detected using the above-assembled dual-function immunochromatographic test strip, and the detection method was as follows:

[0130] The above samples were added to the sample pads respectively, and the reaction time was 10 minutes.

[0131] Fig.10 and Fig.11 The figure shows the physical picture of the GO@PBNP dual-function immunochromatographic test strip of the present invention used to detect Pseudomonas aeruginosa; it shows that the GO@PBNP dual-function immunochromatographic test strip of the present invention detects different concentrations of Pseudomonas aeruginosa, and the test line of the test strip presents different colorimetric and photothermal signals. As the concentration of Pseudomonas aeruginosa increases, the colorimetric and photothermal signals gradually increase, and the naked eye can see that the concentration is 2×10 3 CFU / mL, an obvious signal appeared, indicating that the GO@PBNP-based dual-function immunochromatographic test strip was effective in detecting Pseudomonas aeruginosa.

[0132] In summary, the method provided by the present invention successfully prepared a GO@PBNP dual-function immunochromatographic test strip, which can be widely used for sensitive detection of antigens or antibodies. The above is only a preferred implementation of the present invention. The present invention is not limited to the above implementation. According to the principle of the present invention, this application includes any changes, uses or improvements of the present invention, including changes that are out of the scope disclosed in this application and are made using conventional techniques known in the art, which should fall within the scope of protection of the present invention.

[0133] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A dual-function immunochromatographic test strip, characterized in that: The dual-function immunochromatographic test strip includes a GO@PBNP labeled antibody complex; The method for preparing the GO@PBNP labeled antibody complex comprises the following steps: S1, uniformly mixing the monolayer graphene oxide solution and polyvinyl pyrrolidone, and uniformly dispersing the mixture in hydrochloric acid together with the potassium ferrocyanide solution to obtain a dispersion; S2, continue to add polyvinyl pyrrolidone to the dispersion and mix evenly, react and grow Prussian blue particles under heating conditions, centrifuge to obtain a precipitate, wash it, and then redissolve it in pure water to obtain GO@PBNP; S3, adding the antibody to be labeled to the GO@PBNP, adding a blocking agent after sufficient mixing, centrifuging to obtain a precipitate after the reaction, and re-dissolving the obtained precipitate to prepare a GO@PBNP labeled antibody complex; In step S1, the concentration of the monolayer graphene oxide solution is 1.5 mg / mL~2.5 mg / mL, the molecular weight of polyvinyl pyrrolidone is 10000, the concentration of polyvinyl pyrrolidone is 8 mg / mL~12 mg / mL, the concentration of hydrochloric acid is 0.008 M~0.012 M, the concentration of potassium ferrocyanide solution is 8 mg / mL~12 mg / mL, and the added volume ratio of the monolayer graphene oxide solution, polyvinyl pyrrolidone, hydrochloric acid and potassium ferrocyanide solution is 4~6:12~18:25~35:5~7; In step S2, the molecular weight of polyvinyl pyrrolidone is 40000, the concentration of polyvinyl pyrrolidone is 0.15 g / mL~0.25 g / mL, the added volume of polyvinyl pyrrolidone is 1.5 times~2.5 times the volume of the monolayer graphene oxide solution, the heating temperature is 70 ℃~90 ℃, and the heating time is 18 h~22 h; the centrifugal conditions include: the speed is 3000 r / min~12000 r / min, and the time is 10 min~30 min.

2. The dual-function immunochromatographic test strip according to claim 1, characterized in that: In step S1, the method for preparing a single-layer graphene oxide solution comprises the following steps: The monolayer graphene oxide dispersion is ultrasonically treated, centrifuged, and the precipitate is collected and resuspended in water to obtain a monolayer graphene oxide solution; The thickness of the single-layer graphene oxide in the single-layer graphene oxide dispersion is 0.8 nm to 1.2 nm; the flake diameter is less than 500 nm; the power of the ultrasonic treatment is 500 W to 1000 W; the ultrasonic time is 10 min to 20 min; and the centrifugal speed is 8000 r / min to 12000 r / min.

3. The dual-function immunochromatographic test strip according to claim 1, characterized in that: Step S3 includes the following specific steps: The GO@PBNP was ultrasonicated for 4 min to 6 min, a 0.015 M to 0.025 M borate buffer with a pH of 5.0 to 9.0 was added, and then the antibody to be labeled was added, and a blocking agent was added after sufficient mixing, and the mixture was blocked at room temperature for 0.5 h to 1.5 h. After the reaction, the precipitate was centrifuged and the obtained precipitate was redissolved with a 0.008 M to 0.012 M phosphate buffer with a pH of 7.4 to obtain a GO@PBNP labeled antibody complex; Among them, the concentration of the antibody to be labeled is 10μg / mL~100μg / mL, the concentration of the blocking agent is 1%~10%, and the blocking agent is any one or more of bovine serum albumin, casein, ovalbumin, and skim milk; the centrifugal speed is 8000 r / min~10000 r / min, and the time is 10 min~20 min; the volume of the solution after reconstitution is 1 / 20~1 / 5 of the initial volume.

4. The dual-function immunochromatographic test strip according to claim 3, characterized in that: The antibodies to be labeled include monoclonal antibodies, polyclonal antibodies, nano antibodies, and phage-expressed antibodies.

5. The dual-function immunochromatographic test strip according to claim 1, characterized in that: The dual-function immunochromatographic test strip comprises: Base plate; A nitrocellulose membrane is arranged on the bottom plate, and the nitrocellulose membrane has detection lines and quality control lines arranged at intervals; A glass fiber mat is arranged at one end of the nitrocellulose membrane having a detection line, one end of the glass fiber mat is arranged on the bottom plate, and the other end is arranged on the nitrocellulose membrane, and the GO@PBNP labeled antibody complex is arranged on the glass fiber mat; Absorbent paper is arranged at one end of the nitrocellulose membrane having a quality control line, one end of the absorbent paper is arranged on the bottom plate, and the other end is arranged on the nitrocellulose membrane; A sample pad has one end arranged on the bottom plate and the other end arranged on the glass fiber pad.

6. The dual-function immunochromatographic test strip according to claim 5, characterized in that: The method of placing the GO@PBNP labeled antibody complex on the glass fiber mat comprises: The GO@PBNP labeled antibody complex was sprayed onto the glass fiber mat at a volume of 7 μL / cm~9 μL / cm and vacuum dried for 1.5 h~2.5 h.

7. The dual-function immunochromatographic test strip according to claim 5, characterized in that: The test line is coated with artificially coupled antigen or antibody to be tested, and the quality control line is coated with goat anti-mouse antibody; The method for forming a detection line and a quality control line on the nitrocellulose membrane comprises the following steps: Use 0.01 M to 0.5 M, pH 6.0 to 8.0 PBS solution to adjust the concentration of the coated artificial conjugated antigen or antibody to be detected and goat anti-mouse antibody to 0.01 mg / mL to 10.0 mg / mL; wherein the antibody to be detected includes monoclonal antibody, polyclonal antibody, nano antibody, phage-expressed antibody; The artificially coupled antigen or antibody to be detected is sprayed on the upper part of the nitrocellulose membrane as a detection line, and the goat anti-mouse antibody is sprayed on the lower part of the nitrocellulose membrane as a quality control line; wherein the spraying amount of the artificially coupled antigen or antibody to be detected and the goat anti-mouse antibody is 0.25 μL / cm~0.74 μL / cm; The nitrocellulose membrane sprayed with the test line and the quality control line is dried.

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