Nano-enzyme immunochromatography method and kit for detecting bovine casein

Abstract

The invention discloses a nano-enzyme immunochromatography method and a kit for detecting bovine casein, the kit comprises a test strip and a probe, a nitrocellulose membrane of the test strip is respectively provided with a T line and a C line, the T line is coated with an anti-casein rabbit polyclonal antibody, and the C line is coated with a rabbit anti-M13 antibody; the used probe is a manganese dioxide nano enzyme labeled anti-casein bacteriophage display nano antibody, the nano antibody is named as P19, the amino acid sequence of the nano antibody is shown as SEQ ID NO.1, and the nucleotide sequence for coding the amino acid is shown as SEQ ID NO.2. By utilizing the color of the manganese dioxide nano enzyme and the activity of peroxidase-like enzyme, a colorimetric signal can be amplified through the color development of a catalytic substrate, so that the detection sensitivity of the test strip is improved. The nano-enzyme immunochromatography method provided by the invention has the characteristics of simplicity in operation, low cost, good specificity and the like, and can be applied to detection of bovine casein in food.

Description

Technical Field

[0001] The invention belongs to the technical field of food safety detection, and specifically relates to a nanoenzyme immunochromatography method and a kit for detecting bovine milk casein. Background Art

[0002] Cow's milk is one of the eight major allergenic foods identified by the Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO). Cow's milk allergy may cause allergic reactions in the skin, intestines or respiratory system, leading to symptoms such as vomiting, erythema, urticaria, angioedema, lethargy or asthma, posing a potential threat to the life and health of consumers. Cow's milk protein mainly includes two categories: casein and whey protein. Casein accounts for 80% of the total protein content and is the main protein in cow's milk. It is also the most important allergen that causes cow's milk allergic reactions. Therefore, it is very necessary to effectively monitor and quickly detect cow's milk casein in food.

[0003] The main methods for detecting milk allergens include polymerase chain reaction technology, ultra-high performance liquid phase-tandem mass spectrometry, capillary electrophoresis analysis, etc. However, these methods usually require complex pretreatment, long operation time, high requirements for the environment and operation, and it is difficult to achieve rapid on-site detection. As a rapid detection method, immunochromatography has attracted great interest due to its simplicity, rapidity and high specificity. In recent years, it has been widely used in the detection of targets such as food allergens. Most of the immunochromatography methods reported so far are based on traditional antibodies, which have problems such as long preparation cycle, relatively cumbersome screening process, and large batch differences. Compared with traditional antibodies, nanoantibodies have the advantages of short preparation cycle, high stability, and easy large-scale production. Nanoantibodies can be obtained by constructing an M13 phage display library for panning. M13 phage-displayed nanoantibodies have both nanoantibody characteristics and M13 phage characteristics. They are easier to prepare and purify than protein-shaped antibodies. Their special structure is also conducive to loading labeled materials to achieve detection signal amplification. However, nanoantibodies against bovine casein have not been reported yet.

[0004] Nanozymes are a class of nanomaterials with a size of 1-100 nm that mimic enzyme properties. Compared with natural enzymes, they are highly stable, low-cost, and durable, and are expected to be used as direct substitutes for traditional enzymes. 2 ) Nanozymes have attracted wide attention due to their catalytic activity, biocompatibility and redox properties. The present invention provides an anti-bovine milk casein nanobody, using manganese dioxide nanozyme-labeled M13 phage-displayed nanobody as a probe to establish a simple, economical, sensitive and rapid nanozyme test strip that can be used for the detection of bovine milk casein in food. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and to provide a nanoenzyme immunochromatography method and a kit for detecting bovine casein.

[0006] To achieve the above object, the technical solution provided by the present invention is as follows:

[0007] According to a first aspect of the present invention, the present invention provides a nanoenzyme immunochromatography kit for detecting bovine casein, comprising a test strip and a probe, wherein the test strip comprises a bottom plate, a sample pad, a nitrocellulose membrane (NC membrane) and a water absorbent pad, wherein the water absorbent pad, the nitrocellulose membrane, the sample pad are sequentially pasted on the bottom plate from top to bottom along the length direction of the test strip, and adjacent connections overlap; characterized in that a T line and a C line are provided on the detection area on the nitrocellulose membrane, the T line is coated with an anti-casein rabbit polyclonal antibody, and the C line is coated with a rabbit anti-M13 antibody;

[0008] The probe is an anti-casein phage-displayed nanobody labeled with manganese dioxide nanozyme, the amino acid sequence of the nanobody is shown in SEQ ID NO.1, and the nucleotide sequence encoding the amino acid is shown in SEQ ID NO.2.

[0009] According to the second aspect of the present invention, the present invention provides a bovine milk casein immunochromatographic detection method based on the kit, which comprises the following steps:

[0010] The casein standard sample solutions of different concentrations were mixed with the probe as loading buffer and added to the sample pad of the test strip. After the color development was completed, the nitrocellulose membrane was cut and placed on TMB (3,3',5,5'-tetramethylbenzidine)-H 2 O 2 In the substrate mixed solution, after a certain period of catalytic reaction, a camera is used to take pictures and record the results, and the gray value of the T line is converted to gray value, and a standard curve is established with casein concentration as the horizontal axis and T line gray value as the vertical axis;

[0011] The sample to be tested is pre-treated to prepare a loading buffer solution, and the T-line gray value is obtained by the same detection method as the standard sample solution. The casein concentration in the sample to be tested is obtained by comparing the T-line gray value with the standard curve, thereby achieving quantitative detection of casein in the sample to be tested.

[0012] According to the third aspect of the present invention, the present invention provides a labeled probe for detecting bovine casein, which is an anti-casein phage-displayed nanobody labeled with manganese dioxide nanozyme, the amino acid sequence of the nanobody is shown in SEQ ID NO.1, and the nucleotide sequence encoding the amino acid is shown in SEQ ID NO.2.

[0013] According to a preferred embodiment of the present invention, the preparation method of the manganese dioxide nanozyme labeled probe is as follows: the phage display nanoantibody can be labeled by the manganese dioxide nanozyme through electrostatic adsorption. The 0.40 mg / mL manganese dioxide nanozyme solution is ultrasonicated for 30 min; an excess of anti-casein phage display nanoantibody is added, and the reaction is stirred at room temperature for 2 h; then 10% BSA-PBS is added, and the stirring reaction is continued for 30 min; the reactant is centrifuged at 12000 rpm for 10 min; finally, the precipitate obtained by centrifugation is resuspended in 100 μL, 0.01 M PBS to obtain a phage display nanoantibody probe labeled with manganese dioxide nanozyme.

[0014] According to a preferred embodiment of the present invention, the preparation method of the test strip is as follows: First, the sample pad is soaked in the blocking solution and dried at 37°C overnight. The NC membrane, the absorbent pad and the sample pad are sequentially attached to the PVC liner, overlapping by 1-2 mm. Then, the casein rabbit polyclonal antibody is diluted to 0.8-2.8 mg / mL with PBS, and the anti-M13 antibody is diluted to 0.04 mg / mL with PBS, and the T line and C line are drawn on the NC membrane at 0.5 μL / cm using a film drawing instrument, and the immunochromatographic test strip is obtained after drying at 37°C.

[0015] According to a preferred embodiment of the present invention, the pretreatment of the sample to be tested includes diluting the sample to be tested with 0.01 M PBS, mixing it with the probe labeled with manganese dioxide nanozyme and Tween-20 to obtain a loading buffer. Preferably, the volume percentage of the probe in the loading buffer is 1%, and the volume percentage of Tween-20 is 0.2%.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The nanozyme immunochromatographic test strip detection system provided by the present invention has a dual readout signal. The T line of the test strip appears brown based on the color of the manganese dioxide nanozyme itself. The peroxidase-like activity of the manganese dioxide nanozyme can also catalyze the oxidation of colorless TMB into blue ox-TMB, thereby superimposing the color to amplify the detection signal. The minimum detection limit of the test strip detection system is 24.26 ng / mL, and the linear detection range is 31.25-1000 ng / mL.

[0018] The bovine milk casein nanoenzyme immunochromatographic test strip based on nanoantibodies prepared by the present invention can be applied to the rapid detection of bovine milk casein, is easy to prepare, has good specificity, and is low in cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1The indirect ELISA identification results of nanobody-positive phage clones; (A) is the identification results of phage clone numbers P3-P29, (B) is the identification results of phage clone numbers P31-P48;

[0020] Figure 2 Results of the optimization of the reaction conditions of the nanozyme test strips; (A) is the optimization of the nanozyme concentration, (B) is the optimization of the T-line coated antibody concentration, (C) is the optimization of the probe loading amount, and (D) is the optimization of the Tween-20 concentration;

[0021] Figure 3 This is the optimization result of TMB substrate reaction time;

[0022] Figure 4 The optimization results of adding volume for TMB substrate;

[0023] Figure 5 This is the optimization result of pH value of TMB substrate;

[0024] Figure 6 The specific analysis results of the nanozyme test strips;

[0025] Figure 7 The test results of the test strips before and after catalysis;

[0026] Figure 8 This is the standard curve of the test strip before catalysis;

[0027] Fig. 9 This is the standard curve of the test strip after catalysis. DETAILED DESCRIPTION

[0028] The technical solution adopted by the present invention is described in full and in detail below, and the present invention is described in detail in conjunction with the embodiments. It should be noted that the described examples are only a part of the main embodiments of the present invention. All embodiments produced by technicians in this field without making creative achievements based on the embodiments of the present invention belong to the protection scope of the present invention.

[0029] Example 1 Selection and identification of anti-bovine milk casein nanobodies

[0030] 1. Affinity Panning of Anti-Bovine Casein Nanobodies

[0031] Dilute bovine milk casein with 0.01 M PBS to a final concentration of 30 μg / mL, add to 96 ELISA plates (100 μL / well) and coat overnight at 4°C; wash the plates with PBS, add 300 μL of 3 wt% OVA solution (using 3 wt% OVA and 2% Noprotein Blocking alternately), and block at 37°C for 2 h; after washing the plates, take 10 μL of phage library (library capacity is 2×1010 ) was mixed with 90 µL PBS and reacted at 37°C for 1 h; unbound phages were aspirated, the plate was washed 5 times with 0.1% PBST, and then washed 5 times with PBS and patted dry; 100 μL / well of Gly-HCl elution solution with pH 2.2 was added, and the plate was shaken for 8 min; it was immediately transferred to a 1.5 mL sterile centrifuge tube that had been pre-added with 1 M Tris-HCl (pH 8.0) neutralizing solution. 10 μL of the elution solution was taken out to measure the phage titer, and the remaining was amplified and used for the next round of amplification and panning. A total of four rounds of panning were performed, and the concentration of the coated casein was reduced to 25 μg / mL, 20 μg / mL, and 15 μg / mL, respectively, and the number of PBST washes was increased to 5, 10, and 20 times, respectively.

[0032] 2. Identification of positive phage clones

[0033] Dilute bovine casein to 0.1 μg / mL and 1 μg / mL with 0.01 M PBS, and coat the ELISA plate with 100 μL / well; add 50 μL of phage supernatant (add an equal amount of auxiliary phage as a negative control) and 50 μL of blocking solution, 100 μL / well, and react at 37℃ for 1 hour; wash the plate 5 times with PBST, add Anti-M13 antibody (HRP) (working concentration 1:30000), and react at 37℃ for 1 hour; wash the plate with PBST, add TMB colorimetric solution, and color at 37℃ for 15 min; add stop solution to stop the reaction, and measure the absorbance at 450 nm. The ratio (P / N) of the test sample to the negative control at OD450 is greater than 2.1, which can be judged as positive.

[0034] The results of indirect ELISA identification of positive clones are as follows Figure 1 As shown, the clones with positive identification results were sent to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing, and the nucleotide sequence of the positive clone P19 was shown in SEQ ID NO.2, and the amino acid sequence of P19 was shown in SEQ ID NO.1.

[0035] Example 2 Preparation of anti-bovine casein phage-displayed nanobodies

[0036] A single colony of the positive clone P19 was picked and inoculated into 5 mL of 2×YT / Amp liquid medium, and cultured overnight at 37°C and 220 rpm; the overnight culture was inoculated into 20 mL of 2×YT / Amp liquid medium at a ratio of 1%, cultured with shaking until the logarithmic phase, and M13KO7 helper phage (infection multiplicity cell:phage=1:20) was added, and the culture was allowed to stand at 37°C for 15 min, and cultured with shaking at 220 rpm for 35 min; the culture was centrifuged at 3000 rpm for 10 min at 4°C, and the precipitate was resuspended in an equal volume of 2×YT / Amp-Kan medium and cultured with shaking at 30°C overnight; the supernatant was collected by centrifugation, 1 / 5~1 / 6 volume of PEG-NaCl was added to the supernatant, mixed well, and allowed to stand at 4°C for 2 h; the phage precipitate was collected by centrifugation at 10000 rpm for 10 min, and resuspended in PBS to obtain the purified phage-displayed nanoantibody (i.e., anti-casein phage-displayed nanoantibody).

[0037] Example 3 Establishment of bovine milk casein nanoenzyme immunochromatographic test strip based on nanoantibodies

[0038] 1. Assembly of test strips

[0039] The test strip consists of four parts, including a sample pad, NC membrane, absorbent pad, and PVC backing. First, the sample pad was soaked in the blocking solution and dried at 37°C overnight. The NC membrane, absorbent pad, and sample pad were sequentially attached to the backing, overlapping by 1-2 mm. Then, the casein rabbit polyclonal antibody was diluted to 0.8-2.8 mg / mL with PBS, and the anti-M13 antibody was diluted to 0.04 mg / mL. The test line (T line) and the quality control line (C line) were drawn on the NC membrane using a film drawing instrument at 0.5 μL / cm, and the immunochromatographic test strip was obtained after drying at 37°C.

[0040] 2. MnO 2 Preparation of nanozyme-labeled probes

[0041] Anti-casein phage-displayed nanoantibodies can be electrostatically adsorbed onto MnO 2 Nanozyme labeling. 250 μL MnO 2 The nanozyme solution was added to 400 μL 0.01 M PBS and ultrasonically dispersed in an ice bath for 30 min; 250 μL of the anti-casein phage-displayed nanoantibody amplified and purified in Example 2 was added and stirred at room temperature for 2 h; then 100 μL 10% BSA-PBS was added and the stirring reaction was continued for 30 min to fully block the unbound sites; the reactants were centrifuged at 4°C and 12000 rpm for 10 min; finally, the precipitate obtained by centrifugation was resuspended in 0.01 M PBS to obtain MnO 2 Nanozyme-labeled phage-displayed nanoantibody probes.

[0042] 3. MnO 2 Optimization of reaction conditions of nanoenzyme immunochromatographic test strips

[0043] (1) MnO 2 Optimization of nanozyme concentration: different concentrations of MnO 2 Nanozyme (0.25, 0.30, 0.35, 0.40 and 0.45 mg / mL) coupled probe was used to detect casein, and the MnO 2 The optimal concentration of nanozymes. Figure 2 As shown in A, with the increase of MnO 2 As the concentration of nanozymes increases, the T line signal value gradually increases; when MnO 2 When the concentration of nanozyme is 0.40 mg / mL, the T line signal value is the strongest, and the T / C ratio is also closest to 1:1. 2 As the nanozyme concentration continued to increase, the T line intensity began to decrease. Therefore, it was determined that MnO 2 The optimal concentration of nanozyme was 0.40 mg / mL.

[0044] (2) Optimization of T-line coated antibody concentration: The bovine milk casein rabbit polyclonal antibody was diluted to 0.8 mg / mL, 1.2 mg / mL, 1.6 mg / mL, 2.0 mg / mL, 2.4 mg / mL and 2.8 mg / mL for detection. Figure 2 As shown in Figure B, when the concentration of casein rabbit polyclonal antibody was 2.0 mg / mL, the T line intensity was the highest. As the antibody concentration continued to increase, the T line intensity increased, but the corresponding negative control also had a band. Therefore, the optimal concentration of the T line coating antibody was determined to be 2.0 mg / mL.

[0045] (3) Optimization of probe loading amount: 0.25 μL, 0.50 μL, 1 μL, 2 μL, 3 μL, 4 μL and 5 μL of probe were added to 100 μL of chromatography fluid respectively to optimize the probe loading amount. Figure 2 As shown in Figure C, the intensity of the T line gradually increases with the increase of the probe concentration. 2 When the amount of nanozyme probe added was 1 μL, the T line intensity reached the maximum. Therefore, the optimal loading amount of the probe was determined to be 1 μL.

[0046] (4) Optimization of Tween-20 concentration in loading buffer: Add the optimal volume of probe, and add Tween-20 to the loading buffer at final concentrations (volume percentage) of 0.01%, 0.05%, 0.2%, 0.4% and 0.8%. When the Tween-20 concentration is in the range of 0.01%-0.05%, the chromatography speed and effect of the test strip are greatly reduced, which seriously affects the result judgment. When the concentration reaches 0.2%, the observable T line intensity no longer increases. Therefore, the optimal volume percentage of Tween-20 in the loading buffer is determined to be 0.2%.

[0047] 4. MnO 2 Optimization of catalytic conditions of nanoenzyme immunochromatographic test strips

[0048] (1) TMB-H 2 O 2 Substrate mixed solution (purchased from Bio-Tech Biotech Co., Ltd. (Shanghai, China)) Reaction time: using MnO 2 The peroxidase-like activity of the nanozyme catalyzes the TMB-H 2 O 2 The substrate is used to amplify the detection signal. TMB-H 2 O 2 The substrate reaction time was 1 min, 2 min, 3 min, 5 min and 8 min. Figure 3 As shown in the figure, with the increase of incubation time, the gray value of T line reached the highest at 3 min, so TMB-H 2 O 2 The substrate reaction time was determined to be 3 min.

[0049] (2) TMB-H 2 O 2 Substrate addition volume: set TMB-H 2 O 2 The volume of the substrate mixture solution was 200 μL, 300 μL, 400 μL, and 500 μL. Figure 4 As shown in the figure, when the amount added is 400 μL, the gray value of the T line is the highest and the T / C value is closest to 1:1. Therefore, TMB-H 2 O 2 The volume of substrate mixed solution added was 400 μL.

[0050] (3) TMB-H 2 O 2 Substrate pH value: The pH of the TMB substrate mixed solution was adjusted to 2, 3, 4, 5 and 6 respectively. Figure 5 As shown, the gray value of the T line is the highest when the pH is 3, so the optimal pH is 3.

[0051] 5. Test strip specificity experiment

[0052] In order to verify the specificity of the prepared test strip, other proteins (α-lactalbumin, β-lactoglobulin, bovine serum albumin and ovalbumin) were measured. The concentration of the target casein and other interfering proteins was 2 μg / mL, and PBS buffer solution was used as a negative control group. For the group with the target casein, the T line showed clear and obvious bands, while other proteins and the negative group did not show obvious bands on the T line of the test strip ( Figure 6 ). This shows that the test strip prepared by the present invention exhibits good specificity and can accurately distinguish the target protein from the interfering protein.

[0053] 6. Test strip sensitivity test

[0054] Casein was diluted with 0.01 M PBS to different concentration gradients (0-4000 ng / mL) and tested under optimal conditions. The results were then recorded using a camera, and the grayscale value was converted using ImageJ software. A relationship curve was drawn with casein concentration as the horizontal axis and the corresponding grayscale value as the vertical axis. The minimum detection limit (LOD value) was defined as the concentration value on the standard curve corresponding to the blank mean plus three times the standard deviation. The results are shown in Figure 7 As shown in the figure, the visual detection thresholds of the immunochromatographic test strips without TMB color development solution catalysis and with TMB color development solution catalysis were 125 ng / mL and 31.25 ng / mL, respectively. Figure 8 and Fig. 9 As shown, the LOD of the test strips before and after catalysis were 61.49 ng / mL and 24.26 ng / mL, respectively, and the linear detection ranges were 62.5-1000 ng / mL and 31.25-1000 ng / mL, respectively.

[0055] Example 4 Detection of casein content in food samples

[0056] To verify the accuracy of the prepared test strips, three food samples of infant formula, milk and chocolate were purchased from a local supermarket for spike recovery tests. Weigh 1 g of solid sample (add 10 mL PBS) or aspirate 1 mL of liquid sample (add 9 mL PBS), mix well and centrifuge for 10 minutes (12000 rpm, 4°C) to remove fat. 0 ng / mL, 250 ng / mL, 500 ng / mL and 1000 ng / mL of casein were added to the three samples, respectively, and then the casein content in the samples was detected. The results are shown in Table 1. The recovery rate of infant formula ranged from 97.05% to 106.86%, and the coefficient of variation (CV) ranged from 6.43% to 18.29%; the recovery rate of milk ranged from 87.67% to 110.97%, and the coefficient of variation ranged from 1.14% to 13.71%; the recovery rate of chocolate ranged from 86.28% to 110.91%, and the coefficient of variation ranged from 1.81% to 13.81%; the recovery rates and coefficients of variation of the three samples were all within a reasonable range.

[0057] Table 1 - Spike recovery test results

[0058]

[0059] Note: Milk powder is 5×10 5 times dilution, milk at 1×10 5 times dilution, chocolate at 1×10 2 n=3 means each reaction was repeated 3 times.

[0060] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the attached claims.

Claims

1. A nanoenzyme immunochromatography kit for detecting bovine casein, comprising a test strip and a probe, wherein the test strip comprises a bottom plate, a sample pad, a nitrocellulose membrane and a water-absorbing pad, wherein the water-absorbing pad, the nitrocellulose membrane and the sample pad are sequentially pasted on the bottom plate from top to bottom along the length direction of the test strip, and adjacent joints overlap; It is characterized in that The detection area on the nitrocellulose membrane is provided with a T line and a C line, the T line is coated with anti-casein rabbit polyclonal antibody, and the C line is coated with rabbit anti-M13 antibody; The probe is an anti-casein phage-displayed nanobody labeled with manganese dioxide nanozyme, the amino acid sequence of the nanobody is shown in SEQ ID NO.1, and the nucleotide sequence encoding the amino acid is shown in SEQ ID NO.

2.

2. The nanoenzyme immunochromatography kit for detecting bovine casein according to claim 1, characterized in that: The probe is prepared by the following method: ultrasonically treating the MnO2 nanozyme, adding anti-casein phage-displayed nanoantibodies, and stirring the reaction at room temperature; then adding BSA-PBS and continuing to stir the reaction; centrifuging the reactants and resuspending them in PBS to obtain the probe.

3. The nanoenzyme immunochromatography kit for detecting bovine casein according to claim 1, characterized in that: The test strip is prepared by the following method: a water-absorbing pad, a nitrocellulose membrane and a sample pad are sequentially attached to a backing plate from top to bottom along the length direction of the test strip, with adjacent joints overlapping by 1-2 mm; then, casein rabbit polyclonal antibody is diluted to 0.8-2.8 mg / mL with PBS, and rabbit anti-M13 antibody is diluted to 0.04 mg / mL with PBS, and a film drawing instrument is used to draw T lines and C lines on the nitrocellulose membrane at 0.5 μL / cm, respectively, and the test strip is obtained after drying at 37°C.

4. The nanoenzyme immunochromatography kit for detecting bovine casein according to claim 2, characterized in that: The concentration of the MnO2 nanozyme before adding the anti-casein phage-displayed nanoantibody was 0.40 mg / mL; the precipitate obtained by centrifugation of the reaction was resuspended in 100 μL, 0.01 M PBS for later use.

5. The nanoenzyme immunochromatography kit for detecting bovine casein according to claim 3, characterized in that: The anti-casein rabbit polyclonal antibody was diluted with PBS to a concentration of 2.0 mg / mL, and a T line was drawn on a nitrocellulose membrane at 0.5 μL / cm using a membrane stripper.

6. A bovine milk casein nanoenzyme immunochromatography detection method based on the kit according to claim 1, characterized in that: include: The casein standard sample solutions of different concentrations were mixed with the probe as loading buffer, and then dripped onto the sample pad of the test strip. After the color development was completed, the nitrocellulose membrane was cut and placed in the TMB-H2O2 substrate mixed solution. After the catalytic reaction lasted for a certain period of time, a camera was used to take pictures and record the results. The gray value of the T line was converted to gray value, and a standard curve was established with casein concentration as the horizontal axis and T line gray value as the vertical axis. The sample to be tested is pre-treated to prepare a loading buffer solution, and the T-line gray value is obtained by the same detection method as the standard sample solution. The casein concentration in the sample to be tested is obtained by comparing the T-line gray value with the standard curve, thereby achieving quantitative detection of casein in the sample to be tested.

7. The bovine milk casein nanoenzyme immunochromatography detection method according to claim 6, characterized in that: The volume of the loading buffer is 100 μL, of which the volume of the probe is 1 μL. The loading buffer also contains 0.2% by volume of Tween-20.

8. The bovine milk casein nanoenzyme immunochromatography detection method according to claim 6, characterized in that: The pH of the TMB-H2O2 substrate mixed solution was 3, the volume was 400 μL, and the catalytic reaction time was 3 min.

9. A labeled probe for detecting bovine casein, characterized in that: It is an anti-casein phage-displayed nanobody labeled with MnO2 nanozyme, the amino acid sequence of the nanobody is shown in SEQ ID NO.1, and the nucleotide sequence encoding the amino acid is shown in SEQ ID NO.2.

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