Raman detection method of macromolecular antigen

Abstract

The invention belongs to the technical field of antigen detection, and relates to a Raman detection method of a macromolecular antigen. The method comprises the following steps: adding a capture probe-a gold magnetic material labeled with an antibody and a recognition probe-an M13 bacteriophage combined with colloidal gold and Raman molecules into a to-be-detected sample solution for reaction, forming a'sandwich 'structure by the capture probe, a to-be-detected antigen and the recognition probe, separating by an external magnetic field, dispersing a solid in water, and detecting the sample solution. And measuring the Raman signal intensity of the dispersion liquid on a Raman spectrometer, and calculating the content of the macromolecular antigen in the to-be-measured sample liquid according to a fitting equation representing the relationship between the macromolecular antigen concentration and the Raman signal intensity. The detection method provided by the invention is high in sensitivity, the stability of the gold magnetic material is strong, the binding efficiency of the antibody-anti-M13 phage is high, and the operation is simpler.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of antigen detection, and relates to a Raman detection method for macromolecular antigens. BACKGROUND

[0002] Modern medical diagnosis and environmental monitoring have an increasing demand for high-sensitivity and high-specificity biomarker detection. Traditional immunodetection methods (such as ELISA) are limited by enzyme activity stability, cumbersome operation steps, and insufficient sensitivity (the detection limit is usually at the ng / mL level), and it is difficult to meet the detection needs of trace markers (such as early tumor markers or pathogens). Therefore, developing new signal amplification strategies and rapid detection technologies has become a research hotspot.

[0003] SERS technology can enhance Raman signals by 10 6 -10 14 times through the plasmonic resonance effect of noble metal nanostructures (such as gold / silver nanoparticles), and has the advantages of fingerprint pattern specificity, resistance to photobleaching, and potential for multiplex detection. However, the application of SERS in immunodetection faces the following bottlenecks: poor signal repeatability: uneven aggregation of nanoparticles leads to uncontrollable distribution of hot spots; background interference: non-specific adsorption of biomolecules in complex samples (such as serum) interferes with the signal; limited signal amplification: traditional antibody-enzyme labeling strategies are difficult to achieve multi-level amplification.

[0004] The introduction of magnetic nanoparticles (MNPs) can achieve rapid separation and enrichment of target objects through an external magnetic field, significantly reducing sample matrix interference, but existing magnetic immunosensing technology still relies on enzyme or fluorescent labeling, and the sensitivity is limited. M13 bacteriophage, as a genetically engineered viral vector, has the following characteristics: the surface can display multivalent peptide segments (such as targeting antibodies or biotin-binding proteins), enabling efficient coupling with target objects and signal probes; the one-dimensional fiber structure (about 880 nm long and 6 nm in diameter) can load a large number of Raman reporters (such as DTNB) and noble metal nanoparticles, forming a "signal amplifier"; good biocompatibility and stability, better than synthetic polymer carriers.

[0005] Current detection methods combining SERS and magnetic immunization (such as CN110208450A) mostly use antibody-labeled nanoparticles, with low signal amplification efficiency. Therefore, integrating the high loading capacity of M13 bacteriophage, the purification advantage of magnetic separation, and the ultra-high sensitivity of SERS is expected to break through the existing detection limit. SUMMARY

[0006] The present application aims to overcome the shortcomings of the prior art and provide a detection method for macromolecular antigens to solve the problem of low detection sensitivity of traditional enzyme-linked immunosorbent assay for macromolecular antigens.

[0007] To achieve the above technical purposes, the present application adopts the following technical solutions:

[0008] A detection method of macromolecular antigen, comprising the following steps:

[0009] The capture probe and the recognition probe are added into the sample solution to be detected, and reacted at 25-38℃ for 50-500 min; the capture probe and the recognition probe form a "sandwich" structure with the antigen to be detected; after separation by an external magnetic field, the solid is dispersed in water, and the Raman signal intensity of the dispersion liquid is measured on a Raman spectrometer; the content of the macromolecular antigen in the sample solution to be detected is calculated according to a fitting equation representing the relationship between the concentration of the macromolecular antigen and the Raman signal intensity;

[0010] The capture probe is a gold magnetic material labeled with an antibody, and is prepared by a method comprising the following steps: chitosan is reacted with carboxyl-activated magnetic beads to obtain chitosan-coated magnetic beads; the chitosan-coated magnetic beads are mixed with a gold growth solution to grow gold nanoparticles on the surface of the chitosan-coated magnetic beads, thereby obtaining MBs@CS@AuNPs; the MBs@CS@AuNPs are reacted with an antibody, and after magnetic separation, a blocking reaction is performed, and then magnetic separation is performed to obtain the capture probe MBs@CS@AuNPs@Ab.

[0011] The recognition probe is M13 bacteriophage combined with colloidal gold and Raman molecules, and is prepared by a method comprising the following steps:

[0012] A colloidal gold solution is prepared, wherein the particle size of the gold nanoparticles is 1-20 nm; a Raman signal molecule solution is added to the colloidal gold solution for reaction, and then M13 bacteriophage specifically recognizing the antigen to be detected is added for reaction; and the recognition probe is obtained by centrifugation.

[0013] The recognition probe is added to the sample solution to be detected in liquid form, and the capture probe is added in solid form.

[0014] The titer of the M13 bacteriophage specifically recognizing the antigen to be detected in the recognition probe is 1×10 7 ~ 1×10 11 pfu / mL, and the volume ratio of the recognition probe to the sample solution to be detected is 1-2:1. The mass ratio of the capture probe to the volume of the recognition probe is 1 μg:8-20 μL.

[0015] The preparation method of the capture probe is described in detail as follows:

[0016] The magnetic beads are composite microspheres formed by combining magnetic inorganic particles (such as Fe3O4) with organic polymers, and have carboxyl functional groups on the surface.

[0017] The magnetic beads are carboxyl-activated using a MES buffer with a pH of 5-6.5.

[0018] The chitosan reacts with the carboxyl-activated magnetic beads at a temperature of 10-40℃ for 10-60 min. The mass ratio of chitosan to magnetic beads is 10-100:1.

[0019] The chitosan-coated magnetic beads are mixed with a gold growth solution to react at a temperature of 10-40℃ for 10-120 min. The gold growth solution comprises a chloroauric acid solution and a trisodium citrate solution. The concentration of the chloroauric acid solution is 1-10 wt%, and the concentration of the trisodium citrate solution is 20-50 mmol / L. The volume ratio of the chloroauric acid solution to the trisodium citrate solution is 1:7-10.

[0020] The MBs@CS@AuNPs are reacted with the antibody at a temperature of 10-40℃ for 2-24 h.

[0021] The blocking reaction is carried out at a temperature of 10-40℃ for 30 min-5 h to block the non-specific sites of the gold nanoparticles. The blocking agent can be exemplified by bovine serum albumin (BSA).

[0022] The preparation method of the recognition probe is described in detail as follows:

[0023] The preparation of the colloidal gold solution comprises the following steps: mixing the chloroauric acid and the trisodium citrate, adding a sodium borohydride solution under stirring, and reacting in an ice bath for 10-70 min to obtain the colloidal gold solution.

[0024] The molar ratio of the chloroauric acid, the trisodium citrate, and the sodium borohydride is 1:0.9-1.1:5-15.

[0025] The gold nanoparticles in the colloidal gold solution have a particle size of 1-20 nm, preferably 1-10 nm.

[0026] The colloidal gold solution is added with a Raman signal molecule solution to react at a temperature of 10-40℃ for 5-30 h. The Raman signal molecule is at least one of 4-mercaptobenzoic acid (4-MBA), 4-mercaptopyridine, 4-mercaptophenylamine, 1,4-mercaptobenzene, 4-methylmercaptobenzene, rhodamine 6G, rhodamine B, Nile blue A, crystal violet, 4-mercaptophenylboric acid, 5,5-dithiobisphenol-(2-nitrobenzoic acid), or Nile blue A.

[0027] The M13 bacteriophage that specifically recognizes the antigen to be detected is added to react at a temperature of 10-40℃ for 1-5 h.

[0028] The M13 bacteriophage that specifically recognizes the antigen to be detected is prepared by a method comprising the following steps:

[0029] The antigen to be detected is added to the enzyme label hole, and coated overnight at 1-6 DEG C; the phage library is added to the enzyme label hole, and reacted at 30-39 DEG C for 10-100 min, the plate is washed, and the unbound phage is washed away; the blocking solution is added for blocking, the plate is washed, and the HRP enzyme-labeled anti-M13 phage monoclonal antibody solution is added to the hole, and reacted at 30-39 DEG C for 10-100 min, the plate is washed, tetramethyl benzidine color developing solution is added, color development is carried out at 30-39 DEG C for 5-15 min, then sulfuric acid solution is added to terminate the reaction, and the absorbance value at 450 nm is measured; the acid elution is added to the enzyme label hole with effective absorbance value, and M13 phage eluent specifically recognizing the antigen to be detected is obtained, and the neutralizing solution is used for neutralization until the solution pH value is neutral; then the obtained eluent is amplified, and M13 phage specifically recognizing the antigen to be detected is obtained.

[0030] The fitting equation representing the relationship between the concentration of the macromolecular antigen and the Raman signal intensity is obtained by the following method:

[0031] The capture probe and the recognition probe are added to 3-10 different concentrations of the antigen to be detected standard solution, and reacted at 25-38 DEG C for 50-500 min, the capture probe and the recognition probe form a "sandwich" sandwich structure with the antigen to be detected, after separation by an external magnetic field, the solid is dispersed in water, and the Raman signal intensity of the dispersion liquid is measured on a Raman spectrometer, the value of the Raman signal intensity is taken as the ordinate, and the concentration of the antigen to be detected is taken as the abscissa, a standard curve is established, and the fitting equation of the standard curve is obtained.

[0032] The antigen to be detected is a macromolecular antigen, which can be microorganisms, cancer markers, proteins, polysaccharides and other macromolecular antigens with multiple antigenic determinants. The microorganisms include bacteria, which can be listed as Listeria monocytogenes, Bacillus cereus, Salmonella, Enterobacter cloacae, Shigella, Enterobacter sakazakii, Vibrio parahaemolyticus, Staphylococcus aureus and the like.

[0033] The type of the antibody used is determined by the antigen to be detected, for example, if the antigen to be detected is Salmonella, the antibody is Salmonella antibody.

[0034] The principle schematic diagram of the application is as follows: Figure 1The application is shown as follows: the gold magnetic material labeled by the monoclonal antibody is used as a capture probe and a Raman signal enhancement substrate, the M13 bacteriophage capable of specifically recognizing the antigen is used as a Raman signal amplification carrier, and the M13 bacteriophage can be combined with the colloidal gold labeled by the Raman signal molecule after modification, thereby forming a recognition probe; when the macromolecular antigen exists in the sample to be detected, the capture probe, the macromolecular antigen and the recognition probe form a "sandwich" structure, the Raman signal intensity change is used as a signal output under the action of an external magnetic field, and the content of the macromolecular antigen in the sample to be detected is determined by using the Raman signal change: 1) when the concentration of the macromolecular antigen in the sample to be detected is extremely low or even zero, the capture antibody on the surface of the gold magnetic material cannot be combined with the target antigen, the M13 bacteriophage as the recognition probe and the signal amplifier cannot recognize the target macromolecular antigen, and the "sandwich" structure of the gold magnetic material-target antigen-M13 bacteriophage cannot be formed, and the Raman signal value of the solution is slightly increased or not increased compared with the blank value (the gold magnetic material labeled by the capture antibody); 2) when the concentration of the macromolecular antigen in the solution increases, the gold magnetic material labeled by the capture antibody, the M13 bacteriophage and the target antigen are combined to form the "sandwich" structure of the gold magnetic material-target antigen-M13 bacteriophage, and the proportion of the "sandwich" structure gradually increases, and the colloidal gold-Raman signal molecule is further added to be combined with the M13 bacteriophage in the "sandwich" structure, and the Raman signal value of the solution is greatly changed compared with the blank value (the gold magnetic material labeled by the capture antibody); 3) with the change of the concentration of the macromolecular antigen, the Raman signal of the solution increases linearly and is positively correlated with the concentration of the target, so that the quantitative and sensitive detection of the macromolecular antigen is realized.

[0035] Compared with the prior art, the application has the following beneficial effects:

[0036] (1) The present application proposes to use large size M13 bacteriophage to replace the traditional carrier protein coupled detection antibody complex as a signal recognition probe and Raman signal amplifier in the detection of macromolecular antigens. M13 bacteriophage has unique advantages in signal recognition and signal amplification. The signal recognition advantage of M13 bacteriophage lies in two aspects. First, the multivalent targeting binding ability. The surface of M13 bacteriophage can be genetically engineered to display a large number of targeting molecules, which significantly improves the capture efficiency of the target and reduces the false negative rate. Compared with the one-to-one binding of traditional monoclonal antibodies, the "one antibody multiple markers" characteristic of M13 bacteriophage can enhance the recognition ability of weak affinity targets. Second, high specificity and low background interference: The modification site of M13 bacteriophage coat protein (such as pVIII, pIII) is clear, the pIII protein of the M13 bacteriophage can express antibodies that can recognize antigens, and the pVIII protein can express gold binding peptide-GBP that can bind with colloidal gold. Through directed evolution screening of high-specificity binding peptides, non-specific adsorption is reduced, and cross-reactions are not easily triggered in complex samples. Ultra-large signal molecule loading capacity: The fiber structure of M13 bacteriophage about 880 nm long can provide a large specific surface area, and can load up to 2700 copies of Raman reporter molecules such as metal nanoparticles, forming a dense SERS "hot spot". Compared with traditional spherical carriers, this linear structure is more easily to achieve ordered arrangement of nanoparticles, enhancing the electromagnetic field coupling effect. Modular signal amplification design: The regular linear structure allows M13 bacteriophage to realize dual-function display, i.e. one end is connected to the target recognition molecule and the other end is modified with Raman reporter molecules and metal nanoparticles, realizing "recognition-signal" integration. Stable structure and programmability: M13 bacteriophage has higher tolerance to extreme conditions such as temperature, pH value and organic solvents than traditional enzyme-labeled antibodies, and can realize multi-modal signal output by inserting active peptide gene.

[0037] (2) The present application uses gold nanoparticles coated Fe3O4 to form magnetic composite nanoparticles (MBs@CS@AuNPs) as a new strategy for ultra-sensitive SERS detection. Through the combination of magnetic nanoparticles and gold nanoparticles, the analyte and substrate can be concentrated under the action of a magnetic field. Therefore, the SERS signal can be enhanced by the concentration effect, and the gold magnetic material can be easily dispersed and recovered by a magnetic field. In addition, the combination of magnetic separation and SERS detection has great prospects in food detection applications.

[0038] (3) The gold magnetic material of the present application can further enhance the signal of the Raman molecule when combined with the Raman molecule. On the other hand, the M13 bacteriophage combined with colloidal gold and Raman molecule can amplify the plasmon resonance signal. After the gold magnetic material coupled with antibody captures the macromolecular antigen, the M13 bacteriophage combined with colloidal gold and Raman molecule is added, and the gold nanoparticle modified bacteriophage terminal pIII protein can be combined with the macromolecular antigen to form a "sandwich" structure. Under the action of an external magnetic field, the macromolecular antigen is aggregated, and the filamentous bacteriophage is wound around the bacteria. The collision between the gold magnetic material and the gold nanoparticles on the surface of the bacteriophage can excite localized surface plasmon resonance, increase the probability of forming "hot spots", and cause the enhancement of electromagnetic field, thereby enhancing the Raman signal. In addition, the M13 bacteriophage selected as a signal probe from the antibody library has higher affinity to the antigen. Reducing the amount of antibody and the concentration of signal probe can improve the sensitivity of the Raman detection method. Compared with the traditional immunoassay method, the present application has the advantages of simple operation, no damage to the biological system, simultaneous separation and identification, wide detection range, high sensitivity, rapidness, accuracy, greenness, etc. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 The figure is a schematic diagram of the principle of the method of the present application;

[0040] Figure 2 The figure is a standard curve for rapid detection of E. coli O157:H7 based on gold magnetic material surface-enhanced Raman spectroscopy technology in Example 1;

[0041] Figure 3 The figure is a standard curve for rapid detection of Salmonella based on gold magnetic material surface-enhanced Raman spectroscopy technology in Example 2. DETAILED DESCRIPTION

[0042] In the description of the present application, unless otherwise specified, the numerical range "a~b" represents a shorthand representation of any real combination between a and b, and includes a and b. A plurality includes two, three, four, five or more.

[0043] The technical solutions of the present application will be further described and explained by specific examples and drawings. It should be understood that the specific examples described herein are only used to help understand the present application and are not used to limit the specific application. The drawings used herein are only used to better illustrate the disclosed content and do not limit the scope of protection. If not otherwise specified, the raw materials used in the examples of the present application are commonly used raw materials in the art, and the methods used in the examples are conventional methods in the art.

[0044] The mouse-derived IgG class monoclonal antibodies involved in the examples: anti- salmonella virus monoclonal antibody and anti- E. coli O157:H7 antibody were provided by Meridian.

[0045] Carboxyl magnetic beads: purchased from Shanghai Aorun Micro-nano New Material Technology Co., Ltd.

[0046] Phage library: Phage random heptapeptide library (Ph.D.-7TM Phage Display Peptide Library Kit) and Escherchia coli ER2738 were purchased from New England Biolabs Co.

[0047] Example 1

[0048] I. Preparation of capture probe MBs@CS@AuNPs@Ab:

[0049] (1) Carboxyl activation of magnetic beads: 10 μL of 10 mg / mL carboxyl magnetic beads was taken into a 2 mL centrifuge tube, washed with 500 μL of MES buffer (10 mM MES pH 6, 0.05% Tween-20), and placed in a magnetic stand for 2 min. After the carboxyl magnetic beads were completely separated from the supernatant, the supernatant was removed and washed with 500 μL of MES buffer. After mixing well with a vortex mixer, it was again placed in a magnetic stand for 2 min, and the supernatant was removed to obtain activated carboxyl magnetic beads.

[0050] (2) Preparation of chitosan-modified magnetic beads: 10 μL of 10 mg / mL activated carboxyl magnetic beads solution was added to 1 mL of 0.4 w / v% chitosan solution, and incubated at room temperature for 30 min in a rotary mixer. The obtained chitosan-coated MBs (MBs@CS) were washed with deionized water for three times.

[0051] (3) Preparation of MBs@CS@AuNPs: MBs@CS was added to 200 μL of ultrapure water, followed by the addition of 200 μL of growth solution (20 μL of 5 wt% HAuCl4+ 180 μL of 38.8 mM trisodium citrate solution), and reacted at room temperature for 1 h in a rotary mixer. The MBs@CS@AuNPs were washed with deionized water for 2 times to remove the remaining citrate, chloroauric acid and unbound AuNPs, and then 200 μL of ultrapure water was added, and stored at 4°C until use.

[0052] (4) Preparation of MBs@CS@AuNPs@Ab capture probe: 10 μg of anti-E. coli O157:H7 antibody was added to 5 mL of MBs@CS@AuNPs suspension and stirred for 12 h. After magnetic separation, 50 μL of 0.25 wt% BSA solution was added to cover the non-specific sites of the gold nanoparticles. After stirring for another 1 h, the MBs@CS@AuNPs@Ab composite was magnetically separated and then dispersed in PBS (pH 7.0) and stored in a refrigerator at 4°C for later use.

[0053] II. Preparation of M13 phage conjugated colloidal gold and Raman molecule 4-MBA:

[0054] 1) 20 mL of ultrapure water containing 0.25 mM HAuCl4 and 0.25 mM trisodium citrate was prepared. Under the condition of vigorous stirring (1000 rpm), 0.6 mL of freshly prepared 0.1 mol / L sodium borohydride was quickly added (ice bath). After 2 min, the stirring was stopped and the ice bath was maintained for 45 min to synthesize the colloidal gold solution. The gold nanoparticles had a particle size of about 5 nm.

[0055] 2) 10 mL of colloidal gold solution was mixed with 200 μL of 1 mM 4-mercaptobenzoic acid (4-MBA) solution for 24 h. 4-MBA was bound to the gold nanoparticles through Au-S bonds.

[0056] 3) 1 mL of 10 9 pfu / mL of M13 phage that specifically recognized E. coli O157:H7 was added to the 4-MBA modified gold nanoparticles obtained in step 2) and mixed on a sample mixer at room temperature for 4 h. After centrifugation at 8000 g for 5 min, the supernatant was removed, and the precipitate was resuspended in 200 μL of PBS and named M13@AuNPs.

[0057] M13 phage that specifically recognized E. coli O157:H7 was prepared by the following steps:

[0058] A. Screening of M13 phage that specifically recognized the antigen to be tested from the phage library:

[0059] (1) The antigen to be tested, E. coli O157:H7, was diluted with PBS to a final concentration of 10 5 cfu / mL, and 100 μL was added to each well of the enzyme-labeled hole. The antigen to be tested was coated on the enzyme-labeled hole at 4°C overnight;

[0060] (2) 100 μL of phage library was added to the enzyme-labeled hole, and the number of M13 phage was about 6.2 x 10 11 pfu, and incubated at 37°C for 1 h;

[0061] (3) Wash away unbound M13 phage, wash 3 times with PBST (0.01M, pH 7.4 phosphate buffer with 0.05wt% Tween-20) and 3 times with pH 7.4 PBS;

[0062] (4) Blocking: Add 340μL 5wt% skim milk blocking solution per well, block at 37°C for 1h;

[0063] (5) Wash plate: After the reaction is completed, remove the 96-well plate, quickly discard the coating solution in the enzyme-labeled plate, tap off the residual coating solution, wash 3 times with PBST and 3 times with PBS;

[0064] (6) Add HRP enzyme-labeled anti-M13 phage monoclonal antibody: Add HRP enzyme-labeled anti-M13 phage monoclonal antibody solution to all wells, 100μL / well, incubate at 37°C for 1h;

[0065] (7) Add color developing solution: After the reaction is completed, remove the 96-well plate, quickly dry, wash 3 times with PBST and 3 times with PBS; add tetramethylbenzidine (TMB) color developing solution 100μL / well to all wells, color develop at 37°C for 10min;

[0066] (8) Stop solution: Add 50μL 2M H2SO4 solution to all wells to stop the reaction, immediately read the absorbance value at 450nm with an enzyme-labeled instrument, record and save;

[0067] (9) Add acid elution to the enzyme-labeled well with effective absorbance value (greater than blank value + 3 times standard deviation) in step (8), obtain specific binding M13 phage eluate and neutralize to neutral pH value with neutralizing solution.

[0068] B. M13 phage amplification and purification:

[0069] 1) Take 0.5mL specific binding M13 phage eluate and 5mL LB broth medium into a test tube, take 50μL ER2738 strain and add to the medium, incubate at 37°C, 180rpm for 2.5 hours until the absorbance of the bacterial culture solution measured at 600nm wavelength with a spectrophotometer is 0.3 (OD 600nm =0.3);

[0070] 2) Take 10μL helper phage M13K07 (titer 10 13 pfu / mL) at a multiplicity of infection of 10:1 and add to the ER2738 bacteria, mix well, place in a 37°C incubator, stand for 30min, then shake at 37°C, 220rpm for 45min;

[0071] 3) Take 1 bottle of 20 mL 2xYT medium (preheat in 37 °C incubator for 2 h), add kanamycin to a final concentration of 25 μg / mL;

[0072] 4) Take 5 mL of ER2738 bacteria solution obtained from 2) and add to 20 mL 2xYT medium prepared in 3), incubate at 37 °C, 250 rpm, overnight;

[0073] 5) After the bacteria solution is cooled to room temperature, centrifuge at 4 °C, 8000 rpm, 10 min;

[0074] 6) After centrifugation, add 1 / 5 volume of 20% PEG / NaCl to the supernatant, mix well, and stand for 5 h on ice;

[0075] 7) After the standing is completed, centrifuge (4 °C, 10000 x g, 10 min), and discard the supernatant;

[0076] 8) Resuspend the precipitate with 2 mL of sterile PBS, and aliquot into EP tubes, stand in a 4 °C refrigerator for 2 h, to obtain high-concentration and high-purity M13 phages that specifically recognize E. coli O157:H7, and measure the titer.

[0077] III. Standard curve preparation

[0078] 12 μg of capture probe MBs@CS@AuNPs@Ab, 150 μL of signal probe M13@AuNPs (titer 10 9 pfu / mL) corresponding to E. coli O157:H7, and 100 μL of E. coli O157:H7 standard at different concentrations (10 0 CFU / mL, 10 1 CFU / mL, 10 2 CFU / mL, 10 3 CFU / mL, 10 4 CFU / mL, 10 5 CFU / mL) are mixed, incubated at 37 °C for 100 min, separated by an external magnetic field, and then dispersed in 1 mL of sterile PBS (0.01 M, pH 7.4), dropped onto a glass slide, dried, and then measured for the change in Raman signal of the solution using a Raman spectrometer. The value of the Raman signal intensity is taken as the ordinate, and the concentration of E. coli O157:H7 is taken as the abscissa, to establish a standard curve, and the equation of the standard curve is y = 251.34027x - 194.45396, R 2 = 0.98306, see FIG. 4. Figure 2 .

[0079] The limit of detection for this method is defined as the average Raman signal of 20 first standards (average Raman signal of the solution at standard 0) + 3 times the standard deviation (3 times the standard deviation of three parallel samples of the first standard sample), the required antigen concentration; the limit of detection is calculated to be 2.5 CFU / mL using this standard curve.

[0080] IV. Sample Determination

[0081] 12 μg capture probe MBs@CS@AuNPs@Ab, 150 μL signal probe M13@AuNPs (titer 10) corresponding to E. coli O157:H7 9 The sample (pfu / mL) was mixed with 100 μL of the test sample and incubated at 37 °C for 100 minutes. After separation by an external magnetic field, the solid was dispersed in 1 mL of sterile PBS (0.01 M, pH 7.4), dropped onto a glass slide, dried, and the Raman signal of the solution was measured using a Raman spectrometer. The concentration of E. coli O157:H7 in the test sample was obtained by calculating the average value and then substituting it into a standard curve.

[0082] Example 2

[0083] I. Preparation of capture probes MBs@CS@AuNPs@Ab:

[0084] In Example 2, the capture probe MBs@CS@AuNPs@Ab was prepared by replacing the anti-Escherichia coli O157:H7 antibody with an anti-Salmonella virus monoclonal antibody in Example 1. The rest was the same as in Example 2.

[0085] II. Preparation of M13 phage-conjugated colloidal gold and Raman molecule 4-MBA:

[0086] In the preparation of M13 phage coupled with colloidal gold and Raman molecule 4-MBA in Example 2, M13 phage specifically recognizing Salmonella was used instead of M13 phage specifically recognizing Escherichia coli O157:H7 in Example 1. The only difference between the preparation method of M13 phage specifically recognizing Salmonella and the preparation method of M13 phage specifically recognizing Escherichia coli O157:H7 in Example 1 is that the antigen to be tested is Salmonella, and the M13 phage specifically recognizing Salmonella is obtained through the same panning and expansion steps.

[0087] III. Standard Curve Plotting

[0088] 12 μg capture probe MBs@CS@AuNPs@Ab, 150 μL signal probe M13@AuNPs corresponding to Salmonella (titer 10) 9 pfu / mL) and 100 μL of different concentrations (10 0 CFU / mL, 10 1CFU / mL, 10 2 CFU / mL, 10 3 CFU / mL, 10 4 CFU / mL, 10 5 CFU / mL) of Salmonella were mixed, incubated at 37℃ for 100 minutes, and after separation by an external magnetic field, the solid was dispersed in 1 mL of sterile PBS (0.01 M, pH 7.4), dropped onto a glass slide and dried, and then the change in the Raman signal of the solution was measured by a Raman spectrometer. Taking the value of the Raman signal intensity as the ordinate and the concentration of Salmonella as the abscissa, a standard curve was established, and the equation of the standard curve was y = 268.13789x - 222.27462, R 2 = 0.97928, see attached Figure 2 .

[0089] The minimum detection limit of the method was defined as the average Raman signal of 20 first standards (0 standard, average Raman signal of the solution) + 3 times the standard deviation (3 times the standard deviation of three parallel samples of the first standard sample), and the required antigen concentration; the minimum detection limit calculated by the standard curve was 3.2 CFU / mL.

[0090] Four, sample determination

[0091] 12 μg of the capture probe MBs@CS@AuNPs@Ab, 150 μL of the signal probe M13@AuNPs (titer 10 9 pfu / mL) corresponding to Salmonella, and 100 μL of the sample to be tested were mixed, incubated at 37℃ for 100 minutes, and after separation by an external magnetic field, the solid was dispersed in 1 mL of sterile PBS (0.01 M, pH 7.4), dropped onto a glass slide and dried, and then the change in the Raman signal of the solution was measured by a Raman spectrometer. The concentration of Salmonella in the sample to be tested was obtained by calculating the average value and substituting it into the standard curve.

[0092] Aspects, embodiments, features of the present application should be considered illustrative in all aspects and not limiting the present application, and the scope of the present application is only defined by the claims. Other embodiments, modifications and uses will be apparent to those skilled in the art without departing from the spirit and scope of the claimed application.

[0093] In the preparation method of the present application, the order of the steps is not limited to the order listed, and for those skilled in the art, changes in the order of the steps without creative labor are also within the protection scope of the present application. In addition, two or more steps or actions can be performed simultaneously.

[0094] It should be noted that the embodiments described herein are merely illustrative of the present application and should not be construed as limiting the scope of the present application. Those skilled in the art can make various modifications or additions to the embodiments described herein or adopt similar ways to replace them without departing from the spirit of the present application. It is not necessary or possible to describe all the embodiments herein. Any obvious changes or variations derived from the spirit of the present application are still within the scope of the present application, and any additional limitations are contrary to the spirit of the present application.

Claims

1. A method for detecting macromolecular antigens, characterized in that, Includes the following steps: Capture probes and recognition probes are added to the sample solution to be tested and reacted at 25-38℃ for 50-500 min. The capture probes, the antigen to be tested, and the recognition probes form a "sandwich" structure. After separation by an external magnetic field, the solid is dispersed in water. The Raman signal intensity of the dispersion is measured on a Raman spectrometer. The content of macromolecular antigen in the sample solution is calculated according to the fitting equation that represents the relationship between the concentration of macromolecular antigen and the Raman signal intensity. The capture probe is a gold magnetic material labeled with an antibody, prepared by a method comprising the following steps: reacting chitosan with carboxyl-activated magnetic beads to obtain chitosan-coated magnetic beads; mixing and reacting the chitosan-coated magnetic beads with a gold growth solution to grow gold nanoparticles on the surface of the chitosan-coated magnetic beads to obtain MBs@CS@AuNPs; reacting MBs@CS@AuNPs with an antibody, followed by magnetic separation, a blocking reaction, and magnetic separation to obtain the capture probe MBs@CS@AuNPs@Ab; The recognition probe is an M13 phage that combines colloidal gold and Raman molecules, prepared by a method comprising the following steps: A colloidal gold solution was prepared, in which gold nanoparticles with a particle size of 1–20 nm were added to the colloidal gold solution for reaction. Subsequently, M13 bacteriophage, which specifically recognizes the antigen to be tested, was added for reaction, and the recognition probe was obtained by centrifugation.

2. The method for detecting macromolecular antigens according to claim 1, characterized in that, The titer of M13 phage in the recognition probe that specifically recognizes the test antigen is 1 × 10⁻⁶. 7 ~1×10 11 The pfu / mL concentration is maintained at a volume ratio of 1 to 2:1 between the recognition probe and the test sample, and the mass ratio of the capture probe to the recognition probe is 1 μg: 8 to 20 μL.

3. The method for detecting macromolecular antigens according to claim 1, characterized in that, The magnetic beads were activated with carboxyl groups using MES buffer solution at pH 5–6.5; Chitosan was reacted with carboxyl-activated magnetic beads at a temperature of 10–40 °C for 10–60 min. The mass ratio of chitosan to magnetic beads is 10 to 100:

1.

4. The method for detecting macromolecular antigens according to claim 1, characterized in that, Chitosan-coated magnetic beads were mixed with gold growth solution and reacted at a temperature of 10–40°C for 10–120 min. The gold growth solution includes chloroauric acid solution and trisodium citrate solution. The concentration of chloroauric acid solution is 1-10 wt%, the concentration of trisodium citrate solution is 20-50 mmol / L, and the volume ratio of chloroauric acid solution to trisodium citrate solution is 1:7-10. MBs@CS@AuNPs react with antibodies at a temperature of 10–40°C for 2–24 hours.

5. The method for detecting a macromolecular antigen according to claim 1, characterized in that, The preparation of colloidal gold solution includes the following steps: chloroauric acid and trisodium citrate are mixed, sodium borohydride solution is added under stirring, and the mixture is reacted in an ice bath for 10-70 minutes to obtain colloidal gold solution.

6. The method for detecting macromolecular antigens according to claim 5, characterized in that, The molar ratio of chloroauric acid, trisodium citrate, and sodium borohydride is 1:0.9–1.1:5–15; The particle size of gold nanoparticles in colloidal gold solutions ranges from 1 to 20 nm.

7. The method for detecting macromolecular antigens according to claim 1, characterized in that, A Raman signal molecule solution was added to a colloidal gold solution for reaction at a temperature of 10–40 °C for a time of 5–30 h. The Raman signaling molecules are at least one of the following: 4-mercaptobenzoic acid, 4-mercaptopyridine, 4-mercaptoaniline, 1,4-p-mercaptobenzene, 4-methylmercaptobenzene, Rhodamine 6G, Rhodamine B, Nile Blue A, Crystal Violet, 4-mercaptophenylboronic acid, 5,5-dithiobisphenol-(2-nitrobenzoic acid), and Nile Blue A.

8. The method for detecting a macromolecular antigen according to claim 1, characterized in that, The reaction was carried out using M13 phage, which specifically recognizes the antigen to be tested, at a temperature of 10–40°C for 1–5 hours.

9. The method for detecting a macromolecular antigen according to claim 1, characterized in that, M13 bacteriophages that specifically recognize the target antigen are prepared by a method comprising the following steps: Add the antigen to be tested to the enzyme-labeled wells and coat overnight at 1–6°C. Add a phage library to the wells and incubate at 30–39°C for 10–100 min. Wash the plate to remove unbound phages. Add blocking buffer for blocking and wash the plate. Add HRP-labeled anti-M13 phage monoclonal antibody solution to the wells and incubate at 30–39°C for 10–100 min. Wash the plate and add tetramethylbenzidine chromogenic solution. Incubate at 30–39°C for 5–15 min. Then, add sulfuric acid solution to terminate the reaction and measure the absorbance at 450 nm. Add acid to the wells with effective absorbance to elute and obtain the M13 phage eluent that specifically recognizes the antigen to be tested. Neutralize the solution to neutral pH with neutralizing buffer. Then, amplify the obtained eluent to obtain the M13 phage that specifically recognizes the antigen to be tested.

10. The method for detecting a macromolecular antigen according to claim 1, characterized in that, The fitting equation representing the relationship between macromolecular antigen concentration and Raman signal intensity was obtained through the following method: Capture probes and recognition probes were added to 3–10 standard solutions of the antigen to be tested at different concentrations and reacted at 25–38°C for 50–500 min. The capture probes, the antigen to be tested, and the recognition probes formed a "sandwich" structure. After separation by an external magnetic field, the solid was dispersed in water. The Raman signal intensity of the dispersion was measured on a Raman spectrometer. A standard curve was established with the Raman signal intensity as the ordinate and the concentration of the antigen to be tested as the abscissa. The fitting equation of the standard curve was then obtained.

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