Staphylococcus aureus dual-recognition artificial antibody, preparation method and application thereof

Abstract

The present invention discloses a dual-recognition artificial antibody for Staphylococcus aureus, a preparation method, and its application. The dual-recognition artificial antibody surface contains imprinted pores that match the shape and size of the target bacteria, and the imprinted pores contain glycopeptide antibiotics that can recognize peptidoglycan. The preparation method is specifically as follows: first, the glycopeptide antibiotic is modified on the surface of the base material, and then Staphylococcus aureus ( Staphylococcus aureus,SA ) and functional monomers, followed by polymerization; after the reaction is completed, IN Remove from the polymer product to obtain IN Dual recognition artificial antibodies. The artificial antibodies prepared by the present invention can IN It can perform dual recognition of the morphology of the cell wall and the peptidoglycan of the cell wall, and has the characteristics of high affinity and strong specificity. It can be used for trace amounts in food and biological samples. IN Highly efficient adsorption separation.

Description

Technical Field

[0001] The present invention belongs to the field of functional materials, and in particular relates to a Staphylococcus aureus dual-recognition artificial antibody, a preparation method and an application thereof. Background Art

[0002] Staphylococcus aureus (SA) is a common pathogen that can cause skin and soft tissue infections, pneumonia, bacteremia, and infective endocarditis. Furthermore, SA is a typical foodborne pathogen, secreting enterotoxins that can cause severe food poisoning. In recent years, with the emergence of drug resistance, SA has become one of the most deadly pathogens, resulting in 1.1 million deaths annually.

[0003] When detecting SA in a sample, the sensitivity of the detection is greatly reduced due to the low bacterial content and interference from the sample matrix, necessitating the separation and enrichment of SA. However, existing bacterial separation and enrichment methods still have many shortcomings. For example, physical separation and enrichment methods based on membrane filtration or electrostatic adsorption lack selectivity, and the separation efficiency is easily affected by complex sample matrices. Although immunoaffinity separation based on antigen-antibody recognition has high selectivity, the antibodies are expensive and their structural stability needs to be improved. Therefore, there is an urgent need to develop highly selective, low-cost, and structurally stable SA separation and enrichment methods.

[0004] Artificial antibodies (AIs) are a new type of functional material developed using molecular imprinting technology (MIT), inspired by the natural antibody-antigen recognition mechanism. During the preparation of AIs, a template molecule and functional monomers first bind through intermolecular interactions (such as hydrogen bonds, van der Waals forces, and π bonds), self-assembling to form a template-functional monomer complex. Subsequently, the addition of a crosslinker causes the functional monomers to cross-link and polymerize around the template molecule. Finally, the template molecule is removed, leaving imprinted pores on the polymer that match the template molecule's shape, size, and functional groups. Compared to natural antibodies, AIs not only exhibit superior selectivity but also possess improved structural stability in adverse environments such as high temperature and extreme pH.

[0005] Artificial antibodies' recognition of target bacteria relies on two factors: morphological recognition, which is the degree of fit between the target bacterium's shape and size and the three-dimensional imprinted cavity; and functional group recognition, which is the affinity between the artificial antibody and the chemical structure of the bacterial surface. Traditional artificial antibodies often exhibit significantly reduced affinity for target bacteria in real-world samples with complex matrices. This is primarily due to the lack of specificity of the functional group effects of artificial antibodies for target bacteria. This allows biomolecules such as proteins, amino acids, and nucleic acids with similar groups to be easily adsorbed to the imprinted sites through nonspecific interactions, interfering with the binding of the artificial antibodies to the target bacteria. Summary of the Invention

[0006] In order to solve the above technical problems, the present invention provides a SA dual-recognition artificial antibody, a preparation method and its application, which are used for the separation and enrichment of trace SA in complex samples. SA is a Gram-positive bacterium with a cell wall surface rich in peptidoglycan. Glycopeptide antibiotics (GAs) can specifically recognize the D-alanyl-D-alanine (D-Ala-D-Ala) structure in peptidoglycan. On the basis of ensuring the morphological recognition (shape and size) of SA by traditional artificial antibodies, the present invention introduces glycopeptide antibiotics into the bacterial imprinted pores to construct dual-recognition artificial antibodies (Dual-recognized bacteria imprinted polymers, D-BIPs) that cooperate with bacterial morphological recognition and peptidoglycan recognition. The technical solution of the present invention is as follows:

[0007] A SA dual-recognition artificial antibody, characterized in that the artificial antibody is composed of a base material, a glycopeptide antibiotic and an imprinted polymer, the surface of the imprinted polymer contains imprinted pores that match the shape and size of the target bacteria, the imprinted pores contain glycopeptide antibiotics, and the glycopeptide antibiotics are connected to the base material through molecular spacer arms; the base material is a polystyrene well plate, the glycopeptide antibiotic is any one of vancomycin, norvancomycin, and teicoplanin, or a mixture of two or more in any proportion, and the molecular spacer arm is polyethylene glycol 5000 (H2N-PEG5000-NH2) or polyethyleneimine (PEI) with an amino group at the end.

[0008] The method for preparing the SA dual-recognition artificial antibody comprises the following steps:

[0009] (1) Add dopamine hydrochloride solution to each well of the plate and expose it to air for polymerization for 20 to 25 hours to modify the plate surface;

[0010] (2) Wash the plate with sterile pure water, add the molecular spacer arm solution, and continue the reaction for 15 h to 20 h;

[0011] (3) Glycopeptide antibiotics, EDC, and NHS were dissolved in MES buffer solution in sequence, and the mixture was added to the well plate modified with the molecular spacer arm and incubated for 5 h to 8 h. After the reaction was completed, the well plate was washed with sterile pure water;

[0012] (4) Add 10 7 ~5×10 8 cfu / mL SA solution, continue incubation for 1h to 3h; then wash away excess SA with sterile water;

[0013] (5) Add dopamine hydrochloride solution to the well plate and expose it to air for polymerization reaction for 12 h to 36 h;

[0014] (6) eluting with a template washing solution to remove SA, followed by washing with sterile pure water to obtain the SA dual-recognition artificial antibody;

[0015] The dopamine hydrochloride solution in step (1) and step (5) is prepared by dissolving dopamine in Tris-HCl at pH 8.5, with a dopamine hydrochloride concentration of 1.5 to 12 mg / mL. The volume ratio of the dopamine hydrochloride solution in step (1), the mixed solution in step (3), the SA bacterial solution in step (4), and the dopamine hydrochloride solution in step (5) is 1:1:1:1.

[0016] Preferably, the concentration of the molecular spacer arm solution in step (2) is 10 mg / mL, and the solvent is PB buffer solution with a pH of 8.0.

[0017] Preferably, in step (3), the mass ratio of the glycopeptide antibiotic, EDC and NHS is 3:8:6, the pH of the MES buffer solution is 6.0, and the concentration of the glycopeptide antibiotic in the MES buffer solution is 3 mg / mL.

[0018] Preferably, the concentration of the dopamine hydrochloride solution in step (1) is 5 mg / mL, and the concentration of the dopamine hydrochloride solution in step (5) is 1.5 to 12 mg / mL.

[0019] Preferably, the template washing solution in step (6) is an acetic acid (5%, v / v) aqueous solution containing 1 wt% SDS, and the amount used each time is 200 μL.

[0020] An application based on SA dual-recognition artificial antibodies, wherein the dual-recognition artificial antibodies can dually recognize the morphology of SA and cell wall peptidoglycan.

[0021] Preferably, the dual-recognition artificial antibody can be used for efficient adsorption and separation of SA in food and biological samples.

[0022] The present invention has the following beneficial effects: (1) It improves the affinity of the bacterial imprinting cavity for target bacteria. Existing bacterial imprinting polymers rely on the interaction between functional monomers and chemical groups on the bacterial surface to adsorb target bacteria. These interactions are relatively weak for bacteria, resulting in low affinity of the prepared artificial antibodies for target bacteria, making it difficult to efficiently separate and enrich low-concentration target bacteria. The present invention introduces glycopeptide antibiotics into the bacterial imprinting cavity, utilizing the strong affinity of glycopeptide antibiotics for peptidoglycan to improve the adsorption capacity of artificial antibodies for target bacteria.

[0023] (2) Enhanced the anti-interference ability of bacterial imprinted polymers in identifying target bacteria in complex samples. The affinity of chemical groups on the bacterial surface of existing bacterial imprinted polymers lacks specificity, which makes the recognition of target bacteria by artificial antibodies in complex samples easily interfered with by the sample matrix. Glycopeptide antibiotics have strong specificity for bacterial peptidoglycan. Introducing them into bacterial imprinted pores can form a dual recognition mechanism that synergizes bacterial morphological recognition and peptidoglycan recognition, thereby effectively enhancing the anti-interference ability of artificial antibodies in complex sample matrices and achieving efficient adsorption of trace bacteria in complex samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a schematic diagram of the preparation process of D-BIPs of the present invention;

[0025] Figure 2 Scanning electron micrographs of D-BIPs (A) and NIPs (B) prepared in Example 1;

[0026] Figure 3 Atomic force microscopy images of D-BIPs (A) and NIPs (B) prepared in Example 1;

[0027] Figure 4 This is a graph showing the capture efficiency of D-BIPs, BIPs, and NIPs for SA at different concentrations in Example 2;

[0028] Figure 5 The adsorption kinetics curves of SA by D-BIPs, BIPs and NIPs in Example 3;

[0029] Figure 6 This is an analysis of the selective adsorption effects of D-BIPs, BIPs, and NIPs on different bacteria in Example 4;

[0030] Figure 7 Graph showing the SA adsorption analysis results of D-BIPs, BIPs, and NIPs in actual samples in Example 5 (* indicates a difference in SA adsorption compared with the PBS group, P<0.05). DETAILED DESCRIPTION

[0031] The technical solutions of the present invention will be further described in detail below with reference to specific embodiments and the accompanying drawings. Obviously, the following embodiments are merely illustrative of and explain the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included within the scope of protection intended by the present invention.

[0032] Example 1

[0033] (1) Preparation of a SA dual recognition artificial antibody D-BIPs, such as Figure 1 As shown,

[0034] Step 1: Add 200 μL of 5 mg / mL dopamine hydrochloride solution (solvent: 10 mM Tris-HCl, pH = 8.5) to a 96-well plate and allow the plate to polymerize in air for 24 hours to modify the surface of the 96-well plate.

[0035] Step 2: Wash the 96-well plate three times with sterile water, add 10 mg / mL H2N-PEG5000-NH2 solution (dissolved in 30 mM PB buffer, pH = 8.0), and continue the reaction for 18 h;

[0036] Step 3: Weigh 12 mg of vancomycin, 32 mg of EDC, and 24 mg of NHS, add 4 mL of MES buffer (30 mM, pH 6.0), dissolve completely, and add 200 μL per well to a 96-well plate. Cover with a ziplock bag and shake at 25°C for 6 hours. Finally, rinse three times with sterile water until ready to use.

[0037] Step 4: Add 200 μL of 10 7 cfu / ml of SA solution, and continue incubation for 2 h; then wash away excess SA with sterile water;

[0038] Step 5: Add 200 μL of 3.0 mg / mL dopamine hydrochloride solution (solvent: 10 mM Tris-HCl, pH = 8.5) to a 96-well plate and expose to air for polymerization reaction for 18 h;

[0039] Step 6: The SA was removed by elution three times with 200 μL of 1 wt % SDS in acetic acid (5%, v / v) and then washed three times with sterile water to obtain D-BIPs.

[0040] (2) Preparation of NIPs and BIPs

[0041] NIPs are non-imprinted polymers (NIPs) without bacteria-imprinting pores on their surfaces. The preparation of NIPs follows the same conditions as D-BIPs, except that SA solution is not added.

[0042] BIPs are common bacterial imprinted polymers (BIPs). BIPs are prepared by masking glycopeptide antibiotic molecules within D-BIPs. The D-alanyl-D-alanine structure in peptidoglycan is a specific binding site for glycopeptide antibiotics. Therefore, D-alanyl-D-alanine is used to mask glycopeptide antibiotics within D-BIPs. The specific method is as follows:

[0043] First, D-alanyl-D-alanine was prepared into a 3.0 mg / mL solution using sterile PBS (10 mM, pH 7.4). Subsequently, 200 μL of the D-alanyl-D-alanine solution was added to the ELISA plate containing the dual-recognition artificial antibody and the reaction was shaken at room temperature for 2 h. After that, the excess D-alanyl-D-alanine was washed away with 10 mM PBS (pH 7.4) to obtain BIPs.

[0044] (3) Characterization of D-BIPs and NIPs

[0045] The 96-well plate containing D-BIPs was cut into fragments, fixed to a scanning electron microscope (SEM) sample holder with conductive adhesive, and then gold-sprayed. The surface morphology of the D-BIPs was observed using a Zeiss Sigma-300 SEM. The SEM was set at a scanning acceleration voltage of 1.0 kV and a WD of 5.1 mm. Another fragment was used to examine the bacterial pore structure using a Bruker Dimension Icon atomic force microscope (AFM).

[0046] Scanning electron microscopy results showed that there were imprinted holes on the surface of D-BIPs that matched the morphology and size of SA ( Figure 2 A), while on the surface of non-imprinted polymers (NIPs) in the control group without SA solution, no imprinted holes matching the morphology and size of SA were found ( Figure 2 B); Atomic force microscopy results show that the surface of D-BIPs is relatively rough, with obvious imprinted holes. The diameter of the imprinted holes is about 700nm and the depth is about 400nm ( Figure 3 A), while the control non-imprinted polymer surface is relatively smooth and no imprinted holes are found ( Figure 3 B). The results showed that D-BIPs were successfully prepared.

[0047] (4) Optimization of D-BIPs preparation conditions

[0048] D-BIPs were prepared according to the procedure described in Example 1-(1), with the following changes:

[0049] ① Adjust the concentration of SA bacterial solution in step 4 to 5×107 cfu / ml, and other conditions remained unchanged;

[0050] ② Adjust the concentration of SA bacterial solution in step 4 to 1×10 8 cfu / ml, and other conditions remained unchanged;

[0051] ③ Adjust the concentration of SA bacterial solution in step 4 to 5×10 8 cfu / ml, and other conditions remained unchanged;

[0052] ④ Adjust the concentration of SA bacterial solution in step 4 to 1×10 8 cfu / ml, the concentration of dopamine hydrochloride solution in step 5 was 1.5 mg / ml, and the other conditions remained unchanged;

[0053] ⑤ Adjust the concentration of SA bacterial solution in step 4 to 1×10 8 cfu / ml, the concentration of dopamine hydrochloride solution in step 5 was 6.0 mg / ml, and the other conditions remained unchanged;

[0054] ⑥ Adjust the concentration of SA bacterial solution in step 4 to 1×10 8 cfu / ml, the concentration of dopamine hydrochloride solution in step 5 was 12 mg / ml, and the other conditions remained unchanged;

[0055] ⑦ Adjust the concentration of SA bacterial solution in step 4 to 1×10 8 cfu / ml, the vancomycin in step 3 was replaced by norvancomycin, and the other conditions remained unchanged;

[0056] ⑧ Adjust the concentration of SA bacterial solution in step 4 to 1×10 8 cfu / ml, the vancomycin in step 3 was a mixture of norvancomycin and vancomycin with a molar ratio of 1:1, and the other conditions remained unchanged;

[0057] ⑨ Adjust the concentration of SA bacterial solution in step 4 to 1×10 8 cfu / ml, the polymerization time in step 5 was 12 h, and the other conditions remained unchanged;

[0058] ⑩ Adjust the concentration of SA bacterial solution in step 4 to 1×10 8 cfu / ml, the polymerization time in step 5 was 36 h, and the other conditions remained unchanged.

[0059] (5) Adsorption test of SA by D-BIPs and NIPs prepared under different conditions

[0060] 150 μL of 1×10 3cfu / mL SA in PBS, and after adsorption for 120 minutes, 100 μL of supernatant was taken out and spread on LB solid plate. After incubation at 37 °C for 18 hours, the number of colonies was counted and the adsorption rate of each group was calculated according to formula (1).

[0061]

[0062] Where N0 is the number of colonies on the plate before adsorption, and N is the number of colonies on the plate after adsorption.

[0063] The adsorption rates of SA by D-BIPs and NIPs under different preparation conditions are shown in Table 1

[0064] Table 1 Adsorption effect of SA on D-BIPs and NIPs under different preparation conditions

[0065]

[0066]

[0067] D-BIPs and NIPs prepared under different conditions were used to measure the effect of 1×10 3 The adsorption effect of cfu / mLSA is shown in Table 1. The results show that the concentration of template bacterial solution is 1×10 8 cfu / mL, functional monomer concentration (dopamine) concentration of 3 mg / mL, glycopeptide antibiotic vancomycin, and functional monomer polymerization time of 18 h prepared D-BIPs have the best adsorption effect on SA.

[0068] Example 2

[0069] Adsorption effects of D-BIPs, BIPs and NIPs on SA at different concentrations.

[0070] The SA strain was inoculated into LB medium and cultured at 37°C with shaking for 10 h. After the culture was completed, the bacteria were collected by centrifugation and washed twice with sterile PBS (10 mM, pH = 7.4). 600 The value is adjusted to 1.0, at which point the SA concentration is approximately 10 9 cfu / mL. Dilute the bacterial solution to 10 2 , 10 3 , 10 4 cfu / mL, and 150 μL was added to a 96-well plate containing D-BIPs, BIPs, and NIPs, respectively. After 120 minutes of adsorption, 100 μL was taken out of each well, diluted to an appropriate multiple, and then spread on an LB solid plate. After incubation at 37°C for 18 hours, the number of colonies was counted and the adsorption rate of each group was calculated according to formula (1). The results are detailed in Figure 4 .

[0071] Depend on Figure 4 It can be seen that D-BIPs has a significant effect on the2 , 10 3 and 10 4 The SA adsorption rates of 10 cfu / mL were 100%, 97.35%±4.18% and 93.39%±6.10%, respectively, indicating that D-BIPs can efficiently capture SA at different concentrations. 2 , 10 3 and 10 4 The SA adsorption rate per cfu / mL ranged from 14.37% to 48.24%, significantly lower than that of D-BIPs. This is because the imprinted pores of D-BIPs contain glycopeptide antibiotics, which have both imprinting and peptidoglycan recognition and affinity for SA. In contrast, in the imprinted pores of BIPs, the peptidoglycan recognition sites of glycopeptide antibiotics are masked by D-alanyl-D-alanine. Therefore, BIPs only have an imprinting recognition effect on SA, resulting in a significantly lower bacterial capture efficiency than D-BIPs. NIPs, on the other hand, lack neither imprinting pores nor glycopeptide antibiotics on their surfaces and can only bind bacteria through nonspecific interactions. Therefore, their SA capture capacity is lower than that of both D-BIPs and BIPs, at only 5.13% to 13.49%. These results demonstrate that the dual-recognition artificial antibodies prepared by the present invention have superior SA capture performance to conventional single-recognition artificial antibodies.

[0072] Example 3

[0073] Adsorption kinetics experiments of SA on D-BIPs, BIPs and NIPs.

[0074] The SA strain was inoculated into LB medium and cultured at 37°C with shaking for 10 h. After the culture was completed, the bacteria were collected by centrifugation and washed twice with sterile PBS (10 mM, pH = 7.4). 600 The value is adjusted to 1.0, at which point the SA concentration is approximately 10 9 cfu / mL. Dilute the bacterial solution to 10 3 cfu / mL, and 150 μL was added to a 96-well plate containing D-BIPs, BIPs, and NIPs. After adsorption for 0, 15, 30, 60, 90, and 120 minutes, 100 μL of supernatant was taken out and spread on LB solid plates. After incubation at 37°C for 18 hours, the number of colonies was counted. The adsorption rate of the dual-recognition artificial antibody and its control on SA at different time points was calculated according to formula (1). The results are detailed in Figure 5 .

[0075] Figure 5The D-BIPs prepared in this invention exhibit a rapid adsorption rate for SA, reaching equilibrium in just 30 minutes. In contrast, BIPs and NIPs not only exhibited far lower adsorption capacities than D-BIPs, but also exhibited significantly slower adsorption rates, requiring approximately 120 minutes to reach equilibrium. This is due to the fact that in D-BIPs, not only do the functional monomers form an affinity effect with bacteria, but the glycopeptide antibiotics within the D-BIPs imprinted pores also have a strong affinity for the peptidoglycan of SA cell walls. These two affinity effects synergize to achieve rapid SA capture. In contrast, the glycopeptide antibiotics within the imprinted pores of BIPs are masked, allowing them to bind to bacteria only through their functional monomers, resulting in significantly lower SA adsorption rates and amounts. Similarly, NIPs can only bind to bacteria through nonspecific interactions, resulting in lower SA adsorption rates and amounts than D-BIPs and BIPs. The above results show that the dual-recognition artificial antibody prepared in the present invention has a better affinity for SA than the ordinary single-recognition artificial antibody and can achieve the purpose of rapid adsorption of SA.

[0076] Example 4

[0077] Evaluation of the selective adsorption effect of D-BIPs on different bacteria.

[0078] Staphylococcus aureus (SA), Listeria monocytogenes (LM), Escherichia coli (E. coli), and Salmonella typhimurium (ST) were inoculated into LB medium and cultured at 37°C with shaking for 10 hours. After the culture was complete, the cells were collected by centrifugation and the pellet was washed twice with sterile PBS (10mM, pH=7.4). The OD600 value of the bacterial solution was adjusted to 1.0, at which point the bacterial concentration was approximately 10 9 cfu / mL. Dilute the bacterial solution to 10 3 cfu / mL, and 150 μL was added to a 96-well plate containing D-BIPs, BIPs, and NIPs, respectively. After 180 minutes of adsorption, 100 μL of each was removed and spread onto a solid LB plate. After 18 hours of incubation, the number of colonies was counted. The adsorption rate of each bacterial group was calculated according to formula (1), and the selectivity coefficient k of the dual-recognition artificial antibody for different bacteria was calculated according to formula (2).

[0079]

[0080] Among them, K1 is the adsorption capacity of the material for SA, and K2 is the adsorption capacity of the material for other bacteria. The results are detailed in Figure 6 .

[0081] Figure 6It shows that the D-BIPs prepared by the present invention have a significant effect on 10 3 The capture efficiency of SA reached 94.06% ± 4.68% at cfu / mL, while the capture efficiency for the same concentrations of LM, E. coli, and ST was 21.05% ± 7.42%, 8.74% ± 4.41%, and 10.51% ± 7.34%, respectively. Table 2 shows that the selectivity coefficients of D-BIPs for these three bacteria were 3.98, 6.35, and 6.91, respectively. This demonstrates that the D-BIPs prepared by the present invention possess strong specific recognition ability for SA. Compared to D-BIPs, the selectivity coefficients of BIPs for LM, E. coli, and ST were lower, at 1.67, 2.73, and 3.04, respectively, indicating that the glycopeptide antibiotics in the imprinted pores enhance the specificity of D-BIPs for SA. However, NIPs lack imprinted pores that match the morphology of the target bacteria and therefore lack significant specific recognition for SA, LM, E. coli, and ST.

[0082] Table 2 Selectivity coefficients of D-BIPs, BIPs and NIPs for different bacteria

[0083]

[0084] Example 5

[0085] Detection of the adsorption and separation effect of D-BIPs on SA in actual samples.

[0086] In order to evaluate the adsorption and separation effect of the present invention on SA in various actual samples, the present invention collected samples such as pure milk, orange juice, and human urine for spiked experiments. First, SA was cultured to the logarithmic growth phase, washed three times with sterile PBS, and the OD600 of the bacterial solution was adjusted to 1.0. At this time, the SA concentration was about 10 9 cfu / mL. Take appropriate amount of bacterial solution to spike the collected samples to make the final concentration of spiked samples 10 3 cfu / mL. Subsequently, 150 μL of spiked sample was added to each well of D-BIPs, BIPs, and NIPs, and adsorbed at 4°C for 30 min. After 30 min, 100 μL of supernatant sample was taken from each group and inoculated on SA selective medium (Baird-Parker agar plate). The culture medium was incubated at 37°C for 24 h, and the number of colonies was counted. The adsorption rate of each group was calculated according to formula (1) and compared with that of PBS. The results are detailed in Figure 7 .

[0087] Depend on Figure 7It can be seen that there is no significant difference in the adsorption performance of D-BIPs on SA, whether in PBS standard solution or in actual samples (P>0.05). In contrast, the adsorption of SA by BIPs and NIPs in orange juice, milk and urine samples decreased significantly (P<0.05). This is because the present invention innovatively introduces glycopeptide antibiotics into SA imprinting pores. Glycopeptide antibiotics have a strong specific affinity with the peptidoglycan on the SA cell wall, and even in complex actual samples, they can still maintain efficient adsorption of SA. According to literature reports, BIPs mainly adsorb bacteria through the interaction between groups such as phenolic hydroxyl, amino, and benzene rings and hydroxyl, amino, carboxyl and phosphate groups on the SA surface. These groups are not specific components of the bacterial surface. Therefore, when there is only an imprinting effect, the recognition of target bacteria by BIPs is easily interfered with by the sample matrix, resulting in a decrease in adsorption. The above results indicate that the D-BIPs prepared in the present invention have strong anti-interference ability for the adsorption of SA and can still efficiently adsorb and separate SA even in actual samples with complex components.

Claims

1. A method for preparing a dual-recognition artificial antibody against Staphylococcus aureus, characterized in that: The artificial antibody is composed of a base material, a glycopeptide antibiotic, and an imprinted polymer. The surface of the imprinted polymer contains imprinted pores that match the shape and size of the target bacteria. The imprinted pores contain glycopeptide antibiotics, which are connected to the base material via molecular spacer arms. The base material is a polystyrene well plate. The glycopeptide antibiotic is any one of vancomycin and norvancomycin, or a mixture of the two in any proportion. The molecular spacer arm is polyethylene glycol 5000 (H2N-PEG5000-NH2) or polyethyleneimine (PEI) with an amino group at the end. The preparation method comprises the following steps: (1) Add dopamine hydrochloride solution to each well of the plate and expose it to air for polymerization for 20 h to 25 h to modify the plate surface; (2) Wash the plate with sterile pure water, add the molecular spacer arm solution, and continue the reaction for 15 h to 20 h; (3) Dissolve glycopeptide antibiotics, EDC, and NHS in MES buffer solution in sequence, add the mixture to the well plate modified with the molecular spacer arm, and incubate for 5 h to 8 h. After the reaction is complete, wash the well plate with sterile pure water; (4) Add 10 8 ~5×10 8 cfu / mL Staphylococcus aureus solution, continue incubation for 1 h to 3 h; then wash away excess Staphylococcus aureus with sterile pure water; (5) Add dopamine hydrochloride solution to the well plate and expose to air for polymerization reaction for 18 h to 36 h; wherein the concentration of dopamine hydrochloride solution is 3.0 mg / mL; (6) eluting with a template washing solution to remove Staphylococcus aureus, and then washing with sterile pure water to obtain Staphylococcus aureus dual recognition artificial antibodies; The dopamine hydrochloride solution in step (1) and step (5) is prepared by dissolving dopamine in Tris-HCl at pH=8.5, wherein the concentration of dopamine hydrochloride in step (1) is 1.5-12 mg / mL, and the volume ratio of the dopamine hydrochloride solution in step (1), the mixed solution in step (3), the Staphylococcus aureus solution in step (4), and the dopamine hydrochloride solution in step (5) is 1:1:1:

1.

2. The method for preparing a dual-recognition artificial antibody according to claim 1, wherein The concentration of the molecular spacer arm solution in step (2) is 10 mg / mL, and the solvent is PB buffer solution with a pH of 8.

0.

3. The method for preparing a dual-recognition artificial antibody according to claim 1, wherein In step (3), the mass ratio of the glycopeptide antibiotic, EDC, and NHS is 3:8:6, the pH of the MES buffer solution is 6.0, and the concentration of the glycopeptide antibiotic in the MES buffer solution is 3 mg / mL.

4. The method for preparing a dual-recognition artificial antibody according to claim 1, wherein The concentration of the dopamine hydrochloride solution in step (1) is 5 mg / mL.

5. The method for preparing a dual-recognition artificial antibody according to claim 1, wherein The template washing solution in step (6) is an aqueous solution of acetic acid (5%, v / v) containing 1 wt% SDS, and the amount used each time is 200 μL.

6. A Staphylococcus aureus dual-recognition artificial antibody prepared by the preparation method according to any one of claims 1 to 5.

7. A use of the Staphylococcus aureus dual-recognition artificial antibody according to claim 6, characterized in that: The dual-recognition artificial antibody can dually recognize the morphology and cell wall peptidoglycan of Staphylococcus aureus, and is used for efficient adsorption and separation of Staphylococcus aureus in food samples.

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