Probe targeting staphylococcus aureus and application thereof

Abstract

The invention discloses a probe for targeting staphylococcus aureus and an application of the probe. The amino acid sequence of a nano antibody is as follows: QLQLVESGGLVQAGGSMRLSCAASGRTFSTNTMGWFRQAPGKEREFVAGIRWISGSTQADSVKGRFTISRDNAKNTLFLQMNSLPEDAVYYCAAGPHNIPILRTSAYNYWGQGTQVTVSS, and the amino acid sequence of the nano antibody is as follows: QLQLVEGGGLVQAGGTQVTVSS. According to the invention, a high-performance probe is screened, and a high-sensitivity electrochemical sensor is constructed to realize rapid and accurate screening of staphylococcus aureus in different media; staphylococcus aureus can be qualitatively detected within one minute, and the method has high sensitivity and excellent specificity and accuracy. The method meets the urgent demand of accurately, reliably and quickly detecting the staphylococcus aureus in a complex environment.

Description

A probe targeting Staphylococcus aureus and its application Technical Field

[0001] This invention relates to a probe targeting Staphylococcus aureus and its application, belonging to the field of immunoassay. Background Technology

[0002] Staphylococcus aureus, an important zoonotic pathogen, is widely distributed in the natural environment and medical settings. Infectious diseases caused by it are difficult to treat clinically and have a high recurrence rate, posing a persistent public health risk. Developing rapid and accurate on-site detection technologies is crucial for timely tracing of the source of infection, guiding rational clinical drug use, and interrupting the transmission chain.

[0003] Current detection technologies primarily rely on antibody-based immunological methods, with the core being the performance of the recognition probe. Although monoclonal antibodies are widely used, their inherent limitations severely restrict the actual effectiveness of detection systems: on the one hand, antibody molecules lack stability and are easily affected by environmental factors such as temperature and pH, leading to loss of activity and making them unsuitable for complex on-site storage and use conditions; on the other hand, traditional antibody production has long cycles, high costs, and potential batch-to-batch variations, affecting the quality uniformity and accessibility of detection reagents. More critically, staphylococcal protein A (SpA), widely expressed on the surface of Staphylococcus aureus, non-specifically binds to the Fc fragment of most conventional antibodies. This "reverse" binding severely interferes with the specific recognition of the target antigen, leading to increased background signal, decreased detection specificity, and even false positive results.

[0004] In recent years, nanobodies have been considered promising candidates for next-generation immunoassay probes due to their small size, high stability, and ease of genetic engineering. Existing technologies (such as patent announcement number CN119798426B) have validated the application potential of nanobodies in targeted detection. However, these technologies typically rely on screening paired nanobodies against the same target antigen to construct sandwich detection systems. This process presents challenges such as long development cycles, uncertain screening success rates, and the possibility that the affinity of paired combinations may not be optimal, thus limiting their rapid development and further performance improvements.

[0005] Therefore, there is an urgent need in this field for a novel probe construction strategy that can effectively circumvent the non-specific interference of SpA while overcoming the bottleneck of low efficiency in traditional pairing screening methods. High-quality nanobody libraries constructed based on synthetic biology techniques offer the possibility for the rational design and rapid screening of high-affinity probe units. By focusing on key conserved epitopes of target molecules and combining computational simulations for rational design, it is expected to obtain nanobodies with excellent binding properties. More importantly, it is possible to explore the use of the interaction between SpA and the Fc terminus of antibodies, transforming it from an interfering factor into a tool for constructing a universal detection platform. For example, screening nanobodies can be combined with engineered universal antibody modules that can stably bind to SpA to construct a novel detection probe system that is efficient, stable, and unaffected by SpA. This innovative approach is expected to overcome the limitations of existing detection technologies in terms of accuracy, stability, and cost-effectiveness, providing a new solution for developing next-generation high-performance on-site rapid detection products for Staphylococcus aureus. Summary of the Invention

[0006] Purpose of the invention: The purpose of this invention is to provide a highly sensitive and rapid detection probe targeting Staphylococcus aureus and its application.

[0007] Technical Solution: This invention provides a nanobody targeting Staphylococcus aureus, with the following amino acid sequence: QLQLVESGGGLVQAGGSMRLSCAASGRTFSTNTMGWFRQAPGKEREFVAGIRWISGSTSYADSVKGRFTISRDNAKNTLFLQMNSLKPEDTAVYYCAAGPHNIPILRTSAYNYWGQGTQVTVSS. Pairing experiments show that the phage-displayed nanobody can serve as a universal probe, exhibiting good pairing effects with various antibodies and forming paired antibodies for use in immunosensors.

[0008] The present invention also provides an encoding of the nanobody, the nucleotide sequence of which is shown below: SA2 (SEQ ID NO.5): CAGTTGCAGCTCGTGGAGTCGGGGGGAGGATTGGTGCAGGCTGGGGGCTCCATGAGACTCTCCTGTGCAGCCTCTGGACGCACCTTCAGTACGAATACCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCAGGTATTAGGTGGATTAGTGGTAGCACAAGCTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTCTTTCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTGTATTACTGTGCGGCGGGCCCCCATAATATACCGATACTTCGTACTTCGGCTTATAACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA.

[0009] The present invention also provides the application of the nanobody in the preparation of nanobody probes targeting Staphylococcus aureus.

[0010] The present invention also provides a nanobody probe targeting Staphylococcus aureus, which contains the nanobody.

[0011] The present invention also provides the application of the nanobody or the nanobody probe in the preparation of a Staphylococcus aureus detection kit.

[0012] The present invention also provides a Staphylococcus aureus detection kit containing the nanobody or the nanobody probe.

[0013] It also contains an electrochemical biosensor, which is a three-electrode system: a working electrode, a counter electrode, and a reference electrode. The working electrode is a screen-printed electrode modified with gold nanoparticles, the gold nanoparticles being connected to Fc fragments, and the surface of the Fc fragments being sealed with bovine serum albumin (BSA). The counter electrode is a platinum wire, and the reference electrode is a saturated calomel electrode.

[0014] The present invention also provides the application of the nanobody, the nanobody probe, or the Staphylococcus aureus detection kit in the detection of Staphylococcus aureus.

[0015] The present invention also provides a method for detecting Staphylococcus aureus using the nanobody probe, comprising the following steps: (1) adding the sample solution to the working electrode surface of the electrochemical biosensor, incubating, thoroughly washing the electrode surface with phosphate-buffered saline, then adding the nanobody probe (HRP enzyme-labeled phage-displaying nanobody) to the working electrode surface, incubating, washing, drying, and placing the electrochemical biosensor in a CBS buffer containing catechol and hydrogen peroxide, and connecting it to an electrochemical workstation for square wave voltammetry testing; (2) when phage-displaying nanobody SA-2 is used as the nanobody probe and the current exceeds 3.69 μA, it proves that Staphylococcus aureus is present in the sample solution; when phage-displaying nanobody SA-10 is used as the nanobody probe and the current exceeds 2.09 μA, it proves that Staphylococcus aureus is present in the sample solution; when phage-displaying nanobody SA-31 is used as the nanobody probe and the current exceeds 1.76 μA, it proves that Staphylococcus aureus is present in the sample solution. When the current exceeds 1.89 μA, it proves that Staphylococcus aureus is present in the sample solution; when phage display nanobody SA-46 is used as a nanobody probe and the current exceeds 1.89 μA, it proves that Staphylococcus aureus is present in the sample solution.

[0016] The present invention also provides a method for quantitatively detecting the concentration of Staphylococcus aureus using the nanobody probe, comprising the following steps: (1) adding the sample solution to the working electrode surface of the electrochemical biosensor, incubating, thoroughly washing the electrode surface with phosphate buffered saline, then adding the nanobody probe (HRP enzyme-labeled phage-displaying nanobody) to the working electrode surface, incubating, washing, drying, and placing the electrochemical biosensor in a CBS buffer containing catechol and hydrogen peroxide, and connecting it to an electrochemical workstation for square wave voltammetry testing; (2) when using phage-displaying nanobody SA-2 as the nanobody probe, calculating the concentration of Staphylococcus aureus according to the standard curve Y = 1.12x + 0.23, the limit of detection is 158 CFU / mL; when using phage-displaying nanobody SA-10 as the nanobody probe, calculating the concentration of Staphylococcus aureus according to the standard curve Y = 1.05x - 0.77, the limit of detection is 725 CFU / mL. CFU / mL; When using phage-displayed nanobody SA-31 as a nanobody probe, the concentration of Staphylococcus aureus was calculated according to the standard curve Y = 1.08x-1.07, and the limit of detection was 447 CFU / mL; When using phage-displayed nanobody SA-46 as a nanobody probe, the concentration of Staphylococcus aureus was calculated according to the standard curve Y = 1.07x-1.733, and the limit of detection was 2455 CFU / mL; where x is the concentration of Staphylococcus aureus (CFU / mL) and y is the current (μA).

[0017] The parameters for the square wave voltammetry test are set as follows: step voltage (ΔES) is 0.005 V, scanning potential range is 0–0.6 V, square wave amplitude (ΔESW) is 0.025 V, and frequency (f) is 25 Hz.

[0018] The nucleotide sequence of the gene is shown in any one of SEQ ID NO: 5 to 8.

[0019] Among them, the sequences shown in SEQ ID NO: 5~8 are the nucleotide sequences of the SA-2, SA-10, SA-31 and SA-46 nanobodies, respectively.

[0020] This invention also provides a method for screening the phage-displaying nanobody, comprising the following steps: (1) commissioning a company to construct an immune phage-displaying nanobody: using sterilized Staphylococcus aureus (ATCC25923) as the immune source, healthy alpacas are immunized. Subsequently, lymphocytes are extracted from peripheral blood of the alpacas, and total RNA is extracted from the cells using an RNA kit and transcribed into cDNA. The fragment encoding the VHH gene is amplified by PCR, digested with Sfi I enzyme, and re-ligated with the phage particle pComb3XSS using T4 ligase. The recombinant phage particle is then transferred into Escherichia coli TG1 to construct a phage-displaying nanobody library, with a library size of approximately 1×10⁻⁶. 11 The sequence has low repetition and good diversity.

[0021] (2) Inactivated Staphylococcus aureus was coated onto a solid-phase 96-well ELISA plate. After incubation with recombinant phage for a period of time, non-specifically bound recombinant phage was washed away with PBST, and specifically bound phage was eluted with acid. The eluted phage was amplified, and the titer was measured for the next round of screening or analysis. Screening was carried out according to the "adsorption-washing-elution-amplification" steps. By changing the concentration of PBST, the incubation time of phage, and other conditions during the screening process, the screening was carried out step by step to screen for nanobody phages with stronger affinity and specificity.

[0022] (3) Fifty-five phages were selected for Phage-ELISA identification. Bovine serum albumin (BSA) and ovalbumin (OVA) were used as negative controls, and PBS was used as a blank control. Twenty-two phage-displaying nanobodies with good binding to Staphylococcus aureus were obtained. These phage particles were amplified, plasmids were extracted and sequenced to obtain four previously unreported phage-displaying nanobodies.

[0023] This invention pairs each screened phage-displaying nanobody with an Fc fragment, demonstrating the high performance of the obtained phage-displaying nanobody as a probe. It also screens out multiple pairs of highly sensitive and specific paired antibodies. Based on the recorded data, software is used to fit a standard curve and calculate the limit of detection.

[0024] Furthermore, the present invention uses the above-mentioned paired phage display nanobody and Fc fragment as coating antibody and labeling antibody respectively to develop sandwich sensor. The coating antibody is Fc fragment and the labeling antibody is horseradish peroxide (HRP) enzyme-labeled phage display nanobody. The main steps include: (1) cleaning the screen-printed electrode with ultrapure water and ultrasonically cleaning it in 75% ethanol solution for 30 seconds, and then rinsing it with ultrapure water for later use.

[0025] (2) The screen-printed electrode after treatment was immersed in 1% HAuCl4 solution, connected to a three-electrode system, and gold nanoparticles were deposited on the electrode surface by cyclic voltammetry with 3-9 scans. After cleaning with ultrapure water and drying with nitrogen, it was self-assembled overnight in 250 mM 3-mercaptopropionic acid (MPA) solution in the dark.

[0026] (3) Add 0.01 M PB buffer containing 0.05-0.1 mM EDC / NHS to the electrode surface. After the reaction is complete, add 5-10 µg / mL Fc fragment and 1% BSA dropwise. Wash the electrode again with ultrapure water and dry it with nitrogen. After each modification step, immerse the electrode in 5 mM potassium ferricyanide / potassium ferrocyanide ([Fe(CN)6]) solution. 3- / 4- The modification process was monitored in real time using a base solution (containing 0.1 M KCl) by cyclic voltammetry (CV) or electrochemical impedance spectroscopy (EIS).

[0027] (4) Spiked samples containing different concentrations of inactivated Staphylococcus aureus were divided into concentration ranges of 0–10. 8 The solution was diluted to CFU / mL and dropped onto the surface of a screen-printed electrode. After reacting at 37 °C for 30 minutes, 10 μL of HRP-labeled phage display nanobody was added for incubation.

[0028] (5) Clean the electrode with ultrapure water and place it in a solution containing 2×10 -5 M-catechol and 7×10 -5 In M hydrogen peroxide CBS buffer (from 5 × 10⁻⁶) -2 M citrate and 5×10 -2The sample was mixed with M phosphate buffer (PB) and the pH was adjusted to 5.0 for square wave voltammetry (SWV) detection. The SWV detection parameters were set as follows: step voltage (ΔES) of 0.005 V, scan potential range of 0–0.6 V, square wave amplitude (ΔESW) of 0.025 V, and frequency (f) of 25 Hz. Data during the detection process were recorded, and a calibration curve was plotted based on the correlation between concentration and redox peaks to calculate the linear range and minimum detectable concentration.

[0029] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) Compared with traditional monoclonal antibodies, the phage display nanobody of the present invention has the advantages of high sensitivity, strong stability, low cost, easy modification and high biosafety.

[0030] (2) Traditional monoclonal antibodies are affected by the binding of the Fc end to Staphylococcus aureus SPA, making it difficult to directly detect Staphylococcus aureus. They can only indirectly detect the toxic substances secreted by bacteria, making it difficult to accurately assess the infection or contamination status of samples such as patients, water sources, and food. The phage display nanobody screened in this invention can achieve direct detection of Staphylococcus aureus with high accuracy.

[0031] (3) The sandwich sensor constructed in this invention can achieve a detection limit of 158-2455 CFU / mL, which is much lower than that of existing immunological methods.

[0032] (4) The sandwich sensor constructed in this invention has a fast detection speed and can qualitatively detect Staphylococcus aureus within 1 minute. It has high specificity and accuracy, and can realize multi-scenario, real-time and in-situ detection of Staphylococcus aureus. Attached Figure Description

[0033] Figure 1 shows the results of validating positive phage clones using Phage-ELISA with 55 selected phage clones; the horizontal axis represents the phage-displaying nanobody monoclonal number, and the vertical axis represents the absorbance at 450 nm. Figure 2 shows the results of validating the specificity of 22 positive phages using Phage-ELISA; the horizontal axis represents the phage-displaying nanobody monoclonal number coated on the ELISA plate, and the vertical axis represents the absorbance at 450 nm. Figure 3 shows the calibration curve for detecting Staphylococcus aureus using a sandwich ELISA based on phage-displaying nanobody; the horizontal axis represents the concentration of Staphylococcus aureus added, and the vertical axis represents the absorbance at 450 nm. The absorbance values ​​at nm; Figures 3A-3D represent SA-2, SA-10, SA-31, and SA-46, respectively; Figure 4 shows the identification of cyclic voltammetry peaks during the preparation of the phage-displayed nanobody sensor; the horizontal axis represents the screen-printed electrode loading material; the vertical axis represents the detection current value; Figure 5 shows the identification of impedance values ​​during the preparation of the phage-displayed nanobody sensor; the horizontal axis represents the screen-printed electrode loading material; the vertical axis represents the detection impedance value; Figure 6 shows the calibration curve for the detection of Staphylococcus aureus by the sandwich sensor based on phage-displayed nanobody; the horizontal axis represents the concentration of Staphylococcus aureus added; the vertical axis represents the detection current value; Figures 6A-6D represent the absorbance values ​​at nm; Figure 6A-3 ... Figure 7 shows the identification of the specificity of the sandwich sensor based on phage-displayed nanobodies in detecting Staphylococcus aureus; the horizontal axis represents the concentration of Staphylococcus aureus introduced; the vertical axis represents the detection current value; Figures 7A to 7D represent SA-2, SA-10, SA-31, and SA-46, respectively. Figure 8 shows the identification of the accuracy of the sandwich sensor based on phage-displayed nanobodies in detecting Staphylococcus aureus in different media; the horizontal axis represents the concentration of Staphylococcus aureus introduced; the vertical axis represents the detection current value; Figures 8A to 8D represent SA-2, SA-10, SA-31, and SA-46, respectively. Detailed Implementation

[0034] The main instruments and materials used in this invention embodiment are as follows: HRP (horseradish peroxidase) enzyme-antiphage monoclonal antibody (Anti-M13 Antibody, Beijing Yiqiao Shenzhou Technology Co., Ltd., catalog number: 11973-MM05T), HRP conjugation kit, skim milk powder, tetramethylbenzidine, and isopropyl-β-D-thiogalactoside were all obtained from Sangon Biotech (Shanghai) Co., Ltd., Staphylococcus aureus model strain (Beijing Sanyao Technology Co., Ltd., product number 33025), 5-bromo-4-chloro-3-indole-β-D-galactoside (Tiangen Biotech Co., Ltd.), LB broth (Qingdao Haibo Biotechnology Co., Ltd.), tetrachloroauric acid trihydrate (HAuCl4), 2-morpholinoethanesulfonic acid (MES), hydroxysuccinyl diamine (NHS), and carbodiimide hydrochloride (EDC) were all purchased from Sigma-Aldrich (St. Louis, Missouri, USA), CHI760 electrochemical workstation, 5 mM gold electrode, and CHI150... The Hg / HgCl reference electrode and CHI115 platinum wire electrode were purchased from Chenhua Biotechnology Co., Ltd. (Shanghai, China). E. coil ER2738 and the Fc fragment (refer to the NCBI database: FC fragment sequence number 1534918401; PDB number: 6F2Z_A; the FC fragment can bind to Staphylococcus aureus and, together with nanobodies, form a sandwich method for the detection of Staphylococcus aureus. The amino acid sequence of the FC fragment is: SEQ ID NO.9: dkthtsppspapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvslwclvkgfypsdiavewesngqpennykttppvldsdgsfflysaltvdksrwqqgnvfscsvmhealhnhytqkslslspgk) was preserved by the Precision Translational Medicine Center of West China Hospital. Other analytical-grade chemical reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. The immune phage display nanobody library was commissioned to be constructed by Chengdu Apak Biotechnology Co., Ltd. The SARS-CoV-2 N protein was purchased from Shanghai Beyotime Biotechnology Co., Ltd., catalog number P2328-1mg; the norovirus GII.4 protein was purchased from Beijing Sinocare Medical Technology Co., Ltd., catalog number 41002-V10E; and the recombinant rotavirus capsid protein (VP6) was purchased from Beijing Sinocare Medical Technology Co., Ltd., catalog number 906-BRV0040.

[0035] Example 1: Screening of specific phage-display nanobodies targeting Staphylococcus aureus. Three rounds of screening were carried out according to the steps of "coating-adsorption-washing-elution-amplification-identification": (1) After wetting the 96-well ELISA plate with sterile ultrapure water, it was placed in a clean bench and sterilized by irradiation under ultraviolet light for half an hour; (2) Take 1×10 8 (2) Inactivate Staphylococcus aureus with CFU / mL into an ELISA plate and coat it overnight in a refrigerator at 4 ℃; (3) In a sterile operating table, wash the plate three times with sterile 0.1% TBST washing solution, patting it dry on sterile paper each time, and then add 250-350 μL of filtered sterile 1-3% BSA-TBS blocking solution and block it in a constant temperature incubator at 37 ℃ for 1-2 h; (4) Wash the plate three times with sterile 0.1% TBST washing solution, patting it dry on sterile paper each time, and take 2×10⁻⁶ phage display nanobody libraries. 11 (5) Add 100 μL of sterile 1×TBS to the above enzyme-labeled plate and mix well. Bind at 37℃ for 1 h. Wash the plate three times with 0.1% TBST. After each wash, pat dry on sterile paper and add 100 μL of Gly-HCl (adjust pH=2.2 with hydrochloric acid in 0.2M glycine and autoclave for 15 min). Shake at 37℃ for 8-15 min. Remove the elution product and quickly add an appropriate volume of Tris-HCl neutralization buffer (adjust pH=9.1 with hydrochloric acid in 1M tris(hydroxymethyl)aminomethane and autoclave for 15 min) to neutral. (6) Take 10-15 μL of phage for titer determination and use the rest for phage amplification.

[0036] (7) Streak E. coli ER2738 on LB / Tet (tetracycline) solid medium plates and incubate at 37 ℃ for 12-16 h; (8) Pick single colonies from the plate and inoculate them into LB / Tet liquid medium tubes and incubate at 37 ℃ and 220 rpm for 12-16 h; (9) Take 100-500 μL of the above culture into 40-60 mL of LB / Tet liquid medium, add the eluted phage, and incubate at 37 ℃ and 220 rpm for 4-6 h; (10) Transfer the amplification product to a sterile centrifuge tube, centrifuge at 4 ℃ and 8000 rpm for 10-15 min, and collect the supernatant into a fresh sterile centrifuge tube; (11) Add 8-10 mL (1 / 6 of the supernatant volume) of sterile 10-20% PEG-NaCl solution (50g PEG-8000, 36 ... Dissolve NaCl in ultrapure water by heating, bring the volume to 250 mL, autoclave for 15 min, shake well, and let stand at 4℃ for 12-16 h; (12) Centrifuge at 4℃ and 8000 rpm for 10-15 min, discard the supernatant, resuspend the precipitate with 1 mL sterile PBS buffer, transfer to a sterile centrifuge tube, add 200 μL of 20% PEG-NaCl solution, mix well, and incubate on ice for 2 h; (13) Centrifuge at 4℃ and 12000 rpm for 10-15 min, discard the supernatant, centrifuge briefly again, aspirate the remaining supernatant, resuspend the precipitate with 200 μL of sterile PBS, and centrifuge at 4℃ and 5000 rpm for 1-2 min; (14) Transfer the supernatant to a fresh centrifuge tube. Take 10 μL to determine the phage titer and store at -20 ℃; (15) Steps (1)-(14) are the first round of amplification process. The second and third rounds of selection steps are roughly the same. The amount of phage added in each round is 2×10 11 The PFU and Staphylococcus aureus coating concentrations decreased sequentially by 10. 7 CFU and 10 6 CFU, 1-3% OVA-TBS and 1-3% BSA-TBS blocking solutions were used for alternating blocking. The binding times of the bacteriophages to Staphylococcus aureus were 45 min and 30 min, respectively. The washing solution concentrations were 0.25% TBST and 0.5% TBST, respectively. The selection scheme is shown in Table 1.

[0037] Table 1. Screening process for phage-display nanobodies targeting Staphylococcus aureus

[0038] Example 2, determination of phage titer (1) E. coli ER2738 was streaked onto LB / Tet solid medium plates and cultured at 37 ℃ for 12-16 h; (2) Single colonies were picked from the plates and inoculated into LB / Tet liquid medium, and cultured at 37 ℃ and 220 rpm with shaking until the logarithmic growth phase (OD200). 600nm (3) Take 200 μL of bacterial solution from step (2) into sterile 1.5 mL centrifuge tubes respectively; (4) Dilute and elute the phage to 10 mL. -2 10 -3 10 -4 (5) Take 10 μL of phage solution of different dilutions and add it to the centrifuge tube in step (4), mix thoroughly, and incubate at 37 ℃ for 10-15 min; (6) Inject the bacterial solution infected with phage into the top agar, shake gently to mix, and quickly pour it onto LB / IPTG / X-gal plates and incubate at 37 ℃ for 12-16 h; (7) Pick plates with about 30-300 blue spots to count and calculate the phage titer. The calculation formula is: Colony forming unit (CFU) = number of colonies in plate × dilution factor × 100%.

[0039] Example 3: Screening and Identification of Positive Clones After three rounds of screening, plates with approximately 200 blue spots were selected, and 55 clones were chosen for amplification and phage-ELISA identification. The specific steps are as follows: (1) Single colony clones were inoculated into LB / Tet liquid medium and cultured at 37 ℃ and 220 rpm for 12-16 h; (2) 500 μL of overnight E. coil ER2738 culture was added to 50 mL of liquid medium and mixed well; (3) 1 mL of the medium from (2) was aliquoted into centrifuge tubes; (4) Single blue phage plaques were picked into each centrifuge tube and cultured at 37 ℃ and 220 rpm for 4.5-6 h with shaking; (5) After the culture was completed, the tubes were centrifuged at 8000 rpm for 2 min, and the supernatant was transferred to fresh centrifuge tubes and labeled. The tubes were then left to stand at 4 ℃ for later use; (6) 10 μL of each well was taken from each tube. 8(7) Coat CFU-inactivated Staphylococcus aureus onto an ELISA plate and incubate overnight at 4°C; (8) Wash the plate three times with 0.05% PBST, add 300 μL of 5% skim milk for blocking, and incubate at 37°C for 1-2 h; (9) Wash the plate three times with 0.05% PBST, add 100 µL of phage display nanobody supernatant, and incubate at 37°C for 45-60 min; (10) Wash the plate three times with 0.05% PBST, add 100 µL of HRP-labeled Anti-M13 Antibody (E-Tech, Cat: 11973-MM05T-H) to each well, and incubate at 37°C for 45-60 min; (11) Wash the plate three times with 0.05% PBST, add 100 µL of TMB (tetramethylbenzidine) chromogenic solution, and incubate at 37°C for 8-15 minutes. min; (11) Add 50 µL of 2 M H2SO4 (sulfuric acid) to each well and measure the OD. 450 nm Absorbance; (12) Of the 55 clones selected, 22 phage clones showed that the nanobody clones could bind to Staphylococcus aureus, and positive clones (OD) were selected. 450 >1.5) to proceed to the next step of specificity verification (Figure 1).

[0040] Example 4, Identification of the specificity of positive phage-displaying nanobodies (1) Add 100 μL of 10% concentration to each well. 9 CFU / mL Staphylococcus aureus, 3 μg / mL bovine serum albumin (BSA) protein and 3 μg / mL chicken egg white albumin (OVA) protein were coated onto the ELISA plate, and three parallel control groups were set up. The plate was incubated overnight at 4 ℃; (2) The plate was washed three times with 0.05% PBST, blocked with 300 μL of 5% skim milk, and incubated at 37 ℃ for 2 h; (3) The plate was washed three times with 0.05% PBST, and the supernatant culture medium of the above 22 phage display nanobody monoclonals (100 µL of each monoclonal) was added. The plate was incubated at 37 ℃ for 60 min; (4) The plate was washed three times with 0.05% PBST, and 100 µL of HRP enzyme-antiphage monoclonal antibody was added to each well. The plate was incubated at 37 ℃ for 45-60 min; (5) The plate was washed three times with 0.05% PBST, and 100 µL of TMB chromogenic solution was added. The plate was incubated at 37 ℃ for 15 min. min; (6) Add 50 µL of 2 M H2SO4 to each well and measure the absorbance at OD450 nm.

[0041] (7) Among the 22 positive phage plaques identified, all selected phage-displaying nanobody clones showed good affinity and specificity (Figure 2). Therefore, we sent all phage-displaying antibodies for sequencing. The sequencing comparison results showed that four novel phage-displaying nanobodies (SEQ ID NO.1~4) were screened: >SA2 (SEQ ID NO.1): QLQLVESGGGLVQAGGSMRLSCAASGRTFSTNTMGWFRQAPGKEREFVAGIRWISGSTSYADSVKGRFTISRDNAKNTLFLQMNSLKPEDTAVYYCAAGPHNIPILRTSAYNYWGQGTQVTVSS.

[0042] >SA10 (SEQ ID NO.2): EVQLVESGGGLVQAGGSLRLSCATSGRTFSTFSTYVAGWFRQAPGKEREFVAAIRRTGGRTYYADSVKGRFTISGDNAKNMVYLQMNSLKPEDTATYYCAAANNLGSDYVTVTGKYDYWGQGTQVIVSS.

[0043] SA31 (SEQ ID NO. 3): QLQLVESGGGLVQAGDSLRLSCVLSGRTFSNYGMYWFRQAPGKEREGVAGIAWRGGGTIYADSVKGRFTISRDNAKNTVYLQLNSLKPEDTAVYFCGALVFGGGRWERPAHWGQGTQVTVSS.

[0044] SA46 (SEQ ID NO. 4): QVQLVESGGGLVQPGGSLRLSCITSGSRFRFGAGWYRQAPGKSRERVAAINFGSFTNYADSVKGRFTISRDDAANTLYLQMNNLRPEDTAVYYCNTGNYWGEGTQVTVSS.

[0045] The nucleotide sequences of 4 novel phage-displayed nanobodies are shown below: SA2 (SEQ ID NO.5): CAGTTGCAGCTCGTGGAGTCGGGGGGAGGATTGGTGCAGGCTGGGGGCTCCATGAGACTCTCCTGTGCAGCCTCTGGACGCACCTTCAGTACGAATACCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCAGGTATTAGGTGGATTAGTGGTAGCACAAGCTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTCTTTCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTGTATTACTGTGCGGCGGGCCCCCATAATATACCGATACTTCGTACTTCGGCTTATAACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA; SA10 (SEQ ID NO.6): GAGGTGCAGCTGGTGGAGTCGGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAACCTCTGGACGCACCTTCAGTACCTTCAGTACCTATGTCGCGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCAGCTATTAGGCGGACTGGTGGTCGCACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCGGAGACAACGCCAAGAACATGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCACTTATTACTGTGCAGCAGCTAACAACCTGGGTAGCGACTATGTGACAGTGACGGGAAAGTATGACTACTGGGGCCAGGGGACCCAGGTCATCGTCTCCTCA; SA31 (SEQ ID NO.7): CAGTTGCAGCTCGTGGAGTCGGGGGGAGGCTTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGTACTCTCTGGACGCACCTTCAGTAACTATGGCATGTACTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAAGGTGTAGCAGGTATTGCGTGGCGTGGGGGCGGCACTATTTATGCAGAC TCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTCTATCTACAGCTGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTTCTGTGGAGCACTAGTGTTTCGGCGGTGGTAGGTGGGAAAGACCTGCCCACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCTCA; SA46 (SEQ ID NO.8): CAGGTGCAGCTCGTGGAGTCAGGTGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTATAACCTCTGGAAGCCGCTTCAGATTCGGTGCGGGGTGGTACCGCCAGGCTCCAGGGAAGTCCCGCGAGCGAGTCGCGGCTATTAATTTTGGTAGT TTTACAAATTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACGACGCCGCGAACACCTTGTATCTGCAAATGAACAACCTGAGACCTGAAGACACGGCCGTCTATTACTGTAATACAGGGAATTACTGGGGCGAGGGGACCCAGGTCACCGTCTCCTCA. .

[0046] Example 5: Screening and identification of the universality of phage display nanobodies paired with Fc fragments. Four phage display nanobodies were screened and paired with Fc fragments respectively to prove the universality of the obtained phage display nanobodies as probes to Fc fragments, and to screen out multiple pairs of highly sensitive and highly specific paired antibodies. The main steps included: (1) Coating 100 uL of Fc fragment (10 ug / ml) solution into the enzyme label wells and incubating overnight; (2) Washing the plate 5 times with 0.03% PBST and incubating with 3% skim milk powder at 37℃ for 2 h; (3) Washing the plate 5 times with 0.03% PBST and adding different dilution gradients (0-10) respectively. 8(4) Inactivated Staphylococcus aureus (CFU / mL) was incubated at 37°C for 1 hour; (5) The plate was washed 5 times with 0.03% PBST and then incubated with 2×10⁻⁶ ppm of PBST. 11 (5) PFU-screened phage-displaying nanobodies were incubated at 37°C for 60 min; (6) The plates were washed 5 times with 0.03% PBST, and 100 μL of HRP enzyme-antiphage monoclonal antibody was added to each well. The plates were incubated at 37°C for 60 min; (7) The plates were washed 5 times with 0.03% PBST, and 100 μL of TMB chromogenic solution and 50 μL of sulfuric acid stop solution were added sequentially. The plates were incubated at 37°C for 15 min and then identified; (8) Based on the recorded data, the standard curve was fitted using software to calculate the limit of detection; (9) Based on blank + 3 × standard deviation, the detection performance of phage-displaying nanobodies and Fc fragments as paired antibodies for detecting Staphylococcus aureus was calculated (R). 2 =0.99), with the lowest detection limit being 9550 - 146500 CFU / mL (Figure 3).

[0047] Example 6: Preparation of Phage-Displayed Nanobody Sensor 1. Pretreatment of Screen-Printed Electrode (SPE) (1) The screen-printed electrode was washed three times with ultrapure water and ultrasonically cleaned in 75% ethanol solution for 30 seconds, then rinsed with ultrapure water and set aside. The initial CV value and EIS value of the screen-printed electrode surface were measured and recorded. The obtained product here is SPE.

[0048] 2. Modification of gold nanoparticles (AuNPs): A three-electrode system was constructed by using a pretreated screen-printed electrode as the working electrode, a platinum wire as the counter electrode, and a saturated calomel electrode (SCE) as the reference electrode.

[0049] Gold nanoparticles were deposited on the surface of a screen-printed electrode using cyclic voltammetry in an electrolyte containing 1% HAuCl4 solution (electrolyte composition: 0.1 mol / L potassium chloride + 0.5 mmol / L potassium ferricyanide solution). The initial voltage was set to -0.2 V, the high voltage to 0.6 V, the low voltage to -0.2 V, and the termination voltage to -0.2 V, with 6 scan cycles. The electrode was then washed three times with ultrapure water and dried with nitrogen to obtain SPE@Au. Subsequently, 50 μL of 250 mM 3-mercaptopropionic acid (MPA) solution was added, and the electrode self-assembled overnight in the dark to obtain SPE@AU@MPA.

[0050] 3. SPE@AU@MPA modified with Fc fragment was washed three times with ultrapure water and dried under nitrogen. 50 μL of a mixed solution (0.05 mM EDC and 0.05 mM NHS dissolved in 0.01 M PB buffer) was added to the working electrode surface, and the reaction was carried out at 37°C for 30 minutes. After washing three times with ultrapure water and drying under nitrogen, 50 μL of a 10 μg / mL Fc fragment solution was added, and the reaction was carried out at 37°C for 60 minutes, yielding SPE@AU@MPA@Fc.

[0051] Then, 1% BSA was added dropwise to the surface of the working electrode, and the reaction was carried out at 37°C for 60 minutes. The working electrode was then washed again with ultrapure water and dried with nitrogen. SPE@AU@MPA@Fc@BSA was obtained here.

[0052] After each modification step, the working electrode is immersed in 5 mM potassium ferrocyanide / potassium ferrocyanide ([Fe(CN)6]6) 3- / 4- The modification process was monitored in real time using a base solution (containing 0.1 M KCl) by cyclic voltammetry (CV) or electrochemical impedance spectroscopy (EIS).

[0053] Results: After loading a layer of gold ions onto the surface of the screen-printed electrode, CV (Figure 4) and EIS (Figure 5) characterizations showed that the cyclic voltammetry peak and impedance of the electrode surface significantly increased and decreased, respectively. This is because the conductivity of gold ions is better than that of the carbon material in the screen-printed electrode, thus enhancing the electron transfer efficiency of the electrode surface after loading. However, after sequentially loading MPA, Fc fragments, and BSA, the electron transfer efficiency of the surface decreased, resulting in a significant decrease and increase in the cyclic voltammetry peak and impedance, respectively. These characterizations also demonstrate the success of each material modification step, laying the foundation for subsequent electrochemical testing.

[0054] Example 7. Conjugation of HRP enzyme with phage-displayed nanobodies The conjugation steps of horseradish peroxidase and phage-displayed nanobodies were performed according to the instructions of the HRP enzyme conjugation kit (Sangon Biotech (Shanghai) Co., Ltd.): (1) Take 500 μL of horseradish peroxidase solution into a 5 ml centrifuge tube, add 200 μL of HRP activation buffer, and react slowly at room temperature for 30 minutes on a rotary suspender.

[0055] (2) Add 200 μL of HRP coupling buffer and let stand for 30 minutes. The solution slowly turns back to brownish-yellow.

[0056] (3) Add 10 12CFU phage-displayed nanobody (volume controlled at around 1 mL) is gently mixed with a pipette and then transferred to a dialysis bag. One end of the dialysis bag is first secured with a dialysis clamp, the liquid is filled in, and then the other end is secured. Finally, the dialysis bag is placed in 2 L of coupled dialysis solution. The container containing the dialysis solution is placed on a magnetic stirrer and dialyzed at room temperature for 2 hours, or dialyzed overnight at 4°C.

[0057] (4) After dialysis, transfer the liquid in the dialysis bag to a 5 ml centrifuge tube, add 100 μL of reducing agent, let stand at room temperature for 2 hours, and gently mix with a pipette 2-3 times every half hour to complete the coupling.

[0058] Example 8, Performance evaluation of phage-displayed nanobody sensor (1) Electrodes were prepared according to the method in Example 6. PBS solutions containing different concentrations of inactivated Staphylococcus aureus were prepared according to the concentration range of inactivated Staphylococcus aureus: 0–10. 9 The solution was diluted to CFU / mL and added dropwise to the surface of the screen-printed electrode. The electrode was then incubated at 37 °C for 30 minutes.

[0059] (2) After washing with ultrapure water three times and drying with nitrogen, 10 μL of the HRP enzyme-labeled phage display nanobody prepared in Example 7 was dropped onto the electrode surface and incubated for 30 min.

[0060] (3) Rinse three times with ultrapure water and dry with nitrogen. Connect the sensor to the electrochemical workstation and place it in a solution containing 2×10⁻⁶ nitrogen. -5 M-catechol and 7×10 -5 M hydrogen peroxide CBS buffer (CBS buffer is made of 5 × 10⁻⁶) -2 M citrate and 5×10 -2 The sample was mixed with phosphate buffer (PB) and the pH was adjusted to 5.0 for square wave voltammetry (SWV) detection. The SWV detection parameters were set as follows: step voltage (ΔES) of 0.005 V, scan potential range of 0–0.6 V, square wave amplitude (ΔESW) of 0.025 V, and frequency (f) of 25 Hz. By recording the data during the detection process and combining the correlation between concentration and redox peaks, a calibration curve was plotted to calculate its linear range and minimum detectable concentration.

[0061] (4) Based on blank + 3 × standard deviation, the linear standard curve (R0) for detecting Staphylococcus aureus by the phage-displaying nanobody sensor was calculated to show excellent linearity. 2 =0.99), with a minimum detection limit of 158-2455 CFU / mL, exhibiting outstanding sensitivity (Figure 6).

[0062] Example 9: Detection using a phage-displayed nanobody sensor. The phage-displayed nanobody sensor simultaneously identifies Staphylococcus aureus (10...). 8 CFU / mL), Escherichia coli ATCC25922 (10 8 CFU / mL), Pseudomonas aeruginosa ATCC27853 (10 8 CFU / mL), Salmonella ATCC14028 (10 8 CFU / mL), Shigella ATCC13313 (10 8 CFU / mL), SARS-CoV-2 N protein (1 μg / mL), norovirus GIII.4 protein (1 μg / mL), and recombinant bovine rotavirus major inner shell protein (VP6) (1 μg / mL). The specific steps are as follows: Add 50 μL of 10... 8 Incubate with a CFU / mL solution of Staphylococcus aureus and the above-mentioned control strain or antigen solution for 20 min, rinse with deionized water and dry the surface with nitrogen, then add 50 μL of 1×10⁻⁶ CFU / mL solution of Staphylococcus aureus. 12 After incubating the PFU / mL enzyme-linked phage-displaying nanobody (prepared according to Example 7) for 20 min, rinsed and dried with nitrogen gas, the sensor was finally connected to the electrochemical workstation and placed in a solution containing 9 × 10⁻⁶ PFU / mL enzyme-linked phage display nanobody. -5 M H2O2 (hydrogen peroxide) and 4×10 -5 Square wave voltammetry was performed on MH2Q (catechol) in carbonate buffer solution (CBS), with a measurement range of 0 to +0.6 V.

[0063] The results showed that the sensor produced a significant electrochemical signal when detecting Staphylococcus aureus, but no significant electrochemical signal was generated when detecting other bacteria or viruses. These results demonstrate that the prepared sensor has good selectivity and specificity (Figure 7).

[0064] Example 10: Sample Detection. Staphylococcus aureus (10 μg / L) was added to PBS buffer, wastewater samples (wastewater discharge samples from West China Hospital), milk samples, and nasal swab samples (from clinical laboratory samples of West China Hospital). 8 (CFU / mL) was identified using a phage-displayed nanobody sensor: 50 μL of the above sample was added to the surface of the working electrode, incubated for 20 min, rinsed with deionized water, and dried with nitrogen. Then, 50 μL of 1×10⁻⁶ CFU / mL was added. 12 After incubating the PFU / mL enzyme-linked phage-displaying nanobody (prepared according to Example 7) for 20 min, rinsed and dried with nitrogen gas, the sensor was finally connected to the electrochemical workstation and placed in a solution containing 9 × 10⁻⁶ PFU / mL enzyme-linked phage display nanobody. -5M H2O2 (hydrogen peroxide) and 4×10 -5 Square wave voltammetry was performed in a carbonate buffer solution (CBS) of MH2Q (catechol), with a measurement range of 0 to +0.6 V. The results showed that the sensor can accurately identify Staphylococcus aureus in different media, almost unaffected by the matrix, indicating that the sensor has excellent accuracy and is suitable for rapid screening of Staphylococcus aureus in different scenarios (Figure 8).

Claims

1. A nanobody targeting Staphylococcus aureus, characterized in that, Its amino acid sequence is: QLQLVESGGGLVQAGGSMRLSCAASGRTFSTNTMGWFRQAPGKEREFVAGIRWISGSTSYADSVKGRFTISRDNAKNTLFLQMNSLKPEDTAVYYCAAGPHNIPILRTSAYNYWGQGTQVTVSS.

2. A gene encoding the nanobody of claim 1.

3. The gene according to claim 2, characterized in that, Its nucleotide sequence is: CAGTTGCAGCTCGTGGAGTCGGGGGGAGGATTGGTGCAGGCTGGGGGCTCCATGAGACTCTCCTGTGCAGCCTCTGGACGCACCTTCAGTACGAATACCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTTGTAGCAGGTATTAGGTGGATTAGTGGTAGCACAAGCTATGC AGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTCTTTCTGCAAATGAACAGCCTGAAACCTGAGGACACGGGCCGTGTATTACTGTGCGGCGGGCCCCATAATATACCGATACTTCGTACTTCGGCTTATAACTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCTCA.

4. The use of the nanobody of claim 1 in the preparation of nanobody probes targeting Staphylococcus aureus.

5. A nanobody probe targeting Staphylococcus aureus, characterized in that, It contains the nanobody as described in claim 1.

6. The use of the nanobody of claim 1 or the nanobody probe of claim 5 in the preparation of a Staphylococcus aureus detection kit.

7. A Staphylococcus aureus detection kit, characterized in that, It contains the nanobody of claim 1 or the nanobody probe of claim 5.

8. The Staphylococcus aureus detection kit according to claim 7, characterized in that, It also contains an electrochemical biosensor, which is a three-electrode system: a working electrode, a counter electrode, and a reference electrode; the working electrode is a screen-printed electrode modified with gold nanoparticles, the gold nanoparticles being connected to Fc antibodies; the counter electrode is a platinum wire; and the reference electrode is a saturated calomel electrode.

9. The application of the nanobody of claim 1, the nanobody probe of claim 5, or the Staphylococcus aureus detection kit of claim 7 or 8 in the detection of Staphylococcus aureus.

10. A method for quantitatively detecting Staphylococcus aureus concentration using the nanobody probe of claim 5, comprising the following steps: (1) The sample solution is dropped onto the working electrode surface of the electrochemical biosensor, incubated, and the electrode surface is thoroughly washed with phosphate-buffered saline. Then, the nano-antibody probe described in claim 5 is dropped onto the working electrode surface, incubated, washed, dried, and the electrochemical biosensor is placed in a CBS buffer containing catechol and hydrogen peroxide and connected to an electrochemical workstation for square wave voltammetry testing. (2) The measured current is used to calculate the concentration of Staphylococcus aureus according to the standard curve Y = 1.12x + 0.23, where x is the concentration of Staphylococcus aureus and y is the current.

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