Sensor for displaying nano antibody based on bacteriophage and application of sensor

Abstract

The invention discloses a sensor based on a phage display nano antibody and application of the sensor. The amino acid sequence of the phage display nano antibody is EVQLVESGGLVQAGGSLRLSCATSG RTFSTFSTYVAGWFRQAPGKEREFVAAAIRRTGGRTYYADSVKGRFTISGDNAKNMVYLQMNSLKPEDTATYYCAAANNLGSDYVTVTGKYDYWGQGTQVIVSS, and the amino acid sequence of the phage display nano antibody is shown in the specification. According to the invention, the staphylococcus aureus-targeted specific bacteriophage display nano-antibody is screened as a high-performance probe, and is paired with an Fc fragment to construct a high-quality recognition element, so that the defects of instability and accuracy of a traditional probe are overcome. Staphylococcus aureus can be qualitatively detected within one minute, and the method has high sensitivity and excellent specificity and accuracy.

Description

Technical Field

[0001] This invention relates to a phage-displayed nanobody sensor and its application, belonging to the field of immunoassay. Background Technology

[0002] Staphylococcus aureus ( S. aureus Staphylococcus aureus (S. aureus) is one of the most virulent epidemic pathogens, commonly found in wastewater, food, and clinical environments. It can cause serious illnesses including organ inflammation, sepsis, septic shock, and multiple organ failure, resulting in millions of infections and over 100,000 deaths annually, posing a continuous threat to clinical health, food safety, and environmental safety. Timely detection is an effective means of containing Staphylococcus aureus infections and optimizing treatment. Therefore, there is an urgent need to develop rapid, accurate, and efficient detection tools.

[0003] Currently, culture methods remain the gold standard for Staphylococcus aureus detection due to their high accuracy. However, this method is time-consuming (24-48 hours), prone to false negatives for viable but unculturable cells, and poses safety risks. To improve detection efficiency, molecular or immunological techniques are often used to replace culture methods for Staphylococcus aureus screening. Molecular detection methods offer high sensitivity, as exemplified by the patent "Primer Set for Staphylococcus aureus and Reagent Kit for Detection or Identification of Staphylococcus aureus" (application number: CN202411109634.0), but these methods require complex procedures, specialized instruments, and professional personnel, limiting their speed and versatility. In contrast, immunoassay methods offer advantages such as speed, simplicity, and portability, making them attractive for Staphylococcus aureus detection, as reported in the patent "A Test Strip for Rapid Detection of Methicillin-Resistant Staphylococcus aureus and Its Application" (application number: CN202510255539.X). However, current immunoassays rely on high-cost, unstable, and low-sensitivity monoclonal antibodies as probes, resulting in insufficient detection system performance. Furthermore, the SpA protein on the surface of Staphylococcus aureus binds to the Fc terminus of monoclonal antibodies, limiting current immunoassays to quantitative detection of bacterial secretions and qualitative analysis of the bacteria, severely interfering with detection accuracy. To address these challenges, novel immunoassays based on dual nanobody sandwich structures have been developed, such as the patent "A Nanobody Targeting Staphylococcus aureus and Its Preparation Method and Application" (Authorization Announcement No.: CN119798426B), which boasts high accuracy but still faces limited sensitivity. Therefore, the development of high-performance detection probes targeting Staphylococcus aureus remains necessary.

[0004] Phage-displayed nanobodies possess unique performance advantages, including the absence of Fc fragments, a high-affinity pentammeric conformation, excellent stability, and strong environmental tolerance, making them ideal candidate probes for rapid detection of Staphylococcus aureus. Furthermore, leveraging the SpA-antibody specific interaction, high-performance detection probes can be efficiently assembled by pairing phage-displayed nanobodies with virtually any conventional antibody. This strategy avoids the high cost and low efficiency of traditional monoclonal antibody or nanobody pair screening, providing a feasible pathway for designing next-generation immunoassay methods. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide a highly sensitive phage-displayed nanobody sensor for Staphylococcus aureus and its application.

[0006] Technical Solution: This invention provides a nanobody targeting Staphylococcus aureus, with the following amino acid sequence: EVQLVESGGGLVQAGGSLRLSCATSGRTFSTFSTYVAGWFRQAPGKEREFVAAIRRTGGRTYYADSVKGRFTISGDNAKNMVYLQMNSLKPEDTATYYCAAANNLGSDYVTVTGKYDYWGQGTQVIVSS. 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.

[0007] The present invention also provides a nucleotide sequence encoding the nanobody, the nucleotide sequence of which is shown below: SA2 (SEQ ID NO.5): CAGTTGCAGCTCGTGGAGTCGGGGGAGGATTGGTGCAGGCTGGGGGCTCCATGAGACTCTCCTGTGCAGCCTCTGGACGCACCTTCAGTACGAATACCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCAGGTATTAGGTGGATTAGTGGTAGCACAAGCTATGCAGAC TCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTCTTTCTGCAAATGAACAGCCTGAAACCTGAGGACACGGGCCGTGTATTACTGTGCGGCGGGCCCCCATAATATACCGATACTTCGTACTTCGGCTTATAACTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCTCA; SA10(SEQ ID NO.6): GAGGTGCAGCTGGTGGAGTCGGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAACCTCTGGACGCACCTTCAGTACCTTCAGTACCTATGTCGCGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCAGCTATTAGGCGGACTGGTGGTCGCACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCGGAGACAACGCCAAGAACATGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCACTTATTACTGTGCAGCAGCTAACAACCTGGGTAGCGACTATGTGACAGTGACGGGAAAGTATGACTACTGGGGCCAGGGGACCCAGGTCATCGTCTCCTCA; SA31(SEQ ID NO.7): CAGTTGCAGCTCGTGGAGTCGGGGGGAGGCTTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGTACTCTCTGGACGCACCTTCAGTAACTATGGCATGTACTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAAGGTGTAGCAGGTATTGCGTGGCGTGGGGGCGGCACTATTTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTCTATCTACAGCTGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTTCTGTGGAGCACTAGTGTTCGGCGGTGGTAGGTGGGAAAGACCTGCCCACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA; SA46(SEQ ID NO.8): GAGGTGCAGGTGGTGGAGTCGGGGGGAGGCTTGGTGCAGCCTGGGGGATCTCTGAGACTCTCCTGTGCAGCCTCTACAAGCATCGCCAGGATCGATGTCATGGGCTGGTACCGCCAGGCTCCAGGGAAGCAGCGCGAGTTGGTCGCATCGCTTACAGGTGGTGGTACGACAACCTATGCAGA CTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACACCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCGGAGGACACGGCCGTCTATTACTGTAATAAAAACCGCCCAGGTAGTACGTGGTCACGCGGTTATAATTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCTCA.

[0008] The present invention also provides the application of the nanobody in the preparation of nanobody probes targeting Staphylococcus aureus.

[0009] The present invention also provides a nanobody probe targeting Staphylococcus aureus, which contains the nanobody.

[0010] The present invention also provides the application of the nanobody or the nanobody probe in the preparation of a Staphylococcus aureus detection kit.

[0011] The present invention also provides a Staphylococcus aureus detection kit containing the nanobody or the nanobody probe.

[0012] It also contains a nanobody sensor, 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 blocked by bovine serum albumin (BSA). The counter electrode is a platinum wire, and the reference electrode is a saturated calomel electrode.

[0013] 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.

[0014] The present invention also provides a method for detecting Staphylococcus aureus using the aforementioned nanobody probe, comprising the following steps: (1) The sample solution was dropped onto the working electrode surface of the electrochemical biosensor and incubated. The electrode surface was thoroughly washed with phosphate buffered saline. Then, nanobody probes (HRP enzyme-labeled phage display nanobodies) were dropped onto the working electrode surface, incubated, washed, and dried. The electrochemical biosensor was placed in CBS buffer containing catechol and hydrogen peroxide and connected to the electrochemical workstation for square wave voltammetry testing. (2) When phage-displayed nanobody SA-2 is used as a nanobody probe and the current exceeds 3.69 μA, it proves that Staphylococcus aureus is present in the sample solution; when phage-displayed nanobody SA-10 is used as a nanobody probe and the current exceeds 2.09 μA, it proves that Staphylococcus aureus is present in the sample solution; when phage-displayed nanobody SA-31 is used as a nanobody probe and the current exceeds 1.76 μA, it proves that Staphylococcus aureus is present in the sample solution; when phage-displayed 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.

[0015] The present invention also provides a method for quantitative detection of Staphylococcus aureus using the aforementioned nanobody probe, comprising the following steps: (1) The sample solution was dropped onto the working electrode surface of the electrochemical biosensor and incubated. The electrode surface was thoroughly washed with phosphate buffered saline. Then, nanobody probes (HRP enzyme-labeled phage display nanobodies) were dropped onto the working electrode surface, incubated, washed, and dried. The electrochemical biosensor was placed in CBS buffer containing catechol and hydrogen peroxide and connected to the electrochemical workstation for square wave voltammetry testing. (2) When using phage-displayed nanobody SA-2 as a nanobody probe, the concentration of Staphylococcus aureus was calculated based on the standard curve Y = 1.12x + 0.23, and the limit of detection was 158 CFU / mL; when using phage-displayed nanobody SA-10 as a nanobody probe, the concentration of Staphylococcus aureus was calculated based on the standard curve Y = 1.05x - 0.77, and the limit of detection was 725 CFU / mL; when using phage-displayed nanobody SA-31 as a nanobody probe, the concentration of Staphylococcus aureus was calculated based on 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 based on the standard curve Y = 1.07x - 1.733, and the limit of detection was 2455 CFU / mL. CFU / mL; where x is the concentration of Staphylococcus aureus (CFU / mL) and y is the current (μA). The parameters for the square wave voltammetry test are set as follows: step voltage (ΔES) of 0.005V, scan potential range of 0–0.6V, square wave amplitude (ΔESW) of 0.025V, and frequency (f) of 25 Hz.

[0016] The present invention also provides CDR1, CDR2 and CDR3 encoding genes that encode the phage-displaying nanobody.

[0017] The nucleotide sequence of the gene is shown in SEQ ID NO: 6.

[0018] 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, and screening out multiple pairs of highly sensitive and specific paired antibodies. The main steps include: (1) The Fc fragment was coated in the enzyme label wells and incubated overnight; (2) Wash the plate 5 times with 0.03% PBST, and incubate with 3% skim milk powder at 37℃ for 1-2 h; (3) Wash the plate 5 times with 0.03% PBST, and add different dilution gradients (0-10). 8 Inactivated Staphylococcus aureus (CFU / mL) was incubated at 37°C for 1 hour. (4) Wash the plate 5 times with 0.03% PBST, and add 10 11 -10 12 CFU-selected phage-displaying nanobodies were incubated at 37°C for 45-60 min. (5) Wash the plate 5 times with 0.03% PBST, add 100 μL of anti-M13 secondary antibody to each well, and incubate at 37℃ for 45-60 min; (6) Wash the plate 5 times with 0.03% PBST, add 100 μL TMB colorimetric solution and 50 μL sulfuric acid stop solution in sequence, incubate at 37℃ for 10-15 min and then identify.

[0019] (7) Based on the recorded data, use software to fit the standard curve and calculate the minimum detection limit.

[0020] Furthermore, this invention utilizes the paired phage-displaying nanobodies and Fc fragments as coating and labeling antibodies, respectively, to develop a sandwich sensor. The coating antibody is the Fc fragment, and the labeling antibody is a horseradish peroxide (HRP) enzyme-labeled phage-displaying nanobodies. The main steps include: (1) Clean the screen-printed electrode with ultrapure water, and then place it in a 75% ethanol solution for ultrasonic cleaning for 30 seconds. Rinse it with ultrapure water and set aside for use.

[0021] (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.

[0022] (3) 0.01 M PB buffer containing 0.05-0.1 mM EDC / NHS was added to the electrode surface and reacted at 37°C for 30 minutes. Subsequently, 5-10 µg / mL Fc fragment and 1% BSA were added sequentially and reacted at 37°C for 60 minutes. The electrode was then rinsed again with ultrapure water and dried with nitrogen. After each modification step, the electrode was immersed in 5 mM potassium ferricyanide / potassium ferrocyanide ([Fe(CN)6)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).

[0023] (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 and incubated for 30 minutes.

[0024] (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.

[0025] 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.

[0026] (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.

[0027] (3) The sandwich sensor constructed in this invention can achieve 158-2455 The detection limit for CFU / mL is much lower than that of existing immunological methods.

[0028] (4) The sandwich sensor constructed in this invention can qualitatively detect Staphylococcus aureus within 1 minute. It has high sensitivity, excellent specificity and accuracy, and can realize multi-scenario, real-time and in-situ detection of Staphylococcus aureus. Attached Figure Description

[0029] Figure 1 The results show that 55 phage clones were selected and positive clones were verified by Phage-ELISA; the horizontal axis represents the number of the phage display nanobody monoclonal clone, and the vertical axis represents the absorbance at 450 nm. Figure 2 The results of Phage-ELISA verification of the specificity of 22 positive phages are shown; the horizontal axis represents the number of the phage display nanobody monoclonals coated on the ELISA plate; the vertical axis represents the absorbance at 450 nm. Figure 3 This is a calibration curve for detecting Staphylococcus aureus using a sandwich ELISA based on phage-displayed nanobodies; the horizontal axis represents the concentration of Staphylococcus aureus introduced; the vertical axis represents the absorbance at 450 nm; where, Figure 3 A~ Figure 3D is SA-2, SA-10, SA-31 and SA-46 in order; Figure 4 The identification of cyclic voltammetry peaks during the fabrication of phage-displayed nanobody sensors is based on this data; the horizontal axis represents the electrode loading material printed by screen printing; and the vertical axis represents the detection current value. Figure 5 This is an identification of impedance values ​​during the fabrication of a phage-display nanobody sensor; the horizontal axis represents the electrode loading material printed by screen printing; the vertical axis represents the detection impedance value. Figure 6 This is a calibration curve for detecting Staphylococcus aureus using a sandwich sensor based on bacteriophage-displayed nanobodies; the horizontal axis represents the concentration of Staphylococcus aureus introduced; the vertical axis represents the detection current value; where, Figure 6 A~ Figure 6 D is SA-2, SA-10, SA-31 and SA-46 in order; Figure 7 This is an identification of the specificity of a sandwich sensor based on phage-displayed nanobody for detecting Staphylococcus aureus; the horizontal axis represents the concentration of Staphylococcus aureus introduced; the vertical axis represents the detection current value; where, Figure 7 A~ Figure 7 D is SA-2, SA-10, SA-31 and SA-46 in order; Figure 8 This study evaluates the accuracy of a sandwich sensor based on phage-displayed nanobody in detecting Staphylococcus aureus in different media; the horizontal axis represents the concentration of Staphylococcus aureus introduced; and the vertical axis represents the detection current value. Figure 8 A~ Figure 8 D is SA-2, SA-10, SA-31 and SA-46 in order. Detailed Implementation

[0030] The main instruments and materials used in this embodiment of the invention 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 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 (facility sequence number 1534918401; PDB number: 6F2Z_A in the NCBI database; 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: dkthtsppspapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlng) The keykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvslwclvkgfypsdiavewesngqpennykttppvldsdgsfflysaltvdksrwqqgnvfscsvmhealhnhytqkslslspgk) were 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 immunophage display nanobody library was constructed by Chengdu Apak Biotechnology Co., Ltd. The novel coronavirus N protein was purchased from Shanghai Beyotime Biotechnology Co., Ltd., catalog number P2328-1mg; the norovirus GII.4 protein was purchased from Beijing Sinopharm Shenzhou Technology Co., Ltd., catalog number 41002-V10E; and the recombinant rotavirus capsid protein (VP6) was purchased from Beijing Sinopharm Shenzhou Technology Co., Ltd., catalog number 906-BRV0040.

[0031] Example 1: Screening of phage-displaying nanobodies targeting Staphylococcus aureus Three rounds of screening were conducted following the steps of "coating-adsorption-washing-elution-amplification-identification": (1) After wetting the 96-well microplate with sterile ultrapure water, place it in a clean bench and sterilize it by irradiating it with ultraviolet light for half an hour. (2) Take 1×10 8 CFU / mL of Staphylococcus aureus was inactivated and placed in an ELISA plate and incubated overnight at 4°C. (3) In a sterile operating table, wash three times with sterile 0.1% TBST washing solution, pat dry on sterile paper each time, and then add 250-350 μL of filtered sterile 1-3% BSA-TBS blocking solution and seal in a 37 ℃ constant temperature incubator 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. Take 2×10⁻⁶ nanobody libraries for immunophage display. 11 Add 100 μL of sterile 1×TBS to each well (pfu / well), mix well, and then add to the above-mentioned microplate. Incubate at 37°C for 1 h. (5) Wash the plate three times with 0.1% TBST, patting it dry on sterile paper each time. Add 100 μL of Gly-HCl (adjust pH=2.2 with hydrochloric acid in 0.2M glycine, autoclave for 15 min) elution buffer, and elute at 37 ℃ with constant shaking for 8-15 min. Aspirate 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, autoclave for 15 min) to neutral. (6) Take 10-15 μL of phage for titer determination, and use the rest for phage amplification.

[0032] (7) Streak inoculation on LB / Tet (tetracycline) solid medium plates. E. coli ER2738, incubate at 37 ℃ for 12-16 h; (8) Pick a single colony from the plate and inoculate it into a test tube of LB / Tet liquid medium, and incubate at 37 ℃ and 220 rpm for 12-16 h with shaking. (9) Take 100-500 μL of the above culture into 40-60 mL of LB / Tet liquid medium, add the eluted phage, and culture at 37 ℃ and 220 rpm for 4-6 h with shaking. (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 volume of the supernatant) of sterile 10-20% PEG-NaCl solution (50g PEG-8000, 36g NaCl, dissolved in ultrapure water by heating, brought to a final volume of 250mL, and autoclaved for 15min) to the supernatant, 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 of 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 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) constitute the first round of amplification. The second and third rounds of selection are largely the same, with the amount of phage introduced in each round being 2×10⁻⁶. 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.

[0033] Table 1. Screening process for phage-display nanobodies targeting Staphylococcus aureus

[0034] Example 2: Determination of phage titer (1) Streak inoculation on LB / Tet solid medium plates. E. coli ER2738, incubated at 37 ℃ for 12-16 h; (2) Pick a single colony from the plate and inoculate it into LB / Tet liquid medium, and culture at 37 ℃ and 220 rpm with shaking until the logarithmic growth phase (OD). 600nm ≈ 0.5 - 0.6); (3) Take 200 μL of bacterial solution from step (2) into sterile 1.5 mL centrifuge tubes respectively; (4) Dilute the eluted phage to 10 -2 10 -3 10 -4 Double the amount of agar in a microwave oven until melted; (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 °C for 10-15 min; (6) Inject the bacterial suspension infected with bacteriophage into the top agar, shake gently to mix, and quickly pour it onto LB / IPTG / X-gal plates and incubate at 37 °C for 12-16 h; (7) Pick a plate with about 30 to 300 blue spots to count and calculate the phage titer. The calculation formula is: Colony forming unit (CFU) = number of colonies in the plate × dilution factor × 100%.

[0035] 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 identification by phage-ELISA. The specific steps are as follows: (1) Select a single colony clone and inoculate it into LB / Tet liquid medium, and incubate at 37 ℃ and 220 rpm for 12-16 h; (2) Take 500 μL of overnight solution E.coil Add the ER2738 culture to 50 mL of liquid culture medium and mix well; (3) Dispense 1 mL of the culture medium from (2) into centrifuge tubes; (4) Pick a single blue phage plaque into each centrifuge tube and incubate at 37 ℃ and 220 rpm for 4.5-6 h with shaking. (5) After the culture is completed, centrifuge at 8000 rpm for 2 min, and transfer the supernatant to a fresh centrifuge tube and label it. Let it stand at 4 ℃ for later use.

[0036] (6) Take 10 from each hole 8 CFU-inactivated Staphylococcus aureus was coated onto an ELISA plate and incubated overnight at 4°C. (7) Wash the plate three times with 0.05% PBST, add 300 μL of 5% skim milk to block, and incubate at 37 ℃ for 1-2 h; (8) Wash the plate three times with 0.05% PBST, add 100 µL of phage display nanobody supernatant culture medium, and incubate at 37 ℃ for 45-60 min; (9) 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 ℃ for 45-60 min; (10) Wash the plate three times with 0.05% PBST, add 100 µL TMB (tetramethylbenzidine) colorimetric solution, and incubate at 37 °C for 8-15 min; (11) Add 50 µL of 2 M H2SO4 (sulfuric acid) to each well and measure the OD. 450 nm Absorbance value.

[0037] (12) Of the 55 clones selected, 22 phage clones exhibited nanobody clones that could bind to Staphylococcus aureus. Positive clones (OD) were selected. 450 >1.5) to proceed with the next step of specificity verification ( Figure 1 ).

[0038] Example 4: Identification of the specificity of nanobodies displayed by positive phages (1) Add 100 μL of a 10% concentration to each well. 9 CFU / mL Staphylococcus aureus, 3 μg / mL bovine serum albumin (BSA) protein and 3 μg / mL chicken oocyte albumin (OVA) protein were coated onto the ELISA plate, and three parallel control groups were set up and incubated overnight at 4 ℃; (2) Wash the plate three times with 0.05% PBST, add 300 μL of 5% skim milk to block, and incubate at 37 ℃ for 2 h; (3) Wash the plate three times with 0.05% PBST, add the supernatant culture medium of the above 22 phage display nanobody monoclonals (100 µL per monoclonal), and incubate at 37 °C for 60 min; (4) Wash the plate three times with 0.05% PBST, add 100 µL of HRP enzyme-antiphage monoclonal antibody to each well, and incubate at 37 °C for 45-60 min; (5) Wash the plate three times with 0.05% PBST, add 100 µL TMB colorimetric solution, and incubate at 37 ℃ for 15 min; (6) Add 50 µL of 2 M H2SO4 to each well and measure the absorbance at OD450 nm.

[0039] (7) Among the 22 positive phage plaques identified, all selected phage-displaying nanobody clones showed good affinity and specificity. Figure 2Therefore, we sent all phage display antibodies for sequencing. The sequencing alignment results showed that four novel phage display nanobodies (SEQ ID NO. 1~4) were screened. The sequences of CDR1, CDR2 and CDR3 are shown below: SA2 (SEQ ID NO.1): QLQLVESGGGLVQAGGSMRLSCAASGRTFSTNTMGWFRQAPGKEREFVAGIRWISGSTSYADSVKGRFTISRDNAKNTLFLQMNSLKPEDTAVYYCAAGPHNIPILRTSAYNYWGQGTQVTVSS; CDR1: GRTFSTNT; CDR2: IRWISGST; CDR3: AAGPHNIPILRTSAYNY.

[0040] SA10 (SEQ ID NO.2): EVQLVESGGGLVQAGGSLRLSCATSGRTFSTFSTYVAGWFRQAPGKEREFVAAIRRTGGRTYYADSVKGRFTISGDNAKNMVYLQMNSLKPEDTATYYCAAANNLGSDYVTVTGKYDYWGQGTQVIVSS; CDR1: GRTFSTFSTYV; CDR2: IRRTGGRT; CDR3: AAANNLGSDYVTVTGKYDY.

[0041] SA31 (SEQ ID NO.3): QLQLVESGGGLVQAGDSLRLSCVLSGRTFSNYGMYWFRQAPGKEREGVAGIAWRGGGTIYADSVKGRFTISRDNAKNTVYLQLNSLKPEDTAVYFCGALVFGGGRWERPAHWGQGTQVTVSS; CDR1: GRTFSNYG; CDR2: IAWRGGGT; CDR3: GALVFGGGRWERPAH.

[0042] SA46 (SEQ ID NO.4): QLQLVESGGGMVQPGGSLRLSCAASGFTLSFHDMSWVRQAPGKGPEWVSTIKRGGGMTSYADSVKGRFTISRDNAENKLYLQMNSLKPEDTALYRCARRIDHVAGWGKGYWGQGTQVTVSS; CDR1: GFTLSFHD; CDR2: IKRGGGMT; CDR3: ARRIDHVAGWGKGY.

[0043] Example 5: Identification of the universality of phage display nanobodies paired with Fc fragments The four selected phage-displaying nanobodies were paired with Fc fragments to demonstrate the universality of the obtained phage-displaying nanobodies as probes for pairing with Fc fragments. Multiple pairs of highly sensitive and specific paired antibodies were also screened. The main steps included: (1) Coat 100 μL of Fc fragment (10 ug / ml) solution into the enzyme label wells and incubate overnight; (2) Wash the plate 5 times with 0.03% PBST, and incubate with 3% skim milk powder at 37°C for 2 h; (3) Wash the plate 5 times with 0.03% PBST, and add different dilution gradients (0-10). 8 Inactivated Staphylococcus aureus (CFU / mL) was incubated at 37°C for 1 hour. (4) Wash the plate 5 times with 0.03% PBST, and add 2×10 11 PFU-selected phage-displaying nanobodies were incubated at 37°C for 60 min. (5) Wash the plate 5 times with 0.03% PBST, add 100 μL of HRP enzyme-antiphage monoclonal antibody to each well, and incubate at 37℃ for 60 min; (6) Wash the plate 5 times with 0.03% PBST, add 100 μL TMB colorimetric solution and 50 μL sulfuric acid stop solution in sequence, incubate at 37℃ for 15 min and then identify.

[0044] (7) Based on the recorded data, use software to fit the standard curve and calculate the minimum detection limit.

[0045] (8) Based on blank + 3 × standard deviation, the phage-displaying nanobody and Fc fragment as paired antibodies have excellent detection performance for Staphylococcus aureus (R). 2 =0.99), the lowest detection limit is 9550 - 146500 CFU / mL ( Figure 3 ).

[0046] Example 6: Fabrication of a phage-displayed nanobody sensor 1. Pretreatment of screen-printed electrodes (SPE) (1) The screen-printed electrode was cleaned three times with ultrapure water, and then ultrasonically cleaned in 75% ethanol solution for 30 seconds. It was then rinsed with ultrapure water and set aside for use. The initial CV value and EIS value of the screen-printed electrode surface were measured and recorded. The SPE obtained here is as follows.

[0047] 2. Modification of gold nanoparticles (AuNPs) A three-electrode system was constructed by using a pre-treated screen-printed electrode as the working electrode, a platinum wire as the counter electrode, and a saturated calomel electrode (SCE) as the reference electrode.

[0048] 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.

[0049] 3. Fc fragment modification SPE@AU@MPA 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. The electrode was then washed three times with ultrapure water and dried 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.

[0050] 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.

[0051] After each modification step, the working electrode is immersed in 5 mM potassium ferrocyanide / potassium ferrocyanide ([Fe(CN)6)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).

[0052] Results: After loading a layer of gold ions onto the surface of the screen-printed electrode, CV ( Figure 4 ) and EIS ( Figure 5 Characterization revealed that the cyclic voltammetry peak and impedance of the electrode surface showed significant increases and decreases, respectively. This is because the conductivity of gold ions is better than that of the carbon material in the screen-printed electrode, 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, leading to significant decreases and increases 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.

[0053] Example 7: Conjugation of HRP enzyme with phage-displayed nanobodies The conjugation procedure between horseradish peroxidase and phage-displayed nanobodies was performed according to the instructions of the HRP 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.

[0054] (2) Add 200 μL of HRP coupling buffer and let stand for 30 minutes. The solution slowly turns back to brownish-yellow.

[0055] (3) Add 10 12 CFU 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.

[0056] (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.

[0057] Example 8: Performance Qualification of Phage-Displayed Nanobody Sensor (1) Electrodes were prepared according to the method in Example 6. PBS solutions containing different dilutions 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.

[0058] (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.

[0059] (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.

[0060] (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 ).

[0061] Example 9: Detection using a phage-based sensor displaying nanobodies A phage-based sensor displaying nanobodies can simultaneously identify 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), novel coronavirus 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 in a carbonate buffer solution (CBS) of MH2Q (catechol), with a measurement range of 0 to +0.6 V.

[0062] 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 ).

[0063] Example 10, Sample Testing Staphylococcus aureus (10 μg / L) was added to PBS buffer, wastewater samples (from the West China Hospital wastewater treatment plant), milk samples, and nasal swab samples (from clinical laboratory samples at 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. Finally, the sensor was 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 in MH2Q (catechol) carbonate buffer solution (CBS), 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 various scenarios. Figure 8 ).

Claims

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

2. A gene encoding the nanobody of claim 1.

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

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 or 6 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 4 or 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 detecting Staphylococcus aureus 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 nanoantibody 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) When the current exceeds 2.09 μA, it proves that Staphylococcus aureus is present in the sample solution.

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