A novel nanoantibody electrochemical immunosensor and its preparation method and application

Abstract

The present invention discloses a novel nano-antibody electrochemical immunosensor and its preparation method and application. The sensor comprises a biorecognition element for identifying a target protein, and a substrate electrode for loading the biorecognition element and converting the interaction between the biorecognition element and the target protein into an electrical signal. The biorecognition element is a nano-antibody; the substrate electrode is a glassy carbon electrode modified with carboxyl carbon nanotubes. The nano-antibody is obtained by recombining the gene of a virus containing the target protein with a prokaryotic expression vector to obtain a recombinant vector, which is then expressed prokaryotically. The sensor of the present invention has good affinity, strong specificity, good stability, low detection limit, quantification limit, and high sensitivity.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial identification and detection in the environment, and in particular to a novel nano-antibody electrochemical immunosensor and a preparation method and application thereof. Background Art

[0002] The environment, especially water bodies, harbors a wide variety of microorganisms that can cause a variety of diseases and pose a threat to human health. Pathogens include Escherichia coli, Salmonella, Campylobacter, Helicobacter, and Vibrio, while pathogenic viruses include enterovirus, adenovirus, hepatitis virus, and rotavirus. These pathogens and viruses can cause a variety of diseases, necessitating environmental testing, particularly in water bodies, to screen for potential viral hazards and facilitate subsequent disease prevention and control.

[0003] Because pathogenic microorganisms in sewage are numerous, low in abundance, and pose a high risk, along with numerous interfering substances, commonly used detection methods such as PCR (polymerase chain reaction) suffer from long processing times, high costs, and low safety. Therefore, a detection method with high specificity, simplicity, speed, and safety is needed. However, current electrochemical immunosensors suffer from poor stability and sensitivity, making them limited in practical application. Summary of the Invention

[0004] In order to solve the above problems, the present invention provides a novel nano-antibody electrochemical immunosensor and its preparation method and application.

[0005] In the first aspect, a novel nano-antibody electrochemical immunosensor comprises: a biorecognition element for recognizing a target protein, and a substrate electrode for carrying the biorecognition element and converting the interaction between the biorecognition element and the target protein into an electrical signal;

[0006] The biorecognition element is a nanobody; the base electrode is a glassy carbon electrode modified with carboxyl carbon nanotubes;

[0007] The nanobody is obtained by recombining the gene of a virus containing the target protein with a prokaryotic expression vector to obtain a recombinant vector, and then expressing the recombinant vector in a prokaryotic manner;

[0008] The target protein is the new coronavirus S protein RBD, and the gene sequence of the gene of the virus containing the target protein is shown in SEQ ID NO: 1.

[0009] Description: Immobilizing nanoantibodies on the electrode surface can form a biorecognition interface. When a sample containing the target viral protein (i.e., the new coronavirus S protein in the present invention) contacts the electrode surface fixed with the nanoantibody, the nanoantibody specifically binds to the viral protein to form an antigen-antibody complex. This specific binding event will cause the charge distribution on the electrode surface to change, generating a detectable electrochemical signal, thereby realizing the detection process; through the above method, the characteristics of nanoantibodies can be utilized to improve the detection stability of the sensor, so that the thermal stability and chemical stability of the sensor are better; through the modification of carboxyl carbon nanotubes, a carrier for connecting nanoantibodies to glassy carbon electrodes can be provided. At the same time, the carboxyl carbon nanotubes have an amplifying function for the signal of the glassy carbon electrode, which can improve the detection sensitivity of the sensor.

[0010] The above-mentioned anti-COVID-19 S protein RBD antibodies are well representative, enabling the sensor to detect the new coronavirus.

[0011] Furthermore, the prokaryotic expression vector is a pET25b vector.

[0012] Note: The pET25b vector can efficiently express the inserted gene under the action of relevant types of enzymes, and the insertion position can be adjusted by changing the enzyme cutting site to facilitate recombination. It also has a polyhistidine tag (His-tag) to facilitate purification, so the pET25b vector is selected.

[0013] In a second aspect, the present invention provides a method for preparing a novel nanobody electrochemical immunosensor, comprising:

[0014] S1. Prepare anti-COVID-19 S protein RBD antibodies;

[0015] S2, using carboxylated multi-walled carbon nanotubes to modify glassy carbon electrodes;

[0016] S3. Load the anti-COVID-19 S protein RBD antibody onto the glassy carbon electrode to obtain the sensor.

[0017] Furthermore, the S1, preparation of anti-COVID-19 S protein RBD antibodies includes:

[0018] S1-1, recombining the gene of the virus containing the target protein with the pET25b vector to synthesize a recombinant vector;

[0019] S1-2. Transfer the recombinant vector into E. coli DH5α bacteria for induced expression and purification to obtain anti-COVID-19 S protein RBD antibody.

[0020] Furthermore, the induction expression conditions in S1-2 are: using isopropyl-β-D-thiogalactoside as an inducer, the concentration of isopropyl-β-D-thiogalactoside is 0.4~0.6M, the induction time is 11~13h, and the induction temperature is 15~25°C; during the purification process, the concentration of the imidazole solution used for eluting the anti-new crown S protein RBD antibody is 180~210mM.

[0021] Note: The above-mentioned induced expression condition parameters are suitable for the expression of the above-mentioned anti-COVID-19 S protein RBD antibody. These parameters are more preferred, so that the obtained nanoantibodies have higher concentrations and better purity.

[0022] Furthermore, the S2, using carboxylated multi-walled carbon nanotubes to modify the base electrode comprises:

[0023] S2-1, dispersing the carboxylated multi-walled carbon nanotubes in an N,N-dimethylformamide solution to obtain a dispersion;

[0024] S2-2. Evenly apply the dispersion liquid on the glassy carbon electrode and let it dry to obtain a carboxylated multi-walled carbon nanotube modified base electrode.

[0025] Description: N,N-dimethylformamide solution has good dispersibility and stability, and can stably disperse carboxylated multi-walled carbon nanotubes on the surface of the glassy carbon electrode to prevent agglomeration.

[0026] Furthermore, the S3, loading the anti-COVID-19 S protein RBD antibody onto the glassy carbon electrode to obtain the sensor comprises:

[0027] S3-1. Activate the glassy carbon electrode using a 2-morpholineethanesulfonic acid buffer solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, and then rinse with phosphate buffered saline for later use.

[0028] S3-2, mixing the anti-COVID-19 S protein RBD antibody in phosphate buffered saline to form an antibody solution;

[0029] S3-3. After mixing the antibody solution with the glassy carbon electrode treated in S3-1 and letting it stand, take out the glassy carbon electrode and immerse it in a bovine serum albumin solution for incubation, and then wash the glassy carbon electrode with a phosphate buffered saline solution to obtain the sensor.

[0030] Note: The above steps can first activate the glassy carbon electrode and introduce active group amide bonds on its surface to provide anchor points for subsequent biomolecule fixation, thereby improving the binding efficiency and stability of biomolecules to the electrode surface.

[0031] In the third aspect, the present invention provides an application of a new type of nano-antibody electrochemical immunosensor, which is used to detect the new coronavirus S protein in water.

[0032] The above-mentioned water bodies include water bodies in various environments, including sewage, surface water, drinking water, etc.

[0033] The detection method includes: taking 1 mL of water sample and adding 0.07 ng / mL and 0.1 ng / mL of new coronavirus S protein to the water sample, respectively, placing the sensor of the present invention in the water sample for incubation, recording the peak current response before and after incubation, and calculating the recovery rate of the immunosensor for different concentrations of new coronavirus S protein.

[0034] The beneficial effects of the present invention are:

[0035] The present invention can utilize nano-antibodies to improve the detection stability of the sensor, thereby improving the thermal stability and chemical stability of the sensor. By modifying carboxyl carbon nanotubes, a carrier for connecting the nano-antibodies and the glassy carbon electrode can be provided. At the same time, the carboxyl carbon nanotubes have an amplifying function for the signal of the glassy carbon electrode, thereby improving the detection sensitivity of the sensor. The sensor obtained by the preparation method of the present invention has the advantages of good affinity, strong specificity, good stability, and a low detection limit. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 is a SEM image of a sensor substrate electrode according to an embodiment of the present invention;

[0037] Figure 2 Schematic diagram of the SDS-PAGE electrophoresis identification results of the Nanobodies in the Examples of the present invention;

[0038] Figure 3 This is a schematic diagram of the concentration results of the Nanobodies in the Examples of the present invention;

[0039] Figure 4 This is a schematic diagram of the effect of the inducer concentration on the Nanobody concentration in the examples of the present invention:

[0040] Figure 5 Schematic diagram of the effect of induction time on the concentration of Nanobodies in the Examples of the present invention;

[0041] Figure 6 Schematic diagram of the effect of induction temperature on the concentration of Nanobodies in the Examples of the present invention;

[0042] Figure 7 Schematic diagram of the effect of imidazole elution concentration on Nanobody concentration in the examples of the present invention;

[0043] Figure 8 Schematic diagram of the results of indirect ELISA analysis of the performance of nanoantibodies in recognizing S protein in an embodiment of the present invention;

[0044] Figure 9 Schematic diagram of the results of indirect ELISA analysis of the thermal stability of nanobodies in an embodiment of the present invention;

[0045] Figure 10 Schematic diagram of the results of indirect ELISA analysis of the chemical stability of Nanobodies in an embodiment of the present invention;

[0046] Figure 11 Schematic diagram of the in-depth characterization of the chemical functional groups of MWCNTs and nanobody immunosensors using a Fourier transform infrared spectrometer in an embodiment of the present invention;

[0047] Figure 12 Schematic diagram of the effect of coupling agent concentration on sensor preparation results in an embodiment of the present invention;

[0048] Figure 13 Schematic diagram of the effect of coupling pH value on sensor preparation process in an embodiment of the present invention;

[0049] Figure 14 Schematic diagram of the effect of antibody content on sensor preparation process in an embodiment of the present invention;

[0050] Figure 15 Schematic diagram of the binding kinetics results of the electrochemical immunosensor in an embodiment of the present invention;

[0051] Figure 16 Schematic diagram of the quantitative detection results of the electrochemical immunosensor in an embodiment of the present invention;

[0052] Figure 17 Schematic diagram of the quantitative detection results of the electrochemical immunosensor in an embodiment of the present invention;

[0053] Figure 18 Schematic diagram of the specificity results of the immunosensor in an embodiment of the present invention;

[0054] Figure 19 Schematic diagram of the anti-interference performance results of the immunosensor in an embodiment of the present invention;

[0055] Figure 20 Schematic diagram of the reproducibility results of the immunosensor in an embodiment of the present invention;

[0056] Figure 21 Schematic diagram of the stability results of the immunosensor in an embodiment of the present invention;

[0057] Figure 22 Schematic diagram of the signal response results of the sensor of Comparative Example 1 of the present invention to the new coronavirus S protein;

[0058] Figure 23Schematic diagram of the signal response results of the nanoantibody immunosensor to the new coronavirus S protein in an embodiment of the present invention. DETAILED DESCRIPTION

[0059] In order to further illustrate the approach and effects achieved by the present invention, the technical solution of the present invention will be clearly and completely described below in conjunction with experiments.

[0060] The novel nano-antibody electrochemical immunosensor provided in the embodiments of the present application, and its preparation method and application relate to microbial identification and detection, and can be used to identify and detect microorganisms, especially viruses. Specifically, the nano-antibody electrochemical immunosensor is used to detect the environment (water body).

[0061] In order to solve the problems of long cycle, high cost, low safety, low stability and poor sensitivity of existing detection methods in the background technology, the embodiment of the present application provides a new nano-antibody electrochemical immunosensor and its preparation method and application. The biological recognition element in the sensor is a nano-antibody; the base electrode in the sensor is a glassy carbon electrode modified with carboxyl carbon nanotubes. The sensor has the advantages of strong specificity, high stability and good sensitivity.

[0062] In combination with the above content, the following is a detailed solution of an embodiment of the present invention:

[0063] A novel nano-antibody electrochemical immunosensor comprises: a biorecognition element for identifying a target protein, and a substrate electrode for carrying the biorecognition element and converting the interaction between the biorecognition element and the target protein into an electrical signal;

[0064] The biorecognition element is a nanobody; the base electrode is a glassy carbon electrode modified with carboxyl carbon nanotubes;

[0065] The nanobody is obtained by recombining the gene of the virus containing the target protein with a prokaryotic expression vector to obtain a recombinant vector, and then expressing the recombinant vector in a prokaryotic manner.

[0066] The target protein is the novel coronavirus S protein RBD, and the gene sequence of the virus gene containing the target protein is shown in SEQ ID NO: 1. The prokaryotic expression vector is a pET25b vector.

[0067] The preparation method of the novel nanoantibody electrochemical immunosensor comprises the following steps S1-S3

[0068] S1. Prepare anti-COVID-19 S protein RBD antibodies;

[0069] S1-1, recombining the gene of the virus containing the target protein with the pET25b vector to synthesize a recombinant vector;

[0070] The gene sequence of the gene in the anti-COVID-19 S protein RBD antibody is shown in SEQ ID NO: 1;

[0071] SEQ ID NO: 1 is: >RBD-NB95

[0072] CcatggAACACCATCATCATCATCACCACCAAGTACAATTAGTAGAAAGCGGAGGAGGATTAGTACAAGCAGGAGGAAGCCTAAGACTAAGCTGCGCAGCAAGCGGAAGAACATTCAGCAGCTACAGCATGGGATGGTTCAGACAAGCACAAGGAAAGGAAAGAGAATTCGTAGCAACAATAAACGGAAACGGAAGAGACACATACTACACAAACAGCGT AAAGGGAAGATTCACAATAAGCAGAGACGACGCAACAAACACAGTATACCTACAAATGAACAGCCTAAAGCCAGAAGACACAGCAATATACTACTGCGCAGCAGACAAGGACGTATACTACGGATACACAAGCTTCCCAAACGAATACGAATACTGGGGACAAGGAACACAAGTAACAGTAAGCAGCTGGTCACATCCACAATTCGAAAAGTGATGActcgag.

[0073] S1-2. Transfer the recombinant vector into E. coli DH5α bacteria and induce expression and purify to obtain anti-COVID-19 S protein RBD antibody.

[0074] First, the components and preparation methods of the culture medium involved in the following induction expression and purification are described:

[0075] LB liquid medium: 10 g peptone, 5 g yeast extract, 10 g NaCl, dissolved in 1000 mL distilled water, adjusted to pH 7.0, and sterilized at 121°C for 20 min.

[0076] LB solid medium: 10 g peptone, 5 g yeast extract, 10 g NaCl, 15-20 g (preferably 18 g in this embodiment) agar, dissolved in 1000 mL distilled water, adjusted to pH 7.0, and sterilized at 121° C. for 20 min.

[0077] LB / Amp plate: Add ampicillin (Amp) to a final concentration of 100 μg / mL to sterile LB solid medium in an ultra-clean workbench, shake well, and pour into a sterile culture dish. After solidification, cover the dish and invert it.

[0078] The above-mentioned recombinant vector transfer and induction expression include the following steps ①-③; purification includes the following steps 1)-4);

[0079] ① Preparation of competent cells

[0080] (1) E. coli DH5α was streaked onto LB solid medium and then incubated upside down at 37°C for 12 to 16 hours (preferably 15 hours in this embodiment);

[0081] (2) Pick a single colony from the cultured LB solid medium, inoculate it into a shake tube containing 5 mL of LB liquid medium, and culture with shaking at 37°C and 220 rpm for 12 to 16 hours (in this example, overnight for 15 hours) to obtain a culture;

[0082] (3) Take 500 μL of the culture in (2) and inoculate it into a conical flask containing 50 mL of LB liquid medium. Cultivate with shaking at 37°C and 220 rpm until the OD value reaches 0.4;

[0083] (4) Transfer the culture in the conical flask to a sterilized centrifuge tube and cool the culture to 0°C;

[0084] (5) The sample was then centrifuged at 1000 g for 10 min at 4°C. The supernatant was discarded, the residual liquid on the tube wall was aspirated, and the cells were resuspended in 40 mL of pre-chilled 0.1 M MgCl2-CaCl2 (containing 20 mM MgCl2 and 80 mM CaCl2).

[0085] (6) Centrifuge the sample at 1000 g for 10 min at 4°C, discard the supernatant, aspirate the residual liquid on the tube wall, and then gently resuspend the bacteria in 500 μL of pre-cooled 0.1 M CaCl2;

[0086] (7) Sterile glycerol was added to the bacterial solution at a final concentration of 10%, and after thorough mixing, the mixture was placed in an ice bath for 10 minutes to obtain competent cells. The competent cells were stored in a -80°C refrigerator until use.

[0087] ② Transformation of competent cells

[0088] (1) Thaw the competent cells, then add 10 μL of the ligation product to 100 μL of the competent cells. Mix gently in a centrifuge tube and cool the mixture in an ice bath for 30 minutes.

[0089] (2) Transfer the centrifuge tube containing the mixture to a 42°C water bath for 90 seconds, then quickly transfer the centrifuge tube to an ice bath for 2 minutes;

[0090] (3) Then, add 800 μL of LB liquid medium pre-warmed to 37°C and culture with shaking at 37°C and 150 rpm for 40 minutes; centrifuge and resuspend in PBS to obtain the transformation product;

[0091] (4) Take 100 μL of the transformation product and add it to an LB / Amp plate. After spreading it evenly, centrifuge the tube at 12,000 rpm for 1 minute. Discard part of the supernatant and add the remaining 100 μL of the resuspended cells to the LB / Amp plate. Spread it evenly again, invert it, and incubate it at 37°C for 12 to 16 hours (overnight for 15 hours in this example). This yields the E. coli DH5α / pET25b-Nb95 strain.

[0092] ③ Prokaryotic expression of nanobodies

[0093] (1) Take the E. coli DH5α / pET25b-Nb95 strain, streak it onto an LB / Amp plate, and incubate it upside down at 37°C for 12 to 16 hours (in this example, overnight for 15 hours);

[0094] (2) Pick a single colony from the LB / Amp plate and inoculate it into 5 mL of LB / Amp liquid medium; culture at 37°C, 220 rpm, and shake for 8 hours until the colony reaches the logarithmic growth phase (OD = 0.5-0.6);

[0095] (3) Take 500 μL of the cultured bacteria and inoculate into 50 mL of LB / Amp liquid medium; continue to shake and expand the culture at 37°C and 220 rpm for 2.5 hours until the logarithmic growth phase is reached;

[0096] (4) Then, 0.4-0.6 M (preferably 0.5 M) of IPTG (isopropyl-β-D-thiogalactoside, inducer) was added to the culture, and expression was induced at 15-25° C. (preferably 20° C. in this embodiment) and 180 rpm for 11-13 h (preferably 12 h in this embodiment);

[0097] (5) Transfer the culture after induction of expression in (4) to a 100 mL centrifuge tube, centrifuge at 8000 rpm at 4°C for 12 minutes, and discard the supernatant; then resuspend in 15 mL of 0.01 M PBS (phosphate buffered saline) and divide evenly into two 10 mL centrifuge tubes;

[0098] (6) The aliquoted bacterial solution was subjected to ultrasonic disruption and centrifuged at 8000 rpm for 15 minutes at 4°C. The supernatant was collected to obtain the disrupted bacterial supernatant containing the target protein.

[0099] purification:

[0100] 1) First, drain the preservation solution of the resin (also called medium or filler) in the affinity column used for purification; then add ultrapure water to wash the column 5 times, and add PBS buffer to wash the affinity column 5 times;

[0101] 2) Add the supernatant of the broken cells obtained in the above step ③ to Ni 2+ In the affinity column, collect the flow-through liquid and repeat the column twice to ensure that the target protein is fully attached to the column;

[0102] 3) Add PBS buffer to wash the supernatant remaining on the affinity column 5 times; then add 10 mL of 20 mM imidazole solution to wash the impurities adsorbed on the nickel ion affinity column;

[0103] 4) Add 10 mL of 180-210 mM (preferably 200 mM) imidazole solution to elute the target protein, and collect the imidazole solution in a 1.5 mL centrifuge tube to obtain anti-COVID-19 S protein RBD antibody.

[0104] S2, using carboxylated multi-walled carbon nanotubes to modify glassy carbon electrodes;

[0105] It should be understood that, in addition to the biorecognition element, the sensor usually includes a working electrode (i.e., a base electrode), a counter electrode, and a reference electrode, as well as a connector and an interface; wherein, the base electrode is the main part of the electrochemical immunosensor, usually a conductive material; the base electrode provides a platform for fixing antibodies or other recognition elements. The reference electrode and the counter electrode refer to the fact that when performing electrochemical measurements, a reference electrode and a counter electrode are usually required. The reference electrode provides a stable potential reference, while the counter electrode is used to complete the circuit so that current can flow between the working electrode and the reference electrode. In addition, the sensor needs to be connected to an external device (such as an electrochemical workstation) for signal reading and processing. This is usually achieved through connectors and interfaces.

[0106] Illustratively, in an embodiment of the present invention, the base electrode is a glassy carbon electrode; the counter electrode is a platinum wire electrode; and the reference electrode is an Ag / AgCl electrode.

[0107] Before electrode modification and loading, the glassy carbon electrode needs to be placed in 5mmol / L [Fe(CN)6] 3- / 4-Cyclic voltammetry was performed in a solution (containing 0.1M KCl) in the potential range of -0.2 to 0.6 V at a scan rate of 100 mV / s until a stable CV spectrum was obtained, ensuring that the redox peak potential difference was within 100 mV; after scanning, the treated glassy carbon electrode was obtained and blown dry with N2 for later use.

[0108] S2-1, dispersing the carboxylated multi-walled carbon nanotubes in an N,N-dimethylformamide solution to obtain a dispersion;

[0109] S2-2. Evenly apply the dispersion liquid on the glassy carbon electrode and let it dry to obtain a carboxylated multi-walled carbon nanotube modified base electrode.

[0110] Specifically, 1 mg of carboxylated multi-walled carbon nanotubes was dispersed in 1 mL of organic solution N,N-dimethylformamide and ultrasonicated for 30 minutes, 0.05% of Nafion was added and evenly dispersed to obtain a dispersion; 8 μL of the dispersion was evenly drop-coated on the treated glassy carbon electrode using the drop coating method and allowed to dry at room temperature.

[0111] S3. Load the anti-COVID-19 S protein RBD antibody onto the glassy carbon electrode to obtain the sensor.

[0112] S3-1. Activate the glassy carbon electrode using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide in 2-morpholineethanesulfonic acid buffer, then rinse with phosphate buffered saline for later use.

[0113] Exemplarily, in an embodiment of the present invention, the electrode is first functionalized: the glassy carbon electrode is immersed in a MES (2-morpholineethanesulfonic acid) buffer (pH = 7) containing 4 mM EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) and 8 mM NHS (N-hydroxysuccinimide), incubated at room temperature for 1 hour to complete the activation of the glassy carbon electrode, and the resulting electrode is rinsed with a PBS solution;

[0114] S3-2, mixing the anti-COVID-19 S protein RBD antibody in phosphate buffered saline to form an antibody solution;

[0115] Illustratively, in an embodiment of the present invention, specific monoclonal antibody / specific nanoantibody Nb95 were added to 0.01 M PBS buffer to prepare antibody solutions of 0.04 and 0.03 mg / mL, respectively.

[0116] S3-3. After mixing the antibody solution with the glassy carbon electrode treated in S3-1 and letting it stand, take out the glassy carbon electrode and immerse it in a bovine serum albumin solution for incubation, and then wash the glassy carbon electrode with a phosphate buffered saline solution to obtain the sensor.

[0117] Illustratively, in an embodiment of the present invention, the activated electrode was transferred to an antibody solution, incubated at room temperature for 1 hour, and washed three times with a PBS solution to successfully connect the specific antibody to the loaded glassy carbon electrode; the obtained modified electrode was immersed in a 3% (w / v) BSA solution and incubated for 1 hour to block the remaining active sites, and washed twice with a PBS solution to remove unbound BSA; the sensor was obtained and placed in a 4°C refrigerator for standby use after preparation.

[0118] The sensor is applied to the detection of the novel coronavirus S protein in water, and the detection method is as follows:

[0119] Take 1 mL of water sample, and add 0.07 ng / mL and 0.1 ng / mL of new coronavirus S protein to the water sample, respectively. Place the sensor of the present invention in the water sample for incubation, record the peak current response before and after incubation, and calculate the recovery rate of the immunosensor for different concentrations of new coronavirus S protein.

[0120] Comparative Example 1: Monoclonal antibodies were used instead of the nanobodies in the above examples, and the above steps S2-S3 were performed to obtain the sensor of Comparative Example 1.

[0121] Experimental example:

[0122] Experimental Example 1: Characterization of Nanobodies;

[0123] Experimental Example 2, sensor characterization;

[0124] Experimental Example 3: Application of sensors in water body detection;

[0125] Among them, the characterization of the nanobody in Experimental Example 1 includes SDS-PAGE electrophoresis identification of the nanobody, concentration determination of the nanobody, the influence of induction and purification condition parameters, and characterization of immunoassay performance.

[0126] SDS-PAGE electrophoresis identification of nanoantibodies: Gel preparation: First pour in 12% separation gel, wait for it to solidify, then pour in the concentration gel until the liquid surface overflows, then insert the electrophoresis comb, and wait for the concentration gel to solidify. Loading: Mix 30μL of purified protein solution with 10μL 4x Protein Loading Buffer, boil for 10 minutes, and centrifuge at 12000r / min for 10 minutes. Take 20μL of sample and 5μL of pre-stained protein marker in sequence. Electrophoresis: Run at low voltage (70V) for 30 minutes, waiting for the protein sample to enter the separation gel. Then increase the voltage to 110V and continue running for 1 hour until the bromophenol blue dye reaches the bottom of the gel. Staining: Take out the protein gel, place it in Coomassie Brilliant Blue solution for staining, and stain on a shaker for 2 hours. Decolorization: Recover the staining solution, add decolorization solution, shake on a shaker for decolorization, and change the decolorization solution several times until complete decolorization. The identification results are as follows: the unpurified expression product and the purified target protein (i.e., nanobody) are analyzed together using an SDS-PAGE kit; Figure 2 As shown, there are impurities in the unpurified protein band (a), while the purified target protein (b) has an obvious band at around 17kDa and the band is single. The purification effect is good, which is consistent with the experimental expectations, indicating that the expression and purification effect of the nanobody is good.

[0127] Determination of the concentration of nanoantibodies: Use the BCA kit to determine the concentration of the protein. According to the instructions, add the standard sample and the sample to be tested to a 96-well plate, 25 μL per well, and set up three replicates. After mixing the reagent A and reagent B of the working solution at a ratio of 50:1, add 200 μL to each well. After incubating at 37°C for 30 minutes, measure the absorbance value at 562 nm (OD562) and draw a standard curve. Finally, by comparing the absorbance value of the sample to be tested with the protein concentration of the corresponding absorbance value on the standard curve, the protein concentration of the sample to be tested can be calculated. The concentration of nanoantibodies was determined by the BCA method, and a standard curve was drawn with the concentration of the standard sample (BSA) as the X-axis and the OD562 value as the Y-axis. The results are as follows. Figure 3 As shown, the linear equation is y = 0.7783x + 0.0867 (R2 = 0.9997), which shows a good linear relationship. Substituting the measured sample OD562 value into the calculated concentration of the nanobody, the expression level of the nanobody after purification reached 493 μg / mL.

[0128] Effects of induction and purification parameters:

[0129] Inducer concentration: After adding different concentrations of inducer IPTG (0.25M, 0.5M, 0.75M, 1M) to the bacterial solution to induce expression, the bacteria were collected and broken by ultrasonic wave. 2+After column purification, the expression of the antibody was analyzed by SDS-PAGE, and the concentration of the nanobody was determined by BCA method to determine the optimal induction concentration of IPTG. Figure 4 As shown in the figure, after induction of expression with IPTG at different concentrations of 0.25M, 0.5M, 0.75M, and 1M, the expression amount of the supernatant was analyzed by SDS-PAGE (A) and concentration determination (B). As the IPTG concentration increased, the concentration of the nanoantibody increased accordingly. When the IPTG concentration was 0.5M, the expression effect of the antibody was the best. Therefore, the optimal IPTG concentration of 0.5M was selected for induction expression in subsequent experiments.

[0130] Induction time: During the induction process, 0.5M IPTG was added to the bacterial solution and the expression was induced for 6h, 8h, 10h, 12h, 14h, and 16h respectively. The collected bacteria were broken by ultrasonication and the supernatant was retained. 2+ After column purification, the expression effect of the antibody was analyzed by SDS-PAGE electrophoresis, and the concentration of the nanobody was determined by BCA method to determine the optimal induction expression time. Figure 2-5 As shown in the figure, as the induction time increases, the concentration of nanoantibodies gradually increases. When the induction expression time is 12 hours, the band color is the darkest and the antibody expression is the largest. As the induction time increases, the expression amount decreases. Therefore, the optimal induction expression time is selected as 12 hours in subsequent experiments.

[0131] Induction temperature: After adding 0.5M IPTG to the bacterial solution, the cells were induced at 10℃, 15℃, 20℃, 25℃, and 30℃ for 12 hours, and the collected cells were disrupted by ultrasound. After purification, the expression effect of the antibody in the supernatant was analyzed by SDS-PAGE electrophoresis, and the concentration of the nanoantibody was determined by BCA method to determine the optimal induction expression temperature. Figure 2-6 As shown in the figure, when the temperature increases from 10°C to 20°C, the expression level gradually increases, and further increase in temperature has an inhibitory effect on the expression level of the nanobody, so the optimal induction expression temperature is selected as 20°C.

[0132] Imidazole elution concentration: Gradient elution method was used, using 100mM, 150mM, 200mM and 250mM imidazole solutions. The effect of protein purification was analyzed by SDS-PAGE electrophoresis. The protein concentration was determined using the BCA method to determine the optimal imidazole elution concentration. The results are shown in Figure 2. Figure 2-7 As the imidazole concentration increases, the purity of the target protein gradually increases. When the concentration reaches 200mM, the protein elution effect is the best. Therefore, the optimal imidazole elution concentration is 200mM.

[0133] Characterization of immunoassay performance:

[0134] Indirect ELISA analysis of the performance of nanoantibodies in recognizing S protein: The new crown S protein was diluted to a concentration of 1 μg / mL and added to a 96-well enzyme-labeled plate at 100 μL per well. At the same time, 1 μg / mL of casein (OVA) and bovine serum albumin (BSA) were used as negative controls and incubated at 4 ° C for 12 hours. Pat the enzyme-labeled plate dry and wash the plate 3 times with 0.05% PBST, then add 300 μL of skim milk powder (5%) to each well to block the blank site and incubate at 37 ° C for 2 hours. Pat the enzyme-labeled plate dry and wash the plate 5 times with 0.05% PBST, and then add 100 μL of 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.01563, 0.00782, and 0 μg / mL of nanoantibodies to each well in turn and incubate at 37 ° C for 1 hour. Pat the liquid in the wells dry and wash the plate 5 times with 0.05% PBST. Then add 100 μL of secondary antibody diluted 7500 times to each well, incubate at 37°C for 1 hour, then pat the enzyme-labeled plate dry and wash the plate 5 times with 0.05% PBST. Finally, add 100 μL of TMB colorimetric solution to each well and incubate at 37°C for 20 minutes. Add 50 μL of 2M H2SO4 to each well, terminate the reaction and immediately measure OD450 with a microplate reader. Different proteins were selected to evaluate the specificity of nanoantibodies, and structural analogs of the new crown S protein, BSA and OVA, were used as negative controls. First, the antigens were coated separately: new crown S protein, BSA and OVA, and after blocking with skimmed milk powder, the expressed nanoantibody (Nb95) was added, and the affinity and specificity of Nb95 binding to the new crown S protein were verified by indirect ELISA. The results are as follows. Figure 8 , the nanoantibody only binds to the new coronavirus S protein, but not to BSA and OVA, and as the concentration of the nanoantibody increases, its affinity to the new coronavirus S protein becomes stronger, indicating that the nanoantibody has good specificity.

[0135] Indirect ELISA analysis of the thermal stability of nanoantibodies: Nanoantibodies were used to compare with monoclonal antibodies against the new coronavirus. The purified nanoantibodies and monoclonal antibodies were diluted and heated at different temperatures (4°C, 25°C, 37°C, 50°C, 80°C) for 5 minutes. The ability of the antibodies to bind to the antigen before and after treatment was evaluated by enzyme-linked immunosorbent assay. The ELISA method is the same as 2.3.5.1, except that only 1 μg / mL of the new coronavirus S protein was added during the antigen coating process. The results are as follows: Figure 9As shown in the figure, as the temperature rises, monoclonal antibodies gradually lose their binding ability, while the nanobodies expressed in this study can still maintain a certain ability to bind to antigens at a temperature of 80°C. Traditional antibodies, however, will adhere to each other and form precipitation due to the exposure of their hydrophobic surfaces in high temperature environments, destroying their original functions. In extreme environments, nanobodies will form disulfide bonds between CDR1 and CDR3, allowing them to withstand extreme conditions such as high temperatures. The longer the CDR3, the more stable the antibody structure. Therefore, compared with monoclonal antibodies, nanobodies have better thermal stability.

[0136] Indirect ELISA analysis of the chemical stability of nanobodies: To evaluate the chemical stability of nanobodies, the purified nanobodies and monoclonal antibodies were diluted and incubated in different pH buffers (10, 8, 7, 5, 3) for 2 h, and the ability of the antibodies to bind to antigens before and after treatment was evaluated by enzyme-linked immunosorbent assay. Figure 10 As shown, nanoantibodies still show high stability and affinity at different pH levels, while traditional monoclonal antibodies gradually become inactivated. This is also attributed to the disulfide bonds in the nanoantibody structure, so they can withstand extreme conditions. Monoclonal antibodies cause their structure and function to be destroyed under extreme conditions, so nanoantibodies have better chemical stability.

[0137] Experimental Example 2, sensor characterization;

[0138] The characterization of the sensor includes: characterization of the electrode surface morphology and characterization of the sensor preparation conditions. The electrode surface morphology is characterized by scanning electron microscopy (SEM);

[0139] Electrode surface morphology characterization: SEM was used to characterize the morphology of the electrode loaded with nanoantibody Nb95. The results are as follows: Figure 1 As shown in the figure, when the electrode surface is loaded with MWCNTs, it can be seen that the electrode surface presents a tubular, rough and irregularly arranged fiber strip morphology, indicating that the MWCNTs are successfully modified on the electrode surface. After the subsequent coupling of nanoantibodies, there are spherical rough particles around the carbon nanotubes, indicating that the nanoantibodies are successfully coupled to the MWCNTs / GCE surface. Fourier transform infrared spectroscopy is used to conduct in-depth characterization of the chemical functional groups of MWCNTs and nanoantibody immunosensors, as shown in the figure. Figure 11FT-IR spectroscopy shows that the peak at 3437 cm-1 is attributed to the -OH group, the peak at 1717 cm-1 is attributed to the vibrational stretching of -COOH, and the peak at 1236 cm-1 is due to the vibrational stretching of -C=O, indicating that the carboxylation of carbon nanotubes was successfully modified. After coupling with the nanoantibody, the peaks at 3270 cm-1 and 1503 cm-1 are related to the vibration of -NH, the peak at 1659 cm-1 is due to the vibrational stretching of -C=O, and the peak at 1383 cm-1 is attributed to the stretching vibration of the -CN bond, indicating that the nanoantibody was successfully coupled to the surface of the carboxyl carbon nanotubes, forming an amide bond, indicating that the nanoantibody immunosensor was successfully prepared.

[0140] Characterize sensor preparation conditions;

[0141] Coupling agent concentration: including EDC / NHS concentration. EDC can activate the carboxyl group, so that it can react with small molecules containing amino groups (antibodies, peptides, etc.) to form a stable amide bond. The carboxyl group can react with EDC to form the intermediate product O-acylurea, but this product is unstable and easily hydrolyzed. Therefore, it must react in the presence of NHS to form a stable intermediate NHS ester with amino reaction activity, thereby reducing the occurrence of hydrolysis and improving the reaction efficiency. It then reacts with the primary amine to form a stable amide bond. The concentration of EDC-NHS is also an important influencing factor. Therefore, this experiment selected coupling agents with different concentrations of 1-2mM, 2-4mM, 3-6mM, 4-8mM, and 5-10mM, and measured the electrode peak current response values ​​before and after coupling with different concentrations of EDC-NHS. The calculation formula is: ΔI = Current before coupling - Current after coupling; the results are as follows. Figure 12 As shown in the figure, for monoclonal antibodies (A) and nanoantibodies (B), as the concentration of the coupling agent increases, the difference in peak current before and after coupling gradually increases, reaching the highest point when the EDC-NHS concentration is 4-8mM, and then gradually decreases as the concentration increases. This is because too little EDC-NHS will affect the number of carboxyl groups replaced by NHS ester groups, and the carboxyl groups cannot be fully activated, thereby further affecting the subsequent antibody coupling quantity. Excessive use will make it difficult to clean in the subsequent steps and affect the coupling reaction. Therefore, the concentration of the coupling agent EDC-NHS was selected to be 4-8mM in the experiment.

[0142] Coupling pH: The pH of the buffer solution has a crucial impact on the performance of the electrochemical immunosensor. The coupling reaction must be carried out at an appropriate pH, as excessively high or low pH can significantly affect biomolecules, thereby inhibiting the bioactivity of the antibody. To determine the optimal pH for the coupling process, a series of experiments were conducted within a pH range of 6.0 to 8.0. PBS buffer solutions with pH values ​​of 6, 6.5, 7, 7.5, and 8 were prepared as the medium for the antibody coupling reaction. The peak current response of the electrode before and after antibody coupling was measured under different pH conditions. To obtain a more stable immunosensor, this study optimized the pH of the PBS buffer during the coupling process. Figure 13 The figure shows the electrical signal response of the immunosensor before and after coupling with nanoantibodies and monoclonal antibodies in buffer solutions of different pH values. As can be seen from the figure, for monoclonal antibody (A), as the pH value increases, the peak current difference before and after electrode coupling with the antibody gradually increases, reaching a maximum value at pH 6.5. As the pH value continues to rise, the peak current difference gradually decreases. This is because at low pH, the intermediate O-acylurea formed is very unstable and easily hydrolyzed; however, if the pH is too high, the intermediate O-acylurea may hydrolyze, or lead to a decrease in the production of NHS esters, thereby reducing the reaction rate between the amino group and the intermediate, and thus reducing the amount of antibody coupling. Therefore, for the coupling of monoclonal antibodies, the optimal pH value of this experiment was selected as 6.5. For nanoantibody (B), the peak current difference reached its maximum value at pH 7, and as the pH value continues to rise, the peak current difference gradually decreased. Therefore, for the coupling of nanoantibodies, the optimal pH value of this experiment was selected as 7.

[0143] Antibody content: The content of antibodies on the sensor surface has an important influence on the performance of the sensor. Too little content may lead to a reduction in the binding sites of the immune response, thereby affecting the sensor's detection performance of the target molecule. Therefore, ensuring an appropriate antibody concentration is one of the important factors affecting the performance of the immunosensor. This experiment investigated the effects of different concentrations of nanoantibodies and monoclonal antibodies on the performance of the immunosensor. The selected antibody concentrations were 0.005, 0.01, 0.02, 0.03, 0.04, and 0.05 mg / mL. The activated electrode was placed in antibody solutions of different concentrations and incubated for a period of time, and the changes in DPV peak current before and after antibody coupling were recorded. Figure 14(A, B), when the concentrations of monoclonal antibodies and nanoantibodies reached 0.04 mg / mL and 0.03 mg / mL, respectively, the peak current difference basically reached equilibrium. As the concentration increased further, the peak current difference gradually stabilized. The possible reason is that nanoantibodies are smaller in size and can enter grooves and cracks on the surface of the modified material, making it easier to bind to carboxyl groups and reach saturation. Therefore, the antibody concentration was selected as 0.04 mg / mL during the preparation of the sensor in Comparative Example 1, and the antibody concentration was selected as 0.03 mg / mL during the preparation of the nanoantibody immunosensor.

[0144] Binding kinetics of electrochemical immunosensor: The reaction between the successfully prepared electrochemical immunosensor and the new crown S protein depends on specific binding. In immunoassays, the length of the binding time has an important influence on the degree of completion of the immune reaction, thereby significantly affecting the electrical signal response of the electrochemical immunosensor. This experiment selected room temperature as the incubation temperature, and investigated the DPV peak current response of the sensor of comparative example 1 after incubation with the new crown S protein for 5, 10, 15, 20, 30, 40, and 50 minutes in a new crown S protein antigen solution with a concentration of 0.05 ng / mL at room temperature, and the DPV peak current response value after incubation of the nanoantibody immunosensor with the new crown protein for 3, 5, 7, 10, 15, 20, and 30 minutes. Calculation formula: ΔI = Current before incubation - Current after incubation; the results are as follows: Figure 15 As the incubation time increases, the redox peak current difference gradually increases. After reaching 30 minutes, the immune response basically tends to balance, and the peak current difference gradually stabilizes. Too long an incubation time will affect the binding efficiency of the immunosensor. Therefore, for the sensor of Comparative Example 1, the optimal immune response time is 30 minutes. As shown in Figure (B), for the nanoantibody immunosensor, after the incubation time reaches 20 minutes, the immune response basically reaches balance, and the peak current difference gradually stabilizes with time. Too long an incubation time will affect the binding efficiency of the immunosensor. Therefore, for the nanoantibody immunosensor, 20 minutes is selected as the optimal immune response time in this experiment. Compared with the two immunosensors, the nanoantibody immunosensor has a shorter immune response time and faster detection performance.

[0145] Electrochemical immunosensor quantitative detection: Under the above-mentioned optimal experimental conditions, a series of different concentrations of the new crown S protein (0.02-0.4 ng / mL) were measured. The two electrochemical immunosensors (Comparative Example 1 and the embodiment of the present invention) were placed in different concentrations of the new crown S protein solution for incubation, and the sensors were washed with PBS solution to remove non-specifically bound proteins. The DPV peak current response values ​​of the two sensors after incubation with different concentrations of the new crown S protein solution were recorded. The results are as follows: Figure 16As shown in Figure (A), when the concentration of the new crown S protein is in the range of 0.02 to 0.4 ng / mL, the DPV redox peak current gradually decreases with the increase of the antigen concentration. Figure (B) shows the relationship between the peak current difference before and after the binding of the monoclonal antibody on the surface of the immunosensor to the antigen and the antigen concentration in the concentration range of 0.02 to 0.4 ng / mL. When the concentration is in the range of 0.02 to 0.15 ng / mL, the peak current difference has a good linear relationship with the S protein concentration, and the linear regression is ΔI=6.8×10-4C(ng / mL)+8×10-6(R2=0.9466). The detection limit (LOD) of the sensor of Comparative Example 1 is 10.2 pg / mL (S / N=3), the quantification limit (LOQ) is 33.8 pg / mL (S / N=10), and the sensitivity is 6.8×10-4A / (ng / mL);

[0146] like Figure 17 As shown in Figure A, within the range of 0.02 to 0.25 ng / mL of the new crown S protein concentration, the DPV redox peak current gradually decreases with the increase of the antigen concentration. Figure (B) shows that within the range of 0.02 to 0.15 ng / mL of the new crown S protein concentration, there is a good linear relationship between the peak current difference before and after the binding of the nanoantibody to the antigen and the antigen concentration. The linear regression equation is ΔI = 11.8×10-4C (ng / mL) + 2.6×10-6 (R2 = 0.9741). The detection limit (LOD) of the nanoantibody immunosensor is 7.1 pg / mL (S / N = 3), which is 3.1 pg / mL lower than the detection limit of the sensor in Comparative Example 1. The limit of quantification (LOQ) is 23.7 pg / mL (S / N = 10), which is 29.9% lower than the LOQ of the sensor in Comparative Example 1, and the sensitivity is 11.8×10-4A / (ng / mL).

[0147] The results show that the sensor of the embodiment of the present invention has lower detection limit and quantification limit, higher sensitivity and improved detection performance of the immunosensor compared with the sensor of Comparative Example 1.

[0148] Table 1 lists the methods for detecting SARS-CoV-2 using different electrochemical immunosensors. As can be seen from Table 1, the detection limits of the two immunosensors prepared in this study are comparable to those of the RT-PCR method. However, the RT-PCR method requires multiple steps such as reverse transcription, and the operation requirements are relatively high, making rapid detection impossible. Therefore, the sensor prepared in this study has advantages over traditional RT-PCR in environmental testing. Compared with other immunosensors, this sensor has a lower LOD (7.116 pg / mL). For example, the LOD of the sensor in the study by YAKOH A et al. was 0.11 ng / mL. The immunosensor prepared in this study has a good linear range for the new coronavirus S protein and a lower detection limit. The preparation and operation processes are simpler and the price is cheaper.

[0149] Table 1 Methods for detecting SARS-CoV-2 proteins

[0150]

[0151]

[0152] Specificity of immunosensors: In order to verify the specificity of the two electrochemical immunosensors (Comparative Example 1 and the embodiment of the present invention) in detecting the new crown S protein, this experiment set up skimmed milk powder, casein, ovalbumin and bovine serum albumin as competitive proteins, and explored the specific recognition of the new crown S protein by the two immunosensors. Under the optimal experimental conditions, the two immunosensors were placed in a solution of the new crown S protein and competitive protein with a concentration of 0.1 ng / mL and a mixed solution of the above five substances. After incubation at room temperature for a period of time, they were washed three times with PBS solution to remove the influence of non-specific binding. The DPV peak current response values ​​before and after incubation were recorded. The results are as follows. Figure 18As shown, the sensor of Comparative Example 1 has the largest current response in the new crown S protein solution, with a peak current difference of 75.6μA, which is 0.9, 5.2, 6.8, 6.3, and 8.0 times that of the five mixtures (80.9μA), bovine serum albumin (14.5μA), skimmed milk powder (11.2μA), ovalbumin (12.1μA), and casein (9.48μA), respectively. The difference in peak current response values ​​between the mixed solution of the five substances and the solution containing only the new crown S protein is about 6.6%. The experimental results show that the constructed immunosensor has good specificity; for the nanoantibody immunosensor, the results are shown in Figure B, before and after incubation The nanoantibody immunosensor obtained the highest current response to the new coronavirus S protein, with a peak current difference of 112μA, which was 0.9, 7.9, 9.4, 11.5, and 10.1 times that of the five mixtures (118μA), bovine serum albumin (14.2μA), skimmed milk powder (11.9μA), ovalbumin (9.7μA), and casein (11.1μA), respectively. In the mixed solution of the five substances, the difference in peak current response value with the solution containing only the new coronavirus S protein (5%) was smaller than the difference of the sensor in comparative example 1 (6.6%). The experimental results show that the nanoantibody immunosensor has better specificity than the sensor in comparative example 1.

[0153] Interference Resistance of Immunosensors: Since sewage contains numerous pollutants, including organic and inorganic substances and a large number of pathogenic microorganisms, this experiment tested the interference resistance of the two successfully prepared immunosensors to different pollutants in sewage. These pollutants included bovine serum albumin (5 mg / L, 25 mg / L, and 50 mg / L), sodium chloride (0.35 mg / L, 0.7 mg / L, and 3.5 mg / L), glucose (5 mg / L, 25 mg / L, and 50 mg / L), humic acid (1 mg / L, 5 mg / L, and 25 mg / L), and deoxyribonucleic acid (0.1 mg / L, 0.5 mg / L, and 1 mg / L). 0.05 ng / mL of S protein was added to the above interfering substances, and the DPV peak current responses of the two immunosensors were recorded before and after incubation.

[0154] For the nanoantibody immunosensor, the results are as follows Figure 19As shown in the figure, in bovine serum albumin solution (A), low concentration of BSA slightly increases the peak current difference of the immunosensor, which may be related to the sensor's ability to specifically recognize antigens. As the concentration increases, the peak current difference gradually decreases, but the sensor's recognition ability is still above 89.8%. In sodium chloride salt solution and glucose solution (B, C), as the concentration of interfering substances continues to increase, the sensor's response signal gradually decreases. This may be because during the incubation process, some interfering substances undergo non-specific adsorption with nanoantibodies, occupying the binding sites of the new coronavirus S protein, resulting in a decrease in peak current changes, but the signal response is still between 83.2% and 98.3%, indicating that the immunosensor has good anti-interference properties. In humic acid, the interfering substances have no significant effect on the sensor (D), and the sensor has good specific recognition ability, with the peak current response signal reduced by a maximum of 10.2%. For deoxyribonucleic acid, the results are shown in Figure (E). DNA's ability to recognize the sensor's novel coronavirus S protein is somewhat inhibited. When the DNA concentration is 1 mg / L, the sensor's interference is greatest, with the peak current signal decreasing by 10.4%, but a large amount of antigen can still be detected. This experiment also studied the immune sensor's anti-interference performance in a low-concentration single pollutant system. The results, shown in Figure (F), show that compared to the mixed system, the interference in the single system has less impact on the sensor, with the maximum peak current change being 9.4 μA. Therefore, this nanoantibody immune sensor has good anti-interference performance.

[0155] Reproducibility of immunosensors: Whether an immunosensor is reproducible is one of the important indicators for evaluating sensor performance. It can evaluate the minimum difference in measurement results. In order to detect the reproducibility of the two immunosensors prepared in this study, the experiment was carried out under the same environment. Five immunosensors of the same batch were prepared using the same method. The 0.1 ng / mL new coronavirus S protein was measured. The peak current difference before and after incubation was recorded by the DPV method to compare the consistency between the electrodes. The results are as follows: Figure 20 As shown, the two sensors were tested five times respectively, and the results were relatively stable. It was calculated that the RSD of the sensor of Comparative Example 1 was 4.0%, and the RSD of the nanoantibody immunosensor was 3.1%. Compared with the two, the reproducibility of the nanoantibody immunosensor was slightly better than that of the sensor of Comparative Example 1, and the difference between the electrodes was smaller.

[0156] Stability of immunosensors: Since monoclonal antibodies and nanoantibodies have the activity of binding to antigens, they may be affected by various factors during application, which may cause the protein structure of the antibody to denature. In order to evaluate the stability of the two electrochemical immunosensors, this experiment investigated the stability of the electrodes after being placed at a certain temperature for a period of time; the successfully prepared immunosensors were stored in an environment of 4°C for 20 days, and the DPV method was used to measure the peak current response value of the electrochemical immunosensor every two days and compare it with the original blank value. The results are as follows Figure 21 As shown, for the sensor (A) of Comparative Example 1, the peak current response value decreased by 29.04% after 20 days of storage. For the nanobody immunosensor (B), the results showed that the current response remained high after 20 days, with the peak current response value decreasing by only 8.9%. The immunosensor still retained 91.1% of its electrochemical activity. This is because the disulfide bonds within the nanobody help it maintain conformational stability in harsh environments. Therefore, this sensor has better stability than the sensor of Comparative Example 1.

[0157] Experimental Example 3: Application of sensors in water body detection;

[0158] Detection of actual samples: In the experiment, the sewage sample was first filtered through a 0.22μm filter membrane to ensure the removal of particulate matter and suspended matter in the sample. Then 1mL of the filtered water sample was taken, and 0.07 and 0.1ng / mL of the new coronavirus S protein were added to the water sample, respectively, to ensure that the sample contained the target detection object. The two successfully prepared immunosensors were then placed in the water sample and incubated at room temperature for a period of time. The DPV method was used to record the peak current response before and after incubation, and the recovery rate of the immunosensor for different concentrations of new coronavirus S protein was calculated.

[0159] The sensor of Comparative Example 1 ( Figure 22 ), in the sewage influent sample with a SARS-CoV-2 S protein concentration of 0.1 ng / mL, the immunosensor's signal response to the SARS-CoV-2 S protein varied significantly compared to the buffer solution, while the signal response in the sewage effluent and Yangtze River water samples varied less. This may be because the sewage influent sample contained a large number of interfering substances, which affected the efficiency of the antibody in recognizing the antigen, resulting in a decrease in the peak current difference. SARS-CoV-2 S protein standards of varying concentrations were added to the sewage samples for detection. The experimental results are shown in Table 2. SARS-CoV-2 S protein was detected in all samples, with recoveries ranging from 89.4% to 105.0% and RSDs ranging from 2.4% to 4.8%, indicating that the sensor can be used for detection in actual sewage.

[0160] Table 2 Detection of SARS-CoV-2 S protein in actual water samples

[0161]

[0162] Embodiment of the present invention: For nanoantibody immunosensor ( Figure 23 ), the new coronavirus S protein was detected in sewage inlet, sewage effluent and Yangtze River water samples, and the signal response changed little. The new coronavirus S protein standards of different concentrations were added to sewage samples for detection. The experimental results are shown in Table 3. The new coronavirus S protein was detected in samples of different concentrations, with a recovery rate between 93.1% and 103.7% and an RSD in the range of 1.6% to 4.9%, indicating that the sensor can be used for the detection of actual sewage.

[0163] Table 3 Detection of SARS-CoV-2 S protein in actual water samples

[0164]

[0165]

[0166] The recovery rate of the sensor of Comparative Example 1 is between 89.4% and 105.0%, and the RSD is between 2.4% and 4.8%. The recovery rate of the nanoantibody immunosensor of the embodiment of the present invention is between 93.1% and 103.7%, and the RSD is between 1.6% and 4.9%, indicating that the immunosensor can be used for the detection of actual sewage, and the results of the present invention are more preferred.

Claims

1. A novel nano-antibody electrochemical immunosensor, characterized in that: The sensor includes: a biorecognition element for identifying a target protein, and a substrate electrode for carrying the biorecognition element and converting the interaction between the biorecognition element and the target protein into an electrical signal; The biorecognition element is a nanobody; the base electrode is a glassy carbon electrode modified with carboxyl carbon nanotubes; The nanobody is obtained by recombining the gene of a virus containing the target protein with a prokaryotic expression vector, wherein the prokaryotic expression vector is a pET25b vector, obtaining a recombinant vector, and then expressing the recombinant vector in a prokaryotic manner; the specific preparation method is: S1-1, recombining the gene of the virus containing the target protein with the pET25b vector to synthesize a recombinant vector; S1-2, transfer the recombinant vector to E. coli .DH5α bacteria were induced to express and purified to obtain anti-COVID-19 S protein RBD antibodies; wherein, the induction expression conditions are: using isopropyl-β-D-thiogalactoside as an inducer, the concentration of isopropyl-β-D-thiogalactoside is 0.4-0.6 M, the induction time is 11-13 h, and the induction temperature is 15-25°C; during the purification process, the concentration of the imidazole solution used to elute the anti-COVID-19 S protein RBD antibodies is 180-210 mM; The target protein is the SARS-CoV-2 S protein RBD, and the sequence of the gene of the virus containing the target protein is shown in SEQ ID NO:

1.

2. The method for preparing a novel nanobody electrochemical immunosensor according to claim 1, wherein: include: S1. Prepare anti-COVID-19 S protein RBD antibodies; S2, using carboxylated multi-walled carbon nanotubes to modify glassy carbon electrodes; S3. Load the anti-COVID-19 S protein RBD antibody onto the glassy carbon electrode to obtain the sensor.

3. The method for preparing a novel nanobody electrochemical immunosensor according to claim 2, wherein: Said S1, preparation of anti-COVID-19 S protein RBD antibody comprises: S1-1, recombining the gene of the virus containing the target protein with the pET25b vector to synthesize a recombinant vector; S1-2, transfer the recombinant vector to E. coli .Induced expression and purification were carried out in DH5α bacteria to obtain anti-COVID-19 S protein RBD antibodies.

4. The method for preparing a novel nanobody electrochemical immunosensor according to claim 3, characterized in that: The induction expression conditions in S1-2 are as follows: using isopropyl-β-D-thiogalactopyranoside as an inducer, the concentration of isopropyl-β-D-thiogalactopyranoside is 0.4-0.6 M, the induction time is 11-13 h, and the induction temperature is 15-25°C; During the purification process, the concentration of the imidazole solution used to elute the anti-COVID-19 S protein RBD antibody was 180~210 mM.

5. The method for preparing a novel nanobody electrochemical immunosensor according to claim 3, characterized in that: Said S2, using carboxylated multi-walled carbon nanotubes to modify the base electrode comprises: S2-1, dispersing the carboxylated multi-walled carbon nanotubes in an N,N-dimethylformamide solution to obtain a dispersion; S2-2. Evenly apply the dispersion liquid on the glassy carbon electrode and let it dry to obtain a carboxylated multi-walled carbon nanotube modified base electrode.

6. The method for preparing a novel nanobody electrochemical immunosensor according to claim 3, wherein: Said S3, loading the anti-COVID-19 S protein RBD antibody onto the glassy carbon electrode, to obtain the sensor comprises: S3-1. Activate the glassy carbon electrode using a 2-morpholineethanesulfonic acid buffer solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, and then rinse with phosphate buffered saline for later use. S3-2, mixing the anti-COVID-19 S protein RBD antibody in phosphate buffered saline to form an antibody solution; S3-3. After mixing the antibody solution with the glassy carbon electrode treated in S3-1 and letting it stand, take out the glassy carbon electrode and immerse it in a bovine serum albumin solution for incubation, and then wash the glassy carbon electrode with a phosphate buffered saline solution to obtain the sensor.

7. The use of a novel nanobody electrochemical immunosensor according to claim 1, characterized in that: The sensor is applied to non-disease detection of the novel coronavirus S protein in water.

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