A microbial nanozyme probe, a test strip for detecting salbutamol and application thereof

Abstract

The application discloses a microbial nano-enzyme probe, a method for detecting salbutamol and application, and comprises a salbutamol monoclonal antibody and a signal carrier, wherein the signal carrier is a yeast cell loaded with iridium oxide nanoparticles. The application firstly constructs a probe by using a yeast cell loaded with iridium oxide nanoparticles in the detection of an immunochromatographic test strip, uses [3, 3', 5, 5' tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2)] as a substrate to trigger a color developing reaction, can realize high-sensitivity target detection, and the detection limit is as low as 0.012 ng / mL, which is 55 times higher than the sensitivity of a traditional gold-labeled test strip. In addition, the application has a good application prospect, can provide a universal platform for sensitive, accurate and rapid detection of food-borne stimulants, and can be successfully applied to the detection of salbutamol in pig liver and beef samples.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological detection, and relates to a microbial nanoscale enzyme probe, a method for detecting salbutamol and application, in particular to a yeast cell loaded with iridium oxide nanoparticles as a signal carrier, a probe prepared by labeling an antibody, a test strip using the probe, and application of the test strip to rapid and sensitive detection of salbutamol. BACKGROUND

[0002] Foodborne stimulants in food mainly include β2-receptor agonists, and eating food containing these foodborne stimulants will be harmful to the body and mind of people, which is very detrimental to the healthy development of people's body and mind. Salbutamol (SAL) is a rapid-acting β2-adrenergic receptor agonist, which can selectively stimulate the β2 receptor of bronchial smooth muscle, and is used for the treatment of bronchial asthma in clinical treatment. However, SAL is quickly absorbed in the body and easily accumulates in the body, which can be left in hair, blood or tissue. The residual SAL in animal tissue can have a negative impact on human health and cause some clinical symptoms, such as palpitations, muscle tremors, dizziness, profuse sweating and chills, etc. In the breeding industry, when the dose of SAL is 5-10 times higher than the normal therapeutic dose, it can significantly increase lean meat content and reduce fat deposition, thereby significantly improving economic benefits. At present, several analytical methods for detecting the foodborne stimulant salbutamol have been reported, including liquid chromatography tandem mass spectrometry (LC-MS / MS), high performance liquid chromatography (HPLC), gas chromatography mass spectrometry (GC-MS), enzyme-linked immunosorbent assay (ELISA), surface-enhanced Raman scattering (SERS), and electrochemical detection. Although these conventional methods can provide highly sensitive detection and obtain accurate results, they require expensive instruments, professional operators, tedious sample pretreatment steps and time-consuming processes, which hinder their application in on-site and rapid detection. In order to more sensitively detect salbutamol, especially for on-site rapid detection, it is still a great technical challenge to realize an easy, rapid, convenient and low-cost method.

[0003] In recent years, immunochromatographic test strips have received extensive attention due to their low cost, good selectivity, user-friendliness, and intuitive reading, and have become an important means of on-site rapid detection, mainly for monitoring low concentrations of analytes, such as detecting disease biomarkers, chemical pollutants, bacteria, and viruses, especially in the recent SARS-CoV-2. In addition, most immunochromatographic test strips are based on single-mode reading determination, which is easily affected by external environments (e.g., different operators or non-standard detection processes), leading to inaccurate results, and even false positive or false negative test results. Fortunately, the emergence of dual- or multi-modal sensors perfectly makes up for the shortcomings of single-mode sensors. Dual- or multi-modal sensors are a synergistic combination of at least two strategies, achieved by single- or multi-modal probes, to ensure the accuracy of the results and improve the sensitivity of the detection. So far, various dual-mode readout strategies have been developed on immunochromatographic test strips. Among them, colorimetry is always chosen to be combined with other quantitative analysis due to its simplicity and visuality.

[0004] Gold nanoparticles (AuNPs) have unparalleled biocompatibility and unique optical properties, and are a common signal tag for detecting SAL. However, the poor stability and low sensitivity of immunochromatographic test strips based on gold nanoparticles seriously hinder their further application. Fortunately, as a member of transition metal oxides, iridium (IV) oxide nanoparticles (IrO2 NPs) are preferred materials in numerous scientific and applied fields (e.g., biosensors and electrocatalysis) due to their excellent biocompatibility, outstanding electrical conductivity, and high catalytic activity. Typically, IrO2 NPs are synthesized by the thermal hydrolysis of Ir(IV) ([IrCl6] 2- ) as a precursor. The [IrCl6] 2- solution undergoes a hydroxyl / hydrate mechanism and is finally oxidized by O2 to form IrO2 NPs. However, during the preparation of the probe, due to the extremely small particle size of IrO2 NPs, it is difficult to obtain a complete probe by high-speed centrifugation, resulting in a large amount of monoclonal antibody (mAbs) being wasted. Therefore, it is urgent to find a cleaner, non-toxic carrier to increase the particle size of IrO2 NPs to reduce the loss of mAbs. SUMMARY

[0005] In view of the defects and shortcomings in the prior art, the present application aims to provide a microbial nanoscale enzyme probe, a test strip for detecting salbutamol and an application. The probe is prepared by using yeast cells loaded with iridium oxide nanoparticles as a signal carrier and labeled antibodies, and has high monodispersity, uniform shape, excellent biocompatibility, high affinity and rapid antibody labeling capacity. The probe can quickly label monoclonal antibodies through simple physical adsorption, thereby avoiding the use of complex cross-linking agents. In addition, the probe has high peroxidase activity, can catalyze 3,3',5,5'-tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2) to trigger a color developing reaction, and improves the detection sensitivity and accuracy of the detection results, which has important significance and application value for monitoring food-borne stimulant salbutamol.

[0006] To achieve the above technical effects, the technical scheme adopted by the present application is as follows:

[0007] A microbial nanoscale enzyme probe, wherein the signal carrier of the probe is a yeast cell loaded with iridium oxide nanoparticles; the yeast cell is an ellipsoid with a length of 2-3 microns and a width of 1-2 microns; and the iridium oxide nanoparticles have a particle size of 17-21 nm.

[0008] Optionally, the signal carrier of claim 1 is used to label a monoclonal antibody.

[0009] Specifically, the method comprises the following steps:

[0010] Preparation of an iridium oxide nanoparticle solution, loading of iridium oxide nanoparticles on the inactivated yeast cells in the iridium oxide nanoparticle solution, and then adsorption of a monoclonal antibody to obtain the probe.

[0011] Optionally, the preparation of the iridium oxide nanoparticle solution comprises the following steps: mixing trisodium citrate and K2IrCl6 in a mass ratio of 5:3 to prepare a solution, adjusting the pH of the solution to 7.5, and refluxing under boiling; when the color of the solution changes from brown to steel gray, continue to stir the solution and heat for 30 minutes; after the solution is cooled to room temperature, the pH is adjusted to 7.5 again, and then the solution is heated for 30 minutes until the pH is stable, and the color of the solution changes to deep blue;

[0012] Preparation of an inactivated yeast cell solution: culturing yeast cells in a yeast extract protein peptone glucose culture medium at 28 DEG C for 18 hours, collecting the yeast cells by centrifugation, and diluting the yeast stock solution to OD 600 2;

[0013] Preparation of yeast cells loaded with iridium oxide nanoparticles: mixing the prepared iridium oxide nanoparticle solution and the inactivated yeast cell solution at 37 DEG C for 10 minutes.

[0014] Preparation of microbial nanozyme probes: Monoclonal antibodies were added to yeast cell solutions loaded with iridium oxide nanoparticles, mixed, blocked with bovine serum albumin, and resuspended in ultrapure water by centrifugation.

[0015] Optionally, the monoclonal antibody is mixed with yeast cells loaded with iridium oxide nanoparticles at a ratio of 4 μg: 1 mL.

[0016] The mixing time was 0.8–1.2 h, the blocking time for adding bovine serum albumin was 20–40 min, and the final concentration was 1%.

[0017] The application of any of the microbial nanozyme probes described in this invention in the preparation of biological test strips.

[0018] The microbial nanozyme probe described in any one of the present invention is used in the preparation of test strips for detecting salbutamol.

[0019] The application of any of the microbial nanozyme probes described in this invention in the preparation of test strips for detecting salbutamol in meat products.

[0020] A test strip for detecting salbutamol, wherein the test line on the test strip contains any of the microbial nanozyme probes described in this invention;

[0021] The monoclonal antibody in the microbial nanozyme probe is salbutamol antibody.

[0022] Optionally, the detection line is obtained by streaking with a detection solution, and the preparation of the detection solution includes:

[0023] The microbial nanozyme probe solution was added to the salbutamol sample solution to be tested and incubated for 15 minutes.

[0024] OD of microbial nanozyme probe solution 600 The volume ratio of the microbial nanozyme probe solution to the salbutamol sample solution to be detected is 1:20.

[0025] The minimum concentration of salbutamol that the test strip can detect is 0.022 ng / mL.

[0026] A salbutamol detection solution, wherein the detection solution contains any of the microbial nanozyme probes described in this invention;

[0027] The preparation of the detection solution includes:

[0028] The microbial nanozyme probe solution was added to the salbutamol sample solution to be tested and incubated for 15 minutes.

[0029] OD of microbial nanozyme probe solution 600The volume ratio of the microbial nanoscale enzyme probe solution to the sample liquid to be detected is 1:20.

[0030] The method for detecting salsalate in food is characterized in that the microbial nanoscale enzyme probe of any of the present application is used to prepare a detection liquid with a sample to be detected.

[0031] The test strip coated with the salsalate monoclonal antibody on the detection line is used for color development detection, and the detection response time is 90s.

[0032] When the detection line T line is obviously lighter than the blank control strip observed by naked eye, it is judged as positive.

[0033] Compared with the prior art, the present application has the following advantages and positive effects:

[0034] (1) Simple antibody labeling process. The present application only uses the simple adsorption of the yeast cells loaded with iridium oxide nanoparticles and the antibody to prepare the microbial nanoscale enzyme probe, and the complex cross-linking process (such as the EDC / NHS method) is avoided.

[0035] (2) Microbial nanoscale enzyme probe. The present application first uses the yeast cells loaded with iridium oxide nanoparticles as a double-signal carrier to label the antibody to prepare the microbial nanoscale enzyme probe in the immunochromatographic test strip detection, and the probe has high monodispersity, uniform shape, excellent biocompatibility, high affinity and fast antibody labeling capacity, and has high peroxidase activity, can directly catalyze the color development reaction of 3,3',5,5' tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2) substrates on the test paper, does not need an acidic buffer system, has the advantages of simple operation, rapid color development, etc., and improves the detection sensitivity and the accuracy of the detection results. This work develops a cheap, sensitive, portable and rapid reading analysis system for the detection of salsalate in pig liver and beef samples.

[0036] (3) High sensitivity. The test strip provided by the present application has a minimum detection limit of 0.012 ng / mL for salsalate. The method can detect salsalate with high sensitivity, and can be used as a general method for rapid and convenient detection of additive residues in food.

[0037] (4) High specificity. The present application can highly specifically recognize salsalate, and has no specificity for other food additives.

[0038] (5) Good practical application. The present application can detect salsalate in pig liver and beef, has good application prospect, and can be used as a general detection method for detecting various food-borne stimulants. BRIEF DESCRIPTION OF DRAWINGS

[0039] The accompanying drawings are included to provide a further understanding of the present disclosure and constitute a part of the specification, illustrate embodiments of the present disclosure and together with the specific embodiments described in the following detailed description, help explain the present disclosure. In the drawings:

[0040] Figure 1 Schematic diagram of yeast cell material loaded with iridium oxide nanoparticles prepared by the present application;

[0041] Figure 2 Configuration diagram of the immunochromatographic test strip for rapid detection of salbutamol and detection principle diagram of the immunochromatographic test strip according to the present application;

[0042] Figure 3 Optimization experiment results of the immunochromatographic test strip prepared by the present application;

[0043] Figure 4 Detection sensitivity and specificity of the immunochromatographic test strip prepared by the present application, Control, CLE, TBT, MEL, CAP, STR, RAC and SAL represent blank, clenbuterol hydrochloride, terbutaline sulfate, melamine, chloramphenicol, streptomycin sulfate, ractopamine and salbutamol, respectively;

[0044] Figure 5 Comparison of the detection sensitivity of the immunochromatographic test strip prepared by the present application with that of the test strip based on traditional gold nanoparticles;

[0045] Figure 6 Actual application of the immunochromatographic test strip prepared by the present application;

[0046] Figure 7 Characterization of yeast cells loaded with iridium oxide nanoparticles prepared by the present application;

[0047] Figure 8 Characterization of the novel microbial nanozyme probe according to the present application, a, b and c represent IrO2NPs-mAb, Yeast@IrO2-mAb and AuNPs-mAb, respectively;

[0048] The present application will be described in detail below in combination with the accompanying drawings and specific embodiments. DETAILED DESCRIPTION

[0049] In order to make the purpose, technical solutions and advantages of the present application more clear, the present application will be further described in detail below in combination with embodiments. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0050] The application aims to provide a microbial nanoscale enzyme probe, a test strip for detecting salbutamol and an application. The yeast cell loaded with iridium oxide nanoparticles (Yeast@IrO2) has high catalytic activity, biocompatibility, stability and multiple active sites, and compared with other materials such as magnetic quantum dots, polymer dots, carbon dots and perovskite nanocrystals, the Yeast@IrO2 has simple synthesis, good dispersibility and uniform shape, can quickly label the monoclonal antibody through simple physical adsorption, thereby avoiding the use of complex cross-linking agents.

[0051] The yeast cell loaded with iridium oxide nanoparticles (Yeast@IrO2) has high catalytic activity, biocompatibility, stability and multiple active sites, and compared with other materials such as magnetic quantum dots, polymer dots, carbon dots and perovskite nanocrystals, the Yeast@IrO2 has simple synthesis, good dispersibility and uniform shape, can quickly label the monoclonal antibody through simple physical adsorption, thereby avoiding the use of complex cross-linking agents. In the field of electrochemistry, the iridium oxide nanoparticles have good activity and stability in the acid water oxidation process, and are currently a representative commercial acid water oxidant. Generally, the iridium oxide nanoparticles can only catalyze 3,3',5,5'-tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2) to develop color under acidic conditions. However, in the application, the iridium oxide nanoparticles can be directly added on the test strip to catalyze the TMB and H2O2 substrates to trigger the color development reaction, without the need for an acidic buffer system, and have the advantages of simple operation, rapid color development, etc., improving the detection sensitivity and the accuracy of the detection results. In addition, the Yeast@IrO2 has higher antibody affinity and biological activity than the pure iridium oxide nanoparticles (IrO2 NPs) and the traditional colloidal gold nanoparticles (AuNPs), and is a very promising signal carrier.

[0052] In order to obtain the best determination performance, the inventors optimized the amount of antibody used, the amount of antigen drawn, the volume of microbial nanoscale enzyme probe and the immunization time, and determined the optimal system conditions. The finally prepared test strip is used for detecting the food-borne stimulant salbutamol remaining in food, and the method has been successfully applied to the detection of salbutamol in pig liver and beef, verifying the practicability, sensitivity and accuracy thereof.

[0053] The working principle of the test strip is: based on the competition detection principle, first, the microbial nanoscale enzyme probe is mixed with the sample solution, then it is dropped on the test strip, and through capillary action it moves to the test area of the test paper. When the sample solution is not present, the microbial nanoscale enzyme probe will be captured by the antigen in the detection line, forming a visible band on the detection line. On the contrary, for positive samples, there is a strong competition reaction between the free sotalol and the antigen fixed on the detection line, resulting in a decrease in the intensity of the detection line with the increase of the concentration of sotalol. When the concentration of sotalol is high enough, the microbial nanoscale enzyme probe will only be captured by the control line, and no visible band will appear on the detection line. In the signal enhancement mode, due to the peroxidase-like activity of the microbial nanoscale enzyme probe, the color of the detection line changes from blue-black to yellow-green. When the concentration of sotalol is high enough, after adding the mixed solution of TMB and H2O2, the yellow-green color change on the detection line also becomes weaker or invisible.

[0054] The method for preparing the microbial nanoscale enzyme probe comprises:

[0055] (1) Preparation of iridium oxide nanoparticles: mix trisodium citrate with K2IrCl6 in ultrapure water, adjust the pH of the solution to 7.5, and reflux under boiling. When the color of the solution changes from brown to steel gray, continue to stir the solution and heat for 30 minutes. After the solution cools to room temperature, adjust the pH to 7.5 again, then heat the solution for 30 minutes until the pH is stable, and the final solution color changes to dark blue.

[0056] (2) Preparation of inactivated yeast cells: the yeast cells are cultured in a yeast extract peptone glucose medium at 28°C for 18h, and the yeast stock solution is diluted to OD 600 2. Then the above yeast solution is treated with 10% formaldehyde to obtain inactivated yeast cells. The inactivated yeast cells are treated with a demolding buffer at 80°C for 30 minutes.

[0057] (3) Preparation of yeast cells loaded with iridium oxide nanoparticles: mix the IrO2 NPs solution prepared in step (1) with the yeast solution treated in step (2) at 37°C for 10 minutes. Remove the excess IrO2 NPs to obtain Yeast@IrO2, and resuspend in 5mL ultrapure water for next use.

[0058] (4) Preparation of microbial nanoscale enzyme probe: add sotalol monoclonal antibody to the yeast cell solution loaded with iridium oxide nanoparticles in step (3), mix, block with bovine serum albumin, centrifuge and resuspend in ultrapure water. The mixing ratio of the sotalol monoclonal antibody and the yeast cells loaded with iridium oxide nanoparticles is 4μg:1mL, the mixing time is 0.8-1.2h, the blocking time of adding bovine serum albumin is 20-40min, and the final concentration is 1%.

[0059] Combination Figure 2 The immunochromatographic test strip of the present application is composed of five parts, and nitrocellulose membrane, sample pad, combination pad and water absorption pad are sequentially pasted to the backing plate, wherein the line on the nitrocellulose is coated with salmon amine alcohol-bovine serum albumin conjugate (SAL-BSA) and goat anti-mouse immunoglobulin (IgG) as the detection line T line and the control line C line respectively.

[0060] The experimental reagents used in the present application are commercially available and are not further treated, and the detection instrument equipment is a commonly used instrument.

[0061] Example 1: Preparation of probe of test strip for rapid detection of salmon amine alcohol

[0062] Combination Figure 1 According to the above technical scheme, the present embodiment provides a microbial nanozyme probe and a preparation method thereof, which comprises yeast cells loaded with iridium oxide nanoparticles as a signal carrier, and then adding salmon amine alcohol monoclonal antibody for adsorption to obtain the probe. The preparation method comprises the following steps:

[0063] (1) Preparation of iridium oxide nanoparticles (IrO2 NPs): 50 mg of trisodium citrate and 30 mg of K2IrCl6 were mixed with 50 mL of ultrapure water, the pH of the solution was adjusted to 7.5, and the solution was refluxed under boiling. When the color of the solution changes from brown to steel gray, continue to stir the solution and heat for 30 minutes. After the solution is cooled to room temperature, the pH is re-adjusted to 7.5, and then the solution is heated for 30 minutes until the pH is stable, and the final solution changes to dark blue, and the IrO2 NPs solution is prepared.

[0064] (2) Preparation of inactivated yeast cells: the yeast cells were cultured in yeast extract protein peptone glucose medium at 28℃ for 18h. Subsequently, the yeast cells were collected by centrifugation (5000rpm, 5min) and the yeast stock solution was diluted to OD 600 2. Then the above yeast solution (5mL) was treated with 10% formaldehyde to obtain inactivated yeast cells. The inactivated yeast cells were treated with buffer (consisting of 0.03g NaCl, 0.05g SDS, 0.015g DTT and 0.02g NaOH in 5mL sterile water) at 80℃ for 30min.

[0065] (3) Preparation of yeast cells loaded with iridium oxide nanoparticles: the IrO2 NPs solution prepared in step (1) (5mL) and the yeast solution treated in step (2) (5mL, OD 600 2) were mixed at 37℃ for 10min. The excess IrO2 NPs were removed to obtain Yeast@IrO2, and resuspended in 5mL of ultrapure water for next use.

[0066] (4) Preparation of microbial nanoscale enzyme probe: add the sotalol monoclonal antibody to the yeast cell solution loaded with iridium oxide nanoparticles in step (3), mix, block with bovine serum albumin, centrifuge, and resuspend in ultrapure water.

[0067] Under stirring, put an appropriate amount of 4 μg sotalol monoclonal antibody into 1 mL of the above-prepared Yeast@IrO2 for 1 hour, then add 10% BSA and react for 30 minutes to block the excess unbound sites. Finally, centrifuge the mixture, disperse in 100 μL of ultrapure water, and store in a 4°C refrigerator for further use. The sotalol detection probe used in the following Examples 2-4 is prepared in Example 1.

[0068] Example 2: Preparation of an immunochromatographic test strip for rapid detection of sotalol

[0069] This example gives a high-sensitivity immunochromatographic test strip for rapid detection of sotalol, which comprises a nitrocellulose membrane, a sample pad, a conjugate pad, and an absorbent pad, and a backing plate, with the nitrocellulose membrane pasted on the backing plate, one end of the nitrocellulose membrane covered with the absorbent pad, the other end of the nitrocellulose membrane covered with the sample pad and the conjugate pad in sequence, the non-covered surface of the nitrocellulose membrane provided with a test line and a control line in the transverse direction, and the conjugate pad and the sample pad blocked with blocking solution, respectively.

[0070] The preparation method of the nitrocellulose membrane comprises: 1 mg / mL sotalol-bovine serum albumin conjugate is coated on the test line at a scribing rate of 0.8 μL / cm to form the test line, and 1 mg / mL goat anti-mouse immunoglobulin is coated on the control line at a scribing rate of 1 μL / cm to form the control line; and then dried at 37°C for standby use.

[0071] Preparation of the sample pad: cut a glass fiber membrane into a size of 15 mm long and 3 mm wide, immerse it in blocking solution (2% BSA), and dry it at 37°C for 8 hours to obtain the sample pad, which is then stored in a 4°C refrigerator.

[0072] Preparation of the conjugate pad: cut a glass fiber membrane into a size of 8 mm long and 3 mm wide, immerse it in blocking solution (2% BSA), take it out, and dry it at 37°C for 8 hours to obtain the sample pad, which is then stored in a 4°C refrigerator.

[0073] Preparation of the absorbent pad: cut an absorbent paper into a size of 18 mm long and 3 mm wide to obtain the absorbent pad.

[0074] Assembly of the test strip: first, paste the nitrocellulose membrane on the backing plate, then press the sample pad against the conjugate pad by 1-3 mm, press the conjugate pad against the nitrocellulose membrane by 1-3 mm, and press the absorbent pad against the nitrocellulose membrane by 1-3 mm in sequence to obtain the immunochromatographic test strip for rapid detection of sotalol.

[0075] Example 3: Optimization of the test strip for rapid detection of salbutamol

[0076] To obtain the best detection performance, the system optimized some relevant experimental parameters of Yeast@IrO2-ICA. In a typical competitive immunoassay, the amount of mAbs (2, 3, 4, 5 and 6 pg), the amount of T-line scribe (1, 0.9, 0.8, 0.7, 0.6 and 0.5 pL / cm), the amount of probe (3, 4, 5, 6 and 7 pL) and the immuno time (5, 10, 15 and 20 min) directly affect the sensitivity and visibility of the proposed biosensor. According to the signal intensity of T-line and the competitive inhibition rate, the best parameters of the biosensor were selected as the main criteria. The formula for calculating the competitive inhibition rate is (1-T / T0) x 100%, where T0and T are the T-line intensity of SAL negative and SAL positive solution, respectively.

[0077] See Figure 3 A, with the increase of the amount of anti-SAL-mAbs, the intensity of T-line increased significantly. At the same time, when using 4 pg mAbs, the competitive inhibition rate reached a maximum. Therefore, 4 pg anti-SAL-mAbs was considered as the best choice. See Figure 3 B, when the amount of antigen scribe on T-line was 0.7 pL / cm, the maximum T-line intensity and competitive inhibition rate were obtained. Therefore, 0.7 pL / cm was selected as the best scribe amount of antigen on T-line. With the increase of the probe, the color intensity of T-line gradually enhanced Figure 3 C). After adding 5 pL of probe, the intensity of T-line and C-line was almost the same, and the competitive inhibition rate reached a maximum. Considering the economic cost and response sensitivity, 5 pL was the best volume of Yeast@IrO2-mAb probe. In Figure 3 D, the T-line intensity and competitive inhibition rate remained balanced after 15 minutes, which was considered as the best immuno reaction time in this work.

[0078] Example 4: Sensitivity determination of the test strip for rapid detection of salbutamol

[0079] Detection process: The salbutamol standard was dissolved in ultrapure water, and then serially diluted into different concentrations from 0 to 50 ng / mL, and ultrapure water was used as blank control. 5 pL of Yeast@IrO2-mAb probe was mixed with 100 pL of salbutamol standard solution for incubation, and then the sample pad of the test strip was immersed into the above detection solution with different concentrations, and the mixture migrated to the absorbent pad by capillary force. After 15 min of reaction, the signal intensity of T-line was observed by naked eye, and the quantitative measurement was performed by portable device. In the signal enhancement mode, 6 pL of TMB-H2O2 reaction solution was added between T-line and C-line. After 90 s, the test results were photographed using a camera. At the same time, the final intensity on T-line was quantitatively measured by portable device.

[0080] Detection Results: When the test line (T-line) is visually significantly lighter than the blank control line, the minimum concentration of salbutamol is defined as the visual detection limit (vLOD). When the test line (T-line) completely disappears, the corresponding minimum concentration is taken as the threshold concentration. Competitive Inhibition Rate (IC50) 10 Defined as the detection limit (LOD).

[0081] See Figure 4 A and 4B show photographic images of Yeast@IrO2-ICA with and without substrate TMB and H2O2 enhancement. The intensity of the T-line decreases progressively with increasing SAL concentration. Figure 4 (C and 4D). Without the substrates TMB and H2O2, the vLOD and threshold were 0.045 ng / mL and 3 ng / mL, respectively. With the addition of the substrates TMB and H2O2, the vLOD and threshold were 0.02 ng / mL and 12 ng / mL, respectively. Furthermore, the IC50 was calculated according to the corresponding four-parameter logic equation. 10 The values ​​were 0.022 ng / mL and 0.012 ng / mL, respectively. Notably, the signal enhancement system exhibited higher sensitivity and a wider detection range than the unenhanced ICA, which is attributed to the effect of signal amplification on the T-line. Its sensitivity is 55 times higher than that of conventional colloidal gold nanoparticle-based test strips (AuNPs-ICA) (see...). Figure 5 Therefore, this method can detect salbutamol with high sensitivity and can be used as a general method for rapid and convenient detection of foodborne stimulant residues.

[0082] Example 5: Specificity determination of a rapid detection strip for salbutamol

[0083] Detection Procedure: Clenbuterol hydrochloride (CLE), terbutaline sulfate (TBT), melamine (MEL), chloramphenicol (CAP), streptomycin sulfate (STR), and ractopamine (RAC) were diluted with ultrapure water to a concentration of 100 ng / mL. 100 μL of each solution was used as the detection solution and mixed with 5 μL of Yeast@IrO2-mAb probe for incubation. The sample pad of the test strip was then immersed in the above test solutions. Simultaneously, 100 μL of ultrapure water was used as a blank control. After 15 min, the intensity of the T-line was obtained using a portable device. In signal enhancement mode, 6 μL of TMB-H2O2 reaction solution was added between the T-line and C-line. After 90 s, the test results were captured using a camera. Simultaneously, the final intensity on the T-line was quantitatively measured using a portable device.

[0084] See Figure 4E and 4F, 12 ng / mL SAL can inhibit the color of T-line. In contrast, in the presence of other additives, significant color was observed on the T-line. Meanwhile, Figure 4 G shows that in the presence of SAL only, the T-line intensity is the lowest, regardless of the presence of substrates TMB and H202. However, when other additives were tested, high T-line intensity was observed. Moreover, the results above are consistent with the detection results of competitive ELISA Figure 4 H), only the sample of salbutamol can inhibit the color on the T-line, while significant color can be observed on the T-line for other common additives. This indicates that the test strip of the present application can highly specifically recognize salbutamol, and has high specificity.

[0085] Example 6: Application of test strip for rapid detection of salbutamol

[0086] Detection process: The pork liver and beef were spiked with pretreated salbutamol. Before pretreatment, liquid chromatography-mass spectrometry (LC-MS) was used to confirm whether the blank real sample contained salbutamol. 5 g of sample was taken into a 15 mL centrifuge tube containing 10 mL of 3% trichloroacetic acid aqueous solution, vortexed for 5 min, and then centrifuged (8,000 rpm, 10 min). Subsequently, the supernatant was adjusted to neutral with NaOH (1 M) solution.

[0087] The above-mentioned pretreated real sample solution was diluted and spiked (salbutamol concentration was 0-50 ng / mL), 5 μL of Yeast@IrO2-mAb probe was mixed with 100 μL of salbutamol standard solution for incubation, and then the sample pad of the test strip was immersed in the above-mentioned test solution, and the mixture migrated to the absorbent pad by capillary action. After 15 min of reaction, the signal intensity of the T-line was observed with the naked eye, and quantitatively measured with a portable device. In the signal enhancement mode, 6 μL of TMB-H202 reaction solution was added between the T-line and the C-line. After 90 s, the test results were photographed using a camera. At the same time, the final intensity on the T-line was quantitatively measured with a portable device.

[0088] Detection results: see Figure 6 A and 6B, without adding substrates TMB and H202, the visual detection limit vLOD in beef and pork liver was about 0.45 μg / kg. The threshold concentration of SAL in beef was about 15 μg / kg, and the threshold concentration of SAL in pork liver sample was about 30 μg / kg. In the presence of substrates TMB and H202, the visual detection limit vLOD of beef and pork liver samples was 0.2 μg / kg, and the threshold concentrations were 60 μg / kg and 12 μg / kg, respectively Figure 6 C and 6D). In both modes, the T-line intensity decreased with increasing SAL concentration Figure 6(E and 6F). Therefore, this invention can detect salbutamol in pork liver and beef, and the results are consistent with those of spiked samples, reflecting its good practical application value.

[0089] Example 7: Characterization of yeast cells loaded with iridium oxide nanoparticles

[0090] (1) Transmission electron microscopy and scanning electron microscopy: from TEM images ( Figure 7 As shown in A), the synthesized IrO2 NPs exhibit excellent dispersibility and uniformity, and the average diameter of 100 nanoparticles was obtained from the histogram, which is approximately 17.87 ± 2.95 nm. The characteristic absorption peak of the IrO2 NPs is located at 598 nm. Figure 7 B). From SEM images ( Figure 7 As can be seen in C), the primitive yeast cell was an ellipsoid 2-3 μm long and 1-2 μm wide. Figure 7 As shown in Figure D, the treated yeast cell surface exhibited significant shrinkage, and the particle size increased after loading IrO2 NPs. Furthermore, X-ray energy-dispersive spectroscopy (EDS) elemental mapping... Figure 7 E) shows that C, N, O and Ir are uniformly attached to the yeast cells.

[0091] (2) Zeta potential: in Figure 7 In F, the zeta potentials of yeast cells, IrO2 NPs, and Yeast@IrO2 were -16.4 mV, -5.87 mV, and -7.60 mV, respectively, indicating that IrO2 NPs were successfully loaded onto yeast.

[0092] (3) Fourier transform infrared spectroscopy (FTIR): such as Figure 7 As shown in G, the FTIR measurements further confirm the -COOH (3318, 1654, and 1400 cm⁻¹) values. -1 -NH2 (1654 and 1544 cm) -1 -SH (1051, 1244 and 700cm) -1 ) and -OH (3318, 2929 and 1051 cm) -1 The functional groups are located on the yeast cells. In this sense, the various functional groups on the surface of yeast cells can provide a wide range of anchoring sites for stabilizing various metals and biomolecules. The change in wavenumber after the formation of Yeast@IrO2 indicates that a chemisorption reaction of IrO2 NPs on the surface of yeast cells occurred during self-assembly, with -COOH, -SH, and -NH2 groups participating in the reaction.

[0093] (4) X-ray photoelectron spectroscopy (XPS): The elemental composition and valence state of yeast and Yeast@IrO2 were studied using XPS. Figure 7H, and signals of C 1s, N 1s, O 1s and Ir 4f can be seen in Yeast@IrO2. The successful synthesis of Yeast@IrO2 is proved.

[0094] Example 8: Characterization of novel microbial nanosensor probe

[0095] To prove that the microbial nanosensor probe is successfully prepared, the inventors also did the following experiments (see Figure 8 ):

[0096] (1) Zeta potential: Figure A is the zeta potential diagram of Yeast@IrO2, BSA and Yeast@IrO2-BSA. It can be seen that the zeta potential of Yeast@IrO2 (-7.60 mv) and Yeast@IrO2-BSA (-2.91 mv) has changed obviously, which indicates that the protein is successfully labeled on the surface of Yeast@IrO2.

[0097] (2) Evaluation of the coupling efficiency of IrO2 NPs, Yeast@IrO2 and AuNPs with antibodies: Figure B is the OD 450 versus the concentration (5, 2.5, 1, 0.2, 0.1, 0.02, 0.004 and 0.001 μg / mL) of salbutamol monoclonal antibody. As shown in Figure C, the coupling rate of Yeast@IrO2-mAb is the highest, which is 92.2%. At the same time, it is calculated that the amount of salbutamol monoclonal antibody combined with Yeast@IrO2 is 3.7 x 10 13 molecules / mL. The above results show that compared with IrO2 NPs and AuNPs, Yeast@IrO2 can significantly improve the coupling efficiency with antibodies.

[0098] (3) Evaluation of the affinity of IrO2 NPs, Yeast@IrO2 and AuNPs with antibodies: The affinity constant k a value greater than 10 7 M -1 is considered to have high affinity. Figure D is the calculation results of the affinity constant and dissociation constant (K D ) of IrO2 NPs, Yeast@IrO2 and AuNPs. The k 8 value of Yeast@IrO2-mAb (5.9 x 10 -1 M a ) is higher than that of IrO2 NPs-mAb (3.3 x 10 8 M -1 ) and AuNPs-mAb (3.5 x 10 8 M -1). Yeast@IrO2-mAb (3.4 x 10 -10 M) of K D M) of IrO2 NPs-mAb (8.4 x 10 -10 M) of AuNPs-mAb (9.1 x 10 -10 M). Therefore, these results accurately demonstrate that the binding ability of Yeast@IrO2 to the antibody is stronger than that of AuNPs and IrO2 NPs.

[0099] The preferred embodiments of the present disclosure are described in detail above with reference to the drawings, but the present disclosure is not limited to the specific details of the above-described embodiments. Various simple modifications can be made to the technical solutions of the present disclosure within the scope of the technical concept of the present disclosure, and these simple modifications all belong to the protection scope of the present disclosure.

[0100] In addition, it should be noted that each specific technical feature described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present disclosure will not further describe various possible combinations.

[0101] Furthermore, various different embodiments of the present disclosure can also be combined in any manner, as long as they do not deviate from the idea of the present disclosure, and they should also be considered as disclosed by the present disclosure.

Claims

1. A microbial nanosensor probe, characterized in that, The signal carrier of the probe is a yeast cell loaded with iridium oxide nanoparticles; the yeast cell is an ellipsoid with a length of 2-3 μm and a width of 1-2 μm; and the iridium oxide nanoparticles have a particle size of 17-21 nm; The signal carrier is used to label a monoclonal antibody; specifically, an iridium oxide nanoparticle solution is prepared, and inactivated yeast cells are loaded with iridium oxide nanoparticles in the solution, followed by monoclonal antibody adsorption to obtain the monoclonal antibody; The preparation of the iridium oxide nanoparticle solution comprises: mixing trisodium citrate and K2IrCl6 at a mass ratio of 5:3 to prepare a solution, adjusting the pH of the solution to 7.5, and refluxing under boiling; when the color of the solution changes from brown to steel gray, continue to stir the solution and heat for 30 minutes; after the solution is cooled to room temperature, the pH is adjusted to 7.5 again, and then the solution is heated for 30 minutes until the pH is stable, until the color of the solution changes to dark blue; Preparation of inactivated yeast cell solution: Yeast cells were cultured in yeast extract peptone glucose medium at 28 °C for 18 h, and the yeast cells were collected by centrifugation and the yeast stock solution was diluted OD 600 to 2; The yeast cell loaded with iridium oxide nanoparticles is prepared by mixing the prepared iridium oxide nanoparticle solution with an inactivated yeast cell solution at 37 ℃ for 10 minutes; The microbial nanoscale enzyme probe is prepared by adding a monoclonal antibody to the yeast cell solution loaded with iridium oxide nanoparticles, mixing, blocking with bovine serum albumin, centrifuging, and resuspending in ultrapure water; The monoclonal antibody is mixed with the yeast cell loaded with iridium oxide nanoparticles at a ratio of 4 μg:1 mL, and the mixing time is 0.8-1.2 h; the blocking time of the added bovine serum albumin is 20-40 min, and the final concentration is 1%.

2. Use of the microbial nanoscale enzyme probe of claim 1 for preparing a test strip for detecting salbutamol in meat products.

3. A salbutamol detection solution, characterized by, The detection solution contains the microbial nanoscale enzyme probe of claim 1; The preparation of the detection solution comprises: The microbial nanoscale enzyme probe solution is added to the sample solution to be detected for incubation, and the incubation time is 15 min; OD of the microbial nanosensor probe solution 600 was 2, and the volume ratio of the microbial nanosensor probe solution to the salbutamol sample liquid to be detected was 1:

20.

4. A method for the detection of salbutamol in a food product, characterized in that, The microbial nanoscale enzyme probe of claim 1 is used to prepare a detection solution with a sample to be detected, and a test strip for detecting salbutamol is used for detection; The preparation of the test strip comprises: 1 mg / mL salbutamol-bovine serum albumin conjugate is coated on the detection line at a line speed of 0.8 μL / cm to obtain the detection line; 5 μL of the microbial nanoscale enzyme probe is mixed with 100 μL of a salbutamol standard solution for incubation, and then the sample pad of the test strip is immersed in the detection solution with different concentrations, the mixture migrates to the absorbent pad under the action of capillary force, and after 15 min of reaction, the signal intensity of the T line is observed with the naked eye, and a portable device is used for quantitative measurement; The monoclonal antibody in the microbial nanoscale enzyme probe is a salbutamol antibody; The preparation of the detection liquid comprises: adding a microbial nanoscale enzyme probe solution into a sample liquid of salbutamol to be detected for incubation, and the incubation time is 15 min; the OD 600 2, the volume ratio of the microbial nanoscale enzyme probe solution to the sample liquid of salbutamol to be detected is 1:20; and the minimum concentration of the test strip for detecting salbutamol is 0.022 ng / mL. The test strip with the detection line coated with salbutamol-bovine serum albumin conjugate is used for catalytic colorimetric detection, and the detection response time is 90 s; when the signal intensity of the detection line T line is significantly lower than that of the blank control observed with the naked eye, it is judged that the sample contains salbutamol.

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