Preparation method of electrochemical luminescence sensor for detecting staphylococcus aureus in aquatic products

Abstract

The invention discloses a preparation method of an electrochemical luminescence sensor for detecting staphylococcus aureus in aquatic products, which is characterized in that a double signal amplification strategy of high specific surface area of a zinc-cobalt metal frame (Zn / Co-MOF), a luminescent material gold cluster (Arg / ATT-AuNCs) of a host-guest structure, DNA walker and HCR cascade connection is utilized, and the electrochemical luminescence sensor for detecting the staphylococcus aureus in the aquatic products is obtained under the condition of no co-reactants. The electrochemical luminescence aptamer sensor for detecting the staphylococcus aureus in the aquatic product is constructed. The sensor disclosed by the invention can be used for judging whether detected water contains a target substance or not according to the change of an electrochemical luminescence peak value of staphylococcus aureus in a detection result. The method has the advantages that the sensor is low in cost and simple in detection operation; the staphylococcus aureus can be qualitatively and quantitatively determined; the method has high sensitivity and low detection limit, and provides a thought for detection of other pathogenic bacteria and toxic and harmful substances.

Description

Technical Field

[0001] The invention relates to a method for preparing an electrochemiluminescent sensor for detecting Staphylococcus aureus in aquatic products, and belongs to the technical field of electrochemiluminescent aptamer sensors. Background Art

[0002] Staphylococcus aureus (S. aureus) is a common foodborne pathogen found in seafood. It is one of the main pathogens causing food poisoning, and the enterotoxins it produces pose a threat to human health. It can cause a variety of infections, including food poisoning, skin infections, and more serious systemic infections, and has a high susceptibility and drug resistance. Through testing, contaminated seafood can be effectively prevented from entering the market, ensuring the food safety of consumers.

[0003] Traditional detection methods for foodborne pathogens, such as traditional culture, polymerase chain reaction (PCR) and ELISA, are relatively mature, with high sensitivity and accuracy, low detection limits and wide detection ranges. However, their applications are limited by the high cost of instruments, long time consumption, complex sample processing, inconvenient portability, and inability to continuously monitor and conduct on-site measurements. In order to overcome these limitations, it is necessary to develop a faster, simpler and more effective S. aureus detection method, which has important theoretical and practical significance for protecting the ecological environment, improving food quality and protecting human safety.

[0004] The purpose of the invention is to provide a method for preparing an electrochemiluminescent aptamer sensor that can overcome the above problems and has simple preparation, flexible operation, high sensitivity, high accuracy, good selectivity and stability. The constructed electrochemiluminescent aptamer sensor does not require the participation of co-reactants, directly binds to the target substance Staphylococcus aureus through the aptamer, activates DNA walker cyclic amplification, and generates a hybridization chain reaction under the catalysis of the enzyme to load the signal probe, which can be used for the specific detection of Staphylococcus aureus in aquatic products. Summary of the invention

[0005] The technical solution is: a method for preparing a co-reactant-free electrochemiluminescent aptamer sensor for detecting Staphylococcus aureus, characterized in that: the sensitive interface composition of the aptamer sensor includes a zinc-cobalt metal framework (Zn / Co-MOF) fixing a hairpin 1, and self-luminous gold clusters (Arg / ATT-AuNCs) as luminescent probes, and a DNA walker cascade hybridization chain reaction (HCR) is used to achieve secondary signal amplification.

[0006] The method for preparing a co-reactant-free electrochemiluminescent aptamer sensor for detecting Staphylococcus aureus is characterized by: cleaning of a glassy carbon electrode (d=3 mm), construction of a sensitive interface of the aptamer sensor and process characterization (preparation of Zn / Co-MOF, Arg / ATT-AuNCs), establishment of a working curve of the aptamer sensor, detection of the performance of the aptamer sensor, and detection of actual samples by the aptamer sensor.

[0007] The method for preparing a co-reactant-free aptamer sensor for detecting Staphylococcus aureus is characterized by: optimizing the experimental conditions, mainly including the pH of the test base solution, HCR reaction time, aptamer concentration, hairpin (H2, H3) concentration, shear enzyme (ExoⅢ) concentration and incubation time; the working curve of the prepared aptamer sensor is: y=511.46+1117.05lgx (R 2 =0.994); the performance test of aptamer sensor includes the determination of specific selectivity, reproducibility, stability and the recovery rate of aptamer sensor for aquatic products.

[0008] Electrochemiluminescence detection of target pathogenic bacteria: The electrochemiluminescence detection method is used to detect aquatic product samples using a three-electrode system under optimal experimental conditions. According to the analysis of the electrochemiluminescence intensity value in the test results, it can be found that the peak value of the electrochemiluminescence intensity is inversely proportional to the concentration of the pathogenic bacteria, and the concentration information of Staphylococcus aureus can be obtained based on this.

[0009] The preparation principle of the present invention is: using Arg / ATT-AuNCs as a luminescent group, using Zn / Co-MOF as a DNA anchoring track, combining enzyme-mediated DNA walker and HCR dual signal amplification strategy to detect Staphylococcus aureus in seafood. In the process of bacterial recognition, the target specifically binds to the aptamer chain and releases the walking chain. The walking chain triggers the enzyme cycle of hairpin 1 (H1). Under the catalysis of Exo III, enzyme-cut fragments are generated to induce cascade chain reactions. Through the loading of the luminescent probe, the conversion rate of H1 is significantly improved, thereby increasing the sensitivity and detection accuracy of the sensor. The aptamer sensor prepared by the above steps is simple and sensitive, has good stability and specificity, good reproducibility, and the recovery rate meets the requirements.

[0010] To achieve the above objectives, the following technical scheme is adopted: A method for preparing a co-reactant-free electrochemiluminescent aptamer sensor for detecting Staphylococcus aureus, characterized by: (1) Cleaning and performance testing of the bare glassy carbon electrode before preparing the aptamer sensor. If the peak potential difference in the cyclic voltammetry curve is below 100 mV, and the oxidation peak and the reduction peak are symmetrical, the glassy carbon electrode can be used. Otherwise, the cleaning step should be returned to meet the requirements. (2) Zn / Co-MOF is drop-coated on the surface of the cleaned glassy carbon electrode to modify H1, and then the walking chain (cDNA) collected by centrifugation after adding the target is connected to the H1 chain, and then Exo III is added, and finally the probe H2-AuNCs and hairpin 3 (H3) are added. After the preparation of the aptamer sensor is completed, it is placed in a 4°C refrigerator for storage.

[0011] To achieve the above objectives, the following technical scheme is adopted: a method for preparing a co-reactant-free electrochemiluminescent aptamer sensor for detecting Staphylococcus aureus, characterized in that: (1) the prepared aptamer sensor is detected by electrochemiluminescence method in a working base solution to obtain the relationship between the peak value and the target substance; (2) a series of Staphylococcus aureus concentration standard solutions are prepared and measured by electrochemiluminescence method to further obtain the working curve, detection range and detection limit of the prepared aptamer sensor; (3) a series of common pathogenic bacteria species are cultured to detect the selectivity of the prepared aptamer sensor; (4) the stability of the above aptamer sensor is verified multiple times by electrochemiluminescence method; (5) actual aquatic product samples are analyzed to obtain the recovery rate of the aptamer sensor.

[0012] The present invention uses Zn / Co-MOF as a carrier material to increase the specific surface area of ​​the electrode, so as to load more H1 and prepare an aptamer sensor with a strong response signal and high sensitivity; Arg / ATT-AuNCs can achieve self-luminescence without co-reactants, thereby avoiding the error caused by the participation of co-reactions, improving repeatability, and increasing the detection accuracy of the aptamer sensor. In addition, the sensor preparation method is simple, has high stability and specificity, and can be used for the detection of Staphylococcus aureus in aquatic product samples, laying a foundation for the development of a portable device for rapid detection of Staphylococcus aureus in aquatic products.

[0013] The preparation process of the aptamer sensor is as follows: 50 μL of a mixed solution of 100 nM Aptamer and 100 nM cDNA was heated at 95°C for 5 minutes and then cooled to 25°C to form double-stranded Apt@cDNA. 20 μL of Staphylococcus aureus and 16 μL of the mixed solution were cultured at 37°C for 30 minutes. Staphylococcus aureus was separated by centrifugation (6000 rpm, 5 minutes) and the supernatant containing cDNA was collected.

[0014] 6 μL Zn / Co-MOF (0.5 mg / mL) was added to the electrode surface for modification. After the electrode was naturally dried, 6 μL hairpin 1 (H1) was dropped onto the electrode surface and waited for drying. 6 μL 0.1% BSA was added and incubated at 37°C for 30 min. 6 μL collected cDNA and Exo III were added and incubated at 37°C for 40 min. 6 μL H2-AuNCs and hairpin 3 (H3) were added and incubated at 37°C for 60 min. Finally, the aptamer sensor was constructed. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 (A, B) SEM images of Zn / Co-MOF; (C, D, E, F, G) Element distribution of C, N, O, Co and Zn in Zn / Co-MOF; (H) EDS image of Zn / Co-MOF, with the inset showing the element percentage.

[0016] Figure 2 (A, B) TEM images of Arg / ATT-AuNCs, with the inset showing the particle size distribution; (C) XPS full spectrum of Arg / ATT-AuNCs; (D, E) XPS detailed spectra of S and Au in Arg / ATT-AuNCs; (F) Fourier transform infrared spectrum; (G) UV spectrum; (H) Fluorescence spectrum, with the inset showing the luminescence color under 365 nm UV light.

[0017] Figure 3 (A) CV characterization of electrochemiluminescence sensor without co-reactant. (B) EIS characterization of electrochemiluminescence sensor without co-reactant.

[0018] Figure 4 Optimization of the test base solution pH for electrochemiluminescence sensors without co-reactants.

[0019] Figure 5 Optimization of HCR reaction time of electrochemiluminescence sensor without co-reactant.

[0020] Figure 6 Optimization of aptamer concentration in electrochemiluminescent sensor without co-reactants.

[0021] Figure 7 Concentration optimization of co-reactant-free electrochemiluminescent sensor hairpins (H2, H3).

[0022] Figure 8 Optimization of ExoⅢ concentration in electrochemiluminescent sensor without co-reactant.

[0023] Fig. 9 Optimization of incubation time of co-reactant-free electrochemiluminescent sensor ExoⅢ.

[0024] Fig.10 (A) In 10 1 -10 9 Electrochemiluminescence spectra of Staphylococcus aureus added within the CFU / mL concentration range; (B) Linear relationship between electrochemiluminescence intensity and Staphylococcus aureus concentration.

[0025] Fig.11 (A) Study on the specificity of electrochemiluminescence sensors without co-reactants; (B) Study on the anti-interference ability of electrochemiluminescence sensors without co-reactants.

[0026] Fig.12 Reproducibility study of electrochemiluminescence sensor without co-reactants.

[0027] Fig.13 Stability study of electrochemiluminescence sensor without co-reactants. DETAILED DESCRIPTION

[0028] Example: (1) Preparation of Zn / Co-MOF: Methylimidazole (MIM, 1.968 mg, 24 mmol) was added to methanol (200 mL) to obtain solution A. Co(NO3)2·6H2O (0.594 g, 10 mmol) and Zn(NO3)2·6H2O (1.164 g, 20 mmol) were dissolved in the same volume of methanol to obtain solution B. Solution A and solution B were rapidly mixed and stirred at room temperature for 24 h, washed with methanol 5 times after mixing, precipitated and freeze-dried at -80 °C to obtain powder, and prepared into a 0.5 mg / mL solution. (2) Preparation of Arg / ATT-AuNCs: 6-Aza-2-thiothymine (ATT, 10 mL, 80 mM) was dissolved in sodium hydroxide (NaOH, 0.2 M) and added to a solution of chloroauric acid (HAuCl4·3H2O, 14 mL, 10 mg / mL). The pH was adjusted to 8 at room temperature and then stirred in the dark for 1 h. Subsequently, it was washed twice with isopropanol (8000 rpm, 15 min) and redissolved in an equal amount of ultrapure water to obtain ATT-AuNCs. Arginine (Arg, 2 mL, 40 mM) was added to 18 mL of the prepared ATT-AuNCs solution (pH 10) and incubated at 37°C for 24 h.

[0029] (3) Collection of walking chain cDNA: 50 μL of a mixed solution of 100 nM Aptamer and 100 nM cDNA was heated at 95°C for 5 minutes and then naturally cooled to 25°C. 20 μL of Staphylococcus aureus and 16 μL of the mixed solution were incubated at 37°C for 30 minutes. Staphylococcus aureus was separated by centrifugation (6000 rpm, 5 minutes) and the supernatant containing cDNA was collected.

[0030] (4) Cleaning of glassy carbon electrode: Before modification, the glassy carbon electrode was polished to a mirror surface with 0.3 μm Al2O3 on suede. After polishing, it was washed with deionized water to remove surface dirt, and then moved to an ultrasonic water bath for cleaning for 5 min each time, repeated twice, and then ultrasonically cleaned with anhydrous ethanol and deionized water, and dried under nitrogen environment.

[0031] (5) Test of glassy carbon electrode: In the presence of 0.1 M KCl and 5 mM [Fe(CN)6] 3- / 4- A cyclic voltammetry curve is run in a 0.01 M PBS solution to test the performance of the glassy carbon electrode, with a scan rate of 50 mV / s and a scan potential of -0.1 to 0.6 V. When the redox peak potential difference in the cyclic voltammetry curve is below 100 mV, the glassy carbon electrode can be further used, otherwise, return to step (5) to continue processing the glassy carbon electrode until it meets the requirements.

[0032] (6) Add 6 μL Zn / Co-MOF (0.5 mg / mL) to the surface of the electrode (GCE) for modification. After the electrode is naturally dried, add 6 μL hairpin 1 (H1) to the electrode surface and wait for it to dry. Then add 6 μL 0.1% BSA and incubate at 37°C for 30 min. Then add 6 μL collected cDNA and Exo III and incubate at 37°C for 40 min. Then add 6 μL H2-AuNCs and hairpin 3 (H3) and incubate at 37°C for 60 min to construct the aptamer sensor.

[0033] (7) A three-electrode system was used to optimize the experimental factors such as the pH of the test solution, HCR reaction time, aptamer concentration, hairpin (H2, H3) concentration, Exo III concentration and incubation time by electrochemiluminescence method. The pH range of the test solution was 6.5~10.0, the HCR reaction time range was 15~105 min, the aptamer concentration range was 10 nM~300 nM, the hairpin (H2, H3) concentration range was 0.5~3.5 μM, the Exo III concentration range was 0.5~3.5 U, and the Exo III incubation time range was 10~70 min.

[0034] (8) The concentration gradient quantitative analysis test was performed using electrochemiluminescence under the optimal test conditions. The test base solution pH was 8.5, the HCR reaction time was 60 min, the aptamer concentration was 150 nM, the hairpin (H2, H3) concentration was 2 μM, the Exo III concentration was 2 U, the Exo III incubation time was 40 min, and the concentration range of Staphylococcus aureus used in the test was 1.00 × 10 1 ~1.00×10 9 CFU / mL, the minimum detection limit of lead was 1.16 CFU / mL, and the regression equations were I ECL =511.46+1117.05lgC(S. aureus), R 2 =0.994, see Fig.10 .

[0035] (9) Add 1.00×10 8 CFU / mL of Escherichia coli, Listeria, Vibrio parahaemolyticus and Salmonella were tested by electrochemiluminescence to detect their anti-interference properties; 6 electrodes were selected to test the anti-interference performance of the prepared electrodes at a concentration of 1.00×10 5 The CFU / mL was measured three times to test its repeatability.

[0036] (10) After the aquatic product samples are crushed, diluted, centrifuged, and filtered, Staphylococcus aureus solution is added to conduct actual sample testing and analysis.

[0037] This co-reactant-free electrochemiluminescent aptamer sensor can detect Staphylococcus aureus in aquatic products. The sensor has low cost, simple operation, short detection time, strong anti-interference ability, good stability, strong specificity, and high detection sensitivity, which is in line with the development of rapid detection technology of Staphylococcus aureus in aquatic products in my country and international requirements.

Claims

1. A method for preparing an electrochemiluminescent sensor for detecting Staphylococcus aureus in aquatic products, characterized in that: An aptamer sensor was constructed using chemical bonding, covalent bonding and self-assembly technology for the detection of Staphylococcus aureus, and a zinc-cobalt metal framework was introduced to load the luminescent material arginine / 6-aza-2-thiothymine-gold cluster. The preparation steps of the zinc-cobalt metal framework and arginine / 6-aza-2-thiothymine-gold cluster were as follows: Preparation of the zinc-cobalt metal framework: Add 24 mmol1.986 mg of methylimidazole to 200 mL of methanol to obtain solution A. 10 mmol 0.594 g of cobalt nitrate hexahydrate and 20 mmol 1.164 g of zinc nitrate hexahydrate were dissolved in the same volume of methanol to obtain solution B. Solution A and solution B were quickly mixed and stirred at room temperature for 24 h. After mixing, they were washed with methanol 5 times, precipitated and freeze-dried at -80°C to obtain powder, and prepared into a 0.5 mg / mL solution; Preparation of arginine / 6-aza-2-thiothymine-gold clusters: 10 mL of 80 mM 6-aza-2-thiothymine was dissolved in 0.2 M sodium hydroxide, added to 14 mL of 10 mg / mL chloroauric acid solution, the pH was adjusted to 8 at room temperature, and then stirred in the dark for 1 h. Subsequently, it was washed twice with isopropanol, centrifuged at 8000 rpm for 15 minutes, and redissolved with an equal amount of ultrapure water to obtain 6-aza-2-thiothymine-gold clusters. 2 mL of 40 mM arginine was added to 18 mL of the prepared 6-aza-2-thiothymine-gold cluster solution, pH = 10, and incubated at 37 °C for 24 h.

2. An electrochemiluminescent aptamer sensor with a signal amplification strategy for detecting Staphylococcus aureus in aquatic products, characterized in that: The recognition of 150 nM aptamer chain and Staphylococcus aureus was used to release cDNA and activate DNAwalker. Under the catalysis of 2U exonuclease III, the hybridization chain reaction was triggered by 40 min of shearing. After 60 min of reaction time, cascade signal amplification was achieved, and more arginine / 6-aza-2-thiothymine-gold cluster-labeled 2 μM probes H2 and H3 were loaded, thereby increasing the detection accuracy and sensitivity of Staphylococcus aureus. The preparation steps of cDNA and probe H2 were as follows: Preparation of cDNA: 50 μL of a mixed solution of 100 nM aptamer and 100 nM cDNA was heated at 95°C for 5 minutes, then cooled naturally to 25°C, and 20 μL of Staphylococcus aureus and 16 After incubating 30 μL of the mixed solution at 37°C for 30 minutes, Staphylococcus aureus was separated by centrifugation at 6000 rpm for 5 minutes, and the supernatant containing cDNA was collected; Preparation of probe H2: Take 2.0 mL of arginine / 6-aza-2-thiothymine-gold cluster, add 200 μL of 1.0 μM H2 solution, stir at room temperature for 3 h, add 100 μL of 0.1% bovine serum albumin solution for 1 h, centrifuge, wash, obtain probe H2, and disperse it in 2.0 mL of phosphate buffer with a pH of 7.

4.

3. A method for preparing the co-reactant-free electrochemiluminescent aptamer sensing interface for Staphylococcus aureus detection according to claim 1, characterized in that: The zinc-cobalt metal framework is connected to the aptamer H1 chain; the arginine / 6-aza-2-thiothymine-gold clusters emit light in the absence of co-reactants, and the DNA walker is activated through the recognition of the aptamer chain and Staphylococcus aureus. Under the catalysis of exonuclease III, the hybridization chain reaction cascade signal amplification is triggered, and more H2-arginine / 6-aza-2-thiothymine-gold clusters are loaded, thereby increasing the detection accuracy and sensitivity of Staphylococcus aureus. The preparation steps of the aptamer sensor are as follows: 6 μL of 0.5 mg / mL zinc-cobalt metal framework is added to the electrode surface for modification, and after the electrode is naturally dried, 6 μL of H1 is added to the electrode surface, and waited for drying, 6 μL of 0.1% bovine serum albumin is added and incubated at 37°C for 30 minutes, 6 μL of collected cDNA and exonuclease III are added and incubated at 37°C for 40 minutes to complete the DNA walker, and then 6 μL of H2-arginine / 6-aza-2-thiothymine-gold clusters and H3 were incubated at 37°C for 60 minutes, and finally the aptamer sensor was constructed and the detection was completed in 4-hydroxyethylpiperazineethanesulfonic acid base solution with a pH of 8.5.

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