Staphylococcus aureus signal switch type electrochemical aptamer sensor based on nano enzyme

Abstract

The invention discloses a staphylococcus aureus signal'switch 'type electrochemical aptamer sensor based on nano-enzyme. The staphylococcus aureus signal'switch' type electrochemical aptamer sensor is prepared according to the following preparation method: step 1, synthesis of Pt / C-ZIF-8 nano-enzyme: firstly, preparing ZIF-8 by using a conventional solvothermal method; then preparing Pt NPs according to the following process: adding PVP into an H2PtCl6 solution, then slowly dropwise adding NaBH4 into the solution, and when the solution is dark brown, continuously stirring to generate a Pt NPs dispersion for later use; then mixing the ZIF-8 solution with the Pt NPs dispersion liquid, and continuously stirring to obtain Pt / ZIF-8; the formed Pt / ZIF-8 powder is carbonized, and Pt / C-ZIF-8 nano enzyme is obtained; and step 2, preparing an Apt / Pt / C-ZIF-8 / GCE sensor: dispensing the Pt / C-ZIF-8 suspension on the surface of a polished glassy carbon electrode to prepare a Pt / C-ZIF-8 / GCE, and immersing the Pt / C-ZIF-8 / GCE into a staphylococcus aureus-Apt solution to obtain the Apt / Pt / C-ZIF-8 / GCE sensor. The method is used for high-sensitivity and high-selectivity detection of staphylococcus aureus.

Description

Technical Field

[0001] The invention relates to a preparation method and an application method of a nanozyme-based Staphylococcus aureus signal "switch" type electrochemical aptamer sensor, belonging to the technical field of electrochemical biosensor. Background Art

[0002] Staphylococcus aureus (S. aureus) is a Gram-positive pathogenic bacterium that can cause a wide range of infections and diseases in humans worldwide (such as meningitis, endocarditis, pneumonia, abscesses, and sepsis), and can produce different toxins that cause food poisoning. Therefore, it is very necessary to construct an effective sensor method for detecting S. aureus.

[0003] Traditional laboratory detection methods for Staphylococcus aureus include enzyme-linked immunosorbent assay, polymerase chain reaction, D-chain displacement amplification and ligase chain reaction, etc. These methods are very important, but they usually have disadvantages such as time-consuming, complicated operation or expensive instruments.

[0004] Compared with these traditional methods, nanozyme-based electrochemical sensors have the characteristics of fast response, simple preparation and low cost. However, metal-based nanozymes are prone to aggregation and inactivation, and most nanozymes have weak catalytic ability and lack specific recognition sites, which greatly limits their practical application. Therefore, it is necessary to design suitable carriers to anchor metals to improve the activity and stability of nanozymes, and to design specific recognition probes to improve the selectivity of detection. Summary of the invention

[0005] The technical problem to be solved by the present invention is: to overcome the defects of the prior art, use ZIF-8 derived carbon to immobilize platinum nanoparticles, use aptamers as recognition devices, and propose a nanozyme-based Staphylococcus aureus signal "switch" type electrochemical aptamer sensor for highly sensitive and selective detection of Staphylococcus aureus.

[0006] In order to solve the above technical problems, the present invention proposes the following technical solutions: a nanozyme-based Staphylococcus aureus signal "switch" type electrochemical aptamer sensor is prepared according to the following preparation method:

[0007] Step 1: Synthesis of Pt / C-ZIF-8 nanozyme;

[0008] First, ZIF-8 was prepared by conventional solvothermal method, and then Pt NPs were prepared according to the following process: PVP was added to H2PtCl6 solution, and then NaBH4 was slowly dripped into the solution. When the color of the above solution turned dark brown, the generated PtNPs dispersion was stirred for later use;

[0009] The ZIF-8 solution was mixed with the Pt NPs dispersion and stirred continuously to obtain Pt / ZIF-8;

[0010] The formed Pt / ZIF-8 powder is carbonized to obtain Pt / C-ZIF-8 nanozyme;

[0011] Step 2: Prepare Apt / Pt / C-ZIF-8 / GCE sensor;

[0012] Pt / C-ZIF-8 suspension was drop-coated on the polished surface of glassy carbon electrode to prepare Pt / C-ZIF-8 / GCE, and Pt / C-ZIF-8 / GCE was immersed in Staphylococcus aureus-Apt solution to obtain Apt / Pt / C-ZIF-8 / GCE sensor.

[0013] The technical solution is further limited in that, in step 1: 51.5 mg PVP is added to 2.0 mM H2PtCl6 solution (50.0 mL), and then 3.0 mL NaBH4 (0.15 mol / L) is slowly dripped into the solution, and when the color of the above solution turns dark brown, the generated Pt NPs dispersion is continuously stirred for 3 hours for later use;

[0014] 5.0 mL of ZIF-8 solution (1.0 mg / mL) was mixed with 100.0 mL of Pt NPs dispersion and stirred continuously to obtain Pt / ZIF-8;

[0015] The formed Pt / ZIF-8 powder was carbonized at 800 °C (5 °C / min) for 2 h to obtain Pt / C-ZIF-8 nanozyme;

[0016] In step 2: 10 μL of Pt / C-ZIF-8 suspension (0.5 mg / mL) was drop-coated on the polished surface of the glassy carbon electrode.

[0017] The present invention has the following beneficial effects:

[0018] 1. The present invention synthesized a Pt / C-ZIF-8 nanohybrid with excellent peroxidase mimetic activity, and innovatively used it to prepare a simple and sensitive Staphylococcus aureus "off-on" electrochemical Apt sensor. The experimental results show that the prepared Pt / C-ZIF-8 nanozyme can accelerate the generation of DAP by catalyzing the oxidation of OPD. Once Pt / C-ZIF-8 combines with Apt to form an Apt / Pt / C-ZIF-8 sensor, its activity will be inhibited, resulting in a decrease in the DAP peak current (signal "off"). After the addition of Staphylococcus aureus, the catalytic activity was successfully restored, and the intensity was linearly related to the concentration of Staphylococcus aureus, resulting in an increase in the DAP peak (signal "on"). By optimizing the experimental conditions, the proposed electrochemical Apt sensor based on the signal "off-on" nanozyme was sensitive to Staphylococcus aureus (LOD, 2CFU / mL; linear range, 10-10 9 CFU / mL).

[0019] 2. The present invention prepares ZIF-8 (a zinc-based MOF) modified with platinum nanoparticles (Pt / ZIF-8), and then carbonizes it to form Pt / C-ZIF-8 with high conductivity and abundant active sites. The results show that Pt / C-ZIF-8 nanozymes have significant catalytic activity for 1,2-diaminobenzene (OPD) oxidation and diaminobenzene (DAP). After the Apt of Staphylococcus aureus is directly applied to the surface of Pt / C-ZIF-8, the activity of the related nanozymes is inhibited due to the coverage of the active sites, resulting in the disappearance of the DAP peak (signal "off"). When Staphylococcus aureus is present, this catalytic activity can be restored, and the DAP current will increase relatively, which is called the signal "on" state. Based on this mechanism, the present invention designs a novel nanozyme-based signal "off-on" electrochemical Apt sensor, which realizes simple and sensitive monitoring of Staphylococcus aureus. The sensor can also avoid the immobilization process of Apt and has great application potential in the detection of various pathogens other than Staphylococcus aureus, providing a novel, simple and reliable strategy for detecting various pathogens by simply changing the relevant Apt. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 These are characterization images during the nanozyme preparation process (TEM images of (a) ZIF-8, (b) Pt / ZIF-8, and (c) Pt / c-ZIF-8; (d) size distribution of Pt NPs on the surface of Pt / C-ZIF-8).

[0021] Figure 2 This is the thermogravimetric analysis (TGA) change curve from Pt / ZIF-8 to Pt / C-ZIF-8.

[0022] Figure 3 The four sensors are 5.0mmol / L[Fe(CN)6] 3- / 4- Electrochemical impedance spectroscopy (EIS) under +0.1 mol / L KCl conditions, including (a) bare GCE, (b) ZIF-8 / GCE, (c) Pt / ZIF-8 / GCE and (d) Pt / c-ZIF-8 / GCE.

[0023] Figure 4 are the DPV response curves of DAP on different sensors, including (a) ZIF-8 / GCE, (b) Pt / ZIF-8 / GCE, (c) Pt / c-ZIF-8 / GCE, and (d) Apt / Pt / c-ZIF-8 / GCE before and (e) after incubation with Staphylococcus aureus.

[0024] Figure 5 This is a condition optimization experiment, including (a) the amount of Pt / c-zif-8 nanozyme, (b) the concentration of OPD, (c) the concentration of H2O2, (d) the concentration of Apt, (e) the incubation time of Apt and (f) the incubation time of Staphylococcus aureus. Staphylococcus aureus concentration: 10 3 CFU / mL.

[0025] Figure 6 This is a sensitivity experiment. (a) Apt / Pt / C-ZIF-8 / GCE for 0~10 9 DPV response of Staphylococcus aureus at different CFU / mL concentrations; (b) Linear relationship between ΔI value and the logarithm of Staphylococcus aureus concentration.

[0026] Figure 7 This is a sensor anti-interference experiment. The concentration of Staphylococcus aureus is 10 5 CFU / mL, the concentration of other pathogens was 10 6 CFU / mL. DETAILED DESCRIPTION

[0027] The present invention proposes a nanozyme-based Staphylococcus aureus signal "switch" type electrochemical aptamer sensor (abbreviated as Apt / Pt / C-ZIF-8 / GCE sensor), which is prepared according to the following preparation method:

[0028] Step 1: Synthesis of Pt / C-ZIF-8 nanozyme

[0029] First, ZIF-8 was prepared by conventional solvothermal method, and then Pt NPs were prepared according to the following process: 51.5 mg PVP was added to 2.0 mM H2PtCl6 solution (50.0 mL), and then 3.0 mL NaBH4 (0.15 mol / L) was slowly dripped into the solution. When the color of the above solution turned dark brown, the generated Pt NPs dispersion was stirred for 3 hours for use.

[0030] Next, 5.0 mL of ZIF-8 solution (1.0 mg / mL) was mixed with 100.0 mL of Pt NPs dispersion and stirred continuously to obtain Pt / ZIF-8.

[0031] Finally, the formed Pt / ZIF-8 powder was carbonized at 800 °C (5 °C / min) for 2 h to obtain Pt / C-ZIF-8 nanozyme.

[0032] Step 2: Preparation of Apt / Pt / C-ZIF-8 / GCE sensor

[0033] 10 μL of Pt / C-ZIF-8 suspension (0.5 mg / mL) was drop-coated on the polished glassy carbon electrode (GCE) surface, and the prepared Pt / C-ZIF-8 / GCE was immersed in Staphylococcus aureus-Apt solution to obtain the Apt / Pt / C-ZIF-8 / GCE sensor.

[0034] The method for detecting S. aureus using the above-mentioned Apt / Pt / C-ZIF-8 / GCE sensor was as follows: the formed Apt / Pt / C-ZIF-8 / GCE was incubated with S. aureus solution (50 μL) at different levels, and the response signal was tested by differential pulse voltammetry (DPV) in an acetate buffer solution (0.2 mol / L) containing H2O2 / OPD.

[0035] Characterization of Pt / C-ZIF-8

[0036] Characterization of nanozyme preparation process Figure 1 As shown in the figure, ZIF-8 presents a smooth and uniform dodecahedral structure (~100nm). Compared with ZIF-8, many tiny Pt NPs with an average size of 2.2±0.2nm appeared on the surface of Pt / ZIF-8 and Pt / C-ZIF-8, indicating that the modification of Pt NPs was successful. However, there was no obvious change in Pt / ZIF-8 before and after carbonization, indicating that the carbonization process did not destroy its structure. Figure 2It is the thermogravimetric analysis (TGA) change curve from Pt / ZIF-8 to Pt / C-ZIF-8. The mass percentage curve remains stable before ~380 °C; after this temperature, the mass drops sharply, indicating the formation of Pt / C-ZIF-8. After about 600 °C, the mass loss stabilizes at about 32.5%, indicating the complete formation of Pt / C-ZIF-8.

[0037] Electrochemical impedance spectroscopy (EIS) was used to evaluate the electron transfer kinetics ( 3- / 4- ) of different material-modified electrodes in a solution of [Fe(CN)6] containing KCl (0.1 mol / L) Figure 3 . The charge transfer resistance (Rct) can be reflected by the semicircle diameter value. By fitting the data with an equivalent circuit, it can be seen that the Rct value of ZIF-8 / GCE is the largest, and the Rct value of Pt / C-ZIF-8 is the weakest. The Rct order of multiple sensing electrodes is Pt / C-ZIF-8 / GCE < Pt / ZIF-8 / GCE < bare GCE < ZIF-8 / GCE, indicating that the conductivity of ZIF-8 is weak, the electron transfer rate at ZIF-8 / GCE is low, while the conductivities of Pt NPs and C-ZIF-8 are high, which can accelerate the electron transfer rate.

[0038] Sensing principle and feasibility

[0039] Figure 4 It is the differential pulse voltammetry (DPV) responses of different sensing electrodes in a 0.2 mol / L buffer solution containing OPD and H2O2, where (a) ZIF-8 / GCE, (b) Pt / ZIF-8 / GCE, (c) Pt / c-ZIF-8 / GCE, (d) Apt / Pt / c-ZIF-8 / GCE before incubation with Staphylococcus aureus and (e) after incubation.

[0040] There is no peak current of DAP at ZIF-8 / GCE, indicating that ZIF-8 / GCE can hardly accelerate the formation of DAP by catalyzing the oxidation of OPD. There are obvious peak currents for Pt / ZIF-8 and Pt / C-ZIF-8, and the peak current of Pt / C-ZIF-8 is much larger than that of Pt / ZIF-8 because the conductivity of Pt / C-ZIF-8 is higher than that of Pt / ZIF-8. This shows that the prepared Pt / C-ZIF-8 nanozyme has good simulated peroxidase activity and can catalyze the oxidation of OPD to generate DAP. When Staphylococcus aureus-Apt is drop-coated on the surface of Pt / C-ZIF-8 through electrostatic interaction to form Apt / Pt / C-ZIF-8 / GCE, the catalytic activity of the nanozyme is inhibited and the DAP current decreases (signal "off"). After the addition of Staphylococcus aureus, the DAP current increases again (signal "on"). This is because the specific binding of Apt to Staphylococcus aureus causes Apt to detach from the surface of the nanozyme, thereby restoring the activity of the nanozyme to a certain extent.

[0041] Optimization of experimental conditions for sensing the signal "switch" in Staphylococcus aureus

[0042] In order to improve the detection sensitivity, key conditions such as the amount of Pt / C-ZIF-8, OPD / H2O2 / Apt concentration, and culture time were optimized. The DAP current change (ΔI) before and after the addition of Staphylococcus aureus was used as the response signal. Figure 5 As shown, the optimal parameters were: Pt / C-ZIF-8 dosage was 10 μL; OPD concentration was 1.8 mmol / L; H2O2 concentration was 30.0 mmol / L; Apt concentration was 0.6 μmol / L; Apt incubation time was 20 min; and Staphylococcus aureus incubation time was 28 min.

[0043] Quantitative response of Staphylococcus aureus on nanozyme electrochemical aptasensor

[0044] Under the optimized experimental conditions, the nanozyme Apt sensor was used to detect Staphylococcus aureus at different concentrations. Figure 6 As shown in (a), the DAP current increases with the increase of Staphylococcus aureus concentration, indicating that the presence of Staphylococcus aureus can restore the catalytic activity of Pt / C-ZIF-8 nanozyme and 9 In the range of CFU / mL, the ΔI value is linearly related to the logarithm of the Staphylococcus aureus concentration (e.g. Figure 6 The corresponding regression equation is ΔI(μA)=0.7894logC-0.4288(R 2 =0.9975), and the limit of detection (LOD) was 2 CFU / mL (S / N=3).

[0045] Anti-interference experiment of signal "switch" type nanozyme electrochemical sensor

[0046] Comparison of the responses of Staphylococcus aureus, Escherichia coli, Shigella flexneri, and Salmonella on the nanozyme electrochemical sensor, e.g. Figure 7 As shown, only Staphylococcus aureus (10 5 CFU / mL) can cause a significant decrease in peak current, while other pathogens (10 6 CFU / mL) had almost no ΔI value. The electrochemical response of the sensor in the coexistence of Staphylococcus aureus and other pathogens was further studied, showing that the DAP current did not change significantly when Staphylococcus aureus existed alone. These results indicate that the designed signal "switch" type nanozyme electrochemical Apt sensor has good selectivity and specificity.

[0047] Practical application of signal "switch" type nanozyme electrochemical sensor

[0048] Different concentrations of Staphylococcus aureus solutions were added to milk samples and the corresponding electrochemical responses were measured. The results showed that the spiked recoveries of Staphylococcus aureus were 92.5% to 96.86%, and the RSD values ​​were 2.37% to 3.32%.

[0049] Table 1 Detection of Staphylococcus aureus in milk by nanozyme electrochemical sensor spike method

[0050]

[0051] The present invention has the following beneficial effects:

[0052] 1. The present invention synthesized a Pt / C-ZIF-8 nanohybrid with excellent peroxidase mimetic activity, and innovatively used it to prepare a simple and sensitive Staphylococcus aureus "switch" electrochemical Apt sensor. The experimental results show that the prepared Pt / C-ZIF-8 nanozyme can accelerate the generation of DAP by catalyzing the oxidation of OPD. Once Pt / C-ZIF-8 combines with Apt to form an Apt / Pt / C-ZIF-8 sensor, its activity will be inhibited, resulting in a decrease in the DAP peak current (signal "off"). After the addition of Staphylococcus aureus, the catalytic activity is restored to a certain extent, and the intensity is linearly related to the degree of Staphylococcus aureus, resulting in an increase in the DAP peak (signal "on"). Through the optimization of multiple conditions, the proposed electrochemical Apt sensor based on the signal "off-on" nanozyme is effective for Staphylococcus aureus (LOD, 2CFU / mL; linear range, 10-10 9 CFU / mL).

[0053] 2. The present invention prepares ZIF-8 (a zinc-based MOF) modified with platinum nanoparticles (Pt / ZIF-8), and then performs carbonization treatment to make the formed Pt / C-ZIF-8 have high conductivity and abundant active sites. The results show that the Pt / C-ZIF-8 nanozyme has significant catalytic activity for the oxidation of 1,2-diaminobenzene (OPD) to diaminobenzene (DAP). The Apt of Staphylococcus aureus is directly applied to the surface of Pt / C-ZIF-8. Since the active sites are covered, the activity of the nanozyme is inhibited, resulting in a decrease in the DAP peak (signal "off"). When Staphylococcus aureus is present, this catalytic activity is restored to a certain extent, causing the DAP current to increase accordingly, which is the signal "on". Based on this mechanism, the present invention designs a new type of nanozyme-based signal "off-on" electrochemical Apt sensor, which realizes simple and sensitive monitoring of Staphylococcus aureus. By changing Apt, it can be used to detect the corresponding pathogens, providing a novel, simple and reliable strategy for the detection of other pathogens.

Claims

1. A nanozyme-based Staphylococcus aureus signal "switch" type electrochemical aptamer sensor, characterized in that: It is prepared according to the following preparation method: Step 1: Synthesis of Pt / C-ZIF-8 nanozyme; First, ZIF-8 was prepared by conventional solvothermal method, and then Pt NPs were prepared according to the following process: PVP was added to H2PtCl6 solution, and then NaBH4 was slowly dripped into the solution. When the color of the above solution turned dark brown, the generated Pt NPs dispersion was stirred for later use; The ZIF-8 solution was mixed with the Pt NPs dispersion and stirred continuously to obtain Pt / ZIF-8; The formed Pt / ZIF-8 powder is carbonized to obtain Pt / C-ZIF-8 nanozyme; Step 2: Prepare Apt / Pt / C-ZIF-8 / GCE sensor; Pt / C-ZIF-8 suspension was drop-coated on the polished surface of glassy carbon electrode to prepare Pt / C-ZIF-8 / GCE, and Pt / C-ZIF-8 / GCE was immersed in Staphylococcus aureus-Apt solution to obtain Apt / Pt / C-ZIF-8 / GCE sensor.

2. A nanozyme-based Staphylococcus aureus signal "switch" type electrochemical aptamer sensor according to claim 1, characterized in that: In step 1: 51.5 mg PVP was added to 2.0 mM H2PtCl6 solution (50.0 mL), and then 3.0 mL NaBH4 (0.15 mol / L) was slowly dripped into the solution. When the color of the above solution turned dark brown, the generated Pt NPs dispersion was stirred for 3 hours for use; 5.0 mL of ZIF-8 solution (1.0 mg / mL) was mixed with 100.0 mL of Pt NPs dispersion and stirred continuously to obtain Pt / ZIF-8; The formed Pt / ZIF-8 powder was carbonized at 800 °C (5 °C / min) for 2 h to obtain Pt / C-ZIF-8 nanozyme; In step 2: 10 μL of Pt / C-ZIF-8 suspension (0.5 mg / mL) was drop-coated on the polished surface of the glassy carbon electrode.

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