An electrochemical sensor for detecting mercury ions in seawater and its preparation method

Abstract

The present invention provides an electrochemical sensor for detecting mercury ions in seawater and a preparation method thereof, belonging to the field of marine chemical technology. The sensor uses PEDOT as a conductive layer, Ag nanoparticles as a bactericidal layer, a ferrocene-modified mercury ion aptamer as a recognition element, and a novel SH-C(E4K4)2C-SH polypeptide as a marine anti-pollution material. The sensor can effectively inhibit Escherichia coli and Staphylococcus aureus, and the combined use of silver ions and polypeptides can produce a synergistic inhibitory effect on the growth of Staphylococcus aureus. At the same time, the sensor exhibits high sensitivity, excellent selectivity, and excellent stability, and the detection limit of mercury ions in seawater can reach 1.12pM.

Description

Technical Field

[0001] The present invention belongs to the technical field of marine chemistry, and in particular relates to an electrochemical sensor for detecting mercury ions in seawater and a preparation method thereof. Background Art

[0002] The quantitative analysis and detection of chemical element components in the ocean is an important topic in marine chemistry research. 2+ Timely and accurate monitoring of heavy metal ions represented by Hg in coastal areas is crucial for evaluating and effectively preventing marine heavy metal pollution. 2+ As one of the most toxic and common heavy metal ions, Hg can be converted into refractory metal compounds in organisms, leading to bioaccumulation and causing great harm to the human body. 2+ The maximum permissible concentration of Hg is 10 nM. 2+ Sensitive, rapid and accurate detection is crucial. Currently, the methods commonly used for monitoring metal ions in the ocean include atomic spectroscopy, molecular spectroscopy and mass spectrometry, but the above detection methods have problems such as complex equipment and unsuitability for on-site measurement. Therefore, the exploration of portable, fast-response and highly sensitive Hg 2+ The detection method is very important.

[0003] Electrochemical sensors are widely used in the detection of heavy metals due to their good responsiveness and selectivity. Among them, electrochemical aptamer sensors are biosensors that use aptamers as sensitive recognition elements. Aptamers are single-stranded nucleic acids that can specifically bind to target molecules and have good stability and non-toxicity, which have been widely used. 2+ It can specifically bind to the thymine-thymine (TT) pair to form a stable T-Hg 2+ -T coordination compound. This interaction induces the folding of single-stranded DNA into a double-stranded structure. This unique interaction provides a basis for the design of highly sensitive and selective electrochemical Hg 2+ Sensors offer a new approach.

[0004] However, long-term detection of Hg in seawater using electrochemical sensors 2+ At concentrations above 500 nm, there is a problem of biofouling, which can cause a decrease in sensor accuracy. Preventing biofouling and improving the stability and reliability of electrochemical sensors are currently urgent issues to be addressed. Summary of the Invention

[0005] The object of the present invention is to provide an electrochemical sensor for detecting mercury ions in seawater and a preparation method thereof, thereby achieving stable and reliable detection of mercury ions in seawater while preventing bacterial contamination.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] First, the present invention provides an electrochemical sensor for detecting mercury ions in seawater to prevent bacterial contamination. The electrochemical sensor is an apt pep-Au / Ag / PEDOT / GCE electrode. The preparation method of each individual electrochemical sensor includes the following steps:

[0008] (1) Polish the bare glassy carbon electrode (GCE) with alumina powder and clean the electrode thoroughly;

[0009] (2) Place GCE in 0.02 M EDOT solution and use the it method to deposit at a potential of 1.0 V for 20 s to obtain a PEDOT / GCE electrode;

[0010] (3) After cleaning the PEDOT / GCE electrode, place it in a AgNO3-NaNO3 solution and deposit it at a potential of -1.0 V for 20 s to obtain an Ag / PEDOT / GCE electrode;

[0011] (4) The Ag / PEDOT / GCE electrode was placed in a HAuCl4-KNO3 solution and the CV method was used at a potential between −1.5 and 0.5 V at a rate of 0.05 V s −1 The Au / Ag / PEDOT / GCE electrode was prepared by scanning 6 cycles at a scan rate of 100 nm.

[0012] (5) Hg 2+ The aptamer was diluted to 10 μM with TE buffer solution, and 10 μM Hg 2+ The aptamer was mixed with 1 M TCEP in a volume ratio of 300:7.5 to obtain Hg 2+ Aptamer solution;

[0013] (6) The polypeptide SH-C(E4K4)2C-SH solution and the Hg 2+ The aptamer solutions were mixed to obtain a mixed solution, and the Au / Ag / PEDOT / GCE was immersed in the mixed solution at room temperature overnight to prepare an apt-pep-Au / Ag / PEDOT / GCE electrode.

[0014] Preferably, in step (1), the diameter of the GCE is 3.0 mm; the polishing method is to polish with 0.3 mm and 0.05 mm alumina powder in sequence;

[0015] In the step (2), the amount of the EDOT solution used is 5.0 mL;

[0016] In step (3), the composition of the AgNO3-NaNO3 solution is 0.5 mM AgNO3 and 0.5 M NaNO3;

[0017] In the step (4), the composition of the HAuCl4-KNO3 solution is 0.5 mM HAuCl4 and 0.5 M KNO3;

[0018] In step (6), the concentration of the polypeptide SH-C(E4K4)2C-SH solution is 0.2 mg / mL.

[0019] Preferably, the amino acid sequence of the polypeptide SH-C(E4K4)2C-SH is: SH-CEEEEKKKKEEEEKKKKC-SH.

[0020] Preferably, the Hg 2+ The sequence of the aptamer is 5'-HS-SH-TTCTTTCTTCGCGTTGTTTTGT-Fe-3'.

[0021] Preferably, the bacterial contamination is Gram-negative bacteria contamination and Gram-positive bacteria contamination.

[0022] Preferably, the bacterial contamination is Gram-positive bacteria.

[0023] Preferably, the Gram-negative bacteria is Escherichia coli, and the Gram-positive bacteria is Staphylococcus aureus.

[0024] Secondly, the present invention provides a method for preparing an electrochemical sensor for detecting mercury ions in seawater that is resistant to bacterial contamination, the preparation method comprising the following steps:

[0025] (1) Polish a 3.0 mm diameter bare glassy carbon electrode (GCE) using 0.3 mm and 0.05 mm alumina powders and clean the electrode thoroughly.

[0026] (2) Place GCE in 5 mL of 0.02 M EDOT solution and deposit it at a potential of 1.0 V for 20 s to obtain a PEDOT / GCE electrode;

[0027] (3) After cleaning, the PEDOT / GCE electrode was placed in an AgNO3-NaNO3 solution consisting of 0.5 mM AgNO3 and 0.5 M NaNO3, and deposited at a potential of -1.0 V for 20 s to obtain an Ag / PEDOT / GCE electrode;

[0028] (4) The Ag / PEDOT / GCE electrode was placed in a HAuCl4-KNO3 solution consisting of 0.5 mM HAuCl4 and 0.5 M KNO3, and the CV method was used at a potential of −1.5 to 0.5 V at a rate of 0.05 V s −1 The Au / Ag / PEDOT / GCE electrode was prepared by scanning 6 cycles at a scan rate of 100 nm.

[0029] (5) Hg 2+ The aptamer was diluted to 10 μM with TE buffer solution, and 10 μM Hg 2+ The aptamer was mixed with 1 M TCEP in a volume ratio of 300:7.5 to obtain Hg 2+ Aptamer solution;

[0030] (6) 0.2 mg / mL peptide SH-C(E4K4)2C-SH solution and the Hg 2+ The aptamer solutions were mixed to obtain a mixed solution, and the Au / Ag / PEDOT / GCE was immersed in the mixed solution at room temperature overnight to prepare an apt pep-Au / Ag / PEDOT / GCE electrode.

[0031] Preferably, the amino acid sequence of the polypeptide SH-C(E4K4)2C-SH is: SH-CEEEEKKKKEEEEKKKKC-SH;

[0032] The Hg 2+ The gene sequence of the aptamer is: 5'-HS-SH-TTCTTTCTTCGCGTTGTTTTGT-Fe-3'.

[0033] Secondly, the present invention provides an application of a polypeptide in preparing an electrochemical sensor for inhibiting the growth of Gram-negative bacteria and Gram-positive bacteria, wherein the polypeptide is an SH-C(E4K4)2C-SH polypeptide, and the amino acid sequence of the polypeptide is: H-CEEEEKKKKEEEEKKKKC-SH.

[0034] Preferably, the Gram-negative bacteria is Escherichia coli, and the Gram-positive bacteria is Staphylococcus aureus.

[0035] The beneficial effects of the present invention are:

[0036] The present invention has developed a Hg 2+ Electrochemical sensors for long-term and accurate monitoring of Hg in the ocean 2+ The sensor uses a new green and environmentally friendly peptide as an anti-pollution material for the ocean. The peptide has excellent hydrophilicity and can prevent the non-specific adsorption of bacterial proteins. It uses silver nanoparticles as a sterilization layer and uses ferrocene-modified Hg2+ Aptamer detection of Hg 2+ , in Hg 2+ The presence of induced aptamer folding to form T-Hg 2+ -T structure brings the electrochemical signal indicator ferrocene close to the sensor surface, which can effectively enhance the electrochemical response;

[0037] Secondly, the fabricated sensor utilizes PEDOT as a conductive layer to enhance signal transmission, while incorporating Ag NPs and zwitterionic peptides as antibacterial and antifouling layers. This design not only effectively eliminates bacteria but also prevents the adhesion of live and dead bacteria, as well as proteins, to the electrode surface, resulting in a significant synergistic inhibitory effect on the growth of Staphylococcus aureus.

[0038] In addition, the sensor prepared by the present invention exhibits high sensitivity, excellent stability and 2+ Moreover, the accuracy of the sensor's detection results in seawater is consistent with that in ultrapure water, demonstrating its practicality in real samples.

[0039] In summary, the innovative sensor prepared by the present invention is expected to detect Hg in actual marine environments. 2+ . BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The morphology changes of the GCE electrode after the gradual deposition of PEDOT, Ag NPs, and Au NPs;

[0041] Among them, (A) is the morphological change of PEDOT / GCE electrode, (B) is the morphological change of Ag / PEDOT / GCE electrode, and (C) is the morphological change of Au / Ag / PEDOT / GCE electrode;

[0042] Figure 2 The element changes on the electrode surface after layer-by-layer modification;

[0043] Among them, (A) is the element change on the surface of PEDOT / GCE electrode, (B) is the element change on the surface of Ag / PEDOT / GCE electrode, (C) is the element change on the surface of Au / Ag / PEDOT / GCE electrode, and (D) is the element change on the surface of apt pep-Au / Ag / PEDOT / GCE electrode;

[0044] Figure 3 For the case containing 5.0mm[Fe(CN)6] 3- / 4- DPV signals of various electrodes in PBS;

[0045] Among them, (a) is the DPV signal of GCE electrode, (b) is the DPV signal of PEDOT / GCE electrode, (c) is the DPV signal of Ag / PEDOT / GCE electrode, (d) is the DPV signal of Au / Ag / PEDOT / GCE electrode, (e) is the DPV signal of apt-pep-Au / Ag / PEDOT / GCE electrode, (f) is the DPV signal of Hg 2+ -DPV signal of apt-pep-Au / Ag / PEDOT / GCE electrode;

[0046] Figure 4 The results show the effects of different modified interfaces on the growth of Escherichia coli and Staphylococcus aureus.

[0047] Among them, (A) is the growth curve of Escherichia coli; (B) is the growth curve of Staphylococcus aureus;

[0048] Figure 5 The detection results of the current signal changes of electrochemical sensors with different interfaces after being immersed in Escherichia coli and Staphylococcus aureus solutions;

[0049] Among them, in the E. coli solution, (A1) is the current signal change of the GCE electrode, (B1) is the current signal change of the Ag / PEDOT / GCE electrode, (C1) is the current signal change of the pep-Au / PEDOT-GCE electrode, and (D1) is the current signal change of the apt-pep-Au / Ag / PEDOT / GCE electrode; in the Staphylococcus aureus solution, (A2) is the current signal change of the GCE electrode, (B2) is the current signal change of the Ag / PEDOT / GCE electrode, (C2) is the current signal change of the pep-Au / PEDOT-GCE electrode, and (D2) is the current signal change of the apt-pep-Au / Ag / PEDOT / GCE electrode;

[0050] Figure 6 The fluorescence detection results of the anti-fouling performance of different interfaces after being immersed in Escherichia coli and Staphylococcus aureus solutions respectively;

[0051] Among them, after immersion in E. coli solution, (A1) is the fluorescence image of the interface without any modification, (B1) is the fluorescence image of the Ag / PEDOT interface, (C1) is the fluorescence image of the pep-Au / PEDOT interface, and (D1) is the fluorescence image of the apt-pep-Au / Ag / PEDOT interface; after immersion in Staphylococcus aureus solution, (A2) is the fluorescence image of the interface without any modification, (B2) is the fluorescence image of the Ag / PEDOT interface, (C2) is the fluorescence image of the pep-Au / PEDOT interface, and (D2) is the fluorescence image of the apt-pep-Au / Ag / PEDOT interface;

[0052] Figure 7 Hg prepared by the present invention 2+ Electrochemical sensor (apt-pep-Au / Ag / PEDOT / GCE) for different concentrations of Hg 2+ Detection effect of solution;

[0053] Where A) is Hg 2+ Electrochemical sensor with different concentrations of target Hg 2+ DPV response curve after incubation; (B) Hg 2+ Electrochemical sensor with different concentrations of target Hg 2+ Current change after incubation (ΔI = I0 − I), the inset shows the linear curve.

[0054] Figure 8 Hg prepared by the present invention 2+ Results of tests on the selectivity, reproducibility, stability, and long-term performance of electrochemical sensors;

[0055] Where (A) is Hg 2+ The current changes when the electrochemical sensor detects different metal ions and their mixtures; (B) The current changes when the electrochemical sensor detects Hg using 7 independently prepared biosensors 2+ Test results after testing; (C) is Hg 2+ The electrochemical sensor was prepared in the presence of 5.0 mM [Fe(CN)6] 3− / 4− PBS at 0.10 V s -1 The CV images of Hg were scanned at a scanning speed of 60 cycles; (D) 2+ Stability test results of the electrochemical sensor after 5 weeks of storage in seawater;

[0056] Figure 9 Hg 2+ Test results of the detection performance of electrochemical sensors in seawater;

[0057] Where (A) is Hg 2+Differential pulse voltammetric response of the electrochemical sensor in seawater; (B) is immersed in ultrapure water (blue line) and seawater (red line) with different concentrations of Hg 2+ Hg in 2+ Calibration curve of the electrochemical sensor. DETAILED DESCRIPTION

[0058] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0059] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0060] The materials and reagents used in the present invention are as follows:

[0061] The peptide SH-C(E4K4)2C-SH (amino acid sequence: SH-CEEEEKKKKEEEEKKKKC-SH, SEQ ID NO. 1, purity >95%) was synthesized by BankPeptide, Inc. (Hefei, China). 2+ The aptamer (5'-HS-SH-TTCTTTCTTCGCGTTGTTTTGT-Fe-3') was purchased from Sangon Biotech (Shanghai, China). 3,4-Ethoxybenzene (C6H6O2S, EDOT) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Chloroauric acid (HAuCl4) was obtained from Shanghai Yien Chemical Technology Co., Ltd. (Shanghai, China). HgCl2, K3Fe(CN)6, and K4Fe(CN)6 were purchased from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). Ultrapure water (resistivity ≤18 mωcm) was used throughout the experiments. All other chemicals were of analytical grade and used without further purification.

[0062] The instruments used in the present invention are as follows:

[0063] All electrochemical measurements were performed at room temperature using a CHI660E electrochemical workstation. The morphologies of the various modified surfaces were characterized using a Hitachi S-4800 scanning electron microscope (SEM) (Hitachi, Japan). Fluorescence observation of bacteria attached to the samples was performed using a fluorescence microscope (IX–73, using Cellsens imaging software, Olympus, Japan). The conductivity of the aqueous medium in the test glass tube (OD = 5.0 mm, ID = 4.0 mm) was monitored online using a developed eight-channel capacitively coupled non-contact conductivity detector (manufactured by eDAQ Pty Ltd, Sydney, Australia) including the TERA TERM software.

[0064] Example 1 Preparation of Hg with high efficiency in anti-bacterial pollution function 2+ Electrochemical sensor (apt-pep-Au / Ag / PEDOT / GCE)

[0065] (1) Polish the bare glassy carbon electrode (GCE, 3.0 mm diameter) using 0.3 mm and 0.05 mm alumina powders, respectively, and clean the electrode thoroughly.

[0066] (2) Place GCE in 5.0 mL of 0.02 M EDOT solution and deposit it at a potential of 1.0 V for 20 s to obtain a PEDOT / GCE electrode;

[0067] (3) After cleaning, the PEDOT / GCE electrode was placed in a AgNO3-NaNO3 solution (0.5 mM AgNO3 and 0.5 M NaNO3) and deposited at a potential of -1.0 V for 20 s to obtain an Ag / PEDOT / GCE electrode;

[0068] (4) The Ag / PEDOT / GCE electrode was placed in a HAuCl4-KNO3 solution (0.5 mM HAuCl4 and 0.5 M KNO3) and the CV method was used at a potential range of −1.5 to 0.5 V at a rate of 0.05 V s −1 The scan rate was 6 cycles to prepare Au / Ag / PEDOT / GCE electrode;

[0069] (5) Hg 2+ The aptamer was diluted to 10 μM with TE buffer (10 mM Tris and 1 mM EDTA), and 300 μL of 10 μM Hg 2+ The aptamer was mixed with 7.5 μL of 1 M TCEP;

[0070] (6) The prepared Au / Ag / PEDOT / GCE was placed in a solution containing 0.2 mg / ml peptide SH-C(E4K4)2C-SH and 5.0 μM Hg 2+ The aptamer-peptide mixture was immersed in the aptamer-peptide mixture at room temperature overnight to allow the peptide and aptamer to self-assemble on the electrode surface through Au-S bonds, thereby preparing the apt-pep-Au / Ag / PEDOT / GCE electrode.

[0071] Comparative Example 1 Preparation of Ag / PEDOT / GCE Electrode

[0072] (1) Polish the bare glassy carbon electrode (GCE, 3.0 mm diameter) using 0.3 mm and 0.05 mm alumina powders, respectively, and clean the electrode thoroughly.

[0073] (2) Place GCE in 5.0 mL of 0.02 M EDOT solution and deposit it at a potential of 1.0 V for 20 s to obtain a PEDOT / GCE electrode;

[0074] (3) After cleaning, the PEDOT / GCE electrode was placed in a AgNO3-NaNO3 solution (0.5 mM AgNO3 and 0.5 M NaNO3) and deposited at a potential of -1.0 V for 20 s to obtain an Ag / PEDOT / GCE electrode;

[0075] Comparative Example 2 Preparation of pep-Au / PEDOT-GCE electrode

[0076] (1) Polish the bare glassy carbon electrode (GCE, 3.0 mm diameter) using 0.3 mm and 0.05 mm alumina powders, respectively, and clean the electrode thoroughly.

[0077] (2) Place GCE in 5.0 mL of 0.02 M EDOT solution and deposit it at a potential of 1.0 V for 20 s to obtain a PEDOT / GCE electrode;

[0078] (3) Afterwards, the electrode was placed in a HAuCl4-KNO3 solution (0.5 mM HAuCl4 and 0.5 M KNO3) and the CV method was used at a potential range of −1.5 to 0.5 V at a rate of 0.05 V s −1 The Au / PEDOT / GCE electrode was prepared by scanning 6 circles at a scan rate of .

[0079] (4) The Au / PEDOT / GCE electrode was placed in a 0.2 mg / mL peptide SH-C(E4K4)2C-SH solution and soaked overnight at room temperature to prepare pep-Au / PEDOT-GCE.

[0080] Example 2

[0081] The morphology and elements of the GCE electrodes deposited with PEDOT, Ag NPs, and Au NPs were characterized by scanning electron microscopy (SEM) and mapping. Figure 1 and Figure 2 shown.

[0082] from Figure 1 In the figure, we can see that the electrode surface becomes rough after depositing PEDOT, while after depositing Ag NPs, the Ag NPs are evenly distributed on the electrode surface. The deposited Au NPs are also evenly and densely distributed on the electrode surface. This not only improves the conductivity of the electrode, but also provides a larger surface area for the subsequent immobilization of peptides and ligands.

[0083] Secondly, from Figure 2 In the figure, we can see that PEDOT, Ag NPs, and Au NPs are successfully electrodeposited on the electrode surface, and the N element also demonstrates the successful connection between the peptide and the ligand.

[0084] Example 3

[0085] Through DPV detection, the 3- / 4- DPV signals of various electrodes in PBS, thus for Hg 2+ Characterization of the layer-by-layer assembly process on electrochemical sensor surfaces

[0086] (1) The prepared electrodes were placed in Hg 2+ Soak in the solution for 1 h;

[0087] (2) After rinsing the electrodes, the detection electrode is used as the working electrode, the saturated calomel electrode (SCE) is used as the reference electrode, and the platinum wire electrode is used as the counter electrode;

[0088] (3) In 5.0 mM [Fe(CN)6] 3− / 4− Differential pulse voltammetry (DPV, potential range -0.2 ~ 0.6 V, amplitude 0.05 V) was performed in the Figure 3 shown.

[0089] from Figure 3 In the figure, we can see that the current signal is enhanced after the gradual deposition of PEDOT, Ag NPs, and Au NPs. This is because PEDOT, Ag NPs, and Au NPs all have good electrical conductivity and can also increase the specific surface area of the electrode after deposition. 2+ After the aptamer, the current signal decreases because the peptide and Hg 2+ The poor conductivity of the aptamer hinders the charge transfer between the interface and the solution. 2+ With Hg2+ After the aptamer binds, the electrochemical signal increases again because Hg 2+ The presence of induced aptamer folding to form T-Hg 2+ -T structure, which brings the electrochemical signal indicator ferrocene close to the sensor surface, thereby increasing the electrochemical signal.

[0090] Example 4

[0091] The anti-fouling properties of the prepared different modified interfaces were evaluated using Escherichia coli (Gram-negative bacteria) and Staphylococcus aureus (Gram-positive bacteria).

[0092] First, the growth curves of Escherichia coli and Staphylococcus aureus were determined by real-time online monitoring of the conductivity changes of LB broth using a multi-channel contactless conductivity sensor (CCS).

[0093] (1) Different modified interfaces are placed at 10 6 CFU mL -1 The cells were immersed in the bacterial suspension of Escherichia coli and Staphylococcus aureus for 24 h, and then placed in test tubes filled with LB broth (5 mm NMR tubes, NORELL, USA). All the test tubes were then inserted into the CCS at the same time.

[0094] (2) The growth curve of bacteria is monitored online in real time by CCS to reflect the anti-pollution status of different interfaces. The results are as follows: Figure 4 shown.

[0095] from Figure 4 It can be seen that the positive control (inoculation concentration of 10 6 CFU mL -1 In the absence of antifouling materials, Escherichia coli and Staphylococcus aureus both showed typical S-shaped growth curves;

[0096] Ag / PEDOT / GCE, pep-Au / PEDOT-GCE, and apt-pep-Au / Ag / PEDOT / GCE can all significantly inhibit the growth of Escherichia coli and Staphylococcus aureus;

[0097] At the same time, we can see that for Staphylococcus aureus, the peptide and Ag produced an obvious synergistic inhibitory effect, and had an extremely significant inhibitory effect on the growth of Staphylococcus aureus.

[0098] This may be due to the bactericidal effect of Ag NPs at the pep-Au / Ag / PEDOT interface, which leads to a prolonged hysteresis phase in the bacterial growth curve. Due to the presence of the peptide, a hydration layer tends to form on the interface surface, reducing bacterial adhesion and thus producing a synergistic inhibitory effect on the growth of S. aureus.

[0099] Example 5

[0100] Evaluation of the anti-pollution ability of electrochemical sensors at different interfaces using DPV

[0101] (1) The electrodes with different modified interfaces were immersed in a solution with a concentration of 10 8 CFU mL -1 coli and Staphylococcus aureus in the bacterial suspension for 2 h;

[0102] (2) Observe the changes in DPV response signals before and after immersion to evaluate its antifouling effect. The results are as follows: Figure 5 shown.

[0103] from Figure 5 As can be seen from A1 and A2, when the GCE electrode was immersed in the same concentration of Escherichia coli and Staphylococcus aureus for 2 h, the current signal decreased significantly, indicating that the anti-pollution ability of the GCE electrode was extremely poor;

[0104] from Figure 5 As can be seen from B1 and B2, the current signal decreases slightly after Ag / PEDOT / GCE is immersed. This is because Ag NPs have good bactericidal properties and can kill bacteria on the electrode surface. However, as time goes by, dead bacteria will accumulate on the electrode surface, causing the current signal to decrease slightly.

[0105] from Figure 5 As can be seen from C1 and C2, the current signal of the pep-Au / Ag / PEDOT / GCE electrode decreased slightly before and after immersion, indicating that the designed peptide has good anti-fouling ability;

[0106] from Figure 5 As shown in D1 and D2, the current signal of the pep-Au / Ag / PEDOT / GCE electrode does not change significantly before and after immersion, which is the result of the synergistic effect of AgNPs and peptides. This shows that the prepared sensor has excellent anti-fouling ability.

[0107] Example 6

[0108] Fluorescence microscopy to detect bacteria immersed in electrodes

[0109] (1) Different modified interfaces were exposed to different concentrations of 10 6 CFU mL -1 coli and Staphylococcus aureus for 24 h, and then the interface was gently rinsed with saline to remove unattached bacteria;

[0110] (2) Use the live bacteria / dead bacteria double staining kit (SYTO 9 / PI) to stain for 15 minutes in the dark at room temperature. Observe the bacteria on the interface using a fluorescence microscope to distinguish between live and dead bacteria and evaluate the anti-fouling ability of the modified interfaces. The results are as follows: Figure 6 shown.

[0111] from Figure 6 It can be seen that a large number of live bacteria adhered to the surface of the GCE electrode, whether immersed in E. coli or Staphylococcus aureus solution, indicating that the anti-pollution ability was weak;

[0112] The Ag / PEDOT / GCE electrode exhibited significant red fluorescence, indicating excellent bactericidal activity, but also caused the accumulation of dead bacteria on the electrode surface area;

[0113] However, the pep-Au / Ag / PEDOT / GCE electrode showed weak red and green fluorescence signals, indicating its good anti-fouling ability and ability to effectively prevent bacterial adhesion.

[0114] Example 7

[0115] Detection of Hg prepared by the present invention 2+ Electrochemical sensor (apt-pep-Au / Ag / PEDOT / GCE) for different concentrations of Hg 2+ Solution detection effect

[0116] (1) Hg 2+ Electrochemical sensor (apt-pep-Au / Ag / PEDOT / GCE) with different concentrations of target Hg 2+ incubation;

[0117] (2) Detect and draw the DPV response curve and current change curve, and the results are as follows Figure 7 shown.

[0118] from Figure 7 It can be seen that in the range of 0.001–100 nM, the DPV oxidation peak current increases with the concentration of Hg 2+ The concentration increases with the increase of Hg 2+ The logarithm of the concentration is linear, and the regression equation of the calibration curve is y=8.61x +27.31. The linear correlation coefficient R 2 =0.998, and the detection limit was as low as 0.073 pM (S / N=3).

[0119] Example 8

[0120] This example detects the Hg 2+ Selectivity, reproducibility, and stability of electrochemical sensors

[0121] (1) To study Hg 2+ The selectivity of the electrochemical sensor is to detect a series of metal ions, among which Hg 2+ The concentration was 50 nM, and the concentrations of the other metal ions were all 5 μM. The results were as follows Figure 8 As shown in A;

[0122] (2) Five independently constructed Hg 2+ The reproducibility of the electrochemical sensor was tested, and the results are shown in 8B;

[0123] (3) Using continuous CV scanning to measure Hg 2+ The stability of the electrochemical sensor was evaluated and the results were as follows Figure 8 As shown in C;

[0124] (4) After the sensor is placed in seawater for 1-5 weeks, its DPV peak current signal is detected. The results are as follows: Figure 8 As shown in D.

[0125] from Figure 8 A can be seen that the Hg prepared by the present invention 2+ Electrochemical sensor Hg 2+ The current response signal is significantly higher than that of other metal ions, indicating that the sensor has excellent selectivity.

[0126] from Figure 8 B can be seen that the Hg prepared by the present invention 2+ The relative standard deviation (RSD) of the electrochemical sensor was 2.93%, indicating that Hg 2+ Electrochemical sensors have excellent reproducibility.

[0127] from Figure 8 C can be seen that the Hg prepared by the present invention 2+ After 60 cycles of the electrochemical sensor, the CV curve remained basically unchanged;

[0128] from Figure 8 D can be seen that the Hg prepared by the present invention 2+ After the electrochemical sensor was stored in seawater for 5 weeks, its DPV peak current signal only decreased by 5%, indicating that the sensor has excellent stability.

[0129] Example 9

[0130] This example detects the Hg 2+ Detection performance of electrochemical sensors in seawater

[0131] (1) Prepare Hg with a concentration of 0.001-100 nM using seawater as solvent 2+ solution;

[0132] (2) Using the Hg prepared by the present invention 2+ Electrochemical sensor for different concentrations of Hg 2+ The solution was tested and the results were as follows Figure 9 shown.

[0133] from Figure 9 It can be seen that in the range of 0.001 to 100 nM, the regression equation of the calibration curve is y=8.53x+29.10, and the linear correlation coefficient R 2 The detection limit was 1.12 pM (S / N=3), which was similar to the detection results observed in ultrapure water. 2+ Electrochemical sensors can be used to detect Hg in real oceans 2+ Detection.

Claims

1. An electrochemical sensor for detecting mercury ions in seawater and preventing bacterial contamination, characterized in that: The electrochemical sensor is an apt-pep-Au / Ag / PEDOT / GCE electrode, and the preparation method of each individual electrochemical sensor includes the following steps: (1) Polish the bare glassy carbon electrode (GCE) with alumina powder and clean the electrode thoroughly; (2) Place GCE in 0.02 M EDOT solution and use the it method to deposit at a potential of 1.0 V for 20 s to obtain a PEDOT / GCE electrode; (3) After cleaning the PEDOT / GCE electrode, the electrode was placed in a AgNO3-NaNO3 solution and deposited at a potential of -1.0 V for 20 seconds to obtain an Ag / PEDOT / GCE electrode; (4) The Ag / PEDOT / GCE electrode was placed in a HAuCl4-KNO3 solution and the CV method was used at a potential between −1.5 and 0.5 V at a rate of 0.05 V s −1 The Au / Ag / PEDOT / GCE electrode was prepared by scanning 6 cycles at a scan rate of 100 nm. (5) Hg 2+ The aptamer was diluted to 10 μM with TE buffer solution, and 10 μM Hg 2+ The aptamer was mixed with 1 M TCEP in a volume ratio of 300:7.5 to obtain Hg 2+ Aptamer solution; (6) The polypeptide SH-C(E4K4)2C-SH solution and the Hg 2+ The aptamer solutions were mixed to obtain a mixed solution, and the Au / Ag / PEDOT / GCE was immersed in the mixed solution at room temperature overnight to prepare an apt-pep-Au / Ag / PEDOT / GCE electrode; The amino acid sequence of the polypeptide SH-C(E4K4)2C-SH is: SH-CEEEEKKKKEEEEKKKKC-SH.

2. The electrochemical sensor for detecting mercury ions in seawater and preventing bacterial contamination according to claim 1, wherein: In step (1), the diameter of the GCE is 3.0 mm; the polishing method is to use 0.3 mm and 0.05 mm alumina powders in sequence for polishing; In the step (2), the amount of the EDOT solution used is 5.0 mL; In step (3), the composition of the AgNO3-NaNO3 solution is 0.5 mM AgNO3 and 0.5 M NaNO3; In the step (4), the composition of the HAuCl4-KNO3 solution is 0.5 mM HAuCl4 and 0.5 M KNO3; In step (6), the concentration of the polypeptide SH-C(E4K4)2C-SH in the mixed solution is 0.2 mg / mL.

3. The electrochemical sensor for detecting mercury ions in seawater and preventing bacterial contamination according to claim 2, wherein: The Hg 2+ The sequence of the aptamer is 5'-HS-SH-TTCTTTCTTCGCGTTGTTTTGT-Fe-3'.

4. The anti-bacterial electrochemical sensor for detecting mercury ions in seawater according to claim 3, characterized in that: The bacterial contamination includes Gram-negative bacteria contamination and Gram-positive bacteria contamination.

5. The anti-bacterial electrochemical sensor for detecting mercury ions in seawater according to claim 4, characterized in that: The Gram-negative bacteria is Escherichia coli, and the Gram-positive bacteria is Staphylococcus aureus.

6. A method for preparing an electrochemical sensor for detecting mercury ions in seawater and preventing bacterial contamination, characterized in that: The preparation method comprises the following steps: (1) Polish a 3.0 mm diameter bare glassy carbon electrode (GCE) using 0.3 mm and 0.05 mm alumina powders and clean the electrode thoroughly. (2) Place GCE in 5 mL of 0.02 M EDOT solution and deposit it at a potential of 1.0 V for 20 s to obtain a PEDOT / GCE electrode; (3) After cleaning, the PEDOT / GCE electrode was placed in an AgNO3-NaNO3 solution consisting of 0.5 mM AgNO3 and 0.5 M NaNO3, and deposited at a potential of -1.0 V for 20 s to obtain an Ag / PEDOT / GCE electrode; (4) The Ag / PEDOT / GCE electrode was placed in a HAuCl4-KNO3 solution consisting of 0.5 mM HAuCl4 and 0.5 M KNO3, and the CV method was used at a potential of −1.5 to 0.5 V at a rate of 0.05 V s −1 The Au / Ag / PEDOT / GCE electrode was prepared by scanning 6 cycles at a scan rate of 100 nm. (5) Hg 2+ The aptamer was diluted to 10 μM with TE buffer solution, and 10 μM Hg 2+ The aptamer was mixed with 1 M TCEP in a volume ratio of 300:7.5 to obtain Hg 2+ Aptamer solution; (6) 0.2 mg / mL peptide SH-C(E4K4)2C-SH solution and the Hg 2+ The aptamer solutions were mixed to obtain a mixed solution, and the Au / Ag / PEDOT / GCE was immersed in the mixed solution at room temperature overnight to prepare an apt-pep-Au / Ag / PEDOT / GCE electrode; The amino acid sequence of the polypeptide SH-C(E4K4)2C-SH is: SH-CEEEEKKKKEEEEKKKKC-SH; The Hg 2+ The gene sequence of the aptamer is: 5'-HS-SH-TTCTTTCTTCGCGTTGTTTTGT-Fe-3'.

7. Use of a polypeptide in the preparation of an electrochemical sensor for inhibiting the growth of Gram-negative bacteria and Gram-positive bacteria, characterized in that: The polypeptide is SH-C(E4K4)2C-SH polypeptide, and the amino acid sequence of the polypeptide is: SH-CEEEEKKKKEEEEKKKKC-SH; The electrochemical sensor for inhibiting the growth of Gram-negative bacteria and Gram-positive bacteria is the electrochemical sensor described in claim 1.

8. The use according to claim 7, characterized in that The Gram-negative bacteria is Escherichia coli, and the Gram-positive bacteria is Staphylococcus aureus.

Images