Electrochemical visual dual-mode sensor as well as preparation method and application thereof

Abstract

The invention provides an electrochemical visual dual-mode sensor and a preparation method and application thereof, and belongs to the technical field of environmental monitoring, a working electrode is a disposable screen printing electrode SPCE, and the electrode is activated; the method comprises the following steps: firstly, irradiating a GV solution by using an ice cyan LED lamp to generate < 1 > O2, directly irradiating the GV solution under an acidic condition to enable colorless TMB to generate blue oxTMB, and enabling a blue product to completely disappear when the pH value is equal to 7.0; on the basis, an oxidizing medium Ce < 3 + > is added, and Ce < 3 + > is used as the oxidizing medium to oxidize TMB under a neutral condition; lCys containing biological mercaptan is introduced as a receptor for identifying Hg < 2 + >, and an electrochemical visual dual-mode sensor is constructed. According to the present invention, the detection result visualization is achieved, the linear range of the sensor is wide, the detection limit is low, the sensitivity is high, and the real natural water body detection condition is met.

Description

Technical Field

[0001] The invention provides an electrochemical visualization dual-mode sensor and a preparation method and application thereof, belonging to the technical field of environmental monitoring. Background Art

[0002] Hg 2+ It is the most stable and highly toxic heavy metal ion pollutant in water bodies. It is mainly discharged into natural water bodies through various human production and life activities, which not only harms the aquatic ecosystem but also poses a certain threat to human health.

[0003] Existing Hg 2+ The detection methods mainly include fluorescent biosensor, visual sensor and electrochemical biosensor.

[0004] Fluorescence biosensing mainly uses biological molecules as receptors and fluorescent dyes or nanomaterials as signal outputs. It detects the change in signal intensity of fluorescent substances after the target substance binds to the receptor to achieve quantitative detection of the target. However, this method has the following disadvantages: In order to detect and track the target molecule, it is necessary to establish a relationship with the signal of the fluorescent probe. The traditional method uses fluorescent dyes as fluorescent probes, but fluorescent dyes are limited in their application in biological imaging and precise detection of biological molecules due to their poor resistance to photobleaching, narrow absorption bands, and short fluorescence lifetimes.

[0005] Visual biosensing mainly uses the changes on the surface of substances before and after the reaction substances bind to certain biological receptors, and can directly observe the reaction results with the naked eye. However, the pH value of natural water bodies is mostly neutral or weakly alkaline, and the traditional method of constructing biosensors using photooxidase-catalyzed substrate TMB is mostly colorimetric under low pH conditions (pH = 4). Under neutral conditions (pH = 7), TMB is difficult to be oxidized, and the specific requirements for the detection environment lead to great detection limitations.

[0006] Electrochemical biosensors are sensors that convert the chemical amount of a substance to be measured into an electrochemical signal for detection. They often use highly sensitive and selective biomolecules such as DNA enzymes and antibodies as receptors for the substance to be measured. However, photocatalytic electrochemical DNA biosensors require the addition of an electrochemical indicator Fe[(CN)6] 3 / 4 Indirect characterization is performed by detecting the negatively charged [Fe(CN)6] 3- / 4- The electrostatic repulsion between ions and the negatively charged DNA phosphate backbone is used to determine the oxidative cleavage of DNA on the electrode surface based on the EIS spectrum. The Rct changes are then analyzed in relation to the different concentrations of Hg 2+ The linear relationship between 2+ The quantitative detection of Hg in water by this method is2+ The output signal of the photocatalytic electrochemical DNA biosensor is single, which causes certain inconvenience to the observation of the results, and requires the addition of an external electrochemical indicator for indirect characterization, which has certain limitations. Summary of the invention

[0007] In view of the above technical problems, the present invention provides an electrochemical visualization dual-mode sensor and its preparation method and application, which is based on the "switch-on" electrochemical visualization dual-mode sensor that uses nucleic acid dye to photo-oxidize TMB under neutral conditions to detect Hg in natural environment water. 2+ Detection.

[0008] The present invention selects TMB as an electrochemical indicator and a visual color developer, and utilizes nucleic acid dyes GV and Ce 3+ The photosensitivity of the complex, through the electrochemical signal generated by the oxidation reaction and the change in solution color, has constructed an intuitive, simple, highly sensitive, portable, and Hg detection device in a neutral environment. 2+ Electrochemical visualization dual-mode sensor detection device.

[0009] The specific technical solutions are:

[0010] An electrochemical visualization dual-mode sensor and a preparation method thereof, comprising the following steps:

[0011] Step 1 Pretreatment of screen-printed electrodes;

[0012] The working electrode used was a disposable screen-printed electrode SPCE, and the electrode was activated;

[0013] Step 2: Construction of photocatalytic system;

[0014] First, the GV solution was illuminated with an ice-cyan LED light to produce 1 O2, under acidic conditions, directly illuminate the GV solution, so that the colorless TMB generates blue oxTMB, and when pH = 7.0, the blue product disappears completely. On this basis, the oxidizing medium Ce is added 3 + , Ce 3+ As an oxidizing medium, it can achieve the oxidation of TMB under neutral conditions.

[0015] Step 3 Construction of electrochemical visualization dual-mode sensor;

[0016] Introducing LCys containing biothiols as a marker for the recognition of Hg 2+ receptors to construct an electrochemical visualization dual-mode sensor.

[0017] Add 12 μL of 5.25 mM LCys solution and 6 μL of 400 μM Hg to an appropriate amount of 20 mM TrisHCl buffer solution.2+ The solution was incubated at room temperature in the dark. After the incubation was completed, 3 μL of 1000× photosensitive dye GV, 10 μL of 30 mM oxidizing medium Ce, and 10 μL of 30 mM oxidizing medium Ce were added to the solution. 3+ and 10 μL of 24 mM chromogenic substrate TMB, mix the above solutions and illuminate with ice-blue LED light for 2 minutes to complete the reaction. After the reaction is completed, take visual photos with a digital camera, and measure the absorbance of the solution with a UV-visible spectrophotometer. Finally, use SPCE to select the chronoamperometry method in the electrochemical workstation to measure the electrochemical signal of oxidized TMB, set the voltage to +100 mV, and the amperometric response time to 60 s, so that an electrochemical visualization dual-mode sensor can be constructed.

[0018] The present invention realizes visualization of detection results, the sensor has a wide linear range, a low detection limit, and a high sensitivity, which is more in line with actual natural water body detection conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Examples of the presence or absence of Ce under acidic and neutral conditions 3+ Visualization of the photosensitized system in the presence or absence of light;

[0020] Experimental conditions: GV, 10×; Ce 3+ , 1mM; TMB, 0.8mM; illumination time, 2min;

[0021] Figure 2 Examples with or without Ce at different pH conditions 3+ Histogram of changes in the photosensitized system in the presence or absence of light;

[0022] Experimental conditions: GV, 10×; Ce 3+ , 1mM; TMB, 0.8mM; illumination time, 2min;

[0023] Figure 3 This is a graph showing the effect of active oxygen on the photosensitization system in the example;

[0024] Experimental conditions: GV, 10×; Ce 3+ , 1mM; TMB, 0.8mM; illumination time, 2min; Tris-HCl buffer solution pH, 7.0;

[0025] Figure 4 This is a graph showing the chronoamperometric characterization results of the photosensitive system construction process of the embodiment;

[0026] (a)TMB+irradiation; (b)GV+TMB+irradiation; (c)Ce 3++TMB+irradiation; (d)GV+Ce 3+ +TMB+irradiation; (e)GV+Ce 3+ +TMB;

[0027] Experimental conditions: GV, 10×; Ce 3+ , 1mM; TMB, 0.8mM; illumination time, 2min; Tris-HCl buffer solution pH, 7.0;

[0028] Figure 5 The UV-visible spectroscopic characterization result diagram of the photosensitive system construction process of the embodiment;

[0029] (a)TMB+irradiation; (b)GV+TMB+irradiation; (c)Ce 3+ +TMB+irradiation; (d)GV+Ce 3+ +TMB+irradiation; (e)GV+Ce 3+ +TMB;

[0030] Experimental conditions: GV, 10×; Ce 3+ , 1mM; TMB, 0.8mM; illumination time, 2min; Tris-HCl buffer solution pH, 7.0;

[0031] Figure 6 For example, different Hg 2+ Absorbance result graph at different concentrations;

[0032] Figure 7 The absorbance values ​​of the examples are compared with the values ​​of different Hg 2+ Linear relationship graph between concentrations;

[0033] Figure 8 For example, different Hg 2+ Current response diagram under concentration;

[0034] Fig. 9 The current intensity and different Hg 2+ Linear relationship graph between concentrations;

[0035] Fig.10 Visualization diagram and bar graph of the sensor selectivity investigation of the embodiment;

[0036] Fig.11 It is a visualization diagram and a bar graph of the sensor reproducibility investigation of the embodiment;

[0037] Fig.12 Hg is added to the actual water sample in the example 2+ Visualization of the detection;

[0038] Fig.13 Chronoamperometry characterization diagram for the electrochemical-visualization sensor construction process of the embodiment;

[0039] (a)GV+Ce 3+ +TMB+irradiation; (b)GV+Ce 3+ +TMB+L-Cys+irradiation; (c)GV+Ce 3+ +TMB+L-Cys+Hg 2+ +irradiation;

[0040] Experimental conditions: GV, 10×; Ce 3+ , 1mM; TMB, 0.8mM; Hg 2+ , 8μM; L-Cys, 190μM; illumination time, 2min; Tris-HCl buffer solution pH, 7.0;

[0041] Fig.14 The UV-visible absorption spectrum characterization diagram of the electrochemical-visualization sensor construction process of the embodiment;

[0042] (a)GV+Ce 3+ +TMB+irradiation; (b)GV+Ce 3+ +TMB+L-Cys+irradiation; (c)GV+Ce 3+ +TMB+L-Cys+Hg 2+ +irradiation;

[0043] Experimental conditions: GV, 10×; Ce 3+ , 1mM; TMB, 0.8mM; Hg 2+ , 8μM; L-Cys, 190μM; illumination time, 2min; Tris-HCl buffer solution pH, 7.0. DETAILED DESCRIPTION

[0044] The basic principle of the present invention is: when the pH is 7, the GV+Ce is illuminated by ice-blue LED light. 3+ The system catalyzes the oxidation of TMB (3,3',5,5'-tetramethylbenzidine) to generate blue oxTMB. At this time, the photocatalytic system is in the "on" state. However, LCys has a reducing property that can inhibit the oxidation of TMB, resulting in a decrease in the amount of blue oxTMB generated. 3+ The catalytic oxidation ability of photooxidase is "turned off". 2+ When present, the sulfhydryl group in LCys (L-cysteine) reacts with Hg 2+The high affinity of GV+Ce leads to the inhibition of the reduction of LCys, and the photocatalytic system is "turned on", so that TMB can be oxidized to generate blue oxTMB. 3+ As a photocatalyst, TMB was developed as a colorimetric agent and electrochemical indicator for the detection of Hg in water. 2+ "Switch-on" type electrochemical visualization dual-mode sensor for detection.

[0045] Specific experimental instruments and reagents of this embodiment:

[0046] (1) The main instruments used in the invention process are listed in Table 1. A three-electrode system was used in all electrochemical measurement experiments, in which SPCE was the working electrode, Ag / AgCl electrode was the reference electrode, and platinum wire electrode was the counter electrode.

[0047] Table 1 List of instruments used in this experiment

[0048]

[0049] The resistivity of the ultrapure water used in the present embodiment is 18.25 MΩcm, and the main experimental reagents are listed in Table 2.

[0050] Table 2 List of main experimental reagents

[0051]

[0052]

[0053] (2) Solution preparation

[0054] Preparation of 20mM TrisHCl (pH=7.0): Weigh 1.2114g Tris and add it to a beaker containing about 500mL ultrapure water. Stir to mix well, then adjust the pH to 7.0 with 1M HCl. Finally, transfer the mixed solution to a 500mL volumetric flask, make up to volume, shake well, and store at room temperature.

[0055] Preparation of 1000× GoldView (GV) solution: pipette 50 μL of 10000× GV stock solution and 450 μL of ultrapure water into a 1.50 mL centrifuge tube, shake well, and store in a refrigerator at 4°C away from light for later use.

[0056] Preparation of 24 mM TMB solution: Accurately weigh 0.115 g of TMB powder and add it to a 50 mL centrifuge tube. Then add 20 mL of DMSO, shake thoroughly, and store at room temperature away from light for later use.

[0057] 30mM Ce 3+Solution preparation: Accurately weigh 0.335 g of cerium chloride solid and add it into a 50 mL centrifuge tube, then add 30 mL of 20 mM TrisHCl (pH=7.0) buffer solution, shake thoroughly and store at room temperature.

[0058] 400μM Hg 2+ Solution preparation: Accurately pipette 4598.8 μL of TrisHCl buffer solution (pH=7.0) into a 10 mL glass bottle, add 401.2 μL of mercury standard solution, mix thoroughly, and prepare before use.

[0059] Preparation of 100 mM L-cysteine ​​solution: Accurately weigh 0.1212 g L-cysteine, add 10 mL of TrisHCl buffer solution (pH = 7.0), shake well to dissolve, and prepare before use.

[0060] Preparation of 5.25 mM L-cysteine ​​solution: Accurately pipette 525 μL of 100 mM L-cysteine ​​into a 10 mL centrifuge tube, and add 9475 μL of TrisHCl buffer solution (pH=7.0) thereto. Prepare the solution before use.

[0061] The preparation method of the electrochemical visualization dual-mode sensor of this embodiment includes the following steps:

[0062] Step 1 Pretreatment of screen-printed electrodes;

[0063] The working electrode used was a disposable screen-printed electrode (SPCE) (2 mm in diameter). To ensure the cleanliness of the electrode surface, the SPCE electrode surface was rinsed with ultrapure water. The SPCE was connected to the electrochemical workstation using an electrode connection line, and the electrode was activated at a scan rate of 100 mV / s and a scan potential of 0.3 V until a stable cyclic voltammetry curve was obtained to obtain an activated SPCE. Finally, the activated SPCE was washed with ultrapure water and dried at room temperature for later use.

[0064] Step 2: Construction of photocatalytic system;

[0065] like Figure 1 and Figure 2 First, the GV solution was illuminated with an ice-cyan LED light to produce 1 O2, under acidic conditions, direct illumination of the GV solution can convert colorless TMB into blue oxTMB, and at pH = 7.0, the blue product disappears completely. On this basis, the addition of the oxidizing medium Ce 3+ When pH=7.0, the colorless TMB in the light-exposed reaction solution was oxidized to generate a blue product, indicating that the addition of Ce 3+After that, the change of pH value has little effect on the photosensitizing dye GV. 3+ After that, the absorbance value at pH = 7.0 is 3+ The absorbance value was 74 times of the previous value, indicating that the addition of Ce 3+ After that, more 1 O2, confirmed Ce 3+ It can be used as an oxidizing medium to achieve the oxidation of TMB under neutral conditions.

[0066] like Figure 3 As shown, after adding tryptophan and p-benzoquinone, GV+Ce 3+ The photosensitive oxidation of the system was inhibited, and TMB was not oxidized to blue, proving that the 1 O2 and O2 are GV+Ce 3+ The main source of oxidative activity in photocatalytic systems.

[0067] like Figure 4 and Figure 5 As shown in the figure, the UV spectrophotometer and electrochemical workstation were used to characterize the constructed photosensitization system. Figure 5 , GV or Ce 3+ When present alone, TMB will not be oxidized under the icy blue LED light (curves b and c). 3+ However, without LED light, TMB cannot be oxidized (curve e), which fully illustrates the relationship between GV and Ce. 3+ and light are necessary conditions for constructing the entire photosensitivity system (curve d).

[0068] Based on the above experiments, this embodiment constructs a method for generating a 3D image of the GV and Ce under LED ice-blue light. 3+ The invention relates to a photocatalytic system for catalyzing the oxidation of TMB by a photocatalyst.

[0069] Step 3 Construction of electrochemical visualization dual-mode sensor;

[0070] In this example, LCys containing biothiols was introduced as a 2+ A "switch-on" electrochemical visualization dual-mode sensor for Hg2+ detection in water was constructed by adding 12 μL of 5.25 mM LCys solution and 6 μL of 400 μM Hg2+ to a certain amount of 20 mM TrisHCl buffer solution. 2+ The solution was incubated at room temperature in the dark. After the incubation was completed, 3 μL of 1000× photosensitive dye GV, 10 μL of 30 mM oxidizing medium Ce, and 10 μL of 30 mM oxidizing medium Ce were added to the solution. 3+and 10 μL of 24 mM chromogenic substrate TMB, mix the above solution and illuminate with ice-blue LED light for 2 minutes to complete the reaction. After the reaction is completed, take visual photos with a digital camera and measure the absorbance of the solution with a UV-visible spectrophotometer. Finally, use SPCE to select the chronoamperometry method in the electrochemical workstation to measure the electrochemical signal of oxidized TMB, set the voltage to +100 mV, and the amperometric response time to 60 seconds.

[0071] The performance test of the dual-mode sensor provided in this embodiment is as follows:

[0072] like Figure 4 The results measured by electrochemical amperometry were consistent with those measured by UV spectrophotometer, proving that the photocatalytic electrochemical visualization dual-mode sensor constructed using TMB is feasible.

[0073] like Figures 6 to 9 As shown, Hg in the range of 10nM8μM 2+ The concentration change and absorbance value show a good linear relationship, and the linear equation is A = 0.39584lg C (Hg 2+ )0.32011, where A represents the absorbance value of the solution, and the detection limit (LOD) is 0.109nM. Hg 2+ The concentration change and the current signal also show a good linear relationship, and the linear equation is I = 0.01764lgC (Hg 2+ )0.01135, where I represents the current intensity (μA), and LOD is 0.095nM.

[0074] like Fig.10 and Fig.11 As shown, LCys has a significant effect on Hg 2+ It has strong selectivity and reproducibility, the difference between experimental groups is small, the visualization color is basically consistent, and the relative standard deviation (RSD) of the current response is 2.263% (n=7). Therefore, this dual-mode sensor can be applied to Hg in complex water bodies. 2+ Detection.

[0075] like Fig.12 As shown, the dual-mode sensor detects actual water samples. The sensor can still detect Hg in complex matrices. 2+ , with good anti-interference ability. As shown in Table 3, the results of the standard addition method and the standard detection method AFS are compared. The recovery rate of the standard addition method experiment is in the range of 90.34-101.25%, and the relative standard deviation (RSD) is in the range of 2.27-5.61%. Three water samples with different matrices were tested, namely mineral water, lake water and sea water; before the test, Hg concentrations of 100nM and 4000nM were added 2+The standard solution was added into the actual water sample.

[0076] Table 3 Hg spiked into actual water samples 2+ Test results

[0077]

[0078] Beneficial effects brought by the technical solution of the present invention:

[0079] (1) Visualize the test results;

[0080] The present invention uses an electrochemical workstation and a UV-visible spectrophotometer to measure the Hg 2+ The dual-mode sensor was characterized, e.g. Fig.13 and Fig.14 , Fig.13 The visualization in Figure 2 shows the addition of LCys and Hg 2+ The present invention successfully realizes visualization by observing the color change of the solution, so that non-professionals can also grasp the approximate detection status, which greatly improves the efficiency of information transmission.

[0081] (2) High sensitivity enables accurate detection;

[0082] The sensor developed by the present invention can detect Hg 2+ The linear range of concentration is 10nM-8μM, and the detection limit is 0.095nM / 0.109nM, as shown in Table 4. Compared with other existing technologies, the sensor developed in this embodiment has a wider linear range, a lower detection limit, and a high sensitivity, and has a good practical application prospect for achieving accurate detection.

[0083] Table 4 Sensors in this embodiment and other Hg detection 2+ Comparison of biosensors

[0084]

[0085] (3) Achieve detection under neutral conditions;

[0086] The results of this study show that the maximum reaction rate of the photosensitive system at pH 4 is comparable to that at pH 7, which proves that the addition of Ce 3+ After the addition of ions, effective catalytic oxidation of TMB can be achieved at neutral pH. Figure 2 As shown. Therefore, the present invention broadens the pH application range of the color development reaction, breaks through the shortcoming of the traditional technology that can only be detected under acidic conditions, and is more in line with the actual natural water body detection conditions.

Claims

1. A method for preparing an electrochemical visualization dual-mode sensor, characterized in that: The following steps are involved: Step 1 Pretreatment of screen-printed electrodes; The working electrode used was a disposable screen-printed electrode SPCE, and the electrode was activated; Step 2: Construction of photocatalytic system; First, the GV solution was illuminated with an ice-cyan LED light to produce 1 O2, under acidic conditions, directly illuminate the GV solution, so that the colorless TMB generates blue oxTMB, and when pH = 7.0, the blue product disappears completely; on this basis, the oxidizing medium Ce is added 3+ , Ce 3+ As an oxidizing medium, it can realize the oxidation of TMB under neutral conditions; Step 3 Construction of electrochemical visualization dual-mode sensor; Introducing LCys containing biothiols as a marker for the recognition of Hg 2+ receptors to construct an electrochemical visualization dual-mode sensor.

2. The method for preparing the electrochemical visualization dual-mode sensor according to claim 1, characterized in that: Step 1 The specific method is: rinse the surface of the SPCE electrode with ultrapure water, connect the SPCE to the electrochemical workstation using an electrode connecting wire, and activate the electrode at a scanning speed of 100mV / s and a scanning potential of 0.3V until a stable cyclic voltammetry curve is obtained to obtain an activated SPCE; finally, wash the activated SPCE with ultrapure water and dry it at room temperature for use.

3. The method for preparing the electrochemical visualization dual-mode sensor according to claim 1, characterized in that: Step 3: Add 12 μL of 5.25 mM LCys solution and 6 μL of 400 μM Hg to an appropriate amount of 20 mM TrisHCl buffer solution. 2+ The solution was incubated at room temperature in the dark. After the incubation was completed, 3 μL of 1000× photosensitizer GV, 10 μL of 30 mM oxidizing medium Ce 3+ and 10 μL of 24 mM chromogenic substrate TMB, mix the above solutions and illuminate with ice-blue LED light for 2 minutes to complete the reaction; after the reaction is completed, take visual photos with a digital camera and measure the absorbance of the solution with a UV-visible spectrophotometer; finally, use SPCE to select the chronoamperometry method in the electrochemical workstation to measure the electrochemical signal of the oxidized TMB, set the voltage to +100 mV, and the amperometric response time to 60 s, thereby constructing an electrochemical visualization dual-mode sensor.

4. An electrochemical visualization dual-mode sensor obtained according to the preparation method according to any one of claims 1 to 3.

5. The use of the electrochemical visualization dual-mode sensor according to claim 4, characterized in that: For detecting Hg in water 2 + .

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