CN114199977A - Electrochemical heavy metal rapid detection method based on electric drive pre-enrichment - Google Patents
Electrochemical heavy metal rapid detection method based on electric drive pre-enrichment Download PDFInfo
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Abstract
The invention belongs to the technical field of biosensors, and relates to an electrochemical heavy metal rapid detection method based on electric drive pre-enrichment; firstly, polishing a gold electrode by using aluminum oxide powder, and carrying out ultrasonic treatment and drying on the polished gold electrode by using ethanol and water to obtain a treated gold electrode; fc-labeled Hg2+Modifying the aptamer (Fc-apt) on the surface of the treated gold electrode, and marking as Fc-apt/AuE; modifying MCH on the surface of the membrane, and naturally airing at room temperature to be marked as MCH/Fc-apt/AuE; finally, Hg is aligned by an electrostatic field device2+Incubating, applying 0.5-2.5V voltage to Hg2+Pre-enriching, and applying-0.5 to-2.5V voltage to make the electrode surface self-cleaning. The electrochemical biosensor prepared by the invention has the advantages of rapid detection, pre-enrichment, high sensitivity, self-cleaning and the like, and can be used for integrally detecting Hg2+The sensor of (2) is used for sensitive and rapid analysis of an actual sample.
Description
Technical Field
The invention belongs to the technical field of biosensors, and particularly relates to an electrochemical heavy metal rapid detection method based on electric drive pre-enrichment.
Background
Mercury (Hg)2+) The most dangerous heavy metal pollutants are classified by the World Health Organization (WHO) due to acute toxicity, non-biodegradable nature and high bioaccumulation in the ecosystem. Therefore, has high sensitivity and selectivityThe selective rapid determination of mercury is very important in environmental protection, since the presence of mercury, even at trace levels, can be absorbed by plants and animals and further biochemically in the human body through the food chain, causing some damage to the nervous system and organs. To date, several Hg's have been developed2+The detection method of (1), Hg established at home and abroad2+The detection methods of (3) include inductively coupled plasma mass spectrometry (ICP-MS), Atomic Absorption Spectrometry (AAS), Atomic Fluorescence Spectrometry (AFS), fluorescence, colorimetry, electrochemiluminescence, and electrochemical method (EC). The electrochemical method is an analysis method for detecting a substance to be detected by using electrochemical response signals such as redox reaction. Electrochemical aptamer sensor in Hg2+Has wide prospect in selective analysis; wudan et al utilize T-T mismatch to specifically capture Hg2+With T-Hg2+T forms a DNA double strand, however, T-Hg2+The longer incubation time for the formation of the-T structure is still affected longer. Thus, the present invention introduces an electrostatic field strategy to promote Hg2+The DNA identification is beneficial to the high-efficiency pre-enrichment of the DNA on the electrode, and the Hg is effectively shortened2+And increased Hg2+The analytical performance of electrochemical sensing.
Disclosure of Invention
The invention aims to invent an electrochemical biosensor for detecting Hg, which integrates the advantages of rapid detection, pre-enrichment, high sensitivity, self-cleaning and the like2+。
The invention introduces a voltage driving device which enables two metal plates to generate positive and negative electrostatic fields, assembles an aptamer with one end modified with sulfydryl and the other end modified with ferrocene electrochemical signals on the surface of a gold electrode, and then introduces a target object Hg2+By the principle of electrostatic field, a large amount of Hg with positive charge can be generated2+Moving to the negative pole of the electrostatic field to rapidly combine the proper ligand with Hg2+Formation of T-Hg2+And a T structure, which causes the aptamer to generate self-helix, so that a terminal signal molecule ferrocene approaches to the surface of the sensor to generate redox reaction, thereby obtaining the electrochemical signal of the ferrocene. Finally obtaining the aptamerThe object sensor is used for on-site detection, sensitivity and quick analysis of actual samples.
In order to realize the technical purpose, the method comprises the following specific steps:
(1) sequentially polishing the gold electrode by using aluminium oxide powder with different particle sizes, performing ultrasonic treatment in ethanol and water respectively, and drying in the air to obtain a treated gold electrode;
(2) ferrocene (Fc) labeled Hg2+The aptamer is marked as Fc-apt, the Fc-apt is modified to the surface of the gold electrode treated in the step (1), and the modified gold electrode is marked as Fc-apt/AuE;
(3) modifying the electrode surface of the Fc-apt/AuE material prepared in the step (2) with MCH, and after incubating for a period of time at room temperature, marking the obtained material as MCH/Fc-apt/AuE;
(4) immersing the electrode prepared in the step (3) into Hg with different concentrations2+In standard solution, pre-enriching Hg under different electrostatic field conditions by electric drive2+Enriching to obtain electrochemical biosensor, and recording as Hg2+/MCH/Fc-apt/AuE；
(5) Construction of a standard curve: the electrochemical biosensor (Hg) prepared in the step (4)2+/MCH/Fc-apt/AuE) as working electrode, saturated Ag/AgCl electrode as reference electrode, platinum wire electrode as counter electrode, performing electrochemical detection according to detected current value and corresponding Hg2+Constructing a standard curve by using the logarithm of the concentration;
(6) hg in the sample2+Detection of (2): firstly, preparing a sample solution, immersing the electrochemical biosensor prepared in the step (4) into the sample solution, and obtaining a corresponding current value through electrochemical test; substituting the current value into the standard curve constructed in the step (5) to obtain Hg in the sample2+To realize Hg in an unknown sample2+Detection of (3).
Preferably, in step (1), the diameter d of the gold electrode is 3 mm; the grain diameter of the aluminum oxide powder is 0.3 μm and 0.05 μm in sequence; the ultrasound time was 30 s.
Preferably, in the step (2), the Fc-apt is used in an amount of 6 μ L and at a concentration of 3 μ M; the incubation temperature is 4 ℃, and the incubation time is 10-12 h.
Preferably, in the step (3), the MCH solution is used in an amount of 6 μ L and has a concentration of 0.1 μ M; the incubation period is 40-60 min.
Preferably, in step (4), the Hg is2+The concentration of the standard solution was 1X 10-14-1×10-5M。
Preferably, in step (4), the specific operation of electrically driving pre-enrichment under different electrostatic field conditions is as follows: firstly, pre-enriching for 10-60 min under a 0.5-2.5V electrostatic field; after positive pressure enrichment, the voltage is automatically switched to negative pressure of-0.5 to-2.5V and is continuously applied for 30 to 150 seconds.
Preferably, the device involved in the electrically-driven pre-concentration operation under different electrostatic field conditions in step (4) is: a pressure device for generating an electrostatic field is composed of two parallel metal plates and a constant-voltage driving device based on single-chip microcomputer control;
an electrode frame is arranged between the two parallel metal plates and is used for placing Hg2+A standard solution and an electrode;
the constant voltage driving device consists of a constant voltage power supply, a DAC0832 and STC89C 52; the constant voltage power supply is electrically connected with the DAC0832 and the STC89C 52;
the metal plate is connected with an output port of the DAC0832, and voltage output and conversion are achieved.
Further, in the step (5), the specific conditions for the electrochemical workstation to detect the electrochemical signal are as follows: the test was performed in 0.1M PBS (pH 7.4) buffer; the scanning voltage range is-0.4-0.8V, the amplitude is 0.025V, and the frequency is 25 Hz. The invention has the beneficial effects that:
(1) the electrochemical biosensor of the invention can greatly shorten Hg by the action of an electrostatic field2+Incubation time with Fc-apt bound, enrichment of Hg by electric field2+The binding time with Fc-apt is shortened from 60min to 20min, and the method has the advantage of rapid detection.
(2) According to the invention, by applying a-2V reverse electrostatic field, the effect of self-cleaning the electrode interface is achieved, and the sensitivity of the sensor is improved.
(3) The invention introduces specialForeign body recognition element Hg2+By formation of T-Hg2+the-T structure improves the selectivity of the electrochemical biosensor and reduces Hg2+Meanwhile, interference of other ions exists, and Hg is realized2+The specificity of (3).
(4) The electrochemical biosensor constructed by the invention is used for Hg2+The detection has high sensitivity, good selectivity, good stability and wide linear range of 0.01pM to 10 nM.
Drawings
FIGS. 1(A) and (B) are respectively an EC signal variation graph and an EIS impedance graph of an electrochemical biosensor construction process, in which
a is Au/Apt;
b is Au/Apt/MCH;
c is Au/Apt/MCH/Hg2+Adding an electric field (+2V) for 20 min;
d is Au/Apt/MCH/Hg2+Applying an electric field (+2V) for 20min and applying a reverse electric field (-2V) for 2 min;
e is Au/Apt/MCH/Hg2+；
FIG. 2(A) shows electrochemical biosensor to no target Hg2+(curve a), 20min without electric field (curve b), 60min without electric field (curve c), pre-enrichment in electric field for 20min (curve d); (B) pre-enrichment for 20min under the application of electrostatic field and incubation for 60min Hg without electric field2+A comparison graph of electrochemical signal response of (a); (C) the response graphs of electrochemical signals added with inverse electric fields of 30, 90, 120 and 180s are shown; (D) the time of applying the reverse electric field is the influence on the electrochemical signal.
FIG. 3(A) shows electrochemical biosensors using an applied electrostatic field for different Hg concentrations2+A response map of (2); (B) for applying Hg under an electrostatic field2+Taking a linear relation graph of the logarithm of the concentration and the electrochemical signal; (C) for electrochemical biosensor under no electric field to different concentrations of Hg2+A response map of (2); (D) is Hg under no electric field2+The concentration is plotted as a linear function of logarithm to electrochemical signal.
Fig. 4 is a schematic diagram of a pressure device for generating electrostatic field, wherein 1-DAC0832, 2-STC89C52, 3-constant voltage power supply, 4-metal plate, and 5-electrode holder.
The specific implementation mode is as follows:
the embodiments of the present invention will be described in detail below with reference to the accompanying drawings: the embodiments are performed on the premise of the technical scheme of the invention, and detailed implementation steps and specific operation processes are given, but the scope of the invention is not limited to the following embodiments.
The Fc-apt used in the present invention is purchased from Biotechnology engineering (Shanghai) GmbH, and the base sequence is:
5’-SH-(CH2)6TCATGTTTGTTTGTTGGCCCCCCTTCTTTCTTA-Fc-3 ' (the 5 ' end of the aptamer modified thiol, 3 ' modified signal molecule Fc).
Screening optimization conditions:
(1) as shown in FIG. 2 (B), two sets of parameters were set to compare the enrichment in electrostatic field and the incubation of Hg without the application of electric field2+The time of the step (2) is compared with six groups of control, and the corresponding result is that the peak current change value under the electrostatic field enrichment basically reaches the peak value within 20min and is basically consistent with the result of 60min of incubation without the electric field; therefore, the optimum time for electrostatic field enrichment is 20 min.
(2) As shown in (D) of FIG. 2, six sets of experiments were set to compare the time of applying the reverse electric field after pre-enrichment, and the corresponding results were that the peak current value remained substantially unchanged when 120s was applied, indicating that the optimal self-cleaning time of the electrostatic field was 120s
(3) The applied voltage value of the electrostatic field is optimized, and the applied +2V change value is maximum between +0.5V and + 2.5V; the variation value of-2V is maximum between-0.5V and-2.5V; the best effect of the electrostatic field applied voltage is +/-2V.
Example 1:
(1) the manufacturing process of the electrostatic field induction biosensor comprises the following steps:
the invention also provides a pressure device for generating the electrostatic field; the device consists of two parallel metal plates and a constant-voltage driving device based on single-chip microcomputer control,
an electrode frame is arranged between the two parallel metal plates and is used for placing Hg2+Standard liquid and electricityA pole;
the constant voltage driving device consists of a constant voltage power supply, a DAC0832 and STC89C 52; the constant voltage power supply is electrically connected with the DAC0832 and the STC89C 52;
the metal plates are connected with the output port of the DAC0832 to realize the output and conversion of voltage, and an electrostatic field area can be generated between the two metal plates. The DAC0832 and the STC89C52 are powered by a power supply, and the specific structure is shown in figure 4.
(2) Preparation of Fc-apt: fc-apt was diluted with Tris-HCl buffer to a concentration of 3. mu.M.
(3) Polishing gold electrode (d is 3mm, AuE) with 0.3 μm and 0.05 μm aluminum oxide powder, respectively, ultrasonic treating in ethanol and water for 30s, and drying in air;
(4) the concentration of the sulfhydryl-modified Fc-apt is 3 μ M, the dosage is 6 μ L, the sulfhydryl-modified Fc-apt is fixed on the electrode surface treated in the step (3) through Au-S covalent interaction, and the electrode surface is incubated at 4 ℃ for one night, and then the sensor is expressed as Fc-apt/AuE;
(5) modifying the surface of the electrode Fc-apt/AuE in the step (4) with MCH at the concentration of 0.1M and the dosage of 6 μ L, and incubating for 40min at room temperature to remove unbound active sites, wherein the sensor is marked as MCH/Fc-apt/AuE;
(6) the electrode prepared in step (5) was immersed in 50. mu.L of different concentrations (1X 10)-15，1×10-14，1×10-13.8， 1×10-13.2，1×10-13，1×10-12.5，1×10-12，1×10-11.5，1×10-11，1×10-10.5，1×10-10，1×10-9.5， 1×10-9，1×10-8.5，1×10-8，1×10-8.5，1×10-7，1×10-6，1×10-5) Hg of2+Putting the solution in a centrifugal tube of the solution on an electrode frame in the step (1) for electrostatic field pre-enrichment and self-cleaning; the method comprises the following specific steps: turning on the power supply in the step (1), inputting 2V voltage, timing for 20min, applying-2V voltage, timing for 2min, automatically stopping the program, and outputting the voltage of 0V; after treatment, an electrochemical biosensor, noted Hg, was obtained2+/MCH/Fc-apt/AuE；
Taking the electrochemical biosensor prepared in the step (6) as a working electrode, taking a saturated Ag/AgCl electrode as a reference electrode, taking a platinum wire electrode as a counter electrode, and recording and detecting an electrochemical signal by an electrochemical workstation with the model number of CHI 750E; the test was performed in 0.1M PBS (pH 7.4) buffer.
FIG. 1(A) is a diagram of EC signal change in the sensor construction process, after Fc-apt is assembled on the modified electrode surface, the EC signal is rapidly enhanced by applying electrostatic field; (B) an EIS impedance profile for the sensor construction process, which reduces impedance in the presence of an electrostatic field; both of these aspects demonstrate that the presence of an electrostatic field greatly shortens Hg2+Incubation binding time of (3).
Hg detection based on voltage-driven electrostatic field2+The subsequent research of the pre-enrichment electrochemical biosensor;
(1) the feasibility of an electrochemical biosensor prepared by generating an electrostatic field by electric driving was investigated:
biosensor prepared in example 1 was soaked in Hg at a concentration of 0.1nM2+In the standard solution, as shown in fig. 2: (A) respectively applying electrostatic field for 20min, no electric field for 60min, and no target Hg for the electrochemical biosensor pair2+A graph of electrochemical signal response; (B) pre-enrichment for 20min under the application of electrostatic field and incubation for 60min Hg without electric field2+A comparison graph of electrochemical signal response of (a); as can be seen from the graphs (A) and (B), the existence of the electrostatic field greatly shortens Hg2+Time to binding to Fc-apt. (C) The response graphs of electrochemical signals added with inverse electric fields of 30, 90, 120 and 180s are shown; (D) the time of applying the reverse electric field is the influence on the electrochemical signal. As can be seen from FIGS. C and D, the electrochemical signal gradually decreased with the increase in the time of application, indicating that the application of the reverse electric field can reduce the amount of Hg not bound to Fc-apt2+Influence on the electrode surface.
The above results demonstrate that the sensor is feasible
As shown in fig. 3, (a) is the effect of different concentrations of AFB1 on electrochemical signals; (B) the effect of different concentrations of AFB1 on the electrochemiluminescence signal; as can be seen from fig. 3, as the concentration of AFB1 increases, the EC and ECL signals show gradually decreasing changes, which proves that the sensor is feasible.
(2) Performance study of dual-mode electrochemical-electrochemiluminescence biosensors:
the sensors obtained in example 1 were immersed in water at concentrations of 1X 10, respectively-15，1×10-14，1×10-13.8，1×10-13.2， 1×10-13，1×10-12.5，1×10-12，1×10-11.5，1×10-11，1×10-10.5，1×10-10，1×10-9.5，1×10-9，1×10-8.5， 1×10-8，1×10-8.5，1×10-7，1×10-6，1×10-5Hg of M2+In the standard solution, under the best experimental conditions, an electrochemical workstation with the model of CHI 750E records and detects the change of electrochemical peak current, and Hg is pre-enriched in an electrostatic field2+The ACV peak current to different concentrations of Hg is obtained2+Response (fig. 3 (a)). With Hg2+The concentration increases, the electrochemical signal increases and is in Hg respectively2+The concentration is 1X 10-14-1×10-12M and 1X 10-12-1×10-8The linear relationship of the electrochemical signals obtained in the M range (FIG. 3 (B)). ACV peak current to different concentrations of Hg under no electrostatic field2+The response of (fig. 3(C)) and the linear relationship diagram of (fig. 3 (D)). Proves that the electrochemical biosensor under the electrostatic field has wider linear range and higher sensitivity, and can realize the aim of measuring Hg2+And (4) accurate analysis.
Based on the linear relation, the inventor collects soil samples in agricultural engineering agricultural test fields of Jiangsu university and carries out Hg analysis on actual soil samples of farmland2+The analysis was carried out: adding 0.5g of farmland soil actual sample into 10mL of aqua regia, and carrying out boiling water bath for 2 hours; after cooling to room temperature, the mixture was centrifuged at 8000rpm for 15min, and the pH of the filtered supernatant was adjusted to 7.4 with 1M sodium hydroxide. Then, the solution was diluted with ultrapure water to a volume of 100mL to obtain a diluted solution.
Finally, three portions of the diluted solution are taken, and Hg is added into the diluted solution respectively2+Standard solution ofLiquid (1X 10)-5，5×10-5，1×10-6M) obtaining a labeled solution, then soaking the sensor in the labeled solution, firstly applying +2V for pre-enrichment for 20min, then self-cleaning for 2min under a-2V electrostatic field, and then obtaining a corresponding current value by carrying out electrochemical test on the prepared electrode; the Hg in the sample can be obtained by substituting the current value into the constructed standard curve2+To realize Hg in an unknown sample2+The use of detection.
The analysis results are shown in table 1.
The sensing method is utilized to collect Hg with different concentrations in actual farmland soil samples2+The analysis was performed with a recovery rate between 85.1-116%. The comparison with the official inductively coupled plasma mass spectrometer result shows that the electrochemical biosensor which generates the electrostatic field through electric drive is provided for the actual sample of Hg in farmland soil2+The analysis reliability of (2) is higher.
Description of the drawings: the above embodiments are only used to illustrate the present invention and do not limit the technical solutions described in the present invention; thus, while the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted; all such modifications and variations are intended to be included herein within the scope of this disclosure and the present invention and protected by the following claims.
Claims (8)
1. An electrochemical heavy metal rapid detection method based on electric drive pre-enrichment is characterized by comprising the following steps:
(1) sequentially polishing the gold electrode by using aluminium oxide powder with different particle sizes, performing ultrasonic treatment in ethanol and water respectively, and drying in the air to obtain a treated gold electrode;
(2) ferrocene-labeled Hg2+The aptamer is marked as Fc-apt, and the Fc-apt is modified after the treatment of the step (1)The modified gold electrode is marked as Fc-apt/AuE;
(3) modifying the electrode surface of the Fc-apt/AuE material prepared in the step (2) with MCH, and after incubating for a period of time at room temperature, marking the obtained material as MCH/Fc-apt/AuE;
(4) immersing the electrode prepared in the step (3) into Hg with different concentrations2+In standard solution, pre-enriching Hg under different electrostatic field conditions by electric drive2+Enriching to obtain electrochemical biosensor, and recording as Hg2+/MCH/Fc-apt/AuE；
(5) Construction of a standard curve: and (3) performing electrochemical detection by taking the electrochemical biosensor prepared in the step (4) as a working electrode, a saturated Ag/AgCl electrode as a reference electrode and a platinum wire electrode as a counter electrode, and performing electrochemical detection according to the detected current value and the corresponding Hg2+Constructing a standard curve by using the logarithm of the concentration;
(6) hg in the sample2+Detection of (2): firstly, preparing a sample solution, soaking the electrochemical biosensor prepared in the step (4) in the sample solution, and obtaining a corresponding current value through an electrochemical test; substituting the current value into the standard curve constructed in the step (5) to obtain Hg in the sample2+To realize Hg in an unknown sample2+Detection of (3).
2. The electrochemical heavy metal rapid detection method based on electric-driven pre-enrichment as claimed in claim 1, wherein in step (1), the diameter d of the gold electrode is 3 mm; the grain diameter of the aluminum oxide powder is 0.3 μm and 0.05 μm in sequence; the ultrasound time was 30 s.
3. The electrochemical rapid heavy metal detection method based on electric-driven pre-enrichment as claimed in claim 1, wherein in the step (2), the amount of the Fc-apt is 6 μ L, and the concentration is 3 μ M; the incubation temperature is 4 ℃, and the incubation time is 10-12 h.
4. The method for rapidly detecting the electrochemical heavy metal based on the electric-driven pre-enrichment as claimed in claim 1, wherein in the step (3), the MCH solution is used in an amount of 6 μ L and has a concentration of 0.1 μ M; the incubation period is 40-60 min.
5. The electrochemical rapid heavy metal detection method based on electric-driven pre-enrichment as claimed in claim 1, wherein in the step (4), the Hg is used as the Hg2+The concentration of the standard solution was 1X 10-14-1×10-5M。
6. The electrochemical method for rapidly detecting heavy metals based on electrically driven pre-enrichment as claimed in claim 1, wherein in step (4), the specific operation of electrically driven pre-enrichment under different electrostatic field conditions is as follows: firstly, pre-enriching for 10-60 min under a 0.5-2.5V electrostatic field; after positive pressure enrichment, the voltage is automatically switched to negative pressure of-0.5 to-2.5V and is continuously applied for 30 to 150 seconds.
7. The method for electrochemical rapid detection of heavy metals based on electric-driven pre-enrichment as claimed in claim 6, wherein the devices involved in the electric-driven pre-enrichment operation under different electrostatic fields are: a pressure device for generating an electrostatic field is composed of two parallel metal plates and a constant-voltage driving device based on single-chip microcomputer control;
an electrode frame is arranged between the two parallel metal plates and is used for placing Hg2+A standard solution and an electrode;
the constant voltage driving device consists of a constant voltage power supply, a DAC0832 and STC89C 52; the constant voltage power supply is electrically connected with the DAC0832 and the STC89C 52;
the metal plate is connected with an output port of the DAC0832, and voltage output and conversion are achieved.
8. The electrochemical heavy metal rapid detection method based on electric-driven pre-enrichment as claimed in claim 1, wherein in the step (5), the specific conditions for the electrochemical workstation to detect the electrochemical signal are as follows: the test was performed in 0.1M PBS buffer at pH 7.4; the scanning voltage range is-0.4-0.8V, the amplitude is 0.025V, and the frequency is 25 Hz.
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