CN113109408A - Portable palm electrochemical sensor for detecting enzyme concentration based on chip electrode and preparation method and detection method thereof - Google Patents
Portable palm electrochemical sensor for detecting enzyme concentration based on chip electrode and preparation method and detection method thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of electrochemical analysis and electrochemical sensor preparation, and discloses a portable handheld electrochemical sensor for detecting enzyme concentration based on a chip electrode, which comprises a silicon chip substrate and a three-electrode system consisting of a working electrode, an auxiliary electrode and a reference electrode, wherein the three-electrode system is made into a chip and is used together with a handheld electrochemical workstation, the three-electrode system is wirelessly connected with a mobile phone to control detection, and horseradish peroxidase (HRP) is detected by a voltammetry method taking o-dianisidine (ODA) or o-phenylenediamine (OPD) as a substrate.
Description
Technical Field
The invention relates to the technical field of electrochemical analysis and electrochemical sensor preparation, in particular to a portable handheld electrochemical sensor for detecting enzyme concentration based on a chip electrode, and a preparation method and a detection method thereof.
Background
The portable handheld electrochemical sensor for detecting the enzyme concentration by using the chip electrode realizes the rapid detection of a target object through wireless transmission and mainly comprises the chip electrode, a smart phone and a small electrochemical workstation. The XiaoyuZhu et al adopts ultrasonic-assisted liquid phase to strip a two-dimensional layered nano material to modify an electrode, and combines a smart phone and wireless transmission to realize the rapid detection of a plant growth regulator, namely naphthylacetic acid; hui Zhao et al designs and develops the first global new coronavirus electrochemical sensor, recognizes new coronavirus nucleic acid through probe specificity, and adopts nano materials to enrich signal molecules, thereby playing a role in signal amplification, and realizing the purpose of completing the detection of new coronavirus in a smart phone end in a portable and rapid manner; YunpengZhang et al innovatively designed and developed a highly sensitive graphene chiral sensor modified by acetylcholinesterase, and combined with a smart phone and wireless transmission, realized the real-time rapid detection of methamidophos.
The chip electrode can be suitable for measuring a target object, and various functional materials can be modified and loaded on the chip electrode to construct chip electrode sensors with different functions, such as modifying an aptamer to construct a chip electrode aptamer sensor; the chip electrode biological enzyme sensor constructed by the supported protease has the advantages of wide application range, convenient operation, low cost, small sample consumption, high detection speed, control detection and data acquisition by replacing a PC (personal computer) end with a mobile phone end and the like. The portable detector is small in size and convenient to carry, can be suitable for detection of various complex environments, and has wide application prospect on social production and development.
Therefore, how to design a portable handheld electrochemical sensor for detecting the concentration of the enzyme based on a chip electrode is a problem which needs to be solved by those skilled in the art.
Disclosure of Invention
In view of the above, in order to overcome the defects of large volume, high price, more required samples, heavy and inconvenient equipment carrying and the like of the electrode system of the traditional electrochemical sensor, the invention provides the portable handheld electrochemical sensor for detecting the enzyme concentration based on the chip electrode, the preparation method and the detection method thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
a portable palm electrochemical sensor based on chip electrode for detecting enzyme concentration comprises a silicon chip substrate and a three-electrode system consisting of a working electrode, an auxiliary electrode and a reference electrode;
the surface of the silicon wafer substrate is plated with a gold film, the three-electrode system is integrated on the silicon wafer substrate, the working electrode is a gold disc electrode (phi is 2mm), the auxiliary electrode is a gold sheet electrode, and the reference electrode is an Ag/AgCl electrode.
The chip electrode is fixed by the electrode holder, the electrode holder mainly has the electrode slot of fixed chip electrode and the lead wire stitch of connecting the chip electrode, the end of lead wire stitch is scalable stitch.
Preferably, in the portable palm electrochemical sensor for detecting the enzyme concentration based on the chip electrode, the silicon wafer substrate is a square silicon wafer substrate with the side length of 1cm, and the silicon wafer substrate is provided with a square electrolytic cell with the side length of 0.4 cm.
The invention also discloses a preparation method of the portable palm electrochemical sensor for detecting the enzyme concentration based on the chip electrode, which comprises the following steps:
(1) plating a gold raw material on a silicon wafer substrate;
(2) sequentially ultrasonically cleaning three electrodes of a chip by using nitric acid, ethanol and distilled water to obtain a clean electrode surface;
(3) and after the surface of the electrode is dried, coating Ag/AgCl slurry on a gold substrate serving as a reference electrode, and naturally airing to obtain the electrochemical sensor with the gold disc serving as the working electrode, the gold sheet serving as the auxiliary electrode and the Ag/AgCl serving as the reference electrode. And after the surface of the electrode is dried, coating Ag/AgCl slurry on a substrate serving as a reference electrode, and naturally airing to obtain the electrochemical sensor with Ag/AgCl serving as the reference electrode, a gold disc electrode serving as a working electrode and a gold sheet electrode serving as an auxiliary electrode.
Preferably, in the above method for preparing a portable handheld electrochemical sensor for detecting enzyme concentration based on a chip electrode, the step (1) is specifically:
(1-1) preparing a wafer, and preparing a silicon wafer with the thickness of 500 mu m;
(1-2) cleaning;
(1-3) first photolithography;
(1-4) etching silicon by using KOH, wherein the etching depth is 200 mu m, forming a reaction tank, and removing the photoresist;
(1-5) thermal oxygen growth of SiO2Growing to a thickness of 200nm to form an insulating layer;
(1-6) performing second photoetching;
(1-7) sputtering Cr/Au: cr/250nm/Au/750nm, and the total thickness is 1 mu m, so as to form an electrode layer;
and (1-8) stripping the positive photoresist and stripping Cr/Au.
Preferably, in the above method for preparing a portable palm electrochemical sensor based on chip electrode to detect enzyme concentration, the nitric acid is concentrated nitric acid, and the ethanol is absolute ethanol.
The beneficial effects of the above technical scheme are: the strong oxidizing property of nitric acid is utilized to remove part of insoluble impurities on the surface of the electrode, the main purpose of ethanol is to remove organic matters on the surface of the electrode, and nitric acid and ethanol are easy to remove from the surface of the electrode.
Preferably, in the above method for preparing a portable palm electrochemical sensor for detecting enzyme concentration based on a chip electrode, the ultrasonic cleaning time is 5-8 min/time, and the ultrasonic cleaning is 1-3 times.
The invention also discloses a detection method of the portable palm electrochemical sensor based on the chip electrode to detect the enzyme concentration, which comprises the following steps:
(1) enzyme-catalyzed reaction
Sequentially adding a mixture of 3: 1: 1: 0.1: 4: 0.9 solution of o-dianisidine, H2O2Solution of KHPO at pH 5.84-Na2HPO4Shaking the solution, HRP solution, absolute ethyl alcohol and ultrapure water uniformly, reacting in a water bath at 37 ℃ for 15min, and taking out;
(2) electrochemical detection
Transferring 2.0mL of the reaction solution obtained in the step (1) into another 5mL beaker, adding a B-R buffer solution with the pH value of 2.0, diluting to 5.0mL by using ultrapure water, shaking up, taking out 6-10 mu L of the uniformly mixed solution, transferring to an electrolytic cell of an electrochemical sensor, and recording the voltammetry of the product of the enzymatic reaction on a smart phone wirelessly connected with an EmStat3+ Bluetooth type electrochemical workstation.
Wherein KHPO4-Na2HPO4The solution and the B-R buffer solution are used for ensuring the optimal pH stability of the system and ensuring the solution to have good conductivity.
Preferably, in the above method for detecting a portable handheld electrochemical sensor based on a chip electrode to detect enzyme concentration, the electrochemical workstation in step (2) detects an enzyme-catalyzed reaction product by differential pulse voltammetry or cyclic voltammetry.
Preferably, in the detection method of the portable handheld electrochemical sensor based on the chip electrode to detect the enzyme concentration, the differential pulse voltammetry adopts an initial potential of 0V, a termination potential of-0.55V, a potential increment of 0.004V and a rest time of 2 s.
Preferably, in the detection method of the portable handheld electrochemical sensor based on the chip electrode for detecting the concentration of the enzyme, the initial potential adopted by the cyclic voltammetry is 0V, the final potential is-0.55V, the sweep rate is 0.1V/s, and the rest time is 2 s.
Preferably, in the above method for detecting a portable handheld electrochemical sensor based on a chip electrode to detect enzyme concentration, the electrochemical workstation is connected to a smart phone in a bluetooth wireless connection manner, and the mobile phone is equipped with pstrace5.8 software to control and collect data of the electrochemical workstation.
Preferably, in the above method for detecting an enzyme concentration by using a portable handheld electrochemical sensor based on a chip electrode, the concentration of the ODA solution in step (1) is 2.0 × 10-2moL/L，H2O2The concentration of the solution was 1.2X 10-2mol/L，KHPO4-Na2HPO4The concentration of the solution is 0.1mol/L, and the concentration of the HRP solution is 1.0X 10-7～1.0×10-3mg/mL。
According to the technical scheme, compared with the prior art, the invention discloses a portable handheld electrochemical sensor for detecting enzyme concentration based on a chip electrode and a preparation method and a detection method thereof2O2Oxidizing a substrate, wherein a reaction product of the substrate generates a sensitive reduction peak on a gold electrode, and the peak current is increased along with the increase of the concentration of the HRP; the electrochemical technologies such as Cyclic Voltammetry (CV) and Differential Pulse Voltammetry (DPV) are used for researching the enzyme catalysis reaction condition, the measurement condition of the enzyme catalysis reaction product and the electrochemical parameters, and the result shows that the chip type portable electrochemical biological enzyme sensor has obvious response to the product of the HRP catalysis reaction, can be further used for detecting the HRP enzyme, and has lower detection limit and wider detection range for the enzyme.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a schematic diagram of a chip three-electrode system according to the present invention;
FIG. 2 is a graph showing cyclic voltammetry curves for various electrodes of the present invention; the curve a is a conventional three-electrode system (the working electrode is a gold electrode (phi is 2mm), the auxiliary electrode is a gold electrode, the reference electrode is an Ag/AgCl electrode), and the curve b is a chip three-electrode system (the working electrode is a gold disc electrode (phi is 2mm), the auxiliary electrode is a gold sheet electrode, and the reference electrode is an Ag/AgCl electrode);
FIG. 3 is a diagram showing that the chip electrode catalyzes ODA or OPD and H under different enzyme concentrations2O2A differential pulse voltammogram of a reaction product after reaction and a linear relation graph between peak current and enzyme concentration; wherein the concentrations of a to i in graph A are 5X 10-7，1.0×10-6，2.0×10-6，3.0×10-6，4.0×10-6，5.0×10-6，6.0×10-6，9.0×10-6，1.0×10-5mg/mL, the concentrations of a to h in FIG. C are 0, 1.0X 10-9，1.0×10-8，1.0×10-7，1.0×10-6，1.0×10-5，1.0×10-4，1.0×10-3mg/mL。
In the figure: 1 is a working electrode, 2 is an auxiliary electrode, 3 is a reference electrode and 4 is an electrolytic cell.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Reagent: horseradish peroxidase, o-dianisidine (ODA), hydrogen peroxide, methanol, phosphoric acid, acetic acid, boric acid, sodium hydroxide, potassium hydrogen phosphate, disodium hydrogen phosphate, potassium ferricyanide, and potassium chloride. The horseradish peroxidase is used for catalyzing hydrogen peroxide to oxidize a substrate, o-dianisidine is a reaction substrate catalyzed by the enzyme, hydrogen peroxide is used for oxidizing the substrate, methanol is used for dissolving o-dianisidine, a mixed solution of potassium hydrogen phosphate and disodium hydrogen phosphate is a buffer solution during reaction and used for maintaining the stability of the pH value of a system, a mixed solution of phosphoric acid, acetic acid and boric acid is a supporting electrolyte of a catalytic product, the pH value of the buffer solution and the supporting electrolyte is adjusted by sodium hydroxide, and a mixed solution of potassium ferricyanide and potassium chloride is used as a characterization solution of a characterization electrode.
The instrument comprises the following steps: EmStat3+ bluetooth type electrochemical workstation, smart phone, chip three-electrode system (gold disc working electrode 1, gold sheet auxiliary electrode 2 and Ag/AgCl reference electrode 3), wherein the three-electrode system is integrated on the silicon chip substrate with gold film plated on the surface, working electrode 1 is gold disc electrode (phi is 2mm), auxiliary electrode 2 is gold sheet electrode, reference electrode 3 is Ag/AgCl electrode, the chip electrode is fixed by electrode clamp, electrode clamp mainly has electrode slot for fixing chip electrode and lead pin for connecting chip electrode, the end of lead pin is telescopic pin, the silicon chip substrate is square silicon chip substrate with side length of 1cm, the silicon chip substrate is provided with square electrolytic cell 4 with side length of 0.4 cm.
The preparation and detection method of the portable palm electrochemical sensor based on the chip electrode for detecting the enzyme concentration comprises the following steps:
(1) plating a gold raw material on a silicon wafer substrate according to a designed structure to preliminarily form a three-electrode system; sequentially ultrasonically cleaning three electrodes of a chip by nitric acid (1+1), ethanol (1+1) and distilled water (5-8 min/time) to obtain a clean electrode surface, completely coating 0.5 +/-0.1 mg of Ag/AgCl slurry every square millimeter on a gold sheet substrate serving as a reference electrode after the electrode surface is dried, naturally drying to obtain the reference electrode, and forming a chip three-electrode system by taking the gold sheet electrode as a working electrode and the gold disc electrode as an auxiliary electrode, wherein the structure of the chip three-electrode system is shown in figure 1;
(2) method for detecting horse radish peroxidase
3.0mL of ODA solution and H were added in sequence to a 10mL test tube2O2KHPO solution 1.0mL, pH 5.84-Na2HPO41.0mL of solution, 100 mu L of HRP solution with different concentrations and 4mL of absolute ethyl alcohol, diluting with water to the scale, shaking up, and reacting in water bath at 37 ℃ for 15miTaking out after n; transferring 2.0mL of the reaction solution into another 5mL beaker, adding 1.0mL of B-R buffer solution with pH2.0, diluting to 5.0mL with ultrapure water, shaking up, taking 10 μ L of the solution out, transferring to an electrolytic cell of a chip three-electrode system, and recording the voltammetry curve of the product of the enzyme catalysis reaction on a smart phone wirelessly connected with an EmStat3+ Bluetooth type electrochemical workstation.
The detection parameters set for detecting the enzyme catalysis product by the electrochemical workstation are as follows: the DPV method adopts 0V of initial potential, -0.55V of final potential, 0.004V of potential increment and 2s of rest time; the CV method adopts an initial potential of 0V, a final potential of-0.55V, a sweep rate of 0.1V/s and a rest time of 2 s.
The invention carries out relevant tests on the electrochemical behaviors of different types of three-electrode systems and the sensitivity of the electrochemical sensor of the chip three-electrode system, and the test method specifically comprises the following steps:
(1) electrochemical behavior of different three-electrode systems
Fig. 1 shows the results of cyclic voltammetric scanning and comparison in potassium ferricyanide solution of the same concentration in a chip three-electrode system constructed with a gold disk electrode (Φ is 2mm) as a working electrode, a gold sheet electrode as an auxiliary electrode, and an Ag/AgCl electrode as a reference electrode, and with a conventional three-electrode system (gold disk electrode as a working electrode and a counter electrode, and diameter Φ is 2mm), in which curve a is a conventional three-electrode system and curve b is a chip three-electrode system, and the corresponding electrochemical data are shown in table 1.
TABLE 1 comparison of electrochemical parameters for different three-electrode systems
The chart shows that the difference between the electrochemical parameters of the chip three-electrode system disclosed by the invention and the corresponding electrochemical parameters of the conventional three-electrode system is not large, a pair of good redox peaks appear in CV scanning, and the chip three-electrode system constructed by the invention has good electrochemical behavior and can be used for subsequent electrochemical analysis application.
(2) Sensitivity of chip electrode electrochemical sensor
By DPV investigates the enzyme catalysis ODA or OPD and H of a chip three-electrode system2O2The electrochemical reduction behavior of the reaction product, the results are shown in FIG. 3 for ODA + H2O2The method comprises the following steps: HRP concentration of 1.0X 10-7The lower limit of detection is reached when the concentration of HRP is 5.0X 10-7～1.0×10-5In the range of mg/mL, the reduction peak current and the enzyme concentration have a good linear relationship, and the linear regression equation is that I (A) is 0.106C (mg/mL) -4.013X 10-8(γ＝0.993)；
For OPD + H2O2The method comprises the following steps: HRP concentration of 1.0X 10-9The lower limit of detection is reached when the concentration of HRP is 1.0X 10-8～10×10-4In the mg/mL range, the reduction peak current and ln of the enzyme concentration have a good linear relationship, and the linear regression equation is I (μ a) ═ 0.897lnC (mg/mL) +16.64(γ ═ 0.991). The results show that the manufactured portable palm electrochemical sensor for detecting the concentration of the enzyme by the chip electrode has low detection limit and wider detection range for detecting HRP, and shows good sensitivity.
The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other. The device disclosed by the embodiment corresponds to the method disclosed by the embodiment, so that the description is simple, and the relevant points can be referred to the method part for description.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
1. A portable palm electrochemical sensor based on chip electrode for detecting enzyme concentration is characterized by comprising a silicon chip substrate and a three-electrode system consisting of a working electrode, an auxiliary electrode and a reference electrode;
the surface of the silicon wafer substrate is plated with a gold film, the three-electrode system is integrated on the silicon wafer substrate, the working electrode is a gold disc electrode, the auxiliary electrode is a gold sheet electrode, and the reference electrode is an Ag/AgCl electrode.
2. The portable handheld electrochemical sensor for detecting the concentration of the enzyme based on the chip electrode as claimed in claim 1, wherein the silicon wafer substrate is a square silicon wafer substrate with a side length of 1cm, and the silicon wafer substrate is provided with a square electrolytic cell with a side length of 0.4 cm.
3. A method for preparing a portable palm electrochemical sensor based on a chip electrode for detecting enzyme concentration according to any one of claims 1-2, which is characterized by comprising the following steps:
(1) plating a gold raw material on a silicon wafer substrate;
(2) sequentially ultrasonically cleaning three electrodes of a chip by using nitric acid, ethanol and distilled water to obtain a clean electrode surface;
(3) and after the surface of the electrode is dried, coating Ag/AgCl slurry on a substrate serving as a reference electrode, and naturally airing to obtain the electrochemical sensor with Ag/AgCl serving as the reference electrode, a gold disc electrode serving as a working electrode and a gold sheet electrode serving as an auxiliary electrode.
4. The method for preparing the portable palm electrochemical sensor for detecting the enzyme concentration based on the chip electrode as claimed in claim 3, wherein the nitric acid is concentrated nitric acid, and the ethanol is absolute ethanol.
5. The method for preparing a portable palm electrochemical sensor for detecting the concentration of the enzyme based on the chip electrode as claimed in claim 3, wherein the ultrasonic cleaning time is 5-8 min/time, and the ultrasonic cleaning is 1-3 times.
6. A method for detecting a portable handheld electrochemical sensor based on a chip electrode for detecting enzyme concentration according to any one of claims 1-2, which is characterized by comprising the following steps:
(1) enzyme-catalyzed reaction
Sequentially adding a mixture of 3: 1: 1: 0.1: 4: 0.9 solution of o-dianisidine, H2O2Solution of KHPO at pH 5.84-Na2HPO4Shaking the solution, HRP solution, absolute ethyl alcohol and ultrapure water uniformly, reacting in a water bath at 37 ℃ for 15min, and taking out;
(2) electrochemical detection
Transferring 2.0mL of the reaction solution obtained in the step (1) into another 5mL beaker, adding a B-R buffer solution with the pH value of 2.0, diluting to 5.0mL by using ultrapure water, shaking up, taking out 6-10 mu L of the uniformly mixed solution, dripping the uniformly mixed solution into an electrolytic cell on a chip electrode, and recording the voltammetry curve of the product of the enzyme catalysis reaction on a smart phone wirelessly connected with an EmStat3+ Bluetooth type electrochemical workstation.
7. The method for detecting the portable handheld electrochemical sensor based on the chip electrode to detect the enzyme concentration according to claim 6, wherein the electrochemical workstation in the step (2) detects the enzyme catalysis reaction product by adopting differential pulse voltammetry or cyclic voltammetry.
8. The method for detecting the handheld electrochemical sensor based on the chip electrode to detect the enzyme concentration as claimed in claim 7, wherein the differential pulse voltammetry adopts an initial potential of 0V, a final potential of-0.55V, a potential increment of 0.004V, and a rest time of 2 s.
9. The method for detecting the handheld electrochemical sensor based on the chip electrode to detect the enzyme concentration as claimed in claim 7, wherein the cyclic voltammetry employs an initial potential of 0V, a final potential of-0.55V, a sweep rate of 0.1V/s, and a rest time of 2 s.
10. The method as claimed in claim 6, wherein the concentration of the ODA solution in step (1) is 2.0 x 10-2moL/L，H2O2The concentration of the solution was 1.2X 10-2mol/L，KHPO4-Na2HPO4The concentration of the solution is 0.1mol/L, and the concentration of the HRP solution is 1.0X 10-7～1.0×10-3mg/mL。
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