CN115236162A - For Pb 2+ Double-signal electrochemical biosensing method for detection - Google Patents
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Abstract
The invention belongs to the technical field of biosensors, and particularly relates to a double-signal electrochemical biosensing method for Pb & lt 2+ & gt detection. The invention uses electro-deposition gold nano particles as a substrate material to modify Fc modified Pb 2+ The aptamer is fixed on an electrode interface; when Pb is present 2+ Fc-Apt and Pb 2+ The specific binding is subjected to conformational change, so that an Fc signal molecule is close to an electrode interface to undergo redox reaction, and the existence of Pb is detected by using ACV 2+ Fc signal I of Fc Simultaneous Pb acquisition using SWASV 2+ Square wave stripping voltammetric signal I SWASV By obtaining I Fc And I SWASV Signal realization pair Pb 2+ The double signals are accurately detected; has the advantages of high sensitivity, good selectivity, good stability and wide linear range, wherein I Fc The linear range of detection is 1 pM-10nM SWASV The linear range of detection is 10pM to 1 μ M.
Description
Technical Field
The invention belongs to the technical field of biosensors, and particularly relates to a method for preparing Pb 2+ A double-signal electrochemical biosensing method for detection.
Background
With the continuous acceleration of the modernization and urbanization process of industry and agriculture, the emission of industrial waste, the overuse of agricultural pesticides and veterinary drugs and the generation of domestic garbage, the heavy metal pollution is aggravated. Heavy metals cannot be degraded and instead can be enriched in the organism through the food chain,finally enters the human body to cause serious injury to the human body. In which Pb 2+ After entering the human body, various system functions of the human body, such as cardiovascular system functions, reproductive system functions, immune system functions and the like, can be directly damaged, and even the life of the human body is threatened. Thus, efficient and sensitive Pb was developed 2+ Detection techniques are highly desirable. At present, atomic Absorption Spectroscopy (AAS), electron coupled plasma mass spectrometry (ICP-MS), atomic Emission Spectroscopy (AES) and the like are mainly used for heavy metal detection, and the methods have advantages and disadvantages.
Electrochemical-based methods have received much attention because of their simplicity, rapid response, low cost, ease of on-site testing, and the like. Of these, square Wave Anodic Stripping Voltammetry (SWASV) is a commonly used electrochemical method for Pb 2+ The analytical performance of the electrode is closely related to the electrode interface modification material, the commonly used modification materials reported in the literature at present comprise carbon-based nano materials, metal nano materials and the like, and the electrode interface active site is mainly used for detecting Pb in the detection process 2+ The adsorption of (2) promotes the analytical performance of sensing, but the selectivity is not enough, and the output of single signal makes the testing result easily receive the influence of environmental factor, leads to its precision not enough. Therefore, on the basis of the SWASV method, another electrochemical signal is introduced to verify the detection result of the SWASV method, and Pb is improved 2+ The accuracy in the detection process is crucial.
Disclosure of Invention
In view of the deficiencies of the prior art, the present invention is directed to solving one of the problems; provides an electrochemical detection method with high detection reliability and accuracy for Pb 2+ Detection of (3).
The invention uses electro-deposition gold nano particles (AuNPs) as a substrate material to modify ferrocene (Fc) 2+ An aptamer (Fc-Apt) is immobilized at the electrode interface. When Pb is present 2+ Fc-Apt and Pb 2+ The specific binding is subjected to conformational change, so that Fc signal molecules are close to an electrode interface to undergo redox reaction, and the existence of Pb is detected by alternating current cyclic voltammetry (ACV) 2+ Fc signal of (2) Fc Simultaneous Pb acquisition using SWASV 2+ Square wave stripping voltammetric signal I SWASV By obtaining I Fc And I SWASV Signal realization pair Pb 2+ The accurate detection of the dual signal of (2).
The purpose of the invention is realized by the following technical scheme:
the invention provides a method for preparing Pb 2+ The detected double-signal electrochemical biosensing method comprises the following steps:
(1) Sequentially polishing a Glassy Carbon Electrode (GCE) by using alumina powder with different particle sizes, ultrasonically cleaning the polished electrode in ethanol and water respectively, and drying the cleaned electrode in air to obtain the treated GCE;
(2) Immersing the GCE treated in the step (1) into HAuCl 4 In the solution, an electrode obtained after voltage is applied to the electrode for a certain time and is marked as Au/GCE;
(3) Ferrocene (Fc) modified Pb 2+ The aptamer is recorded as Fc-Apt; modifying the Fc-Apt with a certain concentration to the surface of the Au/GCE treated in the step (2) for incubation, and marking an electrode after incubation as Fc-Apt/Au/GCE;
(4) Modifying Mercaptoethanol (MCH) on the surface of the Fc-Apt/Au/GCE electrode prepared in the step (3), and incubating for a period of time at room temperature to obtain an electrochemical biosensor which is recorded as MCH/Fc-Apt/Au/GCE;
(5) Immersing the electrochemical biosensor constructed in the step (4) into Pb with different concentrations 2+ Incubating in the solution at room temperature to obtain the electrochemical biosensor, and recording as Pb 2+ MCH/Fc-apt/Au/GCE; then with Pb 2+ adopting/MCH/Fc-apt/Au/GCE as a working electrode, pt as a counter electrode and Ag/AgCl as a reference electrode, and selectively detecting Pb by using alternating current cyclic voltammetry (ACV) on an electrochemical workstation 2+ The interface current of the electrochemical biosensor is/MCH/Fc-apt/Au/GCE; since the Fc-Apt on the surface of the sensor is combined with a target object and a conformational change occurs, and an Fc signal molecule is close to an electrode interface, the Fc redox current generated by the Fc-Apt on the surface of the electrode is measured by alternating current voltammetry and is marked as I Fc (ii) a Further, bound Pb was measured by current time curve 2+ Reducing the lead to elemental lead, and detecting the stripping current of the elemental lead by Square Wave Voltammetry (SWV), which is marked as I SWASV (ii) a It I Fc And I SWASV Signal value and Pb 2+ The logarithm of the solution concentration is in positive correlation, pb of each concentration 2+ Will correspond to an I Fc And I SWASV Signal values, based on which the two signal values respectively sum with Pb 2+ Constructing a logarithmic value of the concentration to obtain a standard curve;
(6) Pb in the sample 2+ The detection of (2): processing the sample to obtain a sample solution, immersing the electrode of the electrochemical biosensor in the sample solution, incubating at normal temperature, measuring the current value according to the operation steps of measuring the current value in the step (5), and analyzing the data to obtain I Fc And I SWASV A signal value; will I Fc And I SWASV Respectively substituting the signal values into the standard curves constructed in the step (5), and obtaining Pb in the sample 2+ To realize Pb in unknown sample 2+ The use of detection.
Further, in step (1), the diameter d =3mm of the GCE; the grain sizes of the aluminum oxide powder are 0.3 μm and 0.05 μm in sequence; the time for the ultrasonic cleaning was 30s.
Further, in the step (2), the HAuCl 4 The concentration of the solution is 0.1-3% by weight; the HAuCl 4 The solvent of the solution is ultrapure water and 1M sulfuric acid in a volume ratio of 1:1 and mixing to obtain the product.
Further, in the step (2), the voltage of the voltage treatment for a certain time is-0.1 to-0.5V, and the time is 30 to 60s.
Further, in the step (3), the Fc-apt concentration is 0.5-3 μ M, and the modified dosage is 6 μ L.
Further, in the step (3), the incubation temperature is 0-10 ℃, and the incubation time is 8-16 h.
Further, in the step (4), the concentration of the MCH is 1mM, and the modified dosage is 6 mu L; the incubation time is 20-60 min.
Further, in the step (5), the Pb is 2+ The concentration of the solution was 1X 10 -12 ～1×10 -6 M; the incubation time is 20-60 min.
Further, in the step (5), the voltage adopted in the current-time curve is-0.7 to-1.2V, and the time is 90 to 150s; the solution used for the detection process was PBS buffer with pH =7.4 and a concentration of 0.1M.
Further, in the step (6), the incubation time is 20-60 min.
The invention has the beneficial effects that:
(1) The electrochemical biosensor has the advantages of simple construction, convenient operation, safety, environmental protection and no toxic action on human bodies.
(2) The identification process of the present invention is based on the aptamer and Pb 2+ The selectivity of the electrochemical biosensor is effectively improved by the specific combination of the two.
(3) One kind of material constructed by the present invention for Pb 2+ The double-signal electrochemical biosensing method for detection is characterized in that ACV is added on the basis of SWASV, so that the double-signal pair of Pb is realized 2+ The linear advantages of different detection methods are complementary; meanwhile, the overlapping part of the linear range of the double signals can realize double signal verification, and the Pb is effectively improved 2+ And (4) the detection accuracy.
(4) The electrochemical biosensor constructed by the invention is used for Pb 2+ The detection has high sensitivity, good selectivity, good stability and wide linear range, wherein I Fc The linear range of detection is 1 pM-10nM SWASV The linear range of detection is 10pM to 1 μ M.
Drawings
FIG. 1 is a diagram of the construction and detection mechanism of the dual-signal electrochemical biosensor of the present invention.
FIG. 2 (A) is a diagram of a dual-signal electrochemical biosensor I according to the present invention Fc Signal and Pb 2+ A linear plot of the log concentration; (B) The figure is a double-signal electrochemical biosensor I of the invention SWASV Signal and Pb 2+ Linear plot of log concentration.
FIG. 3 (A) is a graph showing the selective performance of the dual-signal electrochemical biosensor of the present invention, wherein blank is an aqueous solution, and a is Pb 2+ Solution b is Pb 2+ +Al 3+ Solution of c is Pb 2+ +Cd 2+ Solution of d is Pb 2+ +Co 2+ Solution of e is Pb 2+ +Fe 3+ Solution f is Pb 2+ +Mn 2+ Solution g of Pb 2+ +Mg 2+ Solution of h is Pb 2+ +Zn 2+ Solution i is Pb 2+ +Ca 2+ Solution j is Pb 2+ +Cu 2+ Solution of k is Pb 2+ +Hg 2+ Solution of l is Pb 2+ +Al 3+ +Cd 2+ +Co 2+ +Fe 3+ +Mn 2+ +Mg 2+ +Zn 2+ +Ca 2+ +Cu 2+ +Hg 2+ A solution; (B) Is a long-term stable performance diagram of the double-signal electrochemical biosensor.
The specific implementation mode is as follows:
embodiments of the invention are described in detail with reference to the accompanying drawings: the embodiments are performed on the premise of the technical solution of the present invention, and detailed implementation steps and specific operation processes are given, but the scope of the present invention is not limited to the following embodiments.
Reagents mentioned in the present invention: fc-Apt was purchased from Biotechnology engineering (Shanghai) Inc., and has a base sequence of: 5' -SH- (CH) 2 ) 6 GGGTGGGTGGGTGGGT-Fc-3' (the 5' end of the aptamer is modified with a thiol group, and the 3' end is modified with a signal molecule Fc).
Example 1:
according to the construction process described in FIG. 1, the preparation method of the dual-signal electrochemical biosensor comprises the following steps:
(1) Grinding GCE with 0.3 μm and 0.05 μm aluminum oxide powder in sequence, respectively performing ultrasonic treatment in ethanol and water for 30s, and drying in air to obtain treated GCE;
(2) Immersing the GCE treated in the step (1) into HAuCl 4 (0.1% by weight) in the solution, a reducing voltage of-0.2V was applied to the electrode, auNPs were formed on the surface of the GCE electrode after 45 seconds of treatment, and the electrode obtained after the treatment was recorded as Au/GCE;
(3) Modifying 6 mu L of Fc-Apt with the concentration of 2 mu M to the surface of the Au/GCE treated in the step (2), incubating for 12h at 4 ℃, and marking the incubated electrode as Fc-Apt/Au/GCE;
(4) Modifying 6 mu L of MCH with the concentration of 0.1mM on the surface of the Fc-Apt/Au/GCE electrode prepared in the step (3), incubating for 40min at room temperature to obtain an electrochemical biosensor which is marked as MCH/Fc-Apt/Au/GCE;
(5) Taking the electrochemical biosensor constructed in the step (4), and immersing the electrochemical biosensor in 100 μ L of Pb with the concentration of 1pM, 10pM, 100pM, 1nM, 10nM, 100nM and 1 μ M 2+ Incubating at normal temperature for 40min in the solution, wherein the prepared sensor is used as a working electrode, a saturated Ag/AgCl electrode is used as a reference electrode, and a Pt electrode is used as a counter electrode; electrochemical signals were recorded and detected by an electrochemical workstation model CHI750E, and tested in 0.1M PBS (pH = 7.4) buffer. The range of the scanning voltage of the ACV is 0.1-0.8V, the amplitude is 0.025V, and the frequency is 25Hz; the deposition potential of an i-t curve used in the SWASV detection process is-0.9V, and the deposition time is 120s; the SWV has a scan voltage range of-0.8 to-0.3V, an amplitude of 0.025V, and a frequency of 25Hz.
As can be seen from (A) in FIG. 2, following Pb 2+ Increase in concentration, I Fc The signal increases continuously and shows a good linear relationship between 1pM and 10nM, its I Fc The linear regression equation of the signal is represented by formula I Fc ＝1.89598+0.14239logC Pb 2 + ；
With Pb 2+ Increase in concentration of I SWASV The signal increases continuously, showing a good linear relationship between 10pM and 1. Mu.M, its I SWASV The signal equation is represented by formula I SWASV ＝0.88584+0.23599logC Pb 2+ 。
And (3) selecting performance measurement:
(1) The prepared electrochemical biosensor is used for inspecting the selective performance of the dual-signal electrochemical biosensor, namely, the dual-signal electrochemical biosensor is respectively placed in aqueous solution to obtain I Fc And I SWASV Is a blank signal; when the blank is replaced by a different solution (a, b, c, d, e, f, g, h, I, j, k, l), according to the I obtained Fc And I SWASV For testing signals, investigating their selection properties
Fig. 3 (a) is a selected performance test chart of the sensor: wherein blank refers to an aqueous solution and is defined as a blank sample; a is Pb 2+ Solution b 50pM Pb 2+ +500pM Al 3+ Solution, c is 50pM Pb 2+ +500pM Cd 2+ Solution, d is 50pM Pb 2+ +500pM Co 2+ Solution e 50pM Pb 2+ +500pM Fe 3+ Solution, f is 50pM Pb 2+ +500pM Mn 2+ Solution, g is 50pM Pb 2+ +500pM Mg 2+ Solution, h is 50pM Pb 2+ +500pM Zn 2+ Solution i of 50pM Pb 2+ +500pM Ca 2+ Solution, j is 50pM Pb 2+ +500pM Cu 2+ Solution, k 50pM Pb 2+ +500pM Hg 2+ Solution,. L.is 50pM Pb 2+ +500pM Al 3+ +500pM Cd 2+ +500pM Co 2+ +500pM Fe 3+ +500pM Mn 2+ +500pM Mg 2+ +500pM Zn 2+ +500pM Ca 2+ +500pM Cu 2+ +500pM Hg 2+ A solution; it can be seen from the figure that the electrochemical biosensor is only used for the target Pb 2+ When the ion source exists, obvious signal change is generated, and the existence of other interferent ions has no obvious influence on the response signal, so that the sensor has good selection performance.
(2) The long-term stability of the dual-signal electrochemical biosensor is inspected by using the prepared electrochemical biosensor, the electrode is placed in a refrigerator at 4 ℃ for storage after the preparation is finished, and then the prepared dual-signal electrochemical biosensor is respectively used for detecting 100pM of Pb in the following 1 st, 2 nd, 3 th, 4 th, 5 th, 6 th and 7 th days 2+ A solution; examine I in the 7 days Fc And I SWASV The signal of (2) changes.
FIG. 3 (B) shows the long-term stability of the electrochemical biosensor, and it can be seen from the graph that I is the measured time at 7 days Fc Signal response of (2) is 91.6% of the initial value, I SWASV The signal response of (a) was 87.6% of the initial value, indicating that the prepared electrochemical biosensor had good long-term stability.
And (3) detecting an irrigation water sample:
a certain amount of irrigation water sample (from Yangtze river basin of Zhenjiang city, jiangsu province) is filtered by a filter membrane of 0.22 mu m to remove solid impurities, and a sample solution is obtained. Taking three sample solutions after filtration, and respectively adding 1nM, 5 nM and 10nM Pb 2+ Post-adoption implementationExample 1 the electrochemical biosensor prepared in step (4) was tested, and the current values were measured and substituted into the established linear regression equation to obtain Pb in the sample 2+ The detection recovery rate was obtained as shown in Table 1.
Table 1: pb in irrigation water 2+ Detection recovery rate of
As can be seen from Table 1, in the present embodiment, for the actual sample detection process, the constructed dual-signal electrochemical biosensor can sensitively and quantitatively detect Pb in the sample to be detected 2+ And the overlapping part of the linear ranges can realize the verification of double signals, the detection accuracy of the sensor is further improved, professional training is not needed in the detection process, and the operation is simple and convenient.
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 (10)
1. For Pb 2+ The detected double-signal electrochemical biosensing method is characterized by comprising the following steps:
(1) Sequentially polishing the glassy carbon electrode by using aluminium oxide powder with different particle sizes, ultrasonically cleaning the polished glassy carbon electrode in ethanol and water respectively, and drying the cleaned glassy carbon electrode in air to obtain a treated glassy carbon electrode;
(2) Immersing the glassy carbon electrode treated in the step (1) into HAuCl 4 In the solution, an electrode obtained after voltage is applied to the electrode for a certain time and is marked as Au/GCE;
(3) Ferrocene-modified Pb 2+ The aptamer is recorded as Fc-Apt; will be concentrated to a certain degreeModifying the Fc-Apt of the degree to the surface of the Au/GCE treated in the step (2) for incubation, and marking an electrode after incubation as Fc-Apt/Au/GCE;
(4) Modifying mercaptoethanol on the surface of the Fc-Apt/Au/GCE electrode prepared in the step (3), and incubating for a period of time at room temperature to obtain an electrochemical biosensor which is marked as MCH/Fc-Apt/Au/GCE;
(5) Immersing the electrochemical biosensor constructed in the step (4) into Pb with different concentrations 2+ Incubating in the solution at room temperature to obtain the electrochemical biosensor which is recorded as Pb 2+ MCH/Fc-apt/Au/GCE; then with Pb 2+ adopting/MCH/Fc-apt/Au/GCE as a working electrode, pt as a counter electrode and Ag/AgCl as a reference electrode, and selectively detecting Pb by using an alternating current cyclic voltammetry on an electrochemical workstation 2+ The interface current of the electrochemical biosensor is/MCH/Fc-apt/Au/GCE; the Fc-Apt on the surface of the sensor is combined with a target object and generates conformational change due to the fact that an Fc signal molecule is close to an electrode interface, and the Fc redox current generated by the Fc-Apt on the surface of the electrode is measured by alternating current voltammetry and is marked as I Fc (ii) a Further, bound Pb was measured by current time curve 2+ Reducing the lead to elemental lead, and detecting the stripping current of the elemental lead by square wave voltammetry, which is marked as I SWASV (ii) a It I Fc And I SWASV Signal value and Pb 2+ The logarithm of the solution concentration is in positive correlation, pb of each concentration 2+ Will all correspond to an I Fc And I SWASV Signal values, based on which the two signal values respectively sum with Pb 2+ Constructing a logarithmic value of the concentration to obtain a standard curve;
(6) Pb in sample 2+ Detection of (2): treating the sample to obtain a sample solution, immersing the electrode of the electrochemical biosensor in the sample solution, incubating at normal temperature, measuring the current value according to the operation steps of measuring the current value in the step (5), and analyzing the I of the data Fc And I SWASV A signal value; finally, will I Fc And I SWASV Respectively substituting the signal values into the standard curves constructed in the step (5), and obtaining Pb in the sample 2+ To realize Pb in unknown sample 2+ The use of detection.
2. A method for Pb according to claim 1 2+ The detected double-signal electrochemical biosensing method is characterized in that in the step (1), the diameter d =3mm of the glassy carbon electrode; the grain diameter of the aluminum oxide powder is 0.3 μm and 0.05 μm in sequence; the time for the ultrasonic cleaning was 30s.
3. A method for Pb according to claim 1 2+ The detected double-signal electrochemical biosensing method is characterized in that in the step (2), HAuCl is adopted 4 The concentration of the solution is 0.1-3 percent by weight; the HAuCl 4 The solvent of the solution is ultrapure water and 1M sulfuric acid in a volume ratio of 1:1 and mixing to obtain the product.
4. A process for Pb according to claim 1 2+ The detected double-signal electrochemical biosensing method is characterized in that in the step (2), the voltage applied for certain time is-0.1 to-0.5V, and the time is 30 to 60s.
5. A method for Pb according to claim 1 2+ The detected double-signal electrochemical biosensing method is characterized in that in the step (3), the Fc-apt concentration is 0.5-3 mu M, and the dosage is 6 mu L.
6. A method for Pb according to claim 1 2+ The detected double-signal electrochemical biosensing method is characterized in that in the step (3), the incubation temperature is 0-10 ℃, and the incubation time is 8-16 h.
7. A method for Pb according to claim 1 2+ The detected double-signal electrochemical biosensing method is characterized in that in the step (4), the concentration of the mercaptoethanol is 1mM, and the dosage is 6 mu L; the incubation period is 20-60 min.
8. A process for Pb according to claim 1 2+ A dual-signal electrochemical biosensing method for detection, characterized in thatIn the step (5), the Pb is 2+ The concentration of the solution was 1X 10 -12 ～1×10 -6 M; the incubation time is 20-60 min.
9. A process for Pb according to claim 1 2+ The detected double-signal electrochemical biosensing method is characterized in that in the step (5), the voltage adopted in the current-time curve is-0.7 to-1.2V, and the time is 90 to 150s; the solution used for the detection process was PBS buffer with pH =7.4 and a concentration of 0.1M.
10. A method for Pb according to claim 1 2+ The detected double-signal electrochemical biosensing method is characterized in that in the step (6), the incubation time is 20-60 min.
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| CN113588745A (en) * | 2021-07-23 | 2021-11-02 | 江苏大学 | Pb with controllable sensitivity2+Induced double-amplification electrochemiluminescence detection method |
| CN114199977A (en) * | 2021-11-23 | 2022-03-18 | 江苏大学 | Electrochemical heavy metal rapid detection method based on electric drive pre-enrichment |
| CN114354582A (en) * | 2021-12-16 | 2022-04-15 | 江苏大学 | Preparation method of double-signal amplification electrochemiluminescence aptamer sensor and detection of Pb by using sensor2+Application of |
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| CN109142483A (en) * | 2018-07-17 | 2019-01-04 | 上海理工大学 | It is a kind of for detecting the electrochemica biological sensor and detection method of inorganic trivalent arsenic |
| WO2021093530A1 (en) * | 2019-11-11 | 2021-05-20 | 暨南大学 | Lead ion nucleic acid aptamer, electrochemical sensor and preparation method |
| CN112540073A (en) * | 2020-11-13 | 2021-03-23 | 江苏大学 | Preparation method and application of dual-output mode sensor for amplifying electrochemical luminescence signal based on Fc-apt |
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