Pb Detection by Electric Field-Regulated Aptamer Orientation on Sensing Interface 2+ , Hg 2+ Simultaneous Ratio Electrochemical Detection Method

Abstract

The present invention belongs to the technical field of biosensors, and particularly relates to a method for simultaneous ratio electrochemical detection of Pb<supgt;2+< / supgt> and Hg<supgt;2+< / supgt> based on electric field-regulated aptamer orientation at the sensing interface. The present invention designs complementary cDNA labeled with anthraquinone (AQ), and simultaneously combines Pb<supgt;2+< / supgt> aptamer labeled with methylene blue (MB) and Hg<supgt;2+< / supgt> aptamer labeled with ferrocene (Fc), and constructs a sensing interface with a DNA structure synthesized by PCR. During the pre-enrichment process, the electric field first pushes the ions close to the aptamer and keeps them away from the DNA, which is beneficial for subsequent recognition; then, when testing the electrochemical signal, the DNA with the retained direction of the electric field provides a stable current for AQ as the detection reference signal. Thus, a novel dual-ratio electrochemical aptamer sensor is constructed to achieve specific enrichment and accurate detection of Pb<supgt;2+< / supgt> and Hg<supgt;2+< / supgt>; the present invention provides a new method for manufacturing high-performance electrochemical sensors for heavy metal ion detection.

Description

Technical Field

[0001] The present invention belongs to the technical field of biosensors, and particularly relates to a simultaneous ratio electrochemical detection method for Pb 2+ , Hg 2+ based on regulating the orientation of aptamers at the sensing interface by an electric field. Background Art

[0002] With the rapid development of agricultural technology, the extensive use of various fertilizers, micronutrients and pesticides has ensured the good growth and quality of agricultural products. However, the excessive use of agricultural chemicals has exacerbated the pollution of the ecosystem, especially the soil and water. Among them, lead (Pb 2+ ) and mercury (Hg 2+ ) have been listed as the most dangerous heavy metal pollutants by the World Health Organization (WHO) due to their characteristics of non-biodegradability, easy bioaccumulation and posing a serious threat to human health. Therefore, rapid detection of lead and mercury with high sensitivity and accuracy is of great significance in environmental protection.

[0003] So far, there are various detection methods for Pb 2+ and Hg 2+ in the relevant literature. The detection methods established at home and abroad include inductively coupled plasma mass spectrometry (ICP-MS), atomic absorption spectrometry (AAS), atomic fluorescence spectrometry (AFS), fluorescence method, colorimetry, electrochemiluminescence method and electrochemistry method (EC). Among them, the electrochemistry method is an analytical method for detecting the analyte by using electrochemical response signals such as redox reactions. Electrochemical aptamer sensors have broad prospects in the selective analysis of Pb 2+ and Hg 2+ ; researchers have found that T-T mismatch can specifically capture Hg 2+ , form a DNA double strand with T-Hg 2+ -T, and G base can specifically capture Pb 2+ to form a G-quadruplex.

[0004] However, the long incubation time for the formation of G-quadruplex and T-Hg 2+ -T structures and the irregular winding of linear DNA cause relatively complex interference to the sensing interface, which has been affected for a long time. Kelley et al. used atomic force microscopy to study the electrostatic effect of an external electric field on the surface topography of DNA, and the helical direction of DNA will change with the change of the applied voltage. However, the current research methods still have technical problems of long time and poor performance. Summary of the Invention

[0005] Aiming at the problems existing in the prior art, the present invention aims to solve one of the problems; to provide an electrochemical aptamer sensor integrating rapid specific enrichment and multi-target simultaneous detection, so as to realize the simultaneous and accurate analysis of Pb 2+ and Hg 2 + in complex environmental samples such as soil.

[0006] First, a label-free signal probe - anthraquinone (AQ) is prepared and stirred and compounded with an auxiliary linear DNA (cDNA). Through the non-covalent interaction between anthraquinone (AQ) and the amino group (-NH 2 ) carried by cDNA, AQ-cDNA is formed; a gene amplifier (PCR) is used to synthesize with the aptamers Fc-apt and MB-apt to form a stable double-stranded structure PCR-DNA; gold nanoparticles (AuNPs) immobilize the thiol-modified PCR-DNA linear aptamer on the sensing interface; an electrostatic field that can generate charge electron transfer is introduced, so that the negatively charged PCR-DNA has an electrostatic repulsion with the electrostatic field, maintaining the stable structure of PCR-DNA unchanged; among them, a part of PCR-DNA includes a linear cDNA aptamer sequence carrying the electrochemical molecule anthraquinone (AQ), and another part includes a linear aptamer carrying the electrochemical molecule methylene blue (MB) for identifying Pb 2+ , and a linear aptamer carrying the ferrocene (Fc) electrochemical signal molecule for identifying Hg 2+ , thereby constructing a novel dual-ratio electrochemical aptamer sensor.

[0007] Compared with the case without applying an electrostatic field, the binding of PCR-DNA enriched under the electrostatic field to its target substances Pb 2+ and Hg 2+ is more likely to occur, and it has a stable and rigid 2D structure, having higher binding efficiency and anti-interference ability in recognition.

[0008] By using the electric field to regulate the DNA orientation in the sensitive interface, problems such as reduced recognition efficiency and electrical signal perturbation caused by the bending and winding of single-stranded DNA are solved.

[0009] Therefore, the present invention uses the electrostatic field adjustable strategy, and uses the directional movement of ions in the electric field to promote the recognition of Pb 2+ and Hg 2+ to DNA, which is beneficial to its efficient pre-enrichment on the electrode, effectively shortening the incubation time of Pb 2+ and Hg 2+ ; and uses the electrostatic field to regulate the conformational changes of DNA, preventing it from winding, aggregating and arranging irregularly, reducing its steric hindrance effect, and improving the analytical performance of electrochemical sensing for Pb 2+ and Hg 2+ .

[0010] The object of the present invention is achieved by the following technical solutions:

[0011] A method for simultaneous ratio electrochemical detection of Hg 2+ and Pb 2+ based on electric field-regulated aptamer orientation at the sensing interface, comprising the following steps:

[0012] (1) Preparation of a label-free electrochemical probe AQ-cDNA complex solution:

[0013] (a) First, dissolve anthraquinone (AQ) in phosphate buffer solution (PBS), and obtain an AQ dispersion after stirring; then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) solution to activate the carboxylic acid group of anthraquinone in the AQ dispersion to obtain an AQ activation solution;

[0014] (b) Dissolve cDNA in tris(hydroxymethyl)aminomethane hydrochloride buffer solution (Tris-HCl) to obtain a cDNA solution; then mix the AQ activation solution with the cDNA solution, and obtain an AQ-cDNA complex solution after magnetic stirring;

[0015] (2) Preparation of a label-free electrochemical probe PCR-DNA complex:

[0016] The ferrocene-labeled linear aptamer is denoted as Fc-apt, and the methylene blue-labeled linear aptamer is denoted as MB-apt; mix the Fc-apt solution with the MB-apt solution, and denote the mixed solution as the Fc-apt-MB-apt solution; then mix the AQ-cDNA complex solution in step (1) with the Fc-apt-MB-apt solution, and perform an amplification reaction using a gene amplifier (PCR), and denote the solution after the reaction as PCR-DNA;

[0017] (3) Polish a glassy carbon electrode (GCE) successively with alumina powders of different particle sizes, and ultrasonically clean the polished glassy carbon electrode in ethanol and water respectively, and then dry it in air to obtain a treated glassy carbon electrode;

[0018] (4) Modify a gold nanoparticle (AuNPs) solution on the sensing interface of the glassy carbon electrode treated in step (3), and denote the modified material as AuNPs / GCE;

[0019] (5) Modify the PCR-DNA obtained in step (2) on the sensing interface of the AuNPs / GCE prepared in step (4), and incubate and fix it to obtain a dual-ratio electrochemical aptasensor based on Fc-apt-MB-apt, denoted as PCR-DNA / AuNPs / GCE;

[0020] (6) Modify the MCH solution on the electrode surface of the PCR-DNA / AuNPs / GCE material prepared in step (5). After incubating for a period of time at room temperature, the obtained material is denoted as MCH / PCR-DNA / AuNPs / GCE;

[0021] (7) Modify the mixed standard solution containing different concentrations of Pb 2+ and Hg 2+ on the surface of the MCH / PCR-DNA / AuNPs / GCE electrode prepared in step (6), and electro-driven preconcentrate Pb 2+ and Hg 2+ under different electrostatic field conditions. The obtained electrochemical biosensor after enrichment is denoted as Pb 2+ &Hg 2+ / MCH / PCR-DNA / AuNPs / GCE;

[0022] (8) Construction of the standard curve:

[0023] Using the sensor Pb 2+ &Hg 2+ / MCH / PCR-DNA / AuNPs / GCE prepared in step (7) as the working electrode, the saturated Ag / AgCl electrode as the reference electrode, and the platinum wire electrode as the counter electrode, place it in the electrostatic field for electrochemical detection to obtain the final electrochemical detection current value; according to the detected current value and the logarithm of the corresponding concentrations of Pb 2+ and Hg 2+ in the mixed standard solution, construct the standard curves respectively;

[0024] (9) Detection of Pb 2+ and Hg 2+ in actual samples in the actual application scenario of agricultural production: First, prepare the sample solution, drop the sample solution on the surface of the sensor MCH / PCR-DNA / AuNPs / GCE electrode prepared in step (6), place it in the electrostatic field, and obtain the corresponding current value through electrochemical testing; substitute the current value into the standard curve constructed in step (8), and the concentrations of Pb 2+ and Hg 2+ in the actual sample can be obtained, realizing the detection of Pb 2+ and Hg 2+ in the actual sample.

[0025] Preferably, in (a) of step (1), the stirring time is 12 h; the concentration of the AQ dispersion is 0.1 mg mL -1 , the concentrations of the EDC and NHS solutions are 10 mM and 5 mM respectively; the concentration of the PBS buffer solution is 0.1 M; the volume ratio of the AQ dispersion, EDC and NHS solutions is 2:1:1;

[0026] Preferably, in step (1)(b), the concentration of the cDNA solution is 2 - 16 μM; the volume ratio of the AQ activation solution to the cDNA solution is 1 - 4:1; the concentration of the Tris-HCl buffer solution is 0.1 M; the magnetic stirring time after mixing the AQ activation solution and the cDNA solution is 2 h; the final concentration of cDNA in the AQ-cDNA complex dispersion is 1 - 8 μM.

[0027] Preferably, in step (2), the concentrations of both Fc-apt and MB-apt are 20 μM, and Fc-apt and MB-apt are mixed at a volume ratio of 1:1; the volume ratio of the AQ-cDNA complex dispersion to the Fc-apt-MB-apt solution is 1:1; the temperature condition for the PCR amplification reaction is 95 °C, the reaction time is 2 min, and then rapid annealing is carried out at 4 °C for 10 min.

[0028] Preferably, in step (3), the diameter d of the glassy carbon electrode is 3 mm; the particle sizes of the aluminum oxide powders used are 0.3 μm and 0.05 μm in sequence; the ultrasonic time is 30 s for both.

[0029] Preferably, in step (4), the dosage of the gold nanoparticle solution modification is 6 μL and the concentration is 5 μM.

[0030] Preferably, in step (5), the dosage of the PCR-DNA modification is 6 μL and the concentration is 0.5 - 4.0 μM; the incubation temperature is 4 °C and the incubation time is 10 - 12 h.

[0031] Preferably, in step (6), the dosage of the MCH solution is 6 μL and the concentration is 0.1 M; the incubation period is 40 - 60 min.

[0032] Preferably, in step (7), the concentrations of Pb 2+ and Hg 2+ in the mixed standard solution are both 1×10 -13 -1×10 - 8 M, the modification dosage of the mixed standard solution containing different concentrations of Pb 2+ and Hg 2+ is 6 μL; the specific steps of electro-driven preconcentration under different electrostatic field conditions: preconcentration is carried out in an electrostatic field of 0 - 3 V for 10 - 60 min.

[0033] Preferably, in step (8), the specific conditions for the electrochemical workstation to detect electrochemical signals are as follows: The test is carried out in a 0.1 M PBS (pH = 7.4) buffer solution; the scanning voltage range is -0.4 to 0.8 V, the amplitude is 0.025 V, and the frequency is 25 Hz; the conditions for the electrostatic field are 0 to 3 V.

[0034] Preferably, in step (9), the specific conditions for the electrochemical workstation to detect electrochemical signals are as follows: The test is carried out in a 0.1 M PBS (pH = 7.4) buffer solution; the scanning voltage range is -0.4 to 0.8 V, the amplitude is 0.025 V, and the frequency is 25 Hz; the conditions for the electrostatic field are 0 to 3 V.

[0035] Advantages of the present invention:

[0036] (1) The dual-ratio electrochemical aptasensor for Pb 2+ and Hg 2+ based on electric-field-regulated aptamer orientation proposed by the present invention, by introducing the signal molecule AQ carried by cDNA and assembling the Pb 2+ aptamer labeled with MB and the Hg 2+ aptamer labeled with Fc through a PCR instrument, constructs a dual-ratio electrochemical aptasensor capable of simultaneously detecting Pb 2+ and Hg 2+ .

[0037] (2) The electrochemical biosensor of the present invention can greatly shorten the incubation time of Pb 2+ & Hg 2+ with MB-apt and Fc-apt through the action of an electrostatic field. Through electric-field enrichment, its binding time is shortened from 60 min to 30 min, having the advantage of rapid detection.

[0038] (3) The electrochemical biosensor of the present invention makes cDNA have a stable and rigid 2D structure, higher binding efficiency and anti-interference ability through the introduction of an electrostatic field.

[0039] (4) The dual-ratio electrochemical biosensor proposed by the present invention can simultaneously obtain three detection signals, with a large amount of information, high accuracy and credibility.

[0040] (5) The electrochemical biosensor constructed by the present invention is used for detection, with high sensitivity, good selectivity, good stability, and a wide linear range, which are 0.1 pM to 1 nM and 1 pM to 10 nM respectively. Description of the Drawings

[0041] Figure 1 In (A), it is the EC signal change diagram during the construction process of the electrochemical biosensor, where

[0042] a is MCH / PCR-DNA / AuNPs / GCE;

[0043] b is Pb 2+ &Hg 2+ / MCH / PCR-DNA / AuNPs / GCE;

[0044] c is Pb 2+ &Hg 2+ / MCH / PCR-DNA / AuNPs / GCE electrostatic field enrichment for 30 min;

[0045] d is Pb 2+ &Hg 2+ / MCH / PCR-DNA / AuNPs / GCE electrostatic field enrichment for 30 min and detection of electrochemical signal in the electrostatic field;

[0046] Figure 1 In (B) is the Nyquist (EIS) impedance diagram of the construction process of the electrochemical biosensor, where

[0047] a is GCE;

[0048] b is AuNPs / GCE;

[0049] c is PCR-DNA / AuNPs / GCE;

[0050] d is MCH / PCR-DNA / AuNPs / GCE;

[0051] e is Pb 2+ / MCH / PCR-DNA / AuNPs / GCE;

[0052] f is Hg 2+ / MCH / PCR-DNA / AuNPs / GCE;

[0053] g is Pb 2+ &Hg 2+ / MCH / PCR-DNA / AuNPs / GCE;

[0054] h is Pb 2+ &Hg 2+ / MCH / PCR-DNA / AuNPs / GCE electrostatic field enrichment for 30 min;

[0055] i is Pb 2+ &Hg 2+ / MCH / PCR-DNA / AuNPs / GCE electrostatic field enrichment for 30 min and detection of electrochemical signal under the application of the electrostatic field;

[0056] Figure 2(A) shows the response graphs of electrochemical signals I Fc and I MB to different cDNA concentrations; (B) shows the response graphs of electrochemical signal I AQ to different applied electrostatic field voltage values; (C) shows the response graphs of electrochemical signals I 2+ &Hg 2+ when the electrostatic field enrichment time is applied to the target substances Pb Fc and I MB ; (D) shows the influence of the voltage change of the applied electrostatic field during signal acquisition on electrochemical signals I Fc and I MB .

[0057] Figure 3 (A) in shows the response graphs of an electrochemical biosensor under an applied electrostatic field to different concentrations; (B) shows the linear relationship graph between the logarithm of the concentration of Pb 2+ &Hg 2+ and the electrochemical signal. Detailed Embodiments

[0058] Now, various exemplary embodiments of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0059] It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0060] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0061] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are also obvious to those skilled in the art. The specification and examples of the present invention are only exemplary.

[0062] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0063] All the reagents used in the present invention are purchased from Sangon Biotech (Shanghai) Co., Ltd. The sequences are as follows (this application does not involve the submission of a sequence listing, and the above sequences are only for illustration and are all conventional reagent sequences, belonging to primer sequences):

[0064] cDNA:

[0065] 5`-SH-(CH 2 ) 6 -AAGAGAGAAGCTGCAACACACCACCCACCCACCCACCC ACCCA-(CH 2 ) 6 -NH 2 -3`;

[0066] MB-apt(Pb 2+ ):5`-TGGGTGGGTGGGTGGGTGGG-MB-3`;

[0067] Fc-apt(Hg 2+ ):5`-TGGTGTGTTGCAGCTTCTCTCTT-Fc-3`;

[0068] Optimization condition screening:

[0069] (1) As shown in (A) of Figure 2 , a total of six groups of different cDNA concentration controls were made. As the cDNA concentration increased, both I MB and I Fc increased, and the current signal basically reached saturation at 2 μM, indicating that the binding sites of the aptamer had reached saturation. Therefore, the optimal cDNA concentration was selected as 2 μM.

[0070] (2) During the detection process, the voltage intensity of the electrostatic field has a certain effect on changing the structure of the cDNA single strand. As shown in (B) of Figure 2 , a total of six groups of detection of the peak current change of I AQ under different electrostatic fields were conducted. Between the applied voltages of 0 V and 3 V, the current response of I AQ decreased from 0 V to 2.5 V and then approached a plateau. Therefore, the optimal effect of the applied voltage for the electrostatic field test was 2.5 V.

[0071] (3) As shown in (C) of Figure 2 , a total of seven groups of pre-concentration time controls of the target under the electrostatic field were made. As the pre-concentration time extended, IMB and I Fc gradually decreased and reached a plateau after 30 min, indicating that the sensor's recognition of Pb 2+ and Hg 2+ had reached saturation. Therefore, it was shown that the optimal time for electrostatic field enrichment was 30 min

[0072] (4) As shown in (D) of Figure 2 , a total of seven groups of optimizations of the enrichment application voltage of the target under the electrostatic field were carried out. As the pre-enrichment voltage increased, I MB and I Fc gradually decreased and reached a plateau when the voltage intensity was 3 V between 0 V and 4 V, indicating that the optimal enrichment voltage of the sensor for Pb 2+ and Hg 2+ was 3 V. Therefore, the best effect of the enrichment application voltage of the electrostatic field was 3 V.

[0073] Example 1:

[0074] (1) First, AQ was dissolved in 0.1 M PBS and stirred for 12 h to obtain an AQ dispersion with a concentration of 0.1 mg mL -1 ; then, 250 μL of the AQ dispersion was added to 250 μL of a 10 mM EDC and 5 mM NHS solution to activate the carboxylic acid groups of anthraquinone, and stirred at room temperature for 15 min to obtain an AQ activation solution;

[0075] The cDNA was dissolved in a tris(hydroxymethyl)aminomethane hydrochloride buffer solution (Tris-HCl) to obtain a cDNA solution with a concentration of 8 μM. 500 μL of the cDNA solution was injected into the AQ activation solution and stirred for another 2 h to obtain an AQ-cDNA composite solution, in which the concentration of cDNA was 4 μM, and it was stored at 4 °C for later use;

[0076] (2) The Fc-apt solution and the MB-apt solution were mixed at a volume ratio of 1:1 to obtain an Fc-apt-MB-apt solution; the concentration of the Fc-apt solution was 20 μM, and the concentration of the MB-apt solution was 20 μM; then, the AQ-cDNA composite solution prepared in step (1) was mixed with the Fc-apt-MB-apt solution at a ratio of 1:1, heated in a PCR instrument at 95 °C for 2 min, and then rapidly annealed at 4 °C for 10 min. The reaction solution was denoted as PCR-DNA.

[0077] (3) A glassy carbon electrode (GCE) with d = 3 mm was polished successively with 0.3 μm and 0.05 μm alumina powder, ultrasonically treated in ethanol and water for 30 s respectively, and then dried in air;

[0078] (4) Modify the surface of the GCE with a 5 μM gold nanoparticle (AuNPs) solution, with a dosage of 6 μL. The modified material is denoted as AuNPs / GCE;

[0079] (5) The dosage of the PCR-DNA solution synthesized and modified with thiol is 6 μL. It is fixed and modified on the sensing interface of AuNPs / GCE prepared in step (4) through Au-S covalent interaction, and incubated overnight (12 h) at 4 °C. At this time, the sensor is denoted as PCR-DNA / AuNPs / GCE;

[0080] (6) Modify the surface of the electrode PCR-DNA / AuNPs / GCE in step (5) with 6 μL of MCH with a concentration of 0.1 M, and incubate at room temperature for 40 min to remove the unbound active sites. The sensor at this time is denoted as MCH / PCR-DNA / AuNPs / GCE.

[0081] Example 2:

[0082] Performance analysis of simultaneous ratio electrochemical detection of Pb 2+ and Hg 2+ based on the orientation of aptamer at the sensing interface regulated by electric field:

[0083] Dual-ratio electrochemical aptasensor based on electric field regulation ( Figure 2 A) The surface is successively modified with mixed standard solutions containing different concentrations of Pb 2+ and Hg 2+ and placed in a 3 V electrostatic field for preconcentration for 30 min. After washing the electrode with PBS (pH = 7.4) solution, it is placed in a 2.5 V electrostatic field for detection. According to the detected current values and the corresponding concentrations, the performance curve of the sensor is finally obtained. The sensor is the working electrode, the saturated Ag / AgCl electrode is the reference electrode, and the platinum wire electrode is the counter electrode. The electrochemical signals are recorded and detected by an electrochemical workstation of model CHI 750E.

[0084] As the concentrations of Pb 2+ and Hg 2+ increase, the electrochemical signals of I Fc and I MB gradually decrease. The action of the electrostatic field keeps the I AQ signal unchanged, and linear relationships of the ratio electrochemical signals are obtained in the concentration ranges of 1×10 2+ to 1×10 -13 for Pb -9 and 1×10 2+ to 1×10 -12 to 1×10 -8 M for Hg AQ / IFc = 0.89lgC Hg 2+ + 14.11(R 2 = 0.991) and I AQ / I MB = 0.32lgC Pb 2+ + 5.04(R 2 = 0.991)( Figure 3 B).

[0085] It is proved that the electrochemical biosensor under the electrostatic field has a wide linear range and high sensitivity, and can realize the accurate analysis of Pb 2+ and Hg 2+ . The test is carried out in 0.1M PBS (pH = 7.4) buffer solution. The scanning voltage range is -0.8 ~ 0.8V, the amplitude is 0.025V, and the frequency is 25Hz.

[0086] Example 3

[0087] Simultaneous dual-ratio detection of Pb 2+ and Hg 2+ in the actual application scenario of agricultural production based on the electric field-regulated aptamer orientation at the sensing interface

[0088] Based on this linear relationship, the inventor collected paddy field irrigation water, paddy field soil samples and paddy field crayfish in the Nanjing Academy of Agricultural Sciences, Jiangsu Province, and extracted and analyzed Pb 2+ and Hg 2+ in the three actual samples respectively. Three dilutions were taken for each sample, and standard solutions of Pb 2+ and Hg 2+ (1×10 -10 , 5×10 -10 , 1×10 -9 M) were added to the dilutions to obtain the spiked solutions as sample solutions;

[0089] The sample solution was dropped on the surface of the prepared sensor (MCH / PCR-DNA / AuNPs / GCE) electrode, placed in a 2.5V electrostatic field, and the corresponding current value was obtained through electrochemical testing; substituting the current value into the standard curve constructed in Example 2, the concentrations of Pb 2+ and Hg 2+ in the sample can be known, and the detection of Pb 2+ and Hg 2+ in the unknown sample is realized.

[0090] The analysis results are shown in Table 1.

[0091] Table 1 Pb in paddy field irrigation water, paddy field soil and paddy field crayfish2+ and Hg 2+ Determination (n = 3)

[0092]

[0093] Note: "- indicates not detected".

[0094] Using a sensing method, different concentrations of Pb 2+ and Hg 2+ in the actually collected samples of paddy field irrigation water, paddy field soil and paddy field crayfish were analyzed:

[0095] Pb 2+ The recovery rates were respectively between 94.1% - 109%, 92.8% - 104%, 105% - 124%;

[0096] Hg 2+ The recovery rates were respectively between 93.4% - 116%, 108% - 124%, 109% - 135%;

[0097] Comparison with the results of the official inductively coupled plasma mass spectrometer shows that the proposed Pb 2+ 、Hg 2+ simultaneous ratio electrochemical detection sensor for Pb 2+ and Hg 2+ in the samples of actual agricultural application scenarios has relatively high analysis reliability.

[0098] Explanation: The above embodiments are only used to illustrate the present invention and do not limit the technical solutions described in the present invention; therefore, although this specification has described the present invention in detail with reference to the above respective embodiments, those of ordinary skill in the art should understand that the present invention can still be modified or equivalently replaced; and all technical solutions and their improvements that do not depart from the spirit and scope of the present invention should be covered within the scope of the claims of the present invention.

Claims

1. A simultaneous ratiometric electrochemical detection method for Pb 2+ and Hg 2+ based on electric-field-regulated aptamer orientation at the sensing interface It is characterized in that it includes the following steps: (1) Preparation of unlabeled electrochemical probe AQ-cDNA complex solution: (a) First, anthraquinone is dissolved in phosphate buffer solution, and after stirring, an AQ dispersion is obtained; then 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide solution are added to the AQ dispersion to activate the carboxylic acid group of anthraquinone, obtaining an AQ activation solution; (b) cDNA is dissolved in tris(hydroxymethyl)aminomethane hydrochloride buffer solution to obtain a cDNA solution; Then the AQ activation solution is mixed with the cDNA solution, and after magnetic stirring, an AQ-cDNA complex solution is obtained; (2) The ferrocene-labeled linear aptamer is denoted as Fc-apt, and the methylene blue-labeled linear aptamer is denoted as MB-apt; the Fc-apt solution is mixed with the MB-apt solution, and the mixed solution is denoted as the Fc-apt-MB-apt solution; then the AQ-cDNA complex solution in step (1) is mixed with the Fc-apt-MB-apt solution, and an amplification reaction is carried out by PCR. The reaction solution is denoted as PCR-DNA; (3) The glassy carbon electrode is polished successively with alumina powders of different particle sizes. After polishing, the glassy carbon electrode is ultrasonically treated in ethanol and water respectively and then dried in air to obtain a treated glassy carbon electrode; (4) The gold nanoparticle solution is modified on the sensing interface of the glassy carbon electrode treated in step (3). The modified material is denoted as AuNPs / GCE; (5) The PCR-DNA obtained in step (2) is modified on the sensing interface of the AuNPs / GCE prepared in step (4). After incubation and fixation, a dual-ratio electrochemical aptasensor based on Fc-apt-MB-apt is obtained, denoted as PCR-DNA / AuNPs / GCE; (6) The MCH solution is modified on the electrode surface of the PCR-DNA / AuNPs / GCE material prepared in step (5). After incubating at room temperature for a period of time, the obtained material is denoted as MCH / PCR-DNA / AuNPs / GCE; (7) Modify the surface of the MCH / PCR-DNA / AuNPs / GCE electrode prepared in step (6) with a mixed standard solution containing different concentrations of Pb 2+ and Hg 2+ and electro-driven preconcentrate Pb 2+ and Hg 2+ under different electrostatic field conditions. The electrochemical biosensor obtained after enrichment is denoted as Pb 2+ &Hg 2+ / MCH / PCR-DNA / AuNPs / GCE; (8) Construction of the standard curve: Using the sensor Pb 2+ &Hg 2+ / MCH / PCR-DNA / AuNPs / GCE as the working electrode, the saturated Ag / AgCl electrode as the reference electrode, and the platinum wire electrode as the counter electrode, place them in an electrostatic field for electrochemical detection to obtain the final electrochemical detection current value; according to the detected current value and the logarithm of the concentration of Pb 2+ and Hg 2+ in the corresponding mixed standard solution, construct the standard curves respectively; (9) Detection of Pb in the actual sample 2+ and Hg 2+ Detection: First, prepare the sample solution. Drop the sample solution onto the surface of the sensor MCH / PCR-DNA / AuNPs / GCE electrode prepared in step (6), and place it in an electrostatic field to obtain the corresponding current value through electrochemical testing. Then, substitute the current value into the standard curve constructed in step (8) to know the concentration of Pb 2+ and Hg 2+ in the actual sample, and achieve the detection of Pb 2+ and Hg 2+ in the actual sample.

2. The simultaneous ratio electrochemical detection method for Pb 2+ , Hg 2+ based on regulating the orientation of aptamer on the sensing interface by electric field according to claim 1 It is characterized in that In step (1)(a), the stirring time is 12 h; the concentration of the AQ dispersion is 0.1 mg / mL -1 , the concentrations of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide solutions are 10 mM and 5 mM respectively; the concentration of the phosphate buffer solution is 0.1 M; the volume ratio of the AQ dispersion, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide solutions is 2:1:1; in step (1)(b), the concentration of the cDNA solution is 2-16 μM; the volume ratio of the AQ activation solution to the cDNA solution is 1-4:1; the concentration of the tris(hydroxymethyl)aminomethane hydrochloride buffer solution is 0.1 M; the magnetic stirring time after mixing the AQ activation solution and cDNA is 2 h; the final concentration of cDNA in the AQ-cDNA complex solution is 1-8 μM.

3. The Pb 2+ , Hg 2+ simultaneous ratio electrochemical detection method according to claim 1, in step (2), the concentrations of both Fc-apt and MB-apt are 20 μM, and Fc-apt and MB-apt are mixed in a volume ratio of 1:1; the volume ratio of the AQ-cDNA composite solution to the Fc-apt-MB-apt solution is 1:1; The temperature condition for the PCR amplification reaction is 95 °C, the reaction time is 2 min, and then rapid annealing is carried out at 4 °C for 10 min.

4. The simultaneous ratio electrochemical detection method for Pb 2+ , Hg 2+ based on the electric field-regulated aptamer orientation of the sensing interface according to claim 1 It is characterized in that in step (3), the diameter d of the glassy carbon electrode is 3 mm; the particle sizes of the alumina powders used are 0.3 μm and 0.05 μm in sequence; the ultrasonic time is 30 s for both.

5. The simultaneous ratio electrochemical detection method for Pb 2+ , Hg 2+ based on regulating the orientation of aptamer on the sensing interface by electric field according to claim 1 It is characterized in that in step (4), the dosage of the gold nanoparticle solution modification is 6 μL, and the concentration is 5 μM.

6. The Pb 2+ , Hg 2+ simultaneous ratio electrochemical detection method according to claim 1 It is characterized in that In step (5), the dosage of the PCR-DNA modification is 6 μL and the concentration is 0.5 - 4.0 μM; the incubation temperature is 4 °C and the incubation time is 10 - 12 h.

7. The Pb 2+ , Hg 2+ simultaneous ratio electrochemical detection method based on regulating the orientation of aptamer on the sensing interface by electric field as claimed in claim 1 It is characterized in that In step (6), the dosage of the MCH solution is 6 μL and the concentration is 0.1 M; the incubation period is 40 - 60 min.

8. The simultaneous ratio electrochemical detection method of Pb 2+ , Hg 2+ based on the electric field-regulated aptamer orientation of the sensing interface according to claim 1 It is characterized in that In step (7), the concentration of Pb 2+ and Hg 2+ in the mixed standard solution is 1×10 -13 to 1×10 -8 M, and the modification dosage of the mixed standard solution containing different concentrations of Pb 2+ and Hg 2+ is 6 μL; The specific steps of electro-driven preconcentration under different electrostatic field conditions: preconcentration is carried out in an electrostatic field of 0 - 3 V for 10 - 60 min.

9. The simultaneous ratio electrochemical detection method of Pb 2+ , Hg 2+ based on the electric field-regulated aptamer orientation of the sensing interface according to claim 1 It is characterized in that In step (8), the specific conditions for electrochemical detection are: testing is carried out in a PBS buffer solution of 0.1 M and pH = 7.4; the scanning voltage range is -0.4 - 0.8 V, the amplitude is 0.025 V, and the frequency is 25 Hz; the condition of the electrostatic field is 0 - 3 V.

10. The simultaneous ratio electrochemical detection method for Pb 2+ , Hg 2+ based on regulating the orientation of aptamer on the sensing interface by electric field according to claim 1 It is characterized in that In step (9), the specific conditions for electrochemical testing are: testing is carried out in a PBS buffer solution of 0.1 M and pH = 7.4; the scanning voltage range is -0.4 - 0.8 V, the amplitude is 0.025 V, and the frequency is 25 Hz; the condition of the electrostatic field is 0 - 3 V.

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