WO2021093530A1 - 铅离子核酸适配体及电化学传感器与制备方法 - Google Patents

铅离子核酸适配体及电化学传感器与制备方法 Download PDF

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WO2021093530A1
WO2021093530A1 PCT/CN2020/122505 CN2020122505W WO2021093530A1 WO 2021093530 A1 WO2021093530 A1 WO 2021093530A1 CN 2020122505 W CN2020122505 W CN 2020122505W WO 2021093530 A1 WO2021093530 A1 WO 2021093530A1
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lead ion
nucleic acid
solution
lead
electrochemical sensor
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白卫滨
冉国敬
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暨南大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/4163—Systems checking the operation of, or calibrating, the measuring apparatus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • the invention belongs to the technical field of detection, and relates to a lead ion nucleic acid aptamer, an electrochemical sensor based on the lead ion nucleic acid aptamer, and a corresponding preparation method and application.
  • Lead is a common heavy metal in the earth's crust. Due to the long-term large-scale mining and use of lead and the random disposal and discharge of lead-containing waste, the problem of lead pollution in the environment is becoming more and more serious. As a non-degradable heavy metal pollutant, lead can be combined with organisms to accumulate in different forms, and then enter the human body through the enrichment of the food chain and affect the liver, kidneys, bones, nervous system, cardiovascular system, and reproductive system. The internal organs and systems cause irreversible damage. Therefore, analysis and detection of lead content in the environment and food is of great significance to environmental protection and human health.
  • lead ions detection methods not only mainstream large-scale instrument detection methods such as atomic emission spectrometry, inductive coupling Plasma mass spectrometry also has rapid detection methods such as immunoassay, electrochemical anodic stripping voltammetry, and biosensor method.
  • large-scale instrument detection methods such as inductively coupled plasma mass spectrometry are very accurate, they also have disadvantages such as expensive instruments and equipment, more infrastructure, and the need for professional operations.
  • biosensors that use nucleic acid aptamers as target substance recognition molecules and electrochemical methods as detection means not only have good specificity, high sensitivity, low detection limit, fast analysis speed, and simple operation , Easy to miniaturize and portability. But so far, there has been no report on the method of detecting lead ions using electrochemical sensors based on specific nucleic acid aptamers.
  • the first aspect of the present invention provides a lead ion nucleic acid aptamer, which has the characteristics of high affinity, strong specificity, good stability, easy modification and the like.
  • the lead ion nucleic acid aptamer of the present invention is single-stranded DNA (ssDNA) with a length of 33-35 nt, and contains the nucleic acid sequence CGGATCGGTATGGATGGTATT.
  • sequence of the aptamer is shown in SEQ ID NO: 1-11.
  • the 5'end of the aptamer is modified with a polyadenine (Poly A) assembly group -AAAAA, that is, the modified nucleic acid sequence is:
  • the second aspect of the present invention provides a lead ion electrochemical sensor based on nucleic acid aptamer.
  • the sensor uses lead ion specific nucleic acid aptamer as the recognition molecule, which has good specificity, high sensitivity and good stability. And reproducibility.
  • a lead ion electrochemical sensor based on a nucleic acid aptamer and a matching detection method uses a lead ion-specific nucleic acid aptamer as a recognition molecule and a screen-printed electrode as a carrier.
  • the method is rapid in detection and has the prospect of real-time rapid detection on site.
  • the lead ion electrochemical sensor based on nucleic acid aptamer of the present invention contains the lead ion nucleic acid aptamer.
  • the electrode used in the lead ion electrochemical sensor is a screen printed electrode, wherein the working electrode is a gold electrode with a diameter of 4 mm.
  • the third aspect of the present invention provides a method for preparing the lead ion electrochemical sensor.
  • Electric cleaning pretreatment of screen-printed gold electrode connect the screen-printed gold electrode sheet to the electrochemical workstation, and then immerse one end of the working electrode of the electrode sheet in a sulfuric acid solution, and use cyclic voltammetry (Cyclic Voltammetry, CV) to counter the electrode
  • the film is pre-processed by electrical cleaning and scanning, and it is continuously scanned for 4-6 times until the scanning current curve is basically stable, then rinsed with ultrapure water, and left to dry for later use, to obtain the pretreated screen-printed gold electrode, wherein the sulfuric acid solution It is a sulfuric acid solution with a concentration of 0.5M;
  • nucleic acid aptamer solution The dry powder of nucleic acid aptamer is first dissolved into a mother solution with a concentration of 100 ⁇ M in the nucleic acid aptamer assembly solution and stored in the refrigerator at -20°C, and then diluted with the system buffer solution to the required concentration before use .
  • the nucleic acid aptamer assembly solution is a 1M NaCl solution
  • the system buffer solution is a 20mM Tris-HCl solution with a pH of 7.4;
  • Electrode assembly and construction Drop 7-9 ⁇ L of a certain concentration of nucleic acid aptamer solution on the surface of the screen-printed gold electrode to ensure that the droplet can maximize the coverage of the working electrode without spreading, and then put the electrode sheet into the plug. Assemble in a centrifuge tube of wet cotton and in a refrigerator at 4°C overnight;
  • the fourth aspect of the present invention provides the application of the lead ion electrochemical sensor in detecting lead ions.
  • the lead ion detection method based on the lead ion electrochemical sensor of the present invention includes the following steps:
  • the assembled nucleic acid aptamer electrochemical sensor is sequentially incubated to detect a gradient concentration of lead ion solution, and ultrapure water is slowly used after the incubation.
  • the gradient concentrations are 0.1ng/ mL, 0.5ng/mL, 1.0ng/mL, 5.0ng/mL, 10.0ng/mL, 50.0ng/mL, 100.0ng/mL, 500.0ng/mL and 1000.0ng/mL;
  • the linear working equation is established with the logarithmic value of the lead ion solution as horizontal
  • the nucleic acid aptamer assembly concentration of the lead ion electrochemical sensor is 10 ⁇ M
  • the assembly time is 24h
  • the lead ion incubation time is 15min
  • the incubation temperature is 30°C.
  • the present invention Compared with the existing lead ion detection method, the present invention has the following beneficial effects:
  • the present invention uses lead ion-specific nucleic acid aptamers for identification and detection.
  • Nucleic acid aptamers have the advantages of high affinity, good specificity, low synthesis cost, stable properties, easy modification, etc., and are more suitable for heavy metal ions Detection.
  • the lead ion-specific nucleic acid aptamer used in the present invention is modified with an -AAAAA assembly group at the 5'end, and the assembly group can complete assembly and sealing on the electrode surface at one time, and the assembly process is further simplified;
  • the nucleic acid aptamer electrochemical sensor detection method for lead ions established in the present invention uses electrochemical methods for characterization and detection, which not only has a wide detection range, high sensitivity, and good reproducibility, and the detection limit is as low as 0.03ng/mL, And the detection is very fast, DPV scanning analysis only needs 2min, can realize the electrical signal response to the lead ion within 2min, and has a higher matching degree with the real-time rapid detection scene on the spot;
  • the nucleic acid aptamer electrochemical sensor method for detecting lead ions established in the present invention has convenient operation, simple steps, no labeling, and no need to use a complicated signal expansion method to achieve a strong electrical signal value.
  • Both the nucleic acid aptamer and the screen-printed electrode used in the method are low in cost and easy to carry, which can provide a new lead ion analysis and detection method for the field of on-site real-time rapid detection including food safety.
  • Figure 1 shows the CV scanning current (Figure A), DPV scanning current ( Figure B) and EIS scanning characteristic electrical signal (Figure C) of the lead ion nucleic acid aptamer electrochemical sensor of the present invention before and after assembly; (a) is the bare gold electrode before assembly, (b) is the nucleic acid aptamer after assembly.
  • Figure 2 is the CV scanning current ( Figure A), DPV scanning current ( Figure B) and DPV peak current intensity value I p ( Figure B) after the lead ion nucleic acid aptamer electrochemical sensor of the present invention detects a gradient concentration of Pb 2+ C); in the figure, a ⁇ j: after assembly, 0.1ng/mL, 0.5ng/mL, 1ng/mL, 5ng/mL, 10ng/mL, 50ng/mL, 100ng/mL, 500ng/mL and 1000ng/ mL.
  • Figure 3 is a linear working equation curve of a lead ion detection method based on a nucleic acid aptamer electrochemical sensor.
  • Figure 4 shows the (A) DPV scanning current and (B) specificity evaluation results after the nucleic acid aptamer electrochemical sensor is incubated to detect each ion; in the figure, a ⁇ l: after assembly, aluminum ion (Al 3+ ), Silver ion (Ag + ), lead ion (Cd 2+ ), calcium ion (Ca 2+ ), copper ion (Cu 2+ ), cobalt ion (Co 2+ ), chromium ion (Cr 3+ ), manganese ion ( Mn 2+ ), barium ions (Ba 2+ ), mixed ions and lead ions.
  • Al 3+ aluminum ion
  • lead ion (Cd 2+ ) silver ion (Ag + ), lead ion (Cd 2+ ), calcium ion (Ca 2+ ), copper ion (Cu 2+ ), cobalt ion (Co 2+ ),
  • the nucleic acid aptamer sequence used in the following examples was synthesized by Shanghai Shenggong Bioengineering Co., Ltd. and purified by HPLC.
  • the screen-printed gold electrode used was Metrohm DropSens C220BT electrode, and the electrochemical workstation used It is a Shanghai Chenhua CHI 660E electrochemical workstation.
  • the electrolyte used is 5mM K 3 [Fe(CN) 6 ] solution, containing 100mM KCl (electrolyte I) and 5mM K 3 [Fe(CN) 6 ]/5mM K 4 [Fe(CN) 6 ] mixed solution (molar ratio 1:1), containing 100 mM KCl (electrolyte II), the lead ion solution used is lead nitrate Pb(NO 3 ) 2 dissolved in 20 mM Tris-HCl at pH 7.4 The mother liquor and gradient dilutions formed after the system buffer.
  • Example 1 Construction of lead ion electrochemical sensor based on nucleic acid aptamer
  • (1) Electric cleaning pretreatment of the screen-printed gold electrode connect the screen-printed gold electrode sheet to the electrochemical workstation, and then immerse one end of the working electrode of the electrode sheet in a sulfuric acid solution with a concentration of 0.5M, and use CV to electrically clean the electrode sheet.
  • Clean scan pre-processing, CV scan parameters are scan voltage range -0.3V-1.3V, scan speed 0.1V/s, scan segment number 60, continuous scan 4 times, 16 cycles in total, until the scan current curve is basically stable, rinse with ultrapure water, Leave it to dry for later use, and obtain the pretreated screen-printed gold electrode;
  • nucleic acid aptamer solution preparation the dry powder of nucleic acid aptamer A1 (SEQ ID NO:1) is first dissolved in a 1M NaCl solution to a concentration of 100 ⁇ M and then placed in a refrigerator at -20°C Save it and dilute it to 5 ⁇ M with 20mM Tris-HCl system buffer of pH 7.4 before use;
  • Electrode assembly and construction drop 7 ⁇ L of 5 ⁇ M nucleic acid aptamer A1 solution on the surface of the screen-printed gold electrode to ensure that the droplets can maximize the coverage of the working electrode without spreading, and then put the electrode sheet into the plug Assemble in a centrifuge tube with wet cotton and in a refrigerator at 4°C overnight;
  • the electrode sheet is first cleaned with ultrapure water, and then allowed to stand to dry and then scan CV, DPV and EIS in electrolyte I and II.
  • the CV scan parameter is set to : Scanning voltage range -0.3V-0.7V, scanning speed 0.1V/s, scanning interval 1mV;
  • DPV scanning characterization parameter setting scanning voltage range -0.3V-0.7V, potential increment 4mV, pulse amplitude 50mV, pulse width 0.05s, pulse period 0.5s;
  • EIS scan parameters characterized as follows: initial voltage of 0.22V, the frequency of the high frequency section 10 5 Hz, 1Hz low frequency sections, a pulse width of 5mV, the characterization results shown in Figure 1, the electrical verified The chemical sensor was assembled successfully.
  • Example 2 Establishment of a method for detecting lead ions with a nucleic acid aptamer electrochemical sensor
  • the nucleic acid aptamer electrochemical sensor constructed by the detection method has a nucleic acid aptamer assembly concentration of 10 ⁇ M, an assembly time of 24h, a lead ion incubation time of 15min, and an incubation temperature of 30°C.
  • Example 3 Evaluation of specificity of nucleic acid aptamer electrochemical sensor
  • Select lead ion (Cd 2+ ), calcium ion (Ca 2+ ), manganese ion (Mn 2+ ), cobalt ion (Co 2+ ), aluminum ion (Al 3+ ), chromium ion (Cr 3+ ), copper Ions (Cu 2+ ), barium ions (Ba 2+ ) and silver ions (Ag + ) are 9 kinds of ions as interfering ions. Under the optimal detection conditions described in Example 2, the sensor is used to measure the concentration of 500ng/ mL of interfering ion solution and its mixed ion solution were incubated for detection.
  • Example 4 Quantitative detection of lead ions in actual spiked samples
  • the solution to be analyzed is diluted 100 times with ultrapure water, and then mixed with a 20mM Tris-HCl system buffer of pH 7.4 at a volume ratio of 1:1, shaken and mixed well for use; the pretreatment method for river water and tap water samples is 0.22 ⁇ m Filter the membrane, then use ICP-MS to detect whether it contains lead ions, and then take 1mL of the solution to be analyzed, mix it with a 20mM Tris-HCl system buffer of pH 7.4 at a volume ratio of 1:1, shake and mix well and set aside.
  • the standard addition method was used to add lead ion solution to each sample to make the lead ion concentration of 1ng/mL, 5ng/mL and 50ng/mL respectively.
  • the sensor was used to detect the three samples. According to the linear working equation established in Example 2, the actual detection concentration, detection recovery rate and relative standard deviation of the corresponding lead ions are calculated through the linear working equation established in Example 2, and the quantitative detection of lead ions in the above three spiked samples is completed. And use ICP-MS to compare the samples.
  • the test results are shown in Table 1. The test results of the three samples are consistent with the ICP-MS test results.
  • the spiked recovery of the tested samples is between 92.9% and 109.0%, and the relative standard deviation is between 0.78% and 3.29%. between.
  • Example 5 Construction of different nucleic acid aptamer sequences and testing of the detection effect of electrochemical sensors.
  • lead ions based on the original nucleic acid aptamer sequence A1 (SEQ ID NO:1) were constructed Nucleic acid aptamer electrochemical sensor has verified its detection effect on lead ions.

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Abstract

提供了一种铅离子核酸适配体，以及基于该铅离子核酸适配体的电化学传感器，以及相应的制备方法与应用。提供的铅离子核酸适配体为长度33-35nt的单链DNA，且含有核酸序列CGGATCGGTATGGATGGTATT。为包括食品安全在内的现场实时快速检测领域提供了一种新的铅离子检测方法。

Description

铅离子核酸适配体及电化学传感器与制备方法 技术领域
本发明属于检测技术领域，涉及一种铅离子核酸适配体，以及基于该铅离子核酸适配体的电化学传感器，以及相应的制备方法与应用。
背景技术
铅是一种地壳中常见的重金属，由于铅的长期大量开采使用和含铅废弃物的随意丢弃排放，环境中的铅污染问题越来越严重。铅作为一种不可降解的重金属污染物，可以与生物体以不同形式结合蓄积，然后通过食物链富集作用进入到人体当中并对包括肝脏、肾脏、骨骼、神经系统、心血管系统和生殖系统在内的各个器官和系统造成非可逆的损害。因此，分析和检测环境及食品中的铅含量对于环境保护和人体健康都具有重要意义。
环境和生物体中的铅多以铅离子(Lead ions，Pb 2+)及其化合物的形式存在，铅离子的检测方法种类繁多,不仅有主流的大型仪器检测法如原子发射光谱法、电感耦合等离子体质谱法，也有快速检测方法如免疫法、电化学阳极溶出伏安法以及生物传感器法等。电感耦合等离子体质谱法等大型仪器检测法尽管检测十分精准，但是也存在诸如仪器设备昂贵、基础设施较多以及需要专业人员操作等缺点，很难在现场快速检测领域中得到实际使用；而在快速检测方法当中，免疫法和电化学阳极溶出伏安法等在灵敏度、特异性以及检测限上都或多或少存在有一定的不足，所以目前也没有得到较好的发展。与以上方法相比，生物传感器法由于形式多样、特性优良、检测迅速以及成本低廉等特点获得了广泛关注和极大发展，是目前的一个研究热点。而在所有类型的生物传感器当中，以核酸适配体作为靶标物质识别分子，以电化学方式作为检测手段的生物传感器，不仅特异性好、灵敏度高、检测限低、分析速度快，而且操作简便、易于微型化和便携化。但到目前为止，使用基于特异性核酸适配体的电化学传感器检测铅离子的方法还未见报道。
发明内容
本发明的第一个方面，提供了一种铅离子核酸适配体，该适配体具有亲和力高、特异性强、稳定性好、易于修饰等特点。
本发明所述的铅离子核酸适配体为长度33-35nt的单链DNA(single-stranded DNA，ssDNA)，且含有核酸序列CGGATCGGTATGGATGGTATT。
优选地，所述适配体的序列如SEQ ID NO:1-11所示。
进一步地，所述的适配体在其5’端修饰有多聚腺嘌呤核苷酸(Poly adenine,Poly A)组装基团-AAAAA，即修饰后的核酸序列为：
AAAAAGACGACCGGATCGGTATGGATGGTATTGTCGTC(5’-3’)。
本发明的第二个方面，提供了一种基于核酸适配体的铅离子电化学传感器，该传感器以铅离子特异性核酸适配体为识别分子，特异性好，灵敏度高，有良好稳定性和重现性。
基于核酸适配体的铅离子电化学传感器以及配套检测方法，传感器以铅离子特异性核酸适配体为识别分子，以丝网印刷电极为载体，方法检测迅速，具有现场实时快速检测应用前景。
本发明所述的基于核酸适配体的铅离子电化学传感器含有所述的铅离子核酸适配体。
优选地，所述铅离子电化学传感器中所用的电极为丝网印刷电极，其中工作电极是直径4mm的金电极。
本发明的第三个方面提供了所述的铅离子电化学传感器的制备方法。
本发明所述的铅离子电化学传感器的制备方法，其特征在于，包括以下步骤：
S1.丝网印刷金电极电清洁预处理：将丝网印刷金电极片连接至电化学工作站，然后使电极片工作电极一端浸入硫酸溶液中，采用循环伏安法(Cyclic Voltammetry，CV)对电极片进行电清洁扫描预处理，连续扫描4-6次直至扫描电流曲线基本稳定后使用超纯水冲洗，静置晾干备用，即得到预处理后的丝网印刷金电极，其中所述硫酸溶液为浓度为0.5M的硫酸溶液；
S2.核酸适配体溶液配制：核酸适配体干粉首先用核酸适配体组装液溶解成浓度为100μM的母液后于-20℃冰箱中保存，使用前再用体系缓冲溶液稀释到所需浓度。其中核酸适配体组装液为1M NaCl溶液，体系缓冲溶液为pH 7.4的20mM Tris-HCl溶液；
S3.电极组装构建：滴加7-9μL一定浓度的核酸适配体溶液于丝网印刷金电极表面，确保液滴能够最大化地覆盖工作电极而不散开，然后将电极片放入塞有湿棉花的离心管中并于4℃冰箱中过夜组装；
S4.传感器电化学特性表征：电化学传感器完成组装后首先用超纯水清洗电极片，静置晾干后进行CV、差分脉冲伏安法(Differential Pulse Voltammetry，DPV)和交流阻抗法(Electrochemical Impedance Spectroscopy，EIS)扫描，以验证电化学传感器组装成功。其中CV和DPV扫描所使用的电解液为5mM K 3[Fe(CN) 6]，含浓度为100mMKCl的溶液；EIS扫描所使用的电解液为摩尔比1:1的5mM K 3[Fe(CN) 6]/5mM K 4[Fe(CN) 6]，含浓度为100mMKCl的溶液。
本发明的第四个方面提供了根据所述的铅离子电化学传感器在检测铅离子方面的应用。
本发明所述的基于铅离子电化学传感器的铅离子检测方法，包括以下步骤：
S1.采用如权利要求4所述的铅离子电化学传感器对梯度浓度硝酸铅溶液进行检测，建立线性工作方程；
S2.将样品前处理后使用标准加标法对样品进行加标，加标后使用所述的铅离子电化学传感器检测样品中的铅离子，通过所建立的线性工作方程，计算相应的铅离子实际检出浓度，完成对加标样品中铅离子的定量检测。
根据本发明所述的铅离子检测方法的进一步特征，所述步骤S1中，将完成组装后的核酸适配体电化学传感器依次孵育检测梯度浓度的铅离子溶液，孵育完成后用超纯水缓慢冲洗并晾干，然后进行CV和DPV扫描分析，依次记录孵育检测各浓度铅离子溶液后的相关电流信号数据，分析数据及两者关系后建立线性工作方程；所述梯度浓度分别为0.1ng/mL、0.5ng/mL、1.0ng/mL、5.0ng/mL、10.0ng/mL、50.0ng/mL、100.0ng/mL、500.0ng/mL和1000.0ng/mL；所述电流信号数据是指DPV扫描峰电流强度值(Peak current intensity,I p)，每个浓度铅离子溶液依次对应I pn，其中n＝1,2……9；建立的线性工作方程以铅离子溶液的浓度对数值为横坐标，相应的DPV扫描峰电流强度变化值ΔI pn值为纵坐标(n＝1,2…9)，工作方程具体为ΔI p(μA)＝9.645*logC Pb2+(ng/mL)+13.36，其中C Pb2+代表铅离子浓度，方法检测限达到0.03ng/mL(S/N＝3)。
根据本发明所述的基于铅离子电化学传感器的铅离子检测方法的进一步特 征，所述铅离子电化学传感器的核酸适配体组装浓度为10μM，组装时间为24h，铅离子的孵育时间为15min，孵育温度为30℃。
与现有的铅离子检测方法相比，本发明具有以下有益效果：
(1)本发明使用铅离子特异性核酸适配体对其进行识别检测，核酸适配体具有亲和力高、特异性好、合成成本低、性质稳定、易于修饰等优点，更适合用于重金属离子的检测。
(2)本发明使用的铅离子特异性核酸适配体在5’端修饰有-AAAAA组装基团，该组装基团可以在电极表面一次性完成组装和封闭，组装流程进一步简化；
(3)本发明中建立的核酸适配体电化学传感器检测铅离子方法，使用电化学方式进行表征检测，不仅检测范围广、灵敏度高、重现性好，检测限低至0.03ng/mL，而且检测非常迅速，DPV扫描分析仅需2min，可在2min时间内对铅离子实现电信号响应，与现场实时快速检测场景匹配度更高；
(4)本发明中建立的核酸适配体电化学传感器检测铅离子方法，操作方便、步骤简洁、无需标记，无需采用复杂的信号扩大方式即可达到较强的电信号值，除此以外，方法所使用的核酸适配体和丝网印刷电极片均成本较低、便于携带，可为包括食品安全在内的现场实时快速检测领域提供一种新的铅离子分析检测方法。
附图说明
图1为本发明所述的铅离子核酸适配体电化学传感器在组装前后的CV扫描电流(图A)、DPV扫描电流(图B)和EIS扫描表征电信号(图C)；图中，(a)为组装前的裸金电极，(b)为组装后的核酸适配体。
图2为本发明所述的铅离子核酸适配体电化学传感器检测梯度浓度Pb 2+后的CV扫描电流(图A)、DPV扫描电流(图B)以及DPV峰电流强度值I p(图C)；图中，a→j：组装完成后、0.1ng/mL、0.5ng/mL、1ng/mL、5ng/mL、10ng/mL、50ng/mL、100ng/mL、500ng/mL和1000ng/mL。
图3为基于核酸适配体电化学传感器的铅离子检测方法的线性工作方程曲线。
图4为核酸适配体电化学传感器孵育检测各离子后的(A)DPV扫描电流和(B)特异性评价结果；图中，a→l：组装完成后、铝离子(Al 3+)、银离子(Ag +)、铅离子(Cd 2+)、钙离子(Ca 2+)、铜离子(Cu 2+)、钴离子(Co 2+)、铬离子(Cr 3+)、锰离子(Mn 2+)、 钡离子(Ba 2+)、混合离子和铅离子。
具体实施方式
下面结合实施例对本发明作进一步的阐述，但并不对本发明的范围进行限制。
以下实施例中所使用的核酸适配体序列由上海生工生物工程有限公司合成，且经HPLC纯化，所使用的丝网印刷金电极为瑞士万通公司DropSensC220BT型电极，所使用的电化学工作站为上海辰华CHI 660E型电化学工作站，所使用的电解液分别为5mM K 3[Fe(CN) 6]溶液，含100mMKCl(电解液I)和5mM K 3[Fe(CN) 6]/5mM K 4[Fe(CN) 6]混合溶液(摩尔比1:1)，含100mMKCl(电解液II)，所使用的铅离子溶液为硝酸铅Pb(NO 3) 2溶于pH 7.4的20mMTris-HCl体系缓冲液后形成的母液及梯度稀释液。
实施例1：基于核酸适配体的铅离子电化学传感器构建
(1)丝网印刷金电极电清洁预处理：将丝网印刷金电极片连接至电化学工作站，然后将电极片工作电极一端浸入浓度为0.5M的硫酸溶液中，采用CV对电极片进行电清洁扫描预处理，CV扫描参数为扫描电压范围-0.3V-1.3V，扫描速度0.1V/s，扫描段数60，连续扫描4次共16圈至扫描电流曲线基本稳定后使用超纯水冲洗，静置晾干备用，得到预处理后的丝网印刷金电极；
(2)核酸适配体溶液配制：采用核酸适配体A1(序列号：SEQ ID NO:1)的干粉先用浓度为1M的NaCl溶液溶解成浓度为100μM的母液后于-20℃冰箱中保存，使用前再用pH 7.4的20mM Tris-HCl体系缓冲液稀释到5μM；
(3)电极组装构建：滴加7μL浓度为5μM的核酸适配体A1溶液于丝网印刷金电极表面，确保液滴能够最大化地覆盖工作电极而不散开，然后将电极片放入塞有湿棉花的离心管中并于4℃冰箱中过夜组装；
(4)传感器电化学特性表征：电化学传感器完成组装后首先用超纯水清洗电极片，静置晾干后依次在电解液I和II中进行CV、DPV和EIS扫描，CV扫描参数设置为：扫描电压范围-0.3V-0.7V，扫描速度0.1V/s，扫描间隔1mV；DPV扫描表征参数设置为：扫描电压范围-0.3V-0.7V，电位增量4mV，脉冲幅度50mV，脉冲宽度0.05s，脉冲周期0.5s；EIS扫描表征参数设置为：初始电压0.22V，高频区段频率10 5Hz，低频区段频率1Hz，脉冲宽度5mV，表征结果如图1所示，验证了电化学传感器组装成功。
实施例2：核酸适配体电化学传感器检测铅离子方法的建立
用于检测方法构建的核酸适配体电化学传感器的核酸适配体组装浓度为10μM，组装时间为24h，铅离子的孵育时间为15min，孵育温度为30℃。在该条件下，将所述的核酸适配体电化学传感器依次孵育检测梯度浓度为0.1ng/mL、0.5ng/mL、1.0ng/mL、5.0ng/mL、10.0ng/mL、50.0ng/mL、100.0ng/mL、500.0ng/mL和1000.0ng/mL的铅离子溶液，孵育完成后用超纯水缓慢冲洗并晾干，然后进行CV和DPV扫描分析，依次记录孵育检测各浓度铅离子溶液后的DPV扫描峰电流强度值I pn(n＝1,2…9)，结果如图2所示；然后减去组装后初始的DPV扫描峰电流强度值I 0得到DPV扫描峰电流强度变化值ΔI pn(n＝1,2…9)，以铅离子浓度的对数为横坐标，ΔI p值为纵坐标，建立的线性工作方程为ΔI p(μA)＝9.645*logC Pb2+(ng/mL)+13.36，其中C Pb2+代表铅离子浓度，方法的线性工作方程如图3所示，其线性关系良好，R 2为0.9931，检测限达到0.03ng/mL(S/N＝3)，完成了检测方法的建立。
实施例3：核酸适配体电化学传感器特异性评价
选取铅离子(Cd 2+)、钙离子(Ca 2+)、锰离子(Mn 2+)、钴离子(Co 2+)、铝离子(Al 3+)、铬离子(Cr 3+)、铜离子(Cu 2+)、钡离子(Ba 2+)和银离子(Ag +)共9种离子作为干扰离子，在实施例2中所述的最佳检测条件下使用传感器分别对浓度为500ng/mL的干扰离子溶液及其混合离子溶液进行孵育检测。记录传感器孵育不同干扰离子后的DPV扫描I p值，计算其对应变化值ΔI p并与铅离子浓度为5ng/mL时的ΔI p值进行对比，结果如图4所示。从中可以发现，当铅离子浓度仅为5ng/mL时传感器即有明显的电信号响应，而当干扰离子浓度为铅离子浓度的100倍时传感器没有显示出明显的电信号响应，说明了传感器不会对其它重金属离子产生特异反应，传感器特异性较好，方法选择性高。
实施例4：实际加标样品中铅离子的定量检测
实施例中选取鱼肉样品、江水样品和自来水样品共3种样品进行检测，其中鱼肉样品的前处理方式为微波消解，消解定容后先用ICP-MS检测溶液中是否含有铅离子，然后取1mL待分析溶液用超纯水稀释100倍，再以体积比1:1与pH 7.4的20mM Tris-HCl体系缓冲液进行混合，震荡混匀之后备用；江水和自来水样品的前处理方式为过0.22μm滤膜，然后用ICP-MS检测是否含有铅离子，再 取1mL待分析溶液，以体积比1:1与pH 7.4的20mM Tris-HCl体系缓冲液进行混合，震荡混匀之后备用。
在完成制样后，采用标准加标法分别向各样品中加入铅离子溶液使得铅离子加标浓度分别为1ng/mL、5ng/mL和50ng/mL，加标后使用传感器分别检测3种样品中的铅离子，通过实施例二中所建立的线性工作方程，计算相应的铅离子实际检出浓度以及检测回收率、相对标准偏差，完成对上述3种加标样品中铅离子的定量检测，并使用ICP-MS对样品进行对比检测。检测结果如表1所示，3种样品的检测结果与ICP-MS检测结果维持一致，所测样品的加标回收率在92.9％-109.0％之间，相对标准偏差在0.78％-3.29％之间。
表1.实际加标样品检测结果
Figure PCTCN2020122505-appb-000001
实施例5：基于不同核酸适配体序列构建并检验电化学传感器的检测效果通过上述实施例1-4构建了基于原始核酸适配体序列A1(序列号：SEQ ID NO:1)的铅离子核酸适配体电化学传感器并验证了其对铅离子的检测效果。
进一步通过电化学实验检测结果表明，核酸适配体序列A1中间的CGGATCGGTATGGATGGTATT为核心序列，其两端可以与任意相互配对的核苷酸进行搭配形成如A2-A11(序列号：SEQ ID NO:2-11)所示的核酸适配体序列，在符合长度为33-35nt且二级结构为固定单一stem-loop的情况下均能对铅离子达到较好的检测灵敏度，具体检测结果如表2所示。
表2.铅离子核酸适配体及其检测灵敏度
Figure PCTCN2020122505-appb-000002
尽管已在上文的实施例中对本发明作出了一般性说明和具体详细描述，但并不用于限定本发明。对于熟悉本技术领域的人员而言，凡是在本发明原理和技术方案基础上对其进行的修改、改进、组合和替代，均属于本发明要求的保护范围内。

Claims (10)

  1. 一种铅离子核酸适配体，其特征在于：所述适配体为长度33-35nt的单链DNA(ssDNA)，且含有核酸序列CGGATCGGTATGGATGGTATT。
  2. 根据权利要求1所述的铅离子核酸适配体，其特征在于：所述适配体的序列如SEQ ID NO:1-11所示。
  3. 根据权利要求1所述的铅离子核酸适配体，其特征在于：所述的适配体在其5’端修饰有多聚腺嘌呤核苷酸(Poly adenine,Poly A)组装基团-AAAAA。
  4. 一种基于核酸适配体的铅离子电化学传感器，其特征在于：所述铅离子电化学传感器含有如权利要求1至3之一所述的铅离子核酸适配体。
  5. 根据权利要求4所述的铅离子电化学传感器，其特征在于：所述铅离子电化学传感器中所用的电极为丝网印刷电极，其中工作电极是直径4mm的金电极。
  6. 如权利要求5所述的铅离子电化学传感器的制备方法，其特征在于，包括以下步骤：
    S1.丝网印刷金电极电清洁预处理：将丝网印刷金电极片连接至电化学工作站，然后使电极片工作电极一端浸入硫酸溶液中，采用循环伏安法(Cyclic Voltammetry，CV)对电极片进行电清洁扫描预处理，连续扫描4-6次直至扫描电流曲线基本稳定后使用超纯水冲洗，静置晾干备用，即得到预处理后的丝网印刷金电极，其中所述硫酸溶液为浓度为0.5M的硫酸溶液；
    S2.核酸适配体溶液配制：核酸适配体干粉首先用核酸适配体组装液溶解成浓度为100μM的母液后于-20℃冰箱中保存，使用前再用体系缓冲溶液稀释到所需浓度；其中核酸适配体组装液为1M NaCl溶液，体系缓冲溶液为pH 7.4的20mM Tris-HCl溶液；
    S3.电极组装构建：滴加7-9μL一定浓度的核酸适配体溶液于丝网印刷金电极表面，确保液滴能够最大化地覆盖工作电极而不散开，然后将电极片放入塞有湿棉花的离心管中并于4℃冰箱中过夜组装；
    S4.传感器电化学特性表征：电化学传感器完成组装后首先用超纯水清洗电极片，静置晾干后进行CV、差分脉冲伏安法(Differential Pulse Voltammetry，DPV)和交流阻抗法(Electrochemical Impedance Spectroscopy，EIS)扫描，以验证电 化学传感器组装成功。其中CV和DPV扫描所使用的电解液为5mM K 3[Fe(CN) 6]，含浓度为100mMKCl的溶液；EIS扫描所使用的电解液为摩尔比1:1的5mM K 3[Fe(CN) 6]/5mM K 4[Fe(CN) 6]，含浓度为100mMKCl的溶液。
  7. 根据权利要求4所述的铅离子电化学传感器在检测铅离子方面的应用。
  8. 一种基于铅离子电化学传感器的铅离子检测方法，其特征在于，包括以下步骤：
    S1.采用如权利要求4所述的铅离子电化学传感器对梯度浓度硝酸铅溶液进行检测，建立线性工作方程；
    S2.将样品前处理后使用标准加标法对样品进行加标，加标后使用所述的铅离子电化学传感器检测样品中的铅离子，通过所建立的线性工作方程，计算相应的铅离子实际检出浓度，完成对加标样品中铅离子的定量检测。
  9. 根据权利要求8所述的铅离子检测方法，其特征在于：所述步骤S1中，将完成组装后的核酸适配体电化学传感器依次孵育检测梯度浓度的铅离子溶液，孵育完成后用超纯水缓慢冲洗并晾干，然后进行CV和DPV扫描分析，依次记录孵育检测各浓度铅离子溶液后的相关电流信号数据，分析数据及两者关系后建立线性工作方程；
    所述梯度浓度分别为0.1ng/mL、0.5ng/mL、1.0ng/mL、5.0ng/mL、10.0ng/mL、50.0ng/mL、100.0ng/mL、500.0ng/mL和1000.0ng/mL；
    所述电流信号数据是指DPV扫描峰电流强度值(Peak current intensity,I p)，每个浓度铅离子溶液依次对应I pn，其中n＝1,2……9；
    建立的线性工作方程以铅离子溶液的浓度对数值为横坐标，相应的DPV扫描峰电流强度变化值ΔI pn值为纵坐标(n＝1,2…9)，工作方程具体为ΔI p(μA)＝9.645*logC Pb2+(ng/mL)+13.36，其中C Pb2+代表铅离子浓度，方法检测限达到0.03ng/mL(S/N＝3)。
  10. 根据权利要求8所述的基于铅离子电化学传感器的铅离子检测方法，其特征在于，所述铅离子电化学传感器的核酸适配体组装浓度为10μM，组装时间为24h，铅离子的孵育时间为15min，孵育温度为30℃。
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