CdTe量子点标记的DNA电化学传感器的研究

利用碳纳米管和CdTe量子点(QDs)组装的电化学传感器,建立了一种识别DNA的新方法.将氨基修饰的单链DNA探针共价键合固定在带有羧基的碳纳米管修饰的金电极上,然后与CdTe QDs标记的目标DNA进行杂交.利用差分脉冲法(DPV)和循环伏安法对目标DNA的固定和杂交进行表征,通过电活性指示剂柔红霉素(DNR)的DPV峰电流变化,对互补DNA、非互补DNA和单碱基错配DNA序列进行识别.与未标记CdTe QDs的目标DNA相比,标记CdTe QDs的目标DNA序列的电流响应灵敏度明显提高.DNA电化学传感器检测的优化条件:DNR的浓度为1.67×10-5 mol/L,DNA杂交时间为80 min,杂交温度为55 ℃.在1.0×10-13 ～1.0×10-8 mol/L范围,目标DNA浓度的对数值与其响应的DPV信号(还原峰电流)呈线性关系,检出限为3.52×10-14 mol/L(S/N=3,n=9),线性方程为ΔI=50.22+3.567 lgcDNA,相关系数为0.996 6.对1.0×10-10 mol/L的目标DNA样品进行重复测定,相对标准偏差为4.8%(n=5),重复性良好.     

量子点双标记的Fe3O4@Au磁性纳米DNA电致发光型传感器

合成了Fe3O4@Au磁性纳米粒子,并根据单链寡聚核苷酸(ss-DNA)杂交原理,利用量子点电化学发光,构建了DNA电化学传感器.在磁控玻碳电极(MCGCE)表面,将5′-SH-ssDNA捕获探针自组装在Fe3O4@Au磁性纳米粒子上,然后与目标DNA互补的一端杂交形成dsDNA,再与双标记了量子点的5′-NH2-ssDNA-NH2-3′信号探针杂交形成三明治杂交的DNA.应用循环伏安法对DNA的固定与杂交进行了表征.目标DNA浓度在1.0×10-13～1.0×10-11 mol/L范围与其响应的ECL信号呈线性关系,检出限为1.8×10-14mol/L.由于采用量子点双标记法,检测的灵敏度显著提高.     

基于单壁碳纳米管-十二醛复合材料的DNA电化学传感器

将单壁碳纳米管(SWNTs)和十二醛(DA)混合超声分散,得到均匀、稳定的无机-有机纳米复合材料(SWNTs-DA)。将其滴涂在玻碳电极表面晾干得到复合材料修饰电极(SWNTs-DA/GCE),再通过胺醛缩合反应将末端修饰氨基的单链DNA探针共价固定在SWNTs-DA/GCE表面,构建了一种新型的DNA电化学传感器。以[Fe(CN)6]3-/4-为电活性探针,采用循环伏安法和电化学阻抗法对传感器的层层组装过程进行表征。以亚甲基蓝(MB)作为杂交指示剂,考察了传感器分析性能。实验结果表明,MB在传感器上的峰电流值(Ip)与互补序列浓度对数值(lgcS2)在1.0×10-15～1.0×10-10mol/L范围内呈良好的线性关系(r=0.998)。根据3倍信噪比(S/N=3),计算得检出限为2.0×10-16mol/L。选择性实验表明该传感器能对互补序列、三碱基错配序列和非互补序列进行很好的识别。     

基于石墨烯和蒽醌-2-磺酸钠的Pb2+核酸适体电化学传感器

构建了一种基于石墨烯(GR)-蒽醌化合物(AQMS)在核酸适体表面层层组装的Pb2+电化学传感器。将修饰有巯基的Pb2+核酸适体(apt)固定在金电极表面,然后利用GR与apt碱基之间的π-π作用,将GR吸附在apt修饰电极表面,并用于电活性分子AQMS的组装和电化学信号的放大。当目标分析物(Pb2+)存在时,Pb2+诱导apt转变为稳定的G-四联体结构,使得GR连同信号分子AQMS从电极表面脱落,电化学信号降低,从而实现对Pb2+的监测。结果表明,传感器对Pb2+具有很好的特异识别性,且Pb2+浓度在5.0×10!10~5.0×10!8mol/L的范围内,AQMS的峰电流变化值(ΔIpa)与Pb2+浓度对数(lgCPb2+)呈现良好的线性关系。根据3σ,计算得检出限达到6.0×10!11mol/L。     

二茂铁巯基化合物/金纳米粒子/还原石墨烯修饰电化学传感器检测Cu~(2+)

通过Hummer法进一步还原合成还原石墨烯(RGO),Shifft碱反应合成新型二茂铁巯基化合物(FcSH)。利用还原石墨烯吸附性将石墨烯修饰在玻碳电极(GCE)上,在石墨烯表面电沉积金纳米粒子(AuNPs),通过自组装制备还原石墨烯和二茂铁巯基修饰电化学传感器(FcSH/AuNPs/RGO/GCE),该电化学传感器具有大的比表面积和富电子性能。实验显示,在0.01 mol/L HCl中,富集时间为180s,Cu~(2+)浓度在1.0×10~(-12)~1.0×10~(-11)mol/L与1.0×10~(-11)~1.0×10~(-10)mol/L范围内与方波伏安峰电流分别呈现良好的线性关系,检出限为0.94×10~(-12)mol/L。该电化学传感器对Cu~(2+)的检测表现出较好的选择性、高的稳定性和灵敏性,可用于环境中痕量Cu~(2+)的测定。     

分子印迹电化学传感器测定大气中的羟基自由基

对羟基苯甲酸可以作为分子探针被羟基自由基(·OH)氧化,产生具有电化学活性的3,4-二羟基苯甲酸(3,4-DHBA)。根据此原理,本研究以吡咯为单体,3,4-二羟基苯甲酸为模板分子,在纳米TiO_2修饰的玻碳电极上电聚合制备分子印迹聚合物膜修饰电极(3,4-DHBA-PPy/TiO_2/GCE),实现对·OH的间接测定。通过扫描电子显微镜(SEM)和电化学方法对此传感器进行表征,优化了电聚合圈数、电聚合体系p H值、洗脱时间、吸附时间等条件。在优化条件下,标准物3,4-DHBA氧化峰电流大小与其浓度在1.0×10~(-8)~1.0×10~(-6)mol/L范围内具有良好的线性关系,检出限为4.2×10~(-9)mol/L(S/N=3)。将传感器用于大气中·OH浓度的测定,结果良好,是一种低廉、便捷、新颖的检测·OH的方法。     

DNA电化学生物传感器在转基因植物特定序列基因检测中的应用

在玻碳电极(GCE)上采用循环伏安法电聚合硫堇(PTh)得到PTh/GCE修饰电极,并利用聚硫堇层共价结合和静电作用吸附金纳米粒子(AuNP′s)制得AuNP′s/PTh/GCE修饰电极。然后通过将ss-DNA/AuNP′s/PTh修饰电极置于cDNA杂交液中,于42℃杂交制得ds-DNA/AuNP′s/PTh修饰玻碳电极,实现了脱氧核糖核酸(DNA)探针在AuNP′s/PTh修饰的玻碳电极上的固定,制得DNA电化学生物传感器。在[Fe(CN)6]3-/4-溶液中采用微分脉冲伏安法(DPV)及交流阻抗谱技术(EIS)对DNA的固定和杂交进行了表征。试验结果表明:在1.0×10-10～1.0×10-6mol.L-1的浓度范围内,该传感器可对转基因植物外源基因草丁膦乙酰转移酶基因(PAT基因)片段进行检测,检出限(3s)为3.2×10-11mol.L-1。     

电化学发光-分子印迹传感器的制备及其在海洛因检测中的应用

利用掺杂多壁碳纳米管(MWNTs)的Nafion膜在玻碳电极上固定联吡啶钌(Ru(bpy)2+3),制得Ru(bpy)2+3/Nafion/MWCNT修饰电极。为了提高修饰电极的选择性,通过溶胶-凝胶技术,对该电极进一步修饰了溶胶-凝胶分子印迹膜,制得电化学发光-分子印迹传感器。优化了扫速、pH值、富集时间等检测条件,传感器显示出既具电化学发光技术的灵敏性和分子印迹技术的选择性。在优化条件下,即pH 7.0的磷酸盐缓冲溶液中,以100 mV/s扫速,富集5 min,对海洛因进行检测,在1.0×10!10~1.0×10!14mol/L范围内有良好线性关系,检出限可达到4.0×10!15mol/L(S/N=3)。传感器直接应用于唾液和尿液中海洛因的测定,回收率达到97%~104%。     

氧化锆/聚中性红电化学DNA传感器用于转基因植物CaMV35S启动子基因序列的测定

制备了基于氧化锆(ZrO2)/聚中性红(PNR)修饰电极的电化学DNA传感器。探针DNA通过磷酸基和ZrO2的相互作用组装到电极表面。原子力显微镜(AFM)和电化学方法用于电极的表征。PNR在DNA杂交前后峰电流的变化作为杂交信号,用示差脉冲伏安法对转基因植物CaMV35S启动子基因片段进行测定。结果表明:探针DNA和完全互补的DNA片段杂交后,杂交信号明显变小,峰电流的变化值与其浓度的对数在1.0×10-11~1.0×10-8mol/L范围内呈良好的线性关系,检出限为3.46×10-12mol/L(S/N=3)。此外,传感器能区分互补、单碱基错配、完全错配的DNA序列,已用于样品的测定。     

石墨烯修饰的三唑磷分子印迹电化学传感器的制备

采用石墨烯作为电极增敏材料,制备三唑磷(TAP)分子印迹电化学传感器。采用自由基聚合法,在石墨烯修饰电极(GR/GCE)上合成分子印迹聚合物膜(MIP)。利用微分脉冲伏安法、电化学阻抗谱对不同修饰电极进行电化学表征,利用微分脉冲伏安法考察了MIP和非分子印迹聚合物膜(NIP)传感器的电化学性能。在最优实验条件下,TAP浓度在1.0×10~(-7)～2.0×10~(-5)mol·L~(-1)内和MIP膜传感器峰电流呈线性关系,检出限为4.3×10~(-8)mol·L~(-1)(S/N=3)。建立MIP膜传感器的动力学吸附模型,测得结合速率常数k为9.0580 s。     

An Electrochemical DNA Sensor Based on Electrodepositing Aluminum Ion Films on Stearic Acid‐Modified Carbon Paste Electrode and Its Application for the Detection of Specific Sequences Related to Bar Gene and CP4 Epsps Gene

An electrochemical DNA biosensor was fabricated by immobilizing DNA probe on aluminum ion films that were electrodeposited on the surface of the stearic acid‐modified carbon paste electrode (CPE). DNA immobilization and hybridization were characterized with cyclic voltammetry (CV) by using methylene blue (MB) as indicator. MB has a couple of well‐defined voltammetric redox peaks at the CPE. The currents of redox peaks of MB decreased after depositing aluminum ion films on the CPE (Al(III)/CPE) and increased dramatically after immobilizing DNA probe (ssDNA/Al(III)/CPE). Hybridization of DNA probe led to a marked decrease of the peak currents of MB, which can be used to detect the target single‐stranded DNA. The conditions for the preparation of Al(III)/CPE, and DNA immobilization and hybridization were optimized. The specific sequences related to bar transgene in the transgenic corn and the PCR amplification of CP4 epsps gene from the sample of transgenic roundup ready soybean were detected by differential pulse voltammetry (DPV) with this new electrochemical DNA biosensor. The difference between the peak currents of MB at ssDNA/Al(III)/CPE and that at hybridization DNA modified electrode (dsDNA/Al(III)/CPE) was applied to determine the specific sequence related to the target bar gene with the dynamic range comprised between 1.0×10^?7 mol/L to 1.0×10^?4 mol/L. A detection limit of 2.25×10^?8 mol/L of oligonucleotides can be estimated.     

基于DNA四面体纳米结构探针的电化学生物传感器检测DNA甲基化

该文以DNA四面体纳米结构探针(TSP)为捕获探针,将辣根过氧化物酶标记的IgG抗体结合在纳米金颗粒表面(AuNPs-IgG-HRP)作为信号分子,构建了一种新型DNA甲基化电化学传感器。利用一步热变性法组装成TSP后,通过Au—S键固定在修饰纳米金颗粒的金电极表面,经过靶标DNA杂交、5-甲基胞嘧啶(5-mc)抗体及AuNPs-IgG-HRP结合后,用差分脉冲伏安法(DPV)进行检测。采用循环伏安法(CV)和电化学阻抗谱(EIS)对修饰电极的构建过程进行电化学表征。探究了杂交时间、5-mc抗体浓度、IgG-HRP加入体积、氢醌(HQ)和过氧化氢(H2O2)浓度对传感器的影响。在最佳条件下,该传感器对甲基化DNA的线性响应范围为1.0×10-15~1.0×10-10 mol/L,检出限(S/N=3)为4.4×10-16 mol/L。该传感器具有良好的选择性和稳定性,为DNA甲基化检测提供了新方法。     

4‐Aminothiophenol Self‐Assembled Monolayer for the Development of a DNA Biosensor Aiming the Detection of Cylindrospermopsin Producing Cyanobacteria

The development of a DNA biosensor for the detection of cylindrospermopsin, based on self‐assembled monolayers (SAMs) of 4‐aminothiophenol, is investigated. SAMs were characterized by electrochemical reductive desorption. Detection of probe immobilization and hybridization has been achieved by cyclic and square‐wave voltammetry (SWV), using methylene blue (MB) as electroactive indicator. The SWV data obtained in phosphate buffer, with and without NaCl, after MB accumulation, revealed an increase of the redox indicator current peaks after the hybridization step. This behavior is consistent with MB intercalation into DNA, for high ionic strength media and attributed to electrostatic interactions in the absence of salt. Evidence for surface modification is also provided by atomic force microscopy and ellipsometry.     

Electrochemical detection of short sequences related to the hepatitis B virus using MB on chitosan-modified CPE

A novel electrochemical DNA biosensor based on methylene blue (MB) and chitosan-modified carbon paste electrode (CCPE) for short DNA sequences and polymerase chain reaction (PCR) amplified real samples related to the hepatitis B virus (HBV) hybridization detection is presented. Differential pulse voltammetry (DPV) was used to investigate the surface coverage and hybridization event. The decrease in the peak current of MB, an electroactive label, was observed upon hybridization of probe with the target. Numerous factors affecting the target hybridization and indicator binding reaction are optimized to maximize the sensitivity.     

A novel route for immobilization of oligonucleotides onto modified silica nanoparticles

A novel approach for immobilization of probe oligonucleotides that uses zirconium phosphate modified silica nanoparticles is proposed. The surface modification of nanoparticles was carried out in two stages. Initially binding of Zr4+ to the surface of silica nanoparticles and later treated with phosphoric acid for terminal phosphate groups. Oligonucleotide probes modified with amine group at 5'-end were strongly binds to the phosphate terminated silica nanoparticles with imidazole in presence of 0.1 mol L(-1) EDC [N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide], as phosphate groups are more reactive towards amine group. Various studies, i.e., synthesis of silica nanoparticles, their surface modification, probe immobilization, measurement of hybridization and effect of bovine serum albumin (BSA) were carried out during optimization of reaction conditions. The significant reduction in the background signal was observed by treating the probe modified silica nanoparticles with bovine serum albumin prior to hybridization. The probe modified silica nanoparticles were retained their properties and the hybridization was induced by exposure of single-stranded DNA (ssDNA) containing silica nanoparticles to the complementary DNA in solution. The decrease in the fluorescence signal for one mismatch and three mismatch was observed upon hybridization of probe with target DNAs, while there was no response for the random target ssDNA under the same experimental conditions. The intensity of fluorescence signal was linear to the concentration of target DNA ranging from 3.9 x 10(-9) to 3.0 x 10(-6)mol L(-1). A detection limit of 1.22 x 10(-9) mol L(-1) of oligonucleotides can be estimated. The proposed hybridization assay is simple and possesses good analytical characteristics and it can provide an effective and efficient route in the development of DNA biosensors and biochips.     

毒莠定印迹邻氨基苯硫酚/金纳米粒子复合膜安培传感器

在模板分子存在下,在金电极上自组装邻氨基苯硫酚（oATP）,通过电聚合制得毒莠定印迹的oATP/金纳米粒子聚合薄膜及其安培传感器.采用循环伏安法和交流阻抗技术对传感器制备过程进行表征,用紫外光谱法研究了单体与模板间的相互作用.以K3Fe（CN）6为探针,示差脉冲伏安曲线的峰电流与毒莠定浓度在2.0×10^-7-2.4×10 4mol/L范围内呈现良好的线性关系（r=0.9963）,毒莠定的检出限为6.5×10 8mol/L（S/N=3）.将该印迹膜传感器用于环境水样加标回收检测,结果令人满意.     

Electrochemical deoxyribonucleic acid biosensor based on the self-assembly film with nanogold decorated on ionic liquid modified carbon paste electrode

An electrochemical DNA biosensor was fabricated by self-assembling probe single-stranded DNA (ssDNA) with a nanogold decorated on ionic liquid modified carbon paste electrode (IL-CPE). IL-CPE was fabricated using 1-butylpyridinium hexafluorophosphate as the binder and the gold nanoparticles were electrodeposited on the surface of IL-CPE (Au/IL-CPE). Then mercaptoacetic acid was self-assembled on the Au/IL-CPE to obtain a layer of modified film, and the ssDNA probe was further covalently-linked with mercaptoacetic acid by the formation of carboxylate ester with the help of N-(3-dimethylamino-propyl)-N'-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide. The hybridization reaction with the target ssDNA was monitored with methylene blue (MB) as the electrochemical indicator. Under the optimal conditions, differential pulse voltammetric responses of MB was proportional to the specific ssDNA arachis sequences in the concentration range from 1.0×10(-11) to 1.0×10(-6) mol L(-1) with the detection limit as 1.5×10(-12) mol L(-1) (3σ). This electrochemical DNA sensor exhibited good stability and selectivity with the discrimination ability of the one-base and three-base mismatched ssDNA sequences. The polymerase chain reaction product of arachis Arabinose operon D gene was successfully detected by the proposed method, which indicated that the electrochemical DNA sensor designed in this paper could be further used for the detection of specific ssDNA sequence.     

DNA and PNA sensing on mercury and carbon electrodes by using methylene blue as an electrochemical label

Described here are the electrochemical parameters for MB on binding to DNA at hanging mercury drop electrode (HMDE), glassy carbon electrode (GCE), and carbon paste electrode (CPE) in the solution and at the electrode surface. MB, which interacts with the immobilized calf thymus DNA, was detected by using single-stranded DNA-modified HMDE or CPE (ssDNA-modified HMDE or CPE), bare HMDE or CPE, and double-stranded DNA-modified HMDE or CPE (dsDNA-modified HMDE or CPE) in combination with adsorptive transfer stripping voltammetry (AdTSV), differential pulse voltammetry (DPV), and alternating current voltammetry (ACV) techniques. The structural conformation of DNA and hybridization between synthetic peptide nucleic acid (PNA) and DNA oligonucleotides were determined by the changes in the voltammetric peak of MB. The PNA and DNA probes were also challenged with excessive and equal amount of noncomplementary DNA and a mixture that contained one-base mismatched and target DNA. The partition coefficient was also obtained from the signal of MB with probe, hybrid, and ssDNA-modified GCEs. The effect of probe, target, and ssDNA concentration upon the MB signal was investigated. These results demonstrated that MB could be used as an effective electroactive hybridization indicator for DNA biosensors. Performance characteristics of the sensor are described, along with future prospects.     

A PDDA/poly(2,6-pyridinedicarboxylic acid)-CNTs composite film DNA electrochemical sensor and its application for the detection of specific sequences related to PAT gene and NOS gene

2,6-Pyridinedicarboxylic acid (PDC) was electropolymerized on the glassy carbon electrode (GCE) surface combined with carboxylic group-functionalized single-walled carbon nanotubes (SWNTs) by cyclic voltammetry (CV) to form PDC-SWNTs composite film, which was rich in negatively charged carboxylic group. Then, poly(diallyldimethyl ammonium chloride) (PDDA), a linear cationic polyelectrolyte, was electrostatically adsorbed on the PDC-SWNTs/GCE surface. DNA probes with negatively charged phosphate group at the 5' end were immobilized on the PDDA/PDC-SWNTs/GCE due to the strong electrostatic attraction between PDDA and phosphate group of DNA. It has been found that modification of the electrode with PDC-SWNTs film has enhanced the effective electrode surface area and electron-transfer ability, in addition to providing negatively charged groups for the electrostatic assembly of cationic polyelectrolyte. PDDA plays a key role in the attachment of DNA probes to the PDC-SWNTs composite film and acts as a bridge to connect DNA with PDC-SWNTs film. The cathodic peak current of methylene blue (MB), an electroactive label, decreased obviously after the hybridization of DNA probe (ssDNA) with the complementary DNA (cDNA). This peak current change was used to monitor the recognition of the specific sequences related to PAT gene in the transgenic corn and the polymerase chain reaction (PCR) amplification of NOS gene from the sample of transgenic soybean with satisfactory results. Under optimal conditions, the dynamic detection range of the sensor to PAT gene target sequence was from 1.0x10(-11) to 1.0x10(-6) mol/L with the detection limit of 2.6x10(-12) mol/L.     

Developing an electrochemical deoxyribonucleic acid (DNA) biosensor on the basis of human interleukine-2 gene using an electroactive label

Development of an electrochemical DNA biosensor based on a human interleukine-2 (IL-2) gene probe, using a pencil graphite electrode (PGE) as transducer and methylene blue (MB) as electroactive label is described. The sensor relies on the immobilization of a 20-mer single stranded oligonucleotide probe (hIL-2) related to the IL-2 gene on the electrode. The hybridization between the probe and its complementary sequence (chIL-2) as the target was studied by square wave voltammetry (SWV) of MB accumulated on the PGE. In this approach the extent of hybridization is evaluated on the basis of the difference between SWV signals of MB accumulated on the probe-PGE and MB accumulated on the probe-target-PGE. Some hybridization experiments with non-complementary oligonucleotides were carried out to assess whether the suggested DNA sensor responds selectively to the target. Some experimental variables affecting the performance of the biosensor including: polishing of PGE, its electrochemical activation conditions (i.e., activation potential and activation time) and probe immobilization conditions on the electrodes (i.e., immobilization potential and time) were investigated and the optimum values of 1.80 V and 300 s for PGE activation, and −0.5 V and 400 s for the probe immobilization on the electrode were suggested.