植物激素吲哚乙酸电化学生物传感器的研究

本文提出了以绿豆芽叶片组织-二茂铁修饰的碳糊电板(LFMCE)作为植物激素吲哚乙酸传感器的研制，探讨了影响电极性能的因素，初步研究了吲哚乙酸氧化酶处于最优活性的基本条件。实验表明，此电极在NaH2PO4-Na2HPO4(pH5．8)缓冲溶液中对植物生长素有良好的电催化作用，氧化峰电位约在0.42V左右。用循环伏安法测得的氧化峰电流变化值与吲哚乙酸浓度在8-l60μg／mL范围内呈良好的线性关系，检测下限为4．2μg／mL，相关系数为0．9984，在样品回收率的测定中取得满意结果。     

基于核酸适体的电化学生物传感器*

核酸适体是一类体外筛选的、可与目标分子高效、高特异亲合的RNA或DNA寡核苷酸片段,与常规识别分子（如抗体等）相比,核酸适体作为一类新型识别分子具有明显特色和优势,已被广泛应用于生物传感等分子识别和应用研究领域。本文就基于核酸适体的电化学生物传感器（标记型和非标记型）的近期进展作简要评述,包括适体简介、标记型（“信号衰减”型、“信号增强”型、酶标记型和纳米粒子标记型）和非标记型电化学适体生物传感器等内容。     

核酸等温扩增技术在电化学生物传感器中的应用

生物分子检测在临床诊断、基因治疗、基因突变分析等方面变得日益重要,因而,建立简单、快速、灵敏的检测方法具有重要意义。近年,电化学生物传感器因其简单、便携、易操作、成本低等优势在生物分子检测的研究中备受关注。为了提高检测方法的灵敏度,不同的核酸等温扩增技术被应用于电化学生物传感器的构建中。本文简单介绍了电化学生物传感器的工作原理,着重综述了几种主要应用于电化学传感器中的核酸等温扩增技术,同时比较了各方法的优缺点。     

电化学DNA传感器的研制及其医学应用

对近几年发展起来的电化学DNA传感器的研究工作进行了讨沦,详细论述了该类传感器的工作原理、探针固定化技术、杂交指示剂的类型以及在基因诊断、药物分析等方面的医学应用,并展望了应用前景和发展方向。     

DNA电化学传感器在DNA损伤研究中的应用

利用单链DNA分子共价固定在石墨电极表面,采用核酸分子杂交技术,以道诺霉素作为杂交指示剂形成DNA电化学传感器.研究了在不同致突变剂的作用下,特定碱基序列的DNA在电极表面能否杂交及杂交程度的差异,探讨了DNA突变情况及可能的突变机理.     

用于DNA杂交检测的丝网印刷型生物传感器的研究

将单链DNA(ssDNA)固定到丝网印刷碳电极上构成电化学DNA传感器,采用电化学指示剂,建立DNA杂交的检测方法.Co(phen)33+电化学指示剂通过钴盐与配体邻菲罗啉络合制备,采用等离子发射光谱法(ICP-AES)和核磁共振法(NMR)表征功能基团,采用循环伏安法(CV)分析指示剂的电化学特性,并以此为基础研究ssDNA在电极表面的固定及DNA杂交过程.本研究探讨了直接吸附、静电吸附与键合等3种ssD-NA在电极表面的固定方法,结果表明,静电吸附法和键合法具有较高的ssDNA固定量,采用静电吸附法固定探针的电极杂交目标DNA后,Co(phen)33+易于嵌入双链DNA (dsDNA)中,CV峰电流(ip)信号随目标DNA浓度增加.本研究采用静电吸附ssDNA的电极检测DNA杂交,实验表明,当探针固定液中ssDNA浓度为5 mg/L时,目标DNA浓度在6.65×10- 8～4.26× 10-6mol/L范围内,Co(phen)33+在dsDNA修饰电极上ip值与DNA浓度呈良好的线性关系,R2为0.9819.本研究为建立新的微生物分子分型手段提供了初步依据.     

聚中性红修饰玻碳电极葡萄糖生物传感器

以中性红为电子媒介体,电聚合于Nafion修饰的玻碳电极表面,以戊二醛作交联剂固定葡萄糖氧化酶,最后覆盖一层Nafion膜防止酶流失,构建一种新型葡萄糖生物传感器.详细探讨了传感器的电化学性能及对葡萄糖的最佳响应条件.结果表明,30℃时,传感器在pH 7.0的PBS中对葡萄糖的线性响应范围为1.0×10-5~5.0×10-3mol.L-1.该传感器制作简单、性能优良,有潜在应用前景.     

硫堇与DNA分子相互作用的电化学方法研究

采用交流阻抗技术和循环伏安法 ,研究了在硫堇自组装膜修饰金电极上 ,以及在硫堇或DNA吸附修饰的玻碳电极上 ,硫堇与DNA分子的相互作用;硫堇自组装膜修饰金电极与DNA分子作用后 ,阻抗增大 ,表明它们之间发生了作用 ;吸附在玻碳电极上的硫堇 (DNA)与DNA(硫堇 )作用后 ,峰电位和峰电流均发生了变化 ,结合光谱测定结果 ,表明硫堇与DNA分子间存在着嵌插、静电等作用 ,二者作用的反应速度与分子在电极上固定的位置有关;在PBS缓冲液中硫堇 -DNA的表观结合常数为4.9×104L·mol -1 ;交流阻抗技术和循环伏安法是研究小分子与DNA分子间相互作用的经济、快速、简便的方法     

胆固醇电化学生物传感器的研究与进展

胆固醇作为一些疾病的重要生物标志物,开发准确且快速的胆固醇检测技术在生物医学应用中具有重要意义.电化学生物传感技术具有检测速度快、灵敏度高、操作方便等优点,成为研制新型高性能胆固醇传感器的主要研究方向.近年来,材料合成技术的提高,新型材料种类的丰富,促进了胆固醇电化学生物传感器在脑和心脏血管疾病临床分析中蓬勃发展.本文...     

基于电中性锇配合物的低背景DNA电化学传感器

采用紫外光谱和电化学方法研究了一种电中性锇配合物Os(DPPZ)(PC)(H₂O)DPPZ=联吡啶并[3,2-a,2',3'-c]吩嗪, PC=2,6-吡啶二羧酸}与DNA的相互作用. 紫外光谱结果表明, DNA的加入引起配合物特征吸收峰的减色及红移效应, 说明二者之间存在嵌插作用. 循环伏安实验结果表明, 配合物溶液中加入DNA后, 氧化还原峰电流降低且式电位正移, 证实了二者之间的嵌插作用模式. 将该配合物作为杂交指示剂对CaMV35S启动子基因片段进行检测发现, 在单链探针DNA修饰电极上未观察到指示剂的电化学信号, 而在杂交后的双链DNA电极上呈现灵敏的电化学响应, 表明传感器具有较高的信噪比. 定量分析实验结果表明, 在最佳条件下, 杂交指示剂在传感器上的还原峰电流与目标序列浓度在8.0×10^-10~2.8×10^-9 mol/L范围内呈良好的线性关系. 选择性实验结果表明, 该传感器对互补序列、碱基错配序列和非互补序列具有良好的识别能力.     

A Peptide Nucleic Acid (PNA)‐DNA Ferrocenyl Intercalator for Electrochemical Sensing

A ferrocenyl intercalator was investigated to develop an electrochemical DNA biosensor employing a peptide nucleic acid (PNA) sequence as capture probe. After hybridization with single strand DNA sequence, a naphthalene diimide intercalator bearing ferrocene moieties (FND) was introduced to bind with the PNA‐DNA duplex and the electrochemical signal of the ferrocene molecules was used to monitor the DNA recognition. Electrochemical impedance spectroscopy was used to characterize the different modification steps. Differential pulse voltammetry was employed to evaluate the electrochemical signal of the FND intercalator related to its interaction with the complementary PNA‐DNA hybrid. The ferrocene oxidation peaks were utilised for the target DNA quantification. The developed biosensor demonstrated a good linear dependence of FND oxidation peak on DNA concentration in the range 1 fM to 100 nM of target DNA, with a low detection limit of 11.68 fM. Selectivity tests were also investigated with a non‐complementary DNA sequence, indicating that the FND intercalator exhibits a selective response to the target PNA‐DNA duplex.     

Label‐Free and Label Based Electrochemical Detection of Hybridization by Using Methylene Blue and Peptide Nucleic Acid Probes at Chitosan Modified Carbon Paste Electrodes

A chitosan modified carbon paste electrode (ChiCPE) based DNA biosensor for the recognition of calf thymus double stranded DNA (dsDNA), single stranded DNA (ssDNA) and hybridization detection between complementary DNA oligonucleotides is presented. DNA and oligonucleotides were electrostatically attached by using chitosan onto CPE. The amino groups of chitosan formed a strong complex with the phosphate backbone of DNA. The immobilized probe could selectively hybridize with the target DNA to form hybrid on the CPE surface. The detection of hybridization was observed by using the label‐free and label based protocols. The oxidation signals of guanine and adenine greatly decreased when a hybrid was formed on the ChiCPE surface. The changes in the peak currents of methylene blue (MB), an electroactive label, were observed upon hybridization of probe with target. The signals of MB were investigated at dsDNA modified ChiCPE and ssDNA modified ChiCPE and the increased peak currents were observed, in respect to the order of electrodes. The hybridization of peptide nucleic acid (PNA) probes with the DNA target sequences at ChiCPE was also investigated. Performance characteristics of the sensor were described, along with future prospects.     

新型超支状液晶核酸传感器用于p53突变基因的检测

基于核酸杂交链式反应影响液晶取向的原理, 构建了一种新型的超支状液晶核酸传感器用于检测p53突变基因. 本文突破传统构建超支状分子的方式, 采用杂交链式反应方法, 以目标序列p53突变基因作为引发剂, 3种不同的发卡探针Hairpin A, Hairpin B和Hairpin C为单体, 在温和的条件下, 通过改变单体的浓度和反应时间自发杂交组装形成尺寸和分子量可控的超支状DNA(branched-like DNA, bDNA). 借助捕获探针将该超支状DNA连接到液晶传感基底表面, 观察液晶分子取向改变前后的光学信号, 实现了p53基因含249密码子突变序列的快速检测. 本方法有望为核酸诊断的发展提供一种新的方法和思路.     

Electrochemical Quantitative Analysis of Nucleic Acids Using β‐Cyclodextrin Modified Gold Electrode

We present an electrochemical biosensor for the analysis of nucleic acids upon hybridization on the β‐cyclodextrin (β‐CD)‐modified gold electrode. The strategy is based on the following: The 5’‐ferrocene‐labeled single stranded capture probe DNA (5’‐fc‐ss‐DNA) was incorporated into the cavity of thiolated β‐CD which was immobilized on the surface of gold electrode. After hybridization of complementary target DNA, hybridized double stranded DNA (ds‐DNA) was released from the cavity of β‐CD. The difference of electrochemical properties on the modified gold electrode was characterized by cyclic voltametry and surface plasmon resonance. We successfully applied this method to the investigation of the sensor responses due to hybridization on various concentrations of applied target DNA. As a result, the label‐free electrochemical DNA sensor can detect the target DNA with a detection limit of 1.08 nM. Finally, our method does not require either hybridization indicators or other signalling molecules such as DNA intercalaters which most of electrochemical hybridization detection systems require.     

Detection of Human Interleukine‐2 Gene Using a Label‐Free Electrochemical DNA Hybridization Biosensor on the Basis of a Non‐Inosine Substituted Probe

The human interleukine‐2 gene (hIL‐2) is detected with a label‐free DNA hybridization biosensor using a non‐inosine substituted probe. The sensor relies on the immobilization of a 20‐mer antisense single strand oligonucleotide (chIL‐2) related to the human interleukine‐2 gene on the pencil graphite electrode (PGE) as a probe. The guanine oxidation signal was monitored using anodic differential pulse voltammetry (ADPV). The electrochemical pretreatment of the polished PGE at 1.80 V for 5 min is suggested. Then, 5 min immobilization at 0.50 V was found as the optimum condition for immobilization of the probe. The electrochemical detection of hybridization between chIL‐2 and hIL‐2 as a target was accomplished. The selectivity of the biosensor was studied using noncomplementary oligonucleotides. Diagnostic performance of the biosensor is described and the detection limit is found 36 pg/μL.     

New Anthraquinone Derivatives as Electrochemical Redox Indicators for the Visualization of the DNA Hybridization Process

Interactions of dsDNA and ssDNA, at the surface of gold and silver electrodes, with three novel anthraquinone derivatives: 3‐(9′,10′‐dioxo‐9′,10′‐dihydro‐anthracen‐1‐yl)‐7,11‐di(carboxymethyl)‐3,7,11‐triazatridecanedioic acid, (AQ‐1); 1‐(9′,10′‐dioxo‐9′,10′‐dihydro‐anthracen‐1yl)‐9‐carboxymethyl‐5‐methyl‐1,5,9‐triazaundecanoicacid, (AQ‐2); and N‐(2‐(9,10‐dioxo‐9,10‐dihydro‐anthracen‐1‐ylamino)ethyl)‐2‐(1,4,10,13‐tetraoxa‐7,16‐diazacyclooctadecan‐7‐yl)acetamide, (AQ‐3) are studied. These derivatives are well soluble in water and phosphate buffer solutions. The square wave voltammetric behavior of these redox indicators is described and the parameters of interactions with DNA are reported. It is also pointed out that these compounds can be employed as the hybridization indicators. The difference in the binding ability of the particular redox indicator to single and double stranded DNA can be used for the detection of the complementary nucleic acids.     

Nucleic Acid Biosensor for Detection of Human Immunodeficiency Virus Using Aquabis(1,10‐phenanthroline)copper(II) Perchlorate as Electrochemical Indicator

The electrochemical behavior of aquabis(1,10‐phenanthroline)copper(II) perchlorate [Cu(H₂O)(phen)₂]·2ClO₄, where phen=1,10‐phenanthroline, on binding to DNA at a glassy carbon electrode (GCE) and in solution, was described. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) results showed that [Cu(H₂O)(phen)₂]²⁺ had excellent electrochemical activity on the GCE with a couple of quasi‐reversible redox peaks. The interaction mode between [Cu(H₂O)(phen)₂]²⁺ and double‐strand DNA (dsDNA) was identified to be intercalative binding. An electrochemical DNA biosensor was developed with covalent immobilization of human immunodeficiency virus (HIV) probe for single‐strand DNA (ssDNA) on the modified GCE. Numerous factors affecting the probe immobilization, target hybridization, and indicator binding reactions were optimized to maximize the sensitivity and speed of the assay. With this approach, a sequence of the HIV could be quantified over the range from 7.8×10^?9 to 3.1×10^?7 mol·L^?1 with a linear correlation of γ=0.9987 and a detection limit of 1.3×10^?9 mol·L^?1.     

基于锁核酸探针的传感器用于BCR/ABL融合基因检测的研究

基于慢性粒细胞白血病中BCR/ABL融合基因的碱基序列,设计了一种新型发夹结构锁核酸(locked nucleic acids, LNA)探针,把LNA探针通过Au-s键固定在金电极表面构建了特异的生物传感器.LNA探针与目标链DNA杂交,以自行合成的苯甲酸二聚铜配合物([Cu2(C7H5O2)4(C2H6O)2], 简称[Cu(R)2]2+)为杂交指示剂,应用差示脉冲伏安法进行检测,表现出良好的响应信号.该新型锁核酸传感器能较好的区分完全互补链DNA、单碱基错配链DNA.对互补链DNA检测的线性范围为1.0×10-8～1.0×10-6 mol•L-1,检出限为2.0×10-9 mol•L-1.     

Electrochemical Biosensor for the Detection of Interaction Between Arsenic Trioxide and DNA Based on Guanine Signal

The interaction of arsenic trioxide (As₂O₃) with calf thymus double‐stranded DNA (dsDNA), calf thymus single‐stranded DNA (ssDNA) and also 17‐mer short oligonucleotide (Probe A) was studied electrochemically by using differential pulse voltammetry (DPV) with carbon paste electrode (CPE) at the surface and also in solution. Potentiometric stripping analysis (PSA) was employed to monitor the interaction of As₂O₃ with dsDNA in solution phase by using a renewable pencil graphite electrode (PGE). The changes in the experimental parameters such as the concentration of As₂O₃, and the accumulation time of As₂O₃ were studied by using DPV; in addition, the reproducibility data for the interaction between DNA and As₂O₃ was determined by using both electrochemical techniques. After the interaction of As₂O₃ with dsDNA, the DPV signal of guanine was found to be decreasing when the accumulation time and the concentration of As₂O₃ were increased. Similar DPV results were also found with ssDNA and oligonucleotide. PSA results observed at a low DNA concentration such as 1 ppm and a different working electrode such as PGE showed that there could be damage to guanine bases. The partition coefficients of As₂O₃ after interaction with dsDNA and ssDNA in solution by using CPE were calculated. Similarly, the partition coefficients (PC) of As₂O₃ after interaction with dsDNA in solution was also calculated by PSA at PGE. The features of this proposed method for the detection of DNA damage by As₂O₃ are discussed and compared with those methods previously reported for the other type of DNA targeted agents in the literature.