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。     

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.     

基于纳米金胶标记DNA探针的电化学DNA传感器研究

以纳米金胶为标记物,将其标记于人工合成的5-端巯基修饰的寡聚核苷酸片段上,制成了具有电化学活性的金胶标记DNA电化学探针;在一定条件下,使其与固定在玻碳电极表面的靶序列进行杂交反应,利用ssDNA与其互补链杂交的高度序列选择性和极强的分子识别能力,以及纳米金胶的电化学活性,实现对特定序列DNA片段的电化学检测以及对DNA碱基突变的识别.     

A Single‐Surface Electrochemical Biosensor for the Detection of DNA Triplet Repeat Expansion

Molecular diagnostics of inherited neurodegenerative disorders such as fragile X syndrome, myotonic dystrophy or Friedreich ataxia (FRDA) is based on analysis of the length of trinucleotide repetitive sequences in certain loci of genomic DNA. The current methods employ PCR and electrophoretic determination of the amplified DNA fragment size. We have recently shown that length of a triplet repetitive DNA sequence can be determined using a double‐surface electrochemical technique involving multiple hybridization of the expanded triplet repeat with short labeled reporter probe (spanning several trinucleotides). Here we propose a single‐surface sensor employing an analogous principle. Target DNA (tDNA) is adsorbed onto surface of a carbon (pyrolytic graphite or screen‐printed) electrode. Biotin‐labeled reporter probe (RP) is hybridized with the immobilized tDNA followed by binding of streptavidin‐alkaline phosphatase (ALP) conjugate. The ALP catalyzes production of an electroactive indicator (1‐naphthol) which is detected voltammetrically on the same electrode. Signal resulting from this electrochemical enzyme‐linked DNA hybridization assay is normalized to the amount of tDNA immobilized at the transducer surface either by measuring intrinsic tDNA voltammetric response, or using electrochemical labeling of the tDNA with osmium tetroxide 2,2′‐bipyridine complex. Detection of (GAA)_n?(TTC)_n triplet repeat expansion in nanogram quantities of PCR‐amplified tDNAs, including amplicons of patients' genomic DNA, is demonstrated. We show that our technique allow differentiation between normal and pathological alleles of X25 gene related to the FRDA.     

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.     

Studies on the Electrochemical Property of Dinuclear Copper(II) Complex Containing Dimethylglyoxime and Its Interaction with DNA

The electrochemical property of dinuclear copper(II) complex containing dimethylglyoxime [Cu₂(Hdmg)₄] was studied with cyclic voltammetry. Cu₂(Hdmg)₄ had an irreversible oxidation peak in pH 5.0 B–R buffer solution at the stearic acid‐modified carbon paste electrode. Cyclic voltammetry, UV‐vis absorption spectra and viscosity measurements were used to study the interaction of Cu₂(Hdmg)₄ with herring fish sperm DNA. Cu₂(Hdmg)₄ interacts with the bases of DNA to form nonelectroactive adducts. The interaction of Cu₂(Hdmg)₄ with ssDNA was stronger than that of Cu₂(Hdmg)₄ with dsDNA because of the exposed bases of ssDNA. The gene sequence related to the BAR transgene in the transgenic corn was detected using Cu₂(Hdmg)₄ as electroactive indicator.     

Impedance‐Based DNA Biosensor Employing Molecular Beacon DNA as Probe and Thionine as Charge Neutralizer

In this paper, we present an impedance‐based DNA biosensor using thionine intercalation to amplify DNA hybridization signal. Beacon single‐stranded DNA (ssDNA) probe and mercaptoacetic acid were self‐assembled onto a Au electrode by forming Au? S bonds. These beacon ssDNAs were hybridized with the complementary sequences around the loop structure. Then thionine was intercalated into the double‐stranded DNA (dsDNA) immobilized on the Au electrode surface. Due to the neutralization of the negative charges of dsDNA by the intercalated thionine, the electronic transfer resistance (R_et) of the DNA modified Au electrode was significantly diminished. Herein, the decreased value of R_et resulted from the thionine intercalating into dsDNA was employed as the hybridization signal. SDS was used to reduce the unspecific adsorption between ssDNA and thionine. Several experimental conditions, including the surface coverage of ssDNA probe on Au electrode, the hybridization temperature and time were all optimized. Moreover, the hybridization reactions of the unstructured linear ssDNA probe and the structured beacon ssDNA probe with their complementary sequences were compared in this work. The sensitivity of the presented DNA biosensor highlighted that the intercalation of thionine into dsDNA was an efficient approach to amplify the hybridization signal using impedance detection technique. Additionally, in this DNA biosensing protocol, beacon ssDNA has a good ability to distinguish target DNA sequences. This results in a higher specificity than using traditional unstructured DNA probe.     

Studies on the Recognition Interaction of Rhodamine B and DNA by Voltammetry

Introduction Theinteractionofvariousmoleculessuchas anticancerdrugs,metalcomplexesandorganic dyeswithDNAhasattractedaconsiderableinter- estofsomeresearchersinrecentyears.Theinflu- encesofsmallorganicmoleculesontheDNAstruc- tureandfunctionareofgreatimportanceindrug composition,carcinogenicmechanismandgenemu- tation[1_3].Atpresent,manymethodsandmodels havebeenproposedforthestudyoftheinteraction ofDNAwithsmallmolecules,suchasspectropho- tometry,fluorometry,lightscatteringtechniques andelectro…     

Adsorptive Stripping Voltammetric Detection of Single‐Stranded DNA at Electrochemically Modified Glassy Carbon Electrode

Electrochemically modified glassy carbon electrode (GCE) was used to study the electrochemical oxidation and detection of denatured single‐stranded (ss) DNA by means of adsorptive stripping voltammetry. The modification of GCE, by electrochemical oxidation at +1.75 V (vs.SCE) for 10 min and cyclic sweep between +0.3 V and ?1.3 V for 20 cycles in pH 5.0 phosphate buffer, results in 100‐fold improvement in sensitivity for ssDNA detection. We speculated that the modified GCE has a high affinity to single‐stranded DNA through hydrogen bond (specific static adsorption). Single‐stranded DNA can accumulate at the GCE surface at open circuit and produce a well‐defined oxidation peak corresponding to the guanine residues at about +0.80 V in pH 5.0 phosphate buffer, while the native DNA gives no signal under the same condition. The peak currents are proportional to the ssDNA concentration in the range of 0–18.0 μg mL^?1. The detection limit of denatured ssDNA is ca. 0.2 μg mL^?1 when the accumulation time is 8 min at open circuit. The accumulation mechanism of ssDNA on the modified GCE was discussed.     

Thermodynamics for the Formation of Double‐Stranded DNA–Single‐Walled Carbon Nanotube Hybrids

For the first time, the thermodynamics are described for the formation of double‐stranded DNA (ds‐DNA)–single‐walled carbon nanotube (SWNT) hybrids. This treatment is applied to the exchange reaction of sodium cholate (SC) molecules on SWNTs and the ds‐DNAs d(A)₂₀–d(T)₂₀ and nuclear factor (NF)‐κB decoy. UV/Vis/near‐IR spectroscopy with temperature variations was used for analyzing the exchange reaction on the SWNTs with four different chiralities: (n,m)=(8,3), (6,5), (7,5), and (8,6). Single‐stranded DNAs (ss‐DNAs), including d(A)₂₀ and d(T)₂₀, are also used for comparison. The d(A)₂₀–d(T)₂₀ shows a drastic change in its thermodynamic parameters around the melting temperature (T_m) of the DNA oligomer. No such T_m dependency was measured, owing to high T_m in the NF‐κB decoy DNA and no T_m in the ss‐DNA.     

聚合物膜与自组装膜法制备电化学DNA传感器的研究进展

介绍了DNA电化学生物传感器的研究现状、原理和结构,对DNA探针固定(尤其是聚合物与自组装膜法固定DNA)以及应用方面的最新研究进展进行了综述。     

Label‐Free Detection of Nanomolar Unmodified Single‐ and Double‐Stranded DNA by Using Surface‐Enhanced Raman Spectroscopy on Ag and Au Colloids

Unlabelled single‐ and double‐stranded DNA (ssDNA and dsDNA, respectively) has been detected at concentrations ≥10^?9 M by surface‐enhanced Raman spectroscopy. Under appropriate conditions the sequences spontaneously adsorbed to the surface of both Ag and Au colloids through their nucleobases; this allowed highly reproducible spectra with good signal‐to‐noise ratios to be recorded on completely unmodified samples. This eliminated the need to promote absorption by introducing external linkers, such as thiols. The spectra of model ssDNA sequences contained bands of all the bases present and showed systematic changes when the overall base composition was altered. Initial tests also showed that small but reproducible changes could be detected between oligonucleotides with the same bases arranged in a different order. The spectra of five ssDNA sequences that correspond to different strains of the Escherichia coli bacterium were found to be sufficiently composition‐dependent so that they could be differentiated without the need for any advanced multivariate data analysis techniques.     

Cyclodextrin Host‐Guest Recognition Approach for Simultaneous Quantification and Voltammetric Studies of Levodopa and Carbidopa in Pharmaceutical Products

An electrochemical sensor for simultaneous quantification of Levodopa (L‐dopa) and Carbidopa (C‐dopa) using a β‐cyclodextrin/poly(N‐acetylaniline) (β‐CD/PNAANI) modified carbon paste electrode has been developed. Preconcentrating effect of β‐CD as well as its different inclusion complex stability with L‐dopa and C‐dopa was used to construct an electrochemical sensor for quantification of these important analytes. The overlapping anodic peaks of L‐dopa and C‐dopa at 810 mV on bare carbon paste electrode resolved in two well‐defined voltammetric peaks at 450 and 880 mV vs. Ag/AgCl, respectively, with a drastic enhancement of the anodic peak currents. Under optimized conditions, linear calibration curves were obtained in the ranges of 0.5–117 µM and 1.6–210 µM with detection limits down to 0.2 and 0.8 µM for L‐dopa and C‐dopa, respectively. The proposed electrode was successfully applied for the determination of L‐dopa /C‐dopa in pharmaceutical formulations and the results were in close agreement with the labeled values.     

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.     

Voltammetric determination of DNA hybridization using methylene blue and self-assembled alkanethiol monolayer on gold electrodes

An electrochemical DNA biosensor based on the recognition of single stranded DNA (ssDNA) by hybridization detection with immobilized complementary DNA oligonucleotides is presented. DNA and oligonucleotides were covalently attached through free amines on the DNA bases using N-hydroxysulfosuccinimide (NHS) and N-(3-dimethylamino)propyl-N′-ethylcarbodiimide hydrochloride (EDC) onto a carboxylate terminated alkanethiol self-assembled monolayers (SAM) preformed on a gold electrode (AuE). Differential pulse voltammetry (DPV) was used to investigate the surface coverage and molecular orientation of the immobilized DNA molecules. The covalently immobilized probe could selectively hybridize with the target DNA to form a hybrid on the surface despite the bases being attached to the SAM. The changes in the peak currents of methylene blue (MB), an electroactive label, were observed upon hybridization of probe with the target. Peak currents were found to increase in the following order: hybrid-modified AuE, mismatched hybrid-modified AuE, and the probe-modified AuE which indicates the MB signal is determined by the extent of exposed bases. Control experiments were performed using a non-complementary DNA sequence. The effect of the DNA target concentration on the hybridization signal was also studied. The interaction of MB with inosine substituted probes was investigated. Performance characteristics of the sensor are described.     

Effect of Various Intercalators on the Fenton‐Type Oxidative Cleavage of Double‐Stranded DNA

The intensity of the linear dichroism (LD) in the absorption region of DNA (about 260 nm) decreased with time in the presence of [Fe(EDTA)]²⁺ (EDTA=ethylenediaminetetraacetic acid), H₂O₂, and ascorbate. The decrease in the LD signal indicated either an increase in flexibility, a shortening of the DNA stem, or both, owing to oxidative cleavage, and was best described by the difference between the two single‐exponential‐decay curves, thereby suggesting the involvement of two sequential first‐order reactions. The fast reaction was assigned to cleavage of one of two DNA strands, which increased the flexibility of the DNA. The slow reaction corresponded to cleavage at or near the first cleavage site, thereby shortening the DNA stem. The presence of an intercalator, including ethidium, propidium, 9‐aminoacridine, and proflavine, inhibited the first step of the cleavage reaction. One of the possible reasons for the observed inhibition might be a change in the DNA conformation near the intercalation site. Intercalation caused an unwinding and elongation of the DNA and resulted in changes in the location of the H atoms of the sugar moiety, which is known to be the main site at which hydroxyl radicals react.     

Synthesis of Nucleobase‐Functionalized Carbon Nanotubes and Their Hybridization with Single‐Stranded DNA

For the first time ssDNA (25‐aptamer of mixed dA, dT, dG, and dC) was wrapped around functionalized single‐walled carbon nanotubes (SWCNTs), whose external surfaces were attached to multiple triazole‐(ethylene glycol)‐dA ligands. This method of hybridization involved the formation of hydrogen bonds between dT of ssDNA and dA of functionalized SWCNTs. It deviates from the reported π–π stacking between the nucleobases of DNA and the external sidewalls of nanotubes. The structural properties of the functionalized SWCNTs and its ssDNA complex were characterized by spectroscopic (including CD and Raman), thermogravimetric, and microscopic (TEM) methods. The results thus obtained establish a new platform of DNA delivery by use of nanotubes as a new vehicle with great potential in biomedical applications and drug development.