Current signal amplification strategies in aptamer-based electrochemical biosensor:A review

Due to their high specificity and affinity towards various targets,along with other unique advantages such as stability and low cost,aptamers are widely applied in analytical techniques.A typical aptamerbased electrochemical biosensor is composed of a aptamer as the biological recognition element and transducer conve rting the biologic interaction into electrical signals for the quantitative measure ment of targets.Improvement of the sensitivity of a biosensor is significantly important in order to achieve the detection of biomolecules with low abundance,and different amplification strategies have been explored.The strategies either employ nanomaterials such as gold nanoparticles to construct electrodes which can trans fer the biological reactions more efficiently,or attempt to obtain enha nced signal through multi-labeled carriers or utilize enzyme mimics to catalyze redox cycling.This review discusses recent advances in signal amplification methods and their applications.Critical assessment of each method is also considered.     

电化学DNA生物传感器*

对特异DNA序列的检测在基因相关疾病的诊断、军事反恐和环境监测等方面均具有非常重要的意义,DNA传感器的研究就是为了满足对特异DNA序列的快速、便捷、高灵敏度和高选择性检测的需要。近年来涌现出了多种传感策略,根据检测方法的不同可以大致分为光学传感器、电化学传感器、声学传感器等。由于电化学检测方法本身所具有的灵敏、快速、低成本和低能耗等特点,电化学DNA传感器已成为一个非常活跃的研究领域并在近几年中得到了快速发展。本文概括了近年来在DNA传感器的重要分支——电化学DNA传感器领域内的一些重要进展,主要包括DNA探针在传感界面上的固定方法和各种电化学DNA杂交信号的检测方法。     

Simultaneous electrochemical DNA hybridization assay for PAT and FMV 35S gene sequence using quantum dots as labels

An electrochemical method for the simultaneous detection of two different DNA sequences from PAT and FMV 35S gene sequence using CdS and PbS quantum dots （QDs） as labels was described. The QDs were readily functionalized with oligonucleotides as electrochemical DNA probes and selectively hybridized to the complementary sequences immobilized on the microplate. The QDs anchored on the hybrids were dissolved in the solution by the oxidation of HNO3 and further detected by a sensitive differential pulse anodic stripping voltammetric method （DPASV）. The DPASV signals of the oxidation of Cd^2＋ and Pb^2＋ ions present in the solution were different and reflected the identity of corresponding ssDNA targets sequences.     

Parallel Detection of Different DNA Sequences on One Gold Electrode

Motivated by the potential of electrochemical techniques to analyze hybridization events fast and in a simple and cost‐effective way we present here a detection system allowing a parallel electrochemical DNA analysis. For this purpose different probe DNA strands have been immobilized on one electrode. By the use of two different target DNA sequences, both marked with the redox active methylene blue, we can show that hybridization with the complementary probe sh“NA strands can occur without steric hindrance. Each target has been recognized down to 3nM with a very high specificity of the sensor. In addition, we can detect two different ssDNA targets labeled with different redox active molecules, methylene blue and ferrocene, on one sensor surface simultaneously.     

SNPs Feasibility of Nonlabeled Oligonucletides on Using Electrochemical Sensing

We have demonstrated an electrochemical gene chip protocol for the SNPs detection of nonlabeled DNA. Using an array consisting of streptavidin‐modified gold electrodes, probe DNA were attached through the application of a direct electric field. Electrochemical response changes originating from the hybridization of nucleic acids to protein‐bound nucleic acids using soluble mediators in K₃Fe(CN)₆ solution could then be observed. The electrochemical protocol developed showed high sensitivity and good reproducibility in the detection of DNA hybridization. Significant changes in electrochemical signals were also observed when using target DNA with a single base mismatch, indicating the applicability of this method to single nucleotide polymorphisms (SNPs) detection.     

Sequence Specific Electrochemical DNA Detection Based on Solution‐Phase Competitive Hybridization

We report a novel electrochemical method for detecting sequence‐specific DNA based on competitive hybridization that occurs in a homogeneous solution phase instead of on a solution‐electrode interface as in previously reported competition‐based electrochemical DNA detection schemes. The method utilizes the competition between the target DNA (t‐DNA) and a ferrocene‐labeled peptide nucleic acid probe (Fc‐PNA) to hybridize with a probe DNA (p‐DNA) in solution. The neutral PNA backbone and the electrostatic repulsion between the negatively‐charged DNA backbone and the negatively‐charged electrode surface are then exploited to determine the result of the competition through measurement of the electrochemical signal of Fc. Upon the introduction of the t‐DNA, the stronger hybridization affinity between the t‐DNA and p‐DNA releases the Fc‐PNA from the Fc‐PNA/p‐DNA hybrid, allowing it to freely diffuse to the negatively charged electrode to produce a significantly enhanced electrochemical signal of Fc. Therefore, the presence of the t‐DNA is indicated by the appearance or enhancement of the electrochemical signal, rendering a signal‐on DNA detection, which is less susceptible to false positive and can produce more reliable results than signal‐off detection methods. All the competitive hybridizations occur in a homogeneous solution phase, resulting in very high hybridization efficiency and therefore extremely short assay time. This simple and fast signal‐on solution‐competition‐based electrochemical DNA detection strategy has promising potential to find application in fields such as nucleic acid‐based point‐of‐care testing.     

High Sensitive Electrochemical Detection of Sequence‐Specific DNA Using Low Current Voltammetry

In this article, we report on efforts to construct a high sensitive electrochemical sensor with immobilized sandwich‐type DNA borne ferrocene (Fc) head for sequence‐specific DNA detection using ultramicroelectrode and low current voltammetry. Based on the difference in deformability between the bending rigid complementary DNA double helix and its anomalous flexile mismatches, the fully complementary target can be distinguished from mismatched targets including the single‐base mismatched target. Detection limit estimated as the amount of DNA is observed to be 100 fM via low current voltammetry. The method offers great promise of high sensitivity and selectivity simultaneously for effective gene identification.     

Nucleoside–metallacarborane conjugates for multipotential electrochemical coding of DNA

Metallacarboranes as electrochemical labels are proposed. The electrochemical properties of nucleoside conjugates, derivatives of thymidine (T), 2′-deoxycytidine (dC), 2′-deoxyadenosine (dA) and 2′-deoxyguanosine (dG), containing metallacarborane complex of cobalt or iron are described. A multielectrochemical detection using specific metallacarborane tags is shown. The proposed labelling of nucleosides lays the foundations for electrochemical coding of DNA with metallacarborane complexes and simultaneous detection of several DNA targets.     

Ultrasensitive assay of ctDNA based on DNA triangular prism and three-way junction nanostructures

Circulating tumor DNA(ctDNA) refers to a class of acellular nucleic acids carrying genetic features of primary tumor,which can be regarded as a promising noninvasive biomarker for cancer diagnosis.The development of ctDNA assay is an important component of liquid biopsy.In this study,we have fabricated a novel electrochemical strategy for ultrasensitive detection of ctDNA combining the merits of strand displacement amplification and DNA nanostructures.Stable DNA triangular prism is firstly selfassembled and modified on the electrode surface.After target initiated strand displacement polymerization reaction,the generated DNA product helps the formation of three-way junction nanostructure on triangular prism,which localizes electrochemical species.By carefully investigating the electrochemical responses,the limit of detection(LOD) for ctDNA assay as low as 48 amol/L is achieved.This proposed electrochemical biosensor shows great potential for clinical applications.     

Electrochemical Signal Enhancer Fabricated Using Lysine‐rich Peptide for Ultrasensitive Electrochemical DNA Biosensor Analysis

Here we present a novel design of electrochemical signal enhancer to increase the detection sensitivity of electrochemical DNA biosensors. The key element of this enhancer is a lysine‐rich peptide (LRP). Its C‐terminal is conjugated with a planer molecule, being able to intercalate into the base pairs of probe‐target duplexes. The lysine residues of LRP are covalently linked with electrochemical signal indicators, acting as an assembly of electrochemical signal indicators. Experimental results proved the feasibility of the novel design. We have examined the effects of the numbers of lysine residues and the hybridization conditions on the detection sensitivity. The optimization procedures have led to significant sensitivity enhancement, and the LOD (limit of detection) has been determined to be 1.4 amol. This enhancer demonstrates advantages of easy operation, simple instrumentation, and high exemption from environmental influence.     

基于DNA聚合酶I的新型电化学传感器及其胰腺癌K-ras基因点突变检测

本文构建DNA聚合酶I的新型DNA电化学传感器,将捕获探针通过Au-S键固定于Au基底表面,与互补靶序列杂交至点突变前一个碱基,通过DNA聚合酶Ⅰ将dUTP-biotin连接在目标DNA的检测位点,再与avidin-HRP反应,而后测定在TMB溶液中的电化学特性. 结果表明,DNA电化学传感电极的检测电流值与K-ras突变型基因浓度（1.0×10^-15 ~ 1.0×10^-10 mol·L^-1）对数呈良好的线性关系,且灵敏度高,特异性较佳.     

The challenge of long-term stability for nucleic acid-based electrochemical sensors

Nucleic acid-based electrochemical sensors are a versatile technology enabling affinity-based detection of a great variety of molecular targets, regardless of inherent electrochemical activity or enzymatic reactivity. Additionally, their modular interface and ease of fabrication enable rapid prototyping and sensor development. However, the technology has inhibiting limitations in terms of long-term stability that have precluded translation into clinically valuable platforms like continuous molecular monitors. In this opinion, we discuss published methods to address various aspects of sensor stability, including thiol-based monolayers and anti-biofouling capabilities. We hope the highlighted works will motivate the field to develop innovative strategies for extending the long-term operational life of nucleic acid-based electrochemical sensors.     

Electrochemical Monitoring of DNA Hybridization by Multiwalled Carbon Nanotube Based Screen Printed Electrodes

The application of multiwalled carbon nanotube (MWCNT) based screen printed graphite electrodes (SPEs) was explored in this study for the electrochemical monitoring of DNA hybridization related to specific sequences on Hepatitis B virus (HBV) DNA. After the microscopic characterization of bare MWCNT‐SPEs and DNA immobilized ones was performed, the optimization of assay has been studied. The development of screen printing process combined with nanomaterial based disposable sensor technology leads herein a great opportunity for DNA detection using differential pulse voltammetry (DPV) by measuring the guanine oxidation signal observed at +1.00 V in the presence of DNA hybridization between HBV probe and its complementary, target. The detection limit estimated for signal to noise ratios =3 corresponds to 96.33 nM target concentration in the 40 μL samples. The advantages of carbon nanotube based screen printed electrode used for electrochemical monitoring of DNA hybridization are discussed with sensitivity, selectivity and reproducibility in comparison with previous nanomaterial based electrochemical transducers developed for DNA or other biomolecular recognitions.     

Optical and electrochemical DNA nanobiosensors

In the past two decades, nanoscale advanced materials have been explored for biosensing molecules, so new horizons have opened up for identifying and quantifying biomolecules, and possible early diagnosis of diseases.DNA nanobiosensors show promise. This article provides an overview on their optical and electrochemical aspects. We discuss recent progress in this field, describing basic concepts of molecular beacons and quantum dots as optical nano-imaging systems. Also, carbon nanotubes provide a platform for development and advancement of electrochemical DNA nanobiosensors, which are increasingly being implemented as robust tools for detection in biomedical sciences.     

功能化纳米金放大的DNA电化学传感器研究

研究了DNA夹心杂交和直接杂交体系,将功能化纳米金引入到标记有生物素的杂交双链上,制成具有电化学活性和纳米金放大作用的DNA电化学传感器,采用循环伏安法测试.在夹心杂交体系中,靶点DNA浓度与阳极峰电流关系曲线的相对标准偏差为3.0%～13.0%,在浓度为6.9×10^-3～0.14nmol/L范围内得到良好的线性关系,检测限达到2.0×10^-3nmol/L,实现了对单碱基突变的高灵敏检测和序列识别.直接杂交检测限为2.5×10^-4mol/L,分别在2.5×10^-4～5.0×10^-3nmol/L和5.0×10^-3～10nmol/L范围内得到峰电量与浓度的良好线性关系.并比较这两种体系.     

非标记电化学DNA杂交指示剂

电化学DNA杂交检测技术因其快速、灵敏、低消耗和易于操作等优点而在临床医学、环境监测和药物分析等领域受到普遍关注。电化学DNA杂交指示剂是DNA电化学杂交传感器的重要组成部分,能与单链DNA和双链DNA通过不同的模式和作用力进行差异性结合。本文介绍了电化学杂交指示剂的定义及其在DNA电化学传感器中的重要性,根据分子结构上的特征差异,将非标记型电化学杂交指示剂分为有机染料（荧光素）、药物分子和金属配合物三类,并从中选取了各个类型中具有代表性的指示剂对它们的工作原理、研究进展和应用现状进行了比较和评述。对杂交指示剂的设计和开发前景,特别是满足基因芯片应用等方面做出了展望。     

Rapid and sensitive DNA target detection using enzyme amplified electrochemical detection based on microchip

A rapid and sensitive DNA targets detection using enzyme amplified electrochemical detection (ED) based on microchip was described. We employed a biotin‐modified DNA, which reacted with avidin‐conjugated horseradish peroxidase (avidin–HRP) to obtain the HRP‐labeled DNA probe and hybridized with its complementary target. After hybridization, the mixture containing dsDNA‐HRP, excess ssDNA‐HRP, and remaining avidin–HRP was separated by MCE. The separations were performed at a separation voltage of +1.6 kV and were completed in less than 100 s. The HRP was used as catalytic labels to catalyze H₂O₂/o‐aminophenol reaction. Target DNA could be detected by the HRP‐catalyzed reduction with ED. With this protocol, the limits of quantification for the hybridization assay of 21‐ and 39‐mer DNA fragments were of 8×10^?12 M and 1.2×10^?11 M, respectively. The proposed method has been applied satisfactorily in the analysis of Escherichia coli genomic DNA. We selected the detection of PCR amplifications from the gene of E. coli to test the real applicability of our method. By using an asymmetric PCR protocol, we obtained ssDNA targets of 148 bp that could be directly hybridized by the single‐stranded probe and detected with ED.     

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.     

检测痕量Hg~(2+)的DNA电化学生物传感器

通过自组装方法将修饰有二茂铁基团的富T序列DNA分子(DNA-Fc)固定在金电极表面,得到了一种基于DNA修饰电极的电化学汞离子(Hg2+)传感器.当溶液中有Hg2+存在时,Hg2+可与修饰电极上DNA的T碱基发生较强的特异结合,形成T-Hg2+-T发卡结构,使DNA分子构象发生改变,其末端具有电化学活性的二茂铁基团远离电极表面,电化学响应随之发生变化.示差脉冲伏安法(DPV)结果显示:DNA末端二茂铁基团的还原峰在0.26V(vs饱和甘汞电极(SCE))附近,峰电流随溶液中Hg2+浓度的增加而降低;Hg2+浓度范围在0.1nmol·L-1-1μmol·L-1时,电流相对变化率与Hg2+浓度的对数呈现良好的线性关系.该修饰电极对Hg2+的检测限为0.1nmol·L-1,可作为痕量Hg2+检测的电化学生物传感器.干扰实验也表明,该传感器对Hg2+具有良好的特异性与灵敏度.     

Decorated DNA-Based Scaffolds as Lateral Flow Biosensors

Here we develop Lateral Flow Assays (LFAs) that employ as functional elements DNA-based structures decorated with reporter tags and recognition elements. We have rationally re-engineered tile-based DNA tubular structures that can act as scaffolds and can be decorated with recognition elements of different nature (i.e. antigens, aptamers or proteins) and with orthogonal fluorescent dyes. As a proof-of-principle we have developed sandwich and competitive multiplex lateral flow platforms for the detection of several targets, ranging from small molecules (digoxigenin, Dig and dinitrophenol, DNP), to antibodies (Anti-Dig, Anti-DNP and Anti-MUC1/EGFR bispecific antibodies) and proteins (thrombin). Coupling the advantages of functional DNA-based scaffolds together with the simplicity of LFAs, our approach offers the opportunity to detect a wide range of targets with nanomolar sensitivity and high specificity.