An electrochemical aptasensor for thrombin detection based on the recycling of exonuclease III and double-stranded DNA-templated copper nanoparticles assisted signal amplification

In this paper, we report an improved electrochemical aptasensor based on exonuclease III and double-stranded DNA (dsDNA)-templated copper nanoparticles (CuNPs) assisted signal amplification. In this sensor, duplex DNA from the hybridization of ligated thrombin-binding aptamer (TBA) subunits and probe DNA can act as an effective template for the formation of CuNPs on the electrode surface, so copper ions released from acid-dissolution of CuNPs may catalyze the oxidation of ο-phenylenediamine to produce an amplified electrochemical response. In the presence of thrombin, a short duplex domain with four complementary base pairs can be stabilized by the binding of TBA subunits with thrombin, in which TBA subunit 2 can be partially digested from 3′ terminal with the cycle of exonuclease III, so the ligation of TBA subunits and the subsequent formation of CuNPs can be inhibited. By electrochemical characterization of dsDNA-templated CuNPs on the electrode surface, our aptasensor can display excellent performances for the detection of thrombin in a broad linear range from 100 fM to 1 nM with a low detection limit of 20.3 fM, which can also specially distinguish thrombin in both PBS and serum samples. Therefore, our aptasensor might have great potential for clinical diagnosis of biomarkers in the future.     

纳米金颗粒增强信号的电化学生物传感器用于谷胱甘肽和半胱氨酸的检测

构建了一种高灵敏检测谷胱甘肽(GSH)和半胱氨酸(Cys)的新型电化学生物传感器. 先将富含T碱基的DNA1和DNA2探针分别修饰在金电极和纳米金颗粒(AuNPs)上, 再加入Hg²⁺, 通过形成T-Hg²⁺-T结构使AuNPs结合到金电极表面. 当加入GSH(或Cys)后, GSH(或Cys)可以竞争结合T-Hg²⁺-T结构中的Hg²⁺, 使AuNPs离开电极表面. 由于AuNPs上修饰的DNA探针能够静电吸附大量电活性物质六氨合钌(RuHex), 因此该过程可引起计时电量信号的显著变化, 据此实现了GSH(或Cys)的高灵敏检测. 该传感器的检出限达10 pmol/L, 比荧光法或比色法降低了2~3个数量级. 实验结果表明, 该传感器具有较好的选择性.     

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.     

纳米金胶增强葡萄糖生物传感器的构建

将纳米金胶(AuNPs)和羟基磷灰石(HAp)按一定比例混合制备了新型复合膜用于葡萄糖氧化酶(GOD)的固定,构建了高灵敏的葡萄糖传感器。由于纳米金胶的存在,葡萄糖氧化酶的直接电化学性质得以增强,在去除氧气的PBS(pH 7.0)介质中,固定在复合膜内的GOD表现出一对良好的氧化还原峰。在饱和氧气条件下,当加入一定量的葡萄糖时,由于GOD催化葡萄糖氧化消耗溶液中的溶解氧,-0.8 V处溶解氧的还原峰电流降低,且峰电流降低的量与葡萄糖浓度在0.02～1.62 mmol/L范围内呈线性相关,检出限为5.0μmol/L,检测灵敏度达9.91 mA.mol-1.L,可实现对葡萄糖的快速检测。     

Electrochemical DNA nano-biosensor for the detection of genotoxins in water samples简

In the present study, a disposable electrochemical DNA nano-biosensor is proposed for the rapid detection of genotoxic compounds and bio-analysis of water pollution. The DNA nano-biosensor is prepared by immobilizing DNA on Au nanoparticles and a self-assembled monolayer of cysteamine modified Au electrode. The assembly processes of cysteamine, Au nanoparticles and DNA were characterized by cyclic voltammetry （CV）. The Au nanoparticles enhanced DNA immobilization resulting in an increased guanine signal. The interaction of the analyte with the immobilized DNA was measured through the variation of the electrochemical signal of guanine by square wave voltammetry （SWV）. The biosensor was able to detect the known genotoxic compounds： 2-anthramine, acridine orange and 2- naphthylamine with detection limits of 2, 3 and 50 nmol/L, respectively. The biosensor was also used to test actual water samples to evaluate the contamination level. Additionally, the comparison of results from the classical genotoxiciw bioassay has confirmed the applicability of the method for real samoles.     

New DNA Technology and the DNA Biosensor

Abstract

Recently there has been an increasing demand to produce systems for the detection of specific DNA sequences that are amenable to non-specialised laboratories. This demand has led to many innovative and novel approaches to DNA analysis which may collectively be termed ′new DNA technology′. Here, we review these advances in relation to their applicability for the production of a DNA biosensor. The present state of the art is described and future possibilities are considered.     

Amplified inhibition of the electrochemical signal of ferrocene by enzyme-functionalized graphene oxide nanoprobe for ultrasensitive immunoassay

A nanoprobe-induced signal inhibition mechanism was designed for ultrasensitive electrochemical immunoassay at a chitosan-ferrocene (CS-Fc) based immunosensor. The nanoprobe was prepared by covalently loading signal antibody and high-content horseradish peroxidase (HRP) on the graphene oxide (GO) nanocarrier. The immunosensor was prepared through the stepwise assembly of gold nanoparticles (Au NPs) and capture antibody at a CS-Fc modified electrode. After sandwich immunoreaction, the GO-HRP nanoprobes were quantitatively captured onto the immunosensor surface and thus induced the production of a layer of insoluble film through the enzymatically catalytic reaction of the HRP labels. Both the dielectric immunocomplex formed on the immunosensor surface and the enzymatic precipitate with low electroconductivity led to the electrochemical signal decease of the Fc indicator, which was greatly amplified by the multi-enzyme signal amplification of the nanoprobe. Based on this amplified signal inhibition mechanism, a new ultrasensitive electrochemical immunoassay method was developed. Using carcinoembryonic antigen as a model analyte, this method showed a wide linear range over 5 orders of magnitude with a detection limit down to 0.54 pg/mL. Besides, the immunosensor showed good specificity, acceptable reproducibility and stability as well as satisfactory reliability for the serum sample analysis.     

A Simple and Highly Sensitive Electrochemical Biosensor for microRNA Detection Using Target‐Assisted Isothermal Exponential Amplification Reaction

A simple and highly sensitive electrochemical biosensor for microRNA (miRNA) detection was successfully developed by integrating a target‐assisted isothermal exponential amplification reaction (EXPAR) with enzyme‐amplified electrochemical readout. The binding of target miRNA with the immobilized linear DNA template generated a part duplex and triggered primer extension reaction to form a double‐stranded DNA. Then one of the DNA strands was cleaved by nicking endonuclease and extended again. The short fragments with the same sequence as the target miRNA except for the replacement of uridines and ribonucleotides with thymines and deoxyribonucleotides could be displaced and released. Hybridization of these released DNA fragments with other amplification templates and their extension on the templates led to target exponential amplification. Integrating with enzyme‐amplified electrochemical readout, the electrochemical signal decreases with the increasing target microRNA concentration. The method could detect miRNA down to 98.9 fM with a linear range from 100 fM to 10 nM. The fabrication and binding processes were characterized with cyclic voltammetry and electrochemical impedance spectroscopy. The specificity of the method allowed single‐nucleotide difference between miRNA family members to be discriminated. The established biosensor displayed excellent analytical performance toward miRNA detection and might present a powerful and convenient tool for biomedical research and clinic diagnostic application.     

Chemiluminescent detection of DNA hybridization using gold nanoparticles as labels

A chemiluminescent (CL) detection method has been developed for DNA hybridization. The assay relies on a sandwich-type DNA hybridization in which gold nanoparticles modified with alkylthiol-capped oligonucleotide strands are used as probes to monitor the presence of the specific target DNA. The , which is the dissolving product of the gold nanoparticles anchored on the DNA hybrids, serves as an analyte in the H₂O₂–luminol– CL reaction for the indirect measurement of the target DNA. The combination of the remarkable sensitivity of the CL analysis with the large number of released from each DNA hybrid allows a detection limit at levels as low as 0.1 pM of the target DNA. Moreover, with a further silver amplification step, the detection limit will be pushed down to the femtomolar domain.      

A sensitive electrochemical aptasensor for ATP detection based on exonuclease III-assisted signal amplification strategy

A target-induced structure-switching electrochemical aptasensor for sensitive detection of ATP was successfully constructed which was based on exonuclease III-catalyzed target recycling for signal amplification. With the existence of ATP, methylene blue (MB) labeled hairpin DNA formed G-quadruplex with ATP, which led to conformational changes of the hairpin DNA and created catalytic cleavage sites for exonuclease III (Exo III). Then the structure-switching DNA hybridized with capture DNA which made MB close to electrode surface. Meanwhile, Exo III selectively digested aptamer from its 3′-end, thus G-quadruplex structure was destroyed and ATP was released for target recycling. The Exo III-assisted target recycling amplified electrochemical signal significantly. Fluorescence experiment was performed to confirm the structure-switching process of the hairpin DNA. In fluorescence experiment, AuNPs–aptamer conjugates were synthesized, AuNPs quenched fluorescence of MB, the target-induced structure-switching made Exo III digested aptamer, which restored fluorescence. Under optimized conditions, the proposed aptasensor showed a linear range of 0.1–20 nM with a detection limit of 34 pM. In addition, the proposed aptasensor had good stability and selectivity, offered promising choice for the detection of other small molecules.     

磁性纳米粒子在生物传感器中的应用研究进展

磁性纳米粒子是一种新型纳米材料,可应用于各种生物活性物质如蛋白质、DNA等的富集和分离,药物的磁靶向,以及疾病的诊断和治疗等许多领域。由于磁性纳米粒子有着独特的化学和物理性能,已经成功应用到磁控生物传感器、DNA传感器、蛋白质传感器、酶传感器以及其它类型的生物传感器中,并显著提高了生物传感器检测的灵敏度、缩短了生化反应的时间和提高检测的通量,为生物传感器领域开辟了广阔的应用前景。本文概述了磁性纳米粒子在生物传感器中的应用研究进展。     

纳米金颗粒增强信号的表面等离子体共振生物传感器用于甲氧檗因高灵敏检测的研究

报道了一种基于表面等离子体共振(SPR)生物传感器的高灵敏检测抗癌药物甲氧檗因的新方法. 分别在纳米金颗粒和金膜表面修饰富含腺嘌呤(A)的DNA链, 当存在甲氧檗因时, 由于一个甲氧檗因分子可与4个A碱基相结合, 从而使得修饰在纳米金颗粒和金膜表面的DNA形成稳定的双链结构, 进而将功能化纳米金颗粒捕获在金膜表面. 由于纳米金颗粒与金膜之间的电场耦合效应可增强SPR信号, 从而可实现对小分子甲氧檗因的高灵敏、特异性检测. 本方法的检测下限低至0.07 pmol/L, 相对比色法和荧光法而言, 降低了约5～6个数量级. 以4种药物(盐酸小檗碱、青霉素G、硫酸庆大霉素、5-氟尿嘧啶)作为对照考察了该传感器的选择性, 结果表明该传感器具有较好的选择性.     

Staining-free gel electrophoresis-based multiplex enzyme assay using DNA and peptide dual-functionalized gold nanoparticles

We report a simple staining-free gel electrophoresis method to simultaneously probe protease and nuclease. Utilizing gold nanoparticles (Au-NPs) dual-functionalized with DNA and peptide, the presence and concentration of nuclease and protease are determined concurrently from the relative position and intensity of the bands in the staining-free gel electrophoresis. The use of Au-NPs eliminates the need for staining processes and enables naked eye detection, while a mononucleotide-mediated approach facilitates the synthesis of DNA/peptide conjugated Au-NPs and simplifies the operation procedures. Multiplex detection and quantification of DNase I and trypsin are successfully demonstrated.     

Colloid Au-enhanced DNA immobilization for the electrochemical detection of sequence-specific DNA

We use colloidal Au to enhance the DNA immobilization amount on a gold electrode and ultimately lower the detection limit of our electrochemical DNA biosensor. Self-assembly of approximately 16-nm diameter colloidal Au onto a cysteamine modified gold electrode resulted in an easier attachment of an oligonucleotide with a mercaptohexyl group at the 5′-phosphate end, and therefore an increased capacity for nucleic acid detection. Quantitative results showed that the surface densities of oligonucleotides on the Au colloid modified gold electrode were approximately (1–4)×10¹⁴ molecules cm⁻². Hybridization was induced by exposure of the ssDNA-containing gold electrode to ferrocenecarboxaldehyde labeled complementary ssDNA in solution. The detection limit is 5×10⁻¹⁰ mol l⁻¹ of complementary ssDNA, which is much lower than our previous electrochemical DNA biosensors. The Au nanoparticle films on the Au electrode provide a novel means for ssDNA immobilization and sequence-specific DNA detection.     

Enzyme-free surface plasmon resonance aptasensor for amplified detection of adenosine via target-triggering strand displacement cycle and Au nanoparticles

Herein, we combine the advantage of aptamer technique with the amplifying effect of an enzyme-free signal-amplification and Au nanoparticles (NPs) to design a sensitive surface plasmon resonance (SPR) aptasensor for detecting small molecules. This detection system consists of aptamer, detection probe (c-DNA1) partially hybridizing to the aptamer strand, Au NPs-linked hairpin DNA (Au-H-DNA1), and thiolated hairpin DNA (H-DNA2) previously immobilized on SPR gold chip. In the absence of target, the H-DNA1 possessing hairpin structure cannot hybridize with H-DNA2 and thereby Au NPs will not be captured on the SPR gold chip surface. Upon addition of target, the detection probe c-DNA1 is forced to dissociate from the c-DNA1/aptamer duplex by the specific recognition of the target to its aptamer. The released c-DNA1 hybridizes with Au-H-DNA1 and opens the hairpin structure, which accelerate the hybridization between Au-H-DNA1 and H-DNA2, leading to the displacement of the c-DNA1 through a branch migration process. The released c-DNA1 then hybridizes with another Au-H-DNA1 probe, and the cycle starts anew, resulting in the continuous immobilization of Au-H-DNA1 probes on the SPR chip, generating a significant change of SPR signal due to the electronic coupling interaction between the localized surface plasma of the Au NPs and the surface plasma wave. With the use of adenosine as a proof-of-principle analyte, this sensing platform can detect adenosine specifically with a detection limit as low as 0.21 pM, providing a simple, sensitive and selective protocol for small target molecules detection.     

MicroRNAs电化学生物传感器在乳腺癌检测中的研究进展

MicroRNAs是一类内源性非编码小RNA分子,可调控靶基因的表达.特异性microRNAs的失调在诸如癌症、心血管疾病、免疫疾病、神经退行性疾病和皮肤疾病等的发展过程中起着关键作用,常作为疾病早期诊断和预后的生物标志物.电化学生物传感器由于其灵敏、快速、成本低等优势,已经成为传统microRNAs检测方法的一种很有...     

基于切刻内切酶信号放大的电致化学发光DNA生物传感器的研究

利用切刻内切酶的酶切作用实现信号放大,结合量子点高效的电化学发光性能,构建了一种新型电化学发光DNA生物传感器.将捕获探针DNA(c-DNA)通过自组装的方式固定到金电极表面,后与目标DNA(t-DNA)互补杂交形成双链DNA,利用切刻内切酶Nt.BstNBI特异性识别双链上的酶切位点(5'-GAGTC-3'),然后在c-DNA相应的切割位点(识别序列3'端后的4个碱基处)对其进行剪切,释放出目标链,参与下一轮的杂交及酶切,通过目标物的循环利用,实现信号放大.利用N-羟基琥珀酰亚胺(N HS)和1-乙基-3-3-二甲基氨丙基碳化二亚胺(EDC)活化羧基化CdTe量子点表面的羧基,与电极表面残留的c-DNA末端的氨基共价交联,通过测定捕获的量子点的电化学发光信号对目标DNA进行检测.优化后的检测条件为:c-DNA浓度1 μmol/L,杂交时间60 min,Nt.BstNBI浓度0.5 U/μL,酶切反应时间4h.在优化条件下,目标DNA浓度在2.0×10-13～2.0×10-11 mol/L范围内,其对数与电化学发光强度呈线性关系,检出限为7.3×10-14 mol/L.人体血样加标回收率为96.4％～108.0％.     

利用互补核酸杂交富集金胶实现信号扩增的电化学凝血酶蛋白生物传感器研究

介绍了一种利用互补核酸杂交富集金胶实现信号扩增的蛋白质生物传感器. 以凝血酶蛋白为研究对象, 利用凝血酶蛋白相对应的两段核酸适配体, 将适配体Ⅰ固定在磁性颗粒上, 用于特异性地捕获蛋白, 将适配体Ⅱ标记金胶作为检测信标. 由凝血酶蛋白和相对应的两段核酸适配体构建三明治结构的凝血酶蛋白生物传感器. 另外, 再通过信标金胶上过剩的核酸适配体链与另一段标记有金胶的互补核酸进一步杂交, 获得金胶的选择性聚集, 实现了信号扩增. 通过信号扩增, 使此传感器的灵敏度大大提高, 对凝血酶蛋白的检测下限可达到4.52×10-15 mol/L. 平行测定浓度为7.47×10-14 mol/L的凝血酶8次, 其RSD为3.0%. 该生物传感器对凝血酶蛋白有很好的特异性, 其它蛋白如溶菌酶和牛血清白蛋白的存在对于检测没有影响.     

Catalytic amplification based on hole-transporting materials as efficient metal-free electrocatalysts for non-enzymatic glucose sensing

Hole-transporting materials with tunable structures and properties are mainly applied in organic light-emitting diodes as transport layer. But their catalytic properties as signal amplifiers in biological assays are seldom reported. In this paper, a starburst molecule, 4,4,4″-tri(N-carbazolyl)-triphenylamine (TCT), containing a triphenylamine as the central core and three carbazoles as the peripheral functional groups was designed and synthesized. Subsequently, the hole-transporting material based on the TCT polymer, poly(TCT) (PTCT), was achieved via a low-cost electrochemical method and exploited as an efficient metal-free electrocatalyst for non-enzymatic glucose detection. Here, this hole-transporting material served three purposes: electrochemical recognition (owing to hydrogen bonding interaction and the biomimetic microenvironment created by the polymer), electrocatalysis (owing to the hole-transporting capability of triphenylamine and the catalytic property of carbazole), and signal amplification (owing to energy migration along the conductive polymer backbone). The electrocatalytic and sensing performances of the sensor based on PTCT were evaluated in detail. Results revealed that the PTCT film could efficiently catalyze the oxidation of glucose at a less-positive potential (+0.20 V) in the absence of any enzymes. The response to glucose was linear in the concentration range of 1.0–6000 μM, and the detection limit was 0.20 μM. With good stability and selectivity, the proposed sensor could be feasibly applied to detect glucose in practical samples. The encouraging sensing performances suggest that the hole-transporting material is one of the promising biomimetic catalysts for electrocatalysis and relevant fields.     

Graphene oxide-based biosensor for sensitive fluorescence detection of DNA based on exonuclease III-aided signal amplification

Based on the super fluorescence quenching efficiency of graphene oxide and exonuclease III aided signal amplification, we develop a facile, sensitive, rapid and cost-effective method for DNA detection. In the presence of target DNA, the target-probe hybridization forms a double-stranded structure and exonuclease III catalyzes the stepwise removal of mononucleotides from the blunt 3′ termini of probe, resulting in the recycling of the target DNA and signal amplification. Therefore, our proposed sensor exhibits a high sensitivity towards target DNA with a detection limit of 20 pM, which was even lower than previously reported GO-based DNA sensors without enzymatic amplification, and provides a universal sensing platform for sensitive detection of DNA.