Amplified Electrochemical Response of Phenol by Oxygenation of Tyrosinase Coupling with Electrochemical‐chemical‐chemical Redox Cycle

In this work, an novel electrochemical‐chemical‐chemical (ECC) redox cycle was designed in an enzyme‐based sensor for acquiring additional signal amplification. The tyrosinase (Tyr) was entrapped in a sulfonated polyaniline?chitosan (SPAN?CS) composite which was used as a redox capacitor on a glass carbon electrode. Firstly, the substrate, phenol was catalyzed to catechol and further catalyzed to o‐benzoquinone by Tyr. Next, in the presence of Ru(NH₃)₆Cl₂, the reduced state of SPAN(SPAN_red) was reacted with o‐benzoquinone to form it's oxidized state (SPAN_ox) and catechol, then SPAN_ox was reduced back to SPAN_red by Ru(II) in the solution. Finally, the amplified anodic current of catechol was obtained on electrode through above ECC redox cycle system. In addition, the ECC redox cycling led to a high signal‐to‐background ratio. The voltammetric response showed excellent analytical performance to phenol over two linear range of 3.5 to 200.0 nmol L^?1 and 200.0 to 2000.0 nmol L^?1 with a high sensitivity of 2204 μA mM^?1. The detection limit was obtained to be 0.8 nmol L^?1 (S/N=3). Furthermore, the proposed approach exhibited good repeatability, stability and specificity, and could offer practicality in the detection of phenol in tap water.     

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.     

基于氧化还原循环信号放大技术的单分子电化学研究进展

现代分析科学的整体发展对分析方法的灵敏度、选择性以及快速响应等有了更高的要求。在单分子水平上实现对目标分子的检测及控制是化学家们长期以来梦寐以求的一项富有挑战性的前沿领域,也是近年来分析科学很重要的前沿发展方向。用电化学方法直接检测单分子面临的一项挑战是单个分子在氧化还原过程中得失电子产生的电流变化太小,现代仪器无法对如此小的电流进行识别。使电极表面氧化还原过程中的电子交换实现多次循环可以放大产生的电流,从而实现单分子水平的直接电化学分析。本文对近期通过循环电子交换过程放大电流信号的技术和装置进行了综述,将各类方法进行对比,并对单分子电化学未来的发展方向进行了展望。     

Molecular Imprint for Electrochemical Detection of Streptomycin Residues Using Enzyme Signal Amplification

We have designed a new molecularly imprinted co‐polymer (MIP) for the sensitive detection of streptomycin (STR) in food using enzymes as signal amplification. The MIP was fabricated via co‐polymerization of aniline and o‐phenylenediamine on gold substrate in the presence of STR as template. The assay is based on competitive binding of free STR and glucose oxidase‐labeled STR (GOx‐STR) to the imprinters on the MIP. On addition of glucose, hydrogen peroxide is formed that is detected by differential pulse voltammetry. Under optimal conditions, the decrease of the catalytic current is proportional to the STR concentration in the range from 0.01 to 10 ng mL^?1, with a detection limit (LOD) of 7.0 pg mL^?1 STR (at 3s_B). Intra‐ and inter‐assay coefficients of variation (CVs) are<10.5 %. The system was further validated and evaluated with STR‐spiked samples including honey and milk, and the recovery was between 82 and 124.2 %.     

基于杂交链式反应辅助多重信号放大的端粒酶灵敏检测

端粒酶是真核细胞维持端粒长度的关键逆转录酶,其生物活性的高低可以为多种癌症的临床诊断和预后治疗提供有价值的信息.本研究以人宫颈癌细胞(HeLa细胞)裂解液中的端粒酶为研究对象,通过借助杂交链式反应辅助多重信号放大策略,提出了一种新颖、灵敏的检测端粒酶电化学方法.首先将端粒酶的延伸引物自组装在金电极表面,当端粒酶存在时,端粒酶能够催化引物的延伸,产生与发卡环探针H1部分互补的序列,进而引发杂交链式反应,形成由两个发卡环探针(H1和H2)交替杂交而形成的DNA长链.由于H1和H2末端均修饰有生物素,加入链霉亲和素修饰辣根过氧化物酶后,辣根过氧化物酶被被连接到电极表面,催化邻苯二胺氧化生成2,3-二氨基吩嗪,产生显著的电化学信号.实验结果表明,本研究建立的端粒酶电化学检测方法高效、可行,线性范围宽,灵敏度高,可以检测每毫升10个HeLa细胞裂解液中的端粒酶.本方法具有较好的选择性,能有效区分端粒酶和对照蛋白.     

Simple and Sensitive Electrochemical DNA Detection of Primer Generation‐Rolling Circle Amplification

A new electrochemical sequence‐specific DNA detection platform based on primer generation‐rolling circle amplification (PG‐RCA), methylene blue (MB) redox indicator, and indium tin oxide (ITO) electrode is reported. In the presence of a specific target sequence, PG‐RCA, an isothermal DNA amplification technique, produced large amounts of amplicons in an exponential manner. In addition to the standard components, the reaction mixture contained MB, which bound with the PG‐RCA amplicons. End‐point electrochemical measurement by differential pulse voltammetry (DPV) was performed using ITO electrode. The amplicon‐bound MB resulted in a lower DPV signal than free MB due to a smaller diffusion coefficient as well as electrostatic repulsion between the negatively charged amplicon‐bound MB and ITO electrode. With simple assay design (recognition probe) and instrumentation (operating temperature at 37 °C and ITO electrode without the need for probe immobilization), this detection platform is well suited for point‐of‐care and on‐site testing. Real‐time measurement was also achieved by pretreating the ITO electrode with bovine serum albumin.     

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.     

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.     

纳米纤维聚苯胺在电化学电容器中的应用

采用脉冲电流方法（PGM）合成了具有纳米纤维结构的导电聚苯胺（PANI）.扫描电子显微镜对膜层观察表明, PANI膜是由直径约为100 nm的掺杂态聚苯胺纤维交织而成.以纳米纤维状聚苯胺组成电化学电容器,研究了其电化学电容性能,并与恒电流方法（GM） 制备的颗粒状PANI电容器性能进行了比较.结果表明,在相同的沉积电量下,PGM制备的纳米纤维状PANI电化学电容器比颗粒状PANI电化学电容器具有更大的电容容量,其电化学电容器的比电容可高达699 F•g－1,能量密度为54.6 Wh•kg－1.并且该电化学电容器具有良好的充放电性能和循环寿命.     

A Sensitive Electrochemical Immunosensor for α‐Fetoprotein Detection with Colloidal Gold‐Based Dentritical Enzyme Complex Amplification

A sensitive and specific electrochemical immunosensor was developed with α‐fetoprotein (AFP) as the model analyte by using gold nanoparticle label for enzymatic catalytic amplification. A self‐assembled monolayer membrane of mercaptopropionic acid (MPA) was firstly formed on the electrode surface through gold‐sulfur interaction. Monoclonal mouse anti‐human AFP was covalently immobilized to serve as the capture antibody. In the presence of the target human AFP, gold nanoparticles coated with polyclonal rabbit anti‐human AFP were bound to the electrode via the formation of a sandwiched complex. With the introduction of goat anti‐rabbit IgG conjugated with alkaline phosphatase, the dentritical enzyme complex was formed through selective interaction of the secondary antibodies with the colloidal gold‐based primary antibody at the electrode, thus affording the possibility of signal amplification for AFP detection. Current response arising from the oxidation of enzymatic product was significantly amplified by the dentritical enzyme complex. The current signal was proportional to the concentration of AFP from 1.0 ng mL^?1 to 500 ng mL^?1 with a detection limit of 0.8 ng mL^?1. This system could be extended to detect other target molecules with the corresponding antibody pairs.     

Electrochemical Detection of Duplex DNA Using Intercalation‐Triggered Decomplexation of Ferrocene with β‐Cyclodextrin

The redox peak of ferrocenylnaphthalene diimide used as a threading intercalator shifted positively due to the formation of its complex with β‐cyclodextrin. This complex collapsed upon the addition of double‐stranded DNA, and its redox potential shifted negatively. This behavior was applied for the homogenous detection of a polymerase chain reaction (PCR) product from Porphyromonas gingivalis, which is important for the diagnosis of periodontal disease, and its quantitative detection was achieved with a detection limit of 2.7 nM.     

A Novel L‐Cysteine Electrochemical Sensor Using Sulfonated Graphene‐poly(3,4‐Ethylenedioxythiophene) Composite Film Decorated with Gold Nanoparticles

By exploiting the electrostatic interaction between positively charged 3,4‐ethylenedioxythiophene cation radicals and negatively charged sulfonated graphene (SG) sheets, we prepared a poly(3,4‐ethylenedioxythiophene)‐sulfonated graphene (SG‐PEDOT) composite film by a one‐step electrochemical process. The composite was further decorated with gold nanoparticles (AuNPs) and employed as an electrode material for the detection of L ‐cysteine (Cys). The SG‐PEDOT composite film is shown to provide a rough surface for the electrodeposition of AuNPs and to improve substrate accessibility and interaction with Cys. Moreover, the AuNPs‐decorated composite exhibits better electrocatalytic performance than that of a SG‐PEDOT composite only. Under optimum experimental conditions, the amperometric current of the sensor is linearly related to the concentration of Cys in the 0.1 to 382 µM range, and the detection limit is 0.02 µM (at S/N=3). The modified electrode displays favorable selectivity, good stability and high reproducibility. The method was successfully applied to the detection of Cys in spiked human urine.     

Combination of Mass Signal Amplification and Isotope‐Labeled Alkanethiols for the Multiplexed Detection of miRNAs

We report a fast and sensitive method for the multiplexed detection of miRNAs by combining mass signal amplification and isotope‐labeled signal reporter molecules. In our strategy, target miRNAs are captured specifically by immobilized DNAs on gold nanoparticles (AuNPs), which carry a large number of small molecules, called amplification tags (Am‐tags), as the reporter for the detection of target miRNAs. For multiplexed detection, we designed and synthesized four Am‐tags containing 0, 4, 8, 12 isotopes so that they had same molecular properties but different molecular weights. By observing the mass signals of the Am‐tags on AuNPs decorated along with different probe DNAs, four types of miRNAs in a sample could be easily discriminated, and the relative amounts of these miRNAs could be quantified. The practicability of our strategy was further verified by measuring the expression levels of two miRNAs in HUVECs in response to different CuSO₄ concentrations.     

Sensitive Phenol Detection Using Tyrosinase‐Based Phenol Oxidation Combined with Redox Cycling of Catechol

This paper reports sensitive phenol detection using (i) tyrosinase (Tyr)‐based oxidation of phenol to catechol, combined with (ii) electrochemical‐chemical‐chemical (ECC) redox cycling involving Ru(NH₃)₆³⁺, catechol, and tris(2‐carboxyethyl)phosphine (TCEP). Phenol is converted into catechol by Tyr in the presence of dissolved O₂. Catechol then reacts with Ru(NH₃)₆³⁺, generating o‐benzoquinone and Ru(NH₃)₆²⁺. o‐Benzoquinone is reduced back to catechol by TCEP, and Ru(NH₃)₆²⁺ is accumulated over the course of the incubation. When Ru(NH₃)₆²⁺ is electrochemically oxidized to Ru(NH₃)₆³⁺, ECC redox cycling occurs. For simple phenol detection, bare ITO electrodes are used without modifying the electrodes with Tyr. The detection limit for phenol in tap water using Tyr‐based oxidation combined with ECC redox cycling is ca. 10^?9 M, while that using only Tyr‐based oxidation is ca. 10^?7 M.     

Evaluation of the Oxo‐bridged Dinuclear Ruthenium Ammine Complex as Redox Mediator in an Electrochemical Biosensor

The mediation of electron‐transfer by oxo‐bridged dinuclear ruthenium ammine [(bpy)₂(NH₃)Ru^III(µ‐O)Ru^III(NH₃)(bpy)₂]⁴⁺ for the oxidation of glucose was investigated by cyclic voltammetry. These ruthenium (III) complexes exhibit appropriate redox potentials of 0.131–0.09 V vs. SCE to act as electron‐transfer mediators. The plot of anodic current vs. the glucose concentration was linear in the concentration range between 2.52×10^?5 and 1.00×10^?4 mol L^?1. Moreover, the apparent Michaelis‐Menten kinetic (K_M^app) and the catalytic (K_cat) constants were 8.757×10^?6 mol L^?1 and 1,956 s^?1, respectively, demonstrating the efficiency of the ruthenium dinuclear oxo‐complex [(bpy)₂(NH₃)Ru^III(µ‐O)Ru^III(NH₃)(bpy)₂]⁴⁺ as mediator of redox electron‐transfer.     

MoS_2中空纳米球的制备及在超灵敏microRNA电化学生物传感器中的应用

采用模板法制备了二硫化钼中空球纳米材料,利用扫描电子电子显微镜、X射线衍射仪和Raman光谱仪对材料的形貌和结构进行表征.将适配体固定在金纳米粒子和二硫化钼共同修饰的电极上构建了一种新型的微小核糖核酸(microRNA)电化学生物传感器,采用循环伏安、微分脉冲伏安和电化学阻抗等技术对构筑的传感器进行表征.结果表明,microRNA浓度在1.0×10~(-10)~1.0×10~(-16)mol/L范围内峰电流(I)与microRNA浓度的负对数(-lgc)呈良好的线性,目标miRNA的检出限为0.55×10~(-16)mol/L.构建出的传感器具备选择性好、灵敏度高、稳定性强等特性,具有广阔的应用前景.     

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.     

Electrochemical Detection of Steroid Hormones Using a Nickel‐Modified Glassy Carbon Electrode

This paper demonstrates the development of an analytical method for detecting steroid hormones by coupling HPLC to electrochemical detection, using a nickel‐modified glassy carbon electrode. The method was evaluated in terms of sensitivity, linear dynamic range, limit of detection, and response stability. The developed method exhibited good figures of merit for the steroid hormones studied with no evidence of electrode fouling. As an example, the limit of detection (S/N=3) for E3 was 0.10 µM and the response precision (n=5) was 0.6 %. The application of the method for the analysis of a real river water sample is demonstrated.     

Electrochemiluminescence Sensor for Selective Preconcentration and Sensitive Detection of Napropamide Using Water‐Soluble Sulfonated Graphene

A novel electrochemiluminescence (ECL) sensor for napropamide determination was prepared using the water‐soluble sulfonated graphene (sulfonated‐G) as solid‐phase microextraction (SPME) material, based on selective preconcentration of target onto an electrode and followed by luminol ECL detection. The effects of pH, adsorption time, buffer solution and the luminescence agent on ECL intensity were optimized. Under the optimized conditions (pH 6; adsorption time 5 min; buffer solution pH 11.0 Na₂CO₃ aHCO₃; luminescence agent luminol; stirring speed 400 rpm), the lowest detection limits (1.0 µg L⁻¹) and good linear range (r²≥0.99) were obtained for the analyte, indicating the superior performance of Nafion/sulfonated‐G/GCE for detecting napropamide.