基于核酸适体电化学生物传感器用于腺苷的免标记检测

以纳米MnO2作为适体固定的构建平台,制备了一种基于核酸适体的新型腺苷电化学生物传感器.固定于电极表面的适体探针与目标腺苷杂交后使电极界面的结构发生改变,通过[Fe(CN)6]3-/4-氧化还原探针监测传感器表面电子传递电阻的变化,以此作为检测信号进行腺苷的免标记检测.表面电子传递电阻的变化值与腺苷浓度的对数在1.0×...     

Recent Progress on Highly Selective and Sensitive Electrochemical Aptamer-based Sensors

Highly selective, sensitive, and stable biosensors are essential for the molecular level understanding of many physiological activities and diseases. Electrochemical aptamer-based (E-AB) sensor is an appealing platform for measurement in biological system, attributing to the combined advantages of high selectivity of the aptamer and high sensitivity of electrochemical analysis. This review summarizes the latest development of E-AB sensors, focuses on the modification strategies used in the fabrication of sensors and the sensing strategies for analytes of different sizes in biological system, and then looks forward to the challenges and prospects of the future development of electrochemical aptamer-based sensors.     

Electrochemical Detection of DNA Melting Curves by Means of Heated Biosensors

This article reports about the detection of DNA melting curves at heated electrochemical biosensors. Osmium tetroxide‐bipyridine‐labeled target oligonucleotides are hybridized with probe oligonucleotides immobilized on gold electrodes. Then, the gold electrode is successively heated in order to measure a complete melting curve consisting of alternating current voltammetric signals. Melting temperatures ?_m, determined at various ionic strengths and in dependence on different numbers of base pair mismatches, have been compared with those obtained by means of UV spectrophotometry. The proposed method holds great promise for the fast and easy parallel detection of nucleic acids sequences on selectively heated electrode arrays. A stringent hybridization temperature can be easily adjusted in order to discriminate base pair mismatches.     

基于核酸适体检测ATP的酶联分析新方法

基于核酸适体对靶标的特异性识别和辣根过氧化物酶(HRP)的高效催化反应, 发展了一种用于检测三磷酸腺苷(ATP)的酶联核酸适体分析新方法. 核酸适体和靶标的特异性结合导致与核酸适体杂交的短链DNA解链, 解离的DNA通过杂交被固定在另一酶标板的DNA捕获. 解离的DNA预先标记了异硫氰酸荧光素(FITC)基团, FITC特异性结合HRP标记的FITC抗体, HRP作为信号传导元素催化四甲基二苯胺(TMB)底物显色, 通过颜色变化及450 nm波长处吸光度的变化检测ATP. 该方法对ATP具有良好的选择性, 检测不受其它物质如GTP, UTP和CTP的干扰, 且检测能在较复杂的试样(体积分数10%和50%的血清)中进行. 实验结果表明, 在ATP浓度为50~400 nmol/L范围内, 具有良好的线性关系, 检出限为26 nmol/L.     

基于计时库仑技术的可再生型三磷酸腺苷适配体电化学传感器的研究

构建了一种可再生型三磷酸腺苷(ATP)适配体计时库仑电化学传感器.将一条短链DNA通过AuS键自组装固定在电极表面, ATP的核酸适配体与该短链DNA杂交而结合在电极表面.带负电的DNA通过静电吸引结合电解液中的六氨合钌(RuHex)阳离子.当传感器和靶分子ATP孵育后,ATP与核酸适配体结合,使适配体链从电极表面解离,电极表面吸附的DNA量减少,结合RuHex的量随之降低.通过计时库仑技术检测RuHex响应信号降低的量 ,可以对ATP进行定量测定.此传感器的电化学响应信号与ATP浓度对数值呈线性关系,线性检测范围为0.001~100 μmol/L,检出限(S/N=3)为0.5 nmol/L.此传感器检测靶分子ATP后,可以通过简单的操作步骤再生,再生5次后的响应信号为初始信号的90%以上.采用此传感器检测大鼠脑透析液中ATP的含量为(19.2±3.7) nmol/L (n=3).     

用于赭曲霉毒素A检测的电化学适配体生物传感器的研究进展

赭曲霉毒素(Ochratoxin)是一类主要由曲霉菌和青霉菌产生的次生代谢产物,其中赭曲霉毒素A(OTA)的毒性最强。OTA相当稳定,常规的食品加工难以去除,若摄入受OTA污染的食品或药物会对人类造成严重的危害。实现对OTA的灵敏和快速检测是及早发现和处置OTA污染的关键。近年来,核酸适配体因其独特的优点,被作为抗体的替代物用于构建OTA电化学生物传感器。本文介绍了经典的OTA检测方法和基于适配体的电化学生物传感检测方法,从OTA电化学适配体传感器的适配体优化、新型材料应用以及生物信号放大技术的应用等三个方面总结了该生物传感技术的研究现状,并对其未来的发展进行了展望。     

基于核酸适配体的荧光法检测水胺硫磷和丙溴磷

建立了基于适配体的农药水胺硫磷和丙溴磷的荧光检测方法.采用可特异性识别水胺硫磷和丙溴磷、且5 '端标记荧光基团FAM的核酸适配体(F-ssDNA),与3 '末端标记猝灭基团DABCYL的短链序列(Q-ssDNA)互补杂交形成双链结构,荧光基团的荧光被淬灭,荧光信号很弱;此时加入靶分子,特异性结合核酸适配体,引起互补短链序列从双链结构中解离,使适配体荧光信号增强,基于此可实现水胺硫磷、丙溴磷的定量检测.优化后的检测条件为:将终浓度为25 nmol/L F-ssDNA与50 nmol/L Q-ssDNA在25℃孵育20 min,使二者杂交形成双链适配体探针复合物,加入等体积的农药样品孵育60 min,然后检测体系的荧光信号变化值△I.在最佳条件下,△I与水胺硫磷和丙溴磷的浓度均在50～ 500 μmol/L范围内呈线性关系.水胺硫磷的检出限(LOD,3σ)为11.4 μmol/L,相对标准偏差(RSD)为5.8％(n=10);丙溴磷的检出限为14.0 μmol/L,RSD为4.9％(n=l0).用于实际水样中两种农药的检测,加标回收率为85.8％ ～95.3％.     

四种钌（Ⅱ）配合物的中心离子电化学行为的比较

The electrochemical behavior of mononuclear and symmetrical binuclear ruthenium（Ⅱ） complexes [Ru1:Ru(bpy)2DIPB(ClO4)2,Ru2:(bpy)2 Ru(DIPB)Ru(bpy)2(ClO4)4,Ru3:Ru(phen)2DIPB(ClO4)2 and Ru4:(phen)2 Ru(DIPB)Ru (phen)2(ClO4)4] containing binuclear ligand 2,2′-bipyridine(bpy),1,10-phenanthroline(phen) and bridging ligand 1,4-di-[2-imidazo[4,5-f][1,10] phenanthroine benzene(DIPB) on a platinum electrode and the intermetallic interaction of binuclear complexes have been investigated using cyclic voltammetry,cyclic ac voltammetry and differential capacitance techniques,etc.In acetonitrile solution with a concentration of 0.1 mol穌m-3 TBAP,the central ions in four complexes all display single 1e and 2e reversible oxidation- reduction waves on the cyclic voltammograms,with apparent diffusion coefficients of the mononuclear complexed cations being larger than that of the binuclear ones.The apparent diffusion coefficients for complexed cations with auxiliary ligands bpy are also found to be larger than that with ligands phen.It appears that the overlapping of two continuous single-electron processes by cyclic ac voltammetry and differential capacitance techniques occurs during the redox processes of the central ions binuclear complexes.The results show that a weak electronic interaction exits between the two central ruthenium ions in binuclear complexes.     

基于阳离子型共轭聚合物和核酸适体的腺苷检测新方法

建立了一种基于阳离子型共轭聚合物和核酸适体的腺苷检测新方法. 荧光素修饰的短链DNA与腺苷的核酸适体部分互补, 形成双链DNA; 阳离子型共轭聚合物通过静电作用与双链DNA结合, 发生高效率的荧光共振能量转移(FRET). 加入腺苷后, 腺苷与核酸适体发生特异性结合, 导致双链DNA分解成单链, 使静电吸引力下降, 能量转移效率降低. 通过阳离子型共轭聚合物对单双链DNA的高效识别, 可快速简易地检测出腺苷.     

毛细管电泳-电化学检测法测定淮山中薯蓣皂苷和腺苷含量

建立了毛细管电泳-电化学检测(CE-ED)法测定淮山中薯蓣皂苷和腺苷的含量。考察了检测电位、运行缓冲液浓度和pH、分离电压及进样时间等的影响。在优化的实验条件下,对0.1mg/L薯蓣皂苷、腺苷在9min内实现了分离,其线性范围分别为0.1~1 000μg/L和0.1~1 200μg/L;检出限分别为0.04μg/L和0.03μg/L,峰电流的相对标准偏差(RSD,n=6)分别为1.7%、1.5%,迁移时间的RSD分别为0.7%、0.6%。该方法已用于淮山样品中薯蓣皂苷和腺苷的测定,样品加标回收率在98.0%~102.5%之间,RSD≤2.3%。     

A Rapid and Sensitive Aptamer‐Based Electrochemical Biosensor for Direct Detection of Escherichia Coli O111

A sensitive and specific electrochemical biosensor based on target‐induced aptamer displacement was developed for direct detection of Escherichia coli O111. The aptamer for Escherichia coli O111 was immobilized on a gold electrode by hybridization with the capture probe anchored on the electrode surface through Au‐thiol binding. In the presence of Escherichia coli O111, the aptamer was dissociated from the capture probe‐aptamer duplex due to the stronger interaction between the aptamer and the Escherichia coli O111. The consequent single‐strand capture probe could be hybridized with biotinylated detection probe and tagged with streptavidin‐alkaline phosphatase, producing sensitive enzyme‐catalyzed electrochemical response to Escherichia coli O111. The designed biosensor showed weak electrochemical signal to Salmonella typhimurium, Staphylococcus aureus and common non‐pathogenic Escherichia coli, indicating high specificity for Escherichia coli O111. Under the optimal conditions, the proposed strategy could directly detect Escherichia coli O111 with the detection limit of 112 CFU mL^?1 in phosphate buffer saline and 305 CFU mL^?1 in milk within 3.5 h, demonstrated the sensitive and accurate quantification of target pathogenic bacteria. The designed biosensor could become a powerful tool for pathogenic microorganisms screening in clinical diagnostics, food safety, biothreat detection and environmental monitoring.     

Electrochemical Sensor for Selective Detection of Meldonium in Urine by Voltammetry

A highly sensitive electrochemical sensor based on arenediazonium tosylates was designed to detect meldonium in urine. The effect of the concentration of ArN2⁺OTs⁻ arenediazonium tosylate modifier and various substituents was studied. Basic operating parameters for voltammetric meldonium detection were established and the procedure for urine sample preparation was developed. The following values were obtained: limit of detection (LOD) 0.005 mg ⋅ L⁻¹ (P=0.95) and limit of quantification (LOQ) 0.01 mg ⋅ L⁻¹. The relationship between the analytical signal and meldonium concentration in the solution ranging from 0.01 to 400 mg ⋅ L⁻¹ was described by a linear function. The meldonium concentration error did not exceed 18 %. The analysis time for a single urine sample was reduced to 15 minutes.     

Hydroxyapatite Nanoparticles Modified Graphite Electrodes for Electrochemical DNA Detection

Hydroxyapatite nanoparticles (HaNP) modified pencil graphite electrodes (PGEs) were developed for the first time in the literature, and accordingly they were applied for electrochemical monitoring of sequence‐selective DNA hybridization. The experimental conditions for HaNP modification of PGE, and DNA hybridization related to Hepatitis B Virus (HBV) DNA sequence were optimized. The microscopic and electrochemical characterization of HaNP‐PGE in contrast to the unmodified one was utilized. Under optimized experimental conditions, the selectivity of HBV DNA probe immobilized biosensor was tested against to non‐complementary (NC), mismatch (MM) sequences and the mixture of target:NC (1 : 1) or target: MM (1 : 1).     

Studies on the Affinity‐based Biosensors for Electrochemical Detection of HER2 Cancer Biomarker

As cancer diseases are the second main cause of death it is necessary to elaborate fast and efficient early diagnosis methods for their detection. One of the possibilities is the analysis of protein biomarkers, which abnormal concentration in physiological fluids might be an indication of cancer disease progression. Herein, we present the studies on the development of affinity‐based biosensors for electrochemical detection of HER2 protein, which is a common biomarker of breast cancer. The main objective was to verify the possibility of fabrication of HER2‐specific hybrid aptamer‐polyclonal antibody and antibody‐based sandwich sensing layer on gold electrode surface. The effectiveness of each electrode modification step was confirmed using voltammetric and impedimetric techniques in the presence of ferri/ferrocyanide redox couple. It was observed that hybrid construct was unlikely to be formed on the gold electrode due to a higher affinity of secondary polyclonal antibody towards target protein, which resulted in the separation of HER2‐antibody complex from the electrode surface. On the contrary, an antibody‐based sandwich receptor layer allowed for protein discrimination in the range from 1 to 100 ng mL^?1 by the application of TMB/H₂O₂ system and chronoamperometry detection technique. Though, the occurrence of interactions between interfering proteins and antibody‐based layer was noted, it led to at least two times smaller current responses than for HER2 protein.     

Electrochemical Properties of Highly Sensitive and Selective CuO Nanostructures Based Neurotransmitter Dopamine Sensor

In this article, electrochemical properties of CuO nanostructures based dopamine (DA) sensor was investigated. The morphology, structure, optical, and compositional properties of the CuO nanostructures were characterized by using SEM, XRD, UV‐Vis, and XPS techniques. The electrochemical properties were investigated using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques. The CV results indicate that biosensors based on CuO nanostructures exhibit a high selectivity and sensitivity of 0.1975 μA μM^–1 toward DA and effectively avoids the interference of ascorbic acid (AA) and uric acid (UA). The obtained EIS spectra for CuO sensors were analysed using an electrical equivalent circuit to understand the bulk and surface response via the capacitive and resistive parameters. The EIS measurement also leads to the direct determination of parameters like series resistance and ion diffusion phenomena at electrode‐electrolyte interface. The experimental CV and EIS results along with their analysis will have a significant impact on understanding the mechanism of high sensitivity and selectivity performance of CuO based sensors. This study may also lay the basis for efficient characterization of biosensors by coupling both the CV and EIS characterization techniques.     

Highly Selective and Sensitive Electrochemical Determination of Ni(II) in Real Samples Based on Ion‐imprinted Polymer Technology

Nickel is one of the heavy metals which has been considered as a serious pollutant affecting the human health. Therefore, it is necessary to control its concentration in foods and drinks. Recently, ion‐imprinted polymer based electrochemical sensors have been attracted lots of attention due to their valuable selectivity and sensitivity toward target analytes. In this study, Ni(II)‐1,10‐phenanthroline complex was prepared in ethanol and added stepwise to the aqueous solution of dopamine, followed by pH adjustment for self‐polymerization of dopamine in alkaline conditions. During the polymerization process, Ni(II)‐imprinted polydopamine (Ni‐PDA) was formed due to effective interactions between polydopamine and 1,10‐phenanthroline ligands carrying Ni(II) ions. A thin film of Ni‐PDA was anchored on a glassy carbon electrode (GCE) through a simple casting procedure. After drying, the sensor was employed for the determination of Ni(II) ions by means of differential pulse voltammetry. Under optimum conditions, a linear calibration curve was obtained in the range 1–25 μM with the detection limit of 0.39 μM (S/N=3). Due to excellent selectivity to Ni(II) ions, the sensor was not suffered from the excess amount of interfering ions and used for Ni(II) determination in different water and tea samples.     

Electrochemical Biosensors for Detection of Biological Warfare Agents

This review discusses current development in electrochemical biosensors for detection of biological warfare agents. This could include bacteria, viruses and toxins that are aerosoled deliberately in air, food or water to spread terrorism and cause disease or death to humans, animals or plants. The rapid and unequivocal detection and identification of biological warfare agents is a major challenge for any government including military, health and other government agents. Reliable, specific characterization and identification of the microorganism from sampling location, either air, water, soil or others is required. This review will survey different types of electrochemical biosensors has been developed based on the following: i) Immunosensors ii) PCR (DNA base Sensor) iii) Bacteria or whole cell sensor and iv) Enzyme sensor. This article gives an overview of electrochemical biosensor for detection of biological warfare agents. Electrochemical biosensors have the advantages of sensitivity, selectivity, to operate in turbid media, and amenable to miniaturization. Recent developments in immunofiltration, flow injection, and flow‐through electrochemical biosensors for bacteria, viruses, and toxin detection are reviewed. The current research and development in biosensors for biological warfare agents detection is of interest to the public as well as to the defense is also discussed.     

胆酸钠与支撑磷脂双层膜作用的电化学研究

采用电化学方法研究胆酸钠(NaC)与s-BLM的相互作用, 并结合实验结果对NaC与s-BLM的作用机理进行了讨论.     

电化学DNA生物传感器定量检测根癌农杆菌终止子基因片段

通过自组装法及共价法固定单链脱氧核糖核酸(ssDNA),制备了电化学DNA生物传感器。将巯基丙酸(MPA)自组装于金电极表面形成单分子膜,再利用1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)和N-羟基琥珀酰亚胺(NHS)的活化作用将ssDNA探针序列固定于金电极表面。将ssDNA修饰的电极与待测溶液中人工合成的转基因食品中常有的根癌农杆菌终止子(NOS)基因片段进行杂交,在[Fe(CN)6]3-/4-溶液中进行循环伏安和电化学阻抗谱扫描,表征ssDNA固定及杂交过程。优化了ssDNA固定条件。待测溶液中DNA浓度在1.0×10-7～1.0×10-10mol/L范围时,其浓度的对数值和ssDNA/Au电极与dsDNA/Au电极峰电流差值的变化值呈线性相关关系,相关系数为0.9822,检出限为8.1×10-11mol/L。     

Phase Selective Alternating Current Voltammetric Signalling Protocol: Application in Dendritic Co‐polymer Sensor for Anthracene

A sensitive anthracene (AN) sensor was developed with a gold electrode modified with a dendritic star‐copolymer film by in situ electrochemical co‐polymerization of generation 3 propylenethiophenoimine and 3‐hexylthiophene. The sensor's {Au/G3PPT‐co‐P3HT: i. e. gold‐generation 3 poly(propylenethiophenoimine)‐co‐poly(3‐hexylthiophene)} analytical response for anthracene was obtained by phase selective alternating current voltammetric (PSACV) signal transduction. An in‐phase angle of 0° yielded the most sensitive stripping signal and produced the best discrimination between the Faradaic and capacitive currents. The PSACV sensor exhibited a linear range (LR) of 3.48–56.4 nmol/L AN and a limit of detection (LOD) of 2.62 nmol/L AN. The LOD is comparable to the value of 4.4 nmol/L AN reported for glassy carbon electrode modified with graphenated polyaniline sensor. The low LOD value suggests that the AN sensor has promise for monitoring compliance to World Health Organisation (WHO) approved limit for polyaromatic hydrocarbons (PAHs) in wastewater (3.93 nmol/L). The Au/G3PPT‐co‐P3HT sensor is not as sensitive as gas chromatography coupled to tandem mass spectrometry (GC‐MS/MS) and reversed‐phase high performance liquid chromatography (RP‐HPLC) methods. However, the advantage of the PSACV signalling protocol is that real sample test results indicate that the sensor can be used for the determination of AN in oil‐polluted wastewater.