基于纳米MnO2的免标记型DNA电化学生物传感器用于大肠杆菌的测定

以室温固相合成法制备纳米MnO2,通过壳聚糖（CHIT）的成膜效应将纳米MnO2固定在玻碳电极表面。DNA在MnO2/CHIT膜上的固定和杂交通过循环伏安和电化学交流阻抗进行表征。以电化学阻抗免标记法检测目标DNA,固定于电极表面的DNA探针与目标DNA杂交后使电极表面的电子传递电阻增大,以此作为检测信号可以高灵敏度地测定目标DNA。电化学阻抗谱检测大肠杆菌基因片段的线性范围为2.0×10^-11 ～2.0×10^-6mol/L,检出限为1.0×10^-12mol/L。     

DNA在Mg/聚2,6-吡啶二甲酸膜上的固定和杂交及其PAT基因片段的电化学阻抗谱测定

以静电吸附法使Mg²⁺修饰于玻碳电极(GCE)上电聚合的2,6-吡啶二甲酸膜(PDC)上, 制得的Mg/PDC/GCE电极, 成为DNA固定及杂交的良好平台. 应用微分脉冲伏安法和电化学阻抗谱对DNA的固定和杂交进行表征. 以电化学阻抗谱免标记法检测目标DNA比以亚甲基蓝为指示剂的微分脉冲伏安法有更高的灵敏度. 固定于电极表面的DNA探针与互补单链DNA杂交后使电负性的[Fe(CN)₆]^3-/4-的表面电子传递电阻值显著增大, 以此作为检测信号可以高灵敏度地测定目标DNA. 电化学阻抗谱检测转基因植物外源PAT基因片段, 线性范围为1.0×10^-9 ~ 1.0×10^-5 mol/L, 检测限为3.4×10^-10 mol/L.     

基于DNA-甲苯胺蓝生物聚合离子膜的多环有机物电化学传感器的研究

利用DNA与小分子之间的相互作用,以DNA/壳聚糖生物聚合离子膜固定电活性小分子,制备了DNA-甲苯胺蓝/壳聚糖聚合离子复合膜修饰电极,并利用多环有机物与染料分子对DNA特异结合的竞争关系,构筑了多环有机物非试剂添加型DNA电化学生物传感器。以盐酸四环素为模式分子,利用循环伏安法和方波伏安法研究了该修饰电极的电化学特性以及该电极对盐酸四环素的电化学响应,结果表明,DNA和甲苯胺蓝成功地固定在电极表面,电极表面的甲苯胺蓝保持了很好的电化学活性。利用紫外-可见分光光度法研究了电极对盐酸四环素响应的作用机理。该传感器的线性范围为2.5~100μmol·L-1。     

硒化铅纳米粒子DNA电化学传感器检测花椰菜花叶病毒35S启动子基因序列

以水热法合成十六烷基三甲基溴化铵(CTAB)修饰的PbSe纳米粒子。在碳糊电极表面制备的PbSe纳米粒子壳聚糖(CHIT)复合膜上,实现了DNA的固定和杂交,并用循环伏安法和电化学交流阻抗法进行了表征。应用电活性分子亚甲紫(MV)作为杂交指示剂,以微分脉冲伏安法对转基因植物CaMV35S启动子基因片段进行测定,检测范围为5.0×10-11～5.0×10-6mol/L;检出限为1.6×10-11mol/L(3σ)。该传感器能很好地识别DNA互补序列、非互补序列和2碱基错配序列。     

硬脂酸钠在油/水界面自组装膜的电化学研究

应用循环伏安法,以铜离子为探针研究硬脂酸钠在油/水界面上的自组装过程.结果表明,硬脂酸钠在蓖麻油/水界面上自组装,经4种聚集状态而达到完整的有序单层膜.Cu2+离子在涂蓖麻油电极上的氧化是完全受吸附控制,而在表面活性剂硬脂酸钠自组装膜的油/水界面上,则其电极过程受扩散和吸附两者混合控制.循环伏安测试给出,峰电流ip随扫描速率v变化的拟合方程为:ip=9.214?3.889v1/2+0.5809v,r=0.9978.     

以还原氧化石墨烯和纳米二氧化锆为DNA探针固定平台电化学测定转基因玉米中特定基因序列

草胺膦乙酰转移酶基因(PAT)是一种转基因植物的外源DNA片段。 本文以还原氧化石墨烯和纳米二氧化锆(nanoZrO₂)的复合物作为固定DNA探针的平台,建立了一种灵敏地检测PAT基因的方法。 首先,氧化石墨烯直接在电极表面进行电化学还原,然后将一层nanoZrO₂涂覆于其表面,利用DNA中的磷酸基团与nanoZrO₂中氧的亲和作用固定DNA探针。 通过微分脉冲伏安法检测DNA探针与PAT基因片段的杂交,构建了用于检测PAT基因片段的电化学生物传感器。 该传感器具有稳定性好,重复性好的特点,可灵敏地检测转基因玉米中的PAT基因,检测限达2.0×10^-15 mol/L。     

以还原氧化石墨烯和纳米二氧化锆为DNA探针固定平台电化学测定转基因玉米中特定基因序列（英文）

草胺膦乙酰转移酶基因(PAT)是一种转基因植物的外源DNA片段。本文以还原氧化石墨烯和纳米二氧化锆(nano ZrO_2)的复合物作为固定DNA探针的平台,建立了一种灵敏地检测PAT基因的方法。首先,氧化石墨烯直接在电极表面进行电化学还原,然后将一层nano ZrO_2涂覆于其表面,利用DNA中的磷酸基团与nano ZrO_2中氧的亲和作用固定DNA探针。通过微分脉冲伏安法检测DNA探针与PAT基因片段的杂交,构建了用于检测PAT基因片段的电化学生物传感器。该传感器具有稳定性好,重复性好的特点,可灵敏地检测转基因玉米中的PAT基因,检测限达2. 0×10~(-15)mol/L。     

微囊藻属DNA电化学传感器的研制

利用自组装法将巯基修饰的DNA探针与6-巯基-1-己醇(MCH)固定到金电极表面,制备了微囊藻属特定DNA传感器,将该传感器与完全互补的微囊藻DNA序列、完全不互补序列,以及单碱基错配序列进行杂交,以Hoechst 33258为杂交指示剂,应用循环伏安法和线性扫描伏安法研究了该传感器对目标DNA的电化学检测行为.研究表明,当与完全互补DNA杂交后,Hoechst 33258氧化信号有明显的增强.实验对自组装时间、MCH浸泡时间及杂交液离子浓度进行了优化.结果表明,当自组装时间为90 min,MCH浸泡时间为1 h,杂交溶液中NaCl浓度为0.3 mol/L时,电化学信号最好.目标DNA的氧化峰电流值与其浓度在1×10~(-8) ～1×10~(-6) mol/L范围内呈良好的线性关系,检出限为8.1×10~(-9) mol/L.     

多壁碳纳米管/纳米Ag-TiO_2膜DNA电化学生物传感器

基于多壁碳纳米管/纳米Ag-TiO2复合膜制备了高灵敏度的DNA电化学生物传感器。将Ag-TiO2复合物与适量分散于N,N-二甲基甲酰胺中的多壁碳纳米管(MWNT)相混合,形成均匀稳定的混合溶液,将其滴涂于裸碳糊电极表面,制得MWNT/Ag-TiO2修饰碳糊电极。碳纳米管大的比表面积和良好的电子传递性能与Ag-TiO2纳米复合物良好的生物相容性和对DNA极好的吸附能力的协同作用,显著提高了DNA探针的固载和DNA杂交的检测灵敏度。应用循环伏安法和电化学交流阻抗谱分别对传感膜的制备和DNA的固定与杂交进行了表征。以电化学交流阻抗谱法对转基因植物外源草丁膦乙酰转移酶基因片段进行了检测,线性范围为1.0×10-11～1.0×10-6mol/L,检出限为3.12×10-12mol/L。     

基于聚苯胺掺杂乙醇胺固定DNA电化学传感器的研究

本研究以电化学聚合法制备了聚苯胺掺杂乙醇胺修饰电极,并成功固定了DNA探针。文中对修饰电极的制备和DNA的固定杂交条件进行了探讨,并利用循环伏安法测定嵌入双链DNA(dsDNA)分子碱基对中的亚甲基蓝的氧化还原峰电流,识别和测定溶液中互补的单链DNA(ssDNA)片段,从而实现对溶液中不同基因片段的检测。     

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.     

基于PAMAM/聚2,6-吡啶二甲酸膜DNA电化学生物传感器的制备及对禽病毒基因检测的研究

以铂电极上聚合的2,6-吡啶二甲酸(PDC)膜组装G5.0树状高分子(PAMAM)固定ssDNA探针, 制备了一种新型的DNA电化学生物传感器. 用[Fe(CN)6]3－/4－作氧化还原指示剂, 以电化学交流阻抗和循环伏安技术对探针ssDNA的固定和杂交进行了表征. 实验表明, 当ssDNA在复合膜上固定及与其互补序列杂交后, 电极表面的传递电阻(Ret)依次增大. 因此, 可以利用Ret的明显差异, 以此固定探针的修饰电极, 对互补序列DNA进行无标记交流阻抗检测. 基于该生物传感器结合交流阻抗技术对禽病毒基因进行检测, 在优化实验条件下, 靶基因ssDNA-2在2.0×10－11～1.0×10－8 mol•L－1线性范围内, 其浓度与电极表面的电子传递电阻(Ret)之间呈良好的线性关系, 检测限为3.6×10－12 mol•L－1. 表明该方法为病毒灵敏地检测提供了一个有益的传感平台.     

以乙二胺为手臂分子制备的DNA修饰电极及其伏安性能

Carboxyl was formed on the surface of glassy carbon electrode(GCE) by electrochemical oxidation. Ethylenediamine(En) was used as the arm molecule to link carboxyl with dsDNA using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N- hydroxysuccinimide (NHS) as the activators to prepare dsDNA modified electrode(dsDNA/En/GCE). It was shown that dsDNA couM be covalently immobilized on the surface of GCE. ssDNA modified electrode(ssDNA/En/GCE) was obtained via the thermal denaturation of dsDNA/En/GCE. The dsDNA/En/GCE and ssDNA/En/GCE were characterized by voltammetry with methylene blue(MB) as the indicator. The results indicated that the currents of the redox peaks of MB at ssDNA/En/GCE were larger than those at dsDNA/En/GCE, and the currents of the redox peaks at En/GCE were the smallest. The peak-currents of MB at the DNA modified electrode had good reproducibility after multi-denaturation and hybridization cycles.     

Electrochemical genosensor for the detection of interaction between methylene blue and DNA

Described here are the chronocoulometric and voltammetric parameters for methylene blue [3,7-bis(dimethylamino)phenothiazin-5-ium chloride, MB] on binding to DNA at carbon paste electrode (CPE) surface. MB, which interacts with the immobilized calf thymus DNA was detected by using single stranded DNA modified CPE (ssDNA modified CPE), bare CPE and double stranded DNA modified CPE (dsDNA modified CPE) in combination with chronocoulometry and differential pulse voltammetry (DPV) techniques. The effect of ionic strength to the behavior of MB with dsDNA and ssDNA was also studied by means of voltammetry. These results demonstrated that MB could be used as an effective electroactive hybridization indicator for DNA biosensors. Performance characteristics of the sensor are described, along with future prospects.     

A DNA biosensor based on graphene paste electrode modified with Prussian blue and chitosan

A chemically modified graphene paste electrode was prepared by incorporating appropriate amounts of graphene in a paste mixture, followed by electrodepositing Prussian blue (PB) and coating chitosan on the electrode surface. The electrode was able to bind ssDNA, and gave a better voltammetric response for complement DNA than did ordinary carbon paste electrodes. The response of the electrode was characterized with respect to the paste composition, immobilization time of probe DNA on the chitosan and PB modified graphene paste electrode, and the effect of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC). The electrochemical behavior of PB assembled on the graphene paste electrode was investigated. The combination of graphene and PB can enhance the current response of the graphene paste electrode. As a consequence of DNA hybridization, a significant change in the current due to daunomycin intercalated with double-stranded DNA (ds-DNA) on the surface of the graphene paste electrode was observed.     

ssDNA/十八酸修饰碳糊电极的制备及伏安法表征

将石墨粉与十八酸在80 ℃下混合制成表面富含—COOH的基底碳糊电极(SA/CPE), 然后在活化剂N-羟基琥珀酰亚胺(NHS)和1-乙基-3-(3-二甲基氨丙基)碳二亚胺盐酸盐(EDC)存在下将ssDNA固定到电极表面制备ssDNA修饰电极(ssDNA/SA/CPE). 以亚甲基蓝(MB)为指示剂, 用循环伏安法对SA/CPE和ssDNA/SA/CPE进行电化学表征, 发现其在ssDNA/SA/CPE上较在SA/CPE上的氧化峰电流(i_pa)和还原峰电流(i_pc)分别增大1.9倍和1.7倍, 式电势(E_f)负移8 mV. 把ssDNA/SA/CPE放在互补ssDNA溶液中杂交后, MB的i_pa 和i_pc较在SA/CPE上分别增大1.0倍和0.8倍, E_f负移18 mV. 用0.5 mol/L 的NaOH溶液冲洗使电极表面杂交而成的dsDNA变性洗脱, MB的伏安信号几乎与在ssDNA/SA/CPE上一样. i_pc与SA/CPE上固定的ssDNA质量在1.0×10^－7～5.0×10^－6 g范围内成线性关系, 检测限为2.0×10^－9 g (S/N＝3). 这种既廉价又灵敏的电化学生物传感器有望在转基因植物产品检测研究中得到应用.     

Sensitively Electrochemical Sensing for Sequence‐Specific Detection of Phosphinothricin Acetyltransferase Gene: Layer‐by‐Layer Films of Poly‐L‐Lysine and Au‐Carbon Nanotube Hybrid

An electrochemical DNA sensing film was constructed based on the multilayers comprising of poly‐L ‐lysine (pLys) and Au‐carbon nanotube (Au‐CNT) hybrid. A precursor film of mercaptopropionic acid (MPA) was firstly self‐assembled on the Au electrode surface. pLys and Au‐CNT hybrid layer‐by‐layer assembly films were fabricated by alternately immersing the MPA‐modified electrode into the pLys solution and Au‐CNT hybrid solution. Cyclic voltammetry was used to monitor the consecutive growth of the multilayer films by utilizing [Fe(CN)₆]^3?/4? and [Co(phen)₃]^3+/2+ as the redox indicators. The outer layer of the multilayer film was the positively charged pLys, on which the DNA probe was easily linked due to the strong electrostatic affinity. The hybridization detection of DNA was accomplished by using methylene blue (MB) as the indicator, which possesses different affinities to dsDNA and ssDNA. Differential pulse voltammetry was employed to record the signal response of MB and determine the amount of the target DNA sequence. The established biosensor has high sensitivity, a relatively wide linear range from 1.0×10^?10 mol/L to 1.0×10^?6 mol/L and the ability to discriminate the fully complementary target DNA from single or double base‐mismatched DNA. The sequence‐specific DNA related to phosphinothricin acetyltransferase gene from the transgenically modified plants was successfully detected.     

Application of diazo-thiourea and gold nano-particles in the design of a highly sensitive and selective DNA biosensor

An effective procedure for constructing a DNA biosensor is developed based on covalent immobilization of NH_2 labeled,single strand DNA(NH_2-ssDNA) onto a self-assembled diazo-thiourea and gold nanoparticles modified Au electrode(diazo-thiourea/GNM/Au).Gold nano-particles expand the electrode surface area and increase the amount of immobilized thiourea and single stranded DNA(ssDNA) onto the electrode surface.Diazo-thiourea film provides a surface with high conductibility for electron transfer and a bed for the covalent coupling of NH_2-ssDNA onto the electrode surface.The immobilization and hybridization of the probe DNA on the modified electrode is studied by differential pulse voltammetry(DPV) using methylene blue(MB) as a well-known electrochemical hybridization indicator.The linear range for the determination of complementary target ssDNA is from 9.5(±0.1) × 10~(-13) mol/L to1.2(±0.2) x 10~(-9) mol/L with a detection limit of 1.2(±0.1) 10~(-13) mol/L.     

Electrochemical deoxyribonucleic acid biosensor based on the self-assembly film with nanogold decorated on ionic liquid modified carbon paste electrode

An electrochemical DNA biosensor was fabricated by self-assembling probe single-stranded DNA (ssDNA) with a nanogold decorated on ionic liquid modified carbon paste electrode (IL-CPE). IL-CPE was fabricated using 1-butylpyridinium hexafluorophosphate as the binder and the gold nanoparticles were electrodeposited on the surface of IL-CPE (Au/IL-CPE). Then mercaptoacetic acid was self-assembled on the Au/IL-CPE to obtain a layer of modified film, and the ssDNA probe was further covalently-linked with mercaptoacetic acid by the formation of carboxylate ester with the help of N-(3-dimethylamino-propyl)-N'-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide. The hybridization reaction with the target ssDNA was monitored with methylene blue (MB) as the electrochemical indicator. Under the optimal conditions, differential pulse voltammetric responses of MB was proportional to the specific ssDNA arachis sequences in the concentration range from 1.0×10(-11) to 1.0×10(-6) mol L(-1) with the detection limit as 1.5×10(-12) mol L(-1) (3σ). This electrochemical DNA sensor exhibited good stability and selectivity with the discrimination ability of the one-base and three-base mismatched ssDNA sequences. The polymerase chain reaction product of arachis Arabinose operon D gene was successfully detected by the proposed method, which indicated that the electrochemical DNA sensor designed in this paper could be further used for the detection of specific ssDNA sequence.