空壳纳米金修饰的新型DNA传感器

利用新型材料金纳米空球, 通过层层修饰的技术, 分别将壳聚糖、空壳纳米金、L-半胱氨酸、细胞色素c以及ssDNA探针修饰到玻碳电极表面, 制备了一种新型的DNA生物传感器. 以紫外及透射电子显微镜(TEM)表征了空壳纳米金, 以循环伏安法、阻抗谱图等电化学方法研究了传感器的特性, 通过原子力显微镜方法观察了该DNA生物传感器不同层之间的形态差异. 结果表明, 该修饰电极所吸附的ssDNA探针为1.672×10^―10 mol•cm^－2. 在指示剂柔红霉素的帮助下, DNA探针可与互补的DNA进行杂交, 借此以微分脉冲伏安法测定DNA.     

基于锁核酸探针的传感器用于BCR/ABL融合基因检测的研究

基于慢性粒细胞白血病中BCR/ABL融合基因的碱基序列,设计了一种新型发夹结构锁核酸(locked nucleic acids, LNA)探针,把LNA探针通过Au-s键固定在金电极表面构建了特异的生物传感器.LNA探针与目标链DNA杂交,以自行合成的苯甲酸二聚铜配合物([Cu2(C7H5O2)4(C2H6O)2], 简称[Cu(R)2]2+)为杂交指示剂,应用差示脉冲伏安法进行检测,表现出良好的响应信号.该新型锁核酸传感器能较好的区分完全互补链DNA、单碱基错配链DNA.对互补链DNA检测的线性范围为1.0×10-8～1.0×10-6 mol•L-1,检出限为2.0×10-9 mol•L-1.     

基于纳米ZnO/壳聚糖复合膜DNA电化学传感器用于HIV基因的免标记检测

以室温固相合成法制备纳米ZnO,通过壳聚糖(CHIT)的成膜效应将纳米ZnO固定在玻碳电极(GCE)表面,制得的ZnO/CHIT/GCE电极成为DNA固定和杂交的良好平台。DNA的固定和杂交通过电化学交流阻抗进行表征。以电化学交流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。电化学阻抗谱检测人类免疫缺陷病毒(HIV)基因片段的线性范围为2.0×10-11~2.0×10-6mol/L,检出限为2.0×10-12mol/L。     

铂纳米颗粒修饰电化学DNA传感器检测大豆中转基因成分

用电沉积方法将铂纳米颗粒修饰在玻碳电极表面,然后将花椰菜花叶病毒35S启动子ssDNA片段直接吸附在铂纳米颗粒上,制成特异的电化学DNA传感器。用扫描电子显微镜和循环伏安法对修饰铂纳米颗粒电极进行了表征。ssDNA探针与互补目的ssDNA杂交,以[Co(phen)3]3 (phen=1,10-Phe-nanthroline)为杂交指示剂,用方波伏安法进行检测,表现出良好的响应信号。与在裸玻碳电极上修饰的探针相比,测定目的基因的灵敏度显著提高。传感器对互补目的ssDNA检测的线性范围为2.14×10-9～2.14×10-7mol/L;检出限为1.0×10-9mol/L,与3个碱基错配的DNA序列杂交,观察不到明显的杂交信号。样品DNA经HindⅢ非限制性内切酶酶切后测定,杂交检测信号增大。用传感器检测含量不同的转基因大豆DNA和非转基因大豆DNA的混合溶液,杂交前后的电流差与转基因DNA的含量呈良好线性关系。连续5次测量含有100%转基因大豆DNA杂交后的电信号,相对标准偏差为5.89%,固定探针的电极再生后可重复使用8次。     

用于DNA杂交检测的丝网印刷型生物传感器的研究

将单链DNA(ssDNA)固定到丝网印刷碳电极上构成电化学DNA传感器,采用电化学指示剂,建立DNA杂交的检测方法.Co(phen)33+电化学指示剂通过钴盐与配体邻菲罗啉络合制备,采用等离子发射光谱法(ICP-AES)和核磁共振法(NMR)表征功能基团,采用循环伏安法(CV)分析指示剂的电化学特性,并以此为基础研究ssDNA在电极表面的固定及DNA杂交过程.本研究探讨了直接吸附、静电吸附与键合等3种ssD-NA在电极表面的固定方法,结果表明,静电吸附法和键合法具有较高的ssDNA固定量,采用静电吸附法固定探针的电极杂交目标DNA后,Co(phen)33+易于嵌入双链DNA (dsDNA)中,CV峰电流(ip)信号随目标DNA浓度增加.本研究采用静电吸附ssDNA的电极检测DNA杂交,实验表明,当探针固定液中ssDNA浓度为5 mg/L时,目标DNA浓度在6.65×10- 8～4.26× 10-6mol/L范围内,Co(phen)33+在dsDNA修饰电极上ip值与DNA浓度呈良好的线性关系,R2为0.9819.本研究为建立新的微生物分子分型手段提供了初步依据.     

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。     

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.     

基于三维有序金掺杂纳米二氧化钛的DNA生物传感器研究

用模板法在氧化铟锡(ITO)电极上制备具有三维有序多孔结构的金掺杂纳米二氧化钛修饰电极(3DOM GTD/ITO),扫描电镜(SEM)结果表明,制备的修饰电极三维结构规整有序、孔径均一。将标记有二茂铁(Fc)的DNA探针修饰到3DOM GTD/ITO电极上构建了一种新的标记型DNA生物传感器,通过Fc在DNA探针杂交前后的电化学信号变化可识别目标靶序列。采用循环伏安(CV)、示差脉冲(DPV)和交流阻抗(EIS)等方法对DNA探针在电极表面的固定和杂交进行表征。实验结果表明,该DNA生物传感器可以成功地识别乳腺癌基因靶序列,Fc的氧化还原电流与靶序列浓度在8.0×10-7～1.0×10-5 mol/L范围内呈线性关系,线性相关系数为0.9908,检测限为5.2×10-7 mol/L。     

基于富勒烯衍生物修饰玻碳电极的DNA电化学传感器

以富勒烯C₆₀, L-苏氨酸及对苯二甲醛为原料, 在氮气保护下反应得到含醛基官能团的2-(4-醛基苯基)-5-(1-羟乙基)富勒烯吡咯烷衍生物(C₆₀-CHO). 将该材料修饰于玻碳电极表面, 并利用醛基与氨基之间温和、高效的缩合反应, 将5-氨基修饰的寡聚核苷酸共价固定到了C₆₀-CHO修饰的玻碳电极表面, 构建了一种新型的电化学DNA传感器. 以[Fe(CN)₆]^3−/4−为电活性探针, 采用电化学阻抗法对转基因植物CaMV35S启动子基因特征片段进行检测. 实验结果表明, 杂交前后的电子传递电阻差值(DR_et)与目标序列浓度对数(lg C_S2)在1.0×10^－13～1.0×10^－9 mol/L浓度范围内呈良好的线性关系, 线性回归方程为DR_et/(10³ Ω)＝3.471 lg (C_S2/mol/L)＋50.425 (r＝0.9977), 检测限为1.5×10⁻¹⁴ mol/L. 杂交特异性实验进一步表明该传感器对完全互补、碱基错配和非互补序列具有良好的识别能力.     

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。     

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.     

氯霉素分子印迹复合膜的制备及电化学

采用电化学聚合法合成了对氯霉素（CAP）有快速响应和高灵敏度的聚苯胺/聚吡咯分子印迹复合膜修饰电极。 通过微分脉冲伏安法、扫描电子显微镜对制备的分子印迹复合膜的电化学性质及表面形貌进行了表征。 结果表明,以铁氰化钾为电化学探针,该膜对CAP的测定电化学信号响应快速、灵敏度高、选择性和膜再生性能良好。 对CAP检测的线性范围为5.00×10-8～1.05×10-6 mol/L,检测限为2.09×10-9 mol/L。     

血清样品中乙肝病毒的DNA电化学传感器检测

利用自组装单分子膜技术，将巯己基修饰的具有乙肝病毒(HBV)DNA序列特异性的单链DNA探针固定在金电极表面，制得DNA电化学传感器；以电活性的Hoechst 33258为指示剂，考察了该传感器对血清样品中乙肝病毒DNA的响应；探讨DNA电化学传感器在临床检测中的应用；将传感器法与荧光聚合酶链反应(PCR)法进行对比，两者的分析结果具有一致性。     

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.     

脱氧核糖核酸在硬脂酸铝离子膜上固定杂交及BAR基因片段检测

将石墨粉、固体石蜡和硬脂酸按一定比例混合制得表面富含羧基的碳糊电极,然后在电极表面组装荷正电的铝离子膜。在硬脂酸铝离子膜上进行DNA探针的固定和与目标基因的杂交。以亚甲蓝为杂交指示剂,用循环伏安法优化了DNA的固定和杂交条件。应用该电化学生物传感器以微分脉冲伏安法对转基因玉米外源BAR基因片段进行了检测,结果令人满意。     

Detection of bar gene encoding phosphinothricin herbicide resistance in plants by electrochemical biosensor

An electrochemical biosensor for the detection of bar gene coding phosphinothricin herbicide resistance is presented. The detection was based on hybridization reaction between the specific to bar gene 19-mer probe immobilized on the electrode surface and complementary DNA in a sample. Single-stranded DNA probe specific to bar gene was covalently attached by 5'-phosphate end to the surface of carbon paste electrode. Outer layer of a conventional CPE was provided with carboxyl groups of stearic acid. ssDNA was coupled to the electrode through ethylenediamine with the use of water-soluble 1-ethyl-3(3'-dimethylaminopropyl)-carbodiimide and N-hydroxy-sulfosuccinimide as activating reagents. Hybridization reaction at the electrode surface was detected via Co(bpy)(3)(3+), which possess a much higher affinity to the resulting DNA duplex compared to ssDNA probe. Detection limit of the sensor was 0.1 microM of target DNA fragments and its response was linear from 5 to 20 microM. Hybridization event was also detected by measuring guanine peak but this approach presented distinctly higher detection limit (1 muM) and lower reproducibility. Complete time of one measurement with the use of the biosensor including covalent attachment of ethylenediamine (linker) and ssDNA probe to the electrode, hybridization with target and interaction with electroactive indicator was about 70 min.     

DNA functionalized single-walled carbon nanotubes for electrochemical detection

Single-walled carbon nanotubes (SWNTs) were effectively dispersed and functionalized by wrapping with single-stranded DNA (ssDNA). The ssDNA-SWNTs attach strongly on glass substrate and easily form a uniform film, making it possible for electrochemical analysis and sensing. The film was fabricated into a working electrode, which exhibited good electrochemical voltammetric properties, such as flat and wide potential window, well-defined quasi-reversible voltammetric responses, and quick electron transfer for a Fe(CN)6(3-)/Fe(CN)6(4) system, indicating that the ssDNA-SWNTs film should be a good analytical electrode for electrochemical detection or sensing. This was demonstrated by highly selective and sensitive detection of a low concentration of dopamine in the presence of excess ascorbic acid.     

A sensor based on polyaniline nanofibers/ionic liquid-functionalized carbon nanotubes composite for electrocatalytic oxidation of guanine

In this paper, a novel polyaniline (PANI) nanofibers/ionic liquid-functionalized carbon nanotubes (IL-CNTs) composite-modified electrode was prepared, and its application on electrocatalytic oxidation of guanine of sequence-specific DNA was investigated. The surface morphology and the related electrochemical behaviors of the PANI/IL-CNTs composite film were characterized with scanning electron microscopy, electrochemical impedance spectroscopy and cyclic voltammetry, respectively. The PANI/IL-CNTs composite showed a good response current toward the direct electrooxidation of ssDNA due to the synergistic effect between PANI nanofibers and IL-CNTs. Based on this, it was adopted as an excellent sensing platform for highly sensitive determination of guanine. The detection limit was 3.1 × 10^?9 mol/L.     

Lable‐Free Electrochemical DNA Sensor Based on Gold Nanoparticles/Poly(neutral red) Modified Electrode

We present a new strategy for the label‐free electrochemical detection of DNA hybridization based on gold nanoparticles (AuNPs)/poly(neutral red) (PNR) modified electrode. Probe oligonucledotides with thiol groups at the 5‐end were covalently linked onto the surface of AuNPs/PNR modified electrode via S‐Au binding. The hybridization event was monitored by using differential pulse voltammetry (DPV) upon hybridization generates electrochemical changes at the PNR‐solution interface. A significant decrease in the peak current was observed upon hybridization of probe with complementary target ssDNA, whereas no obvious change was observed with noncomplementary target ssDNA. And the DNA sensor also showed a high selectivity for detecting one‐mismatched and three‐mismatched target ssDNA and a high sensitivity for detecting complementary target ssDNA, the detection limit is 4.2×10^?12 M for complementary target ssDNA. In addition, the DNA biosensor showed an excellent reproducibility and stability under the DNA‐hybridization conditions.     

Ultra-sensitive electrochemical DNA biosensor based on signal amplification using gold nanoparticles modified with molybdenum disulfide,graphene and horseradish peroxidase

We have developed an ultra-sensitive electrochemical DNA biosensor by assembling probe ssDNA on a glassy carbon electrode modified with a composite made from molybdenum disulfide, graphene, chitosan and gold nanoparticles. A thiol-tagged DNA strand coupled to horseradish peroxidase conjugated to AuNP served as a tracer. The nanocomposite on the surface acts as relatively good electrical conductor for accelerating the electron transfer, while the enzyme tagged gold nanoparticles provide signal amplification. Hybridization with the target DNA was studied by measuring the electrochemical signal response of horseradish peroxidase using differential pulse voltammetry. The calibration plot is linear in the 5.0?×?10^?14 and 5.0?×?10^?9 M concentration range, and the limit of detection is 2.2?×?10^?15 M. The biosensor displays high selectivity and can differentiate between single-base mismatched and three-base mismatched sequences of DNA. The approach is deemed to provide a sensitive and reliable tool for highly specific detection of DNA.