基于磁性自组装分子印迹传感器测定赤霉素A3

利用共沉淀法合成了磁性Fe3 O4纳米粒子,进一步表面功能化,合成Fe3 O4@Au磁性纳米粒子提高粒子表面的亲和性。在高亲和力的金壳表面自组装L-半胱氨酸-GA3,将其滴涂在磁控玻碳电极表面,电聚合L-半胱氨酸制得对GA3具有特异性识别能力的MIP/Fe3 O4@Au修饰电极。对Fe3 O4@Au磁性纳米粒子的表面形态及粒度分布进行了透射电镜分析,对GA3, MIP及nMIP的结构及成分进行了红外光谱对比分析。利用电化学方法对测试体系的工作条件进行了优化。研究表明,当电聚合圈数为30圈,以乙酸-甲醇(1：8, V/V)作为洗脱液、洗脱时间为5 min、重吸附时间为7 min时,传感器具有较高的稳定性,且对GA3具有较好的识别效果。结果表明,探针离子K3[Fe(CN)6]/K4[Fe(CN)6]的氧化峰电流值与GA3浓度在1.0×10-11~1.0×10-8 mol/L范围内呈线性关系,检出限为2.6×10-12 mol/L。此传感器已应用于啤酒中GA3的检测。     

基于磁性分离-硫化铜纳米粒子标记的DNA流动注射化学发光检测

本文基于磁性粒子(MB)良好的分离、富集能力,研究了硫化铜纳米粒子标记的流动注射-化学发光(FI-CL)DNA检测体系.通过硫化铜标记的探针1与目标DNA及连有磁球的探针2形成三明治结构,实现对目标DNA的捕获、分离与标记;通过其溶解释放出CuS标记颗粒的铜离子,引起化学发光信号增强,实现了目标DNA序列的定性定量检测.该方法对完全互补单链DNA(ssDNA)检测的线性范围为1.0×10-11～1.6×10-9 mol/L,检出限为3.0×10-12 mol/L,对1.0×10-9 mol/L目标DNA测定的相对标准偏差为3.2%(n=11),对目标碱基序列具有良好的识别能力.     

新型磁性纳米电化学DNA生物传感器的研究

利用高分子磁性纳米粒子有效地将磁性分离、富集和化学修饰电极的电化学检测相结合,构建以亚甲基蓝为嵌入式杂交指示剂的电化学DNA生物传感器.此传感器对碱基错配的序列有较好的选择性.传感器对目标序列的响应在1×10-13～1×10-6 mol/L范围内呈线性关系;检出限为4.3×10-14 mol/L.这种新型的高分子磁性纳米粒子电化学DNA生物传感器具有高的灵敏度,其线性范围宽,成本低,为DNA的痕量分析提供了一种新的思路.     

基于工具酶信号放大技术电化学发光法检测凝血酶

利用巯基乙胺将合成的金纳米粒子氨基化;基于纳米粒子负载羧基化的联吡啶钌和巯基DNA制得电化学发光信号探针;采用酶循环信号放大技术,获得大量含新增DNA的溶液来捕获信号探针;以金电极为载体,将巯基DNA自组装到电极表面,依次杂交互补DNA和信号探针,构建电化学发光生物传感器.在优化的条件下,此传感器对凝血酶具有良好的响应,在3.0× 10-13～6.0×10-11 mol/L范围内,凝血酶的浓度与发光强度呈良好的线性关系,检出限为1.8× 10-13 mol/L(3a).采用酶切循环放大技术制备的生物传感器具有灵敏度高,选择性和重现性良好等特点.     

磁性粒子表面自组装辛可宁分子印迹电致化学发光传感器

以辛可宁为模板分子、十二烷基硫醇为功能单体,在Fe3 O4@Au纳米粒子表面自组装辛可宁分子印迹膜,构建了新型磁性粒子-分子印迹电化学发光传感器。通过透射电子显微镜对磁性纳米粒子的粒径分布及形貌进行了表征,使用红外光谱对比分析了辛可宁、分子印迹膜洗脱前和洗脱后的结构及成分。结果表明,在最优的实验条件下(0.012 mol/L 硼砂缓冲溶液(pH 9.5),0.8 mmol/L Ru(bpy)2+3),辛可宁浓度的对数在1×10-10~9×10-8 mol/L范围内,与电化学发光强度变化值有良好的线性关系,检出限为3.5×10-11 mol/L。此传感器灵敏度高、选择性好、易于更新,将其用于血清样品的检测,方法回收率为98.8%~104.7%。     

基于直立碳纳米管大面积金粒子的DNA生物传感器用于早幼粒白血病/维甲酸受体α融合基因检测

基于直立碳纳米管上的大面积金粒子构建了新型的电化学DNA生物传感器,用于急性早幼粒细胞白血病PML/RARα融合基因的检测。首先在直立碳纳米管电极表面溅射金粒子,采用自组装方法将巯基修饰的单链DNA固定到电极上,将氨基修饰的单链DNA和羧基化的CdTe量子点通过酰胺缩合反应生成CdTe修饰的DNA探针,通过与目标DNA的双杂交反应形成三明治结构,利用差分脉冲阳极溶出伏安法检测电极表面捕获的CdTe量子点,从而对DNA进行定量分析。结果表明,电极上Cd2+峰电流与目标DNA浓度(1.0×10-12~1.0×10-8 mol/L)的对数值呈线性关系,线性方程为ipa(μA)=1.626+0.132lgC(mol/L)(R=0.996),检出限为4.0×10-13 mol/L(3σ)。传感器表现出良好的重现性和稳定性。     

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％.     

CdTe量子点标记的DNA电化学传感器的研究

利用碳纳米管和CdTe量子点(QDs)组装的电化学传感器,建立了一种识别DNA的新方法.将氨基修饰的单链DNA探针共价键合固定在带有羧基的碳纳米管修饰的金电极上,然后与CdTe QDs标记的目标DNA进行杂交.利用差分脉冲法(DPV)和循环伏安法对目标DNA的固定和杂交进行表征,通过电活性指示剂柔红霉素(DNR)的DPV峰电流变化,对互补DNA、非互补DNA和单碱基错配DNA序列进行识别.与未标记CdTe QDs的目标DNA相比,标记CdTe QDs的目标DNA序列的电流响应灵敏度明显提高.DNA电化学传感器检测的优化条件:DNR的浓度为1.67×10-5 mol/L,DNA杂交时间为80 min,杂交温度为55 ℃.在1.0×10-13 ～1.0×10-8 mol/L范围,目标DNA浓度的对数值与其响应的DPV信号(还原峰电流)呈线性关系,检出限为3.52×10-14 mol/L(S/N=3,n=9),线性方程为ΔI=50.22+3.567 lgcDNA,相关系数为0.996 6.对1.0×10-10 mol/L的目标DNA样品进行重复测定,相对标准偏差为4.8%(n=5),重复性良好.     

基于二硫化钼/纳米金和硫堇/纳米金信号放大的雌二醇电化学适配体传感器

构建了一种新型的基于二硫化钼/纳米金和硫堇/纳米金信号放大的检测17β-雌二醇的电化学适配体传感器. 利用巯基自组装技术将17β-雌二醇的适配体探针DNA固定在二硫化钼/纳米金修饰玻碳电极表面, 与末端带巯基的部分互补DNA链杂交, 将硫堇/纳米金电化学指示剂自组装在杂交后的双链DNA上, 制备了17β-雌二醇电化学适配体传感器. 二硫化钼/纳米金复合材料增加了电极的有效表面积和DNA探针的固定量. 纳米金作为信号物质载体负载硫堇, 实现了电化学指示剂的信号放大. 加入目标物17β-雌二醇后, 目标物与适配体DNA特异性结合, 导致互补DNA链脱落, 双链上结合的硫堇/纳米金电化学指示剂数量减少, 电化学信号降低. 实验结果表明, 在1.0×10 ^-14~5.0×10 ^-12 mol/L范围内17β-雌二醇浓度与峰电流的线性关系良好, 检出限为4.2×10 ^-15 mol/L(S/N=3). 该传感器可望用于其它环境激素类物质的检测.     

基于聚合酶辅助信号放大的电化学发光DNA传感器研究

构建一种新型基于聚合酶辅助电致化学发光DNA传感器,利用循环链置换聚合反应、辅助目标mRNA循环以及量子点的信号放大,实现超灵敏检测目标mRNA。将巯基修饰的发卡型捕获探针(Capture DNA,CP)通过Au-S键组装到Fe_3O_4@Au表面,并通过磁性组装到磁控玻碳电极上。目标mRNA存在时,目标mRNA打开发卡CP,与之杂交形成dsDNA;然后加入聚合酶、引物链(DNA1)及碱基,引物链开始扩增,将目标mRNA取代,释放的目标mRNA重新结合CP,引发下一轮扩增循环,使信号循环放大,最后加入TGACd Te量子点标记的DNA2,与打开后的CP末端序列通过碱基互补配对结合,进行电化学发光检测。在1×10~(-15)~1×10~(-11)mol/L范围内,目标mRNA浓度的对数与ECL信号呈良好的线性关系,检出限为3.4×10~(-16)mol/L。人体血清样加标回收率为97.2%~102.3%。本方法通过加入聚合酶使目标mRNA循环检测以及结合量子点标记的信号放大协同提高了检测的灵敏度。结果表明,此传感器具有良好的选择性、稳定性和重现性。     

新型"三明治"结构DNA电化学传感器对急性早幼粒细胞白血病相关融合基因的检测

基于急性早幼粒细胞白血病(APL)中PML/RARα融合基因的碱基序列,设计了新型的锁核酸(LNA)修饰寡核苷酸作为捕获探针和信号探针,研究出一种基于"三明治"传感模式的电化学生物传感器对PML/RARα融合相关基因进行检测.靶序列分别与捕获探针和信号探针杂交后形成"三明治"结构.将修饰电极置于含有底物3,3′,5,5′-四甲基联苯胺(TMB)和过氧化氢的测定溶液中,用计时电流法检测靶序列.结果表明,该传感器可定量识别和检测溶液中人工合成的短链APL PML/RARα融合基因片段.经过条件优化,杂交前后电流值与靶标链浓度在1.0×10~(-12) ～2.5×10~(-11) mol/L范围内呈良好的线性关系,检出限为8.5×10~(-13) mol/L.该方法简单、特异性好,有望用于实际样品的检测.     

Study on the electrochemiluminescence behavior of ABEI and its application in DNA hybridization analysis

The electrogenerated chemiluminescence (ECL) behavior of N-(4-aminobutyl)-N-ethylisoluminol (ABEI) was studied and it was found that ABEI could produce emission light when oxidized at a +1.0 V (vs. Ag/AgCl) potential in alkaline solution. The addition of H2O2 markedly improved the ECL sensitivity. The pH value of the solution as well as the H2O2 concentration and working potential all have influences on the ECL response. Under optimal conditions, ABEI can be detected in the range 1.3 x 10(-6)-6.5 x 10(-12) mol L(-1). A detection limit of 2.2 x 10(-12) mol L(-1) for ABEI was obtained at a signal-to-noise ratio of 3. ABEI was then used as a marker to label a known sequence oligonucleotide, which was used as a DNA probe for identifying a target ssDNA immobilized on a PPy modified electrode based on a specific hybridization reaction. The hybridization events were evaluated by the ECL measurements. The results showed that only a complementary sequence could form a double-stranded DNA with the DNA probe and give a strong ECL response. A three-base mismatch sequence and non-complementary sequence have no response. The intensity of the ECL was linearly related to the concentration of the complementary sequence in the range 9.6 x 10(-11)-9.6 x 10(-8) mol L(-1), the detection limit was 3.0 x 10(-11) mol L(-1).     

基于Ru(bpy)■-三乙胺体系的电致化学发光生物传感器检测葡萄糖

联吡啶钌(Ru(bpy)■)拥有优良的电致化学发光(ECL)性能,但其较好的水溶性使其固载面临巨大问题。该文制备了Pt纳米粒子与Ru(bpy)■的复合物(Pt NPs-Ru),将其修饰于电极并进一步固载葡萄糖氧化酶(GOx)制得传感器。基于H2O2对Ru(bpy)■-三乙胺体系ECL信号的猝灭作用,随着葡萄糖浓度的增加,其在GOx的催化下原位产生的H2O2量增多,导致ECL信号逐渐减弱,从而实现葡萄糖的检测。ECL强度与葡萄糖浓度的对数在1.0×10-8~5.0×10-5 mol/L范围内呈良好的线性关系,检出限低至5.2×10-9 mol/L。传感器具有好的稳定性和高的选择性。Pt NPs-Ru复合物为ECL传感器的构建提供了良好平台,为葡萄糖检测提供了新方法。     

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.     

Aptamer-based Electrochemical Biosensors for Highly Selective and Quantitative Detection of Adenosine

A new adenosine biosensor based on aptamer probe is introduced in this article. An amino-labeled aptamer probe was immobilized on the gold electrode modified with an o-phenylenediamine electropolymerized film. When adenosine is bound specifically to the aptamer probe, the interface of the biosensor is changed, resulting in the decrement of the peak current. The response current is proportional to the amount of adenosine in sample. The used electrode can be easily regenerated in hot water. The proposed biosensor represents a linear response to adenosine over a concentration range of 1.0x 10^-7-l.0x10^-4 mol/L with a detection limit of 1.0xl0^-8 mol/L. The presented biosensor exhibits a nice specificity towards adenosine. It offers a promising approach for adenosine assay due to its excellent electrochemical properties that are believed to be very attractive for electrochemical studies and electroanalytical applications.     

基于DNA四面体纳米结构探针的电化学生物传感器检测DNA甲基化

该文以DNA四面体纳米结构探针(TSP)为捕获探针,将辣根过氧化物酶标记的IgG抗体结合在纳米金颗粒表面(AuNPs-IgG-HRP)作为信号分子,构建了一种新型DNA甲基化电化学传感器。利用一步热变性法组装成TSP后,通过Au—S键固定在修饰纳米金颗粒的金电极表面,经过靶标DNA杂交、5-甲基胞嘧啶(5-mc)抗体及AuNPs-IgG-HRP结合后,用差分脉冲伏安法(DPV)进行检测。采用循环伏安法(CV)和电化学阻抗谱(EIS)对修饰电极的构建过程进行电化学表征。探究了杂交时间、5-mc抗体浓度、IgG-HRP加入体积、氢醌(HQ)和过氧化氢(H2O2)浓度对传感器的影响。在最佳条件下,该传感器对甲基化DNA的线性响应范围为1.0×10-15~1.0×10-10 mol/L,检出限(S/N=3)为4.4×10-16 mol/L。该传感器具有良好的选择性和稳定性,为DNA甲基化检测提供了新方法。     

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.     

Ultrasensitive electrogenerated chemiluminescence detection of DNA hybridization using carbon-nanotubes loaded with tris(2,2′-bipyridyl) ruthenium derivative tags

An ultrasensitive electrogenerated chemiluminescence (ECL) detection method of DNA hybridization based on single-walled carbon-nanotubes (SWNT) carrying a large number of ruthenium complex tags was developed. The probe single strand DNA (ss-DNA) and ruthenium complex were loaded at SWNT, which was taken as an ECL probe. When the capture ss-DNA with a thiol group was self-assembled onto the surface of gold electrode, and then hybridized with target ss-DNA and further hybridized with the ECL probe to form DNA sandwich conjugate, a strong ECL response was electrochemically generated. The ECL intensity was linearly related to the concentration of perfect-matched target ss-DNA in the range from 2.4 × 10⁻¹⁴ to 1.7 × 10⁻¹² M with a detection limit of 9.0 × l0⁻¹⁵ M. The ECL signal difference permitted to discriminate the perfect-matched target ss-DNA and two-base-mismatched ss-DNA. This work demonstrates that SWNT can provide an amplification platform for carrying a large number of ECL probe and thus resulting in an ultrasensitive ECL detection of DNA hybridization.