纳米探针芯片技术用于微量乙肝病毒DNA的检测

利用两组探针修饰的微粒:(1)表面标记有可与待测乙肝病毒(HBV) DNA另一端结合的纳米金探针1(信号探针)以及可与信号探针部分结合的纳米金探针2(检测探针);(2)表面标记有可与待测HBV DNA一端结合的磁珠探针(捕捉探针1).检测靶HBV DNA时,磁珠探针与信号探针在液相中可分别与HBV DNA靶序列一端结合最终形成三明治样结构.再以磁场将三明治样复合物从反应液中分离,以DTT溶液将信号探针从纳米金颗粒上洗脱.洗脱后的信号探针数量反映靶基因的多寡,信号探针一段与预先点样的基因芯片上的捕捉探针2结合,检测探针与信号探针另一段相结合,最后用银染液将检测探针显色从而得到靶目标DNA相对定量信息.结果表明,本检测方法的检测灵敏度达到10-15 mol/L水平.检测时间少于1.5 h,检测结果与HBV DNA水平呈现较好的线性关系且无假阳性结果;本方法有望用于乙肝病人血清中HBV DNA的快速筛测及其它微生物基因的检测.     

基于免标记发夹型探针和核酸外切酶Ⅲ的荧光信号放大DNA检测

设计合成了一种长臂发夹型核酸探针,结合核酸外切酶Ⅲ水解反应建立了一种免标记荧光信号放大高灵敏检测DNA的新方法.当不存在靶DNA时,SYBR GreenⅠ荧光染料能够嵌入发夹型探针的茎部而发出很强的荧光,而当存在靶DNA并与发夹型探针杂交后,核酸外切酶Ⅲ从杂交产物的3'端开始水解发夹型探针,释放出靶DNA,并触发下一个酶水解反应,同时SYBR GreenⅠ染料也随发夹型探针水解而释放,导致荧光信号降低,从而实现了对DNA的免标记荧光信号放大高灵敏检测.该方法的检出限低至320 fmol/L,比传统双标的分子信标的方法降低了4~5个数量级,且该方法还具有免标记、简单、快速的特点.     

基于纳米金复合探针-蛋白芯片检测心肌损伤标志物

运用纳米金复合探针结合蛋白芯片,建立了一种检测心肌损伤标志物的新方法.构建了2种纳米金探针:标记有检测抗体和DNA探针1的检测探针和标记有DNA探针2(与DNA探针1的碱基互补配对)的信号探针.当目的抗原存在时,检测探针经检测抗体和抗原,与芯片上捕获抗体结合固定在芯片上,信号探针通过碱基互补配对与检测探针结合使信号放大,最后利用纳米金成核原理染色,通过显微镜观察结果并定量分析.该体系在40 min内可检测多种标志物,其中肌钙蛋白Ⅰ(c TnⅠ)的检出限为10 pg/m L,与临床电化学发光法(ECLIA)灵敏度相当;肌红蛋白(MYO)与新型脂肪酸结合蛋白(HFABP)的检出限分别为640和10 pg/m L,与ECLIA及酶联免疫吸附法(ELISA)相比,灵敏度显著提高.     

基于分子识别作用鉴别人参基因的电化学传感器

本文以人参ITS及518 s基因上的SNP位点为检测对象,利用分子识别作用构建了一种电化学传感器,成功地对人参、西洋参进行了品种鉴别。本文设计合成了一种双标记DNA探针(DLP),该探针的一端标记了4-4-二甲氨基苯基偶氮苯甲酸(dabcyl)作为客体分子,另一端标记了金纳米颗粒作为电化学杂交指示剂。同时使用α-CD/MCNTs/GCE电极作为工作电极。由于DLP的茎环结构,只有在DLP与目标DNA杂交后,DLP上的dabcyl分子进入修饰电极表面的α-CD空腔中,进而DLP被α-CD修饰电极捕获。并且,通过金纳米颗粒的AuCl4-的电化学还原电流信号。可灵敏检测4.6×10-10mol.L-1的目标DNA。     

基因芯片-玻璃表面两种不同DNA探针固定化方法的比较研究

研究了基因芯片相关的DNA探针在芯片表面最佳固定化方法。用两种不同的双功能试剂1,4-苯二异硫氰酸酯和戊二醛分别把5'-端氨基衍生的21-mer寡脱氧核苷酸探针直接共价固定到玻片表面,固定化的寡脱氧核苷酸探针与5'-端FITC标记的互补靶序列进行分子杂交,杂交后用配有CCD的IX70型荧光倒置显微镜成像检测。结果表明,两种固定化方法的效果都比较好,能检测到靶序列的最低终浓度为1.5×10^-9mol/L,优化了探针固定化时间、杂交时间、杂交温度等对DNA芯片分析性能的影响,为构建高灵敏度基因芯片打下良好基础。     

基于银纳米粒子构建荧光传感平台用于核酸检测

报道了基于银纳米粒子构建的荧光传感平台,并用于核酸检测.此荧光传感平台对核酸检测基于以下策略:首先,荧光团标记的单链DNA探针被吸附到银纳米粒子的表面,荧光团与银纳米粒子近距离接触,发生荧光猝灭;加入与探针DNA序列互补的目标DNA,两者杂交形成双链DNA,并从银纳米粒子的表面脱离,荧光得到恢复.这种银纳米粒子构建的荧...     

基于双纳米金探针杂交法检测HBV DNA

利用双纳米金探针结合基因芯片平台建立了一种检测乙肝病毒基因(HBV DNA)的新方法. 根据HBV DNA的保守序列设计捕获探针和信号报告探针, 通过一对互补的纳米金检测探针的双杂交法对HBV DNA进行信号放大, 最后进行银染, 达到对HBV DNA的可视化检测. 该方法的灵敏度高, 可检测10 fmol/L的HBV DNA, 且能在1.5 h内完成检测. 其具有的快速、 高灵敏度及低成本等优势使其有望发展成为一种检测HBV DNA的新方法.     

高灵敏纳米金比色芯片检测乙型肝炎病毒DNA及其变异

构建了新型纳米金比色芯片,利用Taq DNA连接酶的连接特异性,将其与乙型肝炎病毒DNA( HBV-DNA)靶序列完全互补杂交的捕获探针(固定在芯片上)和纳米金修饰的探针连接成一条链,从而将纳米金颗粒固定到芯片点阵上,再通过银染反应放大,形成裸眼可见的显色信息.通过点阵的位置及灰度,即可判断HBV-DNA靶序列的单碱基突变,并得出相对定量信息.本实验对不同浓度的HBV-DNA靶序列进行了检测.结果显示:此技术对单碱基突变有很强的特异性识别能力,并且具有较高的灵敏度(约10 pmol/L),在10～100 pmol/L浓度范围内表现出较好的线性关系.该技术检测时间短(＜1 h)、操作简单、不需要特殊的检测设备,具有很好的临床应用前景.     

核酸适配体介导的荧光铜纳米簇用于免标记快速检测水胺硫磷

基于核酸适配体的靶标识别能力和铜纳米簇（CuNCs）优良的荧光性能,本研究开发了一种免标记荧光探针用于有机磷农药水胺硫磷（ISO）的快速检测。当靶标分子ISO不存在时,溶液中ISO的核酸适配体和互补DNA形成双链结构,从而介导合成荧光CuNCs,表现出高的荧光信号;当待测样品中存在ISO时,ISO与其核酸适配体形成复合物,释放出单链互补DNA。游离的单链DNA不能介导合成CuNCs,导致溶液中的荧光信号减弱。在最佳条件下,检测体系的荧光抑制率与ISO浓度的对数在0.05～25 mg/L浓度范围内呈线性关系,检出限为47μg/L (3σ),其它物质对其检测几乎没有干扰。将此探针用于检测水样中的ISO,回收率为80.3%～108.0%。研究结果表明,本方法可用于检测实际样品中的ISO残留。     

阳离子荧光共轭聚合物结合纳米颗粒用于DNA检测

利用纳米颗粒对目标DNA的富集、分离作用以及阳离子荧光共轭聚合物良好的荧光特性,建立了一种特异性检测DNA的新方法.首先将标记有猝灭基团的DNA捕获探针修饰到纳米颗粒上,捕获互补的DNA分子;然后加入S1核酸酶,除去未捕获到互补DNA的捕获探针;最后用Dnase Ⅰ将颗粒上的双链切断,使猝灭基团从纳米颗粒上解离下来,与阳离子荧光共轭聚合物结合并猝灭其荧光.结果表明,目标核酸的浓度与该聚合物的荧光猝灭程度正相关,且具有良好的特异性,线性响应范围为5.0～40 nmol/L; 检出限为3.7 nmol/L(S/N=3).     

A Novel DNA Probe Based on Molecularly Imprinted Polymer Modified Electrode for the Electrochemical Monitoring of DNA

A DNA probe that was based on methylene blue (MB) imprinted polyvinyl pyridine polymer (MIP) modified carbon paste electrodes were developed for the first time for electrochemical monitoring of DNA. Probes were built up by adsorbing MB onto modified electrodes prior to DNA immobilization. It was shown that DNA strongly immobilizes on MIP modified electrodes when MB was adsorbed in advance of DNA immobilization. The performance of the MB imprinted polymer modified carbon paste electrodes (MIP‐CPE) to rebind the template molecule (MB) were compared to those of control polymer modified (non‐imprinted polymer NIP‐CPE) and bare (CPE) electrodes. Electrochemical signal resulting from the oxidation of guanine moiety of the immobilized probe DNA was high enough on the constructed platform, implicating that probes of this kind could be favorably used for DNA analysis. These probes exhibited high selectivity for its complementary DNA sequences (target). HBV‐DNA hybridization was studied to evaluate the selectivity of the probes for complementary, non‐complementary and mismatch sequences. The detection limit of the probe for the target DNA was 8.72 µg/mL (1.38 µM), which was better than those attained by some earlier DNA sensor studies.     

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.     

Electrochemical Determination of Label Free BRCA Hybridization by Single Use Antioxidant Modified Electrode

A novel DNA probe based on caffeic acid modified disposable pencil graphite electrodes were developed for the first time for the electrochemical determination of breast cancer gene sequence (BRCA) hybridization. Amino‐linked BRCA probe highly immobilized onto the caffeic acid modified electrode by means of the interaction between the amino group of BRCA probe and the carboxyl group of caffeic acid compared to the bare electrode. 44 % signal enhancement in guanine oxidation signal was measured by caffeic acid modified electrode. Besides, these probes exhibited high selectivity towards its complementary DNA sequences (target). Hybridization between probe and target (BRCA1) was studied to evaluate the selectivity of the probes for complementary, non‐complementary and mismatch sequences. The selectivity was also tested in the presence of mixture containing the target and one base mismatch BRCA sequences in the same ratio (1 : 1). It can be said this probe can select its complementary from the mixture.     

Label‐Free and Label Based Electrochemical Detection of Hybridization by Using Methylene Blue and Peptide Nucleic Acid Probes at Chitosan Modified Carbon Paste Electrodes

A chitosan modified carbon paste electrode (ChiCPE) based DNA biosensor for the recognition of calf thymus double stranded DNA (dsDNA), single stranded DNA (ssDNA) and hybridization detection between complementary DNA oligonucleotides is presented. DNA and oligonucleotides were electrostatically attached by using chitosan onto CPE. The amino groups of chitosan formed a strong complex with the phosphate backbone of DNA. The immobilized probe could selectively hybridize with the target DNA to form hybrid on the CPE surface. The detection of hybridization was observed by using the label‐free and label based protocols. The oxidation signals of guanine and adenine greatly decreased when a hybrid was formed on the ChiCPE surface. The changes in the peak currents of methylene blue (MB), an electroactive label, were observed upon hybridization of probe with target. The signals of MB were investigated at dsDNA modified ChiCPE and ssDNA modified ChiCPE and the increased peak currents were observed, in respect to the order of electrodes. The hybridization of peptide nucleic acid (PNA) probes with the DNA target sequences at ChiCPE was also investigated. Performance characteristics of the sensor were described, along with future prospects.     

Label‐Free Bioelectronic Detection of Point Mutation by Using Peptide Nucleic Acid Probes

An electrochemical hybridization biosensor based on peptide nucleic acid (PNA) probes with a label‐free protocol is described. The detection of PNA‐DNA and DNA‐DNA hybridizations were accomplished based on the oxidation signal of guanine by using differential pulse voltammetry (DPV) at carbon paste electrode (CPE). It was observed that the oxidation signals of guanine obtained from the PNA and DNA probe modified CPEs were higher than those obtained from the PNA‐DNA and DNA‐DNA hybrid modified CPEs due to the accessible unbound guanine bases. The detection of hybridization between PNA probe and point mutation containing DNA target sequences was clearly observed due to the difference of the oxidation signals of guanine bases, because the point mutation was guanine nearly at the middle of the sequence. The effect of the DNA target concentration on the hybridization signal was also observed. The PNA probe was also challenged with excessive and equal amount of noncomplementary DNA and also mixtures of point mutation and target DNA.     

A self-assembled deoxyribonucleic acid concatemer for sensitive detection of single nucleotide polymorphism

Polymerase-free and label-free strategies for DNA detection have shown excellent sensitivity and specificity in various biological samples. Herein, we propose a method for single nucleotide polymorphism (SNP) detection by using self-assembled DNA concatemers. Capture probes, bound to magnetic beads, can joint mediator probes by T4 DNA ligase in the presence of target DNA that is complementary to the capture probe and mediator probe. The mediator probes trigger self-assembly of two auxiliary probes on magnetic beads to form DNA concatemers. Separated by a magnetic rack, the double-stranded concatemers on beads can recruit a great amount of SYBR Green I and eventually result in amplified fluorescent signals. In comparison with reported methods for SNP detection, the concatemer-based approach has significant advantages of low background, simplicity, and ultrasensitivity, making it as a convenient platform for clinical applications. As a proof of concept, BRAF^T1799A oncogene mutation, a SNP involved in diverse human cancers, was used as a model target. The developed approach using a fluorescent intercalator can detect as low as 0.1 fM target BRAF^T1799A DNA, which is better than those previously published methods for SNP detection. This method is robust and can be used directly to measure the BRAF^T1799A DNA in complex human serum with excellent recovery (94–103%). It is expected that this assay principle can be directed toward other SNP genes by simply changing the mediator probe and auxiliary probes.     

A Sequence‐Selective Electrochemical DNA Biosensor Based on HRP‐Labeled Probe for Colorectal Cancer DNA Detection

Abstract

A highly‐sensitive sequence‐selective DNA sensor based on HRP‐labeled probe to detect specific K‐ras gene which is highly associated with colorectal cancer has been reported. Capture probe modified with–SH was first chemically adsorbed on the gold electrode through self‐assembly. Then, the hybridization of a complementary nucleic acid (target DNA:K‐ras gene) and HRP labeled oligonucleotide detection probe occurred in a sandwich way. Finally, H₂O₂ electroreduction current catalyzed by HRP was measured amperometrically in the presence of hydroquinon as mediator. The sequence selectivity is double guaranteed by the complementary hybridization of target DNA with capture and detection probes. The experimental conditions were optimized. The linear range is 1.17×10^?11～1.17×10^?7 mol l^?1 with a detection limit of 5.85×10^?12 mol l^?1. The electrode with capture probe can be reused after regeneration in boiling water.     

Electrochemical impedance probing of DNA hybridisation on oligonucleotide-functionalised polypyrrole

We report a new approach for detecting DNA hybridisation using non faradaic electrochemical impedance spectroscopy. The technique was applied to a system of DNA probes bearing amine groups that are immobilized by covalent grafting on a supporting polypyrrole matrix functionalised with activated ester groups.The kinetics of the attachment of the ss-DNA probe was monitored using the temporal evolution of the open circuit potential (OCP). This measurement allows the determination of the time necessary for the chemical reaction of ss-DNA probe into the polypyrrole backbone.The hybridisation reactions with the DNA complementary target and non complementary target were investigated by non faradaic electrochemical impedance spectroscopy. Results show a significant modification in the Nyquist plot upon addition of the complementary target whereas, in presence of the non complementary target, the Nyquist plot is not modified. The spectra, in the form of Nyquist plot, were analysed with the Randles circuit. The transfer charge resistance R₂ shows a linear variation versus the complementary target concentration. Sensitivity and detection limit (0.2 nM) were determined and detection limit was lower of one order of magnitude than that obtained with the same system and measuring variation of the oxidation current at constant potential.