Lead Sulfide Nanoparticle as Oligonucleotides Labels for Electrochemical Stripping Detection of DNA Hybridization

We report a method for the detection of DNA hybridization in connection to lead sulfide (PbS) nanoparticle tags and electrochemical stripping measurement of the lead. A kind of lead sulfide nanoparticle with free carboxyl groups on its surface was synthesized in aqueous solution. The nanoparticle was used as a marker to label a sequence‐known oligonucleotide, which was then employed 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 monitored by the oxidation dissolution of the lead sulfide anchored on the hybrids and the indirect determination of the lead ions by anodic stripping voltammetry (ASV). The detection limit is 0.3 pmol L^?1 of target oligonucleotides. The PbS nanoparticle combining its easy conjugation to the DNA molecule with the highly sensitive stripping voltammetry detection of lead shows its promising application in the electrochemical DNA hybridization analysis assay.     

Electrochemical Detection of DNA Hybridization Based on the Probe Labeled with Carbon‐Nanotubes Loaded with Silver Nanoparticles

A sensitive electrochemical method for the detection of DNA hybridization based on the probe labeled with multiwall carbon‐nanotubes (MWNTs) loaded with silver nanoparticles (Ag‐MWNTs) has been developed. MWNTs were electroless‐plated with a large number of silver nanoparticles to form Ag‐MWNTs. Probe single strand DNA (ss‐DNA) with a thiol group at the 3′‐terminal labeled with Ag‐MWNTs by self‐assembled monolayer (SAM) technique was employed as an electrochemical probe. Target ss‐DNA with a thiol group was immobilized on a gold electrode by SAM technique and then hybridized with the electrochemical probe. Binding events were monitored by differential pulse voltammetric (DPV) signal of silver nanoparticles. The signal difference permitted to distinguish the match of two perfectly complementary DNA strands from the near perfect match where just three base pairs were mismatched. There was a linear relation between the peak current at +120 mV (vs. SCE) and complementary target ss‐DNA concentration over the range from 3.1×10^?14 to 1.0×10^?11 mol/L with a detection limit of 10 fmol/L of complementary target ss‐DNA. The proposed method has been successfully applied to detection of the DNA sequence related to cystic fibrosis. This work demonstrated that the MWNTs loaded with silver nanoparticles offers a great promising approach for sensitive detection of DNA hybridization.     

PAMAM Dendrimers‐Based DNA Biosensors for Electrochemical Detection of DNA Hybridization

Gold electrodes were modified with submonolayers of mercaptoacetic acid (RSH) and further reacted with poly(amidoamine) (PAMAM) dendrimers (generation 4.0) to obtain thin films, on which DNA probe was later immobilized to afford a stable recognition layers. The characterization of the PAMAM/RSH‐modified electrode was investigated by cyclic voltammetry (CV) and electrochemical impedance measurement. Differential pulse voltammogram (DPV) measurement was used to monitor DNA hybridization with daunomycin (DNR) as indicator. Experiments carried out with these novel materials not only showed an improved DNA attachment quantity on the dendrimers‐modified electrodes compared to DNA sensors with oligonucleotides directly immobilized on Au electrodes, but also exhibited a high selectivity, sensitivity and stability for the measurement of DNA hybridization.     

Electrochemical DNA Biosensors Based on Palladium Nanoparticles Combined with Carbon Nanotubes

Palladium nanoparticles, in combination with multi‐walled carbon nanotubes (MWCNTs), were used to fabricate a sensitivity‐enhanced electrochemical DNA biosensor. MWCNTs and palladium nanoparticles were dispersed in Nafion, which were used to modify a glassy carbon electrode (GCE). Oligonucleotides with amino groups at the 5′ end were covalently linked onto carboxylic groups of MWCNTs on the electrode. The hybridization events were monitored by differential pulse voltammetry (DPV) measurement using methylene blue (MB) as an indicator. Due to the ability of carbon nanotubes to promote electron‐transfer and the high catalytic activities of palladium nanoparticles for electrochemical reaction of MB, the sensitivity of presented electrochemical DNA biosensors was remarkably improved. The detection limit of the method for target DNA was 1.2×10^?13 M.     

Fabrication of a Sensitive Impedance Biosensor of DNA Hybridization Based on Gold Nanoparticles Modified Gold Electrode

In this work, we report on the preparation of a simple, sensitive DNA impedance sensor. Firstly gold nanoparticles were electrodeposited on the surface of a gold electrode, and then probe DNA was immobilized on the surface of gold nanoparticles through a 5′‐thiol‐linker. Electrochemical impedance spectroscopy (EIS) was used to investigate probe DNA immobilization and hybridization. Compared to the bare gold electrode, the gold nanoparticles modified electrode could improve the density of probe DNA attachment and the sensitivity of DNA sensor greatly. The difference of electron transfer resistance (ΔR_et) was linear with the logarithm of complementary oligonucleotides sequence concentrations in the range of 2.0×10^?12 to 9.0×10^?8 M, and the detection limit was 6.7×10^?13 M. In addition, the DNA sensor showed a fairly good reproducibility and stability during repeated regeneration and hybridization cycles.     

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.     

Sequence Specific Electrochemical DNA Detection Based on Solution‐Phase Competitive Hybridization

We report a novel electrochemical method for detecting sequence‐specific DNA based on competitive hybridization that occurs in a homogeneous solution phase instead of on a solution‐electrode interface as in previously reported competition‐based electrochemical DNA detection schemes. The method utilizes the competition between the target DNA (t‐DNA) and a ferrocene‐labeled peptide nucleic acid probe (Fc‐PNA) to hybridize with a probe DNA (p‐DNA) in solution. The neutral PNA backbone and the electrostatic repulsion between the negatively‐charged DNA backbone and the negatively‐charged electrode surface are then exploited to determine the result of the competition through measurement of the electrochemical signal of Fc. Upon the introduction of the t‐DNA, the stronger hybridization affinity between the t‐DNA and p‐DNA releases the Fc‐PNA from the Fc‐PNA/p‐DNA hybrid, allowing it to freely diffuse to the negatively charged electrode to produce a significantly enhanced electrochemical signal of Fc. Therefore, the presence of the t‐DNA is indicated by the appearance or enhancement of the electrochemical signal, rendering a signal‐on DNA detection, which is less susceptible to false positive and can produce more reliable results than signal‐off detection methods. All the competitive hybridizations occur in a homogeneous solution phase, resulting in very high hybridization efficiency and therefore extremely short assay time. This simple and fast signal‐on solution‐competition‐based electrochemical DNA detection strategy has promising potential to find application in fields such as nucleic acid‐based point‐of‐care testing.     

Enhanced Electrochemical Detection of DNA Hybridization Based on Au/MWCNTs Nanocomposites

Abstract

The nanocomposites of gold nanoparticles and multi‐walled carbon nanotubes (MWCNTs) have been applied in the enhanced electrochemical detection of DNA hybridization. Gold nanoparticles coated on MWCNTs uniformly were synthesized by simply one step reaction. Target DNA was detected by the peak current difference of differential pulse voltammetry (DPV) signals of the electroactive indicator methylene blue (MB) before and after hybridization on the Au/MWCNTs modified glass carbon electrode (GCE). Due to the excellent electrical conductivity of the novel matrix, the biosensor revealed high sensitivity with the detection level down to 1.0 pM. Excellently selectivity and reproducibility were also discussed.     

功能化纳米金增强的DNA电化学检测和序列分析

用冠以大量二茂铁的纳米金微粒 /抗生蛋白链菌素结合物为标记物 ,将其标记于生物素修饰的寡聚核苷酸片段上 ,制成了具有电化学活性和纳米金放大作用的DNA电化学生物传感器 .首先采用巯基DNA和巯基烷烃混合自组装膜制备了金修饰电极 ,将探针DNA分子固定在了电极表面 ,运用杂交原则结合靶点分子在电极表面形成了双螺旋的DNA链 ,然后借助抗生蛋白链菌素和生物素之间的强亲和作用 ,引入了功能化的纳米金 .通过伏安法测定了修饰在纳米金上的二茂铁的氧化还原电流 ,可以识别和测定溶液中互补的靶点DNA ,17 mer靶点DNA的浓度在 0 .0 0 1～ 10nmol/L范围内有线性关系 ,检测限可达 0 .75× 10 -12 mol/L .     

上转换纳米粒子结合DNA链式扩增技术在阿霉素光控释放中的应用

纳米技术的发展使得纳米材料可以通过不同的表面包覆和修饰而在生物医药中发挥应用。 构建简单、经济、药物释放可控的生物相容性纳米药物仍是纳米生物化学领域的重点。 我们构建的纳米载药体系(DDS)以NaYF₄：Yb/Tm上转换纳米粒子为载体,在其表面通过光致断键型小分子4,5-二甲氧基-2-硝基苯基乙酮(DMNPE)连接一段短单链DNA,利用DNA链式扩增技术(HCR)来调节纳米粒子最终修饰的双链DNA的总量,从而控制对抗癌药物阿霉素(Dox)的担载量,在980 nm激光照射下上转换纳米粒子发射可切断DMNPE连接的近紫外光,协同胞内DNA酶的作用达到对药物的可控释放。 由于近红外光照对生物组织具有较好的穿透能力,此体系能够对病灶位置有更好的光靶向性从而减少药物的毒副作用。     

Electrochemical DNA Hybridization Detection Using DNA Cleavage

We report the new method for detection of DNA hybridization using enzymatic cleavage. The strategy is based on that S1 nuclease is able to specifically cleave only single strand DNA, but not double strand DNA. The capture probe DNA, thiolated single strand DNA labeled with electroactive ferrocene group, was immobilized on a gold electrode. After hybridization of target DNA of complementary and noncomplementary sequences, nonhybridized single strand DNA was cleaved using S1 nuclease. The difference of enzymatic cleavage on the modified gold electrode was characterized by cyclic voltammetry and differential pulse voltammetry. We successfully applied this method to the sequence‐selective discrimination between perfectly matched and mismatched target DNA including a single‐base mismatched target DNA. Our method does not require either hybridization indicators or other exogenous signaling molecules which most of the electrochemical hybridization detection systems require.     

纳米金颗粒增强信号的电化学生物传感器用于谷胱甘肽和半胱氨酸的检测

构建了一种高灵敏检测谷胱甘肽(GSH)和半胱氨酸(Cys)的新型电化学生物传感器. 先将富含T碱基的DNA1和DNA2探针分别修饰在金电极和纳米金颗粒(AuNPs)上, 再加入Hg²⁺, 通过形成T-Hg²⁺-T结构使AuNPs结合到金电极表面. 当加入GSH(或Cys)后, GSH(或Cys)可以竞争结合T-Hg²⁺-T结构中的Hg²⁺, 使AuNPs离开电极表面. 由于AuNPs上修饰的DNA探针能够静电吸附大量电活性物质六氨合钌(RuHex), 因此该过程可引起计时电量信号的显著变化, 据此实现了GSH(或Cys)的高灵敏检测. 该传感器的检出限达10 pmol/L, 比荧光法或比色法降低了2~3个数量级. 实验结果表明, 该传感器具有较好的选择性.     

一种基于磁性纳米粒子PCR的高通量SNP分型方法

利用磁性纳米粒子PCR扩增(MNPs-PCR)和等位基因特异性双色荧光探针(Cy3, Cy5)杂交, 建立了一种单核苷酸多态性(SNP)分型的新方法. 应用该方法对9个样本MTHFR基因的C677T多态进行检测, 野生和突变型样本正错配信号比大于9.0, 杂合型正错配信号比接近1.0, 分型结果经测序验证. 此方法无须产物纯化、浓缩, 扫描分型结果快速、直观, 是一种操作简单、快速、高通量、高灵敏度的分型方法.     

An Electrochemical Aptasensor for Detection of Thrombin based on Target Protein‐induced Strand Displacement

A sensitive electrochemical aptasensor for detection of thrombin based on target protein‐induced strand displacement is presented. For this proposed aptasensor, dsDNA which was prepared by the hybridization reaction of the immobilized probe ssDNA (IP) containing thiol group and thrombin aptamer base sequence was initially immobilized on the Au electrode by self‐assembling via Au? S bind, and a single DNA labeled with CdS nanoparticles (DP‐CdS) was used as a detection probe. When the so prepared dsDNA modified Au electrode was immersed into a solution containing target protein and DP‐CdS, the aptamer in the dsDNA preferred to form G‐quarter structure with the present target protein resulting that the dsDNA sequence released one single strand and returned to IP strand which consequently hybridized with DP‐CdS. After dissolving the captured CdS particles from the electrode, a mercury‐film electrode was used for electrochemical detection of these Cd²⁺ ions which offered sensitive electrochemical signal transduction. The peak current of Cd²⁺ ions had a good linear relationship with the thrombin concentration in the range of 2.3×10^?9–2.3×10^?12 mol/L and the detection limit was 4.3×10^?13 mol/L of thrombin. The detection was also specific for thrombin without being affected by the coexistence of other proteins, such as BSA and lysozyme.     

电化学石英晶体微天平研究界面电场对DNA杂交的影响

采用电化学石英晶体微天平, 现场监测不同界面电场下完全匹配的靶标DNA和不完全匹配的靶标DNA分别与寡聚核苷酸探针分子杂交的过程. 结果表明, 电极表面荷正电时DNA表观杂交效率比电极表面荷负电时高, 但假阳性比较显著; 而电极表面荷负电时能有效地抑制错配杂交. 探讨了引入界面电场后探针分子取向和微观作用力对DNA杂交的影响.     

New Anthraquinone Derivatives as Electrochemical Redox Indicators for the Visualization of the DNA Hybridization Process

Interactions of dsDNA and ssDNA, at the surface of gold and silver electrodes, with three novel anthraquinone derivatives: 3‐(9′,10′‐dioxo‐9′,10′‐dihydro‐anthracen‐1‐yl)‐7,11‐di(carboxymethyl)‐3,7,11‐triazatridecanedioic acid, (AQ‐1); 1‐(9′,10′‐dioxo‐9′,10′‐dihydro‐anthracen‐1yl)‐9‐carboxymethyl‐5‐methyl‐1,5,9‐triazaundecanoicacid, (AQ‐2); and N‐(2‐(9,10‐dioxo‐9,10‐dihydro‐anthracen‐1‐ylamino)ethyl)‐2‐(1,4,10,13‐tetraoxa‐7,16‐diazacyclooctadecan‐7‐yl)acetamide, (AQ‐3) are studied. These derivatives are well soluble in water and phosphate buffer solutions. The square wave voltammetric behavior of these redox indicators is described and the parameters of interactions with DNA are reported. It is also pointed out that these compounds can be employed as the hybridization indicators. The difference in the binding ability of the particular redox indicator to single and double stranded DNA can be used for the detection of the complementary nucleic acids.     

Fabrication of a Sensitive Electrochemical Biosensor for Detection of DNA Hybridization Based on Gold Nanoparticles/CuO Nanospindles Modified Glassy Carbon Electrode

In this work, a sensitive electrochemical DNA biosensor for the detection of sequence‐specific target DNA was reported. Firstly, CuO nanospindles (CuO NS) were immobilized on the surface of a glassy carbon electrode (GCE). Subsequently, gold nanoparticles (Au NPs) were introduced to the surface of CuO NS by the electrochemical deposition mode. Probe DNA with SH (HS‐DNA) at the 5′‐phosphate end was covalently immobilized on the surface of the Au NPs through Au? S bond. Scanning electron microscopy (SEM) was used to elucidate the morphology of the assembled film, and electrochemical impedance spectroscopy technique (EIS) was used to investigate the DNA sensor assembly process. Hybridization detection of DNA was performed with differential pulse voltammetry (DPV) and the methylene blue (MB) was hybridization indicator. Under the optimal conditions, the decline of reduction peak current of MB (ΔI) was linear with the logarithm of the concentration of complementary DNA from 1.0×10^?13 to 1.0×10^?6 mol·L^?1 with a detection limit of 3.5×10^?14 mol·L^?1 (S/N=3). In addition, this DNA biosensor has good selectivity, and even can distinguish single‐mismatched target DNA.     

纳米粒子在电化学DNA生物传感器研究中的应用

简要介绍了电化学DNA生物传感器的原理和分类,对纳米粒子在电化学DNA生物传感器研究中的应用进行了详细评述.     

Electrochemical Behavior of Herbal Antitumor Drug Aloe‐Emodin at Carbon‐Coated Nickel Magnetic Nanoparticles Modified Glassy Carbon Electrode

The electrochemical behavior of aloe‐emodin (AE), an important herbal antitumor drug, was investigated at a carbon‐coated nickel magnetic nanoparticles modified glassy carbon electrode (CNN/GCE). A couple of well‐defined redox peaks was obtained. Some electrochemical parameters of AE at a CNN/GCE, such as the charge number, exchange current density, standard heterogeneous rate constant, were measured. The square wave voltammetry (SWV) response of AE was linear with the concentration over two concentration intervals viz. 6.24×10^?9?1.13×10^?6 M and 1.13×10^?6?1.23×10^?5 M, with a detection limit of 2.08 nM. A fast, simple and sensitive detection and analysis of AE was developed.     

基于分子信标荧光纳米探针的李斯特菌DNA均相检测方法

基于分子信标(MB)识别和荧光纳米粒子探针技术,建立了均相体系中李斯特菌目标DNA的高灵敏检测新方法.首先以羊抗人免疫球蛋白(IgG)标记的异硫氰酸荧光素(FITC)为核材料,成功制备了FITC-IgG@SiO2核壳荧光纳米粒子,有效防止了传统方法中采用单一FITC制备纳米颗粒时泄露严重的问题.随后以FITC-IgG@SiO2荧光纳米粒子和纳米金分别标记单核细胞增生李斯特菌序列特异性分子信标探针5'端和3'端,成功构建了单核细胞增生李斯特菌序列特异性分子信标荧光纳米探针.在实验优化条件下,α(令α=F/F0,F代表MB和目标DNA杂交以后的荧光强度,F0代表MB完全闭合时的荧光强度)与目标DNA浓度在1～200pmol/L浓度范围内呈良好的线性关系,检出下限为0.3pmol/L,相对标准偏差为2.6%(50pmol/L,n=11).将该方法应用于食品样品中单核细胞增生李斯特菌的检测,结果与国标法一致.