Real Time Electrochemical Monitoring of DNA/PNA Dissociation by Melting Curve Analysis

An immobilization‐free electrochemical method is reported for real‐time monitoring of the DNA hybrid dissociation between a ferrocene labeled peptide nucleic acid (PNA) and a fully‐complementary or single‐base‐mismatched DNA. This method takes advantages of electrostatic charge characteristics and interactions among the neutrally charged PNA, the negatively charged DNA and the negatively charged electrode surface made of indium tin oxide (ITO). When a ferrocene labeled PNA (Fc‐PNA) sequence is hybridized to a complementary DNA strand, electrostatic repulsion between the negatively charged PNA/DNA hybrid and the negative ITO surface retards the diffusion of the electroactive Fc to the electrode, resulting in a much reduced electrochemical signal. On the other hand, when the Fc‐PNA is dissociated from the hybrid at elevated temperatures, the neutrally charged Fc‐PNA easily diffuses to the electrode with an enhanced electrochemical signal. Therefore, an electrochemical melting curve of the Fc‐PNA/DNA hybrid can be obtained by measuring the Fc signal with the increasing temperature. This strategy allows monitoring of the dissociation of the DNA hybrid in real time, which might lead to a simple detection method for single nucleotide polymorphism (SNP) analysis.     

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.     

Electrochemically‐controlled DNA Release under Physiological Conditions from a Monolayer‐modified Electrode

An indium tin oxide (ITO) electrode prepared on a flexible polymeric support was modified with an amino‐silane and then functionalized with trigonelline and 4‐carboxyphenylboronic acid covalently bound to the amino groups. The trigonelline species containing quarterized ammonium group produced positive charge on the electrode surface regardless of the pH value, while the phenylboronic acid species were neutral below pH 8 and negatively charged above pH 9 (note that their pK_a=8.4). The total charge on the monolayer‐modified electrode was positive at the neutral pH and negative at pH>9 (note that 4‐carboxyphenylboronic acid was attached to the electrode surface in excess to trigonelline, thus allowing the negative charge to dominate on the electrode surface at basic pH). Single‐stranded DNA molecules were loaded on the modified electrode at pH 7.0 due to their electrostatic attraction to the positively charged surface. By applying electrolysis at −1.0 V (vs. Ag/AgCl reference) electrochemical oxygen reduction resulted in the consumption of hydrogen ions and local pH increase in the vicinity of the electrode surface. The process resulted in the transition to the total negative charge due to the negative charges formed on the phenylboronic acid species. This resulted in the electrostatic repulsion and release of the loaded DNA. The developed approach allowed the electrochemically‐triggered DNA release not only in the aqueous solutions, but also in human serum solution, thus giving promise for future biomedical applications.     

Electrochemical Quantitative Analysis of Nucleic Acids Using β‐Cyclodextrin Modified Gold Electrode

We present an electrochemical biosensor for the analysis of nucleic acids upon hybridization on the β‐cyclodextrin (β‐CD)‐modified gold electrode. The strategy is based on the following: The 5’‐ferrocene‐labeled single stranded capture probe DNA (5’‐fc‐ss‐DNA) was incorporated into the cavity of thiolated β‐CD which was immobilized on the surface of gold electrode. After hybridization of complementary target DNA, hybridized double stranded DNA (ds‐DNA) was released from the cavity of β‐CD. The difference of electrochemical properties on the modified gold electrode was characterized by cyclic voltametry and surface plasmon resonance. We successfully applied this method to the investigation of the sensor responses due to hybridization on various concentrations of applied target DNA. As a result, the label‐free electrochemical DNA sensor can detect the target DNA with a detection limit of 1.08 nM. Finally, our method does not require either hybridization indicators or other signalling molecules such as DNA intercalaters which most of electrochemical hybridization detection systems require.     

DNA hybridization at microbeads with cathodic stripping voltammetric detection

In electrochemical DNA hybridization sensors generally a single-stranded probe DNA was immobilized at the electrode followed by hybridization with the target DNA and electrochemical detection of the hybridization event at the same electrode. In this type of experiments nonspecific adsorption of DNA at the electrode caused serious difficulties especially in the case of the analysis of long target DNAs. We propose a new technology in which DNA is hybridized at a surface H and the hybridization is detected at the detection electrode (DE). This technology significantly extends the choice of hybridization surfaces and DEs. Here we use paramagnetic Dynabeads Oligo(dT)(25) (DBT) as a transportable reactive surface H and a hanging mercury drop electrode as DE. We describe a label-free detection of DNA and RNA (selectively captured at DBT) based on the determination of adenines (at ppb levels, by cathodic stripping voltammetry) released from the nucleic acids by acid treatment. The DNA and RNA nonspecific adsorption at DBT is negligible, making thus possible to detect the hybridization event with a great specificity and sensitivity. Specific detection of the hybridization of polyribonucleotides, mRNA, oligodeoxynucleotides, and a DNA PCR product (226 base pairs) is demonstrated. New possibilities in the development of the DNA hybridization sensors opened by the proposed technology, including utilization of catalytic signals in nucleic acid determination at mercury (e.g. signals of osmium complexes covalently bound to DNA) and solid DEs (e.g. using enzyme-labeled antibodies against chemically modified DNAs) are discussed.     

Improved electrochemical performances of polyaniline nanotubes-poly-L-lysine composite for label-free impedance detection of DNA hybridization

A sensitive label-free DNA hybridization biosensing platform was fabricated based on the synergistic effect of polyaniline nanotubes (PANI_nt) and poly-L-lysine (pLys). The composite of pLys and PANI_nt was coated onto the carbon paste electrode (CPE) to form a uniform and very stable nanocomposite membrane. The pLys in the composite film not only acts as a membrane to retain good electron transfer capability of PANI_nt even at physiological pH, but also possesses fine biocompatibility for bio-analytes. DNA probes with negatively charged phosphate groups were readily linked to the positively charged pLys surface due to the strong electrostatic affinity. The synergistic effect of PANI_nt and pLys could significantly enhance the sensitivity of DNA hybridization recognition. The phosphinothricin acetyltransferase (PAT) gene fragment from transgenic corn and the polymerase chain reaction amplification of the terminator of nopaline synthase gene from the real sample of a kind of transgenic soybean were detected by this DNA electrochemical biosensor via label-free impedance method. This stable composite gives convenient permselectivity properties as a transducer material for the design of modern electrochemical impedance biosensor using [Fe(CN)₆]^3?/4? as an indicator.     

电化学石英晶体微天平实时表征和定量检测短序列DNA

利用电化学石英晶体微天平(EQCM)这一灵敏的质量和电化学传感器测定特定序列DNA。应用自组装膜技术在压电石英晶振表面自组装一带羧基的α－硫辛酸单层膜,通过盐酸1-乙基-3-(3-二甲基氨基丙基)碳二亚胺(EDC)及N-羟基琥珀酰亚胺(NHS)共价固化寡聚核苷酸为探针,用于测定与其碱基序列互补的DNA。实验中EQCM实时监测了α-硫辛酸的自组装过程、探针固化过程及其与cDNA杂交过程。定量得出了探针固化量及cDNA杂交量。在酸性、中性和碱性条件下,分别对固化和杂交过程进行表征,实验发现探针固化及DNA杂交都受pH影响,本文对此现象进行了解释。同时,利用染料Hoechst33258的电化学活性,使其与双链DNA嵌合,通过测量Hoechst33258的电化学信息进一步验证了DNA杂交关键步骤。     

Electrochemically Stimulated Insulin Release from a Modified Graphene‐functionalized Carbon Fiber Electrode

A graphene‐functionalized carbon fiber electrode was modified with adsorbed polyethylenimine to introduce amino functionalities and then with trigonelline and 4‐carboxyphenylboronic acid covalently bound to the amino groups. The trigonelline species containing quarterized pyridine groups produced positive charge on the electrode surface regardless of the pH value, while the phenylboronic acid species were neutral below pH 8 and negatively charged above pH 9 (note that their pK_a=8.4). The total charge on the monolayer‐modified electrode was positive at the neutral pH and negative at pH > 9. Note that 4‐carboxyphenylboronic acid was attached to the electrode surface in molar excess to trigonelline, thus allowing the negative charge to dominate on the electrode surface at basic pH. Negatively charged fluorescent dye‐labeled insulin (insulin‐FITC) was loaded on the modified electrode surface at pH 7.0 due to its electrostatic attraction to the positively charged interface. The local pH in close vicinity to the electrode surface was increased to ca. 9–10 due to consumption of H⁺ ions upon electrochemical reduction of oxygen proceeding at the potential of −1.0 V (vs. Ag/AgCl) applied on the modified electrode. The process resulted in recharging of the electrode surface to the negative value due to the formation of the negative charge on the phenylboronic acid groups, thus resulting in the electrostatic repulsion of insulin‐FITC and stimulating its release from the electrode surface. The insulin release was characterized by fluorescence spectroscopy (using the FITC‐labeled insulin), by electrochemical measurements on an iridium oxide, IrO_x, electrode and by mass spectrometry. The graphene‐functionalized carbon fiber electrode demonstrated significant advantages in the signal‐stimulated insulin release comparing with the carbon fiber electrode without the graphene species.     

A PDDA/poly(2,6-pyridinedicarboxylic acid)-CNTs composite film DNA electrochemical sensor and its application for the detection of specific sequences related to PAT gene and NOS gene

2,6-Pyridinedicarboxylic acid (PDC) was electropolymerized on the glassy carbon electrode (GCE) surface combined with carboxylic group-functionalized single-walled carbon nanotubes (SWNTs) by cyclic voltammetry (CV) to form PDC-SWNTs composite film, which was rich in negatively charged carboxylic group. Then, poly(diallyldimethyl ammonium chloride) (PDDA), a linear cationic polyelectrolyte, was electrostatically adsorbed on the PDC-SWNTs/GCE surface. DNA probes with negatively charged phosphate group at the 5' end were immobilized on the PDDA/PDC-SWNTs/GCE due to the strong electrostatic attraction between PDDA and phosphate group of DNA. It has been found that modification of the electrode with PDC-SWNTs film has enhanced the effective electrode surface area and electron-transfer ability, in addition to providing negatively charged groups for the electrostatic assembly of cationic polyelectrolyte. PDDA plays a key role in the attachment of DNA probes to the PDC-SWNTs composite film and acts as a bridge to connect DNA with PDC-SWNTs film. The cathodic peak current of methylene blue (MB), an electroactive label, decreased obviously after the hybridization of DNA probe (ssDNA) with the complementary DNA (cDNA). This peak current change was used to monitor the recognition of the specific sequences related to PAT gene in the transgenic corn and the polymerase chain reaction (PCR) amplification of NOS gene from the sample of transgenic soybean with satisfactory results. Under optimal conditions, the dynamic detection range of the sensor to PAT gene target sequence was from 1.0x10(-11) to 1.0x10(-6) mol/L with the detection limit of 2.6x10(-12) mol/L.     

Imaging Local Proton Fluxes through a Polycarbonate Membrane by Using Scanning Electrochemical Microscopy and Functionalized Alkanethiols

A new application of scanning electrochemical microscopy (SECM) to probe the transport of protons through membranes is described. Herein, a probe ultramicroelectrode (UME) is modified with a self‐assembled monolayer (SAM) of 11‐mercaptoundecanoic acid to qualitatively image areas within different pH regions above a track‐etched membrane. The current response of the modified electrode in the presence of potassium hexacyanoferrate as electroactive component is different in acidic and alkaline solutions. Depending on the pH value of the solution, the SAM‐covered electrode exposes either a neutral or a negatively charged insulating monolayer at pH 3 or 7, respectively, which leads to an increase/decrease in the faradaic current due to electrostatic interactions between the neutral/charged surface and the charged redox mediator. Therefore, local pH changes in the close vicinity of a membrane‐like substrate lead to different current responses recorded at the tip electrode when scanning above the surface.     

Amine-terminated organosilica nanosphere functionalized prussian blue for the electrochemical detection of glucose

A new glucose biosensor had been developed by immobilizing positively charged gold nanoparticles (PGNs) on organosilica nanosphere functionalized prussian blue (OSiFPB)-modified gold electrode. The OSiFPB compound could not only effectively prevent the leakage of the PB mediator during measurements, but also easily form stable film on the electrode surface with efficient redox-activity and excellent conductivity. Furthermore, with the negatively charged surface of OSiFPB, this film could be used as an interface to adsorb the PGNs, which provided a congenial microenvironment for adsorbing biomolecules and decreased the electron-transfer impedance. So, with glucose oxidase as a model biomolecular, the proposed sensor showed rapid and highly sensitive amperometric response to glucose and this immobilization approach effectively improved the stability of the electron-transfer mediator. This work would be promising for construction of biosensor and bioelectronic devices.     

多铜氧化酶/中介体催化氧还原反应动力学分析

采用分光光度法、Clark型氧电极以及循环伏安法结合旋转圆盘电极技术,分别测定了游离多铜氧化酶在扩散型电子中介体存在时,催化氧还原循环中每一个组成步骤的速率并进行了比较,试图确定这个催化反应的决速步骤。 实验结果表明,漆酶分子内部的电子迁移速率(103/s)最高,酶催化氧气化学还原的速率次之(91/s),酶催化中介体氧化产物在电极上电化学还原的速率再次之(0.19/s或7.8×10-3 cm/s),底物O2气以及氧化态/还原态电子中介体2,2′-连氮-双-(3-乙基苯并噻唑啉-6-磺酸)二铵盐(ABTS)的传质系数分别为1.7×10-3、4.4×10-4 和6.3×10-4 cm/s,相应地酶催化中介体氧化的化学反应速率为0.047/s,酶催化中介体氧化的化学反应步骤以及中介体的传质步骤是影响催化反应速率的关键。 在此基础上,通过系统改变体系中酶的种类、活力以及浓度、中介体种类及浓度、溶液温度及pH值等参数,研究了酶电催化氧还原活力与这些参数之间的依赖关系,进一步确证了前述的结论。     

Electrochemical impedance sensing of DNA hybridization on conducting polymer film-modified diamond

The impedimetric sensing of DNA hybridization on polyaniline/polyacrylate (PANI/PAA)-modified boron-doped diamond (BDD) electrode has been investigated. An ultrathin film of PANI-PAA copolymer was electropolymerized onto the diamond surfaces to provide carboxylic groups for tethering to DNA sensing probes. The electrochemical impedance and the intrinsic electroactivity of the polymer-diamond interface were analyzed after the hybridization reaction with target and non-target DNA. The impedance measurement shows changes in the impedance modulus as well as electron-transfer resistance at the stage of probe DNA immobilization (single-strand), as well as after hybridization with target DNA (double-strand). DNA hybridization increases the capacitance of the polymer-DNA layer and reduces the overall impedance of the DNA-polymer-diamond stack significantly. The polymer-modified BDD electrode shows no detectable nonspecific adsorption, with good selectivity between the complementary DNA targets and the one-base mismatch targets. The detection limit was measured to be 2 x 10(-8) M at 1000 Hz. Denaturing test on the hybridized probe and subsequent reuse of the probe indicates chemical robustness of the sensor. Our results suggest that electropolymerization followed by the immobilization of biomolecules is a simple and effective way of creating a functional biomolecular scaffold on the diamond surface. In addition, label-free electrochemical impedance method can provide direct and noninvasive sensing of DNA hybridization on BDD.     

基于电中性锇配合物的低背景DNA电化学传感器

采用紫外光谱和电化学方法研究了一种电中性锇配合物Os(DPPZ)(PC)(H₂O)DPPZ=联吡啶并[3,2-a,2',3'-c]吩嗪, PC=2,6-吡啶二羧酸}与DNA的相互作用. 紫外光谱结果表明, DNA的加入引起配合物特征吸收峰的减色及红移效应, 说明二者之间存在嵌插作用. 循环伏安实验结果表明, 配合物溶液中加入DNA后, 氧化还原峰电流降低且式电位正移, 证实了二者之间的嵌插作用模式. 将该配合物作为杂交指示剂对CaMV35S启动子基因片段进行检测发现, 在单链探针DNA修饰电极上未观察到指示剂的电化学信号, 而在杂交后的双链DNA电极上呈现灵敏的电化学响应, 表明传感器具有较高的信噪比. 定量分析实验结果表明, 在最佳条件下, 杂交指示剂在传感器上的还原峰电流与目标序列浓度在8.0×10^-10~2.8×10^-9 mol/L范围内呈良好的线性关系. 选择性实验结果表明, 该传感器对互补序列、碱基错配序列和非互补序列具有良好的识别能力.     

纳米金放大计时库仑法的PML/RARα融合基因检测

应用恒电位在金基底表面电化学沉积纳米金,通过Au—S键将巯基修饰DNA探针固定在纳米金表面,与互补靶序列杂交,构建计时库仑电化学DNA传感器,并检测急性早幼粒细胞白血病(APL)PML/RARα融合基因.采用扫描电子显微术(SEM)与电化学交流阻抗技术(EIS)观察纳米金和表征DNA传感器的构筑过程.以氯化六氨合钌([Ru(NH3)6]Cl3,RuHex)作电化学杂交指示剂,由计时库仑法检测人工合成APL的PML/RARα融合基因.结果表明,纳米金能放大RuHex检测信号,杂交前后电量差值(ΔQ)与靶标链DNA浓度的对数(lgC)值在1.0×10-13～1.0×10-9mol.L-1范围内呈线性关系,检出下限3.7×10-14mol.L-1(S/N=3).该法操作简便、特异性好,有望用于实际样品的检测.     

Immobilization of single-stranded deoxyribonucleic acid on gold electrode with self-assembled aminoethanethiol monolayer for DNA electrochemical sensor applications

A synthesized 24-mer single-stranded deoxyribonucleic acid (ssDNA) was covalently immobilized onto a self-assembled aminoethanethiol monolayer modified gold electrode, using water-soluble 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide (EDC). The covalently immobilized ssDNAs were hybridized with complementary ssDNA (cDNA) or yAL(3) gene in solution, forming double-stranded DNAs (dsDNA). Meanwhile, daunomycin as an electrochemical active intercalator in the hybridization buffer solution was intercalated into the dsDNA to form a dsDNA/daunomycin system on the gold electrode surface, which was used for DNA electrochemical sensor. The cathodic waves of daunomycin bound to the double-stranded DNA (dsDNA) by linear sweep voltammetry were utilized to detect the cDNA. The cathodic peak current (i(pc)) of duanomycin was linearly related to the concentrations of cDNA between 0.1 mug ml(-1) and 0.1 ng ml(-1). The detection limit was about 30 pg ml(-1).     

A Host‐Guest‐Recognition‐Based Electrochemical Sensor for Sequence‐Specific DNA Detection

We herein constructed a sensor that converts target DNA hybridization‐induced conformational transformation of the probe DNA to electrochemical response based on host‐guest recognition and nanoparticle label. In the sensor, the hairpin DNA terminal‐labeled with 4‐((4‐(dimethylamino)phenyl)azo)benzoic acid (dabcyl) and thiol group was immobilized on Au electrode surface as the probe DNA by Au‐S bond, and the CdS nanoparticles surface‐modified with β‐cyclodextrins (CdS‐CDs) were employed as electrochemical signal provider and host‐guest recognition element. Initially, the probe DNA immobilized on electrode kept the stem‐loop configuration, which shielded dabcyl from docking with the CdS‐CDs in solution due to the steric effect. After target hybridization, the probe DNA underwent a significant conformational change, which forced dabcyl away from the electrode. As a result, formerly‐shielded dabcyl became accessible to host‐guest recognition between β‐cyclodextrin (β‐CD) and dabcyl, thus the target hybridization event could be sensitively transduced to electrochemical signal provided by CdS‐CDs. This host‐guest recognition‐based electrochemical sensor has been able to detect as low as picomolar DNA target with excellent differentiation ability for even single mismatch.     

Electroactive chitosan nanoparticles for the detection of single-nucleotide polymorphisms using peptide nucleic acids

Here we report an electrochemical biosensor that would allow for simple and rapid analysis of nucleic acids in combination with nuclease activity on nucleic acids and electroactive bionanoparticles. The detection of single-nucleotide polymorphisms (SNPs) using PNA probes takes advantage of the significant structural and physicochemical differences between the full hybrids and SNPs in PNA/DNA and DNA/DNA duplexes. Ferrocene-conjugated chitosan nanoparticles (Chi-Fc) were used as the electroactive indicator of hybridization. Chi-Fc had no affinity towards the neutral PNA probe immobilized on a gold electrode (AuE) surface. When the PNA probe on the electrode surface hybridized with a full-complementary target DNA, Chi-Fc electrostatically attached to the negatively-charged phosphate backbone of DNA on the surface and gave rise to a high electrochemical oxidation signal from ferrocene at ∼0.30 V. Exposing the surface to a single-stranded DNA specific nuclease, Nuclease S1, was found to be very effective for removing the nonspecifically adsorbed SNP DNA. An SNP in the target DNA to PNA made it susceptible to the enzymatic digestion. After the enzymatic digestion and subsequent exposure to Chi-Fc, the presence of SNPs was determined by monitoring the changes in the electrical current response of Chi-Fc. The method provided a detection limit of 1 fM (S/N = 3) for the target DNA oligonucleotide. Additionally, asymmetric PCR was employed to detect the presence of genetically modified organism (GMO) in standard Roundup Ready soybean samples. PNA-mediated PCR amplification of real DNA samples was performed to detect SNPs related to alcolohol dehydrogenase (ALDH). Chitosan nanoparticles are promising biometarials for various analytical and pharmaceutical applications. Figure The electrochemical method for SNP detection using PNA probes and chitosan nanoparticles takes advantage of the significant structural and physicochemical differences between PNA/DNA and DNA/DNA duplexes. Single-stranded DNA specific enzymes selectively choose these SNP sites and hydrolyze the DNA molecules on gold electrode (AuE) surface. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

An electrochemical DNA sensor based on a layers-film construction modified electrode

This work developed a relatively inexpensive and layers-film construction electrochemical sensor for DNA recognition and its performance was investigated. The Fe(3)O(4) magnetic nanoparticles-cysteine were immobilized on the carbon paste electrode (CPE) surface using magnetic force. Multiwalled carbon nanotubes (MWCNTs), gold nanoparticles (GNPs), and chitosan (Chi) were used successively to coat on the electrode surface. The thiolated capture probe was assembled and competitively hybridized with the target nucleic acid and biotinylated response probe. The electrochemical behavior was analyzed by cyclic voltammetry and electrochemical impedance spectroscopy. In addition, the sensor performance was also analyzed by introducing the notion of detection efficiency. The experimental results showed that although the electron transfer capability of the CPE is less strong than that of a metal electrode used in the DNA sensor, the materials modified on the CPE could significantly improve the performance. A detection limit of 1 nM of target DNA and a sensitivity of 2.707 × 10(3) mA M(-1) cm(-2) were obtained. Although the resulting detection limit was not remarkable, further experiments could improve it.     

Immobilization of DNA onto poly(dimethylsiloxane) surfaces and application to a microelectrochemical enzyme-amplified DNA hybridization assay

This paper describes immobilization of DNA onto the interior walls of poly(dimethylsiloxane) (PDMS) microsystems and its application to an enzyme-amplified electrochemical DNA assay. DNA immobilization was carried out by silanization of the PDMS surface with 3-mercaptopropyltrimethoxysilane to yield a thiol-terminated surface. 5'-acrylamide-modified DNA reacts with the pendant thiol groups to yield DNA-modified PDMS. Surface-immobilized DNA oligos serve as capture probes for target DNA. Biotin-labeled target DNA hybridizes to the PDMS-immobilized capture DNA, and subsequent introduction of alkaline phosphatase (AP) conjugated to streptavidin results in attachment of the enzyme to hybridized DNA. Electrochemical detection of DNA hybridization benefits from enzyme amplification. Specifically, AP converts electroinactive p-aminophenyl phosphate to electroactive p-aminophenol, which is detected using an indium tin oxide interdigitated array (IDA) electrode. The IDA electrode eliminates the need for a reference electrode and provides a steady-state current that is related to the concentration of hybridized DNA. At present, the limit of detection of the DNA target is 1 nM in a volume of 20 nL, which corresponds to 20 attomoles of DNA.