MCE‐electrochemical detection for following interactions of ssDNA and dsDNA with methylene blue

The interaction between the organic dye, methylene blue and DNA has been studied by MCE with electrochemical detection. Interaction produces two different signals, one corresponding to free methylene blue and other, for the complex methylene blue–DNA. The hybridization between a ssDNA and a complementary sequence, specific to the severe acute respiratory syndrome virus, has been performed and studied in a thermoplastic olefin polymer of amorphous structure CE‐microchip with an end‐channel gold wire detector. Moreover, studies with a longer dsDNA, an expression vector involved in the transitory or stable expression in mammals cells, pFLAG‐CMV4, has also been performed.     

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.     

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.     

Electrochemical genosensor for the detection of interaction between methylene blue and DNA

Described here are the chronocoulometric and voltammetric parameters for methylene blue [3,7-bis(dimethylamino)phenothiazin-5-ium chloride, MB] on binding to DNA at carbon paste electrode (CPE) surface. MB, which interacts with the immobilized calf thymus DNA was detected by using single stranded DNA modified CPE (ssDNA modified CPE), bare CPE and double stranded DNA modified CPE (dsDNA modified CPE) in combination with chronocoulometry and differential pulse voltammetry (DPV) techniques. The effect of ionic strength to the behavior of MB with dsDNA and ssDNA was also studied by means of voltammetry. These results demonstrated that MB could be used as an effective electroactive hybridization indicator for DNA biosensors. Performance characteristics of the sensor are described, along with future prospects.     

Amperometric detector designs for capillary electrophoresis microchips

Electrochemical (EC) detection is a sensitive and miniaturisable detection mode for capillary electrophoresis (CE) microchips. Detection cell design is very important in order to ensure electrical isolation from the high separation voltage. Amperometric detectors with different designs have been developed for coupling EC detection to CE-microchips. Different working electrode alignment: in-channel or end-channel has been tested in conjunction with several materials: gold, platinum or carbon. The end-channel detector was based on a platinum or gold wire manually aligned at the exit of the separation channel. Thick- (screen-printed carbon electrode) and thin-film (sputtered gold film) electrodes have also been employed with this configuration, but with a different design that allowed the rapid replacement of the electrode. The in-channel detector was based on a gold film within the separation channel. A gold-based dual electrode detector, which combined for the first time in- and end-channel detection, has been also tested. These amperometric detectors have been evaluated in combination to poly(methylmethacrylate) (PMMA) and Topas (thermoplastic olefin polymer of amorphous structure) CE-microchips. Topas is a new and promising cyclic olefin copolymer with high chemical resistance. Relevant parameters of the polymer microchip separation such as precision, efficiency or resolution and amperometric detection were studied with the different detector designs using p-aminophenol and L-ascorbic acid as model analytes in Tris-based buffer pH 9.0.     

Integrating amperometric detection with electrophoresis microchip devices for biochemical assays: recent developments

Recent advances in microfluidic systems, particularly in the Micro Total Analysis System (μTAS) or Lab On a Chip (LOC), drive the current analysis tools and equipment towards miniaturization, rapid at-line testing and mobility. The state-of-the-art microfluidic technology targets a wider range but smaller volumes of analytes, making the analytical procedure relatively easier and faster. This trend together with faster electronics and modern instrumentation systems will make real-time and in situ analysis a definite possibility. This review focuses on microchip capillary electrophoresis with amperometric detection (MCE-AD) for the detection of DNA and other electroactive analytes. The problems associated with the microchip design, in particular the choice of materials and the configuration of electrodes are discussed thoroughly and solutions are proposed. Significant developments in the related areas are also covered and reviewed critically.     

Gold and Silver Micro‐Wire Electrodes for Trace Analysis of Metals

Micro‐wire electrodes were made from gold and silver wires (diameter: 25 μm; length: 3–21 mm) and sealed in a polyethylene holder; micro‐disk electrodes were made from the same wires and polished. The gold electrodes were electrochemically coated with mercury before use; the silver wires were used without coating. Comparative measurements demonstrated that the micro‐wire electrodes had much higher sensitivity, and a much (10–100×) lower limit of detection, than micro‐disk electrodes, and the sensitivity increased linearly with the area and length of the electrodes. Using a gold micro‐wire electrode of 21 mm and a deposition time of 300 s the limit of detection was 0.07 nM Pb in seawater of natural pH, compared to a limit of detection of 10 nM Pb (more than 100×greater) using a gold micro‐disk electrode of the same diameter. Using the silver micro‐wire electrode the limit of detection of lead was improved by a factor of 10 to 0.2 nM in acidified seawater. It is expected that the improved sensitivity of micro‐wire electrodes will lead to successful in situ detection of metals in natural waters.     

Use of micromolded carbon dual electrodes with a palladium decoupler for amperometric detection in microchip electrophoresis

The fabrication and evaluation of micromolded dual carbon ink electrodes and their integration with a fabricated palladium decoupler for use in microchip electrophoresis is described. As opposed to previous work involving carbon-based dual electrodes with microchip electrophoresis, this approach results in electrodes that are amenable to mass production in a manner where the decoupler/electrode alignment is fixed and reproducible. In this work, electrode sizes and spacings were optimized to result in dual carbon electrodes that are 1 microm in height and separated by 100 microm. Fluorescence microscopy was used to investigate leakage around the electrode/channel interface as well as to investigate what effect the dual electrodes have on band broadening phenomena. The performance of the microelectrodes was demonstrated by the separation and selective dual electrode detection of neurotransmitters in the presence of ascorbic acid. It was also found that addition of SDS to the buffer system improved both the LODs and collection efficiencies. This approach, which is the first involving carbon-based dual electrodes with an on-chip palladium decoupler, will be useful for separating and detecting neurotransmitters that are either collected by in vivo sampling or released from cells on-chip.     

Capillary electrophoresis with on-chip four-electrode capacitively coupled conductivity detection for application in bioanalysis

Microchip capillary electrophoresis (CE) with integrated four-electrode capacitively coupled conductivity detection is presented. Conductivity detection is a universal detection technique that is relatively independent on the detection pathlength and, especially important for chip-based analysis, is compatible with miniaturization and on-chip integration. The glass microchip structure consists of a 6 cm etched channel (20 microm x 70 microm cross section) with silicon nitride covered walls. In the channel, a 30 nm thick silicon carbide layer covers the electrodes to enable capacitive coupling with the liquid inside the channel as well as to prevent interference of the applied separation field. The detector response was found to be linear over the concentration range from 20 microM up to 2 mM. Detection limits were at the low microM level. Separation of two short peptides with a pI of respectively 5.38 and 4.87 at the 1 mM level demonstrates the applicability for biochemical analysis. At a relatively low separation field strength (50 V/cm) plate numbers in the order of 3500 were achieved. Results obtained with the microdevice compared well with those obtained in a bench scale CE instrument using UV detection under similar conditions.     

Fabrication and integration of planar electrodes for contactless conductivity detection on polyester-toner electrophoresis microchips

In this report, we describe the microfabrication and integration of planar electrodes for contactless conductivity detection on polyester-toner (PT) electrophoresis microchips using toner masks. Planar electrodes were fabricated by three simple steps: (i) drawing and laser-printing the electrode geometry on polyester films, (ii) sputtering deposition onto substrates, and (iii) removal of toner layer by a lift-off process. The polyester film with anchored electrodes was integrated to PT electrophoresis microchannels by lamination at 120 degrees C in less than 1 min. The electrodes were designed in an antiparallel configuration with 750 microm width and 750 microm gap between them. The best results were recorded with a frequency of 400 kHz and 10 Vpp using a sinusoidal wave. The analytical performance of the proposed microchip was evaluated by electrophoretic separation of potassium, sodium and lithium in 150 microm wide x 6 microm deep microchannels. Under an electric field of 250 V/cm the analytes were successfully separated in less than 90 s with efficiencies ranging from 7000 to 13,000 plates. The detection limits (S/N = 3) found for K+, Na+, and Li+ were 3.1, 4.3, and 7.2 micromol/L, respectively. Besides the low-cost and instrumental simplicity, the integrated PT chip eliminates the problem of manual alignment and gluing of the electrodes, permitting more robustness and better reproducibility, therefore, more suitable for mass production of electrophoresis microchips.     

Use of epoxy-embedded electrodes to integrate electrochemical detection with microchip-based analysis systems

A new method of fabricating electrodes for microchip devices that involves the use of Teflon molds and a commercially available epoxy to embed electrodes of various sizes and compositions is described. The resulting epoxy base can be polished to generate a fresh electrode and sealed against poly(dimethylsiloxane) (PDMS)-based fluidic structures. Microchip-based flow injection analysis was used to characterize the epoxy-embedded electrodes. It was shown that gold electrodes can be amalgamated with liquid mercury and the resulting mercury/gold electrode is used to selectively detect glutathione from lysed red blood cells. The ability to encapsulate multiple electrode materials of differing compositions enabled the integration of microchip electrophoresis with electrochemical detection. Finally, a unique feature of this approach is that the electrode connection is made from the bottom of the epoxy base. This enables the creation of three-dimensional gold pillar electrodes (65?μm in diameter and 27?μm in height) that can be integrated within a fluidic network. As compared with the use of a flat electrode of a similar diameter, the use of the pillar electrode led to improvements in both the sensitivity (72.1 pA/μM for the pillar versus 4.2 pA/μM for the flat electrode) and limit of detection (20 nM for the pillar versus 600 nM for the flat electrode), with catechol being the test analyte. These epoxy-embedded electrodes hold promise for the creation of inexpensive microfluidic devices that can be used to electrochemically detect biologically important analytes in a manner where the electrodes can be polished and a fresh electrode surface is generated as desired.     

灿烂甲酚蓝在DNA修饰金电极上的电化学行为

利用自组装技术将巯基乙醇固定在金电极表面形成巯基乙醇自组装膜修饰金电极, 用乙基-(3-二甲基氨丙基)碳二亚胺盐酸盐(EDC)和N-羟基琥珀酰亚胺(NHS)为偶联试剂, 分别将鲱鱼精单链DNA(ssDNA)和双链DNA(dsDNA)固定于金电极表面形成ssDNA和dsDNA 修饰电极. 考察了灿烂甲酚蓝(BCB)在不同DNA 修饰电极上的电化学行为,结果表明, BCB 在ssDNA 和dsDNA 修饰电极上的吸附常数分别为1.67×10^4和3.22×10^4 L·mol-1, BCB 与ssDNA 主要以静电作用结合, 而与dsDNA作用存在静电和嵌插两种模式. dsDNA 对BCB 具有更高的亲和力, 使BCB 可以作为一种有效的电化学杂交指示剂.     

DNA修饰电极的研究: VIII. 1, 10-菲咯啉存在时钴离子在ssDNA 修饰金电极上 的电化学行为及痕量钴的检测

发现在过量1,10-菲咯啉存在时,Co^3^+^/^2^+在单链DNA(ssDNA)修饰金电极上的电化学响应显著增强。采用紫外光谱和循环伏安法考察了Co^3^+^/^2^+/1,10-菲咯啉体系与sSDNA的相互作用,并利用Co^3^+^/^2^+在1,10-菲咯啉存在时在ssDNA修饰金电极上的高灵敏电化学响应对痕量钴离子进行了测定。     

CtrA gene based electrochemical DNA sensor for detection of meningitis

Electrochemical DNA sensor has been fabricated by immobilizing thiolated single stranded oligonucleotide (ssDNA) probe onto gold (Au) coated glass electrode for meningitis detection using hybridization with complementary DNA (CtrA) in presence of methylene blue (MB). These electrodes (ssDNA/Au and dsDNA/Au) have been characterized using atomic force microscopy (AFM), Fourier transform infrared spectroscopy (FT-IR), electrochemical impedance spectroscopy (EIS) and cyclic voltammetric (CV) technique. The DNA/Au electrode can detect the complementary DNA in the range of 7–42 ng/μl in 5 min (hybridization) with response time 60 s and electrode is stable for about 4 months when stored at 4 °C. The sensitivity of dsDNA/Au electrode is 115.8 μA/ng with 0.917 regression coefficient (R).     

Electrochemical detection of DNA hybridization using methylene blue and electro-deposited zirconia thin films on gold electrodes

A novel electrochemical DNA biosensor based on methylene blue (MB) and zirconia (ZrO₂) thin films modified gold electrode for DNA hybridization detection is presented. Zirconia thin films were electrodynamically deposited onto the bare gold electrode in an aqueous electrolyte of ZrOCl₂ and KCl by cycling the potential between −1.1 and +0.7 V (versus Ag/AgCl) at a scan rate of 20 mV s⁻¹. Oligonucleotide probes with phosphate group at the 5′ end were attached onto the zirconia thin films because zirconia is affinity for phosphoric group. The surface density of the immobilized DNA molecules at the zirconia interface was investigated by fluorescence spectroscopy method. Hybridization was induced by exposure of the ssDNA-containing Au electrode to complementary ssDNA in solution. The decreases in the peak currents of MB, an electroactive label, were observed upon hybridization of probe with the target. The cathodic peak current (i_p) of MB after hybridization with the target DNA was linearly related to the logarithmic value of the target DNA concentration ranging from 2.25×10⁻¹⁰ to 2.25×10⁻⁸ mol l⁻¹. A detection limit of 1.0×10⁻¹⁰ mol l⁻¹ of oligonucleotides can be estimated.     

Rapid and sensitive DNA target detection using enzyme amplified electrochemical detection based on microchip

A rapid and sensitive DNA targets detection using enzyme amplified electrochemical detection (ED) based on microchip was described. We employed a biotin‐modified DNA, which reacted with avidin‐conjugated horseradish peroxidase (avidin–HRP) to obtain the HRP‐labeled DNA probe and hybridized with its complementary target. After hybridization, the mixture containing dsDNA‐HRP, excess ssDNA‐HRP, and remaining avidin–HRP was separated by MCE. The separations were performed at a separation voltage of +1.6 kV and were completed in less than 100 s. The HRP was used as catalytic labels to catalyze H₂O₂/o‐aminophenol reaction. Target DNA could be detected by the HRP‐catalyzed reduction with ED. With this protocol, the limits of quantification for the hybridization assay of 21‐ and 39‐mer DNA fragments were of 8×10^?12 M and 1.2×10^?11 M, respectively. The proposed method has been applied satisfactorily in the analysis of Escherichia coli genomic DNA. We selected the detection of PCR amplifications from the gene of E. coli to test the real applicability of our method. By using an asymmetric PCR protocol, we obtained ssDNA targets of 148 bp that could be directly hybridized by the single‐stranded probe and detected with ED.     

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.     

A high-performance polycarbonate electrophoresis microchip with integrated three-electrode system for end-channel amperometric detection

A fully integrated polycarbonate (PC) microchip for CE with end-channel electrochemical detection operated in an amperometric mode (CE-ED) has been developed. The on-chip integrated three-electrode system consisted of a gold working electrode, an Ag/AgCl reference electrode and a platinum counter electrode, which was fabricated by photo-directed electroless plating combined with electroplating. The working electrode was positioned against the separation channel exit to reduce post-channel band broadening. The electrophoresis high-voltage (HV) interference with the amperometric detection was assessed with respect to detection noise and potential shifts at various working-to-reference electrode spacing. It was observed that the electrophoresis HV interference caused by positioning the working electrode against the channel exit could be diminished by using an on-chip integrated reference electrode that was positioned in close proximity (100 microm) to the working electrode. The CE-ED microchip was demonstrated for the separation of model analytes, including dopamine (DA) and catechol (CA). Detection limits of 132 and 164 nM were achieved for DA and CA, respectively, and a theoretical plate number of 2.5x10(4)/m was obtained for DA. Relative standard deviations in peak heights observed for five runs of a standard solution containing the two analytes (0.1 mM for each) were 1.2 and 3.1% for DA and CA, respectively. The chip could be continuously used for more than 8 h without significant deterioration in analytical performance.     

Design and development of microfabricated capillary electrophoresis devices with electrochemical detection

Our efforts have been focused on developing a self-contained and transportable microfabricated electrophoresis (CE) system with integrated electrochemical detection (ED). The current prototype includes all necessary electrodes “on-chip” and utilizes miniaturized CE and ED supporting electronics custom designed for this purpose. State-of-the-art design/modeling tools and novel microfabrication procedures were used to create recessed platinum electrodes with complex geometries and the CE/ED device from two patterned ultra-flat glass substrates. The electrodes in the bottom substrate were formed by a self-aligned etch and deposition technique followed by a photolithographic lift-off process. The microchannels (20 μm deep×65 μm wide (average)) were chemically etched into the top substrate followed by thermal bonding to complete the microchip device. CE/ED experiments were performed using 0.02 M phosphate buffer (pH 6), an analyte/buffer solution (2.2 mM dopamine, 2.3 mM catechol) and varying separation voltages (0-500 V) with a custom electronics unit interfaced to a laptop computer for data acquisition. Detection limits (S/N=3) were found to be at the micromolar level and a linear detection response was observed up to millimolar concentrations for dopamine and catechol. The microchip CE/ED system injected 50 pl volumes of sample, which corresponded to mass detection limits on the order of 200 amol. For the first time, an integrated “on-chip” multi-electrode array CE/ED device was successfully designed, fabricated and tested. The majority of the electrodes (six out of eight) in the array were capable of detecting dopamine with the amplitude of the signal (i.e., peak heights) decreasing as the electrode distance from the channel exit increased.     

Electrochemical DNA Biosensor Based on Graphene and TiO2 Nanorods Composite Film for the Detection of Transgenic Soybean Gene Sequence of MON89788

Based on graphene (GR), TiO₂ nanorods, and chitosan (CTS) nanocomposite modified carbon ionic liquid electrode (CILE) as substrate electrode, a new electrochemical DNA biosensor was effectively fabricated for the detection of the transgenic soybean sequence of MON89788. By using methylene blue (MB) as hybridization indicator for monitoring the hybridization with different ssDNA sequences, the differential pulse voltammetric response of MB on DNA modified electrodes were recorded and compared. Due to the synergistic effects of TiO₂ nanorods and GR on the electrode surface, the electrochemical responses of MB were greatly increased. Under optimal conditions the differential pulse voltammetric response of the target ssDNA sequence could be detected in the range from 1.0×10^?12 to 1.0×10^?6 mol/L with a detection limit of 7.21×10^?13 mol/L (3σ). This electrochemical DNA biosensor was further applied to the polymerase chain reaction (PCR) product of transgenic soybeans with satisfactory results.