Multi-channel PMMA microfluidic biosensor with integrated IDUAs for electrochemical detection

A novel multi-channel poly(methyl methacrylate) (PMMA) microfluidic biosensor with interdigitated ultramicroelectrode arrays (IDUAs) for electrochemical detection was developed. The focus of the development was a simple fabrication procedure and the realization of a reliable large IDUA that can provide detection simultaneously to several microchannels. As proof of concept, five microchannels are positioned over a large single IDUA where the channels are parallel with the length of the electrode finger. The IDUAs were fabricated on the PMMA cover piece and bonded to a PMMA substrate containing the microfluidic channels using UV/ozone-assisted thermal bonding. Conditions of device fabrication were optimized realizing a rugged large IDUA within a bonded PMMA device. Gold adhesion to the PMMA, protective coatings, and pressure during bonding were optimized. Its electrochemical performance was studied using amperometric detection of potassium ferri and ferro hexacyanide. Cumulative signals within the same chip showed very good linearity over a range of 0–38 μM (R ²?=?0.98) and a limit of detection of 3.48 μM. The bonding of the device was optimized so that no cross talk between the channels was observed which otherwise would have resulted in unreliable electrochemical responses. The highly reproducible signals achieved were comparable to those obtained with separate single-channel devices. Subsequently, the multi-channel microfluidic chip was applied to a model bioanalytical detection strategy, i.e., the quantification of specific nucleic acid sequences using a sandwich approach. Here, probe-coated paramagnetic beads and probe-tagged liposomes entrapping ferri/ferro hexacyanide as the redox marker were used to bind to a single-stranded DNA sequence. Flow rates of the non-ionic detergent n-octyl-β-d-glucopyranoside for liposome lysis were optimized, and the detection of the target sequences was carried out coulometrically within 250 s and with a limit of detection of 12.5 μM. The robustness of the design and the reliability of the results obtained in comparison to previously published single-channel designs suggest that the multi-channel device offers an excellent opportunity for bioanalytical applications that require multianalyte detection and high-throughput assays.

Monolithic integration of optical waveguides for absorbance detection in microfabricated electrophoresis devices.

The fabrication and performance of an electrophoretic separation chip with integrated optical waveguides for absorption detection is presented. The device was fabricated on a silicon substrate by standard microfabrication techniques with the use of two photolithographic mask steps. The waveguides on the device were connected to optical fibers, which enabled alignment free operation due to the absence of free-space optics. A 750 microm long U-shaped detection cell was used to facilitate longitudinal absorption detection. To minimize geometrically induced band broadening at the turn in the U-cell, tapering of the separation channel from a width of 120 down to 30 microm was employed. Electrical insulation was achieved by a 13 microm thermally grown silicon dioxide between the silicon substrate and the channels. The breakdown voltage during operation of the chip was measured to 10.6 kV. A separation of 3.2 microM rhodamine 110, 8 microM 2,7-dichlorofluorescein, 10 microM fluorescein and 18 microM 5-carboxyfluorescein was demonstrated on the device using the detection cell for absorption measurements at 488 nm.     

A three-layer PMMA electrophoresis microchip with Pt microelectrodes insulated by a thin film for contactless conductivity detection

A three-layer poly (methyl methacrylate) (PMMA) electrophoresis microchip integrated with Pt microelectrodes for contactless conductivity detection is presented. A 50 μm-thick PMMA film is used as the insulating layer and placed between the channel plate (containing the microchannel) and the electrode plate (containing the microelectrode). The three-layer structure facilitates the achievement of a thin insulating layer, obviates the difficulty of integrating microelectrodes on a thin film, and does not compromise the integration of microchips. To overcome the thermal and chemical incompatibilities of polymers and photolithographic techniques, a modified lift-off process was developed to integrate Pt microelectrodes onto the PMMA substrate. A novel two-step bonding method was created to assemble the complete PMMA microchip. A low limit of detection of 1.25 μg ml(-1) for Na(+) and high separation efficiency of 77,000 and 48,000 plates/m for Na(+) and K(+) were obtained when operating the detector at a low excitation frequency of 60 kHz.     

A new dynamic electrochemical transduction mechanism for interdigitated array microelectrodes

A dynamic electrochemical transduction mechanism for interdigitated array microelectrodes using an electrical charge pumping method is presented in this paper. In this dynamic transduction mechanism, a charged external capacitor is used as the charge supplier for the electrochemical reaction of the reversible redox species at the interdigitated array electrodes. The charges stored in the capacitor are consumed as the electrochemical reaction current, which causes the capacitor potential decay. The theoretical analysis has shown that the species concentration has a decisive effect on the capacitor potential decay, and therefore the characteristics of the capacitor potential decay are recorded and analyzed to evaluate the concentration of redox species. The new transduction mechanism has the advantages of achieving high sensitivity with small sensor area and simplifying the measurement instrumentation. As a demonstration device, interdigitated array microelectrodes (approximately 0.2 mm(2) electrode surface area) have been fabricated and successfully characterized using p-aminophenol as the redox species under this dynamic mechanism. The detection limit of p-aminophenol was calculated to be approximately 4 x 10(-7) M for the sensor with the new dynamic transduction mechanism.     

Microfluidic integration for electrochemical biosensor applications

Electrochemical biosensors are particularly suitable for miniaturization and integration in microfluidic devices. Applications include the detection of whole cells, cell components, proteins, and small molecules to address tasks in the fields of diagnostics and food and environmental control. Microfluidic setups range from simple channels for sample transport to channels with integrated sensing electrodes to highly sophisticated platforms with additional elements for sample preparation. The design of the microfluidics depends on both the type of detection and on the application and sample material. This review summarizes recent work on electrochemical biosensors with integrated microfluidics with the focus on developments for real sample applications, particularly those including measurements with real sample media.     

Nafion-CNT coated carbon-fiber microelectrodes for enhanced detection of adenosine

Adenosine is a neuromodulator that regulates neurotransmission. Adenosine can be monitored using fast-scan cyclic voltammetry at carbon-fiber microelectrodes and ATP is a possible interferent in vivo because the electroactive moiety, adenine, is the same for both molecules. In this study, we investigated carbon-fiber microelectrodes coated with Nafion and carbon nanotubes (CNTs) to enhance the sensitivity of adenosine and decrease interference by ATP. Electrodes coated in 0.05 mg mL(-1) CNTs in Nafion had a 4.2 ± 0.2 fold increase in current for adenosine, twice as large as for Nafion alone. Nafion-CNT electrodes were 6 times more sensitive to adenosine than ATP. The Nafion-CNT coating did not slow the temporal response of the electrode. Comparing different purine bases shows that the presence of an amine group enhances sensitivity and that purines with carbonyl groups, such as guanine and hypoxanthine, do not have as great an enhancement after Nafion-CNT coating. The ribose group provides additional sensitivity enhancement for adenosine over adenine. The Nafion-CNT modified electrodes exhibited significantly more current for adenosine than ATP in brain slices. Therefore, Nafion-CNT modified electrodes are useful for sensitive, selective detection of adenosine in biological samples.     

In situ control of cellular growth and migration on substrates using microelectrodes

We describe an electrochemical method to direct the growth and migration of mammalian cells on a substrate during cultivation in situ. Exposing the albumin-coated substrate to an oxidizing agent, hypobromous acid, electrochemically generated at the tip of the scanning microelectrode, locally switched the substrate from cytophobic to cell-adhesive. This transformation generated the formation of cellular micropatterns. Since the concentration of the oxidizing agent required for the surface processing did not cause significant damage to the cell cultures, we were able to direct in situ cellular proliferation and migration by drawing adhesive micropatterns over the preexisting cellular pattern.     

氧化铱纳米颗粒修饰碳纤维电极用于水果中pH在线检测

pH检测在农业生产、食品加工、环境保护、疾病诊断等领域有着重要意义.电化学法具有响应速度快、灵敏度高和操作简单等优点,是目前最常用的pH检测方法之一.然而,商品化的pH计存在体积大、质子敏感膜易损等问题,仅能在相对稳定的样本溶液环境中工作,并不适用于植入式pH分析.本文将氧化铱纳米颗粒修饰在碳纤维微电极表面,构建了一种...     

Monolithic integration of well-ordered nanoporous structures in the microfluidic channels for bioseparation

Gel electrophoresis and capillary gel electrophoresis are widely used for the separation of biomolecules. With increasing demand in the miniaturized devices such as lab-on-a-chip, it is necessary to integrate such a separation component into a chip format. Here, we describe a simple approach to fabricate robust three-dimensional periodic porous nanostructures inside the microchannels for the separation of DNA molecules. In our approach, the colloidal crystals were first grown inside the microchannel using evaporation assisted self-assembly process. Then the void spaces among the colloidal crystals were filled with epoxy-based negative tone photoresist (SU-8). UV radiation was used to cure the photoresist at the desired area inside the microchannel. After subsequent development and nanoparticle removal, the well-ordered nanoporous structures inside the microchannel were obtained. Our results indicated that it was possible to construct periodic porous nanostructures inside the microchannels with cavity size around 300 nm and interconnecting pores around 30 nm. The mobility of large DNA molecules with different sizes was measured as a function of the applied electric field in the nanoporous materials. It was also demonstrated that 1 kilo-base pair (kbp) DNA ladders could be separated in such an integrated system within 10 min under moderate electric field.     

Scanning electrochemical microscopy imaging of rhodochrosite dissolution using gold amalgam microelectrodes

Gold/mercury amalgam (Au/Hg) microelectrodes with a diameter of 25 microm were developed for the detection of environmentally relevant analytes such as manganese and iron by scanning electrochemical microscopy (SECM), and applied to investigate the controlled dissolution of manganese carbonate (MnCO(3); rhodochrosite) in acidic conditions. Characterization of the amalgam electrode geometry via approach curves recorded during SECM experiments revealed Au/Hg microelectrodes with sphere cap geometry. Quantitative determination of Mn(2+) has been achieved by calibration of the Au/Hg microelectrode in bulk solution experiments. Subsequent SECM imaging experiments confirm the applicability of amalgam microelectrodes for imaging of Mn(2+) production during dissolution of MnCO(3) at pH 3.9. This study confirms feasibility and provides the fundamental basis of SECM imaging with amalgam microelectrodes to address biogeochemically relevant questions.     

Optimization of acetylcholinesterase immobilization on microelectrodes based on nitrophenyl diazonium for sensitive organophosphate insecticides detection

The immobilization of acetylcholinesterase on platinum microelectrodes modified with p-nitrobenzenediazonium is optimized. In the first step, a layer of p-nitrophenyl groups was deposited on the surface and then reduced to p-aminophenyl groups. Finally, the enzyme was linked to the amino groups on the surface using glutaraldehyde. Each step of the electrode modification was characterized by cyclic voltammetry and electrochemical impedance spectroscopy (EIS) at acidic and neutral pH to modify the electric charges of different bound moieties. The deposition of diazonium groups was attempted by potentiometry, amperometry or CV, but only potentiometry proceeded without passivation of the surface. The use of microelectrodes improved the limit of detection of ethylparaoxon measurements to 20 nM (compared to 100 nM in case of screen-printed electrodes based on the same method of immobilization). The method allowed the production of stable and reproducible amperometric microbiosensors and may be adapted to other enzymes and electrode materials.     

Voltammetric studies on the electrochemical determination of methylmercury in chloride medium at carbon microelectrodes

Electroanalytical techniques have been used to determine methylmercury at low levels in environmental matrices. The electrochemical behaviour of methylmercury at carbon microelectrodes in a hydrochloric acid medium using cyclic, square wave and fast-scan linear-sweep voltammetric techniques has been investigated. The analytical utility of the methylmercury reoxidation peak has been explored, but the recorded peak currents were found to be poorly reproducible. This is ascribed to two factors: the adsorption of insoluble chloromercury compounds on the electrode surface, which appears to be an important contribution to hinder the voltammetric signal of methylmercury; and the competition between the reoxidation of the methylmercury radical and its dimerization reaction, which limits the reproducibility of the methylmercury peak. These problems were successfully overcome by adopting the appropriate experimental conditions. Fast-scan rates were employed and an efficient electrochemical regeneration procedure of the electrode surface was achieved, under potentiostatic conditions in a mercury-free solution containing potassium thiocyanate—a strong complexing agent. The influence of chloride ion concentration was analysed. Interference by metals, such as lead and cadmium, was considered. Calibration plots were obtained in the micromolar and submicromolar concentration ranges, allowing the electrochemical determination of methylmercury in trace amounts. An estuarine water sample was analysed using the new method with a glassy carbon microelectrode.     

Immobilization of DNAzyme catalytic beacons on PMMA for Pb2+ detection

Due to the numerous toxicological effects of lead, its presence in the environment needs to be effectively monitored. Incorporating a biosensing element within a microfluidic platform enables rapid and reliable determinations of lead at trace levels. A microchip-based lead sensor is described here that employs a lead-specific DNAzyme (also called catalytic DNA or deoxyribozyme) as a recognition element that cleaves its complementary substrate DNA strand only in the presence of cationic lead (Pb(2+)). Fluorescent tags on the DNAzyme translate the cleavage events to measurable, optical signals proportional to Pb(2+) concentration. The DNAzyme responds sensitively and selectively to Pb(2+), and immobilizing DNAzyme in the sensor permits both sensor regeneration and localization of the detection zone. Here, the DNAzyme has been immobilized on a PMMA surface using the highly specific biotin-streptavidin interaction. The strategy includes using streptavidin physisorbed on a PMMA surface to immobilize DNAzyme both on planar PMMA and on the walls of a PMMA microfluidic device. The immobilized DNAzyme retains its Pb(2+) detection activity in the microfluidic device and can be regenerated and reused. The DNAzyme shows no response to other common metal cations and the presence of these contaminants does not interfere with the lead-induced fluorescence signal. While prior work has shown lead-specific catalytic DNA can be used in its solubilized form and while attached to gold substrates to quantitate Pb(2+) in solution, this is the first use of the DNAzyme immobilized within a microfluidic platform for real time Pb(2+) detection.     

Carbon nanotube-modified microelectrodes for simultaneous detection of dopamine and serotonin in vivo

Dopamine and serotonin are important neurotransmitters that interact in the brain. While dopamine is easily detected with electrochemical sensors, the detection of serotonin is more difficult because reactive species formed after oxidation can adsorb to the electrode, reducing sensitivity. Carbon nanotube treatments of electrodes have been used to increase the sensitivity, promote electron transfer, and reduce fouling. Most methods have focused on nanotube coatings of large electrodes and slower electrochemical techniques that are not conducive to measurements in vivo. In this study, we investigated carbon-fiber microelectrodes modified with single-walled carbon nanotubes for the co-detection of dopamine and serotonin in vivo. Using fast-scan cyclic voltammetry, S/N ratios for the neurotransmitters increased after nanotube coating. Electrocatalytic effects of nanotubes were not apparent at fast scan rates but faster kinetics were observed with slower scanning. Nanotube-modified microelectrodes showed significantly less fouling after exposure to serotonin than bare electrodes. The nanotube-modified electrodes were used to monitor stimulated dopamine and serotonin changes simultaneously in the striatum of anesthetized rat after administration of a serotonin synthetic precursor. These studies show that nanotube-coated microelectrodes can be used with fast scanning techniques and are advantageous for in vivo measurements of neurotransmitters because of their greater sensitivity and resistance to fouling.     

DMF-exfoliated graphene for electrochemical NADH detection

The electrochemical detection of NADH is of considerable interest because it is required as a cofactor in a large number of dehydrogenase-based biosensors. However, the presence of oxygenated functionalities on the electrode often causes fouling due to the adsorption of the oxidised form, NAD(+). Here we report an electroanalytical NADH sensor based on DMF-exfoliated graphene. The latter is shown to have a very low oxygen content, facilitating the exceptionally stable and sensitive detection of this important analyte.     

Scanning electrochemical microscopy of enzymes immobilized on structured glass-gold substrates

Scanning electrochemical microscopy (SECM) was used to characterize enzyme-modified glass-gold specimens. The exposed gold surface was functionalized with an aminothiol and reacted with carbodiimide-activated glucose oxidase. The specimen surface was examined with SECM, using a 25 μm platinum electrode. Images were acquired showing the topography, electric conductivity, and enzymatic activity of the composite surface. It was found that the hydroxy-groups of the glass surface are as likely to bind to the activated enzyme as the amino-groups on the gold surface.     

用于半胱氨酸检测的电化学生物传感器研究进展

半胱氨酸(Cys)作为人体重要的非必需氨基酸之一,对蛋白质合成、渗透调节、解毒过程、神经系统功能和抗氧化过程均发挥着重要作用,近年来广泛应用于医学临床、食品加工和生化研究领域。因此,发展快速、准确检测半胱氨酸浓度的方法具有重要意义。本文简要介绍了半胱氨酸的基本知识以及半胱氨酸的传统检测方法,对半胱氨酸定量检测的电化学传感器的研制与应用进行了重点阐述,并对其发展前景进行了展望。     

用于褪黑素检测的电化学生物传感器研究进展

褪黑素是人体松果体分泌的一种重要的神经递质,近年来其在控制昼夜节律和提供免疫抗炎特性等生理调节作用方面备受关注。因此,发展可靠、快速检测体内和体外样本中褪黑素浓度的方法,对于探索褪黑素的临床应用和生物学特性具有重要的意义。本文对褪黑素及其传统检测方法进行简要介绍,重点阐述了近几年报道的用于生物样本和药物样本中褪黑素定量检测的电化学传感器,并对褪黑素传感器的未来发展方向进行展望。