Miniaturized probe on polymer SU-8 with array of individually addressable microelectrodes for electrochemical analysis in neural and other biological tissues.

An SU-8 probe with an array of nine, individually addressable gold microband electrodes (100 µm long, 4 µm wide, separated by 4-µm gaps) was photolithographically fabricated and characterized for detection of low concentrations of chemicals in confined spaces and in vivo studies of biological tissues. The probe's shank (6 mm long, 100 µm wide, 100 µm thick) is flexible, but exhibits sufficient sharpness and rigidity to be inserted into soft tissue. Laser micromachining was used to define probe geometry by spatially revealing the underlying sacrificial aluminum layer, which was then etched to free the probes from a silicon wafer. Perfusion with fluorescent nanobeads showed that, like a carbon fiber electrode, the probe produced no noticeable damage when inserted into rat brain, in contrast to damage from an inserted microdialysis probe. The individual addressability of the electrodes allows single and multiple electrode activation. Redox cycling is possible, where adjacent electrodes serve as generators (that oxidize or reduce molecules) and collectors (that do the opposite) to amplify signals of small concentrations without background subtraction. Information about electrochemical mechanisms and kinetics may also be obtained. Detection limits for potassium ferricyanide in potassium chloride electrolyte of 2.19, 1.25, and 2.08 µM and for dopamine in artificial cerebral spinal fluid of 1.94, 1.08, and 5.66 µM for generators alone and for generators and collectors during redox cycling, respectively, were obtained.

Calibration curve-free electrochemical quantitation by micro-nano multi-scale gap devices.

Quantitation without relying on the calibration curve has long been an issue of overcoming analytical problems accompanied with the inherent limitations of the calibration curve fitting errors. Here, we report on a calibration curve-free method for electrochemical quantitation based on a multi-scale gap device (MGD). The MGD is an integrated device having a series of interdigitated electrodes (IDE) with micro-to-nano gap distances. The device shows a gap-dependent redox current of the analyte when subjected to the electrochemical cycling between the two facing electrodes of its componential IDEs. Based on the fact that the current increases as the gap distance decreases, the analyte concentration could be directly estimated: the rate of increase in the current was directly proportional to the analyte concentration. The calibration curve was not necessary for the quantitation. The accuracy of this MGD approach was better than that of an IDE collection of the same gap distance, which was deteriorated at the larger gap distances particularly. The MGD-based quantitation of dopamine, potassium ferricyanide, and aminophenol was demonstrated in a relatively broad range of concentrations (100 nM-5 mM).

C60 Mediated Ion Pair Interaction for Label-Free Electrochemical Immunosensing with Nanoporous Anodic Alumina Nanochannels.

Label-free biosensing based on the nanoporous anodic alumina (NAA) membrane emerged as a versatile biosensing platform in the recent decade. In the present work, we developed a new immunosensing strategy based on the nanochannels of NAA and the ion pair interaction mediated by electrochemistry of C60. The NAA served as the matrix for the immobilization of the capture antibodies. The incubation of target antigens resulted in the formation of the immunocomplexes and thus an increase of the steric hindrance of the nanochannels. Therefore, the concentration of the redox probe transported through the nanochannels decreases, which can be detected at the working electrode modified with C60. Herein, we initially found that the cathodic peak ascribed to the reduction of C60 to C60- was obviously enhanced by the presence of the redox probe K3[Fe(CN)6] and which was contributed to the formation of a ternary ion association complex among C60, tetraoctylammonium bromide, and K3[Fe(CN)6]. Therefore, the transportation of K3[Fe(CN)6] though the NAA-based bionanochannels can be detected by a C60 modified electrode with an amplified signal. Choosing human epididymis protein 4 (HE4) as the model target, a linear range of 1.0 ng mL-1 to 100 ng mL-1 can be established between the peak current obtained from the differential pulse voltammetric response of the platform and the concentration of HE4. The detection limit was 0.2 ng mL-1. This study not only provides a new avenue to develop the other nanochannel-based biosensing platform for a variety of other disease biomarkers but also contributes to the electrochemistry of fullerene.

Epoxy Chip-in-Carrier Integration and Screen-Printed Metalization for Multichannel Microfluidic Lab-on-CMOS Microsystems.

The integration of biosensors, microfluidics, and CMOS instrumentation provides a compact lab-on-CMOS microsystem well suited for high throughput measurement. This paper describes a new epoxy chip-in-carrier integration process and two planar metalization techniques for lab-on-CMOS that enable on-CMOS electrochemical measurement with multichannel microfluidics. Several design approaches with different fabrication steps and materials were experimentally analyzed to identify an ideal process that can achieve desired capability with high yield and low material and tool cost. On-chip electrochemical measurements of the integrated assembly were performed to verify the functionality of the chip-in-carrier packaging and its capability for microfluidic integration. The newly developed CMOS-compatible epoxy chip-in-carrier process paves the way for full implementation of many lab-on-CMOS applications with CMOS ICs as core electronic instruments.

Ultracompact Microwatt CMOS Current Readout With Picoampere Noise and Kilohertz Bandwidth for Biosensor Arrays.

High-density on-chip electrochemical biosensor arrays are advancing toward a crucial role in health monitoring and development of new medicines and medical treatments. Nanopore and ion channel based sensors especially have great potential but present demanding resolution/speed/power/area requirements on instrumentation circuits. This paper presents a pixel-level current readout circuit and new group-cluster architecture to address the circuit challenges in high-performance biosensor arrays. Fabricated in 0.5 µm CMOS, this electrochemical voltammetry circuit achieves 7.2 pArms noise in an 11.5-kHz bandwidth and only consumes 21-µ W power and 0.06 mm2 area per readout channel. Cyclic voltammetry experiments in a potassium ferricyanide solution and patch-clamp whole-cell experiments were performed to validate the circuit's feasibility for electrochemical biosensor applications.

Assessment of cyanide contamination in soils with a handheld mid-infrared spectrometer.

We examined the feasibility of using handheld mid-infrared (MIR) Fourier-Transform infrared (FT-IR) instrumentation for detecting and analysing cyanide (CN) contamination in field contaminated soils. Cyanide spiking experiments were first carried out, in the laboratory, to test the sensitivity of infrared Fourier transform (DRIFT) spectrometry to ferro- and ferricyanide compounds across a range of reference soils and minerals. Both benchtop and handheld diffuse reflectance infrared spectrometers were tested. Excellent results were obtained for the reference soils and minerals, with the MIR outperforming the near-infrared (NIR) range. Spectral peaks characteristic of the -C≡N group were observed near 2062 and 2118cm-1 in the MIR region for the ferro- and ferricyanide compounds spiked into soils/minerals, respectively. In the NIR region such peaks were observed near 4134 and 4220cm-1. Cyanide-contaminated samples were then collected in the field and analyzed with the two spectrometers to further test the applicability of the DRIFT technique for soils containing aged CN residues. The prediction of total CN in dry and ground contaminated soils using the handheld MIR instrument resulted in a coefficient of determination (R2) of 0.88-0.98 and root mean square error of the cross-validation (RMSE) of 21-49mgkg-1 for a CN range of 0-611mgkg-1. A major peak was observed in the MIR at about 2092cm-1 which was attributed to "Prussian Blue" (Fe4[Fe(CN)6]3·xH2O). These results demonstrate the potential of handheld DRIFT instrumentation as a promising alternative to the standard laboratory method to predict CN concentrations in contaminated field soils.

Portable Lock-in Amplifier-Based Electrochemical Method to Measure an Array of 64 Sensors for Point-of-Care Applications.

We present a portable lock-in amplifier-based electrochemical sensing system. The basic unit (cluster) consists of four electrochemical cells (EC), each containing one pseudoreference electrode (PRE) and one working electrode (WE). All four ECs are simultaneously interrogated, each at different frequencies, with square wave pulses superposed on a sawtooth signal for cyclic voltammetry (CV). Lock-in amplification provides independent read-out of four signals, with excellent noise suppression. We expanded a single cluster system into an array of 16 clusters by using electronic switches. The chip with an array of ECs was fabricated using planar technology with a gap between a WE and a PRE of ≈2 µm, which results in partial microelectrode-type behavior. The basic electrode characterization was performed with the model case using a ferricyanide-ferrocyanide redox couple (Fe2+/Fe3+) reaction, performing CV and differential pulse voltammetry (DPV). We then used this system to perform cyclic lock-in voltammetry (CLV) to measure concurrently responses of the four ECs. We repeated this method with all 64 ECs on the chip. The standard deviation of a peak oxidation and reduction current in a single channel consisting of 13 ECs was ≈7.46% and ≈5.6%, respectively. The four-EC configuration in each measured spot allows determination of nonperforming ECs and, thus, to eliminate potential false results. This system is built in a portable palm-size format suitable for point-of-care applications. It can perform either individual or multiple measurements of active compounds, such as biomarkers.

Development of a Novel, Low-Cost, Disposable Wooden Pencil Graphite Electrode for Use in the Determination of Antioxidants and Other Biological Compounds.

The development of portable sensors that can be used outside the lab is an active area of research in the electroanalytical field. A major focus of such research is the development of low-cost electrodes for use in these sensors. Current electrodes, such as glassy-carbon electrodes (GCEs), are costly and require time-consuming preparation. Alternatives have been proposed, including mechanical pencil-lead electrodes (MPEs). However, MPEs themselves possess numerous drawbacks, particularly structural fragility. In this paper, we present a novel pencil-graphite electrode (PGE) fabricated from a regular HB#2 pencil. This PGE is a simple, disposable, extremely low-cost alternative to GCEs ($0.30 per PGE, vs. $190 + per GCE), and possesses the structural stability that MPEs lack. PGEs were characterized by square-wave voltammetry of ferricyanide, gallic acid, uric acid, dopamine, and several foodstuffs. In all cases, PGEs demonstrated sensitivities comparable or superior to those of the GCE and MPE (LOD = 5.62 × 10(-4) M PGE, 4.80 × 10(-4) M GCE, 2.93 × 10(-4) M MPE). Signal areas and peak heights were typically four to ten times larger for the PGE relative to the GCE.

Fast and sensitive optical toxicity bioassay based on dual wavelength analysis of bacterial ferricyanide reduction kinetics.

Global urban and industrial growth, with the associated environmental contamination, is promoting the development of rapid and inexpensive general toxicity methods. Current microbial methodologies for general toxicity determination rely on either bioluminescent bacteria and specific medium solution (i.e. Microtox(®)) or low sensitivity and diffusion limited protocols (i.e. amperometric microbial respirometry). In this work, fast and sensitive optical toxicity bioassay based on dual wavelength analysis of bacterial ferricyanide reduction kinetics is presented, using Escherichia coli as a bacterial model. Ferricyanide reduction kinetic analysis (variation of ferricyanide absorption with time), much more sensitive than single absorbance measurements, allowed for direct and fast toxicity determination without pre-incubation steps (assay time=10 min) and minimizing biomass interference. Dual wavelength analysis at 405 (ferricyanide and biomass) and 550 nm (biomass), allowed for ferricyanide monitoring without interference of biomass scattering. On the other hand, refractive index (RI) matching with saccharose reduced bacterial light scattering around 50%, expanding the analytical linear range in the determination of absorbent molecules. With this method, different toxicants such as metals and organic compounds were analyzed with good sensitivities. Half maximal effective concentrations (EC50) obtained after 10 min bioassay, 2.9, 1.0, 0.7 and 18.3 mg L(-1) for copper, zinc, acetic acid and 2-phenylethanol respectively, were in agreement with previously reported values for longer bioassays (around 60 min). This method represents a promising alternative for fast and sensitive water toxicity monitoring, opening the possibility of quick in situ analysis.

Direct separation of faradaic and double layer charging current in potential step voltammetry.

Double layer charging current in electrochemical systems has been a challenging problem in the last several decades because it causes interference to the accurate measurement of faradaic current. A method for extracting faradaic current and double layer charging current directly from the measured total current in potential step voltammetry is developed by using iterative target transformation factor analysis (ITTFA). The method constructs initial target vectors based on the theoretical formulae of faradaic and charging current, and then calculates the weights of faradaic and charging current in the measured signal via the iterative transformation of the initial vectors. Therefore, the two currents in one experiment can be obtained simultaneously without any assumption. The potential step voltammetric signals of potassium ferricyanide, copper sulfate and paracetamol were analyzed with the proposed method. The results show that the shape of the obtained voltammogram is an ideal sigmoid curve with horizontal straight baseline and plateaus, and the intensity of the signal is greatly enhanced. Therefore, the method provides a new way to measure the pure faradic current in the potential step voltammetric experiment, and may provide an alternative for improving the sensitivity of quantitative analysis.

Nafion® modified-screen printed gold electrodes and their carbon nanostructuration for electrochemical sensors applications.

Screen printed electrodes are frequently used in electroanalytical applications because of their properties such as small size, low detection limit, fast response time, high reproducibility and disposable nature. On the other hand, since the discovery of carbon nanotubes there has been enormous interest in exploring and exploiting their properties, especially for their use in chemical (bio)sensors and nanoscale electronic devices. This paper reports the characterization of gold screen printed electrodes, modified with Nafion(®) and nanostructured with carbon nanotubes and carbon nanofibers dispersed on Nafion(®). The dispersing agent and the nanostructure have a marked effect on the analytical signal that, in turn depends on the intrinsic characteristics of the analyte. Several model analytes have been employed in this study. Anionic, cationic and neutral species such as methylene blue, dopamine, iron (III) sulfate, potassium ferrycianide and urea were considered. The importance for the development of nanostructured sensors relies on the fact that depending on these factors the situation may vary from a notorious enhancement of the signal to a blocking or even decrease.

pH-Sensitive "on-off" switching behavior of layer-by-layer films assembled by concanavalin A and dextran toward electroactive probes and its application in bioelectrocatalysis.

Concanavalin A (Con A) and dextran (Dex) were assembled into {Con A/Dex}(n) layer-by-layer films on electrodes by the biospecific interaction between them. At {Con A/Dex}(n) film electrodes, the cyclic voltammetric (CV) response of different electroactive probes, such as Fe(CN)(6)(3-), Ru(NH(3))(6)(3+), ferrocenecarboxylic acid (Fc(COOH)), and ferrocenedicarboxylic acid (Fc(COOH)(2)), was very sensitive to the solution pH. For example, in pH 4.0 buffers, the films showed good permeability toward Fe(CN)(6)(3-), leading to a well-defined CV peak pair of Fe(CN)(6)(3-) with large peak currents at about 0.17 V vs SCE. In pH 9.0 buffers, however, the CV response of Fe(CN)(6)(3-) was significantly depressed or even could hardly be observed. This pH-sensitive "on-off" switching property of the films toward the probe should be attributed to the different electrostatic interaction between Fe(CN)(6)(3-) and the Con A constituent in the films at different pH and could be further used to control the electrocatalytic reduction of H(2)O(2) by horseradish peroxidase (HRP) with Fe(CN)(6)(3-) as the diffusional electron transfer mediator. Fc(COOH)(2) showed the similar pH-dependent "on-off" behavior at {Con A/Dex}(n) film electrodes, and the corresponding pH-sensitive electrocatalytic oxidation of glucose by glucose oxidase (GOD) with Fc(COOH)(2) as the mediator was also realized. This work provides a new interface system that has the pH-sensitive "on-off" CV property, and the better understanding of the interactions involved in this model system may guide us to develop the novel kind of controllable biosensors based on enzymatic electrocatalysis.

Electricity generation from bio-treatment of sewage sludge with microbial fuel cell.

A two-chambered microbial fuel cell (MFC) with potassium ferricyanide as its electron acceptor was utilized to degrade excess sewage sludge and to generate electricity. Stable electrical power was produced continuously during operation for 250 h. Total chemical oxygen demand (TCOD) of sludge was reduced by 46.4% when an initial TCOD was 10,850 mg/l. The MFC power output did not significantly depend on process parameters such as substrate concentration, cathode catholyte concentration, and anodic pH. However, the MFC produced power was in close correlation with the soluble chemical oxygen demand (SCOD) of sludge. Furthermore, ultrasonic pretreatment of sludge accelerated organic matter dissolution and, hence, TCOD removal rate in the MFC was increased, but power output was insignificantly enhanced. This study demonstrates that this MFC can generate electricity from sewage sludge over a wide range of process parameters.

Application of the avidin-biotin interaction to immobilize DNA in the development of electrochemical impedance genosensors.

Impedance spectroscopy is a rapidly developing technique for the transduction of biosensing events at the surface of an electrode. The immobilization of biomaterial as DNA strands on the electrode surface alters the capacitance and the interfacial electron transfer resistance of the conductive electrodes. The impedimetric technique is an effective method of probing modifications to these interfacial properties, thus allowing the differentiation of hybridization events. In this work, an avidin bulk-modified graphite-epoxy biocomposite (Av-GEB) was employed to immobilize biotinylated oligonucleotides as well as double-stranded DNA onto the electrode surface. Impedance spectra were recorded to detect the change in the interfacial electron transfer resistance (R (et)) of the redox marker ferrocyanide/ferricyanide at a polarization potential of +0.17 V. The sensitivity of the technique and the good reproducibility of the results obtained with it confirm the validity of this method based on a universal affinity biocomposite platform coupled with the impedimetric technique.

Characterization of a 20 kHz sonoreactor. Part II: analysis of chemical effects by classical and electrochemical methods.

A new electrochemical redox probe has been investigated in order to characterize the local production of radicals during the cavitation events. The results have been compared with those obtained with Fe(CN)6(3-)/Fe(CN)6(4-) (electrochemical probe for local mechanical effects) and classical chemical methods such as iodide and Fricke dosimeters (chemical probes for global effects).

Carbon fiber nanoelectrodes modified by single-walled carbon nanotubes.

Microelectrode voltammetry has been considered to be a powerful technique for single biological cell analysis and brain research. In this paper, we have developed a simple method to get highly sensitive carbon fiber nanoelectrodes (CFNE) modified by single-walled carbon nanotubes (SWNTs) on the basis of our previous work. The electrochemical behavior of SWNTs/CFNE was characterized by potassium ferricyanide, dopamine (DA), epinephrine (E), and norepinephrine (NE) using cyclic voltammetry (CV). Compared with CFNE, SWNTs/CFNE has a much larger available internal surface area per external geometric area, which is supported by SEM images. The modified electrodes show very high sensitivity and favorable electrochemical behavior toward these neurotransmitters. The peak current increases linearly with the concentration of DA, E, and NE in the range of 1.0 x 10(-)(7)-1.0 x 10(-)(4), 3.0 x 10(-)(7)-1.0 x 10(-)(4), and 5.0 x 10(-)(7)-1.0 x 10(-)(4) M, respectively. The CV detection limit (S/N = 3) of DA, E, and NE is 7.7 x 10(-)(9), 3.8 x 10(-)(8), and 4.2 x 10(-)(8) M, respectively. The modified electrode exhibited almost the same electrochemical behavior after 15 days, indicating that SWNTs/CFNE is pretty stable and has good reproducibility.

Sensitive spectrophotometric determination of amlodipine and felodipine using iron(III) and ferricyanide.

A simple, accurate, sensitive and economical procedure for the estimation of amlodipine besylate and felodipine, both in pure form and in formulations has been developed. The method is based on the reduction of iron(III) by the studied drugs in acid medium and subsequent interaction of iron(II) with ferricyanide to form Prussian blue. The product exhibits absorption maximum at 760 nm in both cases. Beer's law is obeyed in the concentration ranges 5.0-15.0 and 1.5-5.0 microg/ml, for amlodipine and felodipine, respectively. The molar absorptivities are 1.76 x 10(4) and 4.24 x 10(4) l/mol cm. The corresponding Sandell sensitivities are 23.18 and 9.06 ng/cm(2). The limits of detection as well as quantification are reported. Seven replicate analyses of solutions containing three different concentrations of each drug were carried out and the percent error and the RSD values have been reported. The proposed method was applied to the determination of these drugs in pharmaceutical formulations and the results demonstrate that the method is equally accurate and precise as the official methods as found from the t- and F-values. The reliability of the method was established by recovery studies using standard-addition technique.

CNS neurons express two distinct plasma membrane electron transport systems implicated in neuronal viability.

Trans-plasma membrane electron transport is critical for maintaining cellular redox balance and viability, yet few, if any, investigations have studied it in intact primary neurons. In this investigation, extracellular reduction of 2,6-dichloroindophenol (DCIP) and ferricyanide (FeCN) were measured as indicators of trans-plasma membrane electron transport by chick forebrain neurons. Neurons readily reduced DCIP, but not FeCN unless CoQ(1), an exogenous ubiquinone analog, was added to the assays. CoQ(1) stimulated FeCN reduction in a dose-dependent manner but had no effect on DCIP reduction. Reduction of both substrates was totally inhibited by epsilon-maleimidocaproic acid (MCA), a membrane-impermeant thiol reagent, and slightly inhibited by superoxide dismutase. Diphenylene iodonium, a flavoenzyme inhibitor, completely inhibited FeCN reduction but had no affect on DCIP reduction, suggesting that these substrates are reduced by distinct redox pathways. The relationship between plasma membrane electron transport and neuronal viability was tested using the inhibitors MCA and capsaicin. MCA caused a dose-dependent decline in neuronal viability that closely paralleled its inhibition of both reductase activities. Similarly capsaicin, a NADH oxidase inhibitor, induced a rapid decline in neuronal viability. These results suggest that trans-plasma membrane electron transport helps maintain a stable redox environment required for neuronal viability.

Sequence-specific electrochemical detection of asymmetric PCR amplicons of traditional Chinese medicinal plant DNA.

In this study, an electrochemistry-based approach to detect nucleic acid amplification products of Chinese herbal genes is reported. Using asymmetric polymerase chain reaction and electrochemical techniques, single-stranded target amplicons are produced from trace amounts of DNA sample and sequence-specific electrochemical detection based on the direct hybridization of the crude amplicon mix and immobilized DNA probe can be achieved. Electrochemically active intercalator Hoechst 33258 is bound to the double-stranded duplex formed by the target amplicon hybridized with the 5'-thiol-derivated DNA probe (16-mer) on the gold electrode surface. The electrochemical current signal of the hybridization event is measured by linear sweep voltammetry, the response of which can be used to differentiate the sequence complementarities of the target amplicons. To improve the reproducibility and sensitivity of the current signal, issues such as electrode surface cleaning, probe immobilization, and target hybridization are addressed. Factors affecting hybridization efficiency including the length and binding region of the target amplicon are discussed. Using our approach, differentiation of Chinese herbal species Fritillaria (F. thunbergii and F. cirrhosa) based on the 16-mer unique sequences in the spacer region of the 5S-rRNA is demonstrated. The ability to detect PCR products using a nonoptical electrochemical detection technique is an important step toward the realization of portable biomicrodevices for on-spot bacterial and viral detections.

Continuous monitoring of arylesterase in human serum.

Arylesterase (EC 3.1.1.2) activity in serum was specifically measured using thiophenyl acetate in a mechanized assay at 37 degrees C with 4-bromophenylboronic acid as inhibitor of cholinesterase and hexacyanoferrate-III as indicator. The systematic development of a routine method, apparent limitations of thiophenyl compounds as arylesterase substrates, some kinetic constants of the enzyme, analytical variables such as precision (within-run <2% and between-run <2.5% relative standard deviation) and a preliminary reference interval (19.5-52.4 kU/l) for adults are described.