Sonochemically fabricated enzyme microelectrode arrays for the environmental monitoring of pesticides

This paper describes the development of a novel sonochemically fabricated microelectrode based acetylcholinesterase and polyaniline carbon/cobalt phthalocyanine biosensor for the ultra-sensitive determination of pesticides. Arrays of this type are fabricated using microelectrode templates with population densities of 2 x 10(5) cm(-2). The enzymatic response of the sensors is inhibited upon incubation with the pesticide and in this report it is shown that paraoxon may be determined down to concentrations of 1 x 10(-17) M. This sensitivity has thus far not been achieved and mechanisms accounting for the enhancement of the sensitivity reported here are discussed.     

Sonochemically fabricated microelectrode arrays for biosensors. Part III. AC impedimetric study of aerobic and anaerobic response of alcohol oxidase within polyaniline

A sonochemically fabricated alcohol oxidase enzyme micro-electrode array is reported. Sensors of this type were fabricated by first depositing an insulating polydiaminobenzene film on supporting gold electrodes. Sonication and subsequent ablation exposed discrete areas of the underlying conducting electrode, which collectively act as a microelectrode array. Electropolymerisation of aniline has been used to generate in situ polyaniline containing entrapped alcohol oxidase. The physical and electrochemical properties of these films were studied and reported within this paper. The final composites were shown to behave with microelectrode performance characteristics for the detection of aqueous ethanol concentrations.     

Sonochemically fabricated microelectrode arrays for biosensors--part II. Modification with a polysiloxane coating

A polymer-modified sonochemically fabricated glucose oxidase microelectrode array with microelectrode population densities of up to 2.5 x 10(5) microelectrodes per square centimetres is reported. These microelectrode sensors were formed by first depositing an insulating film on commercial screen printed electrodes which was subsequently sonicated to form cavities of regular sizes in the film. Electropolymerisation of aniline at the microelectrode cavities formed polyaniline protrusions containing entrapped glucose oxidase. Chemical deposition of polysiloxane from dichlorodimethysilane was used to deposit a thin protective and diffusion mass transport controlling coating over the electrodes. The physical and electrochemical properties of these films were studied. The performance of the final glucose oxidase based microelectrode sensor array is reported.     

Sonochemically fabricated acetylcholinesterase micro-electrode arrays within a flow injection analyser for the determination of organophosphate pesticides

This report describes the development of novel sonochemically fabricated, bioengineered acetylcholinesterase and polyaniline carbon/cobalt phthalocyanine biosensors for the ultra-sensitive determination of a number of different pesticides. Arrays of this type typically have population micro-electrode densities of up to approximately 2 x 10(5) cm(-2); these represent the highest micro-electrode population densities reported to date by any fabrication means. The enzymatic response of the sensors is inhibited upon incubation with the pesticide, and we have shown that Dichlorvos, Parathion and Azinphos may be determined down to concentrations of approximately 1 x 10(-17) M, approximately 1 x 10(-16) M and approximately 1 x 10(-16) M, respectively. These lower limits of detection are lower than otherwise achievable by any other analytical approach. Measurements were performed within a custom built flow injection system that operates at a constant flow of 1 ml min(-1). Sensor stability studies were also performed whereby a stabilizer mixture of sucrose and polygalacturonic acid was added to the immobilised enzyme matrix at the working electrode and left to dry. Sixty-five percent of the initial enzyme activity was found to remain after a period of 92 days to allow storage of these electrodes and facilitating transportation if required.     

Listening to the brain: microelectrode biosensors for neurochemicals

Chemical signalling underlies every function of the nervous system, from those of which we are unaware, for example, control of the heart, to higher cognitive functions, such as emotions, learning and memory. Neurotransmitters and neuromodulators mediate communication between neurons and between neurons and non-neural cells such as glia and muscle. In the past, the means for studying the production and release of these signalling agents directly has been limited in its temporal and spatial resolution relative to the dynamics of chemical signalling and the structures of interest in the brain. Now microelectrode biosensors are becoming available that give unprecedented spatial and temporal resolution, enabling, for the first time, direct measurement in real time of the chemical conversations between cells in the nervous system.     

Individually addressable microelectrode arrays fabricated with gold-coated pencil graphite particles for multiplexed and high sensitive impedance immunoassays

A renewable, site-selective immobilization platform of microelectrode array (MEA) for multiplexed immunoassays has been initially developed using pencil graphite particles coated with gold layers as microelectrodes. The graphite particles available on the common pencil were utilized for directing the electro-deposition of gold layers with uniform microstructures which displayed a well-defined sigmoidal voltammetric response. In the concept-of-proof experiments, the resulting MEA platform was modified with functionalized monolayer, on which anti-human IgG antibodies could be stably immobilized in a site-selective way through binding chemistry to selectively capture human IgG antigens from the sample media. The subsequent introduction of anti-human IgG antibodies conjugated with 15 nm electro-active gold nanoparticles to recognize the captured IgG proteins resulted in a significant decrease in the interfacial electron-transfer resistance. High sensitive electrochemical quantification by gold nanoparticle-amplified impedance responses could thus be achieved. Experimental results show that the developed MEA sensor can allow for the detection of human IgG with wide linear range (0.05–100 ng ml⁻¹) and sensitivity over 10³ larger than that of the conventional, bulk gold electrode. The rapid regeneration of the used MEA platform can additionally be realized by a simple electrochemical treatment. The high selectivity of four individually addressable MEA platforms for multiple antigens in a single sample has been further demonstrated in the multiplexed immunoassay experiments. Such a site-selective immobilization strategy of MEA platform may open a new door towards the development of various simple, sensitive, cost-effective, and reusable biological sensors and biochips.     

Graphene microelectrode arrays for neural activity detection

We demonstrate a method to fabricate graphene microelectrode arrays (MEAs) using a simple and inexpensive method to solve the problem of opaque electrode positions in traditional MEAs, while keeping good biocompatibility. To study the interface differences between graphene–electrolyte and gold–electrolyte, graphene and gold electrodes with a large area were fabricated. According to the simulation results of electrochemical impedances, the gold–electrolyte interface can be described as a classical double-layer structure, while the graphene–electrolyte interface can be explained by a modified double-layer theory. Furthermore, using graphene MEAs, we detected the neural activities of neurons dissociated from Wistar rats (embryonic day 18). The signal-to-noise ratio of the detected signal was 10.31 ± 1.2, which is comparable to those of MEAs made with other materials. The long-term stability of the MEAs is demonstrated by comparing differences in Bode diagrams taken before and after cell culturing.     

Networks of neurons coupled to microelectrode arrays: a neuronal sensory system for pharmacological applications

Two main features make microelectrode arrays (MEAs) a valuable tool for electrophysiological measurements under the perspective of pharmacological applications, namely: (i) they are non-invasive and permit, under appropriate conditions, to monitor the electrophysiological activity of neurons for a long period of time (i.e. from several hours up to months); (ii) they allow a multi-site recording (up to tens of channels). Thus, they should allow a high-throughput screening while reducing the need for animal experiments. In this paper, by taking advantages of these features, we analyze the changes in activity pattern induced by the treatment with specific substances, applied on dissociated neurons coming from the chick-embryo spinal cord. Following pioneering works by Gross and co-workers (see e.g. Gross and Kowalski, 1991. Neural Networks, Concepts, Application and Implementation, vol. 4. Prentice Hall, NJ, pp. 47-110; Gross et al., 1992. Sensors Actuators, 6, 1-8.), in this paper analysis of the drugs' effects (e.g. NBQX, CTZ, MK801) to the collective electrophysiological behavior of the neuronal network in terms of burst activity, will be presented. Data are simultaneously recorded from eight electrodes and besides variations induced by the drugs also the correlation between different channels (i.e. different area in the neural network) with respect to the chemical stimuli will be introduced (Bove et al., 1997. IEEE Trans. Biomed. Eng., 44, 964-977.). Cultured spinal neurons from the chick embryo were chosen as a neurobiological system for their relative simplicity and for their reproducible spontaneous electrophysiological behavior. It is well known that neuronal networks in the developing spinal cord are spontaneously active and that the presence of a significant and reproducible bursting activity is essential for the proper formation of muscles and joints (Chub and O'Donovan, 1998. J. Neurosci., 1, 294-306.). This fact, beside a natural variability among different biological preparations, allows a comparison also among different experimental session giving reliable results and envisaging a definition of a bioelectronic 'neuronal sensory system'.     

Ceramic-based multisite microelectrode arrays for simultaneous measures of choline and acetylcholine in CNS

A ceramic-based microelectrode array (MEA) with enzyme coatings for the accurate measurement of acetylcholine (ACh) in brain tissues is presented. Novel design features allow for self-referencing recordings for improved limits of detection and highly selective measurements of ACh and choline (Ch), simultaneously. Design and fabrication features also result in minimal tissue damage during implantation and improved enzyme coatings due to isolated recording sites. In these studies we have used a recombinant human acetylcholinesterase enzyme coating, which has better reproducibility than other commercially available enzymes. The precisely patterned recording site dimensions, low limit of detection (0.2 micro M) and fast response time ( approximately 1s) allow for second-by-second measurements of ACh and Ch in brain tissues. An electropolymerized meta-phenylenediamine (mPD) layer was used to exclude interfering substances from being recorded at the platinum recording sites. Our studies support that the mPD layer was stable for over 24h under in vitro and in vivo recording conditions. In addition, our work supports that the current configuration of the MEAs produces a robust design, which is suited for measures of ACh and Ch in rat brain.     

A low noise multichannel integrated circuit for recording neuronal signals using microelectrode arrays

This paper reports on the development of a fully integrated 32-channel integrated circuit (IC) for recording neuronal signals in neurophysiological experiments using microelectrode arrays. The IC consists of 32 channels of low-noise preamplifiers and bandpass filters, and an output analog multiplexer. The continuous-time RC active filters have a typical passband of 20-2000 Hz; the low and the high cut-off frequencies can be separately controlled by external reference currents. This chip provides a satisfactory signal-to-noise ratio for neuronal signals with amplitudes greater than 50 microV. For the nominal passband setting, an equivalent input noise of 3 microV rms has been achieved. A single channel occupies 0.35 mm(2) of silicon area and dissipates 1.7 mW of power. The chip was fabricated in a 0.7 microm CMOS process.     

Immobilisation of glucose oxidase within metallic nanotubes arrays for application to enzyme biosensors

Arrays of nanoscopic gold tubes were prepared by electroless deposition of the metal within the pores of polycarbonate particle track-etched membranes (PTM). Glucose oxidase (Gox) was immobilised onto preformed self-assembled monolayers (SAMs) (mercaptoethylamine or mercaptopropionic acid) on electroless gold via cross-linking with glutaraldehyde or covalent attachment by carbodiimide coupling. The effectiveness of the different steps in the Gox immobilisation procedure was assessed by contact angle measurements, cyclic voltammetry and X-ray photoelectron spectroscopy. The enzyme loading was estimated by radioactivity measurements. The sensitivity to beta-glucose of these different biosensors has been evaluated. Glucose responses as large as 400 nA mM(-1) cm(-2) have been obtained. To our knowledge, this sensitivity value is amongst the highest values reported in the literature for comparable biosensor systems.     

Sensitivity enhancement of spectral surface plasmon resonance biosensors for the analysis of protein arrays

A novel method for sensitivity enhancement of spectral surface plasmon resonance (SPR) biosensors was presented by reducing the refractive index of the sensing prism in the analysis of protein arrays. Sensitivity of spectral SPR biosensors with two different prisms (BK-7, fused silica) was analyzed by net shifts of resonance wavelength for specific interactions of GST–GTPase binding domain of p21-activated kinase-1 and anti-GST on a mixed thiol surface. Sensitivity was modulated by the refractive index of the sensing prism of the spectral SPR biosensors with the same incidence angle. The sensitivity of a spectral SPR biosensor with a fused silica prism was 1.6 times higher than that with a BK-7 prism at the same incidence angle of 46.2°. This result was interpreted by increment of the penetration depth correlated with evanescent field intensity at the metal/dielectric interface. Therefore, it is suggested that sensitivity enhancement is readily achieved by reducing the refractive index of the sensing prism of spectral SPR biosensors to be operated at long wavelength ranges for the analysis of protein arrays.     

Peptide nanowires for coordination and signal transduction of peroxidase biosensors to carbon nanotube electrode arrays

A strategy of metallizing peptides to serve as conduits of electronic signals that bridge between a redox enzyme and a carbon-nanotube electrode has been developed with enhanced results. In conjunction, a protocol to link the biological elements to the tips of carbon nanotubes has been developed to optimize contact and geometry between the redox enzyme and the carbon nanotube electrode array. A peptide nanowire of 33 amino acids, comprised of a leucine zipper motif, was mutated to bind divalent metals, conferring conductivity into the peptide. Reaction between a thiolate of the peptide with the sulfenic acid of the NADH peroxidase enzyme formed a peptide-enzyme assembly that are fully primed to transduce electrons out of the enzyme active site to an electrode. Scanning electron microscopy shows immobilization and linking of the assembly specifically to the tips of carbon nanotube electrodes, as designed. Isothermal titration calorimetry and mass spectrometry indicate a binding stoichiometry of at least three metals bound per peptide strand. Overall, these results highlight the gain that can be achieved when the signal tranducing units of a biosensor are aligned through directed peptide chemistry.