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.     

High-sensitivity electrochemical enzyme-linked assay on a microfluidic interdigitated microelectrode

A novel enzyme-linked DNA hybridization assay on an interdigitated array (IDA) microelectrode integrated into a microfluidic channel is demonstrated with sub-nM detection limit. To improve the detection limit as compared to conventional electrochemical biosensors, a recyclable redox product, 4-aminophenol (PAP) is used with an IDA microelectrode. The IDA has a modest and easily fabricated inter-digit spacing of 10 μm, yet we were able to demonstrate 97% recycling efficiency of PAP due to the integration in a microfluidic channel. With a 70 nL sample volume, the characterized detection limit for PAP of 1.0 × 10?1? M is achieved, with a linear dynamic range that extends from 1.0 × 10?? to 1.0 × 10?? M. This detection limit, which is the lowest reported detection limit for PAP, is due to the increased sensitivity provided by the sample confinement in the microfluidic channel, as well as the increased repeatability due to perfectly static flow in the microchannel and an additional anti-fouling step in the protocol. DNA sequence detection is achieved through a hybridization sandwich of an immobilized complementary probe, the target DNA sequence, and a second complementary probe labeled with β-galactosidase (β-GAL); the β-GAL converts its substrate, 4-aminophenyl-d-galactopyranoside (PAPG), into PAP. In this report we present the lowest reported observed detection limit (1.0 × 10?1? M) for an enzyme-linked DNA hybridization assay using an IDA microelectrode and a redox signaling paradigm. Thus, we have demonstrated highly sensitive detection of a targeted DNA sequence using a low-cost easily fabricated electrochemical biosensor integrated into a microfluidic channel.     

Separation of submicron bioparticles by dielectrophoresis.

Submicron particles such as latex spheres and viruses can be manipulated and characterized using dielectrophoresis. By the use of appropriate microelectrode arrays, particles can be trapped or moved between regions of high or low electric fields. The magnitude and direction of the dielectrophoretic force on the particle depends on its dielectric properties, so that a heterogeneous mixture of particles can be separated to produce a more homogeneous population. In this paper the controlled separation of submicron bioparticles is demonstrated. With electrode arrays fabricated using direct write electron beam lithography, it is shown that different types of submicron latex spheres can be spatially separated. The separation occurs as a result of differences in magnitude and/or direction of the dielectrophoretic force on different populations of particles. These differences arise mainly because the surface properties of submicron particles dominate their dielectrophoretic behavior. It is also demonstrated that tobacco mosaic virus and herpes simplex virus can be manipulated and spatially separated in a microelectrode array.     

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.     

Microfluidic bioassay system based on microarrays of hydrogel sensing elements entrapping quantum dot-enzyme conjugates

This paper presents a simple method to fabricate a microfluidic biosensor that is able to detect substrates for H(2)O(2)-generating oxidase. The biosensor consists of three components (quantum dot-enzyme conjugates, hydrogel microstructures, and a set of microchannels) that were hierarchically integrated into a microfluidic device. The quantum dot (QD)-enzyme conjugates were entrapped within the poly(ethylene glycol) (PEG)-based hydrogel microstructures that were fabricated within the microchannels by a photopatterning process. Glucose oxidase (GOX) and alcohol oxidase (AOX) were chosen as the model oxidase enzymes, conjugated to carboxyl-terminated CdSe/ZnS QDs, and entrapped within the hydrogel microstructures, which resulted in a fluorescent hydrogel microarray that was responsive to glucose or alcohol. The hydrogel-entrapped GOX and AOX were able to perform enzyme-catalyzed oxidation of glucose and alcohol, respectively, to produce H(2)O(2), which subsequently quenched the fluorescence of the conjugated QDs. The fluorescence intensity of the hydrogel microstructures decreased as the glucose and alcohol concentrations increased, and the detection limits of this system were found to be 50 μM of glucose and 70 μM of alcohol. Because each microchannel was able to carry out different assays independently, the simultaneous detection of glucose and alcohol was possible using our novel microfluidic device composed of multiple microchannels.     

L-lactate measures in brain tissue with ceramic-based multisite microelectrodes

A newly developed multisite array microelectrode for in vivo measurements of L-lactate is presented. The resulting microelectrode is composed of three functional layers. First, Nafion is used to repel interfering electroactive anions, such as ascorbate. Second, L-lactate oxidase immobilized onto the recording sites is used to convert L-lactate to hydrogen peroxide. The H2O2 produced is proportional to L-lactate concentrations and is quantified at the platinum recording sites. Third, a layer of polyurethane is coated over the L-lactate oxidase to adjust the linear range of the microelectrode to one that is compatible with in vivo measurements. This layer reduces the amount of L-lactate that diffuses to the enzyme while not significantly limiting oxygen diffusion. The resulting L-lactate microelectrodes were linear to 20 mM (R2 = 0.997 +/- 0.001) and beyond in some cases with detection limits of 0.078 +/- 0.013 mM (n = 12). The selectivity and response time of these electrodes make them suitable for in vivo measurements in brain tissue. Self-referencing recordings may be utilized to further improve the selectivity of the recordings. However this is not necessary for most applications in the brain, because the resting and stimulated levels of dopamine (DA), norepinephrine (NE), and other potentially interfering cations are two to three orders of magnitude lower than that of in vivo L-lactate, which is in the millimolar range. Preliminary in vivo measures of L-lactate in the brain of anesthetized rats support that the microelectrodes are capable of measuring rapid endogenous changes in vivo.     

A metallic multisite recording system designed for continuous long-term monitoring of electrophysiological activity in slice cultures.

This paper describes a flexible, metallic multielectrode array, made on kapton to fit in a recording chamber for interface-type organotypic cultures. This multisite recording system is designed for continuous multisite monitoring of electrophysiological activity in rat brain organotypic slice cultures. The system is composed of a signal conditioning set-up, which also masters electrical stimulation paradigms and a card containing the microelectrode array. The card comprises a perfusion chamber closed by a rigid and permeable membrane on which the pierced microelectrode array supporting the slice culture is placed. Once closed with a gaseous chamber, the inside of the card remained sterile and free of contamination and could be maintained inside or outside the incubator for electrophysiological analyses. Dimensions of each 28-plated gold microelectrode recording site are 50 microns x 100 microns. The design of the chambers and the card makes it possible to modify both the perfusion medium and the gaseous atmosphere in sterile conditions, allowing thus analyses of long-term effects of pharmacological compounds. Using this array one can perform stimulation and recordings of the electrical activity of the slice. Signals obtained with this reusable system exhibit a good signal-to-noise ratio. This device was tested to follow the evolution and modifications of the evoked and/or spontaneous electrical activity of the same groups of neurones during several days.     

A high density microelectrode array biosensor for detection of E. coli O157:H7

A high density microelectrode array biosensor was developed for the detection of Escherichia coli O157:H7. The biosensor was fabricated from (100) silicon with a 2 microm layer of thermal oxide as an insulating layer, an active area of 9.6 mm2 and consists of an interdigitated gold electrode array. The sensor surface was functionalised for bacterial detection using heterobifunctional crosslinkers and immobilised polyclonal antibodies to create a biological sensing surface. Bacteria suspended in solution became attached to the immobilised antibodies when the biosensor was tested in liquid samples. The change in impedance caused by the bacteria was measured over a frequency range of 100 Hz-10 M Hz. The biosensor was evaluated for E. coli O157:H7 detection in pure culture and inoculated food samples. The biosensor was able to discriminate between cellular concentrations of 10(4)-10(7)CFU/mL and has applications in detecting pathogens in food samples.     

Fabrication of glucose oxidase/polypyrrole biosensor by galvanostatic method in various pH aqueous solutions

The pH effect of pyrrole electropolymerization in the presence of glucose oxidase (GODx) on the performance and characteristic of galvanostatically fabricated glucose oxidase/polypyrrole (Ppy) biosensor is reported. Preparing the GODx/Ppy biosensors in 0.1 M KCl saline solution with various pH containing 0.05 M pyrrole monomer and 0.5 mg/ml GODx at 382 microA/cm2 current density for 100 mC/cm2 film thickness, both the galvanostatic responses and characteristics of these resulted biosensors were obtained. The results revealed that the galvanostatic glucose biosensor fabricated at neutral pH condition exhibited much higher sensitivity than those fabricated at lower or higher pH conditions, and had a good linearity form zero to 10 mM glucose with the sensitivity of 7 nA/mM. Finally, the long-term stability and the kinetic parameters, Michaelis constant and maximum current, of this biosensor were also reported.     

Discovery of a eugenol oxidase from Rhodococcus sp. strain RHA1

A gene encoding a eugenol oxidase was identified in the genome from Rhodococcus sp. strain RHA1. The bacterial FAD-containing oxidase shares 45% amino acid sequence identity with vanillyl alcohol oxidase from the fungus Penicillium simplicissimum. Eugenol oxidase could be expressed at high levels in Escherichia coli, which allowed purification of 160 mg of eugenol oxidase from 1 L of culture. Gel permeation experiments and macromolecular MS revealed that the enzyme forms homodimers. Eugenol oxidase is partly expressed in the apo form, but can be fully flavinylated by the addition of FAD. Cofactor incorporation involves the formation of a covalent protein-FAD linkage, which is formed autocatalytically. Modeling using the vanillyl alcohol oxidase structure indicates that the FAD cofactor is tethered to His390 in eugenol oxidase. The model also provides a structural explanation for the observation that eugenol oxidase is dimeric whereas vanillyl alcohol oxidase is octameric. The bacterial oxidase efficiently oxidizes eugenol into coniferyl alcohol (KM=1.0 microM, kcat=3.1 s-1). Vanillyl alcohol and 5-indanol are also readily accepted as substrates, whereas other phenolic compounds (vanillylamine, 4-ethylguaiacol) are converted with relatively poor catalytic efficiencies. The catalytic efficiencies with the identified substrates are strikingly different when compared with vanillyl alcohol oxidase. The ability to efficiently convert eugenol may facilitate biotechnological valorization of this natural aromatic compound.     

Immobilization of yeast microbodies by inclusion with photo-crosslinkable resins.

Yeast microbodies containing FAD-dependent alcohol oxidase, catalase and D-amino acid oxidase were isolated from methanol-grown cells of Kloeckera sp. 2201 and immobilized intact in matrices formed by a short-time illumination of photo-crosslinkable resin oligomers. The relative activities of catalase, alcohol oxidase and D-amino acid oxidase of the gel-entrapped microbodies were 36, 76 and 31% respectively as compared with those of free microbodies. Immobilization enhance d the stability of catalase to a certain degree, but not that of alcohol oxidase. The pH/activity profiles of catalase and alcohol oxidase of the entrapped organelles showed more narrow pH optima than those of the free counterparts. D-Amino acid oxidase in immobilized microbodies showed a somewhat higher Km value for D-alanine than that in free ones. Immobilized microbodies oxidized two moles of methanol to form two moles of formaldehyde with consumption of one mole of molecular oxygen. Addition of 3-amino-1,2,4-triazole, an inhibitor of catalase, reduced the formation of formaldehyde to half the amount without change in the amount of oxygen consumed, indicating the synergic action of alcohol oxidase and catalase in methanol oxidation in the microbodies of living yeast cells.     

Substructure of crystalline peroxisomes in methanol-grown Hansenula polymorpha: evidence for an in vivo crystal of alcohol oxidase.

The substructural organization of completely crystalline peroxisomes present in Hansenula polymorpha cells grown under methanol limitation in a chemostat was investigated by different cytochemical and ultrastructural techniques. Time-dependent cytochemical staining experiments indicated that activities of the two main constituents of these organelles, namely, alcohol oxidase and catalase, were present throughout the crystalline matrix. Catalase was completely removed from isolated peroxisomes by osmotic shock treatment. After such treatment, the ultrastructure of the crystalline matrix of the organelles remained virtually intact. Because alcohol oxidase activity was still present in this matrix, it was concluded that alcohol oxidase protein is the only structural element of the peroxisomal crystalloids. The molecular architecture of the crystalloids was investigated in ultrathin cryosections which permitted recognition of individual molecules in the crystalline matrix. Depending on the plane of sectioning, different crystalline patterns were observed. Tilting experiments indicated that these images were caused by superposition of octameric alcohol oxidase molecules arranged in a tetragonal lattice. A three-dimensional model of the crystalloid is presented. The repeating unit of this structure is composed of four alcohol oxidase molecules. The crystalloid represents an open structure, which may explain the observed free mobility of catalase molecules.     

Sonochemically fabricated microelectrode arrays for biosensors offering widespread applicability: Part I

A novel and patented procedure is described for the sonochemical fabrication of a new class of microelectrode array based sensor with electrode element populations of up to 2 x 10(5) cm(-2). For some years it has been accepted that microelectrode arrays offer an attractive route for lowering minimum limits of detection and imparting stir (convectional mass transport) independence to sensor responses; despite this no commercial biosensors, to date, have employed microelectrode arrays, largely due to the cost of conventional fabrication routes that have not proved commercially viable for disposable devices. Biosensors formed by our sonochemical approach offer unrivalled sensitivity and impart stir independence to sensor responses. This format lends itself for mass fabrication due to the simplicity and inexpensiveness of the approach; in the first instance impedimetric and amperometric sensors are reported for glucose as model systems. Sensors already developed for ethanol, oxalate and a number of pesticide determinations will be reported in subsequent publications.     

Real-time electrochemical imaging using an individually addressable multi-channel electrode.

We developed a real-time electrochemical imaging method that uses a multiple enzyme-modified microelectrode. The method will enable the investigation of the functions of biological materials and cells. To test its effectiveness, we imaged the two-dimensional concentration distribution for hydrogen peroxide and L-glutamate in a standard solution. The multiple electrode consists of an 8 x 8 array of 30 x 30 microm2 carbon micro electrode. Each electrode was connected to a 64-channel potentiostat that could apply a potential to all electrodes at the same time. The multiple electrode was coated with an Os-polyvinylpyridine based polymer (Os-gel) containing horse radish peroxidase (HRP) to detect hydrogen peroxide, which is a very common product of oxidase enzyme. When measuring glutamate, which is a well-known neurotransmitter in the mammalian central nerve system, we modified the electrode with a bilayer of Os-gel-HRP and GluOx. The detection limit of our method was 1 microM and images of the glutamate concentration-distribution changes induced by local injection of glutamate through microcapillary were obtained in real time.     

Moving known libraries to an addressable array: a site-selective hetero-Michael reaction

A two-step, Michael reaction-based strategy for site-selectively placing molecules by unique electrodes in an addressable microelectrode array has been developed. The strategy is compatible with the use of polypeptide nucleophiles and works with microelectrode arrays having either 1024 electrodes/cm (2) or 12,544 electrodes/cm (2). The chemistry should allow for the transfer of existing molecular libraries to microelectrode array devices for analysis.     

Reconstitution of the ethanol oxidase respiratory chain in membranes of quinoprotein alcohol dehydrogenase-deficient Gluconobacter suboxydans subsp. alpha strains.

The ethanol oxidase respiratory chain of Gluconobacter suboxydan was characterized by using G. suboxydans subsp. alpha, a variant species of G. suboxydans incapable of oxidizing ethanol. The membranes of G. suboxydans subsp. alpha exhibited neither alcohol dehydrogenase, ethanol oxidase, nor glucose-ferricyanide oxidoreductase activity. Furthermore, the respiratory chain of the organism exhibited an extremely diminished amount of cytochrome c and an increased sensitivity of the respiratory activity for cyanide or azide when compared with G. suboxydans. The first-subunit quinohemoprotein and the second-subunit cytochrome c of alcohol dehydrogenase complex in the membranes of G. suboxydans subsp. alpha were shown to be reduced and deficient, respectively, by using heme-staining and immunoblotting methods. Ethanol oxidase activity, lacking in G. suboxydans subsp. alpha, was entirely restored by reconstituting alcohol dehydrogenase purified from G. suboxydans to the membranes of G. suboxydans subsp. alpha; this also led to restoration of the cyanide or azide insensitivity and the glucose-ferricyanide oxidoreductase activity in the respiratory chain without affecting other respiratory activities such as glucose and sorbitol oxidases. Ethanol oxidase activity was also reconstituted with only the second-subunit cytochrome c of the enzyme complex. The results indicate that the second-subunit cytochrome c of the alcohol dehydrogenase complex is essential in ethanol oxidase respiratory chain and may be involved in the cyanide- or azide-insensitive respiratory chain bypass of G. suboxydans.     

Oxidation of formaldehyde by alcohol oxidase of Candida boidinii.

A fromaldehyde oxidase activity was found in cellfree extracts of methanol-grown yeast Candida boidinii. Loss of alcohol oxidase activity in a mutant, 48, led to loss of the formaldehyde oxidase activity, indicating that the same enzyme is probably responsible for both activities. This could be demonstrated with the purified alcohol oxidase which oxidizes, besides lower primary alcohols, formaldehyde to formate. The Km value for formaldehyde is 5.7 mM. It seems that alcohol oxidase is not implicated in formaldehyde oxidation in vivo.     

Covalent enzyme immobilization by poly(ethylene glycol) diglycidyl ether (PEGDE) for microelectrode biosensor preparation

Poly(ethylene glycol) diglycidyl ether (PEGDE) is widely used as an additive for cross-linking polymers bearing amine, hydroxyl, or carboxyl groups. However, the idea of using PEGDE alone for immobilizing proteins on biosensors has never been thoroughly explored. We report the successful fabrication of microelectrode biosensors based on glucose oxidase, d-amino acid oxidase, and glutamate oxidase immobilized using PEGDE. We found that biosensors made with PEGDE exhibited high sensitivity and a response time on the order of seconds, which is sufficient for observing biological processes in vivo. The enzymatic activity on these biosensors was highly stable over several months when they were stored at 4 °C, and over at least 3d at 37 °C. Glucose microelectrode biosensors implanted in the central nervous system of anesthetized rats reliably monitored changes in brain glucose levels induced by sequential administration of insulin and glucose. PEGDE provides a simple, low cost, non-toxic alternative for the preparation of in vivo microelectrode biosensors.     

Microelectrode arrays and application to biosensing devices

An electrochemical microanalytical system consisting of a microelectrode array, a micromachined flow-through assembly, and a multichannel potentiostat were constructed for highly sensitive biosensing. Thin-film platinum microelectrode arrays consisting of four interdigitated microelectrodes (IDAs), which are spaced in the sub-micrometer range, were fabricated using silicon technology. On top of this chip, a micromachined flow-through cell was mounted. Using a home made miniaturized multipotentiostat, amperometric measurements of the individual electrodes at different and changing potentials, respective to a single reference electrode, were performed simultaneously. The signal generation, signal processing and the analytical system were controlled by a computer (PC type) and special software. An improved sensor sensitivity was achieved by multielectrode detection and averaging of the IDA responses.

By applying both the oxidation and reduction potentials of reversible redox molecules to pairs of the interdigitated electrodes, an increased current generation can be observed. Thus the steady state current of mediators such as benzoquinone can be amplified by a factor of 30 compared with conventional electrodes. This measuring principle was applied to determine of the activity of hydrolases by detecting the enzyme generated p-aminophenol in the nanomolar range. By combining both, the averaging and the recycling procedures, the detection limit of amperometric biosensing devices may be lowered by about one and a half orders of magnitude.     

Salicyl alcohol oxidase of the chemical defense secretion of two chrysomelid leaf beetles. Molecular and functional characterization of two new members of the glucose-methanol-choline oxidoreductase gene family

Salicyl alcohol oxidase is an extracellular enzyme that occurs in glandular reservoirs of chrysomelid leaf beetle larvae and catalyzes the formation of salicylaldehyde, a volatile deterrent used by the larvae against predators. Salicyl alcohol is the hydrolysis product of salicin, a plant-derived precursor taken up by the beetle larvae from the leaves of willow and poplar trees. The cDNA encoding salicyl alcohol oxidase from two related species Chrysomela tremulae and Chrysomela populi has been identified, cloned, and expressed in an active form in Escherichia coli. The open reading frame of 623 amino acids begins in both enzymes with an N-terminal signal peptide of 21 amino acids. Sequence comparison has revealed that salicyl alcohol oxidase belongs to the family of glucose-methanol-choline oxidoreductase-like sequences with mostly unknown function. Enzymes of this family share similar overall structure with an essentially identical FAD-binding site but possess different catalytic activities. The data suggest that salicyl alcohol oxidase, essential for the activation of the plant-derived precursor salicin, was originally recruited from an oxidase involved in the autogenous biosynthesis of iridoid monoterpenes and found in related chrysomelid leaf beetle species.