Functional characterization of a conducting polymer-based immunoassay system.

Experiments have been performed to characterize the electrical properties and functionality of a poly(3-hexylthiophene)-coated platinum electrode developed as a sensor for immunoassay read-out. Admittance measurements were performed on the coated electrodes as a function of frequency. The admittance spectra obtained show that the sensor is capacitive in nature. A circuit model is presented for comparison to other conducting polymer systems. Dynamic sensor response is characterized by oxidizing the polymer via a hydrogen peroxide-iodide pathway. Hydrogen peroxide is introduced either by direct injection or through a glucose-glucose oxidase reaction to determine electrode functionality and sensitivity. Sensor response to chemical oxidation is measured as a function of frequency and applied signal amplitude. System response is linear in frequency from 1 Hz to 70 Hz and in excitation amplitude up to approximately 600 mV. System sensitivity is analyzed based on oxidant generation from the enzyme-initiated pathway, sensor baseline drift, and the noise band on the quiescent sensor current.

Measurement of acetylcholine receptor function in microcircuit-coupled myoblasts.

An electrophysiological measurement principle for long-term, noninvasive monitoring of the nicotinic acetylcholine receptor (nAChR) function is described. The measurement is based on the ability to record agonist-induced depolarizations of clonal myoblasts that have formed high impedance seals with extracellular microcircuit electrodes. The technique appears promising for several types of assays and environmental monitoring applications.

Immobilization of flavoproteins on silicon: effect of cross-linker chain length on enzyme activity.

The effect of cross-linker chain length on the activities of choline oxidase (ChO) and glucose oxidase (GOx) immobilized on oxidized silicon wafers has been investigated for the cross-linkers N-succinimidyl 4-maleimido-butyrate (GMBS) and N-succinimidyl 6-maleimidocaproate (EMCS). Enzyme activities were determined with an indirect fluorometric assay based on the production of hydrogen peroxide. Immobilization of ChO or GOx onto oxidized silicon with either cross-linker resulted in an 86-99% loss in enzymatic activity relative to the soluble form of the flavoprotein. However, the different cross-linkers had distinctly different effects on enzyme activity: EMCS-immobilized GOx was four times more active than GMBS-immobilized GOx; EMCS-immobilized ChO had a sevenfold higher activity than GMBS-immobilized ChO.

Deep UV photochemistry of chemisorbed monolayers: patterned coplanar molecular assemblies.

Deep ultraviolet (UV) irradiation is shown to modify organosilane self-assembled monolayer (SAM) films by a photocleavage mechanism, which renders the surface amenable to further SAM modification. Patterned UV exposure creates alternating regions of intact SAM film and hydrophilic, reactive sites. The exposed regions can undergo a second chemisorption reaction to produce an assembly of SAMs in the same molecular plane with similar substrate attachment chemistry. The UV-patterned films are used as a template for selective buildup of fluorophores, metals, and biological cells.

Modulation of a gated ion channel admittance in lipid bilayer membranes.

A future class of amperometric biosensors may utilize gated ion channels such as acetylcholine and glutamate receptors as chemical detection components. In this study, bilayer lipid membranes containing voltage-dependent anion channels (VDAC) were used to model an ion-channel-based biosensor which could continuously monitor AC amperometric changes resulting from induced changes in channel conductance. The in-phase and quadrature components of the induced alternating membrane current were monitored as a function of the applied DC offset voltage which was superimposed on the sinusoidal test voltage. The accuracy and sensitivity of the AC-measured VDAC response was dependent on the magnitude of the AC test voltage relative to the DC offset necessary for channel closure. The VDAC channel appears to be a suitable model protein for AC impedance-based biosensor fabrication.

Fabrication of key components of a receptor-based biosensor.

A three-pronged approach was taken to the development of receptor-based bisensors. First, asymmetric bilayer membranes were developed with one monolayer adaptable to the particular receptor of interest and the other monolayer polymerized to enhance membrane stability. Second, alamethicin and calcium channel complexes were introduced into the stabilized membrane and tested for ion-channel function. Third, a porous support for the receptor-containing membrane was fabricated, which is compatible with silicon technology. Preliminary devices incorporating these components were constructed.