Thomas L. Fare

A DC autonulling bridge for real-time resistance measurement

This paper describes a resistance measurement system based on a Wheatstone bridge topology that overcomes the limitations of a direct differential measurement and is suited for monolithic sensor applications. An analog feedback network provides a wide dynamic range and maximizes noise rejection by automatically driving the bridge to null via a voltage-controlled resistor. The circuit also provides transient monitoring of resistance changes in any sensor that produces a resistance change due to an environmental stimulus. The system stability is discussed and analyzed using Liapunov analysis, SPICE simulations, and a prototype circuit implemented using basic off-the-shelf components produces results similar to the theoretical analysis.

Immobilization of acetylcholinesterase on solid surfaces: chemistry and activity studies

Abstract A method for covalent attachment of the enzyme acetylcholinesterase (E.C. 3.1.1.7, Electric Eel) on two different solid surfaces (glass and platinum) is described. The chemistry includes covalent bond formation between a hydroxyl group on the surface, a thiol-terminal silane, a heterobifunctional crosslinker, and the enzyme. The activity of the immobilized enzyme has been determined spectrophotometrically using two different substrates, acetylcholine and butyrylthiocholine. It is determined that after immobilization, acetylcholinesterase retains at least 50% of its activity on glass and 15% of its activity on platinum surfaces when assayed using butyrylthiocholine as a substrate. The general procedure described here for the immobilization of enzymes should find broad use in the fabrication of enzyme electrodes and in biosensor technology.

Coplanar patterns of self-assembled monolayers for selective cell adhesion and outgrowth

Abstract A recently developed deep UV photolithographic method produces high resolution (1–200 μm feature size) patterns of coplanar self-assembled monolayers (SAMs) which spatially control the adhesion and outgrowth of biological cells. Characterization of these patterned SAMs, using wettability, X-ray photoelectron spectroscopy (XPS), and scanning Auger electron spectroscopy (AES), shows that high resolution adjacent regions of intact SAMs are formed in the same molecular plane. Selective adhesion and outgrowth of neuroblastoma cells, explanted rat hippocampal cells, and human umbilical vein endothelial cells are demonstrated on these SAM patterns. Patterned SAMs might provide new approaches to the study of intercellular communication and development, and allow arrangement of cells on surfaces of sensor devices, prosthetic implants, or tissue repair templates.

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.

Voltage modulation of a gated ion channel admittance in platinum-supported lipid bilayers

Platinum-supported phospholipid bilayers containing voltage-dependent anion channels (VDAC) were used to model physically a chemically sensitive amperometric biosensor which would use gated transmembrane channel proteins. An a.c. electrical technique was used to measure admittance changes caused by channel gating. The magnitude of the responses differed from those observed in bilayer lipid membranes (BLM). However, the bilayers were mechanically stable, and permitted reproducible gating of the VDAC conductance in response to d.c. bias voltages in the range of 0 to −60 mV. The technique may be generally suitable for fabrication of durable biosensors using chemically sensitive protein channels.

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.

Langmuir-Blodgett deposited valinomycin-phospholipid films on platinum: an a.c. impedance response to potassium

Abstract Multilayers of phospholipid and valinomycin films are deposited onto platinum electrodes using the Langmuir-Blodgett technique. The pressure-area isotherms of the mixed lipid and valinomycin monolayers have been used to characterize the films prior to deposition. Coverage of platinum surfaces by the deposited layers is determined using an a.c. electrical impedance technique. The stability of the monolayer, lipid adhesion onto the platinum surface and the surface pressure are parameters that prove to be critical for obtaining high-impedance electrode coatings. Film deposition is carried out for high and low ionic strength subphases at neutral pH. Films deposited at surface pressures of 45 mN m−1 have an impedance on the order of that of artificial bilayer lipid membranes. The film impedance on the platinum electrodes is measured as a function of the potassium concentration. The film impedance decreases when potassium is added to the subphase. Conductance changes induced by the potassium are used to evaluate the percentage uptake of potassium by valinomycin. Mechanisms for the electrical response of the film are discussed in terms of adsorption models.

Autonulling ac bridge for accurate measurement of small impedance variations using MOS components

A self‐contained prototype of an autonulling ac bridge based on phase‐sensitive detection is discussed. The bridge nulling circuit uses an analog feedback network to establish a stable null for a wide range of resistive and capacitive values and requires minimal supporting hardware. Changes in resistance and capacitance in one arm of the bridge are compensated independently using a discrete voltage‐variable resistor and capacitor in the alternate arm for nulling. The feedback voltage to the discrete control elements can be used to transduce signals from pressure, temperature, or chemical sensors. This bridge is especially well suited for microfabrication and is readily compatible with silicon‐based sensors. It offers high noise rejection due to the phase‐sensitive measurement taken differentially across the bridge nodes. State‐space analysis is used to demonstrate the stability of this feedback network design.

Stability analysis of an autonulling AC bridge for use with silicon-based sensors

A novel circuit incorporating integral feedback to automatically null an AC bridge is analyzed and discussed. Stability of the feedback scheme is examined by linearizing the system near the critical point (null) of the bridge using a standard perturbation method. Solutions of the nonlinear coupled differential equations that describe the operation of the autonulling AC bridge were obtained by numerical integration. A prototype was constructed using integrated circuits and discrete components to measure the transient response; test results of the circuit from transient impedance changes are compared to the time-dependent numerical solutions. >

A physiologic-based circuit model of the postsynaptic region at the neuromuscular junction

A metal-oxide-semiconductor field-effect transistor (MOSFET) is used as the circuit analog to simulate a group of excitatory transmitter-gated ion channels. This analog is incorporated into a circuit model of the postsynaptic membrane at the neuromuscular junction. Simulation of the model yields an output representing the overall membrane potential of the postsynaptic region. Simulation is performed for the normal and pathologic (myasthenia gravis) states.