Alan R. Koch

A highly stable and selective biosensor using modified nicotinic acetylcholine receptor (nAChR)

Methods for developing stable, sensitive and selective bilayer lipid membrane (BLM)-based biosensors are discussed. Stable BLMs were formed over micromachined polyimide apertures. Selective sensors were made by incorporating nicotinic acetylcholine receptors (nAChRs) modified with bispecific antibodies (BsAbs). When two BsAbs, attached to one nAChR, encounter antigen (Ag), channels are blocked. Sensitivity to single Ag molecules would be possible by monitoring closure of individual nAChRs.

Highly stable bilayer lipid membranes (BLMs) formed on microfabricated polyimide apertures

Abstract Microfabrication is used to realize a polyimide aperture for highly stable bilayer lipid membranes (BLMs). The microfabrication process used in this work is compatible with standard processes used in microelectronics. The physical features of the aperture, such as diameter, thickness and tapering of the edges are precisely controlled during the microfabrication process which results in high yield and reproducibility of the polyimide apertures. A study of BLM stability using precise measurements of specific capacitance shows that the BLM is stable up to 50 h. Alamethicin channels were successfully incorporated into these membranes further proving their bilayer nature. Vesicles containing reconstituted nicotinic acetylcholine receptors (nAChRs) were fused to the BLMs. The successful incorporation of functioning ion channel activity into highly stable BLMs promises a future class of miniature sensors that are very sensitive with fast response times.

Barium modifies the concentration dependence of active potassium transport by insect midgut

SummaryThe rate of active K+ transport by the isolated lepidopteran midgut shows a rectangular hyperbolic relation to [K+] over the range 20 to 70mm K+ in the absence of any divalent cation. Addition of Ba++ to the hemolymph (K+ uptake) side introduces a linear component to the concentration dependence, such that active K− transport is decreased at [K+] of 55mm or less, but increased transiently at higher [K+]. As [Ba++] is increased over the range 2 to 8mm the linear component increases and the saturating component decreases; in 8mm Ba++ the concentration dependence is dominated by the linear component. The effect of Ba++ cannot easily be accounted for by simple competition with K+ for basal membrane uptake sites. Similar effects might be exercised by other alkali earth cations, since the concentration dependence of active K+ transport possesses a substantial linear component in solutions containing 5mm Ca++ and 5mm Mg++ (the alkali earth metal concentrations of standard lepidopteran saline).

Bispecific antibody modification of nicotinic acetylcholine receptors for biosensing.

Recent results show that bispecific antibodies can be used to tailor the selectivity of nicotinic acetylcholine receptors for biosensing purposes. The nicotinic acetylcholine receptors reconstituted in bilayer lipid membranes are inactivated when two bispecific antibodies, attached to the same receptor, bind to a single antigen molecule. Experiments with patch clamp recording equipment reveal that antigen levels of 10(-8) M completely and irreversibly inactivate small numbers of nicotinic acetylcholine receptors. This approach may lead to the construction of biosensors capable of detecting individual antibody-antigen (Ab-Ag) binding events.

Alkalinization by Manduca sexta anterior midgut in vitro: requirements and characteristics

Abstract Manduca sexta anterior midgut alkalinizes its luminal contents in vivo, achieving pH values up to 12 with rates of up to 81.4 μ Eq OH − h −1 . The alkalinization rate dropped to 3.0 μ Eq h −1 within minutes of mounting the tissue in an Ussing chamber, even though K + secretion (as measured by short-circuit transepithelial current and open circuit voltage) continued at a high rate for several hours. In contrast, cannulation of the anterior midgut in situ resulted in a preparation that alkalinized at rates up to 127 μ Eq h −1 for several hours. Removal of the head or ventral nerve cord did not interrupt alkalinization. However, alkalinization could be blocked by increasing the volume of the saline in which the basal side was bathed. The buffer capacity of the saline had no effect on the maximum pH attained or the rate of base secretion. Doubling of the drip rate from 0.5 to 1.0 ml min −1 had no effect on the maximum rate of OH − secretion but resulted in a reduction of the maximum pH attained from 11 to 8.5. These data suggest release of an alkalinization factor from the basal aspect of the gut that may act by altering the passive H + permeability of the tissue.

Kinetic modeling and analysis of a vesicle system for immunosensor development

A novel mechanism is presented for immunosensor development that uses an immunological competition reaction in a vesicle system. This system consists of a suspension of reconstituted vesicles, channel agonist, protein linker to block the channels, voltage sensitive dye and analyte to be detected. In the proposed mechanism analyte serves a catalytic role as individual analytes competitively displace multiple channel linkers through association with one channel, dissociation and new associations with other channels. When one channel opens on a vesicle a permanent Nernst potential develops for that vesicle leading to fluorescence of voltage sensitive dyes. The time constant of the redistribution from linker-channel form to analyte-channel form is 0.92/k4 (k4 is the off-rate constant for the analyte-channel association) in the region of analyte concentrations less than 10(-9) M. Kinetic analyses show that several factors, including concentration of analyte or linker, number of channels per vesicle, on-rate or off-rate constant of the linker-channel and on-rate constant of analyte-channel complexes have significant effects on the minimum signal response time.