Biosensors are molecular sensors that combine a biological recognition mechanism with a physical transduction technique. They provide a new class of inexpensive, portable instrument that permit sophisticated analytical measurements to be undertaken rapidly at decentralized locations. However, the adoption of biosensors for practical applications other than the measurement of blood glucose is currently limited by the expense, insensitivity and inflexibility of the available transduction methods. Here we describe the development of a biosensing technique in which the conductance of a population of molecular ion channels is switched by the recognition event. The approach mimics biological sensory functions and can be used with most types of receptor, including antibodies and nucleotides. The technique is very flexible and even in its simplest form it is sensitive to picomolar concentrations of proteins. The sensor is essentially an impedance element whose dimensions can readily be reduced to become an integral component of a microelectronic circuit. It may be used in a wide range of applications and in complex media, including blood. These uses might include cell typing, the detection of large proteins, viruses, antibodies, DNA, electrolytes, drugs, pesticides and other low-molecular-weight compounds.

Using novel synthetic lipids, a tethered bilayer membrane (tBLM) was formed onto a gold electrode such that a well-defined ionic reservoir exists between the gold surface and the bilayer membrane. Self-assembled monolayers of reservoir-forming lipids were first adsorbed onto the gold surface using gold−sulfur interactions, followed by the formation of the tBLM using the self-assembly properties of phosphatidylcholine-based lipids in aqueous solution. The properties of the tBLM were investigated by impedance spectroscopy. The capacitance of the tBLM indicated the formation of bilayer membranes of comparable thickness to solvent-free black (or bilayer) lipid membranes (BLM). The ionic sealing ability was comparable to those of classical BLMs. The function of the ionic reservoir was investigated using the potassium-specific ionophore valinomycin. Increasing the size of the reservoir by increasing the length of the hydrophilic region of the reservoir lipid or laterally spacing the reservoir lipid results in an improved ionic reservoir. Imposition of a dc bias voltage during the measurement of the impedance spectrum affected the conductivity of the tBLM. The conductivity and specificity of the valinomycin were comparable to those seen in a classical BLM.

Ion channels, such as gramicidin A, selectively facilitate the transport of ions across biological and synthetic membranes. The conductance properties of ion channels are frequently characterized in synthetic bilayer lipid membranes (BLMs). The instability of BLMs has seriously limited the range of applications for these structures, and tethered bilayer lipid membranes (tBLMs) have addressed the problem through tethering many of the membrane components to a solid surface. In the present study, thin gold substrates have been used to tether thiol-and disulfide-terminated membrane components to form a tBLM electrode to provide a reservoir for ions. This study reports on the ion selectivity and apparent permeability of gramicidin channels in such tethered bilayer membranes. The investigations using electrical impedance spectroscopy indicated that the magnitude of ionic conductance varies substantially in reservoirs with different chemical structures. This study addressed the effect of changing ionic concentration, the effect of changing the species in the bulk solution above the membrane, and the influence of the chemical structure of the reservoir tethers. The effect of two-dimensional packing on membrane conductance was also investigated. The present observations suggested that (a) the reservoir region resistivity has a major influence on the overall conductivity of the membrane and in some instances can dominate conduction, (b) the conduction behavior is nonlinear and exhibits saturation with increasing electrolyte concentration, and (c) that ion pairing in the reduced dielectric ( ∼50) reservoir region is the likely basis for the latter effect. The inferred limiting ionic mobilities of alkali chloride species in the membrane reservoir regions were 3-4 orders of magnitude less than in aqueous solution, indicating that the reservoirs resembled hydrated polymer gels.

The use of polar linkers to tether lipid bilayer membranes to a gold substrate results in a hydrophilic layer between the membrane and the gold surface. The tethering of lipid bilayer membranes to gold substrates using tetraethylene glycol chains results in a polar layer between the membrane and the gold surface. This region may sequester ions and can act as a reservoir for ions transported across the tethered lipid membrane. In the present article, we report on the electrical properties of this ionic reservoir. In particular, the Stern model of ionic distribution is used to describe the interfacial capacitance. The model combines a surface adsorption layer (Helmholtz model) and a dynamic diffuse layer of ions (Gouy-Chapman model) to describe the interfacial capacitance. This model is used to interpret data from measurements of the interfacial capacitance obtained over a range of ionic species and concentrations. Four analogues of the sulfur-tetraethylene glycol tethers have been investigated. These studies show the effects of varying the structure of the linker group and of introducing a passivation layer adjacent to the gold. Studies were also made of the influence of spacer molecules included to vary the "in-plane" two-dimensional packing. The effect of applying a dc bias potential between an external reference electrode and the gold surface was also studied. These measurements were carried out using ac impedance spectroscopy on bilayers assembled using the method of Cornell et al. 6 Most data are successfully modeled as a constant Helmholtz capacitance in series with a diffuse region capacitance that depends on ionic concentration. The dependence on ionic concentration has been modeled by the Gouy-Chapman formalism. At low ionic concentrations (<20 mM), the model becomes inadequate. Deviation from the model also occurs at higher concentrations for more tightly packed membranes, in the absence of tethered spacer molecules. According to the model at very low concentrations of electrolyte, the ionic Debye length intrudes into the hydrocarbon region of the bilayer, violating the Gouy-Chapman assumption of a uniform dielectric medium in the diffuse double layer. The Helmholtz capacitance is insensitive to potential and ionic concentration. This is consistent with Helmholtz capacitance being defined by a hard sphere distance of closest approach of the ions to the gold interface over the range of concentrations studied here. The model suggests that the application of a dc potential alters the permittivity of the diffuse region as a result of water and ions being transported into the reservoir. However, the effective relative permittivity in the reservoir region varies only from 27 to 54, suggesting the reservoir has properties more akin to a dense hydrated gel with restricted ionic mobility than to a bulk electrolyte.