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.

The illumination of oxygen-evolving PSII core complexes at very low temperatures in spectral regions not expected to excite P680 leads to charge separation in a majority of centers. The fraction of centers photoconverted as a function of the number of absorbed photons per PSII core is determined by quantification of electrochromic shifts on Pheo(D1). These shifts arise from the formation of metastable plastoquinone anion (Q(A)(-)) configurations. Spectra of concentrated samples identify absorption in the 700-730 nm range. This is well beyond absorption attributable to CP47. Spectra in the 690-730 nm region can be described by the 'trap' CP47 absorption at 689 nm, with dipole strength of approximately 1 chlorophyll a (chl a), partially overlapping a broader feature near 705 nm with a dipole strength of approximately 0.15 chl a. This absorption strength in the 700-730 nm region falls by 40% in the photoconverted configuration. Quantum efficiencies of photoconversion following illumination in the 690-700 nm region are similar to those obtained with green illumination but fall significantly in the 700-730 nm range. Two possible assignments of the long-wavelength absorption are considered. Firstly, as a low intensity component of strongly exciton-coupled reaction center chlorin excitations and secondly as a nominally 'dark' charge-transfer excitation of the 'special pair' P(D1)-P(D2). The opportunities offered by these observations towards the understanding of the nature of P680 and PSII fluorescence are discussed.

A model for the molecular interaction between cholesterol and phospholipid in bilayer membranes is presented. We propose that cholesterol forms associations with phospholipids with stoichiometries of both 1:1 and 1:2. A hydrogen bond between the beta-OH of cholesterol and the glycerol ester oxygen of a phospholipid is suggested as a likely mechanism for tight binding in a 1:1 complex. A second phospholipid molecule is loosely associated with the complex to form domains of 1:2 stoichiometry, which may coexist with pure phospholipid domains. Interfacial boundary phospholipid separates these two domains. Under conditions in which interfacial phospholipid is maximal, the perturbed phospholipid assumes a composition of 20 mol % cholesterol. To account for the phase behavior and surface properties of cholesterol-lipid membranes, we propose a molecular packing model for linear arrays within the cholesterol-rich domains. In this arrangement, two rows of 1:1 complex run antiparallel with loosely associated phospholipid intercalated between them. The loosely associated phospholipid can pack in the nearly hexagonal manner in which pure crystalline phospholipid is known to pack. The model provides maximal van der Waals contact in the hydrocarbon region of the bilayer and can maintain phospholipids as cholesterol's nearest neighbors at all concentrations up to 50 mol % cholesterol. The model is compatible with the diverse experimental observations compiled by many investigators over the past decade.

The interaction of water with the water oxidizing Mn complex of photosystem II has been investigated using electron spin-echo envelope modulation spectroscopy in the presence of H(2)(17)O. The spectra show interaction of the (17)O with the preparation in the S(2) state induced by 200 K illumination. The modulation is observed only in the center of the multiline spectrum. The inferred hyperfine coupling terms are compatible with water (not hydroxyl) oxygen bound to a particular quasi-axial Mn(III) center in a coupled Mn cluster.

Preparation of a minimum PSII core complex from spinach is described, containing four Mn per reaction center (RC) and exhibiting high O2 evolving activity [approximately 4000 micromol of O2 (mg of chl)(-1) x h(-1)]. The complex consists of the CP47 and CP43 chlorophyll binding proteins, the RC D1/D2 pair, the cytochrome b559 subunits, and the Mn-stabilizing psbO (33 kDa) protein, all present in the same stoichiometric amounts found in the parent PSII membranes. Several small subunits are also present. The cyt b559 content is 1.0 per RC in core complexes and PSII membranes. The total chlorophyll content is 32 chl a and <1 chl b per RC, the lowest yet reported for any active PSII preparation. The core complex exhibits the characteristic EPR signals seen in the S2 state of higher plant PSII. A procedure for preparing low-temperature samples of very high optical quality is developed, allowing detailed optical studies in the S1 and S2 states of the system to be made. Optical absorption, CD, and MCD spectra reveal unprecedented detail, including a prominent, well-resolved feature at 683.5 nm (14630 cm(-1)) with a weaker partner at 187 cm(-1) to higher energy. On the basis of band intensity, CD, and MCD arguments, these features are identified as the exciton split components of P680 in an intact, active reaction center special pair. Comparisons are made with solubilized D1/D2/cyt b559 material and cyanobacterial PSII.

We present the first report of highly efficient persistent spectral hole-burning in active (oxygen-evolving) Photosystem II (PSII) preparations. Samples are poised in the S1 state of the Kok cycle, with the primary quinone (QA) either neutral or photoreduced to QA - via a low-temperature pre-illumination. Remarkably efficient hole-burning is observed within the chlorophyll Q y (0,0) absorption envelope in the wavelength range of 676−695 nm. The hole-burning action spectrum of a sample poised in the S1(QA -) state is dominated by a narrow feature (∼40 cm-1) at 684 nm, where hole depths of 30% are attainable. The photoproduct for spectral holes burnt in this region is distributed across the ∼50 cm-1 absorption feature centered at 683.5 nm, independent of the excitation wavelength within this band. Saturated hole-burning experiments indicate weak electron−phonon coupling near 684 nm but stronger coupling for holes burnt near 690 nm. Selective excitation near 690 nm of samples in the S1(QA) state also results in efficient QA - formation. Negligible hole-burning activity is observed at higher energies (<676 nm). Holewidths extrapolated to zero fluence and temperature are 2.0 ± 0.5 GHz near 685 nm for PSII samples in the S1(QA -) state. Holewidths are twice as large and hole-burning quantum efficiencies are up to an order of magnitude greater (approaching 1%) for samples in the S1(QA) state. We ascribe hole-burning near 684 nm to slow (40−210 ps) excitation transfer from a CP43 chlorophyll to the PSII reaction center, and we ascribe hole-burning at ∼690 nm to excitation transfer from a chlorophyll in CP47. The unusually high hole-burning efficiency that we observe is attributed to a mechanism that involves charge separation in the reaction center that follows excitation transfer from these “slow transfer” states in CP43 and CP47. A key result of this work is the observation that selective excitation in the range 685−695 nm leads to efficient charge separation, as indicated by QA - formation. This indicates the presence of (a relatively weak) P680 absorption in a native PSII, extending to low energy and underlying the CP47 chlorophyll trap absorption.

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.

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.