We analyze the consequences of interactions between the pore and the translocating molecule within the framework of a
continuous diffusion model using the Smoluchowski equation with the radiation boundary conditions. We describe the
solute-pore interaction in terms of the potential of mean force. Several of our analytical findings are quite counterintuitive.
Three of the examples to be discussed here are: (i) "Sticking" to the channel slows down translocation (a
particle spends more time in the channel) but increases the flux; (ii) If the potential well modeling the particle-channel
interaction occupies only a part of the channel length, the average translocation time is non-monotonic in the width of
the potential well, first increasing and then decreasing; (iii) At a finite potential bias applied to the channel, the mean
"up-hill" and "downhill" particle translocation times (and their distributions) are identical.
KEYWORDS: Bacteria, Ions, Sensors, Field effect transistors, Signal detection, Polymethylmethacrylate, Solid state electronics, Electron beam lithography, Molecules, Microfluidics
This paper presents a nanowell device that detects the nano-scale electric field fluctuations due to ion cascade in bacteria. Solid-state nano devices allow for the measurement and analysis of fluctuation on the single cell or molecule scale, which can offer orders of magnitude higher sensitivity than microscopic measurements through conventional sensors. We fabricated a nanowell that is a 150nm wide gap in the middle of a titanium line on LiNbO3 substrate. The noise in the electrical current through this gap is measured. When bacteria are infected by bacteriophage, a large amount of ions are released, which yields spatiotemporal fluctuations of electric potential captured by this nanowell. It was demonstrated that this technology can be used to identify bacteria within minutes using the high specificity of phage/bacteria interaction. The perspective of building a biochip with hundreds of nano devices, immobilized phages and microfluidic channels so as to identify a large variety of bacteria is also discussed in this paper.
Fatal injury of bacteria opens transmembrane ion pathways that create temporary ion clouds around the cells. This ion release transiently charges bacteria yielding spatiotemporal fluctuations of the electrical field which show up like a "fatal scream" in thermal noise. The effect has recently been demonstrated with the specific injuries caused by bacteriophage infections (King, et al, in press) and suggested for identification of bacteria with extraordinary speed and selectivity. Calculations indicate that the detection and identification of a single bacterium can be achieved with natural (wild) phages with reasonable efforts within a time window of 10 minutes. However the potential applicability of the agent-triggered ion cascade reaches much beyond that, including other kinds of injuries, such as those induced by antibiotics, ageing, poisoning, etc. Considerations and open questions about the physical aspects of the fluctuations and their detectability are discussed in this talk.
General porin OmpF forms water-filled channels in the outer membrane of E. coli bacteria. When reconstituted into planar bilayer lipid membranes, these channels can be closed (or “gated”) by high electric fields. We discover that: (i) channel gating is sensitive to the type of cations in the membrane-bathing solution according to their position in the Hofmeister series; (ii) channel gates to a “closed” state that is represented by a set of multiple sub-conformations with at least three distinctly different conformations contributing to the closed-state conductance histogram. Taken together with the nearly symmetric response to the applied voltage of changing polarity and the hysteresis phenomena reported previously by others and reproduced here, these findings suggest that the voltage-induced closure of the OmpF channel is a consequence of reversible denaturation of the protein by the high electric field. If so, the voltage-induced gating of bacterial porins can serve as an instructive model to study the physics of protein folding at the single-molecule level.
Statistical analysis of high-resolution current recordings from a single ion channel reconstituted into a planar lipid membrane allows us to study transport of antibiotics at the molecular detail. Working with the general bacterial porin, OmpF, we demonstrate that addition of zwitterionic β-lactam antibiotics to the membrane-bathing solution introduces transient interruptions in the small-ion current through the channel. Time-resolved measurements reveal that one antibiotic molecule blocks one of the monomers in the OmpF trimer for characteristic times from microseconds to hundreds of microseconds. Spectral noise analysis enables us to perform measurements over a wide range of changing parameters. In all cases studied, the residence time of an antibiotic molecule in the channel exceeds the estimated time for free diffusion by orders of magnitude. This demonstrates that, in analogy to substrate-specific channels that evolved to bind specific metabolite molecules, antibiotics have 'evolved' to be channel-specific. The charge distribution of an efficient antibiotic complements the charge distribution at the narrowest part of the bacterial porin. Interaction of these charges creates a zone of attraction inside the channel and compensates the penetrating molecule's entropy loss and desolvation energy. This facilitates antibiotic translocation through the narrowest part of the channel and accounts for higher antibiotic permeability rates.
The reversible binding of sugar to a single maltoporin channel allows us to study time and ensemble variations in the channel functional properties and interpret them using the language of static and dynamic disorder in protein folding. The channel is a trimer that is characterized by two primary parameters: the rate of sugar binding and the ion conductance. Time-resolved binding of maltohexasose molecules shows that whereas dynamic disorder -- the fluctuations in binding rate or in ionic conductance of a single trimer channel with time -- is relatively small, static disorder -- the heterogeneity of reaction rates or conductances among different trimers -- is highly pronounced. This heterogeneity suggests variations in maltoporin folding. The disorder in conductance shows no measurable correlation with the disorder in binding strength; variations in protein folding that are responsible for variations in protein folding that are responsible for variations in ionic conductance do not seem to affect sugar binding. We find 'cooperativity' in static disroder: conductances of monomers in the same trimer are closely similar compared to the range of possible conductances seen over an ensemble of trimers.
Two well known, biologically inspired non-dynamical models of stochastic resonance, the threshold-crossing model and the fluctuating rate model are analyzed in terms of channel information capacity and dissipation of energy necessary for small-signal transduction. Using analogies to spike propagation in neurons we postulate the average output pulse rate as a measure of dissipation. The dissipation increases monotonically with the input noise. We find that for small dissipation both models give an asymptotically linear dependence of the channel information capacity on dissipation. In both models the channel information capacity, as a function of dissipation, has a maximum at input noise amplitude that is different from that in the standard signal-to-noise ration vs. input noise plot. Though a direct comparison is not straightforward, for small signals the threshold model gives appreciably higher density of information per dissipation than the exponential fluctuating rate model. We show that a formal introduction of cooperativity in the rate fluctuating model permits us to imitate the response function of the threshold model and to enhance performance. This finding may have direct relevance to real neural spike generation where, due to a strong positive feedback, the ion channel currents are adding up in a synchronized way.
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