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A single-molecule manipulation system is described that has used the nanocavity enclosed by the [alpha]-hemolysin ([alpha]HL) protein pore to noncovalently encapsulate a guest molecule. The guest-nanocavity supramolecular system has helped in understanding single-molecule folding and unfolding kinetics.

The most widely used correction of fluorescence intensities for inner filter effects in conventional (900) fluorimeters fails at high absorbance values. We have critically examined this failure, which is caused by the difference between the geometrical parameters (GPs) of the excitation and emission beams in the typical instrument (focused beams) and in the theoretical picture on which the correction is based (collimated beams). We provide two types of experimental measurement of GPs and show that their substitution in the correction equations leads to significant improvements in the linear range of corrected fluorescence. We also demonstrate that mathematical optimizations give greater improvements and that the optimizations yield GPs consistent with experimental measurements. For solutions exhibiting primary inner filter effect only, we have extended the range of linearity of corrected fluorescence to a! (absorbance per cm) up to 5.3; for systems with both primary and secondary inner filter effects we have achieved linearity for aex + aem = 6.7. In all cases linear fits have slopes which agree well with the dilute limit. Different series of one- and two-solute solutions were used to demonstrate effectiveness of our correction methods. We also provide a rationale for the unexpected independence of GPs on excitation and emission bandwidths. [ABSTRACT FROM AUTHOR]

The most widely used correction of fluorescence intensities for inner filter effects in conventional (900) fluorimeters fails at high absorbance values. We have critically examined this failure, which is caused by the difference between the geometrical parameters (GPs) of the excitation and emission beams in the typical instrument (focused beams) and in the theoretical picture on which the correction is based (collimated beams). We provide two types of experimental measurement of GPs and show that their substitution in the correction equations leads to significant improvements in the linear range of corrected fluorescence. We also demonstrate that mathematical optimizations give greater improvements and that the optimizations yield GPs consistent with experimental measurements. For solutions exhibiting primary inner filter effect only, we have extended the range of linearity of corrected fluorescence to aex (absorbance per cm) up to 5.3; for systems with both primary and secondary inner filter effects we have achieved linearity for aex + aem = 6.7. In all cases linear fits have slopes which agree well with the dilute limit. Different series of one and two-solute solutions were used to demonstrate effectiveness of our correction methods. We also provide a rationale for the unexpected independence of GPs on excitation and emission bandwidths. [ABSTRACT FROM AUTHOR]

Engineered protein channels have many potential applications in biosensing at the single-molecule level. A future generation of biosensor could be an array of target-specific ion channels, where each protein pore acts as a sensor element. An important step toward this goal is to create a portable, durable, single-protein channel-integrated chip device. Here we report a versatile, modular chip that contains a single-ion channel for single-molecular bio-sensing. The core of the device is a long-lived lipid membrane that has been sandwiched between two air-insulated agarose layers which gel in situ. A single-protein pore embedded in the membrane serves as the sensor element. The modular device is highly portable, allowing a single-ion channel to continuously function following detachment of the chip from the instrument and independent transportation of the device. The chip also exhibits high durability, which is evidenced from long-duration continuous observation of single-channel dynamics. Once engineered protein pores are installed, the chip becomes a robust stochastic sensor for real-time targeting such as detection of the second messenger IP3. This pluggable biochip could be incorporated with many applicable devices, such as a microfluidic system, and be made into a microarray for both biomedical detection and membrane protein research. [ABSTRACT FROM AUTHOR]