Electrokinetic delivery of single fluorescent biomolecules in fluidic nanochannels

We describe the fabrication of sub-100-nanometer-sized channels in a fused silica lab-on-a-chip device and experiments that demonstrate detection of single fluorescently labeled proteins in buffer solution within the device with high signal and low background. The fluorescent biomolecules are transported along the length of the nanochannels by electrophoresis and/or electro-osmosis until they pass into a two-focus laser irradiation zone. Pulse-interleaved excitation and time-resolved single-photon detection with maximum-likelihood analysis enables the location of the biomolecule to be determined. Diffusional transport of the molecules is found to be slowed within the nanochannel, and this facilitates fluidic trapping and/or prolonged measurements on individual biomolecules. Our goal is to actively control the fluidic transport to achieve rapid delivery of each new biomolecule to the sensing zone, following the completion of measurements, or the photobleaching of the prior molecule. We have used computer simulations that include photophysical effects such as triplet crossing and photobleaching of the labels to design control algorithms, which are being implemented in a custom field-programmable-gate-array circuit for the active fluidic control.

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