Fabrication of integrated fluidic systems and methods to perform single-molecule DNA analysis.

摘要

Stretching DNA from its coiled state into a linear form is an important requirement in DNA-protein interaction studies and DNA sequencing. Immobilization of stretched DNA molecules is required to analyze protein interactions and presence of molecules along the DNA strand. The ability to perform DNA immobilization and stretching in microfabricated fluidic systems is a step towards enhancing the applications of DNA studies. Forces derived from hydrodynamic flow and electric field in channels are used to immobilize and stretch DNA molecules.; Electrodes integrated in fluidic channels are used to stretch DNA molecules using electric field. Fabrication technology for integrating electrodes with Si micro- and nano-channels using polymethylmethacrylate bonding was developed. Bonding is performed at low temperature to form integrated channels without leakage and the channels are hydrophilic, allowing introduction of fluid and biomolecules into the channels by capillary action. A novel DNA immobilization technique called protein assisted DNA immobilization (PADI) was developed to immobilize and stretch hundreds of DNA molecules using hydrodynamic flow in a microchannel. The DNA molecules are not overstretched and the immobilization is performed at physiological pH of 8.0 while maintaining continuous hydration of DNA molecules in the channel. Optical mapping and single-molecule transcription were demonstrated in microchannels using the PADI technique. Precise DNA immobilization and stretching across electrode gaps in microchannels were demonstrated using an ac voltage. Using the ac electric field, large numbers of DNA molecules are immobilized on the electrodes without chemical modifications to the DNA or electrode surface. A mechanism based on induced charge electro-osmosis (ICEO) has been developed to induce motion in suspended particles and move them away from the electrodes. The ICEO induces motion of particles in channels without using fluid flow, unlike dc or ac electro-osmosis, or pressure driven flow. The mechanism is applied to control the motion of DNA molecules in channels using low ac voltage, and a DNA velocity of 24 mum/s has been demonstrated at a distance of 500 mum away from the electrodes.