Microfluidic device for the electrokinetic manipulation of single molecules

We are developing a microfluidic device for three-dimensional electrokinetic manipulation of single fluorescent molecules in solution. The device consists of electrode pairs deposited onto glass cover slips via UV microlithography and ionic sputtering. By positioning two such electrode pairs in a tetrahedral configuration separated by 100 microns and applying appropriate digitally-controlled voltages to each, the apparatus generates an electric field of selected directionality in the central bounded region. Proof of concept is demonstrated by controlling the motion of micron-size latex beads, visualized with an EM-CCD camera. By use of a double Mach-Zehnder interferometer configuration, 40 fs Ti:Sapphire laser pulses (repetition rate 76 MHz) are split into four temporally interleaved pulses (effective rate 304 MHz), which are then focused to the vertices of a tetrahedron (approximately one micron per side) within the central electrode region to generate two-photon-excited fluorescence from single molecules. The time stamp data from this four-focus probe, collected with a custom fast-timing single-photon avalanche diode, enables characterization of particle motion through fluorescence cross-correlation spectroscopy.