Rapid, single-molecule assays in nano/micro-fluidic chips with arrays of closely spaced parallel channels fabricated by femtosecond laser machining.

Brian Canfield,Lloyd Davis,Jason King,William Hofmeister,William Robinson

doi:10.3390/s140815400

Abstract

Cost-effective pharmaceutical drug discovery depends on increasing assay throughput while reducing reagent needs. To this end, we are developing an ultrasensitive, fluorescence-based platform that incorporates a nano/micro-fluidic chip with an array of closely spaced channels for parallelized optical readout of single-molecule assays. Here we describe the use of direct femtosecond laser machining to fabricate several hundred closely spaced channels on the surfaces of fused silica substrates. The channels are sealed by bonding to a microscope cover slip spin-coated with a thin film of poly(dimethylsiloxane). Single-molecule detection experiments are conducted using a custom-built, wide-field microscope. The array of channels is epi-illuminated by a line-generating red diode laser, resulting in a line focus just a few microns thick across a 500 micron field of view. A dilute aqueous solution of fluorescently labeled biomolecules is loaded into the device and fluorescence is detected with an electron-multiplying CCD camera, allowing acquisition rates up to 7 kHz for each microchannel. Matched digital filtering based on experimental parameters is used to perform an initial, rapid assessment of detected fluorescence. More detailed analysis is obtained through fluorescence correlation spectroscopy. Simulated fluorescence data is shown to agree well with experimental values.

Highlights

The necessity of rapidly screening the interactions of biomolecules of interest with extensive libraries of small molecules as part of the pharmaceutical drug discovery pipeline has grown tremendously
Protein analysis methods abound with parallel microfluidic schemes
It is interesting to note that each column demonstrates a slightly different flow velocity, which is directly related to the width of the correlation peak, because of the effects of diffusion that were included in the simulation

Summary

Introduction

The necessity of rapidly screening the interactions of biomolecules of interest with extensive libraries of small molecules as part of the pharmaceutical drug discovery pipeline has grown tremendously. The development of a highly parallel fluidic platform, whether microscale or nanoscale, that integrates the ability to probe all channels simultaneously with single-molecule sensitivity remains an active research field. To this end, a system was sought to link two titer plates through a polymer fluidic interconnect comprised of highly parallel microchannels [13], forming the background to this paper. For microchannel widths less than 5 μm, either the polymer material stuck within the molds, destroying channel separations, or the microchannels became highly deformed during bonding [13] Because of these difficulties, polymer microfluidic devices with the desired dimensions were unavailable. A simulated fluorescence data set is generated and analyzed according to the same conditions and found to agree substantially with experiment

Microfluidic Device Fabrication

Detecting Fluorescent Molecules in a Microfluidic Device

Findings

Conclusions