Nanofluidic Separation Devices and Biomolecular Reactions for the Microbial Description

Lab-on-a-chip: biomolecular reactions, free-solution electrophoresis and quantificationat the single molecule resolution

Stéphane C. Corgié1, Joshua D. Cross2, Samuel Stavis2, Harold Craighead2 and Larry P. Walker1

(1) Biological & Environmental Engineering, Riley Robb Hall, CornellUniversity

(2) Applied & Engineering Physics, Clark Hall, CornellUniversity

Abstract

Currently available gel-based methods provide limited quantitative or qualitative description of microbial community structure. These methods are time consuming,may introduce artifacts that prevent an accurate picture of these communities and require high amounts of nucleic acids prior to detection and quantification. This program aims to designand apply the components of an integrated microfluidic device applied to DNA and RNA analysis of bacterial communities. More specifically, this research program is focused on the following goals:

• improving the sensitivity of DNA detection and decreasing DNA amplification rate for improving the qualitative and quantitative accuracy of microbial population fingerprinting by single molecule detection.
• adapting already existing methods to a micro and nanometric scale and developing applied microfluidic separation devices for bacterial community fingerprinting.

A collection of 25 bacterial species has been constituted. Eubacterial universal primers, targeting the V2-V8 regions of 16S rDNA, were designed for PCR or non-PCR reactions to amplify target genes differing in size or sequence. Non-PCR amplification consists in a hybridization-elongation-ligation targeting the 5’-3’ strand of genomic DNA reaction by a Taq polymerase and a thermostable ligase. Primers were fluorescently labeled with AlexaFuor488 or Texas Red.

Fluidic channels with submicrometer dimensions were used to quantify PCR amplification products of 16S PCR fragment (E. coli, 325 bp) with single molecule resolution.Briefly, 488 nm and 568 nm laser beams were overlapped using a dichroic mirror, directed towards a dual band dichroic, and used to underfill a high numerical aperture objective. The objective focused the beams onto the submicrometer fluidic channel and collected emitted fluorescence from analytes crossing the focal volume.PCR products were analyzed by coincidence detection after 2 and up to 40 amplification cycles, without post-amplification purification or size screening.

Fluidic channels with micrometric dimensions were used for DNA size screening by free solution electrophoresis. Double stranded and single stranded DNA was separated in this 4cm length channels. We have been able to separate fragments ranging from 17 bases to 700 bp. In our devices, the longest is the DNA, the fastest is the elution time. For example, a 325 bp PCR product was eluted after 30s whereas the primers were eluted after 110s. Complementary experiments tend to confirm that the length-dependant charge of the molecules, instead of the MW in matrix electrophoresis, and the coupled fluorophore could be the main parameters affecting the separation.