Liquid-on-liquid mixing for slide-based biological assays
Yeh, Richard Cheng-I
Many parallel biochemical assays rely on thin aqueous films to spread a reactant solution over a wide area decorated with multiple distinct substrates. In this asymmetric, microfluidic geometry, diffusion limits the transport of reactants to substrates. Chemical equilibrium, a requirement for reproducibility of results, can take days to achieve. The liquid-on-liquid mixing (LOLM) method overcomes the diffusion barrier by layering an immiscible spectator fluid, such as mineral oil, on the thin film. Stirring the spectator fluid transmits shear at the liquid-liquid interface into the thin film. The mixing accelerates the march towards equilibrium. This technique increases the speed and sensitivity of immunofluorescence staining of Drosophila larval polytene chromosomes by a factor of 100 in time and concentration, when compared to standard coverslip techniques. Flow visualization experiments reveal the fluid motions in the thin aqueous layer. Using time-lapse video photography to monitor the evolution of a drop of colloidal dye in the thin film, I estimate the time needed to achieve good mixing at various stir rates. The major aim of this technique is improving the hybridization step in DNA microarrays. I printed microarrays and subjected them to hybridizations with varying stir rates, durations, and target DNA concentrations. My data suggest that the mixing produces at best a modest improvement in efficiency, uniformity, sensitivity, and specificity when compared to microarrays incubated with the standard coverslip method.
This work was supported by the Cornell University Center for Biotechnology, a New York State Center for Advanced Technology, supported by the New York State Office for Science, Technology, and Academic Research and industrial sponsors. This material is based upon work supported in part by the STC Program of the National Science Foundation under Agreement No. ECS-9876771, through the Cornell Nanobiotechnology Center (NBTC). This work made use of the computing facility of the Cornell Center for Materials Research (CCMR), supported through the National Science Foundation Materials Research Science and Engineering Centers (MRSEC) program (DMR-0079992). This work made use of a site license for National Instruments LabVIEW software purchased by the Cornell Laboratory of Atomic and Solid-State Physics. I thank Cornell University and in particular the Department of Physics for awarding me a teaching assistantship in the spring of 2004 to allow me to conduct this research.
microfluidics; mixing; immunofluorescence staining; microarray hybridization; immiscible; liquid-on-liquid; LOLM; flow visualization; slide
Previously Published As
J Biochem Biophys Methods. 2005 Jul 29;64(1):59-68.
dissertation or thesis