280941 Investigation of the Impact of Interfacial Adsorption On Droplet PCR Using a Novel Tablet Platform

Wednesday, October 31, 2012: 9:55 AM
413 (Convention Center )
Stephanie Angione, Center for Biomedical Engineering, School of Engineering, Brown University, Providence, RI, Anuj Chauhan, Dept. of Chemical Engineering, University of Florida, Gainesville, FL and Anubhav Tripathi, School of Engineering, Brown University, Providence, RI

We present a novel droplet based tablet platform for temporal polymerase chain reaction in microliter droplets. We utilized this platform to successfully amplify DNA at concentrations as low as 2.0 copies/ul. We further utilized the platform to investigate the effect of performing PCR in small volumes on reaction performance by specifically examining adsorption of reagents at the oil/water interface. We determined that adsorption of Taq polymerase at the biphasic interface reduces yield and impairs reaction performance at standard concentrations. We investigated the effect of polymerase adsorption at the interface by examining various polymerase concentrations for three different droplet sizes, 1ul, 2ul and 4ul compared to full volume batch PCR. Our data shows the well known phenomenon of protein adsorption at the water/oil interface. The only volume which displayed amplification for all the polymerase concentrations examined, 0.01-0.05 units/ul was the full volume 50ul PCR controls. The effect of interfacial adsorption was pronounced for all the droplets investigated although significant differences between the droplet volumes were not observed. The reason not much difference is observed between 1ul, 2ul and 4ul is that the relevant controlling parameter is the surface to volume ratio, which is A/V ~ 3/R for a spherical interface. We examined the data points by developing a mass balance equation to calculate , the interfacial enzyme concentration. To evaluate monolayer formation at the interface the thermodynamic variable,  for polymerase was calculated to be 0.0046 units/mm2, or 3.65x108  molecules/mm2. Here, we have used our experimental data to evaluate the bulk and loss concentrations  for the 1ul, 2ul and 4ul droplets. The results indicate that for droplet applications involving enzymatic reactions like PCR, higher concentrations of enzyme are required. The typical full volume PCR reaction utilizes .02-.025 units/ul of polymerase, whereas our results indicate that for a 1ul droplet, to get maximum amplification .04-.045 units/ul are necessary. We compared calculations for monolayer formation of polymerase using the radius of gyration of native polymerase with that required by our data. We then determined that the enzyme is partially unfolded at the oil/water interface using a random coil approximation for further investigation of monolayer coverage. Thus, enzyme adsorption and unfolding at the interface leads to a lack of activity and lower PCR yield. Thus, microdroplet PCR reactions require additional polymerase to achieve sufficient amplification and we project that for applications utilizing nanodroplets or picodroplets like digital applications, even greater concentrations of polymerase are required to achieve desired results.


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See more of this Session: Biomolecules at Interfaces
See more of this Group/Topical: Engineering Sciences and Fundamentals