2012 AIChE Annual Meeting

(407e) Investigation of the Impact of Interfacial Adsorption On Droplet PCR Using a Novel Tablet Platform

Author

Tripathi, A., Brown University


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.

 

See more of this Session: Biomolecules at Interfaces

See more of this Group/Topical: Engineering Sciences and Fundamentals