title: Assessment of surfactants for efficient droplet PCR in mineral oil using the pendant drop technique.
abstract: Amplification and detection of nucleic acid sequences within integrated microsystems is routinely conducted using the technique of droplet PCR, wherein the polymerase chain reaction (PCR) is performed in microscale water-in-oil droplets (nanoliter to picoliter volumes). During droplet PCR, interactions at the interface of the droplet tend to dominate. Specifically, adsorption of the polymerase at the droplet interface leads to inefficient amplification. To reduce polymerase adsorption, surfactants such as the silicone-based ABIL EM90 have been commonly used. However, these surfactants have been selected largely through trial and error, and have been only somewhat effective. For example, when using ABIL EM90, 8 times (8 ×) the manufacturer prescribed concentration of polymerase was necessary for amplification. In this report, we use the pendant drop technique to measure adsorption and loss of enzyme at droplet interfaces for various surfactant-oil combinations. Dynamic interfacial tension and surface pressure measurements showed that significant polymerase adsorption occurs when using ABIL EM90. In contrast, much lower polymerase adsorption is observed when using Brij L4, a nonionic surfactant with a C12 tail and an oxyethylene headgroup, which has not yet been reported for droplet PCR. These results correlate strongly with droplet PCR efficiency. Brij L4 enables highly efficient PCR at 2 × polymerase concentration, and still enables effective PCR at 1 × polymerase concentration. Overall, this work introduces a methodology for quantitatively assessing surfactants for use with droplet microreactors, and it demonstrates the practical value of this new approach by identifying a surfactant that can dramatically improve the efficiency of droplet PCR.
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Maryland, College Park, MD 20742, United States.
authors.forename: Kunal R
authors.initials: KR
authors.lastname: Pandit
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Maryland, College Park, MD 20742, United States.
authors.forename: Paul E
authors.initials: PE
authors.lastname: Rueger
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Maryland, College Park, MD 20742, United States.
authors.forename: Richard V
authors.initials: RV
authors.lastname: Calabrese
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Maryland, College Park, MD 20742, United States; Fischell Department of Bioengineering, University of Maryland, College Park, MD 20742, United States.
authors.forename: Srinivasa R
authors.initials: SR
authors.lastname: Raghavan
authors.affiliation: Fischell Department of Bioengineering, University of Maryland, College Park, MD 20742, United States. Electronic address: ianwhite@umd.edu.
authors.forename: Ian M
authors.initials: IM
authors.lastname: White
mesh_terms: D000327:Adsorption; D008899:Mineral Oil / Q000737:chemistry*; D015394:Molecular Structure; D010316:Particle Size; D016133:Polymerase Chain Reaction; D013499:Surface Properties; D013501:Surface-Active Agents / Q000737:chemistry*
chemical_list: D013501:Surface-Active Agents; D008899:Mineral Oil
keywords: Brij; Droplet PCR; Pendant drop