Analysis of a commercial surfactant for digital PCR
Anett Reichert, Daniel Kraus, Theresa Heckmann, Karina Weber, Thomas Henkel
Leibniz-Institute of Photonic Technology, Albert-Einstein-Str. 9, 07745 Jena, Germany
Digital droplet-based assays offer promising opportunities for the absolute quantitation of low concentration analytic species. During the last decade digital-PCR (dPCR) became one of the most prominent assays for this class of analytical methods. For performing the assay, the sample volume is split into multiple droplets in such a way that each droplet contains either one or none of the target DNA molecules. Due to the small droplet volume, the PCR reaction runs very efficiently even from a single molecule. During amplification, a fluorescent dye is formed or activated. The positive droplets become fluorescent. Absolute quantitation of the number of target molecules is simplified to the count of fluorescence active droplets in the generated droplet collection. Not regarding the simplicity of the approach, its technical implementation is challenged by stabilizing the droplets collected over the complete assay avoiding unwanted droplet coalescence or crosstalk between the droplet ingredients. This has been solved by utilizing perfluorinated mineral oils as the carrier oil in combination with advanced perfluorinated surfactants, which stabilize the emulsion and avoid crosstalk and DNA exchange between the individual droplets. In this application note we are investigating the usability of the commercially available surfactant dSurf for an exemplary digital PCR-assay.
We kindly thank of Photonic Leibniz Institute Technology for this collaboration, and for sharing the results obtained with their system.
For information about Photonic Leibniz Institute Technology : https://www.leibniz-ipht.de/
Materials and Methods
Microfluidic workstation utilized for droplet generation with quality monitoring by video microscopy.
Droplets were generated at a working pressure of 240 mbar for the dSurf and 140 mbar for the PCR-Mix. The chip was connected with PEEK 1/32” tubing OD x .010” and 2x sleeves 1/16” OD x .033” ID x 1.6”, tubing length: 200 mm. Generated droplets were collected into a 0.2 ml PCR vial. Amplification was performed in a conventional thermocycler with the settings displayed on the right. For readout, the amplified droplet collection was transferred into a cell counting capillary slit chamber for subsequent brightfield and fluorescence image acquisition
Droplet generation for dPCR sample has been performed with the parameter settings at the Fluigent-MFCSTM-EZ” pressure control system as given in the Materials and Methods section. For the investigated surfactant droplet generation characteristics in terms of droplet generation regime, droplet size and frequency were observed as given in Figure 2. The average droplet size was 70 µm with a volume of 180 pl.
Figure 2: Droplet generating process. The droplet generator operating in transition mode between dripping and jetting. No significant differences in the mode of operation as in the droplet sizes and size uniformity can be recognized. The middle circle above the droplet channel has a diameter of 50µm
Figure 4: Evaluation procedure for the dPCR. Starting point are brightfield and fluorescence images of the generated droplets. The processing is done by a self developed software to detect and evaluate the intensity of the droplets. The parameters for detecting the droplet contours are defined in advance. Only droplets of a valid size are included in the result.
The experiments have shown that the dSurf surfactant is suitable for scientific as well as routine dPCR applications. The generated droplets were homogeneous in shape and size. Superior droplet stability of the dSurf surfactant system was observed during the amplification process. A few droplets have dissipated during the experiments, but this can be neglected. The reproducibility of the experiments was also confirmed. Droplet generation with identical parameters leads to identical droplet size and quality. Summarily, dSurf can be employed as a surfactant composition for digital droplet-based assays.
1. Pohl, G. and I.-M. Shih, Principle and applications of digital PCR. Expert review of molecular diagnostics, 2004. 4(1): p. 41-47.
2. Huggett, J.F., S. Cowen, and C.A. Foy, Considerations for digital PCR as an accurate molecular diagnostic tool. Clinical chemistry, 2015. 61(1): p. 79-88.
3. Quan, P.-L., M. Sauzade, and E. Brouzes, dPCR: a technology review. Sensors, 2018. 18(4): p. 1271.