Picoliter DNA sequencing chemistry on an electrowetting‐based digital microfluidic platform

Abstract

The results of investigations into performing DNA sequencing chemistry on a picoliter-scale electrowetting digital microfluidic platform are reported. Pyrosequencing utilizes pyrophosphate produced during nucleotide base addition to initiate a process ending with detection through a chemiluminescence reaction using firefly luciferase. The intensity of light produced during the reaction can be quantified to determine the number of bases added to the DNA strand. The logic-based control and discrete fluid droplets of a digital microfluidic device lend themselves well to the pyrosequencing process. Bead-bound DNA is magnetically held in a single location, and wash or reagent droplets added or split from it to circumvent product dilution. Here we discuss the dispensing, control, and magnetic manipulation of the paramagnetic beads used to hold target DNA. We also demonstrate and characterize the picoliter-scale reaction of luciferase with adenosine triphosphate to represent the detection steps of pyrosequencing and all necessary alterations for working on this scale.

Electrochemiluminescence on digital microfluidics for microRNA analysis

“…This combination (DMF and magnetic particles) has been used for a wide range of applications coupled with detection by electrochemistry , fluorescence (Foudeh et al 2015;Tsaloglou et al 2014), chemiluminescence Ng et al 2015), and bioluminescence (Welch et al 2011). But until now, digital microfluidics has never been combined with the promising technique of electrochemiluminescence (ECL) detection.…”

Building bio-assays with magnetic particles on a digital microfluidic platform

“…In 2011, two different groups (Boles et al [44] and Welch et al [45]) proved the feasibility of using EWOD-DMF to pyrosequence DNA. In both cases, authors implemented a three-enzyme pyrosequencing protocol in which enzymes, deoxyribonucleotide triphosphates (dNTPs) and DNA templates were contained in individual droplets.…”

Sample preparation for many-reactant bioassay on DMFBs using common dilution operation sharing

“…Different from the other LoCs which require prefabricated microchannels and pumps to deliver reactants, a DMFB uses the electrowetting-on-dielectrics (EWOD) effect to control and manipulate fluidics on its surface and offers better programmability and reconfigurability [5]- [7]. Nowadays, there are a number of biochemical applications such as clinical diagnostics, immunoassay, protein crystallization, and DNA sequencing, have already been successfully demonstrated on DMFBs [8]- [11].…”