2.1. Droplet-based bisulfite conversion

In this work, we employed our previously-developed single-tube bisulfite conversion method, methylation on beads (MOB) (Bailey et al. 2010; Keeley et al. 2013) and adapted it into a microfluidic droplet format. The bisulfite conversion process in MOB comprises four key sequential steps with intermediate washing steps (Fig. 2a). First, the DNA sample is introduced to ammonium bisulfite, resulting in sulfonation and hydrolytic deamination of unmethylated cytosine residues. By contrast, methylation protects the cytosine residue from sulfonation and prevents further chemical modification in subsequent steps. Second, as the unique feature in MOB, silica-coated superparamagnetic beads (SSBs) are employed as a solid phase substrate for tight DNA adsorption, thus allowing DNA capture and transport and facile exchange of buffers across downstream reaction steps. Third, following a brief wash, sulfonated DNA is exposed to sodium hydroxide, during which the uracil sulfonate residues undergo alkali desulfonation to yield fully converted uracil residues. Lastly, the processed DNA is purified via successive washing steps and separated from the SSBs, readying recovered DNA for downstream methylation assessment techniques, such as methylation-specific PCR (MSP).

a The process for bisulfite conversion is comprised of four main chemical reactions with three intermediate washing steps to prevent carryover of reagents. b The chemistry is detailed for each step. Deamination and sulphonation convert the unmethylated cytosine into uracil-sulphonate (1). The converted DNA is then bound onto the beads for transportation(2). The uracil-sulfonate is desulfonated resulting in the genomic base uracil (3). The final step is to reverse the binding process to recover the DNA from the beads (4). c Photograph of aqueous reagents loaded onto a single lane of droplet chip. Each reagent is contained in a round well that holds the droplet within it. The wells are connected either by a single open channel (i.e., between I and II) to merge the droplets or a narrow sieve (II through VII) to separate the beads from the droplet by surface tension

Critically, the use of SSBs enables the MOB workflow in the microfluidic droplet format because the beads facilitate a robust transport medium for the surface-bound nucleic acids through a lane of isolated droplets, each of which contains a required MOB reagent. An image of the chip with three parallel lanes is shown in Fig. 2b. Each lane consists of one wide and five circular reservoir chambers, each containing aqueous MOB reagent droplets that are isolated within topographic walls. A hydrophobic coating on the bottom surface of the device and mineral oil pre-loaded in each lane of chip to ensure that the aqueous reagents are maintained in droplet form.

To facilitate a droplet-based MOB workflow, we have designed the device to allow facile manipulation of SSBs to merge droplets, separate from droplets, and disperse within droplets (Fig. 3a). For example, in the chamber containing the bisulfite reagent droplet and the binding buffer (i.e., Fig. 3b, ​,ii and ​andii),ii), the first droplet can be transported across the wide chamber by the SSB cluster due to the high surface tension, resulting in merging of both droplets. In chambers connected by narrow sieve structures (i.e., Fig. 3b, ​,iiii through vii), SSBs can be effectively separated from reagent droplets and transported into downstream reagent droplets. Such separation is possible because the narrow sieve creates a surface energy barrier to keep reagent droplets in the chamber, while the SSB cluster can escape due to the significantly higher magnetic force relative to capillary force at the interface. This design provides an efficient and robust means of sequential buffer exchange necessary for bisulfite conversion. Finally, SSBs within droplets can readily disperse by simply removing the magnet; this dispersion thus allows mixing with-in droplets.

a The steps required for bisulfite conversion are demonstrated in their corresponding reagent droplets. As the beads and DNA are transported across the chip surface, the DNA is exposed to each subsequent reagent of the BSC process. b There are three main modes of transportation for the beads. Droplet merging is done across an open channel, where the lower surface energy allows the droplet to be transported with the bead cluster. The separation process is achieved by moving the beads across a sieve where the droplet is retained while the SSB cluster can move. Dispersion is performed by removing the magnetic force and allowing the magnetic beads to resuspend via Brownian motion

On-chip sample processing begins by first mixing 2 μl of extracted genomic DNA (gDNA) with 13 μl of the Lightning Conversion reagent (Zymo) and 5 μl of SSB (Promega Magnesil KF MD) to form a droplet that is then loaded into the first reservoir (Fig. 3b, step i). The sample is subsequently heated to 95 °C for 8 min in order to denature the sample into single-stranded DNA (ssDNA). The temperature is then lowered to 54 °C for 1 h to allow the bisulfite to complete deamination of unmethylated cytosine and sulfonation to yield uracil-sulfonate. After this process, the droplet is mixed together with a 60 μl droplet of M-binding buffer (step ii) and briefly incubated to allow the DNA to adsorb onto the silica surface of the beads. The SSB are then washed with 40 μl ethanol (step iii) to eliminate any remaining bisulfite reagent before being transported into a 20 μl M-desulphonation buffer droplet (step iv). This alkali solution desulfonates the uracil-sulfonate into stable uracil bases. The SSB are washed twice in 40 μl M-wash buffer droplets (steps v and vii) in order to remove any residual sodium hydroxide from the solution. Finally, the remaining DNA is eluted from the SSB into Tris-EDTA buffer (step vii) and the SSB are decanted into the previous well to allow the droplet containing purified, bisulfite-converted DNA to be retrieved from the chip for downstream analysis (step viii).