Extremely rapid and fully reprogrammable total PCR assays using wire-guided droplet microfluidics

Roberto Reyes1 , Dustin Harshman1, Tu-San Park2 , David J. You2, Jeong-Yeol Yoon (PI) 12 Departments of Biomedical Engineering, Department of Agricultural and Biosystems Engineering, University of Arizona
(HTTP://BIOSENSORS.ABE.ARIZONA.EDU)

Introduction
There have been many attempts to incorporate the whole processes of polymerase chain reaction (PCR) assays into microchannels. However, the problem is that the user cannot change the assay protocol easily once the sample is introduced. In addition, the heat transfer throughout the microchannel device makes the heat isolation (required for thermocycling) very difficult, resulting in poor assay results. The use of droplet microfluidics, including electrowetting or magnetofluidics, enables protocol change and offers better heat isolation. Wireguided droplet microfluidics offers a simpleR method of droplet manipulations.

Methods (cont)

Results
Centrifugation of the sample resulted in mean increase in the concentration of E. coli within the sample droplet by more than three-fold. Wire-guided droplet thermocycling successfully completed 30 cycles of 1500-bp Peptidase D amplification in 10 min and 30 cycles of 160 bp amplification in 6 min 50 s. CFO simulation showed that the 30 cycle 160 bp amplifiation could be done in 2 min 30 s if a 1 uL droplet was used. The results of these sequentially executed processes were analyzed using gel electrophoresis, in which the droplet centriguation greatly improved the positive and intensity over the non-centrigued sample.

WIRE-GUIDED DROPLET MICROFLUIDCS

Utilizing programmable and motorized movement of the wire, small droplets are manipulated, including merging, splitting, movement along a programmed path, and rapid mixing on a flat, preferably superhydrophobic surface.

WIRE-GUIDED DROPLET DNA EXTRACTION

DNA extraction was performed on the same superhydrophobic surfaces as centrifugation using the concentrated sample, in which the needle tip was utilized to extract the precipitated DNA.

[Right] Concentration of E. coli in 10 ul sample before and after droplet centrifugation, revealing a 3.06 fold increase in mean concentration after 3 min. [Bottom] Gel electrophoresis results for sample undergoing droplet centrifugation, DNA extraction, amplification, and PCR thermocycling (30 cycles for 10 min, 1500 bp Peptidase D).

Methods

WIRE-GUIDED DROPLET DNA EXTRACTION

A) Add lysis solution to sample B) Mix sample through vibration C) Extract lysis solution and heat at 80 C for 5 min D) Redeposit lysd sample E) Precipitate DNA with IPA F) Air dry for 1 min using vibration G) Wash with EtOH H) Air dry for 1 min I) Resuspend buffer J) Mix with PCR reagents and proceeed to thermocycling

Figure 1. Complete Wire-guided Droplet Thermocycler Apparatus [Right] Gel electrophoresis results for sample undergoing thermocycling for varying lengths of time ranging from longest (9 min 30 s, top sample) to shortest (4 min 12 s, bottom sample) (160 bp GAPDH sequence)

DROPLET CENTRIFUGATION
A-H) Concentration of sample through dorplet centrifugation. Droplet spins at 2300 RPM and syringe extracts dilute sample from center.

WIRE-GUIDED DROPLET DNA EXTRACTION

Droplet PCR thermocycling executes 30 cycles in 6 min 50 s for a 10 uL droplet through forced convective heat transfer under silicone oil submission. Funding provided by Animal, Plan & Fisheries Quarantine & Inspection Agency (QIA), Republic of Korea