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1. B. Weigl et al. Lab Chip, 2008. 2. R. Safavieh and D. Juncker. Lab Chip, 2013. 3. K. E. Mach et al. Trends in Pharmacological Sciences, 2011.
CAPILLARIC CIRCUITS FOR FAST AND SENSITIVE BACTERIA DETECTION Ayokunle Olanrewaju, Andy Ng, and David Juncker
Micro-‐ and Nano-‐bioengineering Lab, Biomedical Engineering Department, McGill University, Montreal, Canada, [email protected]
INTRODUCTION
There is a strong need for fast and sensiTve bacteria detecTon in rapid response seUngs. ConvenTonal bacteria detecTon methods are slow, require trained operators, and oWen need external equipment. Although microfluidic approaches to bacteria detecTon are acTvely explored, most cannot be readily applied in rapid response seAngs because they need large and expensive equipment for fluid manipulaJon [1]. Capillary-‐driven microfluidics enable liquid delivery using only surface tension forces and without external pumps and valves. Our lab recently developed “capillaric circuits” that deliver mulTple liquids at pre-‐programmed Tmes using only capillary forces [2]. Here we apply these capillaric circuits for fast and sensiJve detecJon of E.coli O157:H7. To do this, we incorporate a bead-‐based pre-‐concentraTon step to isolate small bacteria concentraTons from large volumes and perform a pre-‐programmed sandwich immunoassay with fluorescent readout.
ASSAY OVERVIEW
PRE-‐PROGRAMMED LIQUID DELIVERY RESULTS
CONCLUSIONS Taken together, our results demonstrate fast an sensiJve capture of E.coli O157:H7 using capillaric circuits. We implemented a 15-‐min bead-‐based bacteria pre-‐concentraTon followed by a 1-‐min on-‐chip sandwich immunoassay to detect E.coli O157:H7. A proof of principle assay with 107 cfu/mL of bacteria shows a clear difference between negaTve and posiTve controls. Furthermore, preliminary diluTon experiments suggest that we can detect concentraTons approaching 104 cfu/mL –– the clinical detecTon limit for E.coli O157:H7 in urinary tract infecTons [3]. Ongoing work in our lab is aimed at improving the assay sensiTvity with a chemical signal amplificaTon step. We are also invesTgaTng simpler and less expensive detecTon strategies. This work is a stepping stone towards fast and sensiTve in rapid response seUngs.
1. Add beads to bacteria
suspension
2. Beads bind to bacteria and sediment
3. Add bead & bacteria suspension to microfluidic chip
• 24-‐µm PMMA beads coated with anT-‐E.coli O157:H7 anTbodies. • 15-‐min bead-‐based pre-‐concentraTon followed by 1-‐min on-‐chip sandwich assay. • DetecTon with fluorescently labeled anT-‐E.coli O157:H7 anTbodies • Timing of detecTon anTbody and wash buffer release is automated by capillaric circuit NegaTve control: 1x PBS
PosiTve control: 107 cfu/mL E.coli O157:H7
Bacteria capture diluTon curve Close up of reacTon chamber
BACTERIA CAPTURE ASSAY RESULTS
CAPILLARIC CIRCUIT DESIGN
Devices were fabricated by standard photolithography and replicated in polydimethylsiloxane (PDMS) by soW lithography. To operate the capillaric circuit, a user pre-‐loads wash buffer (0.25 µL) and detecTon anTbody (0.1 µL) in the appropriate channels. Trigger valves stop liquids for > 30 minutes due to an abrupt geometry and surface chemistry change so that the exact Jming of reagent loading is not crucial. When ready for the assay, the user adds 1 µL of sample to the sample channel. The capillary pump wicks the sample and traps beads in the reacTon chamber. When all the sample has been wicked, the retenJon burst valves autonomously deliver reagents in a pre-‐programmed manner according to capillary pressure differences encoded in their channel widths. The wider retenTon burst valve (containing anTbody) drains first followed by the narrower retenTon burst valve (containing wash buffer). AWerwards, assay results are visualized with a fluorescence microscope.
Pre-‐programmed liquid delivery in capillaric circuit was validated with food dye soluTons
DEVICE FABRICATION AND OPERATION