THERMOPLASTIC MICROFLUIDIC PCR TECHNOLOGIES FOR NEAR-PATIENT DIAGNOSTICS

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Date

2017

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Abstract

Microfluidic technologies have great potential to help create portable, scalable,

and cost-effective devices for rapid polymerase chain reaction (PCR) diagnostics in

near patient settings. Unfortunately, current PCR diagnostics have not reached

ubiquitous use in such settings because of instrumentation requirements, operational

complexity, and high cost. This dissertation demonstrates a novel platform that can

provide reduced assay time, simple workflow, scalability, and integration in order to

better meet these challenges.

First, a disposable microfluidic chip with integrated Au thin film heating and

sensing elements is described herein. The system employs capillary pumping for

automated loading of sample into the reaction chamber, combined with an integrated

hydrophilic valve for precise self-metering of sample volumes into the device. With

extensive multiphysics modeling and empirical testing we were able to optimize the

system and achieve cycle times of 14 seconds and completed 35 PCR cycles plus

HRMA in a total of 15 minutes, for successful identification of a mutation in the G6PC

gene indicative of von Gierke’s disease.

Next, a scalable sample digitization method that exploits the controlled pinning

of fluid at geometric discontinuities within an array of staggered microfluidic traps is

described. A simple geometric model is developed to predict the impact of device

geometry on sample filling and discretization, and validated experimentally using

fabricated cyclic olefin polymer devices. Finally, a 768-element staggered trap array is

demonstrated, with highly reliable passive loading and discretization achieved within

5 min.

Finally, a technique for reagent integration by pin spotting affords simplified

workflow, and the ability to perform multiplexed PCR. Reagent printing formulations

were optimized for stability and volume consistency during spotting. Paraffin wax was

demonstrated as a protective layer to prevent rehydration and reagent cross

contamination during sample loading. Deposition was accomplished by a custom pin

spotting tool. A staggered trap array device with integrated reagents successfully

amplified and validated a 2-plex assay, showing the potential of the platform for a

multiplexed antibiotic resistance screening panel.

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