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Microfluidic systems for identification of antimicrobial-resistant pathogens

Microfluidic systems for identification of antimicrobial-resistant pathogens
Microfluidic systems for identification of antimicrobial-resistant pathogens
Antimicrobial resistance (AMR) poses a critical, yet historically understated threat to global public health. Recent mutations in the enterobacteriaceae genome that confer resistance to multiple antibiotics, have made some virtually untreatable.

Currently, antibiotics are prescribed largely on guesswork, with little or no prior evidence as to their efficacy. This not only accelerates the spread of AMR, but endangers patient's lives in the process. To counter this, new tools are required which can rapidly identify a pathogen's susceptibility to an antibiotic prior to administration. Traditional procedures are labour-intensive, taking several days - with sample preparation and cell-culture being the primary bottlenecks.

A novel, proof-of concept system was developed to rapidly determine antibiotic resistance in pathogens using nucleic acid analysis. The system comprised of a plastic microfluidic cartridge, control software, optical reader and *control hardware*.

E. coli were captured, concentrated and lysed within the device; DNA could be amplified on-chip using rapid, isothermal DNA amplification (Recombinase Polymerase Amplification). The assay identified samples containing the CTX-M resistance gene; the limit of detection was 500 copies (in 50l), as measured by the reader's integrated fluorescence microscope.

Each component was validated prior to integration into the final system. Concentrations of E. coli equivalent to a urinary-tract-infection (100,000 Colony Forming Units/ mL) were captured with up to 98% efficiency from 1ml suspensions onto functionalised, 0.5m magnetic, ion-exchange beads. These were isolated on-chip by a NdFeB magnet, providing up to 96% bead-capture and a 100x volume reduction. An oil compartmentalised the aqueous samples within the chip, while thermal control was achieved by coupling a resistive heater to an IR camera. Fluid routing was managed by bespoke, in-line, solenoid-controlled valves, which could operate at 5-bar and for over 4000 cycles without degrading. Bacteria were thermally or chemically lysed post-concentration, and an on-chip metering chamber aliquoted fixed-volumes of buffer (1% uncertainty for 400nl). DNA from lysed bacteria was amplified on-chip with a limit of detection of 250 E. coli per sample in the final assay.

Both sample-preparation and DNA amplification performed well on the prototype system. However, inefficient bacterial elution on-chip post-capture prevented the steps being run sequentially, as insufficient bacteria were released to the lysis region. Future development would fully integrate sample transfer and automate the fluid actuation so as to enable complete sample-in, answer-out capability.
University of Southampton
Jones, Isaac Gerald Frederick
9c1002fd-1ece-45fa-b2c2-2d8b6afee79f
Jones, Isaac Gerald Frederick
9c1002fd-1ece-45fa-b2c2-2d8b6afee79f
Green, Nicolas
d9b47269-c426-41fd-a41d-5f4579faa581

Jones, Isaac Gerald Frederick (2017) Microfluidic systems for identification of antimicrobial-resistant pathogens. University of Southampton, Doctoral Thesis, 248pp.

Record type: Thesis (Doctoral)

Abstract

Antimicrobial resistance (AMR) poses a critical, yet historically understated threat to global public health. Recent mutations in the enterobacteriaceae genome that confer resistance to multiple antibiotics, have made some virtually untreatable.

Currently, antibiotics are prescribed largely on guesswork, with little or no prior evidence as to their efficacy. This not only accelerates the spread of AMR, but endangers patient's lives in the process. To counter this, new tools are required which can rapidly identify a pathogen's susceptibility to an antibiotic prior to administration. Traditional procedures are labour-intensive, taking several days - with sample preparation and cell-culture being the primary bottlenecks.

A novel, proof-of concept system was developed to rapidly determine antibiotic resistance in pathogens using nucleic acid analysis. The system comprised of a plastic microfluidic cartridge, control software, optical reader and *control hardware*.

E. coli were captured, concentrated and lysed within the device; DNA could be amplified on-chip using rapid, isothermal DNA amplification (Recombinase Polymerase Amplification). The assay identified samples containing the CTX-M resistance gene; the limit of detection was 500 copies (in 50l), as measured by the reader's integrated fluorescence microscope.

Each component was validated prior to integration into the final system. Concentrations of E. coli equivalent to a urinary-tract-infection (100,000 Colony Forming Units/ mL) were captured with up to 98% efficiency from 1ml suspensions onto functionalised, 0.5m magnetic, ion-exchange beads. These were isolated on-chip by a NdFeB magnet, providing up to 96% bead-capture and a 100x volume reduction. An oil compartmentalised the aqueous samples within the chip, while thermal control was achieved by coupling a resistive heater to an IR camera. Fluid routing was managed by bespoke, in-line, solenoid-controlled valves, which could operate at 5-bar and for over 4000 cycles without degrading. Bacteria were thermally or chemically lysed post-concentration, and an on-chip metering chamber aliquoted fixed-volumes of buffer (1% uncertainty for 400nl). DNA from lysed bacteria was amplified on-chip with a limit of detection of 250 E. coli per sample in the final assay.

Both sample-preparation and DNA amplification performed well on the prototype system. However, inefficient bacterial elution on-chip post-capture prevented the steps being run sequentially, as insufficient bacteria were released to the lysis region. Future development would fully integrate sample transfer and automate the fluid actuation so as to enable complete sample-in, answer-out capability.

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IGFJones PhD Final Thesis colourURL - Version of Record
Available under License University of Southampton Thesis Licence.
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Published date: October 2017

Identifiers

Local EPrints ID: 415850
URI: http://eprints.soton.ac.uk/id/eprint/415850
PURE UUID: 2d7f21e6-c96f-47ca-b3b9-ee382c60f3d1
ORCID for Nicolas Green: ORCID iD orcid.org/0000-0001-9230-4455

Catalogue record

Date deposited: 27 Nov 2017 17:30
Last modified: 14 Mar 2019 01:43

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