RPA using a multiplexed cartridge for low cost point of care diagnostics in the field

Luck Tosan Ereku; Ruth E Mackay; Pascal Craw; Angel Naveenathayalan; Thomas Stead; Manorharanehru Branavan; Wamadeva Balachandran

doi:10.1016/j.ab.2018.02.010

RPA using a multiplexed cartridge for low cost point of care diagnostics in the field

Anal Biochem. 2018 Apr 15:547:84-88. doi: 10.1016/j.ab.2018.02.010. Epub 2018 Feb 12.

Authors

Luck Tosan Ereku¹, Ruth E Mackay², Pascal Craw³, Angel Naveenathayalan⁴, Thomas Stead¹, Manorharanehru Branavan¹, Wamadeva Balachandran¹

Affiliations

¹ Centre for Electronic Systems Research, Electronic and Computer Engineering, CEDPS, Brunel University London, Kingston Lane, Uxbridge UB8 3PH, UK.
² Mechanical, Aerospace and Civil Engineering, CEDPS, Brunel University London, Kingston Lane, Uxbridge UB8 3PH, UK. Electronic address: ruth.mackay@brunel.ac.uk.
³ Oceans and Atmosphere Flagship, Commonwealth Science and Industrial Research Organisation (CSIRO), Hobart, Tasmania 7001, Australia.
⁴ Mechanical, Aerospace and Civil Engineering, CEDPS, Brunel University London, Kingston Lane, Uxbridge UB8 3PH, UK.

Abstract

A point of care device utilising Lab-on-a-Chip technologies that is applicable for biological pathogens was designed, fabricated and tested showing sample in to answer out capabilities. The purpose of the design was to develop a cartridge with the capability to perform nucleic acid extraction and purification from a sample using a chitosan membrane at an acidic pH. Waste was stored within the cartridge with the use of sodium polyacrylate to solidify or gelate the sample in a single chamber. Nucleic acid elution was conducted using the RPA amplification reagents (alkaline pH). Passive valves were used to regulate the fluid flow and a multiplexer was designed to distribute the fluid into six microchambers for amplification reactions. Cartridges were produced using soft lithography of silicone from 3D printed moulds, bonded to glass substrates. The isothermal technique, RPA is employed for amplification. This paper shows the results from two separate experiments: the first using the RPA control nucleic acid, the second showing successful amplification from Chlamydia Trachomatis. Endpoint analysis conducted for the RPA analysis was gel electrophoresis that showed 143 base pair DNA was amplified successfully for positive samples whilst negative samples did not show amplification. End point analysis for Chlamydia Trachomatis samples was fluorescence detection that showed successful detection of 1 copy/μL and 10 copies/μL spiked in a MES buffer.

Keywords: DNA extraction; Isothermal amplification; Multiplexed cartridge; Point of care; RPA.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Chitosan / chemistry
Chlamydia trachomatis* / genetics
Chlamydia trachomatis* / metabolism
Hydrogen-Ion Concentration
Lab-On-A-Chip Devices*
Membranes, Artificial
Nucleic Acid Amplification Techniques / instrumentation*
Nucleic Acid Amplification Techniques / methods*
Nucleic Acids* / analysis
Nucleic Acids* / chemistry
Nucleic Acids* / genetics
Nucleic Acids* / isolation & purification
Point-of-Care Systems*

Substances

Membranes, Artificial
Nucleic Acids
Chitosan

Abstract

Publication types

MeSH terms

Substances

Grants and funding