Development of an integrated paper-based molecular diagnostic platform

Abstract

The thesis describes the preliminary experimental studies, biochemical assay development and computer aided design work conducted to realise an integrated paper-based molecular diagnostic platform. Research trends in paper-based devices have mostly been dominated by studies to recognise additional functionalities; however, there has been a surge towards research in molecular diagnostics or nucleic acid testing on these devices recently. Molecular diagnostic assays consist of many intricate processes, each, requiring a number of steps and effective biochemical reactions to be successfully executed. A Xerox Phaser 8560 DN solid ink printer was utilised to produce wax printed paper-based devices and CO2 laser cutting was utilised to produce paper-based strips and reaction discs. A simple paper-based DNA extraction method which can be integrated to existing biosensing mechanism was produced and optimized to capture 90% of sample DNA. Recombinase polymerase amplification assays to detect Chlamydia trachomatis was developed and implemented on paper-based reaction discs and a sensitivity on 10 copies/μL was achieved. Commercially available Nucleic acid based lateral flow devices were used to detect amplicons generated by the amplification assay and a simple one-step equipment free process was developed to enable direct detection. A proof-of-concept experimental study to evaluate lysate-in-to-answer-out paper-based molecular diagnostic assay was performed, the sample was extracted on a chitosan modified reaction disc, amplified and detected directly on a lateral flow test strip. A concept design for a low-cost, completely disposable paper-based molecular diagnostic device was developed. The device design and the developed biological assays described herein allows the seqential steps required to perform a lysate-in-to-answer-out diagnostic assay to just five. The work presented here has reduced the complexity of this broad multidisciplinary research study to an engineering problem. Although a sample-in-to-answer-out molecular diagnostic platform is not realised yet, the research studies described herein have helped take a step closer to reality.