Development and integration of simplified real-world to chip interfaces for use in the detection of infectious diseases

Birch, Christopher

Biological sciences
June 2014

Thesis or dissertation

© 2014 Christopher Birch. All rights reserved. No part of this publication may be reproduced without the written permission of the copyright holder.

Bacterial-based infectious disease, such as sexually transmitted infections and hospital-acquired infections, present a worldwide burden on healthcare issues. To control the spread of infection and to inform clinical treatment, rapid point of care (POC) diagnosis is required. Although some are currently available, these are commonly limited by a requirement for sample processing prior to analysis and a requirement for user intervention. Novel real world-to-chip interfaces are required which can receive a sample with little or no pre-processing and should be manufactured with minimal cost. In addition, manufacturing protocols should ideally be developed to allow easy adjustment of design for customising to process a wide variety of sample types and volumes.

Here, focussing on bacterial-based POC diagnostics, a microfluidic platform has been developed which holds the potential to receive a variety of sample types for the detection of infectious organisms by implementing multiple sample-to-chip interfaces. The platform consists of a glass microfluidic device which is incorporated in to a custom-made integrated genetic analyser (IGA) for sample processing.

A series of interfacing substrates were investigated using two types of porous silica and the biopolymer chitosan (α(1→4)-linked 2-amino-2-deoxy-β-D-glucopyranose) as contributing materials. For analysis of urine samples, a porous silica monolith, synthesised from tetramethyl orthosilicate, was developed, capable of receiving and processing human urine samples (≈ 150 μl) for DNA capture and purification. Due to the nature of synthesis, these monoliths hold the potential for resizing and shaping, dependent on the sample volume and for integration to downstream steps, such as polymerase chain reaction (PCR) amplification. The monolith was first optimised structurally using flow systems made from monoliths encased in heat shrink wrap and was incorporated in to a microfluidic device by way of disk. The latter was achieved by sealing the monolith in place with a sondary porous silica phase synthesised from potassium silicate, creating a dual porous silica (DPS) real world interface. The DNA extraction efficiencies of monolithic flow systems and the DPS system were 51 % and 44 % respectively. The DPS was shown to provide DNA of sufficient quality and integrity to support PCR amplification for both Chlamydia trachomatis and Neisseria gonorrhoea target sequences. The system did however lack sensitivity (1.3 x 10¯³ ng DNA μl¯¹ urine), when compared with systems of similar applications in the literature, likely due to large elution volumes (> 20 μl) and/or ethanol carryover. In addition, chitosan was introduced to the silica surface of the monolith as an alternative methodology for DNA extraction by anion exchange. The system provided DNA extraction efficiencies of 40 % and DNA was subsequently amplified by PCR.

Using an alternative application, an investigation was also carried out in to the analysis of small volume blood samples (≤ 6 μl) for use in the same system. This was achieved by implementing a Phusion® blood direct kit in to a glass microfluidic device to amplify gDNA of bacterial target, methicillin resistant Staphylococcus aureus (MRSA). The system was shown to amplify DNA in 6 μl blood (0.083 ng μl¯¹) off-chip. It was then demonstrated to work on-chip in a 12.5 μl glass chamber, using a Peltier system for thermal cycling.

For use in conjunction with all interfaces, an IGA with a built in DNA separation/ detection system, based on plug injection, capillary electrophoresis separation and fluorescence detection has been shown to reproducibly report the presence of target DNA sequences, against that of a custom-made size ladder. The detection of a 107 bp generic Staphylococcus aureus marker and 532 bp sequence from the mecA gene unique to MRSA were detected in 20 and 25 min, respectively. Importantly, the detection system is designed to integrate directly to upstream steps and has a Peltier element fitted for thermal cycling.

The work described here contributes towards a platform which offers the opportunity to tackle a number of diagnostic applications in one fit-for-all instrument.

School of Biological, Biomedical And Environmental Sciences, The University of Hull
Dyer, Charlotte E.; Haswell, S. J. (Stephen John), 1954-
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