Biosensor design utilizing particle-bound enzymes

There is a clear need for affordable point-of-care diagnostics, especially in vulnerable low- and middle-income countries where access to laboratory-based testing is limited. This thesis explores whether recombinant protein technology in combination with a low-cost support matrix could provide a basis for an inexpensive, simple, and robust production process for the bio-sensing element of a diagnostic that would be amenable to manufacture in resource constrained settings. Silica was selected as the solid support for this work, given its biocompatibility and wide-availability (including extraction from natural sources, like sand, as demonstrated here). By employing an affinity binding sequence for silica in fusion with the central assay reagent protein targeting the analyte, simultaneous isolation and immobilisation onto silica carrier particles was achieved directly from lysate. In addition, the incorporation of a coloured fluorescent protein in the fusion enabled the protein production and immobilisation to be followed visually without significant laboratory equipment. Diagnostic sensing activity was retained in the immobilised fusion proteins, even over two months at 20-22 ⁰C in a dried state. A comparable limit of detection was achieved with immobilised reagents as with the soluble form. Taken together with the reduced downstream processing attained by a one-step purification and immobilisation approach, this supports the use of particle-bound reagents in the development of point-of-care tests. To make use of the particle-bound reagents, a novel falling particle biosensor design was explored in this thesis, where the sedimentation of the silica particles was used to drive mixing in an otherwise stationary fluid compartment. The performance of this design was compared against two other formats commonly employed with bio-functionalised particles – (A) a simple suspension in a microcentrifuge tube and (B) a packed bed in a microfluidic channel. The falling particle device outperformed both formats. Overall, this work has demonstrated that the integrated functionality of the fusion proteins could facilitate a production pathway from raw material to end diagnostic, highlighting the use of silica as a protein carrier and presenting a novel biosensor format for utilizing particle-bound enzymes.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2020

Author dc:creator

• Henderson, Cassi Joanna

Advisors dc:contributor.advisor

• Hall, Elizabeth
• Daly, Ronan

• biosensors
• protein engineering
• protein immobilisation
• point-of-care diagnostics
• global health
• silica
• sarcosine
• enzymes
• particle sedimentation

Statement dc:rights

• Figures/data from a publication of which I am the first author have been included in whole and in part in various figures in this thesis (as detailed in the separate pdf). C.J. Henderson, E. Pumford, D.J. Seevaratnam, R. Daly, E.A.H. Hall. (2019) Gene to diagnostic : Self immobilising protein for silica microparticle biosensor , modelled with sarcosine oxidase. Biomaterials, 193, 58–70. doi:10.1016/j.biomaterials.2018.12.003. According to the publisher 'as the author of this Elsevier article, you retain the right to include it in a thesis or dissertation, provided it is not published commercially. Permission is not required, but please ensure that you reference the journal as the original source.' The publication has been cited in the thesis.

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/a62682b9-5f72-48a2-9a22-ba2249d4b7f9/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

en

Author Identifier

0000-0002-5980-5642

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/297857