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.Gates Cambridge Trus
