Gene to Diagnostic: Self immobilizing protein for silica microparticle biosensor, modelled with sarcosine oxidase.
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Hall, L., Pumford, E., Henderson, C., Seevaratnam, D., & Daly, R. (2018). Gene to Diagnostic: Self immobilizing protein for silica microparticle biosensor, modelled with sarcosine oxidase.. Biomaterials, 193 58-70. https://doi.org/10.1016/j.biomaterials.2018.12.003
A rational design approach is proposed for a multifunctional enzyme reagent for point-of-care diagnostics. The biomaterial reduces downstream isolation steps and eliminates immobilization coupling chemicals for integration in a diagnostic platform. Fusion con-structs combined the central functional assay protein (e.g. monomeric sarcosine oxidase, mSOx, horseradish peroxidase, HRP), a visualizing protein (e.g. mCherry) and an in-built immobilization peptide (e.g. R5). Monitoring protein expression in E.coli was facilitated by following the increase in mCherry fluorescence, which could be matched to a color card, indicating when good protein expression has occurred. The R5 peptide (SSKKSGSYSGSKGSKRRIL) provided inbuilt affinity for silica and an immobilization capability for a silica based diagnostic, without requiring additional chemical coupling reagents. Silica particles extracted from beach sand were used to collect protein from crude protein extract with 85-95% selective uptake. The silica immobilized R5 pro-teins were stable for more than 2 months at room temperature. The Km for the silica-R52-mCh-mSOx-R5-6H was 16.5±0.9mM (com-pared with 16.5±0.4 mM, 16.3±0.3 mM, and 16.1±0.4 mM for R52-mCh-mSOx-R5-6H, mSOx-R5-6H and mSOx-6H respectively in solution). The use of the “silica-enzymes” in sarcosine and peroxide assays was shown, and a design using particle sedimentation through the sample was examined. Using shadowgraphy and particle image velocimetry the particle trajectory through the sample was mapped and an hourglass design with a narrow waist shown to give good control of particle position. The hourglass biosensor was demonstrated for sarcosine assay in the clinically useful range of 2.5 to 10 µM in both a dynamic and end point measurement regime.
Is supplemented by: https://doi.org/10.17863/CAM.34176
Royal Society IC160089 The Gates Cambridge Trust The Cambridge Trust
Royal Society (IC160089)
External DOI: https://doi.org/10.1016/j.biomaterials.2018.12.003
This record's URL: https://www.repository.cam.ac.uk/handle/1810/287295