Gene to diagnostic: Self immobilizing protein for silica microparticle biosensor, modelled with sarcosine oxidase.

Abstract

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 constructs 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 proteins were stable for more than 2 months at room temperature. The K_m for the silica-R5₂-mCh-mSOx-R5-6H was 16.5 ± 0.9 mM (compared 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 2.5-10 μM in both a dynamic and end point measurement regime.