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 Km for the silica-R52-mCh-mSOx-R5-6H was 16.5±0.9mM (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 to 10 µM in both a dynamic and end point measurement regime.

A Biosilification Fusion Protein for a ‘Self‐immobilising’ Sarcosine Oxidase Amperometric Enzyme Biosensor

“…Gold-mSOx-R5-MPTMS GCE-mSOx-R5-GA over several weeks (as shown previously [6,14]). For the thickest layer, the LOD is reduced to 8 μM, with a dynamic range to 1700 μM.…”

“…Importantly, this result shows that the biosilification only requires one protein to contain the R5 tag. However, we have previously shown that without the R5, the immobilisation is not stable [6]. The data reported herein provide a basis to further expand the manufacturing techniques for use in low resource areas and to contribute to the challenge of delivering sustainable diagnostics in these environments.…”

“…Figure c and d show the FCA mediated current change for enzyme layers of 123–783 μm. These layers are stable with enzyme activity being retained at room temperature over several weeks (as shown previously ). For the thickest layer, the LOD is reduced to 8 μM, with a dynamic range to 1700 μM.…”

“…Importantly, this result shows that the biosilification only requires one protein to contain the R5 tag. However, we have previously shown that without the R5, the immobilisation is not stable .…”

“…We have previously reported a generic approach to engineer and produce many enzymes in low resource environments, for diagnostics . This has been successfully transferred to laboratories in LMICs and, for example, nucleic acid amplification studies have been undertaken in malaria regions and colorimetric and fluorescent biosensors have been studied , but there is still much scope to simplify designs and further reduce cost and complexity.…”