Abstract
AbstractMonomeric sarcosine oxidase (mSOx) fusion with the silaffin peptide, R5, designed previously for easy protein production in low resource areas, was used in a biosilification process to form an enzyme layer electrode biosensor. mSOx is a low activity enzyme (10–20 U/mg) requiring high amounts of enzyme to obtain an amperometric biosensor signal, in the clinically useful range <1 mM sarcosine, especially since the Km is >10 mM. An amperometric biosensor model was fitted to experimental data to investigate dynamic range. mSOx constructs were designed with 6H (6×histidine) and R5 (silaffin) peptide tags and compared with native mSOx. Glutaraldehyde (GA) cross‐linked proteins retained ∼5 % activity for mSOx and mSOx‐6H and only 0.5 % for mSOx‐R5. In contrast R5 catalysed biosilification on (3‐mercaptopropyl) trimethoxysilane (MPTMS) and tetramethyl orthosilicate (TMOS) particles created a ‘self‐immobilisation’ matrix retaining 40 % and 76 % activity respectively. The TMOS matrix produced a thick layer (>500 μm) on a glassy carbon electrode with a mediated current due to sarcosine in the clinical range for sarcosinemia (0–1 mM). The mSOx‐R5 fusion protein was also used to catalyse biosilification in the presence of creatinase and creatininase, entrapping all three enzymes. A mediated GC enzyme linked current was obtained with dynamic range available for creatinine determination of 0.1–2 mM for an enzyme layer ∼800 nm.
Highlights
There have been fewer innovations in amperometric enzyme biosensors, possibly because the core idea and technology is robust or perhaps because target analytes with “block buster” applications like the glucose biosensor have not been found
There is no indication from this structure that engineering of the C terminal of the enzyme should strongly influence the enzyme activity, so to design SOx constructs suitable for forming a thick enzyme layer on the electrode to produce a functional amperometric biosensor, with a low activity enzyme like Monomeric sarcosine oxidase (mSOx), two basic recombinant enzyme constructs were synthesised, with C terminal modification of mSOx: mSOx-6H, having a hexa-histidine peptide tag and mSOx-R5, having a silaffin tag
In this manuscript we have reported on the further use of the mSOx-R5 fusion protein in creating enzyme layers of variable thickness
Summary
There have been fewer innovations in amperometric enzyme biosensors, possibly because the core idea and technology is robust or perhaps because target analytes with “block buster” applications like the glucose biosensor have not been found. There are numerous examples where new effort is required to allow low cost diagnostics to be a reality in LMICs. In the work reported we will focus on sarcosine (N-methylglycine), which first gained research interest in a complex 3-enzyme system, where monomeric sarcosine oxidase (mSOx) was found to be useful for the enzymatic determination of creatinine. The work reported targets the sarcosine levels required in the clinical range for sarcosinemia (nominally < 1 mM) and considers whether the mSOx-R5 fusion protein provides a self-immobilising protein model that would give access to amperometric enzyme biosensors for low activity enzymes like SOx-6H
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