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Monomeric 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. [ABSTRACT FROM AUTHOR]

Lanthanide metal organic frameworks (MOFs), Eu(BTC) and TbEu(BTC) were assessed to follow uptake of deoxynucleotide triphosphate (dNTPs) in loop mediated isothermal amplification (LAMP) using BST2.0. dNTPs could be determined below 150 μM. A ratiometic measurement of Tb3+:Eu3+ fluorescence in TbEu(BTC) gave a limit of detection of 2.08 μM for dNTPs. dNTP uptake slowed above 8 mM Mg2+. This correlated with end‐point and real‐time LAMP, using PicoGreen and SYBR green respectively. For the P.Mal‐Lau 18S rRNA primer set, with P. malariae a detection limit of 102.5 copies was obtained. Cross reactivity between P. malariae and P. knowlesi was shown with 18S rRNA primer sets. [ABSTRACT FROM AUTHOR]

Mycobacterium acquire iron by producing siderophores called mycobactins, with an extremely high affinity complexation of Fe3+. The iron complex shows distinctive electrochemistry predicting 18 orders of magnitude greater affinity of the mycobactin for Fe3+ than Fe2+. A heterogeneous standard rate constant, ks of the order of 10−5 cm s−1 confirms quasi reversible electrochemistry and based on the equilibrium in the presence of excess solution Fe3+/Fe2+ the oxidation and reduction peaks for the siderophore complex could be calibrated for ferric mycobactin J (FeMJ). FeMJ spiked urine collected from cows showed a matrix effect on the current peak height. For example, 240±15 µM FeMJ was estimated in 600 µM spiked urine. However, in the presence of excess solution Fe(acac)3 the same sample yielded an estimated 580±25 µM FeMJ. [ABSTRACT FROM AUTHOR]