Trimethylamine N‐Oxide Electrochemical Biosensor with a Chimeric Enzyme

Abstract

For the first time, an enzyme-based electrochemical biosensor system for determination of trimethylamine N-oxide (TMAO) is described. It employs an active chimeric variant of TorA in combination with an enzymatically deoxygenating system and a low-potential mediator for effective regeneration of the enzyme and cathodic current generation. TMAO reductase (TorA) is a molybdoenzyme found in marine and most enterobacteria that specifically catalyzes the reduction of TMAO to trimethylamine (TMA). The chimeric TorA, named TorA-FDH, corresponds to the apoform of TorA from Escherichia coli reconstituted with the molybdenum cofactor from formate dehydrogenase (FDH). Each enzyme, TorA and TorA-FDH, was immobilized on the surface of a carbon electrode and protected with a dialysis membrane. The biosensor operates at an applied potential of À 0.8 V [vs. Ag/AgCl (1 M KCl)] under ambient air conditions thanks to an additional enzymatic O 2 -scavenger system. A comparison between the two enzymatic sensors revealed a much higher sensitivity for the biosensor with immobilized TorA-FDH. This biosensor exhibits a sensitivity of 14.16 nA/μM TMAO in a useful measuring range of 2-110 μM with a detection limit of LOD = 2.96 nM (S/N = 3), and was similar for TMAO in buffer and in spiked serum samples. With a response time of 16 � 2 s, the biosensor is stable over prolonged daily measurements (n = 20). This electrochemical biosensor provides suitable applications in detecting TMAO levels in human serum.It can also catalyze other N-oxide compounds, like 4-methyl [a] B.

Electrochemical Trimethylamine N-Oxide Biosensor with Enzyme-Based Oxygen-Scavenging Membrane for Long-Term Operation under Ambient Air

“…rable, despite the additional scavenger membrane. However, the response time of 33 ± 5 s is twice as high, and the sensor sensitivity is two times lower in comparison to our earlier work [17], which can again be attributed to the additional diffusion resistance. This observation was also confirmed by the loading experiments, whereby more MV and TMAO were needed in comparison to a system without membrane.…”

“…An almost maximum catalytic current value was obtained after addition of 500 µM MV. This value is twice that published in our previous work [17], where already 250 µM MV were suffi-cient to generate a maximum current response. In the previous work the solutions where also kept oxygen-free, and therefore the parasitic reaction between MV and oxygen was omitted.…”

“…noted that in our previous work, where the bulk solution was enzymatically deoxygenated by the GOD and Cat directly in solution, no negative effect of the serum was observed [17]. These observations indicate that the serum unspecifically interacts at the membrane surface, thus affecting the diffusion of either or both the mediator and TMAO.…”

“…The cleaned GCEs were then ultrasonicated in acetone for 3 min and for 7 min in Milli-Q water. After drying under N 2 stream, 5 µL of TorA were dropcasted onto the electrodes and incubated for 30 min in a desiccator at 4 • C. The modified electrodes were then covered with the previous optimized oxygen scavenging membrane hold by a dialysis membrane and placed in the electrochemical cell with an O-ring assembly as described elsewhere [17].…”

“…Electrochemical biosensors represent an attractive alternative as analytical methods, in particular for the decentralized tests in small laboratories or on-site monitoring, as they offer a fast response time and high selectivity, as well as the potential for low-cost simple operation in miniaturized devices [14]. To the best of our knowledge only three publications report sensors for TMAO, i.e., a microbial sensor using Pseudomonas aminovarans on an oxygen electrode [15], a sensor with molecularly imprinted polymers on ITO [16] and an enzyme biosensor with an enzyme immobilized on a glassy carbon electrode [17].…”