Abstract
An amperometric trimethylamine N-oxide (TMAO) biosensor is reported, where TMAO reductase (TorA) and glucose oxidase (GOD) and catalase (Cat) were immobilized on the electrode surface, enabling measurements of mediated enzymatic TMAO reduction at low potential under ambient air conditions. The oxygen anti-interference membrane composed of GOD, Cat and polyvinyl alcohol (PVA) hydrogel, together with glucose concentration, was optimized until the O2 reduction current of a Clark-type electrode was completely suppressed for at least 3 h. For the preparation of the TMAO biosensor, Escherichia coli TorA was purified under anaerobic conditions and immobilized on the surface of a carbon electrode and covered by the optimized O2 scavenging membrane. The TMAO sensor operates at a potential of −0.8 V vs. Ag/AgCl (1 M KCl), where the reduction of methylviologen (MV) is recorded. The sensor signal depends linearly on TMAO concentrations between 2 µM and 15 mM, with a sensitivity of 2.75 ± 1.7 µA/mM. The developed biosensor is characterized by a response time of about 33 s and an operational stability over 3 weeks. Furthermore, measurements of TMAO concentration were performed in 10% human serum, where the lowest detectable concentration is of 10 µM TMAO.
Highlights
Cardiovascular diseases (CVDs) are the major cause of death globally
In a first step we searched for conditions to optimize the anaerobic surrounding of the TMAO reductase (TorA) enzyme trapped on the electrode
A high glucose oxidase (GOD) activity in the top layer is needed to ensure the total consumption of the oxygen diffusing from the bulk solution in the layer in front of the electrode surface, and enzyme excess is needed to counterbalance the inactivation of enzymes over time for a long-term use of the oxygen-scavenging property
Summary
Cardiovascular diseases (CVDs) are the major cause of death globally. Over the past decades, public health experts have found a role of the gut microbiome in connection with the diet behavior in the development of such diseases. Alternative oxygen elimination methods employ coupled enzyme reactions such as GOD and Cat in the presence of glucose to transform the dissolved molecular oxygen into water. These are very efficient in ensuring anoxic conditions in Biosensors 2021, 11, 98 Biosensors 2021, 11, 98 vacuum degassing which are not suitable for the simple setups typically used for enzyme electrode measurements. Alternative oxygen elimination methods employ coupled eofn zyme reactions such as GOD and Cat in the presence of glucose to transform the dissolved molecular oxygen into water These are very efficient in ensuring anoxic conditions in bdDstahbrDGoaodiuMeoedxirdMOcod,cydoeiSchuGDtgSxiOhiettceOoyOihemeaonngrmeDnnrteaieihecddaionlndacaelxusalCniutoyonschledcalogtxxeuettatlyeahCxptusswnsgiepeeaotoeeireestiln[oni[nuorr2mw2neilc5tim5ubinoee]ao]c.timrn.edoonibeMonMtsmdipsneoteaoeosoninpdsednrnrezsiodenenenoytsoneoznosromvdeysvsotlonetmseuarhtbrlrtsottau,,saeaiinbocftftstomaiioadhnaeocnrrlsde.anelldetyltToa.hmldhoynsh[elTenu1oegoe[ah7arn1annGbwie]s7bgn.iuon]OtoaGg.rvHriwvitaHDiteOsrneitoa-aone/o-gmDwiCmtlgwteuri/seaeneettoerntvCiidnge/vlmoteguaudtienriolttterco,uui/in,smtconfccagefoontaoeosdelarnrduesrsscetet,ccteoahchahoun,ainherinesninpmetiteiapmidprpnteiiurarrerntgeraaioenpsiirtccicnagediprrttccgleiTieerruchcdoTedosaagcidar,ouwlrlolAcauuercuushaAndt-ccesssabodt,e-wesatabasoaeostsoepaahneefdfss[pteddb2e[oeblar21diigbeiope1t,dof2hrids,bpooesa24eeielgsr4d]tnorieenor]eeessanasdfaeoornodtsonnxerhodtrres---doeers, yogxeyngsecnasvceanvgeinngginmgemmebmrabnreasnwesewreeraepapplipeldiewd hwichhicahsasussruerleolcoaclaol xoyxgyegnenddepeplelteitoionnoonnththee eelelecctrtrooddeessuurrfafacceefoforrnnitirtritieteaannddDDMMSSOOmmeeaassuurreemmeenntt[[2211,2,266,2,277]]. The amperometric measurement was performed at different applied potentials (−0.6 V for O2 and +0.6 V for H2O2) and stirring of the solution
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