Abstract
In this study, detection and measurement of non-esterified fatty acids (NEFA) concentration has been achieved by electrochemical method in one operation step. Multilayer films of poly(dimethyldiallyammonium chloride) (PDA) wrapped multi-wall carbon nanotubes (MWCNTs) and two enzymes acyl-CoA synthetase (ACS) and acyl-CoA oxidase (ACOD) were assembled on a carbon screen printed electrode by the layer-by-layer (LbL) immobilization. The fine polymer–enzyme layers produced by the LbL method, allowed mass transport from the reactant cascading down the layers to accomplish the two-step enzyme reactions. The polymer–CNTs and enzyme modified electrode exhibited good electrocatalytical property retaining enzyme activity. Linear increase of anodic current from H2O2 produced from NEFA oxidation was observed with the increasing concentrations of oleic acid. These results indicate a promising technique for a simple, rapid one-step determination of NEFA for diabetes management.
Highlights
Diabetes poses a major and growing health and socio-economic burden on society [1]
Oleoyl coenzyme A lithium salt (OACoA), palmitoyl coenzyme A lithium salt (PACoA), coenzyme A sodium salt hydrate (CoA), adenosine 5 -triphosphate disodium salt hydrate (ATP), poly(dimethyldiallyammonium chloride) (PDA) (Mw = 200–250 kDa), sodium dihydrogen phosphate dehydrate, disodium hydrogen phosphate and potassium ferricyanide (K3[Fe(CN)6]) were purchased from Sigma-Aldrich (Dorset, UK). 1 mM Oleic acid (OA) was the Wako non-esterified fatty acids (NEFA) standard solution purchased from Wako HR-series NEFA-HR(2) enzymatic NEFA assay kit (Neuss, Germany); acyl-CoA synthetase (ACS) was supplied from this assay kit in a solid mixture with CoA, adenosine -triphosphate disodium salt hydrate (ATP) and a few other reagent (R1a) [16]
The amount of H2O2 produced from the reaction is proportional to the amount of PACoA
Summary
Diabetes poses a major and growing health and socio-economic burden on society [1]. With an increase in people being diagnosed with type 2 diabetes (T2D), there should be more ways to monitor the blood glucose levels and the other metabolism biomarkers associated with T2D. NEFA detection in blood can be dated back to the late 1950s [7,8], the methods developed during this time were either based on colorimetric titration of fatty acids in the presence of a pH indicator [9] or spectrophotometric or radiochemical measurements of complexes of fatty acids with divalent metal ions such as Cu2+, Ni2+ or Co2+ [10]. If H2O2 produced from step 2, acyl-CoA oxidation by ACOD at the presence of oxygen, can be detected electrochemically, it should be possible to determine NEFA concentration electrochemically from the amount of H2O2, similar to the colorimetric method This could provide a coherent method to current electrochemical based glucose biosensor to develop a multiplex biosensor platform for simultaneous measurement of different metabolic biomarkers. Good linear correlation between NEFA concentration and H2O2 oxidation currents was obtained, indicating one step NEFA detection achieved This provides a solid base for further development of a multiplex sensor platform
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