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Molecularly “Wired” Cholesterol Oxidase for Biosensing

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Abstract

The influence of several factors on the activity of cholesterol oxidase (ChOx) transiently exposed to a room temperature ionic liquid (RTIL) was studied. Presence of flavin adenine dinucleotide (FAD, prosthetic group of ChOx) during exposure to RTIL makes the procedure enzyme-friendly, while the use of RTIL (green reagent) makes it environmentally-friendly. Following exposure to RTIL and its subsequent removal, FAD becomes part of the molecular structure of the refolded protein (a molecular “wire”). This makes the procedure used here a molecular one. The factors studied were: FAD presence in RTIL during modification, water presence during exposure to RTIL, and ratio FAD:RTIL during “wiring”. Performance parameters monitored were: enzyme activity before and after “wiring” (expressed as (dA/dt)/mg enzyme, and measured spectrophotometrically), peak current in an amperometric biosensor for cholesterol detection, and linearity of the biosensor response depending on cholesterol concentration. After RTIL removal, the modified enzyme (ME) retained a high percentage of the added FAD, which supplemented that of the native enzyme (functioning as a “wire” and enhancing electron transfer kinetics), and a fraction of the initial activity. Used in an amperometric biosensor, ME showed catalytic activity, linear behavior as a function of cholesterol concentration, and stability.

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Abbreviations

ChOx:

Cholesterol oxidase

HPLC:

High performance liquid chromatography

RTIL:

Room temperature ionic liquids

FAD:

Flavin adenine dinucleotide

[EMIM]BF4 :

1-Ethyl-3-methylimidazolium tetrafluoroborate

LDH:

Lactate dehydrogenase

ME:

Modified enzyme

GC:

Glassy carbon

SCE:

Standard calomel electrode

EtOH:

Ethanol

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Authors and Affiliations

  1. Fairleigh Dickinson University, Metropolitan Campus, 1000 River Rd., Teaneck, NJ, 07666, USA

    Mihaela D. Leonida

  2. Chemlogic, 30 Pond Ave., Newport, RI, 02840, USA

    Benedict Aurian-Blajeni

Authors
  1. Mihaela D. Leonida
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  2. Benedict Aurian-Blajeni
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Correspondence to Mihaela D. Leonida.

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Leonida, M.D., Aurian-Blajeni, B. Molecularly “Wired” Cholesterol Oxidase for Biosensing. Protein J 34, 68–72 (2015). https://doi.org/10.1007/s10930-015-9599-9

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  • DOI: https://doi.org/10.1007/s10930-015-9599-9

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