Microfabricated biosensor for the simultaneous amperometric and luminescence detection and monitoring of Ochratoxin A

Abstract

The low molecular weight hapten, Ochratoxin A (OTA), is a natural carcinogenic mycotoxin produced by Aspergillus and Penicillium fungi and so it commonly appears in wines, other foods, and in the environment. An amperometric biosensor has been developed that uses the immobilized synthetic peptide, NFO4; which possesses a high binding affinity and thus provides for molecular recognition of OTA; simulating the mycotoxin-specific antibody. Biotransducers were produced from a microlithographically fabricated electrochemical cell-on-a-chip that uses the microdisc electrode array working electrode format augmented with microporous graphitized carbon (MGC) that was electrodeposited within a poly(aniline-co-meta-aminoaniline) electroconductive polymer layer. A redox mediator, iron-nickel hexacyanoferrate (Fe|NiHCF) was amperometrically deposited onto the MGC. The device was then dip-coated with monomer cocktail that yielded poly(HEMA-co-AEMA) foam that was prepared in-situ by UV crosslinking and by sequentially freezing followed by freeze drying of the chip to yield a 3-D support for the chelation of Zn(2+) ions (ZnCl2) and the subsequent immobilization of N-terminus his-tagged peptide, NFO4. To conduct the biosensors assay, HRP conjugated OTA was added to the free OTA solutions and together competitively incubated on the biospecific MDEA ECC 5037-Pt|MGC|HCF|Hydrogel-NFO4 biotransducer. The amperometric response to peroxide was determined after 5 min of enzymatic reaction following addition of standard substrate H2O2/luminol. Simultaneous analysis of light emission signals (λmax=425 nm) allowed direct comparison of amperometric and luminescence performance. Using chitosan foam and a luminescence bioassay we obtained maximum inhibition at 10 μg L(-1) and half inhibition occurred at 2.1 μg L(-1). Using poly(HEMA-co-AEMA) hydrogel and an amperometric bioassay (50s) we obtained maximum inhibition at 10 μg L(-1) and half inhibition occurred at 2.8 μg L(-1).

Keywords: Amperometry; Biosensors; Luminescence; NFO4; Ochratoxin A; Peptides.