Peptide conjugated chitosan foam as a novel approach for capture-purification and rapid detection of hapten - Example of ochratoxin A

A novel bioassay for the detection and monitoring of Ochratoxin A (OTA), a natural carcinogenic mycotoxin produced by Aspergillus and Penicillium fungi, has been developed and applied for the screening of red wine. Here we report the immobilization and orientation of NOF4, a synthetic peptide, onto 3-D porous chitosan supports using a N-terminal histidine tag to allow binding to M⁺⁺ ions that were previously adsorbed onto the high surface area biopolymer. Three divalent cations (M⁺⁺=Zn⁺⁺, Co⁺⁺, Ni⁺⁺) were evaluated and were found to adsorb via a Langmuir model and to have binding capacities in the order Zn⁺⁺>Co⁺⁺>Ni⁺⁺. Following Zn⁺⁺ saturation and washing, C-terminus vs. the N-terminus His-tagged NOF4 was evaluated. At 1000μgL^-1 OTA the N-terminus immobilization was more efficient (2.5 times) in the capture of OTA. HRP labeled OTA was added to the antigen solutions (standards or samples) and together competitively incubated on biospecific chitosan foam. The chemiluminescence substrate luminol was then added and after 5min of enzymatic reaction, light emission signals (λ_max=425nm) were analyzed. Calibration curves of %B/B₀ vs. OTA concentration in PBS showed that half-inhibition occurred at 1.17μgL^-1, allowing a range of discrimination of 0.25 and 25μgL^-1. In red wine, the minimum concentration of OTA that the system can detect was 0.5μgL^-1 and could detect up to 5μgL^-1. Assay validation was performed against immunoaffinity column (IAC) tandem reversed-phase high pressure liquid chromatography with fluorescence detection (HPLC-FLD) and provided quite good agreement. The association of chitosan foam and specific peptide represents a new approach with potential for both purification-concentration and detection of small molecules. In the future this assay will be implemented in a solid-sate bioelectronic format.