Ochratoxin A detection in coffee by competitive inhibition assay using chitosan-based surface plasmon resonance compact system
Graphical abstract
Introduction
Ochratoxin A (OTA) is highly toxic metabolite produced by molds of the Aspergillus and Penicillium families, which can grow on foods during harvesting and post-harvest stages and occurs naturally in numerous food products such as spices, coffee, cereals, beans and fruits, and in their processed products [1]. Moreover, OTA occurs in 25% of the world crops and poses a serious health risk due to its teratogenic, hepatotoxic and immunotoxic properties. Ochratoxin A has been categorized as a possible human carcinogenic (group 2B) by International Agency for Research on Cancer (IARC) [[2], [3], [4]]. Considering the public health importance, various chromatographic and immunoassay techniques have been developed for the detection and quantification of ochratoxin A in food commodities during last few decades. The most common techniques used for the detection of OTA are liquid chromatography–mass spectroscopy [5,6], high-performance liquid chromatography [7] and gas chromatography [8]. These all techniques are generally time-consuming, expensive and require trained personnel. It is thus, desirable to develop sensitive, low-cost, efficient and robust system for the detection of OTA in food commodities.
Coffee beverages are highly consumed worldwide and become the second largest traded food commodity. The intake of coffee has greatly been increased during the last two decades and it is estimated that more than 19 million tones will be consumed by 2019 [9]. The safe produce of processed coffee is highly desirable for the consumers as the filamentous fungi contaminate coffee during development, harvesting, transportation and, storage [10]. The OTA naturally occurs in green coffee beans in varying concentrations from 0.1 to 360 μg/kg [[11], [12], [13]]. The European Commission sets the highest maximum level of OTA in soluble and instant coffee at 10 ng/g and in roasted/ground coffee bean at 5 ng/g [14].
Optical biosensing is an emerging technology alternative to traditional analytical techniques due to its high sensitivity, fast screening, the efficiency and the simple assay procedures for detection. Optical transducer in most of the developed sensors are based on the optical waveguide, fiber gratings, guided mode resonance, surface plasmon resonance and interferometer [15]. Among these, surface plasmon resonance (SPR) provides a rapid lab-free tool to study the biological interactions in real time and it is used for the quantification of toxins in complex food matrices [16,17]. Surface plasmon is excited by the Kretschmann geometry when the polarized light at the interface of dielectric metal film undergoes total internal reflection. In general, a gold film is used as sensing substrate for the sensor chip because of its ability to excite the SPR response. The principle of molecular binding in SPR is basically similar to the other immunological techniques such as enzyme-linked immunosorbent assay (ELISA), immunochromatographic technique (ICT). Surface plasmon resonance technique has been used for the detection of OTA in wine, cereals and milk with the limit of detection (LOD) ranging from 0.155 ng/mL to 0.55 ng/mL [18,19].
The sensitivity of SPR depends on the volume, weight, and immobilization of biological molecules on the sensor chip, and causes the changes in resonance angle [20,21]. Therefore, detection of small molecules such as OTA (Mw = 403.8 Da) is difficult due to weak response from the sensor (insignificant change of the refractive index). Hence, selective polymers with gold (Au) nanoparticles, self-assembled monolayers, and molecular imprinted polymers are used for the signal amplification [22]. In this regard, chitosan is one of the most promising biopolymers for the immobilization of small biological molecules on the SPR chip surface because of its good film-forming ability, chemical stability, low-cost, biocompatible and eco-friendly nature [23,24]. Chitosan has been used as a matrix in SPR biosensors for the detection of heavy metal ions [[25], [26], [27]]. There is no report yet for the detection of OTA in coffee by SPR immunoassay on chitosan and carboxymethyl chitosan nanomatrix substrates.
Chitosan has the high binding capacity with biomolecules due to presence of protonated amino acids on its side chain and therefore, reduces the nonspecific binding of biological molecules on SPR chip surface during immobilization [28,29]. However, chitosan is not soluble at physiological pH due to its lower pka of nearly 6.5. In order to increase the hydrophilic properties and solubility, chemical modification, such as carboxymethylation of chitosan is generally conducted [30]. Carboxymethyl chitosan (CMC) is soluble in water and exhibits high hydrodynamic volume, high viscosity, low toxicity, biodegradable and biocompatible nature [31]. During the chitosan modification, hydroxyl (OH−) groups are substituted by carboxymethyl groups (CH2COOH). Therefore, presence of amino and carboxyl groups provides the chemical bases for the covalent attachment of biological molecules. Chemical modification on SPR sensor chip can be done through amine coupling, aldehyde coupling and thiol coupling [32].
In this study, in-house built compact SPR sensor based on chitosan and carboxymethyl chitosan nanomatrix was evaluated for detection of OTA in the buffer and coffee. Competitive inhibition immunoassay was developed for the detection of OTA in coffee as illustrated in Fig. 1. The limit of detection was identified on both chitosan and carboxymethyl chitosan nanomatrix substrates.
Section snippets
Reagents and material
Ochratoxin A and mouse monoclonal to Ochratoxin A antibody (mAb) were purchased from Biomed Diagnostics (Thailand). Anhydrous magnesium sulfate and phosphate buffered saline were acquired from Fluka Co. Ltd (Germany). All other chemicals were of analytical grade and bought from Sigma-Aldrich Co. Ltd, USA. The gold (Au) sensor chip was provided by National Electronics and Computer Technology Center (NECTEC) NSTDA, Thailand. All other chemicals used were of analytical grade.
Fabrication of SPR system
The compact and
Results and discussion
Effective immobilization of ligand on the sensor surface is required towards the detection of OTA in coffee and to increase the sensitivity of SPR immunosensor. Chitosan (CS) and carboxymethyl chitosan (CMC) coated substrates were immobilized with OTA-BSA conjugate and evaluated, through indirect competitive inhibition assay for the analysis of OTA in buffer and coffee samples. All the experiments were performed in ambient conditions.
Conclusion
A rapid and sensitive competitive inhibition SPR immunoassay was developed on 3D printed compact SPR system. SPR chips were coated with chitosan and carboxymethyl chitosan through spin coating technique for the detection of OTA in buffer and coffee. Various analytical conditions were optimized such as regeneration of the surface and immobilization of conjugate on substrate. Buffer and coffee samples spiked with OTA were used in the concentration range between 0–50 ng/mL to stablish a
Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Conflict of interest
The authors declare no conflict of interest.
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