Design of a Boron-Doped Diamond Microcell Grafted with HRP for the Sensitive and Selective Detection of Ochratoxin A

At a Glance

Metadata	Details
Publication Date	2023-03-05
Journal	Chemosensors
Authors	Amani Chrouda, Dhekra Ayed, Manahil Babiker Elamin, Shazalia Mahmoud Ahmed Ali, Laila M. Alhaidari
Institutions	Majmaah University, Centre National de la Recherche Scientifique
Citations	5
Analysis	Full AI Review Included

Executive Summary

This research details the fabrication and characterization of a highly sensitive, label-free electrochemical biosensor utilizing a Boron-Doped Diamond (BDD) microcell for the detection of Ochratoxin A (OTA).

Core Value Proposition: Achieves ultra-trace detection of OTA with a Limit of Detection (LOD) of 10 fM, significantly lower (one million times) than conventional ng/mL methods (e.g., HPLC, ELISA).
Sensor Architecture: A planar BDD working electrode is functionalized sequentially with a Diazonium Salt (DS) film, Single-Walled Carbon Nanotubes (SWCNTs), and covalently immobilized Horseradish Peroxidase (HRP) enzyme.
Transduction Mechanism: HRP oxidizes OTA in the presence of H₂O₂. The resulting oxidized OTA is detected via its reduction signal at -180 mV using Square Wave Voltammetry (SWV).
Performance Metrics: The sensor exhibits a wide linear range (10^-14 M to 0.1 M), high sensitivity (0.8 µA per decade), and excellent reproducibility (RSD of 5%).
Stability and Speed: Demonstrates robust long-term stability, retaining >85% of initial response after 30 days. Rapid analysis time is achieved (<3 minutes per measurement point) using a miniaturized wall-jet flow cell system.
Selectivity and Validation: Shows high selectivity against OTA analogs (Ochratoxin B, Aflatoxin B1, M1) and was successfully validated in complex real samples (corn, wheat, feed stuff) with results correlating well with LC-MS/MS.

Technical Specifications

Parameter	Value	Unit	Context
Limit of Detection (LOD)	10	fM	Ochratoxin A (OTA) detection
Linear Working Range	10^-14 to 0.1	M	OTA concentration range
Sensitivity	0.8	µA per decade	Calibration curve slope
Relative Standard Deviation (RSD)	5	%	Reproducibility
Long-Term Stability	>85	%	Response retained after 30 days (stored at +4 °C)
Measurement Time	<3	minutes	Per measuring point (SWV)
BDD Film Thickness	300	nm	Microcrystalline BDD layer
BDD Boron Concentration	>8000	ppm	Polycrystalline film
BDD Substrate Stack	Si/SiO₂/Si₃N₄	-	Insulating layer thickness: 0.5 mm
BDD Deposition Method	MPECVD	-	Microwave-assisted Plasma-Enhanced Chemical Vapor Deposition
Microcell Cutting Method	Femtosecond Laser	-	5 kHz, 2.5 W, 800 nm, 150 fs
SWV Potential Range	0.1 to -0.4	V	Reduction signal measurement
SWV Frequency	25	kHz	Measurement parameter
HRP Immobilization pH	11.0	-	Carbonate buffer solution
Contact Angle (Bare BDD)	77.4	°	Hydrophobic surface
Contact Angle (DS/SWCNTs)	25.4	°	Hydrophilic surface after modification

Key Methodologies

The biosensor fabrication involves precise surface engineering of the BDD microcell working electrode:

BDD Cleaning and Activation: BDD microcells were cleaned using 10 mL of Piranha solution (H₂SO₄/H₂O₂) for 5 minutes, followed by rinsing and nitrogen drying.
Diazonium Salt (DS) Grafting: 4-phenylenediamine was diazotized (20 mM HCl, 20 mM NaNO₂, 0 °C). DS was electro-addressed onto the BDD surface by scanning potential from 0.6 V to -400 mV (100 mV/s scan rate).
SWCNT Activation: Carboxylic acid groups of SWCNTs-COOH (1 mg/mL) were activated using EDC (1-ethyl-3-(3dimethylaminopropyl)-carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) in DMSO for 30 minutes.
HRP Covalent Immobilization: Activated SWCNTs-COOH were dispersed in 0.1 M carbonate buffer (pH 11) containing HRP (10 g/mL). This solution was injected into the wall-jet flow cell and kept in contact with the DS-functionalized BDD surface for 2 hours, forming stable amide bonds.
Electrochemical Characterization: Cyclic Voltammetry (CV) and Electrochemical Impedance Spectroscopy (EIS) were performed in 0.1 M PBS containing 5 mM Fe³⁺/Fe²⁺ redox probe to monitor surface conductivity changes.
OTA Detection (SWV): OTA samples (prepared in 0.1 M PBS with 1 mM H₂O₂) were analyzed using Square Wave Voltammetry (SWV) in the range of 0.1 V to -0.4 V, measuring the reduction peak of oxidized OTA at -180 mV.
Real Sample Preparation: Commercial samples (corn, wheat, feed) were extracted using acetonitrile/water (60:40 v/v), centrifuged, filtered, diluted, and passed through immunoaffinity columns (IAC) before analysis by the HRP biosensor and LC-MS/MS.

Commercial Applications

The robust, highly sensitive, and miniaturized BDD-based biosensor technology is highly relevant for industries requiring rapid, ultra-trace detection capabilities in complex matrices.

Food Safety and Quality Control:
- Rapid, on-site screening for mycotoxins (OTA) in cereals, coffee, wine, and dried fruits, meeting strict European Commission regulatory limits (2-5 ppb).
- High-throughput analysis systems for agricultural feed and raw materials.
Environmental Monitoring:
- Development of robust sensors for detecting trace organic pollutants and toxins in water sources, leveraging BDD’s wide potential window and stability.
Clinical and Veterinary Diagnostics:
- Point-of-Care (POC) devices for rapid screening of biomarkers or toxins in biological fluids, benefiting from the miniaturization offered by the BDD microcell.
Advanced Sensor Manufacturing (BDD Technology):
- Utilization of BDD’s superior properties (chemical inertness, mechanical hardness, stable background current) for creating high-stability electrochemical transducers.
- Integration of CVD diamond films into microfluidic and flow-cell analytical systems.

View Original Abstract

Ochratoxin A (OTA) is considered the most toxic member of the ochratoxin group. Herein, a novel label-free electrochemical sensor based on the horseradish peroxidase (HRP) enzyme is developed for OTA detection. The HRP enzyme was covalently immobilized on the working electrode of a planar boron-doped diamond (BDD) electrochemical microcell previously covered with diazonium film and grafted with single-walled carbon nanotubes (SWCNTs). Each surface modification step was evaluated by cyclic voltammetry and scanning electron microscopy. Square wave voltammetry was used for the detection of OTA. The linear working range of the biosensors ranged between 10−14 and 0.1 M, with a limit of detection (LOD) of 10 fM, an RSD equal to 5%, and a sensitivity of 0.8 µA per decade. In addition, the sensor showed good selectivity in the presence of OTA analogs; it was validated in samples such as corn, feed, and wheat. The metrological performance of the present sensor makes it a good alternative for OTA detection.

Tech Support

Original Source

DOI: https://doi.org/10.3390/chemosensors11030176

References

1965 - Ochratoxin A, a Toxic Metabolite produced by Aspergillus ochraceus Wilh
2018 - Ochratoxin A and human health risk: A review of the evidence
2020 - Risk assessment of ochratoxin A in food
2019 - Development and validation of a SPE-UHPLC-fluorescence method for the analysis of ochratoxin A in certain turkish wines [Crossref]
2023 - An enhanced immunochromatography assay based on gold growth on the surface of E. coli carrier for the simultaneous detection of mycotoxins [Crossref]
2011 - Determination of ochratoxin A in wine from the southern region of Brazil by thin layer chromatography with a charge-coupled detector [Crossref]
2020 - Recent Advances in Ochratoxin A Electrochemical Biosensors: Recognition Elements, Sensitization Technologies, and Their Applications [Crossref]
2018 - Label free aptasensor for ochratoxin A detection using polythiophene-3-carboxylic acid [Crossref]
2017 - An ultrasensitive amperometric immunosensor for zearalenones based on oriented antibody immobilization on a glassy carbon electrode modified with MWCNTs and AuPt nanoparticles [Crossref]