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	Centre National de la Recherche Scientifique, Institut des Sciences Analytiques
Citations	5
Analysis	Full AI Review Included

Technical Documentation & Analysis: Ultra-Sensitive Boron-Doped Diamond Biosensors for Ochratoxin A Detection

Executive Summary

This research validates the superior performance of Microwave Plasma Chemical Vapor Deposition (MPCVD) Boron-Doped Diamond (BDD) microcells as a robust platform for ultra-sensitive electrochemical biosensing.

Record Sensitivity: Achieved an exceptionally low Limit of Detection (LOD) of 10 fM (4.04 x 10^-6 µg/kg) for Ochratoxin A (OTA), significantly surpassing conventional immunosensors and aptasensors by up to one million times.
Material Validation: Confirms the efficacy of highly boron-doped polycrystalline diamond (PCD/BDD) films (300 nm thick, > 8000 ppm B concentration) for high-performance electrochemical applications.
Wide Dynamic Range: Demonstrated a broad linear working range spanning 13 orders of magnitude (10^-14 M to 0.1 M), crucial for real-world sample analysis.
Miniaturization & Speed: The BDD microcell, integrated into a wall-jet flow cell, enables rapid analysis, requiring less than 3 minutes per measurement point.
Enhanced Stability: The covalent immobilization of Horseradish Peroxidase (HRP) onto the BDD/SWCNT matrix resulted in excellent long-term stability, retaining over 85% of initial response after 30 days of storage.
Fabrication Expertise: The successful creation of complex three-electrode microcells via femtosecond laser micromachining validates advanced diamond processing techniques.

Technical Specifications

The following hard data points were extracted from the study detailing the material and performance metrics of the BDD biosensor:

Parameter	Value	Unit	Context
Diamond Material	Polycrystalline BDD	N/A	Deposited via MPECVD
BDD Film Thickness	300	nm	Working electrode layer
Substrate Thickness	0.5	mm	Si/SiO₂/Si₃N₄ insulating layer
Boron Concentration	> 8000	ppm	High doping level for conductivity
Electrode Configuration	3	N/A	Working, Counter, Pseudo Reference
Micromachining Tool	Femtosecond Laser	N/A	Used for microcell fabrication
Detection Method	Square Wave Voltammetry (SWV)	N/A	Used for OTA reduction signal
Linear Working Range	10^-14 to 0.1	M	Wide dynamic range
Limit of Detection (LOD)	10	fM	Equivalent to 4.04 x 10^-6 µg/kg
Sensitivity	0.8	µA per decade	Calibration curve slope
Reproducibility (RSD)	5	%	Relative Standard Deviation
Measurement Time	< 3	minutes	Per measuring point
Storage Stability	> 85	%	Response retained after 30 days at 4 °C

Key Methodologies

The fabrication and characterization of the BDD electrochemical biosensor involved precise MPCVD growth and multi-step surface functionalization:

BDD Film Deposition: Microcrystalline BDD film (300 nm) with high boron concentration (> 8000 ppm) was deposited via Microwave-Assisted Plasma-Enhanced Chemical Vapor Deposition (MPECVD) onto an insulating Si/SiO₂/Si₃N₄ substrate (0.5 mm thick).
Microcell Fabrication: The three-electrode system (working, counter, pseudo reference) was precisely cut from the BDD film using femtosecond laser micromachining (5 kHz, 2.5 W, 800 nm, 150 fs).
Surface Cleaning and Activation: BDD microcells were cleaned using Piranha solution (H₂SO₄/H₂O₂) followed by rinsing and nitrogen drying.
Diazonium Salt (DS) Grafting: 4-phenylenediamine was diazotated (20 mM HCl, 20 mM NaNO₂ at 0 °C) and electro-addressed onto the BDD working electrode surface via cyclic voltammetry (0.6 V to -400 mV).
SWCNT Functionalization: Carboxylic groups of SWCNTs-COOH (1 mg/mL) were activated using EDC/NHS coupling agents in DMSO.
HRP Covalent Immobilization: Activated SWCNTs-COOH were dispersed in carbonate buffer (pH 11) containing HRP (10 g/mL) and covalently grafted onto the DS-functionalized BDD surface for 2 hours.
Electrochemical Detection: Square Wave Voltammetry (SWV) was performed in 0.1 M PBS with 1 mM H₂O₂, measuring the reduction signal of oxidized OTA at -180 mV.

6CCVD Solutions & Capabilities

6CCVD is uniquely positioned to supply the advanced diamond materials and precision fabrication services required to replicate, scale, and advance this high-performance biosensing technology.

Applicable Materials

The core of this research relies on highly conductive, microcrystalline BDD films. 6CCVD offers materials perfectly matched to these specifications:

Heavy Boron Doped Polycrystalline Diamond (BDD): We supply MPCVD BDD wafers and plates with boron concentrations exceeding the > 8000 ppm requirement, ensuring the high conductivity necessary for electrochemical microcells.
Custom Substrates: While the paper used Si/SiO₂/Si₃N₄, 6CCVD can provide BDD films deposited on various insulating or conductive substrates, including silicon, quartz, or specialized ceramics, tailored to specific device integration needs.
Thickness Control: We offer precise control over BDD film thickness, ranging from 0.1 µm up to 500 µm, allowing researchers to optimize the electrochemical properties and mechanical stability of the microcell structure.

Customization Potential

The fabrication of the BDD microcell requires advanced post-processing capabilities, which are standard offerings at 6CCVD:

Research Requirement	6CCVD Customization Capability	Value Proposition
Microcell Geometry	Precision femtosecond laser cutting and etching services.	Replication of complex three-electrode geometries (Working, Counter, Reference) with micron-level accuracy.
Wafer Size	Plates/wafers up to 125 mm (5 inches) in diameter.	Enables high-throughput fabrication of 29+ microcells per wafer, facilitating scale-up and commercialization efforts.
Electrode Contacts	Custom metalization (Au, Pt, Pd, Ti, W, Cu) via sputtering or evaporation.	While the paper used pseudo-reference electrodes, 6CCVD can integrate stable, defined metal contacts (e.g., Ti/Pt/Au) for enhanced electrical performance and long-term stability.
Surface Finish	Polishing services for PCD (Ra < 5 nm) and SCD (Ra < 1 nm).	Ensures ultra-smooth surfaces critical for uniform functionalization (Diazonium, SWCNT, HRP grafting) and reproducible electrochemical response.

Engineering Support

The successful development of this OTA biosensor hinges on optimizing the BDD material properties (doping level, thickness) and the subsequent surface chemistry.

Application Expertise: 6CCVD’s in-house PhD team specializes in electrochemical diamond applications, including biosensors, water treatment, and electroanalysis. We can assist researchers in selecting the optimal BDD grade and surface termination (e.g., hydrogen- or oxygen-terminated) for similar enzymatic or aptamer-based detection projects.
Process Optimization: We provide consultation on integrating diamond materials into flow cells and microfluidic systems, ensuring seamless transition from lab-scale prototypes to scalable analytical devices.

For custom specifications or material consultation, visit 6ccvd.com or contact our engineering team directly.

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]