Testing of Diamond Electrodes as Biosensor for Antibody-Based Detection of Immunoglobulin Protein with Electrochemical Impedance Spectroscopy

At a Glance

Metadata	Details
Publication Date	2022-12-06
Journal	C – Journal of Carbon Research
Authors	Martin Menzler, Charity S. G. Ganskow, Maximilian Ruschig, Essam Moustafa, V. Sittinger
Institutions	Technische Universität Braunschweig, Fraunhofer Institute for Surface Engineering and Thin Films
Citations	1
Analysis	Full AI Review Included

Technical Documentation & Analysis: Diamond Electrodes for EIS Biosensing

Executive Summary

This research successfully demonstrates the feasibility of preparing highly functionalized Boron-Doped Diamond (BDD) electrodes for Electrochemical Impedance Spectroscopy (EIS) biosensing, targeting rapid and precise virus detection.

Material Validation: Polycrystalline BDD films (6-8 µm thick) were successfully deposited via Hot-Filament CVD (HFCVD) onto Niobium substrates, confirming BDD as a viable high-performance electrode material for biosensors due to its wide potential window and low background current.
Surface Engineering Success: A robust, multi-step functionalization protocol was established, including Atmospheric Pressure Plasma treatment using APTMS to introduce amino (NH₂) groups, followed by the successful immobilization of Fc-modified anti-human IgG antibodies.
Methodology: The study utilized EIS to monitor changes in charge transfer resistance upon antigen binding (Human IgG), employing a frequency range of 0.9 Hz to 29 kHz.
Optimization Required: While functionalization was confirmed, the EIS results did not show a clear correlation between antigen concentration and impedance change. The authors recommend critical optimization steps, including varying BDD surface termination (H vs. O), adjusting electrode size/electrolyte volume ratio, and expanding the low-frequency EIS range.
6CCVD Value Proposition: 6CCVD is uniquely positioned to supply the high-quality, customizable BDD materials and surface engineering required to address the identified optimization challenges, accelerating the path toward a fast and precise virus detection platform.

Technical Specifications

The following hard data points were extracted from the research paper detailing the BDD electrode fabrication and EIS measurement parameters.

Parameter	Value	Unit	Context
Diamond Material Type	Boron-Doped Polycrystalline Diamond (BDD)	N/A	HFCVD deposited on Niobium
Substrate Dimensions	50 x 50 x 2	mm	Niobium plate
Diamond Film Thickness	6 to 8	µm	Polycrystalline layer
Final Working Electrode Size	10 x 10	mm	Laser-cut active area (95 mm²)
Carbon Source Concentration	1.83	vol-%	Methane in Hydrogen
Boron Dopant Concentration	0.49	vol-%	Trimethylborane (TMB) for electrical conductivity
Deposition Temperature	~950	°C	Hot-Filament CVD process
CVD Gas Pressure	20	mbar	Deposition pressure
Electrolyte Volume (EIS)	10 (or 12)	mL	Used in 3-terminal chamber
EIS Frequency Range	0.9 Hz to 29	kHz	Measurement range
AC Amplitude (EIS)	10 ± 10%	mV	Applied signal
DC Bias (EIS)	0	V	Measurement condition
Analyte Concentration Range	1.1 pg/mL to 1.1 µg/mL	N/A	Human IgG antigen tested

Key Methodologies

The experimental procedure involved precise material synthesis, surface functionalization, and electrochemical analysis.

Substrate Preparation: Niobium plates (50 mm x 50 mm) were sandblasted, cleaned, and seeded by dipping in a suspension of nanometer-sized diamond particles.
BDD Deposition (HFCVD): Polycrystalline diamond was grown using HFCVD at ~950 °C and 20 mbar. The gas mixture included 1.83 vol-% CH₄ in H₂, doped with 0.49 vol-% Trimethylborane. The resulting BDD film thickness was 6-8 µm.
Electrode Fabrication: The coated plates were laser-cut to the required 10 mm x 10 mm working electrode size.
Amino Functionalization (NH₂): Electrodes were treated with Atmospheric Pressure Plasma using 3-aminopropyl-trimethoxysilane (APTMS) precursor to introduce NH₂ groups, confirmed via fluorescence analysis using FITC marker.
Antibody Immobilization:
- Linker Addition: N-Succinimidyl 4-maleimidobutyrate was added to the NH₂ surface.
- Antibody Binding: Fc-modified anti-human IgG antibody (containing a free Cysteine) was linked via 1,4-conjugate thiol addition, ensuring well-defined attachment.
Surface Blocking: Unoccupied surface sites were blocked using a 2 %w skim milk powder solution in PBS buffer (pH 7.4).
Electrochemical Analysis (EIS): Measurements were performed in a 3-terminal cell (Ag/AgCl reference, Gold counter) using 1 mM K₃[Fe(CN)₆] redox probe, sweeping frequencies from 0.9 Hz to 29 kHz with a 10 mV AC amplitude.

6CCVD Solutions & Capabilities

6CCVD provides the high-quality, customizable MPCVD diamond materials and engineering expertise necessary to overcome the limitations encountered in this initial study and advance the BDD biosensor platform.

Applicable Materials

To replicate this research and address the identified need for comparative studies and surface optimization, 6CCVD recommends the following materials:

Material Specification	Application in Research Extension	6CCVD Capability
Boron-Doped PCD (BDD)	High-conductivity working electrodes (6-8 µm thickness) for EIS biosensing.	Standard BDD wafers/plates up to 125 mm diameter. Custom doping levels available to optimize conductivity (0.49 vol-% TMB used here).
Hydrogen-Terminated BDD	Required for investigating the effect of surface termination on functionalization and charge transfer kinetics, as suggested in the Discussion.	6CCVD offers precise control over surface termination (H-terminated default or O-terminated via post-processing).
Optical Grade SCD	Ideal for fundamental studies requiring ultra-low defect density and atomically smooth surfaces (Ra < 1nm) to minimize background noise and improve signal clarity.	SCD plates available up to 10 mm x 10 mm, polished to Ra < 1nm.
BDD Substrates (Thick)	For robust, reusable sensor platforms or high-power applications.	Substrates available up to 10 mm thickness.

Customization Potential

The research highlights several areas where custom fabrication is essential for optimization:

Custom Dimensions and Cutting: The paper used 10 mm x 10 mm electrodes laser-cut from 50 mm plates. 6CCVD offers custom laser cutting and shaping services to produce electrodes of any required size and geometry, optimizing the BDD electrode size to electrolyte volume ratio (a key improvement area identified by the authors).
Surface Termination Control: The authors noted the critical need to vary BDD termination (H vs. O). 6CCVD guarantees precise control over surface termination, allowing researchers to systematically test the effect of H-termination (known for faster electron transfer) versus O-termination (often preferred for wet chemical functionalization).
Integrated Metalization: While the paper used a separate Gold wire counter electrode, 6CCVD offers in-house metalization services (Au, Pt, Pd, Ti, W, Cu). This capability allows for the integration of counter and reference electrodes directly onto the diamond substrate, creating compact, high-performance, disposable sensor chips.

Engineering Support

6CCVD’s in-house PhD team specializes in diamond material science and electrochemical applications. We can assist researchers in:

Material Selection and Doping: Optimizing the boron doping concentration and film thickness to achieve the ideal balance between conductivity and surface quality for EIS measurements.
Surface Chemistry Optimization: Consulting on the best approach for achieving stable and high-density functionalization, including pre-treatment methods (e.g., cathodic pre-treatment) and termination control (H/O) for similar Electrochemical Biosensing projects.
Scaling and Manufacturing: Providing expertise for transitioning successful lab-scale designs (10 mm x 10 mm) to larger, cost-effective, inch-size Polycrystalline Diamond (PCD) wafers suitable for industrial production of disposable diagnostic kits.

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

View Original Abstract

To control the increasing virus pandemics, virus detection methods are essential. Today’s standard virus detections methods are fast (immune assays) or precise (PCR). A method that is both fast and precise would enable more efficient mitigation measures and better life comfort. According to recent papers, electrochemical impedance spectroscopy (EIS) has proven to detect viruses fast and precise. Boron-doped diamond (BDD) was used as a high-performance electrode material in these works. The aim of this work was to perform an initial test of BDD-based EIS for biosensing. As an easily available standard biomaterial, human immunoglobulin G (IgG) was used as analyte. Niobium plates were coated via hot-filament activated chemical vapor deposition with polycrystalline diamond, and doped with boron for electrical conductivity. An anti-human IgG antibody was immobilised on the BDD electrodes as a biosensing component. Four different analyte concentrations up to 1.1 µg per litre were tested. During EIS measurements, both impedance over frequency curves and Nyquist plot demonstrated no clear sign of a change of the charge transfer resistance. Thus, no positive statement about a successful biosensing could be made so far. It is assumed that these issues need to be investigated and improved, including the relation of BDD electrode size to electrolyte volume, termination of the BDD electrodes (H, O) for a successful functionalisation and EIS frequency range. The work will be continued concerning these improvement issues in order to finally use virus materials as analyte.

Tech Support

Original Source

DOI: https://doi.org/10.3390/c8040074

References

2020 - Getting Through COVID-19: The Pandemic’s Impact on the Psychology of Sustainability, Quality of Life, and the Global Economy—A Systematic Review [Crossref]
2021 - Current methods and prospects of coronavirus detection [Crossref]
2020 - Ultrasensitive detection of pathogenic viruses with electrochemical biosensor: State of the art [Crossref]
2010 - Impedimetric immunosensors—A review [Crossref]
2017 - A rapid-response ultrasensitive biosensor for influenza virus detection using antibody modified boron-doped diamond [Crossref]
2016 - Highly sensitive detection of influenza virus by boron-doped diamond electrode terminated with sialic acid-mimic peptide [Crossref]
2020 - Avian Influenza Virus Detection by Optimized Peptide Termination on a Boron-Doped Diamond Electrode [Crossref]
2019 - Biomolecular influenza virus detection based on the electrochemical impedance spectroscopy using the nanocrystalline boron-doped diamond electrodes with covalently bound antibodies [Crossref]
2021 - Boron doped diamond thin films for the electrochemical detection of SARS-CoV-2 S1 protein [Crossref]