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6CCVD Research

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

At a Glance

Section titled “At a Glance”
MetadataDetails
Publication Date2022-12-06
JournalC – Journal of Carbon Research
AuthorsMartin Menzler, Charity S. G. Ganskow, Maximilian Ruschig, Essam Moustafa, V. Sittinger
InstitutionsFraunhofer Institute for Surface Engineering and Thin Films, Technische UniversitÀt Braunschweig
Citations1
AnalysisFull AI Review Included

Executive Summary

Section titled “Executive Summary”

This study investigated the feasibility of using Boron-Doped Diamond (BDD) electrodes coupled with Electrochemical Impedance Spectroscopy (EIS) for rapid and precise biosensing, aiming toward future virus detection applications.

  • Core Goal: To establish a BDD-based EIS platform for biosensing using standard human Immunoglobulin G (IgG) as a model analyte, bridging the speed gap between lateral flow assays and the precision of PCR.
  • Electrode Material: Polycrystalline BDD films (6-8 ”m thick) were grown on Niobium substrates via Hot-Filament Activated Chemical Vapor Deposition (HFCVD) and doped with Boron (0.49 vol-% TMB).
  • Functionalization Success: A multi-step functionalization process (plasma APTMS treatment followed by maleimide linking and Fc-Cys engineered antibody immobilization) was successfully verified using fluorescence analysis.
  • EIS Testing: EIS was performed across a wide range of IgG antigen concentrations (1.1 pg/mL to 1.1 ”g/mL) in a ferricyanide electrolyte.
  • Primary Finding (Negative): No clear sign of change in the charge transfer resistance (Rct) was observed in either the impedance vs. frequency plots or the Nyquist plots across the tested antigen concentrations.
  • Conclusion & Outlook: A positive statement regarding successful IgG biosensing could not be made. Future work must address critical parameters, including BDD termination (H or O), the ratio of electrode size to electrolyte volume, and optimization of the EIS frequency range (especially lower frequencies).

Technical Specifications

Section titled “Technical Specifications”
ParameterValueUnitContext
Substrate MaterialNiobiumPlateUsed for BDD deposition
BDD Deposition MethodHFCVDN/ALarge-scale reactor (0.5 m2 coating area)
Substrate Temperature~950°CDeposition temperature
Carbon Source Concentration1.83vol-%Methane in Hydrogen
Boron Dopant Concentration0.49vol-%Trimethylborane (TMB)
CVD Gas Pressure20mbarDeposition environment
BDD Film Thickness6 to 8”mPolycrystalline diamond layer
Active Working Electrode Area95mm2Area exposed to electrolyte
EIS Electrolyte1 mM K3[Fe(CN)6]N/AIn 0.1 M Phosphate-Buffered Saline (PBS)
EIS Cell Volume10 (or 12)mL3-terminal chamber volume
EIS Frequency Range0.9 to 29kHzMeasurement range
EIS AC Amplitude10mVApplied alternating current signal
Analyte (IgG) Concentrations1.1 pg/mL, 1.1 ng/mL, 1.1 ”g/mLN/ATested range (six orders of magnitude)
Antibody Incubation Time3hCys-Antibody incubation at 23 °C

Key Methodologies

Section titled “Key Methodologies”

The BDD electrodes were prepared and functionalized using a multi-step chemical and physical process to ensure specific antibody attachment:

  1. BDD Growth: Niobium substrates were coated with polycrystalline BDD via HFCVD (950 °C, 20 mbar) using Methane/Hydrogen and Trimethylborane doping.
  2. Amino Functionalization: The BDD surface was functionalized with amino (NH2) groups using atmospheric-pressure plasma treatment.
    • Precursor: 3-aminopropyl-trimethoxysilane (APTMS) vaporized via Argon bubbling.
    • Process: Plasma stream directed onto the electrode surface, moving at 1 mm per second.
    • Verification: Successful NH2 addition confirmed by high fluorescence signal after incubation with FITC marker.
  3. Maleimide Linker Addition: Amino-modified electrodes were incubated with N-Succinimidyl 4-maleimidobutyrate (5 mM in dry DMSO, 3 h at 23 °C). This step installed a thiol-reactive maleimide moiety on the surface.
  4. Antibody Immobilization: An Fc-Cys engineered anti-human IgG antibody (0.1 mg/mL in PBS) was immobilized onto the maleimide surface via 1,4-conjugate thiol addition (3 h at 23 °C). This strategy ensures defined, oriented attachment through the C-terminal free Cysteine.
  5. Surface Blocking: The remaining unoccupied surface area was blocked using a 2%w skim milk powder solution (1 h at 23 °C) to minimize non-specific binding of the analyte.
  6. Antigen Binding Verification: Successful antibody immobilization was confirmed by incubating the surface with fluorescently labeled human IgG-AF488 antigen and measuring the resulting fluorescence signal.
  7. Electrochemical Impedance Spectroscopy (EIS): EIS was performed in a 3-terminal cell (Ag/AgCl reference, gold counter electrode) using a ferricyanide redox probe. Impedance was measured across 0.9 Hz to 29 kHz to monitor changes in charge transfer resistance upon antigen binding.

Commercial Applications

Section titled “Commercial Applications”

The research focuses on developing a robust, high-performance electrochemical biosensing platform, leveraging the unique properties of BDD.

  • Point-of-Care (POC) Diagnostics: Creating fast, reliable, and disposable test kits for infectious disease detection (e.g., viruses, bacteria) that surpass the reliability of current lateral flow assays.
  • High-Performance Electrochemical Sensing: Utilizing BDD’s wide potential window, fast charge-transfer kinetics, and low background current for sensitive detection of various biomolecules and pathogens.
  • Custom Antibody Engineering Platforms: Validating the use of Fc-Cys engineered antibodies for oriented, stable immobilization on carbon surfaces, crucial for maximizing sensor sensitivity and specificity.
  • Industrial CVD Manufacturing: Demonstrating the use of large-scale HFCVD reactors for producing cost-effective, high-quality BDD electrodes suitable for mass production of disposable sensors.
  • Biomarker Research: Providing a versatile platform for studying protein-protein interactions (antigen-antibody binding) and measuring low concentrations of specific biomarkers in complex biological fluids (blood, sputum).
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 ”g 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

Section titled “Tech Support”

Original Source

Section titled “Original Source”

References

Section titled “References”
  1. 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]
  2. 2021 - Current methods and prospects of coronavirus detection [Crossref]
  3. 2020 - Ultrasensitive detection of pathogenic viruses with electrochemical biosensor: State of the art [Crossref]
  4. 2010 - Impedimetric immunosensors—A review [Crossref]
  5. 2017 - A rapid-response ultrasensitive biosensor for influenza virus detection using antibody modified boron-doped diamond [Crossref]
  6. 2016 - Highly sensitive detection of influenza virus by boron-doped diamond electrode terminated with sialic acid-mimic peptide [Crossref]
  7. 2020 - Avian Influenza Virus Detection by Optimized Peptide Termination on a Boron-Doped Diamond Electrode [Crossref]
  8. 2019 - Biomolecular influenza virus detection based on the electrochemical impedance spectroscopy using the nanocrystalline boron-doped diamond electrodes with covalently bound antibodies [Crossref]
  9. 2021 - Boron doped diamond thin films for the electrochemical detection of SARS-CoV-2 S1 protein [Crossref]

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