Controlled potential electro-oxidation of genomic DNA

At a Glance

Metadata	Details
Publication Date	2018-01-11
Journal	PLoS ONE
Authors	Vytas Reipa, Donald H. Atha, Sanem Hoşbaş Coşkun, Christopher M. Sims, Bryant C. Nelson
Institutions	National Institute of Standards and Technology, Material Measurement Laboratory
Citations	16
Analysis	Full AI Review Included

6CCVD Technical Documentation: Advanced Boron-Doped Diamond (BDD) for Controlled DNA Electro-Oxidation

Reference Paper: Reipa, V. et al. (2018). Controlled potential electro-oxidation of genomic DNA. PLOS ONE, 13(1): e0190907.

Executive Summary

This research validates the use of high surface area Boron-Doped Diamond (BDD) working electrodes (WE) for creating quantitative reference materials by inducing precise oxidative damage in genomic DNA. This application is critical for standardizing biomarkers of disease risk.

BDD Validation: Confirms BDD as the superior electrode material for establishing a stable and robust oxidative environment via potentiostatic control in aqueous solutions.
Target Application: Produces controlled levels of oxidatively modified DNA lesions (reference/calibration standards) for isotope-dilution Gas Chromatography/Mass Spectrometry (GC/MS/MS) analysis.
Dual Mechanism Control: Demonstrated the ability to selectively induce damage via two distinct mechanisms: direct electron abstraction (0.5 V < E < 1.5 V) and highly efficient hydroxyl radical (*OH) generation (E = 2.0 V).
High Radical Yield: At the critical potential of 2.0 V (vs Ag/AgCl), the BDD electrode efficiently produces highly reactive hydroxyl radicals via water electrolysis, leading to a significant (up to 30-fold) enhancement in purine lesion yields (specifically 8-OH-Gua).
Material Specification: The study utilized double-sided BDD electrodes with a substantial total active area (40 cm²), emphasizing the need for large-format, high-quality CVD diamond components.
Commercial Potential: This technique offers a viable chemical-oxidant-free method to develop dependable DNA damage reference materials, fulfilling a long-standing need in biomedical diagnostics and quality assurance.

Technical Specifications

The core electrochemical setup relies on highly specific materials and precise operational parameters, leveraging the extreme electrochemical stability of BDD.

Parameter	Value	Unit	Context
Working Electrode (WE) Material	Boron-Doped Diamond (BDD)	N/A	High surface area anode, efficient `*OH` generator
WE Total Active Area (S)	40	cm²	Double-sided BDD configuration
Electrolyte Solution	0.01 mol/L Phosphate Buffer (pH 7.0)	N/A	Used for bulk electrolysis
DNA Concentration (ct-DNA)	250	µg/mL	Loaded into the electrochemical cell
Counter Electrode (CE) Material	Highly Doped Silicon (Si)	N/A	Separated by 2000 MW cutoff dialysis bag
Reference Electrode (RE)	Flexible Ag/AgCl (saturated KCl)	N/A	Standard reference for potential citation
Applied Potentials (E)	0.5, 1.0, 1.5, 2.0	V	Fixed potentiostatic treatments (vs Ag/AgCl)
*Critical Potential for `OH`**	E > 1.6 V	V	Onset of H₂O oxidation on BDD
BDD Activation Cycle	-1 V to +2.5 V at 20 mV/s	N/A	Pre-treatment cycle duration: 30 min
Treatment Duration	1	h	Duration of potentiostatic exposure
Max Lesion Ratio (E=2.0 V)	Approx. 30:1	N/A	8-OH-Gua yield compared to 8-OH-Ade yield
DNA Molecular Weight (Average)	Approx. 800,000	N/A	Requires dialysis cutoff (2000 MW) to prevent CE access

Key Methodologies

The experiment successfully employed controlled potential electro-oxidation using CVD diamond to achieve reproducible generation of oxidative DNA lesions.

Electrode Fabrication: High surface area BDD working electrodes (total S = 40 cm²) and highly doped Si counter electrodes (S = 60 cm²) were constructed as double-sided plates. Contact leads were produced using silver epoxy.
Electrode Activation: Prior to DNA introduction, electrodes were electrochemically activated in buffer solution by cycling the potential between -1 V and +2.5 V (at 20 mV/s) for 30 minutes.
Cell Setup and Environment: A three-electrode cell was used for bulk electrolysis. The Si counter electrodes were enclosed in dialysis bags (2000 MW cutoff) to prevent DNA molecules from accessing the CE surface and undergoing reverse reactions.
Solution Equilibration: 2 mL of DNA solution (250 µg/mL in 0.01 mol/L phosphate buffer, pH 6.9) was slowly purged with inert Argon (Ar) gas for 30 minutes at the open circuit potential to establish stable redox conditions.
Potentiostatic Treatment: Fixed oxidizing potentials (0.5 V, 1.0 V, 1.5 V, 2.0 V vs Ag/AgCl) were applied to the BDD WE for 1 hour (h) to induce oxidative damage.
DNA Lesion Analysis: Treated DNA was collected, enzymatically digested, and analyzed for five key oxidative lesions (FapyAde, 8-OH-Ade, 5-OH-5-MeHyd, FapyGua, and 8-OH-Gua) using isotope-dilution GC/MS/MS.

6CCVD Solutions & Capabilities

The successful replication and scaling of this high-impact research rely directly on the superior material quality and advanced fabrication capabilities provided by 6CCVD’s MPCVD diamond platforms.

Applicable Materials

To achieve the highly stable, radical-generating environment demonstrated in this study, high-quality Heavy Boron-Doped Diamond (BDD) is mandatory.

6CCVD Material Recommendation	Required Characteristics & Application
Heavy Boron-Doped Diamond (BDD) Plates	High surface area, metallic conductivity, and high overpotential for oxygen evolution, ensuring efficient hydroxyl radical generation at E = 2.0 V.
Optical Grade SCD (Reference/Control)	Suitable for optical studies or systems requiring extremely low defect density and high purity, useful when scaling down electrode size for mechanistic studies.
Highly Doped Silicon Substrates	Available for counter electrode fabrication, aligning with the material specified in the reference paper.

Customization Potential

The research utilized custom, large-format electrodes (40 cm²) and specific electrical contacts. 6CCVD provides the necessary engineering capabilities to meet or exceed these requirements for industrial scale-up or advanced R&D.

Custom Dimensions and Geometry: 6CCVD specializes in producing high-surface area BDD plates/wafers up to 125mm. Our in-house laser cutting services enable the precise fabrication of electrodes matching the 40 cm² double-sided configuration or unique custom shapes required for specialized electrochemical reactors.
Precision Metalization: The study noted the challenge of electrical contact (using silver epoxy). 6CCVD offers expert in-house metalization services (including Au, Pt, Ti, W, and Cu). This ensures reliable, low-resistance ohmic contacts and robust lead integration, vital for maintaining stable potentiostatic control across large electrode areas.
Polishing Standards: For high-precision applications or micro-patterning of BDD, 6CCVD delivers polishing to ultra-low roughness standards: Ra < 5nm for Inch-size PCD.

Engineering Support

This study highlights a critical need for standardized reference materials in redox biology. 6CCVD’s in-house PhD team provides expert technical consultancy to accelerate similar projects.

Application Expertise: Our engineers can assist partners in selecting the optimal BDD doping levels and reactor geometry necessary to maximize hydroxyl radical generation yield for controlled DNA oxidation projects or other advanced electrochemical synthesis applications.
Scale-Up Consultation: We offer support for transitioning laboratory-scale BDD devices (like the 40 cm² system described) into robust, large-scale production reactors.
Global Supply Chain: 6CCVD ensures reliable global shipping (DDU default, DDP available), providing materials necessary to replicate and advance this research worldwide.

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

View Original Abstract

Exposure of mammalian cells to oxidative stress can result in DNA damage that adversely affects many cell processes. Lack of dependable DNA damage reference materials and standardized measurement methods, despite many case-control studies hampers the wider recognition of the link between oxidatively degraded DNA and disease risk. We used bulk electrolysis in an electrochemical system and gas chromatographic mass spectrometric analysis (GC/MS/MS) to control and measure, respectively, the effect of electrochemically produced reactive oxygen species on calf thymus DNA (ct-DNA). DNA was electro-oxidized for 1 h at four fixed oxidizing potentials (E = 0.5 V, 1.0 V, 1.5 V and 2 V (vs Ag/AgCl)) using a high surface area boron-doped diamond (BDD) working electrode (WE) and the resulting DNA damage in the form of oxidatively-modified DNA lesions was measured using GC/MS/MS. We have shown that there are two distinct base lesion formation modes in the explored electrode potential range, corresponding to 0.5 V < E < 1.5 V and E > 1.5 V. Amounts of all four purine lesions were close to a negative control levels up to E = 1.5 V with evidence suggesting higher levels at the lowest potential of this range (E = 0.5 V). A rapid increase in all base lesion yields was measured when ct-DNA was exposed at E = 2 V, the potential at which hydroxyl radicals were efficiently produced by the BDD electrode. The present results demonstrate that controlled potential preparative electrooxidation of double-stranded DNA can be used to purposely increase the levels of oxidatively modified DNA lesions in discrete samples. It is envisioned that these DNA samples may potentially serve as analytical control or quality assurance reference materials for the determination of oxidatively induced DNA damage.

Tech Support

Original Source

DOI: https://doi.org/10.1371/journal.pone.0190907

References

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