Ultrasensitive Diamond Microelectrode Application in the Detection of Ca2+ Transport by AnnexinA5-Containing Nanostructured Liposomes

At a Glance

Metadata	Details
Publication Date	2022-07-14
Journal	Biosensors
Authors	A. Pasquarelli, Luiz H. S. Andrilli, Maytê Bolean, Claudio R. Ferreira, Marcos Antônio Eufrásio Cruz
Institutions	Universidade de São Paulo, Universidade de Ribeirão Preto
Citations	8
Analysis	Full AI Review Included

Technical Documentation & Analysis: Ultrasensitive BNCD Microelectrodes for Ion Transport

6CCVD Material Scientist Analysis of “Ultrasensitive Diamond Microelectrode Application in the Detection of Ca²⁺ Transport by AnnexinA5-Containing Nanostructured Liposomes”

Executive Summary

This research validates the superior sensitivity and selectivity of Boron-Doped Nanocrystalline Diamond (BNCD) microelectrodes for complex biological ion transport studies, a critical application area for 6CCVD’s advanced MPCVD materials.

High Sensitivity Validation: BNCD microelectrodes successfully tracked minute changes in Ca²⁺ concentration due to binding and transport within proteoliposomes, a measurement impossible using common Ca²⁺ selective electrodes.
Material Advantage: The BNCD devices exhibited excellent electrochemical properties, including a wide water dissociation potential window (~3 V) and very low background current (<1 µA/cm²), essential for high-fidelity biosensing.
Selective Transport Confirmed: The diamond microelectrodes demonstrated the selective transport function of Annexin A5 (AnxA5), showing a significant potential drop (Ca²⁺ uptake rate of 2.5 mV/s) in the presence of Ca²⁺, but negligible response to Mg²⁺ (0.3 mV/s).
Custom Fabrication Requirement: The successful device relied on a complex, multi-layer MPCVD stack (a-Si/i-NCD/BNCD) on a sapphire substrate, requiring precise thickness control and high-resolution lithography (16 µm diameter openings).
6CCVD Value Proposition: 6CCVD specializes in replicating and optimizing these exact custom MPCVD diamond stacks, offering the required BDD material, precise thickness control (down to 0.1 µm), and advanced lithographic patterning necessary for next-generation microelectrode arrays (MEAs).

Technical Specifications

The following hard data points were extracted from the research paper regarding the BNCD microelectrode fabrication and performance:

Parameter	Value	Unit	Context
Diamond Material	Boron-Doped Nanocrystalline Diamond (BNCD)	N/A	Active sensing layer
Substrate	Sapphire Wafer	N/A	Base material for planar array
Buffer Layer (a-Si)	~60	nm	Thermal expansion mismatch buffer
Intrinsic NCD (i-NCD) Thickness	~500	nm	Grown on a-Si layer
BNCD Active Layer Thickness	~250	nm	Grown on i-NCD layer
Passivation Layer	Silicon Nitride (PECVD)	1 µm	Insulation layer
Microelectrode Diameter	16	µm	Opening in passivation layer
Electrode Array Type	Planar Four-Channel	N/A	Used for simultaneous recording
Input Bias Current (Readout)	1	pA	High impedance measurement
Input Impedance (Readout)	10	TΩ	High impedance measurement
Ca²⁺ Uptake Rate (AnxA5)	2.5	mV/s	Selective transport confirmation
Mg²⁺ Uptake Rate (AnxA5)	0.3	mV/s	Confirms Ca²⁺ selectivity
Water Dissociation Window	~3	V	Excellent electrochemical stability
Background Current	<1	µA/cm²	Low noise performance

Key Methodologies

The BNCD microelectrode array fabrication utilized advanced MPCVD growth and high-resolution patterning techniques, all within 6CCVD’s core capabilities.

Substrate Preparation: Cleaning of a blank sapphire wafer.
Buffer Layer Deposition: Deposition of a ~60 nm amorphous silicon (a-Si) layer to buffer thermal expansion mismatch and promote diamond nucleation.
Intrinsic Diamond Growth: Growth of a ~500 nm thick film of intrinsic Nanocrystalline Diamond (i-NCD) via MPCVD.
Boron Doping: Growth of a ~250 nm thick film of Boron-Doped Nanocrystalline Diamond (BNCD) via MPCVD.
Pattern Transfer (Conducting Structures): Optical lithography, metal mask protection, and Reactive Ion Etching (RIE) in an argon-oxygen atmosphere to define the conducting structures.
Passivation Layer Deposition: Deposition of a 1 µm thick silicon nitride passivation layer using Plasma-Enhanced CVD (PECVD).
Microelectrode Opening: Optical lithography and RIE in a tetrafluoromethane (CF₄) atmosphere to transfer the pattern of the microelectrode openings (16 µm diameter) and bonding pads.
Surface Termination: Exposure to mild oxygen plasma for 10 minutes to provide the desired hydrophilic, oxidized surface termination, crucial for potentiometric measurements.

6CCVD Solutions & Capabilities

This research highlights the critical role of highly customized, high-quality MPCVD diamond in advancing ultrasensitive biosensing. 6CCVD is uniquely positioned to supply the materials and fabrication services required to replicate, scale, and extend this work.

Applicable Materials

To replicate or extend the BNCD microelectrode array described in this paper, 6CCVD recommends the following materials:

Material Requirement	6CCVD Solution	Technical Rationale
Boron-Doped NCD (BNCD)	Heavy Boron-Doped PCD (Polycrystalline Diamond)	Provides the quasi-metallic conductivity and electrochemical stability required for potentiometric sensing. We offer precise doping control for optimal Nernstian response.
Intrinsic NCD/SCD	Optical Grade SCD or High Purity PCD	Required for the intrinsic buffer layer (~500 nm) to ensure high material quality and interface control in the multi-layer stack.
Custom Substrate	Custom Substrate Integration	We routinely grow diamond films on non-standard substrates (e.g., Sapphire, SiC, Quartz) to meet specific device integration needs, replicating the sapphire wafer used here.

Customization Potential

The success of this device hinges on precise control over layer thickness, doping, and micro-patterning. 6CCVD offers full customization capabilities to meet these exact engineering requirements:

Custom Layer Stacks & Thickness Control: We specialize in growing complex, multi-layer diamond stacks (e.g., a-Si/i-NCD/BNCD) with thickness precision from 0.1 µm to 500 µm, matching the 500 nm i-NCD and 250 nm BNCD layers used in this study.
High-Resolution Patterning: We offer advanced lithography and RIE services to create custom geometries, including the critical 16 µm diameter microelectrode openings and bonding pads, ensuring high device density and yield.
Passivation and Termination: While the paper used SiN passivation, 6CCVD can integrate various dielectric layers and provides controlled surface termination (e.g., oxygen or hydrogen) necessary for optimizing electrochemical sensitivity and stability.
Metalization Services: Although not the primary focus of the sensing element, device integration often requires metal contacts. 6CCVD offers in-house metalization (Au, Pt, Pd, Ti, W, Cu) for robust electrical connections and bonding pads.

Engineering Support

The investigation of selective Ca²⁺ transport mediated by Annexin A5 is a highly specialized application in Biomimetic Sensing and Ion Channel Research.

6CCVD’s in-house PhD team provides expert consultation on material selection, doping levels, and surface preparation protocols to optimize diamond performance for similar projects, including:

Developing high-density MEAs for in vivo and in vitro electrophysiology.
Optimizing BDD films for specific ion-selective potentiometric measurements.
Designing custom diamond structures for integration into microfluidic or implantable biosensors.

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

View Original Abstract

This report describes the innovative application of high sensitivity Boron-doped nanocrystalline diamond microelectrodes for tracking small changes in Ca2+ concentration due to binding to Annexin-A5 inserted into the lipid bilayer of liposomes (proteoliposomes), which could not be assessed using common Ca2+ selective electrodes. Dispensing proteoliposomes to an electrolyte containing 1 mM Ca2+ resulted in a potential jump that decreased with time, reaching the baseline level after ~300 s, suggesting that Ca2+ ions were incorporated into the vesicle compartment and were no longer detected by the microelectrode. This behavior was not observed when liposomes (vesicles without AnxA5) were dispensed in the presence of Ca2+. The ion transport appears Ca2+-selective, since dispensing proteoliposomes in the presence of Mg2+ did not result in potential drop. The experimental conditions were adjusted to ensure an excess of Ca2+, thus confirming that the potential reduction was not only due to the binding of Ca2+ to AnxA5 but to the transfer of ions to the lumen of the proteoliposomes. Ca2+ uptake stopped immediately after the addition of EDTA. Therefore, our data provide evidence of selective Ca2+ transport into the proteoliposomes and support the possible function of AnxA5 as a hydrophilic pore once incorporated into lipid membrane, mediating the mineralization initiation process occurring in matrix vesicles.

Tech Support

Original Source

DOI: https://doi.org/10.3390/bios12070525

References

2001 - PH Sensing by Surface-Doped Diamond Effect of the Diamond Surface Termination [Crossref]
2007 - PH Sensor on O-Terminated Diamond Using Boron-Doped Channel [Crossref]
2021 - Boron-doped diamond (BDD) electro-oxidation coupled with nanofiltration for secondary wastewater treatment: Antibiotics degradation and biofouling [Crossref]
2021 - Electrochemical degradation of per- and poly-fluoroalkyl substances using boron-doped diamond electrodes [Crossref]
2020 - In vivo feasibility of epiretinal stimulation using ultrananocrystalline diamond electrodes [Crossref]
2021 - Laminin coated diamond electrodes for neural stimulation [Crossref]
2020 - Hybrid diamond/ carbon fiber microelectrodes enable multimodal electrical/chemical neural interfacing [Crossref]
2006 - Electroanalytical applications of boron-doped diamond microelectrode arrays [Crossref]
2016 - Electrochemical Determination of Sulphur-containing Pharmaceuticals Using Boron-doped Diamond Electrodes [Crossref]
2014 - Boron doped diamond biotechnology: From sensors to neurointerfaces [Crossref]