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	Universität Ulm, Universidade de São Paulo
Citations	8
Analysis	Full AI Review Included

Executive Summary

The research details the innovative application of Boron-doped Nanocrystalline Diamond (BNCD) microelectrodes for ultrasensitive potentiometric tracking of Ca²⁺ transport kinetics in biomimetic systems.

Core Innovation: BNCD microelectrodes are utilized to track small, transient changes in Ca²⁺ concentration resulting from ion uptake into AnnexinA5 (AnxA5) proteoliposomes, a process previously undetectable by conventional Ca²⁺ selective electrodes.
Ultrasensitivity Demonstrated: The device successfully measured the potential drop associated with Ca²⁺ internalization, confirming the high sensitivity of the BNCD surface to cumulative electrochemical potential changes.
Selective Transport: AnxA5-mediated ion transport was confirmed to be highly selective for Ca²⁺ over Mg²⁺. The initial Ca²⁺ uptake rate was measured at 2.5 mV/s, significantly higher than the 0.3 mV/s rate observed for Mg²⁺.
Mechanism Confirmation: The potential reduction was confirmed to be due to ion transfer into the vesicle lumen, not just surface binding, as the uptake ceased immediately upon the addition of the chelating agent EDTA.
Biological Relevance: The data supports the hypothesis that AnxA5, when incorporated into the lipid membrane, functions as a hydrophilic pore mediating selective Ca²⁺ transport, crucial for the mineralization initiation process in matrix vesicles (MVs).
Device Configuration: The BNCD chip operates without a specific ion-selective membrane, relying on oxygen termination for stable surface Fermi level pinning and reliable sensitivity to electrolyte potential variations.

Technical Specifications

Parameter	Value	Unit	Context
Background Current (BNCD)	<1	µA/cm²	Excellent property of BNCD microelectrodes
Water Dissociation Potential Window	~3	V	Wide electrochemical window
Surface Fermi Level Pinning	1.7	eV	Above the valence band (oxygen termination)
Microelectrode Array Channels	4	-	Planar BNCD chip design
Microelectrode Opening Diameter	16	µm	Size of sensing area
BNCD Film Thickness	~250	nm	Material stack layer
Intrinsic NCD Film Thickness	~500	nm	Material stack layer
a-Si Buffer Layer Thickness	~60	nm	Buffering thermal expansion mismatch
Passivation Layer Thickness (SiN)	1	µm	PECVD deposition
Sensing Chamber Volume	200	µL	Biological environment volume
Readout Input Impedance	10	TΩ	Front-end electronics specification
Readout Input Bias Current	1	pA	Front-end electronics specification
Output Signal Sample Rate	80	Hz	After digital filtering and decimation
Ca²⁺ Uptake Rate (AnxA5)	2.5	mV/s	Initial potential reduction rate (selective transport)
Mg²⁺ Uptake Rate (AnxA5)	0.3	mV/s	Initial potential reduction rate (non-selective)
Initial Ca²⁺ Concentration	1	mM	Electrolyte solution
Ca²⁺/AnxA5 Molar Ratio	50	-	Experimental condition (guaranteed Ca²⁺ excess)
Liposome Diameter (DPPC:DPPS)	110 ± 10	nm	Characterization by DLS
Proteoliposome Diameter (AnxA5)	140 ± 15	nm	Characterization by DLS

Key Methodologies

The BNCD microelectrode array fabrication and experimental protocol were critical for achieving ultrasensitive potentiometric measurements.

BNCD Microelectrode Fabrication (Planar 4-Channel Array)

Substrate and Buffer: A blank sapphire wafer was cleaned, followed by the deposition of a ~60 nm amorphous silicon (a-Si) layer to manage thermal expansion mismatch and promote diamond nucleation.
Diamond Growth: Sequential growth of a ~500 nm film of intrinsic Nanocrystalline Diamond (NCD) and a ~250 nm film of Boron-doped Nanocrystalline Diamond (BNCD).
Patterning (Conducting Structures): The conducting structures were defined using optical lithography, metal mask protection, and Reactive Ion Etching (RIE) in an argon-oxygen atmosphere.
Passivation: A 1 µm thick passivation layer of silicon nitride (SiN) was deposited using Plasma-Enhanced CVD (PECVD).
Opening Definition: Microelectrode openings (16 µm diameter) and bonding pads were patterned using optical lithography and RIE in a tetrafluoromethane (CF₄) atmosphere.

Device Setup and Signal Conditioning

Assembly: The diced chip was flip-chip bonded onto a printed-circuit carrier plate using conducting silver-epoxy. A glass ring was glued over the device to create a 200 µL sensing chamber.
Reference Electrode: A ring-shaped Ag/AgCl quasi-reference electrode was immersed in the chamber and connected to ground.
Surface Termination: Before use, the device was exposed to mild oxygen plasma for 10 minutes to ensure oxygen termination, which provides a hydrophilic surface and stable pinning of the surface Fermi level, essential for reliable potentiometry.
Readout Electronics: A voltage follower design was used (1 pA input bias current, 10 TΩ input impedance), followed by a 4th order Bessel low-pass filter (1 kHz cutoff). Signals were acquired at 4 kHz/channel and digitally filtered to deliver output sampled at 80 Hz (36 Hz bandwidth).

Potentiometric Measurement Protocol

Equilibration: The microelectrode was allowed to equilibrate in the electrolyte (typically 1 mM Ca²⁺ or Mg²⁺ in deionized water or Tris buffer) until a stable baseline was reached.
Vesicle Addition: 10 µL of liposomes (control) or proteoliposomes (AnxA5-containing) were manually dispensed into the chamber.
Kinetics Tracking: The potential change was tracked over 300 s to observe the second equilibration transient and the long-term potential drop caused by Ca²⁺ uptake.
Selectivity Test: Experiments were repeated using 1 mM Mg²⁺ instead of Ca²⁺ to confirm AnxA5 selectivity.
Inhibition Test: EDTA (up to 2.5 mM) was added to chelate free Ca²⁺ ions, confirming that the potential changes were directly related to the concentration of free ions in solution.

Commercial Applications

The technology and materials described, particularly the BNCD microelectrode platform, have direct relevance across several high-value engineering and scientific sectors:

Advanced Biosensing and Diagnostics:
- Real-Time Ion Monitoring: Development of ultrasensitive sensors for tracking critical divalent ions (Ca²⁺, Mg²⁺) in complex biological fluids, relevant for metabolic studies and disease monitoring.
- Membrane Protein Research: High-resolution tools for studying the kinetics and selectivity of ion channels and transporters incorporated into lipid bilayers, crucial for drug discovery targeting membrane proteins.
Biocompatible Neural Interfacing and Implants:
- Electroactive Implants: Utilizing BNCD’s high biocompatibility and stability for chronic in vivo applications, such as retinal stimulation and neural interfacing (e.g., monitoring cardiac action potentials [18]).
- High-Density MEAs: Fabrication of robust microelectrode arrays for high-time resolution electrophysiology and neurochemical detection (e.g., quantal catecholamine secretion [11, 12]).
Harsh Environment Electrochemistry:
- Wastewater Treatment: Application of BDD/BNCD electrodes for electro-oxidation coupled with nanofiltration for degrading persistent organic pollutants and antibiotics in secondary wastewater [3, 4].
- General Electroanalysis: Use in chemical analysis and drug detection where the wide water dissociation potential window and inertness of diamond are advantageous [8, 9].
Material Science and Sensor Manufacturing:
- Robust Sensor Platforms: Commercialization of diamond-based sensors capable of operating reliably through multiple measuring and cleaning cycles (e.g., sodium hypochlorite cleaning) without material degradation.

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

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