Analytical Determination of Serotonin Exocytosis in Human Platelets with BDD-on-Quartz MEA Devices

At a Glance

Metadata	Details
Publication Date	2024-01-31
Journal	Biosensors
Authors	Rosalía González-Brito, Pablo Montenegro, Alicia Méndez, Ramtin E. Shabgahi, A. Pasquarelli
Institutions	Universität Ulm, Universidad de La Laguna
Citations	2
Analysis	Full AI Review Included

Technical Documentation & Analysis: BDD-on-Quartz MEAs for Serotonin Exocytosis

Executive Summary

This research validates a novel transparent Multi-Electrode Array (MEA) platform utilizing Boron-Doped Diamond (BDD) grown on amorphous quartz, offering significant advantages for neurochemical sensing and cellular analysis.

Novel Platform: Successful development and characterization of BDD-on-Quartz MEAs, overcoming the significant thermal expansion mismatch challenge between diamond and quartz.
Dual Functionality: The transparent quartz substrate enables simultaneous amperometric recording of serotonin exocytosis and high-resolution optical microscopy (DIC/fluorescence) of human platelets.
High Sensitivity: The BDD microelectrodes demonstrated extremely low noise, achieving a detection limit for secretory spikes as small as 1.5-1.8 pA, comparable to opaque BDD-on-Silicon MEAs.
Material Validation: MPCVD-grown BDD proved superior to BDD-on-Glass devices, which exhibited noise levels one order of magnitude higher, confirming BDD as the optimal material for high-performance electrochemical biosensing.
Kinetic Quantification: The MEAs successfully quantified key kinetic parameters (Imax, Q, t_1/2) of serotonin release from human platelets under basal and serotonin-loaded conditions.
Translational Potential: These robust, reusable BDD MEA devices are positioned as promising tools for translational medicine, particularly for characterizing human platelets in neurological disease models.

Technical Specifications

The following hard data points were extracted from the research paper detailing the BDD-on-Quartz MEA device structure and performance.

Parameter	Value	Unit	Context
Substrate Material	Amorphous Quartz	N/A	Transparent carrier for BDD MEA
Electrode Material	Boron-Doped Diamond (BDD)	N/A	Grown via MPCVD
Electrode Array Size	4 x 4 (16 total)	N/A	Microelectrode array configuration
Nominal Electrode Diameter	20	µm	Circular active area
Total Active Surface Area	≈5000	µm²	Sum of all 16 microelectrodes
Electrode Pitch	200	µm	Spacing between microelectrodes
iNCD Seeding Layer Thickness	50	nm	Initial intrinsic NCD layer
BDD Overgrowth Thickness	≈350	nm	Boron-doped layer thickness
Total Diamond Thickness (Spots)	≈1.35	µm	50 nm (iNCD) + 1 µm (iNCD spots) + 350 nm (BDD)
Metalization Stack	Ti (100 nm) / Au (50 nm)	nm	Connecting wires and contact pads
Amperometry Potential	+800	mV	Applied potential vs. Ag/AgCl reference
Maximum Current (Imax, Basal)	6.59 ± 0.35	pA	Serotonin release under basal conditions
Maximum Current (Imax, Loaded)	9.70 ± 0.71	pA	Serotonin release after 10 µM 5-HT loading
Spike Net Charge (Q, Loaded)	0.30 ± 0.02	pC	Quantal size measurement
Time Resolution (t_1/2, Loaded)	28.9 ± 1.22	ms	Spike full width at half maximum (FWHM)

Key Methodologies

The fabrication of the BDD-on-Quartz MEA relies on precise control of the MPCVD process and advanced lithography to manage the thermal expansion mismatch between diamond and quartz.

Substrate Preparation: Cleaning and spin-coating the quartz wafer with a NanoAmando seeding solution.
iNCD Growth (Initial Layer): Growth of a 50 nm thin intrinsic nanocrystalline diamond (iNCD) layer via MWCVD at 2200 W, 800 °C, 30 Torr, using 1.5% CH₄ in H₂ atmosphere (10 min duration).
Patterning (Hard Mask): Creation of a titanium hard mask pattern (60 µm spots) via lift-off lithography.
RIE Etching: Reactive Ion Etching (RIE) in Ar-O₂ atmosphere to etch the unprotected iNCD, creating the ‘footprint’ for the microelectrode spots.
iNCD Spot Overgrowth: Growth of the iNCD spots up to 1 µm thickness using the same MWCVD parameters (190 min duration).
BDD Doping and Overgrowth: Overgrowth of the iNCD spots with ≈350 nm of Boron-Doped Diamond (BDD) in a dedicated MWCVD reactor. Doping was provided by boron wires introduced into the plasma (70 min duration).
Metalization: Lift-off process to create the metal ring contacts and wires using a 100 nm Titanium (Ti) adhesion layer and a 50 nm Gold (Au) conductive layer.
Passivation: Wafer passivation using a polyimide-based photoresist (Durimide® 7505), followed by lithography to define electrode openings and contact pads.

6CCVD Solutions & Capabilities

6CCVD is uniquely positioned to supply the advanced MPCVD diamond materials and custom fabrication services required to replicate, scale, and extend the research presented in this paper. Our expertise in managing complex material stacks and precise doping profiles directly addresses the challenges encountered in BDD MEA fabrication.

Applicable Materials

To replicate the high-performance, low-noise electrochemical sensing demonstrated, the following 6CCVD materials are required:

Research Requirement	6CCVD Material Solution	Key Capability Match
Electrode Material	Heavy Boron-Doped PCD (Polycrystalline Diamond)	High doping uniformity (BDD) for stable, low-noise electrochemical sensing.
Substrate Transparency	Custom BDD-on-Quartz Wafers	Expertise in growing high-quality BDD films on non-native substrates (e.g., amorphous quartz, glass) for optical integration.
Low-Noise Interface	Nanocrystalline Diamond (NCD) Layers	Precise control over the initial NCD/iNCD seeding layer (50 nm) thickness and morphology, critical for managing internal stress and noise.
High-Resolution Imaging	Optical Grade BDD	Transparent BDD films suitable for integration with fluorescence and DIC microscopy systems.

Customization Potential

The success of the BDD-on-Quartz MEA hinges on precise control over dimensions, thickness, and metalization—all core competencies of 6CCVD.

Required Customization	6CCVD Capability	Value Proposition
Substrate & Dimensions	Custom plates/wafers up to 125mm (PCD).	Ability to scale the 6 x 6 mm² chip design to larger wafer formats for high-volume production or larger MEA designs.
Diamond Thickness Control	SCD/PCD thickness control from 0.1 µm to 500 µm.	Precise deposition of the required multi-layer stack (50 nm iNCD, 1 µm iNCD spots, 350 nm BDD overgrowth) to manage thermal stress and optimize electrochemical performance.
Custom Metalization	Internal capability for Au, Pt, Pd, Ti, W, Cu deposition.	Replication of the required Ti (100 nm) / Au (50 nm) connecting wires and contact pads, ensuring low contact resistance.
Surface Finish	Polishing capability: Ra < 5nm (Inch-size PCD).	Providing ultra-smooth surfaces necessary for optimal cell adhesion and minimizing background noise in sensitive amperometric measurements.

Engineering Support

The challenges detailed in the paper—specifically the thermal expansion mismatch between diamond and quartz leading to fragmentation (Figure S2)—require expert material science consultation.

6CCVD’s in-house PhD team specializes in optimizing MPCVD recipes and material stacks to mitigate internal stress and cracking in complex heterostructures. We can assist researchers in material selection and fabrication protocol refinement for similar BDD-based MEA biosensing projects, ensuring stability, reproducibility, and optimal signal-to-noise ratios for ultra-sensitive applications like single-cell exocytosis.

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

View Original Abstract

Amperometry is arguably the most widely used technique for studying the exocytosis of biological amines. However, the scarcity of human tissues, particularly in the context of neurological diseases, poses a challenge for exocytosis research. Human platelets, which accumulate 90% of blood serotonin, release it through exocytosis. Nevertheless, single-cell amperometry with encapsulated carbon fibers is impractical due to the small size of platelets and the limited number of secretory granules on each platelet. The recent technological improvements in amperometric multi-electrode array (MEA) devices allow simultaneous recordings from several high-performance electrodes. In this paper, we present a comparison of three MEA boron-doped diamond (BDD) devices for studying serotonin exocytosis in human platelets: (i) the BDD-on-glass MEA, (ii) the BDD-on-silicon MEA, and (iii) the BDD on amorphous quartz MEA (BDD-on-quartz MEA). Transparent electrodes offer several advantages for observing living cells, and in the case of platelets, they control activation/aggregation. BDD-on-quartz offers the advantage over previous materials of combining excellent electrochemical properties with transparency for microscopic observation. These devices are opening exciting perspectives for clinical applications.

Tech Support

Original Source

DOI: https://doi.org/10.3390/bios14020075

References

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2021 - Electrochemical Quantification of Neurotransmitters in Single Live Cell Vesicles Shows Exocytosis is Predominantly Partial [Crossref]
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