| Metadata | Details |
|---|
| Publication Date | 2024-01-31 |
| Journal | Biosensors |
| Authors | RosalĂa GonzĂĄlez-Brito, Pablo Montenegro, Alicia MĂ©ndez, Ramtin E. Shabgahi, A. Pasquarelli |
| Institutions | Universidad de La Laguna, UniversitÀt Ulm |
| Citations | 2 |
| Analysis | Full AI Review Included |
- Novel Device Development: A new transparent Multi-Electrode Array (MEA) utilizing Boron-Doped Diamond (BDD) selectively grown on an amorphous quartz substrate (BDD-on-quartz MEA) was successfully fabricated and characterized.
- Addressing Material Challenges: The complex fabrication protocol was necessary to manage the large thermal expansion mismatch between BDD and quartz, which typically causes film fragmentation and high tensile stress.
- Performance Validation: The BDD-on-quartz MEA achieved electrochemical performance (low noise, stability) nearly identical to the opaque BDD-on-silicon MEA, while offering transparency for optical observation.
- High-Sensitivity Amperometry: The device enables high signal-to-noise ratio amperometric recordings of serotonin exocytosis from human platelets, detecting secretory spikes with maximum currents (Imax) as low as 1.5-2 pA.
- Key Specifications: The MEA features a 4x4 array of 16 BDD microelectrodes, each with a nominal diameter of 20 ”m and a pitch of 200 ”m.
- Biological Application: The MEA successfully quantified serotonin release kinetics from human platelets under basal and serotonin-loaded conditions, validating its use for studying quantal secretion in small, challenging human cells.
- Future Utility: The transparency allows for simultaneous amperometry and microscopy (e.g., fluorescence imaging), opening perspectives for characterizing exocytotic release modes and clinical applications using human platelets.
| Parameter | Value | Unit | Context |
|---|
| Substrate Material | Amorphous Quartz | N/A | Transparent carrier |
| Electrode Configuration | 4 x 4 (16 total) | N/A | Microelectrode array |
| Nominal Electrode Diameter | 20 | ”m | Active BDD area |
| BDD Spot Diameter (Growth) | 60 | ”m | Selectively grown BDD footprint |
| Electrode Pitch | 200 | ”m | Center-to-center spacing |
| Total Active Surface Area | â5000 | ”m2 | Sum of 16 electrodes |
| Incubation Chamber Volume | ~200 | ”L | Volume provided by glass ring |
| BDD Growth Method | MWCVD | N/A | Microwave Plasma Chemical Vapor Deposition |
| BDD Growth Temperature | 800 | °C | Process temperature |
| MWCVD Power | 2200 | W | Plasma power |
| MWCVD Pressure | 30 | Torr | Growth pressure |
| Carbon Source | 1.5 | % CH4 | In H2 atmosphere |
| iNCD Layer Thickness | 50 | nm | Initial intrinsic nanocrystalline diamond layer |
| Final iNCD Spot Thickness | 1 | ”m | Thickness before doping overgrowth |
| Doped BDD Overgrowth Thickness | ~350 | nm | Boron-doped layer |
| Amperometry Potential | +800 | mV | Applied potential (vs Ag/AgCl) |
| Noise Performance (BDD-on-Glass) | 1 order of magnitude larger | N/A | Compared to BDD-on-silicon/quartz |
| Minimum Detectable Imax | 1.5-2 | pA | Spike current above 2.5 SD threshold |
| Serotonin Release (Loaded Q) | 0.30 ± 0.02 | pC | Quantal charge (net charge) |
| Serotonin Release (Loaded Imax) | 9.70 ± 0.71 | pA | Maximum oxidation current |
- Substrate Seeding: Amorphous quartz wafers were cleaned and spin-coated with a NanoAmando seeding solution.
- Initial iNCD Layer Growth: A 50 nm intrinsic nanocrystalline diamond (iNCD) layer was grown via Microwave Plasma Chemical Vapor Deposition (MWCVD) at 800 °C, 30 Torr, 2200 W, using 1.5% CH4 in H2 (10 min duration).
- Patterning and Etching: A titanium hard mask defining the 60 ”m spot pattern was lifted off. Unprotected iNCD was etched using Reactive Ion Etching (RIE) in an Ar-O2 atmosphere to create the diamond spot footprints.
- iNCD Spot Thickening: The iNCD spots were grown further (190 min) to achieve a final thickness of 1 ”m.
- Boron Doping: A ~350 nm BDD layer was overgrown using the same MWCVD parameters, with boron wires introduced into the plasma for doping (70 min duration).
- Metallization and Contact: Ring-shaped metal contacts and wires (100 nm Titanium / 50 nm Gold) were created via lift-off.
- Passivation: The wafer was passivated using polyimide-based photoresist (DurimideŸ 7505), defining openings for the 20 ”m active electrode areas and contact pads.
- Device Assembly: Chips were diced, bound to PCB carriers, and sealed with a glass ring to form the ~200 ”L incubation chamber.
- Electrochemical Setup: Amperometric measurements were conducted by biasing the BDD electrodes at +800 mV against a single Ag/AgCl sintered pellet reference/counter electrode.
- Data Processing: Signals were acquired using 16 transimpedance amplifiers (1 GΩ feedback), filtered (1 kHz cutoff), and analyzed using custom IGOR-Pro 8 macros to extract kinetic parameters (Imax, Q, t1/2, slope m).
- Neuroscience and Neurochemical Sensing:
- High-resolution, low-noise detection of neurotransmitter release (serotonin, dopamine, catecholamines) from primary cells (e.g., chromaffin cells, PC12 cells).
- Studying the fundamental mechanisms of exocytosis (fusion pore kinetics, quantal size distribution).
- Translational Medicine and Clinical Diagnostics:
- Characterization of human platelet function, which serves as an accessible model for neurological disorders (e.g., Parkinsonâs disease, depression).
- Developing standardized MEA platforms for rapid, quantitative assessment of amine content and release capacity in patient-derived samples.
- Advanced Biosensor Platforms:
- Transparent electrochemical chips enabling simultaneous optical (microscopy, fluorescence) and electrical measurements on living cells, crucial for correlating physical events (cell movement, fusion) with chemical release.
- Materials Engineering and Integration:
- Demonstration of robust fabrication techniques for integrating high-quality BDD films onto non-conventional, thermally mismatched substrates (quartz/glass), relevant for optoelectronic devices and microfluidics.
- Drug Screening and Pharmacology:
- High-throughput screening of compounds that modulate secretory pathways in human cells using the multi-electrode array format.
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.
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