Nanodiamond–Quantum Sensors Reveal Temperature Variation Associated to Hippocampal Neurons Firing

At a Glance

Metadata	Details
Publication Date	2022-07-25
Journal	Advanced Science
Authors	Giulia Petrini, Giulia Tomagra, Ettore Bernardi, Ekaterina Moreva, P. Traina
Institutions	Istituto Nazionale di Fisica Nucleare, Sezione di Torino, Torino e-district
Citations	52
Analysis	Full AI Review Included

Technical Documentation & Analysis: Nanodiamond Quantum Thermometry in Neurons

Executive Summary

This research successfully demonstrates the use of Nitrogen-Vacancy (NV) center nanodiamonds (NDs) as highly sensitive, intracellular quantum thermometers to monitor localized temperature variations associated with neuronal activity.

Core Achievement: First-time detection and quantification of localized temperature variations (up to 1 °C) directly correlated with the modulation of hippocampal neuron firing activity.
Sensing Mechanism: Optically Detected Magnetic Resonance (ODMR) exploiting the temperature-dependent shift of the NV center’s ground state splitting frequency ($D_{gs}$).
Quantitative Results: Neuronal firing potentiation (via Picrotoxin) resulted in a significant temperature increase of +1.02 ± 0.24 °C. Neuronal silencing (via TTX+Cd) resulted in a temperature decrease of -0.50 ± 0.17 °C.
Sensor Performance: The ND sensors exhibited high sensitivity, capable of discriminating temperature variations as low as 0.5 °C, with a projected sensitivity of <0.1 °C.
Biocompatibility: The study confirmed that ND internalization (185 nm average diameter) was non-neurotoxic and did not alter spontaneous firing rates or action potential waveforms.
Future Impact: This technique provides a powerful, non-invasive tool for real-time, subcellular mapping of metabolic activity and localized thermogenesis in neurobiology and pathology research (e.g., cancer, neurodegeneration).

Technical Specifications

Parameter	Value	Unit	Context
Maximum Temperature Variation (Potentiation)	1.02 ± 0.24	°C	Induced by 100 µM Picrotoxin
Minimum Temperature Variation (Silencing)	-0.50 ± 0.17	°C	Induced by TTX + Cd
Bath Temperature (Stability)	37.0 ± 0.1	°C	Controlled incubation chamber
ND Hydrodynamic Diameter (Average)	185	nm	Used for internalization
ND Concentration (Incubation)	0.6	µg/ml	Far below cytotoxicity threshold (> 250 µg/ml)
NV Center Coupling Constant (Bulk)	-75	kHz/°C	Standard reference value
NV Center Coupling Constant (NDs, Calibrated)	-76 ± 4	kHz/°C	Used for temperature estimation
Laser Excitation Wavelength	532	nm	CW, attenuated to 1 mW
Laser Spot Size	~1.2 x 1.3	µm²	Focused beam size
PL Emission Rate (Single ND)	~300	kCounts/s	Detected by SPAD
ODMR Acquisition Time	60	s	Trade-off for fast measurement and precision
Projected Sensitivity	<0.1	°C	High sensitivity potential

Key Methodologies

The experiment relied on precise material engineering of the nanodiamonds and integration into a sophisticated confocal ODMR setup.

Nanodiamond Precursor: NDs (MSY 0-0.25) containing 100-200 ppm natural nitrogen impurities were used as the starting material.
Purification and Oxidation: NDs were oxidized in air (510 °C for 5 h) and wet oxidized (HF:HNO₃ at 160 °C for 2 days) to remove surface impurities and create functional groups.
Monodisperse Isolation: Differential centrifugation was used to isolate monodisperse NDs (205 nm hydrodynamic diameter).
NV Center Creation (Irradiation/Annealing):
- Electron irradiation (15.7 MeV, 2.5·10¹⁹ particles cm^-2) was performed at 870 °C for 80 h.
- Subsequent annealing was performed at 900 °C under argon atmosphere for 1 h, followed by air oxidation at 510 °C for 5 h to maximize NV^- yield (64% yield reported).
Cell Culture and Internalization: Hippocampal neurons (10 DIV) were incubated with 0.6 µg/ml NDs for 5 hours to ensure internalization.
ODMR Setup: A single-photon confocal microscope (Olympus IX73) integrated a 532 nm laser (1 mW), an acousto-optic modulator (AOM) for pulsed control, and a microwave source (Keysight N5172B) amplified to 20 dBm, fed to a planar broadband antenna.
Temperature Measurement: Temperature variation (ΔT) was derived from the shift in the NV center’s resonant frequency ($D_{gs}$) by measuring the change in photoluminescence (ΔF) using the differential ODMR technique.

6CCVD Solutions & Capabilities

This research highlights the critical need for highly controlled, high-purity diamond materials for advanced quantum sensing applications in biological environments. 6CCVD is uniquely positioned to supply the foundational materials and custom fabrication required to replicate and advance this work.

Applicable Materials for Quantum Sensing

The success of NV-ODMR thermometry depends entirely on the quality and consistency of the diamond material and the resulting NV center yield. 6CCVD provides the necessary high-purity precursors:

6CCVD Material Solution	Relevance to Research	Customization Potential
High-Purity Single Crystal Diamond (SCD)	Ideal precursor for creating high-density, stable NV centers via controlled implantation/irradiation. Essential for bulk ODMR calibration and high-coherence studies.	Available in thicknesses from 0.1 µm up to 500 µm, suitable for both thin-film and bulk applications.
Optical Grade Polycrystalline Diamond (PCD)	Can be used as a substrate for large-area integrated quantum sensing platforms, especially when combined with Microelectrode Arrays (MEAs) as suggested in the paper.	Plates/wafers up to 125 mm diameter. Excellent surface finish (Ra < 5 nm) for direct cell culture or subsequent thin-film deposition.
Boron-Doped Diamond (BDD)	While not used for NV centers, BDD is crucial for electrochemical biosensing (e.g., neurotransmitter detection) and could be integrated alongside NV-NDs for multi-modal sensing platforms.	Available as thin films or substrates, offering superior electrochemical stability and sensitivity.

Customization Potential for Advanced Bio-Sensing

The paper mentions the integration of NDs with MEAs and the future goal of functionalizing NDs to subcellular compartments. 6CCVD offers specialized services to facilitate these next-generation experiments:

Custom Substrate Fabrication: We provide large-area PCD substrates (up to 125 mm) that can be laser-cut or patterned to integrate seamlessly with standard MEA systems, enabling synchronized electrical and thermal measurements.
Precision Metalization: The ODMR technique requires precise microwave delivery (using a planar broadband antenna in this study). 6CCVD offers in-house metalization capabilities (Au, Pt, Pd, Ti, W, Cu) for creating custom microwave transmission lines or integrated electrodes directly on diamond substrates.
- Example: Fabricating Ti/Pt/Au contact pads or micro-antennas directly onto a diamond substrate for enhanced ODMR signal delivery in integrated MEA setups.
Surface Engineering: While the paper focused on NDs, future work involving SiV or GeV centers requires high-quality SCD substrates. We offer ultra-low roughness polishing (Ra < 1 nm for SCD) essential for epitaxial growth or precise ion implantation required for these alternative color centers.

Engineering Support

6CCVD’s in-house team of PhD material scientists and quantum engineers specializes in optimizing diamond growth parameters (e.g., nitrogen concentration, purity) to maximize the yield and coherence of color centers.

We can assist researchers in:

Material Selection: Choosing the optimal diamond purity and growth method (SCD vs. PCD) based on target color center (NV, SiV, GeV) and application (nanosensors vs. bulk substrates).
Post-Processing Recipe Development: Consulting on irradiation and annealing protocols to achieve desired NV concentrations and coupling constants, ensuring consistency for high-fidelity biological measurements.
Custom Dimensions and Integration: Designing diamond components that fit specific optical setups (e.g., confocal microscopy, high-NA objectives) and integrated platforms (e.g., MEAs).

For custom specifications or material consultation, visit 6ccvd.com or contact our engineering team directly. We ship globally (DDU default, DDP available).

View Original Abstract

Abstract Temperature is one of the most relevant parameters for the regulation of intracellular processes. Measuring localized subcellular temperature gradients is fundamental for a deeper understanding of cell function, such as the genesis of action potentials, and cell metabolism. Notwithstanding several proposed techniques, at the moment detection of temperature fluctuations at the subcellular level still represents an ongoing challenge. Here, for the first time, temperature variations (1 °C) associated with potentiation and inhibition of neuronal firing is detected, by exploiting a nanoscale thermometer based on optically detected magnetic resonance in nanodiamonds. The results demonstrate that nitrogen‐vacancy centers in nanodiamonds provide a tool for assessing various levels of neuronal spiking activity, since they are suitable for monitoring different temperature variations, respectively, associated with the spontaneous firing of hippocampal neurons, the disinhibition of GABAergic transmission and the silencing of the network. Conjugated with the high sensitivity of this technique (in perspective sensitive to < 0.1 °C variations), nanodiamonds pave the way to a systematic study of the generation of localized temperature gradients under physiological and pathological conditions. Furthermore, they prompt further studies explaining in detail the physiological mechanism originating this effect.

Tech Support

Original Source

DOI: https://doi.org/10.1002/advs.202202014

References

2002 - Quantification of Cyclin B1 and P34cdc2 in Bovine Cumulus‐Oocyte Complexes and Expression Mapping of Genes Involved in the Cell Cycle by Complementary DNA Macroarrays