Non-Neurotoxic Nanodiamond Probes for Intraneuronal Temperature Mapping

At a Glance

Metadata	Details
Publication Date	2017-11-07
Journal	ACS Nano
Authors	David Simpson, Emma Morrisroe, Julia M. McCoey, Alain H. Lombard, Dulini C. Mendis
Institutions	Université Paris-Sud, Centre National de la Recherche Scientifique
Citations	163
Analysis	Full AI Review Included

Non-neurotoxic Nanodiamond Probes for Intraneuronal Temperature Mapping: Technical Analysis

Executive Summary

This research validates the use of Nitrogen-Vacancy (NV) containing nanodiamonds (NDs) as viable, non-neurotoxic, multi-functional quantum probes for live neuronal networks, demonstrating a critical pathway for advanced biosensing.

Non-Neurotoxic Validation: NDs at concentrations up to 20 µg/mL (1.6 µM) showed no statistically significant adverse effects across 25 electrophysiological network parameters (e.g., firing rate, burst rate) over 36 hours.
Intracellular Thermometry: Demonstrated wide-field, background-free imaging and simultaneous mapping of local intracellular temperature distributions within primary cortical neurons using Optically Detected Magnetic Resonance (ODMR).
Quantum Sensing Mechanism: Temperature sensing relies on the shift in the NV center’s ground state crystal field splitting ($D$) with a measured sensitivity factor of -74 kHz/K.
Performance Metrics: Temperature changes were mapped simultaneously from thousands of NDs within seconds (12 s total acquisition time for temperature map), achieving an accuracy of approximately ± 1.2 °C for a single ND spot.
Material Limitation & Future Scope: Current probes utilize lower-quality, HPHT-derived NDs (high Nitrogen and ¹³C content), limiting sensitivity (1 K/√Hz) and preventing detection of single Action Potentials (APs). High-purity, isotopically enriched CVD diamond is required to realize the full magnetic and electric field sensing potential.

Technical Specifications

The following hard data points were extracted relating to the material properties, sensing mechanism, and experimental conditions:

Parameter	Value	Unit	Context
Nanodiamond Type (Source)	brFND-100	N/A	High-Pressure High-Temperature (HPHT) derived.
ND Particle Size (Z-avg)	170	nm	Determined via Dynamic Light Scattering (DLS).
NV Centers per Particle	~500	N/A	Estimated average NV density.
NV Excitation Wavelength	532	nm	Diode-pumped solid-state laser.
NV Fluorescence Collection	650-750	nm	Filtered spectrum.
Neurotoxicity Threshold	< 20	µg/mL	No significant statistical difference in network parameters.
MEA Recording Frequency	20	kHz	Used for extracellular electrical recordings.
ODMR Crystal Field Splitting ($D$)	2.87 (mean 2868.59)	GHz (MHz)	Zero magnetic field splitting.
Temperature Sensitivity ($dD/dT$)	-74	kHz/K	Shift rate of the crystal field splitting.
ODMR Linewidth Limitation	Order MHz	N/A	Due to high Nitrogen (100 ppm) and ¹³C (1 ppt) content.
In Vitro Temperature Sensitivity	1	K/√Hz	Favorable compared to other nanoscale probes.
Temperature Map Acquisition Time	12	s	Total time required for signal-to-noise ratio > 1.
Excitation Power Density	30	W/mm²	Used during optical imaging at 37 °C.
Measured Temperature Shift	-1.5 ± 1.2	°C	Local change measured in a single ND spot.

Key Methodologies

The core experiment involved validating neurotoxicity using Multi-Electrode Arrays (MEAs) and then performing quantum thermometry using wide-field ODMR.

Culture Preparation: Primary mouse cortical neurons (DIV 11-13) were cultured on Multiwell MEAs (gold electrodes, FR-4 substrate) coated with PolyEthyleneImine and Laminin. Cultures were maintained at 37 °C with perfused carbogen (95% O₂, 5% CO₂).
Nanodiamond Application: NDs (brFND-100, 1 mg/mL stock) were dispersed in cell media (tested at 1, 5, 10, 20 µg/mL for toxicity; 6 µg/mL for imaging), sonicated for 5 minutes, and applied during routine media change. Uptake was passive (nonspecific endocytosis).
Neurotoxicity Assessment (MEA): Extracellular recordings were performed at 12, 24, and 36 hours post-application. 25 network parameters (e.g., burst rate, spike amplitude, firing rates) were analyzed using high-pass filtering (300 Hz) and statistical comparison (Bonferroni-corrected p-values).
Quantum Imaging Setup: Neuronal networks were cultured on a custom glass coverslip featuring an evaporated gold microwave resonator, coated with a thin layer of PDMS (10:1 ratio) for insulation.
Wide-Field ODMR Spectroscopy:
- NV centers were excited using a 532 nm laser, and fluorescence (650-750 nm) was collected via a modified inverted microscope.
- Microwaves (MW) were applied at $2.87 \text{ GHz}$ (“on resonance”) and $2.84 \text{ GHz}$ (“off resonance”).
- Background auto-fluorescence was suppressed by subtracting the “on” and “off” photoluminescence images, resulting in background-free imaging and a 50% increase in signal-to-background ratio.
- ODMR spectra were acquired by integrating 100 images (30 ms each) per frequency point (6 seconds total acquisition).
Temperature Mapping:
- Individual ND spots were co-localized at two different environmental temperatures ($37.3 {^\circ}\text{C}$ and $35.2 {^\circ}\text{C}$).
- The ODMR crystal field splitting ($D$) was measured at each temperature by fitting a single Lorentzian peak (four central data points removed for improved precision).
- The shift in $D$ was converted to a local temperature change using the factor $\text{d}D/\text{d}T = -74 \text{ kHz/K}$.

6CCVD Solutions & Capabilities

This research successfully demonstrates the potential of NV-nanodiamonds for high-resolution intracellular sensing. However, the study explicitly identifies that the sensitivity required for detecting critical, short-duration biological events, such as single action potentials (APs), is severely limited by the low material quality of the commercially available, HPHT-derived nanodiamonds.

6CCVD offers the ultra-high purity MPCVD materials necessary to overcome this barrier, enabling next-generation quantum biosensors.

Applicable Materials: Enabling High-Coherence Quantum Sensing

The bulk diamond used in this study limits ODMR linewidths to the MHz range, primarily due to high Nitrogen (N) and naturally occurring ¹³C content. Replicating or exceeding the sensitivity requires high-coherence precursors provided by 6CCVD.

Required Material	6CCVD Product Recommendation	Key Benefit for Research Replication
Ultra-High Purity SCD	Optical Grade SCD (Low-N)	Essential precursor for creating high-coherence, single-crystal nanodiamonds via milling. Low intrinsic nitrogen (N) concentration drastically reduces spectral broadening, moving sensitivity three orders of magnitude closer to detecting single APs.
Isotopically Enriched Diamond	Isotopically Enriched ¹²C SCD	Reduces the noise floor associated with the ¹³C spin bath. This material ($\text{< 1 ppm } ^{13}\text{C}$) is critical for achieving the ultralong spin coherence times necessary for sensitive magnetic and electric field measurements outlined in the paper’s future goals.
Substrates for Device Integration	MPCVD Diamond Substrates (Up to 10mm thickness)	Required for integrating quantum sensors directly onto complex MEA or microfluidic platforms, mimicking the experimental design (gold resonator on glass/diamond).

Customization Potential for Biosensing Platforms

6CCVD’s advanced engineering capabilities are crucial for transitioning quantum sensing from proof-of-concept to functional biological tools.

Custom Dimensions and Etching: While the paper used 170 nm NDs, future work relies on creating thin layers or patterned diamond structures. 6CCVD provides custom-sized plates/wafers up to 125mm (PCD) and precise laser micro-machining to etch NV-containing patterns or structures onto specialized biosensing chips.
Precision Polishing: Achieving robust, low-noise quantum measurements requires extremely low surface roughness. 6CCVD delivers Ra < 1nm polishing for SCD and Ra < 5nm for inch-size PCD, ensuring optimal surface quality for subsequent functionalization and integration with high-density gold microwave resonators.
In-House Metalization: The experiment utilized a gold microwave resonator. 6CCVD offers internal, precise thin-film metalization services including Au, Pt, Pd, Ti, W, and Cu. This is vital for directly fabricating integrated quantum devices (e.g., on-chip MW antennas and electrode arrays) onto diamond substrates.

Engineering Support & Logistics

Our PhD-level engineering team specializes in connecting fundamental material science with complex application requirements.

Material Selection Support: 6CCVD’s in-house PhD team can assist researchers in selecting the optimal diamond specifications (e.g., N concentration, ¹²C enrichment level, specific defect engineering) for similar Intraneuronal Quantum Sensing or NV Thermometry projects.
Global Supply Chain: We ensure reliable global shipping (DDU default, DDP available) of high-value, sensitive materials to maintain experimental timelines worldwide.

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

View Original Abstract

Optical biomarkers have been used extensively for intracellular imaging with high spatial and temporal resolution. Extending the modality of these probes is a key driver in cell biology. In recent years, the nitrogen-vacancy (NV) center in nanodiamond has emerged as a promising candidate for bioimaging and biosensing with low cytotoxicity and stable photoluminescence. Here we study the electrophysiological effects of this quantum probe in primary cortical neurons. Multielectrode array recordings across five replicate studies showed no statistically significant difference in 25 network parameters when nanodiamonds are added at varying concentrations over various time periods, 12-36 h. The physiological validation motivates the second part of the study, which demonstrates how the quantum properties of these biomarkers can be used to report intracellular information beyond their location and movement. Using the optically detected magnetic resonance from the nitrogen-vacancy defects within the nanodiamonds we demonstrate enhanced signal-to-noise imaging and temperature mapping from thousands of nanodiamond probes simultaneously. This work establishes nanodiamonds as viable multifunctional intraneuronal sensors with nanoscale resolution, which may ultimately be used to detect magnetic and electrical activity at the membrane level in excitable cellular systems.

Tech Support

Original Source

DOI: https://doi.org/10.1021/acsnano.7b04850