Imaging of transfection and intracellular release of intact, non-labeled DNA using fluorescent nanodiamonds
At a Glance
Section titled âAt a Glanceâ| Metadata | Details |
|---|---|
| Publication Date | 2016-01-01 |
| Journal | Nanoscale |
| Authors | VladimĂra PetrĂĄkovĂĄ, Veronika Benson, Martin BunÄek, Anna FiĹĄerovĂĄ, M Ledvina |
| Institutions | Czech Academy of Sciences, Nuclear Physics Institute, Czech Technical University in Prague |
| Citations | 76 |
| Analysis | Full AI Review Included |
6CCVD Technical Analysis: Fluorescent Nanodiamonds for Label-Free DNA Transfection and Intracellular Sensing
Section titled â6CCVD Technical Analysis: Fluorescent Nanodiamonds for Label-Free DNA Transfection and Intracellular SensingâSource Paper: Imaging of transfection and intracellular release of intact, non-labeled DNA using fluorescent nanodiamonds (Nanoscale, 2016)
Executive Summary
Section titled âExecutive SummaryâThis research validates Fluorescent Nanodiamonds (FNDs) as a dual-function device, serving both as a highly efficient, non-toxic carrier for nucleic acid (NA) delivery and as an intrinsic, label-free intracellular biosensor.
- Core Achievement: Demonstrated successful delivery and real-time monitoring of non-labeled DNA release within living cells using FNDs.
- Sensing Mechanism: Utilizes Nitrogen-Vacancy (NV) color centers within the diamond lattice as a nanoscopic electric charge sensor.
- Molecular Monitoring: Changes in the local chemical environment (DNA binding and subsequent intracellular release) are detected via a spectral shift in the NV center photoluminescence (PL) ratio (NV-/NV°).
- Transfection System: FNDs were successfully coated with cationic Poly(ethyleneimine) (PEI) to form stable, reversible electrostatic complexes (FND-PEI-DNA) that mediate cellular uptake.
- Performance Metrics: Achieved high transfection efficiency (up to 81% positive cells) and remarkably low associated cytotoxicity (6-17%), surpassing commercial transfection reagents.
- Material Advantage: FNDs provide exceptional characteristics for bio-sensing: unlimited photostability, non-toxicity, and high spatial resolution for monitoring complex molecular events over prolonged periods.
Technical Specifications
Section titled âTechnical SpecificationsâThe following critical parameters govern the fabrication and performance of the FND nanodevice:
| Parameter | Value | Unit | Context |
|---|---|---|---|
| Nanodiamond Diameter (Average) | ~35 | nm | Starting material (Microdiamant MSY 0-0.05) |
| Starting Nitrogen Impurities | 100-200 | ppm | Natural concentration in raw material |
| Proton Irradiation Energy | 15.5 | MeV | Used for vacancy generation |
| Proton Irradiation Fluence | 6 x 1016 | cm-2 | NV creation dosage |
| High-Temperature Annealing | 900 | °C | Post-irradiation treatment (1 hour) |
| Air Oxidation Temperature | 510 | °C | Surface termination (6 hours) |
| Oxidized FND Zeta Potential | -33 | mV | Baseline negative surface charge (Oxygen-terminated) |
| FND-PEI Complex Zeta Potential | +36 | mV | Cationic charge after PEI binding |
| FND-PEI-DNA Complex Zeta Potential | -34 | mV | Charge neutralization upon DNA binding |
| NV° Zero Phonon Line (ZPL) | 575 | nm | Neutral charge state luminescence peak |
| NV- Zero Phonon Line (ZPL) | 636 | nm | Negative charge state luminescence peak |
| FND-PEI-DNA Transfection Efficiency | 81 | % | Percentage of AlexaFluor 488-positive IC-21 cells (120 min) |
| FND-PEI-DNA Cytotoxicity (Max) | 17 | % | Maximum Hoechst-positive cells (120 min) |
Key Methodologies
Section titled âKey MethodologiesâThe experimental success relied on controlled surface functionalization and NV center engineering, summarized below:
- Nanodiamond Purification and Surface Termination: HPHT nanodiamond powder (~35 nm) was purified and oxidized using hot acid baths (HNO3/H2SO4 at 85 °C) to generate a stable, negatively charged, oxygen-terminated surface (-33 mV zeta potential).
- NV Center Creation: Purified NDs were pressed into a target holder and irradiated using a 15.5 MeV proton beam to create lattice vacancies. This was followed by high-temperature annealing (900 °C) and subsequent air oxidation (510 °C) to stabilize the NV centers (NV°/NV- states).
- Cationic Coating (PEI): FNDs were coated with low-molecular-weight branched Poly(ethyleneimine) (PEI) via probe sonication, reversing the surface charge to highly positive (+36 mV) for optimal DNA complexing.
- DNA Cargo Loading: Non-labeled DNA (137-bp fragment or pGFP plasmid) was electrostatically bound to the FND-PEI complex, neutralizing the overall charge back to approximately -34 mV.
- Intracellular Monitoring via PL Spectroscopy: Confocal Raman spectroscopy was utilized with green laser excitation (514 nm or 532 nm) to measure the Photoluminescence (PL) spectra of intracellular FND-PEI-DNA. The ratio shift between the NV° ZPL (575 nm) and NV- ZPL (636 nm) intensity quantitatively tracked the complex molecular event of DNA dissociation in real time.
6CCVD Solutions & Capabilities
Section titled â6CCVD Solutions & CapabilitiesâThis research highlights the growing demand for high-purity diamond materials offering integrated quantum sensing capabilities for complex cellular and molecular analysis. 6CCVD is uniquely positioned to supply the foundational materials and customization services required to replicate and scale this nanodevice technology.
| Requirement/Goal from Paper | 6CCVD Custom Solution & Benefit |
|---|---|
| Stable, High-Contrast NV Sensing | Optical Grade SCD Wafers: 6CCVD provides high-purity Single Crystal Diamond (SCD) synthesized via MPCVD. SCD offers superior crystalline quality, resulting in NV centers with enhanced coherence times and increased PL intensity compared to powdered HPHT material, essential for demanding quantum sensing and single-particle tracking. |
| NV Center Creation & Density Control | Custom Nitrogen Doping: We control nitrogen incorporation during MPCVD growth, eliminating the inherent variability of natural impurities (100-200 ppm) cited in the paper. We offer expert consultation on optimized doping and annealing protocols to achieve targeted NV-/NV° charge ratios for biosensing applications. |
| Precise Surface Charge Engineering | Controlled Surface Termination: 6CCVD delivers highly reproducible SCD surfaces with specific, engineered terminations (Oxygen, Hydrogen, etc.), ensuring the precise and repeatable surface charge (e.g., the -33 mV baseline potential) required for reliable and high-performance PEI binding and sensing reproducibility. |
| Bulk Material & Scalability | Wafers up to 125 mm: While this study used nanodiamond powder, 6CCVD supplies inch-sized PCD and SCD wafers (up to 125 mm) for scaling up bio-sensor array fabrication, high-throughput testing, and integration into large-scale microfluidic systems. |
| Custom Device Integration | Advanced Metalization & Fabrication: For extending this research into microchip integration or electro-optical devices, 6CCVD offers in-house metalization services (Au, Pt, Ti, W, Cu) and precision laser cutting to generate custom, micro-scale NV sensors from larger wafers. |
| Engineering Support | In-House PhD Team: 6CCVDâs technical staff provides direct engineering support for projects requiring high-purity diamond for drug delivery, gene transfection, or electric field sensing. We assist in material selection (SCD vs. PCD), doping recipes, and post-processing treatments. |
For custom specifications or material consultation, visit 6ccvd.com or contact our engineering team directly.
View Original Abstract
Efficient delivery of stabilized nucleic acids (NAs) into cells and release of the NA payload are crucial points in the transfection process. Here we report on the fabrication of a nanoscopic cellular delivery carrier that is additionally combined with a label-free intracellular sensor device, based on biocompatible fluorescent nanodiamond particles. The sensing function is engineered into nanodiamonds by using nitrogen-vacancy color centers, providing stable non-blinking luminescence. The device is used for monitoring NA transfection and the payload release in cells. The unpacking of NAs from a poly(ethyleneimine)-terminated nanodiamond surface is monitored using the color shift of nitrogen-vacancy centers in the diamond, which serve as a nanoscopic electric charge sensor. The proposed device innovates the strategies for NA imaging and delivery, by providing detection of the intracellular release of non-labeled NAs without affecting cellular processing of the NAs. Our system highlights the potential of nanodiamonds to act not merely as labels but also as non-toxic and non-photobleachable fluorescent biosensors reporting complex molecular events.