A New Breakthrough in Quantum Sensing from ACS Nano: Real-Time Visualization of Mitochondrial Free Radicals During Myocardial Cell Hypoxia–Reoxygenation
Product Introduction
Equipment Parameters
I. Research Background: The “bottleneck” Challenge in Free Radical Detection
Free radicals serve as signaling molecules in normal physiological processes, but at excessive concentrations they can induce oxidative stress, damaging mitochondria, proteins, and DNA, thereby directly causing cellular injury.
However, traditional detection methods have a critical flaw:
- Fluorescent probes are prone to photobleaching, so they can only measure the “historical state” and cannot track in real time.
- The overall sample exhibits low-resolution detection, making it impossible to pinpoint mitochondria as the primary source of free radicals.
- Under hypoxic conditions (<1% O₂), conventional ROS probes (such as DHE) yield virtually no detectable signal.
Meanwhile, the NV centers in fluorescent nanodiamonds (FNDs) can directly detect free radicals by responding to ambient magnetic noise, offering non‑bleaching performance, high sensitivity, and nanoscale spatial resolution, making them the optimal tool for overcoming this challenge.
II. Core Technology: Mitochondria-Targeted Nanodiamond Quantum Sensing
The research team implemented a key modification: they functionalized the surface of fluorescent nanodiamonds (FNDs) with anti‑VDAC2 antibodies, enabling precise targeting of the mitochondrial outer membrane.
Detection principle:
Magnetic noise arising from free radicals accelerates the T1 relaxation of NV centers; a shorter T1 indicates a higher concentration of free radicals.
This enables: real-time, in situ, mitochondrial-specific, and non‑invasive quantification of free radicals.
III. Key Findings: The dynamics of hypoxia–reoxygenation‑induced free radicals have been captured for the first time.
1. Hypoxia: Mitochondrial free radicals initially decrease and then increase.
- 6-hour hypoxia: Free radicals are significantly reduced (cells activate the HIF‑1α adaptive mechanism).
- 24-hour hypoxia: a dramatic increase in free radicals (cellular overload and metabolic collapse)
- Traditional DHE probes fail to detect any changes, whereas quantum sensing demonstrates overwhelming superiority.
2. Reoxygenation: Free radicals gradually decline.
- Reoxygenation after 24 hours of hypoxia:
- At 80 minutes, free radicals began to decline significantly.
- After 24 hours, it essentially returns to normoxic levels.
- For the first time, the temporal window of reoxygenation protection has been clearly delineated.
3. Mitochondrial morphology and function are simultaneously impaired.
After hypoxia:
- Mitochondria transition from a tubular network to granular fragments.
- ATP levels have dropped significantly.
- Cell viability was significantly reduced.
- HIF-1α translocates extensively into the nucleus, activating the hypoxic response.
4. Nanodiamonds exhibit excellent biocompatibility.
FND and FNDs-anti-VDAC2 exhibit no significant cytotoxicity in cardiomyocytes and are suitable for long-term in vivo imaging.
IV. Significance of the Study: A Leap from “Undetectable” to “Precise Visualization”
1. Methodological Breakthrough
A mitochondrial-specific, real-time, non‑bleaching radical quantum sensing strategy has been established, outperforming all conventional fluorescent probes.
2. New Understanding of Mechanisms
Clarifying the biphasic changes of mitochondrial free radicals during hypoxia–reoxygenation elucidates the temporal patterns underlying myocardial ischemia-reperfusion injury.
3. Broad application prospects
Can be used for:
- Screening of myocardial protective drugs
- Assessment of Cold Preservation and Reperfusion Injury in Organ Transplantation
- Research on the Mechanisms of Oxidative Stress in Cardiovascular Diseases
- Hypoxia-related tumor and inflammation model assays
V. Conclusion
This study uses Quantum sensing + nanodiamonds , for the first time, we “observed” in real time the mitochondria of a single cardiomyocyte. Hypoxia and Reoxygenation The dynamic behavior of free radicals throughout the entire process. This approach not only addresses the longstanding technical challenges of “inaccurate or undetectable free radical measurements,” but also offers novel research tools and target‑oriented strategies for myocardial protection, organ transplantation, and cardiovascular diseases. Looking ahead, as in vivo applications continue to expand, quantum sensing holds the promise of becoming the gold standard for detecting oxidative stress.
Image and Text Guide
Figure 1. Schematic diagram of the experimental design in this study. Nanodiamonds target mitochondria and sense the generation of reactive oxygen species in the vicinity of cardiomyocytes (H9c2 myoblasts).
(a) The green blocks (561 nm) represent laser pulses, while the red blocks correspond to the photoluminescence (PL) of NV centers. The signals detected during the pulses are used to construct the T1 relaxation curve. (b) Plot the PL intensity as a function of different dark‑time durations to generate the T1 curve. In the presence of free radicals, the time required to reach equilibrium (T1) is shortened. The red and black lines represent the T1 measurements for mitochondria‑targeted nanodiamonds under normoxic and hypoxic conditions, respectively. To ensure accuracy, each pulse sequence was repeated 10,000 times, thereby achieving a favorable signal‑to‑noise ratio.
Figure 2. Particle size distribution of bare FNDs and FND-anti-VDAC2.
The hydrodynamic diameter was determined by dynamic light scattering using the Malvern Zetasizer Nano system.
Figure 3. Effects of hypoxic stress on H9c2 cells.
(a) Representative Z-stack confocal microscopy images before and after different hypoxic treatments (oxygen concentration <1%). (b) Cell viability assay (CellTiter method). Green fluorescence corresponds to Mitotracker Green staining. Error bars represent standard deviation. Statistical significance was assessed using one-way ANOVA, with differences deemed significant at ***p ≤ 0.001.
Figure 4. Assessment of HIF‑1α expression and nuclear translocation in H9c2 cells under different hypoxic conditions.
(a) Representative Z‑stack confocal images before and after different hypoxic treatments (oxygen concentration <1%). Cells were incubated with an anti‑HIF‑1α primary antibody followed by a FITC‑labeled secondary antibody. (b) Average optical density was analyzed in different regions of interest using FIJI. N = normoxia; H = hypoxia. Color coding: green = FITC (HIF‑1α); blue = DAPI. Whiskers represent the minimum and maximum values; intergroup comparisons were performed using two‑way ANOVA: **p ≤ 0.01, ****p ≤ 0.0001.
Figure 5. Uptake of FND by H9c2 cells.
(a) H9c2 cells were co-incubated with 1 μg/mL FNDs or FND-anti-VDAC2 for 6 or 24 hours. Color labeling: green = FITC‑phalloidin (staining actin filaments); blue = DAPI (staining DNA); red = FNDs (or FND-anti-VDAC2). (b) The uptake of FNDs by individual cells was quantified after 6 or 24 hours of incubation. The experiment was independently repeated three times, with 60 cells randomly selected from each group. Error bars represent the standard deviation. A two-way ANOVA was used to assess differences between groups: *p ≤ 0.05, **p ≤ 0.01.
Figure 6. Confocal microscopy using the SP8X system reveals the subcellular localization of naked FNDs/FND-anti-VDAC2 in mitochondria.
(a) H9c2 cells were stained and imaged after incubation with different FNDs for 6 or 24 hours. Green = Mitotracker (labels mitochondria); Red = FNDs. (b) Quantitative analysis of the colocalization between naked FNDs/FND-anti-VDAC2 and mitochondria after incubation for 6 or 24 hours. Error bars represent the standard deviation. Data were analyzed using two-way ANOVA: ****p ≤ 0.0001.
Note: Error bars represent the standard deviation. Data were analyzed using a two-way ANOVA.
Table 1 Mandel coefficients of mitochondria and FND/FND-anti-VDAC2 after different incubation times (from Figure 6b)
Figure 7. Cell viability assay (CellTiter method).
Cells treated with 5% DMSO served as the positive control. Data are from three independent experiments. Error bars represent the standard deviation for each group. One-way ANOVA was used to assess differences among groups: ****p ≤ 0.001; ns = not significant.
Figure 8. Detection of free radical generation during normoxia/hypoxia/reoxygenation:
(a) Dihydroethidium (DHE) method (for detecting peroxides and superoxides); (b) T1 measurement in H9c2 cells under hypoxic conditions for 6 or 24 hours; (c) T1 measurement after 24 hours of hypoxia or 24 hours of reoxygenation; (d) Tracking of free radical levels within 180 minutes after 24 hours of hypoxia followed by reoxygenation. Error bars represent the minimum and maximum values; intergroup comparisons in panels (a), (b), and (d) were performed using one-way ANOVA; in panel (c), intergroup comparisons were conducted using a t-test: ns = not significant, *p≤0.1, ***p≤0.001, ****p≤0.0001.
Download Materials
Beijing Yunshang Intelligent Manufacturing Technology Co., Ltd.
Hotline(24 hours a day)
Headquarters Address: Room 708, Building 2, HICOOL Industrial Park, Nanfaxin, Shunyi District, Beijing
Follow
Customer service WeChat
WeChat Official Account
COPYRIGHT 2026 Beijing Yunshang Intelligent Manufacturing Technology Co., Ltd.
Powered by 300.cn Privacy Policy
YUNS-123456
Smart Customer Service
WhatsApp：864009005667
Company email：support@chinayuns.com
Telephone：400 900 5667