I. Research Background

1. **Huntington’s disease (HD)** is triggered by the aggregation of abnormal polyglutamine (PolyQ) proteins, accompanied by elevated levels of reactive oxygen species (ROS); however, the causal relationship and the local spatial distribution remain unclear.

2. Free radicals have short lifetimes and limited diffusion, and conventional ROS detection methods rely primarily on indirect measurements, making it difficult to achieve high spatial resolution and real-time monitoring.

3. Nitrogen–vacancy (NV) color centers within fluorescent nanodiamonds (FNDs) can directly detect free radicals via their response to magnetic noise; this has been validated at the cellular level, but has not yet been extended to live animal models.

4. The C. elegans Q40::YFP model can stably recapitulate PolyQ aggregation, making it well suited for studying the mechanisms and spatial distribution of oxidative stress in vivo.

II. Experimental Methods and Technical Framework

1. Construction of the nematode model

Wild-type N2, the sod-2/3 double mutant oxidative stress model, the Q0::YFP control, and the Q40::YFP Huntington’s disease model nematodes were selected.

2. Nanodiamond Delivery and Distribution

Fluorescent nanodiamonds (FNDs) coated with bovine serum albumin (BSA) can be delivered into nematodes via feeding, enabling them to traverse intestinal epithelial cells and distribute to tissues such as the body wall and muscles.

3. Quantum Sensing and Detection

T1 relaxation measurements based on FNDs, employing a 650 nm filter to eliminate YFP interference, were used to detect free radicals separately in the body wall muscles and the intestinal region.

4. Phenotype and Activity Validation

Motor performance analysis, ATP content determination, DHE‑fluorescence ROS detection, FND biocompatibility assessment

5. Data Processing

T1 values were calculated using a biexponential fit, and statistical analyses were performed using an unpaired t-test and one-way ANOVA.

III. Key Research Findings

1. Biocompatibility of FNDs

• FNDs can enter nematodes via feeding and distribute to the body wall muscles and the intestine.
• No significant effect on nematode survival or ATP levels → Good biocompatibility

2. Method validation: sod-2/3 double-mutant nematodes

• T1 relaxation measurement results: Whole-body T1 values are significantly reduced.
• Explanation: Overall levels of free radicals are elevated.
• Conclusion: The T1 relaxation quantum sensing method has been validated as a reliable approach for detecting free radicals in living animals.

3. PolyQ aggregation and localized elevation of free radicals

• Q40::The T1 value in the body wall muscle region of YFP‑expressing nematodes was significantly lower than that of the Q0 control group.
• Indicates a significant increase in free radical levels at the PolyQ aggregation site.
• The results exhibit spatial specificity: free radical levels are elevated in localized clusters, rather than uniformly increased throughout the body.

IV. Technical Advantages of the Nanodiamond T1 Relaxation Method

This technology is based on NV.   Color center Directly responding to magnetic noise, it enables in situ detection of unpaired electrons in free radicals without the need for indirect labeling, with sub‑micron spatial resolution. The T1 signal can reflect changes in free radical concentration in real time, is unaffected by YFP fluorescence, and can distinguish oxidative stress differences across various tissues and disease models, thereby enabling precise localization and quantification of free radicals in living animals.

V. Research Conclusions and Significance

This study For the first time, nanodiamond quantum sensing technology has been applied to living animals. , enabling in situ, direct, submicron‑scale detection of free radicals and revealing the spatial specificity of PolyQ aggregation and localized oxidative stress elevation, thereby providing a new tool for research on neurodegenerative diseases and holding potential for extension to other model organisms.

1. Technological Breakthrough

• For the first time, nanodiamond quantum sensing has enabled the direct detection of free radicals in living Caenorhabditis elegans, overcoming the limitations of conventional indirect measurements.

2. Biological Discoveries

• PolyQ aggregation can induce a localized, specific increase in free radicals.
• The spatial distribution characteristics of oxidative stress in the Huntington’s disease model have been elucidated.

3. Methodological Contributions

• A quantum-sensing-based free-radical detection protocol at the in vivo animal level has been established.
• It provides a new experimental tool for studying the mechanisms of oxidative stress in neurodegenerative diseases.

4. Application Prospects

• The technology can be extended to multiple model organisms.
• Provides a promising platform for quantum sensing in the study of in vivo pathophysiological mechanisms and drug evaluation.

Image and Text Guide

Figure 1. Experiments conducted in this study

a)   Schematic of diamond-based quantum sensing presented in this paper. Nanodiamonds are taken up by Caenorhabditis elegans and internalized by cells. Quantum-sensing experiments are conducted near the surface where nanodiamonds colocalize with Q40::YFP. b)   Representative relaxation curves of nanodiamonds in adult wild-type N2 nematodes and sod-2/3 mutant nematodes on the first day; c)   Control group: Q0 nematodes and PolyQ‑stressed Q40 nematodes. The NV color center is induced into the bright ms=0 state. Subsequently, by varying the duration of darkness, we observe whether the NV color center remains in this state or returns to a dark‑state equilibrium between ms=0 and ms=±1. This process is accelerated by free radicals. d) Caenorhabditis elegans were immobilized on an agar pad in a glass-bottom dish for measurement. e)   Place the petri dish on the experimental apparatus.

Figure 2. Expression of polyQ expansions in adult Caenorhabditis elegans on day 1.

a)   Generation Q0, b)   Q40 generation. Expression of polyQ expansions in the muscles of Caenorhabditis elegans leads to locomotor defects, specifically manifested as: c)   The proportion of mobile nematodes among all nematodes; d)   The average bending duration per minute for each nematode; e)   ATP assay results for day‑1 adult worms of the Q0 generation and for Q40‑generation nematodes subjected to polyglutamine (polyQ) stress. Error bars represent the standard deviation from three replicate experiments. Statistical significance was assessed using a t‑test; * indicates p ≤ 0.05, and ** indicates p ≤ 0.01.

Figure 3. Distribution of FND/BSA particles in the following nematodes:

a)   N2 wild-type and GA480 (sod2/3 double mutant) nematodes; b)   OW450 Q0:: YFP nematode; c)   AM141 Q40:: YFP‑labeled nematode. The boundaries of the nematode in the magnified image are indicated by dashed lines, and the magnification shows the region outlined in the red box in the left-hand image.

Figure 4. ATP levels were assessed by cell titer assay, with three replicates performed for each nematode strain:

a)   N2 wild type; b)   GA480 (a double mutant of sod2 and sod3); c)   OW450 Q0:: YFP nematode; d)   AM141 Q40:: YFP‑expressing C. elegans were incubated separately with FND, FND/BSA, or 50% ethanol (positive control). In each figure, data were normalized by setting the mean of the control group to 100% and zero to 0%. Error bars represent standard deviation. Statistical significance was assessed using one‑way ANOVA; results are labeled as follows: ns (not significant), p > 0.05; * indicates p ≤ 0.05; ** indicates p ≤ 0.01; *** indicates p ≤ 0.001.

Figure 5. ROS levels in different day-1 adult strains of Caenorhabditis elegans as measured by the DHE assay.

a)   Confocal images of the DHE channel. Enhanced red fluorescence is observed in mutant nematodes. In Figure (a), the average DHE fluorescence intensity of adult nematodes on day 1 was quantified using FIJI software. b)   Comparison between N2 and sod2/3 mutant nematodes. c)   Comparison between Q0 and Q40 nematodes. The whisker plot shows the minimum and maximum data points; the line dividing the box represents the median, while the top and bottom edges of the box correspond to the upper and lower quartiles, respectively. Each experiment analyzed 10 nematodes, with a total of three independent replicates. Data for each group were normalized by setting the control group mean to 100% and 0 to 0%, followed by analysis using an unpaired t-test. **** indicates p ≤ 0.0001.

Figure 6. Detection of free radicals in different worm strains using T1 relaxation measurements; data are derived from multiple independent experiments, with approximately 30 measurements per group.

a)   Q40 Representative fluorescence images of FND/BSA in worms, with white dashed lines indicating the body wall boundary and the intensity bar at the bottom expressed in photons per second. a1) Magnified image of intestinal granules in panel a (yellow cross marks the location), with a photon count of 1.2 × 10⁷ s⁻¹. a2) Magnified image of granules near the body wall’s muscle layer in panel a, with a photon count of 1 × 10⁷ s⁻¹. The figures show FND/BSA granules within N2, sod2/3 double-mutant worms, as well as Q0 and Q40 worms.   b) Near the body wall muscles and   c) Results of T1 relaxation measurements in the intestine. To distinguish free radical levels in different regions of the worm, FND/BSA particles were measured in the intestinal tissue or the body wall muscle layer of the same individual worm. The dashed lines indicate independent measurements for a single worm: d)   Q0 Worm; e)   Q40 Nematodes. In panels (b) and (c), the whiskers represent the minimum and maximum data points, and the line dividing the box indicates the median. Standard deviations are listed in Table S2 (Supporting Information). The upper and lower edges of the box correspond to the first and third quartiles, respectively. Between-group significance was assessed using an unpaired t-test. * denotes p ≤ 0.05, **** denotes p ≤ 0.0001. In panels (d) and (e), error bars indicate the standard deviation across three independent experiments (a total of 30 nematodes measured). Between-group significance was evaluated using a paired t-test. ns = not significant; * denotes p ≤ 0.05, ** denotes p ≤ 0.01, *** denotes p ≤ 0.001, **** denotes p ≤ 0.0001.