Unraveling Eumelanin Radical Formation by Nanodiamond Optical Relaxometry in a Living Cell

Defect centers in a nanodiamond (ND) allow the detection of tiny magnetic fields in their direct surroundings, rendering them as an emerging tool for nanoscale sensing applications. Eumelanin, an abundant pigment, plays an important role in biology and material science. Here, for the first time, we evaluate the comproportionation reaction in eumelanin by detecting and quantifying semiquinone radicals through the nitrogen-vacancy color center. A thin layer of eumelanin is polymerized on the surface of nanodiamonds (NDs), and depending on the environmental conditions, such as the local pH value, near-infrared, and ultraviolet light irradiation, the radicals form and react in situ. By combining experiments and theoretical simulations, we quantify the local number and kinetics of free radicals in the eumelanin layer. Next, the ND sensor enters the cells via endosomal vesicles. We quantify the number of radicals formed within the eumelanin layer in these acidic compartments by applying optical relaxometry measurements. In the future, we believe that the ND quantum sensor could provide valuable insights into the chemistry of eumelanin, which could contribute to the understanding and treatment of eumelanin- and melanin-related diseases.

All solvents and chemicals were purchased from commercial sources and were used without further purification.

Preparation of RGS-ND
RGS-NDs were prepared according to our previous report with slight adjustments. 1Briefly, 100 µL 1 mg/mL ND water dispersion from Adámas Nanotechnologies was diluted with 811 µL MilliQ water and mixed with 79 µL 2.5 mg/mL L-DOPA.After 3 minutes sonication, 10 µL 10.84 mg/mL NaIO 4 was added, followed by 15 minutes shaking.The resulting RGS-ND was purified by 3 cycles of centrifugation/suspension.

Preparation of cHSA-RGS-ND
3][4] Briefly, 150 mg of HSA was dissolved completely in 15 mL of degassed ethylenediamine-HCl solution (2.5 M, pH 4.75), EDC (4 mmol, 621 mg) was then added and stirred for 2 hours.The reaction was terminated by adding an acetate buffer (1 mL, 4 M, pH 4.75).After reaction, the cHSA was washed twice with acetate buffer (4 M, pH 4.75) and 3 times with deionized distilled water using Vivaspin 20 (30 kDa MWCO) centrifugal concentrator and then lyophilized to obtain cHSA as a white fluffy solid.The cHSA-RGS-NDs were prepared simply by mixing 400 μg cHSA and 100 μL 1 mg/mL RGD-ND for 30 minutes at room temperature, and purified by 3 cycles of centrifugation/suspension.

Transmission Electron Microscopy (TEM)
One drop of a 0.1 mg/mL solution of sample in MilliQ was placed onto an oxygen treated copper grid and dried at room temperature.A Jeol 1400 transmissions electron microscope was used to obtain bright field images Samples were scanned with scan rate 300 kHz and scan sizes between 0.5 and 5 µm.Images were processed with the NanoScope Analysis software (Version 1.9).

T 1 relaxation time measurement
The longitudinal spin relaxation time (T 1 ) of the NV centers in the nanodiamonds was measured using a home-built confocal fluorescence microscope.The NV centers were excited using a 532 nm laser, which was focused onto the sample using an oil-immersion objective (Nikon PLAN 100x oil, N.A. = 1.35).The resulting fluorescence from the NV centers was collected by the same objective and filtered with a 740/75 nm band-pass filter and detected using an avalanche photodiode (APD).
The pulse sequence for the T 1 time measurement is shown in Figure 3a.It For the T 1 time statistics, the ND samples were prepared by placing 10 μL of 0.01 mg/mL -1 samples in the silicone gasket placed on top of an O2plasma cleaned glass coverslip.The samples were dried overnight and T 1 measurements were performed using the home-built confocal microscope described above (See Figure 1, Created with BioRender.com).For the T 1 measurements, only single particles with a moderate count rate (500,000 ± 300,000) were measured.10 mM different pH buffer solutions (pH 3: citrate, pH 4 and 5: acetate, pH 6-9.8: phosphate) were added when needed.A total of 20 single, isolated fluorescence spots were selected for the T 1 measurement and some data points are excluded based on the relevance of fit (R-squared < 0.9).

Cell culture
A549 and J774A.1 cells were cultured in DMEM supplemented with 10% FBS and 1% Penicillin/Streptomycin in a humidified atmosphere at 37 °C and 5% CO 2 .Fresh culture medium was replaced every two days and cells were sub-cultured after reaching 80% confluence.For the UV and NIR irradiation, the initial intracellular free radical load was measured by T1 relaxometry measurements on the cHSA-RGS-ND for 15 min.Subsequently, UV (0.3 mW/cm 2 ) or NIR (350 mW/cm 2 ) were introduced to the cells during the continuous T1 relaxometry measurements, which were performed for extra 10 minutes.

Simulation of spin relaxation times
The model [9][10][11] used to simulate the spin relaxation times is similar to those of previous publications.In this model, we considered a thin layer of surface electrons on the diamond surface and a thicker layer (thickness of 2 nm) of radicals on top of the diamond surface for the RGS-ND.0][11] Both layers reduce the relaxation times of the NV centers due to fluctuations of their electron spins.Without the radicals in the shell of RGS-ND, the relaxation time is given by  other 1 1 where ms is the intrinsic NV relaxation time (with a value similar to that of NVs in bulk diamond).Here the contribution due to the noise originating from the surface electrons takes the same functional form as that from the radicals given in the main text.
The expression for ( ) takes the same form as ( ) for the radicals.Following references [9][10][11] , the correlation time has two contributions, the first originating from flip-flop interactions between electron spins and a second from vibrational relaxation and is given by , where is the volume density of the radicals, GHz is the   vib = 50 intrinsic vibrational spin relaxation, and nm is a parameter  min = 0.2 describing the minimum allowed distance among the radicals.In the case of surface electron spin, we chose nm. min = 0.15 In the Monte Carlo simulation, we randomly choose NDs where the random locations and orientations of the NV centers follow uniform distributions with an exclusion range of 2nm from the diamond surface.
For NDs with a diameter of 27.9 nm, a density 11 nm -3 of the surface electrons in a 0.1 nm thick diamond surface was used to reproduce  0 1 s.This surface electron density was used in the simulation for = 223.9 the radical numbers at different pH values.Using the T 1 times in Fig. 2c of the main text, we determined the number of radicals in the shell of RGS-ND.

Samples preparation of cell with cHSA-RGS-ND for Transmission Electron Microscopy (TEM)
The TEM samples were prepared inline with our previous report. 6Briefly, A549 cells were cultured in a 24-well plate pre-placed with carbon coated sapphire discs (d:3mm) with a density of 50,000 cells/mL.After 4 hours coincubation with cHSA-RGS-ND, sapphire discs were placed between 2 aluminum plates to create a 'sandwich' and were mounted afterwards into a holder (Engineering Office, M. Wohlwend) and immediately fixated in a Wohlwend HPF Compact 01 high-pressure freezer (Engineering Office, M.
Wohlwend) with a pressure of 2100 bar.The frozen samples were then stored in liquid nitrogen.Frozen sapphire discs were carefully removed from the aluminum 'sandwich' and transferred into 1 mL pre-cooled freeze substitution medium (0.2% (w/v) osmium tetroxide, 0.1% (w/v) uranyl acetate, 5% (v/v) distilled water in acetone) and kept in a freeze substitution unit (AFS2,Leica).Samples were then slowly warmed up to 0 °C over a period of 20 hours in the unit.After being warmed up, the freeze-substituted samples were increased to room temperature, then the substitution medium was removed and the discs were washed 3 times with acetone at half an hour intervals.Then the discs were infiltrated sequentially in gradient epoxy resin-acetone mixture (

1. 2 . 8 .
Electron spin resonance (ESR) spectroscopy CW (continuous wave) X-Band (~ 9.4 GHz) ESR spectra were recorded using a MiniScope MS200 from Magnettech.The frequency stability of the spectrometer was controlled with a frequency counter from Hewlett Packard.All cw ESR measurements were performed at ambient conditions with 50 mW micro wave power, a field modulation amplitude of 400 µT and a modulation frequency of 100 kHz.A scanning duration of 600 s/scan was chosen and 16 transients were accumulated for each spectrum.The pH dependent RGS-ND samples with concentration of 1 mg/mL were prepared dispersing RGS-NDs in pH buffer solutions.For the measurements 20 μL sample solution is filled in a 50 µL micropipette capillary from BLAUBRAND and sealed using Critoseal.As the NDs are not really dissolved in the solution, they sediment in these tubes during measurements.Therefore, it is important to place the sedimentation layer of the RGS-NDs reproducibly in the center of the micro wave cavity of the ESR spectrometer.For the processing of the recorded data, the EasySpin 5 package for Matlab and Origin Software was employed.

consisted of a series of 10
μs long laser pulses.The laser pulse polarizes the NV centers in the m s = 0 spin state.After a variable waiting time (τ), the subsequent laser pulse reads out the spin state of the NV centers.The fluorescence photons detected in the first 300-500 ns of the laser pulse contained the spin state information and hence constitute the signal.The T 1 measurement data shown were normalized, i.e., the signal (fluorescence obtained during the first 300 ns) was divided by the reference steady-state fluorescence (fluorescence obtained when the NV center was re-initialized into the m s = 0 spin state).The T 1 measurement sequence was repeated several times with a total acquisition time of 15 minutes.The measured fluorescence data are plotted as a function of the waiting time (τ) between the laser pulses and fitted with a mono-exponential function of the form  0 .+ exp (/ 1 )

1. 2 . 11 .
T 1 relaxation time measurement in living cell J774A.1 cells were seeded in an ibidi 18 well µ-slide (100,000 cells/mL, 100 µL each well).After overnight incubation, cells were washed with DPBS, then 100 µg/mL cHSA-RGS-ND in culture medium were added to the cells (100 µL in each well).Then cells were incubated at 37 °C again for 4 hours, washed 3 times with DPBS and maintained in colorless Leibovitz's L-15 Medium for immediate T 1 relaxation time measurements.T 1 measurements were performed on a home-built confocal microscope as described in a previous report6 but with the following differences.A 40X oil-immersion objective with a 1.4 NA from Olympus (UPLXAPO40XO) was used.Laser pulses for T 1 measurements were created with an arbitrary waveform generator 70001A from Tektronix and a directly modulated 513 nm laser from Toptica Photonics (Ibeam-smart 515.S-15133).The laser beam was filtered by a 513 nm bandpass filter (HQ515/20M, Chroma), then a lambda half plate (AHWP10M-600, Thorlabs) followed by a polarizing beamsplitter (PBS121, Thorlabs) were used to further control the laser power.A piezo stage (P-562.3CD)from Physik Instrumente was implemented for the objective's positioning.Fluorescence pulses were collected by an Excelitas avalanche photodiode (SPCM-AQRH 13) protected by a 590 nm long pass filter (ET590lp, Chroma).Pulse averaging was executed with a FAST ComTec time tagger (MCS6A1T2) and a National Instruments card (6343) allowed us to manage the analogue/digital interfacing between the computer and the microscope.To control the temperature, an objective heater (Objektivheizer 2000) and heated insert (P Lab-TekTM S1) from Pecon was implemented.The entire experiment was controlled by the customized open source software: Qudi 7 .The single particle-tracking algorithm used was inspired from the works of Feng et al.8

Figure S2 .Figure S3 .
Figure S1.a) TEM image and b) AFM image (Topography, Scale bar 11.1 nm, Roughness 1.6 nm, the height of NDs in dashed rectangles are analyzed) of fluorescent nanodiamond purchased from Adámas Nanotechnologies.

Table S1 .
Simulation results based on different shape of NDs