Bioorthogonal Fluorescent Nanodiamonds for Continuous Long-Term

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Biological and Medical Applications of Materials and Interfaces

Bioorthogonal Fluorescent Nanodiamonds for Continuous Long-Term Imaging and Tracking of Membrane Proteins Feng-Jen Hsieh, Shingo Sotoma, Hsin-Hung Lin, Ching-Ya Cheng, TsyrYan Yu, Chia-Lung Hsieh, Chun-Hung Lin, and Huan-Cheng Chang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b03640 • Publication Date (Web): 13 May 2019 Downloaded from http://pubs.acs.org on May 13, 2019

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ACS Applied Materials & Interfaces

Bioorthogonal Fluorescent Nanodiamonds for Continuous Long-Term Imaging and Tracking of Membrane Proteins Feng-Jen Hsieh,a-c,‡ Shingo Sotoma,a,†,‡ Hsin-Hung Lin,a Ching-Ya Cheng,a Tsyr-Yan Yu,a,b ChiaLung Hsieh,a Chun-Hung Linb-d,* and Huan-Cheng Changa,e,f,* a Institute

of Atomic and Molecular Sciences, Academia Sinica, Taipei 106, Taiwan

b Taiwan

International Graduate Program – Chemical Biology and Molecular Biophysics, Academia Sinica, Taipei, 115, Taiwan

c Institute

of Biochemical Sciences, National Taiwan University, Taipei, 106, Taiwan

d Institute

of Biological Chemistry, Academia Sinica, Taipei, 115, Taiwan

e Department

of Chemical Engineering, National Taiwan University of Science and Technology, Taipei 106, Taiwan

f Department

of Chemistry, National Taiwan Normal University, Taipei 106, Taiwan

Keywords: cells, fluorescence imaging, glycoproteins, membrane proteins, nanodiamonds ABSTRACT: Real-time tracking of membrane proteins is essential to gain an in-depth understanding of their dynamics on cell surface. However, conventional fluorescence imaging with molecular probes like organic dyes and fluorescent proteins often suffers from photobleaching of the fluorophores, thus hindering their use for continuous long-term observations. With the availability of fluorescent nanodiamonds (FNDs), which have superb biocompatibility and excellent photostability, it is now possible to conduct the imaging in both short and long terms with high temporal and spatial resolution. To realize the concept, we have developed a facile method (e.g., one-pot preparation) to produce alkynefunctionalized hyperbranched-polyglycerol-coated FNDs for bioorthogonal labelling of azide-modified membrane proteins and azide-modified antibodies of membrane proteins. The high specificity of this labelling method has allowed us to continuously monitor the movements of the proteins of interest (such as integrin α5) on/in living cells over 2 h. The results open a new horizon for live cell imaging with functional nanoparticles and fluorescence microscopy.

INTRODUCTION Membrane proteins play an important functional role in cells through interactions with other intracellular and extracellular components.1,2 Direct, real-time, and longterm visualization of the interactions and associated dynamics is crucial to revealing how membrane protein molecules response to environmental stimuli and perturbations.3 Combining the studies of protein dynamics at both single-molecule and ensemble levels should provide not only comprehensive but also detailed understanding of a protein’s functions and activities in cells without heterogeneities and other complex issues.4,5 Lighting up the proteins of interest is the first and essential step to achieve the goal. Numerous probes, including genetically encoded fluorescent proteins, organic dyes, and light-emitting nanoparticles have been

developed over the past few decades for the purpose.6,7 However, the low photostability (blinking and/or photobleaching) of these fluorophores has prevented their practical use for continuous long-term imaging of intricate membrane protein dynamics.3,7 Atom-like fluorophores hosted in nanoparticles are promising alternatives. The negatively charged nitrogen-vacancy (NV–) centers in fluorescent nanodiamonds (FNDs) have recently emerged as a new type of fluorophores for bioimaging applications.8,9 These centers are capable of emitting bright far-red fluorescence (λmax = 685 nm) and have exceptional photostability, showing neither photobleaching nor photoblinking when imaged by confocal fluorescence microscopy.10 Moreover, the fluorescence emission can be magnetically modulated, thanks to the unique magneto-optical properties of the

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center.11 These features, together with the inherent biocompatibility of the nanomaterial, have made FND a promising nanoprobe for monitoring the behaviors of protein molecules (including membrane proteins) in living cells and organisms at the single-particle level in both short and long terms.12 However, to achieve specific labelling of membrane proteins with the nanoprobes, proper surface modification of FNDs to avoid non-specific interactions is essential. Liu et al. have recently reported single-molecule tracking of transforming growth factor membrane beta (TGF-β) receptors using FNDs in cells.13 Apart from loading TGF-β on FNDs, bovine serum albumin (BSA) was additionally coated on the particles to facilitate their dispersion in cell medium.14 However, concerns about non-specific interactions remain as the stealth effect of the BSA coating is not high.15 Therefore, there is necessity to develop more sophisticated surfacemodification methods to accomplish the mission. Biotin-avidin interactions are one of the most commonly adopted approaches for specific cell targeting and labelling applications; however, the presence of endogenous biotin on cell surface is likely to diminish the specificity if biotinylated FNDs are used for the labelling (Figure S1).16 A way to circumvent this issue is to utilize azide-alkyne-based click chemistry, which stands on highly specific molecular recognition between azide and alkyne, enabling efficient modifications of biomolecules even in their natural environment.17 Using this bioorthogonal chemistry, specific labelling of azidemodified membrane proteins on living cells by fluorescent molecular probes has been demonstrated and reviewed.18-20 The same chemistry is readily applicable for surface modification of carbon nanoparticles like nanodiamonds.21-24 However, to our knowledge, the application of the technique for continuous long-term tracking of membrane proteins with alkyne-functionalized FNDs has not yet been undertaken before and the success should represent a significant breakthrough in the field. To realize the aforementioned concept, we have recently developed a method based on ring-opening polymerization of glycidol for surface modification of FNDs.25-27 The method is facile and the resultant FNDs, with a thin layer of hyperbranched polyglycerol (HPG) coated on the surface, show excellent dispersibility in biological buffers with a significant stealth effect. Moreover, the HPG-modified FNDs (HPG-FNDs) can be easily conjugated with any molecules containing epoxy rings and other functional groups.28 By simply reacting acid-treated FNDs with glycidol and glycidyl propargyl ether, which is composed of an epoxy group and alkyne in the structure, we have been able to produce highly dispersible, stealthy, and clickable FNDs in one pot. Here, we report the synthesis of alkyne-modified HPGFNDs (alkyne-HPGFNDs) and their applications for bioorthogonal labelling and long-term imaging of cell

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Figure 1. Preparation and characterization of alkyneHPGFNDs. (a) Workflow of the alkyne-HPGFND preparation. The proposed structure of the polymer coating is shown to the right. (b) Hydrodynamic diameters of FNDs and alkyne-HPGFNDs in DDW and PBS. (c) Thermogravimetric analysis of bare FNDs, HPGFNDs, and alkyne-HPGFNDs over 25 – 800 °C. (d) FTIR spectra of bare FNDs, HPGFNDs, and alkyne-HPGFNDs over 500 – 4000 cm1. The spectra are shifted vertically for clarity. (e) Twodimensional 1H-13C HSQC NMR spectra of HPGFNDs (red) and alkyne-HPGFNDs (blue).

membrane proteins. The membrane proteins chosen in this study are sialoglycoproteins, which participate in a wide range of biological activities such as cell-cell communications.29 These protein molecules can be readily functionalized with azide moieties by co-culturing cells with azide-modified sugars such as tetraacylated Nazidoacetylmannosamine (ManNAz).30 Additionally, in order to demonstrate more directly the specific labelling ability of this technique, integrin α5 was selected as the model protein. The molecule contains oligosialic acids in the glycan and acts as a key receptor in cell migration.31 We first labelled integrin α5 with azide-modified antiintegrin α5 antibody, which was subsequently reacted with alkyne-HPGFNDs directly on cell membrane. The movements of these membrane protein molecules on and in cells were finally tracked in both short and long terms (1 s – 2 h) by fluorescence imaging.

RESULTS AND DISCUSSION Preparation and characterization of alkyne-HPGFNDs


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The experiments began with the synthesis of alkyneHPGFNDs, which were originally prepared by mixing acidtreated FNDs in glycidyl propargyl ether heated at 120 °C. The FNDs, however, immediately formed aggregates owing to the immiscibility of the particles with the solvent. We thus first grafted a thin layer of HPG on the FND surface by heating the FND-glycidol mixture at 120 °C under a nitrogen atmosphere for 2 h, after which glycidyl propargyl ether was added to the mixture and heated for another 22 h to produce alkyne-HPGFNDs (Figure 1a). The coating of FNDs with HPG significantly improved the dispersibility of the particles in high salt solution, as evidenced by dynamic light scattering measurements for their hydrodynamic sizes. The number-averaged sizes of alkyne-HPGFNDs in deionized distilled water (DDW) and phosphate-buffered saline (PBS) were 110 ± 31 nm and 118 ± 32 nm (Figure 1b), respectively, similar to those of HPGFNDs (111 ± 30 nm and 119 ± 37 nm in Figure S2). In contrast, the particle sizes of unmodified FNDs surged from 94 ± 30 nm to 775 ± 119 nm under the same conditions, proving the need of such surface modification to achieve anticoagulation. Thermogravimetric analysis revealed that ~70% of the total weight of HPGFNDs was contributed by the polymer coating (Figure 1c), whereas the corresponding weight percentage for alkyneHPGFNDs was only ~50%. The result is consistent with the difference in the surface coating where, in the latter case, the polymerization process was hindered as soon as glycidyl propargyl ether was added to the terminals of polyglycerol during the sample preparation. Infrared spectroscopy of alkyne-HPGFNDs also confirmed the effectiveness of the surface modification, showing two new absorption peaks at ~1070 cm-1 (C-OC) and 2730 – 2990 cm-1 (C-H) as well as a diminished absorption band at 1700 – 1830 cm-1 (C=O), when compared with those of bare FNDs (Figure 1d). A similar result was obtained for HPGFNDs. To verify the grafting of alkynyl groups on the surface of HPGFNDs, nuclear magnetic resonance (NMR) spectroscopy based on twodimensional 1H-13C heteronuclear single quantum coherence (HSQC) was carried out using the naturalabundant 1H and 13C nuclei in the samples. As shown in Figure 1e for the HSQC spectrum of alkyne-HPGFNDs, a diamond carbon-hydrogen coupling signal appeared at 2.2/30.1 ppm (1H/13C).25 Additionally, by comparing it with the HSQC spectrum of HPGFNDs, a new cross-peak at 3.7/79.9 ppm (1H/13C) was found, which is a clear evidence for the successful coating of alkynyl HPG on the FND surface.32 Applying the same methods, we have also been able to synthesize and characterize smaller alkyneHPGFNDs, such as the 50 nm ones, with success (Figure S3).

Prior to cell labelling, the targeting ability of alkyneHPGFNDs for azide-modified molecules was examined in solution. In this experiment, an aqueous solution containing Alexa Fluor 488-bound azides either with or without the catalysts (Cu+ ions) was first mixed with the alkyne-HPGFND suspension. After 1-h incubation, unbounded dye molecules were removed by washing and the green fluorescence from Alexa Fluor 488-azides (Figure 2a and 2d) and the red fluorescence from alkyneHPGFNDs (Figure 2b and 2e) were then measured separately for samples deposited on a glass slide. Indeed, co-localization of these two emission signals could be observed when the mixture contained Cu+ ions (Figure 2c), but only FND fluorescence signals were detected in the absence of the catalysts (Figure 2f). A quantification of the green fluorescence signals from the suspensions with or without Cu+ ions showed an intensity difference of ~100-fold between these two samples (Figure S4). By referring to the fluorescence intensities of pure Alexa Fluor 488-azide solution, we estimated that the average numbers of alkynyl groups accessible by the dye molecules on the surface of a single spherical alkyneHPGFND were ~55 (100 nm) or ~12 (50 nm), in agreement with the size difference between these two types of particles.

Specific targeting abilities of alkyne-HPGFNDs in solution



Figure 2. Characterization of the specific targeting ability of alkyne-HPGFNDs by confocal fluorescence microscopy. The images of the reaction between alkyne-HPGFNDs and Alexa Fluor 488-azides with or without Cu+ ions are shown in (a-c) and (d-f), respectively. The nominal diameter of the alkyne-HPGFND particles was 100 nm. Scale bars: 5 µm.

The specific targeting ability of alkyne-HPGFNDs was additionally investigated through selective extraction of azide-modified biotin-conjugated NeutrAvidin (NA/AB) in a protein mixture containing an excessive amount of BSA, which is the major constituent of fetal bovine serum in cell culture (Figure 3a). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) served to characterize the efficiency of the extraction.33 As shown in Figure 3b and 3c, both the ion signals of BSA (m/z: ~66,000 and ~33,000 for singly and doubly charged ions, respectively), NA/AB (m/z: ~15,000 for singly charged monomeric ions), and their complexes (m/z: ~81,000) could be detected for the protein mixture containing NA/AB and BSA with a molar ratio of 1:100. However, after extraction by alkyneHPGFNDs, only the signals of NA/AB remained in the mass spectra (Figure 3d), showing a highly specific targeting ability of alkyne-HPGFNDs. Bioorthogonal labelling of cell membrane proteins In applying alkyne-HPGFNDs to target membrane proteins on living cells, cytotoxicity of the particles is a matter of concern. We first performed the assays with the Cell Counting Kit-8 to measure the activities of dehydrogenases in HeLa cells and human fibroblasts (HFW) as the model cell lines. Our results showed that both 50 nm and 100 nm alkyne-HPGFNDs did not cause any significant negative effect on the viability of HeLa and HFW cells, even when the cells were treated at a very high particle concentration, up to 2 mg/mL (Figure 4a and 4b). We next applied alkyne-HPGFNDs to label azidosialoglycoproteins on the surface of living cells. The experiments started with the derivatization of

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Figure 3. Characterization of the specific targeting ability of alkyne-HPGFNDs by mass spectrometry. (a) Workflow of targeting azide-modified biotin-conjugated NeutrAvidin (denoted as NA/AB) by alkyne-HPGFNDs in a protein mixture. (b–d) MALDI-TOF MS spectra of NA/AB and the protein mixture consisting of NA/AB and BSA with the molar ratio of 1:100 before and after extraction with alkyneHPGFNDs. Only singly charged ions of monomeric NA/AB were detected in the mass spectra. The nominal diameter of the alkyne-HPGFND particles was 100 nm.

sialoglycoproteins with azide groups by adding azidemodified sugars (i.e., ManNAz in Figure 5a) into the culture of HeLa cells for 48-h incubation.30 The azidosialoglycoproteins on the cell surface were then labelled with alkyne-HPGFNDs at 1 mg/mL for 30 min to allow observations by both flow cytometry and confocal fluorescence microscopy. Details of the experiments conducted to optimize the bioorthogonal labelling conditions for living cells with the nanoparticles can be found in Supporting Information (Figure S5 and S6). Not surprisingly, without Cu+ ions, no significant labelling of azido-sialoglycoproteins on the surface of HeLa cells by alkyne-HPGFNDs occurred (Figure S7). To minimize the particle size effect and achieve a higher labelling efficiency, it is preferable to employ alkyne-HPGFNDs of smaller size such as the 50 nm ones, which are sufficiently bright for single-particle detection by using a standard confocal fluorescence microscope. For cells labelled at the same weight concentration (such as 1 mg/mL), there are 8-fold more particles to be used with 50 nm alkyne-HPGFNDs than with 100 nm alkyneHPGFNDs. An advantage of this approach is that it significantly shortens the labelling time from 30 min to 10 min, which markedly reduces the toxic effect of the reagents used in the reactions. Moreover, the labelling is considerably more uniform and homogeneous, even for


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Figure 4. Cytotoxicity tests of alkyne-HPGFNDs. The cell viability assays were carried out for HeLa cells (a) and HFW cells (b) with the concentrations of alkyne-HPGFNDs (50 nm or 100 nm in diameter) varying from 0.1 to 2 mg/mL.

azido-sialoglycoproteins on filopodia (Figure 5b – 5d). To evaluate the general applicability of the alkyneHPGFND particles for bioorthogonal labelling of sitespecific proteins, HFW cells were further experimented to prove the principle. As shown in Figure S8, the results of both confocal fluorescence imaging and flow cytometric analysis confirmed the high specific targeting ability of alkyne-HPGFNDs through click chemistry. Sialoglycoproteins are a group of glycoproteins with sialic acid as a component of the glycan. Integrin α5 is one of such glycoprotein molecules. To achieve specific targeting of integrin α5, HFW cells were first incubated with azide-modified anti-integrin α5 antibody (Azidoα5Ab) and then bioorthogonally labelled by 100 nm alkyne-HPGFNDs (Figure 6a). The specific targeting ability of these particles was finally assessed by flow cytometry and confocal microscopy (Figure 6b and S9). Comparing the fluorescence intensities measured by flow cytometry for ~5000 cells each indicated that there were more azido-sialoglycoprotein-expressing cells labelled by alkyne-HPGFNDs (Figure S8) than untreated cells labelled for integrin α5 through Azido-α5Ab (Figure 6b), a result in line with our expectations. The flow cytometry data presented above provide only a relative quantity of the labeling for these two groups of proteins. Moreover, the sensitivity of the method is limited by backgrounds mainly derived from cell autofluorescence. In order to obtain more quantitative information about the labelling with alkyne-HPGFNDs, we took advantage of the fact that the fluorescent centers of FNDs are deeply embedded in the chemically inert diamond matrix and therefore their fluorescence properties are nearly unaffected by chemical treatments and surface modifications at room temperature.16 More importantly, background-free detection of the fluorescence can be achieved by magnetic modulation of the signals, making it well suited for quantitative measurements even in high backgrounds.34 By directly measuring the fluorescence intensities of 5 × 104 alkyne-

Figure 5. Specific labelling of sialoglycoproteins on cell surface. (a) Workflow of labelling azido-sialoglycoproteins on cell surface with alkyne-HPGFNDs. (b) Z-stacked fluorescence image of azido-sialoglycoprotein-expressing HeLa cells labelled with 1 mg/mL 50 nm alkyne-HPGFND in the presence of Cu+ ions for 10 min. (c, d) Zoom-in Zsectional fluorescence image (c) and fluorescence/brightfield merged image (d) of the cells marked by a white square in (b). Scale bars: 20 µm (b) and 5 µm (c).

HPGFND-labelled cells sonicated to rupture cell membrane and release the particles into solution, we determined that the average amounts of alkyneHPGFNDs bound with azido-sialoglycoproteins and integrin α5 on the surface of HWF cells were 44.0 ± 1.3 and 18.6 ± 0.3 pg/cell, respectively. These weights correspond to 2.4 × 104 and 1.0 × 104 particles/cell, assuming a spherical shape for the 100 nm FND particles. In comparison, the fluorescence intensities of the same cells labelled with alkyne-HPGFNDs in the absence of the catalysts were lower by ~2 orders of magnitude (Figure S10), reflecting a significant stealth effect of the labelling. An important implication of this finding is that the method would allow a measurement for the absolute binding capacities of membrane proteins if the labeling is monovalent.35 To reach this goal, monodisperse particles of smaller size (such as 20 nm in diameter) should be used.36 Long-term imaging and tracking of cell membrane proteins Real-time imaging is an indispensable tool in revealing the details of membrane protein transports on cell surface. Emitting highly stable fluorescence without photobleaching, FND is an ideal candidate to enable imaging and tracking of targeted biomolecules over a long term. To examine if the 100 nm alkyne-HPGFNDs can be used to study the dynamics of membrane proteins on living cells, we focused our attention on integrin α5 only and tracked its movement on the HFW cell surface by



Figure 6. Specific labelling of integrin α5 on cell surface. (a) Workflow of the immunostaining of integrin α5 on a living cell with alkyne-HPGFNDs through the azidemodified anti-integrin α5 antibody (Azido-α5Ab). (b) Flow cytometric analysis of HFW cells labelled either without (blue) or with (red) Azido-α5Ab and then 100 nm alkyneHPGFND. The grey area represents the cell-only group.

epifluorescence imaging. An electron multiplying chargecoupled device (EMCCD) served to achieve uninterrupted observations of the membrane protein molecules in real time (Figure 7a). Although only a single focal plane was chosen to monitor the protein movement, a large number of trajectories (e.g., 2280 in Video S1) were recorded in 5 min, thanks to the high efficiency of the labelling. Shown in Figure 7b is the first frame of Video S1 composed of 2882 epifluorescence images. Analysis of the trajectories with care revealed random and active movement of the alkyne-HPGFND-labelled integrin α5 (Figure 7c). The diffusion coefficients, obtained from mean square displacement (MSD) analysis of all valid trajectories within the time increment of 2 s, lied in the range of 0.041 ± 0.032 µm2/s (See Figure S11 and

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Supporting Information for further discussion and analysis). The values are in good agreement with the previous findings (0.01 – 0.1 µm2/s) for nanogold-labelled integrin β1, β3, and α4 moving on the surface of mouse fibroblasts.37-39 The development of alkyne-HPGFNDs as a new tool for real-time tracking immediately points toward continuous long-term observations of membrane protein movements with the technique. The study is essential to gain a complete understanding of how the protein molecules react with their surroundings, including rare and heterogeneous responses, as well as their endocytosis and recycling processes. Such observations in the past were always hampered by photobleaching of the molecular fluorophores that label membrane proteins of interest on cell surface. With the availability of FNDs, it is now possible to conduct fluorescence imaging with an arbitrarily long time without interruption. A proof of this concept is given in Video S2 for integrin α5 on/in HFW cells, which were first labelled with 100 nm alkyneHPGFNDs and then with Alexa Fluor 488-conjugated wheat germ agglutinin (Alexa488-WGA)40 to assist the visualization by confocal fluorescence microscopy (Figure 8a – 8c). In contrast to the random movement observed in the short term (Figure 7b and 7c), the seemingly aimless transports of integrin α5 actually have predefined destinations over a longer term. For instance, the protein molecules slowly moved toward the front side (indicated by blue arrows in Figure 8d – 8f) of the cell migrating route after 2-h incubation, an event that could not be unveiled by short-term imaging. Moreover, the transport of integrin α5 on the filopodia was readily observed for hours, surpassing the observations through dye labelling. We have also successfully applied the techniques to label integrin β1 on HFW cells (Video S3) and HeLa cells (Video S4) using alkyne-HPGFNDs of different sizes (100 nm and 50 nm) and conducted the imaging and tracking of the protein molecules for more than 10 h. The ability

Figure 7. Real-time tracking of membrane proteins on cell surface. (a) Epifluorescence images of alkyne-HPGFND-labelled integrin α5 on living HFW cells. Grey curve represents the outline of the analyzed cell. (b) Zoom-in image and (c) trajectories of integrin α5 (c) in the white square region of (a). Top 400 longest trajectories are shown in (c). The size of the image in (b) was 176 × 176 pixels and the length of the video providing the images was 5 min (or 2882 frames/video). Scale bar: 50 µm.


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ACS Applied Materials & Interfaces particles were thoroughly washed with methanol and DDW to remove unbound polymers.

Figure 8. Long-term tracking of integrin α5 on/in cells. Time-lapse fluorescence images (0 – 2 h) of living HFW cells labelled with Alexa488-WGA (a – c) and alkyne-HPGFND (d – f). White arrows indicate the migrating cell of particular interest and blue arrows denote the migration of integrin α5 on/in the cells. Scale bar: 20 µm.

to achieve high specificity of the labelling for different membrane proteins demonstrates the general applicability of this nanomaterial-enabled method.

CONCLUSIONS We have developed a facile method to produce alkyneHPGFNDs for highly specific and efficient bioorthogonal labelling of membrane proteins on living cells. Possessing exceptional optical properties, the alkyne-HPGFND particles can be applied to study the dynamics of membrane proteins over an extended period of time, essentially until cell death. Specifically, we have tracked the movements of integrin α5 and β1 on/in living cells in both short and long terms, demonstrating the promising use of alkyne-HPGFNDs for bioimaging applications. Further improvement of the technique to ensure monovalent labeling of the targeted proteins to reveal their intrinsic dynamics, however, is required.39 Through this technological improvement in conjunction with super-resolution fluorescence microscopy of NV centers,41 the alkyne-HPGFND particles are expected to open new avenues for biologists to uninterruptedly track specific membrane proteins on living cells with both high temporal and spatial resolution for the first time.

EXPERIMENTAL SECTIONS Preparation and surface modification of FNDs. The method of producing FNDs by ion irradiation and subsequent annealing has been previously reported and detailed in Supporting Information.42 To prepare alkyneHPGFNDs, FNDs were added to glycidol and heated at 120 °C for 2 h under a nitrogen atmosphere. Glycidyl propargyl ether was then mixed with the suspension and continually reacted for another 22 h under the same conditions in one pot. Finally, the alkyne-HPGFND

Characterization of bare and surface-modified FNDs. Hydrodynamic sizes of bare and surface-modified FNDs were measured with a particle size and zeta potential analyzer. Samples for infrared spectroscopic measurements were prepared in KBr-pellets and the spectra were obtained with a FTIR spectrometer. Thermogravimetric analysis was conducted with a thermal analyzer for samples in air from room temperature to 800 °C. For two-dimensional NMR spectroscopy, surfacemodified FNDs were first dispersed in D2O and then analyzed by a spectrometer operating at 850 MHz. The NMR spectrometer was equipped with a triple resonance probe and a standard pulse sequence, hsqcetgpsisp.2, was used to record the HSQC spectra.43 Click chemistry and quantification of alkynyl groups. Click chemistry between alkyne-HPGFNDs and Alexa Fluor 488-azides was conducted following the protocols of Presolski et al.44 with slight modifications. Briefly, alkyneHPGFNDs were added into an Alexa Fluor 488-azide solution containing 0.15 mM Tris(3hydroxypropyltriazolylmethyl)amine (THPTA), 0.03 mM CuSO4, 5 mM aminoguanidine, and 5 mM sodium ascorbate as the catalysts. After 1-h incubation, the Alexa Fluor 488-azide-bound alkyne-HPGFND particles were washed with DDW, collected by centrifugation, and redispersed in DDW. Fluorescence intensities of the individual samples were measured with a multimode microplate reader to obtain Alexa Fluor 488 and FND concentrations. The number of alkynyl groups per alkyneHPGFND was estimated under the assumption that FNDs are spherical in shape. Extraction of azide-modified proteins and MALDI-TOF MS analysis. Azide-modified biotin-conjugated NeutrAvidin (NA/AB) was prepared by mixing N-[2-[2-[2(2-azidoethoxy)ethoxy]ethoxy] ethyl]biotinamide and NeutrAvidin in DDW together for 30 min and subsequently purified by membrane filtration. To extract azide-modified proteins, alkyne-HPGFNDs were added to protein mixtures containing BSA and NA/AB in PBS. After 1-h incubation with catalysts, the alkyne-HPGFND particles were washed with PBS, collected by centrifugation, and analyzed by MALDI-TOF MS as described by Chang et al.33 Cell culture and labelling. HeLa or HFW cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum and 1% antibiotic-antimycotic at 37 °C with 5% CO2. To perform sialoglycoprotein labelling, cells (1 × 105 cells/well) were seeded in medium containing ManNAz 2 days before



experiments. After removal of culture medium by decantation, alkyne-HPGFNDs and the catalysts (3 mM THPTA, 0.6 mM CuSO4, 5 mM aminoguanidine, and 5 mM sodium ascorbate in PBS) were added to label azidosialoglycoprotein on the surface of living cells.19 Following incubation for 30 min, the FND-labelled cells were thoroughly washed with culture medium to remove non-specifically bound FNDs and analyzed by flow cytometry or confocal microscopy. For the integrin α5 targeting, azide-modified antibody was first prepared by mixing anti-integrin α5 antibody with azido-PEG4-NHS, incubated for 2 h at room temperature, after which a TrisHCl buffer was added to terminate the reaction and obtain azide-modified antibody. Bioorthogonal labelling was then conducted by applying azido-anti-integrin α5 antibody for 1 h and finally with alkyne-HPGFNDs. Flow cytometry, confocal microscopy and epifluorescence microscopy. Cells were detached by trypsinization, transferred to 96-well plates, and analyzed by a flow cytometer featuring a 532 nm laser for excitation and detecting the emission at wavelengths >590 nm. Fluorescence images of the attached cells were acquired by using a laser-scanning confocal microscope system equipped with an oil-immersion objective and a supercontinuum white-light laser for the excitation of fluorescent dye molecules and FNDs at 488 and 561 nm, respectively. The corresponding emissions were detected at 500 – 550 and 670 – 800 nm. Videos were produced from continuously z-scanned images of cells for 1 – 12 h and played at a rate of 30 z-scanned images per second. Each z-stacked image consisted of 15 – 30 z-scans with a step size of ~0.4 μm. For epifluorescence imaging, the same microscope system was used but with a mercury lamp and a bandpass filter (515 – 560 nm) as the excitation source. An EMCCD detected the fluorescence emission at wavelengths >590 nm. The exposure time of obtaining the individual images was 30 ms and each video was produced from the continuously recorded images for 5 min. Quantification of cell-attached FNDs. Cells (5 × 104) labelled with alkyne-HPGFNDs were scraped in DDW and lysed by sonication. Fluorescence intensities of the cell lysates were then measured by using a home-built magnetic modulation system.34 The amounts of alkyneHPGFNDs attached to the cells were determined by comparing the measured fluorescence intensities to that of standard FND solutions.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website.

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AUTHOR INFORMATION Corresponding Author * C.-H.L.: [email protected] * H.-C.C.: [email protected]

Present Addresses † Current

address: Institute for Protein Research, Osaka University, Suita-shi, Osaka 565-0871, Japan.

Author Contributions ‡ These

authors contributed equally to this work.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The authors thank Hau-Ming Jan, Sasikala Muthusamy, Oliver Chen, and Wei-Chun Huang for helpful discussion and ChiFon Chang at the High Field Nuclear Magnetic Resonance Center of Academia Sinica for technical assistance. This work was supported by Academia Sinica and the Ministry of Science and Technology, Taiwan, with grant no. 107-2113-M001-018-MY3.

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Bioorthogonal Fluorescent Nanodiamonds for Continuous Long-Term

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