Investigating Functional DNA Grafted on Nanodiamond Surface Using

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Investigating Functional DNA Grafted on Nanodiamond Surface Using SiteDirected Spin Labeling and Electron Paramagnetic Resonance Spectroscopy Rana D. Akiel, Xiaojun Zhang, Chathuaranga Abeywardana, Viktor Stepanov, Peter Z. Qin, and Susumu Takahashi J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b00790 • Publication Date (Web): 08 Apr 2016 Downloaded from http://pubs.acs.org on April 13, 2016

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The Journal of Physical Chemistry

Investigating Functional DNA Grafted on Nanodiamond Surface Using Site-Directed Spin Labeling and Electron Paramagnetic Resonance Spectroscopy

Rana D. Akiel,1,3 Xiaojun Zhang,1 Chathuranga Abeywardana,1,3 Viktor Stepanov,1,3 Peter Z. Qin,1,** and Susumu Takahashi1,2,3, * 1

Department of Chemistry, University of Southern California, Los Angeles CA 90089, USA

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Department of Physics, University of Southern California, Los Angeles CA 90089, USA

3

Center for Quantum Information Science & Technology (CQIST), University of Southern

California, Los Angeles CA 90089, USA

ACS Paragon Plus Environment

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ABSTRACT Nanodiamonds (NDs) are a new and attractive class of materials for sensing and delivery in biological systems. Methods for functionalizing ND surfaces are highly valuable in these applications, yet reported approaches for covalent modification with biological macromolecules are still limited, and characterizing behaviors of ND-tethered bio-molecules is difficult. Here we demonstrated the use of copper-free click chemistry to covalently attach DNA strands at ND surfaces. Using site-direct spin labeling and electron paramagnetic resonance spectroscopy, we demonstrated that the tethered DNA strands maintain the ability to undergo repetitive hybridizations; and behave similarly to those in solutions, maintaining a large degree of mobility with respect to the ND. The work established a method to prepare and characterize an easily addressable identify tag for NDs. This will open up future applications such as targeted ND delivery and developing sensors for investigating bio-molecules.


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INTRODUCTION Nanodiamonds (NDs) are diamond crystals with sizes ranging from hundreds to a few nanometers. In a majority of cases, NDs contain nitrogen-vacancy (NV) centers, which are atomic-scale fluorescent defects embedded within a diamond lattice. NDs are bio-compatible and possess excellent chemical, mechanical, and photo stability, therefore are emerging as a promising class of agents for applications such as biological imaging and drug delivery.1-8 In addition, single NV centers can be monitored individually using fluorescence detection methods, and are extremely sensitive to their surrounding environments, such as variations in magnetic and electric fields as well as temperature.9-16 As such, NV centers, either in ND or in bulk diamond, are one of the leading candidates in nano-sensor development.13-15, 17 In applications involving ND and NV centers, methods for surface modification are highly desirable, as they allow tailoring of the ND surface for specific tasks, for example, enhancing solubility and cellular uptake as well as target specificity.1,

7, 18

A number of approaches to

modify the ND surface for further functionalization have been investigated, including oxidization,19 hydroxylation,5 fluorination,20 hydrogenation,21-22 and azide-functionalization.23-25 Methods for functionalizing ND with biological molecules (e.g., protein and DNA) have been reported, most of which use physical adsorption based on electrostatic interactions between ND and biological molecules,1,

4, 8, 26-28

with a few methods employing covalent attachment.29-32

However, it remains very difficult to obtain information on behaviors of the bio-molecules, as the presence of diamond presents a challenge to standard techniques (e.g., chromatography, optical spectroscopy) for investigating bio-molecules. Here we report a copper-free click approach that efficiently attaches single-stranded DNAs to NDs (Scheme 1). Using the method of site-directed spin labelling combined with electron


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paramagnetic resonance (EPR) spectroscopy, we showed that the tethered DNA strands maintain the ability to undergo repetitive hybridization with its complimentary stands (Scheme 1), and the ND-tethered duplexes behave similarly to those in solutions, maintaining a large degree of mobility with respect to the ND. The work established a method to prepare and characterize an easily addressable identify tag for NDs. This will open up future applications for directing NDs to specific biological targets, as well as the use of ND as a sensor for investigating structure and dynamics of bio-molecules.


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Scheme 1. DNA bar-coding of nanodiamond. A single-stranded DNA (S1) modified with a 5’NH2 group was first functionalized with DBCO, then reacted with N3-functionalized NDs to obtain tethered S1-ND. Functionalities of S1-ND were investigated through repetitive hybridization with a complementary strand (S2). S2 was labeled with a nitroxide spin label (indicated by the yellow dot), thus allowing characterization by EPR spectroscopy.


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MATERIALS AND METHODS Azide functionalization of ND: ND was finalized with azide following a previously reported procedure.23 Briefly, high-temperature high-pressure (HTHP) ND powders with an average diameter of 100 nm (Engis, Wheeling, IL) was first subjected to acid cleaning in a mixture of 9:1 H2SO4:HNO3 to remove surface impurities. Borane reduction was then performed to homogenize the surface with hydroxyl groups, which were then reacted with 3-bromopropyltrichlorosilane (Sigma-Aldrich, Milwaukee, WI; #437808). Lastly, the sample was treated with a saturated solution of sodium azide (Sigma-Aldrich, Milwaukee, WI; #438456) in DMF to yield azide functionalized

ND

(N3-ND).

The

N3-ND

was

purified

using

multiple

cycles

of

washing/centrifugation in water. Synthesis of S1-DBCO: The reaction follows the scheme shown in Supporting Information (SI) Section S.1. The 5’-amine modified DNA (S1) (sequence: 5’/amino-hexyl/CAA CAT GTT GGG ACA TGT TC) (Integrated DNA Technology, Inc., Coralville, IA) and the DibenzocyclooctyneN-hydroxysuccinimidyl (DBCO-NHS) (Click Chemistry Tool, Inc., Scottsdale, AZ) were used to synthesize S1-DBCO. Typically, the reaction mixture (200 µL) included approximately 120 µM of crude S1, 5 mM of DBCO, 100 µL 500 mM sodium bicarbonate buffer (NaHCO3) (pH 8.8), and 100 µL acetonitrile. The mixture was incubated for ~22 hours at room temperature. Upon completion of the reaction, the product was purified using anion-exchange high performance liquid chromatography (AX HPLC) followed by reverse-phase HPLC (RP HPLC). AX HPLC was carried out on an ÄKTA basic system (GE Healthcare, Inc.) using a DNApac PA-100 column (4 × 250 mm, Dionex Inc., Sunnyvale, CA), with a solvent gradient formed by buffer A: 20 mM Tris–HCl (pH 6.8), 1 mM NaClO4; and buffer B: 20 mM Tris–HCl (pH 6.8), 400 mM NaClO4. RP HPLC was carried out using a ProSphere C18 column (Grace, Inc.), with a


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solvent gradient formed by buffer A: 5% acetonitrile, and 0.1 mM Triethylamine acetate (TEAA); and buffer B: 100% acetonitrile. The purified oligonucleotide was lyophilized, re-suspended in water, and stored at -20oC. The concentration of DBCO-labeled S1 was determined based on its UV absorption at 260 nm. The measurement was performed using a Beckman Coulter DU800 UV-Vis spectrometer. The calculations used an extinction coefficient of 192,700 M-1cm-1, which does not include the very small contribution of DBCO. Copper free click reactions with N3-ND: The reaction schemes are shown in SI Section S.2 and S.3. Typically, N3-ND was suspended in 50 µL of 80%/20% (v/v) acetonitrile/water, and the mixture was subjected to ultrasonication for 30 min to obtain a homogeneous suspension. Then, the proper amount of reagents containing DBCO (i.e., S1-DBCO or DBCO-NHS) was added to the N3-ND mixture, and the volume was adjusted to 100 µL. The reaction mixture included 0.11.0 mg N3-ND, 10-320 µM DBCO-NHS or S1-DBCO, and 80%/20% (v/v) acetonitrile/water. The mixture was incubated for 22 − 24 hours at room temperature while subjected to constant ultrasonication. Temperature was not controlled during the reaction, and with ultrasonication the temperature increased to ~45oC by the end of the incubation. Upon conclusion of the incubation, the reaction mixture was washed to remove unreacted S1DBCO strands that are non-covalently adsorbed at the ND surfaces. The reaction mixture was first subjected to centrifugation to recover the NDs particles as a pellet, then, the recovered ND sample was re-suspended in ~20 µL solution of 80%/20% (v/v) acetonitrile/water. The mixture was homogenized using ultrasonication, then the ND sample was recovered again by centrifugation. The washing cycle was repeated until the UV-Vis absorption spectrum (220–320 nm) of the supernatant showed no detectable signal for DNA.


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Control studies showed that reactions in phosphate buffer saline (PBS) gave a reduced yield as compared to that conducted in 80%/20% (v/v) acetonitrile/water (55% vs. 95% at 170 µM DBCO). Hybridization of S1-ND with complimentary strands: The detailed scheme is shown in SI Section S.7. The S2 strand, which is complementary to the S1 strand, has a sequence of 5’-GAA CAT GTC CCA ACA TGT TG-3’. In the present work, S2 strands have either R5 or R5a nitroxides attached at backbone of either the 4th and 17th nucleotide. Following previous established procedures, the S2 strands were synthesized with the desired modification using solid-phase chemical synthesis (Integrated DNA Technology, Inc. San Diego, CA), and labeled with the corresponding nitroxide radical.33-35 The hybridization reaction was performed by mixing spin-labeled S2 (~100 µM) and S1-ND (~0.15 mg) in 100 mM PBS (pH 7, ~10 µL). The reaction was incubated overnight with constant mixing, then the excess DNA strands were removed following the washing process described above (see more details in SI Section S.6). X-band EPR spectroscopy: X-band continuous-wave (cw) EPR spectroscopy was performed using an EMX system (Bruker Biospin) equipped with a high-sensitivity cavity (ER 4119HS, Bruker Biospin). For each measurement, samples were placed in a quartz capillary (inner diameter: 0.86 mm or 0.64 mm), with the typical sample volume being 1–5 µL. cw EPR spectra were obtained by optimizing the microwave power and magnetic field modulation strength to maximize the amplitude of EPR signals without distorting the lineshape, with a typical parameter set being: microwave power of 2 mW; modulation amplitude of 0.03 mT; and modulation frequency of 100 kHz.


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Fourier-transform infrared spectroscopy: Fourier-transform infrared (FTIR) spectroscopy was performed using a Bruker Vertex 80 FTIR spectrometer equipped with a sample holder placed in a vacuum chamber. To prepare a FTIR sample, dried target materials were weighed and mixed with ~100 mg of KBr, and the powder mixture was pressed into a circular plate of ~10 mm in diameter. For each FTIR measurement, FTIR spectra from five different locations of the pellet were obtained to ensure the homogeneity of the sample distribution in the pellet. All spectra were acquired with a spectral resolution of 2 cm-1 and with 320 averages.

RESULTS AND DISCUSSON We have previously developed a method to covalently attach a small molecule onto ND surfaces using copper-catalyzed click chemistry.23 However, the same reaction yielded suboptimal results when adapted for DNA tethering, as the amount of Cu(I) catalyst required results in significant DNA degradation (data not shown). Therefore, we developed a new method based on the copper-free click reaction strategy,36-37 in which DNA strands functionalized with dibenzocyclooctyne (DBCO) are tethered to azide (N3) functionalized NDs (N3-ND) without the use of Cu(I) catalysts (for details, see Materials & Methods and SI Section S.1-3). In the present case, we employed NDs with an average diameter of 100 nm. As shown in Scheme 1, a singlestranded DNA (S1) is first modified with a 5’-amino (5’-NH2) group during solid-phase chemical synthesis, then functionalized with DBCO by reacting with dibenzocyclooctyne-Nhydroxysuccinimidyl ester. The success of DBCO modification of DNA (S1-DBCO) and its subsequent reaction with a N3-modified nitroxide in solution were demonstrated using a combination of chromatography and EPR spectroscopy (see SI Sections S.1 and S.4). To optimize the copper-free click reaction for NDs, we incubated DBCO with N3-NDs (SI Section S.2) and characterized the reaction using Fourier-transformed infrared (FTIR)


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spectroscopy (Materials & Methods).23 In the absence of DBCO (Figure 1a, blue trace), a pronounced peak can be observed at 2100 cm-1, which corresponds to N3 functional groups at the ND surface.38 Upon incubation with DBCO, the N3 signal clearly reduced (Figure 1a, green trace). By monitoring changes of the 2100 cm-1 peak intensity, we found that the reaction is complete within 22 hours, and the efficiency (i.e., the fraction of N3 reacted) depends on the DBCO concentration and the amount of NDs (which is proportional to the amount of N3) (Figure 1b, blue symbols; see also SI Section S.5). These studies indicated that for reactions with up to 0.3 mg of N3-ND in a volume of 100 µL solution, an efficiency of 90±10% can be achieved when the DBCO concentration is ≥ 170 µM (Figure 1b, blue symbols).


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Figure 1. Copper-free click reaction with N3-ND. (a) Normalized FTIR spectra of N3-NDs. The arrow marks the N3 peaks at 2100 cm-1. The spectra were normalized by the sample weight (see SI Section S.5). (b) Attachment efficiency determined based on intensity changes in the N3 FTIR peaks. To allow proper comparison, each data point was scaled by normalizing the amount of ND to 0.3 mg and the reaction volume to 100 µL. See SI Section S.5 for more details.


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Using procedures developed from the DBCO/ND studies, we performed the copper-free click reaction to covalently attach the DBCO-functionalized S1 DNA strand onto NDs (Figure 1, see also SI Sections S.3 and S.5). Upon extensive wash with 80%/20% (v/v) acetonitrile/water to remove unreacted S1 (see also SI Section S.6), FTIR analyses revealed significant reduction in the N3 peak of NDs in samples reacted with S1-DBCO (Figure 1a, red trace), with the yield reaching as high as 90±10% based on changes of FTIR signals (Figure 1b, red symbol). Additionally, in a control experiment using the S1 strand without DBCO attached, we did not observe any change in the FTIR signal at 2100 cm-1 (see SI Section S.5), demonstrating that changes observed at 2100 cm-1 indeed reports reaction of N3–ND with S1-DBCO. We then applied spin-labeling and EPR spectroscopy to investigate functionalities of S1 attached on NDs, specifically its ability to hybridize with its complementary strand (designated as S2) and the behavior of the attached duplex. The R5 or R5a nitroxide labels, which have been previously used to study DNA and RNA,34-35, 39 was attached to the phosphate group at a selected nucleotide of the S2 strand (see SI Section S.7). Continuous-wave (cw) EPR spectra, which report on rotational dynamics of R5 and R5a, were measured and analyzed to assess the behavior of ND-attached DNAs. Figure 2a shows a set of X-band cw-EPR spectra obtained before and after S1-ND was hybridized with a S2 strand with a R5 attached at the fourth nucleotide [R5(p4)-S2]. Before hybridization, the observed spectrum showed one pronounced signal at ~0.3328 Tesla and broad signals at ~0.3300 Tesla and ~0.3357 Tesla, which are consistent with EPR signals in NDs [substitutional single nitrogen impurities (S=1/2) and other paramagnetic impurities (S=1/2)].23 After hybridization and removal of excess S2 DNA (see also SI Sections S.6 and S.7), the EPR spectrum became clearly different, with additional signals appearing at ~0.3307 Tesla, ~0.3324


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Tesla, and ~0.3339 Tesla (Figure 2a). Detailed analyses revealed that the additional spectral features correspond to EPR signals of the

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N nitroxide radical from the R5(p4)-S2 strand (see

Figure 2a inset). This observation is consistent with formation of DNA duplexes tethered to NDs. Moreover, incubation of N3-ND (i.e., without the S1 strand attached) and R5(p4)-S2 showed only EPR signals of NDs after extensive washing (SI Section S.6), indicating a lack of nonspecific association of the spin-labeled S2 DNA strand to NDs. Therefore, the results strongly indicate that S1 tethered to ND maintains the ability to hybridize to its complementary strand. Furthermore, we estimated the number of tethered DNA molecules in the R5(p4)-S2-S1-ND sample by analyzing the intensity of EPR signals in the observed spectrum. By comparing the observed EPR intensity of the R5(p4)-S2-S1-ND sample (Figure 2a) with that of known concentrations of R5(p4)-S2 in a buffer solution (SI Figure S5a), the number of R5(p4)-S2 DNA on the ND surface was estimated to be 0.27 nmol, corresponding to 1.6 × 1014 R5(p4)-S2 molecules on surfaces of 0.1 mg 100-nm-ND. Using 3.51 g/cm3 for the density of diamond and ~5.2 × 10-16 cm3 for the volume of one 100-nm diameter spherical ND, the number of ND particles in the measured sample was estimated to be ~5.5×1010. Thus, each 100 nm ND has ~2900 of R5(p4)-S2 molecules on the surface, which corresponds to the average separation between the R5(p4)-S2 molecules of ~3.3 nm.


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Figure 2. Hybridization and re-hybridization of DNAs attached to NDs. In all panels, experimental spectra are shown as solid blue lines, and simulated ones as dashed red lines. Parameters used in the analyses are listed in SI Section S.8. (a) EPR spectra of DNA-tethered ND sample (S1-ND) before and after hybridization with the R5-labeled complementary strand R5(p4)-S2. The inset shows the extracted ND (green) and nitroxide (magenta) components from the EPR analysis. Details of the EPR analyses are reported in SI Section S.8. (b) EPR spectra of S1-ND upon denaturation and a second hybridization with R5(p4)-S2. (c) EPR spectra of S1-ND upon re-hybridization with R5a(p4)-S2 and R5(p17)-S2. Note that the R5a(p4)-S2 spectrum was obtained from a third hybridization using the same S1-ND.


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Next we tested the ability of ND-tethered DNAs to undergo repeated hybridization. Upon washing the NDs tethered with the S1-S2 duplex repeatedly in an excess amount of 50%/50% (v/v) acetonitrile/water without added salt, EPR spectroscopy showed that observed spectral signals are mostly from NDs, with residual nitroxide signal being < 3% (Figure 2b). The result strongly indicates successful denaturing of the S1-S2 duplex, which resulted in removal of the nitroxide signal associated with the spin-labeled R5(p4)-S2 strand. Significantly, when the same S1-ND sample was hybridized with R5(p4)-S2 following the protocol described above, we again observed a spectrum with identical spectral features as those found in the first hybridization (Figure 2b). Upon normalizing the EPR signals of the NDs to account for differences in the amount of samples between the two measurements, it was found that > 90% of the nitroxide EPR signal was recovered in the re-hybridization process. This clearly indicates successful rehybridization using DNA-tethered NDs. Furthermore, re-hybridization experiments were carried out with two other labeled S2 strands, one with an R5a label at the 4th nucleotide [R5a(p4)-S2] and the other with an R5 label at the 17th nucleotide [R5(p17)-S2] (see Figure 1 and SI Section S.7). In both cases, nitroxide signals were observed in the re-hybridized samples (Figure 2c). Upon accounting for the amount of NDs present, the amplitudes of the signals were comparable to that observed in the 1st hybridization. Together these results demonstrate that S1 strand tethered on NDs is able to repeatedly hybridize to different S2 strands bearing labels with different identity (i.e., R5 vs R5a) or at different location (i.e., p4 vs p17). As such, through interactions with its complementary strand, the tethered DNA sequence serves as an easily addressable tag for NDs. The nitroxide spectra obtained from the three hybridization experiments show features depending on the identity and location of nitroxide labels (Figure 3), which provide information


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on behaviors of the ND-tethered DNA duplexes. Specifically, R5(p4)-S2 and R5(p17)-S2 both showed the three-sharp-line pattern (Figure 3), which has been previously observed in studies of R5-labeled duplexes in solution.33 This is characteristic of highly mobile nitroxides, and indicates that at either site R5 has little direct contact with the ND surface. As such, the NDtethered duplexes are not lying down on the ND surface, but instead spreading out in solution and undergoing relatively free motions with respect to the ND. This conclusion is supported by multiple additional pieces of evidence. First, the rotational correlation time (τc) of R5(p4)-S2-S1ND was 1.0±0.1 ns, which is comparable to τc of free S1-TEMPO (SI Section S.4) as well as free DNA duplex in solution.40 Second, R5 motions are slightly slower at the p17 site (τc ~ 1.2 ns) than that at p4 (τc ~ 1.0 ns) (Figure 3 and SI Section S.8), which can be expected as p4 is further away from the tethering point (SI Section S.7). Third, when R5 is substituted by R5a at the p4 site, the resulting spectrum becomes broader, and shows splitting at the low-field manifold (Figure 3 and SI Section S.9). These results are strongly indicative of a nitroxide undergoing anisotropic rotation under the restriction of a potential, and arises from the substitution of –H by –Br at the 4-position of the pyrroline ring (Scheme 1), which impedes rotations about bonds connecting the pyrroline ring to the DNA.33, 41 This bears strong similarity to results obtained from R5a-labeled DNA duplexes, including those tethered to a protein (streptavidin) (SI Section S.9).40, 42 The high mobility of the tethered DNA may stem from the use of a relatively long and flexible linker to tether the DNA to the ND surface (Scheme 1, SI Section S.7). To test this notion, we used the same DBCO scheme to attach a nitroxide radical, 4-amino-TEMPO, to N3ND (SI Section S.10). This removed 6 ethylene bonds in the linker connecting the radical to ND, and is expected to reduce the mobility of the nitroxide. The resulting TEMPO-ND spectrum


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clearly was much broader, with a correlation time (τc) of ~4 ns (Figure 3, SI Section S.10), which is considerably slower than that obtained from the tethered DNA. In addition, the EPR analysis shows the much wider linewidth of the nitroxide component in the TEMPO-ND spectrum, indicating possible line broadening due to dipolar interaction (SI Section S.10). Overall, the data support the notion that the flexible linker between the DNA and ND contribute to the high mobility of the tethered duplex.

Figure 3. Extracted nitroxide components from simulations of R5(p4)-S2-S1-ND, R5(p17)-S2S1-ND, R5a(p4)-S2-S1-ND and TEMPO-ND EPR signals. Intensity of the TEMPO-ND spectrum is magnified by 10 times to show details of the EPR spectral lineshape. Details of the EPR analyses are reported in SI Section S.8 and S.10.


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CONCLUSION In summary, we demonstrated efficient covalent attachment of a single-stranded DNA onto surface of NDs using copper-free click-chemistry. The tethered DNA was able to hybridize to its complementary strand repeatedly, thus acting as an “identity tag” for the NDs that can be addressed in an easily and controllable fashion. Taking advantage of this re-usable tag, we carried out spin labeling studies to show that the ND-tethered duplexes behave similarly to those in solution, including those tethered to proteins. The method of DNA tethering to ND, its characterization using spin-labeling and EPR, as well as information learned on the tethered DNAs, will open up future applications for directing NDs to specific biological targets, as well as the use of ND as a sensor for investigating structure and dynamics of bio-molecules.

ASSOCIATED CONTENT Supporting Information. Additional data (sample preparation procedures, FTIR analyses and EPR spectral analyses) are included in the Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]; Phone number: +1 (213) 821-3187 **E-mail: [email protected]; Phone number: +1 (213) 821-2461 Notes The authors declare no competing financial interest. ACKNOWLEDGMENT


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This work was supported in part by the Searle scholars program (ST), the National Science Foundation (DMR-1508661, S.T.; CHE-1213673, P.Z.Q.), and the National Institute of Health (1S10RR028992, P.Z.Q.). REFERENCES (1).

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Figures and Graphical Table of Contents


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Figure 1. Copper-free click reaction with N3-ND. (a) Normalized FTIR spectra of N3-NDs. The arrow marks the N3 peaks at 2100 cm-1. The spectra were normalized by the sample weight (see SI Section S.5). (b) Attachment efficiency determined based on intensity changes in the N3 FTIR peaks. To allow proper comparison, each data point was scaled by normalizing the amount of ND to 0.3 mg and the reaction volume to 100 µL. See SI Section S.5 for more details. 162x256mm (300 x 300 DPI)


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Figure 2. Hybridization and re-hybridization of DNAs attached to NDs. In all panels, experimental spectra are shown as solid blue lines, and simulated ones as dashed red lines. Parameters used in the analyses are listed in SI Section S.8. (a) EPR spectra of DNA-tethered ND sample (S1-ND) before and after hybridization with the R5-labeled complementary strand R5(p4)-S2. The inset shows the extracted ND (green) and nitroxide (magenta) components from the EPR analysis. Details of the EPR analyses are reported in SI Section S.8. (b) EPR spectra of S1-ND upon denaturation and a second hybridization with R5(p4)-S2. (c) EPR spectra of S1-ND upon re-hybridization with R5a(p4)-S2 and R5(p17)-S2. Note that the R5a(p4)-S2 spectrum was obtained from a third hybridization using the same S1-ND. 138x369mm (300 x 300 DPI)



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Figure 3. Extracted nitroxide components from simulations of R5(p4)-S2-S1-ND, R5(p17)-S2-S1-ND, R5a(p4)-S2-S1-ND and TEMPO-ND EPR signals. Intensity of the TEMPO-ND spectrum is magnified by 10 times to show details of the EPR spectral lineshape. Details of the EPR analyses are reported in SI Section S.8 and S.10. 230x225mm (300 x 300 DPI)


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Scheme 1. DNA bar-coding of nanodiamond. A single-stranded DNA (S1) modified with a 5’-NH2 group was first functionalized with DBCO, then reacted with N3-functionalized NDs to obtain tethered S1-ND. Functionalities of S1-ND were investigated through repetitive hybridization with a complementary strand (S2). S2 was labeled with a nitroxide spin label (indicated by the yellow dot), thus allowing characterization by EPR spectroscopy. 276x269mm (300 x 300 DPI)



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Graphical Table of Contents 82x24mm (300 x 300 DPI)


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Investigating Functional DNA Grafted on Nanodiamond Surface Using

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