Grafting nitroxide radicals on nanodiamond surface using click chemistry

Article abstract of DOI:10.1021/jp403183x

We demonstrate grafting of nitroxide radicals on the surface of nanodiamonds (NDs). The surface of NDs is functionalized by azide groups. Nitroxide radicals are covalently bonded using Cu(I)-catalyzed azide/alkyne-click chemistry approach. The reaction is confirmed by infrared spectroscopy. The grafting of nitroxides is also verified by studying the rotational correlational time using electron paramagnetic resonance (EPR) spectroscopy. EPR study estimates that a few hundreds (tens) of nitroxide radicals are grafted on the surface of NDs with 100 nm (25 nm) of the average diameter.

Full text of DOI:10.1021/jp403183x

Article
pubs.acs.org/JPCA
Grafting Nitroxide Radicals on Nanodiamond Surface Using Click
Chemistry
†
†
‡
,†,‡
Ekaterina E. Romanova, Rana Akiel, Franklin H. Cho, and Susumu Takahashi*
†
‡
Department of Chemistry and Department of Physics, University of Southern California, Los Angeles, California 90089, United
States
ABSTRACT: We demonstrate grafting of nitroxide radicals
on the surface of nanodiamonds (NDs). The surface of NDs is
functionalized by azide groups. Nitroxide radicals are
covalently bonded using Cu(I)-catalyzed azide/alkyne-click
chemistry approach. The reaction is confirmed by infrared
spectroscopy. The grafting of nitroxides is also verified by
studying the rotational correlational time using electron
paramagnetic resonance (EPR) spectroscopy. EPR study
estimates that a few hundreds (tens) of nitroxide radicals are
grafted on the surface of NDs with 100 nm (25 nm) of the
average diameter.
INTRODUCTION
as well as tolerance toward many functional groups. Therefore
the click chemistry concept currently is one of the most
versatile methods for grafting molecules on nanoparticle surface
and has been successfully applied to various nanoparticles, such
■
Nanodiamonds (NDs) are fluorescent nanometer-sized par-
ticles with excellent mechanical and chemical stability. NDs
have recently been investigated extensively because of unique
optical and magnetic properties of nitrogen-vacancy (NV)
impurity color centers in NDs. NV centers are attractive for
testing fundamental quantum science
applications of magnetic sensing devices.
biocompatibility and photostability of fluorescent NDs make
them suitable for optical and magnetic imaging of biological
systems and for drug delivery by functionalizing their surface to
35
36
37
38
39,40
as Au, CdSe, Fe O , SiO2, and NDs.
2
3
In this article, we demonstrate grafting of nitroxide radicals
onto the surface of high-temperature high-pressure (HTHP)
NDs with an average particle diameter of 100 and 25 nm. A
covalent attachment of nitroxide radicals provides a way to
stably position paramagnetic molecules on the ND surface for
future applications of NV-based magnetic sensing and single-
molecule magnetic resonance spectroscopy. For the grafting, we
employ surface silanization and bromine−azide conversion to
functionalize the ND surface with azide groups. A click-
chemistry approach is employed to covalently attach alkyne-
contained nitroxide radicals on the azide-functional ND surface.
To the best of our knowledge, the combination of the surface
silanization and click-chemistry for grafting organic molecules
on ND surface has not been explored so far. The reaction
process is confirmed using Fourier-transformed infrared
(FTIR) and electron paramagnetic resonance (EPR) spectros-
copies. We find that efficiency of click-chemistry approach is
∼95% based on FTIR analysis. In addition, using EPR
spectroscopy, we show that the rotational correlation time of
nitroxides grafted on NDs is slower than that of free nitroxide
radicals. The decrease in the correlation time strongly supports
successful grafting of nitroxide radicals on the ND surface. The
estimated number of nitroxides attached on a single 100 nm
(25 nm) ND is ∼300 (20).
1
−4
and for future
5
−8
In addition,
9
−21
attach various types of molecules.
Several approaches to
control surface functionalization for NDs have been inves-
tigated. Examples include treatment of NDs with oxidizing
2
2
23
reagents, silanization of the hydroxylated NDs, fluorina-
2
4
25,26
tion, or hydrogenation
of ND’s surface using fluorine or
hydrogen gases and elevated temperatures, followed by
subsequent amination or thiolation. Most of these approaches
require harsh reaction conditions, such as high temperatures,
use of toxic gases, and sophisticated setups. One of the
common ways to achieve homogeneous surface functionaliza-
tion is employment of silanization technique. Surface
homogenization is often required for a high performance and
reproducibility of subsequent reactions. The silanization
2
7
2
8,29
technique has been widely applied to functionalize metal
30−32
and semiconductor
nanoparticles.
Cu(I)-catalyzed azide−alkyne cycloaddition (CuAAC) re-
action known as click chemistry has been utilized in a variety
33
of approaches to graft various molecules on nanoparticle’s
3
4
surface. The click chemistry proceeds at room temperature
under mild reaction conditions and combines azide- and
alkyne-containing reactants to give stable 1,4-disubstituted-
Special Issue: Curt Wittig Festschrift
1
,2,3-triazole linker between nanoparticle surface and grafted
Received: March 31, 2013
Revised: June 1, 2013
molecule. The main advantage of the CuAAC application on
surfaces is high efficiency, the avoidance of high temperatures,
©
XXXX American Chemical Society
A
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The Journal of Physical Chemistry A
Article
Scheme 1. Cu(I)-Catalyzed Azide/Alkyne-Click Chemistry Approach for Grafting Nitroxide Radicals on the ND Surface (1−6)
EXPERIMENTAL SECTION
in a flask under a nitrogen atmosphere. The flask was sonicated
for 30 min, then heated to 80 °C. One mL of 3-
bromopropyltrichlorosilane was added dropwise and the
resulting solution was heated to 80 °C for 24 h. To separate
the product of bromide-functional NDs from unreacted 3-
bromopropyltrichlorosilane, the reaction mixture was redis-
persed in toluene and centrifuged. This cycle was repeated four
times to obtain 200 mg of pure bromide-functional NDs (2).
■
Materials and Chemicals. We employed two kinds of
HTHP ND powders; one has 100 nm average diameter (Engis,
Wheeling, IL) and the other has 25 nm average diameter (Van
Moppes, Geneva, Switzerland). For the synthesis of the
nitroxide-grafted ND, the following chemicals were used: 3-
bromopropyltrichlorosilane (#437808), 4-hydroxy-TEMPO
(
#176141), propargyl bromide (#P51001), borane tetrahydro-
Then, 200 mg of 2 and saturated solution of NaN (100 mg in
3
furan complex solution (BH -THF, #176192), sodium azide
3
4
0 mL of DMF) were combined in a flask under a nitrogen
(
NaN , #438456), sodium hydride (NaH, #223441), copper(I)
3
atmosphere. The mixture was sonicated for 30 min, then stirred
at 80 °C for 24 h. The reaction mixture was redispersed in DI
water and centrifuged to remove excess of sodium azide. This
cycle was repeated four times to yield 175 mg of azide-
functional NDs (3). Parallel to the synthesis of the azide-
functional NDs, the alkyne-contained nitroxide radicals (5)
were prepared from 4-hydroxy-TEMPO radicals (4). To a
stirring suspension of NaH (175 mg) in 30 mL of dry DMF, 1 g
of 4 was added at 0 °C. The solution was stirred at room
temperature for 30 min, then 0.7 mL of propargyl bromide was
added dropwise at 0 °C, followed by stirring for 3 h at room
temperature. The reaction mixture was purified by column
chromatography (silica gel, 10% ETOAC in hexane) to yield
iodide (Cu(I), #205540), triethanolamine (TEA, #90279) and
hydrochloric acid (HCl, #H1758) purchased from Sigma-
Aldrich (Milwaukee, WI), dimethylformamide (DMF,
#
DX1729), ethyl acetoacetate (ETOAC, #8.09622), sulfuric
acid (H SO , #1.00748), nitric acid (HNO , #NX0412), hexane
2
4
3
(
#HX0290), anhydrous acetonitrile (CH CN, #AX0152), and
3
toluene (#TX0734) purchased from EMD Millipore (Billerica,
MA). Milli-Q water (18 MΩ) was used.
Grafting of Nitroxide Radicals on ND Surface. We
employed Cu(I)-catalyzed azide/alkyne-click chemistry ap-
proach to graft nitroxide radicals on ND surfaces. Our synthesis
procedure is shown in Scheme 1. To obtain the starting ND,
ND surface was hydroxylated by performing a strong acid
treatment, followed by a borane reduction. The acid treatment
consists of refluxing NDs in a 9:1 mixture of concentrated
H SO and HNO at 75 °C for 72 h, followed by 0.1 M NaOH
∼
800 mg of 5. Finally, covalent attachment of 5 and 3 was
achieved by click-reaction in the presence of Cu(I) catalyst to
yield nitroxide-grafted NDs (6). 175 mg of 3 was dispersed in
2
4
3
3
0 mL of anhydrous acetonitrile and sonicated for 30 min. The
solution at 90 °C for 2 h, then by 0.1 M HCl solution at 90 °C
for 2 h. After the acid treatment, the obtained NDs were
repeatedly rinsed with deionized (DI) water, separated by
centrifugation (10 000−15 000 rpm for 15 min using Sorvall
RC-5C Plus) and dried under vacuum. The acid cleaning
removes graphitic and other organic impurities. It also oxidizes
the ND surface, increasing the number of carboxyl groups
click-reaction was performed by the addition of 70 mg of 5, 70
mg of Cu(I), and 1 mL of TEA to the mixture. The reaction
was stirred for 48 h at room temperature, followed by
centrifugation, and the pellet was resuspended in acetonitrile.
This procedure was repeated 10−15 times to remove
nonreacted nitroxide radicals. The resulting solid was dried in
vacuum to yield 6.
Characterization. Infrared (IR) spectra of functionalized
NDs were measured using Bruker Vertex 80 FTIR
spectrometer. In the IR measurement, dried ND powder (0.5
to 1 mg) was mixed with KBr powder (100 mg) in an agate
mortar. The mixture was pressed into a pellet using a 10 ton
load. In our FTIR measurement, FTIR signals from a pure KBr
pellet were subtracted as background signals. Nitroxide radicals
on the ND surface and paramagnetic impurities in and on NDs
were characterized using EPR spectroscopy. We employed X-
band continuous-wave (cw) EPR spectrometer (EMX system,
(
−COOH). Subsequently, 6 mL of 1.0 M BH −THF was
3
added dropwise to a stirring suspension of 250 mg acid-treated
NDs in 30 mL of dry THF. Then, the mixture was refluxed for
2
4 h and hydrolyzed with 2 M HCl after cooling to room
temperature. Resulting NDs were washed with DI water (four
times) and acetone (once) in consecutive washing/centrifuga-
tion cycles to remove the boronic acid produced during the
reaction and dried in vacuum to yield 230 mg of hydroxylated
ND powder (1). To functionalize 1 with an azide group
38
required for click chemistry, a silanization technique was used.
30 mg of 1 and 20 mL of anhydrous toluene were first added
2
B
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Article
Figure 1. FTIR spectra of NDs. (a) FTIR signals of 100 nm NDs after acid treated and in 1, 2, 3, and 6 in Scheme 1. (b) FTIR signals of 25 nm NDs
after acid treated and in 1, 3, and 6. FTIR signals from vibrational modes on the ND surface are indicated by dotted lines.
−1
Figure 2. FTIR spectra of azide moieties at 2100 cm . (a) FTIR signals of 100 nm NDs in 3 and 6 in Scheme 1. (b) FTIR signals of 25 nm NDs in
and 6 in Scheme 1.
3
Bruker Biospin) with a high-sensitivity cavity (ER 4119HS,
Bruker Biospin).
peaks of vibrational modes of hydroxyl groups (O−H) at
1
∼1630 cm− and in 3000−3600 cm . The reaction with
tetrahydrofuran complex typically reduces carbonyl groups to
hydroxyl groups, that is, leading to decrease in the CO IR
signals and increase in the O−H and C−H IR signals. As
shown in 1 in Figure 1, an increase in the C−H stretching
−1 23
A home-built cw/pulsed 230/115 GHz EPR spectrometer
was also employed to characterize nitroxide radicals and
paramagnetic impurities. The high-frequency EPR system
employs a high-power solid-state source consisting 8−10
GHz synthesizer, p-i-n switch, microwave amplifiers, and
frequency multipliers. The output power of the source system
is 100 mW (700 mW) at 230 GHz (115 GHz). 230/115 GHz
excitation is propagated using quasioptical bridge and a
corrugated waveguide and couples to a sample located at the
center of 12.1 T cryogenic-free superconducting magnet. EPR
signals are isolated from the excitation using an induction mode
−1
vibrational signal (near 2992 cm ) was visible, indicating
increase of hydroxyl groups due to the reduction of carboxyl
groups on ND surface. Changes of the O−H signals were not
well-pronounced due to absorption signals of water molecules
on ND surface. In addition, the remaining CO signal in 1
suggested that the reduction step resulted in ND surface
partially covered with hydroxyl groups. This is due to the fact
that acid-treated NDs form strongly bounded agglomerates that
are hard to separate by ultrasonication because of interparticle
4
1
operation. For EPR detection, we employed a super-
heterodyne detection system in which 230/115 GHz is
down-converted into 3 GHz of intermediate frequency (IF),
then down-converted again to in-phase and quadrature
components of dc signals. Details of the system will be
described elsewhere.
23
covalent bonding. Therefore, the reducing agent will have
limited access to ND surface, resulting in an inhomogeneous
surface functionalization.
To obtain Product 2, we used the hydroxyl groups of 1 to
covalently attach 3-bromopropyltrichlorosilane to ND surface.
After the reaction, the binding of silane was confirmed by the
characteristic FTIR signal of the C−Si−O bond at ∼1119 cm−
shown in Figure 1a. Then, 2 were reacted with sodium azide to
obtain azide-functional NDs (3). For both 100 and 25 nm
NDs, the success of the reaction was confirmed by the
RESULTS AND DISCUSSION
■
1
During the process of grafting nitroxide radicals on NDs, we
measured FTIR spectrum of the NDs after each step of the
reaction. Figure 1 shows the FTIR spectra of both 100 (a) and
2
5 nm (b) NDs after the acid treatment and after obtaining
−1
product of 1, 2, 3, and 6 in Scheme 1. The FTIR spectra of the
acid-treated NDs show characteristic carbonyl (CO)
stretching vibration modes at ∼1100 and ∼1760 cm− and
appearance of azide signal at 2100 cm in the FTIR spectra of
3
7,38
3.
Finally, using click-reaction, alkyne-contained nitroxide
1
radicals were covalently attached to 3 through the azide-
C
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Figure 3. cw EPR spectra of the ND powder samples (Product 3 in Scheme 1). (a) 230 GHz EPR spectrum of 100 nm NDs. The spectrum includes
4
2
N spin and the other S = 1/2 impurities signals. A fit to the data using easyspin is shown by the red dotted line. The fit gives gx,y = 2.0029 ± 0.0001
and g = 2.0027 ± 0.0001 and 0.8 ± 0.1 mT of Lorentzian peak-to-peak line width for the S = 1/2 EPR signal, and the intensity ratio of N to the S =
z
1/2 EPR is 1:6 ± 1. (b) X-band EPR spectrum of 100 nm NDs. A fit is shown by the red dotted line. For the fit, we used the g value for the S = 1/2
spin obtained from the 230 GHz EPR measurement. The fit gives 0.4 ± 0.1 mT of Lorentzian peak-to-peak line width and the intensity ratio (N to
the S = 1/2 EPR = 1:8 ± 1). The inset shows the fitted EPR spectrum consisting of N and the S = 1/2 EPR spectra. (c) 230 GHz EPR spectrum of
2
1
5 nm NDs. Only the S = 1/2 EPR is visible. A fit is shown by the red dotted line. The fit gives gx,y = 2.0029 ± 0.0001 and g = 2.0027 ± 0.0001 and
.0 ± 0.1 mT of Lorentzian/0.1 ± 0.1 mT of Gaussian peak-to-peak line width. (d) X-band EPR spectrum of 25 nm NDs. For the fit, we used the g
z
value for the S = 1/2 spin obtained from the 230 GHz EPR measurement. The fit gives 0.6 ± 0.1 mT of Lorentzian peak-to-peak line width.
Figure 4. X-band cw EPR spectra of the ND powder samples (Product 6 in Scheme 1). (a) cw EPR spectrum of 100 nm NDs. The inset shows the
simulated EPR spectrum consisting of ND (N plus S = 1/2 impurities signal) and nitroxide EPR spectra. (b) cw EPR spectrum of 25 nm NDs. The
inset shows the simulated EPR spectra consisting of ND (N only) and nitroxide EPR spectra.
functional groups to give 6. This was confirmed by the
significant reduction of the azide peak at 2100 cm in the
μW, and field modulation amplitude of 0.05 mT at frequency of
100 kHz were used. For 230 GHz EPR measurements,
microwave frequency of 230 GHz, microwave power of ∼10
μW at sample and field modulation amplitude of ∼0.05 mT at
frequency of 20 kHz were used. For measuring free nitroxide
radicals aqueous solution, higher microwave power of ∼5 mW
at sample was used due to attenuation of microwave power
from water. Figure 3 shows 230 GHz/X-band cw EPR spectra
for 100 and 25 nm NDs (Product 3 in Scheme 1). EPR
spectrum in Figure 3a shows that paramagnetic impurities
present in NDs consist of substitutional single nitrogen (N)
−1
3
9,40
FTIR spectra of 6.
Figure 2a,b shows the FTIR peak of the
−1
azide vibrational mode at 2100 cm of Products 3 and 6.
Comparing the areas under the peaks of 3 and 6, we estimated
that approximately 95−98% of azides on the ND surface were
reacted to attach nitroxide radicals. We also tested stability of
the silanized surface against hydrolysis. On the basis of the
FTIR intensity of the azide peak, the silanized surface is stable
for more than 16 h under both water solution (pH 7.00) and
slightly acidic phosphate buffer solution (pH 6.85).
Next, we employed EPR spectroscopy to confirm attachment
of paramagnetic nitroxide radicals (S = 1/2) on the surface of
NDs. All EPR measurements were performed at room
temperature. For X-band EPR measurements, microwave
frequency of ∼9.3 GHz, applied microwave power of ∼50
impurities (S = 1/2, gx,y = 2.0024, g = 2.0024 and the hyperfine
z
coupling of the N impurity to 14N nuclear spin (I = 1) is A =
x
4
A = 82 MHz and A = 114 MHz) and other S = 1/2
y
z
2
2,41
impurities.
By fitting the 230 GHz EPR spectrum, we
extracted the g value of the S = 1/2 impurities (gx,y = 2.0029 ±
D
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Figure 5. X-band and 230 GHz cw EPR spectra of 500 μM free nitroxide radical and nitroxide radicals attached on NDs. All ND EPR spectra were
measured with ∼1.0 mg of dry powder placed in a glass capillary tube, followed by the addition of 5 μL of water. In the case of free nitroxide radicals
(5), 5 μL of 500 μM nitroxide solution was measured. (a) X-band EPR spectra of free nitroxide radicals (5) and the nitroxide-drafted NDs (6). The
fit results to extract the correlation time are shown by the red dotted line. Extracted spectrum and fit of nitroxide from the nitroxide-attached NDs
are shown in the inset. (b) 230 GHz EPR spectrum of 5. Five μL of 500 μM free nitroxide radical placed in a Teflon bucket. The fit result to extract
the correlation time is shown by the red dotted line. (c) X-band EPR spectrum of the nitroxide-drafted NDs (6). Extracted spectrum and fit of
nitroxide from the nitroxide-attached NDs are shown in the inset. The fit result to extract the correlation time is shown by the red dotted line. (d) X-
band EPR spectra of 5 and 6 measured with same measurement parameters. In both samples, the same volume of 5 μL was used. All of the volume is
in the active area of the cavity; therefore, the whole sample contributes to the observed EPR signal. 6 (100 nm NDs) and 6 (25 nm NDs) include 1
mg of dry ND powders. 1:0.13:0.35 of the EPR intensity ratio (5 to 6 (100 nm NDs) to 6 (25 nm NDs)) was obtained with EPR line shape analysis.
0
.0001 and g = 2.0027 ± 0.0001). To verify the g factor, we
pronounced peaks due to the hyperfine coupling of the
nitroxide to 14N nuclear spin (I = 1, Aiso = (A + A + A )/3 =
z
also measured the 100 nm NDs using the X-band EPR
spectrometer. As seen in Figure 3b, we found a good agreement
between the experimental data and the fit. In the fit, the g values
for the N and S = 1/2 spins were fixed to the values obtained
from the 230 GHz EPR measurement and only the intensity
ratio of N to S = 1/2 impurities, and their linewidths were
varied. In 25 nm NDs, only the S = 1/2 impurities EPR signal
was observed (see Figure 3c,d). This is due to lower
concentration of N impurities in 25 nm NDs. From the fit,
x
y
z
48 MHz). Lineshape analysis of the EPR spectrum, that is,
analyzing line width and amplitude of the EPR spectrum, gives
the rotational correlation time τ which indicates the degree of
motion of nitroxides in aqueous solution. With the line shape
42
analysis of the X-band EPR spectrum of 5 using easyspin, we
obtained τ = 25 ± 10 ps. The correlation time was also
estimated for 230 GHz EPR spectrum of 5. Because of the
higher Larmor frequency, 230 GHz EPR measurement is more
sensitive to the correlation time in the tens of picoseconds
range. A fit to the 230 GHz spectrum in Figure 5b shows that
the correlation time is τ = 30 ± 8 ps, consistent with the X-
band measurement. We next studied the nitroxide grafted NDs
gx,y = 2.0029 ± 0.0001 and g = 2.0027 ± 0.0001 for the S = 1/2
z
spins were obtained, which is consistent with 100 nm NDs
measurement. We also verified the g factor by comparing the X-
band EPR spectrum (Figure 3d).
After characterizing Product 3 in Scheme 1, we studied cw
EPR spectra of 100 and 25 nm NDs of Product 6. As shown in
Figure 4a,b, the observed EPR spectra are clearly different from
those of 3 because of the existence of nitroxide radicals grafted
on the ND surface. To verify the results, we simulated EPR
spectrum consisting of ND and nitroxides EPR spectrum. As
shown in the inset of Figure 4a,b, the simulated EPR spectra
agree reasonably well with the experimental results.
(
6). Because the EPR spectrum includes both NDs and
nitroxide radical signals, we first analyzed nitroxide EPR
spectrum of 6 by subtracting the ND spectrum (3) with
constant baseline correction (see the inset of Figure 5a). A fit
was then performed to obtain the correlation time. We
obtained τ = 79 ± 31 ps for the 100 nm nitroxide-attached
NDs (6). Similarly, using the EPR spectrum shown in Figure
5
c, we found τ = 63 ± 32 ps for the 25 nm nitroxide-attached
To confirm covalent attachment of nitroxide molecules on a
ND surface, we investigated dynamics of nitroxide radicals in
aqueous solution using cw EPR measurements. The spectra of
NDs (6). Those slower correlation times of the nitroxide-
attached on the 100 and 25 nm NDs are due to confinement of
nitroxide motions; therefore, the result strongly supports
successful covalent attachment of nitroxide radicals on the
ND surface.
5
in Figure 5a show the X-band EPR spectrum of free nitroxide
radicals in aqueous solution. Because of the motional-narrowing
effect of nitroxide EPR signals, the nitroxide spectrum has three
E
dx.doi.org/10.1021/jp403183x | J. Phys. Chem. A XXXX, XXX, XXX−XXX

The Journal of Physical Chemistry A
Article
Finally, we estimated the number of nitroxide molecules
grafted on the ND surface by comparing X-band EPR spectra of
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5
and 6. As shown in Figure 5d, the intensity of 6 (100 nm
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NDs) is 13% of the 500 μM free nitroxide radicals (5);
therefore, the concentration of nitroxide radicals in 6 is ∼67
μM. Given that the sample volume is ∼5 μL, the number of
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1
4
nitroxide molecules in the sample is ∼2 × 10 . The sample also
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3
contains 1 mg of 100 nm NDs. Using 3.1 g/cm for the density
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4
3
−16
3
of 100 nm NDs and ∼5 × 10 cm for the volume of one
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00 nm diameter spherical ND, the number on ND particles in
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1
1
the sample holder is ∼6 × 10 . Thus, each 100 nm ND has
∼
300 nitroxide radicals on the surface. Similarly, with 3.52 g/
3
44
−18
3
cm for the density of 25 nm NDs and ∼8 × 10 cm for
the volume of one 100 nm diameter spherical ND, we found
that each 25 nm ND has ∼20 nitroxides on the surface.
̈
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CONCLUSIONS
■
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We demonstrated grafting of nitroxide radicals on the ND
surface using click chemistry. The reaction of the grafting was
verified by FTIR spectroscopy. We also employed X-band and
2
30 GHz EPR spectroscopy to verify the covalent attachment
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of nitroxide molecules on a ND surface are more constrained
than those of free nitroxide radicals. The observation strongly
suggests successful grafting of nitroxides on NDs. We also
estimated that the number of nitroxide radicals attached on a
surface of NDs using the EPR method is ∼300 for 100 nm and
(
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AUTHOR INFORMATION
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K.; Wei, P.-K.; Tsao, P.-H.; Chang, H.-C.; Fann, W. Characterization
and Application of Single Fluorescent Nanodiamonds as Cellular
Biomarkers. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 727−732.
■
Corresponding Author
E-mail susumu.takahashi@usc.edu.
*
Notes
(17) Neugart, F.; Zappe, A.; Jelezko, F.; Tietz, C.; Boudou, J. P.;
The authors declare no competing financial interest.
Krueger, A.; Wrachtrup, J. Dynamics of Diamond Nanoparticles in
Solution and Cells. Nano Lett. 2007, 7, 3588−3591.
(18) Huang, H.; Pierstorff, E.; Osawa, E.; Ho, D. Protein-Mediated
ACKNOWLEDGMENTS
■
Assembly of Nanodiamond Hydrogels into a Biocompatible and
We thank X. Zhang for support to perform the X-band EPR
measurements. This work was supported by the Searle scholars
program (S.T.).
Biofunctional Multilayer Nanofilm. ACS Nano 2008, 2, 203−212.
(19) Mochalin, V. N.; Shenderova, O.; Ho, D.; Gogotsi, Y. The
Properties and Applications of Nanodiamonds. Nat. Nanotechnol.
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