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RESEARCH ARTICLE
© 2002 Nature Publishing Group http://biotech.nature.com
Magnetic relaxation switches capable of sensing
molecular interactions
J. Manuel Perez, Lee Josephson, Terrence O’Loughlin, Dagmar Högemann, and Ralph Weissleder*
Published online: 22 July 2002, doi:10.1038/nbt720
Highly sensitive, efficient, and high-throughput biosensors are required for genomic and proteomic data acquisition in complex biological samples and potentially for in vivo applications. To facilitate these studies, we have
developed biocompatible magnetic nanosensors that act as magnetic relaxation switches (MRS) to detect
molecular interactions in the reversible self-assembly of disperse magnetic particles into stable nanoassemblies. Using four different types of molecular interactions (DNA–DNA, protein–protein, protein–small molecule,
and enzyme reactions) as model systems, we show that the MRS technology can be used to detect these
interactions with high efficiency and sensitivity using magnetic relaxation measurements including magnetic
resonance imaging (MRI). Furthermore, the magnetic changes are detectable in turbid media and in wholecell lysates without protein purification. The developed magnetic nanosensors can be used in a variety of biological applications such as in homogenous assays, as reagents in miniaturized microfluidic systems, as affinity ligands for rapid and high-throughput magnetic readouts of arrays, as probes for magnetic force
microscopy, and potentially for in vivo imaging.
Nanometer-sized colloidal particles of metals and semiconductors
have many useful optical, electronic, and magnetic properties as a
result of their small size and composition1. When coupled to affinity
ligands, these materials have been used as chemical sensors. For
example, gold nanoparticles can be synthesized to carry oligonucleotides capable of sensing complementary strands detectable by
color changes2–4. Other nanoparticles include fluorescent quantum
dots5–7, magnetoresistive particles8, and magnetic susceptibility
enhancers9. The above methods have been effective in a variety of
applications10–12, but they have certain limitations. Most methods
require purification or isolation of DNA or RNA before readout or
additional amplification steps. As sample sizes become exceedingly
small in high-throughput testing, these requirements place additional
burdens on the described techniques.
Highly uniform magnetic nanoparticles can be prepared with
defined surface characteristics that allow covalent and stoichiometric attachment of oligonucleotides, nucleic acids, small molecules,
peptides, receptor ligands, proteins, and antibodies13–16. We have
previously observed a unique magnetic phenomenon during the
self-assembly of these nanomagnetic probes into larger nanoassemblies16. During this cooperative process, the superparamagnetic iron
oxide core of individual nanoparticles becomes more efficient at
dephasing the spins of surrounding water protons, enhancing
spin–spin relaxation times (T2 relaxation times) so that the
nanoparticles act as magnetic relaxation switches (MRS). We have
now built on this observation and developed generic homogenous
assays to sense a variety of different molecular interactions with high
sensitivity and selectivity in biological samples with minimal or no
sample preparation. Because the sensing step is not dependent on the
separation of bound and unbound reagents or on the use of light,
experiments can be carried out in turbid samples and suspensions.
These features make MRS particularly suitable for miniaturization,
microfluidic applications, and electronic (magnetoresistive) readouts. Here we show that the technology can be used to sense different
types of reversible molecular interactions (Fig. 1A): DNA–DNA,
protein–protein, protein–small molecule, and enzyme reactions.
Results and discussion
Sensing DNA interactions. The MRS were synthesized as nanoparticles containing a 3 nm superparamagnetic colloidal iron oxide
core caged by an epichlorohydrin-crosslinked and aminated dextran coating, resulting in particle sizes of 45 ± 4 nm15.
Nanoparticles were aminated to allow convenient attachment of
affinity ligands for subsequent assay testing. To sense complementary oligonucleotides, we coupled an average of three 12-bp thiolated oligonucleotides to the nanoparticles using N-succinimidyl
3-(2-pyridyldithio)propionate (SPDP) as a linker. The oligonucleotide sequences attached to the particles were chosen to recognize different DNA sequences. For each target sequence we made
two unique nanoparticle populations (termed P1 and P2) that recognized adjacent sequences covering a 24-bp region (Fig. 1A). P1
and P2 recognized a nonsense DNA sequence and GFP-P1 and
GFP-P2 were chosen to recognize green fluorescent protein (GFP)
mRNA. The nanoparticles were stable in solution without precipitation for months and appeared monodisperse when viewed by
atomic force microscopy (Fig. 1B). However, upon hybridization
with a target sequence, the particles self-assembled to form clusters
of 140 ± 16 nm, each containing four to five particles (Fig. 1C).
This effect was not observed when the nanoparticles were incubated with noncomplementary oligonucleotides.
Center for Molecular Imaging Research, Massachusetts General Hospital and Harvard Medical School, Boston, MA, 02129.
*Corresponding author ([email protected]).
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RESEARCH ARTICLE
Self-assembly of these nanoparticles resulted in significant
(P < 0.001) changes in the spin–spin relaxation times (T2) of
neighboring water molecules as determined by relaxation measurements using a nuclear magnetic resonance (NMR) benchtop
relaxometer. Within several minutes after addition of oligonucleotide to a P1–P2 mixture (10 µg Fe/ml), the T2 relaxation times
decreased by over 20 ms (Fig. 2A). When dithiothreitol (DTT) was
added, T2 relaxation times returned to baseline values, as the
hybridized oligonucleotides were cleaved from the MRS. Likewise,
T2 values returned to baseline when the samples were heated to
70–80°C, and values decreased again upon cooling and annealing.
Interestingly, no visible precipitate formed at this concentration
over a 24 h observation period, indicating that the clusters are stable in solution (Fig. 1C). We observed similar decreases in T2
relaxation times when the experiments were carried out in turbid
solution (Fig. 2C, D).
An alternative to measuring T2 relaxation times with NMR
benchtop relaxometers (500 µl sample volume) is to determine these
values by MRI of 384-well plates (50 µl sample volume), allowing
parallel measurements at higher throughput. In the following experiments, we incubated a P1–P2 mixture (3 µg Fe/ml) with increasing
amounts of complementary or noncomplementary oligonucleotides
and carried out MRI at 1.5 T. Substantial differences between the
samples were readily apparent with detection limits of oligonucleotides in the subfemtomole range (Fig. 2E, F). T2 concentration
decreased linearly with the amount of DNA added in the concentration range shown (Fig. 2F). The sensitivity and throughput of the
assay may be increased further using 1,536-well plates and even
lower sample volume (<50 µl). Additional experiments using 1,536well plates indicated that up to 10,000 data points can be generated
within several minutes.
Selectivity. The magnetic nanoparticles are highly stable when
subjected to temperature fluctuations and different ionic media16.
This stability enabled us to select buffer conditions (25 mM KCl, 50
mM Tris, pH 7.5) under which small differences in base pairing
could be detected by T2 measurements (Table 1). Single-nucleotide
insertion in the center of the target sequence almost completely
abrogated magnetic switching. Similar effects were also seen with
larger and other kinds of single and double inserts (Table 1). To
determine the effect that nucleotide mismatches could have on T2
measurements, we tested additional target sequences containing
both single and multiple mismatches. Again, single-nucleotide mis-
A
Affinity ligand
Enzyme
High T2
Low T2
B
200 nm
C
200 nm
Figure 1. Magnetic relaxation switches. (A) Schematic diagram of the
MRS assay. The superparamagnetic nanoparticles self-assemble in the
presence of a binding target, decreasing the T2 relaxation time of
adjacent water protons (from left to right). Conversely, self-assembled
MRS can be prepared such that they form substrates for specific
enzymes. The readout (from right to left) is then accompanied by an
increase in T2 relaxation time (see Fig. 5B). For DNA–DNA interactions
(see Figs 2–4), an average of three thiolated oligonucleotides (P1 and P2)
oligomerize in the presence of a complementary target. (B, C) Atomic
force microscopy image of nanosensors (10 µg Fe/ml) without (B) and
with (C) a complementary oligonucleotide. (C) Small clusters of four to
five individual magnetic nanoparticles are formed.
matches were detectable whereas double mismatches completely
abrogated magnetic switching (Table 1).
We studied the selectivity of the MRS further by preparing probes
targeting a GFP gene sequence and three variants with a single mismatch (T, C, and G instead of A) and taking T2 measurements with
these four sequences (Fig. 3A). The perfect
match (containing A) caused a decrease in T2
Table 1. Summary of tested oligonucleotide sequences and magnetic measurements
within minutes of oligonucleotide addition.
The single mismatches behaved differently
a
δT2 (ms)
Deviation from
P value
and these differences were readily detected by
normalb
T2 measurements at 40°C. As in the previous
Perfect match
experiments, we also carried out MRI at
TAC-GAG-TTG-AGA-ATC-CTG-AAT-GCG
30 ± 2
NA (match)
NA
room temperature and observed similar differences (Fig. 3B). These data show that
Insertions
selective measurements capable of distinTAC-GAG-TTG-AGA-G-ATC-CTG-AAT-GCG
5±1
83%
0.0001
guishing single-nucleotide mismatches can
TAC-GAG-TTG-AGA-GAG-TGC-ATC-CTG-AAT-GCG
2 ± 0.6
93%
0.0001
be carried out reliably and at various temperTAC-GAG-G-TTG-AGA-ATC-CTG-AAT-GCG
4 ± 2.5
87%
0.0002
TAC-GAG-G-TTG-AGA-ATC-CTG-G-AAT-GCG
2 ± 0.5
93%
0.0001
atures using either NMR or MRI techniques
without the need for melting-curve analysis.
Mismatches
Imaging of cell lysates. To test whether
TAC-GAG-TTG-AGA-CTC-CTG-AAT-GCG
21 ± 1.2
30%
0.0029
MRS could be used to identify a target
GAC-GAG-TTG-AGA-ATC-CTG-AAT-GCG
21 ± 0.6
30%
0.0020
sequence in a higher-throughput format, we
TAC-GAG-TTG-AGA-ATC-CTG-CAT-GCG
15 ± 0.6
50%
0.0030
screened a panel of cell lines for GFP mRNA
TAC-GAG-TTG-AGA-CTC-CTC-AAT-GCG
1 ± 0.6
97%
0.0001
TAC-GAC-TTG-AGA-ATC-CTG-CAT-GCG
9 ± 1.7
70%
0.0002
expression using MRI as a detector. The panel
consisted of primary human and rodent
aδT2 = T2
(t = 0 min) – T2(t = 30 min).
bDeviation = ([δT2
tumor cell lines, one of which was transduced
(perfect match) – δT2(insertion or mismatch)] /δT2(perfect match)) × 100.
NA, not applicable.
with a GFP–encoding herpes simplex virus
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T2 (ms)
B
(HSV) amplicon (Gli-36) while another one was transiently A 70
G
A
T
C
transfected with a GFP–encoding plasmid DNA (COS-1). In
addition, the corresponding parental cell lines were transfected
60
with β-galactosidase as a negative control and included in the
Mismatch
50
panel. Total RNA from these cell lines was isolated and imaged
G
after sensing with GFP-P1 and GFP-P2 (Fig. 4A). The sample in
G: TAA-ACG-GCC-ACA-AGT-TCG-GCG-TGT
40
T
well C3 contained RNA from the Gli-36 cells line whereas well
A: TAA-ACG-GCC-ACA-AGT-TCA-GCG-TGT
C
D4 contained RNA from COS-1 cells. The observed magnetic
Match
T: TAA-ACG-GCC-ACA-AGT-TCT-GCG-TGT
30
changes correlated well with fluorescence measurements of the
A
C: TAA-ACG-GCC-ACA-AGT-TCC-GCG-TGT
cell lines and reverse transcription–PCR (data not shown).
20
Both parental and β-galactosidase expressing Gli-36 and COS30
0
20
10
1 cell lines did not show a substantial difference as compared to
Time (min)
wild-type cell lines.
Although the above experiments were carried out with iso- Figure 3. Specificity of magnetic nanosensors. Temporal change of T2 relaxation
lated RNA, we also examined whether similar measurements times of P1-GFP and P2-GFP (10 µg Fe/ml) with the addition of various target
could be taken in cell lysates. For these experiments we added a oligonucleotides containing single-nucleotide mismatches G, T, and C (53 fmol) in
25 mM KCl, 50 mM Tris-HCl, pH 7.4. The perfect target sequence (containing A) is
mild lysis buffer (20mM Tris, pH 8, 5 mM MgCl2, 0.5% clearly distinguished from single-nucleotide mismatches by benchtop
(vol/vol) NP-40, 200 µg/ml tRNA) to adherent cells before measurements (A) and T2 weighted MRI (B) of samples.
probing with P1 and P2. This buffer has been used previously
to extract RNA from cells without requiring cells to be scraped
off the dish17. Differences in GFP mRNA expression between the
a specific GFP mRNA in a pool of total RNA (1 µg) and in whole-cell
parental and GFP-expressing Gli-36 cell lines were clearly identified
lysate. This level of detection with the currently unoptimized MRS
(Fig. 4B). In additional studies, we quantified this difference using
technology is comparable to traditional fluorescence-based methods
Gli-36 cell lines infected with a different multiplicity of infection of a
for oligonucleotide hybridization carried out with purified total
GFP-bearing amplicon vector. Figure 4C shows the correlation
RNA18. We expect that further optimization (of particle composition
and/or readout methods) will enable the detection of significantly
between cell fluorescence measurements and mRNA measurements
lower amounts of analytes in both cell lysates and in turbid media.
using MRS in cell lysates. In these studies, we have been able to detect
Sensing protein–protein interactions. To extend the observations
described above, we tested whether antibody-mediated interactions
A
B
could be sensed with the MRS. We first prepared avidin–P1 conjugates as a generic reagent that could be used to attach any biotinylated antibody or peptide on the nanosensor. On average, each
nanoparticle contained two avidins (eight binding sites).
Biotinylated anti-GFP polyclonal antibody was then attached to
yield anti-GFP-P1. When the nanosensors were used to probe for
GFP protein, we observed significant T2 relaxation time changes (P
< 0.001; Fig. 5A). These changes were time and dose dependent (Fig.
5A). Incubation with control protein (bovine serum albumin; BSA)
resulted in no significant T2 changes. We made similar observations
D
C
using several alternative model systems: (i) probing phage with a
polyclonal anti-fd bacteriophage–P1 conjugate, (ii) probing CA125
protein with an anti-CA125-P1, and (iii) testing biotin as a model
small-molecular-weight agent against avidin–P1. In all of these
experiments, we observed clustering with concomitant T2 changes
with detection limits in the nanomolar range.
Sensing enzymatic activity. Next, we tested whether MRS could be
used to sense enzymatic activity. For this purpose we developed a
F
E
reverse assay (Fig. 5B) in which MRS nanoassemblies can be converted into its constituent nanoparticles by an enzymatic reaction, leading to an increase in T2 relaxation times. A nanoparticle containing
biotinylated DEVD peptides (P2) was prepared and incubated with
avidin–P1 to form a caspase 3–sensitive MRS nanoassembly. The
DEVD peptide sequence is specifically recognized by caspase 3, thus
serving as an assay for this enzyme. The caspase 3–mediated reaction
was associated with a dose-dependent increase in the T2 relaxation
Figure 2. Sensitivity of magnetic nanosensors. (A, B) Temporal change of
water T2 relaxation time of P1 and P2 (10 µg Fe/ml) with target
time with kinetics similar to those reported with fluorogenic subcomplementary oligonucleotide (53 fmol) in (A) 0.1 M phosphate buffer,
strates (Fig. 5B). In additional experiments, we further confirmed
0.1 M NaCl, pH 7.4 and (B) 10% (vol/vol) Intralipid in PBS, 0.1 M NaCl,
the ability of similar MRS nanoassemblies to monitor the effect of
pH 7.4 using a 0.47 T benchtop NMR relaxometer. T2 relaxation times
restriction endonucleases and methylases on target DNA sequences
decrease with addition of hybridizing DNA in clear (C) and opaque (D)
solutions and return to baseline after addition of DTT, as the
(data not shown). Results from these studies further confirm that
oligonucleotides are cleaved from particles without change in color or
MRS nanoassemblies can act as biosensors for the recognition and
precipitation at this particular iron concentration. (E) T2-weighted MR
real-time monitoring of DNA-cleaving agents.
image of a portion of a 384-well plate containing P1 and P2 and various
MRS determines molecular interactions in a different way than
amounts (0–2.7 fmol) of either complementary or noncomplementary
do electronic biosensors, relying on cooperative switching between
oligonucleotide. (F) Calculated T2 values obtained by MRI(E).
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A
A
Protein sensing
B
Enzyme sensing
Caspase 3
GFP
High T2
High T2
Low T2
Low T2
60
80
BSA
B
0
Figure 4. Detection of mRNA. (A) T2 maps of section of a 384-well plate
containing GFP-P1 and GFP-P2 with total RNA (1 µg) extracted from
various cell lines. Wells C3 and D4 correspond to GFP+ human glioma
GLI-36 (fluorescence intensity (FI) = 33.4 AU) and COS-1 (FI = 17.7 AU)
cells; D3 and E4 correspond to β-galactosidase expressing Gli-36 and
COS-1; and B3 and C4 correspond to wild-type (WT) Gli-36 and COS-1.
All other wells correspond to various human and rodent gliomas,
carcinomas, and normal cell lines (9L and U57 gliosarcomas, ovarian
carcinomas, C6 rodent carcinomas, Jurkat, and HUVEC cells). (B) MR
image of nanosensors with lysed cells from wild-type or GFP+ human
glioma cell lysate (2 h after hybridization). GFP mRNA is readily
detectable by MRI. (C) GFP fluorescence and T2 relaxation time
measurements of GFP mRNA correlate well in whole-cell lysate
experiments.
dispersed and aggregated nanoparticle states to change the effect
nanoparticles exert on the dephasing of proton spins (T2 relaxation times. This mechanism endows MRS with a series of unique
properties: (i) because light (and such measurements as fluorescence, absorbance, turbidity, and chemiluminescence) is not used,
substances interfering with light do not affect assay values and
experiments can be carried out in turbid, light-impermeable, and
heterogeneous samples; (ii) the assay is homogeneous, and does
not use a wash to remove unbound analyte; (iii) the assay does not
require immobilization of the biomolecule (DNA or protein) onto
a glass microscope slide, resulting in faster hybridization kinetics;
and (iv) the assay has the flexibility to sense various bimolecular
interactions including protein–DNA interactions, protein–small
molecule interactions and protein–protein interactions with minimal sample preparation.
In this study, we have shown that MRS can be run in a highthroughput, array-based format using MRI. The design of NMR and
MR spectrometers for high throughput19–22 will undoubtedly expand
the potential of magnetic resonance–based sensor assays. In addition, instrumentation to detect nanoparticle magnetic moments
may use the magnetic nanoparticles described here with superconducting quantum interference device (SQUID)–based systems23,24,
magnetic force microscopy25, or handheld magnetic readers9.
Several features of the MRS could make them attractive as an in
vivo method for imaging molecular markers. The basic label
employed by MRS, a crystal of superparamagnetic iron oxide, has little or no toxicity26 and is in routine clinical use. Nanoparticle-based
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10 20 30
GFP (fmol)
GFP
30
C
40
30
40
Caspase 3
50
T2 (ms)
50
T2 (ms)
T2 (ms)
© 2002 Nature Publishing Group http://biotech.nature.com
60
20
10
30
Time (min)
60
40
Inhibitor
20
40
0
10
Time (min)
20
Figure 5. Detection of antibody-based and enzymatic reactions.
(A) Incubation of anti-GFP-P1 nanosensors with GFP or BSA protein
(control). T2 relaxation time decreases as the nanosensor binds to GFP
protein. Steady state is reached within 15 min. (B) Using a DEVD
peptide containing MRS nanoassembly, caspase 3 activity can be
detected by an increase in T2. This effect is not observed when a
specific caspase 3 inhibitor (N-acetyl-DEVD-CHO) is added. The
nanoassembly was formed by incubating avidin–P1 with biotin–DEVDSS-P2. This is a generic platform by which any biotinylated peptide or
protein cleavage site may be sensed.
sensors using cadmium7 or gold3,11, although superb biosensors
in vitro, have yet to demonstrate the requisite nontoxicity to permit
clinical use. Unlike optically based systems for detecting targets,
including enzymes activated near infrared fluorescent systems27,28,
MRS uses already established instrumentation that can sense
nanoparticle position through tissue regardless of its optical properties. Finally, conjugating membrane-translocating sequences like the
Tat peptide to magnetic nanoparticles allows them to be efficiently
internalized by cells13 and penetrate cellular barriers after injection29.
This allows the possibility of delivering MRS nanoparticles to intracellular molecular targets in cell suspension or in vivo with appropriately designed magnetic nanoparticles. The development of this
analytical technique should have broad biomedical applications
including the development of MR microarray–based biosensors and
molecular target detection using MRI.
Experimental protocol
Preparation of magnetic nanosensors. Thiolated oligonucleotides were coupled to aminated nanoparticles using SPDP as a linker16. The physical properties of the resulting conjugates, denoted P1 (CLIO-SS-(CH2)6-CGC-ATTCAG-GAT) and P2 (TCT-CAA-CTC-GTA-(CH2)3-SS-CLIO) (CLIO, crosslinked iron oxide) have been described16. In addition, two additional conjugates, GFP-P1 (CLIO-SS-(CH2)6-ACA-CGC-TGA-ACT) and GFP-P2 (TGTGGC-CGT-TTA-(CH2)3-SS-CLIO), were synthesized in an identical manner
to P1 and P2. These two conjugates were designed so that they would align to
a complementary sequence in the GFP gene. All probes had an average of
three oligonucleotides per particle16. The relaxivities of all four probes were
similar: R1 = 27.2 ± 0.3 s–1 mM–1 and R2 = 73 ± 6 s–1 mM–1.
Atomic force microscopy. An atomic force microscope (Dimension 3100,
Digital Instrument, Santa Barbara, CA) was used to collect images of P1, P2, and
hybridized probes. After 20 min incubation to allow the particles to bind to the
surface of mica, the surface was washed with buffer and images were acquired
using a silicon nitride cantilever (20–40 nm tip size) in tapping–lift mode.
Measurement of proton relaxation times. T2 relaxation time measurements
were carried out at 0.47 T and 40°C (Bruker NMR Minispec, Billerica, MA)
using solutions with a total iron content of 10 µg Fe/ml. T2 values were
obtained before and after addition of target oligonucleotides (53 fmol) con•
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taining various degrees of mismatches and insertions (Table 1). All experiments and measurements were carried out in triplicate and data were
expressed as mean ± s.d.
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Magnetic resonance imaging. MRI was carried out using a 1.5 T superconducting magnet (Signa 5.0; GE Medical Systems, Milwaukee, WI) using T2weighted spin echo sequences with variable echo times (TE = 25–1000 ms)
and repetition times (TR) of 3,000 ms to cover the spectrum of anticipated
T2 values. Image acquisition parameters and analysis were similar to those
described elsewhere30.
Hybridization of GFP-P1 and GFP-P2 to target in total RNA. Total RNA was
isolated from various cell lines including both parental and GFP-expressing
COS-1 and Gli36 human glioma cells using TRIzol reagent (Gibco BRL/Life
Technologies, Rockville, MD). COS-1 cells were transfected with a GFPexpressing plasmid (phGFP-S65T) using FuGene (Roche Molecular
Biochemical, Indianapolis, IN) while Gli36 human glioma cells were infected
with a GFP-expressing HSV amplicon vector, pHGCX31. GFP-P1 and GFP-P2
probes were added to the RNA preparation, incubated overnight at 37°C, and
imaged by MRI in 384-well plates.
Hybridization of GFP-P1 and GFP-P2 to target in cell lysate. Wild-type and
GFP-expressing cells (150,000 cells/dish) were treated with lysis buffer as
described17. Cell lysate was carefully removed, GFP-P1 and GFP-P2 probes
were added, and samples were incubated overnight at 37°C and imaged by
MRI in 384-well plates.
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Protein–protein interactions. Avidin was attached to CLIO nanoparticles and
preparations were purified by a magnetic separation column (Miltenyi Biotec,
Auburn, CA). Biotinylated polyclonal anti-GFP (Research Diagnostics,
Flanders, NJ) was then attached to the particles. To probe for protein–protein
interactions, GFP (33 fmol) was incubated with anti-GFP-CLIO (10 µg Fe/ml)
and T2 relaxation times were recorded.
Enzymatic cleavage assay. In this assay, a biotinylated peptide substrate for
caspase 3 was synthesized (biotin-GDEVDGC, caspase-3 recognition site
underlined) and coupled to aminated CLIO using SPDP (as stated in preparation of magnetic nanosensors). Equimolar amounts of avidin–CLIO and
biotinylated peptide–CLIO conjugate were incubated in PBS (10 µg Fe/ml)
and T2 was measured before and after addition of 25 ng caspase 3 (1.7 nM) in
the presence or absence of a caspase 3 inhibitor.
Acknowledgments
We thank Y. Saeki and N. Sergey for providing different GFP-expressing cell
lines and V. Ntziachristos for providing automation routines to display T2 maps
from MR images. This work was supported in part by P50 CA86355. J.M.P. is
the recipient of a National Cancer Institute–Comprehensive Minority
Biomedical Branch fellowship.
Competing interests statement
The authors declare that they have no competing financial interests.
Received 12 February 2002; accepted 20 May 2002
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AUGUST 2002
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