MRI detection of thrombin with aptamer functionalized superparamagnetic iron oxide nanoparticles.
ABSTRACT Design of smart MRI contrast agent based on superparamagnetic iron oxide nanoparticles and aptamers has been described for the detection of human alpha-thrombin protein. The contrast agent is based on the assembly of the aptamer functionalized nanoparticles in the presence of thrombin. A detectable change in MRI signal is observed with 25 nM thrombin in human serum. Changes were neither observed with control analytes, streptavidin, or bovine serum albumin, nor with inactive aptamer functionalized nanoparticles.
[show abstract] [hide abstract]
ABSTRACT: Magnetic resonance imaging (MRI) was performed in 20 patients before and after intravenous administration of gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA) in a dose of 0.1 mmol/kg. Twelve of the patients had clinical and histologic diagnoses of cerebral tumor, six had hepatic tumors, one had hepatic cysts, and one had transitional cell carcinoma of the bladder. Contrast enhancement was seen with all tumors, but not with the hepatic cysts. The degree of enhancement was greater than that seen with computed tomography (CT) in 13 cases, equal to it in six, and less in one. Contrast enhancement was detectable as long as 18 hr after injection of Gd-DTPA in one case of cerebral tumor. The margin between cerebral tumor and peritumoral edema could be delineated with contrast-enhanced MRI to the degree possible with contrast-enhanced CT. In the liver isointense enhancement was seen with saturation-recovery (SR), inversion-recovery (IR), and spin-echo (SE) sequences although not with all three sequences simultaneously. In general IR sequences were most sensitive for display of the contrast agent, but the enhancement often decreased the difference between abnormal and normal tissue. No short-term side effects were encountered and no significant change was seen in urea, creatinine, electrolytes, liver function tests, blood coagulation, or urine testing after injection of Gd-DTPA. Although much more work will be required to evaluate this contrast agent, these initial experiences are very promising.American Journal of Roentgenology 09/1984; 143(2):215-24. · 2.78 Impact Factor
[show abstract] [hide abstract]
ABSTRACT: A contrast agent for use in magnetic resonance (MR) imaging of the liver and spleen has been designed in which gadolinium-DTPA is chemically incorporated into the lamellar phase of liposome particles. This agent has excellent in vivo stability and is taken up by liver and spleen of normal mice after intravenous administration. The T1 increased by 110% in liver and 66% in spleen at 4 degrees C. At 37 degrees, the relaxation rate in the liver increased by 180%. This method is an attractive concept for the development of various organ-specific liposomal contrast agents that may be used for either MR imaging or nuclear medicine.Radiology 05/1987; 163(1):255-8. · 5.73 Impact Factor
American Journal of Roentgenology. 01/1987; 149(4):723-726.
MRI Detection of Thrombin with Aptamer Functionalized
Superparamagnetic Iron Oxide Nanoparticles
Mehmet Veysel Yigit,†,‡Debapriya Mazumdar,‡,§and Yi Lu*,†,‡,§,4
Center for Biophysics and Computational Biology, Beckman Institute for Advanced Science and Technology, Department of
Chemistry and Department of Biochemistry, University of Illinois at Urbana–Champaign, 600 S. Mathews Avenue, Urbana, IL
61801. Received October 22, 2007; Revised Manuscript Received November 27, 2007
Design of smart MRI contrast agent based on superparamagnetic iron oxide nanoparticles and aptamers has been
described for the detection of human R-thrombin protein. The contrast agent is based on the assembly of the
aptamer functionalized nanoparticles in the presence of thrombin. A detectable change in MRI signal is observed
with 25 nM thrombin in human serum. Changes were neither observed with control analytes, streptavidin, or
bovine serum albumin, nor with inactive aptamer functionalized nanoparticles.
Magnetic resonance imaging (MRI) is advancing rapidly, as
it provides noninvasive, three-dimensional examination of
biological events in living organisms. A particularly active area
of research in the MRI field is the development of MRI contrast
agents for image enhancement (1–9). Superparamagnetic iron
oxide nanoparticles (SPIOs) are attractive, since they are shown
to be effective in enhancing magnetic resonance image contrast
(4). The applications of SPIOs in MRI have ranged from
nontargeted detection of diseases by accumulating at certain
tissues to targeted detection of biomolecular markers in
cells (10–16). Target-specific MRI detection using SPIOs is
particularly interesting, as it helps monitor several cellular or
molecular processes (16–18). For example, cross-linked dextran-
coated superparamagnetic iron oxide (CLIO) nanoparticles have
been functionalized with different biomolecules and used for
detection of different targets including oligonucleotides (19, 20)
proteins (17, 20, 21), enzymatic activities (22), viruses (23),
and enantiomeric impurities (24). It has been shown that CLIO
nanoparticle assemblies create a distinctive magnetic phenom-
enon called magnetic relaxation switching (MRS), where the
core of a single nanoparticle in the assemblies becomes more
effective in enhancing T2 relaxation time of adjacent water
protons, when compared to dispersed nanoparticles (4, 19). This
mechanism has been widely used in many magnetic detection
schemes either going from a disperse state to an assembled state
of nanoparticles or visa versa (19, 20, 22, 24). For instance, it
has been shown that oligonucleotide functionalized dispersed
CLIO nanoparticles can be used for the sequence-specific
detection of complementary oligonucleotides simply by hybrid-
izing oligonucleotides and assembling CLIO nanoparticles into
clusters (19). This process enhances the T2 relaxation of the
nearby water protons and can be detected by MRI. While this
approach is effective in oligonucleotide detection, it would be
very interesting if this nucleic acid-based approach could be
expanded beyond nucleic acid detection to MRI of even broader
classes of targets.
Aptamers are single-stranded functional nucleic acid mol-
ecules which can bind a variety of chemical and biological
molecules with high affinity and selectivity (25–28). They are
obtained through a combinatorial biology technique called
systematic evolution of ligands by exponential enrichment
(SELEX), by isolating the active species from a large random
pool of DNA or RNA molecules (25, 26). They are often
analogous to antibodies due to their selectivity and sensitivity
in binding to a broad range of molecules (29–32). When
compared to antibodies, aptamers serve several advantages such
as the relative ease with which they can be selected for any
target analyte and their stability against biodegradation and
denaturation. Due to these properties, aptamers are good
candidates for building chemical and biological sensors in many
fields such as medical diagnostics and environmental monitoring.
Therefore, these aptamers have been transformed into fluorescent
(33–47), colorimetric (48–57), and electrochemical sensors
(58–60). Although these aptamer sensors have been widely
investigated in Vitro, their applications in ViVo remain a
significant challenge because light penetration through skin is
difficult and signal interference from cellular components is
common. Recently, we reported a method for combining
adenosine aptamer and CLIO nanoparticles into a system to
detect adenosine in the micromolar range via MRI. The contrast
in MR image of the nanoparticle solution increases as the
adenosine concentration increases in the environment (61).
Herein, we describe a new method for combining magnetic
relaxation switching properties of CLIO nanoparticles with
aptamer technology in order to create MRI contrast agents with
nanomolar detection limit. The advantage of this technique over
other sensing methods is that MRI signal is much less vulnerable
to changes in background colors or fluorescence from biological
media, such as serum and cell suspensions. In contrast to our
previously reported system with adenosine, which depends on
analyte-induced disassembly of particles to produce an increase
in brightness, this method is based on assembly of particles
leading to a decrease in brightness of MR image. This change
in signal from bright to dark is a significant advantage, as this
is preferred in T2-weighted MR imaging. Furthermore, instead
of a metabolite, we demonstrate the detection of a protein in
the current system, as proteins constitute most enzymes and
biomolecular markers in living systems.
To demonstrate the use of aptamer functionalized CLIO
nanoparticles for protein detection we chose to detect thrombin
* Fax: (+1) 217-333-2685. Tel: (+1) 217-333-2619. E-mail yi-lu@
†Center for Biophysics and Computational Biology.
‡Beckman Institute for Advanced Science and Technology.
§Department of Chemistry.
4Department of Biochemistry.
Bioconjugate Chem. 2008, 19, 412–417
10.1021/bc7003928 CCC: $40.75
2008 American Chemical Society
Published on Web 01/04/2008
via MRI . We combined the CLIO nanoparticles with thrombin
aptamers, Thrm-A, which binds to the fibrinogen-recognition
exosite of thrombin, and Thrm-B, which binds to the heparin-
binding exosite of thrombin, as shown in Scheme 1 (62, 63).
The contrast agent designed for thrombin detection is composed
of a 1:1 mixture of Thrm-A and Thrm-B functionalized CLIO
nanoparticles (CLIO-Thrm-A and CLIO-Thrm-B, respectively)
in aqueous solution. In the presence of thrombin, aptamer
sequences fold into a G-quadruplex arrangement in order to bind
to thrombin (64, 65). After attachment of the CLIO nanoparticles
to thrombin, the disperse nanoparticles assemble into aggregates,
changing the magnetic relaxation properties of nearby water
protons, thereby reducing the T2 relaxation time. This event
can be monitored as a decrease in brightness of T2-weighted
MR image of the solution via MRI (24).
To confirm that the aptamer functionalized nanoparticles bind
to thrombin, 1 µM thrombin was added into the 1:1 homoge-
Materials: All DNA samples were purchased from Integrated DNA
Technologies Inc. (Coralville, IA). The thiol-modified DNA molecules
were purified by the standard desalting method. Human alpha thrombin
was purchased from Haematologic Technologies Inc. (Essex Junction,
VT). BSA was purchased from Aldrich (St. Louis, MO). Streptavidin
was purchased from SouthernBiotech (Birmingham, AL). N-Succin-
imidyl-3-(2-pyridylthio)-propionate (SPDP) was purchased from Mo-
lecular Biosciences (Boulder, CO). Cross-linked dextran coated super-
paramagnetic iron oxide nanoparticles (CLIO, 500 µg Fe mL-1) were
synthesized and coupled to SPDP according to literature procedure and
purified with PD-10 column (17). The thiol modified oligos, Thrm-A
(5′ SH-T15-GGTTGGTGTGGTTGG 3′), Thrm-B (5′ SH-TTTTTAGTC-
CGTGGTAGGGCAGGTTGGGGTGACT 3′), CNT-Thrm-A (5′ TCA-
CAGATGAGT-A12-SH 3′), and CNT-Thrm-B (5′ SH-CCCAGGT-
TCTCT 3′) were activated by incubating with eight equivalent of tris
(2-carboxyethyl) phosphine hydrochloride (TCEP). Excess TCEP was
removed by desalting using a SepPak C-18 catridge. TCEP-activated
thiol modified DNA (50 µM final concentration) was mixed with CLIO-
SPDP (400 µg Fe mL-1) in 100 mM phosphate buffer at pH 8.0
overnight. Excess DNA was removed by magnetic separation column
(Miltenyi Biotec, Auburn, CA) from CLIO-DNA conjugates. Sample
preparation and MRI detection: CLIO-Thrm-A and CLIO-Thrm-B were
mixed in 1:1 ratio and diluted in 100 mM NaCl, 25 mM KCl, and 25
mM tris-HCl buffer at pH 7.4. 250 µL of sample (12 µg Fe mL-1)
was aliquoted into the wells of a microplate and varying amounts of
analyte was added in each well. T2-weighted MR images were obtained
on a 4.7 T NMR instrument using a spin–echo pulse sequence with
variable echo time (TE ) 25–100 ms) and repetition time (TR) of 3000
ms. Light-scattering experiments: DLS measurements were performed
using Nicomp 380 ZLS Particle Sizer (Particle Sizing Systems, Santa
Barbara, CA). An intensity-weighted value was used to report the
average particle diameter.
Scheme 1. Schematic Illustration for Thrombin Detection Using MRIa
aThe CLIO nanoparticles (shown as red spheres) have been modified with either Thrm-A, a DNA aptamer (shown as blue lines) that binds to
fibrinogen-recognition exosite of thrombin, or Thrm-B, a DNA aptamer (shown as green lines) that binds to the heparin-binding exosite of thrombin.
Addition of thrombin consisting of both fibrinogen (as blue donuts) and heparin (as green donuts) exosites resulted in aggregation of CLIO nanoparticle
assembly, reducing the T2 relaxation time. The DNA sequences are shown at the bottom. The drawing is not to scale.
Figure 1. Particle size distribution of 1:1 CLIO-Thrm-A and CLIO-
Thrm-B mixture before (light gray bars) and after (dark gray bars)
addition of 50 nM thrombin.
CommunicationsBioconjugate Chem., Vol. 19, No. 2, 2008 413
neous mixture of CLIO-Thrm-A and CLIO-Thrm-B (150 µg
Fe mL-1), which resulted in rapid precipitation in seconds (data
not shown). Similar behavior was not observed when bovine
serum albumin (BSA) or streptavidin was used as an analyte.
This result indicates that the precipitation of nanoparticles is
due to the binding event of analyte and its aptamer. Particle
size analysis also showed that, upon addition of 50 nM thrombin
into a mixture of CLIO-Thrm-A and CLIO-Thrm-B (12 µg Fe
mL-1), the average diameter of CLIO nanoparticles immediately
increased from 58.9 ( 4.4 nm to 259.5 ( 22.5 nm. Figure 1
shows the intensity-weighted particle size distribution of CLIO
nanoparticles obtained with dynamic light scattering (DLS),
which indicates that the nanoparticles are cross-linked by
thrombin molecules, therefore increasing the average diameter.
At this CLIO nanoparticle concentration, precipitation of
nanoparticles was not observed (19). These results strongly
suggest that thrombin binding to aptamers on CLIO nanopar-
ticles induces the assembly of nanoparticles.
After confirming thrombin-induced assembly of nanoparticles
via DLS, we proceeded to check its utility as an MRI contrast
agent. The binding of CLIO-Thrm-A and CLIO-Thrm-B to
thrombin, assembled the nanoparticles into clusters, resulting
in a decrease of the T2 relaxation time of the neighboring water
protons in the medium. We have tested the system at different
thrombin concentrations from 0 to 50 nM. A decrease in
brightness of the MR image of the samples was observed as
the concentration of thrombin was increased (Figure 2A), which
was attributed to a decrease in T2 relaxation time (24). A
Figure 2. (A) Contrast change in T2-weighted MR image in 1:1 CLIO-Thrm-A and CLIO-Thrm-B mixture with 0, 10, 25, and 50 nM thrombin
(first column), BSA (second column), and streptavidin (third column). (B) Contrast change in T2-weighted MR image with 0, 10, 25, and 50 nM
thrombin in CLIO-Thrm-A and CLIO-Thrm-B mixture (first column), and in CNT-CLIO-Thrm-A and CNT-CLIO-Thrm-B mixture (second column).
Figure 3. (A) Contrast change in T2-weighted MR image with 0, 10, 25, and 50 nM thrombin in CLIO-Thrm-A (first column), CLIO-Thrm-A and
CLIO-Thrm-B mixture. (Note: The image is completely dark at 50 nM thrombin) (second column) and in CLIO-Thrm-B (third column). (B)
Particle diameter change with CLIO-Thrm-A, 1:1 CLIO-Thrm-A and CLIO-Thrm-B mixture, or CLIO-Thrm-B with addition of thrombin.
Figure 4. T2-weighted MR image of 1:1 CLIO-Thrm-A and CLIO-
Thrm-B mixture in human serum.
414 Bioconjugate Chem., Vol. 19, No. 2, 2008Communications
noticeable change in contrast was observed even as low as 10
nM thrombin, and a significant change was observed at 50 nM
To ensure that the contrast is solely due to the binding event
and not any other artifact, the system was tested with BSA and
streptavadin. The MR images obtained with these two analytes
did not show a difference in contrast when their concentration
was increased from 0 to 50 nM. This result suggests that the
change in contrast is due to thrombin and not any other effect.
In order to check if the change in contrast is due to aptamer
and analyte binding and not thrombin molecule itself, we have
tested random DNA sequences of different lengths that do not
bind to thrombin. To do so, we prepared a 1:1 mixture of random
DNA sequence (CNT-Thrm-A and CNT-Thrm-B) functionalized
CLIO nanoparticles (CNT-CLIO-Thrm-A and CNT-CLIO-
Thrm-B). The control samples were subjected to the same
procedure as was used in preparing CLIO-Thrm-A and CLIO-
Thrm-B, and then placed into the wells of a microplate.
Thrombin was added to both systems with an increasing
concentration from 0 to 50 nM. The obtained MR images
showed a change in brightness for samples with CLIO-Thrm-A
and CLIO-Thrm-B, but no change with CNT-CLIO-Thrm-A and
CNT-CLIO-Thrm-B (see Figure 2B). This result suggests that
the change in the MR signal is due to active thrombin binding
aptamers and not any other nonspecific interaction of DNA with
thrombin. The two control experiments taken together strongly
indicate that the change in MR signal is solely due to the binding
event of thrombin to the aptamers, which results in assembly
of CLIO nanoparticles into clusters, decreasing the T2 relaxation
time of the environment.
In order to demonstrate that thrombin molecule requires a
mixture of CLIO-Thrm-A and CLIO-Thrm-B to generate a MR
signal, we tried to use only CLIO-Thrm-A or CLIO-Thrm-B to
detect thrombin. As seen in Figure 3A, the MR signal did not
change with neither of these nanoparticle suspensions, but a
clear change in MR signal was observed with the 1:1 mixture
of nanoparticles. The particle size analysis confirms this result,
as an increase in particle diameter was observed when thrombin
was added into the 1:1 mixture of nanoparticles, but such an
increase was not observed with CLIO-Thrm-A or CLIO-Thrm-B
alone (see Figure 3B). MR data and particle size analysis
together suggest that both CLIO-Thrm-A and CLIO-Thrm-B
are necessary for detection of thrombin with magnetic relaxation
To check the utility of this system in biological fluids, we
tested our sample in 50% human serum. A clear change in the
MR signal was observed with 25 nM, and a significant change
was seen with 75 nM thrombin (Figure 4). This result
demonstrates that the system works in human serum without
interference of biological components in serum.
In conclusion, we demonstrated aptamer functionalized su-
perparamagnetic iron oxide nanoparticles for detection of an
analyte, which is dependent upon the binding event of aptamer
conjugated CLIO nanoparticles and the target molecule. The
system demonstrated here is specific to thrombin and the
sensitivity is as low as 10 nM. Similar approaches can be applied
to other aptamer and CLIO nanoparticle systems.
The authors thank Natasha Yeung for helpful discussions and
for comments on the manuscript and Dr. Boris Odintsov for
his help in operating the MRI equipment. This material is based
upon work supported by the National Science Foundation
(DMR-0117792, DMI-0328162, and CTS-0120978), the U.S.
Army Research Office (DAAD19-03-1-0227), and Biomedical
Imaging Center of the Beckman Institute for Advanced Science
and Technology and University of Illinois at Urbana–Cham-
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