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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 dextrancoated 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 phenomenon 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 hybridizing 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 molecules which can bind a variety of chemical and biological * Fax: (+1) 217-333-2685. Tel: (+1) 217-333-2619. E-mail yi-lu@ uiuc.edu. † Center for Biophysics and Computational Biology. ‡ Beckman Institute for Advanced Science and Technology. § Department of Chemistry. 4 Department of Biochemistry.
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
10.1021/bc7003928 CCC: $40.75 2008 American Chemical Society Published on Web 01/04/2008
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Scheme 1. Schematic Illustration for Thrombin Detection Using MRIa
a The 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.
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 heparinbinding exosite of thrombin, as shown in Scheme 1 (62, 63). 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-Succinimidyl-3-(2-pyridylthio)-propionate (SPDP) was purchased from Molecular Biosciences (Boulder, CO). Cross-linked dextran coated superparamagnetic 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-TTTTTAGTCCGTGGTAGGGCAGGTTGGGGTGACT 3′), CNT-Thrm-A (5′ TCACAGATGAGT-A12-SH 3′), and CNT-Thrm-B (5′ SH-CCCAGGTTCTCT 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 CLIOSPDP (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.
Figure 1. Particle size distribution of 1:1 CLIO-Thrm-A and CLIOThrm-B mixture before (light gray bars) and after (dark gray bars) addition of 50 nM thrombin.
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-
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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 CLIOThrm-B mixture in human serum.
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 nanoparticles 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
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noticeable change in contrast was observed even as low as 10 nM thrombin, and a significant change was observed at 50 nM thrombin. 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-CLIOThrm-B). The control samples were subjected to the same procedure as was used in preparing CLIO-Thrm-A and CLIOThrm-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 switching. 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 superparamagnetic 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.
ACKNOWLEDGMENT 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
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Imaging Center of the Beckman Institute for Advanced Science and Technology and University of Illinois at Urbana–Champaign.
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