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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]). 816 nature biotechnology • VOLUME 20 • AUGUST 2002 • http://biotech.nature.com © 2002 Nature Publishing Group http://biotech.nature.com 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 http://biotech.nature.com • AUGUST 2002 • VOLUME 20 • nature biotechnology 817 RESEARCH ARTICLE © 2002 Nature Publishing Group http://biotech.nature.com 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). 818 nature biotechnology • VOLUME 20 • AUGUST 2002 • http://biotech.nature.com RESEARCH ARTICLE 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 http://biotech.nature.com • AUGUST 2002 0 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• VOLUME 20 • nature biotechnology 819 RESEARCH ARTICLE 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. © 2002 Nature Publishing Group http://biotech.nature.com 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. 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