Bioconjugate Chem. 2006, 17, 1607−1611
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TECHNICAL NOTES Multiplex Analysis with Peptide-Encoded Beads Nuria Sanvicens, Paul Galvin,† and Thomas G. Cotter* Cell Development and Disease Laboratory, Department of Biochemistry, Bioscience Research Institute, University College, Cork, and Tyndall National Institute, University College Cork, Lee Maltings, Prospect Row, Cork, Ireland. Received January 17, 2006; Revised Manuscript Received July 3, 2006
Paramagnetic beads have considerable potential as identification tags in biological analysis. For example, magnetic sensor-based arrays using the magnetic field generated by paramagnetic beads to test hybridization between interacting molecules have attracted widespread interest in recent years. However, application of paramagnetic beads as identification tags is still limited, since they do not permit differentiation between samples for multiplex analysis. Here, we report the application of a novel encoding of paramagnetic beads with peptide sequences. This strategy allows DNA samples labeled with peptide-encoded paramagnetic beads to be identified by the selective enzymatic cleavage of each peptide cross-linker.
INTRODUCTION Paramagnetic beads have been extensively used for the preparation, separation, transportation, and detection of biological molecules such as DNA, due to their efficiency, simplicity, and low cost (1-4). More recently, paramagnetic particles have also been used as labels for target DNA in microarray applications, where, in the presence of an external magnetic field, the field from the paramagnetic bead labels can be detected using an array of integrated magnetic sensors (e.g., giant magnetoresistance (GMR) sensors), thereby enabling the detection of DNA hybridization (5-8). However, the range of applications that can utilize paramagnetic beads has been limited due to the difficulty of distinguishing between different particles based on magnetic properties, and hence, they do not permit differentiation between samples for multiplexed analysis (e.g., parallel analysis of a test and control sample in a single reaction as is done using different fluorophores for gene expression analysis). Therefore, methods are required that enable paramagnetic bead based assays to be multiplexed. In this report, paramagnetic beads have been encoded using different peptide sequences, which are each cleavable by a specific enzyme. This novel approach based on an enzymatic cleavage methodology enables differentiation between beads (and hence samples). Using this approach, we have labeled oligonucleotides with different peptide-encoded paramagnetic beads. Subsequent identification of each oligonucleotide was possible through selective enzymatic cleavage of peptide cross-linkers from the paramagnetic beads.
EXPERIMENTAL PROCEDURES Chemicals and Oligonucleotides. EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide), sulfo-NHS (sulfo-N-hydroxysuccinimide), PDITC (4-phenylenediisothiocyanate), 3-amino* To whom correspondence should be addressed. Prof. Thomas G Cotter, Cell Development and Disease Laboratory, Department of Biochemistry, Bioscience Research Institute, University College, Cork, Ireland. E-mail:
[email protected], Phone: +353 21 490 1321, FAX: +353 21 490 1377. † Tyndall National Institute.
propyltrimethoxysilane, proteinase K, and organic solvents were purchased from Sigma Chemical Co. (Poole, U.K.). Oligonucleotides were obtained from MWG Biotech (Ebersberg, Germany). Chymotrypsin was purchased from Calbiochem (La Jolla, CA). Target DNA Labeling with Peptide-Tagged Paramagnetic Beads. Preparation of peptide-tagged paramagnetic beads to was done as follows: 1 µm paramagnetic beads (Roche; Roche Diagnostics Ltd., Sussex, U.K.) coated with streptavidin were washed in twice their volume of PBS (phosphate-buffered saline) buffer giving a final concentration of 5 mg/mL. A magnetic separator was used to remove the beads from solution. Then, 2 pmol of a biotin-terminated oligonucleotide (sequences used were GGGG (peptide G) and GGFG (peptide F); Bachem UK Ltd., Merseyside, U.K.) were added for every 1 µL of bead solution, and the mixture was allowed to react for 2 h at room temperature while rotating at a speed of 30 rpm. Following the conjugation of the peptide to the beads, the free carboxylic end of the peptide was activated by means of EDC chemistry as follows: 20 pmol EDC and 20 pmol sulfo-NHS were added into the bead solution for every picamole of peptide, and the resulting mixture was shaken for 3 h at room temperature. Subsequently, labeling of target DNA with peptide-tagged paramagnetic beads was done as follows: a 1:50 dilution of the solution containing the activated peptide-tagged beads was prepared in 0.15 M borate buffer pH 8.5, and 4 µM of target DNA were added into it and the mixture allowed to react for 3 h at room temperature while rotating at a speed of 30 rpm. Specificity Studies of Oligonucleotide Attachment onto the Peptide-Tagged Bead Surface. Studies were done using oligonucleotide F. Fluorescence intensity was compared between blank solutions containing beads, peptide-encoded tagged beads (peptide G), and samples containing oligonucleotide F modified with cy3 and labeled with peptide-tagged paramagnetic beads (peptide/oligonucleotide ratios 1:1, 1:2.5, 1:5, and 1:10). Fluorescence intensity was measured at 560 nm (excitation at 510 nm) with a FlexStation II instrument (Molecular Devices, Wokingham, U.K.) at room temperature.
10.1021/bc060008j CCC: $33.50 © 2006 American Chemical Society Published on Web 10/25/2006
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Figure 1. Specific binding of a 20mer oligonucleotide to peptideencoded beads. Fluorescence studies demonstrated specific binding of oligonucleotide F-cy3 (5′-GTGACTTCCCCTAAATATAA-3′; modifications: 5′ amino-modifier C12 and 3′ Cy3) to paramagnetic beads encoded with peptide G. A peptide/oligonucleotide ratio of 1:10 rendered the maximum coupling yield with no indication of nonspecific binding (coupling in sample 1:10* was performed without EDC and supho-NHS). The data shown represent mean ( SD (standard deviation) of three independent experiments.
Figure 2. Hybridization and control experiments with peptide-encoded paramagnetic beads. Oligonucleotides G and F were labeled with peptide-encoded beads (G). Substrate contained the oligonucleotide comp-F, which is complementary to oligonucleotide F but not to G. Examples where (i) substrate has no oligonucleotide on it, (ii) beads have no oligonucleotide but the substrate does, (iii) the substrate and the peptide-encoded beads have noncomplementary oligonucleotide sequences (comp-F and G, respectively), and (iv) the substrate and the peptide-encoded beads have complementary oligonucleotide sequences (comp-F and F, respectively). Figures are representative of an experiment performed in triplicate. Images were acquired using a Zeiss Axioskop II Plus epi-fluorescence microscope equipped with an Optronics DEI-750 CCD camera and appropriate filter sets.
Surface Functionalization. Deposition and covalent immobilization of synthetic DNA probes onto the array surface involved substrate cleaning and deposition of a silane anchor layer, followed by the addition of a PDITC homobifunctional linker molecule, to facilitate the covalent binding of the oligonucleotides (see Scheme 2). Substrates were cleaned by sonication in trichlorethylene (10 min), immersion in acetone at 50 °C (10 min), sonication in isopropyl alcohol (10 min), followed by drying in N2. For deposition of the anchor layer, 3-aminopropyltrimethoxysilane (3% v/v) was reacted with the free hydroxyl groups on the surface by sonication in ambient temperature using a in a 95:5 methanol/water solution, then sequentially rinsed in methanol and water and dried in N2. Following this, substrates were cured at 120 °C (15 min). Covalent attachment of the amino-terminated oligonucleotide probes was mediated by activation of the amino silanated substrates with a PDITC homobifunctional crosslinker. For this, the amino silanated substrates were immersed in a 40 mL solution of 1 mM PDITC in 10% v/v anhydrous pyridine/dimethylformamide (DMF, 2 h), followed by thorough sequential rinsing with DMF and dichloroethane, and then drying in N2. Oligonucleotide Probe Patterning and Immobilization over Functionalized Substrate. Deposition of unlabeled amino modified oligonucleotide comp-F was performed using a Staedtler technical drawing pin to print 0.5 mL of a 4 µM oligonucleotide solution in 1% N,N-diisopropylethylamine TrisHCl buffer. Substrates were then placed in a humid atmosphere at 37 °C overnight. After immobilization, substrates were washed in a 0.1% SDS solution at 60 °C (10 min) and dried in N2. Following this, substrates were immersed in 0.1% milk PBS buffer for 2 h to deactivate any remaining unreacted surface-
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bound isothiocyanate groups to prevent nonspecific binding of target DNA or beads during hybridization. Finally, substrates were rinsed thoroughly in water and dried in N2. DNA Hybridization. A solution comprising 5 mg/mL of paramagnetic bead-labeled target DNA suspended in 20 µL hybridization buffer (3× SSPE) was introduced onto the array surface. Hybridization was carried out at room temperature for 3 h. Substrate surface was then rinsed with deionized water to remove nonspecifically bound beads, so that only beads immobilized by selective hybridization of target DNA to complementary probes remained. Enzyme Studies. Cleavage and Removal of Beads. Release of the beads attached to oligonucleotide F through peptide F was done using chymotrypsin, which specifically cleaves the sequence containing phenylalanine while leaving the sequence containing glycine intact (beads tagged with the peptide G remain attached to oligonucleotide F). A solution containing 200 ng of chymotrypsin was prepared in 100 M Tris 10 mM CaCl2 buffer (pH 7.8). Then, 20 µL of the enzyme solution was added onto the chip surface and incubated for 3 h at 37 °C. Following this, the substrate surface was rinsed with deionized water to remove cleaved beads, so that only beads attached to oligonucleotide F through peptide G remained. Selective release of beads attached through peptide F was then visualized by fluorescence imaging. Subsequently, beads attached to oligonucleotide F through peptide G were released using the peptidase proteinase K. For this purpose, a saturated solution of proteinase K was prepared in 100 M Tris 10 mM CaCl2 buffer (pH 7.8). Then, 20 µL of the enzyme solution were added onto the substrate surface and incubated for 3 h at 37 °C. Subsequently, the surface was rinsed with deionized water to remove the remaining beads. Selective release of beads attached through peptide G was then visualized by fluorescence imaging. Images were acquired using an Axioskop II Plus epi-fluorescence microscope equipped with an optronics DEI-750 CCD camera and appropriate filter sets. Imagines were analyzed using Image Pro Express software (Media Cybernetics).
RESULTS AND DISCUSSION Paramagnetic beads were encoded by coating them with one of two different peptide sequences: G or F (Scheme 1, step 1). Each peptide comprised a 4 amino acid sequence with the amino end modified with a biotin molecule (Table 1). In this manner, attachment of the peptides to the surface of streptavidin-coated paramagnetic beads was done by means of a biotin-streptavidin conjugation. In a second step, oligonucleotides were labeled with the peptide-encoded beads. This step comprised the attachment of the oligonucleotide containing a 5′-amino group (Table 1) to the free carboxylic end of the specific amino acid sequence of the peptide-encoded bead using carbodiimide chemistry in a buffered solution (Table 2). In this manner, the peptide remained as a cross-linker between the bead and the target DNA sequence (Scheme 1, step 2). To control the number of DNA oligonucleotide molecules per peptide-encoded bead and to minimize nonspecific attachment onto the bead surface, we optimized the peptide/oligonucleotide ratio in the coupling reaction to 1:10. Figure 1 illustrates how a 1:10 peptide/oligonucleotide ratio yielded the maximum fluorescence intensity from all the proportions assayed. Lack of fluorescence in a sample, in which the coupling reaction of oligonucleotide F modified with cy3 onto the beads had been done without EDC and sulfo-NHS, confirmed specific attachment of the oligonucleotide onto the bead surface. Peptide/oligonucleotide ratios higher than 1:10 rendered nonspecific attachment of the oligonucleotide onto the bead surface (data not shown). Specific hybridization of target DNA labeled with peptideencoded paramagnetic beads to the complementary DNA
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Technical Notes Scheme 1. Scheme of the Strategy for Labeling and Differencing DNA Oligonucleotide Target Sequences with Peptideencoded Paramagnetic Beadsa
a The first step comprises encoding paramagnetic beads with different peptide sequences (G or F in the example). In a subsequent step, DNA oligonucleotide targets are labeled with peptide-encoded paramagnetic beads. In this manner, the peptide sequence remains as a cross-linker between the bead and the target oligonucleotide. Then, each target DNA oligonucleotide can be selectively recognized by cleavage of the peptide sequence with a specific peptidase (peptidase F will cleave peptide F but not G). This strategy allows multiplexed analysis in paramagnetic bead based assays.
Table 1. Sequences of Peptides and DNA Oligonucleotidesa name
sequence
Peptide G Peptide F Oligonucleotide G Oligonucleotide F
Bt-GGGG-COOH Bt-GGFG-COOH 5′-GTACTCTATTTGTAGGTTCTTACGT-3′ 5′-GTGACTTCCCCTAAATATAA-3′ Modification: 5′ amino-modifier C12 Oligonucleotide comp-F 5′-TTA TAT TTA GGG GAA GTC AC-3′ Modification: 5′ amino-modifier C12 a
Bt, biotin; comp, complementary.
Table 2. Scheme of Labeling a DNA Oligonucleotide Sequence with Peptide-Encoded Paramagnetic Beadsa 1 2 a
Bead-SA + Bt-G-COOH f Bead-SA-Bt-G-COOH Bead-SA-Bt-G-COOH + oligonucleotide F-NH2 f Bead-SA-Bio-G-CONH-oligonucleotide-F
SA, streptavidin; Bt, biotin.
sequence attached onto an array surface was investigated. Surface functionalization and oligonucleotide patterning and immobilization over the substrate were carried out as previously described (9) (see Scheme 2). Control studies were performed to investigate nonspecific interaction onto the substrates of target DNA labeled with peptide-encoded beads. Furthermore, hybridization conditions were optimized to achieve specific hybridization of target oligonucleotide (F) to complementary sequences (oligonucleotide comp-F). A concentration of beads of 5 mg/mL produced the best results. We found higher concentrations of beads to produce nonspecific binding onto the substrate and lower concentrations to render poor density of bead spots. Under the optimized conditions, assays were carried out in which either the substrate (Figure 2i) or the beads (Figure 2ii) did not bear DNA. Control experiments demon-
Scheme 2. Scheme of the Surface Functionalization, Oligonucleotide Immobilization, and Hybridization of Complementary Probe Labeled with Peptide-Encoded Paramagnetic Beads
strated that nonspecific binding was minimal. As illustrated in Figure 2, bead spots were only distinctly observed when the oligonucleotide attached to the bead was complementary to the one on the substrate (noncomplementary oligonucleotide G, Figure 2iii; complementary oligonucleotide F, Figure 2iv). The peptidase chymotrypsin selectively hydrolyzes peptide sequences at bonds involving the carboxyl group of aromatic L-amino acids such as phenylalanine (F). Therefore, we chose chymotrypsin as the peptidase for releasing paramagnetic beads tagged with peptide F. Since chymotrypsin does not cleave nonaromatic L-amino acids such as glycine (G), paramagnetic beads encoded with the peptide G should remain unaffected after chymotrypsin treatment. Then, for releasing of beads crosslinked with peptide G, we chose the general peptidase proteinase K. Studies to evaluate the enzymatic activity of chymotrypsin and proteinase K were performed on glass slides, in which for simplification oligonucleotide probes labeled with peptideencoded fluorescent tags were immobilized. Under these conditions, concentration of enzyme, reaction time, and temperature were optimized to achieve maximum activity and selectivity toward each peptide sequence (Figure 3). Subsequently, selective cleavage of peptide-encoded paramagnetic beads from target oligonucleotides by a specific peptidase was investigated. Oligonucleotide F encoded with either beads G or F was hybridized to oligonucleotide comp-F attached onto the substrate surface. In a first step, substrates were treated with chymotrypsin, which only removed beads encoded with peptide F (Figure 4a). Under these conditions, beads encoded with peptide G remained unaltered on the substrate surface. Subsequent treatment with proteinase K cleaved the beads encoded with peptide G. Neither treatment with chymotrypsin nor treatment with proteinase K affected the density of beads on the control spot (beads with no peptide linker). Therefore, our results demonstrate that the enzymatic cleavage approach allows the selective removal of the beads from the substrate surface.
CONCLUSIONS This report proposes a strategy for encoding paramagnetic beads by labeling them with different peptide sequences. It has been demonstrated that samples labeled with encoded paramagnetic beads can be subsequently identified by the selective enzymatic cleavage of each peptide cross-linker. Using this approach, we have been able to differentiate between beads
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Figure 3. Selective cleavage of fluorophores from glass surface. Oligonucleotide F labeled with peptide-tagged fluorophores (fluorophore is streptavidin, Alexa Fluor; Bio Sciences, Ltd., Dun Laoghaire, Ireland) was immobilized onto a glass surface as described in the Experimental Procedures section. Conditions were optimized for selective recognition and hydrolysis of peptide F and peptide G by chymotrypsin and proteinase K, respectively. Both chymotrypsin and proteinase K successfully removed the fluorophores encoded with peptide F and G, respectively, from the glass surface resulting in a significant decrease of fluorescence, while only a slight lost of fluorescence was observed in controls (fluorophores without the peptide). Images were acquired using a Zeiss Axioskop II Plus epi-fluorescence microscope equipped with an Optronics DEI-750 CCD camera and appropriate filter sets.
Figure 4. Demonstration of selective enzymatic cleavage of peptide-encoded paramagnetic beads, where oligonucleotide comp-F was immobilized onto the substrate surface by means of covalent chemistry. (i) Hybridization with Oligonucleotide F labeled with peptide-encoded paramagnetic beads (i.e., Bead F or Bead G) was then performed. In a first step, release of the beads encoded with Peptide F was done using chymotrypsin where beads tagged with Peptide G and control beads (beads with no peptide cross-linker) remained intact. Subsequently, beads encoded with Peptide G were released using the generic enzyme, Proteinase K. In this manner, selective enzymatic cleavage of the peptide linkers enabled differentiation between beads, and thereby enabled differentiation between the attached oligonucleotides. Figures are representative of an experiment done in triplicate. (ii) Controls were done with Oligonucleotide F labeled with paramagnetic beads with no peptide cross-linker. Images were acquired using a Zeiss Axioskop II Plus epi-fluorescence microscope equipped with an Optronics DEI-750 CCD camera and appropriate filter sets
encoding different target DNA oligonucleotides. Interestingly, this strategy is not restricted to encoding of paramagnetic beads, but it can also be used to label supports of any nature (for example, nanocrystals, gold nanoparticles, glass beads). While
some micro- and nanoparticles are distinguishable on the basis of different colors (or if fluorescent, on the basis of different excitation and emission spectra), the sensitivity achievable with sensor systems necessary to enable color imaging is typically
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Technical Notes
much less than that achievable with equivalent monochrome technology. Therefore, the present methodology can be employed to increase the multiplicity in any kind of biological assay. Moreover, in addition to the possibility of simultaneous analysis of multiple samples, this approach allows modulation of the bead characteristics by introducing a large number of distinguishable sequences. Brust and co-workers have already reported that functionalized peptide binding to nanoparticle permits introducing multiple functionalities onto gold nanoparticles (10). We contemplate that properties related to hydrophobicity and charge of the encoded beads could also be modulated in a controlled manner by modifying the nature and the number of the nonrelevant amino acids comprising the peptide sequence. Undoubtedly, this would have an impact on the hybridization efficiency. This approach could also be adapted to create dynamic substrates, in which the surface properties could be selectively modified in a spatial manner, by patterning of different molecules that would be tethered to the substrate by different peptide sequences, and by subsequent selective cleavage of molecules in an ordered manner.
ACKNOWLEDGMENT This study was supported by funds from the European Union (contract number QLK3-CT-2001-01982).
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