LETTER pubs.acs.org/ac
Aptamer Capturing of Enzymes on Magnetic Beads to Enhance Assay Specificity and Sensitivity Qiang Zhao,*,†,‡ Xing-Fang Li,‡ and X. Chris Le‡ † ‡
Research Center for Environmental Science and Engineering, Shanxi University, Taiyuan, China, 030006 Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, AB, Canada, T6G 2G3
bS Supporting Information ABSTRACT: Activity and specificity of enzyme molecules are important to enzymatic reactions and enzyme assays. We describe an aptamer capturing approach that improves the specificity and the sensitivity of enzyme detection. An aptamer recognizing the target enzyme molecule is conjugated on a magnetic bead, increasing the local concentration, and serves as an affinity probe to capture and separate minute amounts of the enzyme. The captured enzymes catalyze the subsequent conversion of fluorogenic substrate to fluorescent products, enabling a sensitive measure of the active enzyme. The feasibility of this technique is demonstrated through assays for human alpha thrombin and human neutrophil elastase (HNE), two important enzymes. Thrombin (2 fM) and 100 fM HNE can be detected. The incorporation of two binding events, substrate recognition and aptamer binding, greatly improves assay specificity. With its simplicity, this approach is applicable to biosensing and detection of disease biomarkers.
E
nzymes are important macromolecules in organisms, catalyzing reactions crucial to biological processes.1,2 While many enzymes are widely used in developing bioanalytical assays, direct analysis of specific enzymes is also important for biochemical and clinical research.3 6 Harnessing the catalytic property of enzymes to amplify analytical signals, chromogenic and fluorogenic enzyme assays are highly sensitive.3 6 However, the chromogenic and fluorogenic assays depend on the specificity of enzymes. For instance, proteases as a large family of important enzymes can cleave the same peptide substrates, compromising the specificity.1,2 Introducing a separation step, e.g., specific capture of enzyme by involving an additional binding event, improves the selectivity of the enzyme assay. An ideal capture molecule should maintain the activity of the enzyme. Recent research using short single-stranded oligonucleotides (aptamers7 9) to bind to specific enzyme molecules (e.g., thrombin10,11 and elastase12) has shown that the enzyme was able to catalyze the specific substrate after its binding of the specific aptamer.13 16 Taking advantage of the specific aptamer-enzyme binding, we have now developed a simple strategy (Figure 1) to improve the specificity and sensitivity of the enzyme assays. The aptamer recognizing the enzyme is attached on the magnetic beads, which selectively capture the enzyme from sample mixture. After magnetic separation, the magnetic beads are dispersed in the solution containing a specific fluorogenic substrate of the enzyme. Detection of the fluorescent products arising from the enzymatic conversion of the substrate provides an overall measure of the active enzyme. r 2011 American Chemical Society
Figure 1. Schematic diagram showing the principle of the aptamer capturing of the target enzyme and the subsequent enzymatic conversion of the fluorogenic substrate to a fluorescent product. The enzyme is captured by the aptamer (shown in blue) on the magnetic beads (MB). Following magnetic separation, the fluorogenic substrate is added and the enzyme reaction yields the fluorescent product. Quantitative determination of the fluorescent product provides a measure for the amount of active enzyme.
The use of magnetic beads facilitates an easy separation and improves the binding kinetics between the capturing probe and the target.17 The immobilization of aptamers increases the local concentration of aptamers on the beads and enhances the binding between aptamers and the target. Therefore, aptamers with low or moderate binding affinity can be used, expanding the application of aptamers that have weak binding. To demonstrate the proof of principle, we initially chose two aptamers against Received: November 17, 2011 Accepted: November 19, 2011 Published: November 19, 2011 9234
dx.doi.org/10.1021/ac203063z | Anal. Chem. 2011, 83, 9234–9236
Analytical Chemistry
LETTER
Figure 2. Assay for human α-thrombin showing a wide dynamic range. The fluorescence intensity is the net increase of fluorescence over the control, obtained by subtracting the background fluorescence. The inset shows that the fluorescent signal from the analysis of low concentrations (as low as 1 pM) of thrombin can be distinguished from the background.
enzymes, both having moderate binding affinity (Kd values being 100 nM for thrombin10,11 and 17 nM for elastase12 ). We chose human α-thrombin as a first example to show our strategy for the specific detection of this enzyme. Thrombin is a central enzyme involved in blood coagulation cascade and platelet aggregation, playing multiple roles in hemostasis, thrombosis, inflammation, tumor growth, and metastasis.18 Figure 2 shows the determination of human α-thrombin by using magnetic beads modified with a 15-mer capture aptamer10 (about 1 105 aptamers on each bead) and a bis(p-tosyl-Gly-Pro-Arg) derivative of Rhodamine 110 as the substrate. Thrombin catalyzes the cleavage of this peptide substrate at the Arg site, generating a fluorescent product. With a 3 h enzyme reaction at 37 °C, the assay for thrombin in a 5 μL solution was achieved for a wide concentration range from 1 pM to 10 nM (about 2 pmol of conjugated aptamer applied in the assay). The relative standard deviation for this assay was less than 5%. Considering the weak binding affinity (Kd ∼ 100 nM) between the aptamer and α-thrombin, the ability to detect trace concentrations of thrombin would not have been possible. Our success is due to the enhanced local concentrations of the immobilized aptamers on the magnetic beads, improving the aptamer binding to the target enzyme. The capture of enzyme by aptamers on the magnetic beads allows for the enrichment of enzyme molecules. Thus, increasing the volume of dilute samples from 5 to 250 μL resulted in the expected improvement in the detection limit by 50-fold, enabling the detection of 2 fM thrombin (Table S1 and Figures S1 and S2 in the Supporting Information). The sensitivity of this assay for thrombin is better than or comparable to the ELISA (detection limit ∼14 pM),19 radio immunoassay (∼54 pM),20 and other aptamer-based assays for thrombin.7 9,13 15,21 25 In addition, a short-time (tens of minutes) enzyme reaction can be applied in a rapid analysis of thrombin at high concentration (Table S1 in the Supporting Information). We then investigated the specificity of the assay for human α-thrombin by testing several other common enzymes (trypsin, plasmin, Factor Xa, elastase, and papain), abundant proteins in blood (human serum albumin, immunoglobulin G, and transferrin),
Figure 3. Analyses of α-thrombin and other nontarget proteins show that the interferences from these nontarget proteins (A) were eliminated by using aptamer to capture the α-thrombin enzyme (B). In both tests, the concentrations of α-thrombin, γ-thrombin, trypsin, plasmin, papain, Factor Xa, IgG, HSA, transferrin, and prothrombin were 10 nM. The concentration of elastase was 38 nM. The sample volume was 5 μL, and the enzymatic reaction was carried out at 37 °C for 1 h.
and proteins sharing similar structure with human α-thrombin (e.g., human prothrombin and γ-thrombin). Without using the aptamer-modified beads for separation, the enzyme assay for α-thrombin experienced interference from trypsin, plasmin, Factor Xa, and γ-thrombin because these enzymes also catalyzed the cleavage of the same tripeptide substrate (Figure 3A).26,27 In contrast, by capturing the target enzyme with affinity aptamer, interferences from the nontarget enzymes were eliminated (Figure 3B). In addition, the presence of 100-fold IgG, HSA, and transferrin did not interfere with the detection of human α-thrombin. The improvement in assay specificity is attributed to two selective events: the aptamer binding for capture and the substrate recognition in enzyme reaction. We further tested thrombin in serum and plasma to examine whether the complex matrix affected the enzyme assay. The inhibitors in serum or plasma could cause a low recovery of the spiked thrombin.28,29Dilution of serum or plasma sample improved the recovery.30 Analysis of α-thrombin spiked to 100-fold diluted human serum or plasma showed 90 109% recovery, suggesting no interference from such a sample matrix. Further analyses of various serum samples demonstrated the ability of the assay for thrombin in human serum at a wide range of concentration (Table S2 in the Supporting Information). The similar wide range of thrombin concentrations (from picomolar to nanomolar) (Table S2 in the Supporting Information) in human 9235
dx.doi.org/10.1021/ac203063z |Anal. Chem. 2011, 83, 9234–9236
Analytical Chemistry serum and plasma has been reported previously,15,19,22,23,31 and the thrombin levels could be indicative of the physiological conditions of individuals.15,19,31 Our results demonstrate the potential of the thrombin assay for future biomarker development. To test the general applicability of our strategy, we detected a second enzyme, human neutrophil elastase (HNE), a serine protease involved in pathogen destruction, inflammatory process regulation, and several diseases.32 We were able to detect 0.1 pM HNE in 250 μL by using a 45-nt DNA aptamer,12 and the results are shown in Figures S3 S5 and Table S3 in the Supporting Information. The sensitivity of the assay for HNE is better than previously reported.33,34 Other common enzymes (e.g., porcine pancreatic elastase, trypsin, thrombin, chymotrypsin, protease K, and lysozyme) and abundant proteins (e.g., hemoglobin, human serum albumin, transferrin) did not cause significant interference in the assay (Figure S6 in the Supporting Information). The success of both assays for thrombin and HNE suggests applicability of the strategy. Our assay offers high sensitivity by capitalizing on preconcentration of the target enzyme molecules by the aptamer-modified magnetic beads, molecular amplification through enzymatic reactions, and highly sensitive detection of fluorescent products of the catalyzed reactions. We are able to detect femtomolar human α-thrombin and human neutrophil elastase and achieve successful determination of thrombin present in human serum at a wide range of concentrations. This assay also exhibits improved specificity by incorporating two binding events: the specific capture by aptamers and the selective substrate recognition by the enzymes. Requiring a single aptamer molecule, this assay is much simpler than the sandwich assays that use two affinity probes (aptamers and/or antibodies).8,9,24 The principle of this assay can be extended to other enzymes, and assay kits, automatic operation, and high throughput format may be developed. Most promising applications include disease diagnosis and fundamental research involving enzyme catalysis and molecular interactions.
’ ASSOCIATED CONTENT
bS
Supporting Information. Experimental details and supporting materials. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected].
’ ACKNOWLEDGMENT We acknowledge the financial support from the Natural Sciences and Engineering Research Council of Canada, the Canadian Institutes of Health Research, the Canada Research Chairs Program, the Alberta Health and Wellness, the National Natural Science Foundation of China (Grant 20905043), and the Research Project Supported by Shanxi Scholarship Council of China. We also thank Prof. Hailin Wang of the Research Center for Eco-Environmental Sciences, Chinese Academy of Science, Beijing, for helpful discussion.
LETTER
(3) Goddard, J.-P.; Reymond, J.-L. Trends Biotechnol. 2004, 22, 363–370. (4) Reymond, J.-L., Ed. Enzyme Assays: Highthroughput Screening, Genetic Selection, and Fingerprinting; Wiley-VCH: Weinheim, Germany, 2006. (5) Guarise, C.; Pasquato, L.; Filippis, V.; De; Scrimin, P. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 3978–3982. (6) Pinto, M. R.; Schanze, K. S. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 7505–7510. (7) Klussmann, S., Ed. The Aptamer Handbook, Functional Oligonucleotides and Their Applications; Wiley-VCH: Weinheim, Germany, 2006. (8) Cho, E. J.; Lee, J.-W.; Ellington, A. D. Annu. Rev. Anal. Chem. 2009, 2, 241–264. (9) Liu, J.; Cao, Z.; Lu, Y. Chem. Rev. 2009, 109, 1948–1998. (10) Bock, L. C.; Griffin, L. C.; Latham, J. A.; Vermaas, E. H.; Toole, J. J. Nature 1992, 355, 564–566. (11) Tasset, D. M.; Kubik, M. F.; Steiner, W. J. Mol. Biol. 1997, 272, 688–698. (12) Lin, Y.; Padmapriya, A.; Morden, K. M.; Jayasena, S. D. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 11044–11048. (13) Mir, M.; Vreeke, M.; Katakis, I. Electrochem. Commun. 2006, 8, 505–511. (14) Centi, S.; Messina, G.; Tombelli, S.; Palchetti, I.; Mascini, M. Biosens. Bioelectron. 2008, 23, 1602–1609. (15) M€uller, J.; Becher, T.; Braunstein, J.; Berdel, P.; Gravius, S.; Rohrbach, F.; Oldenburg, J.; Mayer, G.; Potzsch, B. Angew. Chem., Int. Ed 2011, 50, 6075–6078. (16) Petrera, N. S.; Stafford, A. R.; Leslie, B. A.; Kretz, C. A.; Fredenburgh, J. C.; Weitz, J. I. J. Biol. Chem. 2009, 284, 25620–25629. (17) Nam, J.-M.; Thaxton, C. S.; Mirkin, C. A. Science 2003, 301, 1884–1886. (18) Maragoudakis, M. E., Tsopanoglou, N. E., Eds. Thrombin: Physiology and Disease; Springer Science: New York, 2009. (19) Shuman, M. A.; Majerus, P. W. J. Clin. Invest. 1976, 58, 1249–1258. (20) Pelzer, H.; Schwarz, A.; Heimburger, N. Thromb. Haemostasis 1988, 59, 101–106. (21) German, I.; Buchanan, D. D.; Kennedy, R. T. Anal. Chem. 1998, 70, 4540–4545. (22) Yang, H.; Ji, J.; Liu, Y.; Kong, J.; Liu, B. Electrochem. Commun. 2009, 11, 38–40. (23) Wang, H.; Liu, Y.; Liu, C.; Huang, J.; Yang, P.; Liu, B. Electrochem. Commun. 2010, 12, 258–261. (24) Pai, S.; Roberts, A.; Ellington, A. D. Exper. Opin. Med. Diagn. 2008, 2, 1333–1346. (25) Zhao, Q.; Lu, X.; Yuan, C.; Li, X.-F.; Le, X. C. Anal. Chem. 2009, 81, 7484–7489. (26) Lottenberg, R.; Christensen, U.; Jackson, C. M.; Coleman, P. L. Methods Enzymol. 1981, 80, 341–361. (27) Harris, J. L.; Backes, B. J.; Lenonetti, F.; Mahrus, S.; Ellman, J. A.; Craik, C. S. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 7754–7759. (28) Abildgaard, L. J. Scand. J. Clin. Lab. Invest. 1969, 24, 23–27. (29) Mihalyi, E. J. Gen. Physiol. 1953, 37, 139–156. (30) Huang, Y. C.; Ge, B.; Sen, D.; Yu, H.-Z. J. Am. Chem. Soc. 2008, 130, 8023–8029. (31) Bichler, J.; Heit, J. A.; Owen, W. G. Thromb. Res. 1996, 84, 289–294. (32) Meyer-Hoffert, U.; Wiedow, O. Curr. Opin. Hematol. 2011, 18, 19–24. (33) He, J. L.; Wu, Z.-S.; Zhang, S.-B.; Shen, G.-L.; Yu, R.-Q. Talanta 2010, 80, 1264–1268. (34) Castillo, M. J.; Nakajima, K.; Zimmerman, M.; Powers, J. Anal. Biochem. 1979, 99, 53–64.
’ REFERENCES (1) Lopez-Otín, C.; Bond, J. S. J. Biol. Chem. 2008, 283, 30433–30437. (2) Hedstrom, L. Chem. Rev. 2002, 102, 4501–4524. 9236
dx.doi.org/10.1021/ac203063z |Anal. Chem. 2011, 83, 9234–9236
