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1、2294Chem. Commun., 2011, 47, 22942296This journal iscThe Royal Society of Chemistry 2011 Silver nanocluster aptamers: in situ generation of intrinsically fl uorescent recognition ligands for protein detectionw Jaswinder Sharma, Hsin-Chih Yeh, Hyojong Yoo,z James H. Werner and Jennifer S. Martinez* R
2、eceived 7th September 2010, Accepted 16th November 2010 DOI: 10.1039/c0cc03711g We have synthesized an intrinsically fl uorescent recognition ligand that combines the high fl uorescence quantum yield (50%) of oligonucleotide templated AgNCs with the specifi city and strong binding affi nity of DNA a
3、ptamers for their target proteins, to develop a new strategy for detection of specifi c proteins. Gold and silver nanoclusters, consisting of a few metal atoms, have gained considerable attention due to their unique fl uorescence properties and potential applications in the fi elds of chemical-sensi
4、ng, in vivo biological imaging, and in vitro bioassays.14Oligonucleotide-templatedsilver-nanoclusters (AgNCs) are of particular interest due to their facile synthesis, tunable fl uorescence emission, and high photostability.59 Further, fl uorescent nanoclusters are smaller than biological molecules
5、of interest, and hence potentially more advantageous for biosensing than are quantum dots. While advancements in nanocluster synthesis have been made, the use of metallic nanoclusters for biological- or chemical-sensing is still in its infancy, largely resulting from diffi culty in bioconjugation of
6、 these intriguing fl uorophores.10,11Whereas, one can chemically conjugate a fl uorophore to a biomolecule (i.e., recognition ligand), a simpler method is to use the recognition ligand to directly template the fl uorophore. Herein, we report the development and use of an intrinsically fl uorescent r
7、ecognition ligand, based on a DNA aptamer-templated AgNC, for specifi c and sensitive protein detection. Aptamers are nucleic acids (DNA or RNA) that are evolved to specifi cally bind proteins or low-molecular-weight inorganic or organic substrates.12,13 Because of their specifi city and good bindin
8、g constants, aptamers have been used for nanomaterials assembly and detection of heavy metals, proteins and cancer cells.1418Forexample,Yanandcolleagueshaveused aptamers for detection of specifi c proteins and organization of biomolecules by employing a DNA scaff oldaptamer assembly.19,20Likewise, a
9、 number of beautiful and sensitive detectionstrategieshavealsobeendevelopedusing aptamergold nanoparticle conjugates21and electrochemical detection2225of proteins and small molecules. Still the use of aptamers for biosensing purposes requires covalent labeling of the aptamer with electrochemical, fl
10、 uorescent, or nanoparticle substituents.2023This conjugation is costly (i.e., the time for covalent modifi cation and purifi cation of labeled- from unlabeled-aptamer, and monetary cost of labeling). Herein,wereportthedevelopmentanduseofan intrinsically fl uorescent recognition ligand based on a DN
11、A aptamer-templated AgNC for one pot, single step, purifi cation free, specifi c and sensitive detection of protein. Our intrinsically fl uorescent recognition ligand combines the strong fl uorescence of oligonucleotide templated AgNCs with the specifi city and strong binding affi nity of DNA aptame
12、rs for their target proteins, to develop a new strategy for detection of specifi c proteins (Fig. 1). Here the AgNCaptamer assembly serves as both a fl uorescent label and a specifi c binding ligand. Our in situ method is a single-step that requires no covalent attachment of aptamer or protein to a
13、fl uorophore, and is thus a simple and inexpensive labeling and detection method. As a proof of concept, we use human a-thrombin as a target protein. Thrombin (B36 kDa) is an abundant protein in serum that catalyzes reactions involved in coagulation of blood. Two aptamers have been selected that bin
14、d to human a-thrombin: APT15 (15 mer, KdE 100 nM, binds to the fi brinogen-binding site of thrombin), and APT29 (29 mer, KdE 0.5 nM, binds to the heparin-binding site of thrombin).2527 We created an aptamernanocluster templating chimera combining a thrombin APT29 DNA sequence with a cytosine rich DN
15、A sequence that generates highly fl uorescent AgNCs. Fig. 1Schematic of aptamernanoclusterDNA templated AgNCs for detection of thrombin protein. Sequence of the aptamerAgNC template: 50-AGTCCGTGGTAGGGCAGGTTGGGGTGACTAA- AAACCCTTAATCCCC-30. MPA-CINT, Mail Stop, K771, Los Alamos National Laboratory, Lo
16、s Alamos, NM 87545, USA. E-mail: jenmlanl.gov; Fax: +1 505-665-9030; Tel: +1 505-665-0045 w Electronic supplementary information (ESI) available: Experimental conditions, FCS and TCSPC studies and control experiments. See DOI: 10.1039/c0cc03711g z Current address: Department of Chemistry, Hallym Uni
17、versity, 39 Hallymdaehak-gil, Chuncheon-si, Gangwon-do, 200-702, Republic of Korea. E-mail: hyojonghallym.ac.kr COMMUNICATIONwww.rsc.org/chemcomm | ChemComm Published on 09 December 2010. Downloaded on 08/01/2014 07:13:04. View Article Online / Journal Homepage / Table of Contents for this issue Thi
18、s journal iscThe Royal Society of Chemistry 2011Chem. Commun., 2011, 47, 229422962295 TosynthesizeaptamernanoclusterDNAtemplated AgNCs(aptamerAgNCs),theaptamerNCtemplating DNA chimera and AgNO3were mixed in sodium phosphate buff er (see ESIw, S2 for details). Following reduction with NaBH4 , the apt
19、amerAgNCs showed strong red fl uorescence, with an emission peak at 700 nm. Similar to our previously reported AgNCs,7 we fi nd that the aptamerAgNCs are highly photostable and bright (B60% quantum yield), and physically stable over a range of bioassay conditions (NaCl concen- trations and pH). At a
20、 fi xed 50 mM NaCl concentration, we fi nd that the fl uorescence of aptamerAgNCs is quenched considerably upon addition, and subsequent binding, of thrombin protein to the aptamerAgNCs (Fig. 1, 2a and b) (see ESIw, S1 for sequences and S2 for the reaction conditions). Little fl uorescence quenching
21、 was observed upon addition of denatured thrombin, or thrombin prebound to aptamer (Fig. S5 and S6, ESIw), further confi rming that quenching results from the binding of native thrombin with its aptamer sequence. Additionally, nanoclusters templated on a nonspecifi c (random DNA sequence with the sa
22、me length as the aptamer) DNA nanoclustertemplatingchimera(identicalnanocluster templating sequence as in aptamerAgNCs) showed no quenching upon addition of thrombin. To test for specifi city, we added a number of proteins of varied structure and charge state, including bovine serum albumin (BSA), s
23、treptavidin and platelet derived growth factor (PDGF), to the solutions of aptamerAgNCs. In contrasttothe fl uorescencequenchingobservedupon thrombin binding, no fl uorescence quenching was observed in solutions with the nonspecifi c proteins (Fig. 2a and b). Likewise, these proteins did not bind to
24、 the aptamerAgNCs, as observed by the gel-shift analysis (Fig. 2b and S1, ESIw). Thus the fl uorescence quenching of aptamerAgNCs only occurs upon specifi c protein (thrombin) binding (ESIw, S3). Togaininsightintothequenchingmechanism,we performed fl uorescence correlation spectroscopy (FCS) and tim
25、e-correlated single photon counting (TCSPC) on the aptamer AgNCthrombin mixtures. TCSPC studies (Table 1) show the observed fl uorescence lifetime, even for a heavily quenched solution, is still approximately 3.6 ns. This result indicates that the quenching mechanism is static rather than dynamici.e
26、., the thrombinAgNCaptamer complex forms an essentially non-fl uorescent adduct. FCS measurements (Table 1) of the brightness per cluster also confi rm this conclusion: quenching is due to the disappearance of bright species with a remaining population of clusters (unbound) that are of comparable br
27、ightness (for experimental details see ESIw, S5). While TCSPC and FCS measurements both show fl uorescence quenching is due to the removal of bright species and not dynamic, excited-state quenching, there are a number of explanations for this phenomenon. One possibility is that the fl uorescence que
28、nchingmay be caused bythe enhanced oxidation of aptamerAgNCs upon protein binding (oxidized aptamer AgNCs are non-fl uorescent). However, we fi nd that aptamer AgNCs bound to thrombin did not regain their fl uorescence after addition of extra equivalents of NaBH4, suggesting that oxidation is not th
29、e cause of fl uorescence loss. Likewise, it is possible that the guanine within the thrombin recognition sequenceenhancesthenanocluster fl uorescence.4Thus, protein binding may induce a structural change in the aptamerAgNC chimera, resulting in quenching. A second possibility is that the fl uorescen
30、ce quenching may result from removal of the DNA from the AgNCs likely leading to the aggregation of unprotected silver clusters, and to the sub- sequent formation of large silver nanoparticles. However, both UV-Vis spectra and TEM images, taken before and after protein addition, did not show conside
31、rable diff erences (Fig. S3 and S4, ESIw), ruling out the possibility of AgNCs aggregation. We studied the fl uorescence quenching of the aptamer AgNCsoverawiderangeofthrombinconcentration (11500 nM, Fig. 3). Quenching saturates at roughly 1000 nM. Following the IUPAC criterion, we fi nd that the de
32、tection limit in this quenching-based assay is 1 nM thrombin, which iscomparabletootherreported fl uorescencedetection methods2831(for experimental details see S4, ESIw). In conclusion, we have generated an intrinsically fl uorescent recognition ligand, which consists of a fl uorescent AgNC on an ap
33、tamernanocluster DNA chimera. These aptamer AgNCs are easy to synthesize and use (in situ synthesis and no purifi cation), are highly photostable and bright, and are highly selective for the cognate protein of the aptamer. These attributes make aptamerAgNCs a valuable method for detection of protein
34、s or small molecules. Further, by incorporating Fig. 2(a) Fluorescence emission spectra of AgNCs in the presence of specifi c and nonspecifi c proteins. (b) Photographs showing the fl uorescence (top) and gel-shift analysis (bottom) of AgNCs inter- acting with specifi c and nonspecifi c proteins. La
35、ne 1: aptamerAgNCs; lane2:aptamerAgNCswiththrombinprotein;lane3: aptamerAgNCs with streptavidin; lane 4: aptamerAgNCs with PDGF; lane 5: aptamerAgNCs with BSA. Here DNA is imaged using intrinsic cluster fl uorescence. See Fig. S1 (ESIw) for images of DNA shifting position on thrombin binding, as ima
36、ged by ethidium bromide staining. Table 1FCS and TCSPC studies of aptamerAgNCs Bulk fl uorescencea/kHz Brightness per clusterb/kHz Fluorescence lifetimec/ns AgNCs77.615.403.56 AgNCs + thrombin5.611.503.55 a The number of photoelectrons detected per second (i.e. the total count rate) using a single p
37、hoton counting avalanche photodiode, represents the bulk fl uorescence from each sample. b The average brightness per aptamerAgNCs is obtained by dividing the bulk count rate by the average occupancy of fl uorescent AgNC in the detection volume, obtained from the FCS analysis (S5, ESIw). c Data are
38、fi t with a one-exponential model. Published on 09 December 2010. Downloaded on 08/01/2014 07:13:04. View Article Online 2296Chem. Commun., 2011, 47, 22942296This journal iscThe Royal Society of Chemistry 2011 palettesofnanocluster-templatingsequences,eachwith diff erent excitation and emission spec
39、tra, with diff erent aptamer sequences, a one-pot, high-specifi city, multiplex protein detection scheme can be created. Additionally, this work demonstrates a strategy to circumvent the conjugation of recognition molecules (i.e., aptamers) with fl uorophores, particles or electrochemical moieties,
40、which are required for their use in biosensing applications, and we anticipate this strategy will further help researchers in designing conjugation free biosensors based on metallic nanoclusters. This research is supported by the Los Alamos National Laboratory Directed Research and Development (LDRD
41、) and Directors Postdoctoral Fellowship (J.S.) and Department of Energy, Offi ce of Basic Energy Science, Division of Material Science and Engineering (H.Y). This work was performed at the Center for Integrated Nanotechnologies, a U.S. Department of Energy, Offi ce of Basic Energy Sciences user faci
42、lity. Notes and references 1 J. Zheng, P. R. Nicowich and R. M. Dickson, Annu. Rev. Phys. Chem., 2007, 58, 409431. 2 Y. Bao, C. Zhong, D. M. Vu, J. P. Temirov, R. B. Dyer and J. S. Martinez, J. Phys. Chem. C, 2007, 111, 1219412198. 3 Y. Bao, H.-C. Yeh, C. Zhong, S. A. Ivanov, J. K. Sharma, M. L. Nei
43、dig, D. M. Vu, A. P. Shreve, R. B. Dyer, J. H. Werner and J. S. Martinez, J. Phys. Chem. C, 2010, 114, 1587915882. 4 H.-C. Yeh, J. Sharma, J. Han, J. S. Martinez and J. H. Werner, Nano Lett., 2010, 10, 31063110. 5 C. I. Richards, S. Choi, J.-C. Hsiang and Y. Antoku, J. Am. Chem. Soc., 2008, 130, 503
44、85039. 6 E. G. Gwinn, P. ONeill, A. J. Guerrero, D. Bouwmeester and D. K. Fygenson, Adv. Mater., 2008, 20, 279283. 7 J. Sharma, H.-C. Yeh, H. Yoo, J. H. Werner and J. S. Martinez, Chem. Commun., 2010, 46, 32803282. 8 B. Sengupta, C. M. Richie, J. G. Buckman, K. R. Johnsen, P. T. Goodwin and J. T. Pe
45、tty, J. Phys. Chem. C, 2008, 112, 1877618782. 9 P. R. ONeill, L. R. Velazquez, D. G. Dunn, E. G. Gwinn and D. K. Fygenson, J. Phys. Chem. C, 2009, 113, 42294233. 10 S. Dong and L. Shang, J. Mater. Chem., 2008, 18, 46364640. 11 W. Guo, J. Yuan, Q. Dong and E. Wang, J. Am. Chem. Soc., 2010, 132, 93293
46、4. 12 A. D. Ellington and J. K. Szostak, Nature, 1990, 346, 818822. 13 C. Tuerk and L. Gold, Science, 1990, 249, 505510. 14 G. F. Joyce, Curr. Opin. Struct. Biol., 1994, 4, 331336. 15 C.-W. Liu, C.-C. Huang and H. T. Chang, Anal. Chem., 2009, 81, 23832387. 16 K. Sefah, J. A. Philips, X. Xiong, L. Me
47、ng, D. V. Simaeys, H. Chen, J. Martin and W. T. Tan, Analyst, 2009, 134, 17651774. 17 J. A. Hensen, J. Wang, A.-N. Kawde, Y. Xiang, K. V. Gothelf and G. Collins, J. Am. Chem. Soc., 2006, 128, 22282229. 18 J. E. Smith, C. D. Medley, T. D. Shangguan, C. Lofton and W. Tan, Anal. Chem., 2007, 79, 307530
48、82. 19 C. Lin, E. Katilius, Y. Liu, J. Zhang and H. Yan, Angew. Chem., Int. Ed., 2006, 118, 54225427. 20 R. Chhabra, J. Sharma, Y. Ke, Y. Liu, S. Rinker, S. Lindsay and H. Yan, J. Am. Chem. Soc., 2007, 129, 1030410305. 21 J. Liu and Y. Lu, Angew. Chem., Int. Ed., 2006, 45, 9094. 22 E. E. Ferapontova
49、 and K. V. Gothelf, Langmuir, 2009, 25, 42794283. 23 J. S. Swensen, Y. Xiao, B. S. Ferguson, A. A. Lubin, R. Y. Lai, A. J. Heeger, K. W. Plaxco and H. T. Soh, J. Am. Chem. Soc., 2009, 131, 42624266. 24 Y. Xiao, R. Y. Lai and K. W. Plaxco, Nat. Protoc., 2007, 2, 28752880. 25 Y. Xiang, M. Xie, R. Bash, J. J. L. Chen and J. Wang, Angew. Chem., Int. Ed., 2007, 46, 90549056. 26 L. Bock, L. C. Griffi n, J. A. Latham, E. H. Vermass and J. J. Toole, Nature, 1992, 355, 564566. 27 D. M. Tasset, M. F. Kubik and W. Steiner,