We present here a novel conjugated aptamer-gold nanoparticle (Apt-AuNPs) fluorescent probe and its application for specific detection of recombinant human erythropoietin-α (rHuEPO-α). In this nanobiosensor, 12 nm AuNPs function as both a nano-scaffold and a nano-quencher (fluorescent energy acceptor), on the surface of which the complementary sequences are linked (as cODN-AuNPs) and pre-hybridized with carboxymethylfluorescein (FAM)-labeled anti-rHuEPO-α aptamers. Upon target protein binding, the aptamers can be released from the AuNP surface and the fluorescence signal is restored. Key variables such as the length of linker, the hybridization site and length have been designed and optimized. Full performance evaluation including sensitivity, linear range and interference substances are also described. This nanobiosensor provides a promising approach for a simple and direct quantification of rHuEPO-α concentrations as low as 0.92 nM within a few hours.
References
[1]
Xiao, Y.; Patolsky, F.; Katz, E.; Hainfeld, J.F.; Willner, I. “Plugging into enzymes”: Nanowiring of redox enzymes by a gold nanoparticle. Science 2003, 21, 1877–1881.
Chithrani, B.D.; Ghazani, A.A.; Chan, W.C.W. Determining the size and shape dependence of gold nanoparticle uptake into mammalian cells. Nano Lett 2006, 6, 662–668, doi:10.1021/nl052396o. 16608261
[4]
Stoeva, S.I.; Lee, J.S.; Smith, J.E.; Rosen, S.T.; Mirkin, C.A. Multiplexed detection of protein cancer markers with biobarcoded nanoparticle probes. J. Am. Chem. Soc 2006, 128, 8378–8379, doi:10.1021/ja0613106. 16802785
[5]
Lee, J.S.; Lytton-Jean, A.K.R.; Hurst, S.J.; Mirkin, C.A. Silver nanoparticle-oligonucleotide conjugates based on DNA with triple cyclic disulfide moieties. Nano Lett 2007, 7, 2112–2115, doi:10.1021/nl071108g. 17571909
[6]
Dubertret, B.; Calame, M.; Libchaber, A.J. Single-mismatch detection using gold-quenched fluorescent oligonucleotides. Nat. Biotechnol 2001, 19, 365–370, doi:10.1038/86762. 11283596
[7]
Cao, Y.W.C.; Jin, R.C.; Mirkin, C.A. Nanoparticles as labels for biodiagnostic rsearch. Science 2002, 297, 1536–1540, doi:10.1126/science.297.5586.1536. 12202825
[8]
Nam, J.M.; Thaxton, C.S.; Mirkin, C.A. Nanoparticle-based bio-bar codes for the ultrasensitive detection of proteins. Science 2003, 301, 1884–1886, doi:10.1126/science.1088755. 14512622
[9]
Giljohann, D.A.; Seferos, D.S.; Daniel, W.L.; Massich, M.D.; Patel, P.C.; Mirkin, C.A. In vitro selection of structure-switching signaling aptamers. Angew. Chem. Int. Ed 2010, 49, 3280–3294, doi:10.1002/anie.200904359.
[10]
Ellington, A.D.; Szostak, J.W. In vitro selection of RNA molecules that bind specific ligands. Nature 1990, 346, 818–822, doi:10.1038/346818a0. 1697402
[11]
Tuerk, C.; Gold, L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 1990, 249, 505–510, doi:10.1126/science.2200121. 2200121
Zheng, D.; Seferos, D.S.; Giljohann, D.A.; Patel, P.C.; Mirkin, C.A. Aptamer nano-flares for molecular detection in living cells. Nano Lett 2009, 9, 3258–3261, doi:10.1021/nl901517b. 19645478
[17]
Zhang, J.; Wang, L.; Zhang, H.; Boey, F.; Song, S.; Fan, C. Aptamer-based multicolor fluorescent gold nanoprobes for multiplex detection in homogeneous solution. Small 2010, 6, 201–204, doi:10.1002/smll.200901012. 19957283
[18]
Choi, D.; Kim, M.; Park, J. Erythropoietin: Physico- and biochemical analysis. J. Chromatogr. B 1996, 687, 189–199, doi:10.1016/S0378-4347(96)00308-8.
[19]
Abellan, R.; Ventura, R.; Pichini, S.; Remacha, A.F.; Pascual, J.A.; Pacifici, R.; Giovannandrea, R.D.; Zuccaro, P.; Segura, J. Erythropoietin (EPO) as an indirect biomarker of recombinant human EPO misuse in sport. J. Pharm. Biomed. Anal 2004, 35, 1169–1177, doi:10.1016/j.jpba.2004.02.001. 15336362
[20]
Yu, B.; Cong, H.L.; Liu, H.W.; Li, Y.Z.; Liu, F. Ionene-dynamically coated capillary for analysis of urinary and recombinant human erythropoietin by capillary electrophoresis and online electrospray ionization mass spectrometry. J. Sep. Sci 2005, 28, 2390–2400, doi:10.1002/jssc.200500156. 16342807
[21]
Storhoff, J.J.; Elghanian, R.; Mucic, R.C.; Mirkin, C.A.; Letsinger, R.L. One-pot colorimetric differentiation of polynucleotides with single base imperfections using gold nanoparticle probes. J. Am. Chem. Soc 1998, 120, 1959–1964, doi:10.1021/ja972332i.
[22]
Hill, H.D.; Millstone, J.E.; Banholzer, M.J.; Mirkin, C.A. The role radius of curvature plays in thiolated oligonucleotide loading on gold nanoparticles. ACS Nano 2009, 3, 418–424, doi:10.1021/nn800726e. 19236080
Shen, R.; Guo, L.; Zhang, Z.Z.; Meng, Q.W.; Xie, J.W. Determination of rHuEPO-α by an aptamer-based affinity capillary electrophoresis-laser induced fluorescence detection method. J. Chromatogr. A 2010, 1217, 5635–5641, doi:10.1016/j.chroma.2010.06.072. 20673578
[27]
Otsuka, H.; Akiyama, Y.; Nagasaki, Y.; Kataoka, K. Quantitative and reversible lectin-induced association of gold nanoparticles modified with α-Lactosyl-ω-mercapto-poly(ethylene glycol). J. Am. Chem. Soc 2001, 123, 8226–8230, doi:10.1021/ja010437m. 11516273
[28]
Dulkeith, E.; Morteani, A.C.; Niedereichholz, T.; Klar, T.A.; Feldmann, J.; Levi, S.A.; van Veggel, F.C.; Reinhoudt, D.N.; Moller, M.; Gittins, D.I. Fluorescence quenching of dye molecules near gold nanoparticles: Radiative and nonradiative effects. Phys. Rev. Lett 2002, 89, 203002–203005, doi:10.1103/PhysRevLett.89.203002. 12443474
