Aptamers, specific nucleic acid selected from random sequence pools, are a valid alternative to antibodies or other biomimetic receptors. Aptamer-based biosystems are still immature compared to immunoassays, which reflects the limited availability of aptamer types and for this purpose an optical aptamer-based detection systems label free appear as highly efficient devices with enormous potential. [Ellington, A.D., Szostak, J.W., 1990. Nature 346,818-822; Song S., Wanga L., Lia J., Fana C., Zhaob J., 2008 TrAC 27(2), 108-117] We approach the development of an affinity based detection system for analytes unlimited by size and tossicity, designing a nanoaptasensor sensing surface for microfluidic device, coupled with Surface Enhanced Raman Spectroscopy (SERS). Among the optical detection methods, SERS distinguishes itself with several advantages: the spectrum change brought by a single molecules, is induced by the specific interaction between aptamers and their own proteins and the spectral probe specificity is excellent in comparison to the fluorescence methods. The thrombin aptamer is the first example of ssDNA oligonucleotides that bind a target protein with unknown specificity for nucleic acids. [Bock, L.C., et al., 1992. Nature 355, 564-566] A Thrombin SERS aptasensor is usually realized by a sandwich structure, formed between the immobilized aptamer, the protein target and a secondary aptamer bound to a Raman probe (i.e. gold nanoparticles ), which requires a consuming and inconvenient handling step. [Sassolas A, Blum L. J., Leca-Bouvier B. D. 2011 Biosens Bioelectron., on line] In attempt to over come this drawback we report the preparation of a aptamer terminated sensing surface, allowing a faster and cheaper system and suitable for an analytical label free measurements, in order to perform a direct analisys of biological samples. We develop a multi-step depositional sequence: firstly was prepared a mixed self assembled monolayer (SAM) on a gold thin film, containing a binary mixture of biotinylated alkylthiol (BAT) with the capacity to graft neutravidin proteins and diluent methyl-terminated alkylthiol. The composition depth profile of the mixed SAMs was examined by angle-resolved XPS. Insight in to the order of the SAM system was provided by NEXAFS characterization [Nelson K.E., Gamble L., et al. 2001, Langmuir, 17, 2807-2816]. BAT was produced by a solid-phase synthesis, based on a resin, which protects the thiol group and prevents the formation of non-desired compounds [Prats-Alfonso E. et al. 2006 Tetrahedron 62, 6876-6881]. In a second step, the bioconjugation with an avidin-gold sol is performed. Monodispersed 5 nm gold particles were prepared in aqueous medium and covered with neutravidin, overcoming possible non-specific bindings, which are usually attribuited to the isolectric point (~ 6) of neutravidin [Grabar, K.C et al. 1995 Anal. Chem. 67, 735-743; Morris R.E. and Saelinger C.B. 1984 The journal of Histochemistry and Cytochemistry 32, 124-128]. Gold colloid was prepared by reducing tetrachloroauric(III) acid with sodium citrate controlling growth kinetics and temporal size/shape evolution of gold nanocrystal. The citrate plays a dual role as reducing agent and protection capping group and temperature and pH values of the citrate reaction drastically affect the size and quality of the particles. The morphologies was observed by atom force microscopy (AFM) and the concentration of gold species in the colloid was monitored by UV-vis spectra.[Kimling J. et al.2006 J.Phys.Chem B 110, 15700-15707; Ji X. et al. 2007 J.Am.Chem.Soc. 129, 13939-13948] In the ending step, the Aptamer is bond to the surface thought the avidin-biotin linkage. In this strategy aptamer against thrombin [5'GGT TGG TGT GGT TGG T(15)3'Biotin], marked by a guanine quadruplex (G-quadruplex), is used. The resulting SERS changes involved was observed when the target molecule (Thrombin) interacts with its own aptamer [Nie S.; and Emory S. R. 1997 Science 275, 1102].
Aptamer based SERS sensing surface for direct detection of proteins
Quagliano LG;Foglia S
2011
Abstract
Aptamers, specific nucleic acid selected from random sequence pools, are a valid alternative to antibodies or other biomimetic receptors. Aptamer-based biosystems are still immature compared to immunoassays, which reflects the limited availability of aptamer types and for this purpose an optical aptamer-based detection systems label free appear as highly efficient devices with enormous potential. [Ellington, A.D., Szostak, J.W., 1990. Nature 346,818-822; Song S., Wanga L., Lia J., Fana C., Zhaob J., 2008 TrAC 27(2), 108-117] We approach the development of an affinity based detection system for analytes unlimited by size and tossicity, designing a nanoaptasensor sensing surface for microfluidic device, coupled with Surface Enhanced Raman Spectroscopy (SERS). Among the optical detection methods, SERS distinguishes itself with several advantages: the spectrum change brought by a single molecules, is induced by the specific interaction between aptamers and their own proteins and the spectral probe specificity is excellent in comparison to the fluorescence methods. The thrombin aptamer is the first example of ssDNA oligonucleotides that bind a target protein with unknown specificity for nucleic acids. [Bock, L.C., et al., 1992. Nature 355, 564-566] A Thrombin SERS aptasensor is usually realized by a sandwich structure, formed between the immobilized aptamer, the protein target and a secondary aptamer bound to a Raman probe (i.e. gold nanoparticles ), which requires a consuming and inconvenient handling step. [Sassolas A, Blum L. J., Leca-Bouvier B. D. 2011 Biosens Bioelectron., on line] In attempt to over come this drawback we report the preparation of a aptamer terminated sensing surface, allowing a faster and cheaper system and suitable for an analytical label free measurements, in order to perform a direct analisys of biological samples. We develop a multi-step depositional sequence: firstly was prepared a mixed self assembled monolayer (SAM) on a gold thin film, containing a binary mixture of biotinylated alkylthiol (BAT) with the capacity to graft neutravidin proteins and diluent methyl-terminated alkylthiol. The composition depth profile of the mixed SAMs was examined by angle-resolved XPS. Insight in to the order of the SAM system was provided by NEXAFS characterization [Nelson K.E., Gamble L., et al. 2001, Langmuir, 17, 2807-2816]. BAT was produced by a solid-phase synthesis, based on a resin, which protects the thiol group and prevents the formation of non-desired compounds [Prats-Alfonso E. et al. 2006 Tetrahedron 62, 6876-6881]. In a second step, the bioconjugation with an avidin-gold sol is performed. Monodispersed 5 nm gold particles were prepared in aqueous medium and covered with neutravidin, overcoming possible non-specific bindings, which are usually attribuited to the isolectric point (~ 6) of neutravidin [Grabar, K.C et al. 1995 Anal. Chem. 67, 735-743; Morris R.E. and Saelinger C.B. 1984 The journal of Histochemistry and Cytochemistry 32, 124-128]. Gold colloid was prepared by reducing tetrachloroauric(III) acid with sodium citrate controlling growth kinetics and temporal size/shape evolution of gold nanocrystal. The citrate plays a dual role as reducing agent and protection capping group and temperature and pH values of the citrate reaction drastically affect the size and quality of the particles. The morphologies was observed by atom force microscopy (AFM) and the concentration of gold species in the colloid was monitored by UV-vis spectra.[Kimling J. et al.2006 J.Phys.Chem B 110, 15700-15707; Ji X. et al. 2007 J.Am.Chem.Soc. 129, 13939-13948] In the ending step, the Aptamer is bond to the surface thought the avidin-biotin linkage. In this strategy aptamer against thrombin [5'GGT TGG TGT GGT TGG T(15)3'Biotin], marked by a guanine quadruplex (G-quadruplex), is used. The resulting SERS changes involved was observed when the target molecule (Thrombin) interacts with its own aptamer [Nie S.; and Emory S. R. 1997 Science 275, 1102].I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
