An optical aptamer-based detection system label free appears as highly efficient device with enormous potential. Unfortunately such systems are still immature compared to immunoassays, reflecting the limited availability of aptamer types and the relatively poor knowledge of surface-immobilization technologies for aptamers [1]. We present the preparation of an aptamer terminated sensing surface, allowing a fast and cheap system for assay of analytes unlimited by size and tossicity. The designed platform is suitable for an aptamer-based microfluidic device, coupled with a Surface Enhanced Raman Spectroscopy (SERS). To accomplish this, a multi-step deposition sequence was performed: as a start, a mixed self assembled monolayer (SAM) containing a binary mixture of biotinylated alkylthiol (BAT) [2]. with the capacity to graft neutravidin proteins and diluent methyl-terminated alkylthiol, was prepared on a gold thin film. The chemical and electronic structure of the mixed SAMs was investigated by X-ray Photoelectron Spectroscopy and IRRAS (Infrared Spectroscopy in Reflection Mode) [3]. 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, taking usually place at the isolectric point (~ 6) of neutravidin [4, 5]. The sample morphologies were observed by atom force microscopy (AFM), the size was determined by Dynamic Light Scattering and the concentration of gold species in the colloid was monitored by UV-vis spectra [6, 7]. In the ending step, a sensing aptamer (i. e. thrombin-binding aptamer) is bond to the surface through the avidin-biotin linkage. The resulting SERS changes involved was observed when the target molecule (i.e. thrombin) interacts with its own aptamer [8]. This aptamer terminated sensing surface is fitted for affinity based microfluidic devices, providing controlled fluid transport, rapid affinity assay and cost saving advantages over conventional methods for biological and medical applications. Through such design, the sensing surface overcomes the sandwich structure, formed between the immobilized aptamer, the protein target and a secondary aptamer bound to the Raman probe (i.e. gold nanoparticles ), usually realized in SERS aptasensors, limited by consuming and inconvenient handling step [9]. [1] Song S.; Wang L.; Li J.; Fan C.; Zhao J," Trends in Analytical Chemistry, 27, 2, 2008. [2] Ptrats-Alfonso E., Garcia-Martin F.,Bayo N., Cruz L.J., Pla-Roca M., Samitier J., Errachid A., Albericio F., Tetrahedron, 62, 6876-6881, 2006 [3] Nelson K.E., Gamble L., et al., Langmuir, 17, 2807-2816, 2001. [4] Grabar, K.C et al., Anal. Chem., 67, 735-743, 1995. [5] Morris R.E. and Saelinger C.B.., J.Histochemistry and Cytochemistry, 32, 124-128, 1984 [6] Kimling J. et al., J.Phys.Chem B 110, 15700-15707, 2006 [7] Ji X. et al., J.Am.Chem.Soc. 129, 13939-13948, 2007 [8] Nie S.; and Emory S. R., Science 275, 1102, 1997. [9]. Sassolas A, Blum L. J., Leca-Bouvier B. D., Biosens Bioelectron., on line, 2011
Integrated approach to a sensitive platform for SERS aptamer biosensor, suitable for a microfluidic device
Quagliano LG;Foglia S
2012
Abstract
An optical aptamer-based detection system label free appears as highly efficient device with enormous potential. Unfortunately such systems are still immature compared to immunoassays, reflecting the limited availability of aptamer types and the relatively poor knowledge of surface-immobilization technologies for aptamers [1]. We present the preparation of an aptamer terminated sensing surface, allowing a fast and cheap system for assay of analytes unlimited by size and tossicity. The designed platform is suitable for an aptamer-based microfluidic device, coupled with a Surface Enhanced Raman Spectroscopy (SERS). To accomplish this, a multi-step deposition sequence was performed: as a start, a mixed self assembled monolayer (SAM) containing a binary mixture of biotinylated alkylthiol (BAT) [2]. with the capacity to graft neutravidin proteins and diluent methyl-terminated alkylthiol, was prepared on a gold thin film. The chemical and electronic structure of the mixed SAMs was investigated by X-ray Photoelectron Spectroscopy and IRRAS (Infrared Spectroscopy in Reflection Mode) [3]. 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, taking usually place at the isolectric point (~ 6) of neutravidin [4, 5]. The sample morphologies were observed by atom force microscopy (AFM), the size was determined by Dynamic Light Scattering and the concentration of gold species in the colloid was monitored by UV-vis spectra [6, 7]. In the ending step, a sensing aptamer (i. e. thrombin-binding aptamer) is bond to the surface through the avidin-biotin linkage. The resulting SERS changes involved was observed when the target molecule (i.e. thrombin) interacts with its own aptamer [8]. This aptamer terminated sensing surface is fitted for affinity based microfluidic devices, providing controlled fluid transport, rapid affinity assay and cost saving advantages over conventional methods for biological and medical applications. Through such design, the sensing surface overcomes the sandwich structure, formed between the immobilized aptamer, the protein target and a secondary aptamer bound to the Raman probe (i.e. gold nanoparticles ), usually realized in SERS aptasensors, limited by consuming and inconvenient handling step [9]. [1] Song S.; Wang L.; Li J.; Fan C.; Zhao J," Trends in Analytical Chemistry, 27, 2, 2008. [2] Ptrats-Alfonso E., Garcia-Martin F.,Bayo N., Cruz L.J., Pla-Roca M., Samitier J., Errachid A., Albericio F., Tetrahedron, 62, 6876-6881, 2006 [3] Nelson K.E., Gamble L., et al., Langmuir, 17, 2807-2816, 2001. [4] Grabar, K.C et al., Anal. Chem., 67, 735-743, 1995. [5] Morris R.E. and Saelinger C.B.., J.Histochemistry and Cytochemistry, 32, 124-128, 1984 [6] Kimling J. et al., J.Phys.Chem B 110, 15700-15707, 2006 [7] Ji X. et al., J.Am.Chem.Soc. 129, 13939-13948, 2007 [8] Nie S.; and Emory S. R., Science 275, 1102, 1997. [9]. Sassolas A, Blum L. J., Leca-Bouvier B. D., Biosens Bioelectron., on line, 2011I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
