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Sensing of p53 and EGFR Biomarkers Using High Efficiency SERS Substrates.

Owens P, Phillipson N, Perumal J, O'Connor GM, Olivo M - Biosensors (Basel) (2015)

Bottom Line: In this paper we describe a method for the determination of protein concentration using Surface Enhanced Raman Resonance Scattering (SERRS) immunoassays.We use two different Raman active linkers, 4-aminothiophenol and 6-mercaptopurine, to bind to a high sensitivity SERS substrate and investigate the influence of varying concentrations of p53 and EGFR on the Raman spectra.Perturbations in the spectra are due to the influence of protein-antibody binding on Raman linker molecules and are attributed to small changes in localised mechanical stress, which are enhanced by SERRS.

View Article: PubMed Central - PubMed

Affiliation: Centre for Microscopy and Imaging, National University Ireland, University Road, Galway, Ireland. peter.owens@nuigalway.ie.

ABSTRACT
In this paper we describe a method for the determination of protein concentration using Surface Enhanced Raman Resonance Scattering (SERRS) immunoassays. We use two different Raman active linkers, 4-aminothiophenol and 6-mercaptopurine, to bind to a high sensitivity SERS substrate and investigate the influence of varying concentrations of p53 and EGFR on the Raman spectra. Perturbations in the spectra are due to the influence of protein-antibody binding on Raman linker molecules and are attributed to small changes in localised mechanical stress, which are enhanced by SERRS. These influences are greatest for peaks due to the C-S functional group and the Full Width Half Maximum (FWHM) was found to be inversely proportional to protein concentration.

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Functionalization of a Ag nanopillared substrate with linker and covalently bound antibody. With increasing amounts of target antigen, we observe a shift in the CS peak to longer wavenumbers and a corresponding decrease in the FWHM, resulting from increased mechanical stress in the system.
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biosensors-05-00664-f002: Functionalization of a Ag nanopillared substrate with linker and covalently bound antibody. With increasing amounts of target antigen, we observe a shift in the CS peak to longer wavenumbers and a corresponding decrease in the FWHM, resulting from increased mechanical stress in the system.

Mentions: Substrates were first rinsed gently in 100% ethanol, as an initial cleaning step. These substrates were then incubated in either 4-ATP or 6-MP (1 mM) for one hour. The substrates were then rinsed 5 times in 100% ethanol to remove the excess/unbound linker molecules. One substrate with linker molecule only was set aside at this point. 171 mM and 427.5 mM solutions of EDC and NHS were prepared separately in 0.01 M PBS. 20 µL of Antibody (either anti-p53 or anti-EGFR) solution (0.5 mg/mL) was added to 0.5 mL of EDC and 0.5 mL of NHS. The mixture was placed under vortex for 5 min. The substrates were then covered with the EDC/NHS/Antibody mixture and allowed to incubate for 2 h. The purpose of EDC was to activate the carboxyl terminal on the surface of the antibody structure. NHS greatly improves the coupling efficiency of the reaction. After 2 h the bioconjugated substrates (antibody covalently attached to the linker-coated Ag surface) were rinsed with PBS to remove unbound antibody solution. The reaction was then blocked with EDC/NHS activated Glycine (1 mM Glycine made up in 50:50 by volume mixture of EDC/NHS) and left overnight. The substrates were then rinsed with PBS and at this point an antibody only substrate was set aside from the others. A number of concentrations of protein were prepared by serial dilution and a 20 µL of each protein sample was placed on top of the antibody conjugated substrate and left to incubate for 1 h. After 1 h the substrates were rinsed in PBS buffer to remove the excess unbound protein and Raman spectra were measured immediately. All parts of this protocol were performed at room temperature. A schematic for the substrate functionalisation is given in Figure 2. The model protein-antibody systems measured are shown in Table 1.


Sensing of p53 and EGFR Biomarkers Using High Efficiency SERS Substrates.

Owens P, Phillipson N, Perumal J, O'Connor GM, Olivo M - Biosensors (Basel) (2015)

Functionalization of a Ag nanopillared substrate with linker and covalently bound antibody. With increasing amounts of target antigen, we observe a shift in the CS peak to longer wavenumbers and a corresponding decrease in the FWHM, resulting from increased mechanical stress in the system.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4697139&req=5

biosensors-05-00664-f002: Functionalization of a Ag nanopillared substrate with linker and covalently bound antibody. With increasing amounts of target antigen, we observe a shift in the CS peak to longer wavenumbers and a corresponding decrease in the FWHM, resulting from increased mechanical stress in the system.
Mentions: Substrates were first rinsed gently in 100% ethanol, as an initial cleaning step. These substrates were then incubated in either 4-ATP or 6-MP (1 mM) for one hour. The substrates were then rinsed 5 times in 100% ethanol to remove the excess/unbound linker molecules. One substrate with linker molecule only was set aside at this point. 171 mM and 427.5 mM solutions of EDC and NHS were prepared separately in 0.01 M PBS. 20 µL of Antibody (either anti-p53 or anti-EGFR) solution (0.5 mg/mL) was added to 0.5 mL of EDC and 0.5 mL of NHS. The mixture was placed under vortex for 5 min. The substrates were then covered with the EDC/NHS/Antibody mixture and allowed to incubate for 2 h. The purpose of EDC was to activate the carboxyl terminal on the surface of the antibody structure. NHS greatly improves the coupling efficiency of the reaction. After 2 h the bioconjugated substrates (antibody covalently attached to the linker-coated Ag surface) were rinsed with PBS to remove unbound antibody solution. The reaction was then blocked with EDC/NHS activated Glycine (1 mM Glycine made up in 50:50 by volume mixture of EDC/NHS) and left overnight. The substrates were then rinsed with PBS and at this point an antibody only substrate was set aside from the others. A number of concentrations of protein were prepared by serial dilution and a 20 µL of each protein sample was placed on top of the antibody conjugated substrate and left to incubate for 1 h. After 1 h the substrates were rinsed in PBS buffer to remove the excess unbound protein and Raman spectra were measured immediately. All parts of this protocol were performed at room temperature. A schematic for the substrate functionalisation is given in Figure 2. The model protein-antibody systems measured are shown in Table 1.

Bottom Line: In this paper we describe a method for the determination of protein concentration using Surface Enhanced Raman Resonance Scattering (SERRS) immunoassays.We use two different Raman active linkers, 4-aminothiophenol and 6-mercaptopurine, to bind to a high sensitivity SERS substrate and investigate the influence of varying concentrations of p53 and EGFR on the Raman spectra.Perturbations in the spectra are due to the influence of protein-antibody binding on Raman linker molecules and are attributed to small changes in localised mechanical stress, which are enhanced by SERRS.

View Article: PubMed Central - PubMed

Affiliation: Centre for Microscopy and Imaging, National University Ireland, University Road, Galway, Ireland. peter.owens@nuigalway.ie.

ABSTRACT
In this paper we describe a method for the determination of protein concentration using Surface Enhanced Raman Resonance Scattering (SERRS) immunoassays. We use two different Raman active linkers, 4-aminothiophenol and 6-mercaptopurine, to bind to a high sensitivity SERS substrate and investigate the influence of varying concentrations of p53 and EGFR on the Raman spectra. Perturbations in the spectra are due to the influence of protein-antibody binding on Raman linker molecules and are attributed to small changes in localised mechanical stress, which are enhanced by SERRS. These influences are greatest for peaks due to the C-S functional group and the Full Width Half Maximum (FWHM) was found to be inversely proportional to protein concentration.

Show MeSH