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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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(a) 4-ATP Raman spectrum, on p53 bound substrate; (b) Raman spectrum of 6 Mercaptopurine (6MP) on EGFR bound substrate. For both spectra, 785 nm excitation and 25 point average was used.
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biosensors-05-00664-f005: (a) 4-ATP Raman spectrum, on p53 bound substrate; (b) Raman spectrum of 6 Mercaptopurine (6MP) on EGFR bound substrate. For both spectra, 785 nm excitation and 25 point average was used.

Mentions: The Raman spectrum of 4-ATP from a 25 point acquisition of an area in Figure 4 is shown Figure 5a. Here the spectrum is normalised to prominent 1080 cm−1 peak, corresponding to the CS stretching mode, which has been assigned previously [35]. This peak was found to be particularly responsive to antibody-antigen binding and exhibits the greatest shift according to p53 concentration. Similarly, for 6MP spectra, the prominent thiol peak at 860 cm−1 [36,37] was found to be the most sensitive to influences from protein–antibody binding and hence all spectra were normalised to this peak. Interfering species and non-specific binding have been found to mask the SERS signal from the analyte of interest. In this work, a monolayer of linker molecules is self-assembled onto the Ag surface to prevent non-specific adsorption of molecules and improve Raman efficiencies. Furthermore, by utilising large area scans, statistical reliability and reproducibility of frequency changes are improved by accounting for spot to spot intensity fluctuations typically occurring for low abundance biomolecules. If one looks at the 1080 cm−1 peak for ATP in detail, as in Figure 6a, it can be seen that there is a prominent red shift due first to the ATP, and second to binding of the antibody with further shifts due to the influence of p53-anti p53 binding.


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)

(a) 4-ATP Raman spectrum, on p53 bound substrate; (b) Raman spectrum of 6 Mercaptopurine (6MP) on EGFR bound substrate. For both spectra, 785 nm excitation and 25 point average was used.
© Copyright Policy
Related In: Results  -  Collection

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

biosensors-05-00664-f005: (a) 4-ATP Raman spectrum, on p53 bound substrate; (b) Raman spectrum of 6 Mercaptopurine (6MP) on EGFR bound substrate. For both spectra, 785 nm excitation and 25 point average was used.
Mentions: The Raman spectrum of 4-ATP from a 25 point acquisition of an area in Figure 4 is shown Figure 5a. Here the spectrum is normalised to prominent 1080 cm−1 peak, corresponding to the CS stretching mode, which has been assigned previously [35]. This peak was found to be particularly responsive to antibody-antigen binding and exhibits the greatest shift according to p53 concentration. Similarly, for 6MP spectra, the prominent thiol peak at 860 cm−1 [36,37] was found to be the most sensitive to influences from protein–antibody binding and hence all spectra were normalised to this peak. Interfering species and non-specific binding have been found to mask the SERS signal from the analyte of interest. In this work, a monolayer of linker molecules is self-assembled onto the Ag surface to prevent non-specific adsorption of molecules and improve Raman efficiencies. Furthermore, by utilising large area scans, statistical reliability and reproducibility of frequency changes are improved by accounting for spot to spot intensity fluctuations typically occurring for low abundance biomolecules. If one looks at the 1080 cm−1 peak for ATP in detail, as in Figure 6a, it can be seen that there is a prominent red shift due first to the ATP, and second to binding of the antibody with further shifts due to the influence of p53-anti p53 binding.

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