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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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Semi-log plot of the FWHM data for p53/4-ATP. Overlaid on this plot is data for the EGFR-ATP system.
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biosensors-05-00664-f007: Semi-log plot of the FWHM data for p53/4-ATP. Overlaid on this plot is data for the EGFR-ATP system.

Mentions: The FWHM (Figure 7) and peak centre (Figure 8) data are plotted as a semi-log plot (due to the large variation in concentration range) for all protein concentrations showing an exponential decrease over the range measured. A linear regression line is overlaid to show the trend in the data. We suggest that the binding between the antigen and the antibody induces structural changes in the protein moiety that in turn induce mechanical influences on the 4-ATP linker, in particular, the C–S bonding as given by the 1080 cm−1 peak in the 4-ATP spectrum. Peak broadening in Raman spectra has been previously linked to samples under stress. This effect has been studied in the materials sciences, in particular relating to silicon, graphene and carbon where induced mechanical stress and strain effects have been linked to frequency shifts in the Raman peaks of interest and a change in the FWHM [38,39,40]. In our case, and different to observations for Silicon, etc., we find that a broadening of the 1080 cm−1 peak occurs at lower protein concentrations, indicating that strain in the system is reduced as the protein concentration is lowered. We believe that such changes in strain are due to changes in steric arrangement of the protein-antibody system, and are shown by broadening of the spectra with decreasing protein concentration. Additionally, the peak centre is red shifted with decreasing protein concentration further indicating reduced mechanical stress in the system. If we overlay the data for ATP-EGFR protein we see that the assay is specific to p53; a similar response for FWHM and peak centre for EGFR-ATP is not obtained. Protein interactions and their resulting structural conformations are very complicated processes. Both p53 and EGFR are comprised of electron rich aromatic lateral chains, but p53 is smaller structurally at 53 kDa mol weight compared to 97 kDa for EGFR. This size difference coupled to the difference between the amino and diazonium binding sites for 6MP and 4-ATP respectively influences the efficiency of the linker to antibody to protein binding on the substrate. Based on our data, we believe that the interactions of linker/antibody/protein systems are very specific and only certain combinations work that result in changes in the SERS spectra. The lowest concentration used in this work compares with previous detection limits reported in previous studies using stress based nano sensors [26,27].


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)

Semi-log plot of the FWHM data for p53/4-ATP. Overlaid on this plot is data for the EGFR-ATP system.
© Copyright Policy
Related In: Results  -  Collection

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

biosensors-05-00664-f007: Semi-log plot of the FWHM data for p53/4-ATP. Overlaid on this plot is data for the EGFR-ATP system.
Mentions: The FWHM (Figure 7) and peak centre (Figure 8) data are plotted as a semi-log plot (due to the large variation in concentration range) for all protein concentrations showing an exponential decrease over the range measured. A linear regression line is overlaid to show the trend in the data. We suggest that the binding between the antigen and the antibody induces structural changes in the protein moiety that in turn induce mechanical influences on the 4-ATP linker, in particular, the C–S bonding as given by the 1080 cm−1 peak in the 4-ATP spectrum. Peak broadening in Raman spectra has been previously linked to samples under stress. This effect has been studied in the materials sciences, in particular relating to silicon, graphene and carbon where induced mechanical stress and strain effects have been linked to frequency shifts in the Raman peaks of interest and a change in the FWHM [38,39,40]. In our case, and different to observations for Silicon, etc., we find that a broadening of the 1080 cm−1 peak occurs at lower protein concentrations, indicating that strain in the system is reduced as the protein concentration is lowered. We believe that such changes in strain are due to changes in steric arrangement of the protein-antibody system, and are shown by broadening of the spectra with decreasing protein concentration. Additionally, the peak centre is red shifted with decreasing protein concentration further indicating reduced mechanical stress in the system. If we overlay the data for ATP-EGFR protein we see that the assay is specific to p53; a similar response for FWHM and peak centre for EGFR-ATP is not obtained. Protein interactions and their resulting structural conformations are very complicated processes. Both p53 and EGFR are comprised of electron rich aromatic lateral chains, but p53 is smaller structurally at 53 kDa mol weight compared to 97 kDa for EGFR. This size difference coupled to the difference between the amino and diazonium binding sites for 6MP and 4-ATP respectively influences the efficiency of the linker to antibody to protein binding on the substrate. Based on our data, we believe that the interactions of linker/antibody/protein systems are very specific and only certain combinations work that result in changes in the SERS spectra. The lowest concentration used in this work compares with previous detection limits reported in previous studies using stress based nano sensors [26,27].

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