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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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Generalised schematic showing methods for producing SERS signals from target analytes. The direct method uses metal nanoparticles bound to Raman reporters which are then bound to a specific binding molecule, i.e., an antibody. These can then be used to visualise nanoparticle uptake by cells invitro for example. The second approach uses an immunolabelling strategy that uses SERS to enhance the raman signal directly from biomarker-antibody interactions.
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biosensors-05-00664-f001: Generalised schematic showing methods for producing SERS signals from target analytes. The direct method uses metal nanoparticles bound to Raman reporters which are then bound to a specific binding molecule, i.e., an antibody. These can then be used to visualise nanoparticle uptake by cells invitro for example. The second approach uses an immunolabelling strategy that uses SERS to enhance the raman signal directly from biomarker-antibody interactions.

Mentions: Numerous applications of SERS in biological assays have been reported ranging from multimodal photodynamic and theranostic probes [5,6,7,8], Imaging [9], spectral analysis [10,11,12] to NIR probes capable of multiplexed detection [7,13]. Of the many methods in the literature that specifically deal with bioanalysis, two general approaches are followed. The first uses a direct labelling approach where the target analyte is mixed with a colloid of SERS optimised nanotags which then act to enhance the Raman signal of the Raman Reporter [14,15,16,17,18,19,20]. Significant developments have been made in the design and synthesis of bioconjugated nanotags, with a high degree of control on size and shape which control the plasmon frequency and Raman cross section. A number of potential bioconjugation schemes exist for SERS nanotags. One scheme involves direct immobilisation of Raman reporter molecules and antibodies onto the surface of the metal (typically gold) nanoparticles. Another approach involves conjugation of the antibody via a spacer molecule such as Polyethylene Glycol [20]. The distance to the metal nanoparticle is critical and only those labelled moieties close enough will experience significant SERS enhancement. However, reproducibility and quantification of the Raman responses from this approach can be problematic. The second approach utilises immunoassays that rely on the recognition of biomarkers with antibodies that are conjugated to SERS substrates [1,21,22,23,24]. While direct label-free approaches are more convenient than extrinsic SERS labelling, the design and synthesis of stable and well-defined metal array assemblies is still a significant challenge. In the case of trace protein analysis, low concentration high molecular weight biomolecules have limited SERS sensitivity and selectivity can be hampered by a high degree of overlap of Raman bands [25]. A comparison of these general approaches is given in Figure 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)

Generalised schematic showing methods for producing SERS signals from target analytes. The direct method uses metal nanoparticles bound to Raman reporters which are then bound to a specific binding molecule, i.e., an antibody. These can then be used to visualise nanoparticle uptake by cells invitro for example. The second approach uses an immunolabelling strategy that uses SERS to enhance the raman signal directly from biomarker-antibody interactions.
© Copyright Policy
Related In: Results  -  Collection

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

biosensors-05-00664-f001: Generalised schematic showing methods for producing SERS signals from target analytes. The direct method uses metal nanoparticles bound to Raman reporters which are then bound to a specific binding molecule, i.e., an antibody. These can then be used to visualise nanoparticle uptake by cells invitro for example. The second approach uses an immunolabelling strategy that uses SERS to enhance the raman signal directly from biomarker-antibody interactions.
Mentions: Numerous applications of SERS in biological assays have been reported ranging from multimodal photodynamic and theranostic probes [5,6,7,8], Imaging [9], spectral analysis [10,11,12] to NIR probes capable of multiplexed detection [7,13]. Of the many methods in the literature that specifically deal with bioanalysis, two general approaches are followed. The first uses a direct labelling approach where the target analyte is mixed with a colloid of SERS optimised nanotags which then act to enhance the Raman signal of the Raman Reporter [14,15,16,17,18,19,20]. Significant developments have been made in the design and synthesis of bioconjugated nanotags, with a high degree of control on size and shape which control the plasmon frequency and Raman cross section. A number of potential bioconjugation schemes exist for SERS nanotags. One scheme involves direct immobilisation of Raman reporter molecules and antibodies onto the surface of the metal (typically gold) nanoparticles. Another approach involves conjugation of the antibody via a spacer molecule such as Polyethylene Glycol [20]. The distance to the metal nanoparticle is critical and only those labelled moieties close enough will experience significant SERS enhancement. However, reproducibility and quantification of the Raman responses from this approach can be problematic. The second approach utilises immunoassays that rely on the recognition of biomarkers with antibodies that are conjugated to SERS substrates [1,21,22,23,24]. While direct label-free approaches are more convenient than extrinsic SERS labelling, the design and synthesis of stable and well-defined metal array assemblies is still a significant challenge. In the case of trace protein analysis, low concentration high molecular weight biomolecules have limited SERS sensitivity and selectivity can be hampered by a high degree of overlap of Raman bands [25]. A comparison of these general approaches is given in Figure 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