4 Laser-Induced Synthesis and Processing of Nanoparticles …
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in metallic nanostructures (rough metal surfaces, tips, or cavities in nanoparticles
agglomerations).
In this way the electromagnetic field of the light at the surface can be greatly
enhanced under conditions of surface plasmon excitation; the amplification of both
the incident laser field and the scattered Raman field through their interaction with
the surface constitutes the electromagnetic SERS mechanism.
Applications of SERS effect to the sensor field is powered by its ability to identify chemical species and obtain structural information in a wide variety of fields
including polymer and materials science, biochemistry and biosensing, catalysis,
and electrochemistry One of the most important field in which SERS spectroscopy
has been applied is biochemistry. With the development of proteomics, it is indispensable to develop new detection methods for high-throughput protein analysis.
Most biological methods have the disadvantages of being very time-consuming,
consuming large amounts of materials, and resulting in low product yield, which
would be stumbling blocks for high-efficiency proteomics.
For fluorescence-based methods, broad emission spectra from molecular fluorophores make multiplexing impossible, and the drawback of susceptibility to
photobleaching may greatly weaken their detection limits.
In contrast, SERS-based methods for biomolecules have great advantages over
fluorescence-based methods in terms of photostability and spectral multiplexing.
They are also much more sensitive than chemiluminescence-based methods. More
and more studies have proved the great potential of SERS in protein identification
and detection of protein-ligand interactions.
Here, SERS effect has been obtained by using silver NPs in solution produced
by PLAL and the key role played by the PLAL in obtaining a large magnification is
due to the absence of by-products after the NPs formation. Definitely each Ag NP is
able to interact with the protein directly.
As an analyte, we have chosen an amyloidogenic protein called human Islet
Amyloid PolyPeptide (hIAPP). Highly aggregated hIAPP deposits are considered
the common feature of many degenerative pathologies such as Diabetes Mellitus
type II, Parkinson, and Alzheimer [39–42].
Toxicity is often associated with the irreversible formation of amyloid fibers
which are protein aggregates, rich in β-sheet structures [39]. Mounting experimental
evidences suggest that toxicity is associated with non-specific pores within the cell
membrane of the target [43, 44]. Generally, the pathway of fibril formation is mainly
investigated using kinetic fluorescent ThT assay. From such assay, it is possible to
recognize three different regions: a first region where no fluorescence is detected (the
lag-phase); a second region where fluorescence increases (growth-phase) and a final
region where fluorescence reaches a plateau. These three regions are associated with
three different states of the protein.
Specifically, the presence of unstructured aggregates (oligomeric state) have been
assigned to the first region [45], while the increase of ThT fluorescence in the second
region is attributed to a self-assembling process of the amiloidogenic proteins forming
fibrils [46].
143
in metallic nanostructures (rough metal surfaces, tips, or cavities in nanoparticles
agglomerations).
In this way the electromagnetic field of the light at the surface can be greatly
enhanced under conditions of surface plasmon excitation; the amplification of both
the incident laser field and the scattered Raman field through their interaction with
the surface constitutes the electromagnetic SERS mechanism.
Applications of SERS effect to the sensor field is powered by its ability to identify chemical species and obtain structural information in a wide variety of fields
including polymer and materials science, biochemistry and biosensing, catalysis,
and electrochemistry One of the most important field in which SERS spectroscopy
has been applied is biochemistry. With the development of proteomics, it is indispensable to develop new detection methods for high-throughput protein analysis.
Most biological methods have the disadvantages of being very time-consuming,
consuming large amounts of materials, and resulting in low product yield, which
would be stumbling blocks for high-efficiency proteomics.
For fluorescence-based methods, broad emission spectra from molecular fluorophores make multiplexing impossible, and the drawback of susceptibility to
photobleaching may greatly weaken their detection limits.
In contrast, SERS-based methods for biomolecules have great advantages over
fluorescence-based methods in terms of photostability and spectral multiplexing.
They are also much more sensitive than chemiluminescence-based methods. More
and more studies have proved the great potential of SERS in protein identification
and detection of protein-ligand interactions.
Here, SERS effect has been obtained by using silver NPs in solution produced
by PLAL and the key role played by the PLAL in obtaining a large magnification is
due to the absence of by-products after the NPs formation. Definitely each Ag NP is
able to interact with the protein directly.
As an analyte, we have chosen an amyloidogenic protein called human Islet
Amyloid PolyPeptide (hIAPP). Highly aggregated hIAPP deposits are considered
the common feature of many degenerative pathologies such as Diabetes Mellitus
type II, Parkinson, and Alzheimer [39–42].
Toxicity is often associated with the irreversible formation of amyloid fibers
which are protein aggregates, rich in β-sheet structures [39]. Mounting experimental
evidences suggest that toxicity is associated with non-specific pores within the cell
membrane of the target [43, 44]. Generally, the pathway of fibril formation is mainly
investigated using kinetic fluorescent ThT assay. From such assay, it is possible to
recognize three different regions: a first region where no fluorescence is detected (the
lag-phase); a second region where fluorescence increases (growth-phase) and a final
region where fluorescence reaches a plateau. These three regions are associated with
three different states of the protein.
Specifically, the presence of unstructured aggregates (oligomeric state) have been
assigned to the first region [45], while the increase of ThT fluorescence in the second
region is attributed to a self-assembling process of the amiloidogenic proteins forming
fibrils [46].