J. Bukowska and P. Piotrowski
40
support. It has been proved that electromagnetic enhancement decays with distance
from the surface, but is still measurable at about 10 nm [101]. therefore, in case of
very large biomolecules, spectral information may be confined to some fragments
of the molecule directly interacting with the metal nanoparticle or in a short distance
from the metal surface. this specificity and selectivity of the SERS spectra cause
that in the case of large bio-molecules some fragments of the molecules are not
“seen” in the spectra. on the other hand, this would enable characterization of the
interaction between metal surfaces and biological molecule and determination of its
orientation at the surface.
Also tERS spectra of peptides have been successfully recorded. tERS studies of peptide adsorption on metal surface (gold nanoplate) were carried out on a
model molecule of oxidized glutathione and the structure of the peptide on the Au
surface was determined [102]. the uniformity of the spectra measured at several
points along a line across the gold substrate indicated the consistent orientation of
the peptide. this result was regarded as crucial for possibility of characterizing and
sequencing peptides with the help of this method.
Another problem related to SERS biosensing of proteins is their denaturation
upon contact with metal nanoparticles. thus the biocompatibility of the metal surface must be carefully controlled. the most simple way is to cover the nanoparticle
surface with a self-assembled monolayer (SAm) [103] that prevents direct contact
between the protein and the metal surface. there is a great variety of SAms that
could be used to prevent denaturation of the protein at the metal surface. however,
the most optimal ones have to effectively bind the protein e.g. through electrostatic
or covalent interaction, with simultaneous preservation of the protein properties and
biological activity. The most popular SAMs are built of ω-substituted alkanethiols
hS(Ch 2 ) n X, which form very stable metal-sulfur chemical bond. they create an
interface with a well-defined composition and structure and with easily controlled
properties achieved through the selective use of X groups. Among alkanethiols
with negatively charged terminal groups, carboxylate-functionalized ones are the
most frequently employed [104–109]. Sulfonate group is an example of an anionic
group that can be used alternatively [110]. A great deal of work has been devoted
to design biocompatible surfaces that ensure preserving the native structure of immobilized proteins. In the case of redox protein, communication between protein
and electrode, which enables effective electron transfer, is also a major challenge.
the case of cytochrome c (Cc) is a good illustration of the advantages for using
SERS in studies of redox proteins. Numerous reports have been published on SERS
of Cc immobilized on a silver or gold support modified with various monolayers
[105, 108–112]. to improve intensity of the spectra, they are recorded using excitation laser line in resonance with molecular transition in Cc chromophore (SERRS
spectra). the SERRS spectrum of Cc not only allows identifying the protein immobilized on the surface, but also makes it easy to determine the oxidation state and
spin configuration of iron in heme groups, because Raman bands are well known
markers of these properties [113, 114]. moreover, sensitivity of the SERS spectrum
to the orientation of the adsorbed molecules with respect to the surface causes that
relative orientation of the heme plane may be determined by using so called surface
40
support. It has been proved that electromagnetic enhancement decays with distance
from the surface, but is still measurable at about 10 nm [101]. therefore, in case of
very large biomolecules, spectral information may be confined to some fragments
of the molecule directly interacting with the metal nanoparticle or in a short distance
from the metal surface. this specificity and selectivity of the SERS spectra cause
that in the case of large bio-molecules some fragments of the molecules are not
“seen” in the spectra. on the other hand, this would enable characterization of the
interaction between metal surfaces and biological molecule and determination of its
orientation at the surface.
Also tERS spectra of peptides have been successfully recorded. tERS studies of peptide adsorption on metal surface (gold nanoplate) were carried out on a
model molecule of oxidized glutathione and the structure of the peptide on the Au
surface was determined [102]. the uniformity of the spectra measured at several
points along a line across the gold substrate indicated the consistent orientation of
the peptide. this result was regarded as crucial for possibility of characterizing and
sequencing peptides with the help of this method.
Another problem related to SERS biosensing of proteins is their denaturation
upon contact with metal nanoparticles. thus the biocompatibility of the metal surface must be carefully controlled. the most simple way is to cover the nanoparticle
surface with a self-assembled monolayer (SAm) [103] that prevents direct contact
between the protein and the metal surface. there is a great variety of SAms that
could be used to prevent denaturation of the protein at the metal surface. however,
the most optimal ones have to effectively bind the protein e.g. through electrostatic
or covalent interaction, with simultaneous preservation of the protein properties and
biological activity. The most popular SAMs are built of ω-substituted alkanethiols
hS(Ch 2 ) n X, which form very stable metal-sulfur chemical bond. they create an
interface with a well-defined composition and structure and with easily controlled
properties achieved through the selective use of X groups. Among alkanethiols
with negatively charged terminal groups, carboxylate-functionalized ones are the
most frequently employed [104–109]. Sulfonate group is an example of an anionic
group that can be used alternatively [110]. A great deal of work has been devoted
to design biocompatible surfaces that ensure preserving the native structure of immobilized proteins. In the case of redox protein, communication between protein
and electrode, which enables effective electron transfer, is also a major challenge.
the case of cytochrome c (Cc) is a good illustration of the advantages for using
SERS in studies of redox proteins. Numerous reports have been published on SERS
of Cc immobilized on a silver or gold support modified with various monolayers
[105, 108–112]. to improve intensity of the spectra, they are recorded using excitation laser line in resonance with molecular transition in Cc chromophore (SERRS
spectra). the SERRS spectrum of Cc not only allows identifying the protein immobilized on the surface, but also makes it easy to determine the oxidation state and
spin configuration of iron in heme groups, because Raman bands are well known
markers of these properties [113, 114]. moreover, sensitivity of the SERS spectrum
to the orientation of the adsorbed molecules with respect to the surface causes that
relative orientation of the heme plane may be determined by using so called surface