Advanced biophysical approaches to nano-bio-sensing: potential environmental and biomedical applications based on p53-azurin interaction
Author(s)
Domenici, Fabio
Date Issued
July 18, 2011
Type
Doctoral Thesis
Abstract
The availability of advanced methodologies, especially suited for sensitive and reliable
screening of indicators of both environmental and human pathologies, is a crucial element
for an effective early detection. For this reason, nanoscience and nanotechnology research
combines efforts to improve environmental and biomedical diagnostic, often resorting to
advanced biophysical approaches which have the potential to overcome detection limits of
traditional methods. In particular, signal amplification methods, such as Surface-Enhanced
Raman Scattering (SERS) and Surface Plasmon Resonance (SPR), are now entrenching in
modern environmental and biomedical fields. By taking advantage of the peculiar optical
properties of metal structures at the nanoscale, in fact, they offer the possibility of
developing ultrasensitive nano-bio affinity assays.
Given the importance of health and safety in human society, considerable resources have
been and are being expended in efforts to identify and classify human carcinogens, in order
to reduce the risk of cancer. As the tumour suppressor protein p53 is directly involved in
the chain of biochemical events that follow genotoxic damage, some researchers have tried
to incorporate this information into testing protocols, evidencing a close correlation
between the DNA damaging mechanism and the p53 induction pattern of a given chemical.
Hence, p53 has a huge potential as a biological indicator of environmental conditions
which threaten human health, and the novel biophysical approaches offer the adequate
features to turn such p53 potential into novel, more accurate, quick and efficient methods
to early screen substances for carcinogenicity.
In this connection, we have explored the analytical potential of advanced biophysical
methods in an attempt to more deeply understand the improvement they can provide to
traditional bio-affinity assays; successively, by means of these approaches, we have tried to
reduce the detection threshold of the tumour marker p53.
To probe the presence of p53, we have used the blue copper protein Azurin (Az), whose
physical interaction with the tumour suppressor has been recently proposed in connection
with its demonstrated anticancer activity. By SPR, we have analysed the Az-p53 binding
kinetics and ascertained the strong stability of their specific interaction even in presence of
the protein Mdm2, which is one of the main regulators of p53; then we have exploited the
specific interaction between Az and p53 to realize a novel SERS-based immunoassay
detection method, made up by the synergic combination of SERS methodology and
bioaffinity assays. In this way it has been possible to strongly reduce the current detection
limit for p53 down to levels which are appealing also for screening in human serum.
The importance of investigating the Az-p53 interaction is not limited to analytical
detection aspects. Indeed our SPR measurements performed on the proteins p53, Mdm2
and Az indicate that these three proteins are likely engaged in a ternary structure where the
interaction of Az with p53 modulates the Mdm2-p53 binding kinetics. Since the tumour
repression activity of p53 is mainly down-regulated by Mdm2, the latter result could
provide an useful insight into the possibility of designing new anticancer drugs.
Additional information
Dottorato di ricerca in Scienze ambientali
Subjects
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