Roberts
Group Research
The principal research interest of my group is in the area of bioanalytical
chemistry. Within this reach, the primary aim of our research is to
utilize modern analytical chemistry tools in solving complex biochemical issues
in the fields of chemical carcinogenesis, toxicology, and environmental
chemistry.
DNA Damage from Chemical Carcinogens
An area of research we are
pursuing is the development of chemical biomarkers for early cancer detection
and cancer risk-assessment from chemical carcinogens and other forms of
oxidative stress. Many environmental genotoxins
such as polycyclic aromatic hydrocarbons, heterocyclic and aromatic amines, and
mycotoxins are known to cause cancer even though the
“mode-of-action” is still highly debated. However, what is well
established is that many chemical carcinogens require metabolic activation to
reactive intermediates prior to covalently binding (adducting) DNA.
Incorrect (or inefficient) enzymatic removal of the carcinogen-DNA adduct may
lead to genetic mutation and cancer. From this, there is tremendous
interest in developing biomarkers from this mechanistic pathway to monitor the
dose-response relationship and polymorphic susceptibility of humans and animals
to chemical carcinogens. To measure biomarkers on a real-world level in
humans, highly sensitive and selective analytical tools are
required. Currently we are developing a biomarker method for
probing DNA damage (apurinic sites) as a dosimeter of exposure and
susceptibility to chemical carcinogens and oxidative stress. Our
current approach aims at detecting apurinic sites by combining the selectivity
provided by enzymology, high-performance liquid
chromatography, and triple-quadrupole mass
spectrometry. This project is currently being pursued in collaboration
with the
Quantum Dot Probes
Early Detection of Disease
A
second area of our research centers on utilization of highly luminescent
semiconductor nanoparticles known as quantum dots (QD) as new diagnostic
(biomonitoring) indicators of early-stage disease in living cells. In comparison to organic molecules
traditionally used as diagnostic indicators, QDs have 20 times enhanced
luminescence and 100 times enhanced stability.
In addition, a unique property of QDs is that their emission wavelength
(color) changes as a function of the QD’s size. For example with CdSe/ZnS QDs, 2.8 nm
diameter QDs luminesce green, while 3.4 nm dots luminesce yellow, and 5.6 nm dots
are red. Therefore, by conjugating
different size/color QDs with different bioconjugates (e.g., antibodies)
multiple addressing of intracellular targets can be monitored in the same assay
with the same excitation source, which is crucial for real-world analyses where
sample amounts are often limited.
However, for QDs to be practical a well-designed method needs to be
developed to allow for biocompatibility of the QDs; first by chemically
functionalizing the ZnS shell of the QD, followed by streptavidin-biotin
attachment of antibodies that will selectively target intracellular
components. Once the cellular target has
been labeled by the quantum dot conjugate, laser-induced fluorescence, confocal
microscopy, and Raman spectroscopic tools will be used for detection and
imaging. In particular, studies within
this proposed research will focus on investigation of deleterious DNA
nucleobase modifications that have the potential to ultimately lead to
cancer. Research efforts directly
related to the use of QDs to detect pre-carcinogenic DNA are nonexistent, and
abundant success can be envisioned. In
addition to DNA modifications, concurrent spectroscopic investigation of the in
vivo uptake of bioconjugated QDs will be performed. The first intracellular targets will be carcinogenic
DNA adducts of catechol estrogen quinone and dibenzo[a,l]pyrene diol epoxide
as related to breast and lung cancer, respectively.
Bioweapons Detection
A
related area of research focuses on developing new biosensor methodologies
utilizing highly luminescent quantum dots (QDs) along with modern analytical
techniques. For biosensors to be
effective they must be selective, sensitive, and have a high degree of
reproducibility. In terms of selective
recognition of the target to be sensed, we will utilize antibodies grown
against the target, and/or synthetic aptamers.
The latter is a new class of sensor molecule that is created with
randomized sequences of DNA oligomers, where the sequence that provides the
highest degree of binding to the target molecules is isolated for further
sensing applications. The primary
binding forcers of the DNA aptamers is via ligand and electrostatic
interactions. Of great importance,
aptamers have a higher degree of possible permutations than do conventional
methods of antibody productions, and, what is more, no animals are required to
generate the sensory molecule as is the case with antibodies. In terms of selectivity, we will utilize QDs of
CdSe with a ZnS shell. The uniqueness of QDs is that in comparison
to traditional organic tags for biosensing, QDs are brighter and more photostable. Both of
these features make QDs highly attractive for biosensing, and should allow for
strategies of biosensing never before achievable. However,
likely the most crucial step in developing biosensors in the stringent
requirement of a highly sensitive detection method, and, moreover, the
detection method must be able to provide reliable and unambiguous results. For this we will employ several analytical
methods of analysis to offer a multidimensional conformation of the
results. The spectroscopic and
microscopic methods we will employ are near-field optical scanning microscopy
(NSOM), scanning confocal microscopy, atomic force microscopy, Raman
spectroscopy, and fluorescence spectroscopy.
For quality control measure, and for efficacy in our bioconjugation
strategies, we will utilize capillary electrophoresis and high-performance
liquid chromatography.
Initial
targets for our biosensing strategies are in the recognition of various forms/strains
of the bacteria E. coli. Acting as a
simulant for potential biological weapons of importance, we are investigating
both aptamer and antibody approaches orthogonally in order to compare the above
criteria. In addition, intracellular
targets in E.coli (β-galactosidase) and well as the mammalian cell line of
human embryonic kidney cells (nuclear aberrations) are being investigated. Preliminary results in these areas are
encouraging.
Environmental
A third research interest is in
the area of transport (and ultimate fate) of environmental pollutants.
Many pollutants such as polycyclic aromatic hydrocarbons (PAHs),
products of incomplete combustion of carbon, are very hydrophobic, yet can be
found in a variety of aquatic and other ecological systems. It is
believed that PAHs and other hydrophobic pollutants
are transported into the environment via encapsulation in humic
acids or other vehicles such as clay and silica particles. Once in the
environment, microbial remediation measures are in place to detoxify the
pollutant. However, these processes are not completely understood, and
more information is needed about the environmental parameters, microbial
interactions, and metabolic response. Moreover, there is a growing need
for developing sensitive and selective remote-sensing tools that can facilitate
real-time monitoring of pollutants in situ. Several emerging tools such
as diode lasers, fiber optics, portable spectrometers, and molecular imprinting
make remote sensing possible. We are working towards applying these
technologies to model systems for methods development and mechanistic studies
of microbial remediation, followed by testing the applicability to local
polluted ecosystems in