The main thrust of research performed in our laboratory is to develop new techniques for the label-free detection of biochemical events at the liquid (especially blood, serum and plasma) -solid interface. In terms of perspective, the aim is to generate new technologies for application in bioanalytical chemistry and detection science, notably in the areas of clinical diagnostics, biocompatibility and nanomedicine. The work is highly interdisciplinary in character and involves activities ranging from the design and construction of new instruments to theoretical calculations. Research staff possess multidisciplinary expertise including areas such as analytical chemistry, basic electronics, surface chemistry, biochemistry, computational methods, and materials science.
Ultra-high-frequency acoustic wave detection in bioanalytical chemistry (EMPAS)
Work in this area is divided into several categories, all strongly interrelated. First, we are employing transverse shear-wave sensors to detect, in label-free format, a variety of important biochemical systems. A key feature of our detection research is the design and construction of an ultra-high frequency electromagnetic excitation configuration for application to the study of biological species at interfaces and in the field of clinical diagnostics by acoustic physics (electromagnetic piezoelectric acoustic sensor, EMPAS). Here, a specially fabricated planar spiral coil is brought close to a piezoelectric disk which, in turn, is incorporated into a flow-through stream. The coil sets up a very high frequency electromagnetic field, which couples into the piezoelectric tensor of the disk. Such an arrangement allows acoustic physics to be operated at frequencies of 1 GHz, and is, therefore, extremely sensitive from an analytical chemical standpoint. This revolutionary instrument offers huge advances in the detection of biochemical events because of the high frequency, tunability and advantageous use of quartz as a substrate for surface chemistry. Current research also involves use of the new piezoelectric material, aluminum nitride, in addition to devices based on lithium niobate.
Finally, with respect to acoustic physics, the group is also pioneering the examination of theoretical aspects of the propagation of acoustic waves from devices out into liquids. We have observed the phenomenon of molecular slip at the surfaces of TSM devices when being operated in liquids. An understanding of this effect is crucial to the whole field of acoustic wave sensor technology as employed in many applications. Specifically, we are developing a molecular dynamics simulation to characterize multiple slip layers and shear stresses at the solid-liquid interface. A goal is to use results of these calculations to successfully predict the tertiary structure of surface-attached biomolecules from acoustic network parameters.
Interaction of surfaces of biosensors and medical devices with biological fluids:
non-specific adsorption, anti-fouling, and biocompatibility
A crucial aspect of biosensor technology, which shares many common features with the field of biocompatibility, is the unwanted adsorption, or fouling, by biological species when devices are immersed in blood, serum, and plasma. In our work, we are both interested in methods to prevent such fouling and in novel methods for the attachment of proteins and nucleic acids to various materials with applications in both fields. This involves the synthesis of new silanes and other linker moieties with a view to not only probe binding for biosensor development but also the tandem avoidance of non-specific adsorption from entities present in biological fluids. Research with linker molecules is centered on the physical chemistry of probe-diluent ratio, probe biological activity, and adsorption phenomena. Recently we have demonstrated that a 0.5 nm silane film can be successfully applied to biosensor surface in order to act in an anti-fouling capacity. The nature of this film is being studied by techniques such neutron reflectometry and molecular dynamic calculation with the emphasis on the role played by interfacial water molecules.
With respect to biocompatibility, we are researching the surface modification of polymeric materials used in medical bypass surgery and renal dialysis. Micro-clots are known to form on the surfaces of these materials during medical procedures. These clots can have serious medical consequences in terms of cognitive disability. The analogous ultra-thin films employed for our biosensor work have been shown to be extremely successful in the prevention of thrombi. Experiments in this case involve the real-time measurement of aggregation of platelets from human blood using confocal fluorescence microscopy (in collaboration with the Keenan Research Centre of St. Michael’s Hospital).
The group is also working on the surface modification of stainless steel stents. Stents are expandable cylindrical scaffolds that are implanted within narrowed arteries. They are typically constructed from medical grade stainless steel and can be implanted as bare metal stents or as drug eluting stents depending on patient candidacy. The goal of our research is to develop a biologically active and biocompatible coating for stainless steel stents that will reduce both neointimal hyperplasia and the physiological attack of the stent. An Anti-VEGRF antibody that binds endothelial progenitor cells (EPCs) is being covalently immobilized to the steel surface through coupling with linker silane molecules. These cells are present in the blood stream and have been recently found to participate in the re-endothelialization of damaged arterial vessels and the inhibition of neointimal formation.
Early-stage detection of ovarian cancer
Ovarian cancer is associated with tumors that form in the tissue of the ovary, a component of the female reproductory system. Ovarian cancers are either ovarian epithelial carcinomas (cancer that begins in the cells on the surface of the ovary) or malignant germ cell tumors (cancer that begins in egg cells). Such cells can shed or spread to other organs via the process generally understood to be the mechanism of metastasis. It is anticipated to kill 14,000 women in the US in 2015 with over 20,000 new cases being diagnosed. It is often referred to as the “silent” killer since early symptoms are not radically different from certain normal conditions. The cause of the disease is unknown and is difficult to detect, with the existing CA125 assay widely considered to be inadequate. Accordingly, there is an urgent need for an analytical test that is both sensitive and selective and can be incorporated at low cost into a high-throughput instrumental system to be available in the clinical diagnostic laboratory.
Our research involves the use of 2 promising biomarkers that we propose to detect via a biosensor format, heat shock protein 10 (HSP10) and lysophosphatidic acid (LPA). The former has been shown to be present in all Stage III patients, and LPA levels were found to be elevated in 90% of stage I ovarian cancer patients (LPA blood concentrations in the several micro-molar range) , and 100% of later stage patients. In terms of a probe for HSP10, we employ aptamer technology, where a DNA molecule that can selectively bind the protein has been produced by the standard SELEX protocol. We have achieved expression of the protein in our laboratory, and a modest construct that binds the protein has been generated. This is being refined through production of a number of variants in order to enhance binding to the Kd level of nano-molar. A number of such constructs will be imposed on the biosensor using our proprietary attachment and antifouling protocols.
We have initiated research on LPA detection using gelsolin-actin chemistry. Using fluorescence spectroscopy we have demonstrated that gelsolin, a large protein of 782 amino acids and 6 domains, binds LPA with a Kd value of 6 nM with only the first 3 domains of gelsolin being necessary for both actin and LPA binding, thus reducing the size and complexity of the probe. This binding capability is ideal for detection by a simple spectrofluorometric assay and/or by biosensor. We are imposing the protein-actin complex on the biosensor with a view to monitoring the removal of actin via LPA binding from LPA spiked serum.
Theranostics: detection and removal of endotoxin from biological fluid
For many years the traditional approach in medicine has been to diagnose a disease condition which is often followed by drug and other treatments in order to ameliorate the factors that cause such a condition. However, many of the troubling diseases of our time are highly heterogeneous in their expression, which results in the fact that a number of drug-based therapies are only effective for certain sub-populations of the afflicted. These factors have resulted in the appearance of modern-day “personalized medicine” and one rapidly developing aspect of this approach is theranostic technology, that is, the application of therapy in intimate connection with clinical diagnostics.
In our work, we are producing an off-line biosensor assay of lipopolysaccharide (endotoxin or LPS) for coupling with a cartridge which is designed to remove this molecule from patient blood via hemoperfusion. LPS is responsible for the medical condition of endotoxemia or sepsis and is a major trigger of shock, possibly leading to death. The biosensor is based on the afore-mentioned EMPAS system which incorporates a polymyxin surface-attached probe for operation in tandem with our proprietary anti-fouling chemistry. We are also working on a completely new cartridge for the on-line removal of LPS which involves the use of micro-bead technology and immobilized polymyxin.
High resolution scanning Kelvin nanoprobe technology
In our research into the Kelvin probe area we have constructed a unique tip-based instrument that is not only capable of the measurement of Kelvin signals at 100-nm spatial resolution, but that can also be employed to produce an important tandem topographical map of the surface under study.
This technique, interestingly, had its genesis with the work of Kelvin in the late 1800s. When two plates that are made of different metals are allowed to approach an electrometer connected in the circuit shows the presence of a voltage. This result is associated with the equalization of the Fermi levels, which gives rise to a surface charge and a potential difference (contact potential). Since work function is such a fundamental parameter of matter, which is extremely sensitive to chemical, mechanical, electrical or optical influences, the applications of the Kelvin method are many. These include surface chemistry, materials technology, adsorption, stress, microelectronic technology, crystallographic studies, dopant profiling, surface treatment, metal-semiconductor contacts, laser micromachining, characterization of oxide and thin films, chemical sensors and biosensors, self-assembled monolayers, nucleic acids immobilization, DNA and protein microarrays, biocompatibility of materials, pharmacology and drug discovery, histological studies on living cells etc. and corrosion.
Our research involves three aspects. First the Kelvin nanoprobe instrument is under continual development in terms of capability of spatial resolution and sensitivity to changes in surface potential and topography. Second we are applying the instrument to a variety of applications in materials science. Of particular interest is study of micro-corrosion and the influence of surface fractal physics. Also we are looking at microelectronic devices with an emphasis on dielectric processes. The third area concerns use of Kelvin probe technology for the analysis of DNA and protein microarrays. The latter technology has revolutionized the field of molecular biology in recent years. The scanning Kelvin nanoprobe offers huge advantages over fluorescence spectroscopy in terms of label free operation and sensitivity. In our research we are examining the correlation between structure and surface potential of proteins and DNA deposited onto substrates by robotic printing techniques.