Biological systems respond to environmental stresses and chemical agents by producing unique biochemical signatures, i.e. biomarkers, that - when detected and interpreted correctly - yield enormous insight into the state of the organism. Thus, the central objective of this work is to develop an integrated instrument that detects and identifies biomarkers by acquiring small- volume fluid samples, separating and pre-concentrating the biologically important components in a nanofluidic-microfluidic chip, and then characterizing them using on-line mass spectrometry in a miniature mass spectrometer. In the process, the applicability of molecular identification tools in biological research will be enhanced by significantly decreasing the concentration levels at which specific organic molecules can be detected in complex mixtures. Novel protocols for transferring and ionizing compounds of interest based on high-frequency ac electrospray ionization and desorption electrospray ionization methods will be developed and compared with respect to figures of merit, such as mass transfer efficiency, sensitivity and background interferences. One specific aim is to improve the efficiency of the ionization step, by far the least efficient process in mass spectrometry. Critical performance tests of the analysis system will target biomarkers for oxidative stress using biofluids which mimic cerebrospinal fluid (CSF). The coupling of micro/nano fluidic sample preparation to miniature atmospheric pressure mass spectrometers offers much value to the biological sciences, for example making it possible to realize real-time functional assays of changes in fundamental metabolic, regulatory and signaling processes in response to environmental factors. Fundamental improvements in the performance of mass spectrometers and in microfluidic devices will result from the synergy of this project, making possible future generations of biological instruments of great power and utility. In addition to the direct relevance to biological research, successful instrumentation development will impact medical diagnostics: The same markers relevant to biological oxidation processes are germane to the early detection and prognosis in a host of diseases, including multiple sclerosis, Alzheimer's disease, Niemann-Pick C, amyotrophic lateral sclerosis, heart disease, Parkinson's disease and ischemic stroke, making the results readily translatable to human health studies. Furthermore, the training of highly skilled instrumentation scientists is an emerging national need, and this need will be addressed by formalizing collaboration through (1) research student exchange between Notre Dame and Purdue and (2) larger scale exchanges between Purdue's Center for Analytical Instrumentation Development (CAID) and Notre Dame's Advanced Diagnostics and Therapeutics Initiative. Both institutions aim to (i) train graduate students in instrumentation science (ii) engage in instrumentation development, (iii) facilitate its commercialization, (iv) benefit the regional economy through instrument commercialization. The public can follow these activities at the relevant websites: http://sri.nd.edu/advanced-diagnostics- and-therapeutics/ and www.purdue.edu/dp/caid/.
Biological systems respond to harmful outside agents and environmental stress by altering their basic metabolism, resulting in the production of unique biochemical signatures – biomarkers - that when detected and interpreted correctly can yield enormous insight into the state of the organism. Identifying and detecting molecular biomarkers for conditions resulting from oxidative stress, i.e. exposure to reactive oxygen-containing species, is a key goal across a broad spectrum of biological sciences, one with direct consequences for environmental health and safety. Currently, assays for these biomarkers are carried out at central laboratories with very expensive instrumentation. A fundamental transformation, both in terms of cost and efficacy, can result if we can produce a turn-key, point-of-care molecular diagnostic system that can be used by untrained personnel and can provide diagnosis on-the-spot. For example, such a capability could dramatically improve quality of life in the developing world, which cannot afford both the traditional culturing technique or the more modern molecular diagnostic devices---and has an acute shortage of trained personnel to carry out both. It could also lead to early screening devices in the developed world, providing new tools both for basic biologists as well as health-care professionals. All these applications require low-cost and user-friendly devices that can, nevertheless, produce sufficient accurate molecular detection. In this NSF project, a platform and generic approach have been developed for such transformative low-cost diagnostic devices. Its portable mass spectrometry approach allows the detection of a large library of biomarkers, and its architecture supports continuous monitoring of biological samples. The microfluidic platform to connect the continuous physiological feed to the mass spectrometer is designed for generic application. It can be used for stand-alone single-sample/single-state testing or for continuous monitoring of an organism or a tissue without a portable mass spectrometer. It is designed with several functions in mind: cost, automation, sensitivity, selectivity and assay time. With a multi-discipline team, new technologies and improved old ones are integrated to produce a microfluidic chip system that can accommodate this challenging but important component of the future diagnostic systems. An integrated prototype has been developed and its manufacturing system is being designed by commercialization and innovation entities.
