The unambiguous identification of a rare, diseased cell for the purpose of early diagnosis requires the quantitative determination of many biomarkers simultaneously in individual cells at high throughput. A similar requirement for multi-parameter assay is shared by genomic and proteomic researchers, who need to determine simultaneously the presence and quantity of the many genes, proteins and small molecules involved in the translation of gene to cell activity. We propose a novel solution for a broad spectrum of bio-analytical challenges. The new technology takes advantage of the high resolution and sensitivity of inductively coupled plasma mass spectrometry (ICP-MS), combined with the many available stable isotopes of elements of the periodic table, to simultaneously determine many proteins and gene transcripts in samples through the quantification of stable isotope tags bound to a wide variety of bioaffinity molecules. Beads are an attractive option for supporting surface chemistries for immunoassays and oligonucleotide hybridization assays. One of the advantages of beads is the ability to increase the reaction surface area per volume of the reaction mixture, which provides a reliable means of increasing the capacity and dynamic range potential of an assay, as well as miniaturizing the reaction. We expect that our technology will be able to recognize many thousands of distinguishable analytical beads created by the incorporation of various concentrations and ratios of metal ions. We hope that this integrated massively multiplexed technology based on our prototype flow cytometer-mass spectrometer (FC-MS) and reagent support/encoding system will simplify and enhance the diagnostic, prognostic and therapeutic efficacy available to physicians and their patients, increasing the effectiveness of healthcare while substantially reducing the human and financial costs of treatment. The objective of this application is the development of analytical instrumentation, an advanced element (metal) encoding system and methodology for rapid recognition of functionalized encoded beads - the bead array mass spectrometer (BA-MS). The combination with an elemental detector will provide researchers and clinicians with massively multiplexed analytical capabilities. The focus of this application is on the development of an individual (cytometric) bead analysis at high throughput (employing the FC-MS invented, developed and built in our lab). FC-MS continues to undergo extensive development in our lab and is not yet available elsewhere.
Our Specific Aims i nclude: 1) development of these technologies to the engineering beta-level, suitable for placement in laboratories for routine use and evaluation. These beta instruments will be made available within 12 months of the completion of the proposed effort. 2) development of a statistically significant unambiguous encoding system with massive variability (>105 individual beads) and unsupervised algorithms to recognize encoding in a rapid and dependable manner. 3) development of demonstrative bio-analytical methods using the technology in a multiplexed format and validation against existing fluorochrome bead-based technologies. Our team has long experience in research and pre-commercial analytical instrument development, is completely committed to the goals and is poised to advance rapidly.
Enormous progress is being made on the identification of diagnostic biomarker signatures and the understanding of biomarker interactions. Unfortunately, there are few analytical tools capable of recognizing these signatures, and these have serious limitations for detecting many biomarkers in a single analysis or require expensive consumables (i.e., arrays) that may be prohibitive for clinical application and that require lengthy and expert analysis. To obtain the correct molecular signature of a disease, it is rarely sufficient to determine one or even a few biomarkers. The applicants are developing an innovative solution to this challenge that is receiving considerable enthusiasm from the scientific community. The approach takes advantage of the high resolution of mass spectrometry to distinguish biologically-rare metal atoms that replace the fluorescent dyes in current use. In addition to enabling genomics and proteomics researchers to achieve a vast improvement in the depth and range of cellular analysis, this project will provide a diagnostic tool that will define the new standard-of-care benchmark in hospitals, clinics and research departments world-wide.
