Projects

Bioengineering Research Partnership

The National Institute of Bioimaging and Bioengineering (NIBIB) of the United States National Institutes of Health (NIH) has awarded a five year $ 8.3 million CAD grant to support a research partnership between the National Research Council of Canada’s National Institute for Nanotechnology (NINT), the University of Alberta, Los Alamos National Laboratory (LANL) and the La Jolla Bioengineering Institute (LJBI). This partnership is a multidisciplinary initiative involving engineers, biologists and chemists from academia, government and industry both in the U.S. and Canada for the development of new technology platforms and their applications in drug discovery and biomedical diagnostics.

Dr. Fenniri says the goal of this partnership is to develop and combine innovative technology platforms that could revolutionize drug discovery and systems biology. This will allow the partners to take advantage of and challenge the limits of well-established and powerful technologies such as combinatorial chemistry, array technologies, flow cytometry, and their offshoots.

Combinatorial methods were developed over the past decade for the preparation and high throughput screening of chemical libraries synthesized on spherical supports or 2D microarrays. Systems biology, which encompasses proteomics, transcriptomics, metabolomics and functional genomics, relies on integrated systems-based approaches for measuring the global expression of genes and biochemical networks, pathways and the spatial and temporal relationships that give rise to cause and effect in living systems.

A driving force of recent progress in these areas has been the development of technologies for high throughput and highly parallel molecular analysis. The vision of global analysis of biological systems embodied in systems biology depends on the ability to efficiently perform sensitive, specific, and quantitative measurements of biological systems. New implementations of chromatography and electrophoresis, mass spectrometry and microarray technology are examples of recent technology development aimed at improving the speed and scale of molecular analysis.

Beyond genetic analysis, the need to measure the quantity, modification, and function of proteins is driving an even more diverse collection of molecular analysis tools. The microarray format is envisioned to enable highly parallel analysis of proteins in much the same way it enables the analysis of nucleic acid sequence, with antibodies, purified proteins, and other binding ligands replacing oligonucleotides or nucleic acids.

However, the flat surface microarray format has a number of significant limitations. First, they must be prepared one at a time. Second, the preparation, use, and analysis of flat microarrays is not especially amenable to laboratory automation. Third, technical issues, including optimal surface chemistries for biomolecule immobilization, reporter and assay strategies to allow quantitative measurements, and data analysis formalisms to enable extraction of meaningful quantitative parameters require further attention. Finally, the read-out or analysis of flat microarrays is relatively slow (minutes) and not conducive to the analysis of large numbers of samples.

“This partnership will offer innovative tools and alternative strategies to tackle these massive problems, and will have a direct impact on improving human health” says Fenniri. “An important goal of this partnership is to develop a platform based on spectroscopically barcoded microparticles that fulfill the same function and address the challenging issues associated with 2D arrays”.

A few years ago, Fenniri and co-workers developed a way to "bar code" individual chemical compounds, making it quick, easy and economical to identify the most biologically active ones among thousands of candidates in the drug-screening process. This method relies on the use of specially designed "smart" beads. "In addition to their role as support for chemical library synthesis, we designed them so that they would carry vital information about the chemistry to which they were subjected" Fenniri says. In other words, one can interrogate the beads at the end of the synthesis to reveal the chemical nature of their cargo. "Up until this new development, the beads had no role in library screening. So this is a new paradigm in combinatorial chemistry, as the beads are no longer just compound carriers, but are in addition the repository of vital information about the chemical nature of these compounds" Fenniri adds.

The barcoded beads were intentionally synthesized with built-in spectroscopic barcodes. By incorporating infrared- and Raman-active groups that are chemically inert and readily identifiable with standard infrared or Raman spectrometers, each bead displays a unique vibrational spectrum that can be readily converted into a barcode for rapid identification. The barcodes do not deteriorate even if the beads breakdown since the spectroscopic information is evenly distributed over the entire bead.

This technology comes with numerous challenges. First one has to design spatially addressable microspheres that can be remotely interrogated about their chemical/biological cargo (e.g. drug candidate, protein, or antibody), in high speed and with 100% confidence. “Our expertise in nanotechnology and materials chemistry will be particularly useful in expanding the repertoire of barcoded microspheres and enhancing their detectability by several orders of magnitude”.

However, to implement this strategy, Fenniri and co-workers needed one important piece of the puzzle. That is the ability to scan the barcodes of the microsphere “on the fly’ while flowing in a liquid stream at a speed of a 100-1000 microspheres per second. To solve this problem, Fenniri and co-workers turned to Dr. John Nolan and his colleagues at the National Flow Cytometry Resource at Los Alamos National Labs, where flow cytometry was invented in the 1960s. What started as a phone conversation 3 years ago turned into a novel design for a new instrument, the Raman flow cytometer, which is a vital element of this partnership.

“The adaptation of flow-based methods to Raman spectroscopy will open up new possibilities for molecular and cellular analysis, especially for reporting and encoding in multiplexed assays” says Dr. Nolan, Partnership Principal Investigator, now at the la Jolla Bioengineering Institute. “To demonstrate the new technology, we are focusing on receptors and enzymes involved in bacterial pathogenesis, but the instrumentation and methods we develop should be applicable to a broad range of problems in biology, chemistry, and materials science.”

In terms of particle analysis throughput (up to 10,000 particles/sec), serial sample throughput (1 sample/sec), and the ability to sort particles with specific optical features make flow cytometry an extremely competitive analysis platform. While these superior performance features come at the price of increased complexity, there is a vigorous commercial flow cytometer instrument and reagent industry, and sophisticated instruments are affordable for small research and clinical labs.

“The use of the distinctive Raman spectra of polymer beads as an encoding approach has several advantages. First, the Raman spectral features are sharper than for many other optical phenomena, allowing more information to be encoded in a given spectral space. Second, the Raman spectral features report on the chemical composition of a sample, enabling encoding schemes to be rationally or combinatorially designed.” Says Nolan. “Third, the emulsion polymerization methods used to prepare the first generation of Raman-encoded resins scale up well for manufacturing purposes. These advantages and their impact on large scale molecular analysis are motivating factors for this Partnership” Nolan adds.

Raman spectroscopy is an analysis tool with several unique features. Raman spectroscopy has the advantages of sharp spectral features, concentration-dependent signal intensity, and a signal that derives directly from and provides information about the chemical composition of the sample. However, its use has been limited outside of analytical chemistry because of the relatively weak signals and expensive and complex instrumentation. Several developments are addressing these limitations. First, improved dispersion optics and filters provide improved light collection and reduced background and smaller and cheaper high power light sources are available. Second, improvements in Raman spectral analysis methods and computational power enable complex spectra to be analyzed extremely rapidly. Third, the improved understanding of phenomenon such as surface enhancement of Raman vibrations has led to detection strategies that rival fluorescence for sensitivity. The commercial availability of Raman microscopes and the recent application of SERS in a variety of assay formats including flat microarrays have facilitated the rapid development of Raman based methods in biomolecular analysis.

A specific target area of this Partnership is the detection and treatment of human microbial pathogens. Knowledge of virulence factors and their mode of action provide targets for compound development for countermeasures. This Partnership we will initially focus on applying Raman Flow Cytometry and barcoded bead technology to the development of new reagents for the detection and treatment of microbial pathogens and their toxins, including influenza virus, Bacillus anthracis, Leishmania, Stapholococcus aureus, Clostridium spp, and others. Functional analysis of toxin binding and catalysis will be used to identify new peptide compounds that can be used for diagnostic or therapeutic applications. “The technological platforms to be developed by this Partnership will enable large-scale biomolecular analysis and separations essential in systems biology” notes Fenniri.

This Bioengineering Research Partnership grant from NIH represents an exciting opportunity to bring together biologists, chemists, and engineers to develop new technologies to address key challenges in biomedical research. The Raman Flow Cytometry project will lead to important new analytical capabilities that can be applied to medical diagnostics and therapeutics development. These goals reinforce the mission of the La Jolla Bioengineering Institute as place where biomedical technology development occurs hand in hand with basic biological research.

About LJBI

The La Jolla Bioengineering Institute (LJBI) is a non-profit research organization focused on applying engineering principles to fighting disease and improving human health. The Institute has a strong focus on cardiovascular physiology, tissue engineering, and biomaterials, as well as activities in immunology, host-microbe interactions, and biomedical technology development. Research funding for the Institute comes primarily from competitive research grants from the National Institutes of Health and other federal agencies, as well as from industrial partners. Founded in 2001, and located on La Jolla Cove overlooking the Pacific Ocean, LJBI provides an outstanding environment for cutting edge biomedical research.

About Dr. John Nolan

Dr. John Nolan is a Senior Scientist and Principal Investigator at the La Jolla Bioengineering Institute. His research interests are in the quantitative analysis of biomolecular systems, with the goal of developing a predictive understanding that can be used to design new approaches to detect and treat disease. To help achieve this goal, his group develops methods and instrumentation to enable quantitative, high throughput measurements of molecular composition, interactions, and functions. They have a particular interest in cell membrane-based phenomenon including ligand-receptor interactions, and the compartmentalization, transport, and function of macromolecules within the cell. Prior to LJBI, Dr. Nolan was Director of the NIH National Flow Cytometry Resource at Los Alamos National Laboratory, where he led a group of biologists, physicist and engineers in the development of new flow cytometry instruments and applications. Dr. Nolan received B.S. degrees in Biology and Chemistry from the University of Illinois, and a Ph.D. in Biochemistry from The Pennsylvania State University.

Other partners in this project include Dr. Steven Graves, of the Bioscience Division and National Flow Cytometry Resource at Los Alamos National Laboratory, Dr. Steve Doorn, of the Chemistry Division, Los Alamos National Laboratory, and Union Biometrica, Boston, MA.

Project Description

The ability to make quantitative, high throughput molecular measurements of biological systems is a critical need for many areas of biomedical research. This Bioengineering Research Partnership (BRP) aims to develop a powerful new analytical platform for high throughput screening and selection based on Raman Flow Cytometry. This Partnership will develop new analytical instumentation, optically encoded polymer resins for chemical synthesis and screening, and nanostructured materials with unique optical properties for sensitive reporting and encoding. The new technology will perform Raman spectroscopy on single particles in flow to enable new applications in sensitive multiplexed detection, drug discovery, and diagnostics. The Raman Flow Cytometry instrumentation, and applications will be developed by a Partnership involving engineers, biologists, and chemists from academia, government and industry. In the first year of the Partnership, we will modify a commercial particle sorter to detect individual Raman vibrational bands from single particles and sort these particles based on their optical signature. In Years 2-5, we will develop the ability to collect and analyze complete Raman spectra from single particles. In parallel, the Partnership will develop new encoding and reporting strategies for multiplexed molecular analysis and separation. This Raman Flow Cytometry technology will be applied to the development of therapeutics and diagnostics for bacterial pathogens and their toxins. Raman Flow Cytometry will be an important and general new analytical and separation capability that will impact many areas of basic and applied biomedical research in addition to the applications proposed here.