Active Biochip Research - McDevitt Research Labs UT Austin

Measurements of CD4+ T lymphocytes are essential for evaluating HIV-infected patients. CD4 counts, expressed as either absolute numbers of CD4 cells per milliliter of blood, or as a percentage of total T lymphocytes, have enormous prognostic and therapeutic

implications, and form the basis for most treatment decisions. In developed countries, CD4 counts are performed using flow cytometers, however, these instruments are cost prohibitive in resource-poor countries.

Several efforts have been made to develop alternative, affordable CD4 counts for such countries. Single-purpose flow cytometers for CD4 counting have been developed, but remain largely unaffordable and impractical for use in developing countries. Immunomagnetic separation of CD4 cells from other blood cells, followed by standard cell counting methods using light microscopy, has been implemented in some laboratories, particularly in West Africa. While significantly cheaper, these methods are low throughput and less accurate than the flow cytometry-based methods.

With initial funding from the National Institutes of Health (NIH) and the Gates Foundation, our team has pioneered new LOC methodologies that have allowed for the first time the measurement of HIV immune function in resource-poor settings. By replacing the bead-based microchip with a membrane filter, a new "cell-processing unit" suitable for CD4 lymphocyte counting applications was developed and validated through testing in the United States and Botswana. Recently, the University of Texas at Austin has partnered with LabNow, a new company whose mission is to translate these research discoveries into practical diagnostic test units that will be accessible and affordable for use in important global health care settings...read more.

Saliva Diagnostics (NIH U-01 Funding Mechanism):

Interest in saliva as a diagnostic medium has increased dramatically during the last decade, as saliva and other oral fluids have been shown to reflect systemic fluid levels of therapeutic, hormonal, immunological and toxicological molecules. Oral fluids have also been shown to contain biomarkers associated with infectious and neoplastic diseases. Similarly, the analysis of salivary fluids, like blood-based assays, yields useful diagnostic information for the assessment and monitoring of systemic health and disease states, exposure to environmental, occupational, and abusive substances, as well as for the early identification of harmful agents dispersed by bio-terrorist activities. Action plans originating from the Office of the Surgeon General and the National Institute of Dental and Craniofacial Research (NIDCR) emphasize the need for further research in salivary diagnostics, and advocate that oral fluid-based diagnostics have the potential to provide more accurate and less expensive diagnostic procedures than current methodologies.

Over the past decade, our group has sustained efforts to combine and adapt nano-technology tools for the practical implementation of miniaturized sensors that are suitable for a variety of important application areas, including salivary diagnostics. Towards this end, we have developed a saliva-based assay capable of detecting CRP at ultra-low concentrations. The bead array LOC approach yields a much lower limit of detection (by at least 5 orders of magnitude) than that exhibited by the standard ELISA procedure. Using this assay, we have been able to compare CRP levels between healthy, periodontitis and edentulous patients, and have shown correlation with established macroscopic gold standard methods...read more.

Cardiac Risk Assessment (Philip Morris External Research Program)

This program targets the development of assays and lab-on-a-chip methodologies for the detection and measurement of biomarkers and risk factors that contribute to the development of cardiovascular disease and atherosclerosis. Heart disease is a complex multi-factorial disease that has enormous health, social, and economical impact with projected costs in the United States alone of $393 billion in 2005, including health care services, medications, and lost productivity. Prevention and early detection are crucial in the fight against the disease. The discovery of new early biomarkers of the disease promises to help predict and potentially prevent cardiovascular events. However, in order to fully benefit from these advances, it is imperative that the technological infrastructure be in place for multiplex testing, to accommodate a comprehensive screening approach. The measurement of a number of selected biomarkers of the disease might present unique patterns and associations that would best be exploited when tested simultaneously. Our fluorescence-based platform offers multiplexing capacity and the unique capability of quantitating molecular and cellular content in biological fluids through the chemical or mechanical entrapment of the analytes on either bead- or microsieve-based microchips.

Having demonstrated the functionality of the subcomponent systems for miniaturized sensor systems, it becomes important now to search for effective strategies that would enable the full integration of such promising miniaturized sensors systems in the context of important clinical applications. Only with the early implementation of the mini-assay systems for real world clinical testing will the modular assay system be developed in a manner that will service the future needs of clinicians. Thus, in this program we target the important steps towards integration of multiple lab-on-a-chip functionalities into a single test system. The mini in vitro diagnostics platform here described will be developed using microfabrication methods. Furthermore, concept of creating a modular standard assay "operating system" that can be adapted in a simple and rapid manner to new assays as needed will be incorporated into this development effort. While the ultimate goal of such research endeavors is to develop universal assay systems that can be reprogrammed rapidly for new application, the steps taken here will target the development of a flexible diagnostics tool that can support clinical research and clinical treatment of patients with cardiac heart disease, the number one health problem in developed countries...read more.

HIV Monitoring - Biochip Center for Immune Function Monitoring (Health Resources and Services Administration)

Recent activities from the University of Texas at Austin have targeted the development, testing and deployment of a powerful biochip-based technology suitable for the early detection and monitoring of infectious diseases and related immune function themes. Customized lab-on-a-chip systems developed at UT may quickly find essential roles in point-of-care diagnostics in clinics and hospitals. The benefits of rapid infectious diseases diagnostics and immune function monitoring are a decrease in health care costs, improvement in rapid public health responses to disease outbreaks, and a reduction in the use of unnecessary antibiotics. In addition to the revolutionary and evolutionary benefits that can be expected for commercial health care, it is clear that these same technologies can be utilized in humanitarian settings. Indeed, recent activities have led to the successful completion of highly promising human trials for biochip-based HIV monitoring systems in Boston hospital settings as well as in an HIV reference laboratory in Botswana, Africa. The UT biochip approach allows for multiplexing of theme-specific group of analytes. Further, the lab-on-a-chip systems have been shown to meet or exceed the performance characteristics of numerous well-established macroscopic instruments.

Funds from the Health Resources and Services Administration (HRSA) are being used now to establish an equipment infrastructure that is suitable for clinical measurements of the immune function. Through this program, portable instrumentation pioneered at the University of Texas at Austin will be tested and validated against the established gold standard approaches. Particular emphasis will be placed on assays such as CD4 and viral load measurements of HIV+ patients. The establishment here of a Biochip Center for Immune Function Monitoring will serve an important role in defining the equipment needs for the future generations of cost effective medical instrumentation that is functional at the point of care...read more.

Bioterrorism Screening Tools (RDECOM) and Texas Engineering Experiment Station (TEES)

In October 2001, anthrax spores from two contaminated letters were released into the occupied environment of the U.S. Postal Service Brentwood facility in Washington, DC. Two postal workers died from exposure to the spores and 20 others became ill. This incident reveals the vulnerability of our society to pathogenic bio-aerosols. The development of rapid, reliable and cost effective bio-agent detection systems currently represents one of the most important bio-terrorism priorities for our Nation both for civilian and military sectors.

Recent work from the McDevitt group at The University of Texas at Austin has led to the development of a powerful bio-aerosol detection system that is suitable for the rapid and sensitive detection of bacillus spores in military settings.

The detector system has been made to be compatible with the output of standard bio-aerosol collection systems. The program described here will serve to further strengthen and expand the capabilities of the system to respond to important pathogenic bio-aerosols with strong emphasis on the analysis and treatment of real-world samples. Initial UT development efforts have shown efficacy of this system even within the high background environment (i.e. dust, fluorescent inks, brightness additives, etc.) of postal settings. Clearly, such activities bode well for reduction of false positive and false negative measurements for military applications. Development efforts here will target sensors with that are simple, rapid, and reliable. These systems will utilize assay formats with antibodies probes for agent recognition. Nonpathogenic bacillus spores and bacteria will be used to demonstrate the functionality and optimize the performance properties of this point detector system. The final prototype system will be tested in the Edgewood Chemical Biological Center's Biosensor Laboratory. These sensor prototypes will be fully automated and capable of detecting and identifying bio-agents in air, water or surface samples. Further, the effort will contribute to the immediate training of the next generation of scientists and engineers for the military bio-agent detection area.

Toxic Metal Screening Tools: Single Bead Chromatography (Welch Foundation)

For this program, the McDevitt group is now actively developing novel "single bead chromatographic techniques" that utilize protein subsets to chelate various metal ions. Here, methods are being developed that can be used to quantitate metal ions in solution and provide relative binding constants. To support these activities, protocols are being defined and refined that are suitable for the rapid screening of amino acid libraries to select for peptides that bind particular metals with a high specificity.

The initial progress here has demonstrated the conceptual basis for this new methodology in the context of generating a chemically diverse population of integrated separation and detection units. These diverse chemical chelators are being combined with integrated detection elements so as to fashion integrated single bead microreactors that serve the dual function of separation and detection. These interesting bead ensembles are now being studied in the context of toxic metal cation detection. Further, a variety of interesting scientific issues arise and are being studied in the context of the analyte transport properties within the confines on the dual function bead ensemblies.

DNA Hybridization and Discrimination of Single-Nucleotide Mismatches Using Chip-Based Microbead Arrays (NIH)

The development of a chip-based sensor array composed of individually addressable agarose microbeads has been demonstrated for the rapid detection of DNA oligonucleotides. Here, a "plug and play" approach allows for the simple incorporation of various biotinylated DNA capture

probes into the bead-microreactors which are derivatized in each case with avidin docking sites. The DNA-capture probe containing microbeads are selectively arranged in micromachined cavities localized on silicon wafers. The micro-cavities possess trans-wafer openings, which allow for both fluid flow through the microreactors/analysis chambers as well as optical access to the chemically sensitive microbeads. Collectively, these features allow for the identification and quantitation of target DNA analytes to occur in near-real-time using fluorescence changes that accompany the target sample.

The unique 3-dimensional microenvironment within the agarose bead as well as the microfluidics capabilities of the chip structure afford a fully integrated package that fosters rapid analyses of solutions containing complex mixtures of DNA oligomers. These analyses can be completed at room temperature through the use of appropriate hybridization buffers. For applications requiring analysis of 10 2 DNA sequences, the hybridization times and point mutation selectivity factors exhibited by this lab-on-a-chip bead-array method exceed the operational characteristics in many respects for the commonly utilized planar DNA chip technologies. The utility of this DNA detection microbead array methodology is demonstrated here for the analysis of fluids containing a variety of similar 18-base oligonucleotides. Hybridization times on the order of minutes with point mutation selectivity factors greater than 3,800 and limit of detection values of ~10-14 M are obtained readily with this microbead array system.

Early Detection of Oral Cancer with Lab-on-a-Chip Optical Sensors (NIH)

According to statistics from the American Cancer Society, cancer has recently surpassed heart disease as the leading cause of death in people under 85 years of age. The human and economic impact of cancer is staggering, afflicting an estimated 2 out of every 3 families with 556,500 deaths and annual medical costs approaching $37 billion within the United States in 2003. Beyond prevention, early detection is the most crucial determinant for successful treatment and survival of cancer. Yet current methodologies for cancer diagnosis based upon pathological examination are inadequate for detecting early tumor formation.

Recent activities of the McDevitt laboratory have led to the creation of a series of powerful microchip sensor systems that perform sophisticated chemical and biological tests in complex liquids like blood, saliva, or cellular biopsy suspensions. These minaturized lab-on-a-chip systems have the capacity to replace traditional macroscopic instruments thereby enabling common in vitro diagnostic tests to be completed at the point-of-care. Such systems hold strong promise for a variety of important diagnostic tests including HIV immune function monitoring and cardiac risk assessment. Building upon our recent success with CD3+/CD4+ immune monitoring for HIV patients, we are adapting the same proven membrane-based microchip system for early detection of oral cancer. Our experimental approach integrates several recently identified early tumor biomarkers with traditional morphological examination into a multi-parameter single-cell analysis system. This dual strategy will provide a cancer-risk profile that encompasses a large range of tumor progression phenotypes potentially increasing the method's sensitivity over conventional pathology. Ultimately, this research aims to impact human healthcare through earlier tumor detection and assessment of pre-cancer risk thereby improving patient prognosis and survival.