It has been estimated that one-third of the global population (total of ~2 billion) is infected with Mycobacterium tuberculosis (TB). A majority of those infected (90%) will never contract active TB and will remain asymptomatic. In immunocompromised patients, however, TB is a major cause of death worldwide. For example, TB is the leading cause of death in HIV-positive patients. The World Health Organization estimates that 9 million people contracted active TB in 2006 (~6 million from Southeast Asia and Africa alone) and over 1 million died from the disease. Another 10 million are projected to acquire the disease this year, the most in history. Many TB diagnostic assays are on the market or in development. A majority, however, are not well suited for operation in resource-limited settings. Collectively they are too expensive, too time consuming (24 hrs or more), require specialized equipment, expertise, and power, have low sensitivities (50% in the sputum smear test), or do not work on HIV-positive patients or children (antigen tests). It has been estimated that more rapid and accurate TB diagnostic methods would save 400,000 lives per year. The long-term goal of this research is to develop new infectious disease diagnostic assays that can be deployed in resource-limited settings. We seek to develop assays that can be performed outside of a laboratory setting, that is, without PCR amplification, and with minimal power, instrumentation, and scientific expertise. We propose to accomplish this goal by combining two technologies developed at CU-Boulder-modified DNA aptamers and "materials DNAzymes"-with a conductance-based chip platform. Modified DNA aptamers have proven to have affinities and specificities for protein targets that are as good as or better than antibodies. Compared to antibodies, however, DNA aptamers require less time and cost to discover and produce, and are more thermally stable. Materials DNAzymes are DNA sequences that convert otherwise stable metal complex precursors into metal nanoparticles. This application describes the use of modified DNA aptamer/materials DNAzyme conjugates (materials aptazymes) for the electrical detection of TB lipoarabinomannan (LAM) from urine.
The Specific Aims for the project are as follows.
Aim 1 (Months 1-6). Isolate modified DNA aptamers for TB LAM.
Aim 2 (1-6 months). Isolate materials DNAzymes that mediate the formation of Au nanoparticles from solutions containing [Au(Cl)4]1-.
Aim 3 (Months 6-12). Synthesize a chimera containing a TB DNA aptamer and Au DNAzyme and verify that each sequence functions properly.
Aim 4 (Months 12-24). Determine detection limits and specificities of TB LAM in simulated urine. Upon completion of these aims, we will be poised to use the reagents and technology developed in this project in an RO1 project that will validate LAM in urine as a biomarker of TB infection.
The goal of this project is to develop a new diagnostic platform for the detection of infectious disease, with an initial focus on M. tuberculosis (TB). Th technology is intended for use in resource-limited settings, meaning that it will be rapid, portable, and will require little power or significant clinical/scientific expertise to operate.