Isotachophoresis for Rapid Nucleic Acid Hybridization and Sensitive Detection of Urinary Tract Infections

Stanford University, Stanford CA

Investigators

Abstract

1159092 Santiago Proposed is a study of the theory and practice of a novel assay which integrates on-chip ITP with sequence detection capability of molecular beacons (MBs). MB are synthetic oligonucleotide DNA which fluoresce strongly upon hybridization with their complimentary nucleic acid sequence. ITP is a non-linear electrokinetic technique which can focus target analytes with high selectivity in a sharp electric field gradient. The Santiago group proposes the use of on-chip ITP to achieve 100,000-fold preconcentration and mixing of MBs and rRNA into the same 10 picoliter virtual reaction chamber at an ITP focal zone. This focusing causes ~100,000-fold increase of hybridization rates. The Santiago group has demonstrated the principle of the ITP/MB assay, and here propose a transformative new method which can attain a dramatic, 1000-fold increase in sensitivity over their preliminary work combining ITP and MBs. The proposed 100 fM sensitivity would cover the full clinically relevant range, including presymptomatic UTI infections. They propose a novel two-step assay which initially preconcentrates, mixes, and hybridizes MBs, and subsequently separates un-reacted MBs from hybrids immediately prior to detection to minimize non-specific background fluorescence. They will minimize sample dispersion using geometric modifications to on-chip channels. To improve their design and optimization capabilities, they will, in parallel, develop a novel and unique computational tool for coupling finite rate reactions with non-linear electrokinetic flows. They will validate this computational tool using a set of well-controlled experiments. These computational tools will be incorporated into a free, open-source code with a user-friendly interface, and be portable across major platforms (Windows, MAC, and Unix). This unique code will be made available through downloads, a blog website for updates and commentary, tutorial videos, relevant data bases, documentation (including peer-reviewed journal papers describing the solution methods), and example input and output files. In summary, the proposed study will (1) explore the basic limits and potential of leveraging ITP to create widely applicable on-chip virtual reaction chambers for fast hybridization; (2) create a novel react-then-separate assay which speeds up hybridization rates by 100,000 fold and maximizes sensitivity; (3) demonstrate prototype miniaturized systems for UTI diagnosis from human urine in 10 min; and (4) develop new, free, open-source computational tools for the electrokinetics community. Infectious diseases caused by bacterial pathogens remain one of the most common causes of mortality worldwide. This proposal focuses on urinary tract infections (UTI), the second most common infection in the US. UTI affects all patient demographics; and causes $3.4 billion total medical expenditures, approximately 8 million clinic or emergency department visits, and over 100,000 hospitalizations per year. Similar to most other bacterial infections, diagnosis of UTI requires a centralized clinical microbiology laboratory and trained professionals to perform bacterial culture and phenotyping, which typically takes 1-3 days. Molecular amplification and diagnostic methods exist, but are rarely used for UTI and still require trained personnel in centralized laboratories. This delay time in diagnosis is at least partially responsible for overuse of antibiotics and the associated emergence of drug resistant bacterial strains. To address this need, the Santiago group proposes to develop a prototype demonstration of a low-cost microfluidic device with potential to reduce bacterial diagnostic time from ~3 days to ~10 min including sample loading, on-chip sample preparation, and sequence specific detection. This system will be designed with practical implementation in mind, including ease of use and minimal fluid handling. This work has application to both conventional clinical microbiology laboratories and point-of-care settings, and can potentially eliminate dependence on trained operators. Santiago will use isotachophoresis (ITP) to extract and concentrate nucleic acid bacterial signatures, including 16S ribosomal RNA (rRNA), directly from untreated urine lysate, and detect and quantify its presence using sequence specific molecular beacon probes on a miniaturized device. Websites will be developed to describe the work to the general community and to share models and methods with the scientific community; host a science high school teacher in their laboratory each summer; incorporate assignments based on this research into a Fluids Engineering undergraduate course; host 1-2 undergraduates in the lab; and incorporate research-level material into an advance graduate course.