SLAS

MP48:Novel assay and system for rapid diagnostics of urinary tract infections using on-chip isotachophoresis and molecular beacons

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M. Bercovici,(1,2) G.V. Kaigala,(1,2) K.E. Mach,(2) J.C. Liao,(2) and J.G. Santiago(1)
(1) Mechanical Engineering, Stanford University, Stanford, CA 94305, USA
(2) Department of Urology, Stanford University, Stanford, CA 94305, USA

Urinary tract infection (UTI) is the 2nd most common type of infection in the United States, with approximately 8 million visits to outpatient clinics and emergency departments, and 100,000 hospitalizations each year. The current detection methods are time-consuming, most typically consisting of 1-3 day culture and phenotyping by trained professionals. Overall, medical expenditures for UTI in the US are estimated to be $3.4 billion. A rapid, inexpensive, definitive urine test capable of detecting bacteria would be enormously beneficial in ensuring timely treatment of bacterial infections. Moreover, the ability to quickly rule out certain infections would help reduce unnecessary administration of antibiotics. Such a test could reduce costs and burden on the health care system.

We developed a novel microfluidic technique and system capable of a rapid and high sensitivity detection of bacterial pathogens in urine. Our assay is aimed at replacing the standard 2 3 day culture-based phenotypic identification with an on-chip ~5 min test that could be performed at the point-of-care with minimal sample preparation.

16S rRNA is a well-characterized bacterial-specific biosignature used to detect and identify bacteria. In ITP, only ions with mobilities bracketed by two buffers are focused to achieve both sensitivity and selectivity. We use this property to focus 16S rRNA directly from chemically lysed urine pellets (obtained by centrifugation). We use the same ITP process to simultaneously focus and hybridize molecular beacons with the extracted 16S rRNA. Molecular beacons are hairpin-shaped oligonucliotides consisting of a probe section and a self-complementary stem section which brings together fluorophore and quencher molecules. In the absence of target, the stem sequences hybridize and proximity of quencher and fluorophore inhibit fluorescence. In the presence of the target, the beacon preferentially binds to the target; separating the quencher from the fluorophore to yield significant fluorescent signal. In the ITP focused zone, 16S rRNA mixes with and hybridizes the beacons, producing a sequence-specific fluorescence signal which we use to both identify and quantify bacterial RNA.

Our system consists of a simple cross-channel microfluidic chip, laser excitation, high-voltage supply, and a custom-assembled point-confocal fluorescence system. We have developed a lysing procedure compatible with ITP and validated our assay by performing ITP on patient-derived bacterial isolates grown in culture media as well as infected clinical urine samples. When no target is present (control) the molecular beacons are closed, resulting in a relatively low fluorescent signal. When lysed sample is present, the beacons bind to their target resulting in an increased fluorescent signal. Larger bacteria concentrations result in a larger fraction of hybridized molecular beacons and an increase in fluorescent signal. Using our system and assay, we have successfully detected and quantified bacteria within a few minutes across the clinically relevant range of 5E6-5E8 cfu/ml.

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