This paper describes a microfluidic chip that enables the detection of viable Cryptosporidium parvum by detecting RNA amplified by nucleic-acid sequence-based amplification (NASBA). The mRNA serving as the template for NASBA is produced by viable C. parvum as a response to heat shock. The chip utilizes sandwich hybridization by hybridizing the NASBA-generated amplicon between capture probes and reporter probes in a microfluidic channel. The reporter probes are tagged with carboxyfluorescein-filled liposomes. These liposomes, which generate fluorescence intensites not obtainable from single fluorophores, allow the detection of very low concentrations of targets. The limit of detection of the chip is 5 fmol of amplicon in 12.5 L of sample solution. Samples of C. parvum that underwent heat shock, extraction, and amplification by NASBA were successfully detected and clearly distinguishable from controls. This was accomplished without having to separate the amplified RNA from the NASBA mixture. The microfluidic chip can easily be modified to detect other pathogens. We envision its use in -total analysis systems ( -TAS) and in DNA-array chips utilized for environmental monitoring of pathogens.
Citation: Analytical Chemistry
Issue: No. 13
Pub Type: Journals
DNA-sensor, microfluidic chip, signal amplification by liposomes