Miniaturized Detection Systems - Waterborne Pathogens: Detection Methods and Applications - page 329

methods significant progress has been made in the design and operation
of amplification and detection stages, with some integration of these two
stages. A few systems have presented full integration with sample processing
also performed on-chip but this is an area requiring more development.
For more details about the general state-of-the-art regarding microfluidic
molecular methods we recommend the 2009 review article by Zhang and
Ozdemir 98 and the 2012 review article by Ahmad and Hashsham. 99
10.2.5.1. Waterborne pathogens
While there has been considerable progress developing microfluidic systems
that enable on-chip molecular detection, the number of studies applying
these types of technologies to waterborne pathogens is very low. In terms
of waterborne viruses we found that RT-PCR on a microfluidic device
with integrated amplification and fluorescence detection, which could be
performed within 1 h, was demonstrated for rotavirus in 2011. 100
Work developing a multiplexed PCR system for bacteria was announced
in 2006, with promising early results. 101 In 2010, Ramalingam and col-
leagues presented the simultaneous detection of four waterborne bacteria
in a PDMS PCR array, which utilized capillary flow for sample loading. 102
In 2012, Agrawal and coworkers showed simultaneous detection of E. coli
and S. tyhimurium on a circular microfluidics design manufactured in PDMS
using magnetic nanoparticles to control and concentrate samples. 61
With regard to protozoa, two publications that relate to molecular sens-
ing of Cryptosporidium in miniaturized format describe the performance
of NASBA off-chip 103 ; only the detection of the mRNAs amplicons was
performed on-chip. Esch et al. have developed a fluorescence-based detec-
tion assay chip, relying on a sandwich hybridization of the NASBA prod-
uct between capture probes and reporter probes. 104 The microfluidic device
consists of one channel in a PDMS block bonded to a glass slide with a gold
pad at its center to immobilize the capture probe. The reporter probes were
tagged with carboxyfluorescein-filled liposomes giving out better fluorescent
intensities than usual fluorophores. This technique gave an LOD of 5 f fM of
amplicon per test (12.5 µL). The overall time for the full analysis was 1-2 h,
including the heat shock and implementation of the NASBA procedure. 104
10.2.5.2. Commercial systems
Crypto Detect CARD ™ is a platform, shown in Fig. 10.9(a) , with on-chip
integrated sample preparation features, developed by Rheonix and reportedly
capable of detecting Cryptosporidium in raw water samples (Rheonix 2011).