by Wen-Tso LIU
ounting levels of global terrorist activity have raised concern about such biological threats as contamination of drinking water in populated areas. Each year, an estimated 20 million deaths worldwide result from infectious diseases, the majority of which are transmitted through physical contact with microbe-contaminated water. Even though water and wastewater-treatment systems exist now, early-warning mechanisms would play a crucial role in prevention.
Current diagnostic methods for waterborne pathogens rely mostly on scientists' ability to identify infectious bacteria or indicator organisms in the laboratory using step-by-step biological and biochemical assays. Besides providing poor advance warning for suspect cases, identification procedures can take from 2-4 days (for example, for faecal bacteria) to a few weeks (for example, for species of Mycobacterium). These shortcomings reveal a need to develop more rapid and accurate diagnostic methods to alert people to possible infectious outbreaks in drinking-water sources.
Researchers at the National University of Singapore could be a step closer to providing a solution. Wen-Tso Liu and his colleagues at the Centre for Water Research, and the Nanoscience and Nanotechnology Initiative have been focusing on rapid and sensitive enabling techniques to simultaneously detect multiple biomolecules or bio-Xs such as DNA, RNA and proteins unique to many waterborne pathogens.
One project aims to produce a high-density oligonucleotide (a short polymer of nucleotides, the basic structural units of DNA or RNA) chip or a DNA chip that contains a high-density microarray of at least hundreds of oligonucleotide probes targeting unique genetic information on waterborne pathogens in a small solid-surface area (<1cm²).
The brief experimental procedures involve collection and concentration of microbial cells in water samples, extraction and fluorescent labelling of DNA and RNA from the microbial cells, and allowing the labelled DNA and RNA to react with the DNA chip under optimised conditions. Upon completion, a unique array of DNA-probe (T-P) signals or "DNA fingerprints" can be obtained for individual organisms or a mixture of organisms using a fluorescence-detecting device. By further increasing the range of temperature at 1°C per minute for targeted T-P hybrids, the kinetics of separating the hybrid into T (DNA) and P (probe), or so-called "melting curves," can be monitored.
This approach can resolve different T-P hybrids at a resolution of one nucleotide difference, which is very accurate. This is essential to differentiating perfectly matched T-Ps from mismatched T-Ps caused by harmless bacteria with close but mismatched sequences. This project has successfully identified Bacillus anthracis, the causative agent of anthrax, as distinct from other Bacillus species.
Another project aims to develop a more advanced device than the DNA chip that integrates multiple processes - from sample collection, DNA/RNA extraction and amplification, to detection - on a platform smaller than the size of a palm. The idea is to bring highly complex biological and biochemical testing into a miniaturised setting.
The lab-on-a-chip or "personal laboratory" employs the most recent technologies in microfabrication and microfluidics - techniques that produce micro/nanodevices consisting of interconnected fluid reservoirs and pathways. These devices allow the automated transport of samples through selected pathways by electrokinetic forces generated at designated electrodes, or by the voltage gradient between electrodes in capillary electrophoresis.
Further, it is possible to create the functional equivalent of valves and pumps capable of performing manipulations such as reagent dispensing and mixing, incubation/reaction, and sample partition and analyte (a substance being measured in an analytical procedure) detection through various biosensing and nanoparticle-detection systems.
The research team has produced a prototype containing a microchannel, a reaction chamber and a particle-trapping device used to study fluid movement in microchannels with and without the presence of analytes. This work will be developed further to incorporate bacterial detection systems. The group ultimately aims to use this personal laboratory for rapid, sensitive, accurate and simultaneous detection of many waterborne pathogens in various water sources on-site.
For more information contact Wen-Tso Liu at [email protected]