Theron et al. in Nanotechnology in Water Treatment Applications
Advances in microfluidics and microfabrication technologies have contributed greatly to the miniaturization of biological and chemical analytical systems, allowing the handling of low volume samples, as well as reductions in reagent consumption, waste generation, costs and assay time. Micro-total analysis systems (micro-TAS), sometimes called "lab-on-a-chip", are microfabricated devices capable of performing the functions of large analytical devices in small units. These devices are fabricated in glass, silicon or polymer materials, and integrate different functions and functionalities. Some sophisticated versions can perform sample introduction and handling (e.g.
, cell lysis, dilution and debris removal), separation (e.g.
, electrophoresis, chromatography) and detection, all conducted on the chip. It is believed that micro-TAS will be particularly valuable in DNA and protein analysis, genomics and proteomics, and diagnostics.
Miniaturized immunoassays have been performed successfully with microchips. These immunoaffinity microfluidic devices are considered promising platforms to achieve rapid and sensitive immunological detection of microbial cells. Zhu et al. described a simple approach in which sample trapping and concentration steps were integrated together with whole-cell immunoassay in a silicon-based lab-on-a-chip. The immunoassay was performed by injecting the sample solution, which contained C. parvum
and G. lamblia
, into a microchamber. Subsequently, a solution containing fluorescently-labelled target-specific antibodies was delivered and serves to simultaneously concentrate, trap and label the targeted cells at a trapping region. Following a wash step to remove unbound antibodies, the labelled parasites were detected by epifluorescence microscopy. Compared to conventional immunoassays, the total analysis time was reduced from 2-3 h to 2-5 min, and the total consumption of reagents was reduced 20-fold. In an alternative approach, Liu et al. injected microbeads coated with a primary antivirus antibody into a microfluidic device, which are subsequently trapped in front of a pillar-type filter region. A sample containing target virions was injected into the device and virions were captured on the surface of the microbeads. This was followed by injection of a labelling solution containing a secondary antivirus antibody labelled with QDs to allow detection by epifluorescence microscopy. In comparison to a standard ELISA performed on the same marine iridovirus, the minimal detectable concentration of the target virus was improved from 360 to 22 ng/ml, the detection time was shortened from 3 h to less than 30 min, and the amount of antibody consumed was reduced 14-fold.
Considerable effort has been directed to the development of chip-based systems for miniaturized and rapid PCR. The devices consist of a chip containing wells, channels, electrodes, filters, pumps, valves and heating devices designed for buffer and sample storage, PCR and target DNA detection. Remarkably, a polymeric microchip with a 1.7 microlitre chamber containing a thermocoupler was used to successfully amplify a 500-bp DNA fragment of lambda phage in 15 cycles, in a total amplification time of 240 seconds. By making use of a PCR microchip coupled with a capillary electrophoresis (CE) chip, it was more recently demonstrated that bacterial targets as low as 2-3 cells could be amplified within a 200-nl PCR chamber and the PCR-amplified target DNA was subsequently resolved by CE within 10 min. In order to improve PCR throughput and reduce the analysis time, multi-chamber PCR microfluidics on a single chip has been reported. Also, chip devices with optical windows have been fabricated that allows for measurement of fluorescence intensity during the thermocycling process, thus providing a miniaturized version of real-time PCR. In this regard, Cady et al. developed an integrated miniaturized real-time PCR detection device equipped with a microprocessor, pumps, thermocycler and light emitting diodes (LEDs)-based fluorescence excitation/detection. Monolithic DNA purification and real-time PCR enabled fast detection of L. monocytogenes
) within 45 min.
In spite of their potentially powerful application in diagnostics and environmental monitoring, the 'complete' lab-on-a-chip still requires further development. The bottlenecks blocking the realization of a truly and highly integrated chip include sample preparation and product detection. Since the source of raw template samples is varied and the sample preparation methods are diverse, the miniaturization of conventional sample preparation and functionalities on a chip remains a challenge. As for on-chip detection, the product detection methods have not advanced as rapidly as other aspects of chip development. Consequently, miniaturized ultra-sensitive detectors are required if the sensitivity of the lab-on-a-chip devices is to be improved. Moreover, additional efforts have to be made towards the validation of the methods to demonstrate the reliability of micro-TAS systems.