from Theron et al. in Nanotechnology in Water Treatment Applications

Developments, particularly in the fields of genomics and biotechnology in the last few years, have resulted in a wide range of molecular tools, principally based on the detection of nucleic acid material and its amplification. They offer a novel, more sensitive and specific way of detecting microorganisms. They can also identify organisms that would not be detected with current culture techniques and can be used to track new pathogenic entities, including variants of otherwise harmless microorganisms. There are probably very few groups of microorganisms that have not been detected with these amplification techniques and several test kits have already been commercialized. Despite the success of these molecular methods, several barriers must be overcome before they can be used to routinely assess water quality and the microbiological safety of drinking water. The relationship between detection by molecular approaches and the subsequent viability or infectivity of waterborne enteric pathogens remains a concern. In addition, methods used for purifying and concentrating the target microbes and their nucleic acids from water, so that they are free of contaminants that may interfere with the analysis, need further improvement, consolidation and simplification. Further research is also needed to develop and refine the prototype protocols into collaboratively tested methods that could be used routinely and expeditiously to evaluate the microbiological safety of water.

Owing to recent advances in nanoscience and nanotechnology, various different nanomaterials and devices have been developed that show great promise for diagnostic applications. Subsequently, many different nanotechnology-based diagnostic systems have been reported in the literature and many of these have the potential to become the next generation of diagnostic tools. Moreover, microfluidic chip-based systems such as the lab-on-a-chip technology should have a significant impact on environmental microbial monitoring by permitting detection and identification of targets within minutes at the sampling site with a sensitivity level of a single cell. However, some technical and practical problems need to be solved before their full potential can be realized. These include tight control over the synthesis and functionalization of nanomaterials, as small variations can change their properties and behaviour in diagnostic methods. Also, their implementation into routine functional devices remains a challenge, and note should be taken that many of the diagnostic systems must still be taken from proof-of-concept and evaluated with environmental samples. In this regard, some of the challenges that need to be resolved, in addition to those highlighted above, include sample processing, detection of multiple agents in a single sample, and improving the sensitivity and selectivity of the assays for application to complex environmental samples.

from 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 cells (10⁴-10⁷) 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.

from Theron et al. in Nanotechnology in Water Treatment Applications

In contrast to gold nanoparticles and QDs, magnetic nanoparticles have not been used in many biological applications. Nevertheless, advances in the synthesis of monodispersed magnetic nanoparticles, ranging in size from 2 to 20 nm, has provided a basis from which to explore applications of magnetic nanoparticles in diagnostics. Magnetic nanoparticles are produced from materials that can be strongly attracted by magnets or be magnetized. They can be prepared in the form of single domain or superparamagnetic (Fe₃O₄), greigite (Fe₃S₄), maghemite (gamma-Fe₂O₃), and various types of ferrites (MeO.Fe₂O₃, where Me = Ni, Co, Mg, Zn, Mn, etc.). Bound to biorecognition molecules, magnetic nanoparticles can be used to facilitate the separation, purification and concentration of different biomolecules. To do so, biorecognition molecules such as antibodies can be immobilized on the surface of magnetic nanoparticles through covalent or electrostatic interactions. After reacting these magnetic nanoparticles with sample solutions, targeted molecules can be bound by or captured on the surface of these magnetic nanoparticles. By applying a magnetic field, these nanoparticles can subsequently be concentrated and separated from the bulk solution and identified.

Biofunctional magnetic nanoparticles, in which thiolated vancomycin was attached to FePt nanoparticles, have been used to capture and detect of a wide range of bacteria at very low concentrations within 60 min. These included capturing and detection of Staphylococcus aureus at 8 cfu/ml, S. epidermidis at 10 cfu/ml, Enterococcus faecalis at 26 cfu/ml, and E. coli at 15 cfu/ml. Although the sensitivity achieved using magnetic nanoparticles is comparable to PCR-based assays, the direct capture protocol is faster than PCR when the bacterium count is low since it obviates the need for pre-enrichment of bacteria through culturing. In an alternative approach, Ho et al. reported combining biofunctional magnetic nanoparticles with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to detect pathogenic bacteria in water. Biofunctional nanoparticles were fabricated by attaching human immunoglobulin (IgG), which binds selectively to IgG-binding sites on the cell walls of pathogens, onto the surfaces of magnetite (Fe₃O₄) nanoparticles. Using this assay, both S. saprophyticus and S. aureus were detected at concentrations of 3ˆó10⁵ cfu/ml in aqueous sample solutions. Measuring adenosine triphosphate (ATP) bioluminescence of bacteria captured onto magnetic nanoparticles is another proposed method for detecting microorganisms. E coli was detected in milk by Cheng et al. within a short period (1 h) and with a low detection limit (20 cfu/ml).

Biofunctional magnetic glyconanoparticles have also been engineered by covently binding unmodified monosaccharide d-mannose onto iron oxide nanoparticles. These particles had the ability to recognize mannose-specific receptor sites on E. coli. Magnetic nanoparticles have been developed to sequester DNA in water and capture the DNA-nanoparticles complexes by the application of high-gradient magnetic separation. Modifying magnetite clusters with poly(hexamethylene biguanide)- and polyethyleneimine resulted in strong cationic nanoparticles which enabled the binding with DNA molecules through electrostatic forces. The cationic nanoparticles can also serve as a disinfectant by binding to the negatively charged cell envelopes of bacteria. These particles were colloidally stable in fresh and ocean water for weeks at a pH <= 10.

Magnetic microparticle-antibody conjugates (Dynabeads) are commercially available and kits have been developed for the detection of Legionella species, Cryptosporidium oocysts and Giardia cysts from concentrated water samples. Dynabeads are also available for the detection of E. coli, Salmonella and Listeria species; however the samples must be grown for 6 - 8 h in a pre-enrichment broth. Streptavidin coated Dynabeads allow researchers to design their own magnetic microparticle-antibody conjugates for specialized assays (www.invitrogen.com). Biotinylated organism-specific antibodies will bind covalently onto the streptavidin coated Dynabeads. A wide range of biotin-labeled antibodies are available from companies such as Abcam (www.abcam.com).

Despite the promise shown by biofunctional magnetic nanoparticles, some challenges regarding their widespread use have yet to be overcome. In addition to requiring a robust surface chemistry to attach bioactive molecules onto magnetic nanoparticles without laborious synthetic efforts, more precise control of the numbers and orientations of the molecules on the surfaces of magnetic nanoparticles is also required.