from Theron et al. in Nanotechnology in Water Treatment Applications

Metal nanoparticles, such as gold, silver and iron, constitute one of the most researched branches of nanotechnology due to their electronic, optical, catalytic and thermal properties. Among these, gold nanoparticles are intensively used in a variety of colorimetric and fluorescence biodetection assays. Specific focus has been directed at colloidal gold nanoparticles ranging from 3 to 100 nm in size, since they are rather stable and their properties can be easily tailored by chemical modification of their surfaces.

Gold nanoparticle-based probes have been used in the identification of pathogenic bacteria in a chip-based system. The assay consists of a capture DNA strand immobilized on a glass chip that recognizes the DNA of interest. A separate sequence on the captured target strand is then labelled with an oligonucleotide-functionalized gold nanoparticle probe. After catalytic reduction of silver onto the gold nanoparticle surfaces to amplify the target signal, the capture-strand/target/nanoparticle sandwich is visualized with a flatbed scanner. The target DNA was detected at concentrations of 50 fM, which represents a nearly 100-fold increase in sensitivity over traditional fluorescence-based assays. Gold nanoparticles have also been used in the detection of genomic DNA from Staphylococcus aureus, without enzymatic amplification, at concentrations of 33 fM. The assay depends upon the target-selective binding of gold nanoparticle probes to consecutive regions of a target DNA sequence in solution, followed by transfer to an evanescently illuminated glass microscope slide for light scattering measurements. Whereas the gold particle probes scatter orange light in the presence of target, the probes scatter green light if the target is absent, whilst the target concentration is quantified by measuring the intensity of scattered light. Multiplexed detection of different viruses and bacteria, using gold nanoparticles surface functionalized with a target-specific oligonucleotide and further encoded with a Raman-active dye molecule, has also been demonstrated. Whereas the complementary probe sequence imparts the specificity for the target sequence of interest, the presence of the target is confirmed by silver staining and the identity of the target is revealed by detecting the surface-enhanced Raman scattering (SERS) of the Raman dye near the nanoparticle surface. This assay demonstrated a sensitivity of 1 fM target concentration.

Despite its widespread use in nucleic acid hybridization assays, the use of gold nanoparticles for detection of bacterial pathogens in immunoassays has only recently been described. The assay comprises detection of organism-specific antigens with biotinylated polyclonal antibodies after which gold particles functionalized with a secondary antibiotin antibody are added, and the particles are then visualized under a dark-field stereomicroscope. The immunoassays were demonstrated to reliably detect Helicobacter pylori and E. coli O157:H7 antigens in quantities in the order of 10 ng, which provides a sensitivity of detection comparable to those of conventional dot blot assays. A two dot filter system has also been developed where colloidal gold nanoparticles (2 nm) were bounded onto anti-E.coli O157:H7 antibodies. These monoclonal antibodies were bounded onto a 0.2 microm nitrocellulose filtermembrane through which water was filtered. The same antibodies that captured the bacteria acted as detectors since the gold nanoparticles could be visualized under epifluorescence. This one-step detection method not only had a low detection rate (1 cfu/100 ml) and high specificity but would be inexpensive and easy implemented in the routine testing of water samples.

from Theron et al. in Nanotechnology in Water Treatment Applications

Flow cytometry (FC) detects and quantify light scattering from fluorescent-labeled cells that have crossed a laser beam. A single sample can be analysed within 3-5 min with a quantification limit of approximately 200 cells/ml. FC, although an optical detection method, is used in combination with molecular techniques. Bacterial cells in water have been monitored with flow cytometry through nucleic acid staining or targeting specific cells with antibodies or FISH hybridization.

FC is a valuable tool to differentiate between viable, intermediate and nonviable cells. Baclightbacterial viability kit (Live/Dead kit), widely used in flow cytometry, double stains nucleic acid with SYTO dyes (green fluorescence) and propidium iodide (PI) (red fluorescence). SYTO dyes stain the nucleic acid of all the cells, resulting in green fluorescence. The cells are afterwards stained with PI which can only move into membrane compromised cells, staining the nucleic acid and resulting in red fluorescence. The disadvantage is that cells can be dead without showing membrane damage and hence is this rather an assay representing membrane damage than cell viability. Calculating the nucleic acid content has also been used as an indicator of cell viability. The theory is that cells with higher cell viability reproduces at a higher rate and therefore will contain more copies of their genome. Care must be taken with the interpretation of results obtained from this approach. Bouvier et al. investigated the varied correlation between different nucleic acid contents and metabolic activities of subpopulations from a wide range of environmental communities.

FC combined with nucleic acid staining enable researchers to investigate the growth potential of microbial pathogens in natural waters. Vibrio cholerae, the causative agent of cholera, was shown through FC and SYBR Green nucleic acid staining to grow in different freshwater samples. This contradicted previous opinions that natural waters do not have sufficient nutrients to support the growth of this pathogen. Combining these experiments with assimilable organic carbon (AOC) concentrations it was concluded that V. cholerae would proliferate in water with a minimum AOC of 60 mg/l. The same research group investigated the growth potential of E. coli O157 in freshwater samples using the same methodology. E. coli O157 was able to grow in freshwater samples with low carbon concentrations, once again contradicting previous opinions.

Fluorescence activated cell sorting (FACS) makes FC even more indispensible for detecting and differentiating between microbial pathogens in water. Cells with specific nucleic acid targets can be labeled with FISH probes, quantified and separated with FC-FACS. These cells can then be subjected for further genetic and biochemical analysis. Catalyzed fluorescent reporter disposition-FISH and molecular beacons are now incorporated into FC-FACS to increase stain sensitivity and overcome the problem of sorting cells present in low numbers. FC-FACS-FISH has also been applied to sequence previously unsequenced microorganisms and cultured previously uncultured microorganisms from environmental samples.

from Theron et al. in Nanotechnology in Water Treatment Applications

Currently, the detection of microorganisms is largely based on time-consuming culture methods. However, newer enzymatic, immunological and genetic methods are being developed to replace and/or support classical approaches to microbial detection. Moreover, innovations in nanotechnology and nanosciences are having a significant impact in biodiagnostics, where a number of nanoparticle-based assays and nanodevices have been introduced for biomolecular detection. Accordingly, current and emerging molecular approaches for the detection of microbial pathogens as well as nanobiotechnologies that that will extend the limits of current molecular diagnostics are being developed.

from Theron et al. in Nanotechnology in Water Treatment Applications

Immunological methods are based on the specific recognition between antibodies and antigens, and the high affinity that is characteristic of this recognition reaction. Consequently, many different immunoassay methods have become available for both quantitative and qualitative analysis of pathogenic bacteria in water. These include immunocapture of cells or antigens by enzyme-linked immunosorbent assay (ELISA or EIA), or detection of targeted cells by immunofluorescence (IFA). These assays can be performed by a direct or indirect manner. In a direct immunoassay, the monoclonal or polyclonal antibodies, directed against antigens located on the surface of the target pathogen (such as capsid proteins, cell wall or flagellar antigens), are conjugated with a fluorochrome or fluorescent dye. Alternatively, secondary enzymatically- or fluorescently-labelled antibodies directed against the primary antibodies (now serving as antigens) can be used in an indirect immunoassay. The advantage of this procedure is that the secondary antibodies can easily be obtained from a commercial supplier with a range of conjugated fluorochromes and it leads to signal amplification as several labelled secondary antibodies can bind to a single unlabelled primary antibody. The antigen-antibody complex is detected and quantified by the ability of the enzyme to react with a substrate that produces either a coloured product for colorimetry or emits light for luminometry. The immunoassays are often performed on a solid phase to which the pathogen antigens have been applied, such as a membrane filter or the bottom of a microtitre plate well.

Studies have shown that solid-phase enzyme immunoassays generally are too insensitive for direct detection of microbial pathogens in water, as they require a minimum of 103 to 104 target microbes (or their antigens) for detection. In most situations drinking water and its sources rarely contain high enough levels of most target pathogens for direct immunoenzymatic detection. Nevertheless, enumeration of diluted specific cells can be obtained by means of immunomagnetic separation (IMS). Immunomagnetic separation, also termed immunocapture or antibody capture, is a method that uses paramagnetic synthetic beads or other magnetic particles that have been coated with monoclonal or polyclonal antibodies directed against the target microbes to recover the microbes from the sample by antigen-antibody reactions. The retained microbes can be analyzed directly or after they or their nucleic acids have been released or extracted from the antibody and solid phase by various physical or chemical methods. IMS methods have the advantage of selecting, separating and purifying specific target microbes from other microbes and from solutes, based on the specificity of the antigen-antibody reaction. This is a powerful approach for recovering, enriching, purifying and concentrating the target viruses, bacteria and parasites from the sample matrix. However, it is not applicable to some pathogens because of the lack of antisera or the antigenic diversity of a large pathogen group lacking a common antigen and thus requiring many antisera.

As an alternative to the above assays, agglutination methods can be used to detect pathogens by combining dispersed microorganisms with antibodies (on a slide, for example) and allowing for antigen-antibody reactions to produce agglutination (clumping) that can be scored as negative or various degrees of positive. One modification is latex bead agglutination in which antibodies against a specific microbial antigen are attached to latex beads. The beads are reacted with the sample and should the sample contain the specific antigen, agglutination occurs by the reaction of antigens with antibodies on the beads resulting in the beads clumping together. As with enzyme immunoassays, agglutination tests are too insensitive to directly detect and quantify most waterborne pathogens in drinking water and other aquatic samples. The target microbes must first be cultured in order to obtain a sufficient number of them or a sufficient amount of antigen to detect and antigenically characterize them by agglutination methods.

The use of immunological methods for the detection of specific microorganisms is a rapid and simple technique, the accuracy of which mainly depends on the specificity of the antibody. Nevertheless, its application to the detection of specific microorganisms from environmental water samples is limited. While IFA allows specific identification and detection at a single-cell level, it does not provide information on the physiological status or viability of the detected cells. The ELISA is a rapid, simple and quite sensitive test. However, assay limitations are often associated with the specificity of the antibody used, the concentration of both antibody and antigen, and the solid matrix often leads to non-specific binding of the antigen or of the secondary antibody.

Monoclonal antibodies are better suited for biosensors because of their higher specificity. Polyclonal antibodies recognize different epitopes on the same pathogen. False positives can be generated when these antigens are present in other closely-related non-pathogenic microorganisms. The thermal instability of antibodies, in particular monoclonal antibodies, is another drawback when applying them in environmental biosensors. Single domain antibodies (also referred to as nanobodies) have however been developed that are thermostable, even at temperatures as high as 90 degrees C. Their small size, high solubility and refolding capacity are other features that make them ideally situated for biosensing applications.

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 3 x 10⁵ 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.

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

Due to advances in areas such as genomics and biotechnology, powerful methodologies have been developed to detect both specific pathogens and indicator organisms. Because many microorganisms are not easily cultured or can enter a viable but nonculturable (VBNC) state, the current methods focus on immunological or genetic characteristics to detect the presence of specific waterborne pathogenic microorganisms. Not only do these methods increase the rapidity of analysis, but they are also able to achieve a high degree of sensitivity and specificity without the need for complex cultivation and additional confirmation steps. Consequently, some of these methods permit the detection of specific culturable and/or nonculturable microorganisms within hours, instead of the days required with the traditional methods.

from Theron et al. in Nanotechnology in Water Treatment Applications

Detection of pathogenic organisms provides information as to the safety and public health risks associated with a given water supply; however, it often does little to define the potential sources of the contamination. Generally, because different enteric pathogens are present in the intestines of different animals, the identification of a contamination event as being either of human or animal source would provide information as to the types of pathogens that may be expected, the risk of infection and the treatment that may be required to control transmission of disease. In response, molecular techniques are being developed as means to identify the source of a contaminant. DNA fingerprinting is one tool for microbial source tracking (MST) and consists of a family of techniques that are used to identify the sources of fecal contamination in various water bodies.

The different polymorphism-based procedures are generally coupled to a PCR reaction. In the amplified ribosomal DNA restriction analysis (ARDRA) technology, PCR-amplified 16S rRNA genes are digested with restriction endonucleases and the resulting fragments separated electrophoretically. Presence or absence of the restriction site within two strains cause differences in the length of the DNA restriction fragments, and the complexity of the pattern depends upon the number of target sequences and position of restriction sites. Comparison of the generated patterns to those obtained from a database allows assignment of isolates to species or species clusters in those cases were the banding patterns are highly similar. The separated DNA fragments may also be transferred to filters for hybridization with probes specific for an organism of interest. Two other protocols for generating DNA fingerprints use a single primer to amplify fragments with PCR before examination on agarose gels. PCR amplification of repetitive extragenic palindromic sequences (Rep-PCR) takes advantage of repetitive sequences found in the microbial genome. In the randomly amplified polymorphic DNA (RAPD) or arbitrarily primed PCR technology, a short oligonucleotide primer (about 10 nucleotides), usually with random sequence that is not specific for a particular gene is used as a primer to amplify fragments. These methods yield DNA fingerprints comprised of multiple, differently sized DNA amplification products following separation by gel electrophoresis.

Detection of host-specific 16S rRNA genetic markers, using length heterogeneity PCR (LH-PCR) and terminal restriction fragment length polymorphism (T-RFLP) analysis, also holds promise as an effective method for characterizing a microbial population. The technique distinguishes members of mixtures of bacterial gene sequences by detecting differences in the number of base pairs in a particular gene fragment. Whereas LH-PCR separates PCR products for host-specific genetic markers based on the length of amplicons, T-RFLP uses restriction enzymes on amplified PCR products to determine unique size fragments. Specifically, in T-RFLP, rRNA target gene sequences are PCR-amplified using one or both of the primers with a fluorescent label. The amplification product(s) are then digested with appropriate restriction endonucleases and following electrophoresis of the resultant fragments using an automated DNA sequencer, a fluorescent electrophoretic profile of the digestion patterns is obtained. The use of labelled primers limits the analysis (identification) to only the terminal fragments, thus allowing the study of complex microbial communities. Moreover, the possibility of discriminating fragments with differences as small as single bases gives the method a higher resolution than gel-based profiling techniques.

One of the most promising technologies for microbial source tracking is, however, amplified fragment length polymorphism (AFLP) analysis. AFLP analysis appears to have the same taxonomic range as other fingerprinting techniques, but this technology combines several advantages of these different techniques, which in most cases results in the highest power of discrimination. This technology is based on the selective amplification of a subset of genomic restriction fragments using PCR. For AFLP, purified genomic DNA is digested with two restriction endonucleases, one with an average cutting frequency and a second one with a higher cutting frequency after which oligonucleotide adapters are ligated to the genomic DNA restriction fragments. The sequence of the adapters and the adjacent restriction site serves as oligonucleotide primer binding sites for subsequent amplification of the restriction fragments by PCR. Selective nucleotides extending into the restriction fragments are added to the 3' ends of the adapter-specific PCR primers such that only a subset of the restriction fragments are recognized and amplified. The subset of amplified fragments is then analyzed by denaturing polyacrylamide gel electrophoresis to generate the fingerprint. Since relatively small amounts of DNA are digested and detection of AFLP fragments does not depend on hybridization, the AFLP analysis method is more reproducible and robust than other fingerprinting techniques and it also displays more fragments than other fingerprinting techniques.

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

Nanocantilevers, which are typically made of silicon, silicon nitride or silicon oxide, require only minute changes in compressive or tensile surface stress on either their upper surface or lower surface to cause measurable deflection of the cantilever, and are capable of converting biomolecular recognition reactions into micromechanical motion. Consequently, nanocantilevers offer an opportunity for the development of highly sensitive, miniature and label-free detection systems.

Direct, label-free detection of DNA typically involves the use of silicon cantilevers with a thin gold coating on the top surface that is functionalized with thiolated capture DNA strands. Binding of the capture DNA strands with the introduced target DNA causes deflection of the cantilever, which can be measured accurately using optical detection methods. Using this approach, Fritz et al. demonstrated the hybridization of complementary oligonucleotides with a detection limit of 10 nM and showed that a single mismatch between two 12-mer oligonucleotides is clearly detectable. Similarly promising results were obtained by Hansen et al. with a 10-mer oligonucleotide.

In recent years, cantilever immunosensors have been developed to detect bacteria and viruses. Typically, the devices detect the additional mass loading that results from the interaction between specific antibodies, immobilized on the surface of the cantilever, and antigens on the cell membrane surface. In an early experiment, a cantilever biosensor was constructed and used to detect E. coli O157:H7, following immersion of the cantilever in a suspension containing 10⁶-10⁹ cells/ml. The detection of 16 E. coli O157:H7 cells were demonstrated and no frequency shifts were observed when buffer alone or buffer containing S. enterica serovar Typhimurium was incubated with the cantilevers. A magnetoelastic cantilever immunosensor has been developed that uses a magnetic field to induce oscillation of the sensor. The sensor surface is coated with antibodies to permit specific capture of the desired target agent after which alkaline phosphatase-labelled antibodies to the target are added to amplify the signal, thereby increasing the total mass on the sensor. The sensor was tested with E. coli O157:H7 and a sensitivity of 10² cells/ml were reported. More recently, resonant cantilever biosensors have been developed for detection of Listeria innocua and vaccinia virus, and the detection of a single L. innocua cell and vaccinia virus particle were demonstrated.

Despite being in its early days, cantilevers provide an opportunity for label-free, real-time measurements in fluids in a single-step reaction, and can potentially serve as a powerful platform for sensitive, multiplexed detection of biomolecules. Although cantilevers can be microfabricated by standard low-cost silicon technology, leading to a decrease in production costs and allowing the possibility of integrating multiple functional devices onto the same platform, some challenges must be overcome before cantilever array sensors can come into widespread use. These challenges relate especially to methods for detecting nanoscale motion and the development of immobilization techniques that can efficiently transduce the stress involved in biochemical interaction to the cantilever substrate.

from Theron et al. in Nanotechnology in Water Treatment Applications

Quantum dots
Quantum dots (QDs) are colloidal, luminescent inorganic nanocrystals with unique photochemical properties, which include high quantum yields, large extinction coefficients, pronounced photostability, as well as broad absorption spectra coupled to narrow size-tunable photoluminescent emission spectra. A typical QD has a diameter of 2-8 nm and is usually composed of a core consisting of a semiconductor material, such as CdSe, enclosed in a shell of another semiconductor material with a larger spectral band-gap, such as ZnS. The shell prevents the surface quenching of excitons in the emissive core and thus increases the photostability and quantum yield for emission. Since QDs are usually synthesized from organometallic precursors and salts, they have no intrinsic aqueous solubility. Consequently, the native coordinating organic ligands on the surface of the QDs must either be exchanged or functionalized with a ligand that can impart both solubility and bioconjugation sites, if desired. Strategies for attaching biorecognition molecules to QDs comprise of direct chemical coupling to a functional group displayed on the QD surface or by non-covalent self-assembly in which proteins are engineered to express positively charged domains that interact through electrostatic attractions with the negative surface of modified QDs.

Although functionalized QDs have been used to detect complementary target nucleic acid sequences in chip-based assays and in fluorescent in situ hybridization (FISH) assays, are QDs increasingly being used in immunoassay detection. QD-based immunological assays have been applied to the detection of different bacterial and protozoan pathogens. For these immunoassays, the QDs are conjugated to organism-specific antibodies, or, alternatively, biotinylated organism-specific antibodies are used that are subsequently detected using a QD-streptavidin bioconjugate. These approaches have been used successfully for the detection of Cryptosporidium parvum (43 oocysts in spiked reservoir water), Giardia lamblia (1-5 x 10³ cysts) and Escherichia coli O157:H7 (2 x 10⁷ cells). Moreover, QDs appear to be especially suited for multiplex immunoassays. As a demonstration of the potential of QDs in multiplexed immunoassay formats, the simultaneous detection of E. coli O157:H7 and Salmonella enterica serovar Typhimurium bacteria (10⁴ cells in 2 h), and of C. parvum and G. lamblia (5 x 10³ cysts) in environmental water samples, using different coloured QDs as immunoassay labels, has been reported.

QDs have also been applied in flow cytometry because of their broad absorption spectra and narrow size-tunable photoluminescent emission spectra. The broad absorption band enables semiconductor QDs to simplify and improve the detection of multiple bacterial targets in flow cytometry samples. Each organic dye needs a separate excitation source resulting in multiple excitation sources and complicated experimental setups when monitoring more than one organism. In comparison is only a single excitation source in the UV range needed to excite all the visible colours of CdSe QDs. QDs have been used to simultaneously enumerate pathogenic E. coli O157:H7 and harmless E. coli DH5alpha in water. Cross spectral talk of QDs are also drastically lower than the organic dyes traditionally used in flow cytometry due to their narrow emission spectra. Flow cytometric measurements of QDs have been compared with the widely used fluorochrome, fluorescein isothiocyanate (FITC). The minimum fluorophore concentration for detection was a 100-fold lower when paramagnetic beads were labeled with QDs.

Despite their capability for single molecule and multiplexed detection, it is, however, unlikely that QDs will completely replace traditional organic fluorophores as biological labels. Some of the challenges that have yet to be overcome include financial costs, since QDs are expensive in comparison to organic dyes and there is an initial financial investment required regarding instruments optimized for use with QDs. Non-specific binding and aggregation are two factors that can lower QD fluorescence as in the case of antibody stained Cryptosporidium oocysts. Also, QDs are an order of magnitude larger than organic dyes and thus the extent to which their presence perturbs the recognition event being detected, must be determined. This is particularly important when multiplex assays are desired, since labelling several biomolecules with QDs of different sizes could result in varying degrees of perturbation due to the large differences in the QD sizes. In contrast, most organic dyes are of similar size in spite of their large differences in absorption/emission characteristics.

from Theron et al. in Nanotechnology in Water Treatment Applications

Advances in nanotechnology and nanosciences are having a significant impact on the field of diagnostics, where a number of nanoparticle-based assays have been introduced for biomolecular detection. The promise of increasing sensitivity and speed, and reduced cost and labour makes nanodiagnostics an appealing alternative to current molecular diagnostic techniques. New synthesis, fabrication and characterization methods for nanomaterials, which have dimensions that range from 1 to 100 nm, have evolved to a point that deliberate modulation of their size, shape and composition is possible, thereby allowing control of their properties. Along with these advances has come the ability to tailor their binding affinities for various biomolecules (proteins, nucleic acids and microbial pathogens) through surface modification and engineering. Each of these capabilities allows the design of materials that can potentially be implemented into new biodiagnostic assays that can compete favourably with the current molecular diagnostic methodologies. In this section, the most promising nanomaterials and their use in the detection of nucleic acids and microbial pathogens will be highlighted.

from Theron et al. in Nanotechnology in Water Treatment Applications

Nanowires have been explored as signal transduction motifs in the electrical detection of DNA, proteins and microbial pathogens. Nanowire sensors operate on the basis that the change in chemical potential accompanying a target binding event can act as a field-effect gate upon the nanowire, thereby changing its conductance. The ideal nanowire sensor is a lightly doped, high-aspect ratio, single-crystal nanowire with a diameter between 10 and 20 nm. Recently, detailed protocols for fabrication of silicon nanowire devices, including their covalent modification with biorecognition molecules, have been described.

Real-time, label-free detection of DNA has been demonstrated by Hahm and Lieber using silicon nanowires functionalized with peptide nucleic acid (PNA). The conductance of the PNA-functionalized silicon nanowire bridging two electrodes was measured in the presence of target DNA and mutant DNA with three consecutive base deletions. Introduction of target DNA into the assay caused a rapid and immediate change in conductance, while the effect of mutant DNA was negligible. Furthermore, the conductance changes scale with target concentration, and target DNA at concentrations as low as 10 fM were detected.

Semiconducting silicon nanowires have also been used for the electrical detection of viruses in solutions. Patolsky et al. interfaced nanowires functionalized with antibodies specific for influenza A virus particles with a microfluidic sampling system. The nanowire sensing system was able to detect influenza A at the single-virus level, demonstrating that single virus/nanowire recognition events can be detected by measuring real-time changes in nanowire conductivity. In addition to silicon nanowires, metallic multi-striped nanowires have also shown great promise as potential platforms for multiplex immunoassays. These nanowires are built through submicrometer layering of different metals, e.g., gold, silver and nickel, by electrodeposition within a porous alumina template. Due to the permutations in which the metals can be deposited, a large number of unique yet easily identifiable encoded nanowires can be fabricated. The multi-striped nanowires, when coated with target-specific antibodies, were reported to efficiently and accurately allow multiplex detection of Bacillus globiggi spores, MS2 bacteriophage and ovalbumin protein. The sensitivity of detection for B. globiggi spores, MS2 bacteriophage and ova protein was estimated to be 1 x 10⁵ cfu/ml, 1 x 10⁵ pfu/ml and 5 ng/ml, respectively, and is comparable with results obtained using microarrays.

Nanowires are set apart from other available nanobiotechnologies due to several key features such as direct, label-free, real-time electrical signal transduction, as well as ultrahigh sensitivity, exquisite selectivity and the potential for integration of addressable arrays. However, an intrinsic limitation of nanowires is that the detection sensitivity depends on solution ionic strength. Consequently, for samples with a high ionic strength, diagnostics will require a desalting step before analysis to achieve the highest sensitivity. Furthermore, a practical constraint might be that the synthesis and fabrication of nanowire biosensor devices require some technologies that are not common to most laboratories.

from Theron et al. in Nanotechnology in Water Treatment Applications

Nucleic acid hybridization techniques
The easiest way of detecting specific nucleic acid sequences is through direct hybridization of a probe to microbial nucleic acid extracts. These hybridization techniques rely on the specific binding of nucleic acid probes to complementary DNA or RNA (target nucleic acid). The probes are single strands of nucleic acid with the potential of carrying detectable marker molecules highly specific to complementary target sequences, even if these sequences account for only a small fraction of the target nucleic acid. Either DNA or RNA can serve as a nucleic acid probe, but for a number of reasons (e.g., ease of synthesis and stability), most studies have employed DNA probes. The probes may be used to detect genes in the bacterial genome (Southern blots) or to detect mRNA or rRNA (Northern blots).

According to their length, DNA probes can be grouped as either polynucleotide probes (more than 50 nucleotides) or as oligonucleotide probes (less than 20 nucleotides). The latter are more frequently used since they are short enough to allow for single mismatch discrimination of target nucleic acids and large quantities of oligonucleotides can be rapidly and inexpensively produced. During oligonucleotide synthesis a variety of marker or linker sequences can be introduced to the 5' end of the oligonucleotide. The principle conjugate fluorochromes are derivatives of fluorescein or rhodamine, although labels such as digoxigenin and biotin have also been used. For probes to be effective in nucleic acid hybridization, they must be specific and selective. This requires that sequence data be available. Although such data is available for some waterborne pathogens, the data are not comprehensive for all pathogens of concern. However, the use of bacterial 16S rRNA sequences to develop determinative hybridization probes is now well established. The occurrence of 16S rRNA molecules in high copy numbers of usually more than 1000 in any living cell, as well as the fact that 16S rRNA sequences have been determined for a large fraction of bacterial species, currently confers on these genes a high informative value at phylogenetic level. Comparative analyses of rRNA sequences have indicated that the rRNA molecules comprise highly conserved sequence domains interspersed with more variable regions. Consequently, so-called signature sequence motifs on various taxonomic levels, from domains to subspecies, can be identified. Whereas species-specific probes complement the most variable regions, more general probes target more conserved regions of the molecule. Although several oligonucleotide probes are commercially available, new specific probes may also be designed. The design of such 16S rRNA-targeted probes is greatly facilitated by computer-aided sequence comparisons of large rRNA sequence databases. The principle steps involved in the design of probes are: the alignment of rRNA gene sequences; the identification of sequence idiosyncrasies; the synthesis and labelling of complementary nucleic acid probes; and the experimental evaluation and optimization of the probe specificities and assay sensitivities using cultured reference strains.

Nucleic acid hybridization can be performed in a variety of formats. The procedure typically entails the extraction of whole-cell DNA or RNA from the sample that is subsequently fixed to a nylon or nitrocellulose membrane. Alternatively, bacterial colonies can be replica-plated from agar plates to membranes and their nucleic acids exposed following lysis for subsequent hybridization. Whereas the former direct approach is usually inadequate due to high detection limits and large sample volumes that are impractical for most hybridization protocols without further pathogen concentration, the latter approach is time-consuming and cannot be applied to nonculturable organisms. Consequently, the use of whole-cell fluorescent in situ hybridization (FISH) as a method for the direct detection of bacteria in water samples has increased in popularity. The method typically combines membrane filtration, resuscitation of the bacteria on membranes and hybridization with rRNA-targeted probes. During FISH, the morphology of the cells in the sample has to be stabilized in order to maintain the morphological integrity of the cells under harsh hybridization conditions. The cell walls and membranes have to be permeabilized to allow free penetration of fluorescent oligonucleotides to the intracellular rRNA. This can be achieved with fixatives such as aldehydes and/or alcohols. The membranes are hybridized in hybridization solution containing a fluorescently-labelled oligonucleotide. Following incubation at the hybridization temperature for one to several hours, to allow the probe to bind to complementary rRNA sequences, washing steps are used to remove unbound or the non-specifically bound fluorescent probe, and the hybridized cells are then detected by epifluorescence microscopy. Probes will only bind correctly under defined hybridization conditions and the optimization of hybridization and washing conditions is as important as the probe design. Several probes labelled with spectrally different fluorochromes can be simultaneously used on one sample, while counterstaining of the fixed cells with DAPI (4',6-diamidino-2-phenylindole) allows total counts to be made. Alternatively, depending on the concentration of targeted cells in the sample and to increase resolution, FISH hybridization can be performed in suspension and detection can be performed by means of flow cytometry, which enables quantification of the fluorescence intensities for each target-probe hybrid.

Although FISH is currently considered a highly specific cellular detection method and is relatively easy to perform, the procedure may have some limitations when applied to the detection of nutrient-starved bacterial cells disseminated in drinking waters. Generally, probes give a strong signal only if cells are metabolically active and contain large numbers of ribosomes and target rRNA. Conversely, low cellular ribosome contents may result in weak fluorescent hybridization signals. Another area of concern relates to the viability of the detected cells. Although it has been reported that the rRNA content of bacterial cells can be directly correlated with the growth rate, some reports have indicated that a small number of rRNA molecules can remain for a relatively long period after the loss of culturability, thus resulting in false positives. One possible way to overcome this issue is to couple direct viable count techniques with FISH detection.

from Theron et al. in Nanotechnology in Water Treatment Applications

As a consequence of the speed, specificity and low cost of the PCR, the procedure has become one of the most widely used assays for direct detection of low levels of pathogenic microbes in environmental samples. The PCR assay can be used to selectively amplify, to detectable levels, nucleic acid sequences associated with pathogens that might be present in low numbers in water samples. PCR is a process in which target DNA, synthetic oligonucleotide primers, a thermostable DNA polymerase and the DNA subunits are combined in a microcentrifuge tube and subjected to the temperature changes needed for the DNA duplication to occur. During the PCR process, different temperatures are used to facilitate DNA denaturation, annealing of the oligonucleotide primers to the target DNA and extension of the primers across the target sequence. These cycles are repeated many times, thus resulting in increasingly greater quantities of target sequence. Under ideal conditions, the PCR can generate millions of copies of a single DNA molecule in just 20 to 30 repetitions of the temperature cycle, with each cycle requiring only a few minutes. The PCR-amplified products can be detected by means of techniques such as electrophoresis on agarose gels and after staining of the amplification products by a fluorochrome dye or by hybridization with a labelled probe.

There are several essential steps in the development and application of PCR for successful detection of pathogens in water samples. The key steps include: identification and selection of oligonucleotide primers for target genomic sequences; testing of selected primers for sensitivity, specificity and selectivity; purification and concentration of the pathogens in environmental sample concentrates to enable efficient and reliable enzymatic amplification of low numbers of target genomic sequences; and testing of the methods for their applicability to natural pathogenic strains and actual field samples. By using PCR, a selected gene sequence specific to a group of organisms or a single species can be selectively amplified. Various primers have thus been described to amplify fragments of rRNA operons in order to detect specific organisms or groups of organisms in environmental samples. As in the case of designing oligonucleotide probes for hybridization purposes, selection of oligonucleotide primers for target pathogens requires that sequence data be available. Of particular importance is the type and function of the nucleic acid target; its length and location; and the extent to which its sequence is related to that of other, nontarget but genetically related microbes. It is essential to select oligonucleotides having an appropriate length (20 to 30 nucleotides); desired sequence composition for specificity and selectivity; and appropriate melting and annealing temperatures to prevent the formation of undesirable secondary structures, primer-dimers and other artifacts that would interfere with successful PCR.

There exist many variations of the basic PCR technique. The sensitivity and specificity of the PCR may be improved by adopting a nested approach. Nested PCR involves two consecutive rounds of PCR amplification. The first round of amplification is performed with group- or organism-specific primers, whilst the second round of amplification uses the initially amplified product as the template for another round of annealing and extension with different primers. The use of nested primers provides an additional level of specificity since the second round of PCR amplification can only be performed if the correct sequence (complementary to the nested primers) was amplified during the first round. Alternatively, nucleic acid hybridization can be performed using highly specific oligoprobes that would hybridize only with amplicons from a single pathogen type or strain. However, nested PCR permits a more rapid detection (6 to 8 h) compared to confirmation of the correct sequence amplification by probe hybridization (few days). Another variation of the PCR technique, namely multiplex PCR, allows the simultaneous detection of more than one target organism in a single PCR using multiple pairs of primers designed to be specific for different target organisms. However, multiplex PCR may not perform well with all primer blends as the composition and length of primer oligonucleotide, as well as the size of the amplified fragments, may influence each PCR amplification. Since the PCR method cannot be used directly for the amplification of an RNA target sequence, a complementary DNA copy (cDNA) thereof must first be synthesized. This reaction is catalyzed by the enzyme, reverse transcriptase (RT), which is able to synthesize DNA from the RNA template in the presence of specific primers and DNA subunits. The single-stranded cDNA is a suitable target for PCR amplification by making use of the same or a different set of primers. RT-PCR has emerged as a sensitive and specific approach for the detection of enteric viruses containing RNA genomes.

Although the basic PCR method is both specific and sensitive, such standard PCR reactions are not quantitative. To obtain quantitative data from PCR-based analyses, statistical methods based on most probable number (MPN) estimations have been used. In MPN-PCR, DNA extracts are diluted before PCR amplification and limits are set on the number of genes in the sample by reference to known control dilutions. Another way to quantify PCR-amplified products for comparison is to include an internal control in the PCR reaction. Here, a known amount of target DNA is added to a PCR reaction containing DNA from the mixed microbial population. The known target DNA is complementary to the same primers and thus competes with the target sequences in the PCR reaction mixture. By preparing a dilution series of the known and unknown DNA species, it is possible to quantify the amount of product produced from the complementary gene in the extracted DNA. The known DNA target can be generated by cloning the gene of interest or purifying the PCR-amplified product after which a deletion is introduced to give a differently sized PCR product.

An alternative PCR assay for the direct enumeration of targeted cells was reported by Tani et al., who modified the standard PCR protocol so that nucleic acid sequences can be amplified in situ. This new PCR method was successfully applied to the direct enumeration of E. coli from a freshwater sample. With proper fixation and permeabilization conditions, the oligonucleotide primers and other reaction components are able to diffuse into the cells, and, upon thermal cycling, amplify a specific target sequence present in the cell. PCR products were labelled by digoxigenin during the amplification process and anti-digoxigenin antibodies conjugated with fluorescent dye were used for detection by epifluorescence microscopy. This approach allows direct visualization of the fluorescent amplification products at a single-cell level and consequently, direct enumeration of cells. Even though in situ PCR seems promising, it has not been used for routine detection and enumeration of microorganisms in water, as the results showed a weak fluorescence intensity signal of targeted cells.

Another promising approach for quantifying the number of cells is real-time quantitative PCR, which consists of monitoring fluorescently-labelled PCR products as they are being amplified. The fluorescent signal can be generated by using an intercalating fluorescent dye (e.g., SYBR Green I or SYBR Gold) or a probe system (e.g., TaqMan). The use of intercalating dyes is the simplest and least costly approach and involves adding the fluorescent dye directly to the PCR. These dyes undergo a conformational change to become a more efficient fluorophore on binding to double stranded DNA (dsDNA). Although SYBR Green I-based assays are very sensitive, the primer's specificity for the target is crucial as any dsDNA is detected, including any primer artifacts, which can lead to false positive results. Moreover, multiplex reactions are impractical since the dye binds to all dsDNA. The TaqMan approach depends on oligonucleotide probes complementary to a sequence located between the two primers used for PCR amplification. At one end of the probe a fluorescent reporter dye is conjugated, whilst at the other terminus there is a quencher that may be another fluorophore (also called dark-quencher). In effect the structure possesses two dyes in close proximity and in this configuration the fluorescence of one (the reporter) is quenched by the other through FRET (fluorescence resonance energy transfer). During the extension step of PCR the DNA polymerase degrades the bound TaqMan probe, using its inherent 5'-3' exonucleolytic activity, and thus results in the separation of reporter from quencher and an increase in fluorescence emission of the reporter molecule. Although this approach is less prone to false positive results compared to the use of intercalating fluorescent dyes, it is more expensive due to the requirement of the probe molecule. However, by choosing the fluorophores astutely it is possible to perform multiplex PCR. During real-time PCR, irrespective of the approach used, the accumulation of amplified product is measured automatically at each PCR cycle. The amount of target sequence is deduced from the number of PCR cycles (threshold cycle or Ct) required to cross a fixed point above a baseline, using a standard curve as reference. External quantification standards for the construction of standard curves of Ct versus copy number usually consist of the target sequence cloned into a plasmid or DNA extracted from cultured cells where the concentration or copy number of the target can be determined accurately.

Although there are numerous advantages associated with PCR as detection tool, standard PCR cannot, however, be used to detect the infectious state of an organism - only the presence or absence of pathogen-specific DNA or RNA. Yet, this viability concept is fundamental for interpreting the result in terms of public health when dealing with water samples. The PCR technique must consequently be associated with a viability test. To overcome this limitation, an indirect approach has been developed for assessing the viability of PCR-detected bacteria from water samples. This method is based on the analysis of each sample before and after a culture step in a nonselective medium: an increase in the PCR-amplified product after cultivation indicates the occurrence of bacterial multiplication and thus demonstrates the viability of the detected bacteria. Recently, a PCR-based approach to limit detection to intact (viable) cells with an active metabolism has been reported. The approach is based on the use of ethidium monoazide (EMA), which is suggested to enter only membrane-compromised cells (considered "dead"). Once inside membrane-compromised cells, EMA intercalates into the DNA and is covalently bound to the DNA after exposure of the treated samples to bright visible light, whilst the unbound EMA, which remains free in solution, is simultaneously inactivated by reacting with water molecules. The EMA treatment is followed by extraction of genomic DNA and analysis by PCR. The result of treatment is that only unmodified DNA from intact cells whose DNA was not cross-linked with EMA can be amplified, whereas PCR amplification of modified DNA from membrane-compromised cells is efficiently suppressed. Treatment was thus suggested to lead to the exclusion of cells with damaged membranes from analysis.

Despite its advantages, accurate characterization or identification of microbes by PCR is influenced by the same bias and variations that are inherent in many nucleic acid techniques. The main concerns are biased nucleic acid extraction (e.g., efficiency of extraction or cell lysis if using whole-cell methods), degradation of nucleic acids by nucleases and primer reactivity (i.e. sensitivity, specificity and accessibility). Additionally, a frequently encountered limitation inherent to PCR analysis of environmental samples is the inhibition of the enzymatic reaction. Whereas humic substances are known to inhibit the DNA polymerase enzyme, colloidal matter has a high affinity for DNA. The presence of these elements in a water sample can therefore considerably decrease the amplification yield of PCR applied to the detection of greatly diluted bacteria. Consequently, for PCR or RT-PCR, the extracted target nucleic acid is purified by protocols utilizing, for example, Sephadex, Chelex or CTAB.

from Theron et al. in Nanotechnology in Water Treatment Applications

Significant advances in the detection of sequence-specific nucleic acid hybridization have been achieved using microarrays. Microarrays are glass microslides or nylon membranes containing a high density of immobilized nucleic acids (genomic DNA, cDNA or oligonucleotides) in an ordered two-dimensional matrix. Microarrays can be prepared by synthesizing DNA in situ on a glass surface using combinational chemistry or by robotic microdeposition of cDNAs (0.5- to 2-kb) amplified by PCR. The sample DNA, usually bound to a fluorescent or enzyme label, is exposed to the microarray and hybridizes with the target sequences. The detection of the probe-target hybrid at each spot on the array is achieved either by direct fluorescence scanning or enzyme-mediated detection yielding a semi-quantitative result. Advantages of DNA microarray technology, as compared to other techniques, include the small size of the array allowing for a higher sensitivity, the ability to simultaneously detect diverse individual sequences in complex DNA samples, and the capacity to do comparative analysis of a large number of samples. Indeed, PCR-microarrays have been used to measure relative concentrations of microbes from water, to characterize microbial communities from environmental samples and to detect bacterial pathogens from a variety of sources, including water. However, obstacles such as sample size, matrix-associated inhibitors, nonspecific binding and cross-hybridization must be overcome before microarrays can be used generally for the detection and differentiation of pathogens in environmental samples. Moreover, microarrays cannot distinguish between nonviable, culturable and viable but nonculturable (VBNC) cells since DNA can persist for long periods of time after the death of cells. The ability to discriminate between living and nonliving cells is important in order to interpret the risk associated with the detection of pathogenic microbes (especially in environmental samples). The use of mRNA, which is highly labile with a short half-life, or the use of highly expressed targets may be selected to provide sensitive analyses.

from Theron et al. in Nanotechnology in Water Treatment Applications

Traditionally, prediction of the presence of human enteric pathogens in water has been achieved by monitoring for established microbial "indicators" of fecal pollution. Not necessarily pathogenic themselves, fecal coliforms, total coliforms, E. coli, enterococci and bacteriophages are all examples of organisms that when present are viewed as predictive of the potential presence of enteric pathogens, since they have the same fecal source as the pathogenic organisms. Tests for coliform bacteria are standardized and relatively easy and inexpensive to use. Consequently, they are more rapidly administered than tests determining the presence of individual pathogenic microorganisms in water. Despite being successful in predicting possible health risks in many circumstances, there are many flaws in using microbial indicators. Research has established an inability of many of these indicators to predict the presence of disease-causing viruses (such as hepatitis A and E, coxsackie viruses, echoviruses, adenoviruses and Norwalk viruses), indigenous bacteria (such as Legionella and Helicobacter), as well as parasites (such as Cryptosporidium and Giardia).

Since it would appear that conventional detection methods do not adequately assess the risk of waterborne disease, the need for powerful new tools for the detection of pathogenic microorganisms in water is becoming increasingly more important. Although several different molecular methodologies are available, these have only recently been applied in the field of water science and technology. Moreover, the rapid progress of nanotechnology and advanced nanomaterials production offers significant opportunities not only for the detection and remediation of a broad range of environmental contaminants, but also for the development of new diagnostic assays that may serve as an appealing alternative to current molecular diagnostic techniques.

from Theron et al. in Nanotechnology in Water Treatment Applications

Since their discovery, carbon nanotubes (CNTs) have attracted great attention as nanoscale building blocks for micro- and nanodevices. CNTs can be divided essentially into single-walled carbon nanotubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs) based on the principle of hybridized carbon atom layers in the walls of CNTs. Whereas SWCNTs have diameters ranging from 0.3 to 3 nm, the MWCNTs are composed of a concentric arrangement of many cylinders and can reach diameters of up to 100 nm. CNTs are considered to be ideal for use in biosensors for detecting individual biomolecules and other biological agents. Not only do they have high surface-to-volume and surface-to-weight ratios, but they are also conducting, act as electrodes, and can be derivatized with functional groups that allow immobilization of biomolecules.

The use of nano-size electrodes based on CNTs has the potential to greatly improve the sensitivity in recognizing DNA hybridization events. Li et al. developed DNA microarrays containing sensing pads constructed from MWCNTs and built into a matrix within a silicon nitride template. The upper (open) ends of the tubes act as nanoelectrodes and are functionalized with ssDNA probes. Target DNA that hybridizes to the ssDNA probe on the ends of the electrically conductive MWCNTs is detected using an electrochemical method that relies on guanine oxidation. The hybridization of less than a few attomoles of oligonucleotide targets was demonstrated. In an alternative approach, CNTs coated with alkaline phosphatase enzymes was used for the detection of amplified DNA. This assay employs a magnetic microparticle modified with oligonucleotides that are complementary to one-half of the target DNA sequence, and alkaline phosphatase-coated carbon nanotubes that are modified with oligonucleotides that are complementary to the other half of the target DNA sequence. Binding of the target DNA promotes the formation of a magnetic microparticle/target/carbon nanotube sandwich, which is magnetically separated from the assay medium. After separation, the enzyme substrate alpha-naphthyl phosphate is added to the mixture, resulting in formation of alpha-naphthol product that is ultimately detected at a CNT-modified electrode via chronopotentiometric stripping. This method detected target DNA at concentrations as low as 54 aM.

In addition to their potential in recognizing DNA hybridization events, the use of CNTs displaying ligands for the capturing or recognition of bacterial pathogens has recently been described. The solubilization of SWCNTs via fictionalization with derivatized galactose has been reported and it was subsequently shown that the nanotube-bound galactose could serve as polyvalent ligands that strongly interacted with receptors on E. coli O157:H7, resulting in significant cell agglutination. This work has subsequently been extended to the preparation of immuno-carbon tubes. For this purpose, SWCNTs and MWCNTs are functionalized with bovine serum albumin (BSA) to attain aqueous solubility and then further conjugated with an E. coli O157:H7-specific antibody to form immuno-carbon tubes. Limited quantitative data was provided, but the results suggest that the immuno-carbon tubes are capable of sensitively capturing the target bacteria.

While CNTs currently are not as easily functionalized as QDs or nanoparticles, they offer the distinct advantage of rapid, real-time detection and may thus become viable options as nanostructured biodiagnostic devices. In addition to challenges at the fabrication level (e.g., production of pure and uncontaminated nanotubes is costly, continuous growth of defect-free CNTs to macroscopic lengths is difficult to obtain and dispersion of CNTs onto a polymer matrix is very difficult), another important issue related to the use of CNTs is their toxicity. Although results suggest that chemically modifying CNTs can reduce their cytotoxicity to a certain extent, more research is required to address the effect of CNTs on biological systems, as well as information related to safety issues.

from Theron et al. in Nanotechnology in Water Treatment Applications

In addition to QDs, luminescent dye-doped silica nanoparticles (FloDots), which consist of luminescent organic or inorganic dye molecules dispersed inside a silica matrix, have also been developed for ultra-sensitive bioanalysis and diagnostics. Owing to the silica matrix shielding effect, the doped dye molecules are protected from environmental oxygen, enabling the fluorescence to be constant and thus providing an accurate measurement for bioanalysis. Moreover, the silica matrix provides a versatile substrate for surface immobilization and various biorecognition molecules, including oligonucleotides and antibodies, have been conjugated to FloDots.

FloDots have been used successfully for the detection of DNA hybridization and in immunoassays. Using FloDots functionalized with oligonucleotides as labels for chip-based sandwich DNA assays, Zhao et al. reported a detection limit of 1 fM target DNA. The high sensitivity of the assay can be ascribed to the fact that each FloDot has the fluorescence intensity of thousands of dye molecules; therefore, each gene hybridization is reported by thousands of fluorophores. FloDot-immunoassays, using FloDots conjugated with monoclonal antibodies specific for the O-antigen of E. coli O157:H7, have also been developed to achieve rapid E. coli O157:H7 detection at the single-cell level. The fluorescence intensity emitted by one E. coli O157:H7 cell was sufficient to be detected using a normal spectrofluorometer in a conventional plate-based immunological assay, or to be accurately enumerated using a flow cytometer within 1 min of sample preparation. Although the use of FloDots represents an improvement over conventional fluorophore-based assays, they remain somewhat limited by the fundamental drawbacks of conventional fluorophores, including broad adsorption and emission profiles, which ultimately limit multiplexing capabilities.