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

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

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

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.