Smartphone technology facilitates point-of-care nucleic acid diagnosis: a beginner's guide.

The reliable detection of nucleic acids at low concentrations in clinical samples like blood, urine and saliva, and in food can be achieved by nucleic acid amplification methods. Several portable and hand-held devices have been developed to translate these laboratory-based methods to point-of-care (POC) settings. POC diagnostic devices could potentially play an important role in environmental monitoring, health, and food safety. Use of a smartphone for nucleic acid testing has shown promising progress in endpoint as well as real-time analysis of various disease conditions. The emergence of smartphone-based POC devices together with paper-based sensors, microfluidic chips and digital droplet assays are used currently in many situations to provide quantitative detection of nucleic acid targets. State-of-the-art portable devices are commercially available and rapidly emerging smartphone-based POC devices that allow the performance of laboratory-quality colorimetric, fluorescent and electrochemical detection are described in this review. We present a comprehensive review of smartphone-based POC sensing applications, specifically on microbial diagnostics, assess their performance and propose recommendations for the future.

A novel framework for the cell-free enzymatic production of glucaric acid.

Glucaric acid (GlucA) is a valuable glucose-derived chemical with promising applications as a biodegradable and biocompatible chemical in the manufacturing of plastics, detergents and drugs. Recently, there has been a significant focus on producing GlucA microbially (in vivo) from renewable materials such as glucose, sucrose and myo-inositol. However, these in vivo GlucA production processes generally lack efficiency due to toxicity problems, metabolite competition and suboptimal enzyme ratios. Synthetic biology and accompanying cell-free biocatalysis have been proposed as a viable approach to overcome many of these limitations. However, cell-free biocatalysis faces its own limitations for industrial applications due to high enzyme costs and cofactor consumption. We have constructed a cell-free GlucA pathway and demonstrated a novel framework to overcome limitations of cell-free biocatalysis by i) the combination of both thermostable and mesophilic enzymes, ii) incorporation of a cofactor regeneration system and iii) immobilisation and recycling of the pathway enzymes. The cell-free production of GlucA was achieved from glucose-1-phosphate with a titre of 14.1 ±â€¯0.9 mM (3.0 ±â€¯0.2 g l-1) and a molar yield of 35.2 ±â€¯2.3% using non-immobilised enzymes, and a titre of 8.1 ±â€¯0.2 mM (1.70 ±â€¯0.04 g l-1) and a molar yield of 20.2 ±â€¯0.5% using immobilised enzymes with a total reaction time of 10 h. The resulting productivities (0.30 ±â€¯0.02 g/h/l for free enzymes and 0.170 ±â€¯0.004 g/h/l for immobilised enzymes) are the highest productivities so far reported for glucaric acid production using a synthetic enzyme pathway.

Solid-Binding Peptides in Biomedicine.

Some peptides are able to bind to inorganic materials such as silica and gold. Over the past decade, Solid-binding peptides (SBPs) have been used increasingly as molecular building blocks in nanobiotechnology. These peptides show selectivity and bind with high affinity to a diverse range of inorganic surfaces e.g. metals, metal oxides, metal compounds, magnetic materials, semiconductors, carbon materials, polymers and minerals. They can be used in applications such as protein purification and synthesis, assembly and the functionalization of nanomaterials. They offer simple and versatile bioconjugation methods that can increase biocompatibility and also direct the immobilization and orientation of nanoscale entities onto solid supports without impeding their functionality. SBPs have been employed in numerous nanobiotechnological applications such as the controlled synthesis of nanomaterials and nanostructures, formation of hybrid biomaterials, immobilization of functional proteins and improved nanomaterial biocompatibility. With advances in nanotechnology, a multitude of novel nanomaterials have been designed and synthesized for diagnostic and therapeutic applications. New approaches have been developed recently to exert a greater control over bioconjugation and eventually, over the optimal and functional display of biomolecules on the surfaces of many types of solid materials. In this chapter we describe SBPs and highlight some selected examples of their potential applications in biomedicine.

Efficient capture of pathogens with a zeolite matrix.

We show that a peptide linker sequence expressed as part of a fusion with protein G from Streptococcus binds to natural zeolite giving a complex to which a suitable antibody shows affinity binding. As a consequence, addition of the CRY104 antibody specific for an external surface epitope of Cryptosporidium allows the capture of this protozoan pathogen at high efficiency with the advantage of rapid concentration from water samples. The natural zeolite with the specific antibody attached was incorporated into a single and double zeolite column system. The results reported here show that it is possible to capture Cryptosporidium oocysts with outstanding efficiency (>90 %) from water and water incorporating QC-MUD using a size-sorted natural zeolite. The natural zeolite matrix not only allows for high flow rates but also for flexibility of column design and volume of water for sample collection. The system is versatile and it is possible to prepare columns with more than one specific surface antibody to allow the capture of one or two pathogens simultaneously.

Universal Enzyme-Based Field Workflow for Rapid and Sensitive Quantification of Water Pathogens.

A universal filtration and enzyme-based workflow has been established to allow for the rapid and sensitive quantification of leading pathogens Cryptosporidium parvum, Giardia gamblia, Campylobacter jejuni, and Escherichia coli from tap water samples with volumes up to 100 mL, and the potential to scale up to larger volumes. qPCR limits of quantification as low as four oocysts for Cryptosporidium, twelve cysts for Giardia, two cells for C. jejuni, and nineteen cells for E. coli per reaction were achieved. A polycarbonate filter-based sampling method coupled with the prepGEM enzyme-based DNA extraction system created a single-step transfer workflow that required as little as 20 min of incubation time and a 100 µL reaction mix. The quantification via qPCR was performed directly on the prepGEM extract, bypassing time-consuming, labour-intensive conventional culture-based methods. The tap water samples were shown to contain insoluble particles that inhibited detection by reducing the quantification efficiency of a representative pathogen (C. jejuni) to 30-60%. This sample inhibition was effectively removed by an on-filter treatment of 20% (v/v) phosphoric acid wash. Overall, the established workflow was able to achieve quantification efficiencies of 92% and higher for all four leading water pathogens, forming the basis of a rapid, portable, and low-cost solution to water monitoring.

Applications of flow cytometry in environmental microbiology and biotechnology.

Flow cytometry (FCM) is a technique for counting, examining and sorting microscopic particles suspended in a stream of fluid. It uses the principles of light scattering, light excitation and the emission from fluorescent molecules to generate specific multiparameter data from particles and cells. The cells are hydrodynamically focussed in a sheath solution before being intercepted by a focused light source provided by a laser. FCM has been used primarily in medical applications but is being used increasingly for the examination of individual cells from environmental samples. It has found uses in the isolation of both culturable and hitherto non-culturable bacteria present infrequently in environmental samples using appropriate growth conditions. FCM lends itself to high-throughput applications in directed evolution for the analysis of single cells or cell populations carrying mutant genes. It is also suitable for encapsulation studies where individual bacteria are compartmentalised with substrate in water-in-oil-in-water emulsions or with individual genes in transcriptional/translational mixtures for the production of mutant enzymes. The sensitivity of the technique has allowed the examination of gene optimisation by a procedure known as random or neutral drift where screening and selection is based on the retention of some predetermined level of activity through multiple rounds of mutagenesis.

Molecular diversity and catalytic activity of Thermus DNA polymerases.

Thermus aquaticus DNA polymerase (Taq polymerase) made the polymerase chain reaction feasible and led to a paradigm shift in genomic analysis. Other Thermus polymerases were reported to have comparable performance in PCR and there was an analysis of their properties in the 1990s. We re-evaluated our earlier phylogeny of Thermus species on the basis of 16S rDNA sequences and concluded that the genus could be divided into eight clades. We examined 22 representative isolates and isolated their DNA polymerase I genes. The eight most diverse polymerase genes were selected to represent the eight clades and cloned into an expression vector coding for a His-tag. Six of the eight polymerases were expressed so that there was sufficient protein for purification. The proteins were purified to homogeneity and examination of the biochemical characteristics showed that although they were competent to perform PCR, none was as thermostable as commercially available Taq polymerase; all had similar error-frequencies to Taq polymerase and all showed the expected 5'-3' exonuclease activity. We conclude that the initial selection of T. aquaticus for DNA polymerase purification was a far-reaching and fortuitous choice but simple mutagenesis procedures on other Thermus-derived polymerases should provide comparable thermostability for the PCR reaction.

A portable nucleic acid detection system using natural convection combined with a smartphone.

The development of portable nucleic acid diagnostic devices has the potential to expand the availability of molecular diagnostics into low-resource settings. One of the promising solutions for rapid and simple DNA amplification is the use of Rayleigh-Bernard natural convection which is caused by a buoyancy-driven thermal gradient of liquid when heated from below. This natural convection avoids the use of the complex and sophisticated hardware that is required for precise maintenance of temperature cycles in conventional PCR. We have developed a stand-alone convective PCR (cPCR) device linked to a smartphone for rapid detection of nucleic acids using natural convection heating. The device amplifies multiple DNA samples simultaneously using a custom-made heat block controlled by Bluetooth wireless communication. The entire device is highly portable, user-friendly, battery-operated and can provide target DNA amplification in less than 30 min. A detection limit of 2.8 × 103 copies of a segment of lambda DNA was obtained when the two different fluorescently-tagged amplicons were collected magnetically and detected using the smartphone fluorescence reader. Thus, the combination of cPCR and multiplex fluorescence-based detection on a smartphone provides new opportunities for the development of affordable and portable molecular diagnostic devices for point-of-care situations or remote clinical settings.

Tools and strategies for constructing cell-free enzyme pathways.

Single enzyme systems or engineered microbial hosts have been used for decades but the notion of assembling multiple enzymes into cell-free synthetic pathways is a relatively new development. The extensive possibilities that stem from this synthetic concept makes it a fast growing and potentially high impact field for biomanufacturing fine and platform chemicals, pharmaceuticals and biofuels. However, the translation of individual single enzymatic reactions into cell-free multi-enzyme pathways is not trivial. In reality, the kinetics of an enzyme pathway can be very inadequate and the production of multiple enzymes can impose a great burden on the economics of the process. We examine here strategies for designing synthetic pathways and draw attention to the requirements of substrates, enzymes and cofactor regeneration systems for improving the effectiveness and sustainability of cell-free biocatalysis. In addition, we comment on methods for the immobilisation of members of a multi-enzyme pathway to enhance the viability of the system. Finally, we focus on the recent development of integrative tools such as in silico pathway modelling and high throughput flux analysis with the aim of reinforcing their indispensable role in the future of cell-free biocatalytic pathways for biomanufacturing.

Mixed-mode liquid chromatography for the rapid analysis of biocatalytic glucaric acid reaction pathways.

Glucaric acid (GlucA) has been identified as one of the top 10 potential bio-based chemicals for replacement of oil-based chemicals. Several synthetic enzyme pathways have been engineered in bacteria and yeast to produce GlucA from glucose and myo-inositol. However, the yields and titres achieved with these systems remain too low for the requirements of a bio-based GlucA industry. A major limitation for the optimisation of GlucA production via synthetic enzymatic pathways are the laborious analytical procedures required to detect the final product (GlucA) and pathway intermediates. We have developed a novel method for the simple and simultaneous analysis of GlucA and pathway intermediates to address this limitation using mixed mode (MM) HILIC and weak anion exchange chromatography (WAX), referred to as MM HILIC/WAX, coupled with RID. Isocratic mobile phase conditions and the sample solvent were optimised for the separation of GlucA, glucose-1-phosphate (G1P), glucose-6-phosphate (G6P), inositol-1-phosphate (I1P), myo-inositol and glucuronic acid (GA). The method showed good repeatability, precision and excellent accuracy with detection and quantitation limits (LOD and LOQ) of 1.5-2 and 577 mM, respectively. The method developed was used for monitoring the enzymatic synthesis of the final step in the GlucA pathway, and showed that GlucA was produced from GA with near 100% conversion and a titre of 9.2 g L-1.

Potential use of quantum dots in flow cytometry.

QDs may offer significant advantages in environmental and bead-based applications where the target cells need to be discriminated above background fluorescence. We have examined the possible applications of QDs for flow cytometric measurements (FCM) by studying their excitation - emission spectra and their binding to paramagnetic beads. We labelled beads with either QDs or a commonly-used fluorochrome (FITC) and studied their fluorescence intensity by FCM. Flow cytometric comparisons indicated that the minimum fluorophore concentration required for detection of QDs above autofluorescent background was 100-fold less than for FITC.

Reverse transcriptases: intron-encoded proteins found in thermophilic bacteria.

A number of thermophilic bacteria have been surveyed for possessing reverse transcriptase genes using a degenerate primer approach derived from an alignment of known group II intron encoded reverse transcriptases (RT) from mesophilic prokaryotes and eukaryotes. Six out of 34 thermophilic isolates gave a PCR product that was indicative of an RT internal fragment on sequencing. A putative RT from Bacillus caldolyticus strain EA1 was isolated by genomic walking and cloned into an Escherichia coli expression vector. The recombinant protein proved to be insoluble and was unable to be recovered from the insoluble fraction of lysates of E. coli. The RT was successfully expressed in a baculovirus vector although yields remained low. We followed RT activity during purification using the poly(rC)*p(dG)(12-18), which specifically detects only RNA-dependent DNA polymerase activity. We could not detect incorporation of dTTP into poly(rC) )*p(dG)(12-18) when using uninfected Sf21 lysates and conclude that the substrate is not a template for DNA-dependent DNA polymerase. Although a high level of RT activity was detected in the total cell protein, when compared to the activity detected in the soluble fraction, only about 10% of the activity was soluble. Sequence comparisons showed significant differences between the EA1-IEP and a Geobacillus RT expressed by others. We conclude that it may be necessary to isolate the IEP RT as a ribonucleoprotein to obtain sufficient material for further analysis.

Degenerate oligonucleotide gene shuffling.

Improvement of the biochemical characteristics of enzymes has been aided by misincorporation mutagenesis and DNA shuffling. Shuffling techniques can be used on a collection of mutants of the same gene, or related families of genes can be shuffled to produce mutants encoding chimeric gene products. One difficulty with current shuffling procedures is the predominance of unshuffled ("parental") molecules in the pool of mutants. We describe a procedure for gene shuffling using degenerate primers that allows control of the relative levels of recombination between the genes that are shuffled and reduces the regeneration of unshuffled parental genes. This procedure has the advantage of avoiding the use of endonucleases for gene fragmentation before shuffling and allows the use of random mutagenesis of selected segments of the gene as part of the procedure. We illustrate the use of the technique with a diverse family of beta-xylanase genes that possess widely different G and C contents.

Solid-binding peptides for immobilisation of thermostable enzymes to hydrolyse biomass polysaccharides.

BACKGROUND: Solid-binding peptides (SBPs) bind strongly to a diverse range of solid materials without the need for any chemical reactions. They have been used mainly for the functionalisation of nanomaterials but little is known about their use for the immobilisation of thermostable enzymes and their feasibility in industrial-scale biocatalysis. RESULTS: A silica-binding SBP sequence was fused genetically to three thermostable hemicellulases. The resulting enzymes were active after fusion and exhibited identical pH and temperature optima but differing thermostabilities when compared to their corresponding unmodified enzymes. The silica-binding peptide mediated the efficient immobilisation of each enzyme onto zeolite, demonstrating the construction of single enzyme biocatalytic modules. Cross-linked enzyme aggregates (CLEAs) of enzyme preparations either with or without zeolite immobilisation displayed greater activity retention during enzyme recycling than those of free enzymes (without silica-binding peptide) or zeolite-bound enzymes without any crosslinking. CLEA preparations comprising all three enzymes simultaneously immobilised onto zeolite enabled the formation of multiple enzyme biocatalytic modules which were shown to degrade several hemicellulosic substrates. CONCLUSIONS: The current work introduced the construction of functional biocatalytic modules for the hydrolysis of simple and complex polysaccharides. This technology exploited a silica-binding SBP to mediate effectively the rapid and simple immobilisation of thermostable enzymes onto readily-available and inexpensive silica-based matrices. A conceptual application of biocatalytic modules consisting of single or multiple enzymes was validated by hydrolysing various hemicellulosic polysaccharides.

Improvement of the secretion of extracellular proteins and isolation and characterization of the amylase I (amy1) gene from Ophiostoma floccosum.

UV mutagenesis was applied to improve protein secretion in Ophiostoma floccosum. Amylase activity was used as an indicator for enhanced protein production after repeated rounds of mutagenic treatment. The amylase activity in the culture supernatant of the best mutant (MQ.5.1) was increased by more than 240-fold compared to the initial parental strain. At the same time, the increase in total secreted protein was about six times greater than the parental strain. Secreted proteinase and lipase activities of the parental strain and four key mutants were also investigated. N-terminal sequencing of the five dominant protein bands separated by SDS-PAGE from the culture supernatant was conducted. Two of the proteins identified were subtilisin-like proteinases and one was a pepsin-like proteinase. In addition, one protein was identified as an alpha-amylase and one remained unidentified. A 6.5 kb DNA fragment was isolated by Genomic Walking PCR using primers based on the alpha-amylase amino acid sequence. The amplified fragment contained the entire gene encoding alpha-amylase (amy1) and its regulatory sequences. Analysis showed that multiple transcripts were generated from the single alpha-amylase gene locus.

A Novel Universal Detection Agent for Time-Gated Luminescence Bioimaging.

Luminescent lanthanide chelates have been used to label antibodies in time-gated luminescence (TGL) bioimaging. However, it is a challenging task to label directly an antibody with lanthanide-binding ligands and achieve control of the target ligand/protein ratios whilst ensuring that affinity and avidity of the antibody remain uncompromised. We report the development of a new indirect detection reagent to label antibodies with detectable luminescence that circumvents this problem by labelling available lysine residues in the linker portion of the recombinant fusion protein Linker-Protein G (LPG). Succinimide-activated lanthanide chelating ligands were attached to lysine residues in LPG and Protein G (without Linker) and the resulting Luminescence-Activating (LA-) conjugates were compared for total incorporation and conjugation efficiency. A higher and more efficient incorporation of ligands at three different molar ratios was observed for LPG and this effect was attributed to the presence of eight readily available lysine residues in the linker region of LPG. These Luminescence-Activating (LA-) complexes were subsequently shown to impart luminescence (upon formation of europium(III) complexes) to cell-specific antibodies within seconds and without the need for any complicated bioconjugation procedures. The potential of this technology was demonstrated by direct labelling of Giardia cysts and Cryptosporidium oocysts in TGL bioimaging.

Heterologous protein expression in filamentous fungi.

Filamentous fungi are commonly used in the fermentation industry for the large-scale production of proteins--mainly industrial enzymes. Recent advances in fungal genomics and related experimental technologies such as gene arrays and proteomics are rapidly changing the approaches to the development and use of filamentous fungi as hosts for the production of both homologous and heterologous gene products. The emphasis is moving towards sourcing new genes of interest through database mining and unravelling the circuits related to fungal gene regulation, applying, for example, transcriptomics. However, although heterologous fungal proteins are efficiently expressed, expression of gene products from other organisms is subject to several bottlenecks that reduce yield. Current approaches emphasize the study of pathways involved in protein modification and degradation in general rather than gene-by-gene approaches.

Degenerate oligonucleotide gene shuffling (DOGS) and random drift mutagenesis (RNDM): two complementary techniques for enzyme evolution.

Improvement of the biochemical characteristics of enzymes has been aided by misincorporation mutagenesis and DNA shuffling. Many gene shuffling techniques result predominantly in the regeneration of unshuffled (parental) molecules. We describe a procedure for gene shuffling using degenerate primers that allows control of the relative levels of recombination between the genes that are shuffled, and reduces the regeneration of unshuffled parental genes. This shuffling procedure avoids the use of endonucleases for gene fragmentation prior to shuffling and allows the inclusion of random mutagenesis of selected portions of the chimeric genes as part of the procedure. We illustrate the use of the shuffling technique with a family of beta-xylanase genes that possess widely different G+C contents. In addition, we introduce a new method (RNDM) for rapid screening of mutants from libraries where no adaptive selection has been imposed on the cells. They are identified only by their retention of enzymatic activity. The combination of RNDM followed by DOGS allows a comprehensive exploration of a protein's functional sequence space.

Solid-binding peptides: smart tools for nanobiotechnology.

Over the past decade, solid-binding peptides (SBPs) have been used increasingly as molecular building blocks in nanobiotechnology. These peptides show selectivity and bind with high affinity to the surfaces of a diverse range of solid materials including metals, metal oxides, metal compounds, magnetic materials, semiconductors, carbon materials, polymers, and minerals. They can direct the assembly and functionalisation of materials, and have the ability to mediate the synthesis and construction of nanoparticles and complex nanostructures. As the availability of newly synthesised nanomaterials expands rapidly, so too do the potential applications for SBPs.

Isolation, characterization and expression of the hex1 gene from Trichoderma reesei.

Polymers of the HEX1 protein produce Woronin bodies in filamentous fungi. We have isolated and sequenced the hex1 gene and flanking regions from the industrially exploited fungus Trichoderma reesei. Multiple transcription start sites (TSS) and the 5' untranslated region (UTR) were identified by 5'RACE PCR. There are three hex1 transcript types, two of which originate from two TSSs at approximately -320 and -1335 from the start codon, which are separated by a 500-bp intron within the 5'UTR. The third transcript type results from alternative splicing of the intron within the coding sequence at the 3' end, which results in the inclusion or exclusion of an unconserved histidine-rich coding region. The three transcripts code for two forms of HEX1 protein. N-terminal sequencing of HEX1 separated by 2D gel electrophoresis confirms that there are two forms of HEX1 protein which are modified further by alternative cleavage of the N-terminus. The dominant form of HEX1 is coded by a cDNA with TSS at position -1335. Expression of hex1 on cellulase-inducing medium peaks strongly within 24 h of growth but the protein is expressed at a lower and more consistent level in medium containing glucose. This is the first investigation of expression of the hex1 gene encoding a protein unique to filamentous fungi.