Dmitriy A. Khodakov

Surface modification for PDMS-based microfluidic devices.

This review focuses on advances reported from April 2009 to May 2011 in PDMS surface modifications for the application in microfluidic devices. PDMS surface modification techniques presented here include improved plasma and graft polymer coating, dynamic surfactant treatment, hydrosilylation‐based surface modification and surface modification with nanomaterials such as carbon nanotubes and metal nanoparticles. Recent efforts to generate topographical and chemical patterns on PDMS are also discussed. The described surface modifications not only increase PDMS wettability, inhibit or reduce non‐specific adsorption of hydrophobic species onto the surfaces in the act, but also result in the display of desired functional groups useful for molecular separations, biomolecular detection via immunoassays, cell culture and emulsion formation.

Toehold-mediated nonenzymatic DNA strand displacement as a platform for DNA genotyping.

Toehold-mediated DNA strand displacement provides unique advantages in the construction and manipulation of multidimensional DNA nanostructures as well as nucleic acid sequence analysis. We demonstrate a step change in the use of toehold-mediated DNA strand displacement reactions, where a double-stranded DNA duplex, containing a single-stranded toehold domain, enzymatically generated and then treated as a molecular target for analysis. The approach was successfully implemented for human DNA genotyping, such as gender identification where the amelogenin gene was used as a model target system, and detecting single nucleotide polymorphisms of human mitochondrial DNA. Kinetics of the strand displacement was monitored by the quenched Förster resonance energy transfer effect.

Recent developments in nucleic acid identification using solid-phase enzymatic assays

AbstractThis review (containing 101 refs.) covers recent achievements in the development of new approaches for enzymatically assisted detection of nucleic acids on microarrays. We discuss molecular techniques including the polymerase chain reaction, reverse transcription, allele specific primer extension and a range of isothermal techniques for the amplification and discrimination of nucleic acids. This also includes their implementation into microfluidic systems. These techniques all show great promise for use in the life sciences by allowing for high throughput, cost effective and highly sensitive and specific analysis of nucleic acids. Importantly, they can be potentially integrated into personalized and point-of-care medicine. FigureThis review highlights recent developments in molecular techniques including polymerase chain reaction, reverse transcription, allele specific primer extension and isothermal amplification on microarrays for the amplification and discrimination of nucleic acids. Their use in microfluidic platforms is also discussed.

Protected DNA strand displacement for enhanced single nucleotide discrimination in double-stranded DNA

Single nucleotide polymorphisms (SNPs) are a prime source of genetic diversity. Discriminating between different SNPs provides an enormous leap towards the better understanding of the uniqueness of biological systems. Here we report on a new approach for SNP discrimination using toehold-mediated DNA strand displacement. The distinctiveness of the approach is based on the combination of both 3- and 4-way branch migration mechanisms, which allows for reliable discrimination of SNPs within double-stranded DNA generated from real-life human mitochondrial DNA samples. Aside from the potential diagnostic value, the current study represents an additional way to control the strand displacement reaction rate without altering other reaction parameters and provides new insights into the influence of single nucleotide substitutions on 3- and 4-way branch migration efficiency and kinetics.

High‐performance capillary electrophoretic separation of double‐stranded oligonucleotides using a poly‐ (ethylpyrrolidine methacrylate‐co‐methyl methacrylate)‐coated capillary

Here we describe a capillary electrophoretic method for the separation of double‐stranded oligonucleotides (ds‐ODNs) ranging from 16–20 bp with 2 bp resolution using a low concentration of poly(ethylpyrrolidine methacrylate‐co‐methyl methacrylate) (PEPyM‐co‐PMMA) copolymer physically adsorbed to a capillary surface. Contrary to traditional DNA separations, we show that the ds‐ODN with the highest molecular size eluted first and propose that this phenomena is due to a screening effect by the PEPyM‐co‐PMMA coating on the smaller ds‐ODNs negative charge during elution. Key to the performance of this separation was a sample preparation time of less than 1 h and analysis time of 40 min. Repeatability of intraday migration time for the mixtures was typically < 1% relative standard deviation (n = 3). In addition, we demonstrate that the coating has an acceptable capillary lifetime of over 70 injections.

Sequence selective capture, release and analysis of DNA using a magnetic microbead-assisted toehold-mediated DNA strand displacement reaction

This paper reports on the modification of magnetic beads with oligonucleotide capture probes with a specially designed pendant toehold (overhang) aimed specifically to capture double-stranded PCR products. After capture, the PCR products were selectively released from the magnetic beads by means of a toehold-mediated strand displacement reaction using short artificial oligonucleotide triggers and analysed using capillary electrophoresis. The approach was successfully shown on two genes widely used in human DNA genotyping, namely human c-fms (macrophage colony-stimulating factor) proto-oncogene for the CSF-1 receptor (CSF1PO) and amelogenin.

Bacterial production of transparent exopolymer particles during static and laboratory-based cross-flow experiments

Biofouling of seawater reverse osmosis (SWRO) membranes represents one of the leading causes of performance deterioration in the desalination industry. This work investigates the biofouling potential of microbial communities present in a reverse osmosis (RO) feed tank. As an example, water from the RO feed tank of the Penneshaw desalination plant (Kangaroo Island, South Australia) was used in a static biofilm formation experiment. Cultures of the indigenous biofilms formed during the static experiment showed that α-Proteobacteria and γ-Proteobacteria accounted for nearly 80% of the classes of bacteria present in the RO feed tank. Pseudomonas sp. was identified as the major species and isolated for testing in static and laboratory-based cross flow biofilm formation experiments. Results showed that the volume of TEPs generated by Pseudomonas sp. during the laboratory-based cross-flow experiment was 10 fold higher to that produced during the static experiment for the same time period, while both experiments were inoculated with cell concentrations of the same order of magnitude. The availability of nutrients was also shown to be a key driver in TEP production, particularly for the static experiments. This study provides insights into the phenomenon of biofouling by assessing the production of biofouling precursors from one of the main genera of biofilm-forming bacteria, namely Pseudomonas sp.

Surface modification of poly(dimethylsiloxane) (PDMS) microchannels with DNA capture-probes for potential use in microfluidic DNA analysis systems

Poly(dimethylsiloxane) (PDMS) is an elastomeric material used for microfluidic devices and is especially suited to medical and forensic applications. This is due to its relatively low cost, ease of fabrication, excellent optical transmission characteristics and its ability to support electroosmotic flow, required during electrophoretic separations. These aspects combined with its large range of surface modification chemistries, make PDMS an attractive substrate in microfluidic devices for, in particular, DNA separation. Here, we report the successful wet chemical surface modification of PDMS microchannels using a simple three step method to produce an isothiocyanate-terminated surface. Initially, PDMS was oxygen plasma treated to produce a silanol-terminated surface, this was then reacted with 3-aminopropyltriethoxysilane with subsequent reaction of the now amine-terminated surface with p-phenylenediisothiocyanate. Water contact angle measurements both before and after modification showed a reduction in hydrophobicity from 101o for native PDMS to 94o for the isothiocyante-terminated PDMS. The isothiocyanate-terminated surface was then coupled with an amineterminated single-stranded DNA (ssDNA) oligonucleotide capture probe via a thiourea linkage. Confirmation of capture probe attachment was observed using fluorescent microscopy after hybridization of the capture probes with fluorescently labeled complimentary ssDNA oligonucleotides.

Optimisation of DNA hybridisation and toehold strand displacement from magnetic bead surfaces

Here, we present work on the improved hybridisation and release of specially designed pegylated double stranded DNA strands with a pendant toehold. For this forensically relevant DNA namely amelogenin (AMEL) (a sex determination gene) and the human c-fms (macrophage colony-stimulating factor) proto-oncogene for the CSF-1 receptor (CSF1PO) short tandem repeat (STR) were employed. Magnetic beads functionalised with oligonucleotide capture probes with complementarity to the dsDNA PCR product toehold were fabricated. Characterisation of the beads was achieved through dynamic light (DLS) scattering and attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy showing successful attachment of the oligonucleotides. The time frame of hybridisation between the dsPCR product toehold and the oligonucleotide modified beads was decreased from 6 h to 10 min by pre-incubation of the oligonucleotide functionalised magnetic beads in 2% aqueous sodium dodecyl sulphate (SDS) solution. Fluorescent microscopy was used to determine the time of toehold mediated strand displacement of the captured dsPCR product from the magnetic bead. Initially, displacement was achieved within 6 h. This was subsequently reduced to 3 h by the addition of a rate accelerator, PEG6000 at 10% v/v.

Detection of harmful algal bloom causing microalgae using covalently immobilised capture oligonucleotide probes on glass and poly(dimethylsiloxane) surfaces

Harmful algal bloom (HAB) events have been on the rise in the last few decades with some of the causative microalgae exhibiting toxic properties. Therefore, detection is essential in order to prevent mortality of aquatic life and poisoning events from consumption of these biotoxins. Here, oligonucleotide modified glass and poly(dimethylsiloxane) (PDMS) surfaces have been developed for the detection of the HAB causing microalgae, Alexandrium catenella, in a model system. Our preliminary studies show that the glass surface offers superior stability and analytical response when compared to those prepared from PDMS.