Tracy Melvin

Design and theoretical evaluation of a novel microfluidic device to be used for PCR

The design of a novel, microfluidic chip with an integrated micro peristaltic pump and chambers for DNA amplification is described. This chip contains three reaction chambers stable at 90 °C, 72 °C and 55 °C for PCR amplification, a bi-directional peristaltic pump and optical integrated detection of the droplet. A reactant droplet is to be introduced into the device, pumped back and forth between the chambers by the micro peristaltic pump for sample processing. The static behaviour of the micro pump was modelled theoretically in order to evaluate the optimal dimensions for the pump membranes and to obtain the maximum flow rate. Thermal analysis by the finite element method was performed to optimize the location of the heaters and the temperature uniformity over the three reaction chambers. Transient thermal analysis indicates that the reactant droplet can be heated/cooled in the proposed device in less than 1 s to achieve the desired temperatures.

Investigation for the operation of an integrated peristaltic micropump

An investigation into the pumping flow rates and the time-resolved membrane actuation of a microperistaltic pump integrated within a micro total analysis system (µTAS) is presented. The results include (i) the design of the driver circuit to operate the peristaltic micropump, (ii) Michelson interferometer measurements of the pump displacement and (iii) pump flow rate measurements. The peristaltic micropump, configured with three PZT actuated glass membranes and silicon channels, is integrated within the µTAS device with microfluidic reaction chambers. The micropump pumps a 1 µl droplet back and forth between the reaction chambers.

Oligonucleotide-conjugated Thiazole Orange Probes as “Light-up” Probes for Messenger Ribonucleic Acid Molecules in Living Cells¶

Abstract “Light-up” probes, icosa-α-thymidylate-thiazole orange conjugates, for the in situ time-resolved detection of messenger ribonucleic acid (mRNA) in living cells are evaluated. Upon annealing with polyA in aqueous solutions, the icosa-α-thymidylate-thiazole orange conjugates were shown to be up to 15 times more fluorescent. Microinjection of these probes into adherent fibroblasts resulted in high yields of hybridization and fluorescent signals. Incubation of cells in the presence of these probes resulted in facile internalization of the probe and similar painting of the messenger RNA in the nuclear and cytosolic regions.

Fluorescent Properties of Oligonucleotide-conjugated Thiazole Orange Probes¶

The fluorescence properties of thiazole orange, linked via a ( 1 ) hydrophobic alkyl or a ( 2 ) hydrophilic ethylene glycol chain to the central internucleotidic phosphate group of a pentadeca‐2′‐deoxyriboadenylate (dA15), are evaluated. Linkage at the phosphate group yields two stereoisomers, S‐isomer of the phosphorus chiral center (Sp) and R‐isomer of the phosphorus chiral center (Rp); these are studied separately. The character of the linkage chain and the chirality of the internucleotidic phosphate linkage site influence the fluorescent properties of these thiazole orange–oligonucleotide conjugates (TO‐probes). Quantum yields of fluorescence (Φfl) of between 0.04 and 0.07 were determined for the single‐stranded conjugates. The fluorescence yield increased by up to five times upon hybridization with the complementary sequence (d5′[CACT15CAC3′]); Φfl values of between 0.06–0.35 were determined for the double‐stranded conjugates. The Φfl value (0.17) of thiazole orange, 1‐(N,N′‐trimethylaminopropyl)‐4‐[3‐methyl‐2,3‐dihydro‐(benzo‐1,3‐thiazole)‐2‐methylidene]‐quinolinium iodide (TO‐Pro 1) in the presence of the oligonucleotide duplex (TO‐Pro 1: dA15·d5′[CACT15CAC3′] (1:1)) is much less than that for some of the hybrids of the conjugates. Our studies, using steady‐state and time‐resolved fluorescence experiments, show that a number of discrete fluorescent association species between the thiazole orange and the helix are formed. Time‐resolved studies on the four double‐stranded TO‐probes revealed that the fluorescent oligonucleotide–thiazole orange complexes are common, only the distribution of the species varies with the character of the chain and the chirality at the internucleotidic phosphate site. Those TO‐probes in which the isomeric structure of the phosphate‐chain linkage is Rp, and therefore such that the fluorophore is directed toward the minor groove, have higher Φfl values than the Sp isomer. Of the systems studied, thiazole orange linked by an alkyl chain to the internucleotidic phosphate (Rp isomer) has the highest Φfl and the greatest fraction of the longest‐lived fluorescent thiazole orange species (in the hybrid form).

Optical propulsion of mammalian eukaryotic cells on an integrated channel waveguide

The optical propulsion of mammalian eukaryotic cells along the surface of an integrated channel waveguide is demonstrated. 10μm diameter polymethylmethacrylate (PMMA) spherical particles and similarly sized mammalian eukaryotic cells in aqueous medium are deposited in a reservoir over a caesium ion-exchanged channel waveguide. Light from a fibre laser at 1064nm was coupled into the waveguide, causing the polymer particles or cells to be propelled along the waveguide at a velocity which is dependent upon the laser power. A theoretical model was used to predict the propulsion velocity as a function of the refractive index of the particle. The experimental results obtained for the PMMA particles and the mammalian cells show that for input powers greater than 50mW the propulsion velocity is approximately that obtained by the theoretical model. For input powers of less than ~50mW neither particles nor cells were propelled; this is considered to be a result of surface forces (which are not considered in the theoretical model). The results are discussed in light of the potential application of optical channel waveguides for bioanalytical applications, namely in the identification and sorting of mammalian cells from mixed populations without the need for fluorescence or antibody labels.

Polyglutamine aggregate structure in vitro and in vivo; new avenues for coherent anti-stokes raman scattering microscopy

Coherent anti-Stokes Raman scattering (CARS) microscopy is applied for the first time for the evaluation of the protein secondary structure of polyglutamine (polyQ) aggregates in vivo. Our approach demonstrates the potential for translating information about protein structure that has been obtained in vitro by X-ray diffraction into a microscopy technique that allows the same protein structure to be detected in vivo. For these studies, fibres of polyQ containing peptides (D2Q15K2) were assembled in vitro and examined by electron microscopy and X-ray diffraction methods; the fibril structure was shown to be cross β-sheet. The same polyQ fibres were evaluated by Raman spectroscopy and this further confirmed the β-sheet structure, but indicated that the structure is highly rigid, as indicated by the strong Amide I signal at 1659 cm−1. CARS spectra were simulated using the Raman spectrum taking into account potential non-resonant contributions, providing evidence that the Amide I signal remains strong, but slightly shifted to lower wavenumbers. Combined CARS (1657 cm−1) and multi-photon fluorescence microscopy of chimeric fusions of yellow fluorescent protein (YFP) with polyQ (Q40) expressed in the body wall muscle cells of Caenorhabditis elegans nematodes (1 day old adult hermaphrodites) revealed diffuse and foci patterns of Q40-YFP that were both fluorescent and exhibited stronger CARS (1657 cm−1) signals than in surrounding tissues at the resonance for the cross β-sheet polyQ in vitro.

Multi-modal particle manipulator to enhance bead-based bioassays

By sequentially pushing micro-beads towards and away from a sensing surface, we show that ultrasonic radiation forces can be used to enhance the interaction between a functionalised glass surface and polystyrene micro-beads, and identify those that bind to the surface by illuminating bound beads using an evanescent field generated by guided light. The movement towards and immobilisation of streptavidin coated beads onto a biotin functionalised waveguide surface is achieved by using a quarter-wavelength mode pushing beads onto the surface, while the removal of non-specifically bound beads uses a second quarter-wavelength mode which exhibits a kinetic energy maximum at the boundary between the carrier layer and fluid, drawing beads towards this surface. This has been achieved using a multi-modal acoustic device which exhibits both of these quarter-wavelength resonances. Both 1-D acoustic modelling and finite element analysis has been used to design this device and to investigate the spatial uniformity of the field. We demonstrate experimentally that 90% of specifically bound beads remain attached after applying ultrasound, with 80% of non-specifically bound control beads being successfully removed acoustically. This approach overcomes problems associated with lengthy sedimentation processes used for bead-based bioassays and surface (electrostatic) forces, which delay or prevent immobilisation. We explain the potential of this technique in the development of DNA and protein assays in terms of detection speed and multiplexing.

Efficient self-assembly of DNA-functionalized fluorophores and gold nanoparticles with DNA functionalized silicon surfaces: the effect of oligomer spacers.

Although strategies for the immobilization of DNA oligonucleotides onto surfaces for bioanalytical and top-down bio-inspired nanobiofabrication approaches are well developed, the effect of introducing spacer molecules between the surface and the DNA oligonucleotide for the hybridization of nanoparticle–DNA conjugates has not been previously assessed in a quantitative manner. The hybridization efficiency of DNA oligonucleotides end-labelled with gold nanoparticles (1.4 or 10 nm diameter) with DNA sequences conjugated to silicon surfaces via hexaethylene glycol phosphate diester oligomer spacers (0, 1, 2, 6 oligomers) was found to be independent of spacer length. To quantify both the density of DNA strands attached to the surfaces and hybridization with the surface-attached DNA, new methodologies have been developed. Firstly, a simple approach based on fluorescence has been developed for determination of the immobilization density of DNA oligonucleotides. Secondly, an approach using mass spectrometry has been created to establish (i) the mean number of DNA oligonucleotides attached to the gold nanoparticles and (ii) the hybridization density of nanoparticle–oligonucleotide conjugates with the silicon surface–attached complementary sequence. These methods and results will be useful for application with nanosensors, the self-assembly of nanoelectronic devices and the attachment of nanoparticles to biomolecules for single-molecule biophysical studies.

World-to-chip interconnects for efficient loading of genomic DNA into microfluidic channels

A novel sloped interconnect for the efficient delivery of long genomic DNA fragments into a microfluidic channel is designed, fabricated and tested. Out-of-plane slopes are fabricated in silicon wafers using the deep reactive-ion etch lag phenomenon and a combination of anisotropic and isotropic etching. The final structure is capped with anodically bonded glass. Based upon a series of etch-lag calibration studies, the interconnect was designed using finite element analysis to provide a channel with flow acceleration properties appropriate to straighten DNA molecules. The efficiency of transit of the 48.5 kb DNA fragments (~16.5 µm long when fully extended) through the microfluidic device, established using quantitative real-time polymerase chain reaction, is 95 ± 7.3%

Design and fabrication of a quasi-ordered nanoporous silicon membrane suitable for thermally induced drug release

The design and fabrication of an ordered nanoporous silicon membrane and integrated heater and temperature sensor is described. The methodology for fabrication of the nanoporous structure has been developed for integration within microelectromechanical systems. The structure is fabricated from a 500 µm thick silicon (1 0 0) wafer, which has been etched to provide 4 × 4 mm2 membranes of 50 µm thick. Quasi-ordered nanoporous silicon is created within the membrane, the nanopores are of uniform diameter (typical structures of the order of 105 ±5 nm) and smooth sidewalls to a depth of ~300 nm, in a hexagonal close-packed pattern of 200 nm nearest neighbour. The porosity of typical fabricated samples is 31.5%. On the back side of the membrane, a heater and a temperature sensor are incorporated. Three different heater/temperature sensor designs were considered theoretically and the best design was then fabricated and studied experimentally. The results obtained provide both highly ordered nanoporous silicon fabrication methodology as well as evidence that the porous membrane can be heated without deleterious effect.