Des Brennan

A comparison of calibration methods based on calibration data size and robustness

Abstract Least squares (LS) regression, ridge regression (RR) and partial least squares (PLS) regression have been widely used in statistical calibration of near infrared (NIR) instruments. Comparison of these methods has attracted lots of interest in literature. However, most papers compare calibration methods on the basis of a single experiment and focus on “accuracy” rather than “robustness”. In “real life”, the average accuracy level of various calibration methods may not make that much of a difference, but having an extremely bad prediction may be unacceptable. As well, a calibration set may be very expensive to acquire and methods that work well on small calibration sets may be preferred. In this paper, we compare least squares regression, ridge regression and partial least squares regression in the context of the varying calibration data size. Three data sets used in this study are all NIR-based measurements of fat or protein in milk. For a given calibration data size, 100 simulation experiments are carried out, the average value and 95 percentile of the root mean squared prediction errors of each method are compared. We found that relative performance of the calibration methods depends on data set and calibration data size as well.

Silicon microfluidic flow focusing devices for the production of size-controlled PLGA based drug loaded microparticles.

The increasing realisation of the impact of size and surface properties on the bio-distribution of drug loaded colloidal particles has driven the application of micro fabrication technologies for the precise engineering of drug loaded microparticles. This paper demonstrates an alternative approach for producing size controlled drug loaded PLGA based microparticles using silicon Microfluidic Flow Focusing Devices (MFFDs). Based on the precise geometry and dimensions of the flow focusing channel, microparticle size was successfully optimised by modifying the polymer type, disperse phase (Qd) flow rate, and continuous phase (Qc) flow rate. The microparticles produced ranged in sizes from 5 to 50 μm and were highly monodisperse (coefficient of variation <5%). A comparison of Ciclosporin (CsA) loaded PLGA microparticles produced by MFFDs vs conventional production techniques was also performed. MFFDs produced microparticles with a narrower size distribution profile, relative to the conventional approaches. In-vitro release kinetics of CsA was found to be influenced by the production technique, with the MFFD approach demonstrating the slowest rate of release over 7 days (4.99 ± 0.26%). Finally, MFFDs were utilised to produce pegylated microparticles using the block co-polymer, PEG-PLGA. In contrast to the smooth microparticles produced using PLGA, PEG-PLGA microparticles displayed a highly porous surface morphology and rapid CsA release, with 85 ± 6.68% CsA released after 24h. The findings from this study demonstrate the utility of silicon MFFDs for the precise control of size and surface morphology of PLGA based microparticles with potential drug delivery applications.

Three-dimensional hydrogel structures as optical sensor arrays, for the detection of specific DNA sequences

The fabrication and characterization of surface-attached PEG-diacrylate hydrogel structures and their application as sensing platforms for the detection of specific target sequences are reported. Hydrogel structures were formed by a photopolymerization process, using substrate-bound Eosin Y molecules for the production of free radicals. We have demonstrated that this fabrication process allows for control over hydrogel growth down to the micrometer scale. Confocal imaging revealed relatively large pore structures for 25% (v/v) PEG-diacrylate hydrogels, which appear to lie in tightly packed layers. Our data suggest that these pore structures decrease in size for hydrogels with increasing levels of PEG-diacrylate. Surface coverage values calculated for hydrogels immobilized with 21-mer DNA probe sequences were significantly higher compared to those previously reported for 2- and 3-dimensional sensing platforms, on the order of 10(16)molecules cm(-2). Used as sensing platforms in DNA hybridization assays, a detection limit of 3.9 nM was achieved for hybridization reactions between 21-mer probe and target sequences. The ability of these hydrogel sensing platforms to discriminate between wild-type and mutant allele sequences was also demonstrated, down to target concentrations of 1-2 nM. A reduction in the hybridization time down to a period of 15 min was also achieved, while still maintaining confident results, demonstrating the potential for future integration of these sensing platforms within Lab-on-Chip or diagnostic devices.

Integration of a 3D hydrogel matrix within a hollow core photonic crystal fibre for DNA probe immobilization

In this paper, we demonstrate the integration of a 3D hydrogel matrix within a hollow core photonic crystal fibre (HC-PCF). In addition, we also show the fluorescence of Cy5-labelled DNA molecules immobilized within the hydrogel formed in two different types of HC-PCF. The 3D hydrogel matrix is designed to bind with the amino groups of biomolecules using an appropriate cross-linker, providing higher sensitivity and selectivity than the standard 2D coverage, enabling a greater number of probe molecules to be available per unit area. The HC-PCFs, on the other hand, can be designed to maximize the capture of fluorescence to improve sensitivity and provide longer interaction lengths. This could enable the development of fibre-based point-of-care and remote systems, where the enhanced sensitivity would relax the constraints placed on sources and detectors. In this paper, we will discuss the formation of such polyethylene glycol diacrylate (PEGDA) hydrogels within a HC-PCF, including their optical properties such as light propagation and auto-fluorescence.

Refractive index measurements in a shallow multichannel microfluidic system

Thin film interference of monochromatic radiation in transparent films is used extensively as a non-destructive technique to determine film thickness over extended surface areas. This approach may also be used with liquids where reflection at solid/liquid/solid interfaces creates an interference pattern, dependent on the liquid refractive index. Such a system facilitates refractive index measurements in low volume (nanolitre) samples, without the need for high-index structured materials forming waveguide layers. We use a planar multichannel microfluidic device fabricated with a standard photolithography technique and demonstrate its suitability for nanolitre refractive index measurements. Liquids of varied refractive indices from 1.33 to 1.35 were evaluated in the microfluidic channel, indicating a limit of detection 10?6 refractive index unit (RIU), illustrating how this approach can simultaneously monitor a number of transparent and weakly absorbing liquid samples.

Injection and manipulation of silicon microbeads in a customized microfluidic platform

The first on-chip injection and manipulation of optically encoded, silicon microbeads in a microfluidic platform is reported. Encoded microbeads of different shapes and sizes were fabricated in silicon via standard microfabrication techniques. The optical signature consisted of a series of lithographically defined bar-codes, which can be identified by a laser detection system. In-situ identification of encoded microbeads was possible at microbead velocities ≤ 50 cm per second. The microbeads can also be transported within a channel network in accordance with the encoded optical signature of each bead. The microbead transport is controlled by the laminar flow of a liquid in pressure driven microchannels. Hydrodynamic pulsing facilitated single and multiple injection of microbeads from a reservoir into the laminar fluid stream of a branched microfluidic network. Careful control of the fluid velocity and alteration of the microchannel geometry also enabled manipulation of microbead velocity. The incorporation of five pillars to retain microbeads at a specific location within the microchannel network formed the basis of a reaction chamber for on chip functionalization of microbeads. The principle of hydrodynamic switching was utlized to re-direct the transport of microbeads at a branched microfluidic network. In the final part of this research we verify that this microbead technology is suitable for detection of specific target DNA.

A hybrid approach to device integration on a genetic analysis platform

Point-of-care (POC) systems require significant component integration to implement biochemical protocols associated with molecular diagnostic assays. Hybrid platforms where discrete components are combined in a single platform are a suitable approach to integration, where combining multiple device fabrication steps on a single substrate is not possible due to incompatible or costly fabrication steps. We integrate three devices each with a specific system functionality: (i) a silicon electro-wetting-on-dielectric (EWOD) device to move and mix sample and reagent droplets in an oil phase, (ii) a polymer microfluidic chip containing channels and reservoirs and (iii) an aqueous phase glass microarray for fluorescence microarray hybridization detection. The EWOD device offers the possibility of fully integrating on-chip sample preparation using nanolitre sample and reagent volumes. A key challenge is sample transfer from the oil phase EWOD device to the aqueous phase microarray for hybridization detection. The EWOD device, waveguide performance and functionality are maintained during the integration process. An on-chip biochemical protocol for arrayed primer extension (APEX) was implemented for single nucleotide polymorphism (SNiP) analysis. The prepared sample is aspirated from the EWOD oil phase to the aqueous phase microarray for hybridization. A bench-top instrumentation system was also developed around the integrated platform to drive the EWOD electrodes, implement APEX sample heating and image the microarray after hybridization.

An integrated hybrid system for genetic analysis combining EWOD sample preparation and magnetic detection

Over the last decade microelectronic technologies have delivered significant advances in devices for point of care diagnostics. Complex microfluidic systems integrate components such as valves, pumps etc. to manipulate liquids. In recent years, the drive is to combine biochemical protocols in a single system, delivering sample in answer out. An Electrowetting on Dielectric (EWOD) device offers the possibility to move and manipulate 64nl volumes implementing biochemical processes, while the magnetic sensor facilitates hybridisation detection. We outline an injection molding approach where EWOD and magnetic devices are integrated into a hybrid microfluidic system with the potential to implement sample in answer out biological protocols.

DNA probe immobilisation using 3D hydrogel matrix in a hollow core Photonic Crystal Fibre

In this paper, we show for the first time the integration of a 3D hydrogel matrix within a hollow core Photonic Crystal Fibre (HC-PCF) and we also show the fluorescence propagation of a Cy5-labelled DNA probe immobilisation within the hydrogel formed in two different types of HC-PCF. The 3D hydrogel matrix is designed to bind with the amino-groups of biomolecules, providing higher sensitivity and selectivity than the standard 2D coverage, enabling a greater number of probe molecules to be available per unit area. The HC-PCFs, on the other hand, are designed to maximise the capture of fluorescence to improve sensitivity, providing long interaction lengths, being easily integrated with light sources and detectors, and it can also be implemented in point-of-care or remote systems.