Microfluidic biosensor systems for real-time in vivo clinical bioanalysis

The aim of this thesis was to develop online biosensing systems for dialysate tissue metabo- lite detection in real time, to provide an insight into the health of tissue in various in vivo applications. An autocalibration system was developed using LabSmith programmable components to improve the accuracy of results obtained over long monitoring times. A method of col- lecting dialysate into storage tubes for online analysis while retaining temporal resolution was developed and validated. Microfluidic biosensor systems were developed for online measurement of glucose and lactate. One approach employed the use of biosensors, using a combined needle electrode with enzyme encapsulated in a hydrogel layer. The dynamic range of the biosensors was extended by adding an outer polyurethane layer. An alternative approach used automated syringe pumps and valves to develop a microfluidic system for in-flow enzyme addition to the dialysate stream. The existing rsMD system was applied for detection of tissue ischaemia during and after free flap surgery, by measuring dialysate glucose and lactate levels in real time. The system was able to detect flap failure, both during surgery and afterwards in the intensive therapy unit (ITU), much earlier than traditional methods. The rsMD system was adapted to enable monitoring of lactate levels in two dialysate streams and was applied for monitoring isolated porcine kidneys during two methods of cold preservation and subsequent re-warming. Significant differences in the lactate concentrations were observed between the two techniques. The system was extended for use with human transplant kidneys and with both porcine and human pancreases. A novel 3D printed wearable biosensor system was developed for direct integration with a clinical microdialysis probe. The system considerably improved the lag time and dispersional smearing compared with the existing rsMD system. The device was used in a proof-of-concept study with wireless potentiostats to monitor cyclists during exercise.Open Acces

Non-invasive tissue oximetry:current and prospective applications

Monitoring of tissue perfusion is necessary to timely recognise a lack of oxygen and to prevent tissue and organ damage. This dissertation shows that non-invasive tissue oximetry is a useful alternative during short-term and long-term use of heart-lung machines. Non-invasive tissue oximetry is also useful in monitoring transplant viability. However, the different applications of tissue oximetry require different interpretation strategies due to the presence or absence of vascular autoregulation. For instance, cerebral autoregulation has a direct effect on the oxygen supply to the brain. Therefore, it is important to take all available clinical data into account when applying tissue oximetry

Polymers and Their Application in 3D Printing

Dear Colleagues, Fused filament fabrication, also known as 3D printing, is extensively used to produce prototypes for applications in, e.g., the aerospace, medical, and automotive industries. In this process, a thermoplastic polymer is fed into a liquefier that extrudes a filament while moving in successive X–Y planes along the Z direction to fabricate a 3D part in a layer-by-layer process. Due to the progressive advances of this process in industry, the application of polymeric (or even composite) materials have received much attention. Researchers and industries now engage in 3D printing by implementing numerous polymeric materials in their domain. In this Special Issue, we will present a collection of recent and novel works regarding the application of polymers in 3D printing

Development of a non-invasive method to detect pericellular spatial oxygen gradients using FLIM

PhDExtracellular oxygen concentrations affect cellular metabolism and influence tissue function. Detection methods for these extracellular oxygen concentrations currently have poor spatial resolution and are frequently invasive. Fluorescence Lifetime Imaging Microscopy (FLIM) offers a non-invasive method for quantifying local oxygen concentrations. However, existing FLIM methods also show limited spatial resolution >1 μm and low time-resolved accuracy and precision, due to widefield time-gate. This study describes a new optimised approach using FLIM to quantity extracellular oxygen concentration with high accuracy (±7 μmol/kg) and spatial resolution ( ≅ 0.3 μm). An oxygen sensitive fluorescent dye, tris(2,2′-bipyridyl)ruthenium(II) chloride hexahydrate [Ru(bipy)3]+2, was excited with a multi-photon laser and fluorescence lifetime was measured using time-correlated single photon counting (TCSPC). The system was fully calibrated with optimised techniques developed for avoiding artefacts associated with photon pile-up and phototoxicity, whilst maximising spatial and temporal resolution. An extended imaging protocol (1800 sec) showed no phototoxic effects on cells at dye concentrations of <0.4 mM. Extracellular spatial oxygen gradients were identified around isolated chondrocytes, seeded in three-dimensional agarose gel. The technique was validated by regulating oxygen cellular consumption and thus confirming that the oxygen gradient was governed by cellular consumption. The technique identified a subpopulation of cells exhibiting statistically significant spatial oxygen gradients at the cell perihery. The subpopulation was shown to be significantly larger in cell diameter correlating with what that expected from chondrocytes in the deep zone. This technique provides an exciting opportunity to non-invasively quantify pericellular spatial oxygen gradients from within three-dimensional cellular constructs without prior manipulation of the cells. Thus by examining cellular metabolisms it will advance our understanding of the optimal cellular environment for tissue engineering and regenerative medicine

Genetic Analysis and Cell Manipulation on Microfluidic Surfaces

Personalized cancer medicine is a cancer care paradigm in which diagnostic and therapeutic strategies are customized for individual patients. Microsystems that are created by Micro-Electro-Mechanical Systems (MEMS) technology and integrate various diagnostic and therapeutic methods on a single chip hold great potential to enable personalized cancer medicine. Toward ultimate realization of such microsystems, this thesis focuses on developing critical functional building blocks that perform genetic variation identification (single-nucleotide polymorphism (SNP) genotyping) and specific, efficient and flexible cell manipulation on microfluidic surfaces. For the identification of genetic variations, we first present a bead-based approach to detect single-base mutations by performing single-base extension (SBE) of SNP specific primers on solid surfaces. Successful genotyping of the SNP on exon 1 of HBB gene demonstrates the potential of the device for simple, rapid, and accurate detection of SNPs. In addition, a multi-step solution-based approach, which integrates SBE with mass-tagged dideoxynucleotides and solid-phase purification of extension products, is also presented. Rapid, accurate and simultaneous detection of 4 loci on a synthetic template demonstrates the capability of multiplex genotyping with reduced consumption of samples and reagents. For cell manipulation, we first present a microfluidic device for cell purification with surface-immobilized aptamers, exploiting the strong temperature dependence of the affinity binding between aptamers and cells. Further, we demonstrate the feasibility of using aptamers to specifically separate target cells from a heterogeneous solution and employing environmental changes to retrieve purified cells. Moreover, spatially specific capture and selective temperature-mediated release of cells on design-specified areas is presented, which demonstrates the ability to establish cell arrays on pre-defined regions and to collect only specifically selected cell groups for downstream analysis. We also investigate tunable microfluidic trapping of cells by exploiting the large compliance of elastomers to create an array of cell-trapping microstructures, whose dimensions can be mechanically modulated by inducing uniform strain via the application of external force. Cell trapping under different strain modulations has been studied, and capture of a predetermined number of cells, from single cells to multiple cells, has been achieved. In addition, to address the lack of aptamers for targets of interest, which is a major hindrance to aptamer-based cell manipulation, we present a microfluidic device for synthetically isolating cell-targeting aptamers from a randomized single-strand DNA (ssDNA) library, integrating cell culturing with affinity selection and amplification of cell-binding ssDNA. Multi-round aptamer isolation on a single chip has also been realized by using pressure-driven flow. Finally, some perspectives on future work are presented, and strategies and notable issues are discussed for further development of MEMS/microfluidics-based devices for personalized cancer medicine