Studies of binding reactions are of highest importance in a vast number of areas of biomedicine and biotechnology. A demand for fast and accurate small-volume measurements grows stronger, partly due to the development of therapeutic antibodies. In this report, a novel method for studies of binding reactions of antibodies is described. The use of a microfluidic platform shows promising results in determination of affinity binding constants. Affinities between 1E-09 and 1E-11 M have been determined for four TSH antibodies. Reproducibility tests give a CV below 10%, using different Gyrolab instruments and microfluidic CD:s. The method carries the advantages of using solution-based measurements of unmodified molecules. Also an initial proof-of-concept for measurement of binding reaction rate constants shows further usage of the method. The kinetic association rate constant has been determined to 2E+06 M-1s-1 for one antibody. The possibility of using this method for screening of antibody libraries is also discussed.

Blood vessels are essential for the delivery of nutrients and oxygen to tissues, as well as for the removal of waste products. Patients with tumors, wounds or diabetes all have active angiogenesis, formation and remodeling of blood vessels, a process that is initiated and manipulated by gradients of secreted signaling proteins. This thesis describes the development of new microfluidic in vitro assays where directed migration of single endothelial cells and three dimensional vascular structures can be monitored in real time. Combining these assays with live imaging microscopy we have studied the behavior of endothelial cells in gradients of proangiogenic factors as well as directed sprouting in embryonic kidneys and stem cell cultures. With the 2D assay we have quantified endothelial cell chemotaxis towards FGF2, VEGFA165 and VEGFA121 and we also demonstrate that constant levels of VEGFA165, but not of FGF2, are able to reduce chemokinesis of endothelial cells. In the 3D migration chamber we have studied directed endothelial cell sprouting in mouse embryonic kidneys and embryoid bodies in response to VEGFA gradients. In both models directed angiogenesis is detected towards increasing levels of growth factor. Using the microarray technique on differentiating embryonic stem cells we have been able to identify the gene exoc3l2 as potentially involved in angiogenesis and endothelial cell migration and we present evidence that ExoC3l2 is associated with the exocyst complex; an important regulator of cell polarity. We have also shown that siRNA mediated gene silencing of exoc3l2 results in impaired VEGFR2 phosphorylation as well as loss of directionality in response to a VEGFA gradient. / (Faculty of Medicine)

No description available.

The use of microfluidic tools to develop two novel approaches to surface-based oligonucleotide hybridization assays has been explored. In one of these approaches, immobilized oligonucleotide probes on a glass surface of a microfluidic channel were able to quantitatively hybridize with oligonucleotide targets that were electrokinetically injected into the channel. Quantitative oligonucleotide analysis was achieved in seconds, with nM detection limits and a dynamic range of 3 orders of magnitude. Hybridization was detected by the use of fluorescently labeled target. The fluorescence intensity profile evolved as a gradient that could be related to concentration, and was a function of many factors including hybridization reaction rate, convective delivery speed, target concentration and target diffusion coefficient. It was possible to acquire kinetic information from the static fluorescence intensity profile to distinguish target concentration, and the length and base-pair mismatches of target sequences. Numerical simulations were conducted for the system, and fit well with the experimental data. In a second approach, a solid-phase nucleic acid assay was developed using immobilized Quantum Dot (QD) bioprobes. Hybridization was used to immobilize QDs that had been coated with oligonucleotides having two different sequences. The hybridization of one oligonucleotide sequence conjugated to a QD (a linker sequence) with a complementary sequence that was covalently attached to a glass substrate of a microfluidic channel was shown to be an immobilization strategy that offered flexibility in assay design, with intrinsic potential for quantitative replacement of the sensing chemistry by control of stringency. A second oligonucleotide sequence conjugated to the immobilized QDs provided for the selective detection of target nucleic acids. The microfluidic environment offered the ability to manipulate flow conditions for control of stringency and increasing the speed of analytical signal by introduction of convective delivery of target sequences to the immobilized QDs. This work introduces a stable and adaptable immobilization strategy that facilitates solid-phase QD-bioprobe assays in microfluidic platforms.

This thesis conducts numerical and experimental studies of the nonlinear electrokinetic motion of heterogeneous particles in microfluidic systems and their corresponding applications in laboratory-on-a-chip (LOC) systems. Induced-charge electrokinetic (ICEK) phenomena flow is generated by applying an external electric field to a conducting particle immersed in an aqueous solution. As a result of this field, micro-vortices form around the conducting particle. Using this phenomenon, many shortcomings of classical electrokinetics (e.g. poor mixing, leakage, back flow problem) can be improved. This thesis proposes and investigates a complete 3-D numerical multi-physics method to calculate the induced zeta potential on the conducting surface of a heterogeneous object. To model the ICEK motion of a heterogeneous particle in a DC electric field, the moving grid technique is used to conduct the particle-fluid simulation. It was numerically shown that the vortices form near the conducting surface of a particle. Both transitional and rotational motions of heterogeneous particles are investigated. A set of novel experiments are designed and conducted to investigate several aspecs of ICEK. It is demonstrated for the first time that four vortices form around a conducting sphere in contact with an aqueous solution while the DC electric field is applied. The motions of heterogeneous particles are experimentally studied. The speed of a heterogeneous particle is compared with the same size non-conducting particle under the same experimental conditions and it is shown that the heterogeneous particle moves significantly faster than the non-conducting particle. It is also shown that the micro-vortices on the conducting section of the heterogeneous particle act like an engine and push the particle to move faster. These experiments verify the results of our simulation studies. We introduce three applications for induced-charge electrokinetic phenomena in ths thesis: ICEK micro-valve, ICEK micro-mixer, and ICEK micro-motor, which can be used in microfluidics and lab-on-a-chip devises. This ICEK micro-valve significantly improves many shortcomings of other micro-valves reported in the literature (such as leakage, considerable dead volume and complicated fabrication processes). Our ICEK micro-mixers take the advantages of induced micro-vortices and boost the mixing process in a micro-channel. As a result well mixed homogeneous (100%) mixture could be obtained at the downstream of the mixer. Our proposed no-contact ICEK micro-motor rotates as long as the DC electric field is being applied. This thesis develops a new understanding of several ICEK phenomena and applications related to heterogeneous particles. The 3D numerical model developed in this thesis along with the experimental studies are capable of describing the ICEK motion of a heterogeneous particle and is a considerable step to calculate the ICEK phenomena for real-world applications. This thesis, for the first time, experimentally visualized and verified the induced micro-vortices around conducting particles under applied DC electric field. The proposed ICEK micro-mixers, valve and motor can be used in various LOC devices and applications.

The research presented in this thesis was motivated by the desire to understand the flow field within a new digital microfluidic device currently under development. This required an investigation of the dynamics of a droplet migrating along the surface of another fluid due to interfacial surface tension gradients. The quantitative analysis of the flow field presented in this thesis provides the first known solution for the velocity field in a migrating droplet confined to an interface. The first step towards gaining insight into the flow field was accomplished by using the method of reflections to obtain an analytical model for a submerged droplet migrating near a free surface. The submerged droplet model enabled the analysis of the velocity field and droplet migration speed and their dependence on the fluid properties. In general, the migration velocity of a submerged droplet was found to differ dramatically from the classic problem of thermocapillary migration in an unbounded substrate. A boundary-collocation scheme was developed to determine the flow field and migration velocity of a droplet floating trapped at the air-substrate interface. The numerical method was found to produce accurate solutions for the velocity and temperature fields for nearly all parameters. This numerical scheme was used to judge the accuracy of the flow field obtained by the submerged droplet model. In particular, the model was tested using parameter values taken from a digital microfluidic device. It was determined that the submerged droplet model captured most of the flow structure within the microfluidic droplet. However, for a slightly different choice of parameters, agreement between the two methods was lost. In this case, the numerical scheme was used to uncover novel flow structures.

Quantifying the velocity field near the wall in microfluidic devices is important because surface effects become significant at micro- to nanometer scales. Recent studies have suggested that the "no-slip" boundary condition breaks down in microscale flows of Newtonian liquids, where the amount of slip is usually extrapolated from velocity components measured far from the wall. This doctoral thesis presents a new technique, multilayer nano-particle image velocimetry (MnPIV), for measuring the tangential velocity components at different distances from and within about 500 nm of the wall and its application to measuring slip. The feasibility of MnPIV was demonstrated using synthetic images of plane Couette flow incorporating Brownian diffusion and imaging noise. The errors in MnPIV data were then quantified with Brownian dynamics simulations. Calibration experiments were used to correlate the image intensity of the tracer to its distance from the wall z. Multilayer nPIV was then used to determine the z-positions and distribution of the particles for z < 500 nm in experimental studies of microscale Poiseuille flow. The tracers were divided into three distinct layers based on their image intensities, and the average velocity of each layer was placed at the average z-position sampled by the particles in that layer. The resultant velocity gradients were within 6% on average of analytical predictions for 2D Poiseuille flow. Finally, the results of MnPIV studies of aqueous solutions flowing through microchannels with hydrophilic and hydrophobically-coated fused silica surfaces suggest that if the slip lengths are nonzero for both of these surfaces, they are less than the uncertainty in these results, or 27 nm and 31 nm for the hydrophilic and hydrophobic channels, respectively.

Powerful tools for detailed cellular studies are emerging, increasing the knowledge ofthe ultimate target of all drugs: the living cell. Today, cells are commonly analyzed inensembles, i.e. thousands of cells per sample, yielding results on the average responseof the cells. However, cellular heterogeneity implies the importance of studying howindividual cells respond, one by one, in order to learn more about drug targeting andcellular behavior. In vitro assays offering low volume sampling and rapid analysis in ahigh-throughput manner are of great interest in a wide range of single-cellapplications. This work presents a microwell device in silicon and glass, developed using standardmicrofabrication techniques. The chip was designed to allow flow-cytometric cellsorting, a controlled way of analyzing and sorting individual cells for dynamic cultureand clone formation, previously shown in larger multiwell plates only. Dependent onthe application, minor modifications to the original device were made resulting in agroup of microwell devices suitable for various applications. Leukemic cancer cellswere analyzed with regard to their clonogenic properties and a method forinvestigation of drug response of critical importance to predict long-term clinicaloutcome, is presented. Stem cells from human and mouse were maintainedpluripotent in a screening assay, also shown useful in studies on neural differentiation.For integrated liquid handling, a fluidic system was integrated onto the chip fordirected and controlled addition of reagents in various cell-based assays. The chip wasproduced in a slide format and used as an imaging tool for low-volume sampling withthe ability to run many samples in parallel, demonstrated in a protein-binding assay fora novel bispecific affinity protein. Moving from cells and proteins into geneticanalysis, a method for screening genes from clones in a rapid manner was shown bygene amplification and mutation analysis in individual wells. In summary, a microwelldevice with associated methods were developed and applied in a range of biologicalinvestigations, particularly interesting from a cell-heterogeneity perspective. / QC 20100728

Protein aggregation is commonly encountered during the manufacture of protein-based bioproducts in processing such as protein expression, purification, refolding, shipping and storage (Volkin and Middaugh, 1992; Brange, 2000). Aggregation may shorten the shelf-life of pharmaceutical proteins (Frokjaer and Otzen, 2005) and induce severe hypersensitivity (Rosenberg, 2006). In addition, several diseases ranging from Alzheimer’s disease to cystic fibrosis are associated with protein aggregation in the form of amyloid fibrils and plaques (Dobson, 1999; Luheshi et al., 2008). Hence, studies on protein aggregation, especially those dealing with high concentrations of proteins, are highly demanded in both academic and industrial laboratories. To address the aforementioned issues, physicochemical factors affecting protein aggregation were investigated systematically in this project. Strategies were developed to inhibit protein aggregation during renaturation and to enhance protein stability against aggregation during and after production, especially when dealing with high protein concentrations. ∆5-3-Ketosteroid isomerase (KSI) was used as a model for aggregation studies during protein renaturation due to its intrinsic aggregation properties. KSI was overexpressed as inclusion bodies (IBs) in Escherichia coli (E. coli). Cost- and time-efficient combination of chemical extraction and one-step affinity purification ensured the production of denatured KSI with high purity at high yield. Several key factors, including protein concentration and ionic strength, were determined to greatly influence KSI aggregation during renaturation. Polymer addition (PEG 3000 and Eudragit S-100) was found to alter KSI aggregation behaviour in a polymer-specific manner, as quantified using reversed phase-high performance liquid chromatography (RP-HPLC) analysis. Light scattering for second virial coefficient (SVC) measurement, surface plasmon resonance (SPR), and microfluidics were applied to study the fundamental mechanism of protein aggregation. Lysozyme was further introduced as a control protein for comparison with KSI. A rapid lumped method was established to measure specific refractive index (∂n/∂c) and SVC values for KSI and lysozyme, which provided quantitative and qualitative information on thermodynamic interactions of molecules in solution. SPR and microfluidics were also used to explore protein aggregation properties. To our best knowledge, it is the first time SPR and microfluidics have been used to investigate protein aggregation behaviour. Both SPR and microfluidics present significant potential for assessing protein aggregation and diagnosis or drug screening of protein aggregation related diseases. The chemical and physical stability of proteins needs to be maintained after successful refolding to ensure an acceptably long shelf life, especially at high protein concentration (Chang and Hermsdorf, 2002). The pharmaceutical effects of lectins on cell growth provided incentive for studies to improve their stability. Human galectin-2 (hGal-2, a homodimeric lectin) was used as a study model in this project. Mutations were introduced at one of the two Cys residues (C57A, C57M, and C57S). Only the C57M variant was highly expressed in bacteria in soluble form. No aggregate of this mutant was detected during 3 weeks of storage. hGal-2 C57M also facilitated site-directed introduction of poly(ethylene glycol) (PEG) into the remaining sulfhydryl group (Cys75). Product analysis revealed rather complete conjugation with one PEG chain per protein subunit in homodimer. Neither secondary structure alteration nor the absence of binding ability to a glycoprotein (asialofetuin) was observed. The results document the feasibility of tailoring a human galectin for enhanced stability against aggregation as well as monoPEGylation, which enables further testing of biological properties including functionality as a growth regulator and the serum clearance rate of hGal-2.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 7 uppsatser.