Development of new methods to study cell surface glycosylation

This thesis seeks to develop new methods to study the glycosylation of cell surfaces, with a particular interest in apoptosis. Existing methods in glyco- biology include mass spectrometry and glycan identification by lectin bind- ing. The use of new carbohydrate binding proteins (CBP) and microfluidic techniques will improve the field of glycobiology and its relevance in the area of apoptosis. A method using the Lab in a Trench (LiaT) platform was developed to allow sequential labelling of cell surface glycans using lectins. Cells are captured in the trench, fluorescently labelled lectins are then added and allowed time to bind. The lectins are then released by their corresponding free sugar, allowing probing with subsequent lectins without steric hindrance due to adjacent sugars of interest. This study represents the first sequential labelling of the same cell surface by lectins. It has been established, that exposure of terminal N-acetylglucosamine (GlcNAc) occurs in late apoptosis and has a role in immune recognition and clearance of dead cells. AAL-2 is a recombinantly produced lectin or CBP with an affinity for the terminal sugar N-acetyl glucosamine (GlcNAc). It has a binding profile matching that of the commercial GSL II in flow cy- tometry and western blot, but has the advantage of not requiring additional ions in its buffer. Using flow cytometry, this study has shown that AAL- 2 binds exclusively to late apoptotic cells. GafD is another recombinantly produced CBP with an affinity for terminal O-linked GlcNAc (O-GlcNAc). It was determined by flow cytometry that GafD binds to a subset very late apoptotic cells. The proteins to which GafD binds were identified through isolation of the cell membrane followed by mass spectrometric analysis. The proteins were found to be predominantly cytosolic, indicating a migration of the intracellular membrane to the cell surface during late apoptosis

Microfluidic lab-in-a-trench platform: cell capture, manipulation and application in glycobiology

Glycans are highly complex carbohydrate molecules and, unlike proteins, they are not encoded by the genome. Instead, they are processed and incorporated at specific locations on proteins and lipids in various combinations at post-translational stages by tightly regulated, enzyme-mediated pathways. Their ability to form complexes within themselves and with other biomolecules, such as proteins, makes them a core component of cellular physiology. Examples of glycan-mediated signaling include embryonic development, cell differentiation and growth, cell-cell recognition, contact inhibition and cell signaling. Glycans therefore constitute an advanced class of information molecules and the full potential of glycan bioprofiling has yet to be realized

Seqential glycoprofiling of single cells: A novel approach using lab in a trench

Using Lab in a Trench technologies, we have developed a novel method to glycoprofile single cells by sequential probing with fluorescently labelled lectins. Current microscopic methods to glycoprofile cells are limited by the inability to probe the glycan structures with more than one lectin, as steric hindrance can interfere with lectin binding. Flow cytometry is similiarly limited, and methods such as NMR and Mass Spectrometry do not have the possibility to probe at the level of the individual cell. Our system traps the cells in a shear-free environment, allowing us to exchange fluids about the cell to probe with various lectins. By adding the appropriate free sugars to the fluid, the bound lectin can be eluted, and the cells probed with a lectin with another specificity. This ability to probe individual cells and achieve a complete glycoprofile through lectin staining has not been reported before

A portable centrifugal analyser for liver function screening

Mortality rates of up to 50% have been reported after liver failure due to drug-induced hepatotoxicity and certain viral infections(Gao et al. 2008). These adverse conditions frequently affect HIV and tuberculosis patients on regular medication in resource-poor settings. Here, we report full integration of sample preparation with read-out of a 5-parameter liver assay panel (LAP) on a portable, easy-to-use, fast and cost- efficient centrifugal microfluidic analysis system (CMAS). Our unique, dissolvable-film based centrifugo- pneumatic valving was employed to provide sample-to-answer fashion automation for plasma extraction (from finger-prick of blood), metering and aliquoting into separate reaction chambers for parallelized colorimetric quantification during rotation. The entire LAP completes in less than 20 minutes while using only a tenth the reagent volumes when compared with standard hospital laboratory tests. Accuracy of in-situ liver function screening was validated by 96 separate tests with an average coefficient of variance (CV) of 7.9% compared to benchtop and hospital lab tests. Unpaired two sample statistical t-tests were used to compare the means of CMAS and benchtop reader, on one hand; and CMAS and hospital tests on the other. The results demonstrate no statistical difference between the respective means with 94% and 92% certainty of equivalence, respectively. The portable platform thus saves significant time, labour and costs compared to established technologies, and therefore comply with typical restrictions on lab infrastructure, maintenance, operator skill and costs prevalent in many field clinics of the developing world. It has been successfully deployed in a centralised lab in Nigeria