Chapter One
1.1.4. Glycoarray Technology
The detection and quantification of bacterial species is thus of great importance; microarray technology can be used to probe the mechanisms of bacterial adhesion and aid both diagnostics and the development of new treatments, Figure 1.4. The facile and rapid detection of bacteria is of particular importance in this era of increasing antibiotic resistance. If a correct diagnosis of the infection can be
8 quickly and easily obtained then the prevalence of unnecessary antibiotic prescribing, one of the causes of increased antibiotic resistance, can be reduced.
Figure 1.4: Glycoarray coated with mannose, glucose, N-Acetylglucosamine, galactose and
fucose. Labelled E. coli are seen to bind only to the mannose coated regions, due to the presence
of a mannose specific receptor protein (FimH) on their surface. Such systems can be used as diagnostic tools for the detection of pathogens. Taken from the work of M. D. Disney and P. H.
Seeberger16
A microarray is a solid substrate onto which compounds of interest are immobilised; they are used to assay large amounts of biological material in a high throughput manner.17 The field of microarray technology began with antibody microarrays and quickly became applied to DNA. Today, however, there is great interest in the potential applications of carbohydrate microarrays, sometimes termed glycoarrays, which are surfaces displaying a large number of different carbohydrates, Figure 1.5. These arrays have the potential to not only shorten the timescales involved in biochemical measurements, but also offer miniaturisation, which is as straight forward with solution studies. This is of particular importance when you consider that, despite huge improvements in their production, pure oligosaccharides and glycoconjugates are still typically produced in very small quantities.18
9 Carbohydrate arrays have transformed a number of medical and biological research areas, and can be considered as simplified biomimics of the cell glycocalyx.19
Figure 1.5: Current applications of carbohydrate microarrays, adapted from the work of N.
Laurent, J. Voglmeir and S. L. Flitsch18
The immobilisation of the carbohydrates onto the array surface is critical to the success of the technology. Broadly speaking, the immobilisation techniques can be categorised into covalent bonding or non-covalent (physical) adsorption. Each category can be further divided into site specific (regioselective) and site non- specific, with respect to the attachment of the glycan on the surface, Figure 1.6.18 Due to non-covalent immobilisations relying on the glycans adhering to the surfaces typically via hydrogen bonds, van der Waals interactions or other non-covalent interactions, a large contact area must be available. Therefore the glycans must be sufficiently large, making this technique unsuitable for adhering individual carbohydrates, which would be removed during the washing steps.20 Non-covalent immobilisation has been seen using a nitrocellulose21 or oxidised black polystyrene
10 coating22 to adhere polysaccharides in a non-site-specific fashion, or streptavidin- coated surfaces for adhering biotin conjugated glycans.23 The simplest covalent immobilisations involve the attachment of free glycans onto boronic acid,24 phthalimide25 or azidoaryl26 coated surfaces in a non-site-specific fashion. Techniques for the site-specific covalent attachment of the glycans have been the most extensively developed and there are numerous examples within the literature. Typically, modified sugars are required to bind to the surfaces, for example homobifunctional disuccinimidyl functionalised glass requires ethanolamine functionalised sugars for binding.16
The use of covalent, orthogonal linkers is highly advantageous and they are widely employed,18 including by the Consortium for Functional Glycomics (CFG),27 which employs the amino linkers to react with succinimidyl ester glass slides. The latest version of the CFG array system (version 5.2, 2012)28 employs 20 different spacer arms, ranging from simple Sp0 linkers, comprised of -CH2CH2NH2, to peptide linkers, such as Sp24 and Sp25 (lysine-valine-alanine-asparagine-lysine-threonine and valine-alanine-asparagine-lysine respectively).16 The use of linkers in protein- carbohydrate binding systems allows controlled access to the binding pocket and can be used to improve selectivity. “Click” chemistry inspired routes have also been developed, utilising alkyne or azido- sugars with their complementary surfaces. More recently, thiol-ene type click reactions have gained attention in both their radical and nucleophilic (Michael addition) formats,29 see section 1.2.6. However, often glycans which have been synthesised de novo chemically or chemo- enzymatically must be subjected to further modification and/or coupling in order to react with the desired array surface. In addition to the challenges associated with generating cost-effective, reproducible arrays and minimising non-specific binding
11 interactions, this requirement for functionalised glycans is hindering progress towards a complete microarray technology; where in the future it may be possible to present the entire glycome on one chip or on a series of chips.
The type of surface onto which the glycans are immobilised is also important. Gold has been used to support self-assembled monolayers of alkenethiols, onto which monosaccharides can be immobilised,30, 31 but glass is most commonly used, including amino and epoxy silanised surfaces.32 However, the development of a carbohydrate surface attachment that is compatible with a variety of surfaces would be highly desirable, allowing more extensive characterisation techniques to be utilised and also increasing the applications for the resulting microarrays.
Figure 1.6: Categories for the immobilisation of carbohydrates onto solid supports. Adapted
12 Recent progress in microarray technology has seen the introduction of arraying robots and printers, incorporating scanning devices for efficient monitoring of detection signals. These methods can be divided into contact and non-contact printing. In contact printing, the glycans are printed onto the surface via the touch of a steel pin which has been immersed in a glycan solution. Typically 0.5 nL of solution is printed in each spot. In non-contact printing, a piezoelectric printer delivers approximately 0.3 nL of glycan solution via a glass capillary and electrical impulses, without touching the surface.33
As an alternative to carbohydrate microarrays, lectin microarrays have been developed as a platform for glycan analysis. Instead of functionalising the surface with carbohydrates, lectins (or carbohydrate antibodies) are immobilised and fluorescently labelled glycoproteins are added. This method is being introduced as an alternative to conventional glycan analytical techniques, such as mass spectrometry or liquid chromatography, because it does not require removal of the glycan from the core protein prior to analysing. It enables direct analysis of crude glycoprotein containing samples; however it is not quantitative and is best suited to comparative studies such as differential profiling.34
In addition to the immobilisation of the carbohydrates, the elucidation with biological material can also pose challenges. Typically, fluorescently-labelled proteins are added in order to assess binding, however, the isolation and labelling of proteins often proves to be challenging and it would be preferable to instead incubate the functionalised slides with whole bacteria. This can create the problem of non- specific adhesion to the slides, as bacteria adhere to and colonise all non-living surfaces. This colonisation process begins with the adhesion of a single cell or cell
13 aggregates and continues, if the conditions are favourable, with the growth and division of the adhered cells. The resulting micro-colonies have both advantageous applications (e.g. waste water treatment) but also pose serious disadvantages,35 as in this case where they will reduce the resolution of the assay and give rise to false positive results. A non-fouling or hydrophilic surface could circumvent this problem, by resisting the adhesion of the proteins, bacteria and cells. The development of glass-based carbohydrate microarrays with high carbohydrate-spotting density and a protein-resistant surface (Figure 1.7) has the potential to significantly improve this technology. It is proposed that this can be achieved using linkers composed of polymers of oligo(ethyleneglycol) or N-isopropylacrylamide, which possess both protein resistant36, 37 and stimuli-responsive38 properties, as explained in section 1.2.5 Many polymers have been shown to exhibit attributes that are desirable for biomedical applications, such as antifouling properties,39 making them suitable for many array applications, but the synthesis of well defined, reproducible polymers is crucial to the success of these systems, requiring precision polymerisation methods.
Figure 1.7: Protein resistant surfaces are used to reduce non-specific binding and give higher
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