Immobilisation biomolecules

The motivations for the study of biomolecules immobilised on surfaces, briefly outlined above, also provide an explanation for the increased popularity of microscopy-based techniques. Indeed, while for single molecule and self-assembled layers the use of microscopy techniques is obvious, the commercial biodevices are increasingly micro- and even nanoscale devices. 

Among the many microscopy-based techniques for the study of biomolecules immobilised on surfaces, scanning probe microscopies (SPM) and especially atomic force microscopies (AFM) are arguably the most used techniques because of their molecular and sub-molecular level resolution and in situ imaging capability. Moreover, the invasiveness of AFM, which is less of a problem for the DNA molecules, is essential for another two functions, apart from the mapping of surface nanotopographies, namely the quantification and visualisation of the distribution of chemistry, hydrophobicity and local mechanical properties on surfaces and the fabrication of nanostructures. 

The analysis of biomolecules by AFM is sometimes [3] referred to as surface biology, as opposed to the so-called test-tube biology, because the immobilisation of oligonucleotides on sohd surfaces is central to the design, fabrication and operation of DNA-based microdevices, such as biosensors, DNA micro- and nanoarrays, microPCR and lab-on-a-chip devices. As the analysed biomolecules are in close contact and very often in intimate interaction with the surface, sample preparation for the AFM analysis of surface-immobihsed biomolecules is both critical and dehcate. The biomolecules need to be firmly anchored on the substrate, which has to have a sufficiently minimal or easily discriminated topography [1]. The Kleinschmidt method [6] for the DNA... 

Many other applications for plasma polymers in the Life Sciences have been dted, often in relation to implantable medical devices or materials, with the goal of concealing the device from the bodies defence mechanisms, or improving cell colonisation of the material, e.g. endothelial cell growth into vascular grafts. A number of excellent studies from the group of Hans Griesser (CSIRO, Australia) describe the use of plasma polymers as substrates to which biomolecules can be immobilised. These immobilisations have been demonstrated to enhance the medium-term acceptability of contact lens materials and may prove relevant to implantable devices. 

The format and the preparation of protein microarrays depends on the nature of the immobilised biomolecule and its application. While peptide arrays are manufactured synthetically directly on the support [14], proteins are delivered using either pin-based spotting or liquid microdispensing. To date, the most com-... 

Due to the stability, orientation and abihty to fimctionahse the terminal groups on the molecules they can offer a very versatile method for immobilisation of biomolecules to gold electrode surfaces for biosensor development [25,26]. 

To perform spectroscopy on biomolecules at a surface, it would be favourable to confine the measurement to a thin layer at the surface, in the region where the molecules are immobilised. Typically, this region would range from 5 nm for... 

Stable immobilisation of macromolecular biomolecules on conducting microsurfaces with complete retention of their biological recognition properties is a crucial problem for the commercial development of miniaturised biosensors. Various conducting polymers have been utilised for immobilisation of enzymes at an electrode surface including PPy [74-76, 103-106], polyindole [79], PANI [77, 107, 108], poly (N-methylpyrrole) [65] and copolymers of N-substituted pyrroles [34]. 

A number of biomolecules have been physically immobilised on conducting polymers [66,112, 116-119]. This is the simplest method of enzyme immobilisation. Since the binding forces involved are hydrogen bonds, van der Waals forces, etc., porous conducting polymer surfaces are most commonly used. The pre-adsorption of an enzyme monolayer prior to the electrodeposition of the polymer, [120] and two-step enzyme adsorption on the bare electrode surface and then on PPy film [121] have also been investigated. 

The crosslinking of the bioraolecules via bifunctional reagents, e.g., glutaraldehyde and bovine serum albumin, has been utilised to stabilise the biomolecules. In this case, a crosslinked network of enzyme is formed resulting in the formation of a bigger molecule on the polymer surface [122, 123]. Enzymes have also been immobilised on the surface of electrodeposited polymer by applying the bovine serum albumin and glutaraldehyde procedure followed by the electrodeposition of the polymers [1,124,125]. This method has certain limitations since it may cause a drastic loss in the activity of the biomolecules. 

It has been observed that the surface of the conducting polymer plays an important role in the effective immobilisation of the desired enzyme. The Langmuir-Blodgett (LB) technique can be successfully applied to deposit a desired monolayer with the desired orientation of the biomolecules/enzymes [142-145]. Ramanathan and co-workers [146] have utilised the polyemeraldine base LB films for the immobilisation of GOD. These films have been shown to function as amperometric glucose biosensors and have a linear range from 5 to 50 mM. LB films of PT immobilised with GOD and urease have also been prepared for application to respective biosensors [147, 148]. 

Biosensors require highly active enzymes/biomolecules therefore, the immobilisation methods must be chosen in such a way that they can achieve a high sensitivity and functional stability. This is important for economic reasons also. The measurable activity gives an idea about the biocatalytic efficiency of an immobilised enzyme. The rate of substrate conversion should rise linearly with enzyme concentration. The measured reaction rates depend not only on the substrate concentration and the kinetic constants (Michaelis Menten constant) and (maximum velocity of the reaction) but also on the immobilisation effects. The following effects have been observed [157] due to the immobilisation process ... 

W. Schuhmann in Immobilised Biomolecules in Analysis A Practical Approach, Eds., T. Cass and E Ligler, Oxford University Press, Oxford, 1998, 187. 

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