Protein adsorption/immobilisation

Self-Assembled Monolayers on Gold Thin Film A Surface Model for Protein Adsorption and Immobilisation... 

The rapid initial phase of salivary protein adsorption is followed by a second, comparatively slower phase of protein adsorption onto the protein-coated enamel surface. The second stage of pellicle formation is characterised by a continuous adsorption of biopolymers from saliva. This process involves protein-protein interactions between already adsorbed proteins, immobilised in the pellicle layer, and proteins as well as protein aggregates from saliva. Amino acid and Auger analyses of the pellicle layer formed on buccally carried enamel slabs [18] indicate that the adsorbed proteins reach an initial thickness in about 2-3 min, and stay at that level for about 30 min. The thickness of the pellicle then increases to about three times its initial thickness and reaches a plateau after 30-90min [5, 18, 27], Within 60min, the thickness of the in situ-formed pellicle will further increase to between 100 and lOOOnm [17, 28], dependent on the supply of locally available salivary biopolymers and the prevailing intraoral conditions [17,28,29] (fig. 2). 

Pei et al. used protein A immobilisation in the development of a sensor for fibrin in serum [141]. The immobiUsation gave better results than those obtained with polyethyleneimine and adsorption of BSA, glutaraldehyde was used to cross-Unk the antibodies to the adsorbed BSA layer. Under optimised conditions the analyte could be measured from 10 to 10 g L . The... 

Cole MA, Thissen H, Losic D et al (2007) A new approach to the immobilisation of poly(ethylene oxide) for the reduction of non-spedfic protein adsorption on conductive substrates. Surf Sci 601 1716-1725... 

Despite these improvements, there are other important biosensor limitations related to stability and reproducibility that have to be addressed. In this context, enzyme immobilisation is a critical factor for optimal biosensor design. Typical immobilisation methods are direct adsorption of the catalytic protein on the electrode surface, or covalent binding. The first method leads to unstable sensors, and the second one presents the drawback of reducing enzyme activity to a great extent. A commonly used procedure, due to its simplicity and easy implementation, is the immobilisation of the enzyme on a membrane. The simplest way is to sandwich the enzyme between the membrane and the electrode. Higher activity and greater stability can be achieved if the enzyme is previously cross-linked with a bi-functional reagent. 

The detection of complement proteins has also received much attention. Pei and coworkers described a Pz immunosensor for the detection of complement C4 [134,135]. The authors first used adsorption to immobilise the specific antibody, showing better results than for the use of polyethyle-nieimine [134]. They measured the protein linearly from 0.1 to 10 p.gmL The sensor showed no response to any materials present in serum and the gold surface could be regenerated 15 times for repeated use. Later they self-assembled the antibodies onto a layer of protein A, a method which gave superior results to the previously tested immobilisation procedures [135]. This setup gave a better linear range of 5 x 10 -1 x 10 p.gmL but the crystal was only reusable about ten times. 

The group later published a similar sensor for the detection of complement C6 [136]. Immobilisation of the antibody via polyethyleneimine showed superior results to physical adsorption. Under optimised conditions the protein could be determined linearly from 10 to 10 pg mL . The gold surface could be regenerated 12 times for repeated use before loss of sensitivity due to damage of the gold was observed. 

A protein chip is a glass, plastic or silicon chip onto which different proteins have been attached at separate locations in an ordered manner to form a microscopic array. They are attached mainly by adsorption, absorption, covalent cross-linking or affinity binding. There are two main types of protein arrays analytical arrays and functional protein arrays. In the hrst type the capture molecules are antibodies or antibody mimics which are used to detect the presence and amount of protein in a sample. These are mainly used for diagnostics and expression profiling experiments. The second type of protein array involves immobilising proteins on a chip and using the chip to probe biochemical activity. 

Two principle techniques for electrochemical enzyme deposition have been reported, entrapment in an electrochemically grown polymer and electrochemically aided absorption. A wide range of electrochemically grown polymers have been used. The polymer can function as both an entrapment matrix and as an anti-interference layer (7, 12-20), as a matrix for the immobilisation of the protein with an electron transfer mediator (21-23), and as an electron transfer matrix alone (24, 25), Electrochemically aided adsorption has received comparably less attention (26-30), However, in our experience (31) the latter technique results in larger responses and is more appropriate to microelectrodes. Here we will present results on the electrochemically aided adsorption of GOx and BSA, and also of urease. Furthermore to reduce the interferences at the GOx/ BSA electrode we will describe the deposition of an anti-interference layer of polypyrrole, which is grown on the electrode after the deposition of the proteins. 

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