Biosensors are prized for their ability to couple the breadth of biological interactions to the precision of optic-electronic systems, and as such the sensing surface itself plays a vital role. Careful attention needs to be aid to the interaction of sample and sensor, with expert knowledge and design required at both the macro- and nano-scales for consistent results.
The treatment of the gold surface to create a specific biosensor is known as derivatisation, or functionalization. Many techniques are possible, but the final transducer surface should meet several basic conditions. Most importantly, they must shield the substrate, this both prevents fouling and provides a stable surface for further modification. They must also have consistent properties (such as surface energy, charge, and chemical reactivity) across the entire surface, this vastly increases the reliability of the data produced.
Further requirements may be needed for different protocols and immobilization methods. These may include accessible covalent binding sites, a structurally defined immobilisation matrix, low non-specific binding coatings, or the ability to perform quantitative regeneration of the biosensor. Some of these features are mutually exclusive, and cannot be combined into one sensor surface.
XanTec bioanalytics are market leaders in the functional coating of different surfaces, and so have developed a wide range of different coating technologies. SPR measurements can be performed by sensor chips containing hydrogels, planar coatings, or untreated gold surfaces – although intermediate surfaces can also be functionalised to produce more complex sensor chips.
The majority of experiments utilise sensor chips coated with hydrogels, which are three-dimensional networks of hydrophilic polymers that provide a far greater number of potential binding sites than a planar coating. Sensor chip hydrogels are commonly constructed from carboxymethylated dextran. This hydrogel not only exhibits relatively low non-specific interaction with the sample matrix, but also preserves all ligand structural and other properties following immobilisation. Hydrogels, with their multiple binding sites, are thus well suited for qualitative studies – both examining a wide range of molecular interactions as well as determining analyte concentrations.
Planar coatings are a thin layer added to the top of the sensor chip, unlike hydrogels they are essentially two-dimensional. Their chemistry is more or less identical to the aforementioned hydrogels, but these coatings do not display a bulky hydrogel structure. As such, they are well suited for the analysis of larger analytes such as high MW proteins, aggregates, vesicles, viruses, particles or certain cell types.
Hydrophobic polymer layers are less frequently used and are normally added using a spincoater; which utilises centrifugal force on a rapidly spinning disc to add an extremely thin polymer layer of defined thickness to the chip surface.Polystyrene or Polycarbonate are two polymers which can be used in SPR sensor chips.
Bare gold surfaces can also be treated with ligand solutions, using electrostatic forces or the strong interaction between gold and thiol-containing amino acids to couple macromolecules to the surface. This technique is most often used in the analysis of large particles, such as whole cells or viruses. As coverage of the surface is never total, blocking solutions are usually required to prevent non-specific interactions. Nonetheless, nonspecific binding remains a serious problem associated with such surfaces.
The most common approach involves derivatizing the chip surface with biotin, followed by streptavidin – as streptavidin can bind to four separate biotin molecules, this leaves a number of unoccupied binding sites. Biotinylated biomolecules are then injected, binding to streptavidin and creating a functionalised surface, ready for analysis of analyte binding.
Another relatively popular technique uses the rapid reaction of gold with sulphur to bind thiol-containing molecules to the surface. This is most often used in the assembly of peptides upon the surface to form a receptor layer.
There are three methods which are commonly used to couple the bio-specific ligand to the supporting matrix.
The first, and most common, is covalent coupling of the ligand to the matrix. This usually involves cross-reacting functional groups in the dextran layer to amine or thiol groups on the ligand itself. Several different approaches to achieve this can be seen in the following figure.
Secondly, as mentioned previously, biotinylated ligands can be non-covalently linked to streptavidin-coated surfaces. The streptavidin-biotin binding affinity is extremely high, which means that used chips can be regenerated without breaking the non-covalent streptavidin-biotin linkage, (this linkage, however, cannot be regenerated). This approach is well suited for coupling DNA oligomers or PCR products to the sensor surface.
Lastly, one can use capture antibodies. This tactic is usually appropriate when covalent linkage of an antibody has been observed to reduce its activity, and can be thought of as a western blot in reverse. Firstly, a capture antibody (such as mouse anti-Rabbit-Fc) is covalently linked to the surface matrix. Secondly, the antigen-specific antibody (rabbit anti-Tau, for example) is added, where it binds to the capture antibody. Finally the antigen-containing solution is injected, whereupon the antigen will bind to the second antibody. As the majority of these interactions are non-covalent, regeneration of the chip requires that the loading process be repeated.
The various surface coating methods discussed above have been used intensively to study biomolecular interactions. The examples listed below have been successfully used by XanTec Bioanalytics customers, but many more possibilities exist. To find out more about how to approach your specific situation, get in touch with us today.
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