SKi Sensors are the label free binding chips which are used for binding studies on SKi Pro. They are nanoporous silicon chips, functionalized with chemistries appropriate for different assays and embedded within a polycarbonate flow cartridge. They integrate easily within SKi Pro and, assay depending, may be reused many times.
When performing label-free measurements, having low non-specific binding is arguably the most important characteristic of the sensor surface. Should molecules attach non-specifically to the surface, say through electrostatic interactions or the hydrophobic effect, this could be confused for the specific binding one cares about or give a false-positive in an AC-MS experiment.
SKi Sensors are based on nano-porous silicon, giving 80× the surface area of a standard surface. The porous area is transformed into silica (SiO2) then silanized. A very hydrophilic layer, based on poly-ethylene glycol, is attached which exposes the appropriate chemical moeity, for instance a carboxyl or NTA group.
A good test of the quality of the non-specific binding performance of a surface is to perform binding experiments in the presence of plasma. In Figure 1 a carboxyl chip is prepared with stretpavidin in the sample channel (solid lines) and no protein in the reference channel (dashed lines). Different concentrations of plasma is spiked with bBSA and interacted to the surface. An instantaneous signal is seen, which is due to the so-called refractive index effect which is due to the plasma molecules entering the pores and causing the refractive index to increase. After that the binding occurs.
After the reference channel is subtracted (bottom panel) the binding is near equivalent regardless of the concentration of plasma, showing the high quality of the surface and no signs of any non-specific binding.
Silicon Kinetics offers various types of sensor chips for immobilizing various proteins. On occasion, custom surfaces have been prepared. (Contact us about these inquiries.) Coupling kits for the various kits are also available.
|Sensor||P/N (8 pk.)||Application|
|Carboxyl||S-C-8||General protein coupling|
|Protein A||S-A-8||Polyclonal Abs|
|Protein G||S-G-8||Monoclonal Abs|
Figure 2 shows a typical coupling scheme used with the carboxyl chips. In step 1 the equilibrated surface at neutral pH exposes an ionized carboxyl group. In step 2 a sulfo-succinimide ester is formed by using the SKi Sensor carboxyl activation kit (P/N C-1). The native amines in the protein, usually from lysine residues, displace the sulfo-succinimide group (step 3) which covalently binds the protein as an amide (step 4).