It is well known that bonding between Streptavidin (STV) and biotin is very strong. As a part of my experiment I would like to check if STV binds to biotin which is attached to PEG. The surface preparation protocol goes as follows:

1. Perform APTES (silane) coating on glass coverslip

2. Incubate these coverslips with NHS-PEG-Biotin on the surface. The NHS ester has affinity for amine groups on APTES. Then the PEG-Biotin layer is formed on the glass surface. The NHS-PEG-Biotin (5kDa, around 30nm contour length) is purchased from Laysan Bio.

3. Then add STV on the surface and let it bind to biotin on surface and wash off unbound STV.

As PEG molecules can exist in coiled structures in liquid, STV may not get access to Biotin on PEG. So I would like to check by any method if STV is bound to biotin and I am also interested to know the density of STV molecules on the surface.

I have attempted this in two ways:

a. Measure the adhesion on PEG surface with bare tip: No adhesion in case of NHS-PEG-Biotin. But there is adhesion between tip and surface when STV is added on the surface and washed off. This is an indication for presence of STV

b. AFM tapping mode imaging:

Both the surfaces seems to be very smooth and indistinguishable from each other. It may be due to the nature of PEG that it is deformed when force is applied on the surface during the imaging process. The STV is unable to be detected as PEG is being deformed which is under STV.

Please let me know if there is any better way to do this either using AFM or any other instrument. Also let me know if there are any mistakes in my understanding of my observations.