Application of Catalyst-Free Click Reactions in Attaching
Affinity Molecules to Tips of Atomic Force Microscopy for Detection
of Protein Biomarkers
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Abstract
Atomic force microscopy (AFM) has
been extensively used in studies
of biological interactions. Particularly, AFM based force spectroscopy
and recognition imaging can sense biomolecules on a single molecule
level, having great potential to become a tool for molecular diagnostics
in clinics. These techniques, however, require affinity molecules
to be attached to AFM tips in order to specifically detect their targets.
The attachment chemistry currently used on silicon tips involves multiple
steps of reactions and moisture sensitive chemicals, such as (3-aminopropyl)triethoxysilane
(APTES) and <i>N</i>-hydroxysuccinimide (NHS) ester, making
the process difficult to operate in aqueous solutions. In the present
study, we have developed a user-friendly protocol to functionalize
the AFM tips with affinity molecules. A key feature of it is that
all reactions are carried out in aqueous solutions. In summary, we
first synthesized a molecular anchor composed of cyclooctyne and silatrane
for introduction of a chemically reactive function to AFM tips and
a bifunctional polyethylene glycol linker that harnesses two orthogonal
click reactions, copper free alkyne–azide cycloaddition and
thiol-vinylsulfone Michael addition, for attaching affinity molecules
to AFM tips. The attachment chemistry was then validated by attaching
antithrombin DNA aptamers and cyclo-RGD peptides to silicon nitride
(SiN) tips, respectively, and measuring forces of unbinding these
affinity molecules from their protein cognates human α-thrombin
and human α<sub>5</sub>β<sub>1</sub>-integrin immobilized
on mica surfaces. In turn, we used the same attachment chemistry to
functionalize silicon tips with the same affinity molecules for AFM
based recognition imaging, showing that the disease-relevant biomarkers
such as α-thrombin and α<sub>5</sub>β<sub>1</sub>-integrin can be detected with high sensitivity and specificity by
the single molecule technique. These studies demonstrate the feasibility
of our attachment chemistry for the use in functionalization of AFM
tips with affinity molecules