A Three-Arm Scaffold Carrying Affinity Molecules for Multiplex Recognition Imaging by Atomic Force Microscopy: The Synthesis, Attachment to Silicon Tips, and Detection of Proteins

We have developed a multiplex imaging method for detection of proteins using atomic force microscopy (AFM), which we call multiplex recognition imaging (mRI). AFM has been harnessed to identify protein using a tip functionalized with an affinity molecule at a single molecule level. However, many events in biochemistry require identification of colocated factors simultaneously, and this is not possible with only one type of affinity molecule on an AFM tip. To enable AFM detection of multiple analytes, we designed a recognition head made from conjugating two different affinity molecules to a three-arm linker. When it is attached to an AFM tip, the recognition head would allow the affinity molecules to function in concert. In the present study, we synthesized two recognition heads: one was composed of two nucleic acid aptamers, and the other one composed of an aptamer and a cyclic peptide. They were attached to AFM tips through a catalyst-free click reaction. Our imaging results show that each affinity unit in the recognition head can recognize its respective cognate in an AFM scanning process independently and specifically. The AFM method was sensitive, only requiring 2 to 3 μL of protein solution with a concentration of ∼2 ng/mL for the detection with our current setup. When a mixed sample was deposited on a surface, the ratio of proteins could be determined by counting numbers of the analytes. Thus, this mRI approach has the potential to be used as a label-free system for detection of low-abundance protein biomarkers

Application of Catalyst-Free Click Reactions in Attaching Affinity Molecules to Tips of Atomic Force Microscopy for Detection of Protein Biomarkers

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 α₅β₁-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 α₅β₁-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

AFM image of an intact murine ROS disc.

<p>(A, B) Height (A) and deflection (B) images obtained by contact mode AFM generated using low force. (C, D) Height (C) and deflection (D) images obtained by contact mode AFM generated using higher force. The rim region (1) and nanodomains in the lamellar region (2) are discernible. Height images were scaled to a height range of 38 nm. Scale bar, 250 nm. Illustrations of a disc adsorbed on mica scanned by the AFM tip at low and high forces are shown next to AFM images. (E) A height profile is shown for the cross-section highlighted by a dotted line in panel C.</p

AFM images of <i>X</i>. <i>laevis</i> ROS disc membranes.

<p>(A-G) Representative deflection images of <i>X</i>. <i>laevis</i> ROS disc membranes obtained by contact mode AFM. ROS disc membranes exhibit a varying number of lobes, which are formed by deeply penetrating incisures. Scale bar, 500 nm. (H) Histogram of nanodomain sizes measured in 57 images of <i>X</i>. <i>laevis</i> ROS disc membranes. The data was fit by a Log Gaussian function (<i>n</i> = 14,390).</p

Murine ROS disc membranes imaged at 37°C.

<p>Representative images obtained by tapping mode AFM are shown. Murine ROS disc membranes were prepared at 4°C and imaged at 37°C. Height (left) and amplitude (right) images are shown. Height images were scaled to a height range of 25 nm. Scale bar, 500 nm.</p

<i>X</i>. <i>laevis</i> ROS disc membrane preparation.

<p>(A) The secondary structure of rhodopsin is shown with amino acid residue differences in <i>X</i>. <i>laevis</i> (red) and murine (blue) rhodopsin highlighted. (B) Light microscopy image of purified ROS from the retina of <i>X</i>. <i>laevis</i>. Scale bar, 15 μm. (C) SDS-PAGE of <i>X</i>. <i>laevis</i> (lane 1) and murine (lane 2) ROS disc membrane preparations. The sizes of protein standards are indicated in kDa.</p