Determination of the surface pK(a) of self-assembled monolayers (SAMs) at aqueous buffer/gold interfaces using surface-enhanced Raman scattering (SERS) measurements, namely, SERS titration, is reported for the first time. From the analysis of pH-dependent SERS spectra of 4-mercaptopyridine monolayers on gold, the pK(1/2) (3.9 ± 0.2) was determined. By further correlating the surface pH value with the bulk pH value in terms of the Gouy-Chapmann model, the surface pK(a) (5.3 ± 0.3) was evaluated accordingly. Compared to the conventional contact angle titration, SERS titration has the advantage of giving further insight into the microscopic and dynamic information of the surface-confined functional groups. In addition, such a technique has high sensitivity, micrometer spatial resolution, and chemical selectivity.

We used atomic force microscopy (AFM) to explore the antigen binding forces of individual Fv fragments of antilysozyme antibodies (Fv). To detect single molecular recognition events, genetically engineered histidine-tagged Fv fragments were coupled onto AFM tips modified with mixed self-assembled monolayers (SAMs) of nitrilotriacetic acid- and tri(ethylene glycol)-terminated alkanethiols while lysozyme (Lyso) was covalently immobilized onto mixed SAMs of carboxyl- and hydroxyl-terminated alkanethiols. The quality of the functionalization procedure was validated using X-ray photoelectron spectroscopy (surface chemical composition), AFM imaging (surface morphology in aqueous solution), and surface plasmon resonance (SPR, specific binding in aqueous solution). AFM force-distance curves recorded at a loading rate of 5000 pN/s between Fv- and Lyso-modified surfaces yielded a distribution of unbinding forces composed of integer multiples of an elementary force quantum of approximately 50 pN that we attribute to the rupture of a single antibody-antigen pair. Injection of a solution containing free Lyso caused a dramatic reduction of adhesion probability, indicating that the measured 50 pN unbinding forces are due to the specific antibody-antigen interaction. To investigate the dynamics of the interaction, force-distance curves were recorded at various loading rates. Plots of unbinding force vs log(loading rate) revealed two distinct linear regimes with ascending slopes, indicating multiple barriers were present in the energy landscape. The kinetic off-rate constant of dissociation (k(off) approximately = 1 x 10(-3) s(-1)) obtained by extrapolating the data of the low-strength regime to zero force was in the range of the k(off) estimated by SPR.

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