Diagnosis and monitoring of complex diseases such as cancer require quantitative detection of multiple proteins. Recent work has shown that when specific biomolecular binding occurs on one surface of a microcantilever beam, intermolecular nanomechanics bend the cantilever, which can be optically detected. Although this label-free technique readily lends itself to formation of microcantilever arrays, what has remained unclear is the technologically critical issue of whether it is sufficiently specific and sensitive to detect disease-related proteins at clinically relevant conditions and concentrations. As an example, we report here that microcantilevers of different geometries have been used to detect two forms of prostate-specific antigen (PSA) over a wide range of concentrations from 0.2 ng/ml to 60 microg/ml in a background of human serum albumin (HSA) and human plasminogen (HP) at 1 mg/ml, making this a clinically relevant diagnostic technique for prostate cancer. Because cantilever motion originates from the free-energy change induced by specific biomolecular binding, this technique may offer a common platform for high-throughput label-free analysis of protein-protein binding, DNA hybridization, and DNA-protein interactions, as well as drug discovery.

Generation of nanomechanical cantilever motion from biomolecular interactions can have wide applications, ranging from highthroughput biomolecular detection to bioactuation. Although it has been suggested that such motion is caused by changes in surface stress of a cantilever beam, the origin of the surface-stress change has so far not been elucidated. By using DNA hybridization experiments, we show that the origin of motion lies in the interplay between changes in configurational entropy and intermolecular energetics induced by specific biomolecular interactions. By controlling entropy change during DNA hybridization, the direction of cantilever motion can be manipulated. These thermodynamic principles were also used to explain the origin of motion generated from protein-ligand binding. U nderstanding the mechanisms of how biological reactions produce motion is fundamental to several physiological processes (1-3). Although most past effort (4-6) has focused on studying single molecular motors (7-9), recent experiments (10, 11) by using microcantilever beams have led to observations that multiple DNA hybridization and antigen-antibody reactions can collectively produce nanomechanical motion. The promising prospects of interfacing molecular biology with micro-and nanomechanical systems can best be exploited if we learn how to control and manipulate nanomechanical motion generated by biomolecular interactions. Although an understanding of the origins of this motion would allow such control, it has so far remained elusive. It has been suggested (11) that the motion is induced by changes in surface stress of the cantilever caused by biomolecular binding. Although this may be true, the origin of surface-stress change is not understood. In this paper, we show that cantilever motion is created because of the interplay between changes in configurational entropy and intermolecular energetics induced by specific biomolecular reactions. The entropy contribution can be critical in determining the direction of motion. By using thermodynamic principles in conjunction with DNA hybridization experiments, we demonstrate that both the direction and magnitude of cantilever motion can be controlled. These thermodynamic principles are also used to explain the nanomechanical motion created by protein-ligand binding. Materials and MethodsExperimental Setup and Approach. Fig. 1 illustrates the experiment we used for studying nanomechanical motion created by multiple specific biomolecular reactions. The experimental setup consisted of a transparent fluid cell, within which a gold-coated silicon nitride (Au͞SiN x ) cantilever was mounted. The fluid cell and the V-shaped micromechanical silicon nitride cantilevers were purchased from Digital Instruments (Santa Barbara, CA). The cantilevers used were 200 m long and 0.5 m thick, and each leg was 20 m wide. The gold films originally coated on cantilevers were etched away, and a fresh 25-nm-thick gold coating was deposited. For good adhesion between gold and silicon nitride, a 5-nm-thick chro...