Piconewton Regime Measurements of Biomolecular Interactions by Nanomechanical Force Gauge Ki-Hun Jeong, Michael Pio, Chris G. Keller † , and Luke P. Lee Berkeley Sensor and Actuator Center Department of Bioengineering, University of California, Berkeley, CA, USA † MEMS Precision Instruments, Richmond, CA, USA Abstract  A piconewton regime measurement of biomolecular interactions in an aqueous solution by a novel Nanomechanical Force Gauge (NFG) is presented in this paper. A highly sensitive nanoscale cantilever with a spring constant, which is thousand times smaller than that of an atomic force microscope (AFM) microcantilever, is fabricated by a batch process. The NFG has a capability of direct reading without any optical amplification. The control of nanoscale thickness of a single crystal silicon cantilever is done by a thermal oxidation process. The deflection of the cantilever, corresponding to piconewtons is directly measured by reading the tick movements in the reading scale of the NFG under the microscope. The spring constant of the NFG is calculated by identifying the natural frequency using electrostatic force excitation, and the minimum value of the designed device was 78.6 pN/mm. As an example of the biomolecular applications, the dissociation between a biotinylated bead and avidins is measured, and the mean is 636 pN. The NFG has the potential of 1 pN/mm sensitivity through the nanofabrication technology as well as serving as an inexpensive and powerful substitute for an atomic force microscope in studying bio-molecular interactions. I. INTRODUCTION Highly sensitive microprobes such as atomic force microscopy (AFM), fluorescence detection, and optical trapping have been recently introduced as proper tools to quantitatively characterize molecular interactions at the piconewton level such as single molecular mechanics, cell adhesion, or dissociation strength between biomolecules. In particular, an AFM, which consist of a microcantilever, laser, optical apparatus, and detector, has been used for the studies of the mechanical behavior of biomolecules or living cells in both air and liquid environments. Many efforts have been put into the development of cantilever- based sensors for the detection of physical phenomena and chemical reactions [1, 2, 3]. However, most of the sensors require extra optical components and detectors. In addition, the laser optical alignment is often a cumbersome and time-consuming task for the optical amplification of signals. Unlike an AFM or other microprobes, the NFG introduced in this paper has a capability of direct reading without any laser, detector, and optical amplification. It is a simple and robust device to install and measure. In addition, the NFG `is one of the most inexpensive ways to measure the interaction between biomolecules in the piconewton regime since it is fabricated with a similar cost of an AFM tip. The NFG is easy to set up on microscope and can be measured by the direct reading of the tick movements through the objective lens as illustrated in Fig. 1. Simultaneous force measurement and epifluorescence detection can be also performed directly in an epi- fluorescence microscope without optical amplifications. This paper reports the fabrication, calibration, and demonstration of a NFG with a highly sensitive nanoscale cantilever. The microfabrication of a nanoscale cantilever with lateral flexibility is described using thermal oxidation. The calibration of the device is carried out through measuring the natural frequency by an electrostatic force excitation source. As an example of the biomolecular applications, the measurement of the dissociation force of proteins is demonstrated with the NFG. Fig. 1. An apparatus of piconewton measurement of biomolecular interactions by a Nanomechanical Force Gauge (NFG) with a nanoscale cantilever.