**2.3. Microcantilever in cellulose study**

In cellulose, the interaction energy arises from noncovalent interactions (hydrogen bonding and van der Waals interactions) between tightly packed cellulose chains. To release cellulose chains, surface-adsorbed cellulase must break up noncovalent interactions between cellulose chains through decrystallization, which results in a change in overall interaction energy in the cellulose. If cellulose is deposited onto a microcantilever, such dynamic change in interaction energy will be transduced from the cellulose coating into the microcantilever, and will result in a measurable bending of the microcantilever. Thus, the unique link between the enzymatic decrystallization and the resulting energy alteration in cellulose measured by the microcantilever bending has provided a novel strategy to experimentally examine this unusual enzymatic activity.

Probing the Interaction Between Cellulose and Cellulase with a Nanomechanical Sensor 133

sit for about 1 h. Afterwards a drop of water was added to form the cellulose film. The resulting cellulose-coated microcantilever was soaked in water for additional 4 h, during which the water was replaced every 30 min. Finally the cellulose-coated microcantilever was

AFM imaging (Zhao et al., 2010): To characterize the coverage, morphology, roughness, and thickness of surface coatings, AFM imaging was performed using a Nanoscope IIIa multimode scanning probe microscope from Digital Instruments, Inc. The samples were scanned in contact mode in air using silicon nitride cantilevers (MLCT) manufactured by Veeco (Camarillo, CA), with a nominal spring constant of 0.05 N/m. Images were obtained from at least three different surface areas of the sample with a typical size of 5 μm × 5 μm. The images of both height and deflection modes were captured and the surface morphology

Microcantilever measurement: All the experiments were performed using a modified commercial Nanoscope III scanning probe microscope (Digital Instruments/Veeco, Santa Barbara, CA). The cellulose-coated microcantilever was mounted with the coating facing down in a liquid cell of the AFM with a volume of 50 μL. The solutions were introduced through injection. The cellulose-coated microcantilever was usually allowed to equilibrate in the assay buffer (25 mM sodium acetate, pH 5.5) for at least 2 h prior to any addition. The enzyme solution was prepared in the same buffer 30 min prior to use. Each assay was run against a reference to allow the subtraction of the background signal and to control for the bulk solvent effect. A desktop PC, running programs written in LabView (National Instruments, Austin, TX), was used to record the microcantilever deflection signal from the AFM via a data acquisition board with a maximum data acquisition rate of 300 kHz. The deflection measurement was monitored using a 5 mW laser diode with a wavelength of 680 nm, and a split position sensitive detector. During a measurement, 100,000 data points were taken every 30 seconds at a rate of 100 kHz. The bending of the microcantilever was

The surface of a microcantilever was coated with a model film of cellulose II having a thickness between 10 to 20 nm. It is generally agreed that the natural substrate is often too complicated to be useful for detailed characterizations of cellulases (Kontturi et al., 2006). Various cellulose model surfaces have therefore been developed in the past decade (Kontturi et al., 2006) and utilized extensively in a variety of studies including the investigation of the interaction between cellulose and cellulase. Among all the model surfaces, cellulose II has been used most often and there is much technical information available about it. In addition, the surface of cellulose II is easy to prepare and characterize.

For attaching cellulose to the surface of a microcantilever, an anchoring layer of PVAM was first prepared (Figure 10A). The cellulose dissolved in a hot mixture of NMMO and DMSO was then deposited onto the top of the anchoring layer (Falt et al., 2003; Zhao et al., 2010). The cellulose coating on the microcantilever was characterized with AFM imaging and the

All of these make the cellulose II film an ideal model surface for the current study.

incubated in an oven at 80°C for 1 h to complete the surface coating.

was analyzed using the image-processing software.

obtained by simply averaging the data points.

**3.2. Preparation of a cellulose-coated microcantilever** 

Such adsorbate-induced bending often occurs in the presence of a specific interaction (e.g., decrystallization) between an adsorbate (e.g., cellulase) and a substrate (e.g., cellulose) that can alter the internal energy of the substrate. Since a mere adsorption such as the initial binding of cellulase on cellulose has a very minimum impact on the internal energy of cellulose, it will not induce a measurable bending even though the surface-bound cellulase changes the overall gravity (mass) on the microcantilever. Therefore the microcantilever bending can be attributed primarily to enzymatic actions (e.g., enzymatic decrystallization and hydrolytic cleavage) after the initial binding. Because the extent of bending is linearly proportional to the effective concentration of adsorbed species on the microcantilever (Berger et al., 1997; Desikan et al., 2006; Velanki & Ji, 2006), the real-time measurement of the microcantilever bending will reveal the kinetics for enzymatic actions including enzymatic decrystallization by cellulase. To our knowledge, this is the first use of a nanomechanical sensor to study mechanistic enzymology and heterogeneous enzymatic catalysis that involves a solid substrate (e.g., cellulose).
