**1. Introduction**

124 Cellulose – Medical, Pharmaceutical and Electronic Applications

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## **1.1. Cellulose and cellulose biomass**

A cellulose molecule is a linear polymer of D-anhydroglucopyranose units linked by β-1, 4 glucosidic bonds (Figure 1). On its reducing end, a cellulose molecule has an unsubstituted hemiacetal. On its non-reducing end, it has a hydroxyl group.

**Figure 1.** Molecular structure of a cellulose molecule.

Cellulose is the skeleton structure of almost all green plants. It is particularly abundant in non-food plants like trees and grasses, which typically have 40-60% cellulose, 20-40% hemicellulose, and 10-25% lignin (Lynd et al., 2002; Yang et al., 2007). There are four major polymorphs of cellulose: I, II, III, and IV. Cellulose I, often found in native cellulose, contains allomorphs Iα (bacteria and algae) and Iβ (higher plants) (Kontturi et al., 2006; Pérez & Samain, 2010). Cellulose I, when treated with a concentrated alkaline solution, turns into cellulose II, a thermodynamically more stable crystalline form than cellulose I. Cellulose IIII can be obtained when cellulose microcrystal is subjected to supercritical ammonia. The structure of another allomorph of cellulose III, IIIII is still being debated. Cellulose IVI and IVII are formed when cellulose IIIis heated in glycerol at 260°C (Peter, 2001).

Probing the Interaction Between Cellulose and Cellulase with a Nanomechanical Sensor 127

recalcitrance and high cost of cellulase in biomass conversion, the current process for biofuel production is not yet a viable option for the large-scale production (Dwivedi et al., 2009). Much research and developmental efforts have been dedicated to the improvement of the efficiency of cellulase in biomass conversion. One feasible approach is through the incorporation of new features (mutations) into cellulase that accelerate key steps (e.g., rate limiting step) of the enzymatic process. This approach requires a comprehensive

**Figure 3.** The process of biomass conversion through the enzyme-based biochemical method.

Cellulase (Lynd et al., 2002; Mosier et al., 1999; Wilson & Irwin, 1999), like all glycosyl hydrolase enzymes found in bacteria, fungi, plants and some invertebrate animals, breaks down β-1, 4-glycosidic bonds of cellulose through general acid/base catalysis. There are mainly three kinds of cellulases: exo-β-1, 4-D-glucanase, endo-β-1, 4-D-glucanase and β-Dglucosidase. Each enzyme alone cannot hydrolyze the complex crystalline cellulose efficiently but working synergistically with other types of cellulases can increase the rate of

 Exo-β-1, 4-D-glucanase can access individual cellulose chains from the exposed reducing end or non-reducing end and cleave two to four glucose units at a time to

 Endo-β-1, 4-D-glucanase breaks internal glycosidic bonds of individual cellulose chains to disrupt the network structure of cellulose and expose individual polysaccharide

produce tetrasaccharides or disaccharides (i.e., cellobioses) (Figure 4).

β-D-Glucosidase or cellobiase hydrolyzes cellobiose to release D-glucose units.

**Figure 4.** Schematic illustration of cellulose hydrolysis by exo-β-1, 4-D-glucanase and endo-β-1, 4-D-

Most of fungal cellulases have a domain like structure that contains a catalytic domain (CD) and a carbohydrate binding module (CBM) (Martin, 2000). These two domains are

mechanistic understanding of cellulose hydrolysis by cellulase.

**1.3. Cellulase and enzymatic hydrolysis of cellulose** 

hydrolysis significantly (Dwivedi et al., 2009; Lynd et al., 2002).

chains (Figure 4).

glucanase.

**Figure 2.** (A) Hydrophilic and hydrophobic sites of cellulose. (B) Schematic drawing of the intrasheet hydrogen-bonding network in cellulose Iα.

Various noncovalent interactions such as hydrogen bonding and van der Waals interactions are present in the ultrastructure of cellulose. While the OH-O hydrogen bonding is mostly responsible for cellulose intrasheet interactions, both the weaker CH-O hydrogen bonding and van der Waals interactions contribute to cellulose intersheet interactions (Li Q. & Renneckar, 2011). Figure 2 shows the arrangement of the intrasheet hydrogen bonding network in cellulose Iα and the resulting hydrophilic and hydrophobic sites of the ring plane (Brown & Saxena, 2007). Overall, because of these noncovalent interactions, cellulose chains aggregate into various forms of ultrastructure, which do not melt or dissolve in any common solvents. Such aggregation prevents the potential cleavage sites (i.e., glycosidic bonds) of a cellulose chain from being accessed by cellulase.

#### **1.2. Degradation of cellulose in biomass conversion**

The biomass conversion is the key step to produce biofuel from cellulosic biomass. Such conversion is often accomplished either through biochemical methods or thermochemical methods, where the polysaccharides in cellulosic biomass are hydrolyzed by biochemical agents such as cellulase enzyme, or by thermal treatment such as gasification to produce simple sugars that are fermentable to produce biofuel products (Dwivedi et al., 2009). For biochemical methods (Gray et al., 2006), cellulases are usually employed to convert the solid cellulosic biomass into glucose or small sugar polymers that can be readily fermented with microorganism to produce ethanol. Compared to thermochemical methods which often require a large amount of acid and energy, biochemical methods are more environmentally friendly and economically feasible because of their better conversion efficiencies and milder operating conditions. By far the enzyme-based biochemical methods are considered as the most promising technologies for biomass conversion. However, because of biomass recalcitrance and high cost of cellulase in biomass conversion, the current process for biofuel production is not yet a viable option for the large-scale production (Dwivedi et al., 2009). Much research and developmental efforts have been dedicated to the improvement of the efficiency of cellulase in biomass conversion. One feasible approach is through the incorporation of new features (mutations) into cellulase that accelerate key steps (e.g., rate limiting step) of the enzymatic process. This approach requires a comprehensive mechanistic understanding of cellulose hydrolysis by cellulase.

**Figure 3.** The process of biomass conversion through the enzyme-based biochemical method.

## **1.3. Cellulase and enzymatic hydrolysis of cellulose**

126 Cellulose – Medical, Pharmaceutical and Electronic Applications

hydrogen-bonding network in cellulose Iα.

bonds) of a cellulose chain from being accessed by cellulase.

**1.2. Degradation of cellulose in biomass conversion** 

**Figure 2.** (A) Hydrophilic and hydrophobic sites of cellulose. (B) Schematic drawing of the intrasheet

Various noncovalent interactions such as hydrogen bonding and van der Waals interactions are present in the ultrastructure of cellulose. While the OH-O hydrogen bonding is mostly responsible for cellulose intrasheet interactions, both the weaker CH-O hydrogen bonding and van der Waals interactions contribute to cellulose intersheet interactions (Li Q. & Renneckar, 2011). Figure 2 shows the arrangement of the intrasheet hydrogen bonding network in cellulose Iα and the resulting hydrophilic and hydrophobic sites of the ring plane (Brown & Saxena, 2007). Overall, because of these noncovalent interactions, cellulose chains aggregate into various forms of ultrastructure, which do not melt or dissolve in any common solvents. Such aggregation prevents the potential cleavage sites (i.e., glycosidic

The biomass conversion is the key step to produce biofuel from cellulosic biomass. Such conversion is often accomplished either through biochemical methods or thermochemical methods, where the polysaccharides in cellulosic biomass are hydrolyzed by biochemical agents such as cellulase enzyme, or by thermal treatment such as gasification to produce simple sugars that are fermentable to produce biofuel products (Dwivedi et al., 2009). For biochemical methods (Gray et al., 2006), cellulases are usually employed to convert the solid cellulosic biomass into glucose or small sugar polymers that can be readily fermented with microorganism to produce ethanol. Compared to thermochemical methods which often require a large amount of acid and energy, biochemical methods are more environmentally friendly and economically feasible because of their better conversion efficiencies and milder operating conditions. By far the enzyme-based biochemical methods are considered as the most promising technologies for biomass conversion. However, because of biomass Cellulase (Lynd et al., 2002; Mosier et al., 1999; Wilson & Irwin, 1999), like all glycosyl hydrolase enzymes found in bacteria, fungi, plants and some invertebrate animals, breaks down β-1, 4-glycosidic bonds of cellulose through general acid/base catalysis. There are mainly three kinds of cellulases: exo-β-1, 4-D-glucanase, endo-β-1, 4-D-glucanase and β-Dglucosidase. Each enzyme alone cannot hydrolyze the complex crystalline cellulose efficiently but working synergistically with other types of cellulases can increase the rate of hydrolysis significantly (Dwivedi et al., 2009; Lynd et al., 2002).


**Figure 4.** Schematic illustration of cellulose hydrolysis by exo-β-1, 4-D-glucanase and endo-β-1, 4-Dglucanase.

Most of fungal cellulases have a domain like structure that contains a catalytic domain (CD) and a carbohydrate binding module (CBM) (Martin, 2000). These two domains are

connected by a peptide linker, which is known to maintain the separation between the CD and the CBM (Figure 5). The CD contains the enzyme active site that is responsible for cellulose hydrolysis. The CBM is a contiguous amino acid sequence that anchors the CD onto the surface of cellulose through hydrogen bonding and van der Waals interactions (Boraston et al., 2004; Guillén et al., 2010). Cellulases can be grouped into families according to sequence similarities of their amino acid residues within their CDs and CBMs.

Probing the Interaction Between Cellulose and Cellulase with a Nanomechanical Sensor 129

equilibrium binding models and dynamic binding models have also been proposed to account for the complexities of the binding process including the partially irreversible adsorption of

For cellulose hydrolysis, most recent studies have been focused on elucidation of the mechanism of the hydrolytic cleavage reaction (step 3 in Figure 6) (Divne et al., 1998; Li Y. et al., 2007; Parsiegla et al., 2008). Some of the key amino acid residues involved in cleavage reactions have been identified. The distinctions between endo- and exo-glucanases, and between retention and inversion for the stereo configuration of the products have been made after years of biochemical and biophysical studies. The majority of studies on the hydrolytic cleavage step were done by measuring the concentration of the sugars (P)

Meanwhile, very little success has been achieved in obtaining fundamental knowledge of the enzymatic decrystallization reaction in step 2 (DOE/SC-0095, 2006). In particular, the importance of enzymatic decrystallization was largely unnoticed until very recently (Chundawat et al., 2011; DOE/SC-0095, 2006; Wilson, 2009). Several pieces of biochemical and physical evidence have indicated the presence of such an activity. Back to 1997, Wilson and his coworker showed that cleavage of the β-1, 4-glycosidic linkage in crystalline cellulose is not the rate-limiting step for *T. fusca* endoglucanase E2 (Zhang S. & Wilson, 1997). They speculated that the binding of a cellulose chain from a microfibril into the active site of a cellulase is the rate-limiting step for degradation of crystalline cellulose (Wilson, 2009). Lee and coworkers found indentations and paths on the surface of cotton fibers that had been treated with a cellulase that was incapable of hydrolytic cleavage (Lee et al., 2000). The evidence suggests that such surface modifications on cellulose are likely caused by the

**Figure 7.** Mechanistic models of enzymatic decrystallization. (A) The CBM model: the CBM serves as a wedge to assist the release of a single cellulose chain. (B) The CD model: the protrusion of the CD

The mechanistic model for enzymatic decrystallization proposed by Reilly and coworkers is shown in Figure 7A (Mulakala & Reilly, 2005). In this model, the CBM is inserted under a cellulose chain like a wedge to separate the chain from the cellulose network. Then the released cellulose chain is pulled into the active site of the CD along the top face of the CBM to achieve the decrystallization. In this model, the CBM is essential to enzymatic decrystallization of cellulose. Numerous biochemical studies, however, have shown that in the absence of the CBM, the CD domain alone still retains 20~50% of the hydrolytic activity on crystalline cellulose (Reinikainen et al., 1992; Srisodsuk et al., 1993; Van Tilbeurgh et al.,

domain serves as a wedge to assist the release of a single cellulose chain.

cellulase, multiple types of adsorption site, and so on (Lynd et al., 2002).

released during cleavage of soluble cellodextrins or insoluble cellulose.

decrystallization activity of cellulase.

**Figure 5.** The domain like structure of the cellulase cellobiohydrolase I (CBH I) that is bound to cellulose Iβ microfibril. (Zhong et al., 2008)

Cellulose hydrolysis by cellulase is a multi-step process (Chundawat et al., 2011) that is initiated with the binding of cellulase (E) onto the surface of cellulose (S), shown in Figure 6. After this "initial binding" step, a single cellulose chain will be separated from the cellulose aggregate by cellulase and pulled into the active site of cellulase. This is the "decrystallization" step which forms a pseudo-Michaelis complex (E\*S). E\*S will then undergo "hydrolytic cleavage" to produce cellobiose as the product (P).

**Figure 6.** Mechanism of cellulose hydrolysis by cellulase.

For cellulose hydrolysis, the substrate cellulose is water insoluble and resistant to the attack by biological agents. This makes the formation of E\*S much more difficult compared to those formed with soluble substrates.

#### **1.4. Knowledge gap**

The initial binding (step 1 in Figure 6) has been extensively studied over the years. A Langmuir equation is widely used as a simplified mechanistic model to describe the formation of ES resulting from the initial interaction between the cellulase and the cellulose, which often reaches steady-state within half an hour (Lynd et al., 2002; Zhang Y. H. & Lynd, 2004). Other equilibrium binding models and dynamic binding models have also been proposed to account for the complexities of the binding process including the partially irreversible adsorption of cellulase, multiple types of adsorption site, and so on (Lynd et al., 2002).

128 Cellulose – Medical, Pharmaceutical and Electronic Applications

cellulose Iβ microfibril. (Zhong et al., 2008)

connected by a peptide linker, which is known to maintain the separation between the CD and the CBM (Figure 5). The CD contains the enzyme active site that is responsible for cellulose hydrolysis. The CBM is a contiguous amino acid sequence that anchors the CD onto the surface of cellulose through hydrogen bonding and van der Waals interactions (Boraston et al., 2004; Guillén et al., 2010). Cellulases can be grouped into families according

to sequence similarities of their amino acid residues within their CDs and CBMs.

**Figure 5.** The domain like structure of the cellulase cellobiohydrolase I (CBH I) that is bound to

undergo "hydrolytic cleavage" to produce cellobiose as the product (P).

**Figure 6.** Mechanism of cellulose hydrolysis by cellulase.

those formed with soluble substrates.

**1.4. Knowledge gap** 

Cellulose hydrolysis by cellulase is a multi-step process (Chundawat et al., 2011) that is initiated with the binding of cellulase (E) onto the surface of cellulose (S), shown in Figure 6. After this "initial binding" step, a single cellulose chain will be separated from the cellulose aggregate by cellulase and pulled into the active site of cellulase. This is the "decrystallization" step which forms a pseudo-Michaelis complex (E\*S). E\*S will then

For cellulose hydrolysis, the substrate cellulose is water insoluble and resistant to the attack by biological agents. This makes the formation of E\*S much more difficult compared to

The initial binding (step 1 in Figure 6) has been extensively studied over the years. A Langmuir equation is widely used as a simplified mechanistic model to describe the formation of ES resulting from the initial interaction between the cellulase and the cellulose, which often reaches steady-state within half an hour (Lynd et al., 2002; Zhang Y. H. & Lynd, 2004). Other For cellulose hydrolysis, most recent studies have been focused on elucidation of the mechanism of the hydrolytic cleavage reaction (step 3 in Figure 6) (Divne et al., 1998; Li Y. et al., 2007; Parsiegla et al., 2008). Some of the key amino acid residues involved in cleavage reactions have been identified. The distinctions between endo- and exo-glucanases, and between retention and inversion for the stereo configuration of the products have been made after years of biochemical and biophysical studies. The majority of studies on the hydrolytic cleavage step were done by measuring the concentration of the sugars (P) released during cleavage of soluble cellodextrins or insoluble cellulose.

Meanwhile, very little success has been achieved in obtaining fundamental knowledge of the enzymatic decrystallization reaction in step 2 (DOE/SC-0095, 2006). In particular, the importance of enzymatic decrystallization was largely unnoticed until very recently (Chundawat et al., 2011; DOE/SC-0095, 2006; Wilson, 2009). Several pieces of biochemical and physical evidence have indicated the presence of such an activity. Back to 1997, Wilson and his coworker showed that cleavage of the β-1, 4-glycosidic linkage in crystalline cellulose is not the rate-limiting step for *T. fusca* endoglucanase E2 (Zhang S. & Wilson, 1997). They speculated that the binding of a cellulose chain from a microfibril into the active site of a cellulase is the rate-limiting step for degradation of crystalline cellulose (Wilson, 2009). Lee and coworkers found indentations and paths on the surface of cotton fibers that had been treated with a cellulase that was incapable of hydrolytic cleavage (Lee et al., 2000). The evidence suggests that such surface modifications on cellulose are likely caused by the decrystallization activity of cellulase.

**Figure 7.** Mechanistic models of enzymatic decrystallization. (A) The CBM model: the CBM serves as a wedge to assist the release of a single cellulose chain. (B) The CD model: the protrusion of the CD domain serves as a wedge to assist the release of a single cellulose chain.

The mechanistic model for enzymatic decrystallization proposed by Reilly and coworkers is shown in Figure 7A (Mulakala & Reilly, 2005). In this model, the CBM is inserted under a cellulose chain like a wedge to separate the chain from the cellulose network. Then the released cellulose chain is pulled into the active site of the CD along the top face of the CBM to achieve the decrystallization. In this model, the CBM is essential to enzymatic decrystallization of cellulose. Numerous biochemical studies, however, have shown that in the absence of the CBM, the CD domain alone still retains 20~50% of the hydrolytic activity on crystalline cellulose (Reinikainen et al., 1992; Srisodsuk et al., 1993; Van Tilbeurgh et al., 1986). To resolve this discrepancy, we propose an alternative mechanistic model, where a wedge-like structure at the bottom of the CD can be inserted under the cellulose chain to lift it into the active site of the CD (Figure 7B). Since decrystallization by cellulase has been speculated to be the rate limiting step for the degradation of crystalline cellulose (DOE/SC-0095, 2006; Wilson, 2009), understanding the mechanism of this enzymatic activity becomes essential to a comprehensive understanding of cellulose hydrolysis by cellulase.

Probing the Interaction Between Cellulose and Cellulase with a Nanomechanical Sensor 131

The microcantilever is a highly sensitive nanomechanical sensor that originates from a microfabricated AFM probe. Typical dimensions of a microcantilever, which are similar to AFM probes, are 200 μm long, 1 μm thick, and 20 μm wide (Goeders et al., 2008; Lavrik et al., 2004). The microcantilever is capable of detecting minute changes in interaction energy between individual molecules in the thin film of a polymer coating (cellulose, protein, DNA, or polymer brush) in the form of a measurable bending (10-6 to 10-12 m) of the microcantilever (Moulin et al., 1999; Mukhopadhyay et al., 2005; Shu et al., 2005; Yan et al., 2006; Zhao et al., 2010; Zhou et al., 2006). The microcantilever bending can be measured based on the deflection of a laser beam reflecting from the tip of the microcantilever in the AFM (Figure 9). Overall, the

Dynamic mode (Vashist, 2007): When an analyte is adsorbed on an oscillating microcantilever, the microcantilever will oscillate at a lower frequency. The difference in frequency can be used

Static mode (Vashist, 2007): Adsorbates tend to induce a significant change in surface stress of a microcantilever, which results in a deflection of the microcantilever (Lavrik et al., 2004; Shuttleworth, 1950). The surface stress and the deflection of the microcantilever are

> *Et*

Where Δz is the deflection of the microcantilever at the end of the microcantilever, *ν* and *E* are Poisson's ratio and Young's modulus of the microcantilever, *t* and *l* are the thickness and length of the microcantilever, and ∆*σ* is the differential stress on the microcantilever. Using the static mode microcantilever sensor, Ji and coworkers investigated the conformational change of calmodulin (Yan et al., 2006). Sauers and coworkers successfully detected 2 mercaptoethanol using a gold-coated microcantilever (Datskos & Sauers, 1999). Zhou and coworkers successfully demonstrated the use of the microcantilever bending as a means to probe changes in internal structure of polymer brushes in response to changes in pH and

2 2 3(1 )*l*

(1)

to measure properties of adsorbates, such as viscosity and density, etc.

*z*

microcantilever detection has high specificity, high sensitivity, and quick response.

**2.2. Microcantilever and its applications** 

**Figure 9.** Working scheme of the microcantilever.

quantitatively related (Yan et al., 2006):

electrolyte concentration (Zhou et al., 2006).

## **2. Innovative approach and microcantilever**

#### **2.1. Existing technologies and their technical limitations**

The conventional approaches to study the cellulose hydrolysis by cellulase are based on the measurement of the concentration of glucose or other simple sugars that are produced in the hydrolytic process. These approaches are not suitable for studying the decrystallization process because no new product is formed and released from this process. Spectroscopic techniques such as Fourier transform infrared spectroscopy (Fengel et al., 1995), Raman spectroscopy (Schenzel et al., 2005), and x-ray photoelectron spectroscopy (Ahola et al., 2008; Fardim et al., 2005) have been used to study the structural change of cellulose fibers. All these techniques focus on the global variations of cellulose. Since cellulose decrystallization only occurs on the outer layer within a relative small region of the surface of cellulose, these techniques are not sensitive enough for such study. Quartz crystal microbalance has been used to study the enzymatic hydrolysis of cellulose (Ahola et al., 2008; Rojas Orlando et al., 2007; Turon et al., 2008), however, its suitability for studying decrystallization has not yet been demonstrated.

**Figure 8.** Various shapes of AFM probes.

Atomic force microscopy (AFM) has been used to examine changes of surface morphology of cellulose. Typical size of AFM probe is 200 μm x 40 μm x 1 μm (length x width x thickness) with various shapes (Figure 8). Lee and coworkers used the tapping mode AFM to exam the effects of three different cellulases on the surface of cotton fibers (Lee et al., 2000). Li and coworkers used the AFM to detect structural changes of cellulose microfibril fragments caused by sonication (Li Q. & Renneckar, 2011). With this powerful surface imaging tool, we can investigate the surface change caused by enzymatic activities. However, AFM imaging is mostly limited to the surface analysis at discrete time points. Additionally a high quality image requires that the surface area of cellulose be prepared prior to imaging. Any extensive handling may potentially alter the surface properties and delay the timely analysis under the assay conditions*.* Therefore, AFM imaging is not an ideal approach for analyzing the dynamic impact of cellulase on cellulose. Both scanning electron microscopy and transmission electron microscopy have similar limitations. This has led to the development of the microcantilever technique for cellulase study.

#### **2.2. Microcantilever and its applications**

130 Cellulose – Medical, Pharmaceutical and Electronic Applications

**2. Innovative approach and microcantilever** 

decrystallization has not yet been demonstrated.

**Figure 8.** Various shapes of AFM probes.

**2.1. Existing technologies and their technical limitations** 

1986). To resolve this discrepancy, we propose an alternative mechanistic model, where a wedge-like structure at the bottom of the CD can be inserted under the cellulose chain to lift it into the active site of the CD (Figure 7B). Since decrystallization by cellulase has been speculated to be the rate limiting step for the degradation of crystalline cellulose (DOE/SC-0095, 2006; Wilson, 2009), understanding the mechanism of this enzymatic activity becomes

The conventional approaches to study the cellulose hydrolysis by cellulase are based on the measurement of the concentration of glucose or other simple sugars that are produced in the hydrolytic process. These approaches are not suitable for studying the decrystallization process because no new product is formed and released from this process. Spectroscopic techniques such as Fourier transform infrared spectroscopy (Fengel et al., 1995), Raman spectroscopy (Schenzel et al., 2005), and x-ray photoelectron spectroscopy (Ahola et al., 2008; Fardim et al., 2005) have been used to study the structural change of cellulose fibers. All these techniques focus on the global variations of cellulose. Since cellulose decrystallization only occurs on the outer layer within a relative small region of the surface of cellulose, these techniques are not sensitive enough for such study. Quartz crystal microbalance has been used to study the enzymatic hydrolysis of cellulose (Ahola et al., 2008; Rojas Orlando et al., 2007; Turon et al., 2008), however, its suitability for studying

Atomic force microscopy (AFM) has been used to examine changes of surface morphology of cellulose. Typical size of AFM probe is 200 μm x 40 μm x 1 μm (length x width x thickness) with various shapes (Figure 8). Lee and coworkers used the tapping mode AFM to exam the effects of three different cellulases on the surface of cotton fibers (Lee et al., 2000). Li and coworkers used the AFM to detect structural changes of cellulose microfibril fragments caused by sonication (Li Q. & Renneckar, 2011). With this powerful surface imaging tool, we can investigate the surface change caused by enzymatic activities. However, AFM imaging is mostly limited to the surface analysis at discrete time points. Additionally a high quality image requires that the surface area of cellulose be prepared prior to imaging. Any extensive handling may potentially alter the surface properties and delay the timely analysis under the assay conditions*.* Therefore, AFM imaging is not an ideal approach for analyzing the dynamic impact of cellulase on cellulose. Both scanning electron microscopy and transmission electron microscopy have similar limitations. This has

led to the development of the microcantilever technique for cellulase study.

essential to a comprehensive understanding of cellulose hydrolysis by cellulase.

The microcantilever is a highly sensitive nanomechanical sensor that originates from a microfabricated AFM probe. Typical dimensions of a microcantilever, which are similar to AFM probes, are 200 μm long, 1 μm thick, and 20 μm wide (Goeders et al., 2008; Lavrik et al., 2004). The microcantilever is capable of detecting minute changes in interaction energy between individual molecules in the thin film of a polymer coating (cellulose, protein, DNA, or polymer brush) in the form of a measurable bending (10-6 to 10-12 m) of the microcantilever (Moulin et al., 1999; Mukhopadhyay et al., 2005; Shu et al., 2005; Yan et al., 2006; Zhao et al., 2010; Zhou et al., 2006). The microcantilever bending can be measured based on the deflection of a laser beam reflecting from the tip of the microcantilever in the AFM (Figure 9). Overall, the microcantilever detection has high specificity, high sensitivity, and quick response.

**Figure 9.** Working scheme of the microcantilever.

Dynamic mode (Vashist, 2007): When an analyte is adsorbed on an oscillating microcantilever, the microcantilever will oscillate at a lower frequency. The difference in frequency can be used to measure properties of adsorbates, such as viscosity and density, etc.

Static mode (Vashist, 2007): Adsorbates tend to induce a significant change in surface stress of a microcantilever, which results in a deflection of the microcantilever (Lavrik et al., 2004; Shuttleworth, 1950). The surface stress and the deflection of the microcantilever are quantitatively related (Yan et al., 2006):

$$
\Delta z = \frac{\Im(1-\nu)l^2}{Et^2} \Delta \sigma \tag{1}
$$

Where Δz is the deflection of the microcantilever at the end of the microcantilever, *ν* and *E* are Poisson's ratio and Young's modulus of the microcantilever, *t* and *l* are the thickness and length of the microcantilever, and ∆*σ* is the differential stress on the microcantilever. Using the static mode microcantilever sensor, Ji and coworkers investigated the conformational change of calmodulin (Yan et al., 2006). Sauers and coworkers successfully detected 2 mercaptoethanol using a gold-coated microcantilever (Datskos & Sauers, 1999). Zhou and coworkers successfully demonstrated the use of the microcantilever bending as a means to probe changes in internal structure of polymer brushes in response to changes in pH and electrolyte concentration (Zhou et al., 2006).
