Wenjian+Du+Final+Project

=Using Nano Mechanical Approach to Study Enzyme Catalysis and Cell Biology=

Wenjian Du Department of Chemistry Drexel University, Philadelphia, PA. 19104 December, 2011

Abstract
Micro-cantilever and AFM which employed with cantilever are powerful nano mechanical approach in chemistry, biology, and physical field. AFM is a very powerful nano scale imaging tool and it is becoming more and more popular after it was invented in 1986. It could give 3-d information of sample surface, which make it irreplaceable among the modern characterization methods. AFM using in force curve mode could give the viscosity properties of cells, which could be used to study some cell biology process. Micro-cantilevers using as highly sensitive sensors have many valuable applications. This kind of sensor could characterize the energy change of cellulose layer on just one side of it. AFM could be used to study the morphology change of this cellulose layer. Combination of AFM with micro-cantilever sensors makes it possible to explain the interaction between enzyme and sample in enzyme catalysis process.

Introduction
Atomic force microscope (AFM) is a type of Scanning probe microscopy (SPM) with a very high resolution. It could demonstrate the surface properties of sample on the scale of nanometers. Almost all types of surface, including polymers, ceramics, glasses, even biological samples could be studied by AFM. It could be used for samples in ambient air, in liquid, even for vacuum environments, without little or no sample preparation.

Micro-cantilever of AFM can also be used as a very powerful tool as sensors. By measuring the bending or resonance frequency changes due to the mass loading change on micro-cantilever, it could be used as a micro-balance. Also the bending induced by the morphology change of loading stuff on micro-cantilever could be used to study the physical or chemical change of reactant in a reaction.

To be used as sensors, micro-cantilever has several advantages. It has very high sensitivity because the size of it is extremely small. It is inexpensive compared with other macroscopic analytical tools and can be operated in most labs. They are easily to be made because most of them are made of silicon/silicon nitride or polymer materials. Also it is easy to use micro-cantilevers to from sensor arrays. With all these great abilities, this technology has great potential to be the next generation of highly sensitive sensors.

AFM
AFM was invented by Binning, Quate and Gerber in 1986. The most important part of AFM is micro-cantilever, which located on the end of AFM probe. It has a very tiny tip on the top of it and the spring constant of it is extremely low. It is so flexible that even the van der Waals forces between the cantilever tip and the atoms of the sample surface will bend the micro-cantilever. The forces experienced by cantilever have two different regions, like Fig. 1: 1) repulsion region: if the tip is close to the sample surface, the force between the tip and sample will be repulsion force; 2) attractive region: if the distance between the tip and sample surface keeps increasing, the force between them will be attractive force; 3) if the distance is even larger, they will be separated and cantilever will experience no force.(1) Fig .1 Force-distance relation of AFM micro-cantilever

This force experienced by cantilever will make the cantilever bend according to the hooke’s law. And the deflection of cantilever can be tested by the laser which reflects at the cantilever to the photodiode. Combination of this deflection signal and the position of the tip on the sample surface will provide the information of the surface topography. The working scheme is shown in Fig.2. A 3D surface profile will be given by AFM. With this powerful tool of surface imaging, we could investigate the surface change within a chemical or physical process.

Fig. 2. Working scheme of AFM

1) Contact Mode:
As the first operation mode of AFM, contact mode is widely used. There are two different types of contact mode, and for both of them, the cantilevers are in hard contact with sample. The overall force between the tip and sample will be repulsive. One has constant height of sample scanner; another has constant deflection of micro-cantilever. For the first one, the height of sample platform is constant, so when the cantilever scanning across the sample, it will deflected according to the sample corrugation. For the second one, the deflection of cantilever is constant, so the piezoelectric material in the scanner will change the height of sample platform when the scanning is in progress. Combine the deflection signal (for first one) or the height change of piezoelectric scanner (for the second one) with the x-y position information will give us the topography information of sample.

Because the tip is in hard contact with the sample, so the stiffness of cantilever should be less than the force holding the atoms of sample together, which is on the order of 1-10 nN/nm. Most cantilevers for contact mode have spring constant less than 1 N/m.

2) Non-contact mode:
In non-contact mode, the tip of the cantilever will not contact the sample surface. A stiff cantilever will oscillate at a frequency a little above its resonance frequency. Amplitude of this oscillation is about 5-10 nm. So, cantilever is still working in the attractive regime. By changing the distance between the piezoelectric scanner and the tip, we could keep the resonant frequency or oscillation amplitude constant. So, the position change of scanner along with the position information on sample will let us know what the sample surface looks like.

The advantage of non-contact mode is obvious. Since the tip doesn’t touch the sample, there is no tip or sample degradation effect that is sometimes observed after taking numerous scans with the contact mode AFM.

Usually, the non-contact mode will give the same image like the contact mode. But in the case where there are monolayers of absorbed fluid on the sample surface, they will be different. It is because, for the contact mode AFM, cantilever could penetrate the liquid layer to image the sample surface. But for the non-contact mode AFM, the cantilever will only oscillate above the fluid layer to image the liquid surface.

3) Tapping Mode:
In ambient environment, most samples will have a liquid layer on the surface. If the tip is too close to the surface, it will be stuck to the surface. That’s the reason why tapping mode AFM was developed.

In tapping mode, the cantilever is oscillating up and down at its resonance frequency above the sample surface. The amplitude of this oscillation is about 100 nm. When the tip is close to the sample surface, the amplitude will decrease. To keep the amplitude constant, the piezoelectric material in the scanner will lower the sample stage. The scanner will keep adjusting the height of the stage when the cantilever is scanned over the sample, which will tell us the surface information of the sample.

Micro-cantilever sensors
Micro-cantilever, not only be used as AFM tips, could also be developed to be physical, chemical or biological sensor by detecting the changes in cantilever bending or vibrational frequency. Different shapes are shown in Fig. 3.(2)

Fig. 3. Different types of microcantilever.

When a specific mass of analyte is adsorbed on cantilever or some kind of force is applied on to the cantilever, the weight of cantilever may increase which makes the cantilever oscillates at a lower frequency and the cantilever may deflect. Viscosity, density, and flow rate, etc can be measured by detecting changes in the vibrational frequency. Also, by measuring the deflection of cantilever, people could detect the molecular adsorption if it is only on one side of the cantilever. For biochips, scientists usually coat a layer of receptors on just one side of cantilever. Upon the binding of the analyte (e.g. biological molecules, such as proteins or antigens) with the receptor, the cantilever will deflect. This deflection (usually in nanometers) could be test by optical techniques and will be proportional to the analyte concentration.(3) Micro-cantilever sensors have enormous potential for detection of various analytes in gas, liquid and vacuum media. They have great potentials because of their high specificity, high sensitivity, simplicity, low cost, low analyte requirement, non-hazardous procedure with fewer setps, quick response, and low power requirement.(4) Roberto Raiteri and co-workers use micro-cantilevers to detect the concentration of herbicides in the liquid environment.(5) Micro-cantilever sensors have been employed to detect a concentration of 10^-9 M CrO4^2- in a flow cell.(6) Microcantilever based sensor can measure changes in temperature as small as 10^-5 K and can be used for photo thermal measurement.(7) When the medium viscoelasticity changes, the resonance frequency of cantilever will change.(8) Oden and co-workers have developed a remote infrared (IR) radiation detection sensor.(9) A matchbox-size device which could be used to detect explosives in airport luggage and landmines has been developed by Thundat and his group based on micro-cantilever technique.(10) Micro-cantilever based sensor could even be used to test cancers.(11) After receptors are binded to one side of cantilevers, biochips could be used to detect biological molecules.(12)

Enzyme catalysis
Many and many new enzymes are being discovered and developed in recent years with the development of biology engineering. Enzymes are widely used in several industrial and medical fields, including wound healing, dental, detergency, food processing, biotechnical analysis systems, and for specific organic synthesis.(13-19) Cellulose is the skeleton structure of almost all plants, which make it an unlimited source as nature polymers. Cellulose hydrolysis is of great importance in the conversion of plant biomass to fuels. The scientific research on topic of cellulose hydrolysis has become a hotspot in chemistry, biology and some other fields. Converting cellulose from biomass into biofuels such as cellulosic ethanol is now attracting people both from science and industrial field.

Cellulase, which is a kind of enzyme which could break down the cellulose of plant cell walls into simple sugars, is interesting for paper and fiber applications. Sugars can then be fermented into ethanol and many other products. Optimize cellulase enzyme’s application to produce ethanol from cellulose will increase ethanol production in an environmentally friendly way. The quantity of scientific information on components of cellulose-hydrolyzing enzyme system has expanded dramatically in recent years. Converting cellulose from biomass into biofuels such as cellulosic ethanol is now attracting people both from science and industrial field.

To control enzymes in these and other applications requires knowledge of the different processes occurring when enzyme is in contact with a substrate surface. Enzymatic hydrolysis of cellulose is a very complex system. Before degradation, it is proposed that the enzymatic hydrolysis of crystalline cellulose is initiated with the adsorption of cellulase to the surface of the cellulose. Both the degradation and the adsorption may affect the enzymatic degradation. There are still many factors which will influence the enzymatic degradation of interfacial substrates. And there are many interested research published during recent years(20-25) [3,9,10,13,15–20-25]. But this still face great challenges.

Lee Ida and co-workers treat cotton fibers with two different cellulase enzymes – CBH I and EG II. They find the degrees of surface disruption caused by cellulase treatment are different for these two enzymes. This may because of the different ability of cellulase binding to insoluble, crystalline cellulose.(22) Levy IIan and co-workers study the binding domain of cellulase enzyme and they found that purified protein improved the treated paper’s mechanical properties and will transform filter paper into a more water-repellent paper.(20)

Cells biology
When treated by chemicals, physical properties of cells will change. But it is impossible to monitor this change using regular characterization methods. AFM can be used to study the mechanical properties of cells. This measurement requires the AFM working in the force spectroscopy mode. In this mode, scanning is disabled, and the cantilever approaches the sample at a given spatial position, contact it, and when it reaches the setting point of reflection, it is then withdrawn backward.

We can get elastic properties from the force curve collected from AFM. This curve represents the dependence between the loading force on cantilever (calculated from deflection) and the relative sample position (Fig. 4). If the sample is stiff (such as glass slide), the deflection reflects the sample’s position. The force curve will be a straight line and usually used as a reference line. For soft samples like cells, cantilever will indent into the cells and the force curve has non-linear character. Subtract reference line from this curve will reveal the relation between the loading force and the indentation.

Fig. 4. Idea of the determination of cell elastic properties using AFM.

After got this force curve, Young’s modulus can be calculated. There are two formula describe the relationship between the load force F and the indentation depth δ: For a conical tip: For a paraboloidal tip: Where α is the open angle, R is the radius of curvature of the AFM tip, E’ is the reduced Young’s modulus. The reduced Young’s modulus is given by: For living cells which Esample <<Etip: Where μ (usually has a range of 0~0.5) is the poisson ratio which represents the compressibility of sample. So each indentation could get one force curve which could then be used to calculate for a Young’s Modulus. After tens of modulus calculated from tens of points of one cell, we could get a modulus distribution of a cell. But it is important to be aware that this modulus could only represent modulus of a certain layer which depends on the indentation depth. So, actually, it is hard to use force curve to get absolute modulus of cells, since cell is a very complex system. But in most cases, only the change of modulus would be enough for biological system.

Conclusion
AFM is a very powerful tool when characterize samples. By AFM, people could get 3-D information of the sample surface in most of environment with less sample preparation. AFM could even be used to monitor the changes of mechanical properties of cells and soft materials. Micro-cantilever based sensors are of great interest for scientists because their high sensitivity, quick response, low analyte consume, selectively, and so on. Enzyme catalysis and cell system are very complex and could not be studied by traditional characterization methods. Enzyme catalysis starts from adsorption of enzyme on sample surface, followed by the enzyme hydrolysis. It is also very hard to study the mechanical properties of cells in a biology process. AFM imaging technique and micro-cantilever make it possible to study these. These nano approaches will be promising tools in the future.