Silicon AFM Cantilever For Imaging Of Biomolecules Biology Essay

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Short and sharp cantilevers have both a high resonant frequency and a low spring constant, thus are appropriate for atomic force microscope (AFM) imaging. Silicon on insulator wafers are used to fabricate a small silicon (Si) cantilever integrating with an ultra sharp Si tip. The resolution of images taken with this cantilever is higher than those taken with the commercial cantilever.

Since the invention of the atomic force microscopy (AFM) in 1986, it has become a powerful tool for high resolution imaging of surfaces. The key component of the AFM is a flexible cantilever with a sharp tip is scanned across the surface and the cantilever deflects according to the topography of the sample. The cantilevers have received remarkable attention in the microelectromechanical systems (MEMS) and nanotechnology community due to their simple structure and fabrication process flow, along with their displayed versatility in a wide range of applications. The first cantilevers utilised a V-shaped amorphous SiO2 film etched on a Si wafer. Soon after, cantilevers produced from Si3N4 films on Si wafers were used widely because Si3N4 is more durable than SiO2. Nowadays, the majority of cantilevers are made from silicon with integrated tips (Bhushan, and Fuchs, 2006).

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There are many advantages to using silicon microfabrication technology, such as (1) miniaturisation of devices, (2) the ability to integrate many functional devices on the same platform which achieve the ultimate goal of a "lab on a chip", (3) reduction in production cost due to batch fabrication, (4) accurate control of dimensions, and (5) the ability to fabricate an array of devices with very close physical parameter values (Gupta et al. 2003 and Gupta, Akin, and Bashir, 2004) .

The design of the cantilever needs the suitable choice of spring constant (k), resonant frequency (fo) and the dimensions to satisfy certain measurement requirement. In terms of biological samples, imaging biomolecules requires cantilevers with a low spring constant, a high resonance frequency and a sharp tip. Using short and thin cantilevers increase the resonant frequency and decrease the spring constant, respectively (Fig. 1) (Hosaka et al. 1999). All these features of cantilevers have encouraged the improvement of processes for the fabrication of cantilevers and instrumentation for utilising them (Siffert and Krimmel, 2004 and Grow et al. 2002).

The purpose of this paper is to present in details a simple process to fabricate a silicon cantilever with integrated silicon tip. The cantilever and tip have been made using silicon on insulator (SOI) wafers and many micromachining techniques including photolithography, wet etching and thermal oxidation.

Fig. 1. A contour map of spring constant and resonant frequency due to length and thickness of AFM cantilever (Hosaka et al. 1999).

1. Dimension and structure :

As mentioned earlier, the cantilevers with a low spring constant and a high resonant frequency are suitable for imaging biological samples. With regard to commercial silicon AFM cantilevers, most of them do not achieve these requirements, which are necessary for imaging biological samples. For example, two traditional silicon cantilevers are: the short cantilevers with a size of 90m 35 m 1 m with spring constant of ~ 1.7 N/m and resonant frequency of ~ 150 kHz and the long cantilevers with a size of 300 m 35 m 1 m with spring constant of of ~ 0.03 N/m and resonant frequency of ~ 10 kHz. For the first cantilevers, the high spring constant possibly causes deformation of biological samples and enhances the thermal noise. The second type is usually utilised for imaging in contact mode. However, for imaging biological samples, tapping mode that reduces the lateral force often causing deformation of the biological samples is preferred over contact mode. To overcome these disadvantages, silicon cantilever tips should be designed to have dimensions suitable for imaging biological samples as well as to be compatible with current commercial AFM instruments. This is possible by fabricating cantilever with length about 70 m, width 35 m and thinner than 1 m.

A silicon AFM cantilever with a high resonant frequency is useful to increase the imaging rate, which is appropriate for monitoring the conformational changes, reactions and interactions of biomolecules. In addition, the AFM cantilever with a low spring constant is very crucial to reduce the deformation of the soft sample, such as biomolecules. Furthermore, it considerably enhances the sensitivity of force measurement which has been utilised for investigating the inter- and intra- molecular interactions between macromolecules (Viani et al. 2000). Using very thin cantilever is necessary because the spring constant of the cantilever is proportional to the cube of its thickness (Grow et al. 2002).

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There are many reports used the tip and cantilever that are made of crystalline silicon and low-stress silicon nitride, respectively (Floch, Wrighton and Schmidt, 1997 and Grow et al. 2002). This choice of materials is useful to sharpen the tips by oxidation sharpening without affecting the cantilever. In addition, the thickness of silicon nitride cantilevers can be managed by chemical vapour deposition (CVD). Even though silicon cantilever tips are more complicated to fabricate than silicon nitride cantilever tips, there are many benefits for the utilise of silicon cantilevers and tips: (1) the Si tip that is integrated with Si cantilever is easier to be fabricated in large scale than carbon tip deposited by electron beam deposition, (2) an sharp Si tip can be fabricated easily through thermal oxidation, (3) for imaging biological samples, Si tips can be fabricated with monolayers of protein resistant for imaging biological samples, and (4) cantilevers are fabricated from the single crystal Si have a higher Q factor than the amorphous silicon nitride cantilevers developed by CVD (Yam et al. 2003 and Yam et al. 2004).

2. Fabrication:

Silicon on insulator (SOI) wafers with <100> orientation are utilised as the starting material and the tip and cantilever can be formed by the device layer of the SOI wafers. There are three main methods are used to produce SOI wafers: The Si fusion bonded wafer (SFB), zone melt recrystallised (ZMR) polysilicon and SIMOX (Separated by Implanted Oxygen). With Si fusion bonded wafer, the process begins with an oxide layer formed on a standard silicon wafer. This wafer is bonded to another silicon wafer to form a sandwich. This sandwich is annealed at high temperature in a nitrogen atmosphere that forms a strong bond between the two silicon wafers. In terms of the second technique (ZMR), polysilicon is crystallised with an electron beam or a laser, then is deposited on an oxidised silicon wafer. The third technique is SIMOX, which is recommended to use in this case study. In this process, oxygen ions implant a standard silicon wafer. After that annealed at high temperature. The oxygen and silicon combine together to produce a silicon oxide layer. The thickness and the depth of oxide layer can be controlled by modifying the anneal temperature and the dose and energy of the implant. A chemical vapour deposition (CVD) may be utilised to deposit additional silicon on the top surface (Fig. 2) (Madou, 2001).

Fig. 2. Silicon on insulator are made by SIMOX (Dunn, 1993 cited in Madou, 2001).

The process of fabrication involves four important steps: (1) producing the handle (Fig. 3a, b), (2) defining the cantilever (Fig. 3c, d), (3) producing the cantilever and the tip together, and (4) Oxidation sharpening.

The thickness uniformity is a crucial feature to fabricate thin cantilevers. The thickness of cantilever can be easily controlled when it is fabricated from SOI wafers. This is because the device layer of SOI wafers is thinner than the single crystal Si wafers and has a lower variation of absolute thickness. Because the height of the tip depends on the thickness of the device layer, the height of the tip can be managed by modifying the thickness of the device layer. In this fabrication process, SOI wafers consists of 5m device layer doped with boron, 2 m buried oxide layer and 375 m handle layer. The buried oxide layer of SOI wafers is used as an etch stop (Yu et al. 2006).

Fig. 3. Schematic of the fabrication process from a SOI wafer. The layers with yellow colour are silicon oxide layers and the blue colour layers are silicon (Yu et al. 2006).

2.1 Producing the handle:

Oxide layers are formed by thermal oxidation at the top and bottom sides of the wafer. When the cleaving lines (C-C) and trench (T-T) are formed, these thermal oxide layers are necessary to protect the device layer. By utilising conventional photolithographic technique, the wafer is coated with the photoresist at the bottom side by spin coating and the cleaving lines and trenches can be transferred (Yu et al. 2006). Conventional photolithography technique consists of several steps. In general, most of microfabrication processes begin with surface preparation step. The surface of the wafers are chemically cleaned to remove any contamination. Hexamethyl disilasane vapour (HMDS) is used to promote adhesion of the photoresist to the wafer. The silicon atom in HMDS will bind to oxygen in the hydroxyl group at the wafer surface, and the amine group reacts with hydrogen, releasing ammonia (Fig. 4). The silane layer makes the surface of the wafer hydrophobic, which inhibits water readsorption. As mentioned before, the spin coating is applied to cover the wafer with a positive photoresist. This process produces a uniform thin layer, usually standard resist thicknesses are about

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1m. A bake step is applied at 90 ËšC for 60 s on a hotplate or 30 min in an oven to drive out excess photoresist solvent. For positive resist, the resist is exposed to UV light through a contact mask and the photochemical reaction during the exposure changes the chemical structure of the resist so that it becomes more soluble in developing solution. In development, the exposed parts of photoresist turn to carboxylic acid that is removed by developer, such as TMAH ( tetramethyl ammonium hydroxide) (Franssila, 2010). The photoresist is baked at 120 ËšC for 30- 60 min (Floch, Wrighton and Schmidt, 1997). Hard bake makes the resist tougher, which is useful in wet etching because it develops resist adhesion. Buffered hydrofluoric acid BHF ( it is a mixture of ammonium fluoride NH4F and hydrofluoric acid HF) wet etching is utilised to open the window at the site of cleaving lines and trench on the oxide layer. Buffered HF is usually utilised to etch SiO2 on Si substrate (Madou, 2001). Anisotropic wet etching is used to etch the Si handle layer. In this process, TMAH is preferred to be used rather than KOH to etch the silicon handle layer. The reason is that the oxide layer is etched under KOH, whereas the oxide layer is stable under TMAH. The thickness of the oxide layers is sufficient to survive 12 hours etching at 70 ËšC that is important to etch through the handle layer to produce the trench stopped at the buried oxide layer. After anisotropic wet etching, the cleaving lines has a V- groove shape, as shown in Fig. 3 (b). This design provides the robustness to the wafers in the following steps. With regard to the trench region, there is only a thin membrane is formed. To prevent the damage of the membrane in the following steps, the wafer is fixed on a conventional silicon wafer. After photoresist has played its role as a protective layer, it is removed by immersing the silicon wafers in a piranha solution (H2SO4 : H2O2 :: 3 : 1) (Floch, Wrighton and Schmidt, 1997).

2.2 Defining the cantilever:

In order to define the site of cantilever, a 20 m wide line across the trench (T-T) is patterned on the top layer and etched utilising photolithography for double sided alignment (Yu et al. 2006). According to Franssila (2010), the double sided alignment mechanism depends on imaging process. By storing an image of the mask alignment marks, the silicon wafer is placed between the alignment microscope and the mask. In this case, the alignment marks on the silicon wafer should be aligned with the mask alignment marks, which are stored previously. After this step, the site of the cantilever is easy to determine because the trench edges (T`- T`) can be observed from the device (top) layer side. To etch the cantilever, a 40% solution of KOH in a 65 ˚C is utilised. The addition of small quantities of an antimony to KOH solution reduces the resulting surface roughness significantly. The surface becomes homogeneous and on a microscopical scale with a rms roughness of below 2 nm. No effect on the regular etch rates is noticed (Mihalcea et al. 2001). In this step, there is about 0.5m from the device layer is etched to obtain the cantilever mesa (M- M) with thickness 0.5m. The etched area (E- E) and the mesa are etched together. This step is necessary to maintain the membrane strong before pattering the tip disk.

3.3 Producing the cantilever and tip together:

At the end of defined cantilever, a tip disk is patterned (10 m). In this step, the previous recipe of the etchant is utilised. Using the same etching rate to etch the (M- M) and (E- E) together. When the (E- E) area is completely etched, the etching process is stopped and the cantilever and tip are formed. Controlling the thickness variation of cantilever is not difficult because the etching rate of the etching is about 250 nm/min. After this step, there is still an oxide cap left above the tip (Fig. 4 (a)). The resulting tip has pyramid shape and the etched plane is (411) (Resnik et al, 2003). This shape is attributed to the high concentration of the KOH solution (Zubel, 2000).

3.4 Oxidation sharpening

Currently, there are many techniques have been used to form a sharp silicon tip.

A sharp tip can be fabricated by anisotropic wet etching. However, there is a small overetch after forming an ideal tip which may decrease the sharpness of the tip significantly. Sharpening of the tip can be further developed by thermal oxidation method (Fig. 3 (b) and (c)). Oxidation leads to sharper tip and the fabrication process is easy to be controlled by oxidation time rather than etch time (Franssila, 2010).

In addition, the thermal oxidation is useful to control the thickness of the cantilever. SEM is used to measure the thickness of the cantilever before thermal oxidation. Depending on the date from SEM, the consumption of Si can be calculated. After that, the appropriate time of oxidation can be determinated. Controlling the thickness is not difficult because the oxidation process requires hours (Yu et al. 2006). The rate of oxide growth is not regular because it is relatively low in areas of high curvature, such as the tip apex and it is large in areas of low curvature, such as the tip slopes. Consequently, the apex of the tip will be sharper than before (Liu and Gamble, 1998).

Fig. 4. SEM images of the cantilever and the tip. (a) There is still a cap above the tip. (b) and (c) The tip and cantilever after oxidation for sharpening the tip and controlling the thickness of the cantilever (Yu et al. 2006).

4. AFM tip testing:

To assess the quality of the fabricated tip, it is used to image oligo ethylene glycol (OEG) terminated monolayers on silicon surfaces. Preparing this film is based on hydrosilylation reaction of α-hepta-(ethylene glycol) methyl w- undecenyl ether (EG7) with hydrogen terminated silicon surfaces to form Si-C bonds (Yam et al. 2003). As shown in Fig. 6, the resolution of the image obtained with a sharp and short Si tip fabricated by the previous method is higher than images obtained with commercial Si tips.

Fig. 6. An OEG terminated monolayer is imaged by: (a) a commercial Si tip in contact mode AFM, (b) tapping mode AFM, and (c) the tip fabricated by the method explained above (Yu et al. 2006).

Modification of Si AFM tip allows precise measurement of the molecular interactions with high resolution imaging. The conventional method involves the growth of a self assembled monolayer (SAM) of thiolates on tip coated with a thin layer of gold (20- 100 nm). However, this method

Conclusion

A sample process to fabricate silicon AFM cantilever with integrated silicon tip has presented. This cantilever posses a low spring constant and a high resonant frequency which are crucial for high resolution AFM of biological samples. In this process, several micromachining techniques have been used to achieve these requirements.