**3.1 Microcontact printing (μCP)**

μCP is a direct method for pattern transfer, generating a non-structured, chemically modified surface. The process of μCP is shown in figure 2. Photolithography was used for the fabrication of silicon-based masters in preparing PDMS stamps. Multi-layer molds were made of thick photoresist like SU-8 on silicon or glass wafers by standard lithography techniques. It was subsequently placed at least 30min in an oven at 160°C to make the photoresist adhere to the substrate closely. Release agent DC20 or OTS were always spincoated and drying on the master before pouring PDMS. Liquid PDMS (Sylgard-184 from Dow Corning) was poured onto the mold and clamped by the foil, so that the shape of the mold microstructure was transferred to PDMS membrane. It was subsequently placed at least 2 h in an oven at 80°C. The molded PDMS slab was then peeled off and placed onto a glass slide for handling. After curing, PDMS stamps are soaked in a protein "ink", such as PEI, PLL, or LN. 20 minutes later, the "ink" was blew off by using nitrogen gas. Then the raised regions were brought into conformal contact with a substrate in order to print the ink onto the substrate surface. The material of interest was transferred from the PDMS stamp onto the substrate surface. The microscopy of the PDMS stamps were shown in figure 3.

biocompatibility, chemical stability, optical transparency, air permeability, elasticity. Moreover, the polymer precursors can be aggregated into a mold by UV radiation. PDMS

To cure the PDMS prepolymer in general, a mixture of silicon elastomer and a curing agent (10:1, Sylgard 184 silicone elastomer kit, Dow Corning Corp.) is poured onto the master and placed at 70-80°C for 1 h. The character of the PDMS is closely related to the mixture ratio, curing temperature, and vacuum. Silicon, quartz or glass, and some photoresist are the most common materials to fabricate the masters by standard lithography, transferring the

**3. Soft lithography fabrication methods and it's application in patterning** 

Various soft lithographic technologies have been applied to fabricate high-quality microstructures and nanostructures including micro contact printing (μCP), replica molding (REM), microtransfer molding (μTM), micromolding in capillaries (MIMIC), and solventassisted micromolding (SAMIM). Here, three soft lithographic methods are introduced to fabricate micropatterns onto a surface or MEA: μCP, microfluidic patterning technique and microstencil. The former two can be achieved using the same PDMS stamps and molds. A novel technology was applied to get the high depth-to-width ratio silicon-based mold to fabricate the topographic PDMS microstencil with microfluidic channel (Y.Nam, et al, 2006). Finally, microchannels on MEA with polyimide (PI) guiding the cell growing were also

μCP is a direct method for pattern transfer, generating a non-structured, chemically modified surface. The process of μCP is shown in figure 2. Photolithography was used for the fabrication of silicon-based masters in preparing PDMS stamps. Multi-layer molds were made of thick photoresist like SU-8 on silicon or glass wafers by standard lithography techniques. It was subsequently placed at least 30min in an oven at 160°C to make the photoresist adhere to the substrate closely. Release agent DC20 or OTS were always spincoated and drying on the master before pouring PDMS. Liquid PDMS (Sylgard-184 from Dow Corning) was poured onto the mold and clamped by the foil, so that the shape of the mold microstructure was transferred to PDMS membrane. It was subsequently placed at least 2 h in an oven at 80°C. The molded PDMS slab was then peeled off and placed onto a glass slide for handling. After curing, PDMS stamps are soaked in a protein "ink", such as PEI, PLL, or LN. 20 minutes later, the "ink" was blew off by using nitrogen gas. Then the raised regions were brought into conformal contact with a substrate in order to print the ink onto the substrate surface. The material of interest was transferred from the PDMS stamp onto the substrate surface. The microscopy of the PDMS stamps were shown in figure 3.

polymerization is shown in Figure 1.

Fig. 1. PDMS polymerization

patterns to the PDMS stamp.

**3.1 Microcontact printing (μCP)** 

introduced.

Fig. 2. Schematics of the processes of μCP

Application of Soft Lithography and Micro-Fabrication on Neurobiology 21

MEA was a cell-based biosensor for extracellular electrophysiological investigations of neuronal networks. PDMS microstencil was designed to pattern adhesion molecules at the surface of MEA guiding cultured cells grow along the patterns. PDMS microstencil mold was fabricated by a complex photomask aligning method, shown in figure 4.

HF etching

Spin-coat photoresist

DRIE

DRIE

Spin-coat photoresist

SiO2

thermal oxidation

Si

Si

Si

Si

Si

Si

Fig. 4. Schematics of the fabrication of microstencil mold

d

e

f

a

b

c

work.

525μm silicon was thermal oxidated with 4000 Ǻ SiO2 (see Fig. 4a). The substrate was rst coated with a thin photoresist (AZ AZ9912) for 30 s at 3000 rpm (see Fig. 4b). 8 × 8 SiO2 arrays were fabricated by wet etching as the RIE mask which was the same as the MEA structure (see Fig. 4c). Photoresist was spin-coated to the silicon with SiO2 mask again (see Fig. 4d) and selectively exposed to UV under a chromium photomask. The silicon was etched 30μm by deep reactive ion etching (DRIE) with photoresist in order to construct the microchannel , (see Fig. 5a). Then photoresist was removed by ultrasonication in acetone. The silicon was selectively etched 70μm by DRIE with SiO2 resist, forming the topographic PDMS microstencil mold (see Fig. 5b). The high depth-to-width ratio silicon-based mold was designed to penetrate through the PDMS membrane on the MEA to exposure the electrodes and form the microchannel between the electrodes so that the MEA could also

Fig. 3. Microscopy of the PDMS stamps

The bare areas of substrate surface that the PDMS stamp has not touched can be exposed to another coating material. μCP provides the patterning of self-assembled monolayers(SAMs) of alkanethiols on gold, and the resulting control over the adsorption of adhesive proteins facilitates the patterning of cells on substrates.

μCP enables easy stamp replication, fast printing using parallelization, and low-cost batch production. A conformal contact between the stamp and the surface of the substrate is the key to its success. The polymer stamps also minimize the problems of sample carry-over and cross contamination. Printing has the advantage of simplicity and convenience: Once the stamp is available, multiple copies of the pattern can be produced using straightforward experimental techniques. Printing is an additive process; the waste of material is minimized. Printing also has the potential to be used for patterning large areas.

However, μCP has some limitations that are mainly caused by the use of a soft polymer stamp. The swelling of a stamp during inking often results in an increase in the pattern size by diffusion of the excessive printed molecules on the substrate.

## **3.2 Microfluidic patterning using microchannels**

The difference between μCP and microfluidic patterning is that PDMS stamps are soaked in the "ink" in the former usage, but the stamps contact the substrate forming microchannels delivering the materials for cell adhesion or cell suspension to the desired area in the latter usage because of the elastic nature and hydrophobicity of PDMS. The substrate tilted 45 degree, drop of liquids were injected to the PDMS microchannels by the pipette. Then the substrate was put on the test tube rack vertically for 25 to 30 min. Patterns were formed of after the liquid dried.

While this method has been used primarily for surface attachment of cells, it may be possible to adapt this method to three-dimensional tissue constructs. In many cases three dimensional tissue constructs promote cellular differentiation and more authentic cellular morphology and metabolism.

#### **3.3 Microstencil on MEA**

The former two methods enable patterning adhesion molecules and guiding cultured cells grow physically. But the cells' communication and interactions in co-cultures are difficult to be detected, which is important to research the function of the cells network.

The bare areas of substrate surface that the PDMS stamp has not touched can be exposed to another coating material. μCP provides the patterning of self-assembled monolayers(SAMs) of alkanethiols on gold, and the resulting control over the adsorption of adhesive proteins

μCP enables easy stamp replication, fast printing using parallelization, and low-cost batch production. A conformal contact between the stamp and the surface of the substrate is the key to its success. The polymer stamps also minimize the problems of sample carry-over and cross contamination. Printing has the advantage of simplicity and convenience: Once the stamp is available, multiple copies of the pattern can be produced using straightforward experimental techniques. Printing is an additive process; the waste of material is minimized.

However, μCP has some limitations that are mainly caused by the use of a soft polymer stamp. The swelling of a stamp during inking often results in an increase in the pattern size

The difference between μCP and microfluidic patterning is that PDMS stamps are soaked in the "ink" in the former usage, but the stamps contact the substrate forming microchannels delivering the materials for cell adhesion or cell suspension to the desired area in the latter usage because of the elastic nature and hydrophobicity of PDMS. The substrate tilted 45 degree, drop of liquids were injected to the PDMS microchannels by the pipette. Then the substrate was put on the test tube rack vertically for 25 to 30 min. Patterns were formed of

While this method has been used primarily for surface attachment of cells, it may be possible to adapt this method to three-dimensional tissue constructs. In many cases three dimensional tissue constructs promote cellular differentiation and more authentic cellular

The former two methods enable patterning adhesion molecules and guiding cultured cells grow physically. But the cells' communication and interactions in co-cultures are difficult to be detected, which is important to research the function of the cells network.

Fig. 3. Microscopy of the PDMS stamps

facilitates the patterning of cells on substrates.

Printing also has the potential to be used for patterning large areas.

by diffusion of the excessive printed molecules on the substrate.

**3.2 Microfluidic patterning using microchannels** 

after the liquid dried.

morphology and metabolism.

**3.3 Microstencil on MEA** 

MEA was a cell-based biosensor for extracellular electrophysiological investigations of neuronal networks. PDMS microstencil was designed to pattern adhesion molecules at the surface of MEA guiding cultured cells grow along the patterns. PDMS microstencil mold was fabricated by a complex photomask aligning method, shown in figure 4.

Fig. 4. Schematics of the fabrication of microstencil mold

525μm silicon was thermal oxidated with 4000 Ǻ SiO2 (see Fig. 4a). The substrate was rst coated with a thin photoresist (AZ AZ9912) for 30 s at 3000 rpm (see Fig. 4b). 8 × 8 SiO2 arrays were fabricated by wet etching as the RIE mask which was the same as the MEA structure (see Fig. 4c). Photoresist was spin-coated to the silicon with SiO2 mask again (see Fig. 4d) and selectively exposed to UV under a chromium photomask. The silicon was etched 30μm by deep reactive ion etching (DRIE) with photoresist in order to construct the microchannel , (see Fig. 5a). Then photoresist was removed by ultrasonication in acetone. The silicon was selectively etched 70μm by DRIE with SiO2 resist, forming the topographic PDMS microstencil mold (see Fig. 5b). The high depth-to-width ratio silicon-based mold was designed to penetrate through the PDMS membrane on the MEA to exposure the electrodes and form the microchannel between the electrodes so that the MEA could also work.

Application of Soft Lithography and Micro-Fabrication on Neurobiology 23

However, PDMS microstencil is difficult to practise because it is hard to align with MEA

Planar MEA are developed to study electrogenic tissues such as dissociated neuronal cultures (Hiroaki Oka,et al. 1999). They have been widely used with dissociated cultures for a variety of neuroscience investigation including learning and memory and cell-based biosensors for the detection of neurotoxins (Conrad D. James, et al. 2004). But the neurons grow disorderly and cannot form a network so that the function is not the same as the cells in vitro. Combining the patterning technology and MEA forming neuronal networks is the

μCP, microfluidic patterning technique and microstencil are difficult to operate because the space is too small and the PDMS stamp or stencil can hardly align with MEA. The best and easiest way to forming neuronal networks is to fabricate the microchannels on MEA with

MEA were fabricated using a conventional semiconductor process (Guangxin Xiang, et al. 2007). After cleaning the polished quartz glass wafer, the conductive layer of Au/Ti lm (Au 3000 Ǻ and Ti 700 Ǻ) was sputtered. 8 × 8 electrode arrays were left with the photomask protection by standard photolithography. Then, a combination of SiO2/Si3N4/SiO2 (3000 Ǻ /4000 Ǻ /3000 Ǻ) passivation layers was deposited onto the substrate using plasma enhanced chemical vapor deposition (PECVD), and the insulating layers on the electrodes and the bonding-pads were removed by inductively coupled plasma (ICP) (see Fig. 7a). Finally, Negative photosensitive polyimide (AP2210B, Fujifilm Electronic Materials Inc) was spin-coated to form microchannels having a thickness of 3~4μm and photo-etched by the

standard procedure to expose the microelectrodes and the terminals (see Fig. 7b).

(a) (b)

GABAergic neurons in the striatum and PC12 cells were cultured on MEA with PI microchannels which were coated with poly-l-lysine (PLL) to promote cell adhesion, (see Fig. 8a, 8b). PI microchannels could be seen between the electrodes and the neural cell can grow along the microchannels. However the nerve cell synapse could not formed along the microchannels. Because the depth of microchannels could not match the neurons and the

Fig. 7. Microscopy of the MEA with PI microchannels

and the silicon mold is easy to fracture when lifting off.

**3.4 MEA with microchannels for patterning** 

efficient method to research the neurobiology.

polyimide (PI) guiding the cell growing.

Fig. 5. SEM images of PDMS microstencil mold

The mixture of PDMS prepolymer and curing agent was spin-coated on the mold for 40 s at 4000 rpm. The coated mold was cured for 2 hours at 110◦C in a convection oven. The fullycured PDMS-coated mold was soaked in an acetone ultra-sonication bath until the PDMS layer released from the mold. The detached microstencil was rinsed with IPA and DI water. The upside and downside of PDMS microstencil with microholes and microchannels SEM images was shown in figure 6.

Fig. 6. SEM images of PDMS microstencil

However, PDMS microstencil is difficult to practise because it is hard to align with MEA and the silicon mold is easy to fracture when lifting off.
