Patterning of Crystalline SLayer Proteins

3.3.1. Excimer Laser Patterning

Silicon wafers were cleaned with several solvents and O2 plasma treated (see Subheading 3.2.). Recrystallization of isolated S-layer protein on the silicon wafer was carried out as previously described (see Subheading 3.2.).

Prior to irradiation, the recrystallized S-layer was carefully dried in a stream of high-purity nitrogen gas in order to remove excess water not required for maintaining the structural integrity of the protein lattice (see Note 5). Then, the

Table 1

Supports and Their Modifications

Used in Formation of Crystalline S-Layer Protein Layers3

Table 1

Supports and Their Modifications

Used in Formation of Crystalline S-Layer Protein Layers3

Support

Surface and modifications

SbsB

SbpA

Silicon wafers

Si (native oxide layer) RCA cleaned

-

(100 orientation,

p-type)

Si (native oxide layer)

+

+b

Si (native oxide layer) O2 plasma treated

-

+

Si3N4

+

+

Octadecyltrichlorosilane

+

+

(3-Methacryloyloxypropyl)-trimethoxysilane

+

+

Trimethoxysilane

+

+

Decyldimethylsilane

+

+

Hexamethyldisilane

+

+

2-Aminopropyltrimethoxysilane

+

+

3-Mercaptopropyltrimethoxysilane

+

+

Metallic supports

Gold

+

+

Titanium

+

+

Aluminum

n

+

Palladium

+

+

Polymers

Polyester

+

n

Polypropylene

+

+

Polyethylene terephthalate)

n

+

Poly(methacrylic acid methylester)

n

+

Polycarbonate

+

n

Others

Glass

+

+

Cellulose

+

+

Mica

-

+

Highly oriented pyrolytic graphite

-

+

a+, crystallization; -, no crystallization; n, not tested. bVery large crystalline domains.

a+, crystallization; -, no crystallization; n, not tested. bVery large crystalline domains.

lithographic mask was brought into direct contact with the S-layer-coated silicon wafer (Fig. 5). The whole assembly was irradiated by the ArF excimer laser (see Note 6) in a series of one to five pulses with an intensity of about 100 mJ/cm2 per pulse (pulse duration: 8 ns; 1 pulse/s). Finally, the mask was removed and the S-layer-coated silicon wafer was immediately immersed in buffer.

S-layers that have been patterned by ArF excimer laser radiation may also be used as high-resolution etching masks in nano/microlithography. This application requires enhancement of the patterned protein layer by electro-less

Fig. 5. Schematic drawing of patterning of S-layers by exposure to deep ultraviolet (DUV) radiation. (A) A pattern is transferred onto the S-layer by exposure to ArF excimer laser radiation through a microlithographic mask. (B) The S-layer is specifically removed from the silicon surface in the exposed areas. (C) A scanning force microscopic image of a patterned S-layer on a silicon wafer is shown. Bar = 3 |im. (Modified after ref. 5 with permission from the publisher; © 1999, Wiley-VCH.)

Fig. 5. Schematic drawing of patterning of S-layers by exposure to deep ultraviolet (DUV) radiation. (A) A pattern is transferred onto the S-layer by exposure to ArF excimer laser radiation through a microlithographic mask. (B) The S-layer is specifically removed from the silicon surface in the exposed areas. (C) A scanning force microscopic image of a patterned S-layer on a silicon wafer is shown. Bar = 3 |im. (Modified after ref. 5 with permission from the publisher; © 1999, Wiley-VCH.)

metallization prior to subsequent reactive ion etching. Since S-layers are only 5 to 10 nm thick, and thus much smaller than conventional resists (500-1000 nm mean thickness), proximity effects are strongly reduced, yielding a considerable improvement in edge resolution. For the development of bioanalytical sensors, patterned S-layers may also be used as electrode structures for binding biologically active molecules at specified target areas.

3.3.2. Soft Lithography Patterning

A well-known soft lithography technique, micromolding in capillaries (MIMIC) (19,20), can be used for patterning and self-assembly of 2D S-layer protein arrays on silicon supports. For mold formation, 6-|m-high mesa-struc-

Fig. 6. Fluorescence image of FITC-labeled S-layer protein SbpA patterned at a plasma-treated native silicon oxide support using a PDMS mold. Bar = 50 |im.

ture mold masters were fabricated in photoresist on 4-in. silicon wafers using photolithography. PDMS was used to generate the molds from the masters (21,22). Ten parts of PDMS and one part of elastomer were mixed and degassed in an exsiccator. The PDMS solution was put on the master, which was placed in a Petri dish, and again the solution was degassed until no bubbles were observed. The PDMS mold was baked at 50°C for at least 4 h and subsequently removed from the master and cut to a proper size. Microchannels were formed when the recessed grooves in the PDMS mold were brought into conformal contact with the planar support, typically a native oxide-terminated silicon wafer.

The microchannels were subsequently filled from one end with protein solution (0.1 mg/mL of SbpA in buffer C) by capillary action. The silicon supports (solvent cleaned) were O2 plasma treated before application of the mold in order to increase the wettability of the surface and to improve channel filling (see Note 7). After self-assembly and crystallization of the S-layer protein (30 min to 24 h), the PDMS mold was removed under Milli-Q water, leaving the patterned S-layer arrays on the support. The patterning was detected either by AFM (see Subheading 3.2.) or by epifluorescence microscopy.

For microscopic detection of fluorescence, the protein structures were labeled with FITC (see Note 8). The solid-supported S-layer patterns were incubated with the FITC suspension (1 mg of FITC in 100 |L of DMSO, diluted with 2 mL of buffer D) for 1 h at room temperature in the dark. After labeling, the samples were washed with buffer D and, finally, the patterning was investigated by epifluorescence microscopy (Fig. 6).

The MIMIC technique can be utilized for lateral patterning of simple and moderately complex crystalline S-layer arrays ranging in critical dimension from submicrons to hundreds of microns. Furthermore, the native chemical functionality of the S-layer protein is completely retained, as demonstrated by attachment of human IgG antibody and subsequent binding of anti-human IgG antigen on the patterned S-layer substrates (23). This versatile MIMIC patterning technique can also be combined with immobilization techniques (see Subheading 3.4.), such as for controlled binding of nanoparticles with well-defined locations and orientations.

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