Micropatterning of proteins provides a power tool for studying the interactions between proteins, antibodies, and other biomolecules, [1,2] which can find applications in many fields such as biology, biosensor technology, biomedical devices, and tissue engineering. [3][4][5] Most micropatterning techniques are based on conventional photoresist lithography, soft lithography, and pin spotting. [6][7][8][9][10] Both photoresist-lithography and pin-spotting techniques require high-tech equipment such as mask aligners, electron (e)-beam writers, or pin spotters, and well-trained people to operate them. Conversely, soft lithography uses a polydimethylsiloxane (PDMS) stamp or stencil as a template to create a pattern of proteins or cells on the substrate, which is more convenient.[11] However, fabrication of a PDMS stamp or stencil still relies on photolithography. Furthermore, for most of the work carried out so far, 2D patterns of proteins were generated by using soft-lithography techniques on substrates with planar surfaces. Very recently it has been reported that porous polymer films with well-ordered honeycomb structures can be prepared by using a breath-figure method, a templating method based on evaporative cooling and subsequent water-droplet templating to form an ordered array of breath figures. [12][13][14][15] A variety of polymers and block polymers such as polystyrene (PS), a-D-glucose, b-cyclodextrin and cellulose derivatives, dendronized polymethacrylate block polymer, and linear poly(ethylene oxide) block polymer have been used to fabricate such films. [16][17][18] Nanoparticles, such as gold, silver, and CdSe, are added into the porous films for different applications. [19][20][21] In this work, a porous polymer film was used as a template to guide the patterning of proteins and a 3D pattern of proteins was achieved. A big challenge was how to attach proteins to the designated regions of the polymer film, because most of the polymers used in the breath-figure method are hydrophobic and do not have functional chemical groups for conjugating proteins.A schematic drawing is given in Figure 1 to show how proteins can be patterned by using the porous film as the template. PS and a small amount of amino-terminated PS (PS-mNH 2 ) were dissolved in chloroform. The mixed polymer solution was spread on a glass slide to form a film. PS-mNH 2 is an amphiphilic linear polymer, bearing a hydrophilic amino group at one end and a long hydrophobic alkyl group at the other end, which could be used as a surfactant to be self-assembled at the interfaces between water droplets and waterimmiscible PS solution. During the formation of breath figures, the hydrophobic alkyl group of the amphiphilic PS-mNH 2 was oriented towards the hydrophobic phase (PS) in the solution and the hydrophilic amino group was oriented towards the water droplet. After the water droplets were removed, pores were formed with an amino-terminated pore surface. The surface property of the pores in such a film was different from that of a pure PS film whe...
