Contact Processing with Hydrogel Stamps

 Contact processing (left-hand panel in Figure 1) mimics the deposition of proteins from an aqueous environment to a surface by utilizing a hydrogel swollen with a solution of protein [12,14]. The proteins can diffuse through this hydrophilic matrix and adsorb onto the substrate without uncontrolled spreading. The stamp consists of two parts. The first is a reservoir above the hydrogel containing proteins dissolved in a biological buffer. The second is the hydrogel that makes contact with the substrate and mediates the transport of proteins to the substrate. A stamp that has a hydrogel made of poly(6-acry loyl-b-O-methyl-galactopyranoside), for example, embedded in a fine capillary can pattern proteins with a resolution of 20 mm [14]. Hydrogels having a refined composition and a greater degree of crosslinking exhibited better mechanical resistance, and were patterned by replication of a mold [15]. The latter approach should allow a protein to be patterned on a surface with a resolution better than 20 mm. CP based on hydrogel stamps has interesting features. First, biomolecules remain in a biological buffer until the stamp is removed and the substrate dried. Denaturation of proteins in this case should be minimal, and may be similar to that of proteins adsorbed from solution onto polystyrene microtiter plates. Second, it is straightforward to reuse such stamps for multiple CP experiments [14].

Microcontact Printing

 Microcontact printing of proteins uses PDMS stamps replicated from a mold (middle panel in Figure 1). Inking the stamp with proteins is simple, and analogous to depositing a layer of capture antibody (Ab) on polystyrene for conducting a solid-phase immunoassay. The duration of inking and the concentration of protein in the ink solution determine the coverage of protein obtained on the stamp [16]. Inking a stamp can be local and/or involve multiple types of proteins when the stamp is locally exposed using a microfluidic network (mFN) or microcontainers to one or more solutions of protein [17]. The transfer of proteins can be remarkably homogeneous and effective, depending on the wetting properties of the substrate [18]. The large area of interaction of proteins with substrates and their high molecular weight account for the high-resolution potential of mCP of proteins. At the limit, single protein molecules can be printed as arrays on a surface [16], whereas the diffusion of alkanethiols on noble metals or the reactivity of silanes with themselves limit the practical resolution achieved for microcontact printing self-assembled monolayers on surfaces. Microcontact printing proteins on surfaces ap pears to be limited by the resolution and mechanical stability of the patterns on the stamp. Stamps made of Sylgard 184 and using masters prepared using rapid prototyping or photolithography can have micrometer-sized patterns on fields even larger than 10 cm2 [19]. Microcontact printing proteins with arbitrary patterns and submicrometer resolution benefits from the use of a PDMS elastomer stiffer than Sylgard 184 and masters patterned using electron-beam lithography [8]

Affinity-Contact Printing

 Tailoring the surface chemistry of stamps to ink a particular type of biomolecule is crucial for aCP (right-hand panel in Figure 1). The chemical stability of silicone elastomers is both an advantage for preparing chemically resistant stamps and an obstacle to modifying the surface of PDMS stamps. Exposing PDMS to an oxygen-based plasma forms a glassy silica-like surface layer [20]. The oxidized layer is a few nanometers thick and contains silanol groups (–Si–OH), which are useful for anchoring organosilanes [21]. Oxidized PDMS can thus be derivatized similarly to glass or SiO2 in a few chemical steps using silane monolayers and with crosslinkers for proteins [22]. Affinity-contact printing is the technique of covalently immobilizing ligand biomolecules onto a PDMS stamp, and using them to ink a stamp selectively with receptor molecules. A stamp for aCP is roughly analogous to a chromatography column due to its ability to extract proteins selectively from a mixture, although releasing them involves printing them onto a surface [13]. Bio molecules that are naturally present in crude solutions and have a function on a surface are ideal candidates for applications of aCP. Cell adhesion molecules is one example that has already been demonstrated, but aCP could well be extended to a large variety of bio molecules for which ligands exist. Stamps in aCP are reusable and may include sites of different affinity to capture and print multiple types of protein in parallel [23].

Fig1. Three related methods can pattern proteins from a stamp to a surface. In contact processing (left), a hydrogel stamp mediates the diffusion of proteins from its bulk to a surface. Microcontact printing (center) utilizes an elastomeric stamp inked with proteins to print the proteins on a substrate without having a liquid. The stamp in affinity contact printing (right) is derivatized with capture proteins, which allows it to be selectively inked with target proteins released to a substrate during printing.

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