Low melting point agarose as a protection layer in photolithographic patterning of aligned binary proteins

Abstract

A novel photolithography method to build aligned patterns of two different proteins is presented. Chessboard patterns of 125 microm x 125 microm squares are constructed on a silicon dioxide substrate, using standard photoresist chemistries in combination with low-temperature oxygen plasma etching. Low-melting-point agarose (LMPA) is used to protect underlying protein layers and, at the appropriate stage, the digestive enzyme GELase (EPICENTRE) is used to selectively remove the prophylactic LMPA layers. Two antibodies, mouse-IgG and human-IgG, were immobilized and patterned by this procedure. The patterned antibodies maintained the specificity of their antigen-antibody binding, as demonstrated by fluorescence microscopy. In addition, normalized fluorescence intensity profiles illustrate that the patterned proteins layers are uniform (standard deviations below 0.05). Finally, a trypsin activity test was conducted to probe the effect of the patterning protocol on immobilized enzymes; the results imply that this photolithographic process using LMPA as a protection layer preserves 70% of immobilized enzyme activity.

Protein immobilization techniques for microfluidic assays

“…Naturally or artificially grown oxide on the silicon surface makes silanol-based chemistries compatible with for protein immobilization on silicon. 40,111,112 While powerful, silicon has, however, three major drawbacks for microfluidic design: (1) opaqueness of silicon in the visible spectrum renders various optical imaging techniques irrelevant; (2) incompatibility of silicon with electrokinetic methods owing to the electrical conductivity of the silicon substrate, and (3) expense associated with the sophisticated microfabrication techniques used to micromachine silicon in a cleanroom environment. Therefore, silicon is not as widely used as initially with the exceptions of continued widespread application in electrochemical analysis 113 and SPR.…”

“…However, confining immobilization sites of proteins at an asked time point is not straightforward in a conventional "injection and incubation" microfluidic format. Thus, various localized photochemical (Figure 15(a)), 21,22,42,112,119,188,196 electrochemical ( Figure 15(b)), 130,195,197 or thermal stimuli (Figure 15(c)) 32,198 are employed to initiate protein immobilization at a specific location. Reversely, for off-chip assay steps or surface rejuvenation, protein can be detached from local immobilization sites (i.e., elution) upon stimulus (Figure 15(d)).…”

“…112 First, thermal oxide was grown on a silicon surface. The oxide surface was successively treated with APTES and GA.…”

Innovative Microsystems: Novel Nanostructures to Capture Circulating Breast Cancer Cells

“…39 This seems to be an upper limit for the antibody surface density which, based on initial results, is on the order of 10 11 molecules cm…”

Innovative Microsystems: Novel Nanostructures to Capture Circulating Breast Cancer Cells

“…Shiff-base reaction The immunoassay for functionalizing an oxide surface with antibodies is schematically illustrated in Figure 3. The inner surface of the fabricated microchannel is uniformly functionalized with antibody molecules following a previously reported bio assay [11]. The microchanel is filled with 1% (vol/vol) 3-aminopropyltrioxysilane (APTES)-acetone solution for 15minutes at room temperature.…”