4. Outlook and Future Direction of µIP
The development of µIP has provided fundamental insight into: (1) the spotter-free arraying of ready-to-print biological molecules; (2) the in situ co-synthesis and immobilization of messenger RNA or protein molecules directly from prearrayed DNA; (3) the self-assembly of microarrays with an increased density of up to 25 million per chip, which is 3–4 orders of magnitude higher than that of existing approaches; and (4) high resolution with sub-micrometer features. Despite the success of µIP, several aspects of printing must be improved. First, the current protocol of µIP requires the use of a microchamber-array chip fabricated by top-down microfabrication using soft lithography, which relies on the use of photolithography to generate the master, which is not a universally-affordable technique. In an advanced form of µIP, it may be possible to produce the microchamber-array chip by solution processing, i.e., a solution-based deposition technique, which will help significantly to reduce the cost and increase the availability of µIP. Second, a PDMS-based microchamber-array chip is used for µIP; the limitations of the use of PDMS in cell-free protein synthesis-based microarrays are its propensity to adsorb analytes and its porosity, which allows small molecular components of the cell-free system, such as amino acids, to diffuse into the bulk of PDMS [33]. Minimizing the scale, which increases the surface area of PDMS relative to the sample volume, can thus alter arraying outcomes, as a result of interfacial phenomena. Recently, we have investigated the effectiveness of a PDMS surface for miniaturizing the cell-free in situ synthesis of GFP inside PDMS microchambers with different sizes, and thus, different surface area-to-volume (S/V) ratios, and observed that the yield in the cell-free synthesis of GFP in PDMS microchambers decreased monotonically with decreasing microchamber diameter from 100 down to 25 µm [34]. Therefore, the application of coating techniques to suppress the PDMS interfacial phenomena can increase the suitability of PDMS while obtaining the benefits of µIP for developing ultrahigh-density protein microarrays. In summary, µIP can be applied to simultaneously synthesize and pattern virtually any full-length protein at a micrometer-scale resolution from prearrayed DNAs, and thus, this system is a versatile platform for several applications, including the on-chip molecular screening of mutant protein libraries, the identification of novel protein sequences and the detection of protein to protein interactions for global proteome analysis.