Capillary barriers provide a simple and elegant means for autonomous fluid-flow control in microfluidic systems. In this work, we report on the fabrication of periodic hydrogel microarrays in closed microfluidic systems using non-fluorescent capillary barriers. This design strategy enables the fabrication of picoliter-volume patterns of photopolymerized and thermo-gelling hydrogels without any defects and distortions. Graphical abstract: Large scale patterning of hydrogel microarrays using capillary pinning Selective hydrogel patterning offers a novel way to expand the capability of biological and clinical microarrays, gel-based lab-on-a-chip bioassays, cell patterning techniques, and biomolecule separation technologies. A reduction in size to small volumes enables dramatic increases in the number of analyses and throughput of hydrogel microarrays due to faster mass transport and increased surface-to-volume ratios. Despite the large promise of small volume hydrogel microarrays, their fabrication has remained challenging. Here we demonstrate that picoliter volume microarrays of photopolymerizing and thermo-gelling hydrogel types can be robustly and autonomously fabricated by capillary pinning in microfluidic devices. The method enables patterning in closed microfluidic systems entirely manufactured from non-fluorescent materials. Hydrogels have found widespread use in microfluidic systems due to their unique material properties. They provide excellent sensitivity to chemical and physical stimuli such as pH,1 ionic strength,1 temperature,2 electric field,3 and light.4 Hydrogels have been used for electrokinetic biomolecule separations for over a century, as they provide a dense mesh of a porous three dimensional matrix without a significant effect on electrolyte composition.5 These functionalities have brought hydrogel usage to the fore in wireless biomolecule measurements,6 two dimensional (bio)molecule separation,7 drug delivery,8 microdialysis,9 and biosensing10–13 applications with microfluidic devices. The incorporation of hydrogels in microfluidic systems is commonly accomplished by traditional methods such as optical and soft lithography techniques.14,15 The majority of optical lithography techniques are based on masked photolithography15 and laser patterning.16 Optical lithography has proven to be a well-established and reliable method. However, patterning via conventional photomasks comes at the cost of poor structure resolution in closed platforms due to diffraction of UV light from the microchip walls and uncontrollable free radical diffusion during polymerization.17 Hence, working with conventional lithography photomasks poses serious challenges when the aim is to fabricate hydrogel microarrays on the scale of tens of micrometers without any defects. Soft lithography techniques, including microcontact printing18 and micromolding,19 offer inexpensive, convenient, and scalable templates for patterning. However, these techniques require polymer (polydimethylsiloxane) molds for patterning and therefore are not suitable for hydrogel fabrication in closed microfluidic systems.20 For the hydrogel array applications mentioned above, a high degree of control over the shape and the size distribution of hydrogels down to the micrometer scale is needed. In the past, capillary valves (‘phaseguides’) have been successfully implemented in closed microchips to pattern hydrogel structures by local pinning of the hydrogel precursor on a scale ranging from ~100 of microns to tens of millimeters.21,22 Using this method, a maximum number of ~400 pinned liquid patches, each containing a few microliters of liquid, were patterned. In addition, phaseguide arrays presented in the literature were made of SU-8, a photopatternable polymer, that was chosen for its relatively less hydrophilic character (65 < θ < 85°, θ is the contact angle) in comparison with glass surfaces.23 This approach hinders fluorescence-based biomedical applications because SU-8 is strongly autofluorescent, overlapping with the emission bands of many fluorescent tagging agents.24 Both the large size and the fact that structures are made of SU-8 present limitations on this approach. Further development of the phaseguide technique is thus warranted for applications where large scale patterning of picoliter volume hydrogel patches in large scale areas and/or the use of non-fluorescent polymers in fabrication are necessary. In this work, we fabricated massively parallel hydrogel patterns by capillary pinning followed by photopolymerization or thermo-gelation in closed microfluidic platforms. Capillary pinning barriers were made of fused silica glass, which is a non-fluorescent material. Despite the fact that fused silica provides hydrophilic surfaces (θ < 25 °C) unfavorable for pinning,22 we show that silanization can enable the production of periodic hydrogel patterns on this material. Capillary barriers allow for autonomous and precisely controlled trapping of the hydrogel precursor solution over large areas (cm2), with high reliability and spatial resolution, and without any defects and distortions. For a proof-of-concept demonstration, we fabricated a closed microchip with an array of ~400k hydrogel patterns, sandwiched between ~400k glass pillars, and ~800k capillary barriers using only a 0.5 μl hydrogel precursor. We showed that large scale microarray patterning by capillary barriers is applicable to both photopolymerized and thermo-gelling hydrogel types.