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Biosensing analytical platforms rely on the intimate structure-function relationship of immobilized probes. In this context, hydrogels are appealing semi-wet systems to locally confine biomolecules while preserving their structural integrity and function. Yet, limitations imposed by biomolecule diffusion rates or fabrication difficulties still hamper their broad application. Here, using a self-assembling peptide, a printable and self-adhesive hydrogel was obtained and applied to fabricate arrays of localized bio-functional 3D microenvironments on analytical interfaces. This soft matrix represents a robust and versatile material, allowing fast and selective tuning of analyte diffusion, which is exploited here to run in-gel immunoassays under solution-like conditions in an unprecedented (<10 min) time frame. The developed material overcomes major limitations associated with hydrogels for bioassays, widening the prospects for easy fabrication of multifunctional bio-interfaces for high-throughput, molecular recognition assays., Competing Interests: The authors declare no conflicts of interest., (This journal is © The Royal Society of Chemistry.)