Microcontact printing (μCP) of proteins is widely used for biosensors and cell biology but is constrained to printing proteins adsorbed to a low free energy, hydrophobic surface to a high free energy, hydrophilic surface. This strongly limits μCP as harsh chemical treatments are required to form a high energy surface. Here, we introduce humidified μCP (HμCP) of proteins which enables universal printing of protein on any smooth surface. We found that by flowing water in proximity to proteins adsorbed on a hydrophilized stamp, the water vapor diffusing through the stamp enables the printing of proteins on both low and high energy surfaces. Indeed, when proteins are printed using stamps with increasing spacing between water-filled microchannels, only proteins adjacent to the channels are transferred. The vapor transport through the stamp was modeled, and by comparing the humidity profiles with the protein patterns, 88% relative humidity in the stamp was identified as the threshold for HμCP. The molecular forces occurring between PDMS, peptides, and glass during printing were modeled ab initio to confirm the critical role water plays in the transfer. Using HμCP, we introduce straightforward protocols to pattern multiple proteins side-by-side down to nanometer resolution without the need for expensive mask aligners, but instead exploiting self-alignment effects derived from the stamp geometry. Finally, we introduce vascularized HμCP stamps with embedded microchannels that allow printing proteins as arbitrary, large areas patterns with nanometer resolution. This work introduces the general concept of water-assisted μCP and opens new possibilities for "solvent-assisted" printing of proteins and of other nanoparticles.

Microfluidic probes (MFPs) combine the concepts of microfluidics and of scanning probes and constitute a contact-free and channel-free microfluidic system. Whereas classically the sample is introduced into the microfluidic device, with a MFP, the microfluidic stream is applied to the sample. MFPs use hydrodynamic flow confinement instead of walls to constrain a microfluidic stream between the MFP tip and a substrate. Because MFPs are free to move, they can be used to process large areas and samples in a selective manner. The development of MFP technology is recent and has numerous potential applications in several fields, most notably in the life sciences. In this review, we discuss the concept of MFPs and highlight their application in surface biopatterning, controlling the cellular microenvironments, local processing of tissue slices, and generating concentration gradients of biochemicals. We hope that this manuscript will serve as an interdisciplinary guide for both engineers as they further develop novel MFPs and applications and for life scientists who may identify novel uses of the MFP for their research.

Cells navigate in response to inhomogeneous distributions of extracellular guidance cues. The cellular and molecular mechanisms underlying migration in response to gradients of chemical cues have been investigated for over a century. Following the introduction of micropipettes and more recently microfluidics for gradient generation, much attention and effort was devoted to study cellular chemotaxis, which is defined as guidance by gradients of chemical cues in solution. Haptotaxis, directional migration in response to gradients of substrate-bound cues, has received comparatively less attention; however, it is increasingly clear that in vivo many physiologically relevant guidance proteins – including many secreted cues – are bound to cellular surfaces or incorporated into extracellular matrix and likely function via a haptotactic mechanism. Here, we review the history of haptotaxis. We examine the importance of the reference surface, the surface in contact with the cell that is not covered by the cue, which forms a gradient opposing the gradient of the protein cue and must be considered in experimental designs and interpretation of results. We review and compare microfluidics, contact printing, light patterning, and 3D fabrication to pattern substrate-bound protein gradients in vitro. The range of methods to create substrate-bound gradients discussed herein makes possible systematic analyses of haptotactic mechanisms. Furthermore, understanding the fundamental mechanisms underlying cell motility will inform bioengineering approaches to program cell navigation and recover lost function.