This can be achieved by including additional aspiration-only apertures beyond the extremities of the array in the em X /em -direction, which we term stabilization apertures. multiplexed treatment of a tissue core with -p53 and C-actin antibodies was performed using four adjacent HFCs created with an aperture-array probe (HFC dimensions: 4 0.3 mm 0.25 mm). The ability of these devices and methods to perform multiplexed assays, present sequentially different liquids on surfaces, and interact with surfaces at the centimeter-scale will likely spur new and efficient surface assays. Introduction Compartmentalization is usually central to studying the effect of various (bio)chemical microenvironments on biological entities. Such screening and analysis Phenformin hydrochloride of multiple parameters are useful in (bio)chemical screening, analysis, synthesis, and characterization Phenformin hydrochloride with applications, for instance, in drug discovery, studies of cell-to-cell communication, and tumor marker detection.1?4 Microtiter plates are currently one of the most common substrates for compartmentalized assays in both research and diagnostics. To increase the analytical throughput, the pattern has been to reduce the footprint and volume of each well of the microtiter plates, with the current footprint of standard wells measuring 1.5 mm 1.5 mm (1536 well plates). Further scaling of the microtiter plates is usually hindered by constraints in fabrication together with requirements for liquid and mechanical interfacing and imaging. These limitations have brought on a drive towards substrate-based assays. Such surface formats, called microarrays, use lithographic methods, inkjet printing and pin spotting to produce high-density patterns of specimens and reagents on surfaces.5 These surface assays have the potential to enable high-throughput analytical testing while simplifying read-out and detection. However, their lack of physical compartmentalization hinders multiplexing of liquid reagents and indicates the need for a new set of tools to enable targeted conversation with biological samples such as DNA/protein microarrays, tissue sections, and cell monolayers. Such a tool should ideally be able to (i) interact with the substrate on spatially unique areas at the mm- to cm-scale; (ii) deliver different liquids to a surface in both a parallel and sequential manner; (iii) enable conversation with the surface without physical contact between the tool and the surface, and (iv) operate in a wet environment to avoid drying artifacts. Several techniques have been designed that allow local processing of immersed substrates6?10 and fulfill subsets of the above criteria. However, a versatile method to efficiently interact with immersed, cm-scale substrates in a localized manner remains elusive. Pin spotters and Nr2f1 inkjet systems are established technologies primarily utilized for patterning reagents on a dry surface and are not suitable to implement biological assays on surfaces.11,12 Aqueous two-phase systems applied on immersed substrates by means of an inkjet-like nozzle have been utilized for patterning mammalian cells and bacteria,6,13 but the spatial resolution and the limitations imposed by diffusion between the two phases are not favorable for confining molecular reagents. Rapp et al. exhibited cm-scale patterning of antibodies using selective UV irradiation enabled by a digital micromirror device.14 This method is limited to photoinitiated reactions and does not allow a selective switch of the liquid environment on a surface. Local processing was also exhibited by Kim et al. by conformably sealing microchannels on surfaces but without the ability to stain specific regions of interest.10,15 In addition, mechanical contact can introduce cross-contamination and adverse mechanical stress on the biological sample, which is also the case for another contact-based microfluidic device, namely the chemistrode.16 Atomic force microscope (AFM)-based methods and their derivatives, such as the FluidFM17 and dip-pen lithography,18,19 enable local interaction with biological substrates with high resolution, but their narrow range of operation (which is 150 m 150 m 20 m) is not compatible with cm-scale substrates. Other methods for high-precision interfacing with biological substrates are scanning ion conductance microscopy20,21 and scanning electrochemical microscopy,22,23 both requiring implementation of reference electrodes and maintenance of specific homogeneous buffer conditions. This complicates the application of different processing liquids in (bio)chemical surface-based assays. A encouraging technology for local conversation with immersed substrates is the Phenformin hydrochloride microfluidic probe and its variants Phenformin hydrochloride (MFP).8,9,24 The MFP operates in a noncontact scanning mode and localizes a liquid on a surface by creating a hydrodynamic flow confinement (HFC; Physique ?Physique11a). Using the MFP, interactions with surfaces on length scales ranging from single m to several hundred m have been exhibited. The MFP and its variants have been applied for multiplexed immunohistochemical analysis of tissue sections,25 biopatterning,26 and pharmacology on a single-cell level.27 In the configurations of.