Underpinning transport phenomena for the patterning of biomolecules.

Surface-based assays are increasingly being used in biology and medicine, which in turn demand increasing quantitation and reproducibility. This translates into more stringent requirements on the patterning of biological entities on surfaces (also referred to as biopatterning). This tutorial focuses on mass transport in the context of existing and emerging biopatterning technologies. We here develop a step-by-step analysis of how analyte transport affects surface kinetics, and of the advantages and limitations this entails in major categories of patterning methods, including evaporating sessile droplets, laminar flows in microfluidics or electrochemistry. Understanding these concepts is key to obtaining the desired pattern uniformity, coverage, analyte usage or processing time, and equally applicable to surface assays. A representative technological review accompanies each section, highlighting the technical progress enabled by transport control in e.g. microcontact printing, inkjet printing, dip-pen nanolithography and microfluidic probes. We believe this tutorial will serve researchers to better understand available patterning methods/principles, optimize conditions and to help design protocols/assays. By highlighting fundamental challenges and available approaches, we wish to trigger the development of new surface patterning methods and assays.

Tissue lithography: Microscale dewaxing to enable retrospective studies on formalin-fixed paraffin-embedded (FFPE) tissue sections.

We present a new concept, termed tissue lithography (TL), and its implementation which enables retrospective studies on formalin-fixed paraffin-embedded tissue sections. Tissue lithography uses a microfluidic probe to remove microscale areas of the paraffin layer on formalin-fixed paraffin-embedded biopsy samples. Current practices in sample utilization for research and diagnostics require complete deparaffinization of the sample prior to molecular testing. This imposes strong limitations in terms of the number of tests as well as the time when they can be performed on a single sample. Microscale dewaxing lifts these constraints by permitting deprotection of a fraction of a tissue for testing while keeping the remaining of the sample intact for future analysis. After testing, the sample can be sent back to storage instead of being discarded, as is done in standard workflows. We achieve this microscale dewaxing by hydrodynamically confining nanoliter volumes of xylene on top of the sample with a probe head. We demonstrate micrometer-scale, chromogenic and fluorescence-based immunohistochemistry against multiple biomarkers (p53, CD45, HER2 and ß-actin) on tonsil and breast tissue sections and microarrays. We achieve stain patterns as small as 100 µm × 50 µm as well as multiplexed immunostaining within a single tissue microarray core with a 20-fold time reduction for local dewaxing as compared to standard protocols. We also demonstrate a 10-fold reduction in the rehydration time, leading to lower processing times between different stains. We further show the potential of TL for retrospective studies by sequentially dewaxing and staining four individual cores within the same tissue microarray over four consecutive days. By combining tissue lithography with the concept of micro-immunohistochemistry, we implement each step of the IHC protocol-dewaxing, rehydration and staining-with the same microfluidic probe head. Tissue lithography brings a new level of versatility and flexibility in sample processing and budgeting in biobanks, which may alleviate current sample limitations for retrospective studies in biomarker discovery and drug screening.

Rapid Subtractive Patterning of Live Cell Layers with a Microfluidic Probe.

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one particular implementation of the MFP, the hierarchical hydrodynamic flow confinement (hHFC), multiple liquids are simultaneously brought in contact with a substrate. Local chemical action and liquid shaping using the hHFC, is exploited to create cell patterns by locally lysing and removing cells. By utilizing the scanning ability of the MFP, user-defined patterns of cell monolayers are created. This protocol enables rapid, real-time and spatially controlled cell patterning, which can allow selective cell-cell and cell-matrix interaction studies.

Convection-Enhanced Biopatterning with Recirculation of Hydrodynamically Confined Nanoliter Volumes of Reagents.

We present a new methodology for efficient and high-quality patterning of biological reagents for surface-based biological assays. The method relies on hydrodynamically confined nanoliter volumes of reagents to interact with the substrate at the micrometer-length scale. We study the interplay between diffusion, advection, and surface chemistry and present the design of a noncontact scanning microfluidic device to efficiently present reagents on surfaces. By leveraging convective flows, recirculation, and mixing of a processing liquid, this device overcomes limitations of existing biopatterning approaches, such as passive diffusion of analytes, uncontrolled wetting, and drying artifacts. We demonstrate the deposition of analytes, showing a 2- to 5-fold increase in deposition rate together with a 10-fold reduction in analyte consumption while ensuring less than 6% variation in pattern homogeneity on a standard biological substrate. In addition, we demonstrate the recirculation of a processing liquid using a microfluidic probe (MFP) in the context of a surface assay for (i) probing 12 independent areas with a single microliter of processing liquid and (ii) processing a 2 mm(2) surface to create 170 antibody spots of 50 × 100 µm(2) area using 1.6 µL of liquid. We observe high pattern quality, conservative usage of reagents, micrometer precision of localization and convection-enhanced fast deposition. Such a device and method may facilitate quantitative biological assays and spur the development of the next generation of protein microarrays.