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 mm2 surface to create 170 antibody spots of 50 ×
100 μm2 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.