Thiol-ene Monolithic Pepsin Microreactor with a 3D-Printed Interface for Efficient UPLC-MS Peptide Mapping Analyses.

To improve the sample handling, and reduce cost and preparation time, of peptide mapping LC-MS workflows in protein analytical research, we here investigate the possibility of replacing conventional enzymatic digestion methods with a polymer microfluidic chip based enzyme reactor. Off-stoichiometric thiol-ene is utilized as both bulk material and as a monolithic stationary phase for immobilization of the proteolytic enzyme pepsin. The digestion efficiency of the, thiol-ene based, immobilized enzyme reactor (IMER) is compared to that of a conventional, agarose packed bed, pepsin IMER column commonly used in LC-MS based protein analyses. The chip IMER is found to rival the conventional column in terms of digestion efficiency at comparable residence time and, using a 3D-printed interface, be directly interfaceable with LC-MS.

Photolithographic Patterning of FluorAcryl for Biphilic Microwell-Based Digital Bioassays and Selection of Bacteria.

FluorAcryl 3298 (FA) is a UV-curable fluoroacrylate polymer commonly employed as a chemically resistant, hydrophobic, and oleophobic coating. Here, FA was used in a cleanroom-based microstructuring process to fabricate hydrophilic-in-hydrophobic (HiH) micropatterned surfaces containing femtoliter-sized well arrays. A short protocol involving direct UV photopatterning, an etching step, and final recovery of the hydrophobic properties of the polymer produced patterned substrates with micrometer resolution. Specifically, HiH microwell arrays were obtained with a well diameter of 10 µm and various well depths ranging from 300 nm to 1 µm with high reproducibility. The 300 nm deep microdroplet array (MDA) substrates were used for digital immunoassays, which presented a limit of detection in the attomolar range. This demonstrated the chemical functionality of the hydrophilic and hydrophobic surfaces. Furthermore, the 1 µm deep wells could efficiently capture particles such as bacteria, whereas the 300 nm deep substrates or other types of flat HiH molecular monolayers could not. Capturing a mixture of bacteria expressing red- and green-fluorescent proteins, respectively, served as a model for screening and selection of specific phenotypes using FA-MDAs. Here, green-fluorescent bacteria were specifically selected by overlaying a solution of gelatin methacryloyl (GelMA) mixed with a photoinitiator and using a high-magnification objective, together with custom pinholes, in a common fluorescence microscope to cross-link the hydrogel around the bacteria of interest. In conclusion, due to the straightforward processing, versatility, and low-price, FA is an advantageous alternative to more commonly used fluorinated materials, such as CYTOP or Teflon-AF, for the fabrication of HiH microwell arrays and other biphilic microstructures.

Fabrication of Biomolecule Microarrays Using Rapid Photochemical Surface Patterning in Thiol-Ene-Based Microfluidic Devices.

In many biochip applications, it is advantageous to be able to immobilize biomolecules at specific locations on the surface of solid supports. In this protocol, we describe a photochemical surface patterning procedure based on thiol-ene/yne photochemistry which allows for the simple and rapid selective patterning of biomolecules on thiol-ene solid supports. We describe the preparation of solid supports which are required for the immobilization, including porous monoliths, as well as two different immobilization schemes based on biotin-streptavidin interactions and covalent linkage via free amino groups respectively.