Thiol-ene-based microfluidic chips for glycopeptide enrichment and online digestion of inflammation-related proteins osteopontin and immunoglobulin G.

Proteins, and more specifically glycoproteins, have been widely used as biomarkers, e.g., to monitor disease states. Bottom-up approaches based on mass spectrometry (MS) are techniques commonly utilized in glycoproteomics, involving protein digestion and glycopeptide enrichment. Here, a dual function polymeric thiol-ene-based microfluidic chip (TE microchip) was applied for the analysis of the proteins osteopontin (OPN) and immunoglobulin G (IgG), which have important roles in autoimmune diseases, in inflammatory diseases, and in coronavirus disease 2019 (COVID-19). TE microchips with larger internal surface features immobilized with trypsin were successfully utilized for OPN digestion, providing rapid and efficient digestion with a residence time of a few seconds. Furthermore, TE microchips surface-modified with ascorbic acid linker (TEA microchip) have been successfully utilized for IgG glycopeptide enrichment. To illustrate the use of the chips for more complex samples, they were applied to enrich IgG glycopeptides from human serum samples with antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The dual functional TE microchips could provide high throughput for online protein digestion and glycopeptide enrichment, showing great promise for future extended applications in proteomics and the study of related diseases.

cIAP1/2 antagonization by SMAC mimetic induces non-canonical NF-κB mediated TH 17 cell homotypic interactions and increases their resistance to shear stress.

SMAC antagonization of cIAP1/2 in TH 17 cells upregulates cell adhesion and cytoskeleton genes through the NIK-RelB and p52 axis. SMAC also increases the homotypic interactions of TH 17 cells through a non-canonical NF-κB- and integrin-mediated mechanism resulting in increased ability of TH 17 cells to withstand shear stress.

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.

The FAST Pump, a low-cost, easy to fabricate, SLA-3D-printed peristaltic pump for multi-channel systems in any lab.

With the increasing interest in high throughput screening and parallel assays, laboratories around the world inevitably find themselves in need of driving a multitude of fluid lines to facilitate their large scale studies. The comparatively low cost and no-fluid-contact design of peristaltic pumps make them the go-to systems for such ventures, but using commercially available pumping systems this still becomes a costly endeavor at typically $250-$1000 per pump line. Here we have developed an alternative, a peristaltic pump that can be fabricated in most research laboratories using 3D-printing and readily available off-the-shelf parts. The pump features 8 parallel channels with linear ranges spanning from 0.7 µL/min to 6 mL/min. The pump can be fabricated and assembled by anyone with access to a 3D-printer at a cost of less than $45 per channel and is driven by a stepper motor that connects directly to any computer. This device has the potential to be disruptive in areas such as drug screening and assay development, as well as lab-on-a-chip applications and cell cultivation, where it significantly reduces hardware expenses and allows for construction of more comprehensive fluidic systems at a fraction of current costs.

Transcriptional profiles of human islet and exocrine endothelial cells in subjects with or without impaired glucose metabolism.

In experimental studies, pancreatic islet microvasculature is essential for islet endocrine function and mass, and islet vascular morphology is altered in diabetic subjects. Even so, almost no information is available concerning human islet microvascular endothelial cell (MVEC) physiology and gene expression. In this study, islets and exocrine pancreatic tissue were acquired from organ donors with normoglycemia or impaired glucose metabolism (IGM) immediately after islet isolation. Following single-cell dissociation, primary islet- and exocrine MVECs were obtained through fluorescence-activated cell sorting (FACS) and transcriptional profiles were generated using AmpliSeq. Multiple gene sets involved in general vascular development and extracellular matrix remodeling were enriched in islet MVEC. In exocrine MVEC samples, multiple enriched gene sets that relate to biosynthesis and biomolecule catabolism were found. No statistically significant enrichment was found in gene sets related to autophagy or endoplasmic reticulum (ER) stress. Although ample differences were found between islet- and exocrine tissue endothelial cells, no differences could be observed between normoglycemic donors and donors with IGM at gene or gene set level. Our data is consistent with active angiogenesis and vascular remodeling in human islets and support the notion of ongoing endocrine pancreas tissue repair and regeneration even in the adult human.

Protein Kinase R Is Constitutively Expressed in the Human Pancreas.

Viral infection of the insulin-producing cells in the pancreas has been proposed in the etiology of type 1 diabetes. Protein kinase R (PKR) is a cytoplasmic protein activated through phosphorylation in response to cellular stress and particularly viral infection. As PKR expression in pancreatic beta-cells has been interpreted as a viral footprint, this cross-sectional study aimed at characterizing the PKR expression in non-diabetic human pancreases. PKR expression was evaluated in pancreas tissue from 16 non-diabetic organ donors, using immunohistochemistry, qPCR, and western blot. Immunohistochemistry and western blot showed readily detectable PKR expression in the pancreatic parenchyma. The qPCR detected PKR mRNA in both endocrine and exocrine samples, with a slightly higher expression in the islets. In conclusion, PKR is constitutively expressed in both endocrine and exocrine parts of the pancreas and its expression should not be interpreted as a viral footprint in pancreatic beta cells.

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.

Recent advances in lab-on-a-chip for biosensing applications.

The marriage of highly sensitive biosensor designs with the versatility in sample handling and fluidic manipulation offered by lab-on-a-chip systems promises to yield powerful tools for analytical and, in particular, diagnostic applications. The field where these two technologies meet is rapidly and almost violently developing. Yet, solutions where the full potentials are being exploited are still surprisingly rare. In the context of this review, sensor designs are often fairly advanced, whereas the lab-on-a-chip aspect is still rather simplistic in many cases, albeit already offering significant improvements to existing methods. Recent examples, showing a staggering variety of lab-on-a-chip systems for biosensing applications, are presented, tabularized for overview, and briefly discussed.