PeptiCHIP: A Microfluidic Platform for Tumor Antigen Landscape Identification.

Identification of HLA class I ligands from the tumor surface (ligandome or immunopeptidome) is essential for designing T-cell mediated cancer therapeutic approaches. However, the sensitivity of the process for isolating MHC-I restricted tumor-specific peptides has been the major limiting factor for reliable tumor antigen characterization, making clear the need for technical improvement. Here, we describe our work from the fabrication and development of a microfluidic-based chip (PeptiCHIP) and its use to identify and characterize tumor-specific ligands on clinically relevant human samples. Specifically, we assessed the potential of immobilizing a pan-HLA antibody on solid surfaces via well-characterized streptavidin-biotin chemistry, overcoming the limitations of the cross-linking chemistry used to prepare the affinity matrix with the desired antibodies in the immunopeptidomics workflow. Furthermore, to address the restrictions related to the handling and the limited availability of tumor samples, we further developed the concept toward the implementation of a microfluidic through-flow system. Thus, the biotinylated pan-HLA antibody was immobilized on streptavidin-functionalized surfaces, and immune-affinity purification (IP) was carried out on customized microfluidic pillar arrays made of thiol-ene polymer. Compared to the standard methods reported in the field, our methodology reduces the amount of antibody and the time required for peptide isolation. In this work, we carefully examined the specificity and robustness of our customized technology for immunopeptidomics workflows. We tested this platform by immunopurifying HLA-I complexes from 1 × 106 cells both in a widely studied B-cell line and in patients-derived ex vivo cell cultures, instead of 5 × 108 cells as required in the current technology. After the final elution in mild acid, HLA-I-presented peptides were identified by tandem mass spectrometry and further investigated by in vitro methods. These results highlight the potential to exploit microfluidics-based strategies in immunopeptidomics platforms and in personalized immunopeptidome analysis from cells isolated from individual tumor biopsies to design tailored cancer therapeutic vaccines. Moreover, the possibility to integrate multiple identical units on a single chip further improves the throughput and multiplexing of these assays with a view to clinical needs.

Aqueous and non-aqueous microchip electrophoresis with on-chip electrospray ionization mass spectrometry on replica-molded thiol-ene microfluidic devices.

This work describes aqueous and non-aqueous capillary electrophoresis on thiol-ene-based microfluidic separation devices that feature fully integrated and sharp electrospray ionization (ESI) emitters. The chip fabrication is based on simple and low-cost replica-molding of thiol-ene polymers under standard laboratory conditions. The mechanical rigidity and the stability of the materials against organic solvents, acids and bases could be tuned by adjusting the respective stoichiometric ratio of the thiol and allyl ("ene") monomers, which allowed us to carry out electrophoresis separation in both aqueous and non-aqueous (methanol- and ethanol-based) background electrolytes. The stability of the ESI signal was generally ≤10% RSD for all emitters. The respective migration time repeatabilities in aqueous and non-aqueous background electrolytes were below 3 and 14% RSD (n=4-6, with internal standard). The analytical performance of the developed thiol-ene microdevices was shown in mass spectrometry (MS) based analysis of peptides, proteins, and small molecules. The theoretical plate numbers were the highest (1.2-2.4×104m-1) in ethanol-based background electrolytes. The ionization efficiency also increased under non-aqueous conditions compared to aqueous background electrolytes. The results show that replica-molding of thiol-enes is a feasible approach for producing ESI microdevices that perform in a stable manner in both aqueous and non-aqueous electrophoresis.

Thiol-ene microfluidic devices for microchip electrophoresis: Effects of curing conditions and monomer composition on surface properties.

Thiol-ene polymer formulations are raising growing interest as new low-cost fabrication materials for microfluidic devices. This study addresses their feasibility for microchip electrophoresis (MCE) via characterization of the effects of UV curing conditions and aging on the surface charge and wetting properties. A detailed comparison is made between stoichiometric thiol-ene (1:1) and thiol-ene formulations bearing 50% molar excess of allyls ("enes"), both prepared without photoinitiator or other polymer modifiers. Our results show that the surface charge of thiol-ene 1:1 increases along with increasing UV exposure dose until a threshold (here, about 200J/cm(2)), whereas the surface charge of thiol-ene 2:3 decreases as a function of increasing UV dose. However, no significant change in the surface charge upon storage in ambient air was observed over a period of 14 days (independent of the curing conditions). The water contact angles of thiol-ene 2:3 (typically 70-75°) were found to be less dependent on the UV dose and storing time. Instead, water contact angles of thiol-ene 1:1 slightly decrease (from initial 90 to 95° to about 70°) as a function of UV increasing exposure dose and storing time. Most importantly, both thiol-ene formulations remain relatively hydrophilic over extended periods of time, which favors their use in MCE applications. Here, MCE separation of biologically active peptides and selected fluorescent dyes is demonstrated in combination with laser-induced fluorescence detection showing high separation efficiency (theoretical plates 8200 per 4cm for peptides and 1500-2700 per 4cm for fluorescent dyes) and lower limits of detection in the sub-µM (visible range) or low-µM (near-UV range) level.