Extending the shelf life of HLM chips through freeze-drying of human liver microsomes immobilized onto thiol-ene micropillar arrays.

Microfluidic flow reactors functionalized with immobilized human liver microsomes (HLM chips) represent a powerful tool for drug discovery and development by enabling mechanism-based enzyme inhibition studies under flow-through conditions. Additionally, HLM chips may be exploited in streamlined production of human drug metabolites for subsequent microfluidic organ models or as metabolite standards for drug safety assessment. However, the limited shelf life of the biofunctionalized microreactors generally poses a major barrier to their commercial adaptation in terms of both storage and shipping.

Harnessing T cell exhaustion and trogocytosis to isolate patient-derived tumor-specific TCR.

To study and then harness the tumor-specific T cell dynamics after allogeneic hematopoietic stem cell transplant, we typed the frequency, phenotype, and function of lymphocytes directed against tumor-associated antigens (TAAs) in 39 consecutive transplanted patients, for 1 year after transplant. We showed that TAA-specific T cells circulated in 90% of patients but display a limited effector function associated to an exhaustion phenotype, particularly in the subgroup of patients deemed to relapse, where exhausted stem cell memory T cells accumulated. Accordingly, cancer-specific cytolytic functions were relevant only when the TAA-specific T cell receptors (TCRs) were transferred into healthy, genome-edited T cells.

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.

Digital Microfluidics-Enabled Analysis of Individual Variation in Liver Cytochrome P450 Activity.

The superfamily of hepatic cytochrome P450 (CYP) enzymes is responsible for the intrinsic clearance of the majority of therapeutic drugs in humans. However, the kinetics of drug clearance via CYPs varies significantly among individuals due to both genetic and external factors, and the enzyme amount and function are also largely impacted by many liver diseases. In this study, we developed a new methodology, based on digital microfluidics (DMF), for rapid determination of individual alterations in CYP activity from human-derived liver samples in biopsy-scale.

Cell adhesion and proliferation on common 3D printing materials used in stereolithography of microfluidic devices.

Three-dimensional (3D) printing has recently emerged as a cost-effective alternative for rapid prototyping of microfluidic devices. The feature resolution of stereolithography-based 3D printing is particularly well suited for manufacturing of continuous flow cell culture platforms. Poor cell adhesion or material-induced cell death may, however, limit the introduction of new materials to microfluidic cell culture.

Mechanism of the Oxidation of Heptafulvenes to Tropones Studied by Online Mass Spectrometry and Density Functional Theory Calculations.

We have identified the most likely reaction mechanism for oxidizing heptafulvenes to the corresponding tropones by experimental and theoretical investigations. The experimental studies were done by coupling a three-dimensional printed miniaturized reactor with an integrated electrospray ionization needle to a mass spectrometer. Using the experimentally observed ions as a basis, nine alternative reaction pathways were investigated with density functional theory calculations.

Simple 3D printed stainless steel microreactors for online mass spectrometric analysis.

A simple flow chemistry microreactor with an electrospray ionization tip for real time mass spectrometric reaction monitoring is introduced. The microreactor was fabricated by a laser-based additive manufacturing technique from acid-resistant stainless steel 316L. The functionality of the microreactor was investigated by using an inverse electron demand Diels-Alder and subsequent retro Diels-Alder reaction for testing.

Analysis of steroids in urine by gas chromatography-capillary photoionization-tandem mass spectrometry.

A new heated capillary photoionization (CPI) ion source design was developed to photoionize analytes inside a transfer capillary between a gas chromatograph (GC) and a mass spectrometer (MS). The CPI setup included a wide, oval-shaped vacuum-ultraviolet (VUV) transparent magnesium fluoride (MgF) window to maximize photoionization efficiency and thus sensitivity. The source contained a nitrogen housing around the ionization chamber inlet to avoid undesirable hydrolysis and oxidation reactions with ambient air and to maximize the proportion of formed molecular radical cations of analytes.

Capillary photoionization: interface for low flow rate liquid chromatography-mass spectrometry.

This is the first report on capillary photoionization (CPI) interfacing a liquid chromatograph (LC) and mass spectrometer (MS). A new heated CPI ion source was developed, including a heated transfer capillary, a wide oval-shaped and low-depth ionization chamber with a vacuum ultraviolet (VUV) transparent magnesium fluoride (MgF) window to increase the photoionization efficiency and thus the sensitivity. As both analytes and eluent are first vaporized and then photoionized inside the CPI ion source between the atmosphere and the vacuum of MS, the ion transfer efficiency into the MS and thus the sensitivity is improved.

Interfacing Digital Microfluidics with Ambient Mass Spectrometry Using SU-8 as Dielectric Layer.

This work describes the interfacing of electrowetting-on-dielectric based digital microfluidic (DMF) sample preparation devices with ambient mass spectrometry (MS) via desorption atmospheric pressure photoionization (DAPPI). The DMF droplet manipulation technique was adopted to facilitate drug distribution and metabolism assays in droplet scale, while ambient mass spectrometry (MS) was exploited for the analysis of dried samples directly on the surface of the DMF device. Although ambient MS is well-established for bio- and forensic analyses directly on surfaces, its interfacing with DMF is scarce and requires careful optimization of the surface-sensitive processes, such as sample precipitation and the subsequent desorption/ionization.

Digital microfluidic immobilized cytochrome P450 reactors with integrated inkjet-printed microheaters for droplet-based drug metabolism research.

We report the development and characterization of digital microfluidic (DMF) immobilized enzyme reactors (IMERs) for studying cytochrome P450 (CYP)-mediated drug metabolism on droplet scale. The on-chip IMERs consist of porous polymer (thiol-ene) monolith plugs prepared in situ by photopolymerization and functionalized with recombinant CYP1A1 isoforms (an important detoxification route for many drugs and other xenobiotics). The DMF devices also incorporate inexpensive, inkjet-printed microheaters for on-demand regio-specific heating of the IMERs to physiological temperature, which is crucial for maintaining the activity of the temperature-sensitive CYP reaction.

Thin-Layer Chromatography/Desorption Atmospheric Pressure Photoionization Orbitrap Mass Spectrometry of Lipids.

Desorption atmospheric pressure photoionization (DAPPI) allows surface analysis in the open atmosphere and is thus an appropriate method for the direct coupling of thin-layer chromatography (TLC) and mass spectrometry (MS). Here, the capability of DAPPI-MS for ionizing and detecting lipids, namely, cholesterol, triacylglycerols, 1,2-diol diesters, wax esters, cholesteryl esters, and hydrocarbons, from TLC and high-performance thin-layer chromatography (HPTLC) plates in MS and MS modes was tested. Limits of detection for lipid standards separated using normal-phase (NP)-TLC and NP-HPTLC were established.

A Simple Method for Improving the Spatial Resolution in Infrared Laser Ablation Mass Spectrometry Imaging.

In mass spectrometry imaging of tissues, the size of structures that can be distinguished is determined by the spatial resolution of the imaging technique. Here, the spatial resolution of IR laser ablation is markedly improved by increasing the distance between the laser and the focusing lens. As the distance between the laser and the lens is increased from 1 to 18 m, the ablation spot size decreases from 440 to 44 μm.

Analysis of neonicotinoids from plant material by desorption atmospheric pressure photoionization-mass spectrometry.

Rationale: Neonicotinoids are widely used insecticides which have been shown to affect the memory and learning abilities of honey bees, and are suspected to play a part in the unexplainable, large-scale loss of honey bee colonies. Fast methods, such as ambient mass spectrometry (MS), for their analysis from a variety of matrices are necessary to control the use of forbidden products and study the spreading of insecticides in nature.

Methods: The feasibilities of two ambient MS methods, desorption electrospray ionization (DESI) and desorption atmospheric pressure photoionization (DAPPI), for the analysis of five most used neonicotinoid compounds, thiacloprid, acetamiprid, clothianidin, imidacloprid and thiamethoxam, were tested.

The detection and mapping of the spatial distribution of insect defense compounds by desorption atmospheric pressure photoionization Orbitrap mass spectrometry.

Many insects use chemicals synthesized in exocrine glands and stored in reservoirs to protect themselves. Two chemically defended insects were used as models for the development of a new rapid analytical method based on desorption atmospheric pressure photoionization-mass spectrometry (DAPPI-MS). The distribution of defensive chemicals on the insect body surface was studied.

Solvent jet desorption capillary photoionization-mass spectrometry.

A new ambient mass spectrometry method, solvent jet desorption capillary photoionization (DCPI), is described. The method uses a solvent jet generated by a coaxial nebulizer operated at ambient conditions with nitrogen as nebulizer gas. The solvent jet is directed onto a sample surface, from which analytes are extracted into the solvent and ejected from the surface in secondary droplets formed in collisions between the jet and the sample surface.

Separation of nucleobases, nucleosides, and nucleotides using two zwitterionic silica-based monolithic capillary columns coupled with tandem mass spectrometry.

The capability of employing synthesized zwitterionic silica-based monolithic capillary columns (140 mm × 0.1mm) for separation of highly polar and hydrophilic nucleobases, nucleosides, and nucleotides in hydrophilic interaction chromatography is reported. The suitability of the columns for on-line conjunction with electrospray tandem mass spectrometry was explored.

Analysis of anabolic steroids in urine by gas chromatography-microchip atmospheric pressure photoionization-mass spectrometry with chlorobenzene as dopant.

A gas chromatography-microchip atmospheric pressure photoionization-tandem mass spectrometry (GC-μAPPI-MS/MS) method was developed for the analysis of anabolic androgenic steroids in urine as their trimethylsilyl derivatives. The method utilizes a heated nebulizer microchip in atmospheric pressure photoionization mode (μAPPI) with chlorobenzene as dopant, which provides high ionization efficiency by producing abundant radical cations with minimal fragmentation. The performance of GC-μAPPI-MS/MS was evaluated with respect to repeatability, linearity, linear range, and limit of detection (LOD).

Neurosteroid analysis by gas chromatography-atmospheric pressure photoionization-tandem mass spectrometry.

A new and simple APPI interface employing commercially available hardware is used to combine GC to MS. The feasibility of the method is demonstrated in the analysis of urine samples for neurosteroids as their trimethylsilyl (TMS) derivatives. The effect of different dopants (chlorobenzene, toluene, anisole) on the ionization of the TMS derivatives was investigated.

Capillary photoionization: a high sensitivity ionization method for mass spectrometry.

We present a capillary photoionization (CPI) method for mass spectrometric (MS) analysis of liquid and gaseous samples. CPI utilizes a heated transfer capillary with a vacuum ultraviolet transparent MgF2 window, through which vacuum UV light (10 eV) from an external source enters the capillary. The liquid or gaseous sample, together with dopant, is introduced directly into the heated transfer capillary between the atmosphere and the vacuum of the MS.

Comparison of direct and alternating current vacuum ultraviolet lamps in atmospheric pressure photoionization.

A direct current induced vacuum ultraviolet (dc-VUV) krypton discharge lamp and an alternating current, radio frequency (rf) induced VUV lamp that are essentially similar to lamps in commercial atmospheric pressure photoionization (APPI) ion sources were compared. The emission distributions along the diameter of the lamp exit window were measured, and they showed that the beam of the rf lamp is much wider than that of the dc lamp. Thus, the rf lamp has larger efficient ionization area, and it also emits more photons than the dc lamp.

Feasibility of capillary liquid chromatography-microchip-atmospheric pressure photoionization-mass spectrometry for pesticide analysis in tomato.

A new and sensitive analytical method, using capillary liquid chromatography (capLC) with a microfabricated heated nebulizer chip for atmospheric pressure photoionization and tandem mass spectrometry (μAPPI-MS/MS), was developed for the analysis of selected carbamate pesticides in a tomato matrix. The performance of the instrumental method was evaluated, using seven pesticides, namely oxamyl, methomyl, aldicarb, carbofuran, pirimicarb, thiocarb, and ditalimfos. The limits of detection achieved with the capLC-μAPPI-MS/MS method in the positive ion mode were low, ranging from 0.

Desorption atmospheric pressure photoionization-mass spectrometry in routine analysis of confiscated drugs.

A comprehensive study was made, where desorption atmospheric pressure photoionization (DAPPI) was applied to the direct analysis of confiscated drugs and pharmaceuticals of various forms and matrices. The analyzed samples included herbal products [Catha edulis (khat), Psilocybe mushrooms, opium and Spice], designer drugs in tablet and powder form [e.g.

Feasibility of gas chromatography-microchip atmospheric pressure photoionization-mass spectrometry in analysis of anabolic steroids.

Mass spectrometers equipped with atmospheric pressure ion sources (API-MS) have been designed to be interfaced with liquid chromatographs (LC) and have rarely been connected to gas chromatographs (GC). Recently, we introduced a heated nebulizer microchip and showed its potential to interface liquid microseparation techniques and GC with API-MS. This study demonstrates the feasibility of GC-microchip atmospheric pressure photoionization-tandem mass spectrometry (GC-μAPPI-MS/MS) in the analysis of underivatized anabolic steroids in urine.