Direct analysis of surface chemicals using vibrating sharp-edge spray ionization mass spectrometry.

RATIONALE: Direct analysis of chemicals on a surface using mass spectrometry (MS) is of great importance in forensics, food and drug safety, environmental monitoring, and defense. Solvent extraction-based surface analysis offers a convenient way of controlling the desorption conditions and applying internal standards. To date, it mainly relies on a separate electrospray process to nebulize and ionize the solvents. Here, we report a simple and stand-alone ionization system for the solvent extraction-based surface analysis without the need for high voltage, based on vibrating sharp-edge spray ionization (VSSI). METHODS: We modified the original VSSI device and developed a stand-alone, integrated surface sampling, and ionization system for MS analysis. By incorporating a micropipette-based solvent dispenser with the VSSI device, the new system performs solvent extraction and ionization, and still maintains a small footprint. RESULTS: We demonstrated a four order-of-magnitude linear response for glucose spotted on a glass surface with a limit of detection (LOD) of 0.1 pg/mm2 . We further characterized the performance of this method with a series of compounds and demonstrated a similar LOD to literature values obtained by desorption electrospray ionization. Finally, we applied this method to quantitatively measure the concentration of a pesticide ametryn on spinach surfaces. We demonstrated good linearity (R2 = 0.99) for ametryn with surface densities in the range of 8-800 pg/mm2 and an LOD of 9 pg/mm2 . CONCLUSIONS: We have demonstrated a simple, effective, direct ambient-ionization method that is highly sensitive to molecules on a wide range of surfaces. The flexibility, small footprint, low cost, and voltage-free nature of this method make it an attractive technique for direct surface sample analysis using MS.

Evaluating nanomedicine with microfluidics.

Nanomedicines are engineered nanoscale structures that have an extensive range of application in the diagnosis and therapy of many diseases. Despite the rapid progress in and tremendous potential of nanomedicines, their clinical translational process is still slow, owing to the difficulty in understanding, evaluating, and predicting their behavior in complex living organisms. Microfluidic techniques offer a promising way to resolve these challenges. Carefully designed microfluidic chips enable in vivo microenvironment simulation and high-throughput analysis, thus providing robust platforms for nanomedicine evaluation. Here, we summarize the recent developments and achievements in microfluidic methods for nanomedicine evaluation, categorized into four sections based on their target systems: single cell, multicellular system, organ, and organism levels. Finally, we provide our perspectives on the challenges and future directions of microfluidics-based nanomedicine evaluation.

Mutational analysis reveals the importance of residues of the access tunnel inhibitor site to human P-glycoprotein (ABCB1)-mediated transport.

Human P-glycoprotein (P-gp) utilizes energy from ATP hydrolysis for the efflux of chemically dissimilar amphipathic small molecules and plays an important role in the development of resistance to chemotherapeutic agents in most cancers. Efforts to overcome drug resistance have focused on inhibiting P-gp-mediated drug efflux. Understanding the features distinguishing P-gp inhibitors from substrates is critical. Cryo-electron microscopy has revealed distinct binding patterns, emphasizing the role of the L-site or access tunnel in inhibition. We substituted 5-9 residues of the L-site with alanine to investigate whether the binding of a second inhibitor molecule to the L-site is required for inhibiting drug efflux. We reveal, for the first time, that mutations in the L-site affect the drug efflux activity of P-gp, despite their distance from the substrate-binding pocket (SBP). Surprisingly, after the mutations were introduced, inhibitors such as tariquidar and zosuquidar still inhibited drug efflux by mutant P-gps. Communication between the transmembrane helices (TMHs) and nucleotide-binding domains (NBDs) was evaluated using the ATPase assay, revealing distinct modulation patterns by inhibitors for the mutants, with zosuquidar exhibiting substrate-like stimulation of ATPase. Furthermore, L-site mutations abolished ATP-dependent thermal stabilization. In silico molecular docking studies corroborated the altered inhibitor binding due to mutations in the L-site residues, shedding light on their critical role in substrate transport and inhibitor interactions with P-gp. These findings suggest that inhibitors bind either to the SBP alone, and/or to alternate site(s) when the L-site is disabled by mutagenesis.

Acoustofluidic enzyme-linked immunosorbent assay (ELISA) platform enabled by coupled acoustic streaming.

Bead-based ELISA in microfluidics is a promising platform for reducing the reagent consumption and improving assay throughput due to its fast binding kinetics and low reagent consumption. Current microfluidic bead-based immunoassay mainly relies on magnetic and centrifugal forces to manipulate microparticles to complete an assay protocol. Here we report an acoustic streaming-based microfluidic method that can perform all the fluid and particle operations of bead-based ELISA. A series of sharp-edge structures are used to generate a long-range coupled acoustic streaming that enables simultaneous particle trapping and active molecular exchange in a dead-end microchannel. The device achieved >99% of molecular exchange in 4 min, while maintaining a particle trapping efficiency of 85%. This acoustofluidic-based method demonstrates all three key operations in a bead-based immunoassay: (1) Beads "immobilization"; (2) Active mixing of fluid for bead/target binding; (3) Active molecular exchange for reagent loading and washing. Finally, on-chip quantitative detection of biomarker IL-6 with a limit of detection ∼50 pg/mL is achieved using this platform including an enzymatic signal amplification step. The small footprint, simple setup, and continuous flow operation of the acoustic streaming-based method makes it an attractive platform for continuous flow bead-based immunoassay.

Rapid Solution-Phase Hydrogen/Deuterium Exchange for Metabolite Compound Identification.

Rapid, solution-phase hydrogen/deuterium exchange (HDX) coupled with mass spectrometry (MS) is demonstrated as a means for distinguishing small-molecule metabolites. HDX is achieved using capillary vibrating sharp-edge spray ionization (cVSSI) to allow sufficient time for reagent mixing and exchange in micrometer-sized droplets. Different compounds are observed to incorporate deuterium with varying efficiencies resulting in unique isotopic patterns as revealed in the MS spectra. Using linear regression techniques, parameters representing contribution to exchange by different hydrogen types can be computed. In this proof-of-concept study, the exchange parameters are shown to be useful in the retrodiction of the amount of deuterium incorporated within different compounds. On average, the exchange parameters retrodict the exchange level with ~ 2.2-fold greater accuracy than treating all exchangeable hydrogens equally. The parameters can be used to produce hypothetical isotopic distributions that agree (± 16% RMSD) with experimental measurements. These initial studies are discussed in light of their potential value for identifying challenging metabolite species.

Capillary Vibrating Sharp-Edge Spray Ionization (cVSSI) for Voltage-Free Liquid Chromatography-Mass Spectrometry.

Here, we report a continuous flow-based ionization method, capillary vibrating sharp-edge spray ionization (cVSSI), that nebulizes liquid sample directly at the outlet of a capillary without using high-speed nebulization gas or a high electrical field. cVSSI is built upon the recently reported VSSI principle which nebulizes bulk liquid using vibrating sharp-edges. By attaching a short piece of fused silica capillary on top of the vibrating glass slide in VSSI, liquid is nebulized at the outlet of the capillary as the result of the vibration. Utilizing standard 360-µm OD/100-µm ID capillary, cVSSI works with a wide range of flow rates from 1 µL/min to 1 mL/min. The power consumption is as low as 130 mW. ESI-like MS spectra are obtained for small molecules, peptides, and proteins. Five orders of magnitude linear response for acetaminophen solution is achieved with a limit of detection (LOD) of 3 nM. cVSSI is also demonstrated to be compatible with LC-MS analysis. Two LC-MS applications are demonstrated with cVSSI: (1) separation and detection of a mixture of small molecules and (2) bottom-up proteomics using a protein digest. A mixture of nine common metabolites was appropriately separated and detected using LC-cVSSI-MS. In the bottom-up experiment, 78 peptides were detected using LC-cVSSI-MS/MS with a protein coverage of 100% for cytochrome c, which is comparable with the coverage obtained using LC-ESI-MS. cVSSI offers a means of interfacing LC or other continuous flow-based applications to mass spectrometers with the salient features of voltage-free, flexibility, small footprint, and low power consumption.