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Extracellular vesicles (EVs) play important roles in cell-to-cell communications and carry high potential as markers targeted in disease diagnosis, prognosis, and therapeutic development. The main obstacles to EV study are their high heterogeneity; low amounts present in samples; and physical similarity to the abundant, interfering matrix components. Multiple rounds of separation and purification are often needed prior to EV characterization and function assessment. Herein, we report the offline coupling of asymmetrical flow field-flow fractionation (AF4) and capillary electrophoresis (CE) for EV analysis. While AF4 provides gentle and fast EV separation by size, CE resolves EVs from contaminants with similar sizes but different surface charges. Employing Western Blotting, ELISA, and SEM, we confirmed that intact EVs were eluted within a stable time window under the optimal AF4 and CE conditions. We also proved that EVs could be resolved from free proteins and high-density lipoproteins by AF4 and be further separated from the low-density lipoproteins co-eluted in AF4. The effectiveness of the coupled AF4-CE system in EV analysis was demonstrated by monitoring the changes in EV secretion from cells and by direct injection of human serum and detection of serum EVs. We believe that coupling AF4 and CE can provide rapid EV quantification in biological samples with much reduced matrix interference and be valuable for the study of total EVs and EV subpopulations produced by cells or present in clinical samples.

For decades, there has been immense progress in miniaturizing analytical methods based on electrophoresis to improve sensitivity and to reduce sample volumes, separation times, and/or equipment cost and space requirements, in applications ranging from analysis of biological samples to environmental analysis to forensics. In the field of radiochemistry, where radiation-shielded laboratory space is limited, there has been great interest in harnessing the compactness, high efficiency, and speed of microfluidics to synthesize short-lived radiolabeled compounds. We recently proposed that analysis of these compounds could also benefit from miniaturization and have been investigating capillary electrophoresis (CE) and hybrid microchip electrophoresis (hybrid-MCE) as alternatives to the typically used high-performance liquid chromatography (HPLC). We previously showed separation of the positron-emission tomography (PET) imaging tracer 3'-deoxy-3'-fluorothymidine (FLT) from its impurities in a hybrid-MCE device with UV detection, with similar separation performance to HPLC, but with improved speed and lower sample volumes. In this paper, we have developed an integrated radiation detector to enable measurement of the emitted radiation from radiolabeled compounds. Though conventional radiation detectors have been incorporated into CE systems in the past, these approaches cannot be readily integrated into a compact hybrid-MCE device. We instead employed a solid-state avalanche photodiode (APD)-based detector for real-time, high-sensitivity β particle detection. The integrated system can reliably separate [18F]FLT from its impurities and perform chemical identification via coinjection with nonradioactive reference standard. This system can quantitate samples with radioactivity concentrations as low as 114 MBq/mL (3.1 mCi/mL), which is sufficient for analysis of radiochemical purity of radiopharmaceuticals.

Biologists' preeminent toolbox for separating, analyzing, and visualizing proteins is SDS-PAGE, yet recovering the proteins embedded in these polyacrylamide media as intact species is a long-standing challenge for mass spectrometry. In conventional workflows, protein mixtures from crude biological samples are electrophoretically separated at high-resolution within N,N'-methylene-bis-acrylamide cross-linked polyacrylamide gels to reduce sample complexity and facilitate sensitive characterization. However, low protein recoveries, especially for high molecular weight proteins, often hinder characterization by mass spectrometry. We describe a workflow for top-down/bottom-up mass spectrometric analyses of proteins in polyacrylamide slab gels using dissolvable, bis-acryloylcystamine-cross-linked polyacrylamide, enabling high-resolution protein separations while recovering intact proteins over a broad size range efficiently. The inferior electrophoretic resolution long associated with reducible gels has been overcome, as demonstrated by SDS-PAGE of crude tissue extracts. This workflow elutes intact proteins efficiently, supporting MS and MS/MS from proteins resolved on biologists' preferred separation platform.

Capillary electrophoresis (CE) has been identified as a useful platform for detecting, quantifying, and screening for modulators of protein-protein interactions (PPIs). In this method, one protein binding partner is labeled with a fluorophore, the protein binding partners are mixed, and then, the complex is separated from free protein to allow direct determination of bound to free ratios. Although it possesses many advantages for PPI studies, the method is limited by the need to have separation conditions that both prevent protein adsorption to capillary and maintain protein interactions during the separation. In this work, we use protein cross-linking capillary electrophoresis (PXCE) to overcome this limitation. In PXCE, the proteins are cross-linked under binding conditions and then separated. This approach eliminates the need to maintain noncovalent interactions during electrophoresis and facilitates method development. We report PXCE methods for an antibody-antigen interaction and heterodimer and homodimer heat shock protein complexes. Complexes are cross-linked by short treatments with formaldehyde after reaching binding equilibrium. Cross-linked complexes are separated by electrophoretic mobility using free solution CE or by size using sieving electrophoresis of SDS complexes. The method gives good quantitative results; e.g., a lysozyme-antibody interaction was found to have Kd = 24 ± 3 nM by PXCE and Kd = 17 ± 2 nM using isothermal calorimetry (ITC). Heat shock protein 70 (Hsp70) in complex with bcl2 associated athanogene 3 (Bag3) was found to have Kd = 25 ± 5 nM by PXCE which agrees with Kd values reported without cross-linking. Hsp70-Bag3 binding site mutants and small molecule inhibitors of Hsp70-Bag3 were characterized by PXCE with good agreement to inhibitory constants and IC50 values obtained by a bead-based flow cytometry protein interaction assay (FCPIA). PXCE allows rapid method development for quantitative analysis of PPIs.

High throughput, efficient, and readily adoptable analytical tools for the validation and selection of reliable antibody reagents would impact the life sciences, clinical chemistry, and clinical medicine. To directly quantify antibody-antigen association and dissociation rate constants, kon and koff, in a single experiment, we introduce a microfluidic free-standing kinetic polyacrylamide gel electrophoresis (fsKPAGE) assay. Here, an antibody is immobilized in zones along the length of a single freestanding polyacrylamide gel lane of varying cross-sectional width. Fluorescently labeled antigen is electrophoresed through each immobilized antibody zone, with local cross-sectional area determining the local electric field strength and, thus, the local interaction time between immobilized antibody and electromigrating antigen. Upon crossing, the interaction yields immobilized immunocomplex. The kon is quantified by assessing the amount of immunocomplex formed at each interaction time. To quantify koff, immobilized zones of fluorescently labeled immunocomplex are subjected to a buffer dilution and monitored over time. We determine kon and koff for prostate-specific antigen (PSA) and make a comparison to gold-standard values. The fsKPAGE assay determines kon and koff in a single experiment of less than 20 min, using 45 ng of often limited antibody material and standard laboratory equipment. We see the fsKPAGE assay as forming the basis for rapid, quantitative antibody-screening tools.

Numerous high-performance nanotechnologies have been developed, but their practical applications are largely restricted by the nanomaterials' low stabilities and high operation complexity in aqueous substrates. Herein, we develop a simple and high-reliability hydrogel-based nanotechnology based on the in situ formation of Au nanoparticles in molybdenum disulfide (MoS 2 )-doped agarose (MoS 2 /AG) hydrogels for electrophoresis-integrated microplate protein recognition. After the incubation of MoS 2 /AG hydrogels in HAuCl 4 solutions, MoS 2 nanosheets spontaneously reduce Au ions, and the hydrogels are remarkably stained with the color of as-synthetic plasmonic Au hybrid nanomaterials (Au staining). Proteins can precisely mediate the morphologies and optical properties of Au/MoS 2 heterostructures in the hydrogels. Consequently, Au staining-based protein recognition is exhibited, and hydrogels ensure the comparable stabilities and sensitivities of protein analysis. In comparison to the fluorescence imaging and dye staining, enhanced sensitivity and recognition performances of proteins are implemented by Au staining. In Au staining, exfoliated MoS 2 semiconductors directly guide the oriented growth of plasmonic Au nanostructures in the presence of formaldehyde, showing environment-friendly features. The Au-stained hydrogels merge the synthesis and recognition applications of plasmonic Au nanomaterials. Significantly, the one-step incubation of the electrophoretic hydrogels leads to high simplicity of operation, largely challenging those multiple-step Ag staining routes which were performed with high complexity and formaldehyde toxicity. Due to its toxic-free, simple, and sensitive merits, the Au staining integrated with electrophoresis-based separation and microplate-based high-throughput measurements exhibits highly promising and improved practicality of those developing nanotechnologies and largely facilitates in-depth understanding of biological information.

Intratumor heterogeneity remains a major obstacle to effective cancer therapy and personalized medicine. Current understanding points to differential therapeutic response among subpopulations of tumor cells as a key challenge to successful treatment. To advance our understanding of how this heterogeneity is reflected in cell-to-cell variations in chemosensitivity and expression of drug-resistance proteins, we optimize and apply a new targeted proteomics modality, single-cell western blotting (scWestern), to a human glioblastoma cell line. To acquire both phenotypic and proteomic data on the same, single glioblastoma cells, we integrate high-content imaging prior to the scWestern assays. The scWestern technique supports thousands of concurrent single-cell western blots, with each assay comprised of chemical lysis of single cells seated in microwells, protein electrophoresis from those microwells into a supporting polyacrylamide (PA) gel layer, and in-gel antibody probing. We systematically optimize chemical lysis and subsequent polyacrylamide gel electrophoresis (PAGE) of the single-cell lysate. The scWestern slides are stored for months then reprobed, thus allowing archiving and later analysis as relevant to sparingly limited, longitudinal cell specimens. Imaging and scWestern analysis of single glioblastoma cells dosed with the chemotherapeutic daunomycin showed both apoptotic (cleaved caspase 8- and annexin V-positive) and living cells. Intriguingly, living glioblastoma subpopulations show up-regulation of a multidrug resistant protein, P-glycoprotein (P-gp), suggesting an active drug efflux pump as a potential mechanism of drug resistance. Accordingly, linking of phenotype with targeted protein analysis with single-cell resolution may advance our understanding of drug response in inherently heterogeneous cell populations, such as those anticipated in tumors.

Pores with undulating opening diameters have emerged as an analytical tool enhancing the speed of resistive-pulse experiments, with a potential to simultaneously characterize size and mechanical properties of translocating objects. In this work, we present a detailed study of the characteristics of resistive-pulses of charged and uncharged polymer particles in pores with different aspect ratios and pore topography. Although no external pressure difference was applied, our experiments and modeling indicated the existence of local pressure drops, which modified axial and radial velocities of the solution. As a consequence of the complex velocity profiles, pores with undulating pore diameter and low-aspect ratio exhibited large dispersion of the translocation times. Distribution of the pulse amplitude, which is a measure of the object size, was not significantly affected by the pore topography. The importance of tuning pore geometry for the application in resistive-sensing and multipronged characterization of physical properties of translocating objects is discussed.

With relevance spanning from immunohistochemistry to immunoassays and therapeutics, antibody reagents play critical roles in the life sciences, clinical chemistry, and clinical medicine. Nevertheless, nonspecific interactions and performance reproducibility remain problematic. Consequently, scalable and efficient analytical tools for informed selection of reliable antibody reagents would have wide impact. Therefore, we introduce a kinetic polyacrylamide gel electrophoresis (KPAGE) microfluidic assay that directly measures antibody-antigen association and dissociation rate constants, kon and koff. To study antibody-antigen association, an antigen zone is electrophoresed through a zone of immobilized antibody. Upon crossing, the interaction yields a zone of immobilized immunocomplex. To quantify kon, we assess immunocomplex formation for a range of antigen-antibody interaction times. Here, interaction time is controlled by the velocity of the electromigrating antigen zone, which is determined by the strength of the applied electric field. All species are fluorescently labeled. To quantify koff, an immobilized zone of immunocomplex is subjected to in situ buffer dilution, while measuring the decay in immunocomplex concentration. Two approaches for antibody immobilization are detailed: (i) size-exclusion-based antibody immobilization via a molecular weight cutoff (MWCO) filter fabricated using polyacrylamide gel and (ii) covalent antibody immobilization realized using a photoactive benzophenone methacrylamide polyacrylamide gel. We determine kon and koff for prostate-specific antigen (PSA) and compare to gold-standard values. The KPAGE assay completes in 90 min, requiring 45 ng of often-limited antibody material, thus offering a quantitative antibody screening platform relevant to important but difficult to characterize interaction kinetics.

Methods for identifying chemical inhibitors of protein-protein interactions (PPIs) are often prone to discovery of false positives, particularly those caused by molecules that induce protein aggregation. Thus, there is interest in developing new platforms that might allow earlier identification of these problematic compounds. Capillary electrophoresis (CE) has been evaluated as a method to screen for PPI inhibitors using the challenging system of Hsp70 interacting with its co-chaperone Bag3. In the method, Hsp70 is labeled with a fluorophore, mixed with Bag3, and the resulting bound and free Hsp70 are separated and detected by CE with laser-induced fluorescence detection. The method used a chemically modified CE capillary to prevent protein adsorption. Inhibitors of the Hsp70-Bag3 interaction were detected by observing a reduction in the bound-to-free ratio. The method was used to screen a library of 3443 compounds, and the results were compared to those from a flow cytometry protein interaction assay. CE was found to produce a lower hit rate with more compounds that were reconfirmed in subsequent testing, suggesting greater specificity. This finding was attributed to the use of electropherograms to detect artifacts such as aggregators and to differences in protein modifications required to perform the different assays. Increases in throughput are required to make the CE method suitable for primary screens, but at the current stage of development it is attractive as a secondary screen to test hits found by higher-throughput methods.

We report a novel strategy to immobilize sodium dodecyl sulfate (SDS)-coated proteins for fully integrated microfluidic Western blotting. Polyacrylamide gel copolymerized with a cationic polymer, poly-L-lysine, effectively immobilizes all sized proteins after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and enables SDS-PAGE and subsequent immuno-probing in an automated microfluidic chip. Design of a poly-l-lysine conjugated polyacrylamide gel allows optimization of SDS-protein immobilization strength in the blotting gel region of the microchamber. The dependence of protein capture behavior on both the concentration of copolymerized charges and poly-lysine length is studied and gives important insight into an electrostatic immobilization mechanism. Based on analysis of protein conformation, the immobilized proteins bind with partner antibody after SDS dilution. We demonstrate each step of the microchamber Western blot, including injection, separation, transfer, immobilization, blocking, and immunoblot. The approach advances microfluidic protein immunoblotting, which is directly relevant to the widely-used SDS-PAGE based slab-gel Western blot, while saving sample volume, labor, and assay time.

A protein corona will be formed on nanoparticles (NPs) entering a biological matrix, which can influence particles' subsequent behaviors inside the biological systems. For proteins bound stably to the NPs, they can exhibit different association/dissociation rates. The binding kinetics could affect interaction of the NPs with cell surface receptors and possibly contribute to the outcomes of NPs uptake. In the present study, a method to differentiate the corona proteins based on their relative dissociation rates from the NPs was developed, employing flow field-flow fraction (F4) in combination with centrifugation. The proteins bound to the superparamagnetic iron oxide NPs (SPION) present in an IgG/albumin depleted serum were isolated via collection of the SPIONs by either F4 or centrifugation. They were subsequently analyzed by LC-MS/MS and identified. Because the SPION-protein complexes injected to F4 dissociated continuously under the nonequilibrium separation condition, only the proteins with slow enough dissociation rates would be collected with the NPs in the eluent of F4. However, in centrifugation, proteins with good affinity to the SPIONs were collected regardless of the dissociation rates of the complexes. In both cases, the nonbinding ones were washed off. Capillary electrophoresis and circular dichroism were employed to verify the binding situations of a few SPION-protein interactions, confirming the effectiveness of our method. Our results support that our method can screen for proteins binding to NPs with fast on-and-off rates, which should be the ones quickly exchanging with the free matrix proteins when the NPs are exposed to a new biological media. Thus, our method will be useful for investigation of the temporal profile of protein corona and its evolution in biological matrices as well as for high-throughput analysis of the dynamic feature of protein corona related to particle properties.

Extracellular vesicles (EVs) are cell-derived particles that exhibit diverse sizes, molecular contents, and clinical implications for various diseases depending on their specific subpopulations. However, fractionation of EV subpopulations with high resolution, efficiency, purity, and yield remains an elusive goal due to their diminutive sizes. In this study, we introduce a novel strategy that effectively separates EV subpopulations in a gel-free and label-free manner, using two-dimensional (2D) electrophoresis in a microfluidic artificial sieve. The microfabricated artificial sieve consists of periodically arranged micro-slit-well structures in a 2D array and generates an anisotropic electric field pattern to size fractionate EVs into discrete streams and steer the subpopulations into designated outlets for collection within a minute. Along with fractionating EV subpopulations, contaminants such as free proteins and short nucleic acids can be simultaneously directed to waste outlets, thus accomplishing both size fractionation and purification of EVs with high performance. Our platform offers a simple, rapid, and versatile solution for EV subpopulation isolation, which can potentially facilitate the discovery of biomarkers for specific EV subtypes and the development of EV-based therapeutics.

Bioprocessors for concentrating bioparticles, such as cells and molecules, are commonly needed in bioanalysis systems. In this microfluidic processor, a global flow field generated by ac electroosmosis transports the embedded particles to the regions near the electrode surface. The processor then utilizes electrophoretic and dielectrophoretic forces, which are effective in short range, to trap the target cells and molecules on the electrode surface. By optimizing the operating parameters, we have concentrated various biological objects in a large range of sizes, including Escherichia coli bacteria, lambda phage DNA, and single-stranded DNA fragments as small as 20 bases that have a radius of gyration of only 3 nm.

We have developed and characterized cellular optoporation with visible wavelengths of light using standard uncoated glass cover slips as the absorptive media. A frequency-doubled Nd:YAG laser pulse was focused at the interface of the glass surface and aqueous buffer, creating a stress wave and transiently permeabilizing nearby cells. Following optoporation of adherent cells, three spatial zones were present which were distinguished by the viability of the cells and the loading efficiency (or number of extracellular molecules loaded). The loading efficiency also depended on the concentration of the extracellular molecules and the molecular weight of the molecules. In the zone farthest from the laser beam (> 60 microns under these conditions), nearly all cells were both successfully loaded and viable. To illustrate the wider applicability of this optoporation method, cells were loaded with a substrate for protein kinase C and the cellular contents then analyzed by capillary electrophoresis. In contrast to peptides loaded by microinjection, optoporated peptide showed little proteolytic degradation, suggesting that the cells were minimally perturbed. Also demonstrating the potential for future work, cells were optoporated and loaded with a fluorophore in the enclosed channels of microfluidic devices.

Extracellular vesicles (EVs) are cell-derived, naturally produced, membrane-bound nanoscale particles that are linked to cell-cell communication and the propagation of diseases. Here, we report the design and testing of in-plane nanofluidic devices for resistive-pulse measurements of EVs derived from bovine milk and human breast cancer cells. The devices were fabricated in plane with three nanopores in series to determine the particle volume and diameter, two pore-to-pore regions to measure the electrophoretic mobility and zeta potential, and an in-line filter to prevent cellular debris and aggregates from entering the nanopore region. Devices were tested with and without the channels coated with a short-chain PEG silane to minimize electroosmotic flow and permit an accurate measurement of the electrophoretic mobility and zeta potential of the EVs. To enhance throughput of EVs, vacuum was applied to the waste reservoir to increase particle frequencies up to 1000 min -1 . The nanopores had cross-sections 200 nm wide and 200 nm deep and easily resolved EV diameters from 60 to 160 nm. EVs from bovine milk and human breast cancer cells had similar particle size distributions, but their zeta potentials differed by 2-fold, -8 ± 1 and -4 ± 1 mV, respectively.

Due to its potential for exquisite mass detection limits and resolving power, capillary electrophoresis is used for biochemical measurements on single cells; however, accurate measurements of many physiological parameters require sampling strategies that are considerably faster than those presently available. We have developed a laser-based technique to lyse single, adherent, mammalian cells on millisecond time scales. The cellular contents are then introduced into a capillary where electrophoretic separation and detection are performed. Improved temporal resolution of biological measurements results from the extremely rapid lysis made possible by this method. Additionally, the cell is not perturbed by mechanical or electrical stresses prior to sampling. Such disturbances can alter cellular physiology, resulting in inaccurate measurements. The fast cell lysis, the absence of cellular stresses prior to lysis, and the application to adherent mammalian cells are significant refinements to CE-based measurements on single cells. With this laser-micropipet combination, it will be possible to measure the intracellular concentration of molecules that change on subsecond to second time scales, for example, substrates of many cellular enzymes.

Due to its potential for exquisite mass detection limits and resolving power, capillary electrophoresis is used for biochemical measurements on single cells; however, accurate measurements of many physiological parameters require sampling strategies that are considerably faster than those presently available. We have developed a laser-based technique to lyse single, adherent, mammalian cells on millisecond time scales. The cellular contents are then introduced into a capillary where electrophoretic separation and detection are performed. Improved temporal resolution of biological measurements results from the extremely rapid lysis made possible by this method. Additionally, the cell is not perturbed by mechanical or electrical stresses prior to sampling. Such disturbances can alter cellular physiology, resulting in inaccurate measurements. The fast cell lysis, the absence of cellular stresses prior to lysis, and the application to adherent mammalian cells are significant refinements to CE-based measurements on single cells. With this laser-micropipet combination, it will be possible to measure the intracellular concentration of molecules that change on subsecond to second time scales, for example, substrates of many cellular enzymes.

Gel electrophoresis is a powerful technique for the characterization of sequences, sizes and conformations of nucleic acids due to its remarkable separation efficiency. In parallel, liquid chromatography-mass spectrometry (LC-MS) has established itself as a staple tool for the meticulous characterization and accurate quantification of a multitude of DNA modifications. In this study, we devised an in-gel digestion method for coupling gel electrophoresis with LC-MS/MS. This process involves the enzymatic digestion of DNA within the gel by nucleases and release single nucleosides, which subsequently serve as a preprocessing step for (LC-MS/MS) analysis. We demonstrated that ethylenediaminetetraacetic acid (EDTA) in the routine gel electrophoresis buffer reduced the enzymatic digestion efficiency, while Mg 2+ could mitigate this inhibition. We further showed EDTA-free gel electrophoresis and the process of digestion of genomic DNA and plasmid DNA within a gel was fluorescently imaged, proving the efficient digestion of DNA. By this improvement, the efficiency of an in-gel digestion could reach 60% or more of the control, compared with direct in-solution digestion. The measured abundances of DNA modifications (5-methylcytosine and N 6 -methyladenine) via in-gel digestion are consistent with that measured by in-solution digestion. Collectively, we showed an in-gel digestion method, which is a very useful pretreatment technique for the precise quantification of epigenetic modifications in diverse DNA molecules.

Melamine was sometimes adulterated to dairy products for false protein content increase in developing countries. However, a portable sensor has not been developed for on-spot determination of melamine in dairy products yet. Herein, a distance-based sensor was advanced for the quantification of melamine in dairy products based on chip electrophoretic titration (ET) of moving neutralization boundary (NB) and EDTA photocatalysis. In the chip sensor, EDTA, H2O2, and leucomalachite green (LMG) were added in the anode well. Under UV light, EDTA photocatalyzes H2O2 and colorless LMG as H2O and color malachite green (MG) with one positive charge. When applying an electric field, the MG in the anode well migrated into the channel and was neutralized with the base in the channel, resulting in colorless MG-OH and NB. If the melamine-content dairy sample was added into the EDTA-H2O2-LMG system, H2O2 reacts with melamine, leading to the decrease of MG. Thus, the higher the melamine content in dairy products, the shorter the distance of NB migration under the given time, implying a distance-based sensor of melamine. A series of experiments manifested the validity of ET-NB sensor for detection of melamine. Moreover, the results revealed the numerous merits of ETNB sensor, such as good selectivity, high sensitivity (LOD down to 0.20 μM for milk and 0.10 μM for infant formula vs the FDA safety limits of 20 μM for milk and 8.0 μM for infant formula), good repeatability and recoveries (87-108% for milk, 90-107% for formula). Particularly, the cell phone-like sensor was portable, simple (no any pretreatment), rapid (within 15 min), as well as low cost, to evaluate the quality of dairy products. The developed sensor has great potential in on-spot detection of melamine in dairy products as well as other analytes, at which we are testing in our lab. [ABSTRACT FROM AUTHOR]

Microfluidics have been used to create "body-on-chip" systems to mimic in vivo cellular interactions with a high level of control. Most such systems rely on optical observation of cells as a readout. In this work we integrated a cell-cell interaction chip with online microchip electrophoresis immunoassay to monitor the effects of the interaction on protein secretion dynamics. The system was used to investigate the effects of adipocytes on insulin secretion. Chips were loaded with 190 000 3T3-L1 adipocytes and a single islet of Langerhans in separate chambers. The chambers were perfused at 300-600 nL/min so that adipocyte secretions flowed over the islets for 3 h. Adipocytes produced 80 µM of nonesterified fatty acids (NEFAs), a factor known to impact insulin secretion, at the islets. After perfusion, islets were challenged with a step change in glucose from 3 to 11 mM while monitoring insulin secretion at 8 s intervals by online immunoassay. Adipocyte treatment augmented insulin secretion by 6-fold compared to controls. The effect was far greater than comparable concentrations of NEFA applied to the islets demonstrating that adipocytes release multiple factors that can strongly potentiate insulin secretion. The experiments reveal that integration of chemical analysis with cell-cell interaction can provide valuable insights into cellular functions. [ABSTRACT FROM AUTHOR]

A novel capillary electrophoresis-integrated immobilized enzyme reactor (CE-integrated IMER) is developed using single-step in situ acetylcholinesterase (AChE)-mediated alginate hydrogelation and enzyme encapsulation. Alginate hydrogelation with "egg-box" structure is triggered inside a capillary with releasing of Ca2+ by changing the pH of the sol solution, which is accomplished in situ by AChE-catalyzed hydrolysis reaction of acetylthiocholine to produce acetic acid. AChE and any other enzyme initially contained in the sol solution [e.g., xanthine oxidase (XO)] are efficiently encapsulated as the hydrogel network grows, forming CE-integrated IMERs without any additional manipulation process. The proposed method facilitates the analysis of different kinds of enzymes using the same IMER depending on the substrate injected for CE analysis. Approximately 68% of the original enzyme in the sol mixture can be encapsulated, indicating high loading capacity for the CE-integrated IMERs. The IMERs exhibit excellent intraday and interday stability and batch-to-batch reproducibility, and these characteristics imply the reliability of the proposed IMERs for accurate online enzyme assays. Enzymatic activities and inhibition of immobilized AChE and XO are analyzed, and the results are compared with those using free enzymes. The feasibility of the proposed method for potential application in real sample analysis is demonstrated by the successful application of the IMERs in detecting organophosphorus pesticides in apple juice samples using AChE-catalyzed reactions. The proposed method is a simple, efficient, and universal approach for online CE assays with immobilized enzymes, which can be widely applied in bioanalysis. [ABSTRACT FROM AUTHOR]

Mass spectrometers are sensitive tools used to identify and quantify both small and large analytes using the mass-to-charge ratios (m/z) of ions generated by electrospray ionization (ESI) or other methods. Ionization typically generates protonated or deprotonated forms of the analytes or adducts with adventitious metal ions derived from the spray solvent. The formation of a variety of ionized forms of the analyte as well as the presence of cluster ions complicates the data and can have deleterious effects on the performance of the mass spectrometer, especially under high salt or buffer conditions. To address this, a method involving a dual-electrode nanoelectrospray source has been implemented to rapidly and temporarily desalt the spray solution of interfering cationic and anionic species using electrophoretic transport from the spray tip. Peptides, proteins, and pharmaceutical drugs all showed improved results after the desalting process as measured by the quality of the mass spectra and the limits of detection achieved. Importantly ordinary phosphate buffers could be used to record protein mass spectra by nano-ESI. [ABSTRACT FROM AUTHOR]

We describe a technology to perform sizing and concentration analysis of double stranded DNA with a sensitivity of 10 fg/μL in an operating time of 20 min. The technology is operated automatically on a commercial capillary electrophoresis instrument using electro-hydrodynamic actuation. It relies on a new capillary device that achieves online concentration of DNA at the junction between two capillaries of different diameters, thanks to viscoelastic lift forces. Using a set of DNA ladders in the range of 100-1500 bp, we report a sizing accuracy and precision better than 3% and a concentration quantification precision of ~20%. When the technology is applied to the analysis of clinical samples of circulating cell-free DNA (cfDNA), the measured cfDNA concentrations are in good correlation with those measured by digital PCR. Furthermore, the cfDNA size profiles indicate that the fraction of low molecular weight cfDNA in the range of 75-240 bp is a candidate biomarker to discriminate between healthy subjects and cancer patients. We conclude that our technology is efficient in analyzing highly diluted DNA samples and suggest that it will be helpful in translational and clinical research involving cfDNA. [ABSTRACT FROM AUTHOR]

Lysine methylation in protein is one important epigenetic mechanism that regulates diverse biological processes but is challenging to study due to the large variability in methylation levels and sites. Here, we show that supramolecular hosts such as calixarenes and cucurbiturils can be applied in the background electrolyte (BGE) of capillary electrophoresis (CE) for highly effective separation of post-translationally methylated histone peptides. The molecular recognition event causes a shift in the electrophoretic mobility of the peptide, allowing affinity measurement for binding between the synthetic receptor and various methylated lysine species. Successful separation of the H3 peptides carrying different methylation levels at the K9 position can be achieved using CX4 and CX6 as the BGE additives in CE, enabling monitoring of the activity of the histone lysine demethylase JMJD2E. This reveals the power of combining high resolution CE with synthetic hosts for study of protein methylation, and the method should be capable of analyzing complex biological samples for better understanding of the functions of histone methylation. [ABSTRACT FROM AUTHOR]

Recent advances have shown increasing designs of nucleic acid organizations via controlling the thermodynamics and kinetics of oligonucleotides. Nevertheless, deeper understanding and further applications of these DNA nano-technologies are majorly hampered by the lack of effective analytical methodologies that are competent enough to investigate them. To deliver a potential solution, here we developed an innovative exploration that employed the emerging nanopore technique to characterize DNA organization at the single-molecule level and in completely homogeneous condition without covalent modification. With the help of counting and profiling the translocation-induced current drop of a DNA assembly structure passing through a conical glass nanopore (CGN), we have directly verified the formation of the individual double-helix concatemer generated from our model, hybridization chain reaction (HCR). Due to the ultrasensitivity of the nanopore technology, those concatemers that were difficult to observe on a conventional electrophoresis image were brought to light. The translocation duration time also provided the approximate length and folding information for the concatemers. These advantages were proven also applicable to structures with more sophisticated folding behaviors. Eventually, when coupling with an upstream reaction, CGN was further turned to a universal detector that was capable of even detecting other nucleic acid organization behaviors as well as targets that were unable to generate huge products. All of these results are expected to promote deeper study and applications of the nanopore technique in the field of nucleic acid nanotechnology. [ABSTRACT FROM AUTHOR]

For miniaturization and integration of chemical synthesis and analytics on small length scales, the development of complex lab-on-chip (LOC) systems is in the focus of many current research projects. While application specific synthesis and analytic modules and LOC devices are widely described, the combination and integration of different modules is intensively investigated. Problems for in-line processes such as solvent incompatibilities, e.g., for a multistep synthesis or the combination of an organic drug synthesis with a cellbased biological activity testing system, require a solvent exchange between serialized modules. Here, we present a continuously operating microfluidic solvent exchanger based on the principle of free-flow electrophoresis for miscible organic/ aqueous fluids. We highlight a proof-of-principle and describe the working principle for the model compound fluorescein, where the organic solvent DMSO is exchanged against an aqueous buffer. The DMSO removal performance could be significantly increased to 95% by optimization of the microfluidic layout. Moreover, the optimization of the inlet flow ratio resulted in a minimized dilution factor of 5, and we were able to demonstrate that a reduction of the supporting instrumentation is possible without a significant decrease of the DMSO removal performance. Finally, the compatibility of the developed solvent exchanger for cell based downstream applications was proven. The impedimetric monitoring of HEK293A cells in a continuously operating microfluidic setup revealed no adverse effects of the residual DMSO after the solvent replacement. Our solvent exchanger device demonstrates the power of micro-free-flow electrophoresis not only as a powerful technique for separation and purification of compound mixtures but also for solvent replacement. [ABSTRACT FROM AUTHOR]

While protein electrophoresis conducted in capillaries and microchannels offers high-resolution separations, such formats can be cumbersome to parallelize for single-cell analysis. One approach for realizing large numbers of concurrent separations is open microfluidics (i.e., no microchannels). In an open microfluidic device adapted for single-cell electrophoresis, we perform 100s to 1000s of simultaneous separations of endogenous proteins. The microscope slide-sized device contains cells isolated in microwells located in a ~40 µm polyacrylamide gel. The gel supports protein electrophoresis after concurrent in situ chemical lysis of each isolated cell. During electrophoresis, Joule (or resistive) heating degrades separation performance. Joule heating effects are expected to be acute in open microfluidic devices, where a single, high-conductivity buffer expedites the transition from cell lysis to protein electrophoresis. Here, we test three key assertions. First, Joule heating substantially impacts analytical sensitivity due to diffusive losses of protein out of the open microfluidic electrophoretic (EP) cytometry device. Second, increased analyte diffusivity due to autothermal runaway Joule heating is a dominant mechanism that reduces separation resolution in EP cytometry. Finally, buffer exchange reduces diffusive losses and band broadening, even when handling single-cell lysate protein concentrations in an open device. We develop numerical simulations of Joule heating-enhanced diffusion during electrophoresis and observe ~50% protein loss out of the gel, which is reduced using the buffer exchange. Informed by analytical model predictions of separation resolution (with Joule heating), we empirically demonstrate nearly fully resolved separations of proteins with molecular mass differences of just 4 kDa or 12% (GAPDH, 36 kDa; PS6, 32 kDa) in each of 129 single cells. The attained separation performance with buffer exchange is relevant to detection of currently unmeasurable protein isoforms responsible for cancer progression. [ABSTRACT FROM AUTHOR]

Combining the electrophoresis and conventional Coulter methods, we previously proposed the electrophoretic Coulter method (ECM), enabling simultaneous analysis of the size, number, and zeta potential of individual specimens. We validated the ECM experimentally using standard polystyrene particles and red blood cells (RBCs) from sheep; the latter was the first ECM application to biological particles in biotechnology research. However, specimens are prevented from passing through the ECM module aperture, which prevents accurate determination of the zeta potential of each specimen. This problem is caused by electro-osmotic flow (EOF) due to the high zeta potential at the ECM microchannel surfaces. To significantly improve ECM feasibility for biomedicine, we here propose a method to estimate the zeta potential at the ECM microchannel surfaces separate from the zeta potential of each specimen, by investigating the electric-field dependence of the specimen’s experimental electrophoretic velocity. We minimize the zeta potential at the microchannel surfaces by applying an organic-molecule coating, and we suppress the surface zeta potential and its resultant EOF by optimizing the microchannel geometry. We demonstrate that the ECM can distinguish between different biological cells using the differences in zeta potential values and/or sizes. We also demonstrate that the ECM can determine the number of biomolecules attached to individual cells and identify whether the average cell state in an analyzed vial is alive or dead. The high-performance ECM can detect cellular morphology alterations, improve immunologic test sensitivity, and identify cell states (living, dying, and dead); this information is clinically useful for early diagnosis and its follow-up. [ABSTRACT FROM AUTHOR]

Reversed phase liquid chromatography with mass spectrometry (RPLC-MS) peptide mapping is routinely used for interrogating molecular and structural attributes such as amino acid composition, sequence variants, and post-translational modifications (PTMs) in antibody-derived therapeutics. RPLC has some limitations that often impact the analysis of certain peptides including large hydrophobic peptides, hydrophilic di-/tripeptides and glycopeptides. Capillary zone electrophoresis with mass spectrometry (CZE-MS) has great potential for peptide mapping due to high efficiency and outstanding sensitivity. In this report we demonstrate the utility of CZE-MS as an orthogonal and complementary technique to RPLC-MS for peptide mapping analyses of antibody--drug conjugates (ADCs) and their parent antibodies. This work is based on high-resolution CZE-MS separation recently developed in our group, where a mixed aqueous--organic solvent system containing N,N-dimethylacetamide (DMA) or N,N-dimethylformamide (DMF) was used to improve the separation selectivity. The results described here show several advantages of CZE-MS for the analysis of small hydrophilic di-/tripeptides, large hydrophobic peptides, glycopeptides, and hydrophobic drug-linked peptides. [ABSTRACT FROM AUTHOR]

New technologies are urgently required for reliable drug screening given a worldwide epidemic of prescription drug abuse and its devastating socioeconomic impacts on public health. Primary screening of drugs of abuse (DoA) currently relies on immunoassays that are prone to bias and are not applicable to detect an alarming array of psychoactive stimulants, tranquilizers, and synthetic opioids. These limitations impact patient safety when monitoring for medication compliance, drug substitution, or misuse/ abuse and require follow-up confirmatory testing by more specific yet lower throughput instrumental methods. Herein, we introduce a high throughput platform for nontargeted screening of a broad spectrum of DoA and their metabolites based on multisegment injection--capillary electrophoresis--mass spectrometry (MSI--CE--MS). We demonstrate that MSI--CE--MS enables serial injections of 10 samples within a single run (<3 min/ sample) where multiplexed electrophoretic separations are coupled to high resolution MS with full-scan data acquisition. Unambiguous drug identification was achieved by four or more independent parameters, including comigration with a deuterated internal standard or in silico prediction of electromigration behavior together with accurate mass, most likely molecular formula, as well as MS/MS as required for confirmation testing. Acceptable precision was demonstrated for over 50 DoA at 3 concentration levels over 4 days (median coefficient of variance = 13%, n = 117) with minimal ion suppression, isobaric interferences, and sample carry-over (<1%). This approach offers a rapid yet accurate method for simultaneous detection and identification of DoA at their recommended screening cutoff levels in human urine while allowing for systematic surveillance, specimen verification, and retrospective testing of designer drugs that elude conventional drug tests. [ABSTRACT FROM AUTHOR]

Peptide mapping with mass spectrometry (MS) detection is a powerful technique routinely used for interrogating physicochemical properties of proteins. Peptide mapping benefits from an efficient front-end separation to increase selectivity and reduce complexity prior to MS detection. The most commonly used method for peptide mapping is based on reverse phase liquid chromatography with mass spectrometry. Capillary zone electrophoresis with mass spectrometry (CZE-MS) is an orthogonal technique with growing attention for peptide mapping of biotherapeutic proteins due to its high efficiency and sensitivity. However, that growth has been slow due to poorer peptide resolution and method robustness compared to RPLC. Here we present results from optimization of CZE-MS peptide mapping separation using mixed aqueous--aprotic dipolar solvent (N,N-dimethylacetamide (DMA) and N,N-dimethylformamide (DMF), as the background electrolyte (BGE) to improve the separation performance. Addition of DMA or DMF to the BGE impacts separation selectivity through differential change in pKa of the peptides. The CZE-MS peptide mapping method with the modified BGE produced significant improvement in resolution over the conventional CZE-MS methods. The method was evaluated with both sheathless and sheathflow CE-MS ion sources. [ABSTRACT FROM AUTHOR]