Complete resolution across the neodymium/samarium isotopic envelope with a liquid sampling-atmospheric pressure glow discharge - Orbitrap mass spectrometer.

RATIONALE: Nd and Sm isotope ratios play an important role in geological dating and as nuclear forensic signatures; however, the overlap of the respective 144, 148, 150 Nd/Sm isobars requires prior separations to be performed before analysis on typical MS platforms. The work presented here overcomes these isobaric interferences using ultrahigh-mass resolution to alleviate interference without prior chemical separations. METHODS: A liquid sampling-atmospheric pressure glow discharge ion source was coupled to a standard, QExactive Focus Orbitrap mass spectrometer, providing a mass resolution of ~80 k. A Spectroswiss FTMS booster X2 data acquisition package was used to collect extended transients, providing much higher mass resolution; ~230 k and ~600 k are employed here for Nd and Sm isotopes. RESULTS: While the standard Orbitrap resolution is far greater than typical "atomic" MS platforms, it was insufficient to alleviate all isobars. The use of a resolution of ~230 k resulted in baseline separation across the entire isotopic envelope for both Nd and Sm. Isotope ratios obtained from Nd:Sm mixtures using high-resolution were equivalent to those found for individual-element solutions, while isotope ratios obtained at a resolution of ~80 k (standard for the OEM data system) showed large deviations. CONCLUSIONS: Use of ultrahigh-resolution is an attractive alternative to extensive chemical separations to alleviate severe isobaric interferences. Sufficient mass resolution greatly reduces/eliminates the need for sample manipulations (separations) before analysis while reducing costs and total analysis times.

In-line coupling of capillary-channeled polymer fiber columns with optical absorbance and multi-angle light scattering detection for the isolation and characterization of exosomes.

Extracellular vesicles (EVs) have garnered much interest due to their fundamental role in intracellular communication and their potential utility in clinical diagnostics and as biotherapeutic vectors. Of particular relevance is the subset of EVs referred to as exosomes, ranging in size from 30 to 150 nm, which contain incredible amounts of information about their cell of origin, which can be used to track the progress of disease. As a complementary action, exosomes can be engineered with therapeutic cargo to selectively target diseases. At present, the lack of highly efficient methods of isolation/purification of exosomes from diverse biofluids, plants, and cell cultures is a major bottleneck in the fundamental biochemistry, clinical analysis, and therapeutic applications. Equally impactful, the lack of effective in-line means of detection/characterization of isolate populations, including concentration and sizing, is limiting in the applications. The method presented here couples hydrophobic interaction chromatography (HIC) performed on polyester capillary-channeled polymer (C-CP) fiber columns followed by in-line optical absorbance and multi-angle light scattering (MALS) detection for the isolation and characterization of EVs, in this case present in the supernatant of Chinese hamster ovary (CHO) cell cultures. Excellent correlation was observed between the determined particle concentrations for the two detection methods. C-CP fiber columns provide a low-cost platform (< $5 per column) for the isolation of exosomes in a 15-min workflow, with complementary absorbance and MALS detection providing very high-quality particle concentration and sizing information.

Quantitative Recoveries of Exosomes and Monoclonal Antibodies from Chinese Hamster Ovary Cell Cultures by Use of a Single, Integrated Two-Dimensional Liquid Chromatography Method.

Cultured cell lines are very commonly used for the mass production of therapeutic proteins, such as monoclonal antibodies (mAbs). In particular, Chinese hamster ovary (CHO) cell lines are widely employed due to their high tolerance to variations in experimental conditions and their ability to grow in suspension or serum free media. CHO cell lines are known for their ability to produce high titers of biotherapeutic products such as immunoglobulin G (IgG). An emergent alternative means of treating diseases, such as cancer, is the use of gene therapies, wherein genetic cargo is "packaged" in nanosized vesicular structures, referred to as "vectors". One particularly attractive vector option is extracellular vesicles (EVs), of which exosomes are of greatest interest. While exosomes can be harvested from virtually any human body fluid, bovine milk, or even plants, their production in cell cultures is an attractive commercial approach. In fact, the same CHO cell types employed for mAb production also produce exosomes as a natural byproduct. Here, we describe a single integrated 2D liquid chromatography (2DLC) method for the quantitative recovery of both exosomes and antibodies from a singular sample aliquot. At the heart of the method is the use of polyester capillary-channeled polymer (C-CP) fibers as the first dimension column, wherein exosomes/EVs are captured from the supernatant sample and subsequently determined by multiangle light scattering (MALS), while the mAbs are captured, eluted, and quantified using a protein A-modified C-CP fiber column in the second dimension, all in a 10 min workflow. These efforts demonstrate the versatility of the C-CP fiber phases with the capacity to harvest both forms of therapeutics from a single bioreactor, suggesting an appreciable potential impact in the field of biotherapeutics production.

Initial Characterization and Optimization of the Liquid Sampling-Atmospheric Pressure Glow Discharge Ionization Source Coupled to an Orbitrap Mass Spectrometer for the Determination of Plutonium.

Plutonium measurements are essential to the nuclear forensics and safeguards community. The liquid sampling-atmospheric pressure glow discharge (LS-APGD) microplasma ionization source coupled with an Orbitrap mass spectrometer is a proven platform for uranium isotope ratio determinations. This work expands the LS-APGD-Orbitrap platform capabilities by reporting the first-ever analysis of plutonium with the LS-APGD and the first-ever measurement of elemental plutonium with an Orbitrap mass spectrometer. This coupling has the potential to dramatically reduce the complex sample manipulations required for traditional analysis techniques employed for actinide isotope ratio determinations. As a first step toward the goal of simultaneous uranium and plutonium isotope ratio determinations, the initial characterization and optimization of the platform for the detection of plutonium are reported. Collision-induced dissociation modality settings were optimized to reduce water-related and other molecular clusters containing plutonium, maximizing 242Pu16O2+ responses. A design of experiments study was conducted to optimize the discharge conditions of the dual-electrode LS-APGD toward the responsivity of 242Pu16O2+. The measurement sensitivity was determined from a Pu response curve, yielding a limit of detection of 10 fg (absolute) of total analyte when data was collected and processed with a Spectroswiss FTMS Booster X2 data acquisition system. Additionally, plutonium and uranium were measured in a simultaneous acquisition, and each analyte remained unaffected by the other. It is believed that the LS-APGD-Orbitrap platform could be a valuable addition to the nuclear forensics' toolbox and, indeed, other scientific disciplines and regulatory communities in which rapid, high-resolution plutonium determinations are paramount.

Rapid isolation and quantification of extracellular vesicles from suspension-adapted human embryonic kidney cells using capillary-channeled polymer fiber spin-down tips.

Exosomes, a subset of extracellular vesicles (EVs, 30-200-nm diameter), serve as biomolecular snapshots of their cell of origin and vehicles for intercellular communication, playing roles in biological processes, including homeostasis maintenance and immune modulation. The large-scale processing of exosomes for use as therapeutic vectors has been proposed, but these applications are limited by impure, low-yield recoveries from cell culture milieu (CCM). Current isolation methods are also limited by tedious and laborious workflows, especially toward an isolation of EVs from CCM for therapeutic applications. Employed is a rapid (<10 min) EV isolation method on a capillary-channeled polymer fiber spin-down tip format. EVs are isolated from the CCM of suspension-adapted human embryonic kidney cells (HEK293), one of the candidate cell lines for commercial EV production. This batch solid-phase extraction technique allows 1012 EVs to be obtained from only 100-µl aliquots of milieu, processed using a benchtop centrifuge. The tip-isolated EVs were characterized using transmission electron microscopy, multi-angle light scattering, absorbance quantification, an enzyme-linked immunosorbent assay to tetraspanin marker proteins, and a protein purity assay. It is believed that the demonstrated approach has immediate relevance in research and analytical laboratories, with opportunities for production-level scale-up projected.

Comparison of the capillary-channeled polymer (C-CP) fiber spin-down tip approach to traditional methods for the isolation of extracellular vesicles from human urine.

Capillary-channeled polymer fiber (C-CP) solid-phase extraction tips have demonstrated the ability to produce clean and concentrated extracellular vesicle (EV) recoveries from human urine samples in the small EV size range (< 200 nm). An organic modifier-assisted hydrophobic interaction chromatography (HIC) approach is applied in the spin-tip method under non-denaturing conditions-preserving the structure and bioactivity of the recovered vesicles. The C-CP tip method can employ either acetonitrile or glycerol as an elution modifier. The EV recoveries from the C-CP tip method (using both of these solvents) were compared to those obtained using the ultracentrifugation (UC) and polymer precipitation (exoEasy and ExoQuick) EV isolation methods for the same human urine specimen. The biophysical and quantitative characteristics of the recovered EVs using the five isolation methods were assessed based on concentration, size distribution, shape, tetraspanin surface marker protein content, and purity. In comparison to the traditionally used UC method and commercially available polymeric precipitation-based isolation kits, the C-CP tip introduces significant benefits with efficient (< 15 min processing of 12 samples here) and low-cost (< $1 per tip) EV isolations, employing sample volumes (10 µL-1 mL) and concentration (up to 4 × 1012 EVs mL-1) scales relevant for fundamental and clinical analyses. Recoveries of the target vesicles versus matrix proteins were far superior for the tip method versus the other approaches.

Comparative analysis of trilobal capillary-channeled polymer fiber columns with superficially porous and monolithic phases toward reversed-phase protein separations.

A trilobal capillary-channeled polymer fiber stationary phase is evaluated for its performance for intact protein separations under reversed-phase high-performance liquid chromatography conditions. The separation quality, operational characteristics, and protein dynamic loading capacity on the fiber phases are compared to commercially-available superficially porous and monolithic columns. The trilobal or "y-shaped" polypropylene fiber phase was employed to separate a synthetic mixture of five proteins (having diverse chemistries and molecular weights). The separation quality was evaluated based on the resolution, peak heights/recoveries, peak widths, and peak areas. The present work illustrates the unique ability to operate at higher linear velocities (47.5 mm/s) while maintaining lower back pressures (∼4 MPa), faster separation times (<8 min), and faster gradient rates using the fiber columns while yielding comparable chromatographic performance to the commercial columns. The separations employing the commercial stationary phases operate at lower linear velocities (∼3.0 mm/s), higher back pressures (∼9 MPa), require longer separation times (10 min), and require slightly higher compositions of organic mobile phase to effect protein elution. Likewise, based on breakthrough loading analysis of lysozyme and bovine serum albumin, the trilobal, polypropylene C-CP fiber column stationary phases demonstrate 3-9X greater binding capacities on a bed volume basis versus the commercial columns.

Resolving Severe Elemental Isobaric Interferences with a Combined Atomic and Molecular Ionization Source-Orbitrap Mass Spectrometry Approach: The 87Sr and 87Rb Geochronology Pair.

Many fields of basic and applied sciences, including geochronology, astronomy, metabolism, etc., rely on the ability of mass spectrometry to obtain isotope ratio measurements having a high degree of certainty. The inability to resolve difficult isobaric interferences plagues certain measurements. A combined atomic and molecular (CAM) ionization source has been interfaced to a high-field Orbitrap mass spectrometer to alleviate severe atomic, isobaric interferences. This work examines the geochronologically significant 87Sr and 87Rb isotope pair. The mass difference between 87Sr and 87Rb is approximately 0.3 mDa, requiring a minimum resolving power (R = m/Δm) of ∼290,000, a value ∼30× higher than available with sector-field elemental mass spectrometers. Under ultrahigh-resolution conditions, Sr isotope ratio accuracy and precision were evaluated using NIST Sr SRM 987, yielding precision values of <0.1% relative standard deviation (RSD) for the major isotopes and a calculated LOD of 2 pg mL-1 (120 fg of Sr for a 60 µL injection). In addition to manipulating the signal transient length, the total number of ions in the electrostatic trap and the 87Sr/87Rb concentration ratio were found to influence resolution. Ultimately, the isotopes were baseline-resolved with a calculated mass resolution of >1.7M. At equal 87Sr and 87Rb intensities, 87Sr/86Sr was measured as 0.71294 (a relative error of only 0.37%) with a precision of 0.097% RSD, clearly reflecting the alleviation of the isobaric interference.

A novel method of high-purity extracellular vesicle enrichment from microliter-scale human serum for proteomic analysis.

We have developed a rapid, low-cost, and simple separation strategy to separate extracellular vesicles (EVs) from a small amount of serum (i.e.,<100 µL) with minimal contamination by serum proteins and lipoprotein particles to meet the high purity requirement for EV proteome analysis. EVs were separated by a novel polyester capillary channel polymer (PET C-CP) fiber phase/hydrophobic interaction chromatography (HIC) method which is rapid and can process small size samples. The collected EV fractions were subjected to a post-column cleanup protocol using a centrifugal filter to perform buffer exchange and eliminate potential coeluting non-EV proteins while minimizing EV sample loss. Downstream characterization demonstrated that our current strategy can separate EVs with the anticipated exosome-like particle size distribution and high yield (∼1 × 1011 EV particles per mL of serum) in approximately 15 min. Proteome profiling of the EVs reveals that a group of genuine EV components were identified that have significantly less high-abundance blood proteins and lipoprotein particle contamination in comparison to traditional separation methods. The use of this methodology appears to address the major challenges facing EV separation for proteomics analysis. In addition, the EV post-column cleanup protocol proposed in the current work has the potential to be combined with other separation methods, such as ultracentrifugation (UC), to further purify the separated EV samples.

Rapid isolation of extracellular vesicles from diverse biofluid matrices via capillary-channeled polymer fiber solid-phase extraction micropipette tips.

Extracellular vesicles (EVs) play essential roles in biological systems based on their ability to carry genetic and protein cargos, intercede in cellular communication and serve as vectors in intercellular transport. As such, EVs are species of increasing focus from the points of view of fundamental biochemistry, clinical diagnostics, and therapeutics delivery. Of particular interest are 30-200 nm EVs called exosomes, which have demonstrated high potential for use in diagnostic and targeted delivery applications. The ability to collect exosomes from patient biofluid samples would allow for comprehensive yet remote diagnoses to be performed. While several exosome isolation methods are in common use, they generally produce low recoveries, whose purities are compromised by concomitant inclusion of lipoproteins, host cell proteins, and protein aggregates. Those methods often work on lengthy timescales (multiple hours) and result in very low throughput. In this study, capillary-channeled polymer (C-CP) fiber micropipette tips were employed in a hydrophobic interaction chromatography (HIC) solid-phase extraction (SPE) workflow. Demonstrated is the isolation of exosomes from human urine, saliva, cervical mucus, serum, and goat milk matrices. This method allows for quick (<15 min) and low-cost (<$1 per tip) isolations at sample volume and time scales relevant for clinical applications. The tip isolation was evaluated using absorbance (scattering) detection, nanoparticle tracking analysis (NTA), and transmission electron microscopy (TEM). Exosome purity was assessed by Bradford assay, based on the removal of free proteins. An enzyme-linked immunosorbent assay (ELISA) to the CD81 tetraspanin protein was used to confirm the presence of the known exosomal-biomarker on the vesicles.

Rapid isolation of lentivirus particles from cell culture media via a hydrophobic interaction chromatography method on a polyester, capillary-channeled polymer fiber stationary phase.

Lentiviruses are increasingly used as gene delivery vehicles for vaccines and immunotherapies. However, the purification of clinical-grade lentivirus vectors for therapeutic use is still troublesome and limits preclinical and clinical experiments. Current purification methods such as ultracentrifugation and ultrafiltration are time consuming and do not remove all of the impurities such as cellular debris, membrane fragments, and denatured proteins from the lentiviruses. The same challenges exist in terms of their analytical characterization. Presented here is the novel demonstration of the chromatographic isolation of virus particles from culture media based on the hydrophobicity characteristics of the vesicles. A method was developed to isolate lentivirus from media using a hydrophobic interaction chromatography (HIC) method performed on a polyester, capillary-channeled polymer (PET C-CP) stationary phase and a standard liquid chromatography apparatus. The method is an extension of the approach developed in this laboratory for the isolation of extracellular vesicles (EVs). Quantitative polymerase chain reaction (qPCR) was used to verify and quantify lentiviruses in elution fractions. Load and elution mobile phase compositions were optimized to affect high efficiency and throughput. The process has been visualized via scanning electron microscopy (SEM) of the fiber surfaces following media injection, the elution of proteinaceous material, and the elution of lentiviruses. This effort has yielded a rapid (<10 min), low-cost (< $15 per column, providing multiple separations), and efficient method for the isolation/purification of lentivirus particles from cell culture media at the analytical scale.

Coupling of Laser Ablation and the Liquid Sampling-Atmospheric Pressure Glow Discharge Plasma for Simultaneous, Comprehensive Mapping: Elemental, Molecular, and Spatial Analysis.

The spatial distributions of elemental and molecular species are vital pieces of information for a broad number of applications such as material development and bio/environmental analysis. There is currently no single analytical method that can simultaneously acquire elemental, molecular, and spatial information from a single sample. This paper presents the coupling of an NWR213 laser ablation (LA) system to the liquid sampling-atmospheric pressure glow discharge (LS-APGD) microplasma for combined atomic and molecular (CAM) analysis. The work demonstrates a fundamental balance that must be considered between the extent of fragmentation of molecules and ionization of atoms for CAM analysis. Detailed studies showed that the interelectrode gap to be a critical parameter for controlling the ionization efficiency of atomic and molecular species. Utilizing Design-of-Experiment (DoE) procedures, the discharge current was also found to be a significant parameter to control. Elemental lead, caffeine, and simultaneous lead and caffeine analysis via LA-LS-APGD-MS was made possible through improved understanding of the influence of plasma parameters on the product mass spectra of laser-ablated particles. Finally, a chemical map of elemental lead and molecular caffeine, from lead nitrate and caffeine residues, was generated, demonstrating the comprehensive mapping capabilities of LA-LS-APGD-MS. The practical relevance of the capabilities is demonstrated by mapping glutamic acid from a cryosectioned chicken breast with a thallium spike deposited within the tissue. It is believed that the LA-LS-APGD-MS could be a valuable methodology for the simultaneous mapping of elemental and molecular species from a variety of samples.

Rapid Determination of Uranium Isotopic Abundance from Cotton Swipes: Direct Extraction via a Planar Surface Reader and Coupling to a Microplasma Ionization Source.

The collection of solid particulates and liquids from surfaces by the use of cloth swipes is fairly ubiquitous. In such methods, there is a continuous concern regarding the ability to locate and quantitatively sample the analyte species from the material. In this effort, we demonstrate the initial coupling of an Advion Plate Express plate reader to a liquid sampling-atmospheric pressure glow discharge (LS-APGD) microplasma ionization source with an Orbitrap mass spectrometer to perform uranium isotopic analyses of solution residues on cotton swipes. The Plate Express employs a sampling probe head to engage and seal against the swipe surface. Subsequentially, the analyte residues are desorbed and transported within a 2% HNO3 electrolyte flow to the ionization source. Quantitative recoveries were observed following a single 30 s extraction step, with the absolute mass sampled per extraction being ∼100 ng. While the intrasample variability in the analytical responses for triplicate sampling of the same swipe yield ∼30% RSD, this lack of precision is offset by the ability to determine isotope ratios for enriched uranium specimens with a precision of better than 10% RSD. Pooled, intersample precision (n = 9) was found to be <5%RSD across the various sample compositions. Finally, 235U/238U determinations (ranging from 0.053 to 1.806) were accurate with errors of <10%, absolute. The 234U- and 236U-inclusive ratios were determined with similar accuracy in enriched samples. While the driving force for the effort is in the realm of nuclear nonproliferation efforts, the ubiquitous use of cloth swipes across many application areas could benefit from this convenient approach, including the use of versatile, reduced-format mass spectrometer systems.

Application of polydopamine-coated nylon capillary-channeled polymer fibers as a stationary phase for mass spectrometric phosphopeptide analysis.

Capillary-channeled polymer (C-CP) fibers are demonstrated as a selective stationary phase for phosphopeptide analysis via LC-MS. Taking advantage of the oxidative self-polymerization of dopamine under alkaline conditions, a simple system involving a dilute aqueous solution of 0.2% w/v dopamine hydrochloride in 0.15% w/v TRIS buffer, pH 8.5 was utilized to coat polydopamine onto nylon 6 C-CP fibers. Confirmation of the polydopamine coating on the fibers (nylon-PDA) was made through attenuated total reflection-FTIR (ATR-FTIR) analysis. Imaging using SEM was also performed to examine the morphology and topography of the nylon-PDA. Subsequent loading of Fe3+ to the nylon-PDA matrix was confirmed by SEM/energy dispersive X-ray spectroscopy (SEM/EDX). The Fe3+ -bound nylon-PDA fibers packed in a microbore column format were tested in the off-line preconcentration of phosphopeptides from a 1:100 mixture of ß-casein/BSA digests for MALDI-TOF analysis. The packed column was also installed onto an HPLC system as a platform for the online sample clean-up and enrichment of phosphopeptides from a 1:1000 mixture of ß-casein/BSA protein digests that were determined by subsequent ESI-MS analysis.

Polypropylene capillary-channeled polymer fiber column as the second dimension in a comprehensive two-dimensional RP × RP analysis of a mixture of intact proteins.

Online, comprehensive two-dimensional liquid chromatography (2D-LC) has become an attractive option for the analysis of complex samples of relevance in various fields (e.g., environmental, food, biology, and polymer sciences). In general, the second-dimension (2D) separation plays a more crucial role than the first (1D) in the total performance of LC × LC systems. Speed and efficiency of the 2D separation are of primary importance. These challenges are amplified in the case of protein separations where mass transfer limitations effect elution kinetics. The research presented here leverages the developments of capillary-channeled polymer (C-CP) fiber stationary phases directed at high-throughput and rapid protein separations, evaluating them as 2D material in comprehensive reversed-phase × reversed-phase (RP × RP) 2D-LC versus conventional, packed-bed columns as 2D. The ability to operate fiber columns at high linear velocities (> 75 mm s-1 at < 1000 psi) without sacrifice in resolution is the first step towards potential 2D implementation, alleviating the need for ultrahigh-pressure pumping (i.e., UHPLC) in that stage. What must be realized are fast elution gradients and rapid re-equilibration to minimize cycling times (modulation periods). Different flow rates and gradient programs were evaluated. Rapid elution allows the use of shorter shift-gradient windows. At the optimized gradient recycling time on this instrument, 36 s, there is no significant degradation in peak capacity. Finally, a graphic comparison of the separation efficiency between seven commercial reversed-phase columns and the polypropylene C-CP fiber column is presented for a synthetic, eleven-protein mixture using the recommended operating conditions for each of the 2D columns. The kinetic advantages of the fiber columns are clearly demonstrated.

Solid-phase extraction of exosomes from diverse matrices via a polyester capillary-channeled polymer (C-CP) fiber stationary phase in a spin-down tip format.

Exosomes, a subset of the extracellular vesicle (EV) group of organelles, hold great potential for biomarker detection, therapeutics, disease diagnosis, and personalized medicine applications. The promise and potential of these applications are hindered by the lack of an efficient means of isolation, characterization, and quantitation. Current methods for exosome and EV isolation (including ultracentrifugation, microfiltration, and affinity-based techniques) result in impure recoveries with regard to remnant matrix species (e.g., proteins, genetic material) and are performed on clinically irrelevant time and volume scales. To address these issues, a polyethylene terephthalate (PET) capillary-channeled polymer (C-CP) fiber stationary phase is employed for the solid-phase extraction (SPE) of EVs from various matrices using a micropipette tip-based format. The hydrophobic interaction chromatography (HIC) processing and a spin-down workflow are carried out using a table-top centrifuge. Capture and subsequent elution of intact, biologically active exosomes are verified via electron microscopy and bioassays. The performance of this method was evaluated by capture and elution of exosome standards from buffer solution and three biologically relevant matrices: mock urine, reconstituted non-fat milk, and exosome-depleted fetal bovine serum (FBS). Recoveries were evaluated using UV-Vis absorbance spectrophotometry and ELISA assay. The dynamic binding capacity (50%) for the 1-cm-long (~ 5 µL bed volume) tips was determined using a commercial exosome product, yielding a value of ~ 7 × 1011 particles. The novel C-CP fiber spin-down tip approach holds promise for the isolation of exosomes and other EVs from various matrices with high throughput, low cost, and high efficiency. Graphical abstract.

Exosome isolation and purification via hydrophobic interaction chromatography using a polyester, capillary-channeled polymer fiber phase.

Extracellular vesicles, including microvesicles and exosomes, are lipidic membrane-derived vesicles that are secreted by most cell types. Exosomes, one class of these vesicles that are 30-100 nm in diameter, hold a great deal of promise in disease diagnostics, as they display the same protein biomarkers as their originating cell. For exosomes to become useful in disease diagnostics, and as burgeoning drug delivery platforms, they must be isolated efficiently and effectively without compromising their structure. Most current exosome isolation methods have practical problems including being too time-consuming and labor intensive, destructive to the exosomes, or too costly for use in clinical settings. To this end, this study examines the use of poly(ethylene terephthalate) (PET) capillary-channeled polymer (C-CP) fibers in a hydrophobic interaction chromatography (HIC) protocol to isolate exosomes from diverse matrices of practical concern. Initial results demonstrate the ability to isolate extracellular vesicles enriched in exosomes with comparable yields and size distributions on a much faster time scale when compared to traditional isolation methods. As a demonstration of the potential analytical utility of the approach, extracellular vesicle recoveries from cell culture milieu and a mock urine matrix are presented. The potential for scalable separations covering submilliliter spin-down columns to the preparative scale is anticipated.

Isolation and quantification of human urinary exosomes by hydrophobic interaction chromatography on a polyester capillary-channeled polymer fiber stationary phase.

Exosomes are vesicles secreted by cells having a size range from 30 to 150 nm and carrying genetic materials that are important for intercellular functions, including cancer progression. Mounting evidence shows that tumor cells secrete more exosomes than normal cells. Thus, it is important to be able to efficiently isolate and quantify exosomes for potential use in clinical diagnostics, as well as to develop a deeper understanding of their role in intercellular processes. Current methods for exosome isolation and quantification are time-consuming and expensive. Few of these methods are able to combine exosome isolation and quantification into a singular operation scheme. However, a new efficient, rapid, and low-cost isolation and quantification method for exosomes in human urine samples using polyester (PET) capillary-channeled polymer (C-CP) fibers in a hydrophobic interaction chromatography (HIC) protocol has been developed. The process has been verified via scanning electron microscopy (SEM) before and after the capture of exosomes on the fiber surfaces. Sample load and elution rates were optimized to affect high resolution and throughput. Isolated exosomes were quantified based on a UV absorbance response curve created using a commercial human urine-derived exosome standard with an exosome concentration of 7.32 × 1011 mL-1. The loading capacity of a 30-cm C-CP PET column was ~ 7 × 1011 exosomes. An inter-injection washing method with PBS was developed to improve the reproducibility with a 2.9% RSD achieved for 7 complete isolation cycles. Graphical abstract.

Investigation of hydrophobic substrates for solution residue analysis utilizing an ambient desorption liquid sampling-atmospheric pressure glow discharge microplasma.

A practical method for preparation of solution residue samples for analysis utilizing the ambient desorption liquid sampling-atmospheric pressure glow discharge optical emission spectroscopy (AD-LS-APGD-OES) microplasma is described. Initial efforts involving placement of solution aliquots in wells drilled into copper substrates, proved unsuccessful. A design-of-experiment (DOE) approach was carried out to determine influential factors during sample deposition including solution volume, solute concentration, number of droplets deposited, and the solution matrix. These various aspects are manifested in the mass of analyte deposited as well as the size/shape of the product residue. Statistical analysis demonstrated that only those initial attributes were significant factors towards the emission response of the analyte. Various approaches were investigated to better control the location/uniformity of the deposited sample. Three alternative substrates, a glass slide, a poly(tetrafluoro)ethylene (PTFE) sheet, and a polydimethylsiloxane (PDMS)-coated glass slide, were evaluated towards the microplasma analytical performance. Co-deposition with simple organic dyes provided an accurate means of determining the location of the analyte with only minor influence on emission responses. The PDMS-coated glass provided the best performance by virtue of its providing a uniform spatial distribution of the residue material. This uniformity yielded an improved limits of detection by approximately 22× for 20 µL and 4 x for 2 µL over the other two substrates. While they operate by fundamentally different processes, this choice of substrate is not restricted to the LS-APGD, but may also be applicable to other AD methods such as DESI, DART, or LIBS. Further developments will be directed towards a field-deployable ambient desorption OES source for quantitative analysis of microvolume solution residues of nuclear forensics importance.

Dynamic evaluation of a trilobal capillary-channeled polymer fiber shape for reversed phase protein separations and comparison to the eight-channeled form.

A new, trilobal-shaped capillary-channeled polymer fiber is under development to address the issues of poor A-term performance of the previous eight-channeled form. The trilobal geometry should provide better packing homogeneity due to the fewer potential orientations of the symmetric fiber geometry. Comparisons of separation efficiency and peak shape were made between the two fiber shapes through several dynamic parameters. Column hydrodynamics were investigated with two marker compounds, uracil and bovine serum albumin, with van Deemter plots of those two compounds revealing differences in the packing qualities between the different fiber shapes. Parametric fitting to the van Deemter, Knox, and Giddings equations provides insights into the column physical structures. Separation quality for both shapes was evaluated across differences in fiber packing density, gradient rate, and mobile phase linear velocity for the reversed phase separation of a four protein mixture, containing ribonuclease A, cytochrome c, lysozyme, and myoglobin. The results of this study lay the ground work for future efforts in the use of trilobal fibers for the separation of biomacromolecules.