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Single-cell resolution analysis of complex biological tissues is fundamental to capture cell-state heterogeneity and distinct cellular signaling patterns that remain obscured with population-based techniques. The limited amount of material encapsulated in a single cell however, raises significant technical challenges to molecular profiling. Due to extensive optimization efforts, mass spectrometry-based single-cell proteomics (scp-MS) has emerged as a powerful tool to facilitate proteome profiling from ultra-low amounts of input, although further development is needed to realize its full potential. To this end, we carried out comprehensive analysis of orbitrap-based data independent acquisition (DIA) for limited material proteomics. Notably, we found a fundamental difference between optimal DIA methods for high- and low-load samples. We further improved our low-input DIA method by relying on high-resolution MS1 quantification, thus more efficiently utilizing available mass analyzer time. With our ultra-low input tailored DIA method, we were able to accommodate long injection times and high resolution, while keeping the scan cycle time low enough to ensure robust quantification. Finally, we establish a complete experimental scp-MS workflow, combining DIA with accessible single-cell sample preparation and the latest chromatographic and computational advances and showcase our developments by profiling real single cells.

In the light of the ongoing single-cell revolution, scientific disciplines are combining forces to retrieve as much relevant data as possible from trace amounts of biological material. For single-cell proteomics, this implies optimizing the entire workflow from initial cell isolation down to sample preparation, liquid chromatography (LC) separation, mass spectrometer (MS) data acquisition, and data analysis. To demonstrate the potential for single-cell and limited sample proteomics, we report on a series of benchmarking experiments where we combine LC separation on a new generation of micropillar array columns with state-of-the-art Orbitrap MS/MS detection and high-field asymmetric waveform ion mobility spectrometry (FAIMS). This dedicated limited sample column has a reduced cross section and micropillar dimensions that have been further downscaled (interpillar distance and pillar diameter by a factor of 2), resulting in improved chromatography at reduced void times. A dilution series of a HeLa tryptic digest (5-0.05 ng/μL) was used to explore the sensitivity that can be achieved. Comparative processing of the MS/MS data with Sequest HT, MS Amanda, Mascot, and SpectroMine pointed out the benefits of using Sequest HT together with INFERYS when analyzing sample amounts below 1 ng. Here, 2855 protein groups were identified from just 1 ng of HeLa tryptic digest hereby increasing detection sensitivity as compared to a previous contribution by a factor well above 10. By successfully identifying 1486 protein groups from as little as 250 pg of HeLa tryptic digest, we demonstrate outstanding sensitivity with great promise for use in limited sample proteomics workflows.

In the light of the ongoing single-cell revolution, scientific disciplines are combining forces to retrieve as much relevant data as possible from trace amounts of biological material. For single cell proteomics, this implies optimizing the entire workflow from initial cell isolation down to sample preparation, liquid chromatography (LC) separation, mass spectrometer (MS) data acquisition and data analysis. To demonstrate the potential for single cell and limited sample proteomics, we report on a series of benchmarking experiments where we combine LC separation on a new generation of micro pillar array columns with state-of-the-art Orbitrap MS/MS detection and High-Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS). This dedicated limited sample column has a reduced cross section and micro pillar dimensions that have been further downscaled (inter pillar distance and pillar diameter by a factor of 2), resulting in improved chromatography at reduced void times. A dilution series of a HeLa tryptic digest (5-0.05 ng/μL) was used to explore the sensitivity that can be achieved. Comparative processing of the MS/MS data with Sequest HT, MS Amanda, Mascot and SpectroMine pointed out the benefits of using Sequest HT together with INFERYS when analyzing sample amounts below 1 ng. 2855 protein groups were identified from just 1 ng of HeLa tryptic digest hereby increasing detection sensitivity as compared to a previous contribution by a factor well above 10. By successfully identifying 1486 protein groups from as little as 250 pg of HeLa tryptic digest, we demonstrate outstanding sensitivity with great promise for use in limited sample proteomics workflows.

As an alternative to the conventional packed bed nano LC columns that are frequently used in bottom-up proteomics research, a breakthrough in column manufacturing recently led to the introduction of silicon-based micromachined nano LC chip columns known as micro pillar array columns (μPAC™). These columns have an inherent high permeability and an unprecedented low ‘on-column’ dispersion due to the perfect order of the separation bed .Due to the perfect order, the peak dispersion originating from heterogeneous flow paths in the separation bed is eliminated (no A-term contributions). Sample components remain therefore much more concentrated during separation resulting in unprecedented separation performance. The freestanding nature of the pillars also leads to much lower backpressure allowing a high operational flow rate flexibility with exceptional peak capacities. In addition, the monolithic nature of the beds makes them extremely rugged and guarantees an unprecedented longevity of the columns. Moreover, it also guarantees run-to-run time repeatability that is about an order of magnitude better than packed bed columns, thus facilitating and accelerating protein identification. After the introduction of a 200 cm long column, which is ideally suited to perform comprehensive proteome research, a 50 cm long μPAC™ column is now available which can be used in a more routine research setting. With an internal volume of 3 μL, this column is perfectly suited to perform high throughput analyses with shorter gradient solvent times (30, 60 and 90 minute gradients) and it can be used over a wide range of flow rates, between 100 and 2000 nL/min. Recently performed experiments with 500 ng of HeLa cell digest indicate that an increase in protein identifications up to 50% and a gain of 70% in peptide identifications can be achieved when comparing the 50 cm μPAC™ column to the current state-of-the-art packed bed columns. In the recent months, we have also been developing a column that is deeper and wider to meet the requirements of capillary LC flow rate. New data on these columns will be presented at the conference.

Capitalizing on the massive increase in sample concentrations which are produced by extremely low elution volumes, nano-LC-ESI-MS/MS is currently one of the most sensitive analytical technologies for the comprehensive characterization of complex protein samples.However, despite tremendous technological improvements made in the production and the packing of monodisperse spherical particles for nano-flow HPLC, current state-of-the-art systems still suffer from limits in operation at the maximum potential of the technology.With the recent introduction of the µPAC system, which provides perfectly ordered micro-pillar array based chromatographic support materials, completely new chromatographic concepts for optimization towards the needs of ultra-sensitive proteomics become available.Here we report on a series of benchmarking experiments comparing the performance of a commercially available 50 cm micro-pillar array column to a widely used nano-flow HPLC column for the proteomics analysis of 10 ng tryptic HeLa cell digest.Comparative analysis of LC-MS/MS-data corroborated that micro-pillar array cartridges provide outstanding chromatographic performance, excellent retention time stability, increase sensitivity in the analysis of low-input proteomics samples, and thus repeatedly yielded almost twice as many unique peptide and unique protein group identifications when compared to conventional nano-flow HPLC columns.

We report on a comparative study of the basic separation kinetics of commercial packed bed columns and a micro-pillar array column (mu PAC) working in the 1-10 mu L/min flow rate range, i.e., operating in the area of capillary flow LC. This is done using a basic test mixture of 8 alkylphenones under both isocratic and gradient separation conditions. Care was taken the mu PAC and the packed bed columns have similar volumes (around 10 mu L) and hence also similar t 0-times when compared at the same flow rate. In addition, the isocratic mobile phase composition and gradient programs were selected such to have similar elution windows (in absolute times) for all 4 column types. It was found that the mu PAC produces significantly more theoretical plates (up to 3 times) in the 1-4 mu L/min range, while, the packed bed columns perform better at the higher flow rates because of the relatively large inter-pillar distance in the mu PAC. Under gradient conditions, the mu PAC produces a clearly higher peak capacity than any of the three packed bed columns over the entire range of investigated flow rates, albeit that this is also partly to be owed to the steeper gradient that needed to be used in the mu PAC in order to maintain a similar elution window on all columns. (c) 2020ElsevierB.V. Allrightsreserved.

In bottom-up proteomics, capillaries with internal diameters of 50 to 100 μm and packed with sub-2-μm C18-functionalized particles are routinely used in combination with a high resolution mass spectrometer. Typically, microgram amounts of samples are separated in 30 to 240 min long gradients on columns with lengths up to 75 cm. Unlike conventional LC columns, micro pillar array columns (μPAC™) are fabricated using micromachining technology initially developed for the semiconductor industry. Using this advanced technology, perfectly ordered chromatographic separation beds are fabricated by precisely etching micrometer scale channels out of a silicon wafer. As a result of the perfect order that is introduced into the separation bed, analyte dispersion in the column is reduced to an absolute minimum and column permeability is increased by one order of magnitude. This allows using very long columns (up to 200 cm) at a fraction of the pressure needed to operate packed bed alternatives. To validate the μPAC™ column performance for bottom-up proteomics, tryptic digests from HEK293T cell lysates were prepared over a wide concentration range (100 ng/μl to 3 μg/μl) and separated using a 200 cm μPAC™ column as well as a 40 cm long pulled tip packed bed capillary LC column. Using an Orbitrap Elite mass spectrometer, on average 25% more proteins could be identified when working with the μPAC™ column, and this over the entire concentration range. Peak width data showed that the rate at which the peak width increases with gradient time is much lower on the μPAC™ column. For a 10-hour long gradient, average peak widths below 0.5 min were observed, resulting in consistent identification of more than 5,000 proteins. Combining long solvent gradients and this new type of LC column, substantial improvements in plasma proteome coverage were obtained. Data are available via ProteomeXchange with identifiers PXD011547 and PXD011634.

Monoclonal antibodies are becoming a core aspect of the pharmaceutical industry. Together with a huge therapeutic potential, these molecules come with a structural complexity that drives state-of-the-art chromatography and mass spectrometry (MS) to its limits. This article discusses the use of micro-pillar array columns in combination with mass spectrometry for peptide mapping of monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs). Micro-pillar array columns are produced by a lithographic etching process creating a perfectly ordered separation bed on a silicon chip. As a result of the order existing in these columns, peak dispersion is minimized and highly efficient peptide maps are generated, providing enormous structural detail. Using examples from the author's laboratory, the performance of these columns is illustrated.

In the current paper, an axial rearrangement mixer is studied. The mixer aims to flatten out occasional composition fluctuations originating from the pump. In a first phase, dispersion in a single mixer channel is investigated using pulses introduced by a dedicated injection pulse generator. The mixing in the single channel is studied for different flow rates and a model is presented based on the experimental data. Next, the resulting signal for the whole mixer is studied.

We report on a series of hydrodynamic chromatography separations conducted in micropillar array columns with an interpillar distance spacing of, respectively, 1.00, 0.70, and 0.47 μm. The columns have been produced using state-of-the-art deep-UV lithography and deep reactive ion etching techniques. Despite the fact that the efficiency was smaller than theoretically possible (due to fabrication limitations and significant injection and detection band broadening), it was nevertheless possible to separate mixtures of fluorescein isothiocyanate (used as the t(0) -marker) and 20- and 40-nm polystyrene beads. With the smallest interpillar distance, a resolution of R(s) = 0.5 between the 20- and 40-nm particles could be obtained in 90s over a column length of 4 cm. The selectivity obtained in the pillar array columns was found to be very similar to that observed in packed-bed columns. By detecting the fluorescent signals in a 90-μm-deep detection groove at the end of the column, the signal-to-noise ratio could be enhanced up to 150 times.

We investigated the achievable separation performance of a 9-cm-long and 1-mm-wide pillar array channel (volume = 0.6 μL) containing 5 μm diameter Si pillars (spacing 2.5 μm) cladded with a mesoporous silica layer with a thickness of 300 nm, when this channel is directly interfaced to a capillary LC instrument. The chip has a small footprint of only 4 cm × 4 mm and the channel consists of three lanes that are each 3 cm long and that are interconnected using low dispersion turns consisting of a narrow U-turn (10 μm), proceded and preceded by a diverging flow distributor. Measuring the band broadening within a single lane and comparing it to the total channel band broadening, the additional band broadening of the turns can be estimated to be of the order of 0.5 μm around the minimum of the van Deemter curve, and around some 1 μm (nonretained species) and 2 μm (retained species) in the C-term dominated regime. The overall performance (chip + instrument) was evaluated by conducting gradient elution separations of digests of cytochrome c and bovine serum albumin. Peak capacities up to 150 could be demonstrated, nearly completely independent of the flow rate.

We report on the practical limitations of the current state-of-the-art in micro-fabrication technology to produce the small pillar sizes that are needed to obtain high efficiency pillar array columns. For this purpose, nine channels with a different pillar diameter, ranging from 5 to 0.5 μm were fabricated using state-of the-art deep-UV lithography and deep reactive ion etching (DRIE) etching technology. The obtained results strongly deviated from the theoretically expected trend, wherein the minimal plate height ( H min ) would reduce linearly with the pillar diameter. The minimal plate height decreases from 1.7 to 1.2 μm when going from 4.80 to 3.81 μm diameter pillars, but as the dimensions are further reduced, the minimal plate heights rise again to values around 2 μm. The smallest pillar diameter even produced the worst minimal plate height (4 μm). An in-depth scanning electron microscopy (SEM) inspection of the different channels clearly reveals that these findings can be attributed to the micro-fabrication limitations that are inevitably encountered when exploring the limits of deep-UV lithography and DRIE etching processes. When the target dimensions of the design approach the etching resolution limits, the band broadening increases in a strongly non-linear way with the decreased pillar dimensions. This highly non-linear relationship can be understood from first principles: when the machining error is of the order of 100–200 nm and when the target design size for the inter-pillar distance is of the order of 250 nm, this inevitably leads to pores that will range in size between 50 and 450 nm that we want to highlight with our paper highly non-linear relationship. This highly non-linear relationship can be understood from first principles: when the machining error is of the order of 100–200 nm and when the target design size for the inter-pillar distance is of the order of 250 nm, this inevitably leads to pores that will range in size between 50 and 450 nm.

In the present paper, the feasibility to construct micron-sized silicon pillar channels to be used in HPLC is studied. For this, a channel with flow-through pores of 1 μm and with critical sidewall dimensions below 1 μm was constructed using advanced deep-UV lithographic equipment. Integrating a 3-nL injection system on the chip directly in front of the separation channel and using elongated distribution structures, a very controlled and high aspect ratio sample definition across the relatively wide separation channel was accomplished. The system was evaluated in isocratic ion-pair RP mode, allowing the separation of a mixture of two components with, respectively, 300 and 400 base pairs in 5 s only.

New experimental data of the viscous fingering (VF) process have been generated by studying the VF process in perfectly ordered pillar array columns instead of in the traditionally employed packed bed columns. A detailed quantitative analysis of the contribution of VF to the observed band broadening could be made by following the injected species bands using a fluorescence microscope equipped with a CCD-camera. For a viscosity contrast of 0.16 cP, a plate height increase of about 1 microm can be observed, while for a contrast of respectively 0.5 cP and 1 cP, additional plate height contributions of the order of 5-20 microm were observed. Citing these values is however futile without noting that they also depend extremely strongly on the injection volume of injected sample. It was found that, for a given viscosity contrast of 0.314 cP, the maximal plate height increase varied between 0.5 microm and 18 microm if the injection volume was varied between 3.0 nl and 32.7 nl. These values furthermore also strongly vary with the distance along the column axis.

Background & objectives: Chemotherapy with praziquantel remains the only control measure to Schistosoma mansoni infections to date. The neuropeptide hormone somatostatin gives relief from gastrointestinal disturbances, liverpathology, and reduces egg production in S. mansoni infected mice, suggesting an interaction of somatostatin with the parasite rather than with the host alone. Using antibodies directed to epitopes of the seven somatostatin transmembrane receptors (SSTRs), the presence of SSTRs (or proteins that contain these epitopes) was shown on both worm– and eggstages of S. mansoni. The present study was undertaken to investigate whether SSTRs on S. mansoni displayed homo/heterodimerisation properties as well as agonist induced down–regulation. Results: Somatostatin therapy was effective after two days of treatment with no further reduction in pathology after five days of therapy. Immunohistochemistry performed on parasite sections showed reactivity of the anti–SSTR antibodies to the tegument and internal parts of adult S. mansoni worms. SDS–PAGE–Western blotting identified protein bands of 70–100 and 200–250 kDa molecular weight. Upon carboxymethylation of the sulfhydryl groups of proteins in the worm lysate, a reduction in density of the protein band at 200–250 kDa and an increase in density of the protein band at 70–100 kDa were noted. This suggested that a substantial amount of the proteins detected on the blot are present as a homo/heterodimer. A protein microarray was used to investigate whether somatostatin therapy induced receptor down– or up–regulation on the adult worm of S. mansoni. Slides spotted with primary anti–SSTR antibody were exposed to lysates of worms collected from infected C3H mice that received none, two days or five days somatostatin treatment, followed by a secondary anti– SSTR antibody coupled to a fluorophore. Comparison of the different samples in terms of parasite dilution till when the fluorescence was detectable, and the fluorescence intensity, proved that the proteins detected in the parasite worm have been down–regulated after five days of somatostatin treatment. Conclusion: SSTR–like GPCRs are being expressed by adult S. mansoni worms and extended somatostatin treatment may cause down–regulation of these receptors, thus reducing the therapeutic capacities of this neuropeptide. However, the presence of SSTRs on S. mansoni has not yet been proven on a genetic basis. Cross–reactivity of anti–SSTR antibodies with other G–protein coupled receptors (GPCR) thus cannot yet be excluded.

An important bottleneck of pillar array columns designed for liquid chromatography is that small deviations of the target 'magical distance' at the sidewall region leads to detrimental sidewall effects, due to local differences of linear velocities at the sidewall region versus other locations in the pillar bed. In the present study, we demonstrate that a lateral elongation of the pillar significantly increases the tolerance for offsets of the magical distance. By shifting the sidewall distance 600 nm for 2 pillar aspect ratio (AR) designs (AR = 3 and 9), only minor sidewall effects on the measured plate heights could be observed for the AR=9 columns, while the plate height was roughly doubled when using the wrong versus the correct sidewall distance for the AR = 3 columns. Technologically, this constitutes a huge advantage because small deviations (order of 100 nm) between the set and the finally achieved value for the inter-pillar distance are very common using mid-UV lithography based etching processes. (C) 2014 Elsevier B.V. All rights reserved.

We report on the possibility to realize submicrometer plate heights using chromatographic pillar array columns filled with radially elongated diamond-shaped pillars, even when using a relatively large interpillar distance (2.5 mu m) and axial pillar width (5 mu m). It is demonstrated that the use of high aspect ratio radially elongated pillars which are 15 times wider in the radial than in the axial direction can lead to a fivefold reduction of the minimal plate height compared to beds filled with pillars with a similar interpillar distance but with an aspect ratio around unity (cylinders and diamonds). This increase in performance can be attributed to a decrease in longitudinal dispersion, reflected by a reduction of the B-term by a factor of about 25. Experiments were conducted at room temperature, as well as at elevated temperature (70 degrees C), where the B-term band broadening is known to be more critical. The main advantage of radially elongated pillar beds is that they enable a drastic reduction of the footprint of pillar array columns, allowing design of very long channels with a minimum of turns. Under retained conditions, a four-component laser dye mixture could be separated over a distance of only 1.5 mm.

We report on the possibility to realize submicrometer plate heights using chromatographic pillar array columns filled with radially elongated diamond-shaped pillars, even when using a relatively large interpillar distance (2.5 μm) and axial pillar width (5 μm). It is demonstrated that the use of high aspect ratio radially elongated pillars which are 15 times wider in the radial than in the axial direction can lead to a fivefold reduction of the minimal plate height compared to beds filled with pillars with a similar interpillar distance but with an aspect ratio around unity (cylinders and diamonds).This increase in performance can be attributed to a decrease in longitudinal dispersion, reflected by a reduction of the B-term by a factor of about 25. Experiments were conducted at room temperature, as well as at elevated temperature (70 °C), where the B-term band broadening is known to be more critical. The main advantage of radially elongated pillar beds is that they enable a drastic reduction of the footprint of pillar array columns, allowing design of very long channels with a minimum of turns. Under retained conditions, a four-component laser dye mixture could be separated over a distance of only 1.5 mm.

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We report on a series of hydrodynamic chromatography separations conducted in micropillar array columns with an interpillar distance spacing of, respectively, 1.00, 0.70, and 0.47 μm. The columns have been produced using state-of-the-art deep-UV lithography and deep reactive ion etching techniques. Despite the fact that the efficiency was smaller than theoretically possible (due to fabrication limitations and significant injection and detection band broadening), it was nevertheless possible to separate mixtures of fluorescein isothiocyanate (used as the t(0) -marker) and 20- and 40-nm polystyrene beads. With the smallest interpillar distance, a resolution of R(s) = 0.5 between the 20- and 40-nm particles could be obtained in 90s over a column length of 4 cm. The selectivity obtained in the pillar array columns was found to be very similar to that observed in packed-bed columns. By detecting the fluorescent signals in a 90-μm-deep detection groove at the end of the column, the signal-to-noise ratio could be enhanced up to 150 times.

A non-porous C8 coated silicon pillar channel design is presented that allows the achievement of a high pressure and high efficiency operation for HPLC separations. Performing on-chip injection and detection and hence omitting the related dispersive sources, more than one million theoretical plates were obtained in non-retained conditions. The performance potential was also demonstrated by connecting the column to a commercial capillary HPLC system and injecting and detecting (UVVis) sample plugs off-chip.

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In the present paper, the feasibility to construct micron-sized silicon pillar channels to be used in HPLC is studied. For this, a channel with flow-through pores of 1 μm and with critical sidewall dimensions below 1 μm was constructed using advanced deep-UV lithographic equipment. Integrating a 3-nL injection system on the chip directly in front of the separation channel and using elongated distribution structures, a very controlled and high aspect ratio sample definition across the relatively wide separation channel was accomplished. The system was evaluated in isocratic ion-pair RP mode, allowing the separation of a mixture of two components with, respectively, 300 and 400 base pairs in 5 s only.

We report on the possibility to perform HDC in micropillar array columns and the potential advantages of such a system. The HDC performance of a pillar array column with pillar diameter = 5 microm and an interpillar distance of 2.5 microm has been characterized using both a low MW tracer (FITC) and differently sized polystyrene bead samples (100, 200 and 500 nm). The reduced plate height curves that were obtained for the different investigated markers all overlapped very well, and attained a minimum value of about h(min)=0.3 (reduction based on the pillar diameter), corresponding to 1.6 microm in absolute value and giving good prospects for high efficiency separations. The obtained reduced retention time values were in fair agreement with that predicted by the Di Marzio and Guttman model for a flow between flat plates, using the minimal interpillar distance as characteristic interplate distance.

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Five different flow distributors have been compared as a function of the flow rate for their ability to distribute small sample volumes over the entire width of flat rectangular microfabricated pillar array columns. The investigated designs can be divided in two major categories: (1) bifurcating, radially non-interconnecting distributors and (2) radially interconnecting distributors consisting of diamond-shaped pillars, elongated in the direction perpendicular to the flow, providing a high ratio of radial permeability over axial permeability. The quality of the flow distribution was evaluated experimentally by injecting equal volumes of fluorescent tracer into each of the tested designs and calculating the obtained peak variances using the method of moments. Purely bifurcating distributors perform less well than the best possible radially interconnected distributors, because the former inevitably require the use of wide open channels (d10 microm), wherein a lot of band broadening can occur. By doubling the aspect ratio of the radially stretched pillars from 5 to 10, the measured peak variance drops to 1/8 of the original value. The best results were obtained with a distributor in which the flow is distributed by a bed of anisotropic pillars with an aspect ratio of 10, but our results indicate that a substantial improvement can still be made by increasing the aspect ratio and adding gradually diverging sidewalls to the inlet.

Chemotherapy with praziquantel remains the only control measure to Schistosoma mansoni infections to date. The neuropeptide hormone somatostatin gives relief from gastrointestinal disturbances, liverpathology, and reduces egg production in S. mansoni infected mice, suggesting an interaction of somatostatin with the parasite rather than with the host alone. Using antibodies directed to epitopes of the seven somatostatin transmembrane receptors (SSTRs), the presence of SSTRs (or proteins that contain these epitopes) was shown on both worm- and egg-stages of S. mansoni. The present study was undertaken to investigate whether SSTRs on S. mansoni displayed homo/heterodimerisation properties as well as agonist induced down-regulation.Somatostatin therapy was effective after two days of treatment with no further reduction in pathology after five days of therapy. Immunohistochemistry performed on parasite sections showed reactivity of the anti-SSTR antibodies to the tegument and internal parts of adult S. mansoni worms. SDS-PAGE-Western blotting identified protein bands of 70-100 and 200-250 kDa molecular weight. Upon carboxymethylation of the sulfhydryl groups of proteins in the worm lysate, a reduction in density of the protein band at 200-250 kDa and an increase in density of the protein band at 70-100 kDa were noted. This suggested that a substantial amount of the proteins detected on the blot are present as a homo/heterodimer. A protein microarray was used to investigate whether somatostatin therapy induced receptor down- or up-regulation on the adult worm of S. mansoni. Slides spotted with primary anti-SSTR antibody were exposed to lysates of worms collected from infected C3H mice that received none, two days or five days somatostatin treatment, followed by a secondary anti-SSTR antibody coupled to a fluorophore. Comparison of the different samples in terms of parasite dilution till when the fluorescence was detectable, and the fluorescence intensity, proved that the proteins detected in the parasite worm have been down-regulated after five days of somatostatin treatment.SSTR-like GPCRs are being expressed by adult S. mansoni worms and extended somatostatin treatment may cause down-regulation of these receptors, thus reducing the therapeutic capacities of this neuropeptide. However, the presence of SSTRs on S. mansoni has not yet been proven on a genetic basis. Cross-reactivity of anti-SSTR antibodies with other G-protein coupled receptors (GPCR) thus cannot yet be excluded.

An important bottleneck of pillar array columns designed for liquid chromatography is that small deviations of the target 'magical distance' at the sidewall region leads to detrimental sidewall effects, due to local differences of linear velocities at the sidewall region versus other locations in the pillar bed. In the present study, we demonstrate that a lateral elongation of the pillar significantly increases the tolerance for offsets of the magical distance. By shifting the sidewall distance 600 nm for 2 pillar aspect ratio (AR) designs (AR = 3 and 9), only minor sidewall effects on the measured plate heights could be observed for the AR=9 columns, while the plate height was roughly doubled when using the wrong versus the correct sidewall distance for the AR = 3 columns. Technologically, this constitutes a huge advantage because small deviations (order of 100 nm) between the set and the finally achieved value for the inter-pillar distance are very common using mid-UV lithography based etching processes. (C) 2014 Elsevier B.V. All rights reserved.

We report on the possibility to realize submicrometer plate heights using chromatographic pillar array columns filled with radially elongated diamond-shaped pillars, even when using a relatively large interpillar distance (2.5 mu m) and axial pillar width (5 mu m). It is demonstrated that the use of high aspect ratio radially elongated pillars which are 15 times wider in the radial than in the axial direction can lead to a fivefold reduction of the minimal plate height compared to beds filled with pillars with a similar interpillar distance but with an aspect ratio around unity (cylinders and diamonds). This increase in performance can be attributed to a decrease in longitudinal dispersion, reflected by a reduction of the B-term by a factor of about 25. Experiments were conducted at room temperature, as well as at elevated temperature (70 degrees C), where the B-term band broadening is known to be more critical. The main advantage of radially elongated pillar beds is that they enable a drastic reduction of the footprint of pillar array columns, allowing design of very long channels with a minimum of turns. Under retained conditions, a four-component laser dye mixture could be separated over a distance of only 1.5 mm.

Electrochemical anodization has been applied to grow porous shell layers of 300 nm (30 nm pores) in 5 μm diameter pillar array columns (PACs) with a spacing of 2.5 μm. Using turn structures preceded and followed by the flow distributor structures recently introduced by our group and filled with radially elongated pillars, columns with quasi unlimited channel lengths could be conceived. The uniformity of the porous PAC was assessed by determining local plate heights along the channel, which appeared to be constant. Minimal (absolute) plate heights (H) between 4 and 6 μm were obtained at optimal flow rates when imposing increasing retention factors. Upon measuring the surface area involved in chromatographic retention as an indicator of the available surface area, an increase in the surface area by a factor of about 30 compared to that of non-anodized pillars was found. On reconfiguring a commercial HPLC instrument to enable on-chip injections, 90% of the performance (expressed in theoretical plates) could be maintained for a 1 m column, while for a 25 cm column severe losses were still observed. As the corresponding pressure drop for optimal operation of retained components is on the order of 10 bar per m only, portable and cheaper HPLC devices with high efficiencies become realistically conceivable.

In the current paper, an axial rearrangement mixer is studied. The mixer aims to flatten out occasional composition fluctuations originating from the pump. In a first phase, dispersion in a single mixer channel is investigated using pulses introduced by a dedicated injection pulse generator. The mixing in the single channel is studied for different flow rates and a model is presented based on the experimental data. Next, the resulting signal for the whole mixer is studied.

The achievable separation performance of a 9 cm long pillar array channel containing 5 μm diameter silicon pillars (spacing 2.5 μm) cladded with a meso-porous silica layer with a thickness of 300 nm was investigated, interfacing the chip to a capillary LC instrument. The overall performance was evaluated by conducting gradient elution separations of digests of cytochrome c and bovine serum albumin. Peak capacities of up to 150 could be demonstrated.

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In the 21st century, numerous advances have been made in liquid chromatography (LC) column technology. The best known are columns packed with sub-2-µm porous particles or sub-3-µm superficially particles, and monolithic columns. Another very novel and original development is micro-pillar array columns (µPAC). µPACs are produced by a lithographic etching process to create a perfectly ordered separation bed on a silicon chip. Although the performance in terms of efficiency has been illustrated, the applicability for analysis of real complex samples has yet to be fully demonstrated. This article illustrates that state‑of‑the‑art µPAC columns coated with octadecyl are applicable for a challenging application such as lipidomics. The performance is illustrated with the analysis of human blood plasma lipids.

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Hydrodynamic chromatography of polystryrene microparticles in micropillar array columns