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World Congress on Biosensors 2014

World Congress on Biosensors 2014
Biosensors 2014

Tuesday, 31 July 2012

Multi-Well Plates Ensure High Filtered Sample Integrity


The need for reliable methods to purify samples and improve assays has led Porvair Sciences to develop a range of proprietary multi-well filtration microplates optimised for applications including cell harvesting, DNA separations, binding studies, plasmid isolation, general filtration and sample clean-up.

Filtration plates are used in their simplest form to remove particulate matter from liquid, typically either the particulate matter or the filtrate is needed for further study.

Included within the Porvair range are filtration plates that can simultaneously filter 96 or 384 samples of up to 350µl volume providing great productivity benefits and high filtered sample integrity. For larger volume applications (up to 5 ml / well) a choice of 48-well filtration plates are available.  A broad range of filtration media including glass fibre, nylon, PVDF and polyethylene allows control of filtration plate flow rates and retention characteristics to best suit your application.  Manufactured to the standard ANSI/SBS footprint all Porvair Filtration plates are fully automation compatible.

Each well on Porvair filtration plates has an individual drainage spout ensuring 100% sample transfer and zero crossover contamination.  Manufactured from ultra pure grade polypropylene - all Porvair filtration plates minimise leachates when used with most common solvents ensuring the highest  filtered sample integrity.
Established in 1992, Porvair Sciences Ltd. expertise in microplate technology and manufacturing covers scientific fields including Life Sciences, Drug Discovery, Combinatorial Chemistry, Solid Phase Extraction, Protein Purification, High Throughput Screening, Proteomics and Genomics. Porvair Sciences Ltd. is a wholly owned subsidiary of Porvair plc.

For further information on filtration plates please contact Porvair Sciences on telephone +44-1372-824290, email int.sales@porvair-sciences.com or visit www.porvair-sciences.com/downloads.php?showFrom=6&nR=18 to download a filter plate application guide.


AirClean® Systems, a world leader in ductless fume hood technology, also offers AirMax™ polypropylene total exhaust fume hoods


The AirMax fume hood with Wet Fume Scrubber system is a complete solution for protecting personnel from dangerous chemical exposure while also protecting ductwork and the environment by scrubbing acids and other water soluble gases from fume hood exhaust.
AirMax hoods are constructed of thermoplastics (primarily polypropylene) with all contact surfaces utilizing seamless, thermally-welded joints. This eliminates the potential for corrosion commonly associated with metal fume hoods. The advanced rolled-entry airfoil design prevents reverse flow and eddy currents, while an extended rear baffle effectively removes any heavy gases or evaporation from a spill. Dual-wall design provides ample room for mounting fixtures and services.

The Wet Fume Scrubber system is direct-mounted to the fume hood exhaust, eliminating contaminated ductwork between the fume hood and scrubber. An observation window is provided for media inspection, and the scrubber holding tank with recirculating pump is housed in the included base cabinet.

Typical applications of the AirMax with Wet Fume Scrubber are trace metal analysis, hot acid etching, acid digestions and other high-volume, high-acidity processes.

An optional spray bar for perchloric acid use is available, as is an auto pH dosing system for the holding tank.

AirMax fume hoods are available in 36”, 48”, 60”, 72” and 96” widths and are made in the USA.

For more information on AirMax total exhaust fume hoods, visit http://www.aircleansystems.com/totalexhaust_flame.htm

Monday, 30 July 2012

Just Published: Analytica Chimica Acta


A new issue of this journal has just been published. To see abstracts of the papers it contains (with links through to the full papers) click here:
Selected papers from the latest issue:

A new application of scanning electrochemical microscopy for the label-free interrogation of antibody–antigen interactions: Part 2

30 July 2012, 09:18:36
Publication year: 2012
Source:Analytica Chimica Acta, Volume 741
Joanne L. Holmes, Frank Davis, Stuart D. Collyer, Séamus P.J. Higson
Within this paper we describe the use of scanning electrochemical microscopy (SECM) to fabricate a dotted array of biotinylated polyethyleneimine which was then used to immobilise first neutravidin and then a biotinylated antibody towards a relevant antigen of interest (PSA, NTx, ciprofloxacin). These antigens were selected both for their clinical relevance but also since they display a broad range of molecular weights, to determine whether the size of the antigen used effects the sensitivity of this approach. The SECM was then used to image the binding of both complementary and non-complementary antigens in a label-free assay. Imaging of the arrays before and following exposure to various concentrations of antigen in buffer showed clear evidence for specific binding of the complementary antigens to the antibody functionalised dots. Non-specific binding was also quantified by control experiments with other antigens. This demonstrated non-specific binding across the whole of the substrate, thereby confirming that specific binding does occur between the antibody and antigen of interest at the surface of the dots. The binding of ciprofloxacin was investigated both in simple buffer solution and in a more complex media, bovine milk.

Graphical abstract

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Graphical abstract Highlights

► Employed scanning electrochemical microscopy for label-free detection of antigen binding. ► Demonstrated selective and quantitative binding of three different antigens. ► Detected antigens with a wide range of molecular weights. ► Demonstrated detection of ciprofloxacin in bovine milk.

Sorbent materials for separation and preconcentration of gold in environmental and geological samples – A review

30 July 2012, 09:18:36
Publication year: 2012
Source:Analytica Chimica Acta, Volume 741
Krystyna Pyrzynska
Determination of gold in environmental and geological samples requires very often preconcentration and separation due to the high concentration of interfering matrix components and the low content of this metal. Solid phase extraction technique with different kind of solid sorbents offers for this purpose high enrichment factor, rapid phase separation and the ability of combination with different detection techniques. It can be easily implemented and controlled in flow systems, The recent developments in this area are presented and discussed.

Graphical abstract

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Graphical abstract Highlights

► Gold determination requires very often preconcentration and separation. ► Solid phase extraction technique offers for this purpose high enrichment factor, rapid phase separation and the ability of combination with different detection techniques. ► The recent developments in this area were presented and discussed.

Simultaneous determination of ascorbic acid, dopamine, uric acid and tryptophan on gold nanoparticles/overoxidized-polyimidazole composite modified glassy carbon electrode

30 July 2012, 09:18:36
Publication year: 2012
Source:Analytica Chimica Acta, Volume 741
Cun Wang, Ruo Yuan, Yaqin Chai, Shihong Chen, Fangxin Hu, Meihe Zhang
A novel electrode was developed through electrodepositing gold nanoparticles (GNPs) on overoxidized-polyimidazole (PImox) film modified glassy carbon electrode (GCE). The combination of GNPs and the PImox film endowed the GNPs/PImox/GCE with good biological compatibility, high selectivity and sensitivity and excellent electrochemical catalytic activities towards ascorbic acid (AA), dopamine (DA), uric acid (UA) and tryptophan (Trp). In the fourfold co-existence system, the peak separations between AA–DA, DA–UA and UA–Trp were large up to 186, 165 and 285mV, respectively. The calibration curves for AA, DA and UA were obtained in the range of 210.0–1010.0μM, 5.0–268.0μM and 6.0–486.0μM with detection limits (S/N=3) of 2.0μM, 0.08μM and 0.5μM, respectively. Two linear calibrations for Trp were obtained over ranges of 3.0–34.0μM and 84.0–464.0μM with detection limit (S/N=3) of 0.7μM. In addition, the modified electrode was applied to detect AA, DA, UA and Trp in samples using standard addition method with satisfactory results.

Graphical abstract

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Graphical abstract Highlights

Electropolymerization of Im on the GCE, the PIm modified electrode was denoted as PIm/GCE. Subsequently, the PIm/GCE was washed with doubly distilled water, and then transferred to 0.1M PBS (pH 4.0) for electrochemical oxidation at +1.8V for 250s. The obtained electrode was denoted as PImox/GCE (Fig. A). Then, the deposition of GNPs on PImox/GCE was carried out. The obtained electrode was described as GNPs/PImox/GCE (Fig. B).
► The electropolymerization of imidazole (Im) on GCE was first reported. ► The PIm film can be overoxidized to form the overoxidized polyimidazole (PImox). ► PImox allows dispersing of Au and generates additional electrocatalytic sites. ► The overlapping voltammetric response of AA, DA, UA and Trp is well-resolved.

A highly accurate method for determination of dissolved oxygen: Gravimetric Winkler method

30 July 2012, 09:18:36
Publication year: 2012
Source:Analytica Chimica Acta, Volume 741
Irja Helm, Lauri Jalukse, Ivo Leito
A high-accuracy Winkler titration method has been developed for determination of dissolved oxygen concentration. Careful analysis of uncertainty sources relevant to the Winkler method was carried out and the method was optimized for minimizing all uncertainty sources as far as practical. The most important improvements were: gravimetric measurement of all solutions, pre-titration to minimize the effect of iodine volatilization, accurate amperometric end point detection and careful accounting for dissolved oxygen in the reagents. As a result, the developed method is possibly the most accurate method of determination of dissolved oxygen available. Depending on measurement conditions and on the dissolved oxygen concentration the combined standard uncertainties of the method are in the range of 0.012–0.018mgdm−3 corresponding to the k =2 expanded uncertainty in the range of 0.023–0.035mgdm−3 (0.27–0.38%, relative). This development enables more accurate calibration of electrochemical and optical dissolved oxygen sensors for routine analysis than has been possible before.

Graphical abstract

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Graphical abstract Highlights

► Probably the most accurate method available for dissolved oxygen concentration measurement was developed. ► Careful analysis of uncertainty sources was carried out and the method was optimized for minimizing all uncertainty sources as far as practical. ► This development enables more accurate calibration of dissolved oxygen sensors for routine analysis than has been possible before.

Sensitive and robotic determination of bromate in sea water and drinking deep-sea water by headspace solid-phase micro extraction and gas chromatography–mass spectrometry

30 July 2012, 09:18:36
Publication year: 2012
Source:Analytica Chimica Acta, Volume 741
Hyun-Hee Lim, Ho-Sang Shin
A robotic method has been established for the determination of bromate in sea water and drinking deep-sea water. Bromate in water was converted into volatile derivative, which was measured with headspace solid-phase micro extraction and gas chromatography–mass spectrometry (HS-SPME GC–MS). Derivatization reagent and the HS-SPME parameters (selection of fibre, extraction/derivatization temperature, heating time and; the morality of HCl) were optimized and selected. Under the established conditions, the detection and the quantification limits were 0.016μgL−1 and 0.051μgL−1, respectively, and the intra- and inter-day relative standard deviation was less than 7% at concentrations of 1.0 and 10.0μgL−1. The calibration curve showed good linearity with r 2 =0.9998. The common ions Cl, NO3 , SO4 2−, HPO4 2−, H2PO4 , K+, Na+, NH4 +, Ca2+, Mg2+, Ba2+, Mn4+, Mn2+, Fe3+ and Fe2+ did not interfere even when present in 1000-fold excess over the active species. The method was successfully applied to the determination of bromate in sea water and drinking deep-sea water.

Graphical abstract

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Graphical abstract Highlights

► This study is the first analysis method with HS SPME GC–MS of bromate. ► Selection of reagent and fibre, reaction temperature/time and pH were optimized. ► The best reagent and fiber is 2,6-DMP and CAR-PDMS. ► LOD and LOQ in sea water were 0.016 and 0.051μgL−1 and RSD was less than 7.0%.

Membrane-based microchannel device for continuous quantitative extraction of dissolved free sulfide from water and from oil

30 July 2012, 09:18:36
Publication year: 2012
Source:Analytica Chimica Acta, Volume 741
Kei Toda, Yuki Ebisu, Kazutoshi Hirota, Shin-Ichi Ohira
Underground fluids are important natural sources of drinking water, geothermal energy, and oil-based fuels. To facilitate the surveying of such underground fluids, a novel microchannel extraction device was investigated for in-line continuous analysis and flow injection analysis of sulfide levels in water and in oil. Of the four designs investigated, the honeycomb-patterned microchannel extraction (HMCE) device was found to offer the most effective liquid–liquid extraction. In the HMCE device, a thin silicone membrane was sandwiched between two polydimethylsiloxane plates in which honeycomb-patterned microchannels had been fabricated. The identical patterns on the two plates were accurately aligned. The extracted sulfide was detected by quenching monitoring of fluorescein mercuric acetate (FMA). The sulfide extraction efficiencies from water and oil samples of the HMCE device and of three other designs (two annular and one rectangular channel) were examined theoretically and experimentally. The best performance was obtained with the HMCE device because of its thin sample layer (small diffusion distance) and large interface area. Quantitative extraction from both water and oil could be obtained using the HMCE device. The estimated limit of detection for continuous monitoring was 0.05μM, and sulfide concentrations in the range of 0.15–10μM could be determined when the acceptor was 5μM FMA alkaline solution. The method was applied to natural water analysis using flow injection mode, and the data agreed with those obtained using headspace gas chromatography-flame photometric detection. The analysis of hydrogen sulfide levels in prepared oil samples was also performed. The proposed device is expected to be used for real time survey of oil wells and groundwater wells.

Graphical abstract

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Graphical abstract Highlights

► We have developed a membrane-based microchannel extraction (HMCE) device. ► Extraction efficiency was examined theoretically and experimentally for this device. ► Quantitative extraction can be performed with the HMCE device while partial extraction with other conventional membrane-based devices. ► The HMCE device was applied for the measurement of free sulfide or hydrogen sulfide contained in water and oil samples. ► The presented device is expected to be used for survey of underground fluid such as groundwater and oil in future.

Silica nanoparticles pre-spotted onto target plate for laser desorption/ionization mass spectrometry analyses of peptides

30 July 2012, 09:18:36
Publication year: 2012
Source:Analytica Chimica Acta, Volume 741
Mathieu Dupré, Sonia Cantel, Jean-Olivier Durand, Jean Martinez, Christine Enjalbal
We report on the simple deposition of Stöber silica nanoparticles (SiO2 NPs) on conventional MALDI target plate for high throughput laser desorption/ionization mass spectrometry (LDI-MS) analyses of peptide mixtures with sensitivity in the femtomolar range. This low-cost easily prepared material allowed straightforward LDI experiments by deposition of the studied samples directly onto a pre-spotted MALDI plate. This analytical strategy can be performed in any laboratory equipped with a MALDI-TOF instrument. All key benefits of organic matrix-free technologies were satisfied while maintaining a high level of detection performances (sensitivity and reproducibility/repeatability). In particular, sample preparation was simple and detection in the low mass range was not hampered by matrix ions. Imaging studies were undertaken to query sample dispersion into the inert SiO2 NPs and to help into the search of the best experimental conditions producing homogeneous analyte distribution within the deposit. In contrast to commercial disposable LDI targets designed for single use and requiring an adaptor such as NALDI™, the proposed SiO2 NPs pre-spotting on a MALDI target plate allowed very easily switching between MALDI and LDI experiments. They can be conducted either simultaneously (positions with an organic matrix or SiO2 NPs) or in the row (support prepared in advance, stored and washed after use). The overall cost and versatility of the methodology made it very attractive to MALDI users in many domains (peptidomics, proteomics, metabolomics).

Graphical abstract

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Graphical abstract Highlights

► We present a new pre-spotted MALDI target for peptide detection and sequencing. ► The inert substrate consists of silica nanoparticles easily prepared by sol–gel chemistry. ► This very efficient LDI-promoting support produces ions from few femtomoles of mixtures of peptides. ► Rapid MS profiling and subsequent MS/MS analyses are achieved.

A direct assessment of mycotoxin biomarkers in human urine samples by liquid chromatography tandem mass spectrometry

30 July 2012, 09:18:36
Publication year: 2012
Source:Analytica Chimica Acta, Volume 741
Emmanuel Njumbe Ediage, Jose Diana Di Mavungu, Suquan Song, Aibo Wu, Carlos Van Peteghem, Sarah De Saeger
Detection of mycotoxin biomarkers in urine of humans and animals provides a direct approach for assessing exposure to these mycotoxins as opposed to the indirect approach of food analysis, which in most cases is affected by the heterogeneity of the toxin in the food samples. Seven (7) mycotoxins and their metabolites (total 18 analytes) were selected and an LC–MS/MS method for their determination in human urine was developed and validated. The method consisted of direct analysis of two mycotoxin conjugates, deoxynivalenol-glucuronide and zearalenone-glucuronide without beta glucuronidase digestion of the urine samples. Since high method sensitivity is of utmost importance in such study, critical factors which could improve the analyte recovery and method sensitivity were investigated by a D-optimal experimental design. Urine samples (10mL) were first extracted with 15mL ethyl acetate/formic acid (99/1, v/v) followed by SAX SPE clean-up of the acidified aqueous fraction. Both extracts were combined and analyzed using an LC–MS/MS system operated in the positive ionization mode. A total run time of 28min was adopted with all the 18 analytes eluting within 15min. The method was validated by taking into consideration the guidelines specified in Commission Decision 2002/657/EC and 401/2006/EC. Forty samples obtained from volunteers within the laboratory research group were analyzed as part of a pilot study. All results were expressed per mg creatinine. A total of 9 samples were found contaminated with one or more of the following analytes: DON, OTA, OTα, 4-OH OTA, ZEN, CIT and β-ZOL. One-eighth (5/40) of the samples were contaminated with DON in the range of 3.7–67ngmg−1 creatinine. Samples with detectable levels of DON did not show any co-occurrence of DON-3Glu. One sample was found to be contaminated with 4-OH OTA (<LOQ), co-occurring with only OTA (0.2ngmg−1 creatinine). OTα (up to 4.4ngmg−1 creatinine) was detected in three other samples co-occurring with low levels of OTA (up to 0.3ngmg−1 creatinine) and no 4-OH OTA detected. ZEN was detected in 10% (4/40) of the samples analyzed. Three samples were contaminated with β-ZOL (3.3–20ngmg−1 creatinine), co-occurring with ZEN (<LOQ–10.8ngmg−1 creatinine). The ratio of ZEN/β-ZOL varied for all the three samples. α-ZOL was not detected in any of the 40 samples. CIT was detected in one sample at 4.5ngmg−1 creatinine. This is the first study carried out with a small group of the Belgian population to assess exposure to mycotoxins using biomarkers.

Graphical abstract

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Graphical abstract Highlights

► The method was established for analysis of mycotoxin biomarkers in human urine. ► LLE with acidified ethyl acetate was very efficient as extraction solvent. ► Direct determination of the glucuronides was performed without enzyme digestion. ► Optimization of the analytical parameters was achieved with a D-optimal design. ► The proposed sample preparation method is simple, cheaper and robust.

Sample handling and contamination encountered when coupling offline normal phase high performance liquid chromatography fraction collection of petroleum samples to Fourier transform ion cyclotron resonance mass spectrometry

30 July 2012, 09:18:36
Publication year: 2012
Source:Analytica Chimica Acta, Volume 741
Nicole E. Oro, Randy M. Whittal, Charles A. Lucy
Normal phase high performance liquid chromatography (HPLC) is used to separate a gas oil petroleum sample, and the fractions are collected offline and analyzed on a high resolution Fourier Transform Ion Cyclotron Resonance Mass Spectrometer (FT-ICR MS). The separation prior to MS analysis dilutes the sample significantly; therefore the fractions need to be prepared properly to achieve the best signal possible. The methods used to prepare the HPLC fractions for MS analysis are described, with emphasis placed on increasing the concentration of analyte species. The dilution effect also means that contamination in the MS spectra needs to be minimized. The contamination from molecular sieves, plastics, soap, etc. and interferences encountered during the offline fraction collection process are described and eliminated. A previously unreported MS contamination of iron formate clusters with a 0.8 mass defect in positive mode electrospray is also described. This interference resulted from the stainless steel tubing in the HPLC system. Contamination resulting from what has tentatively been assigned as palmitoylglycerol and stearoylglycerol was also observed; these compounds have not previously been reported as contaminant peaks.

Graphical abstract

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Graphical abstract Highlights

► We collect HPLC fractions of gas oils offline for FT-ICR MS analysis. ► Optimized sample preparation procedures are described. ► MS contamination encountered is described and eliminated. ► Three new contaminants are identified, including iron-formate clusters.

Analysis of steroids in human urine by on line liquid chromatography–gas chromatography–mass spectrometry using the Through Oven Transfer Adsorption Desorption interface and a fraction collector

30 July 2012, 09:18:36
Publication year: 2012
Source:Analytica Chimica Acta, Volume 741
Rosa M. Toledano, Eva M. Díaz-Plaza, José M. Cortés, Inmaculada Blázquez, Ana Vázquez, Jesús Villén, Jesús Muñoz-Guerra
Anti-doping laboratories accredited by the WADA (World Anti-Doping Agency) must have available methods capable of detecting synthetic steroids at concentrations below 10ngmL−1 and, at the same time, be able to quantify endogenous steroids with accuracy. The unequivocal identification of the steroids must be carried out by GC–MS. In this manuscript we describe a new method by on-line coupled liquid chromatography–gas chromatography (LC–GC) using the Through Oven Transfer Adsorption Desorption (TOTAD) interface and mass spectrometer (MS) detector. By this means, all the analyte eluted in the LC stage are automatically transferred to the GC, avoiding the contamination associated with manipulation of the sample. A newly designed fraction collector is described for the first time. The collector permits the different fractions eluted in LC to be stored and sent individually to the GC by means of the TOTAD interface. The detection limits attained, measured in gas chromatography–mass spectrometry (GC–MS) in SCAN mode, vary between 0.5 and 3.4ngmL−1, and the method, once the sample is derivatized, is completely automatic.

Graphical abstract

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Graphical abstract Highlights

► A new method to analyze steroids in human urine by LC–GC–MS has been developed. ► The TOTAD interface with a fraction collector is used in the on line coupling LC–GC. ► A newly designed fraction collector, totally automated, is described for the first time. ► Different LC fractions were analyzed in a sole injection of sample. ► The sensitivity achieved permits the detection limits set by WADA to be attained.

Characterization of CdTe/CdSe quantum dots-transferrin fluorescent probes for cellular labeling

30 July 2012, 09:18:36
Publication year: 2012
Source:Analytica Chimica Acta, Volume 741
Li-Yun Guan, Yong-Qiang Li, Song Lin, Ming-Zhen Zhang, Jun Chen, Zhi-Ya Ma, Yuan-Di Zhao
In this paper, we prepared three types of transferrin-quantum dots conjugates (QDs-Tf) using three different methods (electrostatic interaction, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) coupling, denatured transferrin (dTf) coating). Fluorescence emission spectra, surface characteristics, zeta potentials of quantum dots (QDs) and QDs-Tf fluorescent probes were characterized by spectrophotometer, capillary electrophoresis, and dynamic light scattering. Fluorescent imaging of HeLa cells was also performed by QDs and QDs-Tf fluorescent probes. It was found that the fluorescence imaging performances of QDs-Tf probes prepared by electrostatic interaction and EDC coupling were better compared with the one prepared by dTf coating. Then a real-time single cell detection system was established to quantitatively evaluate cell labeling effects of QDs-Tf fluorescent probes. It was found that for cell labeling efficiency, the proportion of cells labeled by quantum dot probes to a group of cells, QDs-Tf probe prepared by EDC coupling showed the highest labeling efficiency (85.55±3.88%), followed by electrostatic interaction (78.86±9.57%), and dTf coating showed the lowest (40.09±10.2%). This efficiency order was confirmed by flow cytometry results. This study demonstrated the relationship between conjugation methods and the resultant QDs-Tf probes and provided a foundation for choosing appropriate QDs-Tf probes in cell labeling.

Graphical abstract

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Graphical abstract Highlights

► A convenient method was designed to assess cell efficiency of QDs probes for cellular labeling. ► The relationship between conjugation methods and effectiveness was evaluated clearly. ► QDs-Tf probe synthesized by EDC coupling had the highest labeling efficiency, followed by electrostatic interaction, and dTf coating.

In situ labeling and imaging of cellular protein via a bi-functional anticancer aptamer and its fluorescent ligand

30 July 2012, 09:18:36
Publication year: 2012
Source:Analytica Chimica Acta, Volume 741
Jun Ai, Tao Li, Bingling Li, Yuanhong Xu, Dan Li, Zuojia Liu, Erkang Wang
In this article, we reported a novel approach for in situ labeling and imaging HeLa cancer cells utilizing a bifunctional aptamer (AS1411) and its fluorescent ligand, protoporphyrin IX (PPIX). In the presence of potassium ion, AS1411 folded to G-quadruplex structure, binded fluorescent ligand (PPIX) with fluorescent enhancement, and targeted the nucleolin overexpressed by cancer cells. Consequently, bioimaging of cancer cells specifically were realized by laser scanning confocal microscope. The bioimaging strategy with AS1411–PPIX complex was capable to distinguish HeLa cancer cells from normal cells unambiguously, and fluorescence imaging of cancer cells was also realized in human serum. Moreover, the bioimaging method was very facile, effective and need not any covalent modification. These results illustrated that the useful approach can provide a novel clue for bioimaging based on non-covalent bifunctional aptamer in clinic diagnosis.

Graphical abstract

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Graphical abstract Highlights

In this work, we report a novel approach to in situ labeling and imaging of a cellular protein nucleolin utilizing a multifunctional anticancer aptamer combined with its fluorescent ligand.
► AS1411 bind protoporphyrin IX and enhances the fluorescence signal remarkably. ► According to LSCM experiment, HeLa cells were imagined by AS1411–PPIX. ► Aptamer-based bioimaging plays an important role for need not any covalent modification.

Quantum-dot-based homogeneous time-resolved fluoroimmunoassay of alpha-fetoprotein

30 July 2012, 09:18:36
Publication year: 2012
Source:Analytica Chimica Acta, Volume 741
Mei-Jun Chen, Ying-Song Wu, Guan-Feng Lin, Jing-Yuan Hou, Ming Li, Tian-Cai Liu
Quantum dots (QDs) with novel photoproperties are not widely used in clinic diagnosis, and homogeneous time-resolved fluorescence assays possess many advantages over current methods for alpha-fetoprotein (AFP) detection. A novel QD-based homogeneous time-resolved fluorescence assay was developed and used for detection of AFP, a primary marker for many cancers and diseases. QD-doped carboxyl-modified polystyrene microparticles (QPs) were prepared by doping oil-soluble QDs possessing a 605nm emission peak. The antibody conjugates (QPs-E014) were prepared from QPs and an anti-AFP monoclonal antibody, and luminescent terbium chelates (LTCs) were prepared and conjugated to a second anti-AFP monoclonal antibody (LTCs-E010). In a double-antibodies sandwich structure, QPs-E014 and LTCs-E010 were used for detection of AFP, serving as energy acceptor and donor, respectively, with an AFP bridge. The results demonstrated that the luminescence lifetime of these QPs was sufficiently long for use in a time-resolved fluoroassay, with the efficiency of time-resolved Förster resonance transfer (TR-FRET) at 67.3% and the spatial distance of the donor to acceptor calculated to be 66.1Å. Signals from TR-FRET were found to be proportional to AFP concentrations. The resulting standard curve was log Y =3.65786+0.43863·log X (R =0.996) with Y the QPs fluorescence intensity and X the AFP concentration; the calculated sensitivity was 0.4ngmL−1. By assaying test samples against the standard curve, the coefficient of variations was <5%, indicating that QDs were suitable for this homogenous time-resolved fluoroimmunoassay. This work extended the potential applications of QDs in future homogeneous analytical bioassays. In the coming research, hepatitis B surface antigen, another primary marker for hepatocellular carcinoma, will be studied for practical detection using a QD-based homogenous multiplex fluoroimmunoassay.

Graphical abstract

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Graphical abstract Highlights

► QDs-based homogeneous time-resolved fluoroimmunoassay was developed to detect AFP. ► The conjugates were prepared with QDs-doped microspheres and anti-AFP McAb. ► The conjugates were prepared with LTCs and another anti-AFP McAb. ► Excess amounts of conjugates were used for detecting AFP without rinsing. ► The wedding of QPs and LTCs was suitable for HTRFIA to detect AFP.

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2ND BVT WORKSHOP



Dear Colleagues,

I am pleased to invite you for purpose of the 2nd BVT Workshop (www.bvt.cz) focused on high sophisticated biosensors systems and their applications. The meeting is held from 17th to 18th October in new conference room of our company in Strážek, Czech Republic.
        This workshop concentrates to new trends and practical application of biosensors and biological activity measurement. The workshop concetrate to real demonstration of work with BVT devices and sensors. The topics of workshop will be presented as the oral presentations and as the practical measurements.
        I hope you found this invitation interesting and attractive for you. I look forward to seeing you soon in our company in Strážek.

With best regards,


Jan Krejčí
                                                                Chief executive officer

Friday, 27 July 2012

Science and the Olympics

In a few hours the Olympic torch will be lit in London to signify the start of the 30th Olympiad, but as I sat in the stadium on Wednesday evening watching a dress rehearsal for the Opening Ceremony I was struck by the good nature of the many different nationalities there with me. Everyone was positive and looking forward to enjoying the next few weeks together. We all had our national favourites of course, but the overriding feeling was one of: "may the best man (or woman) win" and, beyond that, every participant merited our applause and good wishes.
In twenty-five years working to support the scientific community, I have been impressed by the fact that - more often than not - the academics and researchers I deal with are happy to set aside nationality, ethnicity or religion to work with each other toward a common goal, to achieve success for the greater good of humanity. And once someone has achieved that recognition, there is rarely any petty jealously to be seen.
I hope - and strongly suspect - that the the similarities between this approach to science and research are replicated in the London Olympics and Paralympics and hope you enjoy the competition and atmosphere whether you are visiting Great Britain or watching television from an armchair.
Best wishes, 
David

Just Published: Journal of Chromatography B


A new issue of this journal has just been published. To see abstracts of the papers it contains (with links through to the full papers) click here:
Selected papers from the latest issue:

A sensitive LC–MS/MS assay for the simultaneous analysis of the major active components of silymarin in human plasma

27 July 2012, 13:20:17
Publication year: 2012
Source:Journal of Chromatography B, Volume 902
Bryan J. Brinda, Hao-Jie Zhu, John S. Markowitz
Silymarin, an extract of crushed achenes of the milk thistle plant Silybum marianum is a multi-constituent mixture, 70–80% of which consists of a complex assortment containing the flavonolignans silybin A and B, isosilybin A and B, silydianin, and silychristin, and the flavonoid taxifolin. To date, numerous pharmacological actions of the silymarin extract have been documented in the biomedical literature, including hepatoprotective, anti-inflammatory, anti-tumor, and anti-fibrotic activities. The present study describes a novel liquid chromatographic–tandem mass spectrometric method for simultaneous analysis of silychristin, silydianin, silybin A and silybin B, isosilybin A and isosilybin B, and taxifolin in human plasma employing liquid–liquid extraction. This assay provides excellent resolution of the individual silymarin constituents via utilization of a 100A 250mm×2mm, 5μm C18 column with the mobile phase consisting of 51% methanol, 0.1% formic acid, and 10mM ammonium acetate. The lower limit of quantification was 2ng/ml for each constituent. Calibration curves were linear over the range from 2ng/ml to 100ng/ml for all analytes (r 2 >0.99). The intra- and inter-day accuracies were 91–106.5% and 95.1–111.9%, respectively. The intra- and inter-day precision was within 10.5%. Additionally, recovery, stability, and matrix effects were fully validated as well. This method was successfully applied to human plasma samples from subjects treated with the milk thistle extract Legalon®.

Highlights

► Silymarin extract contains numerous bioactive flavonolignans and flavonoids. ► Analysis of silychristin, silydianin, silybin A and B, isosilybin A and B, and taxifolin. ► Pure standard compounds were utilized for absolute quantification. ► The lower limit of quantification was 2ng/ml for each analyte. ► Method was applied to human plasma samples from subjects treated with Legalon®.

Quantification of human serum transferrin using liquid chromatography–tandem mass spectrometry based targeted proteomics

27 July 2012, 13:20:17
Publication year: 2012
Source:Journal of Chromatography B, Volume 902
Ying Yu, Jinhui Xu, Yuan Liu, Yun Chen
Currently, the absolute quantification of human transferrin (hTRF) is based on several techniques other than mass spectrometry. Although these techniques provide valuable information on protein levels and can be extremely sensitive, they often lack the specificity and reproducibility that can be provided by mass spectrometry. In this study, a liquid chromatography–tandem mass spectrometry (LC/MS/MS) based targeted proteomics assay was developed and validated for the determination of transferrin in human serum. We selected the tryptic peptide 108EDPQTFYYAVAVVK121 as the surrogate analyte for quantification and used a stable isotope-labeled synthetic peptide with this sequence as an internal standard. Sample cleanup and enrichment were achieved using solid phase extraction. The validated calibration range was from 500 to 5000ng/mL. The intra- and inter-day precisions were less than 4.9% and 9.0%, respectively. The bias for the quality control (QC) samples was less than 5.4%. Finally, this assay was successfully applied to the quantitative analysis of transferrin in clinical samples. The obtained values were assessed by independently measuring transferrin in the same samples using a commercially available immunoturbidimetric assay. As a result, the absolute concentrations determined by the LC/MS/MS assay compared well with those obtained with the immunoturbidimetric method; however, the LC/MS/MS assay afforded more reliable transferrin values at low concentrations.

Highlights

► A novel LC/MS/MS-based targeted proteomics assay was developed and validated. ► Protein digestion efficiency and surrogate peptide selection were discussed. ► The LC/MS/MS assay was compared with a commercial immunoturbidimetric method. ► Clinical monitoring of prospective protein biomarkers could be achieved by LC/MS/MS.

Development, validation and application of a sensitive LC–MS/MS method for the quantification of thalidomide in human serum, cells and cell culture medium

27 July 2012, 13:20:17
Publication year: 2012
Source:Journal of Chromatography B, Volume 902
Sandra Roche, Louise Sewell, Justine Meiller, Kasper Pedersen, Rajesh Rajpal, Peter O’Gorman, Martin Clynes, Robert O’Connor
A simple, robust, sensitive and selective liquid chromatography tandem mass spectrometry (LC–MS/MS) method for the quantification of thalidomide was developed and validated. The method was applied to thalidomide quantification in three different types of biological samples. Thalidomide was extracted from human serum (100μL), cells (2.5×105), or cell culture media (100μL) by LLE and separated on a Prodigy C18 (150mm×4.0mm, 5μm i.d.) column with isocratic elution using water/acetonitrile (70/30, v/v) 0.1% formic acid, at a flow rate of 0.5mL/min, with umbelliferone (600ng/mL) as an internal standard. Thalidomide was quantified using a triple quadrupole mass spectrometer operated in multi-reaction-monitoring mode using positive electrospray ionisation. The method was validated in two separate thalidomide concentration ranges; human serum (0.05–20μg/mL) and in vitro cells (0.78–50ng) with an inter-day precision of 1.8% and 1.9% and average accuracy of 100% and 101% in serum and cells respectively. Despite the use of small sample volume, the limit of quantification for thalidomide in serum was determined to be 3ng/mL. The method was successfully employed to measure levels of thalidomide in cancer patient serum and cell culture model systems. Although cellular levels were quantifiable, thalidomide was shown to be unstable under in vitro conditions with a half life of approximately 2h. In patient samples, circulating serum levels showed a broad correlation with dose and uncovered some patient compliance issues.

Highlights

► We developed a sensitive LCMS method for thalidomide with an LOQ of 3ng/mL in serum. ► The sample preparation, LLE, gives higher sample recovery than previous methods. ► In patient serum we detected thalidomide levels ranging from 25ng/mL to 1407ng/mL. ► We quantified thalidomide degradation in vitro with 50% remaining after 2h.

Development and full validation of an UPLC-MS/MS method for the determination of an anti-allergic indolinone derivative in rat plasma, and application to a preliminary pharmacokinetic study

27 July 2012, 13:20:17
Publication year: 2012
Source:Journal of Chromatography B, Volume 902
Mouhssin Oufir, Chethan Sampath, Veronika Butterweck, Matthias Hamburger
The natural product (E,Z)-3-(4-hydroxy-3,5-dimethoxybenzylidene)indolin-2-one (indolinone) was identified some years ago as a nanomolar inhibitor of FcɛRI-receptor dependent mast cell degranulation. To further explore the potential of the compound, we established an UPLC-MS/MS assay for dosage in rat plasma. The method was fully validated according to FDA Guidance for industry. Results of this validation and long term stability study demonstrate that the method in lithium heparinized rat plasma is specific, accurate, precise and capable of producing reliable results according to recommendations of international guidelines. The method was validated with a LLOQ of 30.0ng/mL and an ULOQ of 3000ng/mL. The response versus concentration data were fitted with a first order polynomial with 1/X 2 weighting. No matrix effect was observed when using three independent sources of rat plasma. The average extraction recovery was consistent over the investigated range. This validation in rat plasma demonstrated that indolinone was stable for 190 days when stored below −65°C; for 4 days at 10°C in the autosampler; for 4h at RT, and during three successive freeze/thaw cycles at −65°C. Preliminary pharmacokinetic data were obtained in male Sprague–Dawley rats (2mg/kg BW i.v.). Blood samples taken from 0 to 12h after injection were collected, and data analyzed with WinNonlin. A short half-life (4.30±0.14min) and a relatively high clearance (3.83±1.46L/h/kg) were found.

Highlights

► Indolinone is stable in rat plasma stored below −65°C during 190 days. ► Method in rat plasma for indolinone is specific, selective, precise and accurate. ► Indolinone showed a short half-life and a relatively high clearance in rat plasma.

A fully integrated multi-column system for abundant protein depletion from serum/plasma

27 July 2012, 13:20:17
Publication year: 2012
Source:Journal of Chromatography B, Volume 902
Dariusz J. Janecki, Steven C Pomerantz, Eric J. Beil, Jennifer F. Nemeth
This work details the transformation of a conventional HPLC system to a low back pressure liquid chromatography set-up for automated serum/plasma depletion and fractionation. A Dionex U3000 HPLC was converted to low back pressure operation (125psi max) by replacing all narrow-bore lines to larger inner-diameter tubing. The system was configured to use two immunoaffinity columns, first for depletion of the top 14 most abundant proteins (Seppro IgY14), then for the next 200–300 proteins (Seppro SuperMix). The autosampler was dual-purposed for both injection and fraction collection. Both the flow-through and SuperMix bound proteins were collected in an automated fashion. Three samples could be depleted consecutively before the system required user intervention, and up to nine samples could be depleted within a 24h period. This study documents the validation of the instrument performance with a 90-patient sample set, demonstrating overall CVs for 86 of the 90 samples to be within the 95% confidence intervals. Additionally, there was excellent reproducibility within the same patient (biological replicates) across days.

Highlights

► A conventional HPLC system set-up for automated serum/plasma depletion. ► The system uses two immunoaffinity columns in series (Seppro IgY14 and SuperMix). ► The autosampler is dual-purposed for both injection and fraction collection. ► The system significantly speeds up the sample preparation for proteomic workflow.

Direct urine analysis for the identification and quantification of selected benzodiazepines for toxicology screening

27 July 2012, 13:20:17
Publication year: 2012
Source:Journal of Chromatography B, Volume 902
Sevasti Karampela, Ioanna Vardakou, Ioannis Papoutsis, Artemis Dona, Chara Spiliopoulou, Sotirios Athanaselis, Constantinos Pistos
A simple and rapid LC/MS method with direct injection analysis was developed and validated for the identification and quantification of ten benzodiazepines (flunitrazepam, nordiazepam, diazepam, 7-aminoflunitrazepam, flurazepam, bromazepam, midazolam, alprazolam, temazepam and oxazepam) in human urine using diazepam-d5 as internal standard (IS). The main advantage of the proposed methodology is the minimal sample preparation procedure, as diluted urine samples were directly injected into LC/MS system. Electrospray ionization in positive mode using selected ion monitoring was chosen for the identification and quantification of the analytes. The linear range was 50–1000ng/mL for each analyte, with square correlation coefficient (r 2)≥0.981. Interday and intraday errors were found to be ≤5.72%. The LC/MS method was applied at ten real samples found initially to be positive and negative, using immunoassay technique. Finally the results were confirmed with GC/MS. The method demonstrates simplicity and fast sample preparation, accuracy and specificity of the analytes which make it suitable for replacement of immunoassay screening in urine avoiding thus false negative/false positive results. Using this method, laboratories may overcome the problem of high cost instrumentation such as LC–MS/MS by providing similar sensitivity and specificity with other methods.

Highlights

► We develop simpler and faster sample preparation method for the determination of benzodiazepines in urine. ► We overcome the problem of high cost instrumentation such as LC–MS/MS by providing similar sensitivity and specificity with other methods. ► We overcome false negative/false positive results when using immunoassay methods for benzodiazepines. ► The method is applicable in real clinical and forensic samples.

Liquid chromatography–mass spectrometric determination of losartan and its active metabolite on dried blood spots

27 July 2012, 13:20:17
Publication year: 2012
Source:Journal of Chromatography B, Volume 902
R. Nageswara Rao, S. Satyanarayana Raju, R. Mastan Vali, G. Girija Sankar
A simple and rapid quantitative bioanalytical liquid chromatography–tandem mass spectrometric (LC–MS/MS) method for simultaneous determination of losartan and its active metabolite, losartan carboxylic acid on rat dried blood spots was developed and validated as per regulatory guidelines. Losartan and its metabolite were extracted from dried blood spots using 50% aqueous methanol and separated on Waters XTerra® RP18 (250mm×4.6mm, 5μm) column using mobile phase composed of 40% acetonitrile and 60% aqueous ammonium acetate (10mM). The eluents were monitored using ESI tandem mass spectrometric detection with negative polarity in MRM mode using ion transitions m/z 421.2→179.0, m/z 435.3→157.0 and m/z 427.3→193.0 for losartan, losartan carboxylic acid and Irbesartan (internal standard), respectively. The method was validated over the linear range of 1–200ng/mL and 5–1000ng/mL with lower limits of quantification of 1.0ng/mL and 5.0ng/mL for losartan and losartan carboxylic acid, respectively. Inter and intra-day precision and accuracy (Bias) were below 5.96% and between −2.8 and 1.5%, respectively. The mean recoveries of the analytes from dried blood spots were between 89% and 97%. No significant carry over and matrix effects were observed. The stability of stock solution, whole blood, dried blood spot and processed samples were tested under different conditions and the results were found to be well within the acceptable limits. Additional validation parameters such as influence of hematocrit and spot volume were also evaluated and found to be well within the acceptable limits.

Highlights

► A simple isocratic LC–MS/MS method was developed and validated for losartan and its active metabolite. ► First dried blood spot report on losartan and it active metabolite as sample collection technique. ► Stability of analytes on FTA cards was also studied. ► Good LC resolution was obtained between the analytes and IS.

Characterization of taste-active compounds of various cherry wines and their correlation with sensory attributes

27 July 2012, 13:20:17
Publication year: 2012
Source:Journal of Chromatography B, Volume 902
Yunwei Niu, Xiaoming Zhang, Zuobing Xiao, Shiqing Song, Chengsheng Jia, Haiyan Yu, Lingling Fang, Chunhua Xu
Five cherry wines exhibiting marked differences in taste and mouthfeel were selected for the study. The taste and mouthfeel of cherry wines were described by four sensory terms as sour, sweet, bitter and astringent. Eight organic acids, seventeen amino acids, three sugars and tannic acid were determined by high performance liquid chromatography (HPLC). Five phenolic acids were determined by ultra performance liquid chromatography coupled with mass spectrometry (UPLC–MS). The relationship between these taste-active compounds, wine samples and sensory attributes was modeled by partial least squares regression (PLSR). The regression analysis indicated tartaric acid, methionine, proline, sucrose, glucose, fructose, asparagines, serine, glycine, threonine, phenylalanine, leucine, gallic acid, chlorogenic acid, vanillic acid, arginine and tannic acid made a great contribution to the characteristic taste or mouthfeel of cherry wines.

Highlights

► Four sensory attributes of 5 cherry wines were evaluated. ► Thirty-three compounds were correlated to sensory attributes and wine samples through PLSR. ► Seventeen compounds mostly contributed to sensory attributes of cherry wines.

Pharmacokinetics of conjugated metabolites in rat plasma after oral administration of tectoridin

27 July 2012, 13:20:17
Publication year: 2012
Source:Journal of Chromatography B, Volume 902
Jialin Qu, Jie Gao, Jiahong Sun, Lin Zhang, Toshiaki Makino, Dan Yuan
Tectoridin is a major isoflavone found in the flowers of Pueraria thomsonii Benth. It possesses estrogenic, hypoglycemic, anti-oxidant, and anti-inflammatory activities. In the present study, we evaluated the plasma pharmacokinetic profile of tectoridin in rats. We isolated a new metabolite, tectorigenin-7-O-glucuronide-4′-O-sulfate (Te-7G-4′S), from the bile of rats treated orally with tectoridin and determined its chemical structure by spectral analysis. Furthermore, we developed a selective and accurate method for the simultaneous quantification of tectoridin metabolites, including Te-7G-4′S, tectorigenin-7-O-glucuronide (Te-7G), tectorigenin-7-O-sulfate (Te-7S), and tectorigenin in rat plasma, and measured their plasma concentrations in rats orally administered tectoridin (200mg/kg). Plasma concentrations of Te-7G-4′S, Te-7G, Te-7S, and tectorigenin reached maximal values of 21.4±13.8μmol at 3.50±1.87h, 20.5±9.7μmol at 3.17±1.81h, 14.3±3.3μmol at 5.58±3.07h, and 8.67±3.07μmol at 4.92±2.87h, respectively. Enterohepatic recirculation resulted in double peaks or a flat concentration curve/time profile of the metabolites. Since plasma concentrations of tectorigenin conjugated metabolites were higher than those of the tectorigenin aglycone, it can be concluded that extensive phase II metabolism plays an important role in the pharmacokinetics of tectoridin and tectorigenin in vivo.

Highlights

► A new glucuronide-sulfate diconjugate was isolated and structurally determined for the first time. ► Three conjugate metabolites were simultaneously identified and quantified in rat plasma by HPLC–DAD and LC/TOF/MS. ► It provides helpful information for the potential therapeutic application of tectoridin-containing phytopharmacueticals.

Quantification of HPLC-separated peptides and proteins by spectrofluorimetric detection of native fluorescence and mass spectrometry

27 July 2012, 13:20:17
Publication year: 2012
Source:Journal of Chromatography B, Volume 902
Suraj Saraswat, Bruce Snyder, Dragan Isailovic
Due to relatively low reproducibility of the ionization and differences when using buffers as mobile phases, the quantitative analysis by electrospray ionization mass spectrometry (ESI-MS) can be often challenging. In the present study, the native fluorescence of phenylalanine, tyrosine, and tryptophan was investigated as an improvement tool for the analytical quantification of peptides and proteins by HPLC–ESI-MS. Natively fluorescent amino acids as well as peptides, proteins, and protein digests were successfully separated by HPLC, and quantified with a spectrofluorimetric detector and ESI-MS. The two detectors were connected in series and enabled the sequential measurements of the fluorescence intensities as well as the measurements of the ion signals and mass spectral characterization of separated polypeptides. Fluorescence detector provided better linearity and repeatability of quantification than mass spectrometer, and similar limits of detection for most of biomolecules analyzed. The fluorescence signal was linear over 3–4 orders of magnitude with limits of detection in picomole or high femtomole range, depending on nature and number of natively fluorescent amino acid residues present in the analyzed polypeptides. Hence, native fluorescence of phenylalanine, tyrosine, and tryptophan can be used as a label-free methodology to facilitate quantification of peptides and proteins by LC–ESI-MS.

Highlights

► Sequential native fluorescence–ESI-MS quantification of HPLC-separated polypeptides is enabled. ► Peptides and proteins containing all natively fluorescent amino acids are quantified. ► Figures of merit for spectrofluorimetric and ESI-MS quantifications of natively fluorescent biomolecules are complementary. ► Native fluorescence provides better linearity and repeatability of quantification than ESI-MS. ► This is a label-free technique, which can facilitate quantification of peptides and proteins by LC–ESI-MS.

Diffusion Split-Flow Thin Cell (SPLITT) system for protein separations

27 July 2012, 13:20:17
Publication year: 2012
Source:Journal of Chromatography B, Volume 902
Srinivas Merugu, Himanshu J. Sant, Bruce K. Gale
A diffusion Split-Flow Thin Cell (SPLITT) system was used to partially remove small peptides such as β2 microglobulin (β2M) and parathyroid hormone (PTH) in a continuous manner from an input flow stream while preserving most (over 97%) of the larger protein in the sample, such as albumin. To help determine the operating conditions for this work, a two-dimensional numerical model based on the Navier–Stokes equation and convection–diffusion equations was developed for diffusional SPLITT using COMSOL multiphysics software (COMSOL Inc., MA). These simulations were used to obtain the relationship between important operational parameters and the purification efficiency for proteins of interest. The diffusion-based SPLITT system was fabricated using xurography and was used to demonstrate protein purification based on the differences in size or diffusion coefficient of the sample. The results obtained from the experiments are compared with the mathematical model and show good agreement, while the variations between these results are discussed. The results show that significant portions of small peptides (>25%) can be removed while preserving larger proteins (up to 95%) in the carrier stream. A potential application of this technique is to be used as an additional step in kidney dialysis to remove toxins that are not effectively removed by current dialysis protocols.

Highlights

► Continuous removal of beta-2-microglobulin and parathyroid hormone. ► COMSOL multiphysics model of diffusion SPLITT system. ► The model is used to optimize experimental parameters. ► The technique has the potential to be used as an additional step in traditional dialysis protocols.