| 1. |
- Bar, Laure, et al.
(författare)
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Impact of antigen density on recognition by monoclonal antibodies
- 2020
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Ingår i: Analytical Biochemistry. - : American Chemical Society (ACS). - 0003-2697 .- 1096-0309 .- 0003-2700 .- 1520-6882. ; 92:7, s. 5396-5403
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Tidskriftsartikel (refereegranskat)abstract
- Understanding antigen–antibody interactions is important to many emerging medical and bioanalytical applications. In particular, the levels of antigen expression at the cell surface may determine antibody-mediated cell death. This parameter has a clear effect on outcome in patients undergoing immunotherapy. In this context, CD20 which is expressed in the membrane of B cells has received significant attention as target for immunotherapy of leukemia and lymphoma using the monoclonal antibody rituximab. To systematically study the impact of CD20 density on antibody recognition, we designed self-assembled monolayers that display tunable CD20 epitope densities. For this purpose, we developed in situ click chemistry to functionalize SPR sensor chips. We find that the rituximab binding affinity depends sensitively and nonmonotonously on CD20 surface density. Strongest binding, with an equilibrium dissociation constant (KD = 32 nM) close to values previously reported from in vitro analysis with B cells (apparent KD between 5 and 19 nM), was obtained for an average inter-antigen spacing of 2 nm. This distance is required for improving rituximab recognition, and in agreement with the known requirement of CD20 to form clusters to elicit a biological response. More generally, this study offers an interesting outlook in the understanding of the necessity of epitope clusters for effective mAb recognition.
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| 2. |
- Nordström, Anders, et al.
(författare)
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Nonlinear data alignment for UPLC-MS and HPLC-MS based metabolomics : quantitative analysis of endogenous and exogenous metabolites in human serum.
- 2006
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 78:10, s. 3289-95
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Tidskriftsartikel (refereegranskat)abstract
- A nonlinear alignment strategy was examined for the quantitative analysis of serum metabolites. Two small-molecule mixtures with a difference in relative concentration of 20-100% for 10 of the compounds were added to human serum. The metabolomics protocol using UPLC and XCMS for LC-MS data alignment could readily identify 8 of 10 spiked differences among more than 2700 features detected. Normalization of data against a single factor obtained through averaging the XCMS integrated response areas of spiked standards increased the number of identified differences. The original data structure was well preserved using XCMS, but reintegration of identified differences in the original data reduced the number of false positives. Using UPLC for separation resulted in 20% more detected components compared to HPLC. The length of the chromatographic separation also proved to be a crucial parameter for a number of detected features. Moreover, UPLC displayed better retention time reproducibility and signal-to-noise ratios for spiked compounds over HPLC, making this technology more suitable for nontargeted metabolomics applications.
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| 3. |
- Abikhodr, A. H., et al.
(författare)
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Identifying Mixtures of Isomeric Human Milk Oligosaccharides by the Decomposition of IR Spectral Fingerprints
- 2021
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 93:44, s. 14730-14736
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Tidskriftsartikel (refereegranskat)abstract
- The analysis of glycans presents a significant challenge that arises from their isomeric heterogeneity. While high-resolution ion mobility spectrometry (IMS) has shown the ability to resolve subtly different glycan isomers, their unambiguous assignment remains difficult. Here, we demonstrate an infrared (IR) spectroscopic approach for identifying isomers in a glycan mixture. To display the feasibility of this approach, we have constructed a small database of cryogenic spectra of five lacto-N-fucopentaose (LNFP) and six disaccharide isomers and demonstrated that in the cases where they cannot be separated by IMS, we can use a cryogenic IR spectrum to identify the isomeric components of a mixture.
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| 4. |
- Abrahamsson, Christoffer, et al.
(författare)
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Time-resolved NIR spectroscopy for quantitative analysis of intact pharmaceutical tablets
- 2005
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 1520-6882 .- 0003-2700. ; 77:4, s. 1055-1059
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Tidskriftsartikel (refereegranskat)abstract
- Near-infrared (NIR) spectroscopy is a useful technique for quantitative measurements of intact tablets, but it suffers from limitations due to the fact that changes in the physical properties of a sample strongly affect the recorded spectrum. In this work, time-resolved transmission NIR spectroscopy was utilized to conduct quantitative measurements of intact tablets. The technique enables separation of the absorption properties of the sample from the scattering properties and can therefore handle changes of the physical parameters of the samples in a better way than conventional NIR transmission spectroscopy. The experiments were conducted using a pulsed Ti:sapphire laser coupled into a nonlinear photonic crystal fiber as light source. The light transmitted through the sample was measured by a time-resolving streak camera. A comparison of the results from the time-resolved technique with the results from conventional transmission NIR spectroscopy was made using tablets containing different concentrations of iron oxide and manufactured with different thicknesses. A PLS model made with data from the time-resolved technique predicted samples 5 times better than a PLS model made data from the conventional NIR transmission technique. Furthermore, an improvement to predict samples with physical properties outside those included in the calibration set was demonstrated.
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| 5. |
- Acero Sanchez, Josep Ll., et al.
(författare)
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Electrochemical Genetic Profiling of Single Cancer Cells
- 2017
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 89:6, s. 3378-3385
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Tidskriftsartikel (refereegranskat)abstract
- Recent understandings in the development and spread of cancer have led to the realization of novel single cell analysis platforms focused on circulating tumor cells (CTCs). A simple, rapid, and inexpensive analytical platform capable of providing genetic information on these rare cells is highly desirable to support clinicians and researchers alike to either support the selection or adjustment of therapy or provide fundamental insights into cell function and cancer progression mechanisms. We report on the genetic profiling of single cancer cells, exploiting a combination of multiplex ligation-dependent probe amplification (MLPA) and electrochemical detection. Cells were isolated using laser capture and lysed, and the mRNA was extracted and transcribed into DNA. Seven markers were amplified by MLPA, which allows for the simultaneous amplification of multiple targets with a single primer pair, using MLPA probes containing unique barcode sequences. Capture probes complementary to each of these barcode sequences were immobilized on a printed circuit board (PCB) manufactured electrode array and exposed to single-stranded MLPA products and subsequently to a single stranded DNA reporter probe bearing a HRP molecule, followed by substrate addition and fast electrochemical pulse amperometric detection. We present asimple, rapid, flexible, and inexpensive approach for the simultaneous quantification of multiple breast cancer related mRNA markers, with single tumor cell sensitivity.
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| 6. |
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| 7. |
- Adams, Kelly L., et al.
(författare)
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Steady-State Electrochemical Determination of Lipidic Nanotube Diameter Utilizing an Artificial Cell Model
- 2010
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 1520-6882 .- 0003-2700. ; 82:3, s. 1020-1026
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Tidskriftsartikel (refereegranskat)abstract
- By exploiting the capabilities of steady-state electrochemical measurements, we have measured the inner diameter of a lipid nanotube using Fick’s first law of diffusion in conjunction with an imposed linear concentration gradient of electroactive molecules over the length of the nanotube. Fick’s law has been used in this way to provide a direct relationship between the nanotube diameter and the measurable experimental parameters Δi (change in current) and nanotube length. Catechol was used to determine the Δi attributed to its flux out of the nanotube. Comparing the nanotube diameter as a function of nanotube length revealed that membrane elastic energy was playing an important role in determining the size of the nanotube and was different when the tube was connected to either end of two vesicles or to a vesicle on one end and a pipet tip on the other. We assume that repulsive interaction between neck regions can be used to explain the trends observed. This theoretical approach based on elastic energy considerations provides a qualitative description consistent with experimental data.
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| 8. |
- Adler, Belinda, et al.
(författare)
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Miniaturized and Automated High-Throughput Verification of Proteins in the ISET Platform with MALDI MS
- 2012
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 84:20, s. 8663-8669
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Tidskriftsartikel (refereegranskat)abstract
- A major bottleneck in high-throughput protein production is the validation step, which is why parallel and automated sample processing methods are highly desirable. Also, a miniaturized sample preparation format is preferred, as the reduction of reagent volumes significantly decreases the analysis cost per sample. We have developed an automated and miniaturized protein sequence verification protocol for recombinant proteins utilizing peptide mass fingerprinting and MS/MS analysis. The integrated selective enrichment target (ISET) platform, previously developed in our group, with its dual functionality, being both a sample preparation platform and a MALDI target plate, is employed. All steps including immobilized metal ion affinity chromatography of protein on cobalt-loaded beads, tryptic digestion, and MALDI MS analysis are performed in an array format, without any sample transfers, on the same ISET chip. The automated configuration reduced the sample preparation time significantly. Starting with crude lysate, a full plate of 48 purified, digested samples prepared for MALDI-MS can be generated in 4 h, with only 30 min of operator involvement. This paper demonstrates the utility of the method by parallel analysis of 45 His-tagged human recombinant proteins.
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| 9. |
- Aeppli, Christoph, et al.
(författare)
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Direct compound-specific stable chlorine isotope analysis of organic compounds with quadrupole GC/MS using standard isotope bracketing
- 2010
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Ingår i: Analytical Chemistry. - Columbus, OH : American Chemical Society. - 0003-2700 .- 1520-6882. ; 82:1, s. 420-426
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Tidskriftsartikel (refereegranskat)abstract
- A method has been developed for the direct determination of the stable chlorine isotope composition (delta(37)Cl) of organochlorines that eliminates sample preparation, achieves precision comparable to earlier techniques while improving the sensitivity, and makes use of benchtop gas chromatography-quadrupole mass spectrometry instruments (GCqMS). The method is based on the use of multiple injections (n = 8-10) of the sample, bracketed by a molecularly identical isotopic standard with known delta(37)Cl, determined using off-line thermal ionization mass spectrometry (TIMS). Mass traces of two isotopologues differing by one chlorine isotope were used to calculate delta(37)Cl values. Optimization of mass spectrometry and peak integration parameters as well as method validation was achieved using tetrachloroethene (PCE), p,p'-dichlorodiphenyltrichloroethane (DDT), and pentachlorophenol (PCP), spanning a delta(37)Cl range of -5.5 to +3.2 per thousand vs SMOC. Injecting 1.6-1100 pmol resulted in standard deviations (1sigma) of 0.6-1.3 per thousand, and the delta(37)Cl results agreed with values independently measured with TIMS. The method was tested by determining the Rayleigh fractionation during evaporation of pure liquid PCE, resulting in a chlorine isotopic enrichment factor of epsilon(Cl) = -1.1 +/- 0.4 per thousand. Furthermore, position-specific delta(37)Cl analysis based on analysis of DDT mass fragments was evaluated. The GCqMS-delta(37)Cl method offers a simplified yet sensitive approach for compound-specific chlorine isotope analysis.
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| 10. |
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| 11. |
- Aerts, Jordan, et al.
(författare)
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Zero-Degree Celsius Capillary Electrophoresis Electrospray Ionization for Hydrogen Exchange Mass Spectrometry
- 2023
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Ingår i: Analytical Chemistry. - : Springer Nature. - 0003-2700 .- 1520-6882. ; 95:2, s. 1149-1158
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Tidskriftsartikel (refereegranskat)abstract
- Currently, fast liquid chromatographic separations at low temperatures are exclusively used for the separation of peptides generated in hydrogen deuterium exchange (HDX) workflows. However, it has been suggested that capillary electrophoresis may be a better option for use with HDX. We performed in solution HDX on peptides and bovine hemoglobin (Hb) followed by quenching, pepsin digestion, and cold capillary electrophoretic separation coupled with mass spectrometry (MS) detection for benchmarking a laboratory-built HDX–MS platform. We found that capillaries with a neutral coating to eliminate electroosmotic flow and adsorptive processes provided fast separations with upper limit peak capacities surpassing 170. In contrast, uncoated capillaries achieved 30% higher deuterium retention for an angiotensin II peptide standard owing to faster separations but with only half the peak capacity of coated capillaries. Data obtained using two different separation conditions on peptic digests of Hb showed strong agreement of the relative deuterium uptake between methods. Processed data for denatured versus native Hb after deuterium labeling for the longest timepoint in this study (50,000 s) also showed agreement with subunit interaction sites determined by crystallographic methods. All proteomic data are available under DOI: 10.6019/PXD034245.
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| 12. |
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| 13. |
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| 14. |
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| 15. |
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| 16. |
- Agarwal, A., et al.
(författare)
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Control of the Release of Freely Diffusing Molecules in Single-Cell Electroporation
- 2009
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 81:19, s. 8001-8008
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Tidskriftsartikel (refereegranskat)abstract
- Single-cell electroporation using an electrolyte-filled capillary is an emerging technique for transient pore formation in adherent cells. Because adherent cells do not have a simple and consistent shape and because the electric field emanating from the tip of the capillary is inhomogeneous, the Schwan equation based on spherical cells in homogeneous electrical fields does not apply. We sought to determine experimental and cell parameters that influence the outcome of a single-cell electroporation experiment. A549 cells were exposed to the thiol-reactive dye Thioglo-1, leading to green fluorescence from intracellular thiol adducts. Electroporation causes a decrease with time of the intracellular fluorescence intensity of Thioglo-1-loaded cells from diffusive loss of thiol adducts. The transient curves thus obtained are well-described by a simple model originally developed by Puc et al. We find that the final fluorescence following electroporation is related to the capillary tip-to-cell distance and cell size (specifically, 2(A/pi)(1/2) where A is the area of the cell's image in pixels. This quantity is the diameter if the image is a circle). In separate experiments, the relationship obtained can be used to control the final fluorescence following electroporation by adjusting the tip-to-cell distance based on cell size. The relationship was applied successfully to A549 as Well as DU 145 and PC-3 cells. Finally, F-tests show that the variability in the final fluorescence (following electroporation) is decreased when the tip-to-cell distance is controlled according to the derived relationship in comparison to experiments in which the tip-cell distance is a constant irrespective of cell size.
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| 17. |
- Agarwal, A., et al.
(författare)
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Effect of cell size and shape on single-cell electroporation
- 2007
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 79:10, s. 3589-3596
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Tidskriftsartikel (refereegranskat)abstract
- Single-cell electroporation was performed using electrolyte-filled capillaries on fluorescently labeled A549 cells. Cells were exposed to brief pulses (50-300 ms) at various cell-capillary tip distances. Cell viability and electroporation success were measured. In order to understand the variability in single-cell electroporation, logistic regression was used to determine whether the probabilities of cell survival and electroporation depend on experimental conditions and cell properties. Both experimental conditions and cell properties (size and shape) have a significant effect on the outcome. Finite element simulations were used to compare bulk electroporation to single-cell electroporation in terms of cell size and shape. Cells are more readily permeabilized and are more likely to survive if they are large and hemispherical as opposed to small and ellipsoidal with a high aspect ratio. The dependence of the maximum transmembrane potential across the cell membrane on cell size is much weaker than it is for bulk electroporation. Observed survival probabilities are related to the calculated fraction of the cell's surface area that is electroporated. Observed success of electroporation is related to the maximum transmembrane potential achieved.
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| 18. |
- Agarwal, A., et al.
(författare)
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Simultaneous maximization of cell permeabilization and viability in single-cell electroporation using an electrolyte-filled capillary
- 2007
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 79:1, s. 161-167
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Tidskriftsartikel (refereegranskat)abstract
- A549 cells were briefly exposed to Thioglo-1, which converts thiols to fluorescent adducts. The fluorescent cells were exposed to short (50-300 ms) electric field pulses (500 V across a 15 cm capillary) created at the tip of an electrolyte-filled capillary. Fluorescence microscopy revealed varying degrees of cell permeabilization depending on the conditions. Longer pulses and a shorter cell-capillary tip distance led to a greater decrease in the cell's fluorescence. Live/dead (calcein AM and propidium iodide) testing revealed that a certain fraction of cells died. Longer pulses and shorter cell-capillary tip distances were more deadly. An optimum condition exists at a cell-capillary tip distance of 3.5-4.5 mu m and a pulse duration of 120-150 ms. At these conditions, > 90% of the cells are permeabilized and 80-90% survive.
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| 19. |
- Agmo Hernández, Víctor, et al.
(författare)
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Label-Free Characterization of Peptide-Lipid Interactions Using Immobilized Lipodisks
- 2013
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 85:15, s. 7377-7384
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Tidskriftsartikel (refereegranskat)abstract
- Lipodisks, planar lipid bilayer structures stabilized by PEG-ylated lipids, were in the present study covalently bound and immobilized onto sensors for quartz crystal microbalance with dissipation monitoring (QCM-D) studies. It is shown that the modified sensors can be used to characterize the interaction of lipodisks with α-helical amphiphilic peptides with an accuracy similar to that obtained with well established fluorimetric approximations. The method presented has the great advantage that it can be used with peptides in their native form even if no fluorescent residues are present. The potential of the method is illustrated by determining the parameters describing the association of melittin, mastoparan X, and mastoparan with immobilized lipodisks. Both thermodynamic and kinetic analyses are possible. The presented method constitutes a useful tool for fundamental studies of peptide–membrane interactions and can also be applied to optimize the design of lipodisks, for example, for sustained release of antimicrobial peptides in therapeutic applications.
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| 20. |
- Ahn, Ji-Young, et al.
(författare)
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Sol-Gel Derived Nanoporous Compositions for Entrapping Small Molecules and Their Outlook toward Aptamer Screening
- 2012
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 1520-6882 .- 0003-2700. ; 84:6, s. 2647-2653
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Tidskriftsartikel (refereegranskat)abstract
- This paper reports for the first time the application of sol-gel microarrays for immobilizing nonsoluble small chemicals (Bisphenol-A; BPA). Also, known problems of sol-gel adhesion to conventional microtiter well plate substrates are circumvented by anchoring the sol-gel microspots to a porous silion surface so-called, PS-SG chips. We confirmed low molecular weight chemical immobilization inside a sol-gel network using fluorescein. BPA and the BPA specific aptamer were utilized as a model pair to verify the affinity specific interaction in the PS-SG selection system. The aptamer interacted specifically with BPA in the sol-gel spots, as shown in microarrays forming the letters "L", "U", "N", and "D". Moreover, the bound aptamer was released by heat, recovered, and verified by gel electrophoresis. The developed PS-SG chip platform will be used for screening aptamers against numerous small molecules such as toxins, metabolites, or pesticide residues.
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| 21. |
- Ainla, Alar, 1982, et al.
(författare)
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A Microfluidic Diluter Based on Pulse Width Flow Modulation
- 2009
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 81:13, s. 5549-5556
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Tidskriftsartikel (refereegranskat)abstract
- We demonstrate that pulse width flow modulation (PWFM) can be used to design fasts accurate, and precise multi-stage dilution modules for microfluidic devices. The PWFM stage unit presented here yields 10-fold dilution, but several PWFM stages can be connected in series to yield higher-order dilutions. We have combined two stages in a device thus capable of diluting up to 100-fold, and we have experimentally determined a set of rules that can be conveniently utilized to design multistage diluters. Microfabrication with resist-based molds yielded geometrical channel height variances of 7% (22.9(16) mu m) with corresponding hydraulic resistance variances of similar to 20%. Pulsing frequencies, channel lengths, and flow pressures can be chosen within a wide range to establish the desired diluter properties. Finally, we illustrate the benefits of on-chip dilution in an example application where we investigate the effect of the Ca2+ concentration on a phospholipid bilayer spreading from a membrane reservoir onto a SiO2 surface. This is one of many possible applications where flexible concentration control is desirable.
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| 22. |
- Ainla, Alar, 1982, et al.
(författare)
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A Microfluidic Pipette for Single-Cell Pharmacology
- 2010
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 82:11, s. 4529-4536
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Tidskriftsartikel (refereegranskat)abstract
- We report on a free-standing microfluidic pipette made in poly(dimethylsiloxane) having a circulating liquid tip that generates a self-con-fining volume in front of the outlet channels. The method is flexible and scalable as the geometry and the size of the recirculation zone is defined by pressure, channel number, and geometry. The pipette is capable of carrying out a variety of complex fluid processing operations, such as mixing, multiplexing, or gradient generation at selected cells in cell and tissue cultures. Using an uptake assay, we show that it is possible to generate dose response curves in situ from adherent Chinese hamster ovary cells expressing proton-activated human transient receptor potential vanilloid (hTRPV1) receptors. Using confined superfusion and cell stimulation, we could activate hTRPV1 receptors in single cells, measure the response by a patch-clamp pipette, and induce membrane bleb formation by exposing selected groups of cells to formaldehyde/dithiothreitol-containing solutions, respectively. In short, the microfluidic pipette allows for complex, contamination-free multiple-compound delivery for pharmacological screening of intact adherent cells.
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| 23. |
- Akter, Farhima, et al.
(författare)
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Detection of Antigens Using a Protein-DNA Chimera Developed by Enzymatic Covalent Bonding with phiX Gene A
- 2012
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 84:11, s. 5040-5046
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Tidskriftsartikel (refereegranskat)abstract
- The chemical reactions used to make antibody DNA conjugates in many immunoassays diminish antigen-binding activity and yield heterogeneous products. Here, we address these issues by developing an antibody-based rolling circle amplification (RCA) strategy using a fusion of phi X174 gene A* protein and Z(mab2s) (A*-Zmab). The phi X174 gene A* protein is an enzyme that can covalently link with DNA, while the Z(mab2s) protein moiety can bind to specific species of antibodies. The DNA in an A*-Zmab conjugate was attached to the A* protein at a site chosen to not interfere with protein function, as determined by enzyme-linked immunosorbent assay (ELISA) and gel mobility shift analysis. The novel A*-Zmab-DNA conjugate retained its binding capabilities to a specific class of murine immunoglobulin gamma 1 (IgG1) but not to rabbit IgG. This indicates the generality of the A*-Zmab-based immuno-RCA assay that can be used in-sandwich ELISA format. Moreover, the enzymatic covalent method dramatically increased the yields of A*-Zmab-DNA conjugates up to 80% after a 15 min reaction. Finally, sensitive detection of human interferon-gamma (IFN-gamma) was achieved by immuno-RCA using our fusion protein in sandwich ELISA format. This new approach of the use of site-specific enzymatic DNA conjugation to proteins should be applicable to fabrication of novel immunoassays for biosensing.
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| 24. |
- Al-Amin, Rasel A., PhD student, 1983-, et al.
(författare)
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Sensitive Measurement of Drug-Target Engagement by a Cellular Thermal Shift Assay with Multiplex Proximity Extension Readout
- 2021
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 93:31, s. 10999-11009
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- The ability to monitor target engagement in cellular contexts is a key for successful drug discovery and also valuable in clinical routine. A cellular thermal shift assay (CETSA) provides realistic information about drug binding in cells and tissues, revealing drug-target engagement in clinically relevant samples. The CETSA combined with mass spectrometry (MS) detection can be applied in the early hit identification phase to generate target engagement data for large sets of proteins. However, the analysis is slow, requires substantial amounts of the sample material, and often misses proteins of specific interest. Here, we combined the CETSA and the multiplex proximity extension assay (PEA) for analysis of target engagement of a set of 67 proteins from small amounts of the sample material treated with kinase inhibitors. The results were concordant with the corresponding analyses read out via MS. Our approach allows analyses of large numbers of specific target proteins at high sensitivity in limited sample aliquots. Highly sensitive multiplex CETSA-PEA assays are therefore promising for monitoring drug-target engagement in small sample aliquots in the course of drug development and potentially in clinical settings.
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| 25. |
- Al-Amin, Rasel Abdullah, Researcher, 1983-, et al.
(författare)
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Sensitive protein detection using site-specifically oligonucleotide-conjugated nanobody reagents
- 2022
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 98:28, s. 10054-10061
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Tidskriftsartikel (refereegranskat)abstract
- High-quality affinity probes are critical for sensitive and specific protein detection, in particular for detection of protein biomarkers in the early phases of disease development. Proximity extension assays (PEAs) have been used for high-throughput multiplexed protein detection of up to a few thousand different proteins in one or a few microliters of plasma. Clonal affinity reagents can offer advantages over the commonly used polyclonal antibodies (pAbs) in terms of reproducibility and standardization of such assays. Here, we explore nanobodies (Nbs) as an alternative to pAbs as affinity reagents for PEA. We describe an efficient site-specific approach for preparing high-quality oligo-conjugated Nb probes via enzyme coupling using Sortase A (SrtA). The procedure allows convenient removal of unconjugated affinity reagents after conjugation. The purified high-grade Nb probes were used in PEA, and the reactions provided an efficient means to select optimal pairs of binding reagents from a group of affinity reagents. We demonstrate that Nb-based PEA (nano-PEA) for interleukin-6 (IL6) detection can augment assay performance, compared to the use of pAb probes. We identify and validate Nb combinations capable of binding in pairs without competition for IL6 antigen detection by PEA.
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| 26. |
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| 27. |
- Alhamimi, Said, et al.
(författare)
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Carbon Dioxide Expanded Ethanol Extraction : Solubility and Extraction Kinetics of α-Pinene and cis-Verbenol
- 2016
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 88:8, s. 4336-4345
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Tidskriftsartikel (refereegranskat)abstract
- In general, diffusion rates in extractions are enhanced by increasing the temperature. In this study, we instead add compressed liquid carbon dioxide to the extraction phase to accomplish faster mass transfer. The feasibility of using carbon dioxide expanded ethanol (CXE) as the extraction phase was explored, targeting two medium-polar analytes, α-pinene and cis-verbenol in Boswellia sacra tree resin. Hansen solubility parameters (HSP) were first calculated for the analytes and the extraction phases investigated, ethanol, CXE, and supercritical carbon dioxide (scCO2) containing ethanol as a cosolvent. Second, an extraction method with CXE as the extraction phase was optimized using a Box Behnken design, giving optimal conditions of 40 °C, 9.3 MPa, and 0.31 molar fraction of CO2 in ethanol. Third, the developed method was compared with a supercritical fluid extraction (SFE) method and a conventional solid liquid extraction (SLE) method, showing that CXE enables faster and more efficient extraction than both SFE and SLE. In fact, calculations based on Peleg's equation showed that the initial extraction rate of the new method is up to 10 times faster than SFE when using the highest flow rate tested, 3 mL/min. It was also discovered that it is crucial to cool the makeup solvent in the collection system for efficient analyte collection, at least in modern SFE equipment where pressure is regulated by a backpressure regulator. The use of CXE and pertinently also other CO2-expanded liquids in sample preparation shows a great potential in terms of increasing the extraction rate without elevating the temperature.
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| 28. |
- Allard, Erik, et al.
(författare)
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Comparing capillary electrophoresis : mass spectrometry fingerprints of urine samples obtained after intake of coffee, tea, or water.
- 2008
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 80:23, s. 8946-8955
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Tidskriftsartikel (refereegranskat)abstract
- Metabolomic fingerprinting is a growing strategy for characterizing complex biological samples without detailed prior knowledge about the metabolic system. A two-way analysis system with liquid separation and mass spectrometric detection provides detail-rich data suitable for such fingerprints. As a model study, human urine samples, obtained after intake of coffee, tea, or water, were analyzed with capillary electrophoresis electrospray ionization time-of-flight mass spectrometry (CE−ESI-TOF-MS). In-house-developed software (in Matlab) was utilized to manage and explore the large amount of data acquired (230 CE−MS runs, each with 50−100 million nonzero data points). After baseline and noise reduction, followed by suitable binning in time and m/z, the data sets comprised 9 and 14 million data points in negative and positive ESI mode, respectively. Finally, a signal threshold was applied, further reducing the number to about 100 000 data points per data set. A set of interactive exploratory tools, utilizing principal component analysis (PCA) and analysis of variance (ANOVA) results based on a general linear model, facilitated visual interpretation with score plots (for group assessment) and differential fingerprints (for “hot spot” detection). In the model study highly significant differences due to beverage intake were obtained among the 10 first principal components (p < 10−6 for two of the components in both ESI modes). Especially, the contrasts between “coffee” and “tea or water” indicated several “hot spots” with highly elevated intensities (e.g., for uncharged masses 93, 94, 109, 119, 123, 132, 148, 169, 178, 187, 190, and 193) suitable for further analysis, for example, with tandem MS.
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| 29. |
- Alleso, Morten, et al.
(författare)
-
Near-infrared spectroscopy for cocrystal screening : a comparative study with Raman spectroscopy
- 2008
-
Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 80:20, s. 7755-7764
-
Tidskriftsartikel (refereegranskat)abstract
- Near-infrared (NIR) spectroscopy is a well-established technique for solid-state analysis, providing fast, noninvasive measurements. The use of NIR spectroscopy for polymorph screening and the associated advantages have recently been demonstrated. The objective of this work was to evaluate the analytical potential of NIR spectroscopy for cocrystal screening using Raman spectroscopy as a comparative method. Indomethacin was used as the parent molecule, while saccharin and L-aspartic acid were chosen as guest molecules. Molar ratios of 1:1 for each system were subjected to two types of preparative methods. In the case of saccharin, liquid-assisted cogrinding as well as cocrystallization from solution resulted in a stable 1:1 cocrystalline phase termed IND-SAC cocrystal. For L-aspartic acid, the solution-based method resulted in a polymorphic transition of indomethacin into the metastable a form retained in a physical mixture with the guest molecule, while liquid-assisted cogrinding did not induce any changes in the crystal lattice. The good chemical peak selectivity of Raman spectroscopy allowed a straightforward interpretation of sample data by analyzing peak positions and comparing to those of pure references. In addition, Raman spectroscopy provided additional information on the crystal structure of the IND-SAC cocrystal. The broad spectral line shapes of NIR spectra make visual interpretation of the spectra difficult, and consequently, multivariate modeling by principal component analysis (PCA) was applied. Successful use of NIR/PCA was possible only through the inclusion of a set of reference mixtures of parent and guest molecules representing possible solid-state outcomes from the cocrystal screening. The practical hurdle related to the need for reference mixtures seems to restrict the applicability of NIR spectroscopy in cocrystal screening.
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| 30. |
- Allison, Timothy M., et al.
(författare)
-
Computational Strategies and Challenges for Using Native Ion Mobility Mass Spectrometry in Biophysics and Structural Biology
- 2020
-
Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 92:16, s. 10872-10880
-
Tidskriftsartikel (refereegranskat)abstract
- Native mass spectrometry (MS) allows the interrogation of structural aspects of macromolecules in the gas phase, under the premise of having initially maintained their solution-phase noncovalent interactions intact. In the more than 25 years since the first reports, the utility of native MS has become well established in the structural biology community. The experimental and technological advances during this time have been rapid, resulting in dramatic increases in sensitivity, mass range, resolution, and complexity of possible experiments. As experimental methods have improved, there have been accompanying developments in computational approaches for analyzing and exploiting the profusion of MS data in a structural and biophysical context. In this perspective, we consider the computational strategies currently being employed by the community, aspects of best practice, and the challenges that remain to be addressed. Our perspective is based on discussions within the European Cooperation in Science and Technology Action on Native Mass Spectrometry and Related Methods for Structural Biology (EU COST Action BM1403), which involved participants from across Europe and North America. It is intended not as an in-depth review but instead to provide an accessible introduction to and overview of the topic—to inform newcomers to the field and stimulate discussions in the community about addressing existing challenges. Our complementary perspective (http://dx.doi.org/10.1021/acs.analchem.9b05792) focuses on software tools available to help researchers tackle some of the challenges enumerated here.
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| 31. |
- Allison, Timothy M., et al.
(författare)
-
Software Requirements for the Analysis and Interpretation of Native Ion Mobility Mass Spectrometry Data
- 2020
-
Ingår i: Analytical Chemistry. - : American Chemical Society. - 0003-2700 .- 1520-6882. ; 92:16, s. 10881-10890
-
Tidskriftsartikel (refereegranskat)abstract
- The past few years have seen a dramatic increase in applications of native mass and ion mobility spectrometry, especially for the study of proteins and protein complexes. This increase has been catalyzed by the availability of commercial instrumentation capable of carrying out such analyses. As in most fields, however, the software to process the data generated from new instrumentation lags behind. Recently, a number of research groups have started addressing this by developing software, but further improvements are still required in order to realize the full potential of the data sets generated. In this perspective, we describe practical aspects as well as challenges in processing native mass spectrometry (MS) and ion mobility-MS data sets and provide a brief overview of currently available tools. We then set out our vision of future developments that would bring the community together and lead to the development of a common platform to expedite future computational developments, provide standardized processing approaches, and serve as a location for the deposition of data for this emerging field. This perspective has been written by members of the European Cooperation in Science and Technology Action on Native MS and Related Methods for Structural Biology (EU COST Action BM1403) as an introduction to the software tools available in this area. It is intended to serve as an overview for newcomers and to stimulate discussions in the community on further developments in this field, rather than being an in-depth review. Our complementary perspective (http://dx.doi.org/10.1021/acs.analchem.9b05791) focuses on computational approaches used in this field.
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| 32. |
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| 33. |
- Almstrand, Ann-Charlotte, et al.
(författare)
-
Airway monitoring by collection and mass spectrometric analysis of exhaled particles.
- 2009
-
Ingår i: Analytical chemistry. - : American Chemical Society (ACS). - 1520-6882 .- 0003-2700. ; 81:2, s. 662-8
-
Tidskriftsartikel (refereegranskat)abstract
- We describe a new method for simultaneously collecting particles in exhaled air for subsequent chemical analysis and measuring their size distribution. After forced exhalation, particles were counted and collected in spots on silicon wafers with a cascade impactor. Several phospholipids were identified by time-of-flight secondary ion mass spectrometric analysis of the collected spots, suggesting that the particles originated from the lower airways. The amount of particles collected in ten exhalations was sufficient for characterizing the phospholipid composition. The feasibility of the technique in respiratory research is demonstrated by analysis of the phospholipid composition of exhaled particles from healthy controls, patients with asthma, and patients with cystic fibrosis. We believe this technology will be useful for monitoring patients with respiratory disease and has a high potential to detect new biomarkers in exhaled air.
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| 34. |
- Amini, Nahid, et al.
(författare)
-
Screening and Quantification of Pesticides in Water Using a Dual-Function Graphitized Carbon Black Disk
- 2010
-
Ingår i: Analytical Chemistry. - : ACS. - 0003-2700 .- 1520-6882. ; 82:1, s. 290-296
-
Tidskriftsartikel (refereegranskat)abstract
- A simple platform for combining solid phase extraction (SPE) and surface-assisted laser desorption ionization mass spectrometry (SALDI-MS) of extracted analytes, using disks prepared by embedding graphitized carbon black (GCB-4) particles in a network of polytetrafluoroethylene (PTFE), is presented. The system provides a convenient approach for rapid SALDI-MS screening of substances in aqueous samples, which can be followed by robust quantitative and/or structural analyses by liquid chromatography (LC)/MS/MS of positive samples. The extraction discs are easily transferred between gaskets where the sample extraction and desorption of selected samples is performed and the mass spectrometer. The SPE and SALDI properties of the new GCB-4 disc have been characterized for 15 pesticides with varying chemical properties, and the screening strategy has been applied to the analysis of pesticides in agricultural drainage water. Atrazine and atrazine-desethyl-2-hydroxy were detected in the sampled water by SALDI-MS screening and subsequently confirmed and quantified using LC/MS/MS.
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| 35. |
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| 36. |
- Andersson, Martin, et al.
(författare)
-
A microfluidic control board for high-pressure flow, composition, and relative permittivity
- 2018
-
Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 90:21, s. 12601-12608
-
Tidskriftsartikel (refereegranskat)abstract
- Flow control is central to microfluidics and chromatography. With decreasing dimensions and high pressures, precise fluid flows are often needed. In this paper, a high-pressure flow control system is presented, allowing for the miniaturization of chromatographic systems and the increased performance of microfluidic setups by controlling flow, composition and relative permittivity of two-component flows with CO2. The system consists of four chips: two flow actuator chips, one mixing chip and one relative permittivity sensor. The actuator chips, throttling the flow, required no moving parts as they instead relied on internal heaters to change the fluid resistance. This allows for flow control using miniaturized fluid delivery systems containing only a single pump or pressure source. Mobile phase gradients between 49% to 74% methanol in CO2 were demonstrated. Depending on how the actuator chips were dimensioned, the position of this range could be set for different method-specific needs. With the microfluidic control board, both flow and composition could be controlled from constant pressure sources, drift could be removed, and variations in composition could be lowered by 84%, resulting in microflows of CO2 and methanol with a variation in the composition of 0.30%.
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| 37. |
- Andersson, M, et al.
(författare)
-
Accuracy in multiangle light scattering measurements for molar mass and radius estimations. Model calculations and experiments.
- 2003
-
Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 1520-6882 .- 0003-2700. ; 75:16, s. 4279-4291
-
Tidskriftsartikel (refereegranskat)abstract
- Multiangle light scattering (MALS) is a well-established technique used to determine the size of macromolecules and particles. In this study, different extrapolation procedures used in MALS were investigated with regard to accuracy and robustness in the obtained molar mass and rms radius. Three different mathematical transformations of the light scattering function referred to as the Debye, Zimm, and Berry methods for constructing the Debye plot were investigated for two idealized polymer shapes, homogeneous spheres and random coils, with radii from 25 to 250 nm. The effect of the angular interval used for the extrapolation was investigated, as was the robustness of the different transformations toward errors in the measured light scattering intensity at low angles. For an rms radius less than 50 nm, the relative error in molar mass due to extrapolation was less than 1% independent of the method used. For larger radii, the error increased and the extrapolation procedure became more critical. For random coil polymers, the Berry method was superior in terms of accuracy and robustness. For spheres, the Debye method was superior. The Zimm method was inferior to the others. The different extrapolation methods were evaluated and compared on experimental data from a size exclusion chromatography-MALS analysis of an ultrahigh molar mass poly(ethylene oxide) (PEO). The PEO data qualitatively verified the calculations and stressed the importance of optimizing the extrapolation procedure after careful evaluation of the experimental data. A discussion of how to detect erroneous data in an experimental Debye plot is given.
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| 38. |
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| 39. |
- Angerer, Tina B., 1987, et al.
(författare)
-
Improved Molecular Imaging in Rodent Brain with Time-of-Flight-Secondary Ion Mass Spectrometry Using Gas Cluster Ion Beams and Reactive Vapor Exposure
- 2015
-
Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 87:8, s. 4305-4313
-
Tidskriftsartikel (refereegranskat)abstract
- Imaging mass spectrometry has shown to be a valuable method in medical research and can be performed using different instrumentation and sample preparation methods, each one with specific advantages and drawbacks. Time-of-flight-secondary ion mass spectrometry (TOF-SIMS) has the advantage of high spatial resolution imaging but is often restricted to low mass molecular signals and can be very sensitive to sample preparation artifacts. In this report we demonstrate the advantages of using gas cluster ion beams (GCIBs) in combination with trifluoracetic acid (TFA) vapor exposure for the imaging of lipids in mouse brain sections. There is an optimum exposure to TFA that is beneficial for increasing high mass signal as well as producing signal from previously unobserved species in the mass spectrum. Cholesterol enrichment and crystallization on the sample surface is removed by TFA exposure uncovering a wider range of lipid species in the white matter regions of the tissue, greatly expanding the chemical coverage and the potential application of TOF-SIMS imaging in neurological studies. Ar-4000(+) (40 keV) in combination with TFA treatment facilitates high resolution, high mass imaging closing the gap between TOF-SIMS and matrix-assisted laser desorption ionization (MALDI).
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| 40. |
- Angerer, Tina B., 1987, et al.
(författare)
-
Lipid Heterogeneity Resulting from Fatty Acid Processing in the Human Breast Cancer Microenvironment Identified by GCIB-ToF-SIMS Imaging
- 2016
-
Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 88:23, s. 11946-54
-
Tidskriftsartikel (refereegranskat)abstract
- Breast cancer is an umbrella term used to describe a collection of different diseases with broad inter- and intratumor heterogeneity. Understanding this variation is critical in order to develop, and precisely prescribe, new treatments. Changes in the lipid metabolism of cancerous cells can provide important indications as to the metabolic state of the cells but are difficult to investigate with conventional histological methods. Due to the introduction of new higher energy (40 kV) gas cluster ion beams (GCIBs), time-of-flight secondary ion mass spectrometry (ToF-SIMS) imaging is now capable of providing information on the distribution of hundreds of molecular species simultaneously on a cellular to subcellular scale. GCIB-ToF-SIMS was used to elucidate changes in lipid composition in nine breast cancer biopsy samples. Improved molecular signal generation by the GCIB produced location-specific information that revealed elevated levels of essential lipids to be related to inflammatory cells in the stroma, while cancerous areas were dominated by nonessential fatty acids and a variety of phosphatidylinositol species with further in-tumor variety arising from decreased desaturase activity. These changes in lipid composition due to different enzyme activity are seemingly independent of oxygen availability and can be linked to favorable cell membrane properties for either proliferation/invasion or drug resistance/survival
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| 41. |
- Antfolk, Maria, et al.
(författare)
-
Acoustofluidic, label-free separation and simultaneous concentration of rare tumor cells from white blood cells
- 2015
-
Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 1520-6882 .- 0003-2700. ; 87:18, s. 9322-9328
-
Tidskriftsartikel (refereegranskat)abstract
- Enrichment of rare cells from peripheral blood has emerged as a means to enable noninvasive diagnostics and development of personalized drugs, commonly associated with a prerequisite to concentrate the enriched rare cell population prior to molecular analysis or culture. However, common concentration by centrifugation has important limitations when processing low cell numbers. Here, we report on an integrated acoustophoresis-based rare cell enrichment system combined with integrated concentration. Polystyrene 7 μm microparticles could be separated from 5 μm particles with a recovery of 99.3 ± 0.3% at a contamination of 0.1 ± 0.03%, with an overall 25.7 ± 1.7-fold concentration of the recovered 7 μm particles. At a flow rate of 100 μL/min, breast cancer cells (MCF7) spiked into red blood cell-lysed human blood were separated with an efficiency of 91.8 ± 1.0% with a contamination of 0.6 ± 0.1% from white blood cells with a 23.8 ± 1.3-fold concentration of cancer cells. The recovery of prostate cancer cells (DU145) spiked into whole blood was 84.1 ± 2.1% with 0.2 ± 0.04% contamination of white blood cells with a 9.6 ± 0.4-fold concentration of cancer cells. This simultaneous on-chip separation and concentration shows feasibility of future acoustofluidic systems for rapid label-free enrichment and molecular characterization of circulating tumor cells using peripheral venous blood in clinical practice.
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| 42. |
- Appelblad, Patrik, et al.
(författare)
-
Determination of C-21 ketosteroids in serum using trifluoromethanesulfonic acid catalyzed precolumn dansylation and 1,1’-oxalyldiimidazole postcolumn peroxyoxalate chemiluminescence detection
- 1998
-
Ingår i: Analytical Chemistry. - Washington : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 70:23, s. 5002-5009
-
Tidskriftsartikel (refereegranskat)abstract
- A new procedure for the quantitation of C-21 ketosteroids using trifluoromethanesulfonic acid-catalyzed precolumn dansylation and coupled column liquid chromatographic separation, followed by postcolumn 1,1‘-oxalyldiimidazole peroxyoxalate chemiluminescence detection is presented. In the simultaneous optimization of chromatographic resolution and chemiluminescence intensity, a coupled column chromatographic system and a stopped-flow system were used. An eluent containing 20 mM phosphate buffer at pH 6.7 accomplished an efficient separation of 3α-hydroxy-5β-pregnan-20-one from a mixture containing 10 C-21 ketosteroids. Phosphate buffer also proved to be the most advantageous, among the six buffers tested, for sensitive detection. Experimental design and multivariate data analysis were used to characterize and optimize the postcolumn reaction chemistry in the chromatographic system. A valid full factorial design with excellent predictability showed that the flow rates for both 1,1‘-oxalyldiimidazole and hydrogen peroxide were the factors most strongly affecting the sensitivity of the system. The theoretical plate numbers were above 11 000 for all 10 dansylated ketosteroids. The 3σ detection limit estimated from 3α-hydroxy-5β-pregnan-20-one calibration curve data was 1.6 pmol (n = 4, 125 μL injected) and spiked serum containing 0−74 pmol of this compound showed overall recoveries of 73 ± 9% (n = 12). Quantitation of 3α-hydroxy-5β-pregnan-20-one was finally carried out on 45 serum samples and the results compared to those from a radioimmunoassay (RIA) method. The data acquired with the procedure described in this work compare well with the results from RIA, which confirms the reliability of the new analytical procedure.
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| 43. |
- Appelblad, P.K., et al.
(författare)
-
Sources of uncertainty in isotope ratio measurements by inductively coupled plasma mass spectrometry
- 2001
-
Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 73:13, s. 2911-2919
-
Tidskriftsartikel (refereegranskat)abstract
- A model is presented describing the effects of dead time and mass bias correction factor uncertainties, flicker noise, and counting statistics on isotope ratio measurement precision using inductively coupled plasma mass spectrometry (ICPMS) with a single collector. Noise spectral analysis is exploited to enable estimation of the flicker noise parameters. For the instrument used, the flicker noise component exhibited a fairly weak frequency (f) dependence ( f -0.33±0.12), but was directly proportional to the total number of counts, Q. As white noise, determined by counting statistics, is given by Q0.5, the isotope ratio measurement uncertainties will actually cease to improve when Q exceeds a certain threshold. This would suggest that flicker noise could become the limiting factor for the precision with which isotope ratios can be determined by ICPMS. However, under most experimental conditions, uncertainties associated with mass discrimination and dead time correction factors are decisive. For ratios up to ~22 (115In/113In), optimum major isotope count rates are generally below 0.3 MHz, for which precision in the mass discrimination factor is limiting. The model derived could be used as a starting point for determining optimum conditions and understanding the limitations of single-collector ICPMS for precise isotope ratio measurements.
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| 44. |
- Appelblad, Patrik, et al.
(författare)
-
Perfluorosulfonated Ionomer-Modified Polyethylene. A Material for Simultaneous Solid-Phase Enrichment and Enhanced Precolumn Dansylation of C-21 Ketosteroids in Human Serum
- 2001
-
Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 73:15, s. 3701-8
-
Tidskriftsartikel (refereegranskat)abstract
- A new derivatization procedure has been developed where solid-phase catalysis is utilized to facilitate the formation of hydrazones in precolumn labeling of keto-containing compounds. This procedure has been implemented on a solid-phase enrichment and enhanced derivatization (SPEED) device, prepared from porous polyethylene that has been coated with Nafion and dansylhydrazine. The SPEED devices have been optimized using experimental design and characterized for dansylation of C-21 ketosteroids by multivariate data analysis, using progesterone as the model compound. The reaction temperature and the molar ratio between the steroid and the derivatization reagent were found to be the factors most strongly affecting the reaction. Faster reaction kinetics were achieved when the molar ratio between dansylhydrazine and the steroid was increased. Mass spectroscopic analysis showed that the four derivative peaks eluting when derivatized progesterone was separated on an octadecyl silica stationary phase were due to the syn and anti mono- and bis(hydrazones) formed in the reaction. Using optimal reaction conditions, the derivatives mainly constitute the syn and anti conformers of bis-derivatives. In contrast to solution-based acid catalysis, the SPEED device was remarkably insensitive to water in the reaction mixture. A sample volume of 400 L was found to be the maximum, enabling sample enrichment prior derivatization. Using optimal experimental conditions, picomole amounts of ketosteroids could be derivatized in 10 min at room temperature. Analysis of spiked serum samples containing 0.4-2.0 nmol of progesterone showed overall recoveries of 52-63%. The corresponding 3 detection limit was 1.3 pmol (n = 4, 100 L injected), as estimated from calibration curve data.
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| 45. |
- Araújo, Ana Catarina, et al.
(författare)
-
Activated Paper Surfaces for the Rapid Hybridization of DNA through Capillary Transport
- 2012
-
Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 84:7, s. 3311-3317
-
Tidskriftsartikel (refereegranskat)abstract
- The development of low-cost, accurate, and equipment-free diagnostic tests is crucial to many clinical, laboratory, and field applications, including forensics and medical diagnostics. Cellulose fiber-based paper is an inexpensive, biodegradable, and renewable resource, the use of which as a biomolecule detection matrix and support confers several advantages compared to traditional materials such as glass. In this context, a new, facile method for the preparation of surface functionalized papers bearing single-stranded probe DNA (ssDNA) for rapid target hybridization via capillary transport is presented. Optimized reaction conditions were developed that allowed the direct, one-step activation of standard laboratory filters by the inexpensive and readily available bifunctional linking reagent, 1,4-phenylenediisothiocyanate. Such papers were thus amenable to subsequent coupling of amine-labeled ssDNA under standard conditions widely used for glass-based supports. The intrinsic wicking ability of the paper matrix facilitated rapid sample elution through arrays of probe DNA, leading to significant, detectable hybridization in the time required for the sample liquid to transit the vertical length of the strip (less than 2 min). The broad applicability of these paper test strips as rapid and specific diagnostics in "real-life" situations was exemplified by the discrimination of amplicons generated from canine and human mitochondrial and genomic DNA in mock forensic samples.
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| 46. |
- Aref, Mohaddeseh, et al.
(författare)
-
Potentiometric pH Nanosensor for Intracellular Measurements: Real-Time and Continuous Assessment of Local Gradients
- 2021
-
Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 93:47, s. 15744-15751
-
Tidskriftsartikel (refereegranskat)abstract
- We present a pH nanosensor conceived for single intracellular measurements. The sensing architecture consisted of a two-electrode system evaluated in the potentiometric mode. We used solid-contact carbon nanopipette electrodes tailored to produce both the indicator (pH nanosensor) and reference electrodes. The indicator electrode was a membrane-based ion-selective electrode containing a receptor for hydrogen ions that provided a favorable selectivity for intracellular measurements. The analytical features of the pH nanosensor revealed a Nernstian response (slope of -59.5 mV/pH unit) with appropriate repeatability and reproducibility (variation coefficients of <2% for the calibration parameters), a fast response time (<5 s), adequate medium-term drift (0.7 mV h(-)(1)), and a linear range of response including physiological and abnormal cell pH levels (6.0-8.5). In addition, the position and configuration of the reference electrode were investigated in cell-based experiments to provide unbiased pH measurements, in which both the indicator and reference electrodes were located inside the same cell, each of them inside two neighboring cells, or the indicator electrode inside the cell and the reference electrode outside of (but nearby) the studied cell. Finally, the pH nanosensor was applied to two cases: (i) the tracing of the pH gradient from extra-to intracellular media over insertion into a single PC12 cell and (ii) the monitoring of variations in intracellular pH in response to exogenous administration of pharmaceuticals. It is anticipated that the developed pH nanosensor, which is a label-free analytical tool, has high potential to aid in the investigation of pathological states that manifest in cell pH misregulation, with no restriction in the type of targeted cells.
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| 47. |
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| 48. |
- Arnell, Robert, et al.
(författare)
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Validation of the Tracer-Pulse Method for Multi-Component Liquid Chromatography. A Classical Paradox Revisited
- 2006
-
Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 78:13, s. 4615-4623
-
Tidskriftsartikel (refereegranskat)abstract
- The tracer-pulse method was extended and validated for the determination of multicomponent adsorption isotherms in liquid chromatography. Competitive adsorption isotherms can be determined for any number of solutes, up to the column resolution limit. The basic principle is to equilibrate the column with an eluent containing a mixture of the solutes and then measure the migration velocity of each of them through the column. It is easy to calculate the stationary phase concentrations from these velocities, given the eluent composition. As in frontal analysis, real competitive isotherm data are measured using this method, unlike other methods, which only produce parametric estimates. The method was used to measure the binary isotherms of beta-blockers on a Kromasil C8 column. The data were fitted to competitive bi-Langmuir adsorption isotherm functions and was found to agree well with the results of frontal analysis and the perturbation method. Computer simulations based on the isotherm parameters were performed and displayed very good agreement with the experimental chromatograms. An intriguing and seemingly paradoxical property is visualized and discussed: the fact that the injected molecules are not found in the detected peaks.
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| 49. |
- Arrhenius, Karine, et al.
(författare)
-
Traceable reference gas mixtures for sulfur-free natural gas odorants
- 2014
-
Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 13:1, s. 6695-6702
-
Tidskriftsartikel (refereegranskat)abstract
- The first reference gas mixtures of sulfur-free natural gas odorants that are traceable to the International System of Units (SI) have been produced and their compositions validated. These mixtures, which contain methyl acrylate and ethyl acrylate at amount fractions between 1.1 and 2.1 μmol mol-1, can be used to underpin measurements of sulfur-free odorants, which are increasingly being used to odorize natural gas in transmission networks as they have less harmful properties than traditional sulfur-containing odorants. The reference gas mixtures produced have been shown to be stable in passivated aluminum cylinders for at least 8 months and have been validated (to within 6% or less) by interlaboratory measurements at three National Measurement Institutes. The stability of methyl acrylate and ethyl acrylate in gas sampling bags has been investigated, and the challenges of analyzing 2-ethyl-3- methylpyrazine, which is used as a stabilizer in sulfur-free odorants, are also briefly discussed.
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| 50. |
- Artemenko, Konstantin A., et al.
(författare)
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Two dimensional mass mapping as a general method of data representation in comprehensive analysis of complex molecular mixtures.
- 2009
-
Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 81:10, s. 3738-3745
-
Tidskriftsartikel (refereegranskat)abstract
- A recent proteomics-grade (95%+ sequence reliability) high-throughput de novo sequencing method utilizes the benefits of high resolution, high mass accuracy, and the use of two complementary fragmentation techniques collision-activated dissociation (CAD) and electron capture dissociation (ECD). With this high-fidelity sequencing approach, hundreds of peptides can be sequenced de novo in a single LC-MS/MS experiment. The high productivity of the new analysis technique has revealed a new bottleneck which occurs in data representation. Here we suggest a new method of data analysis and visualization that presents a comprehensive picture of the peptide content including relative abundances and grouping into families. The 2D mass mapping consists of putting the molecular masses onto a two-dimensional bubble plot, with the relative monoisotopic mass defect and isotopic shift being the axes and with the bubble area proportional to the peptide abundance. Peptides belonging to the same family form a compact group on such a plot, so that the family identity can in many cases be determined from the molecular mass alone. The performance of the method is demonstrated on the high-throughput analysis of skin secretion from three frogs, Rana ridibunda, Rana arvalis, and Rana temporaria. Two dimensional mass maps simplify the task of global comparison between the species and make obvious the similarities and differences in the peptide contents that are obscure in traditional data presentation methods. Even biological activity of the peptide can sometimes be inferred from its position on the plot. Two dimensional mass mapping is a general method applicable to any complex mixture, peptide and nonpeptide alike.
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