| 1. |
- Mikkonen, Saara, et al.
(författare)
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Analysis of amino acids in cell culture supernatant using capillary electrophoresis with contactless conductivity detection
- 2021
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Ingår i: Electrophoresis. - : Wiley. - 0173-0835 .- 1522-2683.
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Tidskriftsartikel (refereegranskat)abstract
- CE-C4D methods for the analysis of amino acids (AAs) are presented. Combining the results from two methods with acetic acid and cyclodextrin-based BGEs, 20 proteinogenic AAs could be analyzed using CE. CE-C4D was also, for the first time, applied to analyze free AAs in samples of mammalian cell culture supernatant. After dilution as only sample preparation, combining the results of the two CE methods allowed monitoring the concentration changes of 17 AAs in samples taken during the cultivation of CHO cells.
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| 2. |
- Mikkonen, Saara, et al.
(författare)
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Capillary and microchip electrophoresis method development for amino acid monitoring during biopharmaceutical cultivation
- 2022
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Ingår i: Biotechnology Journal. - : Wiley. - 1860-6768 .- 1860-7314.
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Tidskriftsartikel (refereegranskat)abstract
- The increased use of biopharmaceuticals calls for improved means of bioprocess monitoring. In this work, capillary electrophoresis (CE) and microchip electrophoresis (MCE) methods were developed and applied for the analysis of amino acids (AAs) in cell culture supernatant. In samples from different days of a Chinese hamster ovary cell cultivation process, all 19 proteinogenic AAs containing primary amine groups could be detected using CE, and 17 out of 19 AAs using MCE. The relative concentration changes in different samples agreed well with those measured by high-performance liquid chromatography (HPLC). Compared to the more commonly employed HPLC analysis, the CE and MCE methods resulted in faster analysis, while significantly lowering both the sample and reagent consumption, and the cost per analysis.
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| 3. |
- Mikkonen, Saara, et al.
(författare)
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Computer simulations of sample preconcentration in carrier-free systems and isoelectric focusing in microchannels using simple ampholytes
- 2015
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Ingår i: Electrophoresis. - : Wiley-VCH Verlagsgesellschaft. - 0173-0835 .- 1522-2683. ; 36:19, s. 2386-2395
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Tidskriftsartikel (refereegranskat)abstract
- In this work, electrophoretic preconcentration of protein and peptide samples in microchannels was studied theoretically using the 1D dynamic simulator GENTRANS, and experimentally combined with MS. In all configurations studied, the sample was uniformly distributed throughout the channel before power application, and driving electrodes were used as microchannel ends. In the first part, previously obtained experimental results from carrier-free systems are compared to simulation results, and the effects of atmospheric carbon dioxide and impurities in the sample solution are examined. Simulation provided insight into the dynamics of the transport of all components under the applied electric field and revealed the formation of a pure water zone in the channel center. In the second part, the use of an IEF procedure with simple well defined amphoteric carrier components, i.e. amino acids, for concentration and fractionation of peptides was investigated. By performing simulations a qualitative description of the analyte behavior in this system was obtained. Neurotensin and [Glu1]-Fibrinopeptide B were separated by IEF in microchannels featuring a liquid lid for simple sample handling and placement of the driving electrodes. Component distributions in the channel were detected using MALDI- and nano-ESI-MS and data were in agreement with those obtained by simulation. Dynamic simulations are demonstrated to represent an effective tool to investigate the electrophoretic behavior of all components in the microchannel.
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| 4. |
- Mikkonen, Saara
(författare)
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Electrophoretic focusing in microchannels combined with mass spectrometry : Applications on amyloid beta peptides
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Analysis of low-abundance components in small samples remains a challenge within bioanalytical chemistry, and new techniques for sample pretreatments followed by sensitive and informative detection are required. In this thesis, procedures for preconcentration and separation of proteins and peptides in open microchannels fabricated on silicon microchips are presented. Analyte electromigration was induced by applying a voltage along the channel length, and detection was performed either by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) within the open channel, or by sampling a nL fraction containing the preconcentrated analytes from the channel for subsequent nano-electrospray ionization- (nESI-) or MALDI-MS. Utilizing solvent evaporation from the open system during sample supply, sample volumes exceeding the 25-75 nL channel volume could be analyzed. For preconcentration/separation of components in the discrete channel volume a lid of inert fluorocarbon liquid was used for evaporation control.In Papers I and II, aqueous, carrier-free solutions of proteins and peptides were analyzed, and the method was successfully applied for fast and simple preconcentration of amyloid beta (Aβ) peptides, related to Alzheimer’s disease.The impact of possible impurities in the analysis of carrier-free solutions was investigated in Paper III with the 1D simulation software GENTRANS, and a method for open-channel isoelectric focusing in a tailor-made pH gradient was developed. The latter approach was used in Paper IV for preconcentration and purification of Aβ peptides after immunoprecipitation from cerebrospinal fluid and blood plasma, followed by MALDI-MS from a micropillar chip.Paper V includes simulations of an isotachophoretic strategy for selective enrichment of Aβ peptides. GENTRANS simulations were used to select the electrolyte composition, and 2D simulations in a geometry suitable for on-chip implementation were performed using COMSOL Multiphysics.
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| 5. |
- Mikkonen, Saara, et al.
(författare)
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High-resolution dynamic computer simulation of electrophoresis using a multiphysics software platform
- 2018
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Ingår i: Journal of Chromatography A. - : Elsevier. - 0021-9673 .- 1873-3778. ; 1532, s. 216-222
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Tidskriftsartikel (refereegranskat)abstract
- The modeling and simulation software COMSOL Multiphysics (R) was recently extended with an electrophoretic transport interface. Its performance was investigated by comparison to results obtained using the 1D dynamic electrophoresis simulators GENTRANS and SIMUL5. Simulations of zone electrophoresis, isotachophoresis, isoelectric focusing and of an oscillating electrolyte system were performed. Smooth profiles were essentially identical indicating that the COMSOL electrophoretic transport interface is able to reproduce results of the 1D simulators. Differences in the way the respective numerical schemes handle steep concentration gradients and associated instabilities were observed. The COMSOL electrophoretic transport interface is expected to be useful as a general model for simulations in 1D, 2D or 3D geometries, as well as for simulations combining electrophoresis with other physical phenomena.
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| 6. |
- Mikkonen, Saara, et al.
(författare)
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Inverse cationic ITP for separation of methadone enantiomers with sulfated beta-cyclodextrin as chiral selector
- 2019
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Ingår i: Electrophoresis. - : WILEY. - 0173-0835 .- 1522-2683. ; 40:5, s. 659-667
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Tidskriftsartikel (refereegranskat)abstract
- Chiral ITP of the weak base methadone using inverse cationic configurations with H+ as leading component and multiple isomer sulfated beta-CD (S-beta-CD) as leading electrolyte (LE) additive, has been studied utilizing dynamic computer simulation, a calculation model based on steady-state values of the ITP zones, and capillary ITP. By varying the amount of acidic S-beta-CD in the LE composed of 3-morpholino-2-hydroxypropanesulfonic acid and the chiral selector, and employing glycylglycine as terminating electrolyte (TE), inverse cationic ITP provides systems in which either both enantiomers, only the enantiomer with weaker complexation, or none of the two enantiomers form cationic ITP zones. For the configuration studied, the data reveal that only S-methadone migrates isotachophoretically when the S-beta-CD concentration in the LE is between about 0.484 and 1.113 mM. Under these conditions, R-methadone migrates zone electrophoretically in the TE. An S-beta-CD concentration between about 0.070 and 0.484 mM results in both S- and R-methadone forming ITP zones. With >1.113 mM and < about 0.050 mM of S-beta-CD in the LE both enantiomers are migrating within the TE and LE, respectively. Chiral inverse cationic ITP with acidic S-beta-CD in the LE is demonstrated to permit selective ITP trapping and concentration of the less interacting enantiomer of a weak base.
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| 7. |
- Mikkonen, Saara, et al.
(författare)
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Mass spectrometric analysis of nanoscale sample volumes extracted from open microchannels after sample preconcentration applied on amyloid beta peptides
- 2014
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Ingår i: Analytical and Bioanalytical Chemistry. - : Springer Science and Business Media LLC. - 1618-2642 .- 1618-2650. ; 406:14, s. 3521-3524
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Tidskriftsartikel (refereegranskat)abstract
- A new instrumental concept for extraction of nanovolumes from open microchannels (dimensions 150 mu m x 50 mu m, length 10 mm) manufactured on silicon microchips has been used in combination with a previously developed method for preconcentrating proteins and peptides in the open channels through electromigration. The extracted nanovolumes were further analyzed using nanoelectrospray ionization (nESI) or matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) directly or with subsequent enzymatic protein digestion in a nanodroplet prior to the MS analysis. Preconcentration of the samples resulted in a 15-fold sensitivity increase in nESI for a neurotensin solution, and using MALDI-MS, amyloid beta (A beta) peptides could be detected in concentrations down to 1 nM. The method was also successfully applied for detection of cell culture A beta.
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| 8. |
- Mikkonen, Saara, et al.
(författare)
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Microfluidic Isoelectric Focusing of Amyloid Beta Peptides Followed by Micropillar-Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry
- 2016
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 88:20, s. 10044-10051
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Tidskriftsartikel (refereegranskat)abstract
- A novel method for preconcentration and purification of the Alzheimer’s disease related amyloid beta (Aβ) peptides by isoelectric focusing (IEF) in 75 nL microchannels combined with their analysis by micropillar-matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) is presented. A semiopen chip-based setup, consisting of open microchannels covered by a lid of a liquid fluorocarbon, was used. IEF was performed in a mixture of four small and chemically well-defined amphoteric carriers, glutamic acid, aspartyl-histidine (Asp-His), cycloserine (cSer), and arginine, which provided a stepwise pH gradient tailored for focusing of the C-terminal Aβ peptides with a pI of 5.3 in the boundary between cSer and Asp-His. Information about the focusing dynamics and location of the foci of Aβ peptides and other compounds was obtained using computer simulation and by performing MALDI-MS analysis directly from the open microchannel. With the established configuration, detection was performed by direct sampling of a nanoliter volume containing the focused Aβ peptides from the microchannel, followed by deposition of this volume onto a chip with micropillar MALDI targets. In addition to purification, IEF preconcentration provides at least a 10-fold increase of the MALDI-MS-signal. After immunoprecipitation and concentration of the eluate in the microchannel, IEF-micropillar-MALDI-MS is demonstrated to be a suitable platform for detection of Aβ peptides in human cerebrospinal fluid as well as in blood plasma.
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| 9. |
- Mikkonen, Saara, 1987-
(författare)
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Sample preconcentration in open microchannels : Combinations with MALDI and nano-ESI mass spectrometry and computer simulations
- 2014
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In this thesis a novel concept for preconcentration of biomolecules in open microchannels is presented. The preconcentration is based on electromigration of charged analytes, and detection is performed with matrix-assisted laser desorption/ionization (MALDI) or nano-electrospray ionization (nESI) mass spectrometry (MS).Analysis of minute volumes of low-concentration samples is an important and challenging task within several fields of chemistry, biology and medicine. In bioanalytical chemistry in particular, sample pretreatment procedures including preconcentration must frequently be applied. Due to the often small available sample volumes, it is advantageous to perform these pretreatments in microfluidic devices. Moreover, since MS in many cases is the detection method of choice, there is a requirement for developing suitable interfacing techniques between the microchip and MS.In Paper I, the preconcentration concept is presented; silicon microchips with parallel open channels were used. The channels have a rectangular shape and are 1 cm long, 50-150 µm wide and 50 µm deep, yielding a total channel volume of 25-75 nL. By supplying sample to the channel and applying a voltage over the channel length, charged analytes will migrate towards the oppositely charged electrode and become concentrated. In Paper I, detection was performed by using the open microchannel directly as a MALDI-target. To achieve this, matrix solution was added to the channel after the preconcentration with electrospray matrix deposition. Using this approach, preconcentration of cytochrome c was achieved, and the lowest initial protein concentration successfully detected after preconcentration was 1 nM. The trypsin digest of cytochrome c was also analyzed, and the peptides were preconcentrated at different ends of the channel based on charge.Other means of coupling the preconcentration to MS, by extracting a nanovolume of the preconcentrated sample from the open channel, are presented in Paper II. The extracted samples could either be analyzed directly using nESI- or MALDI-MS, or subjected to further pretreatment (such as enzymatic digestion) in a nanodroplet under a fluorocarbon (FluorinertPTMP) liquid lid prior to MS-analysis. Furthermore, in Paper II the method was applied on an amyloid beta cell culture. This resulted in that peptides not detectable without preconcentration easily could be detected with MALDI-MS in nanodroplets extracted from the microchannels after preconcentration.Paper III includes theoretical simulations of the preconcentration procedure obtained using the electrophoresis simulator GENTRANS. The experimental results from Paper I are compared to simulations of similar systems, and simulations of an isoelectric focusing (IEF) procedure for proteins or peptides in a mixture of amino acids, are presented. The IEF procedure is to be used in the open microchannels in future experimental work.
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| 10. |
- Mikkonen, Saara, et al.
(författare)
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Sample preconcentration in open microchannels combined with MALDI-MS
- 2012
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Ingår i: Electrophoresis. - : Wiley. - 0173-0835 .- 1522-2683. ; 33:22, s. 3343-3350
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Tidskriftsartikel (refereegranskat)abstract
- In this work, a method for preconcentrating samples in 1 cm long, 50-150 μm wide open microchannels is presented. Platinum electrodes were positioned at the channel ends, voltage was applied, and charged analyte was preconcentrated at the oppositely charged side during continuous supply of sample. The preconcentration was initially studied in a closed system, where an influence on the analyte position from a pH gradient, generated by water electrolysis, was observed. In the open channel, the analyte distribution after preconcentration was evaluated using MALDI-MS with the channel as MALDI target. MALDI matrix was applied with an airbrush or by electrospray matrix deposition and by using the latter technique higher degrees of crystallization in the channels were obtained. After preconcentrating a 1 nM cytochrome c solution for 5 min, corresponding to a supplied amount of 1.25 fmol, a signal on the cathodic channel end could be detected. When a solution of cytochrome c trypsin digest was supplied, the peptides were preconcentrated at different positions along the channel depending on their charge.
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