| 1. |
- Ek, Patrik, et al.
(författare)
-
Electrospray Ionization from an Adjustable Gap between two Silicon Chips
- 2009
-
Ingår i: Journal of Mass Spectrometry. - : Wiley. - 1076-5174 .- 1096-9888. ; 44:2, s. 171-181
-
Tidskriftsartikel (refereegranskat)abstract
- In this paper, a silicon chip - based electrospray emitter with a variable orifice size is presented. The device consists of two chips, with a thin beam elevating from the center of each of the chips. The chips are individually mounted to form an open gap of a narrow, uniform width between the top areas of the beams. The electrospray is generated at the endpoint of the gap, where the spray point is formed by the very sharp intersection between the crystal planes of the < 100 > silicon chips. Sample solution is applied to the rear end of the gap from a capillary via a liquid bridge, and capillary forces ensure a spontaneous imbibition of the gap. The sample solution is confined to the gap by means of a hydrophobic treatment of the surfaces surrounding the gap, as well as the geometrical boundaries formed by the edges of the gap walls. The gap width could be adjusted between 1 and 25 μm during electrospray experiments without suffering from any interruption of the electrospray process. Using a peptide sample solution, a shift toward higher charge states and increased signal-to-noise ratios was observed when the gap width was decreased. The limit of detection for the peptide insulin (chain B, oxidized) was approximately 4 nM. We also show a successful interfacing of the electrospray setup with capillary electrophoresis.
|
|
| 2. |
- Hartmann, Michael, et al.
(författare)
-
Non-contact protein microarray fabrication using a procedure based on liquid bridge formation
- 2009
-
Ingår i: Analytical and Bioanalytical Chemistry. - : Springer Science and Business Media LLC. - 1618-2642 .- 1618-2650. ; 393:2, s. 591-598
-
Tidskriftsartikel (refereegranskat)abstract
- Contemporary microarrayers of contact or noncontact format used in protein microarray fabrication still suffer from a number of problems, e. g. generation of satellite spots, inhomogeneous spots, misplaced or even absent spots, and sample carryover. In this paper, a new concept of non-contact sample deposition that reduces such problems is introduced. To show the potential and robustness of this pressure-assisted deposition technique, different sample solutions known to cause severe problems or to be even impossible to print with conventional microarrayers were accurately printed. The samples included 200 mg mL(-1) human serum albumin, highly concentrated sticky cell adhesion proteins, pure high-salt cell-lysis buffer, pure DMSO, and a suspension of 5-mu m polystyrene beads. Additionally, a water-immiscible liquid fluorocarbon, which was shown not to affect the functionality of the capture molecules, was employed as a lid to reduce evaporation during microarray printing. The fluorocarbon liquid lid was shown to circumvent hydrolysis of water-sensitive activated surfaces during long-term deposition procedures.
|
|
| 3. |
|
|
| 4. |
- Bonn, Jonas, et al.
(författare)
-
Electrostatic Sample Nebulization for Improved Sample Vaporization in the Split/Splitless Gas Chromatography Inlet
- 2009
-
Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 81:13, s. 5327-5332
-
Tidskriftsartikel (refereegranskat)abstract
- The split/splitless inlet system has basically the same fundamental drawbacks it had when it was introduced: poor repeatability of the injected amount of sample and discrimination of high-boiling analytes. Hot needle injection improves the repeatability of the sample transfer but suffers from in-needle discrimination. Injection with a fast autosampler, resulting in minimal heating of the needle, solves this problem but usually requires a glass wool packing in the inlet liner to assist in vaporization of the sample. As glass wool has been reported to cause degradation of labile analytes, it cannot be applied as a general remedy for improving incomplete vaporization. In this paper, a novel concept, based on electrostatic nebulization of the injected sample, is presented. The resulting fine droplets promote a more effective heat transfer and a rapid vaporization. Evaluation of the electrospray inlet in the split mode, using a straight, empty glass liner and a cold needle, showed an improvement in peak area repeatability by about 1 order of magnitude, compared with the results obtained when no electrostatic field was applied, Splitless injection of a series of hydrocarbons up to C-28 in the electrospray inlet with an empty, tapered liner, using a cold needle, showed no measurable analyte discrimination. The relative standard deviation in terms of area count for the largest hydrocarbon (C-28) was < 1.5%, compared to similar to 30% for injections where no high voltage was applied.
|
|
| 5. |
- Bonn, Jonas, et al.
(författare)
-
Mixed sorbent phases for thick film open tubular traps
- 2009
-
Ingår i: Journal of Chromatographic Science. - : Oxford University Press (OUP). - 0021-9665 .- 1945-239X. ; 47:4, s. 297-303
-
Tidskriftsartikel (refereegranskat)abstract
- In this work, we present a technique for the preparation of tailor-made sorbent phases for thick film open tubular traps. Solid or liquid polymers are dispersed in a polydimethylsiloxane (PDMS) pre-polymer, which is cross-linked in situ after coating. The technique is evaluated by preparing thick film open tubular traps with PDMS containing solid or liquid poly(ethylene glycol) (PEG). A significant increase in retention for polar analytes is observed, even when only 7.5% PEG is present. The increase in retention for 3-chloro-1,2-propanediol is more than tenfold. The preparation method is simple and no solvents are required. Also, the concept provides great flexibility in terms of phase composition.
|
|
| 6. |
- Chilo, José, 1960-, et al.
(författare)
-
A flexible electronic nose for odor discrimination using different methods of classification
- 2009
-
Ingår i: 2009 16th IEEE-NPSS Real Time Conference - Conference Record. - New York : IEEE. - 9781424444557 ; , s. 317-320, s. 317-320
-
Konferensbidrag (refereegranskat)abstract
- Ovarian cancer is one of the leading causes of death from cancer in women. The lifetime risk is around 1.5%, which makes it the second most common gynecologic malignancy (the first one being breast cancer). To have a definitive diagnose, a surgical procedure is generally required and suspicious areas (samples) will be removed and sent for microscopic and other analysis. This paper describes the result of a pilot study in which an electronic nose is used to "smell" the aforementioned samples, analyze the multi-sensor signals and have a close to real-time answer on the detection of cancer. Besides being fast, the detection method is inexpensive and simple. Experimental analysis using real ovarian carcinoma samples shows that the use of proper algorithms for analysis of the multi-sensor data from the electronic nose yielded surprisingly good results with more than 77% classification rate. The electronic nose used in this pilot study was originally developed to be used as a "bomb dog" and can distinguish between e.g. TNT, Dynamex, Prillit. However, it was constructed to be a flexible multi-sensor device and the individual (16) sensors can easily be replaced/exchanged. This is suggestive for further investigations to obtain even better results with new, specific sensors. In another pilot experiment, headspace of an ovarian carcinoma sample and a control sample were analyzed using gas chromatography-mass spectrometry. Significant differences in chemical composition and compound levels were recorded, which would explain the different response obtained with the electronic nose.
|
|
| 7. |
- Ek, Patrik, et al.
(författare)
-
Electrospray Ionization from a Gap with Adjustable Width
- 2006
-
Ingår i: Rapid Communications in Mass Spectrometry. - : Wiley. - 0951-4198 .- 1097-0231. ; 20:21, s. 3176-3182
-
Tidskriftsartikel (refereegranskat)abstract
- In this paper, we present a new concept for electrospray ionization mass spectrometry, where the sample is applied in a gap which is formed between the edges of two triangular-shaped tips. The size of the spray orifice can be changed by varying the gap width. The tips were fabricated from polyethylene terephthalate film with a thickness of 36 μm. To improve the wetting of the gap and sample confinement, the edges of the tips forming the gap were hydrophilized by means of silicon dioxide deposition. Electrospray was performed with gap widths between 1 and 36 μm and flow rates down to 75 nL/min. The gap width could be adjusted in situ during the mass spectrometry experiments and nozzle clogging could be managed by simply widening the gap. Using angiotensin I as analyte, the signal-to-noise ratio increased as the gap width was decreased, and a shift towards higher charge states was observed. The detection limit for angiotensin I was in the low nM range.
|
|
| 8. |
- Hamberg, Anders, et al.
(författare)
-
C-terminal ladder sequencing of peptides using an alternative nucleophile in carboxypeptidase Y digests
- 2006
-
Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 357:2, s. 167-172
-
Tidskriftsartikel (refereegranskat)abstract
- A method for improved sequence coverage in C-terminal sequencing of peptides, based on carboxypeptidase digestion, is described. In conventional carboxypeptidase digestions, the peptide substrate is usually extensively degraded and a full amino acid sequence cannot be obtained due to the lack of a complete peptide ladder. In the presented method, a protecting group is introduced at the C terminus of a fraction of the peptide fragments formed in the digest, and thereby further degradation of the C-terminally modified peptides are slowed down. The protecting group was attached to the C-terminal amino acid through a carboxypeptidase-catalyzed reaction with an alternative nucleophile, 2-pyridylmethylamine, added to the aqueous digestion buffer. Six peptides were digested by carboxypeptidase Y with and without 2-pyridylmethylamine present in the digest buffer, and the resulting fragments subsequently were analyzed with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Comparison of the two digestion methods showed that the probability of successful ladder sequencing increased, by more than 50% using 2-pyridylmethylamine as a competing nucleophile in carboxypeptidase Y digests.
|
|
| 9. |
- Kempka, Martin, et al.
(författare)
-
Improved method for peak picking in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
- 2004
-
Ingår i: Rapid Communications in Mass Spectrometry. - : Wiley. - 0951-4198 .- 1097-0231. ; 18:11, s. 1208-1212
-
Tidskriftsartikel (refereegranskat)abstract
- A method for peak picking for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) is described. The method is based on the assumption that two sets of ions are formed during the ionization stage, which have Gaussian distributions but different velocity profiles. This gives rise to a certain degree of peak skewness. Our algorithm deconvolutes the peak and utilizes the fast velocity, bulk ion distribution for peak picking. Evaluation of the performance of the new method was conducted using peptide peaks from a bovine serum albumin (BSA) digest, and compared with the commercial peak-picking algorithms Centroid and SNAP. When using the new two-Gaussian algorithm, for strong signals the mass accuracy was equal to or marginally better than the results obtained from the commercial algorithms. However, for weak, distorted peaks, considerable improvement in both mass accuracy and precision was obtained. This improvement should be particularly useful in proteomics, where a lack of signal strength is often encountered when dealing with weakly expressed proteins. Finally, since the new peak-picking method uses information from the entire signal, no adjustments of parameters related to peak height have to be made, which simplifies its practical use.
|
|
| 10. |
- Kloskowski, Adam, et al.
(författare)
-
Thick film traps with an irregular film : Preparation and evaluation
- 2004
-
Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673 .- 1873-3778. ; 1035:2, s. 159-165
-
Tidskriftsartikel (refereegranskat)abstract
- A new method for preparation of sorbent-based ultra-thick film traps for concentration of trace volatile components from gaseous matrices is described. The procedure is based on blowing a prepolymer (polydimethylsiloxane) through a capillary tube, forming an irregular film of stationary phase. Subsequently, the prepolymer is immobilized in a few seconds by heating to 200 °C. Evaluation of the performance of the new traps showed that the loss of efficiency, compared to regular smooth film traps is only on the order of 20–30%. In terms of breakthrough volume, this loss in performance is rather insignificant. The technology is extremely simple and allows a rapid and cheap production of a large number of ultra-thick film traps, even in non-specialized laboratories. The method can be applied to any type of cross-linkable stationary phase, thereby expanding the scope of sorbent-based trapping and preconcentration concept. Many applications are anticipated in trace and ultra-trace analysis in a wide range of fields, such as environmental chemistry, polymers, food and process analysis.
|
|
| 11. |
- Mikkonen, Saara, et al.
(författare)
-
Microfluidic Isoelectric Focusing of Amyloid Beta Peptides Followed by Micropillar-Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry
- 2016
-
Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 88:20, s. 10044-10051
-
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.
|
|
| 12. |
- Pettersson, Johan, et al.
(författare)
-
Automated high-capacity sorption probe for extraction of organic compounds in aqueous samples followed by gas chromatographic analysis
- 2004
-
Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673 .- 1873-3778. ; 1033:2, s. 339-347
-
Tidskriftsartikel (refereegranskat)abstract
- An automated high-capacity sorption device for GC analysis of ultra trace components has been developed. The scope of the presented technique was to combine the simplicity of solid-phase microextraction (SPME) with the high extraction efficiency of the stir bar sorptive extraction technology. Sorptive extractions of water samples were performed using polydimethylsiloxane (PDMS) rubber tubing (120 μl) mounted onto a glass rod. The sampling procedure was carried out by a robotic autoinjector. Since the setup is fully automated, unattended and precise time-controlled extraction of samples is possible and makes quantitation with non-equilibrium extractions feasible. The sorption probes are easy to exchange, which facilitates off-line/in-field sampling. The system was evaluated with a test mixture of 44 environmentally hazardous compounds. Detection limits were found to be in the sub-ppt region. The performance of the system was demonstrated with the analysis of polycyclic aromatic hydrocarbons in urban snow.
|
|
| 13. |
- Pettersson, Johan, et al.
(författare)
-
Method for analysis of polar volatile trace components in aqueous samples by gas chromatography
- 2005
-
Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 77:10, s. 3365-3371
-
Tidskriftsartikel (refereegranskat)abstract
- A new method has been developed for direct analysis of volatile polar trace compounds in aqueous samples by gas chromatography. Water samples are injected onto a short packed precolumn containing anhydrous lithium chloride. A capillary column is coupled in series with the prefractionation column for final separation of the analytes. The enrichment principle of the salt precolumn is reverse to the principles employed in conventional methods such as SPE or SPME in which a sorbent or adsorbent is utilized to trap or concentrate the analytes. Such methods are not efficient for highly polar compounds. In the LiCl pre-column concept, the water matrix is strongly retained on the hygroscopic salt, whereas polar as well as nonpolar volatile organic compounds show very low retention and are eluted ahead of the water. After transfer of the analytes to the capillary column, the retained bulk water is removed by backflushing the precolumn at elevated temperature. For direct injections of 120 μL of aqueous samples, the combined time for injection and preseparation is only 3.5 min. With this procedure, direct repetitive automated analyses of highly volatile polar compounds such as methanol or tetrahydrofuran can be performed, and a limit of quantification in the low parts-per-billion region utilizing a flame ionization detector is demonstrated.
|
|
| 14. |
- Pettersson, Johan, et al.
(författare)
-
Quantitative accuracy in the gas chromatographic analysis of solvent mixtures
- 2003
-
Ingår i: Journal of Chromatography A. - 0021-9673 .- 1873-3778. ; 985:1-2, s. 21-27
-
Tidskriftsartikel (refereegranskat)abstract
- Quantitative accuracy is of great importance in the analysis of bulk mixtures of solvents, particularly when the analysis is related to quality control of very large product volumes like in solvent recovery plants. Serious errors can be made if the effects of density differences between the pure solvents and volume contractions are not properly addressed. In earlier work, the use of an iterative process for correcting such errors has been suggested. However, in the case of volume contractions and mixtures of several solvents, this procedure is difficult to apply. In the present paper, we describe a simple procedure where calibration curves based on mass concentration are utilized. The densities of calibration mixtures of known compositions are determined with a density meter, in order to provide for correction factors caused by volume contractions. Model experiments with mixtures of water, ethanol, acetone and methanol showed a significant improvement in quantitative accuracy, when the suggested calibration strategy was applied.
|
|
| 15. |
- Pettersson, Johan, et al.
(författare)
-
Ultra thick film open tubular traps with an increased inner diameter
- 2004
-
Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673 .- 1873-3778. ; 1047:1, s. 93-99
-
Tidskriftsartikel (refereegranskat)abstract
- In this paper, the concept of open tubular traps, coated with a very thick film of polydimethyldisiloxane for enrichment of trace volatile components has been further explored. From theoretical calculations as well as practical experiments it is demonstrated that it can be advantageous to increase the inner diameter of such traps. For a given sampling flow rate and phase ratio, the plate number of the traps is not dependent on the inner diameter, provided that the linear flow velocity remains sufficiently high to offset the effect of axial diffusion. It is shown that this is due to the basic fact that for a given sampling flow rate, the average linear flow velocity in the trap is inversely proportional to the square of the inner diameter of the trap. However, in contrast to chromatographic separations, the linear flow velocity is not important. Under conditions of a constant phase ratio, an increased inner diameter also increases the amount of sorbent in the trap, which is a key parameter for obtaining high breakthrough volumes. Open tubular traps with an expanded inner diameter have very low pressure drop characteristics, which provides the possibility to construct new, simplified sampling systems.
|
|
| 16. |
- Sjodahl, Johan, et al.
(författare)
-
Separation of oligonucleotides in N-methyl-formamide-based polymer matrices by capillary electrophoresis
- 2007
-
Ingår i: Journal of Separation Science. - : Wiley. - 1615-9306 .- 1615-9314. ; 30:1, s. 104-109
-
Tidskriftsartikel (refereegranskat)abstract
- N-Methylformamide (NMF)-based matrices for capillary electrophoretic separation of nucleic acids have been developed. The use of an organic solvent as liquid base for the separation matrices allowed a hydrophobic polymer, C-16-derivatized 2-hydroxyethyl cellulose (HEC), to be employed as structural element in the sieving medium. With a matrix consisting of 5% w/v of this polymer dissolved in NMF containing 50 mM ammonium acetate, p(dA)(12-18) and p(dA)(40-60) oligonucleoticles were baseline separated. The addition of ammonium acetate to the buffer and separation matrix resulted in enhanced separation efficiency. Furthermore, it was possible to tailor the sieving performance of the separation medium by the use of a binary mixture of C16-derivatized HEC and PVP. Differences in sieving behavior of the various matrices evaluated are discussed.
|
|
| 17. |
- Sjödahl, Johan, et al.
(författare)
-
Chip with twin anchors for reduced ion suppression and improved mass accuracy in MALDI-TOF mass spectrometry
- 2005
-
Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 77:3, s. 827-832
-
Tidskriftsartikel (refereegranskat)abstract
- A new sample target for matrix-assisted laser desorption/ionization mass spectrometry is described. The target consists of pairs of elevated hydrophilic anchor surfaces, positioned in proximity onto a microchip. The anchors are used to obtain separate preparations of sample and external standard, while both anchor surfaces are irradiated simultaneously by the laser pulse. Using a standard, based on six peptides, a 2-fold improvement in mass accuracy is observed. Also, ion suppression is significantly reduced. With a one peptide calibration standard, 22 tryptic fragments from a BSA digest are detected using the twin-anchor concept, whereas only 14 fragments are detected when the sample and standard are laser-ablated as a mixture from a conventional anchor target. A volume of similar to30 pL of sample solution of angiotensin I is transferred to the anchor surface, under a thin layer of a perfluorocarbon, to prevent a concentration bias due to evaporation. With this arrangement, a detection limit of 1.5 amol was achieved with a signal-to-noise ratio of 22:1.
|
|
| 18. |
- Uemura, Suguru, et al.
(författare)
-
Picoliter droplet formation on thin optical fiber tips
- 2006
-
Ingår i: Langmuir. - : American Chemical Society (ACS). - 0743-7463 .- 1520-5827. ; 22:24, s. 10272-10276
-
Tidskriftsartikel (refereegranskat)abstract
- In this paper, we present experimental results on how minute droplets are formed on fiber optic end faces. Results show that reproducible picoliter volumes can be generated when fibers are retracted from an aqueous phase contained under an inert fluorinated immiscible liquid, with a coefficient of variation (CV) of 0.7-2.3%. The droplet formation was analyzed as a function of the fiber diameter, retraction speed, and wettability. Experiments reveal a volume-determining critical equilibrium contact angle between 60 and 75, defining the onset of fiber end-face dewetting. The dynamics of the droplet snap-off progression was characterized using high-speed imaging in order to explain the observed wettability-volume dependency.
|
|
| 19. |
- Villanueva, Walter, et al.
(författare)
-
Microdroplet deposition under a liquid medium
- 2007
-
Ingår i: Langmuir. - : American Chemical Society (ACS). - 0743-7463 .- 1520-5827. ; 23:3, s. 1171-1177
-
Tidskriftsartikel (refereegranskat)abstract
- An experimental and numerical study of the factors affecting the reproducibility of microdroplet depositions performed under a liquid medium is presented. In the deposition procedure, sample solution is dispensed from the end of a capillary by the aid of a pressure pulse onto a substrate with pillar-shaped sample anchors. The deposition was modeled using the convective Cahn-Hilliard equation coupled with the Navier-Stokes equations with added surface tension and gravity forces. To avoid a severe time-step restriction imposed by the fourth-order Cahn-Hilliard equation, a semi-implicit scheme was developed. An axisymmetric model was used, and an adaptive finite element method was implemented. In both the experimental and numerical study it was shown that the deposited volume mainly depends on the capillary-substrate distance and the anchor surface wettability. A critical equilibrium contact angle has been identified below which reproducible depositions are facilitated.
|
|
| 20. |
|
|
| 21. |
|
|
| 22. |
- Aldaeus, Fredrik, et al.
(författare)
-
Multi-step dielectrophoresis for separation of particles
- 2006
-
Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673 .- 1873-3778. ; 1131:1-2, s. 261-266
-
Tidskriftsartikel (refereegranskat)abstract
- A new concept for separation of particles based on repetitive dielectrophoretic trapping and release in a flow system is proposed. Calculations using the finite element method have been performed to envision the particle behavior and the separation effectiveness of the proposed method. As a model system, polystyrene beads in deionized water and a micro-flow channel with arrays of interdigited electrodes have been used. Results show that the resolution increases as a direct function of the number of trap-and-release steps, and that a difference in size will have a larger influence on the separation than a difference in other dielectrophoretic properties. About 200 trap-and-release steps would be required to separate particles with a size difference of 0.2%. The enhanced separation power of dielectrophoresis with multiple steps could be of great importance, not only for fractionation of particles with small differences in size, but also for measuring changes in surface conductivity, or for separations based on combinations of difference in size and dielectric properties.
|
|
| 23. |
- Aldaeus, Fredrik
(författare)
-
New Concepts for Dielectrophoretic Separations and Dielectric Measurements of Bioparticles
- 2006
-
Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis presents two new concepts for separation of micro particles using dielectrophoresis, demonstrated by calculated examples, as well as a new method for obtaining dielectric data on living cells. The thesis is based on four papers. Paper I describes how the trapping efficiency of micro particles may be significantly increased when superpositioned electric fields are employed in a high conductivity medium. Avoiding low conductivity media is important when working with living cells. Calculations were performed to predict trajectories of Escherichia coli bacteria in the system with superpositioned electric fields, and a model was developed which employed two arrays of interdigitated electrodes in a micro channel. Paper II proposes a new concept for separation of micro particles, based on repetitive dielectrophoretic trapping and release in a flow system. Calculations show that the resolution increases as a direct function of the number of trap and release steps, and that a difference in size will have a larger influence on the separation than a difference in dielectrophoretic properties. Polystyrene beads in deionized water were used as a model, and calculations were performed to predict the particle behavior and the separation efficiency. It should be possible to separate particles with a size difference of 0.2 % by performing 200 trap-and-release steps. The enhanced separation power of multi step dielectrophoresis could have significant applications, not only for fractionation of particles with small differences in size, but also for measuring changes in surface conductivity. Paper III presents a new calculation method for predicting dielectrophoretic motion of micro particles. The method is based on a soft sphere method often used in molecular dynamics. Results from the calculations are in good agreement with theoretical predictions as well as initial experimental results, showing that the method provides good efficiency and accuracy. Paper IV describes a new method for measurements of conductivity of living bacteria. To obtain reliable conductivity values, it is important to handle the cells as gently as possible during the measurement process. A standard conductivity meter was used in combination with cross-flow filtration. In this way, repeated centrifugation and resuspension is avoided which otherwise may cause damage to the bacteria. The conductivity of Bacillus subtilis was determined to be 7000 μS/cm by means of the cross-flow filtration method, and the values differ from earlier published values by almost an order of a magnitude. In addition to the work presented in the papers, some experimental dielectrophoresis work in chip-based systems was performed. The behavior of Escherichia coli and polystyrene beads at different voltages and frequencies were studied. Separation of beads with different sizes was achieved on an array of interdigitated electrodes. Using electrodes with a pointed shape, alignment in different directions, pearl-chain formation, rotation, and other dielectrophoretic motion of E. coli were observed.
|
|
| 24. |
- Aldaeus, Fredrik, et al.
(författare)
-
Superpositioned dielectrophoresis for enhanced trapping efficiency
- 2005
-
Ingår i: Electrophoresis. - : Wiley. - 0173-0835 .- 1522-2683. ; 26:22, s. 4252-4259
-
Tidskriftsartikel (refereegranskat)abstract
- One of the major applications for dielectrophoresis is selective trapping and fractionation of particles. If the surrounding medium is of low conductivity, the trapping force is high, but if the conductivity increases, the attraction decreases and may even become negative. However, high-conductivity media are essential when working with biological material such as living cells. In this paper, some basic calculations have been performed, and a model has been developed which employs both positive and negative dielectrophoresis in a channel with interdigitated electrodes. The finite element method was utilized to predict the trajectories of Escherichia coli bacteria in the superpositioned electrical fields. It is shown that a drastic improvement of trapping efficiency can be obtained in this way, when a high conductivity medium is employed.
|
|
| 25. |
- Anderson, M. E., et al.
(författare)
-
Evaluation of generic chiral liquid chromatography screens for pharmaceutical analysis
- 2003
-
Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673 .- 1873-3778. ; 1005:02-jan, s. 83-101
-
Tidskriftsartikel (refereegranskat)abstract
- Two different automated generic liquid chromatography screens for the separation of chiral compounds of pharmaceutical interest have been evaluated. The test set comprised 53 chemically diverse chiral compounds involving 55 enantiomeric pairs from the pharmaceutical industry (i.e. starting materials, synthetic intermediates and drug substances). The first screen utilised four polysaccharide-based columns with five mobile phases and showed enantioselectivity for 87% of the test compounds. The second screen employed three macrocyclic glycopeptide columns with two mobile phases and showed enantioselectivity for 65% of the test compounds. Merging of the two screening procedures resulted in an enantioselectivity for 96% of the chiral compounds. It is anticipated that the systematic use of the automated chiral HPLC screens described in this report will substantially reduce the necessary time for method development of pharmaceutically related chiral analytical methods.
|
|
| 26. |
- Benkestock, Kurt, et al.
(författare)
-
Automated Nano-Electrospray Mass Spectrometry for Protein-Ligand Screening by Noncovalent Interaction Applied to Human H-FABP and A-FABP
- 2003
-
Ingår i: Journal of Biomolecular Screening. - : Elsevier BV. - 1087-0571 .- 1552-454X. ; 8:3, s. 247-256
-
Tidskriftsartikel (refereegranskat)abstract
- A method for ligand screening by automated nano-electrospray ionization mass spectrometry (nano-ESI/MS) is described. The core of the system consisted of a chip-based platform for automated sample delivery from a 96-well plate and subsequent analysis based on noncovalent interactions. Human fatty acid binding protein, H-FABP (heart) and A-FABP (adipose), with small potential ligands was analyzed. The technique has been compared with a previously reported method based on nuclear magnetic resonance (NMR), and excellent correlation with the found hits was obtained. In the current MS screening method, the cycle time per sample was 1.1 min, which is approximately 50 times faster than NMR for single compounds and approximately 5 times faster for compound mixtures. High reproducibility was achieved, and the protein consumption was in the range of 88 to 100 picomoles per sample. Furthermore, a novel protocol for preparation of A-FABP without the natural ligand is presented. The described screening approach is suitable for ligand screening very early in the drug discovery process before conventional high-throughput screens (HTS) are developed and/or used as a secondary screening for ligands identified by HTS.
|
|
| 27. |
- Benkestock, Kurt, et al.
(författare)
-
Electrospray ionization mass spectrometry as a tool for determination of drug binding sites to human serum albumin by noncovalent interaction
- 2005
-
Ingår i: Rapid Communications in Mass Spectrometry. - : Wiley. - 0951-4198 .- 1097-0231. ; 19:12, s. 1637-1643
-
Tidskriftsartikel (refereegranskat)abstract
- Most proteins in blood plasma bind ligands. Human serum albumin (HSA) is the main transport protein with a very high capacity for binding of endogenous and exogenous compounds in plasma. Many pharmacokinetic properties of a drug depend on the level of binding to plasma proteins. This work reports studies of noncovalent interactions by means of nanoelectrospray ionization mass spectrometry (nanoESI-MS) for determination of the specific binding of selected drug candidates to HSA. Warfarin, iopanoic acid and digitoxin were chosen as site-specific probes that bind to the main sites of HSA. Two drug candidates and two known binders to HSA were analyzed using a competitive approach. The drugs were incubated with the target protein followed by addition of site-specific probes, one at a time. The drug candidates showed predominant affinity to site I (warfarin site). Naproxen and glyburide showed affinity to both sites I and II. The advantages of nanoE-SI-MS for these studies are the sensitivity, the absence of labeled molecules and the short method development time.
|
|
| 28. |
- Benkestock, Kurt, 1961-
(författare)
-
Electrospray Ionization Mass Spectrometry for Determination of Noncovalent Interactions in Drug Discovery
- 2008
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Noncovalent interactions are involved in many biological processes in which biomolecules bind specifically and reversibly to a partner. Often, proteins do not have a biological activity without the presence of a partner, a ligand. Biological signals are produced when proteins interact with other proteins, peptides, oligonucleotides, nucleic acids, lipids, metal ions, polysaccharides or small organic molecules. Some key steps in the drug discovery process are based on noncovalent interactions. We have focused our research on the steps involving ligand screening, competitive binding and ‘off-target’ binding. The first paper in this thesis investigated the complicated electrospray ionization process with regards to noncovalent complexes. We have proposed a model that may explain how the equilibrium between a protein and ligand changes during the droplet evaporation/ionization process. The second paper describes an evaluation of an automated chip-based nano-ESI platform for ligand screening. The technique was compared with a previously reported method based on nuclear magnetic resonance (NMR), and excellent correlation was obtained between the results obtained with the two methods. As a general conclusion we believe that the automated nano-ESI/MS should have a great potential to serve as a complementary screening method to conventional HTS. Alternatively, it could be used as a first screening method in an early phase of drug development programs when only small amounts of purified targets are available. In the third article, the advantage of using on-line microdialysis as a tool for enhanced resolution and sensitivity during detection of noncovalent interactions and competitive binding studies by ESI-MS was demonstrated. The microdialysis device was improved and a new approach for competitive binding studies was developed. The last article in the thesis reports studies of noncovalent interactions by means of nanoelectrospray ionization mass spectrometry (nanoESI-MS) for determination of the specific binding of selected drug candidates to HSA. Two drug candidates and two known binders to HSA were analyzed using a competitive approach. The drugs were incubated with the target protein followed by addition of site-specific probes, one at a time. The drug candidates showed predominant affinity to site I (warfarin site). Naproxen and glyburide showed affinity to both sites I and II.
|
|
| 29. |
- Benkestock, Kurt, et al.
(författare)
-
Influence of droplet size, capillary-cone distance and selected instrumental parameters for the analysis of noncovalent protein-ligand complexes by nano-electrospray ionization mass spectrometry
- 2004
-
Ingår i: Journal of Mass Spectrometry. - : Wiley. - 1076-5174 .- 1096-9888. ; 39:9, s. 1059-1067
-
Tidskriftsartikel (refereegranskat)abstract
- It has been suggested in the literature that nano-electrospray ionization (nano-ESI) mass spectrometry better reflects the equilibrium between complex and free protein in solution than pneumatically assisted electrospray ionization (ESI) in noncovalent interaction studies. However, no systematic studies of the effects of ionization conditions have been performed to support this statement. In the present work, different instrumental and sample-derived parameters affecting the stability of noncovalent complexes during analysis by nano-ESI were investigated. In general, increased values of parameters such as drying gas flow-rate, ion-source temperature, capillary tip voltage and buffer concentration lead to a dissociation of ribonuclease A (RNAse)-cytidine 2'-monophosphate (CMP) and cytidine 5'-triphosphate (CTP) complexes. The size of the electrosprayed droplets was shown to be an important issue. Increasing the capillary to cone distance yielded an increased complex to free protein ratio when a hydrophilic ligand was present and the reverse effect was obtained with a hydrophobic ligand. Important in this regard is the degree of sampling of ions originating from late-generation residue droplets, that is, ions present in the droplet bulk. Sampling of these ions increases with longer capillary-cone distance (flight time). Furthermore, when the sample flow-rate was increased by increasing the capillary internal tip i.d. from 4 to 30 mum, a decreased complex to free protein ratio for the RNAse-CTP system was observed. This behavior was consistent with the change in surface to volume ratio for flow-rates between 2 and 100 nl min(-1). Finally, polarity switching between positive and negative ion modes gave a higher complex to free protein ratio when the ligand and the protein had the same polarity.
|
|
| 30. |
- Benkestock, Kurt, et al.
(författare)
-
On-line microdialysis for enhanced resolution and sensitivity during electrospray mass spectrometry of non-covalent complexes and competitive binding studies
- 2002
-
Ingår i: Rapid Communications in Mass Spectrometry. - : Wiley. - 0951-4198 .- 1097-0231. ; 16:21, s. 2054-2059
-
Tidskriftsartikel (refereegranskat)abstract
- Many proteins and macromolecules easily form metal adduct ions which impairs their analysis by mass spectrometry. The present study describes how the formation of undesired adducts can be minimized by on-line microdialysis for non-covalent binding studies of macromolecules with low molecular mass ligands with electrospray ionization mass spectrometry (ESI-MS). The technique was indispensable for protein-ligand studies due to reduction of unwanted adduct ions, and thus gave excellent resolution and a sensitivity improvement of at least 5 times. The core of the analytical system was a modified microdialysis device, which was operated in countercurrent mode. A novel technique based on microdialysis for competitive binding studies is also presented. The noncovalent complex between a protein and a ligand was formed in the sample vial prior to analysis. The complex was injected into an on-line microdialysis system where a competitive ligand was administered in the dialysis buffer outside of the fiber. The second ligand competitively displaced the first ligand through transport via the wall of the dialysis fiber, and the intact complexes were detected by ESI-MS.
|
|
| 31. |
- Bonn, Jonas, 1978-
(författare)
-
Improved Techniques for Sampling and Sample Introduction in Gas Chromatography
- 2008
-
Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Sampling and sample introduction are two key steps in quantitative gas chromatography. In this thesis, a development of a previously described sampling technique as well as a novel concept for sample introduction in gas chromatography are presented. The thesis is based on two papers. Paper I describes a method for preparing physically mixed polymers for use as sorbent phases in open tubular trapping of gaseous analytes. The concept is based on mechanical disintegration and mixing of solid or liquid poly(ethylene glycol), PEG, into poly(dimethylsiloxane), PDMS, in a straightforward manner. The resulting mixture exhibits a higher affinity towards polar analytes, as compared to pure PDMS. Paper II describes a novel approach to liquid sample introduction with the split/splitless inlet, used in gas chromatography. Classical injection techniques struggle with discrimination of high boiling analytes and poor repeatability of the injected amount of analytes. The presented injection technique utilizes high voltage to obtain a spraying effect of the injected liquid. The spraying effect can be achieved with a cold needle, which is unprecedented for gas chromatographic injections. The cold needle spraying results in highly repeatable injections, free from discrimination of high boiling analytes.
|
|
| 32. |
- Chabert, Max, et al.
(författare)
-
Automated microdroplet platform for sample manipulation and polymerase chain reaction
- 2006
-
Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 78:22, s. 7722-7728
-
Tidskriftsartikel (refereegranskat)abstract
- We present a fully automated system performing continuous sampling, reagent mixing, and polymerase chain reaction (PCR) in microdroplets transported in immiscible oil. Sample preparation and analysis are totally automated, using an original injection method from a modified 96-well plate layered with three superimposed liquid layers and in-capillary laser-induced fluorescence endpoint detection. The process is continuous, allowing sample droplets to be carried uninterruptedly into the reaction zone while new drops are aspirated from the sample plate. Reproducible amplification, negligible cross-contamination, and detection of low sample concentrations were demonstrated on numerous consecutive sample drops. The system, which opens the route to strong reagents and labor savings in high-throughput applications, was validated on the clinically relevant quantification of progesterone receptor gene expression in human breast cancer cell lines.
|
|
| 33. |
- Curcio, M., et al.
(författare)
-
Continuous segmented-flow polymerase chain reaction for high-throughput miniaturized DNA amplification
- 2003
-
Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 75:1, s. 1-7
-
Tidskriftsartikel (refereegranskat)abstract
- A continuous segmented-flow method for sequential DNA amplification is described in order to provide a basis for high-throughput genetic analysis. The approach allows an immediate distinction between amplified and nonamplified products. A mixture of sample and reagents are loaded in the form of small segments one after another in a 15-m-long narrow-bore Teflon tube, coiled such as to be repeatedly exposed to three different temperature zones. After having passed the heated zones, the samples are mixed with an intercalating dye by flow injection and sequentially detected on-line by laser-induced fluorescence. The aqueous samples travel as separate segments in a continuous flow of an immiscible, organic. liquid. Perfluorodecalin was shown to be particularly suitable due to its hydrophobicity and inert properties. To reduce carryover between samples, an intermediate water plug between two consecutive samples was required. Selected regions from human genomic DNA were successfully amplified in 300-nL volumes after 30 passes through the heated zones. The total reaction time was similar to45 min, and the detection interval between individual samples was 1 min. Automation and the possibility to further reduce sample volumes, as well as to employ many reaction columns simultaneously, should provide a platform for an extremely high throughput.
|
|
| 34. |
- Curcio, M., et al.
(författare)
-
Multiplex high-throughput solid-phase minisequencing by capillary electrophoresis and liquid core waveguide fluorescence detection
- 2002
-
Ingår i: Electrophoresis. - 0173-0835 .- 1522-2683. ; 23:10, s. 1467-1472
-
Tidskriftsartikel (refereegranskat)abstract
- Minisequencing, solid-phase single-nucleotide primer extension reaction, is a robust method for performing multiplex single-nucleotide polymorphism (SNP) analysis. We have combined this technology with capillary gel electrophoresis in a multicapillary format, using liquid core waveguide (LCW) fluorescence detection. Polymerase chain reaction (PCR) amplification of multiple DNA targets is performed with one primer for each target biotinylated. Separation of the complementary strands, minisequencing and washing steps are carried out using streptavidin-coated magnetic beads. Dideoxynucleotides analogues labelled with different fluorophores are used for the extension of the minisequencing primers. The extended oligonucleotides, the length of which defines the position on the target and the color the identity of the polymorphism, are then separated in a gel-filled array of capillaries, coated on the outside with a layer of a fluoropolymer to provide the liquid core waveguide characteristics. The technology has a potential for extremely high throughputs when a combination of multiplex PCR-minisequencing is used together with a large array of capillaries, four-color detection and high-speed separation.
|
|
| 35. |
- Ek, Patrik, et al.
(författare)
-
Electrospray ionization mass spectrometry from discrete nanoliter-sized sample volumes
- 2010
-
Ingår i: Rapid Communications in Mass Spectrometry. - : Wiley. - 0951-4198 .- 1097-0231. ; 24:17, s. 2561-2568
-
Tidskriftsartikel (refereegranskat)abstract
- We describe a method for nanoelectrospray ionization mass spectrometry (nESI-MS) of very small sample volumes. Nanoliter-sized sample droplets were taken up by suction into a nanoelectrospray needle from a silicon microchip prior to ESI. To avoid a rapid evaporation of the small sample volumes, all manipulation steps were performed under a cover of fluorocarbon liquid. Sample volumes down to 1.5 nL were successfully analyzed, and an absolute limit of detection of 105 attomole of insulin (chain B, oxidized) was obtained. The open access to the sample droplets on the silicon chip provides the possibility to add reagents to the sample droplets and perform chemical reactions under an extended period of time. This was demonstrated in an example where we performed a tryptic digestion of cytochrome C in a nanoliter-sized sample volume for 2.5h, followed by monitoring the outcome of the reaction with nESI-MS. The technology was also utilized for tandem mass spectrometry (MS/MS) sequencing analysis of a 2 nL solution of angiotensin I.
|
|
| 36. |
- Ek, Patrik, 1980-
(författare)
-
Mass Spectrometry with Electrospray Ionization from an Adjustable Gap
- 2008
-
Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In this thesis the fabrication and analytical evaluation of two new electrospray emitters utilized for mass spectrometry analysis is presented. The emitters are based on a new concept, where the spray orifice can be varied in size. The thesis is based on two papers. All present-day nanoelectrospray emitters have fixed dimensions. The range of the applicable flow rate for such an emitter is therefore rather limited and exchange of emitters may be necessary from one experiment to another. Optimization of the signal of the analyte ions is also limited to adjustments of the applied voltage or the distance between the emitter and the mass spectrometer inlet. Furthermore, clogging can occur in emitters with fixed dimensions of narrow orifice sizes. In this thesis, electrospray emitters with a variable size of the spray orifice are proposed. An open gap between two thin substrates is filled with sample solution via a liquid bridge from a capillary. Electrospray is generated at the end point of the gap, which can be varied in width. In Paper I, electrospray emitters fabricated in polyethylene terephthalate have been evaluated. Triangular tips are manually cut from the polymer film. The tips are mounted to form a gap between the edges of the tips. The gap wall surfaces are subjected to a hydrophilic surface treatment to increase the wetting of the gap walls. In Paper II, silicon electrospray chips with high precision are fabricated and evaluated. A thin beam, elevated from the bulk silicon chip is fabricated by means of deep reactive ion etching. The top surfaces of the beams of two chips act as a sample conduit when mounted in the electrospray setup. An anisotropic etching step with KOH of the intersecting <100> crystal planes results in a very sharp spray point. The emitters were given a hydrophobic surface treatment except for the hydrophilic gap walls. For both emitter designs, the gap width has been adjusted during the experiments without any interruption of the electrospray. For a continuously applied peptide mixture, a shift towards higher charge states and increased signal to noise ratios could be observed when decreasing the gap width. The limit of detection has been investigated and the silicon chips have been interfaced with capillary electrophoresis.
|
|
| 37. |
- Ek, Patrik, et al.
(författare)
-
New Method for Fabrication of Fused Silica Emitters with Submicrometer Orifices for Nanoelectrospray Mass Spectrometry
- 2011
-
Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 83:20, s. 7771-7777
-
Tidskriftsartikel (refereegranskat)abstract
- In this paper, we describe a new method for fabrication of nanoelectrospray emitters. The needles were pulled from fused silica capillary tubing, which was melted by means of a plasma, formed by electrical discharges between two pointed platinum electrodes. A key feature of the pulling device is a rotating configuration of the electrodes, which results in an even radial heating of the capillary. The construction of the setup is straightforward, and needles with a variety of shapes can be fabricated, including orifices of submicrometer dimensions. Pulled needles with long tapered tips and an orifice of 0.5 mu m were utilized for electrospray ionization mass spectrometry (ESI-MS) of discrete sample volumes down to 275 pL. The picoliter-sized samples were transferred into the tip of the needle from a silicon microchip by aspiration. To avoid a rapid evaporation of the sample, all manipulations were performed under a cover of a fluorocarbon liquid. The limit of detection was measured to be ca. 20 attomole for insulin (chain B, oxidized).
|
|
| 38. |
- Ek, Patrik, 1980-
(författare)
-
New methods for sensitive analysis with nanoelectrospray ionization mass spectrometry
- 2010
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In this thesis, new methods that address some current limitations in nanoelectrospray mass spectrometry (nESI-MS) analysis are presented. One of the major objectives is the potential gain in sensitivity that can be obtained when employing the proposed techniques. In the first part of this thesis, a new emitter, based on the generation of electrospray from a spray orifice with variable size, is presented. Electrospray is generated from an open gap between the edges of two individually mounted, pointed tips. The fabrication and evaluation of two different types of such emitters is presented; an ESI emitter fabricated from polyethylene terephtalate (Paper I), and a high-precision silicon device (Paper II). Both emitters were surface-treated in a selective way for an improved wetting of the gap and to confine the sample solution into the gap. In the second part of this thesis, different methods for improved sensitivity of nESI-MS analysis have been developed. In Paper III, a method for nESI-MS analysis from discrete sample volumes down to 1.5 nL is presented, using commercially available nESI needles. When analyzing attomole amounts of analyte in such a small volume of sample, an increased sensitivity was obtained, compared to when analyzing equal amounts in conventional nESI-MS analysis. To be able to analyze smaller sample volumes, needles with a narrower orifice and a higher flow resistance were needed. This triggered the development of a new method for fabrication of fused silica nESI needles (Paper IV). The fabrication is based on melting of a fused silica capillary by means of a rotating plasma, prior to pulling the capillary into a fine tip. Using the described technique, needles with sub-micrometer orifices could be fabricated. Such needles enabled the analysis of sample volumes down to 275 pL, and a further improvement of the sensitivity was obtained. In a final project (Paper V), nESI-MS was used to study the aggregation behavior of Aβ peptides, related to Alzheimer’s disease. An immunoprecipitation followed by nESI-MS was employed. This technique was also utilized to study the selectivity of the antibodies utilized.
|
|
| 39. |
- Emmer, Åsa, et al.
(författare)
-
Enzymatic protein digest in chip-based nanovials with immobilized proteolytic enzymes
- 2005
-
Ingår i: Analytica Chimica Acta. - : Elsevier BV. - 0003-2670 .- 1873-4324. ; 542:2, s. 137-143
-
Tidskriftsartikel (refereegranskat)abstract
- In the present work, protein digest reactions in silicon-based microchips, coated with immobilized proteolytic enzymes, have been carried out. The performance of such vials, modified with trypsin or chymotrypsin, was tested with myoglobin as a substrate. Capillary electrophoresis and matrix-assisted laser desorption/ionization mass spectrometry were utilized for analysis of the digests, and the influence of different instrumentation setups. immobilization procedures and reaction conditions are discussed.
|
|
| 40. |
- Emmer, Åsa, et al.
(författare)
-
Wall deactivation with fluorosurfactants for capillary electrophoretic analysis of biomolecules
- 2001
-
Ingår i: Electrophoresis. - 0173-0835 .- 1522-2683. ; 22:4, s. 660-665
-
Tidskriftsartikel (refereegranskat)abstract
- This paper describes the use of fluorosurfactants as buffer additives for capillary electrophoretic separation of proteins and peptides. Due to fluorosurfactant bilayer formation at the capillary inner wall, the surface charge can be adjusted and even reversed. If the running buffer pH is kept at a level where the proteins have the same sign of charge as the wall, electrostatic repulsion will be obtained. The protein wall adsorption can therefore be reduced and the separation performance can be noticeably increased. The separation performance can also be further improved by including mixtures of different types of fluorosurfactants in the running buffer. The buffer system can accordingly be adapted for a certain separation problem. Mechanisms for the use of fluorosurfactants for wall deactivation in capillary electrophoretic protein separations is discussed in the present work and some examples of applications are also presented.
|
|
| 41. |
- Gantelius, Jesper, et al.
(författare)
-
Magnetic bead-based detection of autoimmune responses using protein microarrays.
- 2009
-
Ingår i: New biotechnology. - : Elsevier BV. - 1871-6784. ; 26, s. 269-276
-
Tidskriftsartikel (refereegranskat)abstract
- In the present study, a magnetic bead-based detection approach for protein microarrays is described as an alternative approach to the commonly used fluorescence-based detection system. Using the bead-based detection approach with applied magnetic force, it was possible to perform the detection step more rapidly as a result of the accelerated binding between the captured analyte in the microspot and the detection antibody, which was coupled to the magnetic beads. The resulting strong opacity shift on the microspots could be recorded with an ordinary flatbed scanner. In the context of autoimmunity, a set of 24 serum samples was analyzed for the presence of antibodies against 12 autoantigens using standard fluorescence and magnetic bead-based detection methods. Dynamic range, sensitivity, and specificity were determined for both detection methods. We propose from our findings that the magnetic bead-based detection option provides a simplified and cost effective readout method for protein microarrays.
|
|
| 42. |
- Griss, P., et al.
(författare)
-
Development of micromachined hollow tips for protein analysis based on nanoelectrospray ionization mass spectrometry
- 2002
-
Ingår i: Journal of Micromechanics and Microengineering. - : IOP Publishing. - 0960-1317 .- 1361-6439. ; 12:5, s. 682-687
-
Tidskriftsartikel (refereegranskat)abstract
- Two novel types of micromachined nanoelectrospray emitter tips have been designed, fabricated and tested. The fabrication method of the hollow tips is based on a self-aligning deep reactive ion etch process. The tips consist of either silicon dioxide or silicon and feature orifice diameters of 10 and 18 mum, respectively. The geometrical characteristics of both emitter types are favorable for the generation of stable electrospray ionization, i.e. wetting of the tip shaft is avoided and the base of the Taylor cone is limited to the diameter of the orifice. A silicon dioxide tip was operated in a bench top setup to visually evaluate the electrospray. Both types of tips were also successfully used for the analysis of an insulin sample in an ion trap mass spectrometer.
|
|
| 43. |
- Hanning, A., et al.
(författare)
-
A liquid core waveguide fluorescence detector for multicapillary electrophoresis applied to DNA sequencing in a 91-capillary array
- 2000
-
Ingår i: Electrophoresis. - 0173-0835 .- 1522-2683. ; 21:15, s. 3290-3304
-
Tidskriftsartikel (refereegranskat)abstract
- A new laser-induced fluorescence (LIF) detector for multicapillary electrophoresis is presented. The detection principle is based on waveguiding of the emitted fluorescence from the point of illumination to the capillary ends by total internal reflection (TIR) and imaging of the capillary ends. The capillaries themselves thus act as liquid core waveguides (LCWs). At the illumination point, the capillaries are arranged in a planar array, which allows clean and efficient illumination with a line-focused laser beam. The capillary ends are rearranged into a small, densely packed two-dimensional array, which is imaged end-on with high light collection efficiency and excellent image quality. Wavelength dispersion is obtained with a single prism. Intercapillary optical crosstalk is less than 0.5%, and rejection of stray light is very efficient. The detector is applied to four-color DNA sequencing by gel electrophoresis in a 91-capillary array, with simple fluorescein and rhodamine dyes as fluorophores. Since the imaged two-dimensional array is so compact, the detector has a high potential for very large-scale multiplexing.
|
|
| 44. |
- Hanning, A., et al.
(författare)
-
Laser induced fluorescence detection by liquid core waveguiding applied to DNA sequencing by capillary electrophoresis
- 2000
-
Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 72:15, s. 3423-3430
-
Tidskriftsartikel (refereegranskat)abstract
- A new laser-induced fluorescence detector for capillary electrophoresis (CE) is described. The detector is based on transverse illumination and collection of the emitted fluorescent light via total internal reflection along the separation capillary. The capillary is coated with a low refractive index fluoropolymer and serves as a liquid core waveguide (LCW). The emitted light is detected end-on with a CCD camera at the capillary exit. The observed detection limit for fluorescein is 2.7 pM (550 ymol) in the continuous-flow mode and 62 fM in the CE mode. The detector is applied to DNA sequencing. One-color G sequencing is performed with single-base resolution and signal-to-noise ratio similar to 250 for peaks around 500 bases. The signal-to-noise ratio is similar to 50 for peaks around 950 bases. Full four-color DNA sequencing is also demonstrated. The high sensitivity of the detector is suggested to partly be due to the efficient rejection of scattered laser light in the LCW. The concept should be highly suitable for capillary array detection.
|
|
| 45. |
- Hartmann, Michael, et al.
(författare)
-
Expanding assay dynamics : A combined competitive and direct assay system for the quantification of proteins in multiplexed Immunoassays
- 2008
-
Ingår i: Clinical Chemistry. - : Oxford University Press (OUP). - 0009-9147 .- 1530-8561. ; 54:6, s. 956-963
-
Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: The concurrent detection and quantification of analytes that vary widely in concentration present a principal problem in multiplexed assay systems. Combining competitive and sandwich immunoassays permits coverage of a wide concentration range, and both highly abundant molecules and analytes present in low concentration can be quantified within the same assay. METHODS: The use of different fluorescence readout channels allows the parallel use of a competitive system and a sandwich configuration. The 2 generated assay signals are combined and used to calculate the amount of analyte. The measurement range can be adjusted by varying the competitor concentration, and an extension of the assay system's dynamic range is possible. RESULTS: We implemented the method in a planar protein microarray-based autoimmune assay to detect autoantibodies against 13 autoantigens and to measure the concentration of a highly abundant protein, total human IgG, in one assay. Our results for autoantibody detection and IgG quantification agreed with results obtained with commercially available assays. The use of 2 readout channels in the protein microarray-based system reduced spot-to-spot variation and intraassay variation. CONCLUSIONS: By combining a direct immunoassay with a competitive system, analytes present in widely varying concentrations can be quantified within a single multiplex assay. Introducing a second readout channel for analyte quantification is an effective tool for spot-to-spot normalization and helps to lower intraassay variation.
|
|
| 46. |
- Hartmann, Michael
(författare)
-
Microfluidic Methods for Protein Microarrays
- 2010
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Protein microarray technology has an enormous potential for in vitro diagnostics (IVD)1. Miniaturized and parallelized immunoassays are powerful tools to measure dozens of parameters from minute amounts of sample, whilst only requiring small amounts of reagent. Protein microarrays have become well-established research tools in basic and applied research and the first diagnostic products are already released on the market. However, in order for protein microarrays to become broadly accepted tools in IVD, a number of criteria have to be fulfilled concerning robustness and automation. Robustness and automation are key demands to improve assay performance and reliability of multiplexed assays, and to minimize the time of analysis. These key demands are addressed in this thesis and novel methods and techniques concerning assay automation, array fabrication as well as performance and detection strategies related to protein microarrays are presented and discussed. In the first paper an automated assay format, based on planar protein microarrays is described and evaluated by the detection of several auto-antibodies from human serum and by quantification of matrix metalloproteases present in plasma. Diffusion-rate limited solid phase reactions were enhanced by microagitation, using the surface acoustic wave technology, resulting in a slightly increased signal-to-noise ratio. In the second paper of the thesis, a novel multiplexed immunoassay system was developed by combining a direct immunoassay with a competitive system. This set-up allows quantification of analytes present in widely varying concentrations within a single multiplex assay. In the third paper, a new concept for sample deposition is introduced, addressing contemporary problems of contact or non-contact microarrayers in protein microarray fabrication. In the fourth paper, a magnetic bead-based detection method for protein microarrays is described as a cost-effective alternative approach to the commonly used fluorescence-based confocal scanning systems. The magnetic bead-based detection could easily be performed by using an ordinary flatbed scanner. In addition, applying magnetic force to the magnetic bead-based detection approach enables to run the detection step more rapidly. Finally, in paper five, a microfluidic bead-based immunoassay for multiplexed detection of receptor tyrosine kinases in breast cancer tissue is presented. Since the assay is performed inside a capillary, the amounts of sample and reagent material could be reduced by a factor of 30 or more when compared with the current standard protein microarray assay.
|
|
| 47. |
- Hartmann, Michael, et al.
(författare)
-
Protein microarrays for diagnostic assays
- 2009
-
Ingår i: Analytical and Bioanalytical Chemistry. - : Springer Science and Business Media LLC. - 1618-2642 .- 1618-2650. ; 393:5, s. 1407-1416
-
Tidskriftsartikel (refereegranskat)abstract
- Protein microarray technology has enormous potential for in vitro diagnostics (IVD). Miniaturized parallelized immunoassays are perfectly suited to generating a maximum of diagnostically relevant information from minute amounts of sample whilst only requiring small amounts of reagent. Protein microarrays have become well-established research tools in basic and applied research and the first products are already on the market. This article reviews the current state of protein microarrays and discusses developments and future demands relating to protein arrays in their role as multiplexed immunoassays in the field of diagnostics.
|
|
| 48. |
|
|
| 49. |
- Johansson, LarsErik, et al.
(författare)
-
Determination of conductivity of bacteria by using cross-flow filtration
- 2006
-
Ingår i: Biotechnology letters. - : Springer Science and Business Media LLC. - 0141-5492 .- 1573-6776. ; 28:8, s. 601-603
-
Tidskriftsartikel (refereegranskat)abstract
- An important property of the bacterial surface is its conductivity. To obtain reliable conductivity values, it is essential to handle the cells as gently as possible during the measurement procedure. We have developed a method where a standard conductivity meter is used in combination with cross-flow filtration, thus avoiding repeated centrifugation and resuspension. With this method, the conductivity of Bacillus subtilis was determined to be 7000 mu S/cm, which is a deviation from previously published data by almost an order of a magnitude.
|
|
| 50. |
|
|
