Johan Roeraade

Method for fabrication of microfluidic systems in glass

In this report we describe a new way of fabricating integrated microfluidic elements in glass. By employing a matrix of underpinning posts and a thin wall, surrounding etched flow channels, an efficient sealing of glass chips substrates to thin cover glass can be accomplished. The use of this arrangement enables the overlay sheath of glass to hermetically close the flow channel by fusion bonding while avoiding problems with void or crack formation that are due to dust particles, non-planarity and differences in thermal coefficient of expansion. As an example a structure with a thin cover glass, useful for studying phenomena in capillary electrophoresis utilizing large numerical aperture microscope lenses, was fabricated.

Protein microarrays for diagnostic assays

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.

Improved capillary zone electrophoretic separation of basic proteins, using a fluorosurfactant buffer additive

Abstract In this paper, a new method to reduce the adsorption of basic proteins in capillary zone electrophoresis is described. Small amounts of a cationic fluorosurfactant are added to the running buffer. This leads to a surface charge reversal. Consequently, proteins at a pH below their p I are repelled from the wall. High efficiencies and symmetrical peaks were obtained for a number of model proteins, even when running buffer solutions with a low ionic strength were employed. Reproducibility was excellent. It is believed that the extreme hydrophobic nature of the fluorocarbon chain of the surfactant is a significant factor for the improved performance.

Evaluation of generic chiral liquid chromatography screens for pharmaceutical analysis.

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.

N-methylformamide as a separation medium in capillary electrophoresis

SummaryThe organic solvent N-methylformamide (NMF) has been used as a separation medium in capillary electrophoresis. The advantageous properties of this compound are its high dielectric constant, high solubilizing power and low conductivity, as well as its amphiprotic character. It was shown that, unlike for most organic solvents, the electroosmotic flow is substantial. It was found to be possible to utilize NMF without added electrolyte. Field strengths exceeding 1000 volts/cm could be employed, while a low current was maintained and it was thus possible to obtain rapid analyses. Also, the properties of NMF allowed the analysis of substances with a low solubility in aqueous media. These features are exemplified by separation of carboxylic acids and pharmaceuticals. Excellent reproducibility of migration and no sign of electrical breakdown were observed, even under high field strength conditions.

Development of micromachined hollow tips for protein analysis based on nanoelectrospray ionization mass spectrometry

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 μm, 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.

Parallel reactions in open chip-based nanovials with continuous compensation for solvent evaporation

In an earlier report (Litborn, E., Emmer, Å., Roeraade, J., Anal. Chim. Acta 1999, 401, 11—19), we described a technique for performing chemistry in chip‐based vials. A major problem, solvent evaporation, was partially remedied by using a closed humidity chamber. In this paper we report an improved technique for performing parallel reactions in open, 15 nL volume, chip‐based vials. The evaporation of solvent from the reaction fluid was continuously compensated by addition of solvent via an array of microcapillaries. The suitability of the method was demonstrated by performing eight separate peptide maps of myoglobin in parallel, using the three enzymes trypsin, α‐chymotrypsin and endoproteinase Glu‐C. The total amount of myoglobin utilized to perform the eight digests was less than 100 pmol. The corresponding amount of enzymes was ca. 0.1 pmol per reaction. In order to evaluate the operating limits of the technique, a study of the evaporation of solvents from a series of vials with proportionally smaller volumes operated at different temperatures was performed. The results showed that the concept for continuous compensation of solvent evaporation should be applicable to reaction volumes down to 30 pL.

Automated monitoring of organic trace components in water : I. Continuous flow extraction together with on-line capillary gas chromatography

Abstract A system for automated monitoring of volatile organic trace compounds in water is described. The water is continuously extracted in a fused-silica capillary tube after flow injection of an immiscible solvent. Separation of the phases is accomplished with a porous PTFE membrane separator. The extract flows through a fused-silica sample loop, connected to a fused-silica capillary column or pre-column. The system operation is controlled by a programmable timer, which allows unattended analysis with regular time intervals. For evaluation of the system, dilute water solutions of aromatic and aliphatic hydrocarbons as well as water samples contaminated with trace amounts of a naphtha fraction were employed. Extraction was performed with n-pentane. The results were practically identical to those obtained from batch extractions carried out under comparable conditions. However, a better reproducibility was obtained with the flow extraction procedure. The on-column injection of large sample volumes (150 μ) permitted analyses at the ppt level, as demonstrated with a sample of continuously extracted waste-water from a sewage plant. Significant adsorption of the trace components can occur on surfaces that are not brought into contact with the extractant. It is suggested that only a short length of fused-silica capillary tubing be used as a connection between the sample and the segmenting device.

Characterization of proteinases from Antarctic krill (Euphausia superba)

Fractions of three trypsin-like proteinases, TL I, TL II, and TL III, a chymotrypsin-like proteinase, CL, two carboxypeptidase A enzymes, CPA I and CPA II and two carboxypeptidase B enzymes, CPB I and CPB II, from Antarctic krill (Euphausia superba) have been characterized with respect to purity by the means of capillary electrophoresis, CE, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The masses of the trypsin-like and chymotrypsin-like proteinases were determined to be 25,020, 25,070, 25,060, and 26,260Da for TL I, TL II, TL III, and CL, respectively. The masses of the CPA enzymes are likely 23,170 and 23,260Da, whereas the CPB enzyme masses likely are 33,730 and 33,900Da. The degradation efficiency and cleavage pattern of the trypsin-like proteinases were studied with native myoglobin as a model substrate using CE, MALDI-TOF-MS, and nanoelectrospray mass spectrometry (nESI-MS). The degradation efficiency of the trypsin-like proteinases was found to be approximately 12 and 60 times higher compared to bovine trypsin at 37 degrees C and 1-3 degrees C, respectively. All three fractions of trypsin-like proteinases showed a carboxypeptidase activity in combination with their trypsin activity.

Micro vials on a silicon wafer for sample introduction in capillary electrophoresis

Abstract A multi-sample holder produced by photolithographic and anisotropic etching techniques on a monocrystalline silicon wafer is described. Well defined volumes are created by utilizing the crystal orientations of the silicon material. By using step and repeated lithography, a large number of identical cavities can be obtained on a single chip. The surface is covered with a thin film of gold, which serves as a cathode during electrokinetic injections. Evaluation was carried out by injecting a mixture of pd(A) oligonucleotides (40–60) bases from a 118-nl sample well on to a 50 ωm I.D. polyacrylamide-filled gel column. When compared with injections from classical vials, no additional zone broadening was observed.