Biomedical subjects

T Laurell

Publications and source records attributed to T Laurell.

At least 19 recordsLinked to original sources

PubMed2001

A method for the design and study of enzyme microstructures formed by means of a flow-through microdispenser.

Micrometer-sized enzyme grids were fabricated on gold surfaces using a novel method based on a flow-through microdispenser. The method involves dispensing very small droplets of enzyme solution (approximately 100 pL) during the concomitant relative movement of a gold substrate with respect to the nozzle of a microdispenser, resulting in enzyme patterns with a line width of approximately 100 microm. Different immobilization methods have been evaluated, yielding either enzyme monolayers using functionalized self-assembled thiol monolayers for covalent binding of the enzyme or enzyme multilayers by cross-linking or entrapping the enzymes in a polymer film. The latter immobilization techniques allow the formation of coupled multienzyme structures. On the basis of this feature, coupled bienzyme (glucose oxidase and catalase) or three-enzyme (alpha-glucosidase, mutarotase, and glucose oxidase) microstructures consisting of line patterns of one enzyme intersecting with the patterned lines of the other enzyme(s) were fabricated. By means of scanning electrochemical microscopy (SECM) operated in the generator-collector mode, the enzyme microstructures and their integrity were visualized using the localized detection of enzymatically produced/consumed H2O2. A calibration curve for glucose could be obtained by subsequent SECM line scans over a glucose oxidase microstructure for increasing glucose concentrations, demonstrating the possibility of obtaining localized quantitative data from the prepared microstructures. Possible applications of these enzyme microstructures for multianalyte detection and interference elimination and for screening of different biosensor configurations are highlighted.

Biosensing Techniques↗

PubMed2001

Development of silicon microstructures and thin-film MALDI target plates for automated proteomics sample identifications.

Here we report on the development of a proteomic platform utilizing a piezoelectric flow-through dispensing unit made from silicon microstructures. The use of a novel surface coating, where matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI MS) targets were uniformly precoated with a thin film of matrix/nitrocellulose, made the sample preparation straightforward and enabled the enrichment and analysis of proteins at low levels in proteomics samples. We demonstrate this by analyzing excised spots in a biological sample originating from a human fetal fibroblast cell line that was subjected to 2D gel-electrophoresis. Furthermore, a sample deposition rate below 30 Hz results in an increased analyte density on the dispensed sample spot, rendering signal amplification. In general, the sensitivity for proteins and peptides can be enhanced 10-50 times compared to traditional MALDI sample preparation techniques.

Animals↗

PubMed2001

Determination of diffusion coefficients of electroactive species in time-of-flight experiments using a microdispenser and microelectrodes.

Two novel methods for the determination of diffusion coefficients of redox species combining the special properties of microdispensing devices and microelectrodes are presented. Both are based on the local application of tiny volumes of the redox-active species by means of a dispenser nozzle at a defined distance from the surface of a microelectrode. The microelectrode, which is inserted through the bottom into an electrochemical cell, is held at a constant potential sufficient to oxidize or reduce the electro-active species under diffusional control. The dispenser, which is filled with the electro-active species, can be positioned by means of micrometer screws over the microelectrode. After dispensing a defined number of droplets near the microelectrode surface, the current through the microelectrode is recorded, usually yielding a peak-shaped curve having a defined time delay between the shooting of the droplets and the maximum current. The time that is necessary to attain maximum current, together with the known distance between two dispensing points, can be used to determine the diffusion coefficient of the electroactive species without knowledge of any system parameters, such as concentration of the redox species, diameter of the electroactive surface or number of transferred electrons. A similar method for the determination of diffusion coefficient of redox species involves a second redox species for calibration purposes. A mixture of both species is shot close to the microelectrode surface. Due to the different formal potentials of the redox species that are used, they can be distinguished in sequential experiments by variation of the potentials that are applied to the microelectrode, and it is thus possible to determine the individual transit times of the redox species independently. The difference in the transit times, together with the known diffusion coefficient of one of the redox species, can be used to calculate the unknown diffusion coefficient of the second one.

Journal Article↗

PubMed2001

Silicon microstructures for high-speed and high-sensitivity protein identifications.

Silicon microtechnology has been used to develop a microstructure toolbox in order to enable high accuracy protein identification. During the last 2 years we developed and applied monocrystalline silicon structures and established new automated protein analysis platforms. The development of a high throughput protein platform is presented where fully automated protein identifications are performed. It includes the reduction and alkylation of the protein sample in a standard 96- or 384-well plate format prior to injection of 1 microl samples into the continuous flow based microtechnology platform. The processed sample is transferred to a microchip nanovial array target using piezoelectric microdispensing. Identification is made by MALDI-TOF MS and a database search. After the initial sample reduction and alkylation period of 50 min the platform can digest and process protein samples at a speed of 100 samples in 210 min. An optional configuration of the platform, operating the dispenser in the 'static mode', enables on-target enrichment of low abundant proteins and peptides e.g. from 2DE samples. This makes detection at the low attomole level possible.

Proteins↗

PubMed2001

Analysis of regulatory phosphorylation sites in ZAP-70 by capillary high-performance liquid chromatography coupled to electrospray ionization or matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

A methodology for the rapid and quantitative analysis of phosphorylation sites in proteins is presented. The coupling of capillary high-performance liquid chromatography (HPLC) to electrospray ionization mass spectrometry (ESI-MS) allowed one to distinguish phosphorylation sites based on retention time and mass difference from complex peptide mixtures. The methodology was first evaluated and validated for a mixture of non-, mono-, and dityrosine-phosphorylated synthetic peptides, corresponding to the tryptic fragment 485-496 (ALGADDSYYTAR) of the human protein tyrosine kinase ZAP-70. The limits of detection for the non-, mono- and diphosphorylated peptides were about 15, 40 and 100 fmol, respectively, when using a 300 microm I.D. column. Application of the method was extended to identify phosphopeptides generated from a trypsin digest of recombinant autophosphorylated ZAP-70, in particular with respect to quantifying the status at the regulatory phosphorylation sites Tyr-492 and Tyr-493. Combination of chromatographic and on-line tandem mass spectrometry data allowed one to ascertain the identity of the detected peptides, a prerequisite to analyses in more complex biological samples. As an extension to the methodology described above, we evaluated the feasibility of interfacing capillary HPLC to matrix assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS), using a micromachined piezoelectric flow-through dispenser as the interface. This enabled direct arraying of chromatographically separated components onto a target plate that was precoated with matrix for subsequent analysis by MALDI-TOF-MS without further sample handling.

Amino Acid Sequence↗

PubMed2001

Signal amplification using "spot-on-a-chip" technology for the identification of proteins via MALDI-TOF MS.

The presented "spot-on-a-chip" technology enables easy enrichment of samples in the low nanomolar (1-5 nM) range and provides a fast and reliable automated sample preparation method for performing matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis with high sensitivity and throughput. Through microdispensing, which allows accurate deposition of 60-pL droplets, dilute samples were enriched by making multiple droplet depositions in nanovials. The sample was confined to a defined spot area (300 x 300 microm), and multiple depositions increase the surface density of analyte in the nanovial, thereby providing detection of low attomole levels. The impact of the nanovial geometry with respect to the MALDI-TOF MS resolution for peptides deposited in the microfabricated silicon vials was investigated and the optimal geometry and size were determined. The spot-on-a-chip technology, that is, the combination of microdispensing, micromachined silicon nanovials and on-spot enrichment provides a signal amplification of at least 10-50 times as compared to an ordinary sample preparation. The linearity of the enrichment effect is shown by the analysis of a peptide mixture at the 5 nM level. The signal amplification provided by the spot-on-a-chip enrichment is demonstrated by the analysis of relevant biological samples, interleukin-8 from a spiked cell supernatant, and by successful protein identification of an excised spot from a high-sensitivity silver-stained two-dimensional electrophoresis gel separation.

Adrenocorticotropic Hormone↗

PubMed2001

Disposable polymeric high-density nanovial arrays for matrix assisted laser desorption/ionization-time of flight-mass spectrometry: II. Biological applications.

A novel disposable high-density matrix assisted laser desorption/ionization (MALDI) target plate made either from polymethylmethacrylate (PMMA) or polycarbonate (PC) is presented where thousands (1,200-1,600) of samples can be deposited and subsequently analyzed by MALDI-time of flight (TOF) mass spectrometry. Good reproducibility was obtained across the plate regardless of position on the target plate with a relative standard deviation (RSD) on the peak intensity of typically 30% calculated from data generated by analysis of a 10 nm peptide mixture of angiotensin I, II, III and bradykinin. The nanovial array format combined with microdispensing technology makes it possible to carry out in-vial chemistry on deposited samples. This is demonstrated by the analysis of peptides from beta-casein and subsequent in-vial dephosphorylation of its phosphopeptides at 10 fmol levels by microdispensing of alkaline phosphatase, into the nanovial. The mass spectra obtained from these polymeric targets provides can also be used in high sensitivity applications as shown by peptide mass fingerprinting of human fibroblast proteins separated by two-dimensional gel electrophoresis.

Amino Acid Sequence↗

PubMed2001

Downsizing proteolytic digestion and analysis using dispenser-aided sample handling and nanovial matrix-assisted laser/desorption ionization-target arrays.

An efficient technique for enzymatic digestion of proteins in nanovial arrays and identification by peptide mass fingerprinting using matrix-assisted laser desorption/ionization (MALDI-MS) is presented in this work. Through dispensing of a protein solution with simultaneous evaporation the protein (substrate) is concentrated up to 300 times in-vial. At higher substrate concentrations the catalytic turnover numbers increase according to the Michaelis-Menten kinetics. Therefore, the dispenser-aided nanodigestion is valuable for identification of low-level proteins (10 nM-500 nM) as well as for automatic high efficiency digestions performed in 0.2-10 min. As an example of low-level protein identification, a 10 nM solution of lysozyme C was unambiguously identified after 5 min of nanodigestion. Moreover, only 30 s nanodigestion was sufficient to identify hemoglobin (10 microM), exemplifying the fast catalysis of the nanodigestion technique. The developed silicon flow-through piezoelectric dispenser is adapted for low-volume and preconcentrated samples in the nL-microL range and provides fast, accurate and contact-free sample positioning into the nanovials. In this work, the properties of the nanodigestion concept regarding proteins of different characteristics are explored. Furthermore, the potential of automated protein identification using precoated proteolytic nanovial-arrays is demonstrated.

Animals↗

PubMed2001

The geometric design of micromachined silicon sieve electrodes influences functional nerve regeneration.

A neural interface could be used to control a limb prosthesis. Such an interface can be created by facilitating axonal regeneration through a sieve electrode and then register nerve signals intended to control the prosthesis. A key question is how to design the electrodes to ensure the best possible regeneration. Our previous studies have indicated that regeneration can be achieved using electrodes with square-shaped, 100 x 100 microm, via holes (holes that axons will regenerate through). Other reports have indicated a suitable range of these holes between 40 and 65 microm. In the present study we used silicon sieve electrodes with via holes of either 30 or 90 microm. The transparency, i.e. the percentage of the total via hole area, of these electrodes was either 20 or 30%. The electrodes were inserted into a silicone chamber which was used to bridge a gap in a rat sciatic nerve. After 12 weeks of nerve regeneration electrodes with a hole size of 30 microm and a 30% transparency had the most favourable result as judged by the regained gastrocnemius muscle force and the formation of reactive tissue inside the chamber. The sieve electrode transparency is crucial for ensuring regeneration.

Animals↗

PubMed2001

A miniaturised electrochemical affinity assay based on a wall-free sample droplet and micro-dispensing of the redox-labelled binding partner.

An affinity-assay was developed that is based on the modulation of the diffusion coefficient of a redox-labelled hapten upon complementary recognition of the analyte leading to an increase of molecular weight and hence to a decrease of the diffusion coefficient. The slower diffusion is monitored by means of cyclic voltammetry. In order to demonstrate the feasibility of this assay format, recognition of biotin by streptavidin has been chosen as a model system. Labelling of biotin was achieved by covalent binding of a ferrocene derivative to the biotin unit. To reduce the consumption of expensive compounds and to allow automatisation of the assay a novel miniaturised set-up was developed based on a wall-free sample droplet which forms the electrochemical cell with typical volumes of up to 10 microl. This droplet is dispensed by means of a step-motor driven syringe pump through a specially designed electrode holder spanning the gap between a micro-working electrode and a macroscopic counter electrode. By means of a piezo-driven micro-dispenser a predefined number of nano-droplets (100 pl volume each) containing the redox-labelled hapten are shot into the sample droplet. By this, any physical contact and hence any cross-contamination between the sample and the reagent solution could be avoided. Signal amplification can be achieved by redox recycling between the micro-electrode and the perpendicular positioned macroscopic counter electrode.

Biosensing Techniques↗

PubMed2001

Picodroplet-deposition of enzymes on functionalized self-assembled monolayers as a basis for miniaturized multi-sensor structures.

We are reporting on a novel approach for structured immobilisation of enzymes on gold surfaces modified with monolayers of functionalised alkylthiols. The formation of enzyme spots is achieved by shooting very small volumes of an appropriate enzyme solution (down to 100 pl) onto a thiol-monolayer modified gold surface using a micro-dispenser. Formation of enzyme patterns is obtained by moving the micro-dispenser relative to the modified gold surface using a micro-positioning device. Enzyme spots with typical lateral dimensions of 100 microm are obtained, but also, more complex structures, e.g. lines or meander structures, can be achieved by multiple droplets dispensed during the concomitant movement of the micro-dispenser. The first enzyme layer on top of the functionalised thiol-monolayer is subsequently covalently immobilised using either carbodiimide activation of carboxilic headgroups at the enzyme or via already introduced activated ester functions at the monolayer. Immobilised enzyme activities of glucose oxidase and lactate oxidase patterns have been characterised by means of scanning electrochemical microscopy. The product of the enzyme-catalysed reaction, H(2)O(2), is detected with an micro-electrode in the presence of either or both substrates, glucose and lactate, leading to a visualisation of the corresponding enzyme pattern and the lateral enzymatic activity.

Biosensing Techniques↗

PubMed2001

Microfluidic components for protein characterization.

The use of microfluidic components to create an analytical toolbox for the very rapidly growing field of proteomics is described. This toolbox provides novel generic analytical solutions that are highly adaptable for analysis of various biomolecules, ranging from high to low abundant. The components are fabricated using silicon micromachining and consist of a microchip immobilised enzyme reactor (microIMER), a piezoelectric microdispenser and high-density nanovial target plates. This microtechnology based platform interfaces matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI TOF-MS) to a wide range of upstream sample handling and/or analytical techniques. Examples of applications such as rapid on-line digestion (12 s) and sample preparation of proteins, interfacing to capillary liquid chromatography (100 attomol sensitivity), and in-vial chemistry on femtomol amounts of sample are presented.

Bioreactors↗

PubMed2001

Protein chips based on recombinant antibody fragments: a highly sensitive approach as detected by mass spectrometry.

With the human genome in a first sequence draft and several other genomes being finished this year, the existing information gap between genomics and proteomics is becoming increasingly evident. The analysis of the proteome is, however, much more complicated because the synthesis and structural requirements of functional proteins are different from the easily handled oligonucleotides, for which a first analytical breakthrough already has come in the use of DNA chips. In comparison with the DNA microarrays, the protein arrays, or protein chips, offer the distinct possibility of developing a rapid global analysis of the entire proteome. Thus, the concept of comparing proteomic maps of healthy and diseased cells may allow us to understand cell signaling and metabolic pathways and will form a novel base for pharmaceutical companies to develop future therapeutics much more rapidly. This report demonstrates the possibilities of designing protein chips based on specially constructed, small recombinant antibody fragments using nano-structure surfaces with biocompatible characteristics, resulting in sensitive detection in the 600-amol range. The assay readout allows the determination of single or multiple antigen-antibody interactions. Mass identity of the antigens, currently with a resolution of 8000, enables the detection of structural modifications of single proteins.

Antibodies↗

PubMed2000

Protein identification platform utilizing micro dispensing technology interfaced to matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

An integrated protein microcharacterization/identification platform has been developed. The system has been designed to allow a high flexibility in order to tackle challenging analytical problems. The platform comprises a cooled microautosampler, an integrated system for microcolumn HPLC, and a capillary reversed-phase column that is interfaced to matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) system via a low internal volume flow-through microdispenser. The chromatographic separation is continuously transferred onto a MALDI target plate as discrete spots as the dispenser ejects bursts of droplets of the column effluent in a precise array pattern. A refrigerated microfraction collector was coupled to the outlet of the flow-through microdispenser enabling enrichment and re-analysis of interesting fractions. The use of target plates pre-coated with matrix simplified and increased the robustness of the system. By including a separation step prior to the MALDI-TOF-MS analysis and hereby minimizing suppression effects allowed us to obtain higher sequence coverage of proteins compared to conventional MALDI sample preparation methodology. Additionally, synthetic peptides corresponding to autophosphorylated forms of the tryptic fragment 485-496 (ALGADDSYYTAR) of tyrosine kinase ZAP-70 were identified at sensitivities reaching 150 amol.

Amino Acid Sequence↗

PubMed2000

Integrated microanalytical technology enabling rapid and automated protein identification.

Protein identification through peptide mass mapping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has become a standard technique, used in many laboratories around the world. The traditional methodology often includes long incubations (6-24 h) and extensive manual steps. In an effort to address this, an integrated microanalytical platform has been developed for automated identification of proteins. The silicon micromachined analytical tools, i.e., the microchip immobilized enzyme reactor (mu-chip IMER), the piezoelectric microdispenser, and the high-density nanovial target plates, are the cornerstones in the system. The mu-chip IMER provides on-line enzymatic digestion of protein samples (1 microL) within 1-3 min, and the microdispenser enables subsequent on-line picoliter sample preparation in a high-density format. Interfaced to automated MALDI-TOF MS, these tools compose a highly efficient platform that can analyze 100 protein samples in 3.5 h. Kinetic studies on the microreactors are reported as well as the operation of this microanalytical platform for protein identification, wherein lysozyme, myoglobin, ribonuclease A, and cytochrome c have been identified with a high sequence coverage (50-100%).

Autoanalysis↗

PubMed2000

Capillary liquid chromatography interfaced to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using an on-line coupled piezoelectric flow-through microdispenser

A piezoelectric flow-through microdispenser interfacing capillary liquid chromatography (LC) with matrix-assisted laser desorption/ionization time-of-fight mass spectrometry (MALDI-TOF MS) was developed for the identification of biomolecules. The MALDI target plate was placed on a computer controlled high-resolution x-y stage, on to which the column effluent was deposited as discrete spots, which thereby facilitated tracing of the chromatographic separation. The entire target plate was sprayed with a homogeneous layer of alpha-cyano-4-cinnamic acid mixed with nitrocellulose by using an air-brush. Hence the tedious manual handling of a micropipetter applying matrix solution on top of each fraction collected spot was avoided. The pre-made target plates were stable for at least 3 weeks if kept in darkness at room temperature, which easily allowed re-analysis of dispensed sample spots. The integrated microsystem was characterized and optimized by means of fluidics, dispersion, operational stability and sensitivity parameters. The dispensing unit was developed specifically to match high-resolution capillary LC separations using a dispenser with an internal volume from inlet to the ejecting nozzle of 250 nl. Minimizing dead volumes was crucial in order to maintain the chromatographic resolution. The volume of the ejected droplets was of the order of 60 pl. Successful separations of seven immunoregulating peptides were made: ACTH 1-17, bradykinin, enkephalin, angiotensin III, angiotensin II, angiotensin I and ACTH 18-39. On-line sample dispensing on the target plate in combination with trace enrichment followed by automated MALDI-TOF MS identification is demonstrated, reaching a sensitivity of 100 amol.

Journal Article↗

PubMed1999

Homogeneous sample preparation for automated high throughput analysis with matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry.

This work presents a simple method for obtaining homogeneous sample surfaces in matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) for the automated analysis of peptides and proteins. The sample preparation method is based on applying the sample/matrix mixture onto a pre-deposited highly diluted matrix spot. The pre-deposited crystals act as seeds for the new sample containing crystals which become much smaller in size and more evenly distributed than with conventional methods. This 'seed-layer' method was developed, optimised and compared with the dried-droplet method using peptides and proteins in the 1000-20,000 Da range. The seed-layer method increases the surface homogeneity, spot to spot reproducibility and sample washability as compared with the commonly used dried-droplet method. This methodology is applicable to alpha-cyanohydroxycinnamic acid, sinapinic acid and ferulic acid, which all form homogeneous crystal surfaces. Within-spot variation and between-spot variation was investigated using statistics at a 95% confidence level (n = 36). The statistical values were generated from more than 5000 data points collected from 500 spectra. More than 90% of the sample locations results in high intensity spectra with relatively low standard deviations (RSDs). Typically obtained data showed an RSD of 19-35% within a sample spot as well as in-between spots for proteins, and an RSD of < or = 50% for peptides. Linear calibration curves were obtained within one order of magnitude using internal calibration with a point-RSD of 3% (n = 10). The sample homogeneity allows mass spectra (average of 16 laser shots) to be obtained on each individual sample within 15 sec, whereby a 100 spot target plate can be run in 25 min. High density target plates using the seed-layer method were prepared by spotting approximately 100 picoliter droplets onto the target, resulting in sample spots < or = 500 microns in diameter using a flow-through piezo-electric micro-dispenser. By using this automated sample preparation step lower standard deviations are obtained in comparison to manually prepared samples.

Coumaric Acids↗

PubMed1999

Perforated silicon nerve chips with doped registration electrodes: in vitro performance and in vivo operation.

An in vitro model was developed for the study of signal transduction between a Cu-wire, miming a neural signal source, and recording electrodes on perforated silicon chips. Phosphorous doped electrodes were used to achieve an all silicon device. The model was used to study signal amplitude as a function of the spatial position, and distance to the signal source. Recordings of the signal crosstalk to neighboring electrodes on the chips were made. It was found that the amplitude decreased by a factor of two at a distance of 50 microns between the electrode surface and the signal source. The chip electrode signal crosstalk was found to be 6 dB using an external reference electrode. Improvements were accomplished with an on chip reference electrode giving a crosstalk suppression of 20 dB. Impedance analysis showed that doped silicon electrodes displayed similar characteristics as Cu-electrodes at frequencies above 3 kHz. Sieve electrodes were implanted in the rat sciatic nerve and following a 10-week nerve regeneration period the dorsal and ventral (L5) roots in the spinal cord were stimulated. Compound action potentials were recorded via the chip. Stimulating the regenerated sciatic nerve via the sieve electrode also induced lower leg muscle contraction activity.

Action Potentials↗