Microfluidic Isoelectric Focusing of Amyloid Beta Peptides Followed by Micropillar-Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry.

A novel method for preconcentration and purification of the Alzheimer's disease related amyloid beta (Aß) 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 Aß 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 Aß 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 Aß 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 Aß peptides in human cerebrospinal fluid as well as in blood plasma.

New method for fabrication of fused silica emitters with submicrometer orifices for nanoelectrospray mass spectrometry.

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 µm 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).

Electrospray ionization mass spectrometry from discrete nanoliter-sized sample volumes.

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 2.5 h, 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.

Separation and characterization of aggregated species of amyloid-beta peptides.

We have investigated the use of isoelectric focusing and immunodetection for the separation of low molecular weight species of amyloid-beta (Abeta) peptides from their aggregates. From solutions of Abeta(1-40) or Abeta(1-42) monomeric peptides, low molecular weight material appeared at a pI value of ca. 5, while the presence of aggregates was detected as bands, observed at a pI of 6-6.5. The formation of Abeta aggregates (protofibrils) was verified by a sandwich ELISA, employing the protofibril conformation-selective antibody mAb158. In order to study the aggregation behavior when using a mixture of the monomers, we utilized the IEF separation combined with Western blot using two polyclonal antisera, selective for Abeta(1-40) and Abeta(1-42), respectively. We conclude that both monomers were incorporated in the aggregates. In a further study of the mixed aggregates, we used the protofibril conformation-selective antibody mAb158 for immunoprecipitation, followed by nanoelectrospray mass spectrometry (IP-MS). This showed that the Abeta(1-42) peptide is incorporated in the aggregate in a significantly larger proportion than its relative presence in the original monomer composition. IP-MS with mAb158 was also performed, and compared to IP-MS with the Abeta-selective antibody mAb1C3, where a monomeric Abeta(1-16) peptide was added to the protofibril preparation. Abeta(1-16) is known for its poor aggregation propensity, and acted therefore as a selectivity marker. The results obtained confirmed the protofibril conformation selectivity of mAb158.

Magnetic bead-based detection of autoimmune responses using protein microarrays.

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.

Electrostatic sample nebulization for improved sample vaporization in the split/splitless gas chromatography inlet.

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 approximately 30% for injections where no high voltage was applied.

Mixed sorbent phases for thick film open tubular traps.

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.

Non-contact protein microarray fabrication using a procedure based on liquid bridge formation.

Contemporary microarrayers of contact or non-contact 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-microm 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.

Electrospray ionization from an adjustable gap between two silicon chips.

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 microm 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.

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.

Expanding assay dynamics: a combined competitive and direct assay system for the quantification of proteins in multiplexed immunoassays.

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.

Separation of oligonucleotides in N-methyl-formamide-based polymer matrices by capillary electrophoresis.

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, C16-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 oligonucleotides 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.

Microdroplet deposition under a liquid medium.

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.

Automated microdroplet platform for sample manipulation and polymerase chain reaction.

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.

Picoliter droplet formation on thin optical fiber tips.

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 degrees and 75 degrees , 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.

Electrospray ionization from a gap with adjustable width.

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 microm. 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 microm 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.

Multi-step dielectrophoresis for separation of particles.

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.

C-terminal ladder sequencing of peptides using an alternative nucleophile in carboxypeptidase Y digests.

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.

Determination of conductivity of bacteria by using cross-flow filtration.

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 7,000 microS/cm, which is a deviation from previously published data by almost an order of a magnitude.

Superpositioned dielectrophoresis for enhanced trapping efficiency.

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.