Richardus B.M. Schasfoort

A new approach to immunoFET operation

A new method is presented for the detection of an immunological reaction taking place in a membrane, which covers the gate area of an ISFET. By stepwise changing the electrolyte concentration of the sample solution, a transient diffusion of ions through the membrane-protein layer occurs, resulting in a transient membrane potential, which is measured by the ISFET. The diffusion rate is determined by the immobile charge density in the amphoteric protein layer, which changes upon formation of antibody-antigen complexes. No membrane potential is induced at zero fixed charge density as occurs at a protein characteristic pH. Isoelectric points of embedded proteins can be determined by detecting the zero potential response. Up to now, the authors have studied the membrane adsorption of lysozyme, human serum albumin (HSA) and the immune reaction of HSA with the antibody anti-human serum albumin (alpha HSA). The influence of protein parameters on the amplitude of the transient can be described with an empirical equation. Assuming Langmuir behaviour, the protein concentration in the solution can well be correlated with the concentration in the membrane. This new detection method is unique concerning direct measurements of charge densities and isoelectric points of amphoteric macromolecules adsorbed in the membrane. The simple procedure of one incubation stage followed by one detection stage, without separate washing and labelling techniques, gives direct information about specific charge properties of the macromolecules to be studied.

New approaches for fabrication of microfluidic capillary electrophoresis devices with on-chip conductivity detection.

In practice, microfluidic systems are based on the principles of capillary electrophoresis (CE), for a large part due to the simplicity of electroosmotic pumping. In this contribution, a universal conductivity detector is presented that allows detection of charged species down to the μM level. Additionally, powderblasting is presented as a novel technique for direct etching of microfluidic networks. This method allows creation of features down to 50u2005μm with a total processing time (design to device) of less than one day. The performance of powderblasted devices with integrated conductivity detection is illustrated by the separation of lithium, sodium, and potassium ions and that of fumaric, malic, and citric acid.

The ISFET based heparin sensor with a monolayer of protamine as affinity ligand

The ion-step measuring method was used to determine absolute heparin concentrations in PBS and blood plasma with a Ta2O5 ISFET on to which a monolayer of protamine had been immobilized. Heparin is a highly negatively charged polysaccharide, which is used clinically to delay the clotting of blood. Protamine acts as an affinity ligand for heparin. The response of the ISFET system on a step-wise increase in the electrolyte concentration (a so-called ion-step) is a transient change of the output voltage, which is related to the surface charge density of the ISFET gate oxide. After 2 mins of incubation in a plasma sample containing heparin, the amplitude of the transient ISFET response to an ion-step showed a linear relation to the heparin concentration. In blood plasma, heparin concentrations between 0.3 and 2.0 Units/ml could be determined with an accuracy of +/- 0.08 Units/ml. Heparin concentrations in different plasma samples of heparinized patients were determined and compared with the APTT. No direct relation was found between the APTT and the heparin concentration, but this result was not surprising.

Powder-blasting technology as an alternative tool for microfabrication of capillary electrophoresis chips with integrated conductivity sensors

The fabrication and characterization of a microfluidic device for capillary electrophoresis applications is presented. The device consists of a glass chip which contains a single separation channel as well as an integrated conductivity detection cell. In contrast to most microfluidic glass devices the channels are not wet etched in HF but machined by the newly developed micro powder-blasting technique which allows the creation of microstructures below 100 µm, and additionally makes parallel hole machining at very low costs outside the cleanroom environment possible [1, 2]. The integration of the conductivity detector was achieved by leading two thin-film metal electrodes inside the separation channel. For rapid sample injection the chip is mounted inside an autosampler-based capillary electrophoresis platform. The detection electrodes for conductivity detection are read out by lock-in amplifier electronics. First measurements show the successful separation of various ions in the sub-millimeter range.

Possibilities and limitations of direct detection of protein charges by means of an immunological field-effect transistor

An outline of the requirements for the construction of an immunological field-effect transistor (ImmunoFET) which should operate on the direct potentiometric sensing of protein charges is given. Selectivity of the ImmunoFET can be obtained by immobilizing antibodies on the gate area of the ISFET, enhancing the surface affinity to the corresponding antigens over other molecules in the solution. A theoretical approach is given based on the Donnan equilibrium description, which provides an insight into the potential and ion distribution in the protein layer on the ImmunoFET. It is shown that the Donnan potential and the internal pH shift, induced by the protein charges, compensate each other to a great extent. If the ISFET shows Nernstian behaviour, it is concluded that a direct detection of protein charge is impossible. In order to construct an ImmunoFET, a reference FET (REFET) or ISFET with low sensitivity would satisfy the detection of the partially compensated Donnan potential in the presence of an adsorbed protein layer. However, the application of such an ImmunoFET is limited to samples with low ionic strength.

Mapping of citrullinated fibrinogen B-cell epitopes in rheumatoid arthritis by imaging surface plasmon resonance.

IntroductionRheumatoid arthritis (RA) frequently involves the loss of tolerance to citrullinated antigens, which may play a role in pathogenicity. Citrullinated fibrinogen is commonly found in inflamed synovial tissue and is a frequent target of autoantibodies in RA patients. To obtain insight into the B-cell response to citrullinated fibrinogen in RA, its autoepitopes were systematically mapped using a new methodology.MethodsHuman fibrinogen was citrullinated in vitro by peptidylarginine deiminases (PAD), subjected to proteolysis and the resulting peptides were fractionated by ion exchange chromatography. The peptide composition of the citrullinated peptide-containing fractions was determined by high resolution tandem mass spectrometry. The recognition of these fractions by patient sera was subsequently analyzed by imaging surface plasmon resonance on microarrays.ResultsIn total about two-thirds of the 81 arginines of human fibrinogen were found to be susceptible to citrullination by the human PAD2, the human PAD4 or the rabbit PAD2 enzymes. Citrullination sites were found in all three polypeptide chains of fibrinogen, although the α-chain appeared to contain most of them. The analysis of 98 anti-citrullinated protein antibody-positive RA sera using the new methodology allowed the identification of three major citrullinated epitope regions in human fibrinogen, two in the α- and one in the β-chain.ConclusionsA comprehensive overview of citrullination sites in human fibrinogen was generated. The multiplex analysis of peptide fractions derived from a post-translationally modified protein, characterized by mass spectrometry, with patient sera provides a versatile system for mapping modified amino acid-containing epitopes. The citrullinated epitopes of human fibrinogen most efficiently recognized by RA autoantibodies are confined to three regions of its polypeptides.

Development of an ISFET based heparin sensor using the ion-step measuring method

The ion-step measuring method was used to determine heparin concentrations in PBS and blood plasma. Heparin is a sulphated polysaccharide which is clinically used as a drug to prevent the clotting of blood. The measuring method is based on detection of changes in charge density in a porous membrane which is deposited on the gate of an ISFET. Protamine was used as affinity ligand in the membrane. In PBS a linear relation was found between the heparin concentration and the ISFET response. The incubation time was reduced from 18 h to 15 min by increasing the porosity of the membrane. The results of the measurements in blood plasma show a significant nonspecific binding of plasma components in the membrane. Suggestions are given to prevent this nonspecific adsorption. The results described in this paper show a detection limit for the ion-step measuring method of at least 5 ± 10-11 Mol/l which is promising for future practical applications.

Fabrication of a Microfluidic Chip by UV Bonding at Room Temperature for Integration of Temperature Sensitive Layers

A method for the bonding of a microfluidic device at room temperature is presented. The wafer with the fluidic structures was bonded to a sensor wafer with gold pads by means of adhesive bonding, utilizing an UV-curable glue layer. To avoid filling the fluidic channels with the glue, a stamping process was developed which allows the selective application of a thin glue layer. In this way a microfluidic glass chip was fabricated that could be used for performing surface plasmon resonance measurements without signs of leakage. The advantage of this method is the possibility of integration of organic layers as well as other temperature-sensitive layers into a microfluidic glass device.

Analysis of cell surface antigens by Surface Plasmon Resonance imaging

Surface Plasmon Resonance (SPR) is most commonly used to measure bio-molecular interactions. SPR is used significantly less frequent for measuring whole cell interactions. Here we introduce a method to measure whole cells label free using the specific binding of cell surface antigens expressed on the surface of cancer cells and specific ligands deposited on sensor chips using an IBIS MX96 SPR imager (SPRi). As a model system, cells from the breast cancer cell line HS578T, SKBR3 and MCF7 were used. SPRi responses to Epithelial Cell Adhesion Molecule (EpCAM) antibody and other ligands coated on the sensor chips were measured. SPR curves show a response attributable to the sedimentation of the cells and a specific binding response on top of the initial response, the magnitude of which is dependent on the ligand density and the cell type used. Comparison of SPRi with flow cytometry showed similar EpCAM expression on MCF7, SKBR3 and HS578T cells.

Proteomics-on-a-chip: the challenge to couple lab-on-a-chip unit operations

This review describes a vision of a proteomics-on-a-chip device to separate, detect and identify the proteome. It guides the reader towards a development strategy, avoiding some of the pitfalls. It also describes the current state-of-the-art developments in proteomic analysis including available technologies, current market issues, the elements of an envisaged proteomics-on-a-chip device, the required microfabrication processes and the integration of the elements into one device. Address-flow microfluidics is a tool for connecting separation and detection platforms. The final section contains an expert opinion on the recommended development strategies, benefits of proteomics-on-a-chip in the life sciences and the anticipated market.