Andreas Katerkamp

A label-free affinity sensor with compensation of unspecific protein interaction by a highly sensitive integrated optical Mach–Zehnder interferometer on silicon

Abstract An integrated optical Mach–Zehnder interferometer (IO-MZI) on silicon was specially designed and tested for application as an affinity sensor. In order to obtain the necessary sensitivity, an optimisation of the refractive index and the thickness of the waveguiding layer was carried out. Refractive measurements with ethanol/water mixtures show a sensitivity of about one order of magnitude higher than the IO-MZIs previously described. The compensation of unspecific protein interaction in an affinity sensor set-up was demonstrated by using both branches of the IO-MZI. One branch was coated with a antigenic structure and blocked with a protein mixture whereas the other was only blocked. A sample with a high background of serum proteins was applied and only the sample containing the specific antibody gave a measurable signal.

Remote sensing of tetrachloroethene with a micro-fibre optical gas sensor based on surface plasmon resonance spectroscopy

A miniaturized fibre optical sensor based on surface plasmon resonance spectroscopy is investigated in view of the detection of organic solvent vapours, particularly tetrachloroethene. Surface plasmons are excited on a silver coated multimode fibre by polychromatic light, and the resonant excitation is detected as a resonant absorption band in the measured output spectrum. When the analyte is absorbed in a thin gas-sensitive polysiloxane film deposited on the silver layer the polymer film changes its thickness and its refractive index. These changes result in a wavelength shift of the resonant curve depending on the analyte gas concentration. Theoretical considerations about the sensing effect are made and resonance curves were computer-simulated. Based on these simulations the layout of all sensor parameters was optimized. The sensor shows an excellent response to tetrachloroethene with a response time of two seconds and high reproducibility. When using self-assembling monolayers on the silver surface a long-term stability of more than 3 months can be obtained. Very low cross sensitivities of less than 1% to other solvent vapours like acetone and ethanol are obtained, furthermore, the influence of humidity is very low. This miniaturized fibre optical sensor in combination with an easy-to-handle and non-sophisticated measuring and evaluation unit is excellently suitable for the remote sensing of special organic solvent vapours.

Flow-injection immunosensor for triazine herbicides using Eu( III) chelate label fluorescence detection

An immunochemical flow-injection system for the determination of triazine herbicides based on principles of immunoaffinity chromatography was developed. Triazine herbicide derivatives immobilized on oxirane acrylic beads serve as the affinity column. They are saturated with fluorescently labelled monoclonal anti-herbicide antibodies prior to the assay. The label used is the fluorescent Eu(III) chelate W8044-Eu. A fraction of the fluorescent antibodies is replaced when exposed to analyte and this fluorescence is detected in a postcolumn mode by means of a special laser-based fluorimeter. With the reusable affinity column a detection limit of μg l−1 for the herbicide atrazine was obtained.

Sensing fatty acid binding protein with planar and fiber-optical surface plasmon resonance spectroscopy devices

Abstract The human heart-type fatty acid binding protein (H-FABP) can be used as an early heart infarction marker in clinical diagnostics because its concentration in blood plasma increases about 1.5 to 3 hours after the onset of an acute myocardial infarction. Hence, it is of main interest to determine an infarct with a fast immunosensor. Surface plasmon resonance spectroscopy (SPRS) is one of the most versatile and sensitive direct optical technologies used for biochemical sensors. The aim of this study is the development of a direct optical immunosensor for the detection of H-FABP. Two different SPRS devices have been constructed: the well-known planar configuration and a new promising fiber-optical transducer. Both transducers are compared under the same conditions with regard to the measurement of H-FABP. They show a similar response and an excellent correspondence of the calibration curves. The detection limit of H-FABP in a competitive assay is about 200 ng ml −1 in both cases.

NEW WAYS IN BIOANALYSIS ONE-WAY OPTICAL SENSOR CHIP FOR ENVIRONMENTAL ANALYSIS

An optical immunosensor system consisting of a disposable low-cost sensor chip including a fluidic system and a base unit for optical readout was developed. Near infrared (NIR)-fluorescence markers (Cy5) were excited by an evanescent wave generated on the surface of the sensor chip. The combination of both fluorescence measurements and evanescent wave excitation provides extremely sensitive detection and avoids any washing or separation steps. To demonstrate the feasibility of the system for environmental control assays for the determination of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) were developed. Two different assay formats were applied to determine 2,4-D in the relevant concentration range. Due to the assay formats chosen a direct proportional relationship between analyte concentration and signal intensity was achieved. Within an assay time of 15 min only, the analyte 2,4-D could be determined in a linear concentration range covering three orders of magnitude.

Surface investigations on the development of a direct optical immunosensor.

In the present paper surface studies for the development of a direct optical immunosensor for fast diagnosis of a myocardial infarction are presented. A fatty acid binding protein was detected by monoclonal antibodies. The applied measuring system was the grating coupler BIOS-1. Based on commercially available transducer materials protein immobilisation techniques have been developed and characterised by TOF-SIMS, AFM and EM. Three different label-free assay types were investigated. Only one assay leads to a sensitive and regenerable sensor set-up. It was possible to detect concentrations of the fatty acid binding protein down to 330 ng/ml. The general applicability of a direct optical immunosensor in the field of myocardial infarction diagnosis was demonstrated by this.

Optimisation of a polymer membrane used in optical oxygen sensing

A method for detection of viable cells utilises a sensor based on the optical measurement of oxygen consuming by cells. Changes in the oxygen level were measured via quenching of the fluorescence of an oxygen-sensitive fluorophor (Ru(dpp) 3Cl2). The fluorescence lifetime changing was measured in accord with Stern–Volmer equation, using a phase-shift method. The fluorophor was embedded into a polysulfone membrane that is in contact with the cell medium. The sensitivity of oxygen sensor depends on behaviour of polysulfone membrane. Manufacturing method, type of polysulfone and concentration of fluorophor can also change this behaviour. These parameters were explored to obtain the optimum analytical performance, and the optimum sensitive membrane was chosen for 3 mmol/l concentration of fluorophor, when a linear plot was obtained with R = 0.99987 for a sensitivity of 12.11 ± 0.11 mV/% O2 (n = 5).

Micro-chemical sensors based on fiber-optic excitation of surface plasmons

In this paper the theoretical base and experimental results of a new class of fiber optical supported surface plasmon resonance spectroscopy (SPRS) transducer is given. Surface plasmons were excited by polychromatic light, and the resonant excitation is detected as an intensity minimum in the measured spectra at certain wavelengths. The excitation takes place at the end zone of a multimode fiber coated with a thin surface plasmon resonance supporting metal layer. As examples for the large application field of this transducer a fiber optical immunosensor for the detection of antibodies against bovine serum albumin and a gas sensor for remote detection of tetrachloroethene was constructed and tested successfully. The sensors were constructed following the theoretical predictions for an optimal performance.

Kinetic analysis of immunointeractions with covalently immobilized fatty acid-binding protein using a grating coupler sensor

Application of a grating coupler sensor (GCS) to the real time investigation of the interaction kinetics of covalently immobilized recombinant bovine heart-type fatty acid-binding protein (H-FABP) and corresponding antibody is described. The immobilization of the antigen is performed by activating the matrix hydroxyl groups with p-toluenesulfonyl chloride (TSC) and afterwards coupling the protein by reaction with its nucleophilic aminogroups. Covalent coupling via TSC permits reproducible measurements of immunointeractions on the same grating coupler sensor chip and complete regeneration after each binding cycle with glycine-hydrochloride. We demonstrate the analysis of binding data obtained on a GCS by linearization as well as direct curve fitting using the integrated rate equation for the determination of apparent rate and affinity constants. With both analysis methods we studied H-FABP/monoclonal anti-H-FABP-antibody interactions and obtained an average apparent association rate constant ka = 4.2 X 10(3) M(-1) s(-1) a dissociation rate constant of kd=1.3 X 10(-4) s(-1) and an equilibrium constant of KD=3 X 10(-8) M.

Evaluation of several methods to quantify immobilized proteins on gold and silica surfaces

Abstract In the present study, the application of several methods for the determination of an immobilized protein are presented and the results discussed. Streptavidin was bound covalently on gold and silica surfaces. A radio assay was used as a reference method for the quantification of the immobilized protein. Two photometric, two fluorescence-spectroscopic methods and an immunochemical approach, based on a sandwich-ELISA (enzyme linked immunosorbent assay) format were applied with regard to their feasibility for sensitive protein quantification. The photometric methods were not sensitive enough, but one of the fluorescence based methods and the ELISA could be applied for the detection of low protein concentrations on the surfaces.