Guenter Gauglitz

Optical multiple-analyte immunosensor for water pollution control.

A prototype of a portable optical immunosensor (called river analyser) has been developed. It can be applied for the monitoring of surface water quality. Antibodies carrying a fluorescent label are used for the specific recognition of pollutants, such as frequently applied pesticides. The transduction principle is based on total internal reflection fluorescence (TIRF). The outstanding advantage of the river analyser is, that at least three analytes can be detected simultaneously in one sample. Test cycles and fluid handling are automated and enable unattended measuring.

Characterization of biomembranes by spectral ellipsometry, surface plasmon resonance and interferometry with regard to biosensor application

Phospholipid bilayers with transport proteins and antigen/antibody interfaces are considered to be suitable biosensor systems. The quality of such membranes or interfaces depends on the properties of the layers. Optical methods have proved to be an appropriate tool for characterizing those layers in situ and in a non-destructive manner. Two systems with potential for biosensor applications are characterized by some of these methods: phospholipid bilayer membranes spread from vesicle solution and protein-antigens both adsorbed on planar solid support. The results of spectral ellipsometry, surface plasmon resonance (SPR) and spectral interferometry are compared with respect to quality of characterization, expenditure of sample preparation and measurement, and time resolution. The phospholipid membranes adsorbed show a relatively low refractive index and a relatively high thickness. Bruggeman effective medium approximation is used to calculate the effective layer thickness. This result is compared to SPR measurements. A correlation between thickness and vesicle concentration may be detected. Further, the test protocol of an immunoassay is examined by spectral interferometry and SPR. Thicknesses determined are compared to results obtained by applying spectral ellipsometry. The data measured by ellipsometry are in agreement with the molecular dimensions of the immunoglobulins. Differences between details can be explained by physical considerations.

Optical biosensor for pharmaceuticals, antibiotics, hormones, endocrine disrupting chemicals and pesticides in water: Assay optimization process for estrone as example.

Certain contaminants at trace concentrations in surface waters can have dramatic effects on the hormonal system of organisms in the aquatic environment. Therefore, immunoanalytical methods at a very low limit of detection (LOD) and a low limit of quantification (LOQ) are becoming more and more important for environmental analysis and especially for monitoring drinking water quality. Environmental monitoring of antibiotics, hormones, endocrine disrupting chemicals, and pesticides in real water samples (e.g. surface, ground or drinking water) with difficult matrices places high demands on chemical analysis. Biosensors have suitable characteristics such as efficiency in allowing very fast, sensitive, and cost-effective detection. Here we describe an assay optimization process with a fully automated immunoassay for estrone which resulted in a LOD below 0.20ngL(-1) and a LOQ below 1.40ngL(-1). In contrast to common analytical methods such as GC-MS or HPLC-MS, the biosensor used requires no sample pre-treatment and pre-concentration. The very low validation parameters for estrone are the result of the continuous optimization of the immunoassay. The basis of our sensitive assay is the antibody with a high affinity constant towards estrone. During the optimization process, we reduced the amount of antibody per sample and improved the chip surface modification. Finally, this proceeding led to a calibration routine with an amount of antibody of only 3.0ng per sample (sample volume: 1.0mL). The reduction of the amount of antibody per sample results in better validation parameters (LOD, LOQ, and IC(50)), but this reduction leads to the current device-related limitation of the River Analyser (RIANA). For some endocrine disrupting compounds, no effect levels (NOELs) in the lower nanogram per liter range are reported. This defines the challenge, which analytical methods have to compete with and our RIANA instrument with its improved sensitivity for the detection of a single hormone in the lower nanogram per liter range is a powerful tool in aquatic analytics in addition to the common analytical methods.

Integrated optical fluorescence multisensor for water pollution

An integrated optical multisensor for organic pollutants has been realised, and characterised for a single analyte. The sensor exploits fluorescence immunoassay in the evanescent field of channel waveguides to enable rapid, simultaneous and high-sensitivity fluorescence detection of up to 32 pollutants in water. The chemical modification used to render the surface specific to analytes allows automatic regeneration for immediate reuse. The system has been demonstrated for the key pollutant estrone and a detection limit below 1 ng/L has been achieved.

Total internal reflectance fluorescence (TIRF) biosensor for environmental monitoring of testosterone with commercially available immunochemistry: antibody characterization, assay development and real sample measurements.

Nowadays, little technology exists that can monitor various water sources quickly and at a reasonable cost. The ultra-sensitive, fully automated and robust biosensor River Analyser (RIANA) is capable of detecting multiple organic targets rapidly and simultaneously at a heterogeneous assay format (solid phase: bulk optical glass transducers). Commercialization of such a biosensor requires the availability of commercial high-affinity recognition elements (e.g. antibodies) and suitable commercial haptens (modified target molecules) for surface chemistry. Therfore, testosterone was chosen as model analyte, which is also a task of common analytical methods like gas chromatography-mass spectrometry (GC-MS), because they have to struggle with detecting sub-nanogram per liter levels in environmental samples. The reflectometric interference spectroscopy (RIfS) was used to characterize the commercially available immunochemistry resulting in a high-affinity constant of 2.6+/-0.3 x 10(9)mol(-1) for the unlabeled antibody. After the labeling procedure, necessary for the TIRF-based biosensor, a mean affinity constant of 1.2 x 10(9)mol(-1) was calculated out of RIfS (1.4+/-0.4 x 10(9)mol(-1)) and TIRF (1.0+/-0.3 x 10(9)mol(-1)) measurements. Thereafter, the TIRF-based biosensor setup was used to determine the steroidal hormone testosterone at real world samples without sample pre-treatment or sample pre-concentration. Results are shown for rapid and ultra-sensitive analyses of testosterone in aqueous samples with at a remarkable limit of detection (LOD) of 0.2 ng L(-1). All real world samples, even those containing testosterone in the sub-nanogram per liter range (e.g. 0.9 ng L(-1)), could be determined with recovery rates between 70 and 120%. Therefore, the sensor system is perfectly suited to serve as a low-cost system for surveillance and early warning in environmental analysis in addition to the common analytical methods. For the first time, commercially available immunochemistry was fully characterized using a label-free detection method (RIfS) and successfully incorporated into a TIRF-based biosensor setup (RIANA) for reliable sub-nanogram per liter detection of testosterone in aqueous environmental samples.

Strategies for label-free optical detection.

A large number of methods using direct detection with label-free systems are known. They compete with the well-introduced fluorescence-based methods. However, recent applications take advantage of label-free detection in protein-protein interactions, high-throughput screening, and high-content screening. These new applications require new strategies for biosensors. It becomes more and more obvious that neither the transduction principle nor the recognition elements for the biomolecular interaction process alone determine the quality of the biosensor. Accordingly, the biosensor system has to be considered as a whole. This chapter focuses on strategies to optimize the detection platform and the biomolecular recognition layer. It concentrates on direct detection methods, with special focus on optical transduction. Since even this restriction still leaves a large number of methods, only microrefractometric and microreflectometric methods using planar transducers have been selected for a detailed description and a listing of applications. However, since many review articles on the physical principles exist, the description is kept short. Other methods are just mentioned in brief and for comparison. The outlook and the applications demonstrate the future perspectives of direct optical detection in bioanalytics.

Observation of spectral interferences for the determination of volume and surface effects of thin films

SummaryThe application of a rapid scanning diode array spectrometer allows the time-resolved observation of the interferences caused by multiple reflections at the interfaces of thin films. This spectral interferometry enables the observation of changes in optical pathlengths and allows to separate volume-effects like polymer swelling from surface-effects like adsorption or deposition. Polymer/solvent interactions will give an example for an application of this method.

Application and comparison of algorithms for the evaluation of interferograms

SummaryInterferometry is a valuable tool for the fast and non-destructive determination of the thickness of optically transparent films. Combined with the advantages of fibre optical measurements the technique is used in many fields. This paper describes three methods for the evaluation of interference spectra obtained by measurements of reflectance at thin polymer films in the visible range suitable for on-line process control in the sub second range and for the absolute determination of both film thickness and dispersion.

Biosensor for seven sulphonamides in drinking, ground, and surface water with difficult matrices

Abstract Environmental monitoring of antibiotics and other pharmaceuticals in real water samples with difficult matrices places high demands on chemical analysis. Biosensors have suitable characteristics like their efficiency in a fast, sensitive, and cost‐effective detection of pollutants. In this article, we present a recently developed immunoassay for seven sulphonamides (sulphadiazine, sulphamethoxazole, sulphadimidine, sulphamethizole, sulphadimethoxine, sulphathiazole, and sulphamethoxypyridazine) which can only be detected separately. For the simultaneous determination of multiple sulphonamides in the future we performed measurements with different combinations of binary mixtures. The results of the immunosensor were compared to a mathematical model which was developed in our group. Using an automated biosensor system it was possible for the first time to achieve limits of detection (LOD) below 10 ng L−1 and limits of quantification (LOQ) below 100 ng L−1 without sample pre‐concentration for these sulphonamides. Sulphonamide calibrations with different immobilised analyte derivatives were made in Milli‐Q water. Unstrained spiked and un‐spiked real water samples with complex matrices (drinking, ground, and surface water) were measured. In compliance with the Association of Analytical Communities (AOAC) International most recovery rates obtained were between 70% and 120%. The reproducibility was checked by measuring replica of each sample within independent repetitions. Robustness could be demonstrated by long‐term stability tests of the biosensor surface. These studies show that the biosensor used offers the necessary reproducibility, precision, and robustness required for an analytical method. The measuring data of the binary mixtures show a systematic error compared to the mathematical model at high concentrations of both sulphonamides, because the approximation uses only the standard calibration curves (data of the logistic fit function) as input data. It is also hard to adequately describe the cross‐reactivity and the behaviour of a mixture of polyclonal antibodies.

Design of new integrated optical substrates for immuno-analytical applications

Integrated optical Mach-Zehnder interferometers supply information on changes in refractive index and/or thickness of a film placed as a superstrate on top of one of its surface wave-guides. The internal propagation of light is influenced by the evanescent field reaching into the superstrate. This propagating light interferes with an uninfluenced wave in the second arm after recombination. The result is an intensity modulation depending on the refractive index parameters of the substrate, the waveguide itself and the properties of the superstrate. Taking an antigen layer as the superstrate, its interaction with antibodies changes its thickness by several nanometers. This can be observed by recording the change in intensity of the signal of the interferometer. The sensitivity of such a device depends on particular values of the optical parameters of substrate and waveguide with respect to the given superstrate properties. Computer calculations help to select optimum glass and waveguide fabrication conditions. The numerical results of a variety of assumed conditions have been tested experimentally. The application to the improved detection of triazines is discussed.