Guenther Proll

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.

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.

Reflectometric Interference Spectroscopy

Reflectometry is classified in comparison to the commercialized refractometric surface plasmon resonance (SPR). The advantages of direct optical detection depend on a sophisticated surface chemistry resulting in negligible nonspecific binding and high loading with recognition sites at the biopolymer sensitive layer of the transducer. Elaborate details on instrumental realization and surface chemistry are discussed for optimum application of reflectometric interference spectroscopy (RIfS). A standard protocol for a binding inhibition assay is given. It overcomes principal problems of any direct optical detection technique.

Biosensors for unattended, cost-effective and continuous monitoring of environmental pollution: Automated Water Analyser Computer Supported System (AWACSS) and River Analyser (RIANA)

This work describes our recent progress and achievements in the field of fully automated biosensors (Automated Water Analyser Computer Supported System (AWACSS) and River Analyser (RIANA)) for unattended, cost-effective and continuous monitoring of environmental pollution. We report on ultra-sensitive immunoassays for the hormones progesterone, testosterone and estrone and the pesticides propanil and isoproturon as examples of the outstanding progress made on biosensors in the field of environmental monitoring and water analysis. Most of the bio-active organic pollutants (estrone, progesterone, propanil and isoproturon) were detected at levels as low as 1.0 pg mL−1 or even below. In fact, the reported limits of detection (LOD) were between 0.2 and 6.0 pg mL−1. For the first time, commercially available derivatives and antibodies were incorporated into immunoassays (progesterone and testosterone) for fully automated biosensors. To verify the assay performance for quantifying testosterone, progesterone, and isoproturon in real-world samples using our immunosensors, we spiked river and drinking water at six different levels from 0.9 pg mL−1 to 90 ng mL−1. Nearly all recovery rates could be obtained between 70 and 120% as the AOAC International recommends it chiefly for water analysis.

Total Internal Reflection Fluorescence Sensing - Quality Assurance and Application to Water Analysis

Based on the water directives of the European Union, the results of a biosensor system using total internal reflection fluorescence (TIRF) are demonstrated. The optical principal, the assay type, as well as the calibration and validation parameters are discussed. Results for endocrine disrupting compounds are given, and the validation parameters are outlined. These are applied to a measurement in comparison to a collaborative test in cooperation with accredited water laboratories using high performace liquid chromatography (HPLC) and gas chromatography (GC) techniques.

Low-Volume Label-Free Detection of Molecule-Protein Interactions on Microarrays by Imaging Reflectometric Interferometry.

This system allows the high-throughput protein interaction analysis on microarrays. We apply the interference technology 1λ–imaging reflectometric interferometry (iRIf) as a label-free detection method and create microfluidic flow cells in microscope slide format for low reagent consumption and lab work compatibility. By now, most prominent for imaging label-free interaction analyses on microarrays are imaging surface plasmon resonance (SPR) methods, quartz crystal microbalance, or biolayer interferometry. SPR is sensitive against temperature drifts and suffers from plasmon crosstalk, and all systems lack array size (maximum 96 spots). Our detection system is robust against temperature drifts. Microarrays are analyzed with a spatial resolution of 7 µm and time resolution of ≤50 fps. System sensitivity is competitive, with random noise of <5 × 10−5 and baseline drift of <3 × 10−6. Currently available spotting technologies limit array sizes to ~4 spots/mm2 (1080 spots/array); our detection system would allow ~40 spots/mm2 (10,800 spots/array). The microfluidic flow cells consist of structured PDMS inlays sealed by versatilely coated glass slides immobilizing the microarray. The injection protocol determines reagent volumes, priming rates, and flow cell temperatures for up to 44 reagents; volumes of ≤300 µL are validated. The system is validated physically by the biotinylated bovine serum albumin streptavidin assay and biochemically by thrombin aptamer interaction analysis, resulting in a KD of ~100 nM.

Integrated optical immunofluorescence multisensor for river pollution

A 32-analyte integrated optical immunofluorescence multisensor system has been realized and tested for the first time. The sensor system is based upon bio/immuno-chemistry at the waveguide surface and fluoroimmunoassay in the evanescent fields of the optcal waeguides, to enable rapid, simultaneous and high-sensitivity fluorescence detection of up to 32 pollutants in water, and automatic regeneration for immediate reuse. The system has been demonstrated for estrone and a detection limit of 13 ng/L has been achieved.