Fourier spotting: a novel setup for single-color reflectometry.

Single-color reflectrometry is a sensitive and robust detection method in optical biosensor applications, for example for bioanalysis. It is based on the interference of reflected monochromatic radiation and is label free. We present a novel setup for single-color reflectometry based on the patented technology of Berner et al. from 2016. Tilting areas of micro-mirrors allow us to encode the optical reflection signal of an analyte and reference channel into a particular carrier frequency with the amplitude being proportional to the local reflection. Therefore, a single photodiode is sufficient to collect the signals from both channels simultaneously. A 180∘ phase shift in the tilt frequency of two calibrated micro-mirror areas leads to a superposition of the analyte and reference signal which enables an efficient reduction of the baseline offset and potential baseline offset drift. A performance test reveals that we are able to detect changes of the refractive index n down to Δn < 0.01 of saline solutions as regents. A further test validates the detection of heterogeneous binding interaction. This test compromises immobilized testosterone-bovine serum albumin on a three-dimensional layer of biopolymer as ligand and monoclonal anti-testosterone antibodies as analyte. Antibody/antigen binding induces a local growth of the biolayer and change in the refractive index, which is measured via the local change of the reflection. Reproducible measurements enable for the analysis of the binding kinetics by determining the affinity constant KA = 1.59 × 10- 7 M- 1. In summary, this work shows that the concept of differential Fourier spotting as novel setup for single-color reflectometry is suitable for reliable bioanalysis. Graphical Abstract.

Reflectometric Interference Spectroscopy.

Reflectometry is classified in comparison to the commercialized refractometric surface plasmon resonance. The advantages of direct optical detection depend on a sophisticated surface chemistry resulting 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.

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.

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.

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.

Potential of label-free detection in high-content-screening applications.

The classical approach of high-content screening (HCS) is based on multiplexed, functional cell-based screening and combines several analytical technologies that have been used before separately to achieve a better level of automation (scale-up) and higher throughput. New HCS methods will help to overcome the bottlenecks, e.g. in the present development chain for lead structures for the pharmaceutical industry or during the identification and validation process of new biomarkers. In addition, there is a strong need in analytical and bioanalytical chemistry for functional high-content assays which can be provided by different hyphenated techniques. This review discusses the potential of a label-free optical biosensor based on reflectometric interference spectroscopy (RIfS) as a bridging technology for different HCS approaches. Technical requirements of RIfS are critically assessed by means of selected applications and compared to the performance characteristics of surface plasmon resonance (SPR) which is currently the leading technology in the area of label-free optical biosensors.

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.

Improved strategy for biosensor-based monitoring of water bodies with diverse organic carbon levels.

To protect water resources and to control the water quality it is necessary to develop fast, sensitive, cost-effective, and easy-to-use analytical systems, which are able to measure a variety of contaminants in water. Monitoring water bodies with various matrices can be very difficult. The diverse organic carbon level in water samples (e.g. river water or seawater) causes problems at common analysis and in particular at immunological methods. Here, we demonstrate a new method to overcome the partly occurring matrix problems at quasi-continuous real-world biosensor monitoring. Therefore, we developed an easy matrix referencing method for our fully automated immunoassays that could be adapted to other applications depending on a similar test-format. The method was developed using a synthetic organic carbon standard, and validated using a diluted turf extract. Results for the ultra-sensitive immunoassay for estrone quantification are shown as example. The developed method was verified using immunoassays for testosterone, progesterone, ethinylestradiol, estradiol, and estriol.

Automated water analyser computer supported system (AWACSS) Part I: Project objectives, basic technology, immunoassay development, software design and networking.

A novel analytical system AWACSS (automated water analyser computer-supported system) based on immunochemical technology has been developed that can measure several organic pollutants at low nanogram per litre level in a single few-minutes analysis without any prior sample pre-concentration nor pre-treatment steps. Having in mind actual needs of water-sector managers related to the implementation of the Drinking Water Directive (DWD) (98/83/EC, 1998) and Water Framework Directive WFD (2000/60/EC, 2000), drinking, ground, surface, and waste waters were major media used for the evaluation of the system performance. The instrument was equipped with remote control and surveillance facilities. The system's software allows for the internet-based networking between the measurement and control stations, global management, trend analysis, and early-warning applications. The experience of water laboratories has been utilised at the design of the instrument's hardware and software in order to make the system rugged and user-friendly. Several market surveys were conducted during the project to assess the applicability of the final system. A web-based AWACSS database was created for automated evaluation and storage of the obtained data in a format compatible with major databases of environmental organic pollutants in Europe. This first part article gives the reader an overview of the aims and scope of the AWACSS project as well as details about basic technology, immunoassays, software, and networking developed and utilised within the research project. The second part article reports on the system performance, first real sample measurements, and an international collaborative trial (inter-laboratory tests) to compare the biosensor with conventional anayltical methods.

Automated Water Analyser Computer Supported System (AWACSS) Part II: Intelligent, remote-controlled, cost-effective, on-line, water-monitoring measurement system.

A novel analytical system AWACSS (Automated Water Analyser Computer Supported System) based on immunochemical technology has been evaluated that can measure several organic pollutants at low nanogram per litre level in a single few-minutes analysis without any prior sample pre-concentration or pre-treatment steps. Having in mind actual needs of water-sector managers related to the implementation of the Drinking Water Directive (DWD) [98/83/EC, 1998. Council Directive (98/83/EC) of 3 November 1998 relating to the quality of water intended for human consumption. Off. J. Eur. Commun. L330, 32-54] and Water Framework Directive (WFD) [2000/60/EC, 2000. Directive 2000/60/EC of the European Parliament and of the Council of 23 October 2000 establishing a framework for Community action in the field of water policy. Off. J. Eur. Commun. L327, 1-72], drinking, ground, surface, and waste waters were major media used for the evaluation of the system performance. The first part article gave the reader an overview of the aims and scope of the AWACSS project as well as details about basic technology, immunoassays, software, and networking developed and utilised within the research project. The second part reports on the system performance, first real sample measurements, and an international collaborative trial (inter-laboratory tests) to compare the biosensor with conventional anayltical methods. The systems' capability for analysing a wide range of environmental organic micro-pollutants, such as modern pesticides, endocrine disrupting compounds and pharmaceuticals in surface, ground, drinking and waste water is shown. In addition, a protocol using reconstitution of extracts of solid samples, developed and applied for analysis of river sediments and food samples, is presented. Finally, the overall performance of the AWACSS system in comparison to the conventional analytical techniques, which included liquid and gas chromatographic systems with diode-array UV and mass spectrometric detectors, was successfully tested in an inter-laboratory collaborative trial among six project partners.

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.

Verification of performance with the automated direct optical TIRF immunosensor (River Analyser) in single and multi-analyte assays with real water samples.

In order to verify the reproducibility, precision, and robustness of the optical immunosensor River Analyser (RIANA), we investigated two common statistical methods to evaluate the limit of detection (LOD) and the limit of quantification (LOQ). Therefore, we performed a simultaneous multi-analyte calibration with atrazine, bisphenol A, and estrone in Milli-Q water. Using an automated biosensor, it was possible for the first time to achieve a LOD below 0.020 microg L(-1) using a common statistically based method without sample pre-treatment and pre-concentration for each of the analytes in a simultaneous multi-analyte calibration. This biosensor setup shows values comparable to those obtained by more classical analytical methods. Based on this calibration, we measured spiked and un-spiked real water samples with complex matrices (samples from different water bodies, from ground water sources, and tap water samples). The comparison between our River Analyser and common analytical methods (like GC-MS and HPLC-DAD) shows overall comparable values for all three analytes. Furthermore, a calibration of isoproturon (IPU) (in single analyte mode) resulted in a LOD of 0.016 microg L(-1), and a LOQ of 0.091 microg L(-1). In compliance with guidelines of the Association of Analytical Communities International (AOAC), six out of nine recovery rates (recovery rate: measured concentration divided by real concentration in percent) for three surface water samples with different matrices (spiked and un-spiked) could be obtained between 70 and 120% (recovery rates between 70 and 120%, as demanded by the guidelines of the AOAC International). 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.

Ultra-sensitive fully automated immunoassay for detection of propanil in aqueous samples: steps of progress toward sub-nanogram per liter detection.

The widely-used pesticide propanil is a selective post-emergent general-use acetanilide herbicide registered for control of broadleaf and grass weeds in rice, small grain, and turf. Because broad application and quite heavy use of this herbicide lead to contaminated sites and, consequently, contaminated water, immunoanalytical methods with very low limits of detection (LOD) and low limits of quantification (LOQ) are becoming increasingly important for environmental analysis and, especially, for monitoring drinking-water quality. Environmental monitoring of pesticides, hormones, endocrine-disrupting chemicals, and antibiotics in aqueous samples (e.g. surface, ground, waste, or drinking water) with quite difficult matrices places large demands on chemical analysis. Biosensors have suitable characteristics such as efficiency in enabling very fast, sensitive, and cost-effective detection. Here we describe the steps of progress toward sub-nanogram per liter detection of propanil with a fully automated immunoassay. In contrast with common analytical methods such as GC-MS or HPLC-MS the biosensor used requires no sample pre-treatment and pre-concentration. The basis of our sensitive assay is an antibody with a high affinity constant toward propanil. During the optimization process, we compared different surface modifications (four different immobilized derivatives) and reduced the amount of antibody per sample. In fact, optimization of the assay resulted in an LOD of 0.6 ng L(-1) and an LOQ of 4.5 ng L(-1) without any sample pre-treatment and without pre-concentration. These results for propanil with the RIANA instrument, and its improved sensitivity for detection of a single pesticide at the low nanogram per liter range, show that biosensors can compete with common analytical methods in the field of water analysis.

Immunosensor for estrone with an equal limit of detection as common analytical methods.

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. Biosensors have suitable characteristics such as efficiency in allowing very fast, sensitive, and cost-effective detection. Here we describe a fully automated immunoassay for estrone with a LOD below 0.20 ng L(-1) and a LOQ below 1.40 ng L(-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 basis of our sensitive assay is the antibody with a high affinity constant towards estrone. The very low amount of antibody per sample results in low validation parameters (LOD, LOQ, and IC50), but this assay for estrone represents the current device-related limitation of the River Analyser (RIANA).