(R)evolution of the Standard Addition Procedure for Immunoassays.

A new method to transfer the standard addition procedure for concentration determination to immunoassays with non-linear calibration curves was developed. The new method was successfully applied to simulated data and benchmarked against a state-of-the-art algorithm, showing a significantly improved performance with improvement factors between 2 and 192. The logit function was used to transform the immunoassay signal response of test samples spiked with known analyte concentrations. The relationship between logit(signal) and log-transformed estimated total analyte concentration is linear if the estimated total analyte concentration is correct. Finally, the new method was validated experimentally using different assays in varying, relevant complex matrices, such as serum, saliva, and milk. Different concentrations of testosterone and amitriptyline between 0.05 and 3.0 µg L-1 were quantified using a binding inhibition assay in combination with reflectometric interference spectroscopy (RIfS) as the transduction principle. The sample concentration was calculated using a numerical method. Samples could be quantified with recoveries between 70 and 118%. The standard addition method accounts for individual matrix interference on the immunoassay by spiking the test sample itself. Although the experiments were carried out using RIfS, the method can be applied to any immunoassay that meets the analytical requirements.

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.

Parallelized label-free monitoring of cell adhesion on extracellular matrix proteins measured by single colour reflectometry.

The understanding of the initial cell adhesion to biomaterials is crucial for the survival of implants. The manifold possibilities to tailor an implant surface and the diverse requirements for different implant applications necessitate a timesaving and highly parallelized analytical methodology. Due to its intrinsic advantages (label-free, time-resolved, robust against temperature fluctuations, and particularly the multiplexing possibilities), single colour reflectometry (SCORE) is used for the first time to investigate cell adhesion to different extracellular matrix protein-coated surfaces. The excellent correlation between the novel SCORE technology and well-established reference methods proves that the results obtained by using this direct optical method are able to reflect the cell binding processes at the transducer surface. Additionally, the high time resolution of SCORE revealed the differences in the adhesion behaviour of the cells on the different extracellular matrix protein-coated glass slides during the initial adsorption phase and during the spreading of the cells on the surfaces. Therefore, we conclude that SCORE is a perfectly suited methodology for studying the entire cell adsorption process, including morphological changes, and shows great potential for other cell-based sensing applications.

Comparison of methods for quantitative biomolecular interaction analysis.

In order to perform good kinetic experiments, not only the experimental conditions have to be optimized, but the evaluation procedure as well. The focus of this work is the in-depth comparison of different approaches and algorithms to determine kinetic rate constants for biomolecular interaction analysis (BIA). The different algorithms are applied not only to flawless simulated data, but also to real-world measurements. We compare five mathematical approaches for the evaluation of binding curves following pseudo-first-order kinetics with different noise levels. In addition, reflectometric interference spectroscopy (RIfS) measurements of two antibodies are evaluated to determine their binding kinetics. The advantages and disadvantages of the individual approach will be investigated and discussed in detail. In summary, we will raise awareness on how to evaluate and judge results from BIA by using different approaches rather than having to rely on "black box" closed (commercial) software packages.

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.

A multi-analyte biosensor for the simultaneous label-free detection of pathogens and biomarkers in point-of-need animal testing.

For the first time, a multi-analyte biosensor platform has been developed using the label-free 1-lambda-reflectometry technique. This platform is the first, which does not use imaging techniques, but is able to perform multi-analyte measurements. It is designed to be portable and cost-effective and therefore allows for point-of-need testing or on-site field-testing with possible applications in diagnostics. This work highlights the application possibilities of this platform in the field of animal testing, but is also relevant and transferable to human diagnostics. The performance of the platform has been evaluated using relevant reference systems like biomarker (C-reactive protein) and serology (anti-Salmonella antibodies) as well as a panel of real samples (animal sera). The comparison of the working range and limit of detection shows no loss of performance transferring the separate assays to the multi-analyte setup. Moreover, the new multi-analyte platform allows for discrimination between sera of animals infected with different Salmonella subtypes.

Size does matter! Label-free detection of small molecule-protein interaction.

This review is focused on methods for detecting small molecules and, in particular, the characterisation of their interaction with natural proteins (e.g. receptors, ion channels). Because there are intrinsic advantages to using label-free methods over labelled methods (e.g. fluorescence, radioactivity), this review only covers label-free techniques. We briefly discuss available techniques and their advantages and disadvantages, especially as related to investigating the interaction between small molecules and proteins. The reviewed techniques include well-known and widely used standard analytical methods (e.g. HPLC-MS, NMR, calorimetry, and X-ray diffraction), newer and more specialised analytical methods (e.g. biosensors), biological systems (e.g. cell lines and animal models), and in-silico approaches.

Biosensors paving the way to understanding the interaction between cadmium and the estrogen receptor alpha.

Cadmium is a toxic heavy metal ubiquitously present in the environment and subsequently in the human diet. Cadmium has been proposed to disrupt the endocrine system, targeting in particular the estrogen signaling pathway already at environmentally relevant concentrations. Thus far, the reports on the binding affinity of cadmium towards human estrogen receptor alpha (hERα) have been contradicting, as have been the reports on the in vivo estrogenicity of cadmium. Hence, the mode of interaction between cadmium and the receptor remains unclear. Here, we investigated the interaction between cadmium and hERα on a molecular level by applying a novel, label-free biosensor technique based on reflectometric interference spectroscopy (RIfS). We studied the binding of cadmium to hERα, and the conformation of the receptor following cadmium treatment. Our data reveals that cadmium interacts with the ligand binding domain (LBD) of the ERα and affects the conformation of the receptor. However, the binding event, as well as the induced conformation change, greatly depends on the accessibility of the cysteine tails in the LBD. As the LBD cysteine residues have been reported as targets of post-translational modifications in vivo, we present a hypothesis according to which different cellular pools of ERα respond to cadmium differently. Our proposed theory could help to explain some of the previously contradicting results regarding estrogen-like activity of cadmium.

Kinetic analysis of the estrogen receptor alpha using RIfS.

The label-free time-resolved reflectometric interference spectroscopy has been used to study the interaction of the human estrogen receptor alpha (ERa) and different types of ligands. Different possible sensor surface coatings including various estrogen derivatives were evaluated for their suitability for detection of ERa. The determination of the kinetic and thermodynamic constants was carried out for the interaction in the heterogeneous phase as well as for the interaction in homogeneous phase. In addition, the affinity of 11 ligands ranging from natural hormones and pharmaceuticals to endocrine disrupting chemicals (EDCs) has been determined with this label-free assay format.

A new assay design for clinical diagnostics based on alternative recognition elements.

Herein, we present a new sandwich assay design containing a high affinity polypeptide scaffold as immobilized capture element and an antibody for detection. These polypeptide scaffolds provide a good affinity towards one antigen and can be linked to biosensor surfaces without affecting their binding capabilities. Furthermore, the small peptides are very stable, which allows for regenerating the surface several hundreds of times and thus for reuse of the biosensor. Moreover, these receptors can be synthesized with different affinities towards one antigen, which has been proven by characterizing them using a label-free detection method RIfS (reflectometric interference spectroscopy) for collecting kinetic data. Polypeptide scaffolds with different affinities have been chosen and characterized. Upon these results, sandwich-type assays have been set-up using a fluorescently labelled antibody as detection element. Thereby could be shown, that the working range of the assay can be shifted according to the affinity of the used capturing polypeptide scaffold. The scaffolds with a higher affinity towards the antigen can detect lower concentration, and in contrary, scaffolds with lower affinities can detect higher concentrations. In consequence, using this new sandwich-type assay, we avoid the complex procedure to immobilize antibodies in correct orientation, but simultaneously keep this well-known recognition element in the assay for detection. Furthermore, in addition to all the acknowledged properties of immunoassays, we add the possibility of tuning the working range of assays in distinct manner according to request.

A novel analytical tool for quantification of estrogenicity in river water based on fluorescence labelled estrogen receptor alpha.

A novel combined procedure for estrogen-affinity purification and labelling of estrogen receptor alpha ligand-binding domain with Cy 5.5 cystein reactive dye was established. By using this procedure, mainly functional proteins are recovered. It can be easily adapted to a large variety of other proteins for which ligand-coated affinity materials are available. The labelled receptor was used in a total internal reflection fluorescence-based binding inhibition assay for determination of the impact of pollutants in river water on the receptor. The great advantage compared to conventional methods is that the total effect on the receptor is measured instead of concentrations of single compounds and that even currently unknown ligands are found as well. Therefore, the obtained signal is related to the response of the organism, which is exposed to the water. The limit of detection was found to be 0.139 nM of estradiol equivalents. The assay also provides a highly sensitive tool for pharmaceutical research and can be adapted to diagnostic applications.

Structural information, resolution, and noise in high-resolution atomic force microscopy topographs.

AFM has developed into a powerful tool in structural biology, providing topographs of proteins under close-to-native conditions and featuring an outstanding signal/noise ratio. However, the imaging mechanism exhibits particularities: fast and slow scan axis represent two independent image acquisition axes. Additionally, unknown tip geometry and tip-sample interaction render the contrast transfer function nondefinable. Hence, the interpretation of AFM topographs remained difficult. How can noise and distortions present in AFM images be quantified? How does the number of molecule topographs merged influence the structural information provided by averages? What is the resolution of topographs? Here, we find that in high-resolution AFM topographs, many molecule images are only slightly disturbed by noise, distortions, and tip-sample interactions. To identify these high-quality particles, we propose a selection criterion based on the internal symmetry of the imaged protein. We introduce a novel feature-based resolution analysis and show that AFM topographs of different proteins contain structural information beginning at different resolution thresholds: 10 A (AqpZ), 12 A (AQP0), 13 A (AQP2), and 20 A (light-harvesting-complex-2). Importantly, we highlight that the best single-molecule images are more accurate molecular representations than ensemble averages, because averaging downsizes the z-dimension and "blurs" structural details.

An advanced biosensor for the prediction of estrogenic effects of endocrine-disrupting chemicals on the estrogen receptor alpha.

A label-free and time-resolved biosensor based on reflectometric interference spectroscopy (RIfS) has been developed to evaluate the agonistic or antagonistic effects of potential ligands with unknown behavior. The biosensor utilizes the specific interaction between the estrogen receptor alpha (ER alpha) and short specific peptides. The unique feature of these peptides allows the investigation of the behavior of ligands and the discrimination between the agonistic and antagonistic effects caused by conformational changes of the receptor. Thus, this developed biosensor allows not only the differentiation between ligands and nonligands of a receptor, but also the potential of these ligands to influence conformational changes in the receptor, leading to activation or inhibition of the receptor-dependent pathways. Owing to the robustness of the direct optical detection principle used, the biosensor is applicable to complex biological matrices, even crude cell extracts. Moreover, the reliability of the biosensor, including regeneration steps when performing subsequent measurements, has been verified.

Direct optical detection in fragment-based screening.

Label-free biosensors based on direct optical detection principles are widely used in many different fields of research. Currently the higher level of automation and the increasing throughput of this technology are stimulating the interest of pharmaceutical companies. The information gained with label-free biosensors can be extremely valuable during the drug design process, particularly in combination with complementary techniques, including NMR, mass spectrometry and X-ray crystallography. In this article we focus on the advantages of direct optical biosensors especially in the field of fragment-based drug design, which is a widely used and extremely promising concept. Furthermore, we present optical biosensors as versatile tools for fragment-based screening and the future drug design process.