Günther Proll

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.

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 α (ERα) 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.

Development of a new parallelized, optical biosensor platform for label-free detection of autoimmunity-related antibodies

Autoimmune diseases are characterized by the presence of autoantibodies in serum of affected patients. The heterogeneity of autoimmune relevant antigens creates a variety of different antibodies, which requires a simultaneous detection mode. For this reason, we developed a tool for parallelized, label-free, optical detection that accomplishes the characterization of multiple antigen–antibody interactions within a single measurement on a timescale of minutes. Using 11-aminoundecyltrimethoxysilane, we were able to immobilize proteinogenic antigens as well as an amino-functionalized cardiolipin on a glass surface. Assay conditions were optimized for serum measurements with a single spot antigen chip on a single spot 1-λ detection system. Minimized background signal allows a differentiation between patients and healthy controls with a good sensitivity and specificity. Applying polarized imaging reflectometric interference spectroscopy, we evaluated samples from three APS patients and three control subjects for this proof-of-principle and already obtained good results for β2-glycoprotein I and cardiolipin.

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.

Diagnostic performance study of an antigen microarray for the detection of antiphospholipid antibodies in human serum.

Abstract Background: The parallelization of clinically relevant antigens in a microarray format is of growing importance due to the ability to measure multiple antigen-antibody interactions. With the development of a microarray for the detection of antiphospholipid antibodies we focussed on one important autoimmune disease that is still diagnostically challenging. Reasons are the heterogeneity of the autoantibodies and the unspecific clinical symptoms. Methods: For the covalent immobilization of antigenic structures, glass transducers were coated with 11-aminoundecyltrimethoxysilane (11-AUTMS). In total 35 antiphospholipid syndrome (APS) patients, six patients with lupus erythematosus and 24 healthy controls were investigated on a microarray format using polarized imaging reflectometric interference spectroscopy. Results: The novel surface modification based on the short derivative 11-AUTMS resulted in a selective biosensor allowing a clear differentiation of patient and control samples. It combined proteinogenic as well as phospholipid-derived antigens, namely β2-glycoprotein I (β2-GPI), prothrombin, cardiolipin (CL) and a β2-GPI/CL complex. With optimized regeneration conditions, up to 20 consecutive measurements could be performed on one chip. Sensitivity was determined to be 0.800–0.929, specificity was between 0.733 and 0.969, depending on the respective antigen. Conclusions: Multiplexed determination of serological parameters has a great potential. We have shown that our biosensor is capable of detecting four different APS relevant antibodies in parallel exhibiting a sensitivity and specificity comparable to existing ELISA methods.

Optical biosensor system for the quick and reliable detection of virus infections: VIROSENS

Viral infections are of special threat because they can induce severe courses of disease but only few medical treatments are available. Because of socio-economic and climate changes, increased worldwide mobility and population growth, the risk of newly occurring and quickly spreading viral pathogens has increased. A diagnosis of these diseases at an early stage is essential for a quick risk assessment and a proper health management as well as patient’s treatment in an optimal way. Currently, the diagnosis of such diseases is based on time consuming and costly detection methods that can only be performed by specially trained personnel in laboratories at specific security levels. Aim of the project VIROSENS is the development of a biosensor platform that can specifically detect virus particles as well as virus-specific antibodies out of biological matrices like blood, serum, plasma and other body fluids. For this purpose, a disposable cartridge for such antibody- and virus-arrays is designed and developed within the project. The optical detection of viruses is performed with a portable device that will be benchmarked and evaluated concerning currently used standard detection methods in terms of its analytical performance. Within this project, a novel combination of serological tests and direct detection of virus particles will be developed, which will provide faster and more reliable results than presently available and used test systems.

Allgemeine analytische Prinzipien und Gerätekategorien

Die POCT-Technologie hat – ausgehend von einfachen Handgeraten fur die Blutglukosebestimmung und ersten Blutgasanalysatoren in den 1960er- Jahren – die unterschiedlichsten Methodiken und Anwendungen hervorgebracht. Die Verschiedenheit der Anwendungsfelder erlaubt an dieser Stelle keine umfassende Darstellung der zugrunde liegenden Analysemethoden. Die allgemeinen Prinzipien sollen jedoch kurz erlautert werden. Eine hilfreiche erste Ubersicht gibt dabei der Aufbau dieser Analysesysteme [14]: Detektoreinheit, Transducer-Einheit, Probenaufgabe/Fluidikeinheit, Processing-Einheit.

Analytical methods, biosensor technology

This chapter explains the analytical principles incorporated into the POCT devices on the market. Furthermore, continuous monitoring methods are introduced that are already being used in patient care.

Analytische Verfahren, Biosensortechnologie

Dieses Kapitel erlautert die analytischen Grundprinzipien, die bei den im Markt POCT-Geraten zum Einsatz kommen. Zudem werden kontinuierliche Messmethoden vorgestellt, die bereits heute am Patienten angewandt werden.

Multiplex platforms in diagnostics and bioanalytics.

In recent years, significant progress has been made in the field of multi-parameter analysis for diagnostics and bioanalytical applications. That is not only true for the technological approaches, which have been achieved; it is also the case with respect to new biomarkers available now for integration in multiplex systems providing a higher relevance of information. These efforts have already become important for laboratory medicine in terms of improved screening approaches as well as cost efficiency. This may prospectively also influence the opportunities for stratifying patient cohorts in personalized medicine. The development of new platforms capable of multiplex analysis is a multidisciplinary process, including transduction principles, biofunctionalization, recognition elements, microfluidics, data processing, chemometrics, etc. For example, biochip-based systems still provide a solid basis for such analysis of multiple parameters. However, the need for sample preparation and application—especially at the point of need—is often solved inadequately. Here, microfluidic systems offer simple and fast solutions to integrate sampling and transport to the place of analysis. This is also reflected in the current special paper collection of ABC. In addition to standard methods like reading a fluorescent signal, readout of electrical signals becomes more and more interesting. There are promising developments in the field of micro-ring resonators, which combine optics, micro-electronics, and biology in an amazing technological cross-over approach. Through silicon-based technology, these resonators can be conveniently prepared in large batches and offer the possibility of carrying out several analyses—in parallel and even in smallest space. However, any diagnostic or related bioanalytical technology also depends on the availability of robust and standardized biomarkers used for analysis. These must be sufficiently specific to minimize false-positive and false-negative results. The motto here is: a lot does not help much, especially according to the real demand of the clinical routine lab. The use of multiple biomarkers does not automatically increase specificity. This is particularly significant with the analysis of samples without sufficient initial suspicion, as seen in the screening of actually inconspicuous people. In this case, the probability of obtaining a false-positive result is much larger since the actual number of affected patients Published in the topical collection Multiplex Platforms in Diagnostics and Bioanalytics with guest editors Gunter Peine and Gunther Proll.