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.
We present a portable and easy-to-use biosensor platform, allowing for label-free detection of diagnostic markers in undiluted animal serum. Exemplarily, this is shown for the detection of anti-Salmonella antibodies. 1-lambda-Reflectometry was used as detection method, making the new biosensor platform portable, cheap, and robust. As recognition elements, lipopolysaccharides (LPSs) from Salmonella typhimurium bacteria were immobilized as sensitive layer on the transducer to carry out serological tests via a direct assay format. For this purpose, a new surface preparation protocol has been worked out allowing for immobilization of the LPS via hydrophobic interactions. It has been shown that results obtained by 1-lambda-Reflectometry are equivalent to those obtained by the non-portable Reflectometric Interference Spectroscopy setup. The new sensor platform was calibrated in both matrices, buffer and undiluted serum. Good sensitivity, selectivity and intra chip reproducibility have been observed. Furthermore, inter chip reproducibility was examined and recovery rates were found to be between 99 and 117% in undiluted serum.
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.
The properties of integrated optical phase-modulated Mach-Zehnder interferometers (IO-MZI) are used to set up a new generation of chemical and biochemical sensors working in the mid-infrared. First applications of the MZI principle were introduced in the beginning 1990s. They range from a gas sensor to monitor organic solvent concentrations 1 to setting up an immunoassay for the detection of the herbicide simazine 2 . Most if not all sensors of MZI type operate at wavelengths of the visible or near infrared spectrum. There are several reasons to change this strategy and move into the mid-infrared spectral range (MIR): higher manufacturing tolerances, increased evanescent field penetration depth, signal amplification by surface enhanced infrared absorption effect (SEIRA), species identification by MIR fingerprints.The basis of the planned MIR-MZI is a GaAs waveguide pattern epitaxially grown on a substrate 3 . As a first step towards nanostructuring the waveguide surface, chemical deposition of Au nanoparticles on GaAs transducers was established. For the use of MIR-MZI sensors in bioanalytical assay development, chemical immobilization of molecular recognition elements on GaAs transducers was carried out. The modified surfaces were characterized by atomic force microscopy (AFM), dark field microscopy, contact angle measurements and ellipsometric data as well as by a modified version of Reflectometric Interference Spectroscopy (RIfS) 4 . It was possible to monitor both the immobilization of gold nanoparticles and time-resolved specific binding using a model antibody antigen assay.After successful setup of relevant assays with RIfS, e.g. the detection of bacteria or endocrine disruptors, the assays are designed to be transferred onto the mid-infrared Mach-Zehnder interferometer.
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