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.

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.

A biomimetic sensor has been developed, that allows for quantification of autoantibodies related to the antiphospholipid syndrome (APS). Autoantibodies directed against the β(2)-glycoprotein-I (β(2)GP-I) are known as the best markers for diagnosis of APS, however, detection of such antibodies is still a challenge. The epitopes of β(2)GP-I are exposed upon binding to negatively charged membranes. The surface of the sensor chips was therefore modified with such type of membranes, on which β(2)GP-I molecules were subsequently immobilized as recognition elements. Using the label-free method, reflectometric interference spectroscopy, it was possible to quantify anti-β(2)GP-I antibodies and to calibrate the sensor chip in buffer. A mild regeneration procedure allows for many consecutive measurements without stripping off the membrane in between.

A label-free biosensor has been developed, allowing quantification of cystatin C in human serum. This was achieved by using reflectometric interference spectroscopy as detection method. Cystatin C is a small serum protein that allows detection of renal failure more reliably than established parameters as creatinine. The protein was immobilized on the surface of a glass transducer, forming the sensitive layer of the sensor chip. Based on a binding-inhibition assay, two different types of monoclonal cystatin C antibodies were compared, by their behavior and their obtained working range in buffer and serum as matrix. Both antibodies allowed quantification of the protein in serum as matrix within the required clinical ranges of 0.53-1.02 mg/L. Detected recovery rates are in a range between 84.8% and 116.1%. The developed sensor shows high inner chip reproducibility and low cross-sensitivity.

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.