Nanofabricated pores in 20 nm-thick silicon nitride membranes were used to probe various protein analytes as well as to perform an antigen-antibody binding assay. A two-compartment electrochemical cell was separated by a single nanopore, 28 nm in diameter. Adding proteins to one compartment caused current perturbations in the ion current flowing through the pore. These perturbations correlated with both the charge and the size of the protein or of a protein-protein complex. The potential of this nanotechnology for studying protein-protein interactions is highlighted with the sensitive detection of -human chorionic gonadotropin, a hormone and clinical biomarker of pregnancy, by monitoring in real time and at a molecular level the formation of a complex between hormones and antibodies in solution. In this form, the assay compared advantageously to immunoassays, with the important difference that labels, immobilization, or amplification steps were no longer needed. In conclusion, we present proof-of-principle that properties of proteins and their interactions can be investigated in solution using synthetic nanopores and that these interactions can be exploited to measure protein concentrations accurately.The development of more sensitive assays for proteins is highly desirable as it will have a major impact in proteomics (i.e., for the understanding of the role of proteins in complex processes) and in clinical diagnostics (i.e., for alternative test formats). Classic immunoassays, which are routinely used for protein detection, have a sensitivity that is significantly lower than deoxyribonucleic acid (DNA) assays based on an amplification by means of polymerase chain reaction (PCR).1 An elegant way to boost the sensitivity of protein assays is, hence, to use DNA as a label and employ DNA amplification, for example in immuno-PCR 2 or biobarcode assays, 3 which allow a significant decrease in the detection limits to a few tens of proteins.In parallel to these developments, the detection of single biological molecules has become accessible using ultrasensitive fluorescence microscopy, 4-6 which, together with scanning probe microscopy (SPM), 7 is able to reveal inter-and intramolecular interactions and structural information. 8,9 However, the above methods for protein analysis have inherent limitations, such as a requirement for labels, immobilization, or complicated instrumentation that may be overcome with nanoporesensing.10,11 Some unique advantages of using nanopores are (i) no labeling or immobilization of the analyte is necessary; (ii) the instrumental setup is simple and does not require any moving parts, and (iii) it allows real-time detection of the analyte. Nanopores are therefore well suited for studying proteins and interactions between proteins under native conditions and at the single molecule level.Using the biological pore R-hemolysin, Meller et al. could distinguish DNA analytes which only differ in sequence. 11However, biological pores have practical limitations due to operating pH, temperature, a...

A structure is worth a thousand words: Guided by the X‐ray structure of an S‐selective artificial transfer hydrogenase, designed evolution was used to optimize the selectivity of hybrid catalysts. Fine‐tuning of the second coordination sphere of the ruthenium center (see picture, orange sphere) by introduction of two point mutations allowed the identification of selective artificial transfer hydrogenases for the reduction of dialkyl ketones.

Nature's catalysts are specifically evolved to carry out efficient and selective reactions. Recent developments in biotechnology have allowed the rapid optimization of existing enzymes for enantioselective processes. However, the ex nihilo creation of catalytic activity from a noncatalytic protein scaffold remains very challenging. Herein, we describe the creation of an artificial enzyme upon incorporation of a vanadyl ion into the biotin-binding pocket of streptavidin, a protein devoid of catalytic activity. The resulting artificial metalloenzyme catalyzes the enantioselective oxidation of prochiral sulfides with good enantioselectivities both for dialkyl and alkyl-aryl substrates (up to 93% enantiomeric excess). Electron paragmagnetic resonance spectroscopy, chemical modification, and mutagenesis studies suggest that the vanadyl ion is located within the biotin-binding pocket and interacts only via second coordination sphere contacts with streptavidin.