The detection of molecules and their interactions lies at the heart of all biosensing. Achieving biosensing capability at the single-molecule level is, moreover, a particularly important goal. By resolving individual molecular interactions, single-molecule biosensors would operate at the ultimate detection limit and could enable the measurement of biomolecular properties that are obscured in ensemble measurements. A single-molecule biosensor could, for example, resolve the fleeting interactions between a molecule and its receptor, with immediate applications in clinical diagnostics. We have developed a label-free biosensing platform capable of monitoring single molecules and their interaction kinetics,1 thereby achieving unprecedented sensitivity in the optical domain (see Figure 1). Specifically, the platform is able to resolve interactions between complementary oligonucleotides, enabling the detection of DNA strands with a molecular weight of less than 2.4kDa. Furthermore, by monitoring their interaction kinetics, it becomes possible to discern strands with single nucleotide mismatches. Our device employs small glass microspheres as optical transducers,2, 3 which are capable of increasing the number of interactions between light and analyte molecules. A prism couples a light beam into the microsphere (see Figure 2). The light then remains confined inside the sphere, where it is guided by total internal reflections along a circular optical path, similar to the way in which an acoustic wave is guided along the wall of St. Paul’s Cathedral due to so-called whispering gallery modes (WGMs). These WGMs lead to propagation with little loss, enabling even a whisper to be heard on the other side of the gallery. In the optical case, the light beam can travel many thousands of times around the inside of the microsphere before being scattered or absorbed, thereby making numerous contacts with an analyte molecule bound to the microsphere from a surrounding Figure 1. Illustration of our biosensing approach, which is capable of resolving the specific interaction kinetics between single DNA fragments. The optical transducer, which consists of a gold nanowire attached to a glass microsphere, is assembled in a simple three-step protocol. The surface of the nanowire is further modified with oligonucleotide receptors. The interaction kinetics of an oligonucleotide receptor with DNA fragments in the surrounding aqueous solution can monitored at the single-molecule level.1 (Credit: Joseph Alexander, Rockefeller University.)