Single-molecule biosensing using plasmonic nanoparticles

Optical detection of single molecules mostly relies on their fluorescence because of the high contrast of this technique against the background. Since their invention in the early 1990s, single-molecule fluorescence microscopy... [ view full abstract ]

Optical detection of single molecules mostly relies on their fluorescence because of the high contrast of this technique against the background. Since their invention in the early 1990s, single-molecule fluorescence microscopy and spectroscopy have spread to many fields in chemistry, physics, and biology, and have provided unique access to nanometer scales. However, the majority of native bioorganic molecules such as proteins hardly fluoresce at all. Therefore, their detection in native and unmodified state requires a different approach.

We demonstrate the label-free detection of single proteins using plasmonic nanoparticles. We monitor the scattering signal of hundreds of particles simultaneously in a total-internal-reflection microscope. This approach allows for the statistical analysis of single-molecule interactions without requiring any labeling of the analyte. Single-protein binding events are resolved as step-wise changes in the scattered intensity, see Fig. 1. We study an antibody−antigen interaction and find that the waiting time distribution is concentration-dependent and obeys Poisson statistics. The ability to probe hundreds of nanoparticles simultaneously will provide a sensor with a dynamic range of 7 decades in concentration and will enable the study of heterogeneity in molecular interactions.

We foresee that this approach will be particularly attractive to monitor biomarker concentrations directly in a complex matrix in which labeling is not possible. Moreover, our approach will provide a means to improve the sensor's specificity by distinguishing populations of specific interactions from non-specific ones by statistical analysis of single-molecule kinetic parameters. These advantages will benefit future point-of-care biosensors and may allow for the real-time monitoring of biomarker concentrations on- or in-the-body.

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