Detecting molecules and their interactions lies at the heart of all biosensor devices, which have important applications in health, environmental monitoring and biomedicine. Achieving biosensing capability at the single molecule level is, moreover, a particularly important goal since single molecule biosensors would not only operate at the ultimate detection limit by resolving individual molecular interactions, but they could also monitor biomolecular properties which are otherwise obscured in ensemble measurements. For example, a single molecule biosensor could resolve the fleeting interaction kinetics between a molecule and its receptor, with immediate applications in clinical diagnostics.
We have now developed a label-free biosensing platform that is capable of monitoring single DNA molecules and their interaction kinetics, hence achieving an unprecedented sensitivity in the optical domain, Figure 1. We resolve the specific contacts between complementary oligonucleotides, thereby detecting DNA strands with less than 2.4 kDa molecular weight. Furthermore we can discern strands with single nucleotide mismatches by monitoring their interaction kinetics.
Our device utilizes small glass microspheres as optical transducers[1,2, 3], which are capable of increasing the number of interactions between a light beam and analyte molecules. A prism is used to couple the light beam into the microsphere.
Ourr biosensing approach resolves the specific interaction kinetics between single DNA fragments. The optical transducer is assembled in a simple three-step protocol, and consists of a gold nanorod attached to a glass microsphere, where the surface of the nanorod is further modified with oligonucleotide receptors. The interaction kinetics of an oligonucleotide receptor with DNA fragments in the surrounding aqueous solution is monitored at the single molecule level.
The light remains confined inside the sphere where it is guided by total internal reflections along a circular optical path, similar to an acoustic wave guided along the wall of St. Paul’s Cathedral. These so called whispering gallery modes (WGM) propagate with little loss, so that even a whisper can be heard on the other side of the gallery. In the optical case, the light beam can travel many thousand times around the inside of the microsphere before being scattered or absorbed, thereby making numerous interactions with an analyte molecule, bound to microsphere from surrounding sample solution. The most part of the light intensity, however, remains inside the microsphere, just below the reflecting glass surface, resulting in a relatively weak interaction between the light and the bound molecule.
To enhance this interaction further, we attach tiny 42 nm x 12 nm gold nanorods to the glass surface. When passing a nanorod, the lightwave induces oscillations of conduction electrons, resulting in so called plasmon resonance. These nanorod plasmons greatly enhance the light intensity on the nanorod, so that the interaction of the light with a molecule attached to the nanorod is also enhanced[4-6]. This enhanced interaction results in an increase in sensitivity by more than a factor of one thousand, putting our experiments of single DNA molecule detection within reach.
For the specific detection of nucleic acids, we attach single-stranded DNA to the nanorod and immerse our device in a liquid solution. When a matching, i.e. complementary DNA fragment binds from solution to the “bait” on the nanorod, the enhanced interaction with the light results in an observable shift of the WGM wavelength. Since light propagates in a WGM only for a very precise resonance wavelength or frequency, this shift can be detected with great accuracy. On our current biosensor platform, we detect wavelength shifts with an accuracy of less than one femtometer, resulting in an extremely high sensitivity for biosensing, which we leverage for the specific detection of single 8 mer oligonucleotides as well as the detection of less than 1 kDa intercalating small molecules.
 M. D. Baaske, M. R. Foreman, and F. Vollmer, "Single molecule nucleic acid interactions monitored on a label-free microcavity biosensing platform," Nature Nanotechnology, vol. 9, pp. 933-939, 2014.
 Y. Wu, D. Y. Zhang, P. Yin, and F. Vollmer, "Ultraspecific and Highly Sensitive Nucleic Acid Detection by Integrating a DNA Catalytic Network with a Label-Free Microcavity," Small, vol. 10, pp. 2067-2076, 2014.
 M. R. Foreman, W.-L. Jin, and F. Vollmer, "Optimizing Detection Limits in Whispering Gallery Mode Biosensing," Optics Express, vol. 22, pp. 5491-5511, 2014.
 M. A. Santiago-Cordoba, S. V. Boriskina, F. Vollmer, and M. C. Demirel, "Nanoparticle-based protein detection by optical shift of a resonant microcavity," Applied Physics Letters, vol. 99, Aug 2011.
 M. R. Foreman and F. Vollmer, "Theory of resonance shifts of whispering gallery modes by arbitrary plasmonic nanoparticles," New Journal of Physics, vol. 15, p. 083006, Aug 2013.
 M. R. Foreman and F. Vollmer "Level repulsion in hybrid photonic-plasmonic microresonators for enhanced biodetection" Phys. Rev. A 88, 023831 (2013).
Frank Vollmer, "Detecting single DNA molecule interactions with optical microcavities (Presentation Recording)," Proc. SPIE 9547, Plasmonics: Metallic Nanostructures and Their Optical Properties XIII, 954721 (Presented at SPIE Nanoscience + Engineering: August 13, 2015; Published: 5 October 2015); https://doi.org/10.1117/12.2190649.4519370337001.
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