Single-molecule biosensing

A major advantage of studying biochemical interactions at the level of individual molecules is the possibility to measure properties that cannot be extracted from ensemble data, e.g. distributions of molecular properties (such as affinity or kinetic parameters, variations in space and/or in time) or molecular torsion constants. By constructing histograms of particular molecular observables (such as the time it takes for a protein to unbind from its receptor) one is able to identify and characterize different populations within an ensemble. Moreover, the recording of single-molecule trajectories allows us to follow molecular processes in real time and observe rare and short-lived intermediates. In our group we focus on two complementary techniques to detect biomolecular interactions at the single-molecule level: based on magnetic particles and on plasmonic nanoparticles.

Magnetic particle probes

We use functionalized magnetic particles to capture target molecules from solution and subsequently bind to a substrate. The single-molecule regime is reached by reducing the concentration of target molecules in solution and/or the density of capture molecules on the particles and/or the density of capture molecules on the substrate [1]. The use of magnetic fields allows us to exert a force (or torque) on the magnetic particles and thereby on the individual molecular bonds. By monitoring the motion, the association and dissociation of the particles in a microscope, we get information on the characteristics of the molecular interactions. The force-dependent dissociation yields information on the potential-energy landscape of the interaction and thus on the nature of the interaction, e.g. specific vs. non-specific [2] and single vs multiple bonds [1]. Nonspecific binding can for example originate from the nonparatope regions of antibodies and from the substrate materials used in the assay. We also study molecular systems with tethers such as dsDNA [3]. From the mobility we can distinguish e.g. particles that are bound via a single tether versus particles bound by multiple tethers. These studies lead to novel methods to understand and discriminate between specific and nonspecific interactions, with the goal to develop assay technologies for maximum sensitivity and specificity.

Plasmon mediated detection

Localized surface plasmons are coherent oscillations of the conduction electrons in metal nanoparticles. The electric field associated with a surface plasmon resonance decays rapidly into the surrounding medium and acts as a local probe for single molecules. We recently developed two detection modalities with single-molecule sensitivity: (1) detection of non-absorbing proteins by wavelength shifts of the plasmon resonance of a single gold nanorod [4] and (2) the detection of single fluorescent molecules by their enhanced fluorescence caused by the strong near-field around a metal particle [5]. The first detection modality does not require fluorescent labeling of the analyte and can be applied to any macromolecule that exhibits a refractive index contrast with the environment. In this project we explore the limits of single-molecule plasmon sensors in terms of detection limit, the complexity of the environment and the complexity of the process that is probed. The near-field around a single nanorod is confined to a volume more than 104 times smaller than a diffraction limited spot. This zepto-liter (10-21 L) excitation volume combined with the biocompatibility and far-field optical detection makes single metal particles highly promising probes for single molecules inside living cells.

References

[1] A. Jacob, L.J. van IJzendoorn, A.M. de Jong, and M.W.J. Prins, Quantification of protein-ligand dissociation kinetics in heterogeneous affinity assays, Anal. Chem. 84, 9287-9294 (2012)
[2] M. Kemper, D. Spridon, L.J. van IJzendoorn, and M.W.J. Prins, Interactions between protein coated particles and polymer surfaces studied with the rotating particles probe, Langmuir 28, 8149-8155 (2012).
[3] K. van Ommering, M. Koets, R. Paesen, L.J. van IJzendoorn, and M.W.J. Prins, Bond characterization by detection and manipulation of particle mobility in an optical evanescent field biosensor, J. Phys. D 43, 385501 (2010)
[4] P. Zijlstra, P.M.R. Paulo, and M. Orrit, Optical detection of single non-absorbing molecules using the surface plasmon resonance of a gold nanorod, Nature Nanotech. 7, 379-382 (2012).
[5] H. Yuan, S. Khatua, P. Zijlstra, M. Yorulmaz and M. Orrit, Thousand-fold Enhancement of Single-Molecule Fluorescence Near a Single Gold Nanorod, Angew. Chem. Int. Ed. 52, 1217-1221 (2013).