NANO LETTERS
Nanoparticle-Mediated Nonfluorescent Bonding of Microspheres to Atomic Force Microscope Cantilevers and Imaging Fluorescence from Bonded Cantilevers with Single Molecule Sensitivity
2009 Vol. 9, No. 5 2120-2124
Sanjeevi Sivasankar*,†,§ and Steven Chu‡,§ Department of Physics and Astronomy, Iowa State UniVersity, Ames, Iowa 50011, United States Department of Energy, Washington, D.C. 20585, and Lawrence Berkeley National Laboratory, Departments of Physics and Molecular and Cell Biology, UniVersity of California, Berkeley, California 94720 Received February 25, 2009; Revised Manuscript Received March 17, 2009
ABSTRACT A technique to attach silica and glass microspheres onto silicon or silicon nitride cantilevers using silica nanoparticle sol-gel chemistry is presented and a method to image the fluorescence background from the bonded cantilevers with single molecule sensitivity is described. The silica nanoparticles polymerize to form a highly branched network that covalently link the microsphere and cantilever together. The bonding is carried out at room temperature which preserves the integrity of the cantilevers and their reflective coating. Comparison of cantilever and single dye molecule fluorescence demonstrates that the cantilevers are nonfluorescent at the single molecule level.
Silica and glass microspheres attached to atomic force microscope (AFM) cantilevers are routinely used to study fundamental forces such as van der Waals, electrostatic, hydrophobic, hydration, and steric forces that are important in colloidal stability.1-6 These colloidal probes are also used to study force dependent optical properties of fluorescent materials and fluorescence in confined geometries.7-9 In these probes, a microsphere is attached to a cantilever using adhesives such as epoxy resins10 and UV-curable glues.11,12 It is critical that the adhesive used to prepare the probe is nonfluorescent, stable in different solvents, and that the surfaces are free of contamination.13 However most adhesives have a high fluorescence background, degrade in organic solvents and in aqueous solutions with high ionic strengths, and leach overtime to introduce surface contamination. Colloidal probe microscopy can also be combined with single molecule fluorescence microscopy to study the interaction and dynamics of single biological molecules. * To whom correspondence should be addressed. E-mail: sivasank@ iastate.edu. † Iowa State University. § University of California. ‡ United States Department of Energy. 10.1021/nl900616y CCC: $40.75 Published on Web 03/30/2009
2009 American Chemical Society
Previous attempts at combining fluorescence microscopy with AFM force measurements have used conventional, sharp AFM probes.14-17 A critical obstacle in using a sharp AFM probe for combined single molecule AFM-fluorescence measurements is the low probability of interaction between fluorescent molecules on the substrate and the sharp AFM tip. This arises because the fluorescent molecules are bound to the substrate at low, optically resolvable surface densities which are 2 to 3 orders of magnitude smaller than the typical surface coverage in stand-alone single molecule AFM force measurements. Since the dimensions of both the fluorescent molecules and the radius of curvature of a sharp AFM tip are significantly below the wavelength of visible light, the tip cannot be positioned exactly over the fluorescent molecule using diffraction limited optics. This hurdle can be overcome by increasing the area of contact between the tip and the substrate by using a colloidal probe instead of a sharp AFM tip. However, combined single molecule colloidal probefluorescence experiments require a nonfluorescent method to bond microspheres onto AFM cantilevers and a technique to image the fluorescence background from the cantilever with single molecule sensitivity.
Figure 1. Schematic of the nanoparticle mediated bonding of a microsphere to an AFM cantilever. The silica nanoparticles polymerize into a highly branched 3D network and bond glass and silica microspheres on Si and Si3N4 cantilevers via multiple siloxane linkages. The nanoparticles bind together via the reaction Nanoparticle-Si-OH+HO-Si-NanoparticlefNanoparticle-Si-O-SiNanoparticle + H2O.
Nonfluorescent bonding can be accomplished by directly sintering borosilicate glass microspheres onto AFM cantilevers at temperatures close to the softening point of borosilicate glass.13 The high sintering temperatures (780 °C) however cause cantilevers to lose their reflective coating, get warped, and the pyrex glass base that is present on some cantilevers (such as Olympus Biolevers) to melt. Furthermore, this technique can only be used to fuse microspheres that have a significantly lower softening temperature than the cantilever, for example, glass microspheres but not silica microspheres. In this manuscript, a technique to attach silica and glass microspheres onto silicon or silicon nitride cantilevers at room temperature is presented and a method to image the fluorescence background from the bonded cantilevers is described. The bonding is accomplished using silica nanoparticles which form a gel network at room temperature that covalently links the microsphere to the AFM cantilever via multiple siloxane bonds. This bonding method is an extension of a technique that was recently introduced to join silica and glass for high performance optical applications.18 Since the microspheres are attached without using any glues or epoxies, the cantilevers are nonfluorescent, resistant to organic solvents and high ionic strength solutions. The bonding is carried out at room temperature that does not compromise the integrity of the cantilevers or its reflective coating. The fluorescence background from the cantilever is imaged with single molecule sensitivity using a tip scanning contact mode AFM mounted on a sample scanning confocal microscope where the fluorescence is recorded at each position of the AFM tip as it is scanned over a laser beam. Experimental Section. The silica nanoparticles that are suspended in a potassium hydroxide solution form a gel network at room temperature that covalently links the microsphere to the AFM cantilever via multiple siloxane bonds (Figure 1). The OH- ions in the bonding solution hydrolyze and etch the silica nanoparticles, the microsphere, and the cantilever surface. As a result, the surfaces liberate silicate ions and the pH of the bonding solution gradually decreases. At a pH
