We present a new, general method for the controlled deposition of biological molecules on surfaces, based on a nanopipet operating in ionic solution. The potential applied to the pipet tip controls the flux of biological molecules from the pipet, allowing fine control of the delivery rate. We used the ion current to control the distance of the pipet from the surface of a glass slide and deposited the fluorescently labeled DNA or protein G at a defined location onto the surface. Features of 830 nm size were obtained by depositing the biotinylated DNA onto a streptavidin surface; 1.3 mum size spots were obtained by depositing protein G onto a positively charged glass surface.

We report the pulsed delivery of single-stranded DNA molecules through a nanopipet. The conical geometry of the pipet leads to a localized electric field, since all of the potential drop occurs in the tip region. Pulsatile delivery of DNA molecules can be achieved in an experimentally simple way with high precision by controlling the applied voltage. Single-molecule detection and fluorescence correlation spectroscopy in the nanopipet enable us to determine the number of molecules delivered. Anomalous slow diffusion of the DNA molecules in the pipet has also been observed. This nanopumping technique may have potential applications in local drug delivery and nanofabrication of biomolecules on surfaces in aqueous environments.

SummaryWe have developed a novel light source for use in a scanning near-field optical microscope (SNOM or NSOM) based on a nanopipette whose distance from the sample surface is controlled using scanning ion conductance microscopy. The light source is based on the general principle of the chemical reaction between a fluorophore in the pipette and ligand in the bath, to produce a highly fluorescent complex that is continually renewed at the pipette tip. In these experiments we used fluo-3 and calcium, respectively. This complex is then excited with an Ar + laser, focused on the pipette tip, to produce the light source. This method overcomes the transmission problem of more traditional SNOM probes and has been used to acquire simultaneous high-resolution topographic and optical images of biological samples in physiological buffer. A resolution of ∼ 220 nm topographic and ∼ 190 nm optical was determined through imaging fixed sea-urchin sperm flagella. Live A6 cells were also imaged, demonstrating the potential of this system for SNOM imaging of living cells.

We have developed a novel light source suitable for imaging of biological samples. The method is based on the use of a micropipet filled with fluo-3. A fluorogenic complex is formed when fluo-3 meets calcium in the bath solution. The complex is excited by focusing a laser beam at the pipet tip to produce a submicrometer light source. This source is continually renewed at the tip, eliminating problems with photobleaching, and can be controlled by varying the applied potential. We first characterized the light source using fluorescence correlation measurements in order to optimize its properties. We then recorded an image of a model sample under buffer with submicrometer resolution using ion conductance distance control to demonstrate the feasibility of this approach.