Preparation and Loading of Nanopipettes
Tips & Tricks for Nanoinjection, Nanodeposition and Scanning Ion Conductance Microscopy
- Fig. 1: Backloading principle of a nanopipette.
- Fig. 2: a) Whole nanopipette loaded with colored liquid solution. Properly loaded nanopipette (upper nanopipette) and nanopipette loaded with liquid and containing bubbles (lower nanopipette). b) Detailed view of the tip area of a properly loaded nanopipette. c) Detailed view of the tip area of a loaded nanopipette containing a bubble.
The workgroup "Biomolecular Photonics" at Bielefeld University, Germany, has been using nanopipettes now for over a decade, representing key elements for scanning ion conductance microscopy (SICM), nanoinjection and nanodeposition. As essential parts for these applications, nanopipettes mediate an ionic current through liquid solution loaded inside the hollow nanopipettes and leading it through their tip aperture with diameters from 100 nm down to 10 nm. Because of the extremely small aperture size, appropriate loading of nanopipettes with solution is not straight-forward, but of high importance for a stable ionic current, constituting the basis for reliable and precise measurements.
Application of Nanopipettes
Nanopipettes are versatilely usable small hollow glass capillaries with diameters ranging from 10 to 100 nm. They consist either of borosilicate or quartz glass. In the biosciences they are used e.g. in scanning ion conductance microscopy (SICM) to obtain topographic information of non-conducting surfaces, in nanoinjection to deliver molecules with very high precision into living cells, or in nanodeposition to create small patterns with controllable quantity of molecules per spot. But nanopipettes are also employed to extract volumes using the electrowetting technique as this was shown by the attosyringe and resulting nanobiopsy application. Nanopipettes are fabricated by heating and pulling a cylindrical glass capillary with typical outer diameters of 1 mm and inner diameters of 0.5 to 0.7 mm. The so called pulling process is performed by a "pipette puller". The tip diameter of the resulting nanopipette as well as the taper length can be varied by the heating temperature and different timing of heating and pulling.
Backloading of Nanopipettes
Nanopipettes are typically used in combination with an ionic current flowing from an electrode placed inside the back end of the pipette through the tip aperture to a counter electrode placed outside the pipette. The nanopipette is therefore surrounded by conductive medium and is itself filled with conductive liquid. As a consequence, the liquid has to be loaded inside the nanopipette.
There are mainly two options to fill a nanopipette with liquid: 1. Backloading with a microloader, 2. Passive loading, using an exsiccator. This article will mainly focus on the first option, as the procedure of backloading a nanopipette is faster and requires less equipment. Nevertheless, loading a nanopipette with liquid using an exsiccator has its place in situations where difficult to handle liquids or nanopipettes with special shapes are needed. To perform the backloading of nanopipettes, a typical micropipette (air displacement pipette) with a displacement volume of at least 10 µl in combination with a microloader is needed. The microloader is formed similar to a typical disposable tip suitable for the micropipette, with an extended thin-tapered tip end having a typical length of 60 mm and an outer diameter of 0.3 mm.
At the beginning of the backloading process the microloader is filled with solution and inserted from the back into the nanopipette, as depicted in figure 1A. The tip of the microloader is positioned at a distinct position inside the nanopipette at approx. 1-2 mm from the point where the tapering begins. In a first step, some µl of the solution is loaded into the nanopipette (fig. 1B). After a short time, the solution tracks right down to the tip end (fig. 1C) until the tip is visibly filled with solution (fig. 1D). By further insertion of solution from the microloader into the tip region and successive rejection of the microloader (fig. 1E, F) the bulk of the pipette is loaded till the back end or as far as desired (fig. 1G).
Due to the extremely small tip diameter, the interactions between the inserted solution and the glass surface tend to prevent the solution from entering the tip end due to capillary force, ending up with an incompletely filled pipette (see fig. 2) and hence insufficient conductive properties. As a practical remedy, glass capillaries are equipped with a thin glass filament fused inside the lumen are used to facilitate the filling of the tip. Nevertheless, most difficulties during back loading of nanopipettes are still experienced while filling the tip area. Besides the tip aperture size, the taper form and taper length of a nanopipette plays an important role and hence has a strong influence on the expansion of solution within the tip area to the tip end. Thus, it can be very difficult, if not impossible to fill a nanopipette with a small diameter of 10 to 20 nm and an extended taper length of 4 to 5 mm, even if glass capillaries with filament are used. In the case that during the filling process bubbles are generated by either insufficient filling of the tip area or by transferring bubbles from the microloader originating from inaccurate solution pick up, the filling process can be continued anyway, as most of the bigger bubbles will move to the back end of the nanopipette and are therefore not cumbersome. In contrast, small bubbles will remain at their position within the tip area, causing irregularities in the ionic current afterwards. In this case, the microloader can be inserted again after completion of the filling process, by simultaneously extracting the previously inserted solution and the bubbles, till all of the bubbles are collected into the microloader. The bubbles (and some µl of the solution) are then ejected outside of the nanopipette. The filling process can then be repeated.
Another challenge, especially true for nanoinjection or nanodeposition applications, is the backloading of solution containing further components which tend to generate bubbles, such as proteins. This often impedes the filling process significantly. Here, some tricks can help to fill the nanopipette bubble-free. As the quantity of enclosed air depends on the concentration of the bubble-generating agent, the bubble extent can be divided into strong bubble generation (foam-like) or weak bubble generation. For foam-generating liquids, it is necessary to pick up the solution very slowly with the microloader. Otherwise, the foam will already be present prior the backloading process. Mostly it is the first flush of solution entering the nanopipette which causes the majority of foam. In most cases the filling of the nanopipette can be continued as the foam will stay at the upper surface of the solution within the pipette and will ejected out of the back end when the solution from the microloader is completely transferred. For less bubble-generating solutions with single bubbles, it is advantageous to target and extract the bubbles with the tip of the microloader as described previously.
Considering these simple tricks will lead to reliable usable nanopipettes for each application with stable conductive properties of the nanopipette during the measurements.
Matthias Simonis1 and Simon Hennig1
1 Bielefeld University, Faculty of Physics, Biomolecular Photonics, Bielefeld, Germany