Nanoholes (or zero-mode waveguides) and arrays of these nanostructures have many intriguing optical properties: For instance, they can confine light at dimensions down to a tenth of its wavelength. A team led by Hermann Gaub at Munich has now shown that the light emitted by fluorescent DNA molecules also varies depending on their position inside these nanoapertures – a result that will be important for single-molecule sequencing.

Modern-day DNA sequencing consists of breaking molecules of DNA into fragments of different lengths and adding fluorescent tags to the broken ends so that they can be monitored. Current single-molecule sequencing randomly places molecules in nanoapertures so that many holes are either unoccupied or doubly occupied, explains team member Philip Tinnefeld. However, the problem is that only holes with exactly one molecule can be used for sequencing. What is more, data quality can be substantially improved if single molecules are placed in the nanoapertures' centre.

In their work, the Munich researchers have succeeded in using a single-molecule cut-and-paste technique to place fluorescent DNA molecules in the centre of nanoapertures that were around 375 nm across. By comparing the amount of light emitted by molecules that had been randomly placed anywhere inside a nanoaperture compared to those placed exactly in the centre allowed them to prove that the latter fluoresced much brighter.

Pick and paste

"Our technique allows us to 'pick up' single fluorophore-labelled DNA molecules from a ‘depot’ and deposit them exactly where we want," says Tinnnefeld. "The pick and paste works using molecular forces and the depot and target areas are pre-functionalized by microfluidic systems."

The team used a fast, non-invasive custom-built optical microscope in their experiments. "Since the size of the molecules and nanoapertures are beyond the optical diffraction limit, we used super-resolution techniques in which the position of the microscope cantilever that picks up the DNA molecules is localized by ‘gauss-fitting’ the point-spread function of a DNA molecule’s fluorescent label," Tinnefeld told "Similarly, we localized the nanoaperture’s centre by gauss fitting to the (plasmonic) light transmitted by the nanohole. In this way, we were able to position the DNA molecule to a precision of just 20 nm inside the nanohole."

The centre of the nanoaperture appears to be a hot spot, say the researchers, and it is here that the fluorophores emit most light.

DNA molecule "anchor"?

The centrally placed DNA molecule might also, in principle, be used as an "anchor" to immobilize other molecules of interest, such as enzymes, adds Tinnefeld – something that might help improve the efficiency of a particular bioassay. "Parallel loading of different enzymes on the DNA molecules might also allow for parallel screening of a variety of targets – such as those required in drug testing, for example," he says. "More generally, other single-molecule assays based on, say 'nanoantennas' or other nanoparticle structures could also benefit."

The team now hopes to directly transport molecules like enzymes into nanoapertures using so-called co-expressed force handles – as has already been done on plain surfaces without nanoholes. "This will help make it easier to load nanoapertures because it would avoid us having to use the DNA as an anchor to pull down the enzyme molecules," explains Tinnefeld.

The researchers also plan to use their technique to try and analyze the electrodynamics environment of nanostructures (such as nanoantennas) and localize specific hot spots for enhanced light emission in these structures.

The present work is detailed in Nano Letters.