One of the coolest developments in optics has been the ability to see single molecules. However, although single molecule detection has become routine in research labs, it hasn't gone any further. The key reason is that although it is easy to detect a single molecule, you have to know where to look.

Imagine that you want to analyze someone's blood for an incredibly rare molecule that is indicative of some disease. You get a few microliters of blood, and somewhere in that drop is the single molecule that you want to detect. The chances of actually finding it are virtually nil, because single-molecule detection techniques generally rely on the molecule finding its way to some sensor surface or some other molecule that makes it even more visible. At low concentrations this takes a very long time to happen. The moral of the story is that low-concentration samples, which use diffusion-limited detection, take much too long to process for real-world applications.

What is required is some combination of techniques that retains single molecule sensitivity, but, at the same time, drives the molecule to the right place to be detected. A very large group of Italian researchers have achieved just that by using a combination of hydrophobic surfaces and plasmonics to enhance the signal.

Can you enhance that?

The researchers created hydrophobic surfaces so that when water droplets were placed on the surface, they were stable and didn't roll right off the substrate. This was done by etching a substrate in a manner that created a surface consisting of an array of tall pillars with the right radius and spacing. When the spacing and radius were right, a water droplet would sit still on the surface. The water droplet couldn't spread out because the surface repels the water, while the spacing of the pillars also prevented the drop from rolling, so it couldn't join up with other droplets either.

When a drop of water is placed on the surface, it starts to evaporate. But it has such a massive surface area compared to its volume, that it doesn't take long before the drop is gone entirely. Anything that isn't water is left sitting on top of one or two of the pillars right at the center of where the drop was deposited.

Enhance that some more, will you?

Once the drop has evaporated, everything we want to detect will be sitting on top of a few pillars. To detect a substance, we just need to make our pillars into something that allows single molecule detection. To do this, the researchers deposited metal nanostructures on the tops of the pillars. When light hits the pillars, it excites surface plasmon resonances in the metallic structures. The plasmons generate very large electromagnetic fields right at the surface—in other words, right where the molecules are sitting.

When you bathe molecules in strong light fields, they respond quite vigorously. As a result, it is possible to see fluorescence from single molecules—fluorescence is a measurement of certain electronic excitation states that are available to a molecule. You can even see Raman spectra (a map of the energies of mechanical vibrations and rotations of a molecule).

The researchers demonstrated this in spectacular fashion by making up solutions with femtomolar (10-15 mole) and attomolar (10-18 mole) concentrations of dye molecules, DNA molecules, and proteins. These were deposited in 20 microliter drops on the substrate. After waiting a few minutes, the fluorescence and Raman spectra were measured. In both cases, researchers could measure down to single-molecule sensitivities. The lowest concentrations had just one or two molecules in the droplets, which, in the case of DNA molecules, was verified by using atomic force microscopy. To put this in perspective, if the drops did not evaporate and you waited for the molecule to accidentally drop onto the surface, you would expect to wait for days.

To add to the perspective. If you were to use a nonydrophobic substrate and the same samples, the droplet would spread as it evaporates, resulting in that single molecule hiding somewhere in a 5x5mm2 area: a proverbial needle in a haystack.

This is a really clever development. And, because the majority of the fabrication techniques are already in use for large-scale production, it seems like it will be readily adapted for mass production. Furthermore, fluorescence detection is already well developed for certain biochemical measurements, so all the other key technologies are in place as well. If this is 3-5 years away, then something is very wrong with the world—you could be set up to make these within a year.

Nature Photonics, 2011, DOI:10.1038/NPHOTON.2011.222