A little bit of physics does not harm – Quantum dots in biology

February 9, 2012

Quantum dots (QDots) are  a nice technique to label biological samples in a very non-invasive manner. This technique has emerged in molecular biology and biophysics during the last years since QDots offer a number of advantages such as large spectral range, high brightness, and high photostability when compared to regular fluorophores. Here, I want to talk about some of the fundamentals, possible applications, and examples of the usage of QDots. I am not a physicist, so you will not find too much of the theoretical background in this article.

In order to understand the physical principal behind QDots, it is important to be familiar with the so-called electronic band structure of atoms. QDots are nanometer-sized crystals which consist out of semiconducting  atoms such as silicon. The electrons within these atoms can have quantified energy levels which are called bands. The highest and naturally most stable band is called valence band. However, the electrons within this band can be excited by for example photons that originate from a laser, and therefore reach a higher energy-state. In order to do so, the energy-barrier between the “old” and the “new” band must be overcome first. This barrier is called band gap (see also Fig. 1). Once the energy supply to the electron has been high enough it can “tunnel” the band gap and becomes conductive, meaning it can transform its energy to lower band electrons in other atoms. Now, current is flowing.

Fig. 1: The principle behind and the build up of a QDot. Biological molecules (like antibodies) which are attached to the polymer coating of the QDot can be used to serve as a link between the QDot and a biological surface (1st picture: Wikipedia, 2nd: Jim Zuo / University of Illinois at Urbana-Champaign, 3rd: designed myself, originally from Invitrogen).

However, there is an inverse relationship between this band gap and the size of the semiconducting crystal, i.e.  the smaller the crystal, the bigger the band gap that has to be overcome. This results in the necessity to use higher energies to excite the QDot crystal in the case of a smaller crystal, which also leads to a stronger detectable photon signal when the excited electron falls back to its resting state. For biological applications an easily detectable signal is quite important, because it allows the studying of minimally labeled single molecules. Since photophysical properties of QDots strongly depend on their “custom-synthesized” structure, they are an ideal tool to stain biological samples.

Still one obstacle remains: The required excitation energy for QDots is sometimes very close to the energy of the covalent bonds which link the individual semiconducting atoms with each other. This can lead to bond-breakage and freely diffusing atoms such as the toxic cadmium. Therefore, for the use in in vivo applications most QDots still need to be optimized. Nevertheless, QDots have demonstrated that they are an important tool for the biophysicist in in vitro experiments. One example is given below. In this example QDots are used to examine, whether the enzyme DNA helicase unwinds the DNA double strand (dsDNA) by sliding along the dsDNA or the leading single strand (ssDNA) during DNA replication.

For this experiment the researchers added bulky molecules to one strand of the DNA which served as a “roadblock” for the DNA unwinding helicase. Here, this bulky molecule was a visible QDot. If helicase is attached to dsDNA to unwind it, therefore the enzyme will stop in any case since it will inevitably encounter one of the roadblocks. For the case that helicase only attaches to one strand of the DNA strands (the leading strand) in order to unwind the dsDNA, it will, of course, only stop if it encounters the bulky molecule on the leading strand, but not if this molecule is present on the lagging strand (which helicase presumably does not touch). To test this, the researchers used two different ways to visualize the situation. When DNA replicates it does so in two directions and therefore also two fork-like structures arise (Fig. 2 (A) middle). The bottom part of Fig. 2 (A) shows how this fork-situation and the replication itself can be visualized by the addition of Sytox and dig-dUTP to an isolated DNA strain which is stretched by fluid flow. Sytox is a general DNA stain, so parts of the DNA that have already been replicated occur two times as bright as the non-replicated parts. dig-dUTP is added only during the last 25 minutes of the experiment. This means it is possible to observe into which direction the replication fork extends since this region will contain dig-dUTP and antibodies can bind to it.

Fig. 2: Analysis of the helicase behaviour in the presence of a QDot roadblock on DNA. (A) Sytox staining was used to identify replicated DNA (2x stronger signal = thick red) and an antibody against dig-dUTP (blue marks) was applyed to picture directionality and potential blockade of the replication process. (B, C) Show the combination of the labels and their orientation towards each other (1).

When concentrating on Fig. 2 (B) it becomes clear that helicase can probably only translocate along ssDNA. This becomes obvious when looking at the position of the QDot (the roadblock) and relating the staining signals towards it. In addition it is important top know that helicase can only migrate from the 3′ to 5′ direction. When the QDot is positioned at the “bottom” strand, double stranded DNA is only found left from it (red). Blue signal from dig-dUTP is only found left from red Sytox signal. Therefore, the strand is only replicated into the left direction and blocked to the right. However the helicase is able to bypass QDots which are not bound to its strand (Fig. 2 (B) iv). Compare also the fitting Sytox and dig-dUTP patters to it, which indicate that replication is also found after the QDot and into both directions. The QDot can, however, also be attached to the “top” DNA strand. This situation is analogous to the situation described above. Arresting and bypassing conditions are just inverted (Fig. 2 (C)).

By making use of a visible roadblock, a.k.a. a QDot, it was possible to resolve a little puzzle piece of eukaryote DNA replication on single-molecule scale. The QDot gave the researchers some point of orientation in a complex biological process in which the identification of directionality is essential.

(1) Fu et al. (2011), Selective Bypass of a Lagging Strand Roadblock by the Eukaryotic Replicative DNA Helicase, Cell 146, 931-941.


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