Bench philosophy: Cheers to New Imaging Tools

Carbon dots from beer
by Steven D. Buckingham, Labtimes 03/2016



Carbon quantum dots offer a new way to fluorescently label living tissues and deliver drugs or genetic material. They are photo-stable, have low toxicity and are highly versatile – and you can even find them in your beer.

Recent years have seen a growth in the deployment of carbon nanomaterials such as fullerenes, nanotubes and graphite carbon dots. Carbon quantum dots (CQDs) are among the simplest of these nanomaterials. CQDs are ­quasi-spherical nanoparticles of carbon, some 10 nm or less in diameter. When looking a little closer, you will notice they can take on all sorts of appearances, ranging from nearly amorphous lumps to nanocrystalline structures. These odd ­materials bring some interesting chemical and physical properties, and life scientists are catching on to a range of applications, for which they may be used. With a little bit of modification, you can use carbon quantum dots as biosensors, contrast agents and drug delivery vehicles. You can apply them to monitor how a drug is making its way through a patient’s body and you can exploit them to monitor the incorporation of individual nucleotides into an extending DNA strand. Or you can simply use them as a biologically inert, non-toxic, water-soluble contrast agent that doesn’t fade during illumination.

Making them can be complex, involving some arcane chemistry and clever tricks with lasers. Or you can just extract them from cornflakes, biscuits and even beer. Yes, I am being serious.

Tiny lumps of charcoal

CQDs are a member of a class of carbon-based nanomaterials. Other examples are graphene – huge hollow sphere, tube or ellipsoid of carbon atoms – and the better-known carbon nanotubes. CQDs can take on several different structures, depending on how you make them but usually they are amorphous – just tiny lumps of charcoal. What makes them interesting is their unusual properties. One such odd property is their fluorescence, even though it is not entirely clear what triggers it. And what is more, the wavelength of the signal they emit is tuneable, as it changes with the wavelength of excitation. This is a major advantage, especially when you are trying to work with more than one fluorescent probe at the same time.


This plastic bag filled with Tsingtao beer should yield enough carbon dots for your fluorescent labelling experiments.

CQDs also have some very useful absorbance properties. This is because they have many C=C bonds, whose π-π* transitions absorb short-wavelength light. As I explain below, this means that you can attach a light-emitting probe to the CQD and the dot will absorb the signal until the probe becomes detached. Given you some ideas already? I’ll let you read about these in a moment but to give you something to get going on, CQDs have some weird properties that will get our more imaginative readers very excited. For example, CQDs are electrochemiluminescent – pass a current through them and they glow. Possible new voltage probes, perhaps?

Bottom-up or top-down?

How do you make CQDs? There are two basic methods – “bottom-up” or “top-down”. Making them from the bottom up means finding a chemical synthetic pathway that puts the carbons together in the right pattern. Making them top-down means taking some larger-scale molecular structure and breaking it up, so that you end up with CQDs. Let’s look at that a bit more closely. One bottom-up way of making CQDs is to use pyrolysis or carbonisation of organic precursor molecules. Pyro what?, you may ask. Okay, let me start that again. One way of making CQDs is to melt certain compounds (such as amino acids or sugars) until they go brown. Or you can just put sugar in the microwave: several groups have reported good yield of green fluorescent CQDs after just a few minutes’ microwave irradiation of a mixture of sucrose and diethylene glycol. Other workers have used electrochemical ablation, where a current is passed through a mixture of precursor compounds.

Crude methods

So what kinds of compounds can you start with? The list includes organic salts, such as ascorbic acid, glutamic acid, glycerol and coffee. Yes, coffee. It gets weirder...

So much for bottom-up approaches, what about the top-down ones? The basic idea is to take some carbon and break it up. Many top-down approaches to making CQDs start off with carbon nanotubes, graphite rods, carbon fibre and even soot. One method is to oxidise the starting material at low pH, although this nearly always introduces a negative charge onto the CQDs, which makes them insoluble, plus it can be difficult to completely remove the oxidising agent. Alternatively, you can cleave the carbon precursors electrochemically, in which case it is thought that the exfoliation into dots is caused by electrochemical stress. This method is simple and has a high yield.

If these methods often seem a bit crude, you are right. The devil is in the detail and the trickiest part is making sure you end up with a homogenous product. Many workers use standard purification techniques, such as ultrafiltration, centrifugation and chromatography. But there are some exciting high-tech ways, too. For instance, one group (Shen et al, 2012, Chem. Commun. 48: 3686) used a three-step process involving nanoreactors (spherical structures that grab hold of target molecules on their surface). Sticking the organic precursor onto the nanoreactor is the first step, followed by a pyrolysis step (baking, in other words). Finally, the nanoreactor is dissolved away to release the CQDs.

Let's get Nature to do the work

Does all this sound a bit tricky and technical to you? Okay, then let’s get Nature to do the work for us. And this is where things get even odder. It turns out that there are ready-made CQDs in coffee, fizzy drinks and even beer. Mingquian Tan’s group at the Dalian Institute of Chemical Physics, China, has discovered a niche role in finding ready-made CQDs in all sorts of strange, yet familiar, places. First of all, they found them in Nescafé, a brand of instant coffee. Then they decided to look for them in Tsingtao beer, a popular brand in China, and went on to show that having extracted them, by first stirring to remove gas followed by evaporative condensation and purification on Sephadex gel, they could use them to show up cancer cells and even deliver anti-cancer drugs. The original paper has an interesting account of the history of Tsingtao and a methods section reminiscent of a cocktail recipe (Wang et al. 2015, Analytical Methods 7:8911).

Tan went on to show there are CQDs in several soft drinks and it is emerging that they are lurking in just about anything that involves carbonisation, such as bread, caramel and corn flakes.

Once you have got your dots, then the real fun begins. In this case, the fun consists of thinking up things to do with the surface of the dots, to make them do interesting or clever things. This is made easy with CQDs because they have oxygenated functional groups on their surface, which makes them convenient hydrophilic handles, onto which you may stick different chemicals. These materials can be used to alter the native fluorescence of the dots or they can be used in more sophisticated ways, such as delivering drugs or turning the dots into biosensors. And don’t forget the dots are non-toxic, so this works for in vivo imaging, too.

Surface modifications

Some modifications of the surface are designed just to increase the quantum yield of the dots. The most common is passivation using polyethylene glycol, although this has some negative side effects in that it can make it more difficult to add other chemical moieties and can also increase the size of the dots. Other modifications are just designed to change the colour of the emission, and a body of folklore gradually accumulates to act as a guide as to what to add to get the colour and shade of your choice. For instance, adding zinc sulphide or PEG can alter the emission peak from 510 to 650 nm, meaning you can use simultaneous multiple probes. Remember also that the emission of CQDs changes with the wavelength of the excitation, so you have a lot of room to manoeuvre to get the right contrast conditions.

CQDs can be used to make very sensitive probes for chemical species. You can passivate dots using branched ­polyethyleneimine (BPEI), instead of polyethylene glycol, and make yourself a probe for Cu2+ that can detect down to 6 nM. It is thought that the copper’s interaction with the amino groups of the BPEI quenches the fluorescence of the dots. But this makes for big dots that won’t get into cells, although you can get round this by using alternative routes of synthesis.

CQDs have been used to make a pH sensor. One group accomplished this by attaching an established proton receptor (its name is very long, so we won’t waste paper with it here) to dots and found the intensity was linear with pH over a wide range, enabling them to read pH in the cytosol in real time (Kong et al. 2012, Adv. Mater. 24: 5844).

Different applications

CQDs can be used as drug delivery vehicles. This illustrates interplay between the “native” advantages of dots (their biological inertness, high fluorescent yield and photostability) and the “add on” features supplied by the surface modifications. Several labs have confirmed that loading the anticancer drug, doxorubicin (DOX) onto CQDs is effective at selectively killing cancer cells.

Minqian Tan (of Tsingtao beer fame) made CQDs using a simple hydrothermal approach – they baked citric acid and ­o-phenylenediamine. These dots had (as is common) a negative surface charge, which meant it was trivial for them to adsorb the DOX molecules. But there is an extra feature that comes with this arrangement: as well as delivering the drug to the target cells, the presence of the DOX quenched the dots’ fluorescence. This meant that release of the drug could be monitored as the recovery of fluorescence (Biotechnol Lett DOI 10.1007/s10529-015-1965-3).

In a similar approach, Zheng et al. used another anticancer agent (oxidised oxaliplatin) and were able to track the cellular trafficking of the dots to their target (2014, Adv Mater 26: 3554).

But CQDs are not only good for delivering drugs, they can deliver genes too. CQDs modified with polyethylenimine are negatively charged and this is enough to hold DNA molecules for delivery into cells. Qing Wang and colleagues successfully delivered short-interfering RNA against survivin into the human gastric cancer cell line MGC-803, knocking gene expression down by nearly 94% (J Nanobiotechnol, 2014 12:58). And remember, you can modify the dots, enabling you to monitor the delivery in the same way as monitoring drug delivery – all in real time, of course.

Many advantages

Carbon dots offer quite a few advantages over “traditional” fluorescent probes. For one, they are astonishingly photo-stable, in contrast to the notorious bleaching of other types of fluorescent probe, which bedevils high-resolution in vivo imaging. Whereas traditional organic fluorophores bleach within minutes, CQDs typically lose only about five percent of their fluorescence, even after four hours of irradiation.

They are also very non-toxic. After all, they are just lumps of carbon, the stuff from which we are made. Survival rates in the 90% region have been reported for many cells and tissues, including human hepatocellular carcinoma cells, human breast cancer cell lines and even whole animals. But of course, if they are really going to be useful as drug delivery agents, we have to be sure they will clear from the human body quickly; animal studies have indeed shown this to be the case. Of course, safety concerns go beyond mere toxicity but it is encouraging to note that CQDs have been fed in large quantities to mice and rats without any perceptible change in food intake, body weight, behaviour, kidney or liver function.

With this combination of low toxicity and high photo-stability, coupled with their adaptability to a range of applications, CQDs have the potential to become an important technique in the coming decade. You may have some difficulty, however, explaining why you have ordered Tsingtao beer on the lab budget...





Last Changed: 21.06.2016




Information 4


Information 5


Information 6


Information 7


Information 8