Prevention is better than cure, or so the saying goes. It is then no surprise that investment in medical diagnostics continues to grow apace. Research, innovation and development of better detection methods ultimately leads to simpler resolution, reduced trauma for the patient and faster and more cost-effective treatment. These goals have been driving giant leaps in diagnostic development and one of the most fascinating ‘emergent’ (emergent within diagnostics. Quantum dots have been exploited commercially for over 20 years) diagnostic tools is the quantum dot (or QD for short).
QDs are so called because they are miniscule specks of matter – so small that they are in effect concentrated into a single point (hence ‘dot’). Another way of looking at it, is to think of a QD as being zero-dimensional, an object with no measurable width, length or height. Another alias for QDs is ‘artificial atoms’ – while they are miniscule, they are not always single atom-sized. They are in fact groups of atoms at the nanoscale (typically man-made crystal structures that range from 2nm to 10nm in diameter – that’s between 2 to 10 billionths of a meter!) QDs are nanoscale semiconductors, made from materials such as silicon, cadmium selenide, cadmium sulphide, or indium arsenide, ingredients used extensively in electronic circuits.
What makes QDs special – and frankly, a bit quirky – is that they obey the quantum mechanical principle of ‘quantum confinement’, but in their own unique way. What does this mean and why it is significant?
Figure 1: Fluorescence images of quantum dots suspensions (image taken from: Kosaka, Nobuyuki et al., 2010)
School-level science tells us that if you give an atom energy, you can ‘excite’ it; i.e. you can boost an electron inside it to a higher energy level. When the electron returns to a lower level, the atom ‘gives back’ that excitation energy. It emits a photon of light of a slightly lower energy (and longer wavelength) than the atom originally absorbed during excitation. Atoms emit different colours of light. Since wavelength and frequency determine colour, different atoms emit different colours. Another factor determining atoms’ colour is the way their energy levels are arranged. The energy levels in atoms have set values; they are said to be quantised.
QDs are similar in terms of their quantised energy level behaviour. But, curiously, a QD of a specific material (such as silicon) is capable of producing a different colour depending on its size (Figure 1). This ‘tunable’ light emission means that in general, a small-sized QD emits blue light, whereas a large QD generates red light. Different sized QDs can produce a veritable rainbow of spectral colours (Figure 2: quantum dots’ size vs colour).
Figure 2: The spectral range of colours emitted by QDs of different sizes. Image courtesy of: Jeff Yurek, Nanosys Inc, Milpitas, CA.
Now you see me…
Medical diagnostics – the art and science of finding the cause of someone’s symptoms – relies on finding a ‘culprit’, for example, biomarkers in a small volume of human tissue or fluid (such as blood or urine). This chemical finger-print signals the presence of a
specific medical condition. Immunoassays are a class of tests typically used in point-of-care diagnostics to not only identify the specific biomarker, but to find out how much of it is present in the diagnostic sample. The ‘immuno’ prefix relates to the use of antibodies during assay. A well-known example of such a test is home pregnancy tests (see text box: ‘A fine line’).
‘A fine line’A pregnancy test works by detecting hCG, the ‘pregnancy hormone’ in blood or urine. This hormone is produced by the placenta and is a perfect biomarker. In a pregnancy test, a special reagent is used which reacts and binds to hCG. In a home-use device, as urine and testing dye move across a pregnancy test reagent field, hCG is captured along with the dye to produce a qualitative diagnosis, specifically a clearly visible ‘fine line’.
Antibodies, the body’s built-in defence mechanism, have a useful ability to bind to antigens, substances which trigger an immune response in the body. Adding a chemical probe or label to an antibody, which emits light, or fluoresces (known as a fluorophore), makes it easy to detect what is happening, i.e. what biomarker is present and how much of it there is.
Organic dyes (like fluorescein or rhodamine), or fluorescent proteins (GFP or RFP, for example) are widely used as light-emitting probes or labels. But QDs are also fluorophores and are muscling in on the diagnostics scene. A quick look on the web tells you that fluorescent organic dyes are available which cover a wide range of required colours or wavelengths, so replacing them with QD labels may seem unnecessary. However, the introduction of QDs could make a huge difference in diagnostics because test sensitivity, accuracy and specificity are improved, without the need for change or additional complexity in the existing detection equipment. When it comes to QDs versus organic dyes, here is how they perform:
Brightness •
QDs are typically an order of magnitude brighter than their organic molecule counterparts. This is partly because QDs have a larger capacity for absorbing light (scientifically speaking, they have a larger molar absorption coefficient) and the ratio of photons produced per photon received (known as fluorescent quantum yield) is high in the visible range and infra-red. Organic dyes, on the other hand, have a smaller molar absorption coefficient and high quantum yield in the visible range only. The brightness of organic fluorophores depends on their size, rigidity and covalent structure (strong chemical bonds holding the atoms together in a distinctive shape), making them more susceptible to lose their energy in way that do not produce light (i.e. non-radiative). Increased brightness is particularly useful for in vivo diagnostics, where brighter emissions could compensate for processes where light is lost on its way to the detector such as light scattering, absorption and autofluorescence in biological tissues.
Sharpness •
The emission peaks (i.e. the coloured spectrum emitted by the QDs) produced by QDs are sharp and narrow, with little overlap (they don’t emit at the same wavelengths) between the peaks of multiple QDs excited simultaneously (i.e. their emissions are very distinct from each other); this means they are clearer and easier to spot. Thus it’s easy to measure the light emitted by different QDs emitting light simultaneously, because their emission spectra can be separated out very easily. On the other hand, the spectra of light emitted by organic dies are broader (e.g. two different dyes can emit lots of light of different colours) and can overlap (e.g. two different dyes can emit light of the same colour and with similar intensities) in a significant manner. This means that it might be trickier to distinguish (or filter one spectrum out using an optical filter and clever mathematics) two emission signals from one another.
Optical stability •
A phenomenon called photobleaching, a chemical reaction between the fluorophore and its immediate environment, occurs in most organic molecule fluorophore materials. Over time the fluorophore will degrade, until it loses its ability to fluoresce. Quantum dots are inorganic and therefore much less susceptible to this photobleaching effect; a great advantage for in vivo diagnostics.
Excitation •
In order to emit light, QD dots need to absorb an incident (or excitation) light. The absorbance spectrum of QDs is broad, increasing towards lower wavelengths (the blue hues), and is very similar for all quantum dots. What this means is, first, there is a large choice of incident lights in the lower part of the spectrum that can be used as they will have sufficient energy to excite a QD dot. Secondly, as all QDs have similar absorption spectra (they overlap in the lower end of their absorption spectra with intensities that are not too dissimilar), one excitation light can be used to excite multiple quantum dots (often chosen in the UV range). In contrast, organic dyes have narrow emission spectra, which leaves little choice to which wavelength should be used (i.e. on a simplistic scale, only one type of incident light will trigger a light emission from a particular organic dye). Also, the absorption spectra of organic dyes generally do not overlap enough so one excited be used to excite several simultaneously.
Figure 3: Using QD to improve disgnostics techniques
There are several advantages to multiplexing in assays. In complicated medical conditions an accurate diagnosis may not be possible based on detection of a single analyte only. Multiplexing may unlock the development of rapid and low-cost diagnostic platforms. This is particularly useful for point-of-care devices. In a same sample, it is in theory possible to detect simultaneously multiple analytes labelled with QD of different sizes using a single excitation light source. An example of this profiling approach was used to validate a diagnostic barcode technology (see Figure 3) in which multiple regions of the Hepatitis B virus genome (different parts of the genome that have different physical characteristics) were targeted simultaneously, improving the assay sensitivity significantly from 54.9–66.7% to 80.4–90.5%. By encapsulating QDs of various sizes (varies the emitted wavelength) and quantities (varies the intensity of the emitted light) in a single label, it is possible to produce many more distinctive labels (tens of thousands) than by using single QD labels only (these will only emit one particular color rather than a combination of colours).
Other examples of the benefits of using QDs include DNA sequencing and cancer diagnostics and treatment (see text box: QD in the surgeon’s toolbox).
QD in the surgeon’s toolboxImagine for a moment that you’re a surgeon about to operate on a patient’s breast tumour. Ideally, you’d like to be able to accurately identify the cancerous tissue ‘in real-time’, that is, during the procedure, but current methods don’t quite allow that.
Firstly, you need to inject a fluorophore, for example, an organic dye such as methylene blue, into the target area in order to light up the cancerous cells. Here, we’re relying on a property of the fluorophore to penetrate cancerous tissue more deeply than healthy tissue. Unfortunately, the dyes rapidly migrate to the surrounding tissue. This means all you have to drive your steady hand, is an image taken right after the injection. To be on the safe side, you will need to cut a larger margin around the malignant area, which you’ll examine afterwards to make sure it’s clear of any cancer cells (margins can be as big as 2mm).
QDs migrate slowly and can fluoresce for many hours; allowing for a more accurate and prolonged delineation of the tumour. Also, QDs can be tuned to emit light at a desired wavelength to see through body tissue, for example using infra-red wavelength to see through fat tissue.
…Now you don’t?
QDs show great promise for diagnostics applications. The scientific literature has prolific examples of the benefits of QDs in diagnostics. The obvious question is: why don’t we see more diagnostic or more general medical applications using QDs? One reason might be that, as with any new technology, there may be some reluctance to use it for applications where organic dyes are well established and well-characterised. Organic dyes are also comparatively cheap. Other possible hurdles include:
Little things mean a lot
So, what now for QD-based technology? The answer is, despite the great progress being made, there is still some way to go before commercial QD technology is ubiquitous. However, there are encouraging examples, such as life sciences company Thermofisher’s Qdot® product (www.thermofisher.com). By coupling the Qdot® nanocrystals with other substances, such as specific proteins and small molecules, the ability of the QD to bind to the desired targets is significantly improved. Together, with the unique performance of QDs, a class of ‘super-biomarkers’ are created which are able to outperform conventional dye-based biomarker technology.
A lot of research continues in quantum dot-based biosensors for rapid point-of-care detection (and monitoring) of a wide variety of clinical conditions, without the need to resort to laboratory-based testing. One great example is connected real-time diagnostics company, Ellume’s (www.ellumehealth.com) novel class of biomarker based on hundreds of QDs which is used as an ultra-bright fluorescent tag in their GP surgery and home-based products to assist in the diagnosis of influenza, chlamydia and other conditions. When bound with detector antibodies, substantial improvements in performance are delivered, when compared to existing detectors (Ellume website).
These examples of how QD technology are finding a new level in diagnostics – albeit in small steps – are encouraging. The development of QDs is, to quote the editor of leading nanotechnology website nanowerk.com, Michael Berger: “not based on a few big and bold discoveries or inventions. Rather it is a painstakingly slow journey of gradual development; at result of which will be some truly revolutionary products and applications”. Whenever it happens, there is great potential for the tiny QD to make a huge difference in medical diagnostics.
References
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Ute Resch-Genger, Markus Grabolle, Sara Cavaliere-Jaricot, Roland Nitschke, Thomas Nann. “Quantum dots versus organic dyes as fluorescent labels”. Nature Methods. September 2008. Nature.
Imad Naasani. “The Cancer Surgeon’s Latest Tool: Quantum Dots”. IEEE Spectrum. September 2016. IEEE.
Min Fang, Chun-Wei Peng, Dai-Wen Pang, Yan Li. “Quantum dots for cancer research: current satus, remaining issues, and future perspectives. Cancer Biology and Medicine. September 2012. Springer.
Pisanic TR 2nd, Zhang Y, Wang TH. “Quantum dots in diagnostics and detection: principles and paradigms”. Analyst. June 2014. Royal Society of Chemistry.
Jisung Kim, Mia Biondi, Jordan J. Feld, Warren C. W. Chan. “Clinical validation of quantum dot barcode diagnostic technology”. ACS Nano. April 2016. American Chemical Soviety.
