Imaging techniques for mapping diseases in human cells or tissue often depend on specific biomarkers. However, the number of biomarkers that can be seen at any one time is currently restricted by a limited colour range for fluorescent dyes. Now EU-funded scientists under the multidisciplinary Immuno-NanoDecoder project have developed a molecular nanodevice that can illuminate a larger number of biomarkers in rapid sequence, with the potential to improve diagnosis and characterisation of diseases. “We need a tool to see more proteins without radically changing fluorescence imaging tools,” explains project coordinator Dr Matteo Castronovo, a lecturer in biochemistry in the School of Food Science and Nutrition at the University of Leeds, England. “There are usually four to six colours that can be seen at the same time. This is not a great level of multiplexing. In many cases, watching for fewer than four units or individual biomarkers is not sufficient to gain a proper idea of the [underlying] biological process.” With immunofluorescence imaging, protein biomarkers are stained by using antibodies – natural molecules with a high affinity to a specific protein. The project team developed individual nanodevices called ‘nanoencoders’. These are coupled to antibodies and recognise a specific partner nanodevice, called a ‘nanodecoder’, that carries a particular dye. The coupling allows the nanodecoder to detect specific biomarker presence and its distribution in cells and tissues by making them light up. “Then, once we’ve taken an image using optical fluorescence microscopy, we can dissociate the decoder from its encoder and introduce another decoder into the solution which will stain for another encoder, in a cycled imaging approach. You still have the same colour but you know it’s showing something different,” says Dr Castronovo. In that way, the limited colour range can be substantially extended in its use. Reversible system “The technology, despite being a prototype, is reliable, and it is also reversible so we can actually turn the antibody on and off,” he explains. In other existing methods of multiple biomarker staining using modified antibodies, the antibodies become chemically destroyed in the process. “So, at each imaging cycle, they become dark and you have to stain again. Our great advantage is that the illumination can be switched on and off so we can still come back to the first protein,” says Dr Castronovo. Many innovative imaging technologies have been developed in recent years, but they require new or updated instrumentation, which can be a burden on health and laboratory budgets. But this technology uses existing optical microscopes. “It’s just a molecular trick, to perform in a different way,” Dr Castronovo says. Cross-disciplinary approach The huge challenge of the project, which also received support from the Marie Skłodowska-Curie programme, was its cross-disciplinary approach. “We trained biologists to work in nanoscience and nanoscientists in the field of molecular biology. We trained computational scientists to work in the lab and we allowed experimentalists to understand how computational scientists can impact their work,” Dr Castronovo says. “We have unfolded the potential of the technology by studying the fundamental biochemistry of these molecular objects; and we have developed the technology – how to switch the fluorescence on and off and the minimal structure – we cannot make it smaller. Now we have to optimise imaging conditions and test the technology against different biological samples,” Dr Castronovo concludes.
Immuno-NanoDecoder, nanodevice, imaging, biomarkers, immunofluorescence, fluorescence, cells, tissue, antibodies, protein