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Black phosphorous quantum dots as fluorescent nanosensing platforms for detecting unauthorised genetically modified material

Periodic Reporting for period 1 - UGMNanoSens (Black phosphorous quantum dots as fluorescent nanosensing platforms for detecting unauthorised genetically modified material)

Reporting period: 2018-05-01 to 2020-04-30

"Humans have manipulated DNA for thousands of years with the purpose of selecting and producing organisms with the most desired traits. Dogs are a good example of this “artificial breeding” that dates back many years in human history. Yet it wasn’t until the 1970s that a new era of genetic engineering was launched after scientists figured out how to “cut and paste” genes from one organism to the other in the laboratory. With it came the birth of genetically modified (GM) food: its most controversial application. These inventions, also called “biotech crops”, boomed in the 90s with the commercialisation of the first genetically modified fruit in the US, the Flvr Savr tomato, a variety modified to increase firmness and shelf life.
Biotech crops received strong public opposition when the technology came to Europe in the 90s. The EU established one of the strictest legal frameworks to regulate these genetically modified organisms (GMOs) and to ensure information transparency to the public by the correct labelling of modified ingredients. However, there is a ""zero tolerance"" policy in cases where the modified variety has not been approved in the EU, even if it has been approved in other countries outside of the EU. In other words, unapproved genetic variations are not allowed to enter the European market. Ideally, the detection of genetic material (DNA) would confirm the origin of the food product, but this is practically challenging to carry out throughout each step of the food supply chain because the technologies to detect DNA are costly and time-consuming, needing specialised staff. Strict monitoring and traceability procedures are part of the legislation, however, a series of incidents considered as ‘Unauthorised Genetically Modified (organisms) or UGM emergencies’ by the European Union Reference Laboratory for GM Food and Feed, such as the detection of unauthorised rice in UK, France and Germany, have encouraged the development of better methods for UGM monitoring to protect European consumers.
Although detecting DNA has been almost an exclusive task for the field of molecular biology, materials science has joined in this quest in recent years. Materials have been used for biomolecule separation, isolation and detection. By simplifying detection schemes with ultra-low detection levels using highly conductive materials, for example, analysts can dream of “mix-and-read” kit-like capabilities that allow for decentralised use by non-specialised staff. Graphene, the protagonist of the known 2D material family, and the subject of 2010’s Nobel Prize in physics, is one of the most commonly used materials for DNA detection. As scientists discovered that materials can have unprecedented properties from their downsizing to the atomic thin scale, resembling a sheet of paper, the field of 2D materials has been object of intense attention. They have large aspect ratio, and enhanced or unique physical properties, as well as specific and tuneable surface chemistry and thickness, which can control those properties.
This Action had as overall objective the development of simple DNA detection assays based on novel 2D materials for UGM monitoring, originally focusing on one of the newest and less explored materials based on the phosphorus element: 2D black phosphorus (BP) or phosphorene. Isolated only in 2014, this material has attracted significant attention in the scientific community because they are considered the middle point between graphene and transition metal dichalcogenides in terms of electronic properties. Turning layered sheets of this material into small layered dots of lateral nanodimensions results in changed optical properties: the dots become fluorescent under irradiation with light. By interacting with a labelled DNA strand, these BP dots (black phosphorus quantum dots, or BPQDs) were predicted to serve as “light switches” that would turn off and on upon recognition of target DNA sequences."
The Action projected that, by testing the potential of 2D nanostructures towards detecting UGM genetic material, it would be possible to: on one hand, gain new knowledge on novel materials and their interaction with DNA; and, on the other hand, work actively on an application that is highly relevant in the field of food control at the EU-level.
In general, the Action's activities focused on material-based biosensing, achieving its overall objective of studying the interaction of DNA with a novel 2D material using the “light switch” principle. The research activities initially focused on BPQDs as state-of-the-art materials. However, despite their potential as nanofluorophores for DNA detection, many problems related to their ambient instability drove the Action to take on a contingency strategy by choosing a different layered material for the same purpose. Titanium carbides (MXenes), which are also part of a novel 2D family, were tested under the “light switch” off-on principle using fluorophore-labelled DNA. In this scheme, DNA was the fluorescent element of the sensing scheme, whereas the material behaved as 2D nanoquencher, or an “off-switch” for the light emission. The interaction between these two components was investigated in-depth to be able to understand the system using a myriad of analytical techniques and simulations. MXenes were capable of reducing the emission of labelled DNA by interacting at nanoscale distances. When DNA recognised the target sequence, or the DNA of interest, it remained discreetly away from the MXene’s surface. In such difference of distance, even in very few nanometers, relies the sensing/detection principle, as these shifts can cause important changes in the intensity of the emitted light by labelled DNA. While a similar principle was observed almost a decade ago with graphene oxide, the different chemical composition of MXenes makes up a distinctive system of more dynamic nature with outstanding potential for discriminating single mutations, which was also explored during the Action.
The Action involved training activities, including technical hands-on training and soft-skill training on leadership, writing and communication, to help the fellow in reaching a position of professional maturity. The general output of the Action’s research activities were disseminated in 3 international conferences and 3 peer-reviewed publications. Two more publications are being prepared for submission.
In today’s world, DNA detection has gained imperative necessity, from detecting mutations for early diagnosis of genetic disorders and many types of cancers, to monitoring bacterial resistance and viral loads in infected patients during the recent pandemic. The research work developed during the Action represents an important contribution to the field of DNA detection and, more specifically to the newer, less explored, field of DNA-nanomaterial interfaces. The Action put emphasis on simple mix-and-read kit-like assays to transcend the conventional multi-step, laborious protocols often involved in biosensing and bioassays. These mix-and-read types of assays have point-of-care potential and possibly commercial scalability. The impact of the Action’s findings is rooted in knowledge gain that will serve as the foundation for future device/assay development.