The key idea in this ERC Proof of Concept Grant is to start from the well-known chemical amplification technology of polymerase chain reaction (PCR) – a robust and sensitive DNA quantification method which can theoretically detect up to a few sequences – and apply it to a very sensitive detection of proteins. We have achieved that by designing so-called complexes consisting of an aptamer and adaptor (both are single strand DNAs), which when mixed with a protein in some solution, the aptamer will disassociate from the complex and selectively bind to the protein. Then we amplify and quantify the DNA displaced from complexes by protein molecules with real-time Loop Mediated Isothermal Amplification. Aptamers are short single-strand sequences of DNA/RNA, and they offer many advantages compared to antibodies, including long shelf life and simple workflow, which make them particularly suitable for Point of Care (PoC) devices.
In this grant we have further developed this technology with the aim of creating a PoC label-free ultrasensitive protein sensor, with reduced complexity, time-to-result and cost of the assay in comparison with the traditional methods of protein detection and quantification such as ELISA. Within the scope of this grant, we have two main outcomes:
1. The molecular detection methodology for leptin has been improved to involve the amplification of non-displaced complexes and then finely tuned by testing a number of buffers, pH and temperature protocols.
2. The molecular biology protocols have been translated into a PoC platform design, capable of running DNA amplification protocols compatible with the DNAnudge start-up company proprietary cartridge and portable box technology.
Among the advantages of the proposed method are the low number of reagents needed and the quick preparation which does not require washes and long incubation times, making it a rapid, low cost and easy assay for protein detection. The absence of magnetic beads (which was employed in previous works) makes this method a candidate of choice for the Lab-on-Chip implementations. Furthermore, this approach would help us to overcome the complexities of current methods such as ELISA, in order to enable point-of-care detection.
The development of this novel molecular method will enable quantification of a number of protein targets and create a sensor with exceptional sensitivity and specificity, with many applications. Our initial targets have been a broad range of appetite related hormones/proteins such as leptin, ghrelin, CCK, PYY and GLP-1. Simple and accurate monitoring of these hormones are crucial for many appetite control therapies related to obesity, anorexia and similar. The experiments in the project have been mostly done on quantifying the human leptin.
The modular nature of the system allows for easy assembly and adjustment towards a variety of biomarkers for applications in disease diagnosis and personalised medicine far beyond appetite control. For example, this technique can be used for very sensitive and accurate PoC antigen testing, as well as antibody testing for a variety of conditions, provided a suitable aptamer is available and target-specific sequences are designed. Such a device which would permit detection of very low levels of antibodies is very much sought-after technology in the current COVID-19 pandemic.
