Innovative biosensing methods for nucleic acids detection in point-of-care devices

The analysis of nucleic acids (NA) introduced a disruptive innovation in healthcare, opening towards high throughput diagnosis and personalized therapies in many fields of medicine including infectious diseases, oncology, pharmacogenomics, genetic diseases, diabetes, forensic, and neurological, cardiovascular diseases. Conventional methods for NA analysis rely on the Real-Time PCR (Polymerase Chain Reaction) reaction, that represent the gold standard for NA analysis offering a high level of specificity and sensibility. The Real-Time PCR works through the cyclic amplification of a specific target sequence through a couple of small complementary oligoribonucleotides (primers), a thermostable DNA Polymerase enzyme and a fluorescent probe, able to label the target sequence during its amplification1,2.Thanks to the amplification, the number of copies of the NA target is exponentially increased becoming detectable and quantifiable. Figure 1 reports a typical Real-Time PCR mechanism using specific fluorescent probes for DNA detection.Within this approach PCR-based methods are widely diffusing in a lot of biomedical fields but their massive use is strongly limited by a series of drawbacks, such as the constrain to specialized laboratories, instruments and staff and the need of long and expensive procedures to perform both the DNA preparation and detection. To overpass these limitations, a lot of alternative solutions have been introduced so far mostly based on the biosensing approach. Biosensors are analytical systems relying on the synergy between a sensing element (such as enzymes, antibodies, nucleic acids, aptamers, phages, cells etc.), that recognizes and interacts with specific targets producing an input biochemical signal, and a transducer element, that converts the input into an output (electrical, optical, acoustic, etc.) signal used to detect and quantify the targets concentrations4–6. Depending on the type of sensing element, biosensors can be very versatile and suitable for a lot of analytical applications,as schemed in Figure 2.The interest for biosensors increased significantly over the last fifty years. A Scopus study, indeed, revealed that from 1972 to 2014 the number of publications on biosensors showed an exponential growth5,7. This growing attraction was mainly due to the possibility of biosensors to open towards simple, rapid, low-cost and portable analytical solutions that can really improve the quality of life5,8,9. This is the case of the so called genetic Point-of-Care (PoC), consisting in innovative biosensing technologies that empathize the decentralization and simplification of NA analysis. This technologies, indeed, provide a patient-centred diagnostics that can be performed quickly, easily and directly at the patient bed, in the physician office or at home10,11. The importance of PoC varies depending on the context. As an example, in intensive care units, PoCs are used to immediately perform a diagnosis and aid in life-saving decisions. At low resources settings, PoCs offer easy-to-use procedures that can be performed regardless of the presence of a laboratory and specialized personell12. In primary care settings, PoCs are typically used to prevent unnecessary specialist cares, to guide treatment decisions and to quickly provide reassurance to patients, e.g. excluding a severe disease. Moreover, rapid testing can lead to improved clinical performance as it eliminates potentially long intervals between initial patient examination and discussion of test results13. Objective of my Ph.D. thesis was to develop innovative biosensing methods for the detection of nucleic acids and other targets of biomedical interest suitable for PoC applications. The research has been mainly focused on the molecular biosensing of the nucleic acid kinetoplast (k)DNA of the protozoan Leishmania infantum (L. infantum), the double-stranded (ds)DNA genome of the bacterium Pseudomonas aeruginosa (P. aeruginosa) and the single-stranded (ss)RNA genome of the Sars-CoV-2 virus, which are representative of pathogens causing severe infectious disease on human health. As a side activity, I have worked on the development of biosensing methods for the detection of other two biomolecules of diagnostic interest, i.e. microRNA (miRNA) and anti-Aβ peptide antibodies as important biomarkers of the Alzheimer’s disease onset. The reported research activities will be described in the following chapters of my Ph.D. thesis: 1. State-of-the-art of biosensors for biomedical applications. 2. PCR-free biosensing method for the detection of infectious pathogens nucleic acids in a PoC format. 3. Biosensing methods for the detection of Alzheimer’s disease associated biomarkers.