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“DNA-based NANOdevices for the point-of-care electrochemical detection of AntiBodies”

Periodic Reporting for period 2 - DNA-NANO-AB (“DNA-based NANOdevices for the point-of-care electrochemical detection of AntiBodies”)

Reporting period: 2022-03-01 to 2023-02-28

The present fellowship proposes the development and characterization of electrochemical platforms to monitor the quantitative detection of clinically-relevant antibodies, that will be rapid, specific, convenient and selective enough to be employed directly in complex samples, such as blood serum and whole blood.
The detection of specific antibodies and other diagnostic proteins plays a crucial role in the diagnosis of many diseases, infections and pathologies and despite their widespread use current detection methods are either cumbersome, multi-step and laboratory-bound processes (i.e. ELISA, western blot) or only qualitative or semi-quantitative (i.e. later flow immunoassay). Due to the above considerations, better analytical tools that allow the rapid, inexpensive, and quantitative measurement of clinically-relevant biomarkers, including antibodies, are urgently needed. To address this need, during my Marie-Skłodowska Curie project (“DNA-NANO-AB”), I have developed different DNA-based sensors and devices for the detection of a wide range of molecules, including antibodies, that may be of utility for diagnostic applications.

To do so, I employ the designability and versatility of synthetic DNA sequences those can be used as scaffolds to create antibody-responsive nanodevices and I take advantage of the positive feature of electrochemical detection that include interference-free measurements in complex matrices, low-cost instrumentation and mass-producible sensors.

To reach the proposed objectives I will undertake the following tasks:
1. Rational design and characterization of different DNA-based strategies (conformational change, proximity-based approach, etc.) to improve target measurement efficiency using spectroscopic and electrochemical-based approaches. Different DNA-based elements will be tested as well as several recognition element/antibody couples.
2. Development and optimization of versatile electrochemical platforms for the orthogonal and multiplexed antibodies detection. Systematic study of: i) redox reporters, ii) antigen tags, iii) electrochemical parameters, iv) signal amplification.
3. Development of platforms using disposable screen-printed electrodes produced in house to reduce the fabrication cost of each sensor.
4. Test of representative real samples using the developed electrochemical platform. The platforms will be firstly challenged with blood serum and ultimately with whole blood.

In conclusion “DNA-NANO-AB” has paved the basis for new classes of DNA-based sensors for antibody detection. Specifically, the possibility of coupling the advantages of synthetic nucleic acids with the positive feature of electrochemical detection and the possible amplification processes to enhance the sensibility of the sensors make the sensing platforms developed during this project well suited for point-of-care applications.
During “DNA-NANO-AB” project I have worked on the optimization and development of different classes of DNA-based sensors for clinically-relevant antibodies detection. The systems take the advantages of the unique properties of nucleic acid strands, the positive feature of electrochemical detection that include interference-free measurements in complex matrices, low-cost instrumentation and mass-producible sensors and the possibility to introduce amplification processes to increase the sensitivity of the platforms.
Synthetic DNA sequences were used not as recognition elements for the target antibody but only as a scaffold to attach and conjugate the antibody recognition elements (antigens), redox reporter molecules (for example methylene blue) and molecules that allow attachment to the electrode surface.
The initial general idea has been to engineer DNA-based circuits that exploit co-localization mechanism and a well-known DNA-based reaction named “toehold mediated strand displacement reaction (TMSD)”. The sensing principle of the platforms is based on the activation of the strand displacement reaction exclusively in the presence of the target antibody. This in turn would provide a measurable signal (i.e. optical, electrochemical, etc.) or trigger the in-vitro transcription of a functional RNA output strand.
To reach these objectives, I took advantage of the world-wide recognized expertise in the field of electrochemical sensors of Prof. Plaxco’s group (Outgoing phase supervisor, University of California, Santa Barbara UCSB) with that of DNA Nanotechnology and analytical chemistry led by Prof. Ricci’s group (Return phase supervisor, University of Rome Tor Vergata UTV).

During the timeframe of the project, I have disseminated the main results of my research activity thought:
- publications in peer-review journals;
- oral presentation at national and international congresses;
- scientific workshops;
- invited lectures in international contests as Marie-Skłodowska Curie Ambassador.
- several other outcomes of the research activity started during “DNA-NANO-AB” project will lead to scientific publications in the next year.
The COVID19 pandemic has highlighted the crucial role that diagnostic tests can play in the detection, monitoring, and containment of infectious diseases. Different biomarkers can be used for such monitoring, but antibodies are among the most important as their detection reports on current and past infection, can inform on clinical outcomes, are important in the treatment and monitoring of autoimmune diseases and cancer and are increasingly employed as therapeutic agents, in therapeutic drug monitoring. For all the above reasons, diagnostic tests that are easy to perform, convenient, reliable, and suitable for use at the point of care are crucially needed in the detection, monitoring, and containment of infectious diseases and other clinical emergencies. In this scenario, “DNA-NANO-AB” has proposed to combine the positive features optical, electrochemical and cell-free biosensors with those of programmability, low-cost and biocompatibility of synthetic nucleic acid systems to develop platforms that may be of utility at the point-of-care. I have already demonstrated the development of versatile, rapid, low-cost, sensitive and selective platforms for the multiple detection of clinically-relevant antibodies directly in blood serum sample. I have also explored the emergent research field of cell-free biosensors by coupling it with our antibody-responsive DNA-based nanodevices achieving detection of specific antibodies. I am also currently working on other classes of cell-free biosensors that will provide an even higher sensitivity for target detection, with the aim of using these systems for the detection of other diagnostic molecules present at lower level in clinical samples.