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

Periodic Reporting for period 1 - DNA-NANO-AB (“DNA-based NANOdevices for the point-of-care electrochemical detection of AntiBodies”)

Reporting period: 2020-03-01 to 2022-02-28

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). In response to that, the electrochemical platform we proposed may prove to be well positioned for adaptation to point-of-care diagnostics by allowing a quantitative detection of multiple specific antibodies.
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:
i) Rational design and study of different DNA-based strategies (conformational change, proximity-based approach, etc.) to improve antibodies measurement precision.

ii) Development, exploration and optimization of versatile electrochemical platforms for an orthogonal multiplex antibodies measurement and signal amplification.

iii) Adaptation of the developed platforms onto disposable electrode surface.

iv) Application of the developed electrochemical platforms for the analysis of representative real samples.
During this reporting period, we have worked on the optimization and development of the electrochemical DNA-based platform for clinically-relevant antibodies detection. The system takes the advantages of the unique properties of nucleic acid strands and of the positive feature of electrochemical detection that include interference-free measurements in complex matrices, low-cost instrumentation and mass-producible sensors.
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 general idea has been to exploit a well-known DNA-based reaction named “toehold mediated strand displacement reaction (TMSD)” and to induce its activation exclusively in the presence of the target antibody. Specifically, the system is composed by a DNA capture probe immobilized onto the surface of a disposable electrode, a redox-labeled duplex complex and a pair of antigen-conjugated strand designed to co-localize and form the input strand for the TMSD exclusively upon the binding of the specific antibody to its antigens. In the presence of the antibody the redox-labeled strand is released from the duplex complex so producing a decrease of the electrochemical measurable signal due to its moving far from the electrode surface.
We firstly designed our platform by immobilizing the DNA duplex onto the surface of the electrode but we observed a signal change of 40% between the presence and absence of the target antibody, and so we decided to slightly modified the system design performing. To do so, we performed the reaction between the antibody-responsive circuit and the sample in an Eppendorf tube and then transferred the solution to the disposable electrode surface where a DNA capture strand has been previously immobilized. The platform is sensitive (low nanomolar detection limit), specific (no signal is observed in the presence of non-targeted antibodies), selective (the platform can be employed in complex media, including 90% serum) and versatile. In support to this, we have demonstrated the detection at clinically relevant concentrations of five different antibodies, including three of which are clinically important. Moreover, we have been exploiting the features of the new emerging research field of cell-free diagnostic platform with the programmability of antibody-responsive DNA-based device for the detection of specific antibodies in blood serum. We have already developed an optically-based cell-free biosensor and we are currently working on its adaptation to an electrochemical platform.
The results of the project have been disseminated thought publications in peer-review journal and oral presentations and lectures at international congresses and Universities.
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, we propose here to combine the positive features electrochemical biosensor with those of programmability, low-cost and biocompatibility of synthetic nucleic acid systems to develop a platform that may be of utility at the point-of-care. We have already demonstrated the development of a versatile, rapid, low-cost, sensitive and selective electrochemical platform for the multiple detection of clinically-relevant antibodies directly in blood serum sample. We 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. We are currently working on an electrochemical platform that include the use of cell-free biosensor to increase the sensitivity of the system, so opening the possible application of the platform to the detection of other diagnostic molecules present at lower level in clinical samples.