Nature-Inspired Nanoprobes for the Point-Of-Care Detection of Clinically Relevant Biomarkers for Early Tumor Diagnosis

Project location: ITALY, Rome
Project start date: January 2013 - Project end date: January 2015
Project number: 2012-079
Beneficiary: Università di Roma Tor Vergata, Dipartimento di Scienze e Tecnologie Chimiche

The Department of Chemistry of the University of Rome, Tor Vergata has a strong track record of both teaching and research and in a recent national survey resulted to be among the top-5 chemistry departments in Italy. This is reflected by the large number of peer-reviewed publications, and numerous prizes awarded to graduate students and staff. Research in the department is funded by the Minister of Education, European Commission, National Health Organization, National Research Center and other bodies, with current research funding totaling 22 million euros. The department has filed several patents, and participates in numerous international collaborative projects. There is a strong modern instrumentation base, with facilities for X-ray crystallography, mass spectrometry, molecular graphics confocal microscopy, etc. 
The present project will be carried out in the Analytical Chemistry Group, headed by Prof. G. Palleschi. The group has contributed significantly to the emerging research area of electrochemical immunosensors and biosensors based on the use of enzymes and antibodies becoming one of the reference point in this field. The group has strong links with other teams in the department, and collaborates with experts in electrochemistry, enzymes and biosensors all over the world. Among the others, the group collaborates with; Prof. A. Amine (Morocco), Prof. L. Gorton (Sweden), Prof. A.A. Karyakin (Russia), Prof. G. Evtugyn (Kazan), Prof. M. Mascini (Italy), Prof. C. Brett (Portugal), Prof. C. Elliott (Ireland) etc. The experience of the group is also recognized by the large number of publications produced by the group in the area of sensors in international peer-reviewed journals during the last  5 years (more than 60). The group has coordinated 4 EU projects (FAIR n. CT96 PL1092; INCO n. IC15CT980906; QLK1-CT-2001-01617; INTAS 00-273) and has been involved as partner in other 3 EU projects (FAIR n. CT96 PL1095; IC CT9800119; EVK4-CT-2000-00028). These projects are all based on the use and development of sensors and biosensors (see table 5). The group was also involved in a EU concerted action on “Biosensors for Monitoring the Environment” (n.QLK3 2000-01311). Because of all these activities the laboratory of Analytical Chemistry is now a reference point of many PhD. students and Post-Doc researchers, coming from all over the world. The group is also responsible of four National Projects and has strong collaborations with the major research national centers in Italy as CNR, ENEA, ISS, INRAN, and contracts with major industries as Parmalat, Menarini, Eurolab, Aerosekur etc. 
The group of the Analytical Chemistry has acquired great experience and expertise in the field of clinical analysis, point-of-care testing and DNA based switching sensors. This latter activity has been mostly carried out by Dr. Ricci in collaboration with the lab of Prof- Plaxco of the University of California, Santa Barbara. Dr. Ricci is a senior researcher at the group of Prof. Palleschi and will be responsible for the development of the proposed sensors. 
With over 10 million new cases per year cancer is among the most threatening diseases in the world and despite the impressive progress in clinical medicine severe difficulties remain in treating it. A key factor play an important role in these difficulties: most of the current diagnostic technologies and tumor imaging methods are not adequate for early detection of cancer. 
Cancer diagnostic: the heterogeneity and complexity of many tumors make their diagnosis difficult. Usually cancer is diagnosed through the detection of specific markers (proteins, antibodies, cells, RNA, DNA, etc) in body fluids (serum, hurine, blood etc) and by imaging of body, tissues or cells. Both these steps have their own drawbacks. Current diagnostic tests for the detection of biomarkers (ELISA, western blot etc), despite their great attributes,  remain cumbersome, laboratory-bound technologies based on cost-intensive multi-step processes that typically require hours or days to return an answer to the physician’s hands. These response times are poor compared to the needs of modern healthcare, reducing patient compliance and hindering the efficiency and, in some circumstances, the safety with which many drugs and medical procedures are administered. In response to the limitations of current detection schemes, significant effort has gone into the development of quantitative, single-step approaches in which the binding of a target marker to a recognition element leads to quantifiable changes in adsorbed mass (quartz crystal microbalances), charge (field-effect transistors), steric bulk (microcantilevers) or optical properties (surface-plasmon resonance). Unfortunately, however, while many of these approaches exhibit exceptional sensitivity and convenience, they are also prone to an unacceptable level of false positives arising from the non-specific adsorption of other proteins when deployed in complex clinical samples, such as blood serum and they often require highly trained personnel and specialized laboratories. 
For these reasons, in order to ensure rapid appropriate care for patients simple, rapid, inexpensive, and quantitative tools for the detection of specific cancer markers in clinical samples are still urgently needed.  Tumor imaging technologies also suffer of important limitations. Molecular imaging usually provides a picture of what is happening inside the body at the molecular and cellular level and as such represent a key tool for cancer fight. Usually imaging is achieved through the use of an labelling agent (or probe) that signal the presence of the tumor cell or of other biological processes. The major objective in imaging technology is to decrease the signal-to-noise ratio in order to detect and/or image the smallest possible number of tumor cells, ideally before the angiogenic switch. The present detection threshold for solid tumors is approximately 109 cells (1 g = 1 cm3) growing as a single mass, an amount that is orders of magnitude faraway from that needed to achieve early diagnosis. This is due to some inherent limitations such as the relatively low concentration of cancer marker per cell (usually about 103- 104 molecules per cell, corresponding to a cellular concentration of only 1.7-17 nM) and the use of “always-on” imaging agents that increase background noise and hamper imaging sensitivity and resolution. The possibility to use “activatable” probes that reduce background noise and that could reach better sensitivies, therefore, would be a very important step towards the early diagnosis of cancer. 
Overall goal of this proposal
The Department proposes here to study the fundamentals of nature-inspired mechanisms and use this knowledge to engineer self-regulated theranostic nanomachines capable of providing diagnostic information or delivering targeted therapy. 
Nature inspired nanodevices
Nature uses nanometer-scale, protein and nucleic-acid-based “switches” to sense chemical cues and transduce molecular binding events into specific, high-gain signal outputs. Examples of these switches are calmodulin proteins, which regulate cellular processes via a calcium-triggered conformational change, cytokine receptors, which signal through the cell membrane via a hormone-induced conformational change and riboswitches, which regulate translation via a metabolite-induced conformational change in the mRNA leader sequence. These biomolecular switches shift between two or more conformations in response to the binding of a specific target ligand and this leads to very specific and sensitive output signals.
Reasons for nature uses such switches
-Specific: Structure switching is induced by a specific ligand–biomolecule interaction and thus is largely insensitive to the presence of other molecules present in highly complex environments.
-Rapid and reversible: the switching mechanism is a thermodynamic equilibrium which is rapid and reversible and is thus suitable for real-time monitoring. 
-Versatile and easily tunable: the conformational equilibrium of biomolecular switches depends on target concentration and the switch's thermodynamics. This provides a means by which the dynamic ranges can be rationally optimized by simply tuning the switch equilibrium. 
Inspired by this observation, recent years have seen the development of artificial receptors that similarly employ binding-induced structural changes to couple the presence of a molecular target to a specific optical, electronic or catalytic output. The advantages of switch-based biosensors are numerous. Unlike traditional molecular detection methods, including both immunochemical and chromatographic approaches, switch-based probes require neither processing steps nor exogenous reagents. Likewise, because structure switching is largely immune to the non-specific binding of contaminants, switch-based sensors generally perform well in complex, multi-component samples. To date, however, only a limited number of biomolecules have been identified that, upon target binding, undergo a structural change large enough (> 5 nm) to generate strong output signals. The re-engineering of existing biomolecules so that they can undergo a large-scale conformational change upon target binding has also seen many efforts but this strategy is not convenient in terms of cost and time required for optimization8. These drawbacks have limited the versatility of this approach and until now only few and isolated example exist mostly for the detection DNA targets and whose utility is thus limited to certain applications.
Inspired by nature, which employs nanometer-scale protein and nucleic-acid-based “switches” as devices to detect the inputs from thousands of distinct molecules, including disease markers, in a complex physiological environment, the Department proposes here to exploit the “designability” of nucleic acids to design and develop molecular nanodevices that undergo binding-induced conformational changes (switches) upon target binding and, in doing so, can signal the presence of a cancer marker.
The research team will develop “nature-inspired” nucleic acid based nanoswitches that signal the presence of specific tumor markers by undergoing a binding induced conformational change. Nature uses biomolecular switches to sense specific targets in complex surroundings (such as inside cells). The population-shift model desribes these switches as in equilibrium between a non-binding “off” state and a binding-competent “on” state. This equilibrium is pushed toward the latter by target binding. Inspired by this natural occurring mechanism the research team proposes here to develop a class of nucleic acid based nanoswitches that are designed and engineered so that they are in a thermodynamic equilibrium between two different conformations, a signalling binding-conformation and a non-signaling non-binding conformation. The presence of the target pushes this equilibrium towards the signalling conformation thus providing a measurable outputs. Because the signal is observed only upon target binding the approach will be specific, convenient, and, most critically, selective enough to be employed directly in clinical complex samples (i.e. serum, blood, living cells).  Due to the heterogeneity of the tumor markers this approach will be demonstrated with different classes of targets ranging from Transcription Factors (TF), RNA and cell receptors (epitopes expressed on the surface of tumor cells) or other cellular markers of biological processes such as angiogenic and apoptotic pathways (markers of multiple cancers). For each of these targets the team will design a range of nanoswitches based on different nucleic acid secondary structures  (pseudoknots, triplex, G-quadruplex, etc.) and different switching mechanisms (“sliding switch”, stem-loop, etc).  The versatility and designability of nucleic acids allows to optimize the thermodynamic of the switch to achieve the best analytical performances. Also, it is quite easy to attach different recognition elements (antigens, peptides, etc) to the DNA backbone and use the  probe as a versatile scaffold.  The thermodynamic optimization of the switch will be initially performed in solution with optical reporters (such as fluorophore/quencher pairs) in such a way that upon target binding, the conformational switch will change the distance between the two reporters. The solution-phase nanodevices will be applied to tumor-imaging in cells and tissues. The research team will also develop a Point-of-care diagnostic platform by adapting the above mentioned strategies to electrochemical measurement, a technique far more suitable for low-cost measurement, mass-producible test strips and self-testing with clinical samples such as serum or blood.
In this project the research team will follow a bottom-up/simple-to-complex trajectory (see scheme below). They will first develop nanoswitches for cancer markers starting from proof-of-principle demonstrations with optical fluorescent approaches. This first phase will allow to study the thermodynamic principles of the switches and to set a quantitative approach in order to simulate and optimize the switching thermodynamics (Task 1.1). To this end the etam will also apply computer algorithms (such as mfold). The team intends to study the effect that chemical modifications (labels and antigens) could have on the predicted switching thermodynamic. The team will also study alternative structures and switching mechanisms. Of note, they will start using small antigens easily attachable to DNA sequences and then will move to more difficult recognition elements such as small peptides (Task 1.2).
The team proposes to adapt these switches to electrochemical outputs and multi-array detection, which are more suitable for clinical point-of-care applications (Task 1.3). The team will also test the use of different nanomaterials (graphene, carbon nanotubes, gold nanoparticles) to improve the performance levels of the electrochemical devices. During this phase of the project,  the team also proposes to apply the optimized optical nanoswitches for in-vivo monitoring of tumor markers as a novel tool for imaging diagnostics. The electrochemical devices will instead be applied in the last phase of the project for point-of-care diagnostic analysis of cancer biomarkers and a direct comparison with current methods will be carried out. 
The Nando Peretti Foundation has awarded a grant to this project, for the following target goals:
Impact and added value: The current and ongoing need for better diagnostic tools in clinical analysis makes the proposed research extremely timely. The current state-of-the-art approaches for proteins detection present several disadvantages as detailed in the previous sections. The development of the proposed nanoswitches could significantly impact the safety, compliance and efficacy of therapies and medical procedures and the research team believes that this project will give many benefits from scientific, technological and socio-economic points of view. The research is highly multidisciplinary and will impact several scientific fields in the areas of Physical Sciences (PE) and Life Sciences (LS):
-Analytical Chemistry and Sensors (PE4): the idea of switches that employ binding-induced conformational changes to sense their environment is innovative and, although highly promising, few groups are involved in this research. It will be important for EU excellence to have a strong research project in this area. Implementation of naturally inspired strategies in manmade technologies should likely enhance the performance of many biosensing platforms thus greatly impacting the field of Analytical Chemistry.
-Physical Chemistry of biological systems: the study of DNA switching mechanism and its thermodynamic characterization will be an important step for computational physical chemistry as it will uncover novel DNA/DNA and protein/DNA interactions and switching mechanisms.
-Surface science and nanostructures: the study of novel nanomaterials and different SAMs will have a great impact in the field of surface science and nanostructures as it will give new insights on the behaviour of biomolecular (especially DNA) layers on nanoelectrode materials. 
-Thermodynamics: the study of the thermodynamic of the switching mechanism of DNA secondary structures could uncover important information for this field. 
-Biomaterials synthesis: the study and optimization of novel synthesis protocols to modify DNA probes with different recognition elements (small molecules, peptides etc.) or redox labels will be an important added value for the field of biomaterials synthesis.  
-Synthetic biology and bioengineering: this project will be one of the first examples where a “synthetic biology” approach is applied for a diagnostic goal. This is likely to attract a strong interest from the international scientific community as “synthetic biology” is one of the hottest fields at the moment. 
New Research horizons: In addition to sensing and diagnostic, many other applications can be envisioned for the proposed nanoswitches and the know-how acquired during this project will be important for other adjacent research fields (especially Life Science and Biotechnology). 
-Nanomachines: the successful demonstration of nanoswitches will inspire numerous applications in nanotechnologies, such as the design of new nanomachines. For example, the opening of the nanoswitch by an antibody could be used for targeted drug delivery. This would significantly advance our ability to fight cancers and other important diseases.
-Proteomics: the nanoswitches developed during this project and the technology proposed will improve the possibilities in the “functional proteomic” field and will provide new tools that will respond to endogenous target  directly under complex in-vivo conditions supporting the detection of protein functions.
-Drug screening: the evaluation of the effect that drugs have on antibodies and proteins function directly in biologically relevant samples and/or in-vivo conditions is a crucial information for drug screening and this will impact this important area of research. 
-Bioengineering and system biology: the use of naturally occurring strategies to edit (tune) the dynamic range of DNA and RNA based interactions will make it possible to transmit information to and from living systems and obtain control within cells themselves. This could give the capability to program living systems thus also providing access to otherwise inaccessible information on cellular state18.
-Human’s Cancer imaging: the research will demonstrate the use of novel optical probes in cells and tissues that are activatable by the binding of specific receptors. The team expects that the results of this proposal will open new horizons in fields focused on the diagnosis of cancers directly in patients (diagnostic imaging). 
think global, act local
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