Deliverables
Particles identified in WP2 to have a high dissipative capacity will be tested for the detection of spiked synthetic oligos carrying the mutations of interest in serum in combination with the optimized protocol (Task 3.3). The non-specific binding of the particles to the device surface will be studied and eliminated if present; initial results obtained by P1 showed zero liposomes’ binding on a neutravidin-surface modified with DNAs. Finally, micro-devices optimized in WP1 with respect to their frequency will be used in these studies to select the most sensitive combination that will give the anticipated 103 (aM) to 106-fold (zM) signal enhancement.
Report on the optimum protocol for ctDNA isolation and enrichment): Outcomes from the previous tasks will be combined and two alternative options will be explored. In the first and simplest scenario, captured ctDNA on beads will be directly mixed with the 2 pairs of oligos and the ligase chain reaction will be performed in situ, while the second scenario involves release of captured ctDNAs from the beads and subsequent mixing with the LCR cocktail. In both cases, contaminants are removed, serum is exchanged to buffer and ctDNA targets are specifically captured and enriched at least to the aM range. The protocol will be developed using synthetic targets: a) in buffer, b) spiked-in serum.
DNA-coupled vesicles: synthesis protocols; binding information & acoustic dataVesicles prepared within tasks 2.2 and 2.3 will be tested and characterized for their dissipation capability during binding to surface immobilized DNA using TSBAR devices.
Report on publications, staff exchange, workshops, IPR and technology promotional events M24Results of the project will be made available to the research community via presentations (Conferences, Universities, workshops etc.) and publications of open-access articles in peer-reviewed journals and conference proceedings. Advertisement of CATCH-U-DNA to the general audience will be done through regular publication of articles to journals accessible to the general public. Staff exchange between the project partners will take place on a regular basis. This will be done in order that all scientists, engineers and other staff become aware of each others expertise and of the scientific, technological and market needs related to the project and cancer diagnosis. On a yearly basis, results with potential intellectual property rights (IPR) will be evaluated. The priorities for disseminating activities will be discussed and defined during the first year of the project. Part of our exploitation strategy will be to target potential end-users via publications, presentations and press reports. Major international events such as the Conference on Molecular Diagnostics, microTAS etc. will be attended and technology transfer events will be targeted for further exploitation of the results.
Report on oligos’ design and evaluationSynthetic DNA oligos varying in sequence and size mimicking ctDNAs will be designed by FORTH and UOC. In addition, the two partners will design a series of different oligos to be used as surface probes and particle-bound DNA probes. Optimized surface hybridization conditions (task 4.2) will be used to select the optimum length and sequence composition of each oligo.
Report on optimized surface hybridization conditionsFor the two binding events taking place on the sensor surface (target capturing by the surface-probe and target/ DNA-particle hybridization), the optimum temperature and buffer composition (salt, ionic strength, denaturing agents) will be investigated in order to achieve maximum selectivity and specificity using the oligos designed during the previous task
Optimized protocol for ctDNA selective capturing on magnetic beads in serumTo tackle the selectivity and specificity challenge, we will employ a technology recently developed by Curie. We have already demonstrated that the use of a microfluidic fluidized bed allows a higher efficiency of DNA capture (from serum) compared to conventional DNA extraction columns. Here we will first functionalize commercial magnetic beads with capture oligos that will permit by hybridization the specific capture ctDNA sequences of interest (KRAS mutated gene will be first considered). Steptavidin beads will be used with biotinylated capture oligos. Different sequence lengths will be investigated to provide the best capture specificity and efficiency. To capture the ctDNA of interest, we will use capture oligo complementary of a non mutated region of the gene of interest, the mutated sequence targeted being subsequently enriched by a first round of ligation.
Lipid vesicles: physical samples; synthesis protocols; characterization databaseThis task will be focused on the construction of vesicles having different sizes and compositions in a controlled manner (see a-c in figure to the left). Vesicles will incorporate a DNA probe which can hybridize to a specific region of the target DNA. Assembly protocols will be based upon the broad knowledge accumulated in P2’ laboratory in over 15 years of membrane studies. Research efforts will focus on the assembly of lipid vesicles (liposomes) using standard vesicle preparation (dispersion, extrusion, hydration) and characterization (spectroscopic, microscopic, and thermodynamic) techniques, all available at P2’s lab.
Report on a ligase-based assay for the selective enrichment of ctDNA targetsIn the current task, a ligase-based assay will be investigated for the enrichment of mutant sequences in respect to the normal ones after capturing on magnetic beads. LCR is a DNA template-dependent method performed by a thermostable ligase in the presence of 2 pairs of oligonucleotides. Upon hybridization of the oligos with the template, the ligase connects two adjacent oligos only when perfect matching occurs. This procedure requires cycling of two temperatures for denaturation and ligation and results in exponential accumulation of products without introducing any uncontrolled bias and errors. During LCR protocol optimization the following parameters will be investigated: Oligonucleotides sequence composition and length, number of cycles and temperatures, use of single-base 3’ overhangs, 5’ phosphorylation of oligos, introduction of non-complementary tails and use of cycling conditions near the oligos Tm.
Non-lipid vesicles:physical samples, synthesis protocols, characterization databaseVesicular nanoparticles comprising non-lipidic or lipids/non-lipids components will provide additional avenues for construction of vesicle platforms. In particular, inclusion in the vesicle frameworks of amphiphilic conjugated polymers, dendrimers (d) and hydrophobically-coated nanoparticles, will provide means for tuning of desired properties, particularly vesicle size, shape, and rigidity of the molecular scaffolding. Specific non-lipid vesicles which will be explored include polymerized lipid/polydiacetylene vesicles comprising lipids and polydiacetylene, and vesicles built from bolaamphiphiles, containing two hydrophilic head groups linked at each end through a hydrophobic alkylchain, exhibiting high thermodynamic stability in physiological samples.
Targeted mutation analysis on the ctDNA of the 100 mutation-positive patientsConsiderable body of evidence exists on analysis of ctDNA as a circulating biomarker to guide decisions for personalized treatment.22 Recent studies have shown high concordance (94%), sensitivity (66%) and specificity (100%) between EGFR mutations from ctDNA and tumor tissue in NSCLC.23 Here, an established method for ctDNA analysis will be implemented. The Laboratory of Tumor Biology (UOC) developed technology to analyze high-quality nucleic acid biomarkers from plasma and serum, using qPCR-based methods for the detection of genetic alterations. The developed assays are based on a 5´nuclease TaqMan combined with allele-specific Protein-Nucleic Acid (PNA) probes, in some cases in combination with digital PCR.
Targeted NGS analysis on the tissue samples of the 200 patientsIn the case of solid tumours, formalin fixed paraffin embedded (FFPE) blocks are usually the only tumor material available. Although the DNA obtained from FFPE is highly fragmented and cross-linked due to fixation, it has been demonstrated to yield NGS results comparable to those of fresh frozen tissue, as long as appropriate protocols are employed.21 Based on its long experience, the Lab. of Tumor Biology at the UOC will ensure robust analysis of the DNA from FFPE blocks. The mutations identified by NGS will also be confirmed by alternative methods depending on the type of alteration to be validated. P4 is in close collaboration with the Medical Oncology Dept., Univ. Hospital of Heraklion and runs mutation tests for diagnostic purposes in more than 200 samples per year.
Validation of CATCH-U-DNA using model and clinical samplesTo develop robust assay conditions and standardize the proposed method for mutation detection, we will evaluate the lower level of sensitivity by spiking varying amounts of mixture DNA samples with different mixing ratios between the mutant and wild-type DNA templates into peripheral blood plasma from healthy donors. Partner 4 will be responsible for collecting blood samples from patients with lung or colorectal cancer and identifying particular mutations using next generation sequencing (NGS) or qPCR. Serum from the analyzed clinical samples carrying common mutations occurring in colorectal and lung cancers, such as, KRAS, EGFR, and BRAF will be kept and provided to FORTH partner for the validation of the detection method using the developed acoustic wave platform. The results will be compared to the findings of P4 using the NGS and real-time PCR.
Report on characterization of micro-sensors to biological sensing with frequencyDuring this task, microfabrication processes, advanced silicon micromachining and packaging processes will be developed. Prototypes of the array of acoustic wave micro-sensors and the measurement cell will be delivered, followed by exhaustive characterization. Using impedance and network analysers, the complex electromechanical response of the array will be measured in the frequency range of interest. Cross-sensitivity (interferences) between micro-sensor situated closely in the array will also be studied. Microsensors operation in biological sensing will be tested during the binding of a model system consisting of DNA molecules followed by liposomes; testing will be performed using P3’s already commercially available instrumentation. This testing will provide an acoustic database of dissipation measurements versus frequency for DNAs and liposomes of various features. Results will be used to decide on the most appropriate operation frequency for maximum enhancement of the dissipation response.
Report on publications, staff exchange, workshops, IPR and technology promotional events M42Results of the project will be made available to the research community via presentations (Conferences, Universities, workshops etc.) and publications of open-access articles in peer-reviewed journals and conference proceedings. Advertisement of CATCH-U-DNA to the general audience will be done through regular publication of articles to journals accessible to the general public. Staff exchange between the project partners will take place on a regular basis. This will be done in order that all scientists, engineers and other staff become aware of each others expertise and of the scientific, technological and market needs related to the project and cancer diagnosis. On a yearly basis, results with potential intellectual property rights (IPR) will be evaluated. The priorities for disseminating activities will be discussed and defined during the first year of the project. Part of our exploitation strategy will be to target potential end-users via publications, presentations and press reports. Major international events such as the Conference on Molecular Diagnostics, microTAS etc. will be attended and technology transfer events will be targeted for further exploitation of the results.
The interaction between elastic liposome-structures immersed in the oscillatory shear flow will be studied in scales where thermal fluctuations are significant, by using a novel computational tool (FLUAM) based on the Immersed Boundary Method.20 FLUAM runs in Graphics Processor Units (GPU) cards, in an extremely efficient way (about 100 faster than standard CPU nodes) using Fast Fourier Transforms to solve incompressible fluctuating Navier-Stokes. The soft matter structures immersed in the fluid can be constructed with standard coarse-grained models, on which P6 has extensive experience. Aside from the flow drag on the immersed structures, we expect that part of the dissipation arises from the vibrational modes of the structures and chains, excited by the interaction with flow oscillations. This is indeed a highly non-trivial problem which requires the state-of-the-art simulation techniques. Results will be used by P2 for the rational design of liposome structures of high dissipative capability.
Hydrodynamic code resolving coarse-grained molecular description of liposomes in oscillatory flowThe interaction between elastic liposome-structures immersed in the oscillatory shear flow will be studied in scales where thermal fluctuations are significant, by using a novel computational tool (FLUAM) based on the Immersed Boundary Method.20 FLUAM runs in Graphics Processor Units (GPU) cards, in an extremely efficient way (about 100 faster than standard CPU nodes) using Fast Fourier Transforms to solve incompressible fluctuating Navier-Stokes. The soft matter structures immersed in the fluid can be constructed with standard coarse-grained models, on which P6 has extensive experience. Aside from the flow drag on the immersed structures, we expect that part of the dissipation arises from the vibrational modes of the structures and chains, excited by the interaction with flow oscillations. This is indeed a highly non-trivial problem which requires the state-of-the-art simulation techniques. Results will be used by P2 for the rational design of liposome structures of high dissipative capability.
A biobank will be prospectively created from patients with lung or colorectal cancer (n=100). Tumor tissue and matched plasma/serum will be collected before initiation of front-line therapy or during routine clinical evaluation. Samples will be kept identifiable, coded or traceable. A database will be formed with patients’ information, i.e., stage of disease, treatment administration, response to treatment and patient survival.
Specific objectives, main actions and their results will be disseminated through a website, designed to have two parts: one publicly accessible, where the translation of project’s results into clinical practice will be advertised, and a second one, password protected, for partners’ communication only.
Publications
Author(s):
María Calero, Román Fernández, Pablo García, José Vicente García, María García, Esther Gamero-Sandemetrio, Ilya Reviakine, Antonio Arnau, Yolanda Jiménez
Published in:
Biosensors, Issue 10/12, 2020, Page(s) 189, ISSN 2079-6374
Publisher:
Multidisciplinary Digital Publishing Institute (MDPI)
DOI:
10.3390/bios10120189
Author(s):
Roman Fernandez, Maria Calero, Ilya Reiviakine, Jose Vicente Garcia, Maria Isabel Rocha-Gaso, Antonio Arnau, Yolanda Jimenez
Published in:
IEEE Sensors Journal, 2020, Page(s) 1-1, ISSN 1530-437X
Publisher:
Institute of Electrical and Electronics Engineers
DOI:
10.1109/jsen.2020.3015011
Author(s):
Román Fernández, María Calero, José Vicente García, Ilya Reviakine, Antonio Arnau, Yolanda Jiménez
Published in:
IEEE Sensors Journal, 2020, ISSN 1530-437X
Publisher:
Institute of Electrical and Electronics Engineers
DOI:
10.1109/jsen.2020.3042653
Author(s):
Kleita Michaelidou, Chara Koutoulaki, Konstantinos Mavridis, Eleftherios Vorrias, Maria A. Papadaki, Anastasios V. Koutsopoulos, Dimitrios Mavroudis, Sofia Agelaki
Published in:
Cells, Issue 9/11, 2020, Page(s) 2514, ISSN 2073-4409
Publisher:
Multidisciplinary Digital Publishing Institute (MDPI)
DOI:
10.3390/cells9112514
Author(s):
Maria A Papadaki, Afroditi I Sotiriou, Christina Vasilopoulou, Maria Filika, Despoina Aggouraki, Panormitis G Tsoulfas, Christina A Apostolopoulou, Konstantinos Rounis, Dimitrios Mavroudis, Sofia Agelaki
Published in:
Cancers, Issue 12/6, 2020, Page(s) 1556, ISSN 2072-6694
Publisher:
Multidisciplinary Digital Publishing Institute (MDPI)
DOI:
10.3390/cancers12061556
Author(s):
Marc Meléndez, Adolfo Vázquez-Quesada, Rafael Delgado-Buscalioni
Published in:
Langmuir, Issue 36/31, 2020, Page(s) 9225-9234, ISSN 0743-7463
Publisher:
American Chemical Society
DOI:
10.1021/acs.langmuir.0c01429
Author(s):
Dimitra Milioni, Pablo Mateos-Gil, George Papadakis, Achilleas Tsortos, Olga Sarlidou, Electra Gizeli
Published in:
Analytical Chemistry, Issue 92/12, 2020, Page(s) 8186-8193, ISSN 0003-2700
Publisher:
American Chemical Society
DOI:
10.1021/acs.analchem.0c00366
Author(s):
George Papadakis, Alexandros K. Pantazis, Maria Ntogka, Konstantinos Parasyris, Gesthimani-Ioanna Theodosi, Georgia Kaprou, Electra Gizeli
Published in:
ACS Sensors, Issue 4/5, 2019, Page(s) 1329-1336, ISSN 2379-3694
Publisher:
American Chemical Society
DOI:
10.1021/acssensors.9b00264
Author(s):
Karla Perez-Toralla, Iago Pereiro, Sonia Garrigou, Fahima Di Federico, Charlotte Proudhon, François-Clément Bidard, Jean-Louis Viovy, Valérie Taly, Stéphanie Descroix
Published in:
Sensors and Actuators B: Chemical, Issue 286, 2019, Page(s) 533-539, ISSN 0925-4005
Publisher:
Elsevier BV
DOI:
10.1016/j.snb.2019.01.159
Author(s):
A.K. Pantazis, G. Papadakis, K. Parasyris, A. Stavrinidis, E. Gizeli
Published in:
Sensors and Actuators B: Chemical, Issue 319, 2020, Page(s) 128161, ISSN 0925-4005
Publisher:
Elsevier BV
DOI:
10.1016/j.snb.2020.128161
Author(s):
Adolfo Vázquez-Quesada, Marc Meléndez Schofield, Achilleas Tsortos, Pablo Mateos-Gil, Dimitra Milioni, Electra Gizeli, Rafael Delgado-Buscalioni
Published in:
Physical Review Applied, Issue 13/6, 2020, ISSN 2331-7019
Publisher:
American Physical Society
DOI:
10.1103/physrevapplied.13.064059
Author(s):
Vasilios Raptis, Achilleas Tsortos, Electra Gizeli
Published in:
Physical Review Applied, Issue 11/3, 2019, ISSN 2331-7019
Publisher:
American Physical Society
DOI:
10.1103/physrevapplied.11.034031
Author(s):
Aristea Grammoustianou, Electra Gizeli
Published in:
Handbook of Immunoassay Technologies, 2018, Page(s) 203-239, ISBN 9780128117620
Publisher:
Elsevier
DOI:
10.1016/b978-0-12-811762-0.00009-8
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