The main goal of NAQUANTA is the development of a bio-analytical device for quantification and detection of nucleic acids (NAs). The device consists of two elements: i) an electrochemical sensing system able to quantify and determine the NAs concentration; and, ii) a temperature control system or microheater essential for enzymatic reactions during NAs amplification processes. These two components will be equipped with metal-coated electrospun polymeric fibers electrodes attached on the microfluidics support for sample transport. A hydrogel will act as solid-state electrolyte necessary for NAs detection and amplification and, at the same time as a physical barrier for the components of the detection system. In order to achieve the main goal of the project the following objectives are envisaged:

O1. fabrication of electrodes through attachment of metal-coated electrospun polymeric fibers on solid support with imprinted microchannels. This step is essential for the fabrication of both components of the device;

O2. fabrication of the sensing element. Finding the best electrodes’ architecture, combinations and/or modification, the solid-state electrolyte, and testing its capacity for detection and quantification of NAs.

O3. fabrication of the microheater. Finding the best electrode’s architecture, investigating the spatial distribution of power dissipation and consequently their heating capacity.

The project is carried out by the group of Lab. 10. Functional Nanostructures of the National Institute of Materials Physics

Phase 1. In the first stage of the project, the parameters of the electrospinning process were studied to obtain robust polymer fibers. These polymer fibers were coated with Au, Ag, Pt, and AgCl via magnetron sputtering, resulting in the fabrication of electrodes. These electrodes were transferred onto four different substrates: polyethylene terephthalate, polydimethylsiloxane, filter paper, and chromatography paper. The morphology of the metallized polymer fiber assemblies and their substrates was investigated using scanning electron microscopy, while their chemical and structural composition was analyzed through X-ray photoelectron spectroscopy and X-ray diffraction analysis.

Additionally, microfluidic systems were developed on chromatography paper through 3D printing using wax-based filaments and polymeric materials with wax-like properties. Printing and diffusion tests were conducted to create an ideal hydrophobic barrier that ensures the confinement and diffusion of fluids within a well-defined surface or volume.

Phase 2. In the second stage, the attachment of the metallized polymer fibers to both sides of the microfluidic system enabled the development of electrochemical sensors. Research focused on the design, geometry, and electrode architecture on different faces of the microfluidic system to achieve an optimal sensing configuration and to understand the behavior of electroactive species at the electrode/electrolyte interface. Multiple architectures with increasing complexity were developed. Electrochemical responses were tested using two- and three-electrode configurations. The functionality of the sensors was investigated electrochemically under various conditions of pH, supporting electrolyte composition, and in the presence or absence of redox probes. Electrochemical analysis was performed using cyclic voltammetry and impedance spectroscopy.

For the development of the detection element/system, various chemical molecules capable of interacting with nucleic acids were investigated. Particular attention was given to methylene blue, which can interact with nucleic acids through electrostatic forces (predominantly with single-stranded nucleic acids) and intercalation between base pairs (predominantly with double-stranded nucleic acids). The nucleic acid detection capacity of the system was tested using two strategies. The first strategy involved voltammetric methods using methylene blue as an electroactive marker. The experiments showed a decrease in the electrochemical signal of methylene blue's redox reactions as nucleic acid concentration increased, with these variations depending on the secondary structure (single- or double-stranded). Raman spectroscopy and reflectance studies confirmed the interaction between nucleic acids and methylene blue in both chromatography paper-based microfluidic systems and within polyacrylamide hydrogel matrices. The second approach utilized electrochemical impedance spectroscopy without any markers. The addition of DNA led to an increase in the imaginary component of impedance and, consequently, the total system impedance.

Phase 3. In the final stage, microheaters were fabricated using the Joule heating effect, employing polymer meshes made of various polymers coated with Au and Pt. The heating performance under electric current was studied in relation to fiber density. It was observed that meshes with reduced transmittance had higher conductive surfaces, contributing to efficient heating and rapid heat transfer to the surrounding environment. Two configurations were tested: one involved sealing the fluidic channel (fabricated in paper) between two polyethylene terephthalate (PET) films, and the other integrated a hydrogel into one surface of the paper while removing one PET layer. Experimental results demonstrated the ability to stimulate fluid flow from the hydrogel toward the paper under capillary action, enabling constant fluid volume maintenance.

The devices were tested for their ability to reach temperatures corresponding to PCR amplification cycles. Initially, this was performed in a dry environment potentiostatically by applying voltage values to achieve temperatures for the three PCR stages: denaturation, annealing, and elongation. In a humid environment, to ensure constant power under the influence of fluid volume in the porous matrix, the thermal cycling procedure was performed galvanostatically by applying constant current values that generated heating corresponding to the three PCR stages. Finally, the quantification of PCR amplification products was conducted externally using voltammetric detection systems. Real sample analysis was performed to detect and quantify PCR products of the MTHFR gene, mutations of which can increase the risk of cardiovascular diseases. Nucleic acids extracted from human samples were amplified, and the detection limit was 1.38 ± 0.07 ng/µL MTHFR.

The project involve training of three PhD students and two Post-doc Researchers in biosensing technologies and engineering.

Articles

1. R.J.B. Leote, M. Beregoi, I. Enculescu, V.C. Diculescu, Metallized electrospun polymeric fibers for electrochemical sensors and actuators, Current Opinion in Electrochemistry, 2022, 34:101024. https://doi.org/10.1016/j.coelec.2022.101024; IF 7.664; AIS 1.411; Q1;
2. R.J.B. Leote, C.G. Sanz, V.C. Diculescu, Electrochemical characterization of shikonin and in-situ evaluation of interaction with DNA, Journal of Electroanalytical Chemistry, 2022, 921: 116663. https://doi.org/10.1016/j.jelechem.2022.116663; IF 4.598; AIS 0.568; Q1;
3. M.C. Bunea , V.C. Diculescu, M. Enculescu, D. Oprea, T.A. Enache, Influence of the Photodegradation of Azathioprine on DNA and Cells, International Journal of Molecular Sciences, 2022, 23:14438. https://doi.org/10.3390/ijms232214438; IF 6.208; AIS 1.064; Q1;
4. M.-C. Bunea, T.A. Enache, V.C. Diculescu, In situ Electrochemical Evaluation of the Interaction of dsDNA with the Proteasome Inhibitor Anticancer Drug Bortezomib, Molecules 2023, 28, 3277. https://doi.org/10.3390/molecules28073277; IF 4.6.; AIS 0.660; Q2;
5. D. Botta, I. Enculescu, C. Balan, V.C. Diculescu, Integrated architectures of electrodes and flexible porous substrates for point-of-care testing, Current Opinion in Electrochemistry 2023, 42:101418. https://doi.org/10.1016/j.coelec.2023.101418; IF 8.5; AIS 1.495; Q1.
6. D. Botta, M. Beregoi, I.A. Cepleanu-Pascu, D.N. Crisan, A.-M. Ignat, E. Matei, I. Enculescu, V.C. Diculescu, A fluidic paper-based device integrated with submicronic fiber mesh electrodes for voltammetric quantification of nucleic acids, Advanced Functiona Materials, submitted;
7. D. Botta, M. Beregoi, A. Evanghelidis, I.A. Cepleanu-Pascu, E. Matei, M. Enculescu, I. Enculescu, V.C. Diculescu, Paper-based electrochemical cell integrated with electrospun fiber mesh electrodes and applications for impedimetric detection of nucleic acids, ACS Sensors, submitted.
8. D. Botta, M. Beregoi, A. Evanghelidis, I. Enculescu, V.C. Diculescu, A portable paper-based thermal cycling device for in-situ nucleic acids amplification and detection, in preparation.

Book chapters

1. Victor C. Diculescu, Madalina M. Barsan, and Teodor A. Enache, Ch. 8. Biosensors for Diagnosis, in: Emerging Drug Delivery and Biomedical Engineering Technologies, ed. Dimitrios Lamprou, 1st Edition, 2023, CRC Press, Boca Raton, USA.

1. V.C. Diculescu, New Electrode Architectures Based on Electrospun Polymeric Fibers For (Bio)Sensing Applications, at the 18th International Conference on Electroanalysis - ESEAC 2022, 5 - 9 Iunie 2022, Vilnius, Lituania. Invited talk.
2. D. Botta, Electrochemical Devices with Metallized Electrospun Fiber Meshes Electrodes, at the Biosystems in Toxicology and Pharmacology – Current challenges, online, 8-9 September 2022, Leiria, Portugal. Oral Presentation.
3. V.C. Diculescu, Electrospining for electrochemical applications, at the Biosystems in Toxicology and Pharmacology – Current challenges, online, 8-9 September 2022, Leiria, Portugal. Keynote lecture.
4. Victor Diculescu, Fibre polimerice conductoare si noi arhitecturi de electrod pentru (bio)senzoristica si actuare electrochimica, Workshop-ul „Noi frontiere și provocări ale abordărilor transdisciplinare – Analiza și Controlul Dinamicii Sistemelor Celulare” din cadrul conferinței Smart Diaspora 2023. 10-13 Aprilie 2023, Timișoara, România. Prezentare orală.
5. D. Botta, A. Evanghelidis, M. Beregoi, E. Matei, I. Enculescu, V.C. Diculescu, Microfluidic Devices with Conductive Electrospun Polymeric Fibers, 74th Annual Meeting of the International Society of Electrochemistry, 3 - 8 Septembrie 2023, Lyon, Franța. Poster.
6. V.C. Diculescu, D. Botta, M. Beregoi, A. Evanghelidis, A. Aldea, R. Branco-Leote, E. Matei, I. Enculescu, Electrospun Fibers on 3D Patterned Substrates for Point-of-Care Applications, 74th Annual Meeting of the International Society of Electrochemistry, 3 - 8 Septembrie 2023, Lyon, Franța. Prezentare orală.
7. D. Botta, V. Diculescu, M. Beregoi, A. Evanghelidis, E. Matei, I. Enculescu, An Electrochemical Paper-Based Device for Quantification of PCR-Amplified Nucleic Acids, XXVIII International Symposium on Bioelectrochemistry and Bioenergetics of the Bioelectrochemical Society, 19-23 mai 2024, Alcalá de Henares, Spain. Prezentare orală.
8. D. Botta, M. Beregoi, A. Evanghelidis, E. Matei, I. Enculescu, V.C. Diculescu, Portable Electrochemical Cell on Porous Media for Nucleic Acids Detection, Les Journées d'Electrochimie, 1-5 iulie 2024, Saint-Malo, Franța. Poster.
9. D. Botta, V.C. Diculescu, M. Beregoi, A. Evanghelidis, E. Matei, I. Enculescu, Fabrication of a portable electrochemical cell based on flexible porous materials, 10th International Conference On Advanced Materials (Rocam), 15-18 iulie 2024, București, Romania. Prezentare orală.
10. .V.C. Diculescu, D. Botta, M. Beregoi, A. Evanghelidis, E. Matei, M. Enculescu, I. Enculescu, Fibras poliméricas metalizadas e substratos porosos modelados por impressão 3D para dispositivos portáveis de diagnóstico, SIBAE 2024 – XXVI Congresso da Sociedade Ibero-Americana de Electroquimica, 19-23 May 2024, Lisbon, Portugal. Prezentare Orală.
11. V.C. Diculescu, D. Botta, M. Beregoi, A. Evanghelidis, R.J.B. Leote, A. Aldea, I. Enculescu, Portable Biosensing Devices For Healthcare. 10th International Conference On Advanced Materials (Rocam), 15-18 July 2024, București, Romania. Prezentare orală.
12. V. Diculescu, D. Botta, M. Beregoi, A. Evanghelidis, E. Matei, I. Enculescu. Flexible Polymeric Meshes Assembled with 3D Patterned Porous Substrates for Point-of-Care Testing. Les Journées d'électrochimie 2024. 1-5 July 2024, Saint-Malo, France. Invited talk.
13. V.C. Diculescu, Electrospun Fibers and Paper-Based Biosensors: An Approach for Point-of-Care Diagnostics. IC-ANMBES 2024 – The 7th edition of International Conference on Analytical and Nanoanalytical Methods for Biomedical and Environmental Sciences. 17-20 September 2024. Brasov, Romania. Invited talk.
14. V. Diculescu, D. Botta, I. Enculescu. Fluidic Electrochemical Devices On Paper With Integrated Flexible Electrodes. PRIOCHEM 2024. Simpozionul Internațional „PRIORITĂȚILE CHIMIEI PENTRU O DEZVOLTARE DURABILĂ”. 16-18 October 2024. Bucharest, Romania. Invited talk.

1. EPO. EP24207779/21.10.2024 - ELECTROCHEMICAL DEVICE WITH INTEGRATED FLEXIBLE FIBRILLARY STRUCTURE. Autori: Oana-Daciana Botta, Victor Constantin Diculescu, Ionuț Marius Enculescu, Mihaela Beregoi, Alexandru Ionuț Evanghelidis, Elena Matei
2. OSIM. A00738/25.11.2024 - DISPOZITIV PORTABIL DE TERMOCICLARE PENTRU AMPLIFICAREA ACIZILOR NUCLEICI. Autori: Oana-Daciana Botta, Mihaela Beregoi, Victor Constantin Diculescu, Ionuț Marius Enculescu, Alexandru Ionuț Evanghelidis, Mădălina Maria Ignat Bârsan.