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

The results were disseminated through presentations at meetings and conferences:

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 „Noi frontiere și provocări ale abordărilor transdisciplinare – Analiza și Controlul Dinamicii Sistemelor Celulare” within conferece Smart Diaspora 2023. 10-13 Aprilie 2023, Timișoara, România. Oral presentation.
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 September 2023, Lyon, France. Poster presentation.
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 September 2023, Lyon, France. Oral presentation.

Phase 1. The electrodes and the micro-fluidic system (2022)
Three activities were carried out during the Phase 1 of NAQUANTA implementation.
Activity 1.1.Electrospun polymeric fibers
was dedicated to electrospinning of polymeric solutions such as poly(methyl methacrylate) and nylon. The process parameters were investigated and optimized in order to achieve robust polymeric fiber meshes. The obtained fiber meshes were then subjected to coating with thin layers of Au, Ag, Pt or AgCl by magnetron sputtering deposition, which allowed the fabrication of electrodes.
Activity 1.2. Attachment of the metallized polymeric fibers on solid support
The metallized electrospun polymer fiber meshes were transferred by thermal treatment on four different substrates: polyethylene terephthalate, dimethyl polysiloxane, filter or chromatography paper. The morphologies of the assemblies formed by the metallized polymer fibers and these substrates were investigated by scanning electron microscopy, while the chemical and structural composition was analyzed by X-ray photoelectron spectroscopy and X-ray diffraction analysis.
Activity 1.3. Microfluidic system
The investigations performed within this activity were dedicated to the microfluidic system which was obtained on chromatographic paper by 3D printing with wax filaments and polymeric materials with wax-like properties. Printing and diffusion tests were carried out in order to achieve an ideal hydrophobic barrier that allows confining the fluids in a determined area/volume, restricting at the same time their diffusion throughout the entire paper pad. The attachment of the metallized polymeric fibers on the two sides of the microfluidic system, allowed the development of an electrochemical sensor/cell on chromatographic paper, Figure 1. The electrochemical response was investigated in two- and three-electrode architectures. The functionality of the sensors was investigated electrochemically under different conditions of pH, composition of the supporting electrolyte, but also in the absence and presence of different redox samples. Electrochemical analysis was performed by cyclic voltammetry and electrochemical impedance spectroscopy.

Figure 1. A) Schemes of the vector and CAD drawings of the microfluidic channel with the resulting 3D print on paper, before and after thermal treatment. B) Fluid Diffusion analysis on the 3D patterned chromatographic paper. C) PMMA and Nylon fiber meshes before and after magnetron sputtering deposition of Au, Ag and Pt. D) SEM micrographs of the paper before and after thermal attachment of the metal coated fibers. The close-up views present the morphology of the deposited Au and Pd layers.

Phase 2. The detection system (2022)
The objective of the 2^nd Phase was the fabrication of the electrochemical sensing system for nucleic acids quantification. Three main activities were carried out during this phase.
Activity 2.1. Identifying the most suitable electrode architectures which employed different configurations or geometries
Within the Activity 2.1 the investigations focused the design, geometry and architecture of the electrodes attached to different sides of the support. In this regard, electrodes from metal coated electrospun fiber meshes (obtained by the procedure described in the previous report) were transferred to chromatographic paper substrates, Figure 2A, as well as onto flexible polyethylene terephthalate supports, Figure 2B. In order to obtain an optimal configuration for the sensing element, and to understand the behaviour of the electroactive species at the electrode/electrolyte interface, multiple architectures with incremental complexity were developed.
Activity 2.2. Identifying the solid-state electrolyte
Within the Activity 2.2, two configurations were developed: i) the direct attachment of metalized electrospun fibers onto the different sides of the chromatographic paper, which also plays the role of solid-state electrolyte and ii) attachment of the electrodes on polyethylene terephthalate substrates onto different sides a polyacrylamide hydrogel as solid state electrolyte. Each configuration was first investigated by electrochemical methods, in the absence and presence of redox samples, Figure 2A and B. Particular attention was given to methylene blue, which has the property of interacting with nucleic acids both by electrostatic forces (predominant in the case of single-stranded nucleic acids) and by intercalation between nitrogenous base pairs (predominant in the case of double-stranded nucleic acids). Raman spectroscopy and reflectance studies were performed to demonstrate that the interaction between nucleic acids and methylene blue occurs in the microfluidic systems developed on chromatographic paper, as well as inside the polyacrylamide hydrogel, Figure 2C.

Figure 2. A,B) Photographs and characterization by cyclic voltammetry in solutions of 2 mM potassium hexacyanoferrate of the: A) 3D printed microfluidic systems on chromatographic paper with three Au/PMMA electrodes, and B) polyacrylamide hydrogel system with two Au/PMMA electrodes. C) Imagistic and Raman spectroscopy investigation of the dsDNA interaction with methylene blue on chromatographic paper.

Activity 2.3. Testing the nucleic acids detection and quantification capabilities of the fabricated devices
Within the Activity 2.3, the nucleic acids detection capabilities of the fabricated detection system were tested through two different strategies. In the first strategy, a voltametric detection method was employed through the use of methylene blue as an electroactive marker, Figure 3A. The experiments demonstrated a decrease in the electrochemical redox peaks of methylene blue with increasing concentrations of nucleic acids. Different behaviour were in agreement with the nucleic acids secondary structure (single or double stranded). The second approach consisted in monitoring variations of the impedance of the system, a methodology that presents several advantages including the possibility to detected the nucleic acids without the use of any type of marker. In this case, additions of DNA led to the increase of the imaginary component of the impedance and implicitly of the total impedance of the system, Figure 3B. These experiments were carried out with nucleic acids of different origins, including products of the chain polymerization reaction after the amplification of the MTHFR gene of the human chromosome I, confirming the possibility of detecting nucleic acids by these methods.

Figure 3. Electrochemical detection of dsDNA with the 3D printed microfluidic systems on chromatographic paper. A) Square wave voltammograms of methylene blue before (dotted curve) and after subsequent additions of 2 μL of 10 μg/mL dsDNA. B) Variation of the electrochemical impedance upon consecutive additions of dsDNA.

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. IF 7.664; AIS 1.411; Q1 (in Electrochemistry and in Chemistry, Physical)
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. IF 4.598; AIS 0.568; Q1 (in Chemistry, Analytical)
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. IF 6.208; AIS 1.064; Q1 (in Biochemistry & Molecular Biology)
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. 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. IF 8.5; AIS 1.495; Q1

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.