The goal of this research project is to contribute/boost the RDI activities in areas of general social interest, to increase the capacity of the public research sector, and to respond to the global challenges affecting the Romanian/EU economy by implementing scientific research in the areas of Analytical chemistry, Healthcare, Biophysics and Biochemistry through exploration of Alzheimer disease biomarkers. The project address to the development of (i) new functional biomaterials based on functionalization of the nanostructured materials with Amyloid beta peptides and (ii) an electrochemical biosensor for evaluation of peptide Methionine sulfoxide reductase A enzyme activity, an enzyme related with early detection of Alzheimer disease. Several nanostructured materials will be fabricated and their surfaces will be modified with amyloid beta peptide, especially the sequences of 40 or 42 amino acids. These new surfaces will be tested, in order to evaluate the bioactivity properties, against neuronal cells. The electrochemical biosensor for monitoring, quantification and enzymatic activity evaluation of the Methionine sulfoxide reductase A will be developed at the surface modified dispensable screen printed carbon electrodes. The successful implementation of the project is expected to generate methodologies for new advanced biomaterials and will support the development of a new generation of functional biomaterials and biosensors that could be translated to industrial scale.

Dr. Adrian Enache
Dr. Madalina Maria Barsan
Dr. Victor Diculescu
Dr. Monica Enculescu
Dr. Alexandru Evangelidis
Dr. Mihaela Bacalum
Drd. Ricardo Leote
Drd. Melania Onea

Articles:

1. Melania Onea, Mihaela Bacalum, Andreea Luminita Radulescu, Mina Raileanu, Liviu Craciun, Tiberiu Relu Esanu, Teodor Adrian Enache, Electrochemical evaluation of proton beam radiation effect on the B16 cell culture, Sci Rep 12, 2261 (2022).
2. Oprea D, Sanz CG, Barsan MM, Enache TA. PC-12 Cell Line as a Neuronal Cell Model for Biosensing Applications. Biosensors.; 12(7) 500 (2022).
3. Bunea, M.-C.; Diculescu, V.-C.; Enculescu, M.; Oprea, D.; Enache, T.A. Influence of the Photodegradation of Azathioprine on DNA and Cells. J. Mol. Sci. 23, 14438 (2022)
4. Sanz, C; Aldea, A; Oprea, D; Onea, M; Enache, A.T; Barsan, M.M.; Novel cells integrated biosensor based on superoxide dismutase on electrospun fiber scaffolds for the electrochemical screening of cellular stress, Biosens Bioelectron 220 114858 (2023)

Patent requests:

1. Teodor Adrian Enache, Daniela Bratu Oprea, Mihaela Cristina Bunea, Mihaela Beregoi, Procedeu de detecție a melaninei produsă de culturile celulare B16, A/2022/00283
2. Teodor Adrian Enache, Daniela Bratu Oprea, Mihaela Cristina Bunea, Mihaela Beregoi, Monica Enculescu Substrat nanostructurat pentru creșterea și transplantarea culturilor celulare si procedeul de fabricare. A/2022/00284.

Conferences:

1. A. Enache, M.L. Onea, M. Bacalum, Cells Under Irradiation – Electrochemical Evaluation, XXVIth International Symposium on Bioelectrochemistry and Bioenergetics of the Bioelectrochemical Society, 10.05.21 – 15.05.21, Cluj, Romania
2. Leote, C. Sanz, A. Aldea, M.M. Barsan, V.C. Diculescu, Flexible Bio(sensors) for Point-of-Care Biomedical Applications, 72^nd Annual Meeting of the International Society of Electrochemistry, 29.08.21 – 03.09.21, Jeju, Coreea de Sud
3. L. Onea, T.A. Enache, E. Matei, M. Enculescu, M.I. Enculescu, Bio-Sensing properties of Single ZnO nanowire Field Effect Transistor, 72^nd Annual Meeting of the International Society of Electrochemistry, 29.08.21 – 03.09.21, Jeju, Coreea de Sud
4. Leote, C. Sanz, V. C. Diculescu, In-situ Evaluation of Shikonin Interaction with ds-DNA Electrochemical (poster),  Biosensors73^rd Annual Meeting of the International Society of Electrochemistry (Online)  12-16 September 2022
5. Leote, C. Sanz, M.M. Barsan, V.C. Diculescu, Bienzymatic Biosensor for Pyruvate Kinase Inhibitors Screening for Cancer Treatment (oral), Biosystems in Toxicology and Pharmacology, Online, 8-9 September 2022
6. Leote, C. Sanz, M.M. Barsan, A. Enache, V.C. Diculescu, Flexible Biosensors for Continuous Monitoring of Uric Acid in Sweat (oral), International Society of Electrochemistry Regional Meeting, Prague, Czech Republic 15-19 August 2022
7. Leote, C. Sanz, M. M. Barsan, V. C. Diculescu, Bienzymatic biosensors for detection of pharmaceutical compounds (oral) 18th International Conference on Electroanalysis, ESEAC Vilnius, Lithuania 5-9 June 2022
8. C. Diculescu, New Electrode Architectures Based on Electrospun Polymeric Fibers For (Bio)Sensing Applications (oral), 18th International Conference on Electroanalysis - ESEAC 2022, 5 - 9 Iunie 2022, Vilnius, Lituania
9. Madalina Barsan, Caroline Sanz, Melania Onea, Victor Diculescu, Electrochemical investigations into protein degradation and inhibition (oral), The 6th edition of International Conference on Analytical and Nanoanalytical Methods for Biomedical and Environmental Sciences- IC-ANMBES 2022, 8 – 10 Iunie 2022, Brasov, Romania
10. C. Diculescu, Catalytic degradation of proteins. Electrochemical biosensors for 20S proteasome activity and inhibition, The 13th International Symposium of the Romanian Catalysis Society, 22 – 24 iunie, Govora, Romania

Scientific Report Stage 1

During this stage, according to the work plan, five research activities were carried out with the objectives (i) the manufacture and characterization of nanostructured materials for the development of surfaces modified with amyloid peptides, (ii) the development of protocols for the preparation of amyloid solutions. at various stages of aggregation, (iii) functionalization of nanostructured surfaces with amyloid peptides to grow neuronal cells on these surfaces, (iv) electrochemical evaluation of the sensing element to develop a biosensor to determine methionine sulfoxide reductase (MsrA) activity; and (v) Manufacture of catalytic nanostructures necessary for the development of the transducer for the biosensor dedicated to the determination of MsrA activity. Thus, commercial polymeric membranes have been characterized, metallized and tested in cell cultures. Protocols have also been developed for the preparation of Abeta40 amyloid solutions at various stages of aggregation and morphologically modified surfaces with these peptides have been evaluated. At the same time, synthetic peptides containing methionine were electrochemically characterized and metallic electrophilated polymeric fibers and zinc oxide nanostructures were manufactured in order to develop a biosensor to determine the activity of MsrA enzyme.
All the proposed objectives were achieved and the results obtained were disseminated in three international conferences and a scientific article was sent for publication in the journal Scientific Reports.

Scientific Report Stage 2

In this stage, according to the work plan, five research activities were carried out with the following objectives:
(i) manufacturing and characterization of nanostructured materials;
(ii) functionalization of nanostructured surfaces with amyloid peptides;
(iii) in vitro testing of Abeta-modified surfaces;
(iv) manufacturing the catalytic nanostructures necessary for the development of the biosensor for MsrA;
(v) strategies for modifying the electrode surface and immobilizing the sensitive element;
(vi) evaluation of the chemical changes occurring on the surface of the biosensors.
Thus, commercial polymeric membranes were modified with biocompatible nanometer fiber networks, by electrospinning, and tested using B16 melanoma cell culture. Also, protocols were developed for the modification of surfaces with Abeta fibrils but also with other protein/peptide structures such as collagen and polylysine and the biocompatibility of these surfaces was verified using the L929 cell line. At the same time, oxide nanostructures were manufactured and characterized from an electrochemical and morphological point of view in order to develop a biosensor for determining the activity of the MsrA enzyme by immobilizing a substrate peptide for MsrA on the surface of the zinc oxide structures and the electrochemical evaluation of the surface changes.
All the proposed objectives were achieved and the results obtained were disseminated through 7 presentations at international conferences and 4 scientific articles that were published in ISI indexed journals, in Q1 and Q2 frames. Also, the results obtained in this stage led to the elaboration of the documentation for two national patent applications that were submitted to OSIM.

Scientific Report Stage 3

In this stage, the research activities regarding the evaluation of cell cultures grown on surfaces modified with amyloid fibrils and the development of a biosensor for the detection of the enzyme methionine sulfoxide reductase (MsrA) were continued. Thus, the following activities were carried out:

(i) Functionalization of nanostructured surfaces with Abeta peptides

(ii) In vitro testing of Abeta-modified surfaces

(iii) Strategies for modifying the electrode surface and immobilizing the sensitive element

(iv) Evaluation of chemical changes occurring on the surface of biosensors, optimization and validation

Following the obtained results, it was highlighted that amyloid fibrils do not show toxicity on cell cultures. A biosensor for MsrA was also developed and tested on screen-printed carbon electrodes modified with zinc oxide structures and a substrate peptide of MsrA.

All the proposed objectives were achieved and the results obtained were disseminated through a presentation at an international conference and 2 scientific articles that were published in ISI indexed journals, one accepted, and three under evaluation.