Abstract

The use of biodivices for monitoring the oxidative stress at cellular level has been the focus of recent research lines with emphasis in the use of biosensors for the detection of reactive oxygen species (ROS), such as free radicals of hydroxyl, superoxide and hydrogen peroxide. Superoxide anion radical (O2•-) is the primary species of ROS generated from oxidative stress in a cell, and the enzyme superoxid dismutase (SOD) is an important antioxidant defense to fight against its toxic effect on cells, by catalyzing the dismutation of O2•- to either H2O2 or O2. In this context, the present project aims to develop new disposable SOD biosensors based on metallic electrospun fiber mesh, for in vivo O2•- detection released from cell cultures grown directly on the same mesh. The metallic mesh will be fabricated from electrospun polymeric fibers coated with a metallic layer of interest. Moreover, different types of nanostructured materials, e.g. metal/metal oxide nanostructures, carbon nanomaterials and conductive polymers will be incorporated into the biosensor architecture with the aim to improve the biosensor performance. The metallic electrospun fiber mesh will have the cell culture grown on one side, with the enzyme SOD immobilized at the other side of the mesh. In this way, the O2•−  released from cells by adding an agonist will able to freely diffuse through the mesh pores and reach the enzyme, where the enzyme-catalyzed reaction will occur and will be monitored by fixed potential amperometry. The goal is to monitor the dynamic change of O2•−  concentration in vivo, so as to determine the critical concentration at which cell damage appears.

Main Objective

The main goal of the project is the development of new disposable amperometric biosensors based on the enzyme superoxide dismutase (SOD) immobilized on metallic electrospun fibers, to monitor the dynamic change of superoxide (O2•−) concentration in vivo, so as to determine the critical concentration at which cell damage appears. The second goal regards the optimization of the biosensor assembly, by employing functionalized metallic fiber mesh with different nanostructured materials.

Deliverables

1.Metallic meshes based on electrospun fibers for SOD biosensing and cell culture
2.Identification of the proper immobilization method of the SOD onto the metallic mesh.
3.Optimized SOD biosensor based on metallic meshes.
4.Biocompatible metallic fibers.
5.Functionalized metallic fibers with nanostructured materials
6.Optimized SOD biosensor based on functionalized metallic meshes with nanostructured materials
7.Biocompatible functionalized metallic fibers
8.Optimized SOD biosensor for in vivo monitoring of O2•− in cell cultures. Documentation for a patent request
9.Charts with critical O2•− levels that cause cell damage/death.

1. Madalina Maria Barsan (U-1700-039A-2349)
   https://www.brainmap.ro/madalina-maria-barsan
   Project director CSI 50%
2. Teodor Adrian Enache (U-1700-031U-5399)
   https://www.brainmap.ro/teodor-adrian-enache
   Member Senior researcher CSI 15%
3. Anca Aldea (U-1800-055N-8239)
   https://www.brainmap.ro/anca-aldea
   Member-PhD Student 30%
4. Ricardo Leote (U-1900-062Y-0853)
   https://www.brainmap.ro/ricardo-leote
   Member-PhD Student 30%
5. Caroline G Sanz (U-2100-066A-7621)
   https://www.brainmap.ro/caroline-g-sanz
   Member-Posdoc Researcher 75%
6. Mariana Apostol (U-1900-062A-0316)
   https://www.brainmap.ro/mariana-apostol
   Membru-doctorand 40%
7. Oana Daciana Botta (U-1900-063Y-5281)
   https://www.brainmap.ro/oana-daciana-botta
   Membru-doctorand 35%

Participation in international conferences

1. Disposable SOD Biosensors Based on Metallized Electrospun Polymeric Fibers for the Detection of Superoxide in Cell Culture Media (Poster)
   XXVIth International Symposium on Bioelectrochemistry and Bioenergetics, Cluj-Napoca, Romania (online), 09-13 May 2021
   Caroline G. Sanz, Anca Aldea, Ricardo Leote, Madalina M. Barsan
2. Applications of Conductive Electrospun Polymeric Fibers in DNA Biosensing (Poster)
   XXVIth International Symposium on Bioelectrochemistry and Bioenergetics, Cluj-Napoca, Romania (online), 09-13 May 2021
   Anca Aldea, Elena Matei, Victor C. Diculescu
3. 3D flexible electrodes for in vivo measurements in cell cultures based on conductive electrospun polymeric fibers (Invited Oral Presentation)
   XXVIth International Symposium on Bioelectrochemistry and Bioenergetics, Cluj-Napoca, Romania (online), 09-13 May 2021
   Madalina M. Barsan, Caroline G. Sanz, Ariana Serban, Alexandru Evanghelidis, Victor C. Diculescu
4. Flexible Bio(sensors) for Point-of-Care Biomedical Application (Oral Presentation)
   72nd Annual ISE Meeting of the International Society of Electrochemistry, Jeju Island, Korea (online), 29 August- 3 September 2021
   Ricaro Leote, Caroline G. Sanz, Anca Aldea, Madalina M. Barsan, Victor c. Diculescu
5. Superoxide detection in cell culture media with biosensors based on electrospun fibers
   18th International Conference on Electroanalysis (ESEAC 2022)
   Caroline G Sanz, Anca Aldea, Daniel Crisan, Ricardo J.B. Leote, Melania Onea, Madalina m. Barsan
6. Bioconjufates of mercaptocarboxylic acids functionalized aunp and superoxide dismutase for superoxide monitoring (Oral Presentation)International Society of Electrochemistry ISE Regional Meeting 15-19 August, Prague, Czech Republic
   Caroline G. Sanz, Daniel N. Crișan, Ricardo J.B. Leite, Melania Onea, Madalina M. Barsan
7. New Enzymatic Biosensor Based on Metallized Electrospun Polymeric Fibers and ZnO Nanostructures for Antioxidant Properties Determination
   International Society of Electrochemistry ISE Regional Meeting 15-19 August, Prague, Czech Republic
   Anca Aldea, Caroline G. Sanz, Melania Onea, Madalina M. Barsan

Stage 1 summary:

Fabrication of metal-coated electrospun fibers and immobilization of the enzyme superoxide dismutase (SOD) for the development of electrochemical biosensors based on SOD. (O1, O2). The fabrication of metal coated electrospun fibers based on submicron polymeric electrospun fibers by using a suitable precursor solution and a predetermined experimental set-up is envisaged. The metal coating of the polymer fibers is done by thermal evaporation processes (continued in 2021). Biosensors will be constructed using different immobilization strategies for the SOD enzyme, in order to obtain a robust and sensitive biosensor; the immobilization techniques tested include physical adsorption, covalent bonding, crosslinking with glutaraldehyde, sol-gel methodology, inclusion in a polymeric matrix, etc. (continued in 2021).

Stage 2 summary:

The second stage of the project aims to obtain an optimized biosensor for the electrochemical detection of superoxide, based on the immobilization of the enzyme superoxide dismutase (SOD) on metal-coated electrospun fibers. The optimization of the analytical parameters of the superoxide biosensor involves both the use of different immobilization methods for the enzyme SOD (O2), and the incorporation of nanostructured conductive materials in the biosensor configuration, e.g. metal oxides or carbon based materials (O3). Thus, the metal meshes obtained in Stage 1 will be modified with nanostructured materials in order to improve the performance of the biosensor, by allowing a better electronic communication with the redox center of the enzyme. For the same purpose, the enzyme will be immobilized by means of metal nanoparticles, functionalized so as to allow a covalent bond between the enzyme and the nanoparticles (O2, O3). In parallel, biocompatibility studies of non-functionalized and functionalized fibers with different nanostructured materials (O4) will be performed.

Stage 3 summary

This stage is focused on the application of SOD biosensors based on functionalized metallic fibers for the quantification of superoxide O2•- in cell cultures and monitoring its effect on cells. The optimization of the superoxide biosensor was continued by using nanostructured ZnO films electrochemically deposited on polycaprolactone (PCl) fibers metallized with gold and the immobilization of the SOD enzyme by the cross-linking technique with EDC/NHS agents . The obtained biosensors are evaluated  in vitro, this time for the detection of superoxide generated in situ by injecting xanthine into the electrochemical cell containing the dissolved xanthine-oxidase enzyme. In parallel, biocompatibility studies of functionalized fibers with ZnO and AuNP and SOD structures will be carried out. After investigating the biosensors for the detection of superoxide generated in situ, they will be populated with L929 fibroblast cells and subjected to electrochemical tests for the detection of superoxide generated in cell cultures under the action of a cellular stressing agent (O4). The detection mechanism is based on the proportional release of superoxide by the cells under the action of the cellular stressing agent. Scanning electron microscopy and fluorescence measurements will be performed, before and after the action of the stressing agent, to monitor the effect of the superoxide produced by it on the cells immobilized on the surface of the detection device (O5).

1. Talanta (DOI: 10.1016/j.talanta.2022.123255): Disposable superoxide dismutase biosensors based on gold covered polycaprolactone fibers for the detection of superoxide in cell culture media,
2. Journal of Electroanaltical Chemistry (DOI: 10.1016/j.jelechem.2021.116005) Quantification of cell oxygenation in 2D constructs of metallized electrospun polycaprolactone fibers encapsulating human valvular interstitial cells
3. Microchimica Acta (DOI: 10.1007/s00604-022-05352-z): Bioconjugates of mercaptocarboxylic acids functionalized AuNP and superoxide dismutase for superoxide electrochemical monitoring
4. Biosensors (DOI: 3390/bios12070500): PC-12 Cell Line as a Neuronal Cell Model for Biosensing Applications