DISSEMINATION

Scientific communications:

1. Elena-Bianca Stoica, Ana-Mihaela Gavrila, Andrei Sarbu, Andreea Miron, Marius Ghiurea, Bogdan Trica, Valentin Raditoiu, Razvan Botez, Iulia Elena Neblea, Tanta-Verona Iordache, Electrochemical sensor based on molecularly imprinted polymers for lipopolisaccharides detection, PRIOCHEM XVI, 28-30 Octomber 2020, Bucharest, Romania (oral communication).

Articles:

1. Bianca Elena Stoica, Ana-Mihaela Gavrila, Andrei Sarbu, Horia Iovu, Hugues Brisset, Andreea Miron, Tanta-Verona Iordache, Uncovering the behaviour of carbon screen printed electrodes modified with lipopolysaccharide-molecularly imprinted polymers, trimis spre publicare Electrochemistry Communication, revizie majora (IF = 4.333)

2. Ana-Mihaela Gavrila, Simona Nedelcu-Flor, Andrei Sarbu, Teodor Sandu, Andreea Olaru, Gheorghe Hubca, Dan Donescu, Tanta-Verona Iordache, Synthesis and properties of organosilica particles with quaternary ammonium bearings as bacteriostatic interfaces, trimis spre publicare U.P.B. Scientific Bulletin 2020, ISSN 1223-7027.

In order to support the activity of dissemination, EPI SISTEM Partner has created a web page http://www.tehnoadvice.ro/toxinsens.html where the summary of the 2020 stage will be presented.

PHASE II: 3D PRINTING OF HYBRID FILMS AND THEIR TRANSPOSITION ON SCREEN PRINTED ELECTRODES

PHASE PERIOD: 01.01-31.12.2021

OBJECTIVE

Activity

2.1 Hybrid film printing by 3D technique on plain plastic or ceramic (Responsible: EPI SISTEM, ICECHIM)

Activity 2.1.1 Printing films based on electroactive nanoparticles by 3D technique on plastic or ceramic support

Activity 2.1.2 Study of the adhesion, time and maturation temperature of hybrid films deposited on plastic/ ceramic support

Activity 2.2 Physico-chemical characterizations of the hybrid films (Responsible: UPB, ICECHIM)

Activity 2.2.1 Morphological (SEM / microCT), structural (XPS, FTIR) characterization, contact angle measurements and thermal analysis (TGA / DTG, DSC) of hybrid films

Activity 2.2.2 Determination of LPS specificity of hybrid films (static adsorption via UV-vis)

Activity 2.3 Hybrid films printing by 3D technique on electrodes to obtain Hybrid Biosensors (Responsible: EPI SISTEM, ICECHIM)

Activity 2.3.1 Printing films based on electroactive nanoparticles by 3D technique on electrodes

Activity 2.3.2 Hybrid film optimization in terms of electrode sensitivity for LPS (cyclic voltammetry)

Activity 2.4 Physico-chemical characterizations of hybrid biosensor (Responsible: UPB, ICECHIM)

Activity 2.4.1 Study of films reproducibility by morphological (SEM / microCT), structural (XPS / FTIR) characterization and contact angle measurements of hybrid films deposited on electrodes

Activity 2.4.2 Study of films reproducibility by specificity measurements for LPS (cyclic voltammetry) of hybrid films deposited on electrodes

Activity 2.5 Communication and dissemination of results through national or international conferences (Responsible: ICECHIM, EPI SISTEM)

Activity 2.5.1 Communication and dissemination activities of the results

Activity 2.5.2 Communication and dissemination activities of the results

SUMMARY

           In Stage II/2021, the 3D printing of hybrid films and their transposition on screen-printed electrodes is described. This main objective includes (i) Reproducibility of hybrid film preparation containing electroactive nanoparticles with printable paste and physico-chemical characterization (ii) Hybrid films printing by 3D technique on plain plastic or ceramic support and morpho-structural and thermal characterization (iii) Determination of specificity for LPS of hybrid films (static adsorption via UV-vis) (iv) Hybrid films printing by 3D technique on electrodes to obtain Hybrid Biosensors and physico-chemical characterization (v) electrochemical characterization of hybrid films for reproducibility by specificity measurements for LPS (cyclic voltammetry) of hybrid films deposited on electrodes.

ICECHIM coordinator has successfully prepared the precursor solutions for electroactive nanoparticle hybrid films (MIP-NPs) in printable paste form using as non-imprinted particles NIP VBTAC, NIP SDS, NIP CTAB and NIP BTAC and respectively imprinted particles with LPS, MIP VBTAC, MIP SDS, MIP

CTAB and MIP BTAC. Micro and nanoparticles were synthesized by sol-gel technique, using several stabilizers: benzyl triethylammonium chloride (BTAC), vinyl benzyl-trimethylammonium chloride (VBTAC), cetyltrimethylammonium bromide (CTAB) and sodium dodecyl sulfate (SDS). The electroactive hybrid pastes were subsequently printed by EPI-SISTEM partner on the working electrode surface of a screen-printed sensor (SPCE), thus obtaining the molecularly imprinted hybrid sensors with LPS, noted MIP-NPs-SPCE.

EPI-SISTEM S.R.L. Partner presents the tests regarding the hybrid films printing by 3D technique on plain plastic support. The films made by ICECHIM can be printed in good condition on various types of substrates and retain their electroactive properties after drying. The printing is done with nozzles with an inner diameter of 0.4 mm in conditions in which the shape quality of the electrodes is kept in good shape. Basic functionality is maintained and LPS detection is not affected. EPI SISTEM Partner prepared several sets of functionalized electrodes for LPS detection, namely it printed 40-50 electrodes,

10-15 pieces of each functionalization variant: MIP BTAC, MIP CTAB, NIP BTAC and NIP CTAB.

ICECHIM coordinator together with UPB Partner performed physico-chemical characterizations of hybrid films by FTIR spectroscopic analysis, contact angle measurements, BET porosimetry, morphological-dimensional SEM and DLS, thermal TGA / DTG, determination of LPS specificity of hybrid films by UV-Vis spectroscopy. Scanning electron microscopy (SEM) for sol-gel particles revealed relatively porous spherical particles, less regular, grouped in micro / nanosphere aggregates, ranging in size from a few hundred nanometers to a few microns, for both MIPs and NIPs. The variation of the contact angle depending on the time for the imprinted MIP BTAC and MIP CTAB hybrid pastes and the non-imprinted ones NIP BTAC and NIP CTAB, respectively, on plastic support, demonstrated a hydrophilic character. Dimensional characterization trough dynamic light diffusion analysis (DLS) technique of imprinted particles with LPS MIP and non-imprinted NIP, respectively, led to relatively stable dispersions, with the formation of aggregates and mostly single-mode distributions. The N2 adsorption-desorption isotherms of the imprinted, non-imprinted and extracted particle systems have been shown to be type II with a type IV allure according to IUPAC, with a hysteresis curve characteristic of mesoporous structures. The thermal decomposition mechanisms of the imprinted polymers differ from those of the non-imprinted polymers due to the LPS presence, with higher mass losses in the case of MIPs. Re-adsorption tests were performed to investigate the specificity of MIP particles for LPS by static adsorption via UV-vis. In the case of CTAB and BTAC polymers, the highest adsorption values ​​were observed.

For the reproducibility of hybrid films, ICECHIM Coordinator together with UPB Partner performed physico-chemical characterizations of the hybrid films deposited on electrodes (biosensors) by FTIR spectroscopic analyzes, hybrid films contact angle measurements, SEM morphology of particles and biosensors, rheological characterization of hybrid films. FTIR analysis of the hybrid films deposited on the electrodes (MIP imprinted paste and NIP non-imprinted reference paste) confirmed the incorporation of MIP and NIP particles and zinc oxide in the polymer matrix as well as the presence of carbon paste. Rheological testing of MIP CTAB / NIP CTAB, MIP SDS / NIP SDS, MIP BTAC / NIP BTAC, MIP VBTAC / NIP VBTAC hybrid pastes and the reference paste led to a pseudo-plastic behavior with a Newtonian tendency to higher shear gradients of the reference paste similar to hybrid pastes with electroactive nanoparticles, which confirms the reproducibility of their optimal printing properties. The most efficient hybrid biosensors MIP/NIP BTAC-NPs-SPCE and MIP/NIP CTAB-NPs-SPCE were tested and characterized electrochemically by cyclic voltammetry to optimize the sensitivity for LPS. The electrochemical behavior of the MIP BTAC-NPs-SPCE and NIP BTAC-NPs-SPCE biosensors before and after the LPS addition, revealed that with the addition of LPS on the surface of the functionalized working electrode, the intensity of the oxidation current increases considerably due to a positive offset of the anodic peak potential.

In conclusion, hybrid biosensors both on ceramic substrate and printed on plastic support MIP/ NIP-NPs-SPCE electrochemically tested in the presence and absence of LPS template, proved to be reproducible, good results being obtained for both sensor systems based on of BTAC and CTAB.

DISSEMINATION

Scientific communications:

1.     Ana-Lorena Ciurlică, Ana-Mihaela Gavrilă, Catalin Zaharia, Ionut Cristian Radu, Bianca-Elena Stoica, Iulia Neblea, Andrei Sarbu, Horia Iovu, Tanta-Verona Iordache, Molecularly imprinted polymers for lipopolysaccharides recognition, Bucharest Polymer Conference (BPC), 2nd Edition, 9-11 June 2021, Bucharest, Romania (short oral communication)

2.     Ana-Lorena NEAGU, Ana-Mihaela GAVRILA, Petru EPURE, Bianca-Elena STOICA, Iulia Elena NEBLEA, Catalin ZAHARIA, Horia IOVU, Tanta-Verona IORDACHE, Hybrid Inkjet-Pritable Paste for Screen-Printed Electrodes, Simpozion international Prioritatile Chimiei pentru o Dezvoltare Durabila PRIOCHEM – editia XVII, 27- 29 Octombrie 2021, Bucuresti, Romania (Poster)

3.   Elena-Bianca STOICA, Iulia Elena NEBLEA, Elvira ALEXANDRESCU, Sabina Georgiana BURLACU, Catalin Ionut MIHAESCU, Andrei SARBU, Tanta-Verona IORDACHE, Ana-Mihaela GAVRILA, The development of a MIP-based sensor for electrochemical detection, International Scientific Conference “Applications of Chemistry in Nanosciences and Biomaterials Engineering -NanoBioMat 2021” for young researchers, 25-27 November 2021, Bucharest, Romania(oral communication)

4. 1st (June) and 2nd (December) Doctoral Reports, Ana-Lorena Ciurlica (Neagu) with the thesis title: BIOMIMETIC SENSORS OBTAINING BASED ON MOLECULAR IMPRINTED POLYMERS. Doctoral coordinator: Prof. Dr. Ing. Horia IOVU.

Articles :

1. B.E. Stoica, A.M. Gavrila, A. Sarbu, H. Iovu, H. Brisset, A. Miron, T.V. Iordachec, Uncovering the behavior of screen-printed carbon electrodes modified with polymers molecularly imprinted with lipopolysaccharide, Electrochemistry Communications 124, 106965, 2021; https://doi.org/10.1016/j.elecom.2021.106965 Sended 2020, Published 2021

2. A.M. Gavrila, S. Nedelcu-Flor, A. Sarbu, T. Sandu, A. Olaru, G. Hubca, D. Donescu, T.V. Iordachec, Synthesis and properties of organosilica particles with quaternary ammonium bearings as bacteriostatic interfaces, Scientific Bulletin of UPB Series B, vol 3, 2021, Sended 2020, Published 2021

3. A.M. Gavrila , I.C. Radu, H. Stroescu, A. Zaharia, B.E. Stoica, A.L. Ciurlica, T.V. Iordachec, A. Sarbu, Role of functional monomers upon the properties of bisphenol A molecularly imprinted silica films, Applied Sciences 2021, 11(7), 2956; https://doi.org/10.3390/app11072956 Sended 2020, Published 2021

To support the activity of dissemination results, EPI SISTEM Partner has created a web page http://www.tehnoadvice.ro/toxinsens.htm where the summary of the 2021 stage will be presented.

PHASE III: DEVELOPING THE TECHNOLOGY FOR THE PREPARATION OF THE HYBRID BIOSENSOR AND DEMONSTRATION OF THE FUNCTIONALITY

PHASE PERIOD: 01.01-02.08.2022

OBJECTIVES

Activity 3.1 Developing the technology for the preparation of Hybrid Biosensors (Responsible: P1, P2, CO)

Activity 3.1.1 Reproducibility of the preparation method for the precursor solutions of hybrid films (viscosity, structure) and reproducibility of the specificity and sensitivity of the biosensors (cyclic voltammetry) (CO)

Activity 3.1.2 Reproducibility of the printing method for films based on electroactive nanoparticles through the 3D technique on electrodes (P2)

Activity 3.1.3 Reproducibility of morphological (SEM/microCT) and structural (XPS/FTIR) characteristics of biosensors (P1)

Activity 3.2 – Demonstration of the functionality of the Hybrid Biosensor (Responsible: CO)

Activity 3.2.1 Establishing a reconditioning method for biosensors and demonstrating the versatility of hybrid biosensors for LPS fragments from various sources of pathogenic bacteria

Activity 3.3 Communication and dissemination of results through national or international conferences and journals indexed web of science (Responsible: CO, P2)

Activity 3.3.1 Communication and dissemination activities of the results

Activity 3.3.2 Communication and dissemination activities of the results

SUMMARY

            The research carried out in the Stage III describes (i) the reproducibility of the preparation method of the precursor solutions for the hybrid films improved with nano zinc oxide, (ii) the reproducibility of the specificity and sensitivity of the biosensors by cyclic voltammetry (CV) and pulse-differential voltammetry (DPV), (iii) the reproducibility of the method of printing films based on electroactive nanoparticles through the 3D technique on electrodes, (iv) the reproducibility of the morphological (SEM) and structural (FTIR) characteristics of the obtained biosensors, (v) Demonstration of the functionality of the hybrid biosensor for LPS from various sources of pathogenic bacteria (Escherichia Coli and Salmonella enterica).

ICECHIM coordinator prepared a series of polymer particles molecularly imprinted with LPS (MIP CTAB) and control non-imprinted NIP (NIP CTAB) through the sol-gel technique, in order to reproducibly prepare the precursor solutions. This system was selected due to its higher potential to detect LPS with sensitivity and specificity (according to the results of Stage II). The precursor solutions for hybrid films with electroactive nanoparticles in the form of hybrid paste were prepared using MIP/NIP CTAB and ZnO (nano) particles (20% solid powder and 80% carbon paste).

EPI SISTEM carried out activities related to the optimization of thin hybrid films based on carbon and silver inks useful for printing screen-printed electrodes sensitive to LPS and the primary evaluation of the electrochemical performances of the electrodes. The method used can be 100% inkjet for small series of electrodes and for medium and large series the hybrid method is recommended: screen printing and later inkjet.

ICECHIM coordinator together with the UPB Partner performed physico-chemical characterization of the hybrid films deposited on the electrodes (biosensors) through FTIR spectroscopic analyses, contact angle measurements, SEM morphology of biosensors, rheological characterization of the ZnO-based hybrid pastes (nano). In principle, the analysis carried out reveals both an improvement in the properties of the printed pastes/films, with the addition of ZnO (nano), but also a reproducibility of the important differences recorded between the pastes/films with MIP CTAB vs. NIP CTAB (as they were also registered in Stage II).

The CV and DPV experiments were carried out to study the analytical performance, namely the specificity and selectivity of the hybrid biosensors with the best results, e.g., MIP CTAB-Nano-SPCE and NIP CTAB-Nano-SPCE against LPS in the presence of LPS species such as LPS from Escherichia Coli and LPS from Salmonella enterica. The developed hybrid biosensor showed a strong electrochemical response to LPS from Pseudomonas Aeruginosa, indicating a good specificity of the developed CTAB-Nano-SPCE MIP. The performance of the hybrid biosensors led to excellent electrochemical properties, which allowed a low detection of LPS with a limit of detection (LOD) of only 0.28 μg/mL for the CTAB-Nano-SPCE MIP.

During Stage III/2022, in order to fulfil the deliverables from the implementation plan and to present the obtained results, the following dissemination actions were carried out: 1 published ISI article (2 articles in preparation) and 2 scientific communications at an international conference, updating the project web page. Within the framework of the current project, 1 Doctoral Scientific Report was drawn up and presented as part of a doctoral thesis in the field.

In order to support the activity of disseminating the results, the P2 EPI SISTEM has updated the project web page http://www.tehnoadvice.ro/toxinsens.html where the summary of the 2022 stage will also be presented.

DISSEMINATION

Scientific communications:

1. Ana-Lorena Neagu, Bianca-Elena Stoica, Ana-Mihaela Gavrila, Andreea Miron, Sorin Dolana, Andrei Sarbu, Horia Iovu, Tanta-Verona Iordache, Molecularly imprinted surfaces doped with conductive polymers for Lipopolysaccharide recognition, Applications of Chemistry in Nanosciences and Biomaterials Engineering, NanoBioMat 2022 –Summer Edition, 22-24 Iunie 2022, poster.

2.Ana-Mihaela Gavrila, Ana-Lorena Neagu, Petru Epure, Anamaria Zaharia, Catalin Zaharia, Horia Iovu, Andrei Sarbu, Bianca-Elena Stoica, Sorin-Viorel Dolana, Tanta-Verona Iordache, Molecularly Imprinted Polymers for the Detection of Different Hazardous Analytes, 22nd Romanian International Conference on Chemistry and Chemical Engineering, Sinaia, ROMANIA – September 7 – 9, 2022 (accepted poster)

3. 3^rd Doctoral Report (July), Ana-Lorena Ciurlica (Neagu) with the thesis title: BIOMIMETIC SENSORS OBTAINING BASED ON MOLECULAR IMPRINTED POLYMERS. Doctoral coordinator: Prof. Dr. Ing. Horia IOVU.

Articles :

1. Ana-Mihaela Gavrila, Elena-Bianca Stoica, Tanta-Verona Iordache and Andrei Sârbu, Modern and Dedicated Methods for Producing Molecularly Imprinted Polymer Layers in Sensing Applications, Appl. Sci. 2022, 12, 3080. https://doi.org/10.3390/app12063080

Final Raport

Bacterial pollution has become one of the crucial issues regarding food safety, the aquatic environment as well as drinking or domestic water sources. Bacteria lyse during and after death, releasing cell wall endotoxins in the original environment (water) where they can linger for a very long time. The most frequently reported bacterial endotoxins that appear in drinking water or in the intrahospital environment are lipopolysaccharides (LPS) produced by cyanobacteria and Gram-negative bacteria such as coliforms. The danger presented by these types of endotoxins consists in high resistance to temperature, to usual disinfectants and UV light. Therefore, a first step in preventing the contamination of environments with this type of endotoxins, and implicitly with pathogenic bacteria, is their on-site detection.

As a result, the 255PED/2020 TOXINSENS project was proposed in order to develop innovative, versatile and reusable biosensors for the rapid, qualitative and quantitative detection and monitoring of lipopolysaccharides (LPS – endotoxin derived from Gram-negative bacteria, GNB) in various biological samples, food or water. A biosensor is an analytical device, used for the detection of a chemical substance, which combines a biological component with a physical-chemical detector.

Figure 1. Screen-printed electrode model (screen-printed masks)

Figure 2. The surface of a screen-printed electrode visualized by scanning electron microscopy