PHASE 1:Preliminary synthesis of nanodeposition of LPS-MIP NF on glass substrates by sol-gel derived techniques. Part I.

PHASE BUDGET: 56250 RON

PHASE PERIOD: 10.10.2018 - 31.12.2018

PHASE I MAIN OBJECTIVES

The objectives partially attained in this PHASE refer to:

(O1) Refining the Concept of lipopolysaccharide-molecularly imprinted polymer nanofilms (LPS-MIP NF) that will derive from the primary data, aiming towards Experimental models.

 (O4) Dissemination of project output and Protection of Intellectual Property Rights integrated

into a coherent management plan that focuses on risk management. 

PHASE SPECIFIC OBJECTIVES

Activity 1.1. Preliminary studies on the synthesis of nano-assembled films with LPS-imprinted cavities, deposited on glass supports

Activity 1.2. Physical and chemical characterization of LPS molecularly imprinted films by AFM, FTIR, in order to study the thickness and the morphology. Part I 

PHASE SUMMARY

In the 1^st PAHSE of BACTERIOSENS project, nano-assembled monolayer films imprinted with lipopolysaccharide (LPS-MIP NF), intended for the detection of gram- negative (GNB) were obtained by sol-gel techniques- sprayed directly onto glass supports. The organosilane functional monomer 3- (2-trimethoxysilyl) -propyl methacrylate (MAPTES) and the polyaniline (PANI) were used. NIP-control films and LPS-MIP NF with conductive properties were characterized morphologically and structurally by AFM and FTIR, respectively. Considerable differences were observed between NIP morphologies and LPS-MIP NF, which explains the monomer-template self-assembly phenomenon. FTIR analysis confirmed the presence of the LPS template molecule, of silane monomer and of the dispersed conductive polymer as well as the interaction mechanism of LPS with the polymeric matrix. Morphological analyzes of LPS-MIP NF films revealed a homogeneous morphology with dense zones and mesopores, a composition that leads to an interesting nanometric arrangement. By comparing with the reference NIP film, dense and deep areas may represent the effect of molecular imprinting with LPS.

PHASE I RESULTS  

- website of BACTERIOSENS project

Phase 2: Preliminary synthesis of nano-assembled films, LPS-MIP NF, deposited on glass substrates by sol-gel technique (Part II). Optimization of LPS-MIP NF using screen-printed carbon electrodes to obtain LPS biosensors (Part I).

PHASE BUDGET: 250 000 RON

PHASE PERIOD: 01.01.2019 - 31.12.2019

PHASE 2 MAIN OBJECTIVES

The objectives partially attained in this PHASE refer to:

(O1) Refining the Concept of lipopolysaccharide-molecularly imprinted polymer nanofilms (LPS-MIP NF) that derived from the primary data, aiming towards Experimental models

(O2)Transposing the Experimental Concept of LPS-MIP NF into a Functional Concept of LPS biosensor. This step was issued for optimizing the proposed recipes for LPS-MIP NF. For achieving our objective, the nanofilms were deposited directly on commercial screen printed electrodes

(O4)Dissemination of project output and Protection of Intellectual Property Rights

PHASE 2 SPECIFIC OBJECTIVES

Activity 2.1 Preliminary/detailed studies on the synthesis of nano-assembled films with specific imprinted cavities for LPS deposited on glass supports. Part II.

Activity 2.2 Establishing a heat treatment for films maturation and elaboration of the protocol for reconditioning of imprinted films.

Activity 2.3 Physico-chemical characterization of molecularly imprinted films with LPS by AFM, FT-IR, XRD, SEM methods in order to study the thickness and morphology. Part II.

Activity 2.4 Evaluation of the recognition/re-binding performance of LPS-MIP by LPS re-binding measurements on the imprinted films in relation to the control films.

Activity 2.5 Dissemination and communication of results.

PHASE SUMMARY

In the 2^nd PHASE of BACTERIOSENS project, monostratified nano-assembled films of lipopolysaccharide (LPS) molecularly imprinted polymers were successfully obtained. The films obtained by the sol-gel method (doped with electroconductive substances - ZnO, MgO, PANi and PPy) were deposited on glass supports and on screen-printed carbon electrodes. For preparing the molecularly imprinted films two silanes were used. The films deposited on glass supports were characterized by different techniques - SEM, XRD, FT-IR, and those deposited on screen printed carbon electrodes were characterized electrochemically, in potentiodynamic conditions.

After films deposition on glass support, the drying/curring step of the gel followed. For a homogenous morphology without deep cracks, the films deposited on the glass support and screen printed carbon electrodes were curred for 48 hours at room temperature followed by 48 hours at 55°C in an oven.

Films deposited onto glass support: FT-IR analysis was performed in all the four investigated cases (MIP / NIP with ZnO, MIP / NIP MgO, MIP / NIP PANi, MIP / NIP PPy) and showed differences between MIP and NIP. The spectra of films showed characteristic peaks and differences between the imprinted and non-imprinted polymers and confirmed the presence of LPS in the structure of MIP-ZnO, MIP-PPY and MIP-PANi films. For MIP-MgO the FT-IR analysis confirmed weak interactions between monomers and the LPS molecule. The morphology analysis, SEM, revealed a relatively uniform surface of the films, with small cracks and pores. The differences between imprinted and non-imprinted films were due to the monomers and templates auto-assembly before polycondensation. The crystallographic analysis, XRD, was performed for the films (MIP-PPy / NIP-PPy and MIP-PANi / NIP-PANi) and confirmed their semi-crystalline structure.

Films deposited onto screen-sprinted carbon electrode: The same recipes for films were used to modify the surface of screen-printed carbon electrodes. In this case, only zinc oxide (ZnO), polyaniline (PANi) and polypyrrole (PPy) were used as electro-conductive substances, as the films doped with magnesium oxide (MgO) did not show a good adhesion to the carbon surface (after currying the film detached from the electrode). The films deposited on the screen-printed carbon sensors were electrochemically tested, in potentiodynamic conditions. MIP-ZnO films doped with ZnO showed the most interesting electrochemical results (the voltamogram showing a reversible redox system). Considerable differences could be observed between the initial voltamogram, those obtained after the LPS removal and the voltamograms obtained after contacting the extracted sensors with a LPS solution. These differences were due to the recognition effect and rebinding of the LPS in the imprinted cavities.

 PHASE 2 RESULTS  

Dissemination and communication of results

 In order to carry out the project activities 2 articles were sent for publication and accepted. The primary results and those obtained in stages 1 and 2 of the project, were disseminated through 4 communications presented during a national conference and 3 international conferences. Moreover, a patent application for the films preparation method was also filed.

Patent application

1. T.V. Iordache, E.B. Stoica, A. Sârbu, A.M Gavril?, A.-L. Ciurlic?, A. L. Chiriac, A. Zaharia, T. Sandu, “Suprafete hibride pentru detectia electrochimica a endotoxinelor microbiene si procedeu pentru obtinerea acestora/ hybrid surfaces for electrochemical detection of microbial endotoxins and obtaining method of these”, A2019-0804/27.11.2019

Articles

1. E.B. Stoica, A.M. Gavrila, C. Branger, H. Brisset, A.V. Dyshlyuk, O.B. Vitrik, H. Iovu, A. Sarbu, T.V. Iordache, Evaluation of Molecularly Imprinted Thin Films for Ephedrine recognition – accepted for publication in Materiale Plastice, Registration number – 114 in no. 4/2019, ISI, IF-1.393.
2. E.-B. (Georgescu) Stoica, A.-M (Florea) Gavrila, T.-V. Iordache, A. Sarbu, H. Iovu, T. Sandu, H. Brisset, Molecularly imprinted polymers membrane obtained via wet phase inversion for ephedrine retention, U.P.B. Scientific Bulletin (ISI) – accepted for publication, ISI, IF-

Communications

1. E.B. Stoica (Georgescu), H. Brisset, C. Branger, H. Iovu, A.M. Florea, A.-L. Radu, T. Sandu, A. Zaharia, A. Sârbu, T.V. Iordache, Preparation of a new molecularly imprinted polymers via electropolymerization for ephedrine detection, 6^th NANO TODAY CONFERENCE, 16-20 June 2019, Lisbon, Portugal.
2. E.B. Stoica, A.M. Gavrila, T. Sandu, L. Ciurlica, H. Brisset, C. Branger, H. Iovu, A. Sârbu, A.L. Radu, A. Zaharia, T.V. Iordache, Molecularly imprinted polymers films doped with conductive substances for bacteria detection, 8^th Graduate Student Symposium on Molecular Imprinting, 28-30 August, Berlin, Germany.
3. B.-E. Stoica, A.-L. Ciurlica, T.-V. Iordache, A. Sarbu, A. Zaharia, A.-L. Radu, S. Teodor, S. Apostol, M. Duldner, A.-E. Coman, A.-M. Gavrila, Preparation and characterization of non-imprinted films doped with conducting polymers, Zilele Academice Iesene, 2-4 October 2019, Iasi, Romania.
4. Tanta-Verona Iordache, Ana-Mihaela Gavrila, Andrei Sarbu, Anamaria Zaharia, Anita-Laura Radu, Steluta Apostol, Simona Flor, Adam Paruch, Andreea Olaru, „Preparation and characterization of lipopolysaccharide-imprinted sol-gel films for inactivating gram-negative bacteria”, World Congress on Functional Materials and Nanotechnology (WCFMN), 13-14 May 2019, Spain.

PHASE 3: Optimization of LPS-MIP NF using screen-printed carbon electrodes to obtain LPS biosensors (Part II). Establishing the laboratory technology for obtaining the LPS biosensors.

PHASE BUDGET: 168 750 RON

PHASE PERIOD: 01.01.2020 - 09.10.2020

PHASE 3 MAIN OBJECTIVES

The objectives partially attained in this PHASE refer to:

(O2)Transposing the Experimental Concept of LPS-MIP NF into a Functional Concept of LPS biosensor. This step was issued for optimizing the proposed recipes for LPS-MIP NF. For achieving our objective, the nanofilms will be deposited directly on commercial screen printed electrodes.

(O3) Establishing the technology for obtaining the LPS biosensor (from technology to functional models to technology). Starting from the optimized preparation recipes, which led to an efficient recognition of LPS, the reproduction of the results (i.e. physical, chemical characteristics and recognition) is aimed. This objective also intends to validate the functionality and versatility of biosensors in relevant environments (in the presence of LPS from various pathogenic bacteria).

PHASE 3 SPECIFIC OBJECTIVES

Activity 3.1 Reproducibility of LPS-MIP NF film thickness, morphology and recognition properties in terms of time and detection limit for LPS as well as selectivity.

Activity  3.2   Establishing the laboratory technology for obtaining of the biosensors with LPS.

Activity 3.3 Testing the functionality and versatility of LPS biosensors by selectivity measurements against LPS from Escherichia Coli and Salmonella enterica.

Activity 3.4 Dissemination and communication of results.

PHASE SUMMARY

In stage III, nano-assembled films based on molecularly imprinted polymers with LPS were successfully obtained, using the sol-gel technique. The films were deposited by dripping the precursor solutions directly on carbon screen-printed electrodes, in order to obtain sensors that can be used later in the electrochemical detection of bacterial endotoxins from Gram-negative bacteria. The synthesis of the films was performed using two silanes, MPTES and TEOS. Given the fact that the films will be used for the electrochemical detection, it was necessary to incorporate electro-conductive substances in the structure of the films to increase the conductivity. The electro-conductive substances used were ZnO and polypyrrole (PPy). In order to obtain stable films with high adhesion to the carbon substrate, the hydrolysis time for the precursor solution and the maturation/drying time of the films were modified. Thus, the compatibility with the substrate has considerably increased. The sensors can be used in repeated electrochemical determinations. The obtained films were morpho-structurally analyzed by different techniques to determine the characteristics (FT-IR, film thickness analysis, BET, TGA, SEM, TEM, XRD), as well as from an electrochemical point of view.

Electrochemical tests were performed only for the MIP-ZnO and NIP-ZnO pair. Considerable differences could be observed between the voltammograms of the films before and after the removal of the LPS, as well as after contact with the LPS solution. The differences observed are due to a good self-assembly between monomers and template in the molecular imprinting step. As a result of these differences, it can be said that MIP-ZnO films are able to recognize, re-bind the LPS molecule used in the imprinting stage and have a detection time of 1 minute (after one minute of contact with the LPS solution the signal recorded for LPS was observable) and a low detection limit (166,667 E.U./ mL).

Elaboration of the laboratory technology was possible after multiple tests performed in order to determine the reproducibility, but only for the LPS-MIP-ZnO biosensors.

The selectivity of the films was studied by contacting the sensors with LPS solution from E. coli. Following this experiment, it was found that the films are able to recognize and re-bind the LPS molecule from E. coli, but with a lower specificity than the template. Following multiple stages of washing and electrochemical characterization, the films demonstrated high stability and a high capacity at re-use (at least two reconditioning cycles).

PHASE 3 RESULTS  

In order to carry out the activity 3.4, 2 ISI articles were published and 1 paper was sent for publication. The results obtained in this stage will be presented at PRIOCHEM conference, 28-30 October, in Bucharest.

Articles

1. A.M. Gavrila, T.V. Iordache, C. Lazau, T. Rotariu, I. Cernica, H. Stroescu, M. Stoica, C. Orha, C.E. Bandas, A. Sarbu, Biomimetic sensitive elements for 2,4,6-Trinitrotoluene on multi-layered sensors, Coatings, 2020, 10, 273, pp. 2-18. (F.I.=2.436)
2. A.M. Gavrila, A. Zaharia, L. Paruch, F.X. Perrin, A. Sarbu, A.G. Olaru, A.M. Paruch, T.V. Iordache, Molecularly imprinted films and quaternary ammonium-functionalized microparticles working in tandem against pathogenic bacteria in wastewaters, Journal of Hazardous Materials, 2020, 399, 123026. (F.I.=9.038)
3. B. E. Stoica, A.M. Gavrila,  A. Sarbu, H. Iovu, H. Brisset, T.V. Iordache, „Unconvering the behaviour of carbon screen-printed electrodes with molecularly imprinted polymers specific for Lipopolysaccharides”, Chemical Communications, sent for publication. (F.I.=5.99).

Conference communication

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, Bucharest, Romania.