PN-II-RU-TE-2011-3-0076

 

 

 

 

 

 

VERSATILE MICROELECTRODES SURFACE MODIFICATION METHOD FOR ENZYMATIC MICROBIOSENSOR DEVELOPMENT
WITH APPLICATIONS FOR CELL CULTURES MONITORING

 

 

Ø      Project number: PN-II-RU-TE-2011-3-0076; Contract no. 23/05.10.2011

 

Ø      Project Director: Ph.D Petruta-Madalina BUCUR;

 

Ø      Project category: YOUNG RESEARCH TEAMS PROJECTS - PN-II-RU-TE-2011-2;

 

Ø      Project budget: 662.955 lei;

 

Ø      Contract period: 36 months (05 october 2011- –04.October 2014);

 

Ø      Contracting Authority: Executive Unit for Higher Education, Research, Development and Innovation Funding- (UEFISCDI);

 

Ø      Contractant: National Institute of Research and Development for Biological Sciences;

 

Ø      Contract number: 23/05.10.2011;

 

Ø      Contact: Madalina Bucur.

 

FINANCIAL RESOURCES

 

 

YEAR

TOTAL(lei)

2011

2478

2012

208342

2013

247122

2014

205013

TOTAL (lei)

662955

 

 

 

 

RESEARCH TEAM

 

 

 

Ø      Ph.D Petruta Madalina BUCUR – project director (ResearcherID: B-9624-2011)                      

        

Ø      Ph.D Cristina-Maria RADULESCU  (ResearcherID:B-6576-2011)

    

Ø      Ph.D Bogdan BUCUR (ResearcherID: B-8151-2009)

    

Ø      Ph.D student: Mirela DIACONU (ResearcherID:F-7789-2011)

    

Ø      Ph.D student: Ana CHIRA (ResearcherID: F-7678-2011)

 

 

 

 

ABSTRACT

 

 

The use of microelectrodes and microbiosensors in analytical chemistry has several advantages like a better limit of detection or possibility to obtain information on localized phenomena, but their application is restricted by the difficulties in specific surface modification with electrodes and enzymes immobilization.

This project aims to develop a versatile method for enzymatic microbiosensors development. An electrochemical mediator will be electrodeposited on the surface of the microelectrode and subsequently a FAD dependent enzyme will be immobilized. During the developed of microelectrode modification method there will be chosen strategies that are easily adaptable to other electrode substrates and mediators. The modification methods are directed on microelectrode surface (e.g. electropolymerization takes place only on electrode surface while a co-precipitation leads to product deposition also on microelectrode support). In the end, the microbiosensor will be used for monitoring of cell cultures. Other possible applications of the microelectrode modification methods are in other areas of analytical chemistry like EAFM or SECM or the improvement of fuel-cells.

 

OBJECTIVES

 

Ø      Choice of the electrochemical mediator- (2011);

Ø      Microelectrode modification- (2012);

Ø      Microbiosensor development - (2013);

Ø      Microbiosensor improvement and application for cells culture monitoring- (2014).

 

 

 

SHORT PRESENTATION OF THE SCIENTIFIC RESULTS

 

Year

Brief presentation

2011

It was developed a method for the modification of the electrodes surface with aromatic organic groups based on diazonium chemistry. This method allows the modification of electrodes with mediators and enzymes (by reaction with functional groups introduced) and it is adapted for the surface modification of microelectrodes.

 

Modification principle

 

 

 

 

 

 

 

Synthesis of a viologen derivative

 

 

Electrode characterization by cyclic voltammetry and electrochemical impedance spectroscopy

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2012

The modified electrode was used for the detection of hydrogen peroxyde, a byproduct of the enzymatic reactions catalyzed by FAD dependent oxydases. 

The peak of the immobilized viologen (pink) in comparison with blank electrode (modified only with chloronitrobenzyl).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

The calibration graphs for detection of hydrogen peroxyde by amperometry; the analytical signals obtained using a viologen modified electrode (pink) are significantly higher in comparison with a blank electrode (black).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2013

 

It was developed an enzymatic biosensor based on xanthine oxidase able to quantify the total antioxidant capacity of mixtures and evaluate the oxidative stress. In the two figures is presented the calibration graph for substrate (xanthine) and the reduction of the analytical signal correlated with the antioxidant presence.

 

          

Text Box: spontaneous dismutationText Box: electrode
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 


Detection principle of antioxidant capacity using amperometric quantification of hydrogen peroxide in reduction mode. PB is Prussian blue.

 

 

 

 

 

 

 

 

 

 

 


The schematic diagram of FIA system. PBS- phosphate buffer, AA- antioxidant or real sample, R-reactor, D- detector (electrode in a flow-cell).

 

2014

It was developed an enzymatic microbiosensor based on lactate oxidase able to monitoring the cellular cultures. A microbiosensor has 2 major advantages: it is able to continuously monitor the evolution of analyte concentration and it requires a minute sample volume baying able to investigate even a single cell if it is properly positioned.

 

 

 

 

 

 

                                                                   

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Amperometric signals of hydrogen peroxide recorded with PB-Pt and GA-BSA-PB-Pt-microelectrodes at-50 mV, in PBS pH 6.6.                           Calibration plots for hydrogen peroxide detection recorded with PB-Pt and GA-BSA-PB-Pt-microelectrodes at -50 mV, PBS pH 6.6.

 

 

 

 

 

 

Calibration plot for lactate recorded with a GA-BSA-PB-Pt-microelectrode at -50 mV, PBS pH 6.6. Linear domain between 0.005 and 0.1 mM lactate.

 

 

 

Days

Samples (mM)

Yogurt 1

Yogurt 2

Yogurt 3

Pickled cucumbers

1

2.9±0.12

6.9±0.29

3.0±0.13

5.1±0.19

2

3.2±0.15

7.0±0.29

3.2±0.13

5.1±0.25

3

3.2±0.16

7.1±0.32

3.3±0.17

5.2±0.26

Table 1. Lactic acid determination in culture cell.

* The results are represented as mean value ± standard deviation.

 

 

SCIENTIFIC REPORT OF THE PROJECT.pdf

 

 

DISSEMINATION

 

A. Articles:

 

1) B. BUCUR, “Technological Barriers in the Use of Electrochemical Microsensors and Microbiosensors for in vivo Analysis of Neurological Relevant Substances” Current Neuropharmacology, Volume 10, Number 3, September 2012 , pp. 197-211(15), (F.I.=2.847);

2) R. Penu, I. Vasilescu, S. A.V. Eremia, F. Gatea, G.-L. Radu, S.-C. Litescu “Development of a nanocomposite system and its application in biosensors construction” -Central European Journal of Chemistry”- Central European Journal of Chemistry, June 2013, Volume 11, Issue 6, pp 968-978, (F.I.=1.167);

3) M. P. Bucur, B. Bucur, G. L. Radu „Critical Evaluation of Acetylthiocholine Iodide and Acetylthiocholine Chloride as Substrates for Amperometric Biosensors Based on Acetylcholinesterase” Sensors, 2013, 13, 1603-1613; (F.I. =1.953);

4) M. C. Radulescu, B. Bucur, M.P. Bucur, G. L. Radu „Bienzymatic Biosensor for Rapid Detection of Aspartame by Flow Injection Analysis” Sensors 2014, 14, 1028-1038;

 (FI=2.048);

5) Madalina Petruta Bucur, Bogdan Bucur, Jean-Louis Marty, Gabriel-Lucian Radu, “In vitro investigation of anticholinesterase activity of four biopesticides: spinosad, pyrethrum, neem bark extract and veratrine”- Journal of Pesticide Science, 2014, 39(1), 48–52; (FI=0.508);

6) A. Chira, B. Bucur , M. C. Radulescu, T. Galaon, G.-L. Radu, “Study of Electrochemically Modified Electrode with Synthesized N-benzyl-4,4′-bipyridine with Anti-Fouling Properties for Oxygen and Hydrogen Peroxide Detection”- Int. J. Electrochem. Sci., 2014, 9, 4493 – 4511 (F.I.= 1.956);

7) M. Diaconu, A. Chira, G.L. Radu, “Modulating indium doped tin oxide electrode properties for laccase electron transfer enhancement” Thin Solid Films, 2014, 565, 84-88, (F.I =1.867).

8) A Chira, B. Bucur, M. P. Bucur, G.L. Radu, "Electrode modified with nanoparticles composed of 4,4'-bipyridine-silver coordination polymer for sensitive determination of Hg(II), Cu(II) and Pb(II)" - New Journal of Chemistry, 38, 2014, 5641-5646, DOI: 10.1039/C4NJ01245C, (F.I. =3,159);

9) I. Mihai, A. Vezeanu, C. Polonschii, S. David, S. Gaspar, B. Bucur, C.  Blaszykowski, S. Sheikh, M. Thompson, A.  Vasilescu, “Low-fouling SPR detection of lysozyme and its aggregates” - Analytical Methods, 6, 2014, 7646–7654, (FI=1,93).

 

B. Submitted Articles:

 

1) Madalina-Petruta Bucur, Maria-Cristina Radulescu, Bogdan Bucur and Gabriel Lucian Radu, “Low interferences determination of antioxidant capacity based on xanthine oxidase and mediated amperometric measurements in reduction mode”- Food Chemistry

 

 

 

 

C. Conferences:

 

1.  A. Chira, B. Bucur, G.L. Radu: ”Use of N-(p-Nitrobenzyl)-4.4’-Dipyridine for Electrodes Modification Based on Diazonium Chemistry”, The 63rd Annual Meeting of the International Society of Electrochemistry ISE, PRAGUE, CZECH REPUBLIC, 19-24 August, 2012-poster;

2. M. C. Radulescu, B. Bucur, M. P. Bucur, G. L. Radu “Bi-enzymatic Biosensor for Rapid Detection of Aspartame in Commercial Products” Sixth International Workshop on « Biosensor for food Safety and Environmental Monitoring» , ESSAOUIRA-MOROCCO- 03rd - 05th October, 2013-oral communication;

3. M. C. Radulescu, B. Bucur, M.-P. Bucur, G. L. Radu,” Amperometric biosensor for the determination of aspartame in soft drinks”, Simpozionul international "PRIORITÃÞILE CHIMIEI PENTRU O DEZVOLTARE DURABILÃ" - PRIOCHEM - ed. aIXa  , Bucuresti   24-25 oct 2013-poster;

4. M.P. Bucur, B. Bucur, M.C. Radulescu, G.L. Radu,” Low interferences determination of antioxidant capacity based on xanthine oxidase and mediated amperometric measurements in reduction mode”-SPQ-Analitica-2014-COIMBRA, PORTUGAL 14-15 April 2014-oral communication;

5. A. Chira, B. Bucur, M. C. Radulescu, G.L. Radu, “ Electrochemically Modified Electrodes with Synthesized 4.4’-Bipyridine Derivatives” French-Romanian meeting in the frame of third Edition of International Conferences on Analytical and Nanoanalytical Methods for Biomedical and Environmental Sciences”, Brasov, Romania, June 13-15, 2014 - oral communication;

6. M. C. Radulescu, B. Bucur, M. P. Bucur, G. L. Radu “Carboline analysis with an amperometric biosensor based on monoamine oxidase inhibition” , New Trends on Sensing-Monitoring-Telediagnosis for Life Sciences”, Brasov, Romania, 24-26 July, 2014-oral communication.

 

D. PhD thesis:

 

1. Mirela Diaconu successfully sustained her doctoral thesis in April 2012 with the project: “The Characterization of Enzymatic Redox Systems. Bioanalytical Applications”.

2. Ana Chira successfully sustained her doctoral thesis in September 2014 with the project: “Development of Chemically Modified Electrodes Based on 4,4’-Dipyridine Derivates”.

 

Updated: October 2014