International Centre of Biodynamics · nanomotors- Contract No. 16/05.10.2011, ID:...

International Centre of Biodynamics Bucharest - Romania

www.biodyn.ro

The International Centre of Biodynamics

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ICB Annual Report 2014

Content Content ................................................................................................................................................................... 2

About us ................................................................................................................................................................. 3

Our Mission ............................................................................................................................................................ 4

Important Dates in the Institute’s History ................................................................................................... 5

Director’s Message ............................................................................................................................................... 6

The Managing and Advisory Boards ............................................................................................................ 9

… and us ................................................................................................................................................................ 10

Cell Cultures Laboratory .................................................................................................................................. 12

Electrochemistry Laboratory .......................................................................................................................... 14

Applied Electrochemistry Laboratory.......................................................................................................... 16

Microfabrication & Prototyping Laboratory ............................................................................................. 18

Bioanalysis Laboratory ..................................................................................................................................... 19

(including a mobile laboratory) .................................................................................................................... 19

Electrical Measurements Laboratory ..........................................................................................................20

Impedimetric and Plasmonic Bio Sensors .................................................................................................. 22

Optical Microscopy Laboratory .................................................................................................................... 24

Modeling and Data Analysis Laboratory .................................................................................................. 26

Pilot Laboratory on monitoring fish behavior for quality assessment of aquatic environments ................................................................................................................................................................................. 27

Atomic Force Microscopy Laboratory ......................................................................................................... 28

International projects ....................................................................................................................................... 29

National projects ................................................................................................................................................ 31

Visibility actions .................................................................................................................................................. 32

Emergent Applications fostered by ICB ..................................................................................................... 35

Relevant publications ...................................................................................................................................... 37


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About us

According to the Agreement between UNESCO and Romanian Government, the

International Centre of Biodynamics-ICB has been established through the decision No.

1378/2000 of the Romanian Government, under the aegis of UNESCO, as a non-profit

organisation of general interest and public utility to pursue graduate training, research and

development initiatives in the field of Biodynamics.

ICB focuses on noninvasive, real time

assays and (non) linear data analysis for

appraisal of biosystems or/and of their

dynamics.

In a multidisciplinary approach, it

integrates precise measurements into

detailed models (of the dynamics) of living

systems and biointerfaces, taking into

account the complexity of the interaction

between biological structures and the

measuring systems.

The potential applications of these

methodologies have far reaching

implications for Ecology, Food industry,

Bio-Medicine and Pharmaceutical

industry. Some examples are: fast

detection of analytes, e.g. contaminants

or/and toxic compounds in liquid media,

appraisal the efficacy and cytotoxicity of

chemical compounds, biomass monitoring

etc.


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Our Mission

The International Centre of Biodynamics (ICB) is a flexible, highly dynamic structure active

on the national and international research areas. It initiates and coordinates research &

graduate training programs in the field of Biodynamics, for development of noninvasive,

sensitive and cost effective methods to analyze and control biosystems. The applicative

domains span BioMedicine, Food Industry and Ecology. ICB aims to maintain a balance

between fundamental and applied research.

ICB conducts and is involved in a number of collaborative national and international

research projects having the following headlines:

Characterization techniques for cellular systems, with emphasis on noninvasive multi-

parameter real-time monitoring

Detection of contaminants and adulterates in food products and water

(microorganisms, heavy metals, antibiotics & toxins)

Monitoring cell cycle progression & Biomass assessment

Biomedical applications - pre-clinical, in vitro assays to assess the interaction

mechanisms between selected drugs and cellular structures as well as related cytotoxic effects

Monitoring & Nonlinear Analysis of the external stimuli (e.g. drugs, toxins, pathogens,

EM radiation, pollutants) effects on: (1) evolution of cellular systems and (2) fish behavior

ICB aims to provide a competitive research environment supporting the (re)

integration of valuable researchers and formation of new ones through the Graduate

training in collaboration with the University of Bucharest.


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Important Dates in the Institute’s History

November 1996: The proposal to create the International Centre of Biodynamics, ICB, was

discussed at the UNESCO Centre of Membrane Science and Technology, UNSW, Sydney,

Australia

April 1998: The first International Symposium on Biodynamics

October 1999: The Agreement between Romania and UNESCO to establish the ICB

December 2000: Romanian Government issued the Decision No. 1378/2000 to establish ICB

January 2001: The ICB commences its independent activities; starts AFRAMILK, the first

European Research Project (under FP5) in ICB’s portfolio

March 2001: Romanian Parliament passes the law 110/2001 to ratify the Agreement between

Romania and UNESCO to establish the ICB

December 2001: In Agreement with UNESCO the first Management Board of the ICB is

appointed by Romanian Ministers of Education and Research

April 2004: Appointment of the first International Advisory Board of ICB

April 2005: Starts ROBIOS, the first European Project (under FP6) coordinated by ICB

April 2006: ICB activities start in the new lodging

May 2006: The International Conference on Biosensing and Biodynamics: From Basics to

Applications co-financed by UNESCO

April-May 2008: The IInd European Young Investigator Awardees Symposium EURYIAS 2008-

Self-organization and Selection in Evolution of Matter, Molecules and Life

July 2012: Starts EXTRACELL – the first ERC- like project “Monitoring the extracellular space

with catalytic self-propelled nanomotors”, hosted by ICB

July 2012: Starts BIOSCOPE – the first Complex project “Electro-Plasmonics for the analysis of

the dynamics of cellular processes and biomolecular interactions”, coordinated by ICB


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Director’s Message This Annual Report highlights the major ICB accomplishments during 2014 with emphasis on both the progress of ICB research and the capabilities of ICB staff and laboratories for continuous development.

I acknowledge with pride the landmarks of 2014: The projects supporting ICB activity: o BIOSCOPE, Electro-Plasmonics for the analysis of the dynamics of cellular processes

and biomolecular interactions, Contract No. 11/2012, ID: PN II-ID-PCCE-2011-2-0075, coordinated by ICB; Project Director: Eugen Gheorghiu;

o EXTRACELL- Monitoring the extracellular space with catalytic self-propelled nanomotors, ERC- like project Contract No. 9/02.07.2012, Project Director: Szilveszter Gaspar;

o ANTIOXWIN-Exploiting the antioxidant capacity of black grapes for producing wines with high authentic quality– Contract No. 101/2012, ID: PN-II-PT-PCCA-2011-3.1-1809; Project Director: Alina Vasilescu

o Sensitive quantitation of target microorganisms using dual electro plasmonic analysis and magnetic actuation, Contract No. 82-30.04.2013 , ID: PN-II-RU-PD-2012-3-0467, Project Director: Mihai Sorin David

o SENSMOTION Sensing using the electrochemically-triggered motion of catalytic nanomotors- Contract No. 16/05.10.2011, ID: PNII-RU-TE-2011-3-0237, Project Director: Szilveszter Gaspar

o Tracing proteins through food processing with biosensors-Contract No. 06/05.10.2012, ID: PN-II-RU-TE-2011-3-0302, Project Director: Alina Vasilescu

o TUMORANALYZER- Response of in vitro hypoxic tumor models to potentially therapeutic compounds as revealed by an advanced analytical platform, Romanian‐Swiss Research Programme IZERZ0_142236, 2012, Promoter from Switzerland, École Polytechnique Fédérale de Lausanne: Hubert Girault and from ICB: Eugen Gheorghiu;

o CAPTALLERGENES- Aptamer based sensors for allergen detection, Cooperation Romania-France, PN-II-CT-RO-FR-2012-1-0012, Project Director: Alina Vasilescu;

o “Dynamic interactive nanosystems” DYNANO, FP7-PEOPLE-2011-ITN Grant agreement n°: PITN-GA-2011-289033 Coordinator: Mihai BARBOIU – IEM France

o PROARGUS - Contract No. PIRG08-GA-2010-277126, Marie Curie Reintegration Grant Protein Aggregation: a quantitative assessment MC Researcher: Alina Vasilescu

o “Non-invasive electro-optical methods to assess the effect of nano/micro substrate topography on living cell dynamics”, Academic Cooperation between Romania and Russian Federation, Project Director: Eugen Gheorghiu

New scientific developments o Complementarity of EIS and SPR to Reveal Specific and Nonspecific Binding When

Interrogating a Model Bioaffinity Sensor; Perspective Offered by Plasmonic Based EIS, Anal. Chem


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o Magneto-plasmonic biosensor with enhanced analytical response and stability, Biosensors and Bioelectronics

o Label free sensing platform for amyloid fibrils effect on living cells, Biosensors and Bioelectronics

o Functional and molecular characterization of Aβ42 effect on an in vitro epithelial barrier model, Journal of Alzheimer's Disease

o Enzymatically enhanced motion of nanorods and its exploitation for sensing low molecular weight compounds of biological interest, CatChemCat & Biosensors and Bioelectronics & Nanoscale

o Low-fouling SPR detection of lysozyme and its aggregates, Analytical Methods o Label-free detection of lysozyme in wines using an aptamer based biosensor and SPR

detection, Sensors Actuators B Chemical o Simple DPPH.-Based Electrochemical Assay for the Evaluation of the Antioxidant

Capacity: A Thorough Comparison with Spectrophotometric Assays and Evaluation with Real-World Samples, Electroanalysis

Participation at the World Congress on Biosensors in Melbourne, Australia with 3 oral presentations (one invited lecture) and two posters These results allowed ICB to highly strengthen both the financial and human resources that should pave the way to further increased scientific yield.

Strengthening international cooperation: the chain of seminars hosted by ICB was complemented by research stages within ICB of our research partners in the bilateral projects with Switzerland as well as by research stages of ICB members in the group of Semmelweis University in the field of natural and synthetic vesicles as well as European Institute of Membranes, Montpellier, for the development of lipophilic coatings compatible with SPR and EIS analysis.

These accomplishments could not have been possible without the constant support, motivation and efforts of ICB staff and collaborators as well as of ICB Management and International Advisory Boards that fostered the continuous increase of Centre’s RTD capabilities and visibility during 2014.

Thank you!

Eugen Gheorghiu


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Organizational Chart

ICB Functional Structure

Cell Cultures Laboratory Electrochemistry Laboratory Applied Electrochemistry Laboratory Microfabrication & Prototyping Laboratory Bioanalysis Laboratory (including a mobile laboratory) Electrical Measurements Laboratory Impedimetric and Plasmonic Biosensors Optical Microscopy Laboratory Modeling and Data Analysis Laboratory Pilot Laboratory on Monitoring Fish Behavior Atomic Force Microscopy Laboratory


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The Managing and Advisory Boards The International Advisory Board, the Managing Board and the

Director coordinate the activity of the International Centre of

Biodynamics.

According to ICB statute, the current Managing Board was appointed in 2013 by the International Advisory Board.

Managing Board members (in alphabetical order):

Prof. Dr. Mircea Dumitru - Rector, University of Bucharest

Prof. Dr. Alexandru Morega - University Politehnica of Bucharest, Corresponding Member of Romanian Academy

Prof. Dr. Romeo Resiga - Polytechnic University of Timisoara

Prof. Dr. Tatiana Vassu - University of Bucharest

Prof. Dr. Nicolae Zamfir- Institute for Nuclear Physics and Engineering “Horia Hulubei” Corresponding Member of Romanian Academy

The members and observers of the International Advisory Board (in alphabetical order):

Prof. Dr. Koji ASAMI Institute for Chemical Research, Kyoto University, Japan

Prof. Dr. Anton ANTON Representative of Romanian University Research Council

Prof. Dr. Jean-Michel KAUFFMANN Universite Libre de Bruxelles, Belgium

Prof. Dr. Maciej NALECZ Director, Division of Basic and Engineering Sciences UNESCO –Paris, France Director, UNESCO – CEPES

Minister of Education and Research

Representative of the European Commission, to be nominated


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… and us

Eugen Gheorghiu -2001* ICB Director Professor PhD, Physicist, theoretical physics EIS&SPR assays Modeling & Data analysis Madalina Axinie – 2013* PhD student EIS&SPR biosensing

Loredana Antonescu – 2013* MSc student at Faculty of Biology Electrophysiological analysis

Dumitru Bratu-2001* Engineer Electronics Development of Electrochemical devices

Szilveszter Gáspár-2006* Head of Electrochemistry Laboratory PhD, Chemist Novel applications of electrochemical biosensing platforms

Mihaela Gheorghiu -2001* Head of (Bio) Sensors, (Bio) Surface EIS&SPR biosensing Analyses and Cell culturing Department Professor , Physicist Electro - Optical Analysis of Biointerfaces

Mihai Sorin David-2003* PhD, Biophysics/Biodynamics EIS&SPR biosensing Bioimpedance Measurements, Biosensor Development and Nonlinear Analysis

Cristina Polonschii-2006* PhD, Biophysics/Biodynamics EIS&SPR biosensing Development of analytical methods to interrogate bio interfaces

Alina Vasilescu-2011* PhD, Analytical Chemistry EIS&SPR biosensing Practical applications of Biosensors

Ada Ioana Bunea – 2012* MSc student at Faculty of Chemistry, Drugs and Cosmetics Nanomotors development

Ileana Pavel -2012* MSc student at Faculty of Chemistry, Advanced Materials Nanomotors development Veronica Andrei – 2012* MSc student at Faculty of Chemistry, Advanced Materials Electrochemical sensors


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Iuliana Mihai-2012* MSc student at Faculty of Chemistry, EIS&SPR biosensing Drugs and Cosmetics Optical sensors

Alis Vezeanu-2012* Chemist EIS&SPR biosensing

Luciana Stanica – 2012* PhD student Biologist Electrophysiological analysis

Mihnea Rosu Hamzescu-2012* PhD student EIS&SPR biosensing Diana Ciuculete-2012* MSc student at the University Politehnica of Bucharest Simulation of electromagnetic fields EIS&SPR biosensing Alin Dobre – 2012* PhD Electrical Engineering Engineer Simulation of electromagnetic fields

Dumitru Letea -2007* Mechanical Technician

Florica Moranescu – 2011* Cell culture technician

Cezar Giubalca-2001* Head of the Administrative Office Economist

Valeria Nane-2001* Chief Accountant Economist

* Date of employment at ICB


12 ICB Annual Report 2014

Cell Cultures Laboratory

Team: Mihaela Gheorghiu Luciana Stanica Loredana Antonescu Florica Moranescu

Facilities

- Cell cultures facilities that enable cultivation of different epithelial tissues: cell collection

which contains renal epithelial cells (A6, MDCK-I, MDCK-II, MDCK parental, LLC-PK1),

intestinal epithelial cells (CaCo-2), epithelial cells from a cervical carcinoma (HeLa), cells

derived from an adrenal medulla (PC12); optogenetically engineered HEK 293 cells

containing bacterial channel rhodopsin; HT29 cancer cells expressing Carbonic anhydrase IX

under hypoxic conditions; CO2 incubators (from Sanyo Biomedical Division–U.K), cryogenic

storage facilities and deep freezer, Two microelectrodes Voltage Clamp (TEVC) system,

Osmometer (from Gonotec Germany), Transepithelial, impedance and noise system (TINS),

inverted microscope; microinjection setup and support peripherals (pumps, pipette puller,

Faraday cages, dedicated measurement chambers and antivibration table); hypoxic

chamber, computer controlled LED illuminators.

- other lab utilities (purified air, microbiological hood class II, ultra pure water)

Current Research

In the Cell Cultures Laboratory we have developed a battery of methods to study the effects of

external factors (pharmaceutical compounds or bacteria) assessing several useful parameters.

The methods consider:

A. Analysis of transport and cellular permeability;

B. Toxicity studies at cellular level (changes in transport properties and cellular viability);

C. Changes in cell surface attachment and cell- to cell communication upon interaction

with stressors.

D. Cell behavior under hypoxic conditions

This expertise has been advanced with optogenetically engineered HEK 293, hypoxic

conditions and multisensory approaches towards continuous opto-electrical evaluation and

integration of cells in biosensing platforms.



Accomplishments

Gheorghiu M., David S., Polonschii C., Olaru A., Gáspár S., Bajenaru O., Popescu O. B, Gheorghiu E., Label free sensing platform for amyloid fibrils effect on living cells, Biosens. Bioelectr. (2014) 52, 89–97

M. Gheorghiu, A-M Enciu, B.O. Popescu, E. Gheorghiu Functional and molecular characterization of Aβ1-42 effect on barrier properties, JAD (2014) 38,787–798

M. Gheorghiu, S.M. David, C. Polonschii and E. Gheorghiu, “Advanced electro-optical analytical platform for dynamic evaluation of cell-surface and cell-cell interactions ”, World Congress on Biosensors, Melbourne, Australia, May 2014

Sensitive biosensing of target compounds using bioaffinity and cellular (including biomimetic) platforms , 4th DYNANO Network, 5-9 April 2014

Impact of Controlled Illumination on stable Cell Lines Containing Light Activated Channels (Channel Rhodopsin)



Electrochemistry Laboratory

Team: Szilveszter Gáspár Ada Ioana Bunea Ileana Pavel

Facilities

The resources of the laboratory did not change significantly during 2014 and include the

following equipment: VSP modular potentiostat/ galvanostat (from Bio-Logic S.A., France),

CellTest multi-channel potentiostat/ galvanostat (from Solartron Analytical, UK),

Nanoband Explorer II anodic stripping voltammetry-based heavy metal analyzer (from

TraceDetect, USA), 797 VA Computrance PC controlled system for voltammetry (from

Metrohm AG, Switzerland), Scanning Electrochemical Microscopy (SECM, from Sensolytics,

Germany), trinocular stereo microscope (from World Precision Instruments, USA) equipped

with a Coolpix 995 digital camera (from Nikon, Japan), EG-40 micropipette beveller (from

Narishige, Japan), and 3D micropositioner (HS6 from World Precision Instruments, USA).

Current Research and associated projects

The activities within the Electrochemistry Laboratory continued to be focused on enzyme-

based catalytic nanomotors (and into a smaller extent on developing classic electrochemical

sensors). Our interest in enzyme-based nanomotors resulted in the publication of three

papers: one that details enzyme-modified nanorods which enhance their diffusion

coefficient by self-electrophoresis (1), one that details how to use the enhanced diffusive

motion of enzyme-modified nanorods for sensing (2), and a review which summarizes the

literature on enzymatically induced motion (3). Results were also disseminated during two

important international events: “World Congress on Biosensors” (where our work (4) was an

invited talk) and “International Workshop on micro-and nanomachines” (where our work

(5) got one of the Young Researcher Awards).Our interest in classic electrochemical sensors is

reflected first of all by the collaboration with the laboratory led by Alina Vasilescu which

resulted in two additional papers (6-7).

Accomplishments:

(1) Pavel, I.-A.; Bunea, A.-I.; David, S.; Gáspár, S. Nanorods with Biocatalytically Induced Self-Electrophoresis. ChemCatChem 2014, 6, 866–872.

(2) Bunea, A.-I.; Pavel, I.-A.; David, S.; Gáspár, S. Sensing Based on the Motion of Enzyme-Modified Nanorods. Biosensors and Bioelectronics 2015, 67, 42–48.



(3) Gaspar, S. Enzymatically Induced Motion at Nano- and Microscale. Nanoscale 2014, 6, 7757–7763.

(4) S. Gáspár, A.-I. Bunea, I.-A. Pavel, S. David, at “24th Anniversary World Congress on

Biosensors”, Melbourne, Australia, 27-30 May 2014, “Towards sensing based on the motion

of enzyme-modified nanorods”

(5) I.-A. Pavel, A.-I. Bunea, S. David, S. Gáspár, at “International workshop on micro- and

nanomachines”, Hannover, Germany, 2-5 July 2014, “Enzymatically enhanced movement of

nanorods and its application for sensing”

(6) Mihai, I.; Vezeanu, A.; Polonschii, C.; David, S.; Gáspár, S.; Bucur, B.; Blaszykowski, C.; Sheikh, S.; Thompson, M.; Vasilescu, A. Low-Fouling SPR Detection of Lysozyme and Its Aggregates. Anal. Methods 2014, 6, 7646–7654.

(7) Andrei, V.; Bunea, A.-I.; Tudorache, A.; Gaspar, S.; Vasilescu, A. Simple DPPHC-Based Electrochemical Assay for the Evaluation of the Antioxidant Capacity: A Thorough Comparison with Spectrophotometric Assays and Evaluation with Real-World Samples. Electroanalysis. 2014, 26(12), 2677–2685.



Applied Electrochemistry Laboratory

Team: Alina Vasilescu Alis Vezeanu Veronica Andrei, Iuliana Mihai

Facilities

Main resources are represented by an 8-channel potentiostat from Dropsens, Spain and a

portable SPR system (SPREETA, Nomadics). The group shares with the Electrochemistry

group the VSP modular potentiostat/ galvanostat (from Bio-Logic S.A., France).


For the Applied Electrochemistry group, 2014 marked the end of two projects-the Young

Teams project “Tracing the allergens during food processing using biosensors” and the

bilateral project CAPTALLERGENES. It was also the year when the 2 master students,

Iuliana Mihai and Vera Andrei have graduated and left the group. Their activity at CIB

was marked by significant achievements materialized in presentations at conferences and

several publications. After leaving the group they continued towards a career in research

by enrolling in PhD programs at University of Bucharest. In 2014, the focus of the group was

on proving the practical applications of the developed sensors and biosensors. In

collaboration with colleagues from the Electrochemistry group we developed a simple

electrochemical method for the determination of the total antioxidant capacity of wines,

that was implemented at the ICDVV Valea Calugareasca, a wine research institute who is

our partner in the ANTIOXWIN project. The method was based on antioxidant capacity to

quench the DPPH radical, which is electrochemically active and the assay was conceived as

a flow injection analysis. The method was applied by lab personnel at ICDVV with no

background in electrochemistry or biosensors, for monitoring the variation of TAC during

the alcoholic and malolactic fermentation of wines for a total duration of 3 weeks. This

proved the simplicity and robustness of the concept. Another concept with proven

applicability was the SPR aptasensor for the detection of allergenic protein lysozyme in

wines. Lysozyme was spiked in red wines at levels typically used in wine industry. The results

obtained with the aptasensor compared well to those given by a classic chromatographic

method.



Other notable results were:

the design of a concept for detecting small amounts of lysozyme dimer in mixtures

with excess monomer in the frame of the PROARGUS project;

continued work performed at ICB in the frame of CAPTALLERGENES project and

during the research stages in the French partner lab, IMAGES focused on the

development of an electrochemical aptasensor for lysozyme;

the development of an analytical system comprising 5 modified electrode in the

frame of ANTIOXWIN. It should be emphasized that the project financing for 2014

was drastically reduced compared to the value provided by the contract, which led

to a re-assessment of the activities for 2014 and project prolongation.

Accomplishments:

4 papers (2 published and 2 accepted); 3 book chapters published (2 ISI); 10 participations (8 oral and 2 posters) at international conferences in Greece,

Romania, France and Spain; 2 ongoing research projects, 1 national (ANTIOXWIN, PN-II-PT-PCCA-2011-3.1-1809)

and 1 international (PROARGUS, FP7 PEOPLE-2010-RG-277126). 2 research projects finished in 2014 (CAPTALLERGENES, PN-II-CT-RO-FR-2012-1-

0012 and Young Teams project PN-II-RU-TE-2011-3-0302); Young researcher prize awarded to I. Mihai at New Trends on Sensing - Monitoring -

Telediagnosis for Life Sciences conference, Brasov, July 24-26, 2014;



Microfabrication & Prototyping Laboratory

Team: Mihai Sorin David Alin Dobre Dumitru Bratu Dumitru Letea

Facilites

CNC Shape Cutting Machine-2000 series 8 direction mill (from Sherline Products, USA),

P6700 Spin coater (from Specialty Coating Systems, USA), lathe and rectifier lathe. Physical

Vacuum Deposition PVD 75 (Kurt J Lesker, USA). Modular design configured to suit a

variety of thin film deposition applications, typically for research and development or small

batch production.

Current Applications

The 2000-series 8-direction mill is used for developing user defined parts and accessories

from holders and test fixtures electro-optically compatible measurement cells (static and

flow through), with sizes down to tens of microns.

Physical Vapor Deposition-PVD 75

PVD 75 thermal evaporator from KJ Lesker is currently used

for manufacture of different configuration of electrodes and

for different applications requiring precise deposition of

several layers of materials. For example: SPR – we

manufacture SPR chips by depositing 2 nm of

Chromium/Titanium and 50 nm of Gold; Magnetic SPR – we

prepare metallic “sandwiches” comprising of Chromium,

Cobalt and Gold; Electrode passivation – we deposit thin

layer of insulating materials (e.g. SiO).

Additional facilities: Spin coating machine within glove box, laminar flow hood for

specific surface functionalization, ultrasonic processors and UV ozone cleaning system. The

spin coater is used for controlled deposition of thin polymer layers on solid substrates for

specific applications including electrode preparation for impedance measurements,

investigation of cell adhesion to various materials, controlled local electrical insulation for

electrode design, customized PCB boards, optical coupling for SPR measurements.



Bioanalysis Laboratory (including a mobile laboratory)

Team: Mihaela Gheorghiu Szilveszter Gáspár Mihai Sorin David Cristina Polonschii

Facilities

Anodic Stripping Voltammeter Nanoband Explorer II (from Tracedetect U.S.A) Dual system

EIS/SPR (ICB patent), Glomax Luminometer 20/20 (from Promega U.S.A), pH-meter and

conductometer (from WTW Germany), portable UV-VIS Spectrophotometer Nova 60A

(from Merck, Germany), Mobile autolaboratory equipped with working areas, separate

access and electrical connections.

Current Status

The Mobile and Fixed Bioanalysis Laboratory was accredited in 2010 by RENAR as a third

party testing laboratory, in compliance to the ISO/IEC 17025:2005 clauses and to the

provisions of European directives and national legislation on water quality.

Following the reduction of the commercial activities within the accredited laboratories

during 2011, the activity of this laboratory was suspended in 2012, at our request.



Electrical Measurements Laboratory

Team: Eugen Gheorghiu Mihai Sorin David Cristina Polonschii Alin Dobre Mihnea Rosu Hamzescu Diana Ciuculete

Facilities

Impedance analyzer Agilent 4294A (from Agilent Technologies, USA), Impedance / gain

phase Analyzer Solartron; 1470 E Cell Test System(both from Solatron Analytical, U.K),

Digital Oscilloscope TDS 3052 (from Tektronix SA-France), Signal generators, Phasemeter,

Spectral Analyzer HP 3585; 8 channel low frequency impedance spectrometer (designed

and produced in ICB);

Dedicated working lab and current utilities (purified air, ultra pure water, Faraday and

thermostated chambers, 4 point electrode configurations and wide range of surface

electrodes – circular and/or interdigitated, compatible with complementary optical assays).

Current Research and related accomplished projects

DYNANO (FP7) & CHARPAN - Charged Particle Nanotech (FP6) (Ro)NANOINT -

Controlling the interaction between human and bacterial cells with nanostructured

surfaces. Strategies for achievement "Intelligent" biosurfaces.

- Investigation of lipid film formation in view of lipid sensors development - mimics for actual

biological membranes.

- Investigation of the dynamics of electrical parameters of cellular platforms in relation to

engineered interfaces (with attractive/repulsive chemical and morphological features),

- Investigation of the dynamics of electrical parameters of cellular platforms in relation to

chemical and biological stressors (heavy metals, pathogen cells).

- Monitoring cell cycle progression on non/synchronized cell suspensions – an on line system for

electro-optical evaluation of yeast cell suspensions has been developed.

- Evaluation of functionalization protocols (deposition of thin polymeric layers, biorecognition

compounds or ligand – e.g. thiol layers).

- Development of a multi channel dual SPR impedance set-up.

http://www.biodyn.ro/cgi-bin/biodyn/biodyn.cgi?act=Main-Projects&idm=4&idsm=9&locale=en



Accomplishments:

Complementarity of EIS and SPR to Reveal Specific and Nonspecific Binding When

Interrogating a Model Bioaffinity Sensor; Perspective Offered by Plasmonic Based

EIS, Anal. Chem



Impedimetric and Plasmonic Bio Sensors

Team: Mihaela Gheorghiu Mihai Sorin David Cristina Polonschii Alin Dobre Dumitru Bratu Madalina Axinie

Facilities

-Biacore 3000 system (from Biacore AG Sweden)

-Dual spectrometer EIS/SPR (ICB patent),

-3 channel Spreeta Modules, Contact Angle Meter CAM 100(from KSV, Finland).

-Dedicated working lab and current utilities (purified air / N2, chemical hood and ultra

pure water, thermostated chambers)

-Advanced set-up for monitoring the dynamics of aquatic species and the quality of growth

environment.

Additional facilities: Evolution 600 UV-VIS Spectrophotometer (Thermo Scientific USA) with

variable angle specular module, AFM module Nano Wizzard II(JPK, Germany) Multimode

readers Promega / Turner.


The design of novel (bio) sensing interfaces for selectively recognition of the analyte of

interest, from low molecular compounds to cells, and for providing a concentration-

dependent phenomenon that is easy to translate into useful analytical signal) towards a

new generation of analytical tools. These analytical tools will extend our ability of detecting

compounds of interest:

in very low concentrations,

in complex samples without prior separation,

in small sample volumes (microliters) or exotic environments (such as cellular sub

compartments), and

in a timely manner traditional analytical approaches are very often lacking such

abilities.



The integration of complementary analytical tools (SPR, electrochemistry) with micro-

technologies (fluidics, electronics) for the development of portable, sensitive solutions for

real time detection of target analytes.

Accomplishments

Gheorghiu M., David S., Polonschii C., Olaru A., Gáspár S., Bajenaru O., Popescu O.

B, Gheorghiu E., Label free sensing platform for amyloid fibrils effect on living cells,

Biosens. Bioelectr. (2014) 52, 89–97

Gheorghiu M., Enciu A.M., Popescu O. B, Gheorghiu E., “Functional and molecular

characterization of Aβ42 effect on an in vitro epithelial barrier model”, Journal of

Alzheimer's Disease (2014) 38,787–798

C. Polonschii, S. David, S. Gáspár, M. Gheorghiu, M. Rosu-Hamzescu, E. Gheorghiu,

On the complementarity of EIS and SPR to reveal specific and non-specific binding

when interrogating a model bioaffinity sensor, Anal. Chem. Chem.(2014), 86 (17),

8553–8562.

Supported by:

2 national research projects:

o BIOSCOPE, Electro-Plasmonics for the analysis of the dynamics of cellular processes and biomolecular interactions, Contract No. 11/2012, ID: PN II-ID-

PCCE-2011-2-0075, coordinated by ICB; Project Director: Eugen Gheorghiu; o Sensitive quantitation of target microorganisms using dual electro plasmonic

analysis and magnetic actuation, Contract No. 82-30.04.2013 , ID: PN-II-RU-PD-2012-3-0467, Project Director: Mihai Sorin David

1 international research project:

o Tumoranalyzer- Response of in vitro hypoxic tumor models to potentially therapeutic compounds as revealed by an advanced analytical platform, Romanian‐Swiss Research Programme IZERZ0_142236, 2012, Promoter from Switzerland, École Polytechnique Fédérale de Lausanne: Hubert Girault and from ICB: Eugen Gheorghiu;



Optical Microscopy Laboratory

Team: Mihaela Gheorghiu Mihai Sorin David Luciana Stanica Loredana Antonescu

Facilities

Zeiss Total Internal Reflection Microscope (TIRFM), Zeiss AxioObserver Z1, fully motorized

with xyz nm resolution, equipped with EMCCD ANDOR IXon DU-885K camera, with

facilities for epifluorescence, laser (3 line Ar) and white light TIRF, and various contrast

methods (Ph, DIC) and for cell cultivation (OkoLab controlled environmental cage);

Extended image processing facilities (AxioVision, Andor IQ and Andor IQ Tracker);

dedicated chambers for combined (optical and electric) assays.; Additional filter sets 02, 09,

14. NIKON Eclipse 400 microscope with epiflourescence system and CCD camera for data

acquisition.

Additional facilities:

- Spectrophotometer Evolution 600 (Thermo Scientific, USA), with variable angle specular

reflectance accessory and thermostated carrousel.

- GloMax 20/20 Multimode reader (Promega), with 2 injectors and UV and Blue

fluorescence modules.

- Compatible AFM module fully integrable with advanced optical assessment.


- Dual electro-optical measurements based on dedicated flow cells with optimized

electrode configurations.

- Time lapse assessment of the dynamics of cells (morphology, attachment and metabolism)

and cell organelles (pH, ions, morphology) in response to external stimuli and engineered

bio-interfaces cues; membrane processes in normal and pathologic conditions; manipulation

and characterization of natural and synthetic lipid environments (including cholesterol).

ATP & Cytotoxicity assays; Intracellular Ca2+;

The push - pull microfluidic probe developed by the Swiss Partners within

TUMORANALYZER Project has been integrated within the AxioObserver Z1 set-up and

experiments enabled 1. Precise control of probe positioning with optical feedback; 2. Fluidic

and electric cell stimulation 3. Experimental validation of modelling studies.



Accomplishments

2 manuscripts on cell actuation and imaging to be submitted

Presentations at international meetings:

1. Cell polarity and membrane trafficking Pultusk, Poland, 10-15 May 2014 Poster: Characterization of cell adhesion and migration processes of optogenetically modified cells by electro-optical methods Gheorghiu M., Stănică L., Ghinia M., Popescu O., Tudor Badea T., Bratu D. and Gheorghiu E.

2. Transylvanian Experimental Neuroscience Summer School 2014 The Pike Lake, Romania, June 1st-17th, 2014

3. FEBS-EMBO 2014 Conference 30 August - 4 September 2014 Poster: Evaluationof cellular dynamics of optogenetically modified cells by electro-optical methods, Luciana Stănică, Miruna Ghinia, Mihaela Gheorghiu

4. Oral presentation Advanced electro-optical analytical platform for dynamic evaluation of cell-surface and cell-cell interactions oral presentation Networking Systems Biology workshops in Berlin 12-14 May 2014 as satellites of the conference Systems Biology of Mammalian Cells (SBMC)



Modeling and Data Analysis Laboratory

Team: Eugen Gheorghiu Cristina Polonschii Sorin David Alin Dobre

Current research and associated projects

We are currently developing fast, efficient methods to asses target analytes using periodic

actuation.

Dielectric modeling and nonlinear time series analysis

Strong emphasis is placed on both experimental and theoretical aspects regarding:

Development of new Plasmonic-EIS instrumentation;

Development of microscopic models of dielectric behavior with emphasis on:

(1) Membrane Potential (discussion of GHK approach) and the role effect of ionic channels

(2) non-spherical cells (focusing on yeasts, red blood cells, and gap junction connected cells).

A quantitative approach providing shape evolution of budding yeasts during the cell cycle,

consistent with experimental findings, is available.

Time series analysis of dielectric data yielding quantitative measures of the system

dynamics (revealing changes in the tissue structure and function and the invariants of the

cell cycle

Accomplishments

Two patent applications were granted:

1. Patent RO127854/2014- Method to assess the amount of target analytes by

controlled periodic actuation, Author: E Gheorghiu

2. Patent RO127853/2014- Device to assess the amount of target micro-organisms by

controlled periodic actuation, Authors: E Gheorghiu, S David, C Polonschii and D Bratu



Pilot Laboratory on monitoring fish behavior for quality assessment of aquatic environments

Team: Eugen Gheorghiu Cristina Polonschii Dumitru Bratu Dumitru Letea

Facilities

- A platform consisting in a 3D array of

ultrasound transducers and of supporting

analytic modules to monitor fish positions and

assess fish dynamics (ICB patent);

- Dedicated water tank with adequate

infrastructure for controlling water

temperature, composition, including dissolved

Oxygen (DO);

- A module for controlled dispensing of

nutrients;

- Facilities for complementary video

observations of fish behavior;

- A set of sensors for continuous monitoring of water parameters: pH, DO, conductivity and

temperature.

Current Research

Extending the capabilities of the platform to remotely monitor and analyze the

behavior/dynamics of various fish species (including sturgeons) both in tanks and cages

immersed in aquatic media (lakes, rivers). The monitoring system fosters detection of

behavioral changes of aquatic species triggered by harmful compounds that might incidentally

occur in water (even in very small concentrations, well below the lethal threshold of those

species).



Atomic Force Microscopy Laboratory

Team: Szilveszter Gáspár Mihaela Gheorghiu Sorin David

Facilities

The major piece of equipment of the laboratory is a NanoWizard® II Atomic Force

Microscope JPK AG (Berlin, Germany). The optical microscope Zeiss AxioObserver D1 fully

equipped with phase contrast, fluorescence and dark field has been added in 2012 to the

system for complementary optical observations.


Our AFM instrument has the necessary features to work with biological samples (i.e. soft

samples in liquid), and thus is often used to observe small cellular structures (e.g. tight

junctions, microvilli, etc.) or surface absorbed / immobilized proteins (see Figure below). In

addition to the study of biological samples, it is also used to investigate the quality of thin

metal films deposited by PVD or metal sputtering

AFM analysis showing protofibril adsorption on bare gold (A), PEGCOOH SAM-coated gold

(B) and PEG-COOH/MEG-OH mixed SAM-coated gold (C), subjected to a solution of

lysozyme aggregated for 48 h. Reproduced from: Mihai, I.; Vezeanu, A.; Polonschii, C.;

David, S.; Gáspár, S.; Bucur, B.; Blaszykowski, C.; Sheikh, S.; Thompson, M.; Vasilescu,

Anal. Methods 2014, 6, 7646–7654.



International projects ONGOING Tumoranalyzer-Response of in vitro hypoxic tumor models to potentially

therapeutic compounds as revealed by an advanced analytical platform, Romanian‐Swiss Research Programme IZERZ0_142236, 2012, Promoter from Switzerland, École Polytechnique Fédérale de Lausanne: Hubert Girault and from ICB: Eugen Gheorghiu;

“DYNANO” Dynamic Interactive Nanosystems, FP7-PEOPLE-2011-ITN N°289033 7th FP (2011-2015);

“PROARGUS” Protein Aggregation: a quantitative assessment, PIRG08-GA-2019-277126, Call FP7-PEOPLE-2010-RG (2011-2015) – MC grant researcher- Alina Vasilescu;

CAPTALLERGENES- Aptamer based sensors for allergen detection, Cooperation Romania-France, PN-II-CT-RO-FR-2012-1-0012, Project Director: Alina Vasilescu;

“Non-invasive electro-optical methods to assess the effect of nano/micro substrate topography on living cell dynamics”, Academic Cooperation between Romania and Russian Federation, Project Director: Eugen Gheorghiu

ACCOMPLISHED

“NANOMAGMA” - NANOstructured active MAGnetoplasmonic MAterials”, Funded under 7th FWP (Seventh Framework Programme), Contract no.: 214107 /2008, 2008-2011;

„CHARPAN” – „CHARged PArticle Nanotech” 2005-2009;

„ROBIOS”- Strengthening ROmanian Research and Training Capacities in BIOSensing and Related Areas”, 2005-2008;

„Effective Biosensing Platform for Rapid Detection of Environmental Pollutants”, NATO Reintegration Grant, Grantee: PhD Szilveszter Gaspar, Supervisor PhD. Eugen Gheorghiu, 2006-2008;

Role of membrane organization and dynamics on cell behavior and response to external stimuli; Romania -Flanders Bilateral Agreement, 2005-2006;

Partners: Prof. Dr. Paul Steels, Faculty of Medicine, Limburgs Universitair Centrum; Prof. Dr. Marc de Broe, Faculty of Medical Sciences, University Hospital Antwerpen; Prof. Dr. Maria Luiza Flonta, Faculty of Biology, University of Bucharest;





Novel Impedimetric Affinity Biosensors (IAB) for toxicological applications exploiting E.Coli membrane protein LamB - Romania-Flanders bilateral agreement, 2003-2005 Partners: Prof. Dr. Wim de Coen, Universitaet Antwerpen; Dr. Wim Lauyren, IMEC Belgium; Prof. Dr.Patrik Wagner, Limburg Universitair Cetrum;

Development of an Impedance Assay for Immunocapture - Brancusi Romania -France bilateral agreement, 2003-2005

Partners: Prof. Dr. J.-L. Marty, Universite de Perpignan; Prof. Dr. C. Grommidh, Universite de Montpellier ;

Towards the development of a rapid portable immunoassay device for the detection of Microorganisms and Toxins in Food industry & Ecology - COBASE Grant Program 2001

Partner: Prof. O. Sadik, Dept. of Chemistry, State University, New York, Binghamton;

Impedance Spectroscopy of biological membranes: modeling the epithelial tissues and the dielectric dispersion of the plasma membrane; Romania-Flanders bilateral agreement 2001-2003;

Partners: Prof. Dr. W. Van Driessche, Catholic University, Leuven; Prof. Dr. P. Steels, LUC; Prof. Dr. G. Borghs, IMEC, Belgium;

Biomonitoring quantitative evaluation of biological systems, Romanian-German Bilateral Agreement reference number RUM 003-98;

Partner: Prof. Dr. E. Gersing, Anaesthesiology Clinic, Georg-August University, Goettingen, Germany;

AFRAMILK -Antifraud impedimetric and ultrasonic control system to detect fraud (adulteration) of milk GRD1-2000-25801, within the 5th Framework Program of the European Commission 2001-2004 ;

Partners: Institute for Bioprocess - and Analysis Methods, Germany; Centre for Molecular Electronics, Durham University, UK; SECAD S.A., France; RIKILT, State Institute for Quality Control of Agricultural Products, Holland; MEVGAL DAIRY PRODUCT INDUSTRY S.A., Greece;

Fluid Rheology – Application to biology and medicine, financed by the World Bank

Coordinator: Bucharest Politehnic University (UPB), Faculty of Energetics;

Partners: Gastroentherology Clinic Fundeni Hospital – Bucharest; Institute of non-newtonian fluid mechanics, Univ. of Wales –U.K; Material Science Department, T.U. Darmstadt – Germany; Chemical Engineering Department, Universidad de Huelva, Spain.














National projects

ONGOING Financing period Project title and managing/partnership status

2013-2015 Sensitive quantitation of target microorganisms using dual electro plasmonic analysis and magnetic actuation, Contract No. 82-30.04.2013 , ID: PN-II-RU-PD-2012-3-0467, Project Director: S. David

2012-2015 BIOSCOPE - Electro-Plasmonics for the analysis of the dynamics of cellular processes and biomolecular interactions, Contract No. 11/2012, ID: PN II-ID-PCCE-2011-2-0075, coordinated by ICB; Project Director: E. Gheorghiu;

2012-2014 EXTRACELL- Monitoring the extracellular space with catalytic self-propelled nanomotors, ERC- like project Contract No. 9/02.07.2012, Project Director: S. Gáspár;

2012-2015 o ANTIOXWIN-Exploiting the antioxidant capacity of black grapes for

producing wines with high authentic quality – Contract No. 101/2012, ID: PN-II-PT-PCCA-2011-3.1-1809; Project Director: A. Vasilescu;

2011 – 2014 Tracing proteins through food processing with biosensors (PN II-RU-TE-3-0302), Project suppporting development of Young Teams, Principal Coordinator ICB - director A. Vasilescu

2011 – 2014 SENSMOTION - Sensing using the electrochemically-triggered motion of catalytic nanomotors (PN II-RU-TE-2011-3-0237), Project suppporting development of Young Teams, Principal Coordinator ICB - director S.Gáspár

ACCOMPLISHED*

Financing period Project title and managing/partnership status

2011 – 2013 A new strategy for effective assessment of DNA hybridization, based on periodical magnetic actuation, Post Doctoral Grant- director C. Polonschii

2010-2011 Improvement of Management and Marketing Abilities within the International Centre of Biodynamics (MANMAR) – Structural Funds, Economic Competitveness Improvement, Axis 2.2.4.

2007-2010 Dielectric modeling of biological cells and heterostructures with fast and efficient algorithms for boundary integral method; Principal Project Coordinator ICB

2007-2010

DEMENTJUNCTION - Expression and function of the tight junction proteins – a study in experimental models and dementia diagnosed patients Partnership with “Victor Babes” National R&D Institute



2007-2010 BIOSADN – Development of biosensors based on nucleic acids for the evaluation and monitoring of some toxic agents with applications in bioterrorism; Partnership with Faculty of Chemistry, University of Bucharest

2007-2010

PROPETHAD – Advanced Research towards medical applications of nuclear technologies; Partnership with “Horia Hulubei” National Institute of Physics and Nuclear Engineering

*period 2007-2013. Full list is given on the institution website.

Visibility actions

Oral presentations of ICB representatives

1. A. Vasilescu, I. Mihai, E. Popa, C. Purcarea, “Novel approaches for studying protein aggregates based on electrochemistry”, RO-ICAC 2014, September 18-20, 2014 Targoviste, Romania.

2. A. Vasilescu, I. Mihai, A. Vezeanu, V. Andrei, J-L Marty, Development of an aptasensor for lysozyme detection in wines, XIVème colloque du Groupe Français de Bioélectrochimie, September 22-25, Sete, France

3. A. Vasilescu, I. Mihai, A. Vezeanu, Aptasensor for the detection of lysozyme in food products, French-Romanian Workshop, IC-ANMBES, June 15, 2014, Brasov, Romania

4. V. Andrei, E. Sharpe, A. Vasilescu, S. Andreescu, “Disposable nanoceria based electrochemical sensor for the detection of phenolic wine antioxidants”, New Trends on Sensing - Monitoring - Telediagnosis for Life Sciences conference , July 24-26, Brasov, Romania.

5. V. Andrei, A.Tudorache, E.Branduse, A. Vasilescu Monitoring the total antioxidant capacity as a tool in controlling the wine-making process. Characterization by electrochemical and spectrophotometric methods, New Trends on Sensing - Monitoring - Telediagnosis for Life Sciences conference , July 24-26, Brasov, Romania.

6. A. Vezeanu, A. Vasilescu, Challenges in the development of an aptasensor for lysozyme detection in wine, New Trends on Sensing - Monitoring - Telediagnosis for Life Sciences conference , July 24-26, Brasov, Romania.

7. A. Vasilescu, I. Mihai, A. Vezeanu, V. Andrei, Challenges in the development of biosensors for food allergens: example of gliadin, New Trends on Sensing - Monitoring - Telediagnosis for Life Sciences conference , July 24-26, Brasov, Romania.

8. I. Mihai, A. Vasilescu, New SPR-based aptasensor for accurate detection of allergenic lysozyme in food, New Trends on Sensing - Monitoring - Telediagnosis for Life Sciences conference , July 24-26, Brasov, Romania.



9. S. Gáspár, A.-I. Bunea, I.-A. Pavel, S. David, at “24th Anniversary World Congress on Biosensors”, Melbourne, Australia, 27-30 May 2014, “Towards sensing based on the motion of enzyme-modified nanorods”

10. S. Gáspár, at “Third Edition of the International Conference on Analytical and Nanoanalytical Methods for Biomedical and Environmental Sciences”, Brasov, Romania, 13-15 June 2014, “Biosensing through the diffusive movement of oxidase-modified nanorods”

11. C. Polonschii, S. David and E. Gheorghiu, “A new strategy for effective assessment of DNA hybridization based on periodical magnetic actuation ”, World Congress on Biosensors, Melbourne, Australia, May 2014

12. M. Gheorghiu, S.M. David, C. Polonschii and E. Gheorghiu, “Advanced electro-optical analytical platform for dynamic evaluation of cell-surface and cell-cell interactions ”, World Congress on Biosensors, Melbourne, Australia, May 2014

13. M. Gheorghiu, Advanced electro-optical analytical platform for dynamic evaluation of cell-surface and cell-cell interactions at Networking Systems Biology workshops in Berlin 12-14 May 2014 as satellites of the conference Systems Biology of Mammalian Cells (SBMC)

Posters

1. I. Mihai, A. Vezeanu, V. Andrei, A. Vasilescu “Label-free detection of lysozyme in wines using an aptamer-based biosensor and SPR detection”, “ EUROPT®ODE XII - Conference on Optical Chemical Sensors & Biosensors”, April 13-16 2014, Athens, Greece.

2. 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 aggregates, 19th Transfrontier Meeting of Sensors and Biosensors, September 25-26 2014, Barcelona, Spain

3. I.-A. Pavel, A.-I. Bunea, S. David, S. Gáspár, at "International workshop on micro- and nanomachines", Hannover, Germany, 2-5 July 2014, "Enzymatically enhanced movement of nanorods and its application for sensing"

4. M. Gheorghiu, L. Stănică, M. Ghinia, O. Popescu, T. Badea, D. Bratu and E. Gheorghiu Cell polarity and membrane trafficking Pultusk, Poland, 10-15 may 2014 Characterization of cell adhesion and migration processes of optogenetically modified cells by electro-optical methods

5. Luciana Stănică, Miruna Ghinia, Mihaela Gheorghiu FEBS-EMBO 2014 Conference 30 August - 4 September 2014 Evaluationof cellular dynamics of optogenetically modified cells by electro-optical methods

6. S. David, C. Polonschii, M. Rosu-Hamzescu and E. Gheorghiu, “Towards an optimal, sensitive and robust magneto plasmonic biosensor ”, World Congress on Biosensors, Melbourne, Australia, May 2014



7. E. Gheorghiu, S. David, C. Polonschii, A. Dobre, D. Bratu, M. Gheorghiu “Sensitive

quantitation of target pathogenic cells using periodic actuation”, World Congress on Biosensors, Melbourne, Australia, May 2014

8. 15th IUBMB 24 th FAOBMB - TSBMB International Conference, October 21 26, 2014, Taipei, Taiwan A.Dinischiotu, M. Radu, S.N. Voicu, M. Stan, L. Stanica, D. Bratu, M. Ghinia, T. Badea, M. Gheorghiu ”Impact of Controlled Illumination on Stable Cell Lines Containing Light Activated Channels”

Seminars hosted by ICB o Prof. Rabah Boukherroub, Lille, France o Prof. Pankaj Vadgama, London, UK



Emergent Applications fostered by ICB 1. Sensors and biosensors

Developing of rapid, sensitive, cost effective detection platforms to determine low

concentration of target compounds and for kinetic analysis.

In this context, surface plasmon resonance (SPR) based biosensors represent attractive

solutions in environmental monitoring and for quality-control, due to their sensitivity,

miniaturization amenability and wide range of detectable compounds.

Since 2006, the SPR technology is operational within ICB Biosensors Laboratory through

Biacore 3000 equipment (Biacore AB, Sweden).

The analysis methods already implemented allow:

- identification of drugs residues in some food products (e.g. presence of antibiotics in milk);

- detection of drugs and hormones, present even in low concentrations in food products;

- multianalytes detection (mixture of compounds e.g. with toxicogen potential)

SPR-based techniques enable real time, label-free assessment of the interaction between

an analyte (e.g. chemicals, DNA, RNA, proteins) and a covalently immobilized specific

ligand, simultaneously with a high sensitivity in determination of concentrations and kinetic

dates, and also the possible integration with other analysis and fluidic techniques (FIA) or

with approaches like Lab-On-a Chip.

In recent years, biosensors based on cells and tissues have been intensively used for

identification and characterization of toxic compounds in water and determination of their

cytotoxicity. Such cellular sensors could potentially replace/complement traditional methods

of analysis such as mass spectrometry, high performance liquid chromatography or ELISA

rapid tests, and also biological tests – intensively used for cytotoxicity detection or cell

viability after exposure to harmful substances.

In this respect, ICB has developed cellular platforms for assessment of food quality and

cytotoxicity of water samples. Including specific electrochemical instrumentation

(impedance spectrometer, potentiostat) and a detection unit based on electrochemical

sensors, electrical components and fluidic elements to maintain the temperature at 37°C,

and applying a previously validated measurement method, multiple cell parameters can

be potentially determined.



The ICB proprietary technology based on periodic actuation supports

major sensing advantages of ICB platform versus the current state of

the art, as revealed by our paper in Lab on a Chip (2013), 13, 3192–3198:

1.) Portability in a lab on a chip format;

2.) High sensitivity & specificity

3) Reduced analysis time;

4.) Ability to assess the effect of contaminant mixtures, and from economic point of

view, the necessity to use small volumes of reagents, reducing consumption of cells and

amount of valuable reagents, decreasing significantly the costs of operation.

The analytical platform is versatile, so it can provide relevant information not only for

water monitoring activities, but also in chemical or pharmaceutical industry, efficiently

responding to the Romanian and European socio-economic needs regarding the

environmental quality control and / or biosensing applications in other specified domains.

2. Measurement and control systems for optimization of aquaculture activities

An issue of real interest facing both our country and other European countries is

represented by the strong demand for automated systems that will lead to optimization of

aquaculture activity through increasing productivity and reducing costs. Systems already on

the market do not optimize feeding process as a function of fish behavior, which is one of

the causes that generate many losses both in terms of quantity and quality of food (about

30% of the costs of aquaculture operations).

Taking into consideration these trends and the promising results obtained by ICB in research

and experimental studies undertaken in this direction, some of the most important

applications developed in this field are as follows:

- support system of automation and optimization of feeding

- pilot system for non-invasive , multichannel assessment of behavior (dynamics) of aquatic

species

- integrated, remote monitoring system for water (aquatic environment) quality control

able to activate a specific system based on biosensors whenever the behavior of aquatic

species deviates from a normal pattern;

- (non) linear - analysis method which provides a set of quantitative measures,

characteristic of the behavior of species with high economic value (e.g., sturgeons, carps) in

relation to actual environmental conditions (including feeding conditions).



Relevant publications

Polonschii C., David S., Gáspár S., Gheorghiu M., Rosu-Hamzescu M., Gheorghiu E., “Complementarity of EIS and SPR to Reveal Specific and Nonspecific Binding When Interrogating a Model Bioaffinity Sensor; Perspective Offered by Plasmonic Based EIS”, Anal. Chem.(2014), 86 (17), 8553–8562

Gheorghiu M., David S., Polonschii C., Olaru A., Gaspar S.,Bajenaru O., Popescu O. B, Gheorghiu E.,” Label free sensing platform for amyloid fibrils effect on living cells", Biosensors and Bioelectronics (2014) 52, 89–97

Pavel, I.-A.; Bunea, A.-I.; David, S.; Gáspár, S. Nanorods with Biocatalytically Induced Self-Electrophoresis. ChemCatChem 2014, 6, 866–872.

Bunea, A.-I.; Pavel, I.-A.; David, S.; Gáspár, S. Sensing Based on the Motion of Enzyme-Modified Nanorods. Biosensors and Bioelectronics 2014 accepted.

Gaspar, S. Enzymatically Induced Motion at Nano- and Microscale. Nanoscale 2014, 6, 7757–7763.

Mihai, I.; Vezeanu, A.; Polonschii, C.; David, S.; Gáspár, S.; Bucur, B.; Blaszykowski, C.; Sheikh, S.; Thompson, M.; Vasilescu, A. Low-Fouling SPR Detection of Lysozyme and Its Aggregates. Anal. Methods 2014, 6, 7646–7654.

Andrei, V.; Bunea, A.-I.; Tudorache, A.; Gáspár, S.; Vasilescu, A. Simple DPPHC-Based Electrochemical Assay for the Evaluation of the Antioxidant Capacity: A Thorough Comparison with Spectrophotometric Assays and Evaluation with Real-World Samples. Electroanalysis. 2014, 26(12), 2677–2685.

Vasilescu A, Vezeanu A, Badea M, Electrochemical Impedance Spectroscopy investigations focussed on food allergens, Sensing in Electroanalysis, Vol. 8 (K. Kalcher, R. Metelka, I. Švancara, K. Vytras; Eds.), pp. 59-83.2013/2014 University Press Centre, Pardubice, Czech Republic. ISBN 978-80-7395-782-7 (print); ISBN 978-80-7395-783-4 (pdf)

Andrei V., Ngounou Wetie A.G, Mihai I., Darie C.C, Vasilescu A., Detection of Biomedically Relevant Stilbenes from Wines by Mass Spectrometry, Advances in Experimental Medicine and Biology, 2014; 806, 361-382. DOI:10.1007/978-3-319-06068-2_17

Vasilescu A, Vezeanu A., Liu Y., Hosu I.S., Worden M.R., Peteu S.F, Meat Freshness: Peroxynitrite's Oxidative Role, Its Natural Scavengers, and New Measuring Tools, in Instrumental Methods for the Analysis and Identification of Bioactive Molecules, ACS Symposium Series, vol .1185, 2014, 303-332

Vasilescu A, Mihai I, Vezeanu A, Andrei V, Nedelcu G, Popa E, Purcarea C, Subramanian P, Coffinier Y, Boukherroub R, Szunerits S, A new nanomaterial for the study of protein interactions in New applications of Nanomaterials, Series in Micro and Nanoengineering vol 22, editors: Alina Catrinel Ion, Dan Dascalu, Gabriela Carja, Magdalena Lidia Ciurea, publisher: Editura Academiei Romane, 2014, 19-32, ISBN: 978-973-27-2434-7.

Gheorghiu M., Enciu A.M., Popescu O. B, Gheorghiu E., Functional and molecular characterization of Aβ42 effect on an in vitro epithelial barrier model, Journal of Alzheimer's Disease (2014) 38, 787–798



David S., Polonschii C., Gheorghiu M., Bratu D., Dobre A., Gheorghiu E., Assessment of pathogenic bacteria using periodic actuation, Front Cover, Lab on a Chip (2013), 13, 3192–3198

Bunea A.I., Pavel I.A., David S., Gáspár S., Modification with hemeproteins increases the diffusive movement of nanorods in dilute hydrogen peroxide solutions, Chem. Comm. (2013) 49, 8803-05

Subramanian P, Alesniewski A, Kaminska I, Vlandas A, Vasilescu A., Niedziolka-Jonsson J., Pichonat E, Happy H, Boukherroub R, Szunerits S., Lysozyme detection on aptamer functionalized graphene-coated SPR interfaces, Biosens. Bioelectron. (2013) 50, 239-24,3

Gheorghiu M., David S., Polonschii C., Olaru A., Gáspár S., Bajenaru O., Popescu O. B, Gheorghiu E., Label free sensing platform for amyloid fibrils effect on living cells, Biosens. Bioelectr. (2014) 52, 89–97

Olaru A., Gheorghiu M., Polonschii C., David S. and Gheorghiu E., ”Quality assessment of SPR sensors chips; case study on L1 chips” Biosens. Bioelectr. (2013) 45C, 77-81

M. Gheorghiu, S. David, C. Polonschii, D. Bratu, E. Gheorghiu “Dynamic assessment of Amyloid oligomers – cell membrane interaction by advanced impedance spectroscopy” J. Phys.: Conf. Ser. 434 (2013) 012090

M. Gheorghiu, D. Bratu, A. Olaru, C. Polonschii, E. Gheorghiu “Revealing membrane potential by advanced impedance spectroscopy: theoretical and experimental aspects” J. Phys.: Conf. Ser. 434 (2013) 012087

Gáspár S., Marty J. L., Gheorghiu E., Cytochrome c-Based Amperometric Sensors for Superoxide Detection: Where Their Signal Comes From?, Electroanalysis (2013) 25, No. 2, 448 – 452;

Polonschii C., Bratu D., Gheorghiu E., Appraisal of fish behaviour based on time series of fish positions issued by a 3D array of ultrasound transducers, Aquacultural Eng (2013), 55, 37– 45

Vasilescu A., Gáspár S., Mihai I., Tache A. and Litescu S.C., Development of a label-free aptasensor for monitoring the self-association of lysozyme, Analyst, (2013), 138 (12), 3530 – 3537

Wang. Q, Vasilescu A, Subramanian P, Vezeanu A., Andrei V., Coffinier Y, Li M, Boukherroub R., Szunerits S., Simultaneous electrochemical detection of tryptophan and tyrosine using boron-doped diamond and diamond nanowire electrodes, Electrochem Comm, 35 (2013) 84–87

Vasilescu A., Gáspár S., Schuhmann W, Recent progress in the electrochemical detection of disease-related diagnostic biomarkers in " Detection challenges in clinical diagnostics", editors Vadgama P& Peteu S, publisher: RSC, 2013

A. Vasilescu., E. Sharpe, S. Andreescu “Nanoparticle-Based Technologies for the Detection of Food Antioxidants”, Current Analytical Chemistry, (2012), 8 , 495-5058

S. Gáspár, S. David, C. Polonschii, I. Marcu, M. Gheorghiu, E. Gheorghiu “Simultaneous impedimetric and amperometric interrogation of renal cells exposed to a calculus-forming salt”, Anal Chim Acta. (2012) 713: 115-20

M. Gheorghiu, C. Polonschii, S. David, A. Olaru, E. Gheorghiu “SPR Bioanalytical platform to appraise the interaction between antimicrobial peptides and lipid membranes”, In Optical Nano- and Microsystems for Bioanalytics, Series Chemo- and Biosensors 10 (Series Editor Gerald Urban), Springer (2012) pp 183-210



S. Andreescu, M. Gheorghiu, R. E. Ozel, K. Wallace Methodologies for Toxicity Monitoring and Nanotechnology Risk Assessment, chapter 7 in ACS books series "Biotechnology and

Nanotechnology Risk Assessment: Minding and Managing the Potential Threats around Us" Editor(s) Steven Ripp and Theodore B. Henry, Vol. 1079 Publication Date (Web) October 18, (2011)

S. Gáspár, "Detection of Superoxide and Hydrogen Peroxide from Living Cells Using Electrochemical Sensors" in "Oxidative Stress: Diagnostics, Prevention, and Therapy", vol. 1083, American Chemical Society, 2011, pp. 289–309

S. Peteu, S. Szuneritz, A. Vasilescu, W. Knoll "Nanoscale Architectures for Smart Bio-Interfaces: Advances and Challenges" in "Nanofabrication", editor: Yoshitake Masuda, INTECH, ISBN 978-953-307-912-7, 2011

E. Gheorghiu “Relating membrane potential to impedance spectroscopy”, Journal of Electrical Bioimpedance, (2011) 2, 93–97

T. Sandu, D. Vrinceanu, E. Gheorghiu “Surface Plasmon Resonances of Clustered Nanoparticles”, Plasmonics, (2011) 6, 2, 407–412

S. Gaspar, C. Niculite, D. Cucu, I. Marcu "Effect of calcium oxalate on renal cells as revealed by real-time measurement of extracellular oxidative burst", Biosensors and Bioelectronics (2010) 25, 1729-1734

T. Sandu, D. Vrinceanu, E. Gheorghiu “Linear dielectric response of clustered living cells” PHYS. REV. E (2010) 81, 021913 1-11

C. Polonschii, S. David, S. Tombelli, M. Mascini, M. Gheorghiu "A novel low-cost and easy to develop functionalization platform. Case study: Aptamer-based detection of thrombin by surface plasmon resonance" Talanta 80 (2010) 2157–2164

A. Olaru, M. Gheorghiu, S. David, T. Wohland , E. Gheorghiu "Assessment of the multiphase interaction between a membrane disrupting peptide and a lipid membrane", J. Phys Chem B (2009), 113, 14369

M. Gheorghiu, A. Olaru, A. Tar, C. Polonschii, E. Gheorghiu "Sensing based on assessment of non-monotonous effect determined by target analyte: Case study on pore-forming compounds", Biosens. Bioelectron. 24 (2009) 3517

E. Gheorghiu, M. Gheorghiu, S. David, C. Polonschii "Biodynsensing: sensing through dynamics of hybrid affinity / cellular platforms; towards appraisal of Environmental and Biological Risks of Nanobiotechnology" in NATO Science for Peace and Security Series B: Physics and Biophysics , Magarshak, Yuri; Kozyrev, Sergey; Vaseashta, Ashok K. (Eds.) 2009, ISBN: 978-90-481-2522-7

L. Muresan, M. Nistor, S. Gaspar, I.C. Popescu, E. Csoregi “Multianalyte monitoring using enzyme microstructures and Scanning Electrochemical Microscopy”, Bioelectrochemistry 2009, 76, 81-86

C. Balan, D. Broboana, E. Gheorghiu, L. Vekas “Rheological characterization of complex fluids in electro-magnetic fields” Journal of Non-Newtonian Fluid Mechanics 154 (2008) 22–30



E. Gheorghiu “Appraisal of cellular systems using impedance spectroscopy – theoretical and experimental aspects”, IFMBE Proceedings, Springer, vol 17, 2007, part 4, 81-85

M. Gheorghiu, S. David, C. Polonschii, D. Bratu, E. Gheorghiu “Biosensing and controlled interaction with cellular systems via structured interfaces”, Eur. Cel. Mater. Vol14 , S3, 2007, p 63

M. Gheorghiu, S. David and E. Gheorghiu “Sensing the cell- substrate interaction towards development of “smart” surfaces, IFMBE Proceedings, Springer, vol 17, 2007, part 4, 86-89

C. Balut , M. Vandeven, S. Despa, I. Lambrichts, M. Ameloot, P. Steels, I. Smets “Measurement of cytosolic and mitochondrial pH in living cells during reversible metabolic inhibition”, Kidney Int. 2007, 17978815

S. David, M. Gheorghiu, C. Polonschii and E. Gheorghiu “Dual SPR-Impedance Measurement System for detection of bioaffinity interactions”, IFMBE Proceedings, Springer, vol 17, 2007, part 4, 106-109

C. Polonschii, D. Bratu and E. Gheorghiu “Multi Frequency, Multi Channel, Differential Impedance Analyzer for Rapid Assays”, IFMBE Proceedings, Springer, vol 17, 2007, part 4, 229-231

F. Jans , C. Balut, M. Ameloot , P. Wouters, P. Steels “Investigation of the Ba-Sensitive NH(4)(+) Transport Pathways in the Apical Cell Membrane of Primary Cultured Rabbit MTAL Cells”, Nephron Physiol. 2007 Jun 15;106 (3):p45-p53 17570948

K. Zor, S. Gaspar, M. Hashimoto, H. Suzuki, E. Csoregi “High temporal resolution monitoring of fermentations using an on-line amperometric flow-through microdetector”, Electroanalysis, 2007, 19(1), 43-48.

M. Hedstrom, C.E. Grey, S. Gaspar, B. Mattiasson “Miniaturized on-line digestion system for the sequential identification and characterization of protein analytes” J. Chromatography A, 2007, 1146(1), 17-22.

M. Schuvailo, S. Gaspar, A.P. Soldatkin, E. Csoregi “Ultramicrobiosensor for the selective detection of glutamate”, Electroanalysis, 2007, 19(1), 71-78

M. Gheorghiu, S. David, C. Polonschii, E. Gheorghiu “Sensing at nanoscale via structured interfaces” Eur Biophys J. (2007) 36 S157

C.M. Balut, P. Steels, M. Radu, M. Ameloot, W. Van Driessche, D. Jans "Membrane Cholesterol Extraction decreases Na+ Reabsorption in A6 Renal Epithelia", AJP - Cell, 290 (1), C87-94, 2006

D. Cucu D, J. Simaels, J. Eggermont, W. Van Driessche, W. Zeiske “Opposite effects of Ni2+ on Xenopus and rat ENaC expressed in Xenopus oocytes”, Am J Physiol Cell 289(4) 2005 C946-58

C.M. Balut, D. Jans, W. Van Driessche, P. Steels "How membrane cholesterol depletion regulates Na+ transport in A6 renal epithelial cells ", J Am Soc Nephrol 16, 2005

D. Andreescu, A.K. Wanekaya, A. O. Sadik, J. Wang “Nanostructured polyamic acid membranes as novel electrode materials”, Langmuir, Vol 21. No 15. p. 6891-9 (2005)

D. Cucu, J. Simaels, D. Jans, W. Van Driessche “The transoocyte voltage clamp: a non-invasive technique for electrophysiological experiments with Xenopus laevis oocytes”, Pflügers Arch 44, 2005, 934-942.



M. Gheorghiu, W. Van Driessche “Modeling of basolateral ATP release induced by hypotonic treatment in A6 cells” 2004. Eur Biophys J.vol 33 No 5, 412-420

C.M. Balut, A. Segal, D. Cucu, W. van Driessche, P. Steels "Effects of membrane cholesterol on epithelial sodium channels (ENaC) present in A6 cells and expressed in Xenopus laevis oocytes", Pfluegers Archives, 447, 2004

P. Steels, D. Jans, W. van Driessche, C.M. Balut “Membrane Cholesterol Depletion in A6 cells depresses activated sodium transport and elicits a transient chloride secretion during a hypotonic shock", J Am Soc Nephrol 15, 2004

C. Balan, C.M. Balut, L. Gheorghe, C. Gheorghe, E. Gheorghiu, G. Ursu “Experimental determination of blood permittivity and conductivity in simple shear flow”, Clinical Hemorheology and Microcirculation, Vol. 30, Nr. 3-4, 359 – 364, 2004

E. Gheorghiu, E., D. Andreescu, M. Oporanu, M. Gheorghiu, S. Cazacu, C. Balut, A. Ursu “Impedance spectroscopy in biodynamics: Detection of specific cells (pathogens) using immune coated electrodes”, Songklanakarin J. Sci. Technol., 2002, 24(Suppl.) : 791-797

O.A. Sadik, E. Gheorghiu, H. Xu, D. Andreescu, C. Balut, M. Gheorghiu, D. Bratu, "Differential Impedance Spectroscopy for Monitoring Protein Immobilization and Antibody-Antigen Reactions", Analytical Chemistry, 74 (2002), 3142-3150

E. Gheorghiu, C. Balut, M. Gheorghiu, "Dielectric behaviour of Gap Junction Connected cells: a Microscopic Approach", Physics in Medicine and Biology, 47 No 2 (2002) 341-348.

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