Sensitive and selective detection of neurotoxic insecticides based on cholinesterase biosensors coupled with solid phase extraction (CHOLIN-SP)

PN-III-P2-2.1-PED-2016-0367 (contract 45PED/2017)

 

 

 

 

Abstract:

This applicative project aims to develop a selective and sensitive analytical method useful for screening of neurotoxic insecticides in environmental samples based on a combination of the specific advantages of acetylcholinesterase (AChE) biosensors and solid phase extraction (SPE). The biosensors provide the low-cost and easy to use analytical devices while the sample pretreatment with SPE ensures the required sensibility and results reliability due to interference removal from unknown samples. The successful combination of biosensor with SPE is based on optimization of the enzymatic inhibition buffers that contains optimized percentages of various water miscible organic solvents and choice of the appropriate sorbent-elutant for analyte separation. A heterogeneous oxidation of the organophosporothionate insecticides on the SPE column will be tested for improved sensitivity. The consortium combines the vast experience in the field of cholinesterase biosensors and enzymology of National Institute of R&D for Biological Sciences with the know-how of S.C. CP MED Laboratory S.R.L. in solid-phase extraction, reliable analyses for environmental pollution monitoring and method validation.

 

Key words: Cholinesterase biosensors, solid phase extraction (SPE), neurotoxic insecticides, enzymatic inhibition, environmental monitoring

 

 

 

 

 

 

 

 

Partners:

A) Project coordinator

 

The Centre of Bioanalysis from the National Institute of Research and Development for Biological Sciences has all the equipment necessary: 2 potentiostat-galvanostat Autolab 302N each one with supplementary modules for impedance measurements, low currents for microelectrodes (pA), bi-potentiostat for 2 working electrodes, pH/potentiometric measurements; 1 portable potentiostat galvanostat PG 580 (Uniscan Instruments); 1 UV-Viz spectrometer Zenyth 200rt for ELISA plates with module for 1 cm classic cuvets; thermostate ability; possibility for kinetic measurements; quartz crystal microbalance PAR-QCM922 (1-9 MHz) for simultaneous measurement of frequency and resistance; batch & flow cells; flow analysis system - flexible, various components, electrochemical and spectrometric cells. (http://erris.gov.ro/Bioanalysis_Bioanaliza).

Coordinator team:

Project director dr. Bogdan Bucur- has obtained his Ph.D. in 2005 in a co-direction of University of Bucharest with Perpignan University (France). The subject of his thesis was development of biosensors based on AChE, under the supervision of prof. Jean Louis Marty- a renowned world expert in this field. His research interest are: immobilization of enzymes on electrode surface, new enzymatic biosensors for substrate and inhibition detection with reduced interferences, development of and characterization of chemical modified electrodes, flow methods of analysis, food and environmental analysis. He is coauthor of more than 30 original papers in international peer-reviewed journals and 3 Romanian patents (http://www.researcherid.com/rid/B-8151-2009; H index: 11). He has been a researcher in 7 European contracts, 1 French contract and is the director of a bilateral contract with Perpignan University (France). His has extensive experience in national grants and was director/scientific responsible for 5 research grants.

Prof. dr. eng. Gabriel-Lucian RADU, head of the Bioanalysis Centre and professor at the University Politehnica of Bucharest, Faculty of Applied Chemistry and Material Science. His research interest is concerned on: analytical chemistry and bioanalysis; characterization of biological substances by specific analytical methods and techniques; environmental monitoring (biosensors, electrochemical analysis); food analysis, biotechnology (quality control and quality assurance). He has a great managerial expertise proved by a large number of national and bilateral projects he has coordinated and by his large scientific research expertise, embodied in 15 books, over 100 full scientific papers and over 70 papers in national and international conferences, complemented by a sustained university professor teaching performing and Ph.D. coordinator (http://www.researcherid.com/rid/F-7766-2011; H index: 11). He has been partner in two FP7 projects.

Dr. Petruta Madalina Bucur (Dondoi) has a PhD in Chemistry since 2007 at University of Bucharest in the field of biosensors for environmental monitoring. Her research interest covers different domains of analytical chemistry such as: enzymatic biosensors, electrochemical sensors, piezoelectric analysis, enzymatic biosensor development for substrate or inhibitor detection, different methods for enzyme immobilization and stabilization, analytical devices based on lectin bioaffinity for carbohydrates, use of electrochemical mediators. She improved the organophosphoric insecticides detection limits of biosensors based on AChE. She is author/coauthor of 17 ISI full papers published and 4 Romanian patents (http://www.researcherid.com/rid/B-8151-2009; H index: 5). She was director/scientific responsible for 3 Romanian grant. She was involved in a research team FP5 project ROSEPROMILK, Leonardo da Vinci project Training module for environmental pollution control and more than 10 national project (CERES, CEEX, PNII, TE).

Dr. Maria-Cristina Radulescu (Rizea) has obtained her Ph.D. in Chemistry in 2008. Her domains of competences are analytical chemistry and biochemistry such as: electrochemical sensors, enzymatic biosensors, piezoelectric analysis methods, automated analysis methods, spectrophotometry and in the field of separation based on chromatography. She is coauthor of 11 ISI full paper published (http://www.researcherid.com/rid/B-6576-2011; H index: 4). She worked as researcher in a FP5, a FP7 and a Leonardo da Vinci European projects and as scientific responsible in a Romanian project.

Teoaca Nela (technician).

B) Project SME partner team (S.C. CP MED Laboratory S.R.L.):

S.C. CP MED Laboratory S.R.L. with the equipment: GC/MS Agilent Technologies 5975 Series with numerous columns such as CP-Sil 24 CB (medicines and pesticides), DB-VRX (volatile substances), DV-EUPAH (polycyclic aromatic hydrocarbons), DB5-MS (general use); Atomic Absorption System with Graphite Tube Atomizer and Hydride Generator Agilent Technologies 240 AA with lamps from Hach Company for detection of Zn, Hg, Co, Pb, Mn, Ba, As, ni, Cu, Cd, Cr and Se; UV-Vis Spectrophotometer Hach Company DR 2800 and all the necessary smaller devices, glassware and consumables that are characteristic for an analytical chemistry laboratory. (https://erris.gov.ro/Env-Analyses-Laboratory).

Partner team:

Partner responsible eng. Ligia Milea has vast experience in environmental water analysis and validation of the analytical methods at laboratory and analyte level following national regulatory procedure.

Bogdan Purcareanu (Ph. D. student at University Politechnica of Bucharest with a GS-MS and SPE thematic; 2 years laboratory experience), Gheorghe Trifan (30 years experience in environmental monitoring and volatile substances analysis), Alexandra Marin (3 years experience of environmental samples analysis of heavy metal analysis using validated methods AAS) and Mariana Apostol (one year experience at National Institute of Metrology Bucharest, Romania followed by one year experience in environmental analyses); environmental protection: Ana Stanescu (has a MSc Masters Degree: Environmental Policies and Management, Department of Social Sciences and Law, University of Bristol, UK); results valorization: dr. Sorin Axinte (18 years of experience in spin-off development based on IPR valorization and market transfer of the output obtained in research projects).

 

Financial resources:

Year

Public budget

Own budget

Total

2017

431,186.00

45,540.00

476,726.00

2018

168,814.00

10,510.00

179,324.00

Total

600,000.00

56,050.00

656,050.00

 

Phases:

2017: Development of analytical methods for insecticide detection. Multiparametric study of the couple cholinesterase biosensor-solid phase extraction

In this phase there were tested and optimized biosensors for insecticide detection in buffers that contain also organic solvents miscible with water. The studies were carried out initially spectrometrically in order to measure the influence of the organic solvents on the enzyme free in solution. Subsequently, amperometric measurements were made to separate the incubation phase from the measurement of the enzymatic activity and to investigate the effect of the immobilization matrix.

 

Results for spectrometric measurements

 

Solvent (5 v/v)

% inhibition (negative numbers imply an activation)

With 10 min incubation

without incubation

Isopropanol 5%

63.7

52.3

Methanol 10%

9

-25.8

Acetonitrile 5%

57

35

Ethanol 5%

5

-12.5

 

 

Results for amperometric measurements

organic solvent (% v/v)

5% acetonitrile

5% ethanol

10% ethanol

10 % methanol

5% isopropanol

% inhibition

55.1

-0.82

-12.63

-14.39

-9.69

 

A GC-MS analysis method was develop for the detection of a carbamate insecticide (Methomyl) and 3 variants of an organophosphate insecticide (Chlorpyrifos-methyl, Chlorpyrifos methyl-O-analog and Chlorpyrifos-ethyl). The method was able to separate all compounds and quantify their concentration.

 

 

2018: Method validation by comparison with chromatography technique

The biosensors were developed by immobilization of the acetylcholinesterase together with two stabilizers (hydroxy-ethyl cellulose HEC and poly-ethylene glycol PEG) into a sol-gel microporous matrix in order to mitigate the negative effects of the organic solvents on the enzyme. The sol-gel immobilization process consists in fast deposition on the electrode surface of 1.5 mL a mixture of three different ice-cooled solutions: (i) 25 mL 3% HEC and 15% graphite with TCNQ mediator electrochemical for thiols, (ii) 25 mL enzyme prepared in phosphate buffer pH=7.0 for condensation of the silica and (iii) 25 mL hydrolyzed silica sol-gel precursors.

The biosensors were used with two different concentrations of the organic solvents: (i) 10% ethanol + 3,3 % acetonitrile with a minimum influence on the enzyme activity and (ii) 15% ethanol + 5 % acetonitrile to minimize the insecticide after elution from SPE columns. The tests carried out by biosensors incubation during 10 min in phosphate buffer with 10% ethanol + 3,3 % acetonitrile have demonstrated a minimum impact on the enzyme as the remnant activity was higher than 90 % (a decrease higher than 10 % being considered significant). The inhibition measurement using chlorpyriphos and paraoxon demonstrated only a slight increase of the inhibition recorded in buffer with a mixture of the organic solvents in comparison with buffer, this effect being compensated by a proper calibration.

The alternative analytical technique is based on GS-MS, a high-performance technique that is able to both detect and discriminate a large number of neurotoxic insecticides. The samples were pretreated by SPE and eluted using organic solvents with an automated extractor. Various tests were carried out to study the reproducibility of the measurements, long term sample and reagent storage stability and investigation of real environmental water samples collected from various sites.

 

20180627_175249

 

Dissemination (2017-2018):

 

Articles:

1. A. Chira, B. Bucur, G.-L. Radu: Electrodeposited Organic Layers Formed from Aryl Diazonium Salts for Inhibition of Copper Corrosion, Materials, 10, 235, 2017; DOI: 10.3390/ma10030235

2. Madalina-Petruta Bucur, Bogdan Bucur and Gabriel Lucian Radu: Simple, selective and fast detection of acrylamide based on glutathione S-transferase, RSC Advances, accepted 2018

Conferences:

1. P. Bucur, B. Bucur, Gabriel Lucian Radu: Fast detection of acrylamide based on amperometric measurements using glutathione S-transferase, 8th international workshop on biosensors for food safety and environmental monitoring, Rabat-Morocco- October 12 - 14, 2017 (oral presentation)

2. 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) , 8th International Workshop on Surface Modification for Chemical and Biochemical Sensing SMCBS2017, Zelechw, Poland, November 3-7, 2017 (poster)

3. Madalina Petruta Bucur, Bogdan Bucur, Gabriel-Lucian Radu: Immobilization of cholinesterase in sol-gel protective microporous matrix for insecticide detection in organic-solvents mixtures, Electrochemistry for Nano & Nano for Electrochemistry ElecNano8, 29-31 May 2018, Nancy, France (poster)

4. Maria-Cristina Radulescu, Madalina-Petruta Bucur, Bogdan Bucur, Gabriel Lucian Radu: Carbon fiber composite paper electrodes modified with stable Prussian blue-copper used for bienzymatic determination of sweeteners in food, Electrochemistry for Nano & Nano for Electrochemistry ElecNano8, 29-31 May 2018, Nancy, France