WO2021054917A2

WO2021054917A2 - A method and biosensor for determination and measurement of deltamethrin

Info

Publication number: WO2021054917A2
Application number: PCT/TR2020/050836
Authority: WO
Inventors: Ebru SAATCI; Osman Tolga KASKATI
Original assignee: T.C. Erciyes Universitesi
Priority date: 2019-09-18
Filing date: 2020-09-14
Publication date: 2021-03-25
Also published as: WO2021054917A3

Abstract

The invention relates to a method for the determination and measurement of deltamethrin. Said method comprises measuring deltamethrin concentration amperometrically with a biosensor comprising acetylcholinesterase immobilized on a magnetic bead as a biorecognition element.

Description

A METHOD AND BIOSENSOR FOR DETERMINATION AND MEASUREMENT OF

DELTAMETHRIN

Technical Field

The invention relates to the determination and measurement methods of pesticide carried out with enzyme-based biosensors.

The invention particularly relates to a determination and measurement method carried out with an enzyme-based biosensor to determine and measure deltamethrin.

State of the Art

Pesticides are the chemicals that have economic benefits in hunger, war in terms of protecting the public health and agricultural products when used appropriately, in suitable doses and consciously. Nevertheless, they damage the ecological balance by causing water, soil and air pollution with their residues left in large areas. Moreover, they cause some unwanted situations by negatively affecting the normal functioning of the organisms by accumulating in various organs in time, even in tiny amounts through entering in the living body by various ways (mouth, respiration, and skin). Insecticide is the group commonly used against pests among pesticides. Synthetic pyrethroids constitute 30% of the insecticides used in the world. Synthetic pyrethroids are the nervous system poisons that exhibit their toxic effects on the sodium channels in the mammals and/or insects and affect axons in the peripheral and central nervous system (International Programme on Chemical Safety Environmental Health Criteria 98 Tetrametrin, World Health Organization, Geneva, 1990).

Although pesticides are very strongly associated with many health problems, there are severe deficiencies in following and determining these pollutants. Conventional chromatographic methods such as high performance liquid chromatography, gas chromatography, capillary electrophoresis, mass spectrometry etc. are effective methods for pesticide analysis in foods. However, it has many constraints, such as complex processes, time-consuming preparation steps, the need for expensive devices and specialist personnel. Biosensors based on acetylcholinesterase inhibition have been developed to monitor the pesticide residues for the control and safety of food, which are simple, fast and ultra sensitive devices. These biosensors can substitute the conventional analytical methods by making the tests required to be made on-site and in real-time easier and faster with a substantial decrease in the cost per process by facilitating or eliminating sample preparation. Moreover, these devices can be miniaturized easily and are more sensitive against interventions that can be performed by other compounds in complex matrixes.

Bioreceptor (biocomponent) and transducer constitute two critical parts of biosensors. Enzymes, antibodies, nucleic acids, microorganisms, organelles, and tissue cultures are used as bioreceptors. In addition to this classification type, they are classified as potentiometric, amperometric, piezoelectric, optical, and thermal biosensors according to the measurement method. In enzyme-based biosensors, there is a bioactive part in which the enzyme is immobilized on the working electrode and an appropriate transducer is placed for the detection of the reaction catalyzed by the enzyme. The transducer system is a part of the biosensor, used to determine the decrease in coenzyme and/or substrate concentration or increase in product concentration, resulting from the enzymatic reaction on the bioactive layer. The signal to be detected by the transducer system is related to the analyte concentrations on the bioactive layer-conductive interface (Digkaya, E. 1999. Enzim Sensorleri. Biyosensorler, Biyokimya Lisansustu Yaz Okulu Kitabi. Editor: Telefoncu A., 81-139). Mostly amperometric, potentiometric, chemical radiation and thermal transducers are preferred in the enzymatic sensors (Soldatkin, A. P., Arkhypova, V.N., et all. 2005. Analysis of potato glycoalkaloids by using of enzyme biosensor based on pH-ISFETs. Talanta, 66: 28-33; Jenkins, D. M., and Delwiche, M.J. 2002. Manometric biosensor for on-line measurement of milk urea. Biosensors and Bioelectronics, 17: 557- 563). The enzyme sensors are preferred in many fields, from food to medical, from environment to defense due to their advantages provided by their high precision, specificity, ease of use, and small size.

The enzymatic biosensors developed in state of the art allow for monitoring the phosphorus and carbamate classes of pesticides by inhibiting the enzyme-based reaction between the acetylcholinesterase (AChE) and the substrate acetylhiocholine (AThCh). These compounds inhibit the biocatalytic activity by blocking the serine residue in the catalytic three of AChE (histidine, serine, and aspartic acid) utilizing phosphorylation and carbonylation by binding to the active stearic region of the enzyme (Singh, D.K., Agarwal, R.A., 1983. Inhibition kinetics of certain organophosphorus and carbamate pesticides on acetylcholinesterase from the snail Lymnaea acuminata. Toxicology Letters 19: 313— 319).

For example, in patent application numbered CN102103122 (A), an AChE based biosensor for pesticide determination using electrochemical methods is disclosed. The acetylcholinesterase is immobilized to the chitosan membrane in said biosensor. In this application, specifically, a biosensor used for the determination of deltamethrin is not disclosed.

In the patent application numbered CN101581670 (A), an acetylcholinesterase based fluorescence biosensor for determining organophosphorus pesticide residues in food is disclosed.

Moreover, nanotechnological detection methods are developed in the last years. Generally, iron, gold, silver and silica nanoparticles are used in such applications. There are various disadvantages in relation to the use of mentioned nanoparticles. For example, the iron nanoparticles generally tend to be unstable and aggregate. Moreover, gold and silver nanoparticles cannot be used in large-scale applications since they are expensive.

In the patent application numbered CN106248748 (A), a biosensor which is immobilized on pd-Pt alloy supported MnC nanoparticles using acetylcholinesterase chitosan is disclosed. The determination of pesticides such as mainly methyl parathion and carbofuran etc. is made in the mentioned biosensor.

Gandhali Bapat et al. developed silica nanoparticles to which acetylcholinesterase enzyme (AchE) is bound to determine various pesticides (Advances in Colloid and Interface Science 237 (2016) 1-14).

Deltamethrin from synthetic pyrethroid group, IV. and last generation insecticide has a very high insecticide effect and residual activity when compared with I., II., and III. generation pyrethroid insecticides (Llnal G., Gurkan M. O., 2001. insektisitler Kimyasal Yapilari, Toksikolojileri Ve Ekotoksikolojileri. Ankara Llniversitesi Ziraat Fakultesi Bitki Koruma Bolumij, Ankara, 159 s.). The chemical structure of deltamethrin is shown by Formula 1. Formula 1

Deltamethrin binds to the sodium channels from specific regions and it disrupts the ion permeability in these regions. This way, it creates an acute neurotoxic effect (Zlotkin E., 1999. The insect voltage-gated sodium channel as a target of insecticides Annual Review of Entomology, 44: 429-455). Mainly deltamethrin is widely used in agricultural products and food. Deltamethrin is especially very toxic for aquatic living beings (especially fish). It is mainly applied to the empty silo for the control of warehouse insect pests (http://www.egamceyresagligi.com/depo-zararlilari/). Moreover, since the half-life of deltamethrin is very short, it is difficult to detect the applied concentration directly (Beketov, M.A. 2004. Comparative sensitivity to the insecticides, deltamethrin and esfenvalerate of some aquatic insect larvae ( Ephemeroptera and Odonata ) and Daphnia manga. Russian J Ecol, 35: 200-204). There is no explanation for determining the pyrethroids in the state of the art, particularly deltamethrin with acetylcholinesterase enzyme. For these reasons, precise and specific methods are required to determine and measure deltamethrin.

As a result, an improvement is required in the relevant technical field because of the negativities as mentioned above and existing methods’ insufficiency.

Brief Description of the Invention The present invention is related to a method for determining and measuring deltamethrin and a biosensor for determining and measuring deltamethrin, which fulfills the requirements as mentioned above, eliminates all disadvantages and brings some additional advantages. The main aim of the invention is to provide a fast, precise and specific method and a biosensor to determine and measure deltamethrin.

Another aim of the invention is to provide a method and a biosensor that provides an opportunity to determine deltamethrin from a living organism.

A similar aim of the invention is to provide a method and a biosensor that provides an opportunity to determine deltamethrin from insects in areas subject to deltamethrin such as waters and depots.

Another aim of the invention is to provide a biosensor with an extended life to determine and measure deltamethrin.

A similar aim of the invention is to provide a relatively low-cost, easy to use biosensor to determine and measure deltamethrin.

Another aim of the invention is to provide a method with high measurement precision and a biosensor to determine and measure deltamethrin.

Another aim of the invention is to provide a biosensor which is easy to prepare.

To fulfill the abovementioned aims, the present invention describes a method for determining and measuring deltamethrin; said method comprises measuring deltamethrin concentration amperometrically with a biosensor comprising acetylcholinesterase immobilized on a magnetic bead as a biorecognition element.

In a preferred embodiment of the invention, said magnetic bead is functionalized with protein G.

In another preferred embodiment of the invention, said biosensor comprises antibody produced against estradiol immobilized on protein G and estradiol to which acetylcholinesterase is conjugated.

In another preferred embodiment of the invention, said magnetic bead comprises a core consisting of magnetic materials selected from iron, nickel, cobalt, neonidim-iron-boron, samarian-cobalt or magnetite. In another preferred embodiment of the invention, said core consists of magnetite.

In another preferred embodiment of the invention, said magnetic bead comprises a polymer layer selected from polystyrene, dextran, polyacrylic acid or silica.

In another preferred embodiment of the invention, said polymer layer is polystyrene.

In another preferred embodiment of the invention, the size of said magnetic beads is 1 pm.

In another preferred embodiment of the invention, said biosensor comprises screen- printed graphene electrodes.

In another preferred embodiment of the invention, said electrodes are 8-channel screen printed electrodes.

To fulfill the abovementioned aims, the present invention describes an acetylcholinesterase-based amperometric biosensor comprising a magnetic bead functionalized with protein G and antibody immobilized on protein G, and acetylcholinesterase is conjugated to said antibody through estradiol.

To fulfill the abovementioned aims, the present invention describes a method for the immobilization of acetylcholinesterase onto magnetic beads, functionalized with protein G as the biorecognition element, which comprises the following steps: immobilizing the antibody (anti-estradiol antibody) produced against estradiol to said magnetic bead and conjugating acetylcholinesterase to said antibody through estradiol.

In order to fulfill the abovementioned aims, the present invention describes a process to prepare an amperometric biosensor comprising acetylcholinesterase immobilized on a magnetic bead functionalized with protein G and the biorecognition element, said process comprises the following process steps;

(i) mixing a magnetic bead functionalized with protein G with antibody produced against estradiol (anti-estradiol-antibody) and estradiol conjugated acetylcholinesterase solutions (estradiol-AChE) in an appropriate working buffer;

(ii) incubating the mixture obtained in step (i); (iii) retaining the mixture obtained in step (ii) on magnet for a determined period and washing the same with a buffer solution; and

(iv) adding acetylthiocholine and/or a derivative thereof to the mixture after step (iii).

In a preferred embodiment of the invention, said process comprises the following process steps; monitoring the amperograms of the enzyme reaction at a predetermined potential and for a determined period after the addition of acetylthiocholine and/or a derivative thereof and incubation with the enzyme and finding the % activation amounts over the current values.

In a preferred embodiment of the invention, said potential is 700 mV and said duration is 90 seconds.

In order to fulfill the above mentioned aims, the present invention also describes a method for the determination and measurement of deltamethrin in insects, said method comprises the following steps;

(i) providing samples of insect larvae and/or adults;

(ii) obtaining deltamethrin extracts from insect larvae and/or adult samples by sonication using acetonitrile as an extraction solvent;

(iii) obtaining supernatants from said extracts by centrifugation;

(iv) measuring the current values with an amperometric biosensor wherein obtained supernatants comprise magnetic bead functionalized with protein G, antibody immobilized on protein G, acetylcholinesterase is conjugated to said antibody over estradiol and the substrate is acetylthiocholine and/or a derivative thereof.

In a preferred embodiment of the invention, said insect is flour moth, Tribolium confusum.

The structural and characteristic features of the present invention will be understood clearly by the following drawings and the detailed description made concerning these drawings. Therefore the evaluation shall be made by taking these figures and the detailed description into consideration.

Figures

Figure 1 is a schematic view of preparing the inventive biosensor. Figure 2 is the view of example, functional groups of the state of the art of an inventive magnetic bead.

Figure 3 is the view of a flowchart of deltamethrin extraction from T. confusum larvae.

Figure 4 is the view of a flowchart of deltamethrin extraction from T. confusum adults. Figure 5 is the % activation graph against increasing deltamethrin concentrations.

Figure 6 is the amperogram view of the control (only acetylthiocholine and work mixture) and 2,5-125 pg/ml deltamethrin concentrations.

Figure 7 is the Lineweaver-Burk graph of constant deltamethrin concentrations (0, 10, 25, 50 pg/ml) against acetylthiocholine concentrations (0.1-5 mM). REFERENCE NUMBERS IN THE FIGURES

FIGURE 1 A Magnet B Electrode C Magnetic bead D Protein G E Antibody F Estradiol-AChE G Amperometric measurement FI Acetylthiocholine I Thiocholine J Anodic oxidation

K Dithiobischoline

FIGURE 3 L Subjecting the larvae to pesticide for 24 hours

M Counting and addition N 8 larvae

O Weighing the samples 9,2 ± 0,5 mg P Freezing at -80°C for 10 minutes R 200 mI 85% ACN addition S Homogenization for 3 minutes with glass stick T Sonication

It has been repeated 3 times for 10 seconds with intervals of 10 seconds in ice at 150 W, 20 kHz, 40% amplitude.

V Centrifuge

Y With speed of 5000 g, at 4°C, 10 minutes Z Biosensor application

Q After centrifuge, supernatant has been taken and added to the biosensor mixture. FIGURE 4

X Subjecting the adults to pesticide for 24 hours M Counting and addition a 8 adults

W Removing the adult elytras b Weighing the samples 15 ± 0,5 mg

P Freezing at -80°C for 10 minutes

R 200 mI 85% ACN addition S Homogenization for 3 minutes with glass stick T Sonication

It has been repeated 3 times for 10 seconds with intervals of 10 seconds in ice at 150 W, 20 kHz, 40% amplitude

V Centrifuge

Y With speed of 5000 g, at 4°C, 10 minutes Z Biosensor application

Q After centrifuge, supernatant has been taken and added to the biosensor mixture.

Detailed Description of the Invention

In this detailed description, the inventive method and biosensor to determine and measure deltamethrin are described only for clarifying the subject matter in a manner such that no limiting effect is created. Said method and biosensor provide the determination of deltamethrin from the samples obtained from a living organism. Therefore, the measurement of deltamethrin on insects without decaying is provided.

Acetylcholinesterase (AChE) is termed with different names such as real cholinesterase, erythrocyte cholinesterase and acetylcholinestyl hydrolase and is an enzyme that is included in hydrolases. AChE is found in the brain and erythrocytes in high concentrations and its main aim is to end cholinergic neurotransmission. The reaction is provided by rapidly converting acetylcholine (ACh), which is an essential neurotransmitter, into choline and acetic acid in cholinergic and neuromuscular synapses. AChE is mainly used in determining the organophosphate pesticides. The efficacy mechanism of organophosphates is based on the inhibition of AChE activity. Inhibition of AChE stimulates ACh receptors excessively by causing accumulation of ACh in synapses and neuromuscular junctions. As a result of this, cholinergic syndrome occurs.

With the present invention, deltamethrin changes the activity of acetylcholinesterase enzyme (AChE) under in vitro conditions. In this sense, it is determined that deltamethrin exhibits an activating effect on AChE activity. Thus the activity of AChE in the presence of deltamethrin increased depending on the deltamethrin concentration. Said activation can be measured with a biosensor according to the present invention. Said biosensor is based on monitoring the change in AChE activity by electrochemical methods. The inventive biosensor is an AChE based biosensor, and AChE is used as the biorecognition element.

With reference to Figure 1 , the inventive biosensor works by means of measuring the analytic signal in amperes based on AChE activation in the presence of deltamethrin. The substrate of acetylcholinesterase (AChE) in nature is acetylcholine (ACh). In the inventive biosensor, acetylthiocholine (ATCh), an electro-active substrate of acetylcholinesterase (AChE), is used. AChE catalyzes acetylthiocholine (ATCh) hydrolysis and thus the formation of thiocholine (TCh) and acetic acid. Said reaction is shown in Reaction I at the following.

Acetylthiocholine + H20 + AChE Thiocholine (TCh) + Acetic Acid (Reaction I)

Thiocholine oxidizes quickly with a specific, constant voltage and generates an electric current. In other words, anodic oxidation of thiocholine forms the electric current that causes a measurement of the enzyme-based/catalytic activity. Anodic oxidation is given in Reaction II in the following.

2TCh (reduced) Dithiobischoline (oxidized) + 2H⁺ + 2e^~ (Reaction II)

The current signal’s magnitude is associated with the catalytic/enzyme-based activity under constant substrate concentration conditions. In AChE-based biosensors in state of the art, the pesticide included in the sample forms phosphorylated AChE by binding to the active hydroxyl group of AChE. Phosphorylated AChE does not exhibit catalytic activity for acetylthiocholine.

The invented method for determining and measuring deltamethrin is based on the principle of detecting the changes in the current due to the oxidation or reduction of reactants or products in the environment under constant application voltage. The inventive biosensor is an amperometric biosensor. In amperometric biosensors, the current change, formed due to the biochemical reaction, on the working electrode of the products is measured. The changed current is directly proportional to the analyte concentration in the sample. In the present invention, it is found that the enzymatic activity increases when deltamethrin is in the measured sample. Thus there is an increase in the amount of the current. This increase in the current amount can be measured with an amperometric biosensor according to the present invention. Therefore, the change in AChE activity, based on deltamethrin concentration, provides the selective identification and quantification of deltamethrin.

Amperometric sensors are preferred since they are precise and easy to use. Amperometric biosensors are efficient in the applications in various fields such as environment, industry and clinics etc. and they are sensitive, safe, and cost-effective.

Structure and components of the biosensor

Biorecoanition element

In the inventive biosensor, acetylcholinesterase (AChE) is used as the biorecognition element. It is found that with the present invention, AChE activity increases depending on the deltamethrin concentration. Said biosensor is a biosensor based on the AChE enzyme activation and amperometric measurement of this activation. The biosensor comprises AChE immobilized on magnetic beads.

Magnetic beads

Said magnetic bead comprises a core consisting of magnetic materials selected from iron (e.g., Fe2C>3 ve FesC ), nickel, cobalt, neonidim-iron-boron, samarian-cobalt or magnetite etc. There is a thin polymer layer on said core which defines the surface area to adsorb or bind various molecules. Said polymer layer might be a polymer layer consisting of polystyrene, dextran, polyacrylic acid, or silica. Preferred magnetic beads have a magnetite core and a polystyrene layer.

With reference to Figure 2, said polymer layer could be modified with functional groups (e.g., streptavidin, tosyl groups, amino groups, carboxylic acid, antibody or proteins). Peptides, small molecules, proteins, antibodies and nucleic acids can be bound on the surface of the magnetic bead through said functional groups.

In the inventive biosensors, the binding of antibodies over Fc groups by preferring magnetic beads on which there is protein G, which is functionalized with protein G, is provided. Therefore, the immobilization of AChE that is the biorecognition element is provided rapidly and specifically on the magnetic beads. AChE is bound on the magnetic beads over the antibody. There are magnetic beads sold in the market that are functionalized with protein G. In the inventive biosensor, the immobilization requirement of AChE directly on electrodes by using magnetic beads is eliminated. Thus the electrodes can be used more than once when required.

With reference to Figure 1 , in the inventive biosensor, there are magnetic beads functionalized with protein G and antibody (anti-estradiol-antibody) which is immobilized to protein G. The antibody is bound to the protein G with Fc section. The acetylcholinesterase (AChE) enzyme is conjugated to said antibody over estradiol. Flere, the aim is to bind the enzyme to magnetic beads in a shorter way and without losing its activity. Thus AChE does not lose its main active structure. It is thought that the activation of AChE by deltamethrin realizes with this binding method.

Magnetic beads may generally vary in size from a few nanometers (5-50 nm) to a few micrometers (1-10 pm). Preferably, the size of the magnetic beads is 1 pm. Said beads serve for effective transport, rapid measurement kinetics, binding specificity increase and marker. Said magnetic beads can be easily separated after being collected pulling the same from the fluid medium by utilizing small magnets, and they disperse again after the magnet is removed. The sample contents can be easily washed by performing said collecting and dispersing processes.

Electrodes

The electrodes can be used many times by means of realizing the immobilization of AChE on the magnetic beads instead of electrodes. The biosensor according to the invention preferably comprises screen printed electrodes (SPE: Screen-Printed Electrodes). Said electrodes are the electrodes produced employing different conductive inks on ceramic surfaces or various types of insulative plastics. The working electrode, the reference electrode, and the auxiliary electrode are organized around a common center in a small sized strip. Since SPE’s can be produced cost-effectively and in large scales, they are preferably used in the inventive biosensors.

In the inventive biosensor, preferably graphene printed electrode is used since it is cost- effective. In a preferred embodiment of the invention, the biosensor comprises multi graphene electrodes. For example, 8-channel screen printed electrodes are preferred using multi-electrode since they provide standard and sample measurements simultaneously. Said electrodes have a typical electrochemical cell configuration. Said electrodes are appropriate to develop micro volumes, independent analyses or specific sensors, are also useful in simultaneously realizing multiple analyzes.

The electrodes in the present invention are not used as the support to which AChE (biorecognition element) is immobilized/connected; AChE can be immobilized on magnetic nano or micro-magnetic beads. Thus, said electrodes in the present invention are used as a measurement device without modification to only measure the enzyme-based product.

There is a microprocessor to which the signal outlet of the biosensor is connected. The microprocessor is connected to a screen. The biosensor transmits the current signals to the microprocessor before and after deltamethrin measurement is performed. Therefore, the microprocessor processes the current signal to transform the same into a deltamethrin concentration and shows the concentration changes on the screen to monitor.

In an embodiment of the invention, Dropsens 8-channel potentiostat (DRP-STAT8000), 8- channel connector (DRP-CAC8X), and Dropview software were used.

Preparation of the biosensor

Magnetic beads, functionalized with Protein G, are retained on the graphene electrodes by applying an external magnetic field, and in this manner, they are used as an immobilization platform. The modified magnetic beads immobilize the antibody after catching the same used antibody (anti-estradiol antibody) produced against estradiol and acetylcholinesterase (estradiol-AChE) conjugated with estradiol mixture and AChE is conjugated to the antibody over estradiol. There is antibody (anti-estradiol-antibody) and acetylcholinesterase (estradiol-AChE) conjugated with estradiol, sold in the market, is produced against estradiol. The activation of AChE in acetylcholinesterase (estradiol- AChE) conjugated with estradiol is essential. Therefore, the conjugation must be made appropriately. The immobilization is made effortless, and easy transfer of enzyme-bound magnetic beads is provided by means of performing the immobilization of AChE out of the system by using a magnetic field. Therefore, the inventive biosensor makes measurement easily from pesticides, waters, depots, insects in the regions subjected to deltamethrin etc. A process for preparing an amperometric biosensor according to the present invention comprises the following steps:

- mixing the magnetic bead conjugated with protein G, antibody produced again estradiol (anti-estradiol-antibody) and estradiol conjugated acetylcholinesterase solutions (estradiol-AChE) in an appropriate working buffer;

- incubating the mixture obtained;

- retaining obtained mixture on magnet for a determined period and washing the same with a buffer solution; and

- adding acetylthiocholine and/or a derivative thereof to the mixture.

Said process comprises the following process steps; monitoring the amperograms of the enzyme reaction at a predetermined potential and for a determined period after adding acetylthiocholine and/or a derivative thereof and finding the % activation amounts over the current signal.

In a preferred embodiment of the invention, said enzyme reaction can be monitored at 700 mV for 90 seconds.

Determination and measurement method with biosensor

The sample from which deltamethrin determination and measurement being made can be different samples such as pesticide, water, or depot sample or insects provided from regions exposed to deltamethrin. Deltamethrin extraction from the sample can be performed by using an appropriate extraction method among pesticide extraction methods known in the state of the art. In a preferred embodiment of the invention, sonication is used with solid-liquid extraction. Liquid extraction with acetonitrile is applied to the insects, and the obtained extract is used as a sample in the biosensor environment.

The method, according to the present invention for the determination and measurement of deltamethrin from the insects, comprises the following steps:

- providing samples of insect larvae and/or adults;

- obtaining extracts from insect larvae and/or adults samples by sonication using acetonitrile as an extraction solvent;

- obtaining supernatants from said extracts by centrifugation; measuring the current values with an amperometric biosensor wherein obtained supernatants comprise magnetic bead functionalized with protein G and antibody immobilized on protein G, acetylcholinesterase is conjugated to said antibody over estradiol and the substrate is acetylthiocholine.

In a preferred embodiment of the invention, the sample in deltamethrin determination and measurement is deltamethrin extract obtained from larvae and adults of T. Confusum.

EXAMPLES

Tribolium confusum (T. confusum) is one of the most severe pests for stored grains and processed grain products globally. It can easily reach high population density by continuing its development in unsuitable conditions to develop pests, even in other stored grains. For this reason, invasions will possibly occur in case precautions for fighting are not taken against these species. T. confusum adults and larvae are secondary dangerous. Particularly, they are seen widely in grain storages, mill and flour factories (Aitken, A. D., 1975. Insect travellers. Volume I. Coleoptera (No. 31). Coleoptera. Technical Bulletin, 31. H.M.S.O., London. 190 pp.; Hill, D. S., 1990. Pests of stored products and their control. Belhaven press. London. 274 pp).

The inventive deltamethrin determination method and biosensor applications were made on adults and larvae of T. confusum. The pyrethroid pesticide, which is selected for subjecting the adults and larvae, is Decis 2.5 EC. Effective substance is 25 g/l deltamethrin (weight/volume). Adults and larvae of T. Confusum, exposed to various concentrations of deltamethrin, were grown in the growth rooms of the Biology Department of Erciyes University Faculty of Science. It is kept in glass jars which get air, in a 100 g (95% flour and 5% beer yeast g/g) nutrient medium containing 70% humidity at 27C° until the experiment date.

Deltamethrin extraction from larvae:

With reference to Figure 3, larvae were subjected to different concentrations of pesticides for 24 hours. Then, counting and addition processes were realized. 8 dead larvae were collected from each concentration. Collected larvae were weighed, and the total weight was determined as 9.2 ± 0.5 mg. Samples were frozen at -80 C° for 10 minutes. Subsequently, homogenization was provided with a glass stirrer by adding 200 mI 85% acetonitrile (ACN). Sonication was realized in 3 repetitions with 10 seconds intervals for 10 seconds within ice at 150W, 20 kHz, 40%amplitude. Centrifugation was carried out at 5000 g speed, at 4 C° for 10 minutes. The supernatant was taken after centrifugation and added to the biosensor mixture.

Deltamethrin extraction from adults:

With reference to Figure 4, adults were subjected to different concentrations of pesticide for 24 hours. Then, counting and addition processes were done. 8 dead adults were collected from each concentration. Collected adult elytras were removed. Samples were weighed and the total weight was determined as 15 ± 0.5 mg. Samples were frozen at -80 C° for 10 minutes. Subsequently, homogenization was provided with a glass stirrer by adding 200 mI 85% acetonitrile (ACN). Sonication was realized in 3 repetitions with 10 seconds intervals for 10 seconds within ice at 150W, 20 kHz, and 40% amplitude. Centrifugation was carried out at 5000 g speed, at 4 C° for 10 minutes. The supernatant was taken after centrifugation and added to the biosensor mixture.

Deltamethrin biosensor measurement optimization studies

The AChE enzyme activity changes were analyzed with a serial study depending on the different development stages (larvae and adults) of T. confusum to which deltamethrin is applied. After deltamethrin application to insect larvae and adults in this part, the effect of received supernatant samples on AChE activity is measured compared to the standard group. Acetylthiocholine-chloride (ACTC) solution was used as a substrate in determining the activity of the AChE enzyme.

Deltamethrin Biosensor Standard Serial and Case Study Methods

6 different concentrations were determined for the experiment. A stock solution was prepared by dissolving with acetonitrile such that 2,5% Decis EC (25 g/l deltamethrin (weight/volume)) is in the ratio of 1/3. 1 ml serial solutions were prepared by dissolving the same with reading buffer by taking from this prepared stock solution depending on the concentrations. Serial studies were made by adding predetermined amounts of standard deltamethrin concentrations and samples (deltamethrin extracts obtained from larvae and adults of T. Confusum) in the tubes.

Experiment procedure The mixture was prepared by adding protein G-MB’s (0,3 pg/ml), estradiol-antibody (1/25, Cayman Chemical, 482282 estriol Elisa antiserum 1 vial, 500 dtn), estradiol-AChE (1/25, 482280 estriol acetylcholinesterase tracer: 1 vial, 500 dtn), and working buffer (0,01 M PBS, pH 7,4) into 1ml eppendorf tubes such that the total volume would be 500 mI.

The mixture was incubated with shaking for 90 minutes. The mixture was washed 5 times with 0,01 M PBS (pH 7,4) with 5 minutes waiting period on the magnet.

The prepared mixture was distributed to eppendorf tubes such that each of them was 40 mI. (6 standard: 2,5-125 mg/ml, 1 control, 6 samples)

Deltamethrin (25 g/l) was diluted at a ratio of 1/100. The dilution ratio is 1/100.

1 . Dilution: 100 mI deltamethrin main stock+900 mI acetonitrile

2. Dilution: 100 mI deltamethrin intermediate stock +900 mI 0.01 M PBS (pH 7.4)

10 mI of deltamethrin from the standard series was added to the standard tubes to which 40 mI of the mixture was shared into 1 ml of eppendorf tubes. 10 mI from the supernatant of the adults and larvae samples were added to the sample tubes used for the larvae and adults samples and contained 40 mI mixtures.

Only 40 mI mixture and 10 mI 0,01 M PBS buffer, pH 7,4, was added to the control tube.

0.01 M PBS buffer containing only 40 mI mixture and 10 mI 85% acetonitrile, acetonitrile control mixture containing pH 7.4 was prepared to analyze the effect of acetonitrile on AChE.

The shaking incubation period was 60 minutes for the activation.

Used control and blinds:

Straight blind: 10 mI 2mM acetylthiocholine (ACTC) + 10 mI 0.01 M PBS buffer pH 7,4

Sample Control: 10 mI 2 mM ACTC + 10 mI control mixture

Acetonitrile Control: 10 mI 2 mM ACTC + 10 mI acetonitrile control mixture

Straight ACTC control: 10 mI 2 mM ACTC+ 10 mI 0,01 M PBS buffer, pH 7,4

85% ACN buffered ACTC control: 10 mI 85% ACN sample buffer+ 10 mI 2 mM ACTC Amperometric Measuring Method for Biosensor Samples

10 mI work and control mixtures and 10 mI and 2mM ACTC were loaded on 8-graphene multi-electrodes located on the magnet. The realization of the reaction waited for 15 minutes. Subsequently, the electroactive product was monitored at 700 mV for 90 seconds, and the biosensor response was monitored. The AChE activity was calculated by using the amperometric results obtained after the reading process.

The standard concentration in the environment and the corresponding nA values (current) were found when standards are used while working. The graphene electrodes were washed with distilled water and the reading buffer between readings. The same processes were repeated in the presence of different deltamethrin concentrations, and % activation values were determined. Each activation value was given as an average of the results received after duplicate readings on at least two different SPE magnetic electrodes. The biosensor studies were performed at 25 C°.

Results

In the acetylcholinesterase based amperometric biosensor, deltamethrin was found between standard concentration measurement limits (2.5-125 pg/ml). Current values exhibited a direct proportion, depending on the standard concentration of deltamethrin. AChE enzyme exhibits an activation that is directly proportional to the standard concentrations of deltamethrin.

Current % changes in deltamethrin standard concentrations (2.5-125 pg/ml) were calculated in the acetylcholinesterase based amperometric biosensor. As it is given in Table 1 , % current values exhibited direct proportion, depending on the standard concentration of deltamethrin. AChE enzyme exhibits an activation that is directly proportional to the standard concentrations of deltamethrin. This activation was found 95% at the highest concentration and 2.5% at the lowest concentration. In Figure 5, the % activation graph is given. In Figure 6, amperogram of control and 2,5-7,5-15-30-62,5-125 pg/ml deltamethrin concentrations were given.

Table 1

The current values of T. confusum larvae in various deltamethrin concentrations (0.02 - 0.4 mg/ml petri dish concentration) were measured. Current values exhibited a direct proportion, depending on the concentration of deltamethrin larvae samples. AChE enzyme exhibits an activation that is directly proportional to the concentrations of larvae samples of deltamethrin.

The activation percentages of T. confusum larvae in various deltamethrin concentrations (0.02 - 0.4 mg / ml petri dish concentration) were calculated. As it is given in Table 2, the percentage of the current values exhibited direct proportion, depending on the concentration of deltamethrin larvae samples. AChE enzyme exhibits an activation percentage, which is directly proportional to the concentrations of larvae samples of deltamethrin. 53 % activation was observed at the highest concentration, and 5% activation was observed at the lowest concentration.

Table 2

Current values of T. confusum adults were measured using different deltamethrin doses (0.24 - 1.2 mg/ml petri dish concentration). Current values exhibited a direct proportion, depending on the concentration of deltamethrin adult samples. AChE enzyme exhibits an activation that is directly proportional to the concentrations of adult samples of deltamethrin.

The activation percentages of T. confusum adults in various deltamethrin concentrations (0.24 - 0.96 mg/ml petri dish concentration) were calculated. As it is given in Table 3, the percentage of the current values exhibited direct proportion, depending on the concentration of deltamethrin adult samples. AChE enzyme exhibits an activation percentage which is directly proportional to the concentrations of adult samples of deltamethrin. 112% activation was observed at the highest concentration, and 20% activation was observed at the lowest concentration.

Table 3

It is found with the present invention that deltamethrin activation on acetylcholinesterase is mixed type activation. In Figure 7, the Lineweaver-Burk graph of constant deltamethrin (0, 10, 25, 50 pg/ml) concentrations against acetylthiocholine concentrations (0.1 -0.5-1 -2-3-4- 5 mM) is given. Thus, Km (mM) and Vmax (pmol/min) values obtained against the increasing deltamethrin concentrations are given in Table 4.

Table 4

Claims

1. A method for the determination and measurement of deltamethrin, characterized in that it comprises measuring deltamethrin concentration amperometrically with a biosensor comprising acetylcholinesterase immobilized on a magnetic bead as a biorecognition element.

2. Method according to claim 1 , said magnetic bead is functionalized with protein G.

3. Method according to claim 2, said biosensor comprises antibody produced against estradiol immobilized on protein G and estradiol to which acetylcholinesterase is conjugated.

4. Method according to any of the previous claims, said magnetic bead comprises a core consisting of magnetic materials selected from iron, nickel, cobalt, neonidim-iron- boron, samarian-cobalt or magnetite.

5. Method according to claim 4, said core consists of magnetite.

6. Method according to any of the previous claims, said magnetic bead comprises a polymer layer selected from polystyrene, dextran, polyacrylic acid or silica.

7. Method according to claim 6, said polymer layer is polystyrene.

8. Method according to claim 7, the size of said magnetic beads is 1 pm.

9. Method according to any of the previous claims, said biosensor comprises screen- printed graphene electrodes.

10. Method according to claim 9, said electrodes are 8-channel screen printed electrodes.

11 . An acetylcholinesterase based amperometric biosensor comprising magnetic bead functionalized with protein G and antibody immobilized on protein G, characterized in that; acetylcholinesterase is conjugated to said antibody over estradiol.

12. A method for the immobilization of acetylcholinesterase to magnetic beads functionalized with protein G as the biorecognition element, characterized in that, it comprises the following steps; immobilizing the antibody (anti-estradiol antibody) produced against estradiol to said magnetic bead and conjugating acetylcholinesterase to said antibody over estradiol.

13. A process for the preparation of an amperometric biosensor comprising acetylcholinesterase immobilized on magnetic bead functionalized with protein G as the biorecognition element, characterized in that it comprises the following process steps;

(i) mixing the magnetic bead, functionalized with protein G, antibody produced again estradiol (anti-estradiol-antibody) and estradiol conjugated acetylcholinesterase solutions (estradiol-AchE) with an appropriate working buffer;

(ii) incubating the mixture obtained in step (i);

(iii) retaining the mixture obtained in step (ii) on magnet for a determined period and washing the same with a buffer solution; and

14. A process according to claim 13, said process comprises the following process steps; monitoring the amperograms of the enzyme reaction at a predetermined potential and for a determined period after the addition of acetylthiocholine and/or a derivative thereof and finding the % activation amounts over the current values.

15. A process according to claim 14, said potential is 700 mV, and said duration is 90 seconds.

16. A method for the determination and measurement of deltamethrin in insects, characterized in that it comprises the following steps;

(i) providing samples of insect larvae and/or adults;

(ii) obtaining deltamethrin extracts from insect larvae and/or adults samples by sonication using acetonitrile as an extraction solvent;

(iii) obtaining supernatants from said extracts by centrifugation;

(iv) measuring the current changes with an amperometric biosensor wherein obtained supernatants comprise magnetic bead functionalized with protein G and antibody immobilized on protein G, acetylcholinesterase is conjugated to said antibody over estradiol and the substrate is acetylthiocholine and/or a derivative thereof.

17. Method according to claim 16, said insect is flour moth, Tribolium confusum.