An electrochemical biosensor for use in the determination of glyphosate and the preparation method thereof

Abstract

The invention relates to a disposable electrochemical biosensor for use in the sensitive determination of glyphosate (GLY), a herbicide, and the preparation method thereof. In the electrochemical biosensor of the invention, carboxymethyl cellulose (CMC) / hydroxyapatite (HaNP) nanobiocomposite was modified onto the surface of a disposable pencil graphite electrode (PGE). DNA aptamer (DNA APT), a specific bioreceptor of glyphosate, was immobilised on the CMC / HaNP-PGE surface.

Description

[0001] AN ELECTROCHEMICAL BIOSENSOR FOR USE IN THE DETERMINATION OF GLYPHOSATE AND THE PREPARATION METHOD THEREOF

[0002] Technical Field of the Invention

[0003] The invention relates to a disposable electrochemical biosensor for use in the sensitive determination of glyphosate (GLY), a herbicide, and the preparation method thereof.

[0004] State of the Art

[0005] Pesticides are chemical substances or mixtures of substances used in agriculture to destroy and control pests, unwanted weeds and insects that have the potential to harm crops and plants. Pesticides have been used in agricultural areas for years. However, this widespread use has raised concern in the scientific and industrial communities over the long-term human health risks of pesticides due to their known toxicity. Thus, the determination of pesticides is of great importance both due to their increasing toxic effects on humans and to control their use. Pesticides are classified in different ways according to their appearance, the chemical structure of their active ingredients, the source from which they are obtained, and the pest group they affect. One group of pesticides that are most commonly used and researched are herbicides. Herbicides are the general name of chemical drugs used to save plants from unwanted weeds. The fact that herbicides constitute 50% of the world's use has led to an increase in studies on herbicides. Herbicides are first taken up by plants, then enter the food chain through herbivorous animals, and are subsequently taken up by carnivores and tertiary consumers. Since herbicides are resistant within the food chain, they pose a potential danger to humans, animals and the environment. Therefore, the determination of herbicides, which are one of the most widely used pesticides in the world, is of great importance for humans, animals and the environment.

[0006] Glyphosate has been one of the most used herbicides for weed control in both agricultural and urban areas since its discovery in 1971

[0015] ,

[0016] . Glyphosate (N- phosphonomethyl glycine, GLY) contains substituted glycine and is used as the active ingredient in many organophosphate pesticides [1]. In recent studies, it has been determined that GLY affects the cell cycle. The International Institute for Research on Cancer (IARC) defined GLY as a potential carciogenic agent in 2015 (IARC, 2015). In ongoing studies, it has been determined that GLY has genotoxic

[0015] and cytotoxic

[0017] ,

[0018] effects. It has also been reported in the literature to be an endocrine disruptor and affect the nervous system

[0019] and to affect oocyte quality in mice

[0020] . On the other hand, although the use of GLY in agricultural activities in the following years is controversial, the European Union Commission states that the use of GLY will continue until 2022 (European Union Commission, last access date: 1 1.06.2021 ) and it is still actively sold in our country. In addition, it is known that GLY quickly mixes with underground and surface water resources and contaminates water resources. For this reason, there is a need for analysis platforms to be developed for the selective and sensitive determination of GLY.

[0007] There are many studies in the literature for the determination of GLY with electrochemical biosensor systems. Many of the electrochemical biosensors developed in these studies have fabrication principles that require the use of indicators

[0001] , [2], [3] or the application of complex experimental procedures involving synthesis steps and the use of extra chemical agents [4], [5], [6], [7], To give an example from these studies, a metal hydroxide-loaded graphene nanoprobe modified glassy carbon electrode (GCE) was developed by Zhang et al. [6]. The differential pulse voltammetry (DPV) method was determined based on the interaction between copper (Cu) and GLY. The total time required for electrode preparation and GLY determination is approximately 27 hours. In addition, the GCE used in this study requires the application of extra pre-treatment steps such as sanding and sonication, and the GCE structure is not suitable for creating a disposable (bio)sensor platform. In order to determine GLY and glufosinate together, Prasad and his team [5] immobilised the molecularly imprinted polymer that they specifically synthesized onto the surface of gold nanoparticle modified pencil graphite electrode and performed the voltammetric determination of GLY. In another study, an antibody and carbon nanodot modified screen-printed carbon electrode was developed for the specific determination of GLY [8]. By means of the use of the GLY-specific antibodies used in this study, it was possible to detect GLY sensitively by the electrochemiluminescence method. However, the use of animals is required for antibody production, and antibodies are not stable against environmental factors such as temperature and pH. In some studies, metal electrodes were used [2],[4],[9],

[0010] ,

[0011] ,

[0012] ,

[0013] . Metal electrodes, which can only be used by experts in the field, are difficult to prepare and apply, and they cannot be adapted to chip technology in the future because they are expensive, not disposable, and are not suitable for miniaturisation. Therefore, it is important to develop biosensor systems for the electrochemical determination of GLY that have a robust structure, can be prepared in a practical way, can perform selective and sensitive analyses and are also inexpensive.

[0008] The biosensors used in the state of the art and used in the determination of herbicides are not sensitive and reliable enough, and therefore high accuracy cannot be achieved in the determinations performed by these biosensors. In addition, herbicide biosensors in the state of the art are not only eco-friendly and user-friendly, but also impractical. In addition, herbicide determinations in the state of the art are complicated in terms of the types of electrodes used, types of biomarkers, types of nanomaterials or biomaterials used, and the methods developed for electrochemical determination, and they require high costs because they require the use of extra chemical agents. On the other hand, other disadvantages of the (bio)sensors are that the electrodes require cleaning and preliminary preparation, that the modification / immobilisation steps are long and laborious, and that they additionally require the use of extra chemical agents.

[0009] Due to reasons such as the fact that high accuracy results cannot be obtained as a result of the (bio)sensors in the state of the art that enable the determination of glyphosate herbicides, which is a type of pesticide, are not sensitive and safe enough, the biosensors in the state of the art are not eco-friendly and user-friendly, are not practical, and require high costs, it has become necessary to introduce a biosensor to be used in the determination of GLY, which eliminates all these problems.

[0010] Brief Description and Aims of the Invention

[0011] In the invention, a disposable electrochemical biosensor for use in the sensitive determination of glyphosate (GLY), a herbicide, and the preparation method thereof are described. In the electrochemical biosensor of the invention, carboxymethyl cellulose (CMC) I hydroxyapatite (HaNP) nanobiocomposite was modified onto the surface of a disposable pencil graphite electrode (PGE). DNA aptamer (DNA APT) in G-quadruplex (tetrahedral) structure, which is a GLY-specific bioreceptor, was immobilised on the CMC / HaNP-PGE surface. An aim of the invention is to provide a biosensor for use in the sensitive determination of GLY. A biosensor for use in the sensitive determination of GLY is provided in the invention by means of the nanobiocomposite modification and the specific interaction between DNA APT and GLY. Precise determination ensures reliable results and therefore high accuracy determination results are obtained.

[0012] Another aim of the invention is to provide an environmentally friendly glyphosate (GLY) biosensor. An environmentally friendly glyphosate biosensor is provided in the invention by means of the fact that PGEs can be prepared without any pre-treatment and have a graphite structure, the nanocomposite structure created with biocompatible HaNP and CMC, and the ability to work with low volumes of samples for electrochemical analysis and modification / immobilisation / interaction experimental steps.

[0013] In the invention, a practical glyphosate (GLY) biosensor is provided. A practical GLY biosensor is provided in the invention by means of the use of PGEs and voltammetric analysis.

[0014] A low-cost glyphosate (GLY) biosensor is provided with the invention. A low-cost glyphosate (GLY) biosensor is provided in the invention by means of the use of PGEs and the ability to work with small amounts of sample.

[0015] Description of Drawings

[0016] Figure 1. Difference (Ala) values between the average anodic current values obtained after the immobilisation of 10-50 pg / mL hydroxyapatite (HaNP) on the pencil graphite electrode (PGE) for 15 minutes (n=3).

[0017] Figure 2. (I) (a) pencil graphite electrode (PGE), (b) 0.5 pg / mL carboxymethyl cellulose (CMC) modified PGE, (c) HaNP modified PGE, (d) voltammograms of 0.5 pg / mL CMC modified HaNP-PGE. (II) Average anodic current values (la) measured after 30 minutes modification of 0-5 pg / mL CMC to (A) PGE, (B) HaNP-PGE (n=3).

[0018] Figure 3. (A) PGE, (B) HaNP-PGE (a), average la values (n=3) obtained after immobilization of 0.5 pg / mL CMC for (b) 10, (c) 20, (d) 30, (e) 40 min.

[0019] Figure 4. (A) Nyquist curves obtained as a result of electrochemical impedance spectroscopy (EIS) measurements performed with (a) PGE, (b) HaNP-PGE, (c) CMC- PGE, (d) CMC / HaNP-PGE in 2.5 mM Fe(CN)6-3 / -4prepared redox probe containing 0.1 M potassium chloride (KCI). (B) Equivalent circuit model used to fit impedance data. (C) Average difference (ARct) values between the charge-transfer resistance values obtained by (a) HaNP-PGE, (b) CMC-PGE and (c) CMC / HaNP-PGE (n=3).

[0020] Figure 5. Microscopic images of (A) PGE, (B) HaNP-PGE, (C) CMC-PGE, (D) HaNP / CMC-PGE obtained with scanning electron microscopy (SEM) technique at (a) 2000x, (b) 5000x, (c) 10000x magnification, acceleration potential: 15 kV.

[0021] Figure 6. Histograms expressing mean Ala values obtained as a result of immobilisation of (a) unlabelled, 5' end (b) amino, (c) thiol and (d) phosphate group labelled DNA oligonucleotide (DNA ODN) to CMC / HaNP-PGE for 1 h (n=3).

[0022] Figure 7. Average Ala values obtained by immobilisation of 50-150 pg / mL DNA ODN to CMC / HaNP-PGE (n=3).

[0023] Figure 8. Average Ala values obtained after 100 pg / mL DNA ODN immobilisation to (a) PGE, (b) HaNP-PGE, (c) CMC-PGE, (d) CMC / HaNP-PGE (n=3).

[0024] Figure 9. (A) Voltammograms obtained with CMC / HaNP-PGE and 2 pM denatured DNA APT immobilised CMC / HaNP-PGE. (B) Average la values obtained with (a) CMC / HaNP-PGE, (b) 0.1 pM, (c) 0.5 pM, (d) 1 pM, (e) 2 pM, (f) 4 pM DNA APT immobilised CMC / HaNP-PGE (n=3).

[0025] Figure 10. (A) voltammograms, (B) average la values obtained by cyclic voltammetry (CV) method with (a) CMC / HaNP-PGE after immobilisation of 2 pM denatured DNA APT into CMC / HaNP-PGE for (b) 30 min, (c) 60 min, (d) 90 min (n = 3).

[0026] Figure 11. (A) Voltammograms, (B) average la values obtained by CV method with (a) CMC / HaNP-PGE, (b) 2 pM denatured DNA APT immobilised CMC / HaNP-PGE after interaction of 2 pM denatured DNA APT with 5 pg / mL GLY on CMC / HaNP-PGE surface for (c) 30min, (d) 60min, (e) 90min, (f) 120min (n=3).

[0027] Figure 12. (A) Voltammograms obtained by the CV method after the interaction of (a) 2pM denatured DNA APT immobilised CMC / HaNP-PGE and 2 pM denatured DNA APT and (b) 1 , (c) 2, (d) 3, (e) 4, (f) 5 pg / mL GLY on the CMC / HaNP-PGE surface for 90 min. (B) Average la values obtained by the CV method after 90 min interaction of 1 - 15 pg / mL GLY and 2 pM DNA APT on the CMC / HaNP-PGE surface (n=3). Inset figure: Calibration graph based on average la values (n=3) obtained after the interaction of 1 - 5 pg / mL GLY and 2 pg / mL DNA APT.

[0028] Figure 13. (A) Voltammograms, (B) average la values (n=3) obtained by CV measurements performed after the interaction of (a) CMC / HaNP-PGE and (b) 2pM denatured DNA APT immobilised CMC / HaNP-PGE with 2 pM denatured DNA APT with 5 pg / mL (c) GLY, (d) glufosinate (GFS), (e) 2,4 dichlorophenoxyacetic acid (2,4- D), and (f) (aminomethyl)phosphonic acid (AMPA) on the CMC / HaNP-PGE surface for 90 min.

[0029] Figure 14. (I) Voltammograms obtained by CV measurements of 2 pM DNA APT and 5 pg / mL GLY prepared in (a) soft water, (b) moderately soft water, and (c) hard water after interaction on the CMC / HaNP-PGE surface for 90 min. (II) % changes in the average la values obtained by CV measurements after the interaction of (A) 2 pM DNA APT and 5 pg / mL GLY prepared in (B) soft water, (C) moderately soft water, and (D) hard water on the CMC / HaNP-PGE surface for 90 min (n = 3).

[0030] Detailed Description of the Invention

[0031] The invention relates to a disposable electrochemical biosensor for use in the sensitive determination of glyphosate (GLY), a herbicide, and the preparation method thereof. In the electrochemical biosensor of the invention, carboxymethyl cellulose (CMC) / hydroxyapatite (HaNP) nanobiocomposite was modified onto the surface of a disposable pencil graphite electrode (PGE). DNA aptamer (DNA APT), which is the specific bioreceptor of GLY, was immobilised on the CMC / HaNP-PGE surface.

[0032] In biosensor development, DNA oligonucleotide with the nucleotide sequence of SEQ ID NO: 1 and DNA APT with the nucleotide sequence of SEQ ID NO: 2 are used

[0021] ,

[0033] A disposable electrochemical biosensor that is the subject of the invention for use in the sensitive determination of GLY, a pesticide, comprises carboxymethyl cellulose (CMC) / hydroxyapatite (HaNP) nanobiocomposite modified on disposable pencil graphite electrode (PGE) surface and DNA aptamer (DNA APT), which is a specific bioreceptor of GLY, immobilised on the surface of said nanobiocomposite (CMC / HaNP-PGE).

[0034] In an embodiment of the invention, a disposable electrochemical biosensor that is the subject of the invention for use in the sensitive determination of GLY, a pesticide, comprises carboxymethyl cellulose (CMC) / hydroxyapatite (HaNP) nanobiocomposite modified on disposable pencil graphite electrode (PGE) surface and DNA aptamer (DNA APT) at a concentration of 2 pM, which is a GLY specific bioreceptor immobilised on the surface of said nanobiocomposite (CMC / HaNP-PGE).

[0035] A method of preparing a disposable electrochemical biosensor to be used in the sensitive determination of glyphosate (GLY), which is the subject of the invention, comprises the process steps of: i. Preparation of dilute HaNP solution by diluting the stock hydroxyapatite (HaNP) solution in phosphate buffer solution (PBS) with a pH of 7.40, ii. Immersing the pencil graphite electrode (PGE) into the HaNP solution and waiting, immobilising the HaNP solution onto the PGE surface by passive adsorption method, and then washing the electrodes by dipping them in PBS with a pH of 7.40 in order to prevent non-specific binding, ill. Preparation of carboxymethyl cellulose (CMC) in PBS solution with pH 7.40 and then immersing HaNP-PGE in CMC solution to immobilise CMC onto the HaNP-PGE surface by passive adsorption method, and then, to prevent nonspecific binding, immersing and washing the electrode in PBS with a pH of 7.40, iv. Preparation of DNA APT stock solution in tris hydrochloride (TBS) containing potassium chloride (KCI) and preparation of dilute DNA APT solution by diluting the prepared DNA APT stock solution and applying the denaturation process to the dilute DNA APT solution, v. Immobilising denatured DNA APT onto the CMC / HaNP-PGE surface by passive adsorption method and then washing the electrodes in TBS with pH 7.50 to prevent non-specific binding.

[0036] In an embodiment of the invention, a method of preparing a disposable electrochemical biosensor to be used in the sensitive determination of glyphosate (GLY), which is the subject of the invention, comprises the process steps of: i. Preparation of dilute HaNP solution at a concentration of 10-50 pg / mL by diluting the stock hydroxyapatite (HaNP) solution in 0.05 M phosphate buffer solution (PBS) with a pH of 7.40, ii. Immobilising the HaNP solution onto the PGE surface by passive adsorption method by dipping the pencil graphite electrode (PGE) into 80-120 pL HaNP and keeping it for 5-30 minutes, and then washing the electrodes by immersing them in PBS with a pH of 7.40 for 5 seconds in order to prevent non-specific binding, iii. Preparation of carboxymethyl cellulose (CMC) in PBS solution with pH 7.40 in the concentration range of 0.05-5 pg / mL and dipping the HaNP-PGE into 80-120 pL CMC solution and keeping it for 10-40 minutes to immobilise the CMC solution onto the HaNP-PGE surface by passive adsorption method, and then immersing the electrodes in PBS with a pH of 7.40 and washing for 5 seconds to prevent nonspecific binding, iv. Preparation of stock DNA APT solution as 25 pM in 10-30 mM Tris hydrochloride (TBS) solution containing 10-50mM potassium chloride (KCI) and pH 7.50, then preparing a diluted DNA APT solution of 0.1 -4 pM by diluting the prepared DNA APT stock solution in 10-30 mM Tris hydrochloride (TBS) solution containing 10-50mM potassium chloride (KCI) and pH 7.50, and performing the denaturation process by keeping the dilute DNA APT solution at 95°C for 5 minutes and then at +4°C for 15 minutes, v. Immobilising 10-40 pL denatured DNA APT onto the CMC / HaNP-PGE surface for 30-90 minutes by passive adsorption method and then washing the electrodes in TBS with pH 7.50 for 5 seconds to prevent non-specific binding.

[0037] In an embodiment of the invention, a method of preparing a disposable electrochemical biosensor to be used in the sensitive determination of glyphosate (GLY), which is the subject of the invention, comprises the process steps of: i. Preparation of dilute HaNP solution at a concentration of 30 pg / mL by diluting the stock hydroxyapatite (HaNP) solution in 0.05 M phosphate buffer solution (PBS) with a pH of 7.40, ii. Immobilising the HaNP solution onto the PGE surface by passive adsorption method by dipping the pencil graphite electrode (PGE) into 100 pL HaNP and keeping it for 15 minutes, and then washing the electrodes by immersing them in PBS with a pH of 7.40 for 5 seconds in order to prevent non-specific binding, iii. Preparation of carboxymethyl cellulose (CMC) in PBS solution with pH 7.40 in the concentration range of 0.5 pg / mL and dipping the HaNP-PGE into 100 pL CMC solution and keeping it for 30 minutes to immobilise the CMC solution onto the HaNP-PGE surface by passive adsorption method, and then immersing the electrodes in PBS with a pH of 7.40 and washing for 5 seconds to prevent nonspecific binding, iv. Preparation of stock DNA APT solution as 25 pM in 10 mM Tris hydrochloride (TBS) solution containing 50 mM potassium chloride (KCI) and pH 7.50, then, preparing a dilute DNA APT solution to 2 pM by diluting the prepared DNA APT stock solution in 10 mM Tris hydrochloride (TBS) solution containing 50mM potassium chloride (KCI) and pH 7.50, and performing the denaturation process by keeping the dilute DNA APT solution at 95°C for 5 minutes and then at +4°C for 15 minutes, v. Immobilising 30 pLdenatured DNA APT onto the CMC / HaNP-PGE surface for 60 minutes by passive adsorption method and then washing the electrodes in TBS with pH 7.50 for 5 seconds to prevent non-specific binding.

[0038] In the process of realising GLY-DNA APT specific interaction on the CMC / HaNP-PGE surface, first, GLY stock solution is prepared in 10 mM Tris hydrochloride (TBS) solution with pH 7.50 and containing 50mM potassium chloride (KCI) and dilute GLY solutions are prepared by diluting the stock GLY solution with TBS with pH 7.50. Afterwards, DNA APT immobilised CMC / HaNP-PGE is immersed in 10-40 pL GLY solution and kept for 30-120 minutes, and then the electrodes are washed in TBS with a pH of 7.50 to prevent non-specific binding.

[0039] In an embodiment of the invention regarding the process of realising GLY-DNA APT specific interaction on the CMC / HaNP-PGE surface, a 30 pL volume of 5 pM dilute GLY solution is prepared from the stock GLY solution in TBS with a pH of 7.50, and it is kept for 90 minutes and then the electrodes are washed in TBS with a pH of 7.50 to prevent non-specific binding.

[0040] In another embodiment of the invention regarding the process of realising GLY-DNA APT specific interaction on the CMC / HaNP-PGE surface, a 30 pL volume of 1 -15 pM dilute GLY solution is prepared from the stock GLY solution in TBS with a pH of 7.50, and it is kept for 90 minutes and then the electrodes are washed in TBS with a pH of 7.50 to prevent non-specific binding. In the process of interaction of different herbicides with DNA APT on the CMC / HaNP- PGE surface, first, to realise the interaction of different herbicides with DNA APT on the CMC / HaNP-PGE surface, GLY, one of the organophosphorus compounds, is used together with another organophosphate herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D), glufosinate ammonium (GFS), a glyphosate-like pesticide, and (aminomethyl)phosphonic acid (AMPA), the degradation product of glyphosate and 2,4-D stock solution is prepared in dimethylsulfoxide (DMSO), then, GFS or AMPA stock solutions are prepared in TBS (pH 7.50) and 2,4-D, GFS, AMPA are diluted in TBS with pH 7.50 and denatured 2 pM DNA APT immobilised CMC / HaNP-PGE is immersed in 10-40 pL of 3-7 pg / mL 2,4-D / GFS / AMPA solution and waited for 30-120 minutes and the electrodes are washed in TBS solution with pH 7.50 to prevent nonspecific binding.

[0041] In an embodiment of the invention, in the process of interaction of different herbicides with DNA APT on the CMC / HaNP-PGE surface, first, to realise the interaction of different herbicides with DNA APT on the CMC / HaNP-PGE surface, GLY, is used together with 2,4-D, GFS, AMPA, and 2,4-D stock solution is prepared in dimethylsulfoxide (DMSO), then, GFS or AMPA stock solutions are prepared in TBS (pH 7.50) and 2,4-D, GFS, AMPA are diluted in TBS with pH 7.50 to be 5 pg / mL and denatured 2 pM DNA APT immobilised CMC / HaNP-PGE is immersed in 30 pL of 5 pg / mL 2,4-D / GFS / AMPA solution and waited for 90 minutes and the electrodes are washed in TBS solution with pH 7.50 to prevent non-specific binding.

[0042] To realise the specific interaction of DNA APT-GLY on the CMC / HaNP-PGE surface in different synthetic water samples, first of all, three separate water samples including synthetic ground water samples sodium bicarbonate (NaHCOa), calcium sulphate dihydrate (CaSO4*2H2O), magnesium sulphate (MgSO4) and potassium chloride (KCI) are provided as soft water, moderately soft water and hard water, each with a pH value of 7.50; afterwards, GLY is prepared in the prepared water samples and denatured 2 pM DNA APT immobilised CMC / HaNP-PGE is immersed in 10-40 pL 3-10 pg / mL GLY sample and kept for 30-120 minutes, then the electrodes are washed in TBS solution with a pH of 7.50 to prevent non-specific binding.

[0043] In an embodiment of the invention, the specific interaction of DNA APT-GLY on the CMC / HaNP-PGE surface is carried out in different synthetic water samples, three separate water samples are provided as soft water, moderately soft water and hard water, each with a pH of 7.50; afterwards, GLY is prepared in prepared water samples and denatured 2 pM DNA APT immobilised CMC / HaNP-PGE is immersed in 30 pL 5 pg / mL GLY sample and kept for 90 minutes; and then, the electrodes are washed in TBS solution with a pH of 7.50 in order to prevent non-specific binding.

[0044] Before / after each modification / immobilisation / interaction step, measurements were carried out using the cyclic voltammetry (CV) technique in a redox probe prepared in 0.10 M potassium chloride (KCI) containing 2 mM potassium ferrocyanide (III) [(K3Fe(CNe)] and potassium ferrocyanide (IV) [(K4Fe(CNe)] All measurements were carried out in the (-0.50V)-(+1.15 V) potential range with a scan rate of 50 mV / sec. Voltammetric analyses were performed based on the changes in the average anodic (la) value obtained with 3 consecutive measurements.

[0045] Before / after each modification / immobilisation / interaction step, measurements were carried out with the electrochemical impedance spectroscopy (EIS) technique in a redox probe prepared in 0.10 M KCI containing 2.50 mM K3Fe(CNe) and K4Fe(CNe). All measurements were carried out in the frequency range of 100 mHz-100 kHz (98 logarithmic equal intervals), +0.23 V open circuit potential and 10 mV sinusoidal signal value. Impedimetric analyses were carried out based on the changes in the Rd value by measuring the average charge-transfer resistance (Ret) value obtained by applying the Randles-Sevcik equivalent circuit model with 3 consecutive measurements.

[0046] Scanning electron microscopy (SEM-EDX) analyses were performed with a magnification of 2000x-10000x and an acceleration potential of 15 kV.

[0047] When Figure 1 was examined, it was seen that the highest and most reproducible Ala value was obtained after the modification of 30 pg / mL HaNP to PGE. The average Ala value measured with 30 pg / mL HaNP modified PGE was found to be 35.26 ± 3.56 pA (%RSD=10.10%; n=3).

[0048] When Figure 2 and Tables 1 and 2 are examined, the most reproducible average la value with the highest % decrease after CMC modification to HaNP-PGE was obtained in the presence of 0.50 pg / mL CMC. CMC molecules were bonded to the HaNP / PGE surface by forming hydrogen bonds between the Ca+2ions in the HaNP structure and the carboxyl groups in the CMC structure. When Figure 3, Tables 3 and 4 are examined, they represent the voltammetric results obtained after the modification of 0.50 pg / mL CMC to 30 pg / mL HaNP modified PGE for 10-40 minutes. In this study, average la values measured by PGE and HaNP-PGE are 125.10 ± 7.40 pA (RSD% = 5.92%, n=3) and 89.65 ± 6.01 pA (RSD% = 6.71 %, n=3), respectively. It was observed that after CMC modification to HaNP-PGE compared to PGE, the most reproducible results and the highest decrease in the average la value were obtained after 30 minutes of modification.

[0049] When Table 5 is examined, it is seen that the calculated surface area (Aett) values decrease as the modification increases. When the AEPvalues were examined, it was determined that the behaviour of the semi-reversible PGE became irreversible after HaNP, CMC and CMC / HaNP modification, but the highest AEp value was obtained with CMC / HaNP-PGEs.

[0050] When Figure 4 and Table 6 are examined, HaNP is a nanomaterial containing negatively charged phosphate groups in its structure

[0024] , CMC is an anionic polymer

[0022] , Therefore, modification of PGE with HaNP caused repulsive forces between the negatively charged redox probe and negatively charged phosphate groups, resulting in an increase in Ret and ARct values. Similarly, due to its negative structure, CMC caused an increase in Ret and ARct values after its modification to the PGE surface. After CMC modification to the HaNP-PGE surface, repulsive forces occurred between the negatively charged electrode surface and the negatively charged redox probe, resulting in an increase in Ret and ARct values. In this study, mean ARct values with HaNP-PGE, CMC-PGE and CMC / HaNP-PGE was obtained as 257.00 ± 32.16 Ohm (RSD% = 12.51 %; n=3), 325.13 ± 41.36 Ohm (RSD% = 12.72%; n=3) and 536.50 ± 43.47 Ohm (RSD% = 8.10%; n=3), respectively. It was observed that the highest and most reproducible ARct value was obtained with CMC / HaNP-PGE. When the QISR values were evaluated, it was seen that the QISR value decreased as the modification increased. The voltammetric and impedimetric results obtained for the electrochemical characterisation results are compatible with each other and these results show that CMC / HaNP-PGE can be created successfully.

[0051] When the SEM-EDX results given in Figure 5 and Table 7 are examined, the graphite layered structure of PGE is clearly seen at all magnifications in Figure 5-A. After HaNP modification, HaNP particles in the form of rods and stacks were formed on the surface and the layered graphite structure of PGE was covered (Figure 5-B). The CMC stacks formed on the PGE surface after the modification of PGE with CMC are shown in the images in Figure 5-C, but the CMC stacks are quite different from the HaNP stack image. After CMC modification of HaNP-PGE (Figure 5-D), it was observed that rodlike HaNP particles and the bulk structure of CMC formed together on the PGE surface, the graphite surface took on a flatter appearance, and the angular and sharp-lined appearance of graphite was replaced by a softer and blurred structure.

[0052] When the elemental distribution values given in Table 7 are examined, it is seen that after HaNP modification to PGE (table 7-B), there is an increase in the weight percentage (wt%) and atomic percentage (atomic C (at%)) values of C and P elements, and a decrease in these values of O and H elements. This situation is explained by the presence of P atoms in the HaNP structure on the PGE surface. On the other hand, after the CMC modification to PGE (table 5-C), an increase in the relevant values of the C atom was observed, and this is explained by the presence of C atoms in the structure of CMC after the modification. After the modification of HaNP-PGE with CMC (table 5-D), it was observed that the behaviour of the relevant values of the P atom showed a decreasing behaviour and the relevant values of the O element showed an increasing behaviour. These results show that the successful modification of the HaNP-PGE surface with CMC was achieved, that by coating the HaNP-PGE surface with CMC, the values of P atoms in HaNP decreased, and that the O atoms in the CMC structure increased as a result of the presence of CMC on the HaNP-PGE surface.

[0053] As a result of all these characterisation studies, compatible electrochemical and microscopic results were obtained, and these results show that CMC / HaNP-PGE can be created successfully.

[0054] When Figure 6 and Table 8 are examined, a decrease in the la value occurred after DNA ODN immobilisation on the CMC / HaNP-PGE surface. This decrease is explained by the formation of repulsive forces between the negatively charged redox probe and the electrode surface after the immobilisation of negatively charged DNA ODN to the electrode surface and the resulting decrease in the la value

[0024] , Decreases in lavalues were evaluated as Ala values. The highest Ala value was obtained after unlabelled DNA probe immobilisation. In the invention, by means of the preparation of the DNA ODN stock solution in Tris- EDTA solution at a concentration of 500 pg / mL and pH 8.00, and preparation of 100 pL DNA ODN at 100 pg / mL with PBS (pH 7.40) solution and immobilisation on the CMC / HaNP-PGE surface by passive adsorption method for 60 minutes, the use of DNA ODN, labelled or unlabelled with amino, thiol or phosphate groups at the 5' end, was tested, and the most reproducible Ala value was obtained with unlabelled DNA ODN. Unlabelled DNA ODN has been used in ongoing studies. Based on the results in Figure 7, it can be seen that the highest average Ala value was obtained after 100 pg / mL DNA ODN immobilisation. The average Ala value and RSD% value obtained after 100 pg / mL DNA ODN immobilisation are 30.44 ± 2.86 pA and RSD% = 9.41 % (n=3), respectively. A DNA ODN concentration of 100 pg / mL was determined as optimum for ongoing studies. Figure 8 shows the histograms of the average Ala values obtained by immobilizing 100 pg / mL DNA ODN onto the surface of PGE, HaNP-PGE, CMC-PGE and CMC / HaNP-PGE. When the average Ala values obtained in this study are evaluated, it is observed that there is a decrease in the la value after 100 pg / mL DNA ODN immobilisation to PGE, HaNP-PGE, CMC-PGE and CMC / HaNP-PGE, however, the highest decrease can be achieved with CMC / HaNP-PGE with the most reproducible results (Figure 7-d). The average Ala value obtained after 100 pg / mL DNA ODN immobilisation to CMC / HaNP-PGE was 30.44 ± 2.86 pA (RSD% = 9.41 %; n=3). Based on these results, it is evaluated that nucleic acid analyses can be performed sensitively and reproducibly with CMC / HaNP-PGE. With these studies, which group would be marked at the 5' end of DNA APT was optimised and it was shown that nucleic acid analysis could be performed sensitively with the developed CMC / HaNP- PGEs.

[0055] When Figure 9 is examined, compared to the average la value obtained by CMC / HaNP-PGE, the highest decrease after DNA APT immobilisation was achieved in the presence of 2.00 pM DNA APT and the % decrease value was calculated as 15.08%. The average la value measured at +0.530 V peak potential after 2.00 pM DNA APT immobilisation is 52.74 ± 4.04 pA (RSD%=7.67%, n=3). 2.00 pM DNA APT concentration was determined as optimum and studies were continued to optimise the DNA APT immobilisation time (Figure 10). In this study, after 2.00 pM DNA APT immobilisation on the CMC / HaNP-PGE surface, the maximum decrease was obtained after 60 minutes of immobilisation (Figure 10-A,B-c). The average la value measured at +0.530 V peak potential is 52.74 ± 4.04 pA (RSD%=7.67%; n=3) and the % decrease in the average la value after immobilisation to CMC / HaNP-PGE is 14.86%. 60 minutes immobilisation time was determined as optimum.

[0056] When Figure 1 1 is examined, experimental studies were carried out to optimise the GLY interaction time with 2.00 pM denatured DNA APT. In this study, after the interaction of 2.00 pM denatured DNA APT and 5.00 pg / mL GLY on the CMC / HaNP- PGE surface for 30-120 minutes, the maximum decrease in the average la value was obtained after 90 minutes of interaction time (Figure 1 1 -A,B-e). The average la value obtained after 90 minutes of interaction was 40.03 ± 3.29 pA (RSD% = 8.24%, n=3) and the % decrease in the average la value after the interaction was calculated as 24.10%. An interaction time of 90 minutes was determined as optimum and in the ongoing study (Figure 12), interaction was carried out between 2.00 pM denatured DNA APT and 1.00-15.00 pg / mL GLY for 90 minutes. In this study, a decrease in average la values was observed as a result of the interaction of 1 .00-15.00 pg / mL GLY and 2.00 pM DNA APT on the CMC / HaNP-PGE surface for 90 minutes and the highest decrease was seen in the presence of 5.00 pg / mL GLY (Figure 12-C). A linear decrease in the average la value was obtained in the presence of 1.00-5.00 pg / mL GLY, and a calibration graph was drawn based on the log value of GLY concentration and logla values

[0023] ,

[0024] , Based on this calibration chart, the detection limit (DL) was calculated according to the Miller and Miller method

[0014] , and the calculated DL is 0.04 pg / mL. The calculated sensitivity value is 0.57 pA.mL / pg.cm2. As a result of this study, 5.00 pg / mL GLY concentration was determined as optimum.

[0057] The average la values obtained from the CV measurements following the interaction of 2.00 pM denatured DNA APT with 5.00 pg / mL GLY or 2,4-D / GFS / AMPA on the CMC / HaNP-PGE surface for 90 min. are given in Figure 13. In this study, the average la values obtained after the interaction of 2.00 pM DNA APT with 5.00 pg / mL GLY or GFS / 2,4-D / AMPA on the CMC / HaNP-PGE surface for 90 minutes are 40.03 ± 3.29 pA (RSD% = 8.24%, n=3), 49.06±3.28 pA (RSD% = 6.67%, n=3), 50.21 ± 6, 36 pA (RSD% = 12.67%, n=3) and 52.96 ± 1.46 pA (RSD% = 2.77%, n=3), respectively. Compared to the average la value obtained with DNA APT immobilised CMC / HaNP-PGE (Figure 13-B,b), the highest decrease in the average la value obtained after the interaction was obtained after the interaction with GLY (Figure 13-B,c) and the % decrease rate was 24.09%. On the other hand, compared to the average la value obtained after interaction with GLY, the % changes between the la values obtained in the presence of GFS / 2,4- D / AMPA (Figure 13-B-d,e,f, respectively) were calculated as 22.55%, 25.42% and 32.28% respectively.

[0058] After being prepared in three different ways as soft water, moderately soft water and hard water (pH 7.50)

[0025] , CMC / HaNP-PGE based determination of 5.00 pg / mL GLY from these water samples was performed voltammetrically with aptasensor as previously described, and the changes in the average la values obtained are given in Figure 14. In this study, after interaction with 5.00 pg / mL GLY in soft water, moderately soft water and hard water environments measured with the aptasensor platform developed under optimum conditions, a change of 17.46%, 19.47% and 16.92% occurred, respectively, (Figure 14), and the RSD% values of these changes are 5.74%, 4.08% and 3.90%, respectively (n = 3).

[0059] Table 1. The averages values (n=3) measured after 30 min modification of 0-5 pg / mL CMC to (A) PGE, (B) HaNP-PGE.

[0060] Table 2. % decreases in the average la values (n=3) after 0-5 pg / mL CMC modification to (A) PGE and (B) HaNP-PGE. Table 3. The average la values (n=3) measured after 10-40 min modification of 0.5 pg / mL CMC to (A) PGE and (B) HaNP-PGE. Table 4. % decreases in the average la values (n=3) after 10-40 min modification of 0.5 pg / mL CMC to (A) PGE and (B) HaNP-PGE.

[0061] Table 5. (A) The average la values obtained by PGE, HaNP-PGE and CMC / HaNP- PGE (n=3), (B) Aeff values calculated based on these values and (C) difference between the peak values of anodic current and cathodic currents (AEP).

[0062] Table 6. Ret values obtained by PGE, HaNP-PGE, CMC-PGE and CMC / HaNP-PGE and QISR values calculated based on these values.

[0063] Table 7. Elemental distributions of (A) PGE, (B) HaNP-PGE, (C) CMC-PGE, (D)

[0064] CMC / HaNP-PGE obtained by EDX technique.

[0065]

[0066] Table 8. Average Ala values (n=3) and RSD% values obtained as a result of immobilisation of (a) unlabelled, (b) amino, (c) thiol and (d) phosphate group labelled DNA ODN to CMC / HaNP-PGE for 1 hour

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Claims

CLAIMS1. An electrochemical biosensor for use in the determination of glyphosate comprising modified carboxymethyl cellulose (CMC) I hydroxyapatite (HaNP) nanobiocomposite on the pencil graphite electrode (PGE) surface and DNA aptamer (DNA APT), a specific bioreceptor of glyphosate, immobilised on the surface of said nanobiocomposite (CMC / HaNP-PGE).

2. A biosensor according to Claim 1 , comprising modified carboxymethyl cellulose (CMC) I hydroxyapatite (HaNP) nanobiocomposite on the pencil graphite electrode (PGE) surface and DNA aptamer (DNA APT), a specific bioreceptor of glyphosate, immobilized on the surface of the said nanobiocomposite (CMC / HaNP-PGE) at a concentration of 2 pM.

3. A biosensor according to Claims 1 -2, wherein it is disposable.

4. Method of preparing an electrochemical biosensor for use in the determination of glyphosate, comprising the process steps of: i. Preparation of dilute HaNP solution by diluting the stock hydroxyapatite (HaNP) solution in phosphate buffer solution (PBS) with a pH of 7.40, ii. Immersing the pencil graphite electrode (PGE) into the HaNP solution and waiting, immobilising the HaNP solution onto the PGE surface by passive adsorption method, and then washing the electrodes by dipping them in PBS with a pH of 7.40 in order to prevent non-specific binding, iii. Preparation of carboxymethyl cellulose (CMC) in PBS solution with pH 7.40 and then immersing HaNP-PGE in CMC solution to immobilise CMC onto the HaNP-PGE surface by passive adsorption method, and then, to prevent nonspecific binding, immersing and washing the electrode in PBS with a pH of 7.40, iv. Preparation of DNA APT stock solution in T ris hydrochloride (TBS) containing potassium chloride (KCI) and preparation of dilute DNA APT solution by diluting the prepared DNA APT stock solution and applying the denaturation process to the dilute DNA APT solution,v. Immobilising denatured DNA APT onto the CMC / HaNP-PGE surface by passive adsorption method and then washing the electrodes in TBS with pH 7.50 to prevent non-specific binding.

5. A method according to claim 4, comprising the process steps of: i. Preparation of dilute HaNP solution at a concentration of 10-50 pg / mL by diluting the stock hydroxyapatite (HaNP) solution in 0.05 M phosphate buffer solution (PBS) with a pH of 7.40, ii. Immobilising the HaNP solution onto the PGE surface by passive adsorption method by dipping the pencil graphite electrode (PGE) into 80-120 pL HaNP and keeping it for 5-30 minutes, and then washing the electrodes by immersing them in PBS with a pH of 7.40 for 5 seconds in order to prevent non-specific binding, iii. Preparation of carboxymethyl cellulose (CMC) in PBS solution with pH 7.40 in the concentration range of 0.05-5 pg / mL and dipping the HaNP-PGE into 80-120 pL CMC solution and keeping it for 10-40 minutes to immobilise the CMC solution onto the HaNP-PGE surface by passive adsorption method, and then immersing the electrodes in PBS with a pH of 7.40 and washing for 5 seconds to prevent nonspecific binding, iv. Preparation of stock DNA APT solution as 25 pM in 10-30 mM Tris hydrochloride (TBS) solution containing 10-50mM potassium chloride (KCI) and pH 7.50, then preparing a diluted DNA APT solution of 0.1 -4 pM by diluting the prepared DNA APT stock solution in 10-30 mM Tris hydrochloride (TBS) solution containing 10-50mM potassium chloride (KCI) and pH 7.50, and performing the denaturation process by keeping the dilute DNA APT solution at 95°C for 5 minutes and then at +4°C for 15 minutes, v. Immobilising 10-40 pL denatured DNA APT onto the CMC / HaNP- PGE surface for 30-90 minutes by passive adsorption method and then washing the electrodes in TBS with pH 7.50 for 5 seconds to prevent non-specific binding.

6. A method according to claim 5, comprising the process steps of:i. Preparation of dilute HaNP solution at a concentration of 30 pg / mL by diluting the stock hydroxyapatite (HaNP) solution in 0.05 M phosphate buffer solution (PBS) with a pH of 7.40, ii. Immobilising the HaNP solution onto the PGE surface by passive adsorption method by dipping the pencil graphite electrode (PGE) into 100 pL HaNP and keeping it for 15 minutes, and then washing the electrodes by immersing them in PBS with a pH of 7.40 for 5 seconds in order to prevent non-specific binding, iii. Preparation of carboxymethyl cellulose (CMC) in PBS solution with pH 7.40 in the concentration range of 0.5 pg / mL and dipping the HaNP-PGE into 100 pL CMC solution and keeping it for 30 minutes to immobilise the CMC solution onto the HaNP-PGE surface by passive adsorption method, and then immersing the electrodes in PBS with a pH of 7.40 and washing for 5 seconds to prevent nonspecific binding, iv. Preparation of stock DNA APT solution as 25 pM in 10 mM Tris hydrochloride (TBS) solution containing 50 mM potassium chloride (KCI) and pH 7.50, then, preparing a dilute DNA APT solution to 2 pM by diluting the prepared DNA APT stock solution in 10 mM Tris hydrochloride (TBS) solution containing 50mM potassium chloride (KCI) and pH 7.50, and performing the denaturation process by keeping the dilute DNA APT solution at 95°C for 5 minutes and then at +4°C for 15 minutes, v. Immobilising 30 pLdenatured DNA APT onto the CMC / HaNP-PGE surface for 60 minutes by passive adsorption method and then washing the electrodes in TBS with pH 7.50 for 5 seconds to prevent non-specific binding.

7. An electrochemical biosensor for use in the determination of glyphosate prepared by a method according to any one of claims 4-6.

8. A biosensor according to Claim 7, wherein it is disposable.

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