BIOSENSOR-BASED DIAGNOSTIC KIT AND PRODUCTION METHOD FOR THE DETECTION OF PESTICIDE RESIDUES
Patent Information
- Authority / Receiving Office
- TR · TR
- Patent Type
- Applications
- Current Assignee / Owner
- LALE OĞUZHAN
- Filing Date
- 2025-11-12
- Publication Date
- 2026-06-22
Abstract
Description
BIOSENSOR-BASED DIAGNOSTIC KIT FOR THE IDENTIFICATION OF PESTICIDE RESIDUES. PRODUCTION METHOD TECHNICAL FIELD The invention involves using chemicals to increase productivity in agriculture, particularly in vegetables and plants. Developed to detect residues that form on fruits over time. It relates to an innovative biosensor diagnostic kit and its production method. STATE OF THE ART Today, pesticides used in agricultural production leave residues on products. and when these residues enter the human body, they can cause various health problems. This can be the reason. However, those who detect pesticide or chemical residues... Some test systems and laboratory-based methods exist. However, these existing ones... Most methods involve complex analytical steps and are usually performed in a laboratory. This requires a suitable environment, specialized equipment, and expert personnel. For example, in the market... Some of the kits used require specific solvents, reagent solutions, or other preparations before operation. PCR tests require temperature-controlled equipment, which increases costs and... This means the test can only be done in laboratory conditions. Furthermore, this... These systems are generally not portable and are not suitable for field use. Many methods involve additional chemical mixtures and are time-consuming in the sample preparation process. Because it requires certain processes, it both prolongs the process and becomes susceptible to user errors. This makes it possible to conduct these tests, especially in rural areas. Because of this, producers bring their products to the city to detect pesticide residues. They have to send them to laboratories in their centers. This situation takes time. This leads to product loss, spoilage, and economic damage. Document number WO2018192483A1, “Pesticides in edible agricultural products An electronic method for the non-targeted, multi-indexed and rapid detection of remnants; firstly, First-level accurate testing of over 1200 pesticides widely used worldwide. mass spectrometry database and second-level part ion mass spectrometry data the establishment of the base; two mass testing and verification of the pesticide compound. The use of a spectrometry database and a unique electronic 2 for each pesticide. the creation of an identity card, thus using a physical pesticide as a reference. a standard of a traditional identification method with an electronic standard firstly: modification; secondly: high-resolution mass spectrometry of pesticide residues. Software for automated matching, qualitative identification, and intelligent scanning based on data type. the development and deployment of a software program on a device, so that it can be tested comparisons between samples and pesticide mass spectrometry databases speed, high efficiency and accuracy, reliable data, and making processes informative and automated; and thirdly, a single instance once Two detection systems for preparing and simultaneously checking more than 1200 pesticides. The invention involves the use of a technique for the detection of pesticide residues in edible agricultural products. By performing multi-indexed, non-targeted and rapid detection, identification It has been summarized as "greatly increasing its efficiency." Document number US2015185153A1 states, "Examples of the present invention follow these steps..." It describes a pesticide residue detection method that includes: (a) a pesticide residue to be detected the sample is irradiated with a stimulating light and Raman scattered light passes through the sample (b) collection of Raman scattered light from the collected sample; (b) Raman scattering of light from the sample (c) obtaining the spectrum; and (c) the composition of pesticide residue in the sample and To determine the concentration, a reference Raman spectrum is used. with reference Raman spectra for known pesticides in the library Comparison. This method is particularly suitable for rapid detection of pesticides in food. This can enable the effective, useful, and accurate examination of the remains.” It has been summarized. Document number US2024175069A1,” The present invention relates to specific CYP450 pesticide in a sample based on the use of enzymes CYP2B11 and CYP2B10 It relates to the detection method. Furthermore, the present invention involves a method containing these CYP450 enzymes. including kit and simultaneous pesticide detection, relevant to pesticide detection. It is summarized as "relating to uses." DEFINITION OF INVENTION The present invention eliminates the aforementioned disadvantages and the related technical It is related to a kit developed to bring new advantages to the field and its production. 3 The aim of the invention is a gold nanoparticle (AuNPs) based lateral flow biosensor diagnostic kit. by creating it, eliminating the disadvantages of existing pesticide detection methods. The goal is to remove it. Analytical methods such as HPLC or GC-MS used today. Although it gives accurate results, the laboratory environment, solvents, high cost, and expertise are all factors to consider. It requires personnel. Therefore, these methods cannot be applied in the field and do not yield quick results. It is not possible to obtain them. The AuNPs and aptamer components used in the invention are pesticides. When it interacts with the molecule, it causes a color change, resulting in a change within 5-10 minutes. It makes it visible to the naked eye. Thus, neither the device nor anything else is needed to perform the test. A solvent is needed. The aptamer-based structure is more stable than antibodies and It is low-cost; it increases the accuracy of the test while reducing the risk of false results. This As a result, the developed system is fast, economical, portable and accessible to everyone. It is becoming a usable diagnostic kit. Thus, existing methods by eliminating the problems of laboratory work, costs and expert personnel that it requires This will enable direct, easy, and reliable pesticide detection in the field. This kit has been designed so that it can be used directly without the need for any laboratory infrastructure. It is suitable for use in fields, greenhouses, or market places. Fruit and After applying only a tiny drop of the vegetable extract to the test... This will allow us to get results within 10-15 minutes. In this way, the manufacturer and inspector... Or the consumer will be immediately aware of the presence of chemical residues in the food. Our invention has significant applications, particularly in the fields of agriculture and food safety. It has the potential to be used in agricultural production areas, before or during harvest. It can be used in post-inspection checks as well as residue screening in products destined for export. It can also be used for this purpose. In addition, food inspection agencies and producer associations It is a diagnostic kit that can also be used as a quick preliminary assessment tool. It has the characteristic of being in rural areas without electricity, laboratories, or expert personnel. Not requiring fieldwork provides a major advantage. Thus, small producers can... Employees or local inspection teams will be able to use this kit easily. This In this way, both product quality will be controlled and public health will be protected. Measures will be taken to that end. 4 The developed biosensor-based diagnostic kit offers a solution to all of these problems. The invention brings about the use of any solvent, chemical mixture, or PCR device. without needing anything else, a few drops taken directly from vegetable or fruit extract It works by dripping it onto a test surface. Results are visible in a short time. It is obtained in some way. In this way, the user can be a producer, a supervisor, or a consumer. Furthermore, it can conduct tests in the field without the need for a laboratory environment. One of the most important advantages of the invention is its ease of use and economic benefit. Accessibility is key. While current systems require expensive equipment and reagents, The invention kit has a low-cost, portable, and user-friendly design. An expert... It is a structure that can be implemented by anyone without the need for personnel. DETAILED EXPLANATION OF THE INVENTION This detailed description explains the subject of the invention: the pesticide detection kit and its production method. The preferred alternatives to this structure only lead to a better understanding of the subject. This is explained in a way that is geared towards and does not create any limiting effects. The invention involves the visual detection of color change for pesticide detection in fruits and vegetables. The nanomaterial that enables Gold Nanoparticles (AuNPs) to recognize pesticide molecules DNA aptamer (pesticide-specific), which is a bioreceptor, for conjugation and stability. PBS Buffer (pH 7.4), the medium solution, is used for reduction in AuNP synthesis. Sodium citrate, a stabilization agent, and gold, a source for AuNP synthesis. By passing through the production process steps of substances such as HAuCl₄ (gold chloride) It is characterized by being a pre-designed diagnostic kit. The components used in the invention are: - A nanomaterial that provides color change for visual detection, and 1 mL (0.01 M Gold Nanoparticles (AuNPs) with an average diameter of 20–25 nm (in solution), - A bioreceptor that recognizes the pesticide molecule, containing 2 µM (50 µL) Thiol(-SH) Modified DNA Aptamer (Pesticide-specific), - 10 mL Phosphate-buffered saline (PBS) as the medium solution for conjugation and stability. Tampon (pH 7.4), - Used as a reducing and stabilizing agent in AuNP synthesis, at a concentration of 1 mL (1% w / v). Sodium citrate, 5, used during the reaction. - Gold source for AuNP synthesis, prepared in 1 mL (1 mM) solution. HAuCl₄ (Gold chloride), - Sample flow buffer used at 5 mL (10 mM) and pH balance Tris-HCl, which provides - A flow rate regulating surfactant, present at 0.1% by volume and in 100 µL Tween-20 is used in the pretreatment of sample pads. -A non-specific binding inhibitory protein, present at 1% by volume and in 100 µL BSA (Bovine Serum Albumin) is used in sample pad coating. - The surface on which the test and control lines are fixed, 1 strip measuring 4×60 mm. Nitrocellulose Membrane used as the main surface of the test, - The input area where the sample is fed by the kit, measuring 4x20 mm, for pH and Sample Pad (Glass fiber) with buffer pretreatment applied, -1 piece measuring 4×20 mm, which is the area where the AuNP-probe complex is fixed. Conjugation Pad containing dried AuNP, - A 4x30 mm pad that complements the flow and absorbs excess fluid. Cellulose-based absorbent pads. - A carrier, 60 mm long, made of plastic, in which all the components are placed. Cassette (Test body), - 2-3 drops are taken as a sample to detect pesticide residue, and Sample as homogenized sample (Vegetable / Fruit extract), - This is 20 mL of general-purpose distilled water for washing and dilution purposes. The contents of the invention kit are by weight; - Preferably 12%, but around 8-15% Gold Nanoparticles (AuNPs), - Preferably 8%, but around 5-10% pesticide-specific DNA aptamer. - Nitrocellulose membrane with a test surface of approximately 8-18%, preferably 15%. - Sample pad containing approximately 20-35% glass fiber / cotton, preferably 25%. - Contains approximately 8-15% AuNP-Aptamer complex, preferably 12%. Conjugation Pad, - Absorbent pads with a cellulose base of 3-10%, preferably 7%. - PBS buffer at pH 7.4, preferably 5%, but around 3-8%. - Tween-20 as a surfactant, preferably around 3%, but around 2-4%. - BSA (Bovine Serum Albumin) of around 2-6%, preferably 5%, 6 - Sample solution of Tris-HCl buffer, preferably 5%, but around 3-8%. - Distilled water as a solvent, preferably 3%, but around 2-5%. - A plastic cassette is used as the kit body. The invention is based on the contents mentioned above. • HAuCl₄ solution heated to 95–100 °C, with the addition of 1% sodium citrate. until the color is reduced and the color change is complete, for approximately 15 minutes. The gold nanoparticle synthesis stage involves mixing at a speed of 500 rpm. • Mild infusion in PBS at room temperature and pH 7.4 for 12 hours. by mixing, aptamer binding is ensured and in terms of test specificity The critically important AuNP and DNA Aptamer conjugation step, • The pads were treated with Tris-HCl and 0.1% Tween-20 and 1% BSA. coated and dried at 37°C for 30 minutes, and flow control was ensured. Sample, Conjugation, Preparation of Absorbent Pads stages, • Pesticide-specific trapping sequences and anti-biotin on nitrocellulose membrane Antibodies were applied in a linear fashion, fixed at 37°C for 2 hours, and visually observed. Test and Control Lines that define the main region where the signal originates. implementation phase, • Sample, conjugation, test and absorbent pads should have a 2 mm overlap. aligned and placed inside a plastic cassette, then kept under vacuum for 24 hours. The assembly stage of the biosensor layers, where they are dried. • System performance was measured using PBS buffer and distilled water. and calibration and quality where sensitivity, specificity and accuracy tests are performed. A detection kit is created after passing through the control phase. Every element involved in the invention contributes to the test being accurate, fast and portable. It provides this. Gold nanoparticles (AuNPs) are the most characteristic component of the invention. It causes a color change when it comes into contact with the pesticide molecule, the result of which is visible to the naked eye. It allows them to be seen. In addition, thanks to its high surface area, aptamers can attach strongly to the surface. It helps the DNA aptamer to adhere properly and increases the sensitivity of the test. It is a special structure that recognizes the target pesticide molecule. This allows the test to target only the intended target. It reacts to the pesticide, preventing false positive or negative results. The nitrocellulose membrane forms the surface where the reaction takes place and the sample 7 Ensures the sample moves smoothly along the test line. Sample pad, fruit or It ensures that the sample taken from the vegetable extract passes homogeneously into the system. The conjugation pad contains aptamers bonded to gold nanoparticles. This is the area; interaction begins as the sample passes through this region, and color formation occurs. The absorbent pad absorbs excess liquid, balancing the flow of the test and resulting in clearer results. It allows the reaction to occur. PBS buffer and Tris-HCl keep the system stable, preventing the reaction. It optimizes the conditions. Tween-20, as a surfactant, ensures the sample is properly prepared. BSA allows the flow to occur, while BSA prevents unwanted bindings, thus ensuring the test is successful. It improves accuracy. The plastic cassette holds all these components together, making it portable. It forms a durable structure. Distilled water, on the other hand, is used for preparing mixtures and the system. It is used for cleaning. The invention's production process begins with the synthesis of gold nanoparticles (AuNPs). Specific temperature, time, and process are required until the assembly of the lateral flow biosensor kit. This occurs under the following conditions. Here, first, HAuCl₄ at a concentration of 1 mM is used. 100 mL of the solution is taken and boiled in a glass beaker at 95–100 °C. It is heated to the point of boiling and stirred at a speed of 500 rpm. 1% (w / v) is added to this boiling solution. 10 mL of sodium citrate solution is slowly dripped in at a specific ratio. Approximately 15 minutes. then the color of the solution changes from yellow to wine red, revealing the gold nanoparticles. It is observed that it forms. The mixture is cooled to room temperature (preferably 25 °C) and the resulting The resulting AuNP solution is prepared for the next stage. The pesticide is added to the cooled solution. 50 µL of specific DNA aptamer solution is added. This mixture is then heated slightly at room temperature. It is mixed and incubated for 12 hours. At this stage, the medium is prepared using PBS buffer. The pH is kept constant at 7.4. After the conjugation process is complete... The mixture is centrifuged at 4000 × g for 10 minutes, the supernatant is removed, and the pure mixture is obtained. AuNP–aptamer complex is obtained. Lateral flow biosensor layers. In its preparation, first a sample pad is made, preferably cut from glass fiber and mixed with Tris-HCl. The pad is dipped into a solution containing 0.1% Tween-20 and 1% BSA. Leave on for 5 minutes. After being left to stand, it is dried at 37°C for 30 minutes. Then the conjugation pad... 50 µL of purified AuNP-aptamer solution is dropped onto it and placed in the room. It is dried at [temperature] for 1 hour. It is applied to a nitrocellulose membrane using a micropipette. Test (T-line) and control (C-line) lines are applied; the test line is marked with a pesticide-specific marker. A biotin-containing capture DNA sequence is applied to the DNA line, while an anti-biotin antibody solution is applied to the control line. 8 The membrane is fixed by incubating at 37 °C for 2 hours. Absorbent pad cellulose based It is prepared from the material and dried directly, making it ready for use. All components (sample pad, conjugation pad, nitrocellulose membrane and absorbent) The pad is aligned with the backing plate, leaving an overlap of approximately 2 mm, and It is glued together. Then this structure is placed inside a plastic cassette. Assembly Once completed, the test strips should be left at room temperature in a dry environment for 24 hours. It is dried under vacuum and then stored. The moisture content of the chitin is checked before use. To maintain its balance, it is recommended to keep it in a moistened environment with distilled water. During the test, a sample (approximately 2–3 drops) of extract obtained from the fruit or vegetable is taken. The sample is dropped onto a pad and travels across the membrane by capillary action 5–10 It gives results within minutes. Thus, a color change in the test line is observed. The presence of pesticide residue is determined quickly and practically. The entire production process is in this room. The process is carried out at a temperature of (25 ± 2 °C), and the drying steps are done in a moisture-free incubator. This process is carried out at atmospheric pressure, excluding the centrifugation process. The result is a portable, economical, and reliable pesticide detection kit. All materials and process conditions used in the production process of the invention. It has been optimized to be applicable within a specific range of values. Gold Nanoparticle synthesis using 0.5–2 mM HAuCl₄ solution at 0.5–1.5% w / v This is achieved by reduction with sodium citrate at a certain ratio. The reaction temperature is 95– 100 °C, mixing speed 400–700 rpm and time between 10–20 minutes. It can be modified. The size of the nanoparticles formed under these conditions is between 15–35 nm. It is controlled; as particle size increases, color intensity increases, in this case citrate The ratio is adjusted to 0.8–1.2%. After cooling the resulting AuNP solution, pesticide-specific DNA aptamers were used to treat it. Aptamer concentration is 0.1–1 µM, and incubation time is between 4–24 hours. It is applicable. During conjugation, the medium is kept between pH 7.2–7.6 with PBS buffer. If the binding strength is insufficient, the NaCl concentration should be in the range of 100–300 mM. This can be increased. The purification process is carried out at a centrifuge speed of 3000–6000×g for 5–15 minutes. It is completed by implementing it. 9 During sample pad preparation, Tris-HCl buffer was used at a concentration of 5–20 mM, or 0.05–0.2%. Wet for 2–10 minutes in a solution containing Tween-20 and 0.5–2% BSA and incubate at 30–45 °C for 20– It is dried for 45 minutes. 20–80 µL of AuNP-aptamer mixture is applied to the conjugation pad. It is applied and dried at room temperature for 45–90 minutes. Nitrocellulose membrane Test and control lines are drawn on it with a volume of 0.5–1.5 µL / cm², then 30–45 It is incubated at °C for 1–3 hours. The absorbent pad is prepared from cellulose-based material and It is dried directly. During assembly, all layers are aligned with an overlap of 1–3 mm, ensuring plastic sealing. It is placed inside the cassette. The drying process is carried out at 25–35 °C, with %humidity < 20, for 12– The process takes 36 hours. Test strips can be stored in a dry environment at temperatures between 2–30 °C. During use, lightly moisten the test kit with distilled water and place the vegetable or liquid on top. 50–120 µL (approximately 2–4 drops) of fruit extract sample is added. Reaction 5– The process is completed within 15 minutes, and the color change is observed. If it's gold nanoparticles... If the concentration is increased from 10–12% to 14–15%, the aptamer concentration will decrease from 5–7% to 6–8%. The rates should be increased; Tween-20 should be increased from 0.1% to 0.15% and BSA from 1% to 1.5%. Conversely, if the aptamer ratio is reduced, the capture sequence density in the test line increases. Signal balance is maintained by increasing the concentration from 0.1% to 0.2 mg / mL. Fruit or vegetable When 2–3 drops of the extract are applied to the sample pad, capillary flow occurs. The result is a color change that will be observed within 5-10 minutes. In an alternative application of the invention, the product is derived from produced fruits and vegetables. To enable rapid and portable detection of pesticide residues from samples. For this purpose, the use of AuNPs (Gold Nanoparticle) based lateral flow biosensors. It is based on the principle that in this process, plant samples are first homogenized. It is extracted using the QuEChERS method, which involves acetonitrile and magnesium sulfate, and obtained. The resulting upper phase is filtered and prepared for testing. The obtained extract is pre-treated with pesticides. AuNPs-probe conjugates functionalized with molecule-specific aptamers The sample is placed in a few drops on the sample pad of the test strip. Capillary action allows the molecule to move across the membrane, specifically targeting the molecule. Binding has occurred, and the appearance of a red color on the test line indicates a positive result. It shows. The most important parts during the application are; particle 10 in AuNPs synthesis. preservation of size, aggregation formation during the conjugation process prevention, prevention of contamination risk in sample preparation, buffer Maintaining constant pH and ionic strength of the solution, ambient temperature 20–25 °C The test components must be within the specified range and protected from moisture. Additionally, the test must be accurate. For it to yield results, the control line needs to be visible. All these processes... The process takes approximately 15 minutes, and the results can be seen with the naked eye or a mobile camera. This can be verified with a laboratory-based color analysis system. This application method is laboratory-based. Reliable field detection of pesticide residues without requiring a suitable environment. It is a user-friendly system that enables this. In an alternative application of the invention, AuNPs-based lateral flow is used. The biosensor platform uses different nanomaterials to achieve the same purpose. It is adaptable via bioreceptors and signaling systems; for example, gold Silver nanoparticles (AgNPs) instead of magnetic nanoparticles Similar results can be achieved using nanoparticles (Fe₃O₄) or quantum dots (CdSe / ZnS). Visual or fluorescence-based detection systems can be developed; as an alternative to aptamers. monoclonal antibodies or peptide-based receptors specific to pesticide molecules are integrated. The biorecognition component can be modified. Additionally, the component used in the test line can be altered. PVDF or nanofiber polymer membranes are preferred instead of nitrocellulose membranes. By doing this, flow rate and binding efficiency can be optimized. However, color electrochemical or fluorescence-based systems instead of colorimetric systems based on color change. By adapting the reading mechanisms, the detection of the same target pesticide residue can be done differently. This can be achieved through signaling methods. Alternatively, in AuNPs conjugation. carbodiimide (EDC / NHS) is used instead of the thiol-based DNA probe binding strategy. chemistry, butanol dehydration, or streptavidin-biotin interaction-based binding. The methods can be applied. The LAMP-based replication phase is also optional. PCR, RPA (Recombinase Polymerase Amplification) or isothermal It can be modified with other versions of amplification. Furthermore, the system can handle pesticides as well. in addition to detecting heavy metal ions, mycotoxins or antibiotic residues adaptable. These alternative compositions maintain the basic operating principle of the device. (Biorecognition, signal conversion, visual detection) serve the same purpose by protecting; Thus, the project can be adapted to different target molecules, reducing production cost and accuracy. It can be optimized according to the ratio and field conditions. 11 The invention includes analytical verification, calibration curves, LOD calculations, repeatability, and Inter-person variability analyses were performed using field / hybrid conditions (20–35 °C, (30–75% RH) robustness tests and reference method validation are performed using HPLC. Cross-comparison of MS / GC-MS and agarose gel run-up tests. It can be configured. Nitrocellulose membrane drawing parameters and pad in production. pre-processing stages, AuNPs-aptamer conjugation lot records, and Accelerated / real-time shelf life (stability) regulation and risk management by ISO. ISO 13485 quality system, ISO 14971 risk analysis assessment, CE, IVD conformity. evaluation and disposal of bio and chemical waste in accordance with Environmental Legislation Suitable for configuration. Smartphone and / or image processing-based color intensity. To ensure traceability from the field with a reading and cloud recording module, in rural areas. User training, illustrated quick guides, and troubleshooting flowcharts are included. A structure that allows for increased reliability is possible with the invention. It is being done. In terms of the order and priority of operations in an invention; - Sample Preparation: Edible parts of plant samples are homogenized. This ensures even distribution of pesticides. This step maintains sample integrity for accurate identification. It has the top priority in terms of protection. - Extraction Process (QuEChERS Method): 10 mL acetonitrile to 10 g plant sample added, followed by a salt packet containing magnesium sulfate and sodium acetate. Shaking is performed. This stage involves incorporating pesticides into the organic phase and other processes. It is critical for separating the components. - Centrifugation and Phase Separation: Organic phase (superior phase) separated by centrifugation at 4000 × g for 5 min. It is separated. The resulting pesticide extract is purified and clarified for analysis. - Filtration and Cleaning: The upper phase is passed through a 0.22 µm syringe filter. If necessary. Pure extract is obtained by removing pigments and lipids using SPE or mPFC columns. It is done. - Synthesis of AuNPs (Gold Nanoparticles): HAuCl₄ solution sodium citrate reduction Gold nanoparticles are synthesized by heating the solution using this method. The solution changes from yellow to red. Rotation is an indicator of particle formation. 12 - Aptamer or DNA Probe Conjugation: The surface of the synthesized AuNPs is treated with pesticides. The molecule is modified via the thiol (-SH) group using aptamers specific to the molecule. This step It determines the specificity of the biorecognition system. - Assembly of the Lateral Flow Strip: Sample pad, conjugation pad, nitrocellulose The membrane (test and control lines) and absorbent pad are mounted sequentially on the strip. The form is created. - Test Procedure: Add 2–3 drops of filtered plant extract to the test sample. The sample is dripped onto a pad. The sample moves through the air by capillary action and, in the presence of the target pesticide, moves along the surface. AuNPs exhibit binding to the aptamer complex. - Visual Reading and Result Evaluation: Formation of red color on the test line. While showing a positive result, the appearance of only the control line indicates a negative result. It can be read with the naked eye or with a smartphone-assisted color analysis system. It can be done. - Verification and Recording: The obtained results are tested against HPLC-MS or GC-MS references. These methods are compared, and accuracy and repeatability data are recorded. As critical control points for the invention to achieve its purpose, • Preventing contamination during sample preparation • Conservation of particle size in AuNPs synthesis • Prevention of aggregation after conjugation • Maintaining the buffer pH at around 7.4 • The ambient temperature should be between 20–25 °C during the test. • Maintaining membrane moisture content between 30–50% • It is important that the control line is visible in every test. In terms of the proportions of substances that the invention must contain in order to achieve its purpose, - Plant sample and extraction solvent ratios; 10 mL for every 10 g of plant sample. Acetonitrile is used. This ratio ensures maximum solubility of pesticides while protecting the plant. It maintains the saturation of its texture. - Salt Mixture (QuEChERS Salt Packet): 4 g per 10 mL of extraction solution. Magnesium sulfate (MgSO₄) and 1 g of sodium acetate (CH₃COONa) are added. This ratio is... It accelerates phase separation and effectively removes water from the organic phase. It provides. 13 - Gold Nanoparticle (AuNPs) Synthesis Ratio: 1 mM HAuCl₄ solution to 100 mL It is added to boiling distilled water. Then, 1% (w / v) sodium citrate is added. 10 mL of the solution is added. This ratio is approximately 20–25 nm in diameter monodisperse gold. It enables the production of nanoparticles. - Aptamer Conjugation Ratio: 10 µL of 100 µM thiol- to every 1 mL of AuNPs solution. Modified aptamer is added. The mixture is slowly left to infuse at room temperature for 12–24 hours. They are mixed and incubated. This ratio ensures sufficient aptamer binding to the gold surface. It is optimized to ensure stable conjugation. - Buffer Ratio (PBS + NaCl): 1x PBS (pH 7.4) buffer to the remaining NaCl. Concentrations (0.1 M → 0.5 M) are added step by step. This process creates nanoparticles. It prevents aggregation by balancing the electrostatic interactions on its surface. - Pretreatment Ratios of Pads: Tris-HCl (20 mM, pH 8.0), Tween-20 (0.05%) and BSA (1%) A mixture containing these ratios is used. These ratios regulate fluid flow and prevent non-specific bonding. It reduces. - Line Application Rate on Membrane: Test (T-line) and control (C-line) Streptavidin (0.5 mg / mL) and anti-biotin antibodies were injected into the affected areas at a concentration of 1 µL / mm³. Solutions of (0.3 mg / mL) are applied. This ratio both increases the binding efficiency and... It optimizes color contrast. - Sample Application Rate: 2–3 drops (~100 µL) of extract are applied to the test strip. Capillary flow is complete within 5–10 minutes, and color formation is observed. - Analytical Confirmation Rates: Target pesticide in positive control samples The concentration is diluted to a range of 1 pM – 1 µM. Visual signal The intensity gives a linear response in this range (R² > 0.98). - Stability and Storage Rates: Conjugate solutions are stored at 4°C containing 0.05% NaN₃. The membranes are stored in buffers, while the membranes themselves are kept in an environment with 20–30% relative humidity. The physical properties of the materials used in the invention under normal / ideal conditions aspect, - Gold Nanoparticles (AuNPs): Physical state is colloidal liquid (wine-red color) suspension), with a narrow size distribution, particle size in the 20–25 nm range. It is in colloidal suspension form, with a density of approximately 19.3 g / cm³ (for solid Au) and In colloidal form, it is very low, surface charge (Zeta potential) is around -35 mV, this The value ensures the electrostatic stability of the particles, light absorption property 520 nm 14 maximum absorbance (surface plasmon resonance) at the specified wavelength, resolution It is insoluble in water, but when stabilized with citrate or thiol groups It is found dispersed in colloidal suspension and manifests as color change. When aggregation occurs, the color changes from red to purple; this affects the visual aspect of the test. It forms the basis of the principle. - Aptamer (DNA Oligonucleotide): Physical form is powder or lyophilized, dissolved in water. Soluble. Molecular weight is in the range of 7–12 kDa on average. Color / Odor: colorless. Odorless. Stable for extended periods at -20°C and for short periods at room temperature. pH sensitivity remains most stable in the 7.0–8.0 range. - Nitrocellulose Membrane: Physical state is a thin, white, porous ribbon structure (approximately Thickness: 100–150 µm. Pore size: 8–12 µm, this value reflects the capillary flow of the liquid. It determines its speed. Density 1.35 g / cm³ and hygroscopic, tending to absorb moisture. Its flammability is high, and therefore it is stored in a dry environment. It is highly flammable and easily ignites. It should be kept away from heat and open flames. - Buffer Solutions (PBS, Tris-HCl): Physical state is a colorless liquid, a clear solution. pH The value is around 7.4 for PBS and 8.0 for Tris-HCl. There is no odor. The density is approximately... It is completely soluble in water at a concentration of 1 g / mL and solubility, maintaining ionic balance. - Sodium Citrate (Na₃C₆H₅O₇): Physical state is a white crystalline powder. Solubility in water. Highly soluble. Melting point 150 °C, pH (in 1% solution): 7.5–9.0 It is slightly basic in the range and its function is to reduce Au³⁺ ions and the surface of AuNPs. It stabilizes. - Sodium Azide (NaN₃): Physical state is white crystal, colorless, odorless. Melting point. 275 °C (decomposes and releases gas). Density: 1.85 g / cm³. Solubility in water. It is soluble and used in low concentrations (0.05%) as an antimicrobial preservative. - BSA (Bovine Serum Albumin): In its physical form, it is a powder that is protein-soluble and water-soluble. The color is light yellow-beige. The density is 1.35 g / cm³ and it has long-term stability at 4 °C. It is storable. As a surface blocking agent, it prevents non-specific binding.
Claims
1- The invention is a biosensor-based diagnostic kit for the identification of pesticide residues, and its feature is; by weight; - Preferably 12%, but around 8-15% Gold Nanoparticles (AuNPs), - Preferably 8%, but around 5-10% pesticide-specific DNA aptamer. - Nitrocellulose membrane with a test surface of approximately 8-18%, preferably 15%. - Sample pad containing approximately 20-35% glass fiber / cotton, preferably 25%. - Contains approximately 8-15% AuNP-Aptamer complex, preferably 12%. Conjugation Pad, - Absorbent pads with a cellulose base of 3-10%, preferably 7%. - PBS buffer at pH 7.4, preferably 5%, but around 3-8%. - Tween-20 as a surfactant, preferably around 3%, but around 2-4%. - BSA (Bovine Serum Albumin) of around 2-6%, preferably 5%. - Sample solution of Tris-HCl buffer, preferably 5%, but around 3-8%. - Distilled water as a solvent, preferably 3%, but around 2-5%. - The kit consists of a plastic cassette as its body. 2- It is a kit according to Claim 1, and its characteristics are; - A nanomaterial that provides color change for visual detection, and 1 mL (0.01 M Gold Nanoparticles (AuNPs) with an average diameter of 20–25 nm (in solution), - A bioreceptor that recognizes the pesticide molecule, containing 2 µM (50 µL) Thiol(-SH) Modified DNA Aptamer (Pesticide-specific), - 10 mL Phosphate-buffered saline (PBS) as the medium solution for conjugation and stability. Tampon (pH 7.4), - Used as a reducing and stabilizing agent in AuNP synthesis, at a concentration of 1 mL (1% w / v). Sodium citrate used during the reaction, - Gold source for AuNP synthesis, prepared in 1 mL (1 mM) solution. HAuCl₄ (Gold chloride), - Sample flow buffer used at 5 mL (10 mM) and pH balance Tris-HCl, which provides - A flow rate regulating surfactant, present at 0.1% by volume and in 100 µL Tween-20, used as a pretreatment agent in sample pads, 16 -A non-specific binding inhibitory protein, present at 1% by volume and in 100 µL BSA (Bovine Serum Albumin) is used in sample pad coating. - The surface on which the test and control lines are fixed, 1 strip measuring 4×60 mm. Nitrocellulose Membrane used as the main surface of the test, - The input area where the sample is fed by the kit, measuring 4x20 mm, for pH and Sample Pad (Glass fiber) with buffer pretreatment applied, -1 piece measuring 4×20 mm, which is the area where the AuNP-probe complex is fixed. Conjugation Pad containing dried AuNP, - A 4x30 mm pad that complements the flow and absorbs excess fluid. Cellulose-based absorbent pads. - A carrier, 60 mm long, made of plastic, in which all the components are placed. Cassette (Test body), - 2-3 drops are taken as a sample to detect pesticide residue, and Sample as homogenized sample (Vegetable / Fruit extract), - It contains 20 mL of general-purpose distilled water for washing and dilution purposes. 3- The invention describes a method for a biosensor-based diagnostic kit for the identification of pesticide residues. Its characteristic feature is the lateral flow starting from the synthesis of gold nanoparticles (AuNPs). with specific temperature, time and process conditions until the assembly of the biosensor kit This occurs when, first, 100 mL of HAuCl₄ solution at a concentration of 1 mM is added. It is taken and heated in a glass beaker at a temperature of 95–100 °C until it reaches its boiling point, and Stir at a speed of 500 rpm, then add 1% (w / v) sodium citrate to this boiling solution. 10 mL of the solution is slowly dripped in; after about 15 minutes, the color of the solution changes. It is observed that gold nanoparticles form as the color changes from yellow to wine red. The mixture is cooled to room temperature, preferably 25 °C, and the resulting AuNP solution is then placed in a container. It is then prepared for the next stage, and pesticide-specific DNA is added to the cooling solution. 50 µL of aptamer solution is added, and this mixture is gently stirred at room temperature. It is incubated for 12 hours, during which time the pH of the medium is adjusted to 7.4 using PBS buffer. is kept constant at this level, after the conjugation process is complete, the mixture is 4000 The supernatant is removed by centrifugation at × g for 10 minutes, and the pure AuNP–aptamer is obtained. a complex is obtained in the preparation of lateral flow biosensor layers. First, the sample pad is cut, preferably from glass fiber, and placed in Tris-HCl buffer. The pad is dipped into a solution containing 0.1% Tween-20 and 1% BSA and left for 5 minutes. then dried at 37°C for 30 minutes, then placed on a conjugation pad 17 50 µL of purified AuNP-aptamer solution is added dropwise and left at room temperature for 1 After drying for an hour, a test (T-line) is placed on a nitrocellulose membrane using a micropipette. Control (C-line) lines are applied; the test line is then trapped with pesticide-specific biotin. The DNA sequence is applied to the control line and anti-biotin antibody solution is applied to the membrane 37 Incubate and fix at °C for 1–2 hours (or at 20–25 °C for 12–16 hours), Absorbent The pad is made from cellulose-based material and is ready for use in a dry state. and Sample pad, conjugation pad, nitrocellulose membrane and absorbent pad, back The backing plate will have an overlap of approximately 2 mm at each joint. They are aligned and glued together; then this structure is placed inside a plastic cassette and After assembly, the test strips were placed in a dry environment (with desiccant) in the room. It is dried under vacuum at [temperature] for 24 hours or at 37°C for 1–2 hours and then dehumidified. It is stored in a sealed package and no additional moistening is required before use. The test is performed by adding sample / running buffer according to the manufacturer's instructions. It is the characterization of the situation. 4- It is a method according to claim 3, and all used in the production process structuring. The terms and conditions of the transaction will be applicable within a specific range of values. The feature that enables optimization in this way is; the synthesis of gold nanoparticles in 0.5– A 2 mM HAuCl₄ solution mixed with 0.5–1.5% w / v sodium citrate. This is achieved by reduction, with a reaction temperature of 95–100 °C and stirring. The speed can be varied between 400–700 rpm and the duration between 10–20 minutes, The size of the nanoparticles formed under these conditions is controlled between 15–35 nm. this can be achieved and the color intensity increases as the particle size increases, and this In this case, adjusting the citrate concentration to 5–10%, the resulting AuNP solution After cooling, pesticide-specific DNA aptamer is added, increasing the aptamer concentration. 0.1–1 µM, applicable with an incubation period of 4–24 hours, and During conjugation, the medium pH should be maintained between 7.2–7.6 with PBS buffer. If the binding strength is insufficient, the NaCl concentration can be increased to a range of 50-200 mM. and the purification process should be carried out at a centrifuge speed of 10,000–14,000×g for 10-20 minutes. The process is completed by applying Tris-HCl buffer 5–20 while preparing the sample pad. In a solution containing 0.05–0.2% Tween-20 and 0.5–2% BSA at a concentration of mM, for 2–10 minutes. wetting and drying at 30–45 °C for 20–45 minutes, onto the conjugation pad. Apply 20–80 µL of AuNP-aptamer mixture and leave at room temperature for 45–90 minutes. drying, test and control lines of 0.5–1.5 µL / cm³ on nitrocellulose membrane. applied by volume and then incubated at 30–45 °C for 1–3 hours and absorbent The pad is prepared from cellulose-based material and dried directly before assembly. During this stage, all layers are aligned with an overlap of 1–3 mm in the plastic cassette. The drying process is carried out by placing them inside at 25–35 °C, with %humidity < 20 for 12– It lasts 36 hours. 5- The invention is a biosensor-based diagnostic kit for the identification of pesticide residues, and its use... Its feature is that the test kit is slightly moistened with distilled water during use. Adding 50–120 µL (approximately 2–4 drops) of vegetable or fruit extract to the mixture, The reaction should be completed within 5–15 minutes, and a color change should be observed. If the gold nanoparticle ratio is increased from 10–12% to 14–15%, the aptamer ratio will increase. It is necessary to increase it from 5–7% to 6–8% and Tween-20 from 0.1% to 0.15%. If the BSA is increased from 1% to 1.5% and conversely, the aptamer ratio is decreased, the test pipeline will... Signal balance was achieved by increasing the capture sequence density from 0.10 mg / mL to 0.20 mg / mL. It needs to be preserved and 2-3 drops of fruit or vegetable extract, for example When applied to the sample pad, capillary flow occurs within 5–10 minutes. The color change is observable. 6- An alternative to the invention kit according to any of the above claims. In its application, pesticides are extracted from samples taken from produced fruits and vegetables. Its feature of not enabling the rapid and portable detection of remnants; AuNPs (Gold It is based on the use of nanoparticle-based lateral flow biosensors. In this process, plant samples are first homogenized and then treated with acetonitrile and magnesium. Extracted using the sulfate-containing QuEChERS method, the resulting supernatant is filtered. The resulting extract is prepared for testing, and is then processed using a method specific to the pesticide molecule. test strip containing AuNPs-probe conjugates functionalized with aptamers A few drops are placed on the sample pad, and the sample is drawn in by capillary action. thanks to its ability to specifically bind to the target molecule as it moves across the membrane. This occurs, and the appearance of a red color on the test line indicates a positive result. It is the fact that. 7- An alternative to the Invention according to any of the above Claims In the application, the AuNPs-based lateral flow biosensor platform used is the same To achieve this goal, different nanomaterials, bioreceptors, and signaling devices are used. It is an adaptable kit with various systems, and its feature is that it uses silver nanoparticles instead of gold nanoparticles. nanoparticles (AgNPs), magnetic nanoparticles (Fe₃O₄), or Quantum 19 Similar visual or fluorescence-based detection using dots (CdSe / ZnS). The systems can be improved, here using pesticide molecule-specific solutions instead of aptamers. Biorecognition by integrating monoclonal antibodies or peptide-based receptors. its component is replaceable and also the nitrocellulose used in the test line Instead of membranes, PVDF or nanofiber structured polymer membranes (e.g., nylon 6) flow rate and binding efficiency are optimized by choosing (or polycaprolactone). instead of a colorimetric system based on color change and being able to do so by adapting electrochemical or fluorescence-based reading mechanisms, the same Detection of target pesticide residue can be performed using different signaling methods. It is the fact that.