The invention is related to a method for obtaining a fluorophore dye from edible wild plants, developed to be used in biochemistry, biology, molecular biology and genetics, medicine, pharmacology fields, cell monitoring for bio-analytic and medical diagnosis aims, material technologies and nanotechnology fields.

The invention is particularly related to a method to be used in the fluorescence cell monitoring for obtaining a nano-material, carbon-based fluorophore dye from naturally growing edible wild plants.

State of the Art

Today fluorophore dyes are used for cell imaging in bioanalytical studies and medical diagnosis. These fluorophore dyes can be excited by means of UV light and Fluorescence can be determined by means of measuring devices. The quantum yield and photo-stability of these fluorophore dyes are required to be high. In addition, these fluorophore dyes are required to be dissolved in the buffers used in the cell environment and in body fluids, also to be in sizes that can pass through the cell membrane and bio-compatible.

In the present state of the art, as the fluorophore dye, Propidium iodide, Ethidium bromide, Hoechst dye, 4,6-Diamidino-2-phenylindole dihydrochloride (DAPI), Acridine orange, Fluorescein isothiocyanate (FITC) coloring dyes are used. However these mentioned dyes are all synthetic and imported. Toxic wastes are formed by using these compounds in the fluorescence cell-imaging. This condition creates risks for the human health and the environment. For this reason these dyes lead to toxic or mutagenic disadvantages. Also since these dyes are imported, they increase the cost of cell imaging for medical diagnosis.

In the literature, the following applications concerning the subject matter are found.

In an application with number of CN108485307, a dye with fluorescence properties and its production and application methods of this dye are disclosed. In this 5-azido-8- aminokinolin fluorescence dye, the primary binding agents are H, stiren, butyll-1 , 3-dien-1 , -ylbenzen or 4-vinyl phenol, the secondary binding agent is H or styrene and tertiary binding agent is H or styrene. After this, fluorescence dye is subjected to 1 ,3-dipolar cyclosis with alkyne, a fluorophore that has high quantum yield for triazole formation is provided. This fluorescence dye which is suitable for using in fluorescence dye cell monitoring can be obtained synthetically.

In another application with number of CN106478594, a fluorescence dye, synthesis method thereof and a usage method of a fluorescence probe prepared from this dye is disclosed. The quinolone structure in the probe obtained from this dye is a two-photon fluorophore and it is presented for triazole methodology. In the formula of the specified probe, the primary binders are C2-C10 oil alkyl or C5-C6 heterocyclic ring or substitutions thereof; the substituted group here is halogen, C1-C6 alkyl or methoxyl; and the secondary binder is hydrogen, methoxyl, Ci-Cio alkyl or C2-C6 conjugate alkenyl. These probe cells which are determined to give small damage to the cells are stated as an effective research tool for searching specific physiological actions. However the fluorescence dye here and the prob prepared therefrom are obtained synthetically.

As a result due to above mentioned disadvantages and the insufficiency of the current solutions in terms of the subject, an improvement in the mentioned technical field is required.

Brief Description of the Invention

The present invention is related to a method for obtaining a fluorophore dye from the edible wild plants which fulfills the abovementioned requirements, eliminates all disadvantages and brings some additional advantages.

The main purpose of the invention is to develop a method for obtaining fluorophore dye from the natural carbon sources in an environmentally friendly manner.

A purpose of the invention is to develop a method for obtaining the fluorophore dye in an effective, safe and economic manner.

Another purpose of the invention is to develop a method for obtaining a non-toxic fluorophore dye which enables proper and safe bio-monitoring. Another purpose of the invention is to provide a method which allows for rapid preparation of the fluorophore dye.

Another purpose of the invention is to develop a method for obtaining sustainable products.

Another purpose of the invention is to develop a method for obtaining a carbon based nano-materials which reduces the toxic effect of the medical wastes and has feature of not containing medical wastes, does not create risk to human health and environment.

Another purpose of the invention is to develop a method for contributing reduction of foreign source dependency by providing preparation of CQDs (Carbon Quantum Dots) from edible wild plants that are dispersed in our country and can grow naturally.

Another purpose of the invention is to develop fluorophore dyes which can be stimulated by UV light, can be detected by means of devices making fluorescence measurement, have high quantum yield and photo-stability, can dissolve in buffers used in the cell environment and in body fluids, have sizes that can pass through the cell membrane and is bio-compatible and to develop method for producing thereof.

In order to fulfill this, the inventive method comprises the following steps; collecting, drying and grinding the wild plants; preparing carbon quantum dots from wild plant particles; centrifugation for removing the plant residues within the reaction medium from the medium; microfiltration for removing smaller particles which cannot be removed by centrifugation process and for purifying the carbon quantum dots; membrane dialysis for providing equal dimension dispersion of carbon quantum dots and converting carbon quantum dots into solid powder form by lyophylisation in order to prevent aggregation during drying process.

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

Detailed Description of the Invention

In this detailed description, the method for obtaining a nano-material, carbon-based fluorophore dye from the edible wild plants subject to the invention is described only for clarifying the subject manner and in a manner such that it does not create any limiting effect.

The invention is related to a method for preparing a fluorophore dye by means of obtaining a nano-material, carbon-based carbon quantum dots from the edible wild plants to be used in fluorescence cell imaging, which is characterized by comprising the following steps; a. collecting, drying and grinding edible wild plants, b. preparing carbon quantum dots by carbonization process from the particle of the edible wild plants, c. centrifugation for removing the plant residues within the reaction medium from the medium, d. microfiltration for removing smaller particles which cannot be removed by centrifugation process and for purifying the carbon quantum dots, e. membrane dialysis for providing equal dimension dispersion of carbon quantum dots, f. converting carbon quantum dots into solid powder form by lyophylisation in order to prevent aggregation during drying process.

According to an embodiment of the invention, said method comprises the following steps after step “f”;

- testing cell viability ratios and cytotoxic effects of the obtained carbon quantum dots on the human epithelial cells by means of Alamar Blue method in vitro, and

- performances of obtained carbon quantum dots as a fluorophore dye in fluorescence cell imaging, their intracellular localization and dispersions are determined by studies done by confocal microscopy.

According to an embodiment of the invention, said method comprises protecting carbon quantum dots obtained as the last step.

As a result of the inventive method, means of using edible wild plants as a natural carbon source, for the first time preparation of fluorescence carbon quantum dots to be used in cell monitoring technique is performed by means of totally green-chemistry approach. The edible wild plants to be used for obtaining nanomaterial based fluorophore dye by means of the method within the scope of the invention is selection of an individual or combinations from the group comprising; Malva neglecta Wallr (mallow), Polygonum cognatum Meissn (knotweed, sheep’s sorrel), Sinapis arvensis L. (mustard), Urtica dioica L. (nettle), Chondrilla juncea L. var. juncea L. (hackberry), Rumex patientia L. (patience), Portulaca oleracea L. (purslane), Chenopodium album L. var. album L. (pigweed), Silene vulgaris (Moench) Garcke., Tragopogon latifolius Boiss. var. angustifolius Boiss. (oyster plant), Cichorium intybus L., Berberis crataegina DC. In the first step of the method within the scope of the invention is collecting and drying processes of these plants. The plant particles which are dried and grinded are subsequently used as natural carbon sources for preparing carbon quantum dots.

The studies done in the scope of the invention; edible wild plants that have been converted into powder form are subjected to microwave radiation process for carbonization. In order to determine the optimum conditions of this process, ultra-pure water is added to the edible wild plants that have been converted into powder form, 10, 50, 100 or 500 ml. samples are prepared and these samples are subject to radiation in microwave. Microwave radiation provides carbonization of the plant particles and organic components in the solution environment. In order to study substance amount in water amount which exhibits maximum fluorescence emission, from these samples 0.1 , 0.5, 1.0 and 5.0 grams are taken and are subjected to microwave radiation. For the substance amount showing maximum fluorescence emission, different energy levels are used in microwave as 400, 800 and 1600 W. The effect of such energy change is measured with fluorescence emission. After optimum energy level is determined, as a result of the application durations of the microwave in 10, 20, 40 and 80 minutes, fluorescence emission is measured and optimum microwave radiation duration is determined.

As a result of the optimization processes: 50 ml. of water for each 1 gram of plant and 800 W energy and 20-minute microwave treatment duration are determined. Carbon quantum dots which are prepared in optimum conditions are centrifuged at least 10 minutes at 15000 rpm speed. Thus, plant residues within the reaction solution are removed. After centrifugation, the solution which has been separated from its solid is passed through a micro filter. At this phase, smaller particles which cannot be removed by means of centrifugation can be removed. After this process, obtained solution is purified from its water by using a dialysis membrane with MWCO (molecular weight cut-off) 3.5 kDA value during one day. This process enables uniform size dispersion of the carbon quantum dots. Thus, by means of centrifugation, microfiltration and dialysis membrane processes, the recovery of carbon quantum dots from the medium, their purification and uniform size dispersion are accomplished.

After dialysis process, carbon quantum dots within the water are dried as solid powder form by means of taking thereof to the freeze dryer. The aggregation of carbon quantum dots is prevented by means of lyophylisation during drying process.

In obtaining fluorescence carbon quantum dots with uniform size dispersion and high quantum yield, it is critical to determine the following conditions during phases of synthesis and purification of the carbon quantum dots; duration of microwave radiation, energy level, water-dry matter ratio, centrifugation duration and force, micro filter mesh size and molecular weight cut-off (MWCO) value of the dialysis membrane and dialysis duration, and lyophylisation principles during drying process.

Alamar Blue method is used in testing cell viability ratios and cytotoxic effects of the carbon quantum dots obtained by means of the abovementioned method on the human epithelial cells. It is tested in vitro whether the obtained carbon quantum dots have cytotoxic effects on human epithelial cells or not. Carbon quantum dots, whose toxicity is colourimetrically determined as IC ₅ o by Alamar Blue method, can be used as a fluorophore dye in cell imaging. Performances of obtained carbon quantum dots as a fluorophore dye in fluorescence cell monitoring, their intracellular localization and dispersions are determined by studies done by confocal microscopy.

Obtained carbon quantum dots endure between 0-600 temperatures without deformation in their structures and can be used subsequently by protecting at 40.

In an alternative embodiment of the inventive method, instead of microwave radiation, carbon quantum dots can be obtained by means of hydro and solvo-thermal methods within a longer period of time. These synthesis conditions shall be optimized according to the method. In this case, different toxic solutions, except water, are used. Also obtaining carbon quantum dots in this manner poses risks for the human health and the environment and thus this is not an environmental friendly solution. Together with the method within the scope of the invention, CQDs are synthesized for the first time from some edible wild plants which are dispersed in our country and naturally grow and can be used as a fluorophore dye in fluorescence cell imaging. Thus, using the method within the scope of the invention contributes to decreasing the dependency of our country foreign on foreign sources.