Method for detecting pet microplastics using TPA and kit using same

Abstract

The present invention relates to a method for detecting the concentration of PET microplastics in a sample by decomposing PET microplastics in the sample into TPA and measuring the concentration of the decomposed TPA using fluorescence. Furthermore, the present invention provides a kit for detecting PET microplastics based on this principle. According to the present invention, the concentration of PET microplastics can be estimated from the fluorescence value of hTPA converted from PET, thereby enabling detection of PET microplastics.

Description

PET microplastic detection method using TPA and kit using the same

[0001] The present invention relates to a method for detecting PET microplastics using TPA and a kit for detecting PET microplastics using the same.

[0002]

[0003] Plastics are used in a wide range of applications, from films, synthetic fibers, bottles, tubes, and toys to high-heat-resistant, high-strength materials. Their use is continuously increasing due to their lightweight properties, high physical and chemical durability, excellent processability, and low-cost production.

[0004] The increased use of plastics is leading to a rise in plastic waste. Plastic waste takes a long time to decompose naturally, contributing to environmental pollution. Furthermore, incineration of discarded plastics raises concerns about the massive carbon dioxide emissions that accelerate global warming and climate change. Therefore, efforts are being made to reduce plastic use and pollution, including regulations on plastic use, plastic recycling, increased use of biodegradable plastics, and biodegradation by microorganisms.

[0005] Microplastics are plastic particles less than 5mm in diameter and are ingested by humans through water, air, and seafood. The global average weekly intake of microplastics is approximately 5g, and they include PET, PE, PVC, PS, PP, and PU.

[0006] PET stands for polyethylene terephthalate, one of the most widely used and discarded plastics. As plastic waste increases, the problem of microplastics, which are released when exposed to heat or ultraviolet rays and naturally decompose in the environment, has become increasingly serious. Microplastics pollute the environment and accumulate in living organisms through the food chain. Evidence is emerging that microplastics can penetrate not only marine life but also human bloodstream, potentially causing adverse health effects.

[0007] Conventional methods for detecting microplastics include Fourier transform infrared (FT-IR) spectroscopy and Raman spectroscopy. However, Fourier transform infrared spectroscopy and Raman spectroscopy are expensive in terms of equipment and consumables, and are not widely available domestically. Furthermore, they require expert analysis and take a long time, typically 3-7 days. These limitations, coupled with their low cost and accessibility, make it difficult to detect the widespread presence of microplastics.

[0008]

[0009] The present invention aims to develop a method for detecting microplastics in a quick and simple manner, and to provide a kit for detecting microplastics using the method.

[0010]

[0011] In order to achieve the above-mentioned purpose, the object of the present invention is to provide a method for detecting PET microplastics, comprising: a first step of decomposing PET in a sample into TPA; a second step of measuring the fluorescence of the decomposed TPA; and a third step of predicting the concentration of PET microplastics using the measured fluorescence value.

[0012] Another object of the present invention is to provide a kit for detecting PET microplastics, which includes a sample inlet, a microtube through which a sample moves, an enzyme reaction chamber containing a PET-decomposing enzyme, a filter for separating microplastics, and a fluorescence reaction chamber.

[0013]

[0014] The present invention can detect PET microplastics by converting PET microplastics in a sample into hTPA, measuring the fluorescence of the converted hTPA, and predicting the concentration of PET microplastics based on the fluorescence value. In addition, the present invention provides a PET microplastic detection kit based on the principle of converting PET microplastics in a sample into hTPA and measuring the fluorescence of the converted hTPA.

[0015] According to the present invention, the concentration of PET microplastics can be predicted using the fluorescence value of hTPA converted from PET, thereby enabling PET microplastics to be detected specifically for PET.

[0016]

[0017] Figure 1 shows the results of measuring the fluorescence value of a standard solution according to TPA concentration under different filter conditions.

[0018] Figure 2 shows the results of measuring the fluorescence value of a standard solution at different TPA concentrations by changing the pH.

[0019] Figure 3 shows the results of measuring the fluorescence value of a standard solution at different TPA concentrations by changing the concentrations of FeSO4 and EDTA solutions.

[0020] Figure 4 shows the fluorescence value of a TPA standard solution measured under optimized conditions using pH 7.5, 0.1 M sodium phosphate, 5 mM FeSO4, and 5 mM EDTA.

[0021] Figure 5 shows the results of estimating the concentration (mM) of TPA generated from PET based on the fluorescence value according to the cutinase concentration (mg / ml).

[0022] Figure 6 shows the results of confirming the optimal cutinase concentration according to the concentration of PET.

[0023] Figure 7 shows the results of estimating the concentration (mM) of TPA decomposed in PET using esterase enzyme using fluorescence values.

[0024] Figure 8 shows the results of measuring the fluorescence value (fluorescence value of hTPA) according to the PET concentration after adding cutinase enzyme to PET of 0 to 0.008 g / ml and reacting it.

[0025] Figure 9 shows the results of measuring the fluorescence value according to the PET concentration using cutinase enzyme at 0 to 0.4 g / ml of PET.

[0026] Figure 10 shows the results of measuring terephthalic acid (TPA) produced by cutinase enzyme using high-performance liquid chromatography (HPLC, Shimadzu).

[0027] Figure 11 is a graph comparing the TPA concentration estimation method using cutinase enzyme and the TPA concentration analyzed by HPLC.

[0028] Figure 12 shows the results of measuring the fluorescence value by reacting 0.1 mg / ml of cutinase enzyme with 0.01 g / ml of five different types of plastic powder (PET, PS, PE, PP, TPU) at 55°C for 5 hours.

[0029] Figure 13 shows the results of fluorescence measurement after culturing B. subtilisDCP01 with 0.1 g / ml of five types of plastic powder (PET, PP, PS, PE, PU) at 40°C for 24 hours.

[0030] Figure 14 shows a PET microplastic detection kit of the present invention.

[0031]

[0032] The following describes this specification in more detail.

[0033] Each description and embodiment disclosed in this invention can be applied to other descriptions and embodiments thereof. That is, all combinations of the various elements disclosed in this invention fall within the scope of this invention. Furthermore, the scope of this invention is not limited by the specific descriptions described below.

[0034] Expressions such as “comprising” as used herein should be understood as open-ended terms implying the possibility of including other embodiments, unless specifically stated otherwise in the phrase or sentence in which the expression is included.

[0035] The terms or words used in the description and claims of the present invention should not be interpreted as limited to their usual or dictionary meanings, but should be interpreted as meanings and concepts that conform to the technical idea of ​​the present invention, based on the principle that the inventor can appropriately define the concept of the term to explain his or her own invention in the best way.

[0036]

[0037] As one aspect for achieving the above object, the present invention provides a method for detecting PET microplastics, comprising: a first step of decomposing PET in a sample into TPA; a second step of measuring fluorescence of the decomposed TPA; and a third step of predicting the concentration of PET microplastics using the measured fluorescence value.

[0038] The term "sample" in the present invention means a sample containing PET plastic, and specific examples thereof include soil contaminated with PET plastic, plants grown in soil contaminated with PET plastic, various foods contaminated with PET plastic, food additives contaminated with PET plastic, sea salt, raw water, fish, meat, seaweed, wastewater, waste, vinyl processed with PET plastic, household chemical products such as tea bags and wet tissues, plastic containers, tire dust, air contaminated with PET plastic, and the like, which may contain plastics including microplastics, but are not limited thereto as long as PET plastic is included. In addition, it can be applied to various PET plastic decomposition industries for environmental protection. The PET plastic includes PET microplastics, and the microplastics refer to plastics with a diameter of 5 mm or less. The microplastics include nanoplastics.

[0039]

[0040] The method for detecting PET microplastics of the present invention includes, in step 1, a step of decomposing PET in a sample with TPA.

[0041] The above PET refers to polyethylene terephthalate.

[0042] PET in the above sample can be decomposed or converted into TPA (terephthalate) by an enzyme that decomposes PET.

[0043] In the present invention, the decomposition or conversion into TPA (terephthalate) can utilize an enzyme or a microorganism containing the enzyme.

[0044] The above enzyme may be cutinase.

[0045] The above cutinase can be used at a concentration of 0.001 to 0.3 mg / ml, and specifically, can be used at a concentration of 0.09 to 0.12 mg / ml.

[0046] The above microorganism may be a microorganism containing cutinase, esterase or lipase enzymes, and may specifically be Bacillus subtilis DCP01 strain.

[0047] The above Bacillus subtilis DCP01 strain has plastic decomposition activity and contains cutinase, esterase or lipase, so the Bacillus subtilis DCP01 strain can be used instead of enzymes without using enzymes directly.

[0048] The above Bacillus subtilis DCP01 strain corresponds to a strain deposited by the inventor of the present invention to the Biological Resource Center of the Korea Research Institute of Bioscience and Biotechnology on December 14, 2022 and assigned the accession number KCTC15243BP.

[0049] The above PET can be broken down into its constituents, terephthalate (TPA) and ethylene glycol (EG). However, not all enzymes can break down PET into TPA and EG, and intermediate products such as MHET (mono(2-hydroxyethyl)terephthalate) and BHET (bis(2-hydroxyethyl)terephthalate) are sometimes produced in excess. In the present invention, it was confirmed that intermediate products such as MHET and BHET are hardly produced by cutinase, and are instead converted to TPA.

[0050] The method for detecting PET microplastics of the present invention includes a step of measuring fluorescence of the decomposed TPA in two steps.

[0051] The second step of measuring the fluorescence of the above TPA includes a step of converting the decomposed TPA into hTPA using ferric sulfate (FeSO4) and ethylenediaminetetraacetic acid (EDTA); and a step of measuring the fluorescence of the converted hTPA.

[0052] The above hTPA stands for hydroxy terephthalate, and by measuring the fluorescence of hTPA, the concentration of PET microplastics can be predicted as described below.

[0053] Methods for measuring the fluorescence or concentration of TPA include using HPLC and measuring the fluorescence of hTPA. While HPLC offers the advantage of accurately measuring large amounts of TPA concentration, it also requires expensive equipment, requires complex sample pretreatment, and requires a long processing time for results. Consequently, developing a kit that can be easily used by the general public in a short period of time using HPLC is impossible.

[0054] Another method for measuring TPA fluorescence involves converting TPA to fluorescent hydroxy terephthalate (hTPA) and then measuring its fluorescence. This method has the advantages of being fast and simple to measure.

[0055] In the present invention, a method for measuring the fluorescence of hTPA was selected to develop a kit with good commerciality, and modifications were made so that it can be easily applied by the general public.

[0056] The second step of measuring the fluorescence of the above TPA includes the step 2-1 of converting the decomposed TPA into hTPA using ferrous sulfate (FeSO4) and ethylenediaminetetraacetic acid (EDTA). The principle of converting hTPA from TPA is as follows. The hydroxyl radical generated when the divalent iron ion is oxidized to a trivalent iron ion converts TPA into hTPA, and the fluorescence of the converted hTPA is measured using a fluorometer to measure the fluorescence of TPA.

[0057] The present invention may further include a step of filtering the decomposed TPA. Specifically, by performing the filtration process before converting it to hTPA, large-particle impurities, such as microplastics, within the sample are removed.

[0058] In order to convert the above-decomposed TPA into hTPA and measure fluorescence, the sample in which the decomposition reaction into TPA is completed in PET is filtered through a 0.1 to 0.5 μm filter, specifically, a 0.2 to 0.22 μm filter. Afterwards, the filtered sample is converted into hTPA by adding 3 to 10 mM ferric sulfate (FeSO4) and 3 to 10 mM ethylenediaminetetraacetic acid (EDTA). The filter can be made of, but is not limited to, PE (polyethylene), PP (polypropylene), cotton, PES (polyethersulfone), PVDF (polyvinylidene fluoride), cellulose, Nylon, etc.

[0059] The second step of measuring the above TPA fluorescence includes the step 2-2 of measuring the fluorescence of the converted hTPA. By measuring the fluorescence of the converted hTPA with a fluorometer, the presence or concentration of TPA can be indirectly detected.

[0060] Specifically, the fluorescence of the converted hTPA can be measured using a fluorometer at an excitation filter of 320 to 340 nm and an emission filter of 400 to 430 nm.

[0061] More specifically, fluorescence can be measured with a 96-well plate and microplate reader at an excitation filter of 320 to 340 nm and a bandwidth of 50 to 90 nm, and an emission filter of 400 to 430 nm and a bandwidth of 30 to 80 nm.

[0062] The reaction converting the above TPA into hTPA has a very short reaction time of 10 to 15 minutes, specifically about 10 minutes, and even low concentrations of TPA can be confirmed by measuring the fluorescence of hTPA. Furthermore, the fluorescence value of hTPA can be stably measured for 40 to 60 minutes, or about 50 minutes.

[0063] The method for detecting PET microplastics of the present invention comprises a step of predicting the concentration of PET microplastics from the measured fluorescence value in three steps.

[0064] The concentration of PET microplastics can be predicted from the fluorescence value of the hTPA measured above, thereby detecting PET microplastics in a sample.

[0065] Specifically, Figs. 8 and 9 show fluorescence values ​​according to PET concentration. By substituting the measured hTPA fluorescence value into the y-axis of the graphs shown in Figs. 8 and 9, the PET value corresponding to the x-axis can be determined. Accordingly, the concentration of PET in the sample can be estimated and predicted, thereby enabling the detection of PET microplastics in the sample.

[0066]

[0067] In another aspect, the present invention provides a kit for detecting PET microplastics, comprising a sample inlet, a microtube through which a sample moves, an enzyme reaction chamber containing a PET-decomposing enzyme, a filter for separating microplastics, and a fluorescence reaction chamber.

[0068] The kit (100) for detecting PET microplastics of the present invention is as shown in Fig. 14. Referring to the kit perspective view (10) and the kit cross-sectional view (20) of Fig. 14, the kit (100) includes a sample inlet (1), a microtube (2) through which a sample moves, an enzyme reaction chamber (3) containing a PET-decomposing enzyme, a filter (4) for separating microplastics, and a fluorescence reaction chamber (5). In addition, the sample inlet (1), the microtube (2) through which a sample moves, the enzyme reaction chamber (3) containing a PET-decomposing enzyme, the filter (4) for separating microplastics, and the fluorescence reaction chamber (5) of the kit are attached on a glass plate (6).

[0069] In addition, the above kit (100) may additionally include a device such as a hot plate (7) that induces the activity of PET decomposing enzyme and a fluorescence meter (8) that can measure fluorescence.

[0070] First, a sample containing PET microplastics is introduced into the sample inlet (1). The sample (1) containing PET microplastics moves along the microtube (2) and reacts with the PET decomposing enzyme in the enzyme reaction chamber (3) to produce TPA. At this time, when the kit (100) is placed on a hot plate (7) capable of supplying a heat source, the temperature of the enzyme reaction chamber (3) is maintained at 35 to 60°C. As a result, the PET decomposing enzyme in the enzyme reaction chamber (3) is activated, and the PET contained in the sample is converted into TPA. In this way, the first step of decomposing PET in the sample into TPA is performed in the enzyme reaction chamber containing the PET decomposing enzyme.

[0071] After the enzyme reaction is complete, if more sample buffer is added, the sample moves to the fluorescence measurement chamber (5) through the filter (4) located above the enzyme reaction chamber (3). Among the samples, large PET microplastics do not pass through the 0.1 to 0.5 μm, specifically 0.2 to 0.22 μm filter (4) located above the enzyme reaction chamber (3), and thus remain in the enzyme reaction chamber (3). However, the liquid sample containing TPA passes through the 0.1 to 0.5 μm, specifically 0.2 to 0.22 μm filter (4) and moves along the microtube (2) to the fluorescence measurement chamber (5). After FeSO4 and EDTA are added to the fluorescence measurement chamber (5) and the reaction is performed for about 10 minutes, TPA is converted to hTPA, and the fluorescence of hTPA can be measured by a chemical method. In this way, in the fluorescence reaction chamber, a second step of converting the decomposed TPA into hTPA using ferric sulfate (FeSO4) and ethylenediaminetetraacetic acid (EDTA) is performed. In addition, a third step of measuring the fluorescence of the converted hTPA is additionally performed, so that the PET concentration can be estimated and predicted through the fluorescence value of hTPA, as shown in FIGS. 8 and 9, thereby enabling the detection of PET microplastics in the sample. The microplastics include nanoplastics.

[0072] As described above, the fluorescence of the converted hTPA can be measured using a fluorometer at an excitation filter of 320 to 340 nm and an emission filter of 400 to 430 nm, and specifically, the fluorescence can be measured using a microplate reader on a 96 well plate.

[0073]

[0074] Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings so that those skilled in the art can easily implement the present invention. However, the present invention may be implemented in various different forms and is not limited to the embodiments described herein.

[0075]

[0076] Experimental Example 1. Fluorescence measurement of standard solutions at different TPA concentrations

[0077] 1-1. Fluorescence measurement filter optimization

[0078] TPA (terephthalate) was dissolved in DMSO and diluted with 0.1 M sodium phosphate (pH 7.5) to prepare standard solutions for each concentration. In addition, to measure the concentration of TPA, hTPA, produced by mixing 5 mM ferric sulfate (FeSO4) and 5 mM ethylenediaminetetraacetic acid (EDTA), was measured using a fluorometer (microplate reader). Fluorescence values ​​were measured and compared using a Hidex sense microplate reader using three types of fluorescence filters.

[0079] A) Excitation 360nm (bandwidth 9nm), Emission 465nm (bandwidth 20nm)

[0080] B) Excitation 355nm (bandwidth 40nm), Emission 460nm (bandwidth 20nm),

[0081] C) Excitation 330nm (bandwidth 80nm), Emission 405nm (bandwidth 50nm).

[0082] Figure 1 shows the results of measuring the fluorescence values ​​of standard solutions by TPA concentration under different filter conditions. As a result, the difference in fluorescence values ​​by TPA standard solution concentration was the greatest in filter C). Therefore, the filter was determined to be under condition C.

[0083]

[0084] 1-2. pH optimization

[0085] TPA was dissolved in DMSO and diluted with 0.1 M sodium phosphate (pH 7.5) or 0.1 M sodium phosphate (pH 8.5) to prepare standard solutions for different concentrations. In addition, to measure the concentration of TPA, hTPA produced by mixing 5 mM FeSO4 and 5 mM EDTA was measured using a fluorescence meter (Microplate reader). Fluorescence was measured using a Hidex sense microplate reader using a filter of Excitation 330 nm (bandwidth 80 nm) and Emission 405 nm (bandwidth 50 nm) (C condition), and the fluorescence values ​​of the standard solutions for different TPA concentrations are shown in Figure 2.

[0086] Figure 2 shows the results of measuring the fluorescence value of a standard solution at different TPA concentrations by changing the pH.

[0087] As a result, when comparing pH 7.5 and pH 8.5, the basal level (fluorescence value at 0 mM TPA) was lower at pH 7.5, making fluorescence measurement easier. Therefore, it can be seen that preparing a standard solution using pH 7.5 sodium phosphate is optimal.

[0088]

[0089] 1-3. Optimization of FeSO4 and EDTA concentrations (5 mM, 50 mM)

[0090] TPA was dissolved in DMSO and diluted with 0.1 M sodium phosphate (pH 7.5) to prepare standard solutions at various concentrations. Fluorescence was measured using a Hidex sense microplate reader using an Excitation 330 nm (bandwidth 80 nm), Emission 405 nm (bandwidth 50 nm) filter (C condition) and a 5 mM EDTA and 5 mM FeSO4 solution or a 50 mM EDTA and 50 mM FeSO4 solution, and the fluorescence values ​​of the standard solutions at various TPA concentrations are shown in Figure 3.

[0091] Figure 3 shows the results of measuring the fluorescence value of a standard solution at different TPA concentrations by changing the concentrations of FeSO4 and EDTA solutions.

[0092] As a result, when fluorescence was measured using a 5 mM EDTA and 5 mM FeSO4 solution, more sensitive fluorescence values ​​could be measured than under the 50 mM concentration condition. Therefore, it was found that using a 5 mM EDTA and 5 mM FeSO4 solution was the optimal condition.

[0093]

[0094] 1-4. Fluorescence measurement of standard solutions at different TPA concentrations under optimal conditions

[0095] The fluorescence of the standard solution according to TPA concentration was measured using the pH, filter conditions, 5 mM FeSO4, and 5 mM EDTA solution of 0.1 M sodium phosphate, which were verified through previous experimental results.

[0096] TPA was dissolved in DMSO and diluted with 0.1 M sodium phosphate (pH 7.5) to prepare a standard solution. Fluorescence was measured in a 96-well plate using a microplate reader (Hidex, USA) using an Excitation 330 nm (bandwidth 80 nm), Emission 405 nm (bandwidth 50 nm) filter (C condition) and 5 mM EDTA and 5 mM FeSO4 solution. The fluorescence values ​​of the standard solution according to TPA concentration are shown in Figure 4.

[0097] Figure 4 shows the fluorescence values ​​of a TPA standard solution measured under optimized conditions using pH 7.5, 0.1 M sodium phosphate, 5 mM FeSO4, and 5 mM EDTA. As a result, it was confirmed that the fluorescence value increased as the concentration of TPA increased under the conditions.

[0098]

[0099] Experimental Example 2. Measurement of hTPA decomposed and converted from PET using cutinase enzyme.

[0100] Cutinase was used to convert PET (polyethylene terephthalate) into TPA (terephthalate). When reacted with PET, the cutinase enzyme selectively produces TPA over other intermediate products such as MHET and BHET [MHET: mono(2-hydroxyethyl)terephthalate, BHET: bis(2-hydroxyethyl)terephthalate].

[0101] Cutinase enzyme concentrations were varied in 0.08 g / ml of PET powder, and the reaction was performed in 0.1 M phosphate buffer, pH 7.5, at 55°C for approximately 5 hours to induce TPA production. To measure the concentration of TPA, hTPA produced by mixing 5 mM EDTA and 5 mM FeSO4 solution was measured using a fluorometer (Microplate reader) under Excitation 330 (Bandwidth 80), Emission 405 (Bandwidth 50) filter (C condition).

[0102] Figure 5 shows the results of estimating the concentration (mM) of TPA generated from PET according to the cutinase concentration (mg / ml) using fluorescence values. As a result, when the same amount of PET (0.08 g / ml) was reacted with different cutinase concentrations (mg / ml) for the same period of time and the fluorescence values ​​were measured, it was confirmed that the decomposition efficiency of PET into TPA was highest when the cutinase concentration was 0.1 mg / ml. Therefore, it was confirmed that the optimal cutinase concentration was 0.1 mg / ml when PET was at a concentration of 0.08 g / ml.

[0103] Next, to confirm the optimal cutinase concentration according to the PET concentration, tests were conducted with different cutinase concentrations in different PET concentration ranges. Specifically, in Fig. 6a, experiments were conducted with cutinase concentrations of 0.002 mg / ml, 0.01 mg / ml, and 0.1 mg / ml at PET concentrations of 0 to 0.008 g / ml. In addition, in Fig. 6b, tests were conducted with cutinase concentrations of 0.02 mg / ml, 0.1 mg / ml, and 0.2 mg / ml at PET concentrations of 0 to 0.2 g / ml. Fig. 6 shows the results of confirming the optimal cutinase concentration according to the PET concentration. As a result, as confirmed in Figs. 6a and 6b, it was confirmed that the optimal cutinase concentration was 0.1 mg / ml at a PET concentration of 0 to 0.2 g / ml. In other words, it was confirmed that the optimal cutinase concentration was 0.1 mg / ml regardless of the PET concentration.

[0104]

[0105] Experimental Example 3. Fluorescence measurement of hTPA decomposed and converted from PET using esterase enzyme.

[0106] Next, esterase enzyme was used to decompose PET (polyethylene terephthalate) into TPA (terephthalate). PET is theoretically decomposable by esterase because its monomers are composed of ester bonds.

[0107] Accordingly, it was tested whether PET was actually decomposed into TPA by esterase.

[0108] First, Bacillus subtilisesterase was used at a concentration of 0.05 mg / ml, and the reaction was performed for 24 hours at the optimum reaction temperature of 40℃ with different amounts of PET, and the fluorescence value of the supernatant was measured. Specifically, 0.05 mg / ml of esterase enzyme and 0.1 M phosphate buffer pH 7.5 were mixed with PET powder and the reaction was performed at 40℃ for 24 hours to induce TPA production. In order to measure the concentration of the produced TPA, the produced hTPA was measured using a fluorometer (Microplate reader) under the conditions of Excitation 330 (Bandwidth 80), Emission 405 (Bandwidth 50) filter (C condition).

[0109] Figure 7 shows the results of estimating the concentration (in mM) of TPA decomposed from PET using esterase enzyme, as measured by fluorescence. The results show that fluorescence did not increase at all even as PET concentration increased. This confirms that, contrary to theory, esterase does not actually decompose PET into TPA. Therefore, esterase is not a suitable enzyme for converting PET into TPA.

[0110]

[0111] Experimental Example 4. Fluorescence measurement of TPA decomposed and converted from PET using cutinase enzyme.

[0112] As confirmed in Experiment 2 for the optimal cutinase concentration, cutinase enzyme was added at a concentration of 0.1 mg / ml to 0-0.008 g / ml of PET powder, and cutinase enzyme was added at a concentration of 0.1 mg / ml to 0-0.4 g / ml of PET powder, and the reaction was performed in 0.1 M phosphate buffer, pH 7.5, at 55°C for approximately 5 hours to induce TPA production. To measure the concentration of TPA, hTPA produced by mixing 5 mM EDTA and 5 mM FeSO4 solution was measured using a fluorometer (Microplate reader).

[0113] Figure 8 shows the results of measuring the fluorescence value (fluorescence value of hTPA) according to the PET concentration after adding cutinase enzyme to PET at 0 to 0.008 g / ml and reacting it. As a result, the fluorescence value also increased as the PET concentration increased. Figure 9 shows the results of measuring the fluorescence value according to the PET concentration using cutinase enzyme on PET at 0 to 0.4 g / ml. As a result, the fluorescence value showed a tendency to increase as the PET concentration increased. As confirmed in Figures 8 and 9, the fluorescence value also increased as the PET concentration increased. Therefore, it can be seen that TPA decomposed from PET increases. In addition, the concentration of PET can be predicted using the fluorescence value according to the PET concentration, and thereby the detection of PET microplastics is possible.

[0114]

[0115] Experimental Example 5. Verification of TPA concentration using HPLC method

[0116] Next, to confirm whether the fluorescence measurement value measured in Experimental Example 4 represents the actual concentration of TPA, terephthalic acid (TPA) produced by cutinase enzyme was measured using high-performance liquid chromatography (HPLC, Shimadzu).

[0117] The measurement conditions were based on the terephthalic acid and isophthalic acid test methods of the Ministry of Food and Drug Safety, using a C18 column set to 40℃, and a 230nm ultraviolet absorbance detector as the detector. The sample was mixed with acetonitrile in a 1:1 ratio, 0.1ml was injected, and then, using mobile phase A and mobile phase B, a linear concentration gradient was performed from A:B (100:0) to A:B (0:100) for 20 minutes at a flow rate of 1ml / min, and the measurement was completed in 30 minutes.

[0118] Fig. 10 shows the results of measuring terephthalic acid (TPA) produced by cutinase enzyme using high-performance liquid chromatography (HPLC, Shimadzu). The ratio of mobile phase A: 0.25% phosphoric acid in water and mobile phase B: 0.25% phosphoric acid in Acetonitrile (ACN) was gradually increased over 30 minutes and detected with UV 230 nm, and TPA was detected at a retention time of 16 minutes. Fig. 10(a) TPA standard (7.5 ㎍ / ml), Fig. 10(b) PET 0.1 g / ml cutinase degradation product, and Fig. 10(c) PET 0.2 g / ml cutinase degradation product were measured, and the black line represents the TPA standard (analytical standard, Sigma), and the pink line represents the enzyme reaction sample (PET 0.1 g / ml, 0.2 g / ml).

[0119] Using HPLC, it was confirmed whether the amount of TPA produced estimated by the fluorescence value in the present invention and the actual amount of TPA produced matched. As shown in Fig. 10(a), it can be seen that the TPA standard solution was detected at a retention time of 16 minutes, and the width of the peak is the amount of TPA in the injected standard solution (7.5㎍ / ml 400㎕ = 3㎍). Therefore, the amount of TPA actually produced by PET was also calculated in proportion to this. Fig. 10(b) and (c) show that TPA produced was detected as the concentration of PET increased to 0.1 g / ml and 0.2 g / ml, and the amount of TPA produced was calculated through the width of the peak and is shown in Table 1.

[0120] PET(g / ml)Peak AreaHPLC-TPA Amount(㎍)HPLC-TPA Concentration(mM)Fluorescence EstimatedTPA Concentration(mM)Standard TPA935.167330.361-000000.01626.66342.010.2420.0450.11463.6784.690.5660.3350.22507.97898.040.9680.735

[0121] In addition, the same sample was analyzed for fluorescence by the method of Experimental Example 4 above, and the concentration of TPA was predicted using the fluorescence value, and as a result, it was confirmed that it increased in a similar pattern to the data of HPLC. In addition, the TPA concentration estimation method (detection kit) using cutinase enzyme of Experimental Example 4 compared in Table 1 and the TPA concentration analyzed by HPLC are shown in a graph in Fig. 11. Fig. 11 is a graph comparing the TPA concentration estimation method (detection kit) using cutinase enzyme and the TPA concentration analyzed by HPLC. As a result, it was confirmed that the TPA concentration estimated as in Experimental Example 4 (detection kit) and the TPA concentration measured by HPLC were almost identical. That is, it was confirmed that the TPA concentration estimation method using cutinase enzyme of the present invention is not significantly different from the actual TPA concentration, and thus can be applied to a PET detection kit.

[0122]

[0123] Experimental Example 6. hTPA fluorescence measurements on different types of plastics

[0124] Next, to confirm that PET-specific detection was possible when testing other plastics using the same method, five different plastic powders (PET, PS, PE, PP, TPU) were used for comparison.

[0125] Cutinase enzyme was added to five different types of plastic powders (PET, PS, PE, PP, TPU) at a concentration of 0.1 mg / ml and reacted in 0.1 M phosphate buffer, pH 7.5, at 55°C for approximately 5 hours to induce TPA production. To convert the produced TPA into hTPA and measure fluorescence, the reaction sample was filtered through a 0.2 μm filter, and 5 mM EDTA and 5 mM FeSO4 solutions were mixed and the fluorescence value was measured at Excitation 330 (Bandwidth 80) and Emission 405 (Bandwidth 50).

[0126] Figure 12 shows the results of measuring the fluorescence value by reacting 0.1 g / ml of cutinase enzyme with 0.01 g / ml of five different types of plastic powders (PET, PS, PE, PP, TPU) at 55°C for 5 hours. Except for PET, the fluorescence value of other plastic powders decreased compared to the control group, but that of PET increased by about 4 times or more, indicating that PET detection is possible using cutinase.

[0127]

[0128] Experimental Example 7. Measurement of hTPA fluorescence using a fluorescence measurement method using microorganisms.

[0129] Instead of cutinase, B. subtilisDCP01 was used, and fluorescence was measured after culturing it with 0.1 g / ml of five types of plastic powder (PET, PP, PS, PE, PU) at 40°C for 24 h.

[0130] The above Bacillus subtilis DCP01 strain corresponds to a strain deposited by the inventor of the present invention to the Biological Resource Center of the Korea Research Institute of Bioscience and Biotechnology on December 14, 2022 and assigned the accession number KCTC15243BP.

[0131] Figure 13 shows the results of fluorescence measurement after culturing B. subtilisDCP01 with 0.1 g / ml of five types of plastic powder (PET, PP, PS, PE, PU) at 40℃ for 24 hours. B. subtilisDCP01 was cultured with plastic powder for 24 hours in a medium containing 1% glucose in MSM (minimal salt media), centrifuged, and the supernatant was filtered through a 0.22 μm filter, and the fluorescence value was measured in the same manner as above. Since PET showed a higher fluorescence value than when cultured using other plastic powders, it was found that B. subtilisDCP01 can be used for PET detection.

[0132]

[0133] Although the preferred embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements made by those skilled in the art using the basic concept of the present invention defined in the following claims also fall within the scope of the present invention.

[0134] [Correction pursuant to Rule 91, August 23, 2024]

Claims

1. Step 1: Decomposing PET in the sample into TPA; Step 2: measuring the fluorescence of the above-decomposed TPA; and A method for detecting PET microplastics, comprising the step of predicting the concentration of PET microplastics using the measured fluorescence value.

2. A method for detecting PET microplastics in the first paragraph, wherein the decomposition into TPA uses an enzyme or a microorganism containing the enzyme.

3. A method for detecting PET microplastics, wherein the enzyme in claim 1 is cutinase.

4. A method for detecting PET microplastics, wherein in the second paragraph, the microorganism is a Bacillus subtilis DCP01 strain deposited under the accession number KCTC15243BP.

5. A method for detecting PET microplastics, wherein the cutinase in the third paragraph is used at a concentration of 0.001 to 0.3 mg / ml.

6. In the first paragraph, the second step of measuring the fluorescence of the decomposed TPA is Step 2-1 of converting the above-decomposed TPA into hTPA using iron sulfate (FeSO4) and ethylenediaminetetraacetic acid (EDTA); and A method for detecting PET microplastics, comprising: a step 2-2 of measuring the fluorescence of the above-mentioned converted hTPA.

7. A method for detecting PET microplastics, further comprising a step of filtering the decomposed TPA in the sixth paragraph.

8. A method for detecting PET microplastics in paragraph 6, wherein the iron sulfate (FeSO4) and ethylenediaminetetraacetic acid (EDTA) are each at a concentration of 3 to 10 mM.

9. A method for detecting PET microplastics, wherein in the 6th paragraph, the fluorescence measurement is performed at an excitation filter of 320 to 340 nm and an emission filter of 400 to 430 nm.

10. A kit for detecting PET microplastics, comprising a sample inlet, a microtube through which a sample moves, an enzyme reaction chamber containing a PET-decomposing enzyme, a filter for separating microplastics, and a fluorescence reaction chamber.

11. In the 10th paragraph, in the enzyme reaction chamber containing the PET decomposing enzyme, A kit for detecting PET microplastics, the first step of which is to decompose PET in the sample into TPA.

12. In the 10th paragraph, in the fluorescence reaction chamber, A PET microplastic detection kit comprising a two-step process of converting the above-decomposed TPA into hTPA using iron sulfate (FeSO4) and ethylenediaminetetraacetic acid (EDTA).

13. A kit for detecting PET microplastics, wherein the fluorescence measurement in paragraph 6 is performed on a 96-well plate.

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