KR102229764B1 - Method and apparatus for rapid monitoring of unederwater microplastics based on fluorescence tagging and multifunctional photometry - Google Patents

Abstract

The present invention is to provide a method and an apparatus for rapidly distinguishing and quantifying inorganic particles and microplastic particles using inexpensive equipment. The present invention relates to a method and an apparatus for rapidly monitoring underwater microplastics based on fluorescence tagging and multifunctional photometry, wherein the method includes: a step of metering a sample and adding ethanol; a step of performing microplastic dyeing by thermal reaction after adding a dye to the ethanol-added and metered sample; a step of causing the dyed sample to flow into a composite optical system; a step of measuring transmission light, scattering light, and fluorescence at the same time; a step of performing particle counting and size determination using the measured transmission light and scattering light; and a step of determining whether the counted particles are microplastics or inorganic particles using the measured fluorescence.

Description

A method and apparatus for rapid monitoring of underwater microplastics based on fluorescent labels and complex optical measurement {METHOD AND APPARATUS FOR RAPID MONITORING OF UNEDERWATER MICROPLASTICS BASED ON FLUORESCENCE TAGGING AND MULTIFUNCTIONAL PHOTOMETRY}

The present invention is an underwater microplastic analysis method and apparatus for counting the number of microplastics contained in river water, lake water, effluent water of sewage, etc., and measuring the size and distribution of microplastics using a fluorescent label by fluorescent dyeing and complex photometric measurement. It is about.

Recently, the damage of microplastics has become a big issue both domestically and internationally, and efforts are being focused on reducing the use of microplastics, stopping their use, and monitoring them. Microplastics are defined as plastics of less than 5mm, but generally, the size of microplastics present in sewage effluent, river water, lake water, etc. is known to be about 100 to 300㎛. Considering the applicability and diversity in the future, it is thought that a method to monitor microplastics in the range of 10 to 800㎛ should be secured.

Most of the microplastic analysis technology currently used uses an analysis method that counts by observing with a microscope after pretreatment, and confirms whether it is microplastic by component analysis. FT-IR is mainly used for component analysis, but recently Raman spectroscopy is also used to analyze smaller particles. This analysis method takes a very long time for analysis, and because the price of the analysis method is very expensive, it is difficult to expand the analysis method.

Korean Patent Registration 10-2094373 (microplastic detection device) constructed an optical system in which a detector is positioned in a straight line with a light source using pulsed light, and it is described as measuring fluorescence generated from microplastic without separate pretreatment. PE, PP, PS, acrylic resins, which are the main types of plastics, do not generate fluorescence in the material itself even when irradiated with light, so it is difficult to detect microplastics that do not contain an optical brightener. In addition, this method has a disadvantage that it is difficult to know the size and distribution of microplastics.

In Korean Patent Registration No. 10-2135690, a system for monitoring the water quality around the outlet was devised. Among them, a device that can analyze microplastics is constructed, which consists of an image capture unit and an FT-IR analysis unit. As described above, in the case of a device composed of such image analysis and component analysis, it is difficult to quickly analyze and has a disadvantage of being very expensive.

In the case of Korean Patent Application Laid-Open No. 10-2020-0097087, a microplastic detector equipped with a UV LED was designed to detect microplastics contained in food, but as described above, it is difficult to detect microplastics without a fluorescent whitening agent. .

The technology to count particles contained in water quality samples is mainly used in water purification plants, semiconductor water management, pharmaceutical water management, etc. It is a technology that analyzes the particle size and distribution by measuring the scattered or transmitted light of the sample. In general, when the particles are large, transmitted light is used, and when the particles are small, it is common to use scattered light. The basic principle of counting particles using scattered light and transmitted light is very old, and products are developed and sold with their own technology to increase precision. Currently, RION, PAMAS, Malvern, Chemtrac, etc. manufacture and sell particle counters. When analyzing microplastics by simply using such a particle counter, inorganic particles contained in river water, lake water, and effluent water are also counted, making it difficult to use for analysis of microplastics.

Recently, researches to analyze microplastics with fluorescent dyes and then analyze them with an optical microscope are active [Karakolis, E.G.; Nguyen, B.; You, J.B.; Rochman, C.M.; Sinton, D.; Fluorescent dyes for visualizing microplastic particles and fibers in laboratory-based studies. Environmental Science and Technology Letters 2019, 6(6), 334-340.]. In the case of this analysis method, it is thought that the economic feasibility is high, but it is judged that rapid analysis is difficult, and there are restrictions on use because it is counted with the naked eye.

Patent Document 1: Korean Registered Patent Publication No. 10-2135690 Patent Document 2: Korean Patent Application Publication No. 10-2020-0097087

Non-Patent Document 1: Karakolis, E.G.; Nguyen, B.; You, J.B.; Rochman, C.M.; Sinton, D.; Fluorescent dyes for visualizing microplastic particles and fibers in laboratory-based studies. Environmental Science and Technology Letters 2019, 6(6), 334-340.

The present invention relates to a method and apparatus for rapid monitoring of underwater microplastics based on a fluorescent label and complex optical measurement in order to solve the problems of the prior art as described above, and specific problems to be solved of the present invention are as follows.

That is, first, the present invention is to provide a method and apparatus for quantifying not only the number of microplastics but also the distribution of the particle size by dividing inorganic particles and microplastic particles.

Second, the present invention is to provide a method and apparatus for classifying and quantifying microplastic particles having a size of 10 to 800 μm in consideration of applicability and diversity.

Third, the present invention is to provide a method and apparatus for rapidly classifying and quantifying microplastic particles in water.

Fourth, the present invention is to provide a method and apparatus for classifying and quantifying microplastic particles in water using an inexpensive device capable of reducing manpower and enabling unmanned operation by automating.

In order to achieve the above object, the present inventors have applied various methods to achieve the present invention as a result of conducting a long-term study. After dyeing microplastic particles by adding a fluorescent label dyeing agent to a water sample and applying heat, A method and apparatus for analyzing microplastic particles by performing light measurement in an optical system capable of simultaneously measuring transmitted light, scattered light, and fluorescence was invented.

According to the present invention, a method for rapidly monitoring microplastics in water based on a fluorescent label and a composite photometric includes the steps of: 1) weighing a sample and adding ethanol; 2) adding a dyeing agent to the weighed sample to which ethanol was added, followed by thermal reaction to dye the microplastic; 3) introducing the dyed sample into the composite optical system; 4) simultaneously measuring transmitted light, scattered light, and fluorescence; 5) counting the number of particles and determining the size using the measured transmitted light and scattered light; And 6) determining whether the counted particles are microplastics or inorganic particles using the measured fluorescence; fluorescent labeling and complex photometric-based underwater microplastics rapid monitoring method.

At this time, the fluorescent label dyeing agent is selected from a variety of commercially available dyeing agents, and the adopted fluorescent label dyeing agent only dyes microplastic particles, but does not dye inorganic particles such as sand or mud. You can use various commercially available dyes such as Pyrromethene567, Squaraine, and DID.

The most widely used nile red, for example, is described in the process of dyeing. First, the process of preparing the dyeing agent will be described to dissolve the dyeing agent in a surfactant.

Surfactant may be Tween20, and in this case, first, Nile Red is added to Tween20 in an amount that can be 1 mg/ml, and then the mixture of surfactant and dyeing agent is dispersed for 5 minutes using an ultrasonic disperser to prepare a dyeing agent. will be.

The standard solution is prepared by dispersing 0.1 g of microplastics in ethanol having a concentration of 23.7% to prepare a weighed sample to which ethanol is added so that the sample-ethanol mixture becomes 100 mL.

0.25 mL of a dye is injected into 100 mL of the sample-ethanol mixture in which microplastics are dispersed (a weighed sample to which ethanol is added). The microplastic particles are dyed by heating this solution at a temperature of 70 to 100°C, preferably 80°C for 10 to 40 minutes, preferably for 20 minutes, and reacting with heat. When fine plastic particles are dyed with Nile red like this, fluorescence is generated when irradiating light with a wavelength of 520 to 550 nm. These dyes cannot dye inorganic particles such as sand or mud. When dyeing is completed, the sample is transferred to the flowcell of the composite optical system as shown in FIG. 1 and photometric is performed.

When transmitted light decreases or scattered light increases, it is determined that the number of microplastic particles and inorganic particles increases, and when fluorescence increases, the number of microplastic particles increases.

When using the method and apparatus for rapid underwater microplastic monitoring based on a fluorescent label and complex photometric according to the present invention, the distribution of the particle size as well as the number of microplastics is also performed by distinguishing inorganic particles and microplastic particles contained in water. It shows an effect that can be quantified, and it shows the effect of remarkably increasing applicability and diversity as it can be quantified by classifying and quantifying microplastic particles of 10 to 800㎛ size.

In addition, by using the method and the apparatus according to the present invention, the inorganic particles and microplastic particles contained in water are not only exhibited the effect of rapidly classifying and quantifying, but also the manpower can be reduced and automated It shows the effect of providing a method and apparatus for classifying and quantifying using an inexpensive device capable of unmanned operation.

1 is a schematic diagram of an optical system for implementing a method for rapid monitoring of underwater microplastics based on a fluorescent label and complex optical measurement of the present invention.
2 is a graph showing a result of simultaneously measuring transmitted light, scattered light, and fluorescence of the dyed microplastic in the optical system of the present invention.
3 is a graph showing a result of simultaneously measuring transmitted light, scattered light, and fluorescence of borosilicate solid glass microspheres dyed in the optical system of the present invention.

Hereinafter, a method for rapidly monitoring underwater microplastics based on a fluorescent label and complex optical measurement according to the present invention will be described in detail with reference to the accompanying drawings.

The drawings introduced below are provided as an example in order to sufficiently convey the spirit of the present invention to those skilled in the art.

Accordingly, the present invention is not limited to the drawings presented below and may be embodied in other forms, and the drawings presented below may be exaggerated to clarify the spirit of the present invention.

At this time, if there is no other definition in the technical and scientific terms used, they have the meanings commonly understood by those of ordinary skill in the technical field to which this invention belongs, and the gist of the present invention is unnecessary in the following description and accompanying drawings Descriptions of known functions and configurations that may be blurred will be omitted.

According to an embodiment of the present invention, a method for rapid monitoring of underwater microplastics based on a fluorescent label and a complex photometric,

1) weighing the sample and adding ethanol;

2) adding a dyeing agent to the weighed sample to which ethanol was added, followed by thermal reaction to dye the microplastic;

3) introducing the dyed sample into the composite optical system;

4) simultaneously measuring transmitted light, scattered light, and fluorescence;

5) counting the number of particles and determining the size using the measured transmitted light and scattered light; And

6) determining whether the counted particles are microplastics or inorganic particles by using the measured fluorescence; fluorescent labeling and complex photometric-based rapid monitoring of underwater microplastics.

At this time, the fluorescent label dyeing agent is selected from a variety of commercially available dyeing agents, and the adopted fluorescent label dyeing agent only dyes microplastic particles, but does not dye inorganic particles such as sand or mud. You can use various commercially available dyes such as Pyrromethene567, Squaraine, and DID.

The most widely used nile red, for example, is described in the process of dyeing. First, the process of preparing the dyeing agent will be described to dissolve the dyeing agent in a surfactant.

Surfactant may be Tween20, and in this case, first, Nile Red is added to Tween20 in an amount that can be 1 mg/ml, and then the mixture of surfactant and dyeing agent is dispersed for 5 minutes using an ultrasonic disperser to prepare a dyeing agent. will be.

The standard solution is prepared by dispersing 0.1 g of microplastics in ethanol having a concentration of 23.7% to prepare a weighed sample to which ethanol is added so that the sample-ethanol mixture becomes 100 mL.

0.25 mL of a dye is injected into 100 mL of the sample-ethanol mixture in which microplastics are dispersed (a weighed sample to which ethanol is added). The microplastic particles are dyed by heating this solution at a temperature of 70 to 100°C, preferably 80°C for 10 to 40 minutes, preferably for 20 minutes, and reacting with heat. When fine plastic particles are dyed with Nile red like this, fluorescence is generated when irradiating light with a wavelength of 520 to 550 nm. These dyes cannot dye inorganic particles such as sand or mud. When dyeing is completed, the sample is transferred to the flowcell of the composite optical system as shown in FIG. 1 and photometric is performed.

When transmitted light decreases or scattered light increases, it is determined that the number of microplastic particles and inorganic particles increases, and when fluorescence increases, the number of microplastic particles increases.

To explain the complex optical system in detail, first, a lamp (1) that generates light used for measurement and a collimator (2) that collects light in a straight line are installed. When light is irradiated to the flowcell (3), the light is transmitted through the transmitted light condensing lens ( 10) and the transmitted light filter 11, the amount of light is measured in the transmitted light detecting tube 12. The transmitted light filter 11 uses a band pass filter of a corresponding wavelength or a short pass filter that excludes fluorescence generated from the flow cell 3. When the particles pass through the flow cell (3) and cover the light, the intensity of the transmitted light decreases. In addition, when the particles meet light, scattered light is generated. The scattered light is measured in the scattered light detection tube 9 through the scattered light condensing lens 7 and the scattered light filter 8 of the composite optical system. When the intensity of the scattered light measured when the particles pass through the flow cell 3 is represented with respect to time, a peak in the form of a normal distribution can be obtained as shown in FIG. 2. The scattered light filter 8 used to measure the scattered light uses a band pass filter or a long pass filter like the transmitted light filter 11. The size of the particles can be determined using the height and size of the peak in the form of a normal distribution obtained.

In addition, when the microplastic is dyed with a fluorescent dye, the dyed microplastic particles encounter light and generate fluorescence when the sample is flowed through the flow cell (3). Fluorescence is measured in the fluorescence detection tube 6 through the fluorescence collecting lens 4 and the fluorescence filter 5. When the intensity of fluorescence is measured over time, it is possible to obtain a peak in the form of a normal distribution like scattered light.

The optical path for measuring scattered light and transmitted light is equipped with a filter that does not transmit fluorescence near 590 nm, so that fluorescence cannot be measured.

A method of analyzing microplastics will be described in detail using FIG. 1. The standard sample was used by dispersing a commercially available polystyrene powder NIST standard in ultrapure water. Polyethylene was dyed by adding nile red as a dye to the standard sample. When nile red is irradiated with light having a wavelength of 520 to 550 nm, fluorescence of around 590 nm is generated. Therefore, a lamp (1) with a wavelength of 520 nm was used, and the light was adjusted to focus inside the flow cell (3) by using a collimator (2). The intensity of transmitted light, scattered light, and fluorescence was measured while flowing the sample into the flow cell 3 using a pump. It can be seen that when light is irradiated to the microplastic particles, the transmitted light decreases and the scattered light and fluorescence intensity increase. Therefore, when the transmitted light decreases, the scattered light increases, and the fluorescence increases at the same time, it can be determined as microplastic. In addition, when a decrease in transmitted light and an increase in scattered light occur, but no fluorescence is generated, it can be determined that the particles are other than microplastics. By knowing the flow rate of the incoming sample and the number of times the microplastics are detected, the number of microplastics per unit volume can be calculated.

In addition, the distribution of the microplastic particle size can be obtained by deriving the correlation between the decrease in transmitted light and the increase in scattered light according to the microplastic particle size as a relational formula. This has the advantage of simply knowing the size distribution as well as the number of microplastic particles per unit volume.

Since transmitted light, fluorescence, and scattered light signals measured in a photodetector are measured at a current level of ㎂～㎁, this signal is amplified with an operational amplifier and converted into a voltage. Convert and use.

Nile Red is added to Tween20, a surfactant, in an amount that can be 1 mg/ml, and then the surfactant and dye mixture are dispersed for 5 minutes using an ultrasonic disperser to prepare a dyeing agent. 0.1 g of microplastic is dispersed in ethanol having a concentration of 23.7% to prepare a weighed sample to which ethanol is added so that the sample-ethanol mixture becomes 100 mL.

After injecting 0.25 mL of dyeing agent into 100 mL of the sample-ethanol mixture in which microplastics are dispersed (measured sample to which ethanol is added), dyeing is carried out by heat reaction at 80° C. for 20 minutes.

The dyed solution was introduced into the composite optical system of FIG. 1 to measure transmitted light, scattered light, and fluorescence. When fluorescence is detected as transmitted light decreases or scattered light increases, it is identified as microplastic, counted for a certain period of time, and counted in units/m3. Also, the size of the particles is calculated using the size of the scattered light and the transmitted light.

Table 1 shows the results of the above tests using NIST polystyrene standard particles having a particle size of 10 μm.

The test was carried out in the same manner as in Example 1 using NIST polystyrene standard particles having a particle size of 100 μm, and the results are shown in Table 1.

The test was performed in the same manner as in Example 1 using NIST polystyrene standard particles having a particle size of 500 μm, and the results are shown in Table 1.

The test was performed in the same manner as in Example 1 using NIST polystyrene standard particles having a particle size of 800 μm, and the results are shown in Table 1.

Using NIST polystyrene standard particles having a particle size of 500 μm, dyeing in the same manner as in Example 1 and measuring scattered light, transmitted light, and fluorescence simultaneously in a complex optical system resulted in a decrease in transmitted light, an increase in scattered light, and generation of fluorescence at the same time. Occurred. The results are shown in FIG. 2.

Particle size (㎛) No. Transmitted light (AU) Scattered light (AU) Fluorescence (AU) 0
(No microplastics) One 64480 1880 54 2 64515 1894 35 3 64567 1875 46 4 64428 1886 32 5 64513 1849 47 10 One 64342 2546 452 2 64215 2648 463 3 64289 2597 453 4 63946 2538 457 5 64128 2543 461 100 One 63434 10945 857 2 63541 10894 846 3 63654 10937 851 4 63812 10943 852 5 63729 10938 846 500 One 58406 29862 2546 2 58624 29254 2549 3 58753 29558 2486 4 58642 29648 2553 5 58395 26583 2556 800 One 50444 45892 3416 2 50243 45762 3425 3 49865 45834 3432 4 50346 45791 3426 5 49858 45852 3427

(Comparative Example 1)

The particles to be measured were tested in the same manner as in Example 1, except that borosilicate solid glass microspheres, which are inorganic particles, were used, and the size of the particles was 27 to 30 μm. A decrease in transmitted light and an increase in scattered light occurred, but no fluorescence was observed. The results are shown in Table 2.

(Comparative Example 2)

The test was carried out in the same manner as in Comparative Example 1, except that the size of the particles was set to 250 to 300 μm. A decrease in transmitted light and an increase in scattered light occurred, but no fluorescence was observed. The results are shown in Table 2.

(Comparative Example 3)

The test was carried out in the same manner as in Comparative Example 1, except that the size of the particles was 710 to 850 μm. A decrease in transmitted light and an increase in scattered light occurred, but no fluorescence was observed. The results are shown in Table 2.

(Comparative Example 4)

Using a borosilicate solid glass microsphere having a particle size of 250 to 300㎛, dyeing in the same manner as in Example 1, and measuring scattered light, transmitted light, and fluorescence simultaneously in a complex optical system over time. B Fluorescence was measured at the level of electrical noise and no fluorescence was generated. The results are shown in FIG. 3.

Particle size (㎛) No. Transmitted light (AU) Scattered light (AU) Fluorescence (AU) 27-30 One 63873 3054 55 2 63894 6105 24 3 64546 3068 34 4 64359 3084 24 5 65112 3082 31 250-300 One 61495 18256 54 2 61589 18354 25 3 61357 18559 34 4 61952 18527 26 5 62070 18448 25 710～850 One 50248 46825 36 2 49627 46379 34 3 50446 45864 28 4 50624 45779 26 5 50552 46537 35

number Explanation number Explanation One Lamp 7 Scattered light condensing lens 2 Collimator 8 Scattered light filter 3 Flow cell 9 Scattered light detector 4 Fluorescent condensing lens 10 Transmitted light condensing lens 5 Fluorescent filter 11 Transmitted light filter 6 Fluorescence detector 12 Transmitted light detector tube

Claims (6)

1) weighing the sample and adding ethanol;
2) dyeing microplastics by heat-reacting at 70 to 100°C for 10 to 40 minutes after dissolving in a surfactant and adding a dyeing agent dispersed with an ultrasonic disperser to the weighed sample to which ethanol is added;
3) introducing the dyed sample into the composite optical system;
4) simultaneously measuring transmitted light, scattered light, and fluorescence;
5) counting the number of particles and determining the size using the measured transmitted light and scattered light, characterized in that it is determined that the number of microplastic particles and inorganic particles increases when transmitted light decreases or scattered light increases; And
6) determining whether the counted particles are microplastic or inorganic particles using the measured fluorescence, characterized in that it is determined that the number of microplastic particles increases when the fluorescence increases; a fluorescent label and a composite optical measurement consisting of Based underwater microplastics rapid monitoring method. delete delete delete delete delete

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