US-12625078-B2 - Method for estimating mass of microplastics by using fluorescent staining

US12625078B2US 12625078 B2US12625078 B2US 12625078B2US-12625078-B2

Abstract

Proposed is a method for estimating the mass of microplastics by using fluorescent staining. The method may include staining microplastic samples by using a Nile red solution, and capturing a fluorescent image of each of the stained microplastic samples after classifying the stained microplastic samples by mass. The method may also include analyzing the captured fluorescent image to quantify a fluorescence intensity thereof to build a database (DB) about a correlation between mass and fluorescence intensity of microplastics. The method may further include estimating a mass value of an unknown microplastic sample by comparing a fluorescence intensity of a fluorescent image of the unknown microplastic sample obtained by performing the staining and capturing of the unknown microplastic sample with data stored in the database.

Inventors

Min June YANG
Su Hyeon PARK
Tae Jung Ha

Assignees

PUKYONG NATIONAL UNIVERSITY INDUSTRY-UNIVERSITY COOPERATION FOUNDATION

Dates

Publication Date: 20260512
Application Date: 20230512
Priority Date: 20230217

Claims (8)

1 . A method for estimating mass of microplastics by using fluorescent staining, the method comprising: staining microplastic samples by using a Nile red solution; capturing a fluorescent image of each of the stained microplastic samples after classifying the stained microplastic samples by mass and after weighing the stained microplastics; analyzing the captured fluorescent image to quantify a fluorescence intensity thereof, including obtaining pixel-based numerical values of the fluorescence intensity, thereby building a database (DB) about a quantitative correlation between mass and a fluorescence intensity of microplastics; and estimating a mass value of an unknown microplastic sample by comparing a fluorescence intensity of a fluorescent image of the unknown microplastic sample obtained by performing the staining and the fluorescent image capturing with data stored in the database, wherein in the staining, Nile red is dissolved in an acetone solvent to prepare a Nile red staining solution with a predetermined concentration, microplastic particles with a predetermined mass are stained with the Nile red staining solution, and are cooled after being stirred according to preset temperature, time, and stirring speed conditions to prepare the microplastic samples, wherein in the capturing, the microplastic samples stained completely through the staining are separated from the solution through a vacuum filtration process and are dried, the dried microplastic samples are classified by mass according to a predetermined setting, and the capturing of the fluorescent image of each of the microplastic samples classified by mass is performed, and wherein in the estimating, after an unknown microplastic sample to be measured is stained with Nile red and a fluorescent image thereof is captured, a fluorescence intensity of the fluorescent image is analyzed, and a predetermined reference mass based on the quantitative correlation between mass and fluorescence intensity of microplastics stored in the database is compared with the fluorescence intensity of the unknown microplastic sample to derive the mass value of the unknown microplastic sample.
2 . The method of claim 1 , wherein in the capturing, the fluorescent image of the microplastic sample is captured by using visible blue light with a wavelength of 470 nm.
3 . The method of claim 1 , wherein the pixel-based numerical values comprise a mean gray value (MGV) of pixels, and wherein in the analyzing, by using an image processing software comprising ImageJ, the MGV of the fluorescent image is calculated to obtain a quantitative correlation between mass and fluorescence intensity of microplastics.
4 . The method of claim 1 , wherein: in the estimating, learning of a correlation between mass and fluorescence intensity of microplastics is performed by using deep learning or machine learning, or an artificial intelligence learning algorithm comprising an artificial neural network (ANN), a recurrent neural network (RNN), and a convolutional neural network (CNN), and a process of estimating mass of an unknown microplastic sample based on a result of the learning is automatically performed.
5 . A device for measuring mass of microplastics, the device comprising: an input interface configured to receive a fluorescent image of a microplastic sample to be measured; an image analysis processor configured to estimate mass of microplastics by comparing the fluorescent image input through the input part with data pre-stored in a database; and an output interface configured to output and visually display various data comprising the fluorescent image input through the input part and an analysis result of the image analysis processing part, wherein the image analysis processor is configured to estimate mass of microplastics by using the method of claim 1 .
6 . A non-transitory computer readable recording medium, when executed by one or more processors, configured to perform the method of claim 1 .
7 . The method of claim 1 , wherein the Nile red staining solution comprises a concentration of 100 μg/mL, wherein 1 g of microplastic particles are stained with the Nile red staining solution, and wherein the microplastic samples are prepared by stirring at a temperature of 50° C. for 30 minutes at 150 RPM using a shaking incubator and then cooling with ice water.
8 . The method of claim 1 , wherein in the analyzing, a region of interest (ROI) corresponding to stained microplastics is selected in the fluorescent image, and a fluorescence intensity is quantified by obtaining a mean gray value (MGV) of the ROI.

Description

CROSS REFERENCE TO RELATED APPLICATION The present application claims priority to Korean Patent Application No. 10-2023-0021580, filed Feb. 17, 2023, the entire contents of which are incorporated herein for all purposes by this reference. BACKGROUND Technical Field The present disclosure relates generally to device and method for estimating the mass of microplastics. More particularly, the present disclosure relates to a method for estimating the mass of microplastics by using fluorescent staining, in which by using fluorescent staining, the mass of microplastics can be efficiently quantified with a simpler configuration and at lower cost than the conventional methods of analyzing microplastics in order to solve the problems of the conventional methods for analyzing microplastics with the limitations that most of conventional quantitative analyses of microplastics are performed by a method (FT-IR spectroscopy, raman spectroscopy) of counting microplastics by observing the microplastics with a microscope and by a mass spectrometry method (Py GC-MS) based on thermal decomposition, but due to the very high price of analysis devices for this purpose, it was difficult to use the devices in a general laboratory environment, and further, the conventional analysis method of counting microplastics requires long time for analysis, and the conventional analysis method using thermal decomposition is a destructive analysis method. In addition, the present disclosure relates to a method for estimating the mass of microplastics by using fluorescent staining, in which microplastics are stained with a Nile red solution, the stained microplastics are weighted and photographed with a camera, a DB in which fluorescence intensity corresponding to the mass of the microplastics is quantified through the image analysis of the microplastics is generated, and a mass value of the microplastics is estimated through fluorescence intensity of a measured unknown sample, so that by using fluorescent staining, the method of the present disclosure can more efficiently quantify the mass of microplastics than the conventional methods and can be easily applied even to an experiment in which a large amount of analysis samples occurs by reducing cost and time required for analysis in order to solve the problems of the conventional methods for analyzing microplastics with the limitations that in addition to the very high price of the analysis devices as described above, the method of counting microplastics requires long time for analysis and the method of using thermal decomposition is a destructive analysis method. Description of Related Technology Generally, microplastics refer to pieces (particles) of plastic that are less than 5 mm in size. Recently, as environmental pollution has emerged as a serious problem worldwide, the danger of microplastics has become known, and research on the technology to quickly and accurately perform qualitative and quantitative analyses of microplastics present in water or the body of fish and shellfish is actively being conducted. SUMMARY One aspect is a method for estimating the mass of microplastics by using fluorescent staining, in which by using fluorescent staining, the mass of microplastics can be efficiently quantified with a simpler configuration and at lower cost than the conventional methods of analyzing microplastics in order to solve the problems of the conventional methods for analyzing microplastics with the limitations that most of conventional quantitative analyses of microplastics are performed by a method (FT-IR spectroscopy, raman spectroscopy) of counting microplastics by observing the microplastics with a microscope and by a mass spectrometry method (Py GC-MS) based on thermal decomposition, but due to the very high price of analysis devices for this purpose, it was difficult to use the devices in a general laboratory environment, and further, the conventional analysis method of counting microplastics requires long time for analysis, and the conventional analysis method using thermal decomposition is a destructive analysis method. Another aspect is a method for estimating the mass of microplastics by using fluorescent staining, in which microplastics are stained with a Nile red solution, the stained microplastics are weighted and photographed with a camera, a DB in which fluorescence intensity corresponding to the mass of the microplastics is quantified through the image analysis of the microplastics is generated, and a mass value of the microplastics is estimated through fluorescence intensity of a measured unknown sample, so that by using fluorescent staining, the method of the present disclosure can more efficiently quantify the mass of microplastics than the conventional methods and can be easily applied even to an experiment in which a large amount of analysis samples occurs by reducing cost and time required for analysis in order to solve the problems of the conventional methods for an