Patent Publication Number: US-2021181197-A1

Title: Fluorescence imaging-based device for detecting microorganisms and method for manufacturing the same

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
     This application claims priority to and the benefit of Korean Patent Application No. 10-2019-0166599, filed on Dec. 13, 2019, the disclosure of which is incorporated herein by reference in its entirety. 
     BACKGROUND 
     1. Field of the Invention 
     The present invention relates to a fluorescence imaging-based device for detecting microorganisms, a method for manufacturing the same, and a method for detecting microorganisms using the same. 
     2. Discussion of Related Art 
     Microorganisms harmful to humans are referred to as harmful microorganisms. These harmful microorganisms include pathogenic bacteria as well as food poisoning and high-risk infectious bacteria that cause decay and infection. Such microbial infection may lead to human and animal diseases, causing socially and economically adverse effects. Among these microorganisms,  Escherichia coli  contained in food or drinking water causes a disease such as food poisoning, resulting in serious economic losses. 
     For detecting pathogenic microorganisms, although a method using culture and a biochemical test is used, since it takes about 3 to 5 days, there are limitations in early detection of a pathogenic microorganism without culture. Accordingly, there is a demand for the development of technology that can quickly detect a pathogenic microorganism early, and particularly, if there is an on-site device for detecting microorganisms, which can quickly detect microorganisms in food, drinking water and industrial products, such a device will be effective in early detection of pathogenic microorganisms. 
     PRIOR ART DOCUMENT 
     Patent Document 
     (Patent Document 1) Korean Patent No. 10-2047854 
     SUMMARY OF THE INVENTION 
     Therefore, the inventors developed a device that is able to detect a microorganism in the field, which is able to separate microorganisms present in a detection sample from other materials within a certain reaction time by the intrinsic passive mechanism of the device, and directly observe and detect the separated microorganism using a fluorescence microscope. 
     Therefore, the present invention is directed to providing a fluorescence imaging-based device for detecting microorganisms. 
     The present invention is also directed to providing a method for manufacturing a fluorescence imaging-based device for detecting microorganisms. 
     The present invention is also directed to providing a fluorescence imaging-based method for detecting microorganisms. 
     However, technical problems to be solved in the present invention are not limited to the above-described problems, and other problems which are not described herein will be fully understood by those of ordinary skill in the art from the following descriptions. 
     To attain the purpose of the present invention, the present invention provides a fluorescence imaging-based device for detecting microorganisms, which includes 
     a substrate on which a magnet is disposed; 
     a microfluidic channel layer, which is disposed above the magnet of the substrate, has a microfluidic channel formed on a surface thereof and a separation reaction space in which a detection sample and a high-viscosity liquid are placed; and 
     an absorption layer disposed on the microfluidic channel layer and having an empty space, 
     wherein the empty space of the absorption layer has an area the same or smaller than that of the microfluidic channel layer such that the absorption layer and the microfluidic channel layer are in contact with each other, 
     the detection sample is a sample containing free magnetic particles and magnetic particle-conjugated fluorescence-labeled microorganisms, 
     when the detection sample is injected into a separation reaction space in the microfluidic channel layer, 
     the free magnetic particles in the detection sample reach the absorption layer through a microfluidic channel formed in the microfluidic channel layer along with the high-viscosity liquid, and then are absorbed to be removed, and 
     the magnetic particle-conjugated fluorescence-labeled microorganisms in the detection sample are captured by the magnet in the separation reaction space. 
     In addition, the present invention provides a method for manufacturing a fluorescence imaging-based device for detecting microorganisms, which includes the following steps: 
     (a) preparing a microfluidic channel layer by forming a separation reaction space in which a detection sample and a high-viscosity liquid are to be placed in a material for a microfluidic channel layer, and forming a microfluidic channel on its surface; 
     (b) attaching the microfluidic channel layer to the top of a substrate with a magnet; 
     (c) preparing an absorption layer by forming an empty space in a material for an absorption layer, in which the empty space of the absorption layer has an area the same or smaller than that of the microfluidic channel layer such that the absorption layer is in contact with the microfluidic channel layer; and 
     (d) placing the absorption layer on the microfluidic channel layer attached to the substrate. 
     In addition, the present invention provides a fluorescence imaging-based method for detecting microorganisms, which includes the following steps: 
     injecting a high-viscosity liquid and a detection sample into the fluorescence imaging-based device for detecting microorganisms to separate magnetic particle-conjugated fluorescence-labeled microorganisms from free magnetic particles in the detection sample; and 
     after the completion of the separation, observing the magnetic particle-conjugated fluorescence-labeled microorganisms captured by a magnet in a separation reaction space using a fluorescence microscope. 
     In one embodiment of the present invention, the separation reaction space is an empty space formed in the microfluidic channel layer, and may have a magnet under the empty space. 
     In another embodiment of the present invention, due to an area difference between the separation reaction space formed in the microfluidic channel layer and the empty space formed in the absorption layer, a separation reaction time for separating the magnetic particle-conjugated fluorescence-labeled microorganisms from the free magnetic particles in the detection sample may be adjusted. 
     In still another embodiment of the present invention, the microfluidic channel layer may be a water-soluble polymer. 
     In yet another embodiment of the present invention, the water-soluble polymer may be selected from the group consisting of polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, polyacrylic acid, polyacrylamide, carboxymethyl cellulose, pullulan and hydroxypropyl cellulose. 
     In yet another embodiment of the present invention, the high-viscosity liquid may be a liquid material having a viscosity of 20 to 200 mPa·s at room temperature. 
     In yet another embodiment of the present invention, the high-viscosity liquid may be selected from the group consisting of glycerol, polyethylene glycol, polyvinylpyrrolidone and an aqueous solution thereof. 
     In yet another embodiment of the present invention, the magnetic particle-conjugated fluorescence-labeled microorganisms captured by the magnet may be observed or counted using a fluorescence microscope. 
     In yet another embodiment of the present invention, the microorganism detection device may further include an upper case disposed on the absorption layer and having a columnar injection channel connected to the separation reaction space; and a lower case disposed under the substrate and connected with the upper case. 
     In yet another embodiment of the present invention, the microorganism detection device may be portable. 
     In yet another embodiment of the present invention, Step (a) may be for forming a microfluidic channel by humidifying a material for the microfluidic channel layer to disintegrate surface texture and then drying the material. 
     In yet another embodiment of the present invention, Step (a) may be for forming a microfluidic channel by micro-pattern stamping the material for the microfluidic channel layer. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The above and other objects, features and advantages of the present invention will become more apparent to those of ordinary skill in the art by describing in detail exemplary embodiments thereof with reference to the accompanying drawings, in which: 
         FIG. 1  is a schematic diagram of a fluorescence imaging-based device for detecting microorganisms according to one embodiment of the present invention; 
         FIG. 2  is a diagram illustrating operational changes over time after a detection sample is injected into a fluorescence imaging-based device for detecting microorganisms according to one embodiment of the present invention; 
         FIG. 3  is a photograph showing that a high-viscosity liquid (glycerol) dyed green and free magnetic particles are actually absorbed in an absorption layer, which results from operation of three fluorescence imaging-based devices for detecting microorganisms according to one embodiment of the present invention; 
         FIG. 4  is a flow chart showing a method for manufacturing a fluorescence imaging-based device for detecting microorganisms according to one embodiment of the present invention; 
         FIG. 5  is a diagram showing one example of forming a microfluidic channel on the surface of a microfluidic channel layer; 
         FIG. 6  is a diagram showing that a separation reaction is completed in approximately 15 minutes as a result of testing a total of 26 cartridge-type microorganism detection devices, as an example for verifying the application of a fluorescence imaging-based device for detecting microorganisms according to one embodiment of the present invention; and 
         FIG. 7  shows the result of counting magnetic particle-conjugated fluorescence-labeled  Staphylococcus aureus  of 100 CFU or less within 100 μL, which was captured on the capturing surface of a substrate through fluorescence microimaging, after a cartridge-type fluorescence imaging-based device for detecting microorganisms according to one embodiment of the present invention is operated. 
     
    
    
     DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS 
     Hereinafter, embodiments of the present invention will be described in further detail with reference to the accompanying drawings. The embodiments of the present invention may be modified in a variety of different forms, and it should not be construed that the scope of the present invention is not limited to the following embodiments. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art. Therefore, the shape of the elements in the drawings may be exaggerated to emphasize clearer explanation. In addition, terms and words used in the specification and claims should not be construed as limited to general or dictionary terms meanings, and should be interpreted with the meaning and concept in accordance with the technical idea of the present invention based on the principle that the inventors have appropriately defined the concepts of terms in order to explain the invention in the best way. 
     When describing with reference to the drawings, the same or corresponding components are denoted by the like reference numerals, and duplicated descriptions thereof will be omitted. 
       FIG. 1  is a schematic diagram of a fluorescence imaging-based device for detecting microorganisms according to one embodiment of the present invention. 
     Referring to  FIG. 1 , the fluorescence imaging-based device for detecting microorganisms according to one embodiment of the present invention includes a substrate  20  on which a magnet  30  is disposed, a microfluidic channel layer  10 , which is disposed on the magnet  30  of the substrate  20  and has a microfluidic channel on the surface thereof and a separation reaction space C 1  in which a detection sample and a high-viscosity liquid are placed, and an absorption layer  40  disposed on the microfluidic channel layer  10  and provided with an empty space C 2  therein. 
     The magnet  30  is disposed on the substrate  20 , and serves to capture magnetic particle-conjugated fluorescence-labeled microorganisms to be detected in the separation reaction space C 1  formed in the microfluidic channel layer  10 . As the substrate  20 , a polycarbonate-based, acryl-based or polyethylene-based plastic may be used. The shape of the magnet  30  is not particularly limited, and may be, for example, a round magnet. 
     The microfluidic channel layer  10  is disposed on the magnet  30  disposed on the substrate  20 , and has a microfluidic channel through which a high-viscosity liquid can flow on the surface. In addition, the detection sample and the high-viscosity liquid are placed in the microfluidic channel layer  10 , and as described below, there is the separation reaction space C 1  in which free magnetic particles and magnetic particle-conjugated fluorescence-labeled microorganisms present in the detection sample are separated. When a user injects the high-viscosity liquid and then the detection sample into the microorganism detection device of the present invention, the high-viscosity liquid and detection sample injected herein are placed in the separation reaction space C 1 , and the injected high-viscosity liquid flows through the microfluidic channel formed on the surface of the microfluidic channel layer  10  toward the absorption layer  40 . 
     The separation reaction space C 1  may be an empty space formed in the microfluidic channel layer  10 , and the magnet  30  for capturing magnetic particle-conjugated fluorescence-labeled microorganisms in the detection sample may be disposed under the empty space. The empty space may be formed by cutting out the inside of a material for the microfluidic channel layer (e.g., a double-sided tape formed of a water-soluble polymer) in a round shape. 
     The microfluidic channel layer  10  may be formed of a water-soluble polymer, and examples of the water-soluble polymer usable for the present invention may include, for example, polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, polyacrylic acid, polyacrylamide, carboxymethyl cellulose, pullulan or hydroxypropyl cellulose. 
     The absorption layer  40  is placed on the microfluidic channel layer  10 , and has an empty space C 2  therein. The high-viscosity liquid and detection sample injected into the microorganism detection device of the present invention are placed in the separation reaction space C 1  formed in the microfluidic channel layer  10  through the empty space C 2  formed in the absorption layer  40 . 
     In the microorganism detection device of the present invention, a separation reaction time for separating microorganism-bound magnetic particles (magnetic particle-conjugated fluorescence-labeled microorganisms) from the free magnetic particles (magnetic particles not conjugated to microorganisms) in the detection sample may be adjusted by an area difference between the separation reaction space C 1  formed in the microfluidic channel layer  10  and the empty space C 2  formed in the absorption layer  40 . To adjust the separation reaction time, the empty space C 2  of the absorption layer  40  has an area that is the same or smaller than the microfluidic channel layer  10  such that the absorption layer  40  may be in contact with the microfluidic channel layer  10 . If the empty space C 2  of the absorption layer  40  is larger than the area of the microfluidic channel layer  10 , the high-viscosity liquid and the detection sample flowing through the microfluidic channel of the microfluidic channel layer  10  may be absorbed into the absorption layer  40 . 
     The detection sample may be a liquid sample which is expected to have microorganisms to be detected. The detection sample may be prepared by adding fluorescence-labeled magnetic particles (e.g., nano-sized magnetic particles) to a sample to be detected, and therefore, the free magnetic particles (and other impurities) not bound to the microorganism and magnetic particles bound to the microorganism (fluorescence-labeled) are mixed in the detection sample. 
     As shown in  FIG. 1 , the fluorescence imaging-based device for detecting microorganisms according to one embodiment of the present invention may further include an upper case  60  and a lower case  50 . 
     The upper case  60  is disposed on the absorption layer  40 , and includes a columnar injection channel connected to the separation reaction space C 1  formed in the microfluidic channel layer  10 . The high-viscosity liquid and the detection sample are injected into the separation reaction space C 1  of the microfluidic channel layer  10  through the injection channel. 
     The lower case  50  may be disposed under the substrate  20  and connected with the upper case  60 , and the substrate  20 , the microfluidic channel layer  10  and the absorption layer  40  are placed in the upper and lower cases  50  and  60 . 
       FIG. 2  is a diagram illustrating operational changes over time after a detection sample is injected into a fluorescence imaging-based device for detecting microorganisms according to one embodiment of the present invention. 
     Referring to  FIG. 2 , the operation of the present invention will be described in detail. 
     When the high-viscosity liquid  110  and then the detection sample (magnetic particle-conjugated fluorescence-labeled microorganisms  90  and free magnetic particles  100 ) are injected into the fluorescence imaging-based device for detecting microorganisms according to one embodiment of the present invention, separation between the magnetic particle-conjugated fluorescence-labeled microorganisms and the free magnetic particles occurs due to a magnetic force from time t to time t finish  as the high-viscosity liquid flows through a microfluidic channel on the surface of the microfluidic channel layer  10  due to a capillary phenomenon ( 120 ). At the time t finish , due to a flow force caused by the absorption force of the absorption layer  40 , passive absorption of the free magnetic particles and the detection sample solution occurs ( 130 ), and the magnetic particle-conjugated fluorescence-labeled microorganisms are influenced by a stronger magnetic force, followed by separation and capture onto a capturing surface ( 140 ) (the separation reaction space of the microfluidic channel layer). The magnetic particle-conjugated fluorescence-labeled microorganisms captured by the magnet may be observed or counted using a fluorescence microscope. 
       FIG. 3  is a photograph showing that a high-viscosity liquid (glycerol) dyed green and free magnetic particles are actually absorbed in an absorption layer, which results from the operation of three fluorescence imaging-based devices for detecting microorganisms according to one embodiment of the present invention. 
     The high-viscosity liquid flows through the microfluidic channel of the microfluidic channel layer  10  toward the absorption layer  40 , resulting in absorption into the absorption layer  40 . As the high-viscosity liquid of the present invention, for example, a liquid material having a viscosity of 20 to 200 mPa·s may be used. In another example, as the high-viscosity liquid, a solution selected from the group consisting of glycerol, polyethylene glycol, polyvinylpyrrolidone and an aqueous solution thereof may be used. 
     Hereinafter, a method for manufacturing a fluorescence imaging-based device for detecting microorganisms according to one embodiment of the present invention will be described, and duplicated description of the same parts of the above-described fluorescence imaging-based device for detecting microorganisms will be omitted. 
       FIG. 4  is a flow chart showing a method for manufacturing a fluorescence imaging-based device for detecting microorganisms according to one embodiment of the present invention, and  FIG. 5  is a diagram showing one example of forming a microfluidic channel on the surface of a microfluidic channel layer. 
     Referring to  FIG. 4 , the method for manufacturing a fluorescence imaging-based device for detecting microorganisms according to one embodiment of the present invention includes preparing a microfluidic channel layer by forming a separation reaction space in which a detection sample and a high-viscosity liquid are to be placed in a material for a microfluidic channel layer, and forming a microfluidic channel on its surface (S 100 ), attaching the microfluidic channel layer to the top of a substrate with a magnet (S 200 ), preparing an absorption layer by forming an empty space in a material for an absorption layer (S 300 ), and placing the absorption layer on the microfluidic channel layer attached to the substrate (S 400 ). 
     In S 100 , a microfluidic channel layer  10  having a separation reaction space C 1  is prepared. As a material for the microfluidic channel layer, a water-soluble polymer may be used, and to reinforce portability, a thin, round or polygonal water-soluble double-sided tape (e.g., diameter: 1 to 2 cm) may be used. 
     The separation reaction space C 1  may be formed by cutting the center of the microfluidic channel layer into a round or polygonal shape. The size of the separation reaction space C 1  can be freely changed according to an amount of the detection sample or the separation reaction time of a target detection sample detection sample (time for separating the magnetic particle-conjugated fluorescence-labeled microorganisms from the free magnetic particles in the detection sample). 
     To form a microfluidic channel, a method for forming a microfluidic channel, which is known in the art, may be used without limitation. For example, as shown in  FIG. 5 , surface texture  70  may be disintegrated by humidifying the material for the microfluidic channel layer and then dried, thereby forming a microfluidic channel  80 . In another example, a microfluidic channel may be formed by micro-pattern stamping. 
     In S 200 , the prepared microfluidic channel layer  10  is adhered to the upper surface of a substrate  20  on which a magnet  30  is disposed. 
     In S 300 , an absorption layer  40  is prepared by forming an empty space C 2  in the material for an absorption layer. As the material for the absorption layer, an absorption pad may be used, and for example, a square pad having a thickness of 2 mm or less and a length of 2 to 3 cm may be used. The shape and type of the absorption layer  40  may vary according to a detection sample or other conditions. As the material for the absorption layer, a porous membrane such as paper, cotton or fabric may be used. 
     The empty space C 2  in the absorption layer  40  may be formed by cutting the center of the absorption layer  40  into a round or polygonal shape, like the separation reaction space C 1  of the microfluidic channel layer  10 . As described above, by using the difference in size between the empty space C 2  and the separation reaction space C 1  (the cut empty space) formed in the microfluidic channel layer  10 , the reaction time for separating the detection sample may be adjusted. 
     In S 400 , the absorption layer  40  is placed on the microfluidic channel layer  10  adhered to the substrate  20 . In addition, a microorganism detection device may be manufactured by additionally connecting upper and lower cases  50  and  60  to a combination of the substrate  20 , the microfluidic channel layer  10  and the absorption layer  40 . 
     The above-described microorganism detection device of the present invention has the following features. 
     In the manufacturing process, the microorganism detection device of the present invention may adjust a separation reaction time by adjusting the size (e.g., diameter) difference between C 1  and C 2 , and fix a certain time.  FIG. 6  is a diagram showing that the separation reaction is completed in approximately 15 minutes as a result of testing a total of  26  cartridge-type microorganism detection devices, as an example for verifying the application of a microorganism detection device of the present invention. 
     In addition, since free magnetic particles and the entire liquid sample are removed due to a flow force caused by the absorption force of the absorption layer after the end of the reaction time for passive separation of magnetic particle-conjugated fluorescence-labeled microorganisms, sample microorganisms can be directly observed and counted by fluorescence microscope microimaging without additional and complicated manipulation for detection, resulting in enhancement of ease of use. 
     In addition, by making the area of a capturing surface where the magnetic particle-conjugated fluorescence-labeled microorganisms completely separated from the free magnetic particles are captured smaller than the area of the field of view of the fluorescence microimaging, the captured fluorescence-labeled microorganisms may be directly observed, and even a very small number of microorganisms can be accurately detected by counting. 
     Hereinafter, to help in understanding the present invention, exemplary examples will be suggested. However, the following examples are merely provided to more easily understand the present invention, and not to limit the present invention. 
     EXAMPLES 
     Example 1. Manufacture of Fluorescence Imaging-Based Device for Detecting Microorganisms 
     The center of a double-sided tape based on a water-soluble polymer (polyvinyl alcohol) having a diameter of 0.8 cm serving as a microfluidic channel layer was cut into a round shape (C 1 ), and the cut water-soluble double-sided tape was aligned and adhered to the center of a capturing surface of a transparent plastic substrate (polycarbonate) into which a round magnet (neodymium) was inserted. To cause the capillary phenomenon of a high-viscosity liquid to the surface of the microfluidic channel layer, the texture was disintegrated by humidifying the water-soluble double-sided tape and then dried for recombination, thereby forming a microfluidic channel on the surface ( FIG. 5 ). 
     The center of a square absorption pad having an area of 1 cm 2  was cut into a round shape (C 2 ), and aligned on the water-soluble double-sided tape to be concentric with C 1  (C 1  diameter&lt;C 2  diameter, separation reaction time can be adjusted by a change of the difference in diameter between two circles). After alignment, a cup-shaped injection channel, which can contain a certain volume of liquid detection sample, was placed between the upper case and the lower case, and both cases were assembled, thereby manufacturing a cartridge-type microorganism detection device. 
     Example 2. Observation and Counting of Microorganisms using Fluorescence Imaging-Based Device for Detecting Microorganisms 
     A mixed sample of free magnetic particles and magnetic particle-conjugated fluorescence-labeled microorganisms was prepared by a method for first reacting capture particle-coated magnetic particles with microorganisms and staining the microorganisms by adding fluorescent particles to the reaction product. A high-viscosity liquid (glycerol having a viscosity of 20 mPa·s or more at room temperature) was pre-injected into a cup-shaped injection channel that can hold a liquid detection sample of the microorganism detection device, followed by injection of a detection sample in which free magnetic particles and magnetic particle-conjugated fluorescence-labeled microorganisms are mixed. When the high-viscosity liquid flowed through a microfluidic channel on the surface of the microfluidic channel layer due to the capillary phenomenon while the flow of the detection sample stopped, and came into contact with the outer absorption pad, the entire liquid sample was passively and instantly absorbed into an absorption pad while leaving the magnetic particle-conjugated fluorescence-labeled microorganisms on the opposite side of the region of a transparent substrate on which a magnet was placed (on the magnet), and the separation reaction time ended. During the passively controlled separation reaction time, the magnetic particle-conjugated fluorescence-labeled microorganisms in the high-viscosity liquid were separated from free magnetic particles and captured on the substrate surface due to the influence induced by larger magnetic force generated by the magnet inserted into the substrate, and after the completion of the reaction, free magnetic particles remaining in the high-viscosity liquid were removed with the entire liquid sample by flow force formed by the absorption force of the absorption pad. 
     The area of the surface where the fluorescence-labeled microorganisms are captured by the magnet was designed to be smaller than that of the field of view of the microscopic magnification capable of individually identifying microorganisms (10× or greater objective lens), and thus all microorganisms captured thereon were able to be observed or counted ( FIG. 7 ).  FIG. 7  shows the result of counting magnetic particle-conjugated fluorescence-labeled  Staphylococcus aureus  of 100 CFU or less within 100 μL, which was captured on the capturing surface of a substrate through fluorescence microimaging, after operating a cartridge-type fluorescence imaging-based device for detecting microorganisms. 
     The present invention relates to a fluorescence imaging-based device for detecting microorganisms which works with minimal user control and a method for detecting microorganisms, and enables direct observation and counting of a very few microorganisms within a predetermined fixed detection time. In addition, the microorganism detection device of the present invention can be manufactured in the form of a small cartridge, and thus can be used as an on-site device for detecting microorganisms. 
     It should be understood by those of ordinary skill in the art that the above descriptions of the present invention are exemplary, and the embodiments disclosed herein can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. Therefore, it should be interpreted that the embodiments described above are exemplary in all aspects, and are not limitative.