This application claims priority to and the benefit of Korean Patent Application No. 10-2020-0120625 filed in the Korean Intellectual Property Office on Sep. 18, 2020, the entire contents of which are incorporated herein by reference.
The present disclosure relates to a test strip, a microbial sensor device including the same, and a method of sensing a microorganism.
Severe acute respiratory syndrome (SARS, 2003), influenza A H1N1 virus 2009, Middle East respiratory syndrome coronavirus (MERS-CoV, 2005), and the like are known to be contagious diseases that are associated with the diffusion of pathogens in the air, cause tens of thousands of cases of infections, and kill hundreds of people. To prevent the infections and damage caused by these air-borne pathogens, it is important to detect them rapidly and effectively to prevent them from being diffused rapidly.
To effectively detect pathogens in the air, it is necessary to efficiently collect and analyze an atmospheric sample.
A conventional atmospheric sample is collected using a gravity settling method, an impactor method using a cascade impactor, an electrostatic method, a cyclone, and the like. In this case, heavy and large equipment is required, or it takes a long time to collect an atmospheric sample.
Also, such sampling methods require an additional processing process to analyze a certain hazardous material. In the case of microorganism, the analysis of the hazardous material in the atmospheric sample is performed by a culture method, mass spectrometry, a reverse-transcriptase polymerase chain reaction (RT-PCR), and the like. In this case, the culture method has drawbacks in that it requires a long time, and has limitations on quantification, and the methods using analysis equipment is sensitive and accurate, but has drawbacks in that it requires lots of time and cost due to the complicated pretreatment and expensive equipment. Therefore, these methods are not suitable for monitoring pathogens floating in a site.
The above information disclosed in this Background section is only for enhancement of understanding of the background of the invention, and therefore it may contain information that does not form the prior art that is already known in this country to a person of ordinary skill in the art.
An object of the present invention is to provide a measuring device for collecting and analyzing pathogens in the air to monitor the presence/absence and a concentration of the pathogens in a surrounding environment, and a measuring method thereof.
Another object of the present invention is to provide a device for collecting and detecting air-borne pathogens, which it is easy to carry and handle by means of a down-sized and integrated diagnosis platform according to the present invention, and a method of detecting the air-borne pathogens.
The combined sampling/diagnosis kit is combined with a small air sampling machine and analysis equipment so that it includes a device and a measuring method for performing in situ collection and detection at the same time.
Also, a platform for collecting and detecting a number of hazardous materials using a plurality of different combined sampling/diagnosis kits is suggested.
A test strip according to one aspect of the present invention includes a porous sampling pad through which the air passes to collect a specimen, a conjugation pad which is positioned on a supporter in contact with the sampling pad, and in which a plurality of capture agent-nanoparticle composites specifically binding to a target material are dispensed, a membrane which is positioned on the supporter in contact with the conjugation pad, and includes a test line to which the capture agent-nanoparticle composites to which the target material binds are conjugated when the specimen moves and a control line to which the capture agent-nanoparticle composites are conjugated, and an absorption pad configured to absorb the moving specimen on the supporter.
The sampling pad, the conjugation pad, the membrane, and the absorption pad may be positioned to be connected in this order along a moving direction of the specimen.
A portion of the sampling pad may be positioned on a portion of the conjugation pad so that the sampling pad and the conjugation pad partially overlap each other in a direction vertical to the moving direction.
A portion of the membrane may be positioned between a portion of the conjugation pad and the supporter so that the membrane and the conjugation pad partially overlap each other in a direction vertical to the moving direction.
A portion of the membrane may be positioned between a portion of the absorption pad and the supporter so that the membrane and the absorption pad partially overlap each other in a direction vertical to the moving direction.
When an analysis solution is dispensed in the sampling pad so that the specimen moves towards the conjugation pad along with the analysis solution, the target material in the specimen may bind to any one of the plurality of capture agent-nanoparticle composites.
A plurality of capture antibodies may be fixed in the test line, and one of the plurality of capture antibodies may bind to the target material of the capture agent-nanoparticle composites to which the target material binds.
A plurality of control antibodies may be fixed in the control line, the control line may be positioned behind the test line in a moving direction of the specimen, and one of the plurality of control antibodies may bind to the capture agent of the capture agent-nanoparticle composites.
The capture agent-nanoparticle composites may absorb infrared rays with a first wavelength to emit infrared rays with a second wavelength, and the first wavelength may be longer than the second wavelength.
Each of the conjugation pad, the membrane, and the absorption pad may include a solid-phase capillary support, and the porosity of the sampling pad may be greater than the porosity of the solid-phase capillary support.
A surface of the sampling pad may be treated with a mixed solution of PVP, sucrose, BSA, and Tween 20.
A microbial sensor device according to another aspect of the present invention includes an air sampling device configured to collect air, a combined sampling/diagnosis kit positioned on an upper surface of the air sampling device to collect a specimen from the air sucked by the air sampling device, and an analysis device configured to irradiate the kit with infrared rays with a first wavelength to receive the infrared rays with a first wavelength from the combined sampling/diagnosis kit, thereby detecting a target material from the specimen. The combined sampling/diagnosis kit includes a porous sampling pad configured to collect the specimen from the air passing through the corresponding one air suction port, a conjugation pad which is positioned in contact with the sampling pad, and in which a plurality of capture agent-nanoparticle composites specifically binding to the target material are dispensed, a membrane which is positioned in contact with the conjugation pad, and includes a test line to which the capture agent-nanoparticle composites to which the target material binds are conjugated when the specimen moves, and a control line to which the capture agent-nanoparticle composites are conjugated, and an absorption pad configured to absorb the moving specimen.
The air sampling device includes an air suction port device configured to provide an air path through which air flowing in through an air suction port of the combined sampling/diagnosis kit flows, a hollow air suction device configured to collect the air passing through the air path, and an air suction fan device configured to produce an air suction force. The sampling pad may be positioned in the air path.
The air suction port device may include an air outlet formed in a position corresponding to the air suction port of the combined sampling/diagnosis kit, and an air suction through hole formed to extend from the air outlet, and the sampling pad may be positioned on the air suction through hole.
The hollow air suction device may include a first circular aperture formed on an upper surface thereof, a second circular aperture formed on a lower surface thereof, and a common suction space formed between the first circular aperture and the second circular aperture.
The common suction space includes a circular cylinder space having a predetermined depth from the first circular aperture, and a space having a trapezoid circular cylinder shape between a third circular aperture positioned in a lower portion of the circular cylinder space and the second circular aperture.
The air suction fan device includes a fan configured to produce the air suction force while rotating, and an outlet configured to discharge the air sucked by rotation of the fan.
The combined sampling/diagnosis kit may further include a cartridge configured to accommodate a test strip in order to couple the test strip, which includes the supporter, the sampling pad, the conjugation pad, the membrane, and the absorption pad, to the air sampling device.
A method of detecting a microorganism using the kit coupled to the air sampling device according to still another aspect of the present invention includes collecting a specimen from air sucked by the air sampling device using a porous sampling pad through which the air passes, dispensing an analysis solution in the sampling pad to move the specimen thereto, conjugating a target material included in the specimen to one of a plurality of capture agent-nanoparticle composites in a conjugation pad, conjugating a capture antibody fixed in a membrane to the target material of the capture agent-nanoparticle composites to which the target material binds as the specimen moves, and absorbing the moving specimen using an absorption pad.
The method of detecting a microorganism may further include conjugating at least one of the plurality of capture agent-nanoparticle composites to a control antibody fixed in the membrane.
The present invention can monitor the presence/absence and a concentration of the hazardous microorganisms is situ in a surrounding environment in real time by combining a small air sampling machine, a combined sampling/diagnosis kit, and portable analysis equipment to collect and analyze microorganisms in the air in real time.
Also, the present invention can provide a device for collecting and detecting an air-borne microorganism, which is easy to carry and handle with down-sizing of the equipment, and a method of detecting the air-borne microorganism.
Further, the present invention can be applied to a measuring device and a method of collecting and analyzing contaminant microorganisms in the air, water, and foods, and other contaminants hazardous to the human body in real time to detect the presence/absence and a concentration of the contaminant microorganisms and the other contaminants, thereby making it possible to take rapid action for and prevent the air contaminants.
Because air-borne pathogens causing contagious diseases spread easily, a monitoring system capable of detecting and identifying the pathogens in situ may be helpful to prevent and control the diffusion of the pathogen at an early stage. That is, there is need for a diagnosis platform that is rapid and accurate, and easy to use when the diagnosis platform is coupled to an atmospheric sample collecting machine.
Paper-based lateral flow immunoassay (LFA) chromatography analysis is a platform for in situ diagnosis (hereinafter referred to as “LFA platform”) that may show simple, rapid, and sensitive diagnosis results of various viruses. However, the LFA platform is composed of a method of dispensing a sample in an aqueous solution on a sampling pad and analyzing the sample. That is, a large amount of a sample may be lost while collecting an atmospheric sample in the existing air sampling machine, recovering the sample in a solution phase, and then moving the recovered sample to the LFA platform. This may result in deteriorated accuracy and sensitivity of the diagnosis system.
As the LFA platform according to an exemplary embodiment, an integrated LFA platform capable of spatially combining collection and analysis of the sample in order to minimize the loss of the sample is provided.
That is, the integrated LFA platform according to an exemplary embodiment is an integrated sampling and detection platform for detecting pathogens floating in the air is situ in real time, and may include an air sampling device, a paper-based combined sampling/diagnosis kit, and a portable analysis device.
The integrated LFA platform according to an exemplary embodiment may be easy to carry due to the down-sizing of the air sampling device and the analysis device. Also, because the integrated LFA platform is combined as one platform, detection results may be rapidly obtained by sampling and analyzing a specimen in situ.
A plurality of sampling units are composed in the integrated LFA platform, and it is possible to collect and detect a plurality of different pathogens at the same time by combining the combined sampling/diagnosis kit, in which diagnostic capture agents specific to different pathogens are used, to the platform.
Registered Korean Patent No. 10-2049946 may be referenced to the known technology which is not described in the present disclosure.
Hereinafter, Examples disclosed in this specification will be described in detail with reference to the drawings, where like or similar constituent elements have like or similar reference numerals, and thus a repeated description thereof will be omitted.
The suffixes “module” and/or “unit” of the constituent element used in the following description are given in consideration of the easy drafting of this specification only, or are used interchangeably, and not intended to have the meanings or roles that are distinguished from each other in themselves. Also, in describing the Examples disclosed in this specification, a specific description of the related technology known in the art is judged to make the gist of the Examples disclosed in this specification unclear, a detailed description thereof is omitted. Also, the accompanying drawings are merely intended to easily understand the Examples disclosed in this specification, but the accompanying drawings are not intended to limit the technical scope of the Examples disclosed in this specification. Therefore, it should be understood that the present invention encompasses all changes, modifications, equivalents and substitutions which fall within the scope and spirit of the present invention.
The terms including the ordinal numbers such as first, second, and the like may be used to explain various constituent elements, but the terms are not intended to limit the constituent elements. The terms are used to differentiate one constituent element from another constituent element.
It is said that any constituent element is “connected to” or “in contact with” another constituent element, it should be understood that the any constituent element may be directly connected to or in direct contact with another constituent element, but may be connected to the another constituent element through another constituent element. On the other hand, it is said that any constituent element is “directly connected to” or “in direct contact with” another constituent element, it should be understood that there is no another constituent element between them.
In this application, the term “comprising”, “having” or the like is used to specify that a feature, a number, a step, an operation, a constituent element, a part, or a combination thereof disclosed in this specification is present, but does not exclude the possibility of presence or addition of one or more other features, numbers, steps, operations, constituent components, parts or combinations thereof in advance.
As shown in
The air suction port device 300 provides four paths through which the air sucked through four combined sampling/diagnosis kits 31-34 may be sucked without air leakage. The number of the air flow paths is determined depending on the number of the combined sampling/diagnosis kits. The four combined sampling/diagnosis kits 31-34 are inserted and coupled to an upper portion of the air suction port device 300. The air suction port device 300 includes four air suction through holes 321-324 positioned to correspond to the air suction ports 11-14 of the four combined sampling/diagnosis kits 31-34.
Four outlets 111-114 are formed in the air suction port device 300 in positions corresponding to the air suction ports 11-14, and each of the four air suction through holes 321-324 is formed to extend from the corresponding outlet (one of 111-114) along the z axis.
The four combined sampling/diagnosis kits 31-34 are coupled to the air sampling device 10 through four coupling grooves 16-18, and each of sampling pads of the four combined sampling/diagnosis kits 31-34 is positioned on one corresponding hole of the four air suction through holes 321-324. The sampling pads of the four combined sampling/diagnosis kits 31-34 serve to collect a specimen from the air sucked through the four air suction through holes 311-314, and allow the air passing through the sampling pads to flow to the hollow air suction device 200 through the four air suction through holes 331-334 and the four air suction through holes 321-324.
In
As shown in
The hollow air suction device 200 includes a circular aperture 205 formed on an upper surface 204 thereof, a circular aperture 207 formed on a lower surface 206 thereof, and a common suction space 203 formed between the circular aperture 205 and the circular aperture 207. The common suction space 203 includes a circular cylinder space having a predetermined depth d from the circular aperture 205, and a space having a trapezoid circular cylinder shape between a circular aperture 208 positioned in a lower portion of the circular cylinder space and the circular aperture 207.
The air suction fan device 100 includes a fan 101 configured to produce an air suction force while rotating, and an outlet 102 configured to discharge the air sucked by rotation of the fan 101. As shown in
A plurality of air suction through holes 311-314, 331-334, 321-324 are numbered by positions, and the number indicating the corresponding number is a horizontal axis on the graph of
The vertical axis on the graph of
In
As such, as can be seen in
In an exemplary embodiment as shown in
For convenience of description,
In
In
In
In
In
In
The positions of the plurality of suction ports are determined depending on the positions of the plurality of sampling pads disposed in
The air sampling drive unit 40 may include a power source 41 configured to drive the air suction fan device 100 to collect the air, a control circuit 42, and a flow rate measuring machine 43. The flow rate measuring machine 43 may measure an amount of the air discharged from the air suction fan device 100 and transmit the air into the control circuit 42, and the control circuit 42 may control the power source 43 based on the measured amount of the air. For example, to control an amount of the air collected per unit time based on predetermined conditions, the control circuit 42 regulates the output electric power of the power source 41. The control circuit 42 may control the power source 41 in a negative feedback control mechanism in which the output electric power of the power source 41 is lowered when the amount of the air is increased and the output electric power of the power source 41 is raised when the amount of the air is reduced.
The analysis device 20 may irradiate the combined sampling/diagnosis kit 35 coupled to a coupling groove 26 with infrared rays, and sense the infrared rays emitted from the test line of the kit 35 to determine whether a target material is detected and measure an amount of the detected target material, and may sense the infrared rays emitted from the control line to analyze whether the combined sampling/diagnosis kit 35 effectively performs a detection operations.
The analysis device 20 may transmit an image, which is sensed by the infrared rays emitted from the test line and the control line, to an external terminal 50. In
The analysis device 20 includes a laser 21, an optical filter 22, an infrared camera 23, an image processing unit 24, and an interface 25.
The laser 21 irradiates a membrane of the combined sampling/diagnosis kit 35 with infrared rays with a certain band (e.g., 980 nm wavelength). Specifically, the laser 21 may irradiate a test line and a control line of the membrane with infrared rays with 980 nm wavelength.
The optical filter 22 may transmit wavelengths with a band less than or equal to a certain band (e.g., 850 nm). The optical filter 22 may include a visible ray cut-off filter and an ultraviolet ray cut-off filter.
The infrared camera 23 takes an image of infrared rays passing through the optical filter 22.
The image processing unit 24 processes the image taken by the infrared camera 23 to convert the image into image data, which are transmittable to the external terminal 50.
The interface 25 transmits the image data converted by the image processing unit 24 to the outside. The interface 25 may be connected to the cable 51 to transmit the image data to the external terminal 50 through the cable 51.
It is described that one of the plurality of combined sampling/diagnosis kits 31-34 coupled to the air sampling device 10 to complete the collection of the specimen is inserted into the coupling groove 26 in the analysis device 20 in order to perform the analysis, but the present invention is not limited thereto. For example, an analysis operation may be performed together in the air sampling device 10.
In the air sampling device 10, a reader module includes a laser, an optical filter, and an infrared camera, all of which have a fixed or mobile structure, may be positioned on the combined sampling/diagnosis kits 31-34. Then, after the specimen is collected in the air sampling device 10, it may be determined whether a target material is detected by irradiating a membrane of the combined sampling/diagnosis kit with infrared rays while the combined sampling/diagnosis kit is coupled to the air sampling device 10 without detachment from the air sampling device 10, and taking an image of infrared rays emitted from the combined sampling/diagnosis kit. In the case of the mobile structure, the reader module may be positioned in an empty space of the air sampling device 10 during collection of the air to prevent the reader module from interfering with collection of the air, and may move to an upper portion of the kit when the collection of the air is completed.
Each of the plurality of combined sampling/diagnosis kits 31-34 includes a test strip for a target material to be detected, and a plastic cartridge configured to accommodate a test strip to couple the test strip to an air sampling device.
As shown in
The upper lid 400 includes an opening 410 configured to expose a sampling pad 510 of the test strip 500, an opening 420 configured to expose a membrane 530 of the test strip 500, accommodation spaces 411, 421, and 422 formed in an inner direction of the upper lid 400 to accommodate the test strip 500, and protrusions 431-436 for coupling to the lower lid 600. The lower lid 600 includes an opening 610 configured to expose the sampling pad 510 of the test strip 500, an accommodation space 620 formed in an inner direction of the lower lid 600 to accommodate the test strip 500, and grooves 631-636 inserted and coupled to the protrusions 431-436 for coupling to the upper lid 400.
The plurality of air suction through holes 311-314 previously shown in
The opening 420 is formed in such a size that a test line 531 and a control line 532 in the membrane 530 can be exposed to the outside, and the openings 410 and 610 may be formed to expose the sampling pad 510 to the maximum extent to collect the specimen. In
The test strip 500 may be realized to be adapted to lateral flow diagnosis, and may be manufactured using paper as a main material. The test strip 500 includes a sampling pad 510, a capture agent-nanoparticle conjugation pad 520, a membrane 530 configured to detect a signal, an absorption pad 540, and a supporter 550, and the sampling pad 510, the conjugation pad 520, the membrane 530, and the absorption pad 540 are positioned so that they are connected onto the supporter 550 in this order in a moving direction of the specimen. In a horizontal direction (a direction parallel with the moving direction of the specimen), the conjugation pad 520 is positioned in contact with the sampling pad 510, the membrane 530 is positioned in contact with the conjugation pad 520, and the absorption pad 540 is positioned in contact with the membrane 530. A portion of the sampling pad 510 may be positioned on a portion of the conjugation pad 520 so that the sampling pad 510 and the conjugation pad 520 partially overlap each other in a vertical direction (a direction vertical to the horizontal direction). A portion of the membrane 530 may be positioned between a portion of the conjugation pad 520 and the supporter 550 so that the membrane 530 and the conjugation pad 520 partially overlap each other in a vertical direction. A portion of the membrane 530 is positioned between a portion of the absorption pad 540 and the supporter 550 so that the membrane 530 and the absorption pad 540 partially overlap each other in a vertical direction.
The sampling pad 510 is formed in a porous structure, and thus may be realized using a material through which the air passes and which absorbs an analysis solution, such as glass fibers, polyester fibers, meshes, or the like. Surfaces of the glass fibers, the polyester fibers, or the meshes may be subjected to certain treatment. For example, a surface of the sampling pad 510 may be treated with a mixed solution of PVP, sucrose, BSA, and Tween 20. The sampling pad 510 may collect a specimen including a target material that is an analyte in the sucked air. The target material refers to an analyte whose concentration or presence/absence is to be analysed.
The capture agent-nanoparticle composites are dispensed in the conjugation pad 520, and the diagnostic capture agent includes an antibody, an aptamer, and the like, which specifically recognize and bind to the target material. The nanoparticles may be upconversion infrared absorption/emission nanoparticles. The infrared absorption/emission nanoparticles are doped with a rare earth element to provide upconversion nanoparticles that absorb light energy with long wavelengths and emit light energy with short wavelengths through a pyrolytic synthesis reaction. The capture agent-nanoparticle composites specifically bind to a target material, and emit infrared rays other than visible rays when the capture agent-nanoparticle composites absorb the infrared rays. The emitted infrared rays have a long wavelength, and thus may pass through a sample and do not produce background signals. Therefore, because there is no interference between light absorption and emission, a target material to be detected may be detected with high sensitivity. Upon a detection operation of the target material, the conjugation pad 520 may be pretreated with a solution obtained by mixing PVP, BSA, sucrose, Tween 20, and the like in order to efficiently recover the capture agent-nanoparticle composites from the conjugation pad 520.
When the analysis solution is dispensed in the sampling pad 510 and the specimen is allowed to move to the conjugation pad 520 together with the analysis solution, the composites in the conjugation pad 520 may be conjugated to the target material included in the specimen. The analysis solution is a solution that is dispensed in the sampling pad 510 to detect the target material and analyze an amount of the target material, and may be a buffer solution used to dissolve cells. The conjugation pad 520 may include the composites, which bind to the target material that is an analyte in the specimen, in a dried state. After the analysis solution is allowed to flow and accommodated in the conjugation pad 520, a capture agent in the composites specifically binds to the target material when the target material is included in the specimen.
The capture agent-nanoparticle composites absorb infrared rays to emit infrared rays when irradiated with the infrared rays. In this case, the wavelength of the absorbed infrared rays is not identical to the wavelength of the emitted infrared rays. For example, the capture agent-nanoparticle composites may absorb infrared rays with long wavelengths to emit infrared rays with short wavelengths, wherein the infrared rays with long wavelengths may be infrared rays having a wavelength of 960 to 980 nm, and the infrared rays with short wavelengths may be infrared rays having a wavelength of 750 to 850 nm. The infrared rays having a wavelength of 750 to 850 nm may have enhanced transmittance to bio-materials such as tissues, and the like, thereby preventing an effect by the specimen such as blood, feces, and the like. Also, because the infrared rays have a high transmittance to an opaque mixed solution of the specimen, a target material may be detected from various types of collected specimens. Also, because the infrared rays having a wavelength of 750 to 850 nm do not produce autofluorescence, a signal to noise ratio may be improved. The combined sampling/diagnosis kit according to an exemplary embodiment may solve the problems such as low sensitivity while maintaining convenience and economic feasibility of conventional immunoassay diagnosis kits used in the fields.
When the nanoparticle is further doped with a heterogeneous dopant, distortion of a crystal structure in the nanoparticles may be increased to some extent, thereby enabling the transfer of very sensitive electrons. In this way, the luminance intensity may be further enhanced without any change in size of the nanoparticles themselves.
The nanoparticles according to an exemplary embodiment may include any one or more selected from the group consisting of a fluoride, an oxide, a halide, an oxysulfide, a phosphate, and a vanadate. For example, the nanoparticles may include any one or more selected from the group consisting of NaYF4, NaYbF4, NaGdF4, NaLaF4, LaF3, GdF3, GdOF, La2O3, Lu2O3, Y2O3, and Y2O2S. The rare earth element with which the nanoparticles are doped may include a lanthanide element, and a range of wavelengths of light in which the nanoparticles absorb and emit light may be regulated by regulating the type and concentration of the rare earth element included in the nanoparticles. The nanoparticles having no interference in a range of wavelengths absorbing and emitting the infrared wavelengths may be provided by regulating the type and concentration of the rare earth element. For example, the rare earth element may include any one or more selected from the group consisting of Y, Er, Yb, Tm, and Nd. More specifically, the rare earth element may include 45 to 55 mol % of Y, 43 to 52 mol % of Yb, and 1.5 to 3 mol % of Tm.
The luminance intensity of the nanoparticles may be regulated by regulating the type or concentration of the heterogeneous dopant. Examples of the heterogeneous dopant with which the nanoparticles are further doped may include any one or more selected from the group consisting of Ca, Si, Ni, and Ti.
The nanoparticles doped with the rare earth element and the heterogeneous dopant may be manufactured by doping according to the methods commonly used in the technical field of the present invention, and may, for example, be manufactured using a method disclosed in Qian et al., Small, 5: 2285-2290, 2009; Li et al., Advanced Materials, 20: 4765-4769, 2008; Zhao et al., Nanoscale, 5:944-952, 2013; Li et al., Nanotechnology, 19: 345606, 2008. The above-mentioned documents may be incorporated herein by reference in their entireties.
A bond of the diagnostic capture agent (composed of an antibody, an aptamer, and the like) to the nanoparticles doped with the rare earth element and the heterogeneous dopant includes bonds selected from an ionic bond, a covalent bond, a metallic bond, a coordinate bond, a hydrogen bond, and a Van der Waals bond.
The nanoparticles may include a core layer, a shell layer, and a coating layer. The core layer may be composed of particles doped with a rare earth element. The shell layer is further doped with a heterogeneous dopant to surround the core layer, thereby reducing surface defects and improving surface uniformity. The coating layer is formed by coating an outer surface of a cell layer with a monomer or a polymer, thereby enhancing dispersibility of the nanoparticle in a fluid and facilitating fixation of the diagnostic capture agent. The diagnostic capture agent binds to the coating layer. When the nanoparticles have a core-shell structure, surface defects may be reduced to enhance surface uniformity, and monodispersity may be enhanced to maximize infrared emission efficiency. When the shell layer is further doped with the heterogeneous dopant, infrared luminance intensity may be further improved. When the nanoparticles are surface-treated with a monomer or a polymer, the capture agent-nanoparticle composites may have enhanced dispersibility in an analysis solution and fixation of the antibody in the capture agent-nanoparticle composites may be facilitated.
For example, the core layer is formed in the form of nanoparticles by mixing 1-octadecene, oleic acid, and a rare earth element to form a homogeneous solution, mixing methanol containing sodium hydroxide and fluorine ammonium with the homogeneous solution, agitating the resulting mixture, and then reacting the mixture at a constant temperature for a constant time, and the shell layer is formed on the core layer to have a constant thickness by mixing 1-octadecene, oleic acid, a rare earth element, and a heterogeneous dopant to form a homogeneous solution, mixing methanol containing sodium hydroxide and fluorine ammonium with the homogeneous solution together with the core layer, agitating the resulting mixture, and then reacting the mixture at a constant temperature for a constant time.
The polymer used to form a coating layer may include one or more selected from the group consisting of polyacrylic acid (PAA), polyallylamine (PAAM), 2-aminoethyl dihydrogen phosphate (AEP), polyethylene glycol diacid, polyethylene glycol acid, and polyethylene glycol phosphate ester. The formation of the coating layer may be performed using methods commonly taken in practice in the art. For example, the coating layer may be treated by means of ligand engineering such as ligand exchange or oleic acid oxidation, ligand attraction, layer-by-layer assembly, surface treatment using silanization, surface polymerization, and the like. Also, the coating layer may be surface-treated according to the method disclosed in Photon Upconversion Nanomaterials, Fan Zhang, Springer, 2015, the contents of which may be incorporated herein by reference.
A test line 531 capable of detecting a target material, and a control line 532 capable of determining whether the test strip 500 is normally operated are present in the membrane 530, a capture antibody capable of binding to the target material is fixed onto a membrane 530 in the test line 531, and a control antibody binding to the capture agent-nanoparticle composites is fixed onto the membrane 530 in the control line 532. The test line 531 may be positioned in closer proximity to the conjugation pad 520 than the control line 532.
The capture antibody of the test line 531 is configured to specifically bind to or react with the target material. For example, an antibody, an aptamer, and the like may be used. The control antibody is configured to specifically bind to or react with the capture agent of the composites. For example, an antibody, an aptamer, and the like may be used.
The target material specifically binding to the capture agent in the composites may be allowed to move from the conjugation pad 520 to the membrane 530 so that a portion of the target material may be fixed in the test line 531 by binding to the capture antibody, and a portion of the target material may be fixed in the control line 532 by allowing the capture agent in the composites to react with the control antibody.
The capture antibody reacting with the target material is fixed in the test line 531.
It may be determined whether the target material to be analyzed is included in the collected specimen, and a concentration of the target material may be analyzed by determining whether the infrared rays are emitted from the test line 531, and measuring the luminance intensity.
Because the control antibody reacting with the capture agent in the composites is fixed in the control line 532, it may be judged whether the specimen moves to a position necessary for detection of the target material and whether the capture agent in the composites is operated by determining whether the infrared rays are emitted from the control line 532. Therefore, these results may be used as the criteria for reading validity and effectiveness of analysis.
The absorption pad 540 is a pad that absorbs a fluid in the specimen passing through the membrane 530. The absorption pad 540 may serve as a pump that absorbs the dispensed analysis solution and allows the specimen to continuously move from the sampling pad 510 to the membrane 530. The analysis solution may, for example, be a solution including any one or more selected from the group consisting of phosphate buffer saline (PBS), KCl, NaCl, Tween20, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), NaN3, and the like, but the present invention is not limited thereto.
The conjugation pad 520, the membrane 530, and the absorption pad 540 may include a solid-phase capillary support, and the solid-phase capillary support may be used without limitation as long as it is a porous polymer that may serve as a solid-phase capillary carrier for a chemical components such as antigens, antibodies, aptamers, or haptens, or is a natural or synthetic material, or a naturally occurring material modified by synthesis. In this case, a shape of the solid-phase capillary support is not limited. As described above, for example, the solid-phase capillary support may include one or more selected from the group consisting of a cellulose material, paper, cellulose acetate, nitrocellulose, polyether sulfone, polyethylene, nylon, polyvinylidene fluoride (PVDF), polyester, polypropylene, silica, vinyl chloride, a vinylchloride-propylene copolymer and a vinyl chloride-vinyl acetate copolymer, inactivated alumina, diatomite, MgSO4, cotton, nylon, rayon, silica gel, agarose, dextran, gelatin, and polyacrylamide. As a more specific example, the membrane may include at least one polymer selected from the group consisting of nitrocellulose, polyether sulfone, polyethylene, nylon, polyvinylidene fluoride, polyester, and polypropylene. As an exemplary embodiment, the solid-phase capillary support may also have a shape like a rod, a plate, a tube, a bead, or the like.
The porosity of the sampling pad 510 is very greater than the porosities of the conjugation pad 520, the membrane 530, and the solid-phase capillary support of the absorption pad 540. That is, the sampling pad 510 has such a porosity that the sucked air can pass through the sampling pad 510, and the conjugation pad 520, the membrane 530, and the solid-phase capillary support of the absorption pad 540 have such a porosity that the chemical components can move by means of a capillary phenomenon.
All type of the supporter 550 may be used without limitation as long as they can support and carry the sampling pad 510, the conjugation pad 520, the membrane 530, and the absorption pad 540. In this case, the supporter 550 may be impermeable to a liquid to prevent the specimen from leaking from the analysis solution. For example, the supporter 550 may include glass, polystyrene, polypropylene, polyester, polybutadiene, polyvinyl chloride, polyamide, polycarbonate, epoxide, methacrylate, polymelamine, and the like.
Hereinafter, an operation of the combined sampling/diagnosis kit will be described with reference to
As shown in
As shown in
The secondary composites 511+521 and 512+522 move to the membrane 530 along a lateral flow stream, and bind to the capture antibodies 5311 and 5312 fixed in the test line 531 of the membrane 530. The target materials (antigens) 511 and 512 of the secondary composites 511+521 and 512+522 are bound to the capture antibodies 5311 and 5312 on the test line 531 by means of an antigen-antibody reaction, and fixed in the test line 531.
As shown in
When amounts of the capture agent-nanoparticle composites fixed in the test line 531 and the control line 532 are analyzed using optical or chemical signals, and the like, it may be determined whether the target material is detected in the specimen, and an amount of the detected target material may be measured. In fact, a detection operation in the test line 531 and the control line 532 may be performed within 20 minutes.
An immunoassay-based detection process according to an exemplary embodiment may proceed without detaching the combined sampling/diagnosis kits from the microbial sensor device. There is no effect on the results even when the detection process proceeds in separate manner. Because the collection and detection processes are directly performed without any relocation or time delay, the activity of the collected microorganisms may be maintained. Therefore, there is no need for use of additional buffers and containers or certain sampling conditions for this purpose.
Hereinafter, the implementability and effect of the air sampling device according to an exemplary embodiment will be described by describing Experimental Examples.
The MS2 viruses present in a solution for an experiment may be detected by the combined sampling/diagnosis kit when a concentration of the MS2 viruses is greater than or equal to 106 PFU/mL, indicating that the detection limit of the combined sampling/diagnosis kit is ten-fold lower than that of an enzyme-linked immunosorbent assay (ELISA). For an experiment, a high concentration (1011 PFU (plaque forming unit)/mL) of a virus stock solution was diluted with a cytolytic buffer to prepare various concentrations (e.g., 109 to 105.5 PFU/mL) of an experimental solution including the viruses, and 100 uL of the experimental solution was dispensed in a sampling pad. The concentration is in a range of 108 to 104.5 PFU/100 uL based on the total amount of the analysis solution. A PBS buffer diluted under the same conditions was used for an ELISA method. In the ELISA method, the viruses were detected after the concentration of the viruses is greater than 107 PFU/mL.
As shown in
In the experiment, a protein, a surfactant, or the like may be used to treat a surface of the sampling pad. For example, a surface of the sampling pad may be treated with a single solution or a mixed solution, which contains approximately 0.1 to 5% of polyvinylpyrrolidone (PVP), sucrose, bovine serum albumin (BSA), Tween 20, and the like.
As shown in
As shown in
As shown in
The higher detection ratio means the higher probability that the MS2 viruses recovered from the sampling pad bind to the capture agent-nanoparticle composites in the conjugation pad, move to the membrane, and bind to the test line. Then, when the MS2 viruses are present at the same concentration in the sampling pad, the MS2 viruses bind to the test line with the highest recovery rate in the pad treated with the mixed solution of PVP, sucrose, BSA, and Tween 20, resulting in the highest signaling reaction of the nanoparticles. Sensitivities of signals may be improved with an increasing signaling reaction of the nanoparticles.
Hereinafter, a chamber for an experiment used to collect air in the atmosphere and detect a target material will be described.
As shown in
A chamber control system 3 may be connected to individual configurations of the chamber 2 via wire or wireless communications to receive detection signals S1-S4, and produce and transmit control signals C1-C6 in consideration of the experimental conditions.
The pump 701 supplies external air into the inside of the chamber 2 according to the control signal C1 received from the chamber control system 3. The flow rate measuring system 702 may measure an amount of the air supplied from the pump 701 into the inside of the chamber 2 to produce a detection signal S3 and transmit the detection signal S3 to the chamber control system 3.
The air cylinder 703 stores the air required to produce a bioaerosol. The valve 704 controls an amount of the air supplied through a piping 711 according to the control signal C2 received from the chamber control system 3. The flow rate measuring system 705 may measure an amount of the air supplied through the piping 711 to produce a detection signal S4 and transmit the detection signal S4 to the chamber control system 3.
The sprayer 706 mixes a microbial solution with the air supplied through the piping 711 and sprays the mixture into the chamber 2 according to the control signal C3 received from the chamber control system 3. Then, the aerosol including the microorganisms flows in the chamber 2.
The hygrometer 707 may measure humidity in the chamber 2 to produce a detection signal S1 and transmit the detection signal S1 to the chamber control system 3, and the thermometer 708 may measure a temperature in the chamber 2 to produce a detection signal S2 and transmit the detection signal S2 to the chamber control system 3.
Each of the two fans 709 and 710 operates according to the control signals C5 and C6 to control the flow of the air in the chamber 2.
As such, the bioaerosol may be artificially produced in the chamber 2, and the surrounding environments such as a temperature, humidity, a pressure, and the like may be promoted according to the experimental conditions. In the promoted experimental conditions, the air sampling device 1 may be operated to perform an experiment for detection of a target material using the combined sampling/diagnosis kit coupled to the air sampling device 1.
Zone 1 is the outside of the chamber 2, and Zones 2-5 are the inside of the chamber 2.
In this case, as shown in
In Zone 4 with an environment in which the MS2 viruses are present, when the combined sampling/diagnosis kits are positioned outside the air sampling device 10 (Outside), the air sampling device 10 collects a specimen, but does not detect the MS2 viruses.
In Zone 5 with an environment in which the MS2 viruses are present, when the combined sampling/diagnosis kits are coupled to the inside of the air sampling device 10 (Inside), the air sampling device 10 collects a specimen. As a result, it can be seen that the infrared rays are emitted from the test line T of the combined sampling/diagnosis kit to detect the MS2 viruses.
As shown in
When the capture agent-nanoparticle composites positioned on the conjugation pads of the combined sampling/diagnosis kits are prepared, a diagnostic capture agent that specifically recognizes and binds to avian influenza virus (AIV H1N1) was used. It is revealed that the avian influenza virus present in an aqueous solution (a buffer) (AIV H1N1 in a buffer) may be detected from 103 50% egg infectious dose (EID50/mL), and the avian influenza virus adsorbed in the sampling pad (dried AIV H1N1 in the sampling pad) may be detected from 103.5 EID50/mL.
As shown in
It is revealed that the combined sampling/diagnosis kits manufactured using the diagnostic capture agent, which specifically recognizes and reacts with the avian influenza virus (AIV H1N1) even when non-targeted avian infection-related viruses (avian paramyxovirus (APMV), Newcastle disease virus (NDV), infectious bronchitis virus (IBV)) are present, may selectively detect the avian influenza virus (AIV H1N1) only.
While the microbial sensor device collects an atmospheric sample, a combined collection/detection sensor platform may use a disposable cover to minimize contamination of a surface of equipment and user's infection. The disposable cover is sized to cover a surface of the microbial sensor device except for a suction port through which an atmospheric specimen is sucked, and is allowed to be easily attachable and detachable to the microbial sensor device using a fastening clip, and the like. Also, the disposable cover may be made of materials that filter off external dust and contaminants, such as vinyl, paper, and the like.
—Preparation of Infrared Absorption/Emission Nanoparticles—
(1) Formation of Core
1-octadecene, oleic acid, yttrium acetate hydrate, ytterbium acetate hydrate, and thulium acetate hydrate were mixed (specifically, 0.4 mmol of lanthanides (composed of 50 mol % Y, 48 mol % Yb, and 2 mol % Tm) were mixed with 7 mL of 1-octadecene and 3 mL of oleic acid)), and then heated to 150° C. to form a homogeneous solution, and the homogeneous solution was cooled to 50° C.
5 mL of methanol containing 1 mmol NaOH and 1.6 mmol NH4F was added to the homogeneous solution, and agitated for an hour to form a mixed solution. To remove the methanol, the mixed solution was maintained at 100° C. for 10 minutes, and maintained for 30 minutes under a vacuum state, and then maintained at 290° C. for an hour and 30 minutes under an argon gas.
The nanoparticles after the mixed solution was cooled naturally were precipitated with ethanol, and washed three times with cyclohexane and ethanol to obtain nanoparticles (a core).
(2) Formation of Shell (Formation of UCNPs)
1-octadecene, oleic acid, yttrium acetate hydrate, and calcium acetate hydrate were mixed (specifically, 0.2 mmol of a dopant (a lanthanide (Y) was mixed with 7 mL of 1-octadecene and 3 mL of oleic acid), and then heated to 150° C. to form a homogeneous solution, and the homogeneous solution was cooled to 50° C.
The nanoparticles (a core) prepared in the section “(1) Formation of core” were mixed, and heated to 100° C. to remove the cyclohexane, and then cooled again to 50° C. 5 mL of methanol containing 1 mmol NaOH and 1.6 mmol NH4F, and the homogeneous solution were mixed, and agitated for 30 minutes. To remove the methanol, the mixed solution was maintained at 100° C. for 10 minutes, maintained for 30 minutes under a vacuum state, and then maintained at 290° C. for an hour and 30 minutes under an argon gas. The nanoparticles after the mixed solution was cooled naturally were precipitated with ethanol, and washed three times with cyclohexane and ethanol to obtain nanoparticles having a core-shell structure (Core/Shell, UCNPs)
—Preparation of Capture Agent (Antibody)-Nanoparticle Composites—
(1) Formation of Coating Layer
The nanoparticles (core/shell) were coated with a polymer using a ligand engineering method. The nanoparticles prepared in the section “(2) of Preparation of infrared absorption/emission nanoparticles” were dispersed in 13.4 mL of tetrahydrofuran to prepare a nanoparticle solution, 100 mg of dopamine hydrochloride was dispersed in 600 uL of distilled water, and then added to the nanoparticle solution to form a mixed nanoparticle solution, and the mixed nanoparticle solution was then maintained at 50° C. for 5 hours under an argon gas. The mixed nanoparticle solution was cooled naturally, and 16 uL of hydrochloric acid was then added. Thereafter, the resulting mixture was washed twice with distilled water to obtain nanoparticles (NH2-UCNPs) having an amine group.
(2) Antibody Binding (Formation of Antibody-Nanoparticle Composites)
First, 62 ug of a monoclonal anti-MS2 bacteriophage antibody (a first antibody for capturing MS2 viruses) or a monoclonal anti-AIV nucleoprotein antibody (a first antibody for capturing a nucleoprotein of avian influenza virus) was added to 1 uL of a solution formed by mixing 1.0 mg of N-succinimidyl-S-acetyl-thioacetate (SATA), 86 uL of dimethyl sulfoxide, and 611 uL of 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and reacted at room temperature for 30 minutes, and 1 uL of a solution of 0.5 M hydroxylamine hydrochloride was added, and reacted for another 2 hours. Thereafter, the materials remaining after the reaction were removed using a 30 k filter tube to obtain a thiolated antibody. 0.25 mg of the nanoparticle having an amine group prepared in the section “Preparation of capture agent (antibody)-nanoparticle composites”, 291 uL of distilled water, and 3.7 uL of a 10 mM HEPES buffer solution were mixed to prepare a first solution, and 2.5 mg of sulfosuccinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulfo-SMCC) was added to 100 uL of a 10 mM HEPES buffer solution to prepare a second solution. 1 uL of the second solution was mixed with the first solution, and reacted for 2 hours. Thereafter, the materials remaining after the reaction were removed using a 30 k filter tube to obtain maleimided nanoparticles. The thiolated antibody and the maleimided nanoparticles were added to a HEPES buffer solution, reacted at 4° C. for 24 hours, and then centrifuged to obtain antibody-fixed nanoparticles (antibody-nanoparticle composites).
—Preparation of Diagnosis Kit Using Antibody-Nanoparticle Composites—
A sampling pad was fully immersed in a 10 mM HEPES buffer solution containing 0.3% (w/v) polyvinylpyrrolidone (PVP) or a 10 mM HEPES buffer solution containing 0.3% (w/v) PVP, 2.0% (w/v) bovine serum albumin (BSA), 2.0% (w/v) Tween 20, and 2.5% (w/v) sucrose, dried completely, and cut into pieces with a size of 8 mm×10 mm. The absorption pad was used after moisture was removed. A nitrocellulose membrane was laminated on a plastic card (a supporter) using a laminator, and a second antibody (A polyclonal anti-MS2 antibody in the case of MS2 viruses, and an anti-nucleoprotein antibody having an epitope different from the first antibody in the case of avian influenza virus), which reacted with an antigen included in the specimen, and a third antibody (an anti-rabbit antibody in the case of MS2 viruses, and an anti-mouse antibody in the case of avian influenza virus), which reacted with the first antibody fixed in the antibody-nanoparticle composites, were dispensed in regions of the test line T and the control line C, respectively, using an automatic dispenser, and then dried at room temperature for 48 hours. The conjugation pad was fully immersed in a 10 mM HEPES buffer solution containing 2.0% (w/v) bovine serum albumin (BSA), 2.0% (w/v) Tween 20, 2.5% (w/v) sucrose, and 0.3% (w/v) PVP, and dried using a dryer, and the prepared solution of antibody-nanoparticle composites was dispensed, completely dried in a dryer, and then used.
(2) The sampling pad, the conjugation pad, the membrane positioned on the supporter, and the absorption pad prepared above were overlapped and fixed, as shown in
Although exemplary embodiments of the present invention have been described above in detail, the exemplary embodiments are not intended to limit the scope of the present invention, and various changes and modifications made by those skilled in the art to which the present invention pertains also fall within the scope of the present invention.
Number | Date | Country | Kind |
---|---|---|---|
10-2020-0120625 | Sep 2020 | KR | national |