WO2021145580A1 - Dispositif de détection d'une substance chimique et procédé de détection d'une substance chimique - Google Patents

Dispositif de détection d'une substance chimique et procédé de détection d'une substance chimique Download PDF

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Publication number
WO2021145580A1
WO2021145580A1 PCT/KR2020/018928 KR2020018928W WO2021145580A1 WO 2021145580 A1 WO2021145580 A1 WO 2021145580A1 KR 2020018928 W KR2020018928 W KR 2020018928W WO 2021145580 A1 WO2021145580 A1 WO 2021145580A1
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chemical
detector
aptamer
detection signal
light
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PCT/KR2020/018928
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English (en)
Korean (ko)
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손아정
김다빈
채빌리
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이화여자대학교 산학협력단
고려대학교 산학협력단
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Publication of WO2021145580A1 publication Critical patent/WO2021145580A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks

Definitions

  • the present invention relates to a chemical detection device and a chemical detection method.
  • Plastics Since its invention in the early 1900s, plastics have been widely used in our daily lives. Plastics are used in all fields because they have excellent durability and can be molded into various shapes.
  • plasticizers behave similarly to hormones when absorbed into the human body, which may confuse the endocrine system.
  • a plasticizer is a material that is added to a thermoplastic such as polyvinyl chloride (PVC) to increase the mechanical strength of the plastic. These plasticizers may leach or volatilize into surrounding materials such as water. Plasticizers present in surrounding substances such as water can be transported to humans along the food chain.
  • PVC polyvinyl chloride
  • a detection method that can determine whether chemical substances such as plasticizers are present is needed.
  • a detection method and a detection device that can quickly and accurately detect a chemical.
  • An object of the present invention is to provide a chemical substance detection apparatus and a chemical substance detection method capable of rapidly and accurately detecting a chemical substance.
  • a detector capable of binding to a chemical in a sample; and a detector configured to detect a detection signal generated from the detector, wherein the detector detects a rate of change in intensity of the detection signal generated from the detector when the detector binds to the chemical in the sample to detect a change in the intensity of the detection signal in the sample.
  • a chemical detection apparatus is provided, wherein the amount of the chemical is determined, and the rate of change of the detection signal intensity is measured before the reaction between the chemical and the probe reaches an equilibrium state.
  • the sensing unit comprises: a light source unit irradiating light of a second wavelength band different from the first wavelength band toward the detector; and a photosensitive unit configured to receive the light of the first wavelength band and sense the size of the light of the first wavelength band.
  • the detector may include an aptamer capable of binding to the chemical; and a fluorescent material, wherein the detection signal is the intensity of light in a first wavelength band generated by the fluorescent material.
  • the fluorescent material is provided in combination with the aptamer, and when the chemical material and the detector are combined, the fluorescent material is dissociated from the detector. do.
  • an apparatus for detecting a chemical wherein the chemical includes phthalic compounds.
  • a chemical detection device wherein the detector specifically binds to a phthalate compound.
  • a method for detecting a chemical is provided, wherein an intersubstance reaction is measured before equilibrium is reached.
  • the fluorescent material is provided in combination with the aptamer, and when the chemical material and the detector are combined, the fluorescent material is dissociated from the detector, there is provided a method for detecting a chemical substance do.
  • the detection signal is the intensity of light in a first wavelength band generated by the fluorescent material.
  • a method for detecting a chemical wherein the chemical includes phthalic compounds.
  • a target chemical to be detected can be quickly and accurately detected using the chemical detection apparatus and the chemical detection method.
  • a phthalate compound can be detected in a non-equilibrium state, and thus the phthalate compound can be rapidly detected.
  • FIG. 1 is a perspective view showing a chemical detection apparatus according to an embodiment of the present invention.
  • FIG. 2 is a flowchart illustrating a method for detecting a chemical according to an embodiment of the present invention.
  • FIG. 3 is a graph showing the advantageous effect of the chemical detection apparatus according to an embodiment of the present invention.
  • 4A and 4B are graphs showing the composition of a detector in a chemical detection apparatus according to an embodiment of the present invention.
  • 5A to 5C are graphs showing the chemical substance detection performance of the chemical substance detection apparatus according to an embodiment of the present invention.
  • 6A and 6B are graphs showing chemical substance detection performance of a chemical substance detection apparatus according to an embodiment of the present invention.
  • FIG. 7A to 7F are graphs illustrating chemical substance detection performance of a chemical substance detection apparatus according to an embodiment of the present invention.
  • an apparatus for detecting a chemical and a method for detecting a chemical using a specific binding between a detector and a target chemical The chemical substance detection apparatus and the chemical substance detection method according to an embodiment of the present invention can detect a chemical quickly because the detection operation can be completed before the reaction between the detector and the target chemical reaches an equilibrium state.
  • FIG. 1 is a perspective view showing a chemical detection apparatus according to an embodiment of the present invention.
  • the chemical substance detection device 10 may be used to determine whether a specific chemical substance 21 is present in the sample 20 .
  • the chemical detection device 10 may be an environmental sample such as an air sample, a water sample, or a soil sample.
  • the sample 20 may be provided in a liquid phase.
  • the solid or gaseous sample 20 it may be provided in a dissolved form in a solvent.
  • the chemical substance 21 that can be detected by the chemical substance detection device 10 may be an additive used in plastics.
  • the chemical substance 21 may be a plasticizer, and among the plasticizers, it may be a phthalate compound.
  • Phthalate compounds include ester compounds of phthalic acid.
  • the chemical substance 21 may include, for example, the substances in Table 1 below.
  • the above-mentioned phthalate compounds contained in the chemical (21) can cause endocrine disruption. Phthalate compounds can cause various diseases such as decreased male fertility, diabetes, decreased insulin resistance, breast cancer, and obesity. Since the phthalate compound contained in the chemical substance 21 is not particularly chemically combined with a host plastic such as polyvinylchloride, it can be easily extracted and spread by heating or leaching with an organic solvent. Accordingly, there is a high possibility that phthalate compounds are present in sewage or air. Since the phthalate compound has a great adverse effect on the human body, a treatment for removing the phthalate compound is required, and for this, a rapid detection device/method for checking whether the phthalate compound is present in the sample 20 is required.
  • Chemical detection device 10 includes the detector 100 and the detector 200 for detecting the chemical substance 21 in the sample 20 described above.
  • the detector 100 binds specifically to the chemical substance 21, It is used to check whether the chemical substance 21 is present in the sample 20 .
  • the detector 100 emits a detection signal, and when the detector 100 is combined with the chemical substance 21 , the intensity of the detection signal changes. The presence and amount of the chemical substance 21 may be determined by detecting the change rate of the detection signal intensity.
  • the detector 100 may be provided in a dissolved state in the sample 20 .
  • the detector 100 may be provided in the form of, for example, a liquid reagent, added to the sample 20 and homogeneously mixed with the sample 20 . Since the detector 100 is homogeneously mixed in the sample 20 , a reaction between the chemical substance 21 and the detector 100 present in the sample 20 may occur more rapidly.
  • the detector 100 may include an aptamer 110 and a fluorescent material 120 .
  • the aptamer 110 may be an oligonucleotide or peptide molecule capable of binding to a specific target molecule.
  • the aptamer 110 may have various forms, such as a nucleic acid aptamer such as a DNA aptamer, an XNA aptamer, and an RNA aptamer, or a peptide aptamer.
  • the aptamer 110 may include an aptamer support and an aptamer tag.
  • the aptamer support may form the backbone of the aptamer 110
  • the aptamer tag may specifically bind to the target chemical 21 .
  • the aptamer 110 may specifically bind to, for example, a phthalate compound. Accordingly, even when various substances are mixed together with the chemical substance 21 to be the target in the sample 20, the aptamer 110 can specifically bind to the chemical substance 21 including the phthalate compound. there is. Therefore, the chemical substance detection apparatus according to an embodiment of the present invention has excellent sensitivity for detecting the chemical substance 21 .
  • the aptamer tag is a ribose position, a deoxyribose position, a phosphate position, and a base position of the aptamer 110 . It may be provided at at least one position independently from the (base position).
  • the aptamer 110 and the chemical 21 also have 2'-position sugar modification, 2'-amino (2'-NH2), 2'-fluoro in the above-described aptamer tag.
  • the above-described content of the type of the aptamer 110 , the position of the aptamer tag, and the coupling between the aptamer 110 and the chemical substance 21 are merely exemplary.
  • the type of the aptamer 110 may vary depending on the type of the target chemical 21 , the position of the aptamer tag and the type of bonding between the aptamer 110 and the chemical 21 may also vary.
  • the aptamer 110 may also bind the fluorescent material 120 . Accordingly, the detector 100 mixed with the sample 20 may be provided in a form in which the aptamer 110 and the fluorescent material 120 are combined.
  • the fluorescent material 120 may be a material emitting light of a specific wavelength band.
  • the fluorescent material 120 may be a material that receives light of a first wavelength band and emits light of a second wavelength band different from the first wavelength band.
  • the light of the second wavelength band emitted by the fluorescent material 120 may be used as a detection signal for confirming the presence and amount of the chemical material 21 .
  • the first wavelength band and the second wavelength band do not refer to a specific wavelength band, but refer to an arbitrary band. Accordingly, the first wavelength band and the second wavelength band have only the meaning that they are different from each other.
  • the fluorescent material 120 may be a material that emits light in the above-described second wavelength band only in a state in which it is combined with the aptamer 110 .
  • the fluorescent material 120 may receive light of a first wavelength band and emit light of a second wavelength band in a state coupled to the aptamer tag of the aptamer 110 .
  • light of the second wavelength band may not be emitted from the fluorescent material 120 . Accordingly, the intensity of light in the second wavelength band may vary depending on the amount of binding between the fluorescent material 120 and the aptamer 110 , and this may be used as a detection signal.
  • the fluorescent material 120 may be surrounded by hydrophilic molecules present in the sample 20 after dissociation from the aptamer 110 .
  • the fluorescent material 120 surrounded by the hydrophilic molecule may not emit light in the second wavelength band. Accordingly, even if the dissociated fluorescent material 120 remains without being removed from the sample 20 , light in the second wavelength band is emitted from the dissociated fluorescent material 120 , and there is no risk of being caught as noise in the detection result of the chemical detection device. .
  • Both the fluorescent material 120 and the chemical material 21 may be bound to an aptamer tag, which is a specific binding site of the aptamer 110 .
  • the affinity between the chemical substance 21 and the aptamer tag may be stronger than the binding force between the fluorescent substance 120 and the aptamer tag. Accordingly, when the combination of the fluorescent material 120 and the aptamer 110 meets the chemical material 21, the aptamer 110 and the fluorescent material 120 are dissociated and the chemical material 21 can be bound thereto. there is. Accordingly, the aptamer-chemical compound 100' may be formed.
  • the intensity of the detection signal may be changed by the substitution reaction between the fluorescent material 120 and the chemical material 21 .
  • the amount of the fluorescent material 120 combined with the aptamer 110 decreases, and accordingly, the second wavelength band emitted by the fluorescent material 120 .
  • the intensity of the light may also be reduced.
  • the amount of the chemical substance 21 in the sample 20 may be calculated by checking the amount of change in the intensity of light in the second wavelength band.
  • the fluorescent material 120 may be a material having a lower affinity for the aptamer tag than the chemical material 21 while being able to specifically bind to the aptamer 110 .
  • the fluorescent material 120 satisfying the above-described physical properties may include the compound of Formula 1 below or PoPo3.
  • the fluorescent material 120 When the fluorescent material 120 includes the compound of Formula 1 described above, the fluorescent material 120 receives light of a wavelength of about 520 nm to about 540 nm, and emits light of a wavelength of about 560 nm to about 580 nm. can Accordingly, the amount of the chemical substance 21 in the sample 20 can be calculated by checking the amount of change in the intensity of light having a wavelength of about 560 nm to about 580 nm.
  • the chemical detection apparatus 10 includes a detection unit 200 .
  • the sensing unit 200 detects a detection signal generated from the fluorescent material 120 as described above.
  • the detection unit 200 may specifically detect the amount of change in the detection signal.
  • the amount of change in the detection signal may be detected in the form of a change in the intensity of light of a specific wavelength or a change in the magnitude of a voltage generated by light of a specific wavelength.
  • the sensing unit 200 may include a light source unit 210 and a photosensitive unit 220 to perform the above-described function.
  • the light source unit 210 may irradiate light of the first wavelength band toward the probe 100 mixed with the sample 20 .
  • the energy level of electrons included in the fluorescent material 120 may be increased by the light of the first wavelength band. As electrons located at a high energy level fall to a low energy level, light of a second wavelength band different from the first wavelength band may be emitted.
  • the light source unit 210 may include at least one light source emitting light of the first wavelength band.
  • the light source may be in the form of, for example, a light emitting diode (LED), an organic light emitting diode (OLED), or the like.
  • LED light emitting diode
  • OLED organic light emitting diode
  • the emitted light of the first wavelength band may have a relatively narrow wavelength width. Accordingly, the first wavelength band of the light emitted from the light source unit 210 and the second wavelength band of the light emitted from the fluorescent material 120 overlap, so that noise is not generated.
  • the light source unit 210 may emit light having a wavelength of, for example, about 520 nm to about 540 nm.
  • the light source unit 210 may include a helium (He)-neon (Ne) light source.
  • He helium
  • Ne helium
  • the above-described wavelength band and types of light sources emitted by the light source unit 210 are merely exemplary.
  • the light source unit 210 may include a plurality of light sources capable of emitting light of different wavelength bands in some cases.
  • the plurality of light sources emitting light of different wavelength bands may be independently driven according to the type of the fluorescent material 120 to irradiate light of a wavelength band capable of being received by the fluorescent material 120 .
  • the light source unit 210 may be provided on one side of the container in which the sample 20 is provided.
  • the light source unit 210 may be provided as a lid covering the top of the flask.
  • the light source unit 210 may be detachably provided on the side or bottom surface of the container so that light can reach the sample 20 through the transparent flask outer wall.
  • the shape of the light source unit 210 is not particularly limited.
  • the fluorescent material 120 that receives the light of the first wavelength band irradiated by the light source unit 210 emits light of the second wavelength band. Light of the second wavelength band emitted from the fluorescent material 120 may be detected by the photosensitive unit 220 .
  • the photosensitive unit 220 may absorb and sense light of the second wavelength band.
  • the photosensitive unit 220 may include, for example, a photodiode. Accordingly, the photosensitive unit 220 may absorb the light of the second wavelength band and generate a current in proportion to the intensity of the light of the second wavelength band. That is, as the intensity of light in the second wavelength band increases, the magnitude of the current (voltage) generated from the photodiode may increase.
  • the photosensitive unit 220 measures the intensity of the light of the second wavelength band or the magnitude of the voltage generated by the light of the second wavelength band at the first time point, and measures the intensity of the second wavelength band at a second time point after the first time point.
  • the amount of change in the detection signal may be measured by measuring the intensity of light or the magnitude of a voltage generated by light of the second wavelength band.
  • the detection signal change amount may be determined by Equation 1 below.
  • F high means the intensity of the light of the second wavelength band emitted by the fluorescent material 120 at the first time point T high or the magnitude of the voltage generated by the light of the second wavelength band
  • Flow denotes the intensity of the light of the second wavelength band emitted by the fluorescent material 120 at the second time point T low after the first time point T high or the magnitude of the voltage generated by the light of the second wavelength band .
  • the first time point T high and the second time point T low may have a time difference of about 10 seconds to about 5 minutes.
  • the chemical detection apparatus 10 does not measure the magnitude of the detection signal at any time point, but measures the amount of change in the detection signal magnitude. Accordingly, the intensity of the light of the second wavelength band or the magnitude of the voltage generated by the light of the second wavelength band may be measured repeatedly at relatively short intervals. In addition, it is possible to quickly measure the presence and amount of the chemical substance 21 by measuring the intensity of the light of the second wavelength band or the magnitude of the voltage generated by the light of the second wavelength band at short intervals as described above. . More details on this will be described later.
  • the photosensitive unit 220 measures the amount of change in the detection signal magnitude, it is possible to check the presence and amount of the chemical substance 21 before the reaction between the detector 100 and the chemical substance 21 is balanced.
  • the detection procedure was performed in the order of confirming the amount after separating the specific binding body of the detection substance-chemical substance 21 . This method has a problem in that rapid chemical detection is impossible because the reaction must wait until equilibrium is reached.
  • the presence and amount of the chemical can be quickly checked before the reaction reaches equilibrium. Accordingly, the time required for detecting the chemical substance 21 can be significantly reduced.
  • the photosensitive unit 220 may be provided on one side of a container in which the sample 20 is provided.
  • the photosensitive unit 220 may be provided as a lid covering the top of the flask.
  • the photosensitive unit 220 and the light source unit 210 are thus provided adjacent to each other and can operate sequentially. For example, after the light source unit 210 operates for a first time, the operation of the light source unit 210 may be stopped and then the photosensitive unit 220 may be operated. Through this, it is possible to prevent the light emitted from the light source unit 210 from being directly detected by the photosensitive unit 220 .
  • the sensing unit 200 may further include a processor, a memory, and the like in some cases. They may perform calculation operations such as calculating the amount of change in the magnitude of the detection signal detected by the photosensitive unit 220 and calculating the amount of the chemical substance 21 from the change in the magnitude of the detection signal.
  • the chemical detection apparatus 10 may be operated with only a simple configuration including the detection body 100 and the detection unit 200 . Accordingly, the chemical detection apparatus 10 can be miniaturized and highly portable.
  • the chemical detection apparatus 10 measures the amount of change in the detection signal intensity according to the specific binding between the detection object 100 and the chemical substance 21 , thereby measuring the detection object 100 and the chemical substance 21 . ) can complete the detection of the chemical 21 before the reaction reaches equilibrium. Accordingly, very rapid chemical substance 21 detection is possible.
  • the chemical detection device 10 is very excellent in portability by including a simple configuration. Therefore, by using the chemical substance detection apparatus 10 according to an embodiment of the present invention, it is possible to determine whether the chemical substance 21 is present at the sample collection site.
  • FIG. 2 is a flowchart illustrating a method for detecting a chemical according to an embodiment of the present invention.
  • a first step (S100) of mixing a detector and a sample, a second step (S200) of detecting a detection signal, and a detection signal change amount are calculated and a third step (S300) of confirming the amount of the chemical based on this.
  • the detector may be first prepared prior to mixing the detector and the sample.
  • the detector may be prepared by mixing and reacting the aptamer and the fluorescent material so that the aptamer and the fluorescent material are combined.
  • the aptamer and the fluorescent material may be mixed in a ratio of about 0.9:1 to 1.1:1 by volume.
  • the next prepared detector may be mixed with the sample.
  • the sample may be provided in a liquid form dissolved by a polar solvent such as water.
  • a polar solvent such as water.
  • chemicals in the sample may bind to the detector.
  • the contents are the same as described above.
  • the photosensitive unit of the sensing unit detects the detection signal.
  • the detection signal may be the intensity of light in the second wavelength band or the magnitude of a voltage generated from the second wavelength band. The contents are the same as described above.
  • a third step (S300) is performed.
  • the detection signal change amount is measured.
  • the detection signal change amount is an amount by which the detection signal amplitude changes according to the elapsed time after the detector is mixed in the sample.
  • the contents are the same as described above.
  • the amount of change in the detection signal magnitude is checked before the reaction between the detector and the chemical in the sample reaches equilibrium.
  • a detector To prepare a detector, 20 ⁇ L of PoPo3 having a concentration of about 10 ⁇ M and about 20 ⁇ L of an aptamer having a concentration of about 25 ⁇ M were mixed. The mixture of the fluorescent substance and the aptamer was again dissolved in about 160 ⁇ L of about 0.02 M Tris-HCl (Tris-HCl) buffer. In this case, the aptamer was used as a single-stranded DNA having the sequence of 5'-CTT TCT GTC CTT CCG TCA CAT CCC ACG CAT TCT CCA CAT-3'.
  • FIG. 3 is a graph showing the advantageous effect of the chemical detection apparatus according to an embodiment of the present invention.
  • a buffer solution about 160 ⁇ L of about 0.02 M Tris-HCl buffer
  • a mixed solution of the buffer and the fluorescent material about 20 ⁇ L of about 10 ⁇ M of the fluorescent material and About 160 ⁇ L of about 0.02 M Tris-hydrochloric acid (Tris-HCl) buffer mix
  • the fluorescence signal change ( ⁇ RFU 561 ) of the mixed solution of Example 1 with time ( ⁇ RFU 561 ) was measured.
  • the fluorescence of the mixed solution was measured using a SpectraMax M2 spectrofluorometer.
  • the range of the mixed solution of the buffer and the fluorescent material is that the detection signal has a size of about 42.43 ⁇ 0.80 to about 47.16 ⁇ 1.42, and the detection signal size of the mixed solution according to Example 1 is about 65.80 ⁇ 5.33 to about It was confirmed that it was lower than 72.18 ⁇ 4.75.
  • the hydrophobic dye material not bound to the aptamer is surrounded by the hydrophilic molecules of the buffer, and thus the fluorescent material does not emit a detection signal (light in the second wavelength band). Therefore, according to an embodiment of the present invention, it is possible to accurately measure the amount of change in the detection signal without rinsing the fluorescent material that is dissociated from the aptamer and exists in the sample.
  • 4A and 4B are graphs showing the magnitude of the fluorescence signal according to the composition of the detector in the chemical detection apparatus according to an embodiment of the present invention.
  • the fluorescent material and the aptamer were mixed at various ratios. Specifically, various concentrations of PoPo3 dye (final concentrations 0, about 0.1 ⁇ M, about 1 ⁇ M, about 5 ⁇ M and about 10 ⁇ M) and various concentrations of aptamers (final concentrations 0, about 0.05 ⁇ M, 0.25, 0.5 and 2.5 ⁇ M) ) was mixed with the buffer (about 0.02 M Tris-HCl buffer) to make a total of about 200 ⁇ L. The mixed solution was incubated at room temperature for about 3 minutes without shaking. The fluorescence signal of the mixed solution was measured using a SpectraMax M2 spectrofluorometer.
  • the fluorescence signal (RFU 561nm ) of the mixed solution increased in proportion to the increase in the concentration of the added PoPo3.
  • the coefficient of determination (r2) was obtained between the fluorescence of PoPo3 and the concentration of PoPo3 with respect to the concentration of aptamer.
  • the coefficients of measurement (r2) were 0.88, 0.92, 0.95 and 0.98, respectively.
  • 5A to 5C are graphs showing the chemical substance detection performance of the chemical substance detection apparatus according to an embodiment of the present invention.
  • the CD spectrum before and after binding to the chemical of the single-stranded DNA aptamer was analyzed.
  • About 30 ⁇ L of a phthalate compound was used as a chemical in this example.
  • the aptamer one having the sequence of 5'-CTT TCT GTC CTT CCG TCA CAT CCC ACG CAT TCT CCA CAT-3' was used.
  • the CD spectrum in Figure 5a shows that the aptamer has a typical B-form DNA with a negative band and a positive band at about 240 nm and about 280 nm, respectively. However, it can be seen that the CD spectrum changes at about 280 nm, as indicated by the arrow after the reaction with the phthalate compound. After the addition of about 1 ⁇ g/L of the phthalate compound, the positive CD band at about 280 nm decreased from 0.749 (control: aptamer only) to 0.391. In addition, after the addition of about 10 ⁇ g/L of the phthalate compound, the positive CD band at about 280 nm decreased to about 0.414.
  • the chemical substance detection limit of the chemical substance detection apparatus and/or the chemical substance detection method according to an embodiment of the present invention can be confirmed.
  • a mixed solution (about 40 ⁇ L) consisting of about 1 ⁇ M PoPo3 and about 2.5 ⁇ M aptamer was transferred to a microplate and mixed with about 140 ⁇ L of about 0.02 M Tris-HCl buffer. After reacting PoPo3 with the aptamer for about 3 minutes, 20 ⁇ L of phthalate compounds of various concentrations (about 0.1 ⁇ g/L, about 1 ⁇ g/L, about 5 ⁇ g/L, about 50 ⁇ g/L, and about 200 ⁇ g/L) was added to the microplate.
  • Phthalate compounds include phthalic acid (PA), dimethyl phthalate (DMP), diethyl phthalate (DEP), di-n-butyl phthalate (DBP), diisobutyl phthalate (DIBP), benzyl butyl phthalate (BBP) and di-2- ethylhexyl.
  • PA phthalic acid
  • DMP dimethyl phthalate
  • DEP diethyl phthalate
  • DBP di-n-butyl phthalate
  • DIBP diisobutyl phthalate
  • BBP benzyl butyl phthalate
  • di-2- ethylhexyl di-2- ethylhexyl.
  • the fluorescence of PoPo3 was measured by a SpectraMax M2 spectrofluorimeter at 2-min intervals for 30 min.
  • the decrease in the magnitude of the fluorescence signal of PoPo3 was normalized as the rate of change of fluorescence (- ⁇ RFU 561/ ⁇ t).
  • the fluorescence signal (RFU 561nm ) of PoPo3 decreased in size over time. This decrease is because the affinity of the phthalate compound for the aptamer is stronger than that of PoPo3 for the aptamer. Specifically, over time, PoPo3 bound to the aptamer was replaced with a phthalate compound.
  • the limit of quantitative analysis (LOQ) of the phthalate compound is about 0.1 ⁇ g/L. This covers the concentration of phthalate compounds normally contained in the aquatic environment, from about 0.1 ⁇ g/L to about 1472 ⁇ g/L. Therefore, it can be seen that the chemical detection apparatus/method according to an embodiment of the present invention provides a reliable detection range of phthalate in an aquatic environment.
  • 6A and 6B are graphs showing chemical substance detection performance of a chemical substance detection apparatus according to an embodiment of the present invention.
  • the aptamer specifically binds to the target chemical even when a chemical other than the target chemical is present in the sample.
  • target chemical phthalate compounds PA, DMP, DEP, DBP, DIBP, BBP and DEHP
  • non-phthalate compounds bisphenol A and 4-nonyl phenol
  • the probe was prepared by mixing about 1 ⁇ M PoPo3 and about 2.5 ⁇ M aptamer with about 140 ⁇ L of about 0.02 M Tris-HCl buffer. Next, a phthalate compound and a non-phthalate compound were respectively added to the mixed solution of the detector.
  • the fluorescence change rate was about 77% both when BPA was added and when 4-NP was added. This is a value that is significantly different from 100% of the fluorescence change when the 7 phthalate compounds are added or the fluorescence change when the phthalate compound is added.
  • the chemical substance detection apparatus and/or chemical substance detection method according to an embodiment of the present invention can specifically detect a target chemical substance.
  • FIG. 7A to 7F are graphs illustrating chemical substance detection performance of a chemical substance detection apparatus according to an embodiment of the present invention.
  • the presence or absence of a target chemical can be checked by measuring the amount of change in voltage.
  • the change amount of the voltage may be set in proportion to the change amount of the fluorescence signal. This is because the photodiode can convert the fluorescence signal into current/voltage as described above.
  • phthalate compounds at various concentrations (about 1 ⁇ g/L, about 10 ⁇ g/L, about 50 ⁇ g/L, and about 100 ⁇ g/L) were added to the cuvette so that the total volume was about 300 ⁇ L. made to be
  • the cuvette was placed in the cuvette holder of the analyzer and covered with an opaque cover to prevent ambient light from entering the cuvette. Then, the analyzer was set to integrate the voltage for about 30 seconds, about 1 minute, about 3 minutes, and about 5 minutes in a steady state.
  • the LED light source voltage of the analyzer was constant at about 8.6V during the measurement time. Results were automatically recorded by a data logger (MadgeTech 4, MadgeTech Inc., Merrimack County, NH).
  • the output voltages of the blue and green photodiodes were recorded as CH1 and CH2 respectively by the data recorder. The output voltage was measured three times.
  • the output voltage change ( ⁇ Vout / ⁇ t) gradually decreased over the concentration range of the phthalate compound (about 1 ⁇ g/L to about 100 ⁇ g/L) in the figure.
  • the method for detecting a chemical according to an embodiment of the present invention can be performed within about 30 seconds.
  • the chemical substance detection method according to an embodiment of the present invention can be performed within a significantly shorter detection time than the conventional chemical substance detection method.
  • Phthalate group detection the present invention PA, DMP, DEP, DBP, DIBP, BBP, DEHP 30 sec 0.1-200 Immunoassay DMP, DEP, DBP, BBP, DEHP, DCHP, DnOP, DNP 0.67 h 0.01-100 Individual phthalate detection Immunoassay DEHP ⁇ 6 h 0.001-1,000 DEHP DIBP ⁇ 18 h 10-357 DiBP DBP ⁇ 17 h 0.1-100 DBP DBP ⁇ 21 h 5-250 DBP Apta-sensor DEHP ⁇ 3 h 0.0039-39 DEHP DEHP ⁇ 6 h 0.0005-100 DEHP Other sensors DBP ⁇ 0.5 h 1,391-13,917 DBP DEHP ⁇ 2 h 781-7,030 DEHP

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Abstract

Un mode de réalisation de la présente invention concerne un dispositif de détection de substance chimique comprenant : une sonde qui peut se lier à une substance chimique dans un échantillon ; et une unité de détection qui détecte un signal de détection généré à partir de la sonde, l'unité de détection détectant un taux de variation de l'intensité du signal de détection, qui est généré à partir de la sonde lorsque la sonde se lie à la substance chimique dans l'échantillon, pour déterminer la quantité de substance chimique dans l'échantillon, et le taux de variation de l'intensité du signal de détection est mesuré avant que la réaction entre la substance chimique et la sonde atteigne un état d'équilibre.
PCT/KR2020/018928 2020-01-15 2020-12-22 Dispositif de détection d'une substance chimique et procédé de détection d'une substance chimique WO2021145580A1 (fr)

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KR20180089974A (ko) * 2017-02-02 2018-08-10 한양대학교 산학협력단 프탈레이트(phthalate)에 특이적으로 결합하는 단일가닥 DNA 압타머

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KR20100093502A (ko) * 2009-02-16 2010-08-25 동국대학교 산학협력단 압타머를 이용한 타겟물질의 검출방법
KR20110004961A (ko) * 2009-07-09 2011-01-17 피씨엘 (주) 초상자성나노입자를 이용한 타겟물질의 표지 및 검출방법
KR20180089326A (ko) * 2017-01-31 2018-08-08 한양대학교 산학협력단 비색-형광 혼합 모드를 이용한 환경호르몬 검출 방법
KR20180089974A (ko) * 2017-02-02 2018-08-10 한양대학교 산학협력단 프탈레이트(phthalate)에 특이적으로 결합하는 단일가닥 DNA 압타머

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114397282A (zh) * 2021-12-24 2022-04-26 江苏鑫蓝鑫生物科技有限公司 一种用核酸适配体和g-四链体免标记荧光分析法检测氯霉素的方法
CN114397282B (zh) * 2021-12-24 2023-11-17 江苏鑫蓝鑫生物科技有限公司 一种用核酸适配体和g-四链体免标记荧光分析法检测氯霉素的方法

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