WO2023033582A1 - Method for detecting food-noxious substance - Google Patents

Method for detecting food-noxious substance Download PDF

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WO2023033582A1
WO2023033582A1 PCT/KR2022/013171 KR2022013171W WO2023033582A1 WO 2023033582 A1 WO2023033582 A1 WO 2023033582A1 KR 2022013171 W KR2022013171 W KR 2022013171W WO 2023033582 A1 WO2023033582 A1 WO 2023033582A1
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nucleotide sequence
capture probe
food
metal body
chip
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French (fr)
Korean (ko)
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우민아
최성욱
임민철
장현주
김태용
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한국식품연구원
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
    • C12Q2525/10—Modifications characterised by
    • C12Q2525/205—Aptamer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2531/00—Reactions of nucleic acids characterised by
    • C12Q2531/10—Reactions of nucleic acids characterised by the purpose being amplify/increase the copy number of target nucleic acid
    • C12Q2531/125—Rolling circle
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
    • C12Q2563/107—Nucleic acid detection characterized by the use of physical, structural and functional properties fluorescence
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
    • C12Q2563/137—Metal/ion, e.g. metal label
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
    • C12Q2563/155—Particles of a defined size, e.g. nanoparticles

Definitions

  • the present invention relates to a method for detecting substances harmful to food, and more particularly, an aptamer specific to a substance harmful to food has a selective molecular affinity and binds to a capture probe immobilized on a chip when the substance harmful to food does not exist.
  • an aptamer specific to a substance harmful to food has a selective molecular affinity and binds to a capture probe immobilized on a chip when the substance harmful to food does not exist.
  • it detaches from the capture probe and combines with the food hazardous substance, so circular DNA binds to the capture probe and RCA (rolling circle amplification) reaction occurs to form a metal body for signal amplification, thereby improving detection accuracy. It is about a food harmful substance detection method that can be improved.
  • Pesticides which are harmful substances contained in typical foods, are chemicals that are sprayed to prevent crops from being damaged by weeds, pests, bacteria, etc. during farming. It remains in rivers and causes damage to the surrounding environment and people. Therefore, devices and methods for detecting pesticide residues have been widely developed, as shown in the following patent documents.
  • Patent No. 10-1050421 registered on July 13, 2011
  • Patent No. 10-1050421 registered on July 13, 2011
  • the present invention has been made to solve the above problems,
  • an aptamer specific to a food hazardous substance has a selective molecular affinity and binds to a capture probe fixed on a chip when a food hazardous substance is not present, but deviates from the capture probe when a food hazardous substance is present and is harmful to food. Since it binds to substances, it is possible to form a metal body for signal amplification by binding circular DNA to the capture probe to cause an RCA reaction, and the purpose is to provide a method for accurately detecting food harmful substances.
  • the present invention is implemented by an embodiment having the following configuration in order to achieve the above object.
  • the food hazardous substance detection method according to the present invention is a base sequence that is immobilized on a substrate and serves as a linker, and a base that enables complementary binding with an aptamer having selective molecular affinity for pesticides.
  • RCA reaction step of supplying to a chip and conducting RCA reaction to form a single strand having circular DNA partially bound to the capture probe, linked to the capture probe, and having a base sequence repeating to enable complementary binding with the metal body. and; and a metal body formation step of forming a metal body for signal amplification using the single strand formed through the RCA reaction step as a template.
  • the food hazardous substance detection method includes a detection step of irradiating light to the chip after the metal body forming step and measuring an optical signal to detect the presence and amount of pesticides. It is characterized in that it further includes.
  • the nucleotide sequence and the complementary nucleotide sequence enabling complementary binding with the metal body are composed of a plurality of consecutive adenines, so that the strand is formed from a plurality of thymines, and in the metal body formation step, a solution containing copper ions and ascorbate is injected into the chip after the RCA reaction step to form copper nanoparticles using the plurality of thymines as a template.
  • the nucleotide sequence that enables complementary binding with the metal body and the complementary nucleotide sequence enables complementary binding with the gold probe It consists of a nucleotide sequence complementary to the nucleotide sequence, and the strand is formed with a nucleotide sequence that enables complementary binding with the gold probe.
  • the first reactor Reaction of the gold nanoparticles to which the second reactive group binds to forms a gold probe in which gold is bound to a specific ssDNA injects the gold probe into a chip to bind the gold probe to the strand, silver ions and a reducing agent is additionally supplied to form silver clusters on the surface of the gold nanoparticles.
  • the substrate is a COC (cyclic olefin copolymer) substrate
  • the capture probe is a base of SEQ ID NO: 4 to detect diazinon. It is characterized by having a sequence.
  • the circular DNA in the method for detecting harmful substances in food according to the present invention, has the nucleotide sequence of SEQ ID NO: 7.
  • the circular DNA in the method for detecting harmful substances in food according to the present invention, has the nucleotide sequence of SEQ ID NO: 9.
  • the present invention can obtain the following effects by combining and using the above embodiments and configurations to be described below.
  • an aptamer specific to a food hazardous substance has a selective molecular affinity and binds to a capture probe fixed on a chip when a food hazardous substance is not present, but deviates from the capture probe when a food hazardous substance is present and is harmful to food. Since it binds to a material, it is possible to form a metal body for signal amplification by binding the circular DNA to the capture probe and causing an RCA reaction, thereby improving detection accuracy.
  • 1 is a reference diagram for explaining a principle of detecting pesticides according to an embodiment of the present invention.
  • Figure 2 is a graph showing the results of CD analysis for designing the optimal binding nucleotide sequence of the capture probe.
  • Figure 3 is a reference diagram showing the binding relationship between diazinon aptamer and cDNA.
  • 4 and 5 are fluorescence images for confirming the effect of the solvent when DNA having a linker base sequence is immobilized on a COC chip.
  • Figure 6 is a graph showing the fluorescence change for confirming whether the capture probe is immobilized on the COC substrate.
  • 7 and 8 are graphs showing fluorescence changes for confirming RCA efficiency according to the length of the spacer region of the capture probe.
  • TEM 9 is a transmission electron microscope (TEM) image for confirming that poly(T) is produced on a COC substrate and CuNP is synthesized.
  • Figure 11 is a scanning electron microscope (SEM) image for confirming that ssDNA is formed as an RCA product on a COC substrate to synthesize Au@Ag.
  • 12 is a graph showing absorbance measurement results for confirming colorimetric signals according to the size of gold nanoparticles.
  • FIG. 13 is a graph showing results of detecting diazinon using a detection method according to an embodiment of the present invention.
  • FIG. 14 is a graph confirming that the detection method according to an embodiment of the present invention selectively detects diazinon.
  • a method for detecting harmful substances in food will be described with reference to FIGS. 1 to 14 .
  • a pesticide which is a representative example of food harmful substances
  • the method for detecting harmful substances in food is a chip by immobilizing a capture probe containing a nucleotide sequence immobilized on a substrate to act as a linker and a nucleotide sequence enabling complementary binding with an aptamer having selective molecular affinity for pesticides to a substrate.
  • the chip preparation step prepares a chip by immobilizing a capture probe containing a nucleotide sequence immobilized on a substrate to act as a linker and a nucleotide sequence enabling complementary binding with an aptamer having a selective molecular affinity for pesticides to a substrate.
  • the capture probe is distributed on a polymer (eg, COC, etc.) substrate and UV irradiated, the Since T(10)C(10) is immobilized on the polymer substrate surface and the 5' end portion of the capture probe is immobilized on the polymer substrate, the capture probe can be immobilized on the substrate.
  • a polymer eg, COC, etc.
  • the hybridization step is a step of distributing an aptamer having a molecular affinity selective to the pesticide on the chip to partially bind the capture probe and the aptamer, wherein the capture probe fixed to the substrate has a complementary bond with the aptamer. Since it has a nucleotide sequence that enables the capture probe to partially bind to the aptamer when the aptamer is distributed on the chip after the chip preparation step.
  • the aptamer separation step is a step of distributing the pesticide to be measured on the chip after the hybridization step so that the aptamer separates from the capture probe and binds to the pesticide by selective molecular affinity. Since the aptamer has a selective molecular affinity, that is, it has a stronger molecular affinity for the pesticide than the capture probe, when the pesticide is present in the sample, the aptamer separates from the capture probe and binds to the pesticide.
  • a circular DNA comprising a nucleotide sequence enabling complementary binding with the capture probe, a nucleotide sequence enabling complementary binding with the metal body, and a complementary nucleotide sequence, and A polymerase is supplied to the chip and subjected to an RCA reaction so that the circular DNA partially binds to the capture probe and is linked to the capture probe to form a strand having repeating nucleotide sequences enabling complementary binding with the metal body It is a step.
  • the nucleotide sequence enabling complementary bonding with the metal and the complementary nucleotide sequence are composed of a plurality of consecutive adenines, and the strand is composed of a plurality of thymine (poly(T )), and when the metal to be formed is a gold/silver cluster, the nucleotide sequence that enables complementary bonding with the metal body and the complementary nucleotide sequence is the nucleotide sequence that enables complementary bonding with the gold probe It consists of a nucleotide sequence complementary to, and the strand is formed with a nucleotide sequence that enables complementary binding with the gold probe.
  • the metal body forming step is a step of forming a metal body for signal amplification using the strand formed through the RCA reaction step as a template.
  • a metal body for signal amplification using the strand formed through the RCA reaction step as a template.
  • the strand is formed of a plurality of thymine (poly(T))
  • Cu 2+ and ascorbate are injected into the chip to form copper nanoparticles using poly(T) as a template.
  • a second reactor eg, biotin
  • a first reactor eg, biotin
  • a first reactor eg, biotin
  • streptavidin streptavidin
  • the detection step is a step of irradiating light onto the chip after the metal body formation step and measuring an optical signal to detect the presence and amount of pesticide.
  • a base sequence acting as a linker immobilized on a cyclic olefin copolymer (COC) substrate through UV irradiation (hereinafter referred to as 'linker base sequence') [TTTTTTTTTTCCCCCCCCCC (SEQ ID NO: 1)] and a spacer base sequence [TAATCATGATT ( SEQ ID NO: 2)] and a nucleotide sequence (hereinafter referred to as 'binding nucleotide sequence') [GTCCAAGAGC (SEQ ID NO: 3)] that enables complementary binding with Diazinon aptamer.
  • a capture probe [5'-TTTTTTTTTTCCCCCCCCTAATCATGATTGTCCAAGAGC-3' (SEQ ID NO: 4)] was prepared.
  • Diazinon aptamer [5'-ATCCGTCACACCTGCTCTAATATAGAGGTATTGCTCTTGGACAAGGTACAGGGATGGTGTTGGCTCCCGTAT-3' (SEQ ID NO: 6)] containing a nucleotide sequence complementary to the binding nucleotide sequence [GCTCTTGGAC (SEQ ID NO: 5)] to enable complementary binding with the capture probe. prepared.
  • Linear DNA 1 [5'-(phosphate)-GTAGGTGCTCTTGGACGAACATAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACG containing a nucleotide sequence complementary to the binding nucleotide sequence [GCTCTTGGAC (SEQ ID NO: 5) and poly(A) to enable complementary binding with the capture probe -3' (SEQ ID NO: 7)] was prepared.
  • nucleotide sequence complementary to the binding nucleotide sequence [GCTCTTGGAC (SEQ ID NO: 5)] and a nucleotide sequence complementary to the gold probe to enable complementary binding with the capture probe (hereinafter referred to as 'linked nucleotide sequence') ') [AAGCCATCAGTC (SEQ ID NO: 8)] containing linear DNA 2 [5'-(phosphate)-GTAGGTGCTCTTGGACGAACATATTAGCCTTCAGCGCTCCCAAGCCATCAGTCT-3' (SEQ ID NO: 9)] was prepared.
  • Circular DNA 2 (CirDNA) was obtained in the same manner as in Example 1-3 (2) except that linear DNA 2 was used instead of linear DNA 1.
  • CD Chemical dichroism
  • cDNA 1 to 4 complementary to diazinon aptamer were prepared (cDNA 1 [5'-GTTGGACGGA-3' (SEQ ID NO: 10)], cDNA 2 [5' -GCAATACCTC-3' (SEQ ID NO: 11)], cDNA 3 [5'-GTCCAAGAGC-3' (SEQ ID NO: 12)], cDNA 4 [5'-ATACGGGAGC-3' (SEQ ID NO: 13)].
  • Each cDNA binds to different sites (sites 1 to 4) as shown in FIG. 3.
  • the aptamer sites 1 and 2 have similar spectral patterns of samples A and B, so that cDNA is better than DNZ. Since the spectral patterns of samples B and C are similar at aptamer sites 3 and 4, it can be seen that they bind more strongly with DNZ than cDNA, and at aptamer site 3, the aptamer- of sample A Significant spectral pattern differences between the cDNA complex and the aptamer-DZN complex of sample C show a large difference between the three-dimensional structures of the two complexes, indicating that aptamer site 3 is the optimal site for designing the binding sequence. It can be seen that
  • nucleotide sequence acting as a linker [CCCCCCCCTTTTTTTTTTTTTT (SEQ ID NO: 14)] is included and cy3 is attached to the 5' Confirmation of immobilization DNA [5'-(cy3)-TCATGACCCCCCCCTTTTTTTTTTTTTTTT-3' (SEQ ID NO: 15)] was prepared.
  • the COC substrate is treated with O 2 Plasma (20W (power), 10 (time), 20 sccm (flow rate)), and the DNA for immobilization confirmation is solvent A so that the concentration is 0.5uM. (150 mM sodium phosphate buffer with 0.01% tween 20 (pH 8.5)), solvent B (1x micro spotting solution plus (Array it)), dissolved in a mixture of solvents A and B in a specific ratio, respectively, and using a pin After spotting DNA on the COC substrate using a contact type microarrayer, it was dried at room temperature for 10 minutes.
  • O 2 Plasma 20W (power), 10 (time), 20 sccm (flow rate)
  • solvent A so that the concentration is 0.5uM.
  • solvent B (1x micro spotting solution plus (Array it)
  • UV treatment was performed (wavelength band 254nm, treatment time 30 minutes (by XL-1000 UV cross linker)), and then washed (10min with shaking & DW rinsing with 0.1x SC buffer with 0.01% SDS (pH 7)). , fluorescence images were obtained and the results are shown in FIGS. 4 and 5 .
  • a capture probe prepared in Example 1-1 and a capture probe [5'-TTTTTTTTTTTTCCCCCCCCTAATCATGAGTCCAAGAGC-'3 (SEQ ID NO: 16)] for comparison having different spacer region lengths were prepared.
  • step 3 After staining by adding 20ul of 5x SYBR green 2, the fluorescence value (EX: 480 / Em: 522) was measured, and the results are shown in FIG. 7.
  • step 3 RCA reaction solution (1x buffer 3ul, 1mM dNTP 3ul, 20U/30ul Phi29 2ul, DW 22ul) was added to RCA reaction for 2 hours, 5x SYBR green 2 20ul was added for staining, fluorescence Values (EX: 480 / Em: 522) were measured, and the results are shown in FIG. 8 .
  • the fixation efficiency of the COC substrate is similar according to the type of capture probe, but looking at FIG. 8, it can be seen that the RCA reaction is made in proportion to the amount only in the capture probe (probe B), It can be seen that if the length is too short, it can inhibit RCA due to steric hindrance.
  • Capture probe (400nM) prepared in Example 1-1, circular DNA 1 (200nM) prepared in Example 1-3, 1x RCA buffer, 1.25 mM dNTP mixture, and 30 units of phi29 DNA polymerase were mixed in a tube and 30 RCA reacted for 90 min at °C and inactivated at 65 °C for 10 min. Then, 2mM ascorbate, 0.5mM CuSO 4 and 1x MOPS buffer were added to the RCA result and reacted for 5 minutes to synthesize copper nanoparticles, and then confirmed by transmission electron microscopy (TEM), and the results are shown in FIG. 9 (scale bar is 100 nm).
  • TEM transmission electron microscopy
  • the capture probe (20uM) prepared in Example 1-1 was immobilized on a COC substrate by UV irradiation, and circular DNA 1 (20uL) having different concentrations prepared in Example 1-3 was added and incubated at 30°C for 1 hour. maintained. Thereafter, an RCA solution (40uL) containing 1x RCA buffer, 1 mM dNTP mixture, and 30 units of phi29 DNA polymerase was added, incubated at 30°C for 2 hours, and inactivated at 65°C for 10 minutes.
  • Au@Ag was synthesized into thread-shaped ssDNA, which appears to be an RCA product, only in the right image where circular DNA 2 was mixed and RCA was performed, and after hybridization of the capture probe and circular DNA 2, RCA It can be seen that the reaction is performed, the gold probe is hybridized to the RCA product, and the Au probe is bound to synthesize the Au@Ag cluster.
  • a value of ⁇ Abs of 1 or more indicates that the colorimetric signal increased when RCA was performed. Referring to FIG. 12, it can be seen that the colorimetric signal increase was the largest when 20 nm gold nanoparticles were used.
  • the 20uM capture probe (20uL) prepared in Example 1-1 was fixed to each well of the COC substrate by UV irradiation, and the 20uM aptamer (20uL) prepared in Example 1-2 in the hybridization buffer was added and heated at 34 ° C. incubated for 1 hour. Thereafter, DZN was dissolved in 1x binding buffer, and DZN solutions having various concentrations were added to each well, incubated at 30 ° C for 1 hour, 2uM circular DNA 1 (20uL) prepared in Example 1-3 in hybridization buffer was added, and 30 Incubated for 1 hour at ° C.

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Abstract

The present invention relates to a method for detecting a food-noxious substance and, more specifically, to a method for detecting a food-noxious substance wherein, with selective molecular affinity, a food-noxious substance-specific aptamer is bound to a capture probe fixed to a chip in the absence of the food-noxious substance, but departs from the capture probe and binds to the food noxious substance in the presence of the food-noxious substance, so that a circular DNA binds to the capture probe and undergoes an RCA reaction to form a metal body for signal amplification, whereby detection accuracy can be improved.

Description

식품 유해 물질 검출 방법Food Hazardous Substance Detection Method
본 발명은 식품 유해 물질 검출 방법에 대한 것으로, 더욱 상세하게는 식품 유해 물질에 특이적인 압타머가 선택적인 분자친화력을 가져, 식품 유해 물질이 존재하지 않을 때에는 칩에 고정된 캡처 프로브에 결합하나, 식품 유해 물질의 존재 시에는 캡처 프로브에서 이탈하여 식품 유해 물질과 결합하므로 원형 DNA가 캡처 프로브에 결합하여 RCA(rolling circle amplication) 반응이 일어나 신호증폭을 위한 금속체를 형성하는 것이 가능하여, 검출 정확성을 향상시킬 수 있는 식품 유해 물질 검출 방법에 대한 것이다.The present invention relates to a method for detecting substances harmful to food, and more particularly, an aptamer specific to a substance harmful to food has a selective molecular affinity and binds to a capture probe immobilized on a chip when the substance harmful to food does not exist. In the presence of a hazardous substance, it detaches from the capture probe and combines with the food hazardous substance, so circular DNA binds to the capture probe and RCA (rolling circle amplification) reaction occurs to form a metal body for signal amplification, thereby improving detection accuracy. It is about a food harmful substance detection method that can be improved.
식품에는 여러 유해 물질이 포함될 수 있는데, 대표적인 식품에 포함된 유해 물질인 농약은 농사 지을 때 농작물이 잡초, 해충, 세균 등으로부터 피해를 받는 것을 예방하기 위해 살포하는 약품으로, 사용 후 농작물, 토양, 하천 등에 잔류하여 주변 환경 및 사람에 피해를 발생시킨다. 따라서, 하기 특허문헌처럼 잔류 농약을 검출하기 위한 장치 및 방법이 널리 개발되고 있다.Food may contain various harmful substances. Pesticides, which are harmful substances contained in typical foods, are chemicals that are sprayed to prevent crops from being damaged by weeds, pests, bacteria, etc. during farming. It remains in rivers and causes damage to the surrounding environment and people. Therefore, devices and methods for detecting pesticide residues have been widely developed, as shown in the following patent documents.
<특허문헌><Patent Document>
특허 제10-1050421호(2011. 07. 13. 등록) "잔류 농약 검출용 키트 및 이를 이용한 잔류농약 검출방법"Patent No. 10-1050421 (registered on July 13, 2011) "Kit for detecting pesticide residues and method for detecting pesticide residues using the same"
하지만, 종래의 식품유해물질(농약)을 검출하는 방법은 고가의 장비를 필요로 하고, 빠르고 간편하게 식품유해물질(농약)의 존재 유무를 확인할 수 없었다.However, conventional methods for detecting food hazardous substances (pesticides) require expensive equipment, and could not quickly and simply check the presence or absence of food hazardous substances (pesticides).
본 발명은 상기와 같은 문제점을 해결하기 위해 안출된 것으로,The present invention has been made to solve the above problems,
본 발명은 식품 유해 물질에 특이적인 압타머가 선택적인 분자친화력을 가져, 식품 유해 물질이 존재하지 않을 때에는 칩에 고정된 캡처 프로브에 결합하나, 식품 유해 물질의 존재 시에는 캡처 프로브에서 이탈하여 식품 유해 물질과 결합하므로 원형 DNA가 캡처 프로브에 결합하여 RCA 반응이 일어나 신호증폭을 위한 금속체를 형성하는 것이 가능하여, 식품 유해 물질을 정확하게 검출할 수 있는 방법을 제공하는데 그 목적이 있다.In the present invention, an aptamer specific to a food hazardous substance has a selective molecular affinity and binds to a capture probe fixed on a chip when a food hazardous substance is not present, but deviates from the capture probe when a food hazardous substance is present and is harmful to food. Since it binds to substances, it is possible to form a metal body for signal amplification by binding circular DNA to the capture probe to cause an RCA reaction, and the purpose is to provide a method for accurately detecting food harmful substances.
본 발명은 앞서 본 목적을 달성하기 위해서 다음과 같은 구성을 가진 실시예에 의해서 구현된다.The present invention is implemented by an embodiment having the following configuration in order to achieve the above object.
본 발명의 일 실시예에 따르면, 본 발명에 따른 식품 유해 물질 검출 방법은 기판에 고정화되어 링커 역할을 하는 염기서열과, 농약에 선택적인 분자친화력을 가지는 압타머와 상보적 결합이 가능하도록 하는 염기서열을 포함하는 캡처 프로브를 기판에 고정하여 칩을 준비하는 칩준비단계와; 농약에 선택적인 분자친화력을 가지는 압타머를 상기 칩에 분포시켜, 상기 캡처 프로브와 압타머를 부분적 결합시키는 혼성화단계와; 상기 칩에 측정 대상 농약을 분포시켜 선택적인 분자친화력에 의해 압타머가 캡처 프로브에서 떨어져 농약과 결합하도록 하는 압타머분리단계와; 상기 압타머분리단계 후, 상기 캡처 프로브와 상보적 결합이 가능하도록 하는 염기서열과, 금속체와 상보적 결합이 가능하도록 하는 염기서열과 상보적인 염기서열을 포함하는 원형 DNA, 및 중합효소를 상기 칩에 공급하고 RCA 반응시켜, 원형 DNA가 상기 캡처 프로브에 부분적으로 결합하여 상기 캡처 프로브에 연결되며 상기 금속체와 상보적 결합이 가능하도록 하는 염기서열을 반복하여 가지는 단일 가닥을 형성하는 RCA반응단계와; 상기 RCA반응단계를 통해 형성된 단일 가닥을 템플레이트로 하여 신호 증폭을 위한 금속체를 형성하는 금속체형성단계;를 포함하는 것을 특징으로 한다.According to one embodiment of the present invention, the food hazardous substance detection method according to the present invention is a base sequence that is immobilized on a substrate and serves as a linker, and a base that enables complementary binding with an aptamer having selective molecular affinity for pesticides. a chip preparation step of preparing a chip by immobilizing the capture probe containing the sequence to a substrate; a hybridization step of partially binding the capture probe and the aptamer by distributing an aptamer having a selective molecular affinity to the pesticide on the chip; An aptamer separation step of distributing the pesticide to be measured on the chip so that the aptamer separates from the capture probe and binds to the pesticide by selective molecular affinity; After the aptamer separation step, a circular DNA comprising a nucleotide sequence enabling complementary binding with the capture probe, a nucleotide sequence complementary with a nucleotide sequence enabling complementary binding with the metal body, and a polymerase as described above. RCA reaction step of supplying to a chip and conducting RCA reaction to form a single strand having circular DNA partially bound to the capture probe, linked to the capture probe, and having a base sequence repeating to enable complementary binding with the metal body. and; and a metal body formation step of forming a metal body for signal amplification using the single strand formed through the RCA reaction step as a template.
본 발명의 다른 실시예에 따르면, 본 발명에 따른 식품 유해 물질 검출 방법은 상기 금속체형성단계 후 상기 칩에 빛을 조사하고 광학신호를 측정하여 농약의 존재 및 그 양을 검출하는 검출단계;를 추가로 포함하는 것을 특징으로 한다.According to another embodiment of the present invention, the food hazardous substance detection method according to the present invention includes a detection step of irradiating light to the chip after the metal body forming step and measuring an optical signal to detect the presence and amount of pesticides. It is characterized in that it further includes.
본 발명의 또 다른 실시예에 따르면, 본 발명에 따른 식품 유해 물질 검출 방법에 있어서 상기 금속체와 상보적 결합이 가능하도록 하는 염기서열과 상보적인 염기서열은 복수 개가 연이어진 아데닌으로 이루어져, 상기 가닥은 복수의 티민으로 형성되게 되며, 상기 금속체형성단계에서는 RCA반응단계 후 구리 이온과 아스코베이트를 포함하는 용액을 칩에 주입하여, 상기 복수의 티민을 템플레이트로 하여 구리 나노입자를 형성하는 것을 특징으로 한다.According to another embodiment of the present invention, in the method for detecting harmful substances in food according to the present invention, the nucleotide sequence and the complementary nucleotide sequence enabling complementary binding with the metal body are composed of a plurality of consecutive adenines, so that the strand is formed from a plurality of thymines, and in the metal body formation step, a solution containing copper ions and ascorbate is injected into the chip after the RCA reaction step to form copper nanoparticles using the plurality of thymines as a template. to be
본 발명의 또 다른 실시예에 따르면, 본 발명에 따른 식품 유해 물질 검출 방법에 있어서 상기 금속체와 상보적 결합이 가능하도록 하는 염기서열과 상보적인 염기서열은 금 프로브와 상보적 결합이 가능하도록 하는 염기서열과 상보적인 염기서열로 이루어져, 상기 가닥은 금 프로브와 상보적 결합이 가능하도록 하는 염기서열로 형성되게 되며, 상기 금속체형성단계에서는 제1반응기가 결합된 특정 ssDNA에, 상기 제1반응기와 결합하는 제2반응기가 결합된 금 나노입자를 반응시켜, 특정 ssDNA에 금이 결합된 금 프로브를 형성하고, 상기 금 프로브를 칩에 주입하여 상기 가닥에 금 프로브를 결합시키고, 은 이온과 환원제를 추가로 공급하여, 금 나노입자 표면에 은 클러스터를 형성하는 것을 특징으로 한다.According to another embodiment of the present invention, in the food hazardous substance detection method according to the present invention, the nucleotide sequence that enables complementary binding with the metal body and the complementary nucleotide sequence enables complementary binding with the gold probe It consists of a nucleotide sequence complementary to the nucleotide sequence, and the strand is formed with a nucleotide sequence that enables complementary binding with the gold probe. In the metal body formation step, the first reactor Reaction of the gold nanoparticles to which the second reactive group binds to forms a gold probe in which gold is bound to a specific ssDNA, injects the gold probe into a chip to bind the gold probe to the strand, silver ions and a reducing agent is additionally supplied to form silver clusters on the surface of the gold nanoparticles.
본 발명의 또 다른 실시예에 따르면, 본 발명에 따른 식품 유해 물질 검출 방법에 있어서 상기 기판은 COC(cyclic olefin copolymer) 기판이 사용되며, 상기 캡처 프로브는 다이아지논을 검출하기 위해 서열번호 4의 염기서열을 가지는 것을 특징으로 한다.According to another embodiment of the present invention, in the food hazardous substance detection method according to the present invention, the substrate is a COC (cyclic olefin copolymer) substrate, and the capture probe is a base of SEQ ID NO: 4 to detect diazinon. It is characterized by having a sequence.
본 발명의 또 다른 실시예에 따르면, 본 발명에 따른 식품 유해 물질 검출 방법에 있어서 상기 원형 DNA는 서열번호 7의 염기서열을 가지는 것을 특징으로 한다.According to another embodiment of the present invention, in the method for detecting harmful substances in food according to the present invention, the circular DNA has the nucleotide sequence of SEQ ID NO: 7.
본 발명의 또 다른 실시예에 따르면, 본 발명에 따른 식품 유해 물질 검출 방법에 있어서 상기 원형 DNA는 서열번호 9의 염기서열을 가지는 것을 특징으로 한다.According to another embodiment of the present invention, in the method for detecting harmful substances in food according to the present invention, the circular DNA has the nucleotide sequence of SEQ ID NO: 9.
본 발명은 앞서 본 실시예와 하기에 설명할 구성과 결합, 사용관계에 의해 다음과 같은 효과를 얻을 수 있다.The present invention can obtain the following effects by combining and using the above embodiments and configurations to be described below.
본 발명은 식품 유해 물질에 특이적인 압타머가 선택적인 분자친화력을 가져, 식품 유해 물질이 존재하지 않을 때에는 칩에 고정된 캡처 프로브에 결합하나, 식품 유해 물질의 존재 시에는 캡처 프로브에서 이탈하여 식품 유해 물질과 결합하므로 원형 DNA가 캡처 프로브에 결합하여 RCA 반응이 일어나 신호증폭을 위한 금속체를 형성하는 것이 가능하여, 검출 정확성을 향상시킬 수 있는 효과가 있다.In the present invention, an aptamer specific to a food hazardous substance has a selective molecular affinity and binds to a capture probe fixed on a chip when a food hazardous substance is not present, but deviates from the capture probe when a food hazardous substance is present and is harmful to food. Since it binds to a material, it is possible to form a metal body for signal amplification by binding the circular DNA to the capture probe and causing an RCA reaction, thereby improving detection accuracy.
도 1은 본 발명의 일 실시예에 따라 농약을 검출하는 원리를 설명하기 위한 참고도.1 is a reference diagram for explaining a principle of detecting pesticides according to an embodiment of the present invention.
도 2는 캡쳐 프로브의 최적 결합 염기서열 설계를 위한 CD 분석 결과를 나타내는 그래프.Figure 2 is a graph showing the results of CD analysis for designing the optimal binding nucleotide sequence of the capture probe.
도 3은 다이아지논 압타머와 cDNA들의 결합관계를 나타내는 참고도.Figure 3 is a reference diagram showing the binding relationship between diazinon aptamer and cDNA.
도 4 및 5는 링커 염기서열을 가지는 DNA를 COC 칩에 고정 시 용매가 미치는 효과를 확인하기 위한 형광 이미지.4 and 5 are fluorescence images for confirming the effect of the solvent when DNA having a linker base sequence is immobilized on a COC chip.
도 6는 캡처 프로브가 COC 기판에 고정되는지를 확인하기 위한 형광 변화를 나타내는 그래프.Figure 6 is a graph showing the fluorescence change for confirming whether the capture probe is immobilized on the COC substrate.
도 7 및 8은 캡처 프로브의 Spacer 영역 길이에 따른 RCA 효율을 확인하기 위한 형광 변화는 나타내는 그래프.7 and 8 are graphs showing fluorescence changes for confirming RCA efficiency according to the length of the spacer region of the capture probe.
도 9은 COC 기판에서 poly(T)가 생성되어 CuNP가 합성됨을 확인하기 위한 투과전자현미경(TEM) 이미지.9 is a transmission electron microscope (TEM) image for confirming that poly(T) is produced on a COC substrate and CuNP is synthesized.
도 10은 원형 DNA 1의 농도에 따라 형광 세기가 변화함을 확인하기 위한 그래프.10 is a graph confirming that fluorescence intensity changes according to the concentration of circular DNA 1;
도 11은 COC 기판에서 RCA 생성물로 ssDNA가 형성되어 Au@Ag가 합성됨을 확인하기 위한 주사전자현미경(SEM) 이미지.Figure 11 is a scanning electron microscope (SEM) image for confirming that ssDNA is formed as an RCA product on a COC substrate to synthesize Au@Ag.
도 12는 금 나노입자 크기에 따른 비색 신호 확인을 위한 흡광도 측정 결과를 나타내는 그래프.12 is a graph showing absorbance measurement results for confirming colorimetric signals according to the size of gold nanoparticles.
도 13은 본 발명의 일 실시예에 따른 검출 방법을 이용하여 다이아지논을 검출한 결과를 나타내는 그래프.13 is a graph showing results of detecting diazinon using a detection method according to an embodiment of the present invention.
도 14는 본 발명의 일 실시예에 따른 검출 방법이 다이아지논을 선택적으로 검출함을 확인하기 위한 그래프. 14 is a graph confirming that the detection method according to an embodiment of the present invention selectively detects diazinon.
이하에서는 본 발명에 따른 식품 유해 물질 검출 방법을 도면을 참조하여 상세히 설명한다. 특별한 정의가 없는 한 본 명세서의 모든 용어는 본 발명이 속하는 기술분야의 통상의 지식을 가진 기술자가 이해하는 당해 용어의 일반적 의미와 동일하고 만약 본 명세서에 사용된 용어의 의미와 충돌하는 경우에는 본 명세서에 사용된 정의에 따른다. 또한, 본 발명의 요지를 불필요하게 흐릴 수 있는 공지 기능 및 구성에 대해 상세한 설명은 생략한다. 명세서 전체에서, 어떤 부분이 어떤 구성요소를 "포함"한다고 할 때 이는 특별히 반대되는 기재가 없는 한 다른 구성요소를 제외하는 것이 아니라 다른 구성요소를 더 포함할 수 있는 것을 의미한다.Hereinafter, a method for detecting harmful substances in food according to the present invention will be described in detail with reference to the drawings. Unless there is a special definition, all terms in this specification are the same as the general meaning of the term understood by a person skilled in the art to which the present invention belongs, and if it conflicts with the meaning of the term used in this specification, the present invention Follow the definitions used in the specification. In addition, detailed descriptions of well-known functions and configurations that may unnecessarily obscure the subject matter of the present invention will be omitted. Throughout the specification, when a part "includes" a certain component, it means that it may further include other components without excluding other components unless otherwise stated.
본 발명의 일 실시예에 따른 식품 유해 물질 검출 방법을 도 1 내지 14를 참조하여 설명하기로 한다. 하기에서는 식품 유해 물질의 대표적인 예인 농약을 예로 들어 식품 유해 물질 검출 방법을 설명하기로 한다. 상기 식품 유해 물질 검출 방법은 기판에 고정화되어 링커 역할을 하는 염기서열과, 농약에 선택적인 분자친화력을 가지는 압타머와 상보적 결합이 가능하도록 하는 염기서열을 포함하는 캡처 프로브를 기판에 고정하여 칩을 준비하는 칩준비단계와; 상기 칩에 농약에 선택적인 분자친화력을 가지는 압타머를 분포시켜, 상기 캡처 프로브와 압타머를 부분적 결합시키는 혼성화단계와; 상기 칩에 측정 대상 농약을 분포시켜 선택적인 분자친화력에 의해 압타머가 캡처 프로브에서 떨어져 농약과 결합하도록 하는 압타머분리단계와; 상기 압타머분리단계 후, 상기 캡처 프로브와 상보적 결합이 가능하도록 하는 염기서열과, 금속체와 상보적 결합이 가능하도록 하는 염기서열과 상보적인 염기서열을 포함하는 원형 DNA 및 중합효소를 상기 칩에 공급하고 RCA 반응시켜, 원형 DNA가 상기 캡처 프로브에 부분적으로 결합하여 상기 캡처 프로브에 연결되며 상기 금속체와 상보적 결합이 가능하도록 하는 염기서열을 반복하여 가지는 단일 가닥을 형성하는 RCA반응단계와; 상기 RCA반응단계를 통해 형성된 단일 가닥을 템플레이트로 하여 신호 증폭을 위한 금속체를 형성하는 금속체형성단계와; 상기 금속체형성단계 후 상기 칩에 빛을 조사하고 광학신호를 측정하여 농약의 존재 및 그 양을 검출하는 검출단계;를 포함한다.A method for detecting harmful substances in food according to an embodiment of the present invention will be described with reference to FIGS. 1 to 14 . In the following, a pesticide, which is a representative example of food harmful substances, will be described as an example of a method for detecting food harmful substances. The method for detecting harmful substances in food is a chip by immobilizing a capture probe containing a nucleotide sequence immobilized on a substrate to act as a linker and a nucleotide sequence enabling complementary binding with an aptamer having selective molecular affinity for pesticides to a substrate. A chip preparation step for preparing; a hybridization step of partially binding the capture probe and the aptamer by distributing an aptamer having a molecular affinity selective for pesticides on the chip; An aptamer separation step of distributing the pesticide to be measured on the chip so that the aptamer separates from the capture probe and binds to the pesticide by selective molecular affinity; After the aptamer separation step, a circular DNA containing a nucleotide sequence enabling complementary binding with the capture probe, a nucleotide sequence enabling complementary binding with the metal body and a complementary nucleotide sequence, and a polymerase are transferred to the chip. supplied to and subjected to RCA reaction, wherein the circular DNA partially binds to the capture probe to form a single strand having a base sequence that is linked to the capture probe and allows complementary binding with the metal body; and ; a metal body formation step of forming a metal body for signal amplification using the single strand formed through the RCA reaction step as a template; After the metal body forming step, a detecting step of irradiating the chip with light and measuring an optical signal to detect the presence and amount of pesticide.
상기 칩준비단계는 기판에 고정화되어 링커 역할을 하는 염기서열과, 농약에 선택적인 분자친화력을 가지는 압타머와 상보적 결합이 가능하도록 하는 염기서열을 포함하는 캡처 프로브를 기판에 고정하여 칩을 준비하는 단계이다. 상기 링커 역할을 하는 염기서열이 예컨대 T(10)C(10)로 이루어지고 5' 말단 부위에 위치하는 경우, 상기 캡처 프로브를 고분자(예컨대, COC 등) 기판에 분포시키고 UV 조사를 하면, 상기 T(10)C(10)가 고분자 기판 표면에 고정되어 캡처 프로브의 5' 말단 부분이 고분자 기판에 고정되므로, 상기 캡처 프로브를 기판에 고정할 수 있게 된다.The chip preparation step prepares a chip by immobilizing a capture probe containing a nucleotide sequence immobilized on a substrate to act as a linker and a nucleotide sequence enabling complementary binding with an aptamer having a selective molecular affinity for pesticides to a substrate. It is a step to When the nucleotide sequence serving as the linker consists of, for example, T(10)C(10) and is located at the 5' end, the capture probe is distributed on a polymer (eg, COC, etc.) substrate and UV irradiated, the Since T(10)C(10) is immobilized on the polymer substrate surface and the 5' end portion of the capture probe is immobilized on the polymer substrate, the capture probe can be immobilized on the substrate.
상기 혼성화단계는 농약에 선택적인 분자친화력을 가지는 압타머를 상기 칩에 분포시켜, 상기 캡처 프로브와 압타머를 부분적 결합시키는 단계로, 상기 기판에 고정된 캡처 프로브는 상기 압타머와 상보적 결합이 가능하도록 하는 염기서열을 가지므로, 상기 칩준비단계 후 상기 압타머를 상기 칩에 분포시키면, 상기 캡처 프로브와 압타머는 부분적으로 결합하게 된다.The hybridization step is a step of distributing an aptamer having a molecular affinity selective to the pesticide on the chip to partially bind the capture probe and the aptamer, wherein the capture probe fixed to the substrate has a complementary bond with the aptamer. Since it has a nucleotide sequence that enables the capture probe to partially bind to the aptamer when the aptamer is distributed on the chip after the chip preparation step.
상기 압타머분리단계는 상기 혼성화단계 후, 상기 칩에 측정 대상 농약을 분포시켜 선택적인 분자친화력에 의해 압타머가 캡처 프로브에서 떨어져 농약과 결합하도록 하는 단계이다. 상기 압타머는 선택적인 분자친화력을 가지므로, 즉 캡처 프로브보다 농약에 분자친화력이 강하므로, 시료에 농약이 존재하는 경우 압타머는 캡처 프로브에서 떨어져 농약에 결합하게 된다.The aptamer separation step is a step of distributing the pesticide to be measured on the chip after the hybridization step so that the aptamer separates from the capture probe and binds to the pesticide by selective molecular affinity. Since the aptamer has a selective molecular affinity, that is, it has a stronger molecular affinity for the pesticide than the capture probe, when the pesticide is present in the sample, the aptamer separates from the capture probe and binds to the pesticide.
상기 RCA반응단계는 상기 압타머분리단계 후, 상기 캡처 프로브와 상보적 결합이 가능하도록 하는 염기서열과, 금속체와 상보적 결합이 가능하도록 하는 염기서열과 상보적인 염기서열을 포함하는 원형 DNA 및 중합효소를 상기 칩에 공급하고 RCA 반응시켜, 원형 DNA가 상기 캡처 프로브에 부분적으로 결합하여 상기 캡처 프로브에 연결되며 상기 금속체와 상보적 결합이 가능하도록 하는 염기서열을 반복하여 가지는 가닥을 형성하는 단계이다. 예컨대, 형성하고자 하는 금속체가 구리 나노입자인 경우, 상기 금속체와 상보적 결합이 가능하도록 하는 염기서열과 상보적인 염기서열은 복수 개가 연이어진 아데닌으로 이루어져, 상기 가닥은 복수의 티민(poly(T))으로 형성되게 되며, 형성하고자 하는 금속체가 금/은 클러스터인 경우, 상기 금속체와 상보적 결합이 가능하도록 하는 염기서열과 상보적인 염기서열은 금 프로브와 상보적 결합이 가능하도록 하는 염기서열과 상보적인 염기서열로 이루어져, 상기 가닥은 금 프로브와 상보적 결합이 가능하도록 하는 염기서열로 형성되게 된다.In the RCA reaction step, after the aptamer separation step, a circular DNA comprising a nucleotide sequence enabling complementary binding with the capture probe, a nucleotide sequence enabling complementary binding with the metal body, and a complementary nucleotide sequence, and A polymerase is supplied to the chip and subjected to an RCA reaction so that the circular DNA partially binds to the capture probe and is linked to the capture probe to form a strand having repeating nucleotide sequences enabling complementary binding with the metal body It is a step. For example, when the metal to be formed is a copper nanoparticle, the nucleotide sequence enabling complementary bonding with the metal and the complementary nucleotide sequence are composed of a plurality of consecutive adenines, and the strand is composed of a plurality of thymine (poly(T )), and when the metal to be formed is a gold/silver cluster, the nucleotide sequence that enables complementary bonding with the metal body and the complementary nucleotide sequence is the nucleotide sequence that enables complementary bonding with the gold probe It consists of a nucleotide sequence complementary to, and the strand is formed with a nucleotide sequence that enables complementary binding with the gold probe.
상기 금속체형성단계는 상기 RCA반응단계를 통해 형성된 가닥을 템플레이트로 하여 신호 증폭을 위한 금속체를 형성하는 단계이다. 예컨대, 상기 가닥이 복수의 티민(poly(T))으로 형성되는 경우, Cu2+와 ascorbate를 칩에 주입하면 poly(T)를 주형으로 한 구리 나노입자가 형성되게 된다. 또한, 상기 금속체형성단계에서 금/은 클러스터를 형성하고자 하는 경우, 제1반응기(예컨대, 비오틴(biotin) 등)가 결합된 특정 ssDNA에, 상기 제1반응기와 결합하는 제2반응기(예컨대, 스트렙타피딘(streptavidin) 등)가 결합된 금 나노입자를 반응시켜, 특정 ssDNA에 금이 결합된 금 프로브를 형성하고, 상기 금 프로브를 칩에 주입하여 상기 가닥에 금 프로브를 결합시키고, 은 이온과 환원제를 추가로 공급하여, 금 나노입자 표면에 은 클러스터를 형성할 수 있다.The metal body forming step is a step of forming a metal body for signal amplification using the strand formed through the RCA reaction step as a template. For example, when the strand is formed of a plurality of thymine (poly(T)), Cu 2+ and ascorbate are injected into the chip to form copper nanoparticles using poly(T) as a template. In addition, in the case of forming a gold/silver cluster in the metal body formation step, a second reactor (eg, biotin) coupled to a specific ssDNA to which a first reactor (eg, biotin) is coupled to the first reactor gold nanoparticles coupled with streptavidin, etc.) are reacted to form a gold probe in which gold is bound to a specific ssDNA, the gold probe is injected into a chip to bind the gold probe to the strand, and silver ions and a reducing agent may be additionally supplied to form silver clusters on the surface of the gold nanoparticles.
상기 검출단계는 상기 금속체형성단계 후 상기 칩에 빛을 조사하고 광학신호를 측정하여 농약의 존재 및 그 양을 검출하는 단계로, 검사하고자 하는 시료에 농약이 존재하는 경우 압타머가 캡처 프로브에서 떨어져 결과적으로 금속체가 형성되고 이후 빛을 조사하면, 금속체가 구리나노입자인 경우 발광하는 형광을 측정하고, 금속체가 금/은 클러스터인 경우 흡수되는 빛을 측정하여, 기 설정된 농약의 농도와 형광(또는 흡광도) 값 간의 비례적 상관관계를 고려하여, 농약의 존재 및 그 양을 검출할 수 있게 된다.The detection step is a step of irradiating light onto the chip after the metal body formation step and measuring an optical signal to detect the presence and amount of pesticide. As a result, when the metal body is formed and then irradiated with light, fluorescence emitted when the metal body is a copper nanoparticle is measured, and light absorbed when the metal body is a gold/silver cluster is measured, and the concentration and fluorescence (or Absorbance) Considering the proportional correlation between the values, it is possible to detect the presence and amount of pesticides.
이하, 실시예를 통해서 본 발명을 보다 상세히 설명하기로 한다. 하지만, 이들은 본 발명을 보다 상세하게 설명하기 위한 것일 뿐, 본 발명의 권리범위가 이에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through examples. However, these are only for explaining the present invention in more detail, and the scope of the present invention is not limited thereto.
<실시예 1> 시료의 준비<Example 1> Preparation of samples
1. 캡처 프로브(Capture probe)의 준비1. Preparation of capture probe
UV 조사를 통해 COC(Cyclic olefin copolymer) 기판에 고정화되는 링커 역할하는 염기서열(이하, '링커 염기서열'이라 함)[TTTTTTTTTTCCCCCCCCCC(서열번호:1)]과, 스페이서(Spacer) 염기서열[TAATCATGATT(서열번호:2)]과, 다이아지논 압타머(Diazinon aptamer)와 상보적 결합이 가능하도록 하는 염기서열(이하, '결합 염기서열'이라 함)[GTCCAAGAGC(서열번호:3)]을 차례로 포함하는 캡처 프로브[5'-TTTTTTTTTTCCCCCCCCCCTAATCATGATTGTCCAAGAGC-3'(서열번호:4)]를 준비하였다.A base sequence acting as a linker immobilized on a cyclic olefin copolymer (COC) substrate through UV irradiation (hereinafter referred to as 'linker base sequence') [TTTTTTTTTTCCCCCCCCCC (SEQ ID NO: 1)] and a spacer base sequence [TAATCATGATT ( SEQ ID NO: 2)] and a nucleotide sequence (hereinafter referred to as 'binding nucleotide sequence') [GTCCAAGAGC (SEQ ID NO: 3)] that enables complementary binding with Diazinon aptamer. A capture probe [5'-TTTTTTTTTTCCCCCCCCCCTAATCATGATTGTCCAAGAGC-3' (SEQ ID NO: 4)] was prepared.
2. 다이아지논 압타머(Diazinon aptamer)의 준비2. Preparation of Diazinon aptamer
캡처 프로브와 상보적 결합이 가능하도록, 상기 결합 염기서열과 상보적인 염기서열[GCTCTTGGAC(서열번호:5)]을 포함하는 다이아지논 압타머[5'-ATCCGTCACACCTGCTCTAATATAGAGGTATTGCTCTTGGACAAGGTACAGGGATGGTGTTGGCTCCCGTAT-3'(서열번호:6)]를 준비하였다.Diazinon aptamer [5'-ATCCGTCACACCTGCTCTAATATAGAGGTATTGCTCTTGGACAAGGTACAGGGATGGTGTTGGCTCCCGTAT-3' (SEQ ID NO: 6)] containing a nucleotide sequence complementary to the binding nucleotide sequence [GCTCTTGGAC (SEQ ID NO: 5)] to enable complementary binding with the capture probe. prepared.
3. 원형 DNA 1의 준비3. Preparation of circular DNA 1
(1) 캡처 프로브와 상보적 결합이 가능하도록, 상기 결합 염기서열과 상보적인 염기서열[GCTCTTGGAC(서열번호:5) 및 poly(A)를 포함하는 linear DNA 1[5'-(phosphate)-GTAGGTGCTCTTGGACGAACATAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACG-3'(서열번호:7)]를 준비하였다.(1) Linear DNA 1[5'-(phosphate)-GTAGGTGCTCTTGGACGAACATAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACG containing a nucleotide sequence complementary to the binding nucleotide sequence [GCTCTTGGAC (SEQ ID NO: 5) and poly(A) to enable complementary binding with the capture probe -3' (SEQ ID NO: 7)] was prepared.
(2) 상기 linear DNA 1과 DNA 연결효소(ligase)를 튜브에 넣은 후(50uM linear DNA 1 15uL, 10X buffer 10uL, 1mM ATP 5uL, 50mM Mncl2 5uL, 100U/Ul Ligase 4uL, DW 61uL), 60℃에서 8시간 동안 유지하고 80℃에서 10분 동안 유지하여 리게이션 생성물(Ligation product)을 얻였다. 이후, 잔류하는 linear DNA 1을 제거하기 위해, 리게이션 생성물, 엑소뉴클레아제 I 및 엑소뉴클레아제 III를 혼합하고(Ligation product 200uL, Buffer1 26uL, Buffer2 26uL, Exo1 (20U/uL) 6uL, Exo3 (10U/uL) 2uL), 37℃에서 6시간 동안 및 80℃에서 20분 동안 배양하였다. 생성된 생성물을 정제하여 원형 DNA 1(CirDNA)을 얻었다. 60 It was maintained at ℃ for 8 hours and maintained at 80 ℃ for 10 minutes to obtain a ligation product (Ligation product). Then, in order to remove the remaining linear DNA 1, the ligation product, exonuclease I and exonuclease III were mixed (Ligation product 200uL, Buffer1 26uL, Buffer2 26uL, Exo1 (20U/uL) 6uL, Exo3 (10U/uL) 2uL), incubated at 37°C for 6 hours and at 80°C for 20 minutes. The resulting product was purified to obtain circular DNA 1 (CirDNA).
4. 원형 DNA 2의 준비4. Preparation of circular DNA 2
(1) 캡처 프로브와 상보적 결합이 가능하도록 상기 결합 염기서열과 상보적인 염기서열[GCTCTTGGAC(서열번호:5)] 및 금 프로브와 상보적 결합이 가능하도록 하는 염기서열(이하, '연결 염기서열'이라 함)[AAGCCATCAGTC(서열번호:8)]을 포함하는 linear DNA 2[5'-(phosphate)-GTAGGTGCTCTTGGACGAACATATTAGCCTTCAGCGCTCCCAAGCCATCAGTCT-3'(서열번호:9)]를 준비하였다.(1) a nucleotide sequence complementary to the binding nucleotide sequence [GCTCTTGGAC (SEQ ID NO: 5)] and a nucleotide sequence complementary to the gold probe to enable complementary binding with the capture probe (hereinafter referred to as 'linked nucleotide sequence') ') [AAGCCATCAGTC (SEQ ID NO: 8)] containing linear DNA 2 [5'-(phosphate)-GTAGGTGCTCTTGGACGAACATATTAGCCTTCAGCGCTCCCAAGCCATCAGTCT-3' (SEQ ID NO: 9)] was prepared.
(2) linear DNA 1 대신에 linear DNA 2가 사용된 것을 제외하고는 다른 조건을 실시예 1의 3의 (2)와 동일하게 하여 원형 DNA 2(CirDNA)를 얻었다.(2) Circular DNA 2 (CirDNA) was obtained in the same manner as in Example 1-3 (2) except that linear DNA 2 was used instead of linear DNA 1.
5. 금 프로브의 준비5. Preparation of gold probes
(1) 상기 연결 염기서열과 동일한 염기서열[AAGCCATCAGTC(서열번호:8)]을 포함하고, 바이오틴이 기능화된 금 프로브 DNA[5'-AAGCCATCAGTC-(C6SP)-(biotin)-3']를 준비하였다. (1) Preparing a gold probe DNA [5'-AAGCCATCAGTC-(C6SP)-(biotin)-3'] containing the same nucleotide sequence as the linking nucleotide sequence [AAGCCATCAGTC (SEQ ID NO: 8)] and functionalized with biotin did
(2) 상기 금 프로브 DNA와 streptavidin으로 코팅된 20nm 크기의 금 나노입자를 혼합한 후 상온에서 밤새 반응시켜, streptavidin-biotin 반응에 의해 금 프로브 DNA가 금 나노입자에 결합한 금 프로브를 얻었다.(2) The gold probe DNA and streptavidin-coated gold nanoparticles having a size of 20 nm were mixed and reacted overnight at room temperature to obtain a gold probe in which the gold probe DNA was bound to the gold nanoparticles by a streptavidin-biotin reaction.
<실시예 2> 다이아지논 압타머의 최적 혼성화 사이트 확인<Example 2> Confirmation of optimal hybridization site of diazinon aptamer
1. 다이아지논 압타머 뿐만 아니라 원형 DNA에 결합하는 캡쳐 브로브의 능력은 다이아지논 검출에 중요한 영향을 미치므로, CD(Circular dichroism) 분석을 통해 다이아지논(DZN)과 민감하고 선택적으로 반응하는 압타머 사이트를 확인하여 캡쳐 프로브의 결합 염기서열을 설계하였다.1. Since the ability of the capture probe to bind to circular DNA as well as to diazinon aptamer has an important effect on the detection of diazinon, CD (Circular dichroism) analysis is used to determine the pressure that reacts sensitively and selectively with diazinon (DZN). The binding nucleotide sequence of the capture probe was designed by confirming the tamer site.
2. 구체적으로, 우선 다이아지논 압타머에 상보적인 DNA 4종(cDNA 1 내지 4))을 준비하였다(cDNA 1[5'-GTGTGACGGA-3'(서열번호:10)], cDNA 2[5'-GCAATACCTC-3'(서열번호:11)], cDNA 3[5'-GTCCAAGAGC-3'(서열번호:12)], cDNA 4[5'-ATACGGGAGC-3'(서열번호:13)]. 다이아지논 압타머와 각각의 cDNA를 혼합하여 혼합용액을 준비하고 88℃에서 5분 동안 및 30℃에서 30분 동안 차례로 인큐베이션하고 25℃로 냉각하여 샘플 A(Sample type A)를 준비하고, 샘플 A에 DZN을 추가하고 30℃에서 30분 동안 인큐베이션하여 심플 B((Sample type B)를 준비하고, 압타머와 DZN을 혼합하고 30℃에서 30분 동안 인큐베이션하고 각각의 cDNA를 혼합하여 샘플 C(Sample type C)를 준비하였다. 각 샘플에서 다이아지논 압타머, cDNA, 및 DZN의 최종 농도는 각각 0.5μM, 1μM, 및 1ppm이다. 이후, 샘플 A 내지 C에 대하여 CD spectrometer(J-715, JASCO, Tokyo, Japan)로 분석하여 그 결과를 도 2에 나타내었다.2. Specifically, first, four types of DNA (cDNA 1 to 4) complementary to diazinon aptamer were prepared (cDNA 1 [5'-GTTGGACGGA-3' (SEQ ID NO: 10)], cDNA 2 [5' -GCAATACCTC-3' (SEQ ID NO: 11)], cDNA 3 [5'-GTCCAAGAGC-3' (SEQ ID NO: 12)], cDNA 4 [5'-ATACGGGAGC-3' (SEQ ID NO: 13)]. Prepare a mixed solution by mixing Xenon aptamer and each cDNA, incubate at 88 ° C for 5 minutes and at 30 ° C for 30 minutes, cool to 25 ° C to prepare sample A (Sample type A), and sample A Add DZN and incubate at 30 ° C for 30 minutes to prepare simple B ((Sample type B), mix the aptamer and DZN, incubate at 30 ° C for 30 minutes, mix each cDNA to prepare sample C (Sample type B) C) was prepared. The final concentrations of diazinon aptamer, cDNA, and DZN in each sample were 0.5 μM, 1 μM, and 1 ppm, respectively. Then, for samples A to C, a CD spectrometer (J-715, JASCO, Tokyo) was prepared. , Japan) and the results are shown in FIG.
3. 각 cDNA는 도 3에 도시된 바와 같이 다른 사이트(사이트 1 내지 4)에 결합하는데, 도 2를 보면, 압타머 사이트 1 및 2에서는 샘플 A 및 B의 스펙트럼 패턴이 유사하기 때문에 DNZ보다 cDNA와 강하게 결합함을 알 수 있고, 압타머 사이트 3 및 4에서는 샘플 B 및 C의 스펙트럼 패턴이 유사하기 때문에 cDNA보다 DNZ와 강하게 결합함을 알 수 있으며, 압타머 사이트 3에서는 샘플 A의 압타머-cDNA 복합체와 샘플 C의 압타머-DZN 복합체 사이에 두드러진 스펙트럼 패턴 차이를 가져 두 복합체의 3차원 구조체 사이에 큰 차이를 보임을 알 수 있어, 압타머 사이트 3이 결합 염기서열을 설계를 위한 최적 사이트임을 알 수 있다.3. Each cDNA binds to different sites (sites 1 to 4) as shown in FIG. 3. Referring to FIG. 2, the aptamer sites 1 and 2 have similar spectral patterns of samples A and B, so that cDNA is better than DNZ. Since the spectral patterns of samples B and C are similar at aptamer sites 3 and 4, it can be seen that they bind more strongly with DNZ than cDNA, and at aptamer site 3, the aptamer- of sample A Significant spectral pattern differences between the cDNA complex and the aptamer-DZN complex of sample C show a large difference between the three-dimensional structures of the two complexes, indicating that aptamer site 3 is the optimal site for designing the binding sequence. It can be seen that
<실시예 3> 캡처 프로브 고정화 확인<Example 3> Check capture probe immobilization
1. 링커 염기서열을 가지는 DNA를 COC 기판에 고정 시 용매가 미치는 효과를 확인1. Confirmation of the effect of the solvent when immobilizing DNA having a linker base sequence to a COC substrate
(1) 링커 염기서열을 가지는 DNA를 COC 기판에 고정 시 용매가 미치는 효과를 확인하기 위하여, 링커 역할하는 염기서열[CCCCCCCCCCTTTTTTTTTT(서열번호:14)]을 포함하고, 5'에 cy3가 달린 고정화 확인용 DNA[5'-(cy3)-TCATGACCCCCCCCCCTTTTTTTTTT-3'(서열번호:15)]를 준비하였다.(1) In order to confirm the effect of the solvent when DNA having a linker nucleotide sequence is immobilized on a COC substrate, the nucleotide sequence acting as a linker [CCCCCCCCCCTTTTTTTTTT (SEQ ID NO: 14)] is included and cy3 is attached to the 5' Confirmation of immobilization DNA [5'-(cy3)-TCATGACCCCCCCCCCTTTTTTTTTT-3' (SEQ ID NO: 15)] was prepared.
(2) COC 기판 친수화를 위해 COC 기판을 O2 Plasma처리하고(20W (power), 10 (time), 20 sccm (flow rate)), 농도가 0.5uM이 되도록 상기 고정화 확인용 DNA를 용매 A(150 mM sodium phosphate buffer with 0.01% tween 20 (pH 8.5)), 용매 B(1x micro spotting solution plus (Array it)), 특정 비율로 용매 A와 B가 혼합된 혼합용매 각각에 녹이고, pin을 이용한 contact 방식의 microarrayer를 이용하여 DNA를 COC 기판에 spotting 한 후 상온에서 10분 건조하였다. 이후, UV 처리를 수행하고(파장대 254nm, 처리시간 30분 (by XL-1000 UV cross linker)), 세척한 후(0.1x SC buffer with 0.01% SDS (pH 7)로 10min with shaking & DW rinsing), 형광 이미지를 얻어 그 결과를 도 4 및 5에 나타내었다.(2) To make the COC substrate hydrophilic, the COC substrate is treated with O 2 Plasma (20W (power), 10 (time), 20 sccm (flow rate)), and the DNA for immobilization confirmation is solvent A so that the concentration is 0.5uM. (150 mM sodium phosphate buffer with 0.01% tween 20 (pH 8.5)), solvent B (1x micro spotting solution plus (Array it)), dissolved in a mixture of solvents A and B in a specific ratio, respectively, and using a pin After spotting DNA on the COC substrate using a contact type microarrayer, it was dried at room temperature for 10 minutes. Then, UV treatment was performed (wavelength band 254nm, treatment time 30 minutes (by XL-1000 UV cross linker)), and then washed (10min with shaking & DW rinsing with 0.1x SC buffer with 0.01% SDS (pH 7)). , fluorescence images were obtained and the results are shown in FIGS. 4 and 5 .
(3) 도 4 및 5를 보면, 용매 A 또는 B를 단독으로 사용하여 고정화 확인용 DNA를 스팟팅 했을 경우 DNA가 스팟의 가장자리 또는 중심부에 몰려서 균일도가 낮가 스팟 사이즈가 너무 작아지는 등 고정화 효율이 좋이 않은데 반해, 용매 A와 B를 섞어서 사용했을 경우 DNA가 스팟 내에 고르게 분포되었고 스팟의 사이즈도 일정함을 확인하였으며, 특히 용매 A와 B를 1:1로 섞은 용매를 사용했을 때 고정화 효율이 가장 좋았다. 이후, 실험에서는 캡처 프로브의 용매로 용매 A와 B를 1:1로 섞은 용매를 사용하였다.(3) Referring to FIGS. 4 and 5, when spotting DNA for immobilization confirmation using solvent A or B alone, DNA is concentrated at the edge or center of the spot, resulting in low uniformity and a too small spot size. On the other hand, when mixing solvents A and B, it was confirmed that DNA was evenly distributed in the spot and the size of the spot was constant. It was the best. Then, in the experiment, a solvent in which solvents A and B were mixed at a ratio of 1:1 was used as a solvent for the capture probe.
2. 캡처 프로브가 COC 기판에 고정화될 수 있는지 확인2. Confirm that the capture probe can be immobilized on the COC substrate
(1) COC 기판에 농도별 실시예 1의 1에서 준비된 캡쳐 프로브(단, 3' 말단에 cy3이 컨쥬게이션됨)를 20ul 씩 넣고 건조하고, 자외선 조사를 실시하여 고정하고, 세척하고 건조한 후 cy3의 형광값을 측정(excitation 530nm, emission 575nm)하여, 그 결과를 도 6에 나타내었다.(1) 20 ul each of the capture probe prepared in 1 of Example 1 for each concentration (where cy3 is conjugated to the 3' end) is put on a COC substrate, dried, irradiated with ultraviolet light, fixed, washed, dried, and then cy3 The fluorescence value of was measured (excitation 530 nm, emission 575 nm), and the results are shown in FIG. 6 .
(2) 도 6를 보면, 상기 캡쳐 프로브의 농도가 증가할수록 형광값이 커짐을 알 수 있어, 링커 염기서열을 가지는 캡처 프로브가 COC 기판에 잘 고정됨을 알 수 있다.(2) Referring to FIG. 6, it can be seen that the fluorescence value increases as the concentration of the capture probe increases, indicating that the capture probe having the linker sequence is well immobilized on the COC substrate.
<실시예 4> Spacer 영역 길이에 따른 RCA 효율 확인<Example 4> Confirmation of RCA efficiency according to spacer region length
1. 실시예 1의 1에서 준비된 캡쳐 프로브와 Spacer 영역의 길이를 달리하는 비교용 캡쳐 프로브[5'-TTTTTTTTTTCCCCCCCCCCTAATCATGAGTCCAAGAGC-'3(서열번호:16)]를 준비하였다.1. A capture probe prepared in Example 1-1 and a capture probe [5'-TTTTTTTTTTCCCCCCCCCCTAATCATGAGTCCAAGAGC-'3 (SEQ ID NO: 16)] for comparison having different spacer region lengths were prepared.
2. DW에 녹여진 실시예 1의 1에서 준비된 캡처 프로브 또는 실시예 4의 1에서 준비된 비교용 캡쳐 프로브 20ul를 COC 기판에 도포하고, 건조(RT 16h, dry oven 30min), 고정(UV 30min), 세척(SSC washing 10min) 및 건조(dry oven 30min)를 차례로 수행하였다.2. 20ul of the capture probe prepared in Example 1-1 dissolved in DW or the comparison capture probe prepared in Example 4-1 was applied to a COC substrate, dried (RT 16h, dry oven 30min), fixed (UV 30min) , washing (SSC washing 10 min) and drying (dry oven 30 min) were sequentially performed.
3. 비교용 캡쳐 프로브(probe A), 캡처 프로브(probe B)가 각각 고정된 COC 기판에 50% Hybrid Buffer에 녹여진 실시예 1의 3에서 준비된 500nM 원형 DNA 1 20ul를 도포하여, 30℃에서 1시간 혼성화시키고, 세척(SSC washing 10min) 및 건조(dry oven 30min)를 차례로 수행하였다.3. 1 20ul of 500nM circular DNA prepared in Example 1-3 dissolved in 50% Hybrid Buffer was applied to the COC substrate on which the capture probe (probe A) and the capture probe (probe B) were immobilized, respectively, at 30 ° C. Hybridization was performed for 1 hour, washing (SSC washing 10 min) and drying (dry oven 30 min) were sequentially performed.
4. 3과정 이후, 5x SYBR green 2 20ul를 추가하여 염색한 후, 형광값(EX:480 / Em:522)을 측정하여, 그 결과를 도 7에 나타내었다. 또한, 3과정 이후, RCA 반응용액(1x buffer 3ul, 1mM dNTP 3ul, 20U/30ul Phi29 2ul, DW 22ul)을 추가하여 2시간 동안 RCA 반응시키고, 5x SYBR green 2 20ul를 추가하여 염색한 후, 형광값(EX:480 / Em:522)을 측정하여, 그 결과를 도 8에 나타내었다. 4. After step 3, after staining by adding 20ul of 5x SYBR green 2, the fluorescence value (EX: 480 / Em: 522) was measured, and the results are shown in FIG. 7. In addition, after step 3, RCA reaction solution (1x buffer 3ul, 1mM dNTP 3ul, 20U/30ul Phi29 2ul, DW 22ul) was added to RCA reaction for 2 hours, 5x SYBR green 2 20ul was added for staining, fluorescence Values (EX: 480 / Em: 522) were measured, and the results are shown in FIG. 8 .
5. 도 7를 보면 캡처 프로브 종류에 따른 COC 기판의 고정 효율이 비슷한 것을 확인할 수 있으나, 도 8을 보면 캡쳐 프로브(probe B)에서만 양에 비례하여 RCA 반응이 이루어진 것을 확인할 수 있어, 캡처 프로브의 길이가 너무 짧다면 입체 장애로 인해 RCA를 저해할 수 있음을 알 수 있다.5. Looking at FIG. 7, it can be seen that the fixation efficiency of the COC substrate is similar according to the type of capture probe, but looking at FIG. 8, it can be seen that the RCA reaction is made in proportion to the amount only in the capture probe (probe B), It can be seen that if the length is too short, it can inhibit RCA due to steric hindrance.
<실시예 5> CuNP가 합성됨을 확인 및 RCA 테스트<Example 5> Confirming that CuNP is synthesized and RCA test
1. 실시예 1의 1에서 준비된 캡처 프로브(400nM), 실시예 1의 3에서 준비된 원형 DNA 1(200nM), 1x RCA buffer 및 1.25 mM dNTP mixture 및 30 units of phi29 DNA polymerase를 튜브에 혼합하고 30℃에서 90분 동안 RCA 반응시키고 65℃에서 10분 동안 불활성화시켰다. 이후, 2mM ascorbate, 0.5mM CuSO4 및 1x MOPS 퍼버를 RCA 결과물에 추가하고 5분 동안 반응시켜 구리 나노입자를 합성한 후, 투과전자현미경(TEM)으로 확인하여 그 결과를 도 9(스케일바는 100nm임)에 나타내었다. 1. Capture probe (400nM) prepared in Example 1-1, circular DNA 1 (200nM) prepared in Example 1-3, 1x RCA buffer, 1.25 mM dNTP mixture, and 30 units of phi29 DNA polymerase were mixed in a tube and 30 RCA reacted for 90 min at °C and inactivated at 65 °C for 10 min. Then, 2mM ascorbate, 0.5mM CuSO 4 and 1x MOPS buffer were added to the RCA result and reacted for 5 minutes to synthesize copper nanoparticles, and then confirmed by transmission electron microscopy (TEM), and the results are shown in FIG. 9 (scale bar is 100 nm).
2. 도 9를 보면, RCA 반응이 일어난 좌측 이미지에서만 수 나노미터의 구리나노입자가 생성됨을 확인할 수 있어, 캡쳐 프로브와 원형 DNA 1이 혼성화된 후 RCA 반응을 통해 poly(T)가 생성되어 이를 template로 하여 CuNP가 합성됨을 알 수 있다.2. Referring to FIG. 9, it can be confirmed that copper nanoparticles of several nanometers are generated only in the left image where the RCA reaction has occurred. After the capture probe and circular DNA 1 are hybridized, poly(T) is generated through the RCA reaction, It can be seen that CuNP is synthesized using the template.
3. 실시예 1의 1에서 준비된 캡처 프로브(20uM)를 자외선 조사를 통해 COC 기판에 고정하고, 실시예 1의 3에서 준비된 농도를 달리하는 원형 DNA 1(20uL)를 추가하고 30℃에서 1시간 유지하였다. 이후, 1x RCA buffer, 1 mM dNTP mixture 및 30 units of phi29 DNA polymerase을 포함하는 RCA 용액(40uL)을 추가하고 30℃에서 2시간 인큐베이션시키고 65℃에서 10분 동안 불활성화시켰다. 이후, 4mM ascorbate 및 1mM CuSO4를 RCA 결과물에 추가하고 RT에서 10분 동안 반응시켜 구리 나노입자를 합성한 후, 마이크로 플레이트 리더기를 사용하여 형광 강도를 측정하여 그 결과를 도 10에 나타내었다.3. The capture probe (20uM) prepared in Example 1-1 was immobilized on a COC substrate by UV irradiation, and circular DNA 1 (20uL) having different concentrations prepared in Example 1-3 was added and incubated at 30°C for 1 hour. maintained. Thereafter, an RCA solution (40uL) containing 1x RCA buffer, 1 mM dNTP mixture, and 30 units of phi29 DNA polymerase was added, incubated at 30°C for 2 hours, and inactivated at 65°C for 10 minutes. Thereafter, 4 mM ascorbate and 1 mM CuSO 4 were added to the RCA product and reacted at RT for 10 minutes to synthesize copper nanoparticles, and then the fluorescence intensity was measured using a microplate reader, and the results are shown in FIG. 10 .
4. 도 10을 통해, 원형 DNA 1의 농도 증가에 따라 형광 세기가 증가함을 알 수 있다.4. It can be seen from FIG. 10 that the fluorescence intensity increases as the concentration of circular DNA 1 increases.
<실시예 6> Au@Ag cluster 생성됨을 확인<Example 6> Confirmation of creation of Au@Ag cluster
1. DW에 녹여진 20uM의 실시예 1의 1에서 준비된 캡처 프로브 20ul를 COC 기판에 도포하고, 건조(RT 16h, dry oven 30min), 고정(UV 30min), 세척(SSC washing 10min) 및 건조(dry oven 30min)를 차례로 수행하여, COC 기판에 캡처 프로브를 고정하였다.1. 20uM of the capture probe prepared in 1 of Example 1 dissolved in DW was applied to a COC substrate, dried (RT 16h, dry oven 30min), fixed (UV 30min), washed (SSC washing 10min) and dried ( dry oven 30 min) was performed sequentially, and the capture probe was fixed to the COC substrate.
2. 캡처 프로브가 고정된 COC 기판에 50% Hybrid Buffer에 녹여진 실시예 1의 4에서 준비된 (0 or 1.9uM) 원형 DNA 2(Circular DNA) 20ul를 도포하여, 30℃에서 1시간 혼성화시키고, 세척(SSC washing 10min) 및 건조(dry oven 30min)를 차례로 수행하였다. 이후, RCA 반응용액(1x buffer 3ul, 1mM dNTP 3ul, 20U/30ul Phi29 2ul, DW 22ul)을 추가하여 2시간 동안 RCA 반응시키고, 세척(SSC washing 10min)을 하였다.2. 20ul of circular DNA 2 (Circular DNA) prepared in Example 1-4 dissolved in 50% Hybrid Buffer was applied to the COC substrate on which the capture probe was immobilized, hybridized at 30 ° C for 1 hour, Washing (SSC washing 10min) and drying (dry oven 30min) were sequentially performed. Thereafter, RCA reaction solution (1x buffer 3ul, 1mM dNTP 3ul, 20U/30ul Phi29 2ul, DW 22ul) was added to RCA reaction for 2 hours, followed by washing (SSC washing 10min).
3. 이후, 실시예 1의 5에서 준비된 금 프로브(in 50% hybrid Buffer) 20ul를 도포하고 34℃에서 60분 동안 혼성화시키고, 추가로 Silver enhancement 용액 50ul를 도포하고 상온에서 20분 반응 후, 세척하고 건조한 후, gel-DOC 및 주사전자 현미경(SEM)을 촬영하여 그 결과를 도 11에 나타내었다. 도 11에서 inset 이미지는 gel-DOC 촬영 결과를 나타낸다.3. Then, 20ul of the gold probe (in 50% hybrid buffer) prepared in 5 of Example 1 was applied and hybridized at 34 ° C for 60 minutes, additionally applied with 50ul of silver enhancement solution, reacted for 20 minutes at room temperature, and washed After drying, gel-DOC and scanning electron microscopy (SEM) were taken, and the results are shown in FIG. 11 . The inset image in FIG. 11 shows the gel-DOC imaging result.
4. 도 11을 보면, 원형 DNA 2가 혼합되어 RCA가 진행된 오른쪽 이미지에서만 RCA 생성물로 보이는 실타래 모양의 ssDNA에 Au@Ag가 합성된 것을 확인할 수 있어, 캡쳐 프로브와 원형 DNA 2이 혼성화된 후 RCA 반응이 이루어지고, 금 프로브가 RCA 생성물에 혼성화되고 Au 프로브가 결합하여 Au@Ag cluster가 합성됨을 알 수 있다.4. Referring to FIG. 11, it can be confirmed that Au@Ag was synthesized into thread-shaped ssDNA, which appears to be an RCA product, only in the right image where circular DNA 2 was mixed and RCA was performed, and after hybridization of the capture probe and circular DNA 2, RCA It can be seen that the reaction is performed, the gold probe is hybridized to the RCA product, and the Au probe is bound to synthesize the Au@Ag cluster.
<실시예 7> 금나노입자 크기에 따른 비색 신호 확인<Example 7> Confirmation of colorimetric signal according to the size of gold nanoparticles
1. 금 나노입자의 크기(10, 20, 40nm)에 따른 비색 신호를 비교하기 위하여, 20uM 대신에 10uM 캡처 프로브가 사용되고, Silver enhancement 용액 50ul를 도포하고 상온에서 20분 반응하는 과정을 2회 실시하고, 금 프로브의 금 나노입자가 20nm뿐만 아니라 10 및 40nm를 가지도록 한 것을 제외하고는, 다른 조건을 실시예 6과 동일하게 실험하고, 흡광도(360nm)를 측정하여 그 결과를 도 12에 나타내었다(ΔAbs=Abs(w/cirDNA)/Abs(w/o cirDNA)).1. To compare colorimetric signals according to the size of gold nanoparticles (10, 20, 40nm), a 10uM capture probe is used instead of 20uM, 50ul of silver enhancement solution is applied, and the process of reacting for 20 minutes at room temperature is carried out twice And, except that the gold nanoparticles of the gold probe had 20 nm as well as 10 and 40 nm, the other conditions were the same as in Example 6, and the absorbance (360 nm) was measured, and the results are shown in FIG. (ΔAbs=Abs (w/cirDNA) /Abs (w/o cirDNA) ).
2. ΔAbs가 1 이상의 값은 RCA가 진행되었을 때 비색 신호가 증가했음을 나타내는데, 도 12를 보면, 20nm의 금 나노입자가 사용되었을 때 비색 신호 증가량이 가장 큼을 확인할 수 있다.2. A value of ΔAbs of 1 or more indicates that the colorimetric signal increased when RCA was performed. Referring to FIG. 12, it can be seen that the colorimetric signal increase was the largest when 20 nm gold nanoparticles were used.
<실시예 8> DZN의 검출 및 선택성 확인<Example 8> Detection of DZN and confirmation of selectivity
1. 실시예 1의 1에서 준비된 20uM 캡쳐 프로브(20uL)를 자외선 조사를 통해 COC 기판의 각 웰에 고정하고, 혼성화 버퍼 내의 실시예 1의 2에서 준비된 20uM 압타머(20uL)를 추가하고 34℃에서 1시간 배양하였다. 이후 1x binding buffer에 DZN이 용해되어 다양한 농도를 가지는 DZN 용액을 각 웰에 추가하고 30℃에서 1시간 배양하고, 혼성화 버퍼 내의 실시예 1의 3에서 준비된 2uM 원형 DNA 1(20uL)를 추가하고 30℃에서 1시간 배양하였다. 이후, 1x RCA buffer, 1 mM dNTP mixture 및 30 units of phi29 DNA polymerase을 포함하는 RCA 용액(40uL)을 각 웰에 추가하고 30℃에서 5시간 인큐베이션시키고 65℃에서 10분 동안 불활성화시키고, 4mM ascorbate 및 1mM CuSO4를 각 웰에 추가하고 RT에서 10분 동안 반응시켰다. Alliance Mini HD9 system(UVITEC, USA)을 이용하여 이미지화하고, 이미지 분석 소프트웨어(Nine-Alliance Q9)를 사용하여 CuNPs의 형광을 정량화하여 그 결과를 13에 나타내었다.1. The 20uM capture probe (20uL) prepared in Example 1-1 was fixed to each well of the COC substrate by UV irradiation, and the 20uM aptamer (20uL) prepared in Example 1-2 in the hybridization buffer was added and heated at 34 ° C. incubated for 1 hour. Thereafter, DZN was dissolved in 1x binding buffer, and DZN solutions having various concentrations were added to each well, incubated at 30 ° C for 1 hour, 2uM circular DNA 1 (20uL) prepared in Example 1-3 in hybridization buffer was added, and 30 Incubated for 1 hour at ° C. Then, 1x RCA buffer, 1 mM dNTP mixture, and RCA solution (40uL) containing 30 units of phi29 DNA polymerase were added to each well, incubated at 30 ° C for 5 hours, inactivated at 65 ° C for 10 minutes, and 4 mM ascorbate and 1 mM CuSO 4 were added to each well and reacted at RT for 10 minutes. It was imaged using the Alliance Mini HD9 system (UVITEC, USA), and the fluorescence of the CuNPs was quantified using the image analysis software (Nine-Alliance Q9), and the results are shown in 13.
2. 도 13을 보면, DZN의 농도가 증가함에 따라 CuNPs의 형광 세기가 점진적으로 증가함을 확인할 수 있어(DZN의 농도가 3ppm보다 큰 경우 포화됨), 본원발명의 검출방법은 효과적으로 DZN을 검출할 수 있음을 알 수 있다.2. Referring to FIG. 13, it can be seen that the fluorescence intensity of CuNPs gradually increases as the concentration of DZN increases (it is saturated when the concentration of DZN is greater than 3 ppm), so the detection method of the present invention effectively detects DZN know you can do it.
3. 다양한 농도를 가지는 DZN 대신에 농도가 4ppm으로 고정된 DZN, fenitrothion, abamectin, fipronil 각각을 사용하여 실시예 8의 1과 같이 실험하여 그 결과를 도 14에 나타내었다. 도 14를 보면, DZN을 사용했을 때만 형광 세기 큰 것을 알 수 있어 상기 검출방법은 DZN을 선택적으로 검출 할 수 있음을 알 수 있다.3. Instead of DZN having various concentrations, DZN, fenitrothion, abamectin, and fipronil each having a fixed concentration of 4 ppm were used to conduct the experiment as in Example 8-1, and the results are shown in FIG. 14. Referring to FIG. 14, it can be seen that fluorescence intensity is high only when DZN is used, indicating that the detection method can selectively detect DZN.
이상에서, 출원인은 본 발명의 바람직한 실시예들을 설명하였지만, 이와 같은 실시예들은 본 발명의 기술적 사상을 구현하는 일 실시예일 뿐이며 본 발명의 기술적 사상을 구현하는 한 어떠한 변경예 또는 수정예도 본 발명의 범위에 속하는 것으로 해석되어야 한다.In the above, the applicant has described the preferred embodiments of the present invention, but these embodiments are only one embodiment of implementing the technical idea of the present invention, and any changes or modifications are the same as those of the present invention as long as they implement the technical idea of the present invention. should be construed as falling within the scope.
[규칙 제26조에 의한 보정 23.09.2022]　
[Amendment 23.09.2022 under Rule 26]
[규칙 제26조에 의한 보정 23.09.2022]　
Figure WO-DOC-FIGURE-108
[Amendment 23.09.2022 under Rule 26]
Figure WO-DOC-FIGURE-108
[규칙 제26조에 의한 보정 23.09.2022]　
[Amendment 23.09.2022 under Rule 26]
[규칙 제26조에 의한 보정 23.09.2022]　
[Amendment 23.09.2022 under Rule 26]
[규칙 제26조에 의한 보정 23.09.2022]　
[Amendment 23.09.2022 under Rule 26]
[규칙 제26조에 의한 보정 23.09.2022]　
Figure WO-DOC-FIGURE-112
[Amendment 23.09.2022 under Rule 26]
Figure WO-DOC-FIGURE-112

Claims (7)

  1. 기판에 고정화되어 링커 역할을 하는 염기서열과, 농약에 선택적인 분자친화력을 가지는 압타머와 상보적 결합이 가능하도록 하는 염기서열을 포함하는 캡처 프로브를 기판에 고정하여 칩을 준비하는 칩준비단계와; 농약에 선택적인 분자친화력을 가지는 압타머를 상기 칩에 분포시켜, 상기 캡처 프로브와 압타머를 부분적 결합시키는 혼성화단계와; 상기 칩에 측정 대상 농약을 분포시켜, 선택적인 분자친화력에 의해 압타머가 캡처 프로브에서 떨어져 농약과 결합하도록 하는 압타머분리단계와; 상기 압타머분리단계 후, 상기 캡처 프로브와 상보적 결합이 가능하도록 하는 염기서열과, 금속체와 상보적 결합이 가능하도록 하는 염기서열과 상보적인 염기서열을 포함하는 원형 DNA, 및 중합효소를 상기 칩에 공급하고 RCA 반응시켜, 원형 DNA가 상기 캡처 프로브에 부분적으로 결합하여 상기 캡처 프로브에 연결되며 상기 금속체와 상보적 결합이 가능하도록 하는 염기서열을 반복하여 가지는 단일 가닥을 형성하는 RCA반응단계와; 상기 RCA반응단계를 통해 형성된 단일 가닥을 템플레이트로 하여 신호 증폭을 위한 금속체를 형성하는 금속체형성단계;를 포함하는 것을 특징으로 하는 식품 유해 물질 검출 방법.A chip preparation step of preparing a chip by immobilizing a capture probe containing a nucleotide sequence immobilized on a substrate to act as a linker and a nucleotide sequence enabling complementary binding with an aptamer having selective molecular affinity for pesticides to the substrate; and ; a hybridization step of partially binding the capture probe and the aptamer by distributing an aptamer having a selective molecular affinity to the pesticide on the chip; An aptamer separation step of distributing the pesticide to be measured on the chip so that the aptamer separates from the capture probe and binds to the pesticide by selective molecular affinity; After the aptamer separation step, a circular DNA comprising a nucleotide sequence enabling complementary binding with the capture probe, a nucleotide sequence complementary with a nucleotide sequence enabling complementary binding with the metal body, and a polymerase as described above. RCA reaction step of supplying to a chip and conducting RCA reaction to form a single strand having circular DNA partially bound to the capture probe, linked to the capture probe, and having a base sequence repeating to enable complementary binding with the metal body. and; A metal body formation step of forming a metal body for signal amplification using the single strand formed through the RCA reaction step as a template; food harmful substance detection method comprising a.
  2. 제1항에 있어서, According to claim 1,
    상기 식품 유해 물질 검출 방법은, 상기 금속체형성단계 후 상기 칩에 빛을 조사하고 광학신호를 측정하여 농약의 존재 및 그 양을 검출하는 검출단계;를 추가로 포함하는 것을 특징으로 하는 식품 유해 물질 검출 방법.The food hazardous substance detection method further comprises a detection step of irradiating light onto the chip and measuring an optical signal to detect the presence and amount of pesticide after the metal body forming step. detection method.
  3. 제1항에 있어서,According to claim 1,
    상기 금속체와 상보적 결합이 가능하도록 하는 염기서열과 상보적인 염기서열은 복수 개가 연이어진 아데닌으로 이루어져, 상기 가닥은 복수의 티민으로 형성되게 되며, The nucleotide sequence enabling complementary bonding with the metal body and the complementary nucleotide sequence are composed of a plurality of consecutive adenines, so that the strand is formed of a plurality of thymines,
    상기 금속체형성단계에서는 RCA반응단계 후 구리 이온과 아스코베이트를 포함하는 용액을 칩에 주입하여, 상기 복수의 티민을 템플레이트로 하여 구리 나노입자를 형성하는 것을 특징으로 하는 식품 유해 물질 검출 방법.In the metal body formation step, after the RCA reaction step, a solution containing copper ions and ascorbate is injected into the chip to form copper nanoparticles using the plurality of thymine as a template.
  4. 제1항에 있어서,According to claim 1,
    상기 금속체와 상보적 결합이 가능하도록 하는 염기서열과 상보적인 염기서열은 금 프로브와 상보적 결합이 가능하도록 하는 염기서열과 상보적인 염기서열로 이루어져, 상기 가닥은 금 프로브와 상보적 결합이 가능하도록 하는 염기서열로 형성되게 되며,The nucleotide sequence enabling complementary binding with the metal body and the complementary nucleotide sequence are composed of a nucleotide sequence enabling complementary binding with the gold probe and a complementary nucleotide sequence, and the strand is capable of complementary binding with the gold probe. It is formed with a nucleotide sequence that allows
    상기 금속체형성단계에서는 제1반응기가 결합된 특정 ssDNA에, 상기 제1반응기와 결합하는 제2반응기가 결합된 금 나노입자를 반응시켜, 특정 ssDNA에 금이 결합된 금 프로브를 형성하고, 상기 금 프로브를 칩에 주입하여 상기 가닥에 금 프로브를 결합시키고, 은 이온과 환원제를 추가로 공급하여, 금 나노입자 표면에 은 클러스터를 형성하는 것을 특징으로 하는 식품 유해 물질 검출 방법.In the metal body formation step, the gold nanoparticles to which the second reactor to bind to the first reactor reacts with the specific ssDNA to which the first reactor binds, to form a gold probe in which gold is coupled to the specific ssDNA; A method for detecting harmful substances in food, characterized by forming silver clusters on the surface of gold nanoparticles by injecting a gold probe into a chip to bind the gold probe to the strand, and additionally supplying silver ions and a reducing agent.
  5. 제1항에 있어서,According to claim 1,
    상기 기판은 COC 기판이 사용되며, 상기 캡처 프로브는 다이아지논을 검출하기 위해 서열번호 4의 염기서열을 가지는 것을 특징으로 하는 식품 유해 물질 검출 방법.The substrate is a food hazardous substance detection method, characterized in that the COC substrate is used, and the capture probe has the nucleotide sequence of SEQ ID NO: 4 to detect diazinon.
  6. 제5항에 있어서,According to claim 5,
    상기 원형 DNA는 서열번호 7의 염기서열을 가지는 것을 특징으로 하는 식품 유해 물질 검출 방법.The circular DNA is a method for detecting harmful substances in food, characterized in that it has a nucleotide sequence of SEQ ID NO: 7.
  7. 제5항에 있어서,According to claim 5,
    상기 원형 DNA는 서열번호 9의 염기서열을 가지는 것을 특징으로 하는 식품 유해 물질 검출 방법.The circular DNA is a method for detecting harmful substances in food, characterized in that it has a nucleotide sequence of SEQ ID NO: 9.
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