WO2024036801A1 - 金属纳米簇及其制备方法和检测嗅觉受体激活信号的方法 - Google Patents

金属纳米簇及其制备方法和检测嗅觉受体激活信号的方法 Download PDF

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WO2024036801A1
WO2024036801A1 PCT/CN2022/133780 CN2022133780W WO2024036801A1 WO 2024036801 A1 WO2024036801 A1 WO 2024036801A1 CN 2022133780 W CN2022133780 W CN 2022133780W WO 2024036801 A1 WO2024036801 A1 WO 2024036801A1
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atp
contact treatment
metal
contact
ions
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French (fr)
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任航
刘卫红
郑玉
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汉王科技股份有限公司
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures
    • 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
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • 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
    • 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
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

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  • the present invention relates to the field of analysis and detection. Specifically, the present invention relates to a metal nanocluster and a preparation method thereof and a method for detecting olfactory receptor activation signals. More specifically, the present invention relates to a silver nanocluster and a metal nanocluster. Preparation method, kit and method for detecting olfactory receptor activation signals.
  • odorant/olfactory receptor pairing A comprehensive understanding of the mechanism of odorant/olfactory receptor pairing will help promote the rapid development of the field of olfactory perception.
  • basic research it helps to understand the molecular mechanism of smell and establish a model for animals to perceive the chemical environment.
  • industrial applications it helps in the screening and design of odor enhancers/blockers, and the development of new biogas-sensitive sensing devices.
  • Detection of odorant/olfactory receptor pairing is determined by measuring the activation signal strength of olfactory receptors stimulated by the odorant.
  • Current methods for detecting odorant/olfactory receptor pairings have shortcomings such as complex operations and high detection costs.
  • the present invention aims to solve at least one of the technical problems existing in the prior art to at least a certain extent.
  • the present invention provides a method for detecting olfactory receptor activation signals.
  • the method of the present invention can detect the activation signals of olfactory receptors and has the advantages of easy operation and low detection cost.
  • odorant/olfactory receptor pairing research relies on high-throughput screening methods.
  • the established high-throughput screening methods include the following methods for detecting olfactory receptor activation signals:
  • Ca 2+ is a sensitive dye, Fura-2 or Calcium 6, etc. can combine with free Ca 2+ and produce fluorescence after being excited by a specific wavelength light source. The signal intensity of this fluorescence is proportional to the concentration of free Ca 2+ .
  • the patch clamp method is to detect the activation signal of olfactory receptors by measuring the opening of corresponding ion channels on the cell membrane caused by the activation of olfactory receptors by odorants to generate ion currents.
  • Odorants stimulate living mice or isolated olfactory tissues, isolate activated olfactory neurons, and perform RNA library construction and sequencing analysis to identify activated olfactory receptors.
  • the present invention proposes a silver nanocluster.
  • the silver nanoclusters include: nucleic acid molecules and Ag ions.
  • the nucleic acid molecules have the nucleotide sequence shown in CCCCCCCCCC.
  • the Ag ions and the nucleic acid molecules are cross-linked.
  • the Ag ions are cross-linked with each other.
  • the molar ratio of ions to the nucleic acid molecules is 6:1.
  • the inventor found through experiments that contacting ATP with the silver nanoclusters of the present invention can significantly improve the fluorescence signal of the silver nanoclusters, and the fluorescence signals of metal nanoclusters in contact with different final concentrations of ATP are well differentiated.
  • the silver nanoclusters of the present invention can be used to effectively detect the consumption of ATP, and according to the properties of activated olfactory receptors, protein kinase A (PKA) can be activated and activated PKA can consume ATP (i.e., convert ATP into ADP) , which can be used to detect activation signals of olfactory receptors.
  • PKA protein kinase A
  • the invention proposes a method for preparing metal nanoclusters.
  • the method includes: subjecting metal ions, templates and reducing agents to a first mixing process to obtain the metal nanoclusters; wherein the first mixing process is performed at 20-30°C. The process is carried out for 20 to 40 minutes; the template includes at least one selected from DNA, RNA, protein, polypeptide, high molecular polymer and sulfhydryl small molecule.
  • Metal nanoclusters can be prepared according to the method of the embodiment of the present invention, which has the advantages of simple preparation method; and, the fluorescence signal of the metal nanoclusters of the present invention can be significantly improved after contact with ATP, and the metal nanoclusters in contact with different final concentrations of ATP The fluorescence signal of nanoclusters has good discrimination. Therefore, the metal nanoclusters of the present invention can be used to effectively detect the consumption of ATP, and according to the properties that activated olfactory receptors can activate PKA and activated PKA can consume ATP, they can be used to detect the activation of olfactory receptors. Signal.
  • the present invention proposes a metal nanocluster.
  • the metal nanoclusters are obtained according to the aforementioned method.
  • Metal nanoclusters according to embodiments of the present invention can significantly increase fluorescence signals after contact with ATP, and the fluorescence signals of metal nanoclusters in contact with different final concentrations of ATP have good discrimination. Therefore, the metal nanoclusters of the present invention can be used to effectively detect the consumption of ATP, and according to the properties that activated olfactory receptors can activate PKA and activated PKA can consume ATP, they can be used to detect the activation of olfactory receptors. Signal.
  • the invention provides a kit.
  • the kit includes: metal nanoclusters prepared according to the aforementioned method or the aforementioned metal nanoclusters.
  • the kit according to the embodiment of the present invention can effectively detect the consumption of ATP, and is used to detect the activation signal of the olfactory receptor based on the properties that the activated olfactory receptor can activate PKA and the activated PKA can consume ATP and turn it into ADP. .
  • the invention proposes a method for detecting olfactory receptor activation signals.
  • the method includes: subjecting a first cell to a first contact treatment with ATP, wherein the first cell expresses an olfactory receptor, and the first cell is pre-contacted with an odorant;
  • the first contact treatment product is subjected to a second contact treatment with the aforementioned silver nanoclusters, the aforementioned metal nanoclusters or the aforementioned kit; the first fluorescence value of the second contact treatment product is detected; the result based on the first fluorescence value , in order to determine the activation signal of the olfactory receptor.
  • Methods according to embodiments of the present invention can effectively detect activation signals of olfactory receptors, and have the advantages of easy operation, short detection time, and low detection cost.
  • Figure 1 is the ultraviolet spectrum of silver nanoclusters 1 in Example 1 of the present invention.
  • Figure 2 shows the fluorescence signal detection results after mixing silver nanoclusters 1 with different concentrations of ATP in Example 1 of the present invention
  • Figure 3 shows the fluorescence signal detection results after mixing silver nanoclusters 2 with different concentrations of ATP in Example 1 of the present invention
  • Figure 4 shows the fluorescence signal detection results after mixing silver nanoclusters 3 with different concentrations of ATP in Example 1 of the present invention
  • Figure 5 shows the fluorescence signal detection results after mixing silver nanoclusters 1 with different concentrations of ATP in Example 2 of the present invention
  • Figure 6 is a schematic diagram of silver nanoclusters 1 detecting the activation of olfactory receptors in Example 3 of the present invention
  • Figure 7 is the detection result of the activation signal of olfactory receptor OR1A1 by silver nanocluster 1 in Example 3 of the present invention.
  • Figure 8 is the detection result of the activation signal of olfactory receptor OR2W1 by silver nanocluster 1 in Example 3 of the present invention.
  • Figure 9 is the detection result of the activation signal of olfactory receptor mTAAR7f by silver nanocluster 1 in Example 3 of the present invention.
  • first and second are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. Therefore, features defined as “first” and “second” may explicitly or implicitly include one or more of these features. Further, in the description of the present invention, unless otherwise stated, the meaning of "plurality” is two or more.
  • the terms “optionally,” “optionally,” or “optionally” generally mean that the subsequently described event or condition may but need not occur, and that the description includes instances in which the event or condition occurs, and A situation in which the event or condition did not occur.
  • room temperature generally refers to 20 to 25°C.
  • the present invention proposes a silver nanocluster, a metal nanocluster and a preparation method thereof, a kit and a method for detecting olfactory receptor activation signals, which will be described in detail below.
  • the invention proposes a silver nanocluster.
  • the silver nanoclusters include: nucleic acid molecules and Ag ions.
  • the nucleic acid molecules have the nucleotide sequence shown in CCCCCCCCCC.
  • the Ag ions and the nucleic acid molecules are cross-linked.
  • the Ag ions are cross-linked with each other.
  • the molar ratio of ions to the nucleic acid molecules is 6:1.
  • the silver nanoclusters of the present invention can be used to effectively detect the consumption of ATP, and according to the properties that activated olfactory receptors can activate protein kinase A (PKA) and activated PKA can consume ATP, they can be used to detect Activating signals of olfactory receptors.
  • PKA protein kinase A
  • the invention proposes a method for preparing metal nanoclusters.
  • the method includes: subjecting metal ions, templates and reducing agents to a first mixing process to obtain the metal nanoclusters; wherein the first mixing process is performed at 20-30°C. The process is carried out for 20 to 40 minutes; the template includes at least one selected from DNA, RNA, protein, polypeptide, high molecular polymer and sulfhydryl small molecule.
  • Metal nanoclusters can be prepared according to the method of the embodiment of the present invention, which has the advantages of simple preparation method; and, the fluorescence signal of the metal nanoclusters of the present invention can be significantly improved after contact with ATP, and the metal nanoclusters in contact with different final concentrations of ATP The fluorescence signal of nanoclusters has good discrimination. Therefore, the metal nanoclusters of the present invention can be used to effectively detect the consumption of ATP, and according to the properties that activated olfactory receptors can activate PKA and activated PKA can consume ATP, they can be used to detect the activation of olfactory receptors. Signal.
  • the method further includes: treating the first mixed treatment product at 2 to 6° C. under light-proof conditions for 8 to 20 hours.
  • metal ions can be further reduced into metal atoms, thereby increasing the fluorescence signal intensity of metal nanoclusters.
  • the metal ions and the template are premixed in advance.
  • the metal ions and the template can be thoroughly mixed.
  • the premixing process is performed at room temperature for 20 to 50 minutes.
  • the metal ions and the template are thoroughly mixed.
  • the template is DNA
  • the DNA includes at least one selected from the group consisting of single-stranded DNA, hairpin DNA and double-stranded DNA, preferably single-stranded DNA.
  • the nucleotides of the DNA include at least one selected from the group consisting of cytosine nucleotides, uracil nucleotides, adenine nucleotides, and guanine nucleotides.
  • the DNA is a single strand of cytosine nucleotides.
  • the number of nucleotides in the single strand of cytosine nucleotides is 8 to 15.
  • the DNA has a nucleotide sequence as shown in SEQ ID NO: 1.
  • SEQ ID NO: 1 the fluorescence signal of the prepared metal nanoclusters is relatively strong.
  • the metal ions include at least one selected from gold ions, silver ions, copper ions and platinum ions, preferably silver ions.
  • the metal ions are added to the solution in the form of metal salts.
  • the silver ions are provided by AgNO3 .
  • the molar ratio of the metal ions to the template is (4.8-6.5):1.
  • the fluorescence signal of the prepared metal nanoclusters is relatively strong.
  • the reducing agent is NaBH 4 .
  • the molar ratio of the metal ions and NaBH 4 is (0.5 ⁇ 1.5):1.
  • the fluorescence signal of the prepared metal nanoclusters is relatively strong.
  • the present invention proposes a metal nanocluster.
  • the metal nanoclusters are obtained according to the aforementioned method.
  • Metal nanoclusters according to embodiments of the present invention can significantly increase fluorescence signals after contact with ATP, and the fluorescence signals of metal nanoclusters in contact with different final concentrations of ATP have good discrimination. Therefore, the metal nanoclusters of the present invention can be used to effectively detect the consumption of ATP, and according to the properties that activated olfactory receptors can activate PKA and activated PKA can consume ATP, they can be used to detect the activation of olfactory receptors. Signal.
  • the invention provides a kit.
  • the kit includes: metal nanoclusters prepared according to the aforementioned method or the aforementioned metal nanoclusters.
  • the kit according to the embodiment of the present invention can effectively detect the consumption of ATP, and is used to detect the activation signal of the olfactory receptor according to the properties that the activated olfactory receptor can activate PKA and the activated PKA can consume ATP.
  • the kit further includes ATP.
  • the ATP is provided in the form of a solution.
  • the invention proposes a method for detecting olfactory receptor activation signals.
  • the method includes: subjecting a first cell to a first contact treatment with ATP, wherein the first cell expresses an olfactory receptor, and the first cell is pre-contacted with an odorant;
  • the first contact treatment product is subjected to a second contact treatment with the aforementioned silver nanoclusters, the aforementioned metal nanoclusters or the aforementioned kit; the first fluorescence value of the second contact treatment product is detected; the result based on the first fluorescence value , in order to determine the activation signal of the olfactory receptor.
  • the inventors combined the property of ATP to enhance the fluorescence signal of metal nanoclusters with the olfactory signal transduction mechanism. Specifically, the inventors found that after the olfactory receptors expressed on the cell surface are activated by odorants, they will recruit G proteins to bind to them. , G protein will regulate the effector protein adenylyl cyclase (AC) to generate the second messenger cAMP. The increase in intracellular cAMP concentration can activate PKA. The activated PKA can transfer the phosphate group on ATP, causing ATP to become into ADP, thereby consuming ATP; ATP has the effect of enhancing the fluorescence signal intensity of metal nanoclusters.
  • AC effector protein adenylyl cyclase
  • the fluorescence signal of metal nanoclusters will decrease.
  • the olfactory sense can be determined Receptor activation signal. Therefore, the method of the present invention can effectively detect the activation signal of olfactory receptors. Compared with other detection methods, the method of the present invention has the advantages of easy operation, no need to use expensive equipment, short detection time and low detection cost (96 per block). The reagents required for the well detection plate cost only about 15 yuan) and other advantages.
  • the method further includes: subjecting a second cell to the ATP for a third contact treatment, wherein the second cell expresses the olfactory receptor, and the second cell has not been contacted with the ATP in advance.
  • the odorant is subjected to pre-contact treatment; the third contact treatment product is subjected to the fourth contact treatment with the silver nanocluster, metal nanocluster or kit; and the second fluorescence value of the fourth contact treatment product is detected.
  • the activation signal of the olfactory receptor is obtained based on the difference between the second fluorescence value and the first fluorescence value.
  • the density of the first cells in the first contact system is 2 ⁇ 10 5 cells/ml to 5 ⁇ 10 5 cells/ml.
  • the density of the second cells in the third contact system is 2 ⁇ 10 5 cells/ml to 5 ⁇ 10 5 cells/ml.
  • the working concentration of the ATP in the first contact system or the third contact system is 700-900 ⁇ M.
  • the first contact treatment or the third contact treatment is performed at 35-40°C for 40-80 minutes.
  • activated PKA can convert ATP into ADP, thereby consuming ATP.
  • the activation signal of the olfactory receptor can be determined based on the difference in fluorescence signals.
  • the pre-contact treatment product of the first cells and the odorant is subjected to a first lysis treatment in advance.
  • the activated PKA in the cell can be released from the cell, thereby fully contacting the activated PKA with ATP.
  • the pre-contact treatment product of the second cells without the odorant is subjected to a second lysis treatment in advance.
  • the working concentration of ATP in the second contact system or the fourth contact system is 70-90 ⁇ M.
  • the fluorescence signal intensity of the metal nanoclusters can be further improved, and the fluorescence signal discrimination under different final concentrations of ATP can be improved.
  • the second contact treatment or the fourth contact treatment is performed at room temperature for 5 to 20 minutes.
  • the working concentration of the odorant in the pre-contact system is 0.1-500 ⁇ M.
  • Example 1 Preparation of silver nanoclusters and detection of fluorescence spectra
  • the present invention prepares three types of silver nanoclusters, namely silver nanocluster 1, silver nanocluster 2 and silver nanocluster 3.
  • the specific preparation steps are as follows:
  • SEQ ID NO: 1 Synthesize single-stranded DNA (SEQ ID NO: 1) polymerized by 12 cytosine nucleotides, and prepare a 100 ⁇ M DNA solution.
  • Mix 180 ⁇ L of AgNO 3 solution with a concentration of 1 mM and 300 ⁇ L of a DNA solution with a concentration of 100 ⁇ M (molar ratio of Ag + and DNA 6:1), and shake the resulting mixture on a vortexer for 30 min at room temperature.
  • Preparing silver nanoclusters 2 The only difference from the steps of preparing silver nanoclusters 1 is that the conditions for the second shaking are different, that is, replacing "shake at 25°C for 30 minutes” with “shake at 25°C for 2 minutes” ".
  • Preparing silver nanoclusters 3 The only difference from the steps of preparing silver nanoclusters 1 is that the conditions for the second shaking are different, that is, replacing "shake at 25°C for 30 minutes” with “shake at 4°C for 30 minutes” ".
  • the fluorescence signal, the specific detection results are shown in Figures 2-4. The results showed that the fluorescence signals of the three groups of silver nanoclusters were significantly different.
  • the fluorescence signal of silver nanocluster 2 (oscillation condition was 25°C for 2 minutes) was strong, but the discrimination of adding different concentrations of ATP was poor; 3 (the oscillation condition is 4°C for 30 minutes) has good discrimination when adding different concentrations of ATP, but the overall fluorescence signal is weak; the silver nanocluster 1 (the oscillation condition is 25°C for 30 minutes) has a strong fluorescence signal, and the addition of different concentrations The discrimination of concentration ATP is good.
  • silver nanoclusters 1 in this embodiment are mixed with different final concentrations of ATP, not only the fluorescence signal is strong, but also the discrimination of different concentrations of ATP is good. Therefore, silver nanoclusters 1 can be used to detect ATP consumption and olfactory receptor activation signals.
  • Example 2 Detection of fluorescence spectra of silver nanoclusters 1 under different ATP concentrations
  • each part is 50 ⁇ L.
  • Different amounts of ATP were added to each silver nanocluster 1.
  • the final concentrations of ATP in the seven silver nanoclusters 1 were 0 ⁇ M, 10 ⁇ M, 20 ⁇ M, 40 ⁇ M, 60 ⁇ M, 80 ⁇ M and 100 ⁇ M respectively.
  • it was excited with a 565nm wavelength light source, and then the fluorescence signal of the silver nanocluster emitted in the 640nm band was detected.
  • the specific detection results are shown in Figure 5.
  • Example 3 Using silver nanoclusters 1 to detect activation signals of three olfactory receptors
  • Example 1 In order to explore whether the silver nanoclusters 1 prepared in Example 1 can detect the activation signal of olfactory receptors, the inventor conducted the following tests:
  • OR1A1 having the amino acid sequence shown in SEQ ID NO: 2
  • OR2W1 having the amino acid sequence shown in SEQ ID NO: 3
  • mTAAR7f having the amino acid sequence shown in SEQ ID NO: 3
  • ester (CAS: 1797-74-6) is used to stimulate OR2W1
  • N,N-dimethylcyclohexylamine (CAS: 98-94-2) is used to stimulate mTAAR7f), where the concentration of carvone is 100 ⁇ M respectively. and 300 ⁇ M, the concentrations of allyl phenylacetate were 100 ⁇ M and 300 ⁇ M, respectively, and the concentrations of N,N-dimethylcyclohexylamine were 10 ⁇ M and 40 ⁇ M, respectively.
  • the above-mentioned odorant is brought into contact with the cells in the 96-well plate. Specifically, two concentrations of each odorant are mixed with two portions of the corresponding cells expressing olfactory receptors, and 50 ⁇ L of the odorant is added to each well. (Experimental group), and then mix 50 ⁇ L of CD293 culture medium containing 50mM IBMX with another portion of cells (blank control group). After the cells were stimulated with odorants for 30 minutes, the supernatant was removed. Add 90 ⁇ L RIPA lysis solution (Shanghai Beyotime Biotechnology Co., Ltd., P0013B) to each well to lyse the cells and release PKA.
  • RIPA lysis solution Shanghai Beyotime Biotechnology Co., Ltd., P0013B
  • the detection results of the activation status are shown in Figure 8.
  • the detection results of the activation status of the mouse olfactory receptor mTAAR7f when it is treated with N,N-dimethylcyclohexylamine are shown in Figure 9.
  • the first picture shows the fluorescence intensity of silver nanoclusters
  • the second picture below shows the relative fluorescence intensity obtained by subtracting the fluorescence intensity of the blank control group and the experimental group.
  • the silver nanocluster 1 prepared in the present invention successfully detects the signals of three olfactory receptors activated by their corresponding odorants. Therefore, it is further proved that the silver nanocluster 1 and ATP prepared in this application can be used to detect the activation signal of the olfactory receptor.
  • amino acid sequence of OR1A1 is as follows:
  • amino acid sequence of OR2W1 is as follows:
  • amino acid sequence of mTAAR7f is as follows:
  • references to the terms “one embodiment,” “some embodiments,” “an example,” “specific examples,” or “some examples” or the like means that specific features are described in connection with the embodiment or example. , structures, materials or features are included in at least one embodiment or example of the invention. In this specification, the schematic expressions of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the specific features, structures, materials or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, those skilled in the art may combine and combine different embodiments or examples and features of different embodiments or examples described in this specification unless they are inconsistent with each other.

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Abstract

一种检测嗅觉受体激活信号的方法，包括：将第一细胞和ATP进行第一接触处理，第一细胞表达嗅觉受体，第一细胞预先与气味剂进行预接触处理；将第一接触处理产物与金属纳米簇进行第二接触处理；检测第二接触处理产物的第一荧光值;基于第一荧光值的结果，以便确定嗅觉受体的激活信号；其中，金属纳米簇是通过金属离子、模板和还原剂进行第一混合处理获得的，第一混合处理是在20〜3（TC条件下进行20〜40min,模板包括选自DNA、RNA、蛋白质、多肽、高分子聚合物和统基小分子中的至少之一。

Description

金属纳米簇及其制备方法和检测嗅觉受体激活信号的方法
优先权信息
本公开请求2022年08月15日向中国国家知识产权局提交的、专利申请号为202210975846.1的专利申请的优先权和权益，并且通过参照将其全文并入此处。
技术领域
本发明涉及一种分析检测领域，具体地，本发明涉及一种金属纳米簇及其制备方法和检测嗅觉受体激活信号的方法，更具体地，本发明涉及一种银纳米簇、金属纳米簇及其制备方法、试剂盒和检测嗅觉受体激活信号的方法。
背景技术
气味剂/嗅觉受体配对机理的全面了解有助于推动嗅觉感知领域的快速发展。在基础研究方面：有助于理解嗅觉分子机理，建立起动物感知化学环境的模型。在工业应用方面：有助于进行气味增强剂/阻断剂的筛选、设计，以及新型生物气敏传感设备的研发。气味剂/嗅觉受体配对的检测是通过检测被气味剂刺激的嗅觉受体的激活信号强度确定的。目前检测气味剂/嗅觉受体的配对的方法，存在操作复杂和检测成本高等缺点。
因此，亟需开发一种有效的检测嗅觉受体激活信号的方法。
发明内容
本发明旨在至少在一定程度上解决现有技术中存在的技术问题至少之一。为此，本发明提供了一种检测嗅觉受体激活信号的方法，本发明的方法可检测嗅觉受体的激活信号，且具有操作方便和检测成本低等优点。
本发明是基于发明人的下列发现而完成的：
目前，气味剂/嗅觉受体的配对研究依赖于高通量筛选方法，已经建立的高通量筛选方法有以下几种检测嗅觉受体激活信号的方法：
(1)基于Ca 2+浓度变化的嗅觉检测方法：当气味剂激活嗅觉受体时，会激活cAMP信号通路，使得细胞膜上的钙通道开放，细胞质内的游离Ca 2+快速增加。Ca 2+为敏感染料，Fura-2或
Figure PCTCN2022133780-appb-000001
Calcium 6等均可与游离的Ca 2+结合，在特定的波长光源激发后产生荧光，该荧光的信号强度与游离Ca 2+浓度成正比。
(2)基于cAMP浓度变化的嗅觉检测方法：当气味剂激活嗅觉受体时，会激活cAMP信号通路，进而引起下游蛋白CREB的磷酸化，磷酸化后的CREB可以激活包含CRE元件的 启动子，进而启动报告基因的表达，报告基因的表达量或者活性反应嗅觉受体的活性。此外，
Figure PCTCN2022133780-appb-000002
检测法也可直接测量cAMP的增加。
(3)膜片钳方法是通过测量气味剂激活嗅觉受体引起的细胞膜上相应离子通道打开产生离子流，检测嗅觉受体的激活信号。
(4)气味剂刺激活体小鼠或者离体嗅觉组织，分离被激活的嗅觉神经元，进行RNA建库以及测序分析，可鉴定出被激活的嗅觉受体。
但是，上述几种方法存在操作复杂或者实验试剂和检测设备的价格昂贵等缺点。例如，基于Ca 2+浓度变化的检测方法中的FlexStation3以及FLIPR的价格十分昂贵；膜片钳设备的价格也十分昂贵；基于cAMP浓度变化的嗅觉检测方法中用到的商业化试剂或试剂盒价格也十分昂贵，例如荧光素酶检测试剂盒、分泌型碱性磷酸酶(SEAP)检测试剂盒以及
Figure PCTCN2022133780-appb-000003
cAMP检测试剂盒，96孔检测板标准检测体系下，每块检测板所需试剂成本约为800元人民币，虽然有研究报道表明在不显著影响检测效果的前提下可减少标准检测体系中使用的试剂量，但每块96孔检测板所需试剂成本仍需200元人民币；气味剂刺激活体小鼠或者离体嗅觉组织结合RNA-seq的嗅觉检测方法，无法快速获取检测结果，而且操作复杂。因此，有必要研发一种操作简便、设备要求低和检测成本低的检测嗅觉受体的激活信号的方法。
基于此，在本发明的一个方面，本发明提出了一种银纳米簇。根据本发明的实施例，所述银纳米簇包括：核酸分子以及Ag离子，所述核酸分子具有CCCCCCCCCCCC所示的核苷酸序列，所述Ag离子与所述核酸分子相互交联，所述Ag离子与所述核酸分子的摩尔比为6:1。发明人经过试验发现，将ATP与本发明的银纳米簇接触后，可显著提高银纳米簇的荧光信号，且与不同终浓度ATP接触的金属纳米簇的荧光信号区分度好。因此，采用本发明的银纳米簇可有效地检测ATP的消耗情况，并且，根据激活的嗅觉受体可激活蛋白激酶A(PKA)以及激活的PKA可消耗ATP(即将ATP变成ADP)的性质，可将其用于检测嗅觉受体的激活信号。
在本发明的另一方面，本发明提出了一种制备金属纳米簇的方法。根据本发明的实施例，所述方法包括：将金属离子、模板和还原剂进行第一混合处理，以便得到所述金属纳米簇；其中，所述第一混合处理是在20～30℃条件下进行20～40min；所述模板包括选自DNA、RNA、蛋白质、多肽、高分子聚合物和巯基小分子中的至少之一。根据本发明实施例的方法可制备得到金属纳米簇，该方法具有制备方法简单等优点；并且，本发明的金属纳米簇与ATP接触后可显著提高荧光信号，且与不同终浓度ATP接触的金属纳米簇的荧光信号区分度好。因此，采用本发明的金属纳米簇可有效地检测ATP的消耗情况，并且，根据激活 的嗅觉受体可激活PKA以及激活的PKA可消耗ATP的性质，可将其用于检测嗅觉受体的激活信号。
在本发明的又一方面，本发明提出了一种金属纳米簇。根据本发明的实施例，所述金属纳米簇是依据前述的方法获得的。根据本发明实施例的金属纳米簇与ATP接触后可显著提高荧光信号，且与不同终浓度ATP接触的金属纳米簇的荧光信号区分度好。因此，采用本发明的金属纳米簇可有效地检测ATP的消耗情况，并且，根据激活的嗅觉受体可激活PKA以及激活的PKA可消耗ATP的性质，可将其用于检测嗅觉受体的激活信号。
在本发明的又一方面，本发明提出了一种试剂盒。根据本发明的实施例，所述试剂盒包括：依据前述的方法制备的金属纳米簇或前述的金属纳米簇。根据本发明实施例的试剂盒可有效地检测ATP的消耗情况，并且，根据激活的嗅觉受体可激活PKA以及激活的PKA可消耗ATP变成ADP的性质，用于检测嗅觉受体的激活信号。
在本发明的又一方面，本发明提出了一种检测嗅觉受体激活信号的方法。根据本发明的实施例，所述方法包括：将第一细胞和ATP进行第一接触处理，其中，所述第一细胞表达嗅觉受体，所述第一细胞预先与气味剂进行预接触处理；将第一接触处理产物与前述的银纳米簇、前述的金属纳米簇或前述的试剂盒进行第二接触处理；检测第二接触处理产物的第一荧光值；基于所述第一荧光值的结果，以便确定所述嗅觉受体的激活信号。根据本发明实施例的方法可有效检测嗅觉受体的激活信号，且具有操作方便、检测时间短和检测成本低等优点。
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1为本发明实施例1中银纳米簇1的紫外光谱图；
图2为本发明实施例1中银纳米簇1和不同浓度ATP混合后的荧光信号检测结果；
图3为本发明实施例1中银纳米簇2和不同浓度ATP混合后的荧光信号检测结果；
图4为本发明实施例1中银纳米簇3和不同浓度ATP混合后的荧光信号检测结果；
图5为本发明实施例2中银纳米簇1和不同浓度ATP混合后的荧光信号检测结果；
图6为本发明实施例3中银纳米簇1检测嗅觉受体被激活的示意图；
图7为本发明实施例3中银纳米簇1对嗅觉受体OR1A1激活信号的检测结果；
图8为本发明实施例3中银纳米簇1对嗅觉受体OR2W1激活信号的检测结果；
图9为本发明实施例3中银纳米簇1对嗅觉受体mTAAR7f激活信号的检测结果。
具体实施方式
下面详细描述本发明的实施例。下面描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。
需要说明的是，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括一个或者更多个该特征。进一步地，在本发明的描述中，除非另有说明，“多个”的含义是两个或两个以上。
在本文中所披露的范围的端点和任何值都不限于该精确的范围或值，这些范围或值应当理解为包含接近这些范围或值的值。对于数值范围来说，各个范围的端点值之间、各个范围的端点值和单独的点值之间，以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围，这些数值范围应被视为在本文中具体公开。
为了更容易理解本发明，以下具体定义了某些技术和科学术语。除显而易见在本文件中的它处另有明确定义，否则本文中使用的所有其它技术和科学术语都具有本发明所属领域的一般技术人员通常理解的含义。
在本文中，术语“包含”或“包括”为开放式表达，即包括本发明所指明的内容，但并不排除其他方面的内容。
在本文中，术语“任选地”、“任选的”或“任选”通常是指随后所述的事件或状况可以但未必发生，并且该描述包括其中发生该事件或状况的情况，以及其中未发生该事件或状况的情况。
在本文中，术语“室温”通常是指20～25℃。
本发明提出了一种银纳米簇、金属纳米簇及其制备方法、试剂盒和检测嗅觉受体激活信号的方法，下面将分别对其进行详细描述。
银纳米簇
在本发明的一个方面，本发明提出了一种银纳米簇。根据本发明的实施例，所述银纳米簇包括：核酸分子以及Ag离子，所述核酸分子具有CCCCCCCCCCCC所示的核苷酸序列，所述Ag离子与所述核酸分子相互交联，所述Ag离子与所述核酸分子的摩尔比为6:1。 发明人经过试验发现，将ATP与本发明的银纳米簇接触后，可显著提高银纳米簇的荧光信号，且与不同终浓度ATP接触的金属纳米簇的荧光信号区分度好。因此，采用本发明的银纳米簇可有效地检测ATP的消耗情况，并且，根据激活的嗅觉受体可激活蛋白激酶A(PKA)以及激活的PKA可消耗ATP的性质，可将其用于检测嗅觉受体的激活信号。
制备金属纳米簇的方法
在本发明的另一方面，本发明提出了一种制备金属纳米簇的方法。根据本发明的实施例，所述方法包括：将金属离子、模板和还原剂进行第一混合处理，以便得到所述金属纳米簇；其中，所述第一混合处理是在20～30℃条件下进行20～40min；所述模板包括选自DNA、RNA、蛋白质、多肽、高分子聚合物和巯基小分子中的至少之一。根据本发明实施例的方法可制备得到金属纳米簇，该方法具有制备方法简单等优点；并且，本发明的金属纳米簇与ATP接触后可显著提高荧光信号，且与不同终浓度ATP接触的金属纳米簇的荧光信号区分度好。因此，采用本发明的金属纳米簇可有效地检测ATP的消耗情况，并且，根据激活的嗅觉受体可激活PKA以及激活的PKA可消耗ATP的性质，可将其用于检测嗅觉受体的激活信号。
根据本发明的实施例，所述方法进一步包括：将第一混合处理产物在2～6℃、避光条件下进行8～20h。由此，可进一步将金属离子还原成金属原子，提高金属纳米簇的荧光信号强度。
根据本发明的实施例，所述第一混合处理之前，预先将所述金属离子和模板进行预混合处理。由此，可将金属离子和模板充分混合。
根据本发明的实施例，所述预混合处理是在室温条件下进行20～50min。由此，将金属离子和模板充分混合。
根据本发明的实施例，所述模板为DNA。
根据本发明的实施例，所述DNA包括选自单链DNA、发夹型DNA和双链DNA中的至少之一，优选为单链DNA。
根据本发明的实施例，所述DNA的核苷酸包括选自胞嘧啶核苷酸、尿嘧啶核苷酸、腺嘌呤核苷酸、鸟嘌呤核苷酸中的至少之一。
根据本发明的实施例，所述DNA为胞嘧啶核苷酸单链。
根据本发明的实施例，所述胞嘧啶核苷酸单链中的核苷酸个数为8～15个。
根据本发明的实施例，所述DNA具有如SEQ ID NO:1所示的核苷酸序列。由此，制得的金属纳米簇的荧光信号较强。
CCCCCCCCCCCC(SEQ ID NO:1)。
根据本发明的实施例，所述金属离子包括选自金离子、银离子、铜离子和铂离子中的至少之一，优选为银离子。
根据本发明的实施例，所述金属离子是以金属盐的形式加入至所述溶液中。
根据本发明的实施例，所述银离子是由AgNO 3提供的。
根据本发明的实施例，所述金属离子和模板的摩尔比为(4.8～6.5):1。由此，制得的金属纳米簇的荧光信号较强。
根据本发明的实施例，所述还原剂为NaBH 4。
根据本发明的实施例，所述金属离子和NaBH 4的摩尔比为(0.5～1.5):1。由此，制得的金属纳米簇的荧光信号较强。
金属纳米簇
在本发明的又一方面，本发明提出了一种金属纳米簇。根据本发明的实施例，所述金属纳米簇是依据前述的方法获得的。根据本发明实施例的金属纳米簇与ATP接触后可显著提高荧光信号，且与不同终浓度ATP接触的金属纳米簇的荧光信号区分度好。因此，采用本发明的金属纳米簇可有效地检测ATP的消耗情况，并且，根据激活的嗅觉受体可激活PKA以及激活的PKA可消耗ATP的性质，可将其用于检测嗅觉受体的激活信号。
在本发明的又一方面，本发明提出了一种试剂盒。根据本发明的实施例，所述试剂盒包括：依据前述的方法制备的金属纳米簇或前述的金属纳米簇。根据本发明实施例的试剂盒可有效地检测ATP的消耗情况，并且，根据激活的嗅觉受体可激活PKA以及激活的PKA可消耗ATP的性质，用于检测嗅觉受体的激活信号。
根据本发明的实施例，所述试剂盒进一步包括ATP。
根据本发明的实施例，所述ATP是以溶液的形式提供的。
检测嗅觉受体激活信号的方法
在本发明的又一方面，本发明提出了一种检测嗅觉受体激活信号的方法。根据本发明的实施例，所述方法包括：将第一细胞和ATP进行第一接触处理，其中，所述第一细胞表达嗅觉受体，所述第一细胞预先与气味剂进行预接触处理；将第一接触处理产物与前述的银纳米簇、前述的金属纳米簇或前述的试剂盒进行第二接触处理；检测第二接触处理产物的第一荧光值；基于所述第一荧光值的结果，以便确定所述嗅觉受体的激活信号。
发明人首次将ATP增强金属纳米簇荧光信号的特性和嗅觉信号转导机制进行结合，具 体为：发明人发现，表达在细胞表面的嗅觉受体被气味剂激活后，将招募G蛋白与之结合，G蛋白将调节效应蛋白腺苷酸环化酶(AC)，生成第二信使cAMP，细胞内cAMP浓度的升高可激活PKA，激活后的PKA可转移ATP上的磷酸基团，使ATP变成ADP，从而消耗ATP；ATP具有增强金属纳米簇荧光信号强度的作用，消耗ATP后的金属纳米簇的荧光信号会降低，通过检测第二接触处理产物的第一荧光值，可确定所述嗅觉受体的激活信号。因此，采用本发明的方法可有效检测嗅觉受体的激活信号，并且，相较于其他检测方法，本发明的方法具有操作方便、无需使用昂贵设备、检测时间短和检测成本低(每块96孔检测板所需的试剂成本仅约15元人民币)等优点。
根据本发明的实施例，所述方法进一步包括：将第二细胞和所述ATP中进行第三接触处理，其中，所述第二细胞表达所述嗅觉受体，所述第二细胞预先未与气味剂进行预接触处理；将第三接触处理产物与所述银纳米簇、金属纳米簇或试剂盒进行第四接触处理；检测第四接触处理产物的第二荧光值。
根据本发明的实施例，所述嗅觉受体的激活信号是基于所述第二荧光值和第一荧光值的差值获得的。
根据本发明的实施例，所述第一细胞在第一接触体系中的密度为2×10 5个细胞/ml～5×10 5个细胞/ml。
根据本发明的实施例，所述第二细胞在第三接触体系中的密度为2×10 5个细胞/ml～5×10 5个细胞/ml。
根据本发明的实施例，所述ATP在所述第一接触体系或第三接触体系中的工作浓度为700～900μM。
根据本发明的实施例，所述第一接触处理或第三接触处理是在35～40℃条件下进行40～80min。由此，激活的PKA可将ATP变成ADP，从而消耗ATP，后续可基于荧光信号的差值确定嗅觉受体的激活信号。
根据本发明的实施例，所述第一接触处理之前，预先将所述第一细胞与气味剂的预接触处理产物进行第一裂解处理。由此，可将细胞中激活的PKA从细胞中释放出来，从而使激活的PKA与ATP充分接触。
根据本发明的实施例，所述第三接触处理之前，预先将所述第二细胞未与气味剂的预接触处理产物进行第二裂解处理。
根据本发明的实施例，所述ATP在第二接触体系或第四接触体系中的工作浓度为70～90μM。由此，可进一步提高金属纳米簇的荧光信号强度，以及提高不同终浓度ATP条件下的荧光信号区分度。
根据本发明的实施例，所述第二接触处理或第四接触处理是在室温条件下进行5～20min。
根据本发明的实施例，所述气味剂在预接触体系中的工作浓度为0.1～500μM。
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1：银纳米簇的制备及其荧光光谱的检测
1、本发明制备了三种银纳米簇，分别为银纳米簇1、银纳米簇2和银纳米簇3，具体制备步骤如下：
1)制备银纳米簇1：合成由12个胞嘧啶核苷酸聚合成的单链DNA(SEQ ID NO:1)，配制成100μM DNA溶液。将180μL浓度为1mM的AgNO 3溶液与300μL浓度为100μM的DNA溶液(Ag +和DNA的摩尔比＝6:1)混合，将得到的混合液于室温条件下在涡旋仪上振荡30min。将180μL浓度为1mM的NaBH 4(Ag +和NaBH 4的摩尔比＝1:1)加入至上述混合液中，然后于25℃条件下振荡30分钟，随后将振荡后得到的溶液放置在4℃黑暗中孵育过夜(12h)后，得到银纳米簇1。然后对银纳米簇1进行紫外光谱检测，紫外光谱图参见图1。
2)制备银纳米簇2：与制备银纳米簇1的步骤区别仅在于，第二次振荡的条件不同，即为将“25℃条件下振荡30分钟”替换成“25℃条件下振荡2分钟”。
3)制备银纳米簇3：与制备银纳米簇1的步骤区别仅在于，第二次振荡的条件不同，即为将“25℃条件下振荡30分钟”替换成“4℃条件下振荡30分钟”。
2、三种银纳米簇和不同ATP混合液的荧光光谱的检测
分别取上述三组银纳米簇(银纳米簇1、银纳米簇2和银纳米簇3)各6份，每份取50μL。配置浓度为8mM的ATP储液，加入至上述三组银纳米簇中，每组银纳米簇的6份样品中ATP的终浓度分别为0μM、10μM、20μM、40μM、60μM、80μM。然后在25℃条件下反应10分钟后，用565nm波长光源激发银纳米簇，然后扫描银纳米簇在400-700nm波段的荧光信号，在640nm出现峰值，即采用640nm发射光检测三组银纳米簇的荧光信号，具体的检测结果如图2-图4所示。结果发现，三组银纳米簇的荧光信号具有显著差异，其中，银纳米簇2(振荡条件为25℃振荡2分钟)的荧光信号强，但添加不同浓度ATP的区 分度很差；银纳米簇3(振荡条件为4℃振荡30分钟)添加不同浓度ATP的区分度较好，但整体荧光信号较弱；银纳米簇1(振荡条件为25℃振荡30分钟)的荧光信号强，且添加不同浓度ATP的区分度较好。
由上可知，本实施例中的银纳米簇1与不同终浓度ATP混合后，不仅荧光信号强，而且不同浓度ATP的区分度较好。因此，可采用银纳米簇1检测ATP的消耗情况以及嗅觉受体的激活信号。
实施例2：不同ATP浓度条件下的银纳米簇1的荧光光谱的检测
为了使银纳米簇的初始荧光信号最强，发明人探究了ATP的最佳浓度范围。具体试验步骤如下：
取实施例1合成的银纳米簇1，共取7份，每份50μL。每份银纳米簇1中加入不同添加量的ATP，7份银纳米簇1中ATP的终浓度分别为0μM、10μM、20μM、40μM、60μM、80μM和100μM。然后在25℃条件下反应10分钟后，用565nm波长光源激发，然后检测银纳米簇在640nm波段发射光的荧光信号，具体检测结果如图5所示。结果表明，ATP终浓度在10-80μM范围内，其与银纳米簇的荧光强度呈现正线性相关；ATP终浓度为100μM时，虽然也可使银纳米簇的荧光强度增强，但相比于ATP终浓度为80μM的荧光增强效果略有下降。因此，终浓度为20-160μM的ATP可使银纳米簇的荧光强度显著增强，80μM为最佳终浓度。
实施例3：利用银纳米簇1检测三个嗅觉受体的激活信号
发明人为了探究实施例1制备的银纳米簇1是否能检测到嗅觉受体的激活信号，进行了如下试验：
发明人从人和小鼠嗅觉受体库中选择了OR1A1(具有如SEQ ID NO:2所示的氨基酸序列)、OR2W1(具有如SEQ ID NO:3所示的氨基酸序列)和mTAAR7f(具有如SEQ ID NO:4所示的氨基酸序列)，然后分别构建含有嗅觉受体pCI-CMVp-Rho21-OR1A1、pCI-CMVp-Rho21-OR2W1和pCI-CMVp-Rho21-mTAAR7f表达载体，并利用转染试剂Lipofectamine2000(Invitrogen)将表达载体转染至HEK293细胞中。采用MEM/EBSS培养基(HyClone)+10％FBS(胎牛血清)将上述细胞培养于96多孔板中，培养至细胞密度为5×10 5个细胞/mL，去除细胞上清培养液，其中，每种细胞培养三份。
用含50mM 3-异丁基-1-甲基黄嘌呤(IBMX)的CD293培养液配制不同浓度的气味剂(香芹酮(CAS：2244-16-8)用于刺激OR1A1、苯乙酸烯丙酯(CAS：1797-74-6)用于刺 激OR2W1、N,N-二甲基环己胺(CAS：98-94-2)用于刺激mTAAR7f)，其中，香芹酮的浓度分别为100μM和300μM，苯乙酸烯丙酯的浓度分别为100μM和300μM，N,N-二甲基环己胺的浓度分别为10μM和40μM。
将上述气味剂与96多孔板中的细胞进行接触，具体为，将每种气味剂的两种浓度分别与其对应的表达有嗅觉受体的细胞的其中两份进行混合，每孔加入50μL气味剂(实验组)，然后将50μL的含50mM IBMX的CD293培养液与另一份细胞进行混合(空白对照组)。气味剂刺激细胞30min后，移走上清液。每孔加入90μL RIPA裂解液(上海碧云天生物技术有限公司，P0013B)，使细胞裂解释放PKA。每孔加入10μL浓度为8mM的ATP储液，37℃孵育1h。取5.5μL上清液加入到每孔分装有50μL实施例1制得的银纳米簇1的96多孔板中，室温下孵育10min后，用565nm波长光源激发，然后检测银纳米簇在640nm波段发射光的荧光信号。银纳米簇1检测嗅觉受体被激活的示意图参见图6，人类嗅觉受体OR1A1被香芹酮处理时的激活情况的检测结果参见图7，人类嗅觉受体OR2W1被苯乙酸烯丙酯处理时的激活情况的检测结果参见图8，小鼠嗅觉受体mTAAR7f被N,N-二甲基环己胺处理时的激活情况的检测结果参见图9，其中，图7-图9的上方第一个图为银纳米簇的荧光强度、下方第二个图为空白对照组与实验组的荧光强度相减得到的相对荧光强度。
结果表明，本发明制备的银纳米簇1成功检测到了三个嗅觉受体被其相应气味剂激活的信号。因此，进一步证明了可采用本申请制得的银纳米簇1和ATP对嗅觉受体的激活信号进行检测。
其中，OR1A1的氨基酸序列如下所示：
Figure PCTCN2022133780-appb-000004
OR2W1的氨基酸序列如下所示：
Figure PCTCN2022133780-appb-000005
Figure PCTCN2022133780-appb-000006
mTAAR7f的氨基酸序列如下所示：
Figure PCTCN2022133780-appb-000007
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (23)

  1. 一种银纳米簇，其特征在于，包括：核酸分子以及Ag离子，所述核酸分子具有CCCCCCCCCCCC的核苷酸序列，所述Ag离子与所述核酸分子相互交联，所述Ag离子与所述核酸分子的摩尔比为6:1。
  2. 一种制备金属纳米簇的方法，其特征在于，包括：
    将金属离子、模板和还原剂进行第一混合处理，以便得到所述金属纳米簇；
    其中，所述第一混合处理是在20～30℃条件下进行20～40min；
    所述模板包括选自DNA、RNA、蛋白质、多肽、高分子聚合物和巯基小分子中的至少之一。
  3. 根据权利要求2所述的方法，其特征在于，进一步包括：
    将第一混合处理产物在2～6℃、避光条件下进行8～20h。
  4. 根据权利要求2所述的方法，其特征在于，所述第一混合处理之前，预先将所述金属离子和模板进行预混合处理。
  5. 根据权利要求4所述的方法，其特征在于，所述预混合处理是在室温条件下进行20～50min。
  6. 根据权利要求2所述的方法，其特征在于，所述模板为DNA；
    所述DNA包括选自单链DNA、发夹型DNA和双链DNA中的至少之一。
  7. 根据权利要求2所述的方法，其特征在于，所述DNA的核苷酸包括选自胞嘧啶核苷酸、尿嘧啶核苷酸、腺嘌呤核苷酸、鸟嘌呤核苷酸中的至少之一。
  8. 根据权利要求2所述的方法，其特征在于，所述金属离子包括选自金离子、银离子、铜离子和铂离子中的至少之一。
  9. 根据权利要求2所述的方法，其特征在于，所述金属离子和模板的摩尔比为(4.8～6.5):1。
  10. 根据权利要求2所述的方法，其特征在于，所述还原剂为NaBH 4。
  11. 根据权利要求10所述的方法，其特征在于，所述金属离子和NaBH 4的摩尔比为(0.5～1.5):1。
  12. 根据权利要求2所述的方法，其特征在于，所述DNA为胞嘧啶核苷酸单链；
    所述胞嘧啶核苷酸单链中的核苷酸个数为8～15个。
  13. 一种金属纳米簇，其特征在于，是依据权利要求2～12任一项所述的方法获得的。
  14. 一种试剂盒，其特征在于，包括：
    依据权利要求2～12任一项所述的方法制备的金属纳米簇或权利要求13所述的金属纳米簇。
  15. 根据权利要求14所述的试剂盒，其特征在于，进一步包括ATP；
    所述ATP是以溶液的形式提供的。
  16. 一种检测嗅觉受体激活信号的方法，其特征在于，包括：
    将第一细胞和ATP进行第一接触处理，其中，所述第一细胞表达嗅觉受体，所述第一细胞预先与气味剂进行预接触处理；
    将第一接触处理产物与权利要求1所述的银纳米簇、权利要求13所述的金属纳米簇或权利要求14～15任一项所述的试剂盒进行第二接触处理；
    检测第二接触处理产物的第一荧光值；
    基于所述第一荧光值的结果，以便确定所述嗅觉受体的激活信号。
  17. 根据权利要求16所述的方法，其特征在于，进一步包括：
    将第二细胞和所述ATP中进行第三接触处理，其中，所述第二细胞表达所述嗅觉受体，所述第二细胞预先未与气味剂进行预接触处理；
    将第三接触处理产物与所述银纳米簇、金属纳米簇或试剂盒进行第四接触处理；
    检测第四接触处理产物的第二荧光值。
  18. 根据权利要求17所述的方法，其特征在于，所述嗅觉受体的激活信号是基于所述第二荧光值和第一荧光值的差值获得的。
  19. 根据权利要求17所述的方法，其特征在于，所述第一细胞在第一接触体系中的密度为2×10 5个细胞/ml～5×10 5个细胞/ml；和/或
    所述第二细胞在第三接触体系中的密度为2×10 5个细胞/ml～5×10 5个细胞/ml。
  20. 根据权利要求17所述的方法，其特征在于，所述ATP在第一接触体系或第三接触体系中的工作浓度为700～900μM；和/或
    所述ATP在第二接触体系或第四接触体系中的工作浓度为70～90μM。
  21. 根据权利要求17所述的方法，其特征在于，所述第一接触处理或第三接触处理是在35～40℃条件下进行40～80min；和/或
    所述第二接触处理或第四接触处理是在室温条件下进行5～20min。
  22. 根据权利要求17所述的方法，其特征在于，所述第一接触处理之前，预先将所述第一细胞与气味剂的预接触处理产物进行第一裂解处理；和/或
    所述第三接触处理之前，预先将所述第二细胞未与气味剂的预接触处理产物进行第二裂解处理。
  23. 根据权利要求17所述的方法，其特征在于，所述气味剂在预接触体系中的工作浓度为0.1～500μM。
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