WO2023035452A1 - Surface plasmon resonance sensor, preparation method therefor and application thereof - Google Patents

Surface plasmon resonance sensor, preparation method therefor and application thereof Download PDF

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WO2023035452A1
WO2023035452A1 PCT/CN2021/137299 CN2021137299W WO2023035452A1 WO 2023035452 A1 WO2023035452 A1 WO 2023035452A1 CN 2021137299 W CN2021137299 W CN 2021137299W WO 2023035452 A1 WO2023035452 A1 WO 2023035452A1
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plasmon resonance
noble metal
surface plasmon
boron
boronene
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PCT/CN2021/137299
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French (fr)
Chinese (zh)
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薛冬峰
王鑫
王晓明
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中国科学院深圳先进技术研究院
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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
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6825—Nucleic acid detection involving sensors

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  • the application belongs to the technical field of semiconductors, and in particular relates to a surface plasmon resonance sensor and its preparation method and application.
  • the detection of miRNAs is challenging due to the low content of miRNAs, accounting for only 0.01% of the total RNA mass in a given sample.
  • miRNA detection technologies such as quantitative real-time PCR (qRT–PCR), northern hybridization, and microarray-based hybridization in clinical practice is limited due to difficult amplification, high cost, complex operation, and low sensitivity.
  • the surface plasmon resonance (SPR) technology has the advantages of non-destructive testing, high repeatability and low cost, and has been proven to study molecular interactions by evaluating the refractive index changes on the chip surface.
  • SPR surface plasmon resonance
  • Due to the limited amount of immobilized probe DNA and miRNA on the chip surface it is still challenging to detect biomolecules at extremely low concentrations using traditional SPR techniques. Therefore, it is urgent to search for advanced materials with large adsorption energy and work function increment to improve the performance of SPR biosensors.
  • the existing two-dimensional nanomaterials have weak interactions with biomarkers and poor chemical stability, which affects the performance of SPR biosensors.
  • the purpose of this application is to provide a surface plasmon resonance sensor and its preparation method and application, aiming to solve the problem of weak interaction between existing two-dimensional nanomaterials and biomarkers and poor chemical stability that affect SPR biosensors Performance issues.
  • the present application provides a surface plasmon resonance sensor, the surface plasmon resonance sensor comprising:
  • a surface plasmon resonance chip comprising a first noble metal substrate, a boron layer is bonded to the first noble metal substrate;
  • a noble metal-ssDNA complex is combined on the surface plasmon resonance chip, and the noble metal-ssDNA complex includes a second noble metal substrate and ssDNA combined on the second noble metal substrate.
  • the present application provides a method for preparing a surface plasmon resonance sensor, the preparation method comprising the following steps:
  • the noble metal-ssDNA complex is assembled on the surface plasmon resonance chip to obtain a surface plasmon resonance sensor.
  • the present application provides a nucleic acid detection sensor, which includes the above-mentioned surface plasmon resonance sensor.
  • the present application provides a method for preparing boron nanosheets, comprising the following steps:
  • Ultrasonic stripping is performed on the boron particle precursor solution to obtain a boron nanosheet dispersion
  • the boronene nanosheet dispersion is subjected to centrifugation to obtain boronene nanosheets with a target number of layers.
  • the surface plasmon resonance sensor provided in the first aspect of the present application includes a surface plasmon resonance chip and a noble metal-ssDNA complex bonded on the surface plasmon resonance chip. Since the boron layer is bonded to the first noble metal substrate, the The surface plasmon resonance sensor has the surface plasmon resonance effect of the mutual coupling of the first noble metal-boron-second noble metal, and at the same time, the single-stranded DNA is grafted on the surface of the second noble metal, and the second noble metal coated with the single-stranded DNA and the boron Therefore, the plasmon resonance sensor has high sensitivity and can be used to detect miRNA in samples, and has the advantages of label-free detection, high reusability and low cost.
  • the method for preparing a surface plasmon resonance sensor provided in the second aspect of the present application can form a surface plasmon with a surface plasmon resonance effect by performing film-forming treatment on the prepared boron nanosheet dispersion on the surface of the first noble metal substrate.
  • Resonance chip, the noble metal-ssDNA complex is assembled on the surface plasmon resonance chip, so that the formed surface plasmon resonance sensor has the surface plasmon resonance effect of the mutual coupling of the first noble metal-boronene-second noble metal.
  • the second noble metal surface can effectively monitor the miRNA of the detection sample by modifying the single-stranded DNA that matches the sample to be detected.
  • the nucleic acid detection sensor provided in the third aspect of the present application since the surface plasmon resonance sensor included in the nucleic acid detection sensor contains a noble metal-ssDNA complex, which can effectively monitor the sample by modifying the surface to match the single-stranded DNA of the sample to be detected miRNA.
  • the preparation method of the boron nanosheets provided in the fourth aspect of the present application first grinds the boron powder to obtain a uniform and fine boron particle precursor solution, and then performs ultrasonic stripping on the boron particle precursor solution to obtain Boronene nanosheet dispersions with different layers, and then centrifuge the boronene nanosheet dispersion to obtain boronene nanosheets with a controllable number of layers. Therefore, this preparation method can prepare single-layer or multi-layer boron nanosheets.
  • Fig. 1 is a schematic flowchart of a method for preparing a surface plasmon resonance sensor based on a boron material provided in an embodiment of the present application.
  • the term "and/or” describes the association relationship of associated objects, indicating that there may be three relationships, for example, A and/or B may mean: A exists alone, A and B exist simultaneously, and B exists alone Condition. Among them, A and B can be singular or plural.
  • the character "/" generally indicates that the contextual objects are an "or" relationship.
  • At least one means one or more, and “multiple” means two or more.
  • At least one of the following” or similar expressions refer to any combination of these items, including any combination of single or plural items.
  • at least one item (unit) of a, b, or c or “at least one item (unit) of a, b, and c” can mean: a, b, c, a-b( That is, a and b), a-c, b-c, or a-b-c, where a, b, and c can be single or multiple.
  • sequence numbers of the above-mentioned processes do not mean the order of execution, and some or all steps may be executed in parallel or sequentially, and the execution order of each process shall be based on its functions and The internal logic is determined and should not constitute any limitation to the implementation process of the embodiment of the present application.
  • the weight of the relevant components mentioned in the description of the embodiments of the present application can not only refer to the specific content of each component, but also represent the proportional relationship between the weights of the various components.
  • the scaling up or down of the content of the fraction is within the scope disclosed in the description of the embodiments of the present application.
  • the mass described in the description of the embodiments of the present application may be ⁇ g, mg, g, kg and other well-known mass units in the chemical industry.
  • first and second are only used for descriptive purposes to distinguish objects such as substances from each other, and cannot be understood as indicating or implying relative importance or implicitly specifying the quantity of indicated technical features.
  • first XX can also be called the second XX
  • second XX can also be called the first XX.
  • a feature defined as “first” and “second” may explicitly or implicitly include one or more of these features.
  • the first aspect of the embodiment of the present application provides a surface plasmon resonance sensor, the surface plasmon resonance sensor includes:
  • a surface plasmon resonance chip includes a first noble metal substrate, and a boron layer is bonded to the first noble metal substrate;
  • the noble metal-ssDNA complex, the noble metal-ssDNA complex is combined on the surface plasmon resonance chip, and the noble metal-ssDNA complex includes a second noble metal substrate and ssDNA combined on the second noble metal substrate.
  • the surface plasmon resonance sensor provided in the embodiment of the present application includes a surface plasmon resonance chip and a noble metal-ssDNA complex. Since the surface plasmon resonance chip includes a boron layer on the first noble metal substrate, the noble metal-ssDNA complex Single-stranded DNA is grafted on the surface of the second noble metal, therefore, there is a strong interaction between the second noble metal coated with single-stranded DNA and boron, so that the surface plasmon resonance sensor has the first noble metal-boron-second noble metal Due to the mutually coupled surface plasmon resonance effect, the plasmon resonance sensor has high sensitivity and can be used to detect miRNA in samples, and has the advantages of label-free detection, high reusability, and low cost.
  • the thickness of the boron layer is 1-100 nm. Specifically, the thickness of the boron layer may be 1 nm, 2-5 nm, or 100 nm.
  • the boron layer can be a continuously distributed boron film layer, can be an island-shaped distributed boron film layer, and can also be a continuously distributed boron film layer and an island-shaped distributed boron film layer.
  • an ultra-thin and continuously distributed boron film layer is preferred to enhance the surface plasmon resonance effect between the first noble metal substrate and the boron layer, thereby improving the sensitivity of the plasmon resonance sensor.
  • the boron layer is a hydroxylated boron nanosheet
  • the number of layers of the hydroxylated boron nanosheet is 1-100 layers.
  • the number of layers of the hydroxylated boronene nanosheets may be 1 layer, 2-5 layers, or 100 layers.
  • the preferred boronene nanosheets in this embodiment undergo surface modification treatment with an alkaline solution, so that the obtained hydroxylated boronene nanosheets exhibit significant semiconductor characteristics, thereby enhancing the surface plasmon resonance with the first noble metal substrate Effect, can further improve the sensitivity of the plasmon resonance sensor.
  • the first noble metal substrate includes a noble metal film
  • the noble metal film can be an Au film, an Ag film, a Pt film, a Pd film, or an Au and Ag film.
  • the thickness of the first noble metal film is 2 nm-20 ⁇ m. Specifically, the thickness of the noble metal film may be 2 nm, 20 ⁇ m, or 100 nm.
  • the second noble metal matrix includes at least one of Au and/or Ag nanorods, nanocubes, and nanosheets, but is not limited thereto. Specifically, the second noble metal matrix may include Au nanorods, Au nanocubes, Au nanosheets, Au nanorods and Au nanocubes.
  • the loading amount of the noble metal-ssDNA complex on the surface plasmon resonance chip is 0.01 mol/cm 2 -10 mol/cm 2 , specifically, the amount of the noble metal-ssDNA complex on the surface plasmon resonance The loading on the chip was 5 mol/cm 2 .
  • the single-stranded RNA solution of the sample to be tested flows through the surface of the hydroxylated boron surface plasmon resonance sensor, the single-stranded RNA pairs with the complementary AuNR-ssDNA to form a double strand; the interaction between miRNA and AuNR-ssDNA
  • the effect leads to the release of AuNR-ssDNA from the hydroxylated boron nanosheets, which reduces the AuNR-ssDNA molecules on the surface of the hydroxylated boron surface plasmon resonance sensor, thereby making the detection signal (resonance angle) of the boron surface plasmon resonance sensor Therefore, important information such as the adsorption and dissociation constants of the interaction between the bioreceptor and the sample to be tested can be obtained according to the detected changes in the dynamic signal.
  • the AuNR-ssDNA complex was adsorbed on hydroxylated boron nanosheets. Therefore, single-stranded DNA-coated Au nanorods and hydroxylated The strong interaction between boronene can enhance the surface plasmon effect of the boronene sensor, which can further improve the sensitivity of the hydroxylated boronene surface plasmon resonance sensor, and the surface of the second noble metal substrate can be modified to match the sample to be detected The single-stranded DNA can effectively monitor the miRNA in the sample.
  • the second aspect of the embodiment of the present application provides a preparation method of a surface plasmon resonance sensor, the preparation method comprising the following steps:
  • the preparation method of the surface plasmon resonance sensor provided in the embodiment of the present application can form a surface plasmon resonance sensor with a surface plasmon resonance effect by performing film-forming treatment on the prepared boron nanosheet dispersion liquid on the surface of the first noble metal substrate. Chip, the noble metal-ssDNA complex is assembled on the surface plasmon resonance chip, so that the formed surface plasmon resonance sensor has the surface plasmon resonance effect of the mutual coupling of the first noble metal-boronene-second noble metal.
  • the surface of the second noble metal can effectively monitor the miRNA of the detection sample by modifying the single-stranded DNA that matches the sample to be detected.
  • step S10 the boronene nanosheet dispersion can be prepared according to the following method:
  • a boron particle precursor solution with uniform size and fineness is obtained, which is convenient for the next step of ultrasonic stripping treatment;
  • the organic solvent includes sec-butanol, ethylene glycol, glycerin At least one of alcohol and n-butanol, but not limited thereto.
  • the organic solvent may be sec-butanol.
  • the boron particle precursor solution was ultrasonically stripped to obtain boron nanosheet dispersions with different layers.
  • the boronene nanosheet dispersion is subjected to centrifugal separation to obtain boronene nanosheets with a controllable target layer number; in this embodiment, single-layer boronene nanosheets can be prepared by adjusting the centrifugal speed range to strengthen the interaction between the first noble metal matrix and boron.
  • the surface plasmon resonance effect between the olefin layers can improve the sensitivity of the plasmon resonance sensor.
  • the boronene nanosheets are also subjected to surface modification treatment with an alkaline solution to obtain hydroxylated boronene nanosheets.
  • the alkaline solution includes at least one of sodium hydroxide, potassium hydroxide and lithium hydroxide, but is not limited thereto.
  • the alkaline solution may be sodium hydroxide, or sodium hydroxide and potassium hydroxide.
  • the surface modification treatment of boronene nanosheets and alkaline solution enables the obtained hydroxylated boronene nanosheets to exhibit significant semiconductor characteristics, which in turn can produce stronger surface plasmon resonance with the first noble metal matrix effect; at the same time, hydroxylated boronene nanosheets have stable chemical properties and strong interactions with biomarkers, which solves the problem of weak interactions with biomarkers and poor chemical stability of existing two-dimensional nanomaterials Sexuality leads to problems affecting the performance of SPR biosensors.
  • the film-forming treatment of the borene nanosheet dispersion on the surface of the first noble metal substrate may use an existing film-forming method or an improved film-forming method based on an existing film-forming method, or other film-forming methods.
  • the boronene nanosheet dispersion can be formed into a film on the surface of the first noble metal substrate, it is within the scope disclosed in the embodiments of the present application.
  • the hydroxylated boronene nanosheet dispersion is sprayed according to the set speed by spin coating. Surface plasmon resonance chips with different thicknesses of hydroxylated boronene nanosheets can be obtained by spin-coating on the surface of the first noble metal substrate.
  • the first precious metal may include any one of gold, silver, palladium and platinum, but is not limited thereto. Specifically, the first precious metal may be gold.
  • the target thickness of boronene nanosheets or hydroxylated boronene nanosheets is 1-100nm
  • the specific target thickness of boronene nanosheets can be 1nm, 2nm, 4nm, 6nm, 8nm and other typical but non-limiting thicknesses.
  • the rotational speed of the spin coating is set at 1-20000r/min, and the specific set rotational speed can be 5000r/min, 6000r/min, 8000r/min, 9000r/min, 12000r/min.
  • an ultra-thin surface plasmon resonance chip with hydroxylated boronene nanosheets can be obtained, and then to obtain a high-sensitivity chip. Hydroxyboron surface plasmon resonance sensors lay the groundwork.
  • the single-stranded DNA is bound to the surface of the second noble metal substrate to obtain a noble metal-ssDNA complex.
  • the second noble metal matrix includes at least one of Au/or Ag nanorods, nanocubes, and nanosheets, but is not limited thereto.
  • the second noble metal matrix may be Au nanorods.
  • the preparation method of Au nanorods may include the following steps: fully mix the gold source HAuCl 4 and the surfactant CTAB, and add NaBH 4 under stirring conditions to synthesize gold crystal nuclei; add the gold crystal nuclei to the mixture containing CTAB, HAuCl 4 , AgNO 3 , H 2 SO 4 , and L-AA mixed solution, and grow anisotropically into gold nanorods (AuNR) under stirring conditions.
  • step S40 the noble metal-ssDNA complex is assembled on the surface plasmon resonance chip to obtain a surface plasmon resonance sensor.
  • the specific method includes, according to the loading amount of the noble metal-ssDNA complex on the surface plasmon resonance chip: 0.01mol/cm 2 -10mol/cm 2 , spin-coat an appropriate amount of noble metal-ssDNA complex on a plasmon resonance chip, place it at room temperature for 5min-60min, and then wash it with PBS buffer to obtain a surface plasmon resonance sensor; specific loading It may be 5 mol/cm 2 .
  • the surface plasmon resonance effect of coupling the first noble metal, hydroxylated boronene nanosheets and the second noble metal can be realized, and by grafting single-stranded DNA on the surface, the second noble metal coated with single-stranded DNA and hydroxylated boronene
  • the strong interaction between the nanosheets enables the high sensitivity of the surface plasmon resonance sensor.
  • the embodiment of the present application further provides a nucleic acid detection sensor, which includes the above-mentioned surface plasmon resonance sensor.
  • the nucleic acid detection sensor provided in the embodiment of the present application, since the surface plasmon resonance sensor of the nucleic acid detection sensor is coated with a noble metal-ssDNA complex on the surface of the hydroxylated boronene nanosheet, it can be combined with the sample to be detected through surface modification. Matched single-stranded DNA to effectively monitor miRNA in samples.
  • This embodiment provides a method for preparing a surface plasmon resonance sensor of hydroxylated boronene nanosheets with a thickness of 2 nm, comprising the following steps:
  • a 2 mol/L sodium hydroxide solution was prepared, the boronene dispersion was added to 50 mL of the sodium hydroxide solution, and cold water was sonicated for 2 hours to obtain a hydroxylated boronene solution.
  • centrifuge uses a centrifuge to centrifuge at a set speed of 5000r/min for 10 minutes to remove massive boronene and obtain a small-sized hydroxylated boronene solution, and then use a centrifuge to process the hydroxylated boronene solution at a set speed of 10000r/min Perform centrifugation for 20 minutes to obtain a filter cake of hydroxylated boronene nanosheets, and wash the filter cake of hydroxylated boronene nanosheets with deionized water several times to obtain hydroxylated boronene nanosheets.
  • S30 preparing an AuNRs-ssDNA complex, and coating the AuNRs-ssDNA complex on the surface of a surface plasmon resonance chip to obtain a hydroxylated borene surface plasmon resonance sensor.
  • 300mg HAuCl 4 is used as a gold source, fully mixed with 0.5mol/L CTAB surfactant, and 0.02mol/L NaBH 4 is added under constant stirring to synthesize gold crystal nuclei; , H 2 SO 4 , L-AA mixed solution, anisotropically grow into gold nanorods under constant stirring conditions; fully mix gold nanorods with PSS, stir at room temperature for 10 hours, take out and centrifuge to remove excess PSS , to obtain PSS-modified gold nanorods (AuNRs).
  • This embodiment provides a method for preparing a surface plasmon resonance sensor of hydroxylated boronene nanosheets with a thickness of 4 nm, comprising the following steps:
  • a 2 mol/L sodium hydroxide solution was prepared, the boronene dispersion was added to 50 mL of the sodium hydroxide solution, and cold water was sonicated for 2 hours to obtain a hydroxylated boronene solution.
  • centrifuge uses a centrifuge to centrifuge at a set speed of 5000r/min for 10 minutes to remove massive boronene and obtain a small-sized hydroxylated boronene solution, and then use a centrifuge to process the hydroxylated boronene solution at a set speed of 10000r/min Perform centrifugation for 20 minutes to obtain a filter cake of hydroxylated boronene nanosheets, and wash the filter cake of hydroxylated boronene nanosheets with deionized water several times to obtain hydroxylated boronene nanosheets.
  • S30 Prepare the AuNRs-ssDNA complex, and coat the AuNRs-ssDNA complex on the surface of the surface plasmon resonance chip to obtain a hydroxylated borene surface plasmon resonance sensor.
  • 300mg HAuCl 4 is used as a gold source, fully mixed with 0.5mol/L CTAB surfactant, and 0.02mol/L NaBH 4 is added under constant stirring to synthesize gold crystal nuclei; , H 2 SO 4 , L-AA mixed solution, anisotropically grow into gold nanorods under constant stirring conditions; fully mix gold nanorods with PSS, stir at room temperature for 10 hours, take out and centrifuge to remove excess PSS , to obtain PSS-modified gold nanorods (AuNRs).
  • This embodiment provides a method for preparing a surface plasmon resonance sensor with a thickness of 6nm hydroxylated boron nanosheets, comprising the following steps:
  • a 2 mol/L sodium hydroxide solution was prepared, the boronene dispersion was added to 50 mL of the sodium hydroxide solution, and cold water was sonicated for 2 hours to obtain a hydroxylated boronene solution.
  • centrifuge uses a centrifuge to centrifuge at a set speed of 5000r/min for 10 minutes to remove massive boronene and obtain a small-sized hydroxylated boronene solution, and then use a centrifuge to process the hydroxylated boronene solution at a set speed of 10000r/min Perform centrifugation for 20 minutes to obtain a filter cake of hydroxylated boronene nanosheets, and wash the filter cake of hydroxylated boronene nanosheets with deionized water several times to obtain hydroxylated boronene nanosheets.
  • S30 preparing an AuNRs-ssDNA complex, and coating the AuNRs-ssDNA complex on the surface of a surface plasmon resonance chip to obtain a hydroxylated borene surface plasmon resonance sensor.
  • 300mg HAuCl 4 is used as a gold source, fully mixed with 0.5mol/L CTAB surfactant, and 0.02mol/L NaBH 4 is added under constant stirring to synthesize gold crystal nuclei; , H 2 SO 4 , L-AA mixed solution, anisotropically grow into gold nanorods under constant stirring conditions; fully mix gold nanorods with PSS, stir at room temperature for 10 hours, take out and centrifuge to remove excess PSS , to obtain PSS-modified gold nanorods (AuNRs).
  • the AuNRs-ssDNA complex of 20 mg was coated on the surface of the surface plasmon resonance chip, and washed with PBS buffer to obtain a surface plasmon resonance sensor with a thickness of 6 nm of hydroxylated boronene nanosheets.
  • This embodiment provides a method for preparing a surface plasmon resonance sensor with a thickness of 8nm hydroxylated boron nanosheets, comprising the following steps:
  • a 2 mol/L sodium hydroxide solution was prepared, the boronene dispersion was added to 50 mL of the sodium hydroxide solution, and cold water was sonicated for 2 hours to obtain a hydroxylated boronene solution.
  • centrifuge uses a centrifuge to centrifuge at a set speed of 5000r/min for 10 minutes to remove massive boronene and obtain a small-sized hydroxylated boronene solution, and then use a centrifuge to process the hydroxylated boronene solution at a set speed of 10000r/min Perform centrifugation for 20 minutes to obtain a filter cake of hydroxylated boronene nanosheets, and wash the filter cake of hydroxylated boronene nanosheets with deionized water several times to obtain hydroxylated boronene nanosheets.
  • S30 preparing an AuNRs-ssDNA complex, and coating the AuNRs-ssDNA complex on the surface of a surface plasmon resonance chip to obtain a hydroxylated borene surface plasmon resonance sensor.
  • 300mg HAuCl 4 is used as a gold source, fully mixed with 0.5mol/L CTAB surfactant, and 0.02mol/L NaBH 4 is added under constant stirring to synthesize gold crystal nuclei; , H 2 SO 4 , L-AA mixed solution, anisotropically grow into gold nanorods under constant stirring conditions; fully mix gold nanorods with PSS, stir at room temperature for 10 hours, take out and centrifuge to remove excess PSS , to obtain PSS-modified gold nanorods (AuNRs).
  • the surface plasmon resonance chip is obtained by spin-coating hydroxylated boronene nanosheet dispersions of different thicknesses on the gold film, and then the AuNRs-ssDNA complex is coated on the surface plasmon
  • the surface of the bulk resonance chip is used to obtain boronene surface plasmon resonance sensors with different thicknesses, so as to set the influence of different thicknesses of hydroxylated boronene nanosheets on the performance of the boronene surface plasmon resonance sensor.
  • the refraction angle of the boron surface plasmon resonance sensor decreases with the decrease of mi-RNA, and the less the concentration of mi-RNA, the fewer molecules paired with ssDNA by mi-RNA, Then the less is analyzed, that is, the smaller the refraction angle, therefore, important information such as the adsorption and dissociation constants of the interaction between the bioreceptor and the sample to be tested can be obtained according to the change of the dynamic signal that can be detected.

Abstract

A surface plasmon resonance sensor, a preparation method therefor and an application thereof, belonging to the technical field of semiconductors. The surface plasmon resonance sensor comprises a surface plasmon resonance chip and a noble metal-ssDNA complex. The surface plasmon resonance chip comprises a first noble metal matrix and a borophene layer bonded to the first noble metal matrix. The noble metal-ssDNA complex is bonded to the surface plasmon resonance chip, and comprises a second noble metal matrix and an ssDNA bonded to the second noble metal matrix. Because a first noble metal-borophene-second noble metal coupled surface plasmon resonance effect, the surface plasmon resonance sensor has a high sensitivity and may be used for detecting miRNA of a sample, and has the advantages of label-free detection, high reutilization and low costs.

Description

表面等离子体共振传感器及其制备方法和应用Surface plasmon resonance sensor and its preparation method and application 技术领域technical field
本申请属于半导体技术领域，尤其涉及一种表面等离子体共振传感器及其制备方法和应用。The application belongs to the technical field of semiconductors, and in particular relates to a surface plasmon resonance sensor and its preparation method and application.
背景技术Background technique
由于miRNA的含量很低，在给定样本中只占总RNA质量的0.01％，使得miRNA检测面临挑战性。另外，由于扩增困难、成本高、操作复杂和灵敏度低，使得定量实时PCR(qRT–PCR)、northern杂交以及基于微阵列的杂交等miRNA检测技术在临床实践中的应用受到限制。而表面等离子体激元共振(surface plasmon resonance，SPR)技术具有无损检测、高重复性和低成本等优点，已被证明可以通过评估芯片表面的折射率变化来研究分子间的相互作用。但由于芯片表面上固定探针DNA和miRNA的数量有限，使用传统SPR技术检测极低浓度的生物分子仍然具有挑战性。因此，迫切需要寻找具有较大吸附能和功函数增量的先进材料，以提高SPR生物传感器的性能。The detection of miRNAs is challenging due to the low content of miRNAs, accounting for only 0.01% of the total RNA mass in a given sample. In addition, the application of miRNA detection technologies such as quantitative real-time PCR (qRT–PCR), northern hybridization, and microarray-based hybridization in clinical practice is limited due to difficult amplification, high cost, complex operation, and low sensitivity. The surface plasmon resonance (SPR) technology has the advantages of non-destructive testing, high repeatability and low cost, and has been proven to study molecular interactions by evaluating the refractive index changes on the chip surface. However, due to the limited amount of immobilized probe DNA and miRNA on the chip surface, it is still challenging to detect biomolecules at extremely low concentrations using traditional SPR techniques. Therefore, it is urgent to search for advanced materials with large adsorption energy and work function increment to improve the performance of SPR biosensors.
目前，许多新兴的二维纳米材料包括包括石墨烯、过渡金属二卤化物(TMDs)、拓扑绝缘体、黑磷和MXenes被用于DNA分子传感，然而，由于与生物分子的弱相互作用和较差的化学稳定性，使得这些纳米材料的应用受到限制。Currently, many emerging two-dimensional nanomaterials including graphene, transition metal dichalcogenides (TMDs), topological insulators, black phosphorus, and MXenes are used for DNA molecular sensing, however, due to weak interactions with biomolecules and relatively Poor chemical stability limits the application of these nanomaterials.
因此，现有的二维纳米材料与与生物标记物存在弱相互作用和较差的化学稳定性而影响SPR生物传感器的性能。Therefore, the existing two-dimensional nanomaterials have weak interactions with biomarkers and poor chemical stability, which affects the performance of SPR biosensors.
发明内容Contents of the invention
本申请的目的在于提供一种表面等离子体共振传感器及其制备方法和应用，旨在解决现有的二维纳米材料与生物标记物存在弱相互作用和较差的化学稳定性而影响SPR生物传感器性能的问题。The purpose of this application is to provide a surface plasmon resonance sensor and its preparation method and application, aiming to solve the problem of weak interaction between existing two-dimensional nanomaterials and biomarkers and poor chemical stability that affect SPR biosensors Performance issues.
为实现上述申请目的，本申请采用的技术方案如下：In order to realize the above-mentioned application purpose, the technical scheme adopted in this application is as follows:
第一方面，本申请提供一种表面等离子体共振传感器，所述表面等离子体共振传感器包括：In a first aspect, the present application provides a surface plasmon resonance sensor, the surface plasmon resonance sensor comprising:
表面等离子体共振芯片，所述表面等离子体共振芯片包括第一贵金属基体，在所述第一贵金属基体上结合有硼烯层；A surface plasmon resonance chip, the surface plasmon resonance chip comprising a first noble metal substrate, a boron layer is bonded to the first noble metal substrate;
贵金属-ssDNA复合物，所述贵金属-ssDNA复合物结合在所述表面等离子体共振芯片上，且所述贵金属-ssDNA复合物包括第二贵金属基体和结合在所述第二贵金属基体上的ssDNA。A noble metal-ssDNA complex, the noble metal-ssDNA complex is combined on the surface plasmon resonance chip, and the noble metal-ssDNA complex includes a second noble metal substrate and ssDNA combined on the second noble metal substrate.
第二方面，本申请提供一种表面等离子体共振传感器的制备方法，所述制备方法包括以下步骤：In a second aspect, the present application provides a method for preparing a surface plasmon resonance sensor, the preparation method comprising the following steps:
制备硼烯纳米片分散液；Prepare boron nanosheet dispersion;
将所述硼烯纳米片分散液在第一贵金属基体表面进行成膜处理，获得表面等离子体共振芯片；performing a film-forming treatment on the surface of the boron nanosheet dispersion on the surface of the first noble metal substrate to obtain a surface plasmon resonance chip;
将ssDNA结合在第二贵金属基体上，形成贵金属-ssDNA复合物；Binding ssDNA to a second noble metal substrate to form a noble metal-ssDNA complex;
将所述贵金属-ssDNA复合物组装至所述表面等离子体共振芯片上，得到表面等离子体共振传感器。The noble metal-ssDNA complex is assembled on the surface plasmon resonance chip to obtain a surface plasmon resonance sensor.
第三方面，本申请提供一种核酸检测传感器，该核酸检测传感器包括上述的表面等离子体共振传感器。In a third aspect, the present application provides a nucleic acid detection sensor, which includes the above-mentioned surface plasmon resonance sensor.
第四方面，本申请提供一种硼烯纳米片的制备方法，包括如下步骤：In a fourth aspect, the present application provides a method for preparing boron nanosheets, comprising the following steps:
对硼粉与溶剂的混合物就进行研磨处理，获得硼颗粒前驱体溶液；Grinding the mixture of boron powder and solvent to obtain boron particle precursor solution;
对所述硼颗粒前驱体溶液进行超声剥离处理，获得硼烯纳米片分散液；Ultrasonic stripping is performed on the boron particle precursor solution to obtain a boron nanosheet dispersion;
对所述硼烯纳米片分散液进行离心分离处理，获得目标层数的硼烯纳米片。The boronene nanosheet dispersion is subjected to centrifugation to obtain boronene nanosheets with a target number of layers.
本申请第一方面提供的表面等离子体共振传感器，其包括表面等离子体共振芯片和结合在表面等离子体共振芯片上的贵金属-ssDNA复合物，由于第一贵金属基体上结合有硼烯层，使得该表面等离子体共振传感器具有第一贵金属-硼烯-第二贵金属相互耦合的表面等离子体共振效应，同时通过第二贵金属表面 接枝单链DNA，单链DNA包覆的第二贵金属与硼烯之间具有强相互作用，因此，该等离子体共振传感器具有高灵敏度，可用于检测样品的miRNA，并且具有无标记检测、可重复利用高以及成本低等优点。The surface plasmon resonance sensor provided in the first aspect of the present application includes a surface plasmon resonance chip and a noble metal-ssDNA complex bonded on the surface plasmon resonance chip. Since the boron layer is bonded to the first noble metal substrate, the The surface plasmon resonance sensor has the surface plasmon resonance effect of the mutual coupling of the first noble metal-boron-second noble metal, and at the same time, the single-stranded DNA is grafted on the surface of the second noble metal, and the second noble metal coated with the single-stranded DNA and the boron Therefore, the plasmon resonance sensor has high sensitivity and can be used to detect miRNA in samples, and has the advantages of label-free detection, high reusability and low cost.
本申请第二方面提供的表面等离子体共振传感器的制备方法，其通过将制备的硼烯纳米片分散液在第一贵金属基体表面进行成膜处理，可以形成具有表面等离子体共振效应的表面等离子体共振芯片，将贵金属-ssDNA复合物组装至表面等离子体共振芯片上，使形成的表面等离子体共振传感器具有第一贵金属-硼烯-第二贵金属相互耦合的表面等离子体共振效应，在提高表面等离子体共振传感器灵敏度的同时，第二贵金属表面通过修饰与待检测样品匹配的单链DNA，可以有效监测检测样品的miRNA。The method for preparing a surface plasmon resonance sensor provided in the second aspect of the present application can form a surface plasmon with a surface plasmon resonance effect by performing film-forming treatment on the prepared boron nanosheet dispersion on the surface of the first noble metal substrate. Resonance chip, the noble metal-ssDNA complex is assembled on the surface plasmon resonance chip, so that the formed surface plasmon resonance sensor has the surface plasmon resonance effect of the mutual coupling of the first noble metal-boronene-second noble metal. While improving the sensitivity of the bulk resonance sensor, the second noble metal surface can effectively monitor the miRNA of the detection sample by modifying the single-stranded DNA that matches the sample to be detected.
本申请第三方面提供的核酸检测传感器，由于该核酸检测传感器包括的表面等离子体共振传感器含有贵金属-ssDNA复合物，其通过表面修饰与待检测样品匹配的单链DNA，从而可以有效监测该样品的miRNA。The nucleic acid detection sensor provided in the third aspect of the present application, since the surface plasmon resonance sensor included in the nucleic acid detection sensor contains a noble metal-ssDNA complex, which can effectively monitor the sample by modifying the surface to match the single-stranded DNA of the sample to be detected miRNA.
本申请第四方面提供的硼烯纳米片的制备方法，先通过对硼粉进行研磨处理，获得尺寸均匀且细小的硼颗粒前驱体溶液，然后对硼颗粒前驱体溶液进行超声剥离处理，可以获得不同层数的硼烯纳米片分散液，再对硼烯纳米片分散液进行离心分离处理，可获得目标层数可控的硼烯纳米片，因此，该制备方法可以制备出单层或多层的硼烯纳米片。The preparation method of the boron nanosheets provided in the fourth aspect of the present application first grinds the boron powder to obtain a uniform and fine boron particle precursor solution, and then performs ultrasonic stripping on the boron particle precursor solution to obtain Boronene nanosheet dispersions with different layers, and then centrifuge the boronene nanosheet dispersion to obtain boronene nanosheets with a controllable number of layers. Therefore, this preparation method can prepare single-layer or multi-layer boron nanosheets.
附图说明Description of drawings
为了更清楚地说明本申请实施例中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本申请的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。In order to more clearly illustrate the technical solutions in the embodiments of the present application, the accompanying drawings that need to be used in the descriptions of the embodiments or the prior art will be briefly introduced below. Obviously, the accompanying drawings in the following description are only for the present application For some embodiments, those of ordinary skill in the art can also obtain other drawings based on these drawings without any creative effort.
图1是本申请实施例提供的基于硼烯材料的表面等离子体共振传感器的制备方法的流程示意图。Fig. 1 is a schematic flowchart of a method for preparing a surface plasmon resonance sensor based on a boron material provided in an embodiment of the present application.
具体实施方式Detailed ways
为了使本申请要解决的技术问题、技术方案及有益效果更加清楚明白，以下结合实施例，对本申请进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本申请，并不用于限定本申请。In order to make the technical problems, technical solutions and beneficial effects to be solved in the present application clearer, the present application will be further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described here are only used to explain the present application, and are not intended to limit the present application.
本申请中，术语“和/或”，描述关联对象的关联关系，表示可以存在三种关系，例如，A和/或B，可以表示：单独存在A，同时存在A和B，单独存在B的情况。其中A，B可以是单数或者复数。字符“/”一般表示前后关联对象是一种“或”的关系。In this application, the term "and/or" describes the association relationship of associated objects, indicating that there may be three relationships, for example, A and/or B may mean: A exists alone, A and B exist simultaneously, and B exists alone Condition. Among them, A and B can be singular or plural. The character "/" generally indicates that the contextual objects are an "or" relationship.
本申请中，“至少一个”是指一个或者多个，“多个”是指两个或两个以上。“以下至少一项(个)”或其类似表达，是指的这些项中的任意组合，包括单项(个)或复数项(个)的任意组合。例如，“a,b,或c中的至少一项(个)”，或，“a,b,和c中的至少一项(个)”，均可以表示：a,b,c,a-b(即a和b),a-c,b-c,或a-b-c，其中a,b,c分别可以是单个，也可以是多个。In this application, "at least one" means one or more, and "multiple" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or plural items. For example, "at least one item (unit) of a, b, or c", or "at least one item (unit) of a, b, and c" can mean: a, b, c, a-b( That is, a and b), a-c, b-c, or a-b-c, where a, b, and c can be single or multiple.
应理解，在本申请的各种实施例中，上述各过程的序号的大小并不意味着执行顺序的先后，部分或全部步骤可以并行执行或先后执行，各过程的执行顺序应以其功能和内在逻辑确定，而不应对本申请实施例的实施过程构成任何限定。It should be understood that in various embodiments of the present application, the sequence numbers of the above-mentioned processes do not mean the order of execution, and some or all steps may be executed in parallel or sequentially, and the execution order of each process shall be based on its functions and The internal logic is determined and should not constitute any limitation to the implementation process of the embodiment of the present application.
在本申请实施例中使用的术语是仅仅出于描述特定实施例的目的，而非旨在限制本申请。在本申请实施例和所附权利要求书中所使用的单数形式的“一种”、“所述”和“该”也旨在包括多数形式，除非上下文清楚地表示其他含义。Terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a", "said" and "the" used in the embodiments of this application and the appended claims are also intended to include plural forms unless the context clearly indicates otherwise.
本申请实施例说明书中所提到的相关成分的重量不仅仅可以指代各组分的具体含量，也可以表示各组分间重量的比例关系，因此，只要是按照本申请实施例说明书相关组分的含量按比例放大或缩小均在本申请实施例说明书公开的范围之内。具体地，本申请实施例说明书中所述的质量可以是μg、mg、g、kg等化工领域公知的质量单位。The weight of the relevant components mentioned in the description of the embodiments of the present application can not only refer to the specific content of each component, but also represent the proportional relationship between the weights of the various components. The scaling up or down of the content of the fraction is within the scope disclosed in the description of the embodiments of the present application. Specifically, the mass described in the description of the embodiments of the present application may be μg, mg, g, kg and other well-known mass units in the chemical industry.
术语“第一”、“第二”仅用于描述目的，用来将目的如物质彼此区分开，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。例如，在不脱离本申请实施例范围的情况下，第一XX也可以被称为第二XX，类似地，第二XX也可以被称为第一XX。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。The terms "first" and "second" are only used for descriptive purposes to distinguish objects such as substances from each other, and cannot be understood as indicating or implying relative importance or implicitly specifying the quantity of indicated technical features. For example, without departing from the scope of the embodiments of the present application, the first XX can also be called the second XX, and similarly, the second XX can also be called the first XX. Thus, a feature defined as "first" and "second" may explicitly or implicitly include one or more of these features.
本申请实施例第一方面提供一种表面等离子体共振传感器，该表面等离子体共振传感器包括：The first aspect of the embodiment of the present application provides a surface plasmon resonance sensor, the surface plasmon resonance sensor includes:
表面等离子体共振芯片，表面等离子体共振芯片包括第一贵金属基体，在第一贵金属基体上结合有硼烯层；A surface plasmon resonance chip, the surface plasmon resonance chip includes a first noble metal substrate, and a boron layer is bonded to the first noble metal substrate;
贵金属-ssDNA复合物，贵金属-ssDNA复合物结合在表面等离子体共振芯片上，且贵金属-ssDNA复合物包括第二贵金属基体和结合在第二贵金属基体上的ssDNA。The noble metal-ssDNA complex, the noble metal-ssDNA complex is combined on the surface plasmon resonance chip, and the noble metal-ssDNA complex includes a second noble metal substrate and ssDNA combined on the second noble metal substrate.
本申请实施例提供的表面等离子体共振传感器，其包括表面等离子体共振芯片和贵金属-ssDNA复合物，由于表面等离子体共振芯片包括的第一贵金属基体上结合有硼烯层，贵金属-ssDNA复合物通过第二贵金属表面接枝单链DNA，因此，单链DNA包覆的第二贵金属与硼烯之间具有强相互作用，使得该表面等离子体共振传感器具有第一贵金属-硼烯-第二贵金属相互耦合的表面等离子体共振效应，因此，该等离子体共振传感器具有高灵敏度，可用于检测样品的miRNA，并且具有无标记检测、可重复利用高以及成本低等优点。The surface plasmon resonance sensor provided in the embodiment of the present application includes a surface plasmon resonance chip and a noble metal-ssDNA complex. Since the surface plasmon resonance chip includes a boron layer on the first noble metal substrate, the noble metal-ssDNA complex Single-stranded DNA is grafted on the surface of the second noble metal, therefore, there is a strong interaction between the second noble metal coated with single-stranded DNA and boron, so that the surface plasmon resonance sensor has the first noble metal-boron-second noble metal Due to the mutually coupled surface plasmon resonance effect, the plasmon resonance sensor has high sensitivity and can be used to detect miRNA in samples, and has the advantages of label-free detection, high reusability, and low cost.
在一些实施例中，硼烯层的厚度为1-100nm，具体的，硼烯层的厚度可以为1nm，还可以为2-5nm，还可以为100nm。硼烯层可以为连续分布的硼烯膜层，可以为岛状分布的硼烯膜层，还可以为连续分布的硼烯膜层和岛状分布的硼烯膜层。本实施例优选超薄且连续分布的硼烯膜层，以增强第一贵金属基体与硼烯层之间的表面等离子体共振效应，进而提高等离子体共振传感器灵敏度。In some embodiments, the thickness of the boron layer is 1-100 nm. Specifically, the thickness of the boron layer may be 1 nm, 2-5 nm, or 100 nm. The boron layer can be a continuously distributed boron film layer, can be an island-shaped distributed boron film layer, and can also be a continuously distributed boron film layer and an island-shaped distributed boron film layer. In this embodiment, an ultra-thin and continuously distributed boron film layer is preferred to enhance the surface plasmon resonance effect between the first noble metal substrate and the boron layer, thereby improving the sensitivity of the plasmon resonance sensor.
在一些实施例中，硼烯层为羟基化硼烯纳米片，该羟基化硼烯纳米片的层数为1-100层。具体的，该羟基化硼烯纳米片的层数可以为1层，可以为2-5 层，还可以为100层。本实施例优选的硼烯纳米片经过与碱性溶液进行表面改性处理，使得到的羟基化硼烯纳米片表现出显著的半导体特性，进而增强与第一贵金属基体之间的表面等离子体共振效应，可以进一步提高等离子体共振传感器灵敏度。In some embodiments, the boron layer is a hydroxylated boron nanosheet, and the number of layers of the hydroxylated boron nanosheet is 1-100 layers. Specifically, the number of layers of the hydroxylated boronene nanosheets may be 1 layer, 2-5 layers, or 100 layers. The preferred boronene nanosheets in this embodiment undergo surface modification treatment with an alkaline solution, so that the obtained hydroxylated boronene nanosheets exhibit significant semiconductor characteristics, thereby enhancing the surface plasmon resonance with the first noble metal substrate Effect, can further improve the sensitivity of the plasmon resonance sensor.
在一些实施例中，第一贵金属基体包括贵金属膜，该贵金属膜可以为Au膜，可以为Ag膜，可以为Pt膜，还可以为Pd膜，还可以为Au和Ag膜。第一贵金属膜的厚度为2nm-20μm，具体的，贵金属膜的厚度可以为2nm，可以为20μm，还可以为100nm。第二贵金属基体包括Au和/或Ag纳米棒、纳米立方、纳米片中的至少一种，也不限于此。具体的，第二贵金属基体可以包括Au纳米棒，可以包括Au纳米立方，还可以包括Au纳米片，还可以包括Au纳米棒和Au纳米立方。In some embodiments, the first noble metal substrate includes a noble metal film, and the noble metal film can be an Au film, an Ag film, a Pt film, a Pd film, or an Au and Ag film. The thickness of the first noble metal film is 2 nm-20 μm. Specifically, the thickness of the noble metal film may be 2 nm, 20 μm, or 100 nm. The second noble metal matrix includes at least one of Au and/or Ag nanorods, nanocubes, and nanosheets, but is not limited thereto. Specifically, the second noble metal matrix may include Au nanorods, Au nanocubes, Au nanosheets, Au nanorods and Au nanocubes.
在一些实施例中，贵金属-ssDNA复合物的量在表面等离子体共振芯片上的负载量为0.01mol/cm 2-10mol/cm 2，具体的，贵金属-ssDNA复合物的量在表面等离子体共振芯片上的负载量为5mol/cm 2。 In some embodiments, the loading amount of the noble metal-ssDNA complex on the surface plasmon resonance chip is 0.01 mol/cm 2 -10 mol/cm 2 , specifically, the amount of the noble metal-ssDNA complex on the surface plasmon resonance The loading on the chip was 5 mol/cm 2 .
具体的，当待测样品的单链RNA溶液流经羟基化硼烯表面等离子体共振传感器的表面时，单链RNA与互补的AuNR-ssDNA配对形成双链；miRNA与AuNR-ssDNA之间的相互作用导致AuNR-ssDNA从羟基化硼烯纳米片中释放，使得羟基化硼烯表面等离子体共振传感器表面的AuNR-ssDNA分子的减少，从而使得硼烯表面等离子体共振传感器的检测信号(共振角度)显著减小，因此，可以根据检测出的动态信号的变化来得到生物受体与待测样品相互作用的吸附、解离常数等重要信息。本实施例由于单链DNA与羟基化硼烯纳米片之间的强相互作用，使AuNR-ssDNA复合物被吸附到羟基化硼烯纳米片上，因此，单链DNA包覆的Au纳米棒和羟基化硼烯之间的强相互作用，可以增强硼烯传感器的表面等离子体效应，进而可以进一步提高羟基化硼烯表面等离子体共振传感器的灵敏度，而第二贵金属基体表面通过修饰与待检测样品匹配的单链DNA，可以有效监测样品的miRNA。Specifically, when the single-stranded RNA solution of the sample to be tested flows through the surface of the hydroxylated boron surface plasmon resonance sensor, the single-stranded RNA pairs with the complementary AuNR-ssDNA to form a double strand; the interaction between miRNA and AuNR-ssDNA The effect leads to the release of AuNR-ssDNA from the hydroxylated boron nanosheets, which reduces the AuNR-ssDNA molecules on the surface of the hydroxylated boron surface plasmon resonance sensor, thereby making the detection signal (resonance angle) of the boron surface plasmon resonance sensor Therefore, important information such as the adsorption and dissociation constants of the interaction between the bioreceptor and the sample to be tested can be obtained according to the detected changes in the dynamic signal. In this example, due to the strong interaction between single-stranded DNA and hydroxylated boron nanosheets, the AuNR-ssDNA complex was adsorbed on hydroxylated boron nanosheets. Therefore, single-stranded DNA-coated Au nanorods and hydroxylated The strong interaction between boronene can enhance the surface plasmon effect of the boronene sensor, which can further improve the sensitivity of the hydroxylated boronene surface plasmon resonance sensor, and the surface of the second noble metal substrate can be modified to match the sample to be detected The single-stranded DNA can effectively monitor the miRNA in the sample.
本申请实施例第二方面提供一种表面等离子体共振传感器的制备方法，该制备方法包括以下步骤：The second aspect of the embodiment of the present application provides a preparation method of a surface plasmon resonance sensor, the preparation method comprising the following steps:
S10：制备硼烯纳米片分散液；S10: preparing a boronene nanosheet dispersion;
S20：将硼烯纳米片分散液在第一贵金属基体表面进行成膜处理，获得表面等离子体共振芯片；S20: performing a film-forming treatment on the boronene nanosheet dispersion on the surface of the first noble metal substrate to obtain a surface plasmon resonance chip;
S30：将ssDNA结合在第二贵金属基体上，形成贵金属-ssDNA复合物；S30: binding ssDNA to a second noble metal substrate to form a noble metal-ssDNA complex;
S40：将贵金属-ssDNA复合物组装至所述表面等离子体共振芯片上，得到表面等离子体共振传感器。S40: Assembling the noble metal-ssDNA complex on the surface plasmon resonance chip to obtain a surface plasmon resonance sensor.
本申请实施例提供的表面等离子体共振传感器的制备方法，其通过将制备的硼烯纳米片分散液在第一贵金属基体表面进行成膜处理，可以形成具有表面等离子体共振效应的表面等离子体共振芯片，将贵金属-ssDNA复合物组装至表面等离子体共振芯片上，使形成的表面等离子体共振传感器具有第一贵金属-硼烯-第二贵金属相互耦合的表面等离子体共振效应，在提高表面等离子体共振传感器灵敏度的同时，第二贵金属表面通过修饰与待检测样品匹配的单链DNA，可以有效监测检测样品的miRNA。The preparation method of the surface plasmon resonance sensor provided in the embodiment of the present application can form a surface plasmon resonance sensor with a surface plasmon resonance effect by performing film-forming treatment on the prepared boron nanosheet dispersion liquid on the surface of the first noble metal substrate. Chip, the noble metal-ssDNA complex is assembled on the surface plasmon resonance chip, so that the formed surface plasmon resonance sensor has the surface plasmon resonance effect of the mutual coupling of the first noble metal-boronene-second noble metal. At the same time as the sensitivity of the resonance sensor, the surface of the second noble metal can effectively monitor the miRNA of the detection sample by modifying the single-stranded DNA that matches the sample to be detected.
在步骤S10中，制备硼烯纳米片分散液可以按照如下方法制备获得：In step S10, the boronene nanosheet dispersion can be prepared according to the following method:
先通过对硼粉与有机溶剂的混合物进行研磨处理，获得尺寸均匀且细小的硼颗粒前驱体溶液，以便于下一步的超声剥离处理；其中，有机溶剂包括仲丁醇、乙二醇、丙三醇以及正丁醇中的至少一种，但不限于此。具体的，有机溶剂可以为仲丁醇。对硼颗粒前驱体溶液进行超声剥离处理，以获得不同层数的硼烯纳米片分散液。对硼烯纳米片分散液进行离心分离处理，获得目标层数可控的硼烯纳米片；本实施例可以通过调节离心转速区间制备单层的硼烯纳米片，以增强第一贵金属基体与硼烯层之间的表面等离子体共振效应，进而提高等离子体共振传感器灵敏度。Firstly, by grinding the mixture of boron powder and organic solvent, a boron particle precursor solution with uniform size and fineness is obtained, which is convenient for the next step of ultrasonic stripping treatment; wherein, the organic solvent includes sec-butanol, ethylene glycol, glycerin At least one of alcohol and n-butanol, but not limited thereto. Specifically, the organic solvent may be sec-butanol. The boron particle precursor solution was ultrasonically stripped to obtain boron nanosheet dispersions with different layers. The boronene nanosheet dispersion is subjected to centrifugal separation to obtain boronene nanosheets with a controllable target layer number; in this embodiment, single-layer boronene nanosheets can be prepared by adjusting the centrifugal speed range to strengthen the interaction between the first noble metal matrix and boron. The surface plasmon resonance effect between the olefin layers can improve the sensitivity of the plasmon resonance sensor.
实施例中，硼烯纳米片还通过与碱性溶液进行表面改性处理，获得羟基化硼烯纳米片。其中，碱性溶液包括氢氧化钠、氢氧化钾以及氢氧化锂中的至少 一种，但不限于此。在一实施例中，碱性溶液可以为氢氧化钠，还可以为氢氧化钠和氢氧化钾。本实施例通过将硼烯纳米片与碱性溶液进行表面改性处理，使得到的羟基化硼烯纳米片表现出显著的半导体特性，进而可与第一贵金属基体产生更强的表面等离子体共振效应；同时羟基化硼烯纳米片具有稳定的化学性质和与生物标志物间强的相互作用，解决了现有的二维纳米材料因存在与生物标记物存在弱相互作用和较差的化学稳定性导致影响SPR生物传感器性能的问题。In the embodiment, the boronene nanosheets are also subjected to surface modification treatment with an alkaline solution to obtain hydroxylated boronene nanosheets. Wherein, the alkaline solution includes at least one of sodium hydroxide, potassium hydroxide and lithium hydroxide, but is not limited thereto. In one embodiment, the alkaline solution may be sodium hydroxide, or sodium hydroxide and potassium hydroxide. In this example, the surface modification treatment of boronene nanosheets and alkaline solution enables the obtained hydroxylated boronene nanosheets to exhibit significant semiconductor characteristics, which in turn can produce stronger surface plasmon resonance with the first noble metal matrix effect; at the same time, hydroxylated boronene nanosheets have stable chemical properties and strong interactions with biomarkers, which solves the problem of weak interactions with biomarkers and poor chemical stability of existing two-dimensional nanomaterials Sexuality leads to problems affecting the performance of SPR biosensors.
在步骤S20中，硼烯纳米片分散液在第一贵金属基体表面进行成膜处理可以采用现有成膜方法或基于现有成膜方法改进的成膜方法，或其他性的成膜方法。只要是能够将硼烯纳米片分散液在第一贵金属基体表面成膜均在本申请实施例公开的范围，如实施例中，采用旋涂方法根据设定转速将羟基化硼烯纳米片分散液旋涂在第一贵金属基体表面，可以获得羟基化硼烯纳米片层厚度不同的表面等离子体共振芯片。实施例中，第一贵金属可以包括金、银、钯以及铂中的任意一种，但并不限于此。具体第一贵金属可以为金。其中，硼烯纳米片或羟基化硼烯纳米片的目标厚度为1-100nm，具体硼烯纳米片的目标厚度可以为1nm，2nm，4nm，6nm，8nm等典型但非限制性的厚度。其中，设定旋涂的转速为1-20000r/min，具体设定转速可以为5000r/min，6000r/min，8000r/min，9000r/min，12000r/min。本实施例通过调控旋涂转速和硼烯纳米片或羟基化硼烯纳米片分散液的滴量可以获得超薄的羟基化硼烯纳米片层的表面等离子体共振芯片，进而为获得高灵敏度的羟基化硼烯表面等离子体共振传感器打好基础。In step S20, the film-forming treatment of the borene nanosheet dispersion on the surface of the first noble metal substrate may use an existing film-forming method or an improved film-forming method based on an existing film-forming method, or other film-forming methods. As long as the boronene nanosheet dispersion can be formed into a film on the surface of the first noble metal substrate, it is within the scope disclosed in the embodiments of the present application. As in the embodiment, the hydroxylated boronene nanosheet dispersion is sprayed according to the set speed by spin coating. Surface plasmon resonance chips with different thicknesses of hydroxylated boronene nanosheets can be obtained by spin-coating on the surface of the first noble metal substrate. In an embodiment, the first precious metal may include any one of gold, silver, palladium and platinum, but is not limited thereto. Specifically, the first precious metal may be gold. Wherein, the target thickness of boronene nanosheets or hydroxylated boronene nanosheets is 1-100nm, and the specific target thickness of boronene nanosheets can be 1nm, 2nm, 4nm, 6nm, 8nm and other typical but non-limiting thicknesses. Wherein, the rotational speed of the spin coating is set at 1-20000r/min, and the specific set rotational speed can be 5000r/min, 6000r/min, 8000r/min, 9000r/min, 12000r/min. In this embodiment, by adjusting the rotation speed of the spin coating and the droplet volume of boronene nanosheets or hydroxylated boronene nanosheet dispersions, an ultra-thin surface plasmon resonance chip with hydroxylated boronene nanosheets can be obtained, and then to obtain a high-sensitivity chip. Hydroxyboron surface plasmon resonance sensors lay the groundwork.
在步骤S30中，将单链DNA结合在第二贵金属基体表面，获得贵金属-ssDNA复合物。其中，第二贵金属基体包括Au/或Ag纳米棒、纳米立方、纳米片中的至少一种，但不限于此。在一实施例中，第二贵金属基体可以为Au纳米棒。其中，Au纳米棒的制备方法可以按照包括以下步骤：将金源HAuCl 4与表面活性剂CTAB充分混合，并在搅拌条件下加入NaBH 4，合成金晶核；将金晶核加入到含有CTAB、HAuCl 4、AgNO 3、H 2SO 4、L-AA混合溶液中，并在 搅拌条件下各向异性生长为金纳米棒(AuNR)。将硫基化ssDNA溶液添加到AuNRs溶液中，反应后获得AuNR-ssDNA混合液；将AuNR-ssDNA混合液与NaCl混合后进行离心处理，除去过量的硫基化ssDNA后获得AuNR-ssDNA颗粒；将AuNR-ssDNA颗粒分散处理和超声处理，后进行搅拌处理获得贵金属-ssDNA复合物。 In step S30, the single-stranded DNA is bound to the surface of the second noble metal substrate to obtain a noble metal-ssDNA complex. Wherein, the second noble metal matrix includes at least one of Au/or Ag nanorods, nanocubes, and nanosheets, but is not limited thereto. In one embodiment, the second noble metal matrix may be Au nanorods. Wherein, the preparation method of Au nanorods may include the following steps: fully mix the gold source HAuCl 4 and the surfactant CTAB, and add NaBH 4 under stirring conditions to synthesize gold crystal nuclei; add the gold crystal nuclei to the mixture containing CTAB, HAuCl 4 , AgNO 3 , H 2 SO 4 , and L-AA mixed solution, and grow anisotropically into gold nanorods (AuNR) under stirring conditions. Add the thiolated ssDNA solution to the AuNRs solution, and obtain the AuNR-ssDNA mixture after the reaction; mix the AuNR-ssDNA mixture with NaCl and perform centrifugation to remove excess thiolated ssDNA to obtain AuNR-ssDNA particles; AuNR-ssDNA particles were dispersed and sonicated, and then stirred to obtain noble metal-ssDNA complexes.
在步骤S40中，将贵金属-ssDNA复合物组装至表面等离子体共振芯片上，得到表面等离子体共振传感器，具体方法包括，根据贵金属-ssDNA复合物的量在表面等离子体共振芯片上的负载量为0.01mol/cm 2-10mol/cm 2，将适量贵金属-ssDNA复合物旋涂到等离子体共振芯片上，室温放置5min-60min，后用PBS缓冲液洗涤，得到表面等离子体共振传感器；具体负载量可以为5mol/cm 2。可以实现将第一贵金属、羟基化硼烯纳米片以及第二贵金属形成相互耦合的表面等离子体共振效应，并通过表面接枝单链DNA，单链DNA包覆的第二贵金属与羟基化硼烯纳米片之间的强相互作用，使表面等离子体共振传感器具有很高的灵敏度。 In step S40, the noble metal-ssDNA complex is assembled on the surface plasmon resonance chip to obtain a surface plasmon resonance sensor. The specific method includes, according to the loading amount of the noble metal-ssDNA complex on the surface plasmon resonance chip: 0.01mol/cm 2 -10mol/cm 2 , spin-coat an appropriate amount of noble metal-ssDNA complex on a plasmon resonance chip, place it at room temperature for 5min-60min, and then wash it with PBS buffer to obtain a surface plasmon resonance sensor; specific loading It may be 5 mol/cm 2 . The surface plasmon resonance effect of coupling the first noble metal, hydroxylated boronene nanosheets and the second noble metal can be realized, and by grafting single-stranded DNA on the surface, the second noble metal coated with single-stranded DNA and hydroxylated boronene The strong interaction between the nanosheets enables the high sensitivity of the surface plasmon resonance sensor.
第三方面，本申请实施例还提供了一种核酸检测传感器，该核酸检测传感器包括上述的表面等离子体共振传感器。In a third aspect, the embodiment of the present application further provides a nucleic acid detection sensor, which includes the above-mentioned surface plasmon resonance sensor.
本申请实施例提供的核酸检测传感器，由于该核酸检测传感器包括的表面等离子体共振传感器的羟基化硼烯纳米片表面上涂覆了贵金属-ssDNA复合物，因此，可以通过表面修饰与待检测样品匹配的单链DNA，从而有效监测样品的miRNA。The nucleic acid detection sensor provided in the embodiment of the present application, since the surface plasmon resonance sensor of the nucleic acid detection sensor is coated with a noble metal-ssDNA complex on the surface of the hydroxylated boronene nanosheet, it can be combined with the sample to be detected through surface modification. Matched single-stranded DNA to effectively monitor miRNA in samples.
下面结合具体实施例进行说明。The following will be described in conjunction with specific embodiments.
实施例1Example 1
本实施例提供一种厚度为2nm的羟基化硼烯纳米片的表面等离子体共振传感器的制备方法，包括以下步骤：This embodiment provides a method for preparing a surface plasmon resonance sensor of hydroxylated boronene nanosheets with a thickness of 2 nm, comprising the following steps:
S01：制备羟基化硼烯纳米片分散液。S01: Preparation of hydroxylated boronene nanosheet dispersion.
取500mg硼粉放入研钵中粉碎，加入仲丁醇并沿同一方向均匀研磨1个小 时，使其充分剥离，得到粒径均匀且尺寸较小的的硼颗粒前驱体溶液；将硼颗粒前驱体溶液移入探头超声细胞破碎仪中，在冰水浴中超声处理6小时，得到硼烯分散液。Take 500 mg of boron powder and put it into a mortar for crushing, add sec-butanol and grind it uniformly in the same direction for 1 hour to make it fully peeled off to obtain a boron particle precursor solution with uniform particle size and small size; the boron particle precursor The bulk solution was transferred into a probe ultrasonic cell disruptor, and ultrasonically treated in an ice-water bath for 6 hours to obtain a boronene dispersion.
配制2mol/L的氢氧化钠溶液，将硼烯分散液加入到50mL氢氧化钠溶液中，冷水超声2小时，得到羟基化硼烯溶液。A 2 mol/L sodium hydroxide solution was prepared, the boronene dispersion was added to 50 mL of the sodium hydroxide solution, and cold water was sonicated for 2 hours to obtain a hydroxylated boronene solution.
利用离心机根据设定的转速5000r/min离心处理10min，去除块状的硼烯，获得小尺寸的羟基化硼烯溶液，再利用离心机根据设定的转速10000r/min对羟基化硼烯溶液进行离心处理20min，得到羟基化硼烯纳米片滤饼，将羟基化硼烯纳米片滤饼用去离子水多次洗涤，获得羟基化硼烯纳米片。Use a centrifuge to centrifuge at a set speed of 5000r/min for 10 minutes to remove massive boronene and obtain a small-sized hydroxylated boronene solution, and then use a centrifuge to process the hydroxylated boronene solution at a set speed of 10000r/min Perform centrifugation for 20 minutes to obtain a filter cake of hydroxylated boronene nanosheets, and wash the filter cake of hydroxylated boronene nanosheets with deionized water several times to obtain hydroxylated boronene nanosheets.
向羟基化硼烯纳米片加入乙醇溶液，配成2mol/L的羟基化硼烯纳米片分散液。Add ethanol solution to the hydroxylated boron nanosheets to prepare a 2 mol/L hydroxylated boronene nanosheet dispersion.
S02：将羟基化硼烯纳米片分散液在金膜表面进行成膜处理，获得表面等离子体共振芯片；S02: forming a film-forming treatment of the hydroxylated boronene nanosheet dispersion on the surface of the gold film to obtain a surface plasmon resonance chip;
利用旋涂法以转速9000-12000r/min将羟基化硼烯纳米片分散液旋涂到金膜表面，然后移入手套箱中80℃烘干，得到表面等离子体共振芯片。Spin-coat the dispersion of hydroxylated boronene nanosheets onto the surface of the gold film at a rotational speed of 9000-12000 r/min by a spin-coating method, then move it into a glove box and dry it at 80° C. to obtain a surface plasmon resonance chip.
S30：制备AuNRs-ssDNA复合物，并将AuNRs-ssDNA复合物涂覆到表面等离子体共振芯片表面，得到羟基化硼烯表面等离子体共振传感器。S30: preparing an AuNRs-ssDNA complex, and coating the AuNRs-ssDNA complex on the surface of a surface plasmon resonance chip to obtain a hydroxylated borene surface plasmon resonance sensor.
将300mg HAuCl 4作为金源，与0.5mol/L的CTAB表面活性剂充分混合，在不断搅拌条件下加入0.02mol/L的NaBH 4合成金晶核；将金晶核加入含有CTAB、HAuCl 4、AgNO 3、H 2SO 4、L-AA混合溶液中，在不断搅拌条件下各向异性生长为金纳米棒；将金纳米棒与PSS充分混合，常温下搅拌10小时，取出并离心处理除去多余的PSS，获得PSS修饰的金纳米棒(AuNRs)。 300mg HAuCl 4 is used as a gold source, fully mixed with 0.5mol/L CTAB surfactant, and 0.02mol/L NaBH 4 is added under constant stirring to synthesize gold crystal nuclei; , H 2 SO 4 , L-AA mixed solution, anisotropically grow into gold nanorods under constant stirring conditions; fully mix gold nanorods with PSS, stir at room temperature for 10 hours, take out and centrifuge to remove excess PSS , to obtain PSS-modified gold nanorods (AuNRs).
取25μL的100nM硫基化单链DNA溶液添加到200μL的AuNRs溶液以进行表面接枝，16小时后，将得到的混合溶液与0.25mL10％NaCl混合，得到AuNRs-ssDNA混合液；AuNRs-ssDNA混合液以转速5000r/min离心处理两次，持续20秒以除去过量的硫基化单链DNA，得到AuNRs-ssDNA颗粒，将 AuNRs-ssDNA颗粒重新分散在PBS缓冲液(1M NaCl，100mM PBS，pH＝7)中，得到AuNRs-ssDNA胶体溶液，并将AuNRs-ssDNA胶体溶液超声处理5分钟，然后在室温下搅拌1小时，获得AuNRs-ssDNA复合物。Add 25 μL of 100 nM thiolated single-stranded DNA solution to 200 μL of AuNRs solution for surface grafting. After 16 hours, mix the resulting mixed solution with 0.25 mL of 10% NaCl to obtain AuNRs-ssDNA mixed solution; AuNRs-ssDNA mixed The solution was centrifuged twice at a speed of 5000r/min for 20 seconds to remove excess thiolated single-stranded DNA to obtain AuNRs-ssDNA particles. The AuNRs-ssDNA particles were redispersed in PBS buffer (1M NaCl, 100mM PBS, pH =7), the AuNRs-ssDNA colloidal solution was obtained, and the AuNRs-ssDNA colloidal solution was sonicated for 5 minutes, and then stirred at room temperature for 1 hour to obtain the AuNRs-ssDNA complex.
取20mg的AuNRs-ssDNA复合物涂覆在表面等离子体共振芯片的表面，用PBS缓冲液洗涤，得到羟基化硼烯纳米片厚度为2nm的表面等离子体共振传感器。20 mg of the AuNRs-ssDNA complex was coated on the surface of the surface plasmon resonance chip and washed with PBS buffer to obtain a surface plasmon resonance sensor with a thickness of 2 nm of hydroxylated boronene nanosheets.
实施例2Example 2
本实施例提供一种厚度为4nm的羟基化硼烯纳米片的表面等离子体共振传感器的制备方法，包括以下步骤：This embodiment provides a method for preparing a surface plasmon resonance sensor of hydroxylated boronene nanosheets with a thickness of 4 nm, comprising the following steps:
S01：制备羟基化硼烯纳米片分散液。S01: Preparation of hydroxylated boronene nanosheet dispersion.
取500mg硼粉放入研钵中粉碎，加入仲丁醇并沿同一方向均匀研磨1个小时，使其充分剥离，得到粒径均匀且尺寸较小的的硼颗粒前驱体溶液；将硼颗粒前驱体溶液移入探头超声细胞破碎仪中，在冰水浴中超声处理6小时，得到硼烯分散液。Take 500 mg of boron powder and put it into a mortar for crushing, add sec-butanol and grind it uniformly in the same direction for 1 hour to make it fully peeled off to obtain a boron particle precursor solution with uniform particle size and small size; the boron particle precursor The bulk solution was transferred into a probe ultrasonic cell disruptor, and ultrasonically treated in an ice-water bath for 6 hours to obtain a boronene dispersion.
配制2mol/L的氢氧化钠溶液，将硼烯分散液加入到50mL氢氧化钠溶液中，冷水超声2小时，得到羟基化硼烯溶液。A 2 mol/L sodium hydroxide solution was prepared, the boronene dispersion was added to 50 mL of the sodium hydroxide solution, and cold water was sonicated for 2 hours to obtain a hydroxylated boronene solution.
利用离心机根据设定的转速5000r/min离心处理10min，去除块状的硼烯，获得小尺寸的羟基化硼烯溶液，再利用离心机根据设定的转速10000r/min对羟基化硼烯溶液进行离心处理20min，得到羟基化硼烯纳米片滤饼，将羟基化硼烯纳米片滤饼用去离子水多次洗涤，获得羟基化硼烯纳米片。Use a centrifuge to centrifuge at a set speed of 5000r/min for 10 minutes to remove massive boronene and obtain a small-sized hydroxylated boronene solution, and then use a centrifuge to process the hydroxylated boronene solution at a set speed of 10000r/min Perform centrifugation for 20 minutes to obtain a filter cake of hydroxylated boronene nanosheets, and wash the filter cake of hydroxylated boronene nanosheets with deionized water several times to obtain hydroxylated boronene nanosheets.
向羟基化硼烯纳米片加入乙醇溶液，配成2mol/L的羟基化硼烯纳米片分散液。Add ethanol solution to the hydroxylated boron nanosheets to prepare a 2 mol/L hydroxylated boronene nanosheet dispersion.
S02：将羟基化硼烯纳米片分散液在金膜表面进行成膜处理，获得表面等离子体共振芯片；S02: forming a film-forming treatment of the hydroxylated boronene nanosheet dispersion on the surface of the gold film to obtain a surface plasmon resonance chip;
利用旋涂法以转速8000-9000r/min将羟基化硼烯纳米片分散液旋涂到金膜表面，然后移入手套箱中80℃烘干，得到表面等离子体共振芯片。Spin-coat the dispersion of hydroxylated boronene nanosheets onto the surface of the gold film at a rotational speed of 8000-9000 r/min by a spin-coating method, and then move it into a glove box to dry at 80° C. to obtain a surface plasmon resonance chip.
S30：制备AuNRs-ssDNA复合物，并将AuNRs-ssDNA复合物涂覆到表面 等离子体共振芯片表面，得到羟基化硼烯表面等离子体共振传感器。S30: Prepare the AuNRs-ssDNA complex, and coat the AuNRs-ssDNA complex on the surface of the surface plasmon resonance chip to obtain a hydroxylated borene surface plasmon resonance sensor.
将300mg HAuCl 4作为金源，与0.5mol/L的CTAB表面活性剂充分混合，在不断搅拌条件下加入0.02mol/L的NaBH 4合成金晶核；将金晶核加入含有CTAB、HAuCl 4、AgNO 3、H 2SO 4、L-AA混合溶液中，在不断搅拌条件下各向异性生长为金纳米棒；将金纳米棒与PSS充分混合，常温下搅拌10小时，取出并离心处理除去多余的PSS，获得PSS修饰的金纳米棒(AuNRs)。 300mg HAuCl 4 is used as a gold source, fully mixed with 0.5mol/L CTAB surfactant, and 0.02mol/L NaBH 4 is added under constant stirring to synthesize gold crystal nuclei; , H 2 SO 4 , L-AA mixed solution, anisotropically grow into gold nanorods under constant stirring conditions; fully mix gold nanorods with PSS, stir at room temperature for 10 hours, take out and centrifuge to remove excess PSS , to obtain PSS-modified gold nanorods (AuNRs).
取25μL的100nM硫基化单链DNA溶液添加到200μL的AuNRs溶液以进行表面接枝，16小时后，将得到的混合溶液与0.25mL10％NaCl混合，得到AuNRs-ssDNA混合液；AuNRs-ssDNA混合液以转速5000r/min离心处理两次，持续20秒以除去过量的硫基化单链DNA，得到AuNRs-ssDNA颗粒，将AuNRs-ssDNA颗粒重新分散在PBS缓冲液(1M NaCl，100mM PBS，pH＝7)中，得到AuNRs-ssDNA胶体溶液，并将AuNRs-ssDNA胶体溶液超声处理5分钟，然后在室温下搅拌1小时，获得AuNRs-ssDNA复合物。Add 25 μL of 100 nM thiolated single-stranded DNA solution to 200 μL of AuNRs solution for surface grafting. After 16 hours, mix the resulting mixed solution with 0.25 mL of 10% NaCl to obtain AuNRs-ssDNA mixed solution; AuNRs-ssDNA mixed The solution was centrifuged twice at a speed of 5000r/min for 20 seconds to remove excess thiolated single-stranded DNA to obtain AuNRs-ssDNA particles. The AuNRs-ssDNA particles were redispersed in PBS buffer (1M NaCl, 100mM PBS, pH =7), the AuNRs-ssDNA colloidal solution was obtained, and the AuNRs-ssDNA colloidal solution was sonicated for 5 minutes, and then stirred at room temperature for 1 hour to obtain the AuNRs-ssDNA complex.
取20mg的AuNRs-ssDNA复合物涂覆在表面等离子体共振芯片的表面，用PBS缓冲液洗涤，得到羟基化硼烯纳米片厚度为4nm的表面等离子体共振传感器。20 mg of the AuNRs-ssDNA complex was coated on the surface of the surface plasmon resonance chip, and washed with PBS buffer to obtain a surface plasmon resonance sensor with a thickness of 4 nm of hydroxylated boronene nanosheets.
实施例3Example 3
本实施例提供一种厚度为6nm的羟基化硼烯纳米片的表面等离子体共振传感器的制备方法，包括以下步骤：This embodiment provides a method for preparing a surface plasmon resonance sensor with a thickness of 6nm hydroxylated boron nanosheets, comprising the following steps:
S01：制备羟基化硼烯纳米片分散液。S01: Preparation of hydroxylated boronene nanosheet dispersion.
取500mg硼粉放入研钵中粉碎，加入仲丁醇并沿同一方向均匀研磨1个小时，使其充分剥离，得到粒径均匀且尺寸较小的的硼颗粒前驱体溶液；将硼颗粒前驱体溶液移入探头超声细胞破碎仪中，在冰水浴中超声处理6小时，得到硼烯分散液。Take 500 mg of boron powder and put it into a mortar for crushing, add sec-butanol and grind it uniformly in the same direction for 1 hour to make it fully peeled off to obtain a boron particle precursor solution with uniform particle size and small size; the boron particle precursor The bulk solution was transferred into a probe ultrasonic cell disruptor, and ultrasonically treated in an ice-water bath for 6 hours to obtain a boronene dispersion.
配制2mol/L的氢氧化钠溶液，将硼烯分散液加入到50mL氢氧化钠溶液中，冷水超声2小时，得到羟基化硼烯溶液。A 2 mol/L sodium hydroxide solution was prepared, the boronene dispersion was added to 50 mL of the sodium hydroxide solution, and cold water was sonicated for 2 hours to obtain a hydroxylated boronene solution.
利用离心机根据设定的转速5000r/min离心处理10min，去除块状的硼烯，获得小尺寸的羟基化硼烯溶液，再利用离心机根据设定的转速10000r/min对羟基化硼烯溶液进行离心处理20min，得到羟基化硼烯纳米片滤饼，将羟基化硼烯纳米片滤饼用去离子水多次洗涤，获得羟基化硼烯纳米片。Use a centrifuge to centrifuge at a set speed of 5000r/min for 10 minutes to remove massive boronene and obtain a small-sized hydroxylated boronene solution, and then use a centrifuge to process the hydroxylated boronene solution at a set speed of 10000r/min Perform centrifugation for 20 minutes to obtain a filter cake of hydroxylated boronene nanosheets, and wash the filter cake of hydroxylated boronene nanosheets with deionized water several times to obtain hydroxylated boronene nanosheets.
向羟基化硼烯纳米片加入乙醇溶液，配成2mol/L的羟基化硼烯纳米片分散液。Add ethanol solution to the hydroxylated boron nanosheets to prepare a 2 mol/L hydroxylated boronene nanosheet dispersion.
S02：将羟基化硼烯纳米片分散液在金膜表面进行成膜处理，获得表面等离子体共振芯片；S02: forming a film-forming treatment of the hydroxylated boronene nanosheet dispersion on the surface of the gold film to obtain a surface plasmon resonance chip;
利用旋涂法以转速6000-8000r/min将羟基化硼烯纳米片分散液旋涂到金膜表面，然后移入手套箱中80℃烘干，得到表面等离子体共振芯片。Spin-coat the dispersion of hydroxylated boronene nanosheets onto the surface of the gold film at a speed of 6000-8000 r/min by a spin-coating method, and then move it into a glove box to dry at 80° C. to obtain a surface plasmon resonance chip.
S30：制备AuNRs-ssDNA复合物，并将AuNRs-ssDNA复合物涂覆到表面等离子体共振芯片表面，得到羟基化硼烯表面等离子体共振传感器。S30: preparing an AuNRs-ssDNA complex, and coating the AuNRs-ssDNA complex on the surface of a surface plasmon resonance chip to obtain a hydroxylated borene surface plasmon resonance sensor.
将300mg HAuCl 4作为金源，与0.5mol/L的CTAB表面活性剂充分混合，在不断搅拌条件下加入0.02mol/L的NaBH 4合成金晶核；将金晶核加入含有CTAB、HAuCl 4、AgNO 3、H 2SO 4、L-AA混合溶液中，在不断搅拌条件下各向异性生长为金纳米棒；将金纳米棒与PSS充分混合，常温下搅拌10小时，取出并离心处理除去多余的PSS，获得PSS修饰的金纳米棒(AuNRs)。 300mg HAuCl 4 is used as a gold source, fully mixed with 0.5mol/L CTAB surfactant, and 0.02mol/L NaBH 4 is added under constant stirring to synthesize gold crystal nuclei; , H 2 SO 4 , L-AA mixed solution, anisotropically grow into gold nanorods under constant stirring conditions; fully mix gold nanorods with PSS, stir at room temperature for 10 hours, take out and centrifuge to remove excess PSS , to obtain PSS-modified gold nanorods (AuNRs).
取25μL的100nM硫基化单链DNA溶液添加到200μL的AuNRs溶液以进行表面接枝，16小时后，将得到的混合溶液与0.25mL10％NaCl混合，得到AuNRs-ssDNA混合液；AuNRs-ssDNA混合液以转速5000r/min离心处理两次，持续20秒以除去过量的硫基化单链DNA，得到AuNRs-ssDNA颗粒，将AuNRs-ssDNA颗粒重新分散在PBS缓冲液(1M NaCl，100mM PBS，pH＝7)中，得到AuNRs-ssDNA胶体溶液，并将AuNRs-ssDNA胶体溶液超声处理5分钟，然后在室温下搅拌1小时，获得AuNRs-ssDNA复合物。Add 25 μL of 100 nM thiolated single-stranded DNA solution to 200 μL of AuNRs solution for surface grafting. After 16 hours, mix the resulting mixed solution with 0.25 mL of 10% NaCl to obtain AuNRs-ssDNA mixed solution; AuNRs-ssDNA mixed The solution was centrifuged twice at a speed of 5000r/min for 20 seconds to remove excess thiolated single-stranded DNA to obtain AuNRs-ssDNA particles. The AuNRs-ssDNA particles were redispersed in PBS buffer (1M NaCl, 100mM PBS, pH =7), the AuNRs-ssDNA colloidal solution was obtained, and the AuNRs-ssDNA colloidal solution was sonicated for 5 minutes, and then stirred at room temperature for 1 hour to obtain the AuNRs-ssDNA complex.
取20mg的AuNRs-ssDNA复合物涂覆在表面等离子体共振芯片的表面，用PBS缓冲液洗涤，得到羟基化硼烯纳米片厚度为6nm的表面等离子体共振传 感器。The AuNRs-ssDNA complex of 20 mg was coated on the surface of the surface plasmon resonance chip, and washed with PBS buffer to obtain a surface plasmon resonance sensor with a thickness of 6 nm of hydroxylated boronene nanosheets.
实施例4Example 4
本实施例提供一种厚度为8nm的羟基化硼烯纳米片的表面等离子体共振传感器的制备方法，包括以下步骤：This embodiment provides a method for preparing a surface plasmon resonance sensor with a thickness of 8nm hydroxylated boron nanosheets, comprising the following steps:
S01：制备羟基化硼烯纳米片分散液。S01: Preparation of hydroxylated boronene nanosheet dispersion.
取500mg硼粉放入研钵中粉碎，加入仲丁醇并沿同一方向均匀研磨1个小时，使其充分剥离，得到粒径均匀且尺寸较小的的硼颗粒前驱体溶液；将硼颗粒前驱体溶液移入探头超声细胞破碎仪中，在冰水浴中超声处理6小时，得到硼烯分散液。Take 500 mg of boron powder and put it into a mortar for crushing, add sec-butanol and grind it uniformly in the same direction for 1 hour to make it fully peeled off to obtain a boron particle precursor solution with uniform particle size and small size; the boron particle precursor The bulk solution was transferred into a probe ultrasonic cell disruptor, and ultrasonically treated in an ice-water bath for 6 hours to obtain a boronene dispersion.
配制2mol/L的氢氧化钠溶液，将硼烯分散液加入到50mL氢氧化钠溶液中，冷水超声2小时，得到羟基化硼烯溶液。A 2 mol/L sodium hydroxide solution was prepared, the boronene dispersion was added to 50 mL of the sodium hydroxide solution, and cold water was sonicated for 2 hours to obtain a hydroxylated boronene solution.
利用离心机根据设定的转速5000r/min离心处理10min，去除块状的硼烯，获得小尺寸的羟基化硼烯溶液，再利用离心机根据设定的转速10000r/min对羟基化硼烯溶液进行离心处理20min，得到羟基化硼烯纳米片滤饼，将羟基化硼烯纳米片滤饼用去离子水多次洗涤，获得羟基化硼烯纳米片。Use a centrifuge to centrifuge at a set speed of 5000r/min for 10 minutes to remove massive boronene and obtain a small-sized hydroxylated boronene solution, and then use a centrifuge to process the hydroxylated boronene solution at a set speed of 10000r/min Perform centrifugation for 20 minutes to obtain a filter cake of hydroxylated boronene nanosheets, and wash the filter cake of hydroxylated boronene nanosheets with deionized water several times to obtain hydroxylated boronene nanosheets.
向羟基化硼烯纳米片加入乙醇溶液，配成2mol/L的羟基化硼烯纳米片分散液。Add ethanol solution to the hydroxylated boron nanosheets to prepare a 2 mol/L hydroxylated boronene nanosheet dispersion.
S02：将羟基化硼烯纳米片分散液在金膜表面进行成膜处理，获得表面等离子体共振芯片。S02: Forming a dispersion of hydroxylated boronene nanosheets on the surface of the gold film to obtain a surface plasmon resonance chip.
利用旋涂法以转速5000-6000r/min将羟基化硼烯纳米片分散液旋涂到金膜表面，然后移入手套箱中80℃烘干，得到表面等离子体共振芯片。Spin-coat the dispersion of hydroxylated boronene nanosheets onto the surface of the gold film at a rotational speed of 5000-6000 r/min by a spin-coating method, and then move it into a glove box to dry at 80° C. to obtain a surface plasmon resonance chip.
S30：制备AuNRs-ssDNA复合物，并将AuNRs-ssDNA复合物涂覆到表面等离子体共振芯片表面，得到羟基化硼烯表面等离子体共振传感器。S30: preparing an AuNRs-ssDNA complex, and coating the AuNRs-ssDNA complex on the surface of a surface plasmon resonance chip to obtain a hydroxylated borene surface plasmon resonance sensor.
将300mg HAuCl 4作为金源，与0.5mol/L的CTAB表面活性剂充分混合，在不断搅拌条件下加入0.02mol/L的NaBH 4合成金晶核；将金晶核加入含有CTAB、HAuCl 4、AgNO 3、H 2SO 4、L-AA混合溶液中，在不断搅拌条件下各向异性生长 为金纳米棒；将金纳米棒与PSS充分混合，常温下搅拌10小时，取出并离心处理除去多余的PSS，获得PSS修饰的金纳米棒(AuNRs)。 300mg HAuCl 4 is used as a gold source, fully mixed with 0.5mol/L CTAB surfactant, and 0.02mol/L NaBH 4 is added under constant stirring to synthesize gold crystal nuclei; , H 2 SO 4 , L-AA mixed solution, anisotropically grow into gold nanorods under constant stirring conditions; fully mix gold nanorods with PSS, stir at room temperature for 10 hours, take out and centrifuge to remove excess PSS , to obtain PSS-modified gold nanorods (AuNRs).
取25μL的100nM硫基化单链DNA溶液添加到200μL的AuNRs溶液以进行表面接枝，16小时后，将得到的混合溶液与0.25mL10％NaCl混合，得到AuNRs-ssDNA混合液；AuNRs-ssDNA混合液以转速5000r/min离心处理两次，持续20秒以除去过量的硫基化单链DNA，得到AuNRs-ssDNA颗粒，将AuNRs-ssDNA颗粒重新分散在PBS缓冲液(1M NaCl，100mM PBS，pH＝7)中，得到AuNRs-ssDNA胶体溶液，并将AuNRs-ssDNA胶体溶液超声处理5分钟，然后在室温下搅拌1小时，获得AuNRs-ssDNA复合物。Add 25 μL of 100 nM thiolated single-stranded DNA solution to 200 μL of AuNRs solution for surface grafting. After 16 hours, mix the resulting mixed solution with 0.25 mL of 10% NaCl to obtain AuNRs-ssDNA mixed solution; AuNRs-ssDNA mixed The solution was centrifuged twice at a speed of 5000r/min for 20 seconds to remove excess thiolated single-stranded DNA to obtain AuNRs-ssDNA particles. The AuNRs-ssDNA particles were redispersed in PBS buffer (1M NaCl, 100mM PBS, pH =7), the AuNRs-ssDNA colloidal solution was obtained, and the AuNRs-ssDNA colloidal solution was sonicated for 5 minutes, and then stirred at room temperature for 1 hour to obtain the AuNRs-ssDNA complex.
取20mg的AuNRs-ssDNA复合物涂覆在表面等离子体共振芯片的表面，用PBS缓冲液洗涤，得到羟基化硼烯纳米片厚度为8nm的表面等离子体共振传感器。20 mg of the AuNRs-ssDNA complex was coated on the surface of the surface plasmon resonance chip, and washed with PBS buffer to obtain a surface plasmon resonance sensor with a thickness of hydroxylated boronene nanosheets of 8 nm.
基于不同厚度的羟基化硼烯纳米片的表面等离子体共振传感器实验对比分析：Experimental comparative analysis of surface plasmon resonance sensors based on hydroxylated boronene nanosheets with different thicknesses:
表1Table 1
Figure PCTCN2021137299-appb-000001
Figure PCTCN2021137299-appb-000001
由上述表1的不同厚度的羟基化硼烯纳米片的表面等离子体共振传感器实验对比分析得出以下结论：The following conclusions are drawn from the comparative analysis of the surface plasmon resonance sensor experiments of the hydroxylated boronene nanosheets of different thicknesses in the above table 1:
采用本申请实施例1至实施例4提供的通过在金膜上旋涂不同厚度的羟基化硼烯纳米片分散液，得到表面等离子体共振芯片，再将AuNRs-ssDNA复合物涂覆在表面等离子体共振芯片的表面，以得到不同厚度的硼烯表面等离子体共振传感器，从而设置出不同厚度的羟基化硼烯纳米片对硼烯表面等离子体共振传感器的性能影响，从实验结果可以看出：当注入相同浓度的mi-RNA时， 硼烯表面等离子体共振传感器的折射角度随羟基化硼烯纳米片的厚度减小而增大，说明硼烯表面等离子体共振传感器中的羟基化硼烯纳米片的厚度越薄，其折射角度越大，其灵敏度越高。当注入不同浓度的mi-RNA时，硼烯表面等离子体共振传感器的折射角度随mi-RNA减少而减小，mi-RNA的浓度越少时，mi-RNA跟ssDNA配对的分子就越少，那么解析出来的就越少，即折射角度越小，因此，可以根据可以检测出的动态信号的变化来得到生物受体与待测样品相互作用的吸附、解离常数等重要信息。Using the method provided in Examples 1 to 4 of this application, the surface plasmon resonance chip is obtained by spin-coating hydroxylated boronene nanosheet dispersions of different thicknesses on the gold film, and then the AuNRs-ssDNA complex is coated on the surface plasmon The surface of the bulk resonance chip is used to obtain boronene surface plasmon resonance sensors with different thicknesses, so as to set the influence of different thicknesses of hydroxylated boronene nanosheets on the performance of the boronene surface plasmon resonance sensor. From the experimental results, it can be seen that: When the same concentration of mi-RNA is injected, the refraction angle of the boron surface plasmon resonance sensor increases with the decrease of the thickness of the hydroxylated boron nanosheets, indicating that the hydroxylated boron nanosheets in the boron surface plasmon resonance sensor The thinner the sheet, the larger its refraction angle and the higher its sensitivity. When injected with different concentrations of mi-RNA, the refraction angle of the boron surface plasmon resonance sensor decreases with the decrease of mi-RNA, and the less the concentration of mi-RNA, the fewer molecules paired with ssDNA by mi-RNA, Then the less is analyzed, that is, the smaller the refraction angle, therefore, important information such as the adsorption and dissociation constants of the interaction between the bioreceptor and the sample to be tested can be obtained according to the change of the dynamic signal that can be detected.
以上所述仅为本申请的较佳实施例而已，并不用以限制本申请，凡在本申请的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本申请的保护范围之内。The above descriptions are only preferred embodiments of the application, and are not intended to limit the application. Any modifications, equivalent replacements and improvements made within the spirit and principles of the application should be included in the protection of the application. within range.

Claims (10)

  1. 一种表面等离子体共振传感器，其特征在于，所述表面等离子体共振传感器包括：A surface plasmon resonance sensor, characterized in that the surface plasmon resonance sensor comprises:
    表面等离子体共振芯片，所述表面等离子体共振芯片包括第一贵金属基体，在所述第一贵金属基体上结合有硼烯层；A surface plasmon resonance chip, the surface plasmon resonance chip comprising a first noble metal substrate, a boron layer is bonded to the first noble metal substrate;
    贵金属-ssDNA复合物，所述贵金属-ssDNA复合物结合在所述表面等离子体共振芯片上，且所述贵金属-ssDNA复合物包括第二贵金属基体和结合在所述第二贵金属基体上的ssDNA。A noble metal-ssDNA complex, the noble metal-ssDNA complex is combined on the surface plasmon resonance chip, and the noble metal-ssDNA complex includes a second noble metal substrate and ssDNA combined on the second noble metal substrate.
  2. 如权利要求1所述表面等离子体共振传感器，其特征在于，所述硼烯层厚度为1-100nm；The surface plasmon resonance sensor according to claim 1, wherein the thickness of the boron layer is 1-100nm;
    其中，所述硼烯层包括连续分布的硼烯膜层和/或岛状分布的硼烯膜层。Wherein, the boron layer includes continuously distributed boron film layers and/or island-shaped distributed boron film layers.
  3. 如权利要求1或2所述表面等离子体共振传感器，其特征在于，所述硼烯层为羟基化硼烯纳米片；和/或The surface plasmon resonance sensor according to claim 1 or 2, wherein the boron layer is a hydroxylated boron nanosheet; and/or
    所述羟基化硼烯纳米片的层数为1-100层。The number of layers of the hydroxylated boronene nanosheets is 1-100 layers.
  4. 如权利要求1或2所述表面等离子体共振传感器，其特征在于，所述第一贵金属基体为Au和/或Ag的膜层；和/或The surface plasmon resonance sensor according to claim 1 or 2, wherein the first noble metal substrate is a film layer of Au and/or Ag; and/or
    所述第二贵金属基体包括Au和/或Ag纳米棒、纳米立方、纳米片中的至少一种；和/或The second noble metal matrix includes at least one of Au and/or Ag nanorods, nanocubes, and nanosheets; and/or
    所述第一贵金属基体的厚度为2nm-20μm；和/或The thickness of the first noble metal matrix is 2nm-20μm; and/or
    所述贵金属-ssDNA复合物的量在所述表面等离子体共振芯片上的负载量为0.01mol/cm 2-10mol/cm 2。 The loading amount of the noble metal-ssDNA complex on the surface plasmon resonance chip is 0.01 mol/cm 2 -10 mol/cm 2 .
  5. 一种表面等离子体共振传感器的制备方法，其特征在于，所述制备方法包括以下步骤：A preparation method of a surface plasmon resonance sensor, characterized in that the preparation method comprises the following steps:
    制备硼烯纳米片分散液；Prepare boron nanosheet dispersion;
    将所述硼烯纳米片分散液在第一贵金属基体表面进行成膜处理，获得表面等离子体共振芯片；performing a film-forming treatment on the surface of the boron nanosheet dispersion on the surface of the first noble metal substrate to obtain a surface plasmon resonance chip;
    将ssDNA结合在第二贵金属基体上，形成贵金属-ssDNA复合物；Binding ssDNA to a second noble metal substrate to form a noble metal-ssDNA complex;
    将所述贵金属-ssDNA复合物组装至所述表面等离子体共振芯片上，得到表面等离子体共振传感器。The noble metal-ssDNA complex is assembled on the surface plasmon resonance chip to obtain a surface plasmon resonance sensor.
  6. 如权利要求5所述表面等离子体共振传感器的制备方法，其特征在于，将所述硼烯纳米片分散液在第一贵金属基体表面进行成膜处理的方法包括如下步骤：The method for preparing a surface plasmon resonance sensor according to claim 5, wherein the method of performing film-forming treatment on the surface of the boron nanosheet dispersion on the surface of the first noble metal substrate comprises the following steps:
    将所述硼烯纳米片分散液采用旋涂方法在第一贵金属基体表面成膜处理，获得表面等离子体共振芯片。The boron nanosheet dispersion is formed into a film on the surface of the first noble metal substrate by a spin coating method to obtain a surface plasmon resonance chip.
  7. 如权利要求5或6所述表面等离子体共振传感器的制备方法，其特征在于，制备所述贵金属-ssDNA复合物的方法包括：The method for preparing a surface plasmon resonance sensor according to claim 5 or 6, wherein the method for preparing the noble metal-ssDNA complex comprises:
    将单链DNA与第二贵金属基体进行表面接枝处理，获得贵金属-ssDNA复合物。The single-stranded DNA is grafted onto the second noble metal substrate to obtain a noble metal-ssDNA complex.
  8. 一种核酸检测传感器，其特征在于，所述核酸检测传感器包括权利要求1-4任一项所述的表面等离子体共振传感器。A nucleic acid detection sensor, characterized in that the nucleic acid detection sensor comprises the surface plasmon resonance sensor according to any one of claims 1-4.
  9. 一种硼烯纳米片的制备方法，其特征在于，包括如下步骤：A preparation method of boron nanosheets, characterized in that, comprising the steps of:
    对硼粉与溶剂的混合物进行研磨处理，获得硼颗粒前驱体溶液；Grinding the mixture of boron powder and solvent to obtain a boron particle precursor solution;
    对所述硼颗粒前驱体溶液进行超声剥离处理，获得硼烯纳米片分散液；Ultrasonic stripping is performed on the boron particle precursor solution to obtain a boron nanosheet dispersion;
    对所述硼烯纳米片分散液进行离心分离处理，获得目标层数的硼烯纳米片。The boronene nanosheet dispersion is subjected to centrifugation to obtain boronene nanosheets with a target number of layers.
  10. 如权利要求9所述的硼烯纳米片的制备方法，其特征在于，所述制备方法还包括：The preparation method of boron nanosheet as claimed in claim 9, is characterized in that, described preparation method also comprises:
    将所述硼烯纳米片与碱性溶液进行表面改性处理，获得羟基化硼烯纳米片。The boronene nanosheets are subjected to surface modification treatment with an alkaline solution to obtain hydroxylated boronene nanosheets.
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