WO2022213432A1 - Hollow gold-silver core-shell nanoflower sers nanoprobe hau/agsnfs-atp - Google Patents

Hollow gold-silver core-shell nanoflower sers nanoprobe hau/agsnfs-atp Download PDF

Info

Publication number
WO2022213432A1
WO2022213432A1 PCT/CN2021/090389 CN2021090389W WO2022213432A1 WO 2022213432 A1 WO2022213432 A1 WO 2022213432A1 CN 2021090389 W CN2021090389 W CN 2021090389W WO 2022213432 A1 WO2022213432 A1 WO 2022213432A1
Authority
WO
WIPO (PCT)
Prior art keywords
sers
qualitative
nucleic acid
quantitative
agsnfs
Prior art date
Application number
PCT/CN2021/090389
Other languages
French (fr)
Chinese (zh)
Inventor
陈全胜
许艺
李欢欢
焦天慧
朱阿芳
Original Assignee
江苏大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 江苏大学 filed Critical 江苏大学
Publication of WO2022213432A1 publication Critical patent/WO2022213432A1/en

Links

Images

Classifications

    • 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
    • G01N21/65—Raman scattering
    • G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons
    • 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
    • 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
    • G01N21/65—Raman scattering
    • G01N2021/653—Coherent methods [CARS]
    • G01N2021/656—Raman microprobe

Definitions

  • the invention relates to the field of photochemical analysis technology and biological detection, in particular to a hollow gold-silver core-shell nano-flower SERS nano-probe HAu/AgSNFs-ATP.
  • SERS Surface-Enhanced Raman Scattering
  • SERS refers to the adsorption of molecules on the surface of some specially prepared metal conductors or sols, in the excitation region, due to the enhancement of the electromagnetic field on the surface or near the surface of the sample.
  • SERS overcomes the shortcomings of low sensitivity of Raman spectroscopy, and can obtain structural information that is not easily obtained by conventional Raman spectroscopy. Structural analysis, etc., can effectively analyze the adsorption orientation of the compound at the interface, the change of the adsorption state, and the interface information.
  • SERS has been widely used in materials, chemical, petroleum, polymer, biology, environmental protection, geology and other fields.
  • SERS probes generally refer to composite nanoparticles that use metal nanoparticles as carriers and simultaneously modify molecules with SERS activity and molecules with specific recognition functions (such as antibodies, DNA, etc.). Because it can comprehensively utilize the advantages of surface-enhanced Raman spectroscopy and the properties of noble metal nanoparticles, it has special application value in the field of detection and analysis, which has attracted the attention of scientists and has also been widely developed and applied.
  • Nucleic acid is a biological macromolecular compound composed of many nucleotide monomers. It is an indispensable component of all known life forms and one of the most basic substances of life. Nucleic acid detection is widely used in various aspects, such as early tumor nucleic acid marker detection, rapid identification in criminal investigation, rapid detection of food safety, rapid monitoring of bacteria and viruses in the environment, vaccine safety detection, and screening of blood infectious diseases etc. Therefore, it is of great significance to establish a general nucleic acid rapid detection method, and a general and high-sensitivity nucleic acid rapid detection method has not been reported yet.
  • the purpose of the present invention is to provide a hollow gold-silver core-shell nano-flower SERS nano-probe HAu/AgSNFs-ATP to solve the problems existing in the above-mentioned prior art.
  • the present invention provides a hollow gold-silver core-shell nanoflower SERS nanoprobe HAu/AgSNFs-ATP, and the preparation method of the hollow gold-silver core-shell nanoflower SERS nanoprobe HAu/AgSNFs-ATP is:
  • step (1) Functional modification of HAu/AgSNFs: the HAu/AgSNFs aqueous solution obtained in step (1) is mixed with a C 6 H 7 NS alcohol-water mixed solution, centrifuged and washed after the reaction to obtain the hollow gold-silver core-shell nanoparticle Flower SERS nanoprobe HAu/AgSNFs-ATP.
  • the present invention also provides a SERS-based nucleic acid qualitative and/or quantitative detection sensor
  • the nucleic acid qualitative and/or quantitative detection sensor comprises the hollow gold-silver core-shell nanoflower SERS nanoprobe HAu/AgSNFs- ATP detection zone.
  • the nucleic acid qualitative and/or quantitative detection sensor further includes a sample pretreatment area loaded with polydopamine and an amplification area loaded with an amplification system.
  • the present invention also provides a method for using the SERS-based nucleic acid qualitative and/or quantitative detection sensor, which is:
  • the present invention also provides a nucleic acid qualitative and/or quantitative detection sensor based on magenta sulfite reagent and SERS
  • the nucleic acid qualitative and/or quantitative detection sensor includes a qualitative color development detection area and a quantitative and/or qualitative SERS detection area, wherein, the qualitative color development detection area is loaded with magenta sulfite reagent, and the quantitative and/or qualitative SERS detection area is loaded with the hollow gold-silver core-shell nanoflower SERS nanoprobe HAu/AgSNFs-ATP.
  • the nucleic acid qualitative and/or quantitative detection sensor further includes a sample pretreatment area loaded with polydopamine and an amplification area loaded with an amplification system.
  • the present invention also provides a method for using the nucleic acid qualitative and/or quantitative detection sensor based on the magenta sulfite reagent and SERS, the use method is:
  • the sample to be tested is characterized according to the visual signal, and the visual signal is specifically: a. When no color is displayed in the qualitative color detection area, it indicates that the test result is negative; b. When the qualitative color detection area shows purple, Indicates that the test result is positive;
  • the nucleic acid in the sample to be tested is qualitatively determined according to the standard SERS spectrum obtained in step (1); the nucleic acid in the sample to be tested is quantified according to the prediction model obtained in step (1).
  • the present invention also provides a nucleic acid qualitative and/or quantitative detection sensor based on the nanoprobe HAu/AgSNFs-ATP or the SERS-based nucleic acid or the nucleic acid qualitative and/or quantitative based on the magenta sulfite reagent and SERS.
  • the present invention also provides a nucleic acid qualitative and/or quantitative detection sensor based on the nanoprobe HAu/AgSNFs-ATP or the SERS-based nucleic acid or the nucleic acid qualitative and/or quantitative based on the magenta sulfite reagent and SERS.
  • the invention discloses the following technical effects: the invention proposes a general nucleic acid rapid detection method based on paper-based SERS sensing, and specifically prepares a hollow gold-silver core-shell nanoflower SERS nanoprobe HAu/AgSNFs- ATP can undergo an addition reaction with nucleic acid acid hydrolysis products to generate new functional groups, which can be combined with stoichiometric methods and colorimetric methods to build a steady-state specific pathogenic bacteria visualization and accurate quantitative model. Therefore, the general nucleic acid established by the present invention is The detection method realizes the portable, highly sensitive and specific detection of various nucleic acids, which can meet the needs of on-site detection.
  • Figure 1 shows the transmission electron microscope images of HAu/AgSNFs prepared under different AgNO 3 concentrations, where A is HAu/AgSNFs prepared with 0.1M AgNO 3 , B is HAu/AgSNFs prepared with 0.3M AgNO 3 , and C is 0.5M AgNO 3 Prepared HAu/AgSNFs, D is HAu/AgSNFs prepared with 0.7M AgNO 3 ;
  • Figure 2 shows the SERS spectra of HAu/AgSNFs-ATP prepared under different AgNO 3 concentrations, wherein A is HAu/AgSNFs prepared with 0.1M AgNO 3 , B is HAu/AgSNFs prepared with 0.3M AgNO 3 , and C is 0.5M AgNO 3 3 prepared HAu/AgSNFs, D is 0.7M AgNO 3 prepared HAu/AgSNFs;
  • Figure 3 is a schematic flowchart of a paper-based SERS/colorimetric sensing rapid detection method
  • Fig. 4 is the PLS quantitative model of Staphylococcus aureus
  • Fig. 5 is the PLS quantitative model of Escherichia coli
  • Figure 6 is a PLS quantification model of Salmonella.
  • Step 1 preparation of HAu/AgSNFs: add 18mL AgNO 3 (0.1-1M) under the mixed system of 1mL H 3 NO solution (1 ⁇ 10 -2 -6 ⁇ 10 -2 M) and 1mL NaOH solution (0.01-0.08M). ) solution was stirred for 5-20 min, then 10 mL of HAuCl 4 solution (2 ⁇ 10 -4 -10 ⁇ 10 -4 M) and 200 ⁇ L of C 6 H 5 Na 3 O 7 solution (0.5%-1.5%, w/v) were added ) at 80-110 °C for 5 min to obtain HAu/AgSNFs.
  • Step 2 functional modification of HAu/AgSNFs: add 30 ⁇ L of C 6 H 7 NS alcohol-water mixed solution (0.01-1 M) to 10 mL of HAu/AgSNFs aqueous solution, stir and react for 15 min, centrifuge, wash twice, and finally disperse in the aqueous solution.
  • SERS nanoprobes based on HAu/AgSNFs-ATP were obtained.
  • the TEM images of the HAu/AgSNFs nanoprobes prepared under different AgNO 3 concentrations are shown in Figure 1. It can be seen from Figure 1 that the different AgNO 3 concentrations will affect the morphology of the nanomaterials and indirectly affect its SERS effect.
  • the optimized spectrum of the HAu/AgSNFs-ATP-optimized SERS nanoprobe prepared at the AgNO concentration is shown in Fig . 2. From the TEM image, it can be seen that with the addition of AgNO, the HAu/AgSNFs will gradually change from a spherical shape at the beginning. As petals grow, more hotspots will be produced due to the tip effect, so the SERS effect will be enhanced.
  • Step 3 the preparation of the three-dimensional origami device: use cellulose paper with high mechanical strength to form a corresponding reaction chamber with a diameter of 1 cm by compressing the relevant mold on the cellulose paper, and load the polydopamine material in the pretreatment area, Load 10 ⁇ L of amplification system in the amplification area, and load 20 ⁇ L of magenta sulfite reagent and HAu/AgSNFs-ATP on the qualitative color development detection line and qualitative and/or quantitative SERS detection line of the detection area, respectively.
  • Step 4 Establish a specific detection method for food-borne pathogenic bacteria: prepare pathogenic bacteria with different concentrations, then heat them to crudely extract DNA, and drop the crude extract into the pretreatment area to interfere with the reaction system. The substance is adsorbed, and then the pretreatment area and the amplification area are overlapped by the folding equipment, and the gene amplification is performed at a certain temperature. After the amplification, the amplification area is placed in an acidic condition to perform acid hydrolysis on the amplification product; The amplification area and the detection area are folded and overlapped, and after the corresponding reaction, the visual signal can be obtained in the visual detection line, the SERS spectrum is collected on the SERS detection line, and the quantitative model is obtained by combining the chemometric method.
  • nucleic acid is completely hydrolyzed into base, ribose or deoxyribose and phosphoric acid under strong acid and high temperature; in slightly diluted inorganic acid, the most easily hydrolyzed chemical bond is selectively broken, generally connecting purine and ribose. glycosidic bonds, resulting in apurine nucleic acids.
  • an aldehyde group is formed.
  • the aldehyde group can react with colorless magenta sulfite solution to turn purple, and on the other hand, the aldehyde group can generate an addition reaction with the amino group on the nanoprobe HAu/AgSNFs-ATP.
  • the Raman shift is generally 10-4000cm -1 , which corresponds to the transition of molecular rotation or vibration-rotation energy level, so Raman spectroscopy can also provide information on molecular structure.
  • Raman spectroscopy can also provide information on molecular structure.
  • different compounds give different Raman spectra, which can be used for qualitative analysis and identification of compounds.
  • the same group or chemical bond in different compounds can give Raman bands with similar wave numbers, so functional groups can be identified.
  • Staphylococcus aureus with a concentration of 0-7cfu/mL was heated to crudely extract DNA, and the crude extract was added dropwise to the pretreatment area to adsorb the interfering substances in the reaction system, and then the equipment was folded to separate the pretreatment area and amplification
  • the total volume of the amplification reaction is 20 ⁇ L, which contains a pair of primers P1 and P2 with a concentration of 10 -6 M respectively, and the system also includes dNTPs with a concentration of 5 ⁇ 10 -4 M, 1 ⁇ Isothermopol Buffer, and Bst 2.0 WarmStart DNA polymerase; the amplification reaction temperature is 62 °C, and the reaction time is 30 min.
  • 0.5 mM HCl solution is added to the amplification area and the acid hydrolyzed product is obtained after the reaction at 65 °C for 5 min.
  • the amplification primers are:
  • P2 GCGCGGATCCATCTATAAGTGAC (SEQ ID NO: 2).
  • Extract DNA from quick-frozen food samples by conventional methods drop the extracted DNA into the pretreatment area to adsorb the interfering substances in the reaction system, and then fold the equipment to overlap the pretreatment area and the amplification area for gene amplification and amplification.
  • Conditions and amplification primers were the same as above.
  • the specific method is the same as 2.1.1.1, wherein the amplification primers are:
  • P2 TAACCCGAACTATCTCAATCTGT (SEQ ID NO: 6).
  • the Salmonella PLS quantification model is shown in Figure 6.
  • the training model correlation coefficient (Rc) of the three bacteria ranges from 0.9831 to 0.9893, and the prediction set correlation coefficient (Rp) ranges from 0.9755 to 0.9863.
  • the quantitative models of the three bacteria all have good predictions. Therefore, it is feasible to apply this method to the detection of food-borne pathogens.
  • the minimum detection limit of this method for Staphylococcus aureus is 10 cfu/mL
  • the minimum detection limit for Escherichia coli is 1 cfu/mL
  • the minimum detection limit for Salmonella is 10 cfu/mL.

Abstract

A hollow gold-silver core-shell nanoflower SERS nanoprobe HAu/AgSNFs-ATP, which relates to the field of photochemical analysis technology and biological testing, and a nucleic acid qualitative and/or quantitative measurement sensor based on a fuchsin sulfurous acid reagent and SERS. A hollow gold-silver core-shell nanoflower SERS nanoprobe is prepared by means of nano-controllable self-assembly, and is combined with a chemometrics method and a colorimetric method to build a steady-state nucleic acid visualization and accurate quantitative model. Therefore, a universal nucleic acid testing method is established, thereby realizing portable, highly-sensitive and specific testing of various nucleic acids.

Description

一种中空金银核壳纳米花SERS纳米探针HAu/AgSNFs-ATPA hollow gold-silver core-shell nanoflower SERS nanoprobe HAu/AgSNFs-ATP 技术领域technical field
本发明涉及光化学分析技术与生物检测领域，特别是涉及一种中空金银核壳纳米花SERS纳米探针HAu/AgSNFs-ATP。The invention relates to the field of photochemical analysis technology and biological detection, in particular to a hollow gold-silver core-shell nano-flower SERS nano-probe HAu/AgSNFs-ATP.
背景技术Background technique
表面增强拉曼散射(Surface-Enhanced Raman Scattering，简称SERS)，是指在特殊制备的一些金属良导体表面或溶胶中,在激发区域内,由于样品表面或近表面的电磁场的增强导致吸附分子的拉曼散射信号比普通拉曼散射(NRS)信号大大增强的现象。SERS克服了拉曼光谱灵敏度低的缺点，可以获得常规拉曼光谱所不易得到的结构信息,被广泛用于表面研究、吸附界面表面状态研究、生物大小分子的界面取向及构型、构象研究、结构分析等,可以有效分析化合物在界面的吸附取向、吸附态的变化、界面信息等。目前,SERS己被广泛应用于材料、化工、石油、高分子、生物、环保、地质等领域。Surface-Enhanced Raman Scattering (SERS) refers to the adsorption of molecules on the surface of some specially prepared metal conductors or sols, in the excitation region, due to the enhancement of the electromagnetic field on the surface or near the surface of the sample. A phenomenon in which the Raman scattering signal is greatly enhanced over the normal Raman scattering (NRS) signal. SERS overcomes the shortcomings of low sensitivity of Raman spectroscopy, and can obtain structural information that is not easily obtained by conventional Raman spectroscopy. Structural analysis, etc., can effectively analyze the adsorption orientation of the compound at the interface, the change of the adsorption state, and the interface information. At present, SERS has been widely used in materials, chemical, petroleum, polymer, biology, environmental protection, geology and other fields.
SERS探针通常是指以金属纳米粒子为载体，同时修饰具有SERS活性的分子和具有特异性识别的功能的分子(如抗体、DNA等)的复合型纳米粒子。因其可以同时综合利用表面增强拉曼光谱的优势以及贵金属纳米粒子的特性而在检测分析领域有特殊应用价值，从而备受科学家们关注，也得到了广泛的发展与应用。SERS probes generally refer to composite nanoparticles that use metal nanoparticles as carriers and simultaneously modify molecules with SERS activity and molecules with specific recognition functions (such as antibodies, DNA, etc.). Because it can comprehensively utilize the advantages of surface-enhanced Raman spectroscopy and the properties of noble metal nanoparticles, it has special application value in the field of detection and analysis, which has attracted the attention of scientists and has also been widely developed and applied.
核酸是由许多核苷酸单体聚合成的生物大分子化合物，是所有已知生命形式必不可少的组成物质，是生命的最基本物质之一。核酸的检测广泛应用于各个方面，例如早期肿瘤核酸标志物检测、刑侦工作 中的快速鉴定、食品安全的快速检测、环境中细菌和病毒的快速监测、疫苗安全性检测、血液传染病的筛查等，因此建立通用的核酸快速检测方法具有非常重要的意义，而目前通用的、高灵敏度的核酸快速检测方法还未见报道。Nucleic acid is a biological macromolecular compound composed of many nucleotide monomers. It is an indispensable component of all known life forms and one of the most basic substances of life. Nucleic acid detection is widely used in various aspects, such as early tumor nucleic acid marker detection, rapid identification in criminal investigation, rapid detection of food safety, rapid monitoring of bacteria and viruses in the environment, vaccine safety detection, and screening of blood infectious diseases etc. Therefore, it is of great significance to establish a general nucleic acid rapid detection method, and a general and high-sensitivity nucleic acid rapid detection method has not been reported yet.
因此，亟待开发一种通用的、高灵敏度的核酸快速检测方法，满足各种核酸的实时快速检测要求。Therefore, it is urgent to develop a general and highly sensitive nucleic acid rapid detection method to meet the real-time rapid detection requirements of various nucleic acids.
发明内容SUMMARY OF THE INVENTION
本发明的目的是提供一种中空金银核壳纳米花SERS纳米探针HAu/AgSNFs-ATP，以解决上述现有技术存在的问题。The purpose of the present invention is to provide a hollow gold-silver core-shell nano-flower SERS nano-probe HAu/AgSNFs-ATP to solve the problems existing in the above-mentioned prior art.
为实现上述目的，本发明提供一种中空金银核壳纳米花SERS纳米探针HAu/AgSNFs-ATP，所述中空金银核壳纳米花SERS纳米探针HAu/AgSNFs-ATP的制备方法为：In order to achieve the above object, the present invention provides a hollow gold-silver core-shell nanoflower SERS nanoprobe HAu/AgSNFs-ATP, and the preparation method of the hollow gold-silver core-shell nanoflower SERS nanoprobe HAu/AgSNFs-ATP is:
(1)HAu/AgSNFs制备：在H 3NO溶液和NaOH溶液的混合体系下加入AgNO 3溶液，然后加入一定量的HAuCl 4溶液和C 6H 5Na 3O 7溶液反应得到HAu/AgSNFs水溶液； (1) Preparation of HAu/AgSNFs: AgNO 3 solution was added under the mixed system of H 3 NO solution and NaOH solution, and then a certain amount of HAuCl 4 solution and C 6 H 5 Na 3 O 7 solution were added to react to obtain HAu/AgSNFs aqueous solution;
(2)HAu/AgSNFs的功能化修饰：在步骤(1)得到的HAu/AgSNFs水溶液中与C 6H 7NS醇水混合溶液混合，反应后离心、清洗，得到所述中空金银核壳纳米花SERS纳米探针HAu/AgSNFs-ATP。 (2) Functional modification of HAu/AgSNFs: the HAu/AgSNFs aqueous solution obtained in step (1) is mixed with a C 6 H 7 NS alcohol-water mixed solution, centrifuged and washed after the reaction to obtain the hollow gold-silver core-shell nanoparticle Flower SERS nanoprobe HAu/AgSNFs-ATP.
本发明还提供一种基于SERS的核酸定性和/或定量的检测传感器，所述核酸定性和/或定量检测传感器包括负载有所述的中空金银核壳纳米花SERS纳米探针HAu/AgSNFs-ATP的检测区。The present invention also provides a SERS-based nucleic acid qualitative and/or quantitative detection sensor, the nucleic acid qualitative and/or quantitative detection sensor comprises the hollow gold-silver core-shell nanoflower SERS nanoprobe HAu/AgSNFs- ATP detection zone.
进一步的，所述核酸定性和/或定量检测传感器还包括负载有聚 多巴胺的样品预处理区和负载有扩增体系的扩增区。Further, the nucleic acid qualitative and/or quantitative detection sensor further includes a sample pretreatment area loaded with polydopamine and an amplification area loaded with an amplification system.
本发明还提供一种根据所述的基于SERS的核酸定性和/或定量检测传感器的使用方法，所述使用方法为：The present invention also provides a method for using the SERS-based nucleic acid qualitative and/or quantitative detection sensor, which is:
(1)将已知的不同浓度核酸的酸水解产物分别置于所述定量SERS检测区后，对所述定量SERS检测区进行SERS光谱采集得到SERS信号，用化学计量学方法来建立预测模型；(1) After the known acid hydrolysates of nucleic acids with different concentrations are placed in the quantitative SERS detection area, the SERS spectrum is collected in the quantitative SERS detection area to obtain SERS signals, and a stoichiometric method is used to establish a prediction model;
(2)将待测样品的酸水解产物置于所述定量SERS检测区后，对所述定量SERS检测区进行SERS光谱采集得到SERS信号；(2) after placing the acid hydrolyzate of the sample to be tested in the quantitative SERS detection zone, performing SERS spectrum collection on the quantitative SERS detection zone to obtain a SERS signal;
(3)根据标准SERS图谱对待测样品中的核酸进行定性；根据预测模型对待测样品中的核酸进行定量。(3) qualitatively characterize the nucleic acid in the sample to be tested according to the standard SERS spectrum; quantify the nucleic acid in the sample to be tested according to the prediction model.
本发明还提供一种基于品红亚硫酸试剂与SERS的核酸定性和/或定量检测传感器，所述核酸定性和/或定量检测传感器包括定性显色检测区和定量和/或定性SERS检测区，其中，定性显色检测区负载有品红亚硫酸试剂，定量和/或定性SERS检测区负载有所述的中空金银核壳纳米花SERS纳米探针HAu/AgSNFs-ATP。The present invention also provides a nucleic acid qualitative and/or quantitative detection sensor based on magenta sulfite reagent and SERS, the nucleic acid qualitative and/or quantitative detection sensor includes a qualitative color development detection area and a quantitative and/or qualitative SERS detection area, Wherein, the qualitative color development detection area is loaded with magenta sulfite reagent, and the quantitative and/or qualitative SERS detection area is loaded with the hollow gold-silver core-shell nanoflower SERS nanoprobe HAu/AgSNFs-ATP.
进一步的，所述核酸定性和/或定量检测传感器还包括负载有聚多巴胺的样品预处理区和负载有扩增体系的扩增区。Further, the nucleic acid qualitative and/or quantitative detection sensor further includes a sample pretreatment area loaded with polydopamine and an amplification area loaded with an amplification system.
本发明还提供一种根据所述的基于品红亚硫酸试剂和SERS的核酸定性和/或定量检测传感器的使用方法，所述使用方法为：The present invention also provides a method for using the nucleic acid qualitative and/or quantitative detection sensor based on the magenta sulfite reagent and SERS, the use method is:
(1)将已知的不同浓度核酸的酸水解产物分别置于所述定性和/或定量SERS检测区后，对所述定性和/或定量SERS检测区进行SERS光谱采集得到SERS信号，用化学计量学方法来建立预测模型；(1) After the known acid hydrolysis products of nucleic acids with different concentrations are placed in the qualitative and/or quantitative SERS detection area, respectively, the qualitative and/or quantitative SERS detection area is subjected to SERS spectrum collection to obtain SERS signals. econometric methods to build predictive models;
(2)将待测样品的酸水解产物置于所述定性和/或定量SERS检测区后，对所述定性和/或定量SERS检测区进行SERS光谱采集得到SERS信号；(2) after placing the acid hydrolyzate of the sample to be tested in the qualitative and/or quantitative SERS detection area, perform SERS spectrum collection on the qualitative and/or quantitative SERS detection area to obtain a SERS signal;
(3)将待测样品的酸水解产物置于所述定性显色检测区后获得可视化信号；(3) the acid hydrolyzate of the sample to be tested is placed in the qualitative color development detection zone to obtain a visual signal;
(3)结果判定：(3) Result judgment:
根据所述可视化信号对所述待测样品进行定性，所述可视化信号具体为：a、当定性显色检测区不显示颜色，表示检测结果为阴性；b、当定性显色检测区显示紫色，表示检测结果为阳性；The sample to be tested is characterized according to the visual signal, and the visual signal is specifically: a. When no color is displayed in the qualitative color detection area, it indicates that the test result is negative; b. When the qualitative color detection area shows purple, Indicates that the test result is positive;
根据步骤(1)获得的标准SERS图谱对所述待测样品中的核酸进行定性；根据步骤(1)获得的预测模型对所述待测样品中的核酸进行定量。The nucleic acid in the sample to be tested is qualitatively determined according to the standard SERS spectrum obtained in step (1); the nucleic acid in the sample to be tested is quantified according to the prediction model obtained in step (1).
本发明还提供一种根据所述的纳米探针HAu/AgSNFs-ATP或所述的基于SERS的核酸定性和/或定量的检测传感器或所述的基于品红亚硫酸试剂和SERS的核酸定性和/或定量检测传感器在定性和/或定量检测核酸中的应用。The present invention also provides a nucleic acid qualitative and/or quantitative detection sensor based on the nanoprobe HAu/AgSNFs-ATP or the SERS-based nucleic acid or the nucleic acid qualitative and/or quantitative based on the magenta sulfite reagent and SERS. Application of/or quantitative detection sensor in qualitative and/or quantitative detection of nucleic acid.
本发明还提供一种根据所述的纳米探针HAu/AgSNFs-ATP或所述的基于SERS的核酸定性和/或定量的检测传感器或所述的基于品红亚硫酸试剂和SERS的核酸定性和/或定量检测传感器在制备定性和/或定量检测核酸的产品中的应用。The present invention also provides a nucleic acid qualitative and/or quantitative detection sensor based on the nanoprobe HAu/AgSNFs-ATP or the SERS-based nucleic acid or the nucleic acid qualitative and/or quantitative based on the magenta sulfite reagent and SERS. The application of/or quantitative detection sensor in the preparation of a product for qualitatively and/or quantitatively detecting nucleic acid.
本发明公开了以下技术效果：本发明提出了一种基于纸基SERS传感的通用核酸快速检测方法，具体为本发明制备了一种中空金银核 壳纳米花SERS纳米探针HAu/AgSNFs-ATP，可与核酸酸水解产物发生加成反应，生成新的官能团，与化学计量学方法和比色法结合构建稳态特异性致病菌可视化和精准定量模型，因此本发明建立的通用的核酸检测方法，实现了各种核酸的便携性、高灵敏和特异性检测，能够满足现场化检测需求。The invention discloses the following technical effects: the invention proposes a general nucleic acid rapid detection method based on paper-based SERS sensing, and specifically prepares a hollow gold-silver core-shell nanoflower SERS nanoprobe HAu/AgSNFs- ATP can undergo an addition reaction with nucleic acid acid hydrolysis products to generate new functional groups, which can be combined with stoichiometric methods and colorimetric methods to build a steady-state specific pathogenic bacteria visualization and accurate quantitative model. Therefore, the general nucleic acid established by the present invention is The detection method realizes the portable, highly sensitive and specific detection of various nucleic acids, which can meet the needs of on-site detection.
附图说明Description of drawings
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例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发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，还可以根据这些附图获得其他的附图。In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings required in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some of the present invention. In the embodiments, for those of ordinary skill in the art, other drawings can also be obtained according to these drawings without creative labor.
图1为不同AgNO 3浓度下制备的HAu/AgSNFs的透射电镜图，其中，A为0.1M AgNO 3制备的HAu/AgSNFs，B为0.3M AgNO 3制备的HAu/AgSNFs，C为0.5M AgNO 3制备的HAu/AgSNFs，D为0.7M AgNO 3制备的HAu/AgSNFs； Figure 1 shows the transmission electron microscope images of HAu/AgSNFs prepared under different AgNO 3 concentrations, where A is HAu/AgSNFs prepared with 0.1M AgNO 3 , B is HAu/AgSNFs prepared with 0.3M AgNO 3 , and C is 0.5M AgNO 3 Prepared HAu/AgSNFs, D is HAu/AgSNFs prepared with 0.7M AgNO 3 ;
图2为不同AgNO 3浓度下制备的HAu/AgSNFs-ATP的SERS图谱，其中，A为0.1M AgNO 3制备的HAu/AgSNFs，B为0.3M AgNO 3制备的HAu/AgSNFs，C为0.5M AgNO 3制备的HAu/AgSNFs，D为0.7M AgNO 3制备的HAu/AgSNFs； Figure 2 shows the SERS spectra of HAu/AgSNFs-ATP prepared under different AgNO 3 concentrations, wherein A is HAu/AgSNFs prepared with 0.1M AgNO 3 , B is HAu/AgSNFs prepared with 0.3M AgNO 3 , and C is 0.5M AgNO 3 3 prepared HAu/AgSNFs, D is 0.7M AgNO 3 prepared HAu/AgSNFs;
图3为纸基SERS/比色传感快速检测方法的流程示意图；Figure 3 is a schematic flowchart of a paper-based SERS/colorimetric sensing rapid detection method;
图4为金黄色葡萄球菌的PLS定量模型；Fig. 4 is the PLS quantitative model of Staphylococcus aureus;
图5为大肠杆菌的PLS定量模型；Fig. 5 is the PLS quantitative model of Escherichia coli;
图6为沙门氏菌的PLS定量模型。Figure 6 is a PLS quantification model of Salmonella.
具体实施方式Detailed ways
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, but not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.
为使本发明的上述目的、特征和优点能够更加明显易懂，下面结合附图和具体实施方式对本发明作进一步详细的说明。In order to make the above objects, features and advantages of the present invention more clearly understood, the present invention will be described in further detail below with reference to the accompanying drawings and specific embodiments.
实施例1 SERS纳米探针HAu/AgSNFs-ATP的制备以及纸基SERS传感快速检测方法的建立Example 1 Preparation of SERS nanoprobe HAu/AgSNFs-ATP and establishment of paper-based SERS sensing rapid detection method
步骤一，HAu/AgSNFs制备：在1mL H 3NO溶液(1×10 -2-6×10 -2M)和1mL NaOH溶液(0.01-0.08M)的混合体系下加入18mL AgNO 3(0.1-1M)溶液进行搅拌5-20min，然后加入10mL的HAuCl 4溶液(2×10 -4-10×10 -4M)和200μL C 6H 5Na 3O 7溶液(0.5％-1.5％，w/v)在80-110℃反应5min得到HAu/AgSNFs。 Step 1, preparation of HAu/AgSNFs: add 18mL AgNO 3 (0.1-1M) under the mixed system of 1mL H 3 NO solution (1×10 -2 -6×10 -2 M) and 1mL NaOH solution (0.01-0.08M). ) solution was stirred for 5-20 min, then 10 mL of HAuCl 4 solution (2×10 -4 -10×10 -4 M) and 200 μL of C 6 H 5 Na 3 O 7 solution (0.5%-1.5%, w/v) were added ) at 80-110 °C for 5 min to obtain HAu/AgSNFs.
步骤二，HAu/AgSNFs的功能化修饰：在10mL HAu/AgSNFs水溶液中加入30μL C 6H 7NS醇水混合溶液(0.01-1M)，搅拌反应15min后离心，清洗两次，最后分散在水溶液中得到基于HAu/AgSNFs-ATP的SERS纳米探针。 Step 2, functional modification of HAu/AgSNFs: add 30 μL of C 6 H 7 NS alcohol-water mixed solution (0.01-1 M) to 10 mL of HAu/AgSNFs aqueous solution, stir and react for 15 min, centrifuge, wash twice, and finally disperse in the aqueous solution The SERS nanoprobes based on HAu/AgSNFs-ATP were obtained.
不同AgNO 3浓度下制备的HAu/AgSNFs纳米探针的透射电镜图如图1所示，由图1所可知，AgNO 3浓度的不同会影响该纳米材料的形态，间接会影响其SERS效果，不同AgNO 3浓度下制备的 HAu/AgSNFs-ATP优化SERS纳米探针的光谱优化图如图2所示，从TEM图可以看出，随着AgNO3的添加，HAu/AgSNFs会从一开始的圆球状逐渐长出花瓣，由于尖端效应会产生更多的hotspots，所以其SERS效应会增强。 The TEM images of the HAu/AgSNFs nanoprobes prepared under different AgNO 3 concentrations are shown in Figure 1. It can be seen from Figure 1 that the different AgNO 3 concentrations will affect the morphology of the nanomaterials and indirectly affect its SERS effect. The optimized spectrum of the HAu/AgSNFs-ATP-optimized SERS nanoprobe prepared at the AgNO concentration is shown in Fig . 2. From the TEM image, it can be seen that with the addition of AgNO, the HAu/AgSNFs will gradually change from a spherical shape at the beginning. As petals grow, more hotspots will be produced due to the tip effect, so the SERS effect will be enhanced.
步骤三，三维折纸设备的制备：用具有较高机械强度的纤维素纸通过将相关模具压缩在纤维素纸上形成相应的直径为1cm的反应腔室，将聚多巴胺材料负载在预处理区，将10μL扩增体系负载在扩增区，将20μL品红亚硫酸试剂以及HAu/AgSNFs-ATP分别负载在检测区的定性显色检测线以及定性和/或定量SERS检测线上。 Step 3, the preparation of the three-dimensional origami device: use cellulose paper with high mechanical strength to form a corresponding reaction chamber with a diameter of 1 cm by compressing the relevant mold on the cellulose paper, and load the polydopamine material in the pretreatment area, Load 10 μL of amplification system in the amplification area, and load 20 μL of magenta sulfite reagent and HAu/AgSNFs-ATP on the qualitative color development detection line and qualitative and/or quantitative SERS detection line of the detection area, respectively.
步骤四，食源性致病菌的特异性检测方法建立：制备不同浓度的致病菌，然后分别对其进行加热粗提DNA，将粗提物滴加在预处理区对反应体系中的干扰物质进行吸附，然后折叠设备将预处理区和扩增区重合，在一定温度下进行基因扩增，扩增结束后，将扩增区置入酸性条件下对扩增产物进行酸水解；继续将扩增区和检测区进行折叠重合，进行相应的反应后，在可视化检测线可以得到可视化信号，对SERS检测线进行SERS光谱采集，结合化学计量学方法，得到定量模型。Step 4: Establish a specific detection method for food-borne pathogenic bacteria: prepare pathogenic bacteria with different concentrations, then heat them to crudely extract DNA, and drop the crude extract into the pretreatment area to interfere with the reaction system. The substance is adsorbed, and then the pretreatment area and the amplification area are overlapped by the folding equipment, and the gene amplification is performed at a certain temperature. After the amplification, the amplification area is placed in an acidic condition to perform acid hydrolysis on the amplification product; The amplification area and the detection area are folded and overlapped, and after the corresponding reaction, the visual signal can be obtained in the visual detection line, the SERS spectrum is collected on the SERS detection line, and the quantitative model is obtained by combining the chemometric method.
具体流程示意图如图3所示。The specific flow chart is shown in Figure 3.
检测原理：核酸在强酸和高温下核酸完全水解为碱基，核糖或脱氧核糖和磷酸；在浓度略稀的无机酸中，最易水解的化学键被选择性的断裂，一般为连接嘌呤和核糖的糖苷键，从而产生脱嘌呤核酸。当糖苷键断开后形成醛基，一方面醛基可与无色品红亚硫酸溶液反应显 紫色，另一方面醛基可与纳米探针HAu/AgSNFs-ATP上的氨基产生加成反应生成新的C＝N官能团，从而产生新的SERS信号。Detection principle: nucleic acid is completely hydrolyzed into base, ribose or deoxyribose and phosphoric acid under strong acid and high temperature; in slightly diluted inorganic acid, the most easily hydrolyzed chemical bond is selectively broken, generally connecting purine and ribose. glycosidic bonds, resulting in apurine nucleic acids. When the glycosidic bond is broken, an aldehyde group is formed. On the one hand, the aldehyde group can react with colorless magenta sulfite solution to turn purple, and on the other hand, the aldehyde group can generate an addition reaction with the amino group on the nanoprobe HAu/AgSNFs-ATP. A new C=N functional group, resulting in a new SERS signal.
拉曼位移一般为10-4000cm -1,对应于分子转动或振-转能级的跃迁，因此拉曼光谱一样可提供分子结构的信息。一般来说，不同的化合物给出的拉曼谱图也不同，由此可以对化合物进行定性分析鉴定。另一方面，不同化合物中同一基团或化学键又能给出波数相近的拉曼谱带，因此又可以进行官能团的鉴别。 The Raman shift is generally 10-4000cm -1 , which corresponds to the transition of molecular rotation or vibration-rotation energy level, so Raman spectroscopy can also provide information on molecular structure. Generally speaking, different compounds give different Raman spectra, which can be used for qualitative analysis and identification of compounds. On the other hand, the same group or chemical bond in different compounds can give Raman bands with similar wave numbers, so functional groups can be identified.
实施例2纸基SERS传感快速检测方法的应用Example 2 Application of paper-based SERS sensing rapid detection method
2.1纸基SERS传感快速检测方法在细菌检测中的应用2.1 Application of paper-based SERS sensing rapid detection method in bacterial detection
2.1.1以金黄色葡萄球菌、大肠杆菌、沙门氏菌为例，进行细菌的定性及定量检测2.1.1 Take Staphylococcus aureus, Escherichia coli, and Salmonella as examples to carry out qualitative and quantitative detection of bacteria
2.1.1.1金黄色葡萄球菌2.1.1.1 Staphylococcus aureus
分别将浓度为0-7cfu/mL的金黄色葡萄球菌进行加热粗提DNA，将粗提物滴加在预处理区对反应体系中的干扰物质进行吸附，然后折叠设备将预处理区和扩增区重合，进行基因扩增；扩增反应的总体积为20μL，其中包含一对引物P1和P2浓度分别为10 -6M，体系中还包括浓度为5×10 -4M的dNTPs,1×Isothermopol Buffer,以及Bst 2.0 WarmStart DNA聚合酶；扩增反应温度为62℃，反应时间为30min，扩增结束后，向扩增区加入0.5mM的HCl溶液在65℃反应5min后得到酸水解产物。 Staphylococcus aureus with a concentration of 0-7cfu/mL was heated to crudely extract DNA, and the crude extract was added dropwise to the pretreatment area to adsorb the interfering substances in the reaction system, and then the equipment was folded to separate the pretreatment area and amplification The total volume of the amplification reaction is 20 μL, which contains a pair of primers P1 and P2 with a concentration of 10 -6 M respectively, and the system also includes dNTPs with a concentration of 5×10 -4 M, 1× Isothermopol Buffer, and Bst 2.0 WarmStart DNA polymerase; the amplification reaction temperature is 62 °C, and the reaction time is 30 min. After the amplification, 0.5 mM HCl solution is added to the amplification area and the acid hydrolyzed product is obtained after the reaction at 65 °C for 5 min.
其中，扩增引物为：Among them, the amplification primers are:
P1：GGTTCAAAAGTGAAAGACGGTCTTG(SEQ ID NO：1)；P1: GGTTCAAAAGTGAAAGACGGTCTTG (SEQ ID NO: 1);
P2：GCGCGGATCCATCTATAAGTGAC(SEQ ID NO：2)。P2: GCGCGGATCCATCTATAAGTGAC (SEQ ID NO: 2).
继续将扩增区和检测区进行折叠重合，进行相应的反应后，在可视化检测线可以得到可视化信号，对SERS检测线进行SERS光谱采集，结合化学计量学方法，得到PLS定量模型(如图4所示)。Continue to fold and overlap the amplification area and the detection area, and after the corresponding reaction, a visual signal can be obtained in the visual detection line, and the SERS spectrum is collected on the SERS detection line. Combined with the chemometric method, the PLS quantitative model is obtained (Figure 4 shown).
以常规方法提取速冻食品样本中的DNA，将提取的DNA滴入预处理区对反应体系中的干扰物质进行吸附，然后折叠设备将预处理区和扩增区重合，进行基因扩增，扩增条件与扩增引物同上。扩增结束后，将扩增区置入酸性条件下对扩增产物进行酸水解；继续将扩增区和检测区进行折叠重合，进行相应的反应后，在可视化检测线可以得到可视化信号，对SERS检测线进行SERS光谱采集。Extract DNA from quick-frozen food samples by conventional methods, drop the extracted DNA into the pretreatment area to adsorb the interfering substances in the reaction system, and then fold the equipment to overlap the pretreatment area and the amplification area for gene amplification and amplification. Conditions and amplification primers were the same as above. After the amplification, place the amplification region under acidic conditions to perform acid hydrolysis on the amplification product; continue to fold and overlap the amplification region and the detection region, and after performing the corresponding reaction, a visual signal can be obtained at the visual detection line. SERS detection line for SERS spectrum acquisition.
2.1.1.2饮用水中的大肠杆菌检测2.1.1.2 Detection of Escherichia coli in drinking water
具体方法同2.1.1.1，其中，扩增引物为：The specific method is the same as 2.1.1.1, wherein the amplification primers are:
P1：CGCTCGTTGCGGGACTTAACC(SEQ ID NO：3)；P1: CGCTCGTTGCGGGACTTAACC (SEQ ID NO: 3);
P2：GCTGTCGTCAGCTCGTGTTG(SEQ ID NO：4)。P2: GCTGTCGTCAGCTCGTGTTG (SEQ ID NO: 4).
大肠杆菌PLS定量模型如图5所示。The E. coli PLS quantitative model is shown in Figure 5.
2.1.1.3土壤中的沙门氏菌检测2.1.1.3 Salmonella detection in soil
具体方法同2.1.1.1其中，扩增引物为：The specific method is the same as that in 2.1.1.1, wherein the amplification primers are:
P1：TCTTACCCGCTGTATTTATGC(SEQ ID NO：5)；P1: TCTTACCCGCTGTATTTATGC (SEQ ID NO: 5);
P2：TAACCCGAACTATCTCAATCTGT(SEQ ID NO：6)。P2: TAACCCGAACTATCTCAATCTGT (SEQ ID NO: 6).
沙门氏菌PLS定量模型如图6所示。The Salmonella PLS quantification model is shown in Figure 6.
2.1.1.4总结2.1.1.4 Summary
如图4-6所示，三种细菌的训练模型相关系数(Rc)范围为 0.9831-0.9893，预测集相关系数(Rp)范围为0.9755-0.9863，三种菌的定量模型均具有较好的预测能力，因此应用该方法对食源性致病菌的检测具有一定可行性。As shown in Figure 4-6, the training model correlation coefficient (Rc) of the three bacteria ranges from 0.9831 to 0.9893, and the prediction set correlation coefficient (Rp) ranges from 0.9755 to 0.9863. The quantitative models of the three bacteria all have good predictions. Therefore, it is feasible to apply this method to the detection of food-borne pathogens.
实施例2纸基SERS传感快速检测方法的灵敏度实验Example 2 Sensitivity experiment of paper-based SERS sensing rapid detection method
从图4-图6的SERS定量预测模型可以看出，在线性范围内，该方法对金葡菌的的最低检出限为10cfu/mL，对大肠杆菌的最低检出限为1cfu/mL，对沙门氏菌的最低检出限为10cfu/mL。It can be seen from the quantitative prediction model of SERS in Figures 4-6 that within the linear range, the minimum detection limit of this method for Staphylococcus aureus is 10 cfu/mL, and the minimum detection limit for Escherichia coli is 1 cfu/mL. The minimum detection limit for Salmonella is 10 cfu/mL.
以上所述的实施例仅是对本发明的优选方式进行描述，并非对本发明的范围进行限定，在不脱离本发明设计精神的前提下，本领域普通技术人员对本发明的技术方案做出的各种变形和改进，均应落入本发明权利要求书确定的保护范围内。The above-mentioned embodiments are only to describe the preferred modes of the present invention, but not to limit the scope of the present invention. Without departing from the design spirit of the present invention, those of ordinary skill in the art can make various modifications to the technical solutions of the present invention. Variations and improvements should fall within the protection scope determined by the claims of the present invention.

Claims (9)

  1. 一种中空金银核壳纳米花SERS纳米探针HAu/AgSNFs-ATP，其特征在于，所述中空金银核壳纳米花SERS纳米探针HAu/AgSNFs-ATP的制备方法为：A hollow gold-silver core-shell nanoflower SERS nanoprobe HAu/AgSNFs-ATP, characterized in that the preparation method of the hollow gold-silver core-shell nanoflower SERS nanoprobe HAu/AgSNFs-ATP is:
    (1)HAu/AgSNFs制备：在H 3NO溶液和NaOH溶液的混合体系下加入AgNO 3溶液，然后加入HAuCl 4溶液和C 6H 5Na 3O 7溶液反应得到HAu/AgSNFs水溶液； (1) Preparation of HAu/AgSNFs: AgNO 3 solution was added under the mixed system of H 3 NO solution and NaOH solution, and then HAuCl 4 solution and C 6 H 5 Na 3 O 7 solution were added to react to obtain HAu/AgSNFs aqueous solution;
    (2)HAu/AgSNFs的功能化修饰：在步骤(1)得到的HAu/AgSNFs水溶液中与C 6H 7NS醇水混合溶液混合，反应后离心、清洗，得到所述中空金银核壳纳米花SERS纳米探针HAu/AgSNFs-ATP。 (2) Functional modification of HAu/AgSNFs: the HAu/AgSNFs aqueous solution obtained in step (1) is mixed with a C 6 H 7 NS alcohol-water mixed solution, centrifuged and washed after the reaction to obtain the hollow gold-silver core-shell nanoparticle Flower SERS nanoprobe HAu/AgSNFs-ATP.
  2. 一种基于SERS的核酸定性和/或定量的检测传感器，其特征在于，所述核酸定性和/或定量检测传感器包括负载有权利要求1所述的中空金银核壳纳米花SERS纳米探针HAu/AgSNFs-ATP的检测区。A nucleic acid qualitative and/or quantitative detection sensor based on SERS, wherein the nucleic acid qualitative and/or quantitative detection sensor comprises the hollow gold-silver core-shell nanoflower SERS nanoprobe HAu loaded with the claim 1 / Detection area of AgSNFs-ATP.
  3. 根据权利要求2所述基于SERS的核酸定性和/或定量的检测传感器，其特征在于，所述核酸定性和/或定量检测传感器还包括负载有聚多巴胺的样品预处理区和负载有扩增体系的扩增区。The nucleic acid qualitative and/or quantitative detection sensor based on SERS according to claim 2, wherein the nucleic acid qualitative and/or quantitative detection sensor further comprises a sample pretreatment area loaded with polydopamine and an amplification system loaded with the amplified region.
  4. 一种根据权利要求2或3所述的基于SERS的核酸定性和/或定量检测传感器的使用方法，其特征在于，所述使用方法为：A method of using a SERS-based nucleic acid qualitative and/or quantitative detection sensor according to claim 2 or 3, wherein the method of use is:
    (1)将已知的不同浓度核酸的酸水解产物分别置于所述定量SERS检测区后，对所述定量SERS检测区进行SERS光谱采集得到SERS信号，用化学计量学方法来建立预测模型；(1) After the known acid hydrolysates of nucleic acids with different concentrations are placed in the quantitative SERS detection area, the SERS spectrum is collected in the quantitative SERS detection area to obtain SERS signals, and a stoichiometric method is used to establish a prediction model;
    (2)将待测样品的酸水解产物置于所述定量SERS检测区后，对所述定量SERS检测区进行SERS光谱采集得到SERS信号；(2) after placing the acid hydrolyzate of the sample to be tested in the quantitative SERS detection zone, performing SERS spectrum collection on the quantitative SERS detection zone to obtain a SERS signal;
    (3)根据标准SERS图谱对待测样品中的核酸进行定性；根据所述预测模型对待测样品中的核酸进行定量。(3) Qualifying the nucleic acid in the sample to be tested according to the standard SERS spectrum; quantifying the nucleic acid in the sample to be tested according to the prediction model.
  5. 一种基于品红亚硫酸试剂与SERS的核酸定性和/或定量检测传感器，其特征在于：所述核酸定性和/或定量检测传感器包括定性显色检测区和定量和/或定性SERS检测区，其中，定性显色检测区负载有品红亚硫酸试剂，定量和/或定性SERS检测区负载有权利要求1所述的中空金银核壳纳米花SERS纳米探针HAu/AgSNFs-ATP。A nucleic acid qualitative and/or quantitative detection sensor based on magenta sulfite reagent and SERS, characterized in that: the nucleic acid qualitative and/or quantitative detection sensor comprises a qualitative color development detection area and a quantitative and/or qualitative SERS detection area, Wherein, the qualitative color development detection zone is loaded with magenta sulfite reagent, and the quantitative and/or qualitative SERS detection zone is loaded with the hollow gold-silver core-shell nanoflower SERS nanoprobe HAu/AgSNFs-ATP according to claim 1.
  6. 根据权利要求5所述的基于品红亚硫酸试剂和SERS的核酸定性和/或定量检测传感器，其特征在于，所述核酸定性和/或定量检测传感器还包括负载有聚多巴胺的样品预处理区和负载有扩增体系的扩增区。The nucleic acid qualitative and/or quantitative detection sensor based on magenta sulfite reagent and SERS according to claim 5, wherein the nucleic acid qualitative and/or quantitative detection sensor further comprises a sample pretreatment area loaded with polydopamine and the amplification region loaded with the amplification system.
  7. 一种根据权利要求5或6所述的基于品红亚硫酸试剂和SERS的核酸定性和/或定量检测传感器的使用方法，其特征在于，所述使用方法为：A method of using a nucleic acid qualitative and/or quantitative detection sensor based on magenta sulfite reagent and SERS according to claim 5 or 6, wherein the method of using is:
    (1)将已知的不同浓度核酸的酸水解产物分别置于所述定性和/或定量SERS检测区后，对所述定性和/或定量SERS检测区进行SERS光谱采集得到SERS信号，用化学计量学方法来建立预测模型；(1) After the known acid hydrolysis products of nucleic acids with different concentrations are placed in the qualitative and/or quantitative SERS detection area, respectively, the qualitative and/or quantitative SERS detection area is subjected to SERS spectrum collection to obtain SERS signals. econometric methods to build predictive models;
    (2)将待测样品的酸水解产物置于所述定性和/或定量SERS检测区后，对所述定性和/或定量SERS检测区进行SERS光谱采集得到SERS信号；(2) after placing the acid hydrolyzate of the sample to be tested in the qualitative and/or quantitative SERS detection area, perform SERS spectrum collection on the qualitative and/or quantitative SERS detection area to obtain a SERS signal;
    (3)将待测样品的酸水解产物置于所述定性显色检测区后获得可视化信号；(3) the acid hydrolyzate of the sample to be tested is placed in the qualitative color development detection zone to obtain a visual signal;
    (3)结果判定：(3) Result judgment:
    根据所述可视化信号对所述待测样品进行定性，所述可视化信号具体为：a、当定性显色检测区不显示颜色，表示检测结果为阴性；b、当定性显色检测区显示紫色，表示检测结果为阳性；The sample to be tested is characterized according to the visual signal, and the visual signal is specifically: a. When no color is displayed in the qualitative color detection area, it indicates that the test result is negative; b. When the qualitative color detection area shows purple, Indicates that the test result is positive;
    根据步骤(1)获得的标准SERS图谱对所述待测样品中的核酸进行定性；根据步骤(1)获得的预测模型对所述待测样品中的核酸进行定量。The nucleic acid in the sample to be tested is qualitatively determined according to the standard SERS spectrum obtained in step (1); the nucleic acid in the sample to be tested is quantified according to the prediction model obtained in step (1).
  8. 一种根据权利要求1所述的纳米探针HAu/AgSNFs-ATP或权利要求2-3之一所述的核酸定性和/或定量检测传感器在定性和/或定量检测核酸中的应用。Application of the nanoprobe HAu/AgSNFs-ATP according to claim 1 or the nucleic acid qualitative and/or quantitative detection sensor according to any one of claims 2-3 in qualitative and/or quantitative detection of nucleic acid.
  9. 一种根据权利要求1所述的纳米探针HAu/AgSNFs-ATP或权利要求2-3之一所述的核酸定性和/或定量检测传感器在制备定性和/或定量检测核酸的产品中的应用。A kind of application of the nanoprobe HAu/AgSNFs-ATP according to claim 1 or the nucleic acid qualitative and/or quantitative detection sensor described in one of claims 2-3 in the preparation of a product for qualitative and/or quantitative detection of nucleic acid .
PCT/CN2021/090389 2021-04-06 2021-04-28 Hollow gold-silver core-shell nanoflower sers nanoprobe hau/agsnfs-atp WO2022213432A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202110365969.9A CN113092440B (en) 2021-04-06 2021-04-06 Nucleic acid detection method for food-borne pathogenic bacteria based on hollow gold-silver core-shell nanoflower SERS (surface enhanced Raman scattering) nanoprobes HAu/AgSNFs-ATP (adenosine triphosphate)
CN202110365969.9 2021-04-06

Publications (1)

Publication Number Publication Date
WO2022213432A1 true WO2022213432A1 (en) 2022-10-13

Family

ID=76673789

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/090389 WO2022213432A1 (en) 2021-04-06 2021-04-28 Hollow gold-silver core-shell nanoflower sers nanoprobe hau/agsnfs-atp

Country Status (2)

Country Link
CN (1) CN113092440B (en)
WO (1) WO2022213432A1 (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115627290A (en) * 2022-10-20 2023-01-20 苏州科技大学 Method for improving PCR detection sensitivity

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1865936A (en) * 2006-05-10 2006-11-22 吉林大学 SERS detection method employing nano semiconductor material as substrate
CN105647788A (en) * 2016-01-31 2016-06-08 南京邮电大学 SERS sensor for nucleic acid detection and preparation and multielement detection method thereof
CN107478641A (en) * 2017-03-22 2017-12-15 南京邮电大学 Liquid phase SERS sensor, its preparation method and its purposes for detection of nucleic acids
CN108318471A (en) * 2017-12-26 2018-07-24 中国科学院合肥物质科学研究院 The magnetic test paper and its preparation, application method of detection nitrite anions based on SERS
CN109060764A (en) * 2018-08-23 2018-12-21 安徽中科赛飞尔科技有限公司 A kind of SERS platform preparation method of functionalization and its detection application to ATP
US20190049384A1 (en) * 2015-09-23 2019-02-14 Industry-University Cooperation Foundation Hanyang University Erica Campus High-sensitivity lateral flow immunoassay strip based on surface-enhanced raman scattering and detection method using the same

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6972173B2 (en) * 2002-03-14 2005-12-06 Intel Corporation Methods to increase nucleotide signals by raman scattering
EP2141246A1 (en) * 2008-07-01 2010-01-06 Koninklijke Philips Electronics N.V. Separation-free methods of PCR detection using SERRS
WO2014149071A1 (en) * 2013-03-22 2014-09-25 Duke University Nano-plasmonic molecular probes and methods of use
CN107090506B (en) * 2017-05-19 2021-02-23 鲁东大学 Vancomycin nucleic acid dimer biosensor, preparation method and application thereof
WO2018230803A1 (en) * 2017-06-14 2018-12-20 인천대학교 산학협력단 Method for preparing surface-enhanced raman scattering nanocomposite for detecting pathogens, and method for detecting pathogens by using same and digital pcr
CN109387500B (en) * 2018-09-29 2021-01-12 南京农业大学 Method for detecting escherichia coli based on magnetic graphene oxide composite star @ gold-silver alloy nanoparticles
CN111398241A (en) * 2020-01-15 2020-07-10 武汉市农业科学院 High-flux SERS detection method for food-borne pathogenic bacteria
CN111751540B (en) * 2020-07-20 2023-03-03 烟台大学 SERS lateral flow test strip for nuclease detection and detection method

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1865936A (en) * 2006-05-10 2006-11-22 吉林大学 SERS detection method employing nano semiconductor material as substrate
US20190049384A1 (en) * 2015-09-23 2019-02-14 Industry-University Cooperation Foundation Hanyang University Erica Campus High-sensitivity lateral flow immunoassay strip based on surface-enhanced raman scattering and detection method using the same
CN105647788A (en) * 2016-01-31 2016-06-08 南京邮电大学 SERS sensor for nucleic acid detection and preparation and multielement detection method thereof
CN107478641A (en) * 2017-03-22 2017-12-15 南京邮电大学 Liquid phase SERS sensor, its preparation method and its purposes for detection of nucleic acids
CN108318471A (en) * 2017-12-26 2018-07-24 中国科学院合肥物质科学研究院 The magnetic test paper and its preparation, application method of detection nitrite anions based on SERS
CN109060764A (en) * 2018-08-23 2018-12-21 安徽中科赛飞尔科技有限公司 A kind of SERS platform preparation method of functionalization and its detection application to ATP

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
"Master Thesis", 30 May 2019, JIANGSU UNIVERSITY, CN, article XU, YI: "Detection of 2,4-D Residue in Milk Based on Surface-Enhance Raman Spectroscopy Technology", pages: 1 - 69, XP009540332 *

Also Published As

Publication number Publication date
CN113092440A (en) 2021-07-09
CN113092440B (en) 2022-04-29

Similar Documents

Publication Publication Date Title
Jazayeri et al. Colorimetric detection based on gold nano particles (GNPs): An easy, fast, inexpensive, low-cost and short time method in detection of analytes (protein, DNA, and ion)
Ma et al. SERS aptasensor for Salmonella typhimurium detection based on spiny gold nanoparticles
Shen et al. Recent advances in nanotechnology for simultaneous detection of multiple pathogenic bacteria
Neng et al. Application of surface-enhanced Raman spectroscopy in fast detection of toxic and harmful substances in food
Bostan et al. Ultrasensitive detection of ochratoxin A using aptasensors
Pahlow et al. Isolation and identification of bacteria by means of Raman spectroscopy
CN107796938B (en) Nucleic acid aptamer fluorescent test strip and preparation method and application thereof
Ansari et al. Aptasensors for quantitative detection of Salmonella Typhimurium
Pan et al. Detection of A. alternata from pear juice using surface-enhanced Raman spectroscopy based silver nanodots array
Zhou et al. Colorimetric detection of ractopamine and salbutamol using gold nanoparticles functionalized with melamine as a probe
Bai et al. A comprehensive review of detection methods for Escherichia coli O157: H7
Zhang et al. A ratiometric fluorescent probe for sensitive, selective and reversible detection of copper (II) based on riboflavin-stabilized gold nanoclusters
Sun et al. Raman spectroscopy for food quality assurance and safety monitoring: A review
Yu et al. The universal dual-mode aptasensor for simultaneous determination of different bacteria based on naked eyes and microfluidic-chip together with magnetic DNA encoded probes
Mazur et al. based sensors for bacteria detection
Ahmad et al. Trends in the bacterial recognition patterns used in surface enhanced Raman spectroscopy
Qian et al. Gold nanostars-enhanced Raman fingerprint strip for rapid detection of trace tetracycline in water samples
Chen et al. A fluorescence biosensor for Salmonella typhimurium detection in food based on the nano-self-assembly of alendronic acid modified upconversion and gold nanoparticles
WO2022213432A1 (en) Hollow gold-silver core-shell nanoflower sers nanoprobe hau/agsnfs-atp
CN113720794A (en) Method for sensing and detecting mycotoxin in rice by using gold nanoparticle-based colorimetric aptamer
Lu et al. AFB1 colorimetric aptamer sensor for the detection of AFB1 in ten different kinds of miscellaneous beans based on gold nanoparticles and smartphone imaging
Chen et al. Au@ 4-MBA@ Ag NPs labeled SERS lateral flow immunoassay for ultrasensitive and quantitative detection of Salmonella enteritidis
Huang et al. Zwitterion-modified antifouling swab joint “Snake-Eye” for detection of Salmonella in colored foodstuffs
Yao et al. Rapid and sensitive detection of Hg 2+ with a SERS-enhanced lateral flow strip
Li et al. Detection of pathogen based on the catalytic growth of gold nanocrystals

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21935653

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21935653

Country of ref document: EP

Kind code of ref document: A1