WO2021093220A1 - Biological detection device and detection method using gold nanopore array chip - Google Patents

Biological detection device and detection method using gold nanopore array chip Download PDF

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WO2021093220A1
WO2021093220A1 PCT/CN2020/076879 CN2020076879W WO2021093220A1 WO 2021093220 A1 WO2021093220 A1 WO 2021093220A1 CN 2020076879 W CN2020076879 W CN 2020076879W WO 2021093220 A1 WO2021093220 A1 WO 2021093220A1
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array chip
detection device
gold
sample loading
biological detection
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PCT/CN2020/076879
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French (fr)
Chinese (zh)
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刘钢
党棠
黄丽萍
胡文君
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量准（上海）医疗器械有限公司
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    • 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
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/27—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands using photo-electric detection ; circuits for computing concentration
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the invention belongs to the technical field of biological detection; more specifically, the invention relates to a biological detection device and a detection method using a gold nanopore array chip.
  • Bio testing is a means of measuring the sensitivity of organisms to substances.
  • the application of bioassays is very extensive.
  • amyloid protein aggregates into amyloid fibers through misfolding and deposits in various tissues and organs in the human body.
  • ThT fluorescence detection technology is currently mainly used to monitor amyloid aggregation.
  • the current method is difficult to achieve the advantages of small sample amount, high sensitivity, detection line width, fast test speed, and good stability.
  • the minimum concentration of an amphiphilic polymer that associates in a solvent to form micelles or nanoparticles is the critical micelle concentration.
  • the critical micelle concentration there are many methods to measure the critical micelle concentration. The more commonly used methods are surface tension method, conductivity method, dye method, solubilization method, osmotic pressure method, pulse radiolysis method, fluorescence method, ultrasonic adsorption method, and turbidity method. , PH value method, rheological method, ion selective electrode method and cyclic voltammetry, etc.
  • the light scattering method requires the measured solution to be very clean, and there are many limiting factors; the conductivity method is only suitable for measuring the CMC of ionic surfactants; the fluorescent probe method is used to measure CMC, although there are few limiting factors , But need to configure the benzene solution of the pyrene probe, and the benzene is toxic and the operation process is cumbersome; the equipment needed to measure CMC by the dye method is simple, but the color change is not obvious enough, which will affect the accuracy of the CMC; the turbidity method also depends on the used The problem that hydrocarbon dissolved matter affects the critical micelle concentration of surfactants affects the accuracy of the measured CMC.
  • the technical problem to be solved by the present invention is to provide a biological detection device using gold nanohole array chip with low sample amount, high sensitivity, wide detection line, fast test speed, and good stability in view of the above-mentioned defects in the prior art. And detection method.
  • a biological detection device using a gold nanopore array chip including: a light source, a sample loading unit, a photodetector, and a processor; wherein the sample loading unit includes: a sample loading microcomputer for accommodating a sample to be tested A hole, wherein a gold nanohole array chip is arranged at the bottom of the sample loading microhole; a plurality of nanoholes are formed on one side of the gold nanohole array chip; the light source is used to illuminate the sample loading unit with the sample to be tested, and the photoelectric The detector receives the light transmitted from the sample loading unit and transmits the light to the processor.
  • the photodetector is used to determine the OD value change curve of the sample to be tested over time at a predetermined wavelength.
  • the photodetector is a detection device such as a microplate reader.
  • the plurality of nanoholes does not penetrate the gold nanohole array chip.
  • the plurality of nanoholes are formed in a hole matrix structure.
  • the pore diameter of the nanopore is less than 0 nm.
  • the loading microwells are 96-well plates.
  • the biological detection device is used for C-reactive protein detection, real-time PCR amplification monitoring, amyloid aggregation monitoring, critical aggregation concentration determination, heavy metal ion detection, and virus detection.
  • a detection method is also provided, which adopts the above-mentioned biological detection device using a gold nanopore array chip.
  • the gold nanohole array chip as a new type of nano-plasma optical light sensor chip, has an enhancement effect on the scattered light of the nano-particles, and can effectively reduce the detection limit of the test object. Therefore, the present invention can be advantageously used for the determination of biological samples, and has the advantages of small sample loading, high sensitivity, detection line width, fast test speed, good stability and the like.
  • Fig. 1 schematically shows a structural block diagram of a biological detection device using a gold nanopore array chip according to a preferred embodiment of the present invention.
  • Fig. 2 schematically shows a structural schematic diagram of a sample loading unit of a biological detection device using a gold nanopore array chip according to a preferred embodiment of the present invention.
  • FIG. 3 schematically shows the structure of the gold nanohole array chip in the sample loading unit of the biological detection device using the gold nanohole array chip according to a preferred embodiment of the present invention.
  • Fig. 4 schematically shows a partial perspective view of a biological detection device using a gold nanopore array chip according to a preferred embodiment of the present invention.
  • Fig. 1 schematically shows a structural block diagram of a biological detection device using a gold nanopore array chip according to a preferred embodiment of the present invention.
  • a biological detection device using a gold nanopore array chip includes: a light source 10, a sample loading unit 20, a photodetector 30 and a processor 40.
  • Fig. 2 schematically shows a structural schematic diagram of a sample loading unit of a biological detection device using a gold nanopore array chip according to a preferred embodiment of the present invention.
  • the sample loading unit 20 includes a sample loading microhole 11 for accommodating a sample 13 to be tested, wherein a gold nanohole array chip 12 is arranged at the bottom of the sample loading microhole 11.
  • the loading microwell 11 is a 96-well plate.
  • FIG. 3 schematically shows the structure of the gold nanohole array chip in the sample loading unit of the biological detection device using the gold nanohole array chip according to a preferred embodiment of the present invention.
  • a plurality of nanoholes 22 are formed on one side surface of the gold nanohole array chip 12. After adding the test sample 13, the test sample 13 will enter the nanopore 22.
  • the plurality of nanoholes 22 does not penetrate the gold nanohole array chip 12, that is, the gold nanohole array chip 12 is a blind hole.
  • the pore diameter of the nanopore 22 is less than 200 nm.
  • the plurality of nanoholes 22 are formed in a hole matrix structure.
  • the small holes on the gold nanohole array chip 12 are all nanoholes (for example, the pore size is less than 200nm), and the chip is used as a substrate, glued to the bottom of the sample loading microhole, and the sample to be tested is added, and then the detection (such as Microplate reader).
  • Fig. 4 schematically shows a partial perspective view of a biological detection device using a gold nanopore array chip according to a preferred embodiment of the present invention.
  • the light source is used to illuminate the sample loading unit 20 where the sample 13 to be tested is added, and the photodetector 30 receives the light transmitted from the sample loading unit 20 and transmits the light to the processor 40.
  • the photodetector 30 is used to determine the OD (optical density, representing the optical density absorbed by the detected object) value change curve of the sample to be tested at a predetermined wavelength over time.
  • the photodetector 30 is a detection device such as a microplate reader.
  • the gold nanohole array chip as a new type of nano-plasma optical light sensor chip, has an enhancement effect on the scattered light of the nano-particles, and can effectively reduce the detection limit of the test object. Therefore, the present invention can be advantageously used for the determination of biological samples, and has the advantages of small sample loading, high sensitivity, detection line width, fast test speed, good stability and the like.
  • a detection method is also provided, which adopts the above-mentioned biological detection device using a gold nanopore array chip.
  • the reagents include:
  • the first reagent composition which contains electrolyte, coagulant, surfactant, preservative and buffer;
  • the signal amplification effect of the gold nanopore can reduce the detection line and improve the sensitivity without the need for antibody carrier particles.
  • the device of this application has different degrees of amplification for the scattered light intensity of the complexes of different particle sizes. Based on this difference, this application can be used to monitor the DNA amplification reaction in real time. Compared with real-time fluorescent quantitative PCR technology, it has a relatively low price. Low, more convenient and easy to implement, reduce pollution, and not be affected by fluorescence signal quenching.
  • ThT fluorescence detection technology is mainly used to monitor amyloid aggregation.
  • the application can monitor the inhibitory effect of amyloid aggregation, the aggregation-promoting effect and the deaggregation situation in real time, and has the advantages of convenience and quickness, not affected by ThT, sensitive monitoring, good stability and the like.
  • the minimum concentration of amphiphilic polymers that associate to form micelles or nanoparticles in a solvent is the critical micelle concentration.
  • the light scattering method requires the measured solution to be very clean, and there are many limiting factors; the conductivity method is only suitable for measuring the CMC of ionic surfactants; the fluorescent probe method is used to measure CMC, although there are few limiting factors , But need to configure the benzene solution of the pyrene probe, and the benzene is toxic and the operation process is cumbersome; the equipment needed to measure CMC by the dye method is simple, but the color change is not obvious enough, which will affect the accuracy of the CMC; the turbidity method also depends on the used The problem that hydrocarbon dissolved matter affects the critical micelle concentration of surfactants affects the accuracy of the measured CMC.
  • the device of the present application has a light signal amplification function, and can more accurately determine the critical aggregation concentration of different amphiphilic molecules, and distinguish small differences between different molecules.
  • the detection methods of heavy metal elements include photometry, turbidimetry, spot comparison method, chromatography, spectroscopy, electrochemical analysis, neutron activation analysis, etc.
  • the trace heavy metals that can be processed by chemical precipitation or biological flocculation can be detected by the device of the present application, which has the advantages of sensitive detection, simple operation and the like. It can be used for water quality detection and trace heavy metal detection in food and medicine.
  • the device of the present application has biocompatibility and signal amplification effects on nanoparticles, and can be combined with specific detection technology of immune response to detect biological viruses for the prevention and control of viral diseases.

Abstract

A biological detection device using a gold nanopore array chip and a detection method therefor, the detection device comprising: a light source (10), a sample loading unit (20), a photoelectric detector (30) and a processor (40), wherein the sample loading unit (20) comprises a sample loading micropore (11) used for accommodating a sample (13) to be detected, and a gold nanopore array chip (12) is arranged at the bottom part of the sample loading micropore (11); a plurality of nanopores (22) are formed on one side surface of the gold nanopore array chip (12); and the light source (10) irradiates the sample loading unit (20) that has the sample (13) added thereto, and the photoelectric detector (30) receives light transmitted from the sample loading unit (20) and transmits the light to the processor (40).

Description

一种采用金纳米孔阵列芯片的生物检测装置及检测方法Biological detection device and detection method using gold nanohole array chip 技术领域Technical field
本发明属于生物检测技术领域；更具体地说，本发明涉及一种采用金纳米孔阵列芯片的生物检测装置及检测方法。The invention belongs to the technical field of biological detection; more specifically, the invention relates to a biological detection device and a detection method using a gold nanopore array chip.
背景技术Background technique
生物检测是利用生物对物质的敏感性反应来进行测定的一种手段。生物检测的应用非常广泛。例如，淀粉样蛋白通过错误折叠聚集成为淀粉样纤维，并在人体内多种组织和器官中沉积。对此的检测，目前主要采用ThT荧光检测技术来监控淀粉样蛋白聚集情况。但是，目前的方法很难做到上样量少、灵敏度高、检测上线宽、测试速度快、稳定性好等优点。Biological testing is a means of measuring the sensitivity of organisms to substances. The application of bioassays is very extensive. For example, amyloid protein aggregates into amyloid fibers through misfolding and deposits in various tissues and organs in the human body. For this detection, ThT fluorescence detection technology is currently mainly used to monitor amyloid aggregation. However, the current method is difficult to achieve the advantages of small sample amount, high sensitivity, detection line width, fast test speed, and good stability.
又例如，两亲性的高分子在溶剂中缔合形成胶束或纳米颗粒的最低浓度即为临界胶束浓度。对此的检测，测量临界胶束浓度的方法很多，比较常用的有表面张力法、电导法、染料法、增溶法、渗透压法、脉冲射解法、荧光法、超声吸附法、浊度法、pH值法、流变法、离子选择性电极法和循环伏安法等。不同监测方法的优缺点：光散射法要求所测溶液非常干净，受限制因素较多；而电导法只适于测量离子型表面活性剂的CMC；荧光探针法测量CMC，虽然受限制因素少，但是需要配置芘探针的苯溶液，且苯有毒性而且操作过程繁琐；染料法测量CMC所需的设备简单，但是因颜色变化不够明显，会影响CMC的准确度；浊度法也因所用烃溶解物影响表面活性剂临界胶束浓度的问题，所测CMC的准确度受到影响。For another example, the minimum concentration of an amphiphilic polymer that associates in a solvent to form micelles or nanoparticles is the critical micelle concentration. For this detection, there are many methods to measure the critical micelle concentration. The more commonly used methods are surface tension method, conductivity method, dye method, solubilization method, osmotic pressure method, pulse radiolysis method, fluorescence method, ultrasonic adsorption method, and turbidity method. , PH value method, rheological method, ion selective electrode method and cyclic voltammetry, etc. The advantages and disadvantages of different monitoring methods: the light scattering method requires the measured solution to be very clean, and there are many limiting factors; the conductivity method is only suitable for measuring the CMC of ionic surfactants; the fluorescent probe method is used to measure CMC, although there are few limiting factors , But need to configure the benzene solution of the pyrene probe, and the benzene is toxic and the operation process is cumbersome; the equipment needed to measure CMC by the dye method is simple, but the color change is not obvious enough, which will affect the accuracy of the CMC; the turbidity method also depends on the used The problem that hydrocarbon dissolved matter affects the critical micelle concentration of surfactants affects the accuracy of the measured CMC.
发明内容Summary of the invention
本发明所要解决的技术问题是针对现有技术中存在上述缺陷，提供一种上样量少、灵敏度高、检测上线宽、测试速度快、稳定性好的采用金纳米孔阵列芯片的生物检测装置及检测方法。The technical problem to be solved by the present invention is to provide a biological detection device using gold nanohole array chip with low sample amount, high sensitivity, wide detection line, fast test speed, and good stability in view of the above-mentioned defects in the prior art. And detection method.
根据本发明，提供了一种采用金纳米孔阵列芯片的生物检测装置包括：光源、装样单元、光电检测器和处理器；其中，装样单元包括：用于容纳待测样品的装样微孔，其中在装样微孔底部布置有金纳米孔阵列芯片；金纳米孔阵列芯片的一侧表面上形成有多个纳米孔；光源用于向加入待测样品的装样单元进行照射，光电检测器接收从装样单元传递出来的光线并且将光线传递给处理器。According to the present invention, there is provided a biological detection device using a gold nanopore array chip, including: a light source, a sample loading unit, a photodetector, and a processor; wherein the sample loading unit includes: a sample loading microcomputer for accommodating a sample to be tested A hole, wherein a gold nanohole array chip is arranged at the bottom of the sample loading microhole; a plurality of nanoholes are formed on one side of the gold nanohole array chip; the light source is used to illuminate the sample loading unit with the sample to be tested, and the photoelectric The detector receives the light transmitted from the sample loading unit and transmits the light to the processor.
优选地，光电检测器用于测定预定波长下待测样品随时间的OD值变化曲线。Preferably, the photodetector is used to determine the OD value change curve of the sample to be tested over time at a predetermined wavelength.
优选地，光电检测器是酶标仪等检测设备。Preferably, the photodetector is a detection device such as a microplate reader.
优选地，多个纳米孔未穿透金纳米孔阵列芯片。Preferably, the plurality of nanoholes does not penetrate the gold nanohole array chip.
优选地，多个纳米孔形成为孔矩阵结构。Preferably, the plurality of nanoholes are formed in a hole matrix structure.
优选地，纳米孔的孔径小于0nm。Preferably, the pore diameter of the nanopore is less than 0 nm.
优选地，装样微孔是96孔板。Preferably, the loading microwells are 96-well plates.
优选地，所述生物检测装置用于C反应蛋白检测、实时PCR扩增监控、淀粉样蛋白聚集监控、临界聚集浓度测定、重金属离子检测和病毒检测。Preferably, the biological detection device is used for C-reactive protein detection, real-time PCR amplification monitoring, amyloid aggregation monitoring, critical aggregation concentration determination, heavy metal ion detection, and virus detection.
根据本发明，还提供了一种检测方法，其采用了上述的采用金纳米孔阵列芯片的生物检测装置。According to the present invention, a detection method is also provided, which adopts the above-mentioned biological detection device using a gold nanopore array chip.
在本发明中，金纳米孔阵列芯片作为一种新型纳米等离子光学光感器芯片，对纳米粒子的散射光具有增强作用，可以有效降低待测物的检测限。由此，本发明能有利地用于生物样品测定，具有上样量少、灵敏度高、检测上线宽、测试速度快、稳定性好等优点。In the present invention, the gold nanohole array chip, as a new type of nano-plasma optical light sensor chip, has an enhancement effect on the scattered light of the nano-particles, and can effectively reduce the detection limit of the test object. Therefore, the present invention can be advantageously used for the determination of biological samples, and has the advantages of small sample loading, high sensitivity, detection line width, fast test speed, good stability and the like.
附图说明Description of the drawings
结合附图，并通过参考下面的详细描述，将会更容易地对本发明有更完整的理解并且更容易地理解其伴随的优点和特征，其中：With reference to the accompanying drawings, and by referring to the following detailed description, it will be easier to have a more complete understanding of the present invention and easier to understand its accompanying advantages and features, among which:
图1示意性地示出了根据本发明优选实施例的采用金纳米孔阵列芯片的生物检测装置的结构框图。Fig. 1 schematically shows a structural block diagram of a biological detection device using a gold nanopore array chip according to a preferred embodiment of the present invention.
图2意性地示出了根据本发明优选实施例的采用金纳米孔阵列芯片的生物检测装置的装样单元的结构示意图。Fig. 2 schematically shows a structural schematic diagram of a sample loading unit of a biological detection device using a gold nanopore array chip according to a preferred embodiment of the present invention.
图3意性地示出了根据本发明优选实施例的采用金纳米孔阵列芯片的生物检测装置的装样单元中的金纳米孔阵列芯片的结构示意图。FIG. 3 schematically shows the structure of the gold nanohole array chip in the sample loading unit of the biological detection device using the gold nanohole array chip according to a preferred embodiment of the present invention.
图4示意性地示出了根据本发明优选实施例的采用金纳米孔阵列芯片的生物检测装置的部分透视示意图。Fig. 4 schematically shows a partial perspective view of a biological detection device using a gold nanopore array chip according to a preferred embodiment of the present invention.
需要说明的是，附图用于说明本发明，而非限制本发明。注意，表示结构的附图可能并非按比例绘制。并且，附图中，相同或者类似的元件标有相同或者类似的标号。It should be noted that the drawings are used to illustrate the present invention, but not to limit the present invention. Note that the drawings representing the structure may not be drawn to scale. In addition, in the drawings, the same or similar elements are marked with the same or similar reference signs.
具体实施方式Detailed ways
为了使本发明的内容更加清楚和易懂，下面结合具体实施例和附图对本发明的内容进行详细描述。In order to make the content of the present invention clearer and easier to understand, the content of the present invention will be described in detail below with reference to specific embodiments and drawings.
图1示意性地示出了根据本发明优选实施例的采用金纳米孔阵列芯片的生物检测装置的结构框图。Fig. 1 schematically shows a structural block diagram of a biological detection device using a gold nanopore array chip according to a preferred embodiment of the present invention.
如图1所示，根据本发明优选实施例的采用金纳米孔阵列芯片的生物检测装置包括：光源10、装样单元20、光电检测器30和处理器40。As shown in FIG. 1, a biological detection device using a gold nanopore array chip according to a preferred embodiment of the present invention includes: a light source 10, a sample loading unit 20, a photodetector 30 and a processor 40.
图2意性地示出了根据本发明优选实施例的采用金纳米孔阵列芯片的生物检测装置的装样单元的结构示意图。Fig. 2 schematically shows a structural schematic diagram of a sample loading unit of a biological detection device using a gold nanopore array chip according to a preferred embodiment of the present invention.
如图3所示，装样单元20包括：用于容纳待测样品13的装样微孔11，其中在装样微孔11底部布置有金纳米孔阵列芯片12。As shown in FIG. 3, the sample loading unit 20 includes a sample loading microhole 11 for accommodating a sample 13 to be tested, wherein a gold nanohole array chip 12 is arranged at the bottom of the sample loading microhole 11.
例如，装样微孔11是96孔板。For example, the loading microwell 11 is a 96-well plate.
图3意性地示出了根据本发明优选实施例的采用金纳米孔阵列芯片的生物检测装置的装样单元中的金纳米孔阵列芯片的结构示意图。FIG. 3 schematically shows the structure of the gold nanohole array chip in the sample loading unit of the biological detection device using the gold nanohole array chip according to a preferred embodiment of the present invention.
如图3所示，金纳米孔阵列芯片12的一侧表面上形成有多个纳米孔22。在加入待测样品13后，待测样品13将进入纳米孔22。As shown in FIG. 3, a plurality of nanoholes 22 are formed on one side surface of the gold nanohole array chip 12. After adding the test sample 13, the test sample 13 will enter the nanopore 22.
优选地，多个纳米孔22未穿透金纳米孔阵列芯片12，即金纳米孔阵列芯片12是盲孔。而且优选地，纳米孔22的孔径小于200nm。Preferably, the plurality of nanoholes 22 does not penetrate the gold nanohole array chip 12, that is, the gold nanohole array chip 12 is a blind hole. And preferably, the pore diameter of the nanopore 22 is less than 200 nm.
优选地，多个纳米孔22形成为孔矩阵结构。Preferably, the plurality of nanoholes 22 are formed in a hole matrix structure.
具体地，金纳米孔阵列芯片12上面的小孔都是纳米孔(例如，孔径<200nm)，以该芯片为基底，粘合在装样微孔底部，加入待测样品，再进行检测(如酶标仪)。Specifically, the small holes on the gold nanohole array chip 12 are all nanoholes (for example, the pore size is less than 200nm), and the chip is used as a substrate, glued to the bottom of the sample loading microhole, and the sample to be tested is added, and then the detection (such as Microplate reader).
图4示意性地示出了根据本发明优选实施例的采用金纳米孔阵列芯片的生物检测装置的部分透视示意图。Fig. 4 schematically shows a partial perspective view of a biological detection device using a gold nanopore array chip according to a preferred embodiment of the present invention.
如图4所示，光源用于向加入待测样品13的装样单元20进行照射，光电检测器30接收从装样单元20传递出来的光线并且将光线传递给处理器40。As shown in FIG. 4, the light source is used to illuminate the sample loading unit 20 where the sample 13 to be tested is added, and the photodetector 30 receives the light transmitted from the sample loading unit 20 and transmits the light to the processor 40.
优选地，光电检测器30用于测定预定波长下待测样品随时间的OD(optical density，表示被检测物吸收掉的光密度)值变化曲线。例如，光电检测器30是酶标仪等检测设备。Preferably, the photodetector 30 is used to determine the OD (optical density, representing the optical density absorbed by the detected object) value change curve of the sample to be tested at a predetermined wavelength over time. For example, the photodetector 30 is a detection device such as a microplate reader.
在本发明中，金纳米孔阵列芯片作为一种新型纳米等离子光学光感器芯片，对纳米粒子的散射光具有增强作用，可以有效降低待测物的检测限。由此，本发明能有利地用于生物样品测定，具有上样量少、灵敏度高、检测上线宽、测试速度快、稳定性好等优点。In the present invention, the gold nanohole array chip, as a new type of nano-plasma optical light sensor chip, has an enhancement effect on the scattered light of the nano-particles, and can effectively reduce the detection limit of the test object. Therefore, the present invention can be advantageously used for the determination of biological samples, and has the advantages of small sample loading, high sensitivity, detection line width, fast test speed, good stability and the like.
根据本发明的另一优选实施例，还提供了一种检测方法，其采用了上述的采用金纳米孔阵列芯片的生物检测装置。According to another preferred embodiment of the present invention, a detection method is also provided, which adopts the above-mentioned biological detection device using a gold nanopore array chip.
<具体应用示例><Specific application example>
1、本申请可用于免疫比浊法检测C反应蛋白(CRP)，其采用上述实施例中描述的采用金纳米孔阵列芯片的生物检测装置。试剂包括：1. This application can be used for the immunoturbidimetric method to detect C-reactive protein (CRP), which uses the biological detection device using the gold nanopore array chip described in the above embodiment. The reagents include:
(1)第一试剂组合物，其包含电解质、促凝剂、表面活性剂、防腐剂和缓冲液；(1) The first reagent composition, which contains electrolyte, coagulant, surfactant, preservative and buffer;
(2)第二试剂组合物，不同亲和力的羊抗人CRP多克隆抗体；其中，第一试剂组合物和第二试剂组合物是免疫比浊法检测C反应蛋白的试剂盒，最终和待测物一起加入装样微孔，进行检测。(2) The second reagent composition, goat anti-human CRP polyclonal antibodies with different affinities; wherein the first reagent composition and the second reagent composition are kits for detecting C-reactive protein by immunoturbidimetric method, and the final test The materials are added to the loading microwells for testing.
(3)待测样品(3) Sample to be tested
优点：可以在不需要抗体载体颗粒的情况下，通过金纳米孔的信号放大作用，降低检测线，提高灵敏度。Advantages: The signal amplification effect of the gold nanopore can reduce the detection line and improve the sensitivity without the need for antibody carrier particles.
2、实时PCR(聚合酶链式反应)扩增监控2. Real-time PCR (polymerase chain reaction) amplification monitoring
本申请装置对不同粒径大小的复合物的散射光强具有不同程度的放大作用，基于该差异，本申请可用于实时监控DNA扩增反应，与实时荧光定量PCR技术相比，具有价格相对较低，更简便易行，减少污染，以及不受荧光信号淬灭等影响。The device of this application has different degrees of amplification for the scattered light intensity of the complexes of different particle sizes. Based on this difference, this application can be used to monitor the DNA amplification reaction in real time. Compared with real-time fluorescent quantitative PCR technology, it has a relatively low price. Low, more convenient and easy to implement, reduce pollution, and not be affected by fluorescence signal quenching.
3、淀粉样蛋白聚集监控3. Monitoring of amyloid aggregation
淀粉样蛋白通过错误折叠聚集成为淀粉样纤维,并在人体内多种组织和器官中沉积。目前主要采用ThT荧光检测技术来监控淀粉样蛋白聚集情况。本申请在淀粉样蛋白的研究中，可以实时监测淀粉样蛋白聚集的抑制作用，促聚集效果以及解聚情况，具有方便快捷、不受ThT的影响，监测灵敏，稳定性好等优点。Amyloid aggregates into amyloid fibers through misfolding and deposits in various tissues and organs in the human body. Currently, ThT fluorescence detection technology is mainly used to monitor amyloid aggregation. In the research of amyloid, the application can monitor the inhibitory effect of amyloid aggregation, the aggregation-promoting effect and the deaggregation situation in real time, and has the advantages of convenience and quickness, not affected by ThT, sensitive monitoring, good stability and the like.
4、临界聚集浓度测定4. Determination of critical aggregation concentration
两亲性的高分子在溶剂中缔合形成胶束或纳米颗粒的最低浓度即为临界胶束浓度。测量临界胶束浓度的方法很多，比较常用的有表面张力法、电导法、染料法、增溶法、渗透压法、脉冲射解法、荧光法、超声吸附法、浊度法、pH值法、流变法、离子选择性电极法和循环伏安法等。不同监测方法的优缺点：光散射法要求所测溶液非常干净，受限制因素较多；而电导法只适于测量离子 型表面活性剂的CMC；荧光探针法测量CMC，虽然受限制因素少，但是需要配置芘探针的苯溶液，且苯有毒性而且操作过程繁琐；染料法测量CMC所需的设备简单，但是因颜色变化不够明显，会影响CMC的准确度；浊度法也因所用烃溶解物影响表面活性剂临界胶束浓度的问题，所测CMC的准确度受到影响。本申请装置具有光信号放大作用，可以更精确的测定出不同两亲性分子的临界聚集浓度，区分不同分子之间的微小差异。The minimum concentration of amphiphilic polymers that associate to form micelles or nanoparticles in a solvent is the critical micelle concentration. There are many methods for measuring the critical micelle concentration. The more commonly used methods are surface tension method, conductivity method, dye method, solubilization method, osmotic pressure method, pulse radiolysis method, fluorescence method, ultrasonic adsorption method, turbidity method, pH value method, Rheological method, ion selective electrode method and cyclic voltammetry, etc. The advantages and disadvantages of different monitoring methods: the light scattering method requires the measured solution to be very clean, and there are many limiting factors; the conductivity method is only suitable for measuring the CMC of ionic surfactants; the fluorescent probe method is used to measure CMC, although there are few limiting factors , But need to configure the benzene solution of the pyrene probe, and the benzene is toxic and the operation process is cumbersome; the equipment needed to measure CMC by the dye method is simple, but the color change is not obvious enough, which will affect the accuracy of the CMC; the turbidity method also depends on the used The problem that hydrocarbon dissolved matter affects the critical micelle concentration of surfactants affects the accuracy of the measured CMC. The device of the present application has a light signal amplification function, and can more accurately determine the critical aggregation concentration of different amphiphilic molecules, and distinguish small differences between different molecules.
5、重金属离子检测5. Heavy metal ion detection
重金属元素的检测方法有光度法、比浊法、斑点比较法、色谱法、光谱法、电化学分析法、中子活化分析等。其中可利用化学沉淀或生物絮凝的方法处理的微量重金属均可采用本申请装置进行检测，具有检测灵敏，操作简单等优点。可用于水质检测，食品药品中的微量重金属检测。The detection methods of heavy metal elements include photometry, turbidimetry, spot comparison method, chromatography, spectroscopy, electrochemical analysis, neutron activation analysis, etc. Among them, the trace heavy metals that can be processed by chemical precipitation or biological flocculation can be detected by the device of the present application, which has the advantages of sensitive detection, simple operation and the like. It can be used for water quality detection and trace heavy metal detection in food and medicine.
6、病毒检测6. Virus detection
本申请装置对纳米粒子具有生物兼容性和信号放大作用，可结合免疫反应的特异性检测技术来检测生物病毒，用于病毒性疾病的防控。The device of the present application has biocompatibility and signal amplification effects on nanoparticles, and can be combined with specific detection technology of immune response to detect biological viruses for the prevention and control of viral diseases.
需要说明的是，除非特别指出，否则说明书中的术语“第一”、“第二”、“第三”等描述仅仅用于区分说明书中的各个组件、元素、步骤等，而不是用于表示各个组件、元素、步骤之间的逻辑关系或者顺序关系等。It should be noted that, unless otherwise specified, the terms "first", "second", "third" and other descriptions in the specification are only used to distinguish each component, element, step, etc. in the specification, rather than to indicate The logical relationship or sequence relationship between various components, elements, and steps.
可以理解的是，虽然本发明已以较佳实施例披露如上，然而上述实施例并非用以限定本发明。对于任何熟悉本领域的技术人员而言，在不脱离本发明技术方案范围情况下，都可利用上述揭示的技术内容对本发明技术方案作出许多可能的变动和修饰，或修改为等同变化的等效实施例。因此，凡是未脱离本发明技术方案的内容，依据本发明的技术实质对以上实施例所做的任何简单修改、等同变化及修饰，均仍属于本发明技术方案保护的范围内。It can be understood that although the present invention has been disclosed as above in preferred embodiments, the above-mentioned embodiments are not intended to limit the present invention. For any person skilled in the art, without departing from the scope of the technical solution of the present invention, the technical content disclosed above can be used to make many possible changes and modifications to the technical solution of the present invention, or modified into equivalent changes. Examples. Therefore, any simple modifications, equivalent changes and modifications made to the above embodiments without departing from the technical solution of the present invention based on the technical essence of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims (9)

  1. 一种采用金纳米孔阵列芯片的生物检测装置，其特征在于包括：光源、装样单元、光电检测器和处理器；其中，装样单元包括：用于容纳待测样品的装样微孔，其中在装样微孔底部布置有金纳米孔阵列芯片；金纳米孔阵列芯片的一侧表面上形成有多个纳米孔；光源用于向加入待测样品的装样单元进行照射，光电检测器接收从装样单元传递出来的光线并且将光线传递给处理器。A biological detection device adopting a gold nanohole array chip, which is characterized by comprising: a light source, a sample loading unit, a photodetector, and a processor; wherein the sample loading unit includes: a sample loading micropore for accommodating a sample to be tested, A gold nanohole array chip is arranged at the bottom of the sample loading microhole; a plurality of nanoholes are formed on one side surface of the gold nanohole array chip; the light source is used to illuminate the sample loading unit with the sample to be tested, and the photodetector Receive the light transmitted from the sample loading unit and transmit the light to the processor.
  2. 根据权利要求1所述的采用金纳米孔阵列芯片的生物检测装置，其特征在于，光电检测器用于测定预定波长下待测样品随时间的OD值变化曲线。The biological detection device using a gold nanopore array chip according to claim 1, wherein the photodetector is used to measure the OD value change curve of the sample to be tested over time at a predetermined wavelength.
  3. 根据权利要求1或2所述的采用金纳米孔阵列芯片的生物检测装置，其特征在于，光电检测器是酶标仪等检测设备。The biological detection device using a gold nanopore array chip according to claim 1 or 2, wherein the photodetector is a detection device such as a microplate reader.
  4. 根据权利要求1或2所述的采用金纳米孔阵列芯片的生物检测装置，其特征在于，多个纳米孔未穿透金纳米孔阵列芯片。The biological detection device using the gold nanohole array chip according to claim 1 or 2, wherein the plurality of nanoholes do not penetrate the gold nanohole array chip.
  5. 根据权利要求1或2所述的采用金纳米孔阵列芯片的生物检测装置，其特征在于，多个纳米孔形成为孔矩阵结构。The biological detection device using a gold nanohole array chip according to claim 1 or 2, wherein the plurality of nanoholes are formed in a hole matrix structure.
  6. 根据权利要求1或2所述的采用金纳米孔阵列芯片的生物检测装置，其特征在于，纳米孔的孔径小于0nm。The biological detection device using a gold nanopore array chip according to claim 1 or 2, wherein the pore diameter of the nanopore is less than 0 nm.
  7. 根据权利要求1或2所述的采用金纳米孔阵列芯片的生物检测装置，其特征在于，装样微孔是96孔板。The biological detection device using the gold nanopore array chip according to claim 1 or 2, wherein the sample loading microwells are 96-well plates.
  8. 根据权利要求1或2所述的采用金纳米孔阵列芯片的生物检测装置，其特征在于，所述生物检测装置用于C反应蛋白检测、实时PCR扩增监控、淀粉样蛋白聚集监控、临界聚集浓度测定、重金属离子检测和病毒检测。The biological detection device using gold nanopore array chip according to claim 1 or 2, wherein the biological detection device is used for C-reactive protein detection, real-time PCR amplification monitoring, amyloid aggregation monitoring, critical aggregation Concentration determination, heavy metal ion detection and virus detection.
  9. 一种检测方法，其特征在于采用了根据权利要求1至8之一所述的采用金纳米孔阵列芯片的生物检测装置。A detection method, characterized in that the biological detection device using gold nanohole array chip according to any one of claims 1 to 8 is used.
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