WO2021098142A1 - 激光微阵列芯片扫描仪的浓度梯度荧光校准片及校准方法 - Google Patents

激光微阵列芯片扫描仪的浓度梯度荧光校准片及校准方法 Download PDF

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WO2021098142A1
WO2021098142A1 PCT/CN2020/086375 CN2020086375W WO2021098142A1 WO 2021098142 A1 WO2021098142 A1 WO 2021098142A1 CN 2020086375 W CN2020086375 W CN 2020086375W WO 2021098142 A1 WO2021098142 A1 WO 2021098142A1
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inorganic nano
fluorescent liquid
fluorescent
nano fluorescent
concentration gradient
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PCT/CN2020/086375
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English (en)
French (fr)
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安爽
张冠斌
梁冬
赵淑英
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成都博奥独立医学实验室有限公司
绵阳市人民医院
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Priority to US17/595,036 priority Critical patent/US11946868B2/en
Priority to JP2021572844A priority patent/JP7280390B2/ja
Publication of WO2021098142A1 publication Critical patent/WO2021098142A1/zh

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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
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • 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
    • G01N21/274—Calibration, base line adjustment, drift correction
    • G01N21/278—Constitution of standards
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell plates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N2021/6463—Optics

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  • the invention belongs to the technical field of biochip production equipment, and relates to a concentration gradient fluorescence calibration sheet and a calibration method of a laser microarray chip scanner for calibrating the linear range, sensitivity and resolution of a red and green dual laser microarray chip scanner. .
  • the microarray chip scanner is an indispensable and extremely important detection instrument for microarray chip scanning. Because the light source, light path and detection system of the scanner will change slightly with the extension of the time of use, this will often bring about measurement errors.
  • the fluorescence detection method has extremely high sensitivity and is extremely small when used in trace detection. Errors may lead to completely opposite conclusions, so it is extremely necessary to prepare a concentration gradient calibration sheet for the calibration of microarray chip scanners.
  • the materials commonly used for fluorescence calibration mainly include organic fluorescent materials, polymer-doped organic fluorescent materials, polymer materials, inorganic materials doped with organic fluorescent dyes, and inorganic ion-doped inorganic solid materials.
  • organic fluorescent materials polymer-doped organic fluorescent materials
  • polymer materials polymer materials
  • inorganic materials doped with organic fluorescent dyes and inorganic ion-doped inorganic solid materials.
  • the purpose of the present invention is to overcome the problems existing in the prior art, and provide a concentration gradient fluorescence calibration sheet and calibration method for a laser microarray chip scanner with simple preparation method, short production cycle and low cost.
  • concentration gradient fluorescence calibration sheet for a laser microarray chip scanner, the concentration gradient fluorescence calibration sheet including a glass substrate and an inorganic nano-fluorescent liquid dot matrix arranged on the surface of the glass substrate;
  • the inorganic nano fluorescent liquid dot matrix includes a plurality of inorganic nano fluorescent liquid sub-dot matrices, and the inorganic fluorescent mixed liquid droplets of each inorganic nano fluorescent liquid sub-dot matrix are arranged in A row ⁇ B column.
  • the solution concentration of the inorganic nano fluorescent liquid droplets in the same row is the same, and the solution concentration of the inorganic nano fluorescent liquid droplets in two adjacent rows changes in a multiple relationship, where A is 5-20 and B is 10-40.
  • the glass substrate is a glass substrate with a chemically modified surface or a glass substrate coated with a polymer film on the surface.
  • the inorganic nano fluorescent liquid dot matrix includes 1 to 8 inorganic nano fluorescent liquid sub-dot matrices, and each sub-dot matrix is evenly distributed and arranged on the glass. On the substrate.
  • the matrix spacing of adjacent inorganic nano fluorescent liquid sub-lattices is the same, and the dot pitch of the inorganic nano fluorescent liquid sub-lattices is the same and greater than or Equal to 350 microns.
  • the solution concentration of the first row of inorganic nano fluorescent liquid droplets in the inorganic nano fluorescent liquid sub-array and the penultimate row of inorganic nano fluorescent liquid is the highest concentration. From the concentration of the inorganic nano-fluorescent droplets in the second row to the second-to-last row of the inorganic nano-fluorescent droplets, there is a linear relationship with a positive multiple of increase, and the sensitivity is less than Or equal to 12 concentration gradients.
  • the fluorescence point signal value of the first row of inorganic nano fluorescent droplets and the penultimate row of inorganic nano fluorescent droplets is 65535
  • the fluorescent point signal value of the inorganic nano fluorescent liquid droplet in the second row is 1000-3000
  • the fluorescent point signal value of the inorganic nano fluorescent liquid droplet in the penultimate row is 60000-65535.
  • Another aspect of the present invention provides a method for calibrating a laser microarray chip scanner, which uses the concentration gradient fluorescence calibration sheet of the laser microarray chip scanner to calibrate the scanner under test and divide the dot matrix with inorganic nano fluorescent liquid.
  • the inorganic nano-fluorescent droplet in the first row in the middle is used as an index mark.
  • the fluorescent point signal value of each inorganic nano fluorescent liquid droplet in the inorganic nano fluorescent liquid sub-array is used as a single measurement value of the corresponding solution concentration.
  • the log value of the fluorescence point signal value corresponding to each inorganic nano fluorescent liquid droplet is taken as the abscissa; in order to scan the concentration gradient fluorescence with the scanner under test
  • the logarithm of the signal value of the fluorescent point corresponding to each inorganic nano fluorescent liquid droplet is plotted on the ordinate, and the correlation coefficient R 2 of the linear relationship of the curve is investigated.
  • each scanner of the machine to be tested is calibrated with at least 3 sets of different scanning parameters, when the correlation coefficient R 2 of each set of scanning parameters is greater than 0.90 , It means that the calibration result of the scanner of the machine under test is normal and can be used normally.
  • the present invention provides a concentration gradient fluorescence calibration sheet for a laser microarray chip scanner and a calibration method thereof.
  • the preparation method is simple to operate, has a short production cycle, and is low in cost.
  • the calibration effect is good and has good calibration results. Prospects for promotion and application.
  • Fig. 1 shows a scanning diagram of a non-polar nano fluorescent liquid dot matrix in a concentration gradient fluorescence calibration sheet of a laser microarray chip scanner according to an exemplary embodiment of the present invention.
  • Fig. 2 shows a scanning diagram of a non-polar nano fluorescent liquid sub-array in a concentration gradient fluorescence calibration sheet of a laser microarray chip scanner according to an exemplary embodiment of the present invention.
  • FIG. 3 shows the calibration linear relationship diagram of the first set of parameters in Embodiment 1.
  • FIG. 4 shows the calibration linear relationship diagram of the second set of parameters in Embodiment 1.
  • FIG. 5 shows the calibration linear relationship diagram of the third set of parameters in Example 1.
  • the concentration gradient fluorescence calibration sheet of the laser microarray chip scanner includes a glass substrate and an inorganic nano fluorescent liquid lattice arranged on the surface of the glass substrate.
  • the inorganic nano fluorescent liquid used in the present invention preferably adopts the patented product of Boao Company-Nanobrite inorganic nano fluorescent material, which has fluorescence lifetime, sharp emission spectrum, very stable luminescence (no photobleaching, no flicker), Stokes and anti-flicker.
  • Stokes has the characteristics of large displacement, high chemical stability and low biological toxicity.
  • Chinese Patent CN1231760C “Rare Earth Nanoparticles for Biomaterial Marking, Preparation Method and Use thereof"
  • the present invention is not limited to this.
  • the glass substrate is a glass substrate whose surface is chemically modified or a glass substrate whose surface is coated with a polymer film.
  • the glass substrate with chemically modified surface or the glass substrate coated with polymer film is placed horizontally, and the inorganic nano fluorescent liquid is sprayed on the glass substrate to form an inorganic nano fluorescent liquid dot matrix.
  • FIG. 1 shows a scanning diagram of the electrodeless nano-fluorescent liquid dot matrix in a concentration gradient fluorescence calibration sheet of a laser microarray chip scanner according to an exemplary embodiment of the present invention
  • FIG. 2 shows a laser microarray according to an exemplary embodiment of the present invention Scanning image of the non-polar nano-fluorescence liquid sub-dot matrix in the concentration gradient fluorescence calibration sheet of the chip scanner.
  • the inorganic nano fluorescent liquid dot matrix includes several inorganic nano fluorescent liquid sub-dot matrices, and the inorganic fluorescent mixed liquid droplets of each inorganic nano fluorescent liquid sub-dot matrix are arranged in A row ⁇ B column.
  • A is 5-20
  • B is 10-40.
  • the inorganic nano fluorescent liquid dot matrix includes 1 to 8 inorganic nano fluorescent liquid sub-dot matrices, and each sub-dot matrix is evenly distributed and arranged on the glass substrate.
  • the matrix spacing of adjacent inorganic nano fluorescent liquid sub-lattices is controlled to be the same, and the dot pitch of the inorganic nano fluorescent liquid sub-lattices is the same and greater than or equal to 350 micrometers.
  • the solution concentration of the inorganic nanofluorescent liquid droplets in the first row of the inorganic nanofluorescent liquid sub-array and the solution concentration of the inorganic nanofluorescent liquid droplets in the penultimate row are both the highest concentration.
  • the solution concentration of the droplets is in a linear relationship of positive multiples increasing to the solution concentration of the inorganic nano fluorescent droplets in the penultimate row, and the concentration gradient of the solution in the same inorganic nano fluorescent liquid dot matrix is less than or equal to 12 concentration gradients.
  • the fluorescent point signal value of the inorganic nano fluorescent liquid droplet in the first row and the last one row of inorganic nano fluorescent liquid droplet is the highest value of 65535
  • the fluorescent dot signal value of the inorganic nano fluorescent liquid droplet in the second row is the highest value.
  • the signal value is 1000-3000
  • the fluorescent point signal value of the inorganic nano-fluorescent liquid droplet in the penultimate row is 60000-65535. Since the concentration of the inorganic nano-fluorescent liquid used may be different according to different detection needs, the change ratio and the specific concentration cannot be directly limited, and the range of the limit value of the fluorescence dot is used here for limitation.
  • the present invention provides a method for calibrating a laser microarray chip scanner.
  • the concentration gradient fluorescence calibration sheet of the above-mentioned laser microarray chip scanner is used to calibrate the scanner under test and use inorganic nano-fluorescent liquid.
  • the inorganic nano fluorescent droplets in the first row of the dot matrix are used as index marks.
  • the fluorescent point signal value of each inorganic nano fluorescent liquid droplet in the inorganic nano fluorescent liquid sub-array is used as a single measurement value of the corresponding solution concentration.
  • the concentration gradient fluorescent calibration sheet with a standard machine scanner
  • the The logarithm of the fluorescent point signal value corresponding to each inorganic nano fluorescent droplet is the abscissa; after scanning the concentration gradient fluorescent calibration sheet with the scanner of the machine to be tested, it corresponds to the fluorescence of each inorganic nano fluorescent droplet.
  • the logarithm of the point signal value is plotted on the ordinate, and the correlation coefficient R 2 of the linear relationship of the curve is investigated.
  • At least 3 sets of different scanning parameters are used for calibration for each scanner of the machine under test.
  • the correlation coefficient R 2 of each set of scanning parameters is greater than 0.90, it means that the scanner of the machine under test has a normal calibration result and can be used normally. Otherwise, it is abnormal and cannot be used.
  • the glass substrate of the calibration sheet is a glass substrate with a chemically modified surface, and its size is 75.6 ⁇ 25mm.
  • an inorganic nano-fluorescent liquid is arranged in a dot matrix.
  • the inorganic fluorescent liquid is made of Boao's patent. Product-Nanobrite inorganic nano fluorescent material. Place the chemically modified glass substrate or the glass substrate coated with a polymer film horizontally, and spray the inorganic nano fluorescent liquid on the glass substrate. The inorganic nano fluorescent liquid is uniformly distributed and arranged on the glass substrate in the form of a 5-dot matrix.
  • Each sub-lattice is a 12-row ⁇ 12-column matrix, and the spacing between adjacent sub-lattices is the same, and the dot-spacing of the sub-lattices is the same and greater than or equal to 350 microns.
  • the solution concentration of the inorganic nano fluorescent droplets of each sub-array is the same in the same row. Starting from the second row, the concentration of the fluorescent dye contained in each row increases sequentially, and the inorganic nano fluorescent droplets in the first row are used as the index mark, namely The preparation of the concentration gradient sheet for calibration and measurement of the red and green dual laser microarray chip scanner is completed.
  • the solution concentration multiple relationship of the inorganic nanofluorescent liquid dot matrix is that the first row and the 12th row are both the highest concentration (fluorescence signal value is 65535), and the second row (fluorescence signal value range is 1000 ⁇ 3000) ⁇ Line 11 (fluorescence signal value range is 60000 ⁇ 65535)
  • the solution concentration value of fluorescent liquid droplets is in a linear relationship with a positive multiple of increase.
  • the correlation coefficient of the linear relationship is R 2 >0.90, and the fluorescent point signal value of each inorganic fluorescent mixed liquid droplet is taken as the single measurement value of the corresponding concentration.
  • the logarithm of the fluorescence point signal value of each inorganic fluorescent mixed liquid droplet is the abscissa; after scanning the calibration sheet with the scanner under test, it corresponds to the logarithmic value of the fluorescence point signal value of each inorganic fluorescent mixed liquid droplet Plot the ordinate to investigate the correlation coefficient R 2 of the linear relationship of the curve.
  • the standard scanner and the scanner under test to scan with the following three sets of scanning parameters: the first group: laser intensity -50%, PMT-650; the second group: laser intensity -60%, PMT-650; Three groups: laser intensity -70%, PMT-650.
  • the correlation coefficient R 2 of the three sets of scanning parameters used by the scanner under test is greater than 0.90, indicating that the scanner can be used with normal calibration results.
  • the present invention is not limited to the foregoing specific embodiments.
  • the present invention extends to any new feature or any new combination disclosed in this specification, and any new method or process step or any new combination disclosed.

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Abstract

一种激光微阵列芯片扫描仪的浓度梯度荧光校准片及其校准方法，浓度梯度荧光校准片包括玻璃基片和布置在玻璃基片表面的无机纳米荧光液点阵；无机纳米荧光液点阵包括若干个无机纳米荧光液分点阵，每个无机纳米荧光液分点阵的无机荧光混合液液滴呈A行×B列排列，无机纳米荧光液分点阵中同一行无机纳米荧光液液滴的溶液浓度相同且相邻两行无机纳米荧光液液滴的溶液浓度呈倍数关系变化，其中，A为5～20，B为10～40。该校准方法使用该激光微阵列芯片扫描仪的浓度梯度荧光校准片对待测扫描仪进行校准并以无机纳米荧光液分点阵中第1行的无机纳米荧光液滴作为索引标志。制备方法操作简单、生产周期短且成本低廉，校准效果好。

Description

激光微阵列芯片扫描仪的浓度梯度荧光校准片及校准方法 技术领域
本发明属于生物芯片生产设备技术领域，涉及一种用于校准红、绿双激光微阵列芯片扫描仪的线性范围、灵敏度和分辨力的激光微阵列芯片扫描仪的浓度梯度荧光校准片及校准方法。
背景技术
随着生物技术的迅速发展，电子技术和生物技术相结合诞生了半导体芯片的兄弟产品——生物芯片，这将给我们的生活带来一场深刻的革命，这场革命对于全世界的可持续发展都会起到不可估量的贡献。
我国生物芯片研究始于1997-1998年间，尽管起步较晚，但是技术和产业发展迅速，实现了从无到有的阶段性突破，并逐步发展壮大，生物芯片已经从技术研究和产品开发阶段走向技术应用和产品销售阶段，在表达谱芯片、重大疾病诊断芯片和生物芯片的相关设备研制上取得了较大成就。2008年我国生物芯片市场约为1亿美元，并正以20％以上的速度增长，至2020年生物芯片市场将达到9亿美元。
其中，微阵列芯片扫描仪正是用于微阵列芯片扫描的必不可少且发挥着极其重要作用的检测仪器。由于扫描仪的光源、光路及检测系统随使用时间的延长会出现细微的变化，这往往会带来测量误差，而荧光检测法对灵敏度极高，在用于痕量检测中时极小的检测误差可能导致完全相反的结论，因此制备浓度梯度校准片用于微阵列芯片扫描仪的校准就显得极为必要。
目前，常用作荧光校准的材料主要有有机荧光材料、聚合物掺杂有机荧光 材料、高分子材料、无机材料掺杂有机荧光染料、无机离子掺杂无机固体材料等。无机荧光材料制作校准片虽然操作有些复杂、成本略高，但由于其特殊的抗光漂白特性，在长时间激光照射下其荧光发射强度可保持恒定，且重复性和一致性好的特点。随着生物芯片市场的不断扩大，扫描仪的需求也越来越大，有必要提供一种制作简单、成本低廉的浓度梯度荧光校准片使其更加适合推广应用。
发明内容
本发明的目的在于克服现有技术中存在的问题，提供一种制备方法操作简单、生产周期短且成本低廉的激光微阵列芯片扫描仪的浓度梯度荧光校准片及校准方法。
本发明的一方面提供了激光微阵列芯片扫描仪的浓度梯度荧光校准片，所述浓度梯度荧光校准片包括玻璃基片和布置在玻璃基片表面的无机纳米荧光液点阵；
所述无机纳米荧光液点阵包括若干个无机纳米荧光液分点阵，每个无机纳米荧光液分点阵的无机荧光混合液液滴呈A行×B列排列，所述无机纳米荧光液分点阵中同一行无机纳米荧光液液滴的溶液浓度相同且相邻两行无机纳米荧光液液滴的溶液浓度呈倍数关系变化，其中，A为5～20，B为10～40。
根据本发明激光微阵列芯片扫描仪的浓度梯度荧光校准片的一个实施例，所述玻璃基片为表面进行化学修饰的玻璃基片或表面涂覆有高分子膜的玻璃基片。
根据本发明激光微阵列芯片扫描仪的浓度梯度荧光校准片的一个实施例，所述无机纳米荧光液点阵包括1～8个无机纳米荧光液分点阵，各分点阵均匀分 布排列于玻璃基片上。
根据本发明激光微阵列芯片扫描仪的浓度梯度荧光校准片的一个实施例，相邻无机纳米荧光液分点阵的阵间距相同，所述无机纳米荧光液分点阵的点间距相同且大于或等于350微米。
根据本发明激光微阵列芯片扫描仪的浓度梯度荧光校准片的一个实施例，所述无机纳米荧光液分点阵中第1行无机纳米荧光液液滴的溶液浓度和倒数第1行无机纳米荧光液液滴的溶液浓度均为最高浓度，从第2行无机纳米荧光液液滴的溶液浓度至倒数第2行无机纳米荧光液液滴的溶液浓度呈正数倍比增加的线性关系，且灵敏度小于或等于12个浓度梯度。
根据本发明激光微阵列芯片扫描仪的浓度梯度荧光校准片的一个实施例，所述第1行无机纳米荧光液液滴和倒数第1行无机纳米荧光液液滴的荧光点信号值为65535，所述第2行无机纳米荧光液液滴的荧光点信号值为1000～3000，所述倒数第2行无机纳米荧光液液滴的荧光点信号值为60000～65535。
本发明的另一方面提供了一种激光微阵列芯片扫描仪的校准方法，使用上述激光微阵列芯片扫描仪的浓度梯度荧光校准片对待测机扫描仪进行校准并以无机纳米荧光液分点阵中第1行的无机纳米荧光液滴作为索引标志。
根据本发明激光微阵列芯片扫描仪的校准方法的一个实施例，将无机纳米荧光液分点阵中每个无机纳米荧光液液滴的荧光点信号值作为相应溶液浓度的单次测量值，使用标准机扫描仪扫描所述浓度梯度荧光校准片后，将其对应每个无机纳米荧光液液滴的荧光点信号值的对数值为横坐标；以使用待测机扫描仪扫描所述浓度梯度荧光校准片后，将其对应每个无机纳米荧光液液滴的荧光点信号值的对数值为纵坐标作图，考察得到曲线的线性关系的相关系数R 2。
根据本发明激光微阵列芯片扫描仪的校准方法的一个实施例，对每台待测 机扫描仪采用至少3组不同的扫描参数进行校准，当每组扫描参数的相关系数R 2均大于0.90时，则说明该台待测机扫描仪校准结果正常并且能够正常使用。
与现有技术相比，本发明提供了一种激光微阵列芯片扫描仪的浓度梯度荧光校准片及其校准方法，其制备方法操作简单、生产周期短且成本低廉，校准效果较好且具有良好的推广应用前景。
附图说明
图1示出了根据本发明示例性实施例激光微阵列芯片扫描仪的浓度梯度荧光校准片中无极纳米荧光液点阵的扫描图。
图2示出了根据本发明示例性实施例激光微阵列芯片扫描仪的浓度梯度荧光校准片中无极纳米荧光液分点阵的扫描图。
图3示出了实施例1中第一组参数的校准线性关系图。
图4示出了实施例1中第二组参数的校准线性关系图。
图5示出了实施例1中第三组参数的校准线性关系图。
具体实施方式
本说明书中公开的所有特征，或公开的所有方法或过程中的步骤，除了互相排斥的特征和/或步骤以外，均可以以任何方式组合。
本说明书中公开的任一特征，除非特别叙述，均可被其他等效或具有类似目的的替代特征加以替换。即，除非特别叙述，每个特征只是一系列等效或类似特征中的一个例子而已。
根据本发明的示例性实施例，所述激光微阵列芯片扫描仪的浓度梯度荧光校准片包括玻璃基片和布置在玻璃基片表面的无机纳米荧光液点阵。其中，本发明采用的无机纳米荧光液优选地采用博奥公司专利产品——Nanobrite无机 纳米荧光材料，其具有荧光寿命、发射谱尖锐、发光非常稳定(无光漂白、无闪烁)、Stokes和反Stokes位移大、化学稳定性高和生物毒性低等特点，详见中国专利CN1231760C《用于生物材料标记的稀土纳米粒子、其制备方法及用途》，但本发明不限于此。
优选地，玻璃基片为表面进行化学修饰的玻璃基片或表面涂覆有高分子膜的玻璃基片。制备时，将表面化学修饰的玻璃基片或表面涂有高分子膜的玻璃基片水平放置，在玻璃基片上喷涂无机纳米荧光液形成无机纳米荧光液点阵，
图1示出了根据本发明示例性实施例激光微阵列芯片扫描仪的浓度梯度荧光校准片中无极纳米荧光液点阵的扫描图，图2示出了根据本发明示例性实施例激光微阵列芯片扫描仪的浓度梯度荧光校准片中无极纳米荧光液分点阵的扫描图。
如图1和图2所示，无机纳米荧光液点阵包括若干个无机纳米荧光液分点阵，每个无机纳米荧光液分点阵的无机荧光混合液液滴呈A行×B列排列，其中，A为5～20，B为10～40。优选地，无机纳米荧光液点阵包括1～8个无机纳米荧光液分点阵，各分点阵均匀分布排列于玻璃基片上。在制备时，控制相邻无机纳米荧光液分点阵的阵间距相同，无机纳米荧光液分点阵的点间距相同且大于或等于350微米。
其中，需控制无机纳米荧光液分点阵中同一行无机纳米荧光液液滴的溶液浓度相同且相邻两行无机纳米荧光液液滴的溶液浓度呈倍数梯度递增或递减关系变化。
优选地，无机纳米荧光液分点阵中第1行无机纳米荧光液液滴的溶液浓度和倒数第1行无机纳米荧光液液滴的溶液浓度均为最高浓度，从第2行无机纳米荧光液液滴的溶液浓度至倒数第2行无机纳米荧光液液滴的溶液浓度呈正数 倍比递增的线性关系，且同一无机纳米荧光液分点阵中溶液的浓度梯度小于或等于12个浓度梯度。
根据本发明的一个实施例，第1行无机纳米荧光液液滴和倒数第1行无机纳米荧光液液滴的荧光点信号值为最高值65535，第2行无机纳米荧光液液滴的荧光点信号值为1000～3000，倒数第2行无机纳米荧光液液滴的荧光点信号值为60000～65535。由于使用的无机纳米荧光液浓度可能根据检测需要不同而不同，因此变化倍数和具体浓度无法直接限定，此处采用荧光点限号值的范围进行限定。
基于上述浓度梯度荧光校准片，本发明提供了激光微阵列芯片扫描仪的校准方法，具体使用上述激光微阵列芯片扫描仪的浓度梯度荧光校准片对待测机扫描仪进行校准并以无机纳米荧光液分点阵中第1行的无机纳米荧光液滴作为索引标志。
实际校准时，将无机纳米荧光液分点阵中每个无机纳米荧光液液滴的荧光点信号值作为相应溶液浓度的单次测量值，使用标准机扫描仪扫描浓度梯度荧光校准片后，将其对应每个无机纳米荧光液液滴的荧光点信号值的对数值为横坐标；以使用待测机扫描仪扫描浓度梯度荧光校准片后，将其对应每个无机纳米荧光液液滴的荧光点信号值的对数值为纵坐标作图，考察得到曲线的线性关系的相关系数R 2。
对每台待测机扫描仪采用至少3组不同的扫描参数进行校准，当每组扫描参数的相关系数R 2均大于0.90时，则说明该台待测机扫描仪校准结果正常并且能够正常使用，反之则不正常且不能使用。
下面结合具体实施例对本发明进行进一步的说明，但本发明保护范围不受制于本发明的实施例。
实施例：12浓度(行)×12列浓度梯度荧光校准片的制备
该校准片的玻璃基片为表面做化学修饰的玻璃基片，其尺寸为75.6×25mm，在校准片的表面上呈点阵布置无机纳米荧光液，该无机荧光液采是用博奥公司专利产品－Nanobrite无机纳米荧光材料。将表面化学修饰的玻璃基片或表面涂有高分子膜的玻璃基片水平放置，在玻璃基片上喷涂无机纳米荧光液，无机纳米荧光液呈5点阵的形式在玻璃基片上均匀分布排列，每个分点阵为12行×12列矩阵，相邻分点阵的阵间距相同，分点阵的点间距相同且大于或等于350微米。各分点阵的无机纳米荧光液液滴的溶液浓度同一行均相同，从第2行开始每行所含有的荧光染料浓度依次递增，以第1行的无机纳米荧光液滴作为索引标志，即完成用于红、绿双激光微阵列芯片扫描仪校准测量的浓度梯度片的制备。
具体地，无机纳米荧光液分点阵的溶液浓度倍数关系是第1行和第12行均为最高浓度(荧光信号值为65535)，第2行(荧光信号值范围为1000～3000)～第11行(荧光信号值范围为60000～65535)荧光液液滴的溶液浓度值呈正数倍比增加的线性关系。
所述线性关系的相关系数R 2﹥0.90，取每个无机荧光混合液液滴的荧光点信号值作为相应浓度的单次测量值，使用标准机扫描仪扫描该片校准片后，其对应每个无机荧光混合液液滴的荧光点信号值的对数值为横坐标；使用待测机扫描仪扫描该片校准片后，其对应每个无机荧光混合液液滴的荧光点信号值的对数值为纵坐标作图，考察得到曲线的线性关系的相关系数R 2。
使用该浓度梯度荧光校准片对扫描仪进行校准具体操作如下：
使用标准机扫描仪和待测机扫描仪分别用以下三组扫描参数进行扫描：第一组：激光强度-50％，PMT-650；第二组：激光强度-60％，PMT-650；第三组： 激光强度-70％，PMT-650。
在三组扫描参数所提取的荧光点信号值中剔除信号为饱和的荧光点(信号值为65535)，具体数据如下表1所示。
以使用标准机扫描仪扫描该片校准片后，其对应每个荧光点信号值的对数值为横坐标；以使用待测机扫描仪扫描该片校准片后，其对应每个荧光点信号值的对数值为纵坐标作图，考察得到曲线的线性关系的相关系数R 2，具体数据图像如图1-图3所示。
根据图1至图3可知，该台待测机扫描仪使用3组扫描参数的相关系数R 2均大于0.90，说明此台扫描仪校准结果正常方可使用。
表1 实施例中三组扫描参数的荧光点信号值
Figure PCTCN2020086375-appb-000001
Figure PCTCN2020086375-appb-000002
Figure PCTCN2020086375-appb-000003
Figure PCTCN2020086375-appb-000004
Figure PCTCN2020086375-appb-000005
Figure PCTCN2020086375-appb-000006
Figure PCTCN2020086375-appb-000007
Figure PCTCN2020086375-appb-000008
Figure PCTCN2020086375-appb-000009
Figure PCTCN2020086375-appb-000010
Figure PCTCN2020086375-appb-000011
本发明并不局限于前述的具体实施方式。本发明扩展到任何在本说明书中披露的新特征或任何新的组合，以及披露的任一新的方法或过程的步骤或任何新的组合。

Claims (9)

  1. 一种激光微阵列芯片扫描仪的浓度梯度荧光校准片，其特征在于，所述浓度梯度荧光校准片包括玻璃基片和布置在玻璃基片表面的无机纳米荧光液点阵；
    所述无机纳米荧光液点阵包括若干个无机纳米荧光液分点阵，每个无机纳米荧光液分点阵的无机荧光混合液液滴呈A行×B列排列，所述无机纳米荧光液分点阵中同一行无机纳米荧光液液滴的溶液浓度相同且相邻两行无机纳米荧光液液滴的溶液浓度呈倍数梯度递增或递减关系变化，其中，A为5～20，B为10～40。
  2. 根据权利要求1所述激光微阵列芯片扫描仪的浓度梯度荧光校准片，其特征在于，所述玻璃基片为表面进行化学修饰的玻璃基片或表面涂覆有高分子膜的玻璃基片。
  3. 根据权利要求1所述激光微阵列芯片扫描仪的浓度梯度荧光校准片，其特征在于，所述无机纳米荧光液点阵包括1～8个无机纳米荧光液分点阵，各分点阵均匀分布排列于玻璃基片上。
  4. 根据权利要求1或3所述激光微阵列芯片扫描仪的浓度梯度荧光校准片，其特征在于，相邻无机纳米荧光液分点阵的阵间距相同，所述无机纳米荧光液分点阵的点间距相同且大于或等于350微米。
  5. 根据权利要求1所述激光微阵列芯片扫描仪的浓度梯度荧光校准片，其特征在于，所述无机纳米荧光液分点阵中第1行无机纳米荧光液液滴的溶液浓度和倒数第1行无机纳米荧光液液滴的溶液浓度均为最高浓度，从第2行无机纳米荧光液液滴的溶液浓度至倒数第2行无机纳米荧光液液滴的溶液浓度呈正数倍比递增的线性关系，且同一无机纳米荧光液分点阵中溶液的浓度梯度小于或等于12个浓度梯度。
  6. 根据权利要求5所述激光微阵列芯片扫描仪的浓度梯度荧光校准片，其特征在于，所述第1行无机纳米荧光液液滴和倒数第1行无机纳米荧光液液滴的荧光点信号值为最高值65535，所述第2行无机纳米荧光液液滴的荧光点信号值为1000～3000，所述倒数第2行无机纳米荧光液液滴的荧光点信号值为60000～65535。
  7. 一种激光微阵列芯片扫描仪的校准方法，其特征在于，使用权利要求1至6中任一项所述激光微阵列芯片扫描仪的浓度梯度荧光校准片对待测机扫描仪进行校准并以无机纳米荧光液分点阵中第1行的无机纳米荧光液滴作为索引标志。
  8. 根据权利要求7所述激光微阵列芯片扫描仪的校准方法，其特征在于，将无机纳米荧光液分点阵中每个无机纳米荧光液液滴的荧光点信号值作为相应溶液浓度的单次测量值，使用标准机扫描仪扫描所述浓度梯度荧光校准片后，将其对应每个无机纳米荧光液液滴的荧光点信号值的对数值为横坐标；以使用待测机扫描仪扫描所述浓度梯度荧光校准片后，将其对应每个无机纳米荧光液液滴的荧光点信号值的对数值为纵坐标作图，考察得到曲线的线性关系的相关系数R 2。
  9. 根据权利要求8所述激光微阵列芯片扫描仪的校准方法，其特征在于，对每台待测机扫描仪采用至少3组不同的扫描参数进行校准，当每组扫描参数的相关系数R 2均大于0.90时，则说明该台待测机扫描仪校准结果正常并且能够正常使用。
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