WO2004013627A1 - System and method for analyzing signals in a multi-markers protein chip - Google Patents

System and method for analyzing signals in a multi-markers protein chip Download PDF

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Publication number
WO2004013627A1
WO2004013627A1 PCT/CN2003/000123 CN0300123W WO2004013627A1 WO 2004013627 A1 WO2004013627 A1 WO 2004013627A1 CN 0300123 W CN0300123 W CN 0300123W WO 2004013627 A1 WO2004013627 A1 WO 2004013627A1
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WIPO (PCT)
Prior art keywords
standard
signal
concentration
curve
camera device
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PCT/CN2003/000123
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French (fr)
Chinese (zh)
Inventor
Gengxi Hu
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Shanghai Health Digit Co.Ltd
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Priority to AU2003211848A priority Critical patent/AU2003211848A1/en
Publication of WO2004013627A1 publication Critical patent/WO2004013627A1/en

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    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B30/00—Methods of screening libraries
    • C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
    • G16B25/10—Gene or protein expression profiling; Expression-ratio estimation or normalisation
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression

Definitions

  • Multi-marker protein chip signal analysis system and method thereof Multi-marker protein chip signal analysis system and method thereof
  • the present invention relates to a biochip signal analysis system and method, and in particular, to a signal analysis system and method for a protein chip for detecting multiple markers, particularly multiple tumor markers. Background technique
  • Biochip technology is a high-tech developed rapidly in the field of life sciences in the past 10 years. It mainly refers to the construction of a miniature biochemical analysis system on the surface of a solid chip through micromachining and microelectronic technology to achieve accurate, fast and large information on the tissues, cells, proteins, nucleic acids, sugars and other biological components of living organisms. Amount of detection.
  • the common biochips are divided into three categories: gene chips (Genechip, DNAchip, DNAmicroarray), protein chips (Label on-a-chip), and so on.
  • Gene chips are very similar to the computer chips we call everyday, except that highly integrated are not semiconductor tubes, but thousands of grid-shaped densely arranged gene probes, through DNA fragments of known base sequence. Single-stranded DNA with complementary base sequences is combined to determine the corresponding sequence. In this way, abnormal genes or their products can be identified.
  • the principle of a protein chip is similar to that of a gene chip. The difference is that one is that the molecules fixed on the chip are proteins such as antigens or antibodies. Second, the detection principle is based on the interaction between protein molecules, proteins and nucleic acids, and proteins and other molecules. The protein chip technology appeared relatively late and is still in the development stage.
  • the lab of chips is the ultimate goal of the development of biochip technology. It intensifies the entire process of sample preparation, biochemical reaction, and detection and analysis into a micro-analysis system. Now there are chip laboratories consisting of heaters, micropumps, microvalves, microflow controllers, microelectrodes, electrochemistry and electroluminescence detectors, etc., and biochemical reactions, sample preparation, detection and analysis have appeared. And other partially integrated biochips.
  • the gene chip is the first commercialized product in biochip technology, many products have appeared as signal analysis systems for the gene chip.
  • the most commonly used gene chip signal analysis system is to place the chip in a chip scanner, collect the fluorescence intensity and fluorescence position of each reaction point, and analyze it by related software. Image to obtain biological information.
  • the fluorescence signal analysis system is roughly divided into two types according to different principles: laser confocal fluorescence microscopy and CCD fluorescence microscopy detection. The former has higher detection sensitivity and resolution, but has a longer scan time; the latter has a shorter scan time, but the sensitivity and resolution are not as good as the former.
  • An object of the present invention is to provide an analysis system mainly for detecting a multi-marker protein chip signal of a protein chip for clinical use in the case that an existing chip signal analysis system is mainly used for signal analysis of a gene chip.
  • the object of the present invention is also to provide a multi-marker protein chip analysis method that can simultaneously detect multiple markers, particularly multiple tumor marker protein chip signals, and perform fast, accurate, simple, and quantitative detection.
  • a multi-marker protein chip signal analysis system includes: a base plate, a cassette disposed on the base plate, a skin cavity located on the cassette and communicating with the skin cavity, and a skin cavity tightly arranged on the skin cavity and connected to the skin cavity.
  • Camera device its features are also: a light-tight X- ⁇ -Z- ⁇ positioning adjustment device and a computer analysis processor;
  • the X- ⁇ -Z- ⁇ positioning adjustment device includes: a camera device connector, the camera device connector is fastened to the lower part of the camera device; an XY direction adjusting frame, one end of which is connected to the camera device; a Z direction One end of the adjusting frame is connected with the XY direction adjusting frame, and the other end is fixed on the bottom plate; a triangular supporting frame is used for supporting the XY direction adjusting frame and the z direction adjusting frame;
  • the computer analysis processor has a data acquisition card, in which a corresponding image analysis application program is stored, which is used to store the image data signals collected by the camera device and analyze and process the image data signals.
  • the bottom plate is provided with two horizontal adjustment screws capable of adjusting the horizontal position of the bottom plate on the front side thereof; and a cassette guide bar is provided at an appropriate position on the bottom plate.
  • the cassette is fixed inside the cassette rail.
  • the connector includes a connector body which is gripped and surrounds the lower part of the camera device.
  • a camera device connection block is provided on the connector body.
  • a camera device connection screw is provided on the camera device connection block to connect the camera device. The device is fastened to the lower part of the camera device;
  • the X-Y direction adjusting frame includes an X-axis adjusting piece frame and an X-axis adjusting handle thereof, a Y-axis adjusting frame and a Y-axis adjusting handle thereof;
  • the z-direction adjusting frame includes a Z-axis base plate, a Z-axis adjusting knob provided on the Z-axis base plate, a Z moving plate connected to the X-Y direction adjusting frame, and a Z-axis set knob;
  • the X-Y-Z- ⁇ positioning adjustment device further includes a triangular support frame, which is supported under the X-Y direction adjustment frame and inside the Z-axis substrate of the z-direction adjustment frame.
  • a sample stage is provided in the cassette, and a sample stage level adjustment button capable of adjusting the angle of the sample stage is provided on the casing of the cassette.
  • An analysis method for quantitatively detecting a multi-marker protein chip signal by using the above-mentioned multi-marker protein chip signal analysis system which is characterized in that the analysis method includes the following steps:
  • Step 1 setting parameters on the computer analysis processor, including setting the exposure time of the camera in the signal analysis system and setting the temperature of the camera itself;
  • Step 2 After the required parameters are set, the camera device in the analysis system collects multiple light signals on the protein chip on the sample stage and captures image signals;
  • step three the image signals collected by the camera device are converted into digital signals and sent to a computer analysis processor
  • Step 4 After receiving the image data signal, the computer analysis processor 60 performs the following quantitative detection and analysis processing steps:
  • the fitting manner of the standard curve of "signal-concentration" of each marker may be It is a quadratic curve, a hyperbola, a logarithmic curve, or a combination of various quadratic curves.
  • Standard 1 If the conditions of 1 are not satisfied, remove Standard 1, and Standard 2 respectively.
  • Standard n-1 Standard n performs n-point fitting to obtain three sets of R values and curve monotonicity. The best one set of fit is selected as the output of this fitting.
  • the standard curve uses a signal value and a standard sample concentration as variables.
  • step 3 of the data analysis described above a large template is first used to locate multiple sub-arrays on the chip, and then a small template is used. Position the sample to be tested for each sub-array, position multiple sub-arrays on the chip, locate each signal point, and identify the gray level of the bright point by a computer analysis processor, and convert it into an electrical signal value.
  • the multi-marker protein chip signal analysis system and its analysis method adopt the above technical solution, so that compared with the prior art, it has the following advantages and positive effects:
  • the multi-marker protein chip signal analysis system of the present invention adopts the X-y-z-0 four-dimensional positioning adjustment device and a cooled charge-coupled camera device, which can accurately capture and capture multiple optical signals on the protein chip for images.
  • the multi-marker protein chip signal analysis system of the present invention can accurately analyze and detect the concentration of multiple target proteins in multiple generation samples in the protein chip due to the application of the above-mentioned quantitative analysis method.
  • the protein chip signal analysis system of the present invention has good reproducibility, stability and reliability, simple and intuitive operation, and can simultaneously perform the signals of multiple protein chips (each protein chip has signals of multiple test substances). Read and calculate, saving data analysis time. Brief description of the drawings.
  • FIG. 1 is a block diagram of a multi-marker protein chip signal analysis system in the present invention
  • FIG. 2 is a schematic structural diagram of a multi-marker protein chip signal analysis system in the present invention
  • FIG. 3 is a flowchart of a multi-marker protein chip signal analysis method in the present invention
  • FIG. 4 is an exposure of the chip signal analysis processing in the present invention Time setting dialog;
  • FIG. 5 is a dialog box of temperature setting in the chip signal analysis processing of the present invention.
  • FIG. 6 is a working interface diagram of a computer analysis processor in the present invention.
  • FIG. 7 is a chip signal image before contrast adjustment in chip signal analysis processing of the present invention
  • FIG. 8 is a chip signal image after contrast adjustment in chip signal analysis processing of the present invention
  • FIG. 9 is a secondary standard in chip signal analysis processing of the present invention Flow diagram of curve fitting
  • FIG. 10 is a standard curve diagram after fitting in the chip signal analysis processing of the present invention
  • FIG. 11 is a view of positioning a large template of a corresponding protein chip array in a chip signal analysis process according to the present invention.
  • FIG. 12 is a view of positioning a small template of a corresponding protein chip array in a chip signal analysis process according to the present invention.
  • FIG. 13 is a dialog box of positioning information in the chip signal analysis processing of the present invention.
  • FIG. 14 is a contrast transformation curve diagram of the chip signal analysis processing of the present invention.
  • FIG. 15 is a detection result report sheet output in a report form in the chip signal analysis processing of the present invention.
  • Fig. 16 is a standard signal value limitation table in the process of performing quadratic standard curve fitting on the chip signal analysis processing of the present invention. detailed description:
  • FIG. 1 and FIG. 2 are block diagrams and structure diagrams of a multi-marker protein chip signal analysis system in the present invention.
  • the invention relates to a multi-marker protein chip signal analysis system for providing a signal analysis system that captures a light signal generated by a chemical reaction on a protein chip and emits light.
  • the system includes a base plate 10, a cassette 20, a skin cavity 30, and a camera device. 40.
  • the bottom plate 10 is in the shape of a flat plate.
  • a horizontal adjustment screw 11 capable of adjusting the horizontal position of the bottom plate 10 is respectively provided at both ends of the front side of the bottom plate 10.
  • the cassette 20 is disposed on the bottom plate 10.
  • a cassette guide bar 21 is provided at an appropriate position on the bottom plate 10, and the cassette 20
  • a sample stage is provided, and a sample stage horizontal adjustment knob for adjusting the angle of the sample stage ⁇ is provided on the casing of the cassette 20, and the setting of the ⁇ angle rotation adjustment device for the subject is because the signal direction system of the biochip is Simultaneous shooting and analysis of a large number of signals, and if the location of each signal is not accurate, it will make analysis very difficult.
  • the present invention provides a device for adjusting the angle rotation of the subject on the horizontal plane by ⁇ angle to The angle adjustment function of the sample stage is realized.
  • the ⁇ angle rotation adjustment and positioning device is formed by simplifying the principle of a worm gear, so that an adjustment range of -5 degrees to +5 degrees can be achieved.
  • the leather cavity 30 is fixedly disposed on the cassette 20.
  • the leather cavity 30 communicates with the cassette 20, and a leather cavity tight frame 31 is provided on the leather cavity 30 for connecting with the upper object.
  • the camera device 40 is disposed on the skin cavity 30, and is fixedly connected and communicated with the skin cavity 30 through the skin cavity tight frame 31.
  • the camera device 40 uses a High-resolution cooled CCD camera system (COOL CCD camera); Cooled CCD camera system has higher sensitivity and is used to achieve low-light and very low-light to the conventional CCD camera system is difficult to achieve
  • the camera can be used under low temperature conditions.
  • the cooled CCD camera system can work at a low temperature below 0 ° C. This can reduce the dark current noise, make the captured image noise lower, and improve the image quality. ⁇
  • the optically sealed X-Y-Z- ⁇ positioning adjustment device 50 is composed of a camera device connector 51, an X-Y direction adjustment frame 52, a Z direction adjustment frame 53 and a triangular support frame 54;
  • the camera device connector 51 is fastened to the lower part of the camera device 50;
  • the camera device connector 51 includes a connector body 511, which is in the shape of a grip, which surrounds the lower part of the camera device 50, and
  • the body 511 is provided with a camera device connection block 512, and the connection surface of the camera device connection block 512 is an arc surface adapted to the camera device 50, so that it can be fit outside the camera device 50, and is provided on the camera device connection block 512
  • There is a camera device connection screw 513 and the camera device connector 51 can be fastened to the lower part of the camera device 50 by tightening the connection screw 513;
  • the upper end of the X-Y direction adjusting frame 52 is connected to the camera device 50;
  • the adjusting frame 52 includes an X-axis adjusting piece frame 521 and its X-axis adjusting handle 522, a Y-axis adjusting frame 523 and its Y-axis adjusting handle 524;
  • the Z-direction adjusting frame 53 includes a Z-axis substrate 531, which is fixed on the bottom plate 10, a Z-axis adjustment knob 532 is provided on the Z-axis substrate 531, and an upper end of the Z-axis substrate 531 is connected and provided with a Z moving plate 533 connected to the XY direction adjusting frame 52, and the Z axis is fixed Knob 534.
  • the triangular support frame 54 is disposed on the side of the XY direction adjustment frame 52 and the Z direction adjustment frame 53.
  • the upper plane of the triangular support frame 54 is supported under the XY direction adjustment frame 52, and the side plane of the triangular support frame 54 is Z.
  • the direction adjusting frame 53 is supported on the inside of the Z-axis substrate 531 by the mountain.
  • the computer analysis processor 60 is used for quantitative detection and analysis of a variety of target proteins to be detected on a protein chip; a data acquisition card is set in the computer analysis processor 60, and corresponding images are stored in the data acquisition card
  • the analysis application program is used to store the image data signals collected by the imaging device 40 and analyze and process the image data signals.
  • FIG. 3 is a flowchart of a method for analyzing a signal of a multi-marker protein chip in the present invention.
  • the multi-marker protein chip signal analysis method of the present invention can analyze and detect gene chip signals and detect clinical protein chip signals, and can simultaneously detect multiple markers, especially protein chip signals of multiple tumor markers.
  • the above tumor markers may include AFP, PSA, free-PSA, HGH, ⁇ -hCG, CEA, NSE, CA19-9, CA242, CA15-3, CA125, Ferritin, and the like.
  • a method for analyzing a multi-marker protein chip signal includes the following steps:
  • Step 1 Setting parameters on the computer analysis processor includes setting the exposure time of the camera 40 in the signal analysis system, and setting the temperature of the camera 40 itself;
  • Step 2 After the required parameters are set, the camera device 40 in the analysis system collects multiple optical signals on the protein chip on the sample stage and captures image signals;
  • step three the image signal collected by the camera device 40 is converted into a digital signal and sent to a computer analysis processor;
  • Step 4 After receiving the image data signal, the computer analysis processor 60 performs the following quantitative detection and analysis processing steps:
  • image processing which enlarges the selected image area, increases the contrast of the image, and removes the noise points on the image.
  • Curve uses signal value and standard sample concentration as variables; and the standard curve fitting method may be a quadratic curve, a hyperbola, a logarithmic curve, or a variety of quadratic curves The combination;
  • the “signal-concentration” standard curve for each marker is a quadratic curve
  • the specific process of fitting the quadratic standard curve is:
  • the fitting result is used as the current fitting output.
  • the method for determining whether the curve is monotonic can be verified by a computer algorithm based on the characteristics of the quadratic curve. In this embodiment, The method to determine whether R> 0.98 and the curve is monotonic is:
  • the following is an example of a specific operation process of the computer analysis processor 60 in the present invention for performing quantitative detection and analysis on the signal of the multi-marker protein chip.
  • FIG. 4 and FIG. 5 are descriptions of images acquired by the multi-marker protein chip.
  • the invention can be used to detect 12 tumor markers: AFP, PSA, free-PSA, HGH, ⁇ -hCG, CEA, NSE, CA19-9, CA242, CA15-3, CA125, Ferritin.
  • the chemiluminescence-producing protein chip is placed on the sample stage in the cassette;
  • set the exposure time to a fixed value from 10 seconds to 5 minutes, such as 15 seconds or 60 seconds, as shown in the figure.
  • Settings The exposure time is 30 seconds.
  • the implementation method is as follows: Enter the exposure time m_ExpTime from the exposure time setting dialog shown in Figure 4 and pass it to the variable exposure_time.
  • the function PICM_SetExposure controls the exposure time. ;
  • Set the CCD temperature to any value from -10 ° C to -50 ° C, such as:-20 ° C; set the CCD temperature to -20 ⁇ in the figure; the implementation method is: input from the temperature setting dialog shown in Figure 5
  • the set temperature setTmp is controlled by the function PICM— Set— Temperature (setTmp); click on the “Image Acquisition” menu to obtain the chip image captured by the camera system, and store it in the computer.
  • FIG. 6 to FIG. 8 are working interface diagrams of the computer analysis processor and chip signal images before and after contrast adjustment in the present invention.
  • the computer analysis processor 60 performs image processing on the signal of the multi-marker protein chip, click the "Image Transformation” menu on the working interface of the computer analysis processor, and perform "enlargement", "contrast enhancement", etc. as required.
  • Processing, used to adjust the image display size, contrast, etc. can also use the "fill" function to remove noise in the chip image.
  • the implementation method is as follows: (1) Zoom in
  • Tx the width of the subwindow / the width of the selected image
  • ty the height of the subwindow / the height of the selected image
  • the magnification of the image m_Scale takes the smaller of ⁇ , ty;
  • the noise area with the mouse, and fill the area with the value SpotValue.
  • the calculation method of SpotValue is: outside the selected noise area (3 pixels width), find gray For pixels with a degree value between 40 and 150, take the average of these pixels as the SpotValue. If the number of points is 0, the SpotValue is 80.
  • FIG. 9 is a flow block diagram of the quadratic standard curve fitting in the chip signal analysis processing of the present invention.
  • FIGS. 10 to 15 are standard curve diagrams, protein chip array diagrams, and test result reports related to data analysis and output operations in chip signal analysis and processing of the present invention.
  • Data analysis and output operations are as follows;
  • the method is: use a small template to position each standard array, and adjust the array row, column number, row spacing, column spacing, template size, and template position of the template to make the positioning All the squares of the template are just fixed with all the signal points of a sub-array, and each small square contains only one signal point. Take the average value of the brightest 4 pixels in each small square as the signal value of each point StandardPixAve [i] [SD], and according to the concentration value and signal value, fit the x markers according to Example 3 respectively. standard curve line.
  • Various fitting methods can also be used. combination.
  • Figure 10 A legend of the quadratic standard curve of the CA125 marker in the protein chip, where the X-axis represents the concentration value and the y-axis represents the signal value.
  • a positioning information dialog box will appear, as shown in Figure 13. Click the "Position” button in the dialog box to save the coordinates of the upper left corner of the template as the actual coordinate position of the sub-array LS_ActualPoint [i] [j ], Calculate and search the nearest LS_IdealPoint [i] [j], set it to the array at that position (such as the A1 sub-array), hide the buttons corresponding to the array, and position each sub-array in turn according to this method .
  • the multi-marker protein chip signal analysis system and analysis method of the present invention adopts the above technical solutions and operation steps, which not only can obtain high-quality image resolution, but also can accurately detect the protein chip.
  • Multiple target proteins in multiple samples to be tested that is, the concentration of the marker;
  • the analytical data is reproducible, stable and reliable, and the operation is simple and intuitive;
  • multiple protein chips (each protein chip can be used)
  • the quantitative instrument can also be used in combination with other biochips, so it is extremely practical.

Abstract

A system and method for analyzing signals in a multi-markers protein chip are provided. The system includes a base plate, a cassette on the base plate, a fur cavity in communication with the cassette, a camera provided on the fur cavity, a lighttight X-Y Z-θ positioning adjusting device and a computer analytic processor. The X-YZ-θ positioning adjusting device includes a camera connector, a frame for adjusting X-Y direction one end of which is connected with the camera, a fram for adjusting Z direction and a triangle supporter. The computer analytic processor has a data collecting card in which an application programme for storing and analyzing images is used to store and analyze the image data signals collecting by the camera. The method comprises the steps of. (1) setting the parameters, (2) collecting the signals, (3) transforming the signals, (4) analyzing and processing the signals, wherein the analyzing and processing step comprises processing the images, making the standard curve, analyzing the dataes and outputing the tables. Therefore, a high qualified image resolution can be obtained to detect accurately the concentration of markers and analyze thereof.

Description

多标志物蛋白芯片信号的分析***及其分析方法 技术领域  Multi-marker protein chip signal analysis system and method thereof
本发明涉及一种生物芯片信号分析***和方法， 尤其涉及一种用于检 测多标志物， 特别是多种肿瘤标志物的蛋白芯片的信号分析***及其方法。 背景技术  The present invention relates to a biochip signal analysis system and method, and in particular, to a signal analysis system and method for a protein chip for detecting multiple markers, particularly multiple tumor markers. Background technique
生物芯片技术是近 10年在生命科学领域中迅速发展起来的一项高新技 术。 它主要是指通过微加工和微电子技术在固体芯片表面构建微型生物化学 分析***， 以实现对生命机体的组织、 细胞、 蛋白质、 核酸、 糖类以及其他 生物组分进行准确、 快速、 大信息量的检测。 目前常见的生物芯片分为三大 类： 即基因芯片 （Genechip , DNAchip , DNAmi croarray ) , 蛋白芯片 (Proteinchip)、 芯片实验室 (Lab— on— a— chip) 等。  Biochip technology is a high-tech developed rapidly in the field of life sciences in the past 10 years. It mainly refers to the construction of a miniature biochemical analysis system on the surface of a solid chip through micromachining and microelectronic technology to achieve accurate, fast and large information on the tissues, cells, proteins, nucleic acids, sugars and other biological components of living organisms. Amount of detection. At present, the common biochips are divided into three categories: gene chips (Genechip, DNAchip, DNAmicroarray), protein chips (Label on-a-chip), and so on.
基因芯片和我们日常所说的计算机芯片非常相似， 只不过高度集成的 不是半导体管， 而是成千上万的网格状密集排列的基因探针， 通过已知碱基 顺序的 DNA片段， 来结合碱基互补序列的单链 DNA， 从而确定相应的序列， 通过这种方式来识别异常基因或其产物等。  Gene chips are very similar to the computer chips we call everyday, except that highly integrated are not semiconductor tubes, but thousands of grid-shaped densely arranged gene probes, through DNA fragments of known base sequence. Single-stranded DNA with complementary base sequences is combined to determine the corresponding sequence. In this way, abnormal genes or their products can be identified.
蛋白芯片与基因芯片的原理相似。 不同之处有， 一是芯片上固定的分 子是蛋白质如抗原或抗体等。 其二， 检测的原理是依据蛋白分子、 蛋白与核 酸、 蛋白与其他分子的相互作用。 蛋白芯片技术出现得较晚， 尚处于发展时 期。  The principle of a protein chip is similar to that of a gene chip. The difference is that one is that the molecules fixed on the chip are proteins such as antigens or antibodies. Second, the detection principle is based on the interaction between protein molecules, proteins and nucleic acids, and proteins and other molecules. The protein chip technology appeared relatively late and is still in the development stage.
芯片实验室是生物芯片技术发展的最终目标。 它将样品制备、 生化反 应以及检测分析的整个过程集约化形成微型分析***。 现在已有由加热器、 . 微泵、 微阀、 微流量控制器、 微电极、 电子化学和电子发光探测器等组成的 芯片实验室问世， 并出现了将生化反应、 样品制备、 检测和分析等部分集成 的生物芯片。  The lab of chips is the ultimate goal of the development of biochip technology. It intensifies the entire process of sample preparation, biochemical reaction, and detection and analysis into a micro-analysis system. Now there are chip laboratories consisting of heaters, micropumps, microvalves, microflow controllers, microelectrodes, electrochemistry and electroluminescence detectors, etc., and biochemical reactions, sample preparation, detection and analysis have appeared. And other partially integrated biochips.
生物芯片的关键技术之一就是芯片的信号检测和分析。 由于基因芯片 是生物芯片技术中最先实现商品化的产品， 因而， 作为基因芯片的信号分析 ***已有很多商品出现。 目前最常用的基因芯片信号分析***是将芯片置入 芯片扫描仪中， 通过采集各反应点的荧光强弱和荧光位置， 经相关软件分析 图像， 从而获得有关生物信息。 目前荧光信号分析***根据原理不同大致分 为两种:激光共聚焦荧光显微扫描和 CCD荧光显微照相检测。 前者检测灵敏 度、 分辨率均较高， 但扫描时间长；后者扫描时间短， 但灵敏度和分辨率不 如前者。 这些荧光检测***均有待于进一步完善与发展。 在上述已有的芯片 信号分析***中缺少主要用于检测临床用蛋白芯片的信号分析***和方法， 特别缺少用于检测临床多种肿瘤标志物的蛋白芯片信号分析***和方法。 发明内容： One of the key technologies of the biochip is the signal detection and analysis of the chip. Since the gene chip is the first commercialized product in biochip technology, many products have appeared as signal analysis systems for the gene chip. At present, the most commonly used gene chip signal analysis system is to place the chip in a chip scanner, collect the fluorescence intensity and fluorescence position of each reaction point, and analyze it by related software. Image to obtain biological information. At present, the fluorescence signal analysis system is roughly divided into two types according to different principles: laser confocal fluorescence microscopy and CCD fluorescence microscopy detection. The former has higher detection sensitivity and resolution, but has a longer scan time; the latter has a shorter scan time, but the sensitivity and resolution are not as good as the former. These fluorescence detection systems need to be further improved and developed. Among the above existing chip signal analysis systems, there is a lack of signal analysis systems and methods that are mainly used to detect protein chips for clinical use, and particularly lack of protein chip signal analysis systems and methods that are used to detect a variety of clinical tumor markers. Summary of the invention:
本发明的目的在于， 针对现有的芯片信号分析***主要用于基因芯片 信号分析的情况， 提供一种主要用于检测临床用蛋白芯片的多标志物蛋白芯 片信号的分析***。  An object of the present invention is to provide an analysis system mainly for detecting a multi-marker protein chip signal of a protein chip for clinical use in the case that an existing chip signal analysis system is mainly used for signal analysis of a gene chip.
本发明的目的还在于提供一种可以同时检测多个标志物， 特别是多种 肿瘤标志物的蛋白芯片信号进行快捷、 准确、 简便、 定量检测的多标志物蛋 白芯片信号的分析方法。  The object of the present invention is also to provide a multi-marker protein chip analysis method that can simultaneously detect multiple markers, particularly multiple tumor marker protein chip signals, and perform fast, accurate, simple, and quantitative detection.
本发明的目的是这样实现的：  The object of the present invention is achieved as follows:
一种多标志物蛋白芯片信号的分析***， 包括： 一底板， 安置在底板 上的暗盒， 位于暗盒上且与之连通的皮腔， 通过皮腔紧框设置在皮腔上且与 之连接的摄像装置； 其特点是还包括： 一光密闭的 X-Υ-Ζ- θ定位调节装置 和一计算机分析处理器；  A multi-marker protein chip signal analysis system includes: a base plate, a cassette disposed on the base plate, a skin cavity located on the cassette and communicating with the skin cavity, and a skin cavity tightly arranged on the skin cavity and connected to the skin cavity. Camera device; its features are also: a light-tight X-Υ-Z-θ positioning adjustment device and a computer analysis processor;
所述的 X-Υ-Ζ- θ定位调节装置包括： 一摄像装置连接器， 该摄像装置 连接器紧固在摄像装置下部； 一 X-Y方向调节架， 它的一端与摄像装置连 接； 一 Z方向调节架， 它的一端与 X-Y方向调节架连接， 另一端固定在底 板上； 一三角支撑架， 用以支撑 X-Y方向调节架和 z方向调节架；  The X-Υ-Z-θ positioning adjustment device includes: a camera device connector, the camera device connector is fastened to the lower part of the camera device; an XY direction adjusting frame, one end of which is connected to the camera device; a Z direction One end of the adjusting frame is connected with the XY direction adjusting frame, and the other end is fixed on the bottom plate; a triangular supporting frame is used for supporting the XY direction adjusting frame and the z direction adjusting frame;
所述的计算机分析处理器具有数据采集卡， 其中存储了相应的图像分 析应用程序， 用于存储摄像装置所采集到的图像数据信号并对图像数据信号 进行分析处理。  The computer analysis processor has a data acquisition card, in which a corresponding image analysis application program is stored, which is used to store the image data signals collected by the camera device and analyze and process the image data signals.
在上述的多标志物蛋白芯片信号的分析***中，， 其中， 所述的底板在 其前侧设置两可调节底板水平位置的水平调节螺丝； 在所述的底板的适当位 置处设置暗盒导轨条， 所述的暗盒固定在暗盒导轨条内侧。  In the above-mentioned multi-marker protein chip signal analysis system, wherein the bottom plate is provided with two horizontal adjustment screws capable of adjusting the horizontal position of the bottom plate on the front side thereof; and a cassette guide bar is provided at an appropriate position on the bottom plate. The cassette is fixed inside the cassette rail.
在上述的多标志物蛋白芯片信号的分析***中， 其中， 所述的摄像装 置连接器包括， 一连接器本体， 它呈抓手， 围在摄像装置下部， 在连接器本 体上设有一摄像装置连接块， 摄像装置连接块上设有一摄像装置连接螺丝， 以将摄像装置连接器紧固在摄像装置下部； In the above-mentioned multi-marker protein chip signal analysis system, wherein the imaging device The connector includes a connector body which is gripped and surrounds the lower part of the camera device. A camera device connection block is provided on the connector body. A camera device connection screw is provided on the camera device connection block to connect the camera device. The device is fastened to the lower part of the camera device;
所述的 X-Y方向调节架包括， X轴调整件架及其 X轴调整手柄， Y轴 调整架及其 Y轴调整手柄；  The X-Y direction adjusting frame includes an X-axis adjusting piece frame and an X-axis adjusting handle thereof, a Y-axis adjusting frame and a Y-axis adjusting handle thereof;
所述的 z方向调节架包括， 一 Z轴基板， '设置在 Z轴基板上的 Z轴调 节旋钮， 与 X-Y方向调节架连接的 Z动向板， 以及 Z轴紧定旋钮；  The z-direction adjusting frame includes a Z-axis base plate, a Z-axis adjusting knob provided on the Z-axis base plate, a Z moving plate connected to the X-Y direction adjusting frame, and a Z-axis set knob;
所述的 X-Y-Z- Θ定位调节装置还包括一三角支撑架， 它支撑在 X-Y方 向调节架下和 z方向调节架的 Z轴基板内侧。  The X-Y-Z-Θ positioning adjustment device further includes a triangular support frame, which is supported under the X-Y direction adjustment frame and inside the Z-axis substrate of the z-direction adjustment frame.
在上述的多标志物蛋白芯片信号的分析***中， 其中， 在所述的暗盒 中设置有样品台， 在该暗盒的外壳上设置一可调节样品台角度的样品台水平 调节钮。 '  In the above-mentioned multi-marker protein chip signal analysis system, a sample stage is provided in the cassette, and a sample stage level adjustment button capable of adjusting the angle of the sample stage is provided on the casing of the cassette. '
一种用上述多标志物蛋白芯片信号的分析***对多标志物蛋白芯片信 号进行定量检测的分析方法， 其特点是该分析方法包括以下步骤：  An analysis method for quantitatively detecting a multi-marker protein chip signal by using the above-mentioned multi-marker protein chip signal analysis system, which is characterized in that the analysis method includes the following steps:
步骤一， 在计算机分析处理器上设置参数， 包括， 信号分析***中摄 像装置曝光时间的设置、 摄像装置本身温度的设置；  Step 1: setting parameters on the computer analysis processor, including setting the exposure time of the camera in the signal analysis system and setting the temperature of the camera itself;
步骤二， 所需参数设置后， 分析***中的摄像装置对样品台上蛋白芯 片上的多个光信号进行采集， 并拍摄图像信号；  Step 2: After the required parameters are set, the camera device in the analysis system collects multiple light signals on the protein chip on the sample stage and captures image signals;
步骤三， 对摄像装置所采集的图像信号转化为数字信号后送入计算机 分析处理器；  In step three, the image signals collected by the camera device are converted into digital signals and sent to a computer analysis processor;
步骤四， 计算机分析处理器 60接收图像数据信号后进行如下定量检测 分析处理步骤：  Step 4. After receiving the image data signal, the computer analysis processor 60 performs the following quantitative detection and analysis processing steps:
1 ) 图像处理， 将选定的图像区域进行放大、 增大图像的对比度、 对图 像上有杂点处进行杂点去除； .  1) image processing, enlarging the selected image area, increasing the contrast of the image, and removing noise points where there are noise points on the image;
2) 作标准曲线， 根据芯片上各标准品中各个标志物的已知浓度值以及 对应的已拍摄的图像的信号值这两种参数， 拟合出各个标志物的 "信号-浓 度"的标准曲线；  2) Make a standard curve and fit the "signal-concentration" standard of each marker according to the two parameters of the known concentration of each marker in each standard on the chip and the signal value of the corresponding captured image Curve
3 ) 数据分析， 先采用大模板对芯片上多个子阵列进行定位， 再采用小 模板对每个子阵列的待测样本进行定位， 采集每个待测样本信号中最亮的 4 个象素灰度平均值作为各点的图像最终信号值， 由上述 2)步骤中 "信号- 浓度"二次标准曲线找到待测样本中各标志物的浓度值； 3) Data analysis, first use a large template to locate multiple sub-arrays on the chip, and then use a small template to locate the test samples of each sub-array, and collect the brightest 4 pixel gray levels in each test sample signal The average value is used as the final signal value of the image at each point. "Concentration" quadratic standard curve to find the concentration value of each marker in the test sample;
4)输出报表，将芯片上每个待测样本中各个标志物的浓度测定值以 Excel 表格的形式输出。  4) Output a report, and output the concentration measurement value of each marker in each test sample on the chip in the form of an Excel table.
在上述的多标志物蛋白芯片信号的分析***的分析方法中， 其中， 在 所述的步骤四的 2) 中， 所述的各个标志物的 "信号-浓度"的标准曲线的 拟合方式可为二次曲线、 双曲线、 对数曲线或各种二次曲线的组合。  In the analysis method of the multi-marker protein chip signal analysis system described above, in the step 2) of step 4, the fitting manner of the standard curve of "signal-concentration" of each marker may be It is a quadratic curve, a hyperbola, a logarithmic curve, or a combination of various quadratic curves.
在上述的多标志物蛋白芯片信号的分析***的分析方法中， 其中， 所 述的作各个标志物的 "信号-浓度"二次标准曲线拟合方式具体过程是：  In the analysis method of the multi-marker protein chip signal analysis system described above, the specific process of fitting the “signal-concentration” quadratic standard curve of each marker is:
( 1 ) 获取蛋白芯片上标准品 0、 标准品 1、 标准品 2, …标准品 n-l、 标准品 n的信号值；  (1) Obtain the signal values of standard 0, standard 1, standard 2, 2, standard n-1, and standard n on the protein chip;
(2) 对标准品 0进行修正， 如果标准品 0的信号值超出限定表中的上 限值， 则把标准品 0的值调整为限定表中相应的值;  (2) Correct the standard 0. If the signal value of the standard 0 exceeds the upper limit in the limit table, adjust the value of the standard 0 to the corresponding value in the limit table;
(3) 以标准品 0信号值、 标准品 1 .信号值、 标准品 2信号值，…标准品 n-1信号值、 标准品 n信号值为 X变量， 标准品 0浓度、 标准品 1浓度、 标 准品 2浓度，…标准品 n-1浓度、 标准品 n浓度为 Y变量的 n+1个点进行最 小二乘法的二次曲线拟合 （Y=aX2+bX+c)， 得到曲线的回归系数 R (其中 具 体 应 用 函 数 是. ： =丄 标准品浓度 ζ· = (标准品 ζ· - )2 c =∑ + bXi + c - A)2， R= Fc/ Sc); (3) Take standard 0 signal value, standard 1. signal value, standard 2 signal value, ... standard n-1 signal value, standard n signal value as X variables, standard 0 concentration, standard 1 concentration , Standard 2 concentration, ... standard n-1 concentration, standard n concentration is n + 1 points of the Y variable and perform the least square method quadratic curve fitting (Y = aX2 + bX + c) to obtain the curve Regression coefficient R (where the specific application function is: 丄 = standard concentration ζ · = (standard ζ ·-) 2 c = ∑ + b Xi + c-A) 2 , R = Fc / Sc);
ι=0 ί=0 ί=ο  ι = 0 ί = 0 ί = ο
(4) 如果 R〉0.98 同时曲线单调递增， 则把拟合结果作为本次拟合输 出；  (4) If R> 0.98 and the curve monotonically increases, the fitting result is used as the current fitting output;
(5) 拟合完成， 给出标准曲线。 (5) The fitting is completed and a standard curve is given.
在上述的多标志物蛋白芯片信号的分析***的分析方法中， 其中， 在 所述的二次标准曲线拟合方式具体过程 （4) 中， 判断 R〉0.98且曲线是否单 调的方法是，  In the analysis method of the multi-marker protein chip signal analysis system described above, in the specific process (4) of the quadratic standard curve fitting method described above, the method for determining whether R> 0.98 and whether the curve is monotonic is:
①如果不满足上述的条件， R<0.98或曲线不是单调递增， 进行去 除 1个点， 即 n个点进行拟合； 此时， 如果标准品 n的信号值〉 1.3倍标准 品 n-l， 则把标准品 n去除， 并回到步骤 c进行 n-1点拟合；  ① If the above conditions are not met, R <0.98 or the curve is not monotonically increasing, and one point is removed, that is, n points are fitted. At this time, if the signal value of the standard n is> 1.3 times the standard nl, then Remove the standard n and return to step c to perform n-1 point fitting;
②如果不满足 ① 的条件， 则分别去除标准品 1、 标准品 2 ， 标准品 n— 1， 标准品 n进行 n个点拟合， 得到 3组 R值和曲线单调性的拟 合结果， 选择最佳的 1组拟合作为本次拟合输出。 ② If the conditions of ① are not satisfied, remove Standard 1, and Standard 2 respectively. Standard n-1. Standard n performs n-point fitting to obtain three sets of R values and curve monotonicity. The best one set of fit is selected as the output of this fitting.
在上述的多标志物蛋白芯片信号的分析***的分析方法中， 其中， 在 所述的步骤四的 2) 中， 所述的标准曲线以信号值和标准样品浓度为变量。  In the above analysis method of the multi-marker protein chip signal analysis system, in the step 2) of the fourth step, the standard curve uses a signal value and a standard sample concentration as variables.
在'上述的多标志物蛋白芯片信号的分析***的分析方法中， 其中， 在 所述的步骤四的 3 ) 数据分析中， 先采用大模板对芯片上多个子阵列进行定 位， 再采用小模板对每个子阵列的待测样本进行定位， 对芯片上多个子阵列 进行定位， 对各信号点进行定位， 并通过计算机分析处理器识别亮点灰度， 且转换为电信号值。  In the analysis method of the above-mentioned multi-marker protein chip signal analysis system analysis method, in the step 3) of the data analysis described above, a large template is first used to locate multiple sub-arrays on the chip, and then a small template is used. Position the sample to be tested for each sub-array, position multiple sub-arrays on the chip, locate each signal point, and identify the gray level of the bright point by a computer analysis processor, and convert it into an electrical signal value.
本发明， 多标志物蛋白芯片信号的分析***及其分析方法， 由于采用 了上述的技术方案， 使之与现有技术相比， 具有以下的优点和积极效果： According to the present invention, the multi-marker protein chip signal analysis system and its analysis method adopt the above technical solution, so that compared with the prior art, it has the following advantages and positive effects:
1.本发明多标志物蛋白芯片信号分析***由于采用了 X- y- z-0 四维定 位调节装置及冷却型电荷耦合摄像装置可以准确地采集并摄取蛋白芯片上的 多个光信号， 进行图像拍摄。 1. The multi-marker protein chip signal analysis system of the present invention adopts the X-y-z-0 four-dimensional positioning adjustment device and a cooled charge-coupled camera device, which can accurately capture and capture multiple optical signals on the protein chip for images. Shoot.
2.本发明多标志物蛋白芯片信号分析***由于应用了上述定量分析方 法， 可以准确地分析并检测出蛋白芯片中多个代测样品中多个目标蛋白的浓 度。  2. The multi-marker protein chip signal analysis system of the present invention can accurately analyze and detect the concentration of multiple target proteins in multiple generation samples in the protein chip due to the application of the above-mentioned quantitative analysis method.
3. 本发明所述的根据存储程序数字计算机控制的蛋白芯片信号分析系 统在以上述的方式运行时， 将会获得高质量、 高分辨率的蛋白芯片图像， 并 可以准确地检测出蛋白芯片中多个待测样品中多种目标蛋白， 即标志物的浓 度。  3. When the protein chip signal analysis system according to the present invention controlled by a digital computer is run in the above-mentioned manner, a high-quality, high-resolution protein chip image will be obtained, and the protein chip can be accurately detected. The concentration of multiple target proteins in multiple test samples, ie, the concentration of a marker.
4. 本发明所述的蛋白芯片信号分析***重现性好， 稳定可靠， 操作简 便直观， 并可对多个蛋白芯片 （每个蛋白芯片上有多个被测物质的信号） 的 信号进行同时读取和计算， 节省了数据分析时间。 附图说明 .  4. The protein chip signal analysis system of the present invention has good reproducibility, stability and reliability, simple and intuitive operation, and can simultaneously perform the signals of multiple protein chips (each protein chip has signals of multiple test substances). Read and calculate, saving data analysis time. Brief description of the drawings.
通过以下对本发明多标志物蛋白芯片信号的分析***及其分析方法的 若干实施例结合其附图的描述， 可以进一步理解本发明的目的、 具体结构特 征和优点。 其中， 附图为：  Through the following description of several embodiments of the multi-marker protein chip signal analysis system and analysis method of the present invention in combination with the accompanying drawings, the purpose, specific structural features, and advantages of the present invention can be further understood. The drawings are:
图 1是本发明中多标志物蛋白芯片信号的分析***的方框图； 图 2是本发明中多标志物蛋白芯片信号的分析***的结构示意图； 图 3是本发明中多标志物蛋白芯片信号的分析方法的流程框图； 图 4是本发明对芯片信号分析处理中曝光时间设置的对话框； 1 is a block diagram of a multi-marker protein chip signal analysis system in the present invention; FIG. 2 is a schematic structural diagram of a multi-marker protein chip signal analysis system in the present invention; FIG. 3 is a flowchart of a multi-marker protein chip signal analysis method in the present invention; FIG. 4 is an exposure of the chip signal analysis processing in the present invention Time setting dialog;
图 5是本发明对芯片信号分析处理中温度设置的对话框；  FIG. 5 is a dialog box of temperature setting in the chip signal analysis processing of the present invention;
图 6是本发明中计算机分析处理器的工作界面图；  6 is a working interface diagram of a computer analysis processor in the present invention;
图 7是本发明对芯片信号分析处理中对比度调整前芯片信号图像； 图 8是本发明对芯片信号分析处理中对比度调整后芯片信号图像； 图 9是本发明对芯片信号分析处理中二次标准曲线拟合的流程框图； 图 10是本发明对芯片信号分析处理中拟合后的标准曲线图；  FIG. 7 is a chip signal image before contrast adjustment in chip signal analysis processing of the present invention; FIG. 8 is a chip signal image after contrast adjustment in chip signal analysis processing of the present invention; FIG. 9 is a secondary standard in chip signal analysis processing of the present invention Flow diagram of curve fitting; FIG. 10 is a standard curve diagram after fitting in the chip signal analysis processing of the present invention;
图 11 是本发明对芯片信号分析处理中对应蛋白芯片阵列进行大模板定 位的视图；  FIG. 11 is a view of positioning a large template of a corresponding protein chip array in a chip signal analysis process according to the present invention; FIG.
图 12 是本发明对芯片信号分析处理中对应蛋白芯片阵列进行小模板定 位的视图；  FIG. 12 is a view of positioning a small template of a corresponding protein chip array in a chip signal analysis process according to the present invention; FIG.
图 13是本发明对芯片信号分析处理中定位信息的对话框；  FIG. 13 is a dialog box of positioning information in the chip signal analysis processing of the present invention;
图 14是本发明对芯片信号分析处理中对比度变换曲线图；  FIG. 14 is a contrast transformation curve diagram of the chip signal analysis processing of the present invention; FIG.
图 15 是本发明对芯片信号分析处理中以报表形式输出的检测结果报告 单；  FIG. 15 is a detection result report sheet output in a report form in the chip signal analysis processing of the present invention;
图 16 是本发明对芯片信号分析处理进行二次标准曲线拟合过程中标准 品信号值限定表。 具体实施方式：  Fig. 16 is a standard signal value limitation table in the process of performing quadratic standard curve fitting on the chip signal analysis processing of the present invention. detailed description:
请参见图 1 和图 2 所示， 它们是本发明中多标志物蛋白芯片信号的分 析***的方框图和结构示意图。 本发明一种多标志物蛋白芯片信号的分析系 统， 用于提供一种捕捉蛋白芯片上化学反应发光所产生的光信号的信号分析 ***， 包括， 底板 10、 暗盒 20、 皮腔 30、 摄像装置 40、 光密闭的 X-Y-Z- Θ定位调节装置 50和一计算机分析处理器 60。  Please refer to FIG. 1 and FIG. 2, which are block diagrams and structure diagrams of a multi-marker protein chip signal analysis system in the present invention. The invention relates to a multi-marker protein chip signal analysis system for providing a signal analysis system that captures a light signal generated by a chemical reaction on a protein chip and emits light. The system includes a base plate 10, a cassette 20, a skin cavity 30, and a camera device. 40. The optically sealed XYZ-Θ positioning adjustment device 50 and a computer analysis processor 60.
所述的底板 10呈平板形状， 作为信号分析***中各部件的安置的基板， 在底板 10前侧两端分别设置一可调节底板 10水平位置的水平调节螺丝 11。  The bottom plate 10 is in the shape of a flat plate. As a base plate on which the components of the signal analysis system are arranged, a horizontal adjustment screw 11 capable of adjusting the horizontal position of the bottom plate 10 is respectively provided at both ends of the front side of the bottom plate 10.
所述的暗盒 20安置在底板 10上， 为了方便暗盒 20的在前进方向的前 后调节， 在底板 10的适当位置处设置暗盒导轨条 21， 在所述的暗盒 20中 设置有样品台， 在该暗盒 20 的外壳上设置一可调节样品台 Θ角度的样品台 水平调节钮， 而对被摄物进行 Θ角度旋转调节装置的设置是因为， 由于生物 芯片信号方向***由于对大量信号的同时拍摄和分析， 而若各信号所在位置 不够准确的话， 会使得分析变得十分困难， 为此， 本发明设置了在水平面上 对被摄物进行 Θ角度旋转调节的装置， 以实现样品台的角度可调节功能； 在 本实施例中， 该 Θ角度旋转调节定位装置采用的是蜗轮蜗杆的原理简化后形 成的， 从而可实现- 5度到 +5度的调节范围。 The cassette 20 is disposed on the bottom plate 10. In order to facilitate the forward and backward adjustment of the cassette 20 in the forward direction, a cassette guide bar 21 is provided at an appropriate position on the bottom plate 10, and the cassette 20 A sample stage is provided, and a sample stage horizontal adjustment knob for adjusting the angle of the sample stage Θ is provided on the casing of the cassette 20, and the setting of the Θ angle rotation adjustment device for the subject is because the signal direction system of the biochip is Simultaneous shooting and analysis of a large number of signals, and if the location of each signal is not accurate, it will make analysis very difficult. For this reason, the present invention provides a device for adjusting the angle rotation of the subject on the horizontal plane by Θ angle to The angle adjustment function of the sample stage is realized. In this embodiment, the Θ angle rotation adjustment and positioning device is formed by simplifying the principle of a worm gear, so that an adjustment range of -5 degrees to +5 degrees can be achieved.
所述的皮腔 30固定设置在暗盒 20上， 该皮腔 30与暗盒 20相通， 在 皮腔 30上设置有一用以与上部物件连接的皮腔紧框 31。  The leather cavity 30 is fixedly disposed on the cassette 20. The leather cavity 30 communicates with the cassette 20, and a leather cavity tight frame 31 is provided on the leather cavity 30 for connecting with the upper object.
所述的摄像装置 40设置在皮腔 30上， 并通过皮腔紧框 31 与皮腔 30 固定连接并相通； 针对蛋白芯片信号较弱适当特点， 在本发明中， 摄像装置 40采用的是一高分辨率的冷却型 CCD摄相***（COOL CCD camera); 冷却 型 CCD摄相***具有较高的灵敏度， 是用于在低照度下和非常低照度下对与 常规的 CCD摄像***很难达到的情况下进行摄像，冷却型 CCD摄相***可在 低于 0°C的低温状态下工作， 这样可减少暗电流噪声， 使所拍摄的图像噪声 较低， 从而可提高图像品质。 ·  The camera device 40 is disposed on the skin cavity 30, and is fixedly connected and communicated with the skin cavity 30 through the skin cavity tight frame 31. In view of the appropriate characteristics of weak signal of the protein chip, in the present invention, the camera device 40 uses a High-resolution cooled CCD camera system (COOL CCD camera); Cooled CCD camera system has higher sensitivity and is used to achieve low-light and very low-light to the conventional CCD camera system is difficult to achieve The camera can be used under low temperature conditions. The cooled CCD camera system can work at a low temperature below 0 ° C. This can reduce the dark current noise, make the captured image noise lower, and improve the image quality. ·
所述的光密闭的 X-Y-Z- Θ定位调节装置 50由摄像装置连接器 51、 X-Y 方向调节架 52、 Z方向调节架 53和三角支撑架 54组成； 其中，  The optically sealed X-Y-Z-Θ positioning adjustment device 50 is composed of a camera device connector 51, an X-Y direction adjustment frame 52, a Z direction adjustment frame 53 and a triangular support frame 54;
所述的摄像装置连接器 51紧固在摄像装置 50下部；摄像装置连接器 51 包括， 一连接器本体 511， 该连接器本体 511呈抓手形状， 它围在摄像装置 50下部， 在连接器本体 511上设有一摄像装置连接块 512， 该摄像装置连接 块 512的连接面呈与摄像装置 50相适配的弧面， 从而可贴合在摄像装置 50 外， 在摄像装置连接块 512上设有一摄像装置连接螺丝 513， 旋紧连接螺丝 513就可将摄像装置连接器 51紧固在摄像装置 50下部；  The camera device connector 51 is fastened to the lower part of the camera device 50; the camera device connector 51 includes a connector body 511, which is in the shape of a grip, which surrounds the lower part of the camera device 50, and The body 511 is provided with a camera device connection block 512, and the connection surface of the camera device connection block 512 is an arc surface adapted to the camera device 50, so that it can be fit outside the camera device 50, and is provided on the camera device connection block 512 There is a camera device connection screw 513, and the camera device connector 51 can be fastened to the lower part of the camera device 50 by tightening the connection screw 513;
所述的 X-Y方向调节架 52的上端与摄像装置 50连接；调节架 52包括， X轴调整件架 521及其 X轴调整手柄 522， Y轴调整架 523及其 Y轴调整 手柄 524;  The upper end of the X-Y direction adjusting frame 52 is connected to the camera device 50; the adjusting frame 52 includes an X-axis adjusting piece frame 521 and its X-axis adjusting handle 522, a Y-axis adjusting frame 523 and its Y-axis adjusting handle 524;
所述的 Z方向调节架 53包括， 一 Z轴基板 531， 该 Z轴基板 531固定 在底板 10上， 在 Z轴基板 531上设置一 Z轴调节旋钮 532，在 Z轴基板 531 上端连接设置一与 X-Y方向调节架 52连接的 Z动向板 533， 以及 Z轴紧定 旋钮 534。 The Z-direction adjusting frame 53 includes a Z-axis substrate 531, which is fixed on the bottom plate 10, a Z-axis adjustment knob 532 is provided on the Z-axis substrate 531, and an upper end of the Z-axis substrate 531 is connected and provided with a Z moving plate 533 connected to the XY direction adjusting frame 52, and the Z axis is fixed Knob 534.
所述的三角支撑架 54设置在 X-Y方向调节架 52和 Z方向调节架 53— 侧； 其中， 三角支撑架 54的上平面支撑在 X-Y方向调节架 52下， 三角支 撑架 54的侧平面作为 Z方向调节架 53的靠山而支撑在 Z轴基板 531内侧。  The triangular support frame 54 is disposed on the side of the XY direction adjustment frame 52 and the Z direction adjustment frame 53. The upper plane of the triangular support frame 54 is supported under the XY direction adjustment frame 52, and the side plane of the triangular support frame 54 is Z. The direction adjusting frame 53 is supported on the inside of the Z-axis substrate 531 by the mountain.
所述的计算机分析处理器 60用于对蛋白芯片上的多种待检测目标蛋白 进行定量检测和分析； 在计算机分析处理器 60 中设置有数据采集卡， 在数 据采集卡中存储了相应的图像分析应用程序， 用于存储摄像装置 40所采集 到的图像数据信号并对图像数据信号进行分析处理。  The computer analysis processor 60 is used for quantitative detection and analysis of a variety of target proteins to be detected on a protein chip; a data acquisition card is set in the computer analysis processor 60, and corresponding images are stored in the data acquisition card The analysis application program is used to store the image data signals collected by the imaging device 40 and analyze and process the image data signals.
请结合图 1和图 2参见图 3所示， 图 3是本发明中多标志物蛋白芯片 信号的分析方法的流程框图。  Please refer to FIG. 3 in conjunction with FIG. 1 and FIG. 2. FIG. 3 is a flowchart of a method for analyzing a signal of a multi-marker protein chip in the present invention.
本发明多标志物蛋白芯片信号的分析方法， 能对于基因芯片信号、 检 测临床用蛋白芯片信号进行分析和检测， 并且还可以同时检测多个标志物， 特别是多种肿瘤标志物的蛋白芯片信号进行快捷、 准确、 简便、 定量检测， 上述的肿瘤标志物可以包括 AFP、 PSA、 free-PSA, HGH、 β - hCG、 CEA、 NSE、 CA19- 9、 CA242、 CA15- 3、 CA125、 Ferritin等。.  The multi-marker protein chip signal analysis method of the present invention can analyze and detect gene chip signals and detect clinical protein chip signals, and can simultaneously detect multiple markers, especially protein chip signals of multiple tumor markers. For fast, accurate, simple and quantitative detection, the above tumor markers may include AFP, PSA, free-PSA, HGH, β-hCG, CEA, NSE, CA19-9, CA242, CA15-3, CA125, Ferritin, and the like. .
本发明， 多标志物蛋白芯片信号的分析方法包括以下步骤：  In the present invention, a method for analyzing a multi-marker protein chip signal includes the following steps:
步骤一， 在计算机分析处理器上设置参数， 包括， 信号分析***中摄 像装置 40曝光'时间的设置、 摄像装置 40本身温度的设置；  Step 1. Setting parameters on the computer analysis processor includes setting the exposure time of the camera 40 in the signal analysis system, and setting the temperature of the camera 40 itself;
步骤二， 所需参数设置后， 分析***中的摄像装置 40对样品台上蛋白 芯片上的多个光信号进行采集， 并拍摄图像信号；  Step 2: After the required parameters are set, the camera device 40 in the analysis system collects multiple optical signals on the protein chip on the sample stage and captures image signals;
步骤三， 对摄像装置 40所采集的图像信号转化为数字信号后送入计算 机分析处理器；  In step three, the image signal collected by the camera device 40 is converted into a digital signal and sent to a computer analysis processor;
步骤四， 计算机分析处理器 60接收图像数据信号后进行如下定量检测 分析处理步骤：  Step 4. After receiving the image data signal, the computer analysis processor 60 performs the following quantitative detection and analysis processing steps:
第一， 图像处理， 将选定的图像区域进行放大、 增大图像的对比度、 对图像上有杂点处进行杂点去除。  First, image processing, which enlarges the selected image area, increases the contrast of the image, and removes the noise points on the image.
第二， 作标准曲线， 根据芯片上各标准品中各个标志物的已知浓度值 以及对应的已拍摄的图像的信号值这两种参数， 拟合出各个标志物的 "信号 -浓度"标准曲线； 所述的标准曲线以信号值和标准样品浓度为变量； 并且， 所述的标准曲线拟合方式可为二次曲线、 双曲线、 对数曲线或多种二次曲线 的组合； Second, make a standard curve, and fit the "signal-concentration" standard of each marker according to the two parameters of the known concentration of each marker in each standard on the chip and the signal value of the corresponding captured image. Curve; the standard curve uses signal value and standard sample concentration as variables; and the standard curve fitting method may be a quadratic curve, a hyperbola, a logarithmic curve, or a variety of quadratic curves The combination;
在本实施例中， 所述的作各个标志物的 "信号-浓度"标准曲线采用的 是二次曲线， 该二次标准曲线拟合方式具体过程是：  In this embodiment, the “signal-concentration” standard curve for each marker is a quadratic curve, and the specific process of fitting the quadratic standard curve is:
( 1 ) 获取蛋白芯片上标准品 0、 标准品 1、 标准品 2， …标准品 n-l、 标准品 n的信号值 ( 1-n代表浓度依次升高）；  (1) Obtaining the signal values of standard 0, standard 1, standard 2, ... of standard n-1 and standard n on the protein chip (1-n means that the concentration increases sequentially);
(2) 对标准品 0进行修正， 如果标准品 0的信号值超出限定表中的上 限值， 则把标准品 0的值调整为限定表 (参见图 16)中相应的值 （限定表中的 值是长期实验得到的经验值)；  (2) Correct standard 0. If the signal value of standard 0 exceeds the upper limit in the limit table, adjust the value of standard 0 to the corresponding value in the limit table (see Figure 16). Is the empirical value obtained from long-term experiments);
(3 ) 以标准品 0信号值、 标准品 1信号值、 标准品 2信号值 标准品 n-1 信号值、 标准品 n信号值为 X变量， 标准品 0浓度、 标准品 1浓度、 标准 品 2浓度 标准品 n-1浓度、 标准品 n浓度为 Y变量的 n+1个点进行最小 二乘法的二次曲线拟合 （Y=aX2+bX+c)， 得到曲线的回归系数 R (其中具 体应用函数是： =丄^;标准品浓度 Ζ· (标准品 ^)2 Fc (3) Take the standard 0 signal value, standard 1 signal value, standard 2 signal value standard n-1 signal value, standard n signal value as X variable, standard 0 concentration, standard 1 concentration, standard 2 concentration standard n-1 concentration, standard n concentration is n + 1 points of the Y variable, and a quadratic curve fitting of the least square method is performed (Y = aX2 + bX + c) to obtain the regression coefficient R of the curve (where The specific application function is: = 丄 ^; standard concentration Z · (standard ^) 2 F c
=0 i=0 i  = 0 i = 0 i
， R= Fc/ Sc);  , R = Fc / Sc);
(4) 如果 R>0.98 同时曲线单调递增， 则把拟合结果作为本次拟合输 出； 判断曲线是否单调的方法， 可根据二次曲线的特性， 利用计算机算法验 证， 在本实施例中， 判断 R>0.98且曲线是否单调的方法是， (4) If R> 0.98 and the curve is monotonically increasing, the fitting result is used as the current fitting output. The method for determining whether the curve is monotonic can be verified by a computer algorithm based on the characteristics of the quadratic curve. In this embodiment, The method to determine whether R> 0.98 and the curve is monotonic is:
① 如果不满足上述的条件， 即： R<0.98 或曲线不是单调递增， 迸行去 除 1个点， 即 n个点进行拟合； 此时， 如果标准品 n的信号值>1.3倍标准 品 n-1， 则把标准品 n去除， 并回到步骤 c进行 n-1点拟合；  ① If the above conditions are not met, that is: R <0.98 or the curve is not monotonically increasing, limply remove 1 point, that is, n points for fitting; at this time, if the signal value of the standard n is> 1.3 times the standard n -1, remove the standard n and return to step c to perform n-1 point fitting;
② 如果不满足 ① 的条件， 则分别去除 (即每次仅去除一个点)标准品 1、 标准品 2， ......， 标准品 n— 1， 标准品 n, 进行 n个点拟合， 得到 3组 拟合结果 （R值和曲线单调性）， 选择最佳的 1 组拟合作为本次拟合输出； 而在实际操作中具体方法是： 首先排除拟合后曲线不单调的组， 接着在剩下 的组中选择 R值最大的作为本次拟合输出， 如果 3 组拟合后的曲线都不是 单调的， 则算法放弃最小二乘法的二次曲线拟合， 釆用直线拟合算法拟合结 果作为本次拟合输出；  ② If the conditions of ① are not satisfied, remove (ie remove only one point at a time) standard 1, standard 2, ..., standard n-1, standard n, and perform n-point simulation. Then, the three sets of fitting results (R value and curve monotonicity) are obtained, and the best one set of fittings is selected as the output of this fitting. In practice, the specific method is: firstly exclude the non-monotonic curves after fitting. Group, and then select the largest R value in the remaining groups as the output of this fitting. If the fitted curves of the three groups are not monotonic, the algorithm gives up the least square method of quadratic curve fitting and uses a straight line. The fitting result of the fitting algorithm is used as the output of this fitting;
(5 ) 拟合完成， 给出标准曲线。 第三， 数据分析， 用模板对芯片上多个子阵列进行定位， 在本实施例 中， 先采用大模板对芯片上多个子阵列进行定位， 再采用小模板对每个子阵 列的待测样本进行定位， 对各信号点进行定位， 通过计算机分析处理器识别 亮点灰度， 且转换为点信号值， 然后采集每个待测样本信号中最亮的 4个象 素灰度且以平均值作为各点的图像最终信号值， 由上述 2) 步骤中 "信号- 浓度"二次标准曲线找到待测样本中各标志物的浓度值。 (5) The fitting is completed, and a standard curve is given. Third, data analysis uses a template to locate multiple sub-arrays on the chip. In this embodiment, a large template is used to locate multiple sub-arrays on the chip, and then a small template is used to locate the test samples of each sub-array. Positioning each signal point, identifying the gray level of the bright point by a computer analysis processor, and converting it into a point signal value, and then collecting the brightest 4 pixel gray levels in each sample signal to be measured and using the average value as each point For the final signal value of the image, the concentration value of each marker in the sample to be tested is found from the "signal-concentration" quadratic standard curve in step 2) above.
第四， 输出报表， 将芯片上每个待测样本中各个标志物的浓度测定值 以 Excel表格的形式输出。  Fourth, output a report, and output the concentration measurement value of each marker in each test sample on the chip in the form of an Excel table.
下面就本发明中计算机分析处理器 60对多标志物蛋白芯片信号进行定 量检测分析处理的具体操作处理的实例说明。  The following is an example of a specific operation process of the computer analysis processor 60 in the present invention for performing quantitative detection and analysis on the signal of the multi-marker protein chip.
请结合图 1和图 3参见图 4和图 5所示， 图 4和图 5是对多标志物蛋 白芯片获取图像的说明。 本发明可用于检测 12种肿瘤标志物： AFP、 PSA、 free-PSA, HGH、 β - hCG、 CEA、 NSE、 CA19- 9、 CA242, CA15- 3、 CA125、 Ferritin, 将上述被测的并已产生化学光的蛋白芯片置于暗盒中的样品台上；  Please refer to FIG. 4 and FIG. 5 in conjunction with FIG. 1 and FIG. 3. FIG. 4 and FIG. 5 are descriptions of images acquired by the multi-marker protein chip. The invention can be used to detect 12 tumor markers: AFP, PSA, free-PSA, HGH, β-hCG, CEA, NSE, CA19-9, CA242, CA15-3, CA125, Ferritin. The chemiluminescence-producing protein chip is placed on the sample stage in the cassette;
在计算机分析处理器上设置参数， 以对蛋白芯片进行图像拍摄； 在本 实施例中， 设置曝光时间为 10秒至 5分钟中的一个定值， 如： 15秒或 60 秒， 图中.设置曝光时间为 30秒； 实现方法为： 从图 4所示的曝光时间设置 对话框中输入曝光时间 m_ExpTime， 传给变量 exposure— time， 由函数 PICM—SetExposure (exposure— time, &error )来控制爆光时间；  Set the parameters on the computer analysis processor to image the protein chip. In this embodiment, set the exposure time to a fixed value from 10 seconds to 5 minutes, such as 15 seconds or 60 seconds, as shown in the figure. Settings The exposure time is 30 seconds. The implementation method is as follows: Enter the exposure time m_ExpTime from the exposure time setting dialog shown in Figure 4 and pass it to the variable exposure_time. The function PICM_SetExposure (exposure_time, & error) controls the exposure time. ;
设置 CCD温度为 - 10°C到 - 50°C的任何一个值， 如： - 20°C ; 图中设置 CCD温度为 -20Ό ; 实现方法为： 从图 5所示的温度设置对话框中输入设置 的温度 setTmp, 由函数 PICM— Set— Temperature (setTmp) 来控制 CCD温度； 在点击 "图像获取"菜单获取由摄像***拍摄的芯片图像，. 存贮在 计算机中。  Set the CCD temperature to any value from -10 ° C to -50 ° C, such as:-20 ° C; set the CCD temperature to -20Ό in the figure; the implementation method is: input from the temperature setting dialog shown in Figure 5 The set temperature setTmp is controlled by the function PICM— Set— Temperature (setTmp); click on the “Image Acquisition” menu to obtain the chip image captured by the camera system, and store it in the computer.
请结合图 1和图 3参见图 6至图 8所示， 图 6至图 8是本发明中计算 机分析处理器的工作界面图和对比度调整前后的芯片信号图像。 本发明中计 算机分析处理器 60对多标志物蛋白芯片信号进行图像处理时， 在计算机分 析处理器的工作界面上点击 "图象变换"菜单， 按需要可进行 "放大"、 "对 比度增强"等处理， 用以调整图象显示的大小、 对比度等， 还可用 "填充" 功能去除芯片图象中的杂点。 实现方法如下： ( 1 ) 放大 Please refer to FIG. 6 to FIG. 8 in conjunction with FIG. 1 and FIG. 3. FIG. 6 to FIG. 8 are working interface diagrams of the computer analysis processor and chip signal images before and after contrast adjustment in the present invention. In the present invention, when the computer analysis processor 60 performs image processing on the signal of the multi-marker protein chip, click the "Image Transformation" menu on the working interface of the computer analysis processor, and perform "enlargement", "contrast enhancement", etc. as required. Processing, used to adjust the image display size, contrast, etc., can also use the "fill" function to remove noise in the chip image. The implementation method is as follows: (1) Zoom in
先用鼠标选定要放大的图象区域， 计算 x、 y方向的放大倍数 tx=子窗 口宽度 /选定图象的宽度， ty=子窗口高度 /选定图象的高度， 图象放大倍数 m_Scale取 ΐχ， ty中较小值；  First select the image area to be enlarged with the mouse, and calculate the magnifications in the x and y directions. Tx = the width of the subwindow / the width of the selected image, ty = the height of the subwindow / the height of the selected image, the magnification of the image m_Scale takes the smaller of ΐχ, ty;
计算选定区域放大前的中心位置 Centerl , 则放大后的中心位置为 Center2=Centerl*m— Scale, 图象放大 m— Scale倍后， 视图滚动条需水平滚 动 Scrollpt. X - Center2. x -rectClient. right/2, 垂直滚动 Scrollpt. y = Center2. y-rectClient. bottom/2。  Calculate the center position Centerl before zooming in the selected area, then the center position after zooming is Center2 = Centerl * m— Scale, after the image is enlarged m— Scale times, the view scroll bar needs to scroll horizontally Scrollpt. X-Center2. X -rectClient right / 2, vertical scroll Scrollpt. y = Center2. y-rectClient. bottom / 2.
. (2) 调整对比度  (2) Adjusting the contrast
、 ①用鼠标选定要调节对比度的区域， 计算区域中象素的个数 nValidPixel, 将这 nValidPixel 个象素的灰度值放在 nSort 中， 用函数 heapsort (nSort, nValidPixel)按灰度值大小对 nSort排序， 取第 95%个 象素的灰度值作为上限， dSMax = nSort [ (0. 95*nValidPixel-l) ] , 取第 5% 个象素的灰度值作为下限， dSMin 二 nSort [0. 05*nValidPixel].; ① Use the mouse to select the area where the contrast is to be adjusted, calculate the number of pixels in the area, nValidPixel, place the gray value of these nValidPixel pixels in nSort, and use the function heapsort (nSort, nValidPixel) to size the gray value Sort nSort, take the gray value of the 95th pixel as the upper limit, dSMax = nSort [(0. 95 * nValidPixel-l)], take the gray value of the 5% pixel as the lower limit, and dSMin two nSort [0. 05 * nValidPixel] .;
②计算图象显示时的灰度值 mybits (取值 0-255)， 若图象原始灰度 值 data (0-4095)大于 dSMax,则 mybits=255; 若小于 dSMin, 则 mybits=0; 否 贝 IJ 按 图 143 · 所 示 的 直 线 计 算 mybits 的 值 ， 即 mybits= (int) (*data*dA+dB) ， dA二 255. 0/ (double) (dSMax—dSMin) ， dB= - dA*dSMin。  ② Calculate the gray value mybits (value 0-255) when the image is displayed. If the original gray value data (0-4095) of the image is greater than dSMax, then mybits = 255; if it is less than dSMin, then mybits = 0; no Bei IJ calculates the value of mybits according to the straight line shown in Figure 143 ·, that is, mybits = (int) (* data * dA + dB), dA 2255. 0 / (double) (dSMax—dSMin), dB =-dA * dSMin.
从图 7 可见， 对比度调整前芯片图像模糊不清， 而从图 8可见， 经过 对比度调整后芯片图像变得清晰；  It can be seen from FIG. 7 that the chip image is blurred before the contrast adjustment, and it can be seen from FIG. 8 that the chip image becomes clear after the contrast adjustment;
(3) 填充  (3) Fill
'对于杂点进行填充处理， 先用鼠标选定杂点区域， 用值 SpotValue 来 替填充该区域， SpotValue 的计算方法是： 在选定的杂点区域外 （3个象素 宽度）， 找灰度值在 40~150 之间的象素， 取这些象素的平均值作为 SpotValue, 若点的个数为 0， 则 SpotValue取 80。  'To fill in the noise, first select the noise area with the mouse, and fill the area with the value SpotValue. The calculation method of SpotValue is: outside the selected noise area (3 pixels width), find gray For pixels with a degree value between 40 and 150, take the average of these pixels as the SpotValue. If the number of points is 0, the SpotValue is 80.
请结合图 1和图 3参见图 9和图 16所示， 图 9是本发明对芯片信号分 析处理中二次标准曲线拟合的流程框图。  Please refer to FIG. 9 and FIG. 16 in conjunction with FIG. 1 and FIG. 3, and FIG. 9 is a flow block diagram of the quadratic standard curve fitting in the chip signal analysis processing of the present invention.
从图 9 中可见， 在设定了五个标准品的蛋白芯片中， 标准曲线的拟合 过程如下： a 获取蛋白芯片上标准品 0、 标准品 1、 标准品 2、 标准品 3、 标准品 4的信号值； It can be seen from Figure 9 that in a protein chip with five standards set, the standard curve fitting process is as follows: a Obtain the signal values of standard 0, standard 1, standard 2, standard 3, and standard 4 on the protein chip;
b 对标准品 0进行修正， 如果标准品 0'的信号值超出限定表中的上限 值， 则把标准品 0 的值调整为限定表 (可见图 16)中相应的值， 限定表是长 期实验得到的经验值；  b Correct standard 0. If the signal value of standard 0 'exceeds the upper limit in the limit table, adjust the value of standard 0 to the corresponding value in the limit table (see Figure 16). The limit table is long-term Experimental experience
c 进行以标准品 0信号值、 标准品 1信号值、 标准品 2信号值、 标准 品 3信号值、 标准品 4信号值为 X变量， 标准品 0浓度、 标准品 1浓度、 标 准品 2浓度、 标准品 3浓度、 标准品 4浓度为 Y变量的 5点最小二乘法的二 次曲线拟合 （ Y=aX2+bX+c )， 得到曲线的回归系数 R (具体方法如下： 丄 ¾标准品浓度 ζ· ∑ (aXi + bXi + c - A)2 Υ (标准品 - Af c Performs the X-variable with the standard 0 signal value, standard 1 signal value, standard 2 signal value, standard 3 signal value, standard 4 signal value, and standard 0 concentration, standard 1 concentration, and standard 2 concentration. The standard point 3 concentration and standard 4 concentration are the 5-point least square method of the quadratic curve fitting of the Y variable (Y = aX 2 + bX + c) to obtain the regression coefficient R of the curve (the specific method is as follows: 丄 ¾standard Product concentration ζ · ∑ (aXi + bXi + c-A) 2 Υ (Standard-Af
Α= 4 '·=ο ， Fc= - ， Sc= -o ， R= Fc/ Sc) ; d 如果 R>0. 98 同时曲线单调递增 （判断曲线是否单调的方法： 根据 二次曲线的特性， 利用计算机算法验证)， 则把拟合结果作为本次拟合输出； 如果不满足上述的条件 （即： R<0. 98 或曲线不是单调递增）， 进行去除 1个点 （即 4点） 拟合； 此时， 如果标准品 4的信号值 >1. 3倍标准品 3， 则 把标准品 4去除， 并回到步骤 3进行 4点拟合； 如果不满足 3. 2. 1的条件， 分别去除标准品 1、 标准品 '2、 标准品 3进行 4点拟合， 得到 3组拟合结果 (R值和曲线单调性）， 选择最佳的 1 组拟合作为本次拟合输出 （具体方法 是： 首先排除拟合后曲线不单调的组， 接着在剩下的组中选择 R值最大的作 为本次拟合输出， 如果 3组拟合后的曲线都不是单调的， 则算法放弃最小二 乘法的二次曲线拟合， 采用直线拟合算法拟合结果作为本次拟合输出； Α = 4 '· = ο, Fc =-, Sc = -o, R = Fc / Sc) ; d If R> 0. 98 At the same time, the curve monotonically increases (the method of judging whether the curve is monotonic: according to the characteristics of the quadratic curve, Use computer algorithm to verify), then use the fitting result as the current fitting output; if the above conditions are not met (ie: R <0.98 or the curve is not monotonically increasing), remove 1 point (ie 4 points). At this time, if the signal value of the standard 4 is> 1.3 times that of the standard 3, remove the standard 4 and return to step 3 to perform a 4-point fitting. If the condition of 3.2.1 is not satisfied, Remove standard 1, standard '2, and standard 3 and perform a 4-point fit to obtain 3 sets of fit results (R value and curve monotonicity). Select the best set of fits as the output of this fit ( The specific method is: first exclude the group whose curve is not monotonic after fitting, and then select the largest R value in the remaining groups as the output of this fitting. If the three groups of fitted curves are not monotonic, the algorithm will give up Least squares quadratic curve fitting, using the straight line fitting algorithm to fit the results as the current fitting output
f 拟合完成， 给出标准曲线。  f The fitting is completed and the standard curve is given.
由于蛋白芯片反应本身存在各种不可预料因素， 因此对于步骤 a 中获 得的标准品 0、 标准品 1、 标准品 2、 标准品 3、 标准品 4的信号值， 从各点 独立的角度看， 存在着信号值上下偏差的客观事实， 但从 5个点整体趋势的 角度看， 则有明显的趋势走向。 基于这样的事实， 我们采用最小二乘法的二 次曲线拟合的方法用 Y X2+bX+c 公式来描述趋势图； 同时， 考虑到标准品 各点信号值的特殊情况 （如出现上下偏差较大等情况）， 可采用去点拟合多 步比较的方法或运用多种拟合方法， 以尽量减小这种偏差造成的对拟合的影 响。 Since there are various unpredictable factors in the protein chip reaction itself, for the signal values of standard 0, standard 1, standard 2, standard 3, and standard 4 obtained in step a, from each point of view, There are objective facts about the deviation of the signal values, but from the perspective of the overall trend of the five points, there is a clear trend. Based on this fact, we use the method of quadratic curve fitting of the least squares method to describe the trend graph with the formula YX 2 + bX + c; At the same time, considering the special situation of the signal value of each point of the standard (such as the upper and lower deviation Etc.), you can use a point-fitting multi-step comparison method or multiple fitting methods to minimize the effect on the fit caused by this deviation. ring.
请结合图 1和图 3参见图 10至图 15所示， 图 10至图 15是本发明对 芯片信号分析处理中数据分析和输出操作所相关的标准曲线图、 蛋白芯片阵 列图、 检测结果报告单、 定位信息的对话框、 对比度变换曲线图。 数据分析 和输出操作如下；  Please refer to FIGS. 10 to 15 in conjunction with FIG. 1 and FIG. 3. FIGS. 10 to 15 are standard curve diagrams, protein chip array diagrams, and test result reports related to data analysis and output operations in chip signal analysis and processing of the present invention. Single, positioning information dialog, contrast transformation curve. Data analysis and output operations are as follows;
1 ) 建立指标对话框， 当分析对象涉及 X个指标 （分别为指标 1、 指标 2、 ……、 指标 X ) 时， 在对话框中输入各标准品的浓度值， 依次赋值给变 量 m一指标 1、 m—指标 2、 ……、 m一指标 x， 输入各标准品浓度梯度值， 存到 数组 StandardC [X] [n] (n指浓度梯度的数量） 中， 再从图像中读出各标准 品的信号值， 方法是： 用小模板给每个标准品阵列定位， 调节该模板的阵列 行数、 列数、 行间距、 列间距、 模板大小、 模板位置使定位后的模板所有方 格刚好固定了一个子列阵的所有信号点， 且每个小方格只含有 1个信号点。 取每个小方格中最亮 4 个象素灰度平均值作为各点的信号值 StandardPixAve [i] [SD] , 根据浓度值、 信号值分别按照实施例 3 拟合出 x 种标志物的标准曲线。 对应于各浓度梯度， 拟合出的信号值记为 Huidu [i] [SD] ,对每种肿瘤标志物可使用不同拟合方式，如 x=ay2+by+c， y=a lg (x) +b， y-a+bxc/ ( 1+dxe)等（其中 x代表浓度值， y代表信号值或以 x 代表信号值， y代表浓度值)， 也可运用多种拟合方式的组合。如图 10所示: 蛋白芯片中 CA125标志物的二次标准曲线图例， 其中， X轴代表浓度值， y 轴代表信号值。 1) Establish an indicator dialog box. When the analysis object involves X indicators (respectively indicator 1, indicator 2, ..., indicator X), enter the concentration values of each standard in the dialog box, and assign values to the variable m an indicator in turn. 1. m—indicator 2, ……, m—indicator x, input the concentration gradient value of each standard and store it in the array St a nd a rdC [ X ] [n] (n is the number of concentration gradients), and then from the image The signal value of each standard is read out. The method is: use a small template to position each standard array, and adjust the array row, column number, row spacing, column spacing, template size, and template position of the template to make the positioning All the squares of the template are just fixed with all the signal points of a sub-array, and each small square contains only one signal point. Take the average value of the brightest 4 pixels in each small square as the signal value of each point StandardPixAve [i] [SD], and according to the concentration value and signal value, fit the x markers according to Example 3 respectively. standard curve line. Corresponding to each concentration gradient, the fitted signal value is recorded as Huidu [i] [SD], and different fitting methods can be used for each tumor marker, such as x = ay 2 + by + c, y = a lg ( x) + b, y-a + bxc / (1 + dxe), etc. (where x represents the concentration value, y represents the signal value, or x represents the signal value, and y represents the concentration value). Various fitting methods can also be used. combination. As shown in Figure 10: A legend of the quadratic standard curve of the CA125 marker in the protein chip, where the X-axis represents the concentration value and the y-axis represents the signal value.
2) 检测待测样本中各肿瘤标志物浓度  2) Detect the concentration of each tumor marker in the test sample
若一次只处理单个未知禅本：  To process only a single unknown Zen text at a time:
用小模板给每个未知样本对应的阵列定位 （该模板阵列行、 列， 大小、 位置均可调）， 取每个小方格中最亮 4个象素灰度平均值作为各点的信号值 StandardPixAve [i] [SD] , 根据前述 2作出的标准曲线的方程， 计算出各点 的浓度值。  Use a small template to position the array corresponding to each unknown sample (the template array can be adjusted in rows, columns, size and position), and take the average value of the brightest 4 pixels in each small square as the signal of each point The value StandardPixAve [i] [SD], according to the equation of the standard curve made in the above 2, calculates the concentration value at each point.
若一次性处理多个未知样本：  To process multiple unknown samples at once:
假设样本数是 48个， 排列为 6*8个子阵列， 图 11所示。  Assume that the number of samples is 48 and arranged in 6 * 8 sub-arrays, as shown in Figure 11.
(1)调整大模板的大小， 同时上下左右移动其位置， 使其与图像的各个 阵列大致重叠， 以该模板中 48个方格的左上角坐标位置作为每个子阵列的 理想位置 LS—IdealP0int [i] [j]， 然后用小模板对每个子阵列定位， 如图 12 所示， 子阵列排列为 5*5。 (1) Adjust the size of the large template, and move its position up, down, left, and right, so that it overlaps with each array of the image, and use the upper-left coordinate position of the 48 squares in the template as the sub-array The ideal position LS_IdealP 0 i n t [i] [j], and then use a small template to locate each sub-array, as shown in Figure 12, the sub-arrays are arranged 5 * 5.
(2)小模板定位后，会出现定位信息对话框，见图 13， 点击对话框的 "定 位" 按钮， 保存该模板左上角的坐标， 作为该子阵列的实际坐标位置 LS_ActualPoint [i] [j] ， 计 算 并 搜 索 该 坐 标 距 离 最 近 的 LS_IdealPoint [i] [j] , 设定其为该位置的阵列 （如 A1 子阵列）， 隐藏该阵 列所对应的按钮， 据此方法依次给每个子阵列定位。  (2) After positioning the small template, a positioning information dialog box will appear, as shown in Figure 13. Click the "Position" button in the dialog box to save the coordinates of the upper left corner of the template as the actual coordinate position of the sub-array LS_ActualPoint [i] [j ], Calculate and search the nearest LS_IdealPoint [i] [j], set it to the array at that position (such as the A1 sub-array), hide the buttons corresponding to the array, and position each sub-array in turn according to this method .
(3) ·由记录的 LS— ACtUalPoirrt [i] [j]， 一次性计算出每个子阵列的信 号值， 由信号值得出浓度值， 图 14所示的是浓度对比度变换曲线图； (3) • From the recorded LS-A C t Ua Poirrt [i] [j], calculate the signal value of each sub-array at one time, and calculate the concentration value from the signal. Figure 14 shows the concentration contrast conversion curve ;
(4) 将各个子阵列各个指标的浓度值全部输出在 Excel中， 如图 15所 示： 在 Excel表格中显示出芯片上待测样本中 12个肿瘤标志物浓度值。  (4) Output the concentration values of each index of each sub-array in Excel, as shown in Figure 15: The Excel table displays the concentration values of the 12 tumor markers in the sample to be tested on the chip.
综上所述， 本发明多标志物蛋白芯片信号的分析***及其分析方法， 由于采用了上述的技术方案和操作步骤， 不仅能获得高质量的图像分辨率， 且可以准确地检测出蛋白芯片中多个待测样品中多种目标蛋白， 即标志物的 浓度； 同时， 分析检测数据重现性好， 稳定可靠， 操作简便直观； 另外， 并 可对多个蛋白芯片 （每个蛋白芯片上有多个被测物质的信号） 的信号进行同 时读取和计算， 极大地节省了数据分析时间； 而且， 该信号分折***可靠稳 定， 适用范围大， 不仅可用于蛋白芯片试剂盒临床应用时的定量仪器， 还可 与其它生物芯片结合使用， 因此极为实用。  In summary, the multi-marker protein chip signal analysis system and analysis method of the present invention adopts the above technical solutions and operation steps, which not only can obtain high-quality image resolution, but also can accurately detect the protein chip. Multiple target proteins in multiple samples to be tested, that is, the concentration of the marker; At the same time, the analytical data is reproducible, stable and reliable, and the operation is simple and intuitive; In addition, multiple protein chips (each protein chip can be used) There are multiple signals of the substance under test) to simultaneously read and calculate the signals, which greatly saves the time of data analysis; moreover, the signal analysis system is reliable and stable, and has a wide range of applications, which can be used not only in the clinical application of protein chip kits The quantitative instrument can also be used in combination with other biochips, so it is extremely practical.

Claims

权利要求 Rights request
1. 一种多标志物蛋白芯片信号的分析***， 包括： 一底板 （10)， 安 置在底板（10)上的暗盒（20)， 位于暗盒（20)上且与之连通的皮腔（30)， 通过皮腔紧框 （31 ) 设置在皮腔 （30) 上且与之连接的摄像装置 （40); 其 特征在于还包括： 一光密闭的 X-Y-Z- Θ定位调节装置 （50) 和一计算机分 析处理器 (60)； 1. A multi-marker protein chip signal analysis system, comprising: a base plate (10), a cassette (20) disposed on the base plate (10), and a skin cavity (30) located on and communicating with the cassette (20) ), A camera device (40) disposed on and connected to the skin cavity (30) through the skin cavity tight frame (31); and further comprising: a light-tight XYZ-Θ positioning adjustment device (50) and a Computer analysis processor (60);
所述的 X-Y-Z- Θ定位调节装置 （50) 包括： 一摄像装置连接器 （51 )， 该摄像装置连接器 （51.)紧固在摄像装置 （50) 下部； 一 X-Y方向调节架 (52)， 它的一端与摄像装置 （50)连接； 一 Z方向调节架 （53 )， 它的一 端与 X-Y方向调节架 （52)连接， 另一端固定在底板 （10) 上； 一三角支 撑架 （54)， 用以支撑 X-Y方向调节架 （52) 和 Z方向调节架 （53 );  The XYZ-Θ positioning adjustment device (50) includes: a camera device connector (51), the camera device connector (51.) is fastened to the lower part of the camera device (50); an XY direction adjustment frame (52) Its one end is connected to the camera device (50); a Z-direction adjusting frame (53), one end of which is connected to the XY-direction adjusting frame (52), and the other end is fixed on the base plate (10); a triangular supporting frame (54 ) For supporting the XY direction adjusting frame (52) and the Z direction adjusting frame (53);
所述的计算机分析处理器 （.60) 具有数据采集卡， 其中存储了相应的 图像分析应用程序， 用于存储摄像装置 （40) 所采集到的图像数据 寸 图像数据信号进 分析处理。  The computer analysis processor (.60) has a data acquisition card, in which a corresponding image analysis application program is stored, which is used to store the image data acquired by the camera device (40). The image data signals are processed for analysis.
2. 如权利要求 1 所述的多标志物蛋白芯片信号的分析***， 其特征在 于： 所述的底板 （10)在其前侧设置两可调节底板 （10) 水平位置的水平调 节螺丝 （11 ); 在所述的底板 （10) 的适当位置处设置暗盒导轨条 （21 )， 所 述的暗盒 （20) 固定在暗盒导轨条（21 ) 内侧。  2. The multi-marker protein chip signal analysis system according to claim 1, characterized in that: the bottom plate (10) is provided on its front side with two level adjustment screws (11) capable of adjusting the horizontal position of the bottom plate (10) ); A cassette guide rail (21) is provided at an appropriate position on the bottom plate (10), and the cassette (20) is fixed inside the cassette guide rail (21).
3. 如权利要求 1 所述的多标志物蛋白芯片信号的分析***， 其特征在 于：  3. The multi-marker protein chip signal analysis system according to claim 1, characterized in that:
所述的摄像装置连接器（51 )包括， 一连接器本体（511 )， 它呈抓手， 围在摄像装置 （50) 下部， 在连接器本体 （511 ) 上设有一摄像装置连接块 ( 512)， 摄像装置连接块 （512) 上设有一摄像装置连接螺丝 （513)， 以将 摄像装置连接器 （51 ) 紧固在摄像装置 （50) 下部；  The camera device connector (51) includes a connector body (511) which is gripped and surrounds the lower part of the camera device (50). A camera device connection block (512) is provided on the connector body (511). ), The camera device connecting block (512) is provided with a camera device connection screw (513) to fasten the camera device connector (51) to the lower part of the camera device (50);
所述的 X-Y方向调节架 （52)包括， X轴调整件架 （521 )及其 X轴调 整手柄 (522), Y轴调整架 （523 )及其 Y轴调整手柄（524);  The X-Y direction adjusting frame (52) includes an X-axis adjusting member frame (521) and its X-axis adjusting handle (522), a Y-axis adjusting frame (523) and its Y-axis adjusting handle (524);
所述的 Z方向调节架 （53) 包括， 一 Z轴基板 （531 )， 设置在 Z轴基 板 （531 ) 上的 Z轴调节旋钮（532)， 与 X-Y方向调节架 （52)连接的 Z动 向板（533 )， 以及 Z轴紧定旋钮 (534)； 所述的 X-Y-Z- Θ定位调节装置 （50) 还包括一三角支撑架 （54)， 它支 撑在 Χ-Υ方向调节架 （52) 下和 Ζ方向调节架 （53 ) 的 Ζ轴基板 （531 ) 内 The Z-direction adjusting frame (53) includes a Z-axis substrate (531), a Z-axis adjusting knob (532) provided on the Z-axis substrate (531), and a Z-movement direction connected to the XY-direction adjusting frame (52). Plate (533), and Z-axis set knob (534); The XYZ-Θ positioning adjustment device (50) further includes a triangular support frame (54), which is supported under the X-Υ direction adjustment frame (52) and the Z-axis substrate (531) of the Z-direction adjustment frame (53). Inside
4. 如权利要求 1 所述的多标志物蛋白芯片信号的分析***， 其特征在 于： 在所述的暗盒 （20) 中设置有样品台， 在该暗盒 （20) 的外壳上设置一 可调节样品台角度的样品台水平调节钮。 4. The multi-marker protein chip signal analysis system according to claim 1, characterized in that: a sample stage is provided in the cassette (20), and an adjustable case is provided on a casing of the cassette (20). The stage level adjustment knob for the stage angle.
5. 一种用上述多标志物蛋白芯片信号的分析***对多标志物蛋白芯片 信号进行定量检测的分析方法， 其特征在于该分析方法包括以下步骤：  5. An analysis method for quantitatively detecting a multi-marker protein chip signal by using the above-mentioned multi-marker protein chip signal analysis system, characterized in that the analysis method includes the following steps:
步骤一， 在计算机分析处理器上设置参数， 包括， 信号分析***中摄 像装置 （40) 曝光时间的设置、 摄像装置本身温度的设置；  Step 1: setting parameters on the computer analysis processor, including setting the exposure time of the camera device (40) in the signal analysis system and setting the temperature of the camera device itself;
步骤二， 所需参数设置后， 分析***中的摄像装置对样品台上蛋白芯 片上的多个光信号进行采集， 并拍摄图像信号； '  Step 2: After the required parameters are set, the camera device in the analysis system collects multiple optical signals on the protein chip on the sample stage and captures the image signals;
步骤三， 对摄像装置所采集的图像信号转化为数字信号后送入计 ί机 分析处理器；  Step three: convert the image signal collected by the camera device into a digital signal and send it to a computer analysis processor;
步骤四， 计算机分析处理器 60 接收图像数据信号后进行如下定量捡测 分析处理步骤：  Step 4. After receiving the image data signal, the computer analysis processor 60 performs the following quantitative detection and analysis processing steps:
1 ) 图像处理， 将选定的图像区域进行放大、 增大图像的对比度、 对图 像上有杂点处进行杂点去除；  1) image processing, enlarging the selected image area, increasing the contrast of the image, and removing the noise points where there are noise points on the image;
2) 作标准曲线， 根据芯片上各标准品中各个标志物的已知浓度值以及 对应的已拍摄的图像的信号值这两种参数， 拟合出各个标志物的 "信号-浓 度" 的标准曲线； '  2) Make a standard curve and fit the "signal-concentration" standard of each marker according to the two parameters of the known concentration of each marker in each standard on the chip and the signal value of the corresponding captured image Curve
3 ) 数据分析， 先采用大模板对芯片上多个子阵列进行定位， 再采用小 模板对每个子阵列的待测样本进行定位， 采集每个待测样本信号中最亮的 4 个象素灰度平均值作为各点的图像最终信号值， 由上述 2) 步骤中 "信号- 浓度"二次标准曲线找到待测样本中各标志物的浓度值；  3) Data analysis, first use a large template to locate multiple sub-arrays on the chip, and then use a small template to locate the test samples of each sub-array, and collect the brightest 4 pixel gray levels in each test sample signal The average value is used as the final signal value of the image at each point, and the concentration value of each marker in the sample to be measured is found from the "signal-concentration" quadratic standard curve in step 2) above;
4)输出报表，将芯片上每个待测样本中各个标志物的浓度测定值以 Excel 表格的形式输出。  4) Output a report, and output the concentration measurement value of each marker in each test sample on the chip in the form of an Excel table.
6. 如权利要求 5所述的多标志物蛋白芯片信号的分析***的分析方法， 其特征在于： 在所述的步骤四的 2) 中， 所述的各个标志物的 "信号-浓度" 的标准曲线的拟合方式可为二次曲线、 双曲线、 对数曲线或各种二次曲线的 组合。 6. The method for analyzing a multi-marker protein chip signal analysis system according to claim 5, characterized in that: in the step 2) of step 4, the "signal-concentration" of each marker is The fitting method of the standard curve can be a quadratic curve, a hyperbola, a logarithmic curve, or a variety of quadratic curves. Combination.
7. 如权利要求 5或 6所述的多标志物蛋白芯片信号的分析***的分析 方法， 其特征在于： 所述的作各个标志物的 "信号-浓度"二次标准曲线拟 合方式具体过程是：  7. An analysis method for a multi-marker protein chip signal analysis system according to claim 5 or 6, characterized in that: the specific process of fitting a "signal-concentration" quadratic standard curve fitting method for each marker Yes:
( 1 ) 获取蛋白芯片上标准品 0、 标准品 1、 标准品 2， …标准品 n-l、 标准品 n的信号值；  (1) Obtaining the signal values of standard 0, standard 1, standard 2,… standard n-1, standard n on the protein chip;
(2) 对标准品 0进行修正， 如果标准品 0的信号值超出限定表中的上 限值， 则把标准品 0的值调整为限定表中相应的值；  (2) Correct the standard 0. If the signal value of the standard 0 exceeds the upper limit in the limit table, adjust the value of the standard 0 to the corresponding value in the limit table;
(3 ) 以标准品 0信号值、 标准品 1信号值、 标准品 2信号值 标准品 n-1信号值、 标准品 n信号值为 X变量， 标准品 0浓度、 标准品 1浓度、 标 准品 2浓度 标准品 n-1浓度、 标准品 n浓度为 Y变量的 n+1个点进行最 小二乘法的二次曲线拟合 （Y=aX2+bX+c), 得到曲线的回归系数 R (其中 具 体 应 用 函 数 是 ： =丄;^标准品浓度 ζ· = (标准品 )2 ^ =∑ +fe(. +c-^()2， R= Fc/ Sc); (3) Take the standard 0 signal value, standard 1 signal value, standard 2 signal value standard n-1 signal value, standard n signal value as X variable, standard 0 concentration, standard 1 concentration, standard 2 concentration standard n-1 concentration, standard n concentration is n + 1 points of Y variable, and perform a least squares quadratic curve fitting (Y = aX2 + bX + c) to obtain the regression coefficient R of the curve (where The specific application function is: = 丄; ^ standard concentration ζ · = (standard) 2 ^ = ∑ + fe ( . + C-^ () 2 , R = Fc / Sc) ;
^ =0 · /=0 ^ = 0 · / = 0
(4) 如果 R>0.98 同时曲线单调递增， 则把拟合结果作为本次拟合输 出；  (4) If R> 0.98 and the curve monotonically increases, the fitting result is used as the current fitting output;
(5) 拟合完成， 给出标准曲线。 (5) The fitting is completed and a standard curve is given.
8. 如权利要求 7所述的多标志物蛋白芯片信号的分析***的分析方法， 其特征在于： 在所述的二次标准曲线拟合方式具体过程（4)中， 判断 R〉0.98 且曲线是否单调的方法是，  8. The analysis method of a multi-marker protein chip signal analysis system according to claim 7, wherein: in the specific process (4) of the quadratic standard curve fitting method, it is determined that R> 0.98 and the curve The monotonous approach is,
①如果不满足上述的条件， 即： R<0.98 或曲线不是单.调递增， 进行去 除 1个点， 即 n个点进行拟合； 此时， 如果标准品 n的信号值〉 1.3倍标准 品 n-l， 则把标准品. n去除， 并回到步骤 c进行 n-1点拟合； '  ① If the above conditions are not met, that is: R <0.98 or the curve is not monotonically increasing, remove 1 point, that is, n points to fit; At this time, if the signal value of the standard n is> 1.3 times the standard nl, remove the standard. n, and return to step c to perform n-1 point fitting;
② 如果不满足 ① 的条件， 则分别去除标准品 1、 标准品 2 ， 标准品 n— 1， 标准品 n进行 n个点拟合， 得到 3组 R值和曲线单调性的拟 合结果， 选择最佳的 1组拟合作为本次拟合输出。  ② If the conditions of ① are not satisfied, then remove standard 1, standard 2, standard n-1, and standard n to perform n-point fitting to obtain three sets of R values and curve monotonicity fitting results. Select The best set of fits is used as the output of this fit.
9. 如权利要求 5所述的多标志物蛋白芯片信号的分析***的分析方法， 其特征在于： 在所述的步骤四的 2) 中， 所述的标准曲线以信号值和标准样 品浓度为变量。 9. The method for analyzing a multi-marker protein chip signal analysis system according to claim 5, wherein in the step 2) of the step 4, the standard curve is based on a signal value and a standard sample. Product concentration is a variable.
10. 如权利要求 5 所述的多标志物蛋白芯片信号的分析***的分析方 法， 其特征在于： 在所述的步骤四的 3) 数据分析中， 先采用大模板对芯片 上多个子阵列进行定位， 再采用小模板对每个子阵列的待测样本进行定位， 对芯片上多个子阵列进行定位， 对各信号点进行定位， 并通过计算机分析处 理器识别亮点灰度， 且转换为电信号值。 '  10. The method for analyzing a multi-marker protein chip signal analysis system according to claim 5, wherein: in step 3) 3) data analysis, a large template is first used to perform multiple subarrays on the chip. Positioning, and then using a small template to locate the sample to be tested for each sub-array, positioning multiple sub-arrays on the chip, locating each signal point, and identifying the gray level of the bright point through a computer analysis processor, and converting it into an electrical signal value . '
PCT/CN2003/000123 2002-08-02 2003-02-08 System and method for analyzing signals in a multi-markers protein chip WO2004013627A1 (en)

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