CN113358650B - A 96-well microplate reader - Google Patents
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Abstract
Description
技术领域Technical Field
本发明属于医学检验或化学分析领域，涉及微孔板阅读器，尤其是一种96孔微孔板阅读仪。The invention belongs to the field of medical inspection or chemical analysis, and relates to a microplate reader, in particular to a 96-well microplate reader.
背景技术Background technique
随着ELISA测定技术等生化分析技术的发展，分析技术呈现出高通量、高灵敏度、小剂量消耗和多重标记等特点和趋势，因此以微孔板已成为高通量生化分析领域的标准器件，以酶标仪为代表的检测设备也成为了生化分析的基础仪器。而生化分析技术的进一步拓展和多样化，也对相应的检测仪器提出了更为复杂的要求，传统的酶标仪在众多场景下的应用已经受到了限制。近年来，为了满足高通量生物分析技术不断发展的需求，出现了多功能微孔板检测仪。在牺牲部分性能指标的前提下，这类检测仪以更高密度的微孔板为载体，使得单位面积可兼容更多的样品数目，在节省大量试剂消耗的同时，可以进行更为微量化地分析；增强了自动化分析处理的能力，可以实现集加样、检测、结果传递、数据处理直至微孔板洗涤为一体的全自动化过程；在原本单一功能的基础上，配合新的分析检测技术，可以同步实现多功能的集成检测。然而，现有的酶标仪和微孔板阅读仪的光源通常为钨灯，存在仪器整体体积、质量较大，携带不便，对使用环境要求较高，检测速度慢，价格昂贵，且维护成本较高、难度较大的不足，极大限制了ELISA测定技术等检测手段在基层的应用场景。With the development of biochemical analysis technologies such as ELISA assay technology, analysis technology has shown characteristics and trends such as high throughput, high sensitivity, low dose consumption and multiple labeling. Therefore, microplates have become standard devices in the field of high-throughput biochemical analysis, and detection equipment represented by microplate readers have also become basic instruments for biochemical analysis. The further expansion and diversification of biochemical analysis technology has also put forward more complex requirements for the corresponding detection instruments. The application of traditional microplate readers in many scenarios has been limited. In recent years, in order to meet the needs of the continuous development of high-throughput bioanalysis technology, multifunctional microplate detectors have emerged. Under the premise of sacrificing some performance indicators, this type of detector uses a higher density microplate as a carrier, so that a larger number of samples can be compatible per unit area, while saving a lot of reagent consumption, more micro-quantitative analysis can be performed; the ability of automated analysis and processing is enhanced, and a fully automated process integrating sample addition, detection, result transmission, data processing and microplate washing can be realized; on the basis of the original single function, combined with new analysis and detection technology, multifunctional integrated detection can be realized simultaneously. However, the light source of existing ELISA readers and microplate readers is usually tungsten lamps, which have the disadvantages of being large in size and weight, inconvenient to carry, having high requirements for the use environment, slow detection speed, high price, high maintenance cost and difficulty, which greatly limits the application scenarios of detection methods such as ELISA assay technology at the grassroots level.
发明内容Summary of the invention
本发明的目的在于克服现有技术的不足，提供一种96孔微孔板阅读仪，解决的问题是：酶标仪和微孔板阅读仪不够便携的问题；酶标仪和微孔板阅读仪检测速度慢的问题；酶标仪和微孔板阅读仪结构复杂，制造和维护成本高的问题。The purpose of the present invention is to overcome the shortcomings of the prior art and provide a 96-well microplate reader to solve the following problems: the problem that the microplate reader and the microplate reader are not portable enough; the problem that the microplate reader and the microplate reader have slow detection speed; the problem that the microplate reader and the microplate reader have complex structures and high manufacturing and maintenance costs.
本发明的目的通过下述技术方案实现：一种96孔微孔板阅读仪，该阅读仪包括交互与中央处理模块、光源模块、控温系统、图像传输系统及图像采集系统，所述交互与中央处理模块通过导线同时与光源模块、控温系统和图像采集系统相连，所述光源模块的一面紧贴控温系统的光源散热器，另一面发出特定颜色的光，光线穿过放置在控温系统的微孔板保温架上的96孔微孔板后，被图像传输系统通过光纤聚集，最终被图像采集系统转换成图像信号传输给交互与中央处理模块。The objective of the present invention is achieved through the following technical scheme: a 96-well microplate reader, which includes an interactive and central processing module, a light source module, a temperature control system, an image transmission system and an image acquisition system. The interactive and central processing module is simultaneously connected to the light source module, the temperature control system and the image acquisition system through wires. One side of the light source module is in close contact with the light source radiator of the temperature control system, and the other side emits light of a specific color. After the light passes through a 96-well microplate placed on a microplate insulation rack of the temperature control system, it is gathered by the image transmission system through optical fibers and finally converted into an image signal by the image acquisition system and transmitted to the interactive and central processing module.
进一步地，所述的光源模块按照微孔板96个孔的对应位置分布有96组发光源，每组发光源包含红、绿、蓝发光源各一个，光源模块的发光面顶部盖有匀光板。Furthermore, the light source module has 96 groups of light sources distributed according to the corresponding positions of the 96 holes of the microplate, each group of light sources includes one red light source, one green light source and one blue light source, and the top of the light-emitting surface of the light source module is covered with a light homogenizing plate.
进一步地，所述的控温系统包含导热液循环泵、光源散热器、第一导热液连接管、第二导热液连接管、微孔板保温架、加热线圈及温度传感器，所述导热液循环泵与光源散热器相连，所述光源散热器材质为铜、铝、银等导热性佳的金属，其内部有导热液流路，底面紧贴光源模块，所述第一导热液连接管与第二导热液连接管连接光源散热器与微孔板保温架形成导热液密闭回路，所述微孔板保温架材质为铜、铝、银等导热性佳的金属，内部有导热液流路且在对应96孔微孔板上各孔的位置留有通孔，所述加热线圈环绕紧贴微孔板保温架中上部，所述温度传感器紧贴微孔板保温架表面。Furthermore, the temperature control system includes a thermal liquid circulation pump, a light source radiator, a first thermal liquid connecting pipe, a second thermal liquid connecting pipe, a microplate insulation rack, a heating coil and a temperature sensor. The thermal liquid circulation pump is connected to the light source radiator. The light source radiator is made of metals with good thermal conductivity such as copper, aluminum, silver, etc., and has a thermal liquid flow path inside. The bottom surface is close to the light source module. The first thermal liquid connecting pipe and the second thermal liquid connecting pipe connect the light source radiator and the microplate insulation rack to form a thermal liquid closed loop. The microplate insulation rack is made of metals with good thermal conductivity such as copper, aluminum, silver, etc., and has a thermal liquid flow path inside and through holes are left at the positions corresponding to the holes on the 96-well microplate. The heating coil surrounds and is close to the upper and middle part of the microplate insulation rack, and the temperature sensor is close to the surface of the microplate insulation rack.
进一步地，所述的图像传输系统包含聚光透镜、光纤固定座、光纤束及光纤集束器，所述聚光透镜安装在微孔板保温架96个通孔内与下方光纤束截面的距离为透镜的焦距，每个通孔底部分别安装有光纤固定座，所述光纤束由96根光纤组成，一端同轴连接光纤固定座，另一端在光纤集束器汇聚成一束。Furthermore, the image transmission system includes a focusing lens, a fiber fixing seat, a fiber bundle and a fiber buncher. The focusing lens is installed in 96 through holes of the microplate insulation rack and the distance between it and the cross section of the fiber bundle below is the focal length of the lens. A fiber fixing seat is installed at the bottom of each through hole. The fiber bundle consists of 96 optical fibers, one end of which is coaxially connected to the fiber fixing seat and the other end is gathered into a bundle in the fiber buncher.
进一步地，所述的图像采集系统包含滤光片选择器、滤光片、图像采集透镜及摄像头，所述滤光片选择器上同径均布安装有若干个滤光片，滤光片可调整到与图像传输系统中光线集束器同轴的位置，所述图像采集透镜和摄像头依次与光纤集束器同轴安装，光纤集束器上为滤光片再上方为摄像头，光纤集束器与摄像头的间距为图像采集透镜的焦距。Furthermore, the image acquisition system includes a filter selector, a filter, an image acquisition lens and a camera. The filter selector is provided with a plurality of filters evenly distributed with the same diameter. The filter can be adjusted to a position coaxial with the light beam bundler in the image transmission system. The image acquisition lens and the camera are coaxially installed with the optical fiber bundler in sequence. The optical fiber bundler is provided with a filter and the camera is located above the filter. The distance between the optical fiber bundler and the camera is the focal length of the image acquisition lens.
进一步地，所述的交互与中央处理模块带有一块触摸屏，可对光源模块的颜色、亮度、色温以及控温系统保持的温度进行调制，且能够对图像的RGB参数进行提取，并根据放置标准品的特定位置的参数值计算出标准曲线，再将其他位置的参数值代入标准曲线进行比较得到检测结果，并将图像检测结果呈现给用户或共享给其他智能终端。Furthermore, the interaction and central processing module is equipped with a touch screen, which can modulate the color, brightness, color temperature of the light source module and the temperature maintained by the temperature control system, and can extract the RGB parameters of the image, and calculate the standard curve based on the parameter values of the specific position where the standard is placed, and then substitute the parameter values of other positions into the standard curve for comparison to obtain the detection results, and present the image detection results to the user or share them with other smart terminals.
本发明的原理：ELISA测定技术等部分生化分析技术是通过对微孔板中反应底物的吸光度进行定量分析来获得检测结果的。而吸光度在图像技术中可通过图像的颜色深浅程度来反映，并可通过背景光颜色、色温等参数的调制以及滤光片的使用来提高图像对吸光度反映的准确性和灵敏度。本发明的原理是当ELISA等生化检测步骤完成后，装有反应底物的微孔板放置于微孔板阅读仪的对应位置，光源按照调制的参数发出的光透射过反应底物被透镜汇聚后投射到光纤截面上。光纤可以无损改变光纤光路，并通过光纤的集束缩小成像面积。最终摄像头获取到光纤集束后的图像，并根据图像中相应位置的颜色状况来分析对应反应底物中特定物质的浓度等信息。Principle of the present invention: Some biochemical analysis techniques such as ELISA determination technology obtain the test results by quantitatively analyzing the absorbance of the reaction substrate in the microplate. In image technology, the absorbance can be reflected by the color depth of the image, and the accuracy and sensitivity of the image to the absorbance can be improved by modulating the parameters such as the background light color and color temperature and using filters. The principle of the present invention is that after the biochemical detection steps such as ELISA are completed, the microplate containing the reaction substrate is placed in the corresponding position of the microplate reader, and the light emitted by the light source according to the modulated parameters is transmitted through the reaction substrate and converged by the lens and projected onto the cross section of the optical fiber. The optical fiber can change the optical fiber light path without loss, and reduce the imaging area by bundling the optical fiber. Finally, the camera obtains the image after the optical fiber is bundled, and analyzes the concentration of the specific substance in the corresponding reaction substrate according to the color condition of the corresponding position in the image.
本发明相对于现有技术具有如下的优点及效果：Compared with the prior art, the present invention has the following advantages and effects:
(1)本发明较酶标仪有更高的灵敏度，更短的分析时间，并通过相应的软件具有更为直观和灵活的显示方式，实现诊断报告的实时出具、长期存储和通过网络分享。(1) The present invention has higher sensitivity and shorter analysis time than an ELISA instrument, and has a more intuitive and flexible display mode through corresponding software, thereby realizing real-time issuance, long-term storage and sharing of diagnostic reports through the Internet.
(2)本发明体积小、重量轻，便携性较好，操作简单，更好地满足了POCT的需求，能够更灵活地适应各种环境，可以实现现场和床边检测，更适合于基层医疗单位和家庭用检测使用来应对突发性公共卫生事件。(2) The present invention is small in size, light in weight, highly portable, and easy to operate, which better meets the needs of POCT. It can adapt to various environments more flexibly, can realize on-site and bedside testing, and is more suitable for primary medical units and families to use for testing to respond to sudden public health events.
(3)本发明结构简单、功耗较低，提高了仪器的稳定性和可靠性，同时也降低了仪器的购置和维护成本。(3) The present invention has a simple structure and low power consumption, which improves the stability and reliability of the instrument and also reduces the purchase and maintenance costs of the instrument.
(4)本发明应用场景较广，可用于ELISA、分光光度法等多种检测手段和项目。(4) The present invention has a wide range of application scenarios and can be used in a variety of detection methods and projects such as ELISA and spectrophotometry.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1为本发明的结构示意图，其中：1-交互与中央处理模块、2-摄像头、3-图像采集透镜、4-滤光片、5-滤光片选择器、6-光纤集束器、7-光纤束、8-光纤固定座、9-温度传感器、10-加热线圈、11-微孔板保温架、12-聚光透镜、13-试剂盒、14-第一导热液连接管、15-第二导热液连接管、16-光源模块、17-光源散热器、18-导热液循环泵；Figure 1 is a schematic diagram of the structure of the present invention, wherein: 1-interaction and central processing module, 2-camera, 3-image acquisition lens, 4-filter, 5-filter selector, 6-fiber bundler, 7-fiber bundle, 8-fiber fixing seat, 9-temperature sensor, 10-heating coil, 11-microplate insulation rack, 12-focusing lens, 13-test kit, 14-first heat transfer liquid connecting pipe, 15-second heat transfer liquid connecting pipe, 16-light source module, 17-light source radiator, 18-heat transfer liquid circulation pump;
图2为本发明光源模块的结构示意图，其中：19-匀光板、20-红色发光源、21-绿色发光源、22-蓝色发光源。FIG2 is a schematic diagram of the structure of the light source module of the present invention, wherein: 19 is a light homogenizing plate, 20 is a red light source, 21 is a green light source, and 22 is a blue light source.
具体实施方式Detailed ways
以下结合附图对本发明的实施例做进一步详述；本实施例是描述性的，不是限定性的，不能由此限定本发明的保护范围。The following is a further detailed description of an embodiment of the present invention in conjunction with the accompanying drawings; this embodiment is descriptive rather than restrictive, and the protection scope of the present invention cannot be limited thereby.
实施例1Example 1
如图1和图2所示，本发明提供了一种96孔微孔板阅读仪，包括交互与中央处理模块、光源模块、控温系统、图像传输系统及图像采集系统，交互与中央处理模块1带有一块大触摸屏，通过导线与光源模块16、控温系统的温度传感器9和加热线圈10以及图像采集系统的摄像头2相连，可获取温度传感器9的反馈结果并调制光源模块16和加热线圈10的参数，同时能够处理或与其他智能终端共享采集到的图像结果；光源模块16按照微孔板96个孔的对应位置分布有96组发光源，每组发光源包含红色发光源20、绿色发光源21、蓝色发光源22各一个，发光面顶部盖有匀光板，一面可按照交互与中央处理模块设定的参数发出特定颜色的光线，另一面紧贴控温系统的光源散热器17散发工作产生的热量维持稳定的工作状态；控温系统包含导热液循环泵18、光源散热器17、第一导热液连接管14、第二导热液连接管15、微孔板保温架11、加热线圈10及温度传感器9，导热液循环泵18与光源散热器17相连驱动导热液在光源散热器17内部流路、第一导热液连接管14、微孔板保温架11内部流路以及第二导热液连接管15形成的密闭回路内循环将光源模块16产生的热量传递给微孔板保温架11用于微孔板13的保温，微孔板保温架11上放置微孔板13可为其维持恒温环境并各孔的对应位置留有通孔，加热线圈10环绕紧贴微孔板保温架11中上部用于预热和温度不足时的快速升温，温度传感器9紧贴微孔板保温架11表面用于反馈当前维持的温度；图像传输系统包含聚光透镜12、光纤固定座8、光纤束7及光纤集束器6，光源模块16发出的光线穿过放置在微孔板保温架11上的微孔板13后，被微孔板保温架11的96个通孔内的聚光透镜12聚焦到微孔板保温架11底部安装的光纤固定座8上的96根光纤组成的光纤束7，并由光纤束7调整光路在光纤集束器6处汇聚；图像采集系统包含滤光片选择器5、滤光片4、图像采集透镜3及摄像头2，光纤7在光纤集束器6汇聚形成的图像经滤光片选择器4上选定的滤光片4，由图像采集透镜3调整成像范围，最终被摄像头2捕获并转换成图像信号传输给交互与中央处理模块1。As shown in Figures 1 and 2, the present invention provides a 96-well microplate reader, including an interactive and central processing module, a light source module, a temperature control system, an image transmission system and an image acquisition system. The interactive and central processing module 1 has a large touch screen, which is connected to the light source module 16, the temperature sensor 9 and the heating coil 10 of the temperature control system and the camera 2 of the image acquisition system through wires, and can obtain the feedback result of the temperature sensor 9 and modulate the parameters of the light source module 16 and the heating coil 10, and can process or share the collected image results with other intelligent terminals; the light source module 16 has 96 groups of light sources distributed according to the corresponding positions of the 96 holes of the microplate, and each group The light source includes a red light source 20, a green light source 21, and a blue light source 22. The top of the light-emitting surface is covered with a light-homogenizing plate. One side can emit light of a specific color according to the parameters set by the interaction with the central processing module, and the other side is close to the light source radiator 17 of the temperature control system to dissipate the heat generated by the work to maintain a stable working state; the temperature control system includes a thermal liquid circulation pump 18, a light source radiator 17, a first thermal liquid connecting pipe 14, a second thermal liquid connecting pipe 15, a microplate insulation rack 11, a heating coil 10 and a temperature sensor 9. The thermal liquid circulation pump 18 is connected to the light source radiator 17 to drive the thermal liquid in the internal flow path of the light source radiator 17, the first thermal liquid connecting pipe 14, the second thermal liquid connecting pipe 15, the microplate insulation rack 11, the heating coil 10 and the temperature sensor 9. The closed loop formed by the tube 14, the internal flow path of the microplate insulation rack 11 and the second heat transfer liquid connecting tube 15 transfers the heat generated by the light source module 16 to the microplate insulation rack 11 for insulation of the microplate 13. The microplate 13 is placed on the microplate insulation rack 11 to maintain a constant temperature environment for it and a through hole is left at the corresponding position of each hole. The heating coil 10 surrounds and fits tightly to the upper middle part of the microplate insulation rack 11 for preheating and rapid heating when the temperature is insufficient. The temperature sensor 9 fits tightly to the surface of the microplate insulation rack 11 for feedback of the currently maintained temperature; the image transmission system includes a focusing lens 12, an optical fiber fixing seat 8, an optical fiber bundle 7 and an optical fiber bundler 6. The light source module 16 emits After the light passes through the microplate 13 placed on the microplate insulation rack 11, it is focused by the focusing lens 12 in the 96 through holes of the microplate insulation rack 11 to the fiber bundle 7 composed of 96 optical fibers on the optical fiber fixing seat 8 installed at the bottom of the microplate insulation rack 11, and the optical fiber bundle 7 adjusts the light path to converge at the fiber bundler 6; the image acquisition system includes a filter selector 5, a filter 4, an image acquisition lens 3 and a camera 2. The image formed by the convergence of the optical fiber 7 at the fiber bundler 6 passes through the filter 4 selected on the filter selector 4, and the imaging range is adjusted by the image acquisition lens 3, and finally captured by the camera 2 and converted into an image signal and transmitted to the interaction and central processing module 1.
实施例2Example 2
基于图1所示的96孔微孔板阅读仪检测尿液中的肌酐，步骤如下：The steps for detecting creatinine in urine based on the 96-well microplate reader shown in Figure 1 are as follows:
(1)96孔微孔板阅读仪设定控温系统的温度为37℃，选择滤光片为510nm，光源模块设置为绿光，开启预热，完成背景光校准。(1) Set the temperature of the temperature control system of the 96-well microplate reader to 37°C, select the filter to 510nm, set the light source module to green light, start preheating, and complete the background light calibration.
(2)微孔板选取9个孔分别配置体积为150μL，浓度为0μM、2.5μM、5μM、10μM、12.5μM、20μM、25μM、50μM和100μM的肌酐溶液。(2) Nine wells of a microplate were selected and each well was prepared with a volume of 150 μL and concentrations of 0 μM, 2.5 μM, 5 μM, 10 μM, 12.5 μM, 20 μM, 25 μM, 50 μM and 100 μM creatinine solutions.
(3)将50μL待测尿液样品稀释200倍后取150μL置于微孔板第10孔。(3) Dilute 50 μL of the urine sample to be tested 200 times and place 150 μL in the 10th well of the microtiter plate.
(4)在上述各孔中先后加入50μL 25mM苦味酸溶液以及50μL 0.75M氢氧化钠溶液。(4) Add 50 μL of 25 mM picric acid solution and 50 μL of 0.75 M sodium hydroxide solution to each well.
(5)微孔板放置于阅读仪内使混合溶液在37℃环境下反应10分钟。(5) Place the microplate in a reader and allow the mixed solution to react at 37°C for 10 minutes.
(6)使用阅读仪记录此时的图像，通过各孔图像分析得到各孔数据，利用前九孔数据生成标准曲线并代入第10孔数据，最终得到尿液中的肌酐浓度。(6) Use a reader to record the image at this time, obtain the data of each well through image analysis of each well, use the data of the first nine wells to generate a standard curve and substitute it into the data of the tenth well, and finally obtain the creatinine concentration in urine.
本发明简化了检测的操作流程，提高了仪器使用的便利性；同时提高了检测的速度，并可实时同步观察微孔板各孔的状态；还能够存储大量的检验数据，并对这些数据进行回顾分析或分享给其他智能终端。The present invention simplifies the detection operation process and improves the convenience of instrument use; at the same time, it improves the detection speed and can synchronously observe the status of each well of the microplate in real time; it can also store a large amount of test data, and review and analyze the data or share them with other intelligent terminals.
上述实施例用来解释说明本发明，而不是对本发明进行限制，在本发明的精神和权利要求的保护范围内，对本发明作出的任何修改和改变，都落入本发明的保护范围。The above embodiments are used to illustrate the present invention rather than to limit the present invention. Any modification and change made to the present invention within the spirit of the present invention and the protection scope of the claims shall fall within the protection scope of the present invention.
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