CN109557060B - Based on NH2Method for visually detecting alkaline phosphatase activity in serum by using (E) -Cu-MOF - Google Patents
Based on NH2Method for visually detecting alkaline phosphatase activity in serum by using (E) -Cu-MOF Download PDFInfo
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Abstract
本发明公开了一种基于NH2‑Cu‑MOF的可视化检测血清中碱性磷酸酶活性的方法，先合成NH2‑Cu‑MOF纳米酶材料；再利用NH2‑Cu‑MOF纳米酶和琼脂糖制备检测碱性磷酸酶活性的凝胶检测管；最后将碱性磷酸酶加到凝胶检测管后，将凝胶检测管置于紫外灯下，通过观察颜色变化实现半定量检测；然后用手机或数码相机拍照，通过RGB颜色提取器软件，读取测定照片中RGB值，分析RGB值来指示OPD的氧化程度，从而实现对血清中碱性磷酸酶活性的现场可视化多色荧光定量检测；检测时，先绘制凝胶颜色随碱性磷酸酶活力大小变化的工作曲线，再对血清实际样进行检测。该方法不需要昂贵的仪器和设备，不需要生物酶的参与，无需专业人员操作即可实现对血清中碱性磷酸酶的现场可视化多色荧光测定。The invention discloses a method for visual detection of alkaline phosphatase activity in serum based on NH 2 -Cu-MOF. First, NH 2 -Cu-MOF nano-enzyme material is synthesized; and then NH 2 -Cu-MOF nano-enzyme and agar are used. A gel detection tube for detecting alkaline phosphatase activity is prepared from sugar; finally, after adding alkaline phosphatase to the gel detection tube, the gel detection tube is placed under an ultraviolet lamp, and semi-quantitative detection is realized by observing the color change; Take a picture with a mobile phone or digital camera, read the RGB value in the measured photo through the RGB color extractor software, and analyze the RGB value to indicate the degree of OPD oxidation, so as to realize the on-site visual multicolor fluorescence quantitative detection of alkaline phosphatase activity in serum; When testing, first draw the working curve of the gel color changing with the alkaline phosphatase activity, and then test the actual serum sample. This method does not require expensive instruments and equipment, does not require the participation of biological enzymes, and can realize on-site visual multicolor fluorescence determination of alkaline phosphatase in serum without professional operation.
Description
技术领域technical field
本发明涉及检测碱性磷酸酶活性的方法，具体是一种基于NH2-Cu-MOF的可视化检测血清中碱性磷酸酶活性的方法。The invention relates to a method for detecting alkaline phosphatase activity, in particular to a method for visually detecting alkaline phosphatase activity in serum based on NH 2 -Cu-MOF.
背景技术Background technique
碱性磷酸酶(ALP)是原核生物和真核生物细胞中普遍存在的一种水解酶，催化磷酸基团从含有磷酸酯的多磷酸底物上移除。在人类的身体中，碱性磷酸酶广泛存在于组织(例如骨，肾，肝等)中，在细胞信号转导、细胞分裂、分化、骨骼钙化等多种正常细胞功能中起着重要的作用。另一方面，碱性磷酸酶活性的下调与多种人类疾病密切相关，包括低磷酸酯酶症，原发性胆汁性肝硬化，糖尿病以及各种癌症等。因此，开发可靠简便地检测碱性磷酸酶活性的方法对于临床疾病的诊断和碱性磷酸酶在生理和病理过程中作用的研究具有重要的研究价值。Alkaline phosphatase (ALP) is a hydrolase ubiquitous in prokaryotic and eukaryotic cells that catalyzes the removal of phosphate groups from phosphate-containing polyphosphate substrates. In the human body, alkaline phosphatase is widely present in tissues (such as bone, kidney, liver, etc.) and plays an important role in various normal cell functions such as cell signal transduction, cell division, differentiation, bone calcification, etc. . On the other hand, down-regulation of alkaline phosphatase activity is closely related to a variety of human diseases, including hypophosphatasia, primary biliary cirrhosis, diabetes, and various cancers. Therefore, developing a reliable and simple method for detecting alkaline phosphatase activity has important research value for the diagnosis of clinical diseases and the research on the role of alkaline phosphatase in physiological and pathological processes.
在目前已经公开检测碱性磷酸酶的相关专利中，CN108375616A一种检测碱性磷酸酶的液晶生物传感器及其制备方法，其液晶生物传感器需要使用偏光显微镜获得光学响应图像；CN106248644A一种基于碳点荧光“猝灭-恢复”的碱性磷酸酶的检测方法，需要使用荧光光谱仪获得荧光光谱图；CN107422014A用于检测碱性磷酸酶的修饰电极及制备方法与检测方法，需要利用电化学工作站获得电化学信号来绘制工作曲线。因此现有的碱性磷酸酶活性检测方法主要借助荧光光谱仪、电化学工作站等分析仪器实现定量检测。然而，这些方法对操作人员要求较高的专业技能，特别是所使用的仪器价格较为昂贵且体积庞大，不利于广泛的应用和推广。Among the related patents that have been published to detect alkaline phosphatase, CN108375616A is a liquid crystal biosensor for detecting alkaline phosphatase and its preparation method, and the liquid crystal biosensor needs to use a polarizing microscope to obtain an optical response image; CN106248644A is a carbon dot-based biosensor The detection method of alkaline phosphatase with fluorescence "quenching-recovery" requires the use of a fluorescence spectrometer to obtain a fluorescence spectrum; CN107422014A Modified electrode for detecting alkaline phosphatase and its preparation method and detection method need to use an electrochemical workstation to obtain electricity. chemical signals to draw working curves. Therefore, the existing alkaline phosphatase activity detection methods mainly rely on analytical instruments such as fluorescence spectrometers and electrochemical workstations to achieve quantitative detection. However, these methods require high professional skills for operators, especially the instruments used are expensive and bulky, which is not conducive to wide application and promotion.
此外，现有的可视化检测碱性磷酸酶活性的方法，CN106066325A一种检测碱性磷酸酶的方法，利用反应液颜色随碱性磷酸酶浓度增加而从蓝色变成无色的梯度颜色变化来实现碱性磷酸酶活性定量测定，然而，这种方法的缺点是浓度梯度显色变化单一、分辨率低；还有CN106596532A一种简单低成本的碱性磷酸酶活性的检测方法也存在同样问题。已有专利 CN106596532A一种简单低成本的碱性磷酸酶活性的检测方法采用亲水性纸张设计纸装置，利用纸装置中检测试剂溶液的流动长度反比于样本中碱性磷酸酶的活性大小。然而该装置需要用到生物酶如糖化酶，而生物酶在生产、运输以及使用的过程中，其活性的影响因素较多，而且仅靠直尺测量、人工读取溶液的流动长度，在精确度和准确度上还有待提升；CN107677804A一种骨源性碱性磷酸酶检测试剂盒及其使用方法，骨源性碱性磷酸酶检测试剂盒虽检测时间短，但仍需全自动生化分析仪来实现检测。因此，研制用于血清中碱性磷酸酶的可视化、便携式、颜色变化多元、高分辨率的检测方法具有重要的现实意义。In addition, the existing method for visually detecting the activity of alkaline phosphatase, CN106066325A, a method for detecting alkaline phosphatase, utilizes the gradient color change in which the color of the reaction solution changes from blue to colorless as the concentration of alkaline phosphatase increases. Achieving the quantitative determination of alkaline phosphatase activity, however, the disadvantage of this method is that the concentration gradient color change is single and the resolution is low; and CN106596532A, a simple and low-cost alkaline phosphatase activity detection method, also has the same problem. The existing patent CN106596532A is a simple and low-cost alkaline phosphatase activity detection method using hydrophilic paper to design a paper device, and the flow length of the detection reagent solution in the paper device is inversely proportional to the alkaline phosphatase activity in the sample. However, the device needs to use biological enzymes such as saccharification enzymes, and biological enzymes in the process of production, transportation and use have many factors affecting their activity, and only relying on ruler measurement and manual reading of the flow length of the solution, in the accurate The accuracy and accuracy still need to be improved; CN107677804A A bone-derived alkaline phosphatase detection kit and its use method, although the detection time of the bone-derived alkaline phosphatase detection kit is short, it still needs an automatic biochemical analyzer to achieve detection. Therefore, it is of great practical significance to develop a visual, portable, multivariate, and high-resolution detection method for alkaline phosphatase in serum.
发明内容SUMMARY OF THE INVENTION
本发明的目的是针对现有技术的不足，而提供了一种基于NH2-Cu-MOF的可视化检测血清中碱性磷酸酶活性的方法，该方法不需要昂贵的仪器和设备，不需要生物酶的参与，无需专业人员操作即可通过溶液或凝胶管的荧光颜色变化判断碱性磷酸酶的活力大小，可以实现对血清中碱性磷酸酶的现场可视化多色荧光测定。The purpose of the present invention is to aim at the deficiencies of the prior art, and to provide a method for visual detection of alkaline phosphatase activity in serum based on NH 2 -Cu-MOF, which does not require expensive instruments and equipment, and does not require biological With the participation of the enzyme, the activity of alkaline phosphatase can be judged by the fluorescence color change of the solution or gel tube without professional operation, and the on-site visual multicolor fluorescence determination of alkaline phosphatase in serum can be realized.
实现本发明目的的技术方案是：The technical scheme that realizes the object of the present invention is:
一种基于NH2-Cu-MOF的可视化检测血清中碱性磷酸酶活性的方法，包括如下步骤：A method for visual detection of alkaline phosphatase activity in serum based on NH 2 -Cu-MOF, comprising the following steps:
（1）合成具有一定形貌特征的NH2-Cu-MOF纳米酶材料；(1) Synthesis of NH 2 -Cu-MOF nanozyme materials with certain morphological characteristics;
（2）利用NH2-Cu-MOF纳米酶和琼脂糖制备检测碱性磷酸酶活性的凝胶检测管；(2) Using NH 2 -Cu-MOF nanozyme and agarose to prepare a gel detection tube for detecting alkaline phosphatase activity;
（3）碱性磷酸酶加到凝胶检测管后，凝胶的黄色荧光强度的强弱与邻苯二胺（OPD）的氧化产物2，3-二氨基吩嗪（DAP）的浓度大小成正相关，又由于NH2-Cu-MOF纳米酶本身具有蓝色荧光，因此将凝胶检测管置于紫外灯下，与DAP形成比率荧光，产生高分辨的颜色变化，通过观察颜色变化实现半定量检测；然后用手机或数码相机拍照，通过RGB颜色提取器软件，读取测定照片中RGB值，分析RGB值来指示OPD的氧化程度，从而实现对血清中碱性磷酸酶活性的现场可视化多色荧光定量检测；检测时，先绘制凝胶颜色随碱性磷酸酶活力大小变化的工作曲线，再对血清实际样进行检测。(3) After alkaline phosphatase is added to the gel detection tube, the intensity of the yellow fluorescence of the gel is directly proportional to the concentration of the oxidation product 2,3-diaminophenazine (DAP) of o-phenylenediamine (OPD). Related, and because the NH 2 -Cu-MOF nanozyme itself has blue fluorescence, the gel detection tube is placed under an ultraviolet lamp to form ratio fluorescence with DAP, resulting in high-resolution color change, and semi-quantitative is realized by observing the color change. Detection; then take a picture with a mobile phone or a digital camera, read the RGB value in the measured photo through the RGB color extractor software, analyze the RGB value to indicate the degree of OPD oxidation, so as to realize the on-site visualization of the alkaline phosphatase activity in the serum. Multicolor Fluorescence quantitative detection; when detecting, first draw the working curve of the change of gel color with the activity of alkaline phosphatase, and then detect the actual serum sample.
步骤（1）所述合成NH2-Cu-MOF纳米酶材料，包括如下步骤：The synthesis of the NH 2 -Cu-MOF nanozyme material in step (1) includes the following steps:
（1.1）将Cu(NO3)2·3H2O溶解在去离子水中，Cu(NO3)2·3H2O和去离子水的质量比为1:10；(1.1) Dissolve Cu(NO 3 ) 2 ·3H 2 O in deionized water, and the mass ratio of Cu(NO 3 ) 2 ·3H 2 O to deionized water is 1:10;
（1.2）将溶解有2-氨基对苯二甲酸的DMF溶液和乙醇混合，混合后加入步骤（1.1）的溶液中，再混合均匀；(1.2) Mix the DMF solution dissolved with 2-aminoterephthalic acid and ethanol, add it to the solution in step (1.1) after mixing, and mix it evenly;
DMF和乙醇溶剂的体积比为1:1，Cu(NO3)2·3H2O和2-氨基对苯二甲酸的质量浓度比为2:1-3:1；The volume ratio of DMF and ethanol solvent is 1:1, and the mass concentration ratio of Cu(NO 3 ) 2 ·3H 2 O and 2-aminoterephthalic acid is 2:1-3:1;
（1.3）充分混匀后，将溶液置于特氟龙衬里的高压釜中，在80°C-90°C下反应；(1.3) After fully mixing, place the solution in a Teflon-lined autoclave and react at 80°C-90°C;
（1.4）最后，通过离心收集NH2-Cu-MOF，并用DMF、乙醇和去离子水依次洗后自然烘干，合成NH2-Cu-MOF纳米酶材料。(1.4) Finally, NH 2 -Cu-MOF was collected by centrifugation, washed with DMF, ethanol and deionized water in turn, and then dried naturally to synthesize NH 2 -Cu-MOF nanoenzyme material.
步骤（2）所述凝胶检测管的制备，包括如下步骤：The preparation of the gel detection tube in step (2) includes the following steps:
（2.1）先将琼脂糖溶于沸水中制得琼脂糖凝胶溶液，琼脂糖质量浓度为5g/L-15g/L；(2.1) Dissolve agarose in boiling water to prepare agarose gel solution, the mass concentration of agarose is 5g/L-15g/L;
（2.2）将NH2-Cu-MOF纳米酶的溶液加入到琼脂糖凝胶溶液中，混匀； NH2-Cu-MOF纳米酶浓度为0.3-0.6g/L；(2.2) Add the solution of NH 2 -Cu-MOF nanozyme into the agarose gel solution and mix well; the concentration of NH 2 -Cu-MOF nanozyme is 0.3-0.6g/L;
（2.3）混匀后，取体积为0.050-0.250mL混合液到测试管的盖子里，放置5 min后凝胶凝固，盖上盖子，在4℃下储存。(2.3) After mixing, take a volume of 0.050-0.250 mL of the mixture into the lid of the test tube, leave it for 5 minutes, and then the gel will solidify, cover with the lid, and store at 4°C.
步骤（3）所述检测具体是：检测时，首先绘制凝胶颜色随碱性磷酸酶活力大小变化的工作曲线，将不同活力大小的碱性磷酸酶标准溶液、67μL ppi溶液加入到凝胶测试管中，正立放于37°C恒温培养振荡器中孵育30 min，然后加入80μL OPD溶液，倒立置于37°C恒温培养振荡器中孵育40 min，在紫外灯下拍照，使用ColorSchemer Studio软件读取照片中溶液的RGB值，计算空白溶液和待测液的R /(R+G+B)，并以△R /(R+G+B)值作纵坐标，即可得到凝胶颜色的△R /(R+G+B)值随碱性磷酸酶活力大小变化的变化曲线，即检测血清中碱性磷酸酶活力的工作曲线；The detection in step (3) is as follows: during detection, first draw a working curve of the change of gel color with the activity of alkaline phosphatase, and add alkaline phosphatase standard solution and 67 μL ppi solution with different activities to the gel test. Incubate the tube upright in a constant temperature incubation shaker at 37°C for 30 min, then add 80 μL of OPD solution, place it upside-down in a constant temperature incubation shaker for 40 minutes, take pictures under a UV light, and use ColorSchemer Studio software Read the RGB value of the solution in the photo, calculate the R/(R+G+B) of the blank solution and the solution to be tested, and use the △R/(R+G+B) value as the ordinate to get the gel color The change curve of the △R/(R+G+B) value with the change of alkaline phosphatase activity, that is, the working curve of detecting alkaline phosphatase activity in serum;
其次，对实际样进行检测：吸取60 μL的血清、67μL ppi溶液加入到凝胶测试管中，正立置于37°C恒温培养振荡器中孵育30 min，而后加入80μL OPD溶液，倒立置于37°C恒温培养振荡器中孵育40 min，在紫外灯下拍照，可得颜色变化；使用ColorSchemer Studio软件读取照片中溶液的RGB值，代入工作曲线中即可得到血清中碱性磷酸酶的活力值。Next, test the actual sample: pipette 60 μL of serum and 67 μL of ppi solution into the gel test tube, place it upright in a constant temperature incubation shaker at 37°C for 30 min, then add 80 μL of OPD solution, and place it upside down. Incubate for 40 min in a constant temperature incubation shaker at 37°C, and take pictures under an ultraviolet light to obtain the color change; use ColorSchemer Studio software to read the RGB values of the solution in the photo, and substitute it into the working curve to obtain the alkaline phosphatase in serum. Vitality value.
本发明方法，首先合成具有一定形貌特征的NH2-Cu-MOF纳米酶材料，利用PPi抑制了纳米酶的催化活性，然后再通过碱性磷酸酶对PPi的水解作用，解除PPi的抑制，恢复了NH2-Cu-MOF对OPD的催化氧化作用，结合NH2-Cu-MOF自身的荧光信号，使得加入不同活力大小的碱性磷酸酶溶液后，混合液在紫外光照射下呈现出不同程度的比率荧光，从而实现定量检测碱性磷酸酶活性。In the method of the invention, the NH 2 -Cu-MOF nano-enzyme material with certain morphological characteristics is first synthesized, the catalytic activity of the nano-enzyme is inhibited by PPi, and then the inhibition of PPi is relieved by the hydrolysis of PPi by alkaline phosphatase, The catalytic oxidation effect of NH 2 -Cu-MOF on OPD was restored, combined with the fluorescence signal of NH 2 -Cu-MOF itself, so that after adding alkaline phosphatase solutions with different activities, the mixture showed different effects under ultraviolet light irradiation. The degree of ratiometric fluorescence enables quantitative detection of alkaline phosphatase activity.
此外，结合琼脂糖凝胶，利用NH2-Cu-MOF材料制备了便携式检测血清中碱性磷酸酶的凝胶检测管。由于碱性磷酸酶溶液的黄色荧光强度强弱与OPD的氧化产物DAP的浓度大小成正相关，因此可以通过手机或数码相机，结合专业配色软件或RGB颜色提取器软件分析测定照片中RGB值的方法来指示邻苯二胺的氧化程度，从而实现对碱性磷酸酶活性的多色荧光定量检测。In addition, combined with agarose gel, a portable gel detection tube for detecting alkaline phosphatase in serum was prepared using NH 2 -Cu-MOF material. Since the intensity of yellow fluorescence of alkaline phosphatase solution is positively correlated with the concentration of DAP, the oxidation product of OPD, the method of measuring RGB values in photos can be analyzed by mobile phone or digital camera, combined with professional color matching software or RGB color extractor software. To indicate the degree of oxidation of o-phenylenediamine, so as to realize the multicolor fluorescence quantitative detection of alkaline phosphatase activity.
本发明基于NH2-Cu-MOF纳米酶的多色可视化检测血清中碱性磷酸酶的方法利用了具有三种功能的NH2-Cu-MOF材料，分别是类氧化酶模拟酶、荧光供体和级联反应参与者的功能。多色荧光检测原理是基于碱性磷酸酶对PPi的水解作用可以解除PPi对NH2-Cu-MOF中的Cu的配位作用，恢复了NH2-Cu-MOF对OPD的催化氧化作用，结合材料自身荧光，使得加入不同活力的碱性磷酸酶溶液在紫外光照射下呈现出不同程度的比率荧光信号，体系颜色变化更加丰富，易于肉眼辨别。该方法不需要昂贵的仪器和设备，不需要生物酶参与，利用肉眼可实现半定量分析；利用具有照相功能的便携式手机作为分析工具，可实现定量分析。本发明无需专业人员操作即可通过凝胶管在紫外灯照射下颜色的变化分析血清中碱性磷酸酶的活力大小，通过手机软件读取RGB数值可以实现对血清中碱性磷酸酶的现场可视化多色荧光定量检测。The method for detecting alkaline phosphatase in serum based on the multi-color visualization of NH 2 -Cu-MOF nano-enzyme of the present invention utilizes NH 2 -Cu-MOF materials with three functions, which are an oxidase-like mimetic enzyme and a fluorescent donor. and the function of cascade response participants. The principle of multicolor fluorescence detection is that the hydrolysis of PPi by alkaline phosphatase can relieve the coordination effect of PPi on Cu in NH 2 -Cu-MOF, and restore the catalytic oxidation effect of NH 2 -Cu-MOF on OPD. The self-fluorescence of the material makes the alkaline phosphatase solution with different activities present different ratio fluorescence signals under ultraviolet light irradiation, and the color of the system changes more abundantly, which is easy to distinguish with the naked eye. The method does not require expensive instruments and equipment, and does not require the participation of biological enzymes, and semi-quantitative analysis can be achieved by using the naked eye; quantitative analysis can be achieved by using a portable mobile phone with a camera function as an analytical tool. The invention can analyze the activity of alkaline phosphatase in serum through the change of the color of the gel tube under ultraviolet light irradiation without professional operation, and the on-site visualization of alkaline phosphatase in serum can be realized by reading RGB values through mobile phone software Multicolor fluorescence quantitative detection.
具体实施方式Detailed ways
下面结合实施例对本发明内容作进一步的说明，但不是对本发明的限定。The content of the present invention will be further described below in conjunction with the examples, but it is not intended to limit the present invention.
实施例Example
一种基于NH2-Cu-MOF纳米酶的多色可视化检测血清中碱性磷酸酶活性的方法，包括如下步骤：A method for detecting alkaline phosphatase activity in serum based on multicolor visualization of NH 2 -Cu-MOF nanozyme, comprising the following steps:
（1） 合成具有一定形貌特征的NH2-Cu-MOF纳米酶材料；(1) Synthesize NH 2 -Cu-MOF nanozyme materials with certain morphological characteristics;
将1g Cu(NO3)2·3H2O溶解在10 mL的去离子水中；将溶解有0.4 g 2-氨基对苯二甲酸的10mL DMF和10 mL乙醇混合，加入上述溶液中混合均匀；充分混匀后，将溶液置于特氟龙衬里的高压釜中，在80°C下反应24 h；最后，通过离心收集NH2-Cu-MOF，并用DMF、乙醇和去离子水依次洗后自然烘干。Dissolve 1 g of Cu(NO 3 ) 2 ·3H 2 O in 10 mL of deionized water; mix 10 mL of DMF and 10 mL of ethanol dissolved with 0.4 g of 2-aminoterephthalic acid, add to the above solution and mix well; fully After mixing, the solution was placed in a Teflon-lined autoclave and reacted at 80 °C for 24 h; finally, NH 2 -Cu-MOF was collected by centrifugation, washed with DMF, ethanol and deionized water in turn, and then naturally drying.
（2）利用NH2-Cu-MOF纳米酶和琼脂糖制备检测碱性磷酸酶活性的凝胶检测管；(2) Using NH 2 -Cu-MOF nanozyme and agarose to prepare a gel detection tube for detecting alkaline phosphatase activity;
将300 μL，pH为7.2的Tris-HCl缓冲溶液、100 μL的NH2-Cu-MOF（2.5 g/L）的乙醇溶液加入到1 mL琼脂糖凝胶中，凝胶水浴控制温度在40℃；混匀后，取200 µL混合液到测试管的盖子里，放置5 min后凝胶凝固，盖上盖子，在4℃下储存，凝胶显无色透明状。Add 300 μL, pH 7.2 Tris-HCl buffer solution, 100 μL NH 2 -Cu-MOF (2.5 g/L) ethanol solution to 1 mL agarose gel, and the temperature of the gel water bath is controlled at 40 °C ; After mixing, take 200 µL of the mixture into the lid of the test tube, leave it for 5 minutes, and the gel will solidify. Cover the lid and store at 4°C. The gel is colorless and transparent.
（3）将凝胶检测管置于紫外灯下，用手机拍照，通过RGB颜色提取器软件，读取测定照片中RGB数值，分析RGB值来指示OPD的氧化程度，从而实现对血清中碱性磷酸酶活性的现场可视化多色荧光定量检测。(3) Put the gel detection tube under the ultraviolet lamp, take a picture with a mobile phone, read the RGB value in the measured photo through the RGB color extractor software, and analyze the RGB value to indicate the degree of oxidation of OPD, so as to realize the alkalinity in serum. In-situ visualization of phosphatase activity with multicolor fluorescence quantitative assays.
具体检测时，首先绘制凝胶颜色随碱性磷酸酶活力大小变化的工作曲线：In the specific detection, first draw the working curve of the gel color changing with the alkaline phosphatase activity:
将不同活力大小的碱性磷酸酶标准溶液（0 U/L、5 U/L、10 U/L、30 U/L、60 U/L、90 U/L、120 U/L）、67μL ppi（30 mmol L-1）溶液加入到凝胶测试管中，正立放于37°C恒温培养振荡器中孵育30 min，然后加入80μLOPD (0.4mmol L-1)溶液，倒立置于37°C智城恒温培养振荡器中孵育40 min，在紫外灯下拍照，使用ColorSchemer Studio软件读取照片中溶液的RGB值，计算空白溶液和待测液的R /(R+G+B)，并以△R /(R+G+B)值作纵坐标，即可得到凝胶颜色的△R /(R+G+B)值随碱性磷酸酶活力大小变化的变化曲线，即检测血清中碱性磷酸酶活力的工作曲线，其颜色变化如表一所示：Alkaline phosphatase standard solutions of different activity sizes (0 U/L, 5 U/L, 10 U/L, 30 U/L, 60 U/L, 90 U/L, 120 U/L), 67 μL ppi (30 mmol L -1 ) solution was added to the gel test tube, placed upright in a constant temperature incubation shaker at 37°C and incubated for 30 min, then 80μLOPD (0.4mmol L -1 ) solution was added and placed upside down at 37°C Incubate for 40 min in the Zhicheng constant temperature incubation shaker, take pictures under UV light, use ColorSchemer Studio software to read the RGB values of the solutions in the photos, calculate the R/(R+G+B) of the blank solution and the solution to be tested, and use The △R/(R+G+B) value is taken as the ordinate, and the change curve of the △R/(R+G+B) value of the gel color with the change of the alkaline phosphatase activity can be obtained, that is, the detection of alkali in serum The working curve of sex phosphatase activity, and its color change is shown in Table 1:
表一基于NH2-Cu-MOF纳米酶的多色可视化检测标准溶液中碱性磷酸酶活力的数据Table 1 Data of alkaline phosphatase activity in standard solution based on multicolor visualization of NH 2 -Cu-MOF nanozyme
其次，对实际样进行检测：吸取60 μL的血清、67μL ppi（30 mmol L-1）溶液加入到凝胶测试管中，正立置于37°C智城恒温培养振荡器中孵育30 min，而后加入80μL OPD (0.4mmol L-1)溶液，倒立置于37°C智城恒温培养振荡器中孵育40 min，在紫外灯下拍照，可得颜色变化；使用ColorSchemer Studio软件读取照片中溶液的RGB值，代入工作曲线中即可得到血清中碱性磷酸酶的活力值，如表二所示：Next, test the actual sample: pipette 60 μL of serum and 67 μL ppi (30 mmol L -1 ) solution into the gel test tube, and place it upright in a 37°C Zhicheng constant temperature incubation shaker for 30 min. Then add 80 μL of OPD (0.4 mmol L -1 ) solution, incubate it upside down in a 37°C Zhicheng constant temperature incubation shaker for 40 min, and take pictures under UV light to obtain the color change; use ColorSchemer Studio software to read the solution in the photo The RGB value of the serum can be substituted into the working curve to obtain the activity value of alkaline phosphatase in the serum, as shown in Table 2:
表二基于NH2-Cu-MOF纳米酶的多色可视化检测血清中碱性磷酸酶活力的数据Table 2 Data of alkaline phosphatase activity in serum based on multicolor visualization of NH 2 -Cu-MOF nanozyme
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