CN102818800A - Human bloody urine protein detection method based on chip-level test paper - Google Patents
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Abstract
本发明属于生物医学信息检测领域，涉及一种基于芯片级试纸的人血尿蛋白检测方法，包括下列步骤：（1）在纳米阵列上喷镀导电性优良的金属，制成具有拉曼增强衬底结构的纳米阵列芯片；（2）将不同浓度的人血尿蛋白溶液标准样品滴加在纳米阵列芯片上，反应一定时间后，分别进行拉曼光谱测试，找到某一特征峰信息，加以比对地址信息与强度信息，进行数据统计，构造形成数据库；（3）将待测人血尿蛋白溶液滴加在某种结构的纳米阵列芯片上，反应一定时间后，进行拉曼光谱测试，将获得的拉曼光谱与数据库里存储的信息相互比对，得到待测人血尿蛋白溶液的浓度。本发明能够能够快速、微量、可便携地检测人血尿蛋白。The invention belongs to the field of biomedical information detection, and relates to a human blood urine protein detection method based on a chip-level test paper, which includes the following steps: (1) spraying a metal with excellent conductivity on a nano-array to form a Raman-enhanced substrate Structured nano-array chip; (2) Add standard samples of human blood urine protein solution of different concentrations on the nano-array chip, and after a certain period of time, conduct Raman spectrum test respectively, find a certain characteristic peak information, and compare the addresses (3) Drop the human blood urine protein solution to be tested on a nano-array chip with a certain structure, react for a certain period of time, and conduct a Raman spectrum test to obtain the Raman The Mann spectrum is compared with the information stored in the database to obtain the concentration of the human blood urine protein solution to be tested. The invention can detect human blood urine protein quickly, in a small amount and in a portable manner.
Description
所属技术领域 Technical field
本发明属于生物医学信息检测领域，涉及一种人血尿蛋白检测方法The invention belongs to the field of biomedical information detection, and relates to a human blood urine protein detection method
背景技术 Background technique
血液中常会有定量的对人类生命活动不可或缺的蛋白存在。一部分的蛋白会在肾脏的丝球体中过滤进入尿液中，但又会在肾小管被吸收而回到血液中。因此，若肾脏的机能正常，在尿液中出现的蛋白量只有一点点，但是当肾脏与尿管出现障碍时就会漏出多量的蛋白变成蛋白尿。正常人尿中有微量蛋白，正常范围内定性为阴性，记为（-）。尿中蛋白质含量多达0.15g/24h以上时，称蛋白尿，尿常规定性可出现阳性。尿蛋白是尿液通过酸化加热后混浊而检出的蛋白质。正常人24小时尿蛋白的范围为≦0.15g，常规化验检测为阴性。如检测尿蛋白＞150毫克/日，即尿蛋白阳性时，说明人体排出的尿蛋白量明显增多，属于异常尿蛋白。尿蛋白持续阳性，往往代表肾脏发生了病变，故临床可依据尿蛋白阳性的多少来判定肾病损伤的程度以及肾病治疗的效果。因此，出现异常尿蛋白，一定要有效控制并消除，防止病情恶化进展。Quantitative amounts of proteins that are indispensable to human life activities often exist in the blood. A portion of the protein is filtered into the urine in the glomerulus of the kidney, but is absorbed in the renal tubules and returned to the blood. Therefore, if the function of the kidneys is normal, there is only a small amount of protein in the urine, but when the kidneys and the urinary tract are malfunctioning, a large amount of protein will leak out and become proteinuria. There is a small amount of protein in the urine of normal people, which is qualitatively negative within the normal range and recorded as (-). When the protein content in the urine is as high as 0.15g/24h or more, it is called proteinuria, and the urine routine can be positive. Urinary protein is a protein detected in urine that is cloudy after acidification and heating. The 24-hour urine protein range of normal people is ≦0.15g, and the routine laboratory test is negative. If the urine protein is detected to be more than 150 mg/day, that is, when the urine protein is positive, it means that the amount of urine protein excreted by the human body has increased significantly, which belongs to abnormal urine protein. Continuously positive urine protein often represents kidney disease, so the degree of kidney damage and the effect of kidney disease treatment can be judged clinically based on the positive urine protein. Therefore, abnormal urine protein must be effectively controlled and eliminated to prevent the progression of the disease.
定性试验是用以筛选和粗略估计尿蛋白含量的方法。试验的方法有三种：试纸法、磺柳酸法和加热醋酸法。磺柳酸法和加热醋酸法都是根据浊度反应将无混浊或无沉淀定为阴性（一），将出现混浊或沉淀的定为阳性（+）。磺柳酸法操作简便，灵敏度高，可广泛用于普查，但其对白蛋白的灵敏度高于球蛋白，且影响因素较多，易造成假阴性或假阳性。加热醋酸法对白蛋白和球蛋白的灵敏度基本一致，影响因素少，准确性较高。Qualitative tests are methods for screening and roughly estimating urine protein content. There are three test methods: test paper method, sulfallic acid method and heating acetic acid method. Both the sulfallic acid method and the heating acetic acid method are based on the turbidity reaction. No turbidity or no precipitation is defined as negative (1), and turbidity or precipitation is defined as positive (+). The sulfalinic acid method is easy to operate and highly sensitive, and can be widely used in general surveys. However, its sensitivity to albumin is higher than that of globulin, and there are many influencing factors, which may easily cause false negatives or false positives. The sensitivity of the heating acetic acid method to albumin and globulin is basically the same, with few influencing factors and high accuracy.
众所周知，由于尿蛋白含量相对微量，因此提纯检测过程中需要浓缩，因此现在检测尿蛋白含量的生物医学处理过程中，需要被检查的患者留尿相当一定量，这样对于体弱的病患、年幼的孩子以及年迈的老人都无疑是非常困难和痛苦的。在这样的医学工程背景下，继续寻找一种快速、微量、可便携的检测手段，作为尿蛋白含量的检测系统。As we all know, because the content of urine protein is relatively small, it needs to be concentrated in the process of purification and detection. Therefore, in the biomedical process of detecting urine protein content, a certain amount of urine needs to be retained by the patient. It is undoubtedly very difficult and painful for young children as well as for elderly people. In such a medical engineering background, continue to search for a rapid, trace, and portable detection method as a detection system for urine protein content.
发明内容 Contents of the invention
本发明的目的是提供一种能够快速、微量、可便携地检测人血尿蛋白的方法。本发明的技术方案如下：The purpose of the present invention is to provide a method capable of detecting human blood urine protein quickly, in a small amount and in a portable manner. Technical scheme of the present invention is as follows:
一种基于芯片级试纸的人血尿蛋白检测方法，包括下列步骤：A method for detecting human blood urine protein based on chip-level test paper, comprising the following steps:
（1）在纳米阵列上喷镀导电性优良的金属，制成具有拉曼增强衬底结构的纳米阵列芯片；(1) Sputtering metal with excellent conductivity on the nano-array to make a nano-array chip with a Raman-enhanced substrate structure;
（2）将不同浓度的人血尿蛋白溶液标准样品滴加在纳米阵列芯片上，反应一定时间后，分别进行拉曼光谱测试，找到某一特征峰信息，加以比对地址信息与强度信息，进行数据统计，构造形成数据库；(2) Drop the standard samples of human blood urine protein solution with different concentrations on the nanoarray chip, and after reacting for a certain period of time, conduct Raman spectrum test respectively, find a certain characteristic peak information, compare the address information and intensity information, and carry out Data statistics, structure and form database;
（3）将待测人血尿蛋白溶液滴加在某种结构的纳米阵列芯片上，反应一定时间后，进行拉曼光谱测试，将获得的拉曼光谱与数据库里存储的信息相互比对，得到待测人血尿蛋白溶液的浓度。(3) Drop the human blood urine protein solution to be tested on a nano-array chip with a certain structure, and after a certain period of reaction, conduct a Raman spectrum test, and compare the obtained Raman spectrum with the information stored in the database to obtain The concentration of human blood urine protein solution to be tested.
作为优选实施方式，所述的纳米阵列为零维的量子点颗粒和一维的量子线结构；所述的导电性优良的金属为Ag、Au、Pt或Cu中的一种或多种；利用磁控溅射工艺实现导电性优良的金属的喷镀。As a preferred embodiment, the nano-array is a zero-dimensional quantum dot particle and a one-dimensional quantum wire structure; the metal with excellent conductivity is one or more of Ag, Au, Pt or Cu; The magnetron sputtering process realizes the spraying of metals with excellent electrical conductivity.
本发明提出的基于芯片级试纸的检测人血尿蛋白的方法可为医学工作者和科研工作者探索微量生物医学信息提供新的实验手段，应用范围也相当广泛。可使得检测尿蛋白含量的检测变得更加便捷，更加易于操作。同时实现真正意义上的“芯片级实验室”，并商业化。The method for detecting human blood urine protein based on the chip-level test strip proposed by the present invention can provide a new experimental method for medical workers and scientific researchers to explore trace biomedical information, and has a wide range of applications. The detection of urine protein content can be made more convenient and easier to operate. At the same time, realize the real "chip-level laboratory" and commercialize it.
具体实施方式 Detailed ways
表面增强拉曼散射（SERS）：吸附在粗糙化金属表面的有机物由于表面局域等离子激元被激发所引起的电磁增强(即物理增强)，以及粗糙表面上的原子簇及吸附其上的分子构成拉曼增强的活性点(即化学增强)，这两者的作用使被测定物的拉曼散射产生极大的增强效应。其增强因子可达103～107次方，已发现能产生SERS的金属有Ag、Au、Cu和Pt等少数金属，以Ag的增强效应为最佳，最为常用。此技术具有选择性好和灵敏度高的优点，实际检测限可达10-12克级。Surface-Enhanced Raman Scattering (SERS): Electromagnetic enhancement (that is, physical enhancement) caused by the excitation of organic matter adsorbed on the roughened metal surface due to the excitation of surface localized plasmons, as well as atomic clusters on the rough surface and molecules adsorbed on it It constitutes the active point of Raman enhancement (that is, chemical enhancement), and the action of the two makes the Raman scattering of the analyte produce a great enhancement effect. Its enhancement factor can reach 10 3 to 10 7 powers. It has been found that the metals that can produce SERS include Ag, Au, Cu, and Pt, and a few metals, such as Ag, have the best enhancement effect and are most commonly used. This technique has the advantages of good selectivity and high sensitivity, and the actual detection limit can reach 10 -12 gram level.
下面对本发明的技术方案进行详细说明The technical scheme of the present invention is described in detail below
（1）组装纳米阵列，喷镀贵金属实现拉曼增强衬底结构(1) Assemble nano-arrays and spray precious metals to realize Raman-enhanced substrate structure
在纳米阵列（纳米阵列形貌包括纳米颗粒、纳米线、纳米棒、纳米带等各种形貌的低维度纳米阵列，尤其是零维的量子点颗粒和一维的量子线结构）上（其中纳米材料的材质可以是有机物、无机物或者复合材料），利用磁控溅射工艺实现导电性优良的金属（Ag、Au、Pt、Cu等中的一种或多种）的薄膜图层覆盖，制成纳米阵列芯片。On nano-arrays (nano-arrays including low-dimensional nano-arrays of various shapes such as nanoparticles, nano-wires, nano-rods, and nano-belts, especially zero-dimensional quantum dot particles and one-dimensional quantum wire structures) (where The material of nanomaterials can be organic, inorganic or composite materials), and the magnetron sputtering process is used to realize the thin film coating of metals with excellent conductivity (one or more of Ag, Au, Pt, Cu, etc.), Made of nano-array chips.
（2）待测溶液标准样品滴定与训练程序(2) Standard sample titration and training program of the solution to be tested
将不同浓度（从稀溶液到浓溶液）待测溶液标准样品滴加在纳米阵列芯片上，反应一定时间（5～600s）后，分别作出拉曼光谱测试。找到某一特征峰信息，加以比对地址信息与强度信息，进行数据库数据统计。形成不同反应时间、不同阵列结构、不同溶液浓度之间的数据库信息网络。Add the standard samples of the solution to be tested with different concentrations (from dilute solution to concentrated solution) dropwise on the nano-array chip, and after reacting for a certain period of time (5-600s), make a Raman spectrum test respectively. Find a certain characteristic peak information, compare the address information and intensity information, and perform database data statistics. A database information network between different reaction times, different array structures, and different solution concentrations is formed.
（3）待测样品测量与数据判别输出系统(3) Sample measurement and data discrimination output system
步骤（2）训练出的数据库，可以打印出来成为产品数据手册、可以形成电子版数据库信息表，也可以作为嵌入式代码编入手持式拉曼光谱仪的数据处理芯片，这样只需要使用特定纳米结构的阵列芯片试纸做测试，规定的时间所呈现的拉曼光谱信息，就可以让作为对比信息，将其引入数据库信息中查找，找到对应的数据库节点，就可以很轻松的判断尿蛋白含量的多少。The database trained in step (2) can be printed out as a product data manual, can form an electronic version of the database information table, or can be used as an embedded code to be programmed into the data processing chip of the handheld Raman spectrometer, so that only specific nanostructures are required The array chip test paper is tested, and the Raman spectrum information presented at the specified time can be used as comparison information, which can be introduced into the database information for search, and the corresponding database node can be found, and the urine protein content can be easily judged. .
本发明的一个实施例为：在ZnO纳米棒阵列上，利用磁控溅射工艺实现贵金属Ag的薄膜图层覆盖。将不同浓度（从稀溶液到浓溶液）待测溶液标准样品滴加在纳米阵列芯片上，反应一定时间（5～600s）后，分别作出拉曼光谱测试。找到某一特征峰信息，加以比对地址信息与强度信息，进行数据库数据统计。形成不同反应时间、不同阵列结构、不同溶液浓度之间的数据库信息网络。）待测样品测量与数据判别输出系统.步骤（2）训练出的数据库，可以打印出来成为产品数据手册。这样只需要使用特定纳米结构的阵列芯片试纸做测试，规定的时间所呈现的拉曼光谱信息，就可以让作为对比信息，将其引入数据库信息中查找，找到对应的数据库节点，就可以很轻松的判断尿蛋白含量的多少。An embodiment of the present invention is: on the ZnO nanorod array, the thin film layer coverage of the noble metal Ag is realized by using a magnetron sputtering process. Add the standard samples of the solution to be tested with different concentrations (from dilute solution to concentrated solution) dropwise on the nano-array chip, and after reacting for a certain period of time (5-600s), make a Raman spectrum test respectively. Find a certain characteristic peak information, compare the address information and intensity information, and perform database data statistics. A database information network between different reaction times, different array structures, and different solution concentrations is formed. ) The sample measurement and data discrimination output system to be tested. The database trained in step (2) can be printed out as a product data booklet. In this way, you only need to use the array chip test paper with a specific nanostructure for testing, and the Raman spectral information presented at the specified time can be used as comparative information, and it can be easily imported into the database information to find the corresponding database node. How much to judge the urine protein content.
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| CN105300971A (en) * | 2015-09-21 | 2016-02-03 | 济南大学 | Preparation method of urine protein detection test paper |
| CN109406480A (en) * | 2018-09-10 | 2019-03-01 | 天津大学 | Chip-scale test paper blood sugar detecting method based on SERS technology |
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| CN101042348A (en) * | 2006-03-24 | 2007-09-26 | 中国科学院长春光学精密机械与物理研究所 | Device for nondestructively detecting carotenoid concentration in human body |
| EP2405257A1 (en) * | 2009-03-04 | 2012-01-11 | Hasegawa, Yuki | Assaying substrate with surface-enhanced raman scattering activity |
| WO2012043028A1 (en) * | 2010-09-29 | 2012-04-05 | 株式会社日立ハイテクノロジーズ | Biopolymer optical analysis device and method |
| CN102175664A (en) * | 2011-02-17 | 2011-09-07 | 福建师范大学 | Method for detecting surface enhanced Raman spectra of blood RNA |
| CN102507530A (en) * | 2011-10-26 | 2012-06-20 | 黑龙江省科学院技术物理研究所 | Method using gamma radiation for preparing nano-silver surface-enhanced Raman spectrum substrate |
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| CN109406480A (en) * | 2018-09-10 | 2019-03-01 | 天津大学 | Chip-scale test paper blood sugar detecting method based on SERS technology |
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