【作者】 张媛；

【导师】 张娟；

【作者基本信息】 上海大学 ， 生物与医药（专业学位）， 2022， 硕士

【摘要】 铅离子是一种常见的重金属离子污染物,会对人的身体健康造成严重危害。现有的铅离子检测方法大多操作复杂、费时、成本昂贵且需要大型仪器,不利于快速的现场化检测。因此,开发操作简单、灵敏度高、特异性强的生物传感器用于铅离子的现场化检测是十分迫切的。智能手机包含内置摄像头、中央处理器和数据储存空间,具有数据分析处理能力和信号传输能力,在分析检测中显示出良好的优势。纸基检测装置具有价格低廉、用户友好和生物相容性较好等优点,在分析检测中有着巨大的应用潜力。本论文基于智能手机和纸基装置的优势设计了两种新型比色生物传感器,用于铅离子的现场化检测,主要研究内容如下:1.基于G4 DNAzyme的铅离子纸基比色传感器研究在本部分工作中,我们利用3D打印技术和智能手机的图像拍摄及处理功能,构建了一种简单、快速且低成本的纸基比色传感器。铅离子能够竞争性的结合钾离子诱导的G-四链体并改变其构象,抑制G4 DNAzyme催化TMB的显色反应,整个显色反应过程在纸基上进行。通过3D打印技术制作暗盒并安装LED光源,开发了一款能够分析图片RGB值并进行数据处理的智能手机APP,使用智能手机拍摄显色反应结果,通过软件得到g值(颜色强度),并由软件自动分析出样品中铅离子的浓度。g值与铅离子的浓度呈正相关(R~2=0.9956),检测的线性范围在0.1 n M到100 n M之间,对铅离子具有较高的选择性。该传感器已成功应用于各种茶饮料和牛奶样品中铅离子的现场化检测。总之,智能手机辅助的G4DNAzyme纸基比色传感器在铅离子的现场化检测中具有操作简单、成本低、灵敏度高和特异性好等优势,为智能手机辅助的纸基检测装置在即时检测中的应用提供了新思路。2.基于CRISPR-Cas12a体系的铅离子纸基微流控研究在这部分工作中,我们将智能手机与纸基微流控芯片结合,开发了一种简单、低成本的铅离子现场化检测方法。在铅离子存在的情况下,富含G的DNA序列能够形成G-四链体结构抑制CRISPR-Cas12a体系的激活,从而降低金纳米颗粒表面ss DNA链的切割效率。而表面修饰了ss DNA的金纳米颗粒可被芯片检测区域的DNA纳米花捕获,导致检测区域因金纳米颗粒的存在而发生颜色变化。使用我们开发的手机软件进行拍摄并分析检测区域的R/G值可以直接得出样品中铅离子的浓度。该纸基微流控芯片的线性检测范围为0.1μM-10μM,已成功应用于牛奶中铅离子的现场化检测。总之,我们构建的智能手机辅助的纸基微流控芯片具有成本低、特异性高、抗干扰能力强、适用于现场化检测等优点。更多还原

【Abstract】 Pb(Ⅱ)is a common heavy metal ion pollutant,which can cause serious harm to human health.Most of the existing Pb(Ⅱ)detection methods are complicated to operate,time-consuming,expensive,and dependent on large-scale instruments,and these methods are not suitable for on-site detection.Therefore,it is very urgent to develop a biosensor with simple operation,high sensitivity and strong specificity for the on-site detection of Pb(Ⅱ).Smartphones have built-in cameras,central processing units,and data storage space,with the capabilities of data analysis and processing as well as signal transmission,and give obvious advantages for analysis.Paper-based detection devices own the advantages of low price,user-friendliness and good biocompatibility,exhibiting great application potential in analytical field.In this dissertation,two new colorimetric biosensors are designed by using the advantages of smartphone and paper-based device for on-site detection of Pb(Ⅱ).The main research contents are given as follows:1.Study on paper-based colorimetric sensor for Pb(Ⅱ) detection based on G4 DNAzymeIn this part,we construct a simple,fast,and low-cost paper-based colorimetric sensor using 3D printing technology as well as the image capture and processing capabilities of smartphones.Pb(Ⅱ)can competitively bind the G-quadruplex induced by potassium ion and change its conformation,inhibiting the color reaction of TMB catalyzed by G4 DNAzyme.The whole color reaction process is carried out on the paper.Moreover,a cassette is prepared by 3D printing technology with the installion of an OLED light source and a mobile APP is develop to analyze the RGB value of the reaction results picture captured by a smartphone and process data to give the g value(color intensity).The g value is positively correlated with the concentration of Pb(Ⅱ)(R~2=0.9956),and the detection linear range is between 0.1 n M and 100 n M,with high selectivity for Pb(Ⅱ).The sensor has been successfully applied to detect Pb(Ⅱ)in various tea beverages and milk samples.In conclusion,the smartphone-assisted G4DNAzyme paper-based colorimetric sensor has the advantages of simple operation,low cost,high sensitivity and good specificity in the on-site detection of Pb(Ⅱ).It provides a new idea for the application of smart phone-assisted paper-based detection device in real-time detection.2.Research on paper-based microfluidics for Pb(Ⅱ) detection based on CRISPR-Cas12a systemIn this part,we combine a smartphone with a paper-based microfluidic chip to develop a simple and low-cost method for on-site detection of Pb(Ⅱ).In the presence of Pb(Ⅱ),G-rich DNA sequences can form G-quadruplex structures to inhibit the activation of the CRISPR-Cas12a system,thereby reducing the cleavage efficiency of ss DNA strands on the surface of gold nanoparticles.The surface-modified gold nanoparticles with ss DNA can be captured by the DNA nanoflowers in the detection area of the chip,resulting in a color change in the detection area due to the presence of gold nanoparticles.Using the mobile phone software to analyze the R/G value of the detection area,the concentration of Pb(Ⅱ)in the sample can be directly obtained.The linear detection range of the paper-based microfluidic chip is 0.1μM-10μM,which has been successfully applied to detect Pb(Ⅱ)in various milks.In conclusion,the smartphone-assisted paper-based microfluidic chip has the advantages of low cost,high specificity,strong anti-interference ability,and suitability for on-site detection.更多还原