【作者】 王辉；

【导师】 刘刚； Ashok Mulchandani；

【作者基本信息】 中国农业大学 ， 农业电气化与自动化， 2018， 博士

【摘要】 我国经济迅猛的发展,涉及生产和使用重金属的行业越来越多。由于部分企业的违法排污、污水灌溉以及过量施用含有重金属的化肥和农药,使大量含有重金属的污染物被排放到自然环境中,最终沉积到土壤和水环境中,使重金属含量远高于背景值造成污染。土壤和水是农业生产的必要因素。土壤和水环境中重金属含量过高会导致农作物中重金属超标,进入如生物体内危害生物体的健康。因此,探索研究一种适合于农田土壤和灌溉水中重金属的快速检测方法,具有非常重要的意义。常规的重金属检测方法具有选择性好、准确度高、重复性好的优点,但这些方法的检测设备昂贵、体积大、耗时长以及需要专业人员进行操作,极大地限制在农田环境下使用。电化学法具有检测速度快、灵敏度高、选择性好、价格低廉、易便携化等优点,与不同传感器联用能实现多种重金属的检测,非常适合于农业环境的现场检测分析。目前该方法已成为当前重金属检测研究领域的一个热点。本文针对电化学法快速检测重金属中存在的选择性差、灵敏度低、成本高等问题。利用纳米材料和生物材料的特殊性能,研制新型纳米复合材料修饰的电化学传感器以及生物材料修饰的生物传感器,提高传感器检测重金属离子的选择性和灵敏性。研发出与重金属检测传感器配套使用的便携式重金属检测仪,二者联用实现农田土壤和灌溉水中不同重金属元素的定性分析和定量检测。1.纳米复合材料修饰的丝网印刷传感器检测镉离子和铅离子研究。针对未修饰丝网印刷传感器检测重金属离子的检出限高、灵敏度低等不足,使用氧化石墨烯、羧化多壁碳纳米管、金纳米颗粒三种纳米材料合成一种稳定的纳米复合材料,并利用碳原子间相互作用固定到丝网印刷传感器工作电极表面;循环伏安法电还原纳米复合材料中的氧化石墨烯后,并给电还原后的工作电极原位镀铋膜实现镉离子和铅离子的检测。丝网印刷传感器的表面结构和电化学性能使用扫描电子显微镜、紫外可见光谱仪、循环伏安法、电化学阻抗谱等方法进行研究,结果表明纳米复合材料提高了丝网印刷传感器工作电极的电化学性能。最优检测条件下,该丝网印刷传感器对锅离子和铅离子表现出高的灵敏性,并在1.0～80.0 μg/L的范围内呈现出良好的线性关系,其中镉离子的线性回归方程为I(μA)= 0.3001 + 0.4413C(μg/L),最低检出限为0.7μg/L;铅离子的线性回归方程为I(μA)= 0.6285 + 0.4893C(μg/L),最低检测限为0.3 μg/L。此外,使用该修饰丝网印刷传感器对土壤样本中的镉和铅进行了检测,检测结果与原子吸收光谱法测量结果无显著性差异,具有高的准确性。2.核酸适配体修饰的生物传感器检测铅离子研究。针对核酸适配体传感器的重复性差、成本高等问题,研制了一种核酸适配体和单壁碳纳米管共同修饰的叉指场效应管。使用3-氨丙基三乙氧基硅烷将单壁碳纳米管固定到场效应管表面以及N-羟基琥珀酰亚胺酯1-芘丁酸将双链DNA与单壁碳纳米管键连;利用铅离子与核酸适配体(G4-DNA)特异性结合形成稳定的复合结构,诱导双链DNA解旋来改变单壁碳纳米管内载流子数目,实现铅离子浓度的检测。最优检测条件下,生物传感器的相对电阻随着铅离子浓度升高而增大,并且在1ng/L至100 μg/L范围与铅离子浓度的对数呈现良好的线性关系,其线性回归方程为y=10.1041og[CPb(Ⅱ)]+5.8656,最低检出限为0.39μg/L。最后,使用该生物传感器检测实际土壤样本中的铅离子,表现出良好的重复性和高的检测精度。3.功能化核酸修饰的生物传感器检测汞离子研究。针对汞离子传感器检测灵敏度低、耗时长,研制了一种使用单链DNA内嵌四个硫代磷酸酯修饰的RNA和单壁碳纳米管修饰的叉指场效应管,使用3-氨丙基三乙氧基硅烷将单壁碳纳米管固定在叉指场效应管,并将单链DNA通过氨基修饰在单壁碳纳米管表面。生物传感器通过汞离子诱导切割RNA与DNA结合位点来改变单壁碳纳米管导电性,实现对汞离子浓度的检测。当生物传感器检测含有汞离子的待测样本时,单链DNA会被汞离子切割引起结构变化,进而影响单臂碳纳米管的导电性发生变化。在最优检测条件下,该生物传感器的相对电阻与汞离子浓度在50pmol/L～100nmol/L和100nmol/L～10 μmol/L两个范围内呈现很好的线性关系,其线性回归方程分别为yi=0.957+5.461log[CHg(Ⅱ)和y2=-135.67+32.313log[CHg(Ⅱ)]最低检测限为10pmol/L。此外,利用该生物传感器对多个实际的土壤样本中汞离子进行了测定试验,检测结果表明该传感器具有高的灵敏度和准确性。4.核酸酶修饰的生物传感器检测银离子研究。针对银离子检测传感器检测时间长、抗干扰能力弱,使用单壁碳纳米管修饰的间隙场效应管,并利用N-羟基琥珀酰亚胺酯1-芘丁酸实现与核酸酶与单壁碳纳米之间键连。银离子可以与核酸酶特异性结合实现快速切割含RNA的DNA链,引起DNA链结构改变,使得单壁碳纳米管的导电性发生变化实现银离子的检测。实验采用紫外-可见分光光度计、拉曼光谱、线性伏安法等方法研究生物传感器的各方面性能。最优检测条件下,该生物传感器的相对电阻变化与银离子浓度在10 pmol/L～107 pmol/L的对数呈现良好线性关系,其线性回归方程是y = 7.0744 + 12.007log[CAg(Ⅰ)],最低检测限为5 pmol/L。利用该生物传感器进行了多个水样本中银离子的测定实验,与标准检测结果对比表明出检测速度快、准确的优点。5.便携式重金属检测仪研究。针对文中研发的电化学传感器和生物传感器所需不同激励信号,设计一款可以提供多种检测方法的便携式重金属检测仪。该检测仪器下位机采用超低功耗混合信号处理器MSP430单片机,并结合电源模块、通讯模块、I/V转换模块、恒电位仪、I/V转换电路、滤波电路等外围辅助电路,实现传感器检测重金属过程中电压的产生和电流信号的采集;上位机软件使用C#语言开发,可以输出方波阳极溶出伏安法和线性扫描伏安法,很好地满足了文中研发的重金属检测传感器。更多还原

【Abstract】 With the development of industry and agriculture in China,an increasing number of industries have involved in heavy metals for their productions.Due to the illegal discharge of sewage by some enterprises,sewage irrigation and the excessive use of chemical fertilizers and pesticides containing heavy metals,the soil,and water environment has been contaminated by these heavy metal pollutants,which causes the contents of heavy metals in farmland and irrigation water are much higher than the background value.The heavy metals in farmland and irrigation water are easily absorbed by the crops,and then eat by the organisms that can affect their health.Therefore,it is of great significance to develop a method for the rapid detection of heavy metals in the agricultural environment.The traditional methods using spectrometries have high accuracy and good repeatability for the detection of heavy metals.However,these methods require a complicated operation,expensive equipment,and skilled workers,which limits the application.The electrochemical method has many advantages,such as high detection speed,high sensitivity,good selectivity,low price,and portable device.It can be used together with different sensors to realize the simultaneous detection of multiple heavy metals and is very suitable for the farmland environment.On-site inspection analysis.At present,this method has become a hot spot in the field of heavy metal detection research.This thesis is aimed at the technical difficulties existed in the rapid detection of heavy metals by the electrochemical method,including poor specificity,low sensitivity,and high cost.Explored new nanomaterials and biomaterials synthesis and modification technology,carry out low-cost heavy metal detection electrochemical sensors and biosensors research,improve the detection of heavy metal detection sensor selectivity and sensitivity.The sensors/biosensors combined with the portable electrochemical workstation is to quickly and accurately obtain the qualitativity and quantitativity of heavy metals in farmland and irrigation waters under a complex agricultural environment.1.To trace the concentrations of Cd(Ⅱ)and Pb(Ⅱ),we have developed an electrochemical inexpensive,sensitive and selective sensor using disposable screen-printed carbon electrode(SPE)immobilized with a composite film of reduced graphene oxide/carboxylation multi-walled carbon nanotubes/gold nanoparticle hybrid(RGO-MWNT-AuNP)through π-π bind.This high conductive nanocomposite layer,"RGO-MWNT-AuNP",was characterized by scanning electron microscopy,UV-visible spectrometer,cyclic voltammetry,and electrochemical impedance spectroscopy.Square wave stripping voltammetry was applied to RGO-MWNT-AuNP/SPE to electroplate bismuth film and monitor the heavy metal ions in the tested solution simultaneously.To obtain high current responses,the detecting parameters were optimized.Under optimized conditions,the linear detection range of the sensor was 1.0～80.0 μg/L for lead and cadmium.The linear regression equation was I(μA)= 0.3001 +0.4413C(μg/L))and I(μA)=0.6285 + 0.4893C(μg/L)for cadmium and lead with a detection limit of 0.7 μg/L and 0.3 μg/L,respectively.Finally,the prepared electrode was further employed to detect Cd(Ⅱ)and Pb(Ⅱ)in soil samples with satisfactory results.2.The article describes a reusable biosensor for Pb(II)ions.A duplex DNA with a terminal amino group,which contained a G-quadruplex(G4)aptamer,was covalently conjugated to single-walled carbon nanotubes on a field effect transistor(FET).The detection scheme is based on the despiralization of the DNA duplex because Pb(Ⅱ)can induce the G4 aptamer to form a stabilizing G4/Pb(Ⅱ)complex.This structural change affects the electrical conductivity of SWNTs which serves as the analytical signal.The biosensor was characterized via scanning electron microscopy,Raman,UV-vis,and voltage-current profiles.Under optimized conditions,the relative resistance at 0.02 V increases linearly with the logarithm of the Pb(Ⅱ)concentration in the range from 1 ng/L to 100 μg/L.The linear regression equation was y=10.104 log[CPb(Ⅱ)]+ 5.8656 and the limit of detection is 0.39 ng/L.Compared to other sensors,the sensor demonstrates superior simplicity,sensitivity,and selectivity even in mixtures of heavy metal ions.It was applied to the determination of Pb(Ⅱ)in(spiked)water and soil samples and gave good results.3.Mercury(Hg)and its compounds,originating from a variety of nature and anthropogenic source,are ubiquitous in the natural environment,which cause severe environmental contamination and pose irreversible harm to human health.Fast and accurate sensing approach is of significant importance for mercury detection.Here,a label-free biosensor using Hg(Ⅱ)-induced cleavage of phosphorothioate(PS)modified RNA was exploited.We designed a specific single-stranded DNA embedded four PS-modified RNA(Hg-DPR)to improve the cleavage reaction yield,and then Hg-DPR was covalently linked with single-walled carbon nanotube field effect transistor(SWNTs/FET)via a peptide bond.The Hg-DPR can be efficiently cleaved after exposed to Hg(Ⅱ),which further causes the conductivity of the SWNTs to change.Using the relative resistance change,the Hg-DPR/SWNTs/FET successfully obtained the calibration curves were linear in the range of 50 pmol/L to 100 nmol/L and 100 nmol/L to 10 μmol/L.The linear regression equation was y1=0.957 + 5.461 log[CHg(Ⅱ)]and y2=-135.67 +32.3131og[CHg(Ⅱ)],which the limit of detection was 10 pmol/L.Our work suggests that DPR/SWNTs/FET are promising for low-cost,portable,real-time,heavy metal ion detectors.Additionally,DPR/SWNTs/FET exhibited excellent sensitivity,portability and low-cost for Hg(Ⅱ)detection.4.Silver is a very common heavy metal,and its detection is of significant analytical importance.In this article,we report a label-free field-effect transistor(FET)biosensor using single-walled carbon nanotubes(SWNTs)functionalized with a silver ion(Ag(Ⅰ))specific RNA-cleaving DNAzyme for ultrasensitive detection of silver ions(Ag(Ⅰ)).The complementary strand DNA(CS-DNA)is covalently immobilized on the surface of SWNTs through amide bond formation and then combine with Agzyme.The properties of DNAzyme/SWNT/FET were investigated using different spectroscopy and electrochemical methods.The RNA-base on the CS-DNA can be cleaved at RNA site efficiently when Agzyme specific binding with Ag(Ⅰ).The structure of duplex DNA on the SWNTs changes,which further induces a remarkable changing electrical conductivity of the SWNTs.This proposed biosensor is much more convenient and efficient than that of the previously reported biosensor based on cytosine-Ag-cytosine.After the detecting parameters were optimized,the biosensor presented a high sensitivity and selectivity towards Ag(Ⅰ)at unprecedentedly low concentrations 5 pmol/L with a wide linear range from 10 pmol/L to 107 pmol/L.The linear regression equation was y = 7.0744 + 12.007 log[CAg(1)].Additionally,the prepared biosensor was applied to measure the Ag(1)in the water sample with good performance.5.Research on portable heavy metal device.Aiming at the different detecting signals required by the electrochemical sensors and biosensors developed in the paper,a portable heavy metal device was designed that can provide multiple detection methods.The device used an ultra-low-power mixed signal processor MSP430 MCU,integrates power supply module,communication module,I/V conversion module,potentiostat,I/V conversion circuit,filter circuit and other peripheral auxiliary circuits.The voltage generation and current signal acquisition during the heavy metal detection process.The upper computer software developed using C#language can output square wave anodic stripping voltammetry and linear sweep voltammetry,which was suitable to different sensors.更多还原