微喷涂生物传感器
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摘要
食品是人类赖以生存和发展的物质基础,而食品安全问题是关系到人体健康和国计民生的重大问题。评价一种食品是否安全,就是要依靠一定的检测手段提供科学依据,确定食品中的有毒有害物质的含量和毒性,通过风险评估来考虑其是否对人体造成实际的危害。传统的检测手段所需时间长,需要昂贵的设备,操作复杂,因此发展新型、快捷、经济、准确的快速检测手段,是食品安全的一项重要研究内容。
目前应用较广泛的食品安全快速检测手段主要有荧光分析、快速检测试剂盒、快速检测试纸条、生物传感器等。生物传感器具有体积小、成本低、灵敏度高、选择性好、抗干扰能力强、响应快等优点,近年来成为研究热点之一。本论文主要是研究利用微喷涂技术构建一类新型的生物传感器,论文主要围绕以下几个方面开展了一些工作:
1微喷涂技术条件的优化
本研究是利用微喷涂技术进行传感器的制备,在制备过程中喷涂针头的堵塞和导电线路的不完整性将直接影响到传感器的电化学性能。研究了银浆材料的流变学性质、喷涂针头的内径大小、喷涂压力、等待延滞时间、喷涂针头和基底材料的间距对所制备传感器性能的影响。最后选用的微喷涂参数为:银浆的粘度8000CPS～9000CPS;喷涂针头内径为0.15mm;延滞时间50ms～300ms;喷涂压力20 KPa;喷涂针头和基底材料间距为0.1mm;Z轴回复高度0.2mm～0.5mm。
2微喷涂传感器的构建及性能表征
在电脑上利用AutoCAD,Photoshop等作图软件对传感器的图案进行设计之后,编写好相应的程序,输入到系统中,然后制备三电极系统的传感器,利用银浆制备成导电线路,Ag/AgCl作为参比电极,并在工作电极和辅助电极的表面用导电高分子材料PEDOT/PSS(3,4-ethylenedioxythiophene/polystyrene sulfonic acid)进行修饰。用实体显微镜和原子力显微镜表征了传感器的表面形态特征,实验结果表明传感器表面形态良好。在1 mmol/L的硫堇溶液中用循环伏安法对传感器进行了电化学表征,记录传感器的氧化峰电位E_(pa)、氧化峰电流I_(pa)和半波电位E_(pa/2),采用Epa、Epa/2、Ipa和电荷传递系数α作为评估电极一致性的指标。结果显示传感器具有较好的电化学性能、稳定性、批量一致性。
3微喷涂葡萄糖氧化酶传感器
在本研究中,我们在制备了空白的三电极传感器之后,用微喷涂技术在工作电极的表面滴涂上PEDOT/PSS导电高分子材料,将葡萄糖氧化酶(glucose oxidase,GOD)和电子媒介体铁氰化钾溶解于羧甲基纤维素(carboxymethyl cellulose,CMC)溶液中,然后直接滴涂在工作电极上面,自然晾干,形成生物敏感膜,制备成微喷涂葡萄糖氧化酶传感器。实验结果表明我们用微喷涂技术构建的葡萄糖氧化酶传感器检测葡萄糖溶液具有良好的响应,在0.5mg/L～5.0mg/L葡萄糖浓度范围内有较好的线性相关度,线性相关系数达到了0.9894,传感器的一致性和稳定性良好。
4微喷涂辣根过氧化物酶传感器
在本实验中为了检验所制备传感器的性能,我们又制备了用辣根过氧化物酶(horseradish peroxidase,HRP)进行修饰的生物传感器,用来检测H_2O_2。在实验中,我们在先用高分子导电材料PEDOT/PSS成膜修饰到空白电极上面,然后在导电高分子薄膜之上修饰辣根过氧化物酶,自然晾干之后,整个工作电极用具有选择性的醋酸纤维素半透膜进行封装。经电化学检测之后,实验结果显示,我们制备的微喷涂辣根过氧化物酶传感器对H_2O_2具有非常良好的灵敏性,在0.5～5.0mmol/L浓度范围内的有较好的线性相关度,线性相关系数达到了0.9851。传感器的稳定型非常好。
Food is essential for human's survival and development.Food safety is related to the people's health and the national interest and the people's livelihood.One urgent and important work in this field is to develop a rapid detection technique with the features of convenience, inexpensiveness and precision because the traditional detection techniques need expensive equipment and are time-consuming and operated difficultly.
At present the most widely used rapid detection techniques in food safety include fluorophotometer,rapid detection kit,rapid detection paper,biosensor,and so on.The biosensor is attracting more and more concern as a rapid detection technique for its excellent advantages such as small bulk,low cost,high sensitivity,good selectivity,and rapid responding ability.The research work carried out in this paper would be described as follows:
1 Optimizing of micro-dispensing parameters
In this work we fabricated sensors with the help of micro-dispenser. The blockage of the needle in fabricating process and the breakage of the silver conducting circuit could affect the sensors' performance.Under this research on micro-dispensing condition,the optimal parameters were determined as follows:silver paste viscosity 8000～9000CPS,the inner diameter of the needle 0.15mm,dwell time 50～300ms,dispensing pressure 20KPa,the distance between needle and baseplate 0.1mm,and Z axes return distance 0.2～0.5mm.
2 The fabrication and characterization of micro-dispensing sensor
On the basis of a three-electrode structure designed on computer with the help of software AutoCAD and Photoshop,we fabricated a biosensor using micro-dispenser after writing a programme into the micro-dispenser system.This sensor,using silver conducting circuit, comprised a Ag/AgCl reference electrode,a working electrode and a counter electrode.The working and counter electrodes were modified with the conducting polymer blend poly (3,4-ethylenedioxythiophene/polystyrene sulfonic acid)(PEDOT/PSS). The sensor was characterized by stereomicroscope,AFM and chemical method based on cyclic voltammetry in 1mmol/L thionine solution by recording the potential of anodic peak,current of anodic peak,and half-wave potential.The reproducibility of the sensor was assessed by these three chemical indicators as well as electron transfer coefficient. Experimental results indicated that the sensors demonstrated good electrochemical performance and reproducibility.
3 Micro-dispensing GOD biosensor
The prototype of micro-dispensing GOD biosensor was realized by dispenser on the base of electrode modified with the conducting polymer (PEDOT/PSS),using carboxymethyl cellulose(CMC)solution which contained glucose oxidase(GOD)and potassium ferricyanide. Experimental results showed that the micro-dispensing GOD biosensor had a good response to glucose with an acceptable linear correlation coefficient of 0.9894 in the range of 0.5～5.0 mg/L.The sensor had acceptable reproducibility,stability and accuracy.
4 Micro-dispensing HRP biosensor
We fabricated the micro-dispensing HRP sensor to test the performance of the sensor.In this experiment,the prototype of micro-dispensing HRP biosensor was realized by dispensing the horseradish peroxidase(HRP)on the bare sensor coated with the PEDOT/PSS film.As the final sensor construction step,in order to prevent dissolution of the enzyme and the PEDOT/PSS film in the aqueous environment,the device was encapsulated with a water-resistant semi-permeable membrane of cellulose acetate.The membrane was made up by dip-coating the active area in a solution of cellulose acetate in acetone:THF 60:40.Under the electrochemical detection,the experimental results indicated that the micro-dispensing HRP biosensor had a good response to H_2O_2 and an acceptable linear correlation coefficient of 0.9851 in the range of 0.5～5.0 mmol/L.The sensor had good reproducibility,stability and accuracy.
目前应用较广泛的食品安全快速检测手段主要有荧光分析、快速检测试剂盒、快速检测试纸条、生物传感器等。生物传感器具有体积小、成本低、灵敏度高、选择性好、抗干扰能力强、响应快等优点,近年来成为研究热点之一。本论文主要是研究利用微喷涂技术构建一类新型的生物传感器,论文主要围绕以下几个方面开展了一些工作:
1微喷涂技术条件的优化
本研究是利用微喷涂技术进行传感器的制备,在制备过程中喷涂针头的堵塞和导电线路的不完整性将直接影响到传感器的电化学性能。研究了银浆材料的流变学性质、喷涂针头的内径大小、喷涂压力、等待延滞时间、喷涂针头和基底材料的间距对所制备传感器性能的影响。最后选用的微喷涂参数为:银浆的粘度8000CPS～9000CPS;喷涂针头内径为0.15mm;延滞时间50ms～300ms;喷涂压力20 KPa;喷涂针头和基底材料间距为0.1mm;Z轴回复高度0.2mm～0.5mm。
2微喷涂传感器的构建及性能表征
在电脑上利用AutoCAD,Photoshop等作图软件对传感器的图案进行设计之后,编写好相应的程序,输入到系统中,然后制备三电极系统的传感器,利用银浆制备成导电线路,Ag/AgCl作为参比电极,并在工作电极和辅助电极的表面用导电高分子材料PEDOT/PSS(3,4-ethylenedioxythiophene/polystyrene sulfonic acid)进行修饰。用实体显微镜和原子力显微镜表征了传感器的表面形态特征,实验结果表明传感器表面形态良好。在1 mmol/L的硫堇溶液中用循环伏安法对传感器进行了电化学表征,记录传感器的氧化峰电位E_(pa)、氧化峰电流I_(pa)和半波电位E_(pa/2),采用Epa、Epa/2、Ipa和电荷传递系数α作为评估电极一致性的指标。结果显示传感器具有较好的电化学性能、稳定性、批量一致性。
3微喷涂葡萄糖氧化酶传感器
在本研究中,我们在制备了空白的三电极传感器之后,用微喷涂技术在工作电极的表面滴涂上PEDOT/PSS导电高分子材料,将葡萄糖氧化酶(glucose oxidase,GOD)和电子媒介体铁氰化钾溶解于羧甲基纤维素(carboxymethyl cellulose,CMC)溶液中,然后直接滴涂在工作电极上面,自然晾干,形成生物敏感膜,制备成微喷涂葡萄糖氧化酶传感器。实验结果表明我们用微喷涂技术构建的葡萄糖氧化酶传感器检测葡萄糖溶液具有良好的响应,在0.5mg/L～5.0mg/L葡萄糖浓度范围内有较好的线性相关度,线性相关系数达到了0.9894,传感器的一致性和稳定性良好。
4微喷涂辣根过氧化物酶传感器
在本实验中为了检验所制备传感器的性能,我们又制备了用辣根过氧化物酶(horseradish peroxidase,HRP)进行修饰的生物传感器,用来检测H_2O_2。在实验中,我们在先用高分子导电材料PEDOT/PSS成膜修饰到空白电极上面,然后在导电高分子薄膜之上修饰辣根过氧化物酶,自然晾干之后,整个工作电极用具有选择性的醋酸纤维素半透膜进行封装。经电化学检测之后,实验结果显示,我们制备的微喷涂辣根过氧化物酶传感器对H_2O_2具有非常良好的灵敏性,在0.5～5.0mmol/L浓度范围内的有较好的线性相关度,线性相关系数达到了0.9851。传感器的稳定型非常好。
Food is essential for human's survival and development.Food safety is related to the people's health and the national interest and the people's livelihood.One urgent and important work in this field is to develop a rapid detection technique with the features of convenience, inexpensiveness and precision because the traditional detection techniques need expensive equipment and are time-consuming and operated difficultly.
At present the most widely used rapid detection techniques in food safety include fluorophotometer,rapid detection kit,rapid detection paper,biosensor,and so on.The biosensor is attracting more and more concern as a rapid detection technique for its excellent advantages such as small bulk,low cost,high sensitivity,good selectivity,and rapid responding ability.The research work carried out in this paper would be described as follows:
1 Optimizing of micro-dispensing parameters
In this work we fabricated sensors with the help of micro-dispenser. The blockage of the needle in fabricating process and the breakage of the silver conducting circuit could affect the sensors' performance.Under this research on micro-dispensing condition,the optimal parameters were determined as follows:silver paste viscosity 8000～9000CPS,the inner diameter of the needle 0.15mm,dwell time 50～300ms,dispensing pressure 20KPa,the distance between needle and baseplate 0.1mm,and Z axes return distance 0.2～0.5mm.
2 The fabrication and characterization of micro-dispensing sensor
On the basis of a three-electrode structure designed on computer with the help of software AutoCAD and Photoshop,we fabricated a biosensor using micro-dispenser after writing a programme into the micro-dispenser system.This sensor,using silver conducting circuit, comprised a Ag/AgCl reference electrode,a working electrode and a counter electrode.The working and counter electrodes were modified with the conducting polymer blend poly (3,4-ethylenedioxythiophene/polystyrene sulfonic acid)(PEDOT/PSS). The sensor was characterized by stereomicroscope,AFM and chemical method based on cyclic voltammetry in 1mmol/L thionine solution by recording the potential of anodic peak,current of anodic peak,and half-wave potential.The reproducibility of the sensor was assessed by these three chemical indicators as well as electron transfer coefficient. Experimental results indicated that the sensors demonstrated good electrochemical performance and reproducibility.
3 Micro-dispensing GOD biosensor
The prototype of micro-dispensing GOD biosensor was realized by dispenser on the base of electrode modified with the conducting polymer (PEDOT/PSS),using carboxymethyl cellulose(CMC)solution which contained glucose oxidase(GOD)and potassium ferricyanide. Experimental results showed that the micro-dispensing GOD biosensor had a good response to glucose with an acceptable linear correlation coefficient of 0.9894 in the range of 0.5～5.0 mg/L.The sensor had acceptable reproducibility,stability and accuracy.
4 Micro-dispensing HRP biosensor
We fabricated the micro-dispensing HRP sensor to test the performance of the sensor.In this experiment,the prototype of micro-dispensing HRP biosensor was realized by dispensing the horseradish peroxidase(HRP)on the bare sensor coated with the PEDOT/PSS film.As the final sensor construction step,in order to prevent dissolution of the enzyme and the PEDOT/PSS film in the aqueous environment,the device was encapsulated with a water-resistant semi-permeable membrane of cellulose acetate.The membrane was made up by dip-coating the active area in a solution of cellulose acetate in acetone:THF 60:40.Under the electrochemical detection,the experimental results indicated that the micro-dispensing HRP biosensor had a good response to H_2O_2 and an acceptable linear correlation coefficient of 0.9851 in the range of 0.5～5.0 mmol/L.The sensor had good reproducibility,stability and accuracy.
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