一次性电化学酒精生物传感器的研究
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摘要
近年来,随着交通运输业的迅速发展和人民生活水平的不断提高,机动车和拥有驾驶执照的人员数量快速增长,交通事故频繁发生,其中因驾驶人员饮酒与醉酒造成的交通事故就占20%。
长期以来,由于我国一直没有检测酒后(饮酒8小时之内)驾车的国家标准,又没有一种简便、快速、准确检测血酒精浓度的便携式检测仪。因此当酒后驾车人员面临交通警察处罚时,还“理直气壮”地要法律条例,处罚证据。
目前血酒精浓度(blood alcohol concentration,BAC)测定的主要方法有:呼气法、分光光度法、气相色谱法。呼气法虽然操作简单、价格低廉,但只能定性,不能准确定量检测。分光光度法和气相色谱法均需要血浆或血清样本,受试者要到医院或专业机构抽取静脉血,给受试者及交管部门带来极大不便。综上所述,呼气法、分光光度法、气相色谱法不能满足简便、快速定量检测血酒精浓度的要求。
生物传感器是分析生物技术的一个重要领域,它是一个典型的多学科交叉产物,结合了生命科学、分析化学、物理学和信息科学及其相关技术,能够对所需要检测的物质进行快速分析和追踪。经过30多年的发展,已经成为一个涉及内容广泛、多学科介入和交叉、充满创新活力的领域。生物传感器由于其高选择性、高灵敏度,已经被广泛地应用于临床诊断、食品工业、环境监测等领域。
以酶作为分子识别器件的电化学生物传感器称为酶电极。酶电极是由一个固定化的酶敏感膜和与之密切结合的电极组成的换能系统,它把固化酶和电极结合在一起。要使酶作为电化学生物传感器的敏感膜使用,必须将酶固定在电极表面,即在电极表面覆盖一层敏感膜。在固定过程中,既要保持酶本身固有的特性,又要避免自由酶应用上的缺陷。因此酶的固定化技术决定着酶电极的稳定性、选择性和灵敏度等主要性能。
丝网印刷是印刷业的一项传统工艺,有悠久的历史。20世纪60年代以后广泛用于电子工业生产,作为制作电路板的主要工序。1981年,一项专利文献报道将丝网印刷技术用于厚膜电化学传感器的制作。丝网印刷技术主要优点包括:①可以在表面印制各种图案,设计十分灵活;②印刷过程容易实现自动化;③重现性好;④适用于各种材质;⑤成本低廉,因此能够大批量生产重现性好的生物传感器。由于传感器成本很低,可以一次性使用,即所谓“用过即扔”,对临床检验非常方便。
本研究将电化学酶传感器、纳米技术、丝网印刷技术、临床检验分析技术结合起来制备一次性酒精生物传感器,探讨了pH、缓冲液、温度、干扰物等对传感器响应电流的影响,并用于实际样品酒精浓度检测,表现出了良好的特异性、敏感性、重复性、稳定性,而且响应时间快、线性范围宽,为医学实验室和交通执法部门提供了一种快速检测血酒精浓度的参考。
本研究主要分为以下两个部分:
1.基于交联法固定酶的丝网印刷电极(screen-printed electrode,SPE)酒精生物传感器的制备。方法:1)丝网印刷电极的制备:在0.2mm厚的聚氯乙烯(polyvinyl chloride,PVC)薄膜上首先印刷一层导电银膜,再用碳浆印刷直径为3mm的圆盘作为工作电极,然后用PVC油墨印刷在导电银膜上作为绝缘层。2)Nafion-MB修饰电极的制备:丝网印刷电极在室温下依次用乙醇和超纯水超声清洗10min,然后在空气中晾干。将5%(w/w) Nafion-117溶液用无水乙醇稀释到1%(w/w),然后吸取5μL 1%的Nafion-乙醇溶液滴加在丝网印刷电极的工作区域,在空气中干燥,待电极表面干燥后形成一层Nafion膜。然后将Nafion修饰的丝网印刷电极置于含1mmol/L麦尔多拉蓝(Meldola’s blue, MB)的磷酸盐缓冲液(phosphate buffer solution,PBS) (0.1mol/L,pH8.0)中用循环伏安法以50mV/s的扫描速度进行循环伏安扫描,使MB通过离子交换固定到Nafion膜中。3)酒精生物传感器的制备:把4mg乙醇脱氢酶(Alcohol Dehydrogenase,ADH)、5mg NAD+(Nicotinamide Adenine Dinucleotide)、10mg牛血清白蛋白(bovine serum albumin,BSA)溶于200μL磷酸盐缓冲液(0.1mol/L,pH8.0)中,再加入20μL 2.5%戊二醛溶液充分混合;再吸取5μL此溶液滴加到已制备好的Nafion-MB修饰丝网印刷碳电极(screen-printed carbon electrode,SPCE)工作区域,在空气中干燥,溶剂蒸发后形成一层酶膜,制成酒精生物传感器。制成的酒精生物传感器在使用前被置于4℃冰箱保存。结果:所有测试均在电化学工作站中三电极系统下完成,饱和甘汞电极(saturated calomelelectrode,SCE)作为参比电极,铂丝作为对电极, Nafion-MB-酶修饰丝网印刷电极作为工作电极。该传感器在25℃时,以-0.17V(vs. SCE)作为工作电位,在pH为8.0的磷酸盐缓冲液中响应电流达到最大。传感器检出限为1.1×10-5mol/L,达到95%稳态响应时间不超过30S。用同一支生物传感器对含1mmol/L的酒精的磷酸盐缓冲液(pH8.0)重复测定10次,相对标准偏差(relative standard deviation,RSD)为3.6%。在5个批次印制的丝网印刷电极中随机抽样5个电极制成酒精生物传感器,分别对含1mmol/L的酒精的磷酸盐缓冲液(pH8.0)重复测定3次取其平均值,相对标准偏差为4.3%。酒精生物传感器于4℃冰箱放置10、20、30天后,对相同浓度的底物响应电流分别减少了3.7%、5.2%、8.4%。甲醇、糖、乳酸、抗坏血酸等均不会影响生物传感器对酒精的响应。结论:该酒精生物传感器可以快速检测酒精浓度,表现出良好的特异性、重复性、准确性、稳定性和抗干扰能力,可以为实验室检测酒精浓度提供一个参考。
2.基于Nafion和纳米金(gold nanoparticles,GNPs)固定酶的丝网印刷电极酒精生物传感器的制备。方法:1)纳米金的制备:取一定量的氯金酸溶液加热至沸,迅速加入柠檬酸钠溶液还原。所制备的溶液置于棕色瓶内于4℃保存。纳米金溶胶的颜色为亮红色,其粒径可用透射电子显微镜测试约为20nm。2)MB修饰的丝网印刷电极的制备:用聚酯作为生物传感器的基质材料,在聚酯上首先印刷一层导电银膜,再用混合2%(w/w)MB的碳浆印刷直径为3mm的圆盘作为工作电极,然后印刷UV胶作为绝缘层。银浆和碳浆印刷后,电极在40℃干燥2h以除去所应用油墨中的水分。UV胶印刷后需进行UV固化。3)酒精生物传感器的制备:MB修饰的丝网印刷电极在室温下依次用无水乙醇和超纯水超声清洗10min,然后在空气中晾干。把4mg ADH、5mg NAD+溶于200μL磷酸盐缓冲液(0.1mol/L,pH8.0)中配成酶液。再把100μL酶液和100μL 5%(w/w)Nafion溶液充分混合。然后100μL纳米金溶液被加入以上溶液并充分混合。接着10μL混合溶液被滴加到MB修饰丝网印刷电极的工作区域,在空气中干燥,溶剂蒸发后形成一层酶膜,制成酒精生物传感器。制成的酒精生物传感器在使用前被置于4℃冰箱保存。结果:所有测试均在电化学工作站中三电极系统下完成,饱和甘汞电极作为参比电极,铂丝作为对电极,MB-酶-Nafion-GNPs修饰丝网印刷电极作为工作电极。该传感器在25℃时,以0.0V(vs. SCE)作为工作电位,在pH为8.0的磷酸盐缓冲液中响应电流达到最大。传感器检出限为1.6×10-5mol/L,检测上限达到了8mmol/L,达到95%稳态响应时间不超过40S。用同一个生物传感器在1天内对两个不同的酒精浓度(1和5mmol/L)分别连续检测10次,相对标准偏差为5.49和1.44%。从10批丝网印刷电极中随机抽取10支电极制成酒精生物传感器,然后用这10支不同的生物传感器分别对两个不同的酒精浓度(1和5mmol/L)进行检测,相对标准偏差为6.98和2.49%。酒精生物传感器于4℃冰箱放置10、20、30天后,对1mmol/L酒精响应电流分别减少了3.1%、4.6%、7.7%。甲醇、糖、乳酸、抗坏血酸等均不会影响生物传感器对酒精的响应。结论:该酒精生物传感器可以快速检测血酒精浓度,表现出良好的特异性、重复性、准确性、稳定性和抗干扰能力,可以为实验室检测血酒精浓度提供一个参考。
Blood alcohol determination plays an important role in the forensic medicine and laboratory medicine for several reasons. First drunk driving is a very serious problem in the modern world, which results in lots of fatal accidents every year. Blood alcohol concentration (BAC) values served as the“gold standard”in which drivers were recognized as drunk driving by police officers. It is also necessary for clinical laboratories to detect the acute alcoholism and some syndromes related alcohol abuse. Nowadays, many methods have been used for alcohol measurement, such as spectrometric and chromatographic analysis or breathalyzer where the alcohol concentration or refractivity was detected. However, these methods are time consuming and complex to perform laborious sample pre-treatment .In addition, the expensive analytical apparatus is necessary. Thus, there is an increasing requirement for rapid, accurate and inexpensive methods for alcohol determination.
The disposable amperometric biosensor should be readily applicable to alcohol determination, since biosensors have such favorable analytical characteristics as portability, low cost and potential for fabrication. Several alcohol biosensors have been reported to control the fermentation process in the food and beverages industries, based on immobilization of Alcohol Oxidase (AOD) or Alcohol Dehydrogenase (ADH). In an AOD-based biosensor, O2 consumption or H2O2 production is determined. However, this kind of biosensor is oxygen dependent and low selectivity for alcohol. Concerning ADH-based biosensor, it is possible to directly detect reduced form of Nicotinamide Adenine Dinucleotid (NADH) produced in the enzymatic reaction of alcohol with Nicotinamide Adenine Dinucleotide (NAD+) under catalysis of ADH. However, the electrochemical oxidation of NADH involves the use of a high overpotential at which a few oxidizable substances in the real samples would be oxidized, thus increasing the likelihood of interferences. For this purpose many electron transfer mediators have been used for electrochemical oxidation of NADH, which could be detected at a low oxidizing potential. Among them, Meldola’s Blue (MB) shows some of the most promising characteristics. However, it has been found that MB could not be absorbed stably on the electrode, meaning that the electrode is unstable for practical applications. To improve MB attachment to the electrode, a few methods have been used. However, these methods are complicated. Nafion, a kind of ion exchanger, can absorb MB through ion exchange.
Screen printing technology, due to its low cost and mass production, is a versatile tool for the inexpensive, easy and highly reproducible production of disposable biosensors. Up to now, most of disposable biosensors based on screen printing technology are applied in the forensic medicine and laboratory diagnosis, since it could resolve such critical problems as low cost, fast response and portability.
This paper describes the development of disposable alcohol biosensor based on screen-printed electrode (SPE). The biosensor response for alcohol is investigated in terms of pH, buffer solution, temperature and some interferents. It presents the good specificity, reproducibility, stability, accuracy and provides a fast response. The biosensor is applied for measuring serum alcohol and satisfactory result is obtained.
Our research is mainly divided into two parts as follows:
1. Development of the disposable alcohol biosensor based on the cross-linking method to immobilize ADH and NAD+ on the Nafion-MB modified screen-printed electrode. Methods:1 ) Preparation of screen-printed electrode. A polyvinyl chloride (PVC) film is selected as the support of SPE. The biosensor comprises a 3-mm diameter working area, the insulation layer and an electrical contact site. The biosensors are printed onto the PVC sheet. The different layers including silver conducting basal track, carbon working area and insulation layer are printed one after the other. After every step the film is left to dry for 2 hours in an oven at 40℃to drive off the solvents from the applied ink. 2)Preparation of Nafion-MB modified electrode. The SPE is ultrasonically cleaned with ethanol and ultrapure water for 10 min respectively, and dried at room temperature. A 5% (w/w) Nafion-117 solution is diluted to 1% (w/w) with absolute ethanol. 5μL of 1% (w/w) Nafion is dropped onto the SPE and dried at room temperature. The pretreated electrode is scanned by cyclic voltammetry in the 0.1mol L-1 (pH8.0) phosphate buffer solution (PBS) with 1mmol L-1 MB at room temperature. Eventually, a Nafion-MB modified electrode is ready for use. 3)Preparation of alcohol biosensor. 4mg of ADH、5mg of NAD+ and 10mg of BSA are dissolved in 200μL of 0.1 mol L-1 PBS (pH8.0), then 20μL of 2.5% (w/w) glutaraldehyde solution is added to the mixed enzyme solution with vigorous stirring. 5μL of the mixed solution described above is dropped onto the working area of the electrode and allowed to dry in air at room temperature for 12 hours. The alcohol biosensor is prepared and kept dry in a refrigerator at 4℃before use. Results : All electrochemical measurements are carried out in a conventional three electrode cell which is composed of saturated calomel reference electrode (SCE), a platinum wire counter electrode and a modified working electrode. The maximum response current for alcohol is obtained in 0.1mol L-1 PBS (pH 8.0) at the working potential of -0.17V (vs. SCE), at 25℃. The detection limit of the biosensor is estimated to be 1.1×10-5mol L-1 alcohol at a signal to noise ration of 3. The linear response range of the biosensor to alcohol can be extended at least to 5mmol L-1. The biosensor response time is very short, reaching 95% of the steady-state current within 30s. The reproducibility of the identical biosensor is examined in 0.1mol L-1 PBS (pH8.0) with 1mmol L-1 alcohol at the working potential of -0.17V (vs. SCE), at 25℃. The relative standard deviation is 3.6% for 10 successive assays. 5 electrodes are randomly selected from 5 batches of screen-printed electrodes to fabricate the alcohol biosensors independently. The acceptable batch reproducibility with a relative standard deviation of 4.3% is obtained. The storage stability of biosensor toward the response for alcohol is examined as well. After being stored at 4℃for 10,20,30 days, the response current decreased by 3.7%,5.2%,8.4% of the initial response, respectively. The electroactive substances commonly present in the real samples, such as methanol, glucose, lactic acid, ascorbic acid, all can not influence on the biosensor response for alcohol. Conclusion: The biosensor presents the good specificity, reproducibility, stability, accuracy and provides a fast response. The proposed biosensor may provide a useful screening procedure for the determination of alcohol.
2. Development of the disposable alcohol biosensor based on Nafion combined with gold nanoparticles (GNPs) to immobilize ADH and NAD+ on the MB modified screen-printed electrode. Methods:1)Preparation of gold nanoparticles. Gold nanoparticles are prepared by adding sodium citrate solution to a boiling HAuCl4 aqueous solution. The solution is stored in brown glass bottles at 4℃refrigerator. The average particle diameter is 20nm as determined by transmission electron microscopy. 2)Preparation of MB modified screen-printed electrode. The polyester is selected as the support of the biosensor. The biosensor comprises a 3-mm diameter working area, the insulation layer and an electrical contact site. The biosensors are printed onto the polyester sheet. The different layers including silver ink, carbon ink and UV adhesives are printed one after the other. After printing of silver and carbon ink the polyester sheet is heated for 2h in an oven at 40℃to drive off the solvents from the applied ink. After printing of UV adhesives the ultraviolet curing of UV adhesives is followed. 3)Preparation of alcohol biosensor. The MB modified SPE is ultrasonically cleaned with absolute ethanol and ultrapure water for 10 min respectively, and dried at room temperature. Enzymes solution is obtained by dissolving 4mg of ADH and 6mg of NAD+ in 200μL of 0.1mol/L (pH8.0) phosphate buffer solution (PBS), then 100μL of enzymes solution and 100μL of 5% (w/w) Nafion solution is mixed with vigorous stirring. 100μL of gold nanoparticles solution is added to this mixed solution with vigorous stirring. 10μL of the freshly prepared mixed solution is dropped onto the working area of the SPE and allowed to dry in air at room temperature for 12h. The alcohol biosensor is prepared and kept dry in a refrigerator at 4℃before use. Results:All electrochemical measurements are carried out in a conventional three electrode cell which is composed of saturated calomel reference electrode, a platinum wire counter electrode and a modified working electrode. The maximum response current for alcohol is obtained in 0.1mol L-1 PBS (pH 8.0) at the working potential of 0.0 (vs. SCE), at 25℃. The detection limit of the biosensor is estimated to be 1.6×10-5 mol/L alcohol at a signal to noise ration of 3. The linear response range of the biosensor to alcohol could be extended at least to 8mmol/L. The biosensor response time is very short, reaching 95% of the steady-state current within 40s. The two different alcohol concentrations (1 and 5mmol/L) are detected using the identical biosensor for 10 successive assays within one day respectively. The relative standard deviations are 5.49 and 1.44%, respectively. 10 electrodes are randomly selected from 10 batches of screen-printed electrodes to fabricate the alcohol biosensors independently. 1 and 5mmol/L alcohol concentrations are detected using the ten different biosensors respectively. The relative standard deviations are 6.98 and 2.49%, respectively. The storage stability of biosensor toward the response for alcohol is examined as well. After being stored at 4℃for 10,20,30 days, the response current for 1mmol/L alcohol decreased by 3.1%,4.6%,7.7% of the initial response, respectively. The electroactive substances commonly present in the real samples, such as methanol, glucose, lactic acid, ascorbic acid, all can not influence on the biosensor response for alcohol. Conclusion: The biosensor presents the good specificity, reproducibility, stability, accuracy and provides a fast response. The proposed biosensor may provide a useful screening procedure for the determination of alcohol.
长期以来,由于我国一直没有检测酒后(饮酒8小时之内)驾车的国家标准,又没有一种简便、快速、准确检测血酒精浓度的便携式检测仪。因此当酒后驾车人员面临交通警察处罚时,还“理直气壮”地要法律条例,处罚证据。
目前血酒精浓度(blood alcohol concentration,BAC)测定的主要方法有:呼气法、分光光度法、气相色谱法。呼气法虽然操作简单、价格低廉,但只能定性,不能准确定量检测。分光光度法和气相色谱法均需要血浆或血清样本,受试者要到医院或专业机构抽取静脉血,给受试者及交管部门带来极大不便。综上所述,呼气法、分光光度法、气相色谱法不能满足简便、快速定量检测血酒精浓度的要求。
生物传感器是分析生物技术的一个重要领域,它是一个典型的多学科交叉产物,结合了生命科学、分析化学、物理学和信息科学及其相关技术,能够对所需要检测的物质进行快速分析和追踪。经过30多年的发展,已经成为一个涉及内容广泛、多学科介入和交叉、充满创新活力的领域。生物传感器由于其高选择性、高灵敏度,已经被广泛地应用于临床诊断、食品工业、环境监测等领域。
以酶作为分子识别器件的电化学生物传感器称为酶电极。酶电极是由一个固定化的酶敏感膜和与之密切结合的电极组成的换能系统,它把固化酶和电极结合在一起。要使酶作为电化学生物传感器的敏感膜使用,必须将酶固定在电极表面,即在电极表面覆盖一层敏感膜。在固定过程中,既要保持酶本身固有的特性,又要避免自由酶应用上的缺陷。因此酶的固定化技术决定着酶电极的稳定性、选择性和灵敏度等主要性能。
丝网印刷是印刷业的一项传统工艺,有悠久的历史。20世纪60年代以后广泛用于电子工业生产,作为制作电路板的主要工序。1981年,一项专利文献报道将丝网印刷技术用于厚膜电化学传感器的制作。丝网印刷技术主要优点包括:①可以在表面印制各种图案,设计十分灵活;②印刷过程容易实现自动化;③重现性好;④适用于各种材质;⑤成本低廉,因此能够大批量生产重现性好的生物传感器。由于传感器成本很低,可以一次性使用,即所谓“用过即扔”,对临床检验非常方便。
本研究将电化学酶传感器、纳米技术、丝网印刷技术、临床检验分析技术结合起来制备一次性酒精生物传感器,探讨了pH、缓冲液、温度、干扰物等对传感器响应电流的影响,并用于实际样品酒精浓度检测,表现出了良好的特异性、敏感性、重复性、稳定性,而且响应时间快、线性范围宽,为医学实验室和交通执法部门提供了一种快速检测血酒精浓度的参考。
本研究主要分为以下两个部分:
1.基于交联法固定酶的丝网印刷电极(screen-printed electrode,SPE)酒精生物传感器的制备。方法:1)丝网印刷电极的制备:在0.2mm厚的聚氯乙烯(polyvinyl chloride,PVC)薄膜上首先印刷一层导电银膜,再用碳浆印刷直径为3mm的圆盘作为工作电极,然后用PVC油墨印刷在导电银膜上作为绝缘层。2)Nafion-MB修饰电极的制备:丝网印刷电极在室温下依次用乙醇和超纯水超声清洗10min,然后在空气中晾干。将5%(w/w) Nafion-117溶液用无水乙醇稀释到1%(w/w),然后吸取5μL 1%的Nafion-乙醇溶液滴加在丝网印刷电极的工作区域,在空气中干燥,待电极表面干燥后形成一层Nafion膜。然后将Nafion修饰的丝网印刷电极置于含1mmol/L麦尔多拉蓝(Meldola’s blue, MB)的磷酸盐缓冲液(phosphate buffer solution,PBS) (0.1mol/L,pH8.0)中用循环伏安法以50mV/s的扫描速度进行循环伏安扫描,使MB通过离子交换固定到Nafion膜中。3)酒精生物传感器的制备:把4mg乙醇脱氢酶(Alcohol Dehydrogenase,ADH)、5mg NAD+(Nicotinamide Adenine Dinucleotide)、10mg牛血清白蛋白(bovine serum albumin,BSA)溶于200μL磷酸盐缓冲液(0.1mol/L,pH8.0)中,再加入20μL 2.5%戊二醛溶液充分混合;再吸取5μL此溶液滴加到已制备好的Nafion-MB修饰丝网印刷碳电极(screen-printed carbon electrode,SPCE)工作区域,在空气中干燥,溶剂蒸发后形成一层酶膜,制成酒精生物传感器。制成的酒精生物传感器在使用前被置于4℃冰箱保存。结果:所有测试均在电化学工作站中三电极系统下完成,饱和甘汞电极(saturated calomelelectrode,SCE)作为参比电极,铂丝作为对电极, Nafion-MB-酶修饰丝网印刷电极作为工作电极。该传感器在25℃时,以-0.17V(vs. SCE)作为工作电位,在pH为8.0的磷酸盐缓冲液中响应电流达到最大。传感器检出限为1.1×10-5mol/L,达到95%稳态响应时间不超过30S。用同一支生物传感器对含1mmol/L的酒精的磷酸盐缓冲液(pH8.0)重复测定10次,相对标准偏差(relative standard deviation,RSD)为3.6%。在5个批次印制的丝网印刷电极中随机抽样5个电极制成酒精生物传感器,分别对含1mmol/L的酒精的磷酸盐缓冲液(pH8.0)重复测定3次取其平均值,相对标准偏差为4.3%。酒精生物传感器于4℃冰箱放置10、20、30天后,对相同浓度的底物响应电流分别减少了3.7%、5.2%、8.4%。甲醇、糖、乳酸、抗坏血酸等均不会影响生物传感器对酒精的响应。结论:该酒精生物传感器可以快速检测酒精浓度,表现出良好的特异性、重复性、准确性、稳定性和抗干扰能力,可以为实验室检测酒精浓度提供一个参考。
2.基于Nafion和纳米金(gold nanoparticles,GNPs)固定酶的丝网印刷电极酒精生物传感器的制备。方法:1)纳米金的制备:取一定量的氯金酸溶液加热至沸,迅速加入柠檬酸钠溶液还原。所制备的溶液置于棕色瓶内于4℃保存。纳米金溶胶的颜色为亮红色,其粒径可用透射电子显微镜测试约为20nm。2)MB修饰的丝网印刷电极的制备:用聚酯作为生物传感器的基质材料,在聚酯上首先印刷一层导电银膜,再用混合2%(w/w)MB的碳浆印刷直径为3mm的圆盘作为工作电极,然后印刷UV胶作为绝缘层。银浆和碳浆印刷后,电极在40℃干燥2h以除去所应用油墨中的水分。UV胶印刷后需进行UV固化。3)酒精生物传感器的制备:MB修饰的丝网印刷电极在室温下依次用无水乙醇和超纯水超声清洗10min,然后在空气中晾干。把4mg ADH、5mg NAD+溶于200μL磷酸盐缓冲液(0.1mol/L,pH8.0)中配成酶液。再把100μL酶液和100μL 5%(w/w)Nafion溶液充分混合。然后100μL纳米金溶液被加入以上溶液并充分混合。接着10μL混合溶液被滴加到MB修饰丝网印刷电极的工作区域,在空气中干燥,溶剂蒸发后形成一层酶膜,制成酒精生物传感器。制成的酒精生物传感器在使用前被置于4℃冰箱保存。结果:所有测试均在电化学工作站中三电极系统下完成,饱和甘汞电极作为参比电极,铂丝作为对电极,MB-酶-Nafion-GNPs修饰丝网印刷电极作为工作电极。该传感器在25℃时,以0.0V(vs. SCE)作为工作电位,在pH为8.0的磷酸盐缓冲液中响应电流达到最大。传感器检出限为1.6×10-5mol/L,检测上限达到了8mmol/L,达到95%稳态响应时间不超过40S。用同一个生物传感器在1天内对两个不同的酒精浓度(1和5mmol/L)分别连续检测10次,相对标准偏差为5.49和1.44%。从10批丝网印刷电极中随机抽取10支电极制成酒精生物传感器,然后用这10支不同的生物传感器分别对两个不同的酒精浓度(1和5mmol/L)进行检测,相对标准偏差为6.98和2.49%。酒精生物传感器于4℃冰箱放置10、20、30天后,对1mmol/L酒精响应电流分别减少了3.1%、4.6%、7.7%。甲醇、糖、乳酸、抗坏血酸等均不会影响生物传感器对酒精的响应。结论:该酒精生物传感器可以快速检测血酒精浓度,表现出良好的特异性、重复性、准确性、稳定性和抗干扰能力,可以为实验室检测血酒精浓度提供一个参考。
Blood alcohol determination plays an important role in the forensic medicine and laboratory medicine for several reasons. First drunk driving is a very serious problem in the modern world, which results in lots of fatal accidents every year. Blood alcohol concentration (BAC) values served as the“gold standard”in which drivers were recognized as drunk driving by police officers. It is also necessary for clinical laboratories to detect the acute alcoholism and some syndromes related alcohol abuse. Nowadays, many methods have been used for alcohol measurement, such as spectrometric and chromatographic analysis or breathalyzer where the alcohol concentration or refractivity was detected. However, these methods are time consuming and complex to perform laborious sample pre-treatment .In addition, the expensive analytical apparatus is necessary. Thus, there is an increasing requirement for rapid, accurate and inexpensive methods for alcohol determination.
The disposable amperometric biosensor should be readily applicable to alcohol determination, since biosensors have such favorable analytical characteristics as portability, low cost and potential for fabrication. Several alcohol biosensors have been reported to control the fermentation process in the food and beverages industries, based on immobilization of Alcohol Oxidase (AOD) or Alcohol Dehydrogenase (ADH). In an AOD-based biosensor, O2 consumption or H2O2 production is determined. However, this kind of biosensor is oxygen dependent and low selectivity for alcohol. Concerning ADH-based biosensor, it is possible to directly detect reduced form of Nicotinamide Adenine Dinucleotid (NADH) produced in the enzymatic reaction of alcohol with Nicotinamide Adenine Dinucleotide (NAD+) under catalysis of ADH. However, the electrochemical oxidation of NADH involves the use of a high overpotential at which a few oxidizable substances in the real samples would be oxidized, thus increasing the likelihood of interferences. For this purpose many electron transfer mediators have been used for electrochemical oxidation of NADH, which could be detected at a low oxidizing potential. Among them, Meldola’s Blue (MB) shows some of the most promising characteristics. However, it has been found that MB could not be absorbed stably on the electrode, meaning that the electrode is unstable for practical applications. To improve MB attachment to the electrode, a few methods have been used. However, these methods are complicated. Nafion, a kind of ion exchanger, can absorb MB through ion exchange.
Screen printing technology, due to its low cost and mass production, is a versatile tool for the inexpensive, easy and highly reproducible production of disposable biosensors. Up to now, most of disposable biosensors based on screen printing technology are applied in the forensic medicine and laboratory diagnosis, since it could resolve such critical problems as low cost, fast response and portability.
This paper describes the development of disposable alcohol biosensor based on screen-printed electrode (SPE). The biosensor response for alcohol is investigated in terms of pH, buffer solution, temperature and some interferents. It presents the good specificity, reproducibility, stability, accuracy and provides a fast response. The biosensor is applied for measuring serum alcohol and satisfactory result is obtained.
Our research is mainly divided into two parts as follows:
1. Development of the disposable alcohol biosensor based on the cross-linking method to immobilize ADH and NAD+ on the Nafion-MB modified screen-printed electrode. Methods:1 ) Preparation of screen-printed electrode. A polyvinyl chloride (PVC) film is selected as the support of SPE. The biosensor comprises a 3-mm diameter working area, the insulation layer and an electrical contact site. The biosensors are printed onto the PVC sheet. The different layers including silver conducting basal track, carbon working area and insulation layer are printed one after the other. After every step the film is left to dry for 2 hours in an oven at 40℃to drive off the solvents from the applied ink. 2)Preparation of Nafion-MB modified electrode. The SPE is ultrasonically cleaned with ethanol and ultrapure water for 10 min respectively, and dried at room temperature. A 5% (w/w) Nafion-117 solution is diluted to 1% (w/w) with absolute ethanol. 5μL of 1% (w/w) Nafion is dropped onto the SPE and dried at room temperature. The pretreated electrode is scanned by cyclic voltammetry in the 0.1mol L-1 (pH8.0) phosphate buffer solution (PBS) with 1mmol L-1 MB at room temperature. Eventually, a Nafion-MB modified electrode is ready for use. 3)Preparation of alcohol biosensor. 4mg of ADH、5mg of NAD+ and 10mg of BSA are dissolved in 200μL of 0.1 mol L-1 PBS (pH8.0), then 20μL of 2.5% (w/w) glutaraldehyde solution is added to the mixed enzyme solution with vigorous stirring. 5μL of the mixed solution described above is dropped onto the working area of the electrode and allowed to dry in air at room temperature for 12 hours. The alcohol biosensor is prepared and kept dry in a refrigerator at 4℃before use. Results : All electrochemical measurements are carried out in a conventional three electrode cell which is composed of saturated calomel reference electrode (SCE), a platinum wire counter electrode and a modified working electrode. The maximum response current for alcohol is obtained in 0.1mol L-1 PBS (pH 8.0) at the working potential of -0.17V (vs. SCE), at 25℃. The detection limit of the biosensor is estimated to be 1.1×10-5mol L-1 alcohol at a signal to noise ration of 3. The linear response range of the biosensor to alcohol can be extended at least to 5mmol L-1. The biosensor response time is very short, reaching 95% of the steady-state current within 30s. The reproducibility of the identical biosensor is examined in 0.1mol L-1 PBS (pH8.0) with 1mmol L-1 alcohol at the working potential of -0.17V (vs. SCE), at 25℃. The relative standard deviation is 3.6% for 10 successive assays. 5 electrodes are randomly selected from 5 batches of screen-printed electrodes to fabricate the alcohol biosensors independently. The acceptable batch reproducibility with a relative standard deviation of 4.3% is obtained. The storage stability of biosensor toward the response for alcohol is examined as well. After being stored at 4℃for 10,20,30 days, the response current decreased by 3.7%,5.2%,8.4% of the initial response, respectively. The electroactive substances commonly present in the real samples, such as methanol, glucose, lactic acid, ascorbic acid, all can not influence on the biosensor response for alcohol. Conclusion: The biosensor presents the good specificity, reproducibility, stability, accuracy and provides a fast response. The proposed biosensor may provide a useful screening procedure for the determination of alcohol.
2. Development of the disposable alcohol biosensor based on Nafion combined with gold nanoparticles (GNPs) to immobilize ADH and NAD+ on the MB modified screen-printed electrode. Methods:1)Preparation of gold nanoparticles. Gold nanoparticles are prepared by adding sodium citrate solution to a boiling HAuCl4 aqueous solution. The solution is stored in brown glass bottles at 4℃refrigerator. The average particle diameter is 20nm as determined by transmission electron microscopy. 2)Preparation of MB modified screen-printed electrode. The polyester is selected as the support of the biosensor. The biosensor comprises a 3-mm diameter working area, the insulation layer and an electrical contact site. The biosensors are printed onto the polyester sheet. The different layers including silver ink, carbon ink and UV adhesives are printed one after the other. After printing of silver and carbon ink the polyester sheet is heated for 2h in an oven at 40℃to drive off the solvents from the applied ink. After printing of UV adhesives the ultraviolet curing of UV adhesives is followed. 3)Preparation of alcohol biosensor. The MB modified SPE is ultrasonically cleaned with absolute ethanol and ultrapure water for 10 min respectively, and dried at room temperature. Enzymes solution is obtained by dissolving 4mg of ADH and 6mg of NAD+ in 200μL of 0.1mol/L (pH8.0) phosphate buffer solution (PBS), then 100μL of enzymes solution and 100μL of 5% (w/w) Nafion solution is mixed with vigorous stirring. 100μL of gold nanoparticles solution is added to this mixed solution with vigorous stirring. 10μL of the freshly prepared mixed solution is dropped onto the working area of the SPE and allowed to dry in air at room temperature for 12h. The alcohol biosensor is prepared and kept dry in a refrigerator at 4℃before use. Results:All electrochemical measurements are carried out in a conventional three electrode cell which is composed of saturated calomel reference electrode, a platinum wire counter electrode and a modified working electrode. The maximum response current for alcohol is obtained in 0.1mol L-1 PBS (pH 8.0) at the working potential of 0.0 (vs. SCE), at 25℃. The detection limit of the biosensor is estimated to be 1.6×10-5 mol/L alcohol at a signal to noise ration of 3. The linear response range of the biosensor to alcohol could be extended at least to 8mmol/L. The biosensor response time is very short, reaching 95% of the steady-state current within 40s. The two different alcohol concentrations (1 and 5mmol/L) are detected using the identical biosensor for 10 successive assays within one day respectively. The relative standard deviations are 5.49 and 1.44%, respectively. 10 electrodes are randomly selected from 10 batches of screen-printed electrodes to fabricate the alcohol biosensors independently. 1 and 5mmol/L alcohol concentrations are detected using the ten different biosensors respectively. The relative standard deviations are 6.98 and 2.49%, respectively. The storage stability of biosensor toward the response for alcohol is examined as well. After being stored at 4℃for 10,20,30 days, the response current for 1mmol/L alcohol decreased by 3.1%,4.6%,7.7% of the initial response, respectively. The electroactive substances commonly present in the real samples, such as methanol, glucose, lactic acid, ascorbic acid, all can not influence on the biosensor response for alcohol. Conclusion: The biosensor presents the good specificity, reproducibility, stability, accuracy and provides a fast response. The proposed biosensor may provide a useful screening procedure for the determination of alcohol.
引文
[1]张先恩.生物传感器[M].北京:化学工业出版社.2006.7
[2]林泉,宋文强,彭承琳.生物传感器及其在生物医学中的应用研究[J].2007,28(8):27-28
[3]张先恩.生物传感器[M].北京:化学工业出版社.2006.8
[4] Nanjo M, Guilbault GG.. Enzyme electrode for l-amino acids and glucose [J].Analytica Chimica Acta, 1974,73(2)367-373
[5] Wu LN,Chen J, Du D, et al. Electrochemical immunoassay for CA125 based on cellulose acetate stabilized antigen/colloidal gold nanoparticles membrane[J].Electrochimica Acta, 2006,51(7):1208-1214
[6] Yao T, Musha S. Electrochemical enzymatic determinations of ethanol and -lactic acid with a carbon paste electrode modified chemically with nicotinamide adenine dinucleotide [J]. Analytica Chimica Acta, 1979,110(2):203-209
[7] Papastathopoulos DS, Rechnita GA. A urea-sensing membrane electrode for whole blood measurements [J]. Analytica Chimica Acta, 1975,79:17-26
[8] Marrazza G, Chiti G,1 Mascini M, ea al.Detection of Human Apolipoprotein E Genotypes by DNA Electrochemical Biosensor Coupled with PCR, Clinical chemistry,2000,46(1):31-37
[9]铃木周一.生物传感器[M].霍纪文,姜远海译.北京:科学出版社,1988.69
[10]孙康,李仲辉.主客体葡萄糖、乳糖生物传感器的研制.化学研究与应用,2001,13(4):376-379
[11] Qu HB, Zhang XE,Zhang SZ. Simultaneous determination of maltose and glucose using a dual-electrode flow injection system [J].Food Chemistry, 1995,52(2): 187-192
[12] Zhang XE, Rechnitz GA. Simultaneous determination of glucose and sucrose by a dual-working electrode multienzyme sensor flow-injection system [J]. Electroanalysis,1994,6(5-6):361-367
[13]魏福祥,韩菊,刘庆洲,等.胆碱酯酶生物传感器测定有机磷农药敌敌畏[J].河北科技大学学报,2003,22(4):92-94
[14]刘鹂,瞿丽雅.生物电化学传感器测定农药残留的研究进展[J].安全与环境学报,2005,12(5):113-116
[15] Campanella L, Colapicchioni C, Favero G, et al. Organophosphorus pesticide paraoxon analysis using solid state sensors [J].Sens Actuators B, 1996,331, 33(1) :25-33 .
[16] Ivanov A N, Evtugyn G A, Gyurcsayi R E, et al. Comparative investigation of electrochemical cholinesterase biosensors for pesticide determination[J].Anal Chim Acta, 2000,4042, 404(2) :55~59 .
[17] Probst G, Norman R. New BOD biosensor from Dr.Lange[J].Biosens Bioelectron, 1994,9(9) :12~13 .
[18]鞠熀先.电分析化学与生物传感技术[M].北京:科学出版社.2006,188
[19]鞠熀先.电分析化学与生物传感技术[M].北京:科学出版社.2006,192
[20] Scott DL, Bowden EF. Enzyme-substrate kinetics of adsorbed cytochrome c peroxidase on pyrolytic graphite electrodes [J]. Anal Chem 1994;66(8):1217-23
[21]郑德海.丝网印刷发展趋势[J].今日印刷,1998,5(S1)50-54
[22]张先恩.生物传感器[M].北京:化学工业出版社.2006.237-240
[23] Vijayakumar AR, Cs?regi E, Heller A, et al.Alcohol biosensors based on coupled oxidase-peroxidase systems [J], Anal.Chim.Acta,1996,327 (3)223-234
[24] Gülce H, Gülce A, Kavanoz M, et al. A new amperometric enzyme electrode for alcohol determination [J], Biosens.Bioelectron,2002,17 (6-7) 517-521
[25] Morales A, Céspedes F, Fàbregas EM,et al. Ethanol amperometric biosensor based on an alcohol oxidase-graphite-polymer biocomposite [J], Electrochim.Acta,1998,43 (23) 3575-3579
[26] Cai CX, Xue KH, Zhou YM, et al. Amperometric biosensor for ethanol based on immobilization of alcohol dehydrogenase on a nickel hexacyanoferrate modified microband gold electrode [J], Talanta, 1997,44 (3) 339-347
[27] Sprules SD, Hartley IC,Wedge R, et al. A disposable reagentless screen-printed amperometric biosensor for the measurement of alcohol in beverages [J], Anal.Chim.Acta,1996,329 (3) 215-221
[28]施清照,程琼.介体型乙醇生物传感器的研制及应用[J].分析试验室,1997,16(1):67-69
[29] Santos AS, Freire RS, Kubota LT. Highly stable amperometric biosensor for ethanol based on Meldola’s blue adsorbed on silica gel modified with niobium oxide [J], J.Electroanal.Chem,2003,547 (2) 135-142
[1] Mullor SG, Cabezudo MS, Ordieres AJM, el al. Alcohol biosensor based on alcohol dehydrogenase and Meldola Blue immobilized into a carbon paste electrode [J], Talanta,1996,43 (5):779-784
[2] Santos AS, Freire RS, Kubota LT. Highly stable amperometric biosensor for ethanol based on Meldola’s blue adsorbed on silica gel modified with niobium oxide [J], J.Electroanal.Chem,2003,547 (2):135-142
[3] Gorton L, Torstensson A, Jaegfeldt H, et al. Electrocatalytic oxidation of reduced nicotinamide coenzymes by graphite electrodes modified with an adsorbed phenoxazinium salt [J], meldola blue, J.Electroanal.Chem,1984,161 (1):103-120
[4] Dong SJ, Wang BX, Liu BF. Amperometric glucose sensor with ferrocene as an electron transfer mediator [J], Biosens.Bioelectron,1991,7 (3):215-222
[5] Santos AS, Gorton L, Kubota LT. Electrocatalytic NADH oxidation using an electrode based on Meldola Blue immobilized on silica coated with niobium oxide [J]. Electroanal,2002,14 (12):805-812
[6] Harrison DJ,Turner RFB, Baltes HP. Characterization of perfluorosulfonic acid polymer coated enzyme electrodes and a miniaturized integrated potentiostat for glucose analysis in whole blood [J], Anal Chem,1988,60 (19):2002-2007
[7] Sprules SD, Hartley IC,Wedge R, et al. A disposable reagentless screen-printed amperometric biosensor for the measurement of alcohol in beverages [J], Anal.Chim.Acta,1996,329 (3) 215-221
[8] Erlenkotter A, Kottbus M, Chemnitius GC. Flexible amperometric transducers for biosensors based on screen printed three electrode system [J]. J Electroanal Chem,2000,481(1):82-94
[9] Penton Z. Headspace measurement of ethanol in blood by gas chromatography with a modified autosampler [J]. Clin Chem 1985;31(3):439-441
[10]孙康,李仲辉.主客体葡萄糖、乳糖生物传感器的研制.化学研究与应用,2001,13(4):376-379
[11]周爱儒,查锡良.生物化学[M].北京:人民卫生出版社.2003.57
[12] Rover L, Fernandes JCB, Oliveira-Neto G, Kubota LT, Katekawa E, Serrano SHP. Study of NADH stability using ultraviolet–visible spectrophotometric analysis and factorial design [J]. Anal Biochem 1998,260(1):50-55
[13]周爱儒,查锡良.生物化学[M].北京:人民卫生出版社.2003.57
[14] Scott DL, Bowden EF. Enzyme-substrate kinetics of adsorbed cytochrome c peroxidase on pyrolytic graphite electrodes [J]. Anal Chem 1994;66(8):1217-23
[1]张先恩.生物传感器[M].北京:化学工业出版社.2006.45-49
[2]鞠熀先.电分析化学与生物传感技术[M].北京:科学出版社.2006,195
[3] Karyakin AA,Kotelnikova EA,Lukachova LV,et al. Optimal Environment for Glucose Oxidase in Perfluorosulfonated Ionomer Membranes: Improvement of First-Generation Biosensors [J].Analytical chmeistry,2002,74(7):1597-1603
[4] Dong SJ, Wang BX, Liu BF. Amperometric glucose sensor with ferrocene as an electron transfer mediator [J], Biosens.Bioelectron,1991,7 (3):215-222
[5] Harrison DJ,Turner RFB, Baltes HP. Characterization of perfluorosulfonic acid polymer coated enzyme electrodes and a miniaturized integrated potentiostat for glucose analysis in whole blood [J], Anal Chem,1988,60 (19):2002-2007
[6] Zhao S, Zhang K, Bai Y, et al. Glucose oxidase/colloidal gold nanoparticles immobilized in Nafion film on glassy carbon electrode: Direct electron transfer and electrocatalysis [J]. Bioelechem,2006,69(2):158-163
[7] Chen XG, Qian Y, Zhang SJ, Zou XY. Hydrogen peroxide biosensor based on immobilizing enzyme by gold nanoparticles and thionine. Acta Chim Sinica,2007,659(4):337-343
[8] Li QF, Yuan R, Liu Y, Mo CL, Chai YQ, Huang XQ, et al. A new biosensor for glucose based on Au colloid modified electrode and azure mediation. Chines J Aanl Chem 2005,33(5):631-634
[9] Grabar KC, Freeman RG., Hommer MB, Natan MJ. Preparation and characterization of au colloid monolayers. Anal Chem 1995,67(4):735-43
[10] Sprules SD, Hartley IC,Wedge R, et al. A disposable reagentless screen-printed amperometric biosensor for the measurement of alcohol in beverages [J], Anal.Chim.Acta,1996,329 (3) 215-221
[11] Erlenkotter A, Kottbus M, Chemnitius GC. Flexible amperometric transducers for biosensors based on screen printed three electrode system [J]. J Electroanal Chem,2000,481(1):82-94
[12] Penton Z. Headspace measurement of ethanol in blood by gas chromatography witha modified autosampler [J]. Clin Chem 1985;31(3):439-441
[13] Mullor SG, Cabezudo MS, Ordieres AJM, el al. Alcohol biosensor based on alcohol dehydrogenase and Meldola Blue immobilized into a carbon paste electrode [J], Talanta,1996,43 (5):779-784
[14] Santos AS, Freire RS, Kubota LT. Highly stable amperometric biosensor for ethanol based on Meldola’s blue adsorbed on silica gel modified with niobium oxide [J], J.Electroanal.Chem,2003,547 (2):135-142
[15]周爱儒,查锡良.生物化学[M].北京:人民卫生出版社.2003.57
[16] Rover L, Fernandes JCB, Oliveira-Neto G, Kubota LT, Katekawa E, Serrano SHP. Study of NADH stability using ultraviolet–visible spectrophotometric analysis and factorial design [J]. Anal Biochem 1998,260(1):50-55
[17]周爱儒,查锡良.生物化学[M].北京:人民卫生出版社.2003.57
[18] Scott DL, Bowden EF. Enzyme-substrate kinetics of adsorbed cytochrome c peroxidase on pyrolytic graphite electrodes [J]. Anal Chem 1994;66(8):1217-23
[19] Kennedy JW, Carey RW, Coolen RB, Garber CC, Lee HT, Levine JB, et al. EP5-A: Evaluation of precision performance of clinical chemistry devices; approved guideline. Wayne: NCCLS, 1999.
[20] Powers DM, Boyd JC, Click MR, Miller WG. EP7-A: Interference testing in clinical chemistry; approved guideline. Wayne: NCCLS, 2002.
[21] Carey RN, George H, Hartmann AE, Janzen VK, Levine JB, Miller WG, et al. EP15-A: User demonstration of performance for precision and accuracy; approved guideline. Wayne: NCCLS, 2001.
[22] Krouwer JS, Tholen DW, Garber CC, Goldschmidit HMJ, Kroll MH, Linnet K, et al. EP9-A2:Method comparison and bias estimation using patient samples; approved guideline-second edition. Wayne: NCCLS, 2002.
[1] Sprules SD, Hartley IC,Wedge R, et al. A disposable reagentless screen-printed amperometric biosensor for the measurement of alcohol in beverages [J], Anal.Chim.Acta,1996,329 (3) 215-221
[2] Erlenkotter A, Kottbus M, Chemnitius GC. Flexible amperometric transducers for biosensors based on screen printed three electrode system [J]. J Electroanal Chem,2000,481(1):82-94
[3]邱宪波,袁景淇,唐日泉.便携式血糖仪控制器的研制[J].微型电脑应用.2003,19(4):36-37
[4]王坤,袁景淇.基于PIC单片机的便携式血糖仪的开发[J].中国医疗器械杂志.2006,30(1):29-32
[5]邱宪波,袁景淇,唐日泉.EM78P458单片机及其在便携式血糖仪中的应用[J].测控技术.2003,22(4)60-62
[6]胡军.基于松翰单片机的血糖仪开发[J].化学传感器.2007,27(3):62-67
[7]唐日泉,郭琛,邱宪波.具有存储记忆功能的便携式血糖仪的研制[J].测控技术,2004,23(6)14-15
[8]郭琛,唐日泉,邱宪波.串行时钟芯片HT1380及其在便携式血糖仪中的应用[J].微型电脑应用.2004,20(4):38-40
[9]邹向阳,刘戎.基于SIC单片机的便携式快速血糖仪的设计与实现[J].传感器与微系统.2008,27(2)96-97
[10]胡军.手持式血糖仪控制器开发[J].传感器世界.2007,13(6):10-13
[11]唐日泉,袁景淇,邱宪波.液晶驱动芯片HT1621及其在便携式血糖仪中的应用[J].2003,19(5)22-24
[1]张先恩.生物传感器[M].北京:化学工业出版社.2006.1
[2]鞠熀先.电分析化学与生物传感技术[M].北京:科学出版社.2006,187
[3]林泉,宋文强,彭承琳.生物传感器及其在生物医学中的应用研究[J].医疗卫生装备.207,28(8)27-28
[4] Clark L C ,Lyons C. Electrode systems for continuous monitoring in cardiovascular surgery. Ann N Y Acad Sci ,1962 ,102 :29-33
[5] Updike SJ,Hicks JP. The enzyme electrode [J].Nature.1967,214(5092)986-988
[6] Guilbault GG,Montalvo JG. Urea-specific enzyme electrode[J].J Am Chem Soc.1969,91(8):2164-2165
[7] Divis C.Annals of Microbiology.1975,126A:175-186
[8] Rechnitz GA. A bio-selective membrane electrode prepared with living bacterial cells [J]. Analytica Chimica Acta, 1977, 94(2):377-384
[9] Karube I, Matsunage T, Mitsuda S, et al. Microbial Electrode BOD Sensors[J]. Biotechnology and Bioengineering,1977,19(10): 1535-1547.
[10] Janata J,Moss SD,Johnson CC.Selectvie chemical sensitvie field effect transducers.US Patent 4020830.1977
[11] Mosbach K, Danielsson B. An enzyme thermistor[J]. Biochim Biophys Acta. 1974,364(1):140-145
[12] Voelkl KP,Opita N,Lubbers DW,et al.Anal Chem.1980,301():162-163
[13] Guilbault GG. Determination of Formaldehyde with an Enzyme Coated Piezoelectric Crystal[J], Anal. Chem.1983,55():1682-1684
[14] Liedberg B,Nylander C,Lundstrm I. Development of an optical waveguide interferometric immunosensor[J].Sensors and Actuators B: Chemical,1991,4(3-4):297-299
[15] Cass AEG,Francis DG,Hill HAO,et al. Ferrocene-mediated enzyme electrode for amperometric determination of glucose[J].Anal Chem,1984,56(4):667-671
[16] Refereed Abstracts Book,the first world congress on biosensors.Singpore:Elsevier Science,1990
[17] Refereed Abstracts Book,the second world congress on biosensors.Geneva:Elsevier Science,1992
[18] Refereed Abstracts Book,the third world congress on biosensors.New Oreans:Elsevier Science,1994
[19] Refereed Abstracts Book,the forth world congress on biosensors.Bangkok:Elsevier Science,1996
[20] Refereed Abstracts Book,the fifth world congress on biosensors.Berlin:Elsevier Science,1998
[21] Refereed Abstracts Book,the sixth world congress on biosensors.San Diego:Elsevier Science,2000
[22] Refereed Abstracts Book,the sevevnth world congress on biosensors.Kyoto:Elsevier Science,2002
[23] Refereed Abstracts Book,the eighth world congress on biosensors.Granada:Elsevier Science,2004
[24]张先恩.生物传感器[M].北京:化学工业出版社.2006.6
[25]鞠熀先.电分析化学与生物传感技术[M].北京:科学出版社.2006,188
[26]张先恩.生物传感器[M].北京:化学工业出版社.2006.63
[27] Wollenberger U, Scheller F, Pfeiffer D,et al. Laccase/glucose oxidase electrode for determination of glucose[J].Analytica chimica acta.1986,187:39-45
[28] Blaedel WJ,Engstrom RC. Investigations of the ferricyanide-ferrocyanide system by pulsed rotation voltammetry[J].Anal Chem,1978,50(3):476-479
[29] Yao T, Musha S. Electrochemical enzymatic determinations of ethanol and -lactic acid with a carbon paste electrode modified chemically with nicotinamide adenine dinucleotide[J]. Analytica Chimica Acta, 1979, 10(2): 203-209
[30] Mascini M,Guilbault GG. Urease coupled ammonia electrode for urea determination in blood serum [J].Anal Chem.1977,49(6):795-798
[31] Nanjo M,Guilbault GG. Enzyme electrode sensing oxygen for uric acid in serum and urine [J].Anal Chem,1974,46(12):1769-1772
[32] Guilbault GG,et al.Anal Lett,1980,13(B18):1607
[33] Guilbault GG,Shu F. An electrode for the determination of glutamine[J].Anal Chim Acta.1971,56(3):333-338
[34] Mizutani F,Tsuda K, Karube I, et al. Determination of glutamate pyruvate transaminase and pyruvate with an amperometric pyruvate oxidase sensor[J].Anal Chim Acta.1980,118(1):65-71
[35]张先恩.生物传感器[M].北京:化学工业出版社.2006.80
[36] Hikuma M,et al.Biotechnol Bioengin,1979,21():1945
[37] Karube I, Okada T, Suzuki S,et al. Amperometric determination of ammonia gas with immobilized nitrifying bacteria [J].Anal Chem.1981,53(12):1852-1854
[38] Karube I,et al.Ibid,1982,54():1725
[39]张先恩.生物传感器[M].北京:化学工业出版社.2006.91
[40] Dai Z,Chen J,Yan F,et al.Electrochemical sensor for immunoassay of carcinoembryonic antigen based on thionine monolayer modified gold electrode[J].Cancer detection and prevention.2005,29(3):233-240
[41] Ju HX,Yan GF,Chen F,et al. Enzyme-linked immunoassay of a-1-fetoprotein in serum by differential pulse voltammetry[J].Electroanal.1999,11(2):124-128
[42] Dai Z,Chen J,Yan F,et al. Reagentless amperometric immunosensors based on direct elecrtochemistry of horseradish peroxidase for rapid determination of carcinoma antigen[J].Anal Chem.2003,75(20): 5429-5434
[43] Du D,Yan F,Liu SL,et al. Electrochemical immunosensor for carbohydrate antigen 19-9 based on immunological reaction and its immobilization in titania sol-gel matrix[J].Immuno Methods.2003,283(1-2):67-75
[44] Chen J,Yan F,Wu J,et al. Electrochemical immunoassay of human chorionic gonadotrophin based on its immobilization in gold nanoparticles-chitosan membrane[J].Electroanal. 2006,18(7):670-676
[45]鞠熀先.电分析化学与生物传感技术[M].北京:科学出版社.2006,425
[46] Wang J,Rivas G,Cai X,et al. DNA electrochemical biosensors for environmental monitoring[J].Anal Chim Acta.1997,347(1-2):1-8
[47] Siontorou CG,Nikolelis DP,Miernik A,et al. Rapid methods for detection ofAflatoxin M1 based on electrochemical transduction by self-assembled metal-supported bilayer lipid membranes (s-BLMs) and on interferences with transduction of DNA hybridization[J].Electrochim Acta.1998,43(23):3611-3617
[48] Wang J,Rivas G,Parrado C,et al. Electrochemical biosensor for detecting DNA sequences from the pathogenic protozoan Cryptosporidium parvum[J].Talanta,1997,44(11):2003-2010
[49] Wang J,Rivas G,Cai X. Screen-printed electrochemical hybridization biosensor for the detection of DNA sequences from the Escherichia coli pathogen[J].Electroanalysis.1997,9(5):395-398
[50] Mascini M,Palchetti I,Marrazza G,et al. DNA electrochemical biosensors[J]. Fresenius' Journal of Analytical Chemistry. 2001,369(1):15-22
[51] Wang J,Rivas G,Cai X,et al. Sequence-specific electrochemical biosensing of M. tuberculosis DNA[J].Anal Chim Acta.1997,337(1):41-48
[52] Erdem A,Kerman K,Meric B,et al. DNA Electrochemical Biosensor for the Detection of Short DNA Sequences Related to the Hepatitis B Virus[J].Electroanalysis,1999,11(8):586-588
[53] Ye YK,Zhao JH,Yan F,et al. Electrochemical behavior and detection of hepatitis B virus DNA PCR production at gold electrode[J].Biosens Bioelectron.2003:18(12):1501-1508
[54] Azek F,Grossiord C,Joannes M,et al. Hybridization Assay at a Disposable Electrochemical Biosensor for the Attomole Detection of Amplified Human Cytomegalovirus DNA[J].Anal Biochem.2000,284(1):107-113
[55] Hashimoto K,Ito K,Ishimori Y. Sequence-Specific Gene Detection with a Gold Electrode Modified with DNA Probes and an Electrochemically Active Dye[J].Anal Chem.1994,66(21):3830-3833
[56] Wang J,Rivas G,Cai X,et al. Detection of point mutation in the p53 gene using a /peptide nucleic acid biosensor[J].Anal Chim Acta.1997,344(1-2):111-118
[57] Bardea A,Patolsky F,Dagan A,et al.Chem Commun.1999,21():
[58] Maeda M,Mitsuhashi Y,Nakano K,et al.Anal Sci.1992,8():83
[59] Brett AMO,Serrano SHP,Macedo TA,et al. Voltammetric behavior of nitroimidazoles at a DNA-biosensor[J].Electroanal.1997,9(14):1132-1137
[60] Brabed V. DNA sensor for the determination of antitumor platinum compounds[J].Electrochim Acta.2000,45(18):2929-2932
1. He Q.H, Peng C.L, Wu B.M, et al. Research methods of brain-machine interface technology. Journal of chongqing university,2002;25:106 [何庆华,彭承琳,吴宝明等.脑-机接口技术研究方法.重庆大学学报,2002;25:106]
2. Wan B.K, Gao Y, Zhao L, et al. Brain-machine interface: A new channel for information communicate between human brain and environments. Biomedical engineering foreign medical sciences, 2005;28(1):4 [万柏坤,高扬,赵丽等.脑-机接口:大脑对外信息交流的新途径.国外医学生物医学工程分册,2005;28(1):4]
3. Williams J.C, Rennaker R.L, Kipke D.R, et al. Long-term neural recording characteristics of wire microelectrode arrays implanted in cerebral cortex. Brain Res,1999;4:303
4. Mmaynard E, Hatsopouulos N.G,. Ojakangas C.L et al. Neuronal interactions improve cortical population coding of movement direction.J.Neuroscil,1999;19(18):8083
5. .Eddleston M, Mucke. Molecular profile of reactive astrocytes.Neuroscience,1993;54:15
6. Goss J.R, O’Malley M.E, Zou L, et al. Astrocytes are the major source of nerve growth factor upregulation following traumatic brain injury in the rat.Exper.Neurol,1998;149:301
7. .Logan A, Green J, Hunter A, et al. Inhibition of glial scarring in the injured rat brainby a recombinant human monoclonal antibody to transforming growth factor-beta2.Eur.J.Neurosci,1999;11(7):2367
8. Webster T.J, Ergun C, Doremus R.H, et al. Enhanced functions of osteoclast-like cells on nanophase ceramics.Biomaterials,2001;22:1327
9. Karen A, Moxon, Nader M, et al. Nanostructured surface modification of ceramic-based microelectrodes to enhance biocompatibility for a direct brain-machine interface. IEEE transactions on biomedical engineering,2004;51(6):881
10. Dewes R, Aspinwall D, Simao J, et al. Electrical discharge machining and surface alloying-the process, parameters and state of play. Materials World,2003;11(5):16
11. Timothy A, Sylvain M, Nicholas G, et al. Microelectrode array fabrication by electrical discharge machining and chemical etching. IEEE transactions on biomedical engineering,2004;51(6):890
12. Rousche P.J, Pellinen D.S,. Pivin D.P, et al. Flexible polyimide-based intracortical electrode arrays with bioactive capability. IEEE transactions on biomedical engneering,2001;48:361
13. Yoon T.H, Hwang E.J, Shin DY, et al. A micromachined silicon depth probe for multichannel neural recording. IEEE transactions on biomedical engineering,2000;47:1082
14. Vetter R.J, Williams J.C, Hetke J.F, et al. Chronic neural recording using silicon-substrate microelectrode arrays implanted in cerebral cortex. IEEE transactions on biomedical engineering,2004;51(6):896
15. Donoghue J.P. Connecting cortex to machines: recent advances in brain interfaces. Nature neuroscience supplement,2002;5:1085
16. Kennedy P.R, Bakay R.A,. Moore M.M, et al. Direct control of a computer from the human central nervous system. IEEE Trans Rehabil Eng, 2000;2,19
[2]林泉,宋文强,彭承琳.生物传感器及其在生物医学中的应用研究[J].2007,28(8):27-28
[3]张先恩.生物传感器[M].北京:化学工业出版社.2006.8
[4] Nanjo M, Guilbault GG.. Enzyme electrode for l-amino acids and glucose [J].Analytica Chimica Acta, 1974,73(2)367-373
[5] Wu LN,Chen J, Du D, et al. Electrochemical immunoassay for CA125 based on cellulose acetate stabilized antigen/colloidal gold nanoparticles membrane[J].Electrochimica Acta, 2006,51(7):1208-1214
[6] Yao T, Musha S. Electrochemical enzymatic determinations of ethanol and -lactic acid with a carbon paste electrode modified chemically with nicotinamide adenine dinucleotide [J]. Analytica Chimica Acta, 1979,110(2):203-209
[7] Papastathopoulos DS, Rechnita GA. A urea-sensing membrane electrode for whole blood measurements [J]. Analytica Chimica Acta, 1975,79:17-26
[8] Marrazza G, Chiti G,1 Mascini M, ea al.Detection of Human Apolipoprotein E Genotypes by DNA Electrochemical Biosensor Coupled with PCR, Clinical chemistry,2000,46(1):31-37
[9]铃木周一.生物传感器[M].霍纪文,姜远海译.北京:科学出版社,1988.69
[10]孙康,李仲辉.主客体葡萄糖、乳糖生物传感器的研制.化学研究与应用,2001,13(4):376-379
[11] Qu HB, Zhang XE,Zhang SZ. Simultaneous determination of maltose and glucose using a dual-electrode flow injection system [J].Food Chemistry, 1995,52(2): 187-192
[12] Zhang XE, Rechnitz GA. Simultaneous determination of glucose and sucrose by a dual-working electrode multienzyme sensor flow-injection system [J]. Electroanalysis,1994,6(5-6):361-367
[13]魏福祥,韩菊,刘庆洲,等.胆碱酯酶生物传感器测定有机磷农药敌敌畏[J].河北科技大学学报,2003,22(4):92-94
[14]刘鹂,瞿丽雅.生物电化学传感器测定农药残留的研究进展[J].安全与环境学报,2005,12(5):113-116
[15] Campanella L, Colapicchioni C, Favero G, et al. Organophosphorus pesticide paraoxon analysis using solid state sensors [J].Sens Actuators B, 1996,331, 33(1) :25-33 .
[16] Ivanov A N, Evtugyn G A, Gyurcsayi R E, et al. Comparative investigation of electrochemical cholinesterase biosensors for pesticide determination[J].Anal Chim Acta, 2000,4042, 404(2) :55~59 .
[17] Probst G, Norman R. New BOD biosensor from Dr.Lange[J].Biosens Bioelectron, 1994,9(9) :12~13 .
[18]鞠熀先.电分析化学与生物传感技术[M].北京:科学出版社.2006,188
[19]鞠熀先.电分析化学与生物传感技术[M].北京:科学出版社.2006,192
[20] Scott DL, Bowden EF. Enzyme-substrate kinetics of adsorbed cytochrome c peroxidase on pyrolytic graphite electrodes [J]. Anal Chem 1994;66(8):1217-23
[21]郑德海.丝网印刷发展趋势[J].今日印刷,1998,5(S1)50-54
[22]张先恩.生物传感器[M].北京:化学工业出版社.2006.237-240
[23] Vijayakumar AR, Cs?regi E, Heller A, et al.Alcohol biosensors based on coupled oxidase-peroxidase systems [J], Anal.Chim.Acta,1996,327 (3)223-234
[24] Gülce H, Gülce A, Kavanoz M, et al. A new amperometric enzyme electrode for alcohol determination [J], Biosens.Bioelectron,2002,17 (6-7) 517-521
[25] Morales A, Céspedes F, Fàbregas EM,et al. Ethanol amperometric biosensor based on an alcohol oxidase-graphite-polymer biocomposite [J], Electrochim.Acta,1998,43 (23) 3575-3579
[26] Cai CX, Xue KH, Zhou YM, et al. Amperometric biosensor for ethanol based on immobilization of alcohol dehydrogenase on a nickel hexacyanoferrate modified microband gold electrode [J], Talanta, 1997,44 (3) 339-347
[27] Sprules SD, Hartley IC,Wedge R, et al. A disposable reagentless screen-printed amperometric biosensor for the measurement of alcohol in beverages [J], Anal.Chim.Acta,1996,329 (3) 215-221
[28]施清照,程琼.介体型乙醇生物传感器的研制及应用[J].分析试验室,1997,16(1):67-69
[29] Santos AS, Freire RS, Kubota LT. Highly stable amperometric biosensor for ethanol based on Meldola’s blue adsorbed on silica gel modified with niobium oxide [J], J.Electroanal.Chem,2003,547 (2) 135-142
[1] Mullor SG, Cabezudo MS, Ordieres AJM, el al. Alcohol biosensor based on alcohol dehydrogenase and Meldola Blue immobilized into a carbon paste electrode [J], Talanta,1996,43 (5):779-784
[2] Santos AS, Freire RS, Kubota LT. Highly stable amperometric biosensor for ethanol based on Meldola’s blue adsorbed on silica gel modified with niobium oxide [J], J.Electroanal.Chem,2003,547 (2):135-142
[3] Gorton L, Torstensson A, Jaegfeldt H, et al. Electrocatalytic oxidation of reduced nicotinamide coenzymes by graphite electrodes modified with an adsorbed phenoxazinium salt [J], meldola blue, J.Electroanal.Chem,1984,161 (1):103-120
[4] Dong SJ, Wang BX, Liu BF. Amperometric glucose sensor with ferrocene as an electron transfer mediator [J], Biosens.Bioelectron,1991,7 (3):215-222
[5] Santos AS, Gorton L, Kubota LT. Electrocatalytic NADH oxidation using an electrode based on Meldola Blue immobilized on silica coated with niobium oxide [J]. Electroanal,2002,14 (12):805-812
[6] Harrison DJ,Turner RFB, Baltes HP. Characterization of perfluorosulfonic acid polymer coated enzyme electrodes and a miniaturized integrated potentiostat for glucose analysis in whole blood [J], Anal Chem,1988,60 (19):2002-2007
[7] Sprules SD, Hartley IC,Wedge R, et al. A disposable reagentless screen-printed amperometric biosensor for the measurement of alcohol in beverages [J], Anal.Chim.Acta,1996,329 (3) 215-221
[8] Erlenkotter A, Kottbus M, Chemnitius GC. Flexible amperometric transducers for biosensors based on screen printed three electrode system [J]. J Electroanal Chem,2000,481(1):82-94
[9] Penton Z. Headspace measurement of ethanol in blood by gas chromatography with a modified autosampler [J]. Clin Chem 1985;31(3):439-441
[10]孙康,李仲辉.主客体葡萄糖、乳糖生物传感器的研制.化学研究与应用,2001,13(4):376-379
[11]周爱儒,查锡良.生物化学[M].北京:人民卫生出版社.2003.57
[12] Rover L, Fernandes JCB, Oliveira-Neto G, Kubota LT, Katekawa E, Serrano SHP. Study of NADH stability using ultraviolet–visible spectrophotometric analysis and factorial design [J]. Anal Biochem 1998,260(1):50-55
[13]周爱儒,查锡良.生物化学[M].北京:人民卫生出版社.2003.57
[14] Scott DL, Bowden EF. Enzyme-substrate kinetics of adsorbed cytochrome c peroxidase on pyrolytic graphite electrodes [J]. Anal Chem 1994;66(8):1217-23
[1]张先恩.生物传感器[M].北京:化学工业出版社.2006.45-49
[2]鞠熀先.电分析化学与生物传感技术[M].北京:科学出版社.2006,195
[3] Karyakin AA,Kotelnikova EA,Lukachova LV,et al. Optimal Environment for Glucose Oxidase in Perfluorosulfonated Ionomer Membranes: Improvement of First-Generation Biosensors [J].Analytical chmeistry,2002,74(7):1597-1603
[4] Dong SJ, Wang BX, Liu BF. Amperometric glucose sensor with ferrocene as an electron transfer mediator [J], Biosens.Bioelectron,1991,7 (3):215-222
[5] Harrison DJ,Turner RFB, Baltes HP. Characterization of perfluorosulfonic acid polymer coated enzyme electrodes and a miniaturized integrated potentiostat for glucose analysis in whole blood [J], Anal Chem,1988,60 (19):2002-2007
[6] Zhao S, Zhang K, Bai Y, et al. Glucose oxidase/colloidal gold nanoparticles immobilized in Nafion film on glassy carbon electrode: Direct electron transfer and electrocatalysis [J]. Bioelechem,2006,69(2):158-163
[7] Chen XG, Qian Y, Zhang SJ, Zou XY. Hydrogen peroxide biosensor based on immobilizing enzyme by gold nanoparticles and thionine. Acta Chim Sinica,2007,659(4):337-343
[8] Li QF, Yuan R, Liu Y, Mo CL, Chai YQ, Huang XQ, et al. A new biosensor for glucose based on Au colloid modified electrode and azure mediation. Chines J Aanl Chem 2005,33(5):631-634
[9] Grabar KC, Freeman RG., Hommer MB, Natan MJ. Preparation and characterization of au colloid monolayers. Anal Chem 1995,67(4):735-43
[10] Sprules SD, Hartley IC,Wedge R, et al. A disposable reagentless screen-printed amperometric biosensor for the measurement of alcohol in beverages [J], Anal.Chim.Acta,1996,329 (3) 215-221
[11] Erlenkotter A, Kottbus M, Chemnitius GC. Flexible amperometric transducers for biosensors based on screen printed three electrode system [J]. J Electroanal Chem,2000,481(1):82-94
[12] Penton Z. Headspace measurement of ethanol in blood by gas chromatography witha modified autosampler [J]. Clin Chem 1985;31(3):439-441
[13] Mullor SG, Cabezudo MS, Ordieres AJM, el al. Alcohol biosensor based on alcohol dehydrogenase and Meldola Blue immobilized into a carbon paste electrode [J], Talanta,1996,43 (5):779-784
[14] Santos AS, Freire RS, Kubota LT. Highly stable amperometric biosensor for ethanol based on Meldola’s blue adsorbed on silica gel modified with niobium oxide [J], J.Electroanal.Chem,2003,547 (2):135-142
[15]周爱儒,查锡良.生物化学[M].北京:人民卫生出版社.2003.57
[16] Rover L, Fernandes JCB, Oliveira-Neto G, Kubota LT, Katekawa E, Serrano SHP. Study of NADH stability using ultraviolet–visible spectrophotometric analysis and factorial design [J]. Anal Biochem 1998,260(1):50-55
[17]周爱儒,查锡良.生物化学[M].北京:人民卫生出版社.2003.57
[18] Scott DL, Bowden EF. Enzyme-substrate kinetics of adsorbed cytochrome c peroxidase on pyrolytic graphite electrodes [J]. Anal Chem 1994;66(8):1217-23
[19] Kennedy JW, Carey RW, Coolen RB, Garber CC, Lee HT, Levine JB, et al. EP5-A: Evaluation of precision performance of clinical chemistry devices; approved guideline. Wayne: NCCLS, 1999.
[20] Powers DM, Boyd JC, Click MR, Miller WG. EP7-A: Interference testing in clinical chemistry; approved guideline. Wayne: NCCLS, 2002.
[21] Carey RN, George H, Hartmann AE, Janzen VK, Levine JB, Miller WG, et al. EP15-A: User demonstration of performance for precision and accuracy; approved guideline. Wayne: NCCLS, 2001.
[22] Krouwer JS, Tholen DW, Garber CC, Goldschmidit HMJ, Kroll MH, Linnet K, et al. EP9-A2:Method comparison and bias estimation using patient samples; approved guideline-second edition. Wayne: NCCLS, 2002.
[1] Sprules SD, Hartley IC,Wedge R, et al. A disposable reagentless screen-printed amperometric biosensor for the measurement of alcohol in beverages [J], Anal.Chim.Acta,1996,329 (3) 215-221
[2] Erlenkotter A, Kottbus M, Chemnitius GC. Flexible amperometric transducers for biosensors based on screen printed three electrode system [J]. J Electroanal Chem,2000,481(1):82-94
[3]邱宪波,袁景淇,唐日泉.便携式血糖仪控制器的研制[J].微型电脑应用.2003,19(4):36-37
[4]王坤,袁景淇.基于PIC单片机的便携式血糖仪的开发[J].中国医疗器械杂志.2006,30(1):29-32
[5]邱宪波,袁景淇,唐日泉.EM78P458单片机及其在便携式血糖仪中的应用[J].测控技术.2003,22(4)60-62
[6]胡军.基于松翰单片机的血糖仪开发[J].化学传感器.2007,27(3):62-67
[7]唐日泉,郭琛,邱宪波.具有存储记忆功能的便携式血糖仪的研制[J].测控技术,2004,23(6)14-15
[8]郭琛,唐日泉,邱宪波.串行时钟芯片HT1380及其在便携式血糖仪中的应用[J].微型电脑应用.2004,20(4):38-40
[9]邹向阳,刘戎.基于SIC单片机的便携式快速血糖仪的设计与实现[J].传感器与微系统.2008,27(2)96-97
[10]胡军.手持式血糖仪控制器开发[J].传感器世界.2007,13(6):10-13
[11]唐日泉,袁景淇,邱宪波.液晶驱动芯片HT1621及其在便携式血糖仪中的应用[J].2003,19(5)22-24
[1]张先恩.生物传感器[M].北京:化学工业出版社.2006.1
[2]鞠熀先.电分析化学与生物传感技术[M].北京:科学出版社.2006,187
[3]林泉,宋文强,彭承琳.生物传感器及其在生物医学中的应用研究[J].医疗卫生装备.207,28(8)27-28
[4] Clark L C ,Lyons C. Electrode systems for continuous monitoring in cardiovascular surgery. Ann N Y Acad Sci ,1962 ,102 :29-33
[5] Updike SJ,Hicks JP. The enzyme electrode [J].Nature.1967,214(5092)986-988
[6] Guilbault GG,Montalvo JG. Urea-specific enzyme electrode[J].J Am Chem Soc.1969,91(8):2164-2165
[7] Divis C.Annals of Microbiology.1975,126A:175-186
[8] Rechnitz GA. A bio-selective membrane electrode prepared with living bacterial cells [J]. Analytica Chimica Acta, 1977, 94(2):377-384
[9] Karube I, Matsunage T, Mitsuda S, et al. Microbial Electrode BOD Sensors[J]. Biotechnology and Bioengineering,1977,19(10): 1535-1547.
[10] Janata J,Moss SD,Johnson CC.Selectvie chemical sensitvie field effect transducers.US Patent 4020830.1977
[11] Mosbach K, Danielsson B. An enzyme thermistor[J]. Biochim Biophys Acta. 1974,364(1):140-145
[12] Voelkl KP,Opita N,Lubbers DW,et al.Anal Chem.1980,301():162-163
[13] Guilbault GG. Determination of Formaldehyde with an Enzyme Coated Piezoelectric Crystal[J], Anal. Chem.1983,55():1682-1684
[14] Liedberg B,Nylander C,Lundstrm I. Development of an optical waveguide interferometric immunosensor[J].Sensors and Actuators B: Chemical,1991,4(3-4):297-299
[15] Cass AEG,Francis DG,Hill HAO,et al. Ferrocene-mediated enzyme electrode for amperometric determination of glucose[J].Anal Chem,1984,56(4):667-671
[16] Refereed Abstracts Book,the first world congress on biosensors.Singpore:Elsevier Science,1990
[17] Refereed Abstracts Book,the second world congress on biosensors.Geneva:Elsevier Science,1992
[18] Refereed Abstracts Book,the third world congress on biosensors.New Oreans:Elsevier Science,1994
[19] Refereed Abstracts Book,the forth world congress on biosensors.Bangkok:Elsevier Science,1996
[20] Refereed Abstracts Book,the fifth world congress on biosensors.Berlin:Elsevier Science,1998
[21] Refereed Abstracts Book,the sixth world congress on biosensors.San Diego:Elsevier Science,2000
[22] Refereed Abstracts Book,the sevevnth world congress on biosensors.Kyoto:Elsevier Science,2002
[23] Refereed Abstracts Book,the eighth world congress on biosensors.Granada:Elsevier Science,2004
[24]张先恩.生物传感器[M].北京:化学工业出版社.2006.6
[25]鞠熀先.电分析化学与生物传感技术[M].北京:科学出版社.2006,188
[26]张先恩.生物传感器[M].北京:化学工业出版社.2006.63
[27] Wollenberger U, Scheller F, Pfeiffer D,et al. Laccase/glucose oxidase electrode for determination of glucose[J].Analytica chimica acta.1986,187:39-45
[28] Blaedel WJ,Engstrom RC. Investigations of the ferricyanide-ferrocyanide system by pulsed rotation voltammetry[J].Anal Chem,1978,50(3):476-479
[29] Yao T, Musha S. Electrochemical enzymatic determinations of ethanol and -lactic acid with a carbon paste electrode modified chemically with nicotinamide adenine dinucleotide[J]. Analytica Chimica Acta, 1979, 10(2): 203-209
[30] Mascini M,Guilbault GG. Urease coupled ammonia electrode for urea determination in blood serum [J].Anal Chem.1977,49(6):795-798
[31] Nanjo M,Guilbault GG. Enzyme electrode sensing oxygen for uric acid in serum and urine [J].Anal Chem,1974,46(12):1769-1772
[32] Guilbault GG,et al.Anal Lett,1980,13(B18):1607
[33] Guilbault GG,Shu F. An electrode for the determination of glutamine[J].Anal Chim Acta.1971,56(3):333-338
[34] Mizutani F,Tsuda K, Karube I, et al. Determination of glutamate pyruvate transaminase and pyruvate with an amperometric pyruvate oxidase sensor[J].Anal Chim Acta.1980,118(1):65-71
[35]张先恩.生物传感器[M].北京:化学工业出版社.2006.80
[36] Hikuma M,et al.Biotechnol Bioengin,1979,21():1945
[37] Karube I, Okada T, Suzuki S,et al. Amperometric determination of ammonia gas with immobilized nitrifying bacteria [J].Anal Chem.1981,53(12):1852-1854
[38] Karube I,et al.Ibid,1982,54():1725
[39]张先恩.生物传感器[M].北京:化学工业出版社.2006.91
[40] Dai Z,Chen J,Yan F,et al.Electrochemical sensor for immunoassay of carcinoembryonic antigen based on thionine monolayer modified gold electrode[J].Cancer detection and prevention.2005,29(3):233-240
[41] Ju HX,Yan GF,Chen F,et al. Enzyme-linked immunoassay of a-1-fetoprotein in serum by differential pulse voltammetry[J].Electroanal.1999,11(2):124-128
[42] Dai Z,Chen J,Yan F,et al. Reagentless amperometric immunosensors based on direct elecrtochemistry of horseradish peroxidase for rapid determination of carcinoma antigen[J].Anal Chem.2003,75(20): 5429-5434
[43] Du D,Yan F,Liu SL,et al. Electrochemical immunosensor for carbohydrate antigen 19-9 based on immunological reaction and its immobilization in titania sol-gel matrix[J].Immuno Methods.2003,283(1-2):67-75
[44] Chen J,Yan F,Wu J,et al. Electrochemical immunoassay of human chorionic gonadotrophin based on its immobilization in gold nanoparticles-chitosan membrane[J].Electroanal. 2006,18(7):670-676
[45]鞠熀先.电分析化学与生物传感技术[M].北京:科学出版社.2006,425
[46] Wang J,Rivas G,Cai X,et al. DNA electrochemical biosensors for environmental monitoring[J].Anal Chim Acta.1997,347(1-2):1-8
[47] Siontorou CG,Nikolelis DP,Miernik A,et al. Rapid methods for detection ofAflatoxin M1 based on electrochemical transduction by self-assembled metal-supported bilayer lipid membranes (s-BLMs) and on interferences with transduction of DNA hybridization[J].Electrochim Acta.1998,43(23):3611-3617
[48] Wang J,Rivas G,Parrado C,et al. Electrochemical biosensor for detecting DNA sequences from the pathogenic protozoan Cryptosporidium parvum[J].Talanta,1997,44(11):2003-2010
[49] Wang J,Rivas G,Cai X. Screen-printed electrochemical hybridization biosensor for the detection of DNA sequences from the Escherichia coli pathogen[J].Electroanalysis.1997,9(5):395-398
[50] Mascini M,Palchetti I,Marrazza G,et al. DNA electrochemical biosensors[J]. Fresenius' Journal of Analytical Chemistry. 2001,369(1):15-22
[51] Wang J,Rivas G,Cai X,et al. Sequence-specific electrochemical biosensing of M. tuberculosis DNA[J].Anal Chim Acta.1997,337(1):41-48
[52] Erdem A,Kerman K,Meric B,et al. DNA Electrochemical Biosensor for the Detection of Short DNA Sequences Related to the Hepatitis B Virus[J].Electroanalysis,1999,11(8):586-588
[53] Ye YK,Zhao JH,Yan F,et al. Electrochemical behavior and detection of hepatitis B virus DNA PCR production at gold electrode[J].Biosens Bioelectron.2003:18(12):1501-1508
[54] Azek F,Grossiord C,Joannes M,et al. Hybridization Assay at a Disposable Electrochemical Biosensor for the Attomole Detection of Amplified Human Cytomegalovirus DNA[J].Anal Biochem.2000,284(1):107-113
[55] Hashimoto K,Ito K,Ishimori Y. Sequence-Specific Gene Detection with a Gold Electrode Modified with DNA Probes and an Electrochemically Active Dye[J].Anal Chem.1994,66(21):3830-3833
[56] Wang J,Rivas G,Cai X,et al. Detection of point mutation in the p53 gene using a /peptide nucleic acid biosensor[J].Anal Chim Acta.1997,344(1-2):111-118
[57] Bardea A,Patolsky F,Dagan A,et al.Chem Commun.1999,21():
[58] Maeda M,Mitsuhashi Y,Nakano K,et al.Anal Sci.1992,8():83
[59] Brett AMO,Serrano SHP,Macedo TA,et al. Voltammetric behavior of nitroimidazoles at a DNA-biosensor[J].Electroanal.1997,9(14):1132-1137
[60] Brabed V. DNA sensor for the determination of antitumor platinum compounds[J].Electrochim Acta.2000,45(18):2929-2932
1. He Q.H, Peng C.L, Wu B.M, et al. Research methods of brain-machine interface technology. Journal of chongqing university,2002;25:106 [何庆华,彭承琳,吴宝明等.脑-机接口技术研究方法.重庆大学学报,2002;25:106]
2. Wan B.K, Gao Y, Zhao L, et al. Brain-machine interface: A new channel for information communicate between human brain and environments. Biomedical engineering foreign medical sciences, 2005;28(1):4 [万柏坤,高扬,赵丽等.脑-机接口:大脑对外信息交流的新途径.国外医学生物医学工程分册,2005;28(1):4]
3. Williams J.C, Rennaker R.L, Kipke D.R, et al. Long-term neural recording characteristics of wire microelectrode arrays implanted in cerebral cortex. Brain Res,1999;4:303
4. Mmaynard E, Hatsopouulos N.G,. Ojakangas C.L et al. Neuronal interactions improve cortical population coding of movement direction.J.Neuroscil,1999;19(18):8083
5. .Eddleston M, Mucke. Molecular profile of reactive astrocytes.Neuroscience,1993;54:15
6. Goss J.R, O’Malley M.E, Zou L, et al. Astrocytes are the major source of nerve growth factor upregulation following traumatic brain injury in the rat.Exper.Neurol,1998;149:301
7. .Logan A, Green J, Hunter A, et al. Inhibition of glial scarring in the injured rat brainby a recombinant human monoclonal antibody to transforming growth factor-beta2.Eur.J.Neurosci,1999;11(7):2367
8. Webster T.J, Ergun C, Doremus R.H, et al. Enhanced functions of osteoclast-like cells on nanophase ceramics.Biomaterials,2001;22:1327
9. Karen A, Moxon, Nader M, et al. Nanostructured surface modification of ceramic-based microelectrodes to enhance biocompatibility for a direct brain-machine interface. IEEE transactions on biomedical engineering,2004;51(6):881
10. Dewes R, Aspinwall D, Simao J, et al. Electrical discharge machining and surface alloying-the process, parameters and state of play. Materials World,2003;11(5):16
11. Timothy A, Sylvain M, Nicholas G, et al. Microelectrode array fabrication by electrical discharge machining and chemical etching. IEEE transactions on biomedical engineering,2004;51(6):890
12. Rousche P.J, Pellinen D.S,. Pivin D.P, et al. Flexible polyimide-based intracortical electrode arrays with bioactive capability. IEEE transactions on biomedical engneering,2001;48:361
13. Yoon T.H, Hwang E.J, Shin DY, et al. A micromachined silicon depth probe for multichannel neural recording. IEEE transactions on biomedical engineering,2000;47:1082
14. Vetter R.J, Williams J.C, Hetke J.F, et al. Chronic neural recording using silicon-substrate microelectrode arrays implanted in cerebral cortex. IEEE transactions on biomedical engineering,2004;51(6):896
15. Donoghue J.P. Connecting cortex to machines: recent advances in brain interfaces. Nature neuroscience supplement,2002;5:1085
16. Kennedy P.R, Bakay R.A,. Moore M.M, et al. Direct control of a computer from the human central nervous system. IEEE Trans Rehabil Eng, 2000;2,19