基于乙酰胆碱酯酶的一次性可抛纸基酶传感器应用于杀螟硫磷的检测研究
Study on the Application of Acetylcholinesterase-Based Disposable Paper-Based Sensor to the Detection of Fenitrothion

作者: 丁 睿 * , 刘 帅 * , 叶 露 , 潘 婧 , 鲍 宁 , 刘 扬 :南通大学公共卫生学院,江苏 南通;

关键词: 杀螟硫磷纸基传感器乙酰胆碱酯酶 Fenitrothion Paper-Based Sensor Acetylcholinesterase

摘要:
本研究旨在研发了一种应用于有机磷农药快速、准确、一次性可抛的传感装置。用碳胶/氧化铟锡玻璃(ITO)作为工作电极,碳和银作为对电极和参比电极构成基于三电极系统。纳米金铂–石墨烯材料修饰在工作电极表面用来固定乙酰胆碱酯酶(AChE)和加速电子传递速度。以氯化乙酰胆碱为底物,通过AChE酶催化反应检测底物杀螟硫磷。结果表明,使用差分脉冲伏安法检测杀螟硫磷,其浓度从1.0 × 10−7到1.5 × 10−5 mol/L范围内,氧化峰电流的降低程度与杀螟硫磷浓度的对数成正比,检测限低至5.4 × 10−8 mol/L。该酶传感器有希望成为快速检测有机磷农药的新方法。

Abstract: The purpose is to develop a rapid, accurate, disposable sensing device applied to organophosphrus pesticides (OPs). Using carbon glue/indium tin oxide glass (ITO) as working electrode, carbon glue and silver glue as counter and reference electrode, a three-electrode system was formed. Gold/ platinum-graphene nanocomposites were modified to immobilize acetylcholinesterase (AChE) and enhance electron transfer rate. Acetylcholine chloride was used as substrate to detect fenitrothion by AChE enzyme catalyzed reaction. The results showed that the reduction of oxidation peak current was directly proportional to the logarithm of the concentration of fenitrothion in the range of 1.0 × 10
−7 to 1.5 × 10−5 mol/L by differential pulse voltammetry (DPV). The detection limit was as low as 5.4 × 10−8 mol/L. The enzyme sensor is expected to be a new method for rapid and economical detection of OPs.

1. 引言

酶传感器作为一种环境友好、微型化的传感器,可以识别测定物和通过转化装置输出信号 [1] [2]。在近几年的研究中,常见的报道有:基于葡萄糖氧化酶的检测葡萄糖传感器 [3],基于尿素酶的铵离子传感器 [4] 和基于AChE的有机磷农药传感器 [5] [6] [7]。但传统固体大电极的酶传感器有如下缺点:1) 检测过程中,电极的表面容易被污染;重复使用时候,电极需要被重新打磨和清洁;2) 检测过程中由于生物样品的复杂性,处理过程过于复杂耗时,限制了传感器的应用。因此,研究一种廉价、易得、可抛型的酶传感器有深远的意义 [8]。

基于丝网印刷技术的传感器由于成本低廉、有望工业生产、操作简单 [9] [10] 而用来检测底物 [11] [12]。丝网印刷电极通常包含在同一条纸上打印出来的三电极系统(工作电极、对电极、参比电极),而工作电极通常可用不同的材料打印,比如:碳、金、银 [13]。但是可抛的丝网印刷电极还存在有一些缺点:1) 不能对工作电极进行修饰,如组装纳米材料难度很大 [14] [15] [16];2) 灵敏度、精确度、重现性与传统固体大电极相差很大 [17] [18];3) 对电极和参比电极与工作电极组装在一个平面上,在生物反应的时候很容易被污染 [19]。

本研究采用的纳米材料优势有:1) 石墨烯具有良好的电子特性,丰富的边缘缺陷,较大的比表面积(单层石墨烯理论上可以达到2630 m2∙g−1)以及极强的机械性能 [20] [21] [22] [23]。2) 铂的电催化能力和高灵敏度可以通过与金纳米粒子结合而加强 [24] [25] [26]。3) 酶可以通过Au-N和Pt-N共价键与电极表面稳定结合 [27] [28] [29]。

为了克服丝网印刷电极的上述缺陷,本研究我们成功制备了基于ITO和打印滤纸的纸基电分析传感器。工作电极是将ITO电极为基底,丝网印刷技术用来制作参比电极和对电极,用来检测杀螟硫磷(FT)。有机磷农药可以迅速杀灭虫,价格低廉,在蔬菜、水果等农作物的种植过程中是利用最广泛一种杀虫剂 [30] [31]。但有机磷农药可以抑制通过抑制AChE的活性 [32] [33] 从而导致严重的健康隐患 [34] [35] [36],并且会增加患癌的风险 [37]。杀螟硫磷(即0,0-二甲基-0-硫代磷酸酯,化学式为C9H12NO5PS)是常见有机磷农药中的一种,具有胃肠道接触性毒性 [38] [39]。检测杀螟硫磷的方法有传统的气相色谱法和液相色谱法 [40] [41],但简单又灵敏的电化学AChE传感器因为其廉价、易操作并且能快速检测已经吸引了越来越多学者的关注 [42] [43] [44]。目前就我们所知,基于纸基电极一次性可抛的AChE传感器中的很少有报道 [45] [46]。因此,我们研究了基于AChE/GR-Au-Pt的纸基电极,用来对实际样品进行检测,并且具有很高的灵敏度,很宽的检测范围和很好的重现性。这次研究的主要目的是通过简单、廉价、大量生产的方式来研制能快速、灵敏、稳定地检测有机磷农药的传感器 [47] [48]。

2. 实验与仪器

2.1. 材料和仪器

ITO玻璃(电阻为10 Ω/cm2,膜厚180 nm)从中国深圳的纳铂有限公司购买;面包板、电线和USB接口均是从中国浙江的宁波中益电子设备厂购买;双面碳胶(12 mm宽)从SPI公司购买(West Chester, PA, USA);滤纸(Whatman No. 1)可从Whatman International Co. Ltd. (Maidstone, United Kingdom)购买获得;浓度为0.1 mol/L 磷酸盐缓冲液(PBS)是由0.1mol/L Na2HPO4和0.1 mol/L NaH2PO4混合制得。AChE、氯化乙酰硫代胆碱(ATCl)、丙酮、二氯甲烷、无水硫酸钠、甲醇、亚铁氰化钾、铁氰化钾从Sigma (St. Louis, MO, USA)购买。所有化学品都是分析纯,所有溶液都是用双蒸水制备。碳浆(C10903D)则是从Gwent Electronic Materials (Gwent, United Kingdom)所购买,银浆是中国上海海证有限公司购买;电化学检测在用CHI1242B化学工作站(上海辰华)进行;扫描电镜(SEM)用Quanta 250FEG-Field场发射电镜所得,应用二次电子成像模型,其工作电压为20 keV,激光光斑直径3 mm;EDS使用Apollo-40EDS (Edax, Ltd., USA)应用于二次电子成像模型,放大1250倍数;EIS在Autolab-302N 电化学工作站(Metrohm China Co. Ltd., Switzerland)开路电位下进行测定,检测底液是1.0 mmol/L [Fe(CN)6]3−/4−与0.10mol/LKCl 1:1混合。气相色谱分析检测用于四种蔬菜样品中杀螟硫磷的浓度检测金标准,分别用CP3800 (Varian Associates INC., USA)加入1.44 × 10−6 mol/L的杀螟硫磷进行回收率实验。所有实验均在室温23℃ ± 2℃下进行。

2.2. AChE/GR-Au-Pt修饰的纸基电极的制备过程

基于AChE/GR-Au-Pt的纸基系统的制备如图1中所示:

① ITO导电玻璃(20 mm × 12 mm)分别用丙酮、酒精和双蒸水超声清洗5 min,双面碳胶(12 mm × 12 mm)被贴在干净的ITO玻璃上形成碳胶-ITO。在塑料胶带上打上一个5 mm直径的圆孔,然后将塑料胶带贴于碳胶-ITO上形成完全一样的区域(5 mm直径的圆形工作区域),作为工作电极(a)。

② 5.0 μL的0.5 mg/mLGR溶液滴加入工作电极孔,在室温下干燥形成基于GO工作电极(b)。

③ 20.0 μL1.0 mmol/L氯金酸和1.0 mmol/L氯铂酸的电解质溶液加入到石墨烯的工作电极,电化学沉积Au-Pt纳米粒子,方法参考了文献 [45]:CV的电位区间为−0.1到−0.9V,扫描20圈,扫速50 mV/s,形成GR-Au-Pt纳米复合物(c)。

④ 滤纸通过激光打印机(Hewlett-Packard, Model HP 1010 LaserJet)双面打印,分辨率为600 dpi,黑色区域是通过Adobe illustrator CS4设计然后放入150℃烘箱内加热5个小时,在烘干之后,滤纸上没有碳粉的矩形区域(长1.1 cm宽0.74 cm)作为电化学平台用于酶检测。丝网印刷技术用于构造参比电极和对电极。银浆用于构建参比电极,碳浆用于构建对电极。

⑤ 5 μL的20 U/mL AChE滴加在GR-Au-Pt修饰的工作电极表面,然后在4℃下孵育8小时。在用pH 7.2的PBS溶液冲洗去除游离的AChE之后,基于AChE/GR-Au-Pt的工作电极和打印纸,纸基的参比电极对电极用双面胶结合在一起。

⑥ 20 μL 1 mmol/LATCl滴加在纸基系统上通过DPV来获得电化学响应,DPV的电位区间是从−0.1到+0.7 V (e)~(f)。不使用的时候,基于AChE/GR-Au-Pt的工作电极储存在4℃冰箱。

Figure 1. Schematic diagram of fenitrothion sensor based on AChE/GR-Au-Pt nanocomposite modified PADs

图1. 基于AChE/GR-Au-Pt纳米复合物修饰纸基系统对杀螟硫磷的酶传感器构建示意图

3. 结果和讨论

3.1. 基于石墨烯/金铂纳米粒子的工作电极的表征

SEM和EDS的表征基于AChE/GR-Au-Pt修饰工作电极的SEM图2所示。图2A显示了固定于在工作电极表面的石墨烯的2维结构,可以看出石墨烯典型的褶皱状和薄如纱的形貌。图2B显示的是基于AChE/GR-Au-Pt工作电极表面在放大10,000倍之后的SEM图像,证明了纳米粒子分散在基于石墨烯的工作电极表面。金铂纳米粒子的直径可从图C中观察到,大约在150~400 nm之间。图2D是固定在修饰AChE/GR-Au-Pt工作电极表面的AChE的SEM图。与图2B相比,可以观察到薄薄的一层覆盖物。石墨烯和GR-Au-Pt纳米复合物的相应能谱分析(如图3所示)也证实了工作电极表面金、铂、碳元素的存在(图3A, B, C)。

3.2. 氧化还原探针表征

图4所示,可以观察到一个半圆对应裸的工作电极(Rct = 750 Ω,曲线a),GR修饰在工作电极上之后,电阻值下降(Rct = 500 Ω,曲线b),证明了GR具有良好的导电性能。当Pt纳米粒子或Au纳米粒子修饰到基于GR纸基系统上时,半圆的直径明显减少,表明了界面的电化学阻抗(Rct = 270 Ω,曲线c;Rct = 250 Ω,曲线d)减小。然而,Au-Pt共沉淀的双合金使得半圆的直径进一步减少(Rct = 50 Ω,曲线e),这是因为双金属系统具有更好的类似于导线和电子通道的作用。在AChE固定在GR-Au-Pt修饰纸基系统之后,可以看到Ret显著增加(Rct = 1500 Ω,曲线f),这是因为AChE对电子转移的阻碍作用。

图5表示了不同扫速下,GR-Au-Pt修饰纸基电极在0.1 mmol/L探针溶液检测的CV图。在半无限线性扩散条件和可逆反应下,Randles-Sevcik等式可描述为 [49]:

i p = kAn 3 / 2 AD 1 / 2 C o V 1 / 2

在等式(1)中,ip代表峰值电流,单位安培,A是电极表面积,单位cm2,n是转移电子数目,D电活性材料的扩散系数,Co是电活性材料的体积浓度,单位cm2∙s−1,v是扫速,单位V∙s−1。对铁氰化钾来说,n是1,D是7.6 × 10−6 cm2∙s−1,Co是浓度,ip相对于ν1/2的斜率是4.0 × 10−5 (R2 = 0.9997),如图5B所示,用来计算有效工作表面积大小。AChE/GR-Au-Pt的A计算得出大小为0.53 cm2。研究得出基于GR-Au-Pt修饰纸基系统的电活性表面积三倍大于其物理面积(0.13 cm2)。

代表电子转移速率的常数–标准速率常数(ks)可以通过以下方式计算。ks是快速简单的方式来评估电极反应动力学程度的参数。在Nicholson理论 [49] 中:

ψ = ( D O / D R ) α / 2 K s [ D O π v ( nF / RT ) ] 1 / 2

Figure 2. SEM images (A) GR-modified working electrode and (B) Gr-Au-Pt modified working electrode at a magnification of 10,000 times (C) Gr-Au-Pt modified working electrode at a magnification of 5000 times (D) AChE/Gr-Au-Pt modified working electrode at A magnification of 5000 times

图2. SEM图 (A) GR修饰的工作电极和(B) GR-Au-Pt修饰的工作电极在放大10,000倍的图(C) GR-Au-Pt修饰的工作电极在放大5000倍的图 (D) AchE/GR-Au-Pt修饰的工作电极在放大5000倍的图

Figure 3. Energy spectrum analysis of naked working electrode (A), graphene-modified working electrode (B), and Gr- Au-Pt nanocomposite modified working electrode (C)

图3. 裸的工作电极 (A), GR修饰工作电极 (B), GR-Au-Pt纳米复合物修饰工作电极(C)的能谱分析

Figure 4. EIS diagram of PADs modified with different materials (a) bare, (b) graphene, (c) graphene/Au nanocomposite, (d) graphene/Pt nanocomposite, (e) graphene/Au-Ptnanocomposite, (f) AChE/Au-Pt nanocomposite contains 10 mmo/L [Fe(CN)6]3−/4− (1:1) in 0.1moL /L KCl

图4. 不同材料修饰PADs在0.1 mo/L KCl含有10 mmo/L [Fe(CN)6]3/4 (1:1)溶液中的交流阻抗图 (a) 裸电极, (b) 石墨烯, (c) 石墨烯/金 纳米复合物, (d) 石墨烯/铂 纳米复合材物, (e) 石墨烯/金-铂纳米复合物, (f) 乙酰胆碱酯酶/金-铂纳米复合物

对于[Fe(CN)6]3−/4−这个体系,n是电子数目,α是转移系数,D是电活性材料的扩散系数,ν是扫秒速度(单位V/s)。当△Ep = 67 mV,Ψ = 4,ks的数值为0.039 cm/s,该研究表明GR-Au-Pt纳米材料和工作电极之间有良好电子传递过程,该过程可逆。

Figure 5. CV of AChE/ GR-Au-Pt modified PADS in 0.1mmol /L KCl/1 mmol/L [Fe(CN)6]3−/4− solution (A). The scanning velocity from A to G is: 50, 80, 100, 150, 180, 200 and 250 mV∙S-1 (B)

图5. A图是AChE/GR-Au-Pt修饰PADs在0.1 mmol/L KCl/1 mmol/L [Fe(CN)6]3−/4−溶液中的CV图扫描速度从a到g分别是:50,80,100,150,180,200和250 mV∙s−1 (B)

3.3. 研究基于AChE/GR-Au-Pt的纸基系统应用于杀螟硫磷检测

不同材料修饰的基于AChE的传感器上对于ATCl的电化学响应,通过DPV进行研究如图6中所示,电位范围在−0.2 V到 0.7 V之间,约在+0.26 V处可以观察的氧化电流峰,如图6A。在图6B中所示,峰值电流的改变在基于不同材料修饰的AChE的传感器上对比图。很显然,基于GR的纸基系统的峰值电流高于仅仅在纸基系统上修饰AChE (图6B(a)~(b)),这也许是因为GR具有极好的导电性能,可以加快电子转移 [23] [24]。在Au-Pt纳米粒子电沉积到纸基系统上之后,峰电流进一步增加(图6B(c)~(d))。

Figure 6. (A) DPV of 1 mmol/L ATCl on the electrodes of different modified materials (a) Pad modified AChE, (b) Pad modified AChE/ Graphene, (c) Pad modified AChE/Au-graphene (d) Pad modified AChE/Au-Pt-graphene (B) Peak current comparison histogram of Pads modified by different materials

图6. (A) 1 mmol/L ATCl在不同修饰材料的电极上DPV图(a) PAD修饰AChE, (b) PAD修饰AChE/graphene, (c) PAD修饰AChE/Au-graphene (d) PAD修饰AChE/Pt-graphene, (e) PAD修饰AChE/Au-Pt-graphene(B)不同材料修饰PADs的峰电流比较柱状图

我们可以通过DPV曲线研究pH和固定AChE的量对于峰值电流的影响。氧化峰值电流随着pH的增加而增加并且在pH 7.2达到最大,随后pH进一步增加时,峰电流持续降低(图7A),pH值为7.2。图7B显示了固定在酶生物传感器上的AChE响应的影响。很明显峰值电流随着AChE数量的增加而增加并且在20 U/mL达到最大,当AChE的浓度大于20 U/mL时仍保持不变。这是因为过量的AChE可能阻碍底物和电极之间的电子转移。因此,在这项研究中20 U/mL被选择作为AChE的最佳浓度。

Figure 7. Effect of pH (A) and immobilized enzyme (B) content on peak current values of AChE/GR-Au-Pt-modified PADS

图7. pH值(A)和AChE固定值(B)的含量对AChE/GR-Au-Pt-修饰PADs的峰电流值的影响

图8所示,在上述优化条件下,研究该系统对杀螟硫磷的检测。可在+0.26 V处观察到明显的氧化峰,其来源于胆碱的氧化(TCh)。在滴加杀螟硫磷溶液后(静置30s),纸基电极上电流的改变与杀螟硫磷浓度的对数(log(FT))呈线性相关(图8)。杀螟硫磷的浓度从1.0 × 10−7到2.3 × 10−3 mol/L范围内,氧化峰(Δip)的减少与浓度的对数(log(FT))成比例。线性方程表达为“Δip(μA) = 35.99 + 12.67lgCFT (R2 = 0.9809)”,基于3倍信噪比计算得到杀螟硫磷的检测限为5.4 × 10−8 mol/L。

Figure 8. (A) DPV of 20 μL 1 mmol/L ATCL and different concentrations of fenitrothion on PADS modified by AChE/Au-Pt-GR. From a to g: 0, 0.353, 0.706, 1.42, 1.71, 2.06 and 2.59 × 10−6 mol/L; (B) the linear relation diagram of fenitrothion

图8. (A) DPV图采集于20 μL of 1 mmmol/L ATCl和不同浓度的杀螟硫磷在AChE/Au-Pt-GR修饰PADs;从a到g:0,0.353,0.706,1.42,1.71,2.06和2.59 × 10−6 mol/L (B)杀螟硫磷的线性关系图

在相同的电极上进行6次连续安培测量来研究生物传感器的重复性,得到相对标准偏差(RSD)为4.07%,这证明了很好的可重复性。当基于酶修饰的工作电极修饰不使用时,要存储于4℃冰箱里。28天之后,酶电极的响应电流仅比初始响应减少了8%,表明很好的稳定性。

3.4. 蔬菜样品分析的实际应用

为了研究酶生物传感器在实际样品分析中的应用,基于该传感器被用于检测从当地超市购买的四种常见蔬菜中有机磷农药的残留量(表1)。通过四种蔬菜样品中加入1.44 × 10−6 mol/L杀螟硫磷进行回收实验,将该传感器的结果与气相色谱法的结果进行比较,该方法的回收率从89.3%到103.8%,结果表明实际样品中农药检测的准确性,有实际应用的潜能。

Table 1. Recovery of bothophos measured by this sensor in vegetable samples and compared with gas chromatography

表1. 本传感器在蔬菜样品中检测杀螟硫磷的回收率实验,并且和气相色谱相比。

4. 结论

本研究成功制备一个灵敏的、廉价的、一次性可抛的纸基电极,通过高效固定酶来应用于构造酶生物传感器。通过丝网印刷技术加工过的滤纸上的对电极和参比电极和工作电极结合在一起。新型GR-Au-Pt纳米粒子用于修饰工作电极,可以加速电子转移和固定酶。纸基电极证明可以高效结合蛋白质/酶,应用于在AChE催化反应快速检测杀螟硫磷。构建的AChE-传感器具有微型化,高灵敏度,宽的线性范围,好的稳定性和再现性等优点。研究证明,显示了该新型纸基电极可以成为食品中快速检测有机磷农药的新方法。

基金项目

本论文受到国家自然科学基金(81202249)、江苏省六大人才高峰项目(2015-YY-008)南通市科技项目(MS12019062和MS12017018-2)的支持。

文章引用: 丁 睿 , 刘 帅 , 叶 露 , 潘 婧 , 鲍 宁 , 刘 扬 (2021) 基于乙酰胆碱酯酶的一次性可抛纸基酶传感器应用于杀螟硫磷的检测研究。 纳米技术, 11, 218-229. doi: 10.12677/NAT.2021.113025

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