基于纳米孔单分子技术的抗结核药物异烟肼的检测新方法
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摘要
基于纳米孔单分子检测技术,利用α溶血素突变蛋白(M113R)_7和6-氨基-6-脱氧-β-环糊精(am_7βCD)适配体构建了一种新型传感器((M113R)_7-am_7βCD),建立了快速、高灵敏度的抗结核药物异烟肼(Isoniazid,INH)的单分子检测新方法。优化了通道蛋白、缓冲液p H值、电压等条件。相比于野生型蛋白和(M113N)7突变蛋白,(M113R)_7突变蛋白更利于检测;在中性缓冲溶液和高电压条件下,从cis端加入异烟肼,可获得更高的检测灵敏度。在高于异烟肼浓度40倍的条件下,此传感器对一些常见药物赋形剂(葡萄糖、蔗糖、淀粉)、溶液中可能共存的金属离子以及异烟肼的合成中间体异烟酸均无响应,表明其对异烟肼有良好的选择性。在最佳测试条件下,异烟肼浓度在0.5~30μmol/L范围内呈良好的线性关系,检出限为2 nmol/L。本方法直接应用于药片中异烟肼含量的测定,加标回收率为92.9%~108.2%。
An analytical method was proposed for determination of antituberculosis drug isoniazid at singlemolecule level by protein nanopores equipped with a cyclodextrin adapter. The developed method is rapid,highly sensitive and specific. The influence of channel protein,p H of buffer solution and recording voltage on detection signal was investigated. The results showed that( M113 R)_7 could provide an enhanced resolution for isoniazid recognition compared with that observed in wild-type protein or( M113 N)_7. The addition of isoniazid from cis side during relatively high voltage and neutral p H conditions could improve the detection sensitivity.Some common pharmaceutical excipients( glucose,sucrose and starch),metal ions that may coexist in solution and synthetic intermediates of isoniazid,isonicotinic acid( the concentrations of all analytes were above 40 times of isoniazid) were added to evaluate the selectivity of the sensing platform,and it was found that no obvious response could be observed after adding these analytes mentioned above,showing a good selectivity. The linear range and the detection limit of the sensor were 0. 5-30 μmol/L and 2 nmol/L. The method was directly applied to the rapid detection of isoniazid,with the recoveries of 92.9%-108.2%.
An analytical method was proposed for determination of antituberculosis drug isoniazid at singlemolecule level by protein nanopores equipped with a cyclodextrin adapter. The developed method is rapid,highly sensitive and specific. The influence of channel protein,p H of buffer solution and recording voltage on detection signal was investigated. The results showed that( M113 R)_7 could provide an enhanced resolution for isoniazid recognition compared with that observed in wild-type protein or( M113 N)_7. The addition of isoniazid from cis side during relatively high voltage and neutral p H conditions could improve the detection sensitivity.Some common pharmaceutical excipients( glucose,sucrose and starch),metal ions that may coexist in solution and synthetic intermediates of isoniazid,isonicotinic acid( the concentrations of all analytes were above 40 times of isoniazid) were added to evaluate the selectivity of the sensing platform,and it was found that no obvious response could be observed after adding these analytes mentioned above,showing a good selectivity. The linear range and the detection limit of the sensor were 0. 5-30 μmol/L and 2 nmol/L. The method was directly applied to the rapid detection of isoniazid,with the recoveries of 92.9%-108.2%.
引文
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2 Churchyard G J,Fielding K L,Lewds J J,Goetzea L,Corbett E L,Godfrey-Faussett P,Hayes R J,Chaisson R E,Grant A D.N.Engl.J.Med.,2014,370(4):301-310
3 Evgenév M I,Garmonov S Y,Evgenéva I I,Kurtbelyalova K I.Anal.Chem.,1998,53(1):89-94
4 LIU Li-Jun,ZHENG Xing-Wang,ZHANG Zhu-Jun.Chinese J.Anal.Chem.,2003,31(9):1112-1114刘丽君,郑行望,章竹君.分析化学,2003,31(9):1112-1114
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6 Chinese Pharmacopoeia,China Medical Science and Technology Press,2010:294-296中华人民共和国药典(第2部),中国医药科技出版社,2010:294-296
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9 Roozbahani G M,Chen X H,Zhang Y W,Juarez O,Li D,Guan X Y.Anal.Chem.,2018,90(9):5938-5944
10 Kim Y H,Hang L,Cifelli J L,Sept D,Mayer M,Yang J.Anal.Chem.,2018,90(3):1635-1642
11 Ying Y L,Zhang J,Gao R,Long Y T.Angew.Chem.Int.Ed.,2013,52(50):13154-13161
12 WANG Hui-Feng,HUANG Fei,GU Zhen,HU Zheng-Li,YING Yi-Lun,YAN Bing-Yong,LONG Yi-Tao.Chinese J.Anal.Chem.,2018,46(6):843-850王慧锋,黄飞,顾震,胡正利,应佚伦,颜秉勇,龙亿涛.分析化学,2018,46(6):843-850
13 Guan X,Gu L Q,Cheley S,Braha O,Baley H.Chembiochem,2005,6(10):1875-1881
14 Bayley H,Cremer P S.Nature,2001,413(6852):226-230
15 Bayley H,Braha O,Gu L Q.Adv.Mater.,2000,12(2):139-142
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17 Gu L Q,Braha O,Conlan S,Cheley,Baley H.Nature,1999,398(6729):686-690
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19 Wang L,Yao F,Kang X F J.Anal.Chem.,2017,89(15):7958-7965
20 Guo Y L,Niu A H,Jian F F,Wang Y,Yao F J,Wei Y F,Tian L,Kang X F.Analyst,2017,142(7):1048-1053
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22 Cheley S,Braha O,Lu X,Conlan S,Baley H.Protein Sci.,1999,8(6):1257-1267
23 Lakshmanan M R,Nepokroeff C M,Porter J W.Proc.Natl.Acad.Sci.USA,1972,69(12):3516-3519
24 Teixeira M G,De A J V,Soares C G,Venancio M F,Lopes J F,Nascimento C S,Anconi C P,Carvalho G S,Louren9o C S,De A M V,Fernandes S A,De A W B.J.Phys.Chem.B,2014,118(1):81-93
25 Firnkes M,Pedone D,Knezevic J,Doblinger M,Rant U.Nano Lett.,2010,10(6):2162-2167
26 Mereuta L,Roy M,Asandei A,Lee J K,Park Y.Andricioaei I,Luchian T.Sci.Rep.,2014,4(4):3885-3895
27 Asandei A,Schiopu I,Chinappi M,Seo C H,Park Y,Luchian T.ACS Appl.Mater.Inter.,2016,8(20):13166-13179
28 Piguet F,Discala F,Breton M F,Pelta J,Bacri L,Qukhaled A.J.Phys.Chem.Lett.,2014,5(24):4362-4367
29 Bhamidimarri S P,Prajapati J D,Van D B B,Winterhalter M,Kleinekath9fer U.Biophys.J.,2016,110(3):600-611
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36 Astier Y,Braha O,Bayley H.J.Am.Chem.Soc.,2006,128(5):1705-1710