Hedges Duncan H P, Richardson David J, Russell David A
School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich NR4 7TJ, United Kingdom.
Langmuir. 2004 Mar 2;20(5):1901-8. doi: 10.1021/la035795c.
Cytochrome c has been immobilized onto functionalized, optically transparent indium tin oxide (ITO) electrodes by covalent and electrostatic techniques. Covalent immobilization was achieved by the formation of a disulfide bond between N-succinimidyl 3-(2-pyridyldithio)propionate-(SPDP-) modified cytochrome c and SPDP-silanized ITO. Additionally, ITO electrodes have been modified with the bifunctional reagent 1,12-dodecanedicarboxylic acid (DDCA), resulting in formation of a carboxylic acid-terminated monolayer. Covalent protein attachment to the DDCA-functionalized ITO was achieved with the cross-linker 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride. Electrostatic attachment of the protein involved ion-pair and hydrogen-bond interactions between the terminating carboxylic acid groups of the DDCA-functionalized ITO and the primary amine groups of the lysine residues of cytochrome c. The electrostatic interaction between the cytochrome c and the functionalized ITO resulted in greater rotational mobility of the protein at the electrode surface, leading to ca. 63% electroactivity, as compared to ca. 41% electroactivity for the covalently immobilized protein. The redox state of the electrostatically bound cytochrome c monolayers could be electrochemically switched between ferric and ferrous forms. Electrochemical control of the bound protein was used to regenerate the biosensing surface following binding of nitric oxide (NO). Ligation of NO with the cytochrome c was monitored by measurement of the change of absorbance intensity at 416 nm. Through application of a negative potential, the cytochrome c was reduced from the ferric to the ferrous form, which led to the removal of the ligated NO. Application of a positive potential regenerated the ferric cytochrome c, enabling multiple repeat measurements of NO. Such electrochemical control of proteins immobilized on transparent electrodes enables the optical biosensing of analyte targets without recourse to exogenous reagents.
细胞色素c已通过共价和静电技术固定在功能化的光学透明铟锡氧化物（ITO）电极上。通过在N-琥珀酰亚胺基3-（2-吡啶二硫代）丙酸酯-（SPDP-）修饰的细胞色素c与SPDP-硅烷化的ITO之间形成二硫键来实现共价固定。此外，ITO电极已用双功能试剂1,12-十二烷二羧酸（DDCA）进行了修饰，从而形成了以羧酸为末端的单分子层。使用交联剂1-[3-（二甲基氨基）丙基]-3-乙基碳二亚胺盐酸盐实现蛋白质与DDCA功能化ITO的共价连接。蛋白质的静电连接涉及DDCA功能化ITO的末端羧酸基团与细胞色素c赖氨酸残基的伯胺基团之间的离子对和氢键相互作用。细胞色素c与功能化ITO之间的静电相互作用导致蛋白质在电极表面具有更大的旋转流动性，与共价固定的蛋白质约41%的电活性相比，导致约63%的电活性。静电结合的细胞色素c单分子层的氧化还原状态可以在三价铁和二价铁形式之间进行电化学切换。在一氧化氮（NO）结合后，利用结合蛋白质的电化学控制来再生生物传感表面。通过测量416nm处吸光度强度的变化来监测NO与细胞色素c的结合。通过施加负电位，细胞色素c从三价铁形式还原为二价铁形式，这导致结合的NO被去除。施加正电位可使三价铁细胞色素c再生，从而实现对NO的多次重复测量。这种对固定在透明电极上的蛋白质的电化学控制使得无需借助外源试剂即可对分析物目标进行光学生物传感。
