Improvement of Homogeneity of Analytical Biodevices by Gene Manipulation

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• 作者：Jing-Xue Shi ; Xian-En Zhang ; Wei-Hong Xie ; Ya-Feng Zhou ; Zhi-Ping Zhang ; Jiao-Yu Deng ; Anthony E. G. Cass ; Zhi-Ling Zhang ; Dai-Wen Pang ; and Cheng-Gang Zhang
• 刊名：Analytical Chemistry
• 出版年：2004
• 出版时间：February 1, 2004
• 年：2004
• 卷：76
• 期：3
• 页码：632 - 638
• 全文大小：560K
• 年卷期：v.76,no.3(February 1, 2004)
• ISSN：1520-6882

文摘

Homogeneity is proposed for evaluation of the quality ofanalytical biodevices, such as biosensors and biochips.As a demonstration, glucose oxidase (GOx) was modifiedat its C-terminal with a linker peptide with a cysteineresidue at the end. The fusion structure (GOx-linker-cysteine) enables the enzyme to immobilize on goldsurfaces with a Cys-S-Au bond or to immobilize on asilanized glass surface via disulfide chemistry. With thisfusion structure, the enzyme can be anchored onto thesubstrate with well-controlled orientation, thus forminga homogeneous biological layer on biodevices. The linkerpeptide between GOx and the cysteine acts as a spacer toreduce the steric hindrance caused by the bulky body ofthe enzyme. Biochemistry experiments showed that thisgenetically modified glucose oxidase (shortened to GOxm)retained most of its catalytic characteristics, with K_m andK_cat similar to those of the wild-type GOx. Electrochemistry experiments showed that GOxm-modified electrodegave higher and more stable current responses than theelectrode modified with GOx which has no free -SH onits surface. The coefficients of variation (used for evaluation of the interchangeability of the enzyme device fromthe same batch preparation) were 9.5% for the GOxm goldelectrode and 20.0% for the GOx gold electrode and theGOxm oxygen electrode. The relative errors (used forevaluation of the precision of the individual enzymedevice) were 2.9% for the GOxm gold electrode, 12.0%for the GOx gold electrode, and 11.2% for the GOxmoxygen electrode. Atomic force microscopy images revealed that GOxm formed a self-assembled monolayer ina hexagonal-like lattice packing arrangement on the goldsurface, while GOx formed multilayer assembling oraggregated particles. The homogeneity of the proteinchips, the GOxm array that was prepared through -S-S- formation, and the GOx array that was preparedthrough nonspecific adsorption was evaluated. The coefficients of variation, calculated with the signal level of alldots, were 5.4% for the GOxm array and 81.8% for theGOx array. All experimental results pointed to the fact thatthe homogeneity of the analytical biodevices could beconsiderably improved by using the proposed method.