Improvement of homogeneity of analytical biodevices by gene manipulation.
Homogeneity is proposed for evaluation of the quality of analytical biodevices, such as biosensors and biochips. As a demonstration, glucose oxidase (GOx) was modified at its C-terminal with a linker peptide with a cysteine residue at the end. The fusion structure (GOx-linker-cysteine) enables the enzyme to immobilize on gold surfaces with a Cys-S-Au bond or to immobilize on a silanized glass surface via disulfide chemistry. With this fusion structure, the enzyme can be anchored onto the substrate with well-controlled orientation, thus forming a homogeneous biological layer on biodevices. The linker peptide between GOx and the cysteine acts as a spacer to reduce the steric hindrance caused by the bulky body of the enzyme. Biochemistry experiments showed that this genetically modified glucose oxidase (shortened to GOxm) retained most of its catalytic characteristics, with K(m) and K(cat) similar to those of the wild-type GOx. Electrochemistry experiments showed that GOxm-modified electrode gave higher and more stable current responses than the electrode modified with GOx which has no free -SH on its surface. The coefficients of variation (used for evaluation of the interchangeability of the enzyme device from the same batch preparation) were 9.5% for the GOxm gold electrode and 20.0% for the GOx gold electrode and the GOxm oxygen electrode. The relative errors (used for evaluation of the precision of the individual enzyme device) were 2.9% for the GOxm gold electrode, 12.0% for the GOx gold electrode, and 11.2% for the GOxm oxygen electrode. Atomic force microscopy images revealed that GOxm formed a self-assembled monolayer in a hexagonal-like lattice packing arrangement on the gold surface, while GOx formed multilayer assembling or aggregated particles. The homogeneity of the protein chips, the GOxm array that was prepared through -S-S- formation, and the GOx array that was prepared through nonspecific adsorption was evaluated. The coefficients of variation, calculated with the signal level of all dots, were 5.4% for the GOxm array and 81.8% for the GOx array. All experimental results pointed to the fact that the homogeneity of the analytical biodevices could be considerably improved by using the proposed method.
Shi
Jing-Xue
JX
Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China.
Zhang
Xian-En
XE
Xie
Wei-Hong
WH
Zhou
Ya-Feng
YF
Zhang
Zhi-Ping
ZP
Deng
Jiao-Yu
JY
Cass
Anthony E G
AE
Zhang
Zhi-Ling
ZL
Pang
Dai-Wen
DW
Zhang
Cheng-Gang
CG
D001234:Aspergillus niger / Q000201:enzymology; D015374:Biosensing Techniques / Q000379:methods / Q000592:standards; D003545:Cysteine / Q000737:chemistry; D004563:Electrochemistry; D004566:Electrodes; D004800:Enzymes, Immobilized; D015971:Gene Expression Regulation, Enzymologic; D005898:Glass / Q000737:chemistry; D005947:Glucose / Q000032:analysis; D005949:Glucose Oxidase / Q000737:chemistry* / Q000235:genetics; D006046:Gold / Q000737:chemistry; D007700:Kinetics; D018625:Microscopy, Atomic Force; D010100:Oxygen / Q000737:chemistry; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics
D004800:Enzymes, Immobilized; D011994:Recombinant Proteins; D006046:Gold; D005949:Glucose Oxidase; D005947:Glucose; D003545:Cysteine; D010100:Oxygen