DNA-directed coimmobilization of multiple enzymes on organic-inorganic hybrid DNA flowers

Yali Li; Jing Wang; Fenghong Huang; Yufei Zhang; Mingming Zheng

doi:10.3389/fbioe.2022.951394

DNA-directed coimmobilization of multiple enzymes on organic-inorganic hybrid DNA flowers

Front Bioeng Biotechnol. 2022 Aug 10:10:951394. doi: 10.3389/fbioe.2022.951394. eCollection 2022.

Authors

Yali Li^{1

2}, Jing Wang², Fenghong Huang^{1

2}, Yufei Zhang², Mingming Zheng²

Affiliations

¹ Insititute of Food & Nutrition Science and Technology, Shandong Academy of Agricultural Sciences, Jinan, China.
² Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Hubei Key Laboratory of Lipid Chemistry and Nutrition, Key Laboratory of Oilseeds Processing, Ministry of Agriculture, Wuhan, China.

Abstract

The artificial multienzyme systems developed by mimicking nature has attracted much interest. However, precisely controlled compositions and ratios of multienzymatic co-immobilization systems are still limited by the indistinguishable nature of enzymes. Herein, a strategy for fabricating DNA-directed immobilization of horseradish peroxidase (HRP) and glucose oxidase (GOx) on hybrid DNA nanoflowers (GOx-HRP@hDFs) is presented. The preparation of micron-sized hybrid DNA flowers (hDFs) begins with the predetermined repeatable polymer-like DNA sequences which contained two strands. The hDFs structure is generated through one-pot rolling circle amplification (RCA) and self-assembly with magnesium pyrophosphate inorganic crystals. Based on the rigid-base pairing, GOx and HRP conjugated with sequences complementary to strands would be anchored to the predesigned locations, respectively. By adjusting the loading amount/ratio of enzymes properly, the maximal catalytic efficiency can be precisely regulated. The reaction activity of GOx-HRP@hDFs was 7.4 times higher than that of the free GOx-HRP under the optimal mole ratio (GOx/HRP 4:1). In addition, this multienzyme catalyst system exhibits excellent precision, specificity, reproducibility, and long-term storage stability when applied to real human blood samples. The preceding results validate that GOx-HRP@hDFs are promising candidates for personal diabetes detection.

Keywords: DNA-directed immobilization (DDI); colorimetric detection of glucose; multienzyme coimmobilization; organic-inorganic hydrid DNA flowers (hDFs); rolling circle amplification (RCA).