Self-Assembly Of DNA Hydrogel As Carrier For Immobilization Of Enzyme

Enzyme immobilization is the core technology of biocatalysis. Enzyme immobilization is expected to improve the efficiency of enzyme catalysis, the recovery rate and extend the shelf life of enzyme. It laid the foundation for biomedical applications and industrial application of enzymes. Research and development multifunctional materials for immobilizing enzymes is considered as one of most activated orients. Because of the easy programmability of DNA sequences and convenient chemical synthesis, DNA hydrogels are one of the most important bulk-scale materials because of their several advantages, such as designable responsiveness, biodegradability and high permeability of biomaterials. These DNA hydrogels show potential application in protein-loading, drug release, cell-free protein production and DNA immunotherapy, especially widely used to encapsulate and load nanoscale species such as enzymes, proteins, nanoparticles and microscale cells. In this paper, relying on the advantages of DNA hydrogels such as controlled assembly, DNA-protein hybrid hydrogel was designed and constructed for enzyme immobilization and encapsualtion; Furthermore, enzyme cascade system based on DNA hydrogels was developed. The details are summarized as follows:1. A DNA-protein hybrid hydrogel was constructed based on a super sandwich hybridization reaction and streptavidin-biotin crosslinker, which served as a carrier for immobilization and encapsulation of alcohol oxidase (AOx) and enhanced its biological stability. A dsDNA building block tailored with precise residues was fabricated based on supersandwich hybridization, and then the addition of streptavidin, triggered the formation of the DNA-protein hybrid hydrogel. AOx was encapsulated in the hybrid hydrogel using a synchronous assembly approach. The loading efficiency and the loading capacity is 42%,18 mg/g AOx/hydrogel, respectively. The hydrogel, which formed a flower-like porous structure that was 6.7 ± 2.1 um in size, served as a reservoir system for enzyme encapsulation. The enzyme-encapsulated hydrogel was utilized to extend the duration time for ethanol removal in serum plasma and the enzyme retained 78% activity after incubation with human serum for 24 h. The DNA-protein hybrid hydrogel can mediate the intact immobilization on a streptavidin-modified and positively charged substrate, which is very beneficial to solid-phase biosensing applications. DNA-protein hybrid hydrogel presents improved stability against various denaturation factors, including elevated temperature, freeze-thaw cycles, and organic solvents. This method showed great promise in the field of enzyme sensor.2. Glucose oxidase (GOx) was encapsulated in the G4-hemin dopped DNA hydrogel, which served as enzyme cascade system for efficient catalysis of glucose oxidation and hydrogen peroxide decomposition. A dsDNA building block tailored with G4 single strands was fabricated based on supersandwich hybridization, and then the addition of hemin triggered the formation of the DNA hydrogel. Cationized GOx was encapsulated in the hydrogel using a synchronous assembly approach through electrostatic interactions, and the loading efficiency reach up to 73%. Therefore, an enzyme cascade system containing GOx and G4-hemin, which catalysized glucose oxidation and subsequent hydrogen peroxide decomposition was established in the DNA hydrogel. G4-hemin DNA hydrogel system have 26 times enhanced catalysis ability for hydrogen peroxide decomposition, which efficiently improved the cascade catalytic of glucose oxidation and hydrogen peroxide degradation. The enzyme cascade based DNA hydrogel is expected to be used in the field of bioreactor.