The Effects Of The Surface Properties Of Gold Nanoparticles On The Structure And Activity Of Immobilized HRP

Immobilization of enzymes on the surface of various nanoparticles with specialoptical and electric properties has been demonstrated as a promising way to fabricatesensing devices. The immobilized enzymes often present decreased enzymaticactivity and thus greatly affect the device performance, and this commonly attributesto their conformational change after being loaded on the surface of nanoparticles.Much effort has been focused on investigating the interactions between modelproteins with surface-functionalized nanoparticles with different surface chemicalproperties. Some elaborately designed surface ligands have showed their ability todecrease effectively the negative effect of nanoparticles to certain enzymes. Due tothe lengthy synthetic process of ligands and the special requirements of enzymestructure, the routes to improve enzyme activity through surface modification ofnanoparticles are limited to some extent. Currently, it still remains a challenge todevelop a simple and practical approach to improve activity of the immobilizedenzymes and understand the corresponding mechanism.At present, introduction of additives such as polymers and proteins has beenproven to be effective to improve the activity of enzymes immobilized onmacroscopic or micrometer-sized solid substrates. However, function of theadditives havs not been well understood yet and similar strategy has rarely beenused during immobilization of enzymes on the surface of nanoparticles. In this work, A systematic study of the influence of immobilization on the surface of goldnanoparticles (Au NPs) on the structure and activity of horseradish peroxidase (HRP)as a stating point, the effect of bovine serum albumin (BSA) on the immobilizationof HRP was systematic investigated to understanding the effects of the surfaceproperties of gold nanoparticles on the structure and activity of immobilized HRP.Firstly,25nm Au NPs which prepared via a modified Frens method was usedas a carrier to study the effect of immobilization on the structure and acitivity ofHRP. Only30%of its catalytic activity was kept after being immobilized on thesurface of Au NPs for4hours. The activity and structural changes of immobilizedHRP during immobilization on Au NPs were monitored. The time dependent changein the tertiary structure around the active site of HRP was fully consistent with thedecrease in the activity of the immobilized HRP, but the secondary structure changeof immobilized HRP showed little relevance, indicating that the tertiary structurearound the active site of HRP plays a critical role in maintaining the activity of HRP.In Chapter3, the function of bovine serum albumin (BSA) additive onimmobilized HRP was investigated by addition of BSA during HRP immobilizationon the surface of Au NPs. The addition time and amount of BSA have a significantimpact on the activity of immobilized HRP. The catalytic activity of HRP wassignificantly improved to80%when appropriate amount of BSA was added at theinitial period of the immobilization. Systematic spectral investigation indicated thatthe addition of BSA inhibited the tertiary structure change around the active site ofHRP, which was prerequisite for improved activity of the immobilized HRP.Steady-state kinetic analyses revealed that the addition of BSA reduced the affinityof the immobilized HRP for both substrates. This is detrimental to the activity ofimmobilized HRP. But BSA could effectively improve the turnover rate ofsubstrate-to-product which also contributed to the improvement of catalysis activity.In Chapter4, firstly the steady-state kinetic of HRP in the presence of BSA wasstudied. It revealed that though BSA could improve the turnover rate ofsubstrate-to-product, but reduced affinity to substrates, which has a detrimentaleffect on the activity of HRP. In order to reduce the negative effect of BSA on the activity of immobilized HRP and fully retain the positive role at the same time, theBSA pre-modified Au NPs (Au NPs-BSA) were used as the carrier for HRPimmobilization. The concentration of BSA which used for preparation of AuNPs-BSA had great influence on the structure and activity of immobilized HRP. AuNPs-BSA particles prepared at very low concentrations of BSA will induce thechange in the tertiary structure around the active site thus reduce the activity ofimmobilized HRP. When the concentration of BSA reaches a certain amount, notnecessary to cover the surface of the Au NPs completely, the tertiary structurearound the active site of immobilized HRP is completely stabilized and the activityof immobilized HRP is promoted to more than200%of the free-state. Steady-statekinetic analyses revealed that compared with the method of adding BSA during theimmobilization of HRP on Au NPs, the immobilized HRP prepared by this methodhad better affinity for substrate and had better enhancement effect on the turnoverrate of substrate-to-product of HRP. So the activity of the immobilized HRP hasbeen further improved.Finally, in order to investigate the reason why the activity of immobilized HRPexceeded the free-state in the presence of BSA, long alkyl chain thiol ligandsmodified Au NPs was used to simulate the Au NPs-BSA, and the effects of thenanoparticle surface charge on the immobilized HRP was studied. Three kinds of AuNPs with different surface charge properties were prepared by modifying Au NPswith11-mercaptoundecanoic acid,11-amino-1-undecanethiol and the mixture ofthese two ligands. Spectra characterization of the immobilized HRP showed that thetertiary structures around the active site of HRP were not changed whenimmobilized on these Au NPs. Comparing the activities of immobilized HRP onthree kinds of Au NPs at different pH value, we conclude that the negative charge onthe surface of Au NPs is the reason why the activity of immobilized HRP exceed thefree-state. Steady-state kinetic analyses revealed that the nagetive charge on thesurface of Au NPs could improve the turnover rate of substrate-to-product ofimmobilized HRP, which was prerequisite for improved the activity of theimmobilized HRP. It is supposed that the improvement of turnover rate of substrate-to-product of immobilized HRP in the presence of BSA is benefit fromnegative charged amino acid residue on the surface of BSA. The possible function ofnegative charge was discussed.