Correlation of Kinetics and Conformations of Free and Immobilized Enzymes on Non- and Nanotextured Silicon Biosensor Surfaces

Abstract

Quantification of sensing signals from biocatalyzed events in enzyme-based biosensors are characterized by the kinetic parameters (such as Km). Immobilization of enzymes on surfaces is known to affect Km and decrease the sensor performance. While performance degradation upon immobilization is inevitable, there is a need to minimize it. With the change in structural conformations arising from immobilizations affecting the functionality of the enzymes, understanding the correlation between the kinetic parameters and the conformations would help provide insights leading to possible alleviation measures. We are not aware of any prior work on correlating the enzymatic performance with the secondary structures upon immobilization. Again, while there are reported works on the effect of surface chemical modification on the secondary structures of enzymes, we are not aware of any prior work on the effect of surface physical modification. Here, we report the changes in the kinetic parameters of enzymes (untagged and antibody-tagged) upon surface immobilization on non- and nanotextured silicon surfaces and attempt to understand these parameter changes by correlating with the changes in the secondary structures. The novelty of our work is that we have shown a correlation of the enzymatic performance parameters with the secondary structure. Also, importantly, we have demonstrated a method of partial recovery of the loss of enzyme functionality upon surface immobilization by nanotexturing the surfaces. The significance of our work is that the application of the insights obtained here can be expected to improve the performance parameters of enzyme-based sensors.