Abstract
Muscovite mica with an amino silane-modified surface is commonly used as a substrate in atomic force microscopy (AFM) studies of biological macromolecules. Herein, the efficiency of two different protein immobilization strategies employing either (N-hydroxysuccinimide ester)-based crosslinker (DSP) or benzophenone-based photoactivatable crosslinker (SuccBB) has been compared using AFM and mass spectrometry analysis. Two proteins with different physicochemical properties—human serum albumin (HSA) and horseradish peroxidase enzyme protein (HRP)—have been used as model objects in the study. In the case of HRP, both crosslinkers exhibited high immobilization efficiency—as opposed to the case with HSA, when sufficient capturing efficiency has only been observed with SuccBB photocrosslinker. The results obtained herein can find their application in commonly employed bioanalytical systems and in the development of novel highly sensitive chip-based diagnostic platforms employing immobilized proteins. The obtained data can also be of interest for other research areas in medicine and biotechnology employing immobilized biomolecules.
Highlights
Surface chemistry plays a crucial role in the development of materials with biological functionality [1] intended for various medical and biotechnological applications, including fabrication of sensor chips for biomarker detection [2,3,4], development of medical implants [2,5] and enzyme catalysis for wastewater purification [6], food [7,8,9] and biofuel production [10] etc
As one can see from these images, the number of objects with >1 nm height, visualized after the incubation of either dithiobis(succinimidyl propionate) (DSP)-activated of SuccBB-activated substrate in protein-free water, does not exceed the noise level
To study the protein capturing onto the surface of atomic force microscopy (AFM) substrates, activated with either succinimide crosslinker (DSP) or benzophenone (SuccBB) photoactivatable crosslinker, we have selected two model proteins with different physicochemical properties: horseradish peroxidase (HRP) and human serum albumin (HSA)
Summary
Surface chemistry plays a crucial role in the development of materials with biological functionality [1] intended for various medical and biotechnological applications, including fabrication of sensor chips for biomarker detection [2,3,4], development of medical implants [2,5] and enzyme catalysis for wastewater purification [6], food [7,8,9] and biofuel production [10] etc. This principle is widely used in various types of biosensor systems [20]
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