Quartz crystal microbalance (QCM) with immobilized protein receptors: comparison of response to ligand binding for direct protein immobilization and protein attachment via disulfide linker.
Results from an investigation of the frequency response resulting from ligand binding for a genetically engineered hormone-binding domain of the alpha-estrogen receptor immobilized to a piezoelectric quartz crystal are reported. Two different approaches were used to attach a genetically altered receptor to the gold electrode on the quartz surface: (1) the mutant receptor containing a single solvent-exposed cysteine was directly attached to the crystal via a sulfur to gold covalent bond, forming a self-assembled protein monolayer, and (2) the N-terminal histidine-tagged end was utilized to attach the receptor via a 3,3-dithiobis[N-(5-amino-5-carboxypentyl)propionamide-N',N'-diacetic acid] linker complexed with nickel. Previous studies have shown that these engineered constructs bind 17beta-estradiol and are fully functional. Exposure of the receptor directly attached to the piezoelectric crystal to the known ligand 17beta-estradiol resulted in a measurable frequency response, consistent with a change in conformation of the receptor with ligand binding. However, no response was observed when the receptor immobilized via the linker was exposed to the same ligand. The presence of the linker between the quartz surface and the protein receptor does not allow the crystal to sense the conformational change in the receptor that occurs with ligand binding. These results illustrate that the immobilization strategy used to bind the receptor to the sensor platform is key to eliciting an appropriate response from this biosensor. This study has important implications for the development of QCM-based sensors using protein receptors.
Baltus
Ruth E
RE
Department of Chemical and Biomolecular Engineering and Department of Chemistry and Biology, Clarkson University, Potsdam, New York 13699, USA. baltus@clarkson.edu
Carmon
Kendra S
KS
Luck
Linda A
LA
D000818:Animals; D015374:Biosensing Techniques; D003432:Cross-Linking Reagents / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D004566:Electrodes; D004958:Estradiol / Q000032:analysis*; D006639:Histidine / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D011960:Receptors, Estrogen / Q000737:chemistry* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics
D003432:Cross-Linking Reagents; D004220:Disulfides; D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; C033223:polyhistidine; D006639:Histidine; D004958:Estradiol