Differential impedance spectroscopy for monitoring protein immobilization and antibody-antigen reactions.
This work describes the theoretical and experimental approaches for monitoring the interfacial biomolecular reaction between immobilized antibody and the antigen binding partner using novel differential impedance spectroscopy. The prerequisite of any biosensor is the immobilization of macromolecules onto the surface of a transducer. It is clear that the function of most macromolecules changes from what is observed in solution once immobilization has occurred. In the worst case, molecules entirely lose their binding activity almost immediately after immobilization. Certain conditions (e.g., denaturation, interfacial effects based on ionic strength, surface charge, dielectric constants, etc.) at interfaces are responsible for alterations of binding activity; it is not clear whether a combination of such processes is understood. However, these processes in combination must be reliably modeled in order to predict the outcome for most macromolecules. This work presents the theoretical and practical means for elucidating the surface reactivity of biomolecular reagents using ion displacement model with antibody-antigen (Ab-Ag) reaction as the test case. The Ab-Ag reaction was directly monitored using a dual-channeled, impedance analyzer capable of 1 measurement/s using covalent immobilization chemistry and polymer-modified electrodes in the absence of a redox probe. The evidence of Ab-Ag binding was revealed through the evolution of differential admittance. The surface loading obtained using the covalent immobilization chemistry was 9.0 x 10(16)/cm2, whereas with polymer-modified electrodes, the surface loading was 9.0 x 10(15)/cm2, representing a 10 times increase in surface reactivity. The proposed approach may be applicable to monitoring other surface interfacial reactions such as DNA-DNA interactions, DNA-protein interactions, and DNA-small molecule interactions.
Department of Chemistry, State University of New York-Binghamton, 13902-6016, USA. Osadik@binghamton.edu
O A
OA
Sadik
H
H
Xu
E
E
Gheorghiu
D
D
Andreescu
C
C
Balut
M
M
Gheorghiu
D
D
Bratu
D000465:Algorithms; D000937:Antigen-Antibody Reactions; D001665:Binding Sites; D002138:Calibration; D003429:Cross Reactions; D004563:Electrochemistry; D011506:Proteins / Q000737:chemistry*; D013057:Spectrum Analysis
D011506:Proteins