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Direct electrical control of IgG conformation and functional activity at surfaces

View Article: PubMed Central - PubMed

ABSTRACT

We have devised a supramolecular edifice involving His-tagged protein A and antibodies to yield surface immobilized, uniformly oriented, IgG-type, antibody layers with Fab fragments exposed off an electrode surface. We demonstrate here that we can affect the conformation of IgGs, likely pushing/pulling electrostatically Fab fragments towards/from the electrode surface. A potential difference between electrode and solution acts on IgGs’ charged aminoacids modulating the accessibility of the specific recognition regions of Fab fragments by antigens in solution. Consequently, antibody-antigen affinity is affected by the sign of the applied potential: a positive potential enables an effective capture of antigens; a negative one pulls the fragments towards the electrode, where steric hindrance caused by neighboring molecules largely hampers the capture of antigens. Different experimental techniques (electrochemical quartz crystal microbalance, electrochemical impedance spectroscopy, fluorescence confocal microscopy and electrochemical atomic force spectroscopy) were used to evaluate binding kinetics, surface coverage, effect of the applied electric field on IgGs, and role of charged residues on the phenomenon described. These findings expand the concept of electrical control of biological reactions and can be used to gate electrically specific recognition reactions with impact in biosensors, bioactuators, smart biodevices, nanomedicine, and fundamental studies related to chemical reaction kinetics.

No MeSH data available.


Response (resonance frequency variation, Δf, and resonance frequency distribution full width at half maximum variation, Δ(FWHM)) of prot A+ IgG layer to changes in substrate potential.
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f2: Response (resonance frequency variation, Δf, and resonance frequency distribution full width at half maximum variation, Δ(FWHM)) of prot A+ IgG layer to changes in substrate potential.

Mentions: Once we had assembled a protein A-IgG monolayer, we performed electrochemical QCM measurements to investigate the dependence of its resonance frequency and dissipation upon changes in electrode potential. Having immobilized a layer of molecules on one electrode of a QCM, we measured its response to an electrical potential changing in sign and intensity (±100 ÷ ± 200 mV) inside a liquid cell equipped with a counter and reference electrode. Figure 2 reports the corresponding data. Both variations in resonance frequency and in FWHM (this last parameter being proportional to energy dissipation of the oscillator, i.e. to the compactness of the layer facing the QCM electrode) displayed a similar, although opposite in direction, stepwise behavior upon changing electrode potential. These data suggest qualitatively that the layer is responsive to those changes. One can think of potential-induced conformational changes increasing the apparent visco-elastic load measured by QCM since positive charges, hence molecular domains, are attracted towards the electrode surface when it is negatively biased. The conformation of the molecules on the surface can also affect the amount of trapped water which is moving together with the molecular layer affecting, on its turn, the resonance frequency of the oscillating crystal.


Direct electrical control of IgG conformation and functional activity at surfaces
Response (resonance frequency variation, Δf, and resonance frequency distribution full width at half maximum variation, Δ(FWHM)) of prot A+ IgG layer to changes in substrate potential.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5121884&req=5

f2: Response (resonance frequency variation, Δf, and resonance frequency distribution full width at half maximum variation, Δ(FWHM)) of prot A+ IgG layer to changes in substrate potential.
Mentions: Once we had assembled a protein A-IgG monolayer, we performed electrochemical QCM measurements to investigate the dependence of its resonance frequency and dissipation upon changes in electrode potential. Having immobilized a layer of molecules on one electrode of a QCM, we measured its response to an electrical potential changing in sign and intensity (±100 ÷ ± 200 mV) inside a liquid cell equipped with a counter and reference electrode. Figure 2 reports the corresponding data. Both variations in resonance frequency and in FWHM (this last parameter being proportional to energy dissipation of the oscillator, i.e. to the compactness of the layer facing the QCM electrode) displayed a similar, although opposite in direction, stepwise behavior upon changing electrode potential. These data suggest qualitatively that the layer is responsive to those changes. One can think of potential-induced conformational changes increasing the apparent visco-elastic load measured by QCM since positive charges, hence molecular domains, are attracted towards the electrode surface when it is negatively biased. The conformation of the molecules on the surface can also affect the amount of trapped water which is moving together with the molecular layer affecting, on its turn, the resonance frequency of the oscillating crystal.

View Article: PubMed Central - PubMed

ABSTRACT

We have devised a supramolecular edifice involving His-tagged protein A and antibodies to yield surface immobilized, uniformly oriented, IgG-type, antibody layers with Fab fragments exposed off an electrode surface. We demonstrate here that we can affect the conformation of IgGs, likely pushing/pulling electrostatically Fab fragments towards/from the electrode surface. A potential difference between electrode and solution acts on IgGs’ charged aminoacids modulating the accessibility of the specific recognition regions of Fab fragments by antigens in solution. Consequently, antibody-antigen affinity is affected by the sign of the applied potential: a positive potential enables an effective capture of antigens; a negative one pulls the fragments towards the electrode, where steric hindrance caused by neighboring molecules largely hampers the capture of antigens. Different experimental techniques (electrochemical quartz crystal microbalance, electrochemical impedance spectroscopy, fluorescence confocal microscopy and electrochemical atomic force spectroscopy) were used to evaluate binding kinetics, surface coverage, effect of the applied electric field on IgGs, and role of charged residues on the phenomenon described. These findings expand the concept of electrical control of biological reactions and can be used to gate electrically specific recognition reactions with impact in biosensors, bioactuators, smart biodevices, nanomedicine, and fundamental studies related to chemical reaction kinetics.

No MeSH data available.