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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.


ECAFM results on the supramolecular edifice prot A+ IgGs as a function of substrate potential.(a,b) Topographic images measured at −100 mV (a), and +100 mV (b) vs SCE, respectively. (c) A representative pair of line profiles belonging to the two images. (d) The height difference histogram resulting from line profile analysis on the visible bumps.
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f3: ECAFM results on the supramolecular edifice prot A+ IgGs as a function of substrate potential.(a,b) Topographic images measured at −100 mV (a), and +100 mV (b) vs SCE, respectively. (c) A representative pair of line profiles belonging to the two images. (d) The height difference histogram resulting from line profile analysis on the visible bumps.

Mentions: Second, we characterized also at a microscopic level the changes induced by switching the sign of potential applied to a gold substrate on which the antibody layer was assembled. For doing that we used ECAFM, measuring topography on a single region during two consecutive scans performed at +100 and −100 mV, respectively while keeping the imaging force at the lowest possible value for both scans. Figure 3(a,b) reports two of those scans taken on the same area, showing a marked difference in height in the visible spots, associated to single IgGs. The analysis, performed by measuring corresponding line profiles (c) and building the resulting height difference histogram (d) reveals a difference in the average height of 1.7 nm between the case at +100 mV (Fab fragment repulsion) and that at −100 mV (attraction). These results are consistent with a change of molecular conformation with substrate potential, likely connected with the rise of Fab arms at positive, repulsive potential, and with lowering of them in the negative, attractive case. It is however fair to argue that the reported data do not figure out other possible conformational variations, such as a different molecular tilting induced by changes in substrate potential.


Direct electrical control of IgG conformation and functional activity at surfaces
ECAFM results on the supramolecular edifice prot A+ IgGs as a function of substrate potential.(a,b) Topographic images measured at −100 mV (a), and +100 mV (b) vs SCE, respectively. (c) A representative pair of line profiles belonging to the two images. (d) The height difference histogram resulting from line profile analysis on the visible bumps.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: ECAFM results on the supramolecular edifice prot A+ IgGs as a function of substrate potential.(a,b) Topographic images measured at −100 mV (a), and +100 mV (b) vs SCE, respectively. (c) A representative pair of line profiles belonging to the two images. (d) The height difference histogram resulting from line profile analysis on the visible bumps.
Mentions: Second, we characterized also at a microscopic level the changes induced by switching the sign of potential applied to a gold substrate on which the antibody layer was assembled. For doing that we used ECAFM, measuring topography on a single region during two consecutive scans performed at +100 and −100 mV, respectively while keeping the imaging force at the lowest possible value for both scans. Figure 3(a,b) reports two of those scans taken on the same area, showing a marked difference in height in the visible spots, associated to single IgGs. The analysis, performed by measuring corresponding line profiles (c) and building the resulting height difference histogram (d) reveals a difference in the average height of 1.7 nm between the case at +100 mV (Fab fragment repulsion) and that at −100 mV (attraction). These results are consistent with a change of molecular conformation with substrate potential, likely connected with the rise of Fab arms at positive, repulsive potential, and with lowering of them in the negative, attractive case. It is however fair to argue that the reported data do not figure out other possible conformational variations, such as a different molecular tilting induced by changes in substrate potential.

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.