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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.

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Confocal fluorescence imaging of two separately addressable gold pads biofunctionalized with prot A+ IgG and exposed to a solution of FITC-labelled insulin while kept at different substrate potentials.Image size 100 × 100 μm2. (a,c) pH 5.5; (b,d) pH 9.5. Note the lower intensity of the left pad in (a) (−100 mV). Panels c and d report the normalized integrated fluorescence intensity along columns for the two pads of images a and b.
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f6: Confocal fluorescence imaging of two separately addressable gold pads biofunctionalized with prot A+ IgG and exposed to a solution of FITC-labelled insulin while kept at different substrate potentials.Image size 100 × 100 μm2. (a,c) pH 5.5; (b,d) pH 9.5. Note the lower intensity of the left pad in (a) (−100 mV). Panels c and d report the normalized integrated fluorescence intensity along columns for the two pads of images a and b.

Mentions: The role of the applied electric potential has also been confirmed by performing antigen-binding measurements by means of a different technique, i.e. electrochemically assisted fluorescence microscopy. Two separately addressable gold pads were functionalized with uniformly oriented IgG layers, driven at two different potentials (±100 mV) while incubated with a fluorescently labeled specific antigen (insulin). In a control measurement, both electrodes were kept at +100 mV. After 1 h incubation, the sample was rinsed in buffer and imaged. Figure 6 reports the corresponding fluorescence images along with the computed normalized fluorescence intensity calculated by summing over the rows the collected light intensity in the two images. Once more (Fig. 6a), the role of the positive potential was that of eliciting a higher amount of antigens bound to the antibody layer in the case of incubation at a positive potential (confirming data shown in Fig. 5a).


Direct electrical control of IgG conformation and functional activity at surfaces
Confocal fluorescence imaging of two separately addressable gold pads biofunctionalized with prot A+ IgG and exposed to a solution of FITC-labelled insulin while kept at different substrate potentials.Image size 100 × 100 μm2. (a,c) pH 5.5; (b,d) pH 9.5. Note the lower intensity of the left pad in (a) (−100 mV). Panels c and d report the normalized integrated fluorescence intensity along columns for the two pads of images a and b.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: Confocal fluorescence imaging of two separately addressable gold pads biofunctionalized with prot A+ IgG and exposed to a solution of FITC-labelled insulin while kept at different substrate potentials.Image size 100 × 100 μm2. (a,c) pH 5.5; (b,d) pH 9.5. Note the lower intensity of the left pad in (a) (−100 mV). Panels c and d report the normalized integrated fluorescence intensity along columns for the two pads of images a and b.
Mentions: The role of the applied electric potential has also been confirmed by performing antigen-binding measurements by means of a different technique, i.e. electrochemically assisted fluorescence microscopy. Two separately addressable gold pads were functionalized with uniformly oriented IgG layers, driven at two different potentials (±100 mV) while incubated with a fluorescently labeled specific antigen (insulin). In a control measurement, both electrodes were kept at +100 mV. After 1 h incubation, the sample was rinsed in buffer and imaged. Figure 6 reports the corresponding fluorescence images along with the computed normalized fluorescence intensity calculated by summing over the rows the collected light intensity in the two images. Once more (Fig. 6a), the role of the positive potential was that of eliciting a higher amount of antigens bound to the antibody layer in the case of incubation at a positive potential (confirming data shown in Fig. 5a).

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.


Related in: MedlinePlus