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Probing cytochrome c in living mitochondria with surface-enhanced Raman spectroscopy.

Brazhe NA, Evlyukhin AB, Goodilin EA, Semenova AA, Novikov SM, Bozhevolnyi SI, Chichkov BN, Sarycheva AS, Baizhumanov AA, Nikelshparg EI, Deev LI, Maksimov EG, Maksimov GV, Sosnovtseva O - Sci Rep (2015)

Bottom Line: Here we suggest a novel label-free approach based on the surface-enhanced Raman spectroscopy (SERS) to monitor the redox state and conformation of cytochrome c in the electron transport chain in living mitochondria.We demonstrate that SERS spectra of living mitochondria placed on hierarchically structured silver-ring substrates provide exclusive information about cytochrome c behavior under modulation of inner mitochondrial membrane potential, proton gradient and the activity of ATP-synthetase.Mathematical simulation explains the observed enhancement of Raman scattering due to high concentration of electric near-field and large contact area between mitochondria and nanostructured surfaces.

View Article: PubMed Central - PubMed

Affiliation: Department of Biophysics, Biological Faculty, Moscow State University, Leninskie gory 1/12, Moscow, 119234, Russia.

ABSTRACT
Selective study of the electron transport chain components in living mitochondria is essential for fundamental biophysical research and for the development of new medical diagnostic methods. However, many important details of inter- and intramembrane mitochondrial processes have remained in shadow due to the lack of non-invasive techniques. Here we suggest a novel label-free approach based on the surface-enhanced Raman spectroscopy (SERS) to monitor the redox state and conformation of cytochrome c in the electron transport chain in living mitochondria. We demonstrate that SERS spectra of living mitochondria placed on hierarchically structured silver-ring substrates provide exclusive information about cytochrome c behavior under modulation of inner mitochondrial membrane potential, proton gradient and the activity of ATP-synthetase. Mathematical simulation explains the observed enhancement of Raman scattering due to high concentration of electric near-field and large contact area between mitochondria and nanostructured surfaces.

No MeSH data available.


Results of mathematical simulation.Schematic presentation of a mitochondrion located: (A) on a flat Ag nanostructured surface with Ag nanoparticles and (B) in a cavity on Ag nanostructured surface. Mitochondrion is illuminated by linear-polarized light wave. Blue arrows show polarization of the electric field E. Red arrows indicate: (A) induced total dipole moment of Ag nanoparticles, and (B) normal component (with respect to the substrate surface) of the light-induced dipole moment of Ag nanoparticles. (C) Distribution of Ag nanoparticles with diameters of 40–50 nm on Ag surface used in numerical simulations. (D) Electric field intensity calculated in a plane, 60 nm above the Ag surface (XY-plane), when the structure is normally irradiated by linear-polarized plane light wave with the wavelength of 532 nm. (E) Electric field intensity calculated in a plane, 60 nm above the Ag surface, when the structure is irradiated at 65 degrees by TM-polarized plane light wave with the wavelength of 532 nm. (F) Distribution of the electric field intensity (corresponding to the case (E)) in the plane perpendicular to the Ag surface. The plane passes through the dashed line shown in (E).
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f5: Results of mathematical simulation.Schematic presentation of a mitochondrion located: (A) on a flat Ag nanostructured surface with Ag nanoparticles and (B) in a cavity on Ag nanostructured surface. Mitochondrion is illuminated by linear-polarized light wave. Blue arrows show polarization of the electric field E. Red arrows indicate: (A) induced total dipole moment of Ag nanoparticles, and (B) normal component (with respect to the substrate surface) of the light-induced dipole moment of Ag nanoparticles. (C) Distribution of Ag nanoparticles with diameters of 40–50 nm on Ag surface used in numerical simulations. (D) Electric field intensity calculated in a plane, 60 nm above the Ag surface (XY-plane), when the structure is normally irradiated by linear-polarized plane light wave with the wavelength of 532 nm. (E) Electric field intensity calculated in a plane, 60 nm above the Ag surface, when the structure is irradiated at 65 degrees by TM-polarized plane light wave with the wavelength of 532 nm. (F) Distribution of the electric field intensity (corresponding to the case (E)) in the plane perpendicular to the Ag surface. The plane passes through the dashed line shown in (E).

Mentions: The multiscale inhomogeneous distribution of nanoparticles in the AgNSSs enables excitation of strong electric near-fields in the system. In the experiments external light is normal incident with respect to the sample. However, because many nanoparticles of the AgNSS are located on side walls of different cavities (Fig. 4C,D), they are irradiated by polarized light under the incline conditions with respect to the surface. As a result, the light electric field directed perpendicular to the side walls of the cavities will induce strong nanoparticle’s electric dipole moments. This will lead to induction of strong nanoparticle's electric dipole moments by the light electric field directed perpendicular to the side walls of the cavities. Our approach to explain the observed strong Raman scattering is illustrated schematically in Fig. 5A,B. There are two different possibilities. When a mitochondrion is located on a flat metal surface covered by nanoparticles and is irradiated by normally incident light (Fig. 5A), the mitochondrion has a limited contact area interacting with weak optical near fields generated by the in-plane induced dipole moments (red arrows in Fig. 5A). In this case the Raman signal has to be low. The Raman signal should be much stronger if the mitochondrion is located within a cavity with walls covered by nanoparticles, as in the multiscale AgNSSs (Figs 4 and 5B). Much stronger optical near fields must exist due to the light-induced normal components of the nanoparticle electric dipoles. The distribution of these electric fields can be imagined as electric-field “needles” deeply penetrating into the mitochondrion (Fig. 5B). In this case, the contact area between the mitochondrion and nanoparticle structure is also increased providing better conditions for Raman scattering. We assume that this situation corresponds perfectly to our experimental conditions.


Probing cytochrome c in living mitochondria with surface-enhanced Raman spectroscopy.

Brazhe NA, Evlyukhin AB, Goodilin EA, Semenova AA, Novikov SM, Bozhevolnyi SI, Chichkov BN, Sarycheva AS, Baizhumanov AA, Nikelshparg EI, Deev LI, Maksimov EG, Maksimov GV, Sosnovtseva O - Sci Rep (2015)

Results of mathematical simulation.Schematic presentation of a mitochondrion located: (A) on a flat Ag nanostructured surface with Ag nanoparticles and (B) in a cavity on Ag nanostructured surface. Mitochondrion is illuminated by linear-polarized light wave. Blue arrows show polarization of the electric field E. Red arrows indicate: (A) induced total dipole moment of Ag nanoparticles, and (B) normal component (with respect to the substrate surface) of the light-induced dipole moment of Ag nanoparticles. (C) Distribution of Ag nanoparticles with diameters of 40–50 nm on Ag surface used in numerical simulations. (D) Electric field intensity calculated in a plane, 60 nm above the Ag surface (XY-plane), when the structure is normally irradiated by linear-polarized plane light wave with the wavelength of 532 nm. (E) Electric field intensity calculated in a plane, 60 nm above the Ag surface, when the structure is irradiated at 65 degrees by TM-polarized plane light wave with the wavelength of 532 nm. (F) Distribution of the electric field intensity (corresponding to the case (E)) in the plane perpendicular to the Ag surface. The plane passes through the dashed line shown in (E).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4561893&req=5

f5: Results of mathematical simulation.Schematic presentation of a mitochondrion located: (A) on a flat Ag nanostructured surface with Ag nanoparticles and (B) in a cavity on Ag nanostructured surface. Mitochondrion is illuminated by linear-polarized light wave. Blue arrows show polarization of the electric field E. Red arrows indicate: (A) induced total dipole moment of Ag nanoparticles, and (B) normal component (with respect to the substrate surface) of the light-induced dipole moment of Ag nanoparticles. (C) Distribution of Ag nanoparticles with diameters of 40–50 nm on Ag surface used in numerical simulations. (D) Electric field intensity calculated in a plane, 60 nm above the Ag surface (XY-plane), when the structure is normally irradiated by linear-polarized plane light wave with the wavelength of 532 nm. (E) Electric field intensity calculated in a plane, 60 nm above the Ag surface, when the structure is irradiated at 65 degrees by TM-polarized plane light wave with the wavelength of 532 nm. (F) Distribution of the electric field intensity (corresponding to the case (E)) in the plane perpendicular to the Ag surface. The plane passes through the dashed line shown in (E).
Mentions: The multiscale inhomogeneous distribution of nanoparticles in the AgNSSs enables excitation of strong electric near-fields in the system. In the experiments external light is normal incident with respect to the sample. However, because many nanoparticles of the AgNSS are located on side walls of different cavities (Fig. 4C,D), they are irradiated by polarized light under the incline conditions with respect to the surface. As a result, the light electric field directed perpendicular to the side walls of the cavities will induce strong nanoparticle’s electric dipole moments. This will lead to induction of strong nanoparticle's electric dipole moments by the light electric field directed perpendicular to the side walls of the cavities. Our approach to explain the observed strong Raman scattering is illustrated schematically in Fig. 5A,B. There are two different possibilities. When a mitochondrion is located on a flat metal surface covered by nanoparticles and is irradiated by normally incident light (Fig. 5A), the mitochondrion has a limited contact area interacting with weak optical near fields generated by the in-plane induced dipole moments (red arrows in Fig. 5A). In this case the Raman signal has to be low. The Raman signal should be much stronger if the mitochondrion is located within a cavity with walls covered by nanoparticles, as in the multiscale AgNSSs (Figs 4 and 5B). Much stronger optical near fields must exist due to the light-induced normal components of the nanoparticle electric dipoles. The distribution of these electric fields can be imagined as electric-field “needles” deeply penetrating into the mitochondrion (Fig. 5B). In this case, the contact area between the mitochondrion and nanoparticle structure is also increased providing better conditions for Raman scattering. We assume that this situation corresponds perfectly to our experimental conditions.

Bottom Line: Here we suggest a novel label-free approach based on the surface-enhanced Raman spectroscopy (SERS) to monitor the redox state and conformation of cytochrome c in the electron transport chain in living mitochondria.We demonstrate that SERS spectra of living mitochondria placed on hierarchically structured silver-ring substrates provide exclusive information about cytochrome c behavior under modulation of inner mitochondrial membrane potential, proton gradient and the activity of ATP-synthetase.Mathematical simulation explains the observed enhancement of Raman scattering due to high concentration of electric near-field and large contact area between mitochondria and nanostructured surfaces.

View Article: PubMed Central - PubMed

Affiliation: Department of Biophysics, Biological Faculty, Moscow State University, Leninskie gory 1/12, Moscow, 119234, Russia.

ABSTRACT
Selective study of the electron transport chain components in living mitochondria is essential for fundamental biophysical research and for the development of new medical diagnostic methods. However, many important details of inter- and intramembrane mitochondrial processes have remained in shadow due to the lack of non-invasive techniques. Here we suggest a novel label-free approach based on the surface-enhanced Raman spectroscopy (SERS) to monitor the redox state and conformation of cytochrome c in the electron transport chain in living mitochondria. We demonstrate that SERS spectra of living mitochondria placed on hierarchically structured silver-ring substrates provide exclusive information about cytochrome c behavior under modulation of inner mitochondrial membrane potential, proton gradient and the activity of ATP-synthetase. Mathematical simulation explains the observed enhancement of Raman scattering due to high concentration of electric near-field and large contact area between mitochondria and nanostructured surfaces.

No MeSH data available.