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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.


Related in: MedlinePlus

Characterization of Ag nanostructured surfaces.(A) Optical microphotograph of Ag nanostructured surface in the transmitted light. The inset figure shows AgNSS in reflected light and the reflectivity spectrum of AgNSS obtained with x50 magnification, objective NA 0.75. (B–D) Scanning electron microscopy images of Ag nanostructured surface with different magnifications; white horizontal scale bars are equaled to 10, 1 and 0.2 μm, respectively. Figure B shows overlapping of nanostructured silver rings. Figure C demonstrates hierarchically organized clusters (“bricks”) forming concaved walls of nanostructured silver rings. Figure D shows magnified view of porous nanostructured silver “bricks” covered with smaller spherical silver nanoparticles. Figure E demonstrates TEM image of the hierarchically structured silver showing channels in the porous silver bricks. The channels are filled with nanometer—sized embryocrystals of silver formed by fast thermal decomposition of diaminsilver hydroxide solution from initially sprayed aerosol droplets. The embryocrystals are being moved through the channels onto the surface of porous silver bricks due to capillary forces followed by evaporation of water solvent and the crystal overgrowth up to the sizes of “sesame seeds”. White horizontal scale bar is equaled to 5 nm.
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f4: Characterization of Ag nanostructured surfaces.(A) Optical microphotograph of Ag nanostructured surface in the transmitted light. The inset figure shows AgNSS in reflected light and the reflectivity spectrum of AgNSS obtained with x50 magnification, objective NA 0.75. (B–D) Scanning electron microscopy images of Ag nanostructured surface with different magnifications; white horizontal scale bars are equaled to 10, 1 and 0.2 μm, respectively. Figure B shows overlapping of nanostructured silver rings. Figure C demonstrates hierarchically organized clusters (“bricks”) forming concaved walls of nanostructured silver rings. Figure D shows magnified view of porous nanostructured silver “bricks” covered with smaller spherical silver nanoparticles. Figure E demonstrates TEM image of the hierarchically structured silver showing channels in the porous silver bricks. The channels are filled with nanometer—sized embryocrystals of silver formed by fast thermal decomposition of diaminsilver hydroxide solution from initially sprayed aerosol droplets. The embryocrystals are being moved through the channels onto the surface of porous silver bricks due to capillary forces followed by evaporation of water solvent and the crystal overgrowth up to the sizes of “sesame seeds”. White horizontal scale bar is equaled to 5 nm.

Mentions: To build a model of Raman enhancement of mitochondrial cytochrome c on AgNSSs, we performed their characterization with scanning and transmission electron microscopy combined with local reflectance spectra recording. The obtained AgNSS have highly overlapping silver rings of a complex morphology with multilevel arrangement of AgNPs (Fig. 4A). The AgNSS structure originates from the decomposition of micrometer-sized droplets of ultrasonic mist of diaminsilver (I) hydroxide solution, this phenomenon is known as the coffee-ring effect33. The silver ring formation is accompanied by multiscale structuring due to complex redistribution of nutrient liquid and building block overgrowth (Fig. 4B–D). During silver reduction, larger and smaller gaps appear in the region of ring rims resulting in randomly organized blocks (‘‘bricks of the wall’’) of 200–500 nm (Fig. 4B–D). “Blocks” of highly overlapping rings are covered with interconnected silver particles of 10–50 nm where smaller AgNPs lying over larger AgNPs like sesame seeds resulting in the multiscale structure of AgNSS (Fig. 4D). We consider that the porosity feature and capillary effects initiates the growth of the “sesame seeds” which eventually coveri the surface of silver rings in many points (Fig. 4E). The reflectivity spectra recorded from various parts of AgNSS using x50 magnification are similar to one another and have a wide reflectance dip in the region of 400–850 nm (Fig. 4). The light absorption at a wide wavelength range can be explained by a complex multiscale structure of AgNSS. The similarity of reflectance spectra recorded from spots with various magnifications (data are not shown) is an evidence of AgNSS microscopic homogeneity that is highly important for serial biomedical studies.


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)

Characterization of Ag nanostructured surfaces.(A) Optical microphotograph of Ag nanostructured surface in the transmitted light. The inset figure shows AgNSS in reflected light and the reflectivity spectrum of AgNSS obtained with x50 magnification, objective NA 0.75. (B–D) Scanning electron microscopy images of Ag nanostructured surface with different magnifications; white horizontal scale bars are equaled to 10, 1 and 0.2 μm, respectively. Figure B shows overlapping of nanostructured silver rings. Figure C demonstrates hierarchically organized clusters (“bricks”) forming concaved walls of nanostructured silver rings. Figure D shows magnified view of porous nanostructured silver “bricks” covered with smaller spherical silver nanoparticles. Figure E demonstrates TEM image of the hierarchically structured silver showing channels in the porous silver bricks. The channels are filled with nanometer—sized embryocrystals of silver formed by fast thermal decomposition of diaminsilver hydroxide solution from initially sprayed aerosol droplets. The embryocrystals are being moved through the channels onto the surface of porous silver bricks due to capillary forces followed by evaporation of water solvent and the crystal overgrowth up to the sizes of “sesame seeds”. White horizontal scale bar is equaled to 5 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Characterization of Ag nanostructured surfaces.(A) Optical microphotograph of Ag nanostructured surface in the transmitted light. The inset figure shows AgNSS in reflected light and the reflectivity spectrum of AgNSS obtained with x50 magnification, objective NA 0.75. (B–D) Scanning electron microscopy images of Ag nanostructured surface with different magnifications; white horizontal scale bars are equaled to 10, 1 and 0.2 μm, respectively. Figure B shows overlapping of nanostructured silver rings. Figure C demonstrates hierarchically organized clusters (“bricks”) forming concaved walls of nanostructured silver rings. Figure D shows magnified view of porous nanostructured silver “bricks” covered with smaller spherical silver nanoparticles. Figure E demonstrates TEM image of the hierarchically structured silver showing channels in the porous silver bricks. The channels are filled with nanometer—sized embryocrystals of silver formed by fast thermal decomposition of diaminsilver hydroxide solution from initially sprayed aerosol droplets. The embryocrystals are being moved through the channels onto the surface of porous silver bricks due to capillary forces followed by evaporation of water solvent and the crystal overgrowth up to the sizes of “sesame seeds”. White horizontal scale bar is equaled to 5 nm.
Mentions: To build a model of Raman enhancement of mitochondrial cytochrome c on AgNSSs, we performed their characterization with scanning and transmission electron microscopy combined with local reflectance spectra recording. The obtained AgNSS have highly overlapping silver rings of a complex morphology with multilevel arrangement of AgNPs (Fig. 4A). The AgNSS structure originates from the decomposition of micrometer-sized droplets of ultrasonic mist of diaminsilver (I) hydroxide solution, this phenomenon is known as the coffee-ring effect33. The silver ring formation is accompanied by multiscale structuring due to complex redistribution of nutrient liquid and building block overgrowth (Fig. 4B–D). During silver reduction, larger and smaller gaps appear in the region of ring rims resulting in randomly organized blocks (‘‘bricks of the wall’’) of 200–500 nm (Fig. 4B–D). “Blocks” of highly overlapping rings are covered with interconnected silver particles of 10–50 nm where smaller AgNPs lying over larger AgNPs like sesame seeds resulting in the multiscale structure of AgNSS (Fig. 4D). We consider that the porosity feature and capillary effects initiates the growth of the “sesame seeds” which eventually coveri the surface of silver rings in many points (Fig. 4E). The reflectivity spectra recorded from various parts of AgNSS using x50 magnification are similar to one another and have a wide reflectance dip in the region of 400–850 nm (Fig. 4). The light absorption at a wide wavelength range can be explained by a complex multiscale structure of AgNSS. The similarity of reflectance spectra recorded from spots with various magnifications (data are not shown) is an evidence of AgNSS microscopic homogeneity that is highly important for serial biomedical studies.

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.


Related in: MedlinePlus