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

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 nearfield and large contact area between mitochondria and nanostructured surfaces.Mitochondria are organelles of fundamental importance for cellular energy production, metabolic regulation, aging and cell survival under stress [1][2][3] . Normal function of mitochondria and their pathological changes, including production of reactive oxygen species (ROS), are heavily dependent on the redox state of the electron transport chain (ETC) cytochromes and cytochrome c in particular 4,5 . At present, most of the studies of isolated mitochondria and mitochondria in cells are performed by fluorescent microscopy, absorption spectroscopy and measurements of O 2 consumption 3,[6][7][8] . The fluorescent microscopy with small fluorescent dyes (rhodamin and MitoTracker-family, etc.) or fluorescent proteins (GFP, YFP, RFP) can provide general information about changes in the potential of the inner mitochondrial membrane (Δ Φ ), the mitochondrial volume, and the co-localization of certain mitochondrial components with a molecule of interest

Raman probing of lipids, proteins, and mitochondria in skeletal myocytes: a case study on obesity

“…Many resonance Raman studies are dedicated to mitochondrial cytochromes in cells and organs . One of the reasons is that cytochromes are crucial for the operation of electron transport chain (ETC) in mitochondria.…”

Revisiting preparation routes of SERS materials

“…This deposition mode has several advantages in terms of manufacturing SERS-active structures for promising applications: silver micro-and nanostructures are immobilized onto a substrate and form a rather stable metallic film on cheap materials like glass; there is no need to filter or separate nanoparticles; the nanostructure consists of porous silver sponge only; the temperatures are high enough for the one-stage formation of metallic silver nanostructured layer. The negligible background of the USR substrates allowed to use small laser power that prevents photoinduced damage of biological objects and ensures repeated measurements of SERS even from the same point [7,15]. Thus, this preparation technique is the method of choice for preparation of functional components for new SERS devices.…”

“…Silver nanoparticles and nanostructured materials are a common choice for SERS measurements due to their broad plasmon resonance, high stability, facile fabrication methods and the largest enhancement for special molecules [3][4][5]. A complex morphology such as nanoflowers, nanorices, cubes, multipods and nanodendrites, mesocages is achieved by kinetically controlled, aggregation-based, heterogeneously seeded, template-directed growth, selective etching and colloidal system aging; the polyol technique allows one to synthesize silver nanoparticles of a wide variety of shapes and sizes [6][7][8][9][10][11][12][13][14][15][16]. Despite of all possible advantages, the nanoparticles themselves are likely to aggregate uncontrollably in solutions, making it difficult to reproduce SERS results [5,6,11,14]; nanoparticle sols exhibit limited plasmonic tunability as compared to a new class of nanomaterials -colloidosomes composed of a dielectric core and a concentric metal shells with hybridization of plasmon modes supported by an inner cavity and an outer surface of the nanoshell [5,7,9,10].…”

“…A complex morphology such as nanoflowers, nanorices, cubes, multipods and nanodendrites, mesocages is achieved by kinetically controlled, aggregation-based, heterogeneously seeded, template-directed growth, selective etching and colloidal system aging; the polyol technique allows one to synthesize silver nanoparticles of a wide variety of shapes and sizes [6][7][8][9][10][11][12][13][14][15][16]. Despite of all possible advantages, the nanoparticles themselves are likely to aggregate uncontrollably in solutions, making it difficult to reproduce SERS results [5,6,11,14]; nanoparticle sols exhibit limited plasmonic tunability as compared to a new class of nanomaterials -colloidosomes composed of a dielectric core and a concentric metal shells with hybridization of plasmon modes supported by an inner cavity and an outer surface of the nanoshell [5,7,9,10]. However, SERS still suffers a lack of reproducibility due to the masking of SERS signals by surfactants or by-products, gradients of electric fields near the nanoparticles and variations of molecule positions with respect to the nanoparticles; commonly used linkers, surfactants, reducing agents and their oxidized forms, halide ions can change the characteristic properties of nanoparticles because of their aging, recrystallization or random formation of aggregates [5,[11][12][13][14].…”

Vapor phase SERS sensor based on mesoporous silica decorated with silver nanoparticles

“…At the same time, ordinary materials for SERS with ecological, pharmaceutical, biological and medical applications are commonly designed for the analysis of liquid analytes [1][2][3][4][5][6][7][8][9][10] while a limited number of actual research efforts has focused on vapor phase detection [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29]. The latter challenge evidently widens the promising application area of SERS and therefore requires novel approaches for the design of SERS-active materials.…”