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Electrochemical detection of quinone reduced by Complex I Complex II and Complex III in full mitochondrial membranes

In the last decades enormous advances have been made in characterizing the atomic and molecular structure of respiratory chain supercomplexes. *

However, it still remains a challenge to stitch this refined spatial atomistic description with functional information provided by biochemical studies of isolated protein material. Development of functional assays that detect respiratory chain complexes in their native membrane environment contribute to address the open questions related to the role played by their association and interactions. *

In the article “Electrochemical detection of quinone reduced by Complex I Complex II and Complex III in full mitochondrial membranes” Daniel G. Cava, Julia Alvarez-Malmagro, Paolo Natale, Sandra López-Calcerrada, Iván López-Montero, Cristina Ugalde, Jose Maria Abad, Marcos Pita, Antonio L. De Lacey and Marisela Vélez present a characterization assay in which a functionalized gold electrode is modified with mitochondrial membrane fragments that allows monitoring electrochemically the activity of different respiratory chain complexes immersed in the mitochondrial membrane. *

Daniel G. Cava et al. measure the intensity of the reducing current of the electron mediator CoQ1 at the electrode surface and its variation upon addition of the corresponding enzymatic substrates. The activities of Complex I, Complex II and Complex III were monitored by the way in which they reduce the current, reflecting the amount of quinone reduced by the complexes in the presence of their substrates. *

The authors detect that CoQ1H2 produced by Complex I remains partially trapped within the membrane and is more easily oxidized by Complex III or the electrode than the quinone reduced by Complex II. *

Atomic Force Microscopy (AFM) was used to image the topography of the membrane modified electrode. NanoWorld Pyrex-Nitride Silicon-Nitride AFM probes (PNP-DB, diving board shaped cantilevers, the short AFM cantilever with a typical force constant of 0.48 N/m and 67 kHz resonance frequency) were used. *

The surfaces analysed were the electrodes. The two surfaces imaged are the same previously polished electrodes used for electrochemical measurements. The microscope sample holder was adapted in-home to support the electrodes. Two surfaces were analysed: the polished gold functionalized with 4-aminothiophenol and the electrode after incubation with mitochondria subparticles prepared similarly to the electrodes used for the electrochemical measurements.*

Fig. 2 from Daniel G. Cava et al 2024 “Electrochemical detection of quinone reduced by Complex I Complex II and Complex III in full mitochondrial membranes”
QCM and AFM characterization of modified gold. Panel A shows the frequency (left, black) and dissipation (right red) changes detected on a gold covered quartz crystal previously modified with a 4-ATP after injection in the chamber of the mitochondrial fragments at the time point indicated by the thick arrow. Panel B show AFM images of the surface topography of a modified gold electrode before (left) and after (right)incubation with the mitochondrial membrane. The inset below shows the height profile of the lines indicated in the images.

*Daniel G. Cava, Julia Alvarez-Malmagro, Paolo Natale, Sandra López-Calcerrada, Iván López-Montero, Cristina Ugalde, Jose Maria Abad, Marcos Pita, Antonio L. De Lacey and Marisela Vélez
Electrochemical detection of quinone reduced by Complex I Complex II and Complex III in full mitochondrial membranes
Electrochimica Acta, Volume 484, 20 April 2024, 144042
DOI: https://doi.org/10.1016/j.electacta.2024.144042

The article “Electrochemical detection of quinone reduced by Complex I Complex II and Complex III in full mitochondrial membranes” by Daniel G. Cava, Julia Alvarez-Malmagro, Paolo Natale, Sandra López-Calcerrada, Iván López-Montero, Cristina Ugalde, Jose Maria Abad, Marcos Pita, Antonio L. De Lacey and Marisela Vélez is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third-party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/.

Electrochemically Synthesized Poly(3-hexylthiophene) Nanowires as Photosensitive Neuronal Interfaces

Poly(3-hexylthiophene) (P3HT), a hole-conducting polymer, generates a lot of interest especially because of its excellent optoelectronic properties (such as good electrical conductivity and high extinction coefficient) and good processability, which make this polymer an excellent choice for building organic optoelectronic devices (e.g., organic solar cells). *

P3HT films and nanoparticles have also been used to restore the photosensitivity of retinal neurons. *

For their article “Electrochemically Synthesized Poly(3-hexylthiophene) Nanowires as Photosensitive Neuronal Interfaces” Szilveszter Gáspár, Tiziana Ravasenga, Raluca-Elena Munteanu, Sorin David, Fabio Benfenati, and Elisabetta Colombo investigated the template-assisted electrochemical synthesis of P3HT nanowires doped with tetrabutylammonium hexafluorophosphate (TBAHFP) and their biocompatibility with primary neurons. *

They were able to show that template-assisted electrochemical synthesis can relatively easily turn 3-hexylthiophene (3HT) into longer (e.g., 17 ± 3 µm) or shorter (e.g., 1.5 ± 0.4 µm) P3HT nanowires with an average diameter of 196 ± 55 nm (determined by the used template) and that the nanowires produce measurable photocurrents following illumination. *

The fact that template-assisted electrochemical synthesis combines polymerization, doping, and polymer nanostructuring into one, relatively simple step is the most important advantage of this method. The possibility of easily tuning the length of the produced nanowires represents another important advantage. *

The authors were also able to demonstrate that primary cortical neurons can be grown onto P3HT nanowires drop-casted on a glass substrate without relevant changes in their viability and electrophysiological properties, indicating that P3HT nanowires obtained by template-assisted electrochemical synthesis represent a promising neuronal interface for photostimulation. *

Szilveszter Gáspár et al. proved the biocompability of the obtained P3HT nanowires upon incubation for different periods with primary neuronal cultures. They demonstrated that their presence does not affect the membrane properties of the neurons or the excitability of the neurons as evaluated by patch-clamp experiments. These results show the potential of the described synthesis methodology to fabricate injectable P3HT-based photosensitive nanowires with high biocompatibility, ultimately paving the way for their exploitation for neuronal photostimulation. *

Atomic Force Microscopy (AFM) was used to characterize P3HT nanowires drop-casted onto glass coverslips. *

The Atomic Force Microscopy images were obtained in air and in intermittent contact-mode using line rates as slow as 0.2 Hz and NanoWorld Pointprobe® NCSTR silicon soft-tapping AFM probes (typical values: resonant frequency 160 kHz, force constant 7.2 N m). The ratio between the set-point amplitude and the free amplitude of the AFM cantilever was set to 0.5–0.6. The obtained AFM images were used to determine both the lengths and the diameters of the nanowires. *

Figure 3 from “Electrochemically Synthesized Poly(3-hexylthiophene) Nanowires as Photosensitive Neuronal Interfaces” by Szilveszter Gáspár et al.: AFM images of “long” P3HT nanowires (A) and of “short” P3HT nanowires (B). NanoWorld Pointprobe NCSTR soft-tapping mode probes were used. — Figure 3 from “Electrochemically Synthesized Poly(3-hexylthiophene) Nanowires as Photosensitive Neuronal Interfaces” by Szilveszter Gáspár et al.:
AFM images of “long” P3HT nanowires (A) and of “short” P3HT nanowires (B).

*Szilveszter Gáspár, Tiziana Ravasenga, Raluca-Elena Munteanu, Sorin David, Fabio Benfenati, and Elisabetta Colombo
Electrochemically Synthesized Poly(3-hexylthiophene) Nanowires as Photosensitive Neuronal Interfaces
Materials 2021, 14(16), 4761, Special Issue Advanced Designs of Materials, Devices and Techniques for Biosensing
DOI: https://doi.org/10.3390/ma14164761 (please follow this external link to read the full article.)

Open Access The article “Electrochemically Synthesized Poly(3-hexylthiophene) Nanowires as Photosensitive Neuronal Interfaces” by Szilveszter Gáspár, Tiziana Ravasenga, Raluca-Elena Munteanu, Sorin David, Fabio Benfenati, and Elisabetta Colombo is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Screencast Overview about all NanoWorld AFM probes series reaches 500 views mark

the Pointprobe® Silicon AFM probe series is the most widely used and best-know AFM tips series worldwide
the Arrow Silicon AFM probes series offers optimized positioning through maximized AFM tip visibility
the Ultra-Short Cantilever Series offers six different kinds of AFM tips for High Speed Atomic Force Microscopy ( HS-AFM ) in air and in liquid for up to video rate scanning
and the Pyrex-Nitride Silicon Nitride AFM probe series which is designed for various imaging applications in contact mode and dynamic mode AFM

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