(K-410) Piezoelectric Acousto Nanodevice for Protein Redox Modulation

Redox reactions in proteins,^1–3 are the center of its life-sustaining functionality and stability (e.g., respiration, cell apoptosis, etc.) by governing conformation and aggregation behaviors.^4,5 When it is disturbed, protein misfolding occurs and results in, for example, Alzheimer’s or Parkinson’s diseases. ^6,7 In this abstract, we present a novel nanodevice that can externally control the protein redox states (Fig. 1). Using ultrasound irradiation, surface-engineered (i.e., gold half-coating) piezoelectric barium titanate (BTO) nanoparticles (Acousto Nanodevice) induce the oxidation of protein (cytochrome c as the model protein). The Acousto Nanodevice is expected to bring great clinical implications as it enables the modulation of biological processes or protein conformation.

Fig. 2 shows fabrication of the Acousto Nanodevice, which uses a facile three-step process: (1) depositing a monolayer of BTO nanoparticles (≤ 300 nm; US Research Nanomaterials, Inc.) onto a silicon (Si) substrate by spin coating technique (2) depositing gold onto BTO nanoparticles (half coating) using the sputter coating method (3) lift off the half-coated BTO nanoparticles (BTO-Au) from the Si substrate by sonicating the substrate in 0.1 M PBS.

Following the preparation, we added a low concentration of the Acousto Nanodevice (in the order of 1 nM) in a cuvette that contains 5 μM of reduced cyt c (oxidized state Cytochrome C (Sigma Aldrich) was chemically reduced using sodium dithionite (Sigma Aldrich) for use as a reference.^8,9).  A low-intensity ultrasound (∼ 150 mW/cm²) was then applied to re-oxidize cyt c in a large water tank with the cuvette.

UV-Vis absorption spectrometer (Jasco V-670) was used to confirm the redox state of the cyt c. The surface morphology and elemental analysis of Acousto Nanodevice were characterized using Field Emission Scanning Electron Microscope (JEOL, JSM-7600F) and Energy-dispersive X-ray spectroscopy (JEOL, JSM-7600F) respectively. The acoustic intensity of the applied ultrasound was measured using a fiber-optic hydrophone (Precision Acoustics, UK). The system output was monitored using an oscilloscope (Tektronix MDO3054).

After the ultrasound irradiation, we observed the color change of reduced cyt c from light pink to light orange, indicating oxidized cyt c, as shown in Fig. 3a-b. UV-Vis absorption spectrum also confirmed that the reduced cyt c is successfully oxidized by the Acousto Nanodevice under ultrasound. A blueshift of ∼ 5 nm around the soret region (415 nm to 410 nm; Fig. 3c inset) was observed due to the oxidation of heme (Fe²⁺ to Fe³⁺), suggesting an overall net electron transfer from the reduced cyt c to the Acousto Nanodevice (Fig. 3d). This result also agrees to the reported absorption peaks of oxidized (409 nm) and reduced cyt c (415 nm).¹⁰ In contrast, there were no observable changes in UV-Vis spectrum when we attempted to reduce the oxidized cyt c using the same system (i.e., Acousto Nanodevice under ultrasound) (Fig. 3e). It suggests that there are little electron transfers between oxidized cyt c and the Acousto Nanodevice (Fig. 3f).

To confirm the role of ultrasound and surface metal coating, three control groups and the test group were included (Fig. 4a). The results validate that only the combination of Acousto Nanodevice under ultrasonic irradiation could induce measurable change in the spectra. Based on the various sham controls, we postulate the working mechanism of the Acousto Nanodevice is that the alternating electrical polarization of BTO nanoparticles can tentatively modulate the work function of the metal, thereby inducing unidirectional electron transfer from the protein redox centers to the Acousto Nanodevice (Fig. 4b). The band alignment is proposed to be responsible for the unidirectional electron transfer from protein to the Acousto Nanodevice (Fig. 4c-d).

We presented the Acousto Nanodevice capable of modulating the redox state of a protein (i.e., reduced cyt c) under ultrasonic irradiation. The results clearly demonstrated that protein oxidation occurs only when a half-surface gold-coated Acousto Nanodevice is activated by ultrasound. To our best knowledge, the present work is the first demonstration of modulating the redox state of proteins selectively by an ultrasonic-mediated Acousto Nanodevice. In the future, we look forward to targeting specific redox proteins involved in neurodegenerative diseases.

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) No. 2018R1C1B5086311 (Song) and the National Science Foundation (NSF) No. ECCS-2245090 (Kim).

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