Research ArticleBIOPHYSICS

Graphene biointerfaces for optical stimulation of cells

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Science Advances  18 May 2018:
Vol. 4, no. 5, eaat0351
DOI: 10.1126/sciadv.aat0351
  • Fig. 1 Graphene-based optoelectronic interface for optical stimulation of cells.

    (A) Schematic representation of our hypothesis. (B) Representative scanning electron microscopy (SEM) image of iCell cardiomyocytes (CMs) on reduced graphene oxide (rGO)–coated coverslips. (C) Summary of the normalized cell density for neonatal rat ventricular CM cultures on control, rGO-coated, and graphene-coated coverslips. Data are means ± SEM (n ≥ 100 cells per each condition). ***P < 0.005, unpaired t test. Representative SEM images of iCell CMs on rGO-coated (D) and control (G) coverslips, as well as CMs with an acutely deposited rGO flake (J). Representative traces demonstrating light-induced effects on action potentials in iCell CMs on rGO-coated (E) and control (H) coverslips and with deposited rGO flakes (K). Representative traces demonstrating light-induced effects on contractile activity of human-induced pluripotent stem cell (hiPSC)–derived CMs on rGO-coated (F) and control coverslips (I) and with deposited rGO-flakes (L). Graphene materials are highlighted blue. Light illumination events are indicated by bars. AU, arbitrary units.

  • Fig. 2 Proposed mechanism of optical stimulation via G-biointerfaces.

    (A) Photogenerated electrons from G-biointerfaces are detected using UV-Vis spectroscopy that monitors changes in absorption spectra due to G-biointerface–mediated photocatalytic reduction of resazurin to resorufin (top). Graph shows changes in absorption of a 40 μM resazurin solution (at its absorption peak of 610 nM) on G-biointerfaces (red squares) or noncoated glass coverslips (blue circles) as a function of illumination time (465 nm, 1.8 mW/mm2). (B) No changes in the surface temperature of rGO-coated coverslips during continuous 30-min light exposure (n = 3). Inset: Linear calibration curve was fitted to enable the subsequent extraction of the temperature values from the pipette resistance values. Light in (B) and (C): cyan, 4.3 mW/mm2. Data are presented as mean ± SEM. (C) No changes in the pH values of an electrolytic solution covering rGO-coated coverslips during continuous 30-min light exposure (n = 5). (D) Cartoon illustrating the proposed mechanism of cellular optical stimulation using light-activated G-biointerfaces. (E) Representative trace of light-triggered membrane depolarization in a current-clamped CHO cell cultured on G-coated substrates. (F) Representative trace of light-triggered action potential in a current-clamped hiPSC-derived CM cultured on G-coated substrates. Light in (E) and (F): cyan, 4.5 mW/mm2. Light illumination events are indicated by bars.

  • Fig. 3 Dynamic control of functional activity of CMs on G-biointerfaces.

    (A) Effects of cyan, green, and red light signals on contractile activity of iCell CMs on rGO-coated coverslips. (B) Absorption spectrum of rGO coating. (C) Light-induced effects in iCell CMs on coverslips (n = 14 per each condition) coated with single-layer graphene (SGL), double-layer graphene (DGL), and rGO. Data are means ± SEM. (D) Representative traces of light-induced changes in contractions of hiPSC-derived CMs on rGO-coated coverslips in response to 40-ms 2-Hz light pulses and a 3-s step of light (green, 1.1 mW/mm2). (E) Representative traces demonstrating the effects of light signals of different intensities on iCell CMs on rGO-coated coverslips. (F) Box plots of light-induced changes in the contraction frequency (normalized to values at x = 0) as a function of light intensity. Light illumination events are indicated by bars. AU, arbitrary units.

  • Fig. 4 Biological applications for G-biointerface–enabled optical stimulation.

    (A) Representative contraction traces in iCell CMs on light-illuminated G-coated coverslips in the presence of 20 μM mexiletine. ΔA, a mexiletine-induced decrease in the contraction amplitude at a given frequency; AU, arbitrary units. Dotted lines highlight the time course of use-dependent inhibition of CM contraction by mexiletine. (B) Summary of use-dependent effects of mexiletine in optically stimulated CMs: Inhibition in the absence (black triangles, n = 36) and the presence of mexiletine (blue circles, n = 41). r, Pearson’s correlation coefficient (Pearson’s correlation two-tailed t test). (C) Zebrafish embryo 3 dpf. (D) Summary of G-biointerface–enabled optical stimulation effects (green light, 0.8 mW/mm2) on the heart rates of zebrafish embryos from three groups: Control (n = 12), injected with 0.1 mg/ml (n = 6, G1) and with 0.5 mg/ml (n = 6, G2). Data are presented as mean ± SEM. ***P < 0.005, paired t test. (E) Representative heart contraction traces demonstrating optical stimulation effects (green light, 0.8 mW/mm2) in control (left) and G-treated (0.5 mg/ml; right) zebrafish embryos. (F) Heart periods from G-treated (right) zebrafish embryos stimulated by short 500-ms light pulses (2 mW/mm2). Light illumination events are indicated by bars.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/5/eaat0351/DC1

    fig. S1. Biocompatibility of G-biointerfaces.

    fig. S2. Geometrical characteristics of G-flakes.

    fig. S3. Basal heart rates in control and G-treated zebrafish (n = 7 for each group) 2 hours after injection.

    movie S1. Optical stimulation of contractile activity in mouse embryonic stem cell–derived CMs cultured on rGO-coated coverslips (green light, 4.6 mW/mm2).

    movie S2. Optical stimulation of contractile activity in mouse embryonic stem cell–derived CMs via rGO flakes using the same conditions as in movie S1.

    movie S3. Effects of light illumination on the heart contractions in a 3-dpf zebrafish embryo injected with G-biointerfaces (0.5 mg/ml) dispersed in PBS.

    movie S4. Automated image analysis of CM contractions.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Biocompatibility of G-biointerfaces.
    • fig. S2. Geometrical characteristics of G-flakes.
    • fig. S3. Basal heart rates in control and G-treated zebrafish (n = 7 for each group) 2 hours after injection.
    • Legends for movies S1 to S4

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    Other Supplementary Material for this manuscript includes the following:

    • movie S1 (.avi format). Optical stimulation of contractile activity in mouse embryonic stem cell–derived CMs cultured on rGO-coated coverslips (green light, 4.6 mW/mm2).
    • movie S2 (.avi format). Optical stimulation of contractile activity in mouse embryonic stem cell–derived CMs via rGO flakes using the same conditions as in movie S1.
    • movie S3 (.avi format). Effects of light illumination on the heart contractions in a 3-dpf zebrafish embryo injected with G-biointerfaces (0.5 mg/ml) dispersed in PBS.
    • movie S4 (.avi format). Automated image analysis of CM contractions.

    Files in this Data Supplement:

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