Graphene-Mediated Optical Stimulation
Mechanism of Action
Our technology builds on exciting findings that graphene can efficiently convert light into electricity via a hot-carrier multiplication process on a femto-second timescale without heating the lattice. We take advantage of these unique optoelectronic properties of graphene when designing a graphene-based optical actuator.
When a cell is positioned near an illuminated graphene surface, photo-generated ballistic “hot” electrons in graphene can change the cell membrane potential by displacing cations near the graphene/cell membrane interface due to the capacitive coupling between the cell membrane and the surface of graphene materials.
In other words, a graphene-based optoelectronic platform provides optical stimulation of cells via the external light-generated electric field that interacts with the transmembrane field gradient, leading to depolarization of the cell membrane, and triggering voltage-dependent events (e.g., activation of voltage-gated ion channels and action potential firing) in a physiological manner.
By simply turning the light on/off, we can elicit repetitive and transient cell membrane depolarization, and enable the dynamic optical stimulation of cells positioned near a graphene surface.
GraMOS provides more physiological activation via external direct cell membrane depolarization;
GraMOS does not require genetic modification of cells;
GraMOS can be enabled by a wide range of light wavelengths;
Simplified workflow/ Technological simplicity (neither wires/electrodes nor genetic modifications are required).
no genetic targeting (yet) of graphene-based optical actuators in genetically distinct sub-population of cells.
However, many drug screening assays are using homogeneous populations of cells, and this main advantage of optogenetics is not critical in some applications (e.g., drug screening).
Q & A
Q: Why would we use your consumable G-coverslips or G-plates?
No changes in established cell culture protocols
The only commercial cell substrate for enabling non-genetic optical stimulation of cells
Q: Are your G-substrates biocompatible?
A: Our G-substrates exhibit excellent biocompatibility.
We evaluated biocompatibility of G-coverslips using HEK-293 cells, COS cells, CHO cells, primary murine hippocampal and cortical neurons, hIPSC-derived neurons, primary culture of neonatal rat ventricular cardiomyocytes, mouse embryonic stem cell–derived cardiomyocytes, hiPSC-derived cardiomyocytes.
We discovered that all cell types behave drastically better on G-surfaces in terms of cell heath and proliferation. Furthermore, we exposed cells on graphene substrates to light for 12 hours and determined that these conditions had no effect on cell health and proliferation.
The excellent biocompatibility demonstrated by our G-substrates is in agreement with published studies that utilized graphene materials as support structures in cellular scaffolds. These studies suggested that surface chemistry characteristics, mechanical properties, and micro-scale topographic features of graphene were responsible for favorable cell microenvironment.
Q: Can GraMOS be only used with neurons and cardiomyocytes?
A: GraMOS technology is universally valid in all cell types, because all cells have cell membranes, and all cells have the transmembrane electric field gradient across the cell membrane. By modulating the transmembrane field gradient, GraMOS can modulate voltage-dependent processes in any cell.
Q: Is the tight interaction between G-biointerfaces and live cells required?
A: The free path of photogenerated electrons from graphene is up to 1 micron depending on the media.
Cells and G-biointerfaces do not have to form long-term tight interactions. We demonstrated that transient contact between G-biointerfaces in motion can result in efficient optical stimulation of cell sand tissues.
Q: Do you need additional (non-graphene) light-absorbing photoactive layers for photogeneration of electrons?
A: In other studies, scientists indeed used an additional layer to absorb photons and then transfer them to another location using graphene electrodes. However, in GraMOS, graphene itself is the photoactive layer where free charge carriers are generated in response to light.
Q: How does GraMOS compare with the electric field stimulation (EFS) method?
A: Changing the transmembrane electric potential by applying external electric field (E-field) has been utilized in biological studies for years. When E-field is applied to a pair of metal electrodes immersed in the electrolytic physiological solution with cells or tissues, it triggers electric currents which lead to changes in the membrane potential of cells located between electrodes.
Due to the IP issues, EFS modules cannot be used in voltage imaging assays that were suggested by the CiPA initiative. Additionally, the EFS approach has several inherent technical limitations (e.g., cell damage, concomitant depolarization and hyperpolarization areas) that could negatively impact the assay results.
GraMOS vs. EFS:
stimulation effects are independent of the cell orientation, since light is applied along the Z-axis;
GraMOS triggers depolarization without accompanying hyperpolarization;
all cells can be stimulated uniformly and simultaneously;
Spatial stimulation patterns can be defined via DLP or patterned surfaces.
Q: How does GraMOS compare with optogenetic stimulation?
A: Optogenetic-based optical stimulation technology that requires high expression of exogenous light-sensitive proteins from algae into cells of interest.
Optogenetic stimulation is a complex biophysical phenomenon that inevitably affects cells. This revolutionary method comes with several critical caveats: (1) Optogenetic actuators are pore-bearing transmembrane proteins that conduct ion currents with complex gating kinetics. Thus, their activation might affect some endogenous voltage- and ion-dependent processes in cells and the kinetics of drug effects in drug screening assays; (2) Optogenetics requires viral gene transfer, high expression levels of exogenous transmembrane proteins, and their functional integration into intracellular machinery. Genetic modifications are not desirable during development, differentiation, and maturation of hiPSC-cells, because they might change the outcome of these processes and affect the properties of cells even in the absence of optical stimulation.
GraMOS vs. optogenetics:
Genetically intact cells
Simplified workflow (no cell transfection required)
Physiologically more relevant stimulation mechanism.
Q: What is the difference between GraMOS and graphene electrodes?
A: Graphene can be incorporated into transparent electrodes which allows to combine the direct electrical stimulation of cells with simultaneous optical recordings. In these studies, graphene serves as a passive intermediary delivering electrons that were generated elsewhere.
In GraMOS, graphene is a critical core material where free charge carriers are generated in response to light exposure, which subsequently leads to optical stimulation of cells..
Q: How can the parameters of light signals affect GraMOS?
Intensity: GraMOS effects are proportional to the light intensity in the tested range (up to 10 mW/mm2).
Absorption: Photon absorption in graphene is proportional to the number of graphene layers. Therefore, G-substrates with lower optical density (which means a thicker graphene coating) can absorb more photons, and generate more free charge carriers.
Wavelength: In undoped graphene materials, photon absorption is nearly constant for wavelengths in the range 300 – 2500 nm. Therefore, light signals of different wavelengths but with comparable intensities can produce similar optical stimulation effects.
Duration: Due to very short life time of photogenerated excitons (femtoseconds), there is no “accumulation” of free charge carries from graphene. Therefore, changes in the duration of light signals have no effect on GraMOS.
Q: Are there thermal effects in GraMOS?
A: Nope. We were concerned with such possibility, and performed experiments to get the answers.
GraMOS has optoelectronic rather than photothermal mechanism.
If there were any thermal effects, then the on and off kinetics of stimulation effects would have been gradual and proportional to the duration of light exposure. It is not the case. Instead, we are seeing the “instant” activation. Please, check our paper in Science Advances (2018) for additional experimental details.
Q: Can GraMOS be incorporated in all-optical drug screening assays?
A: All-optical assays combine optical stimulation with optical recording for non-invasive probing of cellular activity. In all-optical GraMOS-enabled assays, we can use (a) G-coverslips / G-plates to stimulate cells, and (b) calcium- and voltage-sensitive fluorescent indicators to detect and quantify light-induced functional activity of cells.
Q: Can you use GraMOS technology to discover use-dependent drugs?
A: Yes. To explore the utility of G-substrates for use-dependent drug screening, we probed the effects of Mexiletine, a class Ib antiarrhythmic drug. To optically stimulate iPS-cardiomyocytes on G-substrates, we exposed them to light of different intensities to drive the contraction frequency of cardiomyocytes from their spontaneous (<1.5 Hz) to various light-induced rates. Using a bright-field optical detection method for monitoring the contractions of cardiomyocytes, we determined that there was a strong, positive correlation between inhibitory effects of Mexiletine (20 µM) and the cardiomyocyte contraction frequency.
Q: Is there an optical cross talk between light signals for activation of G-substrates and light signals for excitation of fluorescent indicators?
A: To enable all-optical assays, two non-overlapping light signals are required: for optical stimulation via G-plates (Ls) and for excitation of fluorescent indicators (Le).
By taking into account the properties of GraMOS and fluorescent indicators, we developed a protocol for selecting the appropriate combination of light parameters (e.g., wavelength and intensity) to avoid optical crosstalk between Ls and Le.
Ls: The efficiency of stimulation via G-substrates is virtually independent of the light wavelength of the same intensity. When a light wavelength for “activation” of G-plates is selected outside the absorption spectrum of fluorescent indicators, then this light will not have any effect on fluorophores (including photobleaching), and will not interfere with optical monitoring.
Le: Since the graphene absorption spectrum is flat, the fluorophore excitation light might trigger optical stimulation, However, it can only happen if the intensity of excitation light exceeds the threshold required for “activation” of G-biointerfaces. Using sub-threshold light intensities to excite fluorophores eliminates the potential effect on optical stimulation.
Q: What light source can be used for GraMOS-enabled all-optical non-genetic drug discovery assays?
A: To enable all-optical assays on G-substrates, you will need to combine two light wavelengths at different light intensities: a) a fluorophore-specific excitation wavelength of light at a sub-threshold (low) intensity, and b) high-intensity light of any wavelength outside fluorophore excitation spectrum.
To offer the complete solution to our customers, we developed our own LED-based light source (PhotonMaker system) that can simultaneously deliver 7 light signals while independently controlling their intensity and temporal parameters. PhotonMaker is compatible with any microscope-based system.
Any other light sources that are capable of simultaneously delivering two light signals with different intensities and different temporal profiles can also be used for GraMOS-enabled all-optical assays.