Q and A
When a cell is positioned near an illuminated graphene surface, photo-generated ballistic “hot” electrons in graphene will change the cell membrane potential, as they displace 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 would provide optical stimulation of cells via the external light-generated e-field that interacts with the transmembrane field gradient, leading to membrane depolarization, and subsequent action potential generation. By simply turning the light on/off, we can control the activation state of cells near a graphene surface.
·does not require genetic modification of cells;
·provides more physiological activation via a capacitive mechanism rather than via exogenous optogenetics-driven ion currents;
·can be enable by a wide range of light wavelengths;
·all cells are activated perpendicular to the surface.
Limitations: no genetic targeting (yet) of optical actuators in genetically distinct sub-population of cells. However, many drug screening assays are using homogenous populations of cells, and this main advantage of optogenetics is not
Q: What is your advantage vs. potential competition?
A: The advantage of G-substrates vs. Electric Field Stimulation (EFS): No need for specialized equipment which allows using our plates on common imaging systems.
The advantage of G-substrates vs Optogenetic Actuators: No need for genetic modifications of cells which saves time and money while providing the results without false negatives and false positives.
Q: Why would we use your consumable G-substrates?
No changes in established protocols
Q: Are these thermal effects?
A: Nope. We were concerned with this possibility, and performed experiments to get the answers.
GraMOS has optoelectronic rather than photothermal mechanism. You can look in our paper to see experimental details
If there were any thermal effects, then on and off development of stimulation effects would have been gradual. Instead, we are seeing the “instant” activation.
Q: Are your G-substrates biocompatible?
A: Our G-substrates exhibit excellent biocompatibility (REF).We evaluated biocompatibility of G-substrates using neonatal rat ventricular (NRV) CMs, and human induced pluripotent stem cell (hiPSC)-derived CMs, primary cortical neurons, hIPSC-derived neurons.
We discovered that all cell types behave drastically better on graphene 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 previous 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.
Do not be troubled by few published studies that reported graphene toxicity towards biological entities. Due to the huge variability in chemical synthesis protocols, solvents, sizes of graphene materials, anything can happen.
Q: Is there optical cross talk between light signals to activate G-substrates and fluorescent indicators?
A: All-optical assays combine optical stimulation with optical recording for non-invasive probing of cellular activity. In our assays, we will use (a) G-plates to stimulate CMs, and (b) calcium- and voltage-sensitive fluorescent indicators to detect and quantify light-induced functional activity of CMs. To enable such assays, two non-overlapping light signals are required: for optical stimulation via G-plates (Ls) and for excitation of fluorescent indicators (Le). Figure 6 demonstrates our strategy 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 are you using for GraMOS-enabled all-optical non-genetic assays?
A: We developed our own LED-based light source that can simultaneously turn on 7 light signals while independently controlling their intensity and temporal parameters.
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 subthreshold (low) intensity, and b) high-intensity light of any wavelength outside fluorophore excitation spectrum.
To provide our customers with the complete solution for all-optical drug discovery stimulation assays, we have designed and constructed a novel LED-based optical stimulation light source that will be compatible with any microscope-based system. This activity was performed under the umbrella of indirect efforts.
Specifically, we have engineered a light source with the possibility of the delivery of up to 7 wavelengths of UV/visible light at once, or in any combination of colors and times (Fig. 10).
In this light source we have also included the thermal stability module so the individual wavelengths of light will stay at the exact predefined frequencies, eliminating the LED source heating, the intensity stability control, and the possibility to deliver multiple individual wavelengths of light in a predefined time sequence.
That allowed our customers to record the calcium or voltage signal from live cells, while simultaneously stimulating the cells using non-overlapping wavelengths of stimulation light.
Q: Can we use this light source for optogenetic experiments?
A: Yes, absolutely. The light intensity required for GraMOS is comparable with the light intensity required for optogenetic actuators.