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Cite This: ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
Ultra-High Signal Detection of Human Embryonic Stem Cells Driven by Two-Dimensional Materials Sophia S. Y. Chan,† Yaw Sing Tan,‡ Kan-Xing Wu,§ Christine Cheung,§,∥ and Desmond K. Loke*,† †
Science Faculty, Singapore University of Technology and Design, Singapore 487372, Singapore Bioinformatics Institute, Agency for Science, Technology and Research (A*STAR), Singapore 138671, Singapore § Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore 308232, Singapore ∥ Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), Singapore 138673, Singapore Downloaded via DURHAM UNIV on August 8, 2018 at 13:52:39 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
‡
S Supporting Information *
ABSTRACT: We observed a unique bioelectric signal of human embryonic stem cells using direct current−voltage measurements facilitated by few-layered 2DMoS2 sheets. A 1.828 mA cell signal was achieved (2 orders of magnitude higher than previous electrical-based detection methods) as well as multiple cell reading cycles demonstrating I ∼ 1.9 mA. Native stem cell proliferation, viability, and pluripotency were preserved. Molecular dynamics simulations elucidated the origin of the 2D-MoS2 sheet-assisted increase in current flow. This paves the way for the development of a broadly applicable, fast, and damage-free stem cell detection method capable of identifying pluripotency with virtually any complementarymetal-oxide-semiconductor circuits.
KEYWORDS: human embryonic stem cells, two-dimensional (2D) materials, current−voltage measurements, molecular dynamics (MD) simulations, nano-scale, chemical design
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destruction of differentiated hESCs (via immunostaining and PCR methods), preventing further cell studies/uses.8−10 EBD methods are based on the detection of electrical-based signals of stem cells. These features favor primarily the detection of pluripotent hESCs. For facile monitoring/validation, these methods can be combined with external complementary metal−oxide−semiconductor (CMOS)-based circuits (e.g., digital-electronic memory, thermoelectric actuator, optoelectronic lighting, etc.). However, these circuits require high cell signal values in the order of hundreds of microamperes to approximately 1 milliampere.11,12 Previous electrochemicalbased methods have demonstrated hESC signals to be around the order of a few to several tens of microamperes.6,7 A difficulty arises from increasing the current flow between the hESCs and metal electrodes while enhancing the cell-electrode adhesion for high device reliability. This has prevented EDB system/hESC detector commercialization. Two-dimensional (2D) materials such as molybdenum disulfide (MoS2) have a molecular formula of MX2 and comprise of a hexagonal metal (M) layer sandwiched between two chalcogen (X) layers.13,14 2D-MoS2 sheets show higher electrical conductivities along the in-plane directions compared to those of the cross-plane directions.13,15 The 2D-MoS2 sheets
t present, there is much interest in human embryonic stem cells (hESCs) as a starting resource to derive functional cells and tissues for regenerative medicine. hESCbased therapy could be used to replace traditional stem cellbased therapies such as transgene-free induced pluripotent stem cell (iPSC)-based therapy,1 nuclear transfer embryonic stem cell-based therapy,2 and other stem cell-based therapies,to avoid immune rejection. hESC-based therapy, based on the autologous transplantation of stem cells derived from patientspecific cells, are generally able to maintain pluripotency.3 Clinical applications of hESC-based therapy can only be viable if the monitoring/validation of pluripotent hESCs can be achieved (e.g., monitoring of pluripotent hESCs prior to differentiation, validating the removed residual pluripotent hESCs after differentiation, etc.). This is required to minimize the risk of teratoma formation from pluripotent hESCs prior to transplantation for safe clinical uses.4,5 Electrical-based detection (EBD) methods (e.g., cyclic voltammetry (CV) and differential pulse voltammetry (DPV)) are excellent candidates for monitoring/validation of pluripotent hESCs.6,7 These methods could be used to replace traditional detection methods, for example, optical-based detection methods (i.e., immunostaining, fluorescence-activated cell sorting (FACS)), biological-based detection methods (i.e., polymerase chain reaction (PCR)), and so on to avoid the time-consuming prelabeling of target proteins with antibodies (via immunostaining and FACS methods) and © XXXX American Chemical Society
Received: May 8, 2018 Accepted: July 18, 2018
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DOI: 10.1021/acsabm.8b00085 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
Letter
ACS Applied Bio Materials
Figure 1. Enhanced current flow of H9-MoS2 over controls. (a) Current−voltage (I−V) characteristics where current (mA) was measured against the applied voltage (V), where pure PBS solution (black line), MoS2 sheets (green line), and the dried H9-PBS cells (blue line) showed a lower current flow compared to that of the dried H9-MoS2 cells (gray line). (b) I−V plots were also measured for samples of H9-PBS (blue line) and H9MoS2 (gray line) after t = 24 and 48 h of incubation as demonstrated by solid and dotted lines, respectively. For both graphs, current was measured for a voltage sweep of V = −5.0 to 5.0 V. (c) Illustration of stem cell-detector structure. Photographs of LED demonstration of current flow at V ∼ 4.0 V for (d,i) H9-MoS2 and (d,ii) H9-PBS devices. (e) I−V measurements for both pure PBS solution (black line) and H9-MoS2 cells (gray line) under wet conditions for a voltage sweep of V = −2.7 to 2.7 V. (f) Measured current for H9-MoS2 cells under wet conditions at I ∼ 1.9 mA for 4,000 reading cycles.
Here, we observed a unique bioelectric signal of hESCs using direct I−V measurements facilitated by few-layered 2DMoS2 sheets. A 1.828 mA cell signal was achieved as well as multiple cell reading cycles at I ∼ 1.9 mA. Native cell proliferation, viability, and pluripotency were preserved. Molecular dynamics (MD) simulations suggest that the interaction/biding between H9 cells and 2D-MoS2 sheets is driven by van der Waals (vdW) interactions between carbon atoms on the cell membrane and sulfur atoms on the sheet and strong electrostatic forces between oxygen atoms on the cell membrane and molybdenum atoms on the sheet, which essentially assisted the increase in current flow. This avoids the energy efficiency drawbacks of previous EBD methods by using a conductive bare 2D-material medium rather than a not so
can exhibit good adhesion with metal electrodes and surface molecules unique to stem cells. These materials/molecules essentially allow the current to pass through the cell, 2D-MoS2 sheet, and electrode smoothly, enabling the detection of hESC bioelectric signal using current−voltage (I−V) measurements. Because each cell has unique surface molecules,6 different bioelectrical signals can also be obtained. Earlier studies have demonstrated the 2D-MoS2 sheets to generally show low cytotoxicity in human lung and kidney epithelial cells.16,17 However, similar cytotoxicity studies have not been explored for 2D-MoS2 sheets in the presence of hESCs. Although 2DMoS2 sheets have demonstrated favorable interactions between biomolecules (e.g., lysozymes, α-peptides),18,19 such interactions have yet to be explored for hESCs/cell membrane. B
DOI: 10.1021/acsabm.8b00085 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
Letter
ACS Applied Bio Materials
Figure 2. Low cytotoxic effects of 2D-MoS2 sheets. Confocal microscopy images of H9 cells incubated in cell media without (a, c, e) (control) and with (b, d, f) 2D-MoS2 sheets (H9-MoS2) (dark regions); images were taken at t = 0 h (a, b), 24 h (c, d), and 48 h (e, f). At t = 0 h, H9 cells maintained rounded and compact colonies with clear, well-defined edges. At t = 24 h, the colonies grew slightly bigger but remained round. Both control and H9-MoS2 after t = 48 h demonstrated pluripotent colonies with clearer colony borders, larger nuclei, and smaller cytoplasmic areas than those at t = 24 h. (g) TEM image of the 2D-MoS2 sheets embedded in the membrane of the H9 cells. Black arrows point to the cell membrane, and white arrows indicate the MoS2 sheets. (h) Photograph of H9 cells with different 2D-MoS2 concentrations. H9 cells were stained and fixed with crystal violet (CV) salt solution with paraformaldehyde (PFA). The samples show higher color intensity (in purple) with a decrease in 2D-MoS2 concentration. The color intensity correlates to the population of H9 cells adhered to the well plate. (i) Plot of percentage of normalized area of H9 cell density in the well plate as a function of 2D-MoS2 concentrations ranging from 0 to 100 μM at 25 μM intervals. The cell areas were computed using Fiji and calculated using eq 1. The error bars show the range of values obtained from calculations using different image samplings (n = 3). (j) Representative images of H9 cells incubated with 25 μM 2D-MoS2 sheets stained for SOX2 (left panel), OCT4 (second panel from the left), and DAPI (second panel from the right), confirming that native pluripotency of H9 cells was unaffected by the addition of 2D-MoS2 sheets. The right panel is an overlay of all of the images.
ing contrast between cells fixed at different incubation times, cell signals were measured for H9-PBS/H9-MoS2 cells at t = 24 and 48 h (Figure 1b). Interestingly, the cell signals for both H9-PBS and H9-MoS2 cells were higher after t = 48 h of incubation than t = 24 h, as illustrated by the dotted and solid lines, respectively, indicating time-dependent cell signal. As the cells grow and proliferate over time, there are more surface molecules present. The increase in cell signal over time can be produced by the increase in surface molecules. These devices can be connected to external CMOS circuits, such as optoelectronic lightings. To demonstrate CMOS compatibility and differences in cell signals, our devices were connected to light-emitting diodes (LEDs) (Figure 1di, dii). When V ∼ 4.0 V was applied, the H9-MoS2 device produced a more intense light than that of the H9-PBS device, denoting the enhancing effect of 2D-MoS2 sheets. For reproducibility, conductivity was also measured for different 2D-TMD sheets (Figure S2).
conductive mixed nanoparticle/biomolecule medium6 as the ultrahigh cell signal detection agent for hESCs. 2D-MoS2 sheets prepared via sonication showed native cellular activity comparable to that of control studies (Figure S1). Solutions of phosphate-buffered saline (PBS) only, MoS2 sheets (MoS2), H9 cells in PBS (H9-PBS), and H9 cells incubated with 2D-MoS2 sheets (H9-MoS2) were drop-cast onto our device and dried (Figure 1c), and I−V measurements were carried out (see Experimental Procedures). A current signal was observed for PBS only due to the presence of free ions, whereas H9 cells demonstrated a native cell signal independent of PBS (Figure 1a). The H9-MoS2 cells produced a significantly higher cell signal (gray lines), indicating that the presence of 2D-MoS2 sheets enhanced the current flow between the H9 cells and metal electrodes (e.g., I ≈ 0.196 mA to 0.424 mA at V = 5.0 V). It can be noted that MoS2 concentration-dependent cell signal was also observed, but upper concentration limits were restricted by lower cell viability with increasing MoS2 concentration. For demonstratC
DOI: 10.1021/acsabm.8b00085 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
Letter
ACS Applied Bio Materials
Figure 3. Interaction between the MoS2 sheet and the lipid bilayer. (a) Close-up snapshot of the phosphatidylcholine (POPC) lipids aggregating below the MoS2 sheet at t = 500 ns. The lipid tails (light blue) appear to be lying parallel to the S atoms at the bottom of the MoS2 sheet with some of the O atoms on the lipid interacting with the exposed Mo atoms. (b) MoS2 sheet used during the simulation with Mo in blue and S in yellow. The side with the gray double arrows indicates the zigzag molybdenum edge. (c) Interaction between MoS2 sheet and the lipid bilayer comprised of POPC lipids over time. The MoS2 sheet approached the lipid bilayer in a surface manner. Lipid aggregation and significant curving of the lipid bilayer was observed around t = 450 ns. Snapshots were taken from t = 0 to 500 ns. For (a) and (c), color coding of the species is as follows: Mo, pink; S, yellow; C, light blue; O, red; P, gold; N, blue. (d) Plot of interatomic distance as a function of simulation time. The dotted green lines demarcate 3−6 Å, the distance range for weak vdW interactions. The distances between a C atom in the lipid bilayer and an S atom in the middle of the MoS2 sheet, and an O atom in lipid bilayer and a Mo atom at the edge of the 2D-MoS2 sheet are shown in purple and yellow, respectively.
It was crucial to further understand whether 2D-MoS2 sheets were capable of preserving native H9 cell activity while simultaneously enhancing current flow between the cell, 2DMoS2 sheet, and metal electrodes. We investigated the dependence of cell morphology, proliferation, and survivability (viability) on incubation time and 2D-MoS2 concentration as well as the pluripotency of H9 cells in the presence of 2DMoS2 sheets. Both control (H9 cells without 2D-MoS2 sheets) and H9MoS2 cells were incubated and fixed for observation at t = 24 and 48 h. Despite growing at a slightly slower rate, H9-MoS2 cells displayed comparable adhesion and cell morphology to
The cell signals of H9-MoS2 cells were also measured in solution; H9-MoS2 cells in PBS solution were dropped onto the device, and I−V measurements were performed under wet conditions as described in the Experimental Procedures. The cells demonstrated high cell signal values (e.g., I ≈ 1.828 mA at V = 2.0 V) (Figure 1e), which is the highest reported in any advanced electrical-based method so far,6 suggesting that measurements can be performed under culture conditions. These cells also exhibited constant cell signal values for multiple reading cycles (i.e., I ∼ 1.9 mA, ∼4,000 cycles) (Figure 1f), indicating that the measurements produce negligible cell damage. D
DOI: 10.1021/acsabm.8b00085 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
Letter
ACS Applied Bio Materials
this is the first TEM demonstration of few-layer 2D-MoS2 sheets embedded in H9 cells under full incubation conditions. To understand the origin of increase in current flow, MD simulations of a single MoS2 sheet and a lipid bilayer comprised of phosphatidylcholine (POPC) were carried out. Early in the simulation, MoS2 approached the lipid bilayer in a surface-first manner, resting above the lipid bilayer. Toward the last part of the calculation (i.e., t = 450−500 ns), lipid aggregation below the substrate and significant curving of the lipid bilayer were observed (Figure 3a, c). By the end of the simulation (t = 500 ns), the substrate was tilted with the zigzag metal edge pointing toward the lipid bilayer (Figure 3b, c). Dynamic interatomic distance measurements demonstrated the spontaneous and favorable formation of weak vdW interactions and strong electrostatic forces between the MoS2 sheet and lipid bilayer (Figure 3d). Weak vdW interactions were observed between C atoms on the lipid bilayer and S atoms in the MoS2 sheet with the C-S interatomic distances fluctuating within the range of 3−6 Å (indicated by the dotted green lines), the distance range for weak vdW interactions.22 On the other hand, strong electrostatic forces were observed between O atoms on the lipid bilayer and Mo atoms at the edge of the MoS2 sheet, which brought them to within a constant distance of ∼2 Å (Figure 3d). Similar interactions/forces were also observed in simulations of the lipid bilayer with a MoS2 bilayer sheet (Figure S4). Both experimental and computational results suggest low cytotoxicity of 2D-MoS2 sheets in the presence of H9 cells while enhancing current flow (between cell, 2D-MoS2, and metal electrode) via spontaneous and favorable interactions/ binding between H9 cells and 2D-MoS2 sheets. H9-MoS2 cells demonstrated a higher intensity of the LED devices compared to that of H9 cells alone. Using TEM, 2D-MoS2 sheets were observed to be embedded within the H9 cell membrane, suggesting stem cell−2D material interaction/binding. Simulations of the lipid bilayer and single MoS2 sheet confirm our observations; both weak vdW interactions and strong electrostatic forces contribute to their binding during the simulation. In addition, the tilt of the MoS2 sheet and curve of the lipid bilayer could possibly assist the translocation of the MoS2 sheet via endocytosis, which is commonly observed for other 2D materials such as graphene.23 This phenomenon would explain the enhanced current flow observed for H9 cells incubated with 2D-MoS2 sheets. In summary, we observed a unique bioelectric signal of hESCs using direct I−V measurements assisted by few-layered 2D-MoS2 sheets. A 1.828 mA cell signal was achieved along with multiple cell reading cycles at I ∼ 1.9 mA. We determined the biocompatibility of 2D-MoS2 sheets with H9 cells using time- and concentration-dependent experiments and showed comparable cell morphology and viability to control without affecting native stem cell pluripotency. Simulation results showed spontaneous weak vdW interactions and strong electrostatic forces between the H9 cell membrane and 2DMoS2 sheet, which was likely to have facilitated enhanced current flow. In principle, this method is applicable to all types of device structures, 2D materials, and stem cells, where an appropriate combination of these variables should provide new opportunities for optimizing hESC detection performance.
control overall. Upon material addition (i.e., at t = 0 h) (Figure 2a, b), the H9 cells maintained rounded and compact colonies with clear well-defined edges. At t = 24 h, the colonies grew slightly bigger but remained round (Figure 2c, d). Both control and H9-MoS2 cells after t = 48 h exhibited stable stem cell-like morphologies: good pluripotent colonies with clearer colony borders as the cells compacted in with larger nuclei and smaller cytoplasmic areas (Figure 2e, f). The addition of few-layer 2DMoS2 sheets did not perturb the native activity or viability of the H9 cells, similar to previous studies on other human cell lines.16,17 Comparable to previous studies,16,17 2D-MoS2 concentration varying from 0 to 100 μM at 25 μM intervals altered H9 cell viability (population of living cells). After being fixed with paraformaldehyde (PFA), H9 cells were stained purple with crystal violet (CV) salt solution; a higher density of purple regions indicated a higher population of H9 cells adhered to the well plate (living) as unattached H9 cells (nonliving) were removed during washing (Figure 2h). Using Fiji (a distribution of ImageJ),20 the areas of purple regions (N) were calculated as a percentage of the control sample using eq 1 and plotted against MoS2 concentration (Figure 2i). N=
area of purple region at x concentration × 100% area of purple region for control (1)
The H9 cells showed a higher density of purple regions with a decrease in the 2D-MoS2 concentration, suggesting concentration-dependent cell viability (e.g., 0−50 μM, >50%, 75−100 μM,
