Cancer Cell Hyperactivity and Membrane Dipolarity Monitoring via

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Cancer Cell Hyperactivity and Membrane Dipolarity Monitoring via Raman Mapping of Interfaced Graphene: Towards Non-Invasive Cancer Diagnostics Bijentimala Keisham, Arron Cole, Phong Nguyen, Ankit Mehta, and Vikas Berry ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b12307 • Publication Date (Web): 14 Nov 2016 Downloaded from http://pubs.acs.org on November 21, 2016

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ACS Applied Materials & Interfaces

Cancer Cell Hyperactivity and Membrane Dipolarity Monitoring via Raman Mapping of Interfaced Graphene: Towards Non-Invasive Cancer Diagnostics Bijentimala Keisham†, Arron Cole‡, Phong Nguyen†, Ankit Mehta‡* & Vikas Berry†* †

Department of Chemical Engineering, University of Illinois at Chicago, 60607 ‡

Department of Neurosurgery, University of Illinois at Chicago, 60612

*[email protected] and [email protected] KEYWORDS. Graphene, Cancer, GBM, Raman, phonon

ABSTRACT:

Ultra-sensitive detection, mapping and monitoring of the activity of cancer cells is critical for treatment evaluation and patient care. Here, we demonstrate that a cancer cell’s glycolysis-induced hyperactivity and enhanced electronegative membrane (from sialic acid) can sensitively modify the second-order overtone of in-plane phonon vibration energies (2D) of interfaced graphene via a hole-doping mechanism. By leveraging ultrathin graphene’s high quantum capacitance and

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responsive phononics, we sensitively differentiated the activity of interfaced Glioblastoma Multiforme (GBM) cells, a malignant brain tumor, from that of human astrocytes at a single-cell resolution. GBM cell’s high surface electronegativity (potential ~310 mV) and hyperacidic-release induces hole-doping in graphene with a 3-fold higher 2D vibration energy shift of approximately 6±0.5 cm-1 than astrocytes. From molecular dipole induced quantum coupling, we estimate that the sialic acid density on the cell membrane increases from one molecule per ~17 nm2 to one molecule per ~7 nm2. Further, graphene phononic response also identified enhanced acidity of cancer cell’s growth medium. Graphene’s phonon-sensitive platform to determine interfaced cell’s activity/chemistry will potentially open avenues for studying activity of other cancer cell types, including metastatic tumors and characterizing different grades of their malignancy.

INTRODUCTION Cancer is one of the leading causes of death worldwide, and its diagnosis is critical to initiate therapies1. Successful cancer treatment relies on early detection at high sensitivity and specificity. Hence, it is imperative to develop sensitive detection technologies that can identify cancer at early stages. Over the past couple of decades, research efforts have leveraged the sensitive quantum mechanical effects and high surface area to volume ratio of nanoparticle to develop several cancer detectors.2– 5

Some of the employed nanomaterials include gold nanoparticles,6–8 quantum dots,9 and carbon

nanotubes.10 These sensors operate on the principle of optical, magnetic, mechanical, chemical and/or physical actuation at interfacing with cancer biomarkers. Similarly, Raman spectroscopy (or surface enhanced Raman spectroscopy) and other spectroscopy methods have been used for determining the chemical fingerprints of cancer biomarkers to


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Figure 1. Schematic depicting the doping mechanism of graphene by a well-attached GBM cell and the resultant modification of phonon dispersion in 2D peak. Doping influences the electronic bands of graphene (solid lines) to become flatter compared to the intrinsic one (dashed lines). differentiate normal and cancerous cells11. They can also provide information on the chemistry of the cell membrane molecules. However, such spectroscopic techniques generally do not provide information about the activity of the cell or its effect on its local environment. There is a critical need of a sensitive platform, which can detect, differentiate, and quantify such cell activity. Here, we measure the change in the frequency of one of graphene's prominent Raman peaks upon interfacing of a single cell to determine if the cell is hyperactive. Graphene – a planar sheet of sp2 hybridized carbon atoms arranged in a honeycomb lattice12–16 – possesses superior properties17–20 to enable this application: (a) graphene is the thinnest material (~0.3 nm, vibration modes are active), (b) high quantum capacitance (highly sensitive to doping), (c) high carrier-phonon coupling with phononic energies sensitive to its Fermi level21,22, and (d) a large interfacial area, providing an ideal platform for cell attachment23,24. When the cell interfaces with graphene, the


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molecular dipoles on the membrane apply an electric field25, which sensitively changes the chargecarrier concentration of graphene due to its large quantum capacitance. Further, the cell also injects ions to the membrane, which can gate graphene. The carrier doping modifies Graphene's Raman peak frequencies, which can be recorded at high spectral, spatial, and temporal resolution. Since the cell’s activity is manifested in its membrane chemistry and ion-exchange, the Raman imaging provides the level of activity and chemical density. Based on this principle, we have developed a facile, non-invasive, Raman-based early-stage cancer diagnostic device. It is important to note that graphene-based electronic sensors for cells do not provide spatially resolved activity of the cell. Also, graphene oxide which comprises of heavy lattice disorders and oxygen functional groups17,26, do not provide a sensitive Raman detection platform due to the lack of clarity of Raman peaks. The detection study here was methodically conducted for cultured Glioblastoma Multiforme (GBM) cells immobilized on graphene substrate. GBM, WHO Grade IV malignant astrocytomas, are the most aggressive and common malignant tumor with a poor prognosis of approximately 14 months27,28. Human astrocytes (normal) were cultured and utilized as a control. EXPERIMENTAL SECTION Monolayer graphene was synthesized via chemical vapor deposition (CVD) process and transferred onto a SiO2/Si chip (300nm SiO2). The U138 human Glioblastoma Multiforme (GBM) and normal human astrocyte (NHA, Lonza) cell lines, employed in our study, were grown and later suspended in Dulbecco's Modified Eagle Medium (DMEM). To interface the cells with graphene, 3 drops of the cell suspension were deposited on the transferred graphene surface. The graphene/cell sample was capped using a cover slip to minimize the evaporation of the media. In order to allow the proper attachment of the cells on the surface, the sample was incubated at 37⁰ C for 2 and half hours. Optical microscopy and Raman spectroscopy were employed to visualize


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and analyze the three samples (probed from the top) including: (1) monolayer graphene on SiO2/Si substrate, (2) monolayer graphene with DMEM on SiO2/Si substrate, and (3) monolayer graphene with cell/DMEM (GBM cell and astrocyte) on SiO2/Si substrate. Further experimental details can be found in supporting information, section S1. RESULTS AND DISCUSSION The CVD graphene was characterized using Raman Spectroscopy and its Raman spectra is represented by three characteristic Raman bands, a small D-band peak around 1350 cm-1, a G-band peak near 1580-1590 cm-1 (corresponding to the in-plane vibration mode) and a 2D-band peak between 2600-2700 cm-1 (second order overtone of in-plane vibration)29, depending on the energy of the excitation laser (D and 2D peaks)29–31. These three primary peaks are associated with phonon vibrational modes in graphene29–32, and are sensitive to the electronic, structural and interfacial properties of graphene. Das et al21 quantitatively correlated the concentration of injected carriers in graphene (dopants/cm2) to the Raman peak positions by monitoring the amount of charge carriers at various gate voltages. The position of G peak increases with the addition of either electrons or holes, however the 2D peak position responds differently to holes and electrons: increases with p-doping and decreases with n-doping21,22,33. Thus, 2D band provides a comprehensive information on the polarity and density of charge carriers in graphene, while the G band only provides the carrier density. Also, it is well-known that graphene exhibits Kohn anomaly for the Raman-active E2g Γ phonon (G band)29–32. Upon extensive doping, this abnormal phonon dispersion behavior (Kohn anomaly) is removed non-adiabatically (beyond the adiabatic Born-Oppenheimer approximation) from the Γ point which leads to stiffening of G peak and saturation (at high doping level)21,33–35. However, the 2D position is not restricted by the carrier concentration level as the 2D phonons are


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not influenced by the non-adiabatic effects (see supporting information, Figure S3). Therefore, the 2D peak position analysis was used in this work. Since the sensor is based on cell activity, it is important to understand the biochemical activity of a normal and cancer cell. Cancer cells undergo anaerobic fermentation (not respiring), where the electron transport mechanism and oxygen-dependency for energy production are disrupted36,37. This conversion of the primary mechanism for energy production folds

results

in

excessive (~200

higher) accumulation of organic acids (via glycolysis) and pH modifications in cancerous

cells and tissues, leading to an acidic microenvironment around the cell36,38–40. Further, cancer cells have increased negative charges on the outer cell coat (glycocalyx) which are due to the presence of sialic acid residues making them more electronegative than normal cells40. The increased acidic efflux from the cell [H+] onto the graphene surface24,41 and the heightened negative charge on the cell surface, [E] induces p-doping on graphene’s surface23,42 (consistent with the observation). Further, these cells can infiltrate the nearby tissues and change their micro-environment27. This property might be leveraged to study the body fluids, including blood and cerebrospinal fluid, and its potential application in cancer diagnostics.


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Figure 2. a) A representative model showing the cell attachment process on graphene. b) Optical images showing a less-adhered GBM cell and a well-adhered cell on graphene/SiO2 surface. Scale bar 10 m. The p-doping on graphene affects the resonance condition of the 2D phonons causing the renormalization of the electronic band, as shown in Figure 1. The absolute electron energies reduce and the electronic bands are pushed further away from the K and K’ points, decreasing the lifetime of the excited quasiparticles and phonon momentum43. This abnormal phonon dispersion process results in an increase in the 2D mode Raman shift caused by 2D Raman scattering involving phonons of higher energies. To interface the cell with the graphene, an aliquot of the cell culture suspension was placed on graphene/SiO2 and incubated at 37⁰ C for 1 to 3 hours. The cells showed strong adherence on the graphene surface when incubated for 2.5 hours. Cells adhered with 1.5-hour incubation time were


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susceptible to detachment when exposed to the Raman laser with power as low as 10 mW. Further, the cell growth medium plays an important role in the cell attachment process. When the cells were suspended in phosphate buffer solution (PBS), they exhibited poor adhesion to the graphene surface, even after 3 hours of incubation. The (DMEM) used here contains fetal bovine serum (FBS), which includes numerous growth factors and proteins that stimulate cell adhesion44. At the microscopic level, the spreading of the cell, which flattens it from the initial circular form is enabled by the polymerization of the cytoskeleton component, actin. Actin filaments act on the plasma membrane and its polymerization force pushes the membrane forward, allowing the cell to establish contact with the substrate45. Figure 2 depicts the representative micrograph of a welladhered GBM cell and a less-adhered cell on the graphene-SiO2 substrate. The cells that are not adhered and still suspended in medium look circular in shape. As the cell attaches to a surface, it flattens out and forms protrusions (and retractile regions)45. The doping activity of the interfaced GBM cells and astrocytes (Lonza) on the graphene platform was analyzed using Raman spectroscopy. A typical cell is 40 – 50 m in size and can be probed with a diffraction-limited spatial resolution of 300 nm Raman spot size (area of 0.7 m2). Figure 3a shows the 2D band Raman spectra of the bare graphene exposed to air (black), graphene interfaced with Astrocyte covered in medium (blue) and graphene interfaced with the GBM cell covered in medium (red). The GBM cells exhibit a large blue shift in the 2D peak position (~6.3 cm-1), indicating a high degree of p-doping in graphene and a significant decrease in 2D signal


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Figure 3. a) Raman spectra showing the 2D peak positions of graphene interfaced with air (black), Astrocyte (blue) and GBM cell (red) covered in medium. (Inset) 2D position mapping of GBM cell, scale 10 m. b) Raman spectra showing the 2D peak positions of graphene interfaced with air (black), cellular growth medium influenced by Astrocyte (blue) and GBM cell (red). (Inset) 2D position mapping of Astrocyte, scale 10 m. All the Raman spectra have been normalized to the intensity of G peak. intensity. On the other hand, the astrocyte cells (control, Lonza Inc.) show a weaker p-doping on graphene (2D band blue shifts by ~2.3 cm-1) and lower reduction in the 2D intensity. Further, the graphene under the cell is more doped than that just outside the cell’s periphery. The overall effective doping outside the GBM cell’s boundary is higher than in pure graphene, and lower than that for graphene interfaced with cell. This is apparent in the spatial plot of 2D peak position, which maps the footprint of the cell (inset of figure 3a). Since, the GBM cancer cells exhibit enhanced [H+] diffusion from the cell and an increased surface electronegativity in comparison to the astrocytes, this confirms that graphene’s 2D phonon vibration mechanism is sensitive to the difference in the activity levels of interfaced astrocytes and GBM cells. Further, the results also show that [H+] can diffuse from the cell into the medium, which can then dope graphene. The doping induced shift in graphene’s G band peak-position is also consistent with the observations mentioned above (See supporting information, section Figure S1). As mentioned above, the cells decrease the pH of the surrounding medium environment by releasing acidic biomolecules. The modified acidity of the medium in turn p-dopes graphene46–49. Since enhanced glycolysis in GBM cells must cause more [H+] release than in astrocytes, to


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confirm increased GBM activity, we compared the media of the cells. Figure 3b shows the 2D peak positions of graphene in air (black), graphene covered in cellular growth medium acidified by an astrocyte (blue) (blue shift ~0.5 cm-1) and that acidified by a GBM cell (red) (blue shift ~ 1.3 cm-1). To minimize the doping effects from the cell, the Raman spectra for graphene-inmedium was acquired from an area > 100 µm away from the cells. Higher 2D peak-shift for GBM’s media confirmed enhanced [H+] release. Further, pure medium when interfaced with graphene caused reduction of p-doping in graphene (See supporting information, section Figure S2). Graphene’s surface is ultrasensitive to the interfacial attachment, which can dope graphene and modify its phononic (Raman) properties. The sensitive doping is a consequence of graphene’s high quantum capacitance given by (for monolayer) 𝐶𝑄 =

4𝑒 2 √𝜋 ℎ𝜗𝐹

√𝑛𝑅 , where e is the electron charge, h

is Planck’s constant, ϑF is the Fermi velocity of the Dirac electron, and nR is the total carrier concentration of graphene50. In the present case, the total carrier concentration of graphene (nR) is a combination of intrinsic carrier concentration of graphene (𝑛𝐺 ), the [H+] doping on graphene sheet from the cell (𝑛[𝐻 +] ), and the induced carrier doping via the electronegative cell membrane: (𝑛[𝐸] ): 𝑛𝑅 = 𝑛[𝐻 +] + 𝑛[𝐸] + 𝑛𝐺 . The quantum coupling of the interfacial sialic acid molecules with graphene enhances the effective electric field due to its dipole moment42,50–52. The effective cell potential doping graphene (𝑉⌊𝐺⌋ ) can be estimated by the following expression: 𝑉⌊𝐺⌋ =

𝑒.𝑛𝑅 𝐶𝑄

.

Further, the effective potential (cellular potential, 𝑉⌊𝐺⌋ ) is the total contribution of (1) the cellmembrane’s dipole potential (𝑉⌊𝐶𝑒𝑙𝑙⌋ ), and (2) the [H+] absorbed on graphene (proton potential, 𝑉⌊𝑃𝑟𝑜𝑡𝑜𝑛⌋ ). The contribution of proton potential towards the total cellular potential is only 10 % (Table 1), while a dominant role is played by the cellular membrane’s dipole potential in the change of graphene’s Fermi level. For calculations, this cellular membrane’s dipole potential is


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assumed to originate from the coverage of sialic acid molecules with a dipole moment of 2.5 Debye.

Table 1. Summary of the doping effects of various chemical environments and potential values associated with GBM cells as well as the Astrocytes. Doping type

Chemical system

Doping (×1012 cm-2)

n

Medium influenced by Astrocytes

-1.00

p

Medium influenced by GBM

5.56

p

Astrocytes

p

GBM cells

Cellular Potential, mV (𝑉⌊𝐺⌋ )

Proton potential, mV(𝑉⌊𝑃𝑟𝑜𝑡𝑜𝑛⌋ )

7.24

114-172

12.3-17.5

23.4

222-310

38.8-78.9

From the calculations, the estimated density of sialic acid molecules on GBM cell, and Astrocyte are 0.15 nm-2 (~ 1 per 7 nm2) and 0.06 nm-2 (~ 1 per 17 nm2) respectively (see supporting information for detail calculation, section S5). Consequently, the effective potential of GBM on graphene are estimated to be two-folds higher than those of Astrocyte (Table 1). These values are in agreement with typical voltage difference of cells across membranes, 10-100 mV53. CONCLUSION In conclusion, graphene’s ultrasensitive second-order-overtone of in-plane phonon vibration makes it ideal for spatially resolved (0.7 m2) detection of the activity of a single cancer cell. The cell potential of GBM cells (range: 222 – 310 mV) was found to be significantly higher than that of astrocytes (114 – 172 mV). This is attributed to the estimated ~2.5 folds higher density of sialic acid in GBM cell membrane. Further, the higher influence of the GBM cell’s glycolysis-induced hyperactivity on the medium (by 2.2 to 6.4 folds) was also confirmed. Futuristically, this phonon-


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energy-sensitivity can be extended to other cancer cells, tissues and in-vivo samples to spatially map, study or detect cell activity. ASSOCIATED CONTENT Supporting Information. Materials and methods, effect of cell interfaced with graphene (Raman G peak), effects of cell growth medium, PBS and high cell density on graphene and detail calculation of density estimate via quantum capacitance are included in the supporting information. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *Emails (V. Berry and A. Mehta): [email protected] and [email protected] Author Contributions V.B and A.M conceived and directed the project, B.K. conducted most of the experiments, A.C. directed the cell cultures, P.N contributed in the calculations. All authors contributed equally towards writing the manuscript. Notes A disclosure has been filed for this technology. ACKNOWLEDGMENT VB and AM would like to acknowledge the financial support from University of Illinois at Chicago. Thanks to Yi Lu for the help in cell cultures.


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Cancer Cell Hyperactivity and Membrane Dipolarity Monitoring via

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