Applications of graphene to cell biology 2

Chapter Early sensor attempts This chapter describes my attempts at developing a graphene-based device that would allow measurements of forces exerted by cells on their substrate. As covered in Section 1.4, getting a better understanding of this process is useful for answering a number of biological processes, and existing techniques have limitations. The basic concept behind of all these approaches is to build a transparent, electronic device. The transparency allows optical imaging of cells with a microscope, while substrate deformation, and therefore forces applied, are transceived into electrical signals. Most of these ideas could be first experimented with using gold electrodes, which are fairly opaque to light [43], and moving to graphene electrodes later on when a proof of concept has been shown to be functional. I describe possible ways of converting forces into electrical signals: capacitive, piezoresistive, using graphene itself as a strain gauge, piezoelectric, and quartz crystal micro-balance principle (QCM). All these approaches, except the one using graphene as a strain gauge, 49 Polymer film Graphene Figure 3.1: Basic layer grid design. Cells and their medium are put on top of the sensor. The polymer film can be PDMS, PDMS with conductive particles, or PVDF, depending on the approach taken. It can be replaced by a Quartz crystal for the QCM approach. use sets of parallel stripes of graphene, perpendicular to each other, separated with a thin film (see Figure 3.1). The thin film can be either a deformable dielectric, a piezoresistive/piezoelectric film, or a quartz crystal for the QCM. These methods are mainly thought to measure forces perpendicular to the surface (normal forces), but will probably also be sensitive to forces parallel to the surface (Shear forces). A summary of the different approaches, with their advantages and disadvantages, is provided in Table 3.1. Some approaches were investigated in details, with some experiments performed, while others did not leave the design stage. One of the main challenge is that electrical signals are very weak when we attempt to shrink the device to the sort of resolution desired for our device (≈ µm resolu50 tion). I even considered looking at larger cells (Amoeba, see Appendix B), but the biological significance of studying such cells is less clear. While ultimately unsuccessful, these experiments led to the more interesting device presented in Chapter 4. 51 52 Forces deform a dielectric, causing a change in capacitance Forces deform a piezoresistive film, causing a change in resistance Forces deform a graphene strip, changing its resistance Forces applied on the film create charges Presence of cells causes changes in resonance of a crystal Capacitive Piezoresistive film Graphene as a strain gauge Piezoelectric film Quartz crystal micro-balance Disadvantages • Very small capacitances are challenging to measure (noise, resolution) • Thin films (desired to increase capacitance) become stiffer, reducing sensitivity • Piezoresistive material needs to be transparent • Mostly sensitive to normal forces only • Very challenging to fabricate: transfer of graphene onto soft material; 2D grid requires 3D interconnects. • Challenges in read-out: Small sensing area have small capacitance; better suited for high frequency signals • Read-out equipment is more complex if both frequency and Q factor are to be measured • May not measure forces, but different aspects of cell-matrix interaction. • Easy to fabricate • Relatively simple read-out • Can potentially measure Shear forces • Easy to fabricate (provided a good piezoresistive material is found) • Very simple read-out to Advantages • Very simple read-out • Sensitive to Shear forces • Easy to fabricate • Potentially sensitive Shear forces • Potentially easy to fabricate • Possibly goes beyond measuring forces Table 3.1: Comparison of force sensor approaches, with their advantages, disadvantages and technological challenges. Description Method 3.1 Capacitive Figure 3.1 shows the basic capacitive sensing design: perpendicular lines of electrodes creates a matrix of capacitors, that can be read individually. Each intersection of graphene stripes forms a parallel plate capacitor, whose capacitance C is expressed by: C= r ·A d , (3.1) where A is the plates area, d the distance between the plates, dielectric constant (8.854 · 10−12 [Fm−1 ]), and r is the is the material-dependent relative permittivity. When a pressure P (in pascals) is applied on the capacitor, the dielectric is compressed, and the distance between the places is reduced by P ∆d = , d E where E is the Young’s modulus of the dielectric. This deformation leads to a change in capacitance ∆C: ·A − ·A ∆C d ∆d ∆d d−∆d d = = −1= ≈ , ·A C d − ∆d d − ∆d d d ∆C ∆d P ≈ = . C d E 3.1.1 (for small ∆d) Numerical values for PDMS dielectric Using PDMS as dielectric ( r ≈ [24]), numerical computations for the base capacitance lead to the results in Table 3.2. Even large area (100 × 100 µm), and small spacing (0.1 µm), produces small capacitances 53 (2.7 pF or 2.7 · 10−12 F), that may be problematic to measure, especially in the presence of other parasitic capacitances. Furthermore, standard measuring equipments usually have an input capacitance in the order of 25 pF [4], an order of magnitude larger than the capacitance expected to be measured. Young’s modulus of soft PDMS is E ≈ 100 kPa (for 1:40 crosslinker:base ratio [39]). Therefore, a force of kPa would cause a variation of only ∼ 1% in the capacitance, which may be hard to detect, considering the small base capacitance we begin with. Even softer PDMS can be used, but obtaining a large capacitance value requires thin PDMS membranes, spun at high speed, that are known to be harder than bulk PDMS [77]. In theory, a chip such as AD7745 from Analog Devices [4] is capable of measuring capacitances changes down to aF (4 · 10−18 F), which may be enough to get significant results for relatively large sensing area. I tried this device, using an evaluation board, and managed to get some capacitive readings with an accuracy of around 60 aF. d (µm)/s (µm) 0.1 0.5 1.0 15.0 0.27 [...]... Vmax 20 0 A Dir: Increasing Vg, Parameter: Hole mobility' Time (minutes) 100 B Time (minutes) A Dir: Increasing Vg, Parameter: Vmax 20 1404 02- 0 921 12- cells 160 150 0 .25 0 .20 0.15 0.16 0.18 0 .20 0 .22 0 .24 0 .26 4 3 2 1 0 24 0 23 0 22 0 23 0 22 0 21 0 20 0 3.5 2. 5 Dirac peak [V] Dirac peak [V] Leakage (uA) Mobility' [uA/V ^2] Mobility' [uA/V ^2] Absmax leakage (uA) Mobility' [uA/V ^2] Mobility' [uA/V ^2] 140 130 300 28 0... read-out part of Figure 4.4) The graphene sheet is connected between 2 resistors of known values, one small resistor RL1 is grounded (bias resistor), while RL2 is connected to the 5 V input voltage (VCC ) The graphene resistance is obtained by a voltage divider equation: RG = (RL1 + RL2 ) · VG , VCC − VG where VG is the measured voltage drop across the graphene sheet The role of RL1 is to bias the graphene. .. resistor, 1 nF capacitor) connected to an Arduino, with the data displayed using a simple Java application 64 d (µm)/A (µm2 ) 9 25 1 2. 95 1.06 4 11.81 4 .25 25 73.78 26 .56 100 29 5.13 106 .25 10000 29 513.33 10 624 .80 Table 3.3: Base capacitance of PVDF-dielectric sensor in aF (10−18 F) using capacitor area A, and a thickness of d between the electrodes “diluted” in a capacitor 35 times larger before being... attached to the device using conductive epoxy The other side of the ribbon is connected to a PCB, that provides BNC connectors for use with standard measurement equipments Device (2x2mm) 72 Chapter 4 Cell sensing device In this chapter, I show that intrinsic graphene, without functionalization, can be used to detect cell metabolism in solution In Section 4.1, I transfer a large piece of graphene onto a... connect 2 electrodes onto the graphene sheet Instead of applying the gate voltage from the back of the device, through a silicon oxide layer, as is commonly done [87], I use the fact that cell media is an electrolyte to gate the device via a platinum electrode (see Section 1.1 .2. 2 for details) This allows us to transfer graphene to transparent substrates (glass coverslips), making it possible to perform... overnight, then removing this conditioned media and adding it to a device confirms that I am detecting a cell by-product Section 4 .2 focuses on an improved device design, that is built to allow measurement of the number of cells in a dish I envision this as a new kind of cell counter, complementing existing techniques such as hemocytometer, automated cell counters or impedance-based systems [11, 34, 59] 4.1... piece of monolayer graphene on copper (Graphene Supermarket, Calverton, NY) Following the transfer method described in [97], one side of the copper foil is plasma etched to remove graphene (oxygen plasma, performed by Graphene Supermarket) I spin coat the graphene side of the copper foil with poly(methyl methacrylate) in anisole (PMMA, MicroChem 495K A4; 4000 RPM for 80 s), then bake it for 2 minutes... device, ready to be transferred to the microscope Figure 4.1: Device fabrication steps, after graphene transfer to a coverslip 76 Gate electrode Sealant Graphene + - + - 1st electrode + + - Glass coverslip 2nd electrode Resistance Resistance Read-out Read-out - Gate Gate Control Control Electrolyte (PBS/DMEM) Figure 4 .2: Electrolyte gating of graphene (for reference, identical to Figure 1.4) Graphene is... capacitance, while cells themselves cause a large shift of the Dirac peak towards more negative values, consistent with an addition of positive charges on the surface of the graphene Furthermore, I show that this behavior cannot be explained by a simple change in pH, and that the device response is proportional to the number of cells Furthermore, conditioning media by putting it in presence of cells overnight,... sheet to a slight positive voltage This allows to gate the graphene to an effective negative voltage, despite the fact that the Arduino is unable to produce such negative voltage output For example, assuming a voltage drop of 0.5 V on RL1 , applying a gate voltage of 0 V through the PWM circuit will lead to an effective gate voltage of −0.5 V Since the range of the PWM circuit output is 0 V → 2 V, the . managed to get some capacitive readings with an accuracy of around 60 aF. d (µm)/s (µm) 1 2 5 10 100 1000 0.1 0 .27 1.06 6.64 26 .56 26 56 .20 26 5 620 .00 0.5 <0.1 0 .21 1.33 5.31 531 .24 53 124 .00 1.0. resistor, 1 nF capacitor) connected to an Arduino, with the data displayed using a simple Java application. 64 d (µm)/A (µm 2 ) 1 4 25 100 10000 9 2. 95 11.81 73.78 29 5.13 29 513.33 25 1.06 4 .25 26 .56. 0.11 0.66 2. 66 26 5. 62 265 62. 00 15.0 <0.1 <0.1 <0.1 0.18 17.71 1770.80 Table 3 .2: Base capacitance of PDMS-dielectric sensor (fF, 10 −15 F), using lines of width s (i.e. the total parallel