Organic Electrochemical Transistor Arrays with Integrated Lipid-Sealed Femtolitre Chambers for Simultaneous Electrical and Optical Detection of Membrane Protein Activity

Abstract

We report a method for producing an array of fifty two ion-sensitive PEDOT:PSS organic electrochemical transistors on a glass coverslip, each featuring an integrated fluoropolymer microwell sealed with lipid bilayer into which membrane proteins can be inserted for simultaneous electrical and fluorescence microscopy studies. To demonstrate capability, we fill the microwells with an `inner' phosphate assay buffer solution containing 20 $μ$M Alexa-488 dye and 50 mM KCl, seal the microwells with lipid bilayer using an aqueous-organic-aqueous liquid exchange technique, and then fill the common flow-cell volume above the sealed microwells with a dye-free `outer' phosphate assay buffer containing 100 mM KCl. We insert $α$-hemolysin, which embeds into the lipid bilayer forming a heptameric pore with diameter ~ 2.6 nm. The pore allows K$^{+}$ ions to diffuse into the microwell and Alexa-488 dye molecules to diffuse out of the microwell producing a corresponding drop in transistor conductance and microwell fluorescence intensity, respectively. These two signals occur at different timescales, consistent with the known size difference between K$^{+}$ ions and Alexa-488 molecules. Our approach to fabricating microwell arrays with PEDOT:PSS OECTs incorporated into the bottom of selected microwells distributed in the array is both scalable and versatile, opening a path to studies using larger arrays and with other membrane proteins embedded in the lipid bilayer sealing the microwells.

Figures (22)

• Figure 1: Experiment Concept and Device Design. ( a,b) Schematics of a single OECT device in the array ( a) before and ( b) after insertion of the $\alpha$-hemolysin pore, whereupon K$^{+}$ ions (orange) enter the well and Alexa-488 molecules (green) leave the well, reducing both the current (red arrow) through the PEDOT:PSS transistor channel and the microwell fluorescence intensity. ( c, d, e) Photographs of a completed $52$ OECT array slide at three different magnifications. The scale bars represent ( c) $2$ mm, ( d) $100~\mu$m, ( e) $20~\mu$m. The purple dashed rectangle in ( e) indicates the edges of the PEDOT:PSS channel. The black dot-dashed line in ( e) corresponds to the cross-section shown schematically in ( a) and ( b).
• Figure 2: Electrical Characterization of our OECTs. ( a) Drain current $I_{d}$ vs source-drain bias $V_{sd}$ characteristics for gate voltages $V_{g}$ ranging from $-0.5$ V (top - brown) through $0.0$ V (green) to $+0.5$ V (bottom - purple) in steps of $0.1$ V for a typical OECT device. The inset shows a schematic of the device configuration for the measurements in Fig. 2 (and Fig. 3) where the microwell is unsealed and filled with KCl solution. ( b) Drain current $I_{d}$ (left-axis/solid lines) and transconductance $g_{m}$ (right-axis/dashed lines) vs gate voltage $V_{g}$ for three different source-drain biases $V_{sd}$. Data in both panels was obtained with [KCl] = $100$ mM and the gate voltage applied via an Ag/AgCl electrode.
• Figure 3: Ion-Sensitivity Testing for our OECTs. ( a, b) Plots of ( a) buffer KCl concentration and ( b) OECT channel conductance vs time for decadal progression of [KCl] for five typical OECTs on a single coverslip. Data offset for clarity: $\textcolor{red}{\blacklozenge}$$\#1$, $0~\mu$S; $\textcolor{blue}{\blacklozenge}$$\#7$, $-160~\mu$S; $\textcolor{green}{\blacklozenge}$$\#31$, $-100~\mu$S; $\textcolor{black}{\blacklozenge}$$\#47$, $-180~\mu$S; $\textcolor{orange}{\blacklozenge}$$\#50$, $-200~\mu$S. ( c, d) Plots of ( c) buffer KCl concentration and ( d) OECT channel conductance vs time for the [KCl] change used in our $\alpha$-hemolysin assays for five typical OECTs on a single coverslip (different coverslip to ( a, b)). Data offset for clarity: $\textcolor{red}{\blacklozenge}$$\#16$, $0~\mu$S; $\textcolor{blue}{\blacklozenge}$$\#22$, $-10~\mu$S; $\textcolor{green}{\blacklozenge}$$\#31$, $-30~\mu$S; $\textcolor{black}{\blacklozenge}$$\#34$, $-40~\mu$S; $\textcolor{orange}{\blacklozenge}$$\#42$, $-50~\mu$S. Data in both panels was obtained with gate voltage held at $\sim0$ V via an Ag/AgCl electrode.
• Figure 4: Effect of K$^{+}$ concentration on bilayer curvature. ( a) Schematic and ( b) and fluorescence image of a bilayer-sealed microwell with equal [K$^{+}$] on both sides of the bilayer, which gives a flat bilayer at the centre of the microwell. ( c) Schematic and ( d) fluorescence images of a bilayer-sealed microwell with low [K$^{+}$] outside the microwell and high [K$^{+}$] inside the microwell, which gives a bilayer that bows outward (positive curvature). ( e) Schematic and ( f) fluorescence images of a bilayer-sealed microwell with high [K$^{+}$] outside the microwell and low [K$^{+}$] inside the microwell, which gives a bilayer that bows inward (negative curvature). All fluorescence images obtained with $20~\mu$M Alexa-488 in the inner solution and dye-free outer solution.
• Figure 5: Simultaneous electrical and optical study of $\alpha$-hemolysin activity. ( a) fluorescence microscopy images of the region surrounding an OECT (Device 40 in ( b)) at four times $t = 0$ s, $450$ s, $900$ s and $1350$ s during the assay. Four microwells are circled to indicate examples of: a device microwell with embedded $\alpha$-hemolysin (purple circle), a non-device microwell with embedded $\alpha$-hemolysin (brown circle), a non-device microwell with embedded $\alpha$-hemolysin that ruptures part way through the assay (red circle), and a non-device microwell that failed to seal with bilayer (cyan circle). The gold regions indicate where metal leads are and the underlying greyscale image has been converted to greenscale for clarity. The dashed purple lines indicate the edges of the PEDOT:PSS channel. ( b) Plots of fluorescence intensity (left column/green) and OECT conductance (right column/black) vs time $t$ for eight selected OECTs on a single coverslip. The intensity/conductance data has an offset of Device 6 $3993$ a.u./$237~\mu$S, Device 28 $2954$ a.u./$260~\mu$S, Device 29 $3382$ a.u./$259~\mu$S, Device 30 $2992$ a.u./$263~\mu$S, Device 31 $3374$ a.u./$231~\mu$S, Device 32 $3724$ a.u./$241~\mu$S, Device 37 $3675$ a.u./$236~\mu$S and Device 40 $4241$ a.u./$274~\mu$S subtracted for clarity. The four downward pointing purple triangles in the plot of fluorescence intensity vs time for Device 40 indicate the time-points for the four fluorescence microscopy images shown in ( a).