Cell-chip coupling for bioelectronic devices. Lab manual

Transcription

1 Ferienpraktikum Nanoelektronik Cell-chip coupling for bioelectronic devices Lab manual September 3 rd 9 th 2012 Forschungszentrum Jülich Francesca Santoro, Sergii Pud, Bernhard Wolfrum (PGI-8/ICS-8, Forschungszentrum Jülich)

2 Microelectrode array recordings from HL-1 Cells 1) Preparation of an Ag/AgCl reference electrode Background: A reference electrode is an electrode, which ideally maintains a constant potential in a solution. Stable reference electrodes are an important tool in all electrochemical measurements, where the potential of a working electrode has to be set versus a fixed reference potential. The potential is generated by the exchange of ions at the interface between electrode and solution. A high stability of electrode reference potentials can be achieved by employing a well-defined redox system with constant concentrations of the participants of the redox reaction. Each redox system is characterized by an individual value on the electrochemical potential scale. The basis of this scale (0.0 V) is formed by the standard hydrogen electrode. All other redox potentials are referenced versus this potential. When performing electrical measurements with cells, the potential of the extracellular solution should be defined by a stable reference potential. The Ag/AgCl-electrode which is used in many electrochemical experiments is a good choice as it is biocompatible under measurement conditions. It is made by electrochemical oxidation of a thin silver wire in hydrochloric acid. The basic reaction is given by: Ag + HCl AgCl + ½ H 2 When an Ag/AgCl electrode is brought into contact with a 1 M potassium chloride solution at 25 C, it develops a potential of around V versus the standard hydrogen electrode. Since the salt concentration in an extracellular solution is maintained roughly at a constant value, the silver chloride electrode exhibits a stable potential for electrical cell measurements. Setup: Voltage source Silver electrode Glass beaker 1M HCL solution Tasks: 1. Grind off electrode for cleaning and roughening. 2. Put the electrode into the socket. 3. Insert the setup upside down into the glass beaker such that the electrode is immersed into the HCl-solution. 4. Switch on power, turn to 1-2 V and wait until silver wire has become brown/black. 5. Get electrode out of the setup. 6. Rinse carefully with MilliQ water

3 2) Recordings of HL-1 cell signals Background: HL-1 cells belong to a cardiac muscle cell line that exhibits contractile activity and can be passaged in culture as well as recovered from frozen stocks. Since these cells are relatively easy to culture and show spontaneous electrical activity, they are an ideal candidate for cell-chip communication experiments. After a few days in culture, confluent layers of HL-1 cells exhibit synchronous beating and propagation of action potentials. In our experiments we want to monitor these action potentials using microelectrode arrays as extracellular recording devices. To this end, cells are plated three or four days prior to the experiments on microelectrode chips which are stored in the cell culture. For our electrical experiments, we use an automatic setup which is controlled by a personal computer. Setup: Microscope MEA-amplifier system with head stage, main stage, and controlling computer MEA-chip & cell culture Ag/AgCl reference electrode Norepinephrine stimulation solution Tasks: 1. Put MEA-chip with the cells carefully into socket. (Make sure that no electrolyte is spilled into headstage!!) 2. Attach the reference electrode and put it into the solution. 3. Optical observation of cells o Maneuver stage with chip under objective o Focus microscope and confirm whether cells are growing on microelectrodes o Can you see beating cells? 4. Electrical recordings of spontaneous HL-1 cell acitivity o Start Med64 and observe cell signals for ~1 minute o Record and analyze 1 minute of data Calculate spike frequency Estimate propagation speed and direction 5. Stimulation of HL-1 cells with norepinephrine o Start recording for 90s o After 30s add 15 µl of norepinephrine solution to cell culture o Describe effects of chemical stimulation on cell activity for the remaining 60s. 6. Note: If time permits we will do a complementary experiment using calcium imaging to optically verify activity of HL-1 cells.

4 3) Measuring the bandwidth of the recording system An action potential (AP) from an HL-1 cell is characterized by frequencies below a few khz. To investigate and analyze the coupling of the cells to the microelectrode array, by monitoring extracellular recorded APs, it is necessary to characterize the recording setup (MEA and amplifier system) in terms of the bandwidth. In this case the bandwidth is defined as the frequencies, which are amplified and recorded by the recording set-up. Therefore, the ratio,where V out is the recorded voltage and V in is the applied voltage, is defined and plotted over the frequency range. To measure the bandwidth a MEA chip, the BIOMAS amplifier system, a Lock-In amplifier and different electrolyte solutions are needed. The MEA chip is inserted into the BIOMAS system as as described in section two for the recording measurements of HL-1 APs. The Lock-In amplifier is used to apply a sinusoidal signal of known amplitude and frequency to the bath-electrode on top of the MEA. The sine out plug of the Lock-In amplifier is connected to the reference electrode (I in Fig. 1). Additionally, to get the recorded signal from the BIOMAS amplifier back into the Lock-In amplifier the Mainamplifier OUTPUT 1 plug is connected to the plug A of the Lock-In (II in Fig. 1). Since the signal is amplified by a factor up to 1000 a voltage divider needs to be pluged in between the cable and input A. This division will be corrected later in the software. II I Voltage divider II to reference electrode

5 Next the BIOMAS MEA software needs to be running. At the bottom you can find Outputs. Setting Main out 1 the channel which is applied to the Mainamplifier OUTPUT 1 plug is selected. The Lock-In amplifier is controlled by a second software. Parameters like frequency, inputs of the Lock-In amplifier can be controlled. The Amplitude has to be set to V. The frequency range has to be set from 1 Hz to 10 khz. In addition, the voltage divider is now corrected by setting Voltage Cor to

6 Tasks: 1) Measure the bandwith of the amplifier system: Explain the results what behavior do you expect from such an amplifier system? What are the limitations imposed on cell measurements by these results

7 4) Electrode impedances The impedance of an electrode characterizes the current/voltage with respect to frequency in a given setup. It is an important factor for extracellular recordings from single cells, since a high electrode impedance imposes a lower limit on the noise characteristics of the measurement and, in combination with leak resistances, may act as a voltage divider effectively attenuating the measurable signal. We will measure the impedance of our microelectrode from the MEA-chip using a commercial potentiostat see image below. A sinusoidal voltage is applied to the microelectrode versus a silver/silver chloride electrode that is immersed in the bath solution and used as a reference. The resulting current is measured and plotted versus frequency. The potentiostat enables us to control the potential of the electrode under investigation (working electrode) with respect to the reference electrode. Thereby a current is driven through a third electrode, the so called counter electrode, keeping the reference electrode current-free. This ensures that the reference electrode is not damaged and that we do not experience an additional voltage drop at the reference electrode, thus enabling a stable potential at the working electrode. However, since we are measuring very small currents below the na range we can connect the counter electrode feed line with the reference electrode in this experiment. The small currents applied at the reference will not cause any significant voltage drop and will also not alter or damage the electrode.

8 Tasks: 1) Put an encapsulated chip into the measurement headstage 2) Connect one of the microelectrodes to the working/sense electrode of the potentiostat (red wires) 3) Connect the leads for reference (blue) and counter electrode (white) to the silver/silver chloride electrode and place it directly above the chip s surface 4) Carefully fill in the PBS electrolyte solution into the chamber of the chip. The reference should be immersed in the electrolyte solution 5) Measure the impedance of at least three individual microlectrodes for a frequency range between 10 Hz and 10 khz. 6) Discuss the results with respect to signal measurements from cells? Can you draw a simplified electrical circuit including the impedances of the cell, the electrode, the cell-chip junction, the amplifer & possible leak paths? 7) Discuss ideal and worst case scenarios for cell recordings what possibilities do you imagine could improve the recording system?