To overcome the issue related to the uneven pressure distribution in the single-channel µpMEA device, a two-suction channel design has been implemented. Figure 4.3a shows an optical image of the two-channel µpMEA device mounted on a metal holder, with a zoom-in view of the perforated electrode region and the PDMS-based microfluidic channel underneath shown in Figure 4.3b. In this design, to achieve a relatively uniform negative pressure distribution within the sensing region, two symmetric vacuum channels have been defined. Additionally, a local delivery channel with a width of 200 µm has been included in the central region of the device. This channel serves multiple purposes, including the introduction of chemical stimuli from the external medium reservoir and enhancement of the contact between the retina and electrodes through applying negative pressure via the open holes patterned on the polyimide layer on top of this channel. The µpMEA features gold electrodes with sizes of 60x60 and 80x80 µm2, as well as graphene electrodes with sizes of 30x30 and 40x40 µm2, which provide good electrical performance. Figure 4.3c shows a typical transparent graphene probe surrounded by opened holes. Figure 4.3d is a schematic of the experimental setup for the microfluidic path and retina recordings. To begin the experiment, the retina is first transferred into the well and inspected for qual- ity using fluorescent imaging. The perfusion cannula, which is connected to an external
Figure 4.2: (a) Optical image of a single channel µpMEA device with gold probes, with a scale bar of 750µm. The white arrow indicates the media flow direction of the single neg- ative pressure fluidic channel. (b) Histogram of action potential firing rates before (Suction OFF) and after (Suction ON) the negative pressure is applied to the vacuum channel. (c) A typical spontaneous single spiking waveform detected from RGCs with single channel µpMEA device. The Black lines are the extracted median waveform. (d) The fluorescent image in Figure 4.2d shows the whole retina on top of theµpMEA device, with the distri-
global delivery path, provides oxygenated fresh Ames media and controls the temperature of the media to be around 36°C to maintain the health of the retina. The suction cannula, connected to a peristaltic pump, prevents overflow of the media inside the well. Negative pressure is then applied to the top and bottom vacuum channels using a syringe pump (#1) to enhance the contact between the retina and the electrodes. After the retina has settled for a while, negative pressure is applied to the middle delivery channel using a syringe pump (#2) to introduce local Ames media. This allows for the controlled delivery of chemical stimuli to the retina for study and analysis.
Figure 4.4a shows an image of the actual experimental setup for the µpMEA device mounted on the stage of a fluorescent microscope. This setup includes theµpMEA device mounted on a metal holder sitting on an inverted microscope, as well as the perfusion and suction cannulas for maintaining the health of the retina and preventing overflow of media from the well. The electrical outputs from the polyimide film are clamped to a zero-insertion-force connector, which allows for easy and reliable connection of the film to the recording chip. Figure 4.4b is a cross-sectional view of the media flow inside the local delivery channel and top reservoir after transferring the retina onto the perforated polyimide sensor film. It is important to note that when negative pressure is applied to the local delivery channel, the medium is not only withdrawn from the local delivery channel but also from the top well. However, the total flow resistance of the through holes on the polyimide film and porous retina should be larger than that of the delivery channel, which is required to ensure that the medium is mainly withdrawn from the reservoir connected to the local delivery channel instead of the media well holding the retina. This can often be achieved if the integrity of the retina tissue is maintained with a good sealing to the etched through holes on the polyimide substrate. In this case, most of the medium flowing through the local delivery channel comes from the external source rather than the retina well on the top. Therefore, pre-inspection of the retina tissue is crucial for this experiment. This helps to ensure that the tissue is healthy and able to withstand the negative pressure applied to
Figure 4.3: (a) Optical image of theµpMEA device featuring a local delivery channel and two negative pressure channels, mounted on a metal microscope holder. (b) Zoom-in image of the perforated electrode sensor assembled with the PDMS-based microfluidic channel.
The thickness of the microfluidic channel is approximately 100 µm, with the top and bot- tom channels representing negative pressure channels and the middle chamber serving as both a negative pressure and local delivery channel. The sensor features 16 electrodes, including gold electrodes of sizes 60x60 and 80x80µm2and graphene electrodes of sizes 30x30 and 40x40 µm2. The scale bar in this image is 300 µm. (c) Optical image of an individual transparent graphene electrode. Scale bar is 40µm. (d) The schematic setup of the electrical activities recording system and microfluidic platform with µpMEA device.
The microfluidic platform primarily consists of two media delivery paths: the global and local delivery path. The global delivery path is used to control the media flowing to the reservoir, where the retina is completely exposed to the surrounding global media. The local delivery path can introduce and confine media flow within the underneath local deliv- ery channel, where the retina is partially in contact with the media. The flow of media in the local delivery channel is controlled by external syringe pump 2. Additionally, external syringe pump 1 is used to apply negative pressure to the retina to achieve better physical contact with the electrodes. The perfusion cannula, equipped with a temperature controller, and the suction cannula are incorporated into the delivery path to maintain the viability of the retina and prevent overflow of media inside the well, respectively. [8]
the local delivery channel.
The position of the retina with respect to the local delivery channel is another crucial factor in this experiment. When transferring the retina into the well, a filter paper with a hole cut open in the middle is used to hold the retina. The optical nerve head (ONH) is placed in the open hole region and visible under the microscope. If the ONH is placed near the local delivery channel, as shown in Figure 4.4c and 4.4d, the uneven structure near the ONH [106] will prevent intimate contact between the retina and polyimide and leave a gap in between. This gap leads to a poor sealing of the etched holes on the local delivery channel, causing most of the medium to be withdrawn from the top well. On the other hand, when the position of the ONH is farther away from the channel, as shown in Figure 4.4e and 4.4f, the local delivery channel is mainly filled with medium from the supply reservoir, which is labeled with blue food dye. This is because the tissue becomes flatter as the distance from the ONH increases, allowing for a better sealing of the etched holes on the local delivery channel. It is essential to carefully control the flow of the medium and avoid introducing bubbles to the local delivery channel, as the width of the channel is only about 200 µm. Any bubble introduced to the local delivery path may block the local delivery channel and stop the continuous flow of the media. If an attempt is made to remove the bubble by increasing the negative pressure to the channel, it may damage the tissue as the retina is fragile. To address these issues, a two to one port is used to switch between the Ames media with and without a highK+ concentration. This allows for more precise control over the flow of the medium and helps to avoid introducing bubbles to the local delivery channel.