The authors gratefully acknowledge Charles University (project GA UK No 176119) for supporting this study. This work was supported by the Charles University research program PROGRES Q 28 (Oncology).

No living animals were involved in this study. The experiment was performed on an ex vivo model consisting of a porcine esophagus and stomach. The stomach and esophagus were purchased from a local butchery as their standard product. This procedure is in accordance with Czech laws, and we prefer it because of the &#34;3R&#34; principle (Replacement, Reduction, and Refinement).
1. Fabrication of the pH sensor assembly
NOTE: Observe precautions for handling electrostatic discharge (ESD) sensitive components throughout the fabrication of the pH sensor assembly. Be careful when working with the soldering iron.
<ol>
	<li>Place the ISFET pH sensor mounted on a printed circuit board (PCB) on a flat surface. Locate the solderable contacts.</li>
	<li>Trim the solderable contacts, so their length is no longer than 3 mm.</li>
	<li>Solder a 15 mm section of fluorinated ethylene propylene (FEP) coated cable to the solderable electrodes of the pH sensor. Do not mechanically or chemically clean the bare die assembly. Try to avoid contamination of the die and PCB with flux during soldering.</li>
	<li>Inspect the pH sensor-cable assembly under a microscope for open circuits and shorts. Then, check the shorts with an open-short tester. A correctly prepared assembly at this stage is shown in Figure 1.</li>
	<li>Clean the pH sensor assembly in an ultrasonic cleaner for 5 min at 70 &#176;C in a 5% solution of flux remover in water. The optimum range of ultrasound power is 50-100 W/l. Do not exceed 100 W/l.</li>
	<li>Rinse the pH sensor assembly in technical grade isopropyl alcohol for at least 3 min and let it dry in an oven at 80 &#176;C for 15 min.</li>
	<li>Place all pH sensors on a flat surface (in case multiple are prepared simultaneously) before proceeding to the next step.</li>
	<li>Mix an appropriate amount of two-part epoxy for encapsulation of the soldered electrodes. Use a minimum of 2 mL to allow thorough mixing. Use black opaque epoxy to allow for inspection later- parts of the sensor exposed to the environment will be seen easier as they will not have opaque epoxy on them</li>
	<li>Transfer the mixed epoxy to a 1 mL syringe with a 0.5 mm flat end needle.</li>
	<li>Coat the soldering area of pH sensors with epoxy. Make sure to coat the whole area of PCB electrodes and the exposed wire.</li>
	<li>Let the epoxy cure either at room or elevated temperature (80 &#176;C max), for this study 50 &#176;C was used with the epoxy listed in the Table of Materials.</li>
	<li>Inspect the coated area under a microscope. If any uncoated metal parts (either PCB electrode or wire) are exposed, repeat steps 1.8-1.11 until there are no visual signs of uncoated metal.</li>
	<li>Trim the wires to the length and angle shown in Figure 2. Coat the ends with solder to avoid fraying.</li>
</ol>
2. Fabrication of the electronic assembly
NOTE: Observe precautions for handling ESD-sensitive components throughout the fabrication of the electronics. Be careful when working with the soldering iron and hot-air gun.
<ol>
	<li>Place the PCB (manufactured based on the supplementary files &#34;pcb1.zip&#34; and schematic diagram &#34;schematic.png&#34;) on a flat surface, components side up.</li>
	<li>Apply solder paste to all the exposed gold-plated pads.</li>
	<li>Place all passive and active components using tweezers according to Figure 3 and the Table of Materials.</li>
	<li>Heat the PCB with the hot air gun to solder the components. Heat the PCB gradually to 150 &#176;C for 2 min to expel residual water from the packages and activate the flux in the solder paste. Then, heat the PCB to 260 &#176;C to solder the components. Let the PCB cool to room temperature, do not move it during the whole soldering process.</li>
	<li>After soldering and cooling down to room temperature, inspect the PCB under a microscope to verify the correct placement of all the components and shorts. If no shorts or incorrect component placement is observed, skip step 2.6.</li>
	<li>Repair any shorts or incorrect component placement with a soldering gun or hot air gun. Go to step 2.5.</li>
	<li>Solder 5 wires to the components (power and programming leads) as shown in Figure 4.</li>
	<li>To connect the PCB to the programmer, connect the wires soldered in step 2.7. to the connector of the programmer.</li>
	<li>Program firmware (see Representative Results for a detailed explanation of which file to use) to the microcontroller. Use the previously described procedure to set up the programming software19. Set the programmer to power the device with a voltage of approximately 2.5 V. De-solder the 5 wires after programming.</li>
	<li>Place the PCB on a flat surface, component side up. Solder the AWG38 copper antenna wire (length of 3 cm) as shown in Figure 5 and wrap it around the edge of the PCB. Fix the antenna wire to the edge of the PCB with a cyanoacrylate adhesive. Solder the other two wire jumpers with SWG38 copper wire as shown in Figure 5. Avoid electrical contact with other components.</li>
	<li>Put the PCB on a flat surface, component side down.</li>
	<li>Solder two battery holders to the opposite part of PCB, as shown in Figure 6.</li>
	<li>Solder the pH sensor assembly to the terminals on the PCB, as shown in Figure 7.</li>
	<li>Insert two AG1 batteries into the battery holders. 
	NOTE: Do not proceed with this step and next steps in this section earlier than 24 h before testing and endoscopic implantation of the sensor.</li>
	<li>Prepare an appropriate amount of epoxy as described in step 1.8. for encapsulation of the device.</li>
	<li>Encapsulate the device with the epoxy using the same procedure described in step 1.9 (syringe with a needle). Let the epoxy cure at room temperature or slightly elevated temperature (do not exceed 50 &#176;C because of the presence of batteries). See Figure 8 for the correct encapsulation results.</li>
	<li>Create a titanium wire hook according to Figure 9. 
	NOTE: Titanium (Grade II) was chosen because of its biocompatibility and track record of use in implantable medical devices. Stainless steel may be used, too. However, the type and heat treatment must be chosen carefully as some stainless steel types are very brittle.</li>
	<li>Attach the wire hook to the device with a drop of fast-curing epoxy (see Figure 10) and let it cure at room temperature or slightly elevated temperature (50 &#176;C maximum). The pH sensor is located on the bottom left side of the implantable device.</li>
	<li>The sensor becomes activated 24 h after the insertion of the batteries. Meanwhile, proceed with step 3. 
	&#8203;NOTE: Pause the protocol now if completion of step 3 within 24 h after insertion of the batteries is possible.</li>
</ol>
3. Fabrication of passive rectenna receiver
<ol>
	<li>Place the PCB (manufactured based on the supplementary file &#34;pcb2.zip&#34;). for the rectenna on a flat surface.</li>
	<li>Solder the components using the solder paste method described in steps 2.2-2.6 or use a soldering gun according to Figure 11A. 
	NOTE: If the experimenter decides to manufacture the rectenna receiver again (it was previously manufactured and matched) or does not want to proceed with receiver matching, use the values of the components previously determined by the experimenter or provided in Figure 11B and skip steps 3.5-3.7.</li>
	<li>Solder the SMA connector to the PCB.</li>
	<li>Inspect the PCB under a microscope. If any shorts or incorrect component placement is observed, fix the issues.</li>
	<li>Attach a vector network analyzer input to the SMA connector.</li>
	<li>Record the S11 Smith chart of the rectenna from 300-500 MHz with 1 kHz resolution bandwidth. Observe the response and record the impedance at 431.7 MHz. Use an impedance matching calculator software to determine the values of matching components. The sample Smith chart is shown in Figure 12A.</li>
	<li>Solder the impedance matching components and inspect under a microscope for short circuits and component placement.</li>
	<li>Measure with spectrum analyzer again and confirm that the voltage standing wave ratio (VSWR) is under 3 between 300-500 MHz (inside the outer cyan circle shown in Figure 12B). If not, either repeat with different matching components or continue with the reduced performance of the rectenna in mind.</li>
	<li>Connect the 433 MHz band antenna to the SMA connector. Connect an oscilloscope to the rectenna output.</li>
	<li>Set the oscilloscope to single-channel operation, rolling time base, DC mode, 500 ms/div time base, and 5 mV/div voltage scale.</li>
</ol>
4. Testing of the device
NOTE: The following steps require the use of chemicals. Study the material safety data sheets of the chemicals beforehand and use proper protective equipment and common lab practices when manipulating them.
<ol>
	<li>Inspect the output of the sensor by observing the signal shown on the oscilloscope. The sample output is shown in Figure 13,14. The device will be active after 24 h past the insertion of the batteries. The period of transmitting the output of the pH sensor varies depending on the file which was programmed to the microcontroller (see Representative Results for a detailed explanation).</li>
	<li>Prepare 2% hydrochloric acid solution (use caution when handling hydrochloric acid). Prepare 100 mM buffer solutions of pH 4 (potassium hydrogen phthalate/hydrochloric acid), pH 7 (potassium dihydrogen phosphate/sodium hydroxide), and pH 10 (sodium carbonate/sodium hydrogen carbonate) using standard laboratory procedures and mark the beakers.</li>
	<li>Verify the pH of all four beakers using a calibrated pH meter. Adjust if needed.</li>
	<li>Submerge the capsule in every beaker and record at least 3 samples. Measure the period between the second and third pulse and fill it in the provided spreadsheet (Supplemental File 1). Determine the calibration coefficients for the pH sensor using the spreadsheet.</li>
	<li>After calibration, measure the time between the second and the third pulse and input it into the spreadsheet to determine the pH of the solution to which the pH sensor is exposed.</li>
</ol>
5. Endoscopic implantation of the sensor
<ol>
	<li>Prepare an ex vivo endoscopic porcine model made up of the stomach and a long segment of the esophagus.</li>
	<li>Grasp the sensor externally with a hemostatic clip, as shown in Figure 15 and Figure 16.</li>
	<li>Insert the endoscope with the sensor in the clip in the standard way into the model.</li>
	<li>Position the clip with the sensor close to the lower esophageal sphincter.</li>
	<li>Rotate the endoscope against the esophageal wall, open the clip and then push toward the esophageal wall. Close the clip and release the clip. The sensor will remain attached to the esophageal wall at the desired location, as shown in Figure 17D and Figure 17E.</li>
	<li>Extract the endoscope.</li>
</ol>
6. Experiment after implantation
NOTE: The following steps require the use of chemicals. Study the material safety data sheets of the chemicals beforehand and use proper protective equipment and common lab practices when manipulating them.
<ol>
	<li>Place the receiver within 10 cm (maximum) of the implanted sensor.</li>
	<li>Inject 50 mL of the solutions with various pH values into the esophagus, as shown in Figure 18, and observe the changes in the sensor&#39;s response. Retract the endoscope after every injection and read the value no earlier than 30 s after injection. Wash the esophagus with 100 mL of deionized water between injecting solutions with different pH.</li>
	<li>Use the spreadsheet (Supplemental File 1) to calculate the pH measured by the sensor.</li>
</ol>

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(139), e57268 (2018).</li><li>Pavelka, M., Roth, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parietal+Cells+Of+Stomach:+Secretion+Of+Acid.">Parietal Cells Of Stomach: Secretion Of Acid.</a> Functional Ultrastructure. , 202-203 (2010).</li><li>Jones, R. D., Neuman, M. R., Sanders, G., Cross, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Miniature+antimony+pH+electrodes+for+measuring+gastroesophageal+reflux.">Miniature antimony pH electrodes for measuring gastroesophageal reflux.</a> The Annals of Thoracic Surgery. 33 (5), 491-495 (1982).</li><li>Waugh, R. W., Buted, R. 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Microsemi ZL70323 MICS-band RF miniaturized standard implant module (MiniSIM) Available from: <a target="_blank" href="https://www.microsemi.com/document-portal/doc_download/135307-zl70323-datasheet">https://www.microsemi.com/document-portal/doc_download/135307-zl70323-datasheet</a> (2015)</li></ol>

The authors have nothing to declare.

This method is suitable for researchers who work on the development of novel active implantable medical devices. It requires a level of proficiency in the manufacturing of electronic prototypes with surface mount components. The critical steps in the protocol are related to the manufacturing of the electronics, especially populating the PCBs, which is prone to operator error in placement and soldering of small components. Then, correct encapsulation is crucial to prolong the lifetime of the device when exposed to moistur...

The Montreal Consensus defines gastroesophageal reflux disease (GERD) as &#34;a condition that develops when refluxing the contents of the stomach causes unpleasant symptoms and/or complications&#34;. It may be associated with other specific complications such as esophageal strictures, Barrett&#39;s esophagus, or esophageal adenocarcinoma. GERD affects approximately 20% of the adult population, mainly in countries with high economic status1.
Ambulatory pH monitoring of pathological reflux (acid exposure time of more than 6%) allows us to distinguish the relationship between symptoms and acidic or non-acidic gastroesophageal reflux2,3. In patients unresponsive to PPI (proton pump inhibitor) therapy, pH monitoring can answer whether it is pathological gastroesophageal reflux and why the patient does not respond to standard PPI therapy. Various pH and impedance monitoring options are currently offered. One of the newer possibilities is wireless monitoring using implantable devices4,5.
GERD is associated with lower esophageal sphincter (LES) disorder, where the contractions shown during esophageal manometry are not pathological but have a reduced amplitude in long-term GERD. LES consists of smooth muscle and maintains tonic contractions due to myogenic and neurogenic factors. It relaxes due to vagal-mediated inhibition involving nitric oxide as a neurotransmitter6.
Electrical stimulation with two pairs of electrodes was proven to increase the contraction time of the LES in a canine reflux model7. The relaxation of the LES including the residual pressure during swallowing was not affected by both low and high frequency stimulation. High-frequency stimulation is an obvious choice because it requires less power and extends the battery life.
Although electrostimulation treatment (ET) of the lower esophageal sphincter is a relatively new concept in the treatment of patients with GERD, this therapy was shown to be safe and effective. This form of treatment has been shown to provide significant and lasting relief from the symptoms of GERD while eliminating the need for PPI treatment and reducing esophageal acid exposure8,9,10.
The current state-of-the-art pH sensor for diagnostics of GERD is the Bravo device11,12. At an estimated volume of 1.7 cm3, it can be implanted directly into the esophagus with or without visual endoscopic feedback and provides 24 h+ monitoring of pH in the esophagus.
Considering that electrostimulation therapy is one of the most promising alternatives for treating GERD not responding to standard therapy8,13, it makes sense to provide the data from the pH sensor to the neurostimulator. The recent research shows a clear path to future development in this field which will lead to rigid all-in-one implantable devices which will reside at the site of neurostimulation14,15. For this purpose, the ISFET (ion-sensitive field-effect transistor) is one of the best types of sensors because of its miniature nature, the possibility of on-chip integration of a reference electrode (gold in this case), and sufficiently high sensitivity. On silicon, the ISFET resembles the structure of a standard MOSFET (Metal Oxide Semiconductor Field Effect Transistor). However, the gate, normally connected to an electrical terminal, is replaced by a layer of active material in direct contact with the surrounding environment. In the case of pH-sensitive ISFETs, this layer is formed by silicon nitride (Si3N4)16.
The main disadvantage of endoscopically implantable devices is the inherent limitation of the battery size, which may lead to a reduced lifetime of these devices or motivate the manufacturers to develop advanced algorithms that will deliver the required effect at a lower energy cost. One of the examples of such an algorithm would be a closed-loop system for on-demand neurostimulation therapy of GERD. Similar to continuous glucose meters (CGM) + insulin pump systems17, such a system would employ an esophageal pH sensor or another sensor to detect the current pressure of the lower esophageal sphincter together with a neurostimulation unit.
The response to the neurostimulation therapy and the requirements for neurostimulation patterns can be individual13. Thus, it is important to develop independent sensors that could be used either for diagnosis and characterization of the dysfunction or to actively participate in calibrating the neurostimulation system according to the individual requirements of the patients18. These sensors should be as small as possible to not affect the normal functionality of the organ.
This manuscript describes a method of design and fabrication of an ISFET based pH sensor with amplitude-shift keying (ASK) transmitter and a small footprint passive rectenna-based receiver. Based on the simple architecture of the solution, the pH data can be received by an external receiver or even the implantable neurostimulator without any significant volume or power penalty. The ASK modulation is chosen because of the nature of the passive receiver, which is only capable of detection of received RF signal power (often called &#34;received signal strength&#34;). The schematic diagram, which is embedded as Supplementary material, shows the construction of the device. It is powered directly from two AG1 alkaline batteries, which provide a voltage between 2.0-3.0 V (based on the state of charge). The batteries power the internal microcontroller, which utilizes its ADC (analog-to-digital converter), DAC (digital-to-analog converter), internal operation amplifier, and FVR (fixed-voltage reference) peripherals to bias the ISFET pH sensor. The resulting &#34;gate&#34; voltage (the gold reference electrode) is proportional to the pH of the surrounding environment. A stable Ids current is provided by a low-side R2 sensing resistor. The source of the ISFET sensor is connected to the non-inverting input of the operational amplifier, while the inverting input is connected to the output voltage of the DAC module set to 960 mV. The output of the operational amplifier is connected to the drain pin of the ISFET. This operational amplifier regulates the drain voltage so that the voltage difference on the R2 resistor is always 960 mV; thus, a constant bias current of 29 &#181;A flows through the ISFET (when in normal operation). The gate voltage is then measured with an ADC. The microcontroller then powers on the RF transmitter via one of the GPIO (general purpose input/output) pins and transmits the sequence. The RF transmitter circuit involves a crystal and matching network which matches the output to 50 &#937; impedance.
For the experiments demonstrated here, we used a pig stomach with a long section of the esophagus mounted in a standardized plastic model. This is a commonly used model for practicing endoscopic techniques such as ESD (endoscopic submucosal dissection), POEM (oral endoscopic myotomy), endoscopic mucosal resection (EMR), hemostasis, etc. Concerning the closest possible anatomical parameters approaching human organs, we used the stomach and esophagus of pigs weighing 40-50 kg.

<table><tbody><tr><td>AG1 battery</td><td>Panasonic</td><td>SR621SW</td><td>Two batteries per one implant</td></tr><tr><td>Battery holder</td><td>MYOUNG</td><td>MY-521-01</td><td></td></tr><tr><td>Copper enamel wire for the antenna</td><td>pro-POWER</td><td>QSE Wire - 0.15 mm diameter, 38 SWG</td><td></td></tr><tr><td>Epoxy for encapsulation</td><td>Loctite</td><td>EA M-31 CL</td><td>Two-part medical-grade ISO10993 compliant epoxy</td></tr><tr><td>FEP cable for pH sensor</td><td>Molex / Temp-Flex</td><td>100057-0273</td><td></td></tr><tr><td>Flux cleaner</td><td>Shesto</td><td>UTFLLU05</td><td>Prepare 5% solution in deionized water for cleaning by sonication</td></tr><tr><td>Hemostatic clip</td><td>Boston Scientific</td><td>Resolution</td><td></td></tr><tr><td>Hot air gun + soldering iron</td><td>W.E.P.</td><td>Model 706</td><td>Any soldering iron capable of soldering with tin and hot-air gun capable of maintaining 260 &amp;deg;C can be used</td></tr><tr><td>Impedance matching software</td><td>Iowa Hills Software</td><td>Smith Chart</td><td>Can be downloaded from http://www.iowahills.com/9SmithChartPage.html - alternatively, any RF design software supports calculation of impedance matching components</td></tr><tr><td>ISFET pH sensor on a PCB</td><td>WinSense</td><td>WIPS</td><td>Order a model pre-mounted on a PCB with on-chip gold reference electrode</td></tr><tr><td>Laboratory pH meter</td><td>Hanna Instruments</td><td>HI2210-02</td><td>Used with HI1131B glass probe</td></tr><tr><td>Microcontorller programmer</td><td>Microchip</td><td>PICkit 3</td><td>Other PIC16 compatible programmers can be also used</td></tr><tr><td>Pig stomach with esophagus</td><td>Local pig farm</td><td>Obtained from approx. 40&amp;ndash;50 kg pig</td><td>It is important that the stomach includes a full length of the esophagus.</td></tr><tr><td>Printed circuit board - receiver</td><td>Choose preferred PCB supplier</td><td>According to pcb2.zip data</td><td>One layer, 0.8 mm thickness, FR4, no mask</td></tr><tr><td>Printed circuit board - sensor</td><td>Choose preferred PCB supplier</td><td>According to pcb1.zip data</td><td>Two-layer with PTH, 0.6 mm thickness, FR4, 2x mask</td></tr><tr><td>Receiver - 0R</td><td>Vishay</td><td>CRCW04020000Z0EDC</td><td>See Figure 12 and Figure 13 for placement</td></tr><tr><td>Receiver - 1.5 pF</td><td>Murata</td><td>GRM0225C1C1R5CA03L</td><td>See Figure 12 and Figure 13 for placement</td></tr><tr><td>Receiver - 100 pF</td><td>Murata</td><td>GRM0225C1E101JA02L</td><td>See Figure 12 and Figure 13 for placement</td></tr><tr><td>Receiver - 33 nH</td><td>Pulse Electronics</td><td>PE-0402CL330JTT</td><td>See Figure 12 and Figure13 for placement</td></tr><tr><td>Receiver - RF schottky diodes</td><td>MACOM</td><td>MA4E2200B1-287T</td><td>See Figure 12 and Figure 13 for placement</td></tr><tr><td>Receiver - SMA antenna</td><td>LPRS</td><td>ANT-433MS</td><td></td></tr><tr><td>Receiver - SMA connector</td><td>Linx Technologies</td><td>CONSMA001</td><td>See Figure 12 and Figure 13 for placement</td></tr><tr><td>Sensor - C1</td><td>Murata</td><td>GRM0225C1H8R0DA03L</td><td>8 pF 0402 capacitor</td></tr><tr><td>Sensor - C2</td><td>Murata</td><td>GRM0225C1H8R0DA03L</td><td>8 pF 0402 capacitor</td></tr><tr><td>Sensor - C3</td><td>Murata</td><td>GCM155R71H102KA37D</td><td>1 nF 0402 capacitor</td></tr><tr><td>Sensor - C4</td><td>Murata</td><td>GRM0225C1H1R8BA03L</td><td>1.8 pF</td></tr><tr><td>Sensor - C5</td><td>Vishay</td><td>CRCW04020000Z0EDC</td><td>Place 0R 0402 resistor or use to match the antenna</td></tr><tr><td>Sensor - C6</td><td>Murata</td><td>GRM155C81C105KE11J</td><td>1 uF 0402 capacitor</td></tr><tr><td>Sensor - C7</td><td>Murata</td><td>GRM155C81C105KE11J</td><td>1 uF 0402 capacitor</td></tr><tr><td>Sensor - C8</td><td>Murata</td><td>GRM022R61A104ME01L</td><td>100 nF 0402 capacitor</td></tr><tr><td>Sensor - IC1</td><td>Microchip</td><td>MICRF113YM6-TR</td><td>MICRF113 RF transmitter</td></tr><tr><td>Sensor - IC2</td><td>Microchip</td><td>PIC16LF1704-I/ML</td><td>PIC16LF1704 low-power microcontroller</td></tr><tr><td>Sensor - R1</td><td>Vishay</td><td>CRCW040210K0FKEDC</td><td>10 kOhm 0402 resistor</td></tr><tr><td>Sensor - R2</td><td>Vishay</td><td>CRCW040233K0FKEDC</td><td>33 kOhm 0402 resistor</td></tr><tr><td>Sensor - R3</td><td>Vishay</td><td>CRCW04021K00FKEDC</td><td>1 kOhm 0402 resistor</td></tr><tr><td>Sensor - R5</td><td>Vishay</td><td>CRCW040210K0FKEDC</td><td>10 kOhm 0402 resistor</td></tr><tr><td>Sensor - X1</td><td>ABRACON</td><td>ABM8W-13.4916MHZ-8-J2Z-T3</td><td>3.2 x 2.5 mm 13.4916 MHz 8 pF crystal</td></tr><tr><td>Titanium wire</td><td>Sigma-Aldrich</td><td>GF36846434</td><td>0.125 mm titanium wire</td></tr><tr><td>Vector network analyzer</td><td>mini RADIO SOLUTIONS</td><td>miniVNA Tiny</td><td>Other vector network analyzers can be used - the required operation frequency is 300&amp;ndash;500 MHz, resolution bandwidth equal or lower than 1 MHz, output power of no more than 0 dBm and dynamic range preferably better than 60 dB for the receiving front-end</td></tr></tbody></table>

construction of a wireless-enabled endoscopically implantable sensor for ph monitoring with zero-bias schottky diode-based receiver

Ambulatory pH monitoring of pathological reflux is an opportunity to observe the relationship between symptoms and exposure of the esophagus to acidic or non-acidic refluxate. This paper describes a method for the development, manufacturing, and implantation of a miniature wireless-enabled pH sensor. The sensor is designed to be implanted endoscopically with a single hemostatic clip. A fully passive rectenna-based receiver based on a zero-bias Schottky diode is also constructed and tested. To construct the device, a two-layer printed circuit board and off-the-shelf components were used. A miniature microcontroller with integrated analog peripherals is used as an analog front end for the ion-sensitive field-effect transistor (ISFET) sensor and to generate a digital signal which is transmitted with an amplitude shift keying transmitter chip. The device is powered by two primary alkaline cells. The implantable device has a total volume of 0.6 cm3 and a weight of 1.2 grams, and its performance was verified in an ex vivo model (porcine esophagus and stomach). Next, a small footprint passive rectenna-based receiver which can be easily integrated either into an external receiver or the implantable neurostimulator, was constructed and proven to receive the RF signal from the implant when in proximity (20 cm) to it. The small size of the sensor provides continuous pH monitoring with minimal obstruction of the esophagus. The sensor could be used in routine clinical practice for 24/96 h esophageal pH monitoring without the need to insert a nasal catheter. The &#34;zero-power&#34; nature of the receiver also enables the use of the sensor for automatic in-vivo calibration of miniature lower esophageal sphincter neurostimulation devices. An active sensor-based control enables the development of advanced algorithms to minimize the used energy to achieve a desirable clinical outcome. One of the examples of such an algorithm would be a closed-loop system for on-demand neurostimulation therapy of gastroesophageal reflux disease (GERD).

A device capable of autonomous pH sensing and wireless transmitting of the pH value was successfully constructed, as shown in Figure 8. The constructed device is a miniature model; it weighs 1.2 g and has a volume of 0.6 cm3. The approximate dimensions are 18 mm x 8.5 mm x 4.5 mm. As shown in Figure&#160;15, Figure&#160;16, and Figure&#160;17, it can be implanted to the proxi...

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Construction of a Wireless-Enabled Endoscopically Implantable Sensor for pH Monitoring with Zero-Bias Schottky Diode-based Receiver

The manuscript presents a miniature implantable pH sensor with ASK modulated wireless output together with a fully passive receiver circuit based on zero-bias Schottky diodes. This solution can be used as a basis in the development of in vivo calibrated electrostimulation therapy devices and for ambulatory pH monitoring.

Ambulatory pH monitoring of pathological reflux is an opportunity to observe the relationship between symptoms and exposure of the esophagus to acidic or ...

construction-wireless-enabled-endoscopically-implantable-sensor-for

Research

JoVE Journal

Bioengineering

2.5K Views.  Charles University. Het protocol toont de fabricage van draadloze implanteerbare pH-sensoren die gegevens verzenden naar een volledig passieve ontvanger, waardoor zeer energie-efficiënte levering van gegevens van het ene implanteerbare apparaat naar het andere mogelijk is. De sensor kan met de hand en met algemeen beschikbare technieken worden vervaardigd. Vervolgens kan de methode van gegevensoverdracht direct worden hergebruikt bij de ontwikkeling van verschillende implanteerbare apparaten.Het is van c...

Video: Construction of a Wireless-Enabled Endoscopically Implantable Sensor for pH Monitoring with Zero-Bias Schottky Diode-based Receiver

Measurement of Bioelectric Current with a Vibrating Probe

Electric fields, generated by active transport of ions, are present in many biological systems and often serve important functions in tissues and organs. For example, they play an important role in directing cell migration during wound healing. Here we describe the manufacture and use of ultrasensitive vibrating probes for measuring extracellular electric currents. The probe is an insulated, sharpened metal wire with a small platinum-black tip (30-35 &mu;m), which can detect ionic currents in the &mu;A/cm2 range in physiological saline. The probe is vibrated at about 200 Hz by a piezoelectric bender. In the presence of an ionic current, the probe detects a voltage difference between the extremes of its movement. A lock-in amplifier filters out extraneous noise by locking on to the probe's frequency of vibration. Data are recorded onto computer. The probe is calibrated at the start and end of experiments in appropriate saline, using a chamber which applies a current of exactly 1.5 &mu;A/cm2. We describe how to make the probes, set up the system and calibrate. We also demonstrate the technique of cornea measurement, and show some representative results from different specimens (cornea, skin, brain).

The manufacture, calibration and use of non-invasive vibrating probes to measure bioelectric current in various biological systems is described.

Electric fields, generated by active transport of ions, are present in many biological systems and often serve important functions in tissues and organs. ...

Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals suffering from chronic medical conditions facile assessment of their immediate physiological state. Furthermore, it could serve as a research tool for analysis of complex, multifactorial medical conditions. In order for such a multianalyte sensor to be realized, it must be minimally invasive, sampling of interstitial fluid must occur without pain or harm to the user, and analysis must be rapid as well as selective.
Initially developed for pain-free drug delivery, microneedles have been used to deliver vaccines and pharmacologic agents (e.g., insulin) through the skin.1-2 Since these devices access the interstitial space, microneedles that are integrated with microelectrodes can be used as transdermal electrochemical sensors. Selective detection of glucose, glutamate, lactate, hydrogen peroxide, and ascorbic acid has been demonstrated using integrated microneedle-electrode devices with carbon fibers, modified carbon pastes, and platinum-coated polymer microneedles serving as transducing elements.3-7,8
This microneedle sensor technology has enabled a novel and sophisticated analytical approach for in situ and simultaneous detection of multiple analytes. Multiplexing offers the possibility of monitoring complex microenvironments, which are otherwise difficult to characterize in a rapid and minimally invasive manner. For example, this technology could be utilized for simultaneous monitoring of extracellular levels of, glucose, lactate and pH,9 which are important metabolic indicators of disease states7,10-14 (e.g., cancer proliferation) and exercise-induced acidosis.15

This article details the construction of a multiplexed microneedle-based sensor. The device is being developed for in situ sampling and electrochemical analysis of multiple analytes in a rapid and selective manner. We envision clinical medicine and biomedical research uses for these microneedle-based sensors.

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals ...

A New Single Chamber Implantable Defibrillator with Atrial Sensing: A Practical Demonstration of Sensing and  Ease of Implantation

Implantable cardioverter-defibrillators (ICDs) terminate ventricular tachycardia (VT) and ventricular fibrillation (VF) with high efficacy and can protect patients from sudden cardiac death (SCD). However, inappropriate shocks may occur if tachycardias are misdiagnosed. Inappropriate shocks are harmful and impair patient quality of life. The risk of inappropriate therapy increases with lower detection rates programmed in the ICD. Single-chamber detection poses greater risks for misdiagnosis when compared with dual-chamber devices that have the benefit of additional atrial information. However, using a dual-chamber device merely for the sake of detection is generally not accepted, since the risks associated with the second electrode may outweigh the benefits of detection. Therefore, BIOTRONIK developed a ventricular lead called the LinoxSMART S DX, which allows for the detection of atrial signals from two electrodes positioned at the atrial part of the ventricular electrode. This device contains two ring electrodes; one that contacts the atrial wall at the junction of the superior vena cava (SVC) and one positioned at the free floating part of the electrode in the atrium. The excellent signal quality can only be achieved by a special filter setting in the ICD (Lumax 540 and 740 VR-T DX, BIOTRONIK). Here, the ease of implantation of the system will be demonstrated.

Dual-chamber implantable cardioverter-defibrillators (ICDs) may improve detection of atrial fibrillation as well as differentiation of tachycardias. However, this advantage is undermined by complications associated with the second electrode, which is required in conventional dual chamber devices. Therefore, BIOTRONIK has developed a new electrode called the LinoxSMART S DX that, when used in conjunction with the Lumax DX ICD, offers dual-chamber detection without the risks associated with the second electrode.

Implantable cardioverter-defibrillators (ICDs) terminate ventricular tachycardia (VT) and ventricular fibrillation (VF) with high efficacy and can protect ...

Medicine

Sensing of Barrier Tissue Disruption with an Organic Electrochemical Transistor

The gastrointestinal tract is an example of barrier tissue that provides a physical barrier against entry of pathogens and toxins, while allowing the passage of necessary ions and molecules. A breach in this barrier can be caused by a reduction in the extracellular calcium concentration. This reduction in calcium concentration causes a conformational change in proteins involved in the sealing of the barrier, leading to an increase of the paracellular flux. To mimic this effect the calcium chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N&#39;,N&#39;-tetra acetic acid (EGTA) was used on a monolayer of cells known to be representative of the gastrointestinal tract. Different methods to detect the disruption of the barrier tissue already exist, such as immunofluorescence and&#160;permeability assays. However, these methods are time-consuming and costly and not suited to dynamic or high-throughput measurements. Electronic methods for measuring barrier tissue integrity also exist for measurement of the transepithelial resistance (TER), however these are often costly and complex. The development of rapid, cheap, and sensitive methods is urgently needed as the integrity of barrier tissue is a key parameter in drug discovery and pathogen/toxin diagnostics. The organic electrochemical transistor (OECT) integrated with barrier tissue forming cells has been shown as a new device capable of dynamically monitoring barrier tissue integrity. The device is able to measure minute variations in ionic flux with unprecedented temporal resolution and sensitivity, in real time, as an indicator of barrier tissue integrity. This new method is based on a simple device that can be compatible with high throughput screening applications and fabricated at low cost.

The Organic Electrochemical Transistor is integrated with live cells and used to monitor ion flux across the gastrointestinal epithelial barrier. In this study, an increase in ion flux, related to disruption of tight junctions, induced by the presence of the calcium chelator EGTA (ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetra acetic acid), is measured.

The gastrointestinal tract is an example of barrier tissue that provides a physical barrier against entry of pathogens and toxins, while allowing the ...

Manufacturing of a Nafion-coated, Reduced Graphene Oxide/Polyaniline Chemiresistive Sensor to Monitor pH in Real-time During Microbial Fermentation

Here, we report the engineering of a solid-state micro pH sensor based on polyaniline-functionalized, electrochemically reduced graphene oxide (ERGO-PA). Electrochemically reduced graphene oxide acts as the conducting layer and polyaniline acts as a pH-sensitive layer. The pH-dependent conductivity of polyaniline occurs by doping of holes during protonation and by the dedoping of holes during deprotonation. We found that an ERGO-PA solid-state electrode was not functional as such in fermentation processes. The electrochemically active species that the bacteria produce during the fermentation process interfere with the electrode response. We successfully applied Nafion as a proton-conducting layer over ERGO-PA. The Nafion-coated electrodes (ERGO-PA-NA) show a good sensitivity of 1.71 &#937;/pH (pH 4 - 9) for chemiresistive sensor measurements. We tested the ERGO-PA-NA electrode in real-time in the fermentation of Lactococcus lactis. During the growth of L. lactis, the pH of the medium changed from pH 7.2 to pH 4.8 and the resistance of the ERGO-PA-NA solid-state electrode changed from 294.5 &#937; to 288.6 &#937; (5.9 &#937; per 2.4 pH unit). The pH response of the ERGO-PA-NA electrode compared with the response of a conventional glass-based pH electrode shows that reference-less solid-state microsensor arrays operate successfully in a microbiological fermentation.

Here, we report the protocol for the fabrication of a Nafion-coated, polyaniline-functionalized, electrochemically reduced graphene oxide chemiresistive micro pH sensor. This chemiresistor-based, solid-state micro pH sensor can detect pH changes in real-time during a Lactococcus lactis fermentation process.

Here, we report the engineering of a solid-state micro pH sensor based on polyaniline-functionalized, electrochemically reduced graphene oxide (ERGO-PA). ...

Autonomous and Rechargeable Microneurostimulator Endoscopically Implantable into the Submucosa

Gastric dysmotility can be a sign of common diseases such as longstanding diabetes mellitus. It is known that the application of high-frequency low-energetic stimulation can help to effectively moderate and alleviate the symptoms of gastric dysmotility. The goal of the research was the development of a miniature, endoscopically implantable device to a submucosal pocket. The implantable device is a fully customized electronic package which was specifically designed for the purpose of experiments in the submucosa. The device is equipped with a lithium-ion battery which can be recharged wirelessly by receiving an incident magnetic field from the charging/transmitting coil. The uplink communication is achieved in a MedRadio band at 432 MHz. The device was endoscopically inserted into the submucosal pocket of a live domestic pig used as an in vivo model, specifically in the stomach antrum. The experiment confirmed that the designed device can be implanted into the submucosa and is capable of bidirectional communication. The device can perform bipolar stimulation of muscle tissue.

The application of high-frequency low-energetic stimulation can alleviate the symptoms of gastric dysmotility. In this research, a miniature, endoscopically implantable and wirelessly rechargeable device which is implanted into a submucosal pocket is presented. Successful both-way communication and stimulation control were achieved during an experiment on live pig.

Gastric dysmotility can be a sign of common diseases such as longstanding diabetes mellitus. It is known that the application of high-frequency ...

Neuroscience

Implantation and Control of Wireless, Battery-free Systems for Peripheral Nerve Interfacing

Peripheral nerve interfaces are frequently used in experimental neuroscience and regenerative medicine for a wide variety of applications. Such interfaces can be sensors, actuators, or both. Traditional methods of peripheral nerve interfacing must either tether to an external system or rely on battery power that limits the time frame for operation. With recent developments of wireless, battery-free, and fully implantable peripheral nerve interfaces, a new class of devices can offer capabilities that match or exceed those of their wired or battery-powered precursors. This paper describes methods to (i) surgically implant and (ii) wirelessly power and control this system in adult rats. The sciatic and phrenic nerve models were selected as examples to highlight the versatility of this approach. The paper shows how the peripheral nerve interface can evoke compound muscle action potentials (CMAPs), deliver a therapeutic electrical stimulation protocol, and incorporate a conduit for the repair of peripheral nerve injury. Such devices offer expanded treatment options for single-dose or repeated dose therapeutic stimulation and can be adapted to a variety of nerve locations.

This is a protocol for the surgical implantation and operation of a wirelessly powered interface for peripheral nerves. We demonstrate the utility of this approach with examples from nerve stimulators placed on either the rat sciatic or phrenic nerve.

Peripheral nerve interfaces are frequently used in experimental neuroscience and regenerative medicine for a wide variety of applications. Such interfaces ...

 Fabricating Epimysial EMG Electrodes for Preclinical Rodent Models 

Development of a Low-cost Epimysial Electromyography Electrode: A Simplified Workflow for Fabrication and Testing

Electromyography (EMG) is a valuable diagnostic tool for detecting neuromuscular abnormalities. Implantable epimysial electrodes are commonly used to measure EMG signals in preclinical models. Although classical resources exist describing the principles of epimysial electrode fabrication, there is a sparsity of illustrative information translating electrode theory to practice. To remedy this, we provide an updated, easy-to-follow guide on fabricating and testing a low-cost epimysial electrode. 
Electrodes were made by folding and inserting two platinum-iridium foils into a precut silicone base to form the contact surfaces. Next, coated stainless steel wires were welded to each contact surface to form the electrode leads. Lastly, a silicone mixture was used to seal the electrode. Ex vivo testing was conducted to compare our custom-fabricated electrode to an industry standard electrode in a saline bath, where high levels of signal agreement (sine [intraclass correlation - ICC= 0.993], square [ICC = 0.995], triangle [ICC = 0.958]), and temporal-synchrony (sine [r = 0.987], square [r = 0.990], triangle [r= 0.931]) were found across all waveforms. Low levels of electrode impedance were also quantified via electrochemical impedance spectroscopy. 
An in vivo performance assessment was also conducted where the vastus lateralis muscle of a rat was surgically instrumented with the custom-fabricated electrode and signaling was acquired during uphill and downhill walking. As expected, peak EMG activity was significantly lower during downhill walking (0.008 &plusmn; 0.005 mV) than uphill (0.031 &plusmn; 0.180 mV, p = 0.005), supporting the validity of the device. The reliability and biocompatibility of the device were also supported by consistent signaling during level walking at 14 days and 56 days post implantation (0.01 &plusmn; 0.007 mV, 0.012 &plusmn; 0.007 mV respectively; p &gt; 0.05) and the absence of histological inflammation. Collectively, we provide an updated workflow for the fabrication and testing of low-cost epimysial electrodes.

Author Spotlight: Epimysial Electrode Fabrication and Testing in ACL Injury Studies

Our purpose was to provide an updated, easy-to-follow guide on the fabrication and testing of epimysial electromyography electrodes. To that end, we provide instructions for material sourcing and a detailed walkthrough of the fabrication and testing process.

Electromyography (EMG) is a valuable diagnostic tool for detecting neuromuscular abnormalities. Implantable epimysial electrodes are commonly used to ...

Spinal Cord Surgery and Optical Shank Implantation in Rodents

Implantation of Optoelectronic Devices in the Rodent Spinal Cord

Neuromodulation can provide diagnostic, modulatory, and therapeutic applications. While extensive work has been conducted in the brain, modulation of the spinal cord remains relatively unexplored. The inherently delicate and mobile spinal cord tissue imposes constraints that make the precise implantation of neural probes challenging. Despite recent advances in neuromodulation devices, particularly flexible bioelectronics, opportunities to expand their use in the spinal cord have been limited by the surgical complexities of device implantation. Here, we provide a series of surgical protocols tailored specifically for the implantation of a custom-made optoelectronic device that interfaces with the spinal cord in rodents. The steps to place and anchor an optical shank on a specific segment of the spinal cord via two different surgical implantation methods are detailed here. These methods are optimized for a diverse range of devices and applications, which may or may not require direct contact with the spinal cord for optical stimulation. To elucidate the methodology, the vertebral anatomy is referenced first to identify prominent landmarks before making a skin incision. The surgical steps to secure an optical shank over the cervical spine in rodents are demonstrated. Procedures are then outlined for securing the optoelectronic device connected to the optical shank in a subcutaneous space away from the spinal cord, minimizing unnecessary direct contact. Behavioral studies comparing animals receiving the implants to those undergoing sham surgeries indicate that the optical shanks did not adversely affect hindlimb or forelimb function seven days post-implantation. The present work broadens the neuromodulation toolkit for use in future studies aimed at investigating various spinal cord interventions.

Author Spotlight: Innovative Methodology for Implanting and Securing Neural Probes in the Rodent Spinal Cord

This protocol details a surgical procedure for performing spinal cord surgery and for implanting and securing an optical shank over the spinal cord in rodents.

Neuromodulation can provide diagnostic, modulatory, and therapeutic applications. While extensive work has been conducted in the brain, modulation of the ...

Real-Time Telemetric Impedance Measurements During Cochlear Implantation

Performing Repeated Intraoperative Impedance Telemetry Measurements during Cochlear Implantation

Impedance measurements are routinely performed during cochlear implantation (CI) after finalized electrode insertion. They may allow conclusions on the electrode's and implant's function. In the postoperative setting, the analysis of impedance changes enables the identification of scarring or inflammation processes around the electrode. Recent studies report associations between impedance telemetry and the site of stimulation. Consequently, repeated impedance measurements during cochlear implant electrode insertion may enable objective feedback on whether the electrode is positioned inside the perilymph or outside the inner ear. With the presented novel method, impedances can be measured in real-time during cochlear implantation. This protocol systematically explains how to perform repeated impedance recordings during CI surgery. These repeated measurements are challenging since they depend on multiple intraoperative methodological factors starting with the draping of the patient. Thus, for successful recordings, a standardized procedure is mandatory. In this article, we comprehensively illustrate the system setup and procedure of performing intraoperative measurements during CI surgery.

Author Spotlight: Advancements in Impedance Monitoring for Cochlear Implant Surgery

Here we present a protocol to conduct repeated impedance telemetry measurements during cochlear implantation (CI). They may allow conclusions on the electrode's and implant's function. Repeated impedance measurements enable objective feedback on whether the electrode is positioned inside the perilymph or outside the inner ear.

Impedance measurements are routinely performed during cochlear implantation (CI) after finalized electrode insertion. They may allow conclusions on the ...

Construction of a Wireless-Enabled Endoscopically Implantable Sensor for pH Monitoring with Zero-Bias Schottky Diode-based Receiver

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