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

Summary

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.

Abstract

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 "zero-power" 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).

Introduction

The Montreal Consensus defines gastroesophageal reflux disease (GERD) as "a condition that develops when refluxing the contents of the stomach causes unpleasant symptoms and/or complications". It may be associated with other specific complications such as esophageal strictures, Barrett'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 reflux^2,3. 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 devices^4,5.

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 exposure^8,9,10.

The current state-of-the-art pH sensor for diagnostics of GERD is the Bravo device^11,12. 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 therapy^8,13, 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 neurostimulation^14,15. 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 "received signal strength"). 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 "gate" voltage (the gold reference electrode) is proportional to the pH of the surrounding environment. A stable I_ds 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 µA 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 Ω 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.

Protocol

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 "3R" 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.

1. Place the ISFET pH sensor mounted on a printed circuit board (PCB) on a flat surface. Locate the solderable contacts.
2. Trim the solderable contacts, so their length is no longer than 3 mm.
3. 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.
4. 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.
5. Clean the pH sensor assembly in an ultrasonic cleaner for 5 min at 70 °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.
6. Rinse the pH sensor assembly in technical grade isopropyl alcohol for at least 3 min and let it dry in an oven at 80 °C for 15 min.
7. Place all pH sensors on a flat surface (in case multiple are prepared simultaneously) before proceeding to the next step.
8. 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
9. Transfer the mixed epoxy to a 1 mL syringe with a 0.5 mm flat end needle.
10. Coat the soldering area of pH sensors with epoxy. Make sure to coat the whole area of PCB electrodes and the exposed wire.
11. Let the epoxy cure either at room or elevated temperature (80 °C max), for this study 50 °C was used with the epoxy listed in the Table of Materials.
12. 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.
13. Trim the wires to the length and angle shown in Figure 2. Coat the ends with solder to avoid fraying.

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.

1. Place the PCB (manufactured based on the supplementary files "pcb1.zip" and schematic diagram "schematic.png") on a flat surface, components side up.
2. Apply solder paste to all the exposed gold-plated pads.
3. Place all passive and active components using tweezers according to Figure 3 and the Table of Materials.
4. Heat the PCB with the hot air gun to solder the components. Heat the PCB gradually to 150 °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 °C to solder the components. Let the PCB cool to room temperature, do not move it during the whole soldering process.
5. 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.
6. Repair any shorts or incorrect component placement with a soldering gun or hot air gun. Go to step 2.5.
7. Solder 5 wires to the components (power and programming leads) as shown in Figure 4.
8. To connect the PCB to the programmer, connect the wires soldered in step 2.7. to the connector of the programmer.
9. 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.
10. 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.
11. Put the PCB on a flat surface, component side down.
12. Solder two battery holders to the opposite part of PCB, as shown in Figure 6.
13. Solder the pH sensor assembly to the terminals on the PCB, as shown in Figure 7.
14. 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.
15. Prepare an appropriate amount of epoxy as described in step 1.8. for encapsulation of the device.
16. 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 °C because of the presence of batteries). See Figure 8 for the correct encapsulation results.
17. 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.
18. 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 °C maximum). The pH sensor is located on the bottom left side of the implantable device.
19. The sensor becomes activated 24 h after the insertion of the batteries. Meanwhile, proceed with step 3.
    ​NOTE: Pause the protocol now if completion of step 3 within 24 h after insertion of the batteries is possible.

3. Fabrication of passive rectenna receiver

1. Place the PCB (manufactured based on the supplementary file "pcb2.zip"). for the rectenna on a flat surface.
2. 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.
3. Solder the SMA connector to the PCB.
4. Inspect the PCB under a microscope. If any shorts or incorrect component placement is observed, fix the issues.
5. Attach a vector network analyzer input to the SMA connector.
6. 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.
7. Solder the impedance matching components and inspect under a microscope for short circuits and component placement.
8. 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.
9. Connect the 433 MHz band antenna to the SMA connector. Connect an oscilloscope to the rectenna output.
10. Set the oscilloscope to single-channel operation, rolling time base, DC mode, 500 ms/div time base, and 5 mV/div voltage scale.

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.

1. 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).
2. 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.
3. Verify the pH of all four beakers using a calibrated pH meter. Adjust if needed.
4. 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.
5. 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.

5. Endoscopic implantation of the sensor

1. Prepare an ex vivo endoscopic porcine model made up of the stomach and a long segment of the esophagus.
2. Grasp the sensor externally with a hemostatic clip, as shown in Figure 15 and Figure 16.
3. Insert the endoscope with the sensor in the clip in the standard way into the model.
4. Position the clip with the sensor close to the lower esophageal sphincter.
5. 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.
6. Extract the endoscope.

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.

1. Place the receiver within 10 cm (maximum) of the implanted sensor.
2. 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'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.
3. Use the spreadsheet (Supplemental File 1) to calculate the pH measured by the sensor.

Representative Results

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 15, Figure 16, and Figure 17, it can be implanted to the proximity of the lower esophageal sphincter with a single hemostatic clip; no special accessories are needed. A detailed view of a dissected esophagus with the sensor implanted is shown in Figure 19.

The passive rectenna receiver has an overall footprint of only 22 mm² even though it is optimized for hand-soldering. When the passive rectenna receiver is put into proximity of the pH sensing device (10 cm) when in an active state (24 h after insertion of batteries until full discharge of the batteries), clear voltage spikes can be observed when the device is transmitting. This is shown in Figure 13. The first two short (75 ms) pulses are synchronization pulses. The distance between the end of the second pulse and the beginning of the third pulse is proportional to the V_gs voltage of the ISFET subtracted by 800 mV (100 ms = 900 mV, 200 ms = 1000 mV, etc.). This voltage linearly translates to the pH of the environment that the sensor is subjected to.

Based on a simple two-point calibration with pH buffers of pH 4 and pH 10 (Table 1), the sensor can return stable and repeatable pH value readings (Table 2). A total of four different solutions with known pH were used-pH 0.6 (160 mM solution of hydrochloric acid in the water, mimicking the stomach acid²⁰) and calibration buffers with pH 4, pH 7, and pH 10. The mean error pH values of the sensor were 0.25 and 0.31 when tested in solutions in beakers and an ex vivo model, respectively. The standard deviations of the errors were 0.30 and 0.36, respectively.

When in the proximity of the transmitter (10 cm), the passive rectenna produces a signal with an amplitude of at least tens of millivolts which can be easily detected by a simple comparator or amplified with an ultra-low-power quiescent current operational amplifier. The effect of a mobile phone antenna with an active GSM call has only a minor negative effect on receiving the data from the sensor, as demonstrated in Figure 14. The mobile phone transmission peaks can be filtered by a simple passive RC/LC (resistor-capacitor/inductor-capacitor) filter as they form a high-frequency part of the signal (their frequency is generally above 500 Hz).

In one of the devices, a short circuit between all three of the ISFET electrodes was intentionally made to show how the device's behavior changes when the device is incorrectly assembled. In this case, no voltage-pH response is observed, and the gate voltage is equal to the drain voltage, which is the battery pack voltage (2-3.2 V). The AD converter, which is referenced to an internal 2.048 V reference, then returns the highest possible value, which translates to 2048 mV. Noise may cause slight fluctuations in the ADC output.

Two variants of firmware that can be programmed to the device were developed and tested. The first one (firmware_10s.zip) is intended for short-term experiments where the pH value is transmitted every 10 s. This provides more data points for the cost of reduced battery life, which is limited to around 24-30 h. The other one (firmware_1min.zip) is intended for long-term experiments. The pH value is transmitted once per min. The lifetime of the sensor with a lower sampling frequency is around 5-6 days. There is also a version of the firmware (firmware-test.zip), which does not include the 24 h delay. This file can be used for testing the correct functionality of the electronics before encapsulation. Alternatively, the delay can be modified by changing the code and recompiling the project. The delay was implemented to allow for a full cure of the epoxy or a possibility when the device is manufactured at a different site than the endoscopic surgery room. With the introduced delay, the useful operating life of the device is maximized.

Figure 1: pH sensor assembly before final trimming Please click here to view a larger version of this figure.

Figure 2: pH sensor assembly after final trimming Please click here to view a larger version of this figure.

Figure 3: Placement diagram for the implantable sensor (see Table of Materials for component values). Pin 1 is marked as a red dot. Please click here to view a larger version of this figure.

Figure 4: Placement of programming wires Please click here to view a larger version of this figure.

Figure 5: Placement of antenna wire and jumper wires Please click here to view a larger version of this figure.

Figure 6: Placement of battery holders Please click here to view a larger version of this figure.

Figure 7: Soldering of the pH sensor assembly to the electronics Please click here to view a larger version of this figure.

Figure 8: Finished encapsulated sensor. (A) side view, (B) back view Please click here to view a larger version of this figure.

Figure 9: Titanium wire hook Please click here to view a larger version of this figure.

Figure 10: Attachment of the wire hook to the implantable device Please click here to view a larger version of this figure.

Figure 11: Placement diagram for the rectenna. (A) with matching components, (B) without matching components, ready to be matched with a vector network analyzer Please click here to view a larger version of this figure.

Figure 12: Smith chart. (A) unmatched rectenna, (B) matched rectenna Please click here to view a larger version of this figure.

Figure 13: Example response of the rectenna to the incoming data from the sensor Please click here to view a larger version of this figure.

Figure 14: Example response when in the presence of RF noise (nearby phone with an active GSM call). (A) 20 cm between the edge of the phone and receiver, (B) 10 cm between the edge of the phone and receiver, (C) 5 cm between the edge of the phone and receiver Please click here to view a larger version of this figure.

Figure 15: Picture of the endoscope with hemostatic clip and implantable pH sensor Please click here to view a larger version of this figure.

Figure 16: Implantable pH sensor grasped with the hemostatic clip in a cap Please click here to view a larger version of this figure.

Figure 17: Implantation of the sensor. (A) insertion of the endoscope with the implantable pH sensor into the model, (B) place of implantation - 3 cm above the gastroesophageal junction, (C) preparation of the clip placement, (D) the clip was successfully placed, (E) view of the ISFET pH sensor, implanted to the proximity of lower esophageal sphincter Please click here to view a larger version of this figure.

Figure 18: Injection of the pH buffer solution through the endoscope channel Please click here to view a larger version of this figure.

Figure 19: Dissected esophagus of the ex vivo model with the implanted sensor Please click here to view a larger version of this figure.

Calibration data
pH value (cal. meter) [-]	Pulse length [ms]	Calc. volt. output [mV]
3.98	400	1200
10.01	710	1510

Table 1: Example calibration data

Measured data
pH value (cal. meter) [-]	Calc. volt. output [mV]	Estimated pH [-]	Error [abs. pH]	Error [%]
0.62	1010	0.28	-0.34	-54%
3.98	1200	3.98	0.00	0%
10.01	1490	9.62	-0.39	-4%
0.62	1020	0.48	-0.14	-23%
7.01	1350	6.90	-0.11	-2%
3.98	1220	4.37	0.39	10%
10.01	1480	9.43	-0.58	-6%
3.98	1210	4.17	0.19	5%
7.01	1350	6.90	-0.11	-2%
		Std. deviation of pH [-]	0.30
		Mean error [-]	0.25

Table 2: Measured data (test with beakers)

Measured data
pH value (cal. meter) [-]	Calc. volt. output [mV]	Estimated pH [-]	Error [abs. pH]	Error [%]
0.62	1010	0.28	-0.34	-54%
3.98	1220	4.37	0.39	10%
7.01	1340	6.70	-0.31	-4%
10.01	1520	10.20	0.19	2%
		Std. deviation of pH [-]	0.36
		Mean error [-]	0.31

Table 3: Measured data (test in an ex vivo model)

Supplemental File 1: spreadsheet.xlsx. Spreadsheet for calibrating and processing of the data from the sensor Please click here to download this File.

Supplemental File 2: pcb1.zip. Gerber manufacturing data for the implantable device Please click here to download this File.

Supplemental File 3: pcb2.zip. Gerber manufacturing data for the receiver Please click here to download this File.

Supplemental File 4: firmware_10s.zip. Firmware for the microcontroller with 10 s transmission period Please click here to download this File.

Supplemental File 5: firmware_1min.zip. Firmware for the microcontroller with 1 min transmission period Please click here to download this File.

Supplemental File 6: firmware-test.zip. Firmware for the microcontroller without 24 h pause before activation Please click here to download this File.

Supplemental File 7: Schematic diagram of the electronics Please click here to download this File.

Discussion

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 moisture and liquids. The implantation method was designed with simplicity in mind. The risk of perforation of the esophagus or other adverse events during the implantation is minimal. Hemostatic clips are widely used in clinical practice; thus, no special training is needed to perform the implantation.

The device can be easily modified to accompany other sensors with voltage output, i.e., resistive sensors and other ISFET sensors. This gives great flexibility to utilize the whole concept in other areas of research and clinical practice; it is not limited to research of novel methods of treatment of GERD in the case of a pH ISFET sensor.

The constructed device is miniature; it weighs 1.2 g and occupies 60% less volume (0.6 cm³) than the closest commercialized implantable pH sensor. Further miniaturization could be achieved by the integration of the ISFET onto the PCB with wires bonded directly to the PCB. This, however, would significantly increase the barrier of entry in terms of required equipment (it would require at least a manual wire bonder). Thus, a more economically viable alternative with a pre-packaged ISFET sensor by the manufacturer was presented.

As for the power source, silver oxide/alkaline/carbon-zinc 1.5 V cells provide better performance and do simplify the circuit design. The use of primary lithium batteries or Li-Ion batteries in this device form factor could lead to potential problems. Small primary lithium batteries have high output resistance, which would cause significant voltage drops, potentially leading to the brown-out of the microcontroller and RF transmitter. Lithium-ion batteries, on the other hand, are incompatible with 3.3 V microcontrollers (their operating voltage is around 3.0-4.2 V), adding complexity to the circuitry (requirement of a regulator or DC/DC step-down converter). For these reasons, two primary 1.5 V button cells are the best readily available type of battery based on the availability, operating voltage, and sufficiently low output resistance.

The sensor exhibits good accuracy for esophageal pH monitoring; the mean error of pH in an ex vivo model was 0.31 with a standard deviation of 0.36. Despite the washing step with deionized water between each buffer addition, a larger deviation in the ex vivo model could have been caused by minor mixing of the different buffer solutions in the esophagus, which may have altered the pH of the solutions. The sensitivity of the used ISFET pH sensor almost follows the Nernstian slope (-58 mV/pH for 25 °C) at -51.7 mV/pH. The sensitivity is higher than reported in antimony-based pH sensors for monitoring GERD (-45 mV/pH)²¹.

The delay of 24 h between the insertion of batteries and the start of the wireless transmission routine was introduced to accommodate for encapsulation epoxy curing and instances where the lab for manufacturing of electronics is present at a different location than the endoscopic surgery room. This delay can be altered by modifying the source code and recompiling the firmware.

Depending on the nature of the experiment, which will be done by the researchers, suitable epoxy (cost versus performance) can be chosen. The initial experiments were done with automotive-grade epoxy, which was suitable for initial experiments but not for in vivo experiments from the point of biocompatibility. For survival experiments, a medical-grade epoxy that is ISO10993 compliant for long-term contact with mucous membranes shall be chosen. Also, coatings that improve biocompatibility (e.g., PTFE or parylene) can further reduce the rejection rate of the implant and/or inflammation/irritation of the implantation site.

The fully passive rectenna receiver can be improved by biasing the detector diodes to improve the sensitivity^22,23. In case that improved immunity against electromagnetic interference or RF noise is required, the diode detector can be further modified by adding a highly selective band SAW filter between the RF input and diode detector²⁴. If longer-range communication is required, an active ASK receiver (or a software-defined receiver - SDR) can be used. In both cases, the center frequency of the receiver shall be set to 431.73 MHz (frequency of the crystal multiplied by 32 by the PLL in the RF transmitter integrated circuit) and the resolution bandwidth of around 150-250 kHz. The RF output frequency is both voltage and temperature-dependent, and drifts up to 50 kHz from the center frequency were observed during normal operation. The output power in the band can then be monitored and used to decode the pH value according to the protocol. The use of an active receiver is recommended for initial testing. If used inside an implantable device, it comes with an increase in complexity and a major energy penalty. It cannot provide the "zero-power" advantage that the Schottky detector provides.

Today, virtually all active implantable medical devices are not designed with interoperability in mind. Their configuration is done manually by a surgeon or practitioner²⁵ and does not cooperate. The implantable device presented in this method together with a passive rectenna receiver, shows a way to realize seamless data transfer from a disposable sensor to another implantable device. While commercially available RF modules for implantable devices based on the heterodyne concept exist, the receiver mode is very power demanding²⁶. With the presented solution, no active receiver in the neurostimulator is required; the circuit can be built to be completely passive. The main advantages of taking real-time patient data into account are to improve the efficacy of the therapy and significantly lower the power consumption. For example, in the case of GERD therapy, a pH sensor presented in the manuscript can be implanted above the lower esophageal sphincter after the implantation of the stimulator to automatically adapt the neurostimulation pattern to maximize the effect of the therapy while minimizing the power consumption. As the implantation of the sensor to the inner esophageal wall is prone to dislocation after several days, it makes more sense to design the sensor as a battery-powered one. Thanks to the higher volumetric energy density of primary batteries, the use of a primary power source is superior to a sensor that contains a wireless power receiving circuit, charging coil, and capacitor-based energy storage. The overall efficiency of the wireless charging is also heavily dependent on the spatial orientation of the coils, which would introduce yet another difficulty to the design. Wireless charging provides benefits to the permanently implanted microneurostimulators, i.e., to the submucosa¹⁴. The battery-powered pH sensor provides a possibility to optimize the energy consumption of such a microneurostimulator. Instead of permanent/regular neurostimulation of the sphincter, the pH sensor can show when the stimulation is needed (i.e., primarily at night and/or which hours of the day) and what power output is the lowest possible to achieve sufficient lower esophageal sphincter pressure. These closed-loop or quasi-closed-loop implantable systems can become a promising alternative to current traditional systems, offering smaller implantable devices with less-invasive implantation and improving the treatment's efficacy.

Materials

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