Overview

There are several significant barriers between our concept, biosensor, and the practical issues that synthetic biology projects generally tend to face. To overcome these and implement the project in the real world, a physical application was necessary. Thus, we built it. This is our "SAMURAI-Device."

Figure0-1

Figure0-2

Figure0-3

Figure0-4

This hardware is designed to automatically collect soil samples from the field, extract nitrogen using an elution, mix and react with a cell-free biosensor, measure the output, and wirelessly transmit the data. It is intended to perform these operations continuously over a long period. Additionally, the use of a lot of mechanical tricks keeps the number of electronic components used to a minimum, limited to low-cost microcontrollers, motors, and sensors, making it both cost-effective and energy-efficient. In other words, it can automatically perform the various operations required by the cell-free biosensor and soil sampling, enabling high-frequency monitoring without burdening the end-user. This meets the project's needs and solves the challenges of implementing synthetic biology in the real world.

Additionally, this device is modular, featuring an automatic soil sampling mechanism, a cost-effective quantitative liquid dispensing tube pump, and an automatic cartridge exchange mechanism that can handle various reaction procedures, expanding the applicability of cell-free systems. This versatility makes it a device of great significance for future iGEM teams and projects.

In the design of this device, the definition of the requirements was determined after Humapnractice with several experts to ensure that our project would be supported. This ensures that from the beginning we have all the features needed to make this project more suitable.

In the development process, repeating trial and error, we eventually made a prototype device that could function in an indoor environment. This prototype had all the major mechanisms in place, and all of its core functions were confirmed to work and function. Further improvements are needed to achieve practical levels of accuracy and durability, but we have demonstrated that the device is fully functional and useful.

Prototypes and mock-ups were used to conduct user testing with several end users and nitrogen sensor development companies. This provided a great deal of feedback on usability and real-world functionality from an end users' perspective, as well as feedback on the mechanics and functionality of the device from a professional perspective. Based on this feedback, we made a number of improvements to the instrument and plans were developed for future improvements.

In order to implement the device and achieve the project goals in the future, we integrated user testing and stakeholder discussions, and compiled a discussion and social implementation roadmap for social implementation throughout the entire project. With necessary improvements, collection of operational data, and integration with biosensors and software, this device can be developed into a total solution to achieve nitrogen fertilizer management in practice.

Finally, we documented all information necessary to recreate and improve this device, including the 3D model of the device, electronic circuitry, and operating code, and posted in an easy-to-understand format.

Our device is going to be a powerful bridge for the implementation of our synthetic biology in the real world.

Requirement Definition

(1)It must have the capability to repeat the process from sampling to data transmission fully automatically.

(2)It should be able to collect soil samples from the ground and perform solution extraction using reagents.

(3)It must be capable of providing the sample solution to a cell-free biosensor, conducting necessary processing and reactions, measurement, and data transmission.

(4)It should be capable of long-term continuous operation in farmland (to reduce the operational burden on end-users).

(5)It must be affordable (to reduce the implementation cost for end-users).

(6)It should have sufficient measurement accuracy.

These requirements were determined by focusing on the realization of our project concept and fully integrating various opinions from experts, users, and stakeholders through Human Practice.

(1)

First, the necessity of conducting frequent measurements (approximately once a day) was raised to achieve the project goal. From our user tests last year (Kyoto 2023 Hardware) and others, it was evident that having farmers perform this high-frequency measurements manually would be a significant burden and unrealistic, and the SAMURAI device must have automated measurement capabilities. Initially, we aimed to achieve long-term continuous measurements using a living whole-cell sensor, but the project was revised to use a cell-free sensor due to the regulations under the Cartagena Protocol and imaginative negative concerns about introducing bacteria into farmland (PLANTX 1st). Furthermore, the need for fully automated measurement and data transmission for efficient operation on farmland was reaffirmed (Symbiobe 1st). requirement(1) was therefore developed.

(2)

Although we initially planned to avoid a soil sampling mechanism due to its lack of precedent and high difficulty, it turned out to be necessary to treat the soil with reagents such as KCl to extract nitrogen for the measurement of plant-available soil nitrogen content through conversations with several experts in soil nitrogen research. (Dr. FunakawaMr. Matsumoto). Therefore, we had to try sampling the soil itself and extracting nitrogen with extraction reagents built into the device. This is requirement(2).

(3)

Our (and almost all) cell-free systems cannot be reused once a reaction is complete due to deterioration or depletion of reagents. This makes continuous sensing with cell-free systems difficult and is one of the disadvantages compared to whole-cell sensors. However, due to the reasons mentioned above, both the adoption of a cell-free system and continuous sensing are necessary for our project. Therefore, we need to overcome this through the mechanism and function of the SAMURAI device, and the entire process from sampling to data transmission must be automated. This is requirement(3).

(4)

As mentioned above in (1), the devices need to operate continuously for a certain length of time because the high-frequency measurements we require would be a burden on farmers if they were to be performed manually.In addition to continuous automatic measurement for this, high durability, reagent holding capability, and ease of maintenance are required. This is requirement(4).

(5)

The device must actually be widely adopted. In fact, there is testimony that how reasonable devices are is the biggest constraint on the adoption of nitrogen sensors in Japan Horiba 1st. Also, affordability is essential in terms of business( Symbiobe 1st Horiba 2nd, Entrepreneurship). In this project, this is especially important because a large number of units will be required. This is requirement(5).

(6)

Finally, of course, there must be measurement capability. While it does not reach the level of laboratory measurement equipment, it must have sufficient accuracy to sense nitrogen concentrations at a level required for precise cultivation management. This aspect is also emphasized by Horiba, a manufacturer of existing nitrate and ammonium sensors (Horiba 2nd). In this case, due to a lack of research in terms of Modeling, Human practice, and Software, we could not obtain a clear benchmark, so we will strive to achieve the highest possible standard, as long as it does not significantly contradict requirement(5). This is requirement(6).

Conceptualization and Design

(1) Soil collection mechanism

(2) Tube pump

(3) Cartridge exchange and measurement mechanism

Soil collection mechanism collects soil from agricultural land, weighs it, and mixes it with a potassium chloride solution to extract nitrogen from the soil. Tube pump transports the eluate/reagents from Soil collection mechanism to the cartridge measurement and exchange mechanism. Cartridge exchange and measurement mechanism automatically carries out a series of processes, including the mixing and reaction of the eluate with the cell-free system, measurement and transmission via electronic sensors from the cell-free system, and the exchange of sensor cartridges.

The actual operation of each part will be described in detail in the "Demonstration of Device Functionality and Practicality" section.

（１）Soil Sampling Mechanism

Feedback from experts revealed that, in order to accurately measure nitrogen levels in farmland soil, it is necessary to directly collect soil and elute nitrogen using KCl solution, rather than collecting water that passes through the soil.

Therefore, we devised a method to efficiently collect and use soil while avoiding the enlargement of the hardware. The mechanism devised for soil sampling using simple movements involves rotating a drill-shaped nozzle in the soil to transfer the soil into the device. This method allowed collecting soil simply by operating a continuous rotation servo motor, enabling us to collect soil while keeping the device compact.

This is a modeling diagram of the drill nozzle. The more turns the drill nozzle has, the smaller the soil particles that can fit between the spiral plates, only fine soil particles are collected even if large gravel is contained in soil. This prevents the inclusion of stones and other things in the sample. Additionally, in clayey soil, where soil particles are fine and tend to clump together, reducing the number of turns decreases resistance from the soil when the nozzle rotates, contributing to more stable motor operation.

Figure1

Next, to ensure measurement accuracy, it is necessary to keep the volume of soil used as a sample. Simply collecting soil using the above method does not keep a consistent volume. Therefore, we created a mechanism to level off the soil volume and transfer it to the reaction chamber. This mechanism performs two functions using a single stepper motor, contributing to energy efficiency. The mechanism consists of a spoon and a cylinder combined vertically. When the cylinder slides horizontally relative to the spoon filled with soil, the soil is leveled off to the spoon's volume. As the cylinder slides, a planar gear also slides in sync, engaging with a gear on the spoon's shaft and the cylinder slides along the circumference. Then, the spoon is automatically flipped over, allowing the soil to be transferred to the reaction chamber located below the mechanism. Please refer to the video below for a detailed image of the operation.

Finally, a reaction chamber that conducts measurements accurately was created. We considered that any soil remaining in the reaction chamber from the previous measurement could affect the results, so we devised a way to ensure that all soil is completely discarded after each measurement. Specifically, we reduced residues by coating the inner walls of the reaction chamber with a powerful hydrogen-repellent material. By using aluminum foil that mimics the structure of lotus leaves which was developed by a Japanese company (TOYAL LOTUS, Toyo Aluminium Corporation) as a hydrogen-repellent material, we ensured that the chamber maintains a high level of water repellency over a long period.

This movie shows the power of the structure of lotus.It has been found to exhibit a high water-repellent effect even against muddy water.

Figure2-1

Figure2-2

Additionally, to reduce measurement errors caused by the soil's moisture content, we devised a structure that ensures the total volume of KCl solution mixed with the soil remains constant. By making an outlet halfway up the wall of the reaction chamber, any excess KCl solution above a certain volume flows out. This design enables accurate measurements regardless of the soil's moisture condition.

Figure3

When the water level rises to the gutter installed on the front wall, any additional water will be discharged.

（２）Tube Pump

To conduct accurate measurements at a low cost, it was necessary to create a tube pump capable of precise liquid transfer, powered by a continuous rotation servo motor. The operating principle of the tube pump is as follows.

Figure4

Past iGEM teams, such as Aachen2015/2017, have created excellent tube pumps, but they used expensive stepper motors and a variety of components, making them difficult to adopt under cost limitation. Therefore, we devised a compact tube pump using only a continuous rotation servo motor, 3D-printed parts, and an inexpensive tube. This pump is much more affordable and compact compared to those made by previous teams, yet it provides sufficiently accurate liquid transfer.

The biggest barrier in creating an affordable tube pump is the inability to use expensive, high-torque motors. The more a tube pump is designed to press the tube, the more torque is required to rotate it. Therefore, to achieve sufficient liquid transfer while pressing the tube firmly and rotating it with the torque of a continuous rotation servo motor, the conventional pump design did not work. To address this, we modified the shape of the rotor that presses the tube from a simple cylindrical shape used in the previous pump to a cylinder with a dent in the middle, as shown in the photo. This adjustment allowed us to achieve sufficient compression while maintaining stable rotation. Additionally, through trial and error, we discovered that the optimal number of rotors to ensure the most accurate liquid transfer in this design is four.

Figure5-1

Left: The specially designed rotating rotor we created

Figure5-2

Right: The final tube pump

（３）Cartridge Exchange and Measurement Mechanism

This mechanism is the part of this device that performs the most functions. It automatically performs everything from replacing the cartridge unit, injecting reagents and sampling liquids, to measurement and disposal. Typically, incorporating many functions autonomously requires numerous electronic components, but this increases the production cost and power consumption during operation inevitably. Therefore, we minimized the use of electronic components and focused on designing shapes of the parts in a way that enables many mechanisms to operate without using electricity.

In the prototype we created, measurements can be conducted using three cartridges at once. This allows simultaneous measurement of both NO₃⁻ and NH₄⁺, while the remaining lane can be used for BLANK measurements to improve accuracy or to handle new detectable substances.

Cartridge Unit

First, we describe the cartridge units used in this mechanism.It is essential that researchers using this mechanism can employ their desired cell-free systems for measurements. Therefore, we devised a cartridge unit that directly utilizes a standard PCR tube. The cartridge has a cylindrical shape with a PCR tube fitted into a cylindrical frame, making it suitable for the device to load and replace automatically. The bottom of the cylinder is open, allowing measurement of outputs such as fluorescence by irradiating excitation light from below. The cell-free biosensor is stored within the PCR tube, either freeze-dried or in a similar state that is suitable for long-term room temperature storage. The top of the PCR tube is sealed with aluminum foil instead of a lid, which blocks light, moisture, and microorganisms. During use, the injector tip simply breaks through the aluminum foil and injects the solution inside, causing the cell-free biosensor to dissolve, react, and output the results.

Figure6-1

Figure6-2

Figure6-3

Rotating Disk

This is the most important component of this multifunctional cartridge exchange and measurement mechanism. By rotating, it enables various functions to operate in a chain without using any additional energy.

In addition to the three cartridge loading holes for the 3-lane measurement, this disk has two specially shaped circumferential walls on its outer edge. The two differently shaped indentations on this circumferential wall act as a switch that activates the mechanism.

As the disk rotates, it moves the cartridge units through the sequence of (1) Loading Site → (2) Solution and Reagent Injection Site → (3) Measurement Site → (4) Disposal Site in a counterclockwise direction.

The following sections will explain the mechanisms that work at each of these four sites in this order.

Figure7-1

Figure7-2

(1)Loading Site

This site features a cartridge unit that autonomously and continuously exchanges cartridges. The mechanism operates without using energy, as the rotating disk facilitates the automatic exchange. It can be easily constructed using 3D-printed parts, common wood and plastic sheets, and sheet magnets.

The cartridge exchange mechanism consists of two main components : (1) the cartridge case (2) the stopper mechanism. The cartridge case is magnetically attached to the wooden fixture on the base.

The cartridge case is designed to hold the cartridge units, accommodating up to 30 units across three lanes for the nitrate sensor. It has a circular shape with a loading hole in one position, equipped with a guiding skirt. A cartridge-pushing component is attached, which moves along the circumference of the case.

The stopper mechanism consists of a part shaped like a clock hand, which has a plate that covers the loading hole of the cartridge case. The loading hole remains blocked by the stopper mechanism, while the next cartridge to be loaded sits above it. When the loading hole of the rotating disk aligns with the recess in the outer wall, the stopper is released at the moment the coordinates of the disk's loading hole and the stopper mechanism coincide. This allows the cartridge to fall into the disk's loading hole.

This mechanism operates by releasing the stopper, which is fixed along the outer wall, as the recess passes by. In other words, the system functions without any additional energy input as thedisk rotates.

However, a new cartridge cannot move to the loading hole of the cartridge case. This is because the cartridge case is installed horizontally, preventing the use of gravitational force. To address this problem, we developed an energy-free automatic cartridge filling mechanism.

The antenna mounted on the rotating disk catches the claw of the cartridge pusher (a soft plastic plate), moving the pusher along the circumference and pushing the cartridge forward. Once the cartridge has moved, the plastic plate bends, releasing its grip on the antenna. This ensures that the cartridge is always kept snugly at the end.

For more details on the mechanism, please refer to the video below.

(2)Solution and Reagent Injection Site

Since the top surface of the PCR tube is bonded with aluminum foil, the sample solution, which is a mixture of soil and KCl, and the reagents used for the reaction must be placed in the tube after breaking the aluminum foil.

For this reason, in this site, we used a mechanism in which an injection nozzle with a pointed tip is dropped from above, breaking through the aluminum foil and allowing the nozzle to be set inside the tube to inject the liquid. In this mechanism, we used an arm that moves up and down along the inside perimeter wall of the rotating disk. When the cartridge reaches this site, the arm descends along the recess in the outer wall, causing the attached nozzle to drop as well.

The dispensing mechanism dispenses an equal volume of solution or reagent from the tubing of the tube pump, which is then pumped directly through the tubing to the nozzle, so that liquid is injected when the nozzle is set and the pump is operated. The dispensing mechanism is explained in detail in the Demonstration of Device Functionality and Practicality chapter. This mechanism can be used to add new reagents or other substances to the cartridge if the same is installed in the period between (2) and (3).

Figure9-1

Figure9-2

(3)Measurement Site

A blue excitation light installed at the base of the rotating disk at the position of site (3) shines and illuminates the PCR tube in the cartridge from below. The output such as fluorescence generated by the light is then read from above by the spectral sensor AS7341. AS7341 is equipped with a light-shielding skirt for measurement to enhance the measurement accuracy. This mechanism becomes available when the cartridge is moved to site (3).

Figure10-1

Figure10-2

The AS7341 is mounted on the base attached to the top.

(4)Disposal Site

When the cartridges come to the site (4), they fall into a hole in the base, which completes the disposal process. The cartridges that fall into the hole enter and collect in a waste case below the hole, and the end users can remove this case to collect the waste cartridges.

https://video.igem.org/w/pKsd3jLuRGJZEPgbC1kX1m

Electronic Components

Here is a description of the electronic components used in this device. Additionally, I have provided explanations for the components that may require further clarification.

Stepper motor

The 28BYJ-48 5V stepper motor is an affordable option that allows for precise angle control during rotation. We use it as the driving power for the rotating disk in the cartridge exchange mechanism, where accurate angle specification is crucial.

Additionally, the cutting mechanism also requires a complete rotation of exactly 360° each time. Therefore, we control the stepper motor to ensure it moves precisely through one full rotation.

Survo motor

In this project, we used two types of servos: the normal servo motor SG90 and the continuous servo motor FS90. The SG90 is employed to flip the reaction chamber for the disposal of the soil contents.

The FS90 is chosen for its affordability, high torque, and ability to specify rotation speed. It is used to rotate the drill nozzle for soil extraction. Additionally, we leverage its variable rotation speed to control the liquid delivery rate of the tube pump, making the flow rate proportional to the rotation speed of the motor.

Arduino Uno R4 WiFi

By using Wi-Fi, we enable remote control capabilities for the device, making it an integral part of the development board for creating a concept centered around remote operation. Additionally, many electronic components are compatible with Arduino, so we recommend this platform to other teams looking to enhance or modify the device.

• Methods for Remote Data Transmission and Reception

In this project, we used the Arduino Uno R4 Wi-Fi, which allows for Wi-Fi connectivity without an additional Wi-Fi module. First, the program specifies the Wi-Fi router to connect to. By executing this program, the Arduino connects to the Wi-Fi network. Next, we programmed it to display the Arduino's IP address on the serial monitor. We then created an HTML interface to design the screen that will be displayed when accessing the IP address. This setup enables users to connect to the IP address shown on the serial monitor from any device. They can send commands via a web browser to execute the program written on the Arduino and display the data obtained from the Arduino on the browser.

Expansion Board

To control multiple servos with a single Arduino, we used the PCA9685 expansion board. Also, we employed the TCA9548A to manage two AS7341 sensors for measurements.

AS7341

This is a relatively inexpensive spectral sensor. In this project, the device's fluorescence detection is carried out using this sensor.

how to get data of AS7341

First, in order to use the AS7341 with the Arduino IDE, download the dedicated library,Adafruit from Library Manager. Once downloaded, copy the program from an example sketch, such as read_all_channels, and connect the AS7341 to the Arduino. Executing this program will display the data acquired from the AS7341 on the IDE's serial monitor. If there are any garbled characters, set the serial monitor's baud rate to 115200. By doing so, the data is shown correctly. You can check the methods defined in the Adafruit AS7341 library at https://github.com/adafruit/Adafruit_AS7341

how to operate two AS7341 simultaneously

The AS7341 communicates with the Arduino using I2C. When using two I2C devices, it's crucial to specify unique addresses in the program to prevent communication conflicts between the devices. However, the Adafruit AS7341 library does not provide a method for address specification, making it difficult to manage multiple sensors.

To resolve this issue, we utilized the TCA9548A multiplexer module. This module allows multiple devices to share the same I2C address without conflict, enabling separate communication with each AS7341 sensor. By using the TCA9548A, we can effectively operate two AS7341 sensors simultaneously without modifying the library or dealing with address conflicts.

Demonstration of Device Functionality and Practicality

（１）Tube Pump

• We demonstrated that the continuous servo motor can be used as a power source to achieve sufficient liquid delivery.

（２）Soil Gathering Mechanism

• We demonstrated the ability to collect soil using typical garden soil.
• We confirmed that the collected soil can be homogenized to a consistent volume.
• We verified that it can be mixed with water in the reaction chamber.
• We showed that water can be extracted from the reaction chamber using a tube pump.
• We confirmed that the contents of the reaction chamber can be disposed of, leaving minimal soil or water remaining inside.

（３）Cartridge Exchange and Measurement Mechanism

• We demonstrated that the cartridge can be automatically exchanged in synchronization with the rotation of the disk.
• We confirmed that liquid can be injected into the cartridge using a tube pump, also in coordination with the disk's rotation.
• We verified that the cartridge can be automatically disposed of as the disk rotates.

（４）Output Measurement Sensor Mechanism

We demonstrated that the AS7341, using blue excitation light, can effectively detect the fluorescence emitted by broccoli.

The details of the actual test procedures and results are described below.

Tube Pump

We pumped the liquid using the Tube Pump we made.

Using a continuous servo motor, we successfully delivered blue water up to approximately 200 mm above the liquid surface.Observing the water droplets exiting the tube, we noted that the drop rate remained consistent. This indicates that, when the pump operates at a constant rotation speed, it can deliver a relatively constant volume of liquid per unit time.However, we were unable to demonstrate the level of precision in liquid delivery typically required in laboratory settings. In the future, we plan to further improve the pump and establish an experimental environment to evaluate its delivery performance, allowing us to demonstrate the accuracy of the tube pump at a high level.

Soil Sampling Mechanism

We successfully demonstrated the operation of the following functional requirements of Soil Sampling Mechanism:

• Sampling soil
• Leveling off the soil to a consistent volume
• Mixing the soil with KCl solution
• Pumping up the mixed solution
• Disposing of the mixed solution with minimal residue left in the reaction chamber

The following is an explanation of the mechanism that this demonstration video proves to work.

(1) In the operation from 0:00 to 0:13, it was demonstrated that the rotation of the continuous servo motor causes the drill nozzle to rotate, enabling soil sampling.

(2) In the operation from 0:13 to 0:24, it was shown that the rotation of the stepping motor activates the soil leveling mechanism, which scrapes the soil to a fixed volume and transfers it into the reaction chamber.

(3) In the operation from 0:24 to 2:03, it was shown that water was introduced into the reaction chamber, demonstrating the mixing of water, which simulates soil and KCl solution. This time, the soil is transferred using a simple pump, but it is originally intended to be transferred by a tube pump.In the actual process of eluting nitrogen from the soil, it is considered to be mixed more because the mixture is left to sit for several hours before being drawn up .

(4) In the operation from 2:03 to 2:53, it was demonstrated that the mixed solution can be drawn out from the reaction chamber by the operation of the tube pump.

(5) In the operation from 2:53 onward, it was shown that by flipping the reaction chamber by the operation of the normal servo motor, the soil and moisture inside were discarded with minimal residue left in the chamber.

Cartridge Exchange and Measurement Mechanism

We successfully demonstrated the operation of the following functional requirements of the cartridge exchange and measurement mechanism. In addition, in this video, the mechanism's operation was initiated remotely via a WiFi signal from a terminal.This also demonstrated that the device can be operated remotely.

• The cartridge is automatically replaced in sync with the rotation of the disk.
• Liquid is injected into the cartridge using the tube pump in sync with the rotation of the disk.
• The cartridge is automatically discarded in sync with the rotation of the disk.

From 0:00 to 0:10, it was shown that the rotating disk by the operation of the stepping motor moved to the loading site, and the cartridge was automatically loaded.

From 0:14 to 0:25, the rotating disk moved to the solution and reagent injection site, by the operation of the stepping motor, where the nozzle dropped rapidly into the cartridge, demonstrating the injection of the solution by the operation of the tube pump. In the video, the injected solution is represented by blue-colored water.

From 0:29 to 0:36, the rotating disk rotated 45° by the operation of the stepping motor, and the cartridge moved to the position of the second injection nozzle, showing that a second reagent can be injected.

From 0:36 to 0:46, the rotating disk moved to the measurement site by the operation of the stepping motor, demonstrating that measurement can be performed in a single series of movements.

At 0:49, it was shown that the cartridge is automatically discarded with the rotation of the rotating disk. From 0:49 onwards, it was demonstrated that the entire sequence of operations could be repeated, indicating that continuous measurement is possible.

In this mechanism, we attempted to create a dispensing mechanism to distribute the liquid pumped from the pump into three lanes for injection, but we failed to demonstrate its function. A discussion on this issue is provided in Discussion.

Additionally, we tested whether the cartridge, sealed with aluminum foil, could be broken by the dropping injection nozzle, and in many cases, it failed to break. Suggestions for improvements on this issue are provided in Discussion.

Output Measurement Sensor Mechanism

It was demonstrated that the measurement sensor mechanism we created could detect the fluorescence of Broccoli.

Below is the video showing the measurement process.

We tested the ability to detect the fluorescence of Broccoli fluorescent aptamer in the laboratory. Broccoli is one of the reporters used in actual sensing. The Broccoli used in this test was prepared under standard in vitro transcription conditions as described in　Experiment. As controls, we used 1.5mM DFHBI-1t and MilliQ water (with zero fluorescence). We verified that the background and output fluorescence could be distinguished, as well as the ability to differentiate between zero fluorescence and fluorescence.

As shown in the video, the samples (Broccoli and Control) were exposed to 470 nm excitation light with the LED bulbs embedded in the device, and the output was measured simultaneously using two AS7341 sensors. To account for sensor measurement error, the same sample was measured three times. Out of the 8 channels on AS7341, values from 2 channels each (445 nm, 480 nm, 515 nm, and 555 nm) closest to the excitation light and fluorescence wavelengths were recorded as the results. Measurements were conducted under four conditions: with DFHBI-1t and MilliQ as a blank, and with or without a blue light-blocking filter of the transilluminator placed between the sample and the sensor.

For comparison of accuracy, the same sample and control were also measured using Qubit.

Control=DFHBI-1t, Blue light-blocking filter = None

Control=DFHBI-1t, Blue light-blocking filter = Present

Control=MiliQ, Blue light-blocking filter = None

Control=MiliQ, Blue light-blocking filter = Present

Qubit

The results indicated that AS7341 could clearly distinguish Broccoli fluorescence, background fluorescence, and zero fluorescence with differences ranging from several to tens of times, depending on the observed indicators, both with and without the filter. This strongly suggests that the device has sufficient measurement capability to read biosensor reporting in actual sensing.

However, Qubit was able to distinguish Broccoli from the background with a difference of about 400 times, and compared to a laboratory fluorometer, the sensitivity is undeniably lower. Therefore, there is room for debate on whether it meets the measurement accuracy ultimately required for the project. This will be discussed in detail in Discussion.

User Test and Interview

In the process of designing and building the device, we interviewed four user-tested end-users and experts to gather their opinions.As follows, we explain the feedback we received and how we reflected it in our design. The user tests were conducted using full scale models.End users experienced activities they would actually need to perform.

Mr. Yoshihiko Morita

A full-time farmer in Kamigamo, Kyoto City, who focuses on fertilizer. This farmer is particularly aware of nitrogen issues.

Figure11-1

Figure11-2

Implementation

• We conducted installation tests using full scale models in farmland.
• We explained activities that end users would perform.

Results

• We found that users can easily install equipment whose size we are considering on the farm. In addition, there were no concerns about excavating the device for maintenance of its underground components regularly.
• In terms of reagent replacement, instead of refilling a fixed tank with reagents, he said that it would be better to replace the entire tank filled with reagents. In terms of the cartridge unit replacement, he said that it would be better to replace the entire cartridge case filled with cartridge units rather than refilling the units within the case.
• There are a few concerns about the soil sampling mechanism using a drill nozzle, particularly when operating in clayey soil. It means when the drill nozzle rotates, the rotational resistance caused by the clay soil may hinder proper soil sampling.

Integration

• Instead of using adhesives to secure the reagent tank and cartridge case, we employed magnets to allow for easy attachment and detachment, enabling full replacement of the components.

• We devised a new mechanism for soil sampling in clayey soil. For a detailed explanation of this mechanism, please refer to the Proof of Concept section on this page. Also, we developed a drill nozzle shape that functions effectively in clayey soil.

Here is the modeling figure of the new soil sampling mechanism. This figure shows inserting a pointed cylinder into the soil and pushing the soil contained within it out of the device to collect the sample. In the future, we plan to verify whether this mechanism makes the torque for soil sampling lower compared to traditional methods in clayey soil.

Figure12

Using the push rod below, we will expel the soil from within the cylinder. When we attempted to collect soil using a traditional helical drill nozzle in clayey soil, we encountered a phenomenon where soil became trapped and solidified between the spiral plates of the nozzle. This prevented normal sampling. We believe that using a nozzle with a quarter of the traditional spiral might improve this situation. In the future, we will investigate which nozzle shape can most effectively reduce the required torque.

Organic nico

A company growing and researching organic farming in Nishikyo Ward, Kyoto City. They perform soil analysis at their own facility.

Figure13

Implementation

• We explained activities that end users would perform by using full scale models.

Results

• There is a concern that our device might have a bad effect on the growth of crops depending on the installation location because of its size.
• No concerns were expressed regarding the previously mentioned methods for reagent replacement and cartridge replenishment.
• We received suggestions on the optimal disposal method for the cartridge. It was found that placing a box under the disposal site in the Cartridge Exchange and Measurement Mechanism and removing the box during maintenance to dispose of the accumulated waste would be user-friendly.
• In terms of the soil sampling mechanism using a drill nozzle, there is a concern about its operation in clayey soil. It means that the rotational resistance caused by the clay soil may hinder proper soil sampling when the drill nozzle rotates.
• There is a concern about the necessity of a permanent sensor. They suggested that in the current fertilization methods, there is a higher demand for collecting soil samples and analyzing nitrogen content at the necessary times during the farming season.

Integration

• We realized that by not only analyzing the soil collected automatically but also by removing the drill nozzle and inserting the soil, and moving it into the hole, we can run the same program used for constant automatic measurements. Therefore, we made the drill nozzle removable.
• To implement the disposal method mentioned earlier, a box was installed at the disposal site in the Cartridge Exchange and Measurement Mechanism. The cartridge disposal was demonstrated using this method. Please watch the video below.

Mr. Seiji Matsumoto

A staff member from the Kyoto Prefectural Agricultural, Forestry, and Fisheries Technology Center. He conducts research and discussions related to agriculture. He is an expert with deep knowledge of actual farming practices.

Implementation

• We explained activities that end users would perform by using full scale models.

Results

• There is a concern that the relationship between the root zone and the position of the collected soil may cause variations in the results.

Integration

• By extending the liquid delivery tubes and wiring that connect the upper and lower parts of the device, we designed and manufactured it to allow for an unlimited depth of the underground section, enabling the setting of the depth of the soil layer to be collected.

We developed a mechanism that maintains a constant volume of the soil and KCl mixture for measurement. This ensures that the ratio of liquid added to the soil remains consistent, preventing significant variations in the measurements. The collected soil is adjusted to a specific volume using the soil cutting mechanism introduced in Conceptualization and Design. Additionally, when the total volume of soil and KCl entering the reaction chamber exceeds a certain threshold, any newly injected KCl is allowed to flow out of the reaction chamber.For more details, please refer to the description of the reaction chamber in Conceptualization and Design.

HORIBA Advanced Techno, Co., Ltd.

A company based in Kyoto that has locations around the world, specializing in the development and manufacturing of analytical and measurement systems. It is a professional in developing and selling nitrate sensors, EC sensors, and other devices currently used in the agricultural field. The company also develops and sells ammonium sensors and fluorometers for laboratory use.

Figure14-1

Figure14-2

Implementation

• Obtain feedback on the design from the sensor manufacturing company.
• Show the device's operating mechanism in person and gather their opinions on it.

Results

• There is a concern regarding the durability of the device. It became clear that it needs to have a lifespan comparable to existing EC sensors, ensuring a similar introduction cost.
• It was determined that, for accurately measuring outputs such as fluorescence, it may be necessary to create a highly precise measurement device. (＊)
• When handling fluorescence as an output, there was a suggestion that it might be better to focus on measuring fluorescence lifetime rather than absolute intensity. (＊)
• Concerns were raised that some form of agitation device may be necessary when mixing the freeze-dried cell-free system with nitrogen-saturated elution liquid. (＊)
• There is a concern that, as measurements accumulate, the impact of contamination from residues of previous measurements may become more pronounced.
• It became clear that ensuring high maintainability, particularly the ease of replacing fragile components is crutial.
• It was suggested that it would be even better if the system could measure not only nitrogen but also phosphorus, pH, and EC.
• In terms of (＊), it was indicated that since they have no prior experience, it is necessary to create a prototype and conduct verification.

Integration

• We conducted a preliminary prototype for the (*) item. As a result, we found that a significantly more advanced and precise measuring device is necessary for accurate fluorescence measurements. Additionally, subsequent validations showed that measuring absolute intensity with the AS7341 was heavily influenced by the excitation light, indicating that measuring fluorescence lifetime might be a more accurate approach.
• We designed an oscillation device utilizing part of the existing mechanism, which can be created without adding new electronic components. To reduce contamination effects, we also designed a reaction chamber for the efficient disposal of soil and KCl mixed samples.
• To mitigate contamination effects, I designed a reaction chamber for the efficient disposal of soil and KCl mixed samples.

The reaction chamber used for mixing soil and KCl has the following properties: by angling part of the wall, it facilitates easier disposal of the soil. Additionally, coating the interior with lotus-effect aluminum foil significantly reduces the amount of soil that adheres to the walls. For more details, please refer to the description of the reaction chamber in Conceptualization and Design.

We will explain the design of the shaking device. The injection nozzle of the solution/reagent injection device described in the Conceptualization and Design section is replaced with a rod-shaped structure for stirring. When there is an incline on the outer edge of the rotating disk, the disk is moved by rotating it back and forth in small increments, causing the stirring rod to move up and down repeatedly inside the cartridge.

Roadmap for Social Implementation

Device Improvement

Based on feedback from interviews, the following improvements for the device towards social implementation have been identified.

• Manufacturing Cost
• Running Cost
• Size
• Durability
• Soil Collection Performance
• Output to Users

Manufacturing Cost

Regarding social implementation, the biggest concern is the cost associated with the introduction of the device. Among these, manufacturing cost directly impacts the overall implementation cost. By reducing manufacturing costs, it becomes easier for end-users to adopt the device, and stakeholders can produce and operate a larger number of devices.

In this project, the manufacturing cost of the prototype device is approximately 10,000 yen, with about 80% of that attributed to electronic components. Considering feedback from stakeholders such as Horiba and Mr. Morita, it was determined that the manufacturing cost needs to be reduced to 5,000 yen per unit for future social implementation of the device. To achieve this, we plan to lower the cost of electronic components by further specializing their functions for this device, thereby significantly reducing the overall price. Specifically, instead of using the expensive Arduino used in the prototype, we will manufacture the device using a low-cost microcontroller that has only the essential performance needed to operate the device.

Running Cost

In the early stages of device design, we did not prioritize running costs. However, during discussions about costs with stakeholders such as Synbiobe, it became clear that running costs are a point that should be considered and improved more than manufacturing costs.

The components of running costs can roughly be divided into two categories: the battery costs for the device and the manufacturing costs of the cell-free cartridge unit and reagents for reactions.

The former, we believe that by enabling a single commercially available 9V battery to perform 30 measurements, the operational costs can be kept at a user-friendly price even for a fixed sensor setup. At this stage, while the device can operate using two 9V batteries, we have not yet verified the battery's longevity during use. Therefore, we will optimize the electronic circuit and conduct repeated trial operations to gather data on power consumption, thereby assessing the feasibility of this goal.

For the latter, most of the costs are associated with the reagents needed for the production of the cell-free cartridge unit. Therefore, we plan to optimize and mass-produce the cell-free cartridge units in the future to reduce the individual price of the cartridge units.

Size

Through interviews with end users, such as Organic Nico, concerns have arisen regarding the size of the device. There is apprehension that if the device is too large, it could hinder the growth of plants during long-term installation and operation. One potential solution to address this concern is to reduce the size of the device. However, the current design is already optimized for its functionality, making it challenging to downsize while retaining its essential design. To achieve some degree of miniaturization, it may be necessary to increase the proportion of electronic components, thereby digitalizing the operational mechanisms. However, this digitalization could lead to increased manufacturing and operational costs.

Therefore, it is considered preferable to address these concerns without reducing the device's size. One method being explored is to reconsider the installation approach of the device. A potential issue is that the above-ground portion of the device may obstruct the growth of leaves in plants like strawberries. To mitigate this, we plan to creatively design the waste collection area of the cartridge, allowing the above-ground portion of the device to be buried in the soil to the necessary height while maintaining ease of maintenance. This way, we can lower the height of the above-ground part of the device while preserving its critical design features.

Durability

Ensuring durability is crucial when operating the device for an extended period in agricultural fields. By securing durability, we can reduce the frequency of device replacements, which leads to lower operational costs and reduced environmental impact. Feedback from Horiba indicated that if the implementation costs of the EC sensor and this device are the same, the lifespan of the device needs to be around five years.

In the prototype of this device, the primary materials used were 3D-printed components and wood. However, we can enhance durability by replacing these with more durable plastics. Additionally, by revisiting the design, we can create a device that is more durable. The plan is to extend the lifespan to around five years while maintaining manufacturing costs.

Soil Sampling Performance

With the current performance of the device, soil can only be sampled from an area approximately 10 cm around the device. Therefore, if there are local differences in nitrogen content due to uneven fertilization, the sampled soil is likely to be unsuitable as a representative sample of the farm's soil nitrogen conditions, making it difficult to assess the overall fertilization status of the farm from the measurement results. Additionally, interviews with Organic Nico revealed the possibility that the current drill nozzle may not function effectively in clay-rich soils.

Considering these factors, to achieve social implementation in the future, it is essential to develop a mechanism that can sample a wider range of soil, thereby enhancing the utility of the soil samples. Furthermore, we aim to create a sampling method suitable for various soil types across different farms, ensuring consistent operation regardless of the farm's conditions.We will develop new and innovative soil sampling mechanisms to achieve these goals.

Output to Users

This device quantifies the output from a cell-free cartridge and uses that data to measure nitrogen levels in the soil. Therefore, the output format is numeric only. However, interviews with end users revealed that for many, directly seeing measurement data is not as important as understanding whether there is too much or too little fertilization. To address this, after transmitting the measurement values to end users, we can use diagnostic software to determine the appropriateness of fertilization, making the information more user-friendly.

During an interview with PLANTX, we learned about the software SAIBAIX, which meets the above functional requirements. By functionally integrating the device with existing software like SAIBAIX, we aim to create a device that fully meets the project goal, enabling end users to manage their fertilization effectively.

Proof of usefulness of the device

When considering social implementation, it is necessary not only to improve the device's performance but also to present the benefits of using the device to the end-users.This section describes how to prove the device's usefulness.

Presenting the benefits of the device implementation

This device, which analyzes soil nitrogen levels and supports optimal fertilization, is expected to increase end-user profits due to the following factors.

(1)Optimizing fertilization improves crop growth, leading to an increase in yield.

(2)Optimizing fertilization improves crop growth, resulting in higher crop quality and a higher unit price.

(3)Optimizing fertilization improves crop growth, reducing the costs required to control insects and disease-causing bacteria.

(4)Optimizing fertilization eliminates excessive fertilization and reduces the amount of fertilizer used, lowering fertilization costs.

If the total profit gains from installing this device on a farm exceeds the manufacturing and operating costs, the device will be beneficial for the end-users. In the early stages of design, we assumed that factors (1), (2), and (4) would contribute mainly to profit increase, but with feedback from Organic nico, we also decided to consider (3) as a factor for profit increase.

Therefore, in order to implement devices socially as truly user-friendly, we will create a reliable criterion for these profit increases and present them to the end-users.

This criterion can be obtained through actual trial operation of the device, but the values are expected to vary widely depending on the type of crop being cultivated. Therefore, we plan to conduct extensive trial operations and collect data on how fertilization optimization promotes plant growth, how this growth enhancement improves the plant's own defense response, and how much the cost of fertilization can be minimized with optimal fertilization, so that the end-users can understand this device better.

As a current estimate of profit increase, there is data from PLANTX showing that production increased fivefold in a typical plant factory through their growth management. Therefore, we expect to obtain data that can encourage the end-users to adopt the device.

Credibility of device sampling

Since this device is a permanent fixture, it is designed to sample soil from a fixed location periodically. Therefore, without investigating whether the soil at that location can function as a representative sample for the entire farm, we cannot determine if the device's measurement results can be used to assess the fertilization conditions. Thus, we will examine how localized fertilization unevenness affects the representativeness of the sample and demonstrate the reliability of the sample.

Disadvantages of Using Biosensors

While we show benefits of using the device, It is necessary to explain how the disadvantages can be mitigated. In this case, the demerits we considered for the installation of the device were not only the installation costs but also the risks associated with operating biosensors in farmlands. All biological elements in this device are designed cell-free and all used cartridge units are contained within a disposal box, isolating them from the environment. Therefore, biological contamination is at low risk. However, it is still unclear how seriously end users will perceive this risk. Thanks to the interviews we did for end users, it became clear that Organic nico and Mr. Morita are not concerned about this risk. Therefore, if we explain disadvantages politely, we do not have to worry about biological contamination resulting from the introduction of the device. On the other hand, the result of the survey for consumers shows that using SAMURAI to grow crops may reduce consumers' willingness to buy crops. For detailed information, please refer to the section on ### Consumer Survey in the Human Practice chapter.This decrease may come from unconscious negative feelings toward biosensers. In order to implement this device, we plan to explain to consumers the merits of cell-free devices and measures for protecting biosafety.

Roadmap

Finally, based on the above issues and discussions, we present a roadmap for addressing these problems and achieving social implementation. We have planned a social implementation roadmap consisting mainly of three stages: device improvement and completion, device commercialization, and device dissemination.

(1)Improvement and Completion

First, we change our prototype device into a practical model, which can be used without problems in the field. We mainly focus on functional improvement and to reach the level of practical manufacturing.

In terms of functional improvement, we will improve design based on feedbacks from users and companies, and examine in detail the accuracy of our sampling and measurements and devise mechanisms to improve accuracy. As mentioned in Proof of Concept, we know that the functionality of the main mechanisms will be demonstrated so that we will brush up them through time-consuming verification and trials.

Furthermore, we will realize manufacturing at a practical level. The steps are as follows. Currently, we are prototyping general modules such as 3D printers and Arduino with limited funding. These modules pose challenges in terms of cost and durability. Practical devices will be produced through commercial manufacturing-level fabrication, including the use of full-scale machine tools, the use of custom electronics, and the specification of injection molded plastics. The plan is to accomplish this by negotiating the use of university machine tools and partnering with sponsoring companies. The use of more durable materials and optimized electronic components will allow significant improvements in durability and cost.

(2)Commercialization

Second, we commercialize practical devices.

For more information about managerial strategy from startup to opt-out, see (Entrepreneurship) page.

From concept to prototype, the device has been shaped by discussions with stakeholders with a history of developing and marketing agricultural and nitrogen sensors, including Symbiobe and Horiba Advanced Tecno Co.Ltd.We plan to create a commercially viable device by utilizing advanced machining equipment, custom electronic components, and injection-molded plastics. Further refinement, standards compliance, and testing for commercialization will follow in the same manner. Commercial success is envisioned and we believe it has the potential.

At this stage, manufacturing, marketing, and deployment will be transferred from iGEM Kyoto to the company, and commercial collaboration with multiple stakeholders, such as those listed in Human Practice, will provide a total solution for nitrogen management, including fertilizer management, and widely demonstrate its safety and effectiveness. The safety and efficacy of the technology will be widely demonstrated.

(3)Dissemination

Even after the goal in Entrepreneurship, a dissemination phase of a total solution for nitrogen fertilizer management, including this device, is considered for the final goal of the project. Utilizing the deployment capabilities of the company, the device will be disseminated through agricultural cooperatives and local governments as a foothold.

Further refinement of the device and fertilizer management technology will also be carried out based on data obtained through actual operation on farmland and feedback obtained through actual use.

Ultimately, we aim to achieve nationwide deployment of the devices and solve the nitrogen problem. We are also considering global use, following the example of Horiba Seisakusho (which develops nitrogen sensors overseas).

Thus, until we reach the stage of solving the nitrogen problem, which is the ultimate goal of the project, the device will combine cell-free biosensors and nitrogen fertilization management as a physical application and strongly promote its application in the real world.

Discussion

In the "Synthetic Biological Significance of the Device" section, as mentioned on the ##Contribution page, we have summarized how this device can be applied and contribute to synthetic biology projects beyond our own.

In the "Device Function" section, we have compiled discussions on proof-of-concept failures and several functions that require particular consideration, even though they were not raised during user testing or discussions on social implementation.

The Significance of the Device in Synthetic Biology

The Significance of the Device's Soil Collection Mechanism

The device's soil collection mechanism will assist teams focused on soil sensing and other approaches in real-world applications. Soil is a critical target in areas such as environmental protection, agriculture, and public health, as demonstrated by projects like the iGEM Team Goettingen 2018. However, due to its non-liquid form, fully automated approaches have been relatively challenging. In contrast, examples like the WM400 automatic water quality monitoring device developed by Yokogawa Electric Corporation in Japan show how continuously operating sensors can help end-users measure water quality daily without feeling burdened. Unfortunately, this is not feasible for soil. Our device builds a mechanism that enables unprecedented automated sampling and elution from soil, allowing other teams to easily replicate it. In this way, it introduces the option of "automatic sampling" for subsequent teams and projects outside iGEM, thereby broadening the scope of synthetic biology applications in real-world soil approaches.

The Significance of the Device's Automatic Cartridge Exchange Measurement Mechanism

As a device capable of continuous, fully automated sensing using cell-free systems, it will assist all teams developing outdoor cell-free biosensors. One of the advantages of whole-cell sensors is their ability to remain alive, allowing for continuous and automatic sensing, as seen in examples like the iGEM Team Edinburgh_OG2019. In practice, the ability of biosensors to survive is significant; for instance, goldfish and mussels are used in wastewater treatment and purification facilities to detect harmful substances, enabling constant measurement without human intervention.

The Significance of the Miniature Tube Pump

The miniature tube pump serves as a valuable asset for all teams aiming to develop cost-effective devices for collecting and sensing water-soluble substances in outdoor environments.

When creating biosensors, there is oftena high likelihood that liquids, such as reagents, need to be moved within the device. To ensure measurement accuracy while striving for an affordable and compact design, it is desirable to incorporate a low-cost, small-scale quantitative liquid delivery pump.

The new tube pump we developed is both inexpensive and easy to manufacture compared to the tube pumps created by previous iGEM Teams Aachen 2015/2017. It can be constructed using only a low-cost continuous rotation servo motor (FS90) and parts that can be printed with a household 3D printer, resulting in a production cost of under 500 yen per unit.

This innovation allows for precise management of sampling liquids and reagents when building affordable biosensor devices, significantly enhancing the measurement accuracy of the sensors and expanding the types of reactions that can be utilized.

Device Function

We present considerations on the dispensing mechanism, which failed in the proof-of-concept, as well as an analysis of the measurement accuracy of the incorporated sensor, As7341.

Dispensing Mechanism

As a non-primary but necessary mechanism, we included a system for dispensing soil extraction solution into three cartridges to enable simultaneous sensing using multiple sensor systems. While automated dispensing mechanisms are common in laboratory automation devices, we aimed to implement this at a very low cost by attempting to split the flow path from the tube pump to the cartridge injector, thus achieving three-way dispensing.

Despite attempts at trial and error and proof-of-concept testing, we were unable to achieve accurate dispensing using this mechanism in this iteration. (The final version of the prototype branching mechanism's data is included in the STL data mentioned later.) The cause is likely that a household 3D printer lacked the precision to print components capable of dividing the pressure exerted for extrusion into three equal parts accurately.

As a solution, considering that the suction pressure of the tube pump is constant, we are considering splitting the flow path between the reaction chamber and the pump or using a different mechanism, as mentioned later, to dispense without relying on flow path branching.

AS7341

This is a simple light spectrum sensor used on this device. In the proof of concept, it successfully distinguished between blue excitation light and green fluorescence, demonstrating the ability to clearly measure the difference between background fluorescence and the fluorescence of one of the reporters, Broccoli fluorescent Aptamer.

The reasons for its adoption include its low cost and the aim to use the same sensor for sensing all reporting formats—fluorescence, luminescence, and color change—of the sensing platform MITSUNARI.

However, the AS7341 is a simplified sensor and a color sensor, which is said to have lower sensitivity compared to monochrome sensors. Therefore, further investigation is needed to determine whether it can accurately measure all formats with sufficient precision.

Due to progress issues in the wet experiments, we were unable to conduct concept validation for anything other than fluorescence, leaving us without conclusive materials for discussion. However, the proof of concept suggested that the fluorescence detection sensitivity of the AS7341 might be 10 to 100 times lower compared to a Qubit sensor. This indicates a potential lack of sensitivity, particularly in detecting luminescence. On the other hand, the light spectrum sensor may be the most suitable type for coloring reporters due to its inherent characteristics, making it difficult to state that we should switch to a different sensor definitively.

We are currently inquiring with Sony Corporation, which manufactures experimental fluorescence imagers using light spectrum sensors. Additionally, through user tests, Horiba Manufacturing suggested that practical trials are necessary. Moving forward, we plan to deepen our verification through actual testing and interviews with experts.

Moreover, the design has room for improvement, including options for external light shielding, the addition of filters, sensor placement closer to the sample, and the possibility of upgrading to sensors more suited for specific reporters than the AS7341. We intend to implement these improvements as the project evolves.

About The Material of The Injection Nozzle

During the prototype development, we created an injection nozzle capable of puncturing aluminum foil using a 3D printer. However, due to the printing limitations of the 3D printer, we were unable to make the nozzle tip sufficiently thin, resulting in frequent failures to pierce the aluminum foil. Therefore, we believe it would be preferable to use glass for the injection nozzle, designed with a sharp tip for better performance.

Creating More Accurate Devices

Creating highly accurate tube pumps and dispensing mechanisms is challenging with a home-use 3D printer, which made it difficult for this device—originally designed with these mechanisms in mind—to achieve precise measurements. As a result, we have redesigned the device to forgo these mechanisms in favor of ensuring higher measurement accuracy. The new design still achieves the concepts of cost and electronic component limitations.

An overview of the redesigned device is shown below.

In the new device, a microchip is used instead of a PCR tube as the cartridge. This microchip is attached to the tip of a 1ml Terumo Syringe, allowing the user to draw liquid into the syringe, which supplies the sample solution and reagents to the cell-free system within the microchip. Since the liquid volume can be adjusted by finely tuning the piston of the Terumo Syringe, it allows for precise liquid volume control without the need for a dispensing mechanism or tube pump.

Figure15

Additionally, since the overall mechanism is expected to be simpler, the above-ground portion of the device is anticipated to be smaller than the current design. This may reduce the likelihood of hindering the growth of crops during installation.

Moving forward, we plan to proceed with the production of this new device.

Conclusion

Our devices are designed to meet every need by precisely solving the challenges of project implementation through thorough integration and meticulous design of human practice. In addition, each whole or part is versatile and has potential for a wider range of applications.

(Overview , Requirements ,Concept and Design , Discussion )

The main mechanisms worked well at the prototype level, demonstrating the practicality and functionality of this device. It strongly suggests that with further improvements, it can be developed into a commercial and practical model in the future.

（Demonstration of Device Functionality and Practicality , Roadmap for Social Implementation, Discussion ）

We conducted multiple user tests and interviews and received a variety of feedback on the device as a whole. This feedback was incorporated either into the actual production of the device or into a map of future improvements and social implementations.

（UsernTest and Interview 、Roadmap for Social Implementation)

All design information, 3D printed data, electronic schematics, parts lists, and assembly information for the final prototypes were fully documented and posted in a format that was easy to reference during replication.

（Data for duplication ）

From the above, we propose this device as a promising soil nitrogen sensing device and a powerful physical solution to apply cell-free biosensors to reality.

Data for duplication

The parts have been given names that identify which module they are from and what they are for.

Any team or project that wants to use and improve part or all of this hardware can easily replicate and improve our devices based on this data and pictures of the devices.

All you have to do is to buy and print the components and assemble them in the arrangement shown in the pictures, supplemented by appropriate auxiliary parts.

This would be easily accomplished with rudimentary woodworking and electronics skills and tools at the level of a typical iGEM hardware team, as well as a home 3D printer.

Cartridge replacement and measurement device_Base
Download

Cartridge replacement and measurement device_Cartridge cover
Download

Cartridge replacement and measurement device_Cartridge pusher(1)
Download

Cartridge replacement and measurement device_Cartridge pusher(2)
Download

Cartridge replacement and measurement device_Magazine Lid
Download

Cartridge replacement and measurement device_Magazine skirt
Download

Cartridge replacement and measurement device_Magazine
Download

Cartridge replacement and measurement device_Part of arm of magazine lid
Download

Cartridge replacement and measurement device_Piston holder(arm+)
Download

Cartridge replacement and measurement device_Piston holder(No arm)
Download

Cartridge replacement and measurement device_Piston roter stopper
Download

Cartridge replacement and measurement device_Piston roter
Download

Cartridge replacement and measurement device_Piston
Download

Cartridge replacement and measurement device_Rotary disc shaft
Download

Cartridge replacement and measurement device_Rotary disc
Download

Cartridge replacement and measurement device_Skirt of AS7341
Download

Cartridge replacement and measurement device_Stepping moter cover
Download

Cartridge replacement and measurement device_Triple injecter
Download

Prototype_Cartridge replacement and measurement device_Dispenser A
Download

Prototype_Cartridge replacement and measurement device_Dispenser B
Download

Prototype_Soil sampling device_Circle gear of sampling bamboo-spear
Download

Prototype_Soil sampling device_Linear gear of sampling bamboo-spear
Download

Prototype_Soil sampling device_Piston of sampling bamboo-spear
Download

Prototype_Soil sampling device_Sampling bamboo-spear
Download

Shaft base of scraping mechanism _Soil sampling device
Download

Soil sampling device_Elution solution tank
Download

Soil sampling device_Gear of Scraping mechanism
Download

Soil sampling device_Guide tube of sampling drill
Download

Soil sampling device_Outer flame (Upper)
Download

Soil sampling device_Outer frame (Lower)
Download

Soil sampling device_Quarter gear of Scraping mechanism
Download

Soil sampling device_Reaction dish
Download

Soil sampling device_Samplimg drill(Single stranded 7.5cycle)
Download

Soil sampling device_Sampling dirill(Double stranded 3.75cycle)
Download

Soil sampling device_Sampling drill(double stranded 7.5cycle)
Download

Soil sampling device_Scraping tube os Scraping mechanism
Download

Soil sampling device＿Spoon of Scraping mechanism
Download

Tube pump_Cover
Download

Tube pump_Frame
Download

Tube pump_Roter(1)
Download

Tube pump_Roter(2)
Download

Tube pump_Shaft cover
Download

Tube pump_Shaft-moter connecter
Download

Tube pump_Shaft
Download