The inspiration for our hardware device is from the COVID-19 rapid test kit, which possesses traits including portability, user-friendliness, immediacy, precision, accessibility, and affordability. Since the human body's inflammatory response can be difficult to detect at an early stage, we came up with the idea of building a rapid test kit for inflammation. To accomplish this, we have combined the advantages of the COVID-19 rapid test kit with the input and feedback from experts and mentors, resulting in the following design principles. The features mentioned above would be achieved by the following components:
We strive to build a device that is simple in design and easy for users to assemble. For more convenient use, we try to reduce the complexity of signal transduction and connectivity by a single USB cable as our connection between our device and the computer. In the future, we will make our machine even easier to use by incorporating Bluetooth, as a substitute for physical wires in the process of data transmission.
Our team aims to develop a device with minimal components, eliminating the need for users to invest excessive time and effort in assembly. While prioritizing short-term usability, we also recognize the imperative of maintaining detection precision. Therefore, to enhance user convenience, we intend to simplify the complexity of signal transduction and connectivity. To achieve this objective, we plan to implement Bluetooth technology for data transmission, replacing physical wires. However, the integration of Bluetooth will be considered as part of our future endeavors. For the time being, we will utilize a USB cable for the connection between the device and the computer.
Designing a device that is convenient, and suitable for home use, there are several services we wish to provide for our users. First of all, testing samples of our users should be easily put into our device for sensing. Second, the device should be stable and the electric circuit should not be easily damaged. Lastly, an easy-to-use software with a clear and simple user interface for our users to understand and track their results.
Since our objective is to create a device that is convenient and suitable for home use, it should be effortless for users to insert their testing samples into our device without disrupting its normal operation. Furthermore, to ensure future accessibility, we have chosen affordable materials for our device, making the overall cost more budget-friendly.
Most test kits we can see in today’s market would not be too large or heavy and hence can expand their use in more areas. Rather than using wood, cardboard, or metals, we decided to use polylactic acid (PLA), as our main material for the device. Besides being light, it also has the advantages of being non-toxic and biodegradable. Moreover, it has been approved for medical use.
The sensing component will be facilitated by an electronic module and electrical circuit capable of receiving the fluorescence or electrical signals emitted by the genetically engineered bacteria. Furthermore, the data recording segment will incorporate the utilization of Arduino or a potentiostat, complemented by our software.
Considering the characteristics and the mechanism of fluorescent protein, we take the following points into account when designing our device:
A photoresistor is an electronic component that is sensitive to light and is used to sense the intensity of light and quantize it into resistance values, which could change significantly (from 10MΩ to 1kΩ) with the change of light. The increase in the brightness of the light source would result in a lower resistance value. Vice versa, when there is no light at all, the resistance value would be quite high. Photoresistors, also known as LDRs (Light Dependent Resistors), are high-resistance insulators with few free electrons. When light hits the photoresistor, photons will be absorbed by the semiconductor lattice, providing energy to the electrons in the lattice. This allows those electrons to gain enough energy to break free from the lattice, making the material conductive and reducing its resistance.
Though the sensitivity of the photoresistor's resistance value varies with the wavelength of light, it still has a limited wavelength range, which is shown in the following figure (Fig.1). The maximum peak sensitivity depends on the material of the photoresistor but typically falls within the visible light spectrum. However, it has drawbacks such as being temperature-sensitive, for example, higher temperatures would result in lower sensitivity, and would have a relatively slow response time. It also requires more time to achieve a balanced resistance value during sensing.
Based on the characteristics of the photoresistor discussed in the previous section, we can calculate the corresponding absorbance values using the following steps:
When a resistor R is connected in series with an LDR and a voltage of 5V is applied across the two terminals, the resistance value and the voltage across the resistance are directly proportional. Since we are measuring the voltage across the LDR, we can derive the following process:
Because the Arduino analog measurement range is 0 to 1024 (corresponding to 0V to 5V), you can convert the above equation into the following format:
From the above table, we obtain γ=0.6 for GL5528. Furthermore, based on the definition of γ, we can derive the following table and relationship diagram:
Based on the table and the relationship diagram above, the equation for converting resistance values to illumination levels is:
We referenced https://www.jvruo.com/archives/110/ to create a program that can generate RGB values corresponding to visible light with a wavelength of X. The 1931 CIE-XYZ standard colorimetric system is a mathematical approach that selects three ideal primary colors to replace the actual three primary colors in the RGB system, thereby transforming the spectral tristimulus values and chromatic coordinates r, g, and b into positive values in the CIE-RGB system.
In simple terms, the algorithm converts the wavelength of light into chromatic coordinates x, y, and z. Then it transforms these chromatic coordinates into the corresponding visible light R, G, and B values for that wavelength. For detailed code, please refer to the attachment.
The size of the whole device is 10cm in length and width, with the height adjusted to match that of the cuvette. The top is a hinged design so that after the cuvette is placed inside, the lid can be closed, creating a completely dark environment during measurements. Another advantage of the hinge is that we do not require extra components, for example, screws, to build and secure our device. In addition, the internal blank space is reserved for the Arduino board and electric circuits. The enclosed design helps prevent unnecessary collisions and tugging, which might result in poor contact.
Initially, we use our code to convert fixed-wavelength light into RGB values, which the Arduino then controls. The exposure time of the light is set to 600ms.
Photodetector: For sensing the light source, we utilize a photoresistor along with a variable resistor to measure light flux (Lux), as previously explained. This allows us to achieve the same effect as commercially available light sensor modules using basic components, aligning with the spirit of our competition – achieving precision instrumentation with simple materials.
In the first version of our device, we placed the LED light, cuvette, and photodetector in a straight line, which means the arrangement would be 180 degrees. While the photodetector also detects excitation light from the LED, this situation is consistent for each sample, and we treat it as a controlled variable.
Owing to the disadvantages mentioned above, we came up with the following design, Version 2, in the hope to improve the performance of our device.
The difference between this version and the previous one lies solely in the 3D-printed casing. When designing this version of the device, our primary consideration was the positioning of the cuvette and the circuitry for measurement. Additionally, we took into account space-saving and ease of placement, which led us to position the cuvette in a corner, leaving approximately one centimeter of space around it for easy cuvette replacement. Furthermore, the 3D-printed casing can be opened on both sides, facilitating adjustments to the electronic components inside. During measurements, it can also cover these materials, preventing unnecessary collisions and tugging, which could otherwise result in poor contact.
In this version of the device, we changed the placement of the LED light and the photosensitive element from a 180-degree configuration to a 90-degree configuration. With the LED light and the photosensitive element no longer in the same line, this design ensures that the photosensitive element receives emission light exclusively from the fluorescent protein. We aim to identify the optimal design for the final device based on the measurement results obtained from these two device versions.
From this graph, we can observe that each line represents a different wavelength band, and they all exhibit similar trends. Therefore, we have selected the wavelength band with the highest light flux as the optimal excitation light measurement band, which, in this case, is 445nm.
In contrast to version 1, version 2 simplifies the device to focus solely on scattered light. The design of having the LED light source and photosensor at a 90-degree angle effectively blocks almost all excitation light. Consequently, we have chosen to adopt the design of version 2.
Based on the aforementioned experimental results, we have decided to employ a 90-degree configuration in our final device and incorporate a novel conversion and calculation theory for which there is limited existing information on the market. After experimental validation, its feasibility has been confirmed, with high accuracy during repeated verification. Furthermore, we found that this particular strain of bacteria performs best at excitation wavelengths between 445nm and 465nm, thereby significantly enhancing the accuracy of our final device. This falls within the range of wavelengths that we had previously identified as being optimal in our earlier research. In addition, to improve the quality of the user experience, we have added a button and an OLED screen to the exterior surface of the device. This allows users to operate the device without the need for a computer. Its low cost, small size, portability, high accuracy, and customizable design make our final device highly user-friendly and offer limitless business opportunities for the future.
We can learn two things from the above result:
In the designs of our initial two test devices, the excitation end employed tri-color LEDs to adjust RGB at different ratios, utilizing a custom-built program to determine the proportions of RGB at varying wavelengths. This was done to stimulate fluorescent proteins effectively. On the receiving end, we developed a theory called "Photoresistor-to-Photon Flux Conversion," which operates on the electrical principles affecting the resistance of the photoresistor based on light intensity changes. After voltage division and computational processing, we derived the photon flux passing through the solution. Multiple tests and adjustments revealed that, compared between 180-degree (V1) and 90-degree (V2) setups, the former gave less accurate data due to the simultaneous measurement of excitation and emitted light. Nonetheless, it still approximated the predicted trend. Consequently, the final device was designed using the 90-degree (V2) approach. Data analysis also confirmed the aforementioned theory and helped us identify the most suitable wavelength, fulfilling our anticipated objectives.
Under the theoretical validations of the first two test devices and assuming that zinc ions effectively inhibit bacterial fluorescence, our final device measures the impact of zinc ions and bacterial concentrations. Compared with the wet lab group, the trend observed from the charts indicated a slight decline in both cases, confirming that zinc ions could effectively inhibit bacterial fluorescence. Additionally, when comparing fluorescent and non-fluorescent bacteria under the same zinc ion concentrations, a slight difference in photon flux was observed. This difference serves as the basis for our device's judgment, enabling us to detect whether a patient shows symptoms of an illness.
In order to unlock the full potential of our device for future teams and various research applications, we've made a strategic choice by using LEDs as our light source. This decision holds EXCEPTIONAL SIGNIFICANCE. It empowers our device to detect not just mCerulean but a wide range of fluorescent proteins (FPs). This versatility stems from our ability to customize the excitation light for any specific FP. The code to control the light source can be readily accessed on our hardware page.
To achieve our goal of understanding the trend of fluorescence intensity changes inhibited by zinc ions, it is essential to identify a standard value that will allow us to estimate the original concentration of zinc ions in the sample. To this end, we propose the following research steps:
Considering the characteristics and the mechanism of the Extracellular Electron Transfer (EET) system, we take the following points into account when designing our device:
EET system operates only under anaerobic conditions, thus, our device must include an anaerobic space for our bacteria.
EET system emits electrons in a small quantity, for us to optimize these electron signals, we must design a sensitive electrode system to sense current intensity.
EET system operates only under anaerobic conditions, hence, we must design our device to be able to contain an anaerobic condition in our bacteria component. To accomplish this, we referred to various studies, and iGEM team projects, and consulted biomedical equipment companies. Ultimately, we opted to use the CoverWell Perfusion Chamber as our carrier for the EET system and biomarkers.
The CoverWell Perfusion Chamber chosen by our team has specifications of 9 mm diameter, 0.5 mm depth, and is colored orange with two external openings for Polytetrafluoroethylene (PTFE) tubes. Furthermore, there is no concern about oxygen entering through the PTFE tubes, as the PTFE tube diameter is as narrow as 1mm. We would be able to introduce the EET system and biomarkers separately through the two openings using syringes and needles.
To optimize our device sensitivity, we utilized the three-electrode system including the working electrode (WE), counter electrode (CE), as well as reference electrode (RE), and cyclic voltammetry to test the current intensity. Compared with a dual-electrode system, adding a reference electrode allows the originally dual-electrode system, capable of measuring only current and voltage variations, to be capable of quantifying actual numerical changes.
Considering that electrode rods in chemistry labs are both expensive and bulky, we ultimately opted for cost-effective and compact screen-printed electrodes. Our team selected screen-printed electrodes with dimensions of 12.6mm*38.0mm, comprising three cost-effective electrode materials: two carbon electrodes (WE & CE) and one silver electrode (RE). They are small in volume and cost-effective with their single-use nature, while still being very effective and sensitive to the sensing of electric current. With screen-printed three electrodes, we can build a portable and lightweight device.
The size of our 3D design is 10cm in length and width, and 7cm in height. The top has a hinge design so that the CoverWell Perfusion Chamber and screen-printed electrodes are well protected, while also making replacements easy and speedy. Another advantage of the hinge is that we do not require extra components, for example, screws, to build and secure our device. We designed two compartments, one for the perfusion chamber and electrodes, and the other for the electronic circuits. The enclosed design helps prevent unnecessary collisions and tugging, which might result in poor contact.
Figure27. shows the insertion of the PTFE tube into the Press Fit Tubing Connectors, which requires cutting a pointed end on the PTFE tube before it can be smoothly inserted into the Press Fit Tubing Connectors. After insertion, the PTFE tube should be cut flat to fit into the opening of the CoverWell Perfusion Chamber.
In Figure28., it demonstrates the attachment of Press Fit Tubing Connectors to the screen-printed electrodes, where two of the Press Fit Tubing Connectors need to be appropriately trimmed to securely adhere to the screen-printed electrodes.
Figure29. and figure30. depict the arrangement of screen-printed electrodes, two Press Fit Tubing Connectors, two PTFE tubes, two syringes, and three alligator clips placed inside our team's fabricated 3D device. Figure29. primarily illustrates the placement of the alligator clips onto the screen-printed electrodes, while figure30. demonstrates the injection mode for the EET system bacterial solution and Biomarkers.
For measuring and testing, our team assembled our EET device, and prepared solutions containing various concentrations of zinc divalent ions. We turned to Assistant Professor Ta-Chung Liu's laboratory to borrow their potentiostat for measurement, as our lab did not have this equipment. We aimed to establish a calibration curve to enable our device to measure unknown Zn2+ ion concentrations. By achieving this result, we can confirm our ability to detect the electrical signals released by the EET system and proceed to broaden our capacities for measuring biomarkers and sensing inflammatory responses. Zn2+ ions were prepared in a solution of LB+CM+Amp, with a total of ten tubes, ranging in concentration from 0mM, 0.2mM, 0.4mM to 1.8mM. The concentrations of 0.6mM and 1mM Zn2+ with LB+CM+Amp were not measured due to spillage during transport, which resulted in an insufficient solution. This could potentially lead to inadequate contact between the electrodes and unequal reaction areas, so we decided not to measure these concentrations.
To enhance the accuracy and credibility of CV (Cyclic Voltammetry) plots and results, the scan rate was set to a slower 100mV/s to avoid distortion of the results in the IviumSoft software. By referencing the reduction potential of the reference electrode in the standard electrode potential table and the reduction potential of zinc ions (Zn(OH)4 2⁻＋2e⁻ ⇌ Zn(s)＋4OH⁻, E°(V)=-1.199), we ultimately set the scanning range from -2V to +2V. The study and discussion of the CV plot for Zn2+ and the creation of the calibration curve were ultimately conducted with the reduction peak at -1V. By employing the technique of overlaying plots (as shown in Figure 31. and Figure 32. below), all concentration results can be placed on the same graph, facilitating a clear and effective comparison of differences in the CV plots. Furthermore, to ensure the accuracy of the results, all the CV plots we used were from the fourth cycle of each measurement.
Ignoring the small noise peaks, which may be due to redox reactions of trace substances or contaminants in the solution, it is evident from Figure 31. that at -1V, there is a consistent trend among different concentrations. Higher concentrations of Zn2+ exhibit more pronounced reduction peaks, with their CV plot lines appearing lower. For example, the highest concentration of 1.8mM Zn2+ is represented by the bottom purple line in Figure32. In summary, the CV overlay plots in this test have successfully and comprehensively validated the performance of Zn2+ at different concentrations, demonstrating a consistent and favorable trend.
From our CV results, we took a closer look at the currents measured at -1V, where our measurements showed a consistent trend among different concentrations. We documented each current value and formed the calibration curve.
From Figure33., we can see that our trendline has a positive correlation, with stronger currents as the Zn2+ concentration increased. The formula of the trendline is y= 0.185*x + 0.256, with a coefficient of determination R2 of 0.971. The coefficient of determination for our trendline is above the threshold of 0.95, which makes it reliable in predicting Zn2+ concentration when we have the current of the solution at -1V.
The EET device system developed by our team has successfully demonstrated its capability to measure concentrations ranging from 0mM to 1.8mM of Zn2+ in an LB+CM+Amp environment. This is supported by the high coefficient of determination (R2) of 0.971 achieved with our established calibration curve. Furthermore, the EET device system not only aligns with the initial preliminary requirements, including portability, lightweight, immediate and precise results, user-friendliness, accessibility, affordability, and potential for future prevalence, but it also achieves the goal of creating an anaerobic environment. By designing this EET device system, we have created something for everyone to use, allowing measurement of various substances, including metal ions, electrical signals, and many other related extensions.
Based on the successful measurement of Zn2+, we can reasonably infer that the EET device similarly possesses the ability to detect the electrical signals released by the EET system and can be further developed to expand our capacities for measuring biomarkers and sensing inflammatory responses. Whether it is sensing electrical signals, biomarkers, or inflammatory responses, all of these aspects require repetitive testing to establish more precise results in future work.
For the future improvement of the EET device, we will continue to adhere to our preliminary requirements. Regarding the screen-printed electrodes, we will seek more stable electrode materials, such as AgCl for the reference electrode material, which is more stable than the current Ag material. In the context of the potentiostat, we will consider factors such as price and size during the selection process, to incorporate it into the EET device (USB-sized ones are already available in the market). Additionally, our plans for future work include the development of appropriate software and the integration of Bluetooth for the device.
In conclusion, through comprehensive design and hardware and software upgrades of the EET device, we aim to achieve the goal of widespread adoption of a portable and precise sensing device for monitoring human inflammatory responses.
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