Our project is to achieve the treatment of diabetes patients by controlling the synthesis of insulin. In our project, we used GAL4 and VP16 proteins. Specifically, pGIP GI-Gal4 and pGIP LOV-VP16 were used to transcribe the "Gal4" and "VP16" proteins at high glucose concentrations, followed by brief blue light irradiation to form the Gal4-VP16 transcription factor. Furthermore, it binds to the promoter 5xUAS to promote insulin synthesis and expression.
In our experiment, the expression level of insulin sharply increased in cells with Gal4-VP16 transcription factor present. Therefore, we need to control the synthesis of the Gal4-VP16 transcription factor, which is controlled by blue light, so that our system can fully synthesize and release insulin when insulin secretion is needed, without allowing excessive insulin expression to lead to low blood sugar levels in the body. So we designed a "blue light instrument" hardware for this purpose.

We first determined the function of the blue light instrument, which is to emit blue light when the glucose concentration is high and turn off blue light when the glucose concentration is low. Therefore, we break down the hardware's functional implementation into three main parts: obtaining glucose concentration, judging the concentration, and turning on or off blue light. Among them, the determination of glucose concentration and whether to turn on blue light is achieved through programming. For hardware design, the first problem we need to solve is to obtain glucose concentration.
We have studied the commonly used methods for obtaining glucose concentration and learned about glucose test strips using enzymatic principles, detection methods using optical principles, gel based separation free point of care (POC) devices and so on. Due to the fact that our instrument needs to be used for laboratory experiments, we have chosen a more accurate glucose test strip detection method.
Our hardware design includes detection instruments and glucose test strips. When we insert one end of a glucose test strip into the sensor interface and immerse the other end into the test solution, the glucose in the solution can undergo a chemical reaction with the glucose oxidase in the test strip, and the glucose is oxidized to generate hydrogen peroxide. Then, the hydrogen peroxide content is detected, and the blood glucose value is measured.

In the thinking one, we achieved the acquisition of glucose concentration. In this step, we need to address the issue of blue light. We need to design an instrument that can emit blue light, which can emit light that can bind Gal4-VP16 transcription factor, and the blue light switch can be controlled by a program. We can use software and transmission systems to convert the judgment of whether the glucose concentration exceeds the threshold into an instruction to turn on blue light.

Our school's 2021 participating team (2021 CSU-CHINA) used a blue light instrument that can be used for experiments. It has a switch that directly controls the blue light and can adjust the brightness. The blue light hardware part of this instrument is very valuable for us to learn from, which proves the feasibility of our second thought.

But at the same time, we also found its shortcomings. It does not have a glucose detection device and is more like a manually controlled blue light source device. In order to complete our thinking and optimize the design and production of blue light instrument, we have communicated and cooperated with Luzhai Technology Company.

From an experimental perspective, we need to ensure the practicality of the blue light instrument in specific experiments. In the initial equipment production, we used the blue light parameters of the 2021 CSU-CHINA team. (the experimental environment and materials related to blue light are basically the same as those of the 2021 CSU-CHINA team)

In addition, we have designed two control methods for the blue light switch. One is to control the glucose concentration according to our thinking, and the other is to manually control it to meet various experimental requirements.

After design and improvement, we have drawn a basic design sketch. It can detect glucose concentration, display glucose concentration, and control blue light.

Subsequently, we refined the images and referred to them to create our blue light instrument.

The following picture is a picture of our blue light instrument. It consists of three parts, the small box below the picture is the glucose detection device (Figure 7), the monitor on the left side of the picture (Figure 8), and the largest device in the upper right corner of the picture is the instrument emitting blue light (Figure 8).

This is a glucose detection device that uses electrochemical technology to sense chemical changes in glucose solution through electrodes, which are then converted into electrical signals, such as electrochemically active enzymes (such as glucose oxidase or glucose dehydrogenase) that can catalyze glucose and produce electrochemical reactions. During this process, the electrons generated by the reaction between glucose and enzymes are captured by the electrode, resulting in the generation of an electric current. The magnitude of the generated current is proportional to the concentration of glucose, and by accurately measuring this current, the device can accurately calculate the concentration of glucose. Thus, it can detect the instantaneous concentration of glucose solution when one end of the test strip is inserted into the glucose detection instrument and the other end is immersed in the glucose solution.

Figure 9.parameters of instrument emitting blue light

Our experiment on the above study was conducted in 293T cell culture dishes, with cells cultured in 24 well plates. The desired plasmid was transfected into the cells at an appropriate density, and after 36-48 hours of constant temperature and CO2 culture, cells with further growth were taken out of the cell chamber, lysed, and subsequently added with luciferase and renilla fluorescent dyes. The expression level of dual luciferase was detected to simulate the expression level of insulin.

How do we conduct experiments with our instruments? On the second day after transfection (about 24 hours later), we took out the cells that needed to be irradiated with blue light from the culture box and exposed them to blue light for 15 minutes in a closed box. The control group was wrapped in tin foil throughout the transfection process to minimize the chance of light stimulation. After exposure to blue light, place the board back in the incubator and continue to culture until fluorescence is measured.

In addition, we prepared solutions with different glucose concentrations to simulate different blood glucose levels in the human body, in order to explore the secretion of insulin at different blood glucose levels.

In the background, we mentioned that our project utilizes pGIP GI-Gal4 and pGIP LOV-VP16 to transcribe the "Gal4" and "VP16" proteins at high glucose concentrations, followed by brief blue light irradiation to form the Gal4-VP16 transcription factor, which then binds to the promoter 5xUAS to promote insulin synthesis and expression. In the laboratory, we replaced the Insulin gene with Luciferase, which allows us to observe the fluorescence intensity to determine the status of the Gal4-VP16 transcription factor, i.e. the effect of blue light on the formation of the Gal4-VP16 transcription factor.
We co transfected 5XUAS luciferase and plasmids expressing GI-Gal4 and LOV-VP16 into 293T cells and cultured the cells in the absence or presence of blue light. As shown below, when cells were exposed to blue light for 15 minutes, luciferase activity increased by approximately 300 times. Therefore, our data confirms that blue light can stimulate the formation of active transcription factors in GI-Gal4 and LOV-VP16.

Figure 10. Blue light can stimulate the formation of an active transcription factor from GI-Gal4 and LOV-VP16

We tested the blue light instrument, measured and corrected its parameters (such as setting different glucose thresholds for experiments, detecting light intensity ranges, etc.), and ensured that it could correctly detect glucose concentration and control blue light (operation video), as well as emit blue light that enabled our experiment to be successfully completed(figure 9).

In the test, we noticed that the high power output of the LED brought a lot of heat. To solve this problem, we modified the device and added a heat sink at the bottom of the circuit board. To further assist with cooling, we have added two Noctua 12V fans. The speed of the fan is regulated by a thermistor, which acts as a thermal sensor. This adjustment allows the fan to respond to the temperature of the system, ensuring that the entire setup remains cool. In addition to maintaining the normal operation of the equipment, this also plays a crucial role in keeping the bacterial culture in the micropores at a consistent temperature.

We have strengthened the sealing of the blue light instrument to prevent interference with the control group cells that do not require light outside the device. And, within the range that does not affect the experimental results, we have increased the number of LED lights on the panel to ensure that every inch of the 24 hole board receives maximum intensity of blue light direct.

In order to enable the device to adapt to more experiments, we have added the function of autonomously adjusting the light intensity. In addition, due to the high energy of blue light, it causes certain damage to our cells. We considered replacing the instrument emitting blue light with instrument emitting red light, but preliminary experiments have shown that only blue light can meet the experimental requirements.

Our blue light instrument has helped us complete the experimental task, but we believe there is still some room for improvement.

Due to the absence of animal and human experiments in this year's project, there was no dynamic environment for glucose. But as the experiment deepens, we will encounter an environment that requires maintaining stable blood glucose dynamics, which requires real-time monitoring of glucose concentration. The test paper detection method we use obviously cannot meet this requirement, and currently many optical detection methods face the problem of low accuracy. We look forward to inventing better detection devices and methods to achieve this goal.

Our project will not stay in the laboratory forever. In the future, our project will be applied clinically and popularized in the daily life of patients with type I diabetes. Therefore, it is necessary to minimize and make blue light instrument as convenient as possible.

Through discussion, we plan to turn the instrument into a wristband and and inject engineered cells containing plasmids such as UAS insulin and transcription factors subcutaneously into patients. The sensor will be connected subcutaneously to detect changes in blood glucose. When the blood glucose concentration exceeds the threshold set by the instrument (i.e. the highest value within the patient's normal blood glucose range), it will automatically trigger the activation of blue light.

The achievement of this goal not only requires exploring the optimal equipment parameters through human experiments, but also requires our blue light instrument to achieve a leap in non-invasive and real-time detection of blood sugar.

Figure 11.Pictures from the Internet can explain our bracelet scheme device

Click here to view the hardware documentation!

1.Li CY, Wu T, Zhao XJ, Yu CP, Wang ZX, Zhou XF, Li SN, Li JD. A glucose-blue light AND gate-controlled chemi-optogenetic cell-implanted therapy for treating type-1 diabetes in mice. Front Bioeng Biotechnol. 2023 Feb 10;11:1052607. doi: 10.3389/fbioe.2023.1052607. PMID: 36845170; PMCID: PMC9954140.
2.Dai J, Zhang H, Huang C, Chen Z, Han A. A Gel Based Separation Free Point of Care Device for Whole Blood Glucose Detection. Anal Chem. 2020 December 15; 92 (24): 16122-16129.] Doi: 10.1021/acs.analchem. 0c03801. Epub 2020 Nov 2. PMID: 33137252.