Introduction
The goal of our project is to create a device that can easily quantify fatigue. There have been attempts to estimate the degree of fatigue by asking the patients about their current conditions, but this method relies on the sensation of fatigue. However, it is difficult to use this feeling of fatigue as an objective indicator because it varies from person to person and several factors, including the environment. By creating such a device that can quantify fatigue in a simple way, we believe that it will be the first step to understanding fatigue that we are not aware of.
In order to create the device, we interviewed Prof. Horita, an assistant professor of the Department of Psychiatry and Neurology at Gifu University and a clinical psychologist (Take a look at our Human Practice). As a result, we set the course of the project by being conscious of a user interface that did not require specialized knowledge.
In our current policy, unlike RT-qPCR, the experimenter who performs the quantification does not need specialized experimental skills or knowledge. On the other hand, to quantify HHV-6, a biomarker, it is necessary to measure the fluorescence of the probe, which requires equipment such as a microplate reader. A design that requires such expensive equipment makes it difficult to achieve the goal of simple stress quantification in the field. Therefore, we devised a simple kit using lateral flow. The lateral flow is a method that is used in combination with Cas-based virus quantification methods such as SHERLOCK and DETECTR.
The mechanism is as follows.
1. First, the FAM-biotin reporter is cleaved by the collateral cleavage activity of Cas12a or Cas13a.
2. When the sample is dropped onto a lateral flow pad, the sample is transferred by capillary action.
3. The cleaved and uncleaved reporters bind to antibodies on different bands, and the more cleaved the reporter, the greater the amount of virus.
Although lateral flow has been used in many qualitative tests, we focused on the possibility of quantifying it by measuring the intensity of the bands using image processing software. In addition, we focused on smartphones so that image processing can be performed using tools that are familiar to the general public. Since a smartphone has both a computer and a camera necessary for image processing, we developed hardware to assist in taking pictures and enclosed it in the kit to make it a simple fatigue quantification kit.
In order to create the device, we interviewed Prof. Horita, an assistant professor of the Department of Psychiatry and Neurology at Gifu University and a clinical psychologist (Take a look at our Human Practice). As a result, we set the course of the project by being conscious of a user interface that did not require specialized knowledge.
In our current policy, unlike RT-qPCR, the experimenter who performs the quantification does not need specialized experimental skills or knowledge. On the other hand, to quantify HHV-6, a biomarker, it is necessary to measure the fluorescence of the probe, which requires equipment such as a microplate reader. A design that requires such expensive equipment makes it difficult to achieve the goal of simple stress quantification in the field. Therefore, we devised a simple kit using lateral flow. The lateral flow is a method that is used in combination with Cas-based virus quantification methods such as SHERLOCK and DETECTR.
The mechanism is as follows.
1. First, the FAM-biotin reporter is cleaved by the collateral cleavage activity of Cas12a or Cas13a.
2. When the sample is dropped onto a lateral flow pad, the sample is transferred by capillary action.
3. The cleaved and uncleaved reporters bind to antibodies on different bands, and the more cleaved the reporter, the greater the amount of virus.
Although lateral flow has been used in many qualitative tests, we focused on the possibility of quantifying it by measuring the intensity of the bands using image processing software. In addition, we focused on smartphones so that image processing can be performed using tools that are familiar to the general public. Since a smartphone has both a computer and a camera necessary for image processing, we developed hardware to assist in taking pictures and enclosed it in the kit to make it a simple fatigue quantification kit.
To implement in the real world
We had initially aimed to create a kit for medical diagnosis and application in the medical field, however, according to Prof. Horita, it became clear that application for medical purposes would be difficult due to legal restrictions. Therefore, we decided to change the way of operation of our system to a device for the purpose of maintaining health in daily life, instead of aiming for medical applications. Since Gifu University holds a health promotion seminar every year, we are thinking of setting up a booth for simple measurement of fatigue as part of the planning of this seminar. We are also planning to use our device at the Gifu University Exhibition, which is also a participatory exhibition, to reflect our device in the real world.
About our Safety
The devices we have designed do not use substances that are toxic to the human body or the natural environment. In addition, we do not use genetically modified organisms in the entire process of searching for targets, binding to targets, and releasing signals, which are important points in biosensors.
On the other hand, since the test requires the collection of saliva, care must be taken in the management of the samples collected from humans. Gifu University requires prior application before conducting any experiments on human subjects, including samples collected from humans. There is an internal code of ethics that must be followed when conducting these experiments, and we need to follow it as well.
Since it is possible to read genetic information such as an individual's genome from saliva, it is necessary to clarify the usage and disposal procedures of the collected saliva. Also, the measured data can be personal information. In order to prevent problems arising from personal information, it is necessary to inform subjects in detail about the use, storage, and disposal of data, and to obtain their approval.
By complying with the university's regulations and providing sufficient informed consent to the subjects, our fatigue quantification kit can be used in projects such as the one described in "To implement in the real world".
Application in the future
Figure1. The device we created
Figure 1 shows a simplified system we developed to measure fluorescence intensity. To reduce the cost, we used cardboard as the material. When we actually measure the fluorescence intensity, we use a blackout curtain to block as much natural light as possible.
The captured image is converted into an HSV model consisting of hue, saturation, and brightness, and the object on each image is divided into nine regions. Then, the average brightness of each region is measured. This operation will be repeated every 30 seconds to enable the measurement of temporal changes in the fluorescence intensity of the regions. The position of the object on the image and the coordinates of the nine regions can always be fixed by using the hardware that we created. The source code is on our GitHub.
Furthermore, by incorporating a filter into the device, it will be possible to extract excitation light <480nm~500nm> and fluorescence <515nm~525nm>, enabling accurate measurement of the fluorescence intensity. By actually measuring the time variation of the fluorescence intensity of specimens and clarifying the relationship between the fluorescence intensity and the concentration of HHV-6, it will be possible to estimate the concentration of HHV-6 from the fluorescence intensity. By using this, we believe that we can build a system to quantify the fatigue level of patients using only this hardware and a smartphone. In addition, although we used cardboard as the material for the hardware this time, we believe that by conducting hardware 3D CG modeling and making it available to the public, it will be possible to create hardware using a 3D printer and standardize the hardware.
References
1) Gootenberg JS, Abudayyeh OO, Kellner MJ, Joung J, Collins JJ, Zhang F (2018) Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6. Science 360:439–444 .
2) Sun Y., Yu L., Liu C., Ye S., Chen W., Li D., Huang W. One-Tube SARS-CoV-2 Detection Platform Based on RT-RPA and CRISPR/Cas12a. J. Transl. Med. 2021;19:74.
3) iGEM Lambert_GA 2020