We must sense the fluorescence of GFP and mCherry expressed in the test kit developed by Wet lab to determine if antibodies to the dengue virus are generated in the serum used for the specimen. The measurement results are used for diagnosis, so the fluorescence must be quantitatively and accurately discriminated. However, there are few advanced instruments such as fluorescence spectrometers in Southeast Asian hospitals today, and the cost of installing them is high. They generally cost more than 2 million yen to install . In addition, fluorescent spectrometers generally require a power source rather than batteries, making them difficult to use in areas where electricity is not available or where the power supply is unreliable. Our project goal is to collect data on dengue serotypes in all locations, analyze the data, and make epidemic forecasts. To achieve this, we first need to have as many people as possible tested in as many areas as possible.In order to encourage more people to take the test, the hardware must do as follows.
Accuracy: With the assumption, the test needs to be accurate.
Reproducibility: The results must be the same no matter where or who performs the test.
Convenience (non-verbal): It should be easy to use for medical professionals and government officials who conduct inspections regardless of age or language.
Communication: If measurement data can be communicated to PCs, etc., regardless of whether it is wired or wireless, it will be easier to collect data for epidemic forecasting.
Mobility: It needs to be small and easy to move around, as it may be difficult to install in some places if it is large in size.
Cost performance: should be inexpensive so that it can be used in various locations (hospitals), regardless of the region. figure1: Image of our premises
To confirm the principle of fluorescence observation, GFP is expressed from all cells as a proof of cell survival, while mCherry is expressed from non-infected cells as a proof that the cells are not infected with the virus, as described in Wet lab. Therefore, the presence or absence of GFP and mCherry in a well can be confirmed through fluorescence to confirm infection of the serum donor with dengue virus. Fluorescent proteins emit specific fluorescence wavelengths upon receiving specific excitation light, and since the excitation and fluorescence wavelengths of GFP and mCherry are different, they can be discriminated by using filters.
Fluorescence can be observed and measured on a laboratory scale using instruments such as a fluorometer, absorbance spectrophotometer, or fluorescence microscope. Generally speaking, fluorescence is used qualitatively rather than quantitatively, as in the case of fluorescent pens and fluorescent paints, where one can only visually determine whether or not fluorescence is present when an excitation light is shone on the material. Considering that the fluorescence in this case is very weak, it is necessary to use equipment such as that in a laboratory, as mentioned above.
However, we do not need such sophisticated measurement equipment. We only need to be able to determine whether there is GFP, mCherry, or no GFP. Our target is an instrument that can determine the presence of fluorescent substances at low concentrations. If the intensity of fluorescence exceeds a certain threshold value, it is judged that fluorescent substances are present, and if not, it is judged that fluorescent substances are not present. As a preliminary step, we are developing an instrument that can determine the amount of fluorescence at a certain level of fluorescence. We are confident that this instrument will lead to the development of an instrument that can easily determine the presence of fluorescence at low concentrations in the future, and that it will be a highly versatile instrument that can be applied to other experiments as well.