I Introduction

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 [1]. 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.

-Core-

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.

-Addition-

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.

II Basic Design

II-1 Outlook

　In making the fluorescence measurement kit, the first step is to measure the fluorescence that can be observed by the naked eye.

II-2 Hardware stage

We decided to divide the hardware creation process into four stages of hierarchy. They are the physical environment, the reading environment, the analysis environment, and the interface/communication environment. Although each of these is interrelated, they can be considered as independent stages to some extent, because it is easier to conduct development in this way.

(1) Physical Environment: Accurate data can be obtained regardless of location. Examples include the type, exposure, and intensity of excitation light, avoiding ambient light, and installing a filter on the camera.

(2) Reading Environment: It always reads the same way on the basis of (1) above. Depending on the sensitivity of the camera's color sensor, it may not be possible to correctly measure either ratio. If there is a significant difference compared to a standard such as Negacon, analysis is possible, so it is necessary to use a reading device such that such a difference exists.

(3) Analysis Environment: Analyze the image data read in (2). Since it is sufficient to confirm whether fluorescence is present in the image, it is sufficient to accurately determine that there is a significant difference between the negative control and the sample at low concentrations as well as at high concentrations.

(4) Interface/communication Environment: This is installed to operate the equipment, output the analysis results, and store the data in a PC or data center. It includes power switch, positive/negative judgment, data output, etc.

Up to this stage, four of the prerequisites (accuracy, reproducibility, convenience, and communication) are included. The remaining two prerequisites (economy and mobility) depend on the materials and components used in the specific design stage.

To get a feedback about our basic design, we had a discussion with a technician in Shimadzu Corporation. More information Discussion

figure2:Image of our hardware

II-3 Measurement method

In the development of this instrument, the fluorescent substance must be adjustable in concentration. This is because we want to move the fluorescence phase from a state that can be observed by the naked eye to a low concentration state that cannot be observed by the naked eye. For this purpose, we used a solvent-based acrylic resin paint as the fluorescent substance (Mr. Color C-175, Creos [2]). The excitation wavelength of this was the fluorescence wavelength. A lacquer solution (Mr. Color Thinner, Creos[3]) was also used to dilute it. From now on, the ratio of paint to thinning solution will be expressed as a ratio such as (amount of paint)/(amount of thinning solution), which will be used to determine whether fluorescence could be observed. The ratio of the undiluted solution to the diluted solution is the undiluted solution: diluted solution = x:30 (0<=x<=10, where x is an integer).Another data data section

figure3:Samples used in the experiments

In order to determine if the prototype measurement is valid, a comparison experiment is needed. In this study, measurements were performed using a plate reader (TECAN Infinite 200 PRO)[4]. Although the above samples were used in the development of the instrument, the plate reader was used to measure the paint itself, not the above samples.

The measurement results are as follows.

figure4:Excitation wavelength = 470 nm, Fluorescence wavelength = 500 nm The graphs obtained with a plate reader generally look like this. The graph is proportional at low concentrations (amount of paint $<=$ 2), and at high concentrations, the graph is affected by concentration quenching [5]. The accuracy of the plate reader is considered to be quite high, and the closer to this measurement result as possible, the better the measurement.

III Prototype

III-1 Outlook

In the prototype, we did not create an interface/communication environment (4) in the hardware stage. Instead, we decided to deepen each of the (1) physical environment, (2) reading environment, and (3) analysis environment, as well as the sequence of events, in order to improve accuracy and reproducibility, which are the core prerequisites for the prototype. The basic environment of the prototype is as follows

(1) Physical Environment

(1)-1 Excitation light

The fluorescent paint used in this study emits fluorescence (at wavelengths in the ultraviolet region), so a blue LED containing fluorescent paint was used as the excitation light.

figure5: Wavelength characteristics of the LED[6] The position of the excitation light was the plane on which the camera was set up, and two were placed diagonally opposite each other. 15 mA of constant-current diode current was applied to the LED bulbs. The direction of the bulb was adjusted so that it would not hit the sample as much as possible.

figure6: position of LEDs and camera = The figure on the left represents an image of the device viewed from the side. The bottom image is a well plate. The image on the right shows the device viewed from the top.

(1)‐2 Shooting Environment

　Since light from the periphery is noise, a 76 $\times$ 128 $\times$ 130 (mm) rectangle was made from construction paper to minimize it as much as possible. However, the side of 76 $\times$ 128(mm) was not attached because of the placement of the sample to be read and the installation of the camera. The inside of the rectangle was made of gray construction paper, and the joints were reinforced with duct tape to prevent noise light from entering the rectangle as much as possible.

(2) Reading Envioronment　

This time, we used a camera. The type is ELECOM UCAM-C820ABBK [7].

The performance is as follows.

Specification
Image receiving element	1/3-inch CMOS sensor
Number of effective pixels	Approx. 2 million pixels
Picture angle	Diagonal 76 degrees
Lens F value	F2.5
Focus method	Auto focus
Shooting distance	5cm to infinity (from the tip of the lens)

figure7: The camera

(3) Analysis Environment

The color for each pixel is represented in hsv color space, the threshold is set from a sample of negacons and undiluted solution, and the percentage of pixels that reach the threshold is compared. For more information, see the Analysis section.

The procedure for creating the basic physical environment is as follows

create a vertical cylinder of 76 $\times$ 128 $\times$ 130(mm) on construction paper. At this time, the sides of 76 $\times$ 128(mm) are not attached.
Cut out a rectangle of 76 $\times$ 128(mm) from the construction paper and make a 20 $\times$ 20(mm) hole for the camera in the center and a hole for the excitation light symmetrically on the diagonal of the rectangle.
join 1 and 2 together with duct tape. Also, assemble the excitation light circuit.

figure8: Electrical circuit we used = V1 is a 9V alkaline battery, R1 is a 200 Ω resistance, D1 and D2 are the blue LEDs.

III-2 Prototype ver.1

In prototype ver. 1, the above basic design was retained in the creation process. The results of the analysis were as follows.

figure9 The result(ver1) = Horizontal axis is the amount of paint, 0 is the negacon and 30 is the undiluted sample. Vertical axis is the ratio of fluorescence pixels(%).The original data is here.

Considerations

　ver.1, the percentage of pixels determined to be fluorescent increases with concentration to some extent. However, at low concentrations, there is no such trend. This is thought to be due to the fact that the blue excitation light appears to be stronger than the fluorescence in the image, and this excitation light is considered to be noise when measuring fluorescence at low concentrations.

It is necessary to suppress noise such as excitation light in order to make a clear difference even when the concentration is thin. Therefore, ver2 and ver3 were created. In the following, the basic design of III-1 was not changed, and improvements were made by adding elements to it.

Before making another prototype, we asked a technician for advice (Discussion).

III-3 prototype ver.2

Improvements

Black drawing paper was pasted on five inner surfaces (except for the surface where the camera is located). This was done to block out as much as possible other light and elements that would be noise in the fluorescence observation.

The results are as follows.

figure10 The result(ver2) = Horizontal axis is the amount of paint, 0 is the negacon and 30 is the undiluted sample. Vertical axis is the ratio of fluorescence pixels(%).The original data is here.

Considerations

We were able to acquire images more clearly than ver1. However, the analysis was not able to read differences in fluorescence due to concentration compared to ver1.

III-4 prototype ver.3

Improvements

The filter was created using cellophane.

There are four filter patterns:(1) blue, (2) green, (3) blue and green (green is for the camera side), and (4) green and blue (blue is for the camera side), with each color consisting of two layers of cellophane. Since blue and green are the same color as the excitation light and fluorescence, respectively, the experiment was conducted with the idea that the colors by themselves or in combination would have the effect of reducing the intensity of the excitation light entering the camera.

The results are as follows.

(1) Blue

figure11:The result(ver.3, (1) Blue) = Horizontal axis is the amount of paint, 0 is the negacon and 30 is the undiluted sample. Vertical axis is the ratio of fluorescence pixels(%).The original data is here. Considerations

The filter is not suitable for (1), since there was almost no difference between the negative control and the original solution.

(2) Green

figure11:The result(ver.3, (2) Green) = Horizontal axis is the amount of paint, 0 is the negacon and 30 is the undiluted sample. Vertical axis is the ratio of fluorescence pixels(%).The original data is here.

Considerations

The fluorescence of (2) was not well quantified at low concentrations and seemed to be in the same state as when there was no filter.

(3) Blue and green (Green is for the camera side)

figure11:The result(ver.3,(3) Blue and green (Green is for the camera side)) = Horizontal axis is the amount of paint, 0 is the negacon and 30 is the undiluted sample. Vertical axis is the ratio of fluorescence pixels(%).The original data is here.

Considerations

The fluorescence of (3) decreased with concentration. This is the same amount of fluorescence as that observed in the plate reader, so it can be said that the reading is relatively good.

(4) Green and blue (Blue is for the camera side)

figure11:The result(ver.3,(4) Green and blue (Blue is for the camera side)) = Horizontal axis is the amount of paint, 0 is the negacon and 30 is the undiluted sample. Vertical axis is the ratio of fluorescence pixels(%).The original data is here.

Considerations

The same trend as in (3) can be read for (4). However, at 30:1, it is perceived much lower than at (3).

From the above, it was found that some filters were able to acquire clearer images and make appropriate measurements than ver1 and ver2. The measurement of (3) or (4) was relatively good, although there is room for further improvement.

IV Conclusion

IV-1 Completed Prototype

The findings from the prototype created in III were used to create the finished product.

figure14: Drowing = The unit is mm. The bottom left figure is a top view. A small square in it shows the camera lens location, and two small circles around it are LED locations. The top left and bottom right figures are side views.

figure15: Completed Prototype

The features of this device include the following.

The inner surface is all black to block out light from the outside.
Filters made of colored cellophane (green for the camera side and blue for the object to be measured) are used. The reason for this is that we thought that (3) would provide better measurement at low concentrations when compared with (3) and (4) in prototype ver. 3.
Blue LED was used as the excitation light.

The results using this method are shown below. In addition, samples with thinner ratios (40, 50, 60, 70, 80, 90, 100, and 200:1) than the 30:1 concentration ratio used earlier were also prepared.

figure16: The result(Completed Prototype)=Horizontal axis is the amount of paint, 0 is the negacon and 30 is the undiluted sample. Vertical axis is the ratio of fluorescence pixels(%).The original data is here.

Considerations

The results of the measurement were generally similar to those obtained with the plate reader, although the scales were different. The results were generally similar to those obtained with the plate reader, although the scale was different. This is consistent with the fact that the amount of fluorescence is proportional to concentration at low concentrations.

IV-2 Conclusion

In this time, ideal measurement results were obtained up to a concentration ratio of 30:1. However, the measurement results could not be obtained with good accuracy at lower concentrations. The following factors may have contributed to this. Poor sensitivity of the camera or automatic processing in the camera caused the fluorescence intensity to be observed to be smaller than it actually was. The analysis program is not appropriate for low concentrations. Inability to accurately quantify the solution. We will try to solve the problem of the camera itself by using other sensors (e.g., photodiodes) instead of the camera in the future. Regarding the analysis program, this time only Hue (hue) of the HSV color space was used for analysis, and Saturation and Value were not used. As for quantification, the viscosity of the undiluted solution is high, so we would like to improve the program by creating a larger quantity or diluting the solution from a solution of one-tenth of a solution.

IV-3 Future prospects

　The following is a list of concerns and their solutions in terms of the six prerequisites: accuracy, reproducibility, convenience, mobility, communication, and cost performance. 　

In terms of accuracy, there are three points.

Concerns

(1) As mentioned in the summary, the camera's internal processing was not able to detect subtle differences.

(2) The fluorescence concentration of GFP and mCherry expressed from the actual cells could not be approached to the pseudo fluorescence concentration.

(3) Noise other than fluorescence (excitation light, light leaking from outside, etc.) could not be accurately cut off.

Solution

(1) Use a sensor such as a photodiode instead of a camera.

(2) Conduct the measurement at lower concentrations than the camera. In this case, be careful about the method of quantification as described in the summary.

(3) Change from the current color film to colored glass filter. However, the measurement results were not necessarily poor even with the current film, so we would like to consider this while taking economic efficiency into account.

These three points are what we need to do in the future to improve accuracy. 　

In terms of reproducibility, there are three points

Concerns

(1) The environment was not completely free of noise.

(2) The temperature and humidity in the area where dengue fever occurs may be very different from our measurement environment.

(3) The intensity of the excitation light was not uniform from place to place. Also, the analysis program did not work well unless the position was fixed.

Solution

(1) We implemented a completely black condition, eliminating all other wavelengths. We would like to create a space where no light can enter from the outside, starting from the selection of materials based on a precise design.

(2) We will make sure that the experimental results do not fluctuate due to changes in the measurement environment, such as humidity and temperature. Confirm that the system will operate accurately under such an environment without malfunctioning.

(3) We need to consider the placement of the sample in a way that the excitation light is uniformly illuminated. We would like to improve the system so that analysis can be performed regardless of the sample position.

Two points in terms of convenience.

Concerns

(1) Local people may not be able to operate the equipment.

(2) The more complicated the device is, the more expensive and time-consuming it will be to repair it in case of damage.

Solution

(1) We want to design an interface that is easy to use. Although we did not go as far as creating software for use in this time, we would like to create control software with a user interface using non-verbal icons that can be used by beginners of any nationality.

(2) We would like to create a device that is simple in structure. We would like to design a simple device and improve it so that it is less expensive and easier to deal with in the event of damage.

Two points in terms of mobility.

Concerns

(1) There are areas where it is difficult to move the device.

(2) Need a power supply for use in various locations.

Solution

(1) Make it smaller and lighter. Smaller and lighter weight will reduce the cost of transportation and improve accessibility.

(2) Make the device battery-powered only.

In terms of communication, it is one point.

Concerns

(1) Communication between the device and other devices will be required.

Solution

(1) Whether to connect directly to 4G or 5G, wireless communication over a short distance, or wired communication needs to be considered after more careful consideration of actual usage conditions.

From a cost performance perspective, this is a single point.

Concern

(1) The price will be too high.

Solution

(1) This time, the completed product cost less than 20,000 yen all-inclusive. However, the accuracy of the device is not yet satisfactory, so we would like to research and develop a device that is cheaper and uses materials that do not impair accuracy.

V Reference

[1] Fluorescence spectrophotometer (RF) by maker, price comparison all about laboratory by aoyama1954 https://bunseki-keisoku.com/article/normal/rf/ (Oct/2/2022)

[2] Mr. Color My hobby https://www.mr-hobby.com/ja/product1/category_7/87.html (Oct/2/2022)

[3] Mr. Color Thinner My hobby https://www.mr-hobby.com/ja/product1/category_7/153.html (Oct/2/2022)

[4] Infinite 200 PRO TECAN https://lifesciences.tecan.co.jp/plate_readers/infinite_200_pro (Oct/2/2022)

[5] Basics of Spectrofluorometer (5) Precautions for Spectrofluorometer JASCO https://www.jasco.co.jp/jpn/technique/internet-seminar/fp/fp5.html (Oct/2/2022)

[6] OSUB5111A-ST Data Sheet Akizuki Denshi Tsusho　 https://akizukidenshi.com/download/ds/optosupply/OSUB5111A-ST.pdf (Oct/2/2022)

[7] UCAM-C820ABBK ELECOM https://www.elecom.co.jp/products/UCAM-C820ABBK.html (Oct/2/2022)