Team:ZJUT-China/Hardware
We built a handheld illuminator and a fluorescence detector, both of which are simple in construction and easy to reproduce.
Fluorescent reporters have enabled numerous novel applications in diagnostics and point-of-care testing over the past few years. However, most of them rely on laboratory instrumentation to visualize and quantify the fluorescence signal. To overcome this challenge, we have developed a handheld illuminator and a fluorescence detector to support field-applications of Cell-Free biosensors for diagnostics. Consisting of low-cost electronic components, light filters and 3D printed cases made of biodegradable polylactic acid, the handheld illuminator and fluorescence detector are designed to be affordable, portable and recyclable. Also, interchangeable LEDs and light filters allow illuminator and detector to be customized to match the fluorescence wavelength of interest.
We developed a handheld illuminator that provides visualization of fluorescence using the naked eye.
Figure 1. (A) 3D model of handheld
illuminator (simplified).(B) All components of the handheld illuminator.
The handheld illuminator combines LEDs and light filters as a simple, practical method to detect fluorescence qualitatively. In addition, detection can be quantitative by using imaging analysis software such as ImageJ. We believe that the handheld illuminator is suitable for all cell-free sensor-based diagnostic methods.
For many diagnostic applications aim simply to determine whether the reporter is present in the sample, providing a positive or negative result to the user.
Figure 2. (A) Working principle
of our fluorescence detector (our design uses a 470 nm LED and 510 nm filter). (B)
Visualization of eGFP by handheld illuminator under normal light. (C) Visualization
of eGFP by handheld illuminator under dark conditions. Both conditions have a
negative control on the right, followed by eGFP at concentrations of 0.55 μM, 1.09
μM, 2.19 μM, 4.38 μM, 8.75 μM, 17.5 μM, and 35 μM, respectively. Note that the
negative control also shows some degree of fluorescence. Since we did not set an
excitation filter between the light source and fluorescent substances, and the 470
nm LED also emits light at wavelengths above 525 nm, we assumed that the
fluorescence from the negative control was the result of reflection or
autofluorescence of the test tube.
The illuminator consists of inexpensive electronic components (e.g., LEDs, battery holder), 3D-printed cases and a light filter, for a total price of no more than $10. Furthermore, the illuminator can easily be assembled with glue or even double-sided tape.
We also designed a fluorescence detector that can be used for quantitative fluorescence detection.
Figure 3. (A) Working principle of
our fluorescence detector. (B) A 3D illustration of the fluorescence detector.
Our fluorescence detector performs well in terms of accuracy and sensitivity, with a detection limit of approximately 0.3 μM (12 μL volume) for eGFP. We believe our fluorescence detector can meet the needs of Cell-Free biosensor-based diagnostics.
Figure 4. (A) Standard curve of
eGFP using our fluorescence detector. (B) Our design of fluorescence
detector.
For the fluorescence detector, we use a 470 nm LED as the light source, a photo-diode was used for measuring fluorescence intensity and converting it into specific voltage. To improve the accuracy of the detector, two light filters are used as excitation filter (470 nm) and emission filter (525nm) respectively. Arduino UNO R3 records data and transmits it to the mobile phone via bluetooth. By the way, we also built a software for analyzing and displaying the results. The flow chart for using the software is shown in the Software.
The characteristics of the photodiode and the circuit of the fluorescence detector are shown in the Supporting Material.
During the development of the fluorescence detector, we tested a variety of photosensitive elements. The process and conclusions of the device iteration are shown in the History.