Introduction

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We aimed to address the need for accessible, field-based environmental sample testing by creating a device that allows individuals, regardless of research experience, to test their water samples for Prymnesium parvum near water sources. Leveraging the versatility and cost-effectiveness of 3D printing, we focused on developing a low-cost prototype as part of our iGEM project. Our efforts culminated in the development of a device called the PrymChip.

The PrymChip is a 3D-printed device specifically designed to excite, detect, and quantify green fluorescence signals, with data processed using a Python script. The primary objective was to create an affordable, field-deployable tool capable of detecting Prymnesium parvum in environmental water samples using the SHERLOCK detection system. However, due to time constraints and limited reagent availability, we were unable to demonstrate algae detection using SHERLOCK within the PrymChip. Instead, we successfully developed a proof-of-concept device, demonstrating its functionality with fluorescein solutions. At this stage, the PrymChip excites green fluorescence signals, captures images via a smartphone, and analyzes the data using a Python script to calculate fluorescein concentration from the captured images. The final cost of the functional device quals to €20.38.

PrymChip is composed of three main components that work together to create a functional and practical tool:

• Detection Box: This section houses the reaction chamber where the test solution is placed. It is also the area where a smartphone is used to capture a photo for analysis.
• Electronics Box: This contains the core electronic components, including the circuit responsible for exciting the sample's fluorescence using blue LED lights.
• Tool Box: A storage compartment designed to hold necessary experimental equipment and reagents for the procedure.

The schematic of the device, with labelled components, is presented below (Figure 1).

Figure 1. Schematic of the PrymChip.

Structure of the Device

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All the 3D-printed parts were designed in TinkerCAD and printed using filament fused deposition modeling (FDM) technology with a Prusa i3 MK3 Printer. The generated STL files were sliced using PrusaSlicer 2.7.1. To support future iGEM teams and facilitate further modifications, we also created the final models of our 3D-printed parts in AutoCAD, allowing for easier adjustments in more advanced 3D modeling software. A detailed description of each box and its elements is provided in the sections below.

Detection Box

The detection box was printed using black filament to optimize fluorescence detection. At its center, there is a dedicated spot for the reaction chamber, where the test solution is placed to detect fluorescence emission. Holes on one of the box walls are designed to accommodate LED holders, ensuring the blue LEDs from the electronics box are securely and accurately positioned (Figure 2).

Figure 2. The detection box with labelled elements.

The lid of the black box features a droplet symbol, indicating its use for detecting fluorescence in liquid solutions. Additionally, the lid includes a hole for a green fluorescence filter, which blocks all colors except green from escaping the box. On top of the filter, there is a magnifying lens to help the smartphone camera better focus on the centre of the chamber (Figure 3). Both the filter and lens are glued to the lid to ensure they stay in place, although the lid was originally designed to hold them securely without adhesives.

Figure 3. The detection box’s cover lid with labelled elements.

The reaction chamber used in the PrymChip was repurposed from a hair spray bottle cap (refer to our Engineering cycle for details on this choice). The cap was cut to the appropriate height, and a designated black cover with a slot for placing a glass cover slip on top was 3D-printed (Figure 4). The glass cover slip was not glued in place due to its fragility, making it easier to replace if damaged—users could simply remove the broken cover slip and replace it with a new one from the set.

Figure 4. The reaction chamber with labeled elements.

The cost of the elements incorporated into the detection box, excluding the 3D-printed parts, is listed in the table below (Table 1).

Table 1. Cost of the elements incorporated into the detection box.

The 3D printing process for this box took 14 hours and 49 minutes, while the printing of the cover lid required 3 hours and 58 minutes. The printing process of a single support that was also included as a part of PrymChip was completed in 1 hour. The black filament used for printing costs 29,99 € per kg, which results in the costs of the printed parts detailed in the table below.

Table 2. Cost of the 3D-printed parts for the detection box.

For printing, we used Jet Black Prusa PETG filament. The general print settings were based on the '0.10mm DETAIL' preset from the Prusa Slicer system. Modifications to the default system presets are listed below:

• Infill: 30%
• Brim: enabled
• Supports: support on build plate only (organic)
• Fill pattern: Rectilinear
• Top fill pattern: Monotonic Lines
• Bottom fill pattern: Monotonic Lines
• Perimeters: 15 mm/s
• Small perimeters: 20 mm/s
• External perimeters: 20 mm/s
• Infill: 40 mm/s
• Top solid infill: 20 mm/s
• Gap fill: 20 mm/s
• Nozzle (first and other layers): 240 ⁰C 
• Bed (first and other layers): 70 ⁰C
• Filament type: PETG
• Retraction length: 5.5 mm
• Retraction speed: 50 mm/s

The parts we designed can be downloaded here. The downloads are listed below:

• Detection Box CAD Model (TinkerCAD)
• Detection Box Cover Lid CAD Model (TinkerCAD)
• Detection Box CAD Model (AutoCAD)
• Detection Box Cover Lid CAD Model (AutoCAD)

Electronics Box

The electronics box serves as the center of the electronic circuit responsible for exciting fluorescence (Figure 5).

Figure 5. The electronics box with labelled elements.

Its yellow color represents the lightning typically associated with electrical power. This symbol is also incorporated on the lid, which features a sliding mechanism for closing the box (Figure 6). 

Figure 6. The electronics box’s cover lid.

The box was designed to store essential electronic circuit components: LED diodes, battery, and the light switch. The circuit consists of three LED lights with built-in resistors that are connected to one another and soldered to ensure circuit stability. After soldering, the connections between the wires were secured with black hot shrink tubes. The LEDs are connected on one side to the light switch and on the other to the battery holder, which is placed on the battery powering the entire system. This electronic circuit is presented in Figure 7.

Figure 7. The electronic circuit included in the PrymChip.

The cost of the elements incorporated in the electronics box, excluding the 3D-printed parts, is detailed in the table below (Table 3).

Table 3. Cost of the elements incorporated into the electronics box.

The 3D printing process for this box took 19 hours and 18 minutes, while the printing of the cover lid took 3 hours and 38 minutes. The yellow filament used for printing cost 29,99 € per kg, resulting in the costs of the printed parts listed in the table below (Table 4).

Table 4. Cost of the 3D-printed parts for the electronics box.

For printing, we used Mango Yellow Prusa PETG filament. The general print settings were based on the '0.10mm DETAIL' preset from the Prusa Slicer system. Modifications to the default system presets are listed below:

• Infill: 30%
• Brim: enabled
• Supports: support on build plate only (organic)
• Fill pattern: Rectilinear
• Top fill pattern: Monotonic Lines
• Bottom fill pattern: Monotonic Lines
• Perimeters: 15 mm/s
• Small perimeters: 20 mm/s
• External perimeters: 20 mm/s
• Infill: 40 mm/s
• Top solid infill: 20 mm/s
• Gap fill: 20 mm/s
• Nozzle (first and other layers): 225 ⁰C 
• Bed (first and other layers): 70 ⁰C
• Filament type: PETG
• Retraction length: 5.5 mm
• Retraction speed: 50 mm/s

The parts we designed can be downloaded here. The downloads are listed below:

• Electronics_box_CAD_model (TinkerCAD)
• Electronics_box_cover_lid_CAD_model (TinkerCAD)
• Electronics_box_CAD_model (AutoCAD)
• Electronics_box_cover_lid_CAD_model (AutoCAD)

Tool Box

The purple box serves as a storage unit for the tools used during experiments, which is why it is referred to as the tool box. The simplest version of this box is an empty compartment, devoid of internal elements, allowing for flexibility in designing and modeling components tailored to specific experimental needs (Figure 8).

Figure 8. The tool box without any internal elements.

These custom elements can either be printed separately or directly integrated into a redesigned version of the box. The box features a sliding cover mechanism and a sprocket wheel on top that serves as an indicator of the box's function (Figure 9).

Figure 9. The cover lid of the purple tool box.

In our experiment the empty tool box was used. It was designed to accommodate essential items, including cover slips, supports, a syringe, a reaction chamber with a lid, and an Eppendorf tube holder (Figure 10).

Figure 10. The tool box with incorporated holders for glass cover slips, a syringe, a reaction chamber and an Eppendorf tube.

The 3D printing process of the empty box took 24 hours and 12 minutes, and printing the cover lid took 6 hours and 9 minutes. The purple filament used for printing cost 29.90 € per kg, which results in the costs of individual parts listed in the table below (Table 5).

Table 5. Cost of the 3D-printed parts for the tool box.

For printing, we used Purple Polymaker PolyLite PETG filament. The general print settings were based on the '0.10mm DETAIL' preset from the Prusa Slicer system. Modifications to the default system presets are listed below:

• Infill: 30%
• Brim: enabled
• Supports: support on build plate only (organic)
• Fill pattern: Rectilinear
• Top fill pattern: Monotonic Lines
• Bottom fill pattern: Monotonic Lines
• Perimeters: 15 mm/s
• Small perimeters: 20 mm/s
• External perimeters: 20 mm/s
• Infill: 40 mm/s
• Top solid infill: 20 mm/s
• Gap fill: 20 mm/s
• Nozzle (first and other layers): 230 ⁰C 
• Bed (first and other layers): 70 ⁰C
• Filament type: PETG
• Retraction length: 5.5 mm
• Retraction speed: 50 mm/s

The parts we designed can be downloaded here. The downloads are listed below:

• Purple Tool Box
• Black Detection Box
• Yellow Electronics Box

How Does It Work?

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The precise protocol for the use of PrymChip is as follows:

• Connect the two components in the following order: black box (detection box) first, followed by the yellow box (electronics box).
• Open the purple box (tool box), remove the black LED holders, and insert them into the slots between the black and yellow boxes.
• Place the LEDs inside the holders.
• Remove the detection chamber from the purple box.
• Prepare your reagents and transfer them into the reaction chamber.
• Use a 500 µL syringe from the purple box or your own pipette.
• Ensure the liquid volume in the reaction chamber is at least 500 µL.
• To prevent liquid loss during transfer, place the chamber lid on the reaction chamber.
• Open the black box and place the closed chamber into its designated slot.
• Close the boxes by sliding their lids back into place.
• Attach the purple box to the yellow box.
• Position a smartphone on the PrymChip and open the camera app to view the reaction chamber.
• If needed, use the black supports for improved smartphone stability.
• Turn on the LEDs by switching on the power.

After the desired reaction time, take a photo of the chamber.

Video 1. Assembly of the PrymChip.

• For qualitative analysis, check if the green fluorescence signal is visible in the photo.
• For quantitative analysis, upload the photo to the PrymChip software, which detects the green fluorescence of fluorescein in the image and converts it into fluorescein concentration.
• After taking the photo, remove the detection chamber from the black box and wash it thoroughly with water and distilled water for future use.

Adjustments to Meet Specific Needs

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Since the PrymChip was constructed as a tool for exciting and detecting fluorescence, we designed our device to be easily adjustable to various experimental needs. First and foremost, we aim to contribute to future iGEM teams by sharing our designed parts, allowing different teams to enhance our idea and utilize our existing components in their experiments. We hope that it will especially be utilized for testing samples in environmental settings.

To accommodate other experimental needs beyond this project, we made the electronics circuit easily adjustable. The battery and switch are universal and can be connected to any type of LED diode. For instance, instead of blue LEDs, red or green LEDs can be used. 

Additionally, the hole made for the switch can be resized or reshaped by remodeling one of our files. The green light filter we utilize can also be exchanged for any other color filter (Figure 11). The designated spot for the chamber can be removed or easily resized or reshaped. The same applies to the cover lid for the chamber.

Figure 11. Detection box’s cover lid with a blue filter.

However, the most adjustable part is the tool box. We proposed an empty box design, which can be printed as is to store various elements, but it can also be modified to incorporate additional holders for various tools. One of our ideas for filling the box is intended for the environmental isolation of algae DNA using the cellulose dipstick method.

There are potentially many other adjustments that can be made, and we would be happy to see these changes implemented in other projects.

Future of PrymChip

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Since the current PrymChip serves as a proof-of-concept, several improvements are needed for it to evolve into a fully functional device capable of detecting Prymnesium parvum in environmental samples. One promising enhancement is the incorporation of nucleic acid isolation via cellulose dipsticks, which could be seamlessly integrated into the PrymChip design. Optimized isolation methods using SHERLOCK reagents should also be tested.

In terms of device design, several modifications can be made. Adding a drawer to the black detection box would simplify the process of inserting solutions, reducing the risk of spillage. Another improvement would involve redesigning the detection box lid to house an external camera that connects to a phone via USB. This would eliminate the need for software adjustments based on calibration curves derived from individual phone cameras, thereby standardizing image capture. However, the current lid option, allowing for phone-based photography, should remain available for those who find it more convenient.

An independent researcher, who was not part of the project, tested PrymChip to evaluate how easy it is to use and to pinpoint potential areas for improvement in future development. The process of detecting fluorescein fluorescence was carried out by Dr Grzegorz Bereta, a postdoctoral fellow at Jagiellonian University with extensive experience in proteins and enzymatic reactions. He worked with fluorescein concentrations ranging from 0.78 µM to 100 µM, which were prepared by a member of our team. The device seems to be effective and operates smoothly. It is well designed, is sturdy, and its size is adequate. The fluorescence detection technique is straightforward and convenient. However, there are a few improvements that could enhance the device's performance - they were described in great detail on our Human Practices page.

The development of a mobile app for image analysis is also essential, as manually transferring photos from the phone to the designated script is time-consuming and inefficient. Additionally, the LED holder mechanism, widely regarded as a problematic feature, requires redesign. It should become an integral part of the device rather than being removable, by using magnets that can be easily integrated into the thick PrymChip walls. Thanks to the removal of convex and concave elements, it may be a more convenient way of transporting and connecting different boxes.