Overview
Because our manganese field test relies on effective imaging of luminescence, sample responses may be difficult to read in a bright room or outdoors. To supply the end user with a device that effectively serves as a portable dark room for use with a smartphone, the team has developed a 3D-printed device that will orient the phone and isolate the samples from outside light when collecting the data. A prototype of the Luminescence Imaging Device (LID) hardware has been designed for use with an iPhone 11, produced and tested. We have also designed a Mobile Phone Application that will guide the user through water sample testing, calculate the concentration of manganese in the water sample(s) tested, and provide a GPS location stamp for the testing location. This will enable more accurate luminescence readings, which will lead to more accurate manganese concentration predictions.
Hardware Design
The Luminescence Imaging Device (LID) hardware was designed in SolidWorks. The device was inspired by a paper by J. Ma et. al. [1], and designed with the help of our collaborator, Mr. Andrew Kronenberger.
The schematic of the LID hardware is provided in Figure 1. The design, which was the result of several iterations of design and prototype testing, includes the following features:
External Housing
- Made of recyclable Prusa PLA (Vanilla White) resin.
- Painted outer surface to block out virtually all ambient light.
- Slot to hold a mobile phone. Although currently designed for an iPhone, this slot may be adjusted in size and the placement of the imaging window adjusted to accommodate most any mobile phone.
- A cool WSU iGEM name plate.
Internal tube holder:
- Printed in white plastic appropriate for luminescence imaging.
- Slightly beveled edges to enable the piece to slide easily into the external housing.
- Placeholders for 8 tubes for simultaneous imaging of standard curve, controls, and water samples.
- Dividers between samples for improved imaging accuracy. This prevents light from bright samples from bleeding over into the imaging area of adjacent tubes.
The LID hardware schematic designed for the iPhone 11 is shown in Figure 1. The STL files for use with most 3D printers can be downloaded using the links below. These files can be viewed in Paint 3D or eDrawings Viewer.
Figure 1: Schematic of the Luminescence Imaging Device (LID) hardware. The version shown is designed for an iPhone 11, but can be easily adapted for use with any mobile phone.
Mobile Phone App
Our team has begun work on a smartphone app for analyzing the results of our luminescent data obtained using the cell-free assay and LID device (Figure 1). Although the luminescence from some water samples may be detectable with the naked eye, quantifying the level of manganese contamination from the unknown water sample(s) requires accurate measurement of the brightness of the response. Utilizing a smartphone to obtain accurate intensity readings for water samples and a “standard curve” of water samples with known manganese levels will allow the end user to accurately calculate the level of manganese contamination in the water samples.
The mobile phone app is currently under construction. We are utilizing a development framework called Kotlin Multi Platform, which allows us to make an Android app and an iPhone app at the same time, using the same code base. Quantitation will be based on open-source code mirroring the quantitation scripts made in Java programming language. Kotlin is based on Java, so incorporating existing scripts should be straightforward.
Although the fully functional app is not currently available, we hope to have a working prototype soon. Mockups of the screenshots from each step of the mobile app workflow are shown in Figure 2. The app is designed to guide the user through each step of image acquisition, confirm that the assay controls meet acceptance criteria, and calculate the concentration of manganese in the unknown water sample(s) using the provided standard curve. The app will also record the GPS location where the testing was performed to ensure documentation of testing site location.
Figure 2: Guided workflow for the mobile app for quantitation of manganese levels in water. When the mobile app is complete, we will make it freely available in the Apple App Store and on Google Play.
Validation
The Luminescence Imaging Device (LID) hardware was using a standard curve of MnCl2 and 2 water samples (Figure 3). The samples were also measured with a plate reader (Figure 3A) to confirm linearity of the dose curve. Standard cell-free reactions were set up in PCR tubes, incubated at 29C for 2 hours and the resulting luminescence was measured in the LID hardware using an iPhone 11. The resulting light image (bottom) shows all 8 sample tubes were clearly visible. The MnCl2 samples showed the expected dose dependent increase in luminescence (middle), clearly evident in the line profile trace (top) and linear regression (Figure 3B, r2 = 0.987).
Figure 3: Comparison of manganese measurements made on a plate reader and the LID device. (A) MnCl2 dose curve measured on a BioTek Synergy H1 plate reader. (B) Images collected using the Luminescence Imaging Device (LID) hardware including a light image of the tubes in place, the luminescence image, and a pixel intensity trace of the luminescent image. (C) Standard curve determined from the image in panel B quantitated in ImageJ.
Manganese levels in the water samples #1003 and #1004 from Mike Ekberg, Miami Conservancy District, and the cold water and wetland samples from Stephen Jacquemin, Wright State University Lake Campus were calculated using the standard curve in Figure 3C. The calculated mM levels were converted to ppm and are listed in (Table 1, LID).
Table 1: MnCl2 measurements from water samples made using a plate reader and the LID hardware. Plate reader: BioTek Synergy H1 plate reader; and LID: the luminescence imaging device and iPhone 11.
As a proof of principle, we showed that all water samples tested exhibited manganese levels higher than the limit of detection for the NanoLuc assay.
We observed that the ppm values calculated using the plate reader and the LID varied were not exactly similar. However the Manganese levels measured in all samples were above the minimum allowable ppm concentration.
Further optimization of the LID will be performed in the future.
Based on these results the 3D printed LID hardware can serve as a suitable alternative to more expensive plate readers.