Overview
Our 2022 team Designed, Built and Tested a biosensor for the detection of manganese contamination from drinking water. The Learning they received based on feedback from peers and judges at the Jamboree was that the sensor implementation was limited by two factors: (1) the use of live E.coli bacteria, and (2) low fluorescent output in response to manganese.
Our 2023 team has improved our sensor on both of these aspects.
- The sensor has been optimized for use in a cell-free assay format to avoid the use of live bacteria and reduce assay time.
- Further, the change to a NanoLuc reporter has dramatically increased the fold-change increase in reporter output in response to manganese, yielding a luminescent output that is easily detectable using a smartphone and, at high manganese contamination levels, by eye. This avoids the need for specialized equipment.
These improvements make the new sensor more applicable for field work.
The WrightState-OH team has developed a comprehensive implementation strategy to provide the end user with a simple, fieldable, and user-friendly method of testing for manganese contamination in water.
Target Audience
Our sensor aims to fill a gap left by other sensors currently on the market. Current methods for evaluating manganese concentrations require sending samples to a testing lab. These labs can have long wait times between receiving samples and returning results. In instances where speed is essential, a faster option is needed. Additionally, it is difficult for private well owners to get official contaminant testing performed on their drinking water. Current testing requires having contact with a local lab, the money to pay for testing, and the ability to get a good water sample. Water sampling often requires long processes of draining the well and waiting until certain parameters are met, which can take from 30 min to multiple hours. Our sensor allows for water testing on site, with minimal waiting, and a more user friendly process.
Implementation
Our goal is to provide a fast, simple, and fieldable manganese sensor to enable easier testing. This will be especially helpful for private well owners and areas with no easy access to a lab for testing. This can also help on-site testing when speed is most important. In order to meet our goals, we have developed a 4-part implementation strategy as follows:
- Adapting to a cell-free biosensor format - COMPLETE
- 3-D Printed Fieldable Device and Mobile App - PROTOTYPE
- Freeze-dried sensor for improved fieldability - PLANNED
- Functionalized silica-based device to remove manganese from drinking water - PLANNED
By converting our sensor to a cell-free system, we have removed the need for culturing live bacteria from the assay design. This change avoids the potential hazards associated with the use of live bacteria and increases the safety of our final product. The change to cell-free format also improves the potential marketability of our sensor, as we are no longer using living E. coli bacteria; we believe people will be more comfortable with a cell-free system, especially when in regards to drinking water quality.
We have developed a 3D printable device for the portable imaging of luminescence from our biosensor. Because our manganese field test relies on effective imaging of luminescence, sample responses may be difficult to read in a bright situation, such as in the field. 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. This will enable more accurate luminescence readings, which will lead to more accurate manganese concentration predictions. A prototype of this device has been designed for use with an iPhone 11, produced and tested. This implementation strategy is illustrated below in Figure 1. This work is described in detail in our Hardware Wiki page.
Figure 1: Implementation strategy for a fieldable NanoLuc biosensor test for manganese contamination in drinking water.
When in the field, there is no guarantee of access to electricity or refrigeration. To combat this concern, our team investigated different methods of preserving our sensor. Based on the work shown in M. Ravalin et. al., we determined that the best method for improving the stability and shelf-life of our biosensor would be to freeze-dry our cell-free sensor for use as a paper-based test. Ravalin et.al. used the freeze-drying approach with their NanoLuc biosensor for the SARS-CoV-2 Spike Protein. A freeze-dried sensor (including the cell-free extract and NanoLuc sensor plasmid) would allow the end user to simply reconstitute the freeze-dried sensor in the water sample to be tested, wait for the appropriate assay time, currently 2 hours, and then use the luminescence measurement device and mobile application to determine manganese levels.
We have Designed a manganese remediation strategy for the production silica-bead based filter for sequestering manganese from drinking water (Figure 2). The gene blocks needed for the approach have been received. The team is planning to perform the initial Build and Test work in the future. Briefly, functionalized silica beads will be used which will bind covalently to a custom metal-binding protein designed to have very high specificity to manganese. Once the protein is bound to the silica beads, the slurry could be loaded in a column or filter for use. Any manganese present in the water passing through the column or filter would be pulled out of solution. Our NanoLuc biosensor could then be used to test the before and after remediation test samples to confirm remediation was successful.
Figure 2: Sequestration strategy schematic. We plan to clone our existing geneblock for a tagged-metal binding protein (MBP) into a pET plasmid backbone, and then express and purify the MBP for conjugation to functionalized silica beads. The selected MBP has been optimized for specificity to manganese. The MBP-beads will then be used in a column-format filter device to purify manganese-contaminated water.
A strategy for sequestering manganese would be beneficial to the end user because manganese can’t be removed from water using standard household water filters. Manganese contamination typically requires installing expensive filtering equipment or digging a completely new well. Our method could help to provide rural areas a method for cleaning manganese from their water, without the need for any costly equipment.