Team:Calgary/Measurement
MEASUREMENT
INTRODUCTION
After e-waste is processed through the first two steps of our recycling pipeline—bioleaching and metal recovery—we wanted to determine a way to determine the quantity of REE ions extracted. This would not only help us in determining the efficacy of our measurement system during this experimental phase, but also be a method used in industry to help determine the concentration of lanthanide ions recovered.
Current metal ion measurement systems, such as ICP-MS and atomic absorption are not readily accessible since they require analytical laboratory access and expertise. To help supplement our REE-extraction system, an easy-to-use and accessible measurement method would be ideal for implementation in industry. Upon our tours of the e-waste recycling facilities E-Cycle Solutions and Quantum Lifecycle, the differences between an industrial workplace and a laboratory were brought to our attention. These recycling plants focused primarily on e-waste sorting, collecting, and dismantling - all of which occur on a large scale and with heavy-duty machinery on an open workfloor. Sending samples away for analysis is extra time and money, and many industrial workplaces are not well-equipped for a proper laboratory space. As such, a portable and easily-usable metal ion detection system would be a major asset to the industrial REE-recycling space.
To further utilize lanmodulin (LanM), we decided to develop a suite of three LanM-based lanthanide-ion detection systems, known as BRET, Lucifer, and Elektra. Lucifer and BRET, both of which are luminescent reporters, were co-developed with a user-oriented luminometer device. The combination of Lucifer/BRET and Lumos are designed to ensure that our REE-measurement proposal is accessible and user-friendly.
When LanM is in an unbound state, it doesn’t fold into a secondary structure (1). However, the protein undergoes a notable conformational change when bound to targeted lanthanide ions (1). Our design philosophy around our bio-measurement systems revolved around leveraging this structural property of LanM. All three of the aforementioned systems rely on LanM’s conformational changes in order to release measurable signals, which can then be analyzed to determine the concentrations of REEs in solution.
Figure 1.Graphical depictions of BRET, Lucifer, Elektra and Lumos measurement systems
SYSTEMS
BRET
The BRET system follows the principles of bioluminescence resonance energy transfer (BRET), which functions by is a non-radiative energy transfer between a luminophore donor and a fluorophore acceptor (2). When the luminophore and fluorophore are within a specific distance from one another, BRET occurs and a luminescent signal within a known wavelength can be observed. An indicator of the optimal distance required for BRET to occur is the Forster distance (R0), which differs for all BRET pairs. The Forster distance is the distance at which 50% of resonance energy transfer occurs (2) Typically, luminophore donors are a luciferase enzyme (2). In order for luciferase to be activated, and therefore the BRET system to work, luciferase undergoes an enzymatic reaction with the substrate furimazine (2). The wavelength emitted by the activity of luciferase can then be detected by a luminometer.
We were inspired to develop this system due to previous research done on a LanM fluorescent sensor, which used the principles of FRET (Forster resonance energy transfer) (3). While the presence of a catalyst results in the release of light in BRET, the fluorescent donor protein in FRET needs to be excited by an incoming light source, which can result in unwelcome effects such as photobleaching, autofluorescence, and increased background noise (4).
So how could we build a viable measurement system? We planned on using NanoLuc, a luciferase enzyme from Promega attached at the C-terminal of LanM, and mCherry, a fluorophore with an emission peak at 610nM, attached at the N-terminal of LanM (2) This BRET pair was selected due to its Forster distance of 5.43nM, which is best aligned with the distance calculated between the fluorophore and luminophore once LanM transitions to a bound state. To further ensure that the BRET pair would be less than 5.43nM apart after LanM folding, protein modelling studies were performed. This analysis optimized the linkers between NanoLuc and LanM, as well as mCherry and LanM, ensuring that these reporter molecules were held at an optimal distance for generating signal emittance.
Figure 2. Graphical depiction of unbound and bound BRET systems
LUCIFER
Lucifer was coined due to the system’s reliance on NanoBiT, which is a split-luciferase complementation system. Split luciferase is often used in protein-protein interaction studies and complementation reporter assays (5). Split luciferase enzymes are one luciferase enzyme that is split into two subunits, where upon interaction between the subunits and incubation with a luciferin substrate, a luminescent signal is generated. The NanoBiT system consists of a Large Bit (18kDa) and a Small Bit (11 amino acids) which are fused to proteins of interest (6). These proteins have very low affinity to one another, so they must be in very close proximity to interact (6). As such, the background luminescence for this system is relatively low, resulting in more accurate measurements on protein-protein interactions.
Similar to the BRET system, the two subunits of the NanoBiT system were attached to opposite ends of the LanM protein. Upon lanthanide binding and a conformational change, these subunits would move closer together. In the presence of luciferase and once in a close enough proximity, a luminescence output would be released and read using a luminometer. To ensure that upon LanM metal-binding and subsequent protein-folding the NanoBiT system was in close enough proximity to release a signal, linker modelling was done. These models helped determine ideal linker size to ensure that the NanoBiT proteins would properly interact. Furthermore, to ensure that the NanoBiT protein would not interfere with the function and folding of LanM itself, molecular docking simulations were run.
ELEKTRA
For Elektra, this measurement system’s output was a change in redox potential of a ruthenium signal molecule (electrochemical signal). The inspiration for this system came from research developing an accurate measurement system for determining the concentration of thrombin in blood. (7)
The general concept for the electrochemical measurement system was based on this paper, even though there are differences between the thrombin measurement system and the LanM-based sensor. In order to adapt this system to our project, it was determined that the basic method of how the reported electrochemical signal was being generated was through an increase in proximity between the ruthenium signal molecule and the gold electrode. As a result of the increased proximity between the two components, the intensity of the electrochemical signal was also increased. (7)
When binding to lanthanides in solution, LanM brings its terminal ends close together (5). This property is leveraged in order to create this measurement system as well.The plan for Elektra would be to immobilize one end of LanM on a gold electrode and a ruthenium signal molecule on the opposing end. Upon binding the lanthanides in solution, the immobilized-LanM complex will undergo a conformational change that localizes the ruthenium signal molecule to the gold electrode. This action would allow an electrochemical signal to be generated and measured. By using cyclic voltammetry and measuring the generated current over a voltage range the specific oxidation potential of the ruthenium molecule can be detected (in the case of the thrombin measurement system, an oxidation potential of 1.20V). The intensity of the current at the ruthenium oxidation potential would correlate directly to the amount of oxidized ruthenium molecules, which in turn corresponds with the amount of lanthanide ions interacting and being detected by the Elektra system.
For more information on the measurement parts, check out our parts pages here.
LUMOS
During the course of our project design, we realized that we required a luminometer in order to make accurate measurements of the signal output for the BRET and Lucifer systems. Unfortunately, our journey to finding a luminometer posed more challenges than expected. This inspired us to develop Lumos, an accessible and economic luminometer system that would allow for BRET and Lucifer measurements. One of the motivating factors behind the development of this system was our desire to make Neocycle easily implementable for current industry users and processes. Luminometers can be expensive machines and may be difficult to navigate for users without thorough laboratory training and intensive sample preparation. Lumos aims to bridge this gap, by providing a user-friendly REE measurement detection device.
To read more about Lumos, please check out the Lumos page here.
To learn more about how the measurement systems and other subprojects followed the engineering design cycle, check out the engineering success page here.
NEXT STEPS
In the future, we plan to characterize our fusion proteins by running a protein binding assay and determining the impact of the terminal amino acids on the binding efficacy of LanM to lanthanides for the three proposed biosensor designs. In addition, we plan to use Lumos to measure the concentration of lanthanides in the 10 to 20 000 pp range, which is the expected concentration range of lanthanides in e-waste (1). We also plan to test interfering metal ions such as Al, Fe, Mn, Cu, Ca, Sn, or Zn in the 100 to 3 000 000 ppb range (1). After characterizing the protein, we will study the dynamic measurement range of these designs on the extracted lanthanides ions from e-waste using our proposed metal separation process.
REFERENCES
Cotruvo J.A, Featherston E.R., Mattocks J.A., Ho J.V., Laremore T.N. 2018. Lanmodulin: A Highly Selective Lanthanide-Binding Protein from a Lanthanide-Utilizing Bacterium. J. Am. Chem. Soc. 140(44): 15056-15061.
Weihs F, Wang J, Pfleger K.D, Dacres H. 2020. Experimental determination of the bioluminescence resonance energy transfer (BRET) Forster distances of NanoBRET and red-shifted BRET pairs. Anal. Chim. Acta. 6, 100059.
Mattocks JA, Ho JV, Contruvo JA. A Selective, Protein-Based Fluorescent Sensor with Picomolar Affinity for Rare Earth Elements. J Am Chem Soc. 2019. 141(7): 2857-2861. https://doi.org/10.1021/jacs.8b12155
Berthold Technologies. 2021. BRET (Bioluminescence Resonance Energy Transfer). Retrieved online from https://www.berthold.com/en/bioanalytic/knowledge/glossary/bret/
Dixon A.S, Schwinn M.K, Hall M.P, Zimmerman K, Otto P, Lubben T, Butler B.L, Binkowski B.F, Machleidt T, Kirkland T.A, Wood M.G, Eggers C.T, Encell L.P, Wood K.V. 2016. NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells. ACS Chem. Biol. 11(2): 400-408.
Promega. 2021. NanoBiT PPI Starter Systems. Retrieved online from https://www.promega.ca/products/protein-interactions/live-cell-protein-interactions/nanobit-ppi-starter-systems/?catNum=N2014
Lin, Z., Chen, L., Zhu, X., Qiu, B., & Chen, G. Signal-on electrochemiluminescence biosensor for thrombin based on target-induced conjunction of split aptamer fragments. 2010 Chemical Communications, 46(30): 5563. doi:10.1039/c0cc00932f
