Team:ULaval/Design
DESIGN
The air quality and the presence of airborne pathogens has become increasingly important in recent years. However, monitoring air quality is tedious; it requires specialized personnel and is generally lengthy and costly, meaning that a contaminant is often discovered too late. With the rise of new diseases, the resurgence of old ones, and the growing concerns of disease transmission in hospitals (Eames et al., 2009; Mirhoseini et al., 2016; Bing-Yuan et al., 2018; Khan et al., 2017),the need to have more efficient methods to monitor their presence is ever more critical.
After talking to experts in our University (Dr. Caroline Duchaine), and conducting a small market study to see if there is indeed a need for a better tool to detect biological particles in air samples, we were blown away by the results. Everyone who answered our survey, ranging from hospitals to airports, even the armed forces, said that they would be very interested in such a tool if it were available and yielded high confidence results. This really showed our team that there were a strong interest and potential market for this kind of device. However, the surveys also highlighted some critical points that we would need to integrate into our design. This gave us the groundwork for our initial design.
Therefore, we decided to use synthetic biology tools to try to develop a platform. From the survey, we surmised that this tool would need to be easy to use, therefore eliminating the need for highly trained personnel, would yield quick results, be versatile, be specific and able to detect a specific microorganism of interest, and be sensitive enough to detect biological particles in minute concentrations in air.
To answer these demands. We designed a complete device that would incorporate every step of the air monitoring process, including sampling, sample preparation, and detection, in a device requiring as little input from to users as possible. Using ToeHold switches as our detection method, combined with already established air sampling method, Loop-mediated isothermal amplification (LAMP), and microfluidics, would allow us the create this all-in-one device.
Figure 1. Analytic pipeline of A. D. N. The process is divided into several steps. 1) A toehold riboswitch is designed for the pathogen of interest with Toeholder, our computational workflow. 2) The air is sampled and nucleic acids are extracted. 3) In the detection chamber, the nucleic acids are put in contact with the toehold. 4) The target sequence is bound by the toehold riboswitch. 5) The toehold riboswitch expresses a reporter fluorescent protein, which confirms the presence of the pathogen, and the fluorescent signal is analyzed.
Detection
The centerpiece of our design is our detection system. Its way of functioning and limitations dictated our following choices. We decided to rely on specialized riboswitches, called ToeHold switches (Green et al. 2014). These single-stranded RNA molecules possess a specific secondary structure, where they are folded on themselves, called a hairpin. This structure has the particularity to block the ribosome’s access to its binding site and the first start codon on the RNA strand, therefore preventing translation of the coded protein further downstream. However, when this secondary structure is in presence of its “trigger” sequence, the hairpin unfolds, therefore giving access to the ribosome binding site and the start codon. By producing ToeHold switches with very precise trigger sequences that are organism-specific, it is possible to detect the presence of a specific DNA or RNA sequence within a given sample. By using GFP as the reporter protein, the CDS downstream of the RBS blocked by the hairpin, we can assess the presence of the sequence of interest by detecting fluorescence in a cell-free system after adding the desired sample. By designing ToeHolds for a large variety of organisms, and prepare them in lyophilized format, the user could simply select their organism of choice, and the material to detect it would be readily available, and highly specific.
Consequently, we created a web-based bioinformatics tool able to design toehold switches by inputting the microorganism genomic sequences. We also modeled the 3D structure of our switches to make sure that the structures were stable over time since spontaneous unfolding would lead to the expression of the reporter protein and false-positive results.
Cell-free media
Air sampling
A major challenge was to overcome the low concentration of microorganisms in the air. Also advises by Prof. Duchaine, we planned on using already available air samples such as the SASS 2300 that can sample 325L of air per minute over an extended period of time. Hence, concentrating the particles into a liquid that can be easily subsampled and analyzed. The constant and automated addition of a liquid to the cyclone of this sampler enables long term sampling. Furthermore, its high sampling rate (liters/minute) is adequate for air quality monitoring in air treatment units of healthcare facilities since a large volume of air usually transit by them at a rapid pace. In fact, to ensure that particles are effectively sampled in the presence of an important airflow, the sampler should be able to match its rate (isokinetic). Its particulate collection range (size aerosols that can be collected) is also fairly large: 0.5 -10µm. Since no data is available on the natural size distribution of bioaerosols containing viruses, using a sampler with a broad range of collections is a better choice than one that targets a subpopulation of aerosol size.
Sample preparation
Since our detection method relies on the presence of the genomic material of the targeted organisms, we had to ensure proper extraction and purification of nucleic acids in our sample. We also had to guarantee that enough material would get to our detector to allow the production of a signal and that it would require little to no input form to user.
To do so, we turned ourselves toward a platform that would be automated, and where most of the reaction would happen on a microfluidics chip.
By integrating all the steps of our device into a microfluidics system, and automating the protocols using simple time-scripted events (ex: raising/lowering a magnetic bar for the bead extraction, raising the temperature to the optimal LAMP temperature, etc.), we were able to design an automated platform to do detection of airborne biological contaminants. The combination of eight circuits in parallel will allow the analysis of up to 1mL of the air sample. Even though we do not believe our design will enable true quantification of the pathogens, we expect to see different rates of signal increase and numbers of circuits yielding positive responses depending on the concentration. As a result, these values could be used to produce a calibration curve for the approximation of the viral load.
First, by using a specific type of sonication apparatus integrated into the machine itself, we would break apart the air particles and the membranes of the microorganisms potentially present in the sample and achieving extraction.
Figure 2. Different sonication patterns from different technologies. We settled on AFA sonication.
To ensure proper extraction and purification we planned of adapting a commercially available DNA or RNA extraction kit using a magnetic bead binding technique. Therefore, our microfluidics chip arbors multiple channels connected to reservoirs or waste, as well as valves, that allow the incorporation or removal of buffers required for the purification of nucleic acids. The advantage of this technique is that, relatively to silica-based techniques, the large majority of the initial nucleic acids present in the air sample is conserved, as we found out during initial tests. This was critical for samples where our target of interest is already in low concentrations.
To make the limit of detection as small as possible, we looked for ways to increase the number of available genetic sequences, without having the need for specialized equipment or products. We settled on using LAMP (Loop-mediated isothermal amplification) to amplify a specific sequence of interest from a given organism of interest. Therefore, by targeting a specific sequence from a specific organism for amplification, we would be able to rapidly amplify a given sequence that would then serve as the “trigger” for our ToeHold further downstream of the system. The LAMP technics holds the advantage of achieving amplification at a stable temperature, as opposed to the Polymerase Chain Reaction (PCR), which would have required the incorporation of a thermal cycler to our device.
By preparing different microfluidics chips that would arbor the magnetic beads in known amount, the lyophilized LAMP media, with organism-specific primers, and sequence specifies Toehold within a cell-free protein expression system, the user would be able to select the pre-made cartridge for the organism of interest, insert it in the automated platform, and after sampling and reaction, be notified of the presence or absence of the targeted organism in a given sample. Thus was born A.D.N.
Preparing plasmids for ToeHold expression
Initial plasmid design relied on Gibson cloning the pBluescript SK II plasmid with gBlocks that carried ToeHold sequences. The use of the pBluescript SK II plasmid was to help with positive clone identification with the use of a Blue/White screen. However, cloning was unsuccessful. We turned ourselves towards a smaller, higher-copy number plasmid after counseling from our advisor, Helene Deveau, called pNZ123. Cloning was still difficult, so we ended up ordering complete plasmids that already arbored the ToeHold switch, with 100bp overlap between the gBlock fragments. This proved successful.
The in vivo tests would be conducted by transforming the ToeHold containing-plasmid into an E. coli cell that expresses the target gene, as well as the T7 polymerase. We decided to try with the commonly used E. coli BL21 (DE3). If the cell becomes fluorescent, the switch is efficiently expressed. Furthermore, as we designed switches to target two inducible genes (AmpR and OxyR, both targeting RNA), we would be able to detect the background levels of fluorescence of those switches that would stay un-triggered until induction of the gene of interest. We expected that DNA-targeting switches would have had a lower fluorescence level than the RNA-targeting ones, as DNA is present in only one copy at once, and RNA can be present in many copies at once.
Test our microfluidics design
Versatility
The device we designed allows the treatment of 1mL of the liquid sample. Therefore, it could not only be used for the treatment of air samples, but also surface samples, for example, using calcium alginate swabs, or any sample containing only very small solids (obstruction risks). Initially, the designed microfluidics device was in two parts, with a silica-based nucleic acid extraction matrix. However, as time went on, experts advised us that making a microfluidic circuit in two parts was too complex. Therefore, we adapted the design to a smaller circuit, where magnetic beads were used instead of the silica membrane. This allowed us to reduce the size of the channels, and made for a cheaper, more simple design and circuit (see Hardware). To test this device, we planned to prepare the various valves, and see if we could efficiently control the flow of a mock sample. After this, we would have sent known amounts of DNA into the sample chamber, and test this sample at various stages, to verify if the extraction step worked. We would assess extraction efficiency by comparing output after magnetic beads chamber VS initial DNA input. We would assess LAMP efficiency by migrating the initial sample VS LAMP amplified sample on gel. Finally, we would test for detection using a fluorescence detector with various concentrations of trigger nucleic acid, to assess efficiently the tool’s sensitivity.
Test the efficacy of the Toehold
In vivo
To test the efficacy of our toehold design and construction, we first created toehold switches targeting constitutive genes of e.coli as the target would be easily detectable. We created toehold switches to target various genes of E. coli, both their DNA and their RNA. Due to the timewise constraint, we could not test all the switches designed in E. coli.
In vitro
After confirming that we could create working toehold switches we intended to test their interaction with the cell-free media. We were awarded a sponsorship by Arbor Bioscience who generously made a considerable amount of free-cell media to our disposition. We isolated mRNA from E. coli expressing the desired target genes using the RNEasy Qiagen kit. myTXTL cell-free expression system protocol was followed, and 5nM of switch-coding DNA was added, as well as 20ng of mRNA. Results are available on the Results Page,and detailed protocol on Experiments Page.
Design and test of Toehold switches targeting human pathogens
Based on the results of our previous switches design, we could infer our ability to create switches for human pathogens. Unfortunately, we cannot easily have access to viable pathogenic viruses because of the biosafety risks that they represent. Therefore, we would confirm their proper function in our cell-free media by ordering synthetic DNA. As for the previous step, fluorimetric curves would allow us to confirm their ability to detect the targeted sequence.
However, without the complete viral particles, it would be impossible to test the efficacy of sample treatment and nucleic acid extraction. Consequently, we had to resort to using phage models.
Preliminary test our design
After confirming our ability to design and assemble toehold switches, we planned on using phage models to test the complete pipeline. The phages PR772 (dsDNA, non-enveloped), MS2 (ssRNA, non-enveloped) and Phi6 (dsARN, enveloped) were selected because they have been previously used in aerosolization studies (Turgeon et al., 2014) and are similar, both in morphology and type of nucleic acids, to the viral particles of interest: Norovirus (ssARN, non-enveloped), poxvirus (dsDNA, enveloped), measles virus (ssARN, enveloped).
Incorporating theses phages in our complete system would test the efficacy of nucleic acids extraction and detection of our method before moving to the detection of human pathogens.
Fields test
Tests with aerosolized pathogenic viral particles in a specialized closed environment called aerosolization chamber could be done. In these chambers, a solution containing the virus is pulverized and the aerosols created can be kept in suspension, all of it under biosafety cabinets that ensure no viral particles can escape. The bioaerosols could then be collected and analyzed by our device. This would yield the advantage of having an important control of the number of particles present in the sampled air, therefore allowing us to test the limit of detection of our device and fine-tune it.
Our final test would be to take our device in healthcare settings and try to detect the targeted human pathogens during outbreaks.
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Research International (2019). Gallery: SASS 2300 Wetted-Wall Air Sampler.
Turgeon, N., Toulouse, M.-J., Martel, B., Moineau, S. and Duchaine, C. (2014). Comparison of Five Bacteriophages as Models for Viral Aerosol Studies. Appl. Environ. Microbiol. 80, 4242–4250.