Name: design to implementation study for development and patient validation of paper-based toehold switch diagnostics
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The authors thank all members of the Green, Pardee and Pena labs as well as all co-authors of previous manuscripts pertaining to the work disclosed in this manuscript. S.J.R.d.S. was supported by a PhD fellowship sponsored by the Foundation for Science and Technology of Pernambuco (FACEPE), Brazil, reference number IBPG-1321-2.12/18, and currently is supported by a Postdoctoral Fellowship sponsored by the University of Toronto, Canada. P.B. is supported by the William Knapp Buckley Award from the Faculty of Pharmacy, University of Toronto. M.K. was supported by the Precision Medicine Initiative (PRiME) at the University of Toronto internal fellowship number PRMF2019-002. This work was supported by funds to K.P from the Canada Research Chairs Program (Files 950-231075 and 950-233107), the University of Toronto&#39;s Major Research Project Management Fund, the CIHR Foundation Grant Program (201610FDN-375469), and to L.P, A.A.G., and K.P through the CIHR/IDRC Team Grant: Canada-Latin/America-Caribbean Zika Virus Program (FRN: 149783), as well as by funds to K.P. from the Canada&#39;s International Development Research Centre (grant 109434-001) through the Canadian 2019 Novel Coronavirus (COVID-19) Rapid Research Funding Opportunity. This work was also supported by funds to A.A.G. from an Arizona Biomedical Research Commission New Investigator Award (ADHS16-162400), the Gates Foundation (OPP1160667), an NIH Director&#39;s New Innovator Award (1DP2GM126892), an NIH R21 award (1R21AI136571-01A1) to K.P./A.A.G, and an Alfred P. Sloan Fellowship (FG-2017-9108). Figure 1 was created with Biorender.com under academic license to K.P.

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K.P. and A.A.G. are co-inventors of paper-based toehold sensor related technologies. Y.G., S.C., and K.P. are co-founders of LSK Technologies, Inc. and are co-inventors of the PLUM-related technologies. M.K., K.P. and A.A.G. are co-founders of En Carta Diagnostics Ltd. Other authors have no conflicts of interest. Patents: PLUM device: Y.G., S.C., and K.P. patent application was filed by University of Toronto and assigned to LSK Technologies Inc. U. S. Provisional Patent Application No. 62/982,323 filed February 27, 2020; Toehold switch patent: A.A.G., P.Y., and J.J. C. "Riboregulator compositions and methods of use", Patent. WO2014074648A3 filed November 6, 2012; Paper-based diagnostic patent: K.P. and J.J.C. "Paper-based synthetic gene networks" U.S. patent pending No. US15/963,831 filed December 6, 2013; Zika patent 1: K.P., A.A.G., M.K.T., D.B., G.L., T.F., and J.J.C., "Portable, Low-Cost Virus Detection and Strain Identification Platform," U. S. Provisional Patent Application No. 62/403,778 filed October 4, 2016; Zika patent 2: K.P., A. A.G., M.K.T., D.B., G.L., T.F., and J.J.C., "Portable, Low-Cost Virus Detection and Strain Identification Platform," U. S. Provisional Patent Application No. 62/341,221 filed May 25, 2016.

The combined paper-based system described here can bring clinically relevant molecular diagnostics, with performance that is functionally comparable to RT-qPCR, to the point-of-need6. Importantly, for remote settings, the availability of diagnostics on-site can decrease the time to results from days to hours. Highlighting the programmability of this approach, the pipeline that has been described can be used to detect virtually any pathogen target. We have paired the molecular tools with a purpose-built portable plate reader, which is compact and compatible with battery operation (8&#8211;9 h) and provides onboard data analysis to enable distributed applications. In other work, we have validated the combined hardware and Zika virus diagnostic platform with 268 patient samples, in parallel with RT-qPCR, and found a diagnostic accuracy of 98.5%24. Taken together, our goal is to enable the technology transfer of this platform to researchers so that it can be repurposed and improved by the community to address unmet diagnostic needs.
The in silico toehold switch design process has been integrated and automated into a pipeline that can be divided into three stages. The first stage generates a pool of toehold switch designs that hybridize to the target sequence in one nucleotide increments. The second stage examines the secondary structure and toehold switch availability and eliminates sensors with in-frame premature stop codons. A scoring function that considers multiple factors (e.g., defect level of the toehold switch, toehold switch availability, and target site accessibility) is then implemented to select top toehold switch designs based on overall scores. In the final stage, a list of sequences is generated for the top toehold switch designs and their corresponding target triggers. The top sensor sequences should be screened for specificity against the human transcriptome and closely related viral genomes using NCBI/Primer-BLAST25. It is also best practice to screen the sensor target sites for sequence conservation in the Zika viral genome to ensure that the sensors will provide broad and robust detection. Several versions of toehold switch design software have been developed and the design algorithm allows users to generate two versions, either series A6&#160;or series B toehold switches6. In this article, the focus has been on the series B toehold switch design.
Following commercial DNA synthesis, the toehold switches can be rapidly assembled, and then tested by performing an initial screen against a synthetic target trigger sequence that corresponds to short regions (200-300 nt) of the target genome. For screening the performance of toehold switch-based sensors, it is ideal to add the target sequence in the form of RNA. In this article, the steps required to add in vitro transcribed trigger RNA have been outlined. However, if available, full-length genome templates such as quantified viral RNA extracts or commercial synthetic RNA genomes or standards can be used. Using full-length RNA genomes for initial toehold switch screening is beneficial as it can inform whether additional factors, such as RNA secondary structure, will affect sensor performance. To optimize the ON/OFF ratio of candidate switches, the toehold switch DNA can be titrated into the cell-free reaction. This step can also serve to identify high-performing toehold switches (fold amplification, or high ON/OFF ratio) and omit leaky toehold switches (high signal in the absence of the target RNA) from downstream characterization steps.
To improve the limit of detection of the top-performing toehold switch candidates, NASBA is used to increase clinically-relevant concentrations of target Zika viral RNA to a level that can be detected by toehold switches6. Different combinations of forward and reverse primer sets are screened to determine the best NASBA primer and toehold switch combinations to enable detection at clinically relevant concentrations. Once an ideal primer set and toehold switch combination has been identified, the assay is taken forward to clinical validation. It is important to note that the toehold switch and NASBA screening stages can be labor and resource-intensive and therefore test development is best suited to well-resourced research sites. Although we have not applied process automation, it is likely that this could accelerate the iterative design, build, and test cycle32. Fortunately, the turnaround time from sensor design and testing to deployment can be remarkably short (less than a week), making this strategy ideal for time-critical situations, such as epidemic outbreaks6.
Even after a biosensor with clinically relevant sensitivity has been developed, there are technical challenges that need to be addressed. Since this protocol involves manual operation and is a multi-step procedure, there is a risk of cross contamination between samples. We do our best to abate this risk through careful laboratory practice. In a recent clinical trial of 268 patient samples, we did not encounter any contamination issues; however, it is an important consideration24. With this in mind, the protocol remains a laboratory assay and requires a skilled user with command of proper molecular biology techniques. An additional consideration for deployment&#160;is the RNA isolation from patient samples. Here we describe RNA isolation using column-based nucleic acid extraction kits. However, in other work, we have demonstrated an effective and simple boiling lysis method (95 &#176;C for 2 min) for low-burden patient sample processing6. This strategy nearly eliminates the cost&#160;associated with RNA extraction and avoids the use of column-based nucleic acid extraction kits, which can pose a logistics challenge in low-resource settings or supply chain issues during crises, such as the COVID-19 pandemic33.
As we have seen during the COVID-19 pandemic, the instruments used to perform RT-qPCR can themselves serve as a bottleneck and limit patient access to testing. This factor, which is also largely financial, leads to a centralized testing mode that&#160;can limit diagnostic access. For example, during the 2015/2016 Zika outbreak, only five national reference labs were available in Brazil, which caused delays in patient testing. Without considering the potential benefit of economies of scale, the current cost of goods for the portable plate reader is ~$500 USD, which even if increased five-fold to account for labor and commercial margin, still provides an affordable instrument. This compares well to RT-qPCR instruments that range in cost from $15,000-$90,000 USD34. Furthermore, the estimated cost per test for the cell-free assay in Latin America is around $5.48 USD, while the cost per test of RT-qPCR in Brazil was ~$10-11 USD at the time of the Zika outbreak36. Beyond the cost of equipment, the portable plate reader has a small footprint (20 cm3), automatic analysis, data upload to the cloud via internet, and can be run on battery power. These features dramatically expand the potential settings where testing can be deployed and concomitantly expands the patient population that can be served.
To date the most common commercial E. coli CFPS platforms are the S30 and PURE systems37; however, a key consideration in improving access to diagnostics in low- and middle-income countries is the limited domestic availability of these reagents. An important step toward resolving this challenge is the development of local CFPS production. The Federici lab has recently made significant progress toward developing a non-commercial platform to implement toehold switch-based sensors in lysate-based cell-free systems, reaching a 2.7 fM LOD with Zika virus RNA14. Not only does this achievement allow the reagents to be made in the country of use, avoiding import tariffs and delays, but labor costs also scale to local rates and thus the overall cost can be significantly lowered. In the work outlined by the Federici group, the cost of producing the CFPS expression reaction (5 &#181;L) in Chile was 6.9 cents (USD)35,38, providing a dual incentive (improved logistics and cost) for implementing lysate-based systems35,38.
The placement of RT-qPCR-comparable testing into distributed diagnostic networks could bring significant advantages over current practices that are dependent on the transportation of samples to centralized RT-qPCR facilities. In peri-urban settings, where Zika cases were concentrated, the physical distance between a patient and the diagnostic facility slows diagnosis and increases the risk that results will not reach the patient at a clinically relevant time. It is our hope that the work presented here can contribute to enabling the research community, through the transfer of knowledge, to create decentralized biotechnologies and portable hardware for human health, agriculture and environmental monitoring.

RT-qPCR has remained the gold standard technology for clinical diagnostics due to its excellent sensitivity and specificity. Although highly robust, the method depends on expensive, specialized equipment and reagents that require temperature-controlled distribution and storage. This presents significant barriers to the accessibility of quality diagnostics globally, particularly in times of disease outbreaks and in regions where access to well-equipped labs is limited1,2. This was observed during the 2015/2016 Zika virus outbreak in Brazil. With only five centralized laboratories available to provide RT-qPCR testing, significant bottlenecks resulted, limiting access to diagnostics. This was especially challenging for individuals in peri-urban settings, who were more severely impacted by the outbreak3,4. In an effort to improve access to diagnostics, the protocol shows a platform that has been developed with the potential to provide decentralized, low-cost, and high-capacity diagnostics in low-resource settings. As a part of this, a diagnostic discovery pipeline was established, coupling isothermal amplification and synthetic RNA switch-based sensors with paper-based cell-free expression systems5,6.
Cell-free protein synthesis (CFPS) systems, in particular E. coli based cell-free systems, are an attractive platform for a wide range of biosensing applications from environmental monitoring7,8 to pathogen diagnostics5,6,9,10,11,12. Comprising the components necessary for transcription and translation, CFPS systems have significant advantages over whole-cell biosensors. Specifically, sensing is not limited by a cell wall and, in general, they are modular in design, biosafe, inexpensive, and can be freeze-dried for portable use. The ability to freeze-dry gene circuit-based reactions onto substrates such as paper or textiles, enables transport, long-term storage at room temperature5, and even incorporation into wearable technology13.
Previous work has demonstrated that E. coli cell-free systems can be used to detect numerous analytes, for example, toxic metals such as mercury, antibiotics such as tetracycline7,14, endocrine-disrupting chemicals15,16, biomarkers such as hippuric acid17, pathogen-associated quorum sensing molecules9,18 and illicit substances such as cocaine17, and gamma hydroxybutyrate (GHB)19. For the sequence-specific detection of nucleic acids, strategies have for the most part relied on the use of switch-based biosensors coupled to isothermal amplification techniques. Toehold switches are synthetic riboregulators (also referred to as simply &#39;switches&#39; in the rest of the text) that contain a hairpin structure that blocks downstream translation by sequestering the ribosomal binding site (RBS) and the start codon. Upon interaction with their target trigger RNA, the hairpin structure is relieved and subsequent translation of a reporter open reading frame is enabled20.
Isothermal amplification can also be used as molecular diagnostics21; however, these methods are prone to nonspecific amplification, which can reduce the specificity and thereby the accuracy of the test to below that of RT-qPCR 22. In the work reported here, isothermal amplification upstream of the switch-based sensors was used to provide combined signal amplification that enables clinically relevant detection of nucleic acids (femtomolar to attomolar). This pairing of the two methods also provides two sequence-specific checkpoints that, in combination, provide a high level of specificity. Using this approach, previous work has demonstrated the detection of viruses such as Zika6, Ebola5, Norovirus10, as well as pathogenic bacteria such as C. difficile23 and antibiotic resistant Typhoid12. Most recently, cell-free toehold switches have been demonstrated for SARS-CoV-2 detection in efforts to provide accessible diagnostics for the COVID-19 pandemic11,12,13.
The following protocol outlines the development and validation of cell-free, paper-based synthetic toehold switch assay for Zika virus detection, from in silico biosensor design, through the assembly and optimization steps, to field validation with patient samples. The protocol begins with the in silico design of RNA toehold switch-based sensors and isothermal amplification primers specific for the Zika viral RNA. Although numerous isothermal amplification methods exist, here the use of nucleic acid sequence-based amplification (NASBA) to increase the concentration of viral RNA target present in the reaction, enabling clinically significant sensitivity was demonstrated. Practically, isothermal amplification methods have the advantage of operating at a constant temperature, eliminating the need for specialized equipment, such as thermal cyclers, which are generally limited to centralized locations.
Next, the process of assembling the synthetic toehold switch sensors with reporter coding sequences through overlap extension PCR, and screening the synthetic toehold switch sensors for optimal performance in cell-free systems using synthetic RNA is described. For this set of Zika virus sensors, we have selected the lacZ gene encoding the &#946;-galactosidase enzyme, which is able to cleave a colorimetric substrate, chlorophenol red-&#946;-D-galactopyranoside (CPRG), to produce a yellow to purple color change that can be detected by eye or with a plate reader. Once top-performing synthetic switches are identified, the process for screening primers for nucleic acid sequence-based isothermal amplification of the corresponding target sequence using synthetic RNA to find sets that provide the best sensitivity is described.
Finally, the performance of the diagnostic platform is validated on-site in Latin America (Figure 1). To determine the clinical diagnostic accuracy, the paper-based cell-free assay is performed using Zika virus samples from patients; in parallel a gold standard RT-qPCR assay is performed for comparison. To monitor colorimetric cell-free assays, we enable on-site quantification of results in regions where thermal cyclers are not available. The hand-assembled plate reader called Portable, Low-Cost, User-friendly, Multimodal (PLUM; hereafter referred to as portable plate reader) is also introduced here24. Initially developed as a companion device for cell-free synthetic toehold switch diagnostics, the portable plate reader offers an accessible way to incubate and read results in a high-throughput manner, providing integrated, computer vision-based software analysis for users.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63223/63223fig1v9.jpg" /> 
Figure 1: Workflow for testing patient samples using paper-based cell-free toehold switch reactions.&#160;<a href="https://www.jove.com/files/ftp_upload/63223/63223fig1v8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>384 well plate covers - aluminum</td>
			<td>Sarstedt</td>
			<td>95.1995</td>
			<td>Used to cover the 384-well plates before they are inserted into the PLUM reader</td>
		</tr>
		<tr>
			<td>384 well plate covers - transparent</td>
			<td>Sarstedt</td>
			<td>95.1994</td>
			<td>Used to cover the 384-well plates before they are inserted into the BioTek plate reader</td>
		</tr>
		<tr>
			<td>384 well plates</td>
			<td>VWR</td>
			<td>CA11006-180</td>
			<td>2 mm paper-based diagnostics are placed into the wells of these plates for quantification</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>BioShop Canada</td>
			<td>AGA001.500</td>
			<td>Gel electrophoresis</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>BioShop Canada</td>
			<td>ALB001.500</td>
			<td>Blocking agent for the Whatman filter paper</td>
		</tr>
		<tr>
			<td>Cell free reactions</td>
			<td>New England Biolabs</td>
			<td>E6800L</td>
			<td>PURExpress</td>
		</tr>
		<tr>
			<td>CPRG</td>
			<td>Roche</td>
			<td>10884308001</td>
			<td>Chlorophenol red-b-D-galactopyranoside</td>
		</tr>
		<tr>
			<td>Disposable Sterile Biopsy Punches</td>
			<td>Integra Miltex</td>
			<td>23233-31</td>
			<td>Used to create 2 mm paper discs that fit into a 384-well plate</td>
		</tr>
		<tr>
			<td>DNAse I</td>
			<td>Thermo Scientific</td>
			<td>K2981</td>
			<td>Digests template DNA following incubation of the in vitro transcription reaction</td>
		</tr>
		<tr>
			<td>DNAse I Kit</td>
			<td>Thermo Scientific</td>
			<td>74104</td>
			<td>DNase I Kit For removing template DNA from IVT RNA</td>
		</tr>
		<tr>
			<td>dNTPs</td>
			<td>New England Biolabs</td>
			<td>N0446S</td>
			<td>Used for PCRs</td>
		</tr>
		<tr>
			<td>electrophoresis system</td>
			<td>Bio-Rad</td>
			<td>1704487</td>
			<td>Used to run the agarose gels</td>
		</tr>
		<tr>
			<td>Gel imaging station</td>
			<td>Bio-Rad</td>
			<td>1708265</td>
			<td>ChemiDoc XRS+ Imaging System</td>
		</tr>
		<tr>
			<td>IVT kit</td>
			<td>New England Biolabs</td>
			<td>E2040S</td>
			<td>Used for in vitro transcribing template (trigger) RNA for switch screening</td>
		</tr>
		<tr>
			<td>Nanodrop One</td>
			<td>Thermo Scientific</td>
			<td>ND-ONE-W</td>
			<td>Used for determining nucleic acid concentrations</td>
		</tr>
		<tr>
			<td>NASBA kit</td>
			<td>Life Sciences Advanced Technologies</td>
			<td>NWK-1</td>
			<td>Isothermal amplification reaction components</td>
		</tr>
		<tr>
			<td>Nuclease free H20</td>
			<td>invitrogen</td>
			<td>10977015</td>
			<td>Added to reaction mixes</td>
		</tr>
		<tr>
			<td>PAGE electrophoresis system</td>
			<td>Biorad</td>
			<td>1658001FC</td>
			<td>Used to cast and run polyacrylamide gels</td>
		</tr>
		<tr>
			<td>pCOLADuet-LacZ DNA</td>
			<td>Addgene</td>
			<td>75006</td>
			<td>https://www.addgene.org/75006/</td>
		</tr>
		<tr>
			<td>Phusion polymerase/reactioin buffer</td>
			<td>New England Biolabs</td>
			<td>M0530L</td>
			<td>Used for PCRs</td>
		</tr>
		<tr>
			<td>Plate reader</td>
			<td>BioTek</td>
			<td>BioTek NEO2</td>
			<td>Multi Mode Plate Reader, Synergy Neo2</td>
		</tr>
		<tr>
			<td>Primers</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td>Custom oligo synthesis</td>
		</tr>
		<tr>
			<td>Q5 polymerase/reaction buffer</td>
			<td>New England Biolabs</td>
			<td>M0491L</td>
			<td>Used for PCRs</td>
		</tr>
		<tr>
			<td>Qiagen PCR Puriifcation Kit</td>
			<td>QIAGEN</td>
			<td>27106</td>
			<td>QIAprep Spin Miniprep Kit</td>
		</tr>
		<tr>
			<td>RNA loading dye</td>
			<td>New England Biolabs</td>
			<td>B0363S</td>
			<td>2X RNA loading dye</td>
		</tr>
		<tr>
			<td>RNA Purification Kit</td>
			<td>QIAGEN</td>
			<td>EN0521</td>
			<td>QIAamp Viral RNA extraction kit</td>
		</tr>
		<tr>
			<td>RNase inhibitor</td>
			<td>New England Biolabs</td>
			<td>M0314S</td>
			<td>Used to prevent contamination of RNases A, B, and C</td>
		</tr>
		<tr>
			<td>RT-qPCR kit</td>
			<td>QIAGEN</td>
			<td>208352</td>
			<td>QuantiNova Probe RT-PCR Kit</td>
		</tr>
		<tr>
			<td>SYBR Gold</td>
			<td>Invitrogen S11494</td>
			<td>S11494</td>
			<td>PAGE gel stain for nucleic acids</td>
		</tr>
		<tr>
			<td>TAE Buffer</td>
			<td>BioShop Canada</td>
			<td>TAE222.4</td>
			<td>Gel electrophoresis buffer</td>
		</tr>
		<tr>
			<td>Thermal Cycler</td>
			<td>Applied Biosystems</td>
			<td>4484073</td>
			<td>Used for temperature cycling and incubating reactions</td>
		</tr>
		<tr>
			<td>Whatman 42 filter paper</td>
			<td>GE Healthcare</td>
			<td>1442-042</td>
			<td>Used to imbed molecular components for paper-based diagnostics</td>
		</tr>
	</tbody>
</table>

design to implementation study for development and patient validation of paper-based toehold switch diagnostics

Access to low-burden molecular diagnostics that can be deployed into the community for testing is increasingly important and has meaningful wider implications for the well-being of societies and economic stability. Recent years have seen several new isothermal diagnostic modalities emerge to meet the need for rapid, low-cost molecular diagnostics. We have contributed to this effort through the development and patient validation of toehold switch-based diagnostics, including diagnostics for the mosquito-borne Zika and chikungunya viruses, which provided performance comparable to gold-standard reverse transcription-quantitative polymerase chain reaction (RT-qPCR) based assays. These diagnostics are inexpensive to develop and manufacture, and they have the potential to provide diagnostic capacity to low-resource environments. Here the protocol provides all the steps necessary for the development of a switch-based assay for Zika virus detection. The article takes readers through the stepwise diagnostic development process. First, genomic sequences of Zika virus serve as inputs for the computational design of candidate switches using open-source software. Next, the assembly of the sensors for empirical screening with synthetic RNA sequences and optimization of diagnostic sensitivity is shown. Once complete, validation is performed with patient samples in parallel with RT-qPCR, and a purpose-built optical reader, PLUM. This work provides a technical roadmap to researchers for the development of low-cost toehold switch-based sensors for applications in human health, agriculture, and environmental monitoring.

Following the computational design pipeline, the construction of three toehold switches was performed by PCR. The PCR products were analyzed using agarose gel electrophoresis (Figure 2). The presence of a clear band around 3,000 bp, roughly the size of the lacZ gene coupled to a toehold switch, typically indicates a successful reaction. Alternatively, a lane without a band, multiple bands, or a band of the incorrect size, indicates a failed PCR. In the case of a failed PCR, the reaction conditions and/or primer sequences should be optimized.
The assembled toehold switches were screened to assess each sensor against its respective in vitro transcribed trigger RNA (Figure 3). While all three sensors displayed an increased OD570 absorbance, switch&#160;27B (Figure 3A) had the most rapid on-rate. Switches 33B and, to a lesser extent, 47B showed an increased OD570 absorbance in the absence of target trigger RNA, indicating that these switches possess some background activity or leakiness (Figure 3B,C)-a trait not wanted in a candidate sensor as it can reduce specificity. To more clearly identify the sensor with the highest ON/OFF signal ratio, the fold change of OD570 absorbance was calculated (see supplemental information section 5) and plotted (Figure 4). From this analysis, it is clear that switch 27B is the sensor that has the best performance with an ON/OFF&#160;ratio of around 60.
The sensitivity of the top-performing toehold switch (27B) was then evaluated by determining the lowest RNA concentration required to activate the toehold switch&#160;when coupled with a NASBA reaction. The graph illustrates that the top-performing Zika sensors can detect RNA at concentrations as low as 1.24 molecules per &#181;L (equivalent to ~2 aM; Figure 5).
After switch 27B was identified and validated, the sensor materials were distributed to the team members in Recife in the Pernambuco state of Brazil. In Brazil, the clinical diagnostic accuracy of the Zika virus diagnostic platform was assessed using Zika virus patient samples, in parallel with RT-qPCR for comparison. To validate the paper-based Zika diagnostic platform, the portable plate reader was used, which is capable of incubating and reading the colorimetric output of paper-based sensors. The color change from yellow to purple is used to identify a positive sample, while a negative sample remains yellow (Figure 6). An additional option for visualizing the results generated by the portable plate reader (Figure 8) is to plot the colorimetric response for each paper-based reaction over time. Samples were tested in triplicate and those that exceeded the threshold (red line set to 1) were considered positive, while samples below the threshold were considered negative (Figure 6 and Figure 7).
Finally, to evaluate and compare the clinical performance of the Zika toehold switch sensor with the current gold standard method for diagnosing Zika virus infection, all patient samples were tested in parallel with RT-qPCR. The amplification plot of two representative patient samples was tested in triplicate for the detection of Zika virus by RT-qPCR (Figure 9). The samples are considered positive when the cycle threshold (Ct) value is &#8804;38; the red line indicates a positive sample and the blue line indicates a negative sample for Zika virus.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63223/63223fig2v6.jpg" /> 
Figure 2: Agarose gel electrophoresis to assess the quality of PCR products. PCR products are analyzed on a 1% agarose gel in 1X TAE, run at 80 V for 90 min. A single clear band typically indicates a successful reaction. Lane 1: 1 kb DNA ladder; Lanes 2-4: 27B switch DNA, 33B trigger DNA, and 47B trigger DNA, respectively.The numbers on the left-hand side represent band size in bp. <a href="https://www.jove.com/files/ftp_upload/63223/63223fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63223/63223fig03v6.jpg" /> 
Figure 3: Prototyping three toehold switches for paper-based Zika sensors. The performance of three paper-based RNA toehold switch sensors was measured at 37 &#176;C for over 130 min. Each graph contains two traces, one represents the switch only control, while the other represents the switch and trigger. The three graphs represent data acquired using toehold switch sensors 27B (A), 33B (B), and 47B (C). Error bars represent the standard error of the mean (SEM) from three replicates. <a href="https://www.jove.com/files/ftp_upload/63223/63223fig03v6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63223/63223fig04v7.jpg" /> 
Figure 4: Top-performing sensors are identified by calculating the fold change in absorbance at 570 nm. Fold change (or maximum ON/OFF ratio) is calculated by measuring the ratio of absorbance (OD570) at 130 min between the switch only control, and the switch plus trigger CFPS assay. Error bars represent SEM from three replicates. <a href="https://www.jove.com/files/ftp_upload/63223/63223fig4v8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63223/63223fig05v7.jpg" /> 
Figure 5: Sensitivity assessment of top-performing switch. In vitro transcribed Zika RNA is titrated into NASBA reactions. After a 1 h incubation, the reactions were added to cell-free PURExpress reactions on paper discs at a ratio of 1:7. The fold change after 130 min at 37 &#176;C is plotted. This figure has been reproduced&#160;from24. <a href="https://www.jove.com/files/ftp_upload/63223/63223fig05v4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63223/63223fig6v9.jpg" /> 
Figure 6: Portable plate reader capture page loaded with captured image data. This figure shows a sample picture of the final image captured by the portable plate reader during a data collection run. The original date/time stamp is visible at the top of the image. Yellow color indicates control or negative reactions, and the purple color indicates a positive reaction. <a href="https://www.jove.com/files/ftp_upload/63223/63223fig6v8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/63223/63223fig7v9.jpg" /> 
Figure 7: Data analysis mode. On the left, users select the data sets they would like to plot; the graphs are then displayed on the right with unique colors for each sample or control set. The dashed red line serves as a threshold for determining positive and negative samples. Samples tested in triplicate that exceed the threshold are considered positive, while samples below the threshold are deemed negative. Error bars represent standard deviation (SD) from three replicates. Ctrl 1 to Ctrl 5 indicates controls. <a href="https://www.jove.com/files/ftp_upload/63223/63223fig7v8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/63223/63223fig8v8.jpg" /> 
Figure 8. PLUM, a portable plate reader. This&#160;portable plate reader acts as a lab-in-a-box and serves as a temperature-controlled plate reader to incubate and monitor colorimetric reactions. This portable device can provide quantitative and high-throughput measurements of the paper-based Zika sensors on-site. <a href="https://www.jove.com/files/ftp_upload/63223/63223fig8v8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/63223/63223fig9v9.jpg" /> 
Figure 9: RT-qPCR plot of the amplification of two patient samples tested in triplicate for the detection of the Zika virus. Samples are considered positive when the cycle threshold (Ct) value is &#8804;38. The dotted red line serves as a threshold for determining positive and negative samples. The red trace indicates a positive sample, and the blue trace indicates a negative sample. &#916;Rn (delta Rn) value represents the normalized magnitude of the fluorescence signal detected by the RT-qPCR instrument for all the samples tested. <a href="https://www.jove.com/files/ftp_upload/63223/63223fig9v8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Protocol File. <a href="https://www.jove.com/files/ftp_upload/63223/Supplementary_protocol_R2_RE_KJ.docx" target="_blank">Please click here to download this File.</a>
Supplemental Figures.&#160;<a href="https://www.jove.com/files/ftp_upload/63223/Supplemental_Figures.zip" target="_blank">Please click here to download these&#160;figures.</a>

Watch this Scientific Journal Video about Design to Implementation Study for Development and Patient Validation of Paper-Based Toehold Switch Diagnostics at JoVE.com

Design to Implementation Study for Development and Patient Validation of Paper-Based Toehold Switch Diagnostics

Access to decentralized, low-cost, and high-capacity diagnostics that can be deployed into the community for decentralized testing is critical for combating global health crises. This manuscript describes how to build paper-based diagnostics for viral RNA sequences that can be detected with a portable optical reader.

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