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.

<ol>
	<li>Urdea, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirements+for+high+impact+diagnostics+in+the+developing+world.">Requirements for high impact diagnostics in the developing world.</a> Nature. 444, 73-79 (2006).</li><li>Yager, P., Domingo, G. J., Gerdes, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Point-of-care+diagnostics+for+global+health.">Point-of-care diagnostics for global health.</a> Annual Review of Biomedical Engineering. 10, 107-144 (2008).</li><li>Faria, N. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mobile+real-time+surveillance+of+Zika+virus+in+Brazil.">Mobile real-time surveillance of Zika virus in Brazil.</a> Genome Medicine. 8 (1), 97 (2016).</li><li>Faria, N. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zika+virus+in+the+Americas:+Early+epidemiological+and+genetic+findings.">Zika virus in the Americas: Early epidemiological and genetic findings.</a> Science. 352 (6283), 345-349 (2016).</li><li>Pardee, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paper-based+synthetic+gene+networks.">Paper-based synthetic gene networks.</a> Cell. 159 (4), 940-954 (2014).</li><li>Pardee, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid,+low-cost+detection+of+Zika+virus+using+programmable+biomolecular+components.">Rapid, low-cost detection of Zika virus using programmable biomolecular components.</a> Cell. 165 (5), 1255-1266 (2016).</li><li>Duyen, T. T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paper-based+colorimetric+biosensor+for+antibiotics+inhibiting+bacterial+protein+synthesis.">Paper-based colorimetric biosensor for antibiotics inhibiting bacterial protein synthesis.</a> Journal of Bioscience and Bioengineering. 123 (1), 96-100 (2017).</li><li>Jung, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+biosensors+for+rapid+detection+of+water+contaminants.">Cell-free biosensors for rapid detection of water contaminants.</a> Nature Biotechnology. 38 (12), 1451-1459 (2020).</li><li>Yang, Y. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+Escherichia+coli-based+system+to+screen+for+quorum-sensing+molecules+interacting+with+quorum+receptor+proteins+of+streptomyces+coelicolor.">Cell-free Escherichia coli-based system to screen for quorum-sensing molecules interacting with quorum receptor proteins of streptomyces coelicolor.</a> Applied and Environmental Microbiology. 75 (19), 6367 (2009).</li><li>Ma, D., Shen, L., Wu, K., Diehnelt, C. W., Green, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-cost+detection+of+norovirus+using+paper-based+cell-free+systems+and+synbody-based+viral+enrichment.">Low-cost detection of norovirus using paper-based cell-free systems and synbody-based viral enrichment.</a> Synthetic Biology. 3 (1), (2018).</li><li>Park, S., Lee, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+coronaviruses+using+RNA+toehold+switch+sensors.">Detection of coronaviruses using RNA toehold switch sensors.</a> International Journal of Molecular Sciences. 22 (4), 1-13 (2021).</li><li>Amalfitano, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+glucose+meter+interface+for+point-of-care+gene+circuit-based+diagnostics.">A glucose meter interface for point-of-care gene circuit-based diagnostics.</a> Nature Communications. 12 (1), 1-10 (2021).</li><li>Nguyen, P. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wearable+materials+with+embedded+synthetic+biology+sensors+for+biomolecule+detection.">Wearable materials with embedded synthetic biology sensors for biomolecule detection.</a> Nature Biotechnology. 39 (11), 1366-1374 (2021).</li><li>Pellinen, T., Huovinen, T., Karp, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cell-free+biosensor+for+the+detection+of+transcriptional+inducers+using+firefly+luciferase+as+a+reporter.">A cell-free biosensor for the detection of transcriptional inducers using firefly luciferase as a reporter.</a> Analytical Biochemistry. 330 (1), 52-57 (2004).</li><li>Salehi, A. S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+protein+synthesis+approach+to+biosensing+hTR&#946;-specific+endocrine+disruptors.">Cell-free protein synthesis approach to biosensing hTR&#946;-specific endocrine disruptors.</a> Analytical Chemistry. 89 (6), 3395-3401 (2017).</li><li>Salehi, A. S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosensing+estrogenic+endocrine+disruptors+in+human+blood+and+urine:+A+RAPID+cell-free+protein+synthesis+approach.">Biosensing estrogenic endocrine disruptors in human blood and urine: A RAPID cell-free protein synthesis approach.</a> Toxicology and Applied Pharmacology. 345, 19-25 (2018).</li><li>Voyvodic, P. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plug-and-play+metabolic+transducers+expand+the+chemical+detection+space+of+cell-free+biosensors.">Plug-and-play metabolic transducers expand the chemical detection space of cell-free biosensors.</a> Nature Communications. 10 (1), 1-8 (2019).</li><li>Yang, W. C., Patel, K. G., Wong, H. E., Swartz, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplifying+and+streamlining+Escherichia+coli-based+cell-free+protein+synthesis.">Simplifying and streamlining Escherichia coli-based cell-free protein synthesis.</a> Biotechnology Progress. 28 (2), 413-420 (2012).</li><li>Gr&#228;we, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+paper-based,+cell-free+biosensor+system+for+the+detection+of+heavy+metals+and+date+rape+drugs.">A paper-based, cell-free biosensor system for the detection of heavy metals and date rape drugs.</a> PLOS One. 14 (3), 0210940 (2019).</li><li>Green, A. A., Silver, P. A., Collins, J. J., Yin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toehold+Switches:+De-Novo-Designed+Regulators+of+Gene+Expression.">Toehold Switches: De-Novo-Designed Regulators of Gene Expression.</a> Cell. 159 (4), 925-939 (2014).</li><li>Craw, P., Balachandran, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isothermal+nucleic+acid+amplification+technologies+for+point-of-care+diagnostics:+a+critical+review.">Isothermal nucleic acid amplification technologies for point-of-care diagnostics: a critical review.</a> Lab on a Chip. 12 (14), 2469-2486 (2012).</li><li>Zyrina, N. V., Antipova, V. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonspecific+Synthesis+in+the+Reactions+of+Isothermal+Nucleic+Acid+Amplification.">Nonspecific Synthesis in the Reactions of Isothermal Nucleic Acid Amplification.</a> Biochemistry (Moscow). 86 (7), 887-897 (2021).</li><li>Takahashi, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+low-cost+paper-based+synthetic+biology+platform+for+analyzing+gut+microbiota+and+host+biomarkers.">A low-cost paper-based synthetic biology platform for analyzing gut microbiota and host biomarkers.</a> Nature Communications. 9 (1), 1-12 (2018).</li><li>Karlikow, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Field+validation+of+the+performance+of+paper-based+tests+for+the+detection+of+the+Zika+and+chikungunya+viruses+in+serum+samples.">Field validation of the performance of paper-based tests for the detection of the Zika and chikungunya viruses in serum samples.</a> Nature Biomedical Engineering. 6 (3), 246-256 (2022).</li><li>Agarwala, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Database+resources+of+the+National+Center+for+Biotechnology+Information.">Database resources of the National Center for Biotechnology Information.</a> Nucleic Acids Research. 44 (1), 7-19 (2016).</li><li>. BLAST: Basic Local Alignment Search Tool Available from: <a target="_blank" href="https://blast.ncbi.nlm.nih.gov/Blast.cgi">https://blast.ncbi.nlm.nih.gov/Blast.cgi</a> (2022)</li><li>Deiman, B., Van Aarle, P., Sillekens, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+and+applications+of+nucleic+acid+sequence-based+amplification+(NASBA).">Characteristics and applications of nucleic acid sequence-based amplification (NASBA).</a> Molecular Biotechnology. 20 (2), 163-179 (2002).</li><li>Zadeh, J. N., Wolfe, B. R., Pierce, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleic+acid+sequence+design+via+efficient+ensemble+defect+optimization.">Nucleic acid sequence design via efficient ensemble defect optimization.</a> Journal of Computational Chemistry. 32 (3), 439-452 (2011).</li><li>JoVE Science Education Database. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+Methods+in+Cellular+and+Molecular+Biology.+DNA+Gel+Electrophoresis.">Basic Methods in Cellular and Molecular Biology. DNA Gel Electrophoresis.</a> Journal of Visualized Experiments: JoVE. , (2021).</li><li>Lanciotti, R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+and+serologic+properties+of+Zika+virus+associated+with+an+epidemic,+Yap+State,+Micronesia,+2007.">Genetic and serologic properties of Zika virus associated with an epidemic, Yap State, Micronesia, 2007.</a> Emerging Infectious Diseases. 14 (8), 1232 (2008).</li><li>Walsh, D. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardizing+automated+DNA+assembly:+Best+practices,+metrics,+and+protocols+using+robots.">Standardizing automated DNA assembly: Best practices, metrics, and protocols using robots.</a> SLAS Technology. 24 (3), 282 (2019).</li><li>Silva, S. J. R., de Magalh&#227;es, J. J. F., de Pena, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+circulation+of+DENV,+CHIKV,+ZIKV+and+SARS-CoV-2+in+Brazil:+An+inconvenient+truth.">Simultaneous circulation of DENV, CHIKV, ZIKV and SARS-CoV-2 in Brazil: An inconvenient truth.</a> One Health. 12, 100205 (2021).</li><li>Kimura, S., Fujinaga, K., Sekiya, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PCR+machine.+Tanpakushitsu+kakusan+koso.">PCR machine. Tanpakushitsu kakusan koso.</a> Protein, Nucleic Acid, Enzyme. 41 (5), 514-517 (1996).</li><li>Arce, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decentralizing+cell-free+RNA+sensing+with+the+use+of+low-cost+cell+extracts.">Decentralizing cell-free RNA sensing with the use of low-cost cell extracts.</a> Frontiers in Bioengineering and Biotechnology. 9, 727584 (2021).</li><li>Silva, S. J. R., Pardee, K., Balasuriya, U. B. R., Pena, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+validation+of+a+one-step+reverse+transcription+loop-mediated+isothermal+amplification+(RT-LAMP)+for+rapid+detection+of+ZIKV+in+patient+samples+from+Brazil.">Development and validation of a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of ZIKV in patient samples from Brazil.</a> Scientific Reports. 11 (1), 1-13 (2021).</li><li>Shimizu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+translation+reconstituted+with+purified+components.">Cell-free translation reconstituted with purified components.</a> Nature Biotechnology. 19 (8), 751-755 (2001).</li><li>Lavickova, B., Maerkl, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple,+robust,+and+low-cost+method+to+produce+the+PURE+cell-free+system.">A simple, robust, and low-cost method to produce the PURE cell-free system.</a> ACS Synthetic Biology. 8 (2), 455-462 (2019).</li></ol>

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.remotexs.ntu.edu.sg/files/ftp_upload/63223/63223fig1v8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.

Access to low-burden molecular diagnostics that can be deployed into the community for testing is increasingly important and has meaningful wider ...

design-to-implementation-study-for-development-patient-validation

Nanyang Technological University

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JoVE Journal

Bioengineering

2.8K Views.  University of Toronto. Ons protocol beschrijft het ontwerp, de assemblage en de validatie van op gencircuits gebaseerde diagnostiek. Deze op papier gebaseerde moleculaire diagnostiek is goedkoop en gevoelig, kan klinisch relevante concentraties van nucleïnezuren detecteren en kan worden ontworpen om vrijwel elke sequentie te detecteren. Dit is een platformtechnologie die door gebruikers kan worden ontworpen voor hun behoeften en het potentieel heeft om diagnostiek van klinische kwaliteit in meer gedecentraliseerde...

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

Using Micro-Electro-Mechanical Systems (MEMS) to Develop Diagnostic Tools

Using Micro-Electro-Mechanical Systems MEMS to Develop Diagnostic Tools

I'm U De Mercy, I'm the principal investigator of Bio Stick MAMs in Medicine Labs. I did my PhD at Stanford University in electrical engineering. I did a postdoc at Harvard Medical School at MGH working on bio acoustic MAMs or mostly bio MAs.And now I'm continuing as a faculty at Harvard Medical School at Harvard MIT Health Sciences and Technology. And continuing with my research that I enjoy very much. The work that I did during my PhD was very much related to droplets and using acoustics to generate precise size droplets and control their location and position to deposit very sensitive polymers.Now we apply similar technologies to encapsulate cells in droplets and then position them on the surfaces for various applications in tissue engineering to print cells on the surfaces to pattern cells. Also, there are applications where you want to see that you can encapsulate few cells or a single cell or from the same population and to see the differences between the same population from one cell to another there being able to encapsulate cells in droplets at high throughput rates like 10, 000 cells per second kind of rates becomes very important and very useful to understand biological problems. So my research currently has two legs, I would say.One is this cell encapsulation work that I just talked about. How do we pack a cell in a droplet that's, and how can we do this repeatedly and reliably and without harming the cells? So after the cell is ejected or encapsulated in the droplet, we should be able to precisely located on a surface the cell should be functional and alive and viable.It shouldn't be harmed by the effects of ejection and so forth. So currently we have a system where we use acoustic drop focused waves to generate these droplets from open pools where we can encapsulate down to single cells in these really small droplets that are comparable to the cell size. That's very exciting in terms of applying this to cell printing and tissue engineering.The other side of my research is again using these MAMs micro electromechanical system technologies to develop low cost diagnostic tools. Mostly this research uses microfluidic approaches where we can introduce whole blood, very small volumes such as blood from a prick finger prick, less than 10 microliters that you can introduce to a chip. And from that blood we can encapsulate or you can capture certain sub-cellular populations from whole blood.Why is this important? Why should this be cheap for global health applications On top of the mountain in Africa, you want to be able to tell for a HIV patient, for instance, how many CD four T lymphocytes this patient has. Because World Health Organization says that below 200 CD four cells per microliter, you have to start treating the patients In the developed world, you use hundreds of thousands dollars of vert flow cytometers to be able to get this data.And it takes of course time to use and skill of course to be able to use these like huge table size machines. Our small chip can be introduced this small finger brick 10 microliters of whole blood and it'll capture the CD four cells using the surface protein affinities. And then you can rapidly count these cells that are captured because you know that there are CD four cells which are attached to CD four proteins antibodies on the surface of the chip.By controlling the flow rates and shear, you can make sure that the specificity and efficiency for these cell types are optimized. And there's of course always non specific binding, but by our sheer approaches we minimize these effects and make sure that between plus minus 10%errors, which is sufficient to make a diagnostic or prognostic decision on top mountain in Africa. So this has interesting applications for global health as well as these low cost techniques that are disposable can impact the developed world because now these fast blood tests which are really low cost can impact the tests that we use in the developed world.If they're optimized to higher levels of efficiency and specificity, which our initial data shows that they can be, then it'll definitely impact our lives. When I first finished my PhD, I knew more about microfluidics and that in MAMs more than anything else I could see, especially with the droplet application, I could see that if I could print cells and encapsulate cells or manipulate the few cells, single cells, that it'll have great applications in biotechnology area. And I was very interested in working on things that will actually impact people's lives.I used to apply these technologies to semiconductor industry, but then I wanted to to be useful directly to people. So I, that's what directed me towards problems in healthcare. And then that's why I made a big shift and came to a hospital, mass general hospital to the postdoc.And there, you know, I got exposed more and more to problems and it seems, and it's all clear that one of the biggest problems of the world today is global health and there comes illnesses like tuberculosis, HIV that kills thousands of people per day. And these people die not because the drugs are not there, but because there aren't enough diagnostic tools which is much more expensive than the existing drugs. So I know the technology side of the things very well.I get, I got exposed to the biological problems and the more I learned about them, the more I could see that I could make an impact. And that's how it all grew from there. And it's still continuing now.Like I seen biotechnology and in medicine there are like so many problems that actually directly cause our result into loss of people's lives and the technologies, the technology aspect of it and being able to apply it to real world medical problems is a great, I think path to follow. This is like how this whole biotechnology area I think is kind of growing and it's becoming very impactful. If you think about it, HIV cancer, those are big killers in the world.And current micro technologies, current approaches could maybe benefit from early detection of cancer by capturing these cells from blood or making it really cheap, which will make it available to masses. So all these things coming together, I think this whole microtechnology applications in healthcare in medicine could impact the future of human beings. So that's how all these things kind of come together I guess.And being in the Harvard MIT health sciences and technology where on one end you have the technology and on the other end you direct aspect, direct access to patients, direct access to doctors in Brigham and Women's Hospital where I work is a great environment. cause every person that you talk to have their problems and you could maybe it's coming from a different background, provide interesting solutions to those existing real world problems. So this is, I think how I see the whole perspective of how these medical problems meet the technology side.And we try to impact people's lives in a positive way. In single cell cap encapsulation or in cell encapsulation, the major challenge is to be able to encapsulate reliably and repeatably single cells. So you're ejecting 10, 000 hundred thousand cells per second.And how do you make sure that each droplet that you eject has a single cell? This statistics where you are changing the droplet size and minimizing the, optimizing the cell size over the droplet size, which goes into the old problem of packing spheres in a volume and what is the most efficient way to do it. So that's the biggest challenge in the cell printing area for from the technological side.The other aspect of it is now you can say print and precisely locate these cells. How can you generate these three dimensional tissues and how can you keep them alive and how can you transplant them? There comes in the biological end of the problems where you want to now exactly mimic the tissue.We have current approaches to be able to print an eyelet, a pancreas eyelet, and to be able to print using smooth muscle cells, a bladder tissue, directly mimicking what is in human or red bladder so that then we can make this tissue and test how well it performs compared to the real world existing native tissues. So that's the major challenge I guess by taking these novel technologies, how can you make this the ideal tissue that can be replaced, that can be transplanted, that's the, you know, biggest problem from the beginning because the control on single cell gives you the capability to precisely locate them. And then how do you kind of grow it from there?So it's something transplantable there where the impact of human life comes. So that's on that end, what the challenges are in the tissue engineering side of the things in southern encapsulation and in diagnostics using microfluidics, the challenge is to be able to capture a cell out of billions of cells. It's like you have a sugar particle in a salt can and you're trying to pull out that sugar particle and that's like one in a billion.So you, you're processing microliters to milliliters of whole blood and you want to be able to specifically and efficiently without any technological problems like clogging or any biological problems like non-specific binding, you want to be able to isolate at one cell out of billion cells in one microliter of whole blood, you have a few millions of cells. And for the CD four T lymphocyte case in that one microliter, we are after about thousand cells per microliter. So it's like one cell in a thousand kind of a problem where we have shown that it's doable.But when you want to go after circulating tumor cells for cancer, then the challenge becomes one in a billion. So to some summarize it, the challenge is how do we capture this one cell out of billions of other cells around it? And what is the technological aspects?What are the flow rates? What is the device design? What, what are the flow rates?What, what are the volumes of blood that has to be processed and how do you make sure that the cells you capture are the ones that you actually wanted to capture? All these aspects kind formed the whole challenge that can be summarized in one sentence, which is how do you capture this one rare cell out of billion others? So it's really a needle in a haystack kind of problem, which is exciting.And I think this is moving the technologies forward to address these problems is the current technological challenge. Being able to bring them to a level where they do the job that they're supposed to do is one thing. And then taking it to the clinic and making it a product has all different expertise such as being able to start a company, being able to patent these things and all those other aspects come into the picture.And I think we as scientists primarily don't have all of those skills that takes the products from desktop to products. So there comes in again, the importance of collaborations with people who have different backgrounds. And there are many technologies out there that I think could be very impactful and useful.They sometimes don't make it to the clinic or to the use of humankind either because it just didn't happen, wasn't the right time or other effects came into the picture that I just talked about. Or sometimes there isn't a direct link that you couldn't see that the technology could actually solve that problem. And the person who deals with the problem, or let's say the biologists or people in medicine are used to doing it one way for many years.And the people on the technology side mostly if you're not having a biotechnology focus, are not aware of the problems in medicine. So bringing these two sides together is a serious interdisciplinary research. And I think in the past years, the whole emphasis both at NIH level and that we see in research labs and universities is to generate interdisciplinary research.So people end up getting PhDs where they have to know multiple fields. For instance, for my PhD, I had to know acoustics, I had to know mems, I had to know microfluidics and I had to apply this for polymers. So now you see kinda very depth of knowledge in one field where you had to know actually three other fields quite well to be able to solve that problem.So I think the answer is in interdisciplinary research combined with people with business skills to make it available to use of the people, it's a whole big process and some inefficiencies that come in at certain locations. Cause that low percent of tech transfer.

Biology

Engineering Platform and Experimental Protocol for Design and Evaluation of a Neurally-controlled Powered Transfemoral Prosthesis

To enable intuitive operation of powered artificial legs, an interface between user and prosthesis that can recognize the user's movement intent is desired. A novel neural-machine interface (NMI) based on neuromuscular-mechanical fusion developed in our previous study has demonstrated a great potential to accurately identify the intended movement of transfemoral amputees. However, this interface has not yet been integrated with a powered prosthetic leg for true neural control. This study aimed to report (1) a flexible platform to implement and optimize neural control of powered lower limb prosthesis and (2) an experimental setup and protocol to evaluate neural prosthesis control on patients with lower limb amputations. First a platform based on a PC and a visual programming environment were developed to implement the prosthesis control algorithms, including NMI training algorithm, NMI online testing algorithm, and intrinsic control algorithm. To demonstrate the function of this platform, in this study the NMI based on neuromuscular-mechanical fusion was hierarchically integrated with intrinsic control of a prototypical transfemoral prosthesis. One patient with a unilateral transfemoral amputation was recruited to evaluate our implemented neural controller when performing activities, such as standing, level-ground walking, ramp ascent, and ramp descent continuously in the laboratory. A novel experimental setup and protocol were developed in order to test the new prosthesis control safely and efficiently. The presented proof-of-concept platform and experimental setup and protocol could aid the future development and application of neurally-controlled powered artificial legs.

Neural-machine interfaces (NMI) have been developed to identify the user's locomotion mode. These NMIs are potentially useful for neural control of powered artificial legs, but have not been fully demonstrated. This paper presented (1) our designed engineering platform for easy implementation and development of neural control for powered lower limb prostheses and (2) an experimental setup and protocol in a laboratory environment to evaluate neurally-controlled artificial legs on patients with lower limb amputations safely and efficiently.

To enable intuitive operation of powered artificial legs, an interface between user and prosthesis that can recognize the user's movement intent is ...

A Tactile Automated Passive-Finger Stimulator (TAPS)

Although tactile spatial acuity tests are used in both neuroscience research and clinical assessment, few automated devices exist for delivering controlled spatially structured stimuli to the skin. Consequently, investigators often apply tactile stimuli manually. Manual stimulus application is time consuming, requires great care and concentration on the part of the investigator, and leaves many stimulus parameters uncontrolled. We describe here a computer-controlled tactile stimulus system, the Tactile Automated Passive-finger Stimulator (TAPS), that applies spatially structured stimuli to the skin, controlling for onset velocity, contact force, and contact duration. TAPS is a versatile, programmable system, capable of efficiently conducting a variety of psychophysical procedures. We describe the components of TAPS, and show how TAPS is used to administer a two-interval forced-choice tactile grating orientation test.
Corresponding Author: Daniel Goldreich

A Tactile Automated Passive-Finger Stimulator TAPS

We describe a computer-controlled device for investigating the sense of touch: the Tactile Automated Passive-finger Stimulator (TAPS). We describe the components of TAPS, and show how TAPS is used to administer a two-interval forced-choice tactile grating orientation test.

Although tactile spatial acuity tests are used in both neuroscience research and clinical assessment, few automated devices exist for delivering ...

Measuring the Switch Cost of Smartphone Use While Walking

This paper presents a study protocol to measure the task-switching cost of using a smartphone while walking. This method involves having participants walk on a treadmill under two experimental conditions: a control condition (i.e., simply walking) and a multitasking condition (i.e., texting while walking). During these conditions, the participants must switch between the tasks related to the experimental condition and a direction determining task. This direction task is done with a point-light walker figure, seemingly walking towards the left or the right of the participant. Performance on the direction task represents the participant&#8217;s task-switching costs. There were two performance measures: 1) correct identification of the direction and 2) response time. EEG data are recorded in order to measure the alpha oscillations and cognitive engagement occurring during the task switch. This method is limited in its ecological validity: pedestrian environments have many stimuli occurring simultaneously and competing for attention. Nonetheless, this method is appropriate for pinpointing task-switching costs. The EEG data allow the study of the underlying mechanisms in the brain that are related to differing task-switching costs. This design allows the comparison between task switching when doing one task at a time, as compared to task switching when multitasking, prior to the stimulus presentation. This allows understanding and pinpointing both the behavioral and neurophysiological impact of these two different task-switching conditions. Furthermore, by correlating the task-switching costs with the brain activity, we can learn more about what causes these behavioral effects. This protocol is an appropriate base for studying the switching cost of different smartphone uses. Different tasks, questionnaires, and other measures can be added to it in order to understand the different factors involved in the task-switching cost of smartphone use while walking.

This study design measures the task-switching cost of using a smartphone while walking. Participants undergo two experimental conditions: a control condition (walking) and a multitasking condition (texting while walking). Participants switch between these tasks and a direction determining task. EEG data as well as behavioral measures are recorded.

This paper presents a study protocol to measure the task-switching cost of using a smartphone while walking. This method involves having participants walk ...

Behavior

Quantitative Static and Dynamic Assessment of Balance Control in Stroke Patients

In patients with stroke, damage to the central nervous system (CNS) can affect the postural stability and increase the risk of falling. Therefore, accurately assessing the balance is important to understand the type, extent, and causes of balance deficit, and to identify individualized interventions. Clinical assessment methods for balance function can be broadly divided into observation, scale assessment, and balance instrument testing. Here, a clinical protocol is presented for static and dynamic balance assessment in stroke patients, which includes three semiquantitative balance function scale assessments (i.e., Berg Balance Scale, Timed Up and Go Test, and Fugl-Meyer Assessment) and three quantitative instrumental balance evaluation (i.e., Stability Assessment Module, Proprioceptive Assessment Module, and Limit of Stability Module). It is recommended that clinicians consider the use of both classic clinical balance scales and instrumental balance measurements when assessing stroke patients to improve the accuracy of assessments, leading to a better individualized treatment plan.

Presented here is a quantitative, clinical balance assessment method suitable for stroke patients with balance disorders.

In patients with stroke, damage to the central nervous system (CNS) can affect the postural stability and increase the risk of falling. Therefore, ...

An Assessment Method and Toolkit to Evaluate Keyboard Design on Smartphones

Keyboard input has played an essential role in human-computer interaction with a vast user base, and the keyboard design has always been one of the fundamental objects of studies on smart devices. With the development of screen technology, more precise data and indicators could be collected by smartphones to in-depth evaluate the keyboard design. The enlargement of the phone screen has led to unsatisfactory input experience and finger pain, especially for one-handed input. The input efficiency and comfort have attracted the attention of researchers and designers, and the curved keyboard with size-adjustable buttons, which roughly accorded with the physiological structure of thumbs, was proposed to optimize the one-handed usage on large-screen smartphones. However, its real effects remained ambiguous. Therefore, this protocol demonstrated a general and summarized method to evaluate the effect of curved QWERTY keyboard design on a 5-inch smartphone through a self-developed software with detailed variables, including objective behavioral data, subjective feedback, and the coordinate data of each touchpoint. There is sufficient existing literature on evaluating virtual keyboards; however, only a few of them systematically summarized and took reflection on the evaluation methods and processes. Therefore, this protocol fills in the gap and presents a process and method of the systematic evaluation of keyboard design with available codes for analysis and visualization.&#160;It needs no additional or expensive equipment&#160;and is easy to conduct and operate. In addition, the protocol also helps to get potential reasons for the disadvantages of the design and enlightens the optimization of designs. In conclusion, this protocol with the open-source resources could not only be an in-class demonstrative experiment to inspire the novice to start their studies but also contributes to improving the user experience and the revenue of input method editor companies.

The presented protocol integrates various evaluation methods and demonstrates a method to evaluate the keyboard design on smartphones. Pairs matched by English characters are proposed as the input material, and the transition time between two keys is used as the dependent variable.

Keyboard input has played an essential role in human-computer interaction with a vast user base, and the keyboard design has always been one of the ...

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An easy-to-use and low-cost sensor platform is highly desired to measure various types of analytes (e.g., biomarkers, hormones, and drugs) quantitatively and specifically. For this reason, dry film photoresist technology - enabling cheap, facile, and high-throughput fabrication - was used to manufacture the microfluidic biosensor presented here. Depending on the bioassay used afterwards, the versatile platform is capable of detecting various types of biomolecules. For the fabrication of the device, platinum electrodes are structured on a flexible polyimide (PI) foil in the only clean-room process step. The PI foil serves as a substrate for the electrodes, which are insulated with an epoxy-based photoresist. The microfluidic channel is subsequently generated by the development and lamination of dry film photoresist (DFR) foils onto the PI wafer. By using a hydrophobic stopping barrier in the channel, the channel is separated into two specific areas: an immobilization section for the enzyme-linked assay and an electrochemical measurement cell for the amperometric signal readout.
The on-chip bioassay immobilization is performed by the adsorption of the biomolecules to the channel surface. The glucose oxidase enzyme is used as a transducer for electrochemical signal generation. In the presence of the substrate, glucose, hydrogen peroxide is produced, which is detected at the platinum working electrode. The stop-flow technique is applied to obtain signal amplification along with rapid detection. Different biomolecules can quantitatively be measured by means of the introduced microfluidic system, giving an indication of different types of diseases, or, in regard to therapeutic drug monitoring, facilitating a personalized therapy.

A microfluidic biosensor platform was designed and fabricated using low-cost dry film photoresist technology for the rapid and sensitive quantification of various analytes. This single-use system allows for the electrochemical readout of on-chip-immobilized enzyme-linked assays by means of the stop-flow technique.

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...

Design, Fabrication, and Administration of the Hand Active Sensation Test (HASTe)

The concept of personalizing neurologic rehabilitation, based on individual impairments, has experienced a recent surge. In parallel, the number of outcome measures of upper extremity motor performance has grown. However, clinicians and researchers lack practical, quantitative measures of the hand&#8217;s natural role as a receptor of the environment. The Hand Active Sensation Test (HASTe), developed by Williams and colleagues in 2006, is a valid and reliable measure of haptic performance. Though not available commercially, the HASTe can be fabricated from inexpensive materials, and it has been used successfully to identify impairments in haptic touch in individuals with stroke. (Williams, 2006). This paper presents the methods of design and fabrication of the HASTe testing kit, as well as a visual screen to be used during administration, and instructions for the tests administration and scoring.

Design, Fabrication, and Administration of the Hand Active Sensation Test HASTe

The Hand Active Sensation Test (HASTe) is a valid and reliable measure of haptic performance, which has been used successfully to identify impaired haptic touch in individuals with stroke. The purpose of this paper is to describe the design, fabrication and administration of the HASTe.

The concept of personalizing neurologic rehabilitation, based on individual impairments, has experienced a recent surge. In parallel, the number of ...

Medicine

Smartphone-Integrated Dengue NS1 Paper-Based Diagnostic Device

Portable Paper-Based Immunoassay Combined with Smartphone Application for Colorimetric and Quantitative Detection of Dengue NS1 Antigen

Dengue virus (DENV) infection, which is transmitted by Aedes mosquitoes, is a major public health concern in tropical and subtropical countries. With an annual incidence of approximately 10 million cases and 20,000-25,000 deaths, particularly among children, there is an urgent need for practical diagnostic tools. The presence of dengue non-structural protein 1 (NS1) during early infection has been linked to cytokine release, vascular leakage, and endothelial dysfunction, making it a potential marker for severe dengue. 
Paper-based immunoassays such as lateral flow assays (LFAs) and microfluidic paper-based analytical devices (PADs) have gained popularity as diagnostic tests due to their simplicity, rapidity, inexpensiveness, specificity, and ease of interpretation. However, conventional paper-based immunoassays for dengue NS1 detection typically rely on visual inspection, yielding only qualitative results. To address this limitation and enhance sensitivity, we proposed a highly portable NS1 dengue detection assay on a Paper-based Analytical Device (PAD), namely, DEN-NS1-PAD, that integrates a smartphone application as a colorimetric and quantitative reader. The development system enables direct quantification of NS1 concentrations in clinical samples. 
Serum and blood samples obtained from patients were utilized to demonstrate the system prototype performance. The results were obtained immediately and can be employed for clinical assessment, both in well-equipped healthcare facilities and resource-limited settings. This innovative combination of a paper-based immunoassay with a smartphone application offers a promising approach for enhanced detection and quantification of dengue NS1 antigen. By augmenting sensitivity beyond the capabilities of the naked eye, this system holds great potential for improving clinical decision-making in dengue management, particularly in remote or underserved areas.

Author Spotlight: Development of a Smartphone-Enhanced Paper-Based Device for Rapid Dengue NS1 Detection

Addressing urgent dengue diagnostic needs, here we introduce a smartphone app-integrated Dengue NS1 Paper-based Analytical Device (DEN-NS1-PAD) for quantifying Dengue NS1 antigen concentration in clinical serum/blood samples. This innovation enhances dengue management by aiding clinical decision-making in various healthcare settings, even resource-limited ones.

Dengue virus (DENV) infection, which is transmitted by Aedes mosquitoes, is a major public health concern in tropical and subtropical countries. With an ...

Immunology and Infection

Evaluating the Function of the Foot Core System in the Elderly

As a complex structure to link the body and the ground, the foot contributes to postural control in human static and dynamic activities. The foot core is rooted in the functional interdependence of the passive, active, and neural subsystems, which combine into the foot core system controlling foot motion and stability. The foot arch (passive subsystem), responsible for load, is considered the functional core of the foot, and its stability is necessary for normal foot functions. The functional abnormalities of the foot have been widely reported in the elderly, such as weakness of toe flexor muscles, abnormal foot postures, and decreased plantar sensory sensitivity. In this paper, a comprehensive approach is introduced for evaluating the foot function based on foot core subsystems. The strength and morphology of the foot intrinsic and extrinsic muscles were used to evaluate the foot muscle (active subsystem) function. The doming strength test was applied to determine the function of foot intrinsic muscles, while the toe flexion strength test focused more on the function of extrinsic muscles. The navicular drop test and foot posture index were applied to evaluate foot arch (passive subsystem) function. For the neural subsystem, the plantar light touch threshold test and two-point discrimination test were used to assess plantar tactile sensitivity at nine regions of the foot. This study provides new insights into the foot core function in the elderly and other populations.

The functional core stability of the foot contributes to the human static posture and dynamic activities. This paper proposes a comprehensive evaluation for the function of the foot core system, which combines three subsystems. It may provide increased awareness and multifaceted protocol to explore the foot function among different populations.

As a complex structure to link the body and the ground, the foot contributes to postural control in human static and dynamic activities. The foot core is ...

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

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