Difference between revisions of "Team:Munich/Description"

 
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<font size=7 color=#51a7f9><b style="color: #51a7f9;">Description</b></font>
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<font size=7 color=#51a7f9><b style="color: #51a7f9">Description</b></font>
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<p class="introduction">
 
<p class="introduction">
Thanks to advances in molecular biology and biochemistry, scientists have been able to consistently detect lower and lower concentration of molecules<sup><a class="myLink">1</a></sup>, to the point that single molecules can be reliably recognized with methods such as polymerase chain reaction (PCR)<sup><a class="myLink">2</a></sup>, fluorescence in situ hybridization (FISH)<sup><a class="myLink">3</a></sup> and enzyme-linked immunosorbent assays (ELISA)<sup><a class="myLink">4</a></sup>. This has opened doors for synthetic biology to create better and more accurate diagnostic tests that use biomarkers like nucleic acids and proteins as targets<sup><a class="myLink">5</a>,<a class="myLink">6</a></sup>. Through such advances, the field of molecular diagnostics developed. Unfortunately, current standard methods require expensive equipment or trained personnel, which generally limits their usability to hospitals or laboratories. Recently, there has been a push to develop new tests that fuse the reliability of standard methods with affordable platforms such as lab-on-a-chip or paper strips  to overcome this restrictions<sup><a class="myLink">7-9</a></sup>. We wanted to help close this gap and set out to engineer a diagnosis principle for the detection of a wide array of targets that could be used without difficult-to-meet technical requirements..              
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Thanks to advances in molecular biology and biochemistry, scientists have been able to consistently detect lower and lower concentration of molecules<sup><a class="myLink" href="#ref_1">1</a></sup>, to the point where single molecules can be reliably recognized using methods such as polymerase chain reaction (PCR)<sup><a class="myLink" href="#ref_2">2</a></sup>, fluorescence in situ hybridization (FISH)<sup><a class="myLink" href="#ref_3">3</a></sup> and enzyme-linked immunosorbent assays (ELISA)<sup><a class="myLink" href="#ref_4">4</a></sup>. This has opened doors for synthetic biology to create better and more accurate diagnostic tests that use biomarkers like nucleic acids and proteins as a target<sup><a class="myLink" href="#ref_5">5</a>,<a class="myLink" href="#ref_6">6</a></sup>. These advances have led to development of the field of molecular diagnostics. Unfortunately, current standard diagnostic methods require expensive equipment or trained personnel, which limits their usability to hospitals or laboratories. Recently, there has been a push to develop new tests that fuse the reliability of standard methods with affordable platforms such as lab-on-a-chip or paper strips  to overcome these restrictions<sup><a class="myLink" href="#ref_7">7-9</a></sup>. We wanted to help seal this gap and thus set out to engineer a diagnosis principle for the detection of a wide array of targets that could be used at the point-of-care.  
 
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<img src="https://static.igem.org/mediawiki/2017/d/dc/T--Munich--Demonstrate_Overview.svg">
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<p>Overview of our three major hardware modules. Shown are, starting from the left: the processing unit, the paper strip and our fluorescence detector.</p>
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<h3>Problem Definition</h3>
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<p>
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Antibiotic resistance is a global public health risk with high severity. Overuse of antibiotics happening probably since discovery of penicillin has led to appearance of multi-resistant strains of pathogens. About 90% of all antibiotics prescription are issued by general practitioners (GP) and most of them account for upper respiratory tract infections (57% of all prescribed antibiotics in Europe)<sup><a class="myLink" href="#ref_11">11</a></sup>. Astonishing is that from 70% of patients in U.S. having sore throat receive antibiotics prescription, while only about 20-30% are likely to have a bacterial infection recommended for antibiotic treatment<sup><a class="myLink" href="#ref_12">12</a></sup>. Besides, results of survey in UK showed that 55% of GPs felt under pressure, mainly from patients, to prescribe antibiotics and 44% admitted that they issued prescription to get a patient to leave the surgery.<sup><a class="myLink" href="#ref_11">11</a></sup>
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<h3>Solution Statement</h3>
 
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<h3>CascAID</h3>
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<p>   
 
<p>   
Our project, which we named Cas13a controlled assay for infectious diseases (CascAID), features the recently identified CRISPR/Cas effector Cas13a<sup><a class="myLink">10</a></sup>. Unlike other proteins in the familiy, Cas13a has the unique ability to bind and cleave specific RNA targets rather than DNA ones.  Moreover, after cleaving its target, Cas13a is able to unspecifically cleave RNA molecules. By using this collateral activity from Cas13a, our system is capable of detecting virtually any RNA target. This is done by changing the crRNA in the protein, that is a short RNA sequence that determines what is recognized as target.</p>
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We set out to develop highly specific highly sensitive rapid <i>in vitro</i> diagnostic (IVD) device that will provide solid foundation for treatment. We strongly believe that point-of-care (POC) IVD device, which is able to distinguish pathogens (including both viral and bacterial), will significantly contribute to resolution of antibiotics overprescription and respectively of antibiotic resistance crisis. Besides that our device is easily configurable and can be quickly adopted to detect nucleic acid sequence of pathogen of choice.
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As a basis for design of our solution we used WHO guideline for POC testing devices known as “ASSURED”<sup><a class="myLink" href="#ref_13">13</a></sup>, which stands for: affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free, deliverable to the person in need. Moreover, we decided to create all-in-one portable solution that includes all steps of analysis: sample processing, nucleic acid amplification and target detection.</p>
 
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<a href="https://www.uni-muenchen.de/index.html"><img src="https://static.igem.org/mediawiki/2017/0/0d/T--Munich--Logo_LMU.png" alt="Diagram for Cas13a's function" width="270"></a>
 
<p>Cas13a binds specific target RNA depending on the crRNA sequence. After activation, Cas13a cleaves RNA indiscriminately.</p>
 
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<h3>CascAID<sup>+</sup></h3>
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<p>   
 
<p>   
We wanted to start our project by showing that Cas13a's collateral activity could be used to detect the presence of specific RNA. For this, we used the RNAse alert system, as done in a recent publication<sup><a class="myLink">11</a></sup>, to detect RNA digestion. In this assay, the presence of RNAse-like activity is detected by an increase in green fluorescence. Our experiments yielded a convincing proof-of-principle which we went on to model. Moreover, CascAID can be used to detect a wide spectrum of pathogens, as our experiments with gram-positive and viral targets suggested. As we wanted to make CascAID available for everyone, we focused on building an inexpensive fluorescence detector to measure the presence of the target. Our detector “Lightbringer” was designed to be able to detect the fluorescence produced by the fluorescein in the Rnase alert system<sup><a class="myLink">12</a></sup>, but we theorize that changing the filters allows detection of other fluorophores. In addition, we experimented with freeze-drying on paper to make CascAID durable and easily portable.
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Our project, named <b>Cas13a controlled assay for infectious diseases (CascAID)</b>, features the recently identified CRISPR/Cas effector Cas13a<sup><a class="myLink" href="#ref_10">10</a></sup>. Unlike other proteins in the familiy, Cas13a has the unique ability to bind and cleave specific RNA rather than DNA targets. Moreover, after cleaving its target, Cas13a is able to unspecifically cleave RNA molecules. By using this collateral activity of Cas13a, our system is capable of detecting virtually any RNA target. This is done by changing the crRNA in the protein, which is a short RNA sequence that determines what is recognized as target.</p>
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<a href="http://www.uni-muenchen.de/studium/lehre_at_lmu/index.html"><img src="https://static.igem.org/mediawiki/2017/9/9a/T--Munich--Logo_LehreLMU.gif" width="200"></a>
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<p>Cas13a can be used to detect specific RNA sequences</p>
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<img width=320 src="https://static.igem.org/mediawiki/2017/0/03/T--Munich--Description_Cas13a_Movie.gif">
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<p>The 3D structure of Cas13a Lbu shows the crRNA (blue) which binds the complementary target RNA (green).</p>
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<p>Picture of the Thermocycler</p>
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<img width=960 src="https://static.igem.org/mediawiki/2017/0/04/T--Munich--Description_Cas13a_Mechanism.svg" alt="Diagram for Cas13a's function">
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<p>Cas13a binds specific target RNA depending on the crRNA sequence. After activation, Cas13a cleaves RNA indiscriminately, which we use for our detection mechanisms. The scheme vizualizes the possibilty to design new crRNAs to sense any kind of target sequence.</p>
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For RNA extraction from the samples we tested three methods: extraction with silica beads, extraction with silica membrane and heat lysis. We custom-built an affordable thermocycler for signal amplification by RT-PCR to improve the detection limit. We explored recombinase polymerase amplification (RPA), an isothermal amplification procedure, to use over more conventional PCR methods as its simplicity makes it the more attractive option.
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<br><b>Our project is divided in the following 3 general parts:</b></p>
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<h3>Sample Processing Unit</h3>
<img src="https://static.igem.org/mediawiki/2017/a/ae/T--Munich--wiki_image_coop_tum_low.svg" border=0 height="20" onmouseover="this.src='https://static.igem.org/mediawiki/2017/1/1e/T--Munich--wiki_image_coop_tum_high.svg'" onmouseout="this.src='https://static.igem.org/mediawiki/2017/a/ae/T--Munich--wiki_image_coop_tum_low.svg'">
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Tackling the challenge of sample pre-processing on field, we started developing a portable fluidic system featuring a temperature control unit for lysis and isothermal PCR (RPA). Conceiving a platform independent of lab infrastructure, we demonstrate the feasibility of controlling fluid flow control with the simplest tools possible using bike tires and air balloons.</p>
 
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<a href="http://www.e14.ph.tum.de/en/home/" target="_blank5" >
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<h3>Paper Strip Reaction Unit</h3>
<a href="https://www.helmholtz-muenchen.de/" target="_blank5" >
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<img src="https://static.igem.org/mediawiki/2017/0/05/T--Munich--wiki_image_coop_helmholtzzentrum_low.svg" border=0 height="20" onmouseover="this.src='https://static.igem.org/mediawiki/2017/6/6b/T--Munich--wiki_image_coop_helmholtzzentrum_high.svg'" onmouseout="this.src='https://static.igem.org/mediawiki/2017/0/05/T--Munich--wiki_image_coop_helmholtzzentrum_low.svg'">
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After pre-processing, the idea was to combine all diagnostic reactions into one easy-to-use format. We chose to embed all the reactions into the format of a paper strip of the size of a typical post-it, where our full readout reaction cascade takes place. This enables to freeze-dry all reaction agents to make them fit for long-term storage. An additional advantage of the paper format are the low sample volumes needed for a reaction assey. To enable transport of the sample-containing fluid to the areas containing the detection mixture, we chose to use the paper-fluidics technology. The whole printing mechanism of the paper fluidics is based around a regular office printer to pattern the paper with hydrophobic wax channels. The detection circuit is first assessed in bulk, the Cas13a is characterized using the RNaseAlert standard, its detection limit is determined and the differentiation between viral and bacterial targets is verified, before the mechanism is transferred into a paper strip application. Three advanced readout methods are designed and explored, all of which propose an amplification cascade following Cas13a target detection. Those readout methods, combined with the fluidics, should give us the possibility to lower the detection limit and improve the on-field use.</p>
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<h3>Detector Unit</h3>
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Starting from the fact that suitable measurement instruments with sufficient sensitivity for field use are too expensive for mass production, we constructed a portable low-cost fluorescence detector, which can be easily assembled with a few standard worldwide available electronic parts and a 3D-printer. Driving the development even further, we pushed the sensitivity of our detector into the range of commercial plate readers, while conserving an assembly cost of under 15$. A detailed documentation of the detector development and sensitivity determination can be found under <a class="myLink" href="/Team:Munich/Measurement">Measurement</a> and <a class="myLink" href="/Team:Munich/Hardware/Detector">Detector</a>.
 +
On top of our hardware technology we provide a software for a crRNA databank, secondary structure verification of crRNAs and off-target verification of designed crRNAs. In combination with the detector unit, we supply a program code to evaluate data acquired with our detector.</p>
 
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<h3>References</h3>
<img src="https://static.igem.org/mediawiki/2017/8/8b/T--Munich--wiki_image_sm_twitter_low.svg" border=0 height="25" onmouseover="this.src='https://static.igem.org/mediawiki/2017/c/cc/T--Munich--wiki_image_sm_twitter_high.svg'" onmouseout="this.src='https://static.igem.org/mediawiki/2017/8/8b/T--Munich--wiki_image_sm_twitter_low.svg'">
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<p>
</a>
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    <ol style="text-align: left">
 +
      <li id="ref_1">Cohen, Limor, and David R. Walt. "Single-Molecule Arrays for Protein and Nucleic Acid Analysis." Annual Review of Analytical Chemistry 0 (2017).</li>
 +
      <li id="ref_2">Nakano, Michihiko, et al. "Single-molecule PCR using water-in-oil emulsion." Journal of biotechnology 102.2 (2003): 117-124.</li>
 +
      <li id="ref_3">Taniguchi, Yuichi, et al. "Quantifying E. coli proteome and transcriptome with single-molecule sensitivity in single cells." science 329.5991 (2010): 533-538.</li>
 +
      <li id="ref_4">Rissin, David M., et al. "Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations." Nature biotechnology 28.6 (2010): 595-599.</li>
 +
      <li id="ref_5">Pardee, Keith, et al. "Rapid, low-cost detection of Zika virus using programmable biomolecular components." Cell 165.5 (2016): 1255-1266.</li>
 +
      <li id="ref_6">Slomovic, Shimyn, Keith Pardee, and James J. Collins. "Synthetic biology devices for in vitro and in vivo diagnostics." Proceedings of the National Academy of Sciences 112.47 (2015): 14429-14435.</li>
 +
      <li id="ref_7">Tang, Ruihua, et al. "A fully disposable and integrated paper-based device for nucleic acid extraction, amplification and detection." Lab on a Chip 17.7 (2017): 1270-1279.</li>
 +
      <li id="ref_8">Vashist, Sandeep Kumar, et al. "Emerging technologies for next-generation point-of-care testing." Trends in biotechnology 33.11 (2015): 692-705.</li>
 +
      <li id="ref_9">Gubala, Vladimir, et al. "Point of care diagnostics: status and future." Analytical chemistry 84.2 (2011): 487-515.</li>
 +
      <li id="ref_10">Abudayyeh, Omar O., et al. "C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector." Science 353.6299 (2016): aaf5573.</li>
 +
      <li id="ref_11">Llor, C., & Bjerrum, L. (2014). Antimicrobial resistance: risk associated with antibiotic overuse and initiatives to reduce the problem. Therapeutic Advances in Drug Safety, 5(6), 229–241.</li>
 +
      <li id="ref_12">Shulman ST1, Bisno AL, Clegg HW, Gerber MA, Kaplan EL, Lee G, Martin JM, Van Beneden C. Clinical practice guideline for the diagnosis and management of group A streptococcal pharyngitis: 2012 update by the Infectious Diseases Society of America. Clin Infect Dis. 2012 Nov 15;55(10):1279-82. doi: 10.1093/cid/cis847.</li>
 +
      <li id="ref_13">Grace Wu & Muhammad H Zaman. Low-cost tools for diagnosing and monitoring HIV infection in low-resource settings. Bulletin of the World Health Organization 2012;90:914-920. doi: 10.2471/BLT.12.102780</li>
 +
    </ol>
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Latest revision as of 22:45, 15 December 2017


Description

Thanks to advances in molecular biology and biochemistry, scientists have been able to consistently detect lower and lower concentration of molecules1, to the point where single molecules can be reliably recognized using methods such as polymerase chain reaction (PCR)2, fluorescence in situ hybridization (FISH)3 and enzyme-linked immunosorbent assays (ELISA)4. This has opened doors for synthetic biology to create better and more accurate diagnostic tests that use biomarkers like nucleic acids and proteins as a target5,6. These advances have led to development of the field of molecular diagnostics. Unfortunately, current standard diagnostic methods require expensive equipment or trained personnel, which limits their usability to hospitals or laboratories. Recently, there has been a push to develop new tests that fuse the reliability of standard methods with affordable platforms such as lab-on-a-chip or paper strips to overcome these restrictions7-9. We wanted to help seal this gap and thus set out to engineer a diagnosis principle for the detection of a wide array of targets that could be used at the point-of-care.

Overview of our three major hardware modules. Shown are, starting from the left: the processing unit, the paper strip and our fluorescence detector.

Problem Definition

Antibiotic resistance is a global public health risk with high severity. Overuse of antibiotics happening probably since discovery of penicillin has led to appearance of multi-resistant strains of pathogens. About 90% of all antibiotics prescription are issued by general practitioners (GP) and most of them account for upper respiratory tract infections (57% of all prescribed antibiotics in Europe)11. Astonishing is that from 70% of patients in U.S. having sore throat receive antibiotics prescription, while only about 20-30% are likely to have a bacterial infection recommended for antibiotic treatment12. Besides, results of survey in UK showed that 55% of GPs felt under pressure, mainly from patients, to prescribe antibiotics and 44% admitted that they issued prescription to get a patient to leave the surgery.11

Solution Statement

We set out to develop highly specific highly sensitive rapid in vitro diagnostic (IVD) device that will provide solid foundation for treatment. We strongly believe that point-of-care (POC) IVD device, which is able to distinguish pathogens (including both viral and bacterial), will significantly contribute to resolution of antibiotics overprescription and respectively of antibiotic resistance crisis. Besides that our device is easily configurable and can be quickly adopted to detect nucleic acid sequence of pathogen of choice.

As a basis for design of our solution we used WHO guideline for POC testing devices known as “ASSURED”13, which stands for: affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free, deliverable to the person in need. Moreover, we decided to create all-in-one portable solution that includes all steps of analysis: sample processing, nucleic acid amplification and target detection.

CascAID+

Our project, named Cas13a controlled assay for infectious diseases (CascAID), features the recently identified CRISPR/Cas effector Cas13a10. Unlike other proteins in the familiy, Cas13a has the unique ability to bind and cleave specific RNA rather than DNA targets. Moreover, after cleaving its target, Cas13a is able to unspecifically cleave RNA molecules. By using this collateral activity of Cas13a, our system is capable of detecting virtually any RNA target. This is done by changing the crRNA in the protein, which is a short RNA sequence that determines what is recognized as target.

The 3D structure of Cas13a Lbu shows the crRNA (blue) which binds the complementary target RNA (green).

Diagram for Cas13a's function

Cas13a binds specific target RNA depending on the crRNA sequence. After activation, Cas13a cleaves RNA indiscriminately, which we use for our detection mechanisms. The scheme vizualizes the possibilty to design new crRNAs to sense any kind of target sequence.


Our project is divided in the following 3 general parts:

Sample Processing Unit

Tackling the challenge of sample pre-processing on field, we started developing a portable fluidic system featuring a temperature control unit for lysis and isothermal PCR (RPA). Conceiving a platform independent of lab infrastructure, we demonstrate the feasibility of controlling fluid flow control with the simplest tools possible using bike tires and air balloons.

Paper Strip Reaction Unit

After pre-processing, the idea was to combine all diagnostic reactions into one easy-to-use format. We chose to embed all the reactions into the format of a paper strip of the size of a typical post-it, where our full readout reaction cascade takes place. This enables to freeze-dry all reaction agents to make them fit for long-term storage. An additional advantage of the paper format are the low sample volumes needed for a reaction assey. To enable transport of the sample-containing fluid to the areas containing the detection mixture, we chose to use the paper-fluidics technology. The whole printing mechanism of the paper fluidics is based around a regular office printer to pattern the paper with hydrophobic wax channels. The detection circuit is first assessed in bulk, the Cas13a is characterized using the RNaseAlert standard, its detection limit is determined and the differentiation between viral and bacterial targets is verified, before the mechanism is transferred into a paper strip application. Three advanced readout methods are designed and explored, all of which propose an amplification cascade following Cas13a target detection. Those readout methods, combined with the fluidics, should give us the possibility to lower the detection limit and improve the on-field use.

Detector Unit

Starting from the fact that suitable measurement instruments with sufficient sensitivity for field use are too expensive for mass production, we constructed a portable low-cost fluorescence detector, which can be easily assembled with a few standard worldwide available electronic parts and a 3D-printer. Driving the development even further, we pushed the sensitivity of our detector into the range of commercial plate readers, while conserving an assembly cost of under 15$. A detailed documentation of the detector development and sensitivity determination can be found under Measurement and Detector. On top of our hardware technology we provide a software for a crRNA databank, secondary structure verification of crRNAs and off-target verification of designed crRNAs. In combination with the detector unit, we supply a program code to evaluate data acquired with our detector.

References

  1. Cohen, Limor, and David R. Walt. "Single-Molecule Arrays for Protein and Nucleic Acid Analysis." Annual Review of Analytical Chemistry 0 (2017).
  2. Nakano, Michihiko, et al. "Single-molecule PCR using water-in-oil emulsion." Journal of biotechnology 102.2 (2003): 117-124.
  3. Taniguchi, Yuichi, et al. "Quantifying E. coli proteome and transcriptome with single-molecule sensitivity in single cells." science 329.5991 (2010): 533-538.
  4. Rissin, David M., et al. "Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations." Nature biotechnology 28.6 (2010): 595-599.
  5. Pardee, Keith, et al. "Rapid, low-cost detection of Zika virus using programmable biomolecular components." Cell 165.5 (2016): 1255-1266.
  6. Slomovic, Shimyn, Keith Pardee, and James J. Collins. "Synthetic biology devices for in vitro and in vivo diagnostics." Proceedings of the National Academy of Sciences 112.47 (2015): 14429-14435.
  7. Tang, Ruihua, et al. "A fully disposable and integrated paper-based device for nucleic acid extraction, amplification and detection." Lab on a Chip 17.7 (2017): 1270-1279.
  8. Vashist, Sandeep Kumar, et al. "Emerging technologies for next-generation point-of-care testing." Trends in biotechnology 33.11 (2015): 692-705.
  9. Gubala, Vladimir, et al. "Point of care diagnostics: status and future." Analytical chemistry 84.2 (2011): 487-515.
  10. Abudayyeh, Omar O., et al. "C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector." Science 353.6299 (2016): aaf5573.
  11. Llor, C., & Bjerrum, L. (2014). Antimicrobial resistance: risk associated with antibiotic overuse and initiatives to reduce the problem. Therapeutic Advances in Drug Safety, 5(6), 229–241.
  12. Shulman ST1, Bisno AL, Clegg HW, Gerber MA, Kaplan EL, Lee G, Martin JM, Van Beneden C. Clinical practice guideline for the diagnosis and management of group A streptococcal pharyngitis: 2012 update by the Infectious Diseases Society of America. Clin Infect Dis. 2012 Nov 15;55(10):1279-82. doi: 10.1093/cid/cis847.
  13. Grace Wu & Muhammad H Zaman. Low-cost tools for diagnosing and monitoring HIV infection in low-resource settings. Bulletin of the World Health Organization 2012;90:914-920. doi: 10.2471/BLT.12.102780