Temperature-Independent, Portable, and Rapid Field Detection of Ebola via a Silk-Derived Lateral-Flow System

Up to 90% of Ebola victims will die without early diagnosis and medical intervention, which can reduce fatalities 50% and are critical to preventing future epidemics. Current detection methods are expensive, time-consuming and utilize complex instrumentation and chemicals that require uninterrupted refrigeration. Successfully maintaining the reagent's “cold-chain” from laboratory to point of use is highly problematic in regions with poor infrastructure, where Ebola is most common. This research sought to devise a rapid, simple and inexpensive Ebola detection platform that can be stored and transported without refrigeration. To begin, current Ebola ELISA reagents were embedded in silk fibroin, which possesses stabilizing properties, allowing storage of otherwise refrigerated reagents at room temperature. To confirm ELISA colorimetric detection of Ebola after prolonged, non-refrigerated storage of the kit's reagents, the Ebola ELISA was conducted in a 96-wellplate format (A450nm) at 0-7days from initial mixing and dilutions. Results indicate Ebola ELISA detection is viable in water dilutions only on the day of mixing. For silk-embedded reagents, successful detection was realized for up to one week of RT storage. Silk-film embedded Ebola ELISA reagents were used to construct a four-channel, paper-based, fluidic detection card, with colorimetric reagents positioned to create timed, visible detection of Ebola antigens. In this new device, that is stable and stored at room temperature, 30µl drops of water were used to dissolve silk-embedded reagents, initiating a timed-flow towards a center detection zone, where a positive (colored) result confirmed the presence of 500pg/ml Ebola(+)control antigens in 30min, at a cost of $25.

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I am a sophomore at Greenwich High School in Connecticut. My decision to focus my project on the development of a novel temperature-independent, rapid, portable and inexpensive diagnostic assay for the detection of the highly infectious and often fatal Ebola virus, with valuable applicability as an ELISA-based diagnostic for other diseases including HIV, Lyme Disease, Yellow Fever, Dengue Fever and certain cancers, was prompted by the devastating loss of life in Africa during the most recent 2014 Ebola outbreak. The consequences will be far-reaching; the concentration of fatalities in stricken areas has left many children orphaned, and the socioeconomic fabric of entire villages destroyed. School closures have impacted over five million children, risking permanent educational dislocation and high risk behaviors such as child labor. Winning would enable me to continue to develop my Ebola Assay Card as a multiple disease diagnostic assay, and to make a meaningful impact on global health through the early detection of often fatal  diseases. I would know that my research saved lives and changed futures. 

Outside of school I volunteer with the Connecticut Chapter of the Special Olympics, as a middle school tutor and youth swim instructor. I am a competitive swimmer and plan to swim in college. I hope to be a doctor like my late grandfather, and work for a global health organization such as Doctors Without Borders. I am inspired by Dr. Kent Brantly, who showed the global community our collective moral obligation to act with courage and compassion. 

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The current outbreak of the deadly Ebola Hemorrhagic Fever (“Ebola”) has killed over 10,000 West Africans, with an estimated 26,000 people infected as of May, 2015. As many as 90% of those stricken will die, although early medical intervention can reduce that number to approximately 50%. Severity of infection and corresponding likelihood of survival are largely dependent on  whether exposure was in the early stages of infection, when the virus is less severe. Consequently, the ability to rapidly, inexpensively and accurately diagnose Ebola and many other deadly diseases on site in the early stages of an outbreak and at points of travel, triage and high risk is essential. 

Current methods of Ebola detection require up to 12 hours to diagnose, as well as a “temperature-controlled supply line” or “cold chain”, the unbroken refrigeration of reagents from manufacture to use, making reliability highly problematic in remote countries and areas with poor infrastructure and intermittent access to power.  

I developed a “stable and stored at room temperature” Ebola Assay Card, applicable as an ELISA-based diagnostic for diseases such as HIV, Lyme and certain cancers, that will allow for water-activated, timed-release detection of Ebola antigens, with detection limits that are analogous to current sandwich ELISA techniques. Reagents become chemically “stabilized” when mixed into silk, which enables them to remain “chemically active” without refrigeration. This Ebola Assay Card will allow for shipment and storage without refrigeration, and provide detection of the Ebola viral antigens based on color change in as little as 30 minutes. 

 

 

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Current methods of Ebola detection utilize enzyme-linked immunosorbent assay ("ELISA") detection kits which cost approximately $1,000 each, require complex instrumentation, trained medical professionals to administer, and up to 12 hours from testing to diagnosis. The kits require the unbroken refrigeration of reagents from point of manufacture to point of use (the "cold chain"), making the ability to diagnose  in remote areas, where refrigeration is often nonexistent or unreliable, highly problematic if not impossible. Uninterrupted refrigeration requirements and lengthy time to confirm diagnosis are particularly challenging in situations such as airports, travel check points and triage situations, where rapid diagnosis is essential in effectively treating, containing and ultimately eradicating the spread of the deadly virus, and other similar infectious and highly contagious diseases. The temperature-controlled supply line, or “cold chain,” is critical in order to prevent reagents from becoming chemically inert, an irreversible process which destroys their effectiveness. However large areas of the world lack sufficient infrastructure and adequate resources (e.g. electricity, refrigerated trucks and reliable on site refrigeration) to maintain the cold chain. It is estimated with early diagnosis and medical treatment, Ebola fatality rates of up to 90% would decline by approximately 50%. 

The epicenter of the current 2014 outbreak has been in the West African countries of Liberia, Guinea and Sierra Leone whose sparse medical infrastructure, belief system and cultural norms around burial customs have sparked an unprecedented global health crisis. Ebola has spread across the world for the first time, fueling fears of a pandemic and concerns that the disease could genetically mutate and eventually become airborne. As of May, 2015 the CDC estimates in excess of 11,000 fatalities, forty times greater than the second most deadly outbreak in 1976 in Zaire in the first documented incidence of the virus with 280 fatalities. Early diagnosis is essential to containing the rate of infection and the spread of the disease; according to the CDC, in late 2014 cases in Liberia were doubling every 15 days, and every 30 days in Sierra Leone. It can take up to 21 days for a person infected with the Ebola virus to develop symptoms, at which point they become increasingly contagious with a diminished chance of survival, as the virus becomes more virulent the longer the incubation period prior to diagnosis, intervention and treatment.

 My research developed a "stable and stored at room temperature" Ebola Assay Card  (EAC), readily applicable in detection of diseases such as HIV, Lyme, Yellow/Dengue Fevers and certain cancers, building on the proven stabilizing properties of silk fibroin, that will allow for water-activated detection of Ebola antigens, with detection limits that are analogous to current sandwich ELISA techniques. This EAC will allow for shipment and storage on site without refrigeration, and provide detection based on color change in less than 30 minutes of Ebola viral antigens in individuals when they are still asymptomatic and/or in less advanced stages of the virus, when the severity of transmission is less acute and the likelihood of recovery is highest.

 

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Bombyx mori silk fibroin was separated from commercially available silk cocoons as per Rockwood et. al, with minor modifications noted below

Fabrication of the Silk Fibroin Solution

*modification: silk cooked for 60 minutes, allowing for a lower molecular weight to permit a faster release of the encased reagents

Fabrication of Silk Fibroin Thin Film Test Strips

To create silk fibroin thin film test strips on 102 filter paper, for design of micro-fluidic channels for the EAC, 2mm strips of filter paper were dipped into the silk fibroin solution for 5 seconds, hung vertically, and allowed to air-dry for 20 minutes. ATR-FTIR analysis of the solution versus the thin film (on filter paper) illustrates uniform coverage of silk fibroin, that is chemically stable.

SEM analysis of the dried silk fibroin thin film test strips further supports uniform coating of the filter paper substrate, with an average thickness of 30µm. 

Channel-fluidics of Silk Fibroin Thin Film Test Strips 

2mm filter paper test strips (with photographic paper backing) consisting of “pink-ink embedded” 2x8mm silk-fibroin dried films were used to study the dissolution of silk, and the subsequent fluidic motion of the ink (and later reagents) through the channel.  Time-lapse mode of a Smartphone was used to create distance traveled versus time for the addition of 15, 20, and 30 µl droplets (Figs. 4b-d). 30µl droplets were most effective, with the farthest delivery of reagents (15 mm) in ~20 seconds.

Flow channel SEMs, after the silk fibroin film has dissolved and after the reagents have passed demonstrate minimal residual silk fibroin to inhibit flow. 

Stabilization of Ebola ELISA Detection via Silk Fibroin Encasement

The Ebola Human Anti-ZEBOV NP IgG ELISA kit (AE-320520-1; Alpha Diagnostic International) used in this study is typical of a sandwich ELISA, whose methodology is shown in Figure 6.  Ebola antigens contained in the patient’s sample adhere to primary antibodies (Fig. 6b),  followed by sandwich with a secondary, detection antibody (Fig. 6c).  A horseradish peroxidase  complex binds to the detection antibody (Fig. 6d), and finally a TMB substrate is added (Fig. 6e) to enable detection typically at 450 nm. In the Ebola Human Anti-ZEBOV NP IgG ELISA kit, a positive result is indicated by a yellow solution. 

Methodology for the Sandwich ELISA (image courtesy of Isbio.com)

Ebola Human Anti-ZEBOV ELISA kit reagents were freshly prepared, as per instruction where all dilutions are made with deionized water.  Additionally, the same dilutions were carried out in the (prepared) silk fibroin solution.  ELISA detection of 1, 2.5, 5, and 10 U/ml calibration standards, as well as a positive control sample, were carried out in a 96-wellplate format using a Molecular Devices Vmax plate reader, with 450nm (-650nm background) detection. 

All safety precautions were observed, including the wearing of protective gloves and goggles

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EAC initial conceptual design: 102 filter paper affixed to photographic card stock, preventing seepage and encouraging flow in the cut flow channels. Ebola primary antibodies are embedded in silk fibroin, and added to the center detection window, to mimic coating of the 96-wellplate in a typical ELISA assay. Anti-human IgG HRP, TMB, and Stop solution reagents are similarly embedded in silk fibroin at positions 2-4, and wet with 30µl water, following the addition of human serum (sample), for timed dissolution and delivery of ELISA reagents to the detection window. Positive result indicated by a yellow color change.

 

Figure 14 (below): Final design of the silk-fibroin based EAC. Water-soluble blue ink is embedded in silk-fibroin solution at the tips of the flow channels.  30ul of water are added to each, in order of shortest to longest flow channel (1, 2, 3, & 4), so  that “color” arrives at the detection zone in the desired order for Ebola ELISA detection.

 

Figure 14(a-c): Successful Detection of Ebola antigens in +control (1U/ml) with EAC prototype, in ~30 minutes

 

Fresh Preparation:  For freshly prepared reagents, both water and silk dilutions demonstrated expected (well-fit) calibration curves; the +control (a ~1U/ml sample) was easily detectable.  It is important to note that the silk-prepared standards produced deeper, more easily recognized color, relative to the water. This was especially helpful for visible detection in the Ebola Assay Card.

 

 

1 Day: Reagents were stored at room temperature, and the same data collection was performed in new wells after 1-day (Fig 8a-c). Both preparations demonstrated well-fit calibrations, however, for the water samples, the 1U/ml standard, which is the same as the +control threshold, was no longer detectable (A450=0.009), rendering the water kit unusable.

 

1 Week:  Following 1 week of storage at room temperature, repeated run of the ELISA kit on new wells highlights the continued well-fit response for the reagents prepared in silk fibroin solution, including detection of the 1U/ml +control, with an A450 of 0.193 (Fig 9a-c).  Conversely, the calibration for the water-diluted reagents is no longer linear, and the 1U/ml +control is again undetectable.

Results

All aspects of the engineering goal were achieved:

  • Ebola Human Anti-ZEBOV NP IgG ELISA reagents found to be stable in silk fibroin, stored at room temperature, up to one week.
  • Sandwich ELISA detection of Ebola antigens was stable for up to one week of room temperature storage, including successful detection of 1U/ml positive result threshold.
  • An Ebola Assay Card (EAC) was designed with commercially available Ebola ELISA reagents embedded within silk fibroin thin films, on paper flow channels. Ebola detection is based on water-initiated, timed flow of these same reagents.
  • With the newly designed EAC, 1U/ml Ebola antigen was visually detected as a yellow color change for prototypes stored at room temperature up to one week. A “stable and stored at room temperature” Ebola Assay Card was successfully developed that can detect Ebola with similar sensitivities to the laboratory ELISA, in only 30 minutes, at a cost of ~ $25 per assay.
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This research successfully devised a novel, temperature-independent, rapid, simple and inexpensive Ebola detection platform based on proven chemistry with applicability for other diseases including HIV, Lyme Disease, Yellow Fever, Dengue Fever and certain cancers. Ebola ELISA reagents were embedded in silk fibroin, which possesses stabilizing properties, allowing storage of otherwise refrigerated reagents at room temperature. To confirm ELISA colorimetric detection of Ebola after prolonged, non-refrigerated storage of the kit's reagents, the Ebola ELISA was conducted in a 96-wellplate format (A450nm) at 0-7days from initial mixing and dilutions. Results indicate Ebola ELISA detection is viable in water dilutions only on the day of mixing. For silk-embedded reagents, successful detection was realized for up to one week of Room Temperature storage. Silk film-embedded Ebola ELISA reagents were used to construct a four-channel, paper-based, fluidic detection card, with colorimetric reagents positioned to create timed, visible detection of Ebola antigens. In this new device, that is stable and stored at room temperature, 30µl drops of water were used to dissolve silk-embedded reagents, initiating a timed-flow towards a center detection zone, where a positive (colored) result confirmed the presence of 500pg/ml Ebola(+)control antigens in 30min, at a cost of $25.

Results

All aspects of the engineering goal were achieved:

  • Ebola Human Anti-ZEBOV NP IgG ELISA reagents were found to be stable in silk-fibroin, when stored at room temperature, for up to 1 week.
  • Sandwich ELISA detection of Ebola antigens was stable for up to 1 week of room temperature storage, including successful detection of 1U/ml positive result threshold. 
  • An Ebola Assay Card (EAC) was designed, where commercially-available Ebola ELISA reagents were embedded within silk fibroin thin films, on paper flow channels. Ebola detection is based on water-initiated, timed flow of these same reagents.
  • With the newly designed EAC, 1U/ml Ebola antigen was visually detected as a yellow color change for prototypes that were stored at room temperature for up to 1 week.
  • A “stable and stored at room temperature” EAC was successfully developed that can detect Ebola with similar sensitivities to the laboratory ELISA in only 30 minutes, at  ~ $25 per assay (anticipated to meaningfully decrease when produced in volume)

Future Research 

  • Evaluation of EAC for increasingly prolonged storage times 
  • Application of silk-fibroin/flow-channel technology to other ELISA-based disease-detecting assays, notably HIV, which is highly infectious but can go undiagnosed for up to 10 years before an individual becomes symptomatic (v. 21 days for Ebola) 
  • Recalibrate an ELISA kit to detect concentration of the Ebola virus/antigens (and other diseased states) emitted in saliva towards the development of a saliva-based disease diagnostic card.
  • Distinct "load point" shapes transcend language barriers, facilitating use

Future Impact: 

  • Greatly improved global health and mitigation of damaging secondary socioeconomic consequences of epidemics and disease through early diagnosis/treatment 
  • Temperature-Independent EAC permits increased yield and access; ~ 25% of current diagnostic kits rendered inactive due to breaks in the cold chain 
  • EACs included in disaster relief shipments, stored on-site in medical centers and travel check points; distributed through pharmacies, hospitals and NGO’s 
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My Inspiration:

  • Dr. Kent Brantly, with whom I never spoke or met, but whose actions during the 2014 Ebola outbreak demonstrated incredible courage, overwhelming compassion and the awareness that as global citizens, what affects one affects us all. Every one of us has a moral obligation and responsibility as members of a global community to act and the ability to be a catalyst for change. 
  • My late grandfather, a doctor and medical researcher, who showed me that living a life that improves the lives others is a life that matters most of all.

As Martin Luther King Jr. wrote in Letter from Birmingham Jail

“In a real sense all life is inter-related. All men are caught in an inescapable network of mutuality, tied in a single garment of destiny. Whatever affects one directly, affects all indirectly. I can never be what I ought to be until you are what you ought to be, and you can never be what you ought to be until I am what I ought to be...This is the inter-related structure of reality.”

 

Acknowledgements:

  • Mr. Andrew Bramante, my Greenwich High School Honors Science Research teacher and mentor. Mr. Bramante supervised my work in the lab and confirmed that all experiments were conducted in accordance with proper safety precautions. The idea for my research, the development of a novel, temperature-independent (“breaking the cold chain”), asymptomatic, rapid, inexpensive, portable, disease diagnostic device for the on-site diagnosis of Ebola, with broad applicability for other ELISA-based assay diagnostic diseases such as HIV, was mine. I conducted all experiments on my own under Mr. Bramante’s supervision. His encouragement, wisdom and tireless dedication to teaching inspired me every day.
  • Mount Sinai Hospital and the kindness of a Greenwich High School parent for arranging access to and permission for the use of their Scanning Electron Microscope (SEM) to students in the Honors Science Research class. I used the SEM for photos incorporated in my research paper to analyze the dried silk fibroin thin film test strips, which further supported uniform coating of the filter paper substrate. I was permitted to use and operate the SEM during my visit to Mount Sinai with Mr. Bramante's supervision.
  • Professor Fio Omenetto, Associate Dean for Research, Tufts University School of Engineering, who graciously met with me several times to discuss the stabilizing properties and potential of silk fibroin (which had never been applied to ELISA reagents before) and who introduced me to his colleague, Dr. Benedetto Marelli.
  • Professor Benedetto Marelli, Postdoctoral Research Associate, Department of Biomedical Engineering, Tufts University with whom I consulted on the modified fabrication process for the thin-film silk (which I based off of a process detailed in a paper published by Dr. Omenetto and Dr. Marelli, credited in my bibliography below) which I made in the Greenwich High School Laboratory with modifications specified in the procedure section of this submission, and who provided additional silk.
  • Alpha Diagnostic International, the manufacturer of the Ebola Human Anti-Zebov NP IgG ELISA kit (AE-320520-1) that was purchased for use in my research. Harnessing a proven chemical reaction (ELISA) and applying it to a novel lateral-flow format requiring only water to activate enabled me to show that the Ebola Assay Card's potential was not just theoretically possible, but a proven and tested diagnostic tool. The next steps will be to test it in the field, and eventually with saliva, and ready it through further development and manufacture for commercial use. 
  • Greenwich High School Research Lab, Greenwich High School - Greenwich, CT

All of my experiments, with the exception of the use of the SEM at Mount Sinai (credited above) took place at the Greenwich High School Research Lab at Greenwich High School. Equipment Used:

Ventilation Hood, Safety Goggles, Safety Gloves

Use of smart phone to film EAC reaction 

Plate Reader, Fourier Transform Infrared (FTIR) Spectroscopy

 

 

References

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  2. Bioactive Silk Protein Biomaterial Systems for Optical Devices
  3. Brian D. Lawrence, Mark Cronin-Golomb, Irene Georgakoudi, David L. Kaplan, and Fiorenzo G. Omenetto. Biomacromolecules 2008 9 (4), 1214-1220
  4. Center for Disease Control and Prevention. CDC, n.d. Web. 12 Nov. 2014. .
  5. BIOTREND Cheikalien GmbH. General Elisa Kit [Material Safety Data Sheet]. Kaufman, P. et al., Visualization and Measurement of Flow in Two-dimensional Paper Networks. Lab Chip. 2010, 10, No. 19. 2614-2617
  6. Fu, E., et al., Controlled reagent transport in disposable 2D paper networks.Lab Chip. 2010, 10, No. 7.  918-920
  7. Lu, Q., Wang, X., Hu, X., Cebe, P., Omenetto, F. and Kaplan, D. L. (2010), Stabilization and Release of Enzymes from Silk Films. Macromol. Biosci., 10: 359–368. doi: 10.1002/mabi.200900388
  8. Lutz, B. R., Liang, T., Fu E., Ramachandran, S., Kauffman, P. Yager, P.et al., Dissolvable Fluidic Time Delays for Programming Multi-step Assays in Instrument-free Paper Diagnostics. Lab Chip. 2013, 13, No. 14.  2840-2847
  9. Lutz, B. R., et al., Two-dimensional paper networks: programmable fluidic disconnects for multi-step processes in shaped paper. Lab Chip. 2011, 11, No. 24.  4274-4278
  10. Murphy, A.R. & Kaplan, D.L. Biomedical applications of chemically modified silk fibroin. J. Mat. Chem. 2009, 19, 6443–6450
  11. Omenetto, F.G. & Kaplan, D.L. New opportunities for an ancient material. Science. 2010, 329, 528–531
  12. Rockwood, Danielle N., Preda, Rucsanda C., Yucel, T., Wang, X., Lovett, M., & Kaplan, D. L., Materials Fabrication from Bombyx mori silk fibroin. Nature Protocols. 2011, Vol 6, No. 10. 1612-1631
  13. "Tufts Silk Portfolio - Tufts Tech Transfer." Tufts Tech Transfer. TUFTS UNIVERSITY OFFICE FOR TECHNOLOGY LICENSING AND INDUSTRY COLLABORATION. Web. 27 Nov. 2014. .
  14. Vepari, C. & Kaplan, D.L. Silk as a biomaterial. Prog. Poly. Sci. 2007, 32, 991–1007
  15. Wolchover, Natalie. "Seed Magazineabout." The Silk Renaissance § SEEDMAGAZINE.COM. Seed Media Group, 17 Sept. 2007. Web. 27 Nov. 2014.
  16. www.unicefusa.org (UNICEF)
  17. www.Aids.gov
  18. Sifferlin, Alexandra. “5 Million Kids Aren’t in School Because of Ebola” Time Magazine. 17 December 2014.
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