HuluSCOPE a POCT device for COVID-19 and other pathogens

HuluSCOPE is a automated fluorescence microscope using single molecule nucleic acid counting and AI image analysis to detect ultrahigh number of pathogens within 15 minutes at €1 per test

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Inspiration

On March 11th, the WHO officially has declared COVID-19 a pandemic. Before the vaccines and drugs approved for COVID-19, correct isolation of positive patients is the only effective way to stop the spread. The key to correctly label positive symptomatic and asymptomatic patients is aggressive testing of SARS-CoV-2 virus via current molecular testing methods like PCR and IgM/G antibody test.

However, the limitations of PCR or antibody tests are also clear, including a lack of speed, sensitivity, multiplexing, and cost-effectiveness. The ideal situation to stop the spreading would be to test everyone on the planet(1,2). To prepare the society for a potential long-term fight against COVID-19 and potential other outbreaks in the future, there is the necessity to build a robust, affordable, rapid, and comprehensive diagnosis method for agile pandemic response. The high abundance of SARS-CoV-2 viral RNA in nasal swab allows faithful detection of the SARS-CoV-2 RNA from extracted total RNA or even individual Human epithelial cells (3–5). HuluFISH is a single molecule RNA counting method without reverse transcription or PCR amplification. With HuluFISH, there is a high possibility we can directly detect SARS-CoV-2 RNA from total RNA extract or fixed individual Human epithelial cells from a nasal swab. Via this way, firstly we can simplify the whole detection process by eliminating PCR amplification and RNA extraction. Secondly, we can harness the multiplexity mechanism of HuluFISH to detect up to 120 species in one round of hybridization (multiplexity is C(n*(n+1)/2, 3) if n is the channel number)(6). This will fill the throughput gap between PCR and next-generation sequencing (NGS) to have a novel categorical technology, faster than NGS, more targets than PCR.

Combining with a dedicated and automated fluorescence microscope, the HuluFISH detection of SARS-CoV-2 RNA can be developed into a Point-of-Care Testing (POCT) device for massive COVID-19 testing for everyone. The recent development in the fluorescence microscope has democratized the complicated optical device under €1000(7–9). On the other hand, microfluidics has transformed the automated and miniaturized nucleic acid extraction and hybridization(10–13). Developing a HuluFISH device by combining fluorescence microscope and microfluidics will eventually automate the SARS-CoV-2 RNA single-molecule counting, as well as that for other viral/bacterial/parasitic DNA/RNA.

What it does

This device HuluSCOPE allows rapid and precise detection of 20-120 dangerous pathogens within 15 minutes of time, from sample to results. It can detect all dangerous pathogens in your body, at the point of care, with 40x cheaper and 20x faster than existing methods.

HuluSCOPE will use our proprietary single-molecule counting method HuluFISH to detect the SARS-CoV-2 RNA from various kinds of samples. At the first step, the HuluFISH probe set for SARS-CoV-2 RNA will be designed from the consensus sequence of the SARS-CoV-2 S protein mRNA sequence from all variants. This Pan-SARS-CoV-2 probe set will be flexible to detect most existed SARS-CoV-2 strains. In the second step, the signal of every single molecule of SARS-CoV-2 RNA will be acquired under a fluorescence microscope, either available commercial ones like Zeiss Cell Observer or Leica SP8, or our prototype HuluSCOPE. Thirdly, each molecule of SARS-CoV-2 RNA will be diffraction limit dots under the microscope. And our Artificial Intelligence (AI) backed HuluREAD algorithm will automatically filter, identify, decode, and count the absolute number of SARS-CoV-2 RNA in acquired images.

How I built it

We have an interdisciplinary team to build the HuluSCOPE with experts in DNA science, optics, and AI.

Challenges I ran into

We still need experts in software engineers in the embedded system for HuluSCOPE and scientists in microfluidics

Accomplishments that I'm proud of

Our team has the HuluSCOPE design ready and validate our HuluFISH technology for single-molecule counting for pathogens in a closely relevant environment.

HuluSCOPE is a POCT device, combining microscope, microfluidics, and embedded system, for automated sample processing, HuluFISH staining, and imaging of individual SARS-CoV-2 RNA genome extracted from clinical samples like nasal swabs. In this hackathon #EUvsVIRUS, we have quickly formed an interdisciplinary team to tackle the design for the 3 critical parts for HuluSCOPE, optics, microfluidics, and embedded control system. Due to the hardware engineering nature of these tasks, we can only finish paper design work for these parts. In the past hours of our hackathon project, we have achieved significantly in the optical and microfluidic design for HuluSCOPE (Figure 2). We will finalize the development of a novel fluorescence microscope dedicated for single-molecule detection of nucleic acid on a glass surface. This is enabled by our patented single-molecule multiplexing nucleic acid hybridization technology HuluFISH and AI-powered image analysis cloud service HuluREAD. HuluSCOPE prototype will have 2 fluorescence channels for automated scanning of 7 different nucleic acid species at single-molecule level on a glass slide. Selected nucleic acid sequences from SARS-CoV-2 will be synthesized and visualized by our HuluFISH single-molecule probes. We will down-grade our current exclusive HuluSCOPE prototype by exchanging lasers and highly sensitive cameras to LED light sources and less sensitive cameras, in order to dramatically reduce the material cost for the final HuluSCOPE, and ensure affordability to the future end-users.

We will develop the microfluidic cassette for automated detection of pathogen nucleic acid or infected host cell. A microfluidic detection chamber will allow us to automate the entire detection process, from nucleic acid extraction/host cell capture to image acquisition. Τhe 50 μm thin detection channel in the microfluidic cassette will speed up the hybridization reaction. Automated nucleic acid extraction and enrichment from nasal swab will be incorporated into the cassette. Enrichment will be done with sequence-specific capture oligonucleotide probes, typically allowing for 50+ fold enrichment(14). With the rapid and efficient hybridization inside a microfluidic chamber, we expect to reach our targeted 1 molecule per microliter detection sensitivity. A dilution series of SARS-CoV-2 RNA will be tested in relevant sample environments to determine the sensitivity limit of our microfluidic system.

What I learned

HuluSCOPE is only possible with a motivated and multidisciplinary team to build such a POCT device as the frontest line protector for us to contain the virus-like SARS-CoV-2.

What's next for HuluSCOPE a POCT device for COVID-19 and other pathogens

The design of the HuluSCOPE started in 2019, and the global spread of COVID-19 has necessitated development advancement. We plan to bring the HuluSCOPE PoC diagnostic device to market readiness as soon as possible. With the broad deployment of this device, we can build a surveillance network with real-time monitoring of COVID-19 to curb the pandemic. The HuluSCOPE will be sold or licensed to clinics and hospitals where it will take 15 minutes to complete the detection of a COVID-19 infection. The PoC device is also suitable for field use, like mobile drive-through testing stations. Combined with the HuluSCOPE, the HuluFISH detection of SARS-CoV-2 RNA can be developed into a PoC device for universal COVID-19 testing.

After the hackathon, PixelBiotech GmbH will continue their efforts in design, prototyping and manufacturing of HuluSCOPE and HuluFISH COVID-19 kit. HuluSCOPE platform will be a revolutionary POCT device with unprecedented sensitivity and multiplexity, providing rapid and accurate diagnosis data for quick medical action in infectious disease treatment and outbreak control.

Reference

  1. Linnarsson S. To stop COVID-19, test everyone [Internet]. Medium. 2020 [cited 2020 Mar 30]. Available from: https://medium.com/@sten.linnarsson/to-stop-covid-19-test-everyone-373fd80eb03b
  2. Tondo L. Scientists say mass tests in Italian town have halted Covid-19 there. The Guardian [Internet]. 2020 Mar 18 [cited 2020 Mar 30]; Available from: https://www.theguardian.com/world/2020/mar/18/scientists-say-mass-tests-in-italian-town-have-halted-covid-19
  3. Zou L, Ruan F, Huang M, Liang L, Huang H, Hong Z, et al. SARS-CoV-2 Viral Load in Upper Respiratory Specimens of Infected Patients. N Engl J Med. 2020 Mar 19;382(12):1177–9.
  4. Wu C, Zheng S, Chen Y, Zheng M. Single-cell RNA expression profiling of ACE2, the putative receptor of Wuhan 2019-nCoV, in the nasal tissue. medRxiv. 2020 Feb 18;2020.02.11.20022228.
  5. Wang W, Xu Y, Gao R, Lu R, Han K, Wu G, et al. Detection of SARS-CoV-2 in Different Types of Clinical Specimens. JAMA [Internet]. 2020 Mar 11 [cited 2020 Mar 28]; Available from: https://jamanetwork.com/journals/jama/fullarticle/2762997
  6. Cheng Y-S, Zhuo Y, Hartmann K, Zou P, Bekki G, Alter H, et al. Autonomous Combinatorial Color Barcoding For Multiplexing Single Molecule RNA Visualization. bioRxiv. 2017 Apr 18;127373.
  7. Hasan MM, Alam MW, Wahid KA, Miah S, Lukong KE. A Low-Cost Digital Microscope with Real-Time Fluorescent Imaging Capability. PLOS ONE. 2016 Dec 15;11(12):e0167863.
  8. Forcucci A, Pawlowski ME, Crannell Z, Pavlova I, Richards-Kortum R, Tkaczyk TS. All-plastic miniature fluorescence microscope for point-of-care readout of bead-based bioassays. J Biomed Opt. 2015 Oct;20(10):105010.
  9. Stewart C, Giannini J. Inexpensive, Open Source Epifluorescence Microscopes. J Chem Educ. 2016 Jul 12;93(7):1310–5.
  10. Oblath EA, Henley WH, Alarie JP, Ramsey JM. A microfluidic chip integrating DNA extraction and real-time PCR for the detection of bacteria in saliva. Lab Chip. 2013 Apr 7;13(7):1325–32.
  11. Karle M, Miwa J, Czilwik G, Auwärter V, Roth G, Zengerle R, et al. Continuous microfluidic DNA extraction using phase-transfer magnetophoresis. Lab Chip. 2010 Nov 9;10(23):3284–90.
  12. Perez-Toralla K, Mottet G, Guneri ET, Champ J, Bidard F-C, Pierga J-Y, et al. FISH in chips: turning microfluidic fluorescence in situ hybridization into a quantitative and clinically reliable molecular diagnosis tool. Lab Chip. 2015;15(3):811–22.
  13. Henry OYF, O’Sullivan CK. Rapid DNA hybridization in microfluidics. TrAC Trends Anal Chem. 2012 Mar 1;33:9–22.
  14. Deviatkin AA, Lukashev AN, Markelov MM, Gmyl LV, Shipulin GA. Enrichment of Viral Nucleic Acids by Solution Hybrid Selection with Genus Specific Oligonucleotides. Sci Rep. 2017 Aug 29;7(1):1–7.

Built With

  • ai
  • fluorescence
  • hulufish
  • huluread
  • huluscope-is-built-with-a-fluorescence-microscope-with-1-channel-and-microfluidic-cassette-for-automated-clinical-sample-lysis
  • microfluidics
  • microscope
  • nucleic-acid-enrichment-and-hybridization
+ 3 more
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