American Association for Clinical Chemistry Emerging Clinical & Laboratory Diagnostics: The Portable Lab April 24-25, 2014

Transcription

1 Barry Lutz University of Washington Department of Bioengineering Key collaborators: Paul Yager, UW BioE Elain Fu, Oregon State University American Association for Clinical Chemistry Emerging Clinical & Laboratory Diagnostics: The Portable Lab April 24-25, 2014

2 Goal: lab quality tests in rapid and easy to use format Immunoassay (ELISA) DNA/RNA test (PCR) 2008 (in my kitchen) Can we reconfigure the materials from lateral flow to automatically carry out sophisticated multi step tests?

3 Early diagnosis of HIV Early diagnosis is important to initiate treatment and prevent transmission of HIV

4 Automated lateral flow ELISA?

5 Adapt ELISA chemistry to paper Commercial reagents for HIV p24 ELISA Shivani Dharmaraja, Nuttada Panpradist, Ian Andrews

6 Adapt ELISA chemistry to paper Chemistry is good. Now, how do we trick the device to delivery these fluids automatically? Shivani Dharmaraja, Nuttada Panpradist, Ian Andrews

7 2D paper network (2DPN) with dried sugar time delays Lutz, et al. Lab on a Chip (2013)

8 2DPN with dry reagents and delivery from a single fluid source Lutz, et al. Lab on a Chip (2011)

9 Dry ELISA reagents stored on card ELISA reagents (HRP, substrate) stable in dry form (3+ months at 45C) Ramachandran, et al. The Analyst (2014)

10 A 1D paper network (1DPN)? An idea: what if we simply touched multiple fluid sources to a straight paper strip? Lisa Lafleur, Sam Byrnes, Peter Kauffman, Shivani Dharmaraja

11 1DPN for paper based HIV ELISA Shivani Dharmaraja

12 1DPN for paper based HIV ELISA Shivani Dharmaraja, Ian Andrews, Nuttada Panpradist

13 Automated lateral flow ELISA ELISA in paper can be fast & sensitive ELISA reagents can be stable without refrigeration ELISA multi step amplification procedure can be automated by programmed wicking Current work: make it very simple for the user Applications: malaria, dengue, influenza, HIV All reagents stored in cartridge User adds sample & one buffer Amplified assay Quantitative readout

14 Multiplexable Autonomous Disposables for Nucleic Acid Amplification Tests in Limited Resource Settings (MAD NAAT) MAD NAAT for LRS Instrument-free fully-disposable paper-based diagnostic platform Detect multiple pathogens by their DNA or RNA Sample-to-result anywhere Simple enough for untrained users Low enough cost to enable wider use of sophisticated medical tests First targets: MRSA (DNA) RSV (RNA) Barry Lutz UW Bioengineering Paul Yager UW Bioengineering Walt Mahoney Epoch Biosciences Bernhard Weigl PATH David Moore GE Global Research Gaetano Borriello UW CS&E Janet Englund Seattle Children s Ferric Fang UW Lab Medicine

15 The user experience Patient: Samantha Byrnes. Director: Lisa Lafleur

16 Isothermal (constant temperature) amplification by Epoch Biosciences S. aureus gene: in tube reaction, real time detection Other assays: MRSA resistance gene (meca): similar performance RNA target (RSV): <10 copy sensitivity, sample preparation compatible with current device (chemistry + heat) Epoch Biosciences

17 Epoch Biosciences isothermal amplification Data: benchtop heating, lateral flow detection, manual procedure (S. aureus DNA) Not shown amplification in paper: signal ~30% of that in tube Assay: Epoch Biosciences. Data: Bhushan Toley, Sujatha Ramachandran

18 Fluid activated on switch fluid activated on switch Mean: 10.2 minutes Std Dev: 42 sec (7%)

19 Fluid activated timing cascade lysis amplification detection wick timer fluid-activated valves amplification detection Bhushan Toley

20 Fast acting pull tab valves Max Wheeler, Josh Bishop, Lisa Lafleur

21 Can we lyse a range of bacteria? Phylogenetic Tree of Eubacteria

22 Enzyme + heat: lyses a broad range of bacteria Staphylococcus aureus (MRSA) Salmonella typhi (typhoid fever) Bordetella pertussis (whooping cough) Streptococcus pyogenes (sepsis) Mycobacterium marinum (TB-like) Gram positive Gram negative Gram negative Gram positive Gram positive Better than Bead Beating: Digestion of DNA packaging? Less DNA fragmentation? Bead Beating Control = 100% to be tested soon Xiaohong Zhang, Erin Heiniger

23 On board chemical temperature control PATH chemical heater Saline activated chemical reaction (same chemistry as meals ready to eat) Phase change material regulates temperature Jered Singleton, Bernhard Weigl (PATH)

24 Cell Phone Interface results & controls image fiducials QR code color standard Visual guides for image capture Analysis onphone or sent for remote analysis Nikki Dell & Gaetano Borriello (UW CSE), and Yager team (UW BioE)

25 Conclusions & status Each chemical step works with high efficiency as separate modules with fresh reagents, now integrating with dry reagents TRL5 prototype sample to result this year Expandable to RNA, other samples (blood) Early device

26 Thanks to. Shivani Dharmaraja (MS) Nuttada Panpradist (PhC) Ian Andrews (ugrad) Lisa Lafleur, Samantha Byrnes, Peter Kauffman, Josh Buser (1DPN concept) BioRad (reagents) Paul Yager (UW BioE, PI) Walt Mahoney (Epoch) Bernhard Weigl (PATH) Gaetano Borriello (UW CSE) David Moore (GE Global Research) Ferric Fang (UW Lab Medicine) Janet Englund (Seattle Childrens) Lisa Lafleur (UW integration lead)