Journal
ACS BIOMATERIALS SCIENCE & ENGINEERING
Volume 7, Issue 12, Pages 5850-5860Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acsbiomaterials.1c01001
Keywords
nanoyeast; scFv; DENV; diagnostics; thermal stability; serotyping
Categories
Funding
- National Breast Cancer Foundation of Australia [CG12-07]
- Australian Research Council [DP160102836, DP210103151]
- UQ Research Training Program Scholarship
- Commonwealth Scientific Industrial Research Organisation for the CSIRO/UQ Centre for Personalised Nanodiagnostics Initiative
- Advance Queensland Industry Research Fellowship [AQIRF104-2020-CV]
- National Health and Medical Research Council [APP1173669]
- CSIRO Synthetic Biology Fellowship
- Probing Biosystems Future Science Platform (FSP)
- Therapeutic Innovation Australia (TIA)
- Australian Government through the National Collaborative Research Infrastructure Strategy (NCRIS) program
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The study demonstrates a novel approach for cost-efficient production of highly specific nanoyeasts for accurate detection of dengue virus.
Bioengineered yeast bio-nanomaterials termed nanoyeasts displaying antibody single-chain variable fragments (scFvs) against diagnostic targets are a promising alternative to monoclonal antibodies (mAbs). A potential limitation for translating nanoyeasts into diagnostic tools is batch-to-batch variability. Herein, we demonstrate a systematic approach for cost-efficient production of highly specific nanoyeasts that enabled accurate dengue virus (DENV) detection by immunoassay (2.5% CV). Yeasts bioengineered to surface express DENV-specific scFvs (up to 66% of the total cell population) were fragmented into nanoyeast fractions trialing sonication, bead beating, and high-pressure disruption methods. Nanoyeast fractions from sonication had optimal target binding, uniform particle size (+/- 89 nm), were stable, and retained diagnostic activity for 7 days at 37 degrees C compared to traditional mAbs that lost activity after 1 day at 37 degrees C. We engineered a panel of nanoyeast scFvs targeting DENV nonstructural protein 1 (NS1): (i) specific for serotyping DENV 1-4 and (ii) cross-reactive anti-DENV scFvs that are suitable for yes/no diagnostic applications. We demonstrate highly specific nanoyeast scFvs for serotyping DENV. We show that nanoyeast scFvs specifically detect NS1 in simulated patient plasma with a limit of detection of 250 ng/mL, the concentration found in infected patients.
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