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Abstract The field of ecology has undergone a molecular revolution, with researchers increasingly relying on DNA‐based methods for organism detection. Unfortunately, these techniques often require expensive equipment, dedicated laboratory spaces and specialized training in molecular and computational techniques; limitations that may exclude field researchers, underfunded programmes and citizen scientists from contributing to cutting‐edge science.It is for these reasons that we have designed a simplified, inexpensive method for field‐based molecular organism detection—FINDeM (Field‐deployableIsothermalNucleotide‐basedDetectionMethod). In this approach, DNA is extracted using chemical cell lysis and a cellulose filter disc, followed by two body‐heat inducible reactions—recombinase polymerase amplification and a CRISPR‐Cas12a fluorescent reporter assay—to amplify and detect target DNA, respectively.Here, we introduce and validate FINDeM in detectingBatrachochytrium dendrobatidis, the causative agent of amphibian chytridiomycosis, and show that this approach can identify single‐digit DNA copies from epidermal swabs in under 1 h using low‐cost supplies and field‐friendly equipment.This research signifies a breakthrough in ecology, as we demonstrate a field‐deployable platform that requires only basic supplies (i.e. micropipettes, plastic consumables and a UV flashlight), inexpensive reagents (~$1.29 USD/sample) and emanated body heat for highly sensitive, DNA‐based organism detection. By presenting FINDeM in an ecological system with pressing, global biodiversity implications, we aim to not only highlight how CRISPR‐based applications promise to revolutionize organism detection but also how the continued development of such techniques will allow for additional, more diversely trained researchers to answer the most pressing questions in ecology.
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Fluorescent molecular rotors as versatile in situ sensors for protein quantitation
Abstract Accurate protein quantitation is essential for many cellular mechanistic studies. Existing technology relies on extrinsic sample evaluation that requires significant volumes of sample as well as addition of assay-specific reagents and importantly, is a terminal analysis. This study exploits the unique chemical features of a fluorescent molecular rotor that fluctuates between twisted-to-untwisted states, with a subsequent intensity increase in fluorescence depending on environmental conditions (e.g., viscosity). Here we report the development of a rapid, sensitive in situ protein quantitation method usingARCAM-1, a representative fluorescent molecular rotor that can be employed in both non-terminal and terminal assays.
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- Award ID(s):
- 2034780
- PAR ID:
- 10539730
- Publisher / Repository:
- Scientific Reports
- Date Published:
- Journal Name:
- Scientific Reports
- Volume:
- 13
- Issue:
- 1
- ISSN:
- 2045-2322
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1038/s41598-023-46571-5
