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Authors: Katzmeier, Florian
Aufinger, Lukas
Dupin, Aurore
Quintero, Jorge
Lenz, Matthias
Bauer, Ludwig
Klumpe, Sven
Sherpa, Dawafuti
Dürr, Benedikt
Honemann, Maximilian
Styazhkin, Igor
Simmel, Friedrich C.
Heymann, Michael
Title: A low-cost fluorescence reader for in vitro transcription and nucleic acid detection with Cas13a
Issue Date: 2019 Zeitschriftenartikel 17 PLOS ONE 14 (2019), No. 12, e0220091
ISSN: 1932-6203
Abstract: Point-of-care testing (POCT) in low-resource settings requires tools that can operate independently of typical laboratory infrastructure. Due to its favorable signal-to-background ratio, a wide variety of biomedical tests utilize fluorescence as a readout. However, fluorescence techniques often require expensive or complex instrumentation and can be difficult to adapt for POCT. To address this issue, we developed a pocket-sized fluorescence detector costing less than $15 that is easy to manufacture and can operate in low-resource settings. It is built from standard electronic components, including an LED and a light dependent resistor, filter foils and 3D printed parts, and reliably reaches a lower limit of detection (LOD) of ≈ 6.8 nM fluorescein, which is sufficient to follow typical biochemical reactions used in POCT applications. All assays are conducted on filter paper, which allows for a flat detector architecture to improve signal collection. We validate the device by quantifying in vitro RNA transcription and also demonstrate sequence-specific detection of target RNAs with an LOD of 3.7 nM using a Cas13a-based fluorescence assay. Cas13a is an RNA-guided, RNA-targeting CRISPR effector with promiscuous RNase activity upon recognition of its RNA target. Cas13a sensing is highly specific and adaptable and in combination with our detector represents a promising approach for nucleic acid POCT. Furthermore, our open-source device may be used in educational settings, through providing low cost instrumentation for quantitative assays or as a platform to integrate hardware, software and biochemistry concepts in the future.
Appears in Collections:04 Fakultät Energie-, Verfahrens- und Biotechnik

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pone.0220091.s001.pdfKatzmeier et al. 2019 Appendix S1128,81 kBAdobe PDFView/Open
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