A low-cost fluorescence reader for in vitro transcription and nucleic acid detection with Cas13a

Florian Katzmeier, Lukas Aufinger, Aurore Dupin, Jorge Quintero, Matthias Lenz, Ludwig Bauer, Sven Klumpe, Dawafuti Sherpa, Benedikt Dürr, Maximilian Honemann, Igor Styazhkin, Friedrich C. Simmel, Michael Heymann

Research output: Contribution to journalArticlepeer-review

46 Scopus citations

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.

Original languageEnglish
Article numbere0220091
JournalPLoS ONE
Volume14
Issue number12
DOIs
StatePublished - 1 Dec 2019

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