Journal Article

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

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

2019

Overview

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.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Acids / Activity recognition / Assaying / Bacterial Proteins - genetics / Biochemistry / Biology / Biology and life sciences / Biosensing Techniques - instrumentation / CRISPR / CRISPR-Associated Proteins - genetics / CRISPR-Cas Systems / Detection equipment / Electronic components / Electronic components industry / Engineering and Technology / Filter paper / Fluorescein / Fluorescence / Foils / Green Fluorescent Proteins / In Vitro Techniques / Infectious diseases / Infrastructure (Economics) / Instrumentation / Instruments / Laboratories / Laboratory equipment / LEDs / Light emitting diodes / Limit of Detection / Low cost / Nucleic acids / Nucleotide sequence / Optics / Physical Sciences / Physics / Research and Analysis Methods / Ribonuclease / Ribonucleic acid / RNA / RNA, Bacterial - analysis / RNA, Bacterial - genetics / Scientific equipment industry / Sensors / Source code / Target detection / Target recognition / Three dimensional printing / Transcription / Transcription (Genetics) / Transcription, Genetic / Viral infections