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

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

A synthetic tubular molecular transport system

Creating artificial macromolecular transport systems that can support the movement of molecules along defined routes is a key goal of nanotechnology. Here, we report the bottom-up construction of a macromolecular transport system in which molecular pistons diffusively move through micrometer-long, hollow filaments. The pistons can cover micrometer distances in fractions of seconds. We build the system using multi-layer DNA origami and analyze the structures of the components using transmission electron microscopy. We study the motion of the pistons along the tubes using single-molecule fluorescence microscopy and perform Langevin simulations to reveal details of the free energy surface that directs the motions of the pistons. The tubular transport system achieves diffusivities and displacement ranges known from natural molecular motors and realizes mobility improvements over five orders of magnitude compared to previous artificial random walker designs. Electric fields can also be employed to actively pull the pistons along the filaments, thereby realizing a nanoscale electric rail system. Our system presents a platform for artificial motors that move autonomously driven by chemical fuels and for performing nanotribology studies, and it could form a basis for future molecular transportation networks

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

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