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.

Biomaterials composed of synthetic cells have the potential to adapt and differentiate guided by physicochemical environmental cues. Inspired by biological systems in development, which extract positional information (PI) from morphogen gradients in the presence of uncertainties, we here investigate how well synthetic cells can determine their position within a multicellular structure. To calculate PI, we created and analyzed a large number of synthetic cellular assemblies composed of emulsion droplets connected via lipid bilayer membranes. These droplets contained cell-free feedback gene circuits that responded to gradients of a genetic inducer acting as a morphogen. PI is found to be limited by gene expression noise and affected by the temporal evolution of the morphogen gradient and the cell-free expression system itself. The generation of PI can be rationalized by computational modeling of the system. We scale our approach using three-dimensional printing and demonstrate morphogen-based differentiation in larger tissue-like assemblies.

16Point-of-care testing (POCT) in low-resource settings requires tools that can operate in-17 dependent of typical laboratory infrastructure. Due to its favorable signal-to-background 18 ratio, a wide variety of biomedical tests utilize fluorescence as a readout. However, 19 fluorescence techniques often require expensive or complex instrumentation and can 20 be difficult to adapt for POCT. To address this issue, we developed a pocket-sized 21 fluorescence detector costing less than $15 that is easy to manufacture and can operate 22 in low-resource settings. It is built from standard electronic components, including 23 an LED and a light dependent resistor, filter foils and 3D printed parts, and reliably 24 detected less than 10 nM fluorescein concentrations (with a lower limit of detection of 25 ≈ 6.8 nM), which is sufficient to follow typical biochemical reactions used in POCT 26 applications. All assays are conducted on filter paper, which allows for a flat detector 27 architecture to improve signal collection. We validate the device by quantifying in vitro 28 RNA transcription and also demonstrate sequence-specific detection of target RNAs in 29 the nanomolar range using a Cas13a-based fluorescence assay. Cas13a is a RNA-guided, 30RNA-targeting CRISPR effector with promiscuous RNase activity upon recognition of 31 its RNA target. Cas13a sensing is highly specific and adaptable and in combination 32 with our detector represents a promising approach for nucleic acid POCT. Furthermore, 33 our open-source device architecture could be a valuable educational tool that integrates 34 hardware, software and biochemistry concepts. 35

Dynamic biomaterials composed of synthetic cellular structures have the potential to adapt and functionally differentiate guided by physical and chemical cues from their environment. Inspired by developing biological systems, which efficiently extract positional information from chemical morphogen gradients in the presence of environmental uncertainties, we here investigate the analogous question: how well can a synthetic cell determine its position within a synthetic multicellular structure? In order to calculate positional information in such systems, we created and analyzed a large number of replicas of synthetic cellular assemblies, which were composed of emulsion droplets connected via lipid bilayer membranes. The droplets contained cell-free two-node feedback gene circuits that responded to gradients of a genetic inducer acting as a morphogen. We found that in our system, simple anterior-posterior differentiation is possible, but positional information is limited by gene expression noise, and is also critically affected by the temporal evolution of the morphogen gradient and the life-time of the cell-free expression system contained in the synthetic cells. Using a 3D printing approach, we demonstrate morphogen-based differentiation also in larger tissue-like assemblies.