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Low cost and user-friendly paper microfluidic devices, combined with DNA-based biosensors with binding capacities for specific molecules, have been proposed for the developing of novel platforms that ease and speed-up the process of cell secretion monitoring. In this work, we present the first cellulose microfluidic paper-based analytical device for the single-step detection of cell secreted Vascular Endothelial Growth Factor through a self-reporting Structure Switching Signaling Aptamer. A three-part Structure Switching Signaling Aptamer was designed with an aptameric sequence specific for VEGF, which provides a quantifiable fluorescent signal through the displacement of a quencher upon VEGF recognition. The VEGF biosensor was integrated in cellulose paper, enabling the homogenous distribution of the sensor in the paper substrate and the detection of as low as 0.34 ng of VEGF in 30 min through fluorescence intensity analysis. As a proof-of-concept, the biosensor was incorporated in a microfluidic paper-based analytical device format containing a VEGF detection zone and a control zone, which was applied for the detection of cell secreted VEGF in the supernatant of mesenchymal stem cells culture plates, demonstrating its potential use in cell biology research., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

The effect of cell-cell contact on gene transfection is mainly unknown. Usually, transfection is carried out in batch cell cultures without control over cellular interactions, and efficiency analysis relies on complex and expensive protocols commonly involving flow cytometry as the final analytical step. Novel platforms and cell patterning are being studied to control cellular interactions and improve quantification methods. In this study, we report the use of surface patterning of fibronectin for the generation of two types of mesenchymal stromal cell patterns: single-cell patterns without cell-to-cell contact, and small cell-colony patterns. Both scenarios allowed the integration of the full transfection process and the continuous monitoring of thousands of individualized events by fluorescence microscopy. Our results showed that cell-to-cell contact clearly affected the transfection, as single cells presented a maximum transfection peak 6 h earlier and had a 10% higher transfection efficiency than cells with cell-to-cell contact., (© 2021 Wiley Periodicals LLC.)

Low cost, easy to use cell viability tests are needed in the pharmaceutical, biomaterial, and environmental industries to measure adverse cellular effects. We present a new methodology to track cell death with high resolution. Adherent cells commonly detach from the surface when they die, but some toxic compounds promote cell adhesion. A methodology that enables both dynamic detachment monitoring but also rapid detection of toxic effects of compounds that promote cell adhesion would constitute a step forward toward high-throughput cytotoxicity measurements. We achieved dynamic digital quantification of cell viability by simple optical imaging using "single cell adhesion dot arrays" (SCADA), fibronectin (FN) dot arrays designed to accommodate a single cell on each fibronectin dot. For cytotoxicity measurements, cell-filled SCADA substrates were exposed to K 2 CrO 4 , HgSO 4 salts, and dimethyl sulfoxide (DMSO). The toxic effect of DMSO and K 2 CrO 4 was dynamically monitored by measuring the cell detachment rate during more than 30 h by quantifying the number of occupied dots in the SCADA array. HgSO 4 inhibited cellular detachment from the surface, and cytotoxicity was monitored using the trypan blue life/death assay directly on the surface. In all cases, the cytotoxicity effects were easily monitored with single cell resolution, and the results were comparable to previous reports. SCADA enabled dynamic measurements at the highest resolution due to the digital measuring in this method. The integration of SCADA substrates into microfluidic platforms will provide a practical tool that will extend to fundamental research and commercial applications.