Cell Patterning Technology on Polymethyl Methacrylate through Controlled Physicochemical and Biochemical Functionalization.

In recent years, innovative cell-based biosensing systems have been developed, showing impact in healthcare and life science research. Now, there is a need to design mass-production processes to enable their commercialization and reach society. However, current protocols for their fabrication employ materials that are not optimal for industrial production, and their preparation requires several chemical coating steps, resulting in cumbersome protocols. We have developed a simplified two-step method for generating controlled cell patterns on PMMA, a durable and transparent material frequently employed in the mass manufacturing of microfluidic devices. It involves air plasma and microcontact printing. This approach allows the formation of well-defined cell arrays on PMMA without the need for blocking agents to define the patterns. Patterns of various adherent cell types in dozens of individual cell cultures, allowing the regulation of cell-material and cell-cell interactions, were developed. These cell patterns were integrated into a microfluidic device, and their viability for more than 20 h under controlled flow conditions was demonstrated. This work demonstrated the potential to adapt polymeric cytophobic materials to simple fabrication protocols of cell-based microsystems, leveraging the possibilities for commercialization.

Paper based microfluidic platform for single-step detection of mesenchymal stromal cells secreted VEGF.

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.

Continuous monitoring of cell transfection efficiency with micropatterned substrates.

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.

Naked eye Y amelogenin gene fragment detection using DNAzymes on a paper-based device.

Nowadays, great efforts are made in developing new technology for rapid detection of specific DNA sequences, for environmental monitoring, forensic analysis and rapid biomedical diagnosis applications. Microfluidic paper-based analytical devices are suitable platforms for the development of point of care devices, due to their simple fabrication protocols, ease of use and low cost. Herein, a methodology for in situ detection of single strand DNA by using a colorimetric assay based on the formation of a DNAzyme within a paper substrate was developed. A DNAzyme that could only be formed in the presence of a specific sequence of the Y human amelogenin gene was designed. The performance of the DNAzyme was followed colorimetrically first in solution and then in paper substrates. The reaction was found to be specific to the Y fragment selected as analyte. The DNAzyme reaction on paper enabled the unequivocal colorimetric identification of the Y single strand DNA fragment both qualitatively, with the naked eye (143 ng), and quantitatively by image analysis (45.7 ng). As a proof of concept, a microfluidic paper-based device, pre-loaded with all DNAzyme reagents, was characterized and implemented for the simultaneous detection of X and Y single strand DNA fragments.