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.

Optical Single Cell Resolution Cytotoxicity Biosensor Based on Single Cell Adhesion Dot Arrays.

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 K2CrO4, HgSO4 salts, and dimethyl sulfoxide (DMSO). The toxic effect of DMSO and K2CrO4 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. HgSO4 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.

Engineering Metal-Phenolic Network Nanoparticles via Microfluidics.

Microfluidics opens new avenues for materials engineering, as it enables scalable synthesis and provides highly controllable environments for reactions. Herein, we leverage microfluidics to engineer the properties of (bioactive) metal-phenolic network nanoparticles (MPN NPs), an emerging and highly modular nanoparticle platform for the incorporation and delivery of bioactive cargo. By varying the microfluidics operating parameters (flow rate ratio, total flow rate, temperature) and NP composition, we assemble MPN NPs, which consist of poly(ethylene glycol), biomacromolecules, metal ions, and polyphenols. Compared to MPN NPs prepared via bulk assembly, the microfluidics-assembled MPN NPs possess a broader tunable size range (i.e., ∼40-330 nm vs ∼45-220 nm for bulk-assembled NPs) and a higher (by ∼30%) protein loading. The bulk-assembled MPN NPs show pH-responsive protein release behavior (e.g., ∼50% at pH 7; ∼25% at pH 9; 48 h). Likewise, the MPN NPs prepared via microfluidics at a flow rate ratio of 1:1 display similar pH-responsive protein release behavior. For the microfluidics-assembled MPN NPs, protein release is also dependent on temperature (e.g., 30% at 4 °C, and ∼50% at 20 and 37 °C). Furthermore, assembly at a 1:1 flow rate ratio overall enables greater tunability of protein release profiles than that at higher flow rate ratios. While bulk-assembled NPs display a higher degree of cell association, NPs assembled via both strategies can be internalized by cells after 24 h. These findings provide new insights into engineering the properties of metal-organic materials via microfluidics, which is expected to advance their development and application.

A method for the controllable fabrication of optical fiber-based localized surface plasmon resonance sensors.

Optical fiber-based Localized Surface Plasmon Resonance (OF-LSPR) biosensors have emerged as an ultra-sensitive miniaturized tool for a great variety of applications. Their fabrication by the chemical immobilization of gold nanoparticles (AuNPs) on the optic fiber end face is a simple and versatile method. However, it can render poor reproducibility given the number of parameters that influence the binding of the AuNPs. In order to develop a method to obtain OF-LSPR sensors with high reproducibility, we studied the effect that factors such as temperature, AuNPs concentration, fiber core size and time of immersion had on the number and aggregation of AuNPs on the surface of the fibers and their resonance signal. Our method consisted in controlling the deposition of a determined AuNPs density on the tip of the fiber by measuring its LSPR signal (or plasmonic signal, Sp) in real-time. Sensors created thus were used to measure changes in the refractive index of their surroundings and the results showed that, as the number of AuNPs on the probes increased, the changes in the Sp maximum values were ever lower but the wavelength shifts were higher. These results highlighted the relevance of controlling the relationship between the sensor composition and its performance.

Selective Ultrasensitive Optical Fiber Nanosensors Based on Plasmon Resonance Energy Transfer.

The facet of optical fibers coated with nanostructures enables the development of ultraminiature and sensitive (bio)chemical sensors. The sensors reported until now lack specificity, and the fabrication methods offer poor reproducibility. Here, we demonstrate that by transforming the facet of conventional multimode optical fibers onto plasmon resonance energy transfer antenna surfaces, the specificity issues may be overcome. To do so, a low-cost chemical approach was developed to immobilize gold nanoparticles on the optical fiber facet in a reproducible and controlled manner. Our nanosensors are highly selective as plasmon resonance energy transfer is a nanospectroscopic effect that only occurs when the resonance wavelength of the nanoparticles matches that of the target parameter. As an example, we demonstrate the selective detection of picomolar concentrations of copper ions in water. Our sensor is 1000 times more sensitive than the state-of-the-art technologies. An additional advantage of our nanosensors is their simple interrogation; it comprises of a low-power light-emitting diode, a multimode optical fiber coupler, and a miniature spectrometer. We believe that the plasmon resonance energy transfer-based fiber-optic platform reported here may pave the way for the development of a new generation of ultraminiature, portable, and hypersensitive and selective (bio)chemical sensors.