Easy Surface Functionalization and Bioconjugation of Peptides as Capture Agents of a Microfluidic Biosensing Platform for Multiplex Assay in Serum.

The development of assays for protein biomarkers in complex matrices is a demanding task that still needs implementation of new approaches. Antibodies as capture agents have been largely used in bioassays but their low stability, low-efficiency production, and cross-reactivity in multiplex approaches impairs their larger applications. Instead, synthetic peptides, even with higher stability and easily adapted amino acid sequences, still remain largely unexplored in this field. Here, we provide a proof-of-concept of a microfluidic device for direct detection of biomarker overexpression. The multichannel microfluidic polydimethylsiloxane (PDMS) device was first derivatized with PAA (poly(acrylic acid)) solution. CRP-1, VEGF-114, and ΦG6 peptides were preliminarily tested to respectively bind the biomarkers, C-reactive protein (CRP), vascular endothelial growth factor (VEGF), and tumor necrosis factor-alpha (TNF-α). Each PDMS microchannel was then respectively bioconjugated with a specific peptide (CRP-1, VEGF-114, or ΦG6) to specifically capture CRP, VEGF, and TNF-α. With such microdevices, a fluorescence bioassay has been set up with sensitivity in the nanomolar range, both in buffered solution and in human serum. The proposed multiplex assay worked with a low amount of sample (25 µL) and detected biomarker overexpression (above nM concentration), representing a noninvasive and inexpensive screening platform.

Shuttle-mediated nanoparticle transport across an in vitro brain endothelium under flow conditions.

The blood brain barrier (BBB) represents a challenge in the development of new nano-delivery systems able to reach the central nervous system (CNS). In order to test the efficacy of these nanocarriers, it is fundamental to use in vitro models that resemble the in vivo cell culture conditions. Here, we demonstrate for the first time the ability of a membranotropic peptide, namely gH625, to transport a cargo-acting as a shuttle-across the BBB layer under flow conditions that mimic the blood flow rate. To this aim, a BBB microfluidic device was designed based on a transparent polyester porous membrane sandwiched between a top and a bottom overlying channel made of poly(methyl methacrylate) (PMMA). Our data clearly indicate that this microfluidic system allows the growth of brain endothelial bEnd.3 cells and the formation of a confluent layer at 7 days of culture that hinders the passage of nanoparticles compared to porous membrane alone. The device was validated at a 5 µL/min working flow rate, where the capability of the model to remain intact after nanoparticle passage was shown. Very interestingly, the decoration with the gH625 peptide enhances the adhesion of nanoparticles to the endothelial layer and the BBB crossing in flow conditions, thus confirming the efficacy of the gH625 as a delivery platform to the brain. Biotechnol. Bioeng. 2017;114: 1087-1095. © 2016 Wiley Periodicals, Inc.

Advances in microfluidic 3D cell culture for preclinical drug development.

Drug development is often a very long, costly, and risky process due to the lack of reliability in the preclinical studies. Traditional current preclinical models, mostly based on 2D cell culture and animal testing, are not full representatives of the complex in vivo microenvironments and often fail. In order to reduce the enormous costs, both financial and general well-being, a more predictive preclinical model is needed. In this chapter, we review recent advances in microfluidic 3D cell culture showing how its development has allowed the introduction of in vitro microphysiological systems, laying the foundation for organ-on-a-chip technology. These findings provide the basis for numerous preclinical drug discovery assays, which raise the possibility of using micro-engineered systems as emerging alternatives to traditional models, based on 2D cell culture and animals.

DoE Analysis of Approaches for Hydrogel Microbeads' Preparation by Millifluidic Methods.

Hydrogel microbeads hold great promise for immune-protective cell transplants and in vitro studies. Millifluidic generation of hydrogel microbeads is a highly efficient and reproducible approach enabling a mass production. This paper illustrates the preparation and characterization of highly controlled and reproducible microbeads made by different types of hydrogel using millifluidic approaches. The optimization of the process was made by a design of experiments (DoE) approach. The microbeads' large-scale production can be potentially used for single cells or clusters encapsulation.

Coins in microfluidics: From mere scale objects to font of inspiration for microchannel circuits.

The fabrication of microfluidic chips remains a complex and expensive process requiring specific equipment and protocols, often if not always limited to the most privileged laboratories. As an alternative to the most sophisticated methods, the present paper describes the fabrication of microfluidic chips by an approach that uses coins as positive master for the rapid production of multigeometry chips. All steps of chip production were carried out using inexpensive approaches by low-cost chemicals and equipment. The chips were validated by different "classic" microfluidic tasks, such as hydrodynamic focusing, droplets generation, micromixing, and on-chip cell culture. The use of coins is not only an efficient method for rapid prototyping but also represents an inspiring possibility for the design of new microfluidic chips. Finally, coin-inspired chips could represent a laboratory experiment doable at a high school level.

Gelatin-Coated Microfluidic Channels for 3D Microtissue Formation: On-Chip Production and Characterization.

Traditional two-dimensional (2D) cell culture models are limited in their ability to reproduce human structures and functions. On the contrary, three-dimensional (3D) microtissues have the potential to permit the development of new cell-based assays as advanced in vitro models to test new drugs. Here, we report the use of a dehydrated gelatin film to promote tumor cells aggregation and 3D microtissue formation. The simple and stable gelatin coating represents an alternative to conventional and expensive materials like type I collagen, hyaluronic acid, or matrigel. The gelatin coating is biocompatible with several culture formats including microfluidic chips, as well as standard micro-well plates. It also enables long-term 3D cell culture and in situ monitoring of live/dead assays.

Confined Gelatin Dehydration as a Viable Route To Go Beyond Micromilling Resolution and Miniaturize Biological Assays.

Nowadays, microfluidic channels of a few tens of micrometers are required and widely used in many fields, especially for surface-processing applications and miniaturization of biological assays. Herein, we selected micromilling as a low-cost technology and proposed an approach capable of overcoming its limitations; in fact, microstructures below 20-30 µm in depth are difficult to obtain, and the manufacturing error is rather high, as it is inversely proportional to the depth. Indeed, the proposed method uses a confined dehydration process of a patterned gelatin substrate fabricated via replica molding onto a micromilled poly(methyl methacrylate) substrate to produce a gelatin master with demonstrated final micrometric features down to 3 µm for the channel depth and, in specific configurations, down to 5 µm for the channel width. Finally, we demonstrated the ability to flux liquids in miniaturized microfluidic devices and fabricated and tested-as an example-micrometric microstructures arrays connected via microchannels for biological assays.

From square to circular polymeric microchannels by spin coating technology: a low cost platform for endothelial cell culture.

Square microchannels are easy to fabricate by means of micromachining or lithographic techniques. However, in vitro vascular microcapillaries--as well as plug production and microparticle alignment--require mainly circular microchannels that can be used also in applications based on open microchannels. Nowadays, a simple, low cost, and versatile method to fabricate circular microchannels is still missing. Here, we report on a fast, inexpensive, flexible and reproducible method to fabricate circular microchannels by coupling spin coating with micromilled square microchannels. The proposed method is based on the balance between the displacement of liquid PDMS induced by centrifugal forces and the surface tension that tends to keep the liquid accumulated especially in the corners, which become therefore rounded. To show the versatility of the described experimental study we prepared a variety of rounded microchannels, including branched and PMMA-PDMS hybrid configuration microchannels. Finally, an endothelial cell layer was formed by culturing brain endothelial bEnd.3 cells inside the proposed circular microchannels. Results demonstrated a more successful adhesion, growth, and homogeneous distribution of the cells along the circular microchannel than those observed in the square microchannel used as a control.