In this paper we report an innovative use of Poly(DiMethyl)Siloxane (PDMS) to design a microfluidic device that combines, for the first time, in one single reaction chamber, DNA purification from a complex biological sample (blood) without elution and PCR without surface passivation agents. This result is achieved by exploiting the spontaneous chemical structure and nanomorphology of the material after casting. The observed surface organization leads to spontaneous DNA adsorption. This property allows on-chip complete protocols of purification of complex biological samples to be performed directly, starting from cells lysis. Amplification by PCR is performed directly on the adsorbed DNA, avoiding the elution process that is normally required by DNA purification protocols. The use of one single microfluidic volume for both DNA purification and amplification dramatically simplifies the structure of microfluidic devices for DNA preparation. X-Ray Photoelectron Spectroscopy (XPS) was used to analyze the surface chemical composition. Atomic Force Microscopy (AFM) and Field Emission Scanning Electron Microscopy (FESEM) were employed to assess the morphological nanostructure of the PDMS-chips. A confocal fluorescence analysis was utilized to check DNA distribution inside the chip.
The presence of residual antibiotics in food is increasingly emerging as a worrying risk for human health both for the possible direct toxicity and for the development of antibiotic-resistant bacteria. In the context of food safety, new methods based on microfluidics could offer better performance, providing improved rapidity, portability and sustainability, being more cost effective and easy to use. Here, a microfluidic method based on the use of magnetic microbeads specifically functionalized and inserted in polymeric microchambers is proposed. The microbeads are functionalized either with aptamers, antibodies or small functional groups able to interact with specific antibiotics. The setup of these different strategies as well as the performance of the different functionalizations are carefully evaluated and compared. The most promising results are obtained employing the functionalization with aptamers, which are able not only to capture and release almost all tetracycline present in the initial sample but also to deliver an enriched and simplified solution of antibiotic. These solutions of purified antibiotics are particularly suitable for further analyses, for example, with innovative methods, such as label-free detection. On the contrary, the on-chip process based on antibodies could capture only partially the antibiotics, as well as the protocol based on beads functionalized with small groups specific for sulfonamides. Therefore, the on-chip purification with aptamers combined with new portable detection systems opens new possibilities for the development of sensors in the field of food safety.
Antibiotics are widely used to both prevent and treat bacterial diseases as well as promote animal growth. This massive use leads to the presence of residual antibiotics in food with severe consequences for human health. Limitations and regulations on the tolerated amount of antibiotics in food have been introduced and analytical methods have been developed. The bioanalytical methods usually employed to detect antibiotic residues, however, are time-consuming, expensive and laboratory-based. Novel methods with improved rapidity, portability and cost that are easy-to-use and sustainable are therefore highly desirable. In the attempt to fulfill this need, a microfluidic system was set up herein for the purification and pre-concentration of tetracyclines from raw milk selected as the case-study. The system includes a polymeric microfluidic chip containing magnetic beads loaded with copper to exploit the preferential interaction of tetracycline with divalent ions. The microfluidic system was demonstrated to efficiently pre-concentrate tetracycline, oxytetracycline and chlortetracycline with similar performances and efficiently purify tetracycline from raw milk without any pre-treatment. The simplified method described in this paper could be easily integrated in a compact and portable device for the in-field detection of tetracyclines, with the economic advantage of preventing food wastes and guaranteeing food safety.
Extracellular vesicles (EVs) are membranous structures that cells massively release in extracellular fluids.EVs are cargo of cellular components such as lipids, proteins, and nucleic acids that can work as a formidable source in liquid biopsy studies searching for disease biomarkers.We recently demonstrated that nickel-based isolation (NBI) is a valuable method for fast, efficient, and easy recovery of heterogeneous EVs from biological fluids.NBI exploits nickel cations to capture negatively charged vesicles.Then, a mix of balanced chelating agents elutes EVs while preserving their integrity and stability in solution.Here, we describe steps and quality controls to functionalize a matrix of agarose beads, obtain an efficient elution of EVs, and extract nucleic acids carried by them.We demonstrate the versatility of NBI method in isolating EVs from media of primary mouse astrocytes, from human blood, urine, and saliva processed in parallel, as well as outer membrane vesicles (OMVs) from cultured Gramnegative bacteria.
Protein biomarkers are increasingly becoming significant analytical tools in the clinical practice. The introduction of new compact systems for sensitive, fast and simplified analysis is currently playing a substantial role in the development of point-of-care solutions driven by diagnostic and prognostic aims. The developed system represents a compact and miniaturized Lab-On-A-Chip (LOC) microdevice addressing the requirement of a rapid, portable, compact and low cost diagnostic platforms. For low-cost sensing systems, POFs are indeed especially advantageous due to their excellent flexibility, easy manipulation, great numerical aperture, large diameter, and thanks to the fact that plastic is able to withstand smaller bend radii than glass.
