PDMS-Based Microdevices for the Capture of MicroRNA Biomarkers

The isolation and analysis of circulating biomarkers, the main concern of liquid biopsy, could greatly benefit from microfluidics. Microfluidics has indeed the huge potentiality to bring liquid biopsy into the clinical practice. Here, two polydimethylsiloxane (PDMS)-based microdevices are presented as valid tools for capturing microRNAs biomarkers from clinically-relevant samples. After an extensive study of functionalized polydimethylsiloxane (PDMS) properties in adsorbing/eluting microRNAs, the best conditions were transferred to the microdevices, which were thoroughly characterized. The channels morphology and chemical composition were measured, and parameters for the automation of measures were setup. The best working conditions were then used with microdevices, which were proven to capture microRNAs on all channel surfaces. Finally, microfluidic devices were successfully validated via real-time PCR for the detection of a pool of microRNAs related to non-small cell lung cancer, selected as proof-of-principle. The microfluidic approach described here will allow a step forward towards the realization of an ecient microdevice, possibly automated and integrated into a microfluidic lab-on-a-chip with high analytical potentialities

PURIFICAZIONE ED AMPLIFICAZIONE DI ACIDI NUCLEICI IN UN DISPOSITIVO MICROFLUIDICO COMPRENDENTE SUPERFICI DI POLIDIMETILSILOSSANO

La presente invenzione si riferisce al settore dei dispositivi microf luidici , del tipo denominato correntemente con il termine LAB-ON-A-CHIP, atti ad effettuare su di un campione un saggio biologico comprendente uno stadio di estrazione/purificazione di acidi nucleici (DNA) dal campione ed uno stadio di amplificazione/rivelazione degli acidi nucleici mediante reazione a catena della polimerasi (PCR e RT-PCR)

Functional surfaces for exosomes capturing and exosomal microRNAs analysis

Exosomes are small extracellular vesicles well-studied both as cell signaling elements and as source of highly informative biomarkers, in particular microRNAs. Standard techniques for exosome isolation are in general scarcely efficient and give low purity vesicles. New techniques combining microfluidics with suitable functionalized surfaces could overcome these disadvantages. Here, different functional surfaces aimed at exosomes capture are developed thank to the functionalization of silicon oxide substrates. Charged surfaces, both positive and negative, neutral and immunoaffinity surfaces are characterized and tested in functional assays with both exosome mimicking vesicles and exosomes purified from cell supernatants. The different surfaces showed promising properties, in particular the negatively-charged surface could capture more than 4 × 108 exosomes per square centimeter. The captured exosomes could be recovered and their biomarker cargo analyzed. Exosomal microRNAs were successfully analyzed with RT-PCR, confirming the good performances of the negatively-charged surface. The best-performing functionalization could be easily moved to microdevice surfaces for developing modular microfluidic systems for on-chip isolation of exosomes, to be integrated in simple and fast biosensors aimed at biomarker analysis both in clinical settings and in research

Surface Characterization of Biomaterial Interfaces

Any biomaterial application involves the creation of at least one interface. For instance, in a vascular graft, interfaces are created between the biomaterial and blood, and between the biomaterial and the walls of the blood vessel. In general very little is known about the basic interactions which give rise to a particular tissue response. Since the primary interaction occurs on the molecular level and in a very narrow interface width (< 1nm), the surface properties on atomic scale take a central position dealing with biomaterials. The analytical methods must be capable of giving detailed information about both structural, chemical and compositional surface properties. Surfaces, particularly those types encountered in biomedical systems, present special problems for analysis: the measurement of only minute amount of material in the surface region, the separation of the surface signal from a preponderant bulk signal, the effective compatibility with the ultra high vacuum enviroment, the insulating nature. In the present work we address some of these issues discussing the role of surface science in the biomaterial research and development. In addition we show how the combination of surface sensitive analytical techniques (AES, SIMS, XPS, AFM) together with biological evaluation methods can contribute in better understanding biomaterial-tissue interactio

Cell transfer of information via miR-loaded exosomes: a biophysical approach

A new communication route among cells was reported in recent years, via extracellular vesicles and their cargo. Exosomes in particular are attracting increasing interest as privileged mediators of this cell communication route. The exosome-mediated transfer of nucleic acids, especially of microRNAs, is particularly promising for their use both as biomarkers of pathologies and as a therapeutic tool. Here, a simplified model of interaction among cells, microRNAs and vesicles is studied using a biophysical approach. A synthetic and fluorescent microRNA (i.e. miR-1246 conjugated with TAMRA) was selected to model cell communication, monitoring its internalization in cells. The fluorescent miR-1246, either naked or included in synthetic or natural vesicles, was incubated with human breast adenocarcinoma cells (MCF7) for different times. A comparison between this human microRNA and its DNA copy or an exogenous microRNA (from Caenorhabditis elegans) allowed assessment of the specificity of the information transfer through microRNAs, and especially associated with exosomes. The uptake of naked miR-1246 was indeed higher both in terms of number of targeted cells and intensity of fluorescence signal with respect to the other nucleic acids tested. The same occurred with miR-1246 loaded exosomes, evidencing a specific uptake only partially due to the lipidic components and present only when the human microRNA was loaded in exosomes, which were themselves derived from the same MCF7 cells

Surface characterization of biomaterial interfaces

Any biomaterial application involves the creation of at least one interface. For instance, in a vascular graft, interfaces are created between the biomaterial and blood, and between the biomaterial and the walls of the blood vessel. In general very little is known about the basic interactions which give rise to a particular tissue response. Since the primary interaction occurs on the molecular level and in a very narrow interface width (<1nm), the surface properties on atomic scale take a central position dealing with bionaterials. The analytical methods must be capable of giving detailed information about both structural, chemical and compositional surface properties. Surfaces, particularly those types encountered in biomedical systems, present special problems for analysis: the measurement of only minute amount of material in the surface region, the separation of the surface signal from a preponderant bulk signal, the effective compatibility with the ultra high vacuum environment, the insulating nature. In the present work we address some of these issues discussing the role of surface science in the biomaterial research and development. In addition we show how the combination of surface sensitive analytical techniques (AES, SIMS, XPS, AFM) together with biological evaluation methods can contribute in better understanding biomaterial-tissue interactio

Tuning Surface Properties via Plasma Treatments for the Improved Capture of MicroRNA Biomarkers

Advanced materials could bring about fundamental improvements in the evolution of innovative analytical devices, i.e., biosensors or lab-on-a-chip devices, in particular in the context of liquid biopsies. Here, plasma deposition processes were tested for the introduction of primary amines on silicon surfaces by tuning the amounts and availability of amino-charged residues. Different binary (CH4/NH3) and ternary (CH4/NH3/H2 and CH4/NH3/N2) mixtures of gases were used as feeds for the plasma treatments. The obtained surfaces were fully characterized for their chemical and physical properties before their use as capture materials in a functional test. Synthetic and fluorescently conjugated microRNA-21 (miR-21) was selected as the target molecule. The capture of miR-21 increased linearly with the increase in amino nitrogen measured on surfaces. The surface showing the most promising performance was further analyzed in different conditions, i.e., varying pH and time of incubation, incubation with different microRNAs, and possible elution of captured microRNAs. The apparent pH range of primary amines present on the surfaces was around 3.5–4. Positively charged surfaces prepared via PE-CVD were, therefore, demonstrated as being suitable materials for the capture of microRNA biomarkers, paving the way for their inclusion in biomedical devices for the purification and analysis of circulating biomarkers