Molecular Imprinted Polymers Coupled to Photonic Structures in Biosensors: The State of Art

Optical sensing, taking advantage of the variety of available optical structures, is a rapidly expanding area. Over recent years, whispering gallery mode resonators, photonic crystals, optical waveguides, optical fibers and surface plasmon resonance have been exploited to devise different optical sensing configurations. In the present review, we report on the state of the art of optical sensing devices based on the aforementioned optical structures and on synthetic receptors prepared by means of the molecular imprinting technology. Molecularly imprinted polymers (MIPs) are polymeric receptors, cheap and robust, with high affinity and selectivity, prepared by a template assisted synthesis. The state of the art of the MIP functionalized optical structures is critically discussed, highlighting the key progresses that enabled the achievement of improved sensing performances, the merits and the limits both in MIP synthetic strategies and in MIP coupling

A Surface Plasmon Resonance Plastic Optical Fiber Biosensor for the Detection of Pancreatic Amylase in Surgically-Placed Drain Effluent

Postoperative pancreatic fistula (POPF), the major driver of morbidity and mortality following pancreatectomy, is caused by an abnormal communication between the pancreatic ductal epithelium and another epithelial surface containing pancreas-derived, enzyme-rich fluid. There is a strong correlation between the amylase content in surgically-placed drains early in the postoperative course and the development of POPF. A simple and cheap method to determine the amylase content from the drain effluent has been eagerly advocated. Here, we developed an amylase optical biosensor, based on a surface plasmon resonance (SPR) plastic optical fiber (POF), metallized with a 60 nm layer of gold and interrogated with white light. The sensor was made specific by coupling it with an anti-amylase antibody. Each surface derivatization step was optimized and studied by XPS, contact angle, and fluorescence. The POF-biosensor was tested for its response to amylase in diluted drain effluents. The volume of sample required was 50 \ub5L and the measurement time was 8 min. The POF-biosensor showed selectivity for amylase, a calibration curve log-linear in the range of 0.8\u201325.8 U/L and a limit of detection (LOD) of ~0.5 U/L. In preliminary tests, the POF-biosensor allowed for the measurement of the amylase content of diluted surgically-placed drain effluents with an accuracy of >92% with respect to the gold standard. The POF-biosensor allows for reliable measurement and could be implemented to allow for a rapid bedside assessment of amylase value in drains following pancreatectomy

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)

Molecularly imprinted polymers by epitope imprinting: a journey from molecular interactions to the available bioinformatics resources to scout for epitope templates

The molecular imprinting of proteins is the process of forming biomimetics with entailed protein-recognition by means of a template-assisted synthesis. Protein-imprinted polymers (pMIPs) have been successfully employed in separations, assays, sensors, and imaging. From a technical point of view, imprinting a protein is both costly, for protein expression and purification, and challenging, for the preservation of the protein's structural properties. In fact, the imprinting process needs to guarantee the preservation of the same protein three-dimensional conformation that later would be recognized. So far, the captivating idea to imprint just a portion of the protein, i.e., an epitope, instead of the whole, proved successful, offering reduced costs, compatibility with many synthetic conditions (solvents, pH, temperatures), and fine-tuning of the peptide sequence so to target specific physiological and functional conditions of the protein, such as post-translational modifications. Here, protein-protein interactions and the biochemical features of the epitopes are inspected, deriving lessons to prepare more effective pMIPs. Epitopes are categorized in linear or structured, immunogenic or not, located at the protein's surface or buried in its core and the imprinting strategies are discussed. Moreover, attention is given to freely available online bioinformatics resources that might offer key tools to gain further rationale amid the selection process of suitable epitopes templates

The Search for Peptide Epitopes for Molecular Imprinting Through Bioinformatics

Epitope imprinting is an effective strategy to prepare molecularly imprinted polymers (MIPs) for protein recognition. Indeed, the idea to use as a template just a fragment of the protein of interest, called the epitope, instead of the whole protein, presents some key advantages for the imprinting process, in particular: cutting the costs for MIP production and avoiding protein unfolding during the imprinting process, so to ultimately improve the quality of the stamped binding sites. How to select an epitope for the imprinting is the strategic question. Here, the bioinformatics tools to search for suitable epitopes for the imprinting process and rational tools to select the most suitable epitope are briefly introduced along with protocols for their practical use

A Review of Apta-POF-Sensors: The Successful Coupling between Aptamers and Plastic Optical Fibers for Biosensing Applications

Aptamers represent the next frontier as biorecognition elements in biosensors thanks to a smaller size and lower molecular weight with respect to antibodies, more structural flexibility with the possibility to be regenerated, reduced batch-to-batch variation, and a potentially lower cost. Their high specificity and small size are particularly interesting for their application in optical biosensors since the perturbation of the evanescent field are low. Apart from the conventional plasmonic optical sensors, platforms based on silica and plastic optical fibers represent an interesting class of devices for point-of-care testing (POCT) in different applications. The first example of the coupling between aptamers and silica optical fibers was reported by Pollet in 2009 for the detection of IgE molecules. Six years later, the first example was published using a plastic optical fiber (POF) for the detection of Vascular Endothelial Growth Factor (VEGF). The excellent flexibility, great numerical aperture, and the large diameter make POFs extremely promising to be coupled to aptamers for the development of a sensitive platform easily integrable in portable, small-size, and simple devices. Starting from silica fiber-based surface plasmon resonance devices, here, a focus on significant biological applications based on aptamers, combined with plasmonic-POF probes, is reported

Solid phase DNA extraction on PDMS and direct amplification

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A Review of Apta-POF-Sensors: The Successful Coupling between Aptamers and Plastic Optical Fibers for Biosensing Applications

Aptamers represent the next frontier as biorecognition elements in biosensors thanks to a smaller size and lower molecular weight with respect to antibodies, more structural flexibility with the possibility to be regenerated, reduced batch-to-batch variation, and a potentially lower cost. Their high specificity and small size are particularly interesting for their application in optical biosensors since the perturbation of the evanescent field are low. Apart from the conventional plasmonic optical sensors, platforms based on silica and plastic optical fibers represent an interesting class of devices for point-of-care testing (POCT) in different applications. The first example of the coupling between aptamers and silica optical fibers was reported by Pollet in 2009 for the detection of IgE molecules. Six years later, the first example was published using a plastic optical fiber (POF) for the detection of Vascular Endothelial Growth Factor (VEGF). The excellent flexibility, great numerical aperture, and the large diameter make POFs extremely promising to be coupled to aptamers for the development of a sensitive platform easily integrable in portable, small-size, and simple devices. Starting from silica fiber-based surface plasmon resonance devices, here, a focus on significant biological applications based on aptamers, combined with plasmonic-POF probes, is reported

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