Molecular beacon decorated silver nanowires for quantitative miRNA detection by a SERS approach.

Advantages of biosensors based on surface enhanced Raman scattering (SERS) rely on improved sensitivity and specificity, and suited reproducibility in detecting a target molecule that is localized in close proximity to a SERS-active surface. Herein, a comprehensive study on the realization of a SERS biosensor designed for detecting miRNA-183, a miRNA biomarker that is specific for chronic obstructive pulmonary disease (COPD), is presented. The used strategy exploits a signal-off mechanism by means of a labelled molecular beacon (MB) as the oligonucleotide biorecognition element immobilized on a 2D SERS substrate, based on spot-on silver nanowires (AgNWs) and a multi-well low volume cell. The MB was properly designed by following a dedicated protocol to recognize the chosen miRNA. A limit of detection down to femtomolar concentration (3 × 10-16 M) was achieved and the specificity of the biosensor was proved. Furthermore, the possibility to regenerate the sensing system through a simple procedure is shown: with regeneration by using HCl 1 mM, two detection cycles were performed with a good recovery of the initial MB signal (83%) and a reproducible signal after hybridization.

Biosensing by Polymer-Coated Etched Long-Period Fiber Gratings Working near Mode Transition and Turn-around Point.

A methodology to enhance the sensitivity of long-period fiber gratings (LPFGs) based on the combination of three different enhancement approaches is presented; the methods here adopted are the working near mode transition (MT) of a cladding mode (CM), working near the turn-around point of a CM and the enhancement of the evanescent field of CMs by reducing the cladding diameter or by increasing the order number of CMs. In order to combine these enhancement methodologies, an electrostatic self-assembly (ESA) process was used to deposit a polymeric overlay, with a chosen thickness, onto the etched fiber. The add-layer sensitivity of the sensor was theoretically calculated, and the demonstration of the real applicability of the developed LPFG as a biosensor was performed by means of an IgG/anti-IgG immunoassay in human serum in a thermostated microfluidic system. The limits of detection (LODs) calculated by following different procedures (three times the standard deviation of the blank and the mean value of the residuals) were 6.9 × 10-8 µg/mL and 4.5 × 10-6 µg/mL, respectively. The calculated LODs demonstrate the effectiveness of the applied methodology for sensitivity enhancement.

Sensitivity Analysis of Sidelobes of the Lowest Order Cladding Mode of Long Period Fiber Gratings at Turn Around Point.

A new methodology to enhance the sensitivity of a long period fiber grating sensor (LPFG) at the Turn Around Point (TAP) is here presented. The LPFG sensor has been fabricated by etching the fiber up to 20.4 µm, until the sidelobes of dispersed LP0,2 cladding mode appeared near TAP in aqueous medium. The dual peak sensitivity of the sidelobes was found to be 16,044 nm/SRIU (surrounding refractive index units) in the RI range from 1.333 to 1.3335.

An integrated device for fast and sensitive immunosuppressant detection.

The present paper describes a compact point of care (POC) optical device for therapeutic drug monitoring (TDM). The core of the device is a disposable plastic chip where an immunoassay for the determination of immunosuppressants takes place. The chip is designed in order to have ten parallel microchannels allowing the simultaneous detection of more than one analyte with replicate measurements. The device is equipped with a microfluidic system, which provides sample mixing with the necessary chemicals and pumping samples, reagents and buffers into the measurement chip, and with integrated thin film amorphous silicon photodiodes for the fluorescence detection. Submicrometric fluorescent magnetic particles are used as support in the immunoassay in order to improve the efficiency of the assay. In particular, the magnetic feature is used to concentrate the antibody onto the sensing layer leading to a much faster implementation of the assay, while the fluorescent feature is used to increase the optical signal leading to a larger optical dynamic change and consequently a better sensitivity and a lower limit of detection. The design and development of the whole integrated optical device are here illustrated. In addition, detection of mycophenolic acid and cyclosporine A in spiked solutions and in microdialysate samples from patient blood with the implemented device are reported.

Immunosuppressant quantification in intravenous microdialysate - towards novel quasi-continuous therapeutic drug monitoring in transplanted patients.

OBJECTIVES: Therapeutic drug monitoring (TDM) plays a crucial role in personalized medicine. It helps clinicians to tailor drug dosage for optimized therapy through understanding the underlying complex pharmacokinetics and pharmacodynamics. Conventional, non-continuous TDM fails to provide real-time information, which is particularly important for the initial phase of immunosuppressant therapy, e.g., with cyclosporine (CsA) and mycophenolic acid (MPA). METHODS: We analyzed the time course over 8 h of total and free of immunosuppressive drug (CsA and MPA) concentrations measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in 16 kidney transplant patients. Besides repeated blood sampling, intravenous microdialysis was used for continuous sampling. Free drug concentrations were determined from ultracentrifuged EDTA-plasma (UC) and compared with the drug concentrations in the respective microdialysate (µD). µDs were additionally analyzed for free CsA using a novel immunosensor chip integrated into a fluorescence detection platform. The potential of microdialysis coupled with an optical immunosensor for the TDM of immunosuppressants was assessed. RESULTS: Using LC-MS/MS, the free concentrations of CsA (fCsA) and MPA (fMPA) were detectable and the time courses of total and free CsA comparable. fCsA and fMPA and area-under-the-curves (AUCs) in µDs correlated well with those determined in UCs (r≥0.79 and r≥0.88, respectively). Moreover, fCsA in µDs measured with the immunosensor correlated clearly with those determined by LC-MS/MS (r=0.82). CONCLUSIONS: The new microdialysis-supported immunosensor allows real-time analysis of immunosuppressants and tailor-made dosing according to the AUC concept. It readily lends itself to future applications as minimally invasive and continuous near-patient TDM.

Discretized superimposed optical fiber long-period gratings.

A novel technique, to the best of our knowledge, for the inscription of superimposed long-period gratings with arbitrary grating pitches is proposed and experimentally validated. The technique is based on the discretization of an ideal continuous sinusoidal refractive index (RI) pattern with a step function. The RI variation is induced by means of the irradiation of a photosensitive fiber with a 248 nm UV laser beam. The nonlinear relation between the induced RI change and the UV fluence was experimentally derived. Two superimposed long-period grating (LPGs) with different grating pitches have been realized with the discretization technique; the transmission spectrum was compared with that of two superimposed LPGs obtained with the traditional square wave RI modulation. The validity of the proposed technique was demonstrated by the better spectral characteristics of the discretized superimposed LPGs.

Polymeric nanoparticles promote endocytosis of a survivin molecular beacon: Localization and fate of nanoparticles and beacon in human A549 cells.

Polymethylmethacrylate core-shell fluorescent nanoparticles promote, in human lung A549 cancer cells, the internalization of a molecular beacon (MB) specific for survivin mRNA, an anti-apoptotic protein overexpressed in cancer cells. AIMS: To design an effective drug delivery system, the knowledge of the uptake mechanism and of the nanoparticles (NPs) and MB fate is required. MATERIALS AND METHODS AND KEY FINDINGS: Experiments with dextran as marker for endocytosis showed that in the presence of NPs the number of endocytic vesicles per cell doubled and their mean size significantly (p < 0.001) increased with respect to controls in absence of NPs, indicating an involvement of NPs in the endocytotic process. By using LysoTracker™ Deep Red, as marker of lysosomes, we found that nanoparticles co-localize with lysosomes. Moreover, a cellular release of nanoparticles detected in the culture medium, suggested a role of lysosomal exocytosis in nanoparticle elimination. The MB fluorescence in proximity of the labeled Endoplasmic Reticulum was indicative that the opening of the MB occurs in proximity of its target mRNA. SIGNIFICANCE: The results show the involvement of endocytotic pathway in the uptake of NPs, which are an appropriate delivery system capable of being eliminated by cells. Furthermore the data confirm that the MB can be considered an effective tool for the intracellular sensing.

Femtomolar Detection by Nanocoated Fiber Label-Free Biosensors.

The advent of optical fiber-based biosensors combined with that of nanotechnologies has provided an opportunity for developing in situ, portable, lightweight, versatile, and high-performance optical sensing platforms. We report on the generation of lossy mode resonances by the deposition of nanometer-thick metal oxide films on optical fibers, which makes it possible to measure precisely and accurately the changes in optical properties of the fiber-surrounding medium with very high sensitivity compared to other technology platforms, such as long period gratings or surface plasmon resonances, the gold standard in label-free and real-time biomolecular interaction analysis. This property, combined with the application of specialty structures such as D-shaped fibers, permits enhancing the light-matter interaction. SEM and TEM imaging together with X-EDS tool have been utilized to characterize the two films used, i.e., indium tin oxide and tin dioxide. Moreover, the experimental transmission spectra obtained after the deposition of the nanocoatings have been numerically corroborated by means of wave propagation methods. With the use of a conventional wavelength interrogation system and ad hoc developed microfluidics, the shift of the lossy mode resonance can be reliably recorded in response to very low analyte concentrations. Repeated experiments confirm a big leap in performance thanks to the capability to detect femtomolar concentrations in human serum, improving the detection limit by 3 orders of magnitude when compared with other fiber-based configurations. The biosensor has been regenerated several times by injecting sodium dodecyl sulfate, which proves the capability of sensor to be reused.

Towards Sustainable H2 Production: Rational Design of Hydrophobic Triphenylamine-based Dyes for Sensitized Ethanol Photoreforming.

Donor-acceptor dyes are a well-established class of photosensitizers, used to enhance visible-light harvesting in solar cells and in direct photocatalytic reactions, such as H2 production by photoreforming of sacrificial electron donors (SEDs). Amines-typically triethanolamine (TEOA)-are commonly employed as SEDs in such reactions. Dye-sensitized photoreforming of more sustainable, biomass-derived alcohols, on the other hand, was only recently reported by using methanol as the electron donor. In this work, several rationally designed donor-acceptor dyes were used as sensitizers in H2 photocatalytic production, comparing the efficiency of TEOA and EtOH as SEDs. In particular, the effect of hydrophobic chains in the spacer and/or the donor unit of the dyes was systematically studied. The H2 production rates were higher when TEOA was used as SED, whereas the activity trends depended on the SED used. The best performance was obtained with TEOA by using a sensitizer with just one bulky hydrophobic moiety, propylenedioxythiophene, placed on the spacer unit. In the case of EtOH, the best-performing sensitizers were the ones featuring a thiazolo[5,4-d]thiazole internal unit, needed for enhancing light harvesting, and carrying alkyl chains on both the donor part and the spacer unit. The results are discussed in terms of reaction mechanism, interaction with the SED, and structural/electrochemical properties of the sensitizers.

Long-period fiber grating: a specific design for biosensing applications.

In this paper, a detailed investigation on the modeling of long-period fiber grating (LPFG) sensors is discussed with the aim of providing a more realistic solution for their use in biosensing. Add-layer sensitivity, i.e., sensitivity of the sensor to an additional layer adhered onto the fiber surface, is quantified and a clear and complete analysis about the influence of the average thickness of the deposited biological sensing layers, as well as the change in refractive index of these layers, on the resonant wavelength of the cladding modes of an LPFG is provided. Add-layer sensitivity of LPFG sensors close to mode transition (MT) and also at turn-around point (TAP) are taken into account. Adsorbed layer thicknesses, as estimated from measured wavelength shifts of the LPFG, are found to have a good match with the values obtained through other measurement techniques.

Complex Active Optical Networks as a New Laser Concept.

Complex optical networks containing one or more gain sections are investigated, and the evidence of lasing action is reported; the emission spectrum reflects the topological disorder induced by the connections. A theoretical description compares well with the measurements, mapping the networks to directed graphs and showing the analogies with the problem of quantum chaos on graphs. We show that the interplay of chaotic diffusion and amplification leads to an emission statistic with characteristic heavy tails: for different topologies, an unprecedented experimental demonstration of Lévy statistics expected for random lasers is here provided for a continuous-wave pumped system. This result is also supported by a Monte Carlo simulation based on the ray random walk on the graph.

Molecular beacon-decorated polymethylmethacrylate core-shell fluorescent nanoparticles for the detection of survivin mRNA in human cancer cells.

One of the main goals of nanomedicine in cancer is the development of effective drug delivery systems, primarily nanoparticles. Survivin, an overexpressed anti-apoptotic protein in cancer, represents a pharmacological target for therapy and a Molecular Beacon (MB) specific for survivin mRNA is available. In this study, the ability of polymethylmethacrylate nanoparticles (PMMA-NPs) to promote survivin MB uptake in human A549 cells was investigated. Fluorescent and positively charged core PMMA-NPs of nearly 60nm, obtained through an emulsion co-polymerization reaction, and the MB alone were evaluated in solution, for their analytical characterization; then, the MB specificity and functionality were verified after adsorption onto the PMMA-NPs. The carrier ability of PMMA-NPs in A549 was examined by confocal microscopy. With the optimized protocol, a hardly detectable fluorescent signal was obtained after incubation of the cells with the MB alone (fluorescent spots per cell of 1.90±0.40 with a mean area of 1.04±0.20µm2), while bright fluorescent spots inside the cells were evident by using the MB loaded onto the PMMA-NPs. (27.50±2.30 fluorescent spots per cell with a mean area of 2.35±0.16µm2). These results demonstrate the ability of the PMMA-NPs to promote the survivin-MB internalization, suggesting that this complex might represent a promising strategy for intracellular sensing and for the reduction of cancer cell proliferation.

Effect of induced inner curvature on refractive index sensitivity in internally tilted long-period gratings.

A new complete analysis of the effect of induced inner curvature on refractive index (RI) sensitivity in internally tilted long-period gratings (ITLPGs) is presented. The responses in terms of RI sensitivity of a standard LPG and different ITLPGs with curvature values between 15 and 19 m-1 were compared. The analysis suggests first, that the larger the induced curvature, the greater the RI sensitivity; and second, that the RI sensitivity exponentially increases with both the curvature and cladding mode order. RI sensitivity greater than 100 nm RIU-1 can be attained with curvature greater than 25 m-1 for LP06 mode. Conversely, the temperature sensitivity of ITLPGs is comparable to standard LPGs for the considered cladding mode order.

SPR-based plastic optical fibre biosensor for the detection of C-reactive protein in serum.

A plastic optical fibre biosensor based on surface plasmon resonance for the detection of C-reactive protein (CRP) in serum is proposed. The biosensor was integrated into a home-made thermo-stabilized microfluidic system that allows avoiding any thermal and/or mechanical fluctuation and maintaining the best stable conditions during the measurements. A working range of 0.006-70 mg L-1 and a limit of detection of 0.009 mg L-1 were achieved. These results are among the best compared to other SPR-based biosensors for CRP detection, especially considering that they were achieved in a real and complex medium, i.e. serum. In addition, since the sensor performances satisfy those requested in physiologically-relevant clinical applications, the whole biosensing platform could well address high sensitive, easy to realize, real-time, label-free, portable and low cost diagnosis of CRP for future lab-on-a-chip applications. 3D sketch (left) of the thermo-stabilized home-made flow cell developed to house the SPR-based plastic optical fibre biosensor. Exemplary response curve (shift of the SPR wavelength versus time) of the proposed biosensor (right) for the detection of C-reactive protein in serum.

A Complete Optical Sensor System Based on a POF-SPR Platform and a Thermo-Stabilized Flow Cell for Biochemical Applications.

An optical sensor platform based on surface plasmon resonance (SPR) in a plastic optical fiber (POF) integrated into a thermo-stabilized flow cell for biochemical sensing applications is proposed. This device has been realized and experimentally tested by using a classic receptor-analyte assay. For this purpose, the gold surface of the POF was chemically modified through the formation of a self-assembling monolayer. The surface robustness of the POF-SPR platform has been tested for the first time thanks to the flow cell. The experimental results show that the proposed device can be successfully used for label-free biochemical sensing. The final goal of this work is to achieve a complete, small-size, simple to use and low cost optical sensor system. The whole system with the flow cell and the optical sensor are extensively described, together with the experimental results obtained with an immunoglobulin G (IgG)/anti-IgG assay.

Sol-Gel-Based Titania-Silica Thin Film Overlay for Long Period Fiber Grating-Based Biosensors.

An evanescent wave optical fiber biosensor based on titania-silica-coated long period grating (LPG) is presented. The chemical overlay, which increases the refractive index (RI) sensitivity of the sensor, consists of a sol-gel-based titania-silica thin film, deposited along the sensing portion of the fiber by means of the dip-coating technique. Changing both the sol viscosity and the withdrawal speed during the dip-coating made it possible to adjust the thickness of the film overlay, which is a crucial parameter for the sensor performance. After the functionalization of the fiber surface using a methacrylic acid/methacrylate copolymer, an antibody/antigen (IgG/anti-IgG) assay was carried out to assess the performance of sol-gel based titania-silica-coated LPGs as biosensors. The analyte concentration was determined from the wavelength shift at the end of the binding process and from the initial binding rate. This is the first time that a sol-gel based titania-silica-coated LPG is proposed as an effective and feasible label-free biosensor. The specificity of the sensor was validated by performing the same model assay after spiking anti-IgG into human serum. With this structured LPG, detection limits of the order of tens of micrograms per liter (10(-11) M) are attained.

Optical fiber nanotips coated with molecular beacons for DNA detection.

Optical fiber sensors, thanks to their compactness, fast response and real-time measurements, have a large impact in the fields of life science research, drug discovery and medical diagnostics. In recent years, advances in nanotechnology have resulted in the development of nanotools, capable of entering the single cell, resulting in new nanobiosensors useful for the detection of biomolecules inside living cells. In this paper, we provide an application of a nanotip coupled with molecular beacons (MBs) for the detection of DNA. The MBs were characterized by hybridization studies with a complementary target to prove their functionality both free in solution and immobilized onto a solid support. The solid support chosen as substrate for the immobilization of the MBs was a 30 nm tapered tip of an optical fiber, fabricated by chemical etching. With this set-up promising results were obtained and a limit of detection (LOD) of 0.57 nM was reached, opening up the possibility of using the proposed nanotip to detect mRNAs inside the cytoplasm of living cells.

A Hetero-Bifunctional Spacer for the Smart Engineering of Carbon-Based Nanostructures.

Efforts have been made in recent years to develop novel functionalisation protocols aimed at imparting multimodality and improved properties to complex carbon-based nanostructures. The incorporation of cleavable bonds to the nanomaterial surface for the controlled release (or exchange) of specific molecules under appropriate chemical and biological settings is relatively unexplored. The design and synthesis of a hetero-bifunctional linker joining a "cleavable" disulfide moiety for the covalent anchoring of a wide range of thiol end-capped (bio)molecules and a "clickable" terminal acetylene group is described. The strategy is based on the well-established copper-mediated acetylene-azide coupling reaction between the acetylene linker and single-walled carbon nanotubes decorated with phenylazido pendant arms. As a result, easily "post-derivatisable" and traceable nanostructured platforms containing a linking group potentially available for a wide range of biological probes are prepared and completely characterised.

A Hetero-Bifunctional Spacer for the Smart Engineering of Carbon-Based Nanostructures.

Invited for this month's cover are collaborators from four different Italian research groups, three at the National Research Council (ICCOM, IFAC, and ISOF) and one at the University of Florence. The cover picture shows a representative cartoon of engineered 1D carbon nanomaterials and their effective surface decoration with (bio)molecules and fluorescent markers. Read the full text of the article at 10.1002/cplu.201402391.

Stimulated anti-Stokes Raman scattering resonantly enhanced in silica microspheres.

Efficient stimulated anti-Stokes Raman scattering has been observed in silica microspherical resonators pumped by CW laser. This process is observed in the normal dispersion regime. The lack of correlation between stimulated anti-Stokes and Stokes scattering spectra indicates that the signal has to be resonant with the cavity.