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.

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.

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.

Electronic Detection of DNA Hybridization by Coupling Organic Field-Effect Transistor-Based Sensors and Hairpin-Shaped Probes.

In this paper, the electronic transduction of DNA hybridization is presented by coupling organic charge-modulated field-effect transistors (OCMFETs) and hairpin-shaped probes. These probes have shown interesting properties in terms of sensitivity and selectivity in other kinds of assays, in the form of molecular beacons (MBs). Their integration with organic-transistor based sensors, never explored before, paves the way to a new class of low-cost, easy-to-use, and portable genetic sensors with enhanced performances. Thanks to the peculiar characteristics of the employed sensor, measurements can be performed at relatively high ionic strengths, thus optimizing the probes' functionality without affecting the detection ability of the device. A complete electrical characterization of the sensor is reported, including calibration with different target concentrations in the measurement environment and selectivity evaluation. In particular, DNA hybridization detection for target concentration as low as 100 pM is demonstrated.

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.

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.

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.

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.

Nucleic acid and peptide aptamers: fundamentals and bioanalytical aspects.

In recent years new nucleic acid and protein-based combinatorial molecules have attracted the attention of researchers working in various areas of science, ranging from medicine to analytical chemistry. These molecules, called aptamers, have been proposed as alternatives to antibodies in many different applications. The aim of this Review is to illustrate the peculiarities of these combinatorial molecules which have initially been explored for their importance in molecular medicine, but have enormous potential in other biotechnological fields historically dominated by antibodies, such as bioassays. A description of these molecules is given, and the methods for their selection and production are also summarized. Moreover, critical aspects related to these molecules are discussed.

Silencing Survivin: a Key Therapeutic Strategy for Cardiac Hypertrophy.

Cardiac hypertrophy, in its aspects of localized thickening of the interventricular septum and concentric increase of the left ventricle, constitutes a risk factor of heart failure. Myocardial hypertrophy, in the presence of different degree of myocardial fibrosis, is paralleled by significant molecular, cellular, and histological changes inducing alteration of cardiac extracellular matrix composition as well as sarcomeres and cytoskeleton remodeling. Previous studies indicate osteopontin (OPN) and more recently survivin (SURV) overexpression as the hallmarks of heart failure although SURV function in the heart is not completely clarified. In this study, we investigated the involvement of SURV in intracellular signaling of hypertrophic cardiomyocytes and the impact of its transcriptional silencing, laying the foundation for novel target gene therapy in cardiac hypertrophy. Oligonucleotide-based molecules, like theranostic optical nanosensors (molecular beacons) and siRNAs, targeting SURV and OPN mRNAs, were developed. Their diagnostic and therapeutic potential was evaluated in vitro in hypertrophic FGF23-induced human cardiomyocytes and in vivo in transverse aortic constriction hypertrophic mouse model. Engineered erythrocyte was used as shuttle to selectively target and transfer siRNA molecules into unhealthy cardiac cells in vivo. The results highlight how the SURV knockdown could negatively influence the expression of genes involved in myocardial fibrosis in vitro and restores structural, functional, and morphometric features in vivo. Together, these data suggested that SURV is a key factor in inducing cardiomyocytes hypertrophy, and its shutdown is crucial in slowing disease progression as well as reversing cardiac hypertrophy. In the perspective, targeted delivery of siRNAs through engineered erythrocytes can represent a promising therapeutic strategy to treat cardiac hypertrophy. Theranostic SURV molecular beacon (MB-SURV), transfected into FGF23-induced hypertrophic human cardiomyocytes, significantly dampened SURV overexpression. SURV down-regulation determines the tuning down of MMP9, TIMP1 and TIMP4 extracellular matrix remodeling factors while induces the overexpression of the cardioprotective MCAD factor, which counterbalance the absence of pro-survival and anti-apoptotic SURV activity to protect cardiomyocytes from death. In transverse aortic constriction (TAC) mouse model, the SURV silencing restores the LV mass levels to values not different from the sham group and counteracts the progressive decline of EF, maintaining its values always higher with respect to TAC group. These data demonstrate the central role of SURV in the cardiac reverse remodeling and its therapeutic potential to reverse cardiac hypertrophy.

Piezoelectric biosensors for aptamer-protein interaction.

Aptamers can be considered as a valid alternative to antibodies or other biomimetic receptors for the development of biosensors and other analytical methods. The production of aptamers is commonly performed by the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) process, which, starting from large libraries of oligonucleotides, allows the isolation of large amounts of functional nucleic acids by an iterative process of in vitro selection and subsequent amplification through polymerase chain reaction. Aptamers are suitable for applications based on molecular recognition as analytical, diagnostic, and therapeutic tools. The use of aptamers as biorecognition element in piezoelectric biosensors will be here reported with particular application to the detection of thrombin.

Biosensors for RNA aptamers-protein interaction.

Aptamers are an alternative to antibodies in their role as biorecognition elements in analytical devices. RNA aptamers, specific for different proteins, have been exploited as biorecognition elements to develop specific biosensors (aptasensors). These recognition elements have been coupled to piezoelectric quartz crystals and surface plasmon resonance (SPR) devices as transducers. The procedure for fixing the aptamer onto these transducers and for monitoring the interaction with the target protein is shown here.

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.

Magnetically driven drug delivery systems improving targeted immunotherapy for colon-rectal cancer.

Colorectal cancer (CRC) is one of the major causes of cancer-associated mortality worldwide. The currently approved therapeutic agents show a rather limited efficacy. We have recently demonstrated that the atypical cadherin FAT1 is a specific marker of CRC and that the FAT1-specific monoclonal antibody mAb198.3 may offer new therapeutic opportunities for CRC, being efficiently internalized by cancer cells and reducing cancer growth in colon cancer xenograft models. In this study we explored the therapeutic efficacy of mAb198.3 using two drug delivery systems (DDS) for improving the targeted treatment of CRC. The mAb198.3 was either directly bound to super-paramagnetic nanoparticles (spmNPs) or embedded into human erythrocyte-based magnetized carriers, named Erythro-Magneto-Hemagglutinin Virosomes (EMHVs) to produce two different novel mAb198.3 formulations. Both DDS were endowed with magnetic properties and were anchored in the target tumor site by means of an external permanent magnet. The antibody loading efficiency of these two magnetically driven drug delivery systems and the overall therapeutic efficacy of these two formulations were assessed both in vitro and in a proof-of-concept in vivo study. We demonstrated that mAb198.3 bound to spmNPs or embedded into EMHVs was very effective in targeting FAT1-positive colon cancer cells in vitro and accumulating in the tumor mass in vivo. Although both in vivo administered mAb198.3 formulations have approximately 200 lower antibody doses needed, these showed to achieve a relevant therapeutic effect, thus reducing cancer growth more efficiently respect to the naked antibody. These results indicate that the two proposed magnetically driven drug delivery systems have a considerable potential as platforms to improve bioavailability and pharmacodynamics of anti-FAT mAb198.3 and raise new opportunities for a targeted therapy of CRC.

Aptamers-based assays for diagnostics, environmental and food analysis.

Aptamers are single stranded DNA or RNA ligands which can be selected for different targets starting from a huge library of molecules containing randomly created sequences. Aptamers have been selected to bind very different targets, from proteins to small organic dyes. In addition to the very important aspect of having an unlimited source of identical affinity recognition molecules available due to the selection process, aptamers can offer advantages over antibodies that make them very promising for analytical applications. The use of aptamers as therapeutic tools is nowadays well established. On the contrary, the analytical application of aptamers in diagnostic devices or in systems for environmental and food analysis, is still under investigation and the scientific community still need further research to demonstrate the advancements brought by this new kind of ligands. This review will focus on these latter applications with particular attention to the detection of food pathogens, terrorism threat agents, thrombin and cytokines.

Development of combined DNA-based piezoelectric biosensors for the simultaneous detection and genotyping of high risk Human Papilloma Virus strains.

BACKGROUND: Human Papilloma Virus (HPV) is a DNA virus belonging to the Papovavirus family. Genital HPV types have been subdivided into medium-low risk, and high-risk (HPV 16 and 18), frequently associated with cervical cancer. Three DNA-based piezoelectric biosensors were here developed for a quick detection and genotyping of HPV. METHODS: We developed a method for the detection and genotyping of HPV in human cervical scraping samples based on coupling DNA piezoelectric sensors with Polymerase Chain Reaction (PCR). The novelty of this work was the design and immobilisation of a degenerate probe (chosen in a conserved region of the viral genome) for the simultaneous detection of 16 virus strains and of two specific probes (chosen in a less-conserved region of the viral genome) for genotyping. RESULTS: The three biosensors were optimised with synthetic oligonucleotides with good reproducibility (HPVdeg CV% (av) 9%, HPV16 CV%(av) 9%; HPV18 CV%(av) 11%) with a detection limit of 50 nM. Cervical scraping samples after PCR amplification (in 40-200 nM range), were tested without the need of label with high selectivity and reproducibility. The results were in agreement with a reference method used in routinary analysis. CONCLUSION: Piezoelectric biosensors have proven to be suitable for detection and genotyping of HPV.

Polyphenol content and antioxidative activity in some species of freshly consumed salads.

Ten genotypes belonging to Lactuca sativa, Cicorium intybus, Plantago coronopus, Eruca sativa, and Diplotaxis tenuifolia and used in fresh mixed salads were investigated for their polyphenol contents. Flavonoids and hydroxycinnamic acids were characterized by high-performance liquid chromatography (HPLC)/diode array detection/mass spectrometry. Quercetin, kaempferol, luteolin, apigenin, and crysoeriol derivatives were identified; hydroxycinnamic acids were all caffeoyl derivatives. The total polyphenol content was obtained through the Folin-Ciocalteu test and from the HPLC data. The amounts ranged between 0.9 and 4.7 mg/g fresh weight. The antiradical activity was determined by the reaction with the stable DPPH* radical. The Fe2+ chelating activity was determined with a spectrophotometric test. From the complex of data, a quite complete picture of the characteristics of the vegetables emerges. A cultivated C. intybus cultivar exhibited the highest polyphenol content, while a wild C. intybus genotype exhibited the highest antiradical activity. In every case, the characteristics of the different salads as functional foods have been pointed out.