SPECTRA: A Novel Compact System for Surface Plasmon Resonance Measurements.

Surface plasmon resonance (SPR) is a common and useful measurement technique to perform fast and sensitive optical detection. SPR instrumentations usually comprise optical systems of mirrors and lenses which are quite expensive and impractical for point-of-care applications. In this work, we presented a novel and compact SPR device called SPECTRA, designed as a spectrophotometer add-on with a grating coupling configuration. The device is conceived as a marketable solution to perform quick SPR measurements in grating configuration without the requirement of complex instrumentation. The device can be customized either in a vertical structure to reach lower incident light angles, or in a horizontal configuration, which is suitable for SPR analysis using liquid solutions. The SPECTRA performance was evaluated through SPR measurements in typical applications. The vertical SPECTRA system was employed to detect different functionalization molecules on gold 720 nm-period grating devices. Meanwhile, the horizontal SPECTRA configuration was exploited to carry out fluid-dynamic measurements using a microfluidic cell with glycerol solutions at increasing concentrations to account for different refractive indexes. The experimental tests confirmed that the SPECTRA design is suitable for SPR measurements, demonstrating its capability to detect the presence of analytes and changes in surface properties both in static and dynamic set-ups.

Label-free efficient and accurate detection of cystic fibrosis causing mutations using an azimuthally rotated GC-SPR platform.

Plasmonic nanosensors are candidates for the development of new sensors with low detection limits, high sensitivity, and specificity for target detection: these characteristics are of critical importance in the screening of mutations responsible for inherited diseases. In this work, we focused our study on the detection of some of the most frequent mutations responsible for cystic fibrosis (CF) among the Italian population. For the detection of the CF mutations we adopted a recently developed and highly sensitive Grating Coupled-Surface Plasmon Resonance (GC-SPR) enhanced spectroscopy method for label-free molecular identification exploiting a conical illumination configuration. Gold sinusoidal gratings functionalized with heterobifunctional PEG were used as sensing surfaces, and the specific biodetection was achieved through the coupling with DNA hairpin probes designed for single nucleotide discrimination. Such substrates were used to test unlabeled PCR amplified homozygous wild type (wt) and heterozygous samples, deriving from clinical samples, for the screened mutations. Hybridization conditions were optimized to obtain the maximum discrimination ratio (DR) between the homozygous wild type and the heterozygous samples. SPR signals obtained from hybridizing wild type and heterozygous samples show DRs able to identify univocally the correct genotypes, as confirmed by fluorescence microarray experiments run in parallel. Furthermore, SPR genotyping was not impaired in samples containing unrelated DNA, allowing the platform to be used for the concomitant discrimination of several alleles also scalable for a high throughput screening setting.

Development of a multiplex sandwich aptamer microarray for the detection of VEGF165 and thrombin.

In this work we have developed a multiplex microarray system capable of detecting VEGF165 and thrombin. We recently described a Sandwich Aptamer Microarray (SAM) for thrombin detection feasible for use in multiplex microarrays; here we describe a new aptasensor for VEGF165 detection employing Vap7 and VEa5, two DNA aptamers recognizing different sites of the protein. The aptamers were modified to be adapted to the solid phase platform of SAM and their capability to simultaneously recognize VEGF165 by forming a ternary complex was analyzed in solution. Having so defined the best tandem arrangement of modified aptamers, we set up the aptasensor for VEGF165, and finally analyzed the multiplex system with the two aptasensors for the simultaneous detection of VEGF165 and thrombin. The results indicate that each sandwich is specific, even when the two proteins are mixed. The system performance is consistent with the behavior evidenced by the biochemical analysis, which proves to be valuable to drive the evaluation and refinement of aptamers prior to or along the development of a detection platform. Since thrombin upregulates VEGF expression, the simultaneous recognition of these two proteins could be useful in the analysis of biomarkers in pathologies characterized by neo-angiogenesis.

Synthesis and chromatography-free purification of PNA-PEO conjugates for the functionalisation of gold sensors.

Peptide Nucleic Acids (PNAs) linked to high molecular weight (MW) poly(ethylene oxide) (PEO) derivatives could be useful conjugates for the direct functionalisation of gold surfaces dedicated to Surface Plasmon Resonance (SPR)-based DNA sensing. However their use is hampered by the difficulty to obtain them through a convenient and economical route. In this work we compared three synthetic strategies to obtain PNA-high MW PEO conjugates composed of (a) a 15-mer PNA sequence as the probe complementary to genomic DNA of ]Mycobacterium tuberculosis, (b) a PEO moiety (2 or 5 KDa MW) and (c) a terminal trityl-protected thiol necessary (after acidic deprotection) for grafting to gold surfaces. The 15-mer PNA was obtained by solid-phase synthesis. Its amino terminal group was later condensed to bi-functional PEO derivatives (2 and 5 KDa MW) carrying a Trt-cysteine at one end and a carboxyl group at the other end. The reaction was carried out either in solution, using HATU or PyOxim as coupling agents, or through the solid-phase approach, with 49.6%, 100% and 5.2% yield, respectively. A differential solvent extraction strategy for product purification without the need for chromatography is described. The ability of the 5 KDa PEO conjugate to function as a probe for complementary DNA detection was demonstrated using a Grating-Coupling Surface Plasmon Resonance (GC-SPR) system. The optimized PEO conjugation and purification protocols are economical and simple enough to be reproduced also within laboratories that are not highly equipped for chemical synthesis.

Optical System Based on Nafion Membrane for the Detection of Ammonia in Blood Serum Samples.

The blood ammonia (NH3) level is one of the most important hepatic biomarkers for the diagnosis and monitoring of liver pathologies and infections. In this work, we developed an optimized optical biosensing method to extract and quantify the ammonia contained in complex-matrix samples emulating the blood serum. First, the approach was tested with solutions of phosphate-buffered saline (PBS) and ammonia chloride. Then, further trials were carried out with solutions of fetal bovine serum (FBS). The ammonia was extracted from the tested samples through a customized cell, and it was optically quantified by exploiting the indophenol reaction. The extraction cell included a cation-exchange membrane in Nafion, which was chemically pre-treated through cleaning procedures of sulfuric acid and hydrogen peroxide to keep a basic pH in the ammonia solution and to avoid contaminants in the membrane. From the NH3 solution, the indophenol reaction produced light-reactive indophenol dye molecules, which were used as colorimetric indicators. Through absorbance measurements of the indophenol dye solution at 670 nm wavelength, we were able to detect and quantify the ammonia level in the samples both with a spectrophotometer and a customized miniaturized read-out system, obtaining a detection limit of 0.029 µmol/mL.

Human thrombin detection through a sandwich aptamer microarray: interaction analysis in solution and in solid phase.

We have developed an aptamer-based microarray for human thrombin detection exploiting two non-overlapping DNA thrombin aptamers recognizing different exosites of the target protein. The 15-mer aptamer (TBA1) binds the fibrinogen-binding site, whereas the 29-mer aptamer (TBA2) binds the heparin binding domain. Extensive analysis on the complex formation between human thrombin and modified aptamers was performed by Electrophoresis Mobility Shift Assay (EMSA), in order to verify in solution whether the chemical modifications introduced would affect aptamers/protein recognition. The validated system was then applied to the aptamer microarray, using the solid phase system devised by the solution studies. Finally, the best procedure for Sandwich Aptamer Microarray (SAM) and the specificity of the sandwich formation for the developed aptasensor for human thrombin were optimized.

A single mutation in the 729 residue modulates human DNA topoisomerase IB DNA binding and drug resistance.

Human DNA topoisomerase I (hTop1p) catalyzes the relaxation of supercoiled DNA and constitutes the cellular target of the antitumor drug camptothecin (CPT). The X-ray crystal structure of the enzyme covalently joined to DNA and bound to the CPT analog Topotecan suggests that there are two classes of mutations that can produce a CPT-resistant enzyme. The first class includes changes in residues that directly interact with the drug, whereas a second class alters interactions with the DNA and thereby destabilizes the drug binding site. The Thr729Ala, that is part of a hydrophobic pocket in the enzyme C-terminal domain, belongs to a third group of mutations that confer CPT resistance, but do not interact directly with the drug or the DNA. To understand the contribution of this residue in drug resistance, we have studied the effect on hTop1p catalysis and CPT sensitivity of four different substitutions in the Thr729 position (Thr729Ala, Thr729Glu, Thr729Lys and Thr729Pro). Tht729Glu and Thr729Lys mutants show severe CPT resistance and furthermore, Thr729Glu shows a remarkable defect in DNA binding. We postulate that the maintenance of the hydrophobic pocket integrity, where Thr729 is positioned, is crucial for drug sensitivity and DNA binding.

Human DNA topoisomerase IB: structure and functions.

Human DNA Topoisomerase I is a 765aa monomeric enzyme composed of four domains: the N-terminal domain, highly charged and responsible for several protein-protein interactions, the core domain that embraces the DNA during catalysis, the highly charged linker domain and the C-teminal domain containing the active site. The enzyme promotes the relaxation of supercoiled DNA by nicking and rejoining one of the strands of the DNA. Its activity is critical for many biological processes including DNA replication, transcription, and recombination. The aim of this review is to analyze the enzyme activity in terms of structure-function relationship.

Sensitive detection of Ochratoxin A in food and drinks using metal-enhanced fluorescence.

Easy, sensitive, rapid and low cost ochratoxin biosensors are strongly demanded in food analysis since Ochratoxin A (OTA) is a widely diffused food contaminant, highly detrimental for human health. In this work, a novel plasmonic based optical biosensor prototype for ochratoxin A is described. It exploits the metal-enhanced fluorescence phenomenon due to the silver film over nanosphere plasmonic substrate. Since ochratoxin A could be present in different food commodities, sensor performances have been tested on three different matrices (dried milk, juices, and wheat mix). Firstly, a common OTA extraction solvent and a labeling and detection protocol were defined for the analyzed matrices. Then, the efficiency of the Ag-FON surfaces in signal amplification for the detection of low ochratoxin A concentrations was defined. Using samples spiked with OTA-AF 647 or with unlabeled OTA we were able to detect the mycotoxin at concentrations lower than E.U. specifications of 0.5 µg/kg in wheat, milk and apple juice. The test performances are comparable to those of ELISA kits but the platform presented here, once optimized, present some perspective advantages, such as: low cost and time consuming, versatility of the protocol for the investigation of different matrices, employment also in non-qualified laboratories, small dimensions that allow its integration in a compact device for OTA on-site detection.

Development and Optimization of a Thrombin Sandwich Aptamer Microarray.

A sandwich microarray employing two distinct aptamers for human thrombin has been optimized for the detection of subnanomolar concentrations of the protein. The aptamer microarray demonstrates high specificity for thrombin, proving that a two-site binding assay with the TBA1 aptamer as capture layer and the TBA2 aptamer as detection layer can ensure great specificity at times and conditions compatible with standard routine analysis of biological samples. Aptamer microarray sensitivity was evaluated directly by fluorescent analysis employing Cy5-labeled TBA2 and indirectly by the use of TBA2-biotin followed by detection with fluorescent streptavidin. Sub-nanomolar LODs were reached in all cases and in the presence of serum, demonstrating that the optimized aptamer microarray can identify thrombin by a low-cost, sensitive and specific method.

Alterations in linker flexibility suppress DNA topoisomerase I mutant-induced cell lethality.

Eukaryotic DNA topoisomerase I (Top1p) catalyzes changes in DNA topology via the formation of a covalent enzyme-DNA intermediate, which is reversibly stabilized by the anticancer agent camptothecin (CPT). Crystallographic studies of the 70-kDa C terminus of human Top1p bound to duplex DNA describe a monomeric protein clamp circumscribing the DNA helix. The structures, which lack the N-terminal domain, comprise the conserved clamp, an extended linker domain, and the conserved C-terminal active site Tyr domain. CPT bound to the covalent Top1p-DNA complex limits linker flexibility, allowing structural determination of this domain. We previously reported that mutation of Ala(653) to Pro in the linker increases the rate of enzyme-catalyzed DNA religation, thereby rendering Top1A653Pp resistant to CPT (Fiorani, P., Bruselles, A., Falconi, M., Chillemi, G., Desideri, A., and Benedetti P. (2003) J. Biol. Chem. 278, 43268-43275). Molecular dynamics studies suggested mutation-induced increases in linker flexibility alter Top1p catalyzed DNA religation. To address the functional consequences of linker flexibility on enzyme catalysis and drug sensitivity, we investigated the interactions of the A653P linker mutation with a self-poisoning T718A mutation within the active site of Top1p. The A653P mutation suppressed the lethal phenotype of Top1T718Ap in yeast, yet did not restore enzyme sensitivity to CPT. However, the specific activity of the double mutant was decreased in vivo and in vitro, consistent with a decrease in DNA binding. These findings support a model where changes in the flexibility or orientation of the linker alter the geometry of the active site and thereby the kinetics of DNA cleavage/religation catalyzed by Top1p.