Salvatore Petralia

Integrated PCR amplification and detection processes on a Lab-on-Chip platform: a new advanced solution for molecular diagnostics.

Abstract Background: Several microdevices have been developed to perform only a single step of a genotyping process, such as PCR or detection by probe hybridization. Here, we describe a Lab-on-Chip (LoC) platform integrating a PCR amplification microreactor with a customable microarray for the detection of sequence variations on human genomic DNA. Methods: Preliminary work was focused on developing the single analytical steps including PCR and labeling strategies of the amplified product by conventional reference systems. The optimized protocols included a 1:4 forward:reverse primer ratio for asymmetric PCR, and Cy5-dCTP multiple incorporation for the generation of a labeled PCR product to be hybridized to complementary probes bound to the chip surface. Results: Final conditions were applied to the fully integrated LoC platform for the detection of the IVSI-110 G>A mutation in the human β-globin (HBB) gene associated with β-thalassemia, used as a model of genetic application, allowing for correct genotyping of 25 samples that were heterozygous, homozygous or wild-type for this mutation. Conclusions: The overall results show that the present platform is very promising for rapid identification of DNA sequence variations in an integrated, cost effective and convenient silicon chip format. Clin Chem Lab Med 2010;48:329–36.

Monitoring photoswitching of azobenzene-based self-assembled monolayers on ultrathin platinum films by UV/Vis spectroscopy in the transmission modeElectronic supplementary information (ESI) available: synthesis and characterization of 1 and its photoisomerization in solution. See http://www.rsc.org/suppdata/jm/b3/b314710j/

The reversible trans ↔ cis photoisomerization of azobenzene-based self-assembled monolayers on ultrathin platinum films has been monitored for the first time by UV-Vis transmission spectroscopy with a conventional spectrophotometer.

Activation of Pituitary Adenylate Cyclase‐Activating Polypeptide Receptors Prevents Apoptotic Cell Death in Cultured Cerebellar Granule Cellsa

Extensive neuronal death occurs during the normal development of the nervous system. This programmed neuronal death is often controlled by survival-promoting signals from other cells and serves to match neuronal number to target size and to rid the nervous system of inappropriate connections.’ Several lines of evidence suggest that pituitary adenylate cyclase-activating polypeptide (PACAP) acts as a neurotrophic factor in the nervous system. PACAP was shown to promote neurite outgrowth> to stimulate cell proliferation,’ and to prevent naturally programmed neuronal cell death.4 In the present study we investigated the relationship between PACAP receptor activation and neuronal apoptosis in cultured cerebellar granule cells, which are a highly homogeneous population of neurons and which express specific PACAP receptors positively coupled to both adenylyl cyclase and phospholipase CS-’ When

Light-triggered DNA release by dynamic monolayer films

We illustrate a simple strategy to immobilize single and double strand DNA on a two-dimensional surface and to trigger their release under physiological conditions, under the exclusive control of light stimuli. A tailored azobenzene derivative has been self-assembled on transparent platinum electrodes to form cationic-terminated monolayer films. These monolayers encourage the binding of DNA with the metal surface through effective electrostatic interactions with the negatively charged polynucleotide backbone. Irradiation of the film with UVA light induces trans to cisisomerization of the photoresponsive azobenzene units leading to significant changes of surface hydrophilicity and decreasing the binding affinity for DNA, which is consequently released into the solution. It is shown that the amount of DNA released can be precisely tuned by controlling the illumination conditions and is strictly related to the photoinduced structural modifications at the film surface. After the release of DNA the functional monolayers can be recycled through illumination with visible light which causes the cis form of the azo-chromophore to revert to the trans form, restoring the initial conditions. Given the non-specific nature of the Coulombic interactions the approach presented herein may, in principle, also be extended from polynucleotides to other negatively charged biomolecules, making these dynamic monolayers appealing model systems from the perspective of nanoscaled devices for biomedical applications where spatiotemporal control of biological material is required.

Is this the real time for genomics

In the last decades, molecular biology has moved from gene-by-gene analysis to more complex studies using a genome-wide scale. Thanks to high-throughput genomic technologies, such as microarrays and next-generation sequencing, a huge amount of information has been generated, expanding our knowledge on the genetic basis of various diseases. Although some of this information could be transferred to clinical diagnostics, the technologies available are not suitable for this purpose. In this review, we will discuss the drawbacks associated with the use of traditional DNA microarrays in diagnostics, pointing out emerging platforms that could overcome these obstacles and offer a more reproducible, qualitative and quantitative multigenic analysis. New miniaturized and automated devices, called Lab-on-Chip, begin to integrate PCR and microarray on the same platform, offering integrated sample-to-result systems. The introduction of this kind of innovative devices may facilitate the transition of genome-based tests into clinical routine.

Hydrogen atom abstraction from resveratrol and two lipophilic derivatives by tert-butoxyl radicals. A laser flash photolysis study

The reactions of tert-butoxyl radicals with resveratrol (1) and two acetylated derivatives (2 and 3) have been investigated by laser flash photolysis techniques in 1∶2 (v/v) benzene–di-tert-butyl peroxide at room temperature. The transient absorption spectra of the phenoxyl radicals generated upon H atom abstraction by tert-butoxyl radicals from the phenols have been detected and assigned. The absolute rate constants for these reactions have been evaluated to be 45 × 107, 25 × 107 and 4 × 107 M−1 s−1 for 1, 2 and 3, respectively. The order of reactivity 1 ≥ 2 ≫ 3 has been rationalized in terms of the position and effect of the acetyl groups on the aromatic rings. Of the three OH groups present in resveratrol, the one in position 4′ appears to be the most reactive due to the large stability of the corresponding phenoxyl radical by conjugation with the rings. However, in our system, the H-atom-donating ability of resveratrol turns out to be inferior to that of α-tocopherol by ca. one order of magnitude.

Photochemical outcome modification of diflunisal by a novel cationic amphiphilic cyclodextrin

The effects of a novel cationic amphiphilic cyclodextrin (SC6CDNH2) on the photoprocesses of the phototoxic non-steroidal anti-inflammatory drug diflunisal (DF) have been investigated in aqueous medium. Association between DF and SC6CDNH2 was indicated by steady-state absorption, induced circular dichroism and fluorescence spectroscopy. Laser flash photolysis and steady-state photolysis experiments revealed a particular sensitivity of the DF photochemistry to the new microenvironment. Reduction of the triplet state generation efficiency, lengthening of its lifetime, changes in the photoionisation pathways, besides remarkable drug photostabilization and modification of the stable photoproducts distribution, were observed. A rationale for these modifications to the photochemistry of DF based on the multifaceted role of SC6CDNH2 in influencing the efficiency of the primary photochemical act and in interfering with secondary radical reactions is proposed. Relation of the overall results to the phototoxic effects displayed by the drug is also commented upon.

Irreversible photo-oxidation of propranolol triggered by self-photogenerated singlet molecular oxygen

The photochemistry of propranolol (PR), 1-isopropylamino-2-(1-naphthyloxy)propan-2-ol, has been studied by combining laser-flash photolysis and steady-state measurements. Like other compounds bearing the naphthalene chromophore, the triplet state is produced quite efficiently (phi(isc) = 0.35) and it is able to transfer its energy to oxygen, sensitizing singlet oxygen formation with phi(delta) = 0.24. PR photodecomposition takes place in an aerated aqueous medium and leads to 6-hydroxy-1,4-naphthoquinone as the sole stable photoproduct. This seems to be consistent with the intermediary formation of the highly photolabile 1,4-naphthoquinone. It is demonstrated that photodegradation occurs via a type II mechanism involving irreversible trapping of self-photogenerated singlet molecular oxygen.

Absolute rate constants and transient intermediates in the free-radical-induced peroxidation of γ-terpinene, an unusual hydrocarbon antioxidant

The peroxidation of γ-terpinene (TH) initiated by tert-butoxyl radicals has been investigated by means of nanosecond laser flash photolysis techniques in acetonitrile at room temperature. The absolute rate constants for the reactions implicated in the peroxidation process have been determined and the main short-lived intermediates, terpenyl and terpenylperoxyl radicals, have been detected. The rate constant for H-atom abstraction from TH by tert-butoxyl radicals, k3, has been found to be 1.4 × 108 M−1 s−1 while the values for the self-quenching of T˙ radicals, 2k4, and the addition of O2 to T˙, k9, are, respectively, 1.2 × 1010 M−1 s−1 and 1.3 × 109 M−1 s−1. The overall results strongly substantiate the peroxidation mechanism recently proposed by Foti and Ingold, J. Agric. Food Chem., 2003, 51, 2758, and provide a solid basis for a better understanding of the unusual antioxidant activity exhibited by this hydrocarbon.

Si Photomultipliers for Bio-Sensing Applications

In this paper, silicon photomultipliers (SiPM) are proposed as optical detectors for bio sensing. Optical transduction is the most used detection mechanism in many biosensor applications, such as DNA microarray and real-time polymerase chain reaction. The performances of a 25 pixels device used for both applications are studied. The results confirm that the SiPM is more sensitive than the traditionally employed detectors. In fact, it is able to experimentally detect 1 nM and 100 fM of fluorophore concentrations in dried samples and solutions, respectively. We present and discuss in details the detector configuration and its characterization as fluorescence detector for bio sensing.