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Il modello di differenziazione delle competenze delle Comunidades autonomas spagnole, previsto nel Titolo VIII della Costituzione del 1978, è spesso stato utilizzato come riferimento nella predisposizione del nostro sistema di autonomia differenziata previsto, ma ad oggi mai concretamente sperimentato, dal nuovo articolo 116, co. 3 Cost. italiana; molti presumevano e auspicavano, inoltre, che tale modello di autonomia differenziata potesse fornire un esempio concreto anche in fase di attivazione dell’omonimo istituto italiano. Questi auspici, a distanza di quasi 20 anni dall’emanazione della Riforma del Titolo V, sembrano però essere stati avventati: la formula definitiva dell’art. 116 co. 3 n.t. si discosta infatti non poco, in definitiva, dal modello spagnolo. Le Comunidades spagnole accedono infatti alla differenziazione con un meccanismo generalizzato, nel quale sono direttamente i singoli statuti di autonomia a recare, come contenuto essenziale, l’elenco delle “competenze assunte nel quadro stabilito dalla Costituzione” ; la scelta delle materie è dunque demandata all’elaborazione dello Statuto di autonomia. Nell’ordinamento italiano, viceversa, la Costituzione attribuisce automaticamente alle Regioni ordinarie una rosa di competenze, lasciando in via eventuale la possibilità di ottenerne ulteriori attivando la procedura di cui all’art. 116 co. 3 Cost. Per questo motivo, in Spagna, le prime differenziazioni si sono presentate già con l’emanazione dei primi Statuti di autonomia, nel 1981; mentre in Italia, a distanza di quasi 20 anni dall’emanazione della Riforma costituzionale, non è ancora stata conclusa positivamente alcuna procedura di differenziazione. Vi è in comune tra i due modelli, invece, il necessario passaggio parlamentare della legge di recepimento della differenziazione; nel sistema spagnolo, tuttavia, manca qualsiasi riferimento a una previa fase di trattative per la predisposizione del contenuto dell’atto di differenziazione, presente invece in quello italiano, dove Stato e Regione devono concludere un’intesa in tal senso. L’elenco delle materie richiedibili dalle Comunidades è previsto dall’art. 148 Cost. spagnola; si tratta di competenze esclusive, di norma comprensive dei profili normativi e amministrativi. È invece opinione comune della dottrina che l’art. 116 co. 3 Cost. italiana consenta una maggiore modulazione nella scelta delle competenze, che possono essere assunte dalle Regioni in via concorrente o in via esclusiva, integralmente o solo nel profilo amministrativo. L’elenco di competenze incamerabili dalle Comunidades è inoltre completato dalla previsione per cui “le materie non espressamente attribuite da questa costituzione allo Stato potranno attribuirsi alle Comunità Autonome in virtù dei rispettivi Statuti” , ma solo “dopo il decorso di cinque anni” ; tale limite temporale non varrebbe però qualora la Comunità autonoma percorresse la procedura speciale dettata dall’art. 151 Cost. spagnola. Questa dualità di procedura trova le proprie radici nella genesi storica della democrazia spagnola: nella delicatissima transizione da un regime dittatoriale allo Stato democratico si dovette predisporre un sistema di differenziazione che permettesse un equilibrato sviluppo dell’autonomia in territori diversissimi tra loro, permettendo alle c.d. nacionalidades, i territori “costituenti entità regionali storiche” , di addivenire subito a forme di autonomia più ampie rispetto al resto della Spagna. Questo condusse, con la firma dei primi patti autonomici nel 1981, alla creazione di regioni “di primo livello” e “di secondo livello”: vennero concesse alle prime alcune ulteriori competenze importanti (sanità, educazione, reti televisive locali), mentre le seconde intrapresero il procedimento ordinario, con competenze più limitate. Una simile situazione non sussiste nell’attuale scenario socio-politico italiano; tuttavia, le profonde differenze socio-economiche tra Regioni del Nord e quelle del Sud del nostro Paese suggeriscono un approccio graduale nella concessione di competenze alle Regioni ordinarie, sulla base di quanto fatto in Spagna nel distinguere tra Comunità di primo livello e di secondo livello. Questa distinzione si è peraltro decisamente affievolita col tempo, per via di un’opera di progressivo livellamento delle competenze concesse a tutte le comunità: lo testimoniano le revisioni dei patti autonomici intervenute negli anni 90’, che, con sporadiche eccezioni , hanno attivato un processo inesorabile di omogeneizzazione verso l’alto delle competenze comunitarie. Un simile rischio è ipotizzabile anche per il processo di attivazione del regionalismo differenziato in Italia: come testimoniato dalle trattative in atto tra alcune Regioni del Nord e lo Stato , forte è il rischio di intese-fotocopia, spesso concertate tra Stato e diverse Regioni contemporaneamente e poco rispettose del principio di differenziazione delle competenze sulla base delle peculiarità dei territori coinvolti. Non bisogna tuttavia dimenticare che molto positivo è stato l’effetto di trascinamento che le Comunità spagnole più avanzate, arrivate prima alla gestione di alcune competenze, hanno avuto sulle altre Comunità, in un processo elastico che, se si verificasse in Italia, potrebbe portare ad una maggiore responsabilizzazione delle Regioni in sofferenza, spinte dall’iniziativa di quelle più avanzate. L’omogeneizzazione tra le comunità ha però avuto un effetto indesiderato: ha stressato, invece di sopire, le istanze identitarie delle Comunidades con maggiori vocazioni indipendentiste, con particolare riguardo ai Paesi Baschi e alla Catalogna. In particolare, la situazione politico-istituzionale recente di quest’ultima ha assunto una dimensione preoccupante per la tenuta del sistema autonomistico e della stessa integrità territoriale spagnola: dall’approvazione, nel 2006, del nuovo statuto autonomico, fino al referendum secessionista dichiarato illegittimo dal Tribunale costituzionale spagnolo e la successiva proclamazione di indipendenza duramente repressa dal Governo centrale del 2017, la vicenda catalana si è posta come la manifestazione più limpida di una tendenza “centrifuga” dell’autonomia sub-nazionale, con punte di secessionismo, manifestatasi in diverse parti d’Europa negli ultimi anni. Il riproporsi di una simile situazione in Italia appare però improbabile. Nella storia dei territori regionali italiani di rado si sono manifestate tendenze indipendentistiche forti come quella catalana . La situazione della Catalogna non è, peraltro, il risultato delle continue richieste di ampliamento di competenze, bensì della tendenza al livellamento delle competenze rispetto al resto della Spagna, che ha scatenato il malcontento generalizzato nella regione. È oggi utile dividere le Comunidades autonomas spagnole in tre categorie, a seconda del grado di decentramento delle funzioni. Le prime sono le c.d. Comunità forali (Paesi Baschi e Navarra), ad alto grado di decentramento. Si differenziano dalle altre perché riscuotono e amministrano direttamente gran parte delle imposte tradizionalmente gestite dal Governo centrale, e versano allo Stato una quota relativamente bassa per finanziare le funzioni esercitate dallo Stato nel loro territorio, similmente alle Regioni a Statuto speciale presenti in Italia. Vi sono poi le Comunidades con grado intermedio di decentramento, come Andalusia, Catalogna e Galizia, e infine le altre, a basso grado di decentramento. Il regime fiscale di queste comunità è piuttosto omogeneo: esse disciplinano e amministrano alcune imposte legate alla ricchezza, ma soprattutto partecipano al gettito di alcune imposte statali; scarso è invece il ricorso all’istituzione di tributi propri, soggetti a limiti molto stringenti. Per questo la dipendenza finanziaria dallo Stato centrale è molto alta. Si tratta di una situazione molto simile a quanto succede in Italia, dove esistono limiti altrettanto stringenti all’imposizione di tributi locali; se dovesse concretizzarsi una qualche forma di autonomia differenziata, le fonti predilette per finanziarla non potrebbero che essere le revisioni al rialzo delle compartecipazioni ai tributi erariali, piuttosto che un incremento della fiscalità propria. Alla luce di questa ricostruzione del modello di autonomia differenziata spagnola, numerosi appaiono i dubbi rispetto alla possibilità, per il modello di differenziazione predisposto dal nostro art. 116.3 Cost., di svilupparsi in maniera similare. Ostano a ciò differenze di genesi storica dei due istituti, oltre che la diversa configurazione politica dell’autonomia delle Comunidades autonomas. Ciò nonostante, diversi sono gli elementi di tale modello cui si potrebbe efficacemente guardare, come spunti per la risoluzione dei numerosi nodi che si trovano sul cammino della differenziazione regionale italiana.

The considerable expansion of smartphone's market represents a great potential in mobile analytical field. Indeed, smartphones have become very similar to miniature computers combining advanced operating systems, internal memory and high-quality camera lenses. These portable, low-cost devices are very suitable for running routine tests, which are currently performed in laboratory environment by trained personnel using benchtop instrumentation. Exploiting smartphone's hardware and connectivity it would be possible to develop compact and portable biosensor suitable for point-of-care and point-of-need analysis not only for healthcare, but also for food safety, environmental monitoring and biosecurity, especially in remote and rural areas. Here, we review the most recent papers on the use of smartphones as platform for the development of analytical devices, focusing on the different techniques that have been combined with smartphone technology. This overview highlights the pros and cons of the different approaches for evaluating the real potential application of these analytical devices.

Lipase is an enzyme that finds application in biodiesel production and for detection of esters and triglycerides in biosensors. Matrix Assisted Pulsed Laser Evaporation (MAPLE), a technique derived from Pulsed Laser Deposition (PLD) for deposition of undamaged biomolecules or polymers, is characterized by the use of a frozen target obtained from a solution/suspension of the guest material (to be deposited) in a volatile matrix (solvent). The presence of the solvent avoids or at least reduces the potential damage of guest molecules by laser radiation but only the guest material reaches the substrate in an essentially solvent-free deposition. MAPLE can be used for enzymes immobilization, essential for industrial application, allowing the development of continuous processes, an easier separation of products, the reuse of the catalyst and, in some cases, enhancing enzyme properties (pH, temperature stability, etc.) and catalytic activity in non-aqueous media. Here we show that MAPLE technique can be used to deposit undamaged lipase and that the complex structure (due to droplets generated during extraction from target) of the deposited material can be controlled by changing the laser beam fluence.

Food allergies are adverse health effects that arise from specific immune responses, occurring upon exposure to given foods, even if present in traces. Egg allergy is one of the most common food allergies, mainly caused by egg white proteins, with ovalbumin being the most abundant. As allergens can also be present in foodstuff due to unintended contamination, there is a need for analytical tools that are able to rapidly detect allergens in food products at the point-of-use. Herein, we report an origami paper-based device for detecting ovalbumin in food samples, based on a competitive immunoassay with chemiluminescence detection. In this biosensor, magnetic microbeads have been employed for easy and efficient immobilization of ovalbumin on paper. Immobilized ovalbumin competes with the ovalbumin present in the sample for a limited amount of enzyme-labelled anti-ovalbumin antibody. By exploiting the origami approach, a multistep analytical procedure could be performed using reagents preloaded on paper layers, thus providing a ready-to-use immunosensing platform. The assay provided a limit of detection (LOD) of about 1 ng mL−1 for ovalbumin and, when tested on ovalbumin-spiked food matrices (chocolate chip cookies), demonstrated good assay specificity and accuracy, as compared with a commercial immunoassay kit.

The presence of hidden allergens in food products, often due to unintended contamination along the food supply chain (production, transformation, processing, and transport), has raised the urgent need for rapid and reliable analytical methods for detecting trace levels of such species in food products. Indeed, food allergens represent a high-risk factor for allergic subjects due to potentially life-threatening adverse reactions. Portable biosensors based on immunoassays have already been developed as rapid, sensitive, selective, and low-cost analytical platforms that can replace analyses with traditional bench-top instrumentation. Recently, aptamers have attracted great interest as alternative biorecognition molecules for bioassays, since they can bind a variety of targets with high specificity and selectivity, and they enable the development of assays exploiting a variety of transduction and detection technologies. In particular, aptasensors based on luminescence detection have been proposed, taking advantage of the development of ultrasensitive tracers and enhancers. This review aims to summarize and discuss recent efforts in the field of food allergen analysis using aptamer-based bioassays with luminescence detection.

The standard method for estimating the chemical oxygen demand (COD) of water bodies uses dichromate as the main oxidant, a chemical agent whose use has been restricted in the European Union since 2017. This method is hazardous, time-consuming, and burdensome to adapt to on-site measurements. As an alternative and following the current trends of sustainable and green chemistry, a method using the less toxic reagent sodium persulfate as the oxidizing agent has been developed. In this method an excess of persulfate, activated through heating in an alkaline solution, oxidizes the chemically degradable organic fraction through a 2-step radical mechanism. The remaining persulfate is evaluated by chemiluminescence (CL) using luminol and a portable charge-coupled device (CCD) camera. The method provided quantitative recoveries and a sample throughput of >60 samples h. It was validated in river water samples by comparison of COD estimations with the standard dichromate method (R = 0.973, p < 0.05) and with a UV–Vis permanganate-based method (R = 0.9998, p < 0.05), the latter being also used for drinking waters. The proposed method is a sustainable and green alternative to the previous used methods. Overall, the method using activated persulfate is suitable for use as COD quantitation/screening tool in surface waters. Considering that its main components are portable, it can be ultimately adapted for in situ analysis at the point of need.

N-substituted acridine-containing 1,2-dioxetanes have been recently proposed as thermochemiluminescence (TCL) universal labels for bioanalytical applications. The TCL properties of these compounds markedly depend on the nature of substituents of the acridine ring. In the attempt to obtain new TCL probes with improved properties and stability, the push-pull approach was adopted, in which both electron withdrawing and electron donating groups are present in the acridine moiety. The results have been useful to better understand the role of the different decorations at the acridine fluorophore in modulating the photophysical properties and the activation parameters of 1,2-dioxetane labels. Moreover, the great versatility and innovation of these molecules make them extremely attractive for bioanalytical applications, in particular as labels for immune- and gene-probe assays.

Microfluidic paper analytical devices (µPADs) represent one of the most appealing trends in the development of simple and inexpensive analytical systems for diagnostic applications at the point of care (POC). Herein, we describe a smartphone-based origami µPAD for the quantitative determination of glucose in blood samples based on the glucose oxidase-catalyzed oxidation of glucose leading to hydrogen peroxide, which is then detected by means of the luminol/hexacyanoferrate(III) chemiluminescent (CL) system. By exploiting the foldable µPAD format, a two-step analytical procedure has been implemented. First, the diluted blood sample was added, and hydrogen peroxide was accumulated, then the biosensor was folded, and a transport buffer was added to bring hydrogen peroxide in contact with CL reagents, thus promoting the CL reaction. To enable POC applicability, the reagents required for the assay were preloaded in the µPAD so that no chemicals handling was required, and a 3D-printed portable device was developed for measuring the CL emission using the smartphone’s CMOS camera. The µPAD was stable for 30-day storage at room temperature and the assay, displaying a limit of detection of 10 µmol L−1, proved able to identify both hypoglycemic and hyperglycemic blood samples in less than 20 min.

Since the introduction of paper-based analytical devices as potential diagnostic platforms a few decades ago, huge efforts have been made in this field to develop systems suitable for meeting the requirements for the point-of-care (POC) approach. Considerable progress has been achieved in the adaptation of existing analysis methods to a paper-based format, especially considering the chemiluminescent (CL)-immunoassays-based techniques. The implementation of biospecific assays with CL detection and paper-based technology represents an ideal solution for the development of portable analytical devices for on-site applications, since the peculiarities of these features create a unique combination for fitting the POC purposes. Despite this, the scientific production is not paralleled by the diffusion of such devices into everyday life. This review aims to highlight the open issues that are responsible for this discrepancy and to find the aspects that require a focused and targeted research to make these methods really applicable in routine analysis.

To accurately diagnose COVID-19 infection and its time-dependent progression, the rapid, sensitive, and noninvasive determination of immunoglobulins A specific to SARS-CoV-2 (IgA) in saliva and serum is needed to complement tests that detect immunoglobulins G and M. We have developed a dual optical/chemiluminescence format of a lateral flow immunoassay (LFIA) immunosensor for IgA in serum and saliva. A recombinant nucleocapsid antigen specifically captures SARS-CoV-2 antibodies in patient specimens. A labelled anti-human IgA reveals the bound IgA fraction. A dual colorimetric and chemiluminescence detection enables the affordable and ultrasensitive determination of IgA to SARS-CoV-2. Specifically, a simple smartphone-camera-based device measures the colour signal provided by nanogold-labelled anti-human IgA. For the ultrasensitive chemiluminescence transduction, we used a contact imaging portable device based on cooled CCD, and measured the light signal resulting from the reaction of the HRP-labelled anti-human IgA with a H2O2/luminol/enhancers substrate. A total of 25 serum and 9 saliva samples from infected and/or recovered individuals were analysed by the colorimetric LFIA, which was sensitive and reproducible enough for the semi-quantification of IgA in subjects with a strong serological response and in the early stage of COVID-19 infection. Switching to CL detection, the same immunosensor exhibited higher detection capability, revealing the presence of salivary IgA in infected individuals. For the patients included in the study (n = 4), the level of salivary IgA correlated with the time elapsed from diagnosis and with the severity of the disease. This IgA-LFIA immunosensor could be useful for noninvasively monitoring early immune responses to COVID-19 and for investigating the diagnostic/prognostic utility of salivary IgA in the context of large-scale screening to assess the efficacy of SARS-CoV-2 vaccines., Highlights • A LFIA for the point-of-care detection of IgA specific to SARS-CoV2 in serum and saliva was developed. • GNP and HRP-mediated optical/chemiluminescence signals were detected by a smartphone CMOS and a portable CCD, respectively. • The one-step colorimetric and two-step CL IgA-LFIA enabled the detection of anti-SARS-CoV-2 IgA in 15 min. • Salivary IgA were identified in four COVID-19 patients by the CL IgA-LFIA after two weeks from diagnosis.

Most of lateral flow immunoassay (LFIA) devices rely on gold nanoparticles (GNP) labeled antibodies or other biospecific proteins, to achieve reagent-less color-based detection. GNP size, GNP-protein conjugation level and its stability are crucial points for the development of precise and accurate methods. In addition, the purification of the GNP-protein conjugates from unreacted protein and GNP, is necessary for adequate analytical performance of the assay. To assist the synthesis and production process of GNP and their protein conjugates, we use for the first time a non-destructive, particle separation-multi-detection approach based on miniaturized flow field flow fractionation (HF5). A separation method was developed to baseline size-separate GNP, GNP-protein, protein and GNP including BSA used as a surface coater in less than 30 minutes. Freshly synthesized GNP were first characterized and then conjugated with two different model antibodies: a mouse immunoglobulin (IgG) and a fluorescein-labeled mouse immunoglobulin (FITC-IgG). The IgG-GNP complexes were fractionated using the HF5 apparatus, able to separate IgG-GNP from free proteins by their hydrodynamic size, allowing purification of the conjugation product. Both IgG-GNPs and GNPs were characterized according to their size by the MALS detector, and according to their Surface Plasmon Resonance and spectrum by UV-Vis detection, improving the results obtained via batch characterization. This simple non-invasive approach is very useful for the LFIA development and optimization: the use of HF5-mutidetection offers a unique tool for this purpose facilitating the industrialization of the process and the relate optimization and standardization.

High polyphenols levels are one of the main quality characters of Extra Virgin Olive Oil (EVOO), leading to high antioxidant activity and contributing to the EVOO health beneficial effects. Analytical methods for the rapid assessment of EVOO polyphenols content, possibly directly at the place of production or distribution, are therefore important for the commercial enhancement of this product. Unfortunately, most analytical methods for quantification of polyphenols in EVOO require laboratory instrumentation and preliminary sample extraction procedures. In this paper we describe a paper-based chemosensor based on the well-known Folin-Ciocalteu colorimetric assay for the rapid (assay time 15 min) quantitative detection of polyphenols in EVOO without any preliminary sample extraction. Use of n-propanol for diluting the EVOO sample allows its direct analysis on paper supports loaded with the Folin-Ciocalteu reagent. The color change of the paper supports is measured using a smartphone camera. A disposable analytical cartridge containing all necessary reagents, including calibration standards, and accessories for performing the assay using a Samsung S8 smartphone have been developed to perform on-site analysis. Measurement of total polyphenol content of EVOO samples gave results in good agreement with the conventional Folin-Ciocalteu assay, and the limit of detection was 30 μg gallic acid equivalents g−1 EVOO. The same approach can be employed to measure polyphenols in other oils of vegetal origin.

Cosmetics analysis represents a rapidly expanding field of analytical chemistry as new cosmetic formulations are increasingly in demand on the market and the ingredients required for their production are constantly evolving. Each country applies strict legislation regarding substances in the final product that must be prohibited or regulated. To verify the compliance of cosmetics with current regulations, official analytical methods are available to reveal and quantitatively determine the analytes of interest. However, since ingredients, and the lists of regulated/prohibited substances, rapidly change, dedicated analytical methods must be developed ad hoc to fulfill the new requirements. Research focuses on finding innovative techniques that allow a rapid, inexpensive, and sensitive detection of the target analytes in cosmetics. Among the different methods proposed, immunological techniques are gaining interest, as they make it possible to carry out low-cost analyses on raw materials and finished products in a relatively short time. Indeed, immunoassays are based on the specific and selective antibody/antigen reaction, and they have been extensively applied for clinical diagnostic, alimentary quality control and environmental security purposes, and even for routine analysis. Since the complexity and variability of the matrices, as well as the great variety of compounds present in cosmetics, are analogous with those from food sources, immunological methods could also be applied successfully in this field. Indeed, this would provide a valid approach for the monitoring of industrial production chains even in developing countries, which are currently the greatest producers of cosmetics and the major exporters of raw materials. This review aims to highlight the immunological techniques proposed for cosmetics analysis, focusing on the detection of prohibited/regulated compounds, bacteria and toxins, and allergenic substances, and the identification of counterfeits.

Despite the currently available pharmacotherapies, today, thirty percent of worldwide deaths are due to cardiovascular diseases (CVDs), whose primary cause is atherosclerosis, an inflammatory disorder characterized by the buildup of lipid deposits on the inside of arteries. Multiple cellular signaling pathways have been shown to be involved in the processes underlying atherosclerosis, and evidence has been accumulating for the crucial role of Notch receptors in regulating the functions of the diverse cell types involved in atherosclerosis onset and progression. Several classes of nutraceuticals have potential benefits for the prevention and treatment of atherosclerosis and CVDs, some of which could in part be due to their ability to modulate the Notch pathway. In this review, we summarize the current state of knowledge on the role of Notch in vascular health and its modulation by nutraceuticals for the prevention of atherosclerosis and/or treatment of related CVDs.

Paper-based lateral-flow immunoassays (LFIAs) have achieved considerable commercial success and their impact in diagnostics is continuously growing. LFIA results are often obtained by visualizing by the naked eye color changes in given areas, providing a qualitative information about the presence/absence of the target analyte in the sample. However, this platform has the potential to provide ultrasensitive quantitative analysis for several applications. Indeed, LFIA is based on well-established immunological techniques, which have known in the last year great advances due to the combination of highly sensitive tracers, innovative signal amplification strategies and last-generation instrumental detectors. All these available progresses can be applied also to the LFIA platform by adapting them to a portable and miniaturized format. This possibility opens countless strategies for definitively turning the LFIA technique into an ultrasensitive quantitative method. Among the different proposals for achieving this goal, the use of enzyme-based immunoassay is very well known and widespread for routine analysis and it can represent a valid approach for improving LFIA performances. Several examples have been recently reported in literature exploiting enzymes properties and features for obtaining significative advances in this field. In this review, we aim to provide a critical overview of the recent progresses in highly sensitive LFIA detection technologies, involving the exploitation of enzyme-based amplification strategies. The features and applications of the technologies, along with future developments and challenges, are also discussed.

Xanthine oxidase (XO) is an important enzyme, expressed at high levels in the vasculature in endothelial cells, that catalyzes the hydroxylation of hypoxanthine to xanthine and xanthine to uric acid. Excessive production of uric acid results in hyperuricemia linked to gout and cardiovascular diseases. Testing inhibition of XO is important for detection of potentially effective drugs or natural products that could be used to treat diseases caused by increased XO activity. In the present study, for the first time, we developed an in vitro chemiluminescent bioassay to determine XO activity in living endothelial cells and the IC50 value of oxypurinol, the active metabolite of the inhibitor drug allopurinol. Intracellular XO activity was measured in less than 20 min with a luminol/catalyst-based chemiluminescence assay able to measure XO with a limit of 0.4 μU/mL. Oxypurinol addition to 5 × 103 cells (ranging from 5.0 to 0.0 μM) caused a linear decrease in XO activity, with an IC50 of 1.0 ± 0.5 μM. The detection system developed was low-cost, rapid, reproducible, and easily miniaturizable so suitable to be used on small quantities of cells.

Hydrogen peroxide is an unavoidable by-product of cell metabolism, but when it is not properly managed by the body it can lead to several pathologies (e.g., premature aging, cardiovascular and neurodegenerative diseases, cancer). Several methods have been proposed for the measurement of intracellular H2O2 but none of them has proven to be selective. We developed a rapid all-in-one chemiluminescent bioassay for the quantification of H2O2 in living cells with a low limit of detection (0.15 μM). The method relies on an adamantylidene-1,2-dioxetane lipophilic probe containing an arylboronate moiety; upon reaction with H2O2 the arylboronate moiety is converted to the correspondent phenol and the molecule decomposes leading to an excited-state fragment that emits light. The probe has been successfully employed for quantifying intracellular H2O2 in living human endothelial, colon and keratinocyte cells exposed to different pro-oxidant stimuli (i.e., menadione, phorbol myristate acetate and lipopolysaccharide). Imaging experiments clearly localize the chemiluminescence emission inside the cells. Treatment of cells with antioxidant molecules leads to a dose-dependent decrease of intracellular H2O2 levels. As a proof of concept, the bioassay has been used to measure the antioxidant activity of extracts from Brassica juncea wastes, which contain glucosinolates, isothiocyanates and other antioxidant molecules.

The present paper describes the development of an integrated lab-on-chip, in which viral DNA amplification with real-time on-chip detection is carried out under constant temperature of 65 °C. The lab-on-chip is composed of a disposable 10-μL polydimethylsiloxane reaction chamber which is thermally and optically coupled to a glass substrate that hosts a thin-film metallic resistive heater and thin-film amorphous silicon diodes which act as temperature and radiation sensors. A loop-mediated isothermal amplification (LAMP) technique was optimized to specifically amplify parvovirus B19 DNA and coupled with Bioluminescent Assay in Real Time (BART) technology to provide real-time detection of target DNA. The experimental results demonstrate the ability of the proposed device to discriminate among different concentrations of viral DNA with an excellent agreement with standard off-chip methods.

There is a direct relationship between fructose intake and serum levels of uric acid (UA), which is the final product of purine metabolism. Recent preclinical and clinical evidence suggests that chronic hyperuricemia is an independent risk factor for hypertension, metabolic syndrome, and cardiovascular disease. It is probably also an independent risk factor for chronic kidney disease, Type 2 diabetes, and cognitive decline. These relationships have been observed for high serum UA levels (>5.5 mg/dL in women and >6 mg/dL in men), but also for normal to high serum UA levels (5-6 mg/dL). In this regard, blood UA levels are much higher in industrialized countries than in the rest of the world. Xanthine-oxidase inhibitors can reduce UA and seem to minimize its negative effects on vascular health. Other dietary and pathophysiological factors are also related to UA production. However, the role of fructose-derived UA in the pathogenesis of cardiometabolic disorders has not yet been fully clarified. Here, we critically review recent research on the biochemistry of UA production, the relationship between fructose intake and UA production, and how this relationship is linked to cardiometabolic disorders.

Human parvovirus B19 (B19V) is a single-stranded DNA virus. The genome encodes a multifunctional non-structural protein (NS), two capsid proteins (VP1, VP2) and other small non-structural proteins (7.5 kDa, 9 kDa, 11 kDa). Within sensitive cells, B19V can achieve a productive replicative cycle or, on the contrary, establish persistence; differences in its expression profile have been yet investigated following in vitro infections by methodologies enabling information on the entire infected cell population. Conversely, the present study reports quantitative data on the production of B19V analytes (DNA, RNAs, and proteins) at single cell-level, underlining cell-to-cell differences through the viral specific macromolecular synthesis process. Microscope imaging assays (in situ hybridization and immunocytochemical assays), exploiting chemiluminescence as principle detection and targeting viral nucleic acids and antigens, have been performed on a permissive cell line following in vitro infection. Chemiluminescence, involving the emission of photons deriving from a chemical reaction, provided the localization and quantitative detection of analytes down to a few molecules within infected cells. In our experimental conditions, B19V transcriptional activity, leading to the production of NS and VP RNAs, has been detected early in the viral cycle (from 12 h post-infection, hpi) and before genome replication, starting at 24 hpi. The analysis of VP RNAs and related proteins suggested an inhibitory effect on capsid protein translation, as a post-transcriptional regulation events. Indeed, high levels of VP transcripts have been detected at early stages of infection while the proteins accumulated within cells only at 48–72 hpi.

The latest advances in molecular biology have made available several biotechnological tools that take advantage of the high detectability and quantum efficiency of bioluminescence (BL), with an ever-increasing number of novel applications in environmental, pharmaceutical, food, and forensic fields. Indeed, BL proteins are being used to develop ultrasensitive binding assays and cell-based assays, thanks to their high detectability and to the availability of highly sensitive BL instruments. The appealing aspect of molecular biology tools relying on BL reactions is their general applicability in both in vitro assays, such as cell cultures or purified proteins, and in vivo settings, such as in whole-animal BL imaging. The aim of this chapter is to provide the reader with an overview of state-of-the-art bioluminescent tools based on luciferase genes, highlighting molecular biology strategies that have been applied so far, together with some selected examples.