Surface plasmon resonance based platforms for clinical and environmental biosensing applications

My PhD Thesis work, developed in Veneto Nanotech Laboratories (Nanofab in Marghera, LaNN in Padova and ECSIN in Rovigo), was aimed at the exploitation of the Surface Plasmon Resonance (SPR) phenomenon for the set-up of biosensing platforms for clinical and environmental applications. In particular, two types of SPR-based platforms were set-up and optimised: the first one was an oligonucleotide-based platform for the detection of Cystic Fibrosis (CF) causing mutations while the second one was an antibody-based platform for the detection of Legionella pneumophila whole cells. Both sensors are based on the same detection strategy, exploiting the advantages of using a highly sensitive Grating Coupled - Surface Plasmon Resonance (GC-SPR) enhanced spectroscopy method, designed using a conical illumination configuration for label-free molecular detection. Concerning DNA platform for Cystic Fibrosis, a strategy for the detection of some of the most frequent mutations responsible for CF among the Italian population is investigated. For the detection of the CF mutations, gold sinusoidal gratings are used as sensing surfaces, and the specific biodetection is achieved through the usage of allele specific oligonucleotide (ASO) DNA hairpin probes, designed for single nucleotide discrimination. Substrates were used to test unlabeled PCR amplified homozygous wild type (wt) and heterozygous samples (wt/mut) - deriving from clinical samples - for the screened mutations. Hybridisation conditions were optimised to obtain the maximum discrimination ratio (DR) between the homozygous wild type and the heterozygous samples. SPR signals obtained from hybridising wild type and heterozygous samples showed 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 parallel discrimination of several alleles also scalable for a high throughput screening setting. Concerning antibody platform for Legionella pneumophila bacteria detection, a strategy for the exploitation of the SPR phenomenon to develop a fully automated platform for fast optical detection of Legionella pneumophila pathogens was investigated. The legal limit of L. pneumophila in a high-risk hospital environment in Italy is 102 CFU/L, and the gold standard for its identification is a time consuming microbiological culture method, that requires up to 7 days. Starting from these considerations a sensitive GC-SPR system was applied to the detection of L. pneumophila to test the detection limit of the developed sensing device in term of detectable bacterium CFU. The detection was accurately set up and precisely optimised firstly through the usage of flat gold functionalised slides to be then translated to sinusoidal gold gratings for label-free GC-SPR detection using ellipsometer, in order to ensure a reproducible and precise identification of bacteria. Through azimuthally-controlled GC-SPR, 10 CFU were detected, while in the case of fluorescence analysis results, a negative readout is obtained if incubating less than 104 CFU. Successful results were obtained when incubating environmental derived samples. This detection platform could be implemented as a prototype in which water and air samples will be sequentially concentrated, injected into a microfluidic system, and delivered to the SPR sensor for analysis. The peculiar Grating Coupled - Surface Plasmon Resonance method applied for this work has therefore revealed to be an accurate and highly sensitive strategy – with multiplexing possibility - for the sensing and detecting of different kind of biomolecules, from DNA fragments to whole bacteria cell

Il ruolo del tratto di numero nella comprensione delle frasi relative oggetto in pazienti afasici italiani

This study investigates the role of number features in the agrammatic comprehension of object relative clauses with preverbal subjects. A sentence-picture matching task was used to assess the performance of four Italian aphasic patients. Results show that the mismatch condition of number features (with the subject singular/plural and the object plural/singular) is computationally the most complex one. In this condition, aphasic patients have greatest difficulties in establishing the right set of syntactic relations among the constituents of the sentence

Il ruolo del tratto di numero nella comprensione delle frasi relative oggetto in pazienti afasici italiani

This study investigates the role of number features in the agrammatic comprehension of object relative clauses with preverbal subjects. A sentence-picture matching task was used to assess the performance of four Italian aphasic patients. Results show that the mismatch condition of number features (with the subject singular/plural and the object plural/singular) is computationally the most complex one. In this condition, aphasic patients have greatest difficulties in establishing the right set of syntactic relations among the constituents of the sentence

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

Sexual Functioning and Opioid Maintenance Treatment in Women. Results From a Large Multicentre Study

Opioid maintenance treatment (OMT) is the most widespread therapy for both females and males opioid addicts. While many studies have evaluated the OMT impact on men’s sexuality, the data collected about the change in women’s sexual functioning is still limited despite the fact that it is now well-known that opioids - both endogenous and exogenous - affect the endocrine system and play an important role in sexual functioning. The present study aims to determine how OMT with buprenorphine (BUP) or methadone (MTD) affects sexual health in women; examining also any possible emerging correlation between sexual dysfunction (SD), type of opioid and patients’ mental health. This multi-center study case recruited 258 female volunteers attending Italian public Addiction Outpatients Centers that were stabilized with OMT for at least 3 months. SD was assessed with the Arizona Sexual Experience Scale. The twelve-item General Health Questionnaire was used to assess participants’ mental health conditions. The results show that 56.6% of women receiving OMT for at least 3 months presented SD without significant differences between MTD e BUP groups. The majority of the subjects with SD have a poorer quality of intimate relationships and worse mental health than the average. To the best of our knowledge, the present study is the largest report on the presence of SDs in women as a side effects of MTD and BUP used in OMT. Since SDs cause difficulties in intimate relationships, lower patients’ quality of life and interfere with OMT beneficial outcomes, we recommend that women undertaking an opioid therapy have routine screening for SD and we highlight the importance to better examine opioid-endocrine interactions in future studies in order to provide alternative potential treatments such as the choice of opioid, opioid dose reduction and hormone supplementation

Surface plasmon resonance based platforms for clinical and environmental biosensing applications

My PhD Thesis work, developed in Veneto Nanotech Laboratories (Nanofab in Marghera, LaNN in Padova and ECSIN in Rovigo), was aimed at the exploitation of the Surface Plasmon Resonance (SPR) phenomenon for the set-up of biosensing platforms for clinical and environmental applications. In particular, two types of SPR-based platforms were set-up and optimised: the first one was an oligonucleotide-based platform for the detection of Cystic Fibrosis (CF) causing mutations while the second one was an antibody-based platform for the detection of Legionella pneumophila whole cells. Both sensors are based on the same detection strategy, exploiting the advantages of using a highly sensitive Grating Coupled - Surface Plasmon Resonance (GC-SPR) enhanced spectroscopy method, designed using a conical illumination configuration for label-free molecular detection. Concerning DNA platform for Cystic Fibrosis, a strategy for the detection of some of the most frequent mutations responsible for CF among the Italian population is investigated. For the detection of the CF mutations, gold sinusoidal gratings are used as sensing surfaces, and the specific biodetection is achieved through the usage of allele specific oligonucleotide (ASO) DNA hairpin probes, designed for single nucleotide discrimination. Substrates were used to test unlabeled PCR amplified homozygous wild type (wt) and heterozygous samples (wt/mut) - deriving from clinical samples - for the screened mutations. Hybridisation conditions were optimised to obtain the maximum discrimination ratio (DR) between the homozygous wild type and the heterozygous samples. SPR signals obtained from hybridising wild type and heterozygous samples showed 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 parallel discrimination of several alleles also scalable for a high throughput screening setting. Concerning antibody platform for Legionella pneumophila bacteria detection, a strategy for the exploitation of the SPR phenomenon to develop a fully automated platform for fast optical detection of Legionella pneumophila pathogens was investigated. The legal limit of L. pneumophila in a high-risk hospital environment in Italy is 102 CFU/L, and the gold standard for its identification is a time consuming microbiological culture method, that requires up to 7 days. Starting from these considerations a sensitive GC-SPR system was applied to the detection of L. pneumophila to test the detection limit of the developed sensing device in term of detectable bacterium CFU. The detection was accurately set up and precisely optimised firstly through the usage of flat gold functionalised slides to be then translated to sinusoidal gold gratings for label-free GC-SPR detection using ellipsometer, in order to ensure a reproducible and precise identification of bacteria. Through azimuthally-controlled GC-SPR, 10 CFU were detected, while in the case of fluorescence analysis results, a negative readout is obtained if incubating less than 104 CFU. Successful results were obtained when incubating environmental derived samples. This detection platform could be implemented as a prototype in which water and air samples will be sequentially concentrated, injected into a microfluidic system, and delivered to the SPR sensor for analysis. The peculiar Grating Coupled - Surface Plasmon Resonance method applied for this work has therefore revealed to be an accurate and highly sensitive strategy – with multiplexing possibility - for the sensing and detecting of different kind of biomolecules, from DNA fragments to whole bacteria cell.Il mio lavoro di Tesi di Dottorato, sviluppato presso i laboratori Veneto Nanotech (Nanofab a Marghera, LaNN a Padova ed ECSIN a Rovigo), ha avuto come obiettivo l’utilizzo della tecnologia di risonanza plasmonica di superficie (SPR – Surface Plasmon Resonance) per lo sviluppo di piattaforme biosensoristiche per applicazioni clinica ed ambientali. In particolare, durante il lavoro di Dottorato sono state messe a punto due piattaforme SPR: la prima piattaforma utilizza sonde oligonucleotidiche a DNA per l'individuazione di mutazioni causanti fibrosi cistica (CF) mentre la seconda utilizza anticorpi per il rilevamento di cellule di Legionella pneumophila. Entrambi i sensori sono basati sulla stessa strategia di rilevamento, ovvero l’utilizzo di una metodologia Grating Coupled – Surface Plasmon Resonance (GC-SPR) progettata utilizzando una configurazione conica di illuminazione ad azimut rotato per la rilevazione diretta – senza passaggi di marcatura, label-free – dell’analita in esame. Per quanto riguarda la piattaforma a DNA per la fibrosi cistica, si è sviluppata una strategia per l'individuazione di alcune delle mutazioni più frequenti responsabili CF tra la popolazione italiana. Per la rilevazione di tali mutazioni le superfici di analisi utilizzate sono grigliati sinusoidali, e la rilevazione specifica delle sequenze di interesse si ottiene attraverso l'utilizzo di oligonucleotidi allele-specifici (ASO – allele specific oligonucleotide) con struttura ad hairpin, disegnati per la discriminazione di un singolo nucleotide. I substrati plasmonici sono stati utilizzati per testare campioni wild-type ed eterozigoti (wt/mut) per le mutazioni in esame, amplificati tramite PCR a partire da campioni clinici. Le condizioni di ibridazione sono state ottimizzate per ottenere il rapporto di discriminazione (DR – discrimination ratio) massimo tra campioni wild-type ed eterozigoti. I segnali SPR ottenuti ibridando campioni wild-type e campioni eterozigoti hanno mostrato DR in grado di identificare univocamente i genotipi corretti, come confermato da esperimenti di fluorescenza in microarray eseguiti in parallelo. Inoltre la genotipizzazione ottenuta tramite SPR non è stata inficiata in campioni contenenti DNA interferente, consentendo quindi di utilizzare la piattaforma per la discriminazione in parallelo dei diversi alleli, e la possibilità futura di scalare il sistema con un approccio di high throughput screening. Per quanto riguarda la piattaforma ad anticorpi per la rilevazione di Legionella pneumophila, la medesima strategia basata su GC-SPR è stata messa a punto per ottenere una rilevazione rapida e sensibile di tale patogeno. Il limite legale di L. pneumophila in ambienti ospedalieri ad alto rischio in Italia è di 102 UFC/L (unità formanti colonia) e la metodologia di riferimento per la sua identificazione è una tecnica di coltura microbiologica che richiede tempi di attesa fino a 7 giorni. Partendo da tali considerazioni un sistema GC-SPR altamente sensibile è stato sviluppato ed applicato per la rivelazione di L. pneumophila: la rivelazione è stata accuratamente impostata ed ottimizzata con un ceppo standard del battere, prima attraverso l'utilizzo di superfici d’oro non nanostrutturate (flat) opportunamente funzionalizzate ed analizzate tramite fluorescenza, e successivamente attraverso reticoli sinusoidali (grating) d’oro analizzati tramite elissometria GC-SPR. Attraverso la metodologia GC-SPR ad azimut rotato è stato possibile rilevare fino a 10 UFC, mentre con l’analisi in fluorescenza non è stato possibile identificare quantitativi di battere inferiori a 104 UFC. Risultati positivi sono stati ottenuti anche incubando campioni di L. pneumophila isolati direttamente dall’ambiente ospedaliero. Questa piattaforma di rilevazione potrà essere implementata come prototipo in cui campioni di acqua e aria potranno venir sequenzialmente concentrati, iniettati in un sistema di microfluidica, ed incubati sulla superficie del sensore SPR per l'analisi, obiettivi questi del progetto POSEIDON (Horizon2020) attualmente in corso. La particolare metodologia GC-SPR ad azimut rotato applicata in questo lavoro di Tesi si è dimostrata essere una strategia accurata e altamente sensibile - con possibilità di multiplexing - per la rilevazione di diversi tipi di biomolecole, a partire da frammenti di DNA fino ad intere cellule batteriche

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