Electrical modeling and experimental studies of sensing systems for biological and biomedical applications

The core of my research activity during the Ph.D. period has been the detection of biological interactions phenomena using electrical transducers, i.e. biosensors. I have studied different aspects of the electrical transduction process, in order to optimize the detection by improving biosensors selectivity and frequency response. I have started my Thesis work by studying the fundamental theories of electrochemical interfaces between biosensor electrodes and liquid samples, e.g., Helmholtz double layer and Warburg frequency dispersion, in order to understand the electron transfer mechanisms in wet environment. The equivalent electrical modeling plays an important role in interpreting experimental electrochemical data. The net flow of electrical charges across an electrochemical interface is the result of several contributions: each of these processes can be modeled using a lumped parameters equivalent electrical circuit with a peculiar electrical impedance. By connecting these equivalent circuits in suitable networks, the frequency response of a complex electrochemical cell can be predicted. During my Ph.D. period I have further developed a simulation system that I started to implement during my Laurea Thesis: with this simulation system the electrical response of an electrode/electrolyte system is predicted using a pseudo-distributed method, i.e. with an interconnection of basic equivalent electrical circuits derived from the geometrical mesh of the simulated system. Each basic equivalent electrical circuit can have different electrical elements and custom topologies. The value of each electrical element, both passive (e.g., resistors and capacitors) and active (e.g., current generators), is determined through mathematical functions elaborated from experimental electrochemical measurements. This mesh-based approach permits to retain the geometrical information of cell and electrodes layout, that is particularly useful when simulating in-flow channel electrodes and microfluidic biosensors. Simulations and equivalent modeling techniques are also useful when designing biosensors layout. During my Ph.D. activity I used commercial biosensors and custom devices: in both cases, the interpretation of experimental data obtained from biosensors with different layouts have been performed by using equivalent electrical circuits modeling techniques, in order to assess the electromagnetic field distribution between electrodes and the influence of parasitics elements, like cross-talk capacitances and tracks intrinsic impedances. During my Ph.D. period I have contributed to develop, in collaboration with Next Step Engineering (University of Padova spin off), an innovative industrial process that allows to create microelectronic/microfluidic hybrid devices within a single, well-established, production line. With this process I have manufactured all the custom devices I used for my experimental activity. Moreover, this industrial process is the object of an Italian patent that is now pending: I have asked for a six months procrastination of my final discussion in order to write and submit the Italian patent for this innovation as one of the inventors. The feasibility of custom biosensors to biomedical and biological applications have been tested using impedance spectroscopy, voltammetric and amperometric measurements: electrical calibration curves have been obtained with standard electrolytes, i.e. solutions with knows electrical conductivity or redox potential, and relevant interferents species have been identified by measuring more complex solutions with various electrolytes and diluted substances. The biological application of custom biosensors have been developed in collaboration with other Departments of the University of Padova and Research Centers: • a genosensor for monitoring DNA hybridization has been developed in collaboration with San Bortolo Hospital (Vicenza, Italy); • an enzyme-modified biosensor for the detection of lactic acid has been studied with the Department of Biomedical Sciences (University of Padova, Italy) and Sapienza University (Roma, Italy); • a biosensor for both monitoring cells growth and studying electropermeabilization has been developed in collaboration with the Department of Biomedical Sciences (University of Padova, Italy). Recently, during the last part of my Ph.D., I studied another application of the electrical transduction of biological signals. In collaboration with Wetware Concepts (University of Padova spin off) and Dr. Quarta from Stanford University, I have contributed to develop a prototype of sensorized glove for the electrical transduction of force signals exerted by human hands. This prototype allows to monitor the functional rehabilitation process of patients with both mild and severe impairments, enabling the quantitative assessment of the functional rehabilitation protocol effectiveness. I have also contributed to further develop the prototype, in collaboration with I.R.C.S.S. San Camillo hospital (Venezia, Italy) and San Bortolo hospital (Vicenza, Italy), into a biofeedback system able to both measure the force exerted by patients hands and to correlate these data with those gathered from other medical equipments, e.g., electroencephalographs and electromyographs

inkjet sensors produced by consumer printers with smartphone impedance readout

Abstract Inkjet printing technology is showing a disruptive potential for low-cost optical and electrochemical biosensors fabrication. This technology is becoming affordable for every laboratory, potentially allowing every research group to implement a biosensors fabrication platform with consumer inkjet printers, commercially available inks and smartphones for readout. In the present work we developed an example of such platform testing several inks, printers, and substrates. We defined and optimized the protocols assessing the printing limits and the fabricated biosensors electrochemical properties in standard solutions. Our platform has a total cost of less than 450 Euro and a single sensor fabrication cost of 0.026 Euro. Finally, we tested the sensitivity of smartphone-performed impedance measurements with printed biosensors surface coverage by Self Assembling Monolayers (SAM), validating them with standard instruments

sarcoidosis like disease mimicking metastases during adjuvant ipilimumab therapy in advanced melanoma patient ct scan and mri help in managing difficult clinical decision

The onset of an autoimmune, sarcoidosis-like reaction during or after treatment with immunomodulatory drugs as Ipilimumab is an atypical but renowned eventuality. Awareness of this scenario and its radiological features helps the Radiologist to avoid misdiagnosis of disease progression. In this case report, we present a patient operated for advanced cutaneous melanoma of the left forearm who developed hilar adenopathies with lung and splenic nodules during therapy with Ipilimumab in adjuvant setting. These findings were at first referred to as disease recurrences. Based on discrepancies between imaging, clinic and blood test findings we decided to put the patient on strict follow-up which showed a spontaneous complete regression on the visceral lesions few months after Ipilimumab withheld

sensor augmented pump and down syndrome a new tool in tricky patients

W e read with great interest the paper by Piccini and cols. (1) published in the July issue of this Journal. Some years ago, we published the first report ever (to the best of our knowledge) of successful treatment of a girl with Down syndrome, Hashimoto's thyroiditis and celiac disease with continuous subcutaneous insulin infusion (2). Since then, her glycemic control was kept constant and, most of the time, in the target range (HbA1c in 2009: 7.75 ± 0.21%; HbA1c in 2010: 7.35 ± 0.19%; HbA1c in 2011: 7.42 ± 0.30%). At the end of 2011, sensor-augmented pump was initiated (Animas® VibeTM, West Chester, PA, USA) because of both a quite high glycemic variability and the parents' request, and her HbA1c kept improving (HbA1c in 2012: 7.30 ± 0.20%; HbA1c in 2013: 7.10 ± 0.28%). CSII has been recognized as effective and safe in pediatric (3) and in adult patients (4), not only in the short run, but even after many years (5). In patients with Down syndrome and type 1 diabetes, glycemic control may sometimes be particularly tricky (6,7). In our patient, as well as in the one of Piccini and cols. (1), CSII was a safe and effective way to manage diabetes. For a successful CSII therapy in a patient with Down syndrome, whose mental function may be impaired, the collaboration of a highly motivated and compliant family is essential, as well as a skilled multidisciplinary diabetes team (8). Given all of this, pump increased the patient's and family's flexibility, as we had previously reported (2). The significant improvement in the glycemic control observed, and the high level of acceptance of CSII therapy observed in both our case and in that of Piccini and cols. is worth the effort of the patient's family and of the diabetes team in ensuring that the patient has a flexible life. Perhaps CSII therapy might be taken into account when considering insulin therapy in patients with Down's syndrome

Dual-regulated lentiviral vector for gene therapy of X-linked chronic granulomatosis

Regulated transgene expression may improve the safety and efficacy of hematopoietic stem cell (HSC) gene therapy. Clinical trials for X-linked chronic granulomatous disease (X-CGD) employing gammaretroviral vectors were limited by insertional oncogenesis or lack of persistent engraftment. Our novel strategy, based on regulated lentiviral vectors (LV), targets gp91(phox) expression to the differentiated myeloid compartment while sparing HSC, to reduce the risk of genotoxicity and potential perturbation of reactive oxygen species levels. Targeting was obtained by a myeloid-specific promoter (MSP) and posttranscriptional, microRNA-mediated regulation. We optimized both components in human bone marrow (BM) HSC and their differentiated progeny in vitro and in a xenotransplantation model, and generated therapeutic gp91(phox) expressing LVs for CGD gene therapy. All vectors restored gp91(phox) expression and function in human X-CGD myeloid cell lines, primary monocytes, and differentiated myeloid cells. While unregulated LVs ectopically expressed gp91(phox) in CD34(+) cells, transcriptionally and posttranscriptionally regulated LVs substantially reduced this off-target expression. X-CGD mice transplanted with transduced HSC restored gp91(phox) expression, and MSP-driven vectors maintained regulation during BM development. Combining transcriptional (SP146.gp91-driven) and posttranscriptional (miR-126-restricted) targeting, we achieved high levels of myeloid-specific transgene expression, entirely sparing the CD34(+) HSC compartment. This dual-targeted LV construct represents a promising candidate for further clinical development

Electrical modeling and experimental studies of sensing systems for biological and biomedical applications

The core of my research activity during the Ph.D. period has been the detection of biological interactions phenomena using electrical transducers, i.e. biosensors. I have studied different aspects of the electrical transduction process, in order to optimize the detection by improving biosensors selectivity and frequency response. I have started my Thesis work by studying the fundamental theories of electrochemical interfaces between biosensor electrodes and liquid samples, e.g., Helmholtz double layer and Warburg frequency dispersion, in order to understand the electron transfer mechanisms in wet environment. The equivalent electrical modeling plays an important role in interpreting experimental electrochemical data. The net flow of electrical charges across an electrochemical interface is the result of several contributions: each of these processes can be modeled using a lumped parameters equivalent electrical circuit with a peculiar electrical impedance. By connecting these equivalent circuits in suitable networks, the frequency response of a complex electrochemical cell can be predicted. During my Ph.D. period I have further developed a simulation system that I started to implement during my Laurea Thesis: with this simulation system the electrical response of an electrode/electrolyte system is predicted using a pseudo-distributed method, i.e. with an interconnection of basic equivalent electrical circuits derived from the geometrical mesh of the simulated system. Each basic equivalent electrical circuit can have different electrical elements and custom topologies. The value of each electrical element, both passive (e.g., resistors and capacitors) and active (e.g., current generators), is determined through mathematical functions elaborated from experimental electrochemical measurements. This mesh-based approach permits to retain the geometrical information of cell and electrodes layout, that is particularly useful when simulating in-flow channel electrodes and microfluidic biosensors. Simulations and equivalent modeling techniques are also useful when designing biosensors layout. During my Ph.D. activity I used commercial biosensors and custom devices: in both cases, the interpretation of experimental data obtained from biosensors with different layouts have been performed by using equivalent electrical circuits modeling techniques, in order to assess the electromagnetic field distribution between electrodes and the influence of parasitics elements, like cross-talk capacitances and tracks intrinsic impedances. During my Ph.D. period I have contributed to develop, in collaboration with Next Step Engineering (University of Padova spin off), an innovative industrial process that allows to create microelectronic/microfluidic hybrid devices within a single, well-established, production line. With this process I have manufactured all the custom devices I used for my experimental activity. Moreover, this industrial process is the object of an Italian patent that is now pending: I have asked for a six months procrastination of my final discussion in order to write and submit the Italian patent for this innovation as one of the inventors. The feasibility of custom biosensors to biomedical and biological applications have been tested using impedance spectroscopy, voltammetric and amperometric measurements: electrical calibration curves have been obtained with standard electrolytes, i.e. solutions with knows electrical conductivity or redox potential, and relevant interferents species have been identified by measuring more complex solutions with various electrolytes and diluted substances. The biological application of custom biosensors have been developed in collaboration with other Departments of the University of Padova and Research Centers: • a genosensor for monitoring DNA hybridization has been developed in collaboration with San Bortolo Hospital (Vicenza, Italy); • an enzyme-modified biosensor for the detection of lactic acid has been studied with the Department of Biomedical Sciences (University of Padova, Italy) and Sapienza University (Roma, Italy); • a biosensor for both monitoring cells growth and studying electropermeabilization has been developed in collaboration with the Department of Biomedical Sciences (University of Padova, Italy). Recently, during the last part of my Ph.D., I studied another application of the electrical transduction of biological signals. In collaboration with Wetware Concepts (University of Padova spin off) and Dr. Quarta from Stanford University, I have contributed to develop a prototype of sensorized glove for the electrical transduction of force signals exerted by human hands. This prototype allows to monitor the functional rehabilitation process of patients with both mild and severe impairments, enabling the quantitative assessment of the functional rehabilitation protocol effectiveness. I have also contributed to further develop the prototype, in collaboration with I.R.C.S.S. San Camillo hospital (Venezia, Italy) and San Bortolo hospital (Vicenza, Italy), into a biofeedback system able to both measure the force exerted by patients hands and to correlate these data with those gathered from other medical equipments, e.g., electroencephalographs and electromyographs.L’argomento principale dell’attività di ricerca che ho svolto durante il mio periodo di Dottorando in Scienza e Tecnologia dell’Informazione è stato la rilevazione di fenomeni di interazione biologica tramite trasduttori elettrici, ovverosia lo studio di dispositivi elettronici per applicazioni biosensoristiche. Ho studiato diversi aspetti del processo di trasduzione elettrica allo scopo di ottimizzare la rilevazione delle interazioni biologiche e migliorare le caratteristiche dei biosensori, quali ad esempio la selettività e la risposta in frequenza. Ho iniziato il mio lavoro di Tesi studiando le classiche teorie delle interfacce elettrochimiche fra elettrodi metallici e campioni liquidi, ad esempio la teoria del doppio strato di Helmholtz e la dispersione in frequenza di Warburg, per approfondire i meccanismi di trasferimento di carica elettrica in ambienti eterogenei. La modellizzazione elettrica a parametri equivalenti dei dati elettrochimici sperimentali è fondamentale per giungere a una loro interpretazione attendibile: il flusso di cariche elettriche attraverso un’interfaccia elettrochimica è il risultato di numerosi contributi, ciascuno dei quali può essere modellizzato utilizzando circuiti elettrici equivalenti con specifiche impedenze. Collegando questi circuiti equivalenti secondo appropriate topologie è possibile simulare la risposta in frequenza di complesse celle elettrochimiche. Durante il mio periodo di Tesi ho continuato a sviluppare il sistema di simulazione che avevo iniziato a implementare durante il mio periodo di Tesi di Laurea Specialistica: con questo sistema è possibile simulare la risposta elettrica di un sistema elettrodo/elettrolita utilizzando un metodo a elementi pseudo-distribuiti, cioè un’interconnessione finita di circuiti elettrici equivalenti locali la cui topologia viene determinata a partire dalla mesh della geometria della cella elettrochimica. Ciascun circuito elettrico locale può essere formato da diversi elementi elettrici, sia attivi che passivi, con una propria topologia. Il valore di ciascun elemento elettrico locale è determinato con funzioni matematiche ricavate da misure elettrochimiche sperimentali. Questo approccio di simulazione basato sulla mesh consente di preservare le informazioni geometriche legate alla forma degli elettrodi e della cella elettrochimica, che risultano particolarmente importanti quando è necessario simulare elettrodi in flusso oppure biosensori con componenti microfluidiche. Le simulazioni e le tecniche di modellizzazione elettrica risultano importanti anche qualora sia necessario progettare il layout di un biosensore. Durante la mia attività di Dottorato ho utilizzato sia biosensori disponibili commercialmente che dispositivi custom: in entrambi i casi, l’interpretazione dei dati sperimentali ottenuti da biosensori con layout differenti è stata eseguite con tecniche di modellizzazione elettrica equivalente, al fine di valutare la distribuzione del campo elettromagnetico fra gli elettrodi e l’influenza degli elementi parassiti del sistema di misura e del dispositivo, quali ad esempio le capacità di cross-talk e le impedenze elettriche dei contatti elettrici. Durante il mio periodo di Dottorato ho contribuito a sviluppare, in collaborazione con lo spin off dell’Università di Padova Next Step Engineering, un innovativo processo di produzione industriale che consente di creare dispositivi ibridi microelettronici/microfluidici idonei ad applicazioni biologiche all’interno di una singola linea produttiva automatizzata. Con questo processo ho prodotto i dispositivi custom che ho utilizzato per la mia attività sperimentale. Il processo di produzione è oggetto di un brevetto italiano attualmente in fase di deposito, di cui sono uno degli inventori, che ho scritto e sottomesso durante i sei mesi di proroga della discussione finale della Tesi che ho richiesto. La possibilità di utilizzare i biosensori elettrochimici custom per applicazioni biomediche e biologiche è stata verificata utilizzando misurazioni di spettroscopia di impedenza elettrochimica, tecniche voltammetriche e amperometriche: le curve di calibrazione dei vari dispositivi sono state ottenute utilizzando elettroliti standard per le varie applicazioni, cioè soluzioni con conducibilità elettrica e potenziali ossido-riduttivi noti, e l’influenza di interferenti in soluzione è stata valutata misurando matrici più complesse composte da vari elettroliti con sostanze disciolte. Le applicazioni biologiche dei biosensori custom sono state sviluppate in collaborazione con altri Dipartimenti dell’Università degli Studi di Padova e con centri di ricerca: • un biosensore per il monitoraggio dell’ibridazione di sequenze di DNA è stato sviluppato in collaborazione con l’Ospedale San Bortolo (Vicenza, Italia); • un biosensore enzimatico per la rilevazione di acido lattico è stato studiato in collaborazione con il Dipartimento di Scienze Biomediche (Università di Padova, Italia) e con il Dipartimento di Scienze Anatomiche e Istologiche (Università Sapienza, Roma, Italia); • un biosensore per monitorare la crescita cellulare e studiare il fenomeno di elettropermeabilizzazione della membrana cellulare è stato sviluppato in collaborazione con il Dipartimento di Scienze Biomediche (Università di Padova, Italia). Nell’ultimo periodo della mia attività di Dottorato ho studiato un’altra applicazione della trasduzione elettrica di segnali biometrici. In collaborazione con lo spin off dell’Università di Padova Wetware Concepts e con il Dr. Marco Quarta dell’Università di Stanford, ho contribuito a sviluppare un prototipo di guanto sensorizzato per la trasduzione elettrica della forza esercitata da mani umane. Questo prototipo permette di monitorare il processo di riabilitazione funzionale di pazienti con deficit sia lievi che severi, permettendo la valutazione quantitativa dell’efficacia dei protocolli di riabilitazione. Inoltre, ho contribuito a sviluppare ulteriormente il prototipo, in collaborazione con l’I.R.C.C.S. Ospedale San Camillo (Venezia, Italia) e con l’Ospedale San Bortolo (Vicenza, Italia), in un sistema basato su biofeedback in grado di misurare la forza esercitata da un paziente e di correlarla con dati provenienti da altri strumenti medici, quali elettroencefalografi ed elettromiografi

Development of an electrode/electrolyte interface model based on pseudo-distributed elements combining COMSOL, MATLAB and HSPICE

This work describes a Combined Simulation System (CSS) able to predict the electrical behaviour of a metal electrode in contact with an electrolyte. The system is based on a mixture of Lumped Parameters Equivalent Electrical Circuits (LPEEC) and Pseudo-Distributed Elements (PDE): a PDE is a network of basic equivalent electrical circuits, e.g. resistor and capacitor parallels, in which a geometrical information can be retained. Electrochemical Impedance Spectroscopy (EIS) measurements have been performed on a standard solution of known electrical conductivity with Micro-Electrodes Array (MEA) devices in order to investigate set-up parasitic elements and electrochemical interfaces parameters. CSS performance has been compared to usual LPEEC fit approach in terms of both results accuracy and solving time

Characterization of Grating Coupled Surface Plasmon Polaritons Using Diffracted Rays Transmittance

A method to sense the excitation of surface plasmon polariton (SPP) on metallic grating device using the transmitted signal will be presented. The grating transmittance signal will be fully characterized varying the light incident angle and azimuthal grating orientation by means of the SPP vector model and rigorous coupled-wave analysis simulation. Simulation results will be compared with experimental measurements obtained with a 635 nm wavelength laser in the transverse magnetic polarization mode. The laser will light grating devices in contact with either air or water through a customized microfluidic chamber. A characterization of the diffracted rays will show the relationship between the grating coupling configuration and the Kretschmann one. In fact, the diffracted ray affected by SPP resonance is transmitted with an output angle which is the same incident angle that should be used to excite SPP in Kretschmann configuration. Lastly, the grating parameters (amplitude and metal thickness) impact on transmittance signal will be analyzed with respect to the order zero reflectance signal

Coadsorption optimization of DNA in binary self-assembled monolayer on gold electrode for electrochemical detection of oligonucleotide sequences

Optimization of the probe adsorption has a major key in the preparation of electrochemical sensors for the detection of oligonucleotide sequences hybridization. The role of a mixed monolayer of ssDNA sequences and MCH coadsorbed on a gold electrode surface was studied in this work. The working electrode was modified by chemisorption using a solution of thiol-tethered 33-mer DNA probe and mercaptohexanol (MCH), in a concentration range from 2 nM to 20 lM. The probe surface density was monitored by means of electrochemical impedance spectroscopy (EIS), differential pulse voltammetry (DPV) and chronocoulometry. From EIS measurements, the charge transfer resistance was obtained as a function of the MCH concentration in the immobilization solution. The time dependence of mixed SAM adsorption was also investigated. The SAM adsorption was characterized regarding the electrode surface coverage with DPV and EIS measurements. Moreover, the probe surface density was investigated with chronocoulometry in Ru\uf0NH3 e3\ufe 6 solution. Sensor behavior and sensitivity showed significant differences as a function of ssDNA/MCH concentration ratio as hybridization detection efficiency decreases while increasing the MCH concentration. The effect of different probe density in the hybridization detection efficiency was determined. Results demonstrated the effective of the coadsorption of ssDNA and thiols to control the SAM property and the probe density. It was therefore shown the importance to identify the correct density of probes on the electrode, below the saturation value, to ensure both a proper hybridization process and having a high hybridization signal