Development and characterization of grating-coupled surface plasmon resonance sensors for medical and biological applications

The core of my research activity during the Ph.D. period has been the study and the development of Surface Plasmon Resonance (SPR) based sensors for the detection of molecules of biological and medical interest. In particular, between the different configurations allowing plasmon excitation, I have focused my research on the study of nanostructured gratings, which allow to achieve a higher sensitivity than the prism coupled sensors and to miniaturize the measurement system. First my activity focused on the development of an opto-electronic bench able to detect plasmonic signal and to transduce it into an electric one. The test bench must allow varying independently some parameters that are fundamental for plasmonic excitation, such as the incident angle of laser beam, the azimuthal angle between the scattering plane and the grating vector, and the incident light polarization. The light modulated by the grating is transduced into electrical current through a photodiodes array and then acquired by a parameters analyzer. I have realized a versatile bench in order to perform measurements of both reflectance, analyzing the light reflected from the grating, and transmittance. The use of a motorized rotation stage has automated the measurement and it is controlled by a custom National Instruments LabVIEW software: in this way only few initial steps must be manually performed. I have analyzed three types of gratings: - Gold sinusoidal grating, optimized for reflectance measurements in incident light polarization modulation, exploiting the sensitivity increase due to a non-zero azimuthal angle. This grating has been provided us by LaNN laboratory (Laboratory of research for Nanofabrication and Nanodevices) of National Council of Research (CNR) of Padova. The grating has been manufactured through lithography (by Laser Interference Lithography-LIL) of a photoresist deposited over a glass (or silicon wafer), nanostructure replica and thermal evaporation of the gold plasmonic layer. First I have analyzed the bare grating, and then I have measured bulk with different refractive indexes in order to estimate sensor sensitivity. Then I have measured if the sensor is able to detect biological molecules, first through tests of avidin detection, exploiting avidin-biotin binding, and then through tests of DNA detection, via complementary Peptide Nucleic Acid (PNA) immobilization. - Gold digital grating, that exploits light extraordinary transmission. This grating has been fabricated by LaNN laboratory of CNR of Padova through Electron Beam Lithography (EBL) technique, and it has been designed in order to realize a simple and compact detection system, since the only sensing parameter considered is incident light polarization. Grating ability to detect surface changes of refractive index has been evaluated by a functionalization process with dodecanethiol, that is a molecule composed of a chain of twelve carbon atoms that forms a layer of well- known thickness and refractive index. - Silver trapezoidal grating, developed thanks to the collaboration with the Spin-Off Next Step Engineering, that has involved me in the last months of my Ph.D.. In fact, I have participated in grating fabrication exploiting the industrial facilities of the Spin-Off, which allow producing high quantity of low-cost devices, suitable to be a disposable sensor. The manufacturing process consists of the development of a stamper obtained through interferential lithography, the replica molding of polymeric substrate and the metal layer deposition through sputtering. These gratings have been optimized for transmittance measurements and their response as a function of incident light and azimuthal angles has been analyzed. Measurements of bulk with different refractive indexes, in order to estimate sensor sensitivity, and then of grating functionalized with different lengths alkanethiols have been performed. All experimental data have been compared with simulations results. In fact the behavior of the gratings has been studied through different simulation methods. In particular the digital gold grating has been studied through Finite Element Method (FEM) implemented in COMSOL Multiphysics; the vector model has been applied for both sinusoidal gold gratings and trapezoidal silver ones. The latter grating has been also analyzed through Rigorous Coupled Wave Analysis (RCWA). As already mentioned, during the last period of my Ph.D., I have collaborated with Next Step Engineering to develop an innovative industrial process that allows creating both grating for plasmonic events detection and electronic/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 and I am one of the inventors. During my PhD I have also developed microfluidic devices through a particular technique of polymer etching, able to create clear-cut profiles without deforming the planar structure, and also through suitable changes of production process adopted by Next Step Engineering, previously mentioned. The former devices have been used with silver gratings for the measurements of bulk with different refractive indexes

Development and characterization of grating-coupled surface plasmon resonance sensors for medical and biological applications

The core of my research activity during the Ph.D. period has been the study and the development of Surface Plasmon Resonance (SPR) based sensors for the detection of molecules of biological and medical interest. In particular, between the different configurations allowing plasmon excitation, I have focused my research on the study of nanostructured gratings, which allow to achieve a higher sensitivity than the prism coupled sensors and to miniaturize the measurement system. First my activity focused on the development of an opto-electronic bench able to detect plasmonic signal and to transduce it into an electric one. The test bench must allow varying independently some parameters that are fundamental for plasmonic excitation, such as the incident angle of laser beam, the azimuthal angle between the scattering plane and the grating vector, and the incident light polarization. The light modulated by the grating is transduced into electrical current through a photodiodes array and then acquired by a parameters analyzer. I have realized a versatile bench in order to perform measurements of both reflectance, analyzing the light reflected from the grating, and transmittance. The use of a motorized rotation stage has automated the measurement and it is controlled by a custom National Instruments LabVIEW software: in this way only few initial steps must be manually performed. I have analyzed three types of gratings: - Gold sinusoidal grating, optimized for reflectance measurements in incident light polarization modulation, exploiting the sensitivity increase due to a non-zero azimuthal angle. This grating has been provided us by LaNN laboratory (Laboratory of research for Nanofabrication and Nanodevices) of National Council of Research (CNR) of Padova. The grating has been manufactured through lithography (by Laser Interference Lithography-LIL) of a photoresist deposited over a glass (or silicon wafer), nanostructure replica and thermal evaporation of the gold plasmonic layer. First I have analyzed the bare grating, and then I have measured bulk with different refractive indexes in order to estimate sensor sensitivity. Then I have measured if the sensor is able to detect biological molecules, first through tests of avidin detection, exploiting avidin-biotin binding, and then through tests of DNA detection, via complementary Peptide Nucleic Acid (PNA) immobilization. - Gold digital grating, that exploits light extraordinary transmission. This grating has been fabricated by LaNN laboratory of CNR of Padova through Electron Beam Lithography (EBL) technique, and it has been designed in order to realize a simple and compact detection system, since the only sensing parameter considered is incident light polarization. Grating ability to detect surface changes of refractive index has been evaluated by a functionalization process with dodecanethiol, that is a molecule composed of a chain of twelve carbon atoms that forms a layer of well- known thickness and refractive index. - Silver trapezoidal grating, developed thanks to the collaboration with the Spin-Off Next Step Engineering, that has involved me in the last months of my Ph.D.. In fact, I have participated in grating fabrication exploiting the industrial facilities of the Spin-Off, which allow producing high quantity of low-cost devices, suitable to be a disposable sensor. The manufacturing process consists of the development of a stamper obtained through interferential lithography, the replica molding of polymeric substrate and the metal layer deposition through sputtering. These gratings have been optimized for transmittance measurements and their response as a function of incident light and azimuthal angles has been analyzed. Measurements of bulk with different refractive indexes, in order to estimate sensor sensitivity, and then of grating functionalized with different lengths alkanethiols have been performed. All experimental data have been compared with simulations results. In fact the behavior of the gratings has been studied through different simulation methods. In particular the digital gold grating has been studied through Finite Element Method (FEM) implemented in COMSOL Multiphysics; the vector model has been applied for both sinusoidal gold gratings and trapezoidal silver ones. The latter grating has been also analyzed through Rigorous Coupled Wave Analysis (RCWA). As already mentioned, during the last period of my Ph.D., I have collaborated with Next Step Engineering to develop an innovative industrial process that allows creating both grating for plasmonic events detection and electronic/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 and I am one of the inventors. During my PhD I have also developed microfluidic devices through a particular technique of polymer etching, able to create clear-cut profiles without deforming the planar structure, and also through suitable changes of production process adopted by Next Step Engineering, previously mentioned. The former devices have been used with silver gratings for the measurements of bulk with different refractive indexes.Il tema principale dell’attività di ricerca che ho svolto durante il mio periodo di Dottorato in Scienza e Tecnologia dell’Informazione è stato lo studio e lo sviluppo di sensori basati sull’effetto di risonanza plasmonica per la rilevazione di molecole di interesse medico e biologico. In particolare, tra le varie configurazioni che permettono l’eccitazione plasmonica, mi sono focalizzata sullo studio dei reticoli nanostrutturati, i quali permettono di raggiungere elevate sensibilità, se paragonati ai dispositivi accoppiati con prisma, e di miniaturizzare e integrare il sistema di misura come obiettivo nel lungo periodo. Inizialmente la mia attività si è concentrata sullo sviluppo di un banco opto-elettronico che permettesse di rilevare il segnale plasmonico e trasdurlo in un segnale elettrico. Il banco doveva essere in grado di variare indipendentemente alcuni parametri determinanti per l’eccitazione plasmonica, ossia l’angolo di incidenza del fascio laser, l’angolo azimutale tra il piano di scattering e il vettore del reticolo, e la polarizzazione della luce incidente. La luce modulata dal reticolo viene poi trasformata in corrente elettrica attraverso un array di fotodiodi, e quindi acquisita attraverso un analizzatore di parametri. Ho mirato a realizzare un banco molto versatile in modo da poter effettuare misure sia di riflettanza, andando ad analizzare la luce riflessa dal reticolo, sia di trasmittanza, analizzando la luce trasmessa dal campione. L’introduzione di uno stadio motorizzato ha permesso di rendere la misura più automatizzata e gestibile via software, attraverso un programma custom sviluppato in LabVIEW, e lasciando manuali solo pochi passaggi iniziali. Ho analizzato tre tipologie diverse di reticoli: - Reticolo d’oro con superficie sinusoidale, ottimizzato per effettuare misure in riflessione con modulazione della polarizzazione della luce incidente, sfruttando l’aumento di sensibilità derivante dall’angolo azimutale non nullo. Tale reticolo è stato fornito dal laboratorio LaNN (Laboratorio di ricerca per la Nanofabbricazione e i Nanodispositivi) del Consiglio Nazionale delle Ricerche (CNR) di Padova. Il reticolo è stato realizzato attraverso litografia interferenziale di uno strato di fotoresist deposto su un vetrino (o silicio), da cui è stato ricavato uno stampo che permette la replica della nano struttura; infine, attraverso un’evaporazione termica, è stato depositato uno strato d’oro. Inizialmente ho analizzato il reticolo in condizione “fresh”; successivamente ho effettuato misure di “bulk” con indici di rifrazione diversi, per poter stimare la sensibilità del sensore. Ho poi misurato la capacità del dispositivo nel rilevare molecole di interesse biologico, dapprima attraverso prove di rilevazione di avidina presente in una soluzione, sfruttando il legame avidina-biotina, poi con prove di rilevazione di singole catene di DNA, attraverso l’immobilizzazione sulla superficie della nanostruttra di acido peptidonucleico (PNA) complementare. - Reticolo d’oro digitale, ideato per sfruttare il fenomeno di trasmissione straordinaria della luce. Tale reticolo è stato realizzato dal laboratorio LaNN del CNR di Padova attraverso la tecnica di litografia a fascio di elettroni (Electron Beam Lithography-EBL) e nasce con l’obiettivo di creare un sistema di rilevazione estremamente semplice, poiché l’unico parametro di sensing, e quindi variabile, è la polarizzazione della luce incidente. La capacità del sistema di discriminare variazioni superficiali di indice di rifrazione è stata valutata funzionalizzando il reticolo con dodecanethiol, ossia una molecola composta da una catena di dodici atomi di carbonio in grado di formare uno strato di dimensioni e indice di rifrazione noti. - Reticolo trapezoidale in argento, nato dalla collaborazione con lo Spin-Off Next Step Engineering, che mi ha coinvolta nell’ultimo periodo di dottorato. Infatti, ho partecipato in prima persona alla realizzazione del sensore, sfruttando le facilities industriali a cui l’azienda ha accesso, permettendo di produrre dispositivi a basso costo e in elevate quantità, quindi adatti ad un utilizzo di tipo “usa e getta”. Il processo di fabbricazione prevede la realizzazione di uno stampo attraverso litografia interferenziale, una fase di replica a stampo su substrato polimerico e la deposizione di uno strato metallico per polverizzazione catodica. Tali sensori sono stati ottimizzati per la misura della luce trasmessa e si è analizzato il comportamento al variare dell’angolo di incidenza e dell’angolo azimutale. Si è quindi misurato il comportamento del sensore in presenza di bulk ad indici di rifrazione diversi per la stima della sensibilità, e successivamente si sono effettuate misure funzionalizzando il campione con alcantioli di diversa lunghezza. I risultati sperimentali sono stati confrontati con quelli ottenuti dalle simulazioni. Infatti si è studiato il comportamento di ogni reticolo attraverso metodi di simulazione diversi. In particolare il reticolo digitale in oro è stato studiato attraverso il metodo degli elementi finiti (FEM) implementato in COMSOL Multiphysics, il modello vettoriale è stato applicato sia per lo studio del reticolo sinusoidale in oro che del reticolo trapezoidale in argento. Quest’ultimo reticolo è stato analizzato anche attraverso il metodo RCWA (Rigorous Coupled Wave Analysis). Come già accennato, durante l’ultimo 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 non solo reticoli per la rilevazione di segnali plasmonici, ma anche dispositivi ibridi elettronici/microfluidici per applicazioni biologiche e mediche, all’interno di una singola linea produttiva automatizzata. Con questo processo ho prodotto i reticoli in argento, 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. Durante il dottorato ho approfondito anche lo sviluppo di dispositivi microfluidici sia attraverso tecniche di incisione polimerica, in grado di creare profili di taglio netti senza deformarne la struttura planare, sia apportando le appropriate modifiche al processo produttivo utilizzato da Next Step Engineering, precedentemente citato. I dispositivi realizzati sono stati utilizzati per le misure di bulk a diversi indici di rifrazione utilizzando i reticoli in argento

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

SigMate: A Comprehensive Software Package for Extracellular Neuronal Signal Processing and Analysis

none6noneMAHMUD M; BERTOLDO A; GIRARDI S; MASCHIETTO M; PASQUALOTTO E; S. VASSANELLIMahmud, Mufti; Bertoldo, Alessandra; Girardi, Stefano; Maschietto, Marta; Pasqualotto, E; Vassanelli, Stefan

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

LOW-COST ENZYME-BASED BIOSENSOR FOR LACTIC ACID AMPEROMETRIC DETECTION Electrical Modeling and Validation for Clinical and Food Processing Applications

In this work we present the preliminary resulting measurements of an enzyme-based biosensor for the amperometric detection of lactic acid (LA). The sensor is based on low-cost gold electrodes on polymeric substrate. The redox catalytic enzyme used for analyte amperometric detection is lactate oxidase (LOx) from Pediococcus sp. This enzyme has been immobilized over electrodes surfaces by direct adsorption methodologies. Analysis of the enzyme-modified electrodes have been carried out by means of Electrochemical Impedance Spectroscopy (EIS) and with the development of an equivalent electrical model, in order to improve the adsorption process. Biosensors performance have been evaluated with Cyclic Voltammetry (CVM) measurements in different lactic acid solutions with concentrations from 1 mu M up to 300 mM. The lactate sensitivity of this disposable biosensor results in about 6.24 mu A mM(-1) cm(-2)

Plasmonic platforms for innovative surface plasmon resonance configuration with sensing applications

An experimental prototype exploiting Grating-Coupled Surface Plasmon Resonance (GCSPR) based on polarization modulation has been assembled and tested for sensing purposes. The plasmonic gratings are azimuthally rotated in order to exploit the symmetry breaking for the excitation of highly sensitive Surface Plasmon Polaritons in conical mounting. By exploiting the optimal-polarization shift, a scan of the incident polarization is performed and reflectivity data are collected. The output signal exhibits a harmonic dependence on polarization and the phase term is considered as a parameter for sensing. Since the optical configuration is fixed during the analysis and the only degree of freedom is represented by the incident polarization, this setup provides a more compact and simplified architecture with respect to other. commercial SPR techniques, however assuring at the same time competitive performances in refractive index sensitivity and resolution. The employed metallic gratings are fabricated by interferential lithography and replicated onto resin substrates by soft-lithography techniques, thus thermally evaporated