Engineering of Vis-NIR Metamaterials towards Revolutionary Ultrasensitive and Versatile Plasmonic Biosensors

By enabling subwavelength light localization and strong electromagnetic field enhancement, plasmonic biosensors have opened up a new realm of possibilities for a broad range of chemical and biological sensing applications owing to their label-free and real-time attributes. Although significant progress has been made, many fundamental and practical challenges still remain to be addressed. For instance, the plasmonic biosensors are nonselective sensing platforms; they are not well-suited to provide information regarding conformation or chemical fingerprint of unknown biomolecules. Furthermore, tunability of the plasmonic resonance in visible frequency regime is still limited; this will prevent their efficient and reproducible exploitation in single-molecule sensitivity. Here, we show that by engineering geometry of plasmonic metamaterials,1 consisting of periodic arrays of artificial split-ring resonators (SRRs), the plasmonic resonance of metamaterials could be tuned to visible-near infrared regimes (Vis-NIR) such that it allows parallel acquisition of optical transmission and highly surface-enhanced Raman (SERS) spectra from large functionalized SRR arrays. The Au SRRs were designed in form of alphabet letters (U, V, S, H, Y) with various line width (from 80 to 30 nm). By tailoring their size and shape, plasmonic resonance wavelength of the SRRs could be actively tuned so that it gives the strongest SERS effect under given excitation energy and polarization for biological and organic molecules. On the other hand, the plasmonic tunability was also achieved for a given SRR pattern by tuning the laser wavelength to obtain the highest electromagnetic field enhancement. The geometry- and laser-tunable channels typically provide an electromagnetic field enhancement as high as 20 times. This will provide the basis of versatile and multichannel devices for identification of different conformational states of Guanine-rich DNA, detection of a cancer biomarker nucleolin, and femtomolar sensitivity detection of food and drink additives. These results show that the tunable Vis-IR metamaterials are very versatile biosensing platforms and suggest considerable promise in genomic research, disease diagnosis, and food safety analysis.

Carbon nanotubes in nanostructured films: Potential application as amperometric and potentiometric field-effect (bio-)chemical sensors

The assembly of carbon nanotubes (CNTs) into nanostructured films is attractive for producing functionalized hybrid materials and (bio-)chemical sensors, but this requires experimental methods that allow for control of molecular architecturcs. In this study, we exploit the layer-by-layer (LbL) technique to obtain two types of sensors incorporating CNTs. In the first, LbL films of alternating layers of multi-walled carbon nanotubes (MWNTs) dispersed in polyarninoamide (PAMAM) dendrimers and nickel phthalocyanine (NiTsPc) were used in amperometric detection of the neurotransmitter dopamine (DA). The electrochemical properties evaluated with cyclic voltammetry indicated that the incorporation of MWNTs in the PAMAM-NT/NiTsPc LbL films led to a 3-fold increase in the peak current, in addition to a decrease of 50 mV in the oxidation potential of DA. The latter allowed detection of DA even in the presence of ascorbic acid (AA), a typical interferent for DA. Another LbL film was obtained with layers of PAMAM and single-walled carbon nanotubes (SWNTs) employed in field-effect-devices using a capacitive electrolyte-insulator-semiconductor structure (EIS). The adsorption of the film components was monitored by measuring the flat-band voltage shift in capacitance-voltage (C-P) curves, caused by the charges from the components. Constant capacitance (ConCap) measurements showed that the EISPAMAM/SWNT film displayed a high pH sensitivity (ca. 54.5 mV/pH), being capable of detecting penicillin G between 10(-4) mol L(-1) and 10(-2) mol L-1, when a layer of penicillinase was adsorbed atop the PAMAM/SWNT film. (C) 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Low-Frequency Noise in Field-Effect Devices Functionalized With Dendrimer/Carbon-Nanotube Multilayers

Low-frequency noise in an electrolyte-insulator- semiconductor (EIS) structure functionalized with multilayers of polyamidoamine (PAMAM) dendrimer and single-walled carbon nanotubes (SWNT) is studied. The noise spectral density exhibits 1/f(gamma) dependence with the power factor of gamma approximate to 0.8 and gamma = 0.8-1.8 for the bare and functionalized EIS sensor, respectively. The gate-voltage noise spectral density is practically independent of the pH value of the solution and increases with increasing gate voltage or gate-leakage current. It has been revealed that functionalization of an EIS structure with a PAMAM/SWNTs multilayer leads to an essential reduction of the 1/f noise. To interpret the noise behavior in bare and functionalized EIS devices, a gate-current noise model for capacitive EIS structures based on an equivalent flatband-voltage fluctuation concept has been developed.

An impedimetric biosensor to test neat serum for dengue diagnosis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Multiscale sensing of antibody - antigen interactions by organic transistors and single-molecule force spectroscopy

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Capacitance spectroscopy and density functional theory

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Biochemical capacitance of geobacter sulfurreducens biofilms

An electrical model able to decouple the electron pathway from microbial cell machinery impedance terms is introduced. In this context, capacitance characteristics of the biofilm are clearly resolved. In other words, the model allows separating, according to the advantage of frequency and spectroscopic response approach, the different terms controlling the performance of the microbial biofilm respiratory process and thus the directly related electricity production process. The model can be accurately fitted to voltammetry measurements obtained under steady-state conditions and also to biofilm discharge amperometric measurements. The implications of biological aspects of the electrochemical or redox capacitance are discussed theoretically in the context of current knowledge with regard to structure and physiological activity of microbial Geobacter biofilms.

Biomolecular interaction analysis of gestrinone-anti-gestrinone using arrays of high aspect ratio SU-8 nanopillars

In this paper, label-free biosensing for antibody screening by periodic lattices of high-aspect ratio SU-8 nano-pillars (BICELLs) is presented. As a demonstration, the determination of anti-gestrinone antibodies from whole rabbit serum is carried out, and for the first time, the dissociation constant (KD = 6 nM) of antigen-antibody recognition process is calculated using this sensing system. After gestrinone antigen immobilization on the BICELLs, the immunorecognition was performed. The cells were interrogated vertically by using micron spot size Fourier transform visible and IR spectrometry (FT-VIS-IR), and the dip wavenumber shift was monitored. The biosensing assay exhibited good reproducibility and sensitivity (LOD = 0.75 ng/mL).

Development and fabrication of optical biosensors based on biophotonic sensing cells (BICELLs)

El objetivo de esta tesis es el desarrollo y caracterización de biosensores ópticos sin marcado basados en celdas sensoras biofotónicas (BICELLs). Éstas son un nuevo concepto de biosensor desarrollado por el grupo de investigación y consiste en la combinación de técnicas de interrogación vertical, junto a estructuras fotónicas producidas usando métodos de micro- y nanofabricación. Varias conclusiones son extraídas de este trabajo. La primera, que se ha definido una BICELL estándar basada en interferómetros Fabry-Perot (FP). Se ha demostrado su capacidad para la comparación de rendimiento entre BICELLs estructuradas y para la realización de inmunoensayos de bajo coste. Se han estudiado diferentes técnicas de fabricación disponibles para la producción de BICELLs. Se determinó que la litografía de contacto a nivel de oblea produce estructuras de bajo coste, reproducibles y de alta calidad. La resolución alcanzada ha sido de 700 nm. El estudio de la respuesta a inmunoensayos de las BICELLs producidas se ha desarrollado en este trabajo. Se estudió la influencia de BICELLs basadas en diferentes geometrías y tamaños. De aquí resulta un nuevo enfoque para predecir el comportamiento de respuesta para la detección biológica de cualquier biosensor óptico estructurado, relacionando su superficie efectiva y su sensibilidad óptica. También se demostró una técnica novedosa y de bajo coste para la caracterización experimental de la sensibilidad óptica, basada en el depósito de películas ultradelgadas. Finalmente, se ha demostrado el uso de BICELLs desarrolladas en esta tesis, en la detección de aplicaciones reales, tales como hormonas, virus y proteínas. ABSTRACT The objective of this thesis is the development and characterization of optical label-free biosensors based on Bio-Photonic sensing Cells (BICELLs). BICELL is a novel biosensor concept developed by the research group, and it consists of a combination of vertical interrogation optical techniques and photonic structures produced by using micro- and nano-fabrication methods. Several main conclusions are extracted from this work. Firstly, a standard BICELL is defined based on FP interferometers, which demonstrated its capacity for accomplishing performance comparisons among different structured BICELLs, as well as to achieve low-cost immunoassays. Different available fabrication techniques were studied for BICELL manufacturing. It is found that contact lithography at wafer scale produce cost-effective, reproducible and high quality structures. The resolution achieved was 700 nm. Study on the response of developed BICELLs to immunoassays is performed within this work. It is therefore studied the influence of BICELLs based on different geometries and sizes in the immunoassay, which resulted in a new approach to predict the biosensing behaviour of any structured optical biosensor relating to its effective surface and optical sensitivity. Also, it is demonstrated a novel and low-cost characterization technique of the experimental optical sensitivity, based on ultrathin-film deposition. Finally, it is also demonstrated the capability of using the developed BICELLs in this thesis for real applications detection of hormones, virus and proteins.

Reversible oxidation of phosphatase and tensin homolog (PTEN) alters its interactions with signaling and regulatory proteins

Phosphatase and tensin homolog (PTEN) is involved in a number of different cellular processes including metabolism, apoptosis, cell proliferation and survival. It is a redox-sensitive dual-specificity protein phosphatase that acts as a tumor suppressor by negatively regulating the PI3K/Akt pathway. While direct evidence of redox regulation of PTEN downstream signaling has been reported, the effect of PTEN redox status on its protein-protein interactions is poorly understood. PTEN-GST in its reduced and a DTT-reversible H2O2-oxidized form was immobilized on a glutathione-sepharose support and incubated with cell lysate to capture interacting proteins. Captured proteins were analyzed by LC-MSMS and comparatively quantified using label-free methods. 97 Potential protein interactors were identified, including a significant number that are novel. The abundance of fourteen interactors was found to vary significantly with the redox status of PTEN. Altered binding to PTEN was confirmed by affinity pull-down and Western blotting for Prdx1, Trx, and Anxa2, while DDB1 was validated as a novel interactor with unaltered binding. These results suggest that the redox status of PTEN causes a functional variation in the PTEN interactome. The resin capture method developed had distinct advantages in that the redox status of PTEN could be directly controlled and measured.

The redoxomics of PTEN: walking a fine line between damage and signaling:mass spectrometry-based approaches to study the effect of oxidation on PTEN function, structure, and protein-protein interactions

The research described in this PhD thesis focuses on proteomics approaches to study the effect of oxidation on the modification status and protein-protein interactions of PTEN, a redox-sensitive phosphatase involved in a number of cellular processes including metabolism, apoptosis, cell proliferation, and survival. While direct evidence of a redox regulation of PTEN and its downstream signaling has been reported, the effect of cellular oxidative stress or direct PTEN oxidation on PTEN structure and interactome is still poorly defined. In a first study, GST-tagged PTEN was directly oxidized over a range of hypochlorous acid (HOCl) concentration, assayed for phosphatase activity, and oxidative post-translational modifications (oxPTMs) were quantified using LC-MS/MS-based label-free methods. In a second study, GSTtagged PTEN was prepared in a reduced and reversibly H2O2-oxidized form, immobilized on a resin support and incubated with HCT116 cell lysate to capture PTEN interacting proteins, which were analyzed by LC-MS/MS and comparatively quantified using label-free methods. In parallel experiments, HCT116 cells transfected with a GFP-tagged PTEN were treated with H2O2 and PTENinteracting proteins immunoprecipitated using standard methods. Several high abundance HOCl-induced oxPTMs were mapped, including those taking place at amino acids known to be important for PTEN phosphatase activity and protein-protein interactions, such as Met35, Tyr155, Tyr240 and Tyr315. A PTEN redox interactome was also characterized, which identified a number of PTEN-interacting proteins that vary with the reversible inactivation of PTEN caused by H2O2 oxidation. These included new PTEN interactors as well as the redox proteins peroxiredoxin-1 (Prdx1) and thioredoxin (Trx), which are known to be involved in the recycling of PTEN active site following H2O2-induced reversible inactivation. The results suggest that the oxidative modification of PTEN causes functional alterations in PTEN structure and interactome, with fundamental implications for the PTEN signaling role in many cellular processes, such as those involved in the pathophysiology of disease and ageing.

Functionalized carbon micro/nanostructures for biomolecular detection

Advancements in the micro-and nano-scale fabrication techniques have opened up new avenues for the development of portable, scalable and easier-to-use biosensors. Over the last few years, electrodes made of carbon have been widely used as sensing units in biosensors due to their attractive physiochemical properties. The aim of this research is to investigate different strategies to develop functionalized high surface carbon micro/nano-structures for electrochemical and biosensing devices. High aspect ratio three-dimensional carbon microarrays were fabricated via carbon microelectromechanical systems (C-MEMS) technique, which is based on pyrolyzing pre-patterned organic photoresist polymers. To further increase the surface area of the carbon microstructures, surface porosity was introduced by two strategies, i.e. (i) using F127 as porogen and (ii) oxygen reactive ion etch (RIE) treatment. Electrochemical characterization showed that porous carbon thin film electrodes prepared by using F127 as porogen had an effective surface area (Aeff 185%) compared to the conventional carbon electrode. To achieve enhanced electrochemical sensitivity for C-MEMS based functional devices, graphene was conformally coated onto high aspect ratio three-dimensional (3D) carbon micropillar arrays using electrostatic spray deposition (ESD) technique. The amperometric response of graphene/carbon micropillar electrode arrays exhibited higher electrochemical activity, improved charge transfer and a linear response towards H2O2 detection between 250&mgr;M to 5.5mM. Furthermore, carbon structures with dimensions from 50 nano-to micrometer level have been fabricated by pyrolyzing photo-nanoimprint lithography patterned organic resist polymer. Microstructure, elemental composition and resistivity characterization of the carbon nanostructures produced by this process were very similar to conventional photoresist derived carbon. Surface functionalization of the carbon nanostructures was performed using direct amination technique. Considering the need for requisite functional groups to covalently attach bioreceptors on the carbon surface for biomolecule detection, different oxidation techniques were compared to study the types of carbon-oxygen groups formed on the surface and their percentages with respect to different oxidation pretreatment times. Finally, a label-free detection strategy using signaling aptamer/protein binding complex for platelet-derived growth factor oncoprotein detection on functionalized three-dimensional carbon microarrays platform was demonstrated. The sensor showed near linear relationship between the relative fluorescence difference and protein concentration even in the sub-nanomolar range with an excellent detection limit of 5 pmol.

Nanofabrication towards biophotonics

This thesis explores methods for fabrication of nanohole arrays, and their integration into a benchtop system for use as sensors or anti-counterfeit labels. Chapter 1 gives an introduction to plasmonics and more specifically nanohole arrays and how they have potential as label free sensors compared to the current biosensors on the market. Various fabrication methods are explored, including Focused Ion Beam, Electron Beam Lithography, Nanoimprint lithography, Template stripping and Phase Shift Lithography. Focused Ion Beam was chosen to fabricate the nanohole arrays due to its suitability for rapid prototyping and it’s relatively low cost. In chapter 2 the fabrication of nanohole arrays using FIB is described, and the samples characterised. The fabricated nanohole arrays are tested as bulk refractive index sensors, before a bioassay using whole molecule human IgG antibodies and antigen is developed and performed on the senor. In chapter 3 the fabricated sensors are integrated into a custom built system, capable of real time, multiplexed detection of biomolecules. Here, scFv antibodies of two biomolecules relevant to the detection of pancreatic cancer (C1q and C3) are attached to the nanohole arrays, and detection of their complementary proteins is demonstrated both in buffer (10 nM detection of C1q Ag) and human serum. Chapter 4 explores arrays of anisotropic (elliptical) nanoholes and shows how the shape anisotropy induces polarisation sensitive transmission spectra, in both simulations and fabricated arrays. The potential use of such samples as visible and NIR tag for anti-counterfeiting applications is demonstrated. Finally, chapter 5 gives a summary of the work completed and discusses potential future work in this area.

Design, fabrication and characterization of resonant waveguide grating based optical biosensors

The absence of rapid, low cost and highly sensitive biodetection platform has hindered the implementation of next generation cheap and early stage clinical or home based point-of-care diagnostics. Label-free optical biosensing with high sensitivity, throughput, compactness, and low cost, plays an important role to resolve these diagnostic challenges and pushes the detection limit down to single molecule. Optical nanostructures, specifically the resonant waveguide grating (RWG) and nano-ribbon cavity based biodetection are promising in this context. The main element of this dissertation is design, fabrication and characterization of RWG sensors for different spectral regions (e.g. visible, near infrared) for use in label-free optical biosensing and also to explore different RWG parameters to maximize sensitivity and increase detection accuracy. Design and fabrication of the waveguide embedded resonant nano-cavity are also studied. Multi-parametric analyses were done using customized optical simulator to understand the operational principle of these sensors and more important the relationship between the physical design parameters and sensor sensitivities. Silicon nitride (SixNy) is a useful waveguide material because of its wide transparency across the whole infrared, visible and part of UV spectrum, and comparatively higher refractive index than glass substrate. SixNy based RWGs on glass substrate are designed and fabricated applying both electron beam lithography and low cost nano-imprint lithography techniques. A Chromium hard mask aided nano-fabrication technique is developed for making very high aspect ratio optical nano-structure on glass substrate. An aspect ratio of 10 for very narrow (~60 nm wide) grating lines is achieved which is the highest presented so far. The fabricated RWG sensors are characterized for both bulk (183.3 nm/RIU) and surface sensitivity (0.21nm/nm-layer), and then used for successful detection of Immunoglobulin-G (IgG) antibodies and antigen (~1μg/ml) both in buffer and serum. Widely used optical biosensors like surface plasmon resonance and optical microcavities are limited in the separation of bulk response from the surface binding events which is crucial for ultralow biosensing application with thermal or other perturbations. A RWG based dual resonance approach is proposed and verified by controlled experiments for separating the response of bulk and surface sensitivity. The dual resonance approach gives sensitivity ratio of 9.4 whereas the competitive polarization based approach can offer only 2.5. The improved performance of the dual resonance approach would help reducing probability of false reading in precise bio-assay experiments where thermal variations are probable like portable diagnostics.

Integration of virus-like particle macromolecular bioreceptors in electrochemical biosensors

Rapid, sensitive and selective detection of chemical hazards and biological pathogens has shown growing importance in the fields of homeland security, public safety and personal health. In the past two decades, efforts have been focusing on performing point-of-care chemical and biological detections using miniaturized biosensors. These sensors convert target molecule binding events into measurable electrical signals for quantifying target molecule concentration. However, the low receptor density and the use of complex surface chemistry in receptors immobilization on transducers are common bottlenecks in the current biosensor development, adding to the cost, complexity and time. This dissertation presents the development of selective macromolecular Tobacco mosaic virus-like particle (TMV VLP) biosensing receptor, and the microsystem integration of VLPs in microfabricated electrochemical biosensors for rapid and performance-enhanced chemical and biological sensing. Two constructs of VLPs carrying different receptor peptides targeting at 2,4,6-trinitrotoluene (TNT) explosive or anti-FLAG antibody are successfully bioengineered. The VLP-based TNT electrochemical sensor utilizes unique diffusion modulation method enabled by biological binding between target TNT and receptor VLP. The method avoids the influence from any interfering species and environmental background signals, making it extremely suitable for directly quantifying the TNT level in a sample. It is also a rapid method that does not need any sensor surface functionalization process. For antibody sensing, the VLPs carrying both antibody binding peptides and cysteine residues are assembled onto the gold electrodes of an impedance microsensor. With two-phase immunoassays, the VLP-based impedance sensor is able to quantify antibody concentrations down to 9.1 ng/mL. A capillary microfluidics and impedance sensor integrated microsystem is developed to further accelerate the process of VLP assembly on sensors and improve the sensitivity. Open channel capillary micropumps and stop-valves facilitate localized and evaporation-assisted VLP assembly on sensor electrodes within 6 minutes. The VLP-functionalized impedance sensor is capable of label-free sensing of antibodies with the detection limit of 8.8 ng/mL within 5 minutes after sensor functionalization, demonstrating great potential of VLP-based sensors for rapid and on-demand chemical and biological sensing.