Localized Surface Plasmon Resonance Biosensors for Real-Time Biomolecular Binding Study

Surface Plasmon Resonance (SPR) and localized surface plasmon resonance (LSPR) biosensors have brought a revolutionary change to in vitro study of biological and biochemical processes due to its ability to measure extremely small changes in surface refractive index (RI), binding equilibrium and kinetics. Strategies based on LSPR have been employed to enhance the sensitivity for a variety of applications, such as diagnosis of diseases, environmental analysis, food safety, and chemical threat detection. In LSPR spectroscopy, absorption and scattering of light are greatly enhanced at frequencies that excite the LSPR, resulting in a characteristic extinction spectrum that depends on the RI of the surrounding medium. Compositional and conformational change within the surrounding medium near the sensing surface could therefore be detected as shifts in the extinction spectrum. This dissertation specifically focuses on the development and evaluation of highly sensitive LSPR biosensors for in situ study of biomolecular binding process by incorporating nanotechnology. Compared to traditional methods for biomolecular binding studies, LSPR-based biosensors offer real-time, label free detection. First, we modified the gold sensing surface of LSPR-based biosensors using nanomaterials such as gold nanoparticles (AuNPs) and polymer to enhance surface absorption and sensitivity. The performance of this type of biosensors was evaluated on the application of small heavy metal molecule binding affinity study. This biosensor exhibited ~7 fold sensitivity enhancement and binding kinetics measurement capability comparing to traditional biosensors. Second, a miniaturized cell culture system was integrated into the LSPR-based biosensor system for the purpose of real-time biomarker signaling pathway studies and drug efficacy studies with living cells. To the best of our knowledge, this is the first LSPR-based sensing platform with the capability of living cell studies. We demonstrated the living cell measurement ability by studying the VEGF signaling pathway in living SKOV-3 cells. Results have shown that the VEGF secretion level from SKOV-3 cells is 0.0137 ± 0.0012 pg per cell. Moreover, we have demonstrated bevacizumab drug regulation to the VEGF signaling pathway using this biosensor. This sensing platform could potentially help studying biomolecular binding kinetics which elucidates the underlying mechanisms of biotransportation and drug delivery.

PMMA based optical fiber Bragg grating for measuring moisture in transformer oil

Water contamination can cause serious problems that compromise in transformer's safe operation and reduce its lifetime. Online monitoring of moisture concentration in transformer oil would permit the control of moisture buildup. This letter presents a direct optical measurement of moisture concentration in transformer oil using a poly(methyl methacrylate) (PMMA)-based optical fiber Bragg grating (POFBG). The refractive index and volume of PMMA-based optical fiber vary with the moisture in the surrounding transformer oil, changing the reflecting wavelength of the grating. A sensitivity of POFBG wavelength change to moisture content of 29 pm/ppm is demonstrated in this letter, indicating detectable water content better than 0.05 ppm.

Label-free oligonucleotide biosensor based on dual-peak long period fiber grating

We report the simplification and development of biofunctionalization methodology based on one-step 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)-mediated reaction. The dual-peak long period grating (dLPG) has been demonstrated its inherent ultrahigh sensitivity to refractive index (RI), achieving 50-fold improvement in RI sensitivity over a standard LPG sensor used in low RI range. With the simple and efficient immobilization of unmodified oligonucleotides on sensor surface, dLPG-based biosensor has been used to monitor the hybridization of complementary oligonucleotides showing a detectable oligonucleotide concentration of 4 nM with the advantages of label-free, real-time, and ultrahigh sensitivity.

Chiroptical spectroscopy of biomolecules using chiral plasmonic nanostructures

This thesis explores the potential of chiral plasmonic nanostructures for the ultrasensitive detection of protein structure. These nanostructures support the generation of fields with enhanced chirality relative to circularly polarised light and are an extremely incisive probe of protein structure. In chapter 4 we introduce a nanopatterned Au film (Templated Plasmonic Substrate, TPS) fabricated using a high through-put injection moulding technique which is a viable alternative to expensive lithographically fabricated nanostructures. The optical and chiroptical properties of TPS nanostructures are found to be highly dependent on the coupling between the electric and magnetic modes of the constituent solid and inverse structures. Significantly, refractive index based measurements of strongly coupled TPSs display a similar sensitivity to protein structure as previous lithographic nanostructures. We subsequently endeavour to improve the sensing properties of TPS nanostructures by developing a high through-put nanoscale chemical functionalisation technique. This process involves a chemical protection/deprotection strategy. The protection step generates a self-assembled monolayer (SAM) of a thermally responsive polymer on the TPS surface which inhibits protein binding. The deprotection step exploits the presence of nanolocalised thermal gradients in the water surrounding the TPS upon irradiation with an 8ns pulsed laser to modify the SAM conformation on surfaces with high net chirality. This allows binding of biomaterial in these regions and subsequently enhances the TPS sensitivity levels. In chapter 6 an alternative method for the detection of protein structure using TPS nanostructures is introduced. This technique relies on mediation of the electric/magnetic coupling in the TPS by the adsorbed protein. This phenomenon is probed through both linear reflectance and nonlinear second harmonic generation (SHG) measurements. Detection of protein structure using this method does not require the presence of fields of enhanced chirality whilst it is also sensitive to a larger array of secondary structure motifs than the measurements in chapters 4 and 5. Finally, a preliminary investigation into the detection of mesoscale biological structure is presented. Sensitivity to the mesoscale helical pitch of insulin amyloid fibrils is displayed through the asymmetry in the circular dichroism (CD) of lithographic gammadions of varying thickness upon adsorption of insulin amyloid fibril spherulites and fragmented fibrils. The proposed model for this sensitivity to the helical pitch relies on the vertical height of the nanostructures relative to this structural property as well as the binding orientation of the fibrils.

The optical properties of metamaterials

This thesis studies the parametric investigation, polarisation dependence and characterization of fishnet structure at near infrared wavelengths. Detailed simulations are performed to understand the behaviour of the structure at near infrared and optical wavelengths. Simulations are performed to obtain negative refractive index of the fishnet structure formed from nanoimprint lithography (NIL) by taking into account the effect of substrate and polymethyl methacrylate (PMMA) beneath it. Two different structures have been designed and fabricated of varying dimensions using NIL and their resonant wavelength measured in the near infrared at 1.45 µm and 1.88 µm. Simulations suggest that a negative refractive index real part with the magnitude -0.24 is found at 1.53 µm and this decrease to a maximum magnitude of -0.57 at 1.9 µm. The PMMA and suppressed pillars are here responsible for the increasing material losses and limiting the value of negative refractive index. An analytical approach has been suggested to characterise fishnet structures at oblique incidence. The expressions for an absorbing medium are rewritten for an alternative definition of refractive index. The expressions are initially validated for a dielectric slab and a metal film. These results provide the possibility that this proposal may yield a general algorithm for obtaining the complex reflection and transmission coefficients for artificial structures. FDTD simulations have been extensively used in this thesis to understand the optical metamaterials and their characterization.

Chemiluminescence DNA-based Nanotechnologies for Bioanalytical Applications

DNA as powerful building molecule, is widely used for the assembly of molecular structures and dynamic molecular devices with different potential applications, ranging from synthetic biology to diagnostics. The feature of sequence programmability, which makes it possible to predict how single stranded DNA molecules fold and interact with one another, allowed the development of spatiotemporally controlled nanostructures and the engineering of supramolecular devices. The first part of this thesis addresses the development of an integrated chemiluminescence (CL)-based lab-on-chip sensor for detection of Adenosine-5-triphosphate (ATP) life biomarker in extra-terrestrial environments.Subsequently, we investigated whether it is possible to study the interaction and the recognition between biomolecules and their targets, mimicking the intracellular environment in terms of crowding, confinement and compartmentalization. To this purpose, we developed a split G-quadruplex DNAzyme platform for the chemiluminescent and quantitative detection of antibodies based on antibody-induced co-localization proximity mechanism in which a split G-quadruplex DNAzyme is led to reassemble into the functional native G-quadruplex conformation as the effect of a guided spatial nanoconfinement.The following part of this thesis aims at developing chemiluminescent nanoparticles for bioimaging and photodynamic therapy applications.In chapter5 a realistic and accurate evaluation of the potentiality of electrochemistry and chemiluminescence (CL) for biosensors development (i.e., is it better to “measure an electron or a photon”?), has been achieved.In chapter 6 the emission anisotropy phenomenon for an emitting dipole bound to the interface between two media with different refractive index has been investigated for chemiluminescence detection.

Changes in peripheral refractive profile after orthokeratology for different degrees of myopia

Purpose: The purpose of this study was to evaluate the effect of orthokeratology for different degrees of myopia correction in the relative location of tangential (FT) and sagittal (FS) power errors across the central 70 of the visual field in the horizontal meridian. Methods: Thirty-four right eyes of 34 patients with a mean age of 25.2 ± 6.4 years were fitted with Paragon CRT (Mesa, AZ) rigid gas permeable contact lenses to treat myopia (2.15 ± 1.26D, range: 0.88 to 5.25D). Axial and peripheral refraction were measured along the central 70 of the horizontal visual field with the Grand Seiko WAM5500 open-field auto-refractor. Spherical equivalent (M), as well as tangential (FT) and sagittal power errors (FS) were obtained. Analysis was stratified in three groups according to baseline spherical equivalent: Group 1 [MBaseline = 0.88 to 1.50D; n = 11], Group 2 [MBaseline = 1.51 to 2.49D; n = 11], and Group 3 [MBaseline = 2.50 to 5.25D; n = 12]. Results: Spherical equivalent was significantly more myopic after treatment beyond the central 40 of the visual field (p50.001). FT became significantly more myopic for all groups in the nasal and temporal retina with 25 (p 0.017), 30 (p 0.007) and 35 (p 0.004) of eye rotation. Myopic change in FS was less consistent, achieving only statistical significance for all groups at 35 in the nasal and temporal retina (p 0.045). Conclusions: Orthokeratology changes significantly FT in the myopic direction beyond the central 40 of the visual field for all degrees of myopia. Changes induced by orthokeratology in relative peripheral M, FT and FS with 35 of eye rotation were significantly correlated with axial myopia at baseline. Keywords: Field

Rutin ameliorates glycemic index, lipid profile and enzymatic activities in serum, heart and liver tissues of rats fed with a combination of hypercaloric diet and chronic ethanol consumption

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Influence of accommodation and refractive status on the peripheral refractive profile

AIM: The aim of the study was to determine, objectively and non-invasively, whether changes in accommodative demand modify differentially the peripheral refraction in emmetropic and myopic human eyes. METHODS: Forty subjects (19 male, 21 female) aged 20-30 years (mean 22.7 (SD 2.8) years), 21 emmetropes (mean spherical equivalent refractive error (MSE) -0.13 (SD 0.29) D) and 19 myopes (MSE -2.95 (SD 1.76) D) participated in the study. Ametropia was corrected with soft contact lenses (etafilcon A, 58% water content). Subjects viewed monocularly a stationary, high contrast (85%) Maltese cross at 0.0, 1.0, 2.0 and 3.0 D of accommodative demand and at 0, 10, 20 and 30 degrees field angle (nasal and temporal) through a +3.0 D Badal optical system. Static recordings of the accommodation response were obtained for each accommodative level, at each field angle, with an objective, open-view, infrared optometer. RESULTS: Peripheral mean spherical equivalent (M) data showed that the emmetropic cohort exhibited relative myopic shifts into the periphery, while the myopic group showed hypermetropic shifts. Increasing accommodative demand did not alter the peripheral refractive profile in either the temporal (p = 0.25) or nasal (p = 0.07) periphery with no differential accommodative effect between refractive groups in either the temporal (p = 0.77) or nasal (p = 0.73) field. Significant shifts in the J(0) astigmatic component were seen in the temporal (p<0.0005) and nasal (p<0.0005) fields with increasing eccentricity. Interaction effects between eccentricity and accommodative demand illustrated that increasing accommodative demand significantly altered the peripheral refractive profile in the temporal J(0) astigmatic component (p<0.0005). The nasal periphery, however, failed to show such an effect (p = 0.65). CONCLUSIONS: Alterations in peripheral refraction augmented by changes in ocular accommodation are relatively unaffected by refractive error for young, healthy human eyes.

Profile of anisometropia and aniso-astigmatism in children; prevalence and association with age, ocular biometric measures, and refractive status

Purpose. We describe the profile and associations of anisometropia and aniso-astigmatism in a population-based sample of children. Methods. The Northern Ireland Childhood Errors of Refraction (NICER) study used a stratified random cluster design to recruit a representative sample of children from schools in Northern Ireland. Examinations included cycloplegic (1% cyclopentolate) autorefraction, and measures of axial length, anterior chamber depth, and corneal curvature. ?2 tests were used to assess variations in the prevalence of anisometropia and aniso-astigmatism by age group, with logistic regression used to compare odds of anisometropia and aniso-astigmatism with refractive status (myopia, emmetropia, hyperopia). The Mann-Whitney U test was used to examine interocular differences in ocular biometry. Results. Data from 661 white children aged 12 to 13 years (50.5% male) and 389 white children aged 6 to 7 years (49.6% male) are presented. The prevalence of anisometropia =1 diopters sphere (DS) did not differ statistically significantly between 6- to 7-year-old (8.5%; 95% confidence interval [CI], 3.9–13.1) and 12- to 13-year-old (9.4%; 95% CI, 5.9–12.9) children. The prevalence of aniso-astigmatism =1 diopters cylinder (DC) did not vary statistically significantly between 6- to 7-year-old (7.7%; 95% CI, 4.3–11.2) and 12- to 13-year-old (5.6%; 95% CI, 0.5–8.1) children. Anisometropia and aniso-astigmatism were more common in 12- to 13-year-old children with hyperopia =+2 DS. Anisometropic eyes had greater axial length asymmetry than nonanisometropic eyes. Aniso-astigmatic eyes were more asymmetric in axial length and corneal astigmatism than eyes without aniso-astigmatism. Conclusions. In this population, there is a high prevalence of axial anisometropia and corneal/axial aniso-astigmatism, associated with hyperopia, but whether these relations are causal is unclear. Further work is required to clarify the developmental mechanism behind these associations.

Entrepreneurial profile of the UK in the light of the Global Entrepreneurship and Development Index

In this research summary, we provide a novel look into the entrepreneurial profile of the UK in an international context. We use a new method – the Global Entrepreneurship and Development Index GEDI – to identify the entrepreneurial strengths and weaknesses of the UK economy, as well as to identify potential bottlenecks that hold back the performance of the UK relative to other advanced economies. We perform a Penalty for Bottleneck analysis to identify the bottlenecks in the UK's entrepreneurial profile. We also explore optimal resource allocation for UK's policy for National Systems of Entrepreneurship.

Patterns of refractive change in myopic subjects during the incipient phase of presbyopia:a preliminary study

Purpose: Changes in refractive error are well documented over the typical human lifespan. However, a relatively neglected period of investigation appears to be during the late fourth decade; this is at the incipient phase of presbyopia (IP), where the amplitude of accommodation is much reduced and approaches the level where a first reading addition is anticipated. Significantly, informal clinical observation has suggested a low incidence of an unexpected abrupt increase in myopia during IP. Methods: We investigated this alleged myopic shift retrospectively by mapping the longitudinal refraction histories of normally-sighted 35-44years old British White patients previously examined in routine optometric practice. The refractive trends in the right eyes of healthy myopic subjects (spherical equivalent refraction, SER =-0.50D: N=39) were analysed relative to that point at which a first near dioptric addition was considered to be clinically useful. Results: A refractive change was evident in some subjects during IP; viz, an abrupt increase in myopic SER of between -0.50 and -0.75D. These individuals (N=8) represented 20% of the study population of myopic incipient presbyopes. Beyond the pivotal point of the first near addition the longitudinal refraction stabilized in these subjects. In contrast, and as the extent of the available longitudinal data would permit, the remaining myopic eyes maintained an approximately stable refractive trend throughout IP and beyond. Conclusions: The anatomical or physiological basis of this specific late (non-developmental) abrupt myopic refractive change is an intriguing issue. Axial (vitreous chamber elongation), corneal (contour) and lenticular (profile and index) power bases, alone or in concert, might be considered candidates for this hitherto unexplored refractive phenomenon. Although necessarily obtained under conventional conditions of central (0deg) fixation, our data might also be a reflection of the recent recognition of the possible influence of the peripheral refraction upon the axial error. Consideration of this material provides an impetus for further research, including ocular biometry, a reappraisal of ciliary zonular functional anatomy, renewed investigation of the AC/A ratio, and the extent of a centripetal refractive influence on myopia development. © 2011 The College of Optometrists.

Analysis of the entrepreneurial profile of students attending higher education in Portugal: the Carland Entrepreneurship Index application

Background The societal changes have created new necessities in terms of health care and of professionals with diverse skills. The institutions of higher education should promote the development of an entrepreneurial profile in students which may boost the exploration of new opportunities. Objectives: To identify the entrepreneurial profile in the students in higher education and its relationship to some personal characteristics and training. Methods Correlational quantitative study, accomplished with 1,604 students from 18 institutions of polytechnic Institutes of Portugal. The collection of data occurred between July and November/2015. The Carland Entrepreneurship Index (CEI) entrepreneurial skills questionnaire was applied, along with the acquisition of socio-demographic variables of the students. Data was analysed with the SPSS 23.0. The study followed the ethical requirements. Results The (CEI) application, allowed us to note that 75.7 % of the students presented an entrepreneurial profile, 20.8 % a Micro entrepreneurial profile and 3.4 % a Macro entrepreneurial. Additionally, we verified that older students (r = 0.193, p < = 0.000), of the male gender (Female = 0.55; Male, M= 0.580 p < 0.000), that had already worked or would like to work for others, showed the greatest entrepreneurial potential (Yes M= 0.60; No 0.54; p < 0.000), along with those who participated in entrepreneurial contents during their training (Did not participate M= 0.53; Participated 0.58; p < 0.000). Conclusions The entrepreneurial profile is related with some social demographic characteristics of the students; however, the educational institutions may have a preponderant role in the development of that profile, which may contribute to a greater contribution in the wellbeing of the populations.

Magnesium-deficient High-fat Diet: Effects On Adiposity, Lipid Profile And Insulin Sensitivity In Growing Rats.

To determine if magnesium deficiency aggravates the effects of a high-fat diet in growing rats in terms of obesity, lipid profile and insulin resistance. The study population comprised 48 newly weaned male Wistar Hannover rats distributed into four groups according to diet, namely, control group (CT; n = 8), control diet provided ad libitum; pair-feeding control group (PF; n = 16), control diet but in the same controlled amount as animals that received high-fat diets; high-fat diet group (HF; n = 12), and magnesium-deficient high-fat diet group (HFMg(-); n = 12). The parameters investigated were adiposity index, lipid profile, magnesium status, insulin sensitivity and the phosphorylation of proteins involved in the insulin-signaling pathway, i.e. insulin receptor β-subunit, insulin receptor substrate 1 and protein kinase B. The HF and HFMg(-) groups were similar regarding gain in body mass, adiposity index and lipid profile, but were significantly different from the PF group. The HFMg(-) group exhibited alterations in magnesium homeostasis as revealed by the reduction in urinary and bone concentrations of the mineral. No inter-group differences were observed regarding glucose homeostasis. Protein phosphorylation in the insulin-signaling pathway was significantly reduced in the high-fat groups compared with the control groups, demonstrating that the intake of fat-rich diets increased insulin resistance, a syndrome that was aggravated by magnesium deficiency. Under the experimental conditions tested, the intake of a magnesium-deficient high-fat diet led to alterations in the insulin-signaling pathway and, consequently, increased insulin resistance.