Evolutions towards a new LSPR particle: Nano-sinusoid Progress in Electromagnetic Research (PIER)

This paper proposes a novel nano-sinusoid particle to be employed in enhanced localized surface plasmon resonance (LSPR) bio-sensing devices. Numerical investigations are carried out to demonstrate advantages o®ered by the proposed nano-particle on LSPR enhancement over other nano-particles including noble nano-triangles and nano-diamonds. Although nano-triangles exhibit high concentration of the electric ¯eld near their tips, when illuminated with a light polarized along the tip axis, they present only one hot spot at the vertex along the polarization direction. To create a structure with two hot spots, which is desired in bio-sensing applications, two nano-triangles can be put back-to-back. Therefore, a nano-diamond particle is obtained which exhibits two hot spots and presents higher enhancements than nano-triangles for the same resonant wavelength. The main drawback of the nano-diamonds is the °uctuation in their physical size-plasmon spectrum relationships, due to a high level of singularity as the result of their four sharp tip points. The proposed nano-sinusoid overcomes this disadvantage while maintaining the bene¯ts of having two hot spots and high enhancement

Plasmonic Nanostructures for Nano-Scale Bio-Sensing

The optical properties of various nanostructures have been widely adopted for biological detection, from DNA sequencing to nano-scale single molecule biological function measurements. In particular, by employing localized surface plasmon resonance (LSPR), we can expect distinguished sensing performance with high sensitivity and resolution. This indicates that nano-scale detections can be realized by using the shift of resonance wavelength of LSPR in response to the refractive index change. In this paper, we overview various plasmonic nanostructures as potential sensing components. The qualitative descriptions of plasmonic nanostructures are supported by the physical phenomena such as plasmonic hybridization and Fano resonance. We present guidelines for designing specific nanostructures with regard to wavelength range and target sensing materials

Localized surface plasmon resonance for biosensing lab-on-a-chip applications

In recent times, metallic nanoparticle plasmonics coupled with applications towards biosensing has gathered momentum to the point where commercial R&D are investing large resources in developing the so-called localized surface plasmon resonance (LSPR) biosensors. Conceptually, the main motivation for the research presented within this thesis is achievement of fully-operational LSPR biosensor interfaced with the state-of-the-art microfluidics, allowing for very precise control of sample manipulation and stable read-out. LSPR sensors are specifficaly engineered by electron beam lithography nanofabrication technique, where nanoparticle interactions are optimized to exhibit increased sensitivity and higher signal-to-noise ratio. However, the overall performance of LSPR lab-on-a-chip device depends critically on the biorecognition layer preparation in combination with surface passivation. As an introduction, the principles of plasmonic biosensing are identified encompassing both Surface Plasmon Resonance (SPR) and Localized SPR. Being successfully implemented into commercial product, the governing physics of SPR is compared to LSPR in chapter 1, together with advantages and disadvantages of both. Chapter 2 describes methods necessary for LSPR biosensor development, beginning with nano-fabrication methods, the modelling tool (COMSOL Multiphisics), while the basics of micro-fabrication in microfluidics conclude this chapter, where passive and active microfluidics networks are discerned. Particularly attractive optical properties are exhibited by closely-coupled nanoparticles (dimers), with the dielectric gap of below tens of nm, which were theoretically predicted to be very suitable as LSPR biosensing substrates. Chapter 3 is subjected to optical characterization (dependence on the size of the dielectric gap) of nanofabricated dimer arrays. The acquired data demonstrate the advantages of the nanofabrication methods presented in chapter 2 and the technique for fast and reliable determination of nanoparticle characteristic parameters. The initial biosensing-like experiments presented in chapter 4 (no integration with microfluidics) proved for the first time, the theoretical predictions of higher sensitivity, yielding additionally the specific response as function of analyte size and dielectric gap between nanoparticles. The overall response of different dimer arrays (various gaps) provides information about adopted conformation of analyte protein once immobilized. Broad resonances of dimers feature higher noise when employing them for the real-time LSPR biosensing. As a way to circumvent such problem, the feasibility of employing far-field interaction within the nanoparticle array to spectrally narrow resonance is investigated in chapter 5 by optimizing the array periodicity and introducing thin waveguiding layers. Finally, the concluding chapter 6 is dedicated to a full assembly of a Lab-on-a-chip (LOC) LSPR biosensor, starting with interfacing plasmonic substrates with compatible active microfluidic networks, allowing the precise sample delivery and multiplexing. The prototype device consisting of 8 individual sensors is presented with typical modes of operation. The bulk refractive index determination of various samples demonstrates the working principle of such device. Finally, various strategies of biorecognition layer formation are discussed within the on-going research

Plasmonic nanostructures for optical biosensing

In the last few decades, an increasing interest for nanotechnologies is spanning more and more fields of application thanks to the unique properties exhibited by metal nanomaterials if stimulated by external electromagnetic radiations. Indeed, a new research field called plasmonics is emerging and fast growing as a result of the recent technological progress and a deeper understanding of such phenomena. Recently, several types of plasmonic nanostructures are being conceived aiming at improving the performance of plasmon-based devices. For instance, sharp nanostructures exhibit higher field enhancement than smooth surfaces thereby representing a remarkable advantage in applications relying on signal amplification such as surface-enhanced Raman spectroscopy and plasmon-enhanced fluorescence. In addition, when nanostructures are ordered in periodic arrays, collective modes can arise as a result of the field coupling among the surface plasmons so as to promote the occurrence of impressive effects such as lattice resonances. Therefore, the possibility to tune the optical response of a nanostructure by tailoring the nanomaterial shape and size, as well as the structure arrangement, is spurring the researchers to explore new approaches, in terms of both nanofabrication and nano applications, in order to go beyond the current limits of many techniques. The aim of this work is to provide an understanding of this growing field of research and to convey the main features in biosensing applications. To date, several biosensor-based approaches including colorimetric and fluorescence analysis have been explored to effectively work alongside – or even replace – the gold standard methods in a wide variety of applications including environmental pollution monitoring and medical diagnostics. In this regard, optical biosensors offer a rapid, affordable, and practical approach in many fields of applications paving the way for point of care tests and high-throughput analysis. Fluorescence-based techniques are of growing interest since their potential high-throughput analysis, point of care applications, and improvable sensitivity through plasmon-enhanced fluorescence effect. On the other hand, when quickness, practicality, and easiness of use are preferred rather than extremely high sensitivity and accuracy, colorimetric biosensors relying on gold nanoparticles are the ideal candidates since their capability to produce a qualitative response in a few minutes visible by naked eye (a portable and handheld spectrophotometer can be employed if a quantitative measurement is required). The performance of colorimetric biosensors have been tested for detecting small molecules, such as 17β-estradiol in tap water down to picomolar level, and SARS-CoV-2 virions in naso-oropharyngeal swabs from hospital patients, whereas two-dimensional patterns of honeycomb-arranged and randomly positioned gold nanoparticles have been implemented in fluorescence-based malaria apta-immunoassays to effectively amplify the signal intensity through plasmon-enhanced fluorescence effect thereby attaining an ultrasensitive limit of detection at femtomolar level for detecting proteins in human whole blood

Plasmonic Nanostructures for Biosensor Applications

Improving the sensitivity of existing biosensors is an active research topic that cuts across several disciplines, including engineering and biology. Optical biosensors are the one of the most diverse class of biosensors which can be broadly categorized into two types based on the detection scheme: label-based and label-free detection. In label-based detection, the target bio-molecules are labeled with dyes or tags that fluoresce upon excitation, indicating the presence of target molecules. Label-based detection is highly-sensitive, capable of single molecule detection depending on the detector type used. One method of improving the sensitivity of label-based fluorescence detection is by enhancement of the emission of the labels by coupling them with metal nanostructures. This approach is referred as plasmon-enhanced fluorescence (PEF). PEF is achieved by increasing the electric field around the nano metal structures through plasmonics. This increased electric field improves the enhancement from the fluorophores which in turn improves the photon emission from the fluorophores which, in turn, improves the limit of detection. Biosensors taking advantage of the plasmonic properties of metal films and nanostructures have emerged an alternative, low-cost, high sensitivity method for detecting labeled DNA. Localized surface plasmon resonance (LSPR) sensors employing noble metal nanostructures have recently attracted considerable attention as a new class of plasmonic nanosensors.;In this work, the design, fabrication and characterization of plasmonic nanostructures is carried out. Finite difference time domain (FDTD) simulations were performed using software from Lumerical Inc. to design a novel LSPR structure that exhibit resonance overlapping with the absorption and emission wavelengths of quantum dots (QD). Simulations of a composite Au/SiO2 nanopillars on silicon substrate were performed using FDTD software to show peak plasmonic enhancement at QD emission wavelength (560nm). A multi-step fabrication process was developed to create plasmonic nanostructures, and the optical characterization of emission enhancement was performed

Harnessing the plasmonic properties of gold nanoparticles: functionalization strategies coupled with novel spectroscopic tools

Metallic plasmonic substrates such as gold nanoparticles (AuNPs) have fascinated researchers due to their usefulness in verious interdisciplinary studies at the interface between applied physics, biochemistry, engineering, and medicine. A good understanding of the physics of these noble nanostructures, particularly the plasmonic and optical properties, can be employed to improve a wide range of sensors and electronic devices. The relevance of molecular recognition and the binding of biological and chemical entities to diagnostics, biosensors, and drug delivery has attracted significant research interest. By addressing material functionalization design and advanced characterization methods, this doctoral work aims to highlight efforts to exploit the surface modification strategies to enhance the responsiveness of nanoparticle substrates for improved detection of health-relevant biomolecules. The self-assembly of small ligands, such as alkanethiols, and oligonucleotides on the surface of AuNPs provided a possible starting route for the preparation of bio-nanomaterials with precise physicochemical properties. The versatile AuNPs were optimized and thoroughly characterized by employing electron microscopy techniques such as transmission electron microscope (TEM), atomic force microscopy (AFM), and scanning electron microscopy (SEM), spectroscopic techniques, including ultraviolet/visible (UV/Vis), dynamic light scattering (DLS), and thermal lens spectrometry (TLS), and biochemical assays (gel electrophoresis, Dot plot, Western plot, and the Enzyme Linked Immunosorbent Assay (ELISA)). Subsequently, the molecular recognition capabilities of functionalized AuNPs were investigated using multiple techniques, including novel detection routes such as the electrophoresis approach coupled with online TLS. This work establishes a versatile platform for AuNP engineering with controlled size and surface functionality. The strategies presented in this thesis aim to improve medical diagnostics to make them affordable for point-of-care scenarios to enhance the quality of human health.wide range of sensors and electronic devices. The relevance of molecular recognition and the binding of biological and chemical entities to diagnostics, biosensors, and drug delivery has attracted significant research interest. By addressing material functionalization design and advanced characterization methods, this doctoral work aims to highlight efforts to exploit the surface modification strategies to enhance the responsiveness of nanoparticle substrates for improved detection of health-relevant biomolecules. The self-assembly of small ligands, such as alkanethiols and oligonucleotides on the surface of AuNPs provided a possible starting route for the preparation of bio-nanomaterials with precise physicochemical properties. The versatile AuNPs were optimized and thoroughly characterized by employing electron microscopy techniques such as transmission electron microscope (TEM), atomic force microscopy (AFM), and scanning electron microscopy (SEM), spectroscopic techniques, including ultraviolet/visible (UV/Vis), dynamic light scattering (DLS), and thermal lens spectrometry (TLS), and biochemical assays (gel electrophoresis, Dot plot, Western plot, and the Enzyme Linked Immunosorbent Assay (ELISA)). Subsequently, the molecular recognition capabilities of functionalized AuNPs were investigated using multiple techniques, including novel detection routes such as the electrophoresis approach coupled with online TLS. This work establishes a versatile platform for AuNP engineering with controlled size and surface functionality. The strategies presented in this thesis aim to improve medical diagnostics to make them affordable for point-of-care scenarios to enhance the quality of human health

Characterisation of self-assembled engineered proteins on gold nanoparticles and their application to biosensing

PhD ThesisThe use of gold nanoparticles (AuNP) has a long and varied history, thought to cover several thousand years. More recently the unique properties of nanoscale materials have stimulated extensive work on nanoparticles and other nanomaterials leading to their use in novel technologies. AuNPs have been of particular interest for bioscience applications due to their biocompatibility and the ease with which biological molecules can be conjugated to their surface. In this study the assembly of engineered proteins, specifically the transmembrane domain of Escherichia coli outer membrane protein A (OmpATM), onto the surface of AuNPs was investigated both in solution and with the particles attached to a SiO2 substrate. AuNPs were adhered to SiO2 surfaces using a novel silane treatment developed by the industrial sponsor and were characterised using spectroscopy, electron and atomic force microscopy. The addition of a single cysteine residue to the OmpATM structure was shown, by UV-Vis and fluorescence spectroscopy, to increase protein binding at equilibrium and form higher stability protein-AuNP complexes in solution. Following this, engineered OmpATM proteins containing tandem antibody-binding domains from Streptococcal protein G were assembled on the AuNP surface and their structure interrogated using neutron and light scattering. This revealed an oriented protein layer where the functional domains extend away from the AuNP surface and are available to bind antibodies. OmpATM-AuNP conjugates were used to develop biosensing assays using both well-established methods, such as lateral flow assays, and novel spectroscopic methods, which use the unique optical properties of AuNPs. Detection of influenza A nucleoprotein, an antigen used to clinically diagnose influenza, was achieved using a bespoke anti-nucleoprotein single-chain antibody domain fused to OmpATM and assembled on 20 nm diameter AuNPs. The results demonstrate that engineered OmpATM proteins conjugated to AuNPs can be used to develop novel diagnostics using a range of read out technologies

Doctor of Philosophy

dissertationPlasmonic nanomaterials have tunable optical properties that are exploited for sensing applications. When designing nanoparticle substrates, trade-offs are often made: sensitivity or stability, customization or cost. To narrow the gap of these trade-offs

Gold nanorods functionalized with DNA oligonucleotide probes for biosensing and plasmon-enhanced fluorescence detection

Gold nanorods display plasmon resonances that are very sensitive to the refraction index close to the particle’s surface. The site-selective functionalization of Plasmon hot-spots with bioreceptors is crucial to develop plasmonic sensors with improved response bycapturing the target species at the most sensitive regions of the particle. Firstly, we used surface immobilized biotin-functionalized gold nanorods for streptavidin sensing.The selective functionalization of the nanorods’ tips was achieved with a CTAB bilayer and using a thiol linker to attach the desired biotin functionality. The sensor performance was characterized by measuring binding kinetic assays. In the recent years, Dengue virus DENV-2 has been reported as the largest dengue epidemic type and early stage detection of this virus would save the life of many patients. Thus, a plasmonic model biosensor was designed for the detection of RNA sequences proposed as disease biomarkers for Dengue virus.For this purpose, we have functionalized gold nanorods with thiolated DNA oligonucleotide probes complementary to a RNA sequence of Dengue virus.As a signal amplification strategy, we have used biotin-labeled oligonucleotide target sequences, in order to bind streptavidin or anti-biotin antibody to increase the surface plasmon response. Plasmon-enhanced fluorescence (PEF) microscopy provides fast, high-contrast, and lowbackground detection of single molecules. The interaction between the localized surface plasmon of gold nanorods and a fluorophore in their vicinity can induce the acceleration of excitation and decay rates thus leading to substantial fluorescence enhancements. In the third part of this Thesis, it was studied the interaction between gold nanorod antennas and a weakly fluorescence dye, TMPyP porphyrin. This interaction was mediated by electrostatic attraction between the tetracationic TMPyP and the DNA oligonucleotide coating on the nanorods’ surface. Preliminary measurements of optical spectroscopy were carried out to characterize the interaction in solution of TMPyP and single or double-stranded DNA oligonucleotides complementary to a RNA sequence of Dengue virus.The apparent equilibrium constants for the complex of TMPyP with single and double-stranded DNA were determined to be Ka= 3.9×107 M-1and 4.5×107 M-1respectively. The spectral changes show a strong specific intercalation of TMPyP with ds-DNA and ss-DNA because of GC-rich sites in the selected sequences. Next, the plasmon-enhanced fluorescence of TMPyP induced by gold nanorods was investigated using confocal fluorescence lifetime microscopy to perform measurements of nanoparticle emission intensity and spectrum, fluorescence correlation spectroscopy, emission intensity time trace and fluorescence decay. The gold nanorods were immobilized on glass and functionalized with a thiolated oligonucleotide coating, while TMPyP molecules are diffusing in solution and stochastically interact with the rod’s surface. The emission intensity traces measured on single particles show strong fluorescence bursts when TMPyP molecules come into close proximity of the nanorod. We have calculated the emission enhancement factors from a comparison with the non-enhanced emission of TMPyP in the same experimental conditions and found surprisingly large enhancement factors of around 60000-fold for TMPyP’s emission.These values of enhancement are two orders of magnitude larger than our calculated highest enhanced fluorescence expected for TMPyP molecule.Os nano-bastonetes de ouro são caracterizados por plasmões de superfície com frequências de ressonância bastante sensíveis ao índice de refração na proximidade da sua superfície. A funcionalização seletiva da superfície destas nanopartículas com bio-receptores é crucial para o desenvolvimento de sensores plasmónicos com resposta melhorada, pois permite a captura de analitos nas regiões mais sensíveis da nanopartícula. Em primeiro lugar foram preparadas superfícies com nano-bastonetes de ouro que depois foram funcionalizados com recetores biotina para ensaios modelo de deteção de estreptavidina. A funcionalização seletiva das extremidades dos nano-bastonetes foi conseguida através da proteção das suas paredes laterais com uma bicamada de tensioativo CTAB e usando uma biotina derivatizada com uma função tiól. O desempenho do sensor foi caracterizado por medidas da cinética de associação biotina-estreptavidina monitorizada por espectroscopia ótica de absorção. Em anos recentes, a infeção pelo vírus do Dengue DENV-2 tem sido relatada como a maior epidemia por este tipo de vírus, e a deteção precoce desta infeção poderia salvar a vida de muitos pacientes. Deste modo, foi desenhado um sensor plasmónico modelo para a deteção de sequências de ARN propostas como bio-marcadores para a infeção pelo vírus do Dengue. Para o efeito, foram funcionalizados nano-bastonetes de ouro com cadeias de oligonucleotídos de ADN complementares a uma sequência do ARN do vírus do Dengue. Como estratégia de amplificação de sinal foram usadas cadeias de oligonucleotídos alvo marcadas com biotina, de modo a ser possível num segundo passo ligar estreptavidina ou anticorpo anti-biotina com o objetivo de aumentar a resposta do plasmão de superfície dos nano-bastonetes de ouro. A fluorescência intensificada por efeito plasmónico permite a deteção rápida e com elevado contraste de molécula única em microscopia de fluorescência. A interação entre os modos localizados de plasmão de superfície de nano-bastonetes de ouro e moléculas fluorescentes na sua proximidade pode induzir a aceleração das taxas de excitação, decaimento radiativo e não-radiativo, e conduzir a uma intensificação de fluorescência.Na terceira parte desta Dissertação, foram investigadas as interações entre nano-antenas de ouro e um cromóforo pouco fluorescente, a porfirina TMPyP. Esta interação foi mediada pela atração eletrostática entre a porfirina tetra-catiónica e o revestimento de ADN na superfície dos nano-bastonetes de ouro. Ensaios preliminares de espectroscopia ótica foram realizados para caracterizar a interação em solução da TMPyP com sequências de ADN de cadeia simples ou duplacomplementares a uma sequência do ARN do vírus do Dengue. A constante aparente de equilíbrio para o complexo da TMPyP com as sequências de ADN de cadeia simples e dupla foram determinadas como sendo Ka= 3.9×107 M-1and 4.5×107 M-1, respetivamente. As alterações dos espectros de absorção e emissão mostram uma forte interação, provavelmente intercalação, daTMPyPcom ods-DNA,etambém com o ss-DNA, devido ao elevado conteúdo em pares GC nas sequências escolhidas. Em seguida, a fluorescência intensificada por efeito plasmónico na TMPyP induzida por nano-bastonetes de ouro foi investigada por microscopia confocal de tempos-de-vida, tendo sido realizadas medidas de intensidade e espectro de emissão de nanopartículas, espectroscopia de correlação de fluorescência, traços temporais de intensidade de emissão e de decaimento de fluorescência.Os nano-bastonetes de ouro foram imobilizados em vidro e funcionalizados com um revestimento de oligonucleotídostiolados, enquanto que as moléculas de TMPyP difundem-se em solução e podem interatuar estocasticamente com a superfície da nanopartícula. Os traços de intensidade de emissão medidos em nanopartículas individuais mostram picos de fluorescência intensos quando as moléculas de TMPyP se aproximam do nano-bastonete de ouro em resultado do efeito de nano-antena.Foram calculados os fatores de emissão intensificada por comparação com a emissão não-intensificada da TMPyP nas mesmas condições experimentais e obtiveram-se valores surpreendentemente elevados de cerca de 60000 vezes para a emissão intensificada da TMPyP. Estes fatores de intensificação são duas ordens de grandeza mais elevados do que as estimativas teóricas calculadas para a intensificação da emissão da TMPyP pelos nanobastonetes de ouro

CONTROL OF AVERAGE SPACING OF OMCVD GROWN GOLD NANOPARTICLES

Metallic nanostructures and their applications is a rapidly expanding field. Nobel metals such as silver and gold have historically been used to demonstrate plasmon effects due to their strong resonances, which occur in the visible part of the electromagnetic spectrum. Localized surface plasmon resonance (LSPR) produces an enhanced electromagnetic field at the interface between a gold nanoparticle (Au NP) and the surrounding dielectric. This enhanced field can be used for metal-dielectric interface- sensitive optical interactions that form a powerful basis for optical sensing. In addition to the surrounding material, the LSPR spectral position and width depend on the size, shape, and average spacing between these particles. Au NP LSPR based sensors depict their highest sensitivity with optimized parameters and usually operate by investigating absorption peak shifts. The absorption peak of randomly deposited Au NPs on surfaces is mostly broad. As a result, the absorption peak shifts, upon binding of a material onto Au NPs might not be very clear for further analysis. Therefore, novel methods based on three well-known techniques, self-assembly, ion irradiation, and organo-metallic chemical vapour deposition (OMCVD) are introduced to control the average-spacing between Au NPs. In addition to covalently binding and other advantages of OMCVD grown Au NPs, interesting optical features due to their non- spherical shapes are presented. The first step towards the average-spacing control is to uniformly form self- assembled monolayers (SAMs) of octadecyltrichlorosilane (OTS) as resists for OMCVD Au NPs. The formation and optimization of the OTS SAMs are extensively studied. The optimized resist SAMs are ion-irradiated by a focused ion beam (FIB) and ions generated by a Tandem accelerator. The irradiated areas are refilled with 3-mercaptopropyl- trimethoxysilane (MPTS) to provide nucleation sites for the OMCVD Au NP growth. Each step during sample preparation is monitored by using surface characterization methods such as contact angle measurements, ellipsometry, X-ray photoelectron iii spectroscopy (XPS), scanning electron microscopy (SEM), atomic force microscopy (AFM), Rutherford backscattering spectroscopy (RBS), UV-Visible spectroscopy, and time-of-flight secondary ion mass spectroscopy (ToF-SIMS)