Deploying aptameric sensing technology for rapid pandemic monitoring

The genome of virulent strains may possess the ability to mutate by means of antigenic shift and/or antigenic drift as well as being resistant to antibiotics with time. The outbreak and spread of these virulent diseases including avian influenza (H1N1), severe acute respiratory syndrome (SARS-Corona virus), cholera (Vibrio cholera), tuberculosis (Mycobacterium tuberculosis), Ebola hemorrhagic fever (Ebola Virus) and AIDS (HIV-1) necessitate urgent attention to develop diagnostic protocols and assays for rapid detection and screening. Rapid and accurate detection of first cases with certainty will contribute significantly in preventing disease transmission and escalation to pandemic levels. As a result, there is a need to develop technologies that can meet the heavy demand of an all-embedded, inexpensive, specific and fast biosensing for the detection and screening of pathogens in active or latent forms to offer quick diagnosis and early treatments in order to avoid disease aggravation and unnecessary late treatment costs. Nucleic acid aptamers are short, single-stranded RNA or DNA sequences that can selectively bind to specific cellular and biomolecular targets. Aptamers, as new-age bioaffinity probes, have the necessary biophysical characteristics for improved pathogen detection. This article seeks to review global pandemic situations in relation to advances in pathogen detection systems. It particularly discusses aptameric biosensing and establishes application opportunities for effective pandemic monitoring. Insights into the application of continuous polymeric supports as the synthetic base for aptamer coupling to provide the needed convective mass transport for rapid screening is also presented.

Uso de aptâmeros na sexagem de sêmen bovino

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Supramolecular functional assemblies with photo or chemical addressability

Supramolecular self-assembly represents a key technology for the spontaneous construction of nanoarchitectures and for the fabrication of materials with enhanced physical and chemical properties. In addition, a significant asset of supramolecular self-assemblies rests on their reversible formation, thanks to the kinetic lability of their non-covalent interactions. This dynamic nature can be exploited for the development of “self-healing” and “smart” materials towards the tuning of their functional properties upon various external factors. One particular intriguing objective in the field is to reach a high level of control over the shape and size of the supramolecular architectures, in order to produce well-defined functional nanostructures by rational design. In this direction, many investigations have been pursued toward the construction of self-assembled objects from numerous low-molecular weight scaffolds, for instance by exploiting multiple directional hydrogen-bonding interactions. In particular, nucleobases have been used as supramolecular synthons as a result of their efficiency to code for non-covalent interaction motifs. Among nucleobases, guanine represents the most versatile one, because of its different H-bond donor and acceptor sites which display self-complementary patterns of interactions. Interestingly, and depending on the environmental conditions, guanosine derivatives can form various types of structures. Most of the supramolecular architectures reported in this Thesis from guanosine derivatives require the presence of a cation which stabilizes, via dipole-ion interactions, the macrocyclic G-quartet that can, in turn, stack in columnar G-quadruplex arrangements. In addition, in absence of cations, guanosine can polymerize via hydrogen bonding to give a variety of supramolecular networks including linear ribbons. This complex supramolecular behavior confers to the guanine-guanine interactions their upper interest among all the homonucleobases studied. They have been subjected to intense investigations in various areas ranging from structural biology and medicinal chemistry – guanine-rich sequences are abundant in telomeric ends of chromosomes and promoter regions of DNA, and are capable of forming G-quartet based structures– to material science and nanotechnology. This Thesis, organized into five Chapters, describes mainly some recent advances in the form and function provided by self-assembly of guanine based systems. More generally, Chapter 4 will focus on the construction of supramolecular self-assemblies whose self-assembling process and self-assembled architectures can be controlled by light as external stimulus. Chapter 1 will describe some of the many recent studies of G-quartets in the general area of nanoscience. Natural G- quadruplexes can be useful motifs to build new structures and biomaterials such as self-assembled nanomachines, biosensors, therapeutic aptamer and catalysts. In Chapters 2-4 it is pointed out the core concept held in this PhD Thesis, i.e. the supramolecular organization of lipophilic guanosine derivatives with photo or chemical addressability. Chapter 2 will mainly focus on the use of cation-templated guanosine derivatives as a potential scaffold for designing functional materials with tailored physical properties, showing a new way to control the bottom-up realization of well-defined nanoarchitectures. In section 2.6.7, the self-assembly properties of compound 28a may be considered an example of open-shell moieties ordered by a supramolecular guanosine architecture showing a new (magnetic) property. Chapter 3 will report on ribbon-like structures, supramolecular architectures formed by guanosine derivatives that may be of interest for the fabrication of molecular nanowires within the framework of future molecular electronic applications. In section 3.4 we investigate the supramolecular polymerizations of derivatives dG 1 and G 30 by light scattering technique and TEM experiments. The obtained data reveal the presence of several levels of organization due to the hierarchical self-assembly of the guanosine units in ribbons that in turn aggregate in fibrillar or lamellar soft structures. The elucidation of these structures furnishes an explanation to the physical behaviour of guanosine units which display organogelator properties. Chapter 4 will describe photoresponsive self-assembling systems. Numerous research examples have demonstrated that the use of photochromic molecules in supramolecular self-assemblies is the most reasonable method to noninvasively manipulate their degree of aggregation and supramolecular architectures. In section 4.4 we report on the photocontrolled self-assembly of modified guanosine nucleobase E-42: by the introduction of a photoactive moiety at C8 it is possible to operate a photocontrol over the self-assembly of the molecule, where the existence of G-quartets can be alternately switched on and off. In section 4.5 we focus on the use of cyclodextrins as photoresponsive host-guest assemblies: αCD–azobenzene conjugates 47-48 (section 4.5.3) are synthesized in order to obtain a photoresponsive system exhibiting a fine photocontrollable degree of aggregation and self-assembled architecture. Finally, Chapter 5 contains the experimental protocols used for the research described in Chapters 2-4.

Rupture forces of split aptamers

Rupture forces of ligand-receptor interactions, such as proteins-proteins, proteins-cells, and cells-tissues, have been successfully measured by atomic force spectroscopy (AFS). For these measurements, the ligands and receptors were chemically modified so that they can be immobilized on the tip and on a substrate, respectively. The ligand interact the receptor when the tip approaches the substrate. This interaction can be studied by measuring rupture force upon retraction. However, this technique is not feasible for measurements involving small molecules, since they form only few H-bonds with their corresponding receptors. Modifying small molecules for immobilization on surfaces may block or change binding sites. Thus, recorded rupture forces might not reflect the full scope of the involved small ligand-receptor interactions.rnIn my thesis, a novel concept that allows measuring the rupture force of small involved ligand-receptor interactions and does not require molecular modification for immobilization was introduced. The rupture force of small ligand-receptor interaction is not directly measured but it can be determined from measurements in the presence and in the absence of the ligand. As a model system, the adenosine mono phosphate (AMP) and the aptamer that binds AMP were selected. The aptamer (receptor) is a single stranded DNA that can partially self-hybridize and form binding pockets for AMP molecules (ligands). The bonds between AMP and aptamer are provided by several H-bonds and pair stacking.rnIn the novel concept, the aptamer was split into two parts (oligo a and oligo b). One part was immobilized on the tip and the other one on the substrate. Approaching the tip to the substrate, oligo a and oligo b partially hybridized and the binding pockets were formed. After adding AMP into the buffer solution, the AMP bound in the pockets and additional H-bonds were formed. Upon retraction of the tip, the rupture force of the AMP-split aptamer complex was measured. In the presence of excess AMP, the rupture force increased by about 10 pN. rnThe dissociation constant of the AMP-split aptamer complex was measured on a single molecular level (~ 4 µM) by varying the AMP concentrations and measuring the rupture force at each concentration. Furthermore, the rupture force was amplified when more pockets were added to the split aptamer. rnIn the absence of AMP, the thermal off-rate was slightly reduced compared to that in the presence of AMP, indicating that the AMP stabilized the aptamer. The rupture forces at different loading rates did not follow the logarithmic fit which was usually used to describe the dependence of rupture forces at different loading rates of oligonucleotides. Two distinguished regimes at low and high loading rates were obtained. The two regimes were explained by a model in which the oligos located at the pockets were stretched at high loading rates. rnThe contribution of a single H-bond formed between the AMP molecule and the split aptamer was measured by reducing the binding groups of the AMP. The rupture forces reduce corresponding to the reduction of the binding groups. The phosphate group played the most important role in the formation of H-bond network between the AMP molecule and the split aptamer. rn

Sensing applications of biofunctionalised plasmonic gold nanoparticles

Plasmonische Metallnanopartikel bündeln, verstärken und beeinflussen Licht auf nanoskopischer Ebene. Diese grundlegende Eigenschaft kommt von koheränten, kollektiven Schwingungen der Leitungsbandelektronen, die von einfallendem Licht resonant angeregt und lokalisierte Oberflächenplasmonenresonanz (LSPR) oder ‚Partikelplasmonen‘ genannt werden. Plasmonen in Metallnanopartikeln wurden bisher z.B. zur Erkennen von pathogenen Biomolekülen, bei der photothermischen Therapie und zur Verbesserung der Effizienz von Solarzellen verwendet. In dieser Arbeit werde ich meinen Fokus auf die Synthese und Funktionalisierung von Goldnanopartikeln zur Anwendung als Sensoren legen.rnrnKürzliche Verbesserungen in der nasschemischen Synthese haben zur Herstellung von Goldnanopartikel mit unterschiedlichen Formen und Größen geführt, die sich in ihren Sensoreigenschaften unterscheiden. Unter den unterschiedlichen Sensorgeometrien sind Goldnanostäbchen die bevorzugte Form zur Biomolekül-Sensorik durch LSPR. Nanostäbchen werden durch eine positiv geladene CTAB-Schicht stabilisiert, die Proteine bei neutralem pH-Wert anziehen kann. Die Adsorption und Desorption von Proteinen an der Nanopartikeloberfläche und damit die Bindungskinetiken von Proteinen kann auf Einzelmolekülebene erforscht werden. Ich zeige hier eine Studie mit hoher örtlicher und zeitlicher Auflösung um einzelne Bindungsereignisse von Fibronectin auf Goldnanostäbchen darzustellen.rnrnGoldnanostäbchen müssen mit spezifischen biologischen Erkennungselementen funktionalisiert werden um eine Analyterkennung oder Proteinwechselwirkung zu erreichen. Ich funktionalisiere Goldnanostäbchen mit kurzen DNA-Sequenzen (Aptamer-Sequenzen und NTA konjugierten Polihymidinen) und habe anhand diese unterschiedlich sensitiven Partikel eine Studie mit verschiedenen Analyten (oder Protein-Protein Wechselwirkungen) erfolgreich durchgeführt.rn rnPlasmonen von Nanopartikel-Clustern koppeln miteinander, was ihre Resonanzenergie ändert. Der kontrollierte Zusammenbau von Nanopartikeln zu Dimeren oder höher geordneten Strukturen wie ‚Core-Satellites‘ können dazu dienen ihre Sensitivität zu erhöhen. Diese Cluster bieten eine hohe Sensitivität auf Grund der Anwesenheit von plasmonischen Hotspots in der Lücke zwischen zwei Partikeln. Die Plasmonkopplung ist ein Phänomen, das abhängig vom Abstand zweier Partikel zueinander ist und bildet somit die Basis von sogenannten Plasmon-Linealen. Ich habe eine Strategie entwickelt um Dimere aus Hsp90 funktionalisierten Goldnanosphären zu bilden. Diese Technik wird nicht durch Ausbleichen oder das Blinken von Farbstoffen limitiert und ich zeige zum ersten Mal wie man dadurch dynamische Proteinkonformationen untersuchen kann.rn

Effect of the REG1 anticoagulation system versus bivalirudin on outcomes after percutaneous coronary intervention (REGULATE-PCI): a randomised clinical trial.

BACKGROUND REG1 is a novel anticoagulation system consisting of pegnivacogin, an RNA aptamer inhibitor of coagulation factor IXa, and anivamersen, a complementary sequence reversal oligonucleotide. We tested the hypothesis that near complete inhibition of factor IXa with pegnivacogin during percutaneous coronary intervention, followed by partial reversal with anivamersen, would reduce ischaemic events compared with bivalirudin, without increasing bleeding. METHODS We did a randomised, open-label, active-controlled, multicentre, superiority trial to compare REG1 with bivalirudin at 225 hospitals in North America and Europe. We planned to randomly allocate 13,200 patients undergoing percutaneous coronary intervention in a 1:1 ratio to either REG1 (pegnivacogin 1 mg/kg bolus [>99% factor IXa inhibition] followed by 80% reversal with anivamersen after percutaneous coronary intervention) or bivalirudin. Exclusion criteria included ST segment elevation myocardial infarction within 48 h. The primary efficacy endpoint was the composite of all-cause death, myocardial infarction, stroke, and unplanned target lesion revascularisation by day 3 after randomisation. The principal safety endpoint was major bleeding. Analysis was by intention to treat. This trial is registered at ClinicalTrials.gov, identifier NCT01848106. The trial was terminated early after enrolment of 3232 patients due to severe allergic reactions. FINDINGS 1616 patients were allocated REG1 and 1616 were assigned bivalirudin, of whom 1605 and 1601 patients, respectively, received the assigned treatment. Severe allergic reactions were reported in ten (1%) of 1605 patients receiving REG1 versus one (<1%) of 1601 patients treated with bivalirudin. The composite primary endpoint did not differ between groups, with 108 (7%) of 1616 patients assigned REG1 and 103 (6%) of 1616 allocated bivalirudin reporting a primary endpoint event (odds ratio [OR] 1·05, 95% CI 0·80-1·39; p=0·72). Major bleeding was similar between treatment groups (seven [<1%] of 1605 receiving REG1 vs two [<1%] of 1601 treated with bivalirudin; OR 3·49, 95% CI 0·73-16·82; p=0·10), but major or minor bleeding was increased with REG1 (104 [6%] vs 65 [4%]; 1·64, 1·19-2·25; p=0·002). INTERPRETATION The reversible factor IXa inhibitor REG1, as currently formulated, is associated with severe allergic reactions. Although statistical power was limited because of early termination, there was no evidence that REG1 reduced ischaemic events or bleeding compared with bivalirudin. FUNDING Regado Biosciences Inc.

Cytoplasmic RNA modulators of an inside-out signal-transduction cascade

A vaccinia virus-based RNA expression system enabled high-level cytoplasmic expression of RNA aptamers directed against the intracellular domain of the β2 integrin LFA-1, a transmembrane protein that mediates cell adhesion to intercellular adhesion molecule-1 (ICAM-1). In two different cell types, cytoplasmic expression of integrin-binding aptamers reduced inducible cell adhesion to ICAM-1. The aptamers specifically target, and thereby define, a functional cytoplasmic subdomain important for the regulation of cell adhesion in leukocytes. Our approach of aptamer-controlled blocking of signaling pathways in vivo could potentially be applied wherever targeted modulation of a signal-transduction cascade is desired.

Bioactive and nuclease-resistant l-DNA ligand of vasopressin

In vitro selection experiments have produced nucleic acid ligands (aptamers) that bind tightly and specifically to a great variety of target biomolecules. The utility of aptamers is often limited by their vulnerability to nucleases present in biological materials. One way to circumvent this problem is to select an aptamer that binds the enantiomer of the target, then synthesize the enantiomer of the aptamer as a nuclease-insensitive ligand of the normal target. We have so identified a mirror-image single-stranded DNA that binds the peptide hormone vasopressin and have demonstrated its stability to nucleases and its bioactivity as a vasopressin antagonist in cell culture.

Targeting cyclin-dependent kinases in Drosophila with peptide aptamers

Two-hybrid technology provides a simple way to isolate small peptide aptamers that specifically recognize and strongly bind to a protein of interest. These aptamers have the potential to dominantly interfere with specific activities of their target proteins and, therefore, could be used as in vivo inhibitors. Here we explore the ability to use peptide aptamers as in vivo inhibitors by expressing aptamers directed against cell cycle regulators in Drosophila. We expressed two peptide aptamers, each of which specifically recognizes one of the two essential cyclin-dependent kinases (Cdks), DmCdk1 and DmCdk2, in Drosophila. Expression of each Cdk aptamer during organogenesis caused adult eye defects typical of those caused by cell cycle inhibition. Co-overexpression of DmCdk1 or DmCdk2 resulted in suppression of the eye phenotypes, indicating that each aptamer interacts with a Cdk target in vivo and suggesting that these peptides disrupt normal eye development by inhibiting Cdk function. Moreover, the specificity of each aptamer for one of the two Cdks as determined in two-hybrid assays was retained in Drosophila. Combined, our results demonstrate that peptide aptamers generated by yeast two-hybrid methods can serve as inhibitory reagents to target specific proteins in vivo.

An artificial cell-cycle inhibitor isolated from a combinatorial library

Understanding the genetic networks that operate inside cells will require the dissection of interactions among network members. Here we describe a peptide aptamer isolated from a combinatorial library that distinguishes among such interactions. This aptamer binds to cyclin-dependent kinase 2 (Cdk2) and inhibits its kinase activity. In contrast to naturally occurring inhibitors, such as p21Cip1, which inhibit the activity of Cdk2 on all its substrates, inhibition by pep8 has distinct substrate specificity. We show that the aptamer binds to Cdk2 at or near its active site and that its mode of inhibition is competitive. Expression of pep8 in human cells retards their progression through the G1 phase of the cell cycle. Our results suggest that the aptamer inhibits cell-cycle progression by blocking the activity of Cdk2 on substrates needed for the G1-to-S transition. This work demonstrates the feasibility of selection of artificial proteins to perform functions not developed during evolution. The ability to select proteins that block interactions between a gene product and some partners but not others should make sophisticated genetic manipulations possible in human cells and other currently intractable systems.

Sephadex-binding RNA ligands: rapid affinity purification of RNA from complex RNA mixtures

Sephadex-binding RNA ligands (aptamers) were obtained through in vitro selection. They could be classified into two groups based on their consensus sequences and the aptamers from both groups showed strong binding to Sephadex G-100. One of the highest affinity aptamers, D8, was chosen for further characterization. Aptamer D8 bound to dextran B512, the soluble base material of Sephadex, but not to isomaltose, isomaltotriose and isomaltotetraose, suggesting that its optimal binding site might consist of more than four glucose residues linked via α-1,6 linkages. The aptamer was very specific to the Sephadex matrix and did not bind appreciably to other supporting matrices, such as Sepharose, Sephacryl, cellulose or pustulan. Using Sephadex G-100, the aptamer could be purified from a complex mixture of cellular RNA, giving an enrichment of at least 60 000-fold, compared with a non-specific control RNA. These RNA aptamers can be used as affinity tags for RNAs or RNA subunits of ribonucleoproteins to allow rapid purification from complex mixtures of RNA using only Sephadex.

Controlling small guanine–nucleotide-exchange factor function through cytoplasmic RNA intramers

ADP-ribosylation factor (ARF) GTPases and their regulatory proteins have been implicated in the control of diverse biological functions. Two main classes of positive regulatory elements for ARF have been discovered so far: the large Sec7/Gea and the small cytohesin/ARNO families, respectively. These proteins harbor guanine–nucleotide-exchange factor (GEF) activity exerted by the common Sec7 domain. The availability of a specific inhibitor, the fungal metabolite brefeldin A, has enabled documentation of the involvement of the large GEFs in vesicle transport. However, because of the lack of such tools, the biological roles of the small GEFs have remained controversial. Here, we have selected a series of RNA aptamers that specifically recognize the Sec7 domain of cytohesin 1. Some aptamers inhibit guanine–nucleotide exchange on ARF1, thereby preventing ARF activation in vitro. Among them, aptamer M69 exhibited unexpected specificity for the small GEFs, because it does not interact with or inhibit the GEF activity of the related Gea2-Sec7 domain, a member of the class of large GEFs. The inhibitory effect demonstrated in vitro clearly is observed as well in vivo, based on the finding that M69 produces similar results as a dominant-negative, GEF-deficient mutant of cytohesin 1: when expressed in the cytoplasm of T-cells, M69 reduces stimulated adhesion to intercellular adhesion molecule-1 and results in a dramatic reorganization of F-actin distribution. These highly specific cellular effects suggest that the ARF-GEF activity of cytohesin 1 plays an important role in cytoskeletal remodeling events of lymphoid cells.

Preparação e caracterização de nanopartículas de prata para aplicação no desenvolvimento de imunoensaio para imunoglobulina G humana

Neste trabalho, anticorpos anti-IgGh foram conjugados às nanopartículas de prata (NPAg) para detectar imunoglobulina G humana (IgGh). Um imunoensaio colorimétrico baseado na diminuição da agregação devido ao aumento da repulsão eletrostática após a interação ligante-alvo. A agregação é induzida pela variação da força iônica e uma mudança da coloração da suspensão coloidal de amarelo para vermelho pode ser observada. Na presença de IgGh, a agregação é inibida e a coloração da suspensão coloidal não se altera. As nanopartículas foram obtidas por meio de cinco procedimentos diferentes e caracterizadas por espectroscopia UV-Vis, espalhamento dinâmico de luz, difração de raios-X e microscopia eletrônica. Glicose e borohidreto de sódio foram utilizados como agentes redutores, enquanto CTAB e β-ciclodextrina foram utilizados como estabilizantes. Citrato de sódio foi utilizado como agente redutor e/ou estabilizante. Nanoesferas de carbono foram obtidas por tratamento hidrotérmico de uma solução aquosa de glicose e também foram utilizadas no preparo das nanopartículas. As nanopartículas foram funcionalizadas com ácido mercaptossuccínico e a conjugação ocorreu devido à interação entre grupos aminas e grupos carboxílicos ionizados, presentes no anticorpo e agente de acoplamento, respectivamente. A estabilidade dos conjugados e o efeito da adição de IgGh foram avaliados para todos os sistemas preparados. As nanopartículas de prata preparadas com borohidreto de sódio e citrato de sódio foram selecionadas para serem aplicadas no desenvolvimento do imunoensaio e as condições experimentais foram avaliadas. Em condições ótimas, observou-se uma correlação linear entre a diminuição da agregação do sistema (NPAg-anti-IgGh) e a concentração de IgGh (0 a 200 ng mL-1). O limite de detecção foi estimado em 25 ng mL-1. O método colorimétrico apresentou boa seletividade para a detecção de IgGh. Além disso, foi obtido um resultado satisfatório ao aplicar o método para determinação do fator IX de coagulação. Foi desenvolvido também um método para determinação de ATP baseado na agregação de nanopartículas de ouro. Aptâmeros foram utilizados como elemento de reconhecimento. Em princípio, o método pode ser aplicável à determinação de outros analitos, por meio da substituição do aptâmero utilizado neste trabalho pelo oligonucleotídeo específico para o alvo de interesse.

Optical fibre sensors with applications in gas and biological sensing

This thesis describes the study of various grating based optical fibre sensors for applications in refractive index sensing. The sensitivity of these sensors has been studied and in some cases enhanced using novel techniques. The major areas of development are as follows. The sensitivity of long period gratings (LPGs) to surrounding medium refractive index (SRI) for various periods was investigated. The most sensitive period of LPG was found to be around 160 µm and this was due to the core mode coupling to a single cladding mode but phase matching at two wavelength locations, creating two attenuation peaks, close to the waveguide dispersion turning point. Large angle tilted fibre gratings (TFGs) have similar behaviour to LPGs, in that they couple to the co-propagating cladding modes. The tilted structure of the index modulation within the core of the fibre gives rise to a polarisation dependency, differing the large angle TFG from a LPG. Since the large angle TFG couple to the cladding mode they are SRI sensitive, the sensitivity to SRI can be further increased through cladding etching using HF acid. The thinning of the cladding layer caused a reordering of the cladding modes and shifted to more SRI sensitive cladding modes as the investigation discovered. In a SRI range of 1.36 to 1.40 a sensitivity of 506.9 nm/URI was achieved for the etched large angle TFG, which is greater than the dual resonance LPG. UV inscribed LPGs were coated with sol-gel materials with high RIs. The high RI of the coating caused an increase in cladding mode effective index which in turn caused an increase in the LPG sensitivity to SRI. LPGs of various periods of LPG were coated with sol-gel TiO2 and the optimal thickness was found to vary for each period. By coating of the already highly SRI sensitive 160µm period LPG (which is a dual resonance) with a sol-gel TiO2, the SRI sensitivity was further increased with a peak value of 1458 nm/URI, which was an almost 3 fold increase compared to the uncoated LPG. LPGs were also inscribed using a femtosecond laser which produced a highly focused index change which was no uniform throughout the core of the optical fibre. The inscription technique gave rise to a large polarisation sensitivity and the ability to couple to multiple azimuthal cladding mode sets, not seen with uniform UV inscribed gratings. Through coupling of the core mode to multiple sets of cladding modes, attenuation peaks with opposite wavelength shifts for increasing SRI was observed. Through combining this opposite wavelength shifts, a SRI sensitivity was achieved greater than any single observed attenuations peak. The maximum SRI achieved was 1680 nm/URI for a femtosecond inscribed LPG of period 400 µm. Three different types of surface plasmon resonance (SPR) sensors with a multilayer metal top coating were investigated in D shape optical fibre. The sensors could be separated into two types, utilized a pre UV inscribed tilted Bragg grating and the other employed a post UV exposure to generate surface relief grating structure. This surface perturbation aided the out coupling of light from the core but also changed the sensing mechanism from SPR to localised surface plasmon resonance (LSPR). This greatly increased the SRI sensitivity, compared to the SPR sensors; with the gold coated top layer surface relief sensor producing the largest SRI sensitivity of 2111.5nm/URI was achieved. While, the platinum and silver coated top layer surface relief sensors also gave high SRI sensitivities but also the ability to produce resonances in air (not previously seen with the SPR sensors). These properties were employed in two applications. The silver and platinum surface relief devices were used as gas sensors and were shown to be capable of detecting the minute RI change of different gases. The calculated maximum sensitivities produced were 1882.1dB/URI and 1493.5nm/URI for silver and platinum, respectively. Using a DFB laser and power meter a cheap alternative approach was investigated which showed the ability of the sensors to distinguish between different gases and flow rates of those gases. The gold surface relief sensor was coated in a with a bio compound called an aptamer and it was able to detect various concentrations of a biological compound called Thrombin, ranging from 1mM to as low as 10fM. A solution of 2M NaCl was found to give the best stripping results for Thrombin from the aptamer and showed the reusability of the sensor. The association and disassociation constants were calculated to be 1.0638×106Ms-1 and 0.2482s-1, respectively, showing the high affinity of the Aptamer to thrombin. This supports existing working stating that aptamers could be alternative to enzymes for chemical detection and also helps to explain the low detection limit of the gold surface relief sensor.

Microarray-formatted clinical biomarker assay development using peptide aptamers to anterior gradient-2

Anterior gradient-2 protein was identified using proteomic technologies as a p53 inhibitor which is overexpressed in human cancers, and this protein presents a novel pro-oncogenic target with which to develop diagnostic assays for biomarker detection in clinical tissue. Combinatorial phage-peptide libraries were used to select 12 amino acid polypeptide aptamers toward anterior gradient-2 to determine whether methods can be developed to affinity purify the protein from clinical biopsies. Selecting phage aptamers through four rounds of screening on recombinant human anterior gradient-2 protein identified two classes of peptide ligand that bind to distinct epitopes on anterior gradient-2 protein in an immunoblot. Synthetic biotinylated peptide aptamers bound in an ELISA format to anterior gradient-2, and substitution mutagenesis further minimized one polypeptide aptamer to a hexapeptide core. Aptamers containing this latter consensus sequence could be used to affinity purify to homogeneity human anterior gradient-2 protein from a single clinical biopsy. The spotting of a panel of peptide aptamers onto a protein microarray matrix could be used to quantify anterior gradient-2 protein from crude clinical biopsy lysates, providing a format for quantitative screening. These data highlight the utility of peptide combinatorial libraries to acquire rapidly a high-affinity ligand that can selectively bind a target protein from a clinical biopsy and provide a technological approach for clinical biomarker assay development in an aptamer microarray format.