Yes / We report on the predicted structural disruption of an adenosine-binding DNA aptamer adsorbed via noncovalent interactions on aqueous graphene. The use of surface-adsorbed biorecognition elements on device substrates is needed for integration in nanofluidic sensing platforms. Upon analyte binding, the conformational change in the adsorbed aptamer may perturb the surface properties, which is essential for the signal generation mechanism in the sensor. However, at present, these graphene-adsorbed aptamer structure(s) are unknown, and are challenging to experimentally elucidate. Here we use molecular dynamics simulations to investigate the structure and analyte-binding properties of this aptamer, in the presence and absence of adenosine, both free in solution and adsorbed at the aqueous graphene interface. We predict this aptamer to support a variety of stable binding modes, with direct base−graphene contact arising from regions located in the terminal bases, the centrally located binding pockets, and the distal loop region. Considerable retention of the in-solution aptamer structure in the adsorbed state indicates that strong intra-aptamer interactions compete with the graphene−aptamer interactions. However, in some adsorbed configurations the analyte adenosines detach from the binding pockets, facilitated by strong adenosine−graphene interactions.

DNA-functionalized gold nanoparticles (DNA-NPs) have enormous potential as building blocks for materials due to their ability to specifically recognize and respond to target molecules and surfaces. The ability of DNA aptamers to adopt different conformations and bind either complementary DNA sequences or analyte molecules allows them to mediate nanoparticle assembly or disassembly, generating selective colorimetric responses. Aptamer-mediated nanoparticle assembly and disassembly is sensitive to the nanoparticle ligand shell composition and structure, yet these topics have not been extensively explored. In this dissertation, a method for determining the ligand shell composition of DNA-NPs is described and a framework for understanding the impact of the DNA assembly arrangement and recognition strand density upon aptamer-mediated nanoparticle assembly and disassembly is developed. Design rules for creating sensors with desired properties are elucidated, leading to creation of sensors with improved detection limits and quantification ranges. A technique was needed to determine the number of DNA strands of any base composition attached to gold nanoparticles (AuNPs) of any core size. A rapid, convenient and inexpensive method to quantify the number of label-free DNA strands attached to AuNPs was therefore developed. This technique was extended to determine two different DNA sequences bound to AuNPs using UV-visible and fluorescence spectroscopy. Based on the results of quantifying the ligand shells of DNA-NPs functionalized with two sequences, disulfide-terminated DNA non-specifically adsorbs and then rearranges to specifically bind the gold surface. The position of the AuNPs and DNA strands within DNA-NP assemblies had a profound influence on their ability to assemble and sense adenosine. Assemblies designed for large inter-AuNP spacing were stable but unable to sense adenosine. Assemblies designed for short inter-AuNP spacing were unstable until the DNA ligand shell was diluted. AuNPs functionalized with the fewest number of aptamers produced assemblies with the lowest detection limit and apparent disassociation constant and the largest analyte quantification range. Increasing the number of aptamer strands per AuNP increased the cooperativity of the AuNP disassembly response to adenosine. This dissertation includes previously unpublished co-authored material.

A galectina-1 (Gal1) é uma lectina, altamente conservada, que reconhece ?- galactosídeos, e está envolvida na regulação da tolerância da imunidade celular e na homeostase. Dados da literatura mostram que esta lectina endógena é amplamente expressa em locais de inflamação e na tumorigênese, participando diretamente dos processos de adesão celular, crescimento tumoral, metástase e angiogênese, ressaltando a relevância de sua detecção em amostras biológicas, e sugerindo que a inibição dirigida da Gal1 pode resultar em benefícios no tratamento de distúrbios inflamatórios e em novas estratégias terapêuticas antitumorais. Entretanto, ainda são escassos os dados sobre inibidores de Gal1 com real impacto terapêutico no bloqueio da atividade biológica dessa lectina. Os aptâmeros são oligonucleotídeos de cadeia simples (DNA ou RNA), que podem se ligar a uma vasta diversidade de alvos, tais como íons, peptídeos, proteínas, moléculas orgânicas e inorgânicas, com alta afinidade e especificidade. Os aptâmeros são selecionados a partir de bibliotecas com sequências randômicas de oligonucleotídeos fita simples (ssDNA) constituídos por uma região central variável, flanqueada por duas regiões de interação com primers para amplificação das sequências via PCR. Esse processo de seleção é denominado de Evolução Sistemática de Ligantes por Enriquecimento Exponencial (SELEX). Neste trabalho foram selecionados e caracterizados aptâmeros de DNA que se ligam a Gal1 humana recombinante e inibem sua atividade lectínica. O processo de seleção dos aptâmeros foi feito através de uma variação da metodologia SELEX, desenvolvida neste trabalho e aqui denominada de \"single vial selection\" (SVS), na qual todas as etapas de seleção dos aptâmeros ocorreram em um único recipiente, de forma rápida e eficiente, evitando etapas cromatográficas, que geralmente são utilizadas no SELEX. Análises com a técnica de termoflúor (TSA) e espectroscopia de fluorescência intrínseca do triptofano permitiram confirmar que os aptâmeros, de fato, se ligam a Gal1, mas em um sítio afastado do CRD. Ensaios de hemaglutinação mostraram que os aptâmeros selecionados conseguiram inibir a ligação da Gal1 com as glicanas da superfície celular, bloqueando a atividade lectínica da proteína. Assim, esse conjunto de resultados mostram que foi possível o desenvolvimento de uma nova classe de inibidores da Gal1 baseada em aptâmeros de DNA, a partir de uma nova metodologia de SELEX, e que não atuam através dos mecanismos clássicos de bloqueio da atividade lectínica via CRD, abrindo nossas possibilidades no desenvolvimento de estratégias diagnósticas e terapêuticas envolvendo esta proteína. / Galectin-1 (Gal1) is a highly conserved lectin that recognizes ?-galactosides, involved in the regulation of cellular immunity tolerance and homeostasis. Data from the literature show that this endogenous lectin is widely expressed in sites of inflammation and tumorigenesis, directly participating in cell adhesion processes, tumor growth, metastasis and angiogenesis, highlighting the relevance of its detection in biological samples, and suggesting that its direct inhibition may result in benefits in the treatment of inflammatory disorders and in novel antitumor therapeutic strategies. However, data on Gal1 inhibitors with real therapeutic impact in blocking the biological activity of this lectin are still scarce. Aptamers are single-stranded oligonucleotides (DNA or RNA), which can bind to a wide variety of targets, such as ions, peptides, proteins, organic and inorganic molecules, with high affinity and specificity. The aptamers are selected from pools of random single-stranded oligonucleotide (ssDNA) sequences consisting of a variable central region, flanked by two sites of primers interaction for PCR amplification. This selection process is called Systematic Evolution of Ligands by EXponential enrichment (SELEX). In this work, DNA aptamers that bind to recombinant human Gal1 and inhibit their lectin activity have been selected and characterized. The aptamers selection process was done through a variation of SELEX methodology, developed in this work and here called \"single vial selection\" (SVS), in which all stages of aptamers selection occurred in a single container, quickly and efficient, avoiding chromatographic steps, which are usually used in SELEX. Analyzes by Thermofluor (TSA) method and intrinsic tryptophan fluorescence spectroscopy have confirmed that aptamers actually bind to Gal1, but at a site away from the CRD. Hemagglutination assay showed that selected aptamers succeeded in inhibiting the Gal1 binding to cell surface glycans, blocking the protein lectin activity. Thus, this set of results showed that it was possible to develop a new class of Gal1 inhibitors based on DNA aptamers and on a new SELEX methodology, that does not act through the classic blocking mechanisms of lectin activity via CRD, opening new possibilities for the development of diagnostic and therapeutic strategies involving this protein.

Philosophiae Doctor - PhD / In this work, DNA aptamer biosensor systems were developed for the detection of l7p-estradiol - an estrogeneous endocrine disrupting chemical (EDC). Endocrine disrupting chemicals are group of compounds that impact negatively on the endocrine system of humans and wildlife. High concentrations of l7p-estradiol in water or food chain disrupts the physiology of the endocrine system of various animal species, leading to feminisation in fish and stimulates the proliferation of cancer cells in humans. Aptasensor systems for the determination of l7pestradiol were prepared with three immobilization platforms: (i) poly(3,4- ethylenedioxythiophene) {PEDOT} doped with gold nanoparticles (AuNPs) to form PEDOTIAuNPs polymeric nanocomposite, (ii) generation 1 poly(propylene thiophenoimine)-copoly( 3 ,4-ethy lenedioxythiophene) dendritic star copolymer (G 1PPT -co-PEDOT), and (iii) generation 2 poly (propylene thiophenoimine)-co-poly(3,4-ethylenedioxythiophene) dendritic star copolymer (G2PPT-co-PEDOT). The morphological properties of the sensor platforms were interrogated by scanning emission microscopy (SEM) and atomic force microscopy (AFM), while their spectroscopic characteristics were studied by Fourier transform infra red spectroscopy (FTIR) and fluorescence spectroscopy. The electrochemical behaviour of the platforms and the aptasensors were studied by electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV) and square wave voltammetry (SWV). The DNA aptamer developed for detecting 17~-estradiol and which was used in the fabrication of all aptamer biosensors in this study is a 76-mer biotinylated aptamer (5'-BiotinGCTTCCAGCTTATTGAATTACACGCAGAGG TAGCGGCTCTGCGCATTCAATGCTGCGCGCTGAAGCGCGGAAGC-3'). AulPEDOTIAuNPslAptamer (platform 1) was obtained by covalently attaching streptavidin to the polymeric nanocomposite platform using carbodiimide chemistry and the aptamer immobilized via streptavidin-biotin interaction. The electrochemical signal generated from the aptamer-target molecule interaction was monitored electrochemically using cyclic voltammetry and square wave voltammetry in the presence of [Fe(CN)6J 3-/4- as a redox probe. The signal current observed was inversely proportional to the concentration of 17Bestradiol. The aptasensor demonstrated specificity toward 17~-estradiol. The detectable concentration range of the 17B estradiol was 0.01 nM-O .09 nM with a detection limit of 3.2 pM. The 76-mer biotinylated aptamer for 17~-estradiol was incorporated into a generation 1 poly(propylenethiophenoimine )-co-poly(3 ,4-ethylenedioxythiophene) dendritic star copolymer modified Au electrode via biotin-avidin interaction (platform 2). The Bode plot shows that the charge transfer dynamics of the nanoelectrode can be frequency modulated while the AulG 1PPTco- PEDOT nanoelectrode exhibited greater semi-conductor behavior (higher phase angle value) than AulG 1PPT due to the incorporation of charged functionalized dendrimer at low frequencies (100 mHz). The biosensor response to 17~-estradiol was based on the decrease in the SWV current as the EDC binds to the ssDNA aptamer on the biosensor. The dynamic linear range of the sensor was 0.01-0.07 nM with a detection limit of7.27 pM. Synthesis of electro synthetic generation G2PPT-co-PEDOT (platform 3) was performed by copolymerization of PEDOT with G2PPT dendrimer modified electrode immersed in a solution of 0.1 M LiCI04 containing 0.1 M EDOT monomer and 0.1 M sodium dodecyl sulphate (SDS) for ten (10) cycles. The electrochemical behaviour of the dendritic star copolymer was investigated with CV and EIS in LiCI04 and phosphate buffer solutions. The results show that the electrochemical deposition of G2PPT-co-PEDOT on gold electrode decreased the electrochemical charge transfer resistance when compared to AuiPEDOTILiCI04 and AuiLiCI04 interfaces. Bode impedimetric analysis indicates that G2PPT-co-PEDOT is a semiconductor. The fabrication of two novel aptasensors (based on platforms 2 and 3) simultaneously on a screen printed micro array electrode of 96-well multichannel electrochemical robotic sensor testing system for the detection of endocrine disrupting l7~-estradiol, was also carried out. The aptasensors responses to l7~-estradiol, based on the decrease in the SWV current, were evaluated.

This project is aimed toward finding DNA aptamers against prostate cancer peptide antigens. DNA aptamers can function to find and indicate the presence of certain molecules in a specimen. These aptamers will be obtained through the process of evolutionary selection, a specific process called SELEX which stands for Systemic Evolution of Ligands by Experimental Enrichment. By conducting several rounds of SELEX, a DNA aptamer will be selected to bind to a known peptide antigen. A biotinylated column will be utilized to stabilize a random library of DNA aptamers, and those peptides that bind to certain aptamers will cause a conformational change leading to the elution of those specific DNA aptamers. This SELEX process will be conducted again on the eluted aptamers to further select for strong binding DNA aptamers. The DNA aptamers that are obtained can further on be sequenced or used for prostate cancer research studies. Another possible usage of aptamers is to diagnose and determine the stage of various different cancer types. Our prediction is that this research will produce a DNA aptamer that will bind to a specific prostate cancer peptide antigen.

No description available.

<p>The development of low-cost, and reliable platforms for on-site detection of pathogenic agents, and toxic environmental traces is still a critical need for real-time monitoring of potential environmental pollution and imminent outbreaks. The biosensors market is projected to attain 31.5 billion by 2024. In this landscape, colorimetric and electrochemical devices continue to have significant relevance, with paper-based platforms leading the point-of-care (POC) segment for pathogen detection and environmental monitoring.</p> <p>Despite the true potential of biosensors in general, they have witnessed a slow rate in commercialization, mainly due to cost restrictions, and concerns related to their reliability and repeatability once scaled-up. This research evaluates the implementation of printing techniques as a strong approach for the fabrication of paper-based and flexible electrochemical biosensors. The results obtained demonstrated the ability to control and predict the variables affecting the sensing performance, achieving high precision of the printing parameters, and allowing optimization, and iterations since very early stages of prototype development.</p> <p>Besides the novel fabrication approach, this work introduces the use of truncated aptameric DNA sequences for whole cell detection of E. coli O157:H7 and heavy metals (Hg2+ and As3+), providing evidence of high stability and robustness under harsh conditions. Results obtained demonstrate their equal or even superior performance when compared to antibodies.</p> <p>We established the use of aptamer-functionalized multilayered label particles (PEI-grafted gold decorated polystyrene) with high stability as label particles. These particles address the well known drawback of non-selective aggregation typical of traditional naked Gold nanoparticles. The outstanding stability of these multilayered labels was demonstrated when used in an enhanced version of the lateral flow assay for detection of E. coli O157:H7 (state of the art for paper-based colorimetric detection of whole cell bacteria), and in a multiplexed paper-based microfluidic device for dual detection of Mercury and Arsenic. This work sets the foundation of the development of a next generation of health care and environmental monitoring devices that are portable, sensitive, quantitative, and can reliably detect multiple targets with one single test.</p>