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Electrochemical Immunosensor based on Cyclodextrin Supramolecular interactions for the detection of human chorionic gonadotropinWilson, Lindsay January 2012 (has links)
>Magister Scientiae - MSc / Glucose oxidase (GOx) and horseradish peroxidase (HRP) are important enzymes for the development of amperometric enzyme linked immunosensors. The selectivity of each enzyme towards its analyte deepens its importance in determining the sensitivity of the resultant immunosensor. In designing immunosensors that have customized transducer surfaces, the incorporation with FAD and iron based enzymes ensures that electron kinetics remains optimal for electrochemical measurement. Various different immobilization strategies are used to produce response signals directly proportional to the concentration of analyte with minimal interferences. The combination of self-assembled monolayers and
supramolecular chemistry affords stability and simplicity in immunosensor design. In this work, two electrochemical strategies for the detection of human chorionic gonadotropin(hCG) is presented. This involves the modification of a gold surface with a thiolated β-cyclodextrin epichlorohydrin polymer (βCDPSH) to form a supramolecular inclusion complex with ferrocene (Fc)-functionalised carboxymethyl cellulose polymer (CMC). Cyclic voltammetry indicated that ferrocene is in close proximity to the electrode surface due to the supramolecular complex formed with βCDPSH. Furthermore, strategy (a) for the detection of hCG used α-antihCG labelled (HRP) as reporter conjugate. Strategy (b) maintained the CMC bifunctionalised with Fc and recognition antibody for hCG hormone. However, the system was functionalised with a HRP enzyme and detection is done by using GOx reporter conjugates for in situ production of hydrogen peroxide. The reduction of H2O2 was used for the amperometric detection of hCG by applying a potential of 200 mV. The sensitivity and limit of detection of both strategies were calculated from calibration plots. For strategy (a) the LOD was found to be 3.7283 ng/mL corresponding to 33.56 mIU/mL and a sensitivity of 0.0914 nA ng-1 mL-1. The corresponding values for strategy (b) are 700 pg/mL (6.3 mIU/mL) and 0.94 nA ng-1 mL-1.
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DESENVOLVIMENTO DE DIFERENTES DISPOSITIVOS ELETROQUÍMICOS A BASE DE OURO APLICADOS COMO SENSORES E BIOSSENSORESSantos, Cleverson Siqueira 06 May 2016 (has links)
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Previous issue date: 2016-05-06 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / This thesis describes the preparation, characterization and application of electrochemical sensors and biosensors, using different modification techniques, based on Au as transducer. The enzymatic biosensor applied on the detection of pesticide carbaryl was built on a gold electrode functionalized with a
monolayer of polyamidoamine dendrimer of fourth generation with a cystamine core (PAMAM-G4) on which it was immobilized the acetylcholinesterase enzyme (AChE), with the aid of glutaraldehyde. After evaluate and determine the best conditions for immobilization of the AChE enzyme (glutaraldehyde
concentration of 1% (v/v) and the concentration of enzyme units 496 U. mL-1) on the monolayer of PAMAM, the catalytic activity of the enzyme was evaluated by chronoamperometry in presence of enzymatic substrate AChI obtaining = 2.9 . −1 . The biosensor response for carbaryl detection was based on the inhibition of the enzymatic activity caused by the pesticide. It was verified that 5 min in the pesticide solution is sufficient to block the enzyme active sites. After determining the
best conditions for the construction of the biosensor, it was applied for carbaryl detection in the concentration range from 1.0 to 9.0 mol. L-1. The detection and quantification limits were found to be 0.0108 mol. L-1 and 0.032 mol. L-1, respectively. In the second chapter, it is reported the development of an immunosensor applied to qualitative detection of antibodies (AB) T. cruzi. The sensor was built on gold electrode modified with the thiol 3-mercaptopropionic acid (MPA) and the antigens (AG) T. cruzi were covalently immobilized on this surface by the reaction with EDC and NHS. The influence of concentration and of immersion time in the AG solution were evaluated using the techniques of cyclic voltammetry and electrochemical impedance spectroscopy in presence of [Fe(CN)6]3-/4- and the results showed that the best conditions were immersion time of 15 minutes and AG concentration of 0.5 g. L-1.The possible sites of non-specific binding were blocked with bovine serum albumin (BSA). The immersion time in the solution (AB) was also evaluated and the results showed that 30 minutes are sufficient for all specific bonds sites were occupied by AB T. cruzi. Selectivity tests in the absence of AB, only in the serum sample, and in the presence of AB Toxoplasma were performed. The results demonstrated that the immunosensor is selective, since it presented charge transfer resistance (Rct) values
in the presence of AB T. cruzi 70% higher than the Rct values in presence of possible interferences.Therefore, the immunosensor presents itself as an alternative to qualitative diagnosis of american trypanosomiasis. In the third chapter, it is reported the modification of carbon graphite electrodes (CG) obtained from dry batteries zinc/carbon with Au microparticles obtained by potentiostatic electrodeposition. The variables involved in the electrodeposition process, such as the gold salt concentration, time and deposition potential were evaluated and optimized using cyclic voltammetry technique in the presence of redox couple [Fe(CN)6]3-/4-. The best conditions for the Au deposition were electrodeposition time of 700 s, potential of +0.3 V and concentration of 10.0 mmol. L-1. In these
conditions, Au spherical particles were obtained with an average size of 420 nm, which were homogeneously deposited on the surface of GC electrode and promoted the increase of the electroactive area (GC electrode showed an area of 0.12 cm2 and GC/Au electrode presented an area of 0.25 cm2). The
GC/Au electrode was applied for the separation and quantification of dopamine and uric acid present in a mixture. The voltammetric results showed that the GC/Au sensor is selective, once the potential peak separation between the DA and UA species was 370 mV. The detection and quantification limits were found to be 1.86 mol. L-1 and 6.09 mol. L-1 for dopamine and uric acid 17.5 mol. L-1 and 58.5 mol. L-1, respectively. In the development of the three electrochemical devices gold electrodes were used. From the results obtained for the three developed electrochemical devices it can be concluded that high electric conductivity, chemical stability, biocompatibility, ability to miniaturization in the form of
microstructures and ease of functionalization of Au electrodes make them suitable conductive matrixes for construction of electrochemical sensors and particularly biosensors. / Esta tese descreve a preparação, caracterização e aplicação de sensores e biossensores eletroquímicos, utilizando diferentes técnicas de modificação baseadas no Au como transdutor. O biossensor enzimático aplicado na detecção do pesticida carbaril foi construído sobre um eletrodo de ouro funcionalizado com
uma monocamada do dendrímero poliamidoamina de quarta geração com núcleo de cistamina (PAMAMG4),sobre o qual foi imobilizada com o auxílio de glutaraldeído a enzima acetilcolinesterase (AChE).Após avaliar e determinar as melhores condições de imobilização da enzima AChE (concentração de
GLUT 1% (v/v) e concentração de unidades enzimáticas 496 U. mL-1) sobre a monocamada de PAMAM,a atividade catalítica da enzima AChE imobilizada foi avaliada por cronoamperometria na presença do substrato enzimático (AChI) obtendo-se o
= 2,9 . −1. A resposta do biossensor na detecção
de carbaril foi baseada na inibição da atividade enzimática causada pelo pesticida. Foi constatado que 5 min de imersão na solução do pesticida foram suficientes para que os sítios ativos da enzima fossem bloqueados. Após determinar as melhores condições de construção do biossensor, este foi aplicado na
detecção de carbaril na faixa de 1,0 a 9,0 mol. L-1. Os limites de detecção e quantificação encontrados foram de 0,0108 mol. L-1 e 0,032 mol. L-1, respectivamente. No segundo capítulo é relatado o desenvolvimento de um imunossensor aplicado na detecção qualitativa de anticorpos (AB) T. cruzi. O sensor foi construído sobre eletrodo de ouro modificado com o tiol ácido 3-mercaptopropiônico (MPA) e sobre esta superfície foram imobilizados covalentemente pela reação com EDC e NHS, os antígenos (AG) T. cruzi. A influência da concentração da solução de AG e do tempo de imersão nesta solução para a
construção do imunossensor foram avaliadas utilizando as técnicas de voltametria cíclica e espectroscopia de impedância eletroquímica na presença de [Fe(CN)6]3-/4- e os resultados demonstraram que as melhores condições foram: tempo de 15 min e concentração de 0,5 g. L-1. Os possíveis sítios de ligações não específicas foram bloqueados com albumina de soro bovino (BSA). O tempo de imersão na solução de AB também foi avaliado. Os resultados mostraram que 30 min são suficientes para que todos os sítios de ligações específicos fossem ocupados pelos AB T. cruzi. Testes de seletividade na ausência de AB,apenas em amostra de soro e também na presença de AB de Toxoplasma foram realizados. Os resultados demonstraram que o imunossensor é seletivo, pois este apresentou valores de resistência de transferência de carga (Rct) na detecção de AB T. cruzi 70% maiores do que na detecção dos possíveis interferentes. Portanto, o imunossensor apresenta-se como uma alternativa no diagnóstico qualitativo de tripanossomíase americana. No terceiro capítulo é descrito a modificação de eletrodos de carbono grafite (CG) obtidos de pilhas secas de zinco/carbono com micropartículas de Au obtidas pela eletrodeposição potenciostática. As variáveis envolvidas no processo de eletrodeposição, tais como concentração do sal de
ouro, potencial e tempo de deposição foram avaliadas e otimizadas utilizando a técnica de voltametria cíclica na presença do par redox [Fe(CN)6]3-/4-. As melhores condições foram: tempo de 700 s, potencial de +0,3 V e concentração de 10,0 mmol. L-1. Nestas condições, foram obtidas partículas de Au esféricas com tamanho médio de 420 nm, as quais se apresentaram homogeneamente dispersas sobre toda a
superfície do eletrodo de CG e promoveram o aumento da área eletroativa, (o eletrodo CG apresentou uma área de 0,12 cm2 e o eletrodo CG/Au uma área 0,25 cm2). O desempenho do eletrodo CG/Au foi avaliado na separação e quantificação de dopamina e ácido úrico presentes em uma mistura. Os resultados
voltamétricos demonstraram que o sensor CG/Au é seletivo, pois apresentou separação de potencial pico de 370 mV entre as duas espécies. Os limites de detecção e quantificação encontrados foram de 1,86 mol. L-1 e 6,09 mol. L-1 para dopamina, e 17,5 mol. L-1 e 58,5 mol. L-1 para ácido úrico,
respectivamente. A partir dos resultados obtidos para os três dispositivos eletroquímicos desenvolvidos pode-se concluir que a elevada condutividade elétrica, estabilidade química, biocompatibilidade, possibilidade de miniaturização na forma de microestruturas e a facilidade de funcionalização dos eletrodos de Au fazem destes matrizes condutoras apropriadas para construção de sensores e particularmente biossensores eletroquímicos.
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Electrochemical immunosensor based on cyclodextrin supramolecular interactions for the detection of human chorionic gonadotropinWilson, Lindsay January 2012 (has links)
>Magister Scientiae - MSc / Glucose oxidase (GOx) and horseradish peroxidase (HRP) are important enzymes for the development of amperometric enzyme linked immunosensors. The selectivity of each enzyme towards its analyte deepens its importance in determining the sensitivity of the resultant immunosensor. In designing immunosensors that have customized transducer surfaces, the incorporation with FAD and iron based enzymes ensures that electron kinetics remains optimal for electrochemical measurement. Various different immobilization strategies are used to produce response signals directly proportional to the concentration of analyte with minimal interferences. The combination of self-assembled monolayers and
supramolecular chemistry affords stability and simplicity in immunosensor design. In this work, two electrochemical strategies for the detection of human chorionic gonadotropin(hCG) is presented. This involves the modification of a gold surface with a thiolated β-cyclodextrin epichlorohydrin polymer (βCDPSH) to form a supramolecular inclusion complex with ferrocene (Fc)-functionalised carboxymethyl cellulose polymer (CMC). Cyclic voltammetry indicated that ferrocene is in close proximity to the electrode surface due to the supramolecular complex formed with βCDPSH. Furthermore, strategy (a) for the detection of hCG used α-antihCG labelled (HRP) as reporter conjugate. Strategy (b) maintained the CMC bifunctionalised with Fc and recognition antibody for hCG hormone. However, the
system was functionalised with a HRP enzyme and detection is done by using GOx reporter conjugates for in situ production of hydrogen peroxide. The reduction of H2O2 was used for the amperometric detection of hCG by applying a potential of 200 mV. The sensitivity and limit of detection of both strategies were calculated from calibration plots. For strategy (a) the LOD was found to be 3.7283 ng/mL corresponding to 33.56 mIU/mL and a sensitivity of 0.0914 nA ng-1 mL-1. The corresponding values for strategy (b) are 700 pg/mL (6.3 mIU/mL) and 0.94 nA ng-1 mL-1.
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Metallic nanoparticles with polymeric shell: A multifunctional platform for application to biosensorNgema, Xolani Terrance January 2018 (has links)
Philosophiae Doctor - PhD (Chemistry) / Tuberculosis (TB) is an airborne disease caused by Mycobacterium tuberculosis (MTB) that
usually affects the lungs leading to severe coughing, fever and chest pains. It was estimated
that over 9.6 million people worldwide developed TB and 1.5 million died from the infectious
disease of which 12 % were co-infected with human immunodeficiency virus (HIV) in the year
2015. In 2016 the statistics increased to a total of 1.7 million people reportedly died from TB
with an estimated 10.4 million new cases of TB diagnosed worldwide. The development of the
efficient point-of-care systems that are ultra-sensitive, cheap and readily available is essential
in order to address and control the spread of the tuberculosis (TB) disease and multidrugresistant
tuberculosis.
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Síntese, caracterização e aplicação do poli (ácido 3-Hidroxifenilacético) no desenvolvimento de biossensor para detecção de marcador cardíaco / Synthesis, characterization and application of poly(3- hydroxyphenylacetic acid) in the development of a biosensor for detection of cardiac markerMartins, Pâmela Oliveira 25 February 2011 (has links)
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / In this work was realized characterization studies of a new material, poly (3-
hidroxyphenylacetic acid) and its application for the construction of an amperometric
immunosensor for detection of Acute Myocardial Infarction (AMI). Initially, it was
carried out the electropolymerization of 3-hidroxyphenylacetic acid at three different
pH's (0.0, 6.5 and 12.0) which could be assessed, electrochemically, the relationship
between the behavior of the polymer and the reaction s pH. It was found that in the acid
solutions (pH 0.0) the formation of electrochemically active material is more evident.
Moreover, using electrochemical techniques, were carried out investigations on the
structure of the polymeric material by using cationic and anionic probes. Monomer and
polymer were characterized by infrared spectroscopy (FTIR), Ultra-Violet (UV/Vis.)
and fluorescence; thermal analysis (DTA and TGA) and structural analysis (XDR).
These studies were extremely important to highlight the main differences between the
starting material (monomer) and electropolymerized material (polymer), and in
particular assess the main characteristics of the polymer in order to enable its use as a
platform to the proposed immunosensor. Studies of the immunosensor were conducted
using two substances that acted as indicators of the reaction between the specific
antibody for the AMI (anti-troponin T) and specific antigen for AMI (troponin T),
hexaaminruthenium chloride and ferro/ferricyanide potassium. The results showed that
the hexaaminruthenium chloride showed the best performance to indicate the formation
of antibody-antigen complex wich occurs in the AMI. / Neste trabalho foram realizados estudos de caracterização de um novo material, o
poli(ácido 3-hidroxifenilacético) e sua aplicação para construção de um imunossensor
amperométrico para detecção do Infarto Agudo do Miocárdio (IAM). Inicialmente, foi
realizada a eletropolimerização do ácido 3-hidroxifenilacético em três pH s diferentes
(0,0; 6,5 e 12,0) onde foi possível avaliar, eletroquimicamente, a relação entre o
comportamento do polímero formado e o pH do meio reacional. Foi possível constatar
que em meio ácido (pH 0,0) a formação do material eletroquimicamente ativo é mais
evidenciada. Além disso, com o auxílio de técnicas eletroquímicas, foram realizadas
investigações sobre a estrutura do material polimérico formado, utilizando sondas
catiônicas e aniônicas. Monômero e polímero foram caracterizados por técnicas
espectroscópicas de Infravermelho (FTIR), Ultra-Violeta (UV/Vis.) e Fluorescência;
análises térmicas (TGA e DTA) e análises estruturais (DRX). Estes estudos foram de
extrema importância para destacar as principais diferenças entre o monômero e o
material eletropolimerizado e, principalmente, avaliar as principais características do
polímero, no sentido de viabilizar a sua utilização como plataforma do imunossensor
proposto. Os estudos do imunossensor foram conduzidos utilizando-se duas substâncias
que atuaram como indicadores da reação entre anticorpo específico para o IAM (antitroponina
T) e antígeno específico para o IAM (troponina T), o cloreto de
hexaaminrutênio II e o ferro/ferricianeto de potássio. Os resultados obtidos mostraram
que o cloreto de hexaaminrutênio II teve melhor desempenho para indicar a formação
do complexo anticorpo-antígeno, característico no evento do IAM. / Mestre em Química
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Electrochemical affinity sensors for biomedical, food and environmental applications / Capteurs électrochimiques d'affinité appliqué dans l'analyse biomédicale, sécurité alimentaire et environnementaleFlorea, Anca Stefana 14 September 2015 (has links)
Les capteurs électrochimiques sont des outils pour la détection fiable, peu coûteux, avec une haute sensibilité et sélectivité, pour la détermination des composés biologiques et chimiques dans les domaines du diagnostic clinique, l'environnement et l'industrie alimentaire. Particulièrement, les Immunocapteurs, alliant une très grande spécificité. Également des nouveaux techniques produisent des résultats similaires, par exemple, les capteurs basés sur la technique des Polymères à empreinte moléculaire, la quelle produise des récepteurs artificiels. La technique devient très important dans les sciences bioanalytiques parce qu'il porte des avantages inhérents sur les récepteurs naturels: une grande stabilité dans des diffèrent environnement et conditions, également comptent avec une grande flexibilité dans la conception, une large gamme de molécules peuvent être utilisées. L'objectif du travail présenté ici est de développer des capteurs électrochimiques avec une très grande affinité et spécificité pour une analyte. Les quelles comprennent des applications très divers comme dans la protection de l'environnement, la sécurité alimentaire et le domaine biomédical. La première partie de la thèse présent l'état actuel de la conception et techniques de fabrication des biocapteurs. Ensuite, les aspects généraux des immuno capteurs électrochimiques et capteurs base sur des aptamères sont présentés ici, ainsi que plusieurs exemples rapportés dans la littérature pour la détection de marqueurs biologiques du cancer. Les avantages de l'intégration nanomatériaux dans les dispositifs de détection sont présentés. Ensuite, plusieurs aspects sur la technique des Polymères à empreinte moléculaire sont introduits. La partie personnelle de contribution est structuré en trois chapitres: en premier temps la méthodologie et les résultats obtenus pour le développement de deux essais biologiques pour la détection du marqueur tumoral Mucinl. Le premier chapitre est dédié sur un capteur à base de billes magnétiques, dans le deuxième chapitre une capteur aptamère base sur des nanoparticules d'or sans aucun marquage et finalement un capteur basée sur la technique des Polymères à empreinte moléculaire, cette protocole a été appliqué pour la détection d'explosifs, des médicaments, des hormones et les pesticides / Electrochemical sensors provide reliable and inexpensive tools for the determination of biological and chemical compounds with high sensitivity and selectivity, in the fields of clinical diagnosis, environment protection and food industry. Immunosensors hold particular promise, combining the high specificity of immuno- reactions with the sensitivity of electrochemical methods. Artificial receptors based on molecularly imprinted technique attracted considerable attention in bioanalytical sciences due to inherent advantages over natural receptors, such as high stability in harsh conditions and freedom of molecular design towards a wide range of molecules. The aim of the thesis presented here was to develop electrochemical affinity sensors based on various recognition receptors for environment monitoring, food safety and biomedical field. The first part of the thesis reviews the current state of knowledge in these fields. General aspects of electrochemical immuno- and apta-sensors are presented herein, together with several examples reported in the literature for the detection of cancer biomarkers. The advantages of integrating nanomaterials in sensing devices are then presented. At last, several aspects of the molecularly imprinted polymers are introduced. The personal contribution part is structured in three chapters, that include the methodology and results obtained for the development of biosensors for the detection of Mucinl tumor marker, the first chapter being focused on bioassays based on magnetic beads and second chapter on a label-free aptasensor based on gold nanoparticles, and finally, a third chapter dedicated to the molecularly imprinted-based sensors for the detection of explosives, drugs, hormones and pesticides
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Разработка электрохимического сенсора для определения грамм-положительных бактерий staphylococcus aureus в модельных суспензиях : магистерская диссертация / Development of an electrochemical sensor for the determination of gram-positive bacteria staphylococcus aureus in model suspensionsСамкова, И. А., Samkova, I. A. January 2016 (has links)
Объектом исследования являются бактерии Staphylococcus aureus в модельных суспензиях.
Цель работы— апробация разработанных алгоритмов определения Staphylococcus aureus с использованием электрохимической системы на основе бесферментных электрохимических иммуносенсоров в модельных суспензиях. Определение чувствительности микробной флоры к антибактериальному препарату.В процессе работы должны быть проведены исследования синтезированных наночастиц магнетита, изучен характер электропревращений модифицированных наночастиц. Должен быть осуществлен выбор рабочего электрода и оптимальных условий анализа для количественного определения Staphylococcus aureus в модельных суспензиях. Должна быть проведена апробация разработанных алгоритмов по определению Staphylococcus aureus в модельных образцах. Определена чувствительность микробной микрофлоры к антибактериальному препарату мазь «Новокомб – 50%».
В результате исследования были синтезированы наночастицы магнетита (Fe3O4), наличие полимерного покрытия подтверждено методом ИК - спектроскопии. В результате проведенных экспериментов был получен электрохимический аналитический отклик от модифицированных наночастиц магнетита. Были выбраны оптимальные условия регистрации аналитического сигнала. В качестве рабочего электрода был выбран планарный платиновый электрод, в качестве метода иммобилизации - метод физической сорбции антител на рабочую зону электрода. Был получен электрохимический отклик от иммунокомплекса антитело-бактерия, меченая наночастицами магнетита. Были выбраны оптимальные условия проведения количественного определения Staphylococcus aureus в модельных суспензиях. При данных условиях была выполнена оценка результатов в отношении таких показателей, как воспроизводимость и специфичность. По результатам апробации алгоритмов на модельных образцах было выявлено, что результаты данного метода коррелируют с методами ИФА и бактериального посева. Точность метода с использованием электрохимического иммуносенсора удовлетворительная.
Данный метод может быть рекомендован для определения чувствительности микробной флоры к антибактериальным препаратам при их разработке, исследовании и на этапах серийного производства и обращения Основные конструктивные и технико-эксплуатационные показатели: предел обнаружения для бактерий Staphylococcus aureus составил 8.7 КОЕ/мл. Относительное стандартное отклонение не превышает 10%.Эффективность метода определяется возможностью его применения для определения чувствительности микробной флоры к антибактериальным препаратам при их разработке, исследовании и на этапах серийного производства и обращения. / Object of research are the bacteria Staphylococcus aureus in model suspensions.
The purpose of testing the developed algorithms work- determining Staphylococcus aureus using electrochemical systems based on electrochemical besfermentnyh immunosensors in model suspensions. Determination of the sensitivity of the microbial flora to antibacterial preparatu.V during operation should be studied synthesized nanoparticles of magnetite, studied character elektroprevrascheny modified nanoparticles. Selection is to be made the working electrode and optimal assay conditions for quantitative determination of Staphylococcus aureus in model suspensions. It must be carried out testing of the developed algorithms to identify Staphylococcus aureus in model samples. Determine the sensitivity of the microbial microflora antimicrobial ointment "Novokomb - 50%."
The study nanoparticles of magnetite (Fe3O4) were synthesized, the presence of the polymer coating was confirmed by IR - spectroscopy. As a result of the experiments was obtained from the electrochemical analytical response modified magnetite nanoparticles. optimal conditions for the registration of the analytical signal were selected. The working electrode was selected planar platinum electrode, as a method of immobilization - the method of physical adsorption of antibodies to the working electrode area. electrochemical response by the bacterium-antibody immunocomplex, labeled magnetite nanoparticles was obtained. the optimal conditions were selected quantitative determination of Staphylococcus aureus in model suspensions. Under these conditions, evaluation of the results was carried out in relation to indicators such as reproducibility and specificity. According to the results of testing of algorithms to model samples it was found that the results of this method correlate with the ELISA and bacterial seeding. Accuracy of the method using an electrochemical immunosensor satisfactory.
This method can be recommended for the determination of the sensitivity of the microbial flora to antibiotics when they are developing, researching and on the stages of mass production and circulation of basic design and technical and operational parameters: detection limit for bacteria Staphylococcus aureus was 8.7 CFU / ml. The relative standard deviation does not exceed 10% g? O FIG method determined by the possibility of its application for the determination of the sensitivity of the microbial flora to antibiotics when they are developing, researching and on the stages of mass production and circulation.
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Label-free surface-enhanced Raman spectroscopy-linked immunosensor assay (SLISA) for environmental surveillancebhardwaj, vinay 02 October 2015 (has links)
The contamination of the environment, accidental or intentional, in particular with chemical toxins such as industrial chemicals and chemical warfare agents has increased public fear. There is a critical requirement for the continuous detection of toxins present at very low levels in the environment. Indeed, some ultra-sensitive analytical techniques already exist, for example chromatography and mass spectroscopy, which are approved by the US Environmental Protection Agency for the detection of toxins. However, these techniques are limited to the detection of known toxins. Cellular expression of genomic and proteomic biomarkers in response to toxins allows monitoring of known as well as unknown toxins using Polymerase Chain Reaction and Enzyme Linked Immunosensor Assays. However, these molecular assays allow only the endpoint (extracellular) detection and use labels such as fluorometric, colorimetric and radioactive, which increase chances of uncertainty in detection. Additionally, they are time, labor and cost intensive. These technical limitations are unfavorable towards the development of a biosensor technology for continuous detection of toxins. Federal agencies including the Departments of Homeland Security, Agriculture, Defense and others have urged the development of a detect-to-protect class of advanced biosensors, which enable environmental surveillance of toxins in resource-limited settings.
In this study a Surface-Enhanced Raman Spectroscopy (SERS) immunosensor, aka a SERS-linked immunosensor assay (SLISA), has been developed. Colloidal silver nanoparticles (Ag NPs) were used to design a flexible SERS immunosensor. The SLISA proof-of-concept biosensor was validated by the measurement of a dose dependent expression of RAD54 and HSP70 proteins in response to H2O2 and UV. A prototype microchip, best suited for SERS acquisition, was fabricated using an on-chip SLISA to detect RAD54 expression in response to H2O2. A dose-response relationship between H2O2 and RAD54 is established and correlated with EPA databases, which are established for human health risk assessment in the events of chemical exposure. SLISA outperformed ELISA by allowing RISE (rapid, inexpensive, simple and effective) detection of proteins within 2 hours and 3 steps. It did not require any label and provided qualitative information on antigen-antibody binding. SLISA can easily be translated to a portable assay using a handheld Raman spectrometer and it can be used in resource-limited settings. Additionally, this is the first report to deliver Ag NPs using TATHA2, a fusogenic peptide with cell permeability and endosomal rupture release properties, for rapid and high levels of Ag NPs uptake into yeast without significant toxicity, prerequisites for the development of the first intracellular SERS immunosensor.
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Détection de polluants dans l'eau potable. Développement d'un immunocapteur sur la base d'un transistor organique à effet de champ à grille électrolytique. / Detection of Water Pollutants using Label-free Electrochemical Immunosensors and Electrolyte Gated Organic Field-Effect TransistorsNguyen, Thi Thuy Khue 22 October 2018 (has links)
Aujourd'hui, avec l'augmentation de la population, la consommation de médicaments et de produits phytosanitaires dans l'agriculture a considérablement augmenté. Cela devient inquiétant car une grande partie de ces molécules, rejetée dans l'environnement, ne sont pas bien éliminées par les stations d'épuration (lorsqu'elles existent). En trop grande quantité, ces produits deviennent des poisons pour tous les organismes vivants, y compris l’Homme.Des méthodes analytiques classiques pour la mesure de ces produits chimiques existent déjà (méthodes séparatives classiques telles que la chromatographie en phase gazeuse, la chromatographie liquide à haute performance, éventuellement couplée à la spectrométrie de masse, etc.). Cependant, même si elles sont extrêmement précises et fiables, ces techniques sont difficiles à appliquer pour la surveillance sur site et sont généralement coûteuses. Pour cette raison, ma thèse se concentre sur de nouvelles approches analytiques pour détecter de petites molécules en milieu aqueux, telles que ces polluants. Dans une première partie de mon travail, j’ai développé un immunocapteur basé sur une complexation compétitive originale et sur une transduction électrochimique (ampérométrique), pour la détection du diclofénac, un anti - inflammatoire non stéroïdien généralement utilisé pour réduire l’inflammation et soulager la douleur. L'électrode de travail a été fonctionnalisée par deux sels de diazonium, l'un utilisé comme sonde moléculaire (un dérivé du diclofénac couplé à une arylamine) et l'autre comme sonde redox (une quinone) également couplée à une arylamine, capable de transduire l'association haptène-anticorps par une variation de son électroactivité ; en particulier, la transduction a été conçue pour délivrer une augmentation de courant lors de la détection du diclofénac (soit une détection « signal-on »). J’ai montré une limite de détection d’environ 20 fM dans l'eau du robinet, ce qui rend ce type de capteur très compétitif. Dans la suite de mon travail, j'ai conservé la même approche de transduction originale (immunoreconnaissance compétitive) mais appliquée à un transistor à effet de champ organique à grille électrolytique (EGOFET) dont le semiconducteur est le poly (N-alkyldiketopyrrolo-pyrrole dithiénylthiéno [3,2-b ] thiophène) (DPP-DTT) et dont l'électrode de grille a été fonctionnalisée par électrogreffage d'un sel de diazonium fonctionnel capable de lier un anticorps spécifique de l'acide 2,4-dichlorophénoxyacétique (2,4-D), un herbicide courant. Le design de la sonde moléculaire a été rationalisée par modélisation moléculaire afin d’optimiser la capture de l’anticorps en surface de grille. Dans la dernière partie de mon travail, je propose une approche qui met à profit à la fois le couplage capacitif de l'EGOFET mais aussi sa sensibilité aux charges électrostatiques accumulées en surface de grille. J'ai immobilisé en surface de grille un peptide court (Gly-Gly-His) connu pur avoir une forte affinité envers les ions cuivre Cu2+. Le peptide a été immobilisé par électro-oxydation directe de l'amine primaire du premier fragment glycine. J’ai démontré que les dispositifs EGOFET, modifiés par GGH, peuvent transduire la complexation de Cu2+ par des variations significatives de leurs caractéristiques de sortie et de transfert, en particulier par un décalage de la tension de seuil (VTh). / Today, with the increase of population, the consumption of drugs and of chemicals in agriculture has dramatically increased. It becomes a worrisome issue because a large amount of these molecules, excreted to the environment, are not well eliminated by water-treatment plants (when they exist) and are therefore released without control into the ecosystem. In too large quantities, these drugs are poisons for living organisms, including humans. Classical analytical methods for the measurement of these chemicals already exist (classical separative methods such as gas chromatography, high-performance liquid chromatography, possibly coupled with mass spectrometry, etc). However, even if extremely precise and reliable, these techniques are difficult to apply for on-site monitoring and are usually costly. For this reason, my thesis focuses on novel analytical approaches to detect small organic molecules such as these pollutants. In a first part of my work, I developped an original immunosensor based on a competitive complexation and on an electrochemical (amperometric) transduction, for detection of diclofenac, which is a non – steroidal anti – inflammatory drug generally employed to protect patients from inflammation and relieve pain. The working electrode was electrografted with two functional diazonium salts, one as molecular probe (a diclofenac derivative coupled with an arylamine) and the other as redox probe (a quinone) also coupled with an arylamine, able to transduce the hapten-antibody association into a change in electroactivity. The transduction was designed to deliver a current increase upon detection of diclofenac (“signal-on” detection). The detection limit is ca. 20 fM in tap water, which is competitive compared to other label-free immunosensors. In the following part of my thesis, I kept the same original transduction approach (competitive immunoassay) but applied to an Electrolyte-Gated Organic Field-Effect Transistor (EGOFET) based on poly(N-alkyldiketopyrrolo-pyrrole dithienylthieno[3,2-b]thiophene) as organic semiconductor whose gate electrode was functionalized by electrografting a functional diazonium salt capable to bind an antibody specific to 2,4-dichlorophenoxyacetic acid (2,4-D), an herbicide well-known to be a soil and water pollutant. Molecular docking computations were performed to design the functional diazonium salt to rationalize the antibody capture on the gate surface. In the last part of my work, I propose an approach which takes profit not only of the capacitive coupling of the EGOFET but also on its sensitivity to electrostatic charges accumulated on the gate surface. To illustrate this in the field of sensors, I used a short peptide (Gly-Gly-His), known to selectively bind copper ions Cu2+. The peptide was immobilized by direct electrooxidation of the primary amine of the first glycine moiety. I demonstrated that GGH-modified EGOFETs can transduce Cu2+ complexation through significant changes of their output and transfer characteristics, in particular their threshold voltage (VTh).
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Development of a novel high resolution and high throughput biosensing technology based on a Monolithic High Fundamental Frequency Quartz Crystal Microbalance (MHFF-QCM). Validation in food controlCalero Alcarria, María del Señor 02 May 2022 (has links)
Tesis por compendio / [ES] La sociedad actual demanda un mayor control en la seguridad y calidad de los alimentos que se consumen. Esta preocupación se ve reflejada en los diferentes planes estatales y europeos de investigación científica, los cuales, plantean la necesidad de innovar y desarrollar nuevas técnicas analíticas que cubran los requerimientos actuales. En el presente documento se aborda el problema de la presencia de residuos químicos en la miel. El origen de los mismos se debe, fundamentalmente, a los tramientos veterinarios con los que se tratan enfermedades y parásitos en las abejas, y a los tratamientos agrícolas con los que las abejas se ponen en contacto cuando recolectan el néctar en cultivos próximos a las colmenas. La Agencia Europea de Seguridad Alimentaria (EFSA) confirma esta realidad al notificar numerosas alertas sanitarias en la miel.
En los últimos años, los métodos de análisis basados en inmunosensores piezoeléctricos se han posicionado como la base de una técnica de cribado muy prometedora, la cual puede ser empleada como técnica complementaria a las clásicas de cromatografía, gracias a su sencillez, rapidez y bajo coste. La tecnología de resonadores High-Fundamental Frequency Quartz Crystal Microbalance with Dissipation (HFF-QCMD) combina la detección directa en tiempo real, alta sensibilidad y selectividad con un fácil manejo y coste reducido en comparación con otras técnicas. Además, está tecnología permite aumentar el rendimiento del análisis mediante el diseño de arrays de resonadores en un mismo sustrato (Monolithic HFF-QCMD). En este documento se presenta el diseño de un array de 24 sensores HFF-QCMD, junto con un cartucho de micro-fluídica que traza diversos microcanales sobre los diferentes elementos sensores, a los que hace llegar la muestra de miel diluida a analizar. El cartucho actúa también como interfaz para realizar la conexión entre el array de resonadores y el instrumento de caracterización de los mismos. Para obtener el máximo partido del array diseñado, se desarrolla un método de medida robusto y fiable que permite elevar la tasa de adquisición de datos para facilitar la toma de registros eléctricos de un elevado número de resonadores de forma simultánea, e incluso en varios armónicos del modo fundamental de resonancia. La gran sensibilidad de la tecnología HFF-QCMD a los eventos bioquímicos a caracterizar se extiende también a otro tipo eventos externos, como son los cambios de temperatura o presión, lo que es necesario minimizar con el fin de reducir el impacto que estas perturbaciones no deseadas provocan en la estabilidad y fiabilidad de la medida. Con este fin, se desarrolla un algoritmo de procesado de señal basado en la Discrete Transform Wavelet (DTW). Finalmente, todos los desarrollos tecnológicos realizados se validan mediante la implementación de un inmunoensayo para la detección simultánea, en muestras de mieles reales, de residuos químicos de naturaleza química muy diferente, a saber, el fungicida tiabendazol y el antibiótico sulfatiazol. / [CA] La societat actual demanda un major control en la seguretat i qualitat dels aliments que es consumeixen. Aquesta preocupació es veu reflectida en els diferents plans estatals i europeus d'investigació científica, els quals, plantegen la necessitat d'innovar i desenvolupar noves tècniques analítiques que cobrisquen els requeriments actuals. En el present document s'aborda el problema de la presència de residus químics en la mel. L'origen dels mateixos es deu, fonamentalment, als tractaments veterinaris amb els quals es tracten malalties i paràsits en les abelles, i als tractaments agrícoles amb els quals les abelles es posen en contacte quan recol·lecten el nèctar en cultius pròxims als ruscos. L'Agència Europea de Seguretat Alimentària (EFSA) confirma aquesta realitat notificant nombroses alertes sanitàries en la mel.
En els últims anys, els mètodes d'anàlisis basades en immunosensors piezoelèctrics s'han posicionat com la base d'una tècnica de garbellat molt prometedora, la qual pot ser emprada com a tècnica complementària a les clàssiques de cromatografia, gràcies a la seua senzillesa, rapidesa i baix cost. La tecnologia de ressonadors High-Fundamental Frequency Quartz Crystal Microbalance with Dissipation (HFF-QCMD) combina la detecció directa en temps real, alta sensibilitat i selectivitat amb un fàcil maneig i cost reduït en comparació amb altres tècniques. A més, està tecnologia permet augmentar el rendiment del anàlisi mitjançant el disseny d'arrays de ressonadors en un mateix substrat (Monolithic HFF-QCMD). En aquest document es presenta el disseny d'un array de 24 sensors HFF-QCMD, juntament amb un cartutx de microfluídica que estableix diversos microcanals sobre els diferents elements sensors, als quals fa arribar la mostra de mel diluïda a analitzar. El cartutx actua també com a interfície per a realitzar la connexió entre l'array de ressonadors i l'instrument de caracterització d'aquests. Per a traure el màxim partit a l'array dissenyat, es desenvolupa un mètode de mesura robust i fiable que permet elevar la taxa d'adquisició de dades per a facilitar la presa de registres elèctrics d'un elevat nombre de ressonadors de manera simultània, i fins i tot en diversos harmònics del mode fonamental de ressonància. La gran sensibilitat de la tecnologia HFF-QCMD als esdeveniments bioquímics a caracteritzar s'estén també a un altre tipus esdeveniments externs, com són els canvis de temperatura o pressió, la qual cosa és necessari minimitzar amb la finalitat de reduir l'impacte que aquestes pertorbacions no desitjades provoquen en l'estabilitat i fiabilitat de la mesura. A aquest efecte, es desenvolupa un algorisme de processament de senyal basat en la Discrete Transform Wavelet (DTW). Finalment, tots els desenvolupaments tecnològics realitzats es validen mitjançant la implementació d'un immunoassaig per a la detecció simultània, en mostres de mel reals, de residus químics de naturalesa química molt diferent, a saber, el fungicida tiabendazol i l'antibiòtic sulfatiazol. / [EN] Currently, society demands greater control over the safety and quality of the food consumed. This concern is reflected in the different states and European plans for scientific research, which establish the necessity to innovate and develop new analytical techniques that meet current requirements. This document addresses the problem of the presence of chemical residues in honey. Its origin is fundamentally due to the veterinary treatments against diseases and parasites in bees, and also to the agricultural treatments with which the bees come into contact when they collect the nectar in crops close to the hives. The European Food Safety Agency (EFSA) confirms this reality by notifying numerous health alerts in honey.
In recent years, analysis methods based on piezoelectric immunosensors have been positioned as the basis of a very promising screening technique, which can be used as a complementary technique to the classic chromatography, thanks to its simplicity, speed and low cost. High-Fundamental Frequency Quartz Crystal Microbalance with Dissipation (HFF-QCMD) resonator technology combines direct real-time detection, high sensitivity and selectivity with easy handling and low cost compared to other techniques. In addition, this technology allows increasing the performance of the analysis through the design of resonator arrays on the same substrate (Monolithic HFF-QCMD). This document presents the design of an array of 24 HFF-QCMD sensors, together with a microfluidic cartridge that establish various microchannels on the different sensor elements, to provide them the diluted honey sample to be analyzed. The cartridge also acts as an interface to make the connection between the array of resonators and the characterization instrument. To get the most out of the designed array, a robust and reliable measurement method has been developed that allows increasing the data acquisition rate to facilitate electrical parameters readout from a high number of resonators simultaneously, and even in several harmonics of the fundamental resonance mode. The great sensitivity of the HFF-QCMD technology to the biochemical events to be characterized also is extended to other types of external events, such as changes in temperature or pressure, which must be minimized in order to reduce the impact that these unwanted disturbances cause in the stability and reliability of the measurement. To this end, a signal processing algorithm based on the Discrete Transform Wavelet (DTW) is developed. Finally, all the technological developments carried out are validated through the implementation of an immunoassay for the simultaneous detection, in real honey samples, of chemical residues of very different chemical nature, namely, the fungicide thiabendazole and the antibiotic sulfathiazole. / The authors would also like to thank Jorge Martínez from the Laboratory of High Frequency Circuits (LCAF) of the Universitat Politècnica de València (UPV) for assistance with profilometry, and Manuel Planes, José Luis Moya, Mercedes Tabernero, Alicia Nuez and Joaquin Fayos from the Electron Microscopy Services of the UPV for helping with the AFM, and SEM measurements. M.Calero is the recipient of the doctoral fellowship BES-2017-080246 from the Spanish Ministry of Economy, Industry and Competitiveness (Madrid, Spain). This research was funded by Spanish Ministry of Economy and Competitiveness with FEDER funds (AGL 2016-77702-R) and European Commission Horizon 2020 Programme (Grant Agreement number H2020-FETOPEN-2016-2017/737212-CATCH-U-DNA - Capturing non-Amplified Tumor Circulating DNA with Ultrasound Hydrodynamics) for which the authors are grateful. Román Fernández is with the Center for Research and Innovation in Bioengineering (Ci2B), Universitat Politècnica de València, València, Spain and with Advanced Wave Sensors S.L., Paterna, València, Spain. (e-mail: rfernandez@awsensors.com); Yolanda Jiménez, Antonio Arnau and María Calero are with the Center for Research and Innovation in Bioengineering (Ci2B), Universitat Politècnica de València, València, Spain; Ilya Reiviakine is with Advanced Wave Sensors S.L., Paterna, Valencia, Spain and with the Department of Bioengineering, University of Washington, Seattle, WA, 98150 USA; María Isabel Rocha-Gaso and José Vicente García are with Advanced Wave Sensors S.L., Paterna, València, Spain. / Calero Alcarria, MDS. (2022). Development of a novel high resolution and high throughput biosensing technology based on a Monolithic High Fundamental Frequency Quartz Crystal Microbalance (MHFF-QCM). Validation in food control [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/182652 / TESIS / Compendio
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