The first part of this research involves the development of a gold-nanoparticle based sandwich type immunosensor to identify trace amounts of human chorionic gonadotropin hormone based on the direct electrochemical detection of Au nanoparticles. The second part of this research is to design a biosensor that can be easily handled, has higher specificity, sensitivity, low-cost, and rapid response and has a better detection of growth hormone (GH). Current bioanalytical techniques have reported the difficulty to detect GH doping. This research aims to address the issue of measuring GH in small volumes, which has been challenging the limits of analytical detection systems. The electrochemical measurements utilize the redox activity of ferro/ferricyanide in cyclic voltammetry and impedance spectroscopy. The detection limit 10 pg/mL was observed for GH in 20 μL sample volume, which indicated that this versatile platform can be easily adapted for decentralized electrochemical immunosensing of clinically important proteins.

The first part of this research involves the development of a gold-nanoparticle based sandwich type immunosensor to identify trace amounts of human chorionic gonadotropin hormone based on the direct electrochemical detection of Au nanoparticles. The second part of this research is to design a biosensor that can be easily handled, has higher specificity, sensitivity, low-cost, and rapid response and has a better detection of growth hormone (GH). Current bioanalytical techniques have reported the difficulty to detect GH doping. This research aims to address the issue of measuring GH in small volumes, which has been challenging the limits of analytical detection systems. The electrochemical measurements utilize the redox activity of ferro/ferricyanide in cyclic voltammetry and impedance spectroscopy. The detection limit 10 pg/mL was observed for GH in 20 μL sample volume, which indicated that this versatile platform can be easily adapted for decentralized electrochemical immunosensing of clinically important proteins.

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>Magister Scientiae - MSc / A novel 3-mercaptopropionic acid capped iridium selenide quantum dots based label free impedimetric immunosensor was successfully constructed. The 3-mercaptopropionic acid capped iridium selenide quantum dots synthesized were studied using HRTEM, revealing the formation of very small sizes, of about 3 nm. The optical Uv-Vis absorption wavelength of the quantum dots is blue-shifted, a phenomenon explained by the effective mass approximation (EMA) for semiconducting materials with sizes below 10 nm. Using cyclic voltammetry it is noted that the quantum dots have interesting electro-catalytical properties. The immunosensor proved to be sensitive towards nodularin, with a very low detection limit of 0.009 ng/mL and is significantly lower than the recent anti-nodularin ELISA kit developed by (Zhou et al., 2011) which has a detection limit of 0.16 ng/mL.Also the dection limit of the immunosensor is below the South African guideline value for microcystin-LR (0-0.8) μg/L (DWAF; 1996). The calibration curve of the 3MPA-GaSe nanocrystal based biosensor was successfully constructed, which exhibited a trend described by Michaelis-Menten, a typical behaviour of enzymatic biosensors. The detection limit of the biosensor is 0.004 nM and is significantly lower than the action limit of 17beta-estradiol, (1.47 x 10-10 M).

Made available in DSpace on 2015-04-11T13:38:27Z (GMT). No. of bitstreams: 1 Michella Bezerra Lima.pdf: 2830330 bytes, checksum: fabc8826135a63e76710a30d853375fa (MD5) Previous issue date: 2008-08-21 / The development of analytical and low cost methods for fast safe diagnosis and control of illnesses in humans has been one of researchers great interests in the last two decades. This attention is also due to simplicity of use and the great sensitivity in the results. Biosensors based on the surface plasmon resonance (SPR) technique combines the inherent specificity of the recognition elements with high sensitivity of physical transducers, enabling selective and sensitive detection of the analytes of interest without labeling and preliminary purification steps. We proposed the development of a biosensor with immobilized monoclonal antibodies on gold surface, for specific recognition of TNF-α using SPR technique. Design. Anti-TNF-α antibody was covalently immobilized on a gold substrate by silane coupling chemistry. After this, TNF-α antigens aliquots were added on the biosensor surface and the signal was measured during three minutes for TNF-α mass quantification recognized by immobilized antibodies. Results.This biosensor presented 24.0 pg.mm-2 (198,3  28,4) of antibodies immobilized on gold surface. TNF-α human was determined from successive injections, with a linear range from 4.3 up to 34.7 pg/mm-2 (R=0.98, SD=2.18, p<0.0001). Conclusion. We performed a preliminary biosensor to quantify TNF-α for planning of diseases treatment, such as auto immune and periodontal diseases, in which TNF-α inhibitor therapy is usually applied. In this cases, it would be necessary the quantification of this cytokine for therapeutic dose adjustment, improving treatment effectiveness and costs reduction. In the next step we intend to optimize the biosensors conditions preparation as well as to test it in human body fluids samples / Os imunossensores, ou biossensores de imunoafinidade são sistemas que exploram a seletividade e especificidade das interações antígeno-anticorpo, para detecção do analito alvo. O objetivo deste trabalho foi o desenvolvimento de um imunossensor, em que anticorpos monoclonais (anti-TNF-α humano) foram imobilizados em superfícies modificadas de ouro, para reconhecimento específico do Fator de Necrose Tumoral Alfa humano (TNF-α). Para a imobilização dessas imunoglobulinas foi necessária a formação de uma monocamada auto-organizada (SAM) sobre a superfície do sensor, composta por 3-aminopropiltrietóxisilano (APTS), sendo testadas quatro concentrações diferentes (1%, 2,5%, 5% e 10%), em dois tipos de solventes, água deionizada e tolueno, para comparação de metodologias para obtenção de melhores respostas. Os métodos de caracterização adotados foram a espectroscopia de Ressonância de Plásmon de Superfície (SPR), para análise da imobilização de anticorpos, na superfície modificada do sensor e a resposta dos sistemas desenvolvidos para detecção do TNF-α; e, Microscopia de Força Atômica (AFM), para verificação topográfica da SAM da superfície sensora. Os resultados mostraram que as melhores condições de desenvolvimento do biossensor desenvolvido neste trabalho ocorreram com a utilização de APTS 2,5% em tolueno, bloqueado com BSA 1%. O biossensor apresentou 24 pg.mm-2 (198,3  28,4 mgrau) de TNF-α humano imobilizados na superfície de ouro, e após injeções sucessivas de 25 pg.mL-1 de antígeno (n=8), em triplicata experimental, obtivemos uma região linear de resposta de 4,3 a 34.7 pg.mm-2 (R=0.98, SD=2.18, p<0.0001). Em análise topográfica das superfícies sensoras, realizada por AFM, foi observado que as amostras preparadas em tolueno, com melhores respostas de reconhecimento do antígeno, apresentaram menor uniformidade na sua estrutura, em razão da maior rugosidade detectada. Foi desenvolvido um imunossensor experimental, que servirá como modelo para o desenvolvimento de biossensor para aplicação clínica futura para quantificação de TNF- humano em fluidos corporais de pacientes portadores de doenças inflamatórias

Al camp alimentari hi ha una creixent demanda de metodologies analítiques que siguin ràpides, selectives i econòmiques, que permetin la detecció de diferents contaminants alimentaris per a garantir la seguretat del producte al llarg de la cadena alimentària de producció. En aquest context, els biosensors electroquímics s'alcen com a candidats ideals com alternativa a la instrumentació analítica convencional per a l'anàlisi fora de l'àmbit del laboratori. En aquesta tesi es van desenvolupar nous dissenys biomoleculars basats en biosensors electroquímics per a la seguretat alimentària abordant tres aspectes crítics dels dispositius biosensors, que convergeixen cap a la simplificació metodològica: la immobilització orientada del biomaterial, la marcació i la transducció del senyal electroquímic. Donada l'experiència del nostre grup d'investigació en la fabricació de compòsits de grafit-epoxi (GEC) i els avantatges electroquímics demostrats d'aquest material, es van construir nous transductors electroquímics basats en compòsits rígids per a la immobilització orientada de biomolècules per aconseguir la simplificació metodològica en la detecció de DNA i immunoespècies. Els nous transductors construïts es van basar en: i) biocompòsits de grafit-epoxi modificats en volum amb la proteïna avidina (Av-GEB), i ii) compòsits amb un element magnètic integrat (m-GEC). Els biocompòsits d'avidina constitueixen una plataforma universal per a la immobilització directa orientada de material biològic biotinilat, mitjançant la forta unió avidina-biotina. El propi transductor actua com a reservori de material biològic i es pot renovar amb un simple procediment de polit, obtenint una nova superfície per cada assaig. Els magneto elèctrodes permeten la integració de partícules magnètiques als procediments biosensors per tal de realitzar les reaccions biològiques de biorreconeixement en solució, reduint la complexitat de la matriu de les mostres i preconcentrant l'anàlit, obtenint així, una detecció més sensible. Es va dissenyar una nova estratègia de detecció de DNA provinent de mostres reals dels bacteris patògens Salmonella i Escherichia coli, mitjançant la incorporació d'encebadors marcats a la mescla de reacció de la PCR. A part d'amplificar la quantitat de DNA, es va aconseguir marcar doblement el producte amplificat amb biotina i digoxigenina, per aconseguir la immobilització posterior a partícules magnètiques recobertes d'estreptavidina o Av-GEB i la unió d'un conjugat enzimàtic (antiDIG-HRP) per a detectar el producte amplificat. Així es van obtenir uns protocols simples, ràpids i molt sensibles. Aquests protocols van proporcionar límits de detecció més baixos en comparació amb la tècnica clàssica de gel d'electroforesi i la PCR quantitativa amb sistema TaqMan. També es van obtenir resultats molt prometedors incorporant a la PCR un encebador magnètic, encebador unit a partícules magnètiques. Així, es va aconseguir l'amplificació directa a la superfície de les partícules magnètiques, simplificant la metodologia. Aquesta estratègia presenta potencial aplicabilitat per a la detecció directa electroquímica de productes amplificats al termociclador de la PCR. A l'últim bloc de la tesi es va desenvolupar una estratègia immunosensora simplificada amb detecció electroquímica, en la que l'haptè es va immobilitzar a partícules magnètiques recobertes de grups químics tosil, formant un enllaç covalent amb la molècula de naturalesa haptènica. Amb aquesta estratègia es va aconseguir quantificar l'àcid fòlic en mostres cegues de llet enriquida, obtenint valors de recuperació propers al 100%; i quantificar l'àcid fòlic en mostres reals de llet enriquida, obtenint valors molt propers als indicats a la composició dels productes. / The control of food quality along the food production chain has become of growing interest since the increasing incidence of food poisoning is a significant public health concern for customers worldwide. The development of new analytical methodologies with the advantages of rapid response, selectivity and low-cost is still a challenge for food hygiene inspection. Biosensors offer an exciting alternative to the traditional methods allowing decentralised analysis. Novel biomolecular approaches in electrochemical biosensing were developed in this thesis. The main features of these novel biosensing devices were focused on the integration of processes and the methodological simplification. These tasks were achieved considering the main critical aspects of biosensors design: the orientated immobilization of the biorecognition element, the labelling process to achieve the analytical signal and finally, the electrochemical transduction. Novel electrochemical transducers based on graphite-epoxy composites (GEC) were developed. The enhanced electrochemical properties of GEC material has been demonstrated previously in our research group. The novel transducers based on rigid composites were used for the simplified and oriented immobilization of biomolecules such as DNA and immunospecies. The new transducers developed were: i) graphite-epoxy biocomposites bulk-modified with avidin (Av-GEB), and ii) magneto electrodes based on graphite-epoxy composite (m-GEC). Avidin biocomposites can be considered as universal platforms for the oriented immobilization of biotinilated biomolecules, through the strong interaction between avidin and biotin. This biocomposite material acts not only as the electrochemical transducer but also as a reservoir for the biological material, being able to be easily renewed by a polish treatment, obtaining a new surface for each assay. The m-GEC magneto electrodes allow the integration of magnetic particles in biosensing procedures. The biorecognition reaction can thus be effectively performed in solution, reducing the matrix effect of the sample and preconcentrating the analyte, as a result these factors enhance the sensitivity of the detection. A sensitive, rapid and user-friendly strategy for the genetic detection of the main food pathogens - Salmonella and Escherichia coli- was designed. This approach was based on a double-tagging PCR by using two labelled primers, followed by electrochemical genosensing of the double-tagged amplicon. During PCR, not only the amplification of the genetic material was achieved, but also the double-labelling of the amplicon ends with both biotin and digoxigenin tags, in order to achieve the immobilization of the amplicon on streptavidin-modified beads or Av-GEB electrodes as well as the binding of an enzymatic conjugate (antiDIG-HRP) for the electrochemical detection. Better results were achieved in terms of limit of detection compared with the classic gel electrophoresis as well as quantitative PCR based on TaqMan probes. Promising results were also obtained with the use of a novel magnetic primer, primer bound to magnetic beads. This approach allows the DNA direct amplification on streptavidin magnetic beads surface. This strategy showed potential applicability for the direct electrochemical detection of the amplified products in the PCR termocycler. Finally, a simple immunosensing strategy with electrochemical detection was developed. In this case, the haptenic molecule was covalently immobilised on tosylactivated magnetic beads. The utility of this strategy was demonstrated for folic acid detection in spiked milk samples, obtaining recovery values around 100%. Moreover, folic acid was also quantified in real enriched milk samples showing results according to the expected labelled values.

The negative epoxy-based photoresist of SU-8 has a variety of applications within microelectromechanical systems (MEMS) and lab-on-a-chip systems. Herein, SU-8 was functionalized with antigenic peptides to HIV-1 gp41 or HIV-2 gp36 and the detection of antibody against HIV-1/2 was carried out by an electrochemical immunoassay combining an alkaline phosphatase conjugated secondary antibody and p-aminophenyl phosphate. The by- product of the reaction (p-aminophenol) was quantitated electrochemically using differential pulse voltammetry, and the current derived from the oxidation of the hydrolysis product increased linearly over a wide primary antibody concentration range (0.001 – 1 μg/mL), with a detection limit of 1 ng mL-1 (6.7 pM) for both HIV-1 and HIV-2. This level of sensitivity is clinically relevant, and feasibility of this approach for clinical sample testing was also evaluated with HIV clinical patient samples.

The negative epoxy-based photoresist of SU-8 has a variety of applications within microelectromechanical systems (MEMS) and lab-on-a-chip systems. Herein, SU-8 was functionalized with antigenic peptides to HIV-1 gp41 or HIV-2 gp36 and the detection of antibody against HIV-1/2 was carried out by an electrochemical immunoassay combining an alkaline phosphatase conjugated secondary antibody and p-aminophenyl phosphate. The by- product of the reaction (p-aminophenol) was quantitated electrochemically using differential pulse voltammetry, and the current derived from the oxidation of the hydrolysis product increased linearly over a wide primary antibody concentration range (0.001 – 1 μg/mL), with a detection limit of 1 ng mL-1 (6.7 pM) for both HIV-1 and HIV-2. This level of sensitivity is clinically relevant, and feasibility of this approach for clinical sample testing was also evaluated with HIV clinical patient samples.