By mixing different stoichiometric ratios of chitosan with alginic acid (AA) and chitosan with p(toluenesulfonicacid) (PTSA), two new polymer networks were prepared. FT-IR spectroscopy results show the protonation of chitosan by AA and PTSA. Elemental analysis (EA) results show the composition of the networks. Thermal gravimetry analysis (TGA) and differential scanning calorimetry (DSC) results were used to characterize the thermal stability of the networks. Then, cholesterol oxidase (ChOx) enzyme were immobilized in these networks and checked for potential use of these enzyme entrapped polymer networks (EEPN) for enzyme immobilization. Additionally, the maximum reaction rate (Vmax) and Michaelis-Menten constant (Km) were evaluated for immobilized ChOx in these two polymer networks. Also, temperature and pH optimization, operational stability, shelf-life and the proton conductivity of these networks were investigated.

Synthesis and characterization of conducting copolymers were achieved by using thiophene-3-yl acetic acid cholesteryl ester (CM) and poly (3-methylthienyl methacrylate) (PMTM). A new polythiophene containing a cholesteryl side chain in the &amp / #946 / -position was chemically polymerized in nitromethane/carbon tetrachloride using FeCl3 as the oxidizing agent. Polymerization was also achieved by constant current electrolysis in dichloromethane. Subsequently, conducting copolymers of thiophene-3-yl acetic acid cholesteryl ester (CM), PCM1 (obtained from chemical polymerization method), PCM4 (obtained from constant current electrolysis) with pyrrole were synthesized. Thiophene functionalized methacrylate monomer (MTM) was synthesized via esterification of the 3-thiophene methanol with methacryloyl chloride. The methacrylate monomer was polymerized by free radical polymerization in the presence of azobis (isobutyronitrile) (AIBN) as the initiator. Graft copolymers of poly (3-methylthienyl methacrylate)/polypyrrole, (PMTM2/PPy) and poly (3-methylthienyl methacrylate)/polythiophene, (PMTM2/PTh) were synthesized by constant potential electrolyses. PMTM2 coated Pt electrodes were utilized as the anode in the polymerization of pyrrole and thiophene. Moreover, oxidative polymerization of PMTM1 was studied by galvanostatic and chemical techniques. Characterizations of the samples were performed by CV, FTIR, NMR, DSC, TGA and SEM analyses. Electrical conductivities were measured by the four-probe technique. Immobilization of invertase in conducting copolymer matrices, poly (3-methylthienyl methacrylate) with pyrrole and thiophene was achieved by constant potential electrolysis using the sodium dodecyl sulfate as the supporting electrolyte. Polythiophene was also used for immobilization matrices. Cholesterol oxidase has been immobilized in conducting copolymer of thiophene-3-yl acetic acid cholesteryl ester with polypyrrole (CM/PPy) and polypyrrole (PPy) by the electropolymerization method. p-Toluene sulfonic acid was used as a supporting electrolyte. Kinetic parameters (Kinetic parameters / Vmax and Michaelis-Menten constant / Km) and operational stability of enzyme electrodes were investigated. Surface morphology of the films was also examined.

No description available.

Phytosterols are steroid chemicals produced by plants for the purposes of membrane function and hormone production. Phytosterols can cause endocrine disruption in aquatic species at very low concentrations and are suspected of contributing to endocrine disruption linked to pulp and paper effluent. Wastewater from the pulp and paper industry is often treated biologically in aerated stabilization basins (ASBs) that expose phytosterols to a range of redox zones. Phytosterol removal in ASBs varies and stigmasterol has even been shown to increase across the treatment system. Little is known about the microbial processes that occur within ASBs and their effect on phytosterol removal. The objective of this research was to assess the biotransformation potential of phytosterols in a simulated ASB treatment system and to improve understanding of the processes that occur within the various redox zones and their impact on the removal of phytosterols. To assess the biotransformation of phytosterols under aerobic conditions, three assays were conducted using a stock aerobic culture fed with pulp and paper wastewater. The assays tested three conditions: phytosterols present as a sole added carbon source, phytosterols with dextrin as an added carbon source, and phytosterols with ethanol as a solubilizing agent and added carbon source. Phytosterol biotransformation was found to be limited by low phytosterol solubility. When solubilized, phytosterol removal occurred in two phases: an initial near-linear removal, followed by accelerated removal during the culture's stationary stage, possibly due to the release of extracellular cholesterol oxidase. The anoxic and anaerobic biotransformation of phytosterols was examined through a series of three semi-batch cultures maintained under nitrate-reducing, sulfate-reducing and fermentative/methanogenic conditions, all developed from stock cultures fed with pulp and paper wastewater. Phytosterol removal was significant in the nitrate-reducing culture, although microbial activity and phytosterol removal declined in later stages. Phytosterol removal was also observed in the sulfate-reducing culture, although there was a significant lag period before removal occurred. No phytosterol removal was observed in the fermentative/methanogenic culture. Phytosterol biotransformation was also examined in the context of a lab-scale ASB fed continuously with pulp and paper wastewater. The steady-state ASB effluent and sediment characteristics were examined over three hydraulic retention times (HRTs). Effluent quality was not significantly affected by a change in HRT but sediment characteristics were significantly affected and, at shorter HRTs, phytosterols accumulated in the sediment. Wastewater bioassays demonstrated the release of phytosterols during the breakdown of solids. This research improves the understanding of biological processes within ASBs and their effect on phytosterol removal.

Made available in DSpace on 2016-06-02T19:19:58Z (GMT). No. of bitstreams: 1 SOUZA_Tamera_2014.pdf: 3828061 bytes, checksum: 5a203182c573895f9074200bb9448449 (MD5) Previous issue date: 2014-02-24 / Financiadora de Estudos e Projetos / The layer-by-layer technique is one of the most used in the immobilization of molecules of biological interest, being largely employed in the fabrication of modified electrodes for biosensors. One of the main challenges in the development of biosensors is to eliminate interfering electroactive species within the detection system, therefore, this problem can be solved with the introduction of inorganic or organic electron mediators. Here, we carried out systematic studies for the application of hemoglobin acting as an electron mediator, and the self-assembly technique was used to immobilize haemoglobin in nanostructured thin films. The LbL films were tested with two polyelectrolytes, obtaining uniform films with activity kept during 15 days, without responses to possible interfering phospholipids. The layer-bylayer technique was also used to immobilize the enzyme cholesterol oxidase, alternated with different polyelectrolytes. The thin films showed a good performance as an amperometric biosensor for the detection of cholesterol at low concentrations (10-5 M), with the functioning of the biosensor attributed to the use of hemoglobin in mediating the effects of excluding interfering ascorbic acid and uric acid. Some tests were also performed in real samples using egg yolk, with good results obtained. The high sensitivity and selectivity of the biosensor have been attributed to the nanostructured thin nature of the films, despite recognition capability of the biomolecules used, with a strong potential to contribute for new device technologies for food analysis. / A técnica de automontagem ou layer-by-layer é uma das mais utilizadas na imobilização de moléculas de interesse biológico. Essa técnica é empregada na fabricação de eletrodos modificados utilizados, como por exemplo, os biossensores. Um dos desafios no desenvolvimento de biossensores é eliminar interferentes de espécies eletroativas dentro do sistema de detecção. Esse problema pode ser solucionado com a introdução de mediadores de elétrons inorgânicos ou orgânicos. Nessa dissertação foram realizados estudos sistemáticos para a aplicação da hemoglobina atuando como mediadora de elétrons. A técnica de automontagem foi utilizada para a imobilização da hemoglobina, testada com dois polieletrólitos, obtendo filmes uniformes, mantendo sua atividade por 15 dias e sem respostas aos possíveis interferentes fosfolipídios. A automontagem também foi utilizada para imobilizar a enzima colesterol oxidase, alternada com diferentes polieletrólitos. Os filmes finos apresentaram um bom desempenho como biossensor amperométrico na detecção de colesterol em baixas concentrações tais como 10-5 M. O bom funcionamento do biossensor deve- se ao emprego da hemoglobina com mediador excluindo os efeitos dos interferentes acido úrico e acido ascórbico. Também foram realizados testes com amostras reais utilizando a gema de ovo obtendo bons resultados. A alta sensibilidade e seletividade dos biossensores foram atribuídas à natureza do filme automontado e à capacidade de reconhecimento das biomoléculas. Este estudo abre caminho para novas tecnologias de dispositivos para análises de alimentos.

Mycobacterium tuberculosis (Mtb), a deadly pathogen, has scourged mankind for many centuries and has remained a major threat to global world health. Tuberculosis, the disease caused by this bacterium, is a major cause of death in developing nations and there is potential for its re-emergence in developed countries. An alarming rise in cases of multidrug-resistant and extremely-drug resistant tuberculosis (MDR-TB and XDR-TB) that do not respond to the customary first-line antibiotics necessitates the urgent need for development of new anti-TB drugs. Mtb becomes engulfed in human macrophages post infection of the host, but persists in the harsh environment of the human lungs by utilization of host cholesterol as a carbon source. The P450s CYP125A1, CYP142A1 and CYP124A1 are responsible for catalysing the side-chain degradation of cholesterol, which is critical for cholesterol to be used in the Mtb β-oxidation pathway for energy production. This PhD thesis focuses on understanding the structure/mechanism of the Mtb cholesterol 27-oxidases with the aim of facilitating the development of novel inhibitors of these P450s, which are crucial for Mtb to infect the host and to sustain infection. CYP142A1 and CYP124A1 were purified through three chromatographic steps with contaminating proteins successfully removed to give highly pure forms of these enzymes following the final purification step. Spectrophotometric titrations indicate that CYP142A1 and CYP124A1 bind tightly to cholesterol and cholestenone (and also to branched-chain methyl lipids for CYP124A1), highlighting their physiological roles in sterol and fatty acid metabolism, respectively. Binding analyses with a range of azole antibiotics revealed tight binding to bifonazole, clotrimazole, miconazole and econazole, and weak binding to fluconazole. Studies with compounds from a fragment screening library revealed weak binding to fragment hits for the cholesterol oxidases, but much tighter binding to these enzymes was found for ‘elaborated’ hits from a previous fragment screen on the Mtb cyclodipeptide oxidase CYP121A1, indicative of improved ligand potency achieved via ‘fragment merging’ strategies, and of structural similarities between these diverse Mtb P450s. Light scattering data indicate that CYP142A1 exists in dimeric form in solution, but becomes monomeric when treated with DTT; while CYP124A1 is completely monomeric. Crystal structures of CYP142A1 and CYP124A1 in complex with cholestenone, econazole and fragment library hits were determined. CYP142A1 crystal structures with econazole and fragment hits revealed heme coordination via the heterocyclic nitrogen in an azole group, and provide important data towards design of superior inhibitor drugs. The binding of cholestenone within the active site channels of CYP124A1 and CYP142A1 revealed an alignment favourable for C27 hydroxylation of the cholestenone side chain, which supports the physiological roles of CYP142A1 and CYP124A1 (as well as CYP125A1) in host cholesterol catabolism.

No description available.

Esta tese descreve o desenvolvimento e aplicação de sensores particulados (Parte A) e nanomecânicos (Parte B). Nanopartículas de poliestireno (PS) decoradas com Poli (etileno glicol), PEG, foram sintetizadas e caracterizadas antes e após adsorção do corante vermelho do Congo (VC). Colesterol oxidase (Chox), a principal enzima na oxidação do colesterol, foi imobilizada sobre PS/PEG e PS/PEG/VC para gerar sensores de colesterol. A bioconjugação entre VC e Chox permitiu respostas lineares para dosagem de colesterol HDL presente em amostras de sangue artificial. A lipase, uma hidrolase com larga aplicação científica e industrial, também foi imobilizada sobre PS/PEG e PS/PEG/VC. Os parâmetros cinéticos da hidrólise de p-nitrofenil butirato determinados para lipase livre e lipase imobilizada mostraram que sobre PS/PEG/VC a velocidade máxima de reação (Vmax) e número de renovação (kcat) aumentaram em comparação com os valores determinados para enzima livre. Lipase imobilizada sobre PS/PEG/VC pôde ser reutilizado por até sete vezes, perdendo um máximo de 10% ou 30% da atividade enzimática original a 40 °C ou 25 °C, respectivamente. Estes efeitos foram atribuídos à bioconjugação entre lipase e VC. Os comportamentos catalíticos da lipase e da Chox na ausência e na presença de grafeno (G) ou grafeno oxidado (GO) foram sistematicamente investigados. Na presença de G, ambas enzimas apresentaram valores de Vmax e de kcat superiores aos das enzimas livres. Já na presença de GO, Chox não apresenteou atividade e lipase apresentou Vmax e de kcat superiores aos da enzima livre. Estes resultados foram atribuídos à bioconjugação com as partículas de G e GO e grupos hidrofílicos presentes no plano basal de GO. Sensores nanomecânicos foram desenvolvidos a partir de microcantileveres (MC). Respostas nanomecânicas frente à variação de umidade relativa do meio foram detectadas utilizando filmes finos de Poli (hidroxietil metacrilato), PHEMA, com três diferentes massas molares médias depositados sobre MC de silício puros, um substrato hidrofílico, e sobre MC revestidos com PS, um substrato hidrofóbico. Os resultados demonstraram que as respostas nanomecânicas dependem não só do tamanho de cadeia de PHEMA, mas também da camada de água interfacial entre PHEMA e MC. Transdução nanomecânica foi usada para detectar e dosar a presença do biomarcador antígeno carcinoembrionário (CEA) presente em amostras de soro. CEA é um biomarcador de interesse clínico-diagnóstico para acompanhamento e prognóstico de câncer de cólon. O biomarcador é primeiro reconhecido pelo anticorpo (MAb 3C1) ancorado a uma nanopartícula de ouro, e posteriormente reconhecido pelo anticorpo (MAb 3C6) ancorado em um MC de silício, o qual serve como um ressonador mecânico de massa de nanopartículas de ouro capturadas. O biosensor desenvolvido é capaz de dosar CEA sérico em concentrações traços, correspondente a 1,0 10-16 g ml-1. / This thesis reports the development and application of particulate (Part A) and nanomechanics sensors (Part B). Poly (ethylene glycol), PEG, decorated polystyrene (PS) nanoparticles were synthesized and characterized before and after adsorption of the dye Congo red (CR). Cholesterol oxidase (Chox), the key enzyme in the oxidation of cholesterol, was immobilized onto PS/PEG and PS/PEG/CR particles for generating cholesterol sensors. The bioconjugation between CR and Chox allowed linear responses for HDL cholesterol content in artificial blood samples. Lipase, a hydrolase with large scientific and industrial applications, was also immobilized onto PS/PEG and PS/PEG/VC nanoparticles. The kinetic parameters of the hydrolysis of p-nitrophenyl butyrate were determined for free lipase and immobilized onto PS/PEG/CR particles. The results showed that maximum reaction velocity (Vmax) and catalytic efficiency (kcat) increased compared to the values determined for the free enzyme. Lipase immobilized onto PS/PEG/CR particles could be recycled seven times, losing maximum 10% or 30% of the original enzymatic activity at 40 °C or 25 °C, respectively. These effects were attributed to bioconjugation between lipase and CR. Catalytic behavior of lipase and Chox in the absence and in the presence of graphene (G) or graphene oxide (GO) was systematically investigated. In the presence of G, both enzymes showed Vmax and kcat values higher than free enzymes. On the other hand, ChOx was inactive and the interactions between GO and lipase showed Vmax and kcat values higher than those of the free enzyme. These results were attributed to bioconjugation of G and GO particles and hydrophilic groups present in the basal plane of GO. Nanomechanical sensors were developed from microcantilevers (MC). Nanomechanical answers against the relative humidity variation of the medium were detected using thin films of poly (hydroxyethyl methacrylate), PHEMA, with three different average molecular masses deposid onto bare silicon microcantilevers, a hydrophilic substrate, and onto polystyrene (PS) coated microcantilevers, which is a hydrophobic substrate. The results found in the present study demonstrate that the micromechanical responses observed are related not only to the polymer molecular weight, but also to the polymer-interface phenomena and environment-polymer interface. Nanomechanics transductions were used to dose and detect the presence of carcinoembryonic antigen in serum samples. CEA is a biomarker of clinical diagnostic interest for monitoring and prognose of colon cancer. Biomarker is first recognized by a surface-anchored antibody to a gold nanoparticle (MAB 3C1) and later recognized by a surface-anchored antibody to a silicon MC (MAB 3C6), that acts as a mechanical resonator for \'weighing\' the mass of the captured nanoparticles. The biosensor developed is able to dose serum CEA in trace concentrations, corresponding to 1.0 10-16 g ml-1.

Esta tese descreve o desenvolvimento e aplicação de sensores particulados (Parte A) e nanomecânicos (Parte B). Nanopartículas de poliestireno (PS) decoradas com Poli (etileno glicol), PEG, foram sintetizadas e caracterizadas antes e após adsorção do corante vermelho do Congo (VC). Colesterol oxidase (Chox), a principal enzima na oxidação do colesterol, foi imobilizada sobre PS/PEG e PS/PEG/VC para gerar sensores de colesterol. A bioconjugação entre VC e Chox permitiu respostas lineares para dosagem de colesterol HDL presente em amostras de sangue artificial. A lipase, uma hidrolase com larga aplicação científica e industrial, também foi imobilizada sobre PS/PEG e PS/PEG/VC. Os parâmetros cinéticos da hidrólise de p-nitrofenil butirato determinados para lipase livre e lipase imobilizada mostraram que sobre PS/PEG/VC a velocidade máxima de reação (Vmax) e número de renovação (kcat) aumentaram em comparação com os valores determinados para enzima livre. Lipase imobilizada sobre PS/PEG/VC pôde ser reutilizado por até sete vezes, perdendo um máximo de 10% ou 30% da atividade enzimática original a 40 °C ou 25 °C, respectivamente. Estes efeitos foram atribuídos à bioconjugação entre lipase e VC. Os comportamentos catalíticos da lipase e da Chox na ausência e na presença de grafeno (G) ou grafeno oxidado (GO) foram sistematicamente investigados. Na presença de G, ambas enzimas apresentaram valores de Vmax e de kcat superiores aos das enzimas livres. Já na presença de GO, Chox não apresenteou atividade e lipase apresentou Vmax e de kcat superiores aos da enzima livre. Estes resultados foram atribuídos à bioconjugação com as partículas de G e GO e grupos hidrofílicos presentes no plano basal de GO. Sensores nanomecânicos foram desenvolvidos a partir de microcantileveres (MC). Respostas nanomecânicas frente à variação de umidade relativa do meio foram detectadas utilizando filmes finos de Poli (hidroxietil metacrilato), PHEMA, com três diferentes massas molares médias depositados sobre MC de silício puros, um substrato hidrofílico, e sobre MC revestidos com PS, um substrato hidrofóbico. Os resultados demonstraram que as respostas nanomecânicas dependem não só do tamanho de cadeia de PHEMA, mas também da camada de água interfacial entre PHEMA e MC. Transdução nanomecânica foi usada para detectar e dosar a presença do biomarcador antígeno carcinoembrionário (CEA) presente em amostras de soro. CEA é um biomarcador de interesse clínico-diagnóstico para acompanhamento e prognóstico de câncer de cólon. O biomarcador é primeiro reconhecido pelo anticorpo (MAb 3C1) ancorado a uma nanopartícula de ouro, e posteriormente reconhecido pelo anticorpo (MAb 3C6) ancorado em um MC de silício, o qual serve como um ressonador mecânico de massa de nanopartículas de ouro capturadas. O biosensor desenvolvido é capaz de dosar CEA sérico em concentrações traços, correspondente a 1,0 10-16 g ml-1. / This thesis reports the development and application of particulate (Part A) and nanomechanics sensors (Part B). Poly (ethylene glycol), PEG, decorated polystyrene (PS) nanoparticles were synthesized and characterized before and after adsorption of the dye Congo red (CR). Cholesterol oxidase (Chox), the key enzyme in the oxidation of cholesterol, was immobilized onto PS/PEG and PS/PEG/CR particles for generating cholesterol sensors. The bioconjugation between CR and Chox allowed linear responses for HDL cholesterol content in artificial blood samples. Lipase, a hydrolase with large scientific and industrial applications, was also immobilized onto PS/PEG and PS/PEG/VC nanoparticles. The kinetic parameters of the hydrolysis of p-nitrophenyl butyrate were determined for free lipase and immobilized onto PS/PEG/CR particles. The results showed that maximum reaction velocity (Vmax) and catalytic efficiency (kcat) increased compared to the values determined for the free enzyme. Lipase immobilized onto PS/PEG/CR particles could be recycled seven times, losing maximum 10% or 30% of the original enzymatic activity at 40 °C or 25 °C, respectively. These effects were attributed to bioconjugation between lipase and CR. Catalytic behavior of lipase and Chox in the absence and in the presence of graphene (G) or graphene oxide (GO) was systematically investigated. In the presence of G, both enzymes showed Vmax and kcat values higher than free enzymes. On the other hand, ChOx was inactive and the interactions between GO and lipase showed Vmax and kcat values higher than those of the free enzyme. These results were attributed to bioconjugation of G and GO particles and hydrophilic groups present in the basal plane of GO. Nanomechanical sensors were developed from microcantilevers (MC). Nanomechanical answers against the relative humidity variation of the medium were detected using thin films of poly (hydroxyethyl methacrylate), PHEMA, with three different average molecular masses deposid onto bare silicon microcantilevers, a hydrophilic substrate, and onto polystyrene (PS) coated microcantilevers, which is a hydrophobic substrate. The results found in the present study demonstrate that the micromechanical responses observed are related not only to the polymer molecular weight, but also to the polymer-interface phenomena and environment-polymer interface. Nanomechanics transductions were used to dose and detect the presence of carcinoembryonic antigen in serum samples. CEA is a biomarker of clinical diagnostic interest for monitoring and prognose of colon cancer. Biomarker is first recognized by a surface-anchored antibody to a gold nanoparticle (MAB 3C1) and later recognized by a surface-anchored antibody to a silicon MC (MAB 3C6), that acts as a mechanical resonator for \'weighing\' the mass of the captured nanoparticles. The biosensor developed is able to dose serum CEA in trace concentrations, corresponding to 1.0 10-16 g ml-1.

A imobilização de moléculas de interesse biológico em superfícies sólidas é essencial para uma série de aplicações biotecnológicas. Dentre as técnicas de imobilização, a automontagem camada por camada por adsorção física possui inúmeras vantagens, incluindo condições brandas e fisiológicas de preparação, capacidade de incorporar diferentes biomoléculas, e controle molecular. Nesta tese foram explorados filmes nanoestruturados de materiais de interesse biológico, bem como modelos de membranas, em que foram empregadas a técnica de automontagem e a preparação de lipossomos. Os lipossomos, que serviram como modelos de membrana, foram imobilizados em filmes automontados e sua integridade estrutural foi mantida. Também foram utilizados para incorporar e estabilizar melanina, e então imobilizados em filmes automontados, com preservação da propriedade fluorescente da melanina. A automontagem também foi utilizada para imobilização das enzimas uricase, fitase e colesterol oxidase, alternadas com camadas de polieletrólitos. Estes filmes mostraram bom desempenho como biossensores amperométricos para uricase e fitase, e como biossensores usando espectroscopia de impedância para a fitase e colesterol oxidase. Tais biossensores foram usados para detectar baixas quantidades de ácido úrico, ácido fítico e colesterol, respectivamente. Não houve efeitos de interferentes nos sensores amperométricos devido à utilização de eletrodos previamente modificados com azul da Prússia, que funcionou como mediador redox. A alta sensibilidade e seletividade dos biossensores foram atribuídas à natureza do filme ultrafino e à capacidade de reconhecimento das biomoléculas, respectivamente. Esta abordagem abre caminho para novas tecnologias de dispositivos para diagnósticos clínicos e análises de alimentos, bem como para entender mecanismos de interação da biomolécula com a membrana celular para o desenvolvimento de agentes terapêuticos. / The immobilization of molecules of biological interest on solid surfaces is essential for a number of biotechnological applications. Among the techniques for immobilization the layer-by-layer (LbL) method based on physical adsorption exhibits several advantages, including mild, physiological conditions for film preparation, ability to incorporate different biomolecules and molecular control. In this thesis, nanostructured films made with materials of biological interest were exploited as model membranes, where use was made of the LbL technique and liposomes. The latter, which served as membrane models, were immobilized in LbL films and had their structural integrity preserved. Liposomes were also used to incorporate and stabilize melanin, which were then deposited on LbL films with the fluorescence of melanin being preserved. The LbL method was also used to immobilize the enzymes uricase, phytase and cholesterol oxidase, alternated with layers of polyelectrolytes. These LbL films were employed in amperometric biosensors with uricase and phytase, and in biosensors based on impedance spectroscopy for phytase and cholesterol oxidase. Low amounts of uric acid, phytic acid and cholesterol were detected, respectively. There was no effect from interferents in the amperometric biosensors because the electrodes were previously modified with a layer of Prussian Blue, which acted as a redox mediator. The high sensitivity and selectivity were attributed to the ultrathin nature of the films and the ability of molecular recognition of the biomolecules, respectively. The approaches used here open the way for novel devices for clinical diagnostics and food quality control, in addition to understanding the interaction mechanisms between the biomolecules and the cell membranes, which is important for developing therapeutic agents.