“…Since the lateral size of the visible image depends upon the convolution effect that arises between the sample and the AFM tip, an increase of about 8 nm in height in the AFM image was found with the uricase-modified APTES/ITO-μEA/glass surface. This increase in the height of the image is in accordance with the size of the uricase molecule ([Akgöl et al 2008]).Figure 3
Contact-mode AFM images of (a) APTES/ITO-μEA/glass and (b) uricase/BS
3
/APTES/ITO-μEA/glass. …”
We fabricated a miniaturized electrochemical uric acid biosensor with a 3-aminopropyltriethoxysilane (APTES)-modified indium tin oxide (ITO) microelectrode array (μEA). The ITO-μEA on a glass plate was immobilized with the enzyme uricase, through a cross-linker, bis[sulfosuccinimidyl]suberate (BS3). The enzyme-immobilized electrode (uricase/BS3/APTES/ITO-μEA/glass) was characterized by atomic force microscopy and electrochemical techniques. The cyclic voltammetry and impedance studies show an effective binding of uricase at the μEA surface. The amperometric response of the modified electrode was measured towards uric acid concentration in aqueous solution (pH 7.4), under microfluidic channel made of polydimethylsiloxane. The μEA biosensor shows a linear response over a concentration range of 0.058 to 0.71 mM with a sensitivity of 46.26 μA mM−1 cm−2. A response time of 40 s reaching a 95% steady-state current value was obtained. The biosensor retains about 85% of enzyme activity for about 6 weeks. The biosensor using μEA instead of a large single band of electrode allows the entire core of the channel to be probed though keeping an improved sensitivity with a small volume of sample and reagents.Electronic supplementary materialThe online version of this article (doi:10.1186/2194-0517-2-5) contains supplementary material, which is available to authorized users.
“…Since the lateral size of the visible image depends upon the convolution effect that arises between the sample and the AFM tip, an increase of about 8 nm in height in the AFM image was found with the uricase-modified APTES/ITO-μEA/glass surface. This increase in the height of the image is in accordance with the size of the uricase molecule ([Akgöl et al 2008]).Figure 3
Contact-mode AFM images of (a) APTES/ITO-μEA/glass and (b) uricase/BS
3
/APTES/ITO-μEA/glass. …”
We fabricated a miniaturized electrochemical uric acid biosensor with a 3-aminopropyltriethoxysilane (APTES)-modified indium tin oxide (ITO) microelectrode array (μEA). The ITO-μEA on a glass plate was immobilized with the enzyme uricase, through a cross-linker, bis[sulfosuccinimidyl]suberate (BS3). The enzyme-immobilized electrode (uricase/BS3/APTES/ITO-μEA/glass) was characterized by atomic force microscopy and electrochemical techniques. The cyclic voltammetry and impedance studies show an effective binding of uricase at the μEA surface. The amperometric response of the modified electrode was measured towards uric acid concentration in aqueous solution (pH 7.4), under microfluidic channel made of polydimethylsiloxane. The μEA biosensor shows a linear response over a concentration range of 0.058 to 0.71 mM with a sensitivity of 46.26 μA mM−1 cm−2. A response time of 40 s reaching a 95% steady-state current value was obtained. The biosensor retains about 85% of enzyme activity for about 6 weeks. The biosensor using μEA instead of a large single band of electrode allows the entire core of the channel to be probed though keeping an improved sensitivity with a small volume of sample and reagents.Electronic supplementary materialThe online version of this article (doi:10.1186/2194-0517-2-5) contains supplementary material, which is available to authorized users.
“…[40][41][42][43][44][45][46] Outros materiais, como capilares monolíticos de sílica, nanotubos de carbono e fulerenos também são utilizados para a imobilização de íons metálicos na presença ou ausência de agentes quelantes. 47 Adsorventes IMAC (matriz com agente quelante imobilizado) podem ser também adquiridos comercialmente.…”
Section: Matrizes Cromatográficas Utilizadas Em Imac E Química De Atiunclassified
Recebido em 16/7/08; aceito em 16/12/08; publicado na web em 11/5/09 IMMOBILIZED METAL-ION AFFINITY CHROMATOGRAPHY (IMAC) OF BIOMOLECULES: FUNDAMENTAL ASPECTS AND TECHNOLOGICAL APPLICATIONS. Immobilized Metal Ion Affinity Cromatography -IMAC -is a group-specific based adsorption applied to the purification and structure-function studies of proteins and nucleic acids. The adsorption is based on coordination between a metal ion chelated on the surface of a solid matrix and electron donor groups at the surface of the biomolecule. IMAC is a highly selective, low cost, and easily scaled-up technique being used in research and commercial operations. A separation process can be designed for a specific molecule by just selecting an appropriate metal ion, chelating agent, and operational conditions such as pH, ionic strength, and buffer type.Keywords: immobilized metal-ion affinity chromatography; purification; biomolecules.
INTRODUÇÃOOs princípios fundamentais da afinidade de biomoléculas por íons metálicos são conhecidos desde o início do século passado. Em 1974, Everson e Parker demonstraram que íons metálicos presentes em metaloproteínas são os principais responsáveis pela adsorção dessas em resinas contendo quelantes imobilizados e exploraram esta afinidade na separação deste tipo de proteínas. 1 A técnica proposta por Everson e Parker popularizou-se com o trabalho publicado por Porath e colaboradores em 1975, quando os autores introduziram o termo cromatografia de afinidade por íons metálicos imobilizados (Immobilized Metal-Ion Affinity Chromatography -IMAC). 2 IMAC explora a interação entre espécies doadoras de elétrons presentes na superfície de biomoléculas em solução e íons metálicos quelatados imobilizados em um suporte sólido. Inúmeras são as aplicações analíticas, preparativas e industriais de IMAC, como a separação e purificação de diferentes biomoléculas (peptídeos, proteínas e ácidos nucléicos), a separação de células a partir de extratos biológicos e estudos de estrutura-função de proteínas.Inicialmente a técnica de IMAC foi extensamente utilizada para purificação de biomoléculas contendo naturalmente grupos doadores de elétrons em resíduos de aminoácidos expostos na superfície (tais como o anel imidazol de histidina), os quais são primariamente responsáveis pela interação biomolécula-íon metálico. Com o advento da tecnologia do DNA recombinante, foi possível a incorporação de caudas (tag) em proteínas que não contêm naturalmente espécies doadoras de elétrons, tal como a fusão de sequência de seis histidinas na porção C ou N-terminal da proteína alvo, conferindo à mesma a possibilidade de purificação por IMAC. 3,4 Apesar de na literatura existirem excelentes artigos de revisão em inglês sobre IMAC 5-14 , nesta revisão, devido à intensa atividade neste campo, pretende-se abordar aspectos fundamentais, aplicações tecnológicas (escala de laboratório e industrial) e científicas com ênfase na purificação de proteínas nativas e recombinantes (com cauda de poli(histidina)). Dentre os aspectos fundamentais do mé...
“…For these reasons, a number of biosensors have been presented using uricase adsorbed to nanomaterials, including ZnO nanoparticles [287], LB films of fatty acids [288], and carbon felt [289]. In agreement with previous strategies, the adsorption performed at pH values in the 6 – 7 range, from a solution containing 0.02 μg·mL −1 – 1 mg·mL −1 , yielded the best results.…”
An important consideration for the development of biosensors is the adsorption of the bio recognition element to the surface of a substrate. As the first step in the immobilization process, adsorption affects most immobilization routes and much attention is given into the research of this process to maximize the overall activity of the bio sensor. The use of nanomaterials, specifically nanoparticles and nanostructured films, offers advantageous properties that can be fine-tuned for interaction with specific proteins to maximize activity, minimize structural changes, and enhance the catalytic step. In the biosensor field, protein-nanomaterial interactions are an emerging trend that span across many disciplines. This review addresses recent publications about the proteins most frequently used, their most relevant characteristics, and the conditions required to adsorb them to nanomaterials. When relevant and available, subsequent analytical figures of merits are discussed for selected biosensors. The general trend amongst the research papers allows concluding that the use of nanomaterials has already provided significant improvements in the analytical performance of many biosensors and that this research field will continue to grow.
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