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Tailor-Made Antibody Immobilization on Magnetic Particles for Biosensors

This study by Roberto Fernández-Lafuente focuses on a one-step concentration protocol for direct covalent immobilization of bio-molecules, such as antibodies and DNA probes, onto paramagnetic particles for use in biosensors. The research aims to develop general, simple, and universal protocols with high loading capacity and a very inert surface for efficient immobilization. Various techniques, including immobilization through amino groups, His-rich regions, and sugar chains, are evaluated for their effectiveness in enhancing stability and specific biorecognition. The results demonstrate successful antibody immobilization on magnetic particles with preserved immunoactivity, offering insights into optimized protocols for biosensor applications.

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Tailor-Made Antibody Immobilization on Magnetic Particles for Biosensors

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  1. Inmovilización orientada de anticuerpos sobre superficies inertes para su uso como biosensores Roberto Fernández-Lafuente Departamento de biocatalisis Instituto de Catálisis. CSIC. Madrid.

  2. One step concentration Magnetic particles begin end of analytical protocol “Design of biosensors: new protocols to achieve a direct covalent immobilization of bio-molecules (antibodies and DNA probes) on paramagnetic particles” • Cell separation • Immunoassay • Isolation of viruses • Isolation of organelles • Molecular genetics • Biocatalysis ??? (insoluble substrates) purification DETECTION OF TRACES DE ANALYTES

  3. + + + + “New general protocols for the conjugation of biomacromolecules to magnetic microspheres” An ideal tailor-made immobilization protocol • Correct orientation: Good specific biorecognization • Stable attachment: Uni- o multipoint attachment • Unspecific adsorption: Second antibody, target molecule, enzyme,… • Spacer arms: Steric hindrances

  4. Correct orientation • Different immobilization protocols: • Simple • Universal • High loading capacity • Very inert surface of magnetic particles Conjugation of antibodies to magnetic particles

  5. Immobilization of Antibodies on magnetic particles • Different Immobilization protocols: • ●Random Immobilization through amino groups • ●His- rich regions • ●Sugar chains

  6. NH2 NH2 NH2 NH2 NH2 NH2 NH2 NH2 Immobilization of Antibodies on magnetic particles: ramdon immobilization through amino groups Br Br Primary amino groups ■Terminal amino groups ■Lys Br Magnetic particles activated with Cyanogenbromide • not correctly oriented ???? • unstable activated supports

  7. Cu+2 NH His His COO- COO- Immobilization of Antibodies on magnetic particles: His-rich regions o Highly activated magnetic particles with IDA – Cu +2 His moieties • Pre-teatment of IgG not necessary • Correctly oriented ???? • High loading capacity • Rapid and simple immobilization • Mild conditions

  8. .. NH2 PGA 1M fosfato pH 7.0 Bloqueo con pH 7,8 Método estándar de inmovilización a soportes epóxido pH 7.0 Inmovilización covalente Adsorción física (hidrofóbica)

  9. His His His His COO - COO - Co +2 COO - COO - His His His His

  10. ¨ NH2 + NH - + ¨ NH3 ¨ NH2 + ¨ NH3 ¨ NH2 O O C C H H ¨ NH2 ¨ + NH3 ¨ NH2 Immobilization of Antibodies on magnetic particles: Sugar chains Very mild oxidation of IgG Very rapid immobilization Highly activated magnetic particles with controlled pK amino groups • Correctly immobilized antibody ??? • Oxidation of sugar chains present in Fc region • Unaltered immunoactivity

  11. Immobilization of Antibodies on magnetic particles RESULTS Immunoactivity expressed • Cyanogenbromide decreases immunoactivity • IDA-Cu chelates-epoxide correctly orientates and mantains immunoactivity • Sugar-chain correctly orientates and mantains immunoactivity

  12. Sugar chain % Immunoactivity IDA-Cu Time (hours) Immobilization of Antibodies on magnetic particles • Unaltered inmunoactivity • Better rate was achieved using sugar than IDA- Cu +2 • Spacer arms: Sugar chain

  13. + + + + + + + Immobilization of Antibodies on magnetic particles Primary amino groups in magnetic particles surface • Excellent ionic exchanger • Approximately 70% proteins • are ionically adsorbed Charged amino in magnetic particles Unspecific adsorption

  14. muestra Analito de interés Soporte altamente activado Adsorción inespecífica de otras biomoléculas presentes en la muestra Inmovilización de anticuerpos a partículas magnéticas Adsorción inespecífica Tratamiento con polímeros???

  15. + + - - + + + + + + + - - + + + Inmovilización de anticuerpos a partículas magnéticas Colapso de las IgG inmovilizadas sobre superficies activadas

  16. Resultados O O O O C C C C H H H H NH3+ NH2 NH2 O O O O = = = = O O = = NH2 NH2 NH2 NH2 NH2 NH3+ NH3+ + + + + - - - - COO- COO- COO- COO- Inmovilización Covalente y Orientada de Anticuerpos sobre Nanopartículas magnéticas aminadas Superficie inerte Tratamiento con polímeros + +

  17. Resultados Inmovilización Covalente y Orientada de Anticuerpos sobre Nanopartículas magnéticas aminadas Estabilidad de los anticuerpos inmovilizados sobre nanopartículas magnéticas aminadas Dextrano-aspártico-aldehído Tampón fosfato sódico 25 mM pH 7

  18. C N - + + + _ _ _ ¨ ¨ ¨ + + + NH3 NH3 NH3 - C N - - Immobilization of Antibodies on magnetic particles Inert surface Primary amino magnetic particles • Covalent attachment • Blocks positive charges Aspartic-aldehyde-dextran • Heterobifunctional polymer (different molecular weights, different percentage of carboxylic groups) • A first Ionic Adsorption plus an additional covalent attachment • Not significant adsorption of protein

  19. Resultados Inmovilización Covalente y Orientada de Anticuerpos sobre Nanopartículas magnéticas aminadas Detección de trazas de HRP con anti-HRP inmovilizandose nanopartículas magnéticas + proteína diana Incubación Anticuerpo inmovilizado sobre partículas magnéticas otras proteínas Generador campo magnético lavados Electroforesis o determinación actividad HRP (100μl)

  20. Resultados IgG Cadena pesada HRP IgG Cadena ligera Calle1 Calle 2 Calle 3 Inmovilización Covalente y Orientada de Anticuerpos sobre Nanopartículas magnéticas aminadas

  21. Xarxa Temàtica de Nanopartícules Magnètiques. Barcelona/Octubre 2.006 Biótica, S.L. Bacteria LIBRE Bacterias capturadas en partículas IgG anti E.coli Partícula magnética

  22. Immobilization of antibodies on magnetic particles Results • Simple and controlled microspheres surface activation • Correctly oriented antibody immobilized • Universal and simple protocol • High yield immobilization • High loading binding • Inert surface: Hetero-bifunctional polymer Excellent immunobiosensor 0.01 μg peroxidase in 5 liters was fully adsorbed on 1 μg of magnetic beads containing anti-peroxidase

  23. Institute of Catalysis. CSIC. Madrid. Spain Prof. Jose Manuel Guisán Dr. Roberto Fernández-Lafuente Dr. Cesar Mateo Dr Manuel Fuentes

  24. Institute of Catalysis. CSIC. Madrid . Spain

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