Moscow Institute of Physics and Technology, Institutsky 9, Dolgoprudny , Russia 2
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1 Graphene oxide linking layers: a versatile platform for biosensing Yu.V. Stebunov 1, O.A. Aftenieva 1, A.V. Arsenin 1, and V.S. Volkov 1,2 1 Moscow Institute of Physics and Technology, Institutsky 9, Dolgoprudny , Russia 2 University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark stebunov@phystech.edu,
2 Outline Introduction to biosensing Label-free biosensing based on SPR SPR sensor chips based on graphene oxide Optical properties Streptavidin-based biosensing assay Antibody-antigen interactions Summary
3 General biosensor scheme IUPAC. Compendium of Chemical Terminology, 2nd ed. (the "Gold Book"), Receptor Physical or chemical changes Electrical signal
4 General biosensor scheme IUPAC. Compendium of Chemical Terminology, 2nd ed. (the "Gold Book"), Receptor Physical or chemical changes Electrical signal Recognition element Transducer Signal analysis Fluidic design Surface immobilization chemistry Detection format (direct binding, sandwich-type binding, competitive binding) Acoustic Calorimetric Electrochemical Optical Increase SNR Extracting information regarding analyte concentration, binding kinetics, etc.
5 Graphene and graphene oxide biosensing IUPAC. Compendium of Chemical Terminology, 2nd ed. (the "Gold Book"), Receptor Physical or chemical changes Electrical signal Electrochemical biosensor FRET Field-effect transistor Zhang, Chem. Eur. J., 2010 Jang, Angew. Chem. Int. Ed., 2010 He, ACS NANO, 2010
6 Graphene and graphene oxide biosensing IUPAC. Compendium of Chemical Terminology, 2nd ed. (the "Gold Book"), Receptor Physical or chemical changes Electrical signal Electrochemical biosensor FRET Field-effect transistor Zhang, Chem. Eur. J., 2010 Jang, Angew. Chem. Int. Ed., 2010 He, ACS NANO, 2010 Commercial companies Nanomedical Diagnostics, Inc. (USA) Sensia (Spain) BlueVine Graphene Industries, Inc. (USA)
7 Graphene and graphene oxide biosensing IUPAC. Compendium of Chemical Terminology, 2nd ed. (the "Gold Book"), Receptor Physical or chemical changes Electrical signal Electrochemical biosensor FRET Field-effect transistor Zhang, Chem. Eur. J., 2010 Jang, Angew. Chem. Int. Ed., 2010 He, ACS NANO, 2010 Commercial companies Nanomedical Diagnostics, Inc. (USA) Sensia (Spain) Graphene = Transducer + Receptor BlueVine Graphene Industries, Inc. (USA)
8 Label-free biosensing Detecting molecules are without fluorescent or isotope labels Receptor Physical or chemical changes Electrical signal
9 Label-free biosensing Detecting molecules are without fluorescent or isotope labels Receptor Physical or chemical changes Electrical signal Label-free advantages Real-time measurements Possibility to measure binding kinetics of biochemical reactions Retains the properties of interacting substances Easy and quick analysis protocols
10 Label-free biosensing Detecting molecules are without fluorescent or isotope labels Receptor Physical or chemical changes Electrical signal Commercial instruments Label-free advantages Real-time measurements Possibility to measure binding kinetics of biochemical reactions Retains the properties of interacting substances Easy and quick analysis protocols GE Healthcare 2) Quartz crystal microbalance ibidi GmbH 1) Surface plasmon resonance NDK, Ltd. 3) Electric cell-substrate impedance
11 Label-free biosensing Detecting molecules are without fluorescent or isotope labels Receptor Physical or chemical changes Electrical signal Commercial instruments Label-free advantages Real-time measurements Possibility to measure binding kinetics of biochemical reactions Retains the properties of interacting substances Easy and quick analysis protocols GE Healthcare 2) Quartz crystal microbalance ibidi GmbH 1) Surface plasmon resonance NDK, Ltd. 3) Electric cell-substrate impedance
12 Sur face plasmon resonance biosensing Kretschmann s scheme SPR biosensor was presented in 1983 by Liedberg, now biosensors are produced by more than 10 commercial companies
13 Sur face plasmon resonance biosensing Kretschmann s scheme k sp 2 Metal film m X 1 X 1 2 M sin - resonance 2 M SPR biosensor was presented in 1983 by Liedberg, now biosensors are produced by more than 10 commercial companies ( ) res Signal is proportional to the RI changes of the media near the metal film 2
14 Sur face plasmon resonance biosensing SPR angle changes Typical sensogram
15 Sur face plasmon resonance biosensing SPR angle changes Typical sensogram Biosensing sensitivity P P n S SRI E C n C S RI - sensitivity to RI changes E - efficiency
16 SPR instruments and sensor chips Instrument biosensor based on SPR Consumables Biosensor chips Reichert + Reichert BiOptix Biacore Optics + microfluidics + electronics BiOptix Biacore
17 SPR instruments and sensor chips Instrument biosensor based on SPR Consumables Biosensor chips Reichert + Reichert BiOptix Biacore Optics + microfluidics + electronics BiOptix Biacore Self-assembled monolayers Hydrogels Planar surface Alkyl-derivatives of thiols, disulfides, and thioethers Low ligand crowding, reduced re-binding For application with high-molecular-weight macromolecules 3D surface (50-200nm) Hydrophilic, protein-compatible polymers Increased amount of immobilized ligand For applications with small molecules
18 SPR instruments and sensor chips Instrument biosensor based on SPR Consumables Biosensor chips Reichert + Reichert BiOptix Biacore Optics + microfluidics + electronics BiOptix Biacore SPR application areas Academic research Drug discovery in pharmacology Medical diagnostics Safety and quality control Ligand fishing Compound screening Fragment-based drug design Hit confirmation and lead optimization Pharmacokinetics Early ADME Immunogenecity Biosimilars Included into guidelines of FDA and EMA
19 SPR instruments and sensor chips Instrument biosensor based on SPR Consumables Biosensor chips Reichert + Reichert BiOptix Optics + microfluidics + electronics SPR application areas Biacore Academic research Drug discovery in pharmacology Medical diagnostics Safety and quality control BiOptix Ligand fishing Compound screening Fragment-based drug design Hit confirmation and lead optimization Pharmacokinetics Early ADME Immunogenecity Biosimilars Disadvantage: Insufficient sensitivity for small ligand large target interactions Biacore Included into guidelines of FDA and EMA
20 Graphene and graphene oxide SPR biosensing Theoretical sensitivity 1. DNA hybridization Zagorodko, Anal. Chem. 86, Aptamers Wu, Optics Express 18, 2010 Biotin-streptavidin binding kinetics Thiol chip Graphene chip 3. Bacteria Sabramanian, Biosen. Bioelectron. 50, 2013 Sabramanian, ACS Appl. Mat. Interfaces 6, Metal nanoparticles Wijaya, Proc. of SPIE 8424, 2012 Zhang, Colloids Surfaces B 116, 2014
21 Novel type of linking layer based on graphene oxide We proposed a novel type of SPR sensor chips based on non-modified graphene oxide linking layers. Graphene oxide chips can directly replace CM chips in existing biosensing assays and provide higher sensitivity of biosensing. Patented technology of GO SPR sensor chips (RU (Feb. 2013), U.S. Appl )
22 Advantages of GO linking layer for SPR analysis Why graphene oxide? Carboxyl groups for biomolecule immobilization Excellent optical properties High surface area of 2D material Easy and cheap fabrication process
23 Advantages of GO linking layer for SPR analysis Why graphene oxide? Carboxyl groups for biomolecule immobilization Excellent optical properties Deposition of GO films Electrodeposition Spin-coating Spray-coating 80% of one-atomic-layer flakes ( um) High surface area of 2D material Uniform GO film Easy and cheap fabrication process
24 Optical properties of sprayed GO films Ellipsometry of GO film Refractive index at 635 nm Graphene: GO: n n gr GO 3, k 1.16 gr 1.82, k GO
25 Optical properties of sprayed GO films Ellipsometry of GO film Sensitivity to RI changes S RI P n n % S RI graphene 7 nm 0 120% S RI GO 14 nm Refractive index at 635 nm 0 S RI (bare gold) 0 106% S RI 20 nm Graphene: GO: n n gr GO 3, k 1.16 gr 1.82, k GO 0 30% S RI 20 nm
26 GO chips for streptavidin-based immobilization P P n S SRI E C n C E - efficiency Measure of the number of binding sites on a sensing surface Streptavidin-coated surface is used for immobilization of biotinylated ligands such as proteins, peptides, nucleic acids, etc GO film with the thickness of 8.8 nm streptavidin selectively binding the molecules with biotin residue D1, biotinylated 56bp oligonucleotide sequence D2, 50bp oligonucleotide sequence complementary to D1
27 Streptavidin adsorption on the CMD and GO chips CMD sensor chip GO sensor chip 1270 RU 3190 RU 100 nm thickness of CMD layer Streptavidin was adsorbed by amino-coupling procedure Signal was 1270 RU Streptavidin was adsorbed without coupling chemistry via pi-stacking Total signal was 3190 RU Efficiency E was 2.5 times higher for GO sensor chip. Sensitivity was 3 times higher.
28 Selectivity and regeneration of GO sensor chips Selectivity Streptavidin based GO sensor chip selectively adsorbes biotinylated ligands 25 times reduced binding for nonspecific interaction Regeneration Hybridized DNA molecules are regenerated by 20-mM NaOH solution The repeatability of the adsorptions in the range of 10 25%
29 Antibody antigen interactions Protein A-coated GO sensor chips was used for orientationspecific binding of antibodies through Fc-region. Protein A immobilization Antigen-antibody interaction BSA (antigen) 1400 RU Anti-BSA antibody
30 Summary Graphene oxide can directly replace existing linking layers in biosensor chips Streptavidin-based and protein A-based immobilization were demonstrated for GO chips Sensitivity GO sensor chips is 3 times higher than for CMD chips
31 Thank you for your attention! Web: nano.phystech.edu
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