title: Immobilization strategy for enhancing sensitivity of immunosensors: L-Asparagine-AuNPs as a promising alternative of EDC-NHS activated citrate-AuNPs for antibody immobilization.
abstract: This paper addresses the question - Is EDC-NHS activated gold nanoparticles modified electrode surface the best available option for antibody immobilization for immunosensor fabrication? Is there any other alternative covalent immobilization strategy for orthogonal orientation of antibody, ensuring enhanced sensitivity of immunosensors? Does EDC-NHS activation of carboxyl functionalized nanoparticles surface really leads to orthogonal or directed immobilization of antibody? Gold nanoparticles synthesized using L-Asparagine as reducing and stabilization agent were employed for orthogonal immobilization of antibody for immunosensor fabrication. Anti-CA125 antibody was used as a model system for immunosensor fabrication. A comparative evaluation of immunosensors fabricated using L-Asparagine stabilized gold nanoparticles and citrate stabilized gold nanoparticles via different immobilization strategies/chemistries was done. The three strategies involved immobilization of Anti-CA125 antibody - (1) after EDC-NHS activation of citrate stabilized gold nanoparticles, (2) directly onto citrate stabilized gold nanoparticles and (3) directly onto L-Asparagine stabilized gold nanoparticles modified electrode surfaces. Comparative evaluation of Impedimetric response characteristics showed 2.5 times increase in sensitivity (349.36 Ω/(IU/mL)/cm(2)) in case of third strategy as compared to first (147.53 Ω/(IU/mL)/cm(2)) and twice that of second strategy (166.24 Ω/(IU/mL)/cm(2)). Additionally, an extended dynamic range of 0-750 IU/mL was observed while for others it was up to 500 IU/mL. Amino acid coated gold nanoparticles ensured orthogonal immobilization, lesser randomization, with 88% of active antibody available for antigen binding as opposed to other two strategies with less than 30% active antibody.
authors.affiliation: Department of Biotechnology, Jaypee Institute of Information Technology, A-10, Sector-62, Noida 201307, Uttar Pradesh, India.
authors.forename: Ragini
authors.initials: R
authors.lastname: Raghav
authors.affiliation: Department of Biotechnology, Jaypee Institute of Information Technology, A-10, Sector-62, Noida 201307, Uttar Pradesh, India. Electronic address: sudha.srivastava@jiit.ac.in.
authors.forename: Sudha
authors.initials: S
authors.lastname: Srivastava
mesh_terms: D055910:Antibodies, Immobilized / Q000737:chemistry*; D001216:Asparagine / Q000737:chemistry*; D015374:Biosensing Techniques; D018394:CA-125 Antigen / Q000737:chemistry / Q000302:isolation & purification*; D019343:Citric Acid / Q000737:chemistry; D006046:Gold / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification*; D053768:Metal Nanoparticles / Q000737:chemistry
chemical_list: D055910:Antibodies, Immobilized; D018394:CA-125 Antigen; C451759:MUC16 protein, human; D008565:Membrane Proteins; D019343:Citric Acid; D001216:Asparagine; D006046:Gold
keywords: Covalent immobilization; Dynamic range; EDC–NHS; Gold nanoparticles; L-Asparagine capped AuNPs; Sensitivity