Immobilization strategy for enhancing sensitivity of immunosensors: L-Asparagine–AuNPs as a promising alternative of EDC–NHS activated citrate–AuNPs for antibody immobilization

• Published in: Biosensors & bioelectronics 2016-04, Vol.78, p.396-403
• Main Authors: Raghav, Ragini, Srivastava, Sudha
• Format: Article
• Language: English
• Subjects: Antibodies; Antibodies, Immobilized - chemistry; Asparagine - chemistry; Biosensing Techniques; CA-125 Antigen - chemistry; CA-125 Antigen - isolation & purification; Citrates; Citric Acid - chemistry; Covalent immobilization; Dynamic range; EDC–NHS; Gold; Gold - chemistry; Gold nanoparticles; Immobilization; Immunosensors; L-Asparagine capped AuNPs; Membrane Proteins - chemistry; Membrane Proteins - isolation & purification; Metal Nanoparticles - chemistry; Nanoparticles; Sensitivity; Strategy; Surface chemistry
• ISSN: 0956-5663; 1873-4235
• DOI: 10.1016/j.bios.2015.11.066

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 (349.36Ω/(IU/mL)/cm2) in case of third strategy as compared to first (147.53Ω/(IU/mL)/cm2) and twice that of second strategy (166.24Ω/(IU/mL)/cm2). Additionally, an extended dynamic range of 0–750IU/mL was observed while for others it was up to 500IU/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. •Comparing strategies for covalent immobilization of antibody (Ab) for immunosensor.•Asn–AuNP better candidate than EDC–NHS activated cit-AuNP for immobilizing Ab.•Orthogonal immobilization of Ab with 88% active Ab for Asn–AuNP-Ab immunosenor.•Asn–AuNP–Ab immunosensor sensitivity 2.5 times that of cit-AuNP–EDC–NHS–Ab.•Immunosensor dynamic range: Asn–AuNP–Ab>cit-AuNP–Ab=cit-AuNP–EDC–NHS–Ab.