Enhanced Electrochemiluminescence from Ruthenium-Tagged Immune Complex at Flexible Chains for Sensitive Analysis of Glutamate Decarboxylase Antibody

Abstract

Herein, a sensitive electrochemiluminescence (ECL) immunosensor is designed by immobilizing ruthenium-tagged immune complexes at flexible poly-ethylene-glycol (PEG) chains on the electrode surface, which offers more freedom for the collision of the ruthenium complex at the electrode during the initial ECL reaction. The electrochemical characterizations confirm the loose structure of the assembled layer with the immune complex, providing an increase in the current and the resultant enhanced ECL emissions. Comparing the sensors with the rigid structure, a 34-fold increase in the maximal ECL emission is recorded when PEG3400 is used as a linker. Using the optimized protocol, the prepared immunosensor exhibits a wide-ranging linear response to the model antibody (glutamate decarboxylase antibody) ranging from 10 pg/mL to 10 ng/mL. The detection limit is almost two orders lower than the value using the classic enzyme-linked immunosorbent assay, which offers a new design to enhance ECL emissions and the resultant analytical performance.

Keywords: GADA; electrochemiluminescence; flexible PEG chain; immunosensors; ruthenium complex.

Figure 1

Schematic illustration of the construction process of the ECL immunosensor with PEG chains.

Figure 2

(A) CVs of the immunosensor fabrication in 0.1 M PBS (pH = 7.4) with the potential range from −0.2 to 0.8 V and (B) EIS characterization of the immunosensor fabrication in 5.0 mM K₃Fe(CN)₆/K₄Fe(CN)₆ at the frequency range from 0.01 Hz to 10 KHz. Blank line: Au/HS-PEG(3400)-COOH; red line: Au/HS-PEG(3400)-COOH/GAD65 antigen; green line: Au/HS-PEG(3400)-COOH/GAD65 antigen/BSA; blue line: Au/HS-PEG(3400)-COOH/GAD65 antigen/BSA/GADA; purple line: Au/HS-PEG(3400)-COOH/GAD65 antigen/BSA/GADA/Ru-GAD65. (C) CVs and (D) ECL intensity of the immunosensor with TGA (blank line) and HS-PEG(3400)-COOH (red line) as the connection chains with 100 mM TPrA in 0.1 M PBS.

Figure 3

(A) CVs of immunosensors with PEG length and (B) EISs of immunosensor with PEG length (black line: 2000 Da; red line: 3400 Da, green line: 5000 Da and blue line: 10,000 Da). (C) ECL responses of immunosensor with PEG length (Mw ~ 2000, 3400, 5000 and 10,000 Da); (D) ECL intensity of immunosensors with different concentrations of SH-PEG(3400)-COOH (black: 1 μM; red: 5 μM; green: 10 μM; blue: 50 μM; cyan: 100 μM; magenta: 1 mM; yellow: 10 mM).

Figure 4

(A) ECL responses of the immunosensor toward diverse concentrations of GADA modified with HS-PEG₍₃₄₀₀₎-COOH; (B) the selectivity of the immunosensor with various autoantibodies (GADA, IAA Ab, IA2 Ab and ZnT8 Ab); (C) the calibration curve of the ECL immunosensor for GADA modified with HS-PEG₍₃₄₀₀₎-COOH; and (D) the calibration curve of the ECL immunosensor for GADA modified with TGA at various concentrations.