Graphene oxide-based SPR biosensor chip for immunoassay applications

Abstract

This work develops a highly sensitive immunoassay sensor for use in graphene oxide sheet (GOS)-based surface plasmon resonance (SPR) chips. This sensing film, which is formed by chemically modifying a GOS surface, has covalent bonds that strongly interact with the bovine serum albumin (BSA), explaining why it has a higher sensitivity. This GOS film-based SPR chip has a BSA concentration detection limit that is 100 times higher than that of the conventional Au-film-based sensor. The affinity constants (K A) on the GOS film-based SPR chip and the conventional SPR chip for 100 μg/ml BSA are 80.82 × 10(6) M(-1) and 15.67 × 10(6) M(-1), respectively. Therefore, the affinity constant of the GOS film-based SPR chip is 5.2 times higher than that of the conventional chip. With respect to the protein-protein interaction, the SPR sensor capability to detect angle changes at a low concentration anti-BSA of 75.75 nM on the GOS film-based SPR chip and the conventional SPR chip is 36.1867 and 26.1759 mdeg, respectively. At a high concentration, anti-BSA of 378.78 nM on the GOS film-based SPR chip and the conventional SPR chip reveals two times increases in the SPR angle shift. Above results demonstrate that the GOS film is promising for highly sensitive clinical diagnostic applications.

Keywords: Bovine albumin serum (BSA); Graphene oxide sheet (GOS); Protein; Surface plasmon resonance (SPR).

Figure 1

SPR biosensor chip using an immunoassay method for detecting a protein using a gold binding. (a) Conventional SPR chip and (b) GOS film-based SPR chip. Intensity of an evanescent field with a depth of approximately 100 ~ 500 nm decays exponentially with increasing distance from the metal. Bimolecular binding, observed within approximately 10 nm of the metal surface, gives rise to a higher signal shift response than that of the interactive process at a distance of 300 nm therefrom. For typical SPR Kretschmann prism coupling that uses a red light to induce the evanescent field, its field intensity is no more than 600 nm in practice.

Figure 2

GOS, terminal groups, and carboxyl groups. (a) Molecular structure of GOS. (b) Modification of terminal groups (-COOH) of monolayers of GOS film by surface-confined ester reactions. (c) Carboxyl groups ending in -COOH cause GOS surface to exhibit affinity for NH2 end of protein.

Figure 3

Time course for value of SPR sensorgrams in analysis of interaction that involves bimolecular association and dissociation.

Figure 4

GOS-BSA interaction. GOS is immobilized on a planar immobilization film, which is a few tens of nanometers thick, and is readily accessible by analytic BSA protein with which it undergoes specific interactions.

Figure 5

SPR sensorgrams obtained in response to BSA, at concentration of 100 μg/ml, flowing over surfaces of films. (a) Interaction with conventional SPR chip based on Au-MOA film, (b) interaction with the Cys-based GOS film-based SPR chip, and (c) interaction with ODT-based GOS film-based SPR chip. Association rate (k_a), dissociation rate (k_d), affinity constant (K_A), and dissociation constant (K_D) were obtained from fitted curves.

Figure 6

Response of sensor film to various concentrations of BSA. Calibration curves for detection of BSA by GOS film-based SPR chip and conventional SPR chip.

Figure 7

GOS-BSA-anti-BSA interaction. GOS-BSA is immobilized on a planar immobilization film that is a few hundreds of nanometers thick and is readily accessible to analytic anti-BSA protein with which it undergoes particular interactions.

Figure 8

Sensorgram of immobilization of BSA 100 μg/ml on sensor chip in real time. Various detected concentrations of anti-BSA on (a) conventional SPR chip and (b) GOS film-based SPR chip.

Figure 9

Equilibrium analysis of binding of anti-BSA protein to a high-affinity BSA protein.