Graphene oxide-based SPR biosensor chip for immunoassay applications
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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 (KA) on the GOS film-based SPR chip and the conventional SPR chip for 100 μg/ml BSA are 80.82 × 106 M-1 and 15.67 × 106 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.
KeywordsGraphene oxide sheet (GOS) Surface plasmon resonance (SPR) Protein Bovine albumin serum (BSA)
As a novel class of two-dimensional carbon nanostructures, graphene oxide sheets (GOSs) have received considerable attention in recent years in the fields of plasmonics [1, 2, 3] and surface plasmon resonance (SPR) biosensors [4, 5, 6, 7, 8, 9, 10, 11], following both experimental and theoretical scientific discoveries. GOSs have remarkable optical [12, 13, 14, 15, 16, 17, 18, 19] and biosensing [20, 21, 22, 23, 24, 25, 26, 27, 28] properties and are expected to have a wide range of applications. A GOS has a high surface area and sp2 within an sp3 matrix that can confine π-electrons [12, 13, 14, 29]. GOSs contain oxygen at their surfaces in the form of epoxy (-O), hydroxyl (-OH), carboxyl (-COOH), and ether functional groups on a carbon framework [30, 31, 32, 33, 34, 35]. The direct bandgap behavior of a GOS can be controlled by modification by oxidation that generates photoluminescence (PL) [16, 17, 18, 19] and makes available chemically functionalized GOS with biological applications. Surface chemical modifications significantly influence the performance of surface chemistry-derived devices such as optoelectronic devices, luminescent devices, biosensors, and biomaterials. This work develops a novel method for detecting immunological diseases, in which terminal groups (-COOH) are modified and carboxyl groups on GOS surfaces are activated. The carboxyl groups of a GOS film can be converted into amine-reactive groups to increase its surface area sensing. Furthermore, modifying the oxygen-containing functional groups on the surface of GOS can increase its bandgap and its dielectric constant, thereby improving its surface plasmon resonance (SPR) properties.
Designed configuration for sensing
Figure 1a presents a conventional SPR sensing chip and a biomolecule binding mechanism. 8-Mercaptooctanoic acid (MOA; Sigma-Aldrich Co. LLC., St. Louis, MO, USA) is activation of carboxylic acid-terminated thiol self-assembled monolayers (SAMs) on a modified Au surface. MOA binds to the Au surface through their thiol linker (-SH end) resulting monolayers, which are terminated with carboxylic acid (-COOH). The MOA can be further functionalized to immobilize a bovine serum albumin (BSA; Sigma, Chemical Company, St. Louis, MO, USA) protein. Anti-BSA protein interactions are performed as well.
Figure 1b shows a GOS film-based SPR chip with its biomolecule binding mechanism. Two binding mechanisms are functionalized SAMs on amino-modified Au surfaces by solutions of cystamine (Cys; Alfa Aesar Co., Ward Hill, MA, USA) with a concentration of 5 mM and octadecanthiols (ODT, C18H37SH; Sigma-Aldrich Co. LLC.) with a concentration of 10 mM formation of Au-S bonds that immobilize a GOS. Concentration of the GOS diluted in water was 2 mg/ml. The GOS film sensing surface detects BSA protein concentrations in a range of 100 pg/ml to 100 μg/ml and their interaction with anti-BSA. Moreover, analysis is performed of the kinetics of protein-protein interactions at physical contacts that are established between two proteins, owing to biochemical events, protein affinity adsorption forces, and protein binding forces.
Preparation of modified GOS films
Kinetic analysis of bimolecular interactions at surface
where ka and kd are the association and dissociation rate constants for the formation and dissociation of the complex [PT].
where the Langmuir absorption coefficient (Kabs) is defined as Kabs = ka/kd.
where K x is the wave-vector parallel to the surface form which light is reflected, K0 is the wave-vector in a vacuum, and Ksp is the SP wave-vector that is parallel to the interfaces between the metal and the dielectric. θeq is the SPR angle at equilibrium, εp is the refractive index of the prism, and εm and εd are the metal and dielectric constants of the sample, respectively.
Results and discussion
Analysis of sensitivity of interaction between GOS and BSA
Kinetic analysis of interaction between GOS and BSA
Biomolecular interaction analysis using BSA and anti-BSA
In summary, a GOS film was developed for binding with proteins based on SPR analysis for the purpose of immunoassay sensing. The GOS film-based SPR chip herein had a BSA concentration detection limit of as low as 100 pg/ml, which was 1/100th that of the conventional SPR chip. Additionally, in immunoassay detection, the GOS film-based SPR chip was highly sensitive at a low concentration of 75.75 nM, exhibiting an SPR angle shift of 1.4 times that of the conventional chip, and exhibited an SPR angle shift of two times that of the conventional chip at a high concentration of 378.78 nM. Finally, we believe that the fact that the GOS can be chemically modified to increase its SPR sensitivity can be exploited in clinical diagnostic protein-protein interaction applications, especially in cases in which tumor molecular detection is feasible.
The authors would like to thank the Ministry of Science and Technology of the Republic of China, Taiwan, for financially supporting this research under Contract No. MOST 103-2221-E-003 -008, NSC 102-2221-E-003-021, NSC 100-2325-B-182-007, and NSC 99-2218-E-003-002-MY3.
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