Synthesis of carboxyl-go and preparation of sensing chip procedures

In this study, bioaffinity composites of carboxyl-GO (Figure 1A) were synthesized using the modified Hummer method. In this process, hydroxyl and carbonyl groups were converted to carboxylic acid (R–COOH) by chloroacetic acid (ClCH2CO2H, 99%, Sigma-Aldrich, USA), which acts as an alkylating agent under basic conditions.23 Carboxylate ions are conjugated bases of carboxylic acid, RCOO-, and are negatively charged ions with high ionic adsorption26 and enhanced hydrophilic27 properties with biomolecules. The fabrication and characterization of biosensor chips with Au film were used as the excited SPR sensing substrate, which immobilized the carboxyl-GO sheets at the Au surface to enhance SPR energy coupling and improve protein binding affinity. Cystamine (Cys) 5 mM (cystamine dihydrochloride 98%, Alfa Aesar, United Kingdom) was used as the linker at room temperature for 24 hrs to bond the Au surface thiol coupling into the carboxyl-GO on the sensing chip. The surface morphology of the functional carboxyl-GO-based SPR chip at the carboxylic acid groups formed an organic compound shell shape as shown in scanning electron microscope (SEM) (Figure 1B) and transmission electron microscope (TEM) images (Figure 1C). The activator was a mixture of N-hydroxysuccinimide (NHS, 98%, Sigma-Aldrich, USA) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC, ≥98%, Sigma-Aldrich, USA) solution (100 μL of 0.4 M EDC with 100 μL of 0.1 M NHS). Surface activation of the carboxyl groups containing carboxyl-GO sheets was achieved by reaction with EDC/NHS. In brief, 200 μL of EDC/NHS activation solution was injected into the carboxyl-GO film and stored at 4 °C for 1 hr. The carboxylic acid on the surface of the carboxyl-GO sheets facilitated immobilization of covalent binding with a concentration of anti-PAPPA2 of 25 μg/mL (molecular weight of approximately 150 kDa, HPA018430, Sigma-Aldrich, USA), and the mixture was stored at 4 °C for 3 hrs. Finally, the non-reacted surface active sites were blocked by injecting 200 μL of 1 M ethanolamine (EA, ≥98%, Sigma-Aldrich, USA) into the carboxyl-GO film at 4 °C for 30 mins. Cleaning with deionized water was performed after each of these steps. Covalent coupling provided stable and strong binding of the biomolecules to the carboxyl-GO sheets. Most proteins have amine groups on their surface, so they can be directly conjugated to carboxyl-GO sheets containing reactive groups as shown in Figure 1D. The carboxyl-GO-based SPR sensing chip had a composition of BK7/Cr/Au/Cys/carboxyl-GO as shown in Figure 1E.

Chemical modification of graphene-like 2D biosensing materials (A) surface modification of GO with carboxyl-groups (carboxyl-GO), (B) SEM images of carboxyl-GO sheets, (C) TEM image of carboxyl-GO composites clearly reveals a typical organic matrix-mediated shell structure. (D) Protein immobilization on carboxyl-GO sheets forming highly efficient covalent bonds, (E) SPR sensing system for carboxyl-GO-based immunosensor technology.

Abbreviations: GO, graphene oxide; carboxyl-GO, carboxyl- graphene oxide; CCD, charge-coupled device; SEM, scanning electron microscope; TEM, transmission electron microscope; SPR, surface plasmon resonance.