Nanoquantification of RUNX2 by a 1,1'-carbonyldiimidazole-diamond mediated sandwich assay for osteogenic differentiation

Abstract

Adipose tissue-derived stem cells (ADSCs) possess the capability to modulate the immune response and alleviate inflammation, rendering them a promising therapeutic option for various conditions, including autoimmune diseases, cardiovascular diseases, and tissue injuries. The osteogenic differentiation in ADSCs plays a pivotal role in fracture healing, bone growth, and the overall bone turnover process, governed by intricate interactions. Runt-related Transcription Factor 2 (RUNX2) is a key player in mineralized tissue generation and is typically found in the early stages of osteogenic differentiation. The objective of this study was to develop a high-affinity sandwich biosensor for the quantification of RUNX2. 1,1'-Carbonyldiimidazole-modified nanodiamond was immobilized on an amine-modified interdigitated electrode surface, followed by the use of a capture antibody to facilitate antigen interaction. A sandwich assay was conducted with the antibody, and the limit of detection for RUNX2 was calculated as 0.1 ng/mL, with a regression value (R²) of 0.9914 over a linear range of 1-2000 ng/mL. Furthermore, biofouling experiments with a nonimmune antibody, BSA, and TNF-α did not yield any current responses, indicating the specific detection of RUNX2. Additionally, RUNX2-spiked serum exhibited an increasing current response at all concentrations, confirming the selective detection of RUNX2.

Keywords: Antibody orientation; Nanodiamond; Runt-related transcription factor; Sandwich pattern.

Fig. 1

Schematic illustration of RUNX2 identification on nanodiamond-modified electrode surface. IDE surface was initially treated with KOH and then APTES was attached. Furthermore, CDI-coated nanodiamond was attached and then antibody was immobilized on the nanodiamond through CDI-amine interaction. Finally, the sandwich assay was conducted by introducing a detection antibody.

Fig. 2

Imaging of nanodiamond with FESEM and FETEM observation. (a) FESEM with 500 nm; (b) FESEM with 200 nm; (c) FETEM with 50 nm; (d) FETEM with 20 nm. The nanodiamond had a spherical shape with uniform distribution and the size range was identified as approximately 20 ± 5 nm; (e) EDX confirmed the presence of ∼94 % carbon and ∼6 % of nitrogen and oxygen.

Fig. 3

Comparison experiment of antibody with and without nanodiamond. (a) without nanodiamond; (b) with nanodiamond; (c) Current difference of antibody attachment without nanodiamond; (d) Current difference of antibody attachment with nanodiamond. Antibody attachment with nanodiamond shows a higher current response. Data were averaged by triplicate values.

Fig. 4

(a) Detection of RUNX2 on capture antibody immobilized surface. Various concentrations of RUNX 2 were dropped on antibody attached surfaces and the currents were recorded. Furthermore, with increasing concentrations of RUNX 2, the current levels were increased. Figure inset represents the diagrammatic scheme. (b) Sandwich assay of antibody-RUNX2-antibody. A constant concentration of detection monoclonal antibody was dropped on capture antibody-RUNX2 immobilized electrode surfaces. With increasing concentrations of RUNX 2, the current levels were increased. The figure inset represents the diagrammatic scheme.

Fig. 5

(a) Comparison of RUNX2 detection with capture antibody-RUNX2 and Capture antibody-RUNX2-detection antibody complexes. The current response was much higher with capture antibody-RUNX2-detection antibody interaction than with capture antibody-RUNX. The figure inset represents the diagrammatic scheme. Data were averaged with triplicate values. (b) The current difference for each RUNX2 concentration was calculated and plotted in an Excel sheet. The detection limit was calculated as 0.1 ng/mL and 1 ng/mL on capture antibody-RUNX and sandwich assay with the R² values of 0.9663 and 0.9914, respectively.

Fig. 6

(a) Biofouling experiments with nonimmune antibody and control proteins. Control experiments did not show any current response compared with specific molecule, confirming the specific detection of RUNX2. (b) Selective experiments were performed by using RUNX2-spiked human serum. All the concentrations of RUNX2-spiked serum were recognized by anti-RUNX2 antibody, indicating the selective detection of RUNX2 by sandwich pattern. Data were averaged by triplicate values.