Biocompatible graphene-zirconia nanocomposite as a cyto-safe immunosensor for the rapid detection of carcinoembryonic antigen

Graphene-based materials have gained remarkable attention in numerous disciplines owing to their unique electrochemical properties. Out of various hybridized nanocomposites, graphene-zirconia nanocomposite (GZ) was distinctive due to its biocompatibility. Zirconia nanoparticles serve as spacers that reduce the stacking of graphene and improve the electrochemical performance of the material. Considering that lungs and skin suffer the greatest exposure to nanoparticles, this study aimed to evaluate the cytotoxicity of the as-synthesized GZ nanocomposites on MRC5 (lung cells) and HaCaT (skin cells) via morphological observation and cell viability assay using 3-(4,5 dimethylthiazol-2-yl)-(2,5-diphenyltetrazolium bromide) tetrazolium (MTT). GZ-treated cells showed a comparable proliferation rate and morphology with untreated cells under microscopic evaluation. Based on MTT results, the IC50 values of GZ were > 500 µg/ml for MRC5 and HaCaT cells. The excellent biocompatibility was the supremacy of GZ over other nanocomposites applied as electrode materials in biosensors. GZ was functionalized with biolinker for the detection of carcinoembryonic antigen (CEA). The proposed immunosensor exhibited good responses towards CEA detection, with a 4.25 pg/ml LOD and correlation coefficient of R2 = 0.99 within a linear working range from 0.01 to 10 ng/ml. The performance of the immunosensor to detect CEA present in human serum was also evaluated. Good recovery of CEA was found, suggesting that the proposed immunosensor possess a high affinity to CEA even in a complex biological matrix, rendering it a promising sensing platform for real sample analysis and open a new way for the detection of cancer-associated proteins.


Optimization of Fabrication Parameters and Sensing Conditions
The sensor response can be amplified through optimization of the fabrication parameters, and sensing conditions viz., the GZ: PYSE proportion, the Ab concentration, the Ab immobilization time, the active-site blocking time, and the Ab-CEA hybridization time. In addition to signal amplification, the optimization steps as well aim to reduce wastage and prevent unproductive states caused by overly dense electrode surfaces that hindered antigenantibody hybridization.
To determine the optimum GZ: PYSE proportion, electrodes functionalized with different GZ: PYSE nanocomposite (1:2, 1:4, 1:8, and 1:16) were immobilized with 15 µg/ml of Ab for 75 minutes, followed by active-site blocking with 1% skim milk for 20 minutes. The functionalized electrodes were subsequently incubated with 0.5 ng/ml of CEA for 50 minutes. As shown in Fig. S2(a), rRct increased gradually following the adjustment of GZ: PYSE proportion from 1:2 to 1:4, and reduced thereafter. The inferior sensor response at low PYSE proportion was suggestive of inadequate PYSE on the electrode surface. As a result, the amount of immobilized Ab was not efficient for hybridization [5]. On the contrary, high PYSE proportion incorporated abundant amide linkages on the electrode surface and resulted in a thick layer of immobilized Ab. Consequently, the overly dense film of immobilized Ab hindered the hybridization of Ab-CEA due to steric limitations [6]. The finding was validated with statistical analysis using SPSS. ANOVA test showed that GZ: PYSE proportion was a significant parameter affecting the sensor response (p < 0.05), while the paired-sample test showed that each adjustment of the PYSE proportion induced a statistically significant change in rRct (p < 0.05). Since GZ: PYSE proportion of 1:4 was the most optimum ratio among all electrodes, it was adopted in subsequent analyses.
Likewise, the optimum concentration of Ab was investigated by immobilizing Ab of different concentrations (1 µg/ml, 5 µg/ml, 10 µg/ml, and 15 µg/ml) onto the electrode for 75 minutes, followed by active-site blocking with 1% skim milk for 20 minutes and CEA-incubation for 50 minutes. As shown in Fig. S2(b), rRct increased significantly following higher Ab concentration, reached a maximum at 10 µg/ml and levelled off thereafter. This result is consistent with the finding from the optimization of GZ: PYSE discussed earlier: low concentration of Ab was indicative of overly sparse immobilized Ab for effective CEA detection, whereas high concentration of immobilized Ab induced steric hindrances which would, in turn, limit the Ab-CEA immuno-complex that can be formed [7] [8]. Similarly, the ANOVA test endorsed that Ab concentration has a significant influence on rRct (p < 0.05), while the paired-sample test confirmed that each adjustment of the Ab concentration was statistically significant on rRct (p < 0.05). Since maximum rRct was observed for electrodes immobilized with 10 µg/ml of Ab, this concentration was adopted in subsequent analyses.
Subsequently, the influence of Ab immobilization time was considered for optimum Ab density. Electrodes were immobilized with 10 µg/ml of Ab for varying durations (25 minutes, 50 minutes, 75 minutes and 100 minutes) followed by active-site blocking for 20 minutes and CEA-incubation for 50 minutes. As shown in Fig. S2(c), the rRct for 25 minutes of immobilization time was the lowest among all electrodes. It was stipulated that a certain threshold of time was required for the proteins to form specific binding during incubation [9] and 25 minutes of immobilization time was below-threshold. Afterwards, rRct was observed to increase when the immobilization time was adjusted to 50 minutes; nonetheless, the subsequent increase to 75 minutes and 100 minutes led to a lower rRct, implying less efficient Ab-CEA hybridization. Although extended incubation time furnished ample interval for Ab to be immobilized well on the sensor, prolonged immobilization time caused steric limitation and competitive binding, which hindered the formation of Ab-CEA immuno-complex [10]. As supported by the ANOVA and paired-sample test, adjustment of the Ab immobilization time was statistically significant on rRct (p < 0.05). Since 50 minutes of immobilization generated the most amplified sensor response, it was selected as the optimum immobilization time.
The active-site blocking step was optimized by varying the blocking time. Functionalized electrodes were immobilized with 10 µg/ml of Ab for 50 minutes followed by active-site blocking with 1% skim milk for various blocking times (no blocking, 10 minutes, 20 minutes and 30 minutes). All the electrodes were later subjected to incubation with CEA for 50 minutes. As shown in Fig. S2(d), the electrode without the blocking step presented high rRct due to the presence of non-specific binding [11]. The incubated CEA is bound not only to the Ab but also to the PYSE bi-linker due to the presence of amides on the exterior of CEA, causing inaccurately high sensor response. When the blocking time was increased, rRct reduced and reached a plateau after 20 minutes, manifesting that the protein binding sites were saturated with blocking buffer. This observation was supported by the result from the paired-sample test in which the rRct difference from 20 minutes to 30 minutes was statistically insignificant (p > 0.05), indicating that further increase of blocking time imposed no effect on rRct.
ANOVA result also supported the fact that optimization of blocking time is statistically meaningful. Hence, 20 minutes of blocking time were selected to suppress the non-specific bindings on the electrode surface.
The Ab-CEA hybridization time was investigated by varying the incubation time with the CEA solution.
Electrodes were immobilized with 10 µg/ml of Ab for 50 minutes followed by active-site blocking with 1% skim milk for 20 minutes. The electrodes were incubated with CEA for varying durations (25 minutes, 50 minutes, 75 minutes and 100 minutes). Amplified rRct was observed as the hybridization time was increased from 25 minutes to 75 minutes (Fig. S2(e)), depicting the greater Ab-CEA immuno-complex formation. However, prolonged hybridization time (100 minutes) did not result in further change, which implied that the equilibrium of immunoreaction and the formation of Ab-CEA immuno-complex was saturated [12]. This observation was validated with the aid of the paired-sample test which showed a statistically significant rRct difference from 25 minutes to 75 minutes (p < 0.05) but a statistically insignificant rRct change from 75 minutes to 100 minutes (p > 0.05). As such, 75 minutes of hybridization time was considered the plateau and was adopted for subsequent analyses.

S.5 Study of Intra-Assay and Inter-Assay Variation
An intra-assay variation study was performed to assess the closeness of agreement between triplicated measurements provided by the sensor within a single detection. Meanwhile, an inter-assay variation study was done to investigate the closeness of agreement between measurements provided by the sensor from three individual/independent detections. The degree to which responses of the sensor vary is known as the coefficient of variation (CVs), as presented in Table S.1.