Nanoporous Graphene Integrated onto Bimodal Waveguide Biosensors for Detection of C-Reactive Protein

Abstract

Despite the outstanding progress in photonic sensor devices, a major limitation for its application as label-free biosensors for biomedical analysis lies in the surface biofunctionalization step, that is, the reliable immobilization of the biorecognition element onto the sensor surface. Here, we report the integration of bottom-up synthesized nanoporous graphene onto bimodal waveguide interferometric biosensors as an atomically precise biofunctionalization scaffold. This combination leverages the high sensitivity of bimodal waveguide interferometers and the large functional surface area of nanoporous graphene to create highly sensitive, selective, and robust biosensors for the direct immunoassay detection of C-reactive protein (CRP), an inflammatory biomarker widely used in the clinical diagnosis of infections and sepsis. The limit of detection was determined at 3 ng/mL, which is well below the clinical cutoff levels required for the diagnostic detection of CRP in patient samples. This innovative approach holds promise for transforming diagnostics, environmental monitoring, and various fields requiring precise biomolecular detection.

Figure 1

Bottom-up on-surface synthesis of nanoporous graphene. (a) Molecular structure of the DP-DBBA used as a precursor on this synthesis. 7–13-GNR was obtained after the Ullmann coupling and subsequent cyclodehydrogenation reactions induced at steps T₁ and T₂, respectively. At T₃ the GNRs interconnect leading to the generation of the NPG structure. (b) Topographic STM images of the synthesized NPG. STM image parameters: overview (195 × 195 nm², I_t= 1.1 nA and V_s= 2.0 V) and zoom-in (17.5 × 17.5 nm², I_t= 2.4 nA and V_s= 0.8 V).

Figure 2

(a) Schematic of the polymer-free wet-transfer method used to integrate NPG onto the BiMW sensors. After separating the mica from the gold film-NPG, the latter is picked by the BiMW chip. (b) Photograph of the BiMW chip covered with NPG and a thin gold film over the sensing area. (c) Optical microscopy image of an NPG-coated waveguide after the thin gold layer has been removed. The NPG is visible due to a change in contrast. (d) Characteristic Raman spectra of bare BiMW (blue), NPG-BiMW (red), and Au(111)-NPG growth substrate (brown). The G- and D-bands are observed at 1601.0 and 1332.4 cm^–1, respectively.

Figure 3

(a) Schematic of the BiMW device showing waveguides with varied lengths of the sensing area window (4.5, 9.3, and 13.1 mm). (b) Cross-section schematics of the NPG-BiMW device with the SiO₂ spacer layer between the Si₃N₄ waveguide and the NPG coating. The refractive index of each material is indicated. The schematic is not at scale. (c) Output signal intensity measured for NPG-BiMW devices fabricated with variable thickness of the SiO₂ spacer layer and lengths of the sensing area. Data points correspond to the mean and standard deviation of five different waveguides. (d) Temporal evolution of the interference pattern induced by a bulk RI change on three waveguides of the NPG-BiMW chip with different sensing lengths: 13.1, 9.3, and 4.5 mm. Arrows indicate the sample entrance to and exit from the sensing area.

Figure 4

Bulk sensitivity calibration curve of the SiO₂-coated BiMW sensor (green) and the NPG-BiMW sensor (orange). The data points correspond to the phase shift (ΔΦ) value obtained with triplicate measurements of increasing DMSO dilutions (0.4–1.2% DMSO).

Figure 5

(a) Schematics of the NPG biofunctionalized structure and the subsequent covalent immobilization of antibodies through EDC/NHS cross-linking to COOH groups introduced at the NPG structure. The surface is blocked with ethanolamine to avoid unspecific bindings. (b) Overlaid real-time sensorgrams for the detection of CRP at the same concentration and of the negative control (BSA) for specificity test. (c) Statistical comparison between specific and unspecific samples measured in the same NPG-BiMW sensor device. (d) Calibration curve for CRP detection at different concentrations (0.05–1 μg/mL). Each data point corresponds to the mean ± SD of the duplicate measurements performed on different NPG-BiMW sensor devices.