Ultra‑Sensitive, Label‑Free Immunoassay Using Gold Nanoparticle–Graphene Oxide Hybrid Plasmonic Biosensors

Background

Carbon‑based nanomaterials—graphene, carbon nanotubes, and graphene oxide (GO)—have become staples in biosensing due to their exceptional electrical, optical, and surface chemistry. GO’s two‑dimensional structure and oxygen‑rich functional groups (epoxides, hydroxyls, carboxyls) confer high protein affinity and enable covalent coupling, making it an attractive substrate for electrochemical, fluorescence, SPR, and LSPR sensors. Gold nanoparticles (AuNPs) bring tunable plasmonic absorption, biocompatibility, and facile surface chemistry, allowing them to act as signal amplifiers or optical tags. When AuNPs are decorated on GO, the hybrid inherits the rapid binding of GO and the intense LSPR of AuNPs, leading to enhanced sensitivity in immunoassays, DNA detection, and enzymatic assays. Despite extensive electrochemical and SERS studies, no rapid, colorimetric, or label‑free immunoassays based on naked‑eye AuNP‑GO hybrids have been reported to date. This study fills that gap by developing a chemisorption‑based, LSPR‑enhanced immunoassay that operates in aqueous buffer and tolerates common interferents.

Methods/Experimental

Materials

Graphite (Graphene Supermarket), GO (modified Hummer’s method, 0.1–1 µm flakes, 1.1 nm thick), cystamine dihydrochloride (Cys, 96 %), HAuCl₄·3H₂O (99.99 %), sodium citrate, BSA, anti‑BSA (rabbit IgG), NHS, and EDC were all obtained from Sigma‑Aldrich or Alfa Aesar. All reagents were used without further purification.

Synthesis of AuNPs

AuNPs were synthesized by citrate reduction: 15 mL of 1 mM HAuCl₄ was refluxed at 550 °C, 1100 rpm; 1.8 mL of 38.8 mM sodium citrate was added. After 5 min, the mixture was boiled at 400 °C, 900 rpm for 30 min and cooled to room temperature. This procedure yields ~15 nm spherical AuNPs; using 0.8 mL citrate produces ~60 nm particles. The reaction: HAuCl₄ + C₆H₅O₇Na₃ → Au(s) + CO₂ + HCOOH.

Preparation of GO‑BSA Antigen Target

GO sheets (0.1 g L⁻¹) were activated with EDC (400 µM) and NHS (100 µM) (1:1 volume ratio). BSA (20 µL, 100 µg mL⁻¹) was covalently coupled via amide bond formation and purified by repeated centrifugation. Figure 1 illustrates the GO‑BSA conjugation workflow.

GO‑BSA interaction. The carboxyl group of GO sheets can be activated using EDC/NHS reaction and preparation of the GO based on antigen target for GO‑BSA

Preparation of AuNP‑antiBSA Probe

AuNPs were functionalized with Cys (10 mM, 2 h, RT) to introduce exposed –NH₂ groups. Anti‑BSA (20 µL, 100 µg mL⁻¹) was then coupled via amide bonds without additional activation, followed by centrifugation to remove unbound antibody. Serial dilutions from 100 µg mL⁻¹ to 1 pg mL⁻¹ produced a range of AuNP‑antiBSA probes (Fig. 2a). AuNP‑GO‑antiBSA composites were assembled by EDC/NHS activation of GO carboxyls and covalent attachment of Cys‑modified AuNPs, then antibody coupling (Fig. 2b).

AuNP‑antiBSA and AuNP‑GO‑antiBSA interactions. Preparation of the a AuNPs and b AuNP‑GO based on antibody probe

Characterization Techniques

Structural and optical characterization employed FE-TEM, HR‑TEM, SEM (JEOL JSM‑7800F), UV‑vis spectroscopy (U‑2900, Hitachi), Raman microscopy (MRI, Protrustech), FTIR (Bruker Vertex 80v ATR), and XPS (National Synchrotron Radiation Research Center). AuNP size, distribution, and surface chemistry were confirmed across all samples.

Results and Discussion

Morphology and Composition

SEM and TEM revealed uniform 60‑nm AuNPs evenly distributed on wrinkled GO sheets (Fig. 3). XPS confirmed the presence of C–C (sp²), C–C (sp³), C–O, C=O, and O–C=O functional groups on GO, while Au 4f peaks verified AuNP integration.

Surface morphology analysis of the nanocomposite. a SEM images of a GO sheet and b TEM image of AuNP‑GO composite. c The TEM image of the ERGO AuNP‑GO film

Spectroscopic Characterization of GO

High‑resolution C 1s XPS, Raman, and ATR‑FTIR spectra confirm the characteristic oxygenated functional groups of GO (sp² C, epoxides, hydroxyls, carbonyls, carboxyls). Raman peaks at 1355 cm⁻¹ (D), 1614 cm⁻¹ (G), and 2714 cm⁻¹ (2D) corroborate the layered structure (Fig. 4).

Spectral analysis of GO sheets. a XPS high‑resolution scan in the C1s region, b Raman, and c FTIR

Protein Interaction and LSPR Response

UV‑vis spectra of AuNPs (15 nm and 60 nm) show LSPR peaks at 520 nm and 540 nm, respectively. GO alone displays π–π* absorption at 230 nm and n–π* at 300 nm. After functionalization, AuNP‑Cys‑antiBSA conjugates exhibit dual peaks at 540 nm (AuNP) and 755 nm (AuNP‑Cys‑antiBSA) with a concentration‑dependent shift (Fig. 5). Calibration curves (y = 2.43 + 0.25 x, R² = 0.91 for 540 nm; y = 2.56 + 0.31 x, R² = 0.96 for 760 nm) confirm linearity across 1.45 nM to 145 fM anti‑BSA.

Analysis of UV‑vis absorption spectra for AuNP with antiBSA interaction response. a SP absorption spectra of AuNPs, b GO‑bound BSA, c AuNP‑anti‑BSA probe, and d Calibration curves for AuNP with antiBSA interaction response at dilution different concentrations of antiBSA protein from 1.45 nM ~ 145 fM

AuNP‑GO‑antiBSA hybrids further amplify the LSPR signal. Upon addition of GO‑BSA targets, the 540‑nm peak shifts linearly with anti‑BSA concentration (1.45 nM–145 fM), yielding a LOD of 145 fM (Fig. 6). Calibration for the AuNP‑GO probe (f(x) = 0.918 + 0.124 x, R² = 0.94) and for GO alone (f(x) = 0.791 + 0.057 x, R² = 0.954) demonstrate superior sensitivity of the hybrid system.

Analysis of UV‑vis absorption spectra for immune response. a AuNP‑bound GO and GO‑bound BSA, b AuNPs and GO‑bound anti‑BSA, c AuNP‑GO‑antiBSA probe, and d AuNP‑GO‑antiBSA probe and GO‑BSA target for immune response

Interference studies with 10 ng ml⁻¹ hCG showed minimal cross‑reactivity (R² = 0.89 for AuNP‑GO; 0.73 for GO alone), confirming assay robustness. The sensor retained performance after four regeneration cycles, indicating high surface stability.

Conclusions

We have engineered a biocompatible AuNP‑GO hybrid that leverages LSPR to achieve label‑free detection of anti‑BSA at 145 fM. The nanocomposite exhibits high bioaffinity, rapid response, and resilience to common interferents, making it a compelling platform for point‑of‑care immunoassays. Future work will explore multiplexed detection, integration into microfluidic devices, and adaptation to clinically relevant biomarkers.

Abbreviations

Ag:

Silver

AntiBSA:

Bovine serum albumin antibody

Au:

Gold

AuNP:

Gold nanoparticle

BSA:

Bovine serum albumin

Cys:

Cystamine

EDC:

1‑Ethyl‑3‑(3‑dimethylaminopropyl)carbodiimide

FEG‑TEM:

Field‑emission gun transmission electron microscope

FTIR:

Fourier‑transform infrared spectrometer

GO:

Graphene oxide sheet

HR‑TEM:

High‑resolution transmission electron microscope

LOD:

Limit of detection

LSPR:

Localized surface plasmon resonance

MOA:

8‑Mercaptooctanoic acid

NHS:

N‑Hydroxysuccinimide

Pd:

Palladium

POCT:

Point‑of‑care testing

Pt:

Platinum

RGO:

Reduced graphene oxide

SAMs:

Self‑assembled monolayers

SERS:

Surface‑enhanced Raman scattering

UV‑vis:

Ultraviolet‑visible

XPS:

X‑ray photoelectron spectroscopy

ZnO:

Zinc oxide