The biological and physiological changes in the human body constitute a process characterized by the aging phenomenon, influencing alterations in body composition and the onset of chronic systemic inflammation, as indicated by elevated levels of inflammatory markers (Santiago et al., 2018). Monocytes and macrophages play pivotal roles as both effectors and regulators in inflammation and the innate immune response, which is the immediate branch of the immune system (Geissmann et al., 2010). Monocytes play a crucial role in the production and release of polypeptide inflammatory mediators such as interleukin-1 (IL-l), IL-6, and tumor necrosis factor-alpha (TNF-α) (Ballou and Lozanski, 1992). Among them, TNF-α, a cytokine recognized as a key regulator of inflammatory responses, is primarily produced by activated macrophages, T-lymphocytes, and natural killer (NK) cells (Bradley, 2008; Horiuchi et al., 2010). An earlier study demonstrated that various diseases in this life stage lead to alterations in the immune system, disrupting cytokine balance (Santiago et al., 2018). Measuring TNF-α levels serves as a valuable indicator for tracking different diseases and pathological states.

Generally, conventional laboratory-based analytical methods for cytokine species and TNF-α detection, such as commercial kits based on enzyme-linked immunosorbent assay (ELISA), high-performance liquid chromatography (HPLC), gas chromatography (GC), mass spectrometry (MS), enzyme-linked immunosorbent spot (ELI-spot) assays, antibody array assays, bead-based assays, and real-time polymerase chain reaction (qPCR) usually require complex and time-consuming procedures (Campbell et al., 2021; Charlermroj et al., 2021; Choi et al., 2019; Cox et al., 2006; Schröder et al., 2010; Won et al., 2012). For the past few decades, research attention has been directed toward the development and improvement of rapid, portable, inexpensive, and user-friendly analysis techniques for point-of-need testing (Makarona et al., 2016; Parolo and Merkoçi, 2013; Weihs et al., 2021). Immunochemical bioanalytical methods are considered the most suitable for screening and producing rapid, highly specific, and sensitive results (Di Nardo et al., 2021). Barhoumi et al. (2018) developed a sensitive electrochemical immunosensor for the detection of the TNF-α cytokine in human saliva utilizing a fabricated immunosensor based on gold substrates. The developed immunosensor demonstrated excellent performance for TNF-α antigen detection within the range of 1 pg/mL to 30 pg/mL. This new immunosensor may represent a promising bioanalytical tool for monitoring heart failure through saliva analysis (Barhoumi et al., 2018). Al-Nasralla et al. (2023) reported a study on the relationship between the levels of cytokines (IL-10 and TNF-α) and prostate-specific antigen (PSA) in the sera of patients, measured using the ELISA technique as an immunological analysis method (Al-Nasralla et al., 2023). Moreover, Oh et al. (2014) developed a label-free LSPR biosensing technique to detect cell-secreted TNF-α cytokines in clinical blood samples (Oh et al., 2014). This cellular functional immune analysis requires a minimal blood sample volume and a total assay time shorter than that of the conventional ELISA. Interestingly, an immunoassay is a bioanalytical technique where the response between an antigen (Ag) and an antibody (Ab) determines the analyte's quantification (Darwish, 2006). The ability to detect the rate of complex formation and the efficiency of Ag–Ab complex formation are the primary factors that determine the efficacy of an immunoassay. The lateral flow immunoassay technique (LFIA) has emerged as one of the most effective immunoassay-based analytical platforms for decentralized or point-of-need testing strategies that require minimal to no supporting infrastructure (Di Nardo et al., 2021).

Recently, gold nanotechnology has identified highly effective catalysts with unique properties (Wang et al., 2019). Researchers have discovered that as nanotechnology advances, gold nanoparticles (AuNPs) will exhibit distinct properties from their larger counterparts and bulk gold when their size is further decreased to that of nanoclusters (Zheng et al., 2021). Owing to their ultra-small size, they become innovative functional materials for diagnostic applications and the detection of biomarkers and bioimaging (Bai et al., 2020). Sha et al. (2019) demonstrated a fluorescence turn-on biosensor fabricated for the sensitive detection of 5-hydroxytryptamine with transferrin-encapsulated AuNCs. The developed sensor was also successfully applied for the detection of 5-HT in human serum (Sha et al., 2019). Yang et al. (2013) utilized poly-diallyldimethylammonium chloride as a linker and stabilizer to develop a multifunctional boron nitride–gold nanocluster (AuNCs) composite. The evolving composite could be applied as an electrochemical or fluorescent label for immunosensing in a highly sensitive interleukin-6 detection process (Yang et al., 2013).

Streptavidin, a purified protein from the bacterium Streptomyces avidinii, is known for its high selectivity, protein stability, and compatibility with various chemical and enzymatic biotinylation methods. These characteristics contribute to the popularity of streptavidin–biotin systems, which are widely utilized in molecular science with various experimental designs (Bayer et al., 1986; Dundas et al., 2013). The combination of AuNCs, which possess intense fluorescence properties, and streptavidin-conjugation is envisioned for the detection of low-concentration analytes. Kurdekar et al. (2018) evaluated the glutathione (GSH) conjugation in binding to AuNCs with streptavidin to develop an AuNCs immunoassay for the early and sensitive detection of HIV infection. The conjugation between AuNCs and streptavidin may offer attractive multifunctional features for the development of AuNC-based assays. However, few studies have focused on using an immunoassay-based AuNCs analytical platform to measure inflammatory cytokines. In this study, the synthesis of AuNCs and the conjugation of AuNCs with streptavidin, along with their characterizations and spherical morphology were investigated. The differences in TNF-α antigen concentrations by using AuNCs detection was determined. Additionally, a new immunoassay-based AuNCs analytical platform for detecting of the TNF-α antigen and quantifying of TNF-α levels was developed. This assay may serve as a viable alternative for TNF-α measurement with high speed, sensitivity and qualities, ensuring its broad applications.