Aptamers are single-stranded oligonucleotides that can fold into distinct three-dimensional (3-D) structures, including loops, hairpins, pseudoknots, branches, and quadruplexes [44]. These 3-D structures are essential for enabling aptamers to bind to their targets with high specificity and selectivity [45]. Unlike antibodies, aptamers can theoretically be produced against any desired target [46], including proteins, whole cells [47], toxins, non-immunogenic targets, or even small molecules [48]. Additional advantages of aptamers over antibodies include lower production costs, high stability, minimal batch-to-batch variation, extended shelf life [49], and ease of modification via various chemical strategies [50]. Due to these advantages, aptamers have been widely used in colorimetric assays, including LFAs [51], as an alternative to antibodies for detecting pathogenic microorganisms in food and water samples [52]. While antibody-based LFAs remain widely used due to their well-established performance and high affinity toward target antigens [53,54], aptamers offer several practical advantages for PoC diagnostics. In contrast to antibodies, which require biological production systems and controlled storage conditions, aptamers are chemically synthesized, enabling scalable production with lower costs and improved stability under varying environmental conditions [40,54,55]. These characteristics make aptamers attractive for diagnostic applications in resource-limited settings [53,55]. However, despite their promising attributes, aptamer-based detection systems still face several challenges, including the complexity of the Systematic Evolution of Ligand by Exponential Enrichment (SELEX) selection process, potential cross-reactivity with structurally similar targets, and matrix interference in complex food and environmental samples [56,57]. The SELEX process typically involves multiple iterative rounds of binding, separation, and amplification, which can be time-consuming and labor-intensive, often requiring several cycles to obtain high-affinity aptamers [58,59]. In addition, amplification bias during PCR may result in the preferential enrichment of sequences with higher amplification efficiency rather than true binding affinity, thereby compromising the overall selection quality [58]. Nanomaterial-assisted SELEX strategies, such as graphene oxide-based platforms, have been explored to enhance target binding efficiency and improve the selection of high-affinity aptamers, particularly for small-molecule detection. However, it still follows the lengthy SELEX process [60]. Furthermore, the selection process can be influenced by experimental conditions, which may affect reproducibility and limit the robustness of aptamer performance across different studies [58,59]. Although recent advances such as microfluidic SELEX and high-throughput screening approaches have improved selection efficiency and reduced processing time, challenges related to scalability and large-scale production of aptamers remain [61,62]. In recent years, in silico selection of aptamers has proven to be more cost-effective and highly efficient in producing target-specific aptamers in a short period [60].

Overall, aptamer-based LFAs offer several advantages over conventional antibody-based systems. A summary of the key advantages and limitations of aptamer- and antibody-based LFAs is presented in Table 2. These include lower production costs, enhanced stability under varying environmental conditions, ease of chemical modification, and minimal batch-to-batch variation. These features make aptamers particularly attractive for PoC applications, especially in resource-limited settings. However, several limitations remain, including the complexity and time-intensive nature of the SELEX process, potential cross-reactivity with structurally similar targets, and reduced performance in complex sample matrices due to interference effects. In contrast, antibody-based systems benefit from well-established production protocols and consistently high binding affinity. Still, they are limited by higher production costs, batch-to-batch variability, and dependence on cold-chain storage. Therefore, while aptamers represent a promising alternative to antibodies, further optimization of selection processes and assay design is required to fully realize their potential in practical diagnostic applications.

LFA test results are typically translated into an optical signal that can be interpreted with the unaided eye. The visible signal is generated by colored detection probes conjugated to the biorecognition molecules (in this case, aptamers) after binding to immobilized targets at the test and control lines [63]. Examples of such probes include latex beads [64], and graphene oxide [40]. Detection probes must be detectable at low concentrations and retain their functional properties after modification with biorecognition molecules [65]. Traditional detection probes often exhibit limited sensitivity and may be susceptible to photobleaching. Therefore, nanomaterial-based labels have been introduced to overcome these limitations.

NPs are materials with at least one dimension in a size range between 1 and 100 nm. They can be broadly classified into organic and inorganic nanomaterials, resulting in a wide range of NPs such as metallic, polymeric, lipid-based, and carbon-based NPs. Nanomaterials possess a large surface area-to-volume ratio, which allows efficient conjugation with various biomolecules for specific applications [48]. Among these nanomaterials, AuNPs, silver NPs, and QDs have been widely used as visual detection labels for qualitative assays [34]. However, some nanomaterials require specialized readout instruments. For example, QDs in fluorescence-based LFAs require an additional excitation device [7]. In contrast, AuNPs have gained significant popularity because they enable visual detection without additional equipment [66]. Moreover, AuNPs exhibit long-term stability and high biocompatibility [67]. The broader application of AuNPs in bioimaging and colorimetric assays as contrast agents, signaling probes, and fluorescence probes have been extensively reviewed elsewhere [4,68], therefore, only their application as detection probes in LFAs will be discussed herein.

AuNPs are commonly used as chromogenic labels in colorimetric diagnostic systems, including LFAs and in-solution colorimetric assays [69,70]. Due to their localized surface plasmon resonance (LSPR), AuNPs appear red in solution, which makes them particularly useful in the development of colorimetric detection systems that can be interpreted with the naked eye [71]. Their optical properties are influenced by their shape and size, both of which play important roles in determining the efficiency of LFAs [68]. Spherical AuNPs are preferred for LFAs because they exhibit good colloidal stability over a wide size range, stable optical properties, and reduced nonspecific interactions [72].

AuNPs-based LFAs have been widely applied for bacterial detection in biological, food, and environmental samples [73]. For example, Figure 3A illustrates an AuNPs-based LFA for the detection of whole E. coli O157:H7 in solution. In this study, AuNPs with different shapes (spherical, flowerlike, and popcornlike) were used as signal probes (Figure 3B), all of which produced detectable signals that were dependent on AuNP size and shape. These findings demonstrate that pathogens can be rapidly and cost-effectively detected using LFAs [74]. Most AuNPs-based colorimetric biosensors developed for the detection of pathogenic bacteria have focused on milk [75] and meat samples. However, other food matrices such as seafood [76], salads and fresh fruits [77], home-prepared foods [78], and water samples have also been investigated.

Food contamination can occur not only on farms but also during processing stages, storage, and transport. During cold storage, psychotropic bacteria such as Pseudomonas and Acinetobacter spp. commonly grow in milk [79], while contamination with S. aureus, E. coli, and Campylobacter may occur if hygienic practices are not properly maintained [12]. Food products are highly susceptible to microbial contamination and must therefore be monitored at every stage of processing. Rapid colorimetric assays can be used on-site at different production stages to help prevent food-borne outbreaks [12]. Although many assays employ antibodies for their excellent target selectivity and affinity [80], antibodies also have several limitations. These include limited specificity between target antigens and homologous unrelated epitopes, high production costs, thermal and chemical instability, and the inability to target non-antigenic molecules [81].

Aptamers, on the other hand, exhibit low immunogenicity and can bind to their target molecules with high selectivity [82]. In addition, their thermal stability reduces the need for cold storage, which makes them particularly suitable for PoCT in LICs. The use of aptamers, either alone or in combination with antibodies in LFAs, has demonstrated improvements over conventional LFIAs, as reviewed elsewhere [53]. Figure 4A and Figure 4B illustrate that aptamers can be used in competitive and sandwich formats, respectively. An aptamer-AuNPs-based LFAs have been used for the detection of S. typhimurium [83,84] as well as in multiplex system capable of simultaneously detecting S. typhimurium, E. coli O157:H7, and S. aureus in 30 food samples including milk, chicken, and frozen food products [85].

AuNPs-Based in-Solution Colorimetric Assays. AuNPs typically appear red in solution but can change from red to purple, blue or black when changes occur in their refractive index or when they aggregate [86]. The salt-induced aggregation mechanism of AuNPs is fundamental to many colorimetric detection strategies. Citrate-reduced AuNPs are stable in solution because electrostatic repulsion between the negatively charged citrate ions maintains their dispersion. In the absence of stabilizing agents, such as aptamers, adding salt (NaCl) reduces electrostatic repulsion between AuNPs, promoting aggregation. This results in a shift in LSPR, leading to a visible color change from red to blue. In aptamer-functionalized systems, the presence of target molecules can induce conformational changes in the aptamer, reducing its ability to attach and stabilize AuNPs, thereby causing AuNPs instability in the presence of NaCl. This mechanism enables visual detection of target analytes [87,88]. The color change can be visually detected with the naked eye and indicate alterations in NP size and interparticle distance, which affect their light-absorption properties. This phenomenon has been widely explored for use in colorimetric detection assays for both chronic [89] and infectious diseases [69].

In-solution colorimetric assays have been applied for the rapid detection of food-borne bacteria [90] and for the diagnosis of coronavirus disease 2019 [91]. For example, the binding of graphene-oxide-coated AuNPs immunosensor to S. typhimurium or E. coli as target analytes resulted in visible color change within 5 minutes. The limit of detection (LoD) for S. typhimurium was 10³ CFU/mL [90]. Similarly, AuNPs-based immunosensor have demonstrated considerable promise for the rapid detection of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) [91]. The interaction between SARS-CoV-2 antigens (or virus particles) and antibodies resulted in a color change to blue within 5 minutes. The LoD for SARS-CoV-2 antigens and viral particles were reported to be 1 ng/mL and 1000 particles/mL, respectively [92].

The dispersion and aggregation behavior of AuNPs generally follows two strategies, as illustrated in Figure 5. The first strategy (Figure 5A) relies on the displacement of folded aptamers from the AuNPs surface. For example, an aptamer-AuNPs biosensor was developed to detect retinol-binding protein 4, a biomarker for the early diagnosis of type 2 diabetes mellitus, resulting in the detachment of the aptamer from the AuNP surface [93]. In the second strategy (Figure 5B), two single-stranded aptamers that bind to different regions on the target analyte were conjugated to AuNPs. On binding to the analyte, they formed a sandwich structure that brings the AuNPs into proximity [93]. In both strategies, changes in the interparticle spacing led to AuNP aggregation in the presence of NaCl, resulting in a visible color change from red to purple or blue [94]. Using this principle, several AuNP-based aptasensors have been developed for the detection of food- and water-borne pathogens across various matrices, including water, milk, and meat samples.

A two-step label-free aptasensor by Kim and colleagues further demonstrated the simplicity of aptamer-based detection systems. In their strategy, aptamers were first incubated with the samples, and the resulting supernatant was subsequently used for testing. In a negative sample, the aptamers bind to the AuNPs, thereby protecting them from salt-induced aggregation. However, if the target bacterium is present in the sample, it binds to the aptamers, leaving the AuNPs unprotected and susceptible to aggregation. This strategy does not need complex chemical modifications; however, centrifugation is required during the preparation step, which may limit its use in low-resource areas [95]. Nevertheless, the aptamer-AuNPs-based assays remain promising tools for on-site testing by end users. A comparison of representative studies, including detection limits, assay time, multiplexing capability, and validation in real samples, is presented in Table 3. AuNPs-based aptasensors demonstrated rapid assay time (10-30 minutes) and low LoDs (57-10⁵ CFU/mL) across multiple food matrices, highlighting their potential for rapid on-site detection of food- and water-borne pathogens.

The performance of the aptamer-conjugated AuNPs-based detection system is strongly influenced by several experimental parameters, which affect both aptamer functionality and AuNPs behavior. Factors such as pH play a critical role in maintaining the structural conformation of aptamers; changes in pH conditions may disrupt folding and reduce binding affinity towards target molecules. Similarly, ionic strength, particularly salt concentration, directly affects AuNPs stability, which is used in AuNPs-based colorimetric assays. Increased salt concentration can induce aggregation of AuNPs, leading to a visible color change from red to blue, while optimal ionic conditions are required to maintain assay stability and sensitivity [100]. Temperature also influences both the stability of the aptamer-target interaction and the kinetics of the assay, potentially affecting detection efficiency under varying environmental conditions. In addition, the concentrations of aptamers and AuNPs must be carefully optimized, as insufficient probe density may lead to weak signal generation. In contrast, high concentrations can promote non-specific interactions and reduce assay specificity. Furthermore, intrinsic NP properties, including size and surface characteristics, significantly impact optical behavior and detection sensitivity [101]. Collectively, these factors highlight the importance of optimization of experimental conditions to ensure reliable and reproducible performance of aptamer-AuNPs-based colorimetric assays [102].

Despite these advances, several opportunities remain for further improving the practical implementation of aptamer-AuNPs-based detection systems. Future research should focus on the integration of smartphone-based detection platforms and artificial intelligence (AI)-driven data analysis to enable real-time monitoring and improved diagnostic accuracy, particularly in resource-limited settings [103,104]. In addition, nanozyme-based signal amplification strategies offer a promising approach to enhance assay sensitivity and stability, thereby addressing limitations associated with conventional enzyme-based systems and expanding the capabilities of colorimetric detection [105]. The development of multiplexed lateral flow platforms capable of simultaneously identifying multiple pathogens will further improve diagnostic efficiency and outbreak response capacity [106]. However, despite these advances, challenges related to standardization, scalability, and regulatory approval must be addressed to facilitate the successful translation of these technologies from laboratory research to real-world applications, especially at PoC and field environments [107]. Addressing these gaps will be essential for the effective deployment of aptamer-AuNPs-based diagnostic systems and for strengthening global food and water safety, especially in LICs.