A Simple and Visual Colorimetric Aptasensor Based on AuNPs for the Rapid Detection of Sulfamethazine in Environmental Samples

TL;DR

This paper presents a simple and visual method to detect sulfamethazine in environmental samples using gold nanoparticles and an aptamer.

Contribution

A novel AuNP-based colorimetric aptasensor is developed for rapid and visual detection of sulfamethazine in environmental samples.

Findings

The aptasensor has a wide linear range of 0.05 to 0.4 µg/mL and a detection limit of 0.039 µg/mL.

Molecular dynamics simulations confirmed stable aptamer-SMZ binding, ensuring high selectivity.

Spike-and-recovery experiments showed high accuracy and good agreement with HPLC results.

Abstract

Sulfamethazine (SMZ) is widely used in livestock production, and its residues can enter water and soil environments, posing potential risks to human health and ecosystems. This study focuses on environmental samples and constructs an AuNP-based colorimetric aptasensor using the SMZ1S aptamer for the rapid visual detection of SMZ. Under optimized conditions, the aptasensor showed a wide linear range from 0.05 to 0.4 µg/mL and a limit of detection of 0.039 µg/mL. Molecular dynamics simulations have demonstrated that the aptamer’s binding to SMZ is stable, providing a theoretical basis for the high selectivity of the aptasensor. Spike-and-recovery experiments yielded recoveries of 87.3–105.5%, 88.6–102.8%, and 87.5–103.4% for SMZ in lake water, tap water, and soil samples, respectively, with relative standard deviations of 5.9–8.3%, 8.0–10.6%, and 4.8–9.6%, showing good agreement with high-performance liquid chromatography (HPLC) results (R2 ≥ 0.981). Overall, the proposed aptasensor provides a simple and effective approach for rapid detection of SMZ in environmental samples.

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Full text

1. Introduction

Sulfamethazine (SMZ) is a sulfonamide antibacterial drug widely used to prevent and treat infections in pigs, cattle, chickens, and other livestock. However, sulfonamides (SAs) are not fully absorbed in animals; approximately 30–90% of these drugs are excreted in their original form or as metabolites [1,2], ultimately entering soil and water environments through fecal deposition on fields, sewage discharge, or aquaculture activities. Studies have shown that sulfonamide antibiotics are consistently detected in aquatic environments at measurable concentrations [3]. For instance, the average concentration of sulfonamide antibiotics in surface water of the Fenhe River Basin in China was reported to be 33.74 ng/L [4]. In various regions across China, the average surface water concentration of SMZ has been found to exceed 100 ng/L, with peak levels in marine and riverine environments approaching or even surpassing 1000 ng/L [5,6]. These residues accumulate in the environment, posing potential ecological toxicity and, through the food chain, may enter the human body, leading to poisoning, allergic reactions, and even an increased risk of cancer [7,8]. Therefore, precise surveillance of SMZ residues in aquatic systems and surrounding soils is crucial for safeguarding human health and ensuring ecological sustainability.

At present, chromatographic analysis [9,10,11] and immunoassay-based methods [12,13,14,15] are the primary approaches employed for the detection of small-molecule drug residues. Among chromatographic techniques, high-performance liquid chromatography–tandem mass spectrometry (HPLC-MS/MS) [16,17] is widely regarded as the gold standard for the analysis of trace environmental contaminants. A study investigating antibiotics in surface water from the Fenhe River basin reported sulfonamides as one of the major contamination categories, and method validation demonstrated that the limits of detection for different sulfonamide target compounds ranged from 0.014 to 0.319 μg/L, with good linearity observed over the corresponding concentration ranges (R^2^ > 0.995) [4]. Nevertheless, such methods typically rely on complex sample pretreatment procedures, such as solid-phase extraction, and require trained personnel and sophisticated laboratory-based instrumentation, resulting in a total analysis time exceeding 1.5 h per sample. By contrast, immunoassay-based methods exhibit lower procedural complexity. A representative immunoassay based on silver nanocluster fluorescence quenching has been reported for the detection of sulfadimidine, achieving a limit of detection of 0.05 μg/L with a linear dynamic range of 0.14–71.71 μg/L [18]. However, the analytical workflow of this method still involves multiple incubation steps, leading to a total assay time of approximately 3 h and dependence on laboratory-based microplate readers. In addition, immunoassays commonly face challenges such as difficulties in antibody optimization, limited stability, and relatively high costs associated with reagent preparation and use, which collectively restrict their broader application.

Recently, aptamer-based sensing in combination with nanomaterials has become a rapidly growing area for the detection of biological and chemical indicators [19,20]. Aptamers are short single-stranded oligonucleotides with high affinity and specificity toward their targets, generated via the SELEX technique performed in vitro [21,22,23]. Compared with antibodies or enzymes commonly employed in biosensor design, aptamers exhibit superior stability, specificity, low molecular weight, ease of modification, and lower cost. They are strong candidates for the development of small-molecule detection aptasensors [24,25] and play an important role in pathogenic biosensing and diagnostic technologies. In previous work, this research team utilized cobalt oxyhydroxide (CoOOH) nanomaterials to develop a fluorescent aptasensor for the detection of SMZ [26]. Although the aptasensor demonstrated a low limit of detection (LOD), its linear detection range was narrower than that reported in previous studies, and it did not allow for visual detection.

Gold nanoparticles (AuNPs) possess unique catalytic activity, high electron density, and distinctive dielectric and optical properties, enabling them to bind effectively to biological macromolecules. Moreover, their large specific surface area enhances the sensitivity of detection systems, making them widely used in the construction of optical and colorimetric biosensors [27]. For example, Ding et al. [28] reported an innovative enrofloxacin aptasensor constructed from an AuNP–aptamer complex, in which AuNPs were prepared by the seed-mediated method and subsequently functionalized with the aptamer. This platform enabled the selective recognition of ENR and provided a broad detection range (0.05–100 μg·mL^−1^). Likewise, Xiao et al. [29] designed a colorimetric biosensor for azlocillin detection by integrating DNA aptamers as recognition elements with unmodified AuNPs as optical indicators. The presence of azlocillin leads to its specific recognition by the aptamer, resulting in AuNP aggregation and a clear color shift from red to blue, with a detection limit reaching 11.6 nM. The former strategy improves probe density and target recognition efficiency by immobilizing aptamers on the AuNP surface, thereby achieving higher sensitivity and selectivity. However, this strategy relies on a precisely controllable surface functionalization process, and the immobilization mode, surface coverage, and conformational restriction effects of aptamers on AuNPs may influence nanoparticle aggregation behavior and signal response, thus introducing additional interfacial regulatory factors [30]. In contrast, while the latter strategy may exhibit slightly lower sensitivity in trace analyte detection, it reduces variables associated with surface functionalization. Its detection signal is mainly generated by AuNP aggregation behavior induced by molecular recognition events in solution, and the system construction process is relatively straightforward, showing application potential in rapid application scenarios where detection sensitivity requirements are moderate and operational simplicity is emphasized.

Based on these considerations, in the present study, the SMZ-specific aptamer SMZ1S, previously screened by our group, was employed as the recognition element to construct a colorimetric AuNP-based aptasensor. This sensor offers a wide detection linear range and rapid colorimetric detection of SMZ in environmental samples, including lake water, tap water, and soil.

2. Materials and Methods

2.1. Materials and Reagents

The SMZ1S aptamer (5′-CGTTAGACG-3′), which specifically recognizes SMZ, was identified in our laboratory [19] and synthesized by Sangon Biotechnology (Shanghai, China). SMZ, sulfapyridine (SPD), sulfadimethoxine (SDM), and sulfa-5-methoxine (SME) were purchased from Sigma (St. Louis, MO, USA). Ofloxacin (OFL), chloramphenicol (CAP), doxycycline (DOX), tetracycline (TET), HAuCl_4_·4H_2_O, and NaCl were obtained from Aladdin Biotechnology Inc. (Shanghai, China). Trisodium citrate was supplied by Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Analytical-grade chemicals, unless otherwise specified, were supplied by Beijing Chemical Reagent Corporation (Beijing, China).

2.2. Instrumentation

The absorbance of AuNPs was measured at 520 nm using a Varioskan™ LUX Multimode Microplate Reader (Thermo Fisher Scientific Inc., Waltham, MA, USA). The average diameter of the AuNPs was determined by dynamic light scattering (DLS) using a NanoBrook Omni instrument (Holtsville, NY, USA). Ultrapure water used in the experiments was obtained from a Millipore system (Bedford, MA, USA).

2.3. Synthesis and Characterization of AuNPs

AuNPs were prepared according to a reported protocol [31]. Briefly, 0.5 mL of 1% HAuCl_4_·4H_2_O was added to 50 mL of deionized water and boiled under vigorous stirring. Then, 1.15 mL of 1% trisodium citrate was quickly introduced, the heating was stopped, and stirring continued for 5 min until a stable burgundy solution formed. The suspension was cooled to room temperature, transferred to a dark glass container, stored at 4 °C, and characterized by UV-vis spectroscopy and particle size analysis. This preparation method typically produces gold nanoparticles with a diameter of 10–30 nm; however, the particle size can be tuned by varying reaction parameters [32].

2.4. Procedure for Detection of SMZ

The colorimetric aptasensor detection system was prepared in a total volume of 400 µL, consisting of 10 µL of SMZ at various concentrations, 10 µL of SMZ1S (final concentration 50 nM), 360 µL of AuNPs solution, and 20 µL of NaCl solution (final concentration 25 mM). The detection procedure was as follows: First, SMZ solutions of different concentrations were mixed with SMZ1S and incubated at 25 °C for 20 min to allow binding. Subsequently, 360 µL of AuNPs solution was added to the mixture and incubated at 25 °C for 6 min. Finally, the NaCl solution was quickly added, and the mixture was further incubated at 25 °C for 10 min. The interactions among SMZ, SMZ1S, and AuNPs were evaluated by measuring changes in the UV–visible absorption spectra and particle size of AuNPs under different combinations.

2.5. Evaluate the Performance of the Aptasensor

To better reflect real environmental conditions and assess the potential impact of interfering substances on the sensor, lake water that had been simply pretreated was used as the blank matrix for the analytical performance experiments. The performance evaluation of the aptasensor included studies on sensitivity and selectivity. To evaluate sensitivity, SMZ solutions at different concentrations (0.05, 0.1, 0.2, 0.4, 0.8, 1.2, 1.6, and 3.2 µg/mL) were incubated with SMZ1S for 20 min, followed by the addition of AuNPs for 6 min, and finally NaCl for 10 min. The absorbance ratio (A_678_/A_519_) was measured, and the average of seven replicates was used as the vertical coordinate, with SMZ concentration as the horizontal coordinate. The LOD of the aptasensor was calculated using the relation 3·SD/S [33]. To evaluate selectivity, various potential interfering substances, including sulfonamide drugs (SPD, SDM, SME) and other antibiotics (OFL, TET, DOX, CAP), each at a final concentration of 1 µg/mL, were incubated with SMZ (1 µg/mL) and SMZ1S for 20 min, followed by AuNPs for 6 min and NaCl for 10 min. Absorbance values at 678 nm and 519 nm were measured, and the absorbance ratio (A_678_/A_519_) was calculated. The selectivity of the aptasensor was determined by comparing the absorbance ratios of SMZ with those of the interfering substances.

2.6. Molecular Docking and Molecular Dynamics Simulation

Molecular docking and molecular dynamics (MD) simulations were employed to investigate the interaction between the aptamer (SMZ1S) and SMZ. The three-dimensional structure of the aptamer was generated using standard nucleic acid modeling procedures, and the molecular structure of SMZ was obtained from the PubChem database. Blind docking was performed using Xdock (version: 1.0) [34] to identify potential binding conformations.

MD simulations were carried out using GROMACS (version: 2025.4) [35]. A 100 ns simulation of the apo-aptamer was first performed to obtain a stable conformation, followed by a 100 ns simulation of the aptamer–SMZ complex to evaluate binding stability. All simulations were conducted in aqueous solution at 298.15 K and 1 atm [36,37], with long-range electrostatic interactions treated using the PME method. Binding free energy and per-nucleotide energy decomposition were calculated from the MD trajectories using the MM/GBSA approach [38,39] to evaluate the contributions of individual bases to SMZ binding.

2.7. Application to Real Samples

To verify the applicability of the colorimetric aptasensor, 30 representative samples were collected from farms in Chongqing, including 8 tap water samples, 12 lake water samples, and 10 soil samples. All samples were analyzed using both the aptasensor and high-performance liquid chromatography (HPLC). Spike recovery tests were conducted with SMZ at concentrations of 50, 100, 150, and 200 ng/mL for the three types of environmental samples.

For tap water, SMZ at different concentrations was first added to the samples, followed by centrifugation at 10,000 rpm for 10 min. The supernatant was filtered through a 0.22 μm ultrafiltration membrane, and the filtrate was used for spiking and recovery tests. For lake water, SMZ was added to the samples, impurities were removed by filtration and centrifugation for 10 min, and the supernatant was subsequently filtered through a 0.22 μm ultrafiltration membrane for spiking and recovery tests. For the soil, samples were first dried at 60 °C, ground evenly, and then any impurities were removed. Then, 1 g of soil was mixed with 1 mL of SMZ solution at different concentrations, vortexed and shaken for 5 min, and left to stand overnight. Supernatant samples were prepared by centrifugation at 10,000 rpm for 10 min and subsequent filtration with a 0.22 μm ultrafiltration membrane, after which they were applied to spiking and recovery tests. Each experiment was repeated five times per concentration to calculate the recovery rate and relative standard deviation (RSD). All samples were confirmed to be free of SMZ by HPLC.

3. Results and Discussion

3.1. Principles of Colorimetric Aptasensor for Detection of SMZ

As depicted in Figure 1, when SMZ is absent, SMZ1S can adsorb onto the AuNP surface by interacting with gold ions, forming a stable SMZ1S-AuNP complex that keeps AuNPs dispersed even in high-salt environments [40,41]. When SMZ is present, SMZ1S specifically binds to SMZ to form a more stable SMZ1S-SMZ complex, preventing the aptamer bases from being exposed and adsorbing onto the AuNP surface. As a result, the availability of SMZ1S for adsorption onto the AuNP surface is reduced. Upon addition of NaCl, the unprotected nanoparticles aggregate, producing a 678 nm aggregation peak, while the remaining AuNPs stabilized by unbound SMZ1S maintain the 519 nm dispersion peak, leading to a decrease in the 519 nm peak and the appearance of two distinct peaks in the spectrum [42].

3.2. Validation of the Principle

Figure 2 shows the UV-vis absorbance spectra of citrate-capped AuNPs under different conditions. Curve 1 represents the characteristic absorption peak at 519 nm of the AuNPs. Upon addition of NaCl, the absorbance at 519 nm significantly decreases, and a new peak appears at 678 nm (Curve 2), indicating nanoparticle aggregation. In the absence of SMZ, SMZ1S adsorbs onto the AuNP surface and stabilizes the particles, so that the absorption peak at 519 nm remains unchanged after adding NaCl (Curve 3). SMZ alone does not affect the AuNP absorption peak (Curve 4), and the subsequent addition of NaCl to this system leads to typical salt-induced aggregation behavior (Figure S4). When both SMZ and SMZ1S are present, specific binding between SMZ and SMZ1S reduces the aptamer available for AuNP stabilization. After adding NaCl, some nanoparticles aggregate to produce the 678 nm peak, while the remaining AuNPs protected by unbound SMZ1S stay dispersed, resulting in a spectrum that simultaneously displays the 519 nm and 678 nm peaks (Curve 5).

3.3. Characterization of AuNPs

Dynamic light scattering (DLS) characterization indicated that the average diameter of AuNPs was approximately 3 nm (Figure 3A), and increased significantly after NaCl addition (Figure 3B). When SMZ1S adsorbed onto the AuNP surface, the diameter increased to 17.6 nm (Figure 3C), whereas SMZ alone had no effect on particle size (Figure 3D). In the presence of both SMZ and SMZ1S, the formation of the SMZ1S-SMZ complex led to loss of AuNP protection, and subsequent NaCl addition caused AuNP aggregation and a further increase in particle size (Figure 3E).

3.4. Optimization of the Aptasensor

To increase the sensitivity of the SMZ1S aptamer to SMZ, the concentrations of NaCl and SMZ1S, along with their incubation durations, were systematically optimized. As illustrated in Figure S1A, when the final concentration of NaCl was ≥25 mM, the A_678_/A_519_ ratio tended to stabilize. Similarly, when the NaCl incubation time was ≥10 min (Figure S1B), the A_678_/A_519_ ratio reached its maximum, indicating complete aggregation of the AuNPs. Therefore, a final NaCl concentration of 25 mM and an incubation time of 10 min were selected as the optimal conditions for subsequent experiments.

As shown in Figure S1C, the A_678_/A_519_ ratio gradually decreased with increasing SMZ1S concentration and remained stable when the final concentration of SMZ1S was ≥50 nM. This is because higher concentrations of SMZ1S allow more aptamer molecules to adsorb onto the AuNP surface, stabilizing the AuNPs against aggregation, even in the presence of high concentrations of NaCl. The incubation time between SMZ1S and AuNPs was also optimized (Figure S1D). When the incubation time was ≥6 min, the A_678_/A_519_ ratio reached equilibrium, indicating complete coverage of AuNPs by SMZ1S. Accordingly, a final SMZ1S concentration of 50 nM and an incubation time of 6 min were adopted for subsequent experiments.

3.5. Sensitivity and Specificity of the Aptasensor

To better mimic actual environmental conditions, the overall performance of the aptasensor was evaluated in a simply pretreated lake water matrix, including sensitivity and selectivity.

Under the optimal experimental conditions, the sensitivity of the aptasensor was evaluated. As shown in Figure 4A, with increasing SMZ concentration, the color of the AuNPs changed from wine-red to light red and eventually to blue, accompanied by a gradual increase in the absorbance ratio (A_678_/A_519_). When the SMZ concentration reached 1.6 µg/mL, the color change in the AuNPs became less noticeable, and the A_678_/A_519_ value approached a plateau. Meanwhile, A_678_/A_519_ exhibited a linear increase within the SMZ concentration range of 0.05–0.4 µg/mL, with a linear regression equation of A_678_/A_519_ = 0.3438 CSMZ + 0.0031 (R^2^ = 0.99). Using the formula 3·SD/S, the LOD was calculated to be 0.039 µg/mL.

In our previous work, a fluorescence-based aptasensor incorporating rhodamine B and AuNPs was developed to achieve highly sensitive detection of SMZ. However, in the present study, the sensing strategy was intentionally simplified by eliminating fluorescent labels and auxiliary signal reporters, enabling direct colorimetric detection based on AuNP aggregation. Although the limit of detection obtained here is higher than that reported in our previous fluorescence-based method, the current approach offers advantages in terms of operational simplicity, low cost, and visual readout, which are more suitable for rapid screening of SMZ in environmental samples.

To investigate the selectivity of the colorimetric aptasensor, different potential interfering substances were added to the sensing system. As shown in Figure 4B, the absorbance ratio (A_678_/A_519_) of the SMZ-containing system was 0.376, whereas the A_678_/A_519_ values of systems containing other categories of antibiotics were all below 0.05. Three sulfonamide drugs (SME, SDM, SPD), which possess structures and functional groups similar to those of SMZ, produced A_678_/A_519_ values higher than 0.05 but still significantly lower than that of SMZ. These results indicate that although structurally similar sulfonamides may theoretically cause some degree of interference in SMZ recognition, their actual responses are much weaker than that of SMZ. Therefore, even if trace amounts of SME, SDM, or SPD coexist in environmental samples, their potential impact on SMZ detection is limited and does not significantly compromise the selectivity or practical applicability of the proposed aptasensor.

3.6. Recognition Mechanism Based on Molecular Dynamics Simulation

To evaluate the conformational stability and recognition performance of the aptamer in solution, a 100 ns molecular dynamics (MD) simulation was conducted on the aptasensor (Figure 5), following the paradigm of using computational tools to explore aptamer conformational landscapes [43]. The results showed that the aptamer maintained a stable overall conformation throughout the simulation, with its conformational ensemble dominated by a single preferred state, indicating high structural rigidity in solution (Figure 5A–D). This observed structural rigidity is a favorable trait for aptasensor design, as excessive flexibility can complicate target recognition and signal transduction [44]. Further Structural analysis revealed a hairpin architecture composed of a rigid stem region and a relatively flexible loop region, forming a groove at the stem-loop junction that provides a stable structural framework for small-molecule recognition (Figure 5C,E,F).

Based on this representative conformation [45], MD simulations of the aptamer–SMZ complex were conducted to elucidate the binding mode of SMZ and its impact on the aptamer structure [46] (Figure 6). The SMZ ligand remained stably confined within the stem-loop junction groove throughout the simulation, without inducing significant global conformational changes in the aptamer (Figure 6A,B,F), indicating a dynamically stable complex. A schematic diagram of the hydrogen bonding and hydrophobic interactions is shown in Figure S3. Interface analysis revealed that SMZ predominantly interacts with nucleotides in the loop and stem-loop junction regions (Figure 6E), stabilized by persistent yet dynamic hydrogen bonds and other non-covalent interactions (Figure 6C). Free energy landscape analysis further demonstrated that SMZ binding shifts the conformational ensemble toward a more localized low-energy basin (Figure 6D), suggesting thermodynamic stabilization of a specific functional conformation. This ligand-induced conformational stabilization is a key mechanism underlying aptamer-based sensing, as similarly demonstrated in studies that combine rational design with simulation validation [47].

To further clarify the energetic driving forces of the interaction, binding free energy was calculated using the MM/GBSA method based on the MD trajectories (Figure S2). The total binding free energy of the aptamer–SMZ complex was −14.23 kcal/mol (Figure S2A), indicating a thermodynamically favorable interaction. Energy decomposition analysis showed that the binding is predominantly driven by van der Waals interactions, with relatively minor electrostatic contributions, suggesting that recognition relies mainly on shape complementarity and hydrophobic packing. Per-nucleotide energy decomposition further identified loop-region nucleotides T3, T4, A5, and A7 as the major contributors to binding (Figure S2B), consistent with the interface analysis and confirming the key role of the binding pocket.

3.7. Validation of the Aptasensor

The applicability of the colorimetric aptasensor to real matrices was assessed through recovery experiments, where SMZ at concentrations of 0.05, 0.1, 0.15, and 0.2 µg/mL was spiked into lake water, tap water, and soil samples. According to Table 1, the aptasensor exhibited recovery rates of 88.6–102.8% in tap water, 87.3–105.5% in lake water, and 87.5–103.4% in soil, with corresponding RSDs of 8.0–10.6%, 5.9–8.3%, and 4.8–9.6%. The results were positively correlated with those obtained by HPLC results (R^2^ ≥ 0.981). These findings indicate that the aptasensor exhibits good reproducibility and is suitable for detecting SMZ residues in environmental samples.

4. Conclusions

In this study, we established a simple and sensitive aptasensor method for the determination of SMZ levels in environmental samples. This aptasensor enables both qualitative and quantitative detection through changes in the color and absorbance of AuNPs, with a linear detection range of 0.05–0.4 µg/mL and a detection limit of 0.039 µg/mL. The method was further validated by HPLC, showing good correlation, indicating the feasibility and practicality of the proposed aptasensor for SMZ determination in environmental matrices. Based on previous related studies on FRET- and CoOOH-based platforms [26,41], this aptamer also demonstrates potential for application on other detection platforms. It could be employed in electrochemical or other nanomaterial-assisted sensing strategies to enable multiple signal transduction modes, thereby providing additional feasible approaches for rapid and sensitive detection of SMZ in environmental samples, serving as a reference for the development of future sensing platforms.