Sensitive Silver-Enhanced Microplate Apta-Enzyme Assay of Sb³⁺ Ions in Drinking and Natural Waters

Abstract

The toxic effects of antimony pose risks to human health. Therefore, simple analytical techniques for its widescale monitoring in water sources are in demand. In this study, a sensitive microplate apta-enzyme assay for Sb³⁺ detection was developed. The biotinylated aptamer A₁₀ was hybridized with its complementary biotinylated oligonucleotide T₁₀ and then immobilized on the surface of polysterene microplate wells. Streptavidin labeled with horseradish peroxidase (HRP) bound to the biotin of a complementary complex and transformed the 3,3′,5,5′-tetramethylbenzidine substrate, generating an optical signal. Sb³⁺ presenting in the sample bounded to an A₁₀ aptamer, thus releasing T₁₀, preventing streptavidin-HRP binding and, as a result, reducing the optical signal. This effect allowed for the detection of Sb³⁺ with a working range from 0.09 to 2.3 µg/mL and detection limit of 42 ng/mL. It was established that the presence of Ag⁺ at the stage of A₁₀/T₁₀ complex formation promoted dehybridization of the aptamer A₁₀ and the formation of the A₁₀/Sb³⁺ complex. The working range of the Ag⁺-enhanced microplate apta-enzyme assay for Sb³⁺ was determined to be 8–135 ng/mL, with a detection limit of 1.9 ng/mL. The proposed enhanced approach demonstrated excellent selectivity against other cations/anions, and its practical applicability was confirmed through an analysis of drinking and spring water samples with recoveries of Sb³⁺ in the range of 109.0–126.2% and 99.6–106.1%, respectively.

1. Introduction

At present, environmental pollution by heavy metals and metalloids is an extremely important problem. Antimony, as one of most dangerous pollutants of the biosphere, poses a significant problem due to its contamination of the environment, especially water bodies [1]. Antimony has trivalent and pentavalent states; however, the toxicity and poisonousness of Sb(III) is much higher [2]. The main sources of antimony causing its widespread pollution are the production of semiconductors and batteries, textiles, glass and ceramics and the action of coal-fired power plants and metal mining enterprises [3]. Another way Sb³⁺ enters into the human body is its release from polyethylene terephthalate packages, whose production requires antimony (III) oxide [4,5]. Upon entry to the human body through the respiratory tract, skin contact or the food chain, antimony degrades protein and carbohydrate metabolism and damages the liver, heart and nervous system [6], which confirms the demand for monitoring drinking water to assess the content of heavy metal ions [7], in particular antimony. In this regard, the maximum permissible concentration of total antimony in drinking water is 6 ng/mL, as established by the U.S. Environmental Protection Agency (EPA) [8], but the World Health Organization set the maximum permissible level of antimony in drinking water at 20 ng/mL [9].

The analytical techniques used for Sb³⁺ detection are atomic absorption spectrometry [10], inductively coupled plasma with mass spectrometry [11,12] and electrochemical analysis [13]. Although these methods are characterized by high sensitivity and selectivity, the need for expensive sophisticated equipment and long-term preparation of samples restricts their application. Therefore, simple and sensitive techniques for Sb³⁺ detection are an important area of development.

Currently, colorimetric methods deserve special attention as they offer a visual assessment of color change, allowing an easy readout of analytical signals. Colorimetric techniques for Sb³⁺ detection using indicator dyes [14,15,16] and nanoparticles [17,18,19] were developed. The application of dyes such as bis [2-(5-chloro-2-pyridylazo)-5-diethylaminophenolato]cobalt(III), bromopyrogallol red and pyrene causes the formation of complexes with Sb³⁺ and is followed by photo- or fluorometric detection. However, despite the simplicity and cost-effectiveness of these methods, they have insufficient selectivity and reproducibility and cannot ensure the detection of Sb³⁺ concentrations at the MPC level. Nanoparticle-based colorimetric methods are more usable, but they are sensitive to interfering components of matrixes [20]. Alternatively, the colorimetric method based on aptamers, due to their unique properties, can significantly expand the possibilities of determining heavy metal ions. Aptamers are short single-stranded oligonucleotides that are characterized by high affinity and specificity for their specific analytes, and their low production cost makes aptamers relevant analytical reagents [21,22]. Aptamer-based assays have confirmed effectiveness for the detection of heavy metal ions, as summarized in the reviews [23,24].

In particular, a homogeneous colorimetric technique for the detection of Sb³⁺ based on the aggregation of aptamer-modified gold nanoparticles, accompanied by a rapid color transition from pink to blue, was developed [19]. Aptamer A₁₀ was used as the receptor molecule, forming a complex with Sb³⁺ due to interaction with hydroxyl groups of adenine residues [25,26]. However, the detection limit of this assay was 10 ng/mL, which does not reach the established MPC levels. In this regard, an important and urgent task is to find new and better analysis formats that provide the required level of detectable concentrations. One such method is a simple microplate format of an assay for highly sensitive determination. Some microplate methods using aptamers and nanozymes have been previously reported and developed for the determination of various analytes [27,28]. The application of aptamers in these methods provides high specificity and reproducibility, as well as excellent storage stability, and the use of nanozymes allows for increased assay sensitivity. Thus, these advantages have been used to develop approaches for the analysis of the commonest heavy metal ions [29,30,31]. Several microplate colorimetric aptamer-based methods have been developed, including one for the detection of cadmium using gold-nanoparticle-modified MoS₂ [29] and one for detecting lead ions, involving graphene/Fe₃O₄-AuNP composites and the recognition element [31], and rolling circle amplification as a signal amplifier has been proposed for mercury ion detection [30]. However, to the best of our knowledge, the creation of a microplate aptamer-based assay for the determination of Sb³⁺ ions has not previously been reported.

In this study, a microplate apta-enzyme assay for Sb³⁺ detection with a low detection limit is reported for the first time. The proposed microplate method is based on the destruction of a complex of complementary aptamers due to the affinity of aptamer A₁₀ toward antimony ions. Compared with the nanozyme-based approaches described above, this assay is implemented using simple and readily available materials and combines the widely used horseradish peroxidase as a label and specific aptamer recognition. To reach high sensitivity, the assay conditions were varied, including the amount of immobilized biotinylated complementary complex, concentration of streptavidin and incubation time. In addition, the effect of Ag⁺ on the analytical performance of Sb³⁺ detection was investigated. The ability of the developed assay to selectively distinguish Sb³⁺ from other cations and anions and its practical applicability for drinking and spring water sample testing were studied.

2. Results and Discussion

2.1. Principle of Sb³⁺ Detection

The scheme of the designed microplate apta-enzyme assay using Sb³⁺-specific aptamer A₁₀ and complementary oligonucleotide T₁₀ is shown on Figure 1. In this assay, the aptamer A₁₀ and complementary T₁₀ oligonucleotide, both biotinylated at the 5′ end, were used. Firstly, streptavidin was adsorbed in the microplate wells (Figure 1, 1), and then the preliminary formed complementary biotinylated A₁₀/T₁₀ complex was immobilized (Figure 1, 2) through high-affinity biotin–streptavidin interaction. The binding of the A₁₀/T₁₀ complex to the immobilized streptavidin was made possible by the biotinylated end of A₁₀ or T₁₀, and both variants stored the Sb³⁺ binding ability of the A₁₀ aptamer. Next, incubation with HRP-streptavidin (Figure 1, 4) led to its binding to the free biotin of the A₁₀/T₁₀ complex. At the next stage, the addition of TMB substrate promoted the development of blue color (Figure 1, 5), which turned yellow after stopping the reaction with sulfuric acid (Figure 1, 6).

The presence of Sb³⁺ led to a switching of the A₁₀ aptamer structure, resulting in the formation of the A₁₀/Sb³⁺ complex. At the same time, the bonds of the immobilized A₁₀/T₁₀ complex were broken [19,25]. In the case of immobilization of the A₁₀/T₁₀ complex via the biotinylated end of T₁₀, the resulting A₁₀/Sb³⁺ complex was removed after washing the microplate. If the A₁₀/T₁₀ complex was immobilized via the biotinylated end of A₁₀, the formed A₁₀/Sb³⁺ complex remained on the microplate, and T₁₀ was removed during washing. In both cases, washing the microplates reduced the number of HRP-streptavidin binding sites, causing a decrease in the following transformation of the TMB substrate and the registered intensity of the blue color.

2.2. Characterization of the Apta-Assay Components via CD Spectroscopy

The CD spectrum of the aptamer A₁₀ had a large positive peak at 270 nm and a negative minimum at 248 nm (Figure 2a, black line), which is consistent with previous findings [32,33]. Thus, the conformation of the single-stranded oligonucleotide was characterized by a large positive band in the range of 260–270 nm due to base stacking and a negative band at 240 nm due to polynucleotide helicity [34,35]. The CD spectrum of the oligonucleotide T₁₀ had a positive peak near 275 nm and a less intense band at 227 nm (Figure 2a, red line). The positive peaks at 282 and 260 nm and a negative peak at 247 nm (Figure 2a, blue line) confirmed the formation of the A₁₀/T₁₀ complex, which accords with the spectrum of the B-form conformation of the double-stranded oligonucleotide [36]. To confirm the formation of the A₁₀/Sb³⁺ complex, the spectra of the A₁₀ aptamer (Figure 2b), the T₁₀ oligonucleotide (Figure 2d) and the A₁₀/T₁₀ complex (Figure 2c) with Sb³⁺ were recorded. The intensity of the positive band at 218 nm decreased after the addition of Sb³⁺ to the A₁₀ aptamer, and the intensity of the band at 275 nm increased significantly (Figure 2b), similarly to the change in the CD spectrum for the A₁₀/T₁₀ complex with Sb³⁺ (Figure 2c). The interaction of Sb³⁺ with the T₁₀ oligonucleotide was not associated with spectral changes (Figure 2d). The results of CD spectroscopy confirm the feasibility of the proposed assay and demonstrate its applicability for the detection of Sb³⁺.

2.3. Optimization of the Microplate Apta-Enzyme Assay Conditions

To optimize the conditions of the microplate apta-enzyme assay for sensitive Sb³⁺ detection, the concentrations of streptavidin and the A₁₀/T₁₀ complex as well as incubation time were varied. First, the effect of the concentration of the A₁₀/T₁₀ complex on the analytical signal in the absence of Sb³⁺ was investigated. According to Figure 3a, as the concentration of the A₁₀/T₁₀ complex increased up to 5 µg/mL, the optical density at 450 nm (OD₄₅₀) gradually grew. After this, OD₄₅₀ no longer increased, which indicates the saturation value for the A₁₀/T₁₀ complex. Therefore, 5 μg/mL was chosen as the optimal concentration of the A₁₀/T₁₀ complex. In addition, consistent immobilization of the biotinylated A₁₀ aptamer (2.5 μg/mL) and biotinylated T₁₀ oligonucleotide (2.5 μg/mL) to assemble the A₁₀/T₁₀ complex into the microplate wells was tested. However, it was found that the immobilization of the pre-formed complex showed better binding of A₁₀/T₁₀ and reduced analysis time due to the absence of an additional step.

At the next stage, the effect of the concentration of streptavidin, which provides the required number of binding sites for the A₁₀/T₁₀ complex, was investigated (Figure 3b). The streptavidin concentration of 5 μg/mL showed the highest difference between OD₄₅₀ values in the absence of Sb³⁺ and in its presence at a fixed concentration, 10 µg/mL. The ΔOD₄₅₀ data for a concentration of 5 μg/mL were compared with the data for 1 and 10 μg/mL using Student’s t-test, which confirmed the significance of differences (p < 0.05). In addition, the comparison of detection limits of Sb³⁺ showed the lowest value (42 ng/mL) for 5 μg/mL immobilized streptavidin, which was chosen as the optimal concentration.

Finally, the incubation time of Sb³⁺ solutions was studied, which influenced the disruption of the A₁₀/T₁₀ complex and the formation of the A₁₀/Sb³⁺ complex. The OD₄₅₀ reached a plateau at 45 min, and this value was selected as an optimal time for incubation of the Sb³⁺ solution.

As a result, the optimal conditions for the microplate apta-enzyme assay were defined as follows: 5 μg/mL streptavidin, 5 μg/mL A₁₀/T₁₀ complex and 45 min of incubation time for Sb³⁺.

2.4. Analytical Performance of the Microplate Apta-Enzyme Assay of Sb³⁺

Under the optimal conditions, solutions with different concentrations of Sb³⁺ were tested (Figure 4a). The color intensity reached the maximum in the absence of Sb³⁺, and the color of solution was bright yellow. With an increase in the concentration of Sb³⁺, the intensity of the color weakened accordingly. However, the presence of a background signal, even at high concentrations of Sb³⁺, was observed, which is possibly due to the nonspecific effect of the biotinylated T₁₀ oligonucleotide remaining on the microplate wells. The optical densities at 450 nm in response to Sb³⁺ concentrations varying from 0.5 ng/mL to 10 µg/mL are shown in Figure 4a. A linear relationship (Figure 4b) between the OD₄₅₀ value and the logarithmic concentrations of Sb³⁺ was acquired, which corresponded to 40–10,000 ng/mL with a correlation coefficient R² = 0.9906. The detection limit was calculated as the blank signal (in the absence of Sb³⁺) minus 3σ (where σ is the standard deviation of the signal) and determined to be 42 ng/mL, which is higher than the MPCs recommended for drinking water.

2.5. Ag⁺-Enhanced Microplate Apta-Enzyme Assay for Sb³⁺ Detection

Since the obtained detection limit did not reach the maximum permissible concentration for Sb³⁺ in drinking water, the other metal ions were considered as enhancing agents to provide high sensitivity. It is known that the introduction of metal ions into duplexes of oligonucleotides leads to new properties of the formed metal–aptamer systems. Thus, it was found that the addition of Ag⁺ ions to the A₁₀/T₁₀ complex led to the formation of a metallo-DNA system without changes in the canonical hybridization of the Watson–Crick duplex [37]. At the same time, the affinity of adenine–thymine binding was reduced [38], which thereby promoted the dehybridization of the A₁₀ aptamer and more efficient formation of the A₁₀/Sb³⁺ complex.

First, the effect of Ag⁺ and Sb³⁺ on the catalytic activity of horseradish peroxidase was tested to exclude artifacts that may change the assay results. The absence of the inhibition of this process in the presence of these metal ions was shown. To determine the optimal amount of Ag⁺, the addition of different concentrations of Ag⁺ was tested and detection limits of Sb³⁺ were calculated (Figure 5). The most pronounced increase in the detection limit of Sb³⁺ was observed at a Ag⁺ concentration of 10 µg/mL, which was chosen as the optimal concentration of the enhancing agent. The significance of the increase in the analytical signal at a concentration of 10 μg/mL was compared with the values at 5 and 20 μg/mL using Student’s t-test, which confirmed the significance of the deviation (p < 0.05).

The calibration curve for the Ag⁺-enhanced microplate apta-enzyme assay was plotted in the range of 0.5 ng/mL–10 µg/mL (Figure 6a). A linear relationship (Figure 6b) between OD₄₅₀ and the logarithmic concentrations of Sb³⁺ was acquired, which corresponded to 13.7–1111 ng/mL and was regressed to y = 0.22 − 0.058x (R² = 0.9822).The detection limit for the enhanced assay was determined to be 1.9 ng/mL, which is 22 times lower than that of the previous variant. In addition, the achieved detection limit turned out to be 3–10 times lower than the recommended values of MPC for drinking water established by international organizations.

2.6. Selectivity of Ag⁺-Enhanced Microplate Apta-Enzyme Assay

The selectivity of the Ag⁺-enhanced microplate apta-enzyme assay of Sb³⁺ was investigated. The target ion was compared with other toxic cations, such as Pb²⁺, Hg²⁺, Cd²⁺, Cu²⁺, Zn²⁺, As³⁺, Ni²⁺, Co²⁺, Cr³⁺, Sn⁴ and Mo²⁺. In addition, cations (Ca²⁺, Na⁺, Mg²⁺) and anions (Cl⁻, NO³⁻, SO₄²⁻) presenting in high amounts in water were tested using the developed assay. As follows from Figure 7, the results show that most of the ions tested did not have a noticeable effect on the determination of Sb³⁺. However, insignificant nonspecific changes were observed in the presence of Ni²⁺, Sn⁴⁺ and Mo²⁺ ions, which may have an effect on the determination of Sb³⁺ ions. The effect of these ions was observed at 0.1 μg/mL, while the allowable concentration of nickel and molybdenum ions in drinking water is 0.07 and 0.01 μg/mL, respectively. In the case of Sn⁴⁺, its presence in this form is practically impossible and, in addition, the Sn⁴⁺ content in drinking water is not standardized. Thus, the proposed system showed satisfactory selectivity with respect to antimony ions.

2.7. Water Sample Analysis

The effectiveness of the developed Ag⁺-enhanced assay was studied via analysis of drinking and spring water samples. Since the results of the standard ICP-MS reference method do not show the presence of the analyte, the water samples were spiked with different concentrations of Sb³⁺. The calculated analytical recoveries were within acceptable ranges of 109–126% and 99.6–112% for drinking and spring water, respectively (

Table 1. Detection of Sb³⁺ in water samples using Ag⁺-enhanced microplate apta-enzyme assay (n = 3).

Samples	Added, ng/mL	Found, ng/mL	Recovery, %
Drinking water	10.0	10.9 ± 0.3	109.0 ± 2.8
	50.0	63.1 ± 0.2	126.2 ± 0.3
	100	115.0 ± 0.2	115.0 ± 0.2
Spring water	10.0	11.2 ± 0.3	112.0 ± 2.7
	50.0	49.8 ± 0.7	99.6 ± 1.4
	100	106.1 ± 1.1	106.1 ± 1.0

), which is consistent with published results for the determination of heavy metals in analytical practice [39,40,41]. Thus, the development of a colorimetric assay [41] based on the regulation of the oxidase-mimicking activity of Mn₃O₄ nanoparticles by oligonucleotides for mercury and cadmium ions in actual environmental water samples achieved a detection rate in the range of 85.7 to 105.21% for mercury ions and 81.7 to 113.4% for cadmium ions. The work [40] demonstrates a mean recovery of samples in the range of 94.6–124% via an ultrasensitive aptamer-based biosensor for arsenic(III) detection. Thus, the results indicate the potential application of the Ag⁺-enhanced microplate apta-enzyme assay for Sb³⁺ detection in water samples.

2.8. Comparison with Other Methods

The comparison of different methods for Sb³⁺ detection in

Table 2. Parameters of the detection of Sb³⁺ for various methods.

Method	LOD, ng/mL	Probe	Ref.
Sophisticated Instrumental Methods
Inductively coupled plasma–optical emission spectrometry	24.9–32.3	water, basal culture medium, anaerobic sludge plus basal medium	[44]
Ion-assisted photochemical vapor generation with inductively coupled plasma mass spectrometry	0.0047	lake and river water	[12]
Bulk optode coupled with spectrophotometry using 6-(4-(2,4-dihydroxyphenyl)diazenyl)phenyl)−2-oxo-4-phenyl-1,2-dihydro pyridine-3-carbo-nitrileas a ionophore	0.85	tap, domestic, sea, ground, lake water, blood plasma, urine	[48]
Surface-enhanced Raman scattering using silvered porous silicon and phenylfluorone	1	-	[45]
T-shaped slotted quartz tube–atom trap–flame atomic absorption spectrometry	0.75	mineral water	[42]
Excimer fluorescence using pyrene as a sensing probe	160	-	[16]
Hydride generation coupled with atmospheric pressure glow discharge atomic emission spectrometry	0.14	groundwater	[43]
Surface-enhanced Raman spectroscopy using dithiothreitol-functionalized two-dimensional Au@Ag array	1	natural water	[46]
Low-tech methods
Colorimetric method based on the development of a yellow potassium iodoantimonite complex	600	water samples from mine adits	[47]
Colorimetric detection using gold nanoparticles modified with poly-adenine aptamer	10	drinking water	[19]
Ag+-enhanced microplate apta-enzyme assay using poly-adenine aptamer	1.9	drinking and spring water	This work

demonstrates the advantages of the developed Ag⁺-enhanced microplate apta-enzyme assay in relation to both instrumental and colorimetric methods. The LOD of the proposed method (1.9 ng/mL) is comparable to that of flame atomic absorption (emission) spectrometry [42,43], inductively coupled plasma mass spectrometry [12,44], excimer fluorescence [16] and surface-enhanced Raman scattering [45,46], which require the use of expensive equipment. In addition, the proposed method demonstrates more advantageous sensitivity compared to the very small number of low-tech methods available for the determination of Sb³⁺ [19,47].

3. Materials and Methods

3.1. Chemicals and Materials

The aptamer 5′-biotin-AA-AAA-AAA-AA-3′ (A₁₀) and the complementary oligonucleotide 5′-biotin-TT-TTT-TTT-TT-3′ (T₁₀) were synthesized by Syntol (Moscow, Russia). Aqueous solutions of Sb³⁺, Ag⁺, Hg²⁺, Pb²⁺, Cd²⁺, Cu²⁺, Zn²⁺, As³⁺, Ni²⁺, Co²⁺, Cr³⁺, Sn⁴⁺, Mo²⁺, Cl^- and SO₄^2- were purchased from the Center of Standardization of Samples and High-Purity Substances (Saint Petersburg, Russia). The streptavidin and horseradish-peroxidase-labeled streptavidin (HRP-Streptavidin) were from IMTEK (Moscow, Russia). The bovine serum albumin and Tween-20 were obtained from Sigma-Aldrich (St. Louis, MO, USA). The 3,3′,5,5′-tetramethyl benzidine (TMB), sulfuric acid and salts for buffer solutions were purchased from Chimmed (Moscow, Russia). All solutions were prepared using the Simplicity Water Purification System from Millipore (Bedford, MA, USA). The 96-well transparent polystyrene high bind 9018 plates were obtained from Corning Costar (Corning, NY, USA).

3.2. Formation of A₁₀/T₁₀ and A₁₀/Ag⁺/T₁₀ Complexes

The biotinylated A₁₀ aptamer and T₁₀ oligonucleotide were dissolved at the final concentration of 5 µg/mL in 20 mM Tris-HCl buffer containing 150 mM NaCl, 1 mM MgCl₂ and 1 mM CaCl₂. To form a complementary aptamer complex, the reaction mixture was heated at 95 °C for 5 min and kept at room temperature (RT) for 15 min.

To obtain the A₁₀/Ag⁺/T₁₀ complex, Ag⁺ solution at the final concentration of 10 µg/mL was added to the mixture of A₁₀ aptamer and T₁₀ oligonucleotide (2.5 µg/mL for both compounds) in the same buffer solution, followed by heating at 95 °C for 5 min and cooling to RT for 15 min.

3.3. Circular Dichroism Measurement

Circular dichroism (CD) spectra were recorded using the CD spectrometer Chirascan from Applied Photophysics (Leatherhead, UK). The CD spectra were collected at RT in the range of 200–320 nm with 3 repetitions. The investigated aptamer and the complementary complex were preliminary prepared in 20 mM Tris-HCl buffer containing 150 mM NaCl, 1 mM MgCl₂ and 1 mM CaCl₂, annealed at 95 °C for 5 min and cooled down to RT.

3.4. Microplate Apta-Enzyme Assay

A total of 100 μL of streptavidin at a concentration of 5.0 μg/mL in 50 mM phosphate-buffered saline, pH 7.4 (PBS), was applied to the wells of the microplate and incubated overnight at 4 °C. Then, the microplate was washed three times with 10 mM PBS containing 0.05% detergent Tween-20 (PBST). The microplate wells were covered with 150 μL of 1% BSA (blocking buffer) prepared in 50 mM PBS for 30 min at 37 °C in a thermostat TC-1/80 (Russia). After three washes with PBST, 100 μL of freshly prepared A₁₀/T₁₀ complementary complex, 5 µg/mL in 20 mM Tris-HCl buffer, pH 7.6, was added in the wells. The microplates were incubated for 1 h at 37 °C and then washed with PBST three times. At the next stage, 100 μL of solutions containing different concentrations of Sb³⁺ were added into the wells. After 1 h of incubation at 37 °C and triple washing, 100 μL of horseradish-peroxidase-labeled streptavidin (1:5000 dilution in PBST) was added to the wells and incubated again for 1 h at 37 °C. The unbound HRP-labeled streptavidin was removed and washed three times with PBST and once with distilled water. The final steps were the addition of 100 mL of TMB substrate solution and incubation for 15 min at RT. The catalytic reaction was stopped by the addition of 50 μL of 1 M H₂SO₄ per well. Then, the optical density at 450 nm as a colored product of peroxidase-induced oxidation of TMB was measured using an EnSpire multimode plate reader from PerkinElmer (USA). The plotting of (optical density—Sb³⁺ concentration) dependence was carried out using Origin 9 software (OriginLab Corporation, Northampton, MA, USA).

The Ag⁺-enhanced microplate apta-enzyme assay was carried out using the A₁₀/Ag⁺/T₁₀ complex, as described above.

3.5. Preparation and Analysis of Water Samples

Samples of drinking and spring water with the pH adjusted to 8.5 were filtered through a syringe filter with a pore size of 0.2 µm (Sartorius, Germany). According to ICP-MS data (quadrupole mass spectrometer Nexion 300D, Perkin Elmer (Waltham, MA, USA)), the used water samples were reliably free of Sb³⁺. The method’s description is provided in [49]. The results of the water analysis using the ICP-MS technique have been published in our previous works on heavy metal detection; see supplementary information in [49,50]. A series of Sb³⁺-spiked water samples were prepared at concentrations of 10, 50 and 100 ng/mL with the addition of a Sb³⁺ standard solution (0.1 mg/mL in the Milli-Q water). Then, spiked water samples were analyzed with the developed microplate apta-enzyme assay.

4. Conclusions

A sensitive microplate apta-enzyme assay for Sb³⁺ detection was proposed for the first time. The main principle of this approach is based on the formation of a specific complex of the A₁₀ aptamer with Sb³⁺, leading to a reduction in the recorded transformation of the TMB substrate. It was found that Ag⁺ promoted dehybridization of the A₁₀ complex of the complementary oligonucleotide and the formation of the A₁₀/Sb³⁺ complex. Under optimized conditions, the results show that the analytical performance was enhanced by up to 22 times in the presence of Ag⁺. The achieved detection limit, 1.9 ng/mL, is 3–10 times lower than the established MPC values. The main benefits of the developed assay are its (i) low detection limit; (ii) cost-efficiency and (iii) simplicity. Additionally, this assay relies on available materials, such as conjugates of streptavidin with horseradish peroxidase and combinations of biotinylated aptamers. Finally, the proposed assay was applied in spiked drinking water samples with satisfactory results.