Introduction

Doxycycline is a broad-spectrum antibiotic molecule synthetically derived from oxytetracycline and part of the tetracycline family. The structure of doxycycline comprises a hydronaphthacene center (Fig. 1), consisting of four combined rings, it is different from other tetracycline antibiotics (Table 1) as it possesses a beta keto group. By attaching to the 30 S ribosomal subunit and preventing the addition of new amino acids to the expanding peptide chain, doxycycline hinders the translation of proteins. As compared to tetracycline, which is only 60–70% absorbed in the intestine, doxycycline exhibits near-complete absorption as it has 3–5 times more lipophilicity compared to the same-class antibiotics1. While tetracycline is primarily removed through the kidneys, doxycycline removal is via feces and therefore is also suitable for patients with kidney impairment2. Dermatological disorders and Gram-positive and Gram-negative bacterial infections are frequently treated with doxycycline. Apart from that, it is also used to promote animal growth and act as an antibacterial agent in the animal husbandry industry. Due to its known antiviral effects against Chikungunya and Dengue3,4, as well as a broad-spectrum antibiotic activity, it was a routinely prescribed antibiotic during the COVID-19 pandemic for the alleviation of symptoms and prophylactic treatment of secondary infections5.

Fig. 1
figure 1

Structure of tetracycline.

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Table 1 Properties of Doxycycline and tetracycline6,7,8,9.
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As only a part of the ingested antibiotics is metabolized in the human system, a large proportion (~ 60%) of tetracycline antibiotics is reported to be released in human feces in their original form10 and enter wastewater treatment plants. Sewage treatment plants, which are primarily designed to remove organics and nutrients, may or may not be able to remove these pharmaceutical residues optimally leading to the entry of these antibiotics in surface and groundwater along with the release of inefficiently treated wastewater. Moreover, their regular introduction into the environment makes them pseudo-persistent even if they don’t have half-lives as high as the persistent pollutants11. As a result, the pharmaceutical residues are often detected in nanogram to microgram levels in wastewater treatment plants, surface, and groundwater12. DOX was found in 34 out of 98 samples in urban watershed samples of South-East Queensland, Australia with a maximum concentration of 0.4 µg/L13. The formation of antibiotic-resistant species could result from horizontal gene transfer caused by the presence of these suboptimal doses of antibiotics. Therefore, it is important to accurately estimate and monitor the antibiotics in different water/wastewater matrices to design suitable treatment regimes.

The methods for detecting doxycycline that are accessible are high-performance liquid chromatography-based determination14, potentiometry15, and sequential injection chromatography16. The drawbacks of these methods include complex sample preparation and the requirement of highly trained personnel to operate the sophisticated instruments. Low-cost, fast, reliable methods that can be operated by less trained professionals and provide on-site quantification are required. Aptamer-based assays are becoming more and more popular in this field for the development of sensors because of their stability, affordability, and lower size compared to typical antibodies17. Aptamers are single-stranded DNA/RNA molecules obtained by the standard process named systematic evolution of ligands by exponential enrichment (SELEX), that fold into unique secondary and tertiary structures to bind to their target with extreme specificity and affinity18. Aptamer, in particular, is a promising candidate for the development of highly selective and sensitive sensors of different types, like electrochemical19,20, fluorescent21,22, or colorimetric23,24 due to its simplicity of manipulation and flexibility in application25. Out of these aptamer-based sensors, fluorescent biosensing is particularly favored and valued because of its special benefits, which include high sensitivity, high selectivity, stability, and affordability with simple and rapid operation, making them particularly suitable for field or point-of-care analysis. Fluorescent aptasensors can be primarily classified into two modes: labeled and label-free fluorescent aptasensors, based on the signal reporter of each. Because label-free fluorescent aptasensors avoid covalently labeling fluorophores or quenchers to aptamers, they overcome the laborious, time-consuming, and financially ineffective method of labeling aptamers with fluorophores (covalent) and quenchers. Further, the labeling might also negatively affect the aptamer’s binding affinity with the target and might result in reduced sensitivity26. Free dye molecules or cationic polymers are used in label-free fluorescent aptasensors, which transduce fluorescence signals when they intercalate into the aptamer sequence. To create label-free fluorescent aptasensors, a variety of dye molecules are available, such as OliGreen, SYBR Green I (SGI), DAPI, Thiazole orange, Pico Green, Rhodamine B(RhB), and others27.

Rhodamine B (RhB), a xanthene dye, exhibits a high fluorescence quantum yield, stability in aqueous environments, and a strong capacity to interact effectively with nucleic acid aptamers, rendering it appropriate for the identification of diverse DNA and RNA structures28 and for the creation of a fluorescence-based aptasensor. RhB demonstrates positive cooperativity and gradually unwinds the DNA helix while binding to DNA by groove interaction, leading to a substantial enhancement in fluorescence emission29 due to its strong stacking and hydrogen-bonding interactions with ssDNA without requiring duplex formation, whereas dyes like SYBR Green30,31 or DAPI32,33 require duplex or groove-binding environment to fluoresce efficiently which comes as a limitation to create a fluorescent aptasensor using single stranded DNA. This research employed a label-free fluorescent aptasensor utilizing RhB and apt40 for the detection of doxycycline. RhB operates as a signal reporter with a high affinity for binding in minor grooves, producing robust fluorescence emission at a wavelength of 579 nm when excited at 546 nm in the absence of DOX. In the presence of target doxycycline, it binds to the aptamer with increased affinity and multiple interactions, inducing conformational changes in the aptamer structure, which brings RhB molecules in the proximity of the aptamer-DOX complex, promotes non-radiative energy transfer, resulting in a reduction of RhB’s fluorescence emission and producing a quantifiable “turn-off” signal that correlates with the concentration of DOX. This was the foundation for the very specific and selective fluorescence detection of doxycycline. The developed aptasensor also provides high specificity by binding to DOX only while rejecting other interfering contaminants/pharmaceuticals to combine. Consequently, this aptasensor was also used to detect doxycycline in wastewater, yielding positive outcomes. By employing a portable fluorescence reader device, the developed aptasensor has the potential to be a portable sensing tool, and by replacing the aptamer sequence, the system may be easily modified for the detection of various environmental and biological analytes, highlighting its innovation, versatility, and practical relevance.

Experimental section

Chemicals and instrumentation

All chemicals were of analytical or molecular biology grade and used without further purification.TCEP (Tris(2-carboxyethyl) phosphine) (≥ 98%), azithromycin (AZT) (≥ 98%), cefoperazone sodium salt (Cefo) (≥ 98%), doxycycline hyclate (DOX) (≥ 98%), ciprofloxacin (Cipro) (≥ 98%) and 40-mer ssDNA with the sequence 5’-[ThiC6] GAG AAC AGT GTC CCA GTC GCT AGT TTT CTC TGC CTC CGTT (apt40) (≥ 95% purity, HPLC purified) were purchased from Sigma Aldrich. Himedia was the source of the tetracycline hydrochloride (TET) (≥ 98%). Tris Buffer (≥ 99%) and Hydrochloric Acid (HCl) (35–38%) were obtained from LOBA Chemie Pvt Ltd. Tris buffer was dissolved in 7–8% HCl to obtain Tris-HCl (pH 7.4). Rhodamine B (RhB) (≥ 95%, spectroscopic grade) and Phosphate Buffer Saline (PBS, pH 7.4) were procured from Sisco Research Laboratories Pvt Ltd. The 10 µM aptamer stock was prepared in Tris-HCl buffer and stored at -20 °C in aliquots. This stock solution was diluted before use as per the requirement. The UV-visible investigations were performed using a Shimadzu UV-1900i UV-visible spectrophotometer. The Shimadzu RF-6000 spectrofluorometer with slits at 5 nm for both emission and excitation were utilized to capture the fluorescence spectra in the wavelength range of 551–800 nm. Doxycycline was quantified by excitation at 546 nm and emission at 579 nm in Tris-HCl buffer (pH 7.4).

Molecular Docking study of apt40, DOX, and RhB

The apt40-RhB and apt40-DOX bindings were investigated by simulating molecular docking with Auto Dock Tools (version 1.5.6). The secondary structure of apt40 was predicted by the M-fold online web service34, and the 3D structure of the aptamer was obtained using the online application RNAcomposer35. The sdf files for RhB and DOX were obtained from PubChem and converted using Open Babel to pdb format. Once the water molecules were extracted from the DNA, the aptamer with RhB and DOX were completely inserted into the grid box for global docking. The auto dock program’s default settings were used to assign the other parameters, and the docking conformations with the lowest binding energy were selected. The docked conformations were visualized using Pymol software.

Aptasensor fluorescence analysis

All experiments were performed three times in controlled laboratory conditions at ambient temperature. An array of fluorescent tests was conducted to study the sensing capability of the aptamer. Two hundred microliters of 25 nM aptamer solution were combined with 500 µL of 8 µM RhB (resulting in a final concentration of 4 µM) and diluted to 1000 µL using Tris HCl buffer. A 200 µM DOX stock solution in Tris-HCl buffer was made, and this stock was diluted as required to obtain the final concentrations in the range of 0.13 µM to 100 µM. The decrease in fluorescence intensity at different concentrations of DOX was measured.

Furthermore, to test the selectivity of the developed aptasensor, several interfering antibiotics, including tetracycline (TET), cefoperazone (Cefo), ciprofloxacin (Cipro), and azithromycin (AZT), were quantified using the same approach for DOX at the final concentration of 90 µM of apt40 + RhB solution. Finally, the aptasensor’s practical application was validated by examining wastewater samples. The treated wastewater samples were collected from a moving-bed biofilm-based sewage treatment plant in Jaipur, India, and filtered with a 0.22 μm Whatman filter paper before use. The samples were spiked with 23–60 µM of DOX and evaluated using the same procedure as the DOX analysis in Tris-HCl buffer.

Effect of experimental conditions and study of quenching mechanism

To maximize the performance of the designed aptasensor, several factors that might affect sensor performance were assessed. These factors included the type of solvent (PBS Saline buffer, deionized water, and Tris HCl buffer), the aptamer concentration (100–6 nM), the concentration of RhB (0–5 µM), the incubation time of RhB (1–5 min), and the DOX (0–10 min).

Different concentrations of DOX were mixed with the solution in Tris-HCl buffer to study the quenching mechanism between the dye-DNA complex and doxycycline. After 11 min of incubation of each concentration, fluorescence measurements were carried out with the excitation wavelength of 546 nm and emission wavelength of 579 nm. UV/Visible spectra were also recorded for apt40, RhB, DOX, apt40+RhB, and apt40+RhB+DOX at room temperature.

The limit of detection calculation

The limit of detection (LOD) value was determined by using the Eq. 129, and the limit of quantification (LOQ) was computed by applying the formula as stated in Eq. 2.

$$\:LOD=\frac{3\sigma\:}{k}$$
(1)
$$\:LOQ=3\times\:LOD$$
(2)

where σ and k represent the standard deviation of the mean blank signal and the slope of the linear regression curve, respectively36.

Results and discussion

Principle of fluorescence-based sensing

The schematic representation of the sensor preparation and detection process is illustrated in Figure 2. The doxycycline binding aptamer is used in this design to recognize the target. RhB, a water-soluble fluorescent dye37 that binds with the DNA sequence via groove-binding38, showed a strong fluorescence emission upon binding with apt40. The quantification of DOX can be done by fluorescence quenching of RhB due to the electron transfer from RhB (donor) to DOX (acceptor), occurring due to the target-induced structural changes within the apt40+dye complex.

Fig. 2
figure 2

General procedure of fluorescence sensing of doxycycline.

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The proof-of-concept of this assay was confirmed by the fluorescence and UV/Vis spectrum, as shown in Fig. 3. The inset diagram of Fig. 3(a) shows the fluorescence of individual components-apt40, RhB, DOX and apt40 + DOX mixture. It has been observed that each component exhibits negligible fluorescence in the measured region, which confirms that neither apt40 nor DOX contributes significantly to the background emission. The apt40 + DOX mixture also shows no notable fluorescence, indicating that DOX binding with the aptamer is not the only factor to generate the signal. In contrast, a strong emission peak appears only upon the binding of RhB with apt40, which may be due to the shielding of RhB from water and other quenchers in the Tris-HCl buffer. On binding with RhB, the aptamer undergoes a conformational change that traps RhB in a more rigid microenvironment by intercalating the dye within the aptamer structure, which reduces rotational and vibrational motion of RhB through π–π stacking and hydrogen bonding, leading to enhanced quantum yield and fluorescence39 which further confirms the strong interaction between RhB and the aptamer. In contrast, the fluorescence intensity of RhB in the dye-DNA complex was noticeably lower in the presence of target doxycycline than in the solution which may be attributed to the fact that (i) the binding of DOX with apt40 induces conformational changes in the aptamer structure which disrupts the intercalated dye environment and leads to the partial dye release or altered stacking interactions, thereby decreasing FL emission and (ii) The proximity of DOX to RhB within the aptamer framework facilitates non-radiative energy transfer from the excited RhB fluorophore to DOX, resulting in fluorescence quenching. Therefore, the decrease in fluorescence emission at 579 nm can be used for quantifying doxycycline. The mechanism of fluorescence quenching was examined by the analysis of UV-Vis absorption spectra. Quenching may transpire via two principal mechanisms: (1) Static quenching occurs when the creation of a complex between the fluorophore and a tiny molecule induces chemical alterations in the solution, leading to a displacement in the absorption peak location of the UV-Vis spectrum. (2) Dynamic quenching occurs when random collisions with a tiny molecule deactivate the excited state of the fluorophore, without altering its chemical state, hence maintaining the UV-Vis absorption peak location. The results shown in Fig. 3(b) indicate that the absorption peaks of the UV-Vis spectrum for the apt40 + RhB + DOX complex at 554 nm coincided with those of the spectra of RhB and apt40+RhB, signifying the dynamic quenching process. The effectiveness of dynamic quenching is influenced by the dipole-dipole interaction between the donor and acceptor molecules. In the present case, the carboxyl (-COOH) group of rhodamine B can potentially form hydrogen bonds with the hydroxyl (-OH) and amino (-NH2) moieties of doxycycline, as shown in Fig. 3(c), which may contribute to the observed quenching phenomenon40,41.

Fig. 3
figure 3

(a) Performance of aptasensor in Tris HCl for DOX determination; (b) UV/visible spectrum of RhB, apt40, Dox, apt40 + RhB and apt40 + RhB + DOX; (c) Schematic illustration of DOX binding with carboxyl (-COOH) group of Rhodamine B.

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However, environmental conditions, like pH and ionic strength in wastewater matrices, may affect the electrostatic interactions between the aptamer and dye. Nonetheless, since the RhB–aptamer interaction is mostly reinforced by π–π stacking and hydrogen bonding rather than electrostatic forces, the complex demonstrates significant stability and consistent fluorescence response at near-neutral circumstances (pH 7.4). The system’s resilience enhances its application in real-world wastewater sensing conditions, where ionic fluctuations are prevalent but do not substantially interfere with the aptamer–dye system.

Molecular Docking study

Molecular docking was performed to identify the precise binding locations of the aptamer with dye and doxycycline. Docking analysis on apt40-RhB and apt40-DOX was carried out using Autodock Tools. Grid box centre (X: Y:Z)::(-4.625:11.349:-4.234) Grid box dimension (X: Y:Z)::(70:70:70) for apt40-RhB complex. Grid box centre (X: Y:Z)::(0.977:13.682:6.284) Grid box dimension (X: Y:Z)::(62:80:80) for apt40-DOX complex. As shown in Fig. 4(a), the simulation studies demonstrate that RhB (shown in yellow) prefers the minor grooves of DNA when intercalating with the aptamer. The more slender and deeper shape of the DNA minor grooves provides multiple opportunities for van der Waals interactions with the dye’s surface42. The RhB is intercalated within the apt40 groove through strong π–π stacking and hydrogen bonding interactions within adjacent nucleobases. The carboxylic group of RhB is exposed to the outside of the minor groove, where it interacts with the solvent environment, and is orthogonal to the remainder of the molecule, as seen in Fig. 4(b). The carboxyl moiety of RhB forms an intermolecular hydrogen bond with the DNA backbone, with a bond length of 2.1Å, allowing the dye to intercalate within the aptamer with the binding energy of − 3.08 kcal/mol thus stabilizing the complex and restricting the intramolecular rotations of the dye which suppress the non-radiative decay and accounts for the enhance fluorescence observed in the Fig. 3(a). As may be seen from the global viewpoint shown in Fig. 4(c), apt40 folds to create a “pocket” for DOX and forms multiple hydrogen bonds and hydrophobic contacts with nucleobases. Figure 4(d) shows the key points of interaction between the aptamer and DOX, which include hydrogen bonds at G8(2.2 Å), C6(2.2 Å), A5(2.6 Å), T27(2.2 Å), and T26 (3.1 Å), as well as N-O bonds with T26(2.2 Å) with binding free energy of -8.43 kcal/mol. It has been observed that the DOX binding with apt40 induces conformational changes in the aptamer, which disrupts the intercalation of RhB, which weakens the π–π stacking and charge-transfer stabilization of RhB, leading to the fluorescence quenching and decreased UV absorbance intensity as observed experimentally.

Fig. 4
figure 4

(a) Molecular docking of apt40 with RhB; (b) Interaction diagram of apt40 with RhB; (c) Molecular docking of apt40 with DOX; (d) Interaction diagram of apt40 with DOX.

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Optimization of experimental conditions

The biosensor signal originates from the dye-DNA complex’s fluorescent property, and the highest fluorescence intensity is correlated with the best detection sensitivity. Therefore, several crucial variables need to be optimized to create an apt40+RhB complex, including pH, the solvents used to make the solution, the concentrations of aptamer and RhB, the reaction time between the dye and aptamer, and the duration of DOX’s incubation with aptamer. Ensuring background fluorescence and optimizing the reaction efficiency can lead to more sensitive detection of the apt+RhB reaction result. The pH of the sensing medium plays a crucial role in affecting fluorescence quenching and the binding between aptamers and their targets by altering charge distribution, ionic strength, and the conformation of the aptamers43. This study maintained a pH of 7.4 to ensure optimal stability and fluorescence response of the aptamer. This regulated setting reduces structural variations of the aptamer and ensures stable quenching behaviour44,45. Thus, it is imperative to optimize the solvent used in the reaction. The feasibility examination of the suggested approach was carried out in PBS (Phosphate-buffered saline, 0.05 µM, pH 7.4), Tris-HCl buffer (50 mM, pH 7.4), and (DI)deionized water. Figure 5 demonstrates that the fluorescence intensity of the apt40+RhB solution is maximised in deionized water, followed by observations in Tris-HCl buffer and PBS buffer, respectively. In contrast, the deionized solution showed minimal quenching of fluorescence intensity by doxycycline hyclate, while the PBS buffer showed considerable quenching. Tris-HCl solution showed the greatest significant decrease in fluorescence intensity with the addition of doxycycline hyclate. Therefore, Tris–HCl was used for the ensuing experimental protocols. This enhanced responsiveness may be mainly ascribed to many crucial variables. The Tris-HCl buffer creates an optimum ionic microenvironment that retains the natural conformation of the aptamer, thereby enhancing the specificity and strength of interactions between the dye and the aptamer. Conversely, phosphate ions in PBS may cause electrostatic interference with the negatively charged DNA backbone, thereby altering aptamer conformation and diminishing signal transmission efficacy. Furthermore, Tris-HCl buffer demonstrates intrinsically decreased background fluorescence and less non-specific adsorption relative to PBS, hence reducing signal noise and enhancing analytical accuracy46,47; Moreover, its enhanced physicochemical stability and buffering capability around neutral pH ensure consistent sensor performance in conditions representative of real environmental and wastewater samples, which makes it particularly suited for fluorescence-based aptasensing applications. Consequently, Tris-HCl was chosen for further tests, since it was considered the optimal solvent for biosensor development.

Fig. 5
figure 5

Choice of solvent for the proposed aptasensor.

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RhB at a concentration of 4 µM was tested with different concentrations of aptamer in Tris-HCl at intervals of 1 min for 5 min to determine the ideal reaction time of RhB with apt40 for fast and selective determination of DOX. As aptamer concentration is decreased, as illustrated in Figure 6(a), fluorescence intensity rises to 12.5 nM. However, when aptamer concentration is further lowered to 6 nM, fluorescence intensity also decreases due to the reduced availability of aptamer for RhB binding. The aptamer-dye complex forms easily after mixing, but it stabilizes after five minutes without changing the fluorescence intensity, as seen in Fig. 6(b). As a result, the ideal aptamer concentration was established as 25 nM because of its slightly higher initial FL intensity and better signal stability over time as can be seen in Fig. 6(b). Five-minute reaction time was found to be ideal for the binding of dye with ssDNA, as the FL Intensity stabilizes in 5 min (Fig. 6(b)).

Fig. 6
figure 6

(a) Impact of the aptamer concentration on the fluorescence intensity of RhB(4µM) after 5 min; (b) variation of fluorescence intensity of RhB(4µM) concerning time with different concentrations of the aptamer.

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The fluorescence intensity of the aptamer-dye complex was assessed to identify the optimal RhB concentration for biosensor development, with RhB concentrations of 0, 1, 2, 3, 4 and 5 (Fig. 7(a)) being evaluated. The results indicated a signal rise from 0 to 3 µM RhB, followed by stability at 4 µM, and a subsequent decline at higher RhB concentrations. Thus, with a constant aptamer concentration of 25 nM, the ideal RhB concentration was determined to be 4 µM. Subsequently, a 5-minute reaction time, the ideal concentrations of 25 nM aptamer and 4 µM RhB in Tris-HCl buffer were used to evaluate the effect of doxycycline incubation duration on the aptamer-dye complex. The fluorescence intensity was seen to diminish over a period of six minutes, after which it stabilized as shown in Fig. 7(b). Thus, six minutes was established to be the optimal DOX-aptamer response time.

Fig. 7
figure 7

(a) Effect of the RhB concentration on the fluorescence intensity and emission wavelength with aptamer concentration of 25 nM; (b) Reaction time optimization of doxycycline.

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DOX determination by using the proposed aptasensor

The fluorescence intensity of ssDNA+RhB at 579 nm gradually decreased as the concentration of DOX was increased, as seen in Fig. 8(a). The dye-DNA complex fluorescence intensity and DOX concentration have an antagonistic relationship in the range of 0.13 µM – 100 µM (R2 = 0.995), as shown in Fig. 8(b). The addition of DOX successfully quenches the fluorescence, demonstrating the accuracy of the fluorescent detection of DOX. The LOD value of the proposed sensor was determined to be 114.03 nM and the LOQ of 342.09 nM.

Fig. 8
figure 8

(a) The established biosensor’s fluorescence spectra at varying doxycycline hyclate concentrations (0.13 µM to 100 µM); (b) The straight-line correlation between DOX concentration[µM] and the reduced fluorescence intensity.

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Table 2 Comparison study of the developed sensor for DOX.
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A summary of doxycycline detection techniques is given in Table 2 for comparison with the previously developed and published sensors. The advantages of the proposed aptasensor in this work are its simple sample pretreatment, the reduced limit of detection (LOD), broad linear range, which is the typical range of its presence in wastewater, enhanced accuracy, and quick measurement process.

Specificity and reproducibility of the developed fluorescent aptasensor

The specificity was carried out to verify the possible interference originating from other contaminants present in the actual sample matrix. The selectivity of the developed biosensor was determined by cross-referencing it with complex samples that also include drugs besides doxycycline. Under ideal circumstances, 90 µM of several contaminants were tested, including ciprofloxacin, azithromycin, cefoperazone sodium salt, and tetracycline, an antibiotic from the same family.

The tertiary structure of the aptamer creates a binding site specifically suited to the unique size, charge distribution, and functional groups of doxycycline. Structural similarities exist between doxycycline and tetracycline; however, subtle variations in their structures influence steric fit and hydrogen bonding. In contrast, the distinct molecular framework of other pharmaceuticals (Fig. 9(a)) prevents binding to the doxycycline aptamer pocket, leading to no conformational changes and no substantial fluorescence signal55, as shown in Fig. 9(b). These results demonstrate that the constructed biosensor is unaffected by the aforementioned contaminants, suggesting that it has outstanding anti-interference capabilities. To verify the suggested sensors’ repeatability, each test was run three times (n = 3) for every concentration of DOX. The biosensor’s effectiveness and reliability were validated by three independent measurements, with the standard deviation of repeated measurements at the same antibiotic concentration assessed to be between 0.1% and 2%, as seen in Fig. 10(a). The stability of the produced fluorescent aptasensor was assessed by storing it at 4 °C in the dark to prevent photobleaching and conformational degradation and testing it over three consecutive days. The signal variation between the first and last day was under 5%, as seen in Fig. 10(b). The findings unequivocally demonstrate that the sensor maintains its functional integrity and can be utilized for several detection cycles without substantial loss of sensitivity and selectivity towards DOX. Consequently, the sensor has the ability for regeneration and reutilization throughout actual operating cycles.

Fig. 9
figure 9

(a) Chemical structures of different tested pollutants; (b) Comparison of the fluorescence signal of apt40 + RhB in the system with different antibiotics and salts at 90 µM.

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Fig. 10
figure 10

(a) Reproducibility study curve for 35 µM (n = 3); (b) Response vs. number of days curve for stability study for 40 µM of DOX (n = 3).

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The detection performance of apt40+RhB for Doxycycline hyclate in actual samples

The Tris-HCl buffer was used to conduct the DOX standard test for this investigation. A spike and recovery test was conducted to confirm that the fluorescence biosensor could detect DOX in actual samples. The wastewater sample was procured from a full-scale sewage treatment plant in Jaipur, India, where it was treated utilizing UV disinfection as the last step and moving bed bioreactor (MBBR) technology as a secondary phase. Prior to usage, the water sample was filtered through a 0.22 μm Whatman filter paper. As indicated in Table 3, the spike and recovery tests were conducted for the concentrations of 23–60 µM DOX, yielding a recovery range of 96.70–100%. This indicates that the actual complex matrices have no discernible effect on the biosensor, and doxycycline in wastewater may be detectable via the proposed biosensor.

Table 3 Spike and recovery test results.
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Conclusion

In this work, a dye-doped fluorescent aptasensor with high sensitivity, broad range, selectivity, and application for the detection of antibiotics in wastewater matrix was developed. The developed protocol is quick and easy with a response time of 11 min. RhB, the fluorescence probe, can bind with the doxycycline binding aptamer via hydrogen bonding to create a strong fluorescence emission at 579 nm. After adding the target doxycycline, the aptamer could detect and bind to doxycycline selectively via numerous hydrogen bonds and N-O bonds, while the formation of hydrogen bonds between the carboxylic, phenolic, and amino groups of RhB and DOX leads to the quenching of fluorescence emission of the dye-DNA complex. The fluorescence sensor for DOX has a limit of detection (LOD) of 114.03 nM while operating within the specified range of 0.13 µM to 100 µM. The limit of detection (LOD) of the developed fluorescent aptasensor was found to be better when compared to most of the methods that have been devised for DOX detection. Additionally, the biosensor shows great application in evaluating DOX in real-world analysis, with 96% -100% recoveries in wastewater and a high sensitivity for DOX detection, which qualifies it for monitoring water contamination.