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Anti-OTC antibody-conjugated fluorescent magnetic/silica and fluorescent hybrid silica nanoparticles for oxytetracycline detection

  • Viswanathan Kaliyaperumal EMAIL logo , Fatimah Oleyan Al-Otibi EMAIL logo , Ruth Sophila John , Raedah Ibrahim Alharbi and Dhinakar Raj Gopal
Published/Copyright: July 17, 2024
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Open Chemistry
From the journal Open Chemistry Volume 22 Issue 1

Abstract

This study presents two alternative fluorescent nanoparticle-based oxytetracycline (OTC) detection methods in milk samples. Rhodamine 6G-coated fluorescent hybrid silica nanoparticles and fluorescent magnetic/silica nanoparticles functionalized with anti-OTC antibodies were used in this test. The sandwich test format was utilized to compare anti-OTC antibody-conjugated fluorescent magnetic/silica nanoparticles with OTC/OTC antibody-conjugated fluorescent hybrid silica nanoparticles in an Eppendorf tube with magnetic separators. The magnetic separator helps to quickly retain all of the OTC captured by fluorescent magnetic core–shell nanoparticles in the milk sample. As a result, the assay time was dramatically shortened. The obtained linear range was 1.34 × 10−6 to 2.10 × 10−8 (M) (R 2 = 0.9954), the detection limit was 4.76 ng/mL, and the total assay time was 90 min. This approach was used to determine the OTC concentration in milk samples, and the maximum percentage (%) of interference was less than 3.0%, with a recovery rate of greater than 97.0%. This approach offers a high potential for residue detection in milk samples. With a total analysis period of less than 90 min, this approach provided the best way to determine the capture and detector nanoparticles’ response.

Keywords: OTC detection; magnetic separator; fluorescent hybrid silica nanoparticles; fluorescent magnetic silica nanoparticles; sandwich assay

1 Introduction

Food quality and safety have gained a lot of attention recently because chemical contaminants such as drug residues, pesticides, mycotoxins, and so on can be harmful to people health. As a result, monitoring drug residues in food is crucial. Antibiotics such as aminoglycosides, tetracyclines, macrolides, and sulphonamides are frequently used to prevent bacterial infections in the livestock business [1,2,3,4]. Excessive drug use in the livestock industry results in significant levels of antibiotic residues in milk, eggs, and edible tissues generated from the parent drug ingredient or metabolites/conjugates [5,6,7,8,9].

The European Medicines Agency constantly monitors the antibiotic content in food items, and it has already set many standards to monitor drug concentrations in veterinary goods and other food stuffs. Various preconcentration/extraction techniques such as liquid–liquid extraction, solid-phase extraction (SPE), liquid–liquid microextraction, solid-phase microextraction, and magnetic solid-phase extraction (MSPE) were initially used to evaluate antibiotic concentrations in foods and isolate antibiotic residues from various samples [10,11,12]. Following the introduction of chromatographic principles, the researchers developed liquid chromatography–tandem mass spectrometry (LC–MS/MS), ultrahigh-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS), and high-performance liquid chromatography with evaporative light-scattering detection (HPLC-ELSD) techniques. Later, surface-enhanced Raman spectroscopy, biosensors, capillary electrophoresis, and enzyme-linked immunosorbent assay (ELISA) [13,14,15] were developed. The use of nanoparticles for antibiotic residue detection provided several advantages, including a larger surface area that allows for higher biomolecule loading, biomolecule adsorption on nanoparticles which improves the stability, and easy separation from the reaction mixture using a centrifugal/magnetic field [16,17,18]. Tetracycline antibiotics are widely utilized in veterinary medicine because they are effective against both Gram positive and Gram negative microbial infections. Overuse of oxytetracycline (OTC) in farm animals leads to the accumulation of OTC residues in food items such as meat, milk, and chicken eggs, and researchers have made swift efforts to develop a sensing device for the enhanced detection of OTC in contaminated food products [19,20,21,22,23].

Internationally, the Codex Alimentarius, Food and Agricultural Organization, and World Health Organization periodically monitor the maximum residue levels for each medicine or pesticide in food items. Nowadays, technological advancements drive researchers to develop more sensitive and faster screening methods for residue detection in food products, with a wide detection range, high sensitivity, selectivity, and multiple detections in a single test in a short period of time. Microbiological techniques are quick, cheap, and easy to employ, but their sensitivity and specificity are low. Although instrumental analytical methods are exact and quantitative, they require extremely sophisticated, expensive equipment and elaborate sample pre-treatments. Methods for immunological analysis save time while giving high sensitivity and specificity [24–27]. Previously, a multicolour fluorescence sensing platform (CDs-Cit-Eu) [28], a nitrogen-doped and europium-based dual-response fluorescent probe [29], multicolour carbon dots as dual detection probes [30], and a ratiometric fluorescence sensor based on lanthanum-doped carbon dots [31] were described. The development of a drug residue detection system based on a dual method has received more attention (Figure 1).

Figure 1 Structure of the OTC drug.
Figure 1

Structure of the OTC drug.

The purpose of this research is to create a simple Eppendorf test to identify OTC residue in milk utilizing a dual detection approach. For this study, rhodamine 6G hybrid silica nanoparticles were synthesized and coupled with anti-OTC antibodies. The fluorescent magnetic core–shell nanoparticles were created using the sol–gel process and were linked with anti-OTC antibodies. Anti-OTC antibody-conjugated fluorescent magnetic/silica and anti-OTC antibody-conjugated fluorescent hybrid silica nanoparticles were used in magnetic separators to capture and detect OTC from milk. Sandwich reactions were carried out. The results demonstrate that this method can quickly detect OTC in milk with high sensitivity and specificity. This approach is especially effective for detecting OTC because of its unique properties. (1) The magnetic core with a fluorescent dye covered with a silica layer in a capture probe effectively prevents fluorophore leakage, and the surface functionalized with specific antibodies can bind to specific antigens, form immune complexes, and produce distinct signals based on analyte concentrations. (2) The anti-OTC antibody-linked capture probe binds directly to the OTC molecule, enabling rapid OTC detection. Various concentrations of OTC in a sample produce varying rates of electron shifting into the capture probe, resulting in fluorescence quenching of the tris(2,2′-bipyridyl)dichlororuthenium(ii) hexahydrate fluorescent dye. (3) The nanoparticles include rhodamine 6G dye molecules, which provide a strong signal and allow for straightforward quantification. (4) In this multiple probe detection method, when anti-OTC antibodies in a detection probe bind preferentially to OTC, they produce good intensity values at extremely low concentrations of the analyte in real samples. (5) The dye emission spectrum separations between fluorophores offer excellent reproducibility between analyses.

2 Materials and methods

2.1 Reagents

Reagents used in this study such as ferric chloride, ferrous chloride, oleic acid, tetraethyl orthosilicate (TEOS), Tris(2,2′-bipyridyl)dichlororuthenium(ii) hexahydrate, 3-aminopropyltriethoxysilane (APTES), rhodamine 6G, and N-hydroxysuccinimide (NHS) were procured from Sigma-Aldrich. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), bovine serum albumin (BSA), and o-phenylenediamine were obtained from TCI Chemicals. Anti-OTC antibodies were procured from Thermo Fisher Scientific.

2.2 Preparation of fluorescent magnetic/silica nanoparticles

Based on prior research [32,33], fluorescent magnetic/silica nanoparticles were created. In brief, a conical flask containing 10 ml of each of 1 M ferric chloride and 2 M ferrous chloride solutions were deoxygenated with nitrogen. To create oleic acid-decorated magnetic particles, 1 mL of oleic acid was added with 10 mL of ammonium hydroxide in a conical flask. It was heated with agitation at 80°C for 30 min. The flask was then allowed to cool to room temperature before being separated using a magnet and cleaned with distilled water and allowed to dry at 70°C. Finally, for the preparation of fluorescent magnetic/silica nanoparticles, 500 mg of oleic acid-coated iron oxide, 10 mL of Milli-Q water, and 10 mL of ethanol were added in a conical flask and mixed for 10 min. After that, 1.0 mL of TEOS, 5 mL of Tris(2,2′-bipyridyl)dichlororuthenium(ii) hexahydrate fluorescent dye, and 1 mL of ammonium hydroxide solution were added, and the reaction mixture was mixed thoroughly for 3 h at room temperature. Then, 0.2 mL of APTES was added and stirred again for 1 h at room temperature to form the amine group on the surface, and the particles were separated using a magnet.

2.3 Synthesis procedure for fluorescent hybrid silica nanoparticles

To generate fluorescent hybrid silica nanoparticles, we followed a slightly modified procedure from our earlier study [34]. In a 50 mL conical flask, 20 mL of ethanol, 10 mL of 2% polyvinylpyrrolidone (PVP) solution, and 1 mL of TEOS were combined. They were mixed for 20 min, added with 5 mL of 6 × 10–3 mol/L rhodamine 6G fluorescent dye and 1 mL of ammonia hydroxide solution and stirred for 3 h, and then added with 1 mL of APTES and stirred for another 1 h to modify the surface.

The materials were examined by Fourier transform infrared (FTIR) analysis (PerkinElmer Spectrum One FTIR instrument), emission spectroscopy (multi-mode reader TECAN), scanning electron microscopy (SEM, Quanta 200 FEG), and dynamic light scattering (DLS) in the Sophisticated Analytical Instrumentation Facility at the Indian Institute of Technology, Chennai, using readily accessible methods.

2.4 Anti-OTC antibody conjugation

To conjugate anti-OTC antibodies with nanoparticles, 5 mL of NHS (1 × 10−3 mol/L), 5 mL of EDC (8 × 10−4 mol/L), 5 mL of anti-OTC antibodies (1.11 mg/mL), and 5 mL of amine group-modified nanoparticles (25 mg/mL) in a 25 mL vial and stirred for 2 h at room temperature. After that, the solution was centrifuged at 10 min at 8,000 rpm, the supernatant was collected from the vial, and the antibody-coated nanoparticles were dissolved in 10 mL of PBS solution (0.1 M) and stored at 4°C

2.5 Quantification of OTC in samples

To 50 μL of 0.11 mg/mL anti-OTC antibody-conjugated fluorescent magnetic/silica nanoparticles, 50 μL serially diluted OTC samples at concentrations ranging from 5.5 × 10−3 to 8.25 × 10−11 (M) were added to an Eppendorf tube and agitated for 20 min (speed: 250 rpm/min) before being kept under magnetic separators to remove unbound OTC. The fluorescence quenching of capture probe fluorescent nanoparticles during interaction with OTC was studied (excitation (λ ex) 455 nm; emission (λ em) 600 nm). To create the sandwich complex, the anti-OTC-conjugated fluorescent magnetic silica/OTC complex was blocked with 10 mL of 2% BSA solution and magnetically separated for 30 min. Secondary fluorescent hybrid silica nanoparticles containing anti-OTC antibodies (50 µL at 0.12 mg/mL) were added and stirred for 20 min. Finally, the sandwich complex was magnetically separated and washed with PBS buffer. The fluorescence of anti-OTC-coupled rhodamine 6G and silica particles was measured (525 nm excitation; 550 nm emission), and the calibration curve was plotted as OTC concentration versus fluorescence intensity.

2.6 Comparative analysis: ELISA vs developed method

A reference ELISA was used to compare the generated assay. About 100 µL of anti-OTC antibodies (capture antibody diluted 1:5,000 in a carbonate buffer to a final concentration of 1 µg/mL) were coated in microplate wells. After covering the plates with sticky sealing tape, they were kept at 4°C for the whole night in a plastic bag containing wet paper towels. The following day, plates were gently agitated for 5 min while being washed three times with 300 µL of Wash buffer per well in an orbital shaker. By dumping into a sink and blotting with paper towels, the buffer was eliminated. After adding 300 µL of blocking buffer to each well, the plates were capped and left to stand at room temperature for 2 h while being stirred. In order to add them to the plate, the normal OTC samples were created. For the test, working dilutions of 8.20 × 10−11–1.10 × 10−2 (M) OTC were utilized. After the blocking stage, 100 µL of each standard and sample were added to the plate in triplicate after the plates had been cleaned three times using the Wash buffer as previously mentioned. The only solution used as a blank was the Blocking buffer. The plates were shaken during the 2-h RT incubation period. Following that, the plates were poured into a sink and cleaned three times with Wash buffer. Following the emptying and washing of the above solution, 100 µL of anti-OTC–HRP (HRP = horseradish peroxidase) was added to each well. (The mixture was diluted 1:100,000 in PBST-BSA to a final concentration of 5 ng/mL.) Plates were sealed, shielded from light, and incubated with agitation at room temperature for 1 h. Each well was added with 100 µL of 3,3ʹ,5,5ʹ-tetramethylbenzidine (TMB) substrate following the removal of the anti-OTC–HRP solution and washing. After 5 min, the reaction was halted by adding 100 µL of TMB Stop solution to each well and letting it equilibrate for an additional 5 min. After wiping the microplate’s underside with 70% ethanol, the plate was read using a microplate reader at 450 nm, and the wavelength was corrected at 550 nm right away.

3 Results and discussion

Dual detection techniques were developed to identify OTC residue in milk samples. Fluorescent magnetic silica and fluorescent hybrid silica were synthesized for the test. Two fluorescent dyes, rhodamine 6G and [Ru(bpy)3]2+, were chosen for the dual detection test. PVP and TEOS 3D networks were developed for the hybrid fluorescent silica production based on the gelation of organic and inorganic components. PVP was easily dissolved in a water–ethanol solvent, promoting the ring opening of cyclic alkenyl monomers and polymerization between TEOS via a radical mechanism, and the binding processes were increased by heat. The water-soluble hydrogel was created, then the rhodamine 6G dye was added, and hybrid silica was formed by the sol–gel reaction, with ammonia acting as a catalyst. The developed approach yielded the most beneficial inorganic component of organic–inorganic hybrid materials with fluorescently readily changeable functional molecules, as well as a functional group for silane molecule binding. Figure 2 shows the characterizations of the particles evaluated using SEM (Figure 2a), particle size analysis (Figure 2b), FTIR spectroscopy (Figure 2c), and fluorescence emission spectroscopy (Figure 2d). DLS and SEM examinations confirmed that the hybrid fluorescent silica nanoparticles were 75−85 nm in size. The emission spectra revealed that the particles had been properly coated with rhodamine 6G dye. FTIR spectra of hybrid shell-coated rhodamine 6G nanoparticles showed characteristic bands at 3429.06 cm–1 (OH), 1653.69 cm–1 (PVP), 1465.23 cm–1 (Rhod), and 1082 cm–1 (Si–O–Si). For OTC capture, fluorescent magnetic/silica nanoparticles were produced. Using a co-precipitation method, oleic acid-coated magnetic nanoparticles were created. Their surfaces were then further changed by coating the fluorescent dye with a silica layer created using TEOS. The silica layer coating changed the polarity of the magnetic nanoparticles, and the layer made the particles hydrophilic and ideal for surface alterations, as illustrated in Figure 3. The size of the synthesized particles was examined using SEM and DLS, and the results verified that the size was around 110–120 nm (Figure 3a and b, respectively). Using the hysteresis loop, the magnetic sensitivity of the produced nanoparticles was investigated. Saturation magnetization and remanent magnetization measurements were typically used to calculate the magnetic sensitivity of the nanoparticles. The produced nanoparticles amply demonstrated that their magnetism value was roughly 20.69 emu/g, making them ideal for use in magnetic capture experiments (Figure 3c), and the emission spectra are shown in Figure 3d. FTIR spectral analysis was carried out to corroborate the particle characterization (Figure 3e). The fluorescent magnetic core–shell nanoparticles exhibited bands at 3432.54 cm–1 (OH), 1443.91 cm–1 (ruthenium(ii) dye), 1088.88 cm–1 (Si–O–Si), and 586.75 cm–1 (Fe–O). The surface of the synthesized particles was further changed using APTES, which consists of three functional reactive ethoxy groups and one amine group. Through the processes of hydrolysis and condensation, the APTES molecules form amine groups and siloxane bonds (Si–O–Si) with the nanoparticles. The antibodies were bound to the surface of the nanoparticles using EDC–NHS chemistry. The primary amines in the nanoparticles react with NHS–EDC at physiological pH to generate Sulfo-NHS plus EDC (carbodiimide) crosslinking, which is notably more stable than the other intermediate. The antibodies were then conjugated. The antibody binding concentration on the surface of nanoparticles was evaluated using the 280 nm wavelength.

Figure 2 Shows the results of the physical characterization of the fluorescent hybrid silica nanoparticles obtained by (a) scanning electron microscopy (SEM), (b) particle size measurements using DLS, (c) FTIR surface characterization, and (d) emission spectra of the fluorescent hybrid silica nanoparticles (excitation wave length (λ) = 525 nm).
Figure 2

Shows the results of the physical characterization of the fluorescent hybrid silica nanoparticles obtained by (a) scanning electron microscopy (SEM), (b) particle size measurements using DLS, (c) FTIR surface characterization, and (d) emission spectra of the fluorescent hybrid silica nanoparticles (excitation wave length (λ) = 525 nm).

Figure 3 Characterization results of fluorescent magnetic and silica nanoparticles results of measuring particle size using DLS and scanning electron microscopy (SEM) are shown in (a) and (b). (d) Emission spectra of fluorescent magnetic/silica nanoparticles (excitation wave length (λ) = 455 nm), and (c) findings of an FTIR surface characterisation research. (e) Fluorescent magnetic/silica nanoparticle magnetization curves before and after coating with silica and over-the-counter antibodies.
Figure 3

Characterization results of fluorescent magnetic and silica nanoparticles results of measuring particle size using DLS and scanning electron microscopy (SEM) are shown in (a) and (b). (d) Emission spectra of fluorescent magnetic/silica nanoparticles (excitation wave length (λ) = 455 nm), and (c) findings of an FTIR surface characterisation research. (e) Fluorescent magnetic/silica nanoparticle magnetization curves before and after coating with silica and over-the-counter antibodies.

To mobilize the antibodies, 1.11 mg/mL anti-OTC antibodies were mixed with the nanoparticles and swirled overnight at 4°C. The unbound antibodies were then recovered by centrifugation and surface-blocked with 2% BSA solution. According to the calibration curve, the surface of the nanoparticles contains 6.25 × 10−6 mol/mg anti-OTC antibodies in fluorescent magnetic silica nanoparticles. The surface of the fluorescent hybrid silica nanoparticles contains 7.5 × 10−6 mol/mg OTC antibodies. Initially, assay parameters such as nanoparticle quantity and incubation duration were optimized for OTC detection for the assay validation. Each experiment required a maximum of 1.2 mg of anti-OTC antibody-conjugated fluorescent hybrid silica particles and 1 mg of anti-OTC antibody-conjugated fluorescent magnetic/silica nanoparticles. The anti-OTC antibody-conjugated fluorescent magnetic/silica nanoparticles required a maximum of 10 min for OTC enrichment from the samples, while the anti-OTC antibody-conjugated fluorescent hybrid silica nanoparticles required a maximum of 20 min for detection. The BSA blocking concentration was also optimized, the findings indicating that 2% BSA blocking solution is sufficient for nanoparticle surface blocking while minimizing background interferences. The OTC stock standard was created between 8.20 × 10−11 and 1.10 × 10−2 (M) to investigate the performance of the devised test. The anti-OTC antibody-conjugated fluorescent magnetic silica particles were exposed to varied concentrations of OTC for the experiment. Anti-OTC-conjugated fluorescent hybrid silica nanoparticles were used as the detection probe in the sandwich evaluation, and the results are displayed in Figure 4. The assay’s linearity ranged from 1.34 × 10−6 to 2.10 × 10−8 (M) (R 2 = 0.9954). The minimum detectable concentration was 4.76 ng/mL, and the cumulative values (CVs) between 1.1 ng/mL and 476 µg/mL obtained for the inter-/intra-assay were 5.05, 6.05, 5.50, 6.34, 5.11, and 5.21%. The presented approach was extremely reproducible, as seen by the results, which indicated that the CVs were less than 10%. The anti-OTC antibody-conjugated fluorescent magnetic/silica nanoparticles were used as a capture probe. The antibody-conjugated nanoparticles formed the strongest non-covalent binding complex with OTC, and the complex simultaneously transferred the electrons and quenched the fluorescence. The quenching kinetics was investigated using the Stern–Volmer equation: F 0/F = 1 + K SV[Q], where F and F 0 stand for the fluorescence intensities measured in the presence and absence of a quencher, respectively, K SV is the Stern–Volmer quenching constant, and [Q] is the concentration of the quencher. Based on this, the calculated K SV value is shown in Table 1. Amoxicillin was used to conduct a cross-reactivity study; the elevated concentration of amoxicillin was added to 78 µg/mL OTC and measured (Figure 4).

Figure 4 Results of the assay that was run (a) Sandwich testing results of OTC detected with fluorescent hybrid silica nano particles coated with anti-OTC antibodies – each data point represents the mean ± standard deviation of all three sub assays at each tested concentration (b). The anti-OTC conjugated fluorescent magnetic/silica nano particles’ quenching effects on fluorescence.
Figure 4

Results of the assay that was run (a) Sandwich testing results of OTC detected with fluorescent hybrid silica nano particles coated with anti-OTC antibodies – each data point represents the mean ± standard deviation of all three sub assays at each tested concentration (b). The anti-OTC conjugated fluorescent magnetic/silica nano particles’ quenching effects on fluorescence.

Table 1

Stern–Volmer quenching kinetics

OTP concentration [Q] (M) Average fluorescence F 0/F F 0/F − 1 K sv = F 0/F − 1/[Q]
Blank 248.92
2.10 × 10−8 225.5 1.10 0.1 4.76 × 106
4.20 × 10−8 215.2 1.16 0.16 3.73 × 106
8.93 × 10−8 200.15 1.24 0.24 5.80 × 106
1.68 × 10−7 170 1.46 0.46 2.76 × 106
3.36 × 10−7 148.92 1.67 0.67 2.00 × 106
6.61 × 10−7 125.86 1.98 0.98 1.48 × 106
1.34 × 10−6 100.13 2.49 1.49 1.04 × 106
2.69 × 10−6 88.68 2.81 1.81 6.72 × 105
5.37 × 10−6 70.24 3.54 2.54 4.74 × 105
1.05 × 10−5 49.63 5.02 4.02 3.82 × 105
6.38 × 10−4 40.15 6.20 5.20 4.95 × 105

The results showed that the greatest changes were only 2.48%, and the assay results are displayed in Table 2. These findings revealed that the cross-reactivity in this proposed approach was negligible. For the real-sample analysis, the milk samples were spiked with different concentrations of OTC, and the assay was done; the findings showed that 100% of the spiked drug was recovered from the milk sample, as shown in Table 3. A comparative study was carried out to investigate their sensitivity and selectivity with commonly used ELISA kits. The sample analysis done in the devised approach is strongly associated with the ELISA results, as shown in Figure 5. The results revealed that the suggested approach is extremely suitable for detecting drug residues in milk samples, as well as offering miniaturization, integration, and multiplicity.

Table 2

Cross-reactivity study results (OTC concentration of 78 μg/mL was fixed as constant and different concentrations of amoxicillin were added)

Parameters Concentration of OTC (78 μg/mL) spiked in milk samples and then mixed with different concentrations of amoxicillin (mg/mL) (n = 3)
305 mg/mL 156 mg/mL 78 mg/mL
Fluorescence intensity (a.u.) 20.581 ± 1.5 21.025 ± 1.5 21.890 ± 1.2
Percentage of interference 5.87 3.85 0
Table 3

Real-sample analysis results

Parameters Concentration of OTC spiked in milk (n = 3)
2.5 mg/mL 312 μg/mL 78 μg/mL
Fluorescence intensity (a.u.) 34.402 ± 1.1 29.958 ± 0.86 21.806 ± 1.2
Percentage of recovery (%) 101 100.35 104.6
Percentage of interference 1.31 0 4.6
Figure 5 Correlation study findings of OTC-spiked milk samples using the suggested and commercial ELISA methods.
Figure 5

Correlation study findings of OTC-spiked milk samples using the suggested and commercial ELISA methods.

The proposed method is highly equivalent to existing OTC detection methods that have previously been reported such as colorimetric biosensing using the Fe3O4 nanoparticle assay (45 nM) [35], magnetic nanobead-based competitive enzyme-linked aptamer assay (14.7 ng/mL) [36], carbon dots and Fe3O4 MNP-based assay (9.5 nM) [37], magnetic nanotube-based assay (8.1 nM) [38], magnetic nanoparticle-based HPLC–UV detection system (50 μg/L) [39], magnetic nanoparticle-based immunofluorescence assay (50 ng/mL) [40], luminescent silicon-based nanoparticle-based assay (18 nM) [41], and lateral flow assay (5 ppb) [42], among which the low detection limit was exhibited by the colorimetric aptasensor (6.92 nM) [43] and HPLC-photodiode array detector (0.062 µg/g) [44]. Fluorescence assays utilizing single-emission probes are widely employed in diverse biochemical techniques. The primary determinant of assay efficiency in these approaches is the fluorescence intensity signal. However, the fluorescence intensity of the detection probe can be readily impacted by an array of external factors, such as the probe concentration, excitation light intensity, detection environment, and equipment efficiency. When compared to the single-mode detection method in this instance, the dual-mode detection method can eliminate the fluctuations in fluorescence intensity brought about by the previously mentioned factors because the capture probe used in the dual detection method is fluorescent, enabling self-calibration (in situations where knowledge of absolute concentrations is necessary) while retaining favourable selectivity, high sensitivity, and results that are visible to the naked eye. The capture probe conjugated fluorescent magnetic nanoparticles after bound with the specific analyte, and the fluorescence signal was quenched by the OTC, and it varied based on the concentration of the analyte and its rate was studied using the Stern–Volmer quenching kinetics equation. This technique has the advantage that the signal ratio is independent of the donor and acceptor absolute concentrations, in contrast to the approach that uses a quencher as the acceptor (capture probe). The capture probe fluorescence changes help to understand specific analyte binding, but in the sensors and other commonly used assays it needs specific approaches. This assay took less assay time than ELISA for OTC detection when compared to the standard ELISA approach. On the other hand, the newly developed test only needed a short incubation time (90 min) to achieve these limits, whereas the ELISA required extensive incubation times (∼6 h). With no enzymatic step, a stable signal, an excellent signal-to-noise ratio, a flexible platform, and a short incubation time, this new immunoassay is incredibly sensitive. With no extra tools or expertise needed, the test format is simple. For high-throughput screening, it works better, much like ELISA.

4 Conclusions

A dual fluorescent nanoparticle system was created in this study to identify OTC residues in milk samples utilizing particular antibody-conjugated fluorescent nanoparticles. According to the results, the approach was very sensitive and selective for OTC in milk samples. The nanoparticles provided a high volume surface for antibody mobilization and were useful for OTC enrichment. The dual fluorescent probe allowed for simultaneous measurement of the capture and detection probes, and their fluorescence variations were very detectable after OTC binding. The external magnetic separation cut the assay time in half, and the secondary nanofluorescent probe was ideal for detecting OTC at low concentrations. As a result, the dual immunofluorescence approach detecting OTC residues in milk was effective, quick, and promising of parallel processes for additional antibiotic detection in milk samples.

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Acknowledgements

The Researchers Supporting Project number (RSP2024R114) of King Saud University in Riyadh, Saudi Arabia, is acknowledged with gratitude from the authors.

  1. Funding information: The Researchers Supporting Project number (RSP2024R114) of King Saud University in Riyadh, Saudi Arabia, is acknowledged with gratitude from the authors.

  2. Author contributions: Viswanathan Kaliyaperumal – methodology, software, validation, original draft preparation, review and editing supervision, visualization and project in charge. Oleyan Al-Otibi – project administration, report preparation, over all supervision and funding acquisition. Ruth Sophila John and Raedah Ibrahim Alharbi. formal analysis, data curation and investigation, Dhinakar Raj Gopal – Study director and review and editing the reports, All authors have read and agreed to the published version of the manuscript.

  3. Conflict of interest: The authors declare that none of the work reported in this study could have been influenced by any known competing financial interest or personal relationships.

  4. Ethical approval: The conducted research is not related to either human or animal use.

  5. Data availability statement: The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

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Received: 2024-04-01
Revised: 2024-06-10
Accepted: 2024-06-24
Published Online: 2024-07-17

© 2024 the author(s), published by De Gruyter

This work is licensed under the Creative Commons Attribution 4.0 International License.

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  6. Sonochemical synthesis of gold nanoparticles mediated by potato starch: Its performance in the treatment of esophageal cancer
  7. Computational study of ADME-Tox prediction of selected phytochemicals from Punica granatum peels
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https://doi.org/10.1515/chem-2024-0066
Keywords for this article
OTC detection; magnetic separator; fluorescent hybrid silica nanoparticles; fluorescent magnetic silica nanoparticles; sandwich assay
Creative Commons
BY 4.0

Articles in the same Issue

  1. Regular Articles
  2. Porous silicon nanostructures: Synthesis, characterization, and their antifungal activity
  3. Biochar from de-oiled Chlorella vulgaris and its adsorption on antibiotics
  4. Phytochemicals profiling, in vitro and in vivo antidiabetic activity, and in silico studies on Ajuga iva (L.) Schreb.: A comprehensive approach
  5. Synthesis, characterization, in silico and in vitro studies of novel glycoconjugates as potential antibacterial, antifungal, and antileishmanial agents
  6. Sonochemical synthesis of gold nanoparticles mediated by potato starch: Its performance in the treatment of esophageal cancer
  7. Computational study of ADME-Tox prediction of selected phytochemicals from Punica granatum peels
  8. Phytochemical analysis, in vitro antioxidant and antifungal activities of extracts and essential oil derived from Artemisia herba-alba Asso
  9. Two triazole-based coordination polymers: Synthesis and crystal structure characterization
  10. Phytochemical and physicochemical studies of different apple varieties grown in Morocco
  11. Synthesis of multi-template molecularly imprinted polymers (MT-MIPs) for isolating ethyl para-methoxycinnamate and ethyl cinnamate from Kaempferia galanga L., extract with methacrylic acid as functional monomer
  12. Nutraceutical potential of Mesembryanthemum forsskaolii Hochst. ex Bioss.: Insights into its nutritional composition, phytochemical contents, and antioxidant activity
  13. Evaluation of influence of Butea monosperma floral extract on inflammatory biomarkers
  14. Cannabis sativa L. essential oil: Chemical composition, anti-oxidant, anti-microbial properties, and acute toxicity: In vitro, in vivo, and in silico study
  15. The effect of gamma radiation on 5-hydroxymethylfurfural conversion in water and dimethyl sulfoxide
  16. Hollow mushroom nanomaterials for potentiometric sensing of Pb2+ ions in water via the intercalation of iodide ions into the polypyrrole matrix
  17. Determination of essential oil and chemical composition of St. John’s Wort
  18. Computational design and in vitro assay of lantadene-based novel inhibitors of NS3 protease of dengue virus
  19. Anti-parasitic activity and computational studies on a novel labdane diterpene from the roots of Vachellia nilotica
  20. Microbial dynamics and dehydrogenase activity in tomato (Lycopersicon esculentum Mill.) rhizospheres: Impacts on growth and soil health across different soil types
  21. Correlation between in vitro anti-urease activity and in silico molecular modeling approach of novel imidazopyridine–oxadiazole hybrids derivatives
  22. Spatial mapping of indoor air quality in a light metro system using the geographic information system method
  23. Iron indices and hemogram in renal anemia and the improvement with Tribulus terrestris green-formulated silver nanoparticles applied on rat model
  24. Integrated track of nano-informatics coupling with the enrichment concept in developing a novel nanoparticle targeting ERK protein in Naegleria fowleri
  25. Cytotoxic and phytochemical screening of Solanum lycopersicum–Daucus carota hydro-ethanolic extract and in silico evaluation of its lycopene content as anticancer agent
  26. Protective activities of silver nanoparticles containing Panax japonicus on apoptotic, inflammatory, and oxidative alterations in isoproterenol-induced cardiotoxicity
  27. pH-based colorimetric detection of monofunctional aldehydes in liquid and gas phases
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  29. Metformin inhibits knee osteoarthritis induced by type 2 diabetes mellitus in rats: S100A8/9 and S100A12 as players and therapeutic targets
  30. Effect of silver nanoparticles formulated by Silybum marianum on menopausal urinary incontinence in ovariectomized rats
  31. Synthesis of new analogs of N-substituted(benzoylamino)-1,2,3,6-tetrahydropyridines
  32. Response of yield and quality of Japonica rice to different gradients of moisture deficit at grain-filling stage in cold regions
  33. Preparation of an inclusion complex of nickel-based β-cyclodextrin: Characterization and accelerating the osteoarthritis articular cartilage repair
  34. Empagliflozin-loaded nanomicelles responsive to reactive oxygen species for renal ischemia/reperfusion injury protection
  35. Preparation and pharmacodynamic evaluation of sodium aescinate solid lipid nanoparticles
  36. Assessment of potentially toxic elements and health risks of agricultural soil in Southwest Riyadh, Saudi Arabia
  37. Theoretical investigation of hydrogen-rich fuel production through ammonia decomposition
  38. Biosynthesis and screening of cobalt nanoparticles using citrus species for antimicrobial activity
  39. Investigating the interplay of genetic variations, MCP-1 polymorphism, and docking with phytochemical inhibitors for combatting dengue virus pathogenicity through in silico analysis
  40. Ultrasound induced biosynthesis of silver nanoparticles embedded into chitosan polymers: Investigation of its anti-cutaneous squamous cell carcinoma effects
  41. Copper oxide nanoparticles-mediated Heliotropium bacciferum leaf extract: Antifungal activity and molecular docking assays against strawberry pathogens
  42. Sprouted wheat flour for improving physical, chemical, rheological, microbial load, and quality properties of fino bread
  43. Comparative toxicity assessment of fisetin-aided artificial intelligence-assisted drug design targeting epibulbar dermoid through phytochemicals
  44. Acute toxicity and anti-inflammatory activity of bis-thiourea derivatives
  45. Anti-diabetic activity-guided isolation of α-amylase and α-glucosidase inhibitory terpenes from Capsella bursa-pastoris Linn.
  46. GC–MS analysis of Lactobacillus plantarum YW11 metabolites and its computational analysis on familial pulmonary fibrosis hub genes
  47. Green formulation of copper nanoparticles by Pistacia khinjuk leaf aqueous extract: Introducing a novel chemotherapeutic drug for the treatment of prostate cancer
  48. Improved photocatalytic properties of WO3 nanoparticles for Malachite green dye degradation under visible light irradiation: An effect of La doping
  49. One-pot synthesis of a network of Mn2O3–MnO2–poly(m-methylaniline) composite nanorods on a polypyrrole film presents a promising and efficient optoelectronic and solar cell device
  50. Groundwater quality and health risk assessment of nitrate and fluoride in Al Qaseem area, Saudi Arabia
  51. A comparative study of the antifungal efficacy and phytochemical composition of date palm leaflet extracts
  52. Processing of alcohol pomelo beverage (Citrus grandis (L.) Osbeck) using saccharomyces yeast: Optimization, physicochemical quality, and sensory characteristics
  53. Specialized compounds of four Cameroonian spices: Isolation, characterization, and in silico evaluation as prospective SARS-CoV-2 inhibitors
  54. Identification of a novel drug target in Porphyromonas gingivalis by a computational genome analysis approach
  55. Physico-chemical properties and durability of a fly-ash-based geopolymer
  56. FMS-like tyrosine kinase 3 inhibitory potentials of some phytochemicals from anti-leukemic plants using computational chemical methodologies
  57. Wild Thymus zygis L. ssp. gracilis and Eucalyptus camaldulensis Dehnh.: Chemical composition, antioxidant and antibacterial activities of essential oils
  58. 3D-QSAR, molecular docking, ADMET, simulation dynamic, and retrosynthesis studies on new styrylquinolines derivatives against breast cancer
  59. Deciphering the influenza neuraminidase inhibitory potential of naturally occurring biflavonoids: An in silico approach
  60. Determination of heavy elements in agricultural regions, Saudi Arabia
  61. Synthesis and characterization of antioxidant-enriched Moringa oil-based edible oleogel
  62. Ameliorative effects of thistle and thyme honeys on cyclophosphamide-induced toxicity in mice
  63. Study of phytochemical compound and antipyretic activity of Chenopodium ambrosioides L. fractions
  64. Investigating the adsorption mechanism of zinc chloride-modified porous carbon for sulfadiazine removal from water
  65. Performance repair of building materials using alumina and silica composite nanomaterials with electrodynamic properties
  66. Effects of nanoparticles on the activity and resistance genes of anaerobic digestion enzymes in livestock and poultry manure containing the antibiotic tetracycline
  67. Effect of copper nanoparticles green-synthesized using Ocimum basilicum against Pseudomonas aeruginosa in mice lung infection model
  68. Cardioprotective effects of nanoparticles green formulated by Spinacia oleracea extract on isoproterenol-induced myocardial infarction in mice by the determination of PPAR-γ/NF-κB pathway
  69. Anti-OTC antibody-conjugated fluorescent magnetic/silica and fluorescent hybrid silica nanoparticles for oxytetracycline detection
  70. Curcumin conjugated zinc nanoparticles for the treatment of myocardial infarction
  71. Identification and in silico screening of natural phloroglucinols as potential PI3Kα inhibitors: A computational approach for drug discovery
  72. Exploring the phytochemical profile and antioxidant evaluation: Molecular docking and ADMET analysis of main compounds from three Solanum species in Saudi Arabia
  73. Unveiling the molecular composition and biological properties of essential oil derived from the leaves of wild Mentha aquatica L.: A comprehensive in vitro and in silico exploration
  74. Analysis of bioactive compounds present in Boerhavia elegans seeds by GC-MS
  75. Homology modeling and molecular docking study of corticotrophin-releasing hormone: An approach to treat stress-related diseases
  76. LncRNA MIR17HG alleviates heart failure via targeting MIR17HG/miR-153-3p/SIRT1 axis in in vitro model
  77. Development and validation of a stability indicating UPLC-DAD method coupled with MS-TQD for ramipril and thymoquinone in bioactive SNEDDS with in silico toxicity analysis of ramipril degradation products
  78. Biosynthesis of Ag/Cu nanocomposite mediated by Curcuma longa: Evaluation of its antibacterial properties against oral pathogens
  79. Development of AMBER-compliant transferable force field parameters for polytetrafluoroethylene
  80. Treatment of gestational diabetes by Acroptilon repens leaf aqueous extract green-formulated iron nanoparticles in rats
  81. Development and characterization of new ecological adsorbents based on cardoon wastes: Application to brilliant green adsorption
  82. A fast, sensitive, greener, and stability-indicating HPLC method for the standardization and quantitative determination of chlorhexidine acetate in commercial products
  83. Assessment of Se, As, Cd, Cr, Hg, and Pb content status in Ankang tea plantations of China
  84. Effect of transition metal chloride (ZnCl2) on low-temperature pyrolysis of high ash bituminous coal
  85. Evaluating polyphenol and ascorbic acid contents, tannin removal ability, and physical properties during hydrolysis and convective hot-air drying of cashew apple powder
  86. Development and characterization of functional low-fat frozen dairy dessert enhanced with dried lemongrass powder
  87. Scrutinizing the effect of additive and synergistic antibiotics against carbapenem-resistant Pseudomonas aeruginosa
  88. Preparation, characterization, and determination of the therapeutic effects of copper nanoparticles green-formulated by Pistacia atlantica in diabetes-induced cardiac dysfunction in rat
  89. Antioxidant and antidiabetic potentials of methoxy-substituted Schiff bases using in vitro, in vivo, and molecular simulation approaches
  90. Anti-melanoma cancer activity and chemical profile of the essential oil of Seseli yunnanense Franch
  91. Molecular docking analysis of subtilisin-like alkaline serine protease (SLASP) and laccase with natural biopolymers
  92. Overcoming methicillin resistance by methicillin-resistant Staphylococcus aureus: Computational evaluation of napthyridine and oxadiazoles compounds for potential dual inhibition of PBP-2a and FemA proteins
  93. Exploring novel antitubercular agents: Innovative design of 2,3-diaryl-quinoxalines targeting DprE1 for effective tuberculosis treatment
  94. Drimia maritima flowers as a source of biologically potent components: Optimization of bioactive compound extractions, isolation, UPLC–ESI–MS/MS, and pharmacological properties
  95. Estimating molecular properties, drug-likeness, cardiotoxic risk, liability profile, and molecular docking study to characterize binding process of key phyto-compounds against serotonin 5-HT2A receptor
  96. Fabrication of β-cyclodextrin-based microgels for enhancing solubility of Terbinafine: An in-vitro and in-vivo toxicological evaluation
  97. Phyto-mediated synthesis of ZnO nanoparticles and their sunlight-driven photocatalytic degradation of cationic and anionic dyes
  98. Monosodium glutamate induces hypothalamic–pituitary–adrenal axis hyperactivation, glucocorticoid receptors down-regulation, and systemic inflammatory response in young male rats: Impact on miR-155 and miR-218
  99. Quality control analyses of selected honey samples from Serbia based on their mineral and flavonoid profiles, and the invertase activity
  100. Eco-friendly synthesis of silver nanoparticles using Phyllanthus niruri leaf extract: Assessment of antimicrobial activity, effectiveness on tropical neglected mosquito vector control, and biocompatibility using a fibroblast cell line model
  101. Green synthesis of silver nanoparticles containing Cichorium intybus to treat the sepsis-induced DNA damage in the liver of Wistar albino rats
  102. Quality changes of durian pulp (Durio ziberhinus Murr.) in cold storage
  103. Study on recrystallization process of nitroguanidine by directly adding cold water to control temperature
  104. Determination of heavy metals and health risk assessment in drinking water in Bukayriyah City, Saudi Arabia
  105. Larvicidal properties of essential oils of three Artemisia species against the chemically insecticide-resistant Nile fever vector Culex pipiens (L.) (Diptera: Culicidae): In vitro and in silico studies
  106. Design, synthesis, characterization, and theoretical calculations, along with in silico and in vitro antimicrobial proprieties of new isoxazole-amide conjugates
  107. The impact of drying and extraction methods on total lipid, fatty acid profile, and cytotoxicity of Tenebrio molitor larvae
  108. A zinc oxide–tin oxide–nerolidol hybrid nanomaterial: Efficacy against esophageal squamous cell carcinoma
  109. Research on technological process for production of muskmelon juice (Cucumis melo L.)
  110. Physicochemical components, antioxidant activity, and predictive models for quality of soursop tea (Annona muricata L.) during heat pump drying
  111. Characterization and application of Fe1−xCoxFe2O4 nanoparticles in Direct Red 79 adsorption
  112. Torilis arvensis ethanolic extract: Phytochemical analysis, antifungal efficacy, and cytotoxicity properties
  113. Magnetite–poly-1H pyrrole dendritic nanocomposite seeded on poly-1H pyrrole: A promising photocathode for green hydrogen generation from sanitation water without using external sacrificing agent
  114. HPLC and GC–MS analyses of phytochemical compounds in Haloxylon salicornicum extract: Antibacterial and antifungal activity assessment of phytopathogens
  115. Efficient and stable to coking catalysts of ethanol steam reforming comprised of Ni + Ru loaded on MgAl2O4 + LnFe0.7Ni0.3O3 (Ln = La, Pr) nanocomposites prepared via cost-effective procedure with Pluronic P123 copolymer
  116. Nitrogen and boron co-doped carbon dots probe for selectively detecting Hg2+ in water samples and the detection mechanism
  117. Heavy metals in road dust from typical old industrial areas of Wuhan: Seasonal distribution and bioaccessibility-based health risk assessment
  118. Phytochemical profiling and bioactivity evaluation of CBD- and THC-enriched Cannabis sativa extracts: In vitro and in silico investigation of antioxidant and anti-inflammatory effects
  119. Investigating dye adsorption: The role of surface-modified montmorillonite nanoclay in kinetics, isotherms, and thermodynamics
  120. Antimicrobial activity, induction of ROS generation in HepG2 liver cancer cells, and chemical composition of Pterospermum heterophyllum
  121. Study on the performance of nanoparticle-modified PVDF membrane in delaying membrane aging
  122. Impact of cholesterol in encapsulated vitamin E acetate within cocoliposomes
  123. Review Articles
  124. Structural aspects of Pt(η3-X1N1X2)(PL) (X1,2 = O, C, or Se) and Pt(η3-N1N2X1)(PL) (X1 = C, S, or Se) derivatives
  125. Biosurfactants in biocorrosion and corrosion mitigation of metals: An overview
  126. Stimulus-responsive MOF–hydrogel composites: Classification, preparation, characterization, and their advancement in medical treatments
  127. Electrochemical dissolution of titanium under alternating current polarization to obtain its dioxide
  128. Special Issue on Recent Trends in Green Chemistry
  129. Phytochemical screening and antioxidant activity of Vitex agnus-castus L.
  130. Phytochemical study, antioxidant activity, and dermoprotective activity of Chenopodium ambrosioides (L.)
  131. Exploitation of mangliculous marine fungi, Amarenographium solium, for the green synthesis of silver nanoparticles and their activity against multiple drug-resistant bacteria
  132. Study of the phytotoxicity of margines on Pistia stratiotes L.
  133. Special Issue on Advanced Nanomaterials for Energy, Environmental and Biological Applications - Part III
  134. Impact of biogenic zinc oxide nanoparticles on growth, development, and antioxidant system of high protein content crop (Lablab purpureus L.) sweet
  135. Green synthesis, characterization, and application of iron and molybdenum nanoparticles and their composites for enhancing the growth of Solanum lycopersicum
  136. Green synthesis of silver nanoparticles from Olea europaea L. extracted polysaccharides, characterization, and its assessment as an antimicrobial agent against multiple pathogenic microbes
  137. Photocatalytic treatment of organic dyes using metal oxides and nanocomposites: A quantitative study
  138. Antifungal, antioxidant, and photocatalytic activities of greenly synthesized iron oxide nanoparticles
  139. Special Issue on Phytochemical and Pharmacological Scrutinization of Medicinal Plants
  140. Hepatoprotective effects of safranal on acetaminophen-induced hepatotoxicity in rats
  141. Chemical composition and biological properties of Thymus capitatus plants from Algerian high plains: A comparative and analytical study
  142. Chemical composition and bioactivities of the methanol root extracts of Saussurea costus
  143. In vivo protective effects of vitamin C against cyto-genotoxicity induced by Dysphania ambrosioides aqueous extract
  144. Insights about the deleterious impact of a carbamate pesticide on some metabolic immune and antioxidant functions and a focus on the protective ability of a Saharan shrub and its anti-edematous property
  145. A comprehensive review uncovering the anticancerous potential of genkwanin (plant-derived compound) in several human carcinomas
  146. A study to investigate the anticancer potential of carvacrol via targeting Notch signaling in breast cancer
  147. Assessment of anti-diabetic properties of Ziziphus oenopolia (L.) wild edible fruit extract: In vitro and in silico investigations through molecular docking analysis
  148. Optimization of polyphenol extraction, phenolic profile by LC-ESI-MS/MS, antioxidant, anti-enzymatic, and cytotoxic activities of Physalis acutifolia
  149. Phytochemical screening, antioxidant properties, and photo-protective activities of Salvia balansae de Noé ex Coss
  150. Antihyperglycemic, antiglycation, anti-hypercholesteremic, and toxicity evaluation with gas chromatography mass spectrometry profiling for Aloe armatissima leaves
  151. Phyto-fabrication and characterization of gold nanoparticles by using Timur (Zanthoxylum armatum DC) and their effect on wound healing
  152. Does Erodium trifolium (Cav.) Guitt exhibit medicinal properties? Response elements from phytochemical profiling, enzyme-inhibiting, and antioxidant and antimicrobial activities
  153. Integrative in silico evaluation of the antiviral potential of terpenoids and its metal complexes derived from Homalomena aromatica based on main protease of SARS-CoV-2
  154. 6-Methoxyflavone improves anxiety, depression, and memory by increasing monoamines in mice brain: HPLC analysis and in silico studies
  155. Simultaneous extraction and quantification of hydrophilic and lipophilic antioxidants in Solanum lycopersicum L. varieties marketed in Saudi Arabia
  156. Biological evaluation of CH3OH and C2H5OH of Berberis vulgaris for in vivo antileishmanial potential against Leishmania tropica in murine models
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