Micromeria biflora mediated gold and silver nanoparticles for colourimetric detection of antibiotics and dyes degradation

2.1 Samples collection

Enough aerial parts of M. biflora plant was collected from Swat, during the flowering season (June to August 2021) from Swat valley, KPK province, Pakistan. The plant was taxonomically identified at the Botany Department, Peshawar University, Pakistan. The plant was washed to clean from any dust or other contaminants. The samples were then air dried in shade, powdered, kept in dry and cleaned, plastic bags, properly labelled, and stored in refrigerator until analysis. Approval from ethical committee, Kohat University of Science & Technology was obtained for working with biological samples including blood plasma and urine.

2.2 Aqueous extract preparation

For extraction, an established procedure from literature was followed (Jamila et al., 2020). Briefly, deionized water (500 mL) as extraction solvent was used to soak 25 g aerial parts of M. biflora powder. The mixture was stirred well at 500 rpm for 2 h and 40 °C temperature. The extract so obtained was filtered, and used to synthesize MBAuNPs and MBAgNPs.

2.3 Chemicals, reagents, and instrumentation

Gold(III) chloride salt as trihydrate (HAuCl₄·3H₂O), nitrate salt of silver (AgNO₃), sodium borohydride (NaBH₄), dyes [methylene blue (MB), Congo red (CR), Rhodamine (RdB), methyl orange (MO), and ortho- and para-nitrophenol (ONP, PNP)], antibiotic standards [levofloxacin (LFX), amoxicillin (AMX), and azithromycin were all purchased from Merck (Darmstadt, Germany) and Sigma-Aldrich (Steinheim, Germany). Aqueous plant extract and all reagent solutions were prepared with ultrapure deionized water obtained from Milli-Q apparatus (Millipore, MA, USA).

Instruments used in this research study included UV–1800 spectrophotometer (Shimadzu, Japan) for absorption spectra (200 to 800 nm) of the reaction mixture, FT-IR spectrometer (Bruker) for functional groups identification (400–4000 cm⁻¹), field emission-scanning electron microscope (FE-SEM, S-4800, Hitachi, Japan), transmission electron microscope (Phillips CM12, Eindhoven, Netherlands) and atomic force microscopy (AFM) for size and morphology assessment.

2.4 Synthesis and characterization of MBAuNPs and MBAgNPs

The MBAuNPs and MBAgNPs were synthesised using M. biflora aqueous extract under different conditions involving ratios (1:5 to 1:15) of corresponding salt solutions (HAuCl₄·4H₂O and AgNO₃) and aqueous extract, sunlight, incubation at 25 °C in dark, stirring, heating, pH (1–12) and time intervals (0 to 180 min). Synthesis of corresponding NPs were preliminary checked for any visible colour change from yellow (extract) to brown (AgNPs) and maroon/deep purple (AuNPs) (Fig. 1), which were then confirmed by UV–Vis analysis. The synthesized NPs from the solutions were collected via centrifugation and washing with water. The product so obtained was kept in sealed vials until required for analysis. The synthesized NPs were characterized by FE-SEM, FT-IR, AFM, and TEM techniques for functional group identification (involved in NPs synthesis), and size and morphology analysis.

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Fig. 1

Colourimetric visible changes in the synthesis of MBAuNPs and MBAgNPs at sunlight.

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2.5 Dyes degradation by MBAuNPs and MBAgNPs

Dyes degradation was evaluated using synthesized NPs (where they act as catalysts) following reported literature (Jamila et al., 2020). Briefly, 0.1 mM, 2.5 mL each of dyes and nitrophenols were mixed with 0.1 mM, 0.5 mL of NaBH₄. In another experiment, 3 mg of NPs were mixed with 2.5 mL of solutions of dyes and nitrophenols and 0.5 mL NaBH₄. The Ultraviolet–Visible spectra (200–800 nm) were recorded at a time intervals of 0 to180 min. The catalytic efficiency of NPs and degradation of dyes and nitrophenols was noted by discoloration and decrease in SPR absorbance. This decrease was calculated as reduction/decolorization via equation (1) given below.

Where; A_t means absorbance of dye at “t” time and A₀ means absorbance of dye at 0 min.

2.6 Colourimetric antibiotics detection by MBAuNPs and MBAgNPs

In antibiotic detection in real samples, a slightly modified method (Makropoulou et al., 2020) was used. Depending on the solubility, the working solutions were set by taking the selected antibiotics and dissolving either in water or ethanol. The UV spectra of antibiotics working solutions (2.5 mL), a mixture of antibiotics (2.5 mL) and NPs (3 mg), and a mixture of antibiotics working solutions, NPs (3 mg) and real samples (blood plasma, urine, tape water, 1.5 mL) were recorded at 0 to180 min. To evaluate the percent removal of the applied antibiotics, the following equation was used.

Where C and C₀ are the final and initial concentrations of the applied antibiotics, respectively.

3.1 Synthesis and characterization of MBAuNPs and MBAgNPs

M. biflora mediated NPs were synthesized using various ratios of extract and corresponding salt solutions under sunlight, stirring, incubation, and heating techniques. Applying these conditions to mixtures of 1:5, 1:10, and 1:15 ratios, for MBAuNPs, no visible colour change was noticed, and from UV–Vis study there was no surface plasmon resonance band seemed, which indicates that no NPs in the subject mixtures/ratios are produced at stirring, incubation, and heating as depicted from Fig. 2 (A-C). The prominent SPR peak appeared at λ_max 300–350 nm shown in Fig. 2 is attributed to aggregation of particles of sizes larger than 100 nm (Dzimitrowicz et al., 2019). Proceeding the synthesis in sunlight, MBAuNPs were produced at a slow rate in ratio of 1:5 with the more intense SPR at λ_max 300–350 nm than at λ_max 541 nm (Fig. 2D). However, in 1:10 and 1:15 ratios, significant MBAuNPs were produced as indicated by visible colourimetric change in color from light yellow to purple, and appearance of SPR band at 541 nm (Fig. 2E & F). An intense absorption peak at λ_max 541 nm was observed for MBAuNPs produced at the ratio of 1:15, indicating the presence of reasonable AuNPs.

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Fig. 2

Successive UV–Vis spectra of MBAuNPs using aqueous extract in ratios of (A) 1:5, (B) 1:10 and (C) 1:15 at stirring, heating, incubation, (D) 1:5 sunlight, (E) 1:10 sunlight, and (F) 1:15 sunlight.

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In MBAgNPs synthesis, the colorimetric change from yellow to brown at the ratios of 1:5, 1:6, and 1:7 keeping in sunlight play a crucial role in preparation of MBAgNPs, which exhibited SPR at λ_max 431 nm (Fig. 3A & B). A 1:6 ratio demonstrated an intense and sharp SPR band, which indicate the production of significant and well dispersed AgNPs. In a ratio of 1:7, a bathochromic/red shift from 431 to 579 nm and broadened SPR band was observed. Furthermore, due to boosted interactions between MB aqueous extract with excessive amounts of phytocompounds and silver ions, this aggregation ultimately resulted in the decrease of stable MBAgNPs (Lee et al., 2019; Rani et al., 2020). Proceeding with increased salt concentration to 1:10, 1:12, and 1:15, at sunlight, the AgNPs showed weak SPR band (Fig. 3C) and produced at a slow rate, which indicate its formation in small amount. Following the AgNPs synthesis with all the subject ratios at stirring, incubation, and heating, no SPR excitations at 420–450 nm were seen, which corresponded to no AgNPs production (Fig. 3D).

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Fig. 3

Successive UV–Vis spectra of MBAuNPs using aqueous extract in ratios of (A) 1:5 & 1:6 sunlight (B) 1:7 & 1:9 sunlight, (C) 1:10, 1:12 & 1:15 sunlight, (D) 1:5 to 1:15 at stirring, heating, and incubation.

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M. biflora is a rich source of chemical constituents/secondary metabolites such as phenolics, tannins, flavonoids, and coumarins (Chandra et al. 2013). The hypothesised mechanism for the NPs synthesis by the interaction of these secondary metabolites with metal ions include interaction of OH/COOH/NH groups, enol synthesis by liberating reactive hydrogen, and then oxidation of hydroxyl to carbonyl group through adsorption on the metal surface (Kumar et al., 2017). The metal ions M^+/++ thus are reduced to M⁰, and eventually NPs creation. According to Lee et al. (2019), nanomaterials synthesized via these bioactive components, which have hydroxyl groups as reducing agents follow the mechanism as shown below. $$A{u}^{+}+R-OH\to A{u}^0+R=O+H^{+}$$ $$R-OH+A{g}^{+}\to A{g}^0+H^{+}+R=O$$

Depending upon the extent and position of both hydroxyl and carbonyl functional groups in the plant constituents, the NPs synthesis and their biological activities are greatly affected. Regarding the synthesis of MBAuNPs, the interaction of Au⁺ with phenolics of M. biflora extract, the colorimetric colour change from yellow to purple indicate the reduction of Au⁺ to Au, and hence, MBAuNPs production. MBAuNPs synthesized under sunlight were significantly produced in 1:10 and 1:15 ratios, exhibiting surface plasmon resonance (SPR) band at 541 nm, as indicated by previous literature (Dzimitrowicz et al., 2019; Rani et al., 2020). In 1:15 ratio, MBAuNPs were produced in the highest extent. Hence, it is the optimal ratio displaying a sharp absorption peak at λ_max 541 nm. This indicates that the salt solution (HAuCl₄) is rapidly reduced by the chemical constituents of M. biflora to produce homogeneous nuclei, which results in tiny AuNPs rather than aggregates.

Concerning MBAgNPs in 1:6 ratio under sunlight, which display a sharp peak at λ_max 431 nm, shows that this ratio is the optimal condition for the synthesis of MBAgNPs. However, proceeding to higher ratios, a bathochromic/red shift from 431 to 579 nm and a broadened SPR band was observed, which indicates salt induced MBAgNPs aggregation. Also due to greater interactions between M. biflora aqueous extract with excessive amounts of reducing phytoconstituents and accessible silver ions, this aggregation finally resulted in the reduction of stable MBAgNPs which is consistent to the published literature (Lee et al., 2019). Similarly, Rani et al. (2020) has reported about Ostwald slow ripening process to enhance the production of AgNPs and result in size enlargement. Owing to several properties, many of the NPs have been used for the pollutants detection; dyes, antibiotics (Zhang et al., 2020; Aguilar-Pérez et al., 2021).

The functional groups of the phytochemicals (tannins, flavonoids, coumarins) present in M. biflora, which involve in the synthesis of M. biflora nanoparticles (MBNPs) were evaluated using FT-IR technique. The FT-IR spectra of plant extract (MBAE) are labelled by comparing with literature (Fig. 4A & B). The broad and intense absorption bands at 3500–3200 cm⁻¹ is the stretching vibration assigned to hydroxyl (–OH), and or amine (–NH) groups however the absorption frequencies at 1700–1540 cm⁻¹ and 1030–13010 cm⁻¹ are attributed to –C = O and –C–O stretching vibrations. It has been observed that the subject absorption bands in the spectra of MBAuNPs and MBAgNPs either reduced or disappeared, thus concluding the contribution of the different functionalities in MBNPs synthesis (Jamila et al., 2020).

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Fig. 4

FT-IR spectra of (A) MBAE and MBAuNPs, and (B) MBAE and MBAgNPs.

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The morphology and size of the subject MBNPs were analyzed using TEM, SEM and AFM techniques. These techniques showed that the size of MBAuNPs is 26.73 nm, and the surface shape represented nanorods as well as spherical shapes (Fig. 5A-C). Regarding MBAgNPs, the size by the subject techniques was found to be 53.81 nm with spherical shape (Fig. 5D-F).

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Fig. 5

Size and morphology by (A, D) SEM (B, E), TEM (C, F), AFM images of the synthesized MBAuNPs and MBAgNPs.

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3.2 Catalytic activity of MBAuNPs and MBAgNPs in dyes degradation

In the current study, CR, MB, MO, RdB, ONP, and PNP were reduced using NaBH₄ and MBNPs. The combined UV–Vis spectra of dyes reduced with NaBH₄, and MBAuNPs and AgNPs (Fig. 6) explained that dyes without MBNPs have a little variability in absorbance with no noticeable change in colour was observed, which indicates a slow rate of degradation of dyes reduction. However, MBAuNPs added dyes exhibited spectra where certain absorption peaks disappeared within 5 min after the addition of MBNPs. The results of the subject dyes (Fig. 6) showed that MB (cationic dye), which displays λ_max at 653 nm, 591 nm, and 290 and inefficiently reduced by NaBH₄, the absorbance at 653 and 290 nm weakened with a concurrent increase at 255 nm after the MBNPs addition (Fig. 6A). Similarly, the absorption maxima of CR (λ_max at 487 and 349 nm), RdB (550 nm), and MO (λ_max at 462 nm) decrease and shift to blue shift with the addition of MBNPs (Fig. 6B-D). The ortho-nitrophenol and para-nitrophenols (Fig. 6E & F) also have shown high degradation rate for ONP followed by PNP. MBAuNPs reduced CR to 80–85 %, MB 70–75 %, RdB 15–20 %, MO 65–80 %, ONP 80–85 %, and PNP 80–85 %. Regarding the % degradation of dyes by MBAgNPs (Fig. 7), the dyes were reduced as follows: CR (75–80 %), RdB (15–20 %), MB (60–70 %), MO (80–85 %), PNP (80–85 %), and ONP (83–85 %).

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Fig. 6

Successive UV–Vis absorption spectra for the catalytic potential of MBAuNPs in (A) CR, (B) MB, (C) MO, (D) RdB, (E) ONP, (F) PNP in dyes/nitrophenols degradation/reduction process.

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Fig. 7

Successive UV–Vis absorption spectra for the catalytic potential of MBAgNPs in (A) CR, (B) MB, (C) MO, (D) RdB, (E) ONP, (F) PNP in dyes/nitrophenols degradation/reduction process.

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Dyes are the organic synthetic pollutants, which release into the running water by various industries (Sadia et al., 2023). These dyes are polluting water and absorbed by soil thus affecting the environment, human health, and agriculture. Therefore, to control and cope this issue, recently, NPs of well enough catalytic efficiency have played key role as effective catalysts in the discolouration and elimination by decreasing the activation energy (Jamila, 2020; Khan et al., 2021; Yıldırım et al., 2022). The synthetic reducing agent (NaBH₄), which is usually used to reduce dyes due to the large redox potential differences in the dye and BH₄⁻ ions, inefficiently reduce dyes. Therefore, due to the adsorption of dyes on NPs, the electron transfer from NaBH₄ to dyes becomes easy and dye molecules decolorize. This is attributed to the high catalytic locations, lower the activation energy, and high volume to surface ratio of NPs. This diagram depicts MB-AuNPs' catalytic characteristics (Wang et al., 2021).

In this analysis, the λ_max of dyes are decreased, which indicates the degradation of dyes. For example, for CR (Figs. 6 and 7A), showing λ_max at 487 and 349 nm, the absorbance at the subject λ_max decreased and the solution become faint. In MB dye (Figs. 6 and 7B) having λ_max 653 and 290 nm, the absorbance decreased with an appearance of another absorption peak at 255 nm. This simultaneous decrease/increase in the λ_max indicates dyes degradation.

3.3 Antibiotics detection by MBAuNPs and MBAgNPs

The most used antibiotics selected for this study including LFX, AMX, and AZM in water, blood plasma and urine samples were detected using both type of NPs as a probe. From the results (Figs. 8 and 9), it was found that upon the addition of MBNPs to the antibiotics solution, the concentration (absorbance) of the mixtures decreases. AMX solution displays absorption peaks at λ_max 228 nm and 277 nm. Upon the addition of MBNPs, antibiotics are adsorbed on their surface and the λ_max of antibiotics decreases. Regarding the other antibiotics, it can also be seen that the same changes/decrease in λ_max occurs. In this study, it was found that compared to AZM, AMX and LFX capture by MBNPs was significant, and the removal % was in the range of 75–85 %. Proceeding the experiments with aqueous medium (tap water), blood plasma and urine samples, the recorded spectra obtained were irregular, and indicated that the λ_max of antibiotics decreases, which in turn shows decrease in concentrations of antibiotics.

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Fig. 8

Successive UV–Vis absorption spectra of MBAuNPs interaction with (A-C) LFX (D-F) AMX, and (G-I) AZM in in blood, urine and water.

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Fig. 9

Successive UV–Vis absorption spectra of MBAgNPs interaction with (A-C) LFX (D-F) AMX, and (G-I) AZM in in blood, urine and water.

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Antibiotics are also considered as the environmental pollutants from different pharmaceutical industries, various sewage treatment plants, hospitals, and others that can have an impact on microbial ecosystems. Therefore, it is crucial to assess and develop methods for their removal from the environment. Due to the outstanding affinity and optical absorbance of NPs with biomaterials, these have served as an effective technique to identify a variety of chemicals such as -agonists, dopamine, antibiotics, proteins, and DNA. Also, in current study, MBAuNPs and MBAgNPs were used for antibiotics detection in this study. The antibiotics (levofloxacin, amoxicillin, and azithromycin) have a high affinity for M. biflora phytoconstituents due to hydrogen-bonding interactions as previously reported by Lai et al. (2017). This results in cross-linking of AuNPs and AgNPs, leading to clear changes in absorbance features and colour. As given in Figs. 8 and 9, a colorimetric assay based on MBNPs as a probe is designed to detect antibiotics including LFX, AMX, and AZM in real samples including water, blood plasma and urine. From the results a decrease in the λ_max of antibiotics was found, which is attributed to the interaction via hydrogen bonding of the carboxyl and hydroxyl (COOH and OH) functional groups with antibiotics.

From experiments, it was found that the decrease in the absorbance (concentration) of the LFX, AMX, and AZM solutions and MBNPs occurs. This shows that the concentration decrease because of the antibiotics adsorption on the MBNPs then ultimately lead to removal of the studied antibiotics from real samples as also suggested by published studies (Lai et al., 2017; Abedalwafa et al., 2019a; Makropoulou et al., 2020).