A novel successful strategy for detection of antibiotics and toxic heavy metals based on Fluorescence silver/graphene quantum dots nanocomposites

doi:10.21203/rs.3.rs-1670686/v1

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Research Article

A novel successful strategy for detection of antibiotics and toxic heavy metals based on Fluorescence silver/graphene quantum dots nanocomposites

https://doi.org/10.21203/rs.3.rs-1670686/v1

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posted 01 Jun, 2022

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Antibiotic residue and toxic heavy metals in aquaculture have a hazardous impact on human health and environmental safety. So the biggest challenge is designing a powerful detecting tool without harming the fisheries and the environment. A novel dual-function silver/graphene quantum dots ([email protected]) based fluorescence nano-sensor was developed to investigate unprecedented sensing strategies for sensitive and selective detection of antibiotics and heavy metals to ensure that they are present in the authorized percentage. Here, the fluorescence nanocomposites ([email protected]) achieve a new successful sensitive and rapid detection for Oxytetracycline (OTC) and Erythromycin (ERY) antibiotics with detection limits 2.714 nM and 3.306 nM, respectively. The proposed strategy provides an efficient detection way of tracing heavy metals Hg, Cd, Pb with a detection limit of less than 5ppm. Nanoprobe was characterized by UV/VIS spectroscopy, X- ray Diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), and Transmission electron microscopy (TEM). The results were compared with graphene quantum dots (GQDs), graphene oxide quantum dots (GOQDs), and green synthesized silver nanoparticles (AgNPs) which were synthesized from new extracts of aquatic plants and seaweeds from Edku & Marriott Lake. This novel Fluorescence quenching-based technique is sensitive, selective, less time-consuming, and does not need any expensive preparations to replace the commonly used chromatographic detecting techniques.

Silver/graphene quantum dots

Nanoprobe

Nanosensors

Aquaculture

Antibiotics

optical sensors

The continuous growth of the world population is contributed to increased demand for food and drugs administration. Furthermore, industrialization and urbanization are inevitable, accompanied by environmental pollutants [1]. Nowadays, nanostructured materials have unique properties that make them ideal candidates for developing more advanced applications as biosensors and chemical sensors for detecting environmental pollutants such as heavy metal ions, phenolic chemicals, poly-aromatic hydrocarbons, pesticides, and antibiotics [2]. In the present work, we focused only on aquaculture pollutions. Fishes and other aquatic creatures are significant animal protein providers and other essential elements in people's diets. Marine animals and plants account for 17% of animal protein fed globally, over 40% of the world's population [3]. According to Food and agriculture organization (FAO) estimations, aquaculture is one of the world's fastest-growing food-producing areas (FAO, 2020) [4]. The excessive use of antibiotics in aquaculture increases rapidly. About 63,000 tons of antibiotics were used in 2010, and this quantity is expected to be 107,000 tons in 2030 [5]. The introduction of this high quantity of antibiotics into aquatic ecosystems may cause increase the possibility of antibiotic-resistant bacteria. This produces a great problem for our life, where, currently, over 700,000 people die each year from illnesses caused by antibiotic-resistant bacteria. If left uncontrolled, this number may rise to 10 million by 2050 [6].

On the opposite hand, Pollutions of aquaculture by heavy metals are a global issue. This pollution can be done through industrial or home items and pesticides and fertilizers containing heavy metals [7]. The contamination with heavy metals of Hg^+ 2, As ^+ 3, Pb^+ 2, Cd^+ 2, and Cu^+ 2 is a severe threat to human health and the environment. Because of their hazardous effects on plants, animals, and humans, they are non-biodegradable and accumulate in the food chain even at extremely low concentrations. Presently, the determination of metal ion levels in the water can be done by chromatographic and spectroscopic techniques. However, they are expensive and need extensive technical training due to their complexity. Newly, nanosensors provide a new generation of determination of heavy metals with a limit of detection (LOD) up to 0.01 parts per billion (ppb).

Since the Nano-size item reveals different properties of its bulk materials due to variations in its surface-volume ratio, the study and application of silver nano particles (AgNPs) has increased day by day in recent years. They are easily produced from moderately stable silver (I) salts using a variety of reducing agents. [8] Optical, electrical, and thermal capabilities, as well as strong electrical conductivity and biological qualities, are all properties of AgNPs. [9]

Silver nanoparticles (AgNPs) are fascinating nanomaterials with applications in colorimetric sensors, antimicrobial materials, chemical catalysis, and electrochemical sensor aspects [10] Recently, Ag NPs have been widely used for variable applications because of their distinctive surface Plasmon resonance (SPR) [11]. The interaction occurring between the electrons on the surface of Ag NPs with electromagnetic radiation is called localized surface Plasmon resonance (LSPR). This type of interaction generates strong excitation and scattering spectra, around 400 nm in the visible range of the spectrum, so materials based on silver nanoparticles are suitable as optical sensors for detecting antibiotics and pesticides and are considered colorimetric sensors for heavy metals. Different methods can synthesize metal NPs such as Ag NPs; most use harmful chemical substances [12].

In recent years’ researchers have focused on the green synthesis of Ag NPs, which minimize the use of chemical agents, making nanoparticles more environmentally friendly, non-toxic, and cost-effective. Moreover, diverse substances may be used as reducing agents and stabilizers to synthesize Ag NPs. Carbon-based nanomaterials, especially quantum dots (QDs), have recently attracted significant attention as one of these materials.

Graphene quantum dots (GQDs) are nanosized fluorescent carbon compounds having one dimension smaller than 10 nm in size. They can gain much attention as sensors or probes because of their low toxicity, low cost, good stability, high fluorescent activity [13], and extensive surface modification. GQDs play an essential role not as reducing agents and prevent aggregation and oxidation of Ag NPs only but also have an antibacterial property of their own.

The present report will focus on the novel biosynthesis of Ag NPs by various and rare aquatic plants and seaweeds extracts from Edku & Marriott Lake. Edku and Marriott Lakes are two of the five wetland lakes in Egypt's Northern Mediterranean basin. This AgNps can be used as a precursor for [email protected] nanocomposite synthesis. Herein we use fluorescent GQDs as stabilizers and reducing agent to produce [email protected] nanocomposite with smaller particle size ranged from 2 nm to 9 nm. Advanced novel dual function optical sensors that would be promising from [email protected] nanocomposite are studied. We demonstrate new effective analytical biosensors-based nanomaterials that successfully identify, measure, and confirm the presence of antibiotic residue to ensure that they are found in the authorized percentage. Moreover, demonstrate their ability as chemical sensors to detect heavy metal ions in aqueous solutions. It is important to mention that this novel strategy does not recognize one type of antibiotic, but it recognizes more than one type and also determines the different concentrations of each of them and this is not achieved by others. We explain the unprecedented sensing strategy of the fluorescence nanocomposite [email protected] to dispense with the standard time-consuming and expensive techniques. Moreover, to invent advanced environmentally friendly sensors for different future fields. This study will be concentrated on the possibility of applying the successful nano-probe to aquaculture.

Chemicals and Materials

All chemicals were marked as reagent grade and used as received without further purification. Silver nitrate, extra pure (AgNO3, 99.5%); citric acid monohydrate, Cr(NO3)3.6H2O, Cu(NO3)2.3H2O, Hg(NO3)2. H2O, Cd(NO3)2.4H2O, Pb(NO3)2, KNO3, Co(NO3)2.6 H2O all were purchased from (Sigma-Aldrich) and (Merck Chemical Company). Nitric acid (HNO3,72%), sodium hydroxide (NaOH, 99%) pellets extra pure from Oxford Lab Fine Chem. India, Erythromycin, Floroquin, and Penicillin were obtained from (Medical professions for veterinary products Co. MUVCO). Oxytetracycline hydrochloride was purchased from (Pharma SWEDE). The tested antibiotics were prepared by calculating the equivalent active materials and weighing the respective powder.

Preparation of seaweeds & aquatic plant extracts from Edku & Marriott lakes

The seaweeds samples were kept in a polyethylene bag with natural seawater to maintain the seaweeds samples fresh. Preparation of aquatic plant extracts firstly by washing the collecting plants with tap water to remove both epiphytes and necrotic plants and then distilled water to remove any associated debris. The plant samples were dried in an oven at 60oC or shading at room temperature for 2 weeks, and the dried plants' materials were crushed as powder. 1 g of plant powder was weighed and mixed with double distilled water in a clean conical flask at room temperature for about 24 hours, then heated at 60oC with stirring by magnetic stirrer for 15 min. The extract was filtered using Whitman filter paper and stored filtrate in the refrigerator at 4oC to keep them longer.

Green synthesis of Ag NPs by plant extracts from Lake of Edku & Marriott.

10 ml of the filtrate (plant extract) was added to 90 ml of silver nitrate solution (1mM) in a clean conical flask, then stirred by the magnetic stirrer at room temperature and pH 7.2 until the color changed from yellow to dark brown. This indicates that the silver nitrate (AgNO3) was reduced to Ag nanoparticles (Ag NPs) by alkaloids, flavonoids, and phenolic compounds, which could reduce and stabilize agents. This change may occur immediately or after hours or days, depending on the type of plant as it happened. Finally, the obtained colloidal Ag NPs were centrifuged at 10,000 rpm, and the supernatant was discarded. Ag NPs were washed by distilled water several times for further purification for characterization and applications.

Synthesis of graphene quantum dots (GQDs) & graphene oxide quantum dots (GOQDs)

A typical process of GQDs is by using 4 gm of citric acid were put in a 10 ml beaker and heated to 200oC in a furnace for 5 min. After 5 min, citric acid was liquefied and the formation of GQD changed the color of the liquid from colorless to pale yellow and after 30 minutes it changed to orange. If the heating was continued for 2 hrs., the orange color was converted to black solid due to the formation of GOQDs. The obtained orange liquid for preparing GQDs was added drop by drop into 200 mL of NaOH solution (0,2M) under vigorous stirring by a magnetic stirrer. After neutralizing to pH 7.0 with NaOH, the aqueous solution of GQDs was stable and ready for characterization, and for the synthesis of [email protected] nanocomposite, Acetic acid or Nitric acid can be used to adjust pH 7.0. The black solid was dissolved with 100 mL of NaOH (0,2M) solution; the aqueous solution of GOQDs was then neutralized.

Synthesis of AgNPs/GQDs nanocomposite

[email protected] nanocomposite can be synthesized by mixing silver nanoparticles (which were previously prepared by green method from aquatic plants of Idku & Marriott lakes) with GQDs by volume ratio of 2:3 respectively, under ultra-sonication for 30 min; then the mixture was centrifuged and dried.

[email protected] nanocomposite can be prepared using GQDs as reducing and stabilizing agents. 10 ml of the previously prepared GQDs were diluted by 80 ml of double-distilled water. Then pH was adjusted from 7 to 11 by the stock solution of NaOH (0.2M). 1ml of silver nitrate aqueous solution (0.1M) was added to GQDs in a clean 250 ml beaker, then the mixture was heated to 85oC under vigorous stirring by a magnetic stirrer for 2 hr. The black color of the solution converts to transparent and [email protected] nanocomposite was precipitated obviously. Finally, the solution was cooled at room temperature, and the product obtained was purified by centrifugation at 10,000 rpm and washing with deionized water several times (This method has been performed and characterized for applications as Nanoprobe).

Fluorescence measurements

A stock solution of varying concentrations of antibiotics (Oxytetracycline hydrochloride, penicillin, Erythromycin, and Floroquin) was first prepared. For Erythromycin powder (Each 100 gm contain 20 gm Erythromycin thiocyanate, MUVCO, Ismailia, A.R.E.), was prepared by dissolving 0.2 gm at 1L deionized water to prepare stock solution of 0.04 gm/L from the active material. For Oxytetracycline hydrochloride powder (Each 1 gm contains 200 mg of tetracycline hydrochloride, SWEDE Pharma, A.R.E.) was prepared by mixing 0.2 gm with 1L deionized water to prepare 0.04 gm/L from the active material, but for Floroquin solution (10%, FloroMed, each 100 ml contain 10 gm of the raw material) was prepared by mixing 0.4 ml with 1L deionized water to obtain 0.04 gm/L from the active material. On the other hand, Penicillin (United Co. for CHEM & Med (ACCMA), A.R.E.) was mixed with deionized water. As we see, the concentrations of the active material of the all antibiotics are constant to get accurate results. All these solutions were diluted to form the concentration series 0.04, 0.02, 0.01, 0.005, 0.00125 gm/L. Similarity stock solution of series metals ion Cr(NO3)3.6H2O, Cu(NO3)2.3H2O, Hg(NO3)2. H2O, Cd(NO3)2.4H2O, Pb(NO3)2, KNO3, Co(NO3)2.6 H2O was prepared with different concentrations of 5,10,15,30,60,125 and 500 ppm.

A bout 0.1 ml of particular concentration of the antibiotic solution was added into 0.1 ml of [email protected] nanocomposite solution and gently mixed for 5 min at room temperature. The solution was then ready for PL (Photoluminescence Spectroscopy) measurements using Perkin Elmer, model LS55 fluorescence Spectrophotometer) at an excitation wavelength of 330 nm. The same technique was also repeated for the determination of the heavy metals.

Characterization

Instruments

X-ray diffraction (XRD) patterns were recorded on an X-ray diffraction spectrophotometer to investigate the crystalline structures of the synthesized materials using Cu Kα radiation (λ = 1.5406 Å, operating voltage 45 kV, and current 40 mA) at a step size of 0.02° and 2θ range of 20-90°.

The morphology of the nanoparticles was characterized by a (TEM) transmission electron microscope (JEM-1400 Plus, Japan). The nanoparticles size was calculated using the image obtained. Sample was prepared as follows: nanoparticles were dissolved in distilled water, then sonication for 30 min for complete dispersion, then a single drop of nanoparticle solution was put onto the carbon-coated copper grid (400 mesh) and dried at room temperature.

FTIR spectra were recorded using (Nicolet 6700, Nicolet, United States). All spectra were determined in a range of 4000–400 cm-1 using pellets obtained by mixing KBr (Sigma Aldrich) with purified powder of the prepared nanoparticles.

UV–Vis absorption spectrum was analyzed using (Thermal Scientific, model: EVO300).

Transmission electron microscope (TEM) analysis

TEM was used to clarify the morphologies and the particles size of the prepared nanomaterials. TEM was performed for the green synthesized Nano silver from plants of Edku and Marriott. The TEM images are illustrated in Table 1, which shows that the nanoparticles were primarily spherical, with an average size of 14 -30 nm. TEM images of GQDs, graphene oxide quantum dots and [email protected] nanocomposite are illustrated in Fig. 1. As illustrated in Fig. 1-a, 1-b, TEM images for GQDs and graphene oxide show homogeneous dots with a diameter size distribution range between 1.5 and 4 nm. For [email protected] nanocomposites were illustrated in Fig. 1-c. GQDs were coated on the surfaces of Ag NPs to get particles with good crystallinity nature with an average diameter of 1-9 nm. It is believed that GQDs were successfully coated on Ag NPs by a chemical bond to form a nanocomposite as shown Fig.1-d.

FTIR analysis

FTIR enables the in-situ analysis for identification and confirmation of nanoparticles formation and investigates the surface adsorption of functional groups on nanoparticles. FTIR analysis was performed for the synthesized nanoparticles and nanocomposites at the wavenumber region of 4000-500 cm^-1^,and the results are illustrated in Fig. 2. The band observed at a wavenumber of 445-470 cm^-1indicates the presence of silver Ag. The functional groups of the plant extract were also observed in the FTIR spectrum of Ag NPs. Where the axial stretching frequency of (-N-H) was observed at 3277 cm^-1, the (-C-H-) group at 2917 cm^-1, carbonyl (-C=O) stretching vibration at 1613 cm^-1, and the (-C-O) at 1051 cm^-1indicating the presence of functional groups (alkaloids, flavonoids, and phenolic compounds) which work as reducing and stabilizing agents. Furthermore, the polyphenol compounds also reduce Ag⁺ to Ag NPs. The observed groups corresponding to aromatic structures and the band of polyphenols are observed at 1516 cm^-1 and 1320 cm^-1, respectively. The FTIR spectra of GQDs and GO show characteristic bands at wavenumbers of 1384, 1658, and 1642 cm^-1 corresponding to the bands of -COO, C-O, C=C, respectively, which indicate the occurrence of complete carbonization of the citric acid. The CH stretching peak of C (=O)-H is observed at 2553 cm^-1. In the FTIR spectrum of GQD, (C-O-C) stretching vibration can't be detected so there is no characteristic feature of graphene oxide (GO). However, a strong vibration band is observed at 3450-3508 cm^-1, indicating the presence of the OH stretching group.

X-Ray diffraction (XRD)

XRD pattern were performed to investigate the crystalline structures of the synthesized nanomaterials. XRD of the synthesized silver, given in Fig. 3-c, revealed peaks at (2θ) of 37.8^o, 45^o, 65^o, and 78^o characterized for (111), (200), (230), and (311) planes, respectively, of the cubic crystalline face-centered cubic structure (FCC) of silver then compared with the standard powder diffraction card of JCPDS (Joint Committee on Powder Diffraction Standards), silver file No. 04-0783. The average crystallite size D of the silver particles have been estimated from the diffract gram by using the Debye-Scherer's Formula, D = 0.9λ/ β Cos θ, where λ is the wavelength of the X-rays used for diffraction and β is full width at half maximum (FWHM) of a peak [14]. XRD pattern indicates that the synthesized Ag NPs were composed of pure silver with no presence of silver oxide. Furthermore, XRD of GQDs and GO_QDs represented in Fig. 3-a, 3-b show an amorphous structure referring to the formation of highly disordered carbon atoms [15]. The XRD pattern of [email protected] composite also shows an amorphous structure with some minor peaks at the diffraction peaks characterized for Ag nanoparticles.

UV-VIS Spectroscopy

The UV-Visible spectra of the investigated samples are represented in Fig. 4-a. As illustrated, for two different plants a characteristic absorption band of about 380-410 nm is observed due to the surface Plasmon absorption of Ag NPs [16]. The UV-VIS spectrum of GQDs and GO_QDs in aqueous medium were represented in Fig. 4-b, 4-c respectively exhibit an absorption peak at 330 nm and 355 nm due to the absorption of the graphitic structure and the presence of π- π* transition of Sp² carbon (-C-C-) bonds [17]. Interestingly, slightly red shift of U.V-visible absorption spectra and change of the color of the solution (the brown color became darker) was recorded due to the alkaline medium of NaOH and high pH during preparations. The [email protected] nanocomposites were illustrated at Fig.4-d as shown the typical absorption peaks of both GQD at 330 nm and Ag NPs at 400 nm which attributed to LSPR of silver nanoparticles with slight shifts in their positions due to their interaction.

Photoluminescence (PL) spectra of the green synthesized Ag NPs were showed at Fig.5. Fluorescence study was investigated to evaluate the optical properties of the Ag NPs in an aqueous medium. The exciting wavelength was 200 nm. The peaks observed at 430 nm were attributed to the relaxation of the electronic motion of surface Plasmon; the change of the intensity from one plant to another may be attributed to the component of the plant and the capping agents coating the nanoparticles.

The optical properties of the synthesized GQDs and GO_QDs were also studied using PL spectroscopy. The fluorescence spectra of GQDs and GO_QDs at different exciting wavelengths were demonstrated in Fig. 6-a, 6-b respectively. Fig.6-a illustrate GQDs fluorescence peaks at different excitation wavelengths 350, 300, and 280 nm, the PL fluorescence peaks were observed at the same wavelength 500 nm without any shift. However, Fig. 6-b shows the fluorescence of the GO_QDs on 580 nm using different excitation wavelengths of 400 and 350 nm.

A comparison of fluorescence spectra between GQDs &GO_QDs excited at 350 nm was demonstrated at Fig. 6-c. It was found that the intensity of GQDs were more than the intensity of GO_QD at the same excitation wavelength 350 nm. So it is important to mention that it is possible to benefit from GQDs in the development of the chemical sensors, as we will discuss in the research.

The optical properties of [email protected] were studied using PL spectrum at a different excitation wavelength of 330, 340, 350, and 370 nm, and the results obtained are represented in Fig.7-a. the results show fluorescence peaks at around 430, 480, and 540 nm. This is attributed to the LSPR (Localized Surface Plasmon Resonance) absorption peak of Ag nanoparticles besides the surface defects of the graphitic structure, where the GQDs show fluorescence peak at 480 nm at excitation wavelength 330 nm, see Fig. 7-b. The most important observation is that for the same excitation wavelength of 330 nm, the fluorescence peak of [email protected] appears at a higher intensity than those observed for Ag NPs and GQDs. That makes the [email protected] a promising material for many novel applications.

It is important to mention that (Fig.7-b) shows a comparison of fluorescence spectra of [email protected] nanocomposites to GQDs, which illustrates a significant increase in fluorescence. The adsorption of graphene on the Ag (111) surface results in weak bonding, which preserves the electronic structure [18, 19]. The distance between the plasmonic nanoparticles and the fluorescence material can determine whether PL is enhanced or quenched [20, 21]. The mentioned procedure (using GQDs as reducing and stabilizing agents.) didn't introduce any extra stabilizer in the [email protected] system, so the plasmonic nanoparticle Ag was directly associated with the fluorescent material GQDs. This enhances the fluorescence because it can facilitate the ejection of LSPR (Localized Surface-Plasmon Resonance) excited electrons on Ag into the fluorescence material GQDs [22]. The process involves the following mechanism:

Step (1): LSPR excited electrons would oscillate on the surface of the plasmonic nanoparticle Ag NPs under visible light.
Step (2): With the increase of electron density, electron transfer from Ag to the conduction band of GQDs.
Step (3): Then the electron returns to the ground state, so the huge amounts of hot electron transfer eventually lead to fluorescence enhancement. All these steps were explained in Fig. 8.

It is essential to mention that using the plasmonic particles as (Ag NPs) to improve the fluorescence of GQDs presents great benefits as LSPR-enhanced fluorescence can be used to achieve high sensitivity for variable applications as probes:

a) [email protected] for detection of antibiotics

High concentration residue of antibiotics in aquaculture and water is a serious problem. The most common commercially available antibiotics in Egyptian aquaculture are Oxytetracycline hydrochloride, Erythromycin, Penicillin, and Floroquin. Therefore, they were selected to study their luminescent sensing performances with [email protected] nanocomposite as a chemo-sensor. The results obtained demonstrated fluorescence quenching response for Oxytetracycline hydrochloride and Erythromycin more than the other antibiotics but with different degrees Fig. 9-a. The results also showed that Oxytetracycline hydrochloride and Erythromycin exhibit a more significant fluorescence quenching effect. Fig. 9-b, 9-c shows the relationship between the concentration and fluorescence intensity for Oxytetracycline and Erythromycin, respectively. As the concentration increases, fluorescence quenching occurs. Moreover, that demonstrates the unprecedented sensitivity and selectivity of [email protected] nanocomposite. So it is considered a new successful sensing probe not for one type of antibiotics, but it can differentiate between different types as well. So Oxytetracycline and Erythromycin were selected to define their detection efficiency by the [email protected] nanocomposite by determining the relationship between the concentrations of the antibiotics and the fluorescence intensity of the composite. The obtained results in Fig 10-a, showed that the fluorescence is inversely proportional to the antibiotics concentrations. That means the luminescence intensities steadily decrease, and the fluorescence quenching occurs with the antibiotic's continuous addition (concentration increase) according to Stern-Volmer equation [23]:

I_o/I = 1+K_qT_o [Q] (1)

Where I_o is the intensity, or rate of fluorescence, without the antibiotic, I is the intensity, or rate of fluorescence, in the presence of the antibiotic, K_q is the quencher rate coefficient, T_o is the lifetime of the emissive excited state without a quencher present, and [Q] is the concentration of the antibiotic. Higher I_o/I indicate that the probe has excellent sensitivity and selective detection of the antibiotic.

The selectivity test for the investigated [email protected] nanocomposites for different antibiotics especially Oxytetracycline hydrochloride and Erythromycin was shown in Fig. 10-b. The fluorescence quenching percentages plot illustrates the sensitivity of the synthesized [email protected] for detection of antibiotics (Oxytetracycline & Erythromycin) more than any other antibiotics.

Depending on the quenching mechanisms, the quenching process can be classified as static or dynamic. It was represented in Fig. 8. During the static quenching process, the quencher and the fluorescence probe create a bound non-fluorescent complex. It is known that the number of these complexes tends to increase with the increasing concentration of quencher molecules [24]. But the corresponding fluorescence lifetime (fluorescence decay rate) is independent of the quencher concentrations [Q] [25]. Whereas in the case of the dynamic quenching, the quencher molecule collides with the fluorophore in the excited state and is attributed to the change of the quencher concentrations. So that the change of Ksv and corresponding lifetime value T decrease with the increase of [Q] quencher concentrations as following equation:

Ksv = Kq. T (2)

Where Ksv is Stern-Volmer constant, T is the rate of fluorescence decay. According to the Fluorescence plot represented in Fig. 11-a, 11-b, a good linear relationship between fluorescence ratio I_o/I and the Concentration [Q] of the antibiotics is obtained with linear range 2µM -50 µM for Oxytetracycline and 3µM-60 µM for Erythromycin. The corresponding regression coefficient R² for both Oxytetracycline and Erythromycin are 0.9628 and 0.9481 respectively so that confirms the linear relationship. The change of PL intensity of the prepared nanocomposite in the presence of antibiotics with variable concentrations was observed. It is obvious that the fluorescence was quenched gradually with the increase of concentration of antibiotics. The complete data was discussed in the Table 2.

b) AgNPs/GQDs for detection of heavy toxic metals

Fluorescence experiments were designed to identify heavy metals sensing capabilities of [email protected] nanocomposite. The fluorescence intensities in the presence of heavy metals are lower than the fluorescence intensities of the free nanocomposite. That confirms the successful conjugation of the nanocomposite with the heavy metal ion to produce detectable signals. The data presented in Fig.12 show that the Co²⁺, K⁺, Cu²⁺, and Cr³⁺ ions with nearly the same fluorescence quenching intensities are higher than those observed for Hg²⁺, Pb²⁺, and Cd²⁺. This means that the nanocomposite is more sensitive for Hg²⁺, Pb²⁺, and Cd²⁺. than the other investigated ions. So [email protected] can provide a new successful Nanoprobe for precise detection of specific heavy metals.

Results presented in Fig. 13 Show that the photoluminescence intensity decrease with the increase of the concentrations of heavy metals ions Hg²⁺, Pb²⁺, and Cd²⁺, so it is considered ions concentration dependence and that demonstrated in the three independent experiments, which are reflected in values corresponding to PL fluorescence intensities. With a high concentration of Hg²⁺, Pb²⁺, and Cd²⁺, a significant quenching is observed Fig. 13-a, 13-.b and 13-c respectively. These show a high sensitivity of the novel fluorescent sensor [email protected] nanocomposite. The [email protected] based optical sensor is employed for detecting different heavy metals in an aqueous solution with detection limits less than 5 ppm with good sensitivity and selectivity for Hg²⁺, Pb²⁺, and Cd²⁺ ions recognition.

It is essential to mention that [email protected] nanocomposite can detect several metal ions M⁺ and M²⁺ with smaller concentrations less than 5 ppm via fluorescence quenching. The fluorescence quenching of [email protected] nanocomposite is also sensitive for several antibiotics. This means that the [email protected] sample is an excellent and novel dual-function chemo-sensing material for antibiotics and heavy metals.

The selectivity test results for the researched [email protected] nanocomposites for Hg²⁺, Pb²⁺, and Cd²⁺ are shown in Fig. 14. The fluorescence quenching percentages plot shows that the synthesized [email protected] are sensitive to specific metals more than others. Quenching efficiency was studied and presented in Fig. 15 for the proposed fluorescence sensor to highlight variable future directions. The results explain the fluorescence intensity of [email protected] nanocomposites in the absence and presence of the quencher (Hg²⁺, Pb²⁺, and Cd²⁺ ions). The fluorescence intensity decreases rapidly with increasing the concentrations of metal ions. The sensitivity of fluorescence quenching can be effectively related to Stern–Volmer constant (K_SV), using the following equation;

I₀/I = 1 + K_SV[Q] (3)

Where I_o and I are the fluorescent intensities of solute molecules in the absence and presence of quencher, respectively, and [Q] is known as the analyte concentration [26]. The higher K_SV value for these heavy metal analyte can be attributed to the efficient interaction of these heavy metal ions with the proposed sensor, which ultimately leads to a significant Fluorescence quenching. A strong linear relationship between the fluorescence ratio I_o/I and the concentration of the analyte [Q] was illustrated with the linear range at 5-250 ppm for Hg²⁺, according to the Fluorescence plot shown in Fig. 15. The correlation coefficients R² for Hg²⁺, Pb²⁺, and Cd²⁺ are 0.9043, 0.9768, and 0.9533, respectively, indicating that the linear association exists with a limit of detection of 5 ppm. And the results were illustrated in Table 3.

The linear range and the limit of detection (LOD) were calculated to study the detection performance of [email protected] for different antibiotics and heavy metals then the results were compared with the previously reported Nanoprobes and the results were listed in Table 4-5. It can be seen that our sensing system is superior to other fluorescent Nanoprobes toward detecting both antibiotics and toxic heavy metals. So, it is considered a dual-functional nanosensors.

In summary, we have successfully developed a novel strategy to produce Nanoprobe with dual functions and demonstrate their benefit not only in terms of simplicity and cost efficiency but also in terms of environmental friendliness. This work represents synthesis of [email protected] by two different methods. One of them was green synthesis by using new extracts of aquatic plants and seaweeds from Edku & Marriout Lake. The proposed nanosensors provides distinctive detection for both antibiotics and heavy metals. The detection is based on the nanocomposite's fluorescence quenching. The quenching percentages express the sensitivity of the synthesized [email protected] Nanoprobe with Oxytetracycline and Erythromycin being the most sensitive antibiotics. The examined Nanoprobe’s duty includes not only the detection of antibiotics but also the identification of harmful heavy metals and their concentrations such as Hg²⁺, Pb²⁺, and Cd²⁺ ions .

It is obvious through the results that upon increasing the concentration, the fluorescence of the nanocomposite gradually decreases; this reflects that the present sensing system is highly sensitive. The present notion can be regarded as an important step towards developing the promising probe to be the ideal sensor.

[email protected]: silver/graphene quantum dots; OTC: Oxytetracycline; ERY: Erythromycin antibiotics; XRD: X- ray Diffraction; FTIR: Fourier-transform infrared spectroscopy; TEM: Transmission electron microscopy; GQDs: graphene quantum dots; GOQDs: graphene oxide quantum dots; FAO: Food and agriculture organization; LOD: limit of detection; ppb: parts per billion; AgNPs: silver nano particles; SPR: surface Plasmon resonance; LSPR: localized surface Plasmon resonance; AgNO3: silver nitrate; PL: Photoluminescence Spectroscopy; FCC: face-centered cubic structure; JCPDS: Joint Committee on Powder Diffraction Standards; D: the average crystallite size of the silver particles; λ: the wavelength of the X-rays used for diffraction; β: full width at half maximum (FWHM) of a peak; I_o: the intensity, or rate of fluorescence, without the antibiotic; I: the intensity, or rate of fluorescence, in the presence of the antibiotic; K_q: the quencher rate coefficient; T_o: the lifetime of the emissive excited state without a quencher present; Q: the concentration of the antibiotic; Ksv: Stern-Volmer constant, T: the rate of fluorescence decay; DPV: Differential Pulse Voltammetry; AdSV: Amperometry and adsorptive Stripping Voltammetry; AdSDPV: Adsorptive stripping Differential pulse Voltammetry; CV: Cyclic Voltammetry; SWV: Square Wave Voltammetry.

Acknowledgements

The authors thank the central laboratory and laboratories of National Institute of Oceanography and Fisheries for providing the required equipments to make the appropriate measurements.

Authors’ contributions

Mahmoud A. Mousa and Amira H.E. Moustafa finished study design. Mamdouh A. Fahmy and Dina G. Ebrahim finished experimental studies. Amira H.E. Moustafa Dina G. Ebrahim and Hanaa H. Abdelrahman finished data analysis. Mahmoud A. Mousa and Amira H.E. Moustafa and Hanaa H. Abdel Rahman finished manuscript editing. All authors read and approved the final manuscript.

Funding

No funds, grants, or other support was received.

Availability of data and materials

The data used and analyzed during the current study are available from the

corresponding authors upon reasonable request.

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable

Competing interests

The authors declare that they have no competing interests.

Author details

¹ Faculty of Science, Benha University, Banha, Cairo, Egypt

² Faculty of Science, Alexandria University, Alexandria, Cairo, Egypt

³ Marine Chemistry Department, Environmental Division, National Institute of

Oceanography and Fisheries (NIOF), Alexandria, Egypt

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Table 1 is available in the Supplementary Files section

Table 2

Silver/graphene quantum dots were quenched by Oxytetracycline and Erythromycin.

Limit of Detection (LOD)	Linear range	Correlation Coefficient R²	Linear regression equations	Antibiotics
2.0 nM	2.0 -50 µM	0.9628	I_o/I = 0.0242[Q] + 1.1721	Oxytetracycline
3.3 nM	3.0-60 µM	0.9481	I_o/I = 0.0153[Q] + 1.1099	Erythromycin

The limit of detection is defined by the equation LOD=3 δ/K where δ is the standard deviation (n=10) and K is the slope of the calibration plot.

Table 3

Silver/graphene quantum dots quenched by Hg⁺², Pb⁺², and Cd⁺².

Limit of Detection (LOD)	Linear range ppm	Correlation Coefficient R²	Linear regression equations	Analyte
5ppm	5-250	0.9043	I₀/I = 0.002[Q] + 1.1384	Hg⁺
5ppm	5-250	0.9768	I₀/I = 0.0013[Q] + 1.0867	Pb⁺²
5ppm	5-250	0.9533	I0/I =0.0016 [Q] + 1.0997	Cd⁺²

Table 4

Comparison of sensing performance of variables nanoprobes for detection of antibiotics.

Reference	Year	Limit of detection (LOD)	Linear range (µM)	Method	Detected antibiotic	Probe
[1]	2020	100 nM	2-100 µM	Fluorescence	Oxytetracycline	N-GQDs
[2]	2016	6 nM	5 µM -100µM	Differential Pulse Voltammetry (DPV)	Oxytetracycline	GO
[3]	2012	5.3 nM	Up to 8 µM	Amperometry and adsorptive Stripping Voltammetry (AdSV)	Oxytetracycline	C-nanotubes
[4]	2021	400 nM	400 µM to 460 µM	Adsorptive stripping Differential pulse Voltammetry (AdSDPV)	Tetracycline	CNT- Fe₃O₄@SiO₂
[5]	2021	16 nM	29µM-135µM	Cyclic Voltammetry (CV)	Tetracycline	Graphene-Au
[6]	2021	415 nM	5 µM to100 µM	Square Wave Voltammetry (SWV)	Oxytetracycline	Carbon
[7]	2017	12 nM		Fluorescence	Erythromycin	SiO₂/Au
This work		2.7 nM 3.3 nM	2 µM to50 µM 3 µM to 60 µM	Fluorescence	Oxytetracycline Erythromycin	[email protected]

Table 5

Comparison of sensing performance of variables nanoprobes for detection of some toxic heavy metals such as Hg⁺², Pb⁺², and Cd⁺².

Reference	Year	Detection range	Method	Detected analyte	Probe
[8]	2014	Up to 10 ppm	Electrochemical	Pb⁺²	Graphene oxide
[9]	2018	1-10 ppm	Colorimetric	Hg⁺²	AgNPs
[10]	2019	1-10 ppm	Electrochemical	Cd⁺²	AgNPs
[11]	2017	10 ppm	Electrochemical	Cd⁺²	AuNPs/Graphene
[12]	2017	10 ppm	Colorimetric	Hg⁺²	Alkanethiols/graphene
This work		5ppm	Fluorescence	Hg⁺², Cd⁺², Pb⁺²	Ag/GQDs

No competing interests reported.

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A novel successful strategy for detection of antibiotics and toxic heavy metals based on Fluorescence silver/graphene quantum dots nanocomposites

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