VANH as a potent glycopeptide antibiotic has an extraordinary role not only in the prophylaxis but also in the therapy of various gram-positive bacterial life-threatening infections. In synthesis or analysis, the majority of laboratories worldwide are moving nowadays towards green chemistry to decrease impacts on the environment and to improve the health safety of analysts. So, a novel ultrasensitive and simple AgNPs-enhanced fluorescence technique was presented for VANH rapid analysis in its pharmaceutical formulation and biological fluids yielding satisfactory recovery results comparable to those of the reported HPLC technique .
The synthesized Ag-NPs were characterized by UV-visible spectroscopy and TEM micrograph. As illustrated in Fig. 6, the Ag-NPs exhibited a characteristic spectrum with an intense absorption maximum at 415 nm due to the surface plasmon excitation. It was observed that VANH absence from the reaction system resulted in absence of any absorption peak in the visible region (400–700 nm). Also, the formation of Ag-NPs in presence of VANH was confirmed as presented in Fig. 7 by the TEM micrograph which reveals that the Ag-NPs were spherical in shape with smooth surface morphology and size of 10.74 ± 2.44 nm.
Unlike conventional fluorimetric methods, the proposed method is highly sensitive enough to measure VANH concentrations at ultra-trace quantities and consequently, can be adapted for VANH monitoring and studying its bioequivalence in biological fluids. The proposed method is regarded as a green fluorimetric technique appropriate for VANH analysis in miscellaneous matrices at a low cost due to its dependence mainly on water as a cheap and eco-friendly solvent.
6.1. Method optimization
To obtain optimum results of the proposed technique for the determination of VANH, the following variables were studied:
6.1.1. Effect of concentration and volume of AgNO3 solution
Several experiments were performed on differently concentrated solutions of AgNO3 using the same concentration of VANH in each trial at other optimal reaction conditions (Table 1). As a result, it was observed that AgNO3 (3×10−3 M) solution was the best one for optimum results, after which the increase in the concentration of AgNO3 solution resulted in a significant decrease in the fluorescence intensity of Ag-NPs due to the formation of AgCl white precipitate. Afterward, different volumes of AgNO3 solution (3×10−3 M) were tried at the same reaction conditions. The results revealed that 1.2 mL was the best volume for optimum results, after which the fluorescence intensity of Ag-NPs was almost of the same values with the increase in AgNO3 volume (Fig. 8a).
6.1.2. Effect of stabilizer type, concentration, and volume
Ag-NPs are liable to agglomerate during their synthesis. Thus, Ag-NPs were stabilized by one of two stabilizers: electrostatic stabilizers or steric stabilizers to prevent their agglomeration . Electrostatic stabilizers such as sodium citrate, act by adsorption on the nanoparticles’ surface forming an electrical double layer that causes columbic repulsion between the nanoparticles and consequently preventing their agglomeration. While steric stabilizers such as PVP, are characterized by making a protective cap on the nanoparticles’ surface and therefore preventing their agglomeration. In this study, it was observed that using the PVP gave higher fluorescence values than sodium citrate. Thus, PVP was chosen to stabilize Ag-NPs and prevent their agglomeration.
Several trials were performed on different concentrations of PVP solution in a similar way to that of AgNO3 solution. As a result, it was noticed that the PVP (0.14%) solution was the best-concentrated one for optimum results, after which the increase in the concentration of PVP solution led to a slight decrease in the fluorescence intensity of Ag-NPs. Then, different volumes of PVP solution (0.14%) were tried at the same reaction conditions. The results revealed that 1 mL was the best volume for optimum results, after which the fluorescence intensity of Ag-NPs slightly decreased with the increase in PVP volume (Fig. 8b).
6.1.3. Effect of concentration and volume of NaOH solution
During the reduction process of silver ions to Ag-NPs by VANH, the H+ ions were produced in the reaction medium. Hence, NaOH solution was added to provide enough alkalinity to the reaction medium and to consume the produced H+ ions resulting in hastening of the reaction and promoting the reduction process required for Ag-NPs formation. Consequently, the effect of NaOH solution should be well studied by testing differently concentrated solutions of NaOH in a similar way to that of AgNO3 solution. After several trials, it was found that NaOH (5×10−3 M) solution was the best-concentrated one for optimum results, after which the increase in NaOH concentration resulted in a significant decrease in the fluorescence intensity of Ag-NPs due to the formation of Ag2O black precipitate. Also, different volumes of NaOH solution (5×10−3 M) were tested at the same reaction conditions. The results revealed that 1.2 mL was the best volume for optimum results, after which the increase in NaOH volume resulted in a gradual small decrease in the fluorescence intensity of Ag-NPs (Fig. 8c).
6.1.4. Effect of reaction temperature and heating time
It was observed that the reaction system of the proposed method required heating at 90°C in a water bath for a certain time to obtain optimum fluorescence values of Ag-NPs. After which the increase in reaction temperature resulted in a significant decrease in the fluorescence intensity of Ag-NPs due to silver precipitation. Subsequently, different heating times at 90°C were tested in a similar way to that of the AgNO3 solution. It was found that heating at 90°C for 20 minutes was the best time for optimum fluorescence intensity results, after which the Ag-NPs fluorescence remained constant indicating the end of reaction for Ag-NPs synthesis (Fig. 8d).
6.1.5. Effect of Britton-Robinson buffer (pH and volume)
After several trials, it was found that Ag-NPs can’t be formed in presence of buffer solutions. Consequently, Britton-Robinson buffer solution was added after the formation of Ag-NPs to obtain stable fluorescence intensity values.
So, the effects of pH ranging from 2 to 12 and volume of added buffer ranging from 0.25 to 2.75 mL were studied versus the fluorescence intensity at other optimal reaction conditions (Table 1). It was found that the fluorescence intensity of Ag-NPs gradually increased up to pH 6 at which maximum fluorescence intensity was achieved. At high pH values (> 6), the fluorescence intensity of Ag-NPs decreased gradually as a result of Ag-NPs aggregation under alkaline conditions . So, the choice of pH 6 was crucial to obtain optimum and stable fluorescence intensity values (Fig. 8e). Also, the results revealed that 1 mL of Britton-Robinson buffer solution (pH=6) was the best volume for optimum results, after which the fluorescence intensity of Ag-NPs was almost of the same values with the increase in buffer volume (Fig. 8f).
6.1.6. Effect of diluting solvent
Upon dilution with different solvents such as bi-distilled water, acetonitrile, methanol, acetone, ethanol, and 2-propanol, bi-distilled water was observed to give the highest fluorescence intensity value (Fig. 8g). Hence, bi-distilled water was the diluting solvent of choice throughout this study.