Puerarin-coated gold nanoparticles (PUE-AuNPs) synthesized via green synthetic route: a new colorimetric probe for the detection of ciprofloxacin

Puerarin-coated gold nanoparticles (PUE-AuNPs) synthesized via green synthetic route, a new colorimetric sensor, efficiently detected the ciprofloxacin (CP) in tap water and cow milk samples. The PUE-AuNPs were characterized by UV-visible, FTIR, AFM, and DLS techniques and were found to be spherical with an average size of approximately 19–20 nm. FTIR spectrum confirms that functional groups such as −OH, −C=O, −CO and −C=C were responsible for the reduction of gold (III) chloride trihydrate. These functional groups acted as capping agents to form AuNPs. The PUE-AuNPs sensor was proved to be selective and sensitive for the detection of CP through colorimetric method within the concentration of 1 to 1000 μM and the limit of detection was 51 μM. This colorimetric sensor is simple, cost-effective, and selective towards CP detection in environmental (tap water and milk) samples.

CP was consistently observed in a number of water environments due to these various entry routes [11].In Switzerland, CP was detected in the range 249-405 ng/L and 45-568 ng/L in domestic sewage and at WWTPs respectively, 0.6-2 μg/L and 0.02 μg/L of CP was also detected in wastewaters and surface streams across the US.Moreover, CP in the range 0.7-124.5 μg/L was found in wastewater of a Swiss hospital [6].Due to the aforementioned concerns, various methods have been employed so far, to detect the ciprofloxacin in many environmental samples, such as fluorescence spectroscopy [12], cyclic voltammetry [13], high-performance liquid chromatography with fluorescence [14], β-galactosidase-based colorimetric assay [15], amperometric, and electrophoresis with conductivity analysis [16], and liquid chromatographymass spectrometry method [17] (Table 1).Although these approaches offer sensitive and precise multianalytical detection, they also require a lot of time, costly instruments, tricky sampling, and utilization of detrimental natural solvents in massive portions as well as particularly skilled professional operators.Therefore, there is a dire need to develop a simpler, quicker, and more reliable method for the detection of CP in environmental samples.Spectrophotometric-based colorimetric detection methods have many benefits such as simple use, high sensitivity, high reliability, and being inexpensive.In the present study, we account a green synthetic route to synthesize gold nanoparticles (AuNPs) for the first time using puerarin (PUE) (an isoflavonoid, isolated from Pseudocalymma elegans) as a reducing and stabilizing agent.The morphology of the PUE-AuNPs was characterized by atomic force microscopy (AFM) and scanning electron microscopy (SEM).Puerarin [8-(β-D-glucopyranosyl-7-hydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one] is a white powder and is soluble in methanol (Figure 1b) [18].It possesses several pharmacological properties such as antiinflammatory, anticancer, antioxidant, antidiabetic, and cardiac-and neuro-protective.Previously, puerarin-coated nanoparticles were synthesized through different methods like anionic polymerization [19], emulsion solvent evaporation [20], solvent evaporation [21], and precipitation [22].We have planned a cost-effective, simple, and fast colorimetric chemosensing platform for the detection of CP.The developed colorimetric method is based on the vigorous interaction among the Au and NH/OH group of CP and thus forming Au-N/O bond.The CP binds on the surface of PUE-AuNPs, which results in a high degree of aggregation as confirmed by UV-visible and FTIR spectroscopy, as well as AFM and DLS analysis.The proposed method described here is practically applicable for the detection of CP in environmental (tap water and milk) samples.The sensor was proved to be selective and sensitive for the detection of CP in presence of other drugs.or treatment.The stability of nanoparticles is the important factor in the field of application, that is why the synthesized AuNPs were stored at 8 °C over a period of 1 month, and the stability was studied by UV-visible spectrophotometer (Figure 2b).

FTIR spectroscopy of PUE-AuNPs
The FTIR analysis identified the functional groups present in puerarin that were responsible for the reduction of Au +3 ion to Au 0 .The result showed that hydroxyl (OH), carbonyl groups (C = O), C-O and -C=C present in puerarin were responsible for the AuNPs synthesis.The spectrum of PUE-AuNPs was studied in 4000 to 500 cm -1 (Figure 3).The IR spectrum of PUE displayed band at 3330, 3227 cm -1 for hydroxyl stretching vibration  1452, 1378, and 1041 cm -1 regions.The sharp peak at 3330 of hydroxyl group became a broad peak of 3396 cm -1 and the short peak of aromatic C-H stretching changed from 2901 to 2925 cm -1 .The peaks also shifted from 1568, 1513, 1444 to 1452 and 1378 cm -1 .At 1627, carbonyl peak shifted from 1681 to1632 cm -1 .The stretching of the C-O moved from 1053 cm -1 to 1041.These shifting of vibrations in the IR spectrum of PUE-AuNPs as compared to PUE spectrum indicate the contributions of carbonyl, alkene, and polyol groups in the synthesis and stabilization of PUE-AuNPs (Figure 4).Similar observations were also reported in previous literature [25].

Particle size, surface charge, and elemental analysis of PUE-AuNPs
An atomic force microscopy (AFM) and a zeta-sizer were used for the determination of size and morphology of nanoparticles.AFM images further support the results of SEM by showing sphere-shaped of PUE-AuNPs having size in the range of 19-20 nm (Figures 5a and 5b).A zeta-sizer was used to determine the size, and the PUE-AuNPs size distribution profile had an average diameter of 53.21 nm with polydispersity index (PDI) of 0.383 (Figure 5c).Zeta potential (surface charge) was also used to determine the intensity of nanoparticle interactions with its surroundings.Electrostatic repulsions between particles showed the stability of the particles.The surface charge value of PUE-AuNPs was found to be -4.47 mV (Figure 5d).Similar observations were reported for the silver and gold nanoparticles synthesized from the Polystyrene-block-poly (2-vinylpyridine) [26] and Pyrazinium thioacetate ligand [27].

Application study as a chemosensor 3.2.1. Colorimetric detection of ciprofloxacin with PUE-AuNPs using UV-visible spectroscopy
In order to evaluate the chemosensing potential the PUE-AuNPs were used with 1mM a series of sixteen drugs solution in 1:1 (v/v) mixture.The solution changed from colorless to pink (Figure 6a).Only CP was detected through spectrophotometric detection method of PUE-AuNPs.It offers a tool for easily detecting CP visually by naked eye.The wavelength change was also recognized at 528 nm to 538 nm by adding CP solution to PUE-AuNPs.These effects that appeared in PUE-AuNPs and CP were due to the changing of binding between the attached groups i.e. hydroxyl (OH) and carbonyl (C = O) of PUE-AuNPs and carboxylic (COOH), carbonyl (C = O) and amino groups of CP (Figure 6a) [26].Ciprofloxacin's interaction with PEU-AuNPs is schematically illustrated in (Figure 6b).The remaining analyzed drug solutions did not cause any visible changes in the color and wavelength of PUE-AuNPs (including flurbiprofen, paracetamol, omeprazole, zuclopenthixol, azithromycin, metronidazole, esomeprazole, levofloxacin, mefenamic acid, levetiracetam, diclofenac sodium, cephradine,  penicillin G. procaine, amoxicillin trihydrate, piroxicam beta cyclodextrin, cefotaxime and ciprofloxacin) as shown in Figure 6c.

Analytical efficiency of PUE-AuNPs
To examine the analytical efficiency of PUE-AuNPs, several concentration of CP were used to explain the sensitivity in results of PUE-AuNPs based CP sensing.By using UV-visible spectroscopy, numerous CP concentrations were examined against PUE-AuNPs for measuring the sensitive optical response.According to the surface plasmon resonance (SPR) spectrum the absorbance showed strong linearity to the CP concentration in the range of 1 to 1000 µM with the regression constant R 2 = 0.9994 (Figure 6d).The detection limit (LOD) for CP and the quantification limit (LOQ) are equal to 51 19

(f) (g)
µM and 154 µM, respectively.Moreover, the linear range and LOD of the proposed probe were also compared with other reported methods (Table 1).In addition, Job's plot experiment could be used to determine the quantitative relationship between the PUE-AuNPs and CP complexes.Different mole ratios of PUE-AuNPs and CP were taken for plotting the spectra by using UV-visible spectrophotometer.The results showed that the stable complex of PUE-AuNPs-CP was formed at 0.3 and 0.7 mole ratios of CP and PUE-AuNPs, respectively (Figure 6e) ideally reported in the literature [28].

Detection of CP in environmental samples
In order to check sensitivity and selectivity of the detector system, practical application was optimized through using a PUE-AuNPs-based nanosensor.We developed the efficacy of CP recognition in environmental samples of tap water taken from Federal Urdu University of Arts Science and Technology, Karachi and cow milk taken from the local market of Karachi.The standard protocol (mentioned in Section 2.5) was used to spike the samples with PUE-AuNPs (1 mM).In tap water (Figure 6f), the enhancement of absorbance was observed when tap water was spiked with PUE-AuNPs and CP, while a broad peak with a shoulder appeared when tap water was spiked in PUE-AuNPs (Table 2a, Figure 7a).However, cow milk sample (Figure 6g) did not show any enhancement and broadening of peak by the addition of PUE-AuNPs and CP (Table 2b, Figure 7b).Such findings indicated the practical use of this detector sensor and we expect that the PUE-AuNPs device proved to be significant in tap water and other environmental samples.

Interaction mechanism of PUE-AuNPs with CP Chemical interaction by FTIR analysis
The chemical interaction of PUE-AuNPs with CP was studied by FTIR spectroscopy (Figure 8a).The CP-related bands (Figure 8b) occurred at 3434.8a).Enhancement of stretching intensities of -NH and -OH in PUE-AuNPs-CP spectra (Figure 8a) were observed, whereas few peaks appeared to be sharp and dislocated.The obtained results may have suggested that gold nanoparticles (PUE-AuNPs) interacted with OH/NH group of CP, which lead to the crosslinking of two moieties (Figure 4), whereas amine and carboxylic groups might also contribute in nonelectrostatic interaction with the surface of nanoparticles.Our results are very much aligned with previous studies reported in the literature [27].

Molecular recognition of PUE-AuNPs-CP complex via AFM, zeta-sizer, and zeta potential
It was necessary to study the nanoparticles at subnanometric and atomic resolution using the latest atomic force microscopy (AFM) technique.The PUE-AuNPs-CP complex micrographs were screened to evaluate the morphology and dispersity changes that have taken place as it developed a complex with ciprofloxacin.Due to irregular nanoparticles and aggregation, PUE-AuNPs-CP showed a significant shift in size, shape, and dispersity.PUE-AuNPs-CP showed average size of 63-71 nm (Figures 8c and 8d).Dynamic light scattering (DLS) was used to perform zeta-sizer and zeta potential.The CP complex with PUE-AuNPs were distributed at a size of 153 nm and the polydispersity was 0.204 (Figure 8e), while the -4.43 mV was surface charge of PUE-AuNPs-CP complex (Figure 8f).The results are consistent with the previous reported findings [24].

Conclusion
The present work describes the one pot method for the preparation of puerarin-coated gold nanoparticles (PUE-AuNPs), which was found to be green and nontoxic as compared to previous known traditional methods.It has shown to be an effective quantitative colorimetric protocol for the detection of an antibacterial drug, i.e. ciprofloxacin (CP).The proposed sensor was efficiently used in tap water and cow milk samples for the detection of CP.Detailed characterization of nanoparticles, i.e. size, morphology, as well as interaction of PUE-AuNPs with CP has also been carried out by various spectroscopic and microscopic techniques (AFM, FTIR, UV-visible) and zeta-sizer.In contrast to the presence of other drugs, the PUE-AuNPs-based sensor specifically and selectively detected CP with linear correlation following concentration in the range of 1-1000 μM with a limit of detection of 51 μM.Low cost, easiest preparative method, and excellent selectivity of the proposed ciprofloxacin detector revealed the potential for on the spot alarming process of CP in environmental (tap water and cow milk) samples.

Figure 4 :
Figure 4: Schematic illustration of the strategy for the synthesis, chemosensing and probable mechanism of induced aggregation of PUE-AuNPs

Figure 4 .
Figure 4. Schematic illustration of the strategy for the synthesis, chemosensing, and probable mechanism of CP-induced aggregation of PUE-AuNPs.

Figure 7a .
Figure 7a.Graphical representation of PUE-AuNPs based detection of the CP from environmental (tap water) sample.

Figure 7b . 23 Figure 7a .
Figure 7b.Graphical representation of PUE-AuNPs based detection of the CP from environmental (Cow milk) sample.

Figure 7b .
Figure 7b.Graphical representation of PUE-AuNPs based detection of the CP from environmental (Cow milk) sample.

Figure 7a .
Figure 7a.Graphical representation of PUE-AuNPs-based detection of the CP from environmental (tap water) sample.

Table 1 .
Comparison of the established PUE-AuNPs-based probe for the detection of CP with other previously reported methods.