CdS quantum dots embedded in PVP: Inorganic phosphate ion sensing in real sample and its antimicrobial activity

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 234 (2020) 118256

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CdS quantum dots embedded in PVP: Inorganic phosphate ion sensing in real sample and its antimicrobial activity Swastika Dhar a, Buddhadeb Sen b, Subhra Kanti Mukhopadhyay c, Trinetra Mukherjee, Formal analysis c, Asoke Prasun Chattopadhyay a,⁎, Sadhan Pramanik d a

Department of Chemistry, Kalyani University, Kalyani, Nadia 741235, India Department of Chemistry, The University of Burdwan, Burdwan 713104, India Department of Microbiology, The University of Burdwan, , Burdwan 713104, India d Department of Chemistry, Hooghly Womens College, Hooghly 712103, India b c

a r t i c l e

i n f o

Article history: Received 1 November 2019 Received in revised form 12 March 2020 Accepted 13 March 2020 Available online 16 March 2020 Keywords: CdS Quantum dots Chemosensor Phosphate ion sensing Antimicrobial activity Cell image

a b s t r a c t Polyvinyl-pyrrolidone capped spherical cadmium sulphide quantum dots (CdS-PVP QDs), 2–6 nm in size, were developed as a selective turn-on fluorescence nanosensor for monohydrogen phosphate ion (HPO2− 4 ) in aqueous medium. Fluorescence intensity of CdS-PVP QDs significantly increased with addition of HPO2− 4 ions, whereas the other common inorganic ions had very little effect on the fluorescence intensity. The proposed sensor may be efficiently used for the detection of HPO2− 4 ions at a low level of concentration up to 213 nM in real urine sample. Cell imaging study indicates that the CdS-PVP QDs are cell permeable and can detect the intracellular distribution of HPO2− 4 ions under fluorescence microscope. The CdS-PVP QDs showed considerable activity against Staphylococcus aureus also. © 2020 Elsevier B.V. All rights reserved.

1. Introduction Quantum dots (QDs) have emerged as important and interesting material due to their superior properties over organic dyes including broad excitation spectra, and narrow symmetric and tunable emission spectra [1]. QDs have been used as a new class of sensor materials for detecting ions [2] and small molecules [3] owing to their potential applications in many fields such as biology [4] and medicine [5]. Selective and reliable sensing of anions is generally difficult because anions often display high energy of hydration, tautomerism and low surface-charge density, which makes the recognition of anions less effective. Phosphate is a ubiquitous contaminant of ground and surface water [6,7]. Owing to its nature, it can be present as inorganic and/or organic phosphorus. Through biological action in the environment, all phosphorus is eventually converted to inorganic forms. The presence of phosphate in drinking water is a matter of concern. A very high concentration of phosphate is still often found in natural waters and sediments due to the use of several detergents and fertilizers [7]. The

⁎ Corresponding author. E-mail address: [email protected] (A.P. Chattopadhyay).

https://doi.org/10.1016/j.saa.2020.118256 1386-1425/© 2020 Elsevier B.V. All rights reserved.

maximum permissible concentration of phosphate in drinking water recommended by the World Health Organization is 1 mg L−1 [8]. Phosphate measurements are also important for clinical diagnosis of various disorders. Hyper-parathyroidism, hypertension [9], vitamin-D deficiency, mineral and bone disorder [10] and franconia syndrome [11] are some of the clinical conditions where determination of phosphate concentration in body fluids is necessary. Phosphate levels in body fluids can also provide useful information about several diseases such as kidney failure [10]. The adverse effects of abnormally elevated blood level of phosphate (hyperphosphatemia) such as calcium phosphate deposit can lead to kidney damage. Phosphate is a known genetic component of cells responsible for production of proteins in living systems. In biological organisms, inorganic phosphate is essential for storage and supply of energy (as ATP) and for the formation of bone-sugar-phosphate backbone of nucleic acids (RNA and DNA) [12]. Adenosine triphosphate (ATP) in cells has recently been analyzed by Chen et al. [13] Phosphate ions and its derivatives play an important role in energy and signal transduction [14,15]. Therefore the determination of accurate levels of inorganic phosphate is important in elucidating chemical processes occurring within a living body. There are several methods for the estimation of phosphate ion concentration in the literature, such as classical methods [16],

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conductometry [17], voltammetry [18], spectrophotometry [19] etc. Although some of these methods are quantitative, most of them require expensive instrumentation, complicated sample preparation, damage to environment, coexistence of interfering ions and are not compatible for in vivo studies. During the past decades, development of fluorescent chemosensors have become an important research field in chemistry because of their simplicity, high selectivity and sensitivity, together with the advantages of spatial and temporal resolution to analyze and measure the amount of biologically important species both in vitro and in vivo [20]. There have been several reports of using QDs as probes to detect phosphate in biological system [21] including monohydrogenphosphate [22], dihydrogen-phosphate [23], poly-phosphate [24], pyrophosphate [25] and several phosphate-containing compounds [26] like ADP, ATP etc., but the use of CdS-PVP quantum dots as a fluorescent turn-on sensor for inorganic phosphate ion is still rare. The present work deals with the synthesis of polyvinylpyrrolidone capped cadmium sulphide quantum dots (CdS-PVP QDs) which have been duly characterized by DLS, AFM, TEM and HRTEM analysis. The CdS-PVP QDs behave as fluorosensor highly selective for monohydrogen phosphate ion (HPO2− 4 ) with significant enhancement in fluorescence intensity. In the pH range 7.2–7.4, i.e. at biological pH, phosphate ions (PO3− 4 ) exist as mono- hydrogen phosphate ions (HPO2− 4 ) predominantly. For this reason we have used HPO2− instead of PO 3− ions throughout the experiment for 4 4 more convenient result stimulated by the literature survey [22,27]. These cell permeable CdS nanoparticles in PVP showed considerable activity against Staphylococcus aureus. To the best of our knowledge, these CdS-PVP QDs are a novel type of cell permeable inorganic phosphate ion sensor, applicable in real sampling analysis. 2. Experimental section 2.1. Synthesis of CdS QDs (L) CdS nanoparticles were prepared by ordinary sol-gel method at room temperature [28]. At first, 290 mg of PVP was dissolved in 25 ml mili-Q water. Then 3.6 mg of Na2S was added to 5 ml of this PVP solution

taken in a separate beaker. 12 mg of Cd(OCOCH3)2.2H2O was then dissolved to the other 20 ml of the PVP solution. Then the Na2S solution was added to the cadmium salt solution drop by drop with a micropipette with constant and uniform stirring. After complete addition of Na2S solution a yellow coloured transparent solution of CdS QDs was obtained. This yellow coloured solution was stored in a sealed bottle under cold condition which has been duly characterized by FTIR, DLS, AFM studies and finally by TEM and HRTEM analysis. 2.2. Fluorescence measurement The stock solution was prepared by adding CdS-PVP (10−4 M) in Tris-HCl buffer of pH 7.2 at 25 °C. For fluorescence titrations, the 500 μL CdS-PVP (10−4 M) in Tris-HCl buffer was taken with varying con−4 centration of HPO2− M) because in this pH range inorganic 4 ions (10 phosphate mainly exists as HPO2− ions. Fluorescence measurements 4 were performed using 5 nm × 5 nm slit width. All the fluorescence spectra were taken after 10 min of mixing to get the optimized spectra. All the fluorescence experiment was repeated three times for more consistent results. To study the effect of pH, 0.15 M Tris-HCl buffer solution was used by adjusting the pH with dilute NaOH or HCl. 2.3. Ab-initio calculations To understand the mechanism of binding of phosphate and other ions to the PVP capped CdS nanoparticles, a Density Functional Theoretic (DFT) study was carried out. First, the CdS nanoparticles were modelled by a 3 layer CdS cluster. A 3 layer ONIOM model [29] was constructed, with 20 top layer atoms, 39 mid layer atoms and 40 low layer atoms. The top layer is described at DFT level, with SV basis set [30] and B3LYP functional [31]. The middle layer is treated at PM6 [32] level, and the lowest layer is described by a universal force field (UFF) [33]. All calculations are carried out in water, with the solvent treated by Onsager's self-consistent reaction field (SCRF) method [34], with polarized continuous medium (PCM) approximation [35] At first, the geometry is optimized, with the constraint that only the top layers are allowed to relax. The optimized geometry is shown in Fig. S1. Then, an octamer of polyvinyl-pyrrolidone (PVP) molecules

Fig. 1. FTIR spectra of PVP (top), CdS-PVP nanoparticles (middle) and CdS-PVP with added phosphate ions (bottom).

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nutrient broths were inoculated with Bacillus thuringienses, Staphylococcus aureus and Escherichia coli and sterile potato dextrose broth was inoculated with Candida albicans. The 24 h old cultures were spread on sterile agar plates. The plates were kept for 10 min to allow adsorption of the inoculums, and then cork borer was used to cut holes into the agar medium. Next, 100 μl of each samples were poured into separate holes and 10−4 M Cd(OCOCH3)2.2H2O in PVP solution (290 mg PVP in 25 ml double distilled water) was used in one hole as control (See ES3† for details). The plates were incubated at 37 °C. 2.5. Cell imaging study

Fig. 2. Powder XRD spectrum of CdS-PVP nanoparticles.

were treated at the DFT level of theory, in water as solvent. Next, the PVP octamer was placed on top of the 3 layer CdS model, and the geometry was optimized of the PVP octamer alone, keeping the CdS geometry fixed. This part was also done at a 3 layer ONIOM level, with the PVP octamer being added to the top (high) layer, treated at the DFT level. Solvent effect, with water as solvent, was considered at SCRF (PCM) level as before. Finally, 10 HPO2− 4 ions were added to the above geometry, and geometry of the ions along with the CdS-PVP complex were optimized at 3 layer ONIOM level. The top CdS layer and the PVP octamer were treated at high (DFT) level as before. The next CdS layer and the HPO2− 4 ions were considered at PM6 level, and the bottommost layers of CdS were described by a UFF. Only the geometry of the PVP octamer was allowed to relax along with the 10 HPO2− ions. Theoretical FTIR 4 plots of PVP octamer, CDS and CDS-PVP complex, and the CdS-PVPHPO2− 4 were calculated. 2.4. Method for agar cup assay to determine antimicrobial property of CdS Agar diffusion method is frequently used in testing the antimicrobial properties of any chemical compounds. In the hole-plate/agar cup assay, few millimeter diameter holes are aseptically cut in the inoculated agar medium with a metal puncher and filled with the test samples. Sterile

The intracellular phosphate ions imaging behavior of CdS-PVP QDs on Candida albicans cells was studied at biological pH 7.2 using TrisHCl buffer with an inverted fluorescence microscope (Leica DM 1000 LED), digital compact camera (Leica DFC 420C), and an image processor (Leica Application Suite v 3.3.0). Candida albicans cells (IMTECH No. 3018) and Bacillus sp. (strain isolated in our laboratory as a biopesticide agent for controlling looper pest of tea and identified on the basis of 16S rDNA gene sequence homology) cells from exponentially growing culture in potato dextrose broth (pH 5.2, incubation temperature 37 °C) and nutrient broth (pH 7.2, incubation temperature 29 °C) respectively were washed by suspending them in phosphate buffer saline and centrifuged at 3000 rpm for 10 min, washed twice with 0.15 M Tris-HCl buffer (pH 7.2). Then cells were treated with CdS nanoparticle solution (10−4 M) for 1 h. After incubation, the cells were again washed with 0.15 M Tris-HCl buffer (pH 7.2) and observed under a Leica DM 1000 Fluorescence microscope with UV filter. Cells without HPO2− 4 ions salt treatment but incubated with probe were used as control. After that the cells were incubated with varying concentration of the HPO2− 4 ions. 2.6. Measuring of phosphate ion in real urine sample Urine samples were prepared following the method reported previously [27]. Fresh urine samples were obtained from 3 healthy volunteers throughout the day in a 24-hour period and were stored in a refrigerator at 4 °C. 4 mL samples were taken from the stored urine with filtration through Whatman 41 filter paper to remove particulate matters. The samples were diluted 25 fold with 0.15 M Tris-HCl buffer solution at pH 7.2 and then equilibrated for 30 min at room temperature. The accuracy and precision of each measurement were evaluated by spiking 10 μM concentrations of inorganic phosphate ions into the urine samples under the optimized condition [22].

Fig. 3. TEM image (A) and HRTEM image (B) of CdS nanoparticles.

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No. of nanoparticles

25

20

15

10

5

0

1

2

3

4

5

6

7

Size of nanoparticles (nm)

(A)

(B)

Fig. 4. (A) Size distribution of CdS nanoparticles based on the TEM image. (B) SAED pattern of CdS-PVP nanoparticles.

3. Results and discussion FTIR spectra of PVP molecules, CdS-PVP nanocomposite and CdSPVP with added phosphate ions are shown in Fig. 1 from top to bottom in that order. FTIR spectra show that stretching frequency of amide carbonyl group of PVP at 1662 cm−1 decreases by ~11 cm−1 on interaction with CdS, indicating possible interaction of carbonyl oxygen atom of C_O and. CdS quantum dots which makes the N atom slightly electropositive (Nδ+–C-Oδ-). That stretching frequency is further reduced by 21 cm−1 on addition of phosphate ions. At pH ~7.2, the added phosphate ions re2− main as HPO2− 4 ions. These HPO4 ions presumably activate the amide carbonyl through interaction, consequently the amide carbonyls in the ensemble get more involved in co-ordination with CdS. This is evident from lowering of amide carbonyl stretching by ~21 cm−1 in presence of PO3− 4 ions. The X-ray powder diffraction (XRD) pattern of crystalline CdS-PVP nanoparticals, shown in Fig. 2, are in good agreement with that of pure cubic phase CdS (JCPDS 65-2887). The three peaks correspond to the three crystal planes of (111), (220) and (311) respectively [36]. Characterization of CdS-PVP nanoparticles was also done by TEM and HRTEM which confirmed the spherical shapes of nanoparticles. TEM (Fig. 3A), HRTEM (Fig. 3B) and the particle size distribution bar plot (Fig. 4A) shows that particles are spherical, crystalline and their diameters remain between 2 and 6 nm. Majority of these CdS nanoparticles have diameter 4. Fig. 4B shows the electron diffraction patterns (SAED) of an area containing some nanoparticles. The SAED pattern shows a set of rings instead of spots due to the random orientation of the nanoparticles. The three rings correspond to the (111), (220) and (311) planes of the cubic CdS phase respectively. This result was consistent with that obtained from powder XRD analysis. AFM study of CdS-PVP nanoparticles also confirms the synthesis of spherical CdS-PVP nanoparticles (Fig. S3, in Supplementary Information).

remained the same for pH range of 5 to 11, maintaining the centre of the bands at same position in this pH range. From the spectra it is clearly evident that the emission intensity increased whenever pH of the solution was changed from 4 to 5, then it remained the same both in magnitude of intensity and in band position. During the pH change of 4 to 5, the negative charge density may increase around the CdS-PVP NPs and thus the fluorescence intensity increased and the same did not change after successive change of pH (from 5 to 11) as the NPs are stable in this pH range. 3.2. Effect of temperature Effect of temperature on fluorescence emission intensity of the CdSPVP nanoparticles was checked to assess the stability of the nanoparticles. It was found that the NPs are very much stable in the temperature range of 5–70 °C. Effect of temperature on fluorescence emission intensity is shown in Fig. S5 (Supporting Information). The fluorescence

3.1. Effect of pH Fig. S4 (Supplementary Information) shows the effect on fluorescence of CdS-PVP nanoparticles at different pH varying from pH 4–11 in steps of 1 unit. The emission band centered at 428 nm of the NPs was at a lower intensity at pH 4. The emission intensity increased and

Fig. 5. Fluorescence titration graph of CdS nanoparticles embedded in PVP (500 μL) in the presence of different concentrations of HPO2− 4 ions (0, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 450, 500, 550 μL).

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Fig. 6. (Left) Change in colour of CdS QDs in PVP (A) in absence of HPO2− ions (B) in presence of HPO2− ions; (Right) Calibration graph of linearity range (1.25 × 10−6 to 4 4 2.00 × 10−5 mol L−1) for the detection of HPO2− 4 ions from the corresponding fluorescence titration data at λem = 428 nm in 0.15 M Tris-HCl buffer solution.

emission intensity was more or less same in the temperature range, clearly indicating that the interaction of PVP with the CdS as per our proposition remain unchanged in this temperature range.

fluorescence data were recorded three times in each case, with almost similar results obtained.

3.3. Fluorescence studies of CdS QDs

3.4. Selectivity study

On excitation at 290 nm, CdS QDs in PVP exhibit weak emission intensity at 428 nm. On addition of 1 × 10−4 M HPO2− 4 ions (0–550 μL), fluorescence intensity at 428 nm increased significantly and systematically by about 9 fold (Fig. 5). This spectral feature for the addition of HPO2− ions was evidenced by the fluorescence colour change from 4 colourless to blue in presence of UV light (Fig. 6). The linearity of this experimental method of detection of HPO2− 4 ions was verified (vide Fig. 6) and it was found to be 1.25 × 10−6 to 2.00 × 10−5 mol L−1 of HPO2− 4 ions within a very short response time (10–15 s). It was found that the CdS nanoparticles in PVP have excellent stability, tested between pH 5 and 10, making them promising for real applications. Photostability of the nanoparticles is also promising. All the

The fluorescence response of CdS nanoparticles in PVP towards different ions was investigated with 1 × 10−2 M of alkali (K+), alkaline earth (Mg2+, Ba2+), transition metal ions (Zn2+, Mn2+, Cu2+, Co2+), 2− 2− − 2− 2− anions (Cl−, CO2− 3 , Cr2O7 , MoO4 , NO2 , S2O3 , SO4 ) and also 2− HPO4 ions (Fig. S6, Supporting Information). The result shows that CdS-PVP QDs have excellent selectivity to HPO2− ions in comparison 4 to other cations and anions. Initially 0.5 ml 10−4 M CdS nanoparticles in PVP were added to 1.5 ml of 0.15 M Tris-HCl buffer solution and mixed well for taking blank emission spectroscopy of CdS-PVP. Then different ions were added to test the selectivity. Fig. S6 shows that the presence of HPO2− 4 ions enhances significantly the fluorescence intensity of CdS-PVP in comparison to other ions.

Scheme 1. Proposed mechanism of fluorescence enhancement in CdS-PVP-HPO2− 4

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Scheme 2. Proposed mechanism of complexation of CdS-PVP with HPO2− 4 ions.

3.5. Interference study To verify further the practical applicability of PVP capped CdS NPs as a HPO2− 4 selective fluorescence sensor, we performed competition experiments at the same condition (Fig. S7, Supporting Information).

There is no obvious change in emission spectra on addition of the selected competitive ions to CdS NPs and HPO2− ion solution (Fig. S7). 4 These results demonstrate that other surveyed ions are noninterfering with the CDS NPs. Thus it can be said that our CdS NPs can be practically and efficiently used for HPO2− 4 ion detection.

2− Fig. 7. DFT optimized structure of CdS-PVP-complex (left) and CdS-PVP-HPO2− 4 ions (right). 20 top layer Cd and S atoms, PVP octamer and 10 HPO4 are shown in ball and stick. Color code is S = yellow, Cd = ochre, O = red, P = orange, C = ash, N = blue. 3rd layer Cd, S atoms are faint dots. 2nd layer atoms are bolder. Model and methodology proposed for the calculations, and consequently on the results.

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3.6. Binding constant calculation The binding constant value was determined from the emission intensity data following the modified Benesi-Hildebrand equation: [37].

1=ð Fx −F0 Þ ¼ 1=ð Fmax − F0 Þ þ ð1=K ½CÞð1=ð Fmax −F0 Þ where F0, Fx, and Fmax are the emission intensities of probe, CdS QDs 2− considered in the absence of HPO2− 4 ions, at an intermediate HPO4 ions concentration, and at a concentration of complete interaction, respectively. K is the association constant and [C] is the concentration of HPO2− ions. K value (1.11 × 104 M−1 for CdS QDs) was calculated 4 from the intercept/slope using the plot of (Fmax \\F0)/ (Fx-F0) against [C]−1 (Fig. S8, Supporting Information). From the value of K, it is seen that CdS QDs has a stronger binding affinity towards the HPO2− 4 ions. 3.7. Effect of salinity The effect of salinity on the binding of CdS-PVP nanoparticles with the HPO2− was checked with the help of fluorescence spectroscopy 4 (Fig. S9, Supporting Information). It was found from the fluorescence spectra that the emission intensity did not change when the salinity of the solution (of CdS-PVP NPs and HPO2− 4 ) was varied from 0 mM to 100 mM by adding NaCl. Thus it is clearly evident that the increased salinity does not have any effect on CdS-PVP-HPO2− 4 ensemble. 3.8. Detection limit calculation To calculate the detection limit, the calibration curve (Fig. S10, Supporting Information) in the lower region were drawn. From the slope of the curve (S) and the standard deviation of seven replicate measurements of the zero level (σzero) the detection limit was estimated using the equation 3σ/S [38]. In the present study, the detection limit or the limit of detection (LOD) of CdS QDs for HPO2− ions was found 4 to be 213 nM. 3.9. Mechanism The mechanism of selectivity of phosphate ions by the CdS-PVP QDs, through the formation of a CdS-PVP-HPO2− complex, is shown in 4 Scheme 2. Probably the CdS nano-particles embedded in PVP solution exhibits high selectivity for HPO2− due to the complex formation to4 wards the HPO2− 4 ions reflected from the high binding constant value. From the aforementioned results, a possible sensing mechanism of this sensor can be deduced (Scheme 1). In the absence of hydrogenphosphate, the slightly electropositive nitrogen atom of amide group can act as electron acceptor and lower the probability of the electron-hole recombination process resulting in the low fluorescence intensity of the CdS-PVP QDs. In contrast, in the presence of hydrogenphosphate, the slightly electropositive nitrogen atom of amide group may interact with the negative charges of hydrogenphosphates and some electrostatic interaction between the amide nitrogen atom and oxygen atoms of hydrogenphosphate group forms, obstructing the electron acceptor property of the nitrogen atom of amide functionality of PVP unit by reducing the electropositive nature of the nitrogen atom. Consequently, the possibility of the electron-hole recombination increases, and the fluorescence intensity of the CdS-PVP-HPO2− 4 ensemble is enhanced.

Fig. 8. Theoretical FTIR spectra of PVP (red), CdS-PVP (green) and CdS-PVP-HPO2− 4 (blue) obtained. Based on the agreement between experimental and theoretical FTIR spectra (Fig. 1 and Fig. 8), the following scheme (Scheme 2) is proposed for structure of CdSPVP and CdS-PVP-HPO2− 4 ions.

CdS-PVP complex with 10 monohydrogen phosphate ions (HPO2− 4 ) is shown in Fig. S14, all in Supporting Information. The optimized structures of CdS-PVP octamer and CdS-PVP-HPO2− ions are shown in 4 Fig. 7 below. HPO2− ions clearly disrupt the CdS-PVP links by getting 4 closer to CdS. Theoretical FTIR spectra of PVP, CdS-PVP and CdS-PVPphosphate systems are shown in Fig. 8 below. We have highlighted the CO stretching frequency of amide moiety only. Although the frequencies do not match quantitatively with experimental data, the change from PVP (red) to CdS-PVP (green) to CdS-PVP-phosphate (blue) shows the same trend as in Fig. 8 below. This reposes our faith on the model and methodology proposed for the calculations, and consequently on the results obtained. Based on the agreement between experimental and theoretical FTIR spectra (Fig. 1 and Fig. 8), Scheme 2 is proposed for structure of CdS-PVP and CdS-PVP-HPO24 ions. Cell imaging study. The imaging behavior of CdS-PVP on Candida albicans cells was studied at biological pH ~7.2 using the Tris-HCl buffer. Cells incubated with CdS-PVP were used as control and the images of the cells were recorded by fluorescence microscopy following excitation at 290 nm. On addition

3.10. Ab initio calculations The optimized structure of the PVP octamer is shown in Fig. S11 in Supporting Information. Geometry optimized structure of CdS-PVP octamer is shown in Fig. S12. Frontier molecular orbitals (MOs) of the PVP octamer are shown in Fig. S13. Geometry optimized structure of

Fig. 9. Fluorescence microscopic picture of Candida cells treated with CdS-PVP under UV excitation and 100× magnification. Are cell permeable (viz. Fig. 9).

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Table 1 Antimicrobial activity of CdS nanoparticle in PVP on the different microorganisms. Organisms

Cds-PVP

Control

Bacillus thuringiensis Staphylococcus aureus Escherichia coli Candida albicans

Negative Positive Negative Positive

Negative Negative Negative Negative

of CdS-PVP, the Candida cells displayed very weak fluorescence, but with the incubation of HPO2− ions they showed strong fluorescence 4 demonstrating that CdS-PVP are cell permeable (viz.Fig. 9). 3.11. Antimicrobial activity Antimicrobial activity of CdS-PVP was tested on a number of pathogenic bacterial and fungal species by agar cup assay. The CdS nanopaticles showed considerable activity against Staphylococcus aureus (Fig. S15, Supporting Information). This is very much significant because at present many S. aureus strains are becoming multi-drug resistant and scientists in the medical field are desperately searching for a potent antimicrobial agent against such resistant strains. 10−4 M Cd(OCOCH3)2.2H2O in PVP (290 mg PVP in 25 ml double distilled water) was used as control during the experiment. The control didn't show any anti-microbial activity [39]. The antimicrobial activity of CdS on different microorganisms is listed in the Table 1. Each experiment was repeated thrice in each case and similar results were obtained. The experiment of cytotoxicity was done by inoculating S. aureus cells in nutrient broth containing concentrations of nanoparticles in the range of 0, 0.2, 0.4, 0.8 and 1.6 mM and incubated for 12 h at 37 ± 1 °C. The effect of probe on the growth of the bacteria was observed by taking the optical density of cell cultures at 600 nm. Percentage of cytotoxicity of a particular probe at a particular concentration can be calculated by the formula: (x-y/x)*100, where x is the OD of the control broths where no CdS-PVP has been added and y is the OD of the culture where CdS-PVP at a particular concentration has been added. Decrease in cell density with increasing concentrations of the CdS nanoparticle in PVP indicates high level of cytotoxicity of the nanoparticles (Fig. S16). This indicates that the probe has high antimicrobial activity against S. aureus (Table 2). 3.12. Real sample analysis The phosphate urine test measures the amount of phosphate in a sample of urine collected over 24 h (24-hour urine test). The kidneys help control the amount of phosphate in the body. Extra phosphate is filtered by the kidneys and passes out of the body in the urine. If there is not enough phosphate, less is found in the urine. Kidney problems can cause high or low levels of phosphate in the urine. In order to demonstrate the feasibility of the proposed sensor in real samples, the urine samples from 3 healthy volunteers were used as a clinical sample model. There was a huge change in fluorescence intensity observed in the urine sample (Fig. 10). Tap water was taken from the Burdwan University campus, filtered through filter paper and used for the experiment without any further treatment. Further experiment was conducted by spiking the urine samples with known amount of HPO2− 4 using the standard addition technique. All the experiment was Table 2 Antimicrobial activity of CdS nanoparticle in PVP against S. aureus. Conc. of probe Cds-PVP

Optical density at 600 nm

% of cytotoxicity

0.0 0.2 0.4 0.8 1.6

1.6 0.45 0.45 0.37 0.08

– 71.88 71.88 76.87 95

mM mM mM mM mM

Fig. 10. Change in fluorescence intensity of CdS-PVP nanocomposite in presence of HPO2− 4 ions added and HPO2− 4 ions present in the urine samples [volunteer IDs (V -1, 2 and 3)].

repeated for three times and the satisfactory results obtained by the proposed sensor were summarized in Table 3. The results revealed the accuracy of the developed method or the determination of HPO2− 4 ions concentration in the case of natural water and real urine samples [22,27]. To validate our proposed method of estimation of HPO2− in 4 real samples we compared our obtained result with the result obtained from a standard method. Here in this case we choose the standard colorimetric estimation of phosphate by the formation of phosphomolybdate with added ammonium molybdate followed by reduction with hydrazine in acidic medium. Molybdate ions and orthophosphate condense in acidic medium to give molybdophosphoric (phosphomolybdic) acid, which upon selective reduction (perhaps with hydrazinium sulphate) produces a blue colour, due to molybdenum blue with unsure composition. The generated blue colour intensity is proportional to the quantity of phosphate and follows Lambert–Beer's law at 820 nm. From the UV–Vis spectra of known concentrations, a calibration curve is obtained (Fig. S17, Supporting Information). From this calibration curve, molar absorptivity is calculated to be 26,440 L mol−1cm−1 at 820 nm. Now from the absorbance of the spiked real samples the concentrations are known. Our present method is in good agreement with the standard colorimetric method. To have a better understanding a data validation table is given below (Table 4). 4. Conclusion A new fluorescent nanosensor for inorganic phosphate (HPO2− 4 ) ions has been developed, which is highly selective for phosphate ions with little interference from other ions. This is probably the first report of an inorganic HPO2− ions selective fluorescent nanosensor duly 4 established by the spectroscopic studies. The sensor CdS-PVP nanoparticles exhibit high selectivity for HPO2− 4 ions not only in abiotic systems Table 3 Estimation of HPO2− 4 ions in water and urine sample by spike test. Sample

HPO2− 4 added (μM)

HPO2− 4 estimated (μM)

Error (%)

HPO2− 4 found (μM)

Distilled water Tap water Volunteer-1 Volunteer-2 Volunteer-3

10

10.1 ± 1

1

0

10 10 10 10

10.2 ± 1 16.22 ± 2 16.94 ± 3 17.79 ± 1

2 – – –

0 6.22 ± 2 6.94 ± 3 7.79 ± 1

S. Dhar et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 234 (2020) 118256 Table 4 Comparison of two different analytical techniques. Sample

HPO2− 4 estimated (μM) (Present method)

HPO2− 4 estimated (μM) (Colorimetric method)

Distilled water Tap water Volunteer-1 Volunteer-2 Volunteer-3

10.1 ± 1 10.2 ± 1 16.22 ± 2 16.94 ± 3 17.79 ± 1

10.219 10.442 16.089 17.065 17.617

± ± ± ± ±

1 2 1 2 2

but also in living cells, presumably due to the complex formation tendency towards the HPO2− 4 ions. The scheme proposed, Scheme 1, is supported by fairly large-scale ab-initio DFT calculations. Cytotoxicity study indicates that the probe has high antimicrobial activity against S. aureus. Moreover the cell permeability probe is also useful for the detection of HPO2− ions in real urine samples. The simplicity of analysis and the 4 competitive LOD provide a compatible approach for routine monitoring of phosphate ions in various urine samples. CRediT authorship contribution statement Swastika Dhar: Conceptualization, Formal analysis, Writing original draft. Buddhadeb Sen: Formal analysis. Subhra Kanti Mukhopadhyay: Writing - original draft. Asoke Prasun Chattopadhyay: Writing - original draft. Trinetra Mukherjee: Formal analysis. Sadhan Pramanik: Conceptualization, Formal analysis, Writing - original draft. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments The authors gratefully acknowledge the financial assistance from Minor Research Project from University Grants Commission (vide letter No. F.PSW-019/13-14 (ERO) dated 18.03.14), New Delhi, India. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.saa.2020.118256. Input files and associated data from ab-initio calculations can be obtained on request to the corresponding author. References [1] A.P. Alivisatos, Science 271 (1996) 933–937. [2] (a)J. Chen, A. Zheng, Y. Gao, C. He, G. Wu, Y. Chen, X. Kai, C. Zhu, Spectrochim., Acta A 69 (2008) 1044–1052. (b) M. Algarra, B.B. Campos, B. Alonso, M.S. Miranda, A.M. Martínez, C.M. Casado, J.C.G. Esteves da Silva, Talanta 88 (2012) 403–407; (c) J. Wang, X. Zhou, H. Ma, G. Tao, Spectrochim. Acta A 81 (2011) 178–183; (d) X. Wang, Y. Lv, X. Hou, Talanta 84 (2011) 382–386. [3] (a) C.P. Huang, S.W. Liu, T.M. Chen, Y.K. Li, Sens. Actuators B: Chem. 130 (2008) 338–342; (b) S. Huang, Q. Xiao, R. Li, H.L. Guan, J. Liu, X.R. Liu, Z.K. He, Y. Liu, Anal., Chim. Acta 645 (2009) 73–78. [4] (a) G.L. Wang, Y.M. Dong, H.X. Yang, Z.J. Li, Talanta 83 (2011) 943–947; (b) P. Wu, Y. He, H.F. Wang, X.P. Yan, Anal. Chem. 82 (2010) 1427–1433. [5] (a) J. Liang, S. Huang, D. Zeng, Z. He, X. Ji, X. Ai, H. Yang, Talanta 69 (2006) 126–130; (b) A.H. Gore, U.S. Mote, S.S. Tele, P.V. Anbhule, M.C. Rath, S.R. Patil, G.B. Kolekar, Analyst 136 (2011) 2606–2612. [6] R. Gächter, B. Wehrli, Environ. Sci. Technol. 32 (1998) 3659–3665. [7] (a) W. Yao, F.J. Millero, S. Millero, J. Environ. Sci. Technol. 30 (1996) 536–541; (b) C. Bard, W.H. Freeman and Co, New York, 1996. (c) R. Gächter, J.M. Nagatiah, C. Stamn, Environ. Sci. Technol. 32 (1998) 1865–1869. [8] World Health Organization (WHO), Guidelines for Drinking Water Quality, World, Health Organization, Geneva, Switzerland, 1993.

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