One pot fabrication of fluorescein functionalized manganese dioxide for fluorescence “Turn OFF–ON” sensing of hydrogen peroxide in water and cosmetic samples

In recent decades, H2O2 has been promoted as a health indicator because its moderate to high levels can cause some health problems. Herein, we developed a new fluorescent nanoprobe for rapid, selective and sensitive detection of H2O2. The fluorescent nanoprobe is composed of fluorescein dye (FLS) as a fluorescent probe and MnO2 nanosheets (MnO2 NS) as a quencher. In this study, H2O2 can reduce MnO2 NS in the synthesized composite and release FLS, causing sufficient recovery of fluorescent signal related to the concentration of H2O2. The nanoprobe, with λex/λem at 495/515 nm, has a linear range of 0.04–30 μM, with a limit of detection (LOD) of 7.5 nM and a limit of quantitation (LOQ) of 21 nM. The mean relative standard deviation (RSD) was 2.6% and the applicability of the method was demonstrated by the determination of H2O2 in water and cosmetic samples.


Introduction
Hydrogen peroxide (H 2 O 2 ), a colorless liquid usually produced as aqueous solutions of various strengths, is used principally for bleaching cotton and other textiles and wood pulp, in the manufacture of other chemicals, as a rocket propellant, in cosmetics and for medicinal purposes. 1 From a biological point of view, H 2 O 2 is formed in humans and other animals as a short-lived product in biochemical processes and is toxic to cells. The toxicity is due to oxidation of proteins, membrane lipids and DNA by the peroxide ions, so it can be a serious health hazard as a high level of H 2 O 2 can precipitate cancer in the duodenum of mice aer drinking water administration at 0.1% (w/w). 2 The systemic effects of H 2 O 2 result from its interaction with the intracellular catalase enzyme accompanied by the liberation of oxygen and water upon its decomposition. One milliliter of 3% hydrogen peroxide liberates 10 mL of oxygen. When the liberated oxygen exceeds the maximum blood solubility, intravascular oxygen embolism may occur. 3 Repeated exposures to hydrogen peroxide vapor may cause chronic irritation of the respiratory tract and partial or complete lung collapse. Also, inhalation or ingestion of high concentrations of hydrogen peroxide can result in seizures, cerebral infarction, or cerebral embolism that may end in permanent neurological decits or death. 4 Therefore, the analytical methodology must be available for the determination of H 2 O 2 to investigate its physiological functions and diagnosing diseases.
The integration of uorescent nanoprobes and other effective nanostructures in a cross-linked matrix has been widely utilized in the fabrication of typical sensors. The quenching effect induced by nanomaterials towards up-conversion nanoparticles and luminescent probes has already been used to improve uorescence sensing platform. [21][22][23][24] Nevertheless, a crucial drawback of expanding the practical application of these sensing frameworks is the narrowed range of materials for switching the uorescence of probes.
Manganese dioxide (MnO 2 ) has possessed a considerable amount of current interest because manganese(II) is the twelh most common element on the planet and the third most abundant transition element aer iron and titanium. 25 Manganese(II) ions act as cofactors in many functional enzymes with diverse mechanisms and a cornerstone in the oxygen-evolving units of photosynthetic tissues. 26 Additionally, MnO 2 has structural diversity; nanosheets, nanorods, nanospheres, nanobelts, nanowires, nanotubes, nanobers and so on, which moreover expand its applications in a varied range of elds. Among the various MnO 2 nanostructures, nanosheets provide adequate specic surface areas and high surface-to-volume ratios, permitting facile physicochemical interaction between reactants and its active sites. MnO 2 NS can be internally reduced to Mn(II), which in turn is considered to be friendly from the environmental and health points of view. 27 It is worth to mention that, most of the reported MnO 2 NS based uorescent sensors offer some limitations such as an expensive reagent, time-consuming, low sensitivity, and poor MnO 2 NS dispersity and complicated synthesized process. [28][29][30][31][32] Fluorescein (FLS) FLS has attracted great interest in the fabrication of nanoprobes because FLS has been commercially available and so, avoiding complex preparation of emissive nanomaterials. Moreover, FLS has many functional groups that can be easily functionalized with MnO 2 NS. Besides, the aqueous solubility of FLS enables the determination of several analytes in watery environment. 33 FLS modied MnO 2 NS was reported for analysis that relied on the distance between them, which was regulated with adsorption or desorption from MnO 2 NS. 34 In the proposed sensing system, we successfully synthesized MnO 2 NS and uorescein nanoprobe via a template-free, one step sonically treatment. The synthesized MnO 2 NS, in turn quench the relative uorescence intensity (RFI) of uorescein dye through uorescence resonance energy transfer mechanism (FRET).
In addition to being an efficient nanoquencher for the uorescence nanoprobe, the synthesized MnO 2 NS can also act as recognition agent for H 2 O 2 as the latter can provoke the decomposition of the MnO 2 NS which are selectively reduced into Mn 2+ , accompanying the dependent recovery of uorescence intensity of FLS dye.

Reagent and materials
Double distilled water (DDW) was used along the whole work. Fluorescein, glycine, ascorbic acid, sucrose, urea and ferric chloride were purchased from Alpha chemical, Mumbai. Cadmium nitrate and zinc sulfate were purchased from El-Nasr pharma, Egypt. Hydrogen peroxide, potassium permanganate and sodium thiosulfate were purchased from Adco pharma, Egypt. Maltose and copper sulfate were purchased from Lab Chemicals Trade -LCT, Egypt. Potassium hydrogen phosphate, magnesium chloride, calcium chloride, and ferrous sulfate were purchased from Oxford Laboratory Chemicals, India. Glutathione, cysteine, glucose and glucose oxidase enzyme R2 GOD 2701018 were purchased from Biomed Pharmaceutical industry, Egypt. Other reagents and chemicals were purchased from Modern Cairo For Chemicals -Chema Chems, Egypt. Oxygen water bottles (10%, v/v) were purchased from Liza Company, Egypt. Bleach cream was purchased from Ox Light, My Way Skin Clinic Limited, Egypt.
The standard solution of H 2 O 2 was prepared by diluting 5.5 mL H 2 O 2 to 500 mL with DDW. Phosphate buffer solution (PBS) was prepared via mixing 80 mL of Na 2 HPO 4 0.5 M (35.5 g Na 2 HPO 4 /500 mL DDW) and 30 mL of NaH 2 PO 4 0.5 M (30 g NaH 2 PO 4 /500 mL DDW) that has been reconstituted to 500 mL by DDW and adjusted to pH 7 by adding appropriate amounts of the 0.5 M NaH 2 PO 4 solution. 35 (0.04 M H 3 BO 3 , 2.04 g/100 mL) was mixed with (0.04 M H 3 PO 4 , 2.8 mL of 85% H 3 PO 4 /100 mL), and (0.04 M CH 3 COOH, 2.3 mL/100 mL) and set to the proper pH with NaOH to obtain Britton-Robinson (B. R) buffer. 36

Instrumentation
An Adwa AD11P pH-meter (Romania) was used to measure pH values. The UV-Vis and luminescence measurements were carried out by Shimadzu UV-Vis (1601/PC, Japan) and a SCINCO FS/2 FluoroMate (Korea) spectrometers, respectively. Fouriertransform infrared (FT-IR) spectra were carried out by Nic-olet™ iS™10 FTIR, Slovenia in the range of 400-4000 cm À1 . The surface morphology images of FLS@MnO 2 NS was done by scanning electron microscope (SEM), Hitachi and Transmission Electron Microscope (TEM, JEM-100CX II, USA). The phase crystallinity prole of FLS@MnO 2 NS was studied utilizing a Philips X-ray diffractometer (1710 PW, Cu Ka radiation l ¼ 1.5405 A, 40 kV voltage, 30 mA current, and 0.06 min À1 scanning rate, UK). Elemental analysis was performed using OXFORD INA energy dispersive X-ray instrument (EDX). The powder X-ray diffraction (PXRD) was scanned by Philips X-ray diffractometer PW 1710 supplied with 40 kV operating applied voltage, 30 mA current, 0.06 min À1 scanning rate in the 2q range of (4-60 ) and Cu Ka radiation (l ¼ 1.5405 A).

Preparation of uorescein modied manganese dioxide nanosheets (FLS@MnO 2 NS)
The FLS@MnO 2 NS were synthesized by a facile ultrasonic coprecipitation route. Scheme 1 shows a schematic illustration of the synthesis process. Briey, FLS (0.01 g) and of Na 2 S 2 O 3 (0.03 g) were dissolved in 200 mL PBS pH 7 and then 0.2 g KMnO 4 was added to the solution at room temperature. The output mixture was sonicated for 30 min until the entire discharge of the pink color of permanganate and a brown colloid was formed. Subsequently, the brown colloid allowed to centrifugation (4000 rpm for 30 min) and separation of supernatant, and then the brown precipitate was collected and washed with DDW and absolute ethanol three times. Aer that, the precipitate was dried at 60 C for 3 h in an electric oven and a puried 10 mg was dispersed in 10 mL DDW (1 mg mL À1 ) for further characterization and application.

Detection assay of H 2 O 2 using FLS@MnO 2 NS
350 mL FLS@MnO 2 NS aqueous solution (0.35 mg L À1 ) and 500 mL various concentrations of H 2 O 2 were added to 150 mL B. R. (pH 6.0). Aer 12 min at room temperature, the uorescence spectra were observed under excitation of 495 nm.

Application to real samples
1.0 mL of oxygen water 10% v, v that is equivalent to 0.89 M, was diluted with DDW before application of the proposed uorometric method.
0.1 g of bleach cream was dissolved in 20 mL of ethyl alcohol and stirred until complete dissolution, and the volume was completed to 100 mL calibrated ask. Different aliquots of the prepared solution were taken and analyzed by the proposed method.
Ibrahimia conduit water (Assuit, Egypt) was ltered four times through a qualitative lter paper to remove the insoluble matters, preserved in high-quality clean plastic container, and stored at 4 C. 37,38 The conduit water samples were spiked with known concentrations of H 2 O 2 (5, 10, 15, 20, 25 mM) and were analyzed using the general procedure.

Strategy of H 2 O 2 detection using FLS@MnO 2 NS
The master plan for H 2 O 2 determination was established on the capability to modulate the quenching of FLS luminescence that induced by MnO 2 NS (Scheme 2).
An organic uorophore, FLS as an energy donor, was adsorbed on the exterior surfaces of MnO 2 NS, the nanosheets structure was primarily formulated thanks to the sonically reduction of permanganate by Na 2 S 2 O 3 in PBS (pH 7). Since MnO 2 NS has a wide absorption band ranging from 390 to 600 nm that remarkably interferes with the emission of FLS, the uorescence of FLS can be efficiently faded by MnO 2 NS.
Small quantities of H 2 O 2 can mediate the redox pathway by which MnO 2 turned into Mn 2+ leading to the decomposition of the MnO 2 NS accompanied by uorescence restoration (Fig. 1).
The aforementioned in situ redox can be exemplied as the following equation: 31,39

Characterization of FLS@MnO 2 NS
The morphology of MnO 2 NS and the formed FLS@MnO 2 NS was characterized by TEM which revealed a lamellar nanostructure with large irregular folds, showing 2D morphology with a huge surface area. Furthermore, FLS/MnO 2 NS were observed with thicker and less transparent akes that may due to FLS conjugation MnO 2 NS. These multiple folds provide high relevance due to their high surface area available for short transport paths for electrons and ions 40 (Fig. 2).
Moreover, the surface morphology of the formed FLS@MnO 2 NS was examined by SEM and the micrographs demonstrated high morphological purity. The lm surface is compact and well wrapped with ne and disparate shaped grains (Fig. 3). Also, it Scheme 2 Principle for H 2 O 2 detection using FLS@MnO 2 NS. is seen the surface looks highly porous which offers a large surface area. The high porosity and large surface area of lms provide facile oncoming in the redox process and result in a high packing density of the active material. Nano-sized material limits the electron diffusion path which offers helpful support in the redox reaction. Such type of morphology leads to the porous volume, which provides the structural foundation for the high specic performance and the nanocomposite can be used as a cheap high potential catalyst in organic oxidation reactions. 41 The phase purity and crystal structure of MnO 2 NS were inspected by PXRD. As presented in Fig. 3, the diffraction peaks which appeared at 2q ¼ 12. Comparison of the PXRD patterns of unreacted FLS, untreated FLS/KMnO 4 and FLS@MnO 2 NS showed the absence of FLS peak in the FLS@MnO 2 NS (Fig. 4), indicating an interaction between FLS and the MnO 2 NS. 44 The crosslinking of MnO 2 NS and FLS was examined by FTIR spectra of MnO 2 nanostructure (with and without FLS incorporation) and the results are shown in Fig. 1S. † The two characteristic bands between 600 and 400 cm À1 attribute to the stretching collision of O-Mn-O and were blue-shied in the nanocomposite sample by FLS. 45 Furthermore, the absorption peaks at 899 and 1009 cm À1 represent the surface -OH groups of Mn-OH for MnO 2 NS which become wider in the nanocomposite product indicating the presence of FLS. 46 In the high-frequency region, a broadband around 3400 cm À1 is observed which can be assigned to the stretching vibrations of adsorbed molecular water in MnO 2 NS product and maximize thanks to stretching vibrations of the -OH group of FLS in nanostructure. 47 The dominant peak at 1731 cm À1 can be assigned to the carbonyl stretching mode and the peaks at 1203 and 1117 cm À1 may be caused by -C-O-H stretching, implying the existence of residual hydroxyl groups 44 (Fig. 1S †).  The EDX analysis of synthesized FLS@MnO 2 NS showed the presence of Mn and O in the sample (Fig. 2S †). The chemical composition analysis using EDX conrmed the presence of Mn and O in the nanocomposite samples (Table 1S †) and was similar to the earlier studies of other researchers. 48 UV-Vis spectra (Fig. 3S †) illustrate that unreacted FLS has a maximum absorbance of 490 nm. The pink-colored solution aer adding KMnO 4 , before sonication, exhibits maximum absorbance at 540 nm. Aer sonication, the pink-colored product gradually converted into brown colloid indicating the synthesis of FLS@MnO 2 NS that acquire blue shiing from 590 to 495 nm that interfere with FLS emission (515 nm) leading to the respected quenching effect.

Optimization of the experimental conditions
Many buffers were checked to show the performance of FLS@MnO 2 NS and the best results were obtained using B. R. buffer. Fig. 4Sa † shows the effect of the media pH on the uorescence enhancement of FLS@MnO 2 NS in the presence of H 2 O 2 .
A rise in pH from 3 to 5 results in the increased uorescence enhancement efficiency of the FLS@MnO 2 NS at 515 nm aer the addition of H 2 O 2 , further increase in pH from 5 to 8 leads to a plateau, whereas a further increase in pH from 8 to 10 leads to a gradual decrease. Consequently, we selected 6.0 as the optimal pH for our study using B. R. as a buffering system.
The effect of the concentration of FLS@MnO 2 NS on the uorescence enhancement efficiency is displayed in Fig. 4Sb. † The uorescence enhancement efficiency progressively increased with the concentration up to 0.3 mg mL À1 . Exceeding that, the uorescence intensity didn't affect. Therefore, 0.35 mg mL À1 was used as the optimal concentration for further performance.
The inuence of incubation time on the uorescence intensity of the system is shown in Fig. 4Sc. † The uorescence enhancement became slow until reaching a steady state at 12 min. A further increase of time didn't lead to any further perceptible enhancement. So, 12 min was chosen as the optimum incubation time.

Calibration plot, LOD and LOQ
Aer successfully fabricated the FLS@MnO 2 NS, the chance of H 2 O 2 detection has then explored; we applied the developed procedure and inspected the uorescence response signals at serial diverse H 2 O 2 concentrations. At rst, the uorescence intensity of blank (replacing H 2 O 2 loaded sample with doubledistilled water) is nearly negligible at the selected conditions while it was progressively increased with H 2 O 2 incorporation. As the concentration of H 2 O 2 increases, the uorescence intensity increases (Fig. 5Sa †).
The addition of 30 mM H 2 O 2 led to a considerable enhancement, which indicated an almost complete recovery of free unreacted FLS. The recovery of uorescence was related to the reduction of MnO 2 which led to the degradation of the  0.998) (Fig. 5Sb †) with a mean relative standard deviation (RSD) of 2.6%. Besides, the LOD [in terms of 3.3Â standard deviation of the regression line (s)/slope(S)] was 7 nM and the LOQ [in terms of 10 Â (s)/(S)] was 21 nM.

Selectivity study
Possible interfering matters including various chemical, environmental and biological species were incubated with a solution of FLS@MnO 2 NS at the selected experimental conditions. As shown in Fig. 5, only H 2 O 2 can dissociate the nanocomposite, accompanied by discoloration of the brown, accordingly regenerate and enhance the uorescence intensity. This investigation revealed that 300 mM of a wide range of electrolytes and weakly reducing bio-agents didn't produce notable optical responses as well as didn't degrade the nanostructure as the color almost didn't change. It is worth mentioning that although some reports deduced the reduction of MnO 2 NS by ascorbic acid but in our study the proposed method not affected signicantly by ascorbic acid that may be attributed to the low acidity that not sufficient to oxidize ascorbic acid by MnO 2 NS.

Real samples analysis
To test the analytical validity of this approach, the method was applied for the determination of H 2 O 2 in pharmaceutical oxygen water, cosmetic cream and natural water using the procedures described in Section 2.5. As can be seen in Fig. 6S, † the emission spectrum for the analyzed pharmaceutical, cosmetic and natural samples is very identical to that exhibited in Fig. 5Sa. † This displays that the species existing in the inspected samples do not interfere in the estimation of H 2 O 2 by the suggested approach. The analysis details of the real sample are signalized in (Table 1) The recoveries of H 2 O 2 fall in 86.8-106.5%, implying that the synthesized nanoprobe can be efficiently used to determine H 2 O 2 in real samples, the low value of RSD% denotes the precision and feasibility of this method for determination of the respected analyte in real samples. To approve the accuracy of the proposed method, we statistically compare the results deduced by the current approach and other reported uorometric method for the determination of H 2 O 2 that was intended to be used to oxidize non-uorescent coumarin to highly uorescent 7-hydroxycoumarin. 49 As can be seen from Table 1, all the calculated t-values are below the critical t value of 2.571 for 95% condence level and 5 degrees of freedom. Therefore, the accuracy of the method for the determination of the studied analytes is conrmed.

Stability of FLS@MnO 2 NS
The stability potential of the nanoprobe is of important value from an analytical point of view. The more stable a probe is, the more is its capacity for broad applications. Subsequently, the time-stability of the FLS@MnO 2 NS was examined aer storing under normal conditions. Aer storing for one month, the material was collected by centrifugation and washed with double distilled water and ethanol, then dried in an oven at 60 C for 3 h and measure the absorbance signal of the dispersed solution. Fig. 7S † indicated that the absorbance response of FLS@MnO 2 NS was decreased slightly and no obvious change in color or morphology aer storing for one month under normal conditions.

Determination of glucose via enzymatic degradation to H 2 O 2
The proposed approach also proceeded for glucose sensing to examine the generality of the FLS@MnO 2 NS. From the standpoint of biochemistry, glucose can be catalytically oxidized by glucose oxidase enzyme and disintegrated into gluconic acid and H 2 O 2 , thus, glucose can be detected via the sensing of bioenzymatically developed H 2 O 2 . According to reported method, 50 and with slight modication, different concentrations of glucose solution (5, 10, 15, 20 and 25 mM) was successively added to 100 mL B. R. buffer (pH ¼ 6.0) and 100 mL of glucose oxidase enzyme, followed by successively mixing and Then, the dispersion of the prepared FLS@MnO 2 NS (350 mL) was added into the above solution. The resulting mixture was successively incubated at room temperature for 12 min for the further uorescence determination. As shown in Fig. 8S, † as the glucose concentration increased, the uorescence intensity increased. The uorescence intensity at 515 nm was rectilinear correlated to the glucose concentration (5-25 mM, R 2 ¼ 0.9784) proving that the FLS@MnO 2 NS is generalizable and can be utilized to detect various H 2 O 2 generating syntheses.

Comparison of the proposed method with other methods
Comparing the results in the proposed method with other published methods; from Table 2, it can be seen that the FLS@MnO 2 NS can serve as a probe for the detection of H 2 O 2 in a more wide concentration range, and can determinate them in a low concentration.
It is worth to mention that the proposed uorometric method, if compared to other reported methods, has low detection limit and low % RSD, indicating higher sensitivity and reliability of the proposed uorometric method for analysis of H 2 O 2 . Moreover, the applicability of the uorometric method was extended for the determination of the target analyte in different matrices.

Homogeneity of FLS@MnO 2 NS and reproducibility of the synthesis procedure
The homogeneity of FLS@MnO 2 NS and reproducibility of synthesis procedure were performed through analysis of ve independent batches of FLS@MnO 2 NS spectrophotometrically at 495 nm. Moreover, 3 mM H 2 O 2 was repeatedly assayed by the proposed uorimetric method in ve separate sets using ve independent batches of FLS@MnO 2 NS. The % RSD did not exceed 3.89% which conrms that the synthesis procedure of FLS@MnO 2 NS is homogenous and reproducible (Fig. 6).

Conclusion
In summary, we have developed FLS@MnO 2 NS platform via an uncomplicated one-step solution-phase passageway, by the reduction of potassium permanganate with sodium thiosulphate at room temperature with no aid of catalysts or templates and demanding no expensive and precise equipment, guarantees higher purity of the products, exceedingly diminishes the production cost and hence offers a great chance for analytical scale-up preparation of nanostructured materials. As a further matter, it is attractive that the as-synthesized FLS@MnO 2 NS can be used as an effective nanoprobe for the detection of H 2 O 2 with higher selectivity and sensitivity and applied to determine the respected analyte in real samples with  acceptable results. The proposed nanoprobe has great potential for analytical and clinical investigation. In the meantime, this nanostructure is also generalizable and can be readily continued to sense several H 2 O 2 generating substances as a logic gate application. The proposed protocol may provide a new insight to develop low-cost and sensitive methods for food, environmental, biological and clinical diagnostics applications.