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Dual-emission ratiometric probe combining carbon dots and CdTe quantum dots for fluorometric and visual determination of H2O2
Sensors & Actuators: B. Chemical 296 (2019) 126665
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Dual-emission ratiometric probe combining carbon dots and CdTe quantum dots for fluorometric and visual determination of H2O2 ⁎
Rafael C. Castro, José X. Soares, David S.M. Ribeiro , João L.M. Santos
T
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LAQV, REQUIMTE, Department of Chemical Sciences, Laboratory of Applied Chemistry, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira no 228, 4050-313 Porto, Portugal
A R T I C LE I N FO
A B S T R A C T
Keywords: Quantum dots Ratiometric assay CdTe Carbon dots Hydrogen peroxide Visual assay
In this work, blue-emitting carbon quantum dots and distinctly sized CdTe semiconductor quantum dots were combined to implement a ratiometric probe for the monitoring of H2O2. By implementing a sensing scheme that combines multiple nanoprobes, excited at the same wavelength and emitting at different ones, which exhibit also dissimilar reactivity, it was possible to minimize detrimental factors associated with the use of a single photoluminescence wavelength such as fluctuations in excitation source or measured signal, fluorophore concentration, matrix effects and background fluorescence. The developed ratiometric probe was applied on the implementation of a conventional fluorometric assay and on a visual assay relying on the red, green and blue (RGB)-based colour changes promoted by increasing H2O2 concentrations. For the fluorometric determination assay, a good linear relationship between the fluorescence intensity ratio (FICD434/FIMPA587) and H2O2 concentration within the range of 0.0100–0.2125% (w/w) was obtained (R = 0.9994, n = 7). The detection limit was about 0.00793% (w/w). The obtained results in the determination of H2O2 in contact lens solutions were in agreement with those provided by the reference procedure, with relative deviations between −4.71 and 1.89%. Regarding the RGB-visual assay, a linear working range for hydrogen peroxide concentrations up to 0.150% (w/w) was verified (n = 9) with a correlation coefficient of 0.9966, which confirms its potential as valuable analytical tool for on-the-spot semi-quantitative detection of H2O2.
1. Introduction The capacity of hydrogen peroxide for triggering oxidative processes has been widely explored as: i) food additive for controlling the growth of microorganisms; ii) hair bleaching or dyeing and fixing of hair perm; iii) household cleaning agents, including contact lens disinfection; and iv) tooth bleaching [1]. Being a harmful chemical with confirmed carcinogenicity and toxicity and considering the large quantities as it is released into the environment, it has become imperative to monitor hydrogen peroxide effectiveness and safety in several applications namely in industrial, environmental, biochemical and clinical fields. This has accentuated the need for rapid, simple, selective and sensitive methodologies for H2O2 determination, relying some of the most used procedures on fluorescence-based measurements. Indeed, the oxidizing capacity of hydrogen peroxide was explored to form either a fluorescent product from a non-fluorescent compound or to quench the emission of native fluorescent probes. In this regard, 4-amino-1H-1,5-benzodiazepine-3-carbonitrile [2], p-
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hydroxyphenylacetic acid [3], acetaminophen [4] homovanillic acid [5] and 2′,7′-dichlorodihydrofluorescein [6] are examples of some nonfluorescent reagents, which can be found in the scientific literature, that were applied in H2O2 determination based on the formation of fluorescent compounds upon redox reaction with H2O2. These reactions were usually catalysed by an enzyme, namely peroxidase or horseradish peroxidase (HRP). An indirect fluorometric method involving the strong oxidizing ability of the Fenton reaction was also employed to oxidize non-fluorescent coumarin to fluorescent 7-hydroxycoumarin, being H2O2 quantified as consequence of its concentration dependent fluorescence enhancement [7]. The fluorometric scopoletin-horseradish peroxidase method, relying on the quenching of scopoletin fluorescence upon reaction with H2O2, catalysed by HRP, has been also often used for H2O2 determination [8–11]. A similar fluorescence quenching method was based on the reaction of H2O2 with 3,3′-diethyloxadicarbocyanine iodide to form a non-fluorescent compound in acidic medium [12]. A significant drawback of the aforementioned methods is the use of HRP
Corresponding authors. E-mail addresses: [email protected] (D.S.M. Ribeiro), joaolms@ff.up.pt (J.L.M. Santos).
https://doi.org/10.1016/j.snb.2019.126665 Received 15 March 2019; Received in revised form 27 May 2019; Accepted 5 June 2019 Available online 15 June 2019 0925-4005/ © 2019 Elsevier B.V. All rights reserved.
Sensors & Actuators: B. Chemical 296 (2019) 126665
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2. Experimental
enzymes, which are expensive, instable in solution and require reaction conditions that restrain their applicability [13]. An advantageous alternative to the referred methods could be the implementation of fluorescence-based sensing schemes relying on the use of semiconductor quantum dots (QDs) as fluorophores. QDs exhibit unique optical properties such as size-dependent and tunable photoluminescence (PL), long-term photostability, high quantum yield (QY) and long excited-state decay lifetimes that assure simplicity, versatility and reliability in a variety of analytical applications. Additionally, their broad excitation bands enable the simultaneous excitation of multicolour QDs by using a unique light source [14–17]. In this regard, only a few examples have been reported dealing with the direct interaction between QDs and H2O2 that typically result in a quenching of the fluorescence of the PL probe in the presence of the oxidizing agent [18–20]. Others sensing strategies relying on indirect photoluminescence detection of H2O2 were also proposed aiming to obtain an improvement of sensitivity and selectivity of the QDs-based fluorometric methodology. Two illustrative examples are the use of H2O2 for preventing the surface passivation of 3-mercaptopropionoic acid (MPA)–capped CdTe QDs [21] and thioglycolic acid (TGA)–capped CdTe QDs [22] by reduced glutathione and hepatitis B core antibody labelled with horseradish peroxidase, respectively. All of the abovementioned QDs-based PL sensing strategies for H2O2 relied on the measurement of the fluorescence emission at a single wavelength which, although practical and easy to implement, could be hindered by several factors, such as fluctuations in excitation source, probe concentration, matrix effects in complex samples and background fluorescence [23–25]. These aspects can lead to inaccurate and irreproducible results impairing the detection and quantification of the target analyte. The addition of a second fluorophore for the development of a ratiometric fluorescent method can be used to circumvent these adverse effects. Dual-emission ratiometric fluorescent probe systems consisting in a mixture of two distinct fluorophores, one as the analyte-dependent fluorophore and the other as an internal standard, have been used for the determination of a myriad of analytes such as, metal ions [23–30], proteins [31], glucose [32,33], aspirin [34] and 2,4,6-trinitrotoluene [35]. Practical sensing devices, based on the change of materials macroscopic properties [36] or naked eye colour visualization [37,38], is crucial for the development of in situ analytical methods. Considering that on a dual-emission ratiometric fluorescent probe, one emission intensity is constant and the other can be quenched in the presence of the analyte, the colour modifications endured by the probe can be easily and obviously perceived by naked eye under irradiation with UV lamp. In fact, these kind of ratiometric probes can be applied as naked eye visual sensing platforms as is the example proposed by Xia et al in which they developed a red, green and blue (RGB)-based colour change probe for the visual analysis of formaldehyde [39]. In the present work, two distinct photoluminescent nanoparticles were exploited to implement a ratiometric assay for the determination of H2O2 in real samples. This dual-emission nanosystem involved the combination of a blue-emitting carbon dot, which due to their inertness behaviour was used as internal standard, and an orange-emitting CdTe QDs capped with 3-mercaptopropionic acid (MPA) as analyte-dependent fluorophore. The addition of H2O2 in the mixture induces the depassivation of the MPA-CdTe QDs, decreasing thus the orange-emission PL while the photoluminescence of the blue-emitting CDs remained constant. The exploitation of this effect was used to develop an analytical method for the determination of H2O2 using a conventional fluorometric assay. Additionally, a RGB-type sensing scheme based on the colour modulation of the two nanomaterials is also proposed. The PL variations and fluorescent colour changed from orange to blue were used for the detection and quantification of H2O2 in lens care solutions.
2.1. Reagents and solutions All solutions and standards were prepared from chemicals of analytical reagent grade without any pre-treatment process and using water purified from a Milli-Q system (conductivity ≤ 0.1 μS cm−1). All reagents used for the synthesis of semiconductor QDs were purchased from Sigma-Aldrich (St. Louis, MO, USA), namely, cadmium chloride hemi(pentahydrate) (CdCl2·2.5H2O, 99%), sodium tellurite (Na2TeO3, 100 mesh, 99%), sodium borohydride (NaBH4, 99%), sodium 2-mercaptoethanesulfonate (MES, C2H5NaO3S2, 98.0%), glutathione reduced (GSH, C10H17N3O6S, 99.0%), 3-mercaptopropionic acid (MPA, C3H6O2S, 99%) and sodium citrate tribasic dehydrate (C6H5Na3O7·2H2O, 99%). Citric acid (C6H8O7, ≥99.5%) and ethylenediamine (N2C2H6, ≥99.5%), both acquired from Sigma-Aldrich (St. Louis, MO, USA) were the reagents used for the synthesis of carbonbased nanoparticles. QDs and CDs intermediate solutions were prepared by direct dilution, with deionized water, in a ratio of one to twenty (1:20) and one to fifty (1:50), respectively. Hydrogen peroxide intermediate solution of 0.1% (w/w) was daily prepared by appropriate dilution of H2O2 stock solution acquired from Sigma-Aldrich (30% w/w, St. Louis, MO, USA) with deionized water. The working standard solutions of H2O2 were prepared by proper dilution of the above intermediate solution. Six different commercially available lens care solutions were prepared by diluting with deionized water an appropriate volume of the sample, in order to obtain a hydrogen peroxide content included in the analytical range of the proposed methodology. All the samples were analysed without any pretreatment process. 2.2. Equipment The synthesis of CdTe QDs were carried out in a single mode CEM Discover SP® synthesizer operated through a computer using Synergy™ software (Matthews, NC, US). This equipment was equipped with an automated pressure control/sensing system (ActiVent™), an active cooling system (PowerMAX™) and an integrated infrared (IR) sensor for the strict control of irradiation time, reaction temperature and pressure. The emission spectra of the fluorometric probes were obtained with a Jasco FP-6500 spectrofluorometer (Easton, MD, USA) and a multimode microplate reader (Cytation™ 3, BioTek™). All photographs were acquired with a Sony Cyber-shot DSC-HX10 V digital camera. The ratiometric fluorescent probes were photographed in a CN-6 UV darkroom with hand-held UV lamp 254 nm and 365 nm (6 W, model VL6.LC) 2.3. Procedure for the synthesis of MPA capped CdTe QDs and CDs The two different sized MPA capped CdTe QDs were synthesized according to the procedure proposed by Ribeiro et al [40]. This synthetic methodology consisted in a one-step synthetic route assisted by microwave irradiation. The experimental conditions for the synthesis of QDs, such as precursors concentration, pH, irradiation time were used exactly as recommended in the prior work [40]. For the syntheses of the yellow and orange-emitting CdTe QDs the temperatures were 105 °C and 115 °C, respectively. For the syntheses of CDs, our previously optimized conditions were used [unpublished results]. In short, citric acid (0.5 g) and ethylenediamine (0.3 mL) were mixed with water yielding a final concentration of citric acid of 10% (m/v). Then the pH of the reaction mixture was set to 3.8 by the addition of HCl (1 mol L−1). The reaction mixture was transferred into a 40 mL Teflon-lined autoclave and heated at 260 °C for 5 h. After cooling to room temperature, the solution was extensively dialyzed against Mili-Q water in a dialysis membrane (Spectra/Por 6 2
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1000 MWCO, Spectrum Labs™) for 5 days.
photoluminescence of the QDs was gradually lessened with increasing H2O2 concentrations until its complete disappearance. In effect, the thiol groups of MPA attached on the QDs surface through CdeS bonding can be oxidized to form an organic disulfide product (RS–SR) causing its detachment from the surface of the PL probe. As a consequence of QDs depassivation, the surface traps increased which impaired electron-hole recombination and therefore the QDs PL intensity is inhibited [42–44]. The observed blue shift can be ascribed to a probable slight QDs size reduction caused by the irreversible oxidation at the QDs surface [45,46]. This explanation was confirmed by performing the measurement of the excitation spectra of the QDs in the absence and presence of 0.050% H2O2. The results (Fig S4 – Supplementary material) demonstrated a decrease of the absorbance and a slight blue shift (2 nm) of the absorption band for the first excitonic transition with the addition of H2O2, which was accentuated (blue shift of 4 nm and 8 nm) by increasing the reaction time up to 2 and 5 min, respectively. Moreover, the linear Stern-Volmer plots that describes the PL response of the two different sizes CdTe QDs upon interaction with H2O2 demonstrated a higher sensitivity of the 588 nm emitting QDs (OQDs) (F0/F = 57.4 × [H2O2] + 1.31) relatively to the 562 nm emitting one (Y-QDs) (F0/F = 37.0 × [H2O2] + 0.89) (Figs. S2C) and S3C)).
2.4. Conventional fluorometric assay procedure Before the preparation of the ratiometric probe solution, the respective as-prepared nanoparticle solutions were diluted in deionized water. Thus, QDs and CDs intermediate solutions were prepared by direct dilution of the crude solutions with deionized water in a ratio of 1:20 (1 mL in 20 mL) and 1:50 (0.5 mL in 25 mL), respectively. The two dual-emission ratiometric probes for the detection of H2O2 consisted in a mixture of CDs and QDs in a ratio of 1:5. In this sense, 3.2 mL and 16 mL of CDs and QDs intermediate solutions, respectively, were mixed into a 50 mL graduated tube. Afterwards, 960 μL of the fluorescent probe solution and required amounts of deionized water and H2O2 were sequentially added completing the final volume of 2 mL. Immediately after the addition of H2O2, the solution was shaken and transferred into a 1 cm quartz cell and the emission spectra (λem, from 385 to 700 nm) were recorded with the excitation wavelength fixed at 360 nm. The slit widths of excitation and emission were 5.0 and 10.0 nm, respectively. 2.5. RGB-visual assay procedure
3.2. Dual-emission CDs/CdTe-QDs combined probe for H2O2 detection The ratiometric fluorescent probes prepared using the abovementioned conditions were photographed upon irradiation at 365 nm with a digital camera. RGB-type images were cropped to 460 × 160 pixel dimension image and aligned to the centre of the vial containing the probe. The cropped RGB-images were imported to Matlab version R2009b (MathWorks, Natick, MA, USA) and divided into three matrixes corresponding three channels (red, green and blue). Each matrix, with 460 rows and 160 columns, contained at each entry the intensity value (between 0–255) of the corresponding channel. Ratio between the pixel intensity at blue and red channels were obtained by dividing at each pixel the intensities on the blue and on the red channel. Average pixel intensity were calculated by the mean of mean values on each column.
3.1. Preliminary assays
3.2.1. Optimization of the CDs and CdTe-QDs mixture The blue emitting CDs (B-CDs) were mixed with each of the MPACdTe QDs (Y-QDs and O-QDs) in order to obtain two distinct dualemission ratiometric probes (B-CDs/Y-QDs and B-CDs/O-QDs) which were evaluated for application in the fluorometric determination of H2O2. In this sense, CDs and QDs were mixed in different proportions in order to enhance the efficiency of the dual-emission ratiometric probe for the target analyte. Therefore, for both probes tested, the CDs and QDs were mixed (for a final volume of 2000 μL) in a proportion of 1:1, 1:2.5, 1:4 and 1:5 (CDs:QDs), by fixing 160 μL of the diluted solution of CDs (50×) and by varying the amount of the semiconductor nanoparticle with the addition of 160, 400, 640, 800 μL of the diluted solution of QDs (20×). The PL spectra of the blue emitting CDs, yellow and orange emitting CdTe QDs and the corresponding ratiometric probes are shown in Figs. 1 and 2. Upon excitation at 360 nm, the blue emitting CDs and yellow and orange emitting CdTe QDs showed a maximum emission at 434, 560, and 587 nm, respectively. This way, the corresponding combining probes exhibited a dual emission PL band under a single-wavelength excitation (360 nm) at, namely, 434/560 and 434/587 nm. The obtained results demonstrated that by increasing the amount of CdTe QDs, the PL intensity of the QDs gradually increased, as expected, while the PL intensity of the CDs was slightly inhibited (Figs. 1B) and 2 B)). In fact, the CdTe QDs can act as an inner filter by absorbing a considerable portion of excitation photons and consequently the emission intensity of the CDs was quenched. In order to obtain an improved reactivity and sensitivity of the dual-emission probe, the ratio CDs:QDs of 1:5 was selected for further assays.
For the development of the ratiometric fluorescence probe, CDs were chosen as internal standard because they showed a remarkable chemical inertness upon interaction with H2O2. In fact, upon the presence of increasing concentrations of H2O2, up to 0.26% (w/w), the PL emission intensity, as well as the observed colour under a UV lamp, remains unchangeable (Fig. S1 – Supplementary material). On the other hand, two MPA-CdTe QDs of different size were tested to be used as the analyte-dependent fluorophore. In both QDs the fluorescence intensity is gradually quenched in the presence of increasing H2O2 concentrations (Figs. S2 and S3 – Supplementary material). Additionally, a slightly blue shift of the maximum emission wavelength was observed in both cases. The observed colour under a UV lamp corroborated the abovementioned fluorometric results, in which the yellow and orange
3.2.2. Stability of the dual-emission ratiometric probe The stability of the probe solutions is an important parameter that could affect the reproducibility and accuracy of successive fluorometric measurements performed during sample analysis. For this reason, the stability in aqueous solution of the PL intensity of the 434/560 nm and 434/587 nm arrangements was thoroughly studied. Thus, for both dualemission probes the fluorescence intensity ratios (FICD434/FIMPA560 and FICD434/FIMPA587) were monitored throughout 360 min (Fig. 3 and 4). In the case of the 434/560 nm combination, the signal remained constant up to 60 min (log (time) = 1.78) and thereafter decreased for longer period of time (Fig. 3B). Regarding the 434/587 nm probe, the analytical signal persisted unchanged over 20 min (log (time) = 1.30) and then the stability decreased by prolonging the time (Fig. 4B). This
2.6. Reference procedure To validate the results obtained by the conventional fluorometric and the image processing-based assays, the hydrogen peroxide determination were also performed by the reference methodology recommended by the European Pharmacopoeia [41]. The reference procedure involved a permanganometric titration of hydrogen peroxide in acidic medium. The potassium permanganate solution was standardized by titration with arsenic hexaoxide in acidic medium. The hydrogen peroxide amount in the samples was calculated taking into account that each mL of 0.02 mol L−1 KMnO4 solution was equivalent to 1.701 mg of H2O2. 3. Results and discussion
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Fig. 1. PL spectra (A and B) and the corresponding photographs taken under 365 nm UV lamp (C and D) of blue emitting CDs (I), yellow emitting QDs (II) and B-CDs/Y-QDs (434/ 560 nm) ratiometric probe in the CDs:QDs proportions of 1:1 (III), 1:2.5 (IV), 1:4 (V) and 1:5 (VI). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
being MPA-CdTe QDs used as the recognition element and the CDs employed as reference fluorophore. The obtained results revealed a linear relationship between the fluorescence intensity ratios (FICD434/FIMPA560 and FICD434/ FIMPA587) and the concentration of H2O2 in the range up to 0.1000% (w/w) and 0.2125% (w/w), when using B-CDs/Y-QDs and B-CDs/OQDs, respectively. The regression equations obtained from the interaction of H2O2 and B-CDs/Y-QDs and B-CDs/O-QDs probes can be expressed as FICD/ FIMPA = 10.0 ( ± 0.2) × [H2O2] + 0.37 ( ± 0.01) (R = 0.9972) and FICD/FIMPA = 20.6 ( ± 0.4) × [H2O2] + 0.73 ( ± 0.04) (R = 0.9972), respectively. From the obtained results it was observed that the linear regression sensitivity obtained for B-CDs/O-QDs probe was twice as high as for the B-CDs/Y-QDs one. In addition, both ratiometric probes also allowed the visual detection of H2O2. In fact, the changes in fluorescence intensity ratios (FICD434/FIMPA560 and FICD434/FIMPA587) with the increasing of H2O2 concentration can also be observed through the continuous variation of the colour of the fluorescence emission from yellow to blue (Fig. 7 (A)) and from orange to blue (Fig. 7(B)) in the case of the B-CDs/ Y-QDs and B-CDs/O-QDs probes, respectively. Indeed, the combination of both Y-QDs and O-QDs, as sensing probes, and B-CDs, as reference, allowed the obtaining of a wide range of colour variations which can be clearly detected by the naked eye. This can be used to demonstrate the advantage of the ratiometric probes relatively to the single fluorescence quenching of the respective
results revealed a good stability of the optical properties of both probes considering the kinetics of the reaction with the target analyte. Additionally, the analytical response (FI ratio) of ratiometric probe solution was monitored upon the addition of 0.0500% (w/w) H2O2 solution. The obtained results revealed that the reaction between the dual-emission probe and hydrogen peroxide occurred promptly. Prolonging the reaction time up to 25 min, FICD/FIMPA increased gradually due to the PL quenching of the MPA-CdTe QDs which is caused by the oxidation of capping thiol groups attached to the QDs surface (Fig. 5). Consequently, the surface depassivation promoted the occurrence of dangling bonds (mid-gap energy states) that act as surface traps and prevent radiative electron-hole recombination. Consequently, in order to guarantee a good compromise between stability and reactivity the fluorescence response was measured immediately after the addition of H2O2. 3.2.3. Fluorometric determination of H2O2 The PL properties of the 434/560 nm and 434/587 nm probes were assessed upon interaction with H2O2 at a concentration range of 0–0.26% (w/w) (Fig. 6). As expected, the two characteristic emission bands showed different responses. In both cases, the PL intensity corresponding to the CdTe QDs band at 560 and/or 587 nm were effectively inhibited by the addition of increasing concentrations of H2O2, while the 434 nm emission band of CDs was unaffected in the presence of H2O2. Hence, the combination of both nanomaterials demonstrated to be a valuable approach for the ratiometric determination of H2O2,
Fig. 2. PL spectra (A and B) and the corresponding photographs taken under 365 nm UV lamp (C and D) of blue emitting CDs (I), orange emitting QDs (II) and B-CDs/O-QDs (434/ 587 nm) ratiometric probe in the CDs:QDs proportions of 1:1 (III), 1:2.5 (IV), 1:4 (V) and 1:5 (VI). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 3. (A) PL spectra of the dual-emission B-CDs/Y-QDs (434/560 nm) ratiometric probe during 360 min. (B) Fluorescence intensity ratio of the (434/560 nm) ratiometric probe as a function of time (on logarithmic scale).
QDs Figs. S2(A) and S3(A) – Supplementary material). For each individual QDs the small slight colour changes associated with the increase of H2O2 concentration was much more difficult to perceive by naked eye when they were assayed alone than when they were combined with the CDs. In fact, the combination with the blue-emitting CDs, which exhibits a bright emission band at 434 nm, affords a reference colour that facilitates visual readout. Thus, the large separation between 434 and 560 nm and/or 434 nm and 587 nm emission peaks enables that colour changes caused by the addition of a small concentration of H2O2 could be easily discerned by the naked eye (Fig. 7). Moreover, the larger separation of the emission peaks of the B-CDs/ O-QDs combination probe (434/587 nm) relatively to the B-CDs/Y-QDs one (434/560 nm) makes it easier to detect a distinguishable colour change from the background. Therefore, the B-CDs/O-QDs combined system demonstrated to be a more efficient ratiometric probe for H2O2 because showed a more sensitive RGB-type visual read out as well as a higher sensitivity in the PL detection of H2O2. For that reason, it was chosen for the further assays.
(FICD 434) = 24.9 (± 0.4) × C + 0.94 (± 0.04) (FIMPA 587) Where in (FICD434/FIMPA587) was the ratio between the fluorescence intensity obtained by CDs and CdTe QDs and C was the H2O2 concentration. The obtained correlation coefficient was 0.9994 (n = 7) which indicates good linearity. The detection limit calculated from the equation of the calibration curve according to Miller and Miller [47] was about 0.00793% (w/w), far below the declared content of H2O2 in lens care solutions (3% (w/w)). 3.3.2. Applicability in real samples Prior to the application with real samples, the selectivity of the developed ratiometric fluorescent assay for the H2O2 determination in lens care solutions was evaluated in the presence of excipients, such as, sodium dihydrogen phosphate, sodium chloride and edetate disodium. No influence on the analytical signal (FICD434/FIMPA587 variation ≤4%) was verified by the aforementioned compounds, which was expected considering the pronounced real sample dilution that had to be performed before the analysis. The applicability of the proposed ratiometric fluorescent assay was assessed by applying it in the determination of H2O2 in commercially available lens care solutions and the obtained results were compared with those provided by the reference procedure recommended by the European Pharmacopoeia [41]. The results compiled in Table 1 show the good agreement between both procedures as the relative deviations were between –4.71 and 1.89%. The variance ratio F-test and a paired Student’s t-test was used to evaluate precision and accuracy, respectively [47]. Regarding the accuracy, the paired Student’s t-test confirmed no significant differences between the proposed fluorometric assay and the reference procedure since the calculated value (t = 1.51) was lower than the critical tabulated value (t = 2.57), for a confidence level of 95% (n = 6). In terms of precision, the variance ratio F-test also showed no significant
3.3. Analytical features and application Under the optimum conditions, the analytical performance of the ratiometric fluorescent assay for H2O2 detection was demonstrated through the evaluation of the linear working range, limit of detection, precision and accuracy. Moreover, the influence of some possible interfering species was tested before application in real samples. 3.3.1. combination of both Y-QDs and O-QDs By conducting the analysis according to the optimized conditions, the fluorescence intensity ratio (FICD434/FIMPA587) showed a good linear relationship with the concentration of H2O2 in the range of 0.0100–0.2125% (w/w). The corresponding regression equation can be expressed as:
Fig. 4. (A) PL spectra of the dual-emission B-CDs/O-QDs (434/587 nm) ratiometric probe during 360 min. (B) Fluorescence intensity ratio of the (434/587 nm) ratiometric probe as a function of time (on logarithmic scale). 5
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Fig. 5. PL spectra (A) and the effect of the reaction time on fluorescence intensity ratio (FICD/FIMPA) (B) of the dual-emission B-CDs/OQDs (434/587 nm) ratiometric probe solution upon the addition of a 0.0500% (w/w) H2O2. Dashed line and solid line corresponds to the fluorescence signal of the ratiometric probe in the absence and presence of 0.0500% (w/w) H2O2, respectively.
differences between the results obtained by both procedures (Fcalculated = 1.28 and Ftabulated = 5.05, for a confidence level of 95%). The assessment of the intramethod precision, through the repeated analysis of each commercial lens care solutions, shown good repeatability considering the calculated concentration ranges for a confidence level of 95%. 3.3.3. RGB-visual determination of H2O2 As abovementioned, a B-CDs/O-QDs combined system provided a more sensitive RGB-type visual read out for the quantification of H2O2. Making use of a conventional UV lamp and a digital camera, images of the standard solutions with a known concentration of H2O2 and real sample were acquired without any special treatment for removing the shade of the incident light (Fig. 8(A)). By decomposing the image on its corresponding RGB channels, the intensity (between 0 and 255) at each pixel of blue and red channels were extracted (Fig. S5 – Supplementary material). The ratio between the pixel intensity at blue and red channels (Blue:Red channels), obtained by dividing at each pixel the intensities on the blue and on the red channel, was suitable for the quantification. In fact, a linear response was obtained between the average of the intensity ratios and the concentration of H2O2 (Fig. 8(B)), which can be expressed as Iblue/Ired = 13.26 ( ± 0.98) × [H2O2] + 0.27 ( ± 0.07) (R = 0.9966, n = 9). The RGB-visual detection method shown worst intramethod
Fig. 7. Fluorescence images of B-CDs/Y-QDs (A) and B-CDs/O-QDs (B) ratiometric probes upon interaction with H2O2 at different concentrations levels. All photographs were taken under 365 nm UV lamp. (For interpretation of the references to colour in this figure text, the reader is referred to the web version of this article.)
precision, when compared with the same ratiometric probe quantified by a conventional fluorometer (error between 0.09-0.44 vs. 0.04-0.09 for image-based and fluorometer-based detection, respectively). Moreover, the accuracy of the RGB-visual detection was also worst as this method provided systematically lower values than those found with
Fig. 6. PL spectra of B-CDs/Y-QDs (A) and B-CDs/O-QDs (B) combined probes upon the interaction with H2O2 at different concentrations. Linear relationship between the fluorescence intensity ratios (FICD/FIMPA) and H2O2 concentration using B-CDs/Y-QDs (C) and B-CDs/O-QDs (D) ratiometric probes. 6
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Table 1 Comparison of the analytical results obtained in the determination of H2O2 in commercial lens care solutions. Sample
1
2
a b
Batch
A B C D E F
Declared amount (%, w/w)
3
3
Amount found (%, w/w)a Reference procedure
Fluorometric assay
R.D %b
RGB-visual assay
R.D %b
3.35 3.34 3.32 2.94 2.92 2.95
3.26 3.18 3.33 2.92 2.85 3.01
−2.85 −4.71 0.25 −0.78 −2.46 1.89
3.08 2.92 3.23 2.80 2.76 2.85
−8.12 −12.68 −2.69 −4.87 −5.42 −3.43
± ± ± ± ± ±
0.02 0.05 0.02 0.04 0.05 0.02
± ± ± ± ± ±
0.04 0.08 0.09 0.06 0.04 0.06
± ± ± ± ± ±
0.09 0.44 0.32 0.14 0.24 0.29
Mean ± t(0.05) (Student’s t-test)×(S/√n). Relative deviation of the proposed ratiometric assay regarding the reference procedure.
as an alternative analytical tool for monitoring hydrogen peroxide in household cleaning samples. In comparison with the reference method, the H2O2 titrimetric method preconized by European Pharmacopoeia, the proposed ratiometric assays allowed to circumvent the operational errors associated with the visual detection of the titration end-point. In comparison with the other QD-based fluorometric methods, the straightforward nature of our method provides a simpler method when compared with indirect sensing schemes [21,22], and the ratiometric nature of the present method provides a more robust method when compared with direct sensing schemes based on a single fluorophore [18–20], due to the minimization of the variations in excitation source, in the probe concentration, and in the emission readout. As the emission readout is not focused into a single fluorophore, the analytical figures of merits of this method, namely LOD and linearity range, were not as great as other reported methods. Nevertheless, the limitations did not hamper the application of our method as the LOD (0.00793% (w/ w)) was far below the declared content of the real sample (3% (w/w)). Together with the use of the combined probe for the quantification of
the reference procedure. The modest precision and accuracy were attributed to the shadows resultant from the incident UV light (darker areas on the RGB-type image, Fig. 8A). Nevertheless, the image processing-based method do not require special equipment and provided an expeditious procedure suitable for a semi-quantitative detection of H2O2.
4. Conclusions A dual-emission CDs/QDs combined system was successfully developed for the ratiometric detection of hydrogen peroxide. The addition of H2O2 to the combined probe solution induced the depassivation of the MPA-CdTe QDs, decreasing its PL emission (587 nm) while the PL of the blue-emitting CDs (434 nm) remained constant (reference) due to its chemical inertness towards the analyte. This ratiometric probe was successfully applied in the development of ratiometric fluorescent assay for H2O2 determination in lens care solutions since sensitive, selective, accurate and precise results were obtained which could be considered
Fig. 8. A) RGB-type image and Blue:Red-type image of standards with known concentration of H2O2 and samples. B) Calibration curve for the RGB-visual assay. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 7
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H2O2 by measuring the ratiometric fluorescence intensity, the visual detection of the analyte was also accomplished. In fact, the interaction between H2O2 and the dual-emission CDs/QDs probe can be surveyed by means of the significant colour variation of the emitted photoluminescence. Despite not having such good results in terms of accuracy and precision as in the fluorometric assay, it was demonstrated that the proposed RGB-visual methodology can be used efficiently for semiquantitative detection of H2O2. In fact, the combination of the minimization of the detrimental factors associated with the use of a single fluorophore with the RGB-visual detection enables the development of spot test analysis which provides the additional advantages of minimize the reagents consumption, reduce the waste generation, and increase the sample throughput.
[16]
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Acknowledgments
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David S.M. Ribeiro thanks FCT (Fundação para a Ciência e Tecnologia) and POPH (Programa Operacional Potencial Humano) for his Post-Doc grant ref. SFRH/BPD/104638/2014. José X. Soares thanks FCT (Fundação para a Ciência e Tecnologia) and POPH (Programa Operacional Potencial Humano) for his Ph.D. grant Ref. SFRH/BD/ 98105/2013, and also the Biotech Health Programme (Doctoral Programme on Cellular and Molecular Biotechnology Applied to Health Sciences), Reference PD/00016/2012. Authors are grateful for the research grant supported by European Union (FEDER funds POCI/01/ 0145/ FEDER/007265) and financing from National Funds (FCT/MEC, Fundação para a Ciência e Tecnologia and Ministério da Educação e Ciência) under the Partnership Agreement PT2020UID/QUI/50006/ 2013. Financing from Programa INTERREG V A Espanha Portugal (POCTEP) under the project FOODSENS is greatly appreciated.
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Appendix A. Supplementary data [29]
Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.snb.2019.126665.
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David S. M. Ribeiro is a Researcher at REQUIMTE (LAVQ) Associate Laboratory, Faculty of Pharmacy, University of Porto. He obtained his PhD degree in Pharmaceutical Sciences, speciality in Analytical Chemistry (2012) and MSc degree in Analytical Chemistry – Quality Control (2007) from Faculty of Pharmacy, University of Porto. He is carrying out research on the development of new analytical methodologies combining the sensitivity offered by nanomaterials, as quantum dots, and the versatility, low cost and portability of flow-based analytical techniques for chemical analysis.
uric acid using H2O2-sensitive quantum dots/dual enzymes, Biosens. Bioelectron. 67 (2015) 129–133. D. Jin, M.-H. Seo, B.T. Huy, Q.-T. Pham, M.L. Conte, D. Thangadurai, et al., Quantitative determination of uric acid using CdTe nanoparticles as fluorescence probes, Biosens. Bioelectron. 77 (2016) 359–365. Y. Li, B. Li, J. Zhang, H2O2‐ and pH‐sensitive CdTe quantum dots as fluorescence probes for the detection of glucose, Luminescence 28 (2012) 667–672. L. Jing, S.V. Kershaw, Y. Li, X. Huang, Y. Li, A.L. Rogach, et al., Aqueous based semiconductor nanocrystals, Chem. Rev. 116 (2016) 10623–10730. Y. Zhang, J. He, P.-N. Wang, J.-Y. Chen, Z.-J. Lu, D.-R. Lu, et al., Time-dependent photoluminescence blue shift of the quantum dots in living cells: effect of oxidation by singlet oxygen, J. Am. Chem. Soc. 128 (2006) 13396–13401. J.N. Miller, J.C. Miller, Statistics and Chemometrics for Analytical Chemistry, 6th ed., Prentice Hall/Pearson, England, 2010.
José X. Soares is a PhD student at REQUIMTE (LAVQ) Associate Laboratory, Faculty of Pharmacy, University of Porto. He obtained his MSc degree Pharmaceutical Sciences from Faculty of Pharmacy, University of Porto in 2011. He is carrying out research on the synthesis of carbon based nanoparticles and semiconductors quantum dots. João L.M. Santos is Auxiliary Professor at the Faculty of Pharmacy, University of Porto, and research scientist at REQUIMTE Associate Laboratory. He received his PhD in Analytical Chemistry in 2000. His research activities have been focused in the development of automated flow-based methodologies, mainly multicommutation, multipumping and single reaction interface flow systems. His current interests include nanotechnology and nanomaterials, namely quantum dots and metallic nanoparticles for photoluminescent detection and nanodiagnostics.
Rafael C. Castro is currently a junior researcher and student in Faculty of Pharmacy of the University of Porto. His research interest is focused on the synthesis of nanomaterials and its application on chemical analysis.
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Dual-emission ratiometric probe combining carbon dots and CdTe quantum dots for fluorometric and visual determination of H2O2 ⁎
Rafael C. Castro, José X. Soares, David S.M. Ribeiro , João L.M. Santos
T
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LAQV, REQUIMTE, Department of Chemical Sciences, Laboratory of Applied Chemistry, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira no 228, 4050-313 Porto, Portugal
A R T I C LE I N FO
A B S T R A C T
Keywords: Quantum dots Ratiometric assay CdTe Carbon dots Hydrogen peroxide Visual assay
In this work, blue-emitting carbon quantum dots and distinctly sized CdTe semiconductor quantum dots were combined to implement a ratiometric probe for the monitoring of H2O2. By implementing a sensing scheme that combines multiple nanoprobes, excited at the same wavelength and emitting at different ones, which exhibit also dissimilar reactivity, it was possible to minimize detrimental factors associated with the use of a single photoluminescence wavelength such as fluctuations in excitation source or measured signal, fluorophore concentration, matrix effects and background fluorescence. The developed ratiometric probe was applied on the implementation of a conventional fluorometric assay and on a visual assay relying on the red, green and blue (RGB)-based colour changes promoted by increasing H2O2 concentrations. For the fluorometric determination assay, a good linear relationship between the fluorescence intensity ratio (FICD434/FIMPA587) and H2O2 concentration within the range of 0.0100–0.2125% (w/w) was obtained (R = 0.9994, n = 7). The detection limit was about 0.00793% (w/w). The obtained results in the determination of H2O2 in contact lens solutions were in agreement with those provided by the reference procedure, with relative deviations between −4.71 and 1.89%. Regarding the RGB-visual assay, a linear working range for hydrogen peroxide concentrations up to 0.150% (w/w) was verified (n = 9) with a correlation coefficient of 0.9966, which confirms its potential as valuable analytical tool for on-the-spot semi-quantitative detection of H2O2.
1. Introduction The capacity of hydrogen peroxide for triggering oxidative processes has been widely explored as: i) food additive for controlling the growth of microorganisms; ii) hair bleaching or dyeing and fixing of hair perm; iii) household cleaning agents, including contact lens disinfection; and iv) tooth bleaching [1]. Being a harmful chemical with confirmed carcinogenicity and toxicity and considering the large quantities as it is released into the environment, it has become imperative to monitor hydrogen peroxide effectiveness and safety in several applications namely in industrial, environmental, biochemical and clinical fields. This has accentuated the need for rapid, simple, selective and sensitive methodologies for H2O2 determination, relying some of the most used procedures on fluorescence-based measurements. Indeed, the oxidizing capacity of hydrogen peroxide was explored to form either a fluorescent product from a non-fluorescent compound or to quench the emission of native fluorescent probes. In this regard, 4-amino-1H-1,5-benzodiazepine-3-carbonitrile [2], p-
⁎
hydroxyphenylacetic acid [3], acetaminophen [4] homovanillic acid [5] and 2′,7′-dichlorodihydrofluorescein [6] are examples of some nonfluorescent reagents, which can be found in the scientific literature, that were applied in H2O2 determination based on the formation of fluorescent compounds upon redox reaction with H2O2. These reactions were usually catalysed by an enzyme, namely peroxidase or horseradish peroxidase (HRP). An indirect fluorometric method involving the strong oxidizing ability of the Fenton reaction was also employed to oxidize non-fluorescent coumarin to fluorescent 7-hydroxycoumarin, being H2O2 quantified as consequence of its concentration dependent fluorescence enhancement [7]. The fluorometric scopoletin-horseradish peroxidase method, relying on the quenching of scopoletin fluorescence upon reaction with H2O2, catalysed by HRP, has been also often used for H2O2 determination [8–11]. A similar fluorescence quenching method was based on the reaction of H2O2 with 3,3′-diethyloxadicarbocyanine iodide to form a non-fluorescent compound in acidic medium [12]. A significant drawback of the aforementioned methods is the use of HRP
Corresponding authors. E-mail addresses: [email protected] (D.S.M. Ribeiro), joaolms@ff.up.pt (J.L.M. Santos).
https://doi.org/10.1016/j.snb.2019.126665 Received 15 March 2019; Received in revised form 27 May 2019; Accepted 5 June 2019 Available online 15 June 2019 0925-4005/ © 2019 Elsevier B.V. All rights reserved.
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2. Experimental
enzymes, which are expensive, instable in solution and require reaction conditions that restrain their applicability [13]. An advantageous alternative to the referred methods could be the implementation of fluorescence-based sensing schemes relying on the use of semiconductor quantum dots (QDs) as fluorophores. QDs exhibit unique optical properties such as size-dependent and tunable photoluminescence (PL), long-term photostability, high quantum yield (QY) and long excited-state decay lifetimes that assure simplicity, versatility and reliability in a variety of analytical applications. Additionally, their broad excitation bands enable the simultaneous excitation of multicolour QDs by using a unique light source [14–17]. In this regard, only a few examples have been reported dealing with the direct interaction between QDs and H2O2 that typically result in a quenching of the fluorescence of the PL probe in the presence of the oxidizing agent [18–20]. Others sensing strategies relying on indirect photoluminescence detection of H2O2 were also proposed aiming to obtain an improvement of sensitivity and selectivity of the QDs-based fluorometric methodology. Two illustrative examples are the use of H2O2 for preventing the surface passivation of 3-mercaptopropionoic acid (MPA)–capped CdTe QDs [21] and thioglycolic acid (TGA)–capped CdTe QDs [22] by reduced glutathione and hepatitis B core antibody labelled with horseradish peroxidase, respectively. All of the abovementioned QDs-based PL sensing strategies for H2O2 relied on the measurement of the fluorescence emission at a single wavelength which, although practical and easy to implement, could be hindered by several factors, such as fluctuations in excitation source, probe concentration, matrix effects in complex samples and background fluorescence [23–25]. These aspects can lead to inaccurate and irreproducible results impairing the detection and quantification of the target analyte. The addition of a second fluorophore for the development of a ratiometric fluorescent method can be used to circumvent these adverse effects. Dual-emission ratiometric fluorescent probe systems consisting in a mixture of two distinct fluorophores, one as the analyte-dependent fluorophore and the other as an internal standard, have been used for the determination of a myriad of analytes such as, metal ions [23–30], proteins [31], glucose [32,33], aspirin [34] and 2,4,6-trinitrotoluene [35]. Practical sensing devices, based on the change of materials macroscopic properties [36] or naked eye colour visualization [37,38], is crucial for the development of in situ analytical methods. Considering that on a dual-emission ratiometric fluorescent probe, one emission intensity is constant and the other can be quenched in the presence of the analyte, the colour modifications endured by the probe can be easily and obviously perceived by naked eye under irradiation with UV lamp. In fact, these kind of ratiometric probes can be applied as naked eye visual sensing platforms as is the example proposed by Xia et al in which they developed a red, green and blue (RGB)-based colour change probe for the visual analysis of formaldehyde [39]. In the present work, two distinct photoluminescent nanoparticles were exploited to implement a ratiometric assay for the determination of H2O2 in real samples. This dual-emission nanosystem involved the combination of a blue-emitting carbon dot, which due to their inertness behaviour was used as internal standard, and an orange-emitting CdTe QDs capped with 3-mercaptopropionic acid (MPA) as analyte-dependent fluorophore. The addition of H2O2 in the mixture induces the depassivation of the MPA-CdTe QDs, decreasing thus the orange-emission PL while the photoluminescence of the blue-emitting CDs remained constant. The exploitation of this effect was used to develop an analytical method for the determination of H2O2 using a conventional fluorometric assay. Additionally, a RGB-type sensing scheme based on the colour modulation of the two nanomaterials is also proposed. The PL variations and fluorescent colour changed from orange to blue were used for the detection and quantification of H2O2 in lens care solutions.
2.1. Reagents and solutions All solutions and standards were prepared from chemicals of analytical reagent grade without any pre-treatment process and using water purified from a Milli-Q system (conductivity ≤ 0.1 μS cm−1). All reagents used for the synthesis of semiconductor QDs were purchased from Sigma-Aldrich (St. Louis, MO, USA), namely, cadmium chloride hemi(pentahydrate) (CdCl2·2.5H2O, 99%), sodium tellurite (Na2TeO3, 100 mesh, 99%), sodium borohydride (NaBH4, 99%), sodium 2-mercaptoethanesulfonate (MES, C2H5NaO3S2, 98.0%), glutathione reduced (GSH, C10H17N3O6S, 99.0%), 3-mercaptopropionic acid (MPA, C3H6O2S, 99%) and sodium citrate tribasic dehydrate (C6H5Na3O7·2H2O, 99%). Citric acid (C6H8O7, ≥99.5%) and ethylenediamine (N2C2H6, ≥99.5%), both acquired from Sigma-Aldrich (St. Louis, MO, USA) were the reagents used for the synthesis of carbonbased nanoparticles. QDs and CDs intermediate solutions were prepared by direct dilution, with deionized water, in a ratio of one to twenty (1:20) and one to fifty (1:50), respectively. Hydrogen peroxide intermediate solution of 0.1% (w/w) was daily prepared by appropriate dilution of H2O2 stock solution acquired from Sigma-Aldrich (30% w/w, St. Louis, MO, USA) with deionized water. The working standard solutions of H2O2 were prepared by proper dilution of the above intermediate solution. Six different commercially available lens care solutions were prepared by diluting with deionized water an appropriate volume of the sample, in order to obtain a hydrogen peroxide content included in the analytical range of the proposed methodology. All the samples were analysed without any pretreatment process. 2.2. Equipment The synthesis of CdTe QDs were carried out in a single mode CEM Discover SP® synthesizer operated through a computer using Synergy™ software (Matthews, NC, US). This equipment was equipped with an automated pressure control/sensing system (ActiVent™), an active cooling system (PowerMAX™) and an integrated infrared (IR) sensor for the strict control of irradiation time, reaction temperature and pressure. The emission spectra of the fluorometric probes were obtained with a Jasco FP-6500 spectrofluorometer (Easton, MD, USA) and a multimode microplate reader (Cytation™ 3, BioTek™). All photographs were acquired with a Sony Cyber-shot DSC-HX10 V digital camera. The ratiometric fluorescent probes were photographed in a CN-6 UV darkroom with hand-held UV lamp 254 nm and 365 nm (6 W, model VL6.LC) 2.3. Procedure for the synthesis of MPA capped CdTe QDs and CDs The two different sized MPA capped CdTe QDs were synthesized according to the procedure proposed by Ribeiro et al [40]. This synthetic methodology consisted in a one-step synthetic route assisted by microwave irradiation. The experimental conditions for the synthesis of QDs, such as precursors concentration, pH, irradiation time were used exactly as recommended in the prior work [40]. For the syntheses of the yellow and orange-emitting CdTe QDs the temperatures were 105 °C and 115 °C, respectively. For the syntheses of CDs, our previously optimized conditions were used [unpublished results]. In short, citric acid (0.5 g) and ethylenediamine (0.3 mL) were mixed with water yielding a final concentration of citric acid of 10% (m/v). Then the pH of the reaction mixture was set to 3.8 by the addition of HCl (1 mol L−1). The reaction mixture was transferred into a 40 mL Teflon-lined autoclave and heated at 260 °C for 5 h. After cooling to room temperature, the solution was extensively dialyzed against Mili-Q water in a dialysis membrane (Spectra/Por 6 2
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1000 MWCO, Spectrum Labs™) for 5 days.
photoluminescence of the QDs was gradually lessened with increasing H2O2 concentrations until its complete disappearance. In effect, the thiol groups of MPA attached on the QDs surface through CdeS bonding can be oxidized to form an organic disulfide product (RS–SR) causing its detachment from the surface of the PL probe. As a consequence of QDs depassivation, the surface traps increased which impaired electron-hole recombination and therefore the QDs PL intensity is inhibited [42–44]. The observed blue shift can be ascribed to a probable slight QDs size reduction caused by the irreversible oxidation at the QDs surface [45,46]. This explanation was confirmed by performing the measurement of the excitation spectra of the QDs in the absence and presence of 0.050% H2O2. The results (Fig S4 – Supplementary material) demonstrated a decrease of the absorbance and a slight blue shift (2 nm) of the absorption band for the first excitonic transition with the addition of H2O2, which was accentuated (blue shift of 4 nm and 8 nm) by increasing the reaction time up to 2 and 5 min, respectively. Moreover, the linear Stern-Volmer plots that describes the PL response of the two different sizes CdTe QDs upon interaction with H2O2 demonstrated a higher sensitivity of the 588 nm emitting QDs (OQDs) (F0/F = 57.4 × [H2O2] + 1.31) relatively to the 562 nm emitting one (Y-QDs) (F0/F = 37.0 × [H2O2] + 0.89) (Figs. S2C) and S3C)).
2.4. Conventional fluorometric assay procedure Before the preparation of the ratiometric probe solution, the respective as-prepared nanoparticle solutions were diluted in deionized water. Thus, QDs and CDs intermediate solutions were prepared by direct dilution of the crude solutions with deionized water in a ratio of 1:20 (1 mL in 20 mL) and 1:50 (0.5 mL in 25 mL), respectively. The two dual-emission ratiometric probes for the detection of H2O2 consisted in a mixture of CDs and QDs in a ratio of 1:5. In this sense, 3.2 mL and 16 mL of CDs and QDs intermediate solutions, respectively, were mixed into a 50 mL graduated tube. Afterwards, 960 μL of the fluorescent probe solution and required amounts of deionized water and H2O2 were sequentially added completing the final volume of 2 mL. Immediately after the addition of H2O2, the solution was shaken and transferred into a 1 cm quartz cell and the emission spectra (λem, from 385 to 700 nm) were recorded with the excitation wavelength fixed at 360 nm. The slit widths of excitation and emission were 5.0 and 10.0 nm, respectively. 2.5. RGB-visual assay procedure
3.2. Dual-emission CDs/CdTe-QDs combined probe for H2O2 detection The ratiometric fluorescent probes prepared using the abovementioned conditions were photographed upon irradiation at 365 nm with a digital camera. RGB-type images were cropped to 460 × 160 pixel dimension image and aligned to the centre of the vial containing the probe. The cropped RGB-images were imported to Matlab version R2009b (MathWorks, Natick, MA, USA) and divided into three matrixes corresponding three channels (red, green and blue). Each matrix, with 460 rows and 160 columns, contained at each entry the intensity value (between 0–255) of the corresponding channel. Ratio between the pixel intensity at blue and red channels were obtained by dividing at each pixel the intensities on the blue and on the red channel. Average pixel intensity were calculated by the mean of mean values on each column.
3.1. Preliminary assays
3.2.1. Optimization of the CDs and CdTe-QDs mixture The blue emitting CDs (B-CDs) were mixed with each of the MPACdTe QDs (Y-QDs and O-QDs) in order to obtain two distinct dualemission ratiometric probes (B-CDs/Y-QDs and B-CDs/O-QDs) which were evaluated for application in the fluorometric determination of H2O2. In this sense, CDs and QDs were mixed in different proportions in order to enhance the efficiency of the dual-emission ratiometric probe for the target analyte. Therefore, for both probes tested, the CDs and QDs were mixed (for a final volume of 2000 μL) in a proportion of 1:1, 1:2.5, 1:4 and 1:5 (CDs:QDs), by fixing 160 μL of the diluted solution of CDs (50×) and by varying the amount of the semiconductor nanoparticle with the addition of 160, 400, 640, 800 μL of the diluted solution of QDs (20×). The PL spectra of the blue emitting CDs, yellow and orange emitting CdTe QDs and the corresponding ratiometric probes are shown in Figs. 1 and 2. Upon excitation at 360 nm, the blue emitting CDs and yellow and orange emitting CdTe QDs showed a maximum emission at 434, 560, and 587 nm, respectively. This way, the corresponding combining probes exhibited a dual emission PL band under a single-wavelength excitation (360 nm) at, namely, 434/560 and 434/587 nm. The obtained results demonstrated that by increasing the amount of CdTe QDs, the PL intensity of the QDs gradually increased, as expected, while the PL intensity of the CDs was slightly inhibited (Figs. 1B) and 2 B)). In fact, the CdTe QDs can act as an inner filter by absorbing a considerable portion of excitation photons and consequently the emission intensity of the CDs was quenched. In order to obtain an improved reactivity and sensitivity of the dual-emission probe, the ratio CDs:QDs of 1:5 was selected for further assays.
For the development of the ratiometric fluorescence probe, CDs were chosen as internal standard because they showed a remarkable chemical inertness upon interaction with H2O2. In fact, upon the presence of increasing concentrations of H2O2, up to 0.26% (w/w), the PL emission intensity, as well as the observed colour under a UV lamp, remains unchangeable (Fig. S1 – Supplementary material). On the other hand, two MPA-CdTe QDs of different size were tested to be used as the analyte-dependent fluorophore. In both QDs the fluorescence intensity is gradually quenched in the presence of increasing H2O2 concentrations (Figs. S2 and S3 – Supplementary material). Additionally, a slightly blue shift of the maximum emission wavelength was observed in both cases. The observed colour under a UV lamp corroborated the abovementioned fluorometric results, in which the yellow and orange
3.2.2. Stability of the dual-emission ratiometric probe The stability of the probe solutions is an important parameter that could affect the reproducibility and accuracy of successive fluorometric measurements performed during sample analysis. For this reason, the stability in aqueous solution of the PL intensity of the 434/560 nm and 434/587 nm arrangements was thoroughly studied. Thus, for both dualemission probes the fluorescence intensity ratios (FICD434/FIMPA560 and FICD434/FIMPA587) were monitored throughout 360 min (Fig. 3 and 4). In the case of the 434/560 nm combination, the signal remained constant up to 60 min (log (time) = 1.78) and thereafter decreased for longer period of time (Fig. 3B). Regarding the 434/587 nm probe, the analytical signal persisted unchanged over 20 min (log (time) = 1.30) and then the stability decreased by prolonging the time (Fig. 4B). This
2.6. Reference procedure To validate the results obtained by the conventional fluorometric and the image processing-based assays, the hydrogen peroxide determination were also performed by the reference methodology recommended by the European Pharmacopoeia [41]. The reference procedure involved a permanganometric titration of hydrogen peroxide in acidic medium. The potassium permanganate solution was standardized by titration with arsenic hexaoxide in acidic medium. The hydrogen peroxide amount in the samples was calculated taking into account that each mL of 0.02 mol L−1 KMnO4 solution was equivalent to 1.701 mg of H2O2. 3. Results and discussion
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Fig. 1. PL spectra (A and B) and the corresponding photographs taken under 365 nm UV lamp (C and D) of blue emitting CDs (I), yellow emitting QDs (II) and B-CDs/Y-QDs (434/ 560 nm) ratiometric probe in the CDs:QDs proportions of 1:1 (III), 1:2.5 (IV), 1:4 (V) and 1:5 (VI). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
being MPA-CdTe QDs used as the recognition element and the CDs employed as reference fluorophore. The obtained results revealed a linear relationship between the fluorescence intensity ratios (FICD434/FIMPA560 and FICD434/ FIMPA587) and the concentration of H2O2 in the range up to 0.1000% (w/w) and 0.2125% (w/w), when using B-CDs/Y-QDs and B-CDs/OQDs, respectively. The regression equations obtained from the interaction of H2O2 and B-CDs/Y-QDs and B-CDs/O-QDs probes can be expressed as FICD/ FIMPA = 10.0 ( ± 0.2) × [H2O2] + 0.37 ( ± 0.01) (R = 0.9972) and FICD/FIMPA = 20.6 ( ± 0.4) × [H2O2] + 0.73 ( ± 0.04) (R = 0.9972), respectively. From the obtained results it was observed that the linear regression sensitivity obtained for B-CDs/O-QDs probe was twice as high as for the B-CDs/Y-QDs one. In addition, both ratiometric probes also allowed the visual detection of H2O2. In fact, the changes in fluorescence intensity ratios (FICD434/FIMPA560 and FICD434/FIMPA587) with the increasing of H2O2 concentration can also be observed through the continuous variation of the colour of the fluorescence emission from yellow to blue (Fig. 7 (A)) and from orange to blue (Fig. 7(B)) in the case of the B-CDs/ Y-QDs and B-CDs/O-QDs probes, respectively. Indeed, the combination of both Y-QDs and O-QDs, as sensing probes, and B-CDs, as reference, allowed the obtaining of a wide range of colour variations which can be clearly detected by the naked eye. This can be used to demonstrate the advantage of the ratiometric probes relatively to the single fluorescence quenching of the respective
results revealed a good stability of the optical properties of both probes considering the kinetics of the reaction with the target analyte. Additionally, the analytical response (FI ratio) of ratiometric probe solution was monitored upon the addition of 0.0500% (w/w) H2O2 solution. The obtained results revealed that the reaction between the dual-emission probe and hydrogen peroxide occurred promptly. Prolonging the reaction time up to 25 min, FICD/FIMPA increased gradually due to the PL quenching of the MPA-CdTe QDs which is caused by the oxidation of capping thiol groups attached to the QDs surface (Fig. 5). Consequently, the surface depassivation promoted the occurrence of dangling bonds (mid-gap energy states) that act as surface traps and prevent radiative electron-hole recombination. Consequently, in order to guarantee a good compromise between stability and reactivity the fluorescence response was measured immediately after the addition of H2O2. 3.2.3. Fluorometric determination of H2O2 The PL properties of the 434/560 nm and 434/587 nm probes were assessed upon interaction with H2O2 at a concentration range of 0–0.26% (w/w) (Fig. 6). As expected, the two characteristic emission bands showed different responses. In both cases, the PL intensity corresponding to the CdTe QDs band at 560 and/or 587 nm were effectively inhibited by the addition of increasing concentrations of H2O2, while the 434 nm emission band of CDs was unaffected in the presence of H2O2. Hence, the combination of both nanomaterials demonstrated to be a valuable approach for the ratiometric determination of H2O2,
Fig. 2. PL spectra (A and B) and the corresponding photographs taken under 365 nm UV lamp (C and D) of blue emitting CDs (I), orange emitting QDs (II) and B-CDs/O-QDs (434/ 587 nm) ratiometric probe in the CDs:QDs proportions of 1:1 (III), 1:2.5 (IV), 1:4 (V) and 1:5 (VI). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 3. (A) PL spectra of the dual-emission B-CDs/Y-QDs (434/560 nm) ratiometric probe during 360 min. (B) Fluorescence intensity ratio of the (434/560 nm) ratiometric probe as a function of time (on logarithmic scale).
QDs Figs. S2(A) and S3(A) – Supplementary material). For each individual QDs the small slight colour changes associated with the increase of H2O2 concentration was much more difficult to perceive by naked eye when they were assayed alone than when they were combined with the CDs. In fact, the combination with the blue-emitting CDs, which exhibits a bright emission band at 434 nm, affords a reference colour that facilitates visual readout. Thus, the large separation between 434 and 560 nm and/or 434 nm and 587 nm emission peaks enables that colour changes caused by the addition of a small concentration of H2O2 could be easily discerned by the naked eye (Fig. 7). Moreover, the larger separation of the emission peaks of the B-CDs/ O-QDs combination probe (434/587 nm) relatively to the B-CDs/Y-QDs one (434/560 nm) makes it easier to detect a distinguishable colour change from the background. Therefore, the B-CDs/O-QDs combined system demonstrated to be a more efficient ratiometric probe for H2O2 because showed a more sensitive RGB-type visual read out as well as a higher sensitivity in the PL detection of H2O2. For that reason, it was chosen for the further assays.
(FICD 434) = 24.9 (± 0.4) × C + 0.94 (± 0.04) (FIMPA 587) Where in (FICD434/FIMPA587) was the ratio between the fluorescence intensity obtained by CDs and CdTe QDs and C was the H2O2 concentration. The obtained correlation coefficient was 0.9994 (n = 7) which indicates good linearity. The detection limit calculated from the equation of the calibration curve according to Miller and Miller [47] was about 0.00793% (w/w), far below the declared content of H2O2 in lens care solutions (3% (w/w)). 3.3.2. Applicability in real samples Prior to the application with real samples, the selectivity of the developed ratiometric fluorescent assay for the H2O2 determination in lens care solutions was evaluated in the presence of excipients, such as, sodium dihydrogen phosphate, sodium chloride and edetate disodium. No influence on the analytical signal (FICD434/FIMPA587 variation ≤4%) was verified by the aforementioned compounds, which was expected considering the pronounced real sample dilution that had to be performed before the analysis. The applicability of the proposed ratiometric fluorescent assay was assessed by applying it in the determination of H2O2 in commercially available lens care solutions and the obtained results were compared with those provided by the reference procedure recommended by the European Pharmacopoeia [41]. The results compiled in Table 1 show the good agreement between both procedures as the relative deviations were between –4.71 and 1.89%. The variance ratio F-test and a paired Student’s t-test was used to evaluate precision and accuracy, respectively [47]. Regarding the accuracy, the paired Student’s t-test confirmed no significant differences between the proposed fluorometric assay and the reference procedure since the calculated value (t = 1.51) was lower than the critical tabulated value (t = 2.57), for a confidence level of 95% (n = 6). In terms of precision, the variance ratio F-test also showed no significant
3.3. Analytical features and application Under the optimum conditions, the analytical performance of the ratiometric fluorescent assay for H2O2 detection was demonstrated through the evaluation of the linear working range, limit of detection, precision and accuracy. Moreover, the influence of some possible interfering species was tested before application in real samples. 3.3.1. combination of both Y-QDs and O-QDs By conducting the analysis according to the optimized conditions, the fluorescence intensity ratio (FICD434/FIMPA587) showed a good linear relationship with the concentration of H2O2 in the range of 0.0100–0.2125% (w/w). The corresponding regression equation can be expressed as:
Fig. 4. (A) PL spectra of the dual-emission B-CDs/O-QDs (434/587 nm) ratiometric probe during 360 min. (B) Fluorescence intensity ratio of the (434/587 nm) ratiometric probe as a function of time (on logarithmic scale). 5
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Fig. 5. PL spectra (A) and the effect of the reaction time on fluorescence intensity ratio (FICD/FIMPA) (B) of the dual-emission B-CDs/OQDs (434/587 nm) ratiometric probe solution upon the addition of a 0.0500% (w/w) H2O2. Dashed line and solid line corresponds to the fluorescence signal of the ratiometric probe in the absence and presence of 0.0500% (w/w) H2O2, respectively.
differences between the results obtained by both procedures (Fcalculated = 1.28 and Ftabulated = 5.05, for a confidence level of 95%). The assessment of the intramethod precision, through the repeated analysis of each commercial lens care solutions, shown good repeatability considering the calculated concentration ranges for a confidence level of 95%. 3.3.3. RGB-visual determination of H2O2 As abovementioned, a B-CDs/O-QDs combined system provided a more sensitive RGB-type visual read out for the quantification of H2O2. Making use of a conventional UV lamp and a digital camera, images of the standard solutions with a known concentration of H2O2 and real sample were acquired without any special treatment for removing the shade of the incident light (Fig. 8(A)). By decomposing the image on its corresponding RGB channels, the intensity (between 0 and 255) at each pixel of blue and red channels were extracted (Fig. S5 – Supplementary material). The ratio between the pixel intensity at blue and red channels (Blue:Red channels), obtained by dividing at each pixel the intensities on the blue and on the red channel, was suitable for the quantification. In fact, a linear response was obtained between the average of the intensity ratios and the concentration of H2O2 (Fig. 8(B)), which can be expressed as Iblue/Ired = 13.26 ( ± 0.98) × [H2O2] + 0.27 ( ± 0.07) (R = 0.9966, n = 9). The RGB-visual detection method shown worst intramethod
Fig. 7. Fluorescence images of B-CDs/Y-QDs (A) and B-CDs/O-QDs (B) ratiometric probes upon interaction with H2O2 at different concentrations levels. All photographs were taken under 365 nm UV lamp. (For interpretation of the references to colour in this figure text, the reader is referred to the web version of this article.)
precision, when compared with the same ratiometric probe quantified by a conventional fluorometer (error between 0.09-0.44 vs. 0.04-0.09 for image-based and fluorometer-based detection, respectively). Moreover, the accuracy of the RGB-visual detection was also worst as this method provided systematically lower values than those found with
Fig. 6. PL spectra of B-CDs/Y-QDs (A) and B-CDs/O-QDs (B) combined probes upon the interaction with H2O2 at different concentrations. Linear relationship between the fluorescence intensity ratios (FICD/FIMPA) and H2O2 concentration using B-CDs/Y-QDs (C) and B-CDs/O-QDs (D) ratiometric probes. 6
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Table 1 Comparison of the analytical results obtained in the determination of H2O2 in commercial lens care solutions. Sample
1
2
a b
Batch
A B C D E F
Declared amount (%, w/w)
3
3
Amount found (%, w/w)a Reference procedure
Fluorometric assay
R.D %b
RGB-visual assay
R.D %b
3.35 3.34 3.32 2.94 2.92 2.95
3.26 3.18 3.33 2.92 2.85 3.01
−2.85 −4.71 0.25 −0.78 −2.46 1.89
3.08 2.92 3.23 2.80 2.76 2.85
−8.12 −12.68 −2.69 −4.87 −5.42 −3.43
± ± ± ± ± ±
0.02 0.05 0.02 0.04 0.05 0.02
± ± ± ± ± ±
0.04 0.08 0.09 0.06 0.04 0.06
± ± ± ± ± ±
0.09 0.44 0.32 0.14 0.24 0.29
Mean ± t(0.05) (Student’s t-test)×(S/√n). Relative deviation of the proposed ratiometric assay regarding the reference procedure.
as an alternative analytical tool for monitoring hydrogen peroxide in household cleaning samples. In comparison with the reference method, the H2O2 titrimetric method preconized by European Pharmacopoeia, the proposed ratiometric assays allowed to circumvent the operational errors associated with the visual detection of the titration end-point. In comparison with the other QD-based fluorometric methods, the straightforward nature of our method provides a simpler method when compared with indirect sensing schemes [21,22], and the ratiometric nature of the present method provides a more robust method when compared with direct sensing schemes based on a single fluorophore [18–20], due to the minimization of the variations in excitation source, in the probe concentration, and in the emission readout. As the emission readout is not focused into a single fluorophore, the analytical figures of merits of this method, namely LOD and linearity range, were not as great as other reported methods. Nevertheless, the limitations did not hamper the application of our method as the LOD (0.00793% (w/ w)) was far below the declared content of the real sample (3% (w/w)). Together with the use of the combined probe for the quantification of
the reference procedure. The modest precision and accuracy were attributed to the shadows resultant from the incident UV light (darker areas on the RGB-type image, Fig. 8A). Nevertheless, the image processing-based method do not require special equipment and provided an expeditious procedure suitable for a semi-quantitative detection of H2O2.
4. Conclusions A dual-emission CDs/QDs combined system was successfully developed for the ratiometric detection of hydrogen peroxide. The addition of H2O2 to the combined probe solution induced the depassivation of the MPA-CdTe QDs, decreasing its PL emission (587 nm) while the PL of the blue-emitting CDs (434 nm) remained constant (reference) due to its chemical inertness towards the analyte. This ratiometric probe was successfully applied in the development of ratiometric fluorescent assay for H2O2 determination in lens care solutions since sensitive, selective, accurate and precise results were obtained which could be considered
Fig. 8. A) RGB-type image and Blue:Red-type image of standards with known concentration of H2O2 and samples. B) Calibration curve for the RGB-visual assay. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 7
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H2O2 by measuring the ratiometric fluorescence intensity, the visual detection of the analyte was also accomplished. In fact, the interaction between H2O2 and the dual-emission CDs/QDs probe can be surveyed by means of the significant colour variation of the emitted photoluminescence. Despite not having such good results in terms of accuracy and precision as in the fluorometric assay, it was demonstrated that the proposed RGB-visual methodology can be used efficiently for semiquantitative detection of H2O2. In fact, the combination of the minimization of the detrimental factors associated with the use of a single fluorophore with the RGB-visual detection enables the development of spot test analysis which provides the additional advantages of minimize the reagents consumption, reduce the waste generation, and increase the sample throughput.
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Acknowledgments
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David S.M. Ribeiro thanks FCT (Fundação para a Ciência e Tecnologia) and POPH (Programa Operacional Potencial Humano) for his Post-Doc grant ref. SFRH/BPD/104638/2014. José X. Soares thanks FCT (Fundação para a Ciência e Tecnologia) and POPH (Programa Operacional Potencial Humano) for his Ph.D. grant Ref. SFRH/BD/ 98105/2013, and also the Biotech Health Programme (Doctoral Programme on Cellular and Molecular Biotechnology Applied to Health Sciences), Reference PD/00016/2012. Authors are grateful for the research grant supported by European Union (FEDER funds POCI/01/ 0145/ FEDER/007265) and financing from National Funds (FCT/MEC, Fundação para a Ciência e Tecnologia and Ministério da Educação e Ciência) under the Partnership Agreement PT2020UID/QUI/50006/ 2013. Financing from Programa INTERREG V A Espanha Portugal (POCTEP) under the project FOODSENS is greatly appreciated.
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Appendix A. Supplementary data [29]
Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.snb.2019.126665.
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David S. M. Ribeiro is a Researcher at REQUIMTE (LAVQ) Associate Laboratory, Faculty of Pharmacy, University of Porto. He obtained his PhD degree in Pharmaceutical Sciences, speciality in Analytical Chemistry (2012) and MSc degree in Analytical Chemistry – Quality Control (2007) from Faculty of Pharmacy, University of Porto. He is carrying out research on the development of new analytical methodologies combining the sensitivity offered by nanomaterials, as quantum dots, and the versatility, low cost and portability of flow-based analytical techniques for chemical analysis.
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José X. Soares is a PhD student at REQUIMTE (LAVQ) Associate Laboratory, Faculty of Pharmacy, University of Porto. He obtained his MSc degree Pharmaceutical Sciences from Faculty of Pharmacy, University of Porto in 2011. He is carrying out research on the synthesis of carbon based nanoparticles and semiconductors quantum dots. João L.M. Santos is Auxiliary Professor at the Faculty of Pharmacy, University of Porto, and research scientist at REQUIMTE Associate Laboratory. He received his PhD in Analytical Chemistry in 2000. His research activities have been focused in the development of automated flow-based methodologies, mainly multicommutation, multipumping and single reaction interface flow systems. His current interests include nanotechnology and nanomaterials, namely quantum dots and metallic nanoparticles for photoluminescent detection and nanodiagnostics.
Rafael C. Castro is currently a junior researcher and student in Faculty of Pharmacy of the University of Porto. His research interest is focused on the synthesis of nanomaterials and its application on chemical analysis.
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