Sensors & Actuators: B.

Chemical 296 (2019) 126665

Contents lists available at ScienceDirect

Sensors and Actuators B: Chemical

journal homepage: www.elsevier.com/locate/snb

Dual-emission ratiometric probe combining carbon dots and CdTe quantum T

dots for fluorometric and visual determination of H2O2
⁎ ⁎
Rafael C. Castro, José X. Soares, David S.M. Ribeiro , João L.M. Santos
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: In this work, blue-emitting carbon quantum dots and distinctly sized CdTe semiconductor quantum dots were
Quantum dots combined to implement a ratiometric probe for the monitoring of H2O2. By implementing a sensing scheme that
Ratiometric assay combines multiple nanoprobes, excited at the same wavelength and emitting at different ones, which exhibit also
CdTe dissimilar reactivity, it was possible to minimize detrimental factors associated with the use of a single photo-
Carbon dots
luminescence wavelength such as fluctuations in excitation source or measured signal, fluorophore concentra-
Hydrogen peroxide
Visual assay
tion, matrix effects and background fluorescence. The developed ratiometric probe was applied on the im-
plementation 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 hydroxyphenylacetic acid [3], acetaminophen [4] homovanillic acid

The capacity of hydrogen peroxide for triggering oxidative pro-
cesses 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 dis-
infection; 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 im-
perative 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 measure-
ments. Indeed, the oxidizing capacity of hydrogen peroxide was ex-
plored 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-
[5] and 2′,7′-dichlorodihydrofluorescein [6] are examples of some non-
fluorescent reagents, which can be found in the scientific literature, that
were applied in H2O2 determination based on the formation of fluor-
escent 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-fluor-
escent coumarin to fluorescent 7-hydroxycoumarin, being H2O2 quan-
tified as consequence of its concentration dependent fluorescence en-
hancement [7]. The fluorometric scopoletin-horseradish peroxidase
method, relying on the quenching of scopoletin fluorescence upon re-
action 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 io-
dide 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: dsmribeiro@gmail.com (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.
R.C. Castro, et al.
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 photo-
luminescence (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 multi-
colour 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 photo-
luminescence detection of H2O2 were also proposed aiming to obtain an
improvement of sensitivity and selectivity of the QDs-based fluoro-
metric 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 irre-
producible results impairing the detection and quantification of the
target analyte. The addition of a second fluorophore for the develop-
ment of a ratiometric fluorescent method can be used to circumvent
these adverse effects. Dual-emission ratiometric fluorescent probe sys-
tems 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 macro-
scopic 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 ea-
sily 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-depen-
dent fluorophore. The addition of H2O2 in the mixture induces the
depassivation of the MPA-CdTe QDs, decreasing thus the orange-emis-
sion PL while the photoluminescence of the blue-emitting CDs remained
constant. The exploitation of this effect was used to develop an analy-
tical 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
Sensors & Actuators: B. Chemical 296 (2019) 126665
2. Experimental
2.1. Reagents and solutions
All solutions and standards were prepared from chemicals of ana-
lytical 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%), so-
dium 2-mercaptoethanesulfonate (MES, C2H5NaO3S2, 98.0%), glu-
tathione 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 ethyle-
nediamine (N2C2H6, ≥99.5%), both acquired from Sigma-Aldrich (St.
Louis, MO, USA) were the reagents used for the synthesis of carbon-
based nanoparticles.
QDs and CDs intermediate solutions were prepared by direct dilu-
tion, 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 di-
lution 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 hy-
drogen peroxide content included in the analytical range of the pro-
posed methodology. All the samples were analysed without any pre-
treatment 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 multi-
mode microplate reader (Cytation™ 3, BioTek™). All photographs were
acquired with a Sony Cyber-shot DSC-HX10 V digital camera. The ra-
tiometric fluorescent probes were photographed in a CN-6 UV dark-
room with hand-held UV lamp 254 nm and 365 nm (6 W, model VL-
6.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 syn-
thetic 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 ethylene-
diamine (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 -
R.C. Castro, et al.
1000 MWCO, Spectrum Labs™) for 5 days.
2.4. Conventional fluorometric assay procedure
Before the preparation of the ratiometric probe solution, the re-
spective 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 com-
pleting 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
The ratiometric fluorescent probes prepared using the above-
mentioned 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.
2.6. Reference procedure
To validate the results obtained by the conventional fluorometric
and the image processing-based assays, the hydrogen peroxide de-
termination were also performed by the reference methodology re-
commended by the European Pharmacopoeia [41]. The reference pro-
cedure involved a permanganometric titration of hydrogen peroxide in
acidic medium. The potassium permanganate solution was standar-
dized by titration with arsenic hexaoxide in acidic medium. The hy-
drogen peroxide amount in the samples was calculated taking into ac-
count that each mL of 0.02 mol L−1 KMnO4 solution was equivalent to
1.701 mg of H2O2.
3. Results and discussion
3.1. Preliminary 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 pre-
sence 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 concentra-
tions (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
Sensors & Actuators: B. Chemical 296 (2019) 126665
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 con-
sequence of QDs depassivation, the surface traps increased which im-
paired 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 per-
forming the measurement of the excitation spectra of the QDs in the
absence and presence of 0.050% H2O2. The results (Fig S4 – Supple-
mentary 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 (O-
QDs) (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)).
3.2. Dual-emission CDs/CdTe-QDs combined probe for H2O2 detection
3.2.1. Optimization of the CDs and CdTe-QDs mixture
The blue emitting CDs (B-CDs) were mixed with each of the MPA-
CdTe QDs (Y-QDs and O-QDs) in order to obtain two distinct dual-
emission 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 nano-
particle with the addition of 160, 400, 640, 800 μL of the diluted so-
lution 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 emis-
sion 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.
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 dual-
emission 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
R.C. Castro, et al.
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 gra-
dually 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 occur-
rence 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 re-
activity 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 cor-
responding to the CdTe QDs band at 560 and/or 587 nm were effec-
tively 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,
4
Sensors & Actuators: B. Chemical 296 (2019) 126665
Fig. 1. PL spectra (A and B) and the corre-
sponding 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 re-
ferred 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/O-
QDs, 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 detec-
tion 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 var-
iation 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
Fig. 2. PL spectra (A and B) and the corre-
sponding 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 re-
ferred to the web version of this article.)
R.C. Castro, et al.
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 in-
dividual QDs the small slight colour changes associated with the in-
crease of H2O2 concentration was much more difficult to perceive by
naked eye when they were assayed alone than when they were com-
bined with the CDs. In fact, the combination with the blue-emitting
CDs, which exhibits a bright emission band at 434 nm, affords a re-
ference 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 con-
centration 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.
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 in-
terfering 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
Sensors & Actuators: B. Chemical 296 (2019) 126665
(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 con-
centration. 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 ex-
pected 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 differ-
ences between the proposed fluorometric assay and the reference pro-
cedure 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
R.C. Castro, et al. Sensors & Actuators: B. Chemical 296 (2019) 126665

Fig. 5. PL spectra (A) and the effect of the re-

action time on fluorescence intensity ratio
(FICD/FIMPA) (B) of the dual-emission B-CDs/O-
QDs (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 repeat-
ability 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
Fig. 7. Fluorescence images of B-CDs/Y-QDs (A) and B-CDs/O-QDs (B) ratio-
shade of the incident light (Fig. 8(A)). By decomposing the image on its
metric probes upon interaction with H2O2 at different concentrations levels. All
corresponding RGB channels, the intensity (between 0 and 255) at each photographs were taken under 365 nm UV lamp. (For interpretation of the re-
pixel of blue and red channels were extracted (Fig. S5 – Supplementary ferences to colour in this figure text, the reader is referred to the web version of
material). The ratio between the pixel intensity at blue and red channels this article.)
(Blue:Red channels), obtained by dividing at each pixel the intensities
on the blue and on the red channel, was suitable for the quantification.
precision, when compared with the same ratiometric probe quantified
In fact, a linear response was obtained between the average of the in-
by a conventional fluorometer (error between 0.09-0.44 vs. 0.04-0.09
tensity ratios and the concentration of H2O2 (Fig. 8(B)), which can be
for image-based and fluorometer-based detection, respectively).
expressed as Iblue/Ired = 13.26 ( ± 0.98) × [H2O2] + 0.27 ( ± 0.07)
Moreover, the accuracy of the RGB-visual detection was also worst as
(R = 0.9966, n = 9).
this method provided systematically lower values than those found with
The RGB-visual detection method shown worst intramethod

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
R.C. Castro, et al. Sensors & Actuators: B. Chemical 296 (2019) 126665

Table 1
Comparison of the analytical results obtained in the determination of H2O2 in commercial lens care solutions.
Sample Batch Declared amount (%, w/w) Amount found (%, w/w)a

Reference procedure Fluorometric assay R.D %b RGB-visual assay R.D %b

1 A 3 3.35 ± 0.02 3.26 ± 0.04 −2.85 3.08 ± 0.09 −8.12

B 3.34 ± 0.05 3.18 ± 0.08 −4.71 2.92 ± 0.44 −12.68
C 3.32 ± 0.02 3.33 ± 0.09 0.25 3.23 ± 0.32 −2.69
2 D 3 2.94 ± 0.04 2.92 ± 0.06 −0.78 2.80 ± 0.14 −4.87
E 2.92 ± 0.05 2.85 ± 0.04 −2.46 2.76 ± 0.24 −5.42
F 2.95 ± 0.02 3.01 ± 0.06 1.89 2.85 ± 0.29 −3.43

a
Mean ± t(0.05) (Student’s t-test)×(S/√n).
b
Relative deviation of the proposed ratiometric assay regarding the reference procedure.

the reference procedure. The modest precision and accuracy were at-
tributed to the shadows resultant from the incident UV light (darker
areas on the RGB-type image, Fig. 8A). Nevertheless, the image pro-
cessing-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 de-
veloped for the ratiometric detection of hydrogen peroxide. The addi-
tion 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
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 emis-
sion 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

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
R.C. Castro, et al.
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 photo-
luminescence. 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 semi-
quantitative detection of H2O2. In fact, the combination of the mini-
mization 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.
Acknowledgments
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 re-
search 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.
Appendix A. Supplementary data
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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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.
Sensors & Actuators: B. Chemical 296 (2019) 126665
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.
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 develop-
ment 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 photo-
luminescent detection and nanodiagnostics.