Electronic Supplementary Material
Quantum Dot-Modified Paper-Based Assay for Glucose Screening
Gema M. Durán1, 2, Tomás E. Benavidez3, Ángel Ríos1 and Carlos D. García3*
1Department
of Analytical Chemistry, University of Castilla - La Mancha, Avenue Camilo José Cela
s/n, Ciudad Real 13004, Spain
2IRICA
(Regional Institute of Applied Scientific Research), Avenue Camilo José Cela s/n, Ciudad
Real 13004, Spain
3Department
of Chemistry, Clemson University, 219 Hunter Laboratories, Clemson, S.C. 29634,
USA
MATERIAL AND METHODS
Preparation of CdSe/ZnS QD. The synthesis of the CdSe/ZnS core/shell QD was performed using
a modified version of the process reported by Peng et al [1]. Briefly, 0.06 g of CdO, 0.22 g HPA,
and 7 g of TOPO were loaded in a 250 mL three-neck flask clamped in a heating mantle and air in
the system was pumped off and replaced with N2. The mixture was stirred and heated at 300-310
°C for 15 min, and CdO was dissolved in HPA and TOPO. The solution was cooled down to 270 °C
and 2.5 mL of the solution of Se/TOP was swiftly injected. After the injection, the temperature was
adjusted to 250 °C for nucleus growth during 20 min and a change in the color of the solution to red
was observed. To make ZnS shell on the CdSe, 3 mL of a solution of Zn/S/TOP (0.58 g of ZnEt 2,
0.087 mL of (TMS)2S and 3.4 mL of TOP) was added dropwise to the mixture under vigorous
stirring. The mixture was kept to 90 ºC for 4 h to improve the crystallinity of ZnS shell. After cooling
the solution down to room temperature, the QD were diluted with 10 mL of chloroform anhydrous.
Finally, the synthesized QD were purified by adding 10 mL of methanol to 10 mL of the QD solution;
QD were precipitated, collected by ultracentrifugation and washed with methanol four times. The
*
To whom correspondence should be addressed (cdgarci@clemson.edu)
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obtained nanocrystals were then dispersed in 10 mL of anhydrous chloroform and stored in
darkness until use.
Detection Scheme. The mechanism used for the detection of glucose is schematically shown in
Scheme S1. Here, the glucose present in the sample is converted by the enzyme (GOx) into Dglucono-δ-lactone and H2O2 [2,3]. The latter is then used to etch the surface of the QD and change
their optical properties, resulting in a decrease in their fluorescence.
Scheme S1: Schematic representation of the reaction mechanism used to detect glucose by fluorescence quenching of QD.
Although this mechanism has been reported for the analysis of multiple analytes it is important to
note that the selectivity of the process can be limited by secondary processes affecting the surface
of the QD [4].
RESULTS AND DISCUSSION
Effect of QD concentration and pH dependence on the fluorescence properties of QDmodified paper-based assay. In order to improve the fluorescence intensity and the homogeneity
distribution of QD on the paper substrate, the effect of the QD concentration and pH were
investigated. Figure S1 shows the trend in the fluorescence intensity after applying different
volumes of QD solution on paper substrates under the UV lamp (365 nm excitation wavelength).
The amount of QD incorporated in the paper substrates ranged from 2.6 to 15.7 pmol
(corresponding to 3 and 18 µL). As it can be noted in the inset picture, as the concentration of QD
increased, the paper surface was progressively covered with QD and the minimum amount of QD
required to cover the entire paper surface was found to be 7.8 pmol (9 µL).
2
On the other hand, the pH dependence is a key factor in the obtained fluorescence intensity after
activation under UV-vis lamp (365 nm excitation wavelength). For this purpose, solutions of different
pH values were used, including deionized water, sodium hydroxide (0.1 mol L-1) and hydrochloric
acid (0.1 mol L-1). In addition, different concentrations of QD were used in all cases. As it can be
also observed in Figure S1, when hydrochloric acid was used a strong fluorescence quenching was
observed at all concentrations of QD. This observation is in agreement with previous reports stating
that strong acids have the ability to break up the core of the nanocrystal and thus the fluorescence
of QD. On the other hand, in presence of sodium hydroxide, a strong enhancement of fluorescence
intensity was observed in all cases. This can be explained in terms of Cd-OH formation and
elimination of surface defects. Finally, slight differences between pH = 7 and pH = 9 were observed.
At pH values lower than 5 and deionized water, only a slight increase in the fluorescence intensity
was observed. This fact may be due to the initially formation of small Cd 2+ ions which enhances the
fluorescence of QD [5]. Taking into account these results, an amount of QD of 7.8 pmol (9 µL) and
a neutral pH = 7 were selected as optimum and used for the subsequent experiments. This value
also matches the optimal pH for the enzyme (GOx), therefore favouring the production of H 2O2.
Figure S1. Effect of QD volume on the fluorescence intensity at pH = 7. Note that the arrow indicates the selected volume
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used in the subsequent preparation of the paper-based assay. The inset picture shows the fluorescence intensity of QD in
the paper spots and the evolution with the time.
One of the most remarkable advantages of QD with respect to other chromogenic agents is their
stability [6]. In order to evaluate the evolution of the signal intensity of the QD deposited on paper
with respect to storage time, the fluorescence intensity at different concentrations of QD at different
pH was investigated. As it can be observed in Figure S1, only slight variations in the fluorescence
intensity were observed, accounting for an average of less than 1% during the first 7 days and only
7% during a 10-month period.
Photoactivation. It has been already stabilised that when quantum dots are irradiated with light, a
fluorescence enhancement is produced [7]. This phenomenon is commonly known as
“photoactivation” and has been attributed to a combination of effects induced by heat-induction by
light (photoannealing), the adsorption of water molecules on the surface of QD and even photooxidation [7]. To date, different explanations have been reported in bibliography and the existence
of different pathways for the photoactivation phenomenon has been proposed. However, most of
these reports are referred to solution media. Taking into account that a similar process was
observed when the QD are deposited on the paper substrates, experiments were carried out to
select the most appropriate conditions for photoactivation. For this purpose, once the modified QD
papers were prepared different photoactivation pathways were used. In the first case, the QD
modified papers with buffer solution were dried under ambient conditions and then exposed to UV
light (365 nm). Although this would be the simplest process to activate the paper spots, no effective
photoactivation was observed. These results highlight the potential role of water molecules in the
photoactivation process of the QD. On the other hand, the QD-modified papers were exposed to
direct UV light (365 nm) immediately after the addition of the buffer solution. In this case, a
progressive enhancement of the fluorescence intensity was observed, reaching a maximum after 20
min (data not shown). Therefore, a photoactivation time of 20 min was selected as the most
appropriate and used for the remaining experiments.
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Effect of H2O2 and pH on the fluorescence intensity of QD modified paper devices. It has been
reported that H2O2 causes fluorescence quenching of QD and therefore, QD can be used as
indicators for H2O2-dependent enzymatic reactions in solution [8-10]. To demonstrate the possibility
to apply this concept to paper-based assays, we first investigated the influence of the H2O2 on the
fluorescence emission of QD-modified paper devices at relevant pH values. For these experiments,
3 µL of a solution containing different percentages of H 2O2 was dispensed onto the QD modified
paper devices and allowed to react at ambient conditions. The resulting QD modified paper devices
were read under UV-vis lamp (365 nm). As shown in Figure S2, the fluorescence intensity of QD modified paper devices significantly decreased with the addition of increasing H2O2 percentages (in
the 0.05 - 3 % range). However, only slight differences were observed at different pH values. The
figure also shows that the quenching of the fluorescence by H2O2 in 0.1 mol L-1 NaOH (pH = 13),
showed a very narrow dynamic range, therefore limiting the applicability of the approach. On the
other hand, the data obtained at pH = 9 and 7 showed significant differences, yielding a broader
color gradient. Based on these results, a pH = 7 was selected as optimum and used for the paperbased assay.
Figure S2: Effect of the concentration of H2O2 on the fluorescence intensity of QDs-modified paper substrates using
buffer solutions of different pH values.
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In order to optimize the reaction time between QD and H2O2 and maximize the signal intensity, the
evolution of the fluorescence was investigated in the 5-15 min range. The results are summarized in
Figure S3. Although the results show initial changes in the entire concentration range investigated,
the effect of H2O2 concentration was accentuated after 15 min when an abrupt exponential decay
was observed. Therefore, 15 min of reaction was selected as optimum to obtain the best results in
the QDs-H2O2 system.
Figure S3: Effect of the concentration of H2O2 on the fluorescence intensity of QDs-modified paper substrates at pH = 7
at different reaction time.
Effect of enzyme concentration and reaction time. One of the most commonly used enzymes for
analytical applications is GOx. This enzyme has been considered a model and used thoroughly for
the development of numerous biosensors [2]. Although a variety of immobilization strategies have
been proposed, a saturated layer of GOx can be obtained on a variety of substrates in less than 60
min by simple adsorption at the isoelectric point of the enzyme (pI = 4.2) [11]. GOx oxidizes glucose
to gluconolactone and H2O2 in the presence of O2 from air. Since the fluorescence of the QD
deposited on the paper substrates can be affected by the presence of H 2O2, the hypothesis of the
project was that they could be used as indicators for enzymatic reactions via their change in
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fluorescence. In order to maximize the signal of the resulting optical biosensor, the conditions to
immobilize the enzyme were first investigated. In the first case, the effect of the concentration of the
solution containing GOx was investigated. For this purpose, 3 L of solutions containing different
concentrations of enzyme (0, 10, 20, 40, and 80 mg mL -1) were dispensed on the paper substrates
upon the addition of QD. The performance of the resulting paper-based assay was evaluated by
measuring the change in fluorescence after the addition of 3 L of a sample containing 50 mg dL-1
of glucose in 10 mmol L-1 phosphate buffer (pH = 7). As it can be observed in Figure S4, an enzyme
concentration of 20 mg mL-1 provided the maximum signal intensity and the minimized the color
gradient. The latter was attributed to a potential agglutination of the QD in the presence of high
concentrations of the enzyme ( 40 mg mL-1).
Figure S4: Effect of enzyme concentration (0, 10, 20, 40 and 80 mg mL-1) on the glucose measurements with the
concentration of glucose at 50 mg dL-1.
As the fluorescence quenching is attributed to the partial degradation of the surface of the QD, the
effect of the reaction time was also investigated in the 1-30 min range. According to the results
(data not shown), a reaction time of 20 min provided a stable change in the fluorescence. This
rather slow response time (compared with other glucose sensing systems in solution) could be
attributed to the slow reaction between the H 2O2 released and the QD. It is important to note that,
although this could be a limitation for cases where immediate feedback is required, the resulting
color was stable for 10 month (Figure S1).
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