Clean photoinduced generation of free reactive oxygen species by silica films embedded with CdTe–MTA quantum dots

Abstract

CdTe quantum dots capped with mercaptopropionic acid, 3.5 nm in size, were entrapped in sol–gel films prepared with tetramethyl orthosilicate under mineral acidic catalysis in the presence of Triton X-100 as a non-ionic surfactant. The follow-up of the sol–gel process was performed in real-time both with fluorescent crystal violet as a molecular rotor and with quantum dots. Clusters of nanoparticles 500 nm in size become homogeneously distributed in the films, but preserving initial photoluminescence quantum yields (21%), and the emission spectrum had increased excited state lifetimes (65 ± 4 ns) and photostability. Films photoactivation inside a multi-pumping flow system enabled reproducible generation of reactive oxygen species as determined by chemiluminescence using the alkaline luminol reagent, thus opening future developments for clean and environmentally friendly analytical applications.

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Introduction

Quantum dots (QDs) in colloidal solution have been largely exploited as fluorescent reagents for analytical and biological applications.^1,2 The enhanced photostability, quantum efficiency of luminescence and tenability of emission also turn them into attractive alternatives to conventional organic fluorophores in contexts ranging from the improvement of optoelectronic and solar cell devices to sensing and (bio)imaging equipment.^3–6 Those properties can be patterned through elemental composition, crystallinity, size and shape, defects and impurities. The QDs combining zinc or cadmium with oxygen, sulfur, selenium or tellurium as II–VI semi-conductive compounds are particularly popular due to a direct band gap allowance and the numerous available recipes for synthesis. However, several hazardous effects upon humans and the environment are particularly cumbersome. Cadmium, for instance, has no recognized physiological role in humans yet dermal, pulmonary and gastrointestinal exposure causes serious kidney, bone and pulmonary damage, including cancer.⁷ Regulatory agencies like the Environmental Protection Agency (EPA) and the Occupational Safety and Health Administration impose limits of 5 μg L⁻¹ to drinking water and 100 ppb to workplace air, respectively, in order to restrain biohazards.⁸ In turn, engineered nanoparticles in monodisperse colloidal sols exhibit peculiar aggregation, adsorptive and reactive behaviours which include increased metal bioavailability and toxicity to biota.^9,10 Thus, usages based on immobilized QDs and recycling of out of use materials are of great interest, being also beneficial to provide cost-effective analytical applications, reduce wastes and to mitigate environmental concerns upon discarding. Additionally, the same structured material becomes accessible for multiple purposes, including continuous reactive surfaces, separation units or as reusable sensors.¹¹

Many efforts have been undertaken to incorporate QDs into silica sol–gel matrices while preserving the initial photoluminescence properties.^12–17 Several two-step procedures, i.e. separate preparation of nanostructured particles followed by incorporation in the silica bulk medium, are typically used to immobilize QDs in glass films. In this way, luminescent materials with good mechanical characteristics, high transparency, low UV light sensitivity and high thermal stability could be obtained. The alternative, in situ preparation of nanocrystals in molten glasses, evidenced low versatility for the synthesis process, inevitable particle oxidation, volatilization of metal chalcogenides during glass densification and limited ability to obtain core/shell nanostructures.¹⁸ Room temperature procedures were attempted to synthesise II–VI^19,20 and IV–VI QDs²¹ during the hydrolysis-condensation of silica alkoxides in a simpler manner, but particle size heterogeneity and high-concentration defects were pointed out as the main disadvantages. Thus, bottom-up approaches are used to prepare hydrophobic or hydrophilic quantum dots aiming in the first step at the careful passivation of unsatisfied dangling bonds at nanoparticles surfaces which could quench radiative exciton recombination. Only thereafter, QDs are added to the pre-hydrolysed, water–alcohol–silica alkoxide monomer sol. After that, hydrolysis and condensation proceeds resulting in the final gel immobilization, regardless of the morphology intended for the final product. The use of 3-aminopropyltrimethoxysilane (3-APTMS) or tetraethoxysilane (TEOS) monomers, or optimized mixtures of both monomers, hydrolysed in water–alcohol medium under basic catalysis conditions is often referenced. Both monomers and reaction conditions propitiate long gel times (>24 h) with the amine group acting as a basic catalyst leading to the final xerogel. The use of the more common tetramethyl orthosilicate (TMOS) as the starting monomer and the use of a mineral acid catalyst is sometimes referenced to justify unsuccessful trials were immediate flocculation of quantum dots with concomitant luminescent loss occurs.^22–24 Nevertheless, Mulvaney's²⁵ group has succeeded in incorporating trialkylphosphine capped CdSe quantum dots into xerogels derived from TMOS in a basic medium using octylamine as the catalyst. The catalyst reduced the gel time from the hours scale to minutes scale, but only 5–10% of the native photoluminescence quantum yield was observed.²⁵

Herein, it is evidenced that CdTe quantum dots capped with mercaptopropionic acid could be easily entrapped in sol–gel films prepared by means of TMOS under mineral acidic catalysis. However, a non-ionic surfactant must be added to the initial sol in order to preserve native photoluminescence properties. The corresponding films provide a clean long-term ability to produce constant amounts of reactive oxygen species (ROS)^26–32 for chemiluminescent analytical applications in simple flow-systems.

Experimental section

Chemicals and materials

All solutions were prepared with deionized water (18 MΩ cm) and analytical grade reagents. The following chemicals provided by Sigma-Aldrich® were used: absolute ethanol (99.8%), (3-aminopropyl)trimethoxysilane (3-APTMS, 99%), crystal violet (CV, 90%), cadmium chloride (CdCl₂, 99%), luminol, 3-mercaptopropionic acid (MPA, 99%), methanol (99.8%), nitric acid (70%), tellurium powder (200 mesh, 99.8%), tetraethyl orthosilicate (TEOS, 98%), tetramethyl orthosilicate (TMOS, 98%), Triton™ X-100, sodium borohydride (NaBH₄, 99%) and sodium hydroxide.

A 1 mol L⁻¹ sodium hydroxide stock solution was prepared by dissolving 40.0 g of NaOH in 1000 mL of deionized water.

A luminol stock solution with the concentration of 1.0 × 10⁻² mol L⁻¹ luminol was prepared by dissolving 177.1 mg of luminol in 100 mL of an equimolar NaOH solution. The stock solution was kept at low temperature and protected from the light, when not in use. Whenever necessary, working solutions were prepared from this by simple dilution.

Synthesis of CdTe–MPA quantum dots

The molar ratio of Cd²⁺ : Te²⁻ : MPA was fixed at 1 : 0.05 : 1.7 and the resulting CdTe QDs capped with MPA were synthesized with some modifications in accordance with the method described by Zou et al.³³ The first stage consisted of the complete reduction of 2.99 × 10⁻⁴ mol of tellurium with 4.7 × 10⁻³ mol of NaBH₄ in N₂ saturated water to produce NaHTe. The resulting solution was then transferred into a second flask containing 6.59 × 10⁻³ mol of CdCl₂ and 1.02 × 10⁻² mol of MPA in 120 mL of N₂ saturated water. The pH of the solution was adjusted to 11.5 with a 1.0 mol L⁻¹ NaOH solution. The aimed size of CdTe QDs was obtained by fixing the refluxing time to 1 h. Finally, nanoparticles in the crude solution were precipitated in absolute ethanol and subsequently separated by centrifugation, dried under vacuum and kept in amber flasks.

Procedure to implement SiO₂ films with embedded quantum dots

The SiO₂ xerogel was prepared by mixing 2 mL of TMOS, 2 mL of Triton™ X-100, 8 mL of methanol and 1 mL of HNO₃ (1 mol L⁻¹) into a 50 mL Teflon beaker, then covered with a glass plate and strongly stirred over 10 hours. About 200 mg of MPA capped CdTe QDs were dispersed in 3 mL of an ionic solution Cd²⁺/MPA with a molar ratio of 1 : 3,³⁴ which was previously adjusted to pH 10 using 1 mol L⁻¹ of NaOH solution. As the initial step to produce silica films with the QDs entrapped both, previous sols were mixed and stirred for 10 min.

The spin-coating technique was used to produce thin films over soda lime glass slides. In order to activate the silanol groups at the surface of the glass, they were immersion into a fresh aqueous solution of nitric acid 1 : 1 (v/v) for 24 hours followed by profuse surface rinsing with water, acetone, and finally, oven dried at 100 °C. The coating procedure consisted of the injection of 1 mL of the silica mixture over 1 s on the glass surface spinning at 500 rpm, and further increasing of the spin rate to 2500 rpm for 2 min in order to obtain films with a homogeneous thickness of about 25 μm. Film aging and drying was performed at room temperature for 48 hours further.

Instrumentation

The separation of the QDs after synthesis was performed by means of a ThermoElectron Jouan BR4I refrigerated centrifuge (Waltham MA, USA). A Laurell WS-650-23B Spin Coater was employed for silica film deposition on the surface of glass slides. A multi-mode microplate reader (Cytation 3, BIO-TEK) was used in the photoluminescence monitoring of the sol–gel process.

A double-beam Jasco V-660 spectrophotometer (Easton, MD, USA) was used for acquisition of absorption spectra of quantum dots dispersed in solution. A Fluorolog Tau-3 Lifetime spectrofluorometer (Horiba Jobin Yvon, NJ, USA) was used to acquire emission spectra and to perform measurements of the photoluminescence lifetimes by frequency phase modulation using Ludox as a reference standard (τ = 0.00 ns). The measurements of absolute photoluminescence quantum yields were carried out in a Quantaurus QY C11347-11 spectrometer (Hamamatsu) equipped with an integrating sphere to measure all luminous flux.

The morphology and size of the nanoparticles were investigated by high-resolution transmission electron microscopy (HRTEM) using a JEOL JEM 3010 electron microscope (Tokyo, Japan), operated at an acceleration voltage of 300 kV.

The SEM/EDS analysis of silica films with embedded QDs was performed using a high resolution (Schottky) environmental scanning electron microscope with X-ray microanalysis and electron backscattered diffraction analysis: Quanta 400 FEG ESEM/EDAX Genesis X4M.

Fluorescence microscopy was carried out by means of a Tecnai-20 electron microscope (Philips-FEI) system comprising of an inverted epifluorescence microscope (Eclipse TE300, Nikon, Tokyo, Japan) equipped with 10X air objectives, a Polychrome II monochromator (TILL Photonics, Martinsried, Germany) and a CCD camera (C6790; Hamamatsu Photonics, Hamamatsu, Japan).

Flow manifold with in-line generation and detection of ROS

The flow manifold was implemented in a configuration aiming the highest efficiency in generation of reactive oxygen species (ROS) and to enhance the sensitivity of chemiluminescence detection (Fig. 1).

Fig. 1 Schematic of the multi-pumping flow manifold. Pi, solenoid micro-pumps; X, confluence point; PAU, photoactivation unit inside a polycarbonate box; D, chemiluminescence detector; L, 0.5 mmol L⁻¹ of luminol in 25.0 mmol L⁻¹ NaOH solution; LED lamp; W, waste.

It comprised of three 120SP solenoid micro-pumps (Bio-Chem Valve Inc. Boonton, NJ, USA) delivering 10 μL per stroke. These micro-pumps were actuated through a homemade electronic circuit based on the ULN 2803A chip (TOSHIBA) connected to a laptop by means of an USB-6009 interface device (National instruments). The software controlling the micro-pumps and data acquisition was developed in LAB VIEW 8.5. The different components of the flow system were connected with 0.8 mm i.d. polytetrafluoroethylene (PTFE) tubing, acrylic homemade end-fittings, and a confluence point. A chemiluminescence detector, model CL-2027 (Jasco, Easton, MD, USA), equipped with a flow cell consisting of a helical 0.8 mm i.d. PTFE tube (100 μL inner volume) placed in front of the photomultiplier tube was used as the detection unit. In the outset of the flow manifold, special attention was paid to the construction of the photoactivation unit (PAU). It consisted of a small perspex column (40 mm length × 2.5 mm i.d.) packed with 100 mg of crushed silica embedded with CdTe QDs placed in front of a 6 W LED lamp (Parathorm R50 40 daylight) emitting white light with a luminous flux of 240 lumen. At each end of the column, a glass wool stopper was placed to avoid loss of the immobilized material. Because both free radicals and luminescence signal lifetimes are reduced, the flow paths between the extremities of the column (placed in PAU) to confluence point X and to the entrance of the flow-through detection cell were kept as short as possible (about 3 cm).

A 0.4 s time interval for alternate activation of the micro-pumps (1.50 mL min⁻¹, frequency of 2.5 Hz) was initially set in order to fill the tube with the corresponding solutions, luminol at P₂ and water at P₁ and P₃. The analytical signal baseline was established during activation of P₃. Analysis started with activation of P₁ for 6 s followed by P₂ for another equal period of time. Finally, the detection flow-cell was cleaned and the baseline signal re-established through activation of P₃ over 32 s.

Results and discussion

Characterization of the synthesized CdTe–MPA QDs

Size is one of the most important aspects dictating the unique quantum confinement observed for QDs particles smaller than the exciton Bohr radius.⁶ This confinement is characterized by a blue shift in the bandgap energy when the nanoparticle size decreases. In accordance, smaller QDs exhibit photoluminescence in the blue region while larger ones emit in the red or even in the near infrared regions. By fixing a particular size of QDs, the observed quantum yields will depend on the minimization of surface defects that could act as temporary traps for photo-induced charges thereby reducing the experimental quantum yields. In the experimental protocol used, careful passivation of the QDs surface, assuring adequate solution stability, was accomplished with mercaptopropionic acid. The best results were obtained when the molar ratio of Cd²⁺ : Te²⁻ : MPA was fixed at 1 : 0.05 : 1.7, enabling the synthesis of red emitting nanoparticles with 22% quantum yield. The absorption and photoluminescence spectra of the synthesized MPA-capped CdTe QDs are depicted in Fig. 2.

Fig. 2 Normalized UV-vis absorption (A) and photoluminescence (B) spectra of the synthesized CdTe QDs passivated with mercaptopropionic acid (excitation wavelength 400 nm).

It shows a broad absorption band with a maximum for the first excitonic transition located at 587 nm, and a narrow and symmetric emission band with a maximum intensity at 606 nm, corresponding to an energy bandgap of 2.06 eV. The obtained Full Width at Half Maximum (FWHM) value was 57 nm, indicating that the synthesized QDs were nearly monodisperse and homogeneous. The average particle size of the QDs was estimated according to the empirical formula proposed by Yu et al.:³⁵

D = (9.8127 × 10⁻⁷)λ³ − (1.7147 × 10⁻³)λ² + (1.0064)λ − (194.84)

with λ (nm) corresponding to the wavelength for the first excitonic absorption peak of the nanoparticle. In accordance, the estimated average particle size was of approximately 3.5 nm. The HRTEM image (Fig. 3) of CdTe–MPA QDs showed monodispersed nanoparticles with nearly spherical shapes whose average size is in accordance with the diameter value calculated by the empirical formula proposed by Yu et al.³⁵ (3.5 nm). Additionally, the existence of lattice planes confirmed the good crystalline structure of the synthesized CdTe QDs.

Fig. 3 HRTEM image of the MPA-capped CdTe nanocrystals with lattice planes clearly defined.

Thus, the synthesized nanoparticles had adequate properties to allow either the sensitive screening of the immobilization process in silica films and, at the same time, the evaluation of useful applications.

Entrapment of quantum dots in SiO₂ films

The production of silica xerogels by the sol–gel technique has significant advantages such as, the mild chemical conditions used, preparation at room temperature, versatility to tailor the network structure, thickness, pore size and its distribution.³⁶

Table 1 shows the sixteen formulations (F₁–F₁₆) assayed where the precursor, concentration of catalyst, surfactant volume and the water : Si ratio R were varied. In the study, the volumes of co-solvent, methanol, and of the silica monomer were fixed respectively at 8, and 2 mL. The non-ionic surfactant was used as a stabilizer agent to prevent surface deterioration of the QDs and colloidal particle agglomeration during the sol–gel process. The inexistence of specific interactions between the surfactant and QDs was evidenced in previous studies and justified by charge absence in the oxyethylene groups at the polar head.^37,38 On other hand, use of a surfactant in formulation of silica films by the sol–gel technique reduces shrinkage during the aging and drying processes and fracture occurrences after immersion of the film in water once it lowers surface tension inside the pores.³⁹ Thus, the concentration of surfactant stayed in all formulations above the μM range required for critical micellar formation. The longer times for gelation observed for tetraethoxysilane (TEOS) and 3-aminopropyltrime-thoxysilane (3-APTMS) formulations, relative to tetramethoxysilane (TMOS), were expected due to their respective slower hydrolysis rates.^40,41 The gelation times also increased with the increase in R, due to the greater dilution of hydrolyzed precursors in solution, thus lowering the rate at which the condensation process occurs. Xerogels F₃, F₄, F₉, F₁₀, F₁₅ and F₁₆ prepared with higher volumes of the alkaline QDs sol had a translucent appearance, denoting a different network structure. While the hydrolysis and condensation process catalyzed at acidic pH leads to a continuously growing network of small, condensed, oligomers, the raise in pH conditions leads to prompt silica oligomer precipitation. In such conditions, the Ostwald ripening process prevails and is responsible for growth of the silica particles, which finally aggregate to form an often translucent network.⁴²

Table 1 Composition of sols for preparation of films with entrapped CdTe–MPA quantum dots

Films		F1	F2	F3	F4	F5	F6	F7	F8	F9	F10	F11	F12	F13	F14	F15	F16
TMOS	(mL)	2	2	2	2	2	2	—	—	—	—	—	—	—	—	—	—
TEOS	(mL)	—	—	—	—	—	—	2	2	2	2	2	2	—	—	—	—
3-APTMS	(mL)	—	—	—	—	—	—	—	—	—	—	—	—	2	2	2	2
Methanol	(mL)	8	8	8	8	8	8	8	8	8	8	8	8	8	8	8	8
Triton X100	(mL)	2	2	2	2	0.1	0.1	2	2	2	2	0.1	0.1	2	2	0.1	0.1
HNO3 (1 mol L^−1)	(mL)	1	—	1	—	1	—	1	—	1	—	1	—	1	—	1	0
HNO3 (5 mol L^−1)	(mL)	—	1	—	1	—	1	—	1	—	1	—	1	—	1	—	1
QDs sol	(mL)	4	4	8	8	4	4	4	4	8	8	4	4	4	4	8	8
R ratio Si/water		34	34	67	67	34	34	50	50	100	100	50	50	41	41	81	81
Gelation time	(h)	10	10	>12	>12	10	10	12	12	>15	>15	12	12	>15	>15	<24	<24

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In formulations F₅, F₆, F₁₁, F₁₂, F₁₅ and F₁₆, the lower volume of Triton X-100 used rendered transparent, brittle, xerogels that shrunk significantly in the aging/drying process. To evaluate the optimum concentration of nitric acid catalyst in the initial precursor mixture, the exchange of QDs sol by a similar volume of a solution containing crystal violet at the concentration of 0.5 g L⁻¹ was performed. Crystal violet behaves as a non-fluorescent compound in low viscosity medium due to energetic internal conversion enabled by free rotation of dimethylamine functional groups.^43,44 However, with increases in medium viscosity, such rotational movements become restrained and fluorescence emerges. Thus, its use was equated in order to provide real time monitoring of the micro-environment during gel formation. A steady increase of fluorescence was noticed during the hydrolysis-condensation process regardless of the precursor used in the formulations (Fig. 4A).

Fig. 4 (A) crystal violet fluorescence (λ_exc = 589 nm, λ_em = 636 nm) during hydrolysis-condensation of formulations F₁ (I), F₂ (II), F₇ (III) and F₁₃ (VI) stated in Table 1. (B) Quantum dots fluorescence (λ_exc = 400 nm, λ_em = 606 nm) during hydrolysis-condensation of formulations F₁ (Ia), F₇ (IIIa) and F₁₃ (VIa).

A relative comparison of the micro-environment quality of gels produced with TMOS, TEOS or 3-APTMS is not possible, due to changes in the molecular rotor behaviour with medium hydrophilic/hydrophobic properties.⁴⁵ Nevertheless, gels formed after addition of higher concentrations of nitric acid catalyst (curve II, Fig. 4A) and showed much lower intensity of fluorescence relative to formulations prepared with less concentrated acid. In the former, extended segregation of the aqueous phase resulted in smaller pores with lower amounts of entrapped probe. In contrast, the lower concentration of catalyst enabled better entrapment of the molecular rotor inside the silica network, and so, the steepest increase of fluorescence (curve I, Fig. 4A). In Fig. 4B, the screening of QDs fluorescence for formulations F₁, F₇ and F₁₃ (curves Ia, IIIa and via, respectively) is also depicted. In these experiments, the quantum dot sols were added in the beginning of the hydrolysis and condensation process when hydrolysis and condensation reactions, leading to liberation of alcohols to the bulk medium, are significant. A drop in fluorescence is observed during the first 90 min after which the micro-environment overtakes of the initial values. In accordance to the results described by others,^22–24 deterioration of particle surfaces and the irreversible loss of photoluminescence was avoided by the addition of QDs dispersed in an alkaline ionic solution containing cadmium and MPA in the optimized 1 : 3 proportion. On other hand, the initial fluorescence drop almost vanished when the addition of QDs mixture was performed in later stages of the gelation process, when alcohol evaporation was almost complete and the viscosity of the medium was high.

Micrographs obtained by SEM on aged and dried films from formulations F₁ clearly show uniform spherical aggregates (clusters) of CdTe QDs homogenously dispersed into the silica film (Fig. 5A) with diameters of about 500 nm (Fig. 5B). These aggregates were probably formed during the phase segregation concomitant to network growing and are probably located in pores at the interface between the vesicles of surfactant and the silica phase. Fluorescence microscopy imaging (Fig. 5D) corroborated previous observations since it showed spotted fluorescence emission regularly distributed along the film. A related procedure using surfactant was developed by the Murase group that named it the inverse micro-emulsion technique, where the protective effect of silica on QDs luminescence was also evidenced.⁴⁶

Fig. 5 SEM (A–C) and fluorescence microscopy (D) images of films produced according to formulation F₁ where TMOS was used as a precursor in the sol–gel process. Micrograph C shows a 25 μm thick uniform film after scratching the surface with a scalpel. In (D), intensity of emission decreases according to the deepness of the aggregate in the film.

However, this approach has only succeeded to obtain powders using TEOS as the precursor and large volumes of organic solvents.

In addition, EDS studies were conducted on the film considering both microscope localized clusters of QDs and regions without QDs. The EDS spectra (Fig. 6) confirmed presence of Cd, Te and S in film regions with QDs only. The Na and Cl peaks observed can be attributed to the precursors used in the CdTe–MPA QDs synthesis (CdCl₂ and NaOH).

Fig. 6 EDS spectra of the clusters with CdTe–MPA QDs embedded in the silica (solid line) and of regions without the CdTe–MPA QDs (dashed line).

Comparison between optical properties of the CdTe QDs before and after immobilization

The optical properties of the QDs before and after immobilization in silica films were compared in regards to the respective photoluminescence spectra, the photoluminescent lifetimes and quantum yield values. The photoluminescence spectrum of the immobilized QDs obtained after film immersion in a buffer solution at pH 11 is depicted in Fig. 7.

Fig. 7 Normalized photoluminescence spectrum of the CdTe–MPA QDs dispersed in an aqueous solution (A) and upon their immobilization in silica (B) (excitation wavelength 400 nm). The shoulder on the left side of (B) corresponds to background radiation from the soda-lime glass support.

A slight red shift was observed (Fig. 7B) in the emission peak of the immobilized QDs relatively to QDs dispersed in aqueous solution (from 606 to 611 nm), while no significant differences were observed in the FWHM values. The slight red shift observed can be explained by the vicinity of CdTe QDs in the formed agglomerates and partial energy transfer inside each agglomerate.⁴⁷

In Table 2, the observed photoluminescence lifetime values of the QDs are shown.

Table 2 Quantum yield (QY) and lifetime values of CdTe–MPA QDs with size 3.5 nm before and after the sol–gel process

	τ (ns)	QY (%)
CdTe–MPA in water	49.69 ± 0.96	22.2 ± 0.2
CdTe–MPA, in Cd^2+, MPA solution	22.85 ± 0.68	15.2 ± 0.2
CdTe–MPA QDs in silica film	65 ± 4	21.5 ± 0.4

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Firstly, it was observed that a decrease of about 54% in photoluminescence lifetimes (from 49.69 to 22.85 ns) of nanoparticles in the Cd²⁺, MPA ionic solution at pH 10 occurred relative to simple dispersion in water due to adsorption of Cd²⁺ cations onto the surface. In the authors opinion, this adsorption is reversed during the sol–gel process due to both strong coulombic forces between the cadmium cation and free silanol groups, and the equilibrium shift promoted by the excess MPA in the QDs initial sol used in film formulation. Thus, when the immobilization process ends, the lifetimes increased about 185% from 22.85 to 65.13 ns (Table 2) due to the absence of significant dynamic quenching.⁴⁸

A careful analysis of the quantum yield values also show that the photoluminescence properties of the QDs dispersed in aqueous solution were not affected by nanoparticle immobilization. In fact, the CdTe–MPA QDs dispersed in aqueous solution showed a quantum yield of 22.2% and 21.5% at the end of the immobilization process. These demonstrate that QD immobilization by a sol–gel process allows obtaining materials with luminescent properties similar to those observed for QDs dispersed in aqueous solution, with additional advantages arising from a much cleaner and more environmentally-friendly utilization in distinct analytical applications.

In order to verify the photoluminescence stability of the films, these were assayed during 180 min immersed in a buffer solution of pH 11 and profusely rinsed with water between the measurements at intervals of 10 min, setting the excitation and emission wavelengths at 400 nm and 611 nm, respectively. The results are depicted in Fig. 8 and show only a slight decrease of photoluminescence intensity in the first 40 min, followed by constant readings at higher time values.

Fig. 8 Photoluminescence intensity (PL) of the immobilized QDs in silica over 180 min (λ_exc = 400 nm, λ_em = 606 nm). Irradiation power was about 180 μW cm⁻².

Clean generation of reactive oxygen species

The immobilization of nanoparticles into a solid support can be an expeditious way to assure their continuous re-utilization for analytical purposes, thus avoiding the increased toxicity associated with single-use aqueous solutions, assuming their optical properties and reactivity are not impaired. As the photoluminescence properties of QDs in aqueous solution were not altered upon their immobilization in films (similar QY and PL spectra), it was also necessary to demonstrate that the reactivity of the immobilized QDs was unaffected.

The reactivity of the as-prepared films was assessed by using them as continuous multi-use reactors in a flow-based analytical methodology. In these assays, the main aim was to prove, for the first time, that the films preserved the ability to generate reactive oxygen species upon irradiation with visible light as it was already shown to occur for QD colloidal solutions.^29,31,32 According to these reports, QDs exposed to an electromagnetic radiation with an energy equal or higher than the semiconductor bandgap energy promote electron delocalization from the valence band to the conduction band, thus leading to the formation of an exciton (electron–hole pair). The formed excitons possess redox properties that can trigger chemical reactions with surrounding compounds having redox energies within the crystallite band gap. Some of the above mentioned reports^31,32 indicate the superoxide radical O₂⁻˙ as the main reactive oxygen species formed since the conduction band potential of CdTe QDs is sufficient for oxygen reduction (−0.15 eV). In turn, the generated superoxide radical O₂⁻˙ can oxidize a chemiluminescent probe and so enable the basis for analytical chemiluminescence. In the present work, this reaction scheme was implemented in the multi-pumping flow system represented in Fig. 1, wherein a column packed with 100 mg of crushed films was placed in front of a white LED lamp. Luminol, prepared in an alkaline medium, was used as the luminescence probe (CL) for ROS detection and only water was made to flow through the packed reactor. First, the influence of luminol concentration on the analytical signal was studied in a range of 0.0125–2.5 mmol L⁻¹, for a constant concentration of NaOH at 10 mmol L⁻¹ and halting the flow during the irradiation step after 10 min. This flow time stop was used because preliminary assays indicated that the repeatability and magnitude of luminescence signals were significantly affected without a stopped flow strategy.⁴⁹ The obtained results (Fig. 9A) reveal an increase of the analytical signal magnitude with luminol concentration up to 0.50 mmol L⁻¹.

Fig. 9 Influence of luminol and NaOH concentrations on the analytical signal for a stopped flow of water inside the column after 10 min.

Similarly, the influence of NaOH concentrations on the analytical signal was tested between 1.0 up to 50.0 mmol L⁻¹. For concentration values between 1.0 and 25.0 mmol L⁻¹, a remarkable increase on the analytical signal was observed (Fig. 9B). For higher concentrations, no significant differences were observed on the CL intensity. Therefore, 0.50 mmol L⁻¹ luminol solutions were prepared in an alkaline medium with a NaOH concentration of 25 mmol L⁻¹.

As already mentioned, the influence of the irradiation time was important in terms of repeatability and magnitude of the analytical signals. In fact, the response time of silica films used as sensors can be ascribed to the porous structure, the mechanical stability of the silica network and the permeability to a given compound.^36,50,51 Therefore, the influence of the irradiation time was studied by exploiting a stopped flow approach,⁴⁹ halting the water flow stream in the photoactivation unit (Fig. 1) for 0, 2, 5 and 10 min. Without irradiation, no signal was detected since, as expected, the QDs were not photo-activated and excitons were not formed. Then, the obtained results showed an accentuated increase on the CL intensity when increasing the irradiation time between 2 and 5 min. No further signal increase was noticed for 10 min irradiation time.

Once the reactivity of the immobilized QDs in silica was proven, it was also important to demonstrate that this new material can be used continuously as a multi-use reactor for the generation of ROS. Therefore, under the optimized conditions, the stability in the generation of ROS was evaluated for 42 consecutive analytical determinations performed during 210 min and repeated in three different days. The obtained results, in Fig. 10, reveal a high repeatability of the signals with a relative standard deviation, RSD, of 1.29%.

Fig. 10 Signals generated continuously during 210 min and repeated on three different days.

It can be noted that the generation of hazardous wastes associated with QDs nanoparticles and their core metals was minimized. In fact, the use of QDs immobilized into a solid support for the generation of ROS allowed a re-utilization of the same reactive surface, and consequently, no additional production of hazardous wastes. On the contrary, in the scientific works reported in the literature that use QDs dispersed in an aqueous solution for the generation of ROS,^29,31,32 a given amount of CdTe–MPA QDs was consumed per determination, thus producing hazardous wastes that can be problematic after long-time use. As an example, a comparison of this proposed methodology with those reported in the literature when consecutive analytical determinations are performed during 210 min, no consumption of CdTe QDs is required, while in the works performed by Ribeiro et al.,²⁹ Sasaki et al.³¹ and Rodrigues et al.,³² the amount of QDs consumed during 210 min would be 11 300 μg (22 mL of 1.00 μmol L⁻¹ CdTe–MPA dispersion), 1036 μg (49 mL of 0.10 μmol L⁻¹ GSH–CdTe dispersion) and 351 μg (22 mL of 0.15 μmol L⁻¹ GSH–CdTe dispersion), respectively.

Conclusions

The production of silica films by the sol–gel process using TMOS, plus Triton X100 as starting monomer and non-ionic surfactant, respectively, provides effective immobilization of CdTe–MPA QDs, rendering materials with the intrinsic properties of the starting nanoparticles. Additionally, the sol–gel process via acidic catalysis allowed the production of films with high transparency, which is an important factor to emphasize the optical properties of the immobilized nanoparticles and pave the way to chemosensing applications. Herein, the reactivity of the immobilized QDs was proved as reactive oxygen radicals could be produced upon visible light irradiation, thus also demonstrating robust long-term use in chemiluminescent analytical applications. Additionally, the use of the immobilized material for ROS generation inside an automated flow system allowed the carrying out of multiple determinations without the production of hazardous wastes in a more environmentally friendly approach.

Acknowledgements

This work received financial support from the European Union (FEDER funds through COMPETE) and National Funds (FCT, Fundação para a Ciência e Tecnologia) through projects UID/QUI/50006/2013, NORTE-07-0124-FEDER-000067 (QREN-NANOQUÍMICA) and FCT/CAPES no 354/13. To all financing sources the authors are greatly indebted. Gustavo C. S. Souza, acknowledges financial support from CAPES/Brasil Coordenação de Aperfeiçoamento de Pessoal de Nível Superior and FACEPE/Fundação de Amparo à Ciência do Estado de Pernambuco, grant no 3044/14-9. David S. M. Ribeiro thanks the FCT for a post-doc Grant (SFRH/BPD/104638/2014) cosponsored by the FSE (European Social Fund) and national funds of the MEC (ministry of Education and Science).

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