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Quantification of thyroxine by the selective photoluminescence quenching of l -cysteine–ZnS quantum dots in aqueous solution containing hexadecyltrimethylammonium bromide
Journal of Luminescence 156 (2014) 16–24
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Journal of Luminescence journal homepage: www.elsevier.com/locate/jlumin
Quantification of thyroxine by the selective photoluminescence quenching of L-cysteine–ZnS quantum dots in aqueous solution containing hexadecyltrimethylammonium bromide Sarzamin Khan a, Leonardo S.A. Carneiro a, Eric C. Romani b, Dunieskys G. Larrudé b, Ricardo Q. Aucelio a,n a b
Chemistry Department, Pontifícia Universidade Católica do Rio de Janeiro, 22451-900 Rio de Janeiro-RJ, Brazil Physics Department, Pontifícia Universidade Católica do Rio de Janeiro, 22451-900, Rio de Janeiro-RJ, Brazil
art ic l e i nf o
a b s t r a c t
Article history: Received 6 December 2013 Received in revised form 1 July 2014 Accepted 3 July 2014 Available online 16 July 2014
The determination of L-thyroxine is proposed based on the photoluminescence quenching effect caused on the L-cysteine modified ZnS quantum dots (L-cysteine ZnS QDs) aqueous dispersion. Under optimum conditions, the analytical response followed a Stern–Volmer model and the experimental conditions were adjusted to enable a robust and reproducible photoluminescence signal. The linear response observed in the quantum dots aqueous dispersion covered the L-thyroxine concentration from the LOQ (2.0 10 8 mol L 1) to 4.0 10 6 mol L 1. The approach was tested in the determination of L-thyroxine in pharmaceutical formulations used to treat patients with thyroid gland disorder. The percent recoveries in controlled samples were between 93.3 and 103%. Analyte fortified saliva was also evaluated as a possible sample for L-thyroxine monitoring of a patient under treatment. It was identified a static type of photoluminescence quenching caused by L-thyroxine. & 2014 Elsevier B.V. All rights reserved.
Keywords: Quantum dots photoluminescence L-thyroxine L-cysteine modified ZnS quantum dots Saliva
1. Introduction The normal thyroid gland is a discrete soft body made up of a large number of vessels that produce, store, and release two key hormones: triiodothyronine, also called T3 and thyroxine or T4, where the numbers 3 and 4 refer to the number of iodine molecules attached to each hormone. Thyroid cells are the primary cells in the body capable of absorbing iodine, an essential nutrient obtained through food, iodized salt, or supplements. A healthy thyroid produces a proportion of about 20% of T3 and 80% of T4, where T3 is the biologically active hormone that is used by the cells. When needed, the body converts the inactive T4 into the active T3 by removing one atom of iodine from the molecule [1]. Thyroid hormones have a number of functions such as helping cells to convert oxygen into energy and the brain to function properly, it guarantees a normal bone growth and the processing of carbohydrates, enabling a proper sexual development and functioning [2]. A patient that presents low secretion of thyroid hormones or even the lack of it must be treated with the administration of thyroxine (Fig. 1A) in order to enable a regular
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Correspondent author. Fax: þ 55 21 3527 1637. E-mail address: [email protected] (R.Q. Aucelio).
http://dx.doi.org/10.1016/j.jlumin.2014.07.003 0022-2313/& 2014 Elsevier B.V. All rights reserved.
maintenance of the triiodothyronine levels. Levothyroxine (L-thyroxine) is the artificial thyroxine hormone mostly used for the therapy of thyroid dysfunction and it is commercially available under several brand names. L-thyroxine is a non-fluorescent substance at room temperature. Its absorption profile is due to the two weakly conjugated benzene rings, thus it presents the characteristic and more intense B1 (at about 235 nm and close to the lower limit of the spectrum) and a weaker B2 (at about 330 nm) absorption bands of benzene (Fig. 1B). The presence of iodine in the molecular structure forces, through spin–orbital coupling (internal heavy atom effect), the transference of the molecular population from the excited singlet state to the triplet state by means of intersystem crossing. Thus, the long lifetime radiative return to the ground state is not favored when compared to the non-radiative processes that do not favor the natural photoluminescence of thyroxine. The indirect detection of thyroid hormones (T3 and T4) was carried out from either the inhibition of the luminol–iron (II) chemiluminescence or the enhancement of the elecrtochemiluminescence of Tris(2,2-bipyridyl)ruthenium(III)–NADH system. Limits of detection (LOD) of respectively 1.2 10 7 mol L 1 and 5 10 8 mol L 1 were achieved [3,4]. Capillary electrophoresis has also been utilized for the determination of thyroxine in pharmaceutical formulations using electrochemical detection on
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QDs) was studied in aqueous dispersions. The photoluminescence quenching effect caused by L-thyroxine was more effective in aqueous systems containing hexadecyltrimethylammonium bromide (CTAB), which also improved stability of the measured signal over time. The approach using the L-cysteine–ZnS probe was used to quantify L-thyroxine in a pharmaceutical formulation. Analyte fortified saliva was also evaluated as a possible matrix for Lthyroxine monitoring of a patient under treatment.
2. Material and methods 2.1. Apparatus
Fig. 1. Electronic absorption spectra of thyroxine; (a) 3.9 10 7, (b) 9.8 10 7, (c) 3.8 10 6, and (d) 6.0 10 5 mol L 1.
a carbon disk electrode [5]. The use of high performance liquid chromatography (HPLC) for the separation and detection of both T3 and T4 in dietary supplements enabled quantifications down to 0.002 μg mL 1 (2.9 10 9 mol L 1) after the pre-column derivatization of the analytes with 4-fluoro-7-nitrobenzofuran [6]. The indirect detection of thyroxine in urine has been achieved by the spectrophotomeric detection of iodide (at 226 nm) after separation in an anion exchange column [7]. More recently, isotopedilution liquid chromatography/tandem mass spectrometry method was applied for the determination of thyroxine in saliva (at the pg mL 1 level) using (13C6)-T4 as internal standard [8]. The indirect fluorescence detection of thyroxine has been achieved by measuring the quenching effect on 7-hydroxycoumarin and Eu(III)-(pyridine-2,6-dicarboxylate) Tris complex. LOD value of 3.4 10 8 mol L 1 has been achieved [9]. Semiconductor nanoparticles (quantum dots or QDs) are grown to be in the nanoscale size to achieve the effect of quantum confinement, which enables unique optical properties such as wide absorption profile, very intense size-dependent luminescence and high photostability. For instance, CdS QDs and to a less extent the ZnS QDs have been studied since they can be easily synthesized in aqueous medium under mild conditions. In addition many surface modifications in QDs (nanoparticle capping with organic ligands) have been proposed to enhance optical properties, stability in aqueous dispersions and selectivity in interaction with chemical species in solution. These modified nanoparticles can be homogeneously dispersed and stabilized in water, and therefore they are very attractive as analytical probes to selective sense chemical species in aqueous systems [10–12]. In this work, the effect of L-thyroxine on the photoluminescence of L-cysteine modified ZnS quantum dots (L-cysteine–ZnS
A Perkin-Elmer Lambda 19, UV/vis/NIR double beam spectrophotometer (1 cm quartz cuvettes) was used to obtain electronic absorption spectra from L-thyroxine and to evaluate the extinction spectra from the QDs dispersions. All photoluminescence measurements were made using a Perkin-Elmer model LS 55 luminescence spectrometer with solutions placed in 1 cm optical pathlength quartz cuvettes. Photoluminescence spectra were acquired using the FL-Winlabs software and measurements were performed with 10 nm excitation and emission spectral bandpass and 1500 nm/min scan rate. Excitation was made at 312 nm with signal measurement at 424 nm. A thermostatic system with stirring (PTP-1 Fluorescence Peltier System with a PCB1500 Water Peltier System, Perkin-Elmer) was used to keep the solutions in the cuvette at specific constant temperatures and to allow stirring of the CTAB organized L-cysteine–ZnS QDs working dispersion. Photoluminescence lifetime measurements were conducted using a Model HJY 5000M time-resolved fluorescence spectrometer IBH 5000F (Horiba Jobin Yvon, NJ, USA) with excitation using a nanoLED source at 372 nm. Lifetimes were obtained after mathematical deconvolution of the fluorescence decay from the source pulse profile. Transmission electron microscopy (TEM) was made on JEOL 2010 Transmission Electron Microscope under 200 kV accelerating voltage. Dynamic light scattering (DLS) measurements of the quantum dots were recorded in a Zetasizer Nano ZS (Malvern, UK) equipment, operating at 25 1C using a He–Ne laser (633 nm) with measurement range from 0.6 nm to 6 mm and the analyzed range from 2 to 500 nm. For pH measurements, the pH-meter (MS Tecnopon, model MPA-210, Brazil) was employed. 2.2. Reagents, samples and other materials L-cysteine hydrochloride monohydrate, L-thyroxine, zinc acetate dehydrated, sodium phosphate monobasic, sodium phosphate dibasic heptahydrate, hexadecyltrimethylammonium bromide (CTAB) were purchased from Sigma-Aldrich (USA). Sodium dodecyl sulfate (SDS) and triton X-100 were from Merck, Germany. CTAB and SDS were further recrystallized in ethanol in order to eliminate impurities. Analytical grade methanol was from Merck. The pharmaceutical formulation (containing of 200 mg thyroxine per tablet) was obtained from a local drugstore. Utrapure water was purified in a Milli-Q system from Millipore (Simplicity model 185, USA) and used to prepare all aqueous solutions. Solid phase extraction (SPE) was made on a 6 mL volume cartridge containing 1 g of C18 sorbent (Cole Parmer, USA).
2.3. Synthesis of L-thyroxine cysteine–ZnS quantum dots The ZnS nanoparticles modified with L-cysteine were synthesized following similar procedures described in the literature [13]. In typical synthesis, an amount of 0.0214 g (1 10 4 mol) of zinc acetate dihydrated and 0.0172 g (1 10 4 mol) of L-cysteine
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hydrochloride monohydrated were dissolved in 100 mL of deionized water. Then, the solution was stirred for 15 min and its pH value adjusted round to 9.0 by the addition of small volumes of a 0.1 mol L 1 NaOH solution. The resultant solution was transferred to a 150 mL three necked round bottom flask (reactor) where a solution containing 2 10 4 mol of Na2S was slowly injected (in the form of aqueous solution) through a syringe into the reactor, under nitrogen protection. Then, the mixture was refluxed (about 100 1C, under inert atmosphere) for specific period of time. Aliquots of the reaction mixture were taken out at different intervals of time, through a syringe in order to check their absorption and photoluminescence characteristics. The transparent and colorless colloidal dispersion of ZnS QDs was treated with ethanol to precipitate the nanoparticles, then, the solid mass was re-dispersed in a phosphate buffer solution (0.01 mol L 1 and pH 8.0). These nanoparticles were kept under refrigeration being stable for more than 10 months, indicated by transparency of the dispersion and by its high photoluminescence intensity. 2.4. Microscopy and spectroscopy of the L-cysteine–ZnS QDs The morphology and structure of functionalized ZnS nanoparticles were studied by scanning transmission electron microscopy (Fig. 2), which indicates a large number of assemblies of L-cysteine–ZnS nanoparticles with a fairly uniform size and shape. As can be observed, nanoparticles are well dispersed and the size range varied from 5.1 to 6.6 nm. The particle size distribution of L-cysteine–ZnS was also measured by DLS (Fig. 2A). The hydrodynamic diameter was in the range from 12.7 to 17.2 nm with an average diameter of 14.0 nm. The DLS measurements indicated that L-cysteine–ZnS nanoparticles are prone to certain degree of aggregation, occurrence that is probably due to the use of
non-buffered dispersion during DLS analysis that may have promoted the removing of part of the L-cysteine stabilizer from the surface of the QDs (Fig. 2A). ZnS nanoparticles contain few units in their nanocrystal cells, therefore they possess a stable structure and optical properties that do not vary significantly as the reaction time is increased [14]. In addition, contrary to what is observed for other semiconductor luminescent nanoparticles, the excitation profile of the L-cysteine– ZnS QDs is narrower [15]. The electronic optical absorption of the synthesized L-cysteine–ZnS QDs presented a broad profile with the first excitonic at 290 nm (Fig. 3A), photoluminescence maximum observed at 424 nm (related to the 312 nm of the sharp excitation band) and the full width at half maximum value for the photoluminescence emission band of 36 nm (Fig. 3B). The photoluminescence intensity of nanoparticles centered at 424 nm sharply increased with the increasing reflux time, reaching a maximum value at 120 min. The photoluminescence intensity then decreased as the reflux time surpassed 120 min as indicated in Fig. 2C. At longer refluxing times, the cloudy appearance of the dispersion indicated the aggregation of the crystals leading to the formation of bigger particles and consequently less intense photoluminescence. 2.5. Photoluminescence measurements from the L-cysteine–ZnS QDs aqueous dispersions For the determination of L-thyroxine in samples, the L-cysteine– ZnS QDs dispersion, prepared in phosphate buffer (0.01 mol L 1; pH, 8.0) and containing CTAB at concentration of 5 10 5 mol L 1, was transferred to a quartz cuvette. Volumes of either L-thyroxine standards or samples containing L-thyroxine were added to the QDs dispersion at room temperature and under constant stirring. Measurements were made 5 min after the stirring was turned off
Fig. 2. STEM image of L-cysteine–ZnS QDs and DLS histogram of (A) L-cysteine–ZnS QDs aqueous dispersion and (B) L-cysteine–ZnS QDs aqueous dispersion in the presence of thyroxine.
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diluted sodium hydroxide solution (5 10 4 mol L 1). Standard solutions of lower concentration were prepared by further dilution of this stock solution with ultrapure water. The surfactant stock solution (5.0 10 3 mol L 1) was prepared by dissolving appropriate amounts of CTAB in ultrapure water. For the preparation of an L-thyroxine pharmaceutical formulation sample, a pool of ten commercial tablets was pulverized. Mass aliquots of this powder were dissolved in ethanol. The sample was passed through a Teflon syringe filter to retain the non-soluble excipients, then, the solution was passed through a C18 solid phase extraction (SPE) column to retain the L-thyroxine. The cartridge was washed with deionized water (to remove remaining water soluble tablets recipients) and the L-thyroxine was eluted with 1 mL of methanol. The methanol was evaporated and the remaining residue was re-dissolved in the L-cysteine–ZnS QDs buffered dispersion. The saliva sample (10 mL collected in a graduated cylinder) was collected from an euthyroid volunteer that did not receive any other medical treatment nor ingested food or beverages prior the sample collection. The volunteer rinsed his mouth for 5 min with ultrapure water. The saliva was fortified with known amount of thyroxine and mixed with 5 mL of ethanol. Then, the mixture was vortex mixed for 30 s and immediately centrifuged for 15 min at 3000 rpm. After centrifugation, the supernatant was passed through C18 SPE column and washed with ultrapure water. After elution with 1 mL methanol, the eluate was evaporated and the residue re-suspended in the L-cysteine–ZnS QDs buffered dispersion. The same procedure was repeated for blank measurements.
3. Results and discussion 3.1. Optimization of conditions to achieve the photoluminescence quenching of the L-cysteine–ZnS QDs by L-thyroxine L-cysteine is an amino acid that has been used for synthesis of water compatible fluorescent probes [13]. Such ligand can bind to the surface of the ZnS nanoparticles through the sulfur atom of the mercapto group, while the carboxylic acid group provides water compatibility.
Fig. 3. (A) Electronic absorption spectrum. (B) Photoluminescence excitation and emission spectra. (C) Photoluminescence spectra of the cysteine–ZnS QDs synthesized using different refluxing times: (a) 10, (b) 20, (c) 40, (d) 60, (e) 80, (f)100, (g) 130 and (h) 120 min.
(equilibration time). Photoluminescence measurements were made at 424 nm upon excitation at 312 nm. In order to obtain reliable quantitative results, measurements were also taken from a L-cysteine–ZnS QDs dispersion without the addition of L-thyroxine (control). 2.6. Preparation of solutions and samples The stock solution of analyte (1.0 10 2 mol L 1) was prepared by dissolving specific amounts of the drug standard in a
3.1.1. Amount of cysteine–ZnS quantum dots in the dispersion The quenching effect promoted by L-thyroxine (fixed at 9.5 10 7 mol L 1 final concentration) on the L-cysteine–ZnS QDs photoluminescence was investigated in dispersions containing different amounts of nanoparticles. The amount of nanoparticles was varied by introducing different volumes of the synthesized L-cysteine-ZnS QDs stock dispersion (from 0.250 to 2 mL) into the 10 mL final volume of aqueous dispersion, which enabled a range between 1.6 10 4 to 1.3 10 3 mol L 1 (2.4–20.0 μg mL) of zinc concentration in the QDs precursor solution (Fig. 4A). Higher amounts of L-cysteine–ZnS QDs decreased the sensitivity of the photoluminescence quenching response due to inner filter effect caused by the high concentration of QDs in the dispersion. In contrast, the lower concentrations of QDs resulted in robust and sensitive fluorescence quenching response as indicated by the higher L0/L ratio values (where L0 is the photoluminescence measured from the dispersion in absence of L-thyroxine and L is the photoluminescence measured from the dispersion in the presence of L-thyroxine). Dispersions (10 mL total volume) containing 0.8 mL of the quantum dots stock dispersion was chosen due to the high sensitivity in the quenching response.
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3.1.2. Influence of pH on the L-cysteine–ZnS QDs photoluminescence quenching mediated by L-thyroxine The effect of pH of the aqueous dispersion on the interaction of L-thyroxine and quantum dots was studied in order to find the appropriate pH for the sensitive probing of L-thyroxine. The pH was varied in the range from 6.5 to 9.0 (using phosphate buffer or borate buffer at 0.01 mol L 1 final concentration to cover such pH range). The experiments were performed in dispersion with the different pH values either in the presence or in the absence
Fig. 4. (A) Effect of the amount of the synthesized nanoparticles on the photoluminescence quenching of the L-cysteine–ZnS QDs aqueous dispersion. Signal variation expressed as L0/L (where L0 and L are respectively the photoluminescence e of the quantum dots dispersion before and after the addition of 9.5 10 7 mol L 1 of thyroxine). (B) Influence of pH value of the aqueous dispersion on the photoluminescence quenching of L-cysteine–ZnS QDs (3.8 10 7 mol L 1 final concentration of L-thyroxine). Results obtained in triplicate.
7 L-thyroxine (3.8 10 mol L 1). The signal profile (Fig. 4B) indicated a more effective photoluminescence quenching at the pH values of 8.0 and 8.5. The pH value selected for the QDs working aqueous dispersion was 8.0 (achieved by the phosphate buffer).
3.1.3. Effect of surfactants on the photoluminescence quenching caused by L-thyroxine The effect of different surfactants such the cathionic acetyltrimethyl ammonium bromide (CTAB), the anionic sodium dodecyl sulfate (SDS) and the non-ionic Triton X-100 on the L-thyroxine mediated photoluminescence quenching was investigated. Concentration of surfactants was varied from 8.0 10 6 to 1.0 10 3 mol L 1. The presence of SDS caused total suppression of photoluminescence of the system probably due to the electrostatic repulsion between the negative charge on the surface of nanoparticles and the anionic head of SDS, thus at high concentration of SDS, quenching of the photoluminescence from quantum dots was observed. When using Triton X-100, stable photoluminescence measurements were not achieved probably due to adsorption of surfactant onto the surface of nanoparticles, changing the surface properties and probably promoting the removal of L-cysteine. The improvement in the photoluminescence quenching efficiency was obtained in the presence of CTAB at concentration of 5 10 5 mol L 1, which is below the critical micelle concentration (CMC) of this surfactant (about 1 mmol L 1) as indicated in Fig. 5A. As the surface of ZnS QDs capped with cysteine is negatively charged due to the desprotonation of the carboxylic group at pH 8.0, the CTAB (a cationic surfactant) is prone to interact to nanoparticles surface, via electrostatic interaction. In the presence of this cationic surfactant, the photoluminescence from the QDs dispersion was slightly enhanced and became stable, which may due to partial incorporation of nanoparticles into the protective environment of such surfactant micelles. The presence of nanoparticles in micelle prevents the adsorption of other molecules on the surface of the nanoparticles, thus, the excited electron from the conducting band may combine with the vacancy at the valence band with less probability of electron transfer to other molecules nearby. Better analyte induced photoluminescence quenching was observed below the critical micelle concentration (CMC) is probably due to the slight positive surface changes, which facilitates interactions between nanoparticles and the L-thyroxine molecule. At higher concentration of CTAB, the surfactant may block the access to L-thyroxine to the nanoparticles and the resulting in a less effective photoluminescence quenching. Thus, a final concentration
Fig. 5. (A) Effect of CTAB on the photoluminescence quenching of L-cysteine-ZnS QDs at fixed concentration of L-thyroxine (final concentration in aqueous dispersion, 1.2 10 6 mol L 1). (B) Photoluminescence measured from L-cysteine–ZnS QDs dispersion at different temperatures. Results obtained in triplicate.
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L0/L
Photoluminescence
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Time (min)
Time (min)
Fig. 6. (A) Photoluminescence stability of the L-cystein–ZnS QDs dispersion. (B) Photoluminescence stability of the L-cystein-ZnS QDs dispersion after addition of L-thyroxine (final concentration in solution 4.0 10 7 mol L 1).
of 5 10 5 mol L 1 of CTAB was incorporated into the QDs water working dispersion. 3.1.4. Effect of temperature The effect of temperature on the photoluminescence intensity measured from the L-cysteine–ZnS QDs dispersion was studied at temperatures ranging from 18 to 45 1C. The fluorescence measured from quantum dot was found to be inversely dependent on temperature with a fairly constant signal intensity found between 18 and 25 1C (Fig. 5B). The decrease in signal intensity measured from the QDs dispersion is probably due to the increase of the kinetic energy of the components of the system, which disrupts the interaction between the analyte and the L-cysteine ZnS QDs, leading to a less effective photoluminescence quenching. Therefore, room temperature (about 25 1C) was selected in all fluorescence measurement for sensing of L-thyroxine. 3.1.5. Stability of photoluminescence intensity and reaction time Under the selected conditions to enable effective interaction between L-thyroxine and the L-cysteine ZnS QDs in the dispersion, an evaluation of the reproducibility and stability of the measured photoluminescence was made. First, the photoluminescence from the control working dispersion of QDs (L-cysteine–ZnS QDs buffered dispersion containing CTAB before the addition of L-thyroxine) was measured in function of the time (measured every 10 min up to 120 min). The signal was found to be stable during the whole time of the experiment (less than 3% random variation of signal) as indicated in Fig. 6A. The photoluminescence from this same QDs dispersion was also monitored in function of the time after the addition of a fixed amount of L-thyroxine. Measurements were made every 2 min up to 30 min starting 2 min after the addition of L-thyroxine (1 min of mixing and 1 min of equilibration of the solution). The photoluminescence quenching was immediate and after 5 min, the signal becomes stable up for more than 30 min (Fig. 6B). For the analytical method, it was established all measurements to be made after 5 min of the addition of L-thyroxine into the quantum dots dispersion. 3.2. Mechanism of interaction Several mechanisms have been proposed to explain QDs photoluminescence signal reduction, including inner filter effect and non-radiative processes caused by electron transfer, surface
adsorption, complexation among others [16]. As previously mentioned, L-thyroxine has a wide UV–vis absorption profile with maximum at 235 nm and 330 nm. Taking into consideration a 6 10 5 mol L 1 solution of L-thyroxine, the absorbance (Fig. 1B line d) is 0.87 at 235 nm and 0.09 at 330 nm and 0.008 at 312 nm (the wavelength chosen for the excitation of L-cysteine–ZnS QDs). Thus, no inner filter effect caused by L-thyroxine is expected at the excitation wavelength of 312 nm considering concentration levels 5 L-thyroxine below 1.0 10 mol L 1. Since no changes in the absorption spectral profile of the Lcysteine–ZnS QDs (characteristic profile in Fig. 3B) takes places in the presence of L-thyroxine, it is concluded that no aggregation (chemical degradation that can lead to the reducing of photoluminescence) of QDs are taking place. If aggregation has occurred, a measurable decrease in transmittance would be observed because of the significant increasing in light scattering measured from the dispersions. This is an indication that photoluminescence quenching should be promoted by an effective interaction between the analyte and the QDs and not by light filter effect due to the increase of light extinction. Aggregation of nanoparticles induced by L-thyroxine was also ruled out since no changes in particle distribution were observed by microscopy. In addition, DLS measurement after the addition of L-thyroxine into the QDs dispersion (Fig. 2B) showed size distribution profile similar to those observed in the system without L-thyroxine (Fig. 2A). In order to determine if the mechanism of photoluminescence quenching is whether static or dynamic, a study of the dependence of photoluminescence analytical curve sensitivity upon the temperature was made [17]. From the Stern–Volmer plots constructed at two different temperatures (298 and 308 K) it was observed that the sensitivity decreases as the temperature increased (Fig. 7A). These results indicate the L-thyroxine and cysteine–ZnS interactions associative in nature and characteristic of a static type of quenching. In addition, photoluminescence lifetime experiments have indicated the similar profile for measurements made from QDs dispersions in the presence and also in the absence of L-thyroxine (Fig. 7B). It is valid to point out that the photoluminescence of semiconductor nanoparticles is known to decay in a multi-exponential manner, thus, QDs emission consists of more than one lifetime component [10]. Thus, the photoluminescence lifetimes that characterized the dispersion of cysteine– ZnS QDs in CTAB, no matter the presence of L-thyroxine, were: 4 71; 17 72 and 85 74 ns. As no change in lifetime profile of the QDs dispersion is observed in the presence of L-thyroxine, results
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L0/L
Table 1 Optimized experimental conditions for the thyroxine determination using the L-cysteine–ZnS QDs. Experimental parameter
Optimized Value
Type of quantum dots Phosphate buffer solution pH Time required to perform measurement Concentration estimated for QDsa Temperature CTAB concentration
Cysteine–ZnS nanoparticles 0.01 mol L 1 8.0 5 min 4.5 10 4 mol L 1 25 1C 1.2 10 6 mol L 1
a
Estimated by the concentration of Zn2 þ in the QDs precursor solution.
Intensity(counts)
Photoluminescence
Concentration of L-thyroxine(10-7 mol L-1)
Wavelenght (nm)
Time (ns)
indicate that a static quenching is taking place when analyte interacts with QDs. A possible mechanism can be proposed based on the experimental results. In absence of CTAB, no quenching effect was observed since non-protonated L-thyroxine is repelled by the negatively charged cysteine that covers the surface of the ZnS QDs. When CTAB is added, this cationic surfactant interact with QDs neutralizing the negative charges of the capping agent, thus enabling L-thyroxine to form a complex with Zn atoms at the surface of QDs. Thyroxine withdraw electrons from the QDs conducting band resulting in photoluminescence static quenching. As L-thyroxine present four iodine groups any molecular fluorescence that might be stimulated by such electron transfer is also quenched by internal heavy atom effect that transfers the electronic population to the triplet state. Since environment is not free from dissolved oxygen, triplet state radiative decay is not favored. 3.3. Analytical characteristics of the photoluminescence quenching mediated by L-thyroxine 3.3.1. Detection and quantification limits and precision of measurements Under the optimized experimental conditions (Table 1) a Stern–Volmer model (Eq. 1) could be readily used to establish a linear relationship between measured photoluminescence, L, and the concentration of L-thyroxine, [L-thyroxine], in the dispersion.
L0/L
Fig. 7. (A) The Stern–Volmer curves for the quenching of cysteine–ZnS QDs aqueous dispersion in the presence of L-thyroxine at 298 K (■) and 308 K (▲). (B) Photoluminescence lifetime profile of cysteine-ZnS QDs aqueous dispersions in the absence (▲) and in the presence (★ of thyroxine (3.6 10 6 mol L 1).
Concentration of L-thyroxine(10-7 mol L-1) Fig. 8. (A) Photoluminescence emission spectra of L-cysteine–ZnS QDs aqueous dispersion in the presence of different concentrations of L-thyroxine: (a) 0, (b) 1.1 10 7, (c) 2.0 10 7,(d) 3.0 10 7, (e) 3.9 10 7, (f) 4.9 10 7, (g) 9.8 10 7, (h) 2.0 10 6, (i) 2.9 10 6, (j) 3.8 10 6, and (k) 4.0 10 6 mol L 1. (B) Stern–Volmer-type calibration curve for the determination of thyroxine using L-cysteine–ZnS QDs as probe.
L0 is the photoluminescence made from the probe dispersion before the addition of L-thyroxine and Ksv is the Stern–Volmer constant. L0 =L ¼ 1 þ K sv ½L thyroxine
ð1Þ
The results show that L-thyroxine quenches the photoluminescence of the L-cysteine–ZnS QDs in a concentration dependent pattern from 1.1 10 7 to 4.0 10 6 mol L 1 (Fig. 8A). Thus, analytical curves were constructed by adding increasing concentrations of L-thyroxine on the QDs dispersion and then, measuring the photoluminescence decreasing. A typical analytical curve (L0/L versus
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concentration of L-thyroxine) is shown in Fig. 8B, showing a linear range of the analytical response in the concentration range from 1.1 10 7 to 4.0 10 6 mol L 1 of L-thyroxine (final concentration in the dispersion) with correlation coefficient of 0.9926. The equation model of the analytical curve was L0/L¼3.3 105 [L-thyroxine]þ 1.01. Since at these concentration levels L-thyroxine does not significantly absorb light at 312 nm, no correction for inner-filter effect was necessary for the Stern–Volmer model. The limit of detection (LOD) was calculated as the concentration of L-thyroxine that changes the photoluminescence signal in the measurement cell by L0 3sL0, where sL0 is the standard deviation of ten replicate measurements of the photoluminescence intensity of the L-cysteine–ZnS dispersion before the addition of L-thyroxine. Similarly, the limit of quantification (LOQ) was calculated as the L0 10sL0. The LOD and the LOQ were 6.2 10 8 mol L 1 (48.3 ng mL 1) and 2.0 10 8 mol L 1 (15.4 ng mL 1), respectively. The precision of the L-thyroxine measurement using the proposed probe was calculated as the variation of the L0/L value taking into consideration ten independent solutions (in two different L-thyroxine concentrations). In order to do this, the following equation was used: s(L0/L) ¼L0/L [(sL/L)2 þsL0/L0)2]1/2. The s(L0/L), in percentage values, was 2.8% and 4.2% at, respectively the 3.9 10 7 mol L 1 and 2.9 10 6 mol L 1 concentration levels. 3.3.2. Selectivity studies For practical applications of the proposed method for the determination of L-thyroxine in biological samples (saliva) and in pharmaceutical formulations, the effect of some possible relevant interfering substances was evaluated. The chosen substances were the ones commonly found in pharmaceutical formulations and in biological fluids (including several amino acids). Changes in fluorescence intensity due to the presence of these chemical species were expressed in percent values (Table 1). All the tested substances imposed variations in the photoluminescence intensity measured from the probe (at the specified concentrations) under 4% (variation considered not relevant). In contrast, the presence of a much less amount of L-thyroxine (0.4 μmol L 1) caused about 10% decreasing of the photoluminescence measured from the probe. The interference caused by two different complex matrices was also evaluated. One matrix was composed by a mixture of amino acids (cysteine, histidine, phenylalanine, valine, methionine, lysine and threonine), each amino acid with a final concentration of 5 10 6 mol L 1. The other matrix consisted on a mixture of common pharmaceutical excipients (lactose, silicon dioxide, citric acid, calcium chloride, Table 2 Effect of some potential interfering substances on the photoluminescence measured from the L-cysteine–ZnS QDs organized aqueous dispersion. Potential interferent
Concentration (μ mol L 1)
Change of photoluminescence (%)
Cysteine Histidine Tyrosine Phenylalanine Valine Methionine Lysine Threonine Lactose Silicon dioxide Citric acid Calcium Magnesium Potassium Sodium Chloride
150 150 150 150 150 150 150 150 100 100 100 100 100 100 100 100
þ 4.0 þ 3.1 þ 1.4 þ 0.55 þ 0.55 0.55 þ 1.13 1.42 2.6 þ 1.8 þ 2.0 þ 4.1 þ 3.8 þ 2.1 þ 3.1 þ 3.0
23
magnesium sulfate, potassium chloride, sodium chloride) each one at a final concentration of 0.1 10 6 mol L 1. It was observed that the photoluminescence measured from L-cysteine–ZnS QDs dispersion in absence and in the presence of L-thyroxine is not significantly different when either the amino acids mixture or the pharmaceutical excipient mixture is present. For biological samples, proteins can affect the photoluminescence of L-cysteine–ZnS QDs but due to pretreatment of samples (protein precipitation and SPE) such interference is minimized. Such samples also contain salts, but the study to evaluate the influence of NaCl on the photoluminescence quenching of the probe indicated that no interference is expected in samples containing up to 2 10 3 mol L 1 of NaCl. 3.4. Analytical application of the L-cysteine–ZnS QDs dispersion for the determination of L-thyroxine The proposed photoluminescence L-cysteine–ZnS probe has been applied for the determination of L-thyroxine in pharmaceutical formulation (containing the artificial thyroxine hormone as active component) and in analyte fortified human saliva, simulating a sample from a patient with a non-active thyroid gland that was medicated with L-thyroxine. Three different portions (0.57 g) from ten grinded pharmaceutical tablets were selected. The L-thyroxine content in these portions were dissolved in methanol, filtered in a 0.45 mm syringe filter and then diluted in water. A volume (1 mL) of the sample solution was placed into the L-cysteine–ZnS QDs aqueous dispersion in order to achieve a theoretical final concentration of 9.8 10 7 mol L 1 of L-thyroxine in the QDs dispersion. The recoveries (Table 2) achieved using the proposed method at was 97.07 5.2% taking into consideration the value of L-thyroxine indicated in the pharmaceutical formulation instructions (Table 3). In order to evaluate the applicability of the method in simple clinical assays, the analysis of saliva samples (fortified with Lthyroxine at concentration of 2.9 10 7 mol L 1) were performed. The saliva was mixed with 5 mL of ethanol, fortified with the known amount of L-thyroxine then immediately centrifuged for 15 min at 3000 rpm. After centrifugation, the supernant was passed through C18 SPE column and washed with deionzed water. After elution with 1 mL methanol, the eluate was evaporated and the residue re-suspended with the nanoparticle dispersion (made in phosphate buffer pH 8.5). From the photoluminescence quenching magnitude, the concentration of thyroxine was obtained with recovery of 86.07 5.1%. The relatively low recoveries might be due to loss of L-thyroxine in pretreatment of saliva Table 3. Photoluminescence spectra (Fig. 9) indicates that original components from saliva matrix does not interfere with the determination of thyroxine as it does not fluoresce under the condition of the experiment and it does not affect the photoluminescence signal from the L-cysteine–ZnS dispersion when it is not fortified with thyroxine. The effectiveness of cysteine functionalized nanoparticles as probe for substances of clinical and biological interest has been demonstrated in earlier works, for instance, aqueous dispersion of Table 3 Applications of the cysteine–ZnS QDs for determination of L-thyroxine in pharmaceutical formulation and saliva. Sample
L-thyroxine level
200 μg per tablet 7 L-thyroxine fortified 2.9 10 saliva mol L 1 Levotiroxina sódica
Portion 1
Portion 2
Portion 3
Average result (%)
93.3
94.8
103
97.0 7 5.2
88.9
90.1
86.5
86.0 7 5.1
24
S. Khan et al. / Journal of Luminescence 156 (2014) 16–24
of the surfactant CTAB. The overall quenching followed a Stern– Volmer model. The proposed optical probe has been applied for the determination of L-thyroxine in pharmaceutical formulation and in analyte fortified human saliva. The poor optical properties of L-thyroxine make the proposed indirect determination approach attractive when compared to the already reported methods.
a b
c
Acknowledgments
d
Fig. 9. (a) Photoluminescence of the aqueous L-cysteine–ZnS QDs dispersion; (b) photoluminescence of saliva re-suspended in the aqueous L-cysteine–ZnS QDs dispersion; (c) photoluminescence of saliva fortified with L-thyroxine (4.0 10 7 mol L 1) re-suspended in the aqueous L-cysteine–ZnS QDs dispersion. (d) Fluorescence emission spectra from saliva re-suspended in water.
cysteine–CdTe QDs has been applied for the selective determination of cardiolipin in the presence of others phospholipids [18]. Moreover, cysteine–ZnS QDs and cysteine–CdS QDs have also been used as luminescent probes for determination of nucleic acids and mannitol [19,20]. The proposed method for the determination of thyroxine brings important advantages over the methods already reported in the literature because of the poor optical properties of the analyte. Most of the thyroxine determination methods rely on the indirect detection using metallic luminescent complexes that are very sensitive to other species thus prone to interferences [3,4]. On the other hand, chemical derivatization approaches uses highly toxic and expensive derivatization reagents. The heavy metal-free QDs dispersion (L-cysteine–ZnS QDs/CTAB system) is prepared with cheap and readily available reagents, providing a fairly selective interaction with thyroxine using a simple analytical approach. 4. Conclusion The photoluminescence intensity of a L-cysteine–ZnS QDs aqueous dispersion is quenched by L-thyroxine in the presence
The authors wish to thank Brazilian agencies FINEP and FAPERJ for financial support. Scholarships from TWAS-CNPq (Khan S.), FAPERJ (Aucelio R.Q.) and CNPQ (Aucelio R.Q.) are acknowledged. Authors thank Dr. M. Cremona and Dr. S. Louro (Dept of Physics PUC-Rio) for the luminescence lifetime measurements. References [1] M.J. Shomon, The Thyroid Hormone Breakthrough: Overcoming sexual and Hormonal Problems at Every Age, First ed., Harper Collins Publishers, New York, 2009. [2] J.D. Baxter, P Webb, Nat. Rev. Drug. Discov. 8 (2009) 308. [3] A. Waseem, M. Yaqoob, A. Nabi, J. Lumin. 21 (2006) 174. [4] A. Waseem, M. Yaqoob, A. Nabi, G.M. Greenway, Anal. Lett. 40 (2007) 1071. [5] Y. Sun, X.Y. Zhao, P. Li, G.Y. Shi, T.S. Zhou, J. Sep. Sci. 33 (2010) 2417. [6] Y. Sawabe, T. Tagami, K. Yamasaki, S. Taguchi, J. Health Sci. 57 (2011) 47. [7] L. Bhavana, V.J. Ajimon, S.L. Radhika, M. Sindhu, C.S.P. Iyer, J. Chromatogr. B 803 (2004) 363. [8] T. Higashi, T. Ichikawa, C. Shimizu, S. Nagai, S. Inagaki, J.Z. Min, H. Chiba, S. Ikegawa, T. Toyo'oka, J. Chromatogr., B 879 (2011) 1013. [9] E. Gök, C. Öztürk, N. Akbay Goek, C. Oeztuerk, N. Akbay, J. Fluoresc. 18 (2008) 781. [10] L.M. Devi, D.P.S. Negi, Nanotechnology 22 (2011) 1. [11] Y. Shang, L. Qi, F.Y. Wu, Microchim. Acta 177 (2012) 333. [12] J.M. Carvalho, K.C. Leandro, A.R. da Silva, R.Q. Aucelio, Anal. Lett. 46 (2013) 207. [13 M. Koneswaran, R. Narayanaswamy, Sens. Actuators B 139 (2009) 104. [14] A. Mandal, A. Dandapat, G. De, Analyst 137 (2012) 765. [15] B. Gilbert, F Huang, Z. Lin, C. Goodell, H. Zhang, J.F. Banfield, Nano Lett. 6 (2006) 605. [16] A.H. Gore, U.S. Mote, S.S. Tele, P.V. Anbhule, M.C. Rath, S.R. Patil, G.B. Kolekar, Analyst 136 (2011) 2606. [17] A. Chatterjee, A. Priyam, D. Ghosh, S. Mondal, S.C. Bhattacharya, A Saha, J. Lumin. 132 (2013) 545. [18] W. Zhao, Y Fung, W.O., M.P.L. Cheung, Anal. Sci. 25 (2010) 879. [19] Y Li, J. Chen, C. Zhu., L Wang, D Zhao, S. Zhuo, Y. Wu, Spectrochim. Acta Part A 60 (2004) 1719. [20] D. Ghosh, S Ghosh, A. Saha, Anal. Chim. Acta 675 (2010) 165.
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Journal of Luminescence journal homepage: www.elsevier.com/locate/jlumin
Quantification of thyroxine by the selective photoluminescence quenching of L-cysteine–ZnS quantum dots in aqueous solution containing hexadecyltrimethylammonium bromide Sarzamin Khan a, Leonardo S.A. Carneiro a, Eric C. Romani b, Dunieskys G. Larrudé b, Ricardo Q. Aucelio a,n a b
Chemistry Department, Pontifícia Universidade Católica do Rio de Janeiro, 22451-900 Rio de Janeiro-RJ, Brazil Physics Department, Pontifícia Universidade Católica do Rio de Janeiro, 22451-900, Rio de Janeiro-RJ, Brazil
art ic l e i nf o
a b s t r a c t
Article history: Received 6 December 2013 Received in revised form 1 July 2014 Accepted 3 July 2014 Available online 16 July 2014
The determination of L-thyroxine is proposed based on the photoluminescence quenching effect caused on the L-cysteine modified ZnS quantum dots (L-cysteine ZnS QDs) aqueous dispersion. Under optimum conditions, the analytical response followed a Stern–Volmer model and the experimental conditions were adjusted to enable a robust and reproducible photoluminescence signal. The linear response observed in the quantum dots aqueous dispersion covered the L-thyroxine concentration from the LOQ (2.0 10 8 mol L 1) to 4.0 10 6 mol L 1. The approach was tested in the determination of L-thyroxine in pharmaceutical formulations used to treat patients with thyroid gland disorder. The percent recoveries in controlled samples were between 93.3 and 103%. Analyte fortified saliva was also evaluated as a possible sample for L-thyroxine monitoring of a patient under treatment. It was identified a static type of photoluminescence quenching caused by L-thyroxine. & 2014 Elsevier B.V. All rights reserved.
Keywords: Quantum dots photoluminescence L-thyroxine L-cysteine modified ZnS quantum dots Saliva
1. Introduction The normal thyroid gland is a discrete soft body made up of a large number of vessels that produce, store, and release two key hormones: triiodothyronine, also called T3 and thyroxine or T4, where the numbers 3 and 4 refer to the number of iodine molecules attached to each hormone. Thyroid cells are the primary cells in the body capable of absorbing iodine, an essential nutrient obtained through food, iodized salt, or supplements. A healthy thyroid produces a proportion of about 20% of T3 and 80% of T4, where T3 is the biologically active hormone that is used by the cells. When needed, the body converts the inactive T4 into the active T3 by removing one atom of iodine from the molecule [1]. Thyroid hormones have a number of functions such as helping cells to convert oxygen into energy and the brain to function properly, it guarantees a normal bone growth and the processing of carbohydrates, enabling a proper sexual development and functioning [2]. A patient that presents low secretion of thyroid hormones or even the lack of it must be treated with the administration of thyroxine (Fig. 1A) in order to enable a regular
n
Correspondent author. Fax: þ 55 21 3527 1637. E-mail address: [email protected] (R.Q. Aucelio).
http://dx.doi.org/10.1016/j.jlumin.2014.07.003 0022-2313/& 2014 Elsevier B.V. All rights reserved.
maintenance of the triiodothyronine levels. Levothyroxine (L-thyroxine) is the artificial thyroxine hormone mostly used for the therapy of thyroid dysfunction and it is commercially available under several brand names. L-thyroxine is a non-fluorescent substance at room temperature. Its absorption profile is due to the two weakly conjugated benzene rings, thus it presents the characteristic and more intense B1 (at about 235 nm and close to the lower limit of the spectrum) and a weaker B2 (at about 330 nm) absorption bands of benzene (Fig. 1B). The presence of iodine in the molecular structure forces, through spin–orbital coupling (internal heavy atom effect), the transference of the molecular population from the excited singlet state to the triplet state by means of intersystem crossing. Thus, the long lifetime radiative return to the ground state is not favored when compared to the non-radiative processes that do not favor the natural photoluminescence of thyroxine. The indirect detection of thyroid hormones (T3 and T4) was carried out from either the inhibition of the luminol–iron (II) chemiluminescence or the enhancement of the elecrtochemiluminescence of Tris(2,2-bipyridyl)ruthenium(III)–NADH system. Limits of detection (LOD) of respectively 1.2 10 7 mol L 1 and 5 10 8 mol L 1 were achieved [3,4]. Capillary electrophoresis has also been utilized for the determination of thyroxine in pharmaceutical formulations using electrochemical detection on
S. Khan et al. / Journal of Luminescence 156 (2014) 16–24
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QDs) was studied in aqueous dispersions. The photoluminescence quenching effect caused by L-thyroxine was more effective in aqueous systems containing hexadecyltrimethylammonium bromide (CTAB), which also improved stability of the measured signal over time. The approach using the L-cysteine–ZnS probe was used to quantify L-thyroxine in a pharmaceutical formulation. Analyte fortified saliva was also evaluated as a possible matrix for Lthyroxine monitoring of a patient under treatment.
2. Material and methods 2.1. Apparatus
Fig. 1. Electronic absorption spectra of thyroxine; (a) 3.9 10 7, (b) 9.8 10 7, (c) 3.8 10 6, and (d) 6.0 10 5 mol L 1.
a carbon disk electrode [5]. The use of high performance liquid chromatography (HPLC) for the separation and detection of both T3 and T4 in dietary supplements enabled quantifications down to 0.002 μg mL 1 (2.9 10 9 mol L 1) after the pre-column derivatization of the analytes with 4-fluoro-7-nitrobenzofuran [6]. The indirect detection of thyroxine in urine has been achieved by the spectrophotomeric detection of iodide (at 226 nm) after separation in an anion exchange column [7]. More recently, isotopedilution liquid chromatography/tandem mass spectrometry method was applied for the determination of thyroxine in saliva (at the pg mL 1 level) using (13C6)-T4 as internal standard [8]. The indirect fluorescence detection of thyroxine has been achieved by measuring the quenching effect on 7-hydroxycoumarin and Eu(III)-(pyridine-2,6-dicarboxylate) Tris complex. LOD value of 3.4 10 8 mol L 1 has been achieved [9]. Semiconductor nanoparticles (quantum dots or QDs) are grown to be in the nanoscale size to achieve the effect of quantum confinement, which enables unique optical properties such as wide absorption profile, very intense size-dependent luminescence and high photostability. For instance, CdS QDs and to a less extent the ZnS QDs have been studied since they can be easily synthesized in aqueous medium under mild conditions. In addition many surface modifications in QDs (nanoparticle capping with organic ligands) have been proposed to enhance optical properties, stability in aqueous dispersions and selectivity in interaction with chemical species in solution. These modified nanoparticles can be homogeneously dispersed and stabilized in water, and therefore they are very attractive as analytical probes to selective sense chemical species in aqueous systems [10–12]. In this work, the effect of L-thyroxine on the photoluminescence of L-cysteine modified ZnS quantum dots (L-cysteine–ZnS
A Perkin-Elmer Lambda 19, UV/vis/NIR double beam spectrophotometer (1 cm quartz cuvettes) was used to obtain electronic absorption spectra from L-thyroxine and to evaluate the extinction spectra from the QDs dispersions. All photoluminescence measurements were made using a Perkin-Elmer model LS 55 luminescence spectrometer with solutions placed in 1 cm optical pathlength quartz cuvettes. Photoluminescence spectra were acquired using the FL-Winlabs software and measurements were performed with 10 nm excitation and emission spectral bandpass and 1500 nm/min scan rate. Excitation was made at 312 nm with signal measurement at 424 nm. A thermostatic system with stirring (PTP-1 Fluorescence Peltier System with a PCB1500 Water Peltier System, Perkin-Elmer) was used to keep the solutions in the cuvette at specific constant temperatures and to allow stirring of the CTAB organized L-cysteine–ZnS QDs working dispersion. Photoluminescence lifetime measurements were conducted using a Model HJY 5000M time-resolved fluorescence spectrometer IBH 5000F (Horiba Jobin Yvon, NJ, USA) with excitation using a nanoLED source at 372 nm. Lifetimes were obtained after mathematical deconvolution of the fluorescence decay from the source pulse profile. Transmission electron microscopy (TEM) was made on JEOL 2010 Transmission Electron Microscope under 200 kV accelerating voltage. Dynamic light scattering (DLS) measurements of the quantum dots were recorded in a Zetasizer Nano ZS (Malvern, UK) equipment, operating at 25 1C using a He–Ne laser (633 nm) with measurement range from 0.6 nm to 6 mm and the analyzed range from 2 to 500 nm. For pH measurements, the pH-meter (MS Tecnopon, model MPA-210, Brazil) was employed. 2.2. Reagents, samples and other materials L-cysteine hydrochloride monohydrate, L-thyroxine, zinc acetate dehydrated, sodium phosphate monobasic, sodium phosphate dibasic heptahydrate, hexadecyltrimethylammonium bromide (CTAB) were purchased from Sigma-Aldrich (USA). Sodium dodecyl sulfate (SDS) and triton X-100 were from Merck, Germany. CTAB and SDS were further recrystallized in ethanol in order to eliminate impurities. Analytical grade methanol was from Merck. The pharmaceutical formulation (containing of 200 mg thyroxine per tablet) was obtained from a local drugstore. Utrapure water was purified in a Milli-Q system from Millipore (Simplicity model 185, USA) and used to prepare all aqueous solutions. Solid phase extraction (SPE) was made on a 6 mL volume cartridge containing 1 g of C18 sorbent (Cole Parmer, USA).
2.3. Synthesis of L-thyroxine cysteine–ZnS quantum dots The ZnS nanoparticles modified with L-cysteine were synthesized following similar procedures described in the literature [13]. In typical synthesis, an amount of 0.0214 g (1 10 4 mol) of zinc acetate dihydrated and 0.0172 g (1 10 4 mol) of L-cysteine
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hydrochloride monohydrated were dissolved in 100 mL of deionized water. Then, the solution was stirred for 15 min and its pH value adjusted round to 9.0 by the addition of small volumes of a 0.1 mol L 1 NaOH solution. The resultant solution was transferred to a 150 mL three necked round bottom flask (reactor) where a solution containing 2 10 4 mol of Na2S was slowly injected (in the form of aqueous solution) through a syringe into the reactor, under nitrogen protection. Then, the mixture was refluxed (about 100 1C, under inert atmosphere) for specific period of time. Aliquots of the reaction mixture were taken out at different intervals of time, through a syringe in order to check their absorption and photoluminescence characteristics. The transparent and colorless colloidal dispersion of ZnS QDs was treated with ethanol to precipitate the nanoparticles, then, the solid mass was re-dispersed in a phosphate buffer solution (0.01 mol L 1 and pH 8.0). These nanoparticles were kept under refrigeration being stable for more than 10 months, indicated by transparency of the dispersion and by its high photoluminescence intensity. 2.4. Microscopy and spectroscopy of the L-cysteine–ZnS QDs The morphology and structure of functionalized ZnS nanoparticles were studied by scanning transmission electron microscopy (Fig. 2), which indicates a large number of assemblies of L-cysteine–ZnS nanoparticles with a fairly uniform size and shape. As can be observed, nanoparticles are well dispersed and the size range varied from 5.1 to 6.6 nm. The particle size distribution of L-cysteine–ZnS was also measured by DLS (Fig. 2A). The hydrodynamic diameter was in the range from 12.7 to 17.2 nm with an average diameter of 14.0 nm. The DLS measurements indicated that L-cysteine–ZnS nanoparticles are prone to certain degree of aggregation, occurrence that is probably due to the use of
non-buffered dispersion during DLS analysis that may have promoted the removing of part of the L-cysteine stabilizer from the surface of the QDs (Fig. 2A). ZnS nanoparticles contain few units in their nanocrystal cells, therefore they possess a stable structure and optical properties that do not vary significantly as the reaction time is increased [14]. In addition, contrary to what is observed for other semiconductor luminescent nanoparticles, the excitation profile of the L-cysteine– ZnS QDs is narrower [15]. The electronic optical absorption of the synthesized L-cysteine–ZnS QDs presented a broad profile with the first excitonic at 290 nm (Fig. 3A), photoluminescence maximum observed at 424 nm (related to the 312 nm of the sharp excitation band) and the full width at half maximum value for the photoluminescence emission band of 36 nm (Fig. 3B). The photoluminescence intensity of nanoparticles centered at 424 nm sharply increased with the increasing reflux time, reaching a maximum value at 120 min. The photoluminescence intensity then decreased as the reflux time surpassed 120 min as indicated in Fig. 2C. At longer refluxing times, the cloudy appearance of the dispersion indicated the aggregation of the crystals leading to the formation of bigger particles and consequently less intense photoluminescence. 2.5. Photoluminescence measurements from the L-cysteine–ZnS QDs aqueous dispersions For the determination of L-thyroxine in samples, the L-cysteine– ZnS QDs dispersion, prepared in phosphate buffer (0.01 mol L 1; pH, 8.0) and containing CTAB at concentration of 5 10 5 mol L 1, was transferred to a quartz cuvette. Volumes of either L-thyroxine standards or samples containing L-thyroxine were added to the QDs dispersion at room temperature and under constant stirring. Measurements were made 5 min after the stirring was turned off
Fig. 2. STEM image of L-cysteine–ZnS QDs and DLS histogram of (A) L-cysteine–ZnS QDs aqueous dispersion and (B) L-cysteine–ZnS QDs aqueous dispersion in the presence of thyroxine.
S. Khan et al. / Journal of Luminescence 156 (2014) 16–24
19
diluted sodium hydroxide solution (5 10 4 mol L 1). Standard solutions of lower concentration were prepared by further dilution of this stock solution with ultrapure water. The surfactant stock solution (5.0 10 3 mol L 1) was prepared by dissolving appropriate amounts of CTAB in ultrapure water. For the preparation of an L-thyroxine pharmaceutical formulation sample, a pool of ten commercial tablets was pulverized. Mass aliquots of this powder were dissolved in ethanol. The sample was passed through a Teflon syringe filter to retain the non-soluble excipients, then, the solution was passed through a C18 solid phase extraction (SPE) column to retain the L-thyroxine. The cartridge was washed with deionized water (to remove remaining water soluble tablets recipients) and the L-thyroxine was eluted with 1 mL of methanol. The methanol was evaporated and the remaining residue was re-dissolved in the L-cysteine–ZnS QDs buffered dispersion. The saliva sample (10 mL collected in a graduated cylinder) was collected from an euthyroid volunteer that did not receive any other medical treatment nor ingested food or beverages prior the sample collection. The volunteer rinsed his mouth for 5 min with ultrapure water. The saliva was fortified with known amount of thyroxine and mixed with 5 mL of ethanol. Then, the mixture was vortex mixed for 30 s and immediately centrifuged for 15 min at 3000 rpm. After centrifugation, the supernatant was passed through C18 SPE column and washed with ultrapure water. After elution with 1 mL methanol, the eluate was evaporated and the residue re-suspended in the L-cysteine–ZnS QDs buffered dispersion. The same procedure was repeated for blank measurements.
3. Results and discussion 3.1. Optimization of conditions to achieve the photoluminescence quenching of the L-cysteine–ZnS QDs by L-thyroxine L-cysteine is an amino acid that has been used for synthesis of water compatible fluorescent probes [13]. Such ligand can bind to the surface of the ZnS nanoparticles through the sulfur atom of the mercapto group, while the carboxylic acid group provides water compatibility.
Fig. 3. (A) Electronic absorption spectrum. (B) Photoluminescence excitation and emission spectra. (C) Photoluminescence spectra of the cysteine–ZnS QDs synthesized using different refluxing times: (a) 10, (b) 20, (c) 40, (d) 60, (e) 80, (f)100, (g) 130 and (h) 120 min.
(equilibration time). Photoluminescence measurements were made at 424 nm upon excitation at 312 nm. In order to obtain reliable quantitative results, measurements were also taken from a L-cysteine–ZnS QDs dispersion without the addition of L-thyroxine (control). 2.6. Preparation of solutions and samples The stock solution of analyte (1.0 10 2 mol L 1) was prepared by dissolving specific amounts of the drug standard in a
3.1.1. Amount of cysteine–ZnS quantum dots in the dispersion The quenching effect promoted by L-thyroxine (fixed at 9.5 10 7 mol L 1 final concentration) on the L-cysteine–ZnS QDs photoluminescence was investigated in dispersions containing different amounts of nanoparticles. The amount of nanoparticles was varied by introducing different volumes of the synthesized L-cysteine-ZnS QDs stock dispersion (from 0.250 to 2 mL) into the 10 mL final volume of aqueous dispersion, which enabled a range between 1.6 10 4 to 1.3 10 3 mol L 1 (2.4–20.0 μg mL) of zinc concentration in the QDs precursor solution (Fig. 4A). Higher amounts of L-cysteine–ZnS QDs decreased the sensitivity of the photoluminescence quenching response due to inner filter effect caused by the high concentration of QDs in the dispersion. In contrast, the lower concentrations of QDs resulted in robust and sensitive fluorescence quenching response as indicated by the higher L0/L ratio values (where L0 is the photoluminescence measured from the dispersion in absence of L-thyroxine and L is the photoluminescence measured from the dispersion in the presence of L-thyroxine). Dispersions (10 mL total volume) containing 0.8 mL of the quantum dots stock dispersion was chosen due to the high sensitivity in the quenching response.
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3.1.2. Influence of pH on the L-cysteine–ZnS QDs photoluminescence quenching mediated by L-thyroxine The effect of pH of the aqueous dispersion on the interaction of L-thyroxine and quantum dots was studied in order to find the appropriate pH for the sensitive probing of L-thyroxine. The pH was varied in the range from 6.5 to 9.0 (using phosphate buffer or borate buffer at 0.01 mol L 1 final concentration to cover such pH range). The experiments were performed in dispersion with the different pH values either in the presence or in the absence
Fig. 4. (A) Effect of the amount of the synthesized nanoparticles on the photoluminescence quenching of the L-cysteine–ZnS QDs aqueous dispersion. Signal variation expressed as L0/L (where L0 and L are respectively the photoluminescence e of the quantum dots dispersion before and after the addition of 9.5 10 7 mol L 1 of thyroxine). (B) Influence of pH value of the aqueous dispersion on the photoluminescence quenching of L-cysteine–ZnS QDs (3.8 10 7 mol L 1 final concentration of L-thyroxine). Results obtained in triplicate.
7 L-thyroxine (3.8 10 mol L 1). The signal profile (Fig. 4B) indicated a more effective photoluminescence quenching at the pH values of 8.0 and 8.5. The pH value selected for the QDs working aqueous dispersion was 8.0 (achieved by the phosphate buffer).
3.1.3. Effect of surfactants on the photoluminescence quenching caused by L-thyroxine The effect of different surfactants such the cathionic acetyltrimethyl ammonium bromide (CTAB), the anionic sodium dodecyl sulfate (SDS) and the non-ionic Triton X-100 on the L-thyroxine mediated photoluminescence quenching was investigated. Concentration of surfactants was varied from 8.0 10 6 to 1.0 10 3 mol L 1. The presence of SDS caused total suppression of photoluminescence of the system probably due to the electrostatic repulsion between the negative charge on the surface of nanoparticles and the anionic head of SDS, thus at high concentration of SDS, quenching of the photoluminescence from quantum dots was observed. When using Triton X-100, stable photoluminescence measurements were not achieved probably due to adsorption of surfactant onto the surface of nanoparticles, changing the surface properties and probably promoting the removal of L-cysteine. The improvement in the photoluminescence quenching efficiency was obtained in the presence of CTAB at concentration of 5 10 5 mol L 1, which is below the critical micelle concentration (CMC) of this surfactant (about 1 mmol L 1) as indicated in Fig. 5A. As the surface of ZnS QDs capped with cysteine is negatively charged due to the desprotonation of the carboxylic group at pH 8.0, the CTAB (a cationic surfactant) is prone to interact to nanoparticles surface, via electrostatic interaction. In the presence of this cationic surfactant, the photoluminescence from the QDs dispersion was slightly enhanced and became stable, which may due to partial incorporation of nanoparticles into the protective environment of such surfactant micelles. The presence of nanoparticles in micelle prevents the adsorption of other molecules on the surface of the nanoparticles, thus, the excited electron from the conducting band may combine with the vacancy at the valence band with less probability of electron transfer to other molecules nearby. Better analyte induced photoluminescence quenching was observed below the critical micelle concentration (CMC) is probably due to the slight positive surface changes, which facilitates interactions between nanoparticles and the L-thyroxine molecule. At higher concentration of CTAB, the surfactant may block the access to L-thyroxine to the nanoparticles and the resulting in a less effective photoluminescence quenching. Thus, a final concentration
Fig. 5. (A) Effect of CTAB on the photoluminescence quenching of L-cysteine-ZnS QDs at fixed concentration of L-thyroxine (final concentration in aqueous dispersion, 1.2 10 6 mol L 1). (B) Photoluminescence measured from L-cysteine–ZnS QDs dispersion at different temperatures. Results obtained in triplicate.
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L0/L
Photoluminescence
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Time (min)
Time (min)
Fig. 6. (A) Photoluminescence stability of the L-cystein–ZnS QDs dispersion. (B) Photoluminescence stability of the L-cystein-ZnS QDs dispersion after addition of L-thyroxine (final concentration in solution 4.0 10 7 mol L 1).
of 5 10 5 mol L 1 of CTAB was incorporated into the QDs water working dispersion. 3.1.4. Effect of temperature The effect of temperature on the photoluminescence intensity measured from the L-cysteine–ZnS QDs dispersion was studied at temperatures ranging from 18 to 45 1C. The fluorescence measured from quantum dot was found to be inversely dependent on temperature with a fairly constant signal intensity found between 18 and 25 1C (Fig. 5B). The decrease in signal intensity measured from the QDs dispersion is probably due to the increase of the kinetic energy of the components of the system, which disrupts the interaction between the analyte and the L-cysteine ZnS QDs, leading to a less effective photoluminescence quenching. Therefore, room temperature (about 25 1C) was selected in all fluorescence measurement for sensing of L-thyroxine. 3.1.5. Stability of photoluminescence intensity and reaction time Under the selected conditions to enable effective interaction between L-thyroxine and the L-cysteine ZnS QDs in the dispersion, an evaluation of the reproducibility and stability of the measured photoluminescence was made. First, the photoluminescence from the control working dispersion of QDs (L-cysteine–ZnS QDs buffered dispersion containing CTAB before the addition of L-thyroxine) was measured in function of the time (measured every 10 min up to 120 min). The signal was found to be stable during the whole time of the experiment (less than 3% random variation of signal) as indicated in Fig. 6A. The photoluminescence from this same QDs dispersion was also monitored in function of the time after the addition of a fixed amount of L-thyroxine. Measurements were made every 2 min up to 30 min starting 2 min after the addition of L-thyroxine (1 min of mixing and 1 min of equilibration of the solution). The photoluminescence quenching was immediate and after 5 min, the signal becomes stable up for more than 30 min (Fig. 6B). For the analytical method, it was established all measurements to be made after 5 min of the addition of L-thyroxine into the quantum dots dispersion. 3.2. Mechanism of interaction Several mechanisms have been proposed to explain QDs photoluminescence signal reduction, including inner filter effect and non-radiative processes caused by electron transfer, surface
adsorption, complexation among others [16]. As previously mentioned, L-thyroxine has a wide UV–vis absorption profile with maximum at 235 nm and 330 nm. Taking into consideration a 6 10 5 mol L 1 solution of L-thyroxine, the absorbance (Fig. 1B line d) is 0.87 at 235 nm and 0.09 at 330 nm and 0.008 at 312 nm (the wavelength chosen for the excitation of L-cysteine–ZnS QDs). Thus, no inner filter effect caused by L-thyroxine is expected at the excitation wavelength of 312 nm considering concentration levels 5 L-thyroxine below 1.0 10 mol L 1. Since no changes in the absorption spectral profile of the Lcysteine–ZnS QDs (characteristic profile in Fig. 3B) takes places in the presence of L-thyroxine, it is concluded that no aggregation (chemical degradation that can lead to the reducing of photoluminescence) of QDs are taking place. If aggregation has occurred, a measurable decrease in transmittance would be observed because of the significant increasing in light scattering measured from the dispersions. This is an indication that photoluminescence quenching should be promoted by an effective interaction between the analyte and the QDs and not by light filter effect due to the increase of light extinction. Aggregation of nanoparticles induced by L-thyroxine was also ruled out since no changes in particle distribution were observed by microscopy. In addition, DLS measurement after the addition of L-thyroxine into the QDs dispersion (Fig. 2B) showed size distribution profile similar to those observed in the system without L-thyroxine (Fig. 2A). In order to determine if the mechanism of photoluminescence quenching is whether static or dynamic, a study of the dependence of photoluminescence analytical curve sensitivity upon the temperature was made [17]. From the Stern–Volmer plots constructed at two different temperatures (298 and 308 K) it was observed that the sensitivity decreases as the temperature increased (Fig. 7A). These results indicate the L-thyroxine and cysteine–ZnS interactions associative in nature and characteristic of a static type of quenching. In addition, photoluminescence lifetime experiments have indicated the similar profile for measurements made from QDs dispersions in the presence and also in the absence of L-thyroxine (Fig. 7B). It is valid to point out that the photoluminescence of semiconductor nanoparticles is known to decay in a multi-exponential manner, thus, QDs emission consists of more than one lifetime component [10]. Thus, the photoluminescence lifetimes that characterized the dispersion of cysteine– ZnS QDs in CTAB, no matter the presence of L-thyroxine, were: 4 71; 17 72 and 85 74 ns. As no change in lifetime profile of the QDs dispersion is observed in the presence of L-thyroxine, results
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S. Khan et al. / Journal of Luminescence 156 (2014) 16–24
L0/L
Table 1 Optimized experimental conditions for the thyroxine determination using the L-cysteine–ZnS QDs. Experimental parameter
Optimized Value
Type of quantum dots Phosphate buffer solution pH Time required to perform measurement Concentration estimated for QDsa Temperature CTAB concentration
Cysteine–ZnS nanoparticles 0.01 mol L 1 8.0 5 min 4.5 10 4 mol L 1 25 1C 1.2 10 6 mol L 1
a
Estimated by the concentration of Zn2 þ in the QDs precursor solution.
Intensity(counts)
Photoluminescence
Concentration of L-thyroxine(10-7 mol L-1)
Wavelenght (nm)
Time (ns)
indicate that a static quenching is taking place when analyte interacts with QDs. A possible mechanism can be proposed based on the experimental results. In absence of CTAB, no quenching effect was observed since non-protonated L-thyroxine is repelled by the negatively charged cysteine that covers the surface of the ZnS QDs. When CTAB is added, this cationic surfactant interact with QDs neutralizing the negative charges of the capping agent, thus enabling L-thyroxine to form a complex with Zn atoms at the surface of QDs. Thyroxine withdraw electrons from the QDs conducting band resulting in photoluminescence static quenching. As L-thyroxine present four iodine groups any molecular fluorescence that might be stimulated by such electron transfer is also quenched by internal heavy atom effect that transfers the electronic population to the triplet state. Since environment is not free from dissolved oxygen, triplet state radiative decay is not favored. 3.3. Analytical characteristics of the photoluminescence quenching mediated by L-thyroxine 3.3.1. Detection and quantification limits and precision of measurements Under the optimized experimental conditions (Table 1) a Stern–Volmer model (Eq. 1) could be readily used to establish a linear relationship between measured photoluminescence, L, and the concentration of L-thyroxine, [L-thyroxine], in the dispersion.
L0/L
Fig. 7. (A) The Stern–Volmer curves for the quenching of cysteine–ZnS QDs aqueous dispersion in the presence of L-thyroxine at 298 K (■) and 308 K (▲). (B) Photoluminescence lifetime profile of cysteine-ZnS QDs aqueous dispersions in the absence (▲) and in the presence (★ of thyroxine (3.6 10 6 mol L 1).
Concentration of L-thyroxine(10-7 mol L-1) Fig. 8. (A) Photoluminescence emission spectra of L-cysteine–ZnS QDs aqueous dispersion in the presence of different concentrations of L-thyroxine: (a) 0, (b) 1.1 10 7, (c) 2.0 10 7,(d) 3.0 10 7, (e) 3.9 10 7, (f) 4.9 10 7, (g) 9.8 10 7, (h) 2.0 10 6, (i) 2.9 10 6, (j) 3.8 10 6, and (k) 4.0 10 6 mol L 1. (B) Stern–Volmer-type calibration curve for the determination of thyroxine using L-cysteine–ZnS QDs as probe.
L0 is the photoluminescence made from the probe dispersion before the addition of L-thyroxine and Ksv is the Stern–Volmer constant. L0 =L ¼ 1 þ K sv ½L thyroxine
ð1Þ
The results show that L-thyroxine quenches the photoluminescence of the L-cysteine–ZnS QDs in a concentration dependent pattern from 1.1 10 7 to 4.0 10 6 mol L 1 (Fig. 8A). Thus, analytical curves were constructed by adding increasing concentrations of L-thyroxine on the QDs dispersion and then, measuring the photoluminescence decreasing. A typical analytical curve (L0/L versus
S. Khan et al. / Journal of Luminescence 156 (2014) 16–24
concentration of L-thyroxine) is shown in Fig. 8B, showing a linear range of the analytical response in the concentration range from 1.1 10 7 to 4.0 10 6 mol L 1 of L-thyroxine (final concentration in the dispersion) with correlation coefficient of 0.9926. The equation model of the analytical curve was L0/L¼3.3 105 [L-thyroxine]þ 1.01. Since at these concentration levels L-thyroxine does not significantly absorb light at 312 nm, no correction for inner-filter effect was necessary for the Stern–Volmer model. The limit of detection (LOD) was calculated as the concentration of L-thyroxine that changes the photoluminescence signal in the measurement cell by L0 3sL0, where sL0 is the standard deviation of ten replicate measurements of the photoluminescence intensity of the L-cysteine–ZnS dispersion before the addition of L-thyroxine. Similarly, the limit of quantification (LOQ) was calculated as the L0 10sL0. The LOD and the LOQ were 6.2 10 8 mol L 1 (48.3 ng mL 1) and 2.0 10 8 mol L 1 (15.4 ng mL 1), respectively. The precision of the L-thyroxine measurement using the proposed probe was calculated as the variation of the L0/L value taking into consideration ten independent solutions (in two different L-thyroxine concentrations). In order to do this, the following equation was used: s(L0/L) ¼L0/L [(sL/L)2 þsL0/L0)2]1/2. The s(L0/L), in percentage values, was 2.8% and 4.2% at, respectively the 3.9 10 7 mol L 1 and 2.9 10 6 mol L 1 concentration levels. 3.3.2. Selectivity studies For practical applications of the proposed method for the determination of L-thyroxine in biological samples (saliva) and in pharmaceutical formulations, the effect of some possible relevant interfering substances was evaluated. The chosen substances were the ones commonly found in pharmaceutical formulations and in biological fluids (including several amino acids). Changes in fluorescence intensity due to the presence of these chemical species were expressed in percent values (Table 1). All the tested substances imposed variations in the photoluminescence intensity measured from the probe (at the specified concentrations) under 4% (variation considered not relevant). In contrast, the presence of a much less amount of L-thyroxine (0.4 μmol L 1) caused about 10% decreasing of the photoluminescence measured from the probe. The interference caused by two different complex matrices was also evaluated. One matrix was composed by a mixture of amino acids (cysteine, histidine, phenylalanine, valine, methionine, lysine and threonine), each amino acid with a final concentration of 5 10 6 mol L 1. The other matrix consisted on a mixture of common pharmaceutical excipients (lactose, silicon dioxide, citric acid, calcium chloride, Table 2 Effect of some potential interfering substances on the photoluminescence measured from the L-cysteine–ZnS QDs organized aqueous dispersion. Potential interferent
Concentration (μ mol L 1)
Change of photoluminescence (%)
Cysteine Histidine Tyrosine Phenylalanine Valine Methionine Lysine Threonine Lactose Silicon dioxide Citric acid Calcium Magnesium Potassium Sodium Chloride
150 150 150 150 150 150 150 150 100 100 100 100 100 100 100 100
þ 4.0 þ 3.1 þ 1.4 þ 0.55 þ 0.55 0.55 þ 1.13 1.42 2.6 þ 1.8 þ 2.0 þ 4.1 þ 3.8 þ 2.1 þ 3.1 þ 3.0
23
magnesium sulfate, potassium chloride, sodium chloride) each one at a final concentration of 0.1 10 6 mol L 1. It was observed that the photoluminescence measured from L-cysteine–ZnS QDs dispersion in absence and in the presence of L-thyroxine is not significantly different when either the amino acids mixture or the pharmaceutical excipient mixture is present. For biological samples, proteins can affect the photoluminescence of L-cysteine–ZnS QDs but due to pretreatment of samples (protein precipitation and SPE) such interference is minimized. Such samples also contain salts, but the study to evaluate the influence of NaCl on the photoluminescence quenching of the probe indicated that no interference is expected in samples containing up to 2 10 3 mol L 1 of NaCl. 3.4. Analytical application of the L-cysteine–ZnS QDs dispersion for the determination of L-thyroxine The proposed photoluminescence L-cysteine–ZnS probe has been applied for the determination of L-thyroxine in pharmaceutical formulation (containing the artificial thyroxine hormone as active component) and in analyte fortified human saliva, simulating a sample from a patient with a non-active thyroid gland that was medicated with L-thyroxine. Three different portions (0.57 g) from ten grinded pharmaceutical tablets were selected. The L-thyroxine content in these portions were dissolved in methanol, filtered in a 0.45 mm syringe filter and then diluted in water. A volume (1 mL) of the sample solution was placed into the L-cysteine–ZnS QDs aqueous dispersion in order to achieve a theoretical final concentration of 9.8 10 7 mol L 1 of L-thyroxine in the QDs dispersion. The recoveries (Table 2) achieved using the proposed method at was 97.07 5.2% taking into consideration the value of L-thyroxine indicated in the pharmaceutical formulation instructions (Table 3). In order to evaluate the applicability of the method in simple clinical assays, the analysis of saliva samples (fortified with Lthyroxine at concentration of 2.9 10 7 mol L 1) were performed. The saliva was mixed with 5 mL of ethanol, fortified with the known amount of L-thyroxine then immediately centrifuged for 15 min at 3000 rpm. After centrifugation, the supernant was passed through C18 SPE column and washed with deionzed water. After elution with 1 mL methanol, the eluate was evaporated and the residue re-suspended with the nanoparticle dispersion (made in phosphate buffer pH 8.5). From the photoluminescence quenching magnitude, the concentration of thyroxine was obtained with recovery of 86.07 5.1%. The relatively low recoveries might be due to loss of L-thyroxine in pretreatment of saliva Table 3. Photoluminescence spectra (Fig. 9) indicates that original components from saliva matrix does not interfere with the determination of thyroxine as it does not fluoresce under the condition of the experiment and it does not affect the photoluminescence signal from the L-cysteine–ZnS dispersion when it is not fortified with thyroxine. The effectiveness of cysteine functionalized nanoparticles as probe for substances of clinical and biological interest has been demonstrated in earlier works, for instance, aqueous dispersion of Table 3 Applications of the cysteine–ZnS QDs for determination of L-thyroxine in pharmaceutical formulation and saliva. Sample
L-thyroxine level
200 μg per tablet 7 L-thyroxine fortified 2.9 10 saliva mol L 1 Levotiroxina sódica
Portion 1
Portion 2
Portion 3
Average result (%)
93.3
94.8
103
97.0 7 5.2
88.9
90.1
86.5
86.0 7 5.1
24
S. Khan et al. / Journal of Luminescence 156 (2014) 16–24
of the surfactant CTAB. The overall quenching followed a Stern– Volmer model. The proposed optical probe has been applied for the determination of L-thyroxine in pharmaceutical formulation and in analyte fortified human saliva. The poor optical properties of L-thyroxine make the proposed indirect determination approach attractive when compared to the already reported methods.
a b
c
Acknowledgments
d
Fig. 9. (a) Photoluminescence of the aqueous L-cysteine–ZnS QDs dispersion; (b) photoluminescence of saliva re-suspended in the aqueous L-cysteine–ZnS QDs dispersion; (c) photoluminescence of saliva fortified with L-thyroxine (4.0 10 7 mol L 1) re-suspended in the aqueous L-cysteine–ZnS QDs dispersion. (d) Fluorescence emission spectra from saliva re-suspended in water.
cysteine–CdTe QDs has been applied for the selective determination of cardiolipin in the presence of others phospholipids [18]. Moreover, cysteine–ZnS QDs and cysteine–CdS QDs have also been used as luminescent probes for determination of nucleic acids and mannitol [19,20]. The proposed method for the determination of thyroxine brings important advantages over the methods already reported in the literature because of the poor optical properties of the analyte. Most of the thyroxine determination methods rely on the indirect detection using metallic luminescent complexes that are very sensitive to other species thus prone to interferences [3,4]. On the other hand, chemical derivatization approaches uses highly toxic and expensive derivatization reagents. The heavy metal-free QDs dispersion (L-cysteine–ZnS QDs/CTAB system) is prepared with cheap and readily available reagents, providing a fairly selective interaction with thyroxine using a simple analytical approach. 4. Conclusion The photoluminescence intensity of a L-cysteine–ZnS QDs aqueous dispersion is quenched by L-thyroxine in the presence
The authors wish to thank Brazilian agencies FINEP and FAPERJ for financial support. Scholarships from TWAS-CNPq (Khan S.), FAPERJ (Aucelio R.Q.) and CNPQ (Aucelio R.Q.) are acknowledged. Authors thank Dr. M. Cremona and Dr. S. Louro (Dept of Physics PUC-Rio) for the luminescence lifetime measurements. References [1] M.J. Shomon, The Thyroid Hormone Breakthrough: Overcoming sexual and Hormonal Problems at Every Age, First ed., Harper Collins Publishers, New York, 2009. [2] J.D. Baxter, P Webb, Nat. Rev. Drug. Discov. 8 (2009) 308. [3] A. Waseem, M. Yaqoob, A. Nabi, J. Lumin. 21 (2006) 174. [4] A. Waseem, M. Yaqoob, A. Nabi, G.M. Greenway, Anal. Lett. 40 (2007) 1071. [5] Y. Sun, X.Y. Zhao, P. Li, G.Y. Shi, T.S. Zhou, J. Sep. Sci. 33 (2010) 2417. [6] Y. Sawabe, T. Tagami, K. Yamasaki, S. Taguchi, J. Health Sci. 57 (2011) 47. [7] L. Bhavana, V.J. Ajimon, S.L. Radhika, M. Sindhu, C.S.P. Iyer, J. Chromatogr. B 803 (2004) 363. [8] T. Higashi, T. Ichikawa, C. Shimizu, S. Nagai, S. Inagaki, J.Z. Min, H. Chiba, S. Ikegawa, T. Toyo'oka, J. Chromatogr., B 879 (2011) 1013. [9] E. Gök, C. Öztürk, N. Akbay Goek, C. Oeztuerk, N. Akbay, J. Fluoresc. 18 (2008) 781. [10] L.M. Devi, D.P.S. Negi, Nanotechnology 22 (2011) 1. [11] Y. Shang, L. Qi, F.Y. Wu, Microchim. Acta 177 (2012) 333. [12] J.M. Carvalho, K.C. Leandro, A.R. da Silva, R.Q. Aucelio, Anal. Lett. 46 (2013) 207. [13 M. Koneswaran, R. Narayanaswamy, Sens. Actuators B 139 (2009) 104. [14] A. Mandal, A. Dandapat, G. De, Analyst 137 (2012) 765. [15] B. Gilbert, F Huang, Z. Lin, C. Goodell, H. Zhang, J.F. Banfield, Nano Lett. 6 (2006) 605. [16] A.H. Gore, U.S. Mote, S.S. Tele, P.V. Anbhule, M.C. Rath, S.R. Patil, G.B. Kolekar, Analyst 136 (2011) 2606. [17] A. Chatterjee, A. Priyam, D. Ghosh, S. Mondal, S.C. Bhattacharya, A Saha, J. Lumin. 132 (2013) 545. [18] W. Zhao, Y Fung, W.O., M.P.L. Cheung, Anal. Sci. 25 (2010) 879. [19] Y Li, J. Chen, C. Zhu., L Wang, D Zhao, S. Zhuo, Y. Wu, Spectrochim. Acta Part A 60 (2004) 1719. [20] D. Ghosh, S Ghosh, A. Saha, Anal. Chim. Acta 675 (2010) 165.