Quantification of thyroxine by the selective photoluminescencequenching of L-cysteine–ZnS quantum dots in aqueous solutioncontaining hexadecyltrimethylammonium bromide

Sarzamin Khan a, Leonardo S.A. Carneiro a, Eric C. Romani b,Dunieskys G. Larrudé b, Ricardo Q. Aucelio a,n

a Chemistry Department, Pontifícia Universidade Católica do Rio de Janeiro, 22451-900 Rio de Janeiro-RJ, Brazilb Physics Department, Pontifícia Universidade Católica do Rio de Janeiro, 22451-900, Rio de Janeiro-RJ, Brazil

a r t i c l e i n f o

Article history:Received 6 December 2013Received in revised form1 July 2014Accepted 3 July 2014Available online 16 July 2014

Keywords:Quantum dots photoluminescenceL-thyroxineL-cysteine modified ZnS quantum dotsSaliva

a b s t r a c t

The determination of L-thyroxine is proposed based on the photoluminescence quenching effect causedon the L-cysteine modified ZnS quantum dots (L-cysteine ZnS QDs) aqueous dispersion. Under optimumconditions, the analytical response followed a Stern–Volmer model and the experimental conditionswere adjusted to enable a robust and reproducible photoluminescence signal. The linear responseobserved 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-thyroxinein pharmaceutical formulations used to treat patients with thyroid gland disorder. The percentrecoveries in controlled samples were between 93.3 and 103%. Analyte fortified saliva was also evaluatedas a possible sample for L-thyroxine monitoring of a patient under treatment. It was identified a statictype of photoluminescence quenching caused by L-thyroxine.

& 2014 Elsevier B.V. All rights reserved.

1. Introduction

The normal thyroid gland is a discrete soft body made up of alarge number of vessels that produce, store, and release two keyhormones: triiodothyronine, also called T3 and thyroxine or T4,where the numbers 3 and 4 refer to the number of iodinemolecules attached to each hormone. Thyroid cells are the primarycells in the body capable of absorbing iodine, an essential nutrientobtained through food, iodized salt, or supplements. A healthythyroid produces a proportion of about 20% of T3 and 80% of T4,where T3 is the biologically active hormone that is used by thecells. When needed, the body converts the inactive T4 into theactive T3 by removing one atom of iodine from the molecule [1].

Thyroid hormones have a number of functions such as helpingcells to convert oxygen into energy and the brain to functionproperly, it guarantees a normal bone growth and the processingof carbohydrates, enabling a proper sexual development andfunctioning [2]. A patient that presents low secretion of thyroidhormones or even the lack of it must be treated with theadministration of thyroxine (Fig. 1A) in order to enable a regular

maintenance of the triiodothyronine levels. Levothyroxine(L-thyroxine) is the artificial thyroxine hormone mostly used forthe therapy of thyroid dysfunction and it is commercially availableunder several brand names.

L-thyroxine is a non-fluorescent substance at room tempera-ture. Its absorption profile is due to the two weakly conjugatedbenzene rings, thus it presents the characteristic and more intenseB1 (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), thetransference of the molecular population from the excited singletstate to the triplet state by means of intersystem crossing. Thus,the long lifetime radiative return to the ground state is not favoredwhen compared to the non-radiative processes that do not favorthe natural photoluminescence of thyroxine.

The indirect detection of thyroid hormones (T3 and T4) wascarried out from either the inhibition of the luminol–iron (II)chemiluminescence or the enhancement of the elecrtochemilumi-nescence of Tris(2,2-bipyridyl)ruthenium(III)–NADH system. Lim-its of detection (LOD) of respectively 1.2�10�7 mol L�1 and5�10�8 mol L�1 were achieved [3,4]. Capillary electrophoresishas also been utilized for the determination of thyroxine inpharmaceutical formulations using electrochemical detection on

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Journal of Luminescence

http://dx.doi.org/10.1016/j.jlumin.2014.07.0030022-2313/& 2014 Elsevier B.V. All rights reserved.

n Correspondent author. Fax: þ55 21 3527 1637.E-mail address: aucelior@puc-rio.br (R.Q. Aucelio).

Journal of Luminescence 156 (2014) 16–24

a carbon disk electrode [5]. The use of high performance liquidchromatography (HPLC) for the separation and detection of bothT3 and T4 in dietary supplements enabled quantifications down to0.002 μg mL�1 (2.9�10�9 mol L�1) after the pre-column deriva-tization of the analytes with 4-fluoro-7-nitrobenzofuran [6]. Theindirect detection of thyroxine in urine has been achieved by thespectrophotomeric detection of iodide (at 226 nm) after separa-tion in an anion exchange column [7]. More recently, isotope-dilution liquid chromatography/tandem mass spectrometrymethod 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 achievedby measuring the quenching effect on 7-hydroxycoumarin andEu(III)-(pyridine-2,6-dicarboxylate) Tris complex. LOD value of3.4�10�8 mol L�1 has been achieved [9].

Semiconductor nanoparticles (quantum dots or QDs) are grownto be in the nanoscale size to achieve the effect of quantumconfinement, which enables unique optical properties such aswide absorption profile, very intense size-dependent lumines-cence and high photostability. For instance, CdS QDs and to a lessextent the ZnS QDs have been studied since they can be easilysynthesized in aqueous medium under mild conditions. In addi-tion many surface modifications in QDs (nanoparticle cappingwith organic ligands) have been proposed to enhance opticalproperties, stability in aqueous dispersions and selectivity ininteraction with chemical species in solution. These modifiednanoparticles can be homogeneously dispersed and stabilized inwater, and therefore they are very attractive as analytical probes toselective sense chemical species in aqueous systems [10–12].

In this work, the effect of L-thyroxine on the photolumines-cence of L-cysteine modified ZnS quantum dots (L-cysteine–ZnS

QDs) was studied in aqueous dispersions. The photoluminescencequenching effect caused by L-thyroxine was more effective inaqueous systems containing hexadecyltrimethylammonium bro-mide (CTAB), which also improved stability of the measured signalover time. The approach using the L-cysteine–ZnS probe was usedto quantify L-thyroxine in a pharmaceutical formulation. Analytefortified saliva was also evaluated as a possible matrix for L-thyroxine monitoring of a patient under treatment.

2. Material and methods

2.1. Apparatus

A Perkin-Elmer Lambda 19, UV/vis/NIR double beam spectro-photometer (1 cm quartz cuvettes) was used to obtain electronicabsorption spectra from L-thyroxine and to evaluate the extinctionspectra from the QDs dispersions. All photoluminescence mea-surements were made using a Perkin-Elmer model LS 55 lumines-cence spectrometer with solutions placed in 1 cm opticalpathlength quartz cuvettes. Photoluminescence spectra wereacquired using the FL-Winlabs software and measurements wereperformed with 10 nm excitation and emission spectral bandpassand 1500 nm/min scan rate. Excitation was made at 312 nm withsignal measurement at 424 nm. A thermostatic system withstirring (PTP-1 Fluorescence Peltier System with a PCB1500 WaterPeltier System, Perkin-Elmer) was used to keep the solutions inthe cuvette at specific constant temperatures and to allow stirringof the CTAB organized L-cysteine–ZnS QDs working dispersion.Photoluminescence lifetime measurements were conducted usinga Model HJY 5000M time-resolved fluorescence spectrometer IBH5000F (Horiba Jobin Yvon, NJ, USA) with excitation using ananoLED source at 372 nm. Lifetimes were obtained after mathe-matical deconvolution of the fluorescence decay from the sourcepulse profile.

Transmission electron microscopy (TEM) was made on JEOL2010 Transmission Electron Microscope under 200 kV acceleratingvoltage. Dynamic light scattering (DLS) measurements of thequantum 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 analyzedrange from 2 to 500 nm. For pH measurements, the pH-meter (MSTecnopon, model MPA-210, Brazil) was employed.

2.2. Reagents, samples and other materials

L-cysteine hydrochloride monohydrate, L-thyroxine, zinc acet-ate dehydrated, sodium phosphate monobasic, sodium phosphatedibasic heptahydrate, hexadecyltrimethylammonium bromide(CTAB) were purchased from Sigma-Aldrich (USA). Sodium dode-cyl sulfate (SDS) and triton X-100 were from Merck, Germany.CTAB and SDS were further recrystallized in ethanol in order toeliminate impurities. Analytical grade methanol was from Merck.The pharmaceutical formulation (containing of 200 mg thyroxineper tablet) was obtained from a local drugstore. Utrapure waterwas purified in a Milli-Q system from Millipore (Simplicity model185, USA) and used to prepare all aqueous solutions. Solid phaseextraction (SPE) was made on a 6 mL volume cartridge containing1 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 synthe-sized following similar procedures described in the literature [13].In typical synthesis, an amount of 0.0214 g (1�10�4 mol) of zincacetate dihydrated and 0.0172 g (1�10�4 mol) of L-cysteine

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.

S. Khan et al. / Journal of Luminescence 156 (2014) 16–24 17

hydrochloride monohydrated were dissolved in 100 mL of deio-nized water. Then, the solution was stirred for 15 min and its pHvalue adjusted round to 9.0 by the addition of small volumes of a0.1 mol L�1 NaOH solution. The resultant solution was transferredto a 150 mL three necked round bottom flask (reactor) where asolution containing 2�10�4 mol of Na2S was slowly injected(in the form of aqueous solution) through a syringe into thereactor, 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 differentintervals of time, through a syringe in order to check theirabsorption and photoluminescence characteristics. The transpar-ent and colorless colloidal dispersion of ZnS QDs was treated withethanol to precipitate the nanoparticles, then, the solid mass wasre-dispersed in a phosphate buffer solution (0.01 mol L�1 andpH 8.0).

These nanoparticles were kept under refrigeration being stablefor more than 10 months, indicated by transparency of thedispersion and by its high photoluminescence intensity.

2.4. Microscopy and spectroscopy of the L-cysteine–ZnS QDs

The morphology and structure of functionalized ZnS nanopar-ticles were studied by scanning transmission electron microscopy(Fig. 2), which indicates a large number of assemblies ofL-cysteine–ZnS nanoparticles with a fairly uniform size and shape.As can be observed, nanoparticles are well dispersed and the sizerange varied from 5.1 to 6.6 nm. The particle size distribution ofL-cysteine–ZnS was also measured by DLS (Fig. 2A). The hydro-dynamic diameter was in the range from 12.7 to 17.2 nm with anaverage diameter of 14.0 nm. The DLS measurements indicatedthat L-cysteine–ZnS nanoparticles are prone to certain degree ofaggregation, occurrence that is probably due to the use of

non-buffered dispersion during DLS analysis that may havepromoted the removing of part of the L-cysteine stabilizer fromthe surface of the QDs (Fig. 2A).

ZnS nanoparticles contain few units in their nanocrystal cells,therefore they possess a stable structure and optical propertiesthat do not vary significantly as the reaction time is increased [14].In addition, contrary to what is observed for other semiconductorluminescent nanoparticles, the excitation profile of the L-cysteine–ZnS QDs is narrower [15]. The electronic optical absorption of thesynthesized L-cysteine–ZnS QDs presented a broad profile with thefirst excitonic at 290 nm (Fig. 3A), photoluminescence maximumobserved at 424 nm (related to the 312 nm of the sharp excitationband) and the full width at half maximum value for the photo-luminescence emission band of 36 nm (Fig. 3B).

The photoluminescence intensity of nanoparticles centered at424 nm sharply increased with the increasing reflux time, reach-ing a maximum value at 120 min. The photoluminescence inten-sity then decreased as the reflux time surpassed 120 min asindicated in Fig. 2C. At longer refluxing times, the cloudy appear-ance of the dispersion indicated the aggregation of the crystalsleading to the formation of bigger particles and consequently lessintense photoluminescence.

2.5. Photoluminescence measurements from the L-cysteine–ZnS QDsaqueous 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-thyroxinestandards or samples containing L-thyroxine were added to theQDs 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 presenceof thyroxine.

S. Khan et al. / Journal of Luminescence 156 (2014) 16–2418

(equilibration time). Photoluminescence measurements weremade at 424 nm upon excitation at 312 nm. In order to obtainreliable quantitative results, measurements were also taken from aL-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 pre-pared by dissolving specific amounts of the drug standard in a

diluted sodium hydroxide solution (5�10�4 mol L�1). Standardsolutions of lower concentration were prepared by further dilutionof this stock solution with ultrapure water. The surfactant stocksolution (5.0�10�3 mol L�1) was prepared by dissolving appro-priate amounts of CTAB in ultrapure water.

For the preparation of an L-thyroxine pharmaceutical formula-tion sample, a pool of ten commercial tablets was pulverized. Massaliquots of this powder were dissolved in ethanol. The sample waspassed through a Teflon syringe filter to retain the non-solubleexcipients, then, the solution was passed through a C18 solidphase extraction (SPE) column to retain the L-thyroxine. Thecartridge was washed with deionized water (to remove remainingwater soluble tablets recipients) and the L-thyroxine was elutedwith 1 mL of methanol. The methanol was evaporated and theremaining residue was re-dissolved in the L-cysteine–ZnS QDsbuffered dispersion.

The saliva sample (10 mL collected in a graduated cylinder) wascollected from an euthyroid volunteer that did not receive anyother medical treatment nor ingested food or beverages prior thesample collection. The volunteer rinsed his mouth for 5 min withultrapure water. The saliva was fortified with known amount ofthyroxine and mixed with 5 mL of ethanol. Then, the mixture wasvortex mixed for 30 s and immediately centrifuged for 15 min at3000 rpm. After centrifugation, the supernatant was passedthrough C18 SPE column and washed with ultrapure water. Afterelution with 1 mL methanol, the eluate was evaporated and theresidue re-suspended in the L-cysteine–ZnS QDs buffered disper-sion. The same procedure was repeated for blank measurements.

3. Results and discussion

3.1. Optimization of conditions to achieve the photoluminescencequenching of the L-cysteine–ZnS QDs by L-thyroxine

L-cysteine is an amino acid that has been used for synthesis ofwater compatible fluorescent probes [13]. Such ligand can bind tothe surface of the ZnS nanoparticles through the sulfur atom of themercapto group, while the carboxylic acid group provides watercompatibility.

3.1.1. Amount of cysteine–ZnS quantum dots in the dispersionThe quenching effect promoted by L-thyroxine (fixed at 9.5�

10�7 mol L�1final concentration) on the L-cysteine–ZnS QDs

photoluminescence was investigated in dispersions containingdifferent amounts of nanoparticles. The amount of nanoparticleswas varied by introducing different volumes of the synthesizedL-cysteine-ZnS QDs stock dispersion (from 0.250 to 2 mL) into the10 mL final volume of aqueous dispersion, which enabled a rangebetween 1.6�10�4 to 1.3�10�3 mol L�1 (2.4–20.0 μg mL) of zincconcentration in the QDs precursor solution (Fig. 4A). Higheramounts of L-cysteine–ZnS QDs decreased the sensitivity of thephotoluminescence quenching response due to inner filter effectcaused by the high concentration of QDs in the dispersion. Incontrast, the lower concentrations of QDs resulted in robust andsensitive fluorescence quenching response as indicated by thehigher L0/L ratio values (where L0 is the photoluminescencemeasured from the dispersion in absence of L-thyroxine and L isthe photoluminescence measured from the dispersion in thepresence of L-thyroxine). Dispersions (10 mL total volume) con-taining 0.8 mL of the quantum dots stock dispersion was chosendue to the high sensitivity in the quenching response.

Fig. 3. (A) Electronic absorption spectrum. (B) Photoluminescence excitation andemission spectra. (C) Photoluminescence spectra of the cysteine–ZnS QDs synthe-sized using different refluxing times: (a) 10, (b) 20, (c) 40, (d) 60, (e) 80, (f)100,(g) 130 and (h) 120 min.

S. Khan et al. / Journal of Luminescence 156 (2014) 16–24 19

3.1.2. Influence of pH on the L-cysteine–ZnS QDs photoluminescencequenching mediated by L-thyroxine

The effect of pH of the aqueous dispersion on the interaction ofL-thyroxine and quantum dots was studied in order to find theappropriate pH for the sensitive probing of L-thyroxine. The pHwas varied in the range from 6.5 to 9.0 (using phosphate buffer orborate buffer at 0.01 mol L�1

final concentration to cover such pHrange). The experiments were performed in dispersion with thedifferent pH values either in the presence or in the absence

L-thyroxine (3.8�10�7 mol L�1). The signal profile (Fig. 4B) indi-cated a more effective photoluminescence quenching at the pHvalues of 8.0 and 8.5. The pH value selected for the QDs workingaqueous dispersion was 8.0 (achieved by the phosphate buffer).

3.1.3. Effect of surfactants on the photoluminescence quenchingcaused by L-thyroxine

The effect of different surfactants such the cathionic acetyltri-methyl ammonium bromide (CTAB), the anionic sodium dodecylsulfate (SDS) and the non-ionic Triton X-100 on the L-thyroxinemediated 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 photolumines-cence of the system probably due to the electrostatic repulsionbetween the negative charge on the surface of nanoparticles andthe anionic head of SDS, thus at high concentration of SDS,quenching of the photoluminescence from quantum dots wasobserved. When using Triton X-100, stable photoluminescencemeasurements were not achieved probably due to adsorption ofsurfactant onto the surface of nanoparticles, changing the surfaceproperties and probably promoting the removal of L-cysteine.

The improvement in the photoluminescence quenching effi-ciency was obtained in the presence of CTAB at concentration of5�10�5 mol L�1, which is below the critical micelle concentra-tion (CMC) of this surfactant (about 1 mmol L�1) as indicated inFig. 5A. As the surface of ZnS QDs capped with cysteine isnegatively charged due to the desprotonation of the carboxylicgroup at pH 8.0, the CTAB (a cationic surfactant) is prone tointeract to nanoparticles surface, via electrostatic interaction. Inthe presence of this cationic surfactant, the photoluminescencefrom the QDs dispersion was slightly enhanced and became stable,which may due to partial incorporation of nanoparticles into theprotective environment of such surfactant micelles. The presenceof nanoparticles in micelle prevents the adsorption of othermolecules on the surface of the nanoparticles, thus, the excitedelectron from the conducting band may combine with the vacancyat the valence band with less probability of electron transfer toother molecules nearby.

Better analyte induced photoluminescence quenching wasobserved below the critical micelle concentration (CMC) is probablydue to the slight positive surface changes, which facilitates interac-tions between nanoparticles and the L-thyroxine molecule. Athigher concentration of CTAB, the surfactant may block the accessto L-thyroxine to the nanoparticles and the resulting in a lesseffective photoluminescence quenching. Thus, a final concentration

Fig. 4. (A) Effect of the amount of the synthesized nanoparticles on the photo-luminescence quenching of the L-cysteine–ZnS QDs aqueous dispersion. Signalvariation expressed as L0/L (where L0 and L are respectively the photoluminescencee 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 dispersionon the photoluminescence quenching of L-cysteine–ZnS QDs (3.8�10�7 mol L�1

final concentration of L-thyroxine). Results obtained in triplicate.

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.

S. Khan et al. / Journal of Luminescence 156 (2014) 16–2420

of 5�10�5 mol L�1 of CTAB was incorporated into the QDs waterworking dispersion.

3.1.4. Effect of temperatureThe effect of temperature on the photoluminescence intensity

measured from the L-cysteine–ZnS QDs dispersion was studied attemperatures ranging from 18 to 45 1C. The fluorescence measuredfrom quantum dot was found to be inversely dependent ontemperature with a fairly constant signal intensity found between18 and 25 1C (Fig. 5B). The decrease in signal intensity measuredfrom the QDs dispersion is probably due to the increase of thekinetic energy of the components of the system, which disruptsthe interaction between the analyte and the L-cysteine ZnS QDs,leading to a less effective photoluminescence quenching. There-fore, room temperature (about 25 1C) was selected in all fluores-cence measurement for sensing of L-thyroxine.

3.1.5. Stability of photoluminescence intensity and reaction timeUnder 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 measuredphotoluminescence was made. First, the photoluminescence fromthe control working dispersion of QDs (L-cysteine–ZnS QDs buf-fered dispersion containing CTAB before the addition of L-thyrox-ine) was measured in function of the time (measured every 10 minup to 120 min). The signal was found to be stable during thewhole time of the experiment (less than 3% random variation ofsignal) as indicated in Fig. 6A.

The photoluminescence from this same QDs dispersion wasalso monitored in function of the time after the addition of a fixedamount of L-thyroxine. Measurements were made every 2 min upto 30 min starting 2 min after the addition of L-thyroxine (1 min ofmixing and 1 min of equilibration of the solution). The photo-luminescence quenching was immediate and after 5 min, thesignal becomes stable up for more than 30 min (Fig. 6B). For theanalytical method, it was established all measurements to bemade after 5 min of the addition of L-thyroxine into the quantumdots dispersion.

3.2. Mechanism of interaction

Several mechanisms have been proposed to explain QDsphotoluminescence signal reduction, including inner filter effectand non-radiative processes caused by electron transfer, surface

adsorption, complexation among others [16]. As previously men-tioned, L-thyroxine has a wide UV–vis absorption profile withmaximum at 235 nm and 330 nm. Taking into consideration a6�10�5 mol L�1 solution of L-thyroxine, the absorbance (Fig. 1Bline 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 theexcitation wavelength of 312 nm considering concentration levelsL-thyroxine below 1.0�10�5 mol L�1.

Since no changes in the absorption spectral profile of the L-cysteine–ZnS QDs (characteristic profile in Fig. 3B) takes places inthe presence of L-thyroxine, it is concluded that no aggregation(chemical degradation that can lead to the reducing of photo-luminescence) of QDs are taking place. If aggregation has occurred,a measurable decrease in transmittance would be observedbecause of the significant increasing in light scattering measuredfrom the dispersions. This is an indication that photoluminescencequenching should be promoted by an effective interactionbetween the analyte and the QDs and not by light filter effectdue to the increase of light extinction. Aggregation of nanoparti-cles induced by L-thyroxine was also ruled out since no changes inparticle distribution were observed by microscopy. In addition,DLS measurement after the addition of L-thyroxine into the QDsdispersion (Fig. 2B) showed size distribution profile similar tothose observed in the system without L-thyroxine (Fig. 2A).

In order to determine if the mechanism of photoluminescencequenching is whether static or dynamic, a study of the depen-dence of photoluminescence analytical curve sensitivity upon thetemperature was made [17]. From the Stern–Volmer plots con-structed at two different temperatures (298 and 308 K) it wasobserved that the sensitivity decreases as the temperatureincreased (Fig. 7A). These results indicate the L-thyroxine andcysteine–ZnS interactions associative in nature and characteristicof a static type of quenching. In addition, photoluminescencelifetime experiments have indicated the similar profile for mea-surements made from QDs dispersions in the presence and also inthe absence of L-thyroxine (Fig. 7B). It is valid to point out that thephotoluminescence of semiconductor nanoparticles is known todecay in a multi-exponential manner, thus, QDs emission consistsof more than one lifetime component [10]. Thus, the photolumi-nescence lifetimes that characterized the dispersion of cysteine–ZnS QDs in CTAB, no matter the presence of L-thyroxine, were:471; 1772 and 8574 ns. As no change in lifetime profile of theQDs dispersion is observed in the presence of L-thyroxine, results

Time (min) Time (min)

Pho

tolu

min

esce

nce

L 0/L

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).

S. Khan et al. / Journal of Luminescence 156 (2014) 16–24 21

indicate that a static quenching is taking place when analyteinteracts with QDs.

A possible mechanism can be proposed based on the experi-mental results. In absence of CTAB, no quenching effect wasobserved since non-protonated L-thyroxine is repelled by thenegatively charged cysteine that covers the surface of the ZnSQDs. When CTAB is added, this cationic surfactant interact withQDs neutralizing the negative charges of the capping agent, thusenabling L-thyroxine to form a complex with Zn atoms at thesurface of QDs. Thyroxine withdraw electrons from the QDsconducting band resulting in photoluminescence static quenching.As L-thyroxine present four iodine groups any molecular fluores-cence that might be stimulated by such electron transfer is alsoquenched by internal heavy atom effect that transfers the electro-nic population to the triplet state. Since environment is not freefrom dissolved oxygen, triplet state radiative decay is not favored.

3.3. Analytical characteristics of the photoluminescence quenchingmediated by L-thyroxine

3.3.1. Detection and quantification limits and precision ofmeasurements

Under the optimized experimental conditions (Table 1) aStern–Volmer model (Eq. 1) could be readily used to establish alinear relationship between measured photoluminescence, L, andthe concentration of L-thyroxine, [L-thyroxine], in the dispersion.

L0 is the photoluminescence made from the probe dispersionbefore the addition of L-thyroxine and Ksv is the Stern–Volmerconstant.

L0=L¼ 1þKsv½L� thyroxine� ð1ÞThe results show that L-thyroxine quenches the photolumines-

cence of the L-cysteine–ZnS QDs in a concentration dependentpattern from 1.1�10�7 to 4.0�10�6 mol L�1 (Fig. 8A). Thus, analy-tical curves were constructed by adding increasing concentrations ofL-thyroxine on the QDs dispersion and then, measuring the photo-luminescence decreasing. A typical analytical curve (L0/L versus

L 0/L

Concentration of L-thyroxine(10-7 mol L-1)

Time (ns)

Inte

nsity

(cou

nts)

Fig. 7. (A) The Stern–Volmer curves for the quenching of cysteine–ZnS QDsaqueous dispersion in the presence of L-thyroxine at 298 K (■) and 308 K (▲).(B) Photoluminescence lifetime profile of cysteine-ZnS QDs aqueous dispersions inthe absence (▲) and in the presence (★ of thyroxine (3.6�10�6 mol L�1).

Table 1Optimized experimental conditions for the thyroxine determination using theL-cysteine–ZnS QDs.

Experimental parameter Optimized Value

Type of quantum dots Cysteine–ZnS nanoparticlesPhosphate buffer solution 0.01 mol L�1

pH 8.0Time required to perform measurement 5 minConcentration estimated for QDsa 4.5�10�4 mol L�1

Temperature 25 1CCTAB concentration 1.2�10�6 mol L�1

a Estimated by the concentration of Zn2þ in the QDs precursor solution.

Pho

tolu

min

esce

nce

L 0/L

Wavelenght (nm)

Concentration of L-thyroxine(10-7 mol L-1)

Fig. 8. (A) Photoluminescence emission spectra of L-cysteine–ZnS QDs aqueousdispersion 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 determina-tion of thyroxine using L-cysteine–ZnS QDs as probe.

S. Khan et al. / Journal of Luminescence 156 (2014) 16–2422

concentration of L-thyroxine) is shown in Fig. 8B, showing a linearrange of the analytical response in the concentration range from1.1�10�7 to 4.0�10�6 mol L�1 of L-thyroxine (final concentrationin the dispersion) with correlation coefficient of 0.9926. The equationmodel of the analytical curve was L0/L¼3.3�105 [L-thyroxine]þ1.01.Since at these concentration levels L-thyroxine does not significantlyabsorb light at 312 nm, no correction for inner-filter effect wasnecessary for the Stern–Volmer model.

The limit of detection (LOD) was calculated as the concentra-tion of L-thyroxine that changes the photoluminescence signal inthe measurement cell by L0�3sL0, where sL0 is the standarddeviation of ten replicate measurements of the photolumines-cence intensity of the L-cysteine–ZnS dispersion before the addi-tion of L-thyroxine. Similarly, the limit of quantification (LOQ) wascalculated 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 ngmL�1),respectively. The precision of the L-thyroxine measurement using theproposed probe was calculated as the variation of the L0/L value takinginto consideration ten independent solutions (in two differentL-thyroxine concentrations). In order to do this, the following equationwas used: s(L0/L)¼L0/L� [(sL/L)2þsL0/L0)2]1/2. The s(L0/L), in percentagevalues, was 2.8% and 4.2% at, respectively the 3.9�10�7 mol L�1 and2.9�10�6 mol L�1 concentration levels.

3.3.2. Selectivity studiesFor practical applications of the proposed method for the

determination of L-thyroxine in biological samples (saliva) and inpharmaceutical formulations, the effect of some possible relevantinterfering substances was evaluated. The chosen substances werethe ones commonly found in pharmaceutical formulations and inbiological fluids (including several amino acids). Changes influorescence intensity due to the presence of these chemicalspecies were expressed in percent values (Table 1).

All the tested substances imposed variations in the photolumi-nescence intensity measured from the probe (at the specifiedconcentrations) under 4% (variation considered not relevant). Incontrast, the presence of a much less amount of L-thyroxine(0.4 μmol L�1) caused about 10% decreasing of the photolumines-cence measured from the probe. The interference caused by twodifferent complex matrices was also evaluated. One matrix wascomposed by a mixture of amino acids (cysteine, histidine,phenylalanine, valine, methionine, lysine and threonine), eachamino acid with a final concentration of 5�10�6 mol L�1. Theother matrix consisted on a mixture of common pharmaceuticalexcipients (lactose, silicon dioxide, citric acid, calcium chloride,

magnesium sulfate, potassium chloride, sodium chloride) each oneat a final concentration of 0.1�10�6 mol L�1. It was observed thatthe photoluminescence measured from L-cysteine–ZnS QDs dis-persion in absence and in the presence of L-thyroxine is notsignificantly different when either the amino acids mixture orthe pharmaceutical excipient mixture is present.

For biological samples, proteins can affect the photolumines-cence 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 theinfluence of NaCl on the photoluminescence quenching of theprobe indicated that no interference is expected in samplescontaining up to 2�10�3 mol L�1 of NaCl.

3.4. Analytical application of the L-cysteine–ZnS QDs dispersion forthe determination of L-thyroxine

The proposed photoluminescence L-cysteine–ZnS probe hasbeen applied for the determination of L-thyroxine in pharmaceu-tical formulation (containing the artificial thyroxine hormone asactive component) and in analyte fortified human saliva, simulat-ing a sample from a patient with a non-active thyroid gland thatwas medicated with L-thyroxine.

Three different portions (0.57 g) from ten grinded pharmaceu-tical tablets were selected. The L-thyroxine content in theseportions were dissolved in methanol, filtered in a 0.45 mm syringefilter and then diluted in water. A volume (1 mL) of the samplesolution was placed into the L-cysteine–ZnS QDs aqueous disper-sion in order to achieve a theoretical final concentration of9.8�10�7 mol L�1 of L-thyroxine in the QDs dispersion. Therecoveries (Table 2) achieved using the proposed method at was97.075.2% taking into consideration the value of L-thyroxineindicated in the pharmaceutical formulation instructions (Table 3).

In order to evaluate the applicability of the method in simpleclinical assays, the analysis of saliva samples (fortified with L-thyroxine at concentration of 2.9�10�7 mol L�1) were per-formed. The saliva was mixed with 5 mL of ethanol, fortified withthe known amount of L-thyroxine then immediately centrifugedfor 15 min at 3000 rpm. After centrifugation, the supernant waspassed through C18 SPE column and washed with deionzed water.After elution with 1 mL methanol, the eluate was evaporated andthe residue re-suspended with the nanoparticle dispersion (madein phosphate buffer pH 8.5). From the photoluminescence quench-ing magnitude, the concentration of thyroxine was obtained withrecovery of 86.075.1%. The relatively low recoveries might be dueto loss of L-thyroxine in pretreatment of saliva Table 3. Photo-luminescence spectra (Fig. 9) indicates that original componentsfrom saliva matrix does not interfere with the determination ofthyroxine as it does not fluoresce under the condition of theexperiment and it does not affect the photoluminescence signalfrom the L-cysteine–ZnS dispersion when it is not fortified withthyroxine.

The effectiveness of cysteine functionalized nanoparticles asprobe for substances of clinical and biological interest has beendemonstrated in earlier works, for instance, aqueous dispersion of

Table 2Effect of some potential interfering substances on the photoluminescence mea-sured from the L-cysteine–ZnS QDs organized aqueous dispersion.

Potentialinterferent

Concentration(μmol L�1)

Change of photoluminescence(%)

Cysteine 150 þ4.0Histidine 150 þ3.1Tyrosine 150 þ1.4Phenylalanine 150 þ0.55Valine 150 þ0.55Methionine 150 �0.55Lysine 150 þ1.13Threonine 150 �1.42Lactose 100 �2.6Silicon dioxide 100 þ1.8Citric acid 100 þ2.0Calcium 100 þ4.1Magnesium 100 þ3.8Potassium 100 þ2.1Sodium 100 þ3.1Chloride 100 þ3.0

Table 3Applications of the cysteine–ZnS QDs for determination of L-thyroxine in pharma-ceutical formulation and saliva.

Sample L-thyroxinelevel

Portion1

Portion2

Portion3

Averageresult (%)

Levotiroxina sódica 200 μg pertablet

93.3 94.8 103 97.075.2

L-thyroxine fortifiedsaliva

2.9�10�7

mol L�188.9 90.1 86.5 86.075.1

S. Khan et al. / Journal of Luminescence 156 (2014) 16–24 23

cysteine–CdTe QDs has been applied for the selective determina-tion of cardiolipin in the presence of others phospholipids [18].Moreover, cysteine–ZnS QDs and cysteine–CdS QDs have also beenused as luminescent probes for determination of nucleic acids andmannitol [19,20]. The proposed method for the determination ofthyroxine brings important advantages over the methods alreadyreported in the literature because of the poor optical properties ofthe analyte. Most of the thyroxine determination methods rely onthe indirect detection using metallic luminescent complexes thatare very sensitive to other species thus prone to interferences[3,4]. On the other hand, chemical derivatization approaches useshighly toxic and expensive derivatization reagents. The heavymetal-free QDs dispersion (L-cysteine–ZnS QDs/CTAB system) isprepared with cheap and readily available reagents, providing afairly selective interaction with thyroxine using a simple analyticalapproach.

4. Conclusion

The photoluminescence intensity of a L-cysteine–ZnS QDsaqueous dispersion is quenched by L-thyroxine in the presence

of the surfactant CTAB. The overall quenching followed a Stern–Volmer model. The proposed optical probe has been applied forthe determination of L-thyroxine in pharmaceutical formulationand in analyte fortified human saliva. The poor optical propertiesof L-thyroxine make the proposed indirect determination approachattractive when compared to the already reported methods.

Acknowledgments

The authors wish to thank Brazilian agencies FINEP and FAPERJfor 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 PhysicsPUC-Rio) for the luminescence lifetime measurements.

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c

d

a b

Fig. 9. (a) Photoluminescence of the aqueous L-cysteine–ZnS QDs dispersion;(b) photoluminescence of saliva re-suspended in the aqueous L-cysteine–ZnS QDsdispersion; (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.

S. Khan et al. / Journal of Luminescence 156 (2014) 16–2424