Spectrochimica Acta Part A 60 (2004) 385–389

Resonance light scattering spectroscopy study of interaction betweengold colloid and thiamazole and its analytical application

Xiaoling Liu, Hong Yuan, Daiwen Pang, Ruxiu Cai∗

College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, Hubei, People’s Republic of China

Received 19 March 2003; received in revised form 19 March 2003; accepted 5 June 2003

Abstract

In this paper, we used resonance light scattering (RLS) spectroscopy to study the interaction between thiol-containing pharmaceutical-thiamazole and gold colloid. At pH 5.2, the resonance light scattering spectrum of gold nanoparticles has a maximum peak at 555 nm and theRLS intensity is enhanced by trace amount of thiamazole due to the interaction between thiamazole and gold colloid. The binding of colloidalgold to thiamazole results in ligand-induced aggregation of colloidal gold, which was characterized by RLS spectrum, ultraviolet–visible(UV–Vis) spectrum, and transmission electron microscopy (TEM). Based upon the study, we proposed a highly sensitive, gold colloid-basedassay using RLS spectrum to detect pharmaceuticals for the first time. The mechanism of binding interaction between Au colloid and thiamazolewas also discussed.© 2003 Elsevier B.V. All rights reserved.

Keywords: Resonance light scattering; Colloidal gold; Thiamazole

1. Introduction

Colloidal gold have been studied greatly in recent yearsdue to their interesting physical properties[1–3] andpromising applications[4–10]. One aspect of these fantas-tic researches is focused on the phenomena of aggregationor flocculation of gold nanoparticles in solution[11–14].For example, Sastry et.al have investigated the aggregationprocess on carboxylate-functionalized colloid, which dis-play flocculation behavior as a function of pH[15]. TheMirkin’s group has detected single-nucleotide sequencemismatches and DNA, for which the temperature of thesingle particle-aggregate transition is indicative of the de-gree of DNA complementarity[16–18]. Similar reversibleaggregations are used for the analyses of several metal ions[19–21], and antibodies[22]. In these experiments, Particleaggregation process results in visible color changes of goldcolloid solution, which can be characterized by transmissionelectron microscopy (TEM) and absorbance spectroscopy.However, when the amounts of material available for analy-ses are limited, methods with high sensitivity and simplicity

∗ Corresponding author. Tel.:+86-27-8768-4184;fax: +86-27-8764-7617.

E-mail address: dlxfxc@whu.edu.cn (R. Cai).

is needed to find out. An appropriate solution to the prob-lem is to use resonance light scattering (RLS) technique tostudy the gold aggregation process.

The Pasternack’s group has firstly reported that RLStechnique can be used to investigate the formation of ag-gregated choromophores in complex systems[23,24]. Res-onance light scattering occurs when the incident beam isclose in energy to an absorption band produced by an oscil-lating dipole. This phenomenon is amplified when dipolesare strongly coupled, to the level where it can be detectedby a common spectrofluorimeter. Since then, the techniquehas been applied to a large variety of aggregation systemsincluding porphyrin[25–27], dye–nucleic acids[28,29]anddye–protein[30–32]aggregates. In these experiments, RLSnot only meets sensitivity and selectivity criteria but alsooffers the additional benefits of simplicity and versatility.

Though RLS technique is proved an effective methodto investigate aggregate systems, the study on colloidalgold aggregation using RLS technique remains an unex-plored area. In our paper, RLS technique was utilized toinvestigate colloidal gold aggregation, which is induced bytrace amounts of thiamazole (Fig. 1, 1,3-dihydro-1-methyl-1-methylimidazole-2-thiol). This aggregation was alsoinvestigated by TEM and absorbance spectroscopy. Fur-thermore, the analytical capabilities of the RLS technique

1386-1425/$ – see front matter © 2003 Elsevier B.V. All rights reserved.doi:10.1016/S1386-1425(03)00235-X

386 X. Liu et al. / Spectrochimica Acta Part A 60 (2004) 385–389

Fig. 1. Chemical structure of thiamazole.

are discussed. These researches provide evidence that thecombination of RLS technique and colloidal gold may bean effective method to detect thiol-containing substances,such as amino acid or proteins.

2. Experimental

2.1. Materials

All reagents were of analytical grade or the bestgrade commercially available unless otherwise mentioned.HAuCl4·4H2O, sodium citrate, sodium borohydride, con-centrated HCl and HNO3 were all purchased from Shang-hai First Reagent Plant. Thiamazole (Standard) purchasedfrom Material Medical and Biologic Product VerificationInstitution of China were directly dissolved in water toprepare stock solution of 9.71× 10−3 mol l−1 and storedat 0–4 ◦C. The working solutions of 2.68× 10−5 mol l−1

were obtained by diluting the stock solutions with water. Allaqueous solutions were prepared in doubly deionized water.

2.2. Preparation of Au nanoparticles

Colloidal gold (Au) particles of different sizes were pre-pared following the reported procedure[33,34]. Briefly, 13nm-diameter gold nanoparticles were prepared as follows:aqueous HAuCl4 (1 mM) was heated to boiling with vigor-ous stirring and followed by a rapid addition of an excessof 38.8 mM aqueous sodium citrate. The preparation of2.6 nm-diameter colloidal Au is: 1 ml of 1% HAuCl4 wasadded to 90 ml of H2O at room temperature, 2.00 ml of 38.8mM sodium citrate and 1.00 ml of fresh 0.075% sodiumborohydride were added in turn after mixed well. Both ofthem were filtered through a 0.45�m Gelman membranefilter and stored in a dark bottle at 4◦C. For the goldnanoparticles reduced by citrate ions, transmission electronmicroscopy (TEM) indicated a particle size of 13.8±1.8nm (100 particles sampled) and UV–Vis spectrum showedan absorbance maximum at 520 nm, while 2.6 nm-diametercolloidal Au solution yielded an absorbance maximumat 510 nm. The approximate extinction coefficient at 520nm for 13 nm diameter particles was determined to beλ520≈ 2.08× 108 M−1 cm−1 using a reported method[35].The concentration of the gold nanoparticle solution wasdetermined using the absorbance values in conjunction withthe calculated extinction coefficient (11.8 nM final particleconcentration for this preparation). The concentration of 2.6nm-diameter colloidal Au solution was∼1 �M accordingto the method described above.

2.3. Instrumentation

RLS experiments were conducted on a Shimadzu modelRF-5301PC spectrofluorimeter with right angle geometryusing a 1 cmquartz cell. The excitation and the emissionslits were set to 1.5 nm with sensitivity high and responsetime 0.25 s. The excitation and emission monochromaterwavelength were adjusted to scan simultaneously throughthe range from 220 to 700 nm. Gold colloid solution (0.6ml) and an appropriate volume of thiamazole solution wereplaced into a 3 mlcolorimetric tube and mixed gently. Themixture was diluted to 3 ml with water and placed in theair for 15 min prior to scan. The enhancement of the RLSintensity (�I) is the difference in intensities between sam-ple (I) and blank (I0) under the same conditions atλ =555 nm.

The electronic absorption spectra were recorded withUVIKON-941 spectrophotometer (Kontron Instruments).TEM was performed on JEM-100CXII (80 KV), Japan.One drop of sample solution was cast onto a copper (Cu)grid for TEM characterization. The evaporation of solventleaves the nanoparticles on the surface. A HP 7500a induc-tively coupled plasma (ICP)-MS (Agilent) system was usedfor the determination of the atomic gold concentration inthe gold nanoparticle solution.

3. Results and discussion

In general, two processes occur when light passes througha solution of aggregates. If the solvent itself is nonabsorb-ing, then energy is removed from the incident light throughabsorption and scattering by the aggregates. The extent towhich a particle absorbs and scatters light depends on itssize, shape, and index of refraction relative to the surround-ing medium (both real and imaginary parts)[24]. Herein,as trace thiamazole is introduced into the gold colloid so-lution, it acts as a ligand to induce aggregation of the goldnanoparticles. Au nanoparticle aggregation leads to visiblecolor changes in gold colloid solution. These color changesarise from a combination of absorption and scattering oflight by the nanoparticle solutions[16–18].

3.1. RLS spectrum characteristic

Resonance light scattering, a Rayleigh scattering phe-nomenon at a wavelength near or at the absorption bands ofthe scattering molecules, can provide a high signal level andgood selectivity. It is owing to the fact that the refractive in-dex varies steeply near absorption band, and additionally theimaginary part of the molecular polarizability starts to man-ifest its independent contribution to the scattering process[36]. Fig. 2 shows the RLS spectra of 13 nm-diameter goldcolloid solution, thiamazole solution and mixture solutionsof gold colloid with different concentrations of thiamazole.It can be seen that thiamazole has a weak RLS signal at a

X. Liu et al. / Spectrochimica Acta Part A 60 (2004) 385–389 387

Fig. 2. Resonance light-scattering spectra of (a) water; (b) 4.38 moll−1 thiamazole (c) gold; (d) gold+ 3.51× 10−7 mol l−1 thiamazole;(e) gold+ 7.02× 10−7 mol l−1 thiamazole; (f) gold+ 1.05× 10−6 moll−1 thiamazole after placing for 15 min. All of the concentrations of 13nm-diameter gold colloid solutions were 2.36 nM, at pH 5.2.

concentration of 4.38 mol l−1. The gold colloid solutiondisplays an obvious RLS peak at about 560 nm close to itsabsorption band. It is clear that the RLS intensity is highlydependent on the quantity of thiamazole, indicating that theextent of flocculation depends upon the concentration ofthiamazole. As shown inFig. 2, low levels of thiamazoleleads to aggregation and strong electromagnetic couplingbetween nanoparticles, resulting in a greatly amplified RLSresponse, whereas the shape and wavelength position of thelight-scattering spectra profile were not effected. The colorof colloidal Au solution turned into different colors suchas red, mauve, violet and blue with increased thiamazoleconcentration.

In the system of 13 nm-sized colloidal Au, the RLS in-tensity is enhanced rapidly following the addition of thia-mazole. The more thiamazole, the longer it takes to reachthe maximum RLS intensity.Fig. 3shows the effect of timeon the RLS intensity. When the maximum concentration ofthiamazole was added, it takes about 15 min to reach themaximum RLS value (stable for at least 1 h). It should benoted that when the color of Au colloid-thiamazole solu-tion system turned blue, the RLS intensity plateaued, imply-

Fig. 3. Effect of time on the resonance light-scattering intensity of 13nm-diameter gold colloid solution+ thiamazole, at pH 5.2. The finalconcentration of gold and thiamazole are 3.93 nM and 1.75× 10−6 moll−1, respectively andλRLS = 555 nm.

Fig. 4. UV–Vis spectra against water of (a) gold; (b) gold+ 3.51× 10−7

mol l−1 thiamazole; (c) gold+ 7.02× 10−7 mol l−1 thiamazole;(d) gold+ 1.40× 10−6 mol l−1 thiamazole; (e) gold+ 1.75× 10−6 moll−1 thiamazole; (f) gold+ 1.93× 10−6 mol l−1 thiamazole at pH 5.2. Theconcentrations of these gold colloid solution are all 3.93 nM and the sizeof the original gold colloid is 13 nm.

ing that the aggregates have reached its maximum volumein solution. The particles precipitate following overnightincubation.

3.2. Absorption characteristic

The phenomenon of resonance light scattering is alwaysassociated with a strong electronic coupling between adja-cent chromophores, the size and the geometry of the result-ing aggregate, and intense molar absorbance of themonomeric constituents. So, it is important to observe themolecular absorption spectra of Au colloid–thiamazolesystems to understand the interaction mechanism.

As shown inFig. 4, the UV–visible spectrum of the 13nm-diameter gold colloid solution exhibits an absorbancemaximum at 520 nm as a result of resonant excitation ofsurface plasma. Following the addition of trace thiama-zole, the intensity of the plasmon absorbance at 520 nmreduces and an absorption peak appears at about 700 nm.With the increase of the thiamazole concentration, the in-tensity of the red-shifted absorption peak increases andshifts to longer wavelengths while the plasmon absorbancefrom individual particles decreases. The appearance of theabsorbance band (atλ ≈ 700 nm) results from the closecontact of gold nanoparticles. The more particles in contact,the longer the range of the plasmon couple. Long-rangecoupling can lead to red-shifted absorbance that is severalhundred nanometers from that of the individual particles[37].

The phenomena above are similar to the interactionsbetween gold or silver nanoparticles and thiol-containingamino acids[12,38]. However, there are differences insome aspects. First, the time for aggregation in our workis much shorter, which may be due to their differentspecial configurations and active groups. Second, the ag-gregate in our experiments is stabilized by dispersioninteractions between the 1-methylimidazole groups ofthiamazole.

388 X. Liu et al. / Spectrochimica Acta Part A 60 (2004) 385–389

Fig. 5. Electron microscopy of 13 nm-diameter gold colloid. (a) goldcolloid solution without thiamazole; (b) colloid Au solution (final con-centration is 3.93× 10−9 mol l−1) with addition of thiamazole (finalconcentration is 1.93× 10−6 mol l−1), placed for about 1 h.

3.3. Transmission electron microscopy

The above experiment provides evidence for the state ofdispersion of the 13 nm-diameter gold nanoparticles andaggregation of the thiamazole-capped gold nanoparticles.The two kinds of states can also be seen from the TEMmicrographs presented inFig. 5. It is clear that the numberof individual gold nanoparticles decreases because of theaddition of thiamazole, while each particle in aggregate statecan still be observed as a separate entity.

3.4. Effects of gold nanoparticle’s preparationmethod and concentration on the RLS intensity

Gold nanoparticles studied here include 2.6 nm-diametergold nanoparticles reduced by NaBH4 and 13 nm-diametergold nanoparticles reduced by Na3Cit, respectively. The ef-fects of preparation method of gold nanoparticles and goldcolloid solution concentrations on the scattering intensitywere studied in thiamazole (4.38× 10−8–2.28× 10−6 moll−1) standard assay. Good linear relationship was obtainedin Fig. 6. For both the 13 nm and 2.6 nm-diameter goldcolloid systems, the range of linearity is wider for the highconcentration of gold colloid solution at the expense of sen-sitivity. With the decreases of the gold colloid concentra-tion, the linear range is narrowed because the gold colloidsolution has aggregated so sharply that sedimentation takesplace quickly. However, the sensitivity is increased. Thoughthe sensitivity of the 2.6 nm-diameter Au colloid systems isvery high, it takes much longer time to stabilize, which may

Fig. 6. Effects of the preparation and concentration of gold colloid solutionon the RLS intensity: a, b, c, d represent 13 nm-diameter gold colloidsolutions with the concentrations being 1.57, 2.36, 3.14 and 3.93 nM,respectively. e, f, g represent the 2.6 nm-diameter colloidal Au solutionwith approximate concentrations being 0.33, 0.67 and 1�M, respectively;Each sample was placed for 15 min before scanning andλRLS = 555 nm.

be due to the difference of sizes.Fig. 7 shows the relation-ship between relative RLS intensity and the ratio of thiama-zole/13 nm-diameter Au at a fixed thiamazole concentration.It is obvious that Au colloid solution with the higher particleconcentration is less prone to aggregate at a fixed thiama-zole concentration. This phenomenon may be due to the lackof thiamazole per particle to induce aggregation. Thus, it ispossible to use gold colloid solution of lower concentrationto analyze thiamazole with the sensitivity being high.

3.5. Analytical application of RLS techinique

In our reaction systems, there is no evidence of a pH differ-ence or change. The enhancement of RLS intensity of goldcolloid by thiamazole reaches a maximum after 15 minutesand plateaued for at least one hour. We chose 13 nm-diametercolloidal gold solution (2.36 nM) as optimized condition toconstruct the calibration graph for thiamazole. As shownin Fig. 6 (line b), a linear calibration graph was obtainedwhen the thiamazole concentration ranges 0–1.05× 10−6

Fig. 7. Relative RLS intensity as a function of thiamazole/13 nm-diametergold colloid (mol/l) ratio, with the same concentration of thiamazole(1.44× 10−7 mol l−1) and the Au colloid concentration being 1.57, 2.36,3.14 and 3.93 nM, respectively. Each sample was placed for 15 minbefore scanning andλRLS = 555 nm.

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mol l−1. The linear response can be fitted to the equation�I = −4.37+ 6.03× 10−8 C (mol l−1). The detection limitwas found to be 2.01× 10−9 mol l−1 (calculated to be the3Sb/m criterion, wherem is the slope for the range of the lin-earity used andSb is the standard deviation (n = 11) of thesignal from sample solution). The reproducibility of the sys-tem was tested by repeated measurements on sample solu-tion (n = 11) (RSD= 1.86%). Several inorganic cations andsome organic compounds, such as metal ions, amino acids,urea, glucose, and starch, were investigated for their inter-ference in this system. If the relative intensity changed bymore than 5%, the substance added was considered to havecaused interference. The results indicate that the selectivityof this method is good also.

4. Conclusion

Resonance light-scattering spectroscopy was successfullyused to investigate the process of gold aggregation inducedby a kind of thiol-containing pharmaceutical-thiamazole.The thiol portion of thiamazole covalently binds to thesurface of the Au particle, destabilizes the particles andleads to aggregation and strong electromagnetic couplingbetween nanoparticles. The intensity of RLS was enhancedsignificantly accordingly. The RLS technique can be alsoused to detect a minimum of 2.01× 10−9 mol l−1 of thia-mazole under certain conditions. Probe stability is essentialfor realizing high sensitivity and selectivity in such as-says. Therefore, when combining with colloidal gold-basedassay, RLS technique may have potential applications indetermining thiol-containing substances.

Acknowledgements

We thank the Scientific and Technologic Committee ofWuhan city in China (20016009107) for its financial support.We are also grateful to Zuting Pan for his help.

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