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Spectrochimica Acta Part A 75 (2010) 1497–1500

Contents lists available at ScienceDirect

Spectrochimica Acta Part A: Molecular andBiomolecular Spectroscopy

journa l homepage: www.e lsev ier .com/ locate /saa

ltrasensitive determination of serum albumin using resonanceight scattering based on ZnS-polyacrylic acid nanoparticles

i Peng, Chunya Li, Hongbo Xue, Guoqing Zhan ∗

ey Laboratory of Analytical Chemistry of the State Ethnic Affairs Commission, College of Chemistry and Materials Science,outh-Central University for Nationalities, Wuhan 430074, China

r t i c l e i n f o

rticle history:eceived 12 October 2009eceived in revised form 6 January 2010ccepted 3 February 2010

a b s t r a c t

ZnS-polyacrylic acid (ZnS-PAA) was prepared by an in situ polymerization method using nano-ZnS ascore in the presence of acrylic acid (AA), and ZnS-PAA nanoparticles was characterized by ultravioletspectrometry (UV) and transmission electron microscopy (TEM). Based on the significant increase of

eywords:esonance light scatteringanoparticlesnSerum albumin

the resonance light scattering (RLS) intensity with the interaction between nanoparticles and serumalbumin, RLS method was developed for the sensitive determination of serum albumin (BSA andHSA). Under optimum conditions, the change of the intensity (�I) of the RLS spectra at � = 392 nmwas linearly proportional to the concentration of BSA and HSA. The linear range was 1–100 ng mL−1

for HSA and 1–120 ng mL−1 for BSA, and the limit of detection (LOD) was 0.4 ng mL−1 for HSA and0.5 ng mL−1 for BSA. This method proved to be very sensitive, rapid, simple and tolerant of most interfering

substances.

. Introduction

Quantitative determination of proteins is very important in lifeciences, clinical medicine and biochemistry. Several assays forroteins have been reported. The simplest photometric method ishe absorption measurement at 280 nm. However, the selectivitys not satisfactory. Thus, other methods with greater simplic-ty and lower detection limit have been developed, such as theowry [1], Bromocresol Green (BCG) [2] and Coomassie Brilliantlue (CBB) [3,4], but these suffer from limited sensitivity andften tedious procedures. To overcome these limitations, the flu-rimetric methods using covalent and noncovalent probes haveeen widely used for the determination of proteins, becausehey possess high sensitivity and selectivity [5,6]. In addition,hemiluminescence [7] and electrochemical [8] methods are alsomployed to determine proteins successfully. These methodsreatly promoted the trace protein separation and purification,ut the attendant problem is that the majority of the sensi-ive analysis method is very difficult to adapt for trace proteinetermination.

In 1993, since its first introduction to the quantitative deter-ination of biomacromolecules [9], resonance light-scattering

RLS) technique has gradually gained regards from analyticalhemists [10]. It is characterized by high sensitivity, convenience

∗ Corresponding author. Tel.: +86 027 62767591.E-mail address: zhan-g-q@163.com (G. Zhan).

386-1425/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.saa.2010.02.005

© 2010 Elsevier B.V. All rights reserved.

in performance and simplicity in apparatus (usually common spec-trofluorometer). Recent studies have shown RLS is a valuabletechnique for quantification of proteins since the enhanced RLSsignals can be easily measured by using a common spectrofluorom-eter for aggregated species or large particles in nanometer scalenear UV absorption bands. Generally, these quantification meth-ods are based on the bindings of biological dyes [11], artificialsynthetic prophyins [12], and surfactants [13] to proteins and onthe reaction of dye–metal ion complexes with protein [14] owingto the strongly enhanced RLS signals of the bindings. Comparedto traditional organic fluorescent probe, it has many advantages,such as optical stability, high sensitivity and so on. In recent years,nanoparticles used as probes for protein determination by RLS haveattracted great interests because of their excellent optical prop-erties and chemical stability. For example, highly sensitive andselective detection of standard proteins and the study of their func-tionality have been achieved by attachment of gold nanoparticlesfollowed by silver enhancement and RLS detection [15]. In anotherstudy, gold particles were used for sensitive detection of DNAhybridization on cDNA microarrays by RLS detection [16]. Recently,it has been reported that CdS-PAA [17] and PbS-PAA [18] were usedfor the determination of protein with resonance light scatteringmethod. Herein, we prepare functionalized ZnS-PAA nanoparticles

using thioacelamide (TAA) instead of Na2S. It was found that theZnS-PAA nanoparticles are more homogeneously than CdS-PAA andPbS-PAA nanoparticles, and when used as a new biological probe forthe detection of protein, it has lower LOD and relatively higher con-centration of coexisting substances, compared to the methods of

1 Acta Part A 75 (2010) 1497–1500

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Fig. 1. RLS spectra of 0.1 �g mL−1 BSA (1), ZnS-PAA nanoparticles (2), ZnS-PAA

Under the same conditions, this phenomenon can also be observedin the interaction of ZnS-PAA nanoparticles and HSA. Based on thisphenomenon, a new method for the determination of BSA and HSAcould be established.

498 L. Peng et al. / Spectrochimica

dS-PAA and PbS-PAA. Therefore, the method may have its practicalse.

. Experimental

.1. Apparatus and reagents

Resonance light scattering spectra were recorded by a LS-55 flu-rescent/luminescent spectrometer (PE Corporation, USA). In allxperiments, a 1-cm path-length quartz cuvette was used to mea-ure the resonance light scattering spectra. All pH values wereeasured with pHS-3 meter (Shanghai, China). Transmission elec-

ron microscopy (TEM) images of the ZnS-PAA nanoparticles beforend after interaction with BSA were acquired on a Tecnai G2 20-EM (FEI Company, Netherlands). All optical measurements werearried out at room temperature under ambient conditions.

Bovine serum albumin and human serum albumin were pur-hased from Shanghai Bio Life Science & Technology Co. Ltd., andere used without further purification. Solutions of 1 mg mL−1

erum albumin were prepared by adding 0.1000 g BSA (or HSA)nto 100 mL volumetric flask, and diluted to the mark withouble-distilled water, then stored in a refrigerator. The storedolution was diluted to 10 �g mL−1 with double-distilled water.ritton–Robinson (B–R) buffer solution was used to control thecidity of the tested solutions. Other chemicals were of analyti-al grade. All solutions were prepared with double-distilled water.uman serum samples were kindly donated by the People’s Hos-ital of Hubei Province.

.2. Experimental methods

.2.1. Preparation of ZnS-PAA nanoparticlesDouble-distilled water (193 mL) was purged with N2 in a 250 mL

hree-necked round bottomed flask with a gas inlet system. 1.0 mLf 0.1 mol L−1 Zn(Ac)2 were added to the flask under vigorous stir-ing. Then 1.0 mL of 0.1 mol L−1 thioacelamide (TAA) were droppednto the flask in 1 h to obtain ZnS nanoparticles. The as-prepared ZnSanoparticles were used as core to prepare ZnS-PAA nanoparticlessing in situ polymerization method with 3.3 mL of 0.01 g mL−1 KPSnd 2.0 mL acrylic acid under vigorous stirring for 3 h. The obtainednS-PAA nanoparticles were stored in room temperature and didot show any evidence of precipitation for several weeks.

.2.2. MeasurementsIn a 10 mL colorimetric tube, 1.0 mL of Na2HPO4–citric acid

uffer solution (pH 3.80), 3.0 mL of ZnS-PAA nanoparticles colloidalolutions and desired amounts of serum albumin solution weredded, and diluted with double-distilled water to the mark andixed thoroughly with gentle shake. With another incubation time

f 20 min, the RLS intensities were measured with a LS-55 fluores-ent/luminescent spectrometer. The RLS intensity for the mixturef ZnS-PAA and serum albumins (IRLS) and for the ZnS-PAA (I0

RLS)ere measured, the enhancement of RLS intensities is expressed

s �I = IRLS − I0RLS.

. Results and discussion

.1. Investigation of the interaction between ZnS-PAAanoparticles and serum albumin

As shown in Fig. 1, RLS spectra of ZnS-PAA nanoparticles andSA solution was investigated with synchronous scanning in theavelength range from 250 nm to 550 nm. It can be seen that ZnS-

AA nanoparticles has a RLS peak at about 392 nm. When BSA wasdded to this system, the shape of the RLS spectra was similar

composite nanoparticless + 0.01 �g mL−1 BSA (3), ZnS-PAA composite nanoparti-cles + 0.05 �g mL−1BSA (4) and ZnS-PAA composite nanoparticles + 0.12 �g mL−1

BSA (5). ZnS–PAA nanoparticles 1.5 × 104 mol L−1; pH 3.8.

to the RLS spectra of ZnS-PAA nanoparticles with no change ofthe maximal wavelength, but the RLS spectra intensity increasedsignificantly, which indicated that ZnS-PAA nanoparticles haveinteracted with BSA resulting in the formation of ZnS-PAA–BSAcomplex. As reported by Jiang et al. [19], the strongest reso-nance scattering peak of ZnS-PAA–BSA system is located at 392 nm,corresponding to the absorption valley of curve 1 caused byinelastic absorption shown in Fig. 2, which shows the absorptionspectra of ZnS nanoparticles in the presence of various con-centrations of BSA. It can be seen that the absorbance of ZnSnanoparticles shows a slight increase with increasing amounts ofBSA.

A comparison of Figs. 1 and 2 reveals that the RLS peak lieson the red side of the absorption peak of ZnS-PAA–BSA. Accord-ing to the light scattering theory [20–22], this phenomenon can beexplained by an RLS peak resulting from the absorption of ZnS-PAA.At � = 392 nm, the enhancement of RLS spectra intensity of ZnS-PAAnanoparticles was represented as �I = IRLS − I0

RLS, where I0RLS and

IRLS are the intensities of ZnS-PAA nanoparticles with and withoutBSA respectively. The change of the RLS spectra intensity (�I) of theRLS spectra gave a linear relationship with the concentration of BSA.

Fig. 2. Absorption spectra of BSA (1) 10 �g mL−1, and ZnS-PAA composite nanopar-ticles in the presence of BSA (2) 0.0 �g mL−1, (3) 0.05 �g mL−1, (4) 0.1 �g mL−1.ZnS–PAA nanoparticles 1.5 × 104 mol L−1; pH 3.8.

L. Peng et al. / Spectrochimica Acta Part A 75 (2010) 1497–1500 1499

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Fig. 3. TEM image of ZnS-PAA composite nanoparticles before (a) and after (b)

.2. TEM image

TEM was used to investigate the interaction between ZnS-AA nanoparticles and BSA. Typical micrographs of the ZnS-PAAanoparticles without (part a) and with (part b) 0.1 �g mL−1 BSAre shown in Fig. 3. The TEM images show that the size of ZnS-AA–BSA complex was slightly larger than ZnS-PAA nanoparticles,nd the shape of ZnS-PAA–BSA complex were not as round asnS-PAA nanoparticles. It was presumed that ZnS-PAA nanopar-icles have interacted with BSA and the interaction resulted in thenhancement of the RLS spectra.

.3. Optimization of the experimental conditions

.3.1. Effect of pHDifferent buffers system such as B–R, potassium hydrogen

hthalate–hydrochloric acid, trisodium citrate–hydrochloric acidnd Na2HPO4–citric acid have been used to investigate theirnfluence on the RLS intensity of ZnS-PAA–BSA solution. A goodensitivity can be obtained in a Na HPO –citric acid buffer solu-

2 4ion. In addition, it was found that the acidity of the solution greatlympacted on the interaction between ZnS-PAA nanoparticles andSA. As shown in Fig. 4, a maximal change of the RLS spectra inten-ity can be obtained at about pH 3.80.

ig. 4. Effect of pH on the RLS intensity. CBSA = 0.1 �g mL−1; ZnS–PAA nanoparticles.5 × 104 mol L−1.

ction with 0.1 �g mL−1 BSA. ZnS–PAA nanoparticles 1.5 × 104 mol L−1; pH 3.8.

3.3.2. Effect of the ZnS-PAA nanoparticles concentrationIt is well known that the number of the nanoparticles per unit

volume is associated with the aggregation degree. The RLS inten-sity is dependent on the aggregation number of nanoparticles. Theeffect of ZnS-PAA nanoparticles concentration on the RLS intensityis shown in Fig. 5. It was found that the appropriate concentrationof ZnS-PAA nanoparticles was 1.5 × 10−4 mol L−1. At lower ZnS-PAA nanoparticles concentration, the RLS intensity and sensitivitydecrease with decreasing concentration of ZnS-PAA nanoparticlespossibly due to the incomplete interaction of ZnS-PAA compos-ite nanoparticles with protein generating a lower aggregationnumber. However, the RLS intensities declined gradually whenZnS-PAA nanoparticles concentration was >1.5 × 10−4 mol L−1.Higher concentration of ZnS-PAA nanoparticles means fewer serumalbumin molecules adsorbed on ZnS-PAA nanoparticles, reduc-ing the aggregation number of serum albumin with ZnS-PAAnanoparticles per unit volume, resulting in a decrease of RLSintensity.

3.3.3. Effect of the added sequence of reagents

Effect of the mixing sequence of ZnS-PAA nanoparticles, BSA

solution and buffers on RLS intensity was investigated. ThoughZnS-PAA nanoparticles interacted with serum albumin rapidly atroom temperature, the mixing sequence was also very important.

Fig. 5. Effect of the concentration of ZnS-PAA composite nanoparticles on the RLSintensity. CBSA = 0.1 �g mL−1; pH 3.8.

1500 L. Peng et al. / Spectrochimica Acta Part A 75 (2010) 1497–1500

Table 1Linear calibration equation and detection limit for serum albuminsa.

Serum albumin Linear range (ng mL−1) Linear regression equation (C, ng mL−1) R Detection limit (ng mL−1)

BSA 1–120 �IRLS = 3.1323 + 0.8224C 0.998 0.5HSA 1–100 �IRLS = 2.1486 + 0.4898C 0.999 0.4

a ZnS–PAA nanoparticles 1.5 × 104 mol L−1; pH 3.8.

Table 2Tests for the interference of coexisting substancesa.

Coexisting substance Coexisting concentration(�g mL−1)

Change ofRLS (%)

l-Arginine 300 10Methonine 100 5.2Proline 100 3.2Serine 100 5.3Valine 100 5.3Glucose 100 8.6Co2+ 5.0 2.1Cu2+ 10 8.4Ca2+ 10 9.6Mn2+ 10 9.4Pb2+ 20 2.6

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Table 3Results for the determination of HSA samplesa.

Sample no. Content of protein(this method/g L−1, n = 5)

Recovery(%, n = 5)

R.S.D. (%)

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a BSA 0.1 �g mL−1; ZnS-PAA nanoparticles 1.5 × 10−4 mol L−1; pH 3.80.

t was found that the favorable formation of ZnS-PAA–BSA complexas accomplished with the mixing sequence of ZnS-PAA compositeanoparticles, followed by buffers and BSA solution. It is necessary

or the interaction of ZnS-PAA nanoparticles with protein to con-rol the optimum pH, so the buffer should be mixed with ZnS-PAAanoparticles before adding protein.

.3.4. Effect of the reaction timeEffect of the reaction time was also investigated. It found that the

aximum resonance light-scattering intensity was reached whenhe incubation time of the mixed solutions was 20 min. The reso-ance light-scattering intensity remained unchanged for more thanh. Thus, a reaction time of 20 min was adopted in this assay.

.3.5. Calibration curvesUnder the optimal experimental conditions, the �I of the ZnS-

AA–serum albumin was measured. A good linear relationshipetween �I and the concentration of serum albumin was obtained.he correlation coefficient, linear regression equation, linear rangend detection limit are given in Table 1.

.3.6. Effect of coexisting substancesThe influence of various ions, amino acids and other compounds

f interest were studied. The solutions of a fixed concentrationf 0.1 �g mL−1 BSA and each foreign substances with variousoncentrations are mixed prior to the detection. The results areummarized in Table 2. It can be seen from this table that most ofhe substances tested scarcely interfered with the determination.herefore, the method may possess practical applications.

.3.7. Analytical applicationA sample of 50 �L HSA was added into a 50 mL volumetric flask,

iluted to the mark with double-distilled water and then stored inrefrigerator. A 1.0 mL portion of the stored solution was diluted

o 10 mL with double-distilled water. According to the procedureescribed above, HSA in human serum samples were determinedy the present method, the obtained results were shown in Table 3.

t demonstrated that the proposed method is a simple and sensitiveor the detection of serum albumin.

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a ZnS–PAA nanoparticles 1.5 × 104 mol L−1; pH 3.8.

4. Conclusions

A method for the serum albumin determination based on thesignificant enhancement of RLS signals due to the interaction ofZnS-PAA nanoparticles with BSA and HSA was developed. Underthe optimal conditions, the interaction of ZnS-PAA nanoparti-cles with BSA shows much stronger RLS intensity in contrastwith the weak RLS signal of ZnS-PAA composite nanoparticles orBSA alone. The enhanced RLS intensity implies the formation ofZnS-PAA–BSA complex. Considering the results, a highly sensitiveZnS-PAA composite nanoparticles based assay for the serum albu-min determination with RLS technique was proposed. The methodwas applied to the determination of HSA in human serum samplessatisfactorily. Thus, the proposed assay with RLS technique maybe promising for the serum albumin determination in biologicalsamples.

Acknowledgements

The authors acknowledge the financial support from the Nat-ural Science Foundation of Hubei Province (No. 2007ABA127),Natural Science Foundation of South-Central University for Nation-alities (YZY06011) and South-Central University for Nationalities(XTZ09005).

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