resonance light scattering spectrometric study of poly diallyldimethylammonium...

Research Article

Received: 26 February 2008, Revised: 15 August 2008, Accepted: 22 August 2008, Published online 3 April 2009 in Wiley Interscience

(www.interscience.wiley.com) DOI 10.1002/bio.1087

Luminescence 2009; 24: 155–161 Copyright © 2009 John Wiley & Sons, Ltd.

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John Wiley & Sons, Ltd.Resonance light scattering spectrometric study of poly diallyldimethylammonium chloride–antibody–antigen systemJuan Li, Xiaozhou Li, Jieli Tang, Jing Kang and Yihua Zhang*

ABSTRACT: The interaction of antigen (Ag) and antibody (Ab) with poly diallyldimethylammonium chloride (PDDA) in aqueoussolutions has been studied by optical absorption and resonance light-scattering (RLS) spectroscopies. The formation of thethree-component-complex is due to aggregates of Ab or Ag with PDDA by electrostatic interaction and aggregates of Ab withAg by immunoreaction. The influences of some experimental factors, including incubation time, pH value, concentration of PDDAand concentration of Ab, on the aggregation process have also been studied. A linear relationship between the concentrationof Ag and the RLS intensity was found. Under the optimal conditions, for a given concentration of Ab (4.6 mg/mL), theenhancement of RLS intensity is in proportion to the concentration of Ag in the range 0.03–0.83 mg/mL. The RLS could, incombination with immunoassay, be a rapid and sensitive detection method for Ag. Copyright © 2009 John Wiley & Sons, Ltd.

Keywords: resonance light-scattering (RLS); immunoreaction; poly (diallyldimethylammonium chloride) (PDDA); antibody (Ab);antigen (Ag)

Introduction

Since determination of proteins plays a critical role in the field ofchemistry, biochemistry, clinical medicine and immunodiagnostics,there have been numerous reports of new techniques for detectionof proteins. Conventional methods for detection of proteins suchas Biuret (1), Lowry (2) and Bradford (3), are time-consumingand cumbersome.

Resonance light scattering (RLS), an elastic scattering, occurswhen the incident beam is close to an absorption band. Dependingon the size of the photon-interacted particles (d) and thewavelength of incident light beam (λ0), light scattering can beclassified as Mie (d >> λ0), Tyndall (d ≈ λ0) or Rayleigh (d ≤ 0.05λ0)scattering. In terms of the wavelength, differences in the incidentand the scattered beams, and light scattering can also be classifiedas elastic scattering, inelastic scattering and quasi-elastic scattering(4). For particles with size less than 20 × the wavelength of incidentbeams (20d ≤ λ0), their scattered light can be described asRayleigh scattering. When Rayleigh scattering occurs within theabsorption band of the molecule, Rayleigh scattering will absorbthe light energy and bring about an absorption-rescatteringprocess. This is called the resonance enhancement effect ofRayleigh scattering. This process can greatly enhance the scatteringintensity. Therefore RLS is a sensitive technique for monitoringmolecular assemblies. Pasternack and co-workers first establishedthe RLS technique to study the biological macromolecules bymeans of an ordinary fluorescence spectrometer (5–7). RLS studieshave attracted great interest among researchers (8–11). RLS hasemerged as a very attractive technique to monitor molecularassemblies and characterize the extended aggregates of chro-mophores (5–9). In recent years, RLS has been used to determinepharmaceutical (12, 13) and various biological macromolecules,such as proteins (14–21), amino acids (22), nucleic acids (23–32),glucose (33) and metal ions (34). However, the disadvantage ofthe RLS method is its low selectivity.

Immunoassay is of great importance in a number of areas. Thecommon immunoassays are enzyme-linked immunosorbent assay(ELISA), radioimmunoassay (RIA) and electrophoretic immunoassay,which have good sensitivity and specificity, but need labeledreagents and multi-step operations (35).

Poly(diallyldimethylammonium chloride) (PDDA) (Fig. 1), is acationic polyelectrolyte that is not sensitive to pH change. It hasa line-type framework and cationic character (36, 37).

Herein, we assessed the possibility of coupling the RLS methodwith immunoassay to establish a rapid and sensitive method todetect Ag. The reason for the enhancement of RLS intensity is theaggregation of Ab or Ag with PDDA by electrostatic interactionand aggregation of Ab with Ag by immunoreaction. The formationof the three-component-complex is the main reason for theenhancement of RLS intensity.

Experimental

Chemicals

Rabbit anti-goat IgG (Ab), goat anti-human IgG (Ag), humanserum albumin (HSA) and bovine serum albumin (BSA) wereobtained from Beijing Dingguo Biotechnology Co. Ltd. Before theexperiment, the Ab needed further purification. PDDA (averageMw 100 000–200 000, 20 wt% in water) was obtained from Aldrichin United States. It was diluted to 5.0 × 10−4 mol/L (the Mw isconsidered to be 1.00 × 105 g/mol in the study) as stock solution

* Correspondence to: Yihua Zhang, College of Chemistry, Jilin University, Chang-chun 130012, People’s Republic of China. E-mail: [email protected]

College of Chemistry, Jilin University, Changchun 130012, People’s Republicof China

J. Li et al.

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using deionized water. The Ag was also prepared by directlydiluting in double-deionized water. Experiments were made inphosphate buffer.

Apparatus

The absorption spectra were recorded on a Cintra 10e UV–visiblespectrometer. The light scattering measurements were made onan RF-5301PC spectrofluorometer. The scattered light intensitywas monitored using right-angle geometry. The pH values weremeasured using a Delta 320 pH-meter.

Procedures

Aliquots of 30 μL of PDDA, 1 mL PBS (pH 6.6) solution, 20 μL givenconcentration (700 μg/mL) of Ab and an appropriate amount ofAg were added into a 5 mL vial, then diluted with deionizedwater to 3 mL and mixed thoroughly with gentle shaking. Thevial was incubated for 30 min at 37°C. The RLS spectrum wasobtained by scanning simultaneously the excitation and emissionmonochromator (namely, Δλ = 0 nm) with the wavelength rangeof 220–750 nm. The spectral bandwidths of the excitation andemission monochromators were both kept at 5.0 nm. The RLSintensity was measured at 300 nm. The enhanced RLS intensity(ΔIRLS) of the PDDA–Ab–Ag system is represented as

ΔIRLS = IRLS − I0RLS

Here IRLS is the RLS intensity of the system PDDA–Ab–Ag, and I0RLS

is the RLS intensity of PDDA–Ab.

Results and discussion

Spectral characteristics

The RLS spectra and absorption spectra of the Ab–Ag system areshown in Fig. 2a. It can be seen that the RLS intensity of Ab andAg are very weak in the whole scanning wavelength region. Incontrast, with the addition of trace amount of Ag to Ab solution,a remarkably enhanced RLS with a maximum peak at 306 nmwas observed under the same circumstances. The formation ofAb–Ag complex is the main reason for the enhancement of RLSintensity. Meanwhile from the absorption spectra of Ab and Ag,it can be seen that the absorption band of molecule can bringabout the resonance enhancement effect of Rayleigh scattering.This process can much enhance the scattering intensity.

As shown in Fig. 2b, when the RLS intensities of Ag, Ab andPDDA were considered alone, they were weak. When Ab or Agwas mixed with PDDA, the RLS intensity of the solution wasenhanced. When both Ab and Ag were added into the solution

containing PDDA, the RLS intensity of the solution was furtherenhanced. The reason for the enhancement of RLS intensity isthe aggregation of Ab or Ag with PDDA by electrostatic interactionand aggregates of Ab with Ag by immunoreaction. Thus, it can beconcluded that Ag reacted with Ab and PDDA and produced anew compound whose RLS intensity was much higher than thatof Ag, Ab or PDDA when they existed separately. Moreover, thedimension of the formazan particles may be much less than theincident wavelength, and thus the enhanced RLS signal occurs underthe given conditions. Compared with the system only containingAb–Ag, the sensitivity was further enhanced. The combinationbetween PDDA, Ab and Ag can be shown as Scheme 1.

Scheme 1. The combinative mode of the PDDA-Ab-Ag system.

Figure 1. The structure of PDDA.

Figure 2. (a) The RLS spectra of Ag (curve 1), Ab (curve 2) and Ab–Ag (curve 3).The concentrations of Ab and Ag are 9.3 and 1.3 μg/mL, respectively. The absorp-tion spectra of Ab (curve 4) and Ag (curve 5). (b) The RLS spectra of PDDA (curve 1),Ab (curve 2), Ag (curve 3), PDDA–Ab (curve 4), PDDA–Ag (curve 5), Ab–Ag (curve 6)and PDDA–Ab–Ag (curve 7). Concentrations: PDDA, 5 × 10−6 mol/L; Ab, 1.6 μg/mL;Ag, 0.75 μg/mL.

RLS spectrometric study of PDDA–Ab–Ag system

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Optimization of the general procedures

As described above, the RLS signal was enhanced by the formationof a three-component complex. Thus, factors displaying effectson the formation of the three-component complex should beconsidered.

Effect of incubation time

The effect of incubation time on RLS intensity was investigatedin a period of 120 min immediately after mixing PDDA, Ab andAg in phosphate buffer at pH 6.6. As shown in Fig. 3, the RLS

intensity of the system reaches a plateau in 30 min and is stablefor at least 2 h. As stated above, the stability of the RLS intensityis practical for the determination of Ag.

Effect of pH values

The pH of solution plays an important role in the aggregation ofPDDA, Ab and Ag. Thus, the optimization of solution pH is studiedover the pH range from 5.6 to 8.2. Figure 4 shows that the RLSintensity of the system greatly depends on the pH values of thesolution and reaches a maximum at pH 6.6. In solution PDDAtakes a positive charge. It can be seen that, when the pH value is

Figure 3. The effect of incubation time on IRLS.

Figure 4. The effect of pH on IRLS intensity. The concentrations of PDDA, Ab and Ag are 5.0 × 10−6 mol/L,1.6 μg/mL and 0.75 μg/mL, respectively.

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lower than 6.6, the ΔIRLS is very low. This is because that the pHvalue is near the isoelectric point of Ab and Ag, Ab or Ag maytake a less negative charge. As the pH increases, Ab or Ag maytake a more negative charge, and the electrostatic interactionbetween PDDA and Ab or Ag is stronger. Therefore, in the study,pH 6.6 is selected.

Effect of PDDA concentration

The effect of PDDA concentration was investigated and is shownin Fig. 5. The RLS intensity of the PDDA–Ab and PDDA–Ab–Ag

systems increases along with the increase in PDDA concentration.However, when the concentration of PDDA is higher than5.0 × 10−6 mol/L, the ΔIRLS of the system decreases. At the con-centration of 5.0 × 10−6 mol/L of PDDA, the system yields thehighest sensitivity. A concentration of 5.0 × 10−6 mol/L is suitable.

Effect of the concentration of Ab

Another important parameter in this method is the concentrationof Ab. The optimization curve for Ab concentration is shown inFig. 6. The effect of the concentration of Ab (from 0.6 to 19 μg/mL)

Figure 5. The effect of PDDA concentration on IRLS. The concentrations of Ab and Ag are 1.6 μg/mL and0.75 μg/mL, respectively.

Figure 6. The concentration of Ab effects on IRLS. (a) Ab–Ag system; (b) PDDA–Ab–Ag system. The concentrationof Ag is 1.3 μg/mL.



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for Ab–Ag and PDDA–Ab–Ag systems was investigated. As shownin Fig. 6, the RLS intensity of the system increases along with theincrease of Ab concentration. For the Ab–Ag system, when theconcentration of Ab is low, the enhancement of the RLS is notobvious and the sensitivity is poor. However, when the concentrationof Ab is increased to 4.6 μg/mL, compared with the Ab–Ag system,the enhancement of the RLS of the PDDA–Ab–Ag system isobvious and the sensitivity is much enhanced. So in this study,the Ab concentration of 4.6 μg/mL is chosen to detect Ag.

Effect of coexisting substances

The experiments on the effect of coexisting substances werecarried out and the results are shown in Table 1. The results

indicate that the method is free from interference from mostcommon metal ions and glucose.

Calibration curves

It can be see from Fig. 7 that the RLS intensity of the PDDA andAg system changes little with the concentration of Ag in therange 0.0025–1 μg/mL. This indicates that PDDA alone cannotenhance the RLS intensity of the Ag. However, when the Ab isadded into the system, the PDDA–Ab–Ag complex is formed byelectrostatic interaction and immunoreaction. The ΔIRLS value isproportional to the concentration of Ag. Under the optimalcondition, RLS intensities are measured. As shown in Fig. 8, theincrease in concentration for Ag in the PDDA and Ab solution

Table 1. Influence of foreign substances

Foreign substances Concentration coexisting (μg/mL) Relative error (%)

Cu2+, Cl− 5 −3.9%Ca2+, Cl− 12.5 −2.9%Fe2+, Cl− 5 −12.3%Mg2+, Cl− 12.5 −2.01%Zn2+, Cl− 5 −4.3%Al3+, SO4

2− 5 3.9%Mn2+, SO4

2− 12.5 2.3%Cr3+, NO3

− 12.5 −4.8%Na+, Cl− 12.5 3.7%K+, Cl− 17.5 4.9%Glucose 17.5 2.3%BSA 12.5 3.6%HAS 12.5 6.1%

The concentration of the Ag is 0.25 μg/mL.

Figure 7. The RLS intensity of PDDA and different concentration of Ag.

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led to the gradual enhancement in RLS intensity, exhibiting aconcentration-dependent relationship. The production of RLSand its intensity correlate with the formation of the aggregateand its particle dimension in solution. When the RLS intensityreaches a maximum, it may decrease with further addition of Agbecause of the dissolution of immunocomplex. It can be seenthat, for a given concentration of Ab (4.6 μg/mL), the ΔIRLS valueis directly proportional to the concentration of Ag in the range0.03–0.83 μg/mL. The calibration equations were represented as

ΔIRLS = −4.71 + 241.558C, r = 0.994 (C, μg/mL)

ConclusionThe technique of RLS is noted for its high sensitivity. Therefore,in recent years, it has enjoyed widespread use in the quantitativeanalysis of protein. However, this technique has suffered fromdrawbacks such as the interference of other components. Inthis paper, a novel assay of Ag based on the measurement ofenhanced RLS signals resulting from the electrostatic interactionand immunoreaction of the PDDA–Ab–Ag system is proposed.This assay coupled the high sensitivity of the RLS technique andthe high specificity of the immunoreaction. Compared with thetraditional assay, this new assay is much simpler and easier tooperate. Comparison of the system of PDDA–Ab–Ag and Ab–Agis also studied. The results indicate that the system with PDDA–Ab–Ag was more suitable for detection of Ag.

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