Spectrochimica Acta Part A 60 (2004) 747–750
Quantitative determination of proteins at nanogram levels by the resonance light-scattering technique with macromolecules nanoparticles of PS–AA Leyu Wang, Hongqi Chen, Ling Li, Tingting Xia, Ling Dong, Lun Wang∗ College of Chemistry and Materials Science, Anhui Normal University, Wuhu 241000, PR China Received 6 April 2003; accepted 18 June 2003
Abstract The polystyrene–acrylic acid (PS–AA) nanoparticles have been prepared by ultrasonic polymerization, characterized by FT–IR and TEM. It is the first report on the determination of proteins with macromolecules nanoparticles of PS–AA by resonance light-scattering (RLS). At pH 6.9, the RLS of macromolecules nanoparticles of PS–AA can be enhanced by proteins. Based on this, a novel quantitative assay of proteins at the nanogram levels has been proposed. At pH 6.9, the RLS signals of PS–AA were greatly enhanced by proteins in the region of 250–700 nm characterized by the peak at 342 nm. Under optimal conditions, the linear ranges of the calibration curves were 0.02–11.0 g ml−1 , 0.04–10.0 g ml−1 and 0.03–10.0 g ml−1 for ␥-globulin (␥-IgG), bovine serum albumin (BSA) and human serum albumin (HSA), respectively. The detection limits were 16.0 ng ml−1 , 19.0 ng ml−1 , and 15.0 ng ml−1 for ␥-IgG, BSA and HSA, respectively. The method has been applied to the analysis of total proteins in human serum samples collected from the hospital and the results were in good agreement with those reported by the hospital, which indicates that the method presented here is not only sensitive, simple, but also reliable and suitable for practical application. © 2003 Elsevier B.V. All rights reserved. Keywords: Resonance light scattering; Proteins; Macromolecules nanoparticles; PS–AA
1. Introduction Since its first introduction to the quantitative determination of biomacromolecules in 1996 [1,2], resonance light-scattering (RLS) technique has gradually gained regards from analytical chemists [3,4]. It is characterized by high sensitivity, convenience in performance and simplicity in apparatus (usually common spectrofluorometer). By using RLS spectroscopy, organic dye probes used in spectrophotometric assays for protein and nucleic acids can become much more sensitive. For instance, the dynamic range for bovine serum albumin of the RLS method with bromophenol blue was reported to be 0.34–18.7 mg l−1 while the spectrophotometric procedure can be used just for the protein concentrations greater than 10 mg l−1 [2]. It is also true for ethyl violet in the determination of DNA ∗ Corresponding author. Tel.: +86-553-3869303; fax: +86-553-3869303. E-mail address:
[email protected] (L. Wang).
1386-1425/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/S1386-1425(03)00285-3
[5]. Recent studies have shown resonance light scattering (RLS) is a valuable technique for quantification of proteins since the enhanced RLS signals can be easily measured by using a common spectrofluorometer for aggregated species or large particles in nanometer scale near UV absorption bands [6]. Generally, these quantification methods are based on the bindings of biological dyes [7,8], artificial synthetic prophyins [9], and surfactants [10] to proteins and on the reaction of dye–metal ion complexes with protein [11] owing to the strongly enhanced RLS signals of the bindings. In this paper, we prepared the functionalized macromolecules nanoparticles of PS–AA. They are highly resistant to photobleaching and show bright and steady resonance light scattering. The IRLS of PS–AA can be enhanced by ␥-globulin, and the extent of the resonance light scattering intensity enhancement is proportional to the concentration of proteins. So, a new determination method with high sensitivity for proteins in blood serum samples has been developed. Compared with the former RLS technique, the present method have many advantages, such as high sensitivity
748
L. Wang et al. / Spectrochimica Acta Part A 60 (2004) 747–750
(nanogram levels of detect limits), simplicity of operation (one-step assay and without effect of ion strength), commonality of spectrofluorometer and reagents, high stability of macromolecules nanoparticles of PS–AA, and reproducibility.
2. Experimental 2.1. Apparatus The spectrum and the intensity of RLS were obtained with a Hitachi F-4500 fluorescence spectrometer (Tokyo, Japan) with a plotter unit and a quartz cell. The excitation and emission bands widths were 2.5 nm. A VCX500 (Sonic, USA) ultrasonic processor was used in the ultrasonic polymerization of nanoparticles. The pH measurements were made with a model pHS-3C pH-meter (Dazhong Analytical Instruments Factory, Shanghai, China). Transmission electron microscopy (TEM) images of the nanoparticles were acquired on a Hitachi H-600 (Tokyo, Japan) transmission electron microscope. Colloidal solutions of the nanoparticles in water were dropped on to 50 Å thick carbon coated copper grids with the excess solution being immediately wicked away. 2.2. Reagents Styrene, acrylic acid and potassium persulfate (KSP) were acquired from Acros. Stock solutions of proteins were prepared by dissolving commercial human serum albumin (HSA, Sigma), bovine serum albumin (BSA, Sigma) and ␥-globulin (␥-IgG, Sigma) in deionized water. All working concentrations of proteins were 100 g ml−1 . All reagents were of analytical grade or the best grade commercially available. Human serum samples were obtained from the Second Renming Hospital of Wuhu. The serum samples were diluted 1000-fold with doubly deionized water to prepare stock solutions and stored at 0–4 ◦ C. Water used throughout was doubly distilled.
The PS–AA nanoparticles was used to detect the proteins via changes in the relative RLS intensity of the system. To a 10 ml volumetric flask, 1.0 ml of phosphate buffer solution, a certain volume of colloids, an appropriate volume of sample or protein working solution were added, the mixture was diluted to 10.0 ml with water and stirred thoroughly. The RLS spectra were obtained with the excitation and emission synchronously through the wavelength range of 250–700 nm. Based on these spectra, the intensity of RLS was measured with the excitation and emission wavelength around 342 nm.
3. Results and discussion 3.1. TEM images of nanoparticles Fig. 1 shows the TEM image of PS–AA nanoparticles. The diameter of the PS–AA nanoparticles is about 35 nm. In addition, the TEM image of PS–AA is also shown in Fig. 1(A) when ␥-globulin has been added into PS–AA colloidal solution. The IR spectrum, has a peak at ν(C==O) = 1718 cm−1 , characteristic of the acrylic acid capped onto the outer surface of the nanoparticles. 3.2. Spectral characteristics and reaction The reaction between PS–AA and proteins at 30 ◦ C occurs rapidly within 20 min. The scattering intensity is stable for at least 3.0 h. The addition sequence of reagents does not affect the intensity of RLS. Fig. 2 is the light-scattering spectra of PS–AA–␥-globulin binding system. It can be seen that the scattering signal of PS–AA in wavelength region of 250–700 nm is weak and has a characteristic peak at 338 nm. However, the scattering signal of PS–AA has been strongly enhanced by ␥-globulin. The maximum scattering wavelength shifts red about 4 nm after mixing PS–AA with ␥-globulin. The results suggest that there are some interaction between PS–AA and ␥-globulin.
2.3. Procedures The PS–AA colloids were prepared as follows. Into a 1 l three-necked round-bottomed flask, 400 ml of deionized water and 400 ml of acetone were added and stirred. Under vigorous stirring and ultrasonic radiation, 1.2 ml of styrene, 0.9 ml acrylic acid and 0.004 g KSP were added into the flask. The polymerization reaction has taken place for 2 h at 60 ◦ C in a high-purity nitron atmosphere. And then, the PS–AA nanoparticles with carboxyl on the surface were acquired. The acetone was removed by rotary evaporation. The polar carboxylic acid group renders the nanoparticles water-soluble. The free carboxyl group is also available for coupling to various biomolecules, such as proteins and peptides. Excess styrene and acrylic acid were removed by repeated centrifugation [12,13].
Fig. 1. TEM images of PS–AA nanoparticles in the: (A) presence and (B) absence of ␥-globulin.
L. Wang et al. / Spectrochimica Acta Part A 60 (2004) 747–750
Fig. 2. Light-scattering spectra of nano-PS–AA–␥-globulin binding system; concentration of ␥-globulin (g ml−1 ): (1) 0.0, (2) 0.3, (3) 1.0, (4) 1.5, (5) 1.8, (6) 2.0, (7) 3.0, (8) 4.0, (9) 5.0; PS–AA nanoparticles, 6.0 × 10−3 mol l−1 ; pH, 6.9.
749
Fig. 5. Effect of NaCl content on RLS intensity: ␥-IgG, 1.0 g ml−1 ; PS–AA nanoparticles, 6.0 × 10−3 mol l−1 ; pH, 6.9.
(represented by the concentration of styrene existing in single molecules) and reached a maximum in the range (5.0–10.0) × 10−3 mol l−1 . Hence, in this work the concentration of PS–AA was chosen as 5.0 × 10−3 mol l−1 . The reaction between PS–AA and proteins at 30 ◦ C occurs rapidly within 20 min. The scattering intensity is stable for at least 3.0 h. So, a 20 min incubation time was adopted in the study. Fig. 3. Effect of pH on the IRLS : ␥-IgG, 1.0 g ml−1 ; PS–AA nanoparticles, 6.0 × 10−3 mol l−1 ; I, in the presence of ␥-IgG; I0 , in the absence of ␥-IgG; I = I − I0 .
3.3. Optimization of the general procedures It was found that the RLS intensity depends on the pH values of the aqueous medium. As Fig. 3 shows the maximum and constant IRLS (IRLS = I − I0 , I0 , the intensity of RLS of PS–AA; I, the intensity of RLS PS–AA–␥-globulin system) was obtained at pH 6.9. So, a pH of 6.9 was recommended for use. The effect of the concentration of the PS–AA colloidal solutions has also been investigated. As shown in Fig. 4, IRLS increased with the increase of PS–AA concentration
Fig. 4. Effect of the concentration of PS–AA nanoparticles on the RLS intensity.
3.4. Effect of ionic strength The effect of NaCl content on this assay was examined at pH 6.9. The NaCl content did not affect the RLS intensity of reagent blank in the absence of protein. In the presence of protein, the RLS intensity remained constant with increasing salt concentration, as shown in Fig. 5. If the interaction of proteins and reagent was a result of electrostatic binding, the addition of NaCl will reduce the binding of the reagent
Table 1 Tests for the interference of co-existing substances Co-existing substance
Co-existing concentration (g ml−1 )
Change of RLS (%)
Calf thymus DNA Fish sperm DNA ␣-Aminoacetic acid Aminoacetic acid Glucose Citric acid Ba(II), NO3 − Al(III), Cl− Cu(II), SO4 2− Fe(II), SO4 2− Ag(I), NO3 − Mg(II), Cl− Ca(II), Cl− Pb(II), NO3 − Hg(II), NO3 − Zn(II), Cl−
2500.0 2500.0 2500.0 2500.0 2500.0 2500.0 10.0 5.0 5.0 5.0 0.8 10.0 5.0 0.5 2.0 10.0
−6.0 −5.9 −2.3 −4.7 −6.0 1.7 −2.9 2.6 3.6 −3.7 3.4 −4.8 −2.4 1.0 4.3 −4.7
␥-IgG, 1.0 g ml−1 ; PS–AA nanoparticles, 6.0 × 10−3 mol l−1 ; pH, 6.9.
750
L. Wang et al. / Spectrochimica Acta Part A 60 (2004) 747–750
to protein and result in a decreased RLS signal. However, the ionic strength has no effect on the assay. The few effect of NaCl content on the RLS intensity manifested that the interaction of proteins and reagent was mainly a result of non-electrostatic binding (probably hydrogen bond binding). 3.5. Interferences of co-existing foreign substances In order to test the applicability of the method for the determination of total proteins in the human body fluid samples, the effects of several amino acids, salts, nucleic acids, glucose and citric acid are performed. The solutions of a fixed concentration of ␥-globulin (1.0 g ml−1 ) and each foreign substance with various concentrations are mixed prior to the detection. Table 1 shows that a lot of metal ions, amino acids, nucleic acids, glucose and citric acid do not interfere in the determination. However, such ions as Hg(II) and Pb(II) can be allowed only at very low concentration. Even though, the interference of co-existing substances in real samples can be minimized by diluting the samples with water. So, it is possible to use the method for the determination of the proteins in the human body fluid samples without separating the interfering materials.
respectively. The determination of protein is very important in clinical analysis and biochemistry. By using the present method, proteins in human serum samples were measured (shown in Table 3). For each sample, six parallel experiments were conducted, and the maximum relative standard deviation (R.S.D.) was 2.4%. The recovery of the samples was between 96 and 105%. Compared with the clinical data, the results are very satisfactory.
4. Conclusions A new method of protein determination with the limit of determination at nanogram levels is proposed by using a common spectrofluorometer to detect the intensity of RLS. The proposed method is not affected by ion strength. The results of determination for human body fluid samples were in agreement with the clinical data. This method has the priority of high sensitivity, simplicity, high selectivity and high stability. So, we think this method will become a valuable tool for the study on the properties of proteins both in chemistry and biochemistry. Further studies in polymer nanoparticles will open up the way to the application of organic nano-materials in analytical chemistry and biochemistry.
3.6. Calibration curves and sample determinations The calibration graphs for HSA, BSA and ␥-globulin were constructed by following the standard assay procedure. As shown in Table 2, the linear ranges for HSA, BSA and ␥-globulin are 0.03–10.0 g ml−1 , 0.04–10.0 g ml−1 and 0.02–11.0 g ml−1 , respectively, and the coefficients of correlation are above 0.9981 (n = 6). The limits of detection are correspondingly 0.015, 0.019 and 0.016 g ml−1 , Table 2 Analytical parameters for the determination of proteins Protein ␥-IgG BSA HAS
Linear range (g ml−1 )
Regression equation (C; g ml−1 )
LOD (g ml−1 )
r
0.02–11.0 0.04–10.0 0.03–10.0
IRLS = 868.7 + 870.7C IRLS = 861.9 + 719.0C IRLS = 859.4 + 932.5C
0.016 0.019 0.015
0.9988 0.9983 0.9981
1 2 3 a b
Content of protein The clinical data (mg ml−1 )b
This method (mg ml−1 , n = 6)
74.0 77.9 76.4
73.8 78.2 76.1
Recovery R.S.D. (%, n = 6) (%) 96–100 99–105 97–101
PS–AA nanoparticles, 6.0 × 10−3 mol l−1 ; pH, 6.9. The data was obtained from hospital.
The Education Commission Natural Science Foundation (2003kj145) and Natural Science Foundation (03044904) of Anhui Province, and the Foundation of Anhui Normal University for Young Teacher (2002xqn45) supported this work.
References
Table 3 Analytical results for human serum samplesa Sample no.
Acknowledgements
2.1 2.4 1.9
[1] C.Z. Huang, K.A. Li, S.Y. Tong, Anal. Chem. 68 (1996) 2259. [2] C.Q. Ma, K.A. Li, S.Y. Tong, Anal. Biochem. 239 (1996) 86. [3] Y.F. Li, C.Z. Huang, X.L. Hu, Chinese J. Anal. Chem. 26 (1998) 1508. [4] Y.T. Wang, K.A. Li, S.Y. Tong, Chem. J. Chinese Univ. 21 (2000) 1491. [5] T.J. Li, H.X. Shen, Chem. J. Chinese Univ. 19 (1998) 1570. [6] R.F. Pasternack, C. Bustamante, P.J. Collings, A. Giannetteo, E.J. Gibbs, J. Am. Chem. Soc. 115 (1993) 5393. [7] G. Yao, K.A. Li, S.Y. Tong, Anal. Chim. Acta 398 (1999) 319. [8] P. Feng, X.L. Hu, C.Z. Huang, Anal. Lett. 32 (1999) 1323. [9] C.Z. Huang, Y.F. Li, J.G. Mao, D.G. Tan, Analyst 123 (1998) 1401. [10] W. Lu, P. Feng, Y.F. Li, C.Z. Huang, Anal. Lett. 35 (2002) 227. [11] L.J. Dong, R.P. Jia, Q.F. Li, X.G. Chen, Z.D. Hu, Analyst 126 (2001) 707. [12] W.C.W. Chan, S. Nie, Science 281 (1998) 2016. [13] L.Y. Wang, L. Wang, F. Gao, Z.Y. Yu, Z.M. Wu, Analyst 127 (2002) 977.