Determination of proteins by the resonance light scattering technique with chromium hydroxide nanoparticles

Li Zhengping, Liu Wei, Zheng Chao, Xu Wenjie
(College of Chemistry and Environmental Science, Hebei University, Baoding 071002)

Received on Jul. 5, 2005.

Abstract Chromium hydroxide nanoparticles have been prepared by forced hydrolysis method and used as a new kind of resonance light scattering (RLS) probe for the quantitative analysis of proteins. At pH 5.9, the interaction between chromium hydroxide nanoparticles and proteins results in great enhancement of RLS signals in the wavelength range from 250 to 700 nm characterized by the peak around 341 nm. Based on this, a sensitive method for protein determination is established. The linear range of the calibration curve is 0.08 -1.6 mg/mL and the detection limit (3s) is 18.6 ng/mL for bovine serum albumin (BSA). The RLS method exhibits lower variation in response signal for the same weight of BSA, human serum albumin (HSA) and g-globulin (g-G) and has been successfully applied to the determination of the total protein in human serum samples. The result is coincident with that of the Coomassie Bright Blue (CBB) method, which indicates that the proposed method here is very simple, sensitive and reliable. As a new RLS method, it also shows significant potential for the determination of biotical analytes by using nanoparticles as probes.
Keywords chromium hydroxide nanoparticles, bovine serum albumin (BSA), resonance light scattering (RLS)

1. INTRODUCTION
The determination of proteins is a vital problem in molecular biology, biochemistry and clinical diagnosis. The standard methods for protein determination are the Lowry ^[1] and the Bradford ^[2] assays. The basis of these methods is color variation of the dyes after binding to proteins. However, their disadvantages are low sensitivity and complicated procedures. Afterwards, researchers have put forward many new methods such as colloidal gold and colloidal silver staining ^[3], fluorescence assays ^[4] and chemiluminescence methods ^[5].
    Since Pasternack et al. ^[6] applied the resonance light scattering (RLS) technique to study the aggregation of porphyrin and chlorophyll, this technique has gradually drawn many researchers' attention because of high sensitivity and operational simplicity. Huang et al. ^[7] found that RLS signals of some dyes were strongly enhanced after adding nucleic acids, and they firstly set up the RLS method for DNA assay by using a common spectrofluorometer. As a new spectral analysis technique, the RLS technique has been widely used to quantitatively determine proteins ^{[8, 9]} in aqueous solution in view of the enhanced RLS signals. However, all the RLS assays are based on the interaction between proteins and organic dyes ^{[9, 10]}.
    Recently, the nanoparticles have begun to receive more and more attention as the light scattering probes due to their excellent physical and chemical characteristics. Light scattering nanoparticles can be characterized with great advantages for several reasons: firstly, the nanoparticles have high light scattering powers, which allows these particles to be easily detected; secondly, compared with organic dyes, the nanoparticles are generally easy to prepare, low cost and nonpoisonous; thirdly, proteins, DNA probes and other biological molecules can be readily attached to the nanoparticles without altering their biological activity. Zhu et al. have established the light scattering method for determination of proteins by their quenching effect on large particle scattering of colloidal silver chloride ^[11]. The functionalized HgS nanoparticles have also applied to determine g-globulin at nanogram level by its enhancement effect on the RLS ^[12]. However, the response signals of proteins were all obtained by compared with the high background in the quenching method, and the background was very high and the response RLS intensity of g-globulin was little in the method with the functionalized HgS nanoparticles. So these methods have to be applied under rigid experimental conditions.
    In this paper, it was found that chromium hydroxide nanoparticles had low RLS intensity under appropriate conditions. The interaction of the nanoparticles and proteins by the electrostatic binding can result in a great enhancement of the RLS intensity. Based on this, we explored here a sensitive RLS method for protein determination with convenient operation.

2. EXPERIMENTAL
2.1 Apparatus
The RLS intensity and spectra were measured and recorded by a Hitachi F-4500 Fluorescence Spectrophotometer (Tokyo, Japan) equipped with a 1-cm quartz cell. The concentration of chromium hydroxide nanoparticles was determined by a WYX-402 Atomic Absorption Spectrophotometer (Shenyang Analytical Device Plant, Shenyang, China). A pHS-3C digital pH meter (Shanghai Weiye Instruments Plant, Shanghai, China) was used to measure the pH values of the solutions. A QL-901 vortex mixer (Jiangsu Clinical Instruments Plant, Haimen, China) was employed to blend the solution.
2.2 Reagents
Stock solutions of proteins were prepared by dissolving the proteins in water at the concentration of 100 mg/mL, the working solutions were prepared by diluting stock solutions with water according to requirements. In this study, proteins involved bovine serum albumin (BSA) (Sigma Co.), human serum albumin (HAS) (Sigma Co.), insulin (Sigma Co.), gelatin (Tianjin, H&Y Bio. Co.), chicken egg albumin (CEA) (Sigma Co.) and g-globulin (g-G) (Sigma Co.). Human serum samples were obtained from Hebei Agricultural University Hospital.
    The colloidal solution of chromium hydroxide (Cr(OH)₃) nanoparticles was prepared according to the literature ^[13] with slight modification. Briefly, 180 mL of pH 9.5 NaOH solution was added into a 250 mL three-necked round-bottomed flask and heated to boiling, and then 10 mL of 6.0×10^-3 mol/L CrCl₃ was dropped slowly into the flask. Afterwards the mixed solution was refluxed at 102^oC for 5 hours. Finally the colloidal solution was naturally cooled to room temperature and filtrated with 0.45 mm ultrafiltration membrane. The colloidal solution was stored at 0 - 4^oC. The concentration of filtrated colloidal solution was determined with atomic absorption spectrophotometry (AAS). The obtained chromium hydroxide colloidal solution was diluted to 7.5×10^-5 mol/L (calculated with Cr). The chromium hydroxide colloidal solution was dropped onto 50-Å-thick copper grids with the excess solution immediately volatilized away. The TEM images of the nanoparticles were acquired on a JEM -1200EX II electron microscope (Japan). It can be seen from Fig.1, the average size of chromium hydroxide nanoparticles was about 50 nm. Although the nanoparticles on the copper grids without solvent were aggregated, the chromium hydroxide colloidal solution was stable dispersed solution, which can be stable at least two weeks at 0 - 4^oC.
   All reagents were of analytical grade, and doubly distilled water was used throughout.
2.3 Procedures
In a 10 mL volumetric flask, 0.6 mL of chromium hydroxide colloidal solution (containing 7.5×10^-5 mol/L Cr), 0.5 mL of HAc-NaAc buffer solution (pH 5.9, 0.1 mol/L) and a certain volume of protein working solution were added, the mixture was diluted to 10.0 mL with water and stirred thoroughly. After incubation at room temperature for 30 min, the RLS spectra were obtained by synchronously scanning with the same excitation and emission wavelength by F-4500 Spectrophotometer in the wavelength range of 200 - 700 nm. The RLS intensity was measured at the wavelength 341 nm and the slit width and PMT voltage of the measurements were 5 nm and 400V, respectively.

Fig. 1 The TEM image of Cr(OH)₃colloidal solution after ultrafiltration

3. RESULTS AND DISCUSSION
3.1 Spectral characteristics of RLS
Figure 2 shows the light scattering spectra of Cr³⁺ solution and Cr(OH)₃ colloidal solution in the absence and in the presence of BSA. CrCl₃ solution is a real solution, the light scattering is only Rayleigh scattering, and so the light scattering intensity is very weak. It can be seen from Fig.2 that the CrCl₃-BSA solution almost has the same light scattering intensity as the CrCl₃ solution, which means the interaction between Cr³⁺ and protein cannot cause the increase of light scattering intensity. Due to the Tyndall effect, the light scattering intensity of Cr(OH)₃ colloidal solution is stronger than that of Cr³⁺ solution. When BSA is mixed with Cr(OH)₃ colloid solution, the light scattering intensity of Cr(OH)₃ colloidal solution is strongly enhanced by BSA and reaches maximum at 341.0 nm.

Fig. 2 Resonance light scattering spectra for Cr³⁺ (4.5×10^-6 mol/L) solution and Cr(OH)₃ colloidal solution (4.5×10^-6 mol/L) in the absence of BSA and in the presence of BSA (1.0 mg/mL) in the pH 5.9 HAc-NaAc buffer (5.0 mmol/L); 1.Cr³⁺, 2.Cr³⁺-BSA, 3. Cr(OH)₃, 4. Cr(OH)₃-BSA

    According to the RLS theory ^[14], RLS effect is observed as increased scattering intensity at /or very near the wavelength of absorption of an aggregated molecular species. The effect can be dramatically enhanced when strong electronic coupling exists among the chromophores. Cr(OH)₃ nanoparticles are the aggregates of Cr(OH)₃ molecules at nanoscale dimensions. As shown in Fig.3, Cr(OH)₃ colloidal solution has a maximum absorption peak at 340.0 nm and a wide absorption band. Compared with Fig.2, the light scattering of Cr(OH)₃ colloidal solution reaches maximum at 326.0 nm, which can be considered as RLS of Cr(OH)₃ nanoparticles. When BSA is added into Cr(OH)₃ colloidal solution, BSA molecules may interact with Cr(OH)₃ nanoparticles through electrostatic binding, which leads to the aggregation of nanoparticles and size increase of scattering particles. On the other hand, when the interparticle distance in these aggregates decreases to less than approximately the average particle diameter, the aggregation of the nanoparticles can result in electric dipole-dipole interaction and coupling between the plasmons of neighboring particles in the formed aggregates ^[15]. Therefore, it can be concluded that the size increase of scattering particles and the electronic coupling of Cr(OH)₃ nanoparticles originated from the interaction between BSA and Cr(OH)₃ nanoparticles can produce greatly enhanced RLS and form a new RLS peak at longer wavelength (341.0 nm).

Fig. 3 UV-Vis spectrum of the Cr(OH)₃ colloidal solution after ultrafiltration

Fig. 4 Effect of pH on the RLS of Cr(OH)₃ colloidal solution (7.5×10^-6 mol/L) in the absence of BSA (1) and in the presence of BSA (2.0 mg/mL) (2) in the HAc-NaAc buffer (0.010 mol/L)

3.2 Optimization of the general procedure
The effect of pH values on the RLS intensity of the system was studied by using HAc-NaAc buffer to adjust the pH value of the solution. As shown in Fig.4, the RLS intensity of Cr(OH)₃ is approximately kept a constant in the pH range from 4.4 to 6.2, but the RLS intensity produced by BSA gradually increases with increasing the pH value and reaches its maximum at pH 5.9. Afterwards the RLS intensity decreases at pH 6.2. The results indicate that the pH value greatly affects the interaction between Cr(OH)₃ nanoparticles and BSA. According to the literature ^[13], the surface of Cr(OH)₃ nanoparticles is positively charged, and BSA is also positively charged when the pH value of solution is lower than the isoelectric point (pI = 4.7) of BSA. Therefore, when pH value is lower than 4.7, the interaction between Cr(OH)₃ nanoparticles and BSA is rather weak, the RLS intensity is correspondingly low. When pH value is greater than 4.7, BSA is negatively charged, the electrostatic binding of BSA and colloids results in Cr(OH)₃ nanoparticles aggregation through BSA molecules, which causes the RLS intensity gradually increases. Above pH 5.9, the RLS intensity decreases probably because the Cr(OH)₃ colloids is destroyed at higher pH value.
    At pH 5.9, the influence of the concentration of HAc-NaAc buffer on the enhanced RLS intensity was investigated. Different volume of buffer were respectively added into Cr(OH)₃ and Cr(OH)₃-BSA solution. The result shows that the RLS intensity produced by BSA has a maximum at 5.0 mmol/L HAc-NaAc buffer. With the increasing of buffer concentration, ionic strength of solution is raised, which results in decreasing of the RLS intensity. The phenomena suggest the interaction of Cr(OH)₃ nanoparticles and BSA is based on electrostatic binding. Therefore, 5.0 mmol/L HAc-NaAc (pH 5.9) was used to control the pH value of the sample solution.
    The effect of the concentration of the Cr(OH)₃ colloidal solution on the RLS intensity of 1.0 mg/mL BSA was studied. When the concentration of Cr(OH)₃ colloidal solution is raised from 3.0×10^-6 mol/L to 2.25×10^-5 mol/L, the RLS intensity of Cr(OH)₃ is gradually increased, the RLS intensity of Cr(OH)₃-BSA is slightly increased and then presents the decreased trend. The RLS signal reaches maximum at the concentration of 4.5×10^-6 mol/L. When the concentration of Cr(OH)₃ is relatively high, the BSA molecules will be fully saturated by Cr(OH)₃. The complexes of BSA and Cr(OH)₃ nanoparticles will slowly aggregates and precipitates, which results in the RLS intensity of BSA gradually decreasing when the concentration of Cr(OH)₃ is greater than 4.5×10^-6 mol/L. Therefore, 4.5×10^-6 mol/L was selected as the optimum concentration of the Cr(OH)₃ colloidal solution.
    The influence of the incubation time of the Cr(OH)₃-BSA solution on the RLS intensity was also investigated. The RLS signals of both the Cr(OH)₃-BSA solution and Cr(OH)₃ colloidal solution were stable after 30 min and showed a slight change of less than 10% until 120 min. The result proves that the complex can form immediately when Cr(OH)₃ colloidal solution and BSA are mixed, and has a good stability. According to the results mentioned above, the RLS intensity was measured after incubation for 30 min.
3.3 Interference of foreign coexisting substance
In order to study the potential interference of various metal ions and amino acids, the standard solution containing 1.0 mg/mL BSA was premixed with the foreign substance. Then, the RLS intensity was detected according to the general procedure and compared with that of the standard solution itself. The tolerance concentration of metal ions and amino acids are presented in Table 1. From Table 1, it can be seen that the proposed method can tolerate most of the metal ions at higher concentration (greater than1.0×10^-7 mol/L) and the amino acids are tolerated at the concentration from 1.0 to 100.0 mg/mL. The tolerated concentrations of these coexisting substances are generally greater than that in biological samples. So these substances do not interfere with the determination of BSA.

Table 1 Effect of coexisting substances

^a Concentration is expressed by ×10^-7 mol/L for metal ions and ×1 mg/mL  for amino acids, respectively.
Cr(OH)₃ nanoparticles: 4.5×10^-6 mol/L; BSA: 1.0 mg/mL; pH 5.9

3.4 Calibration curves
According to above general procedure, the calibration curves for BSA determination were constructed under the optimum conditions. There is a linear relationships between the enhanced RLS intensity (DI_RLS) and BSA concentration (C) in the range of 0.08-1.6 mg/mL. The linear regression equation of this assay is DI_RLS = -0.17+39.12 C, the correlation coefficient is 0.9991 and the detection limit (3s) is 18.6 ng/mL.
3.5 Sample determinations

Table 2 RLS signals of different protein

1.0 mg/mL of protein was used for each measurement. The results were average value of three measurements.

    Table 2 displayed the variation of RLS signal response for different proteins under the above optimal conditions. Compared with the RLS signal of BSA, the response change of human serum albumin (HSA) and g-globulin was little. And the RLS signal was decreased about 1-fold for chicken egg albumin and about 3-fold for insulin and gelatin, which indicates that the RLS signal response basically depends on the molecular weight of various proteins.
    Figure 5 shows the relationship between RLS intensity and protein molecular weight when the different proteins are at the same weight (1.0 mg/mL). From Fig.5, it can be seen that the RLS intensity linearly increases with increasing protein molecular weight in the molecular weight range of 40000-60000. However, the RLS intensity increases very little with increasing protein molecular weight when the molecular weight is less than 40000 or greater than 60000. Therefore, the proposed RLS method is suitable to determine the total protein in serum samples, in which the albumin and globulin exhibit lower variation in response signal at the same molecular weight.
    The proposed method was applied to determine the total protein content of human serum samples. The fresh serum samples were diluted with doubly distilled water and determined with the RLS method and the Bradford assay ^[2]. Table 3 displays the determination results for human serum samples, which are in good agreement with that of the Bradford assay. Therefore, the proposed method is practical and adaptable for the determination of total protein in serum samples.

Fig.5 Relationship between RLS intensity and protein molecular weight

氢氧化铬纳米粒子共振光散射技术测定蛋白质
李正平，刘玮，郑超, 许文杰
（保定河北大学化学与环境科学学院，071002）
摘要  用强制水解的方法制备的氢氧化铬纳米粒子可以作为一种新的共振光散射探针对蛋白质进行定量分析。在pH5.9的条件下，氢氧化铬纳米粒子与蛋白质相互作用导致共振光散射信号在250-700nm之间有很大的增强，并且在341nm处散射值达到最大。基于此现象，建立了一种较灵敏的检测蛋白质的方法。测定牛血清蛋白的标准曲线的线性范围是0.08- 1.6mg/mL，检出限是18.6ng/mL。用共振光散射方法对相同量的牛血清蛋白，人血清蛋白和g-球蛋白进行测定，其相应的信号变化不大。此方法被成功的用于人血清样品中总蛋白含量的测定，其结果与考马斯亮蓝方法的测定结果一致，这表明该方法简单，灵敏，可靠。作为一种新的以纳米粒子为探针的共振光散射方法，在对生命物质进行测定研究方面将具有很大的发展潜力。
关键词  氢氧化铬纳米粒子，牛血清蛋白，共振光散射