Sensitive determination of protein using terbium-metalloporphyrin as a fluorescence probe in AOT microemulsion

Sensitive determination of protein using terbium-metalloporphyrin as a fluorescence probe in AOT microemulsion

Journal of Molecular Liquids 199 (2014) 67–70 Contents lists available at ScienceDirect Journal of Molecular Liquids journal homepage: www.elsevier...

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Journal of Molecular Liquids 199 (2014) 67–70

Contents lists available at ScienceDirect

Journal of Molecular Liquids journal homepage: www.elsevier.com/locate/molliq

Sensitive determination of protein using terbium-metalloporphyrin as a fluorescence probe in AOT microemulsion Dan Wu ⁎, Ru Li, Dawei Fan, Yong Zhang, Qin Wei Key Laboratory of Chemical Sensing & Analysis in Universities of Shandong, School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, China

a r t i c l e

i n f o

Article history: Received 28 July 2014 Received in revised form 13 August 2014 Accepted 17 August 2014 Available online 27 August 2014 Keywords: Fluorescence probe Terbium-metalloporphyrin AOT microemulsion Protein

a b s t r a c t The quantitative determination of biomolecules is a basic requisite in biochemistry, food analysis, and clinical analysis. In this paper, a highly sensitive and selective fluorescence method was developed to determine proteins using a metalloporphyrin, terbium-meso-vanillin-metalloporphyrin (T(3-MO-4HP)P-Tb), as a probe. In the presence of bis(2-ethylhexyl)sulfosuccinate sodium salt (AOT) microemulsion, protein could enhance the fluorescence intensity of porphyrin. The experimental conditions, such as pH, medium, and probe concentration, were investigated. Under the optimal conditions, the enhanced fluorescence intensity was in proportion to the concentration of proteins in the range of 0.12–6.75 μg·mL−1 for bovine serum albumin (BSA), 0.14–12.00 μg·mL−1 for ovalbumin (Ova) and 0.15–7.60 μg·mL−1 for human serum albumin (HSA). The detection limits were 0.075 μg·mL−1 for BSA, 0.080 μg·mL−1 for Ova and 0.087 μg·mL−1 for HSA, respectively. The proposed method was also applied to the determination of protein in real samples with satisfactory results. This fluorescent method with high sensitivity and low detection has potential application in food science for the determination of proteins. © 2014 Elsevier B.V. All rights reserved.

1. Introduction The quantitative analysis of protein is a basic requisite in biochemistry, food analysis, and clinical analysis, etc. [1–5]. So the detection of trace amount of protein in real samples is of great importance. Numerous analysis techniques have been developed for the detection of protein, including Lowry assay [6], Bradford assay [7], Kjeldahl method [8], and Silver staining method. However, these traditional methods have some disadvantages, such as complicated operation, lower precision, and large sample consumption. To overcome these limitations, many methods have been studied in recent years such as Rayleigh scattering technique [9–15], chemiluminescence technique [16], spectrophotometric [17,18] and fluorescent methods [19–26], etc. In comparison with other methods mentioned above, fluorescent method has attracted considerable attention owing to their high sensitivity, good selectivity and less sample consumption. Determination of protein using fluorescent method is a kind of high technology which has been well developed around the world in recent years. However, the intrinsic fluorescence of protein is weak for the analysis of proteins at low concentrations. Therefore, exploiting sensitive, stable and simple fluorescence probes for determination of proteins has been a focus.

⁎ Corresponding author at: School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, Shandong Province, China. Tel.: +86 531 82767872; fax: +86 531 82765969. E-mail address: [email protected] (D. Wu).

http://dx.doi.org/10.1016/j.molliq.2014.08.021 0167-7322/© 2014 Elsevier B.V. All rights reserved.

Porphyrin as a spectral probe has been widely used for the analysis of metal ions and biomacromolecules [27]. Compared with common metals, rare earth elements exhibit more abundant physicochemical properties and electrochemical properties due to their unique 3d–4f electronic structure. Therefore, rare earth metalloporphyrin can be used as a potential drug for tumor and new organic optoelectronic material. In our previous work, a newly synthesized metalloporphyrin, terbium-meso-vanillin-metalloporphyrin (T(3-MO-4HP)P-Tb), had been investigated as labels for the determination of nucleic acid [28]. In this study, T(3-MO-4HP)P-Tb is used as a spectral probe for the determination of protein to extend our work. The chemical structure of T(3-MO-4HP)P-Tb is presented in Fig. 1. The fluorescence intensity of T(3-MO-4HP)P-Tb was increased after the addition of protein in the presence of AOT microemulsion. Based on this phenomenon, we developed a new method for determination of protein. 2. Experimental section 2.1. Apparatus and reagents Intensity and spectra of fluorescence were recorded with a LS-55 spectrofluorimeter (PerkinElmer) in a 1.0 cm quartz cell. All pH measurements were made using a PHS-3C acidity meter. BSA, Ova, HSA were purchased from Sigma and used as received. The concentration of their working solution was 100 μg·mL−1. T(3-MO4HP)P-Tb solution (5.00 × 10−4 mol·L−1) was prepared by dissolving 0.0478 g of porphyrin with DMF and brought to 100 mL in a volumetric

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Fig. 2. The excitation (1–4) and emission (1′–4′) spectra of the porphyrins with different concentrations of BSA. (1), (1′): porphyrin (1.00 × 10−6 mol·L−1); (2), (2′): porphyrin (1.00 × 10−6 mol·L−1) + AOT microemulsion + buffer solution; (3), (3′): porphyrin (1.00 × 10 − 6 mol·L − 1 ) + 1.50 μg·mL − 1 BSA + AOT microemulsion + buffer solution; (4), (4′): porphyrin (1.00 × 10 − 6 mol·L − 1 ) + 2.50 μg·mL − 1 BSA + AOT microemulsion + buffer solution. Fig. 1. The molecular structure of T(3-MO-4HP)P-Tb.

flask with doubly distilled water. AOT microemulsion was prepared according to the mass ratio given below: AOT:n-butanol:n-heptane: H2O = 1.0:1.0:1.0:97.0. Unless otherwise noted, all reagents and solvents used in this study were analytical grade. 2.2. Experiment procedure A 10 mL test tube is charged with the following solution (in the order shown): 0.20 mL of T(3-MO-4HP)P–Tb solution (5.00 × 10−5 mol·L−1), 30 μL of BSA solution (100 μg·mL−1), 0.1 mL 1% AOT microemulsion, 0.2 mL of HAc–NaAc buffer solution (pH = 5.80). The mixture is diluted to the mark, mixed thoroughly and stood for 20 min at the room temperature. The enhancement of the fluorescence intensity is taken as the intensity difference between sample and blank under the same conditions. ΔF is defined as followed: ΔF = F − F0, where F and F0 are the fluorescence intensity of the sample and blank at λem = 661 nm. 3. Results and discussion 3.1. Fluorescence spectra The excitation and emission spectra of porphyrin–protein system were shown in Fig. 2. It could be seen that all the samples have the same maximum excitation peak at 421 nm and maximum emission peak at 661 nm. The fluorescence intensity of porphyrin could be enhanced greatly by BSA, and the increased intensity was proportional to the concentration of protein. The existence of surfactant had little impact on maximum excitation peak and maximum emission peak of porphyrin. Therefore, the porphyrin could be used as a fluorescence probe for quantitative determination of BSA.

Triton X-100, AOT, Tween-80, cetytrimethyl ammonium bromide (CTMAB), Triton X-10 microemulsions, as well as in the corresponding micelle solution. The results were presented in Fig. 4. It could be seen that AOT microemulsion had a great impact on ΔF for this system. Thus, the AOT microemulsion was chosen as the best medium and the concentration of it was 0.20 g·L−1. This could be due to the effective collisions between AOT microemulsion and porphyrin molecules, which reduced the fluorescence intensity of porphyrin. After adding protein, the interaction of protein with porphyrin molecules reduced shielding effect of the solvent on the porphyrins, resulting in the enhancement of fluorescence intensity.

3.4. Selection of the concentration of T(3-MO-4HP)P-Tb In terms of experimental procedure, the concentration of T(3-MO4HP)P-Tb was varied to obtain the optimum concentration. The result was shown in Fig. 5. When the concentration of T(3-MO-4HP)P–Tb was larger, it would reduce shielding effect of the AOT microemulsion on the porphyrins, and porphyrins were easily aggregated and quite difficult to react with protein. The ΔF enhanced gradually and kept stable in concentration range of 5.00 × 10−7 mol·L−1 ~ 1.50 × 10−6 mol·L−1. Thus, the concentration of T(3-MO-4HP)P-Tb of 1.00 × 10−6 mol·L−1 was used in further study.

3.2. Effect of pH The effect of acidity on ΔF was investigated, as shown in Fig. 3. It could be concluded that the ΔF was maximum and constant at pH 5.80. Therefore, the pH of 5.80 was chosen in subsequent experiments and the amount was 0.90 mL. 3.3. Selection of medium Surfactants can be used as solubilizer and sensitizer for organic complexes, and have a great role on the enhancement of fluorescence intensity of the system. The measurement of ΔF was performed in

Fig. 3. The effect of acidity on fluorescence intensity. Condition: cT(3-MO-4HP)P–Tb = 1.00 × 10−6 mol·L−1; cBSA = 2.00 μg·mL−1; cAOT microemulsion = 0.20 g·L−1.

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Table 1 Analytical parameters of different kinds of protein systems. Protein

Linear range (μg·mL−1)

Regression equation

Correlation coefficient (r)

Detection limit (μg·mL−1)

BSA Ova HSA

0.12–6.75 0.14–12.00 0.15–7.60

ΔF = 12.46 + 66.91ρ ΔF = 2.79 + 59.94ρ ΔF = 4.36 + 57.83ρ

0.992 0.997 0.996

0.075 0.080 0.087

curve and detection limit because of their different isoelectric points and structures. Compared with the detection limit for literature reports regarding protein detection [4,10], the proposed method has a lower detection limit.

Fig. 4. The effect of different medium on the intensity (1)Triton X-100 microemulsion; (2) AOT microemulsion; (3)Tween-80 microemulsion; (4)CTMAB microemulsion; (5)TX-10 microemulsion; (1′)Triton X-100 micelle; (2′)AOT micelle; (3′)Tween-80 micelle; (4′) CTMAB micelle; (5′)TX-10 micelle. Condition: cT(3-MO-4HP)P-Tb = 1.00 × 10−6 mol·L−1; cBSA = 2.00 μg·mL−1; 0.90 mL of buffer solution at pH 5.80.

3.5. Addition order and stability The addition order of reagents had influence on determination of protein by the fluorescent technology. In this system, porphyrin-BSA– AOT microemulsion–HAc–NaAc buffer solution (pH = 5.80) obtained better results. The results were ascribed to not only a strong combination between porphyrin and BSA under the condition of the addition order of porphyrin preceding BSA, but also the sensitizing effect of microemulsion. The system remained constant and reached maximal after the addition of buffer solution owing to the formation of microenvironment suitable for the reaction of BSA. Under the conditions described above, porphyrin could bind rapidly with protein at room temperature and ΔF would reach the maximum instantly in 10 min and remained stable in nearly 2 h. In this study, the optimal stability time was chosen for 20 min.

3.6. Calibration curve and sensitivity Standard solutions of proteins in different volumes were added to a series of 10 mL comparison tubes, respectively. The other procedures followed the previously described procedure. All of the analytical parameters are shown in Table 1. Detection limit (DL) is calculated by the equation DL = KS0/s, where K is a constant related to the confidence level, S0 is the standard deviation of the blank measurements (n = 11, K = 3) and s is the slope of the calibration curve. It can be seen from Table 1 that different proteins have different linear range, working

3.7. Effect of coexistence substances The possible influence of coexistence substances on the determination of proteins was investigated by adding different concentration of substances. The tolerance limit was taken as the maximum concentration of the foreign ions causing about ± 10%. It could be seen from Table 2 that most of coexistence substances had less interference on the determination of BSA. Minority heavy metal ions had relatively large interference on the determination of BSA. 3.8. Analysis of real samples The proposed method was applied to the determination of protein in milk powder. 0.2 g of the sample was dissolved into 4 mL of HAc–NaAc buffer solution. After 5 min, the aqueous phase was transferred into a 100 mL calibrated flask and diluted to the mark with doubly distilled water. An appropriate amount of the sample solution was transferred to 10 mL color comparison tube after being mixed thoroughly and determined as described above. The protein contents of milk powder samples were measured 5 times to obtain the precision. The relative standard deviation (RSD) was calculated and the results are shown in Table 3. The accuracy was also studied through a recovery experiment. 1.00 μg⋅mL−1 of the standard solution was added to two milk powder samples. These experiments were repeated five times. The recovery, referring to the average recovery, was calculated and the results are also shown in Table 3. It can be seen from Table 3 that RSD was in the range of 1.0%–1.3% and the recovery was between 99.3% and 100%. All these presented sufficient precision and high accuracy. 4. Conclusions A novel probe for the determination of protein in the presence of AOT microemulsion was presented in this paper. The proposed method is simple, convenient, and can be used for the determination of real

Table 2 Influence of coexistence substances on the system (3.00 μg·mL−1 BSA).

Fig. 5. The effect of porphyrins concentration on the intensity. Condition: cBSA = 2.00 μg·mL−1; cAOT microemulsion = 0.20 g·L−1; 0.90 mL of buffer solution at pH 5.80.

Coexistence substances

Concentration Relative Coexistence error substances (μg·mL−1) (%)

Pb2+ Fe3+ Cu2+ K+

0.10 40.0 100 300

−9.8 9.2 10.7 −6.9

Cd2+

0.20

9.5

Thymine Uracil Starch RNA

0.50 20.0 25.0 50.0

9.9 9.5 5.5 −6.4

Aminoacetic acid

10.0

7.7

Co2+ Mn2+ Ni2+ Human albumin L-tyrosine

Guanine Adenine DNA Lactic glycogen

Concentration Relative (μg·mL−1) error (%) 28.0 80.0 20.0 0.50

−8.2 −6.8 5.2 9.2

0.15

9.5

25.0 50.0 50.0 25.0

8.7 7.3 −6.8 −5.9

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Table 3 Results for the determination of protein in milk powder. Sample

Found (n = 5) (μg·mL−1)

Mean (n = 5) (μg·mL−1)

RSD (%)

Added (μg·mL−1)

1

1.21, 1.23, 1.19, 1.22, 1.21 0.91, 0.90, 0.91, 0.92, 0.89

1.21

1.33

1.00

0.91

1.02

1.00

2

Recovery (%) 99.3 100

samples without pretreatment. This novel probe has a potential application prospect in studying the interaction between lanthanon and porphyrin. These results can also provide guidance for studying the mechanism between protein and molecule ligands. Acknowledgments This study was supported by the Natural Science Foundation of China (nos. 21375047, 21377046, 21103071), and all the authors express their deep thanks. References [1] [2] [3] [4]

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