A highly sensitive assay for spectrofluorimetric determination of reduced glutathione using organic nano-probes

Spectrochimica Acta Part A 61 (2005) 2533–2538

A highly sensitive assay for spectrofluorimetric determination of reduced glutathione using organic nano-probes Leyu Wang∗ , Lun Wang, Tingting Xia, Guirong Bian, Ling Dong, Zhenxiang Tang, Fei Wang College of Chemistry and Materials Science, Anhui Normal University, Wuhu 241000, China Received 27 May 2004; accepted 9 July 2004

Abstract In this study, the new nanometer-sized fluorescent particles (1-pyrenemethylamine nanoparticles) have been prepared by reprecipitation method under ultrasonic radiation. These nanoparticles have the potential to overcome problems encountered by organic small molecules by combining the advantages of high photobleaching threshold, high quantum yield, long fluorescence lifetime, good chemical stability, and wide excitation spectral properties. These nanoparticles will be able to be directly used as fluorescent nanoparticles probe without modification. A new fluorimetric method for the determination of reduced glutathione (GSH) has been developed with these nanoparticles. Under optimal conditions, the organic nanoparticles reacted with GSH and o-phthalaldehyde (OPA) to give a highly fluorescent derivative in Na2 CO3 –HCl buffer (pH = 9.0). The fluorescence excitation and emission wavelengths of fluorescent derivative were located at 345 and 400 nm, respectively. The relative fluorescence intensity (RF) was linear in the range of the GSH concentration from 8.0 × 10−7 to 1.1 × 10−4 mol l−1 . Limit of detection of 7.1 × 10−8 mol l−1 was achieved for the reduced glutathione. The method was validated and applied to the analysis of three synthetic samples containing reduced glutathione. © 2004 Elsevier B.V. All rights reserved. Keywords: Fluorescence probe; Reduced glutathione; Organic nanoparticles

1. Introduction The tripeptide glutathione (l-␥-glutamyl-l-cysteinglycine, GSH) is the most abundant low-molecular-mass thiol in cells of many different organisms [1]. It plays an essential role in many important biological phenomena, including the synthesis of proteins and DNA, enzyme activity, metabolism and in protection of cells against toxic effects of oxidizing agents, free radicals, ionizing agents and certain exogenous compounds [2,3]. Since evidence for GSH deficiency has been found in a variety of diseases, including diabetes [4], human immunodeficiency virus (HIV) infection [5], cystic fibrous [6], acute respiratory distress syndrome [7] and chronic renal failure [8,9], an analytical method for measuring low levels of GSH in biological fluids would be desirable.

∗

Corresponding author. Tel.: +86 553 3869303; fax: +86 553 3869303. E-mail address: [email protected] (L. Wang).

1386-1425/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.saa.2004.07.041

Increased interest in glutathione as primary player in many cellular functions and detocification mechanisms has led to the development of several different methods for the measurement of glutathione. A number of methods for the quantification of glutathione in biological samples are documented in the literature [10–16]. Some of these methods are electrochemistry [10], spectrophotometry [11,12], enzymatic method [13,14] and spectrofluorimetry [15,16], whereas others utilize high performance liquid chromatography (HPLC) [17–19] and gas chromatography (GC) [20,21]. The enzymatic assays are laborious and complicated, rendering them not suitable for routine analysis. GC and HPLC provide high degree of specificity and sensitivity, however, they are expensive and not readily available in most analytical laboratories. In recent years, spectrofluorimetric method is widely used in the field of biological science for its sensitivity, simplicity and low cost. In the spectrofluorimetric determination of GSH, some fluorescence reagents have been proposed, such as bimanes [22,23], o-phthaldialdehyde (OPA) [24,25], Nsubstituted maleimides [26,27] and so on. All these reagents,

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have advantages of high sensitivity and selectivity, which is favorable for the determination of trace GSH in biological sample. To the best of our knowledge, none of the published methods for the determination of glutathione in biological samples combine all the desired features for a rapid, reliable, sensitive and a simple assay. In this study, the validation and applicability of a method with organic nanoparticles as fluorescence probe for the quantification of reduced glutathione is described. Biocompatible colloidal fluorescence semiconductor nanopaticles (NPs) have been widely used for fluorescence probes in biochemical and biomedical applications [28–32]. The luminescent colloidal semiconductor nanoparticles have the potential to overcome problems encountered by organic small molecules in certain fluorescent tagging applications by combining the advantages of high photobleaching threshold, good chemical stability and readily tunable spectral properties. However, these fluorescent nanoparticles described recently are mainly based on fluorescent inorganic nanoparticles, such as ZnS, ZnSe, CdS, CdTe and CdSe. To make these nanoparticles water-soluble and biocompatible, these nanoparticles must be modified with organic small molecules. The limitation of selection of fluorescent inorganic nanoparticles and the complication of modification procedures limited their application. In this paper, we prepared the highly fluorescent nanoparticles, 1-pyrenemethylamine nanoparticles, under ultrasonic radiation. These nanoparticles will be able to be directly used as fluorescent nanoparticles probe without modification. They also have longer fluorescence decay lifetime and stronger fluorescence than organic small molecule (1-pyrenemethylamine). In the presence of GSH, the derivatization of primary amino groups of 1-pyrenemethylamine nanoparticles with OPA forms a highly fluorescent derivative at pH 9.0. The fluorescence response of the derivative is linear over a range of the GSH concentration from 8.0 × 10−7 to 1.1 × 10−4 mol l−1 with good precision. The detection limit (DL) for reduced glutathione is 7.1 × 10−8 mol l−1 . The present method has been applied to the determination of GSH in synthetic samples with satisfactory results.

Shanghai, China) pH meter was used for accurate adjustment of pH. 2.2. Reagents and chemicals A stock standard solution of o-phthalalhyde (OPA) (Sigma) was prepared by dissolving it in 95% ethanol to a final concentration of 1.0 × 10−3 mol l−1 . Stock standard solutions of GSH and cysteine were prepared by directly dissolving commercially available reagents in water at a concentration of 1.0 × 10−3 mol l−1 . Working solutions were prepared by diluting the stock solutions in water. Solutions can be stored at 0–4 ◦ C temperature. An amount of 0.025 mol l−1 solution of 1-pyrenemethylamine (Sigma, assay ≥ 95%) was prepared by directly dissolving commercially available reagents in acetone and stored as standard solution. All reagents were of analytical grade or the best grade commercially available. Water used throughout was doubly distilled. 2.3. Procedures The nanoparticles were prepared as follows: under vigorous ultrasonic irradiation, 2 ml of 1-pyrenemethylamine solution (0.025 mol l−1 ) was dripped slowly into 250 ml water. Excess 1-pyrenemethylamine solution was removed by repeated centrifugation [28,29] and the nanoparticles were acquired. The amino groups of the out surface of nanoparticles render the nanoparticles water-soluble. The free amino group is also available for binding to various biomolecules, such as nucleic acids, proteins and GSH. In a set of 10 ml volumetric flasks, a certain volume of nanoparticles colloidal solution, 1.5 ml of OPA, 2.0 ml of buffer and appropriate volume standard solutions of GSH were added and diluted to the mark. Fluorescence measurements for both the sample solution (F) and the blank solution (F0 ) (prepared in a similar manner without GSH) were made after the mixture was stood in 30 ◦ C water bath for 20 min with the following settings of the spectrofluorimeter: excitation wavelength (λex ), 345 nm; excitation slit, 5 nm; emission wavelength (λem ), 400 nm; emission slit, 5 nm.

2. Experimental 2.1. Apparatus

3. Results and discussion

A VCX 500 (Sonic, USA) ultrasonic processor was used in the ultrasonic synthesis of nanoparticles. Transmission electron microscopy (TEM) images of the nanoparticles were acquired on a H-600 (Hitachi, Japan) transmission electron microscope. Fluorescence spectra and relative fluorescence intensities were measured on a model F-4500 fluorescence spectrophotometer (Hitachi, Japan) equipped with a xenon lamp, dual monochromators, a 1 cm × 1 cm quartz cell. The slit-width for both excitation and emission was set at 5 nm. A model pHS-3C (Dazhong Analytical Instruments Factory,

3.1. Spectral characteristics of fluorescence The fluorescence spectrum in colloids displayed an excitation maximum at 345 nm and an emission maximum at 400 nm, respectively (Fig. 1). In our research, it was found out that the excitation and emission maxima of nanoparticles are similar to those of 1-pyrenemethylamine, but the fluorescence emission and excitation intensities are significantly enhanced. In the presence of o-phthalaldehyde (OPA), the nanoparticles reacted with GSH in Na2 CO3 –HCl buffer

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Fig. 3. Spectra of fluorescence lifetime of 1-pyrenemethylamine and nanoparticles. Fig. 1. Spectra of excitation and fluorescence emission of 1-pyrenemethylamine and nanoparticles. Concentration of 1-pyrenemethylamine and nanoparticles is 2.4 × 10−7 mol l−1 (λem /λex = 400/345 nm).

(pH = 9.0) and gave an strong fluorescent derivative. Fluorescence spectra of the derivative are listed in Fig. 2. The fluorescence maximum for the derivative was at excitation and emission wavelengths of 345 nm and 432 nm, respectively. The fluorescence intensity of derivative was enhanced greatly by increasing the GSH concentration, which was convenient for the measurement of GSH. Therefore, the 1pyrenemethylamine nanoparticles can be served as a probe for GSH. Fig. 4. TEM image of nanoparticles.

3.2. Fluorescence lifetime and TEM image Fig. 3 gives the fluorescence decay lifetime spectra of 1pyrenemethylamine and nanoparticles. In the spectra of fluorescence decay lifetime, each grid of horizontal axis represents 0.5 ␮s. The full width of fluorescence decay lifetime spectrum at half maximum represents the fluorescence lifetime. From Fig. 3, it can be seen that the fluorescence decay lifetime of nanoparticles is two times long as that of 1pyrenemethylamine, and TEM image of nanoparticles shows that the average size of nanoparticles is about 25 nm (Fig. 4).

Fig. 2. Fluorescence spectra of 1-pyrenemethylamine nanoparticles– OPA–GSH system (λem /λex = 432/345 nm). Colloids: 2.4 × 10−7 mol l−1 ; OPA: 1.5 × 10−4 mol l−1 ; pH 9.0; GSH (×10−5 mol l−1 ): 1, 0.3; 2, 0.9; 3, 1.5; 4, 3.0; 5, 6.0; 6, 9.0.

3.3. Optimization of general procedure Effects of colloidal solution concentration on the fluorescence intensity have been studied. The results shown in Fig. 5 indicated that a maximum and constant value of fluorescence was observed over the colloids concentration range of (2.2–3.5) × 10−7 mol l−1 (represented by the concentration of 1-pyrenemethylamine existing in single molecule). In this work, a colloids concentration of 2.4 × 10−7 mol l−1 was recommended. The effect of OPA concentration was also investigated with constant concentration of colloids and GSH at pH 9.0. The results are shown in Fig. 6. The experimental results indicated that the maximum and constant fluorescence

Fig. 5. Effect of the concentration of nanoparticles on the fluorescence intensity.

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fluence of incubation time on fluorescence intensity was also investigated. The results showed that the maximal fluorescence intensity was reached when the solutions were mixed, and incubated for 20 min in 30 ◦ C water bath. The fluorescence remained constant for at least 2.5 h at room temperature. Therefore, a 20 min incubation time was adopted in this work. The adding sequence of colloids, OPA, GSH and buffer solution had no obvious effect on the fluorescence system. However, considering the consistent manipulation, in all experiments, colloids and OPA were first mixed, then buffer solution was added, and finally the GSH was added. Fig. 6. Effect of the concentration of OPA on the fluorescence intensity, colloids: 2.4 × 10−7 mol l−1 ; pH 9.0.

Fig. 7. Effect of pH on the F; colloids: 2.4 × 10−7 mol l−1 ; OPA: 1.5 × 10−4 mol l−1 ; pH 9.0; GSH: 1.0 × 10−6 mol l−1 ; F = F − F0 ; F: in the presence of GSH; F0 : in the absence of GSH.

intensity of derivative occurred when OPA concentration was in the range 1.2–1.8 × 10−4 mol l−1 . In this work, a OPA concentration of 1.5 × 10−4 mol l−1 was recommended. The effect of pH on the fluorescence of system was also studied (Fig. 7). The experimental results indicated that maximum and constant fluorescence increase was produced in the pH range of 8.3–10.0. Therefore, a pH of 9.0 was recommended for use in this work. This value was obtained by addition of 2.0 ml buffer solution per 10 ml of the final solution. The in-

3.4. Calibration graph, sensitivity, and precision The calibration graph for the determination of GSH was constructed. Under optimal conditions, the fluorescence intensity in the presence of GSH was proportional to the concentration of GSH with a good linear relationship. The calibration curve was as follows: F = 88.1 + 13.9 C (␮mol l−1 ). Linear relationship was obtained from 8.0 × 10−7 to 1.1 × 10−4 mol l−1 GSH. A correlation coefficient of 0.9979 (n = 11) was also acquired. The limit of detection (LOD) was given by the equation: LOD = KS0 /S, where K is a numerical factor chosen according to the confidence level desired (here, a value of 3 for K was used). S0 is the standard deviation of the blank measurements (n = 6) and S is the slope of the calibration curve. In this study, the 3␴ limits of detection for GSH is 7.1 × 10−8 mol l−1 . The precision was established by repeated determination (n = 6) using 3.5 × 10−5 mol l−1 GSH. The relative standard derivations (R.S.D.) did not exceed 2.5%. 3.5. Tolerance of foreign substances When the amount of GSH was 1.0 × 10−6 mol l−1 , the influence of some ions, proteins, glucose, nucleic acids and cysteine as co-existing substances on the determination of GSH was studied, and the results were given in Table 1. It can be seen from the table that most of the substances tested scarcely interfered with the determination. Hg(II), Ag(I) and

Table 1 Tests for the interference of coexisting substancesa Coexisting substance

Coexisting concentration (␮g ml−1 )

Change of RF (%)

Coexisting substance

Coexisting concentration (␮g ml−1 )

Change of RF (%)

Calf thymus DNA Fish sperm DNA Aminoacetic acid Cysteine Glucose Human serum albumin Gamma globulin Bovine serum albumin Ag(I), NO3 − Fe(III), Cl− Fe(II), SO4 2−

10.0 10.0 10.0 1.0 × 10−5 mol l−1 10.0 5.0 5.0 10.0 2.0 5.0 5.0

−3.4 −4.6 +4.2 −5.1 −3.8 −3.3 +3.7 −4.4 −5.9 −3.7 −3.9

Cr(III), NO3 − Hg(II), NO3 − Ca(II), Cl− Co(II), NO3 − Mg(II), Cl− Pb(II), NO3 − Zn(II), Cl− Al(III), Cl− Cu(II), NO3 − K(I), Cl− Na(I), Cl−

5.0 2.0 2.0 2.0 2.4 2.0 10.0 3.0 10.0 25.0 25.0

−7.1 −5.7 −3.1 −3.2 2.0 −7.5 −1.5 −4.4 −2.5 −2.8 −1.9

a

GSH, 1.0 × 10−6 mol l−1 (0.615 ␮g ml−1 ); OPA, 1.5 × 10−4 mol l−1 ; nanoparticles, 2.4 × 10−7 mol l−1 ; pH, 9.0.

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Table 2 Results and determination for synthetic samples GSH in samples (×10−5 mol l−1 )

Coexisting substances and concentration (␮g ml−1 )

GSH founda (×10−6 mol l−1 )

Recovery (%)

R.S.D (%)

2.5 3.5 4.5

Cysytein (4.0 × 10−6 mol l−1 ); HSA (5.0); glucose (5.0); Co(II) (5.0); Zn(II) (5.0) Cysytein (8.0 × 10−6 mol l−1 ); Cu(II) (5.0); Zn(II) (5.0); gamma globulin (5.0) Aminoacetic acid (5.0); HSA (6.0); Ca(II) (6.0); Mg(II) (6.0); gamma globulin (5.0)

2.43 3.35 4.34

97.2 95.7 96.4

1.9 2.5 2.3

OPA, 1.5 × 10−4 mol l−1 ; nanoparticles, 2.4 × 10−7 mol l−1 ; pH, 9.0. a Average of six determinations.

Pb(II) can be tolerated at somewhat low levels, but their content in biological fluids or real samples is finally far below the tolerance listed when the sample is diluted for determination. Furthermore, protein and nucleic acids only give less interference on the method. Cysteine interferes the detection when its concentration is higher than 10 times concentration of GSH. However, cysteine content is very low in whole blood. Thiol-containing compounds in whole blood include GSH, Cys, etc. As the GSH content is higher than 90% of the total thiol-containing compounds in blood, thiol compounds in whole blood can be regarded as GSH [33]. The experimental results indicate that the method has a high selectivity. So the method is practical and it should be able to be used to determine GSH in whole blood (Table 2).

pected with further study in the future. We envisage that the development of organic nanoparticles for biological labeling or fluorescent probes will open up new possibilities for many multicolor experiments and diagnostics. It is expected that this kind of nanoparticles as effective biological labels will have more and more applications in biochemistry and life science research.

Acknowledgments The Special foundation of Anhui Normal University (2004x2x02) Education Commission Natural Science Foundation for Young Teacher of Anhui Province (2004jq121) and the National Natural Science Foundation of China (20375001) supported this work.

4. Conclusion In this paper, a new fluorescence method for the determination of reduced glutathione with organic nanoparticles was reported. The organic nanoparticles of 1pyrenemethylamine have been prepared by reprecipitation method. 1-pyrenemethylamine is not soluble in water, however, the out surface of the nanoparticles have abundant amino groups that rend the nanoparticles water-soluble. The particles were characterized by using TEM and fluorescence analysis. The particles averaged 25 nm in diameter, showed relatively efficient fluorescence, a great reduction in photobleaching, colloidal stability and low nonspecific adsorption. They also have longer fluorescence lifetime than 1pyrenemethylamine. They are easily prepared by relatively simple procedures. At weak basic medium, without any modification, these nanoparticles have been used as a fluorescent probe for fluorometric determination of GSH. Because fluorescent organic small molecules are very abundant and commercial available, and some of the fluorescent organic small molecules have functional groups, which makes the organic nanoparticles will be easily acquired and be able to react with biomolecules without any modification. The proposed method provided an easy approach of combining nanotechnology with biomolecules for biochemical analysis and biotechnology applications. However, the proposed approach is still in an early phase of its development, and considerable improvement in sensitivity and limit of detection can be ex-

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