Quenching electrochemiluminescential analysis of pico-molar level glutathione

Tu Yifeng, Qi Yingying, Wu Ying, Di Junwei
(College of Chemistry and Chemical Engineering, Suzhou University, Suzhou, 215006, China)

Received Aug. 11, 2003; Supported by the National Natural Science Foundation of China (No. 20275025).

Abstract Based on the study of highly sensitized electrochemiluminescence (ECL) of luminol by iodide, in neutral medium, because of the production of free radicals, the authors have developed a sensitive quenching ECL method to determine the reductive glutathione(GSH) due to its elimination for the free radicals. The detect limit of this method has decreased to pico-molar level. It is more sensitive than the methods ever reported .
Keywords Electrochemiluminescence; Luminol; Quenching; Glutathione

1. INTRODUCTION
The electrochemiluminescence(ECL)^[1] is in prospect to be used in trace analysis, immunoassay, biochemical analysis and some other fields^[2,3]. In general, the ECL behaviour of luminol in alkaline medium is superior in intensity to that of in neutral medium^[4]. But for the object to determine the bioactive molecules, the neutral medium would be more suitable for keeping their original properties. The authors have already studied the ECL behaviour of luminol in neutral medium^[5]. Although it revealed that the ECL of luminol in neutral condition was weaker than that in alkaline medium, it has been indicated that some redox species such as oxygen, hydrogen peroxide and iodide can enhance the luminous intensity of luminol in neutral medium on a large scale. It provides an excellent basis to determine the glutathione(GSH)^[6] by quenching effect for iodide enhanced ECL and furthermore to develop a sensitive analytical method of glutathione at pico-molar level of concentration.
    The glutathione widely distributes in natural world. It is an important bioactive peptide for eliminating the free radicals, detoxicating, postponing the aging and relieving the fatigue. It is also the participant of many enzymatic reactions as the role of coenzyme to transfer the electrons. The studies on its determination and the reaction mechanism have attracted great attentions. The improvement on its sensitive determination is especially expected. The ECL quenching method for determination of glutathione, which will be described in this paper, can detects it with a limit concentration at pico-molar level. It is the most sensitive one in the previously reported methods. The probable mechanism of this method is also discussed.

2. EXPERIMENTS
2.1 Instruments and chemicals
A BAS-100A electrochemical analyzer (Bioanalysis System Inc., USA) was used as a potentiostat with a XFD-8C ultralow frequency signal generator (Ningbo Radio Factory, P.R.China) as the outer pulse signal source. A PMT-II faint light meter, made by the authors, was used to detect the luminous intensity of ECL. The scheme of whole installation is presented in our previous paper^[7].
    A three-electrode cell was used in experiments. The working electrode and the auxiliary electrode were made of platinum. The working electrode was firstly polished with Al₂O₃ powder and then washed with 0.1mol/L NaOH and then water in ultrasonic tank. A silver wire worked as a reference electrode. The glass cell was prepared in advance. It was covered with a silver film that formed in a silver mirror reaction merely reserved a hole to keep transparence for light. And then it was covered with the black paint. The hole was situated in the center of the window of photoelectric multiplier (PMT). The cell and the PMT were sealed in a black box.
    Luminol was purchased from Fluka. The reductive glutathione is the product of Shanghai Chemicals Cooperation. The solution of reductive glutathione was prepared by dissolving it in deoxygenated water. Other chemicals are all analytical reagents and water used in all experiments is the sub-boiling distillate water, prepared in a quartz apparatus.
2.2 The method of determination
The determination was carried out in the phosphate buffer solution (pH 6.7) which containing 4.0×10^-4 mol/L of luminol and 4.0×10^-5 mol/L of iodide. The quenched ECL intensity was recorded after certain volume of glutathione solution had been injected into this solution.

3. RESULTS AND DISCUSSIONS
3.1 The optimized conditions for determination of glutathione
The different kinds of buffer solution could not only result in the difference of luminous intensity, but also the sensitization efficiency of sensitizers. So it must be selected carefully. From the results of experiments, the phosphate buffer (pH 6.7) was the best one for iodide enhancing ECL of luminol and for occurrence of the quenching effect from glutathione. The aforementioned concentrations of luminol and iodide contained in buffer solution were selected for providing a proper luminous intensity. They were profitable for detecting and recording the ECL background and the decreasing after the glutathione was injected into the solution.
    In a single pulse period, the potential of pulse signal was set at the constantly lower voltage of 0.0V for 5s, and then it would raise to the higher voltage of 1.5V for 1s, which resulted in the generating of ECL during this stage. The longer period of low potential was sufficient for achieving the equilibrium of reactants by diffusion. The ECL intensity could be stable under this condition.
3.2 The ECL response of reductive glutathione
Under aforementioned conditions, this method is sensitive for the determination of glutathione. When the glutathione was injected into the solution, the ECL luminous intensity remarkably decreased immediately. Fig. 1 shows the ECL curves of luminol, after sensitization from iodide and then quenched by glutathione.
    The quenched ECL intensity linearly responded to the concentration of reductive glutathione within a wide range. It leaped over eight orders of magnitude from 10^-5 mol/L to 10^-13 mol/L with a detecting limit of 3.38×10^-13mol/L. The attached equation in Fig. 1 is the calibration equation of glutathione on this range and Tab.1 shows the calibration equations of glutathione in several orders of magnitude and the linear regression coefficients.

Fig.1 The ECL curves of luminol, sensitized by iodide and quenched by glutathione

Table 1 The calibration equations of glutathione in several orders of magnitude and the linear regression coefficients

3.3 The mechanism of quenching effect of glutathione for ECL
The iodide enhanced ECL of luminol could be described as a procedure of producing and reacting of free radicals.

GSH + R^· GS^· + RH (The R^· standing for all of free radicals)

    There are two experimental evidences to prove that the iodides were oxidized to form free radicals. Firstly, the ECL intensity of luminol-I^-1 solution was obviously come under the influence of amplitude of rectangle pulse (See Fig. 2).
    It presented a regular pattern that the ECL intensity would quasi-linearly increase within the potential of 1.5V but never emit light if the potential was higher than 1.5V. This phenomenon could be understood as that different products were produced under different potential. From the studies of luminol's ECL in neutral medium^[8], it was known that the luminol could emit the light after the first-stage oxidation at the potential of 0.75-0.9V and be followed by other chemical oxidation especially from some free radicals. So, it could be considered that the pulse potential provided the energy to oxidize the iodide to free radical but not the iodine. It is reasonable that the adaptive potential could result in the production of free radicals, but higher potential might lead to quickly annihilation of free radicals of iodide to produce the iodine due to high concentration, it could not go on to aid the luminescence of luminol's first-stage oxidized product in this condition.
    Another hand, it had shown a special phenomenon that the influence of iodide to the ECL intensity would be different within different concentration ranges. The ECL intensity will trend to decrease if there is higher concentration of iodide other than it could sensitize the ECL at lower concentrations (See Fig.3).
    The believable explanation is that the annihilation of iodide free radicals might be in greater degree at higher concentrations of iodide because of the higher production of free radicals.

Fig. 2 The response of ECL upon the amplitude of pulse

Fig. 3 The variation of ECL intensity with the concentration of iodide

4. CONCLUSION
The experimental results have indicated that the glutathione is an excellent eliminator for free radicals. It results in the sensitive quenching effect for that of the iodide sensitized electrochemiluminescence of luminol in neutral medium. It is hopeful for developing a method for the analysis of glutathione.

REFERENCES
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[4] Kulmala S, Ala-Kleme T, Kulmala A et al. Anal. Chem., 1998, 70: 1112.
[5] Tu Y F, Huang B Q, Guo W Y et al. Chinese J. Anal. Chem. (Fenxi Huaxue), 2002, 30 (6): 729.
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