The reaction between fullerenol (F-ol) and cytochrome C (Cyt C) could be carried out to form a nonphosphorescence compound using tween-80 as photosensitizer which causes the sharp quenching of room temperature phosphorescence (RTP) of F-ol. Bearing this in mind, a novel solid substrate room temperature phosphorimetry (SSRTP) for the determination of trace Cyt C has been proposed in this study. Under the optimum conditions, the linear range of this method is
In recent years, the studies have been found that the incidence of leukemia and the execution of chemotherapy drugs to leukemia cells are closely related to cell apoptosis [
The study showed that F-ol and Cyt C could emit weak RTP on nitrocellulose membrane (NCM) using Pb2+ as perturber when the system was heated at 50°C for 15 min. The RTP signal of F-ol was enhanced sharply and the emission wavelength of F-ol blue shifted for 22.2 nm in the presence of tween-80. When adding 9.6 ag Cyt C in tween-80-F-ol system, the RTP signal of F-ol was quenched sharply (
Phosphorescent measurements were carried out on Perkin Elmer LS-55 luminescence spectrophotometer with a solid surface analysis apparatus (Perkin-Elmer Corporation, USA). The instrument’s main parameters are as follows: Ex. Slit: 10 nm; Em. Slit: 8 nm; and a scan speed: 1500 nm min−1. The pHS-3B precision acidometer (Shanghai Medical Laser Instrument Plant), 85-1 constant temperature magnetic stirrer (Beijing Taike Instruments Company), AE240 electronic analytical balance (Mettler-Toledo Instruments Shanghai Company), and 0.50-
Filter paper was purchased from Xinhua Paper Corporation (Hangzhou, China). NCM, acetyl cellulose membrane (ACM), and polyamide membrane (PAM) were purchased from Luqiaosijia Biochemical Plastic (Hangzhou, China). Precut into wafers (diameter
To a 25-mL colorimetric tube, certain amount of Cyt C, 1.00 mL F-ol, and 2.00 mL tween-80 was added, diluted to 25 mL with KH2PO4-Na2HPO4 buffer solution (pH 6.08), and mixed homogeneously. The colorimetric tube was kept at 50°C for 15 min and cooled down by flowing water for 5 min to stop the reaction. The NCM wafers were immersed in 1.00 mol L−1 Pb2+ solution for 10 s then dried at
The phosphorescence spectra of Cyt C-F-ol-tween-80 were scanned by the experimental method. As shown in Figure
RTP spectra of Cyt C-F-ol-tween-80 system (Curves 1–7 are the excitation spectra, and curves 1′–7′ are the emission spectra. 1.1′, 2.2′, 3.3′, 4.4′, 5.5′, 6.6′, and 7.7′ are the numbers of the excitation spectra and the emission spectra. Two sets of data corresponding 1′–7′ are emission wavelength and intensity of RTP, resp.). 1.1′ NCM, 2.2′ 9.6 ag spot−1 Cyt C, 3.3′ 4.4′ + 9.6 ag spot−1 Cyt C, 4.4′ 1.00 mL F-ol, 5.5′ 7.7′ + 9.6 ag spot−1 Cyt C, 6.6′ 7.7′ + 0.040 ag spot−1 Cyt C, 7.7′ 4.4′ + 2.00 mL tween-80.
For the system containing 2.40 ag Cyt C spot−1, the effect of the volumes and concentrations of reagents, reaction acidity, reaction temperature and time, oxygen, temperature and time for drying, standing time, solid substrates, ion perturbers, photosensitizers, and buffer solutions on
The measurement condition.
The measurement condition | The |
Optimal | |
---|---|---|---|
F-ol ( |
1.0, 0.10, 0.010, 0.0010 |
21.2, 34.1, 15.7, 7.2 |
|
tween-80 (1%) |
0.5, 1.0, 2.0, 3.0, 5.0 |
8.22, 11.9, 34.5, 28.7, 23.5 |
2.0% |
Pb2+ ( |
0.10, 0.25, 0.75, 1.00, 1.20 | 14.8, 16.2, 20.5, 33.8, 20.9 | 1.00 mol L−1 |
pH of the system | 1.99, 3.42, 4.98, 5.76, 6.08, 6.63, 7.42, 8.31, 10.05 | 4.6, 17.4, 33.2, 33.9, 34.1, 33.6, 33.4, 21.4, 12.9 | 4.98–7.42 |
Reaction temperature (°C) | 30, 40, 50, 60, 70, 80, 90 | 10.5, 23.7, 34.3, 24.0, 15.6, 12.3, 5.2 | 50°C |
Reaction time (min) | 5, 10, 15, 20, 25 | 5.1, 13.9, 33.5, 25.6, 19.1 | 15 min |
Temperature for drying (°C) | 50, 60, 70, 80, 90, 95 | 10.9, 13.1, 18.3, 26.1, 33.6, 21.7 | 90°C |
Time for drying (min) | 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 | 16.4, 20.2, 22.9, 27.7, 33.0, 28.2 | 2 min |
Standing time (min) | 10, 20, 30, 40, 50, 60, 70, 80 | 33.1, 33.7, 33.9, 34.0, 34.2, 33.8, 21.4, 12.9 | 10–60 min |
Effects of solid substrates (B), ion perturbers (C), photosensitizers (D), and buffer solution (E) on the
The
Comparison of analytic methods (Note:
Method | Linear range |
Regression equation |
|
DL |
RSD |
---|---|---|---|---|---|
Paper method |
|
|
0.9994 |
|
2.3–3.0 |
Rayleigh scattering spectroscopy [ |
|
|
0.9987 |
|
0.63–0.82 |
Voltammetric method [ |
|
|
0.999 |
|
|
Protein blotting method [ |
0.2–600 p mol L−1 |
|
0.9989 |
|
2.3 |
Table
Cyt C was determined by this method (6.0 fg Cyt C mL−1) and the method in [
The effect of coexistent materials.
Paper method | Reference [ | ||
---|---|---|---|
Coexistent ions |
Allowed concentration |
Er (%) | Allowed concentration |
Cu2+ | 0.10 | 2.0 | Interfere |
Ni2+ | 0.10 | 1.3 | Interfere |
H2C2O4 | 0.52 | 2.5 | 0.0025 |
Mn2+( |
0.82 | −1.1 | 0.008 |
Sr2+ | 0.88 | 0.3 | — |
H2 |
1.60 | 0.1 | 0.015 |
Ba2+ | 1.80 | 0.7 | 0.010 |
Ag+ | 1.80 | −0.8 | 0.010 |
H |
1.98 | 1.9 | — |
Zn2+( |
2.00 | 1.2 | 0.015 |
Ca2+ | 2.00 | 3.4 | 0.030 |
Mg2+ | 2.10 | −1.7 | 0.020 |
C1− | 2.25 | 2.2 | 0.040 |
Glucose | 2.70 | −1.5 | 0.060 |
Sucrose | 3.20 | 3.0 | 0.15 |
Drawn from Table
1.00 mL serum of childhood leukemia patients A, B, C, D, E, and F (from fasting) was taken and diluted to fg level with KH2PO4-Na2HPO4 buffer solution of pH 6.08. The Cyt C content of the samples was determined by this method described above, and a standard addition recovery rate experiment was also conducted. This result was compared with ELISA method and listed in Table
The analytical results of Cyt C in human serum (added is 1.00 mg Cyt C L−1.
Sample | This method | ELISA method | |||
---|---|---|---|---|---|
Found |
Percent recovery |
RSD |
Found |
Er (%) | |
Serum A | 0.41 | 102.6 | 4.4 | 0.42 | 2.4 |
Serum B | 6.67 | 100.8 | 0.25 | 6.50 | 1.5 |
Serum C | 19.33 | 97.7 | 3.8 | 19.61 | 1.4 |
Serum D | 41.00 | 100.7 | 1.1 | 40.68 | 0.79 |
Serum E | 50.98 | 101.1 | 1.9 | 49.00 | 4.0 |
Serum F | 84.01 | 98.3 | 0.91 | 81.12 | 2.5 |
According to the method described [
In the weak acid circumstances, −OOC– in Cyt C molecule could react with H+ to form HOOC– [
The reaction between −OOC– in the Cyt C molecule and H+.
F-ol could emit weak RTP on NCM using Pb2+ as perturber when heated at 50°C for 15 min (Figure
Interaction between tween-80 and F-ol.
In the micellae compound, on the one hand, the sorption took place between the polyoxyethylene of tween-80 and the conjugate structure of F-ol; on the other hand, hydrophile group (such as –OH) was introduced to the surface of F-ol, forming amphiphilic structure with hydrophobic group itself. Thus, the surface tension of solution reduced and the interface state of the system was changed [
Reaction between Cyt C molecule and F-ol molecule.
The content of Cyt C had linear relationship with the
In order to prove the reaction probability between F-ol and Cyt, the infrared spectra of F-ol, Cyt C, and F-ol-Cyt compounds were scanned by Nicolet-360 infrared spectrometer (KBr pellet) ranging from 200 cm−1 to 4000 cm−1. The results are listed in Table
Infrared spectra data.
Compounds | –NH2 | –OH | –CH2 | –CH– | C=O | C=C | –NH | C–C | C–S | C–O |
---|---|---|---|---|---|---|---|---|---|---|
F-ol | 3427 | 2960 | 1532 | 1670 | 1573 | |||||
Cyt C | 3468 | 3421 | 2950 | 1527 | 1662 |
1665 | 1618 | 1210 | ||
F-ol-Cyt C | 3463 | 2936 | 1523 | 1654 |
1660 | 1612 | 1558 | 1206 | 1092 |
Table
The SSRTP had high sensitivity, good selectivity, and has been applied to the determination of Cyt-C-Fe (III) in human serum and the prediction of human diseases, which promoted the research progress of ultratrace biological active substances analysis. Moreover, the research on the relationship between Cyt-C-Fe (III) and human diseases was carried out and the forecast of human diseases according to the content of Cyt-C-Fe (III) was realized, which expanded the application field of this research. Besides, the mechanism of SSRTP for the determination of Cyt-C-Fe (III) was discussed, which laid a theoretical foundation for the exploitation of new prediction technique of human diseases and the new development of SSRTP.
This project is supported by the Fujian Province Natural Science Foundation (no. 2010J01053), Fujian Province Education Committee (JK2010035, JA10203, JA10277, and JA11311), Fujian Provincial Bureau of Quality and Technical Supervision (no. FJQI2011006), and Scientific Research Program of Zhangzhou Institute of Technology Foundation (nos. ZZY1101, ZZY1106, and ZZY1014). At the same time, the authors are very grateful to precious advices raised by the reviewers.