A Novel Solid Substrate Room Temperature Phosphorimetry for the Determination of Trace Cytochrome C and Forecast of Human Diseases

1 Department of Food and Biological Engineering, Zhangzhou Institute of Technology, Zhangzhou 363000, China 2 Department of Chemistry and Environmental Science, Zhangzhou Normal College, Zhangzhou 363000, China 3 Department of Food Safety Verification, Fujian Provincial Bureau of Quality and Technical Supervision, Zhangzhou 363000, China 4 Department of Biochemistry, Fujian Education College, Fuzhou 350001, China

New SSRTP for the chlorpromazine hydrochloride [22], quercetin [23], rhamnose [24], and carvedilol [25] detection promoted the progress in study of trace drugs analysis.However, SSRTP for the determination of trace Cyt C and forecast of human diseases has been rarely reported based on the enhancing effect of tween-80 on RTP of F-ol.
The study showed that F-ol and Cyt C could emit weak RTP on nitrocellulose membrane (NCM) using Pb 2+ 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 (ΔI p = 119.6),which was 9.4 times stronger than that (ΔI p = 12.7) without tween-80.According to the facts above, a new SSRTP has been established for the determination of trace Cyt C and the forecast of human diseases resorted to the relativity between the content of Cyt C and cell apoptosis.This research not only develops a new photosensitizer and a new method for improving the sensitivity of trace determination of Cyt C, but also opens up a new application field of F-ol in life sciences, which provides a new technique for the forecast of human diseases in clinic.

Experimental
2.1.Apparatus and Reagents.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-μL flat head microinjector (Shanghai Medical Laser Instrument Plant) were used to introduce solution.Preparation of Cyt C (Sigma Company) working solution is as follows: 1.00 mg mL −1 Cyt C primary standard solution was diluted to 100.00, 10.00, and 1.00 fg mL −1 as working solution; 1.0 × 10 −5 mol L −1 F-ol (synthesized by the method mentioned in [26]), KH 2 PO 4 -Na 2 HPO 4 buffer solution of pH 6.08, and 2.0% (W/V) tween-80 were also prepared.Cyt C is standard reagent and all other reagents are of analytically pure.The water used was thrice subboiling distillation.
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 (Φ) = 1.5 cm) and a ring indentation (Φ = 4.0 mm) were made at the center of each sheet with a standard pinhole plotter for use.

Experimental Method.
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

Results and Discussion
3.1.Phosphorescence Spectra.The phosphorescence spectra of Cyt C-F-ol-tween-80 were scanned by the experimental method.As shown in Figure 1, F-ol and Cyt C could emit weak RTP on NCM whose λ max ex /λ max em were 478.8/645.2nm and 468.6/634.4nm while I p were 79.5 and 55.0 (curve 4.4 � , 2.2 � ), respectively.The RTP signal of F-ol was enhanced sharply when tween-80 existed and λ max em blue shifted for 22.2 nm (λ max ex /λ max em = 457.0/623.0nm, I p = 213.7,curve 7.7 � ), which hinted that a new micellar compound was generated in this system.When 9.6 ag Cyt C was added in this system, the RTP signal of F-ol was quenched sharply (λ max ex /λ max em = 456.0/621.9nm, I p = 94.a nonphosphorescence compound was generated from the reaction between F-ol and Cyt C.

Optimum Measurement Condition.
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 ΔI p of the system were tested, respectively (Table 1 and Figure 2).The results show that the ΔI p of the system reached the maximum when 1.00 mL of 1.0 × 10 −5 mol L −1 F-ol, 2.00 mL of 2% tween-80, and 1.00 mol L −1 Pb 2+ were used; pH of the reaction system, reaction temperature and time, temperature and time for drying, solid substrate, ion perturber, photosensitizer, and buffer solution were 4.98-7.42,50 Under the optimal reaction conditions mentioned above, whether desiccated N 2 was passed, the ΔI p of the system almost stayed invariable and possessed good repeatability within 10-60 min after being cooled down by flowing water for 5 min.At this point, the pH value of reaction solution was 6.08.Maybe that the effect of the change of the sixth porphyrin ligand in haem to activity of Cyt C was in 1.99-5.76and 6.63-10.0pH large of phosphate buffer solution resulting in deactivation of Cyt C [27].Thereinto, the activity of Cyt C was the largest at pH 6.08.

Working Curve, Linear
Range, and DL.The ΔI p of the system had linear relationship with the content of Cyt C. The linear range, the regression equation of working curve, the correlation coefficient (r), RSD % (7 fold parallel determinations for 0.040 and 9.60 ag spot −1 (sample volume: 0.40 μL spot −1 , corresponding concentration: 1.0 × 10 −16 -2.4 × 10 −14 g mL −1 ) Cyt C), and the DL (calculated by 3 Sb/k, 3 Sb/k referred to the quotient between triple of the blank reagent's standard deviation and the slope of the working curve, Sb referred to the standard deviation of 11 parallel analysis of the blank reagent) of this method were compared with those of [4,5,11], and the results are listed in Table 2.
Table 2 shows that the sensitivity of this method was higher than that of [4,5,11], so the method can be used to the determination of trace Cyt C in real sample.

Interference Experiment.
Cyt C was determined by this method (6.0 fg Cyt C mL −1 ) and the method in [4] (0.1 mg Cyt C mL −1 ), respectively.When the relative error (Er) was ±5%, the allowed concentrations of coexistent materials (ion) were compared with those in [4], and the results are listed in Table 3.
Drawn from Table 3, allowed multiples of coexistent materials (ions) of this method were larger than those in [3] showing good selectivity.

Sample Analysis.
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 KH 2 PO 4 -Na 2 HPO 4 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 4.
According to the method described [1], when the Cyt C content is in the range of 0-37.5 mg L −1 , apoptosis inhibitory rate of cell increases gradually, which will cause the number of acute myeloid leukemia cell lines HL-60 cells to increase and lead morbidity to increase; when Cyt C content is larger than 37.5 mg L −1 , the increase of apoptosis rate of cell is not obvious, but necrosis cells can induce interleukin to die.The maximum concentration of Cyt C in the serum of systemic inflammatory response and multiple organ dysfunction syndrome patients is up to 0.21 mg L −1 [28].Seen from Table 4, the number of acute myeloid leukemia cell lines HL-60 cells for patients A, B, and C was still increasing, which shows that the illness was aggravated, while serum analysis of patients D, E, and F shows that their state of illness has been under controlled.Thus, this method was not only applied to the determination of the content of Cyt C in human serum, but also used to predict diseases, which indicated the clinical application value of the new method.

Mechanism of Reaction.
In the weak acid circumstances, − OOC-in Cyt C molecule could react with H + to form HOOC- [27].The reaction could be expressed as Scheme 1.

The measurement condition
The ΔI p of Cyt C-F-ol-tween-80 system Optimal F-ol (×10 −4 mol L −1 ) (mL) F-ol could emit weak RTP on NCM using Pb 2+ as perturber when heated at 50 • C for 15 min (Figure 1, curve 4.4 � ).The RTP signal of F-ol was enhanced sharply when tween-80 existed and λ max em had a blue shift for 22.2 nm (Figure 1, curve 7.7 � ).It maybe that tween-80-F-ol micellae formed by the interaction between tween-80 and F-ol (Scheme 2) causing F-ol emitting strong and steady RTP.
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 Fol, forming amphiphilic structure with hydrophobic group itself.Thus, the surface tension of solution reduced and the interface state of the system was changed [29].After such management, it not only had the solubilization effect but also made tween-80 well dispersed and adsorbed in the side-wall of F-ol.The polyoxyethylene of tween-80 extended to F-ol and densely covered on the surface of F-ol. to form a certain thickness of hydrophilic phase [30] and then promoted the formation of tween-80-F-ol micellae, which not only could cause the λ max em to blue shifted, but also could augment the solubility of F-ol in water.As a result, F-ol could emit strong and steady RTP.Then the reaction was carried out between HOOC-in Cyt C molecule and -OH in F-ol molecule [18], then a nonphosphorescence compound F-ol-Cyt C formed (Scheme 3) causing the RTP of F-ol quenching sharply (Figure 1, curve 5.5 � ).
The content of Cyt C had linear relationship with the ΔI p of the system, and the ΔI p was 9.4 times stronger than that without tween-80.The change of the ΔI p and λ max em could not only prove that the reaction between Cyt C and F-ol could be carried out, but also show that tween-80 had a promoting effect for the reaction to form a nonphosphorescence compound causing the RTP signal to quenching.Therefore, the content of Cyt C can be determined by the SSRTP.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 5.
Table 5 showed that a new characteristic absorption peak of ν -C-O in F-ol-Cyt C appeared, and the characteristic absorption peaks of other groups had little change, which proved the possibility of the reaction occurrence between Fol and Cyt C to form F-ol-Cyt C.

Conclusion
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.

Scheme 1 :
Scheme 1: The reaction between − OOC-in the Cyt C molecule and H + .

Table 1 :
The measurement condition.

Table 3 :
The effect of coexistent materials.

Table 4 :
The analytical results of Cyt C in human serum (added is 1.00 mg Cyt C L −1 .n = 6)