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

Authors: Zhi-Yong Zheng; Ma-Lin Cui; Jia-ming Liu; Shu-Lian Jiang; Li Jiao; Li-Hong Zhang; Xuan Lin; Shao-Qin Lin
Journal: ISRN Spectroscopy
 (2012)

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## Abstract

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 is1.0×10-16
–2.4×10-14  gmL-1, which is directly proportional to ΔIp of Cyt C-F-ol–tween-80 system, and the detection limit (DL) is 3.0×10−17 gmL-1. It has been applied to the determination of Cyt C in human serum and forecast of diseases, and the result matches with the enzyme-linked fluorescence immunoassay (ELISA). Meanwhile, the reaction mechanism of SSRTP for the determination of Cyt C and the enhancing effect of tween-80 on RTP of F-ol were also discussed.


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## Body

## 1. Introduction

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 [1]. Studies on the clinical examination of Cyt C and the researches about its proapoptotic activity regulating molecular mechanism and the release mechanism have become a focus [2]. There are many methods for the determination of Cyt C, such as synchronous fluorescence spectroscopy (linear range: 3.6×10-5–1.92×10-3 g mL−1) [3], CdTe/CdS quantum dots resonance rayleigh scattering spectroscopy (DL: 1.1×10-9 g mL−1) [4], electrochemical probe voltammetric method (DL: 1.6×10-4 g mL−1) [5], cyclic voltammetry [6], adsorption method (DL: 5.0×10-3 g mL−1) [7], ED/Au/I− modified ultramicroelectrode method (DL: 4.0×10-6 g mL−1) [8], capillary zone electrophoretic method (DL: 7.4×10-13 g mL−1) [9], amperometric method (DL: 4.2×10-5 g mL−1) [10], protein blotting method (DL: 5.0×10-5 g mL−1) [11], and ELISA (DL: 1.0×10-9 g mL−1) [12]. All these methods cannot meet the needs of determining the low content of Cyt C in biological samples due to low sensitivity. Therefore, searching for a high sensitive and accurate method for the determination of Cyt C in biological samples and discussing the relativity between the content of Cyt C and cell apoptosis have become research focus with high academic value and great significance. There have been many reports on the synthesis of multihydroxyl C60 derivatives, F-ol derivatives, aminophenol derivatives, dendritic fullerene derivatives [13–15], the fluorescent property of water-soluble F-ol [16], and the determination of alkaline phosphatase [17, 18], glucose [19], As (V) [20], and Mn2+ [21] based on phosphorescent property of F-ol, showing broad prospects of F-ol in analytical application. 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 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 (ΔIp=119.6), which was 9.4 times stronger than that (ΔIp=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.


## 2. 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]), KH2PO4-Na2HPO4 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.


### 2.2. 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 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 90±1°C for 2 min. A 0.40 μL drop of test solution was suspended onto the indentation center of membrane wafers by a 0.50-μL flat head microinjector, and the NCM was dried at 90±1°C for 2 min. A blank test was conducted simultaneously. The phosphorescence spectra were scanned; the emission phosphorescence intensity of the blank reagent (Ip1) and the test solution (Ip2) were recorded. Then, ΔIp (=Ip1-Ip2) was calculated.


## 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]), KH2PO4-Na2HPO4 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.


## 2.2. 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 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 90±1°C for 2 min. A 0.40 μL drop of test solution was suspended onto the indentation center of membrane wafers by a 0.50-μL flat head microinjector, and the NCM was dried at 90±1°C for 2 min. A blank test was conducted simultaneously. The phosphorescence spectra were scanned; the emission phosphorescence intensity of the blank reagent (Ip1) and the test solution (Ip2) were recorded. Then, ΔIp (=Ip1-Ip2) was calculated.


## 3. 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 Figure1, F-ol and Cyt C could emit weak RTP on NCM whose λexmax/λemmax were 478.8/645.2 nm and 468.6/634.4 nm while Ip 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 λemmax blue shifted for 22.2 nm (λexmax/λemmax = 457.0/623.0 nm, Ip = 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 (λexmax/λemmax = 456.0/621.9 nm, Ip = 94.1, curve 5.5′), with the ΔIp being 119.6, which was 9.4 times stronger than that without tween-80 (Ip = 66.8, curve 3.3′, ΔIp = 12.7). Thus, 456/622 nm was chosen as the working wavelength for the determination of trace Cyt C. Besides, Cyt C caused the RTP signal of F-ol quenching and λemmax blue shifting from 645.2 nm to 622.0 nm (Δλemmax = 23.3, curve 3.3′); the reason may be that a nonphosphorescence compound was generated from the reaction between F-ol and Cyt C.Figure 1
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.


### 3.2. 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 ΔIp of the system were tested, respectively (Table 1 and Figure 2). The results show that the ΔIp 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 Pb2+ 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°C, 15 min, 90°C, 2 min, NCM, Pb2+, tween-80, and KH2PO4-Na2HPO4, respectively. Under the optimal reaction conditions mentioned above, whether desiccated N2 was passed, the ΔIp 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 1.99–5.76 and 6.63–10.0 pH 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.Table 1
The measurement condition.
The measurement condition
TheΔIp of Cyt C-F-ol-tween-80 system
Optimal
F-ol (×10-4 mol L−1) (mL)
1.0, 0.10, 0.010, 0.00100.50, 1.00, 1.50, 2.00, 2.50
21.2, 34.1, 15.7, 7.211.6, 33.7, 26.1, 19.0, 17.5
1.0
×
10
-
5 mol L−1  1.00 mL
tween-80 (1%)(mL)
0.5, 1.0, 2.0, 3.0, 5.01.00, 1.50, 2.00, 2.50, 3.00, 4.00
8.22, 11.9, 34.5, 28.7, 23.58.6, 20.9, 33.5, 28.3, 26.1, 17.8
2.0%2.00 mL
Pb2+  (10-4 mol L−1)
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 minFigure 2
Effects of solid substrates (B), ion perturbers (C), photosensitizers (D), and buffer solution (E) on theΔIp of Cyt C-F-ol-tween-80 system (Barss a, b, c, and d were PAM, ACM, NCM, and paper, resp.. The e, f, g, h, and I were I− (NaI), Li+ (LiNO3), Hg2+ (Hg(NO3)2), Cd2+ (Cd(NO3)2), and Pb2+ (Pb(Ac)2), resp.. Barss j, k, l, m, n, and o were sodium lauryl sulfonate, tween-80, ethyltrimethylammonium bromide, Triton X-100, sodium dodecyl benzene sulfonate, and polyvinyl alcohol, resp.. Barss p, q, and w were Na2CO3-NaHCO3, KH2PO4-Na2HPO4, and Tris-HCl, resp.. The ΔIp of Cyt C-F-ol-tween-80 system reached the maximum when the solid substrates, ion perturber, photosensitizers, and buffer solution were NCM, Pb2+, tween-80, and KH2PO4-Na2HPO4, resp.. The concentrations of ion perturber and photosensitizer were 1.0 × 10−4 mol L−1 and 2%, resp.).


### 3.3. Working Curve, Linear Range, and DL

TheΔIp 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 × 10−14g 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
Comparison of analytic methods (Note:y and x in [11] were peak area and analyte concentration (pM), resp.).
Method
Linear range(g mL−1)
Regression equation
r
DL(g mL−1)
RSD(%)
Paper method
1.0
×
10
-
16
–2.4×10-14
Δ
I
p
=
1.609
+
12.39
m
Cyt C
(ag spot−1) (Sb = 0.048, n = 6)
0.9994
3.0
×
10
-
1
7
2.3–3.0
Rayleigh scattering spectroscopy [4]
3.6
×
10
-
4
–1.31×10-6
I
=
27.4
+
721.98
C (mg L−1) (n = 5)
0.9987
10.8
×
10
-
9
0.63–0.82
Voltammetric method [5]
1.0
×
10
-
6
–16×10-6
i
pa
=
15.0
-
0.269
C
0.999
1.6
×
10
-
7
Protein blotting method [11]
0.2–600 p mol L−1
y
=
(
1.6149
×
10
5
)
x
+
3.4579
×
10
4
0.9989
7.4
×
10
-
1
3
2.3Table2 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.


### 3.4. 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.Table 3
The effect of coexistent materials.
Paper method
Reference [4]
Coexistent ions(material)
Allowed concentration(mg L−1)
Er (%)
Allowed concentration(mg L−1)
Cu2+
0.10
2.0
Interfere
Ni2+
0.10
1.3
Interfere
H2C2O4
0.52
2.5
0.0025
Mn2+(SO42-)
0.82
−1.1
0.008
Sr2+
0.88
0.3
—
H2PO4-
1.60
0.1
0.015
Ba2+
1.80
0.7
0.010
Ag+
1.80
−0.8
0.010
HPO42-
1.98
1.9
—
Zn2+(SO42-)
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
0.15Drawn from Table3, allowed multiples of coexistent materials (ions) of this method were larger than those in [3] showing good selectivity.


### 3.5. 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 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 4.Table 4
The analytical results of Cyt C in human serum (added is 1.00 mg Cyt C L−1. n=6)
Sample
This method
ELISA method
Found(mg L−1)
Percent recovery(%)
RSD(%)
Found(mg L−1)
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
2.5According 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.


### 3.6. 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.Scheme 1
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 1, curve 4.4′). The RTP signal of F-ol was enhanced sharply when tween-80 existed and λemmax 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.Scheme 2
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 [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 λemmax 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′).Scheme 3
Reaction between Cyt C molecule and F-ol molecule.The content of Cyt C had linear relationship with theΔIp of the system, and the ΔIp was 9.4 times stronger than that without tween-80. The change of the ΔIp and λemmax 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
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
16621548
1665
1618
1210
F-ol-Cyt C
3463
2936
1523
16541545
1660
1612
1558
1206
1092Table5 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 F-ol and Cyt C to form F-ol-Cyt C.


## 3.1. Phosphorescence Spectra

The phosphorescence spectra of Cyt C-F-ol-tween-80 were scanned by the experimental method. As shown in Figure1, F-ol and Cyt C could emit weak RTP on NCM whose λexmax/λemmax were 478.8/645.2 nm and 468.6/634.4 nm while Ip 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 λemmax blue shifted for 22.2 nm (λexmax/λemmax = 457.0/623.0 nm, Ip = 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 (λexmax/λemmax = 456.0/621.9 nm, Ip = 94.1, curve 5.5′), with the ΔIp being 119.6, which was 9.4 times stronger than that without tween-80 (Ip = 66.8, curve 3.3′, ΔIp = 12.7). Thus, 456/622 nm was chosen as the working wavelength for the determination of trace Cyt C. Besides, Cyt C caused the RTP signal of F-ol quenching and λemmax blue shifting from 645.2 nm to 622.0 nm (Δλemmax = 23.3, curve 3.3′); the reason may be that a nonphosphorescence compound was generated from the reaction between F-ol and Cyt C.Figure 1
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.


## 3.2. 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 ΔIp of the system were tested, respectively (Table 1 and Figure 2). The results show that the ΔIp 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 Pb2+ 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°C, 15 min, 90°C, 2 min, NCM, Pb2+, tween-80, and KH2PO4-Na2HPO4, respectively. Under the optimal reaction conditions mentioned above, whether desiccated N2 was passed, the ΔIp 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 1.99–5.76 and 6.63–10.0 pH 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.Table 1
The measurement condition.
The measurement condition
TheΔIp of Cyt C-F-ol-tween-80 system
Optimal
F-ol (×10-4 mol L−1) (mL)
1.0, 0.10, 0.010, 0.00100.50, 1.00, 1.50, 2.00, 2.50
21.2, 34.1, 15.7, 7.211.6, 33.7, 26.1, 19.0, 17.5
1.0
×
10
-
5 mol L−1  1.00 mL
tween-80 (1%)(mL)
0.5, 1.0, 2.0, 3.0, 5.01.00, 1.50, 2.00, 2.50, 3.00, 4.00
8.22, 11.9, 34.5, 28.7, 23.58.6, 20.9, 33.5, 28.3, 26.1, 17.8
2.0%2.00 mL
Pb2+  (10-4 mol L−1)
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 minFigure 2
Effects of solid substrates (B), ion perturbers (C), photosensitizers (D), and buffer solution (E) on theΔIp of Cyt C-F-ol-tween-80 system (Barss a, b, c, and d were PAM, ACM, NCM, and paper, resp.. The e, f, g, h, and I were I− (NaI), Li+ (LiNO3), Hg2+ (Hg(NO3)2), Cd2+ (Cd(NO3)2), and Pb2+ (Pb(Ac)2), resp.. Barss j, k, l, m, n, and o were sodium lauryl sulfonate, tween-80, ethyltrimethylammonium bromide, Triton X-100, sodium dodecyl benzene sulfonate, and polyvinyl alcohol, resp.. Barss p, q, and w were Na2CO3-NaHCO3, KH2PO4-Na2HPO4, and Tris-HCl, resp.. The ΔIp of Cyt C-F-ol-tween-80 system reached the maximum when the solid substrates, ion perturber, photosensitizers, and buffer solution were NCM, Pb2+, tween-80, and KH2PO4-Na2HPO4, resp.. The concentrations of ion perturber and photosensitizer were 1.0 × 10−4 mol L−1 and 2%, resp.).


## 3.3. Working Curve, Linear Range, and DL

TheΔIp 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 × 10−14g 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
Comparison of analytic methods (Note:y and x in [11] were peak area and analyte concentration (pM), resp.).
Method
Linear range(g mL−1)
Regression equation
r
DL(g mL−1)
RSD(%)
Paper method
1.0
×
10
-
16
–2.4×10-14
Δ
I
p
=
1.609
+
12.39
m
Cyt C
(ag spot−1) (Sb = 0.048, n = 6)
0.9994
3.0
×
10
-
1
7
2.3–3.0
Rayleigh scattering spectroscopy [4]
3.6
×
10
-
4
–1.31×10-6
I
=
27.4
+
721.98
C (mg L−1) (n = 5)
0.9987
10.8
×
10
-
9
0.63–0.82
Voltammetric method [5]
1.0
×
10
-
6
–16×10-6
i
pa
=
15.0
-
0.269
C
0.999
1.6
×
10
-
7
Protein blotting method [11]
0.2–600 p mol L−1
y
=
(
1.6149
×
10
5
)
x
+
3.4579
×
10
4
0.9989
7.4
×
10
-
1
3
2.3Table2 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.


## 3.4. 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.Table 3
The effect of coexistent materials.
Paper method
Reference [4]
Coexistent ions(material)
Allowed concentration(mg L−1)
Er (%)
Allowed concentration(mg L−1)
Cu2+
0.10
2.0
Interfere
Ni2+
0.10
1.3
Interfere
H2C2O4
0.52
2.5
0.0025
Mn2+(SO42-)
0.82
−1.1
0.008
Sr2+
0.88
0.3
—
H2PO4-
1.60
0.1
0.015
Ba2+
1.80
0.7
0.010
Ag+
1.80
−0.8
0.010
HPO42-
1.98
1.9
—
Zn2+(SO42-)
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
0.15Drawn from Table3, allowed multiples of coexistent materials (ions) of this method were larger than those in [3] showing good selectivity.


## 3.5. 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 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 4.Table 4
The analytical results of Cyt C in human serum (added is 1.00 mg Cyt C L−1. n=6)
Sample
This method
ELISA method
Found(mg L−1)
Percent recovery(%)
RSD(%)
Found(mg L−1)
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
2.5According 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.


## 3.6. 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.Scheme 1
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 1, curve 4.4′). The RTP signal of F-ol was enhanced sharply when tween-80 existed and λemmax 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.Scheme 2
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 [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 λemmax 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′).Scheme 3
Reaction between Cyt C molecule and F-ol molecule.The content of Cyt C had linear relationship with theΔIp of the system, and the ΔIp was 9.4 times stronger than that without tween-80. The change of the ΔIp and λemmax 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
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
16621548
1665
1618
1210
F-ol-Cyt C
3463
2936
1523
16541545
1660
1612
1558
1206
1092Table5 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 F-ol and Cyt C to form F-ol-Cyt C.


## 4. 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.



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*Source: 918093-2012-12-26.xml*