Fullerol–fluorescein isothiocyanate phosphorescent labeling reagent for the determination of glucose and alkaline phosphatase

Analytical Biochemistry 404 (2010) 223–231

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Fullerol–fluorescein isothiocyanate phosphorescent labeling reagent for the determination of glucose and alkaline phosphatase Jia-Ming Liu a,*, Hong-Xin Wang b, Li-Hong Zhang c, Zhi-Yong Zheng c, Shao-Qin Lin d, Li-Ping Lin a, Xin-Xing Wang a, Chang-Qing Lin c, Jian-Qin Liu a, Qi-Tong Huang a a

Department of Chemistry and Environmental Science, Zhangzhou Normal College, Zhangzhou 363000, People’s Republic of China Xiamen Le’an High School, Xiamen 361021, People’s Republic of China Department of Food and Biological Engineering, Zhangzhou Institute of Technology, Zhangzhou 363000, People’s Republic of China d Department of Biochemistry, Fujian Education College, Fuzhou 350001, People’s Republic of China b c

a r t i c l e

i n f o

Article history: Received 14 March 2010 Received in revised form 5 May 2010 Accepted 20 May 2010 Available online 25 May 2010 Keywords: Bioactive substances Fullerol–fluorescein isothiocyanate Wheat germ agglutinin Affinity adsorption solid substrate room temperature phosphorimetry

a b s t r a c t The active –OH group in fullerol (F-ol) could react with the dissociated –COOH group in fluorescein isothiocyanate (FITC) to form F-ol–(FITC)n, which could emit room temperature phosphorescence (RTP) signal of F-ol and FITC on acetate cellulose membrane (ACM), respectively. Their RTP signals were enhanced by N,N-dimethylaniline (DMA). The labeling reaction between the –NCS group of FITC in DMA–F-ol– (FITC)n and the –NH2 group in wheat germ agglutinin (WGA) produced DMA–F-ol–(FITC)n–WGA, which could further take affinity adsorption (AA) reaction with bioactive substances (BS), such as glucose and alkaline phosphatase (AP), to produce DMA–F-ol–(FITC)n–WGA–BS. Both of these two products could maintain the good RTP characteristics of F-ol and FITC. Based on the facts above, a new phosphorescent labeling reagent, DMA–F-ol–FITC, was developed, and a new affinity adsorption solid substrate room temperature phosphorimetry (AASSRTP) for the determination of BS was established. This method was applied to the determination of BS in human serum and the diagnosis of diseases, with the results agreeing very well with those of enzyme-linked immunosorbent assay (ELISA). The mechanism of DMA–F-ol– (FITC)n labeling of WGA and AASSRTP for the determination of BS is discussed. Ó 2010 Elsevier Inc. All rights reserved.

With the further development of science and technology, the research on fullerene, bioactive substances (BS)1 (e.g., a-fetoprotein variant [AFP-V], glucose, alkaline phosphatase [AP]), and lectin has made great progress. Some original research has been done on the synthesis and reaction characteristics of fullerene [1–3]. In these studies, the luminescent characteristics of fullerene (as C50Cl10) and its ramification (as fullerol [F-ol]) have been found [1]. Furthermore, the repeat rigid structure of F-ol and the signal amplification effect of catalytic reaction were used to establish solid substrate room temperature phosphorimetry (SSRTP) for the determination * Corresponding author. E-mail address: [email protected] (J.-M. Liu). 1 Abbreviations used: BS, bioactive substances; AFP-V, a-fetoprotein variant; AP, alkaline phosphatase; F-ol, fullerol; SSRTP, solid substrate room temperature phosphorimetry; 3.5-GPD-P, 3.5-generation polyamidoamine dendrimers–porphyrin; 4.0-GPD, 4-generation polyamidoamine dendrimers; R-SiO2, silicon dioxide nanoparticle-containing rhodamine 6G; AASSRTP, affinity adsorption solid substrate room temperature phosphorimetry; AA, affinity adsorption; DL, detection limit; DMA, N,N-dimethylaniline; FITC, fluorescein isothiocyanate; RTP, room temperature phosphorescence; WGA, wheat germ agglutinin; BSA, bovine serum albumin; TBAH, tetrabutylammonium hydroxide; ACM, acetate cellulose membrane; PAM, polyamide membrane; NCM, nitrocellulose membrane; RSD, relative standard deviation; ELISA, enzyme-linked immunosorbent assay; BDD, B diasonagraph detection; CXFPD, computer–X-ray–faultage–photography determination. 0003-2697/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2010.05.018

of trace Mn2+ [4] and AP [5]. These studies not only offered new applications of F-ol but also showed that lectin has been used as a glycosyl probe and has been an important tool in the research on the glycoprotein, glycolipid, and glycosyl chain structure [6]. The reaction between the active –COOH or –NH2 in lectin and – NH2 or –COOH in luminescence molecules could be carried out. Through this reaction, many phosphorescent labeling reagents (e.g., 3.5-generation polyamidoamine dendrimers–porphyrin (3.5GPD-P) [7], 4-generation polyamidoamine dendrimers (4.0-GPD) [8,9], silicon dioxide nanoparticle-containing rhodamine 6G (R-SiO2) [10]) and the products of labeled lectin have been developed. The new affinity adsorption solid substrate room temperature phosphorimetry (AASSRTP) for the determination of trace BS has been established based on affinity adsorption (AA) reaction between the labeling products and BS, which not only opened new applications of lectin but also developed SSRTP. Bioactive substances play a key role in many physiological and pathological processes and have close relationships with the occurrence and therapy of many diseases [11]. Therefore, the technique for the determination of glucose, AP, and AFP-V in human serum with high sensitivity and accuracy is very important to diagnose and treat human diseases [7–10,12]. Especially, the discussion about the determination method of BS that related to the serious

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diseases not only has important clinic application value but also has become a frontier study subject of life science. There have been many methods for the determination of glucose such as gas chromatography (linear range = 0.153– 0.294 g g1) [13], high-performance liquid chromatography with evaporative light scattering detector (detection limit [DL] < 1.2  108 g g1) [14], catalytic method of enzyme (DL = 6.0  105 g ml1) [15], catalytic fluorimetry (DL = 5.9  109 g ml1) [16], Fourier transform–near infrared spectroscopy (linear range = 5.0  103 to 9.0  103 g ml1) [17], modified electrode method (DL = 9.0  107 g ml1) [18], and visible absorption spectrophotometry (linear range = 1.8  102 to 3.1  103 g ml1) [19]. There also have been many methods for the determination of the content of AP such as the lectin method [20], immunity prize method [21], affinity chromatography method [22], fluorescence method (DL = 4.0  109 IU ml1) [23], capillary electrophoresis– ultraviolet spectroscopy method (DL = 7.6  1010 g ml1) [24], chemiluminescence analysis method (DL = 1.6  109 g ml1) [25], and tyrosinase and horseradish peroxidase bienzyme biosensor method (DL = 1.4  107 g ml1) [26]. But all of these methods are of low sensitivity. Obviously, further improving sensitivity and selectivity of analytical methods for the determination of glucose and AP not only will promote the development of the research of BS and the prediction of human diseases but also will have great academic research value and application foreground in life science. Hence, N,N-dimethylaniline (DMA)–F-ol–fluorescein isothiocyanate (FITC)n phosphorescent labeling reagent was developed in this study based on the academic thought that the active –OH group in F-ol could react with the dissociated –COOH group in FITC to form F-ol–(FITC)n and that DMA could increase the room temperature phosphorescence (RTP) of F-ol and FITC. At the same time, the feasibility of this method to determine trace glucose and affinity absorption reaction between the product of wheat germ agglutinin (WGA) labeled by DMA–F-ol–(FITC)n and glucose was studied. Furthermore, it was expanded to the study of the method for the determination of trace AP. Besides, the discussion of reaction mechanism and the possibility of human disease prediction were carried out. The analytical application and mechanism study not only could show the characteristics of AASSRTP, such as flexibility, sensitivity, accuracy, selectivity, and repeatability, but also could settle the theoretical basement for the determination of other BS. The potential application prospects of AASSRTP are exhibited as well. Materials and methods Apparatus and reagents Phosphorescent measurements were carried out on an LS-55 luminescence spectrophotometer with a solid surface analytical apparatus (PerkinElmer, Boston, MA, USA). The instrument’s main parameters are as follows: delay time 0.1 ms, gate time 2.0 ms, cycle time 20 ms, flash count 1, excitation slit 10 nm, emission slit

15 nm, and scan speed 1500 nm min1. A KQ-250B ultrasonic washing machine (Ultrasonic Machine, Kunshan, China), an AE240 electronic analytical balance (Mettler–Toledo Instruments, Shanghai, China), and a 0.50-ll flat head microinjector (Medical Laser Instrument, Shanghai, China) were also used. Glucose, AP, WGA, and bovine serum albumin (BSA) all were purchased from Sigma (St. Louis, MO, USA) and stored at 0 to 4 °C. They were diluted to 1.00 ng ml1 glucose (diluted with 0.10 mol L1 Na2CO3–NaHCO3 buffer solution, pH 9.40), 1.00 pg ml1 AP (diluted with 0.10 mol L1 Na2CO3–NaHCO3 buffer solution gradually), 700.0 ng ml1 WGA (diluted with 0.067 mol L1 KH2PO4–Na2HPO4 buffer solution gradually, pH 7.4), and 10 mg ml1 BSA (diluted with 0.10 mol L1 Na2CO3–NaHCO3 buffer solution gradually), respectively. In addition, 1.0  105 mol L1 Fol (C60(OH)25, fullerol with 24–26 hydroxy groups, was synthesized directly by the reaction of fullerene with aqueous NaOH and H2O2 in the presence of tetrabutylammonium hydroxide [TBAH] as the catalyst [27]), 1.0  105 mol L1 FITC, 1.0% (v/v) DMA, 0.050 mol L1 Tris–HCl buffer solution, Tris–HCl–0.1% Tween 20 washing buffer solution, 1.0 mol L1 Pb(Ac)2, and 2.0 mol L1 HAc were also used in this experiment. All reagents were analytical except BSA, which was a biological reagent. The water was thrice distilled. Acetate cellulose membrane (ACM), polyamide membrane (PAM), and nitrocellulose membrane (NCM) were purchased from Luqiaosijia Biochemical Plastic (Hangzhou, China). The paper sheet was precut into wafers (1.5 cm diameter) and indented (0.4 cm diameter) before use.

Experimental method Glucose was first determined by the AA reaction of the direct method. Here 0.40 ll of glucose of different concentrations was suspended onto the indentation of ACM wafers by a 0.50-ll flat head microinjector and then stored at 4 °C overnight. The substrate wafer was immersed in BSA solution at 37 °C for 0.5 h and then washed with washing buffer solution by ultrasonic oscillation three times repeatedly (20 ml of washing solution at a time, wash for 3 min). It was sipped up with filter paper, a 0.40-ll drop of 700 ng ml1 WGA was suspended onto the same indentation of ACM, and then it was set at 37 °C for 2 h. Then the AA reaction between glucose and WGA occurred [28], and the glucose–WGA product was obtained (Scheme 1). The glucose–WGA was washed three times by ultrasonic oscillation and sipped up with filter paper, on which 0.40 ll of labeling reagent (2.00 ml of 1% DMA–0.20 ml of 1.0  105 mol L1 F-ol– 7.00 ml of 1.0  105 mol L1 FITC) was dropped, before it was set at 37 °C for 2 h. Then the labeling reaction between F-ol– FITC–DMA and glucose–WGA occurred [28] and the glucose– WGA–F-ol–FITC–DMA product was obtained (Scheme 2). F-ol–(FITC)n labeling reagent was formed in the reaction between active –OH in F-ol and the dissociated –COOH in FITC (Scheme 3).

Scheme 1. AA reaction of direct method.

F-ol–FITC phosphorescent labeling reagent / J.-M. Liu et al. / Anal. Biochem. 404 (2010) 223–231

225

Scheme 2. Labeling reaction.

Scheme 3. Formation of F-ol–(FITC)n labeling reagent.

The glucose–WGA–F-ol–FITC–DEA was washed three times repeatedly and sipped up with filter paper. The product was immersed in Pb(Ac)2 solution for 10 s and dried at 90 ± 1 °C for 2 min. Its phosphorescence spectra were scanned, and the emission phosphorescence intensity of the blank reagent (DEA–Fol–FITC–WGA) (Ip1, where Ip was the maximal phosphorescence intensity of the corresponding kmax em ) and the sample (DEA–F-ol– FITC–WGA–glucose) (Ip2) was recorded. Each sample was measured six times. Then DIp (= Ip2  Ip1) was calculated. As for the AA reaction, the sandwich method had higher sensitivity and wider analytical range than the direct method, so the sandwich method was chosen to determine glucose simultaneously. A 0.40-ll drop of 700 ng ml1 WGA was suspended onto the indentation of ACM (0.4 cm diameter) and stored at 4 °C overnight. Glucose (0.40 ll) of different concentrations was suspended onto the indentation of ACM wafers. The following steps were conducted by the direct method, and WGA–glucose–WGA–F-ol–FITC– DEA was obtained. Its phosphorescence spectra were scanned, and the emission phosphorescence intensity of the blank reagent (DEA–F-ol–FITC–WGA + WGA) (Ip1) and the sample (WGA–glucose–WGA–F-ol–FITC–DEA) (Ip2) was recorded. Each sample was measured six times. Then DIp (= Ip2  Ip1) was calculated.

In addition, WGA was labeled by DMA–F-ol–FITC, and then AP was determined by the direct method. The procedures of the determination for AP were similar to those of the determination for glucose by the direct method.

Results and discussion RTP spectra of the AA product The phosphorescence spectra of the DMA–F-ol–FITC–WGA–glucose system were scanned according to the experimental method (Fig. 1A [curves 1–6 and 10 –60 are the excitation spectra and emission spectra of F-ol, respectively], Fig. 1B [curves 1–6 and 10 –60 are the excitation spectra and emission spectra of FITC, respectively], max and Table 1). Results showed that F-ol (kmax ex /kem = 541.6/ 0 709.7 nm, Ip = 41.0 [Fig. 1A, curve2.2 ]) and FITC (kmax ex / 0 kmax em = 481.4/648.0 nm, Ip = 61.4 [Fig 1B, curve 2.2 ]) could emit max RTP on ACM. The RTP intensity of F-ol (kmax ex /kem = 542.2/ 0 712.0 nm, Ip = 103.9, DIp = 62.9 [Fig. 1A, curve 3.3 ]) and FITC max (kmax ex /kem = 481.4/648.3 nm, Ip = 127.8, DIp = 66.4 [Fig. 1B, curve 0 3.3 ]) increased in the presence of DMA. When WGA was added

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F-ol–FITC phosphorescent labeling reagent / J.-M. Liu et al. / Anal. Biochem. 404 (2010) 223–231

tion (WGA–glucose–WGA–F-ol–FITC–DMA) could preserve good properties of RTP of F-ol and FITC. The experiment above indicated that no matter whether the AA reaction was carried out by the sandwich method or the direct method, it could both show the significant property that AASSRTP combined the specific property of the AA reaction with the high sensitivity of SSRTP. To further examine the feasibility of AASSRTP and expand its application, a study of this method for the determination of trace AP was conducted. The phosphorescence spectra of the DMA–Fol–FITC–WGA–AP system were scanned. In the presence of 700 fg max AP, the Ip values of F-ol (kmax ex /kem = 541.9/710.1 nm, Ip = 226.5) max and FITC (kmax /k = 481.6/648.0 nm, Ip = 397.3) increased sharply ex em max max max and kmax /k was still unchanged (k ex em ex /kem values were 541.8/ 709.3 nm for F-ol and 482.1/647.9 nm for FITC). Their DIp values were 117.4 and 256.8, respectively, indicating that the AA reaction product (DMA–F-ol–FITC–WGA–AP) preserved the good RTP characteristics of F-ol and FITC. Simultaneously, a linear relationship between the DIp of the system and the content of AP was found. Thus, AASSRTP could be used to determine trace AP. Optimal determination conditions

Fig. 1. (A) Phosphorescence spectra of F-ol. (B) Phosphorescence spectra of FITC.

to the system of F-ol–FITC–DMA, the Ip of F-ol and FITC increased max slightly, but kmax ex /kem almost stayed invariable (Fig. 1A and B, curve max 4.40 ). The Ip of F-ol (kmax ex /kem = 541.5/710.6 nm, Ip = 225.0 [Fig. 1A, max curve 6.60 ]) and FITC (kmax ex /kem = 482.0/647.8 nm, Ip = 342.2 0 [Fig. 1B, curve 6.6 ]) increased sharply in the presence of 500 pg max glucose. The kmax ex /kem also remained invariable, and the DIp values were 115.9 and 201.6 (Fig. 1A and B, curve 6.60 ), respectively, showing that the product of AA reaction (glucose–WGA–F-ol– FITC–DMA) could preserve good properties of RTP of F-ol and FITC. The phosphorescence spectra of the WGA–glucose–WGA–F-ol– FITC–DMA system were scanned. From the spectra, we could see that the results of the sandwich method were similar to those of the direct method; the Ip increased sharply in the presence of max 500 pg glucose, kmax ex /kem also stayed invariable, and DIp values were 125.6 for F-ol and 173.9 for FITC. The product of the AA reac-

For the system containing 40.0 fg of glucose spot1 (sample volume was 0.40 ll/spot, corresponding concentration was 100 pg ml1), the effects of the volumes and concentrations of reagents, solid substrate (ACM, PAM, and NCM), sensitizer (DMA, sodium lauryl sulfonate (SLS), poly(sodium acrylate) (PANa), and cetylpyridinium bromide (CPB)), the species and concentrations of ion perturber (Ca2+, Mg2+, Pb2+, and Sr2+), time and temperature for drying, and oxygen on the DIp of the system were tested (Table 2). The results showed that the DIp of F-ol and FITC reached the maximum when 0.40 ml of 1.0  105 mol L1 F-ol, 5.00 ml of 1.0  105 mol L1 FITC, 1.00 ml of 700.0 ng ml1 WGA, ACM as solid substrate, 1.50 ml of 1.0% DMA as sensitizer, and 1.0 mol L1 Pb2+ as ion perturber were used, the test samples were dried at 90 °C for 2 min, and whether drying N2 was passed or not. Under the optimal conditions above, the DIp of the system almost stayed invariable and had good repeatability within 50 min after being cooled by flowing water for 5 min. Working curve, DL, and precision WGA labeled with F-ol–FITC–DMA was used to determine glucose or AP according to the two AA methods mentioned above. The results showed that the contents of glucose and AP had linear correlation with the DIp of the system. The working curve, correlation coefficient (r), DL (calculated by 3SDb/k, where SDb refers to the standard deviation of 11 parallel analyses of the blank reagent and k is the slope of the working curve), and relative standard

Table 1 Phosphorescence characteristics. System

kex (nm)

kem (nm)

Ip

1.10 ACM 2.20 1.10 + 0.40 ml F-ol + 5.00 ml FITC

411.7 541.6 481.4 542.2 481.4 541.9 482.1 542.3 482.0 541.5 482.0

582.9 709.7 648.0 712.0 648.3 709.3 647.9 709.4 648.0 710.6 647.8

40.8 41.0 61.4 103.9 127.8 109.1 140.5 114.9 155.4 225.0 342.2

3.30 2.20 + 1.50 ml DMA 0

0

4.4 3.3 + 700 ng WGA 5.50 1.00 pg glucose + 700 ng WGA–F-ol–FITC–DMA 6.60 5.00 pg glucose + 700 ng WGA–F-ol–FITC–DMA

DIp

Luminescence molecule

62.9 66.4 5.2 12.8 5.8 14.9 115.9 201.6

F-ol was from the DMA–F-ol–FITC FITC was from the DMA–F-ol–FITC F-ol was from the DMA–F-ol–FITC FITC was from the DMA–F-ol–FITC F-ol was from the DMA–F-ol–FITC FITC was from the DMA–F-ol–FITC F-ol was from the DMA–F-ol–FITC FITC was from the DMA–F-ol–FITC F-ol was from the DMA–F-ol–FITC FITC was from the DMA–F-ol–FITC

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F-ol–FITC phosphorescent labeling reagent / J.-M. Liu et al. / Anal. Biochem. 404 (2010) 223–231 Table 2 Optimization of the various parameters. Reagent

Concentrations and volumes

DIp in DMA–F-ol–(FITC)n–WGA–glucose system

Optimal

F-ol (105 mol L1)

10.0, 1.0, 0.10, 0.01

43.9, 50.7, 32.6, 25.7 (FITC)

F-ol (ml)

0.20, 0.40, 0.60, 0.80

1.0  105 mol L1 F-ol 0.40 ml F-ol

FITC (105 mol L1)

10.0, 1.0, 0.10, 0.010

FITC (ml)

1.0, 3.0, 5.0, 7.0

DMA (%, v/v)

0.50, 1.0, 2.0, 3.0

DMA (ml)

0.50, 1.0, 1.5, 2.0

WGA (ng ml1)

500, 600, 700, 800, 900

WGA (ml)

0.10, 0.50, 1.0, 1.5, 2.0

Solid substrate

ACM, PAM, NCM

Sensitizer

DMA, SLS, PANa, CPB

Ion perturber

Ca2+, Mg2+, Pb2+, Sr2+

CPb2+ (mol L1)

0.10, 0.50, 1.0, 1.5, 2.0, 2.5

Time for drying (min)

0.5, 1.0, 1.5, 2.0, 2.5

Temperature for drying (°C)

60, 70, 80, 90, 95

Pass drying N2 (min)

10, 15, 20, 25, 30, 35, 40

Not pass drying N2 (min)

10, 15, 20, 25, 30, 35, 40

Stability of the reaction system (min)

10, 20, 30, 40, 50, 60, 70

31.4, 40.1, 31.8, 26.5 (F-ol) 41.5, 50.3, 33.9, 26.4 (FITC) 33.9, 40.5, 24.6, 20 (F-ol) 41.3, 50.8, 28.6, 20.5 (FITC) 35.6, 40.0, 28.8, 18.7 (F-ol) 16.7, 39.1, 50.3, 48.7 (FITC) 18.8, 31.5, 40.4, 36.5 (F-ol) 28.9, 50.7, 40.4, 30.7 (FITC) 26.8, 40.4, 30.6, 20 (F-ol) 26.3, 40.6, 50.7, 47 (FITC) 18.7, 29.7, 40.5, 33 (F-ol) 21.2, 29.6, 50.5, 26.7, 18.4 (FITC) 17.5, 24.9, 40.8, 25.5, 16.8 (F-ol) 23.3,29.0, 50.4, 25.7, 14.6 (FITC) 17,6, 25.7, 40.3, 23.9, 16.5 (F-ol) 50.5, 28.9, 15.5 (FITC) 40.4, 22.9, 18.7 (F-ol) 50.2, 37.3, 44.8, 47.8 (FITC) 40.6, 22.8, 26.1, 34.9 (F-ol) 40.6, 35.8, 50.8, 45.9 (FITC) 32.7, 28.4, 40.7, 38.3 (F-ol) 38.5, 43.3, 50.7, 45.6, 41.3, 35.9 (FITC) 25.7, 30.3, 40.3, 36.5, 32.9, 28.8 (F-ol) 12.5, 25.1, 37.8, 50.3, 45.5 (FITC) 10.1, 21.3, 30.2, 40.3, 36.3 (F-ol) 33.7, 39.3, 44.9, 50.7, 45.7 27.2, 31.9, 36.3, 40.8, 37.6 50.8, 50.6, 50.9, 50.5, 50.7, 50.9, 50.6 (FITC) 40.7, 40.5, 40.9, 40.6, 40.8, 40.6, 40.8 (F-ol) 50.4, 50.8, 50.6, 50.7, 50.8, 50.8, 50.6 (FITC) 40.5, 40.9, 40.7, 40.8, 40.9, 40.9, 40.7 (F-ol) 50.8, 50.6, 50.9, 50.5, 50.7, 46.7, 41.6 (FITC) 40.7, 40.5, 40.9, 40.6, 40.8, 35.6, 30.8 (F-ol)

1.0  105 mol L1 FITC 5.00 ml FITC 1.0% DMA 1.5 ml DMA 700.0 ng ml1 WGA 1.00 mL WGA ACM DMA Li+ 0.50 mol L1 2.0 min 90 °C

Not pass dying N2 50 min

Note. Under the conditions of 0.40 ml F-ol, 5.00 ml 1.0  105 mol L1 FITC, 1.5 ml 1.0% DMA, 1.00 ml 700.0 ng ml1 WGA, and 1.0 mol L1 Pb2+, ACM as solid substrate, drying at 90 °C for 2 min, and with drying N2 or without it, the effects of different concentrations of F-ol on the DIp of the system were studied. According to the DIp of the system, the optimal concentration was 1.0  105 mol L1 F-ol. Similarly, the optimal values of other parameters were selected by single factor optimization.

Table 3 Comparison of the methods. Method

Analytical range

Regression equation

r

DL (g ml1)

RSD (%)

Labeling reagent

Current method (direct method)

0.40–200 (fg spot1)

DIp = 4.958 + 0.5479 mglucose fg spot1 (n = 6, SDb = 0.021) DIp = 15.66 + 0.9269 mglucose fg spot1 (n = 6, SDb = 0.040) DIp = 20.25 + 0.5171 mglucose fg spot1 (n = 6, SDb = 0.023) DIp = 23.01 + 0.7443 mglucose fg spot1 (n = 6, SDb = 0.042) DIp = 10.60 + 0.3793 mAP ag spot1 (n = 6, SDb = 0.045) DIp = 20.76 + 0.8435 mALP ag spot1 (n = 6, SDb = 0.034) DIp = 8.1430 + 0.8594 mglucose fg spot1 (n = 5, SDb = 0.040) DIp = 15.653 + 0.9299 mglucose fg spot1 (n = 5, SDb = 0.040) DIp = 3.792 + 0.4955 mglucose fg spot1 (n = 7, SDb = 0.040) DIp = 12.70 + 0.6527 mglucose fg spot1 (n = 7, SDb = 0.040) DIp = 4.279 + 0.6603 mAP ag spot1 (n = 5, SDb = 0.010) DIp = 16.24 + 0.8857 mAP ag spot1 (n = 5, SDb = 0.040)

0.9990

2.8  1013

3.7–4.8

0.9992

3.3  1013

3.4–4.2

0.9987

1.5  1013

2.9–4.3

0.9990

1.0  10

13

2.3–3.9

0.9987

9.8  1016

3.4–4.2

0.9991

4.5  1016

3.7–4.6

0.9994

3.0  10

13

2.1–3.2

0.9991

3.0  10

13

1.6–2.9

0.9974

6.1  1013

3.6–4.2

F-ol was from the DMA–F-ol–(FITC)n FITC was from the DMA–F-ol–(FITC)n F-ol was from the DMA–F-ol–(FITC)n FITC was from the DMA–F-ol–(FITC)n F-ol was from the DMA–F-ol–(FITC)n FITC was from the DMA–F-ol–(FITC)n Porphyrin was from the 3.5-GPD-P 3.5-GPD was from the 3.5-GPD-P 4.0-GPD

0.9986

4.6  1013

3.1–4.7

0.9994

1.1  10

15

3.9–3.1

0.9993

3.5  1016

3.6–4.2

Current method (sandwich method)

0.10–200 (fg spot1)

Current method (direct method)

1.0–280 (ag spot1)

Ref. [7] (sandwich method)

Ref. [9] (direct method) Ref. [9] (sandwich method) Ref. [10] (direct method) Ref. [10] (sandwich method)

4.0–320 (fg spot1)

1.0–240 (fg spot1) 0.6–240 (fg spot1) 2.0–320 (ag spot1) 1.0–360 (ag spot1)

R-SiO2

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deviation (RSD,%) were compared with those of Refs. [7–10]. Results are listed in Table 3 (3.5-GPD-P, 4.0-GPD, and R-SiO2). Results showed that the labeling reagent used in this method was different from that in Refs. [7,9,10]. The DL of this method reached the same level as that in Refs. [7,9,10], whereas the contents of glucose and AP were at mg and ng levels [29,30] in human serum, respectively. Thus, this method could be used to determine glucose and AP and had wide application prospects.

The regression equations of lnIp  t, r, and the phosphorescent lifetime (s) are listed in Table 4.Results showed that both the sandwich method and direct method had long phosphorescence lifetimes, and the phosphorescence lifetime of the sandwich method was longer than that of the direct method. Hence, the time-resolved phosphorescence spectrometry for the determination of trace glucose and AP could be established based on the phosphorescence lifetime of F-ol or FITC under the suitable condition.

Phosphorescence lifetime

Interference experiment

For the system containing 40.0 fg of glucose spot1 and 40.00 ag of AP spot1, phosphorescent lifetime of immunoreaction products could be calculated by the phosphorescence decay method [31].

Glucose was determined by this method (100 pg glucose ml1) and the methods in Ref. [7] (150 pg glucose ml1) and Ref. [32] (0.10 mg glucose ml1). When relative error was within ±5%, the

Table 4 Phosphorescence lifetimes of products. Method

max kmax ex /kex (nm)

Regression equation of phosphorescence delay curve (ms)

r

s (ms)

Luminescence molecule

Direct method (glucose)

542/710 482/648 542/710 482/648 542/710 482/648

lnIp = 4.2604  0.01601t lnIp = 4.8102  0.01128t lnIp = 4.3110  0.014531t lnIp = 4.9775  0.01040t lnIp = 4.2604  0.01601t lnIp = 4.8102  0.01128t

0.9950 0.9972 0.9944 0.9966 0.9950 0.9972

62.5 88.7 68.8 96.2 62.5 88.7

F-ol was from the DMA–F-ol–(FITC)n FITC was from the DMA–F-ol–(FITC)n F-ol was from the DMA–F-ol–(FITC)n FITC was from the DMA–F-ol–(FITC)n F-ol was from the DMA–F-ol–(FITC)n FITC was from the DMA–F-ol–(FITC)n

Sandwich method (glucose) Direct method (AP)

Table 5 Effects of coexistent materials. Current method

Ref. [7]

Ref. [32]

Coexistent materials

Allowed concentration (ng ml1)

Allowed multiple

Relative error (%)

Allowed multiple

Allowed multiple

Interference degree

Saccharides Serum protein Glutamic acid Uric acid Ascorbic acid Fructose

30.0 65.0 86.0 80.0 75.0 4.0

300 650 860 800 750 40

3.6 4.2 4.5 3.1 4.3 2.7

90 500 450 500 450 13

5 20 20 20 20 1

No interference No interference No interference No interference No interference Critical interference

Table 6 Analysis results of glucose in serum. Serum and age (years)

Obtained (mg ml1)

max kmax ex /kem (nm)

RSD (%)

Added (mg ml1)

Recovery (mg ml1)

Recovery (%)

ELISA method (mg ml1)

A (40)

0.734 0.739 0.896 0.912 1.048 1.057 0.477 0.491 0.461 0.457 0.402 0.411 1.346 1.365 1.542 1.567 1.627 1.638

542/711 482/648 542/711 482/648 542/711 482/648 542/711 482/648 542/711 482/648 542/711 482/648 542/711 482/648 542/711 482/648 542/711 482/648

4.6 4.2 3.8 3.5 2.7 2.2 4.6 4.1 4.6 4.0 3.9 3.2 3.8 3.4 2.7 2.1 3.5 3.0

1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

0.991 0.998 0.987 0.994 1.02 1.04 0.993 1.00 1.03 1.05 0.997 0.991 0.989 0.995 1.04 1.00 1.03 1.05

99.1 99.8 98.7 99.4 102 104 99.3 100.0 103 105 99.7 99.1 89.9 99.5 104 100 103 108

0.741 0.747 0.889 0.904 1.027 1.042 0.458 0.481 0.455 0.446 0.391 0.403 1.372 1.384 1.561 1.576 1.641 1.655

B (40) C (40) D (45) E (45) F (45) G (50) H (50) I (50)

Note. Sandwich method (n = 7). Nine female volunteers, 48.2 ± 1.5 years of age and weighing 51.2 ± 2.4 kg without a history of low blood pressure or heart or liver disease, were banned from alcohol and tobacco and did not use any drugs before or during tests.

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allowed maximum concentration of interfering species is listed in Table 5. From Table 5, we could know that the allowed multiple of coexistence was larger than that in Refs. [7,32]. Although the allowed multiple of fructose was smaller than that in other coexistent materials, the content in the organism was low. Thus, the presence of fructose had no disturbance with the determination of glucose, which showed high selectivity of this method. Sample analysis Serum (1.00 ml) from nine persons (A, B, C, D, E, F, G, H, and I who were hollow) was diluted to 500.00 ml with Na2CO3–NaHCO3 buffer solution (pH 9.40), and then 1.00 ml of this solution was further diluted to 100.00 ml. This test solution was used for the determination of glucose. Then 1.00 ml of serum was diluted by Na2CO3–NaHCO3 buffer solution (pH 9.40) until the concentration of AP reached the level of fg ml1. After that, 1.00 ml of test solution was taken to determine the contents of glucose and AP in human serum according to the experimental method. Meanwhile, the addition standard recovery experiment was also conducted. To investigate the dependability and clinical value of AASSRTP, glucose (glucose kit from Zhongsheng Beijing Biology Technique, http://www.zhongsheng.com.cn) and AP (AP kit from Zhongsheng Beijing Biology Technique) were determined by enzyme-linked immunosorbent assay (ELISA) and a Hitachi series automatic biochemistry analyzer in Zhangzhou Hospital of Chinese Medicine in Fujian Province. Results are listed in Tables 6 and 7. As seen from Table 6, phosphorescence excitation/emission wavelengths of either F-ol or FITC were used to determine trace glucose in the serum sample, and the results agreed well with those of ELISA. The recovery rates of glucose in serum from persons A, B, C, D, E, F, G, H, and I were 97.9 to 103%, and the RSDs were 2.9 to 4.7%, showing that this method was of good accuracy and high precision. The level of glucose in fasting serum of healthy humans was 0.702 to 1.098 mg ml1, the level of glucose in serum of hypoglycemic sufferers was lower than 0.504 mg ml1, and the level of glucose in serum of diabetics was equal to or higher than 1.26 mg ml1. Thus, we could diagnose that A, B, and C were healthy people; D, E, and F were hypoglycemic sufferers; and G, H, and I were diabetics. The forecast coincided with the results of

clinical detection and diagnosis in the Zhangzhou Hospital of Chinese Medicine. As seen from Table 7, the results of determining the content of AP in human serum by this method coincided well with those obtained by ELISA. They showed that the AP content of persons A, B, and C was in normal range (13.1–49.2 ng ml1); the AP content of persons D, E, and F was lower than 13.1 ng ml1; and the AP content of persons G, H, and I was higher than 49.2 ng ml1. According to the decrease of AP content in liver patients’ serum and the increase of AP content in osteopathy patients’ serum, we could forecast that D, E, and F might be liver patients, whereas G, H, and I might be osteopathy patients. This forecast was tallied with the clinical detection and the diagnostic results of Zhangzhou Hospital of Chinese Medicine in Fujian Province. According to the results of clinical detection and diagnosis, persons D, E, F, G, H, and I were treated. After 4 weeks, the content of AP determined by this method was 355.3 lg L1, indicating that it had reached the healthy level. Meanwhile, no pathology was found after B diasonagraph detection (BDD) and computer–X-ray–faultage–photography determination (CXFPD). As mentioned above, we use AASSRTP to determine the contents of glucose and AP. Results were tallied with the clinical detection and the diagnostic results of Zhangzhou Hospital of Chinese Medicine in Fujian Province (Tables 6 and 7), indicating that AASSRTP could replace ELISA for the determination of glucose and AP in routine method. According to the detection results of AASSRTP, doctors diagnosed and treated persons D, E, F, G, H, and I and the results were satisfactory, showing that AASSRTP is of clinical value. The determination principle of ELISA (glucose kit) in Zhangzhou Hospital of Chinese Medicine is as follows. Glucose oxidase could catalyze O2 to oxidize glucose into gluconate, and H2O2 was produced simultaneously. In the presence of 4-amino antipyrine and phenol, catalase could catalyze H2O2 to oxidize primary pigment to produce colored compound, and then absorbance of the sample was determined. The content of glucose in human plasma was calculated based on the fact that DA was proportional to the content of glucose. The glucose kit has low sensitivity, colors that is not steady, and weak reproducibility; thus, AASSRTP has been applied to the determination of glucose and AP in routine method at Zhangzhou Hospital of Chinese Medicine.

Table 7 Analysis results of AP in human serum. Sample

Found (ng ml1)

Measurement Wavelengths max (kmax ex /kem , nm)

RSD (%)

Added (ng ml1)

Recovery (ng ml1)

Recovery (%)

ELISA (ng ml1)

PHC

BDD

CXFPD

A

15.5 15.2 26.6 26.2 34.8 34.4 10.8 11.5 11.7 11.7 12.7 12.4 51.6 51.3 53.6 53.2 57.1 56.8

542/709 482/648 542/709 482/648 542/709 482/648 542/709 482/648 542/709 482/648 542/709 482/648 542/709 482/648 542/709 482/648 542/709 482/648

3.9 3.4 2.1 2.8 3.2 3.7 3.5 3.1 2.8 2.4 4.1 4.5 2.9 2.4 4.2 3.9 4.1 4.5

10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0

10.3 10.2 10.0 10.2 9.8 10.0 9.9 10.1 9.7 9.9 10.2 10.3 9.8 10.2 9.9 10.0 9.6 9.5

103 102 100 102 98.0 100 99.0 101 97.0 99.0 102 103 98.0 102 99.0 100 96.0 95.0

15.8 15.7 27.0 26.7 34.1 33.9 11.0 10.8 11.9 12.3 12.9 12.7 52.5 52.3 53.3 53.0 56.5 55.7



















+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

B C D E F G H I

Note. Direct method (n = 6). Nine male volunteers, 52.4 ± 4.5 years of age and weighing 55.5 ± 5.2 kg without a history of low blood pressure or heart or kidney disease, were banned from alcohol and tobacco and did not use any drugs before or during tests. PHC, primary hepatic carcinoma; +, has pathology; , has no pathology.

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Mechanism of AASSRTP for determination of AP using DMA–F-ol–FITC F-ol and FITC could emit RTP on ACM using 1.0 mol L1 Pb2+ as ion perturber. The RTP of F-ol increased sharply in the presence of DMA (Fig. 1A and B). The possible reasons are as follows: on the one hand, the molecule structures of F-ol and FITC were modified simultaneously by DMA [33], leading to the RTP enhancement of F-ol and FITC; on the other hand, the radiation of charge transfer complex formed by DMA matching with F-ol or FITC played a leading role, increasing the probability of excited singlet state transferring to triplet state [34] and causing the RTP to enhance.

When F-ol and FITC coexisted, the RTP of FITC increased (Fig. 1B). The reason might be that the F-ol–(FITC)n complex containing several FITC molecules formed in the reaction between the active –OH group in F-ol and the dissociated –COOH group in FITC (Scheme 3); this could increase the number of molecules in the biological target and enhance the RTP signal of the system. DMA could enhance the RTP signal of F-ol or FITC, and the DIp of the system was larger than that without DMA (Fig. 1A and B). When WGA was labeled with DMA–F-ol–FITC, the reaction was similar to the labeling reaction between SiO2–FITC–S-CH2–COOH and WGA [28]; labeling product F-ol–(FITC)n–WGA was obtained

Scheme 4. Reaction of DMA–F-ol–FITC labeling WGA.

Scheme 5. AA reactions of DMA–F-ol–FITC–WGA and AP for the direct method.

F-ol–FITC phosphorescent labeling reagent / J.-M. Liu et al. / Anal. Biochem. 404 (2010) 223–231

by the combination of –NCS in FITC and –NH2 in WGA. The labeling reaction could be expressed as in Scheme 4. The specific AA reaction could be carried out between DMA– F-ol–FITC–WGA and AP in the presence of 700 fg AP [10]. The reaction was expressed as in Scheme 5. The DMA–F-ol–FITC–WGA–AP product led to sharp enhancement of the RTP signals of F-ol and FITC. Meanwhile, the DIp of the system was directly proportional to the content of AP. Thus, trace AP was determined by AASSRTP based on WGA labeled by DMA–F-ol–FITC. Conclusion DMA–F-ol–(FITC)n was developed based on the academic thought that the active –OH group in F-ol could react with the dissociated –COOH group in FITC to form the F-ol–(FITC)n complex containing several FITC molecules and that DMA could increase the RTP signal. Simultaneously, AASSRTP for the determination of trace glucose or AP and the new method to predict the human diseases were put forward. Besides, these methods had been successfully applied to determine glucose or AP in real samples, and the results coincided with those of clinical detection and diagnosis in Zhangzhou Hospital of Chinese Medicine in Fujian Province. The results of this study provide a new method for the exploitation and application of new phosphorescent labeling reagent, the establishment of AASSRTP, and the prediction of human diseases. Meanwhile, it promotes the research progress of F-ol, lectin, SSRTP, and life science. Acknowledgments This work was supported by the Fujian Province Science Foundation (2009J1017 and 2008J0313), the Fujian Education Office Science and Technology Item Programme (JA08252, JB08262, and JB09278), and the Scientific Research Program of Zhangzhou Institute of Technology Foundation (ZZY0942 and ZZY0952). We are also very grateful to precious advice provided by the reviewers and the editors of this journal. References [1] S.Y. Xie, F. Gao, X. Lu, R.B. Huang, C.R. Wang, X. Zhang, M.L. Liu, S.L. Deng, L.S. Zheng, Capturing the labile fullerene[50] as C50Cl10, Science 304 (2004) 699. [2] F. Gao, S.Y. Xie, R.B. Huang, L.S. Zheng, Significant promotional effect of CCl4 on fullerene yield in the graphite arc-discharge reaction, Chem. Commun. 21 (2003) 2676–2677. [3] F. Gao, S.Y. Xie, Z.J. Ma, Y.Q. Feng, R.B. Huang, L.S. Zheng, The graphite arcdischarge in the presence of CCl4: chlorinated carbon clusters in relation with fullerenes formation, Carbon 42 (2004) 1954–1963. [4] J.M. Liu, X.J. Cui, F. Gao, L.M. Li, X.C. Huang, M.L. Yang, F.M. Li, H. Wu, Solid substrate–room temperature phosphorescence method for the determination of trace Mn(II) based on oxidizing reaction of hydrogen peroxide using a,abipyridine as sensitizer, J. Fluoresc. 17 (2007) 49–55. [5] J.M. Liu, F. Gao, H.H. Huang, L.Q. Zeng, X.M. Huang, G.H. Zhu, Z.M. Li, Determination of trace alkaline phosphatase by solid-substrate roomtemperature phosphorimetry based on Triticum vulgare lectin labeled with fullerenol, Chem. Biodivers. 5 (2008) 606–616. [6] X. Ying, D.W. Hu, Y.C. Low, Y.X. Fang, D.B. Li, One-step method of labeling Triticum vulgare lectin and cellulose with colloidal gold probe, Electron Microsc. Soc. 2 (2000) 179–183. [7] J.M. Liu, Z.B. Liu, G.H. Zhu, X.L. Li, X.M. Huang, F.M. Li, X.M. Shi, L.Q. Zeng, Determination of trace glucose and forecast of human diseases by affinity adsorption solid substrate–room temperature phosphorimetry based on Triticum vulgaris lectin labeled with dendrimers–porphyrin dual luminescence molecule, Talanta 74 (2008) 625–631.

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