Fluorescein-5-isothiocyanate – Ag013736 Inhibitor

Fullerol-ﬂuorescein isothiocyanate-concanavalin agglutinin phosphorescent sensor for the detection of alpha-fetoprotein and forecast of human diseases
Jia-ming Liu a,⇑, Li-ping Lin a, Shu-Lian Jiang a,b, Ma Lin Cui a, Li Jiao a, Xiao Yang Zhang a, Li-hong Zhang c,
Zhi Yong Zheng c, Xuan Lin a, Shao-qin Lin d

h i g h l i g h t s

● Fullerol-ﬂuorescein isothiocyanate and N,N-dimethylaniline (F-FITC- DMA) was exploited.
● Phosphorescent sensor has been designed using reaction between F- FITC-DMA and Con A.
● The sensor could be applied to the detection of AFP-V and the forecast of diseases.
● Mechanisms of labeling Con A and the determination of AFP-V were discussed.
● The coupling technique of sensor, labeling method and phosphorimetry was developed.

Keywords:
Phosphorescent sensor Alpha-fetoprotein variant
Forecast and diagnosis of human diseases Fullerol-ﬂuorescein isothiocyanate Phosphorescent labeling reagent

a b s t r a c t

Based on the reaction of the active –OH group in fullerol (F) with the dissociated –COOH group in ﬂuo- rescein isothiocyanate (FITC) to form an F-FITC and the enhanced effect of N, N-dimethylaniline (DMA) on phosphorescence signal of F-FITC, a new phosphorescent labeling reagent (DMA-F-FITC) was developed. What’s more, a phosphorescent sensor for the determination of alpha-fetoprotein variant (AFP-V) has been designed via the coupling technique of the high sensitivity for afﬁnity adsorption–solid sub- strate-room temperature phosphorimetry (AA-SS-RTP) with the strong speciﬁcity reaction between DMA-F-FITC-Con A and AFP-V. The DMA-F-FITC increased the number of luminescent molecules in the biological target which improved the sensitivity of phosphorescent sensor. The proposed sensor was responsive, simple, selective and sensitive, and it has been applied to the determination of trace AFP-V in human serum and the forecast of human diseases using phosphorescence emission wavelength of F or FITC, with the results agreed well with those obtained by enzyme-linked immunoassay (ELISA). Mean- while, the mechanisms for the labeling reaction and the sensing detection of AFP-V were discussed.

Introduction

Hepatocellular carcinoma (HCC) is one of the most familiar malignant diseases all over the world. HCC is closely related to the content of AFP-V. It was reported by Sato et al. [1] that the cir- rhosis patients whose AFP-V content increased would mostly be diagnosed as suffering hepatocellular carcinoma in 3–18 months. If AFP-V is used to forecast hepatocellular carcinoma, the accuracy can reach 94% [2]. At present, the available methods for the diagno- sis of HCC including immunohistochemical staining [3], ﬂuorescence diagnosis [4], magnetic resonance imaging [5] and three-dimensional conformal radiotherapy [6]. There are many methods for the determination of AFP-V reported both at home and aboard, such as afﬁnity-electrophoresis enzyme immunoassay (detection limit (L.D) = 1.0 10—9 g mL—1) [7], afﬁnity-absorbent assay (LD = 9.0 10—10 g mL—1) [8], time resolved ﬂuoroimmuoas- say (LD = 1.0 10—10 g mL—1) [9], antibody afﬁnity-blotting meth- od (LD < 4.0 10—8 g mL—1) [10], etc. Though the LDs of above methods are at ng level, they have been rarely applied to clinic diagnosis [10]. Obviously, it is of high academic research value and application prospect to search for a new method with high sensitivity and accuracy to determine AFP-V and forecast HCC in the ﬁeld of life sciences. In recent years, lectin has been used as a glycosyl probe in the research of the glycoprotein, glycolipide, glycosyl-chain structure and alkaline phosphatase (ALP) [11]. For example, ELISA has been used to determine ALP [12]. Therefore, the exploitation of a high efﬁcient labeling reagent of lectin is the key of biology analysis. The fullerene (C50Cl10) and its derivatives have the luminescent characteristics [13]. It has been reported that the ﬂuorescence sig- nal of C60 was enhanced sharply by modifying it with DMA [14]. There have been also many reports on the synthesis of multi-hy- droxyl C60 derivatives, F derivatives, aminophenol derivatives and dendritic fullerene derivatives [15–17] as well as the ﬂuorescent property of water-soluble F [18]. The ALP [19,20], glucose [21], As (V) [22] and Mn2+ [23] were detection based on phosphorescent property of F, showing the broad analytical application prospects of F. Inspired by these reports, we designed this phosphorescent sen- sor for the determination of trace AFP-V and the forecast of human diseases based on the ampliﬁcation effect of DMA on room temper- ature phosphorescence (RTP) of F and FITC. In this work, new DMA-F-FITC phosphorescent labeling reagent and phosphorescent sensor were developed. The sensitivity (LD: 1.2 10—13 g mL—1 for F and 9.0 10—14 g mL—1 for FITC, sandwich way) of this phosphorescent sensor was higher than those of Refs. [7–10]. New sensor was suitable for the determination of ultra- trace AFP-V in serum and the clinical diagnosis for HPC. Compared with ELISA, this sensor except a batch of phosphorescent sensor were prepared for use under room temperature, it has many merits also, such as the higher sensitivity, needs only microliter samples; many operations, including suspending samples, washing, drying and the measurement of phosphorescence can be easily conducted on many kinds of substrates, are similar to those of ELISA showing its broad application prospect. This study not only opened up the analytical application of sensor, F and lectin, but also provided a new way for the development of SS-RTP. Experimental Apparatus and reagents Phosphorescent measurements were carried out on a Perkin–El- mer LS-55 luminescence spectrophotometer with a solid surface analysis apparatus (Perkin Element Corporation of US). The instru- ment’s main parameters were as follows: delay time: 0.10 ms; gate time: 2.0 ms; cycle time: 20 ms; ﬂash count: 3.0; excitation (Ex) slit: 10 nm; emission (Em): 15 nm; scan speed: 1500 nm min—1. KQ-250B ultrasonic washing machine (Kunshan Ultrasonic Ma- chine Company) and AE240 electronic analytical balance (Met- tler-Toledo Instruments Company Limited) were also used. A 0.50-lL ﬂat head micro-injector (Shanghai Medical Laser Instru- ment Plant, China) was used to introduce the solution of lL level. AFP-V, bovine serum albumin (BSA) and Con A were all purchased from Sigma Corporation and stored at 0–4 °C. They were diluted to 1.00 pg mL—1 and 100.00 pg mL—1 AFP-V (diluted with 0.10 mol L—1 Na2CO3–NaHCO3 buffer solution of pH value being 9.40) 700.0 ng mL—1 Con A (diluted with 0.067 mol L—1 KH2PO4– Na2HPO4 buffer solution gradually of pH value being 7.4) and 10 mg mL—1 BSA (diluted with 0.10 mol L—1 Na2CO3–NaHCO3 buf- fer solution), respectively. 1.0 10—5 mol L—1 F (C60 Fullerol with 24–26 hydroxy groups was synthesized directly by the reaction of fullerene with aqueous NaOH and H2O2 in the presence of tetra- butylammonium hydroxide as the catalyst [24]), 1.0 10—5 - mol L—1 FITC, 0.10 mol L—1 DMA, 0.10 mol L—1 Na2CO3–NaHCO3 buffer solution, 0.067 mol L—1 KH2PO4–Na2HPO4 buffer solution, 0.050 mol L—1 Tris (trihydroxymethyl aminomethane)-HCl buffer solution, Tris–HCl-0.1% Tween-20 washing buffer solution, 1.0 mol L—1 Pb(Ac)2 solution and 2.0 mol L—1 HAc were also used in this experiment. Preparation of1-ethyl-3-3-dimethylaminopro- pylcarbodiimide hydrochloride (EDC, Alfa Company)-N-hydrox- ysucci-nimide (NHS, Alfa Company) coupling agent solution: the mixture of 5 mM EDC and 5 mM NHS was prepared with 40% eth- anol. All reagents were AR grade except that BSA was a biological reagent. The water for experiment was thrice-distilled water. Polyamide membrane (PAM), acetyl cellulose membrane (ACM) and nitrocellulose membrane (NCM) were purchased from luqiao- sijia biochemical plastic plant. The paper sheets were pre-cut into wafers for preparation (diameter was 1.5 cm) and indented (diam- eter was 0.4 cm.) before use. Experimental method Preparation of phosphorescent sensor A 0.40 lL drop of different concentrations Con A (diluted with KH2PO4–Na2HPO4 buffer solution of pH = 7.4, gradually) was sus- pended onto the indentation of ACM wafers by a 0.50-lL ﬂat head micro-injector, and then stored at 4 °C overnight. The substrate wafer was immersed in BSA solution at 37 °C for 0.5 h, then washed with washing buffer solution by ultrasonic oscillation for 3 times repeatedly (20 mL of washing solution at a time, wash for 3 min), sipped up with ﬁlter paper. Took ACM out, and then 0.40 lL labeling reagents (1.50 mL of 1% (V/V) DMA-0.40 mL of 1.0 10—5 mol L—1 F-5.00 mL of 1.0 10—5 mol L—1 FITC ethanol solution) were added on the center indentation of ACM. Then, a 0.40 lL drop of EDC-NHS solution was suspended onto the center indentation of ACM, after reacting at 37 °C for 2 h, the labeling product (DMA-F-FITC-Con A) was obtained by the reaction be- tween Con A and DMA-F-FITC. Then took out the labeling product and repeatedly washed for three times by ultrasonic oscillation to eliminate the remained marker reagent on ACM, sipped up with ﬁl- ter paper and phosphorescent sensor was obtained for use. The phosphorescent sensor not only could be batch prepared, but it was used under room temperature. The optimal concentration of Con A was examined by the measurement results of the afﬁnity adsorption (AA) reaction between DMA-F-FITC-Con A phosphores- cent sensor and different concentrations of AFP-V. Afﬁnity adsorption reaction and sensor detection for AFP-V The type of AA reaction used in this paper was sandwich way. A 0.40 lL drop of Con A was suspended onto the indentation of ACM wafers membrane (U = 4 mm) by a 0.50 lL micro-injector, then (200 r min—1) for 5 min, supernatant was diluted to the pg level with Tris–HCl buffer solutions, and stored at 4 °C for use. Results and discussion Phosphorescence spectra of DMA-F-FITC-Con A system Firstly, this paper used a sandwich method for AA reaction. The phosphorescence spectra of DMA-F-FITC-Con A-AFP-V-Con A sys- tem were scanned (Figs. 1a and b and Table 1). Results indicated that F (kmax ¼ 709:7; Ip ¼ 41:0; kmax is 542.0 nm.) and FITC (kmax ¼ 648:0; Ip ¼ 61:4; kmax is 481.0 nm.) could emit strong and stable RTP on ACM, respectively. The phosphorescence intensity of F (kmax ¼ 712:0; Ip ¼ 103:9; DIp ¼ 62:9; kmax is 542 nm.) and FITC ðkmax ¼ 648:3; Ip ¼ 127:8 nm;DIp ¼ 66:4; kmax ¼ 481:0 nm:Þ enhanced in the presence of DMA. When Con A was added to DMA-F-FITC system, the Ip of F (kmax ¼ 709:3; Ip ¼ 109:1; kmax is 542.0 nm.) and FITC (kmax ¼ 647:9; Ip ¼ 140:5; kmax is 481.0 nm.) in- creased slightly, while kmax=kmax was still unchanged. The Ip of F Fig. 1a. RTP spectra of DMA-F-FITC-Con A-AFP-V-Con A system by the sandwich way (Curves 1–6 are the emission spectra, Ip is phosphorescent intensity of F.) stored at 4 °C overnight. The substrate wafer was immersed into 10 mg mL—1 BSA solution for 0.5 h at 37 °C, and then washed with washing buffer solution for three times (20 mL washing solution and 3 min were needed each time by ultrasonic oscillation), sipped up with ﬁlter paper. A 0.40 lL drop of different concentrations AFP-V (diluted gradually with Na2CO3–NaHCO3 buffer solution of pH 9.12) and EDC-NHS solution were suspended on the center indentation of the ACM wafer, respectively, after set at 37 °C for 2 h, washed for three times, sipped up by ﬁlter paper (The washing and sipping procedure below was conducted in the same way.). Then 0.40 lL of DMA-F-FITC-Con A was suspended onto the same indentation of ACM; and stored at 37 °C for 2 h, washed for three times again, sipped up by ﬁlter paper; the s ACM was then im- mersed in Pb(Ac)2 solution for 10 s, then took it out and then dried at 90 ± 1 °C for 2 min. The phosphorescence spectra of the system were scanned. The phosphorescence intensity of the blank reagent (DMA-F-FITC-Con A + Con A) was deﬁned as Ip1 (Ip was the maxi- mal phosphorescence intensity of corresponding kmax) and that of the sample (DMA-F-FITC-Con A-AFP-V-Con A) was deﬁned as Ip2. Each sample was measured for seven times simultaneously. Then, the DIp (=Ip2–Ip1) was calculated. Analysis of sample 0.50 mL of 0.050 mol L—1 Tris–HCl buffer solution (containing 0.50 mol L—1 NaCl) was added into 1.00 mL of six human serum samples (A, B, C, D, E and F), mixed homogeneously for 10 min. After centrifugalization, the supernatant was deserted. Thereafter, 1.00 mL buffer solution was added and mixed homogeneously for 10 min again and stored at 4 °C before use. After that, the adsor- bent Sepharose 4B was added, and supernatant was deserted after centrifugalizating. 1.00 mL Tris–HCl was added again and shaken in an oscillator (300 r min—1) for 20 min. After centrifugalization the presence of 600 pg AFP-V, while kmax was still unchanged, their values of the DIp were 163.9 and 221.2, respectively, indicating that AA reaction products (DMA-F-FITC-Con A-AFP-V-Con A) could preserve the good RTP characteristics of F and FITC. Therefore, 542/ 710 or 481/648 nm could be chosen as the working wavelength. This fact provided the possibility for the determination of the AFP-V by AA-SS-RTP with sandwich way and indicated that DMA-F-FITC-Con A could be used as sensitive RTP sensor response to AFP-V. The phosphorescence spectra of DMA-F-FITC-ConA-AFP-V sys- tem were also scanned by the direct way. Results indicated that the phosphorescence spectra of the direct way were similar to that of the sandwich way, and the Ip of the system increased sharply in Fig. 1b. RTP spectra of DMA-F-FITC-Con A-AFP-V-Con A system by the sandwich way (Curves 1–6 are the emission spectra, Ip is phosphorescent intensity of FITC.).the presence of 600 pg AFP-V, while kmax was still unchanged, the corresponding DIp were 157.0 and 204.8, respectively, and the con- tent of AFP-V was linear with the DIp of the system. Thus, the AFP- V can be detected by the direct way at the working wavelength of 542 /710 or 481/648, showing the ﬂexibility of DMA-F-FITC-ConA as RTP sensor. This fact provides the possibility for the determina- tion of AFP-V by AA-SS-RTP with direct way. This study show that AA-SS-RTP by either the sandwich way or the direct way could combine the high sensitivity of SS-RTP with strong speciﬁcity of the AA reaction between AFP-V and DMA-F-FITC-Con A very well. Optimum determination conditions Concentration and volume of reagents For the system containing 40.0 fg spot—1 AFP-V, the effect of vol- umes and concentrations of reagents on the DIp was investigated in a by univariate approach, respectively. Results show that the DIp of the system reached the maximum and remained stable when 0.40 mL of 1.0 10—5 mol L—1 F, 5.0 mL of 1.0 10—5 mol L—1 FITC and 1.50 mL of 1.0% DMA were used (Table 2). Seen from Table 2, the DIp of the system gradually enhanced with the increasing of the concentration and volume of Con A, this is a necessary result of speciﬁc reaction between Con A and AFP-V; the DIp of the system reached the maximum when the concentra- tion and volume of Con A was 700.0 ng mL—1 and 1.00 mL, perhaps resulting from the yield of the speciﬁc reaction reached the highest degree. While the DIp of the system was nearly unchanged exceed- ing 700.0 ng mL—1 and 1.00 mL, the reason may be that excessive Con A has been washed and speciﬁc reaction was end. Solid substrate For the system containing 40.0 fg AFP-V spot—1, when the solid substrates were ACM, PAM and NCM, corresponding DIp of F were 30.1, 12.6 and 8.4, and the DIp of FITC were 45.8, 23.5 and 11.0, respectively. Results show that the DIp of FITC and F were both the highest when using ACM as the solid substrate. Compared with PAM and NCM, the DIp of the system reached the maximum on ACM probably for the reason that the speed of permeability and diffusion of F and FITC on ACM was suitable [25], which is easy to suspend and carry out the soaking and drying of heavy atom perturbers, and it can preserve good rigidity after being washed for many times. Thus, ACM was chosen as solid substrate in this experiment. Sensitizer For the system containing 40.0 fg AFP-V spot—1, when the sensi- tizers were DMA, sodium lauryl sulfonate, poly sodium acrylate and cetyl pyridinium bromide, corresponding DIp of F were 30.1, 12.5, 16.6 and 24.4, and DIp of FITC were 45.8, 32.9, 39.3 and 45.5, respectively. Results show that the DIp of F and FITC reached the maximum when DMA was used. The possible reasons are as follows. On the one hand, the RTP signals of F and FITC enhanced through modifying their molecular structure with DMA [14]; on the other hand, the leading role of the radiation of the above charge transfer complex increased the transition probability from excited singlet state to triplet state [26], resulting in the RTP enhancing. Ion perturbation For the system containing 40.0 fg AFP-V spot—1, when the ion perturbers were 1.00 mol L—1 of Hg2+, Ba2+, Pb2+ and Ag+, Table 3 Comparison of the methods mentioned above. Method Analytical range fg The regression equation of working r DL fg spot—1 (pg LOQ fg spot—1 RSD (%) Luminescent spot—1 (pg mL—1) Curve (fg spot—1, n = 7) mL—1) (pg mL—1) n =6 molecule Sensor (direct 0.40–240 DIp = 6.027 + 0.6335 0.9991 0.14 0.46 3.2–4.1 F way) (1.00–600) mAFP-V, (Sb = 0.030) (0.35) (1.2) DIp = 10.67 + 0.8143 0.9987 0.11 0.36 3.8–4.9 FITC mAFP-V, (Sb = 0.030) (0.28) (0.92) Sensor 0.10–240 DIp = 19.21 + 0.6062 0.9989 0.049 0.16 3.5–4.4 F (sandwich (0.25–600) mAFP-V, (Sb = 0.010) (0.12) (0.40) way) D Ip = 19.45 + 0.8364 0.9990 0.036 0.12 4.1–5.0 FITC mAFP-V, (Sb = 0.010) 0.090 0.30 Table 4 Speciﬁcity of this sensor. ex ex corresponding DIp of F were 26.3, 22.1, 30.1 and 17.5, and DIp of FITC were 40.0, 33.7, 45.8 and 26.6, respectively. When the concen- trations of Pb2+ were 0.10, 0.30, 0.50, 0.70, 1.0 and 1.2 (mol L—1), corresponding DIp of F were 26.7, 29.9, 30.1, 30.0, 29.9 and 29.9, and DIp of FITC were 40.3, 43.7, 45.8, 45.3, 45.5 and 45.5, respec- tively. Results show that the DIp reached the maximum and re- mained stable when 1.00 (mol L—1) Pb2+ was used. In contrast to Hg2+, Ba2+ and Ag+, Pb2+ has the largest heavy atom perturber effect on the DIp of the system via increasing the transition probabilities of the luminescent molecules from singlet state to triplet state [27]. The DIp of the system enhanced gradually with the increasing of the concentration of Pb2+ until 1.0 mol L—1. When the concentra- tion of Pb2+ was over 1.0 mol L—1 the DIp of the system decreased because the precipitation of Pb(Ac)2 on ACM weakened the heavy atom effect [27]. Drying temperature and time For the system containing 40.0 fg AFP-V spot—1, the effect of drying temperature and time on the DIp of the system was exam- ined, respectively. Results show that when the drying tempera- tures were 60, 70, 80, 90 and 95 (°C), corresponding DIp of F were 20.1, 23.5, 26.8, 30.1 and 27.8, and DIp of FITC were 30.6, 35.7, 40.8, 45.8 and 41.5, respectively. When the drying time was 0.5, 1.0, 1.5, 2.0 and 2.5 (min) at 90 °C, corresponding DIp of F were 7.5, 15.1, 22.6, 30.3 and 25.7, and the DIp of FITC were 11.5, 23.0, 34.5, 45.8 and 41.5, respectively. Obviously, the DIp of the system reached the maximum and remained stable at 90 °C for 2 min. It may be that the removal of water molecules on ACM minimized their quenching effect to the greatest extent. Oxygen and humidity For the system containing 40.0 fg AFP-V spot—1, the effect of oxygen and humidity on the DIp of the system was examined. Re- sults show that when drying N2 were passed for 5, 10, 15, 20, 25, 30 and 35 (min), corresponding DIp of F and FITC were 30.2, 30.1, 29.9, 30.1, 30.2, 29.8, 30.1 and 45.7, 45.8, 45.6, 45.8, 45.6, 45.7, 45.8, respectively. When without passing drying N2, corresponding DIp of F were 30.1, 30.3, 30.0, 30.2, 30.3, 30.2, 30.3 and 45.9, and DIp of FITC 45.5, 45.8, 45.7, 45.9, 45.6 and 45.7, respectively. Thus, the DIp of the system remained stable in the presence of drying N2 for 5–35 min. The reason might be that the effect of oxygen and humidity on the RTP was eliminated completely [25]. Stability of RTP emission Under the optimum conditions, when the standing time was 10, 20, 30, 40, 50, 60 and 70 (min), corresponding DIp of F were 30.1, 30.1, 30.1, 30.1, 30.1, 29.9 and 29.9, and DIp of FITC were 45.8, 45.8, 45.8, 45.8, 45.8, 45.7 and 45.3, respectively. The results show that the DIp of the system remained stable and had good repeat- ability within 50 min. But the DIp of the system declined when the standing time was over 50 min, possible due to the deliques- cence of Con A-AFP-V-Con A-F-FITC-DMA-ACM. Working curve, detection limit and precision Under the optimum conditions, the content of AFP-V was linear with DIp of the system. Thus, the AFP-V could be determined by AA-SS-RTP using DMA-F-FITC-Con A as the RTP sensor according to the two AA ways mentioned above. The working curve, correla- tion coefﬁcient (r), LD (calculated by 3Sb/k, Sb is the standard devi- ation of blank reagent; k is the slope of regression equation. n = 11), the limit of quantiﬁcation (LOQ, calculated by 10 Sb/k, n = 11) and RSD (%) of the RTP sensor are listed in Table 3. From Table 3, we could see that the labeling reagent used in this sensor was different from those in Refs. [7–10], and the DL of this method was lower than those of Refs. [7–10]. The sensitivity of this new sensor was much higher, the possible reasons were as follows: ﬁrstly, DMA-F-FITC increased the number of luminescent mole- cules on the biological target to enhance the sensitivity of SS-RTP further; secondly, DMA-F-FITC preserved the good characteristics including durable luminescence, long phosphorescence lifetime and high quantum yield; thirdly, the perturbation effect of an external heavy atom (Pb2+) increased transition probability from single state to triple state of F-FITC, which sharply enhanced the DIp of the system. The F-FITC-DMA could be directly used without any pretreatment, which simpliﬁed the operation. Besides, both the sandwich way and the direct way had high sensitivity, wide linear range and good precision, while sensitivity of the sandwich way was higher than that of the direct way. This method not only offers a new technology for the determination of ultra-trace AFP-V, but also shows that using DMA-F-FITC-Con A as the RTP sensor is an effective way to further improve the sensitivity of immunoassay. Table 5 Phosphorescence lifetime of AA reaction products. Method Measurement wavelengths kmax=kmaxðnmÞ Regression equation of phosphorescence delay curve (ms) r s (ms) Luminescent molecule Sensor (direct way) 542/710 ln Ip = 4.2622–0.01795s —0.9904 55.71 F 482/648 ln Ip = 4.8104–0.01154s —0.99702 86.65 FITC Sensor (sandwich 542/710 ln Ip = 4.3124–0.01496s —0.9947 66.84 F way) 482/648 ln Ip = 4.9745–0.009466s —0.9972 105.64 FITC Table 6 The analytical results of AFP-V in human serum (BDD is B-diasonagraph detection. CXFPD is computer-X-ray-faultage-photography determination. n = 6). Table 7 Analytical results of AFP-V in treated human serum (n = 3). Serum Obtained kmax=kmaðnmÞ RSD Added Recovery Percent recovery ELISA method PHC BDD CXFPD ex ex (Age) (lg L—1) (%) (lg L—1) (lg L—1) (%) (lg L—1) A(40) 320.3 542/710 3.3 10.00 10.2 102 316.4 – – – 324.9 481/648 2.9 10.00 9.94 99.4 327.5 – – – B(40) 262.1 542/710 1.8 10.00 10.3 10.3 257.7 – – – 283.7 481/648 2.6 10.00 10.0 100 280.1 – – – C(40) 340.2 542/710 3.0 10.00 10.1 101 337.8 – – – 344.7 481/648 3.4 10.00 9.97 99.7 348.2 – – – Speciﬁcity examination The speciﬁcity of this sensor was examined by the direct way, and the control experiment was conducted using DMA — F — FITC — Ab0IgE (Ab0IgE is antibody of IgE.) as RTP sensor. Seen from Table 4, the Ip of the system had no correlation with the content of AFP-V in the control group, indicating that there was no speciﬁc reactivity occurred between Ab0IgE and AFP-V, while the Ip of the system was linear with the content of AFP-V in the experiment team, showing the speciﬁcity of this method and the sensitive response of the RTP sensor (DMA-F-FITC-ConA) to AFP-V. Phosphorescence lifetime For the system containing 40.00 fg AFP-V spot—1, the phospho- rescence lifetime (s) could be determined by phosphorescence delay assay [28], the regression equation of ln Ip–s, r and s are listed in Table 5. The results show that sandwich way and direct way both had long s, and the s of sandwich way was longer than that of direct way. Obviously, under the optimum condition the time resolved technique for the determination of trace AFP-V had been estab- lished based on long s of F or FITC. Determination of AFP-V in human serum and forecast of human diseases 1.00 mL sample solution was taken and the AFP-V content of the samples was determined with present sensor, and the standard recovery experiment was also conducted. Compared with the clin- ical detection of ELISA by the Zhangzhou Hospital of Chinese Med- icine and the results are listed in Tables 6 and 7. (Note: (+) standsScheme 4. Speciﬁc AA reaction between AFP-V and F-(FITC)2-Con A. for having space-occupying lesions, while ( ) stands for having no space-occupying lesions.) As shown in Table 6, the RSD of this sensor was 1.5–4.3% and the recovery rate was 98.4–103%. The content of AFP-V determined with the phosphorescence excitation/emission wavelength of either F or FITC in human serum agreed well with those of clinical detection and diagnosis using ELISA. According to the contents of AFP-V in serum were higher or lower than normal value 355.6 lg L—1, we could forecast that A, B and C might be the suf- fered from PHC, while D, E and F were healthy people. The forecast was coincided with the results of clinical detection and diagnosis in the Zhangzhou Hospital of Chinese Medicine. A, B and C were treated for eight weeks, and ﬁnally the content of AFP-V in serum detected by proposed sensor were lower than 355.3 lg L—1 (Ta- ble 7), indicating that it had reached the healthy level. Meanwhile, no space occupying lesion of PHC was found by B-diasonagraph detection and CT detection. This improved the credibility of fore- casting human diseases by this method. The results not only show that the AA-SS-RTP using DMA-F-FITC-Con A as the RTP sensor was suitable for the determination of AFP-V and diagnosis of human diseases, but also reﬂect a brighter clinical application prospects. In addition, the phosphorescence excitation/emission wavelength of F or FITC could be chosen to determine the content of AFP-V, which improved the ﬂexibility of sensor application. Scheme 5. Speciﬁc AA reaction between AFP-V and F-(FITC-Con A)2. Mechanism of sensing detection and labeling reaction Under the perturbation effect of Pb2+, F and FITC could emit strong and stable RTP on ACM. The phosphorescence intensity of F and FITC increased sharply in the system, the reason may be that the active -OH group of F could react with the dissociated –COOH group of FITC to form F-(FITC)2 complex containing several FITC molecules [20], which could increased the number of FITC mole- cules on biological target and further enhance the RTP signal of F and FITC (Scheme 1): When F-(FITC)2 was used to label Con A (expressed as (H2N)2- Con A-(COOH)2), EDC ﬁrstly coupled with –COOH of Con A to form Con A-COO-EDC, which reacted with NHS to generate Con A-COO- NHS [29] (Scheme 2.), and at last Con A-COO-NHS reacted with the -NCS of FITC in F-(FITC)2 to produce the labeling product F-(FITC)2- Con A and F-(FITC-Con A)2 containing –CO–NH– [19] (Scheme 3), The labeling product F-(FITC)2-Con A and F-(FITC-Con A)2 could destroy the symmetry structure of F, which caused F to emit strong ﬂuorescence at room temperature [30]. Under the perturbation ef- fect of Pb2+, they could emit strong RTP of F. Remarkably, F-(FITC)2- Con A and F-(FITC-Con A)2 could be used as the excellent RTP sensor. When 600 pg AFP-V was added, the speciﬁc AA reactions be- tween –COOH of Con A in F-(FITC)2-Con A or F-(FITC-Con A)2 and –NH2 in AFP-V carried out, and formed F-(FITC)2-Con A-AFP-V or F- (FITC-Con A)2-AFP-V (Schemes 4 and 5): The F-(FITC)2-Con A-AFP-V and F-(FITC-Con A)2-AFP-V pre- served the good RTP characteristics of F and FITC, and the DIp of the system was linear with the content of AFP-V. Thus, AFP-V could be determined by AA-SS-RTP using F-(FITC)2-Con A or F-(FITC-Con A)2 as the RTP sensor. Conclusion In this paper, based on the strong and stable RTP of DMA-F-FITC as well as highly speciﬁc immunological reaction between AFP-V and Con A, a DMA-F-FITC-Con A sensor was designed and AA-SS- RTP for the determination of trace AFP-V was established. The development and application of the new DMA-F-FITC-Con A sensor could improve the ﬂexibility of AA-SS-RTP and provided a new application ﬁeld for AA-SS-RTP, F and lectin. The study results not only had potential academic value and application prospects for the development of phosphorescent labeling technique, SS- RTP and AA, but also opened up the new ways for exploiting sen- sors and the development of AA-SS-RTP. At the same time, they promoted the linkages and development among sensor science, F science, lectin science, analytical science and clinical medicine. Ackowledgement This work was supported by Fujian Province Natural Science Foundation (Grant No. 2010J01053 and JK2010035), Fujian Prov- ince Education Committee (JA11164, JA11311, JA10203 and JA10277), Fujian provincial bureau of quality and technical supervision (FJQI 2011006) and Scientiﬁc Research Program of Zhangzhou Institute of Technology Foundation (ZZY1215, ZZY 1106 and ZZY 1014). 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