Affinity Adsorption Solid Substrate-room Temperature Phosphorimetry Research Articles

Fullerol-fluorescein isothiocyanate-concanavalin agglutinin phosphorescent sensor for the detection of alpha-fetoprotein and forecast of human diseases

Based on the reaction of the active –OH group in fullerol (F) with the dissociated –COOH group in fluorescein 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 substrate-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). Meanwhile, the mechanisms for the labeling reaction and the sensing detection of AFP-V were discussed.

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

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 –NH 2 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.

Analysis application in biological field and prediction of human diseases with dual luminescence molecular of 3.5-generations polyamidoamine dendrimers–porphyrin

A new phosphorescence-labelling reagent (3.5-G-D–P labelling reagent) was developed, based on 3.5-generation polyamidoamine dendrimers (3.5-G-D) as internal acceptor to capture porphyrin (P) molecular. In the disturber of heavy atom, 3.5-G-D–P could emit room temperature phosphorescence (RTP) of 3.5-G-D and P on the surface of polyamide membrane (PAM), respectively. Products (3.5-G-D–P–WGA) of 3.5-G-D–P labelling triticum vulgaris lectin (WGA) could emit strong and stable RTP signal on the surface of PAM, and it also could take specific affinity adsorption reaction (AA) with alkaline phosphatase (ALP). The product of the AA reaction (3.5-G-D–P–WGA–ALP) could keep the RTP characteristics of 3.5-G-D–P very well, and the Δ I p of the system was linear correlation to the content of ALP. The Δ I p of the system with Tween-80 was once for P and twice for 3.5-G-D more than that without Tween-80. Thus, the affinity adsorption solid substrate-room temperature phosphorimetry (AA-SS-RTP) for the determination of trace ALP has been established using Tween-80–3.5-G-D–P to label WGA. The detection limit (LD) of this method was 0.12 fg spot −1 for 3.5-G-D and 0.18 fg spot −1 for P with direct method, 0.14 fg spot −1 for 3.5-G-D and 0.17 fg spot −1 for P with sandwich method, respectively, and the sensitivity was obviously high. This research showed that either using 3.5-G-D or P excitation/emission wavelength to determine the content of ALP in human serum, the results were coincided with ELISA, and the flexibility of AA-SS-RTP was obviously improved and the applicability was wider. Meanwhile, the reaction mechanism of determining ALP by direct method AA-SS-RTP was discussed.

Determination of trace α‐fetoprotein variant by affinity adsorption solid substrate‐room temperature phosphorimetry

The 3.5-generation dendrimers (3.5G-D)-porphyrin (P) dual luminescent molecule (3.5G-D-P) was used to label concanavalin agglutinin (Con A); the product of the reaction is 3.5G-D-P-Con A. A new method for the determination of trace AFP-V by affinity adsorption solid substrate-room temperature phosphorimetry (AA-SS-RTP) was established, based on the room temperature phosphorescence (RTP) property of the product on polyamide membrane (PAM) substrate and the specific affinity adsorption (AA) reaction between 3.5G-D-P-Con A and alpha-fetoprotein variant (AFP-V), which caused the RTP of the system to be sharply enhanced, the DeltaIp was linearly correlated to the content of AFP-V. The sensitivity of the method was obviously high. It could accurately detect the content of AFP-V in serum. The results were tallied well with those obtained by the ELISA method.

Determination of Glucose by Affinity Adsorption Solid Substrate‐room Temperature Phosphorimetry Based on Triticum Vulgare Lectin Labeled with Silicon Dioxide Nanoparticle Containing Fluorescein Isothiocyanate

Abstract In the presence of heavy atom perturber Pb2+, silicon dioxide nanoparticle containing fluorescein isothiocyanate (FITC‐SiO2) could emit a strong and stable room temperature phosphorescence (RTP) signal on the surface of acetyl cellulose membrane (ACM). It was found in the research that a quantitative specific affinity adsorption (AA) reaction between triticum vulgare lectin (WGA) labeled with luminescent nanoparticle and glucose (G) could be carried on the surface of ACM. The product (WGA‐G‐WGA‐FITC‐SiO2) of the reaction could emit a stronger RTP signal, and the ΔIp had linear correlation to the content of G. According to the facts above, a new method to determine G by affinity adsorption solid substrate room temperature phosphorimetry (AA‐SS‐RTP) was established, based on WGA labeled with FITC‐SiO2. The detection limit (LD) of this method calculated by 3Sb/k was 0.47 pg·spot−1 (corresponding to a concentration value 1.2×10−9 g·mL−1, namely 5.3×10−9 mol·L−1), the sensitivity was high. Meanwhile, the mechanism for the determination of G by AA‐SS‐RTP was discussed.

Determination of Bioactive Matter by Affinity Adsorption Solid Substrate–Room Temperature Phosphorimetry Based on Lectin Labeled with Self-ordered Ring of Eosin Y

A new phosphorescent labeling reagent named self-ordered ring (ESOR) of eosin Y (E) was developed. And the application of the determination of bioactive matter by affinity adsorption solid substrate-room temperature phosphorimetry (AA-SS-RTP) based on ESOR labeling lectin was studied. Results showed that pink and homogeneous ESOR could be formed by E on polyamide membrane (PAM) in the presence of cetyltrimethylammonium bromide (CTAB) and ammonia water. ESOR could emit strong and stable room temperature phosphorescence (RTP) signal of E in the presence of heavy atom perturber. Specific affinity adsorption (AA) reactions could be carried out between the products of concanavalin agglutinin (Con A), triticum vulgaris lectin (WGA) labeled with ESOR and alpha-fetoprotein variant (AFP-V), alkaline phosphatase (ALP), glucose (G), respectively. Not only did the products of the affinity adsorption reactions preserve good RTP characteristic of E, but also the DeltaIp (DeltaIp=Ip2-Ip1, Ip1 is the RTP intensity of blank reagent, Ip2 is the RTP intensity of sample) of these products was proportional to the content of AFP-V, ALP and G, respectively. According to the facts above, a new method of AA-SS-RTP for the determination of AFP-V, ALP and G was established, based on ESOR labeling lectin. Detection limits (LD) of this method were 0.040 fg spot(-1) for AFP-V, 0.045 fg spot(-1) for ALP and 0.090 fg spot(-1) for G. And it has been successfully applied to the determination of AFP-V in human serum as well as the survey and forecast of human diseases. This method had high sensitivity, good repeatability, long RTP lifetime and little background interference with lamdamaxem at the long wavelength area. Meanwhile, the mechanism for the determination of trace AFP-V by AA-SS-RTP based on Con A labeled with ESOR was also discussed.

Determination of Trace Alkaline Phosphatase by Solid‐Substrate Room‐Temperature Phosphorimetry Based on Triticum vulgare Lectin Labeled with Fullerenol

Fullerenol (F) shows a strong and stable room-temperature phosphorescence (RTP) signal on the surface of nitrocellulose membrane (NCM) at lambda ex max/ lambda em max =542.0/709.4 nm. When modified by dodecylbenzenesulfonic acid sodium salt (DBS), fullerenol emits a stronger signal. It was also found that quantitative specific affinity-adsorption reaction can be carried out between Triticum vulgare lectin (WGA) labeled with DBS-F and alkaline phosphatase (ALP) on the surface of NCM, and the product obtained (WGA-ALP-WGA-F-DBS) emits a strong and stable RTP signal. Furthermore, the content of ALP was proportional to the DeltaI(p) value. Based on the facts above, a new method for the determination of trace amounts of ALP by affinity-adsorption solid-substrate room-temperature phosphorimetry (AA-SS-RTP) was established, using fullerenol modified with DBS to label WGA. The detection limit was 0.011 fg spot(-1) (corresponding concentration: 2.8x10(-14) g ml(-1), namely 2.8x10(-16) mol l(-1)). This method with high sensitivity, accuracy, and precision has been successfully applied to the determination of the content of ALP in human serum survey and forecast human disease, and the results are tallied with those using alkaline phosphatase kits. The mechanism for the determination of ALP using AA-SS-RTP was also discussed.

A New 4.0‐Generation Dendrimer Phosphorescence Labeling Reagent and Its Application to Determination of Trace Alkaline Phosphatase by Affinity Adsorption Solid Substrate‐room Temperature Phosphorimetry

AbstractA Triton X‐100‐4.0G‐D (4.0G‐D refers to a 4.0‐generation dendrimer) was brought forward as a new phosphorescence labeling reagent. Two types of specific affinity adsorption (AA) reactions (direct method and sandwich method) were carried out between the labeling product of Triton X‐100‐4.0G‐D‐Wheat germ agglutinin (WGA) and alkaline phosphatase (ALP), the product of AA reaction preserved the good characteristics of room temperature phosphorescence (RTP) of 4.0G‐D and ΔIP of the product was proportional to the content of ALP. According to the fact stated above, a new method for the determination of trace ALP by affinity adsorption solid substrate‐room temperature phosphorimetry (AA‐SS‐RTP) was established on the basis of WGA labeled with the Triton X‐100‐4.0G‐D. The detection limits were 0.20 ag·spot−1 (corresponding concentration: 5.0×10−16 g·mL−1, namely 5.0×10−18 mol·L−1) for a direct method and 0.14 ag·spot−1 (corresponding concentration: 3.5×10−16 g·mL−1, namely 3.5×10−18 mol·L−1) for a sandwich method, respectively. For their high sensitivity, good repeatability and high accuracy, the direct method and sandwich method have been successfully applied to determine the content of ALP in human serum, and the results were coincided with the clinical detection results of the enzyme‐linked immunosorbent assay method by the Zhangzhou Hospital of Traditional Chinese Medicine. Meanwhile, the mechanism for the determination of trace ALP by AA‐SS‐RTP was discussed.

Determination of glucose by affinity adsorption solid substrate-room temperature phosphorimetry based on concanavalin agglutinin labeled with fluorescein using 1.5-generation dendrimers as sensitizer

In the presence of the heavy atom perturber Pb(Ac)2, fluorescein (HFin) can emit strong and stable room temperature phosphorescence (RTP) on the surface of a nitrocellulose membrane (NCM) at λexmax/λemmax = 480/648 nm. It can be spiked with the 1.5-generation polyamidoamine dendrimers (abbreviated: D-1.5) to emit stronger RTP. It was found that a quantitative specific affinity adsorption (AA) reaction between concanavalin agglutinin (abbreviated: Con A) labeled with D-1.5-HFin and N-acetylglucosamine (G) could be carried out on the surface of NCM. The product of the reaction (D-1.5-HFin- Con A-G) could emit strong and stable RTP, and the ΔIp was proportional to the content of G. According to the above facts, a new method for determination of G by affinity adsorption solid substrate-room temperature phosphorimetry (AA-SS-RTP) was established, based on Con A labeled with fluorescein using D-1.5 dendrimers molecules as sensitizer. The linear range of the sandwich method was 0.040–60 pg G spot−1 (corresponding concentration range: 0.10–150 ng mL−1; sample volume: 0.40 µL spot−1). The regression equation of the working curve was ΔIp = 6.880 + 5.610 mG pg spot−1, r = 0.9987. The working solutions containing 0.10 and 150 ng mL−1 G were determined repeatedly 11 times, respectively. The corresponding RSDs were 2.9 and 3.8%. The detection limit of this method calculated by 3Sb/k was 0.021 pg spot−1 (5.2 × 10−11 g mL−1). Compared with the direct method (detection limit: 0.078 pg spot−1, linear range: 0.40–40 pg G spot−1), the sensitivity was obviously improved and the linear range was wider. This method has been successfully applied to the determination of G in human plasma, as well as to the supervision and forecast of human diseases, for it is of good sensitivity, accuracy and precision.

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

In this paper, 3.5-generation polyamidoamine dendrimers (3.5G-D)-porphyrin (P) dual luminescence molecule (3.5G-D-P) was developed as a new phosphorescence-labeling reagent. Meanwhile, the room temperature phosphorescence (RTP) characteristics of 3.5G-D-P and its product of labeling triticum vulgaris lectin (WGA) on the surface of polyamide membrane (PAM) were studied. Results showed that in the presence of heavy atom perturber LiAc, 3.5G-D and P of 3.5G-D-P molecule could emit strong and stable RTP on the PAM. And the Tween-80 would spike thoroughly the phosphorescence signal of 3.5G-D and P; moreover, specific affinity absorption (AA) reaction between the products (Tween-80-3.5G-D-P-WGA) of WGA labeled with Tween-80-3.5G-D-P and glucose (G) was carried out. The products of the AA reaction could keep good RTP characteristics of 3.5G-D and P dual luminescence molecule, and the Δ I p was linear correlation to the content of G. According to the facts above, a new method of affinity adsorption solid substrate-room temperature phosphorimetry (AA-SS-RTP) for the determination of trace G was established, basing on WGA labeled with Tween-80-3.5G-D-P dual luminescence molecule. The detection limit of this method was 0.13 fg spot −1 (1.7 × 10 −12 mol l −1, 3.5G-D) and 0.14 fg spot −1 (2.2 × 10 −12 mol l −1, P). Determination of G in human serum using excitation/emission wavelength of either 3.5G-D or P, the result was coincided with enzyme-linked immunosorbent assay (ELISA). Not only the sensitivity and accuracy of this method were higher, but also the flexibility of AA-SS-RTP was obviously improved and the applicability was wider.

Determination of alkaline phosphatase based on affinity adsorption solid-substrate room temperature phosphorimetry using rhodamine 6G–dibromoluciferin luminescent nanoparticle to label lectin and prediction of diseases

In the presence of heavy atom perturber LiAc, the silicon dioxide nanoparticle containing rhodamine 6G (R) and dibromoluciferin (D) (R-D-SiO 2) can emit strong and stable solid-substrate room temperature phosphorescence signal of R (λ ex/λ em = 481/648 nm) and D (λ ex/λ em = 457/622 nm) on the surface of acetyl cellulose membrane (ACM). R-D-SiO 2 is used to label triticum vulgare lectin (WGA). Then two types of affinity adsorption reactions, R-D-SiO 2-WGA- alkaline phosphatase (ALP) (direct method) and WGA-ALP-WGA-R-D-SiO 2 (sandwich method), are carried out on ACM. The conditions and the analytical characteristics for the determination of ALP using affinity adsorption solid-substrate room temperature phosphorimetry (AA-SS-RTP) were studied. For a 0.40-μl drop of sample, results show that the detection limits of the sandwich method are 0.16 ag spot −1(457/622 nm) and 0.17 ag spot −1(481/648 nm), and the detection limits of the direct method are 0.41 ag spot −1 (457/622 nm) and 0.44 ag spot −1 (481/648 nm). The contents of ALP in human serum correlated well with those obtained by enzyme-linked immunoassay. This study shows that AA-SS-RTP whether by the sandwich method or the direct method, can combine very well the characteristics of both high sensitivity of SS-RTP and specificity of the immunoreaction. Simultaneously, whether the phosphorescence excitation/emission wavelength of either R or D in R-D-SiO 2 is chosen to determine ALP, this can promote the agility and widen the adaptability of AA-SS-RTP.