8-Quinolineboronic acid as a potential phosphorescent molecular switch for the determination of alpha-fetoprotein variant for the prediction of primary hepatocellular carcinoma

8-Quinolineboronic acid phosphorescent molecular switch (8-QBA-PMS) in the “off” state emitted weak room temperature phosphorescence (RTP) of 8-QBA on the acetylcellulose membrane (ACM) with the perturbation of Pb²⁺. When 8-QBA-PMS was used to label concanavalin agglutinin (Con A) to form 8-QBA-PMS-Con A based on the reaction between -OH of 8-QBA-PMS and -COOH of Con A, 8-QBA-PMS turned “on” automatically due to its structure change, and RTP of the system increased 2.7 times. Besides, -NH₂ of 8-QBA-PMS-Con A could carry out affinity adsorption (AA) reaction with the -COOH of alpha-fetoprotein variant (AFP-V) to form the product Con A-AFP-V-Con A-8-QBA-PMS containing -NH-CO- bond, causing the RTP of the system to further increase. Moreover, the amount of AFP-V was linear to the ΔI_p of the system in the range of 0.012-2.40 (fg spot⁻¹). Thus, a new affinity sensitive adsorption solid substrate room temperature phosphorimetry using 8-QBA-PMS as labelling reagent (8-QBA-PMS-AASSRTP) for the determination of AFP-V was proposed with the detection limit (LD) of 9 × 10⁻¹⁵ g mL⁻¹. It had been used to determine AFP-V in human serum with the results agreeing with enzyme-link immunoassay (ELISA), showing promise for the prediction of PHC due to the intimate association between AFP-V and primary hepatocellular carcinoma (PHC). The mechanism of the promethod was also discussed.     

Determination of trace alpha-fetoprotein variant by affinity adsorption solid substrate-room temperature phosphorimetry and its application to the forecast of human diseases

In the presence of ion perturber LiAc, 4-generation polyamidoamine dendrimers (4G-D) could emit strong and stable room temperature phosphorescence (RTP) signal at on nitrocellulose membrane (NCM), and Triton X-100 could sharply enhance the RTP signal of 4G-D. Triton X-100-4G-D was used to label concanavalin agglutinin (Con A) to get the labeling product Triton X-100-4G-D-Con A. Quantitative specific affinity adsorption (AA) reaction between Triton X-100-4G-D-Con A and α-fetoprotein variant (AFP-V) could be carried out on the surface of NCM, whose product Triton X-100-4G-D-Con A-AFP-V could emit strong and stable RTP and its ΔI_p was proportional to the content of AFP-V. According to the facts above, a new affinity adsorption solid substrate-room temperature phosphorimetry (AA-SS-RTP) for the determination of trace AFP-V by Con A labeled with Triton X-100-4G-D was established. Detection limits of this method were 0.23 fg spot⁻¹ (direct method, corresponding concentration: 5.8 × 10⁻¹³ g mL⁻¹) and 0.13 fg spot⁻¹ (sandwich method, corresponding concentration: 3.2 × 10⁻¹³ g mL⁻¹). It has been successfully applied to determine the content of AFP-V in human serum and forecast human diseases, for its high sensitivity, long RTP lifetime, good repeatability, high accuracy and little background perturbation with at the long wavelength area. Meanwhile, the mechanism for the determination of trace AFP-V using AA-SS-RTP was also discussed.     

Solid Substrate Room Temperature Phosphorimetry for the Determination of Trace Mercury Based on the Reaction between Alkaline Phosphatase and Mercury Using Triton X-100 as a Sensitizer

A new solid substrate room temperature phosphorimetry (SSRTP) for the determination of trace mercury has been established, using Triton X‐100 as a sensitizer. The regression equation of working curve was ΔI_p=11.40m(Hg²⁺)+1.569 (ag·spot^?1, n=6, ΔI_p=I_p1?I_p2, I_p1 and I_p2 referred to the phosphorescence intensity of the blank reagent and the test solution, respectively), and correlation coefficient (r) was 0.9984. The RSD valus of the determination of 0.016 and 8.0 ag·spot^?1 Hg²⁺ were 4.1% and 1.7% (n=8), respectively, indicating that the method had good repeatability. The limit of detection (LOD) calculated by 3Sb/k was 7.0 zg·spot^?1 Hg²⁺ (corresponding concentration: 1.8×10^?17 g·mL^?1, Sb=0.025, n=11). This method has high sensitivity, selectivity and precision, which was applied to determination of trace mercury in water samples with the result being agreed very well with that of dithizone extraction spectrophotometry.     

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

A 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 △Ip 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 appfied 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.     

2-Carboxy-1-naphthyl phosphate as a substrate for the fluorimetric determination of alkaline phosphatase

A new substrate, 2-carboxy-1-naphthyl phosphate (CNP), was developed for the fluorimetric determination of alkaline phosphatase (ALP) activity. The product of the enzyme reaction is 1-hydroxy-2-naphthoic acid (HNA), which is a strong fluorescent product. The amount of HNA generated is proportional to ALP activity. Optimal conditions for the determination of ALP were investigated. The linear range and detection limit for the determination of ALP are 0.01-4.8 U/L and 7.44 mU/L, respectively. This method is simple, practical and can be successfully applied to assess ALP in human serum with good accuracy and precision. The results were evaluated by comparison with a standard colorimetric assay using p-nitrophenyl phosphate as ALP substrate.     

A New Fluorescein Isothiocyanate-Piperidine Self-ordered Ring Phosphorescent Probe for the Determination of Trace Protein

The ? COOH in fluorescein isothiocyanate (FITC) reacted with ? NH? in piperidine (P) to form FITC‐P on the center of indentation of polyamide membrane (PAM) when drying for 2 min at (92±1)°C. Then, the FITC‐P diffused outward from the indentation center and formed the round SOR‐P‐FITC (containing the FITC‐P self‐ordered rings). Thus, multi‐FITC accumulated on SOR‐P‐FITC, leading to the enhancement of RTP signal on bio‐target, whose I_p increased 2.0 times compared with non‐generated SOR. When bovine serum albumin (BSA) was added to the center of SOR‐P‐FITC, ? NCS of FITC in SOR‐P‐FITC reacted with ? NH₂ of BSA to form SOR‐P‐FITC‐BSA, which caused the RTP signal of FITC to enhance sharply. The ΔI_p of the system was 3.4 times higher than that without β‐CD and 4.0 times higher than that without SOR‐P‐FITC formed. Its ΔI_p was linear to the content of BSA. Therefore, a new solid substrate‐room temperature phosphorimetry (SS‐RTP) for the determination of trace protein was established using SOR‐P‐FITC as a phosphorescent probe. Under the optimum condition, the linear range of this method was 0.040–16.0 ag·spot^?1 with a detection limit (LD) of 8.5 zg·spot^?1 (0.40 µL sample solution per spot, the corresponding concentration was 2.1×10^?17 g·mL^?1), and the regression equation of working curve was ΔI_p=3.848+4.240m_BSA (ag·spot^‐1), n=6, correlation coefficient (r) was 0.9993. This method with high sensitivity had been applied to determining the content of trace protein in the water samples, and the results coincided well with those obtained with pyrocatechol violet‐Mo(VI) method (P.V.M.M.). At the same time, the mechanism of SS‐RTP using SOR‐P‐FITC as a phosphorescent probe (SOR‐P‐FITC‐SS‐RTP) was discussed.     

Catalytic solid substrate-room temperature phosphorimetry for the determination of trace As(V) based on oxidising reaction between hydrogen peroxide and fullerenol using tween-80 as sensitizer

A new catalytic solid substrate-room temperature phosphorimetry (SS-RTP) for the determination of trace arsenic(V) has been established. It is based on the fact that fullerenol (F-ol) emitted strong and stable room temperature phosphorescence (RTP) on nitric acid cellulose membrane (NCM) substrate. H₂O₂ could oxidise F-ol to cause the quenching of RTP. As(V) could catalyse H₂O₂ to oxidise F-ol and decrease the RTP signal of F-ol sharply. After adding tween-80 in the system, its ΔI _p enhanced 7.7 times compared with the without-tween-80 levels. Under the optimum conditions, the linear dynamic range of this method was 0.016?11.2 ag spot^?1 with a detection limit (LD) of 9.3 zg spot^?1 (corresponding concentration: 2.3 × 10^?17 g mL^?1). This sensitive, simple and selective method has been successfully applied to the determination of trace As(V) in human hair and tea samples. The reaction mechanism for SS-RTP is also discussed.     

4-Amino-1-naphthylphosphate as a substrate for the amperometric detection of alkaline phosphatase activity and its application for immunoassay

Immunosensors and biochemical array detection systems based on electrochemical transducers have many advantages such as low detection limit, fast response, simple design and ease of miniaturization. However, further development of such sensors will depend on the availability of suitable substrates that can be converted by a labeling enzyme to an electrochemically active product. Here, we report the synthesis of 4-amino-1-naphthylphosphate and it’s application as a new substrate for alkaline phosphatase. The electrochemical and enzymatic properties of this compound were investigated and compared with the properties of other aromatic 1,4-dihydroxy and 1,4-hydroxy-amine derivatives. The product of the enzyme reaction was 4-aminonaphthol, which was rapidly converted in the presences of air to 1,4-iminonaphthoquinone. This compound could then be detected in an amperometric flow injection assay (AFIA) with −200 mV versus Ag/AgCl potential application. The analytical range for mouse IgG, in an alkaline phosphatase amplified sandwich immuoassay with amperometric detection, was 0.01-100 μg ml⁻¹.     

Development and validation of an LC‐APCI‐MS/MS method for the determination of phenethyl isothiocyanate in human plasma

Phenethyl isothiocyanate (PEITC) is a promising chemopreventive agent present in cruciferous vegetables. This paper describes the development of a method for the determination of PEITC in human plasma by liquid chromatography/tandem mass spectrometry (LC‐MS/MS). Atmospheric‐pressure chemical ionization was found more suitable for ionization of PEITC than electrospray ionization. Because of the lability of PEITC, a combination of low temperature and acidification was applied to minimize the degradation during the sample collection and preparation procedure. A simple protein precipitation with acetonitrile was used for the preparation of plasma samples. The analyte and 1‐phenylpropyl isothiocyanate as internal standard (IS) were subjected to chromatographic analysis on a C₁₈ column (50 × 2.1 mm, 5 µm) using 85% methanol as mobile phase at a flow rate of 0.3 mL/min. The total analysis time for each chromatograph was 3 min and the results were linear over the studied range (5.00–250 ng/mL). The intra‐ and inter‐day precision values were acceptable as per US Food and Drug Administration guidelines. This method was successfully applied in the determination of PEITC concentrations in plasma samples from healthy chinese Volunteers. Copyright © 2014 John Wiley & Sons, Ltd.     

Validation of a new HPLC‐UV method for determination of the antibiotic linezolid in human plasma and in bronchoalveolar lavage

A rapid and selective HPLC‐UV method was developed for the quantification of linezolid (LNZ) in human plasma and bronchoalveolar lavage (BAL) at the concentrations associated with therapy. Plasma samples were extracted by solid‐phase extraction followed by evaporation to dryness and reconstitution in mobile phase solution. The chromatographic separation was carried out on a C₁₈ column with an isocratic mobile phase consisting of dihydrogen phosphate buffer 50 mm (pH 3.5) and acetonitrile (60:40 v/v). The detection was performed using a photodiode array. Under these conditions, a single chromatographic run could be completed within 12 min. The method was validated by estimating the precision and the accuracy for inter‐ and intra‐day analysis in the concentration range of 25–25600 ng/mL. The method was linear over the investigated range with all the correlation coefficients R > 0.999. The intra‐ and inter‐day precision was within 8.90% and the accuracy ranged from ?4.76 to +5.20%. This rapid and sensitive method was fully validated and could be applied to pharmacokinetic study for the determination of LNZ levels in human plasma and BAL samples. Copyright © 2013 John Wiley & Sons, Ltd.     

A highly sensitive coupling technique for the determination of trace quercetin based on solid substrate room temperature phosphorimetry and poly (vinyl alcohol) complex imprinting

Al³⁺ could react with quercetin (Q) to form [AlQ]³⁺ complex which could be used as a template for the preparation of poly (vinyl alcohol)–[AlQ]³⁺ complex imprinting (PVA-C-I). The [AlQ]³⁺ not only had good matching ability and selectivity with the cavity of PVA-C-I, but also could react with the fluorescein isothiocyanate anion (FITC⁻) on the outside of cavity by electrostatic interaction to form ion-association complex [AlQ]³⁺·[(FITC)⁻]₃. The ion-association complex could emit strong and stable room temperature phosphorescence (RTP) on polyamide membrane (PAM) and the ΔI_p of the system had linear relationship with the content of Q, showing the highly selective identification of PVA-C-I to Q. Thus, a new coupling technique for the determination of trace Q based on solid substrate room temperature phosphorimetry and poly (vinyl alcohol) complex imprinting (PVA-C-I-SSRTP) was established. The linear range and limit of detection (LOD) of this method were 0.010–2.0 (×10⁻¹² g mL⁻¹) and 2.0 × 10⁻¹⁴ g mL⁻¹, respectively, showing wide linear range and high sensitivity of PVA-C-I-SSRTP. This method was used to determine the content of Q in waste water, and the results are consistent with those by spectrofluorimetry. Meanwhile, the mechanism for the determination of Q using PVA-C-I-SSRTP was also discussed.     

Solid substrate-room temperature phosphorimetry for the determination of trace protein using ion association complex [Cu(BPY)2]·[(Fin)2] as a phosphorescent probe

Fluorescein (HFin) emitted strong and stable room temperature phosphorescence (RTP) on filter paper after set at 50 °C for 10 min using Li⁺ as the ion perturber. HFin existed as Fin⁻ when the pH value was in the range of 5.45–7.36. Fin⁻ could react with [Cu(BPY)₂]²⁺ (BPY: α,α-bipyridyl) to produce ion association complex [Cu(BPY)₂]²⁺·[(Fin)₂]²⁻, which could enhance the RTP signal of Hfin. In the presence of bovine serum albumin (BSA), the –COOH group of Fin⁻ in the [Cu(BPY)₂]²⁺·[(Fin)₂]²⁻ could react with the –NH₂ group of BSA to form the ion association complex [Cu(BPY)₂]²⁺·[(Fin-BSA)₂]²⁻, which contained –CO–NH– bond. This complex could sharply enhance the RTP signal of Hfin and the ΔI_p was directly proportional to the content of BSA. According to the facts above, a new solid substrate-room temperature phosphorimetry (SS-RTP) for the determination of trace protein had been established using the ion association complex [Cu(BPY)₂]²⁺·[(Fin)₂]²⁻as a phosphorescent probe. This method had wide linear range (0.40 × 10⁻⁹–280 × 10⁻⁹ mg l⁻¹), high sensitivity (the detection limit (LD) was 1.4 × 10⁻¹⁰ mg l⁻¹), good precision (RSD: 3.4–4.9%) and high selectivity (the allowed concentration of coexistent ions or coexistent materials was high). It had been applied to the determination of the content of protein in 10 kinds of real samples, and the result agreed well with pyrocatechol violet-Mo (VI) method (P.V.M.M.), which indicated it had high accuracy. Meanwhile, reaction mechanism for the determination of trace protein with [Cu(BPY)₂]²⁺·[(Fin)₂]²⁻ phosphorescent probe was also discussed. The academic thought of this research could not only be used to develop many kinds of ion association complex phosphorescent probes, but also provided a new way to promote the sensitivity of SS-RTP.