Direct Fluorescent Method Research Articles

Survey of Common Infectious Diseases in Urban Foxes (Vulpes spp.) in Southeastern Iran.

The red fox (Vulpes vulpes) is one of the most common species of wild Canidae and is relatively abundant in Iran. Foxes (Vulpes spp.) transmit many zoonotic diseases, the most important of which are visceral leishmaniasis, rabies, hydatidosis, toxocariasis, and trichinellosis. In this study, visceral leishmaniasis, rabies, ectoparasites, canine gastrointestinal helminths, dermatophytosis, distemper, parvovirus infection, and heartworm infections were evaluated among live-trapped and rescued foxes injured by traffic road accidents referred to the teaching hospital of Kerman, Iran, veterinary faculty. Skin scraping and direct microscopic examination were used to detect ectoparasites and dermatophytosis. Immunochromatography rapid kits were used to detect dirofilariasis, parvovirus infection, and distemper. Necropsy was used to check for gastrointestinal parasites. Rabies and visceral leishmaniosis were screened for with direct fluorescent antibody test and ELISA methods, respectively. Gastrointestinal helminth infections, including Toxocara canis, Taenia taeniaeformis, Dipylidium caninum, Joyeuxiella echinorhyncoids, Toxascaris leonina, Taenia hydatigena, Echinococcus granulosus, Rictolaria spp., Oxynema spp., Macracanthorhynchus hirudinaceus, and Physaloptera spp., were detected. Skin scrapings showed dermatophytosis and various ectoparasites, including Rhipicephalus sanguineus, Ctenocephalides canis and Ctenocephalides felis, and Sarcoptes scabiei, in foxes with dermal lesions. Distemper and parvovirus infection (26.66%) were the common viral diseases, and rabies infection rate was quite high (16.66%). Dirofilariasis and leishmaniasis were detected in 10% of the population. This study showed that urban foxes which often cohabit with humans and domestic animals are carriers of many different pathogens. This interaction may facilitate indirect cross-species transmission of zoonotic disease. Periodic health monitoring and multidisciplinary cooperation for the diagnosis, control, and prevention of these zoonoses is highly recommended.

Evolution of a natural dihydropteroate synthase and development of a signal amplified fluorescence method for detection of 44 sulfonamides in milk

In this study, the recognition mechanisms and affinities of a natural dihydropteroate synthase for 44 sulfonamides were studied respectively. Then the key contact amino acid Ser222 was mutated to Arg222 by using site-directed mutagenesis method to produce a mutant. Results showed that the binding energies and affinities for the 44 drugs were generally improved. Then this mutant was used as recognition reagent to develop a direct competitive fluorescence method on 96-well microplate for determination of the 44 drugs in milk. Due to the used signal amplified fluorescent tracer, the limits of detection for the 44 drugs were in the range of 0.025–0.65 ng/mL, and the sensitivities were improved for about 6–86 folds in comparison with the conventional fluorescent tracer. After comprehensive comparison, the present method showed generally better performances than the previously reported immunoassays for sulfonamides. Therefore, this method could be used as an efficient tool for the routine screening of sulfonamides residues in large number of food samples.

Supramolecular complexation between cucurbit[7]uril and folate and analytical applications

Folate (FA) plays a key role in the biosynthesis of amino acids, purines, and pyrimidines in the human body, and intracellular folate metabolism has become an attractive target of tumor chemotherapy. In this work, an inclusion interaction was found between FA and cucurbit[7]uril (CB[7]), and the formation of a CB[7]-FA 2:1 supramolecular inclusion complex was confirmed by fluorescence spectra, UV-Vis absorption spectroscopy, 1H NMR, and molecular modeling calculations. In addition, FA is generally determined through the indirect fluorescent method because it shows weak fluorescence in aqueous solution. Therefore, a simple, direct fluorescence probe method for rapidly measuring FA was investigated, and the linear equation of FA was ΔF = 14.691C + 37.366 within the concentration ranges of 0.82 ~ 18.31 µg mL–1. The proposed direct fluorescence method was applied to the determination of spiked plasma. We demonstrated that this method could provide an experimental basis for the targeted administration of the CB[7]-FA complex, and it could be extended as a promising fluorescence detection method for drugs in vivo.

Development of a molecularly imprinted microspheres-based microplate fluorescence method for detection of amantadine and rimantadine in chicken

ABSTRACT In this study, molecularly imprinted microspheres of a type capable of recognising amantadine and rimantadine were first synthesised, and three fluorescent tracers based on dansyl chloride, fluorescein isothiocyanate and 5-carboxytetramethylrhodamine were also synthesised. These reagents were used to develop and optimise a direct competitive fluorescence method on conventional 96-well microplate for detection of the two analytes. Results showed that this method achieved simple operation procedure, rapid assay process (30 min), high sensitivity (limits of detection 0.04–0.05 ng mL−1) and acceptable recycle performance (five times). After optimisation of several parameters, this method was used to detect amantadine and rimantadine in chicken muscle samples. Their recoveries from standards fortified blank samples were in the range of 62.3–93.7%. The analysis results for some real chicken samples were consistent with a confirmatory LC-MS/MS method. Therefore, this method could be used as a rapid, simple and accurate tool for routine screening the residues of amantadine and rimantadine in a large number of chicken muscle samples.

Rapid and Sensitive Detection of Viable but Non-culturable Salmonella Induced by Low Temperature from Chicken Using EMA-Rti-LAMP Combined with BCAC

In this study, a rapid and sensitive real-time loop-mediated isothermal amplification (Rti-LAMP) assay was developed for quantitative and evaluation of viable but non-culturable (VBNC) Salmonella. Four micrograms per milliliter of ethidium bromide monoazide (EMA) could significantly inhibit DNA amplification derived from dead cells in Rti-LAMP assays. The EMA-Rti-LAMP was used to monitor the culturable and VBNC Salmonella cells induced by 4 °C and − 20 °C, as direct fluorescence method (DEM) and plate counting method as controls. When 1.3 × 104 CFU/mL Salmonella was 5 cycles of freeze-thaw, the cells were all dead. However, Salmonella in 1.3 × 106 CFU/mL gradually transferred into VBNC state reaching 6.0 × 102 CFU/mL (0.05%) after 5 cycles of freeze-thaw. Keeping Salmonella 1.3 × 104 CFU/mL and 1.3 × 106 CFU/mL in 0.85% NaCl at 4 °C, the culturable cells persistently decreased in plate counting. Meanwhile, the VBNC cells generated gradually from 0 to 4.2 × 103 CFU/mL and 1.3 × 105 CFU/mL detected by both EMA-Rti-LAMP and DEM up to 110-day storage, respectively. While in − 20 °C, 1.3 × 104 CFU/mL Salmonella sharply inactivated during 20 days, but 1.3 × 106 CFU/mL increasingly transferred into VBNC state reaching 3.5 × 104 CFU/mL detected by both EMA-Rti-LAMP and DEM up to 110-day storage. The results indicated that the EMA-Rti-LAMP had similar accuracy with DEM in rapidly detecting viable including VBNC cells, and the former had specificity but the latter did not. The EMA-Rti-LAMP combined with bentonite-coated activated carbon (BCAC) treatment could detect as low as 35 CFU/g VBNC Salmonella derived from contaminated chicken, and the entire assay completed in 5 h. Furthermore, four identical samples were Salmonella positive from 24 retail frozen chicken samples detected by plate culture (GB4789.4-2016), BCAC-Rti-LAMP, and BCAC-EMA-Rti-LAMP. The BCAC-EMA-Rti-LAMP had one more sample for Salmonella positive than that of plate culture, but less two samples than that of BCAC-Rti-LAMP. Noticeably, the BCAC-EMA-Rti-LAMP had much more accuracy as plate counting than that of BCAC-Rti-LAMP in detection of viable Salmonella derived from chicken. These results strongly suggested that the BCAC-EMA-Rti-LAMP assay could be a rapid and sensitive method for detection of viable Salmonella including VBNC cells in chicken without enrichment.

Direct quantification of surface coverage of antibody in IgG-Gold nanoparticles conjugates

It is of paramount importance to be able to accurately quantify surface coverage of antibodies on gold nanoparticles (AuNP) so as to optimise the sensitivity of AuNP-based immunosensors. Herein, we developed a fluorescence-based method to directly quantify rabbit immunoglobulin G (IgG) used as antibody model bound to AuNP. Rabbit IgG was first labelled with fluorescein-5-isothiocyanate (FITC) prior to conjugation to AuNP via either physisorption or chemisorption. IgG-conjugated AuNP were treated with NaCN to dissolve the AuNP and restore the fluorescence emission that was quenched in the presence of the metallic colloids, followed by quantification of fluorescein by spectrofluorimetry. This direct assay gave about 4 IgG bound to each 15-nm diameter AuNP for both immobilization strategies. This surface coverage value was in good agreement with that determined from the theoretical value calculated from the Localized Surface Plasmon Resonance (LSPR) band shift. For comparison, we also applied two indirect methods based on the quantitation of excess IgG remaining in the supernatant using fluorescence assay or enzyme-linked immunosorbent assay (ELISA). The indirect assays, either fluorescence or ELISA, commonly used to assess the antibody coverage on AuNP, overestimated the IgG surface coverage to a large extent, since up to 3 to 4 times higher coverages were measured. Therefore, the direct fluorescence method reported in this paper appears as a valuable method for quantification of surface coverage of antibody on AuNP.

Fluorescent Determination of Succinylcholine Chloride by Naphthalimide/ Stilbazolium Dye ⊂ CP5A.

Succinylcholine Chloride (SCC), a short-acting neuromuscular relaxant, is non-fluorescent in aqueous solutions. This property makes it impossible to be determined by direct fluorescent method. Naphthalimide dye (NA) exhibits very strong fluorescence emissions in aqueous solution, after complexing with carboxylatopillar[5]arene (CP5A) in aqueous solutions, the fluorescent quenched intensity of complex was observed. On the contraty, stilbazolium dye (SA) exhibits weak fluorescence emissions in aqueous solution, after being included by CP5A, a fluorescence enhancement was observed. However, adding SCC to the NA⊂ CP5A or SA⊂ CP5A complex solution led to the recovery of the fluorescence intensity, in the meantime, the color of SA⊂ CP5A solution changed from dark yellow to light yellow. The competitive supramolecular interaction between SCC, NA and SA for CP5A was studied by spectrofluorometry, 1H NMR. Herein, a FID (fluorescence indicator displacement) system to detect SCC based on NA⊂ CP5A and SA⊂ CP5A complex was developed.

Supramolecular interaction of transition metal complexes with albumins and DNA: Spectroscopic methods of estimation of binding parameters

Binding parameters of metal complexes with albumins and DNA since the middle of 1990s are considered and summarized. The most widespread spectroscopic methods of estimation of binding parameters are discussed – direct fluorescent methods, indirect fluorescent methods (by fluorescence quenching parameters), and ways of estimation of binding constants by other optical spectroscopic methods. The methods and approaches to calculations used for the determination of binding constants are discussed. The data on the already found binding constants and kinetic parameters is systematized.

Measurements of serum non-ceruloplasmin copper by a direct fluorescent method specific to Cu(II).

Meta-analyses indicated the breakdown of copper homeostasis in the sporadic form of Alzheimer's disease (AD), comprising copper decreases within the brain and copper increases in the blood and the pool not bound to ceruloplasmin (non-Cp Cu, also known in the literature as "free" copper). The calculated non-Cp Cu (Walshe's) index has many limitations. A direct fluorescent method for non-Cp Cu detection has been developed and data are presented herein. The study included samples from 147 healthy subjects, 36 stable mild cognitive impairment (MCI) and 89 AD patients, who were tested for non-Cp Cu through the direct method, total serum copper, ceruloplasmin concentration and o-dianisidine ceruloplasmin activity. The indirect non-Cp Cu Walshe's index was also calculated. The direct method was linear (0.9-5.9 μM), precise (within-laboratory coefficient variation of 9.7% for low and 7.1% for high measurements), and had a good recovery. A reference interval (0-1.9 μM) was determined parametrically in 147 healthy controls (27-84 years old). The variation of non-Cp Cu was evaluated according to age and sex. Non-Cp Cu was 1.5 times higher in AD patients (regarding the upper value of the reference interval) than in healthy controls. Healthy, MCI and AD subjects were differentiated through the direct non-Cp Cu method [areas under the curve (AUC)=0.755]. Considering a 95% specificity and a 1.91 μmol/L cut-off, the sensitivity was 48.3% (confidence interval 95%: 38%-58%). The likelihood ratio (LR) was 9.94 for positive test results (LR+) and 0.54 for negative test result (LR-). The direct fluorescent test reliably and accurately measures non-Cp Cu, thereby determining the probability of having AD.

Roneparstat (SST0001), an Innovative Heparanase (HPSE) Inhibitor for Multiple Myeloma (MM) Therapy: First in Man Study

▪Background: HPSE is an endo-ß-d-glucuronidase that trims the heparan sulfate (HS) chains of proteoglycans, releasing biologically active fragments of HS. HPSE activity impacts cell signaling, gene expression and promotes extracellular matrix remodeling within the tumor microenvironment; high HPSE expression is associated with enhanced tumor growth, angiogenesis and metastases in several cancer types. As a result of its tumor promoting activities, HPSE is a promising new and unexploited target for anti-cancer therapy. There is a single enzymatically active HPSE in humans and HPSE knockout mice appear to be healthy, thus therapeutic neutralization of HPSE activity would likely have limited negative side effects. In MM preclinical models HPSE was shown to be a master regulator of aggressive tumor behavior. Preclinical evidence also indicates that HPSE promotes chemoresistance suggesting it plays a pivotal role in regulating myeloma response to therapy (Ramani VPC et al., AACR 2014, Abstract nr. 1708). In preclinical studies, bortezomib or melphalan were found to enhance HPSE expression and secretion. High HPSE expressing MM cells were less susceptible to the cytotoxic effects of those drugs. Likewise, a very significant increase in HPSE gene expression following chemotherapy was observed in patient-derived tumor samples, indicating a potential role for HPSE in regulating myeloma response to therapy. Roneparstat (SST0001), a 100% N-acetylated and glycol split heparin, is a potent HPSE inhibitor devoid of any significant anticoagulant activity. In an in vivo model of disseminated myeloma, Roneparstat in combination with either bortezomib or melphalan, significantly decreased both the number of animals with detectable tumor and the tumor burden when compared with animals treated with either of these drugs alone. In addition, studies in animal models of MM indicated that the mechanism of action of Roneparstat was consistent with it having anti-HPSE activity in vivo (reduced angiogenesis and diminished expression of HGF, VEGF and MMP-9 and diminished HPSE induced shedding of syndecan-1, a HS proteoglycan known to be a potent promoter of myeloma growth).Patients and Methods: A First in Man, multicenter, international, phase I clinical study is currently ongoing in advanced heavily pre-treated refractory MM patients (pts) who have exhausted all available anti-MM therapies. Roneparstat is administered subcutaneously, with a starting flat dose defined according to ICH S9 guidelines. A schedule DX5W1,W2 Q28D is being tested. Each cohort plans 3 + 3 pts. A direct fluorescence method (Heparin Red assay) is used in pharmacokinetic studies along with aPTT, used as a surrogate (indirect) measurement of Roneparstat plasma concentration. The pharmacodynamic effect of the drug on the coagulation cascade and any antitumor effect are also evaluated.Results: 15 pts have been enrolled to date. 5 cohorts (doses ranging from 25 to 200 mg/day) have been evaluated, while a 400 mg cohort has just been opened. Five pts have received 1 cycle of therapy, six pts 2 cycles, one 3 cycles, one 5 cycles, one 9 cycles; one patient is currently on treatment, one is not evaluable. Roneparstat administration was found to be safe with only minimal transient side effects. No DLTs and no bleeding complications have been observed. Roneparstat has been well tolerated both systemically and locally. The only side effect observed was minor reactions (redness, bruising) at the injection site (in 6 pts, all grade 1). A decrease > 50% in the serum monoclonal component was observed in one patient, lasting for 6 cycles.Conclusions: Preclinical studies in MM lines and animal models have demonstrated Roneparstat as a potent anti-myeloma compound, particularly when used in combination with other drugs. In the ongoing Phase I escalating dose study (n. pts = 15), Roneparstat administration (at a dose of up to 200 mg/day) was found to be safe with only minimal local side effects. Based on these results, Roneparstat, at a defined dose, in combination with other anti-myeloma agents, will be evaluated in relapsed/resistant MM pts. DisclosuresGalli:sigma-tau Research Switzerland SA: Consultancy. Einsele:Novartis: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Amgen/Onyx: Consultancy, Honoraria, Speakers Bureau. Barbieri:sigma-tau Research Switzerland SA, Mendrisio, Switzerland: Employment. Paoletti:sigma-tau Research Switzerland SA, Mendrisio, Switzerland: Employment. Pace:sigma-sau Industrie Farmaceutiche Riunite SpA, Pomezia (RM), Italy: Employment. Sanderson:Sigma-tau Research S.P.A.: Consultancy, Research Funding. Nagler:Novaratis Pharmaceuticals Corporation: Consultancy, Honoraria, Research Funding.

Monitoring translocation of multisubunit RNA polymerase along the DNA with fluorescent base analogues.

Here we describe a direct fluorescence method that reports real-time occupancies of the pre- and post-translocated state of multisubunit RNA polymerase. In a stopped-flow setup, this method is capable of resolving a single base-pair translocation motion of RNA polymerase in real time. In a conventional spectrofluorometer, this method can be employed for studies of the time-averaged distribution of RNA polymerase on the DNA template. This method utilizes commercially available base analogue fluorophores integrated into template DNA strand in place of natural bases. We describe two template DNA strand designs where translocation of RNA polymerase from a pre-translocation to a post-translocation state results in disruption of stacking interactions of fluorophore with neighboring bases, with a concomitant large increase in fluorescence intensity.

Rapid and sensitive determination of clenbuterol in porcine muscle and swine urine using a fluorescent probe

The feed additive Clenbuterol hydrochloric acid (CLB) is non-fluorescent, thus it is difficult to quantify through direct fluorescent method. Palmatine (PAL) can react with cucurbit[7]uril (CB[7]) to form stable complexes as a fluorescent probe. Significant quenching of the fluorescence intensity of the CB[7]–PAL complex was observed with the addition of CLB. Based on the significant quenching of the supramolecular complex fluorescence intensity, a novel spectrofluorimetric method with high convenience, selectivity and sensitivity was developed for the determination of CLB. The fluorescence quenching values (ΔF) showed good linear relationship with CLB concentrations from 0.011μgmL−1 to 4.2μgmL−1 with a detection limit 0.004μgmL−1. In this research, an ultrasound treatment replaced the former time-consuming shake method to form stable complexes. The proposed spectrofluorimetric method had been successfully applied to the determination of CLB in porcine muscle and swine urine with good precision and accuracy. The competing reaction and the supramolecular interaction mechanisms between the CLB and PAL as they fight for occupancy of the CB[7] cavity were studied using spectrofluorimetry, 1H NMR, and molecular modeling calculations. Interestingly, results indicate that two stable CB[7]–CLB complexes were formed.

The role of cell-free DNA measured by a fluorescent test in the management of isolated traumatic head injuries

BackgroundTraumatic brain injury (TBI) is a major cause of death and disability. In this study a new method to measure cell free DNA (CFD) for the management of TBI is tested. Our hypothesis was that CFD concentrations correlate to the magnitude of brain damage, and may predict the outcome of injured patients.MethodsTwenty eight patients with isolated head injury were enrolled. Their demographic and clinical data were recorded. CFD levels were determined in patients' sera samples by a direct fluorescence method developed in our laboratory.ResultsMean admission CFD values were lower in patients with mild TBI compared to severe injury (760 ± 340 ng/ml vs. 1600 ± 2100 ng/ml, p = 0.03), and in patients with complete recovery upon discharge compared to patients with disabilities (680 ± 260 ng/ml vs. 2000 ± 2300 ng/ml, p = 0.003). Patients with high CFD values had a relative risk to require surgery of 1.5 (95% CI 0.83 to 2.9) a relative risk to have impaired outcome on discharge of 2.8 (95% CI 0.75 – 10), and a longer length of stay (12 ± 13 days vs. 3.4 ± 4.8 days, p = 0.02). CFD values did not correlate with CT scan based grading.ConclusionsCFD levels may be used as a marker to assess the severity of TBI and to predict the prognosis. Its use should be considered as an additional tool along with currently used methods or as a surrogate for them in limited resources environment.

Increased expression of the intercellular adhesion molecule-1 (ICAM-1) on peripheral blood neutrophils in acute pancreatitis

PurposeConsidering the important role of neutrophils’ activation in the pathogenesis of acute pancreatitis (AP), the aim of our study was to evaluate the expression of leukocytes’ adhesion molecules in patients with AP. Patients/methodsThirty-five patients (16 women and 19 men; age 32–77 years, median 56 years) with AP were prospectively included into our study. The absolute number of leukocytes was estimated by haematologic analyser. Surface neutrophils antigens (CD) were assayed by the direct fluorescence method for whole blood, using a flow cytometer. ResultsAt the day 1, significant increase of ICAM-1 expression was found in patients with severe AP (S-AP) (7280mm−3 vs 2850mm−3 in healthy control; p<0.05). In the days 2, 3 and 5 it sharply decreased and peaked again to 4860mm−3 at the day 10. In patients with mild AP (M-AP), not significant elevation of ICAM-1 quickly returned to normal level. In both forms of AP, neutrophil CD62L (L-selectin) expression reached the highest level at the day 1 (8800mm−3 and 9020mm−3, respectively in M-AP and S-AP, in comparison to 3400mm−3 in control; p<0.05). Expression of CD69 (neutrophils’ marker of early activation) significantly increased in both M-AP and S-AP. ConclusionsWe have found an early and significant increase of peripheral blood neutrophil CD54/ICAM-1 expression, specific for S-AP but not for M-AP. It may provide a good marker predicting severe course of pancreatitis.

Determination of paraquat in water samples using a sensitive fluorescent probe titration method

Paraquat (PQ), a nonselective herbicide, is non-fluorescent in aqueous solutions. Thus, its determination through direct fluorescent methods is not feasible. The supramolecular inclusion interaction of PQ with cucurbit[7]uril was studied by a fluorescent probe titration method. Significant quenching of the fluorescence intensity of the cucurbit[7]uril-coptisine fluorescent probe was observed with the addition of PQ. A new fluorescent probe titration method with high selectivity and sensitivity at the ng/mL level was developed to determine PQ in aqueous solutions with good precision and accuracy based on the significant quenching of the supramolecular complex fluorescence intensity. The proposed method was successfully used in the determination of PQ in lake water, tap water, well water, and ditch water in an agricultural area, with recoveries of 96.73% to 105.77%. The fluorescence quenching values (ΔF) showed a good linear relationship with PQ concentrations from 1.0 × 10−8 to 1.2× 10−5 mol/L with a detection limit of 3.35 × 10−9 mol/L. In addition, the interaction models of the supramolecular complexes formed between the host and the guest were established using theoretical calculations. The interaction mechanism between the cucurbit[7]uril and PQ was confirmed by 1H NMR spectroscopy.

Immunoassay Arrays Fabricated by Dip-Pen Nanolithography with Resonance Raman Detection

Here, we report the first use of resonance Raman scattering for the detection of miniaturized microscale arrays fabricated by dip-pen nanolithography. Antibody arrays for prostate-specific antigen (PSA) were printed, and a sandwich immunoassay was carried out. An enzyme-linked detection antibody was used to provide an insoluble and stable colored microdot in the recommended size range for microarray readers, which could be read with resonance Raman scattering. This gives quantitative detection as well as an improved detection limit and a larger dynamic range than that previously achieved by direct fluorescent detection methods. By Raman mapping across the arrayed area, the microdots were easily detected with very little background signal from surrounding areas. Levels of PSA as low as 25 pg/mL were detected using this method, which could be extended to a large number of useful biomarkers.

Competitive supramolecular interaction of carbachol and berberine with cucurbit[7]uril and its analytical application

Carbachol (CCH) is non-fluorescent in aqueous solution. This property makes its determination through direct fluorescent method impossible. Berberine (BER) exhibits very weak fluorescence emissions in aqueous solution. However, In acidic medium and room temperature, BER can react with CB[7] to form stable complex and the fluorescence intensity of the complex was greatly enhanced. The dramatic quenching of the fluorescence intensity of CB[7]–BER complex was observed with the addition of CCH. The competitive supramolecular interaction between CCH and BER for occupancy of the cucurbit[7]uril (CB[7]) cavity was studied by spectrofluorometry, 1H NMR, and molecular modeling calculation. Based on the significant quenching of the supramolecular complex fluorescence intensity, a simple, rapid, and sensitive fluorescence probe method for the determination of CCH in pharmaceutical dosage forms and in biological fluids was developed. The linear range of the method was from 0.01 μg mL− 1 to 1.5 μg mL− 1. The limit of detection was 0.003 μg mL− 1. This shows that the proposed method has promising potential for quality control, forensic identification, drug abuse or therapeutic monitoring, pharmacokinetics, and clinical application.

Supramolecular Interaction of Gliclazide with Cucurbit[7]uril and its Analytical Application

Gliclazide (GLZ) is non-fluorescent in aqueous solution. This property makes its determination through direct fluorescent methods impossible. Palmatine (PAL) exhibits very weak fluorescence emissions in aqueous solution. However, in acidic media at room temperature, PAL can react with cucurbit[7]uril (CB[7]) to form a stable complex and the fluorescence intensity of the complex is greatly enhanced. Dramatic quenching of the fluorescence intensity of the CB[7]–PAL complex was observed with the addition of GLZ. The competing reactions and the supramolecular interaction mechanisms between GLZ and PAL as they fight for occupancy of the CB[7] cavity were studied using spectrofluorimetry, 1H NMR spectroscopy, and molecular modelling calculations. The association constants of the complexes formed between the host and the guest were determined. Based on the significant quenching of the supramolecular complex fluorescence intensity, a fluorescent probe method of high sensitivity was developed to determine GLZ in its pharmaceutical dosage forms and in human plasma with good precision and accuracy. The linear range of the method was from 0.003 to 2.100 μg mL–1. The limit of detection was 0.001 μg mL–1. This shows that the proposed method has promising potential for therapeutic monitoring and pharmacokinetics and for clinical application.

Study on the supramolecular interaction of astemizole with cucurbit[7]uril and its analytical application

Astemizole (AST) is non-fluorescent in aqueous solution. This property makes its determination through direct fluorescence methods difficult. Reaction and supramolecular interaction mechanisms, between AST and palmatine (PAL) as they compete for occupancy of the cucurbit[7]uril (CB[7]) cavity, were studied using spectrofluorimetry, 1H NMR, and molecular modeling calculations. The association constants of the complexes formed between the host and the guest were determined. Based on the significant quenching of the supramolecular complex fluorescence intensity, a fluorescent probe method of high sensitivity and selectivity was developed to determine AST in its pharmaceutical dosage forms and in urine samples with good precision and accuracy. The linear range of the method was from 0.02 μg mL−1 to 2.2 μg mL−1. The detection limit was 0.007 μg mL−1. This shows that the proposed method has promising potential for therapeutic monitoring and pharmacokinetics and for clinical application.

Determination of Pesticide Residue Cartap Using a Sensitive Fluorescent Probe

The insecticide cartap (CP) is non-fluorescent in aqueous solutions. This property makes its determination through direct fluorescent method difficult. In acidic medium and at room temperature, palmatine (PAL) can react with cucurbit[7]uril (CB[7]) to form stable complexes, and the fluorescence intensity of the complex is greatly enhanced. Significant quenching of the fluorescence intensity of the CB[7]-PAL complex was observed with the addition of cartap. Based on the significant quenching of the supramolecular complex fluorescence intensity, a new spectrofluorimetric method with high sensitivity and selectivity was developed to determine cartap in aqueous solution. The fluorescence quenching values (ΔF) showed good linear relationship with cartap concentrations from 0.009 to 2.4 μg mL−1 with a detection limit 0.0029 μg mL−1. The proposed method had been successfully applied to the determination of cartap residues in grain and vegetable with recoveries of 87.4-103%. In addition, the association constants of the complexes formed between the host and the guest were determined. The competing reaction and the supramolecular interaction mechanisms between the cartap and PAL as they fight for occupancy of the CB[7] cavity were studied using spectrofluorimetry, 1H NMR and molecular modeling calculations.