The Development of A Fluorescence Polarization Immunoassay for Aflatoxin Detection

A fluorescence polarization immunoassay （FPIA） was developed for the analysis ofaflatoxins （AFs） using an anti-aflatoxin B1 （AFB1） monoclonal antibody and a novel fluorescein-labeled AFB1 tracer. The FPIA showed an IC50 value of 23.33 ng/mL with a limit of detection of 13.12 ng/mL for AFB1. The cross-reactivities of AFB1, AFB2, AFG1, AFG2, AFM1, and AFM2 with the antibody were 100%, 65.7%, 143%, 23.5%, 111.4%, and 2%, respectively. The group-specificity of anti-AFB1mAb indicated that the FPIA could potentially be used in a screening method for the detection of total AFs, albeit not AFG2 and AFM2. The total time required for analyzing 96 samples in one microplate was less than 5 rain. This study demonstrates the potential usefulness of the FPIA as a rapid and simple technique for monitoring AFs.

Super-resolution fluorescence polarization microscopy

Fluorescence polarization is related to the dipole orientation of chromophores,making fuores-cence polarization microscopy possible to_reveal structures and functions of tagged cellularorganelles and biological macromolecules.Several recent super resolution techniques have beenapplied to fluorescence polarization microscopy,achieving dipole measurement at nanoscale.In this review,we summarize both difraction limited and super resolution fluorescence polari-zation microscopy techniques,as well as their applications in biological imaging.

Antibody Production for a Rapid Fluorescence Polarization Immunoassay of Estrone

Estrone has been identified as a potential endocrine-disrupting chemical （EDC）[1]. Estrone is usually quantified by gas chromatography-mass spectrometry （GC-MS）, GC-MS/MS, high performance liquid chromatography （HPLC）, HPLC- MS, and HPLC-MS/MS, etc.[2-3]. Meanwhile, several immunoassays based on radioimmunoassay, enzyme linked immunosorbent assay （ELISA） or chemiluminescence immunoassay （CLIA） for determination of estrone in real samples have been developed[2＇4]. Although these methods are sensitive, they need multistage separation and are thus time-consuming and laborious. A very promising way for the simplification of immunoassays for routine applications is a shift from heterogeneous methods （with separation） to homogeneous assays （without separation）[5]. Fluorescence polarization immunoassay （FPIA） is one of the homogeneous techniques that meets the requirements of a simple, reliable, fast, and cost-effective analysis[6]. Therefore, the present study is focused on the development of FPIA in order to analyze estrone based on antibody production.

Determining a Diagnostic Cut-Off on Fluorescence Polarization Assay(FPA)for Bovine Brucellosis in Carchi,Ecuador

Serology is the foundation of any brucellosis control and eradication program worldwide, thus it is important to define accuracy diagnostics assays and cut-off of those assays, due to variations from country to country and even among specific areas in the country. The variation of cutoff values depended on: prevalence of disease, vaccination status, animal management, and control and eradication programs. Therefore, a cut-off for the diagnosis of bovine brucellosis through fluorescence polarization assay (FPA) in Carchi—Ecuador was determined. The survey has been carried out in Carchi province of Ecuador, who is considered a province of high prevalence of brucellosis and the vaccination status is unknown due to the lack of registers. Sera samples (n = 200) were obtained from individual cows from randomly selected herds. Blood sera were tested through Fluoresce Polarization Assay (FPA) and competitive enzyme-linked inmunosorbent assay (cELISA) as confirmatory test, and then receiver operating characteristic (ROC) analysis was done. The sensitivity and specificity values of FPA were 88.7% and 92.50% respectively using a cut-off of 89.90 mP. Moreover, the area under the curve showed that 92.2% is the probability accuracy of the test. The advantage of the FPA is that it is a test with good characteristics of sensitivity and specificity as well as a simple and quick test.

Fluorescence polarization assay improves the rapid detection of human brucellosis in China

Background:Brucellosis is an infectious-allergic zoonotic disease caused by bacteria of the genus Brucella.Early diagnosis is the key to preventing,treating,and controlling brucellosis.Fluorescence polarization immunoassay(FPA)is a new immunoassay for relatively rapid and accurate detection of antibodies or antigens based on antigen-antibody interaction.However,there is no report on FPA-based detection of human brucellosis in China.Therefore,this study is to evaluate the value of FPA for the diagnosis of human brucellosis in China.

A high throughout assay for human papillomavirus genotypes with fluorescence polarization

Objective To develop a simple,cheap,quick,accurate and practical method for a high throughout genotypes assay of human papillomavirus (HPV) DNA. Methods Crude DNA was extracted by a simplified proteinase K digesting method. HPV common conservative primers: GP5+/6+ system was used to amplify HPV DNA in 127 samples of condylomata acuminatum (CA) and cervical scrapes by PCR,then the PCR product was assayed using a template directing terminator incorporation (TDI) and genotypes were detected with fluorescence polarization (FP). Major HPVs type-specific probes (HPV6,11,16,18,31,33,35 and 58) designed by us were hybridized with the specific PCR products and a special fluorescent ddNTP terminator was directly added to the end of the probe under direction of specific PCR products. The results were measured with FP and compared with the results of the DNA sequence. Results Compared with the results of DNA sequencing,the results detected with fluorescence polarization were all correct. The proposed method could detect more than one type of HPV infection,but DNA sequencing method could not. The positive rate of HPV was 100% in 78 CA biopsies. Among them,there were 14 HPV double infections [HPV6B and 11 (9 cases),HPV11 and 16 (4),HPV11 and 18 (1)],5 HPV triple infections [HPV6B,11 and 16 (4),HPV11,16 and 18 (1)],and one HPV quadruple infection (HPV6B,11,16 and 18). The positive rate of HPV was 77% in the 49 cervical scrapes. Six HPV double infections [HPV6B and 11 (2),HPV11 and 16 (1),HPV6B and 16 (1),HPV16 and 18 (1),HPV18 and 58 (1)], 3 HPV triple infections [HPV6B,11 and 16 (2),HPV11,16 and 18 (1)] and one HPV quadruple infection (HPV6B,11,16 and 18) were detected in cervical cancer scrapes.Conclusions The proposed method allowed a high throughout,special,simple,rapid,automatic and economical detection of HPV-DNA genotyping without a use of labeling probes. It can detect multiple HPV genotype infection and will be and useful tool in HPV genotype screening.

Investigation of Interaction of Some Chalcones and Cyclic Chalcone Analogues with Outer Mitochondrial Membrane by UV-VIS and Fluorescence Spectroscopy

Interaction of the synthetic chalcones (1b,1c) and their cyclic analogues (2b,2c) with bovine (BSA) and human serum albumin (HSA) as well as with rat liver mitochondria (RLM) was studied by fluorescence spectroscopy. The maxima of emission fluorescence spectra were changed only in the case of 2b and 2c during interaction with BSA, HSA as well as mitochondrial outer membrane showing a slight hypsochromic shift and decrease of fluorescence. Interaction of the methoxy-(1b,2b) and the dimethylamino-substituted (1c,2c) compounds with outer mitochondrial membrane were studied by fluorescence polarization. Fluorescence polarization of 1b in the presence of the two proteins and mitochondria was found to be unchanged. Under similar conditions (2b,1c,2c) showed continuously increasing fluorescence polarization signal during the 30 minute period of investigations. Since fluorescence polarization supposes that as a result of binding these substances to proteins and lipids. Compound 2c displayed a continuous increase of fluorescence polarization signal in the presence of proteins (BSA, HSA), yeast cytoplasm (YC) and mitochondria (YM and RLM). This compound displayed a significant cytotoxic effect. This pattern of interaction with proteins might be one of the contributing vectors of the observed cytotoxicity against several human carcinoma cell lines.

Structure and Orientation of 1-(Hydroxyl)-5-(dodecyloxy)-naphthalene LB Films by Polarized Fluorescence Spectroscopy

ＩｎｔｒｏｄｕｃｔｉｏｎＩｎｒｅｃｅｎｔｙｅａｒｓ，Ｌａｎｇｍｕｉｒ－Ｂｌｏｄｇｅｔ（ＬＢ）ｆｉｌｍｓｈａｖｅａｔｒａｃｔｅｄｍｕｃｈａｔｅｎｔｉｏｎｂｅｃａｕｓｅｏｆｔｈｅｉｒｐｏｓｉｂｉｌｉｔｉｅｓａｓｆｕｎｃｔｉｏｎａｌｍｏｌｅｃｕｌａｒｄｅｖ...

Inclusion Behavior of Dimer β-CyclodextrinBridged with Aspartic Acid Derivative

The (β-cyclodextrin (CD) dimer bridged with aspartic acid (ASP) derivative, FITC-ASP(NH-(-CD)2 (Host, FITC=fluorescein-4-isothiocyanate), was synthesized. Fluorescence polarization study showed that the novel host formed an inclusion compound, [FITC-ASP(NH-(-CD)2]ATA, for which Kd was determined to be 5.0×10-6 mol/L by Beacon 2000 Analyzer, when ATA (Guest) = Adm-Trp-Arg-Arg-NH2 (Adm = 1-adamantanecarboxylic acid, Trp = tryptophan, Arg = arginine), where Kd is the dissociation constant in aqueous solution at 298 K.

Super-resolution dipole orientation mapping via polarization demodulation

Fluorescence polarization microscopy(FPM)aims to detect the dipole orientation of fluorophores and to resolve structural information for labeled organelles via wide-field or confocal microscopy.Conventional FPM often suffers from the presence of a large number of molecules within the diffraction-limited volume,with averaged fluorescence polarization collected from a group of dipoles with different orientations.Here,we apply sparse deconvolution and least-squares estimation to fluorescence polarization modulation data and demonstrate a super-resolution dipole orientation mapping(SDOM)method that resolves the effective dipole orientation from a much smaller number of fluorescent molecules within a sub-diffraction focal area.We further apply this method to resolve structural details in both fixed and live cells.For the first time,we show that different borders of a dendritic spine neck exhibit a heterogeneous distribution of dipole orientation.Furthermore,we illustrate that the dipole is always perpendicular to the direction of actin filaments in mammalian kidney cells and radially distributed in the hourglass structure of the septin protein under specific labelling.The accuracy of the dipole orientation can be further mapped using the orientation uniform factor,which shows the superiority of SDOM compared with its wide-field counterpart as the number of molecules is decreased within the smaller focal area.Using the inherent feature of the orientation dipole,the SDOM technique,with its fast imaging speed(at sub-second scale),can be applied to a broad range of fluorescently labeled biological systems to simultaneously resolve the valuable dipole orientation information with super-resolution imaging.

A simple fluorescence anisotropy assay for detection of bisphenol A using fluorescently labeled aptamer

Bisphenol A(BPA)is one of the environmental endocrine disruptors(EDCs),and BPA contamination in environment can cause high risks to human health.Rapid determination of BPA on sites is in high demand in environmental analysis.Taking advantage of aptamers as affinity ligands and fluorescence anisotropy(FA)analysis,we developed a simple and rapid FA assay for BPA by employing a single tetramethylrhodamine(TMR)labeled short 35-mer DNA aptamer against BPA.The assay is based on the BPA-binding induced conformation change of TMR-labeled aptamer and alteration of interaction between TMR and guanine bases,resulting in change of FA signals.We screened the FA change of aptamer probes having TMR label on a specific site of the aptamer upon BPA addition.The aptamer with a TMR label on the 22nd T base showed large FA-decreasing response to BPA and maintained good binding affinity to BPA.By using this TMR-labeled aptamer,we achieved FA detection of BPA with a detection limit of 0.5μmol/L under the optimized conditions.This assay was selective towards BPA and enabled the detection of BPA spiked in tap water sample,showing the potential applications on water samples.

Aptamer structure switch fluorescence anisotropy assay for aflatoxin B1 using tetramethylrhodamine-guanine interaction to enhance signal change

Fluorescence anisotropy(FA)assay in homogenous solution is simple,sensitive and reproducible.Here,we reported an aptamer structure switch FA assay for detection of aflatoxin B1(AFB1),one of the most toxic mycotoxins,by using tetra methylrhodamine(TMR)-labeled aptamer probe and its complementary DNA(cDNA)with tandem G bases extension,to meet the demand in sensitive and selective detection of AFB1,The hybridization of aptamer and cDNA drew TMR close to the repeated guanine(G)bases,and a high FA value was induced due to TMR-G inte raction and re stricted local rotation of TMR.In the presence of AFB1,aptamer bound to AFB1 instead of the cDNA due to competition.Thus,the TMR-G interaction was eliminated,and FA value of TMR decreased.This assay enabled the detection of AFB1 with detection limit of 125 pmol/L and dynamic range from 125 pmol/L to 31.2 nmol/L.

Microviscosity in modified Nafion membranes measured by fluorescence probes

Sodium or quaternary ammonium modified Nafion membranes in forms of Nafion-Na+, Nafion-NMe+4:, Nafion-NEt+4 and Nafion-NBu+4: have bean prepared by neutralizing Nafion-H+ membrane with NaOH or tetraalkylammonium hydroxide aqueous solution. Intramolecular excimer formation and fluorescence polarisation methods using 1,3-di(1-pyrenyl)propane and acridine orange-as probes respectively have been employed for measurements of the microviscosity within those modified Nafion membranes. Results show that the probes are located in the fluorocarbon/water interface in the cluster of the membranes and the microviscosities around the probe molecules an in the range of ca. 120–1200 cp and increase in the order of Nafion-Na+ <Nafion-NMe+4<Nafion-NEt+4<Nafion-NBu+4. Furthermore, the microviscosity values of these membranes in dry form an higher than those in wet form.

Influence of Metal Cations and Cholesterol on Lipid-amphotericin Membrane

Amphotericin B（AmB） has been widely used in antifungal therapy. AraB molecules combine with cholesterol to form pores that can be toxic to human cells, thus greatly limiting its clinical application. The interaction between Arab and the cell membrane may be influenced by potassimn, sodium and calcium ions. Lq this study, the bilayer in large unilamellar lipid-drug liposomes with or without cholesterol was employed as a model membrane. N-（7-Nitrobenz-2-oxa-1,3-diazol-4-yl）-1,2-dipalmitoyl-sn-glycero-3-phosphoetheanolamine（N-BD-PE） and 1-palmi-toyl-2-[6（7-nitrobenz-2-oxa-1,3-diazol-4-yl）aminodoclecanoyl]-sn-glysero-3-phosphocholine（6-NBD-PC） are two kinds of fluorescent lipid probes, and the NBD group is attached to the polar lipid headgroup in the former, but to the sn-2 fatty acyl chain in the latter. The effect of these metal cations on the lipid-drug membrane was monitored by red edge excitation shift（REES）, fluorescence polarization, and the fluorescence lifetime of lipid probes in hydrophilic and hydrophobic areas of the membrane. These ions have different effects on the lipid-AraB membrane. Cholesterol can strengthen the packing ability of the membrane, which is influenced differently by potassium, sodium and calcium ions. Moreover, the influence of these ions on the membrane may be relative to the method of ion transportation through the membrane. This study is significant to understand the reduction of AraB＇s cellular toxicity.