Development of a rapid, specific fluorescence polarization immunoassay for the herbicide chlorsulfuron

A strategy for design of a derivative of chlorsulfuron, which mimics half of the herbicide molecule, was proposed. The 1-[(2-chloro)phenylsulfonyl]monoamidosuccinic acid was synthesized as a derivative of chlorsulfuron for conjugation to carrier proteins. Rabbits were immunized and the resulting polyclonal antibodies were assessed by the fluorescence polarization technique. The antibodies were highly specific to chlorsulfuron. Cross-reactivity to the structurally similar sulfonylurea and urea herbicides chlorbromuron, amidosulfuron, chlortoluron, isoproturon, diuron and linuron was less than 0.1%. A rapid fluorescence polarization immunoassay (FPIA) for chlorsulfuron detection in water samples was developed and optimized. The detection limit of chlorsulfuron in 50 μl of sample was 10 ng ml⁻¹. Total time for the measurement of 10 samples is 7 min. The proposed FPIA is suitable for rapid testing for pesticide contamination where the highest sensitivity is not critical or in combination with pre-concentration techniques.     

Long-wavelength fluorescence polarization immunoassay for surfactant determination

A new homogeneous fluoroimmunoassay method based on the use of dynamic long-wavelength fluorescence polarization is presented here for the first time. This methodology, which is applied to the determination of linear alkylbenzenesulfonates (LASs) in water samples, involves the use of a new long-wavelength tracer synthesized from the oxazine dye Nile Blue (NB) via a carbodiimide method. This tracer exhibits fluorescent properties at λ_ex 626 and λ_em 674 nm. The variation of fluorescence polarization with time is followed using the T-format configuration of the spectrofluorimeter and the analytical parameter used is the initial rate, which is measured in only 0.7 s. The dynamic range of the calibration graph is 0.05-4.7 mg/L, with a detection limit of 0.03 mg/L. The precision, expressed as relative standard deviation was assayed at 0.05 and 1 mg/L, giving values in the range 7.6-9.1%. Other anionic, cationic and non-ionic surfactants were tolerated at much higher concentration levels than that of the analyte. The method has proven its practical usefulness for the analysis of water samples, in which only a solid phase extraction step is necessary. Recoveries ranged from 80.8 to 119.8%, with a mean value of 100.8%.     

A novel competitive fluorescence immunoassay for the determination of dibutyl phthalate

A novel, sensitive, and specific competitive fluorescence immunoassay has been developed for the quantitative determination of dibutyl phthalate (DBP) using an antibody-coated plate format. Hapten was synthesized in order to produce polyclonal antibodies against dibutyl phthalate. Polyclonal antisera to dibutyl phthalate were generated in rabbits and used to construct the fluorescence immunoassay for measurement of dibutylphthalate. The assay had a detection limit of about 0.02 μg L⁻¹, a dynamic range of approximately 0.1–300 μg L⁻¹. Other similar phthalate compounds do not interfere significantly in the analysis using this immunoassay technique, and the cross-reactivity rates were less than 10%. The study demonstrated that the developed antiserum and fluorescence immunoassay procedure can be used to detect dibutyl phthalate in environmental samples such as tap water, river water, drinking water, and leachate from plastic drinking water bottles.     

Fluorescein labeling of estrogen for application of fluorescence polarization binding assays for antibody and receptor

The fluorescence polarization binding assay (FPBA) using fluorescein-labeled estrogen tracer is a homogeneous assay applicable to both estrogen antibody and estrogen receptor-binding assays. Two estrogen-ethylendiamine fluoresceinthiobamyl (E-EDF) tracers were synthesized; estrogen-6-EDF (E-6-F) derived from 6-ketoestradiol 6-(o-carboxymethyl) oxime and estrogen-17-EDF (E-17-F) was from 17β-estradiol 17-hemisuccinate. In both FPBAs using antibody and receptor, E-6-F tracer (Rf_365nm=0.58) showed a better binding response than E-17-F (Rf_365nm=0.70) indicating that the 17-position of estrogen seems to play an essential role as a binding site for antibody or receptor. In the optimized conditions of FPBA for E₂ using E-6-F tracer, antibody binding (K_d=9.4×10⁻⁹ M) is 50 times sensitive than receptor binding (K_d=4.6×10⁻⁸ M). Binding responses of estrogen and its related chemicals by FPBA indicate that antibody binding assay is able to screen the structural similarity of estrogen showing some response with methyltestosterone (K_i=2.1×10⁻⁵ M). On the other hand, the receptor assay is able to screen for estrogenic chemicals such as tamoxifen (K_i=4.5×10⁻⁹ M) and diethylstilbesterol (K_i=8.1×10⁻⁷ M). Therefore, E-6-F tracer is useful as a tracer for FPBA that is able to screen for chemicals structurally similar to estrogen using antibody, and that is able to screen for chemicals functionally similar to estrogen using receptor binding assay.     

Rotating-disk sorptive extraction of nonylphenol from water samples

In this study the sorption of nonylphenol was implemented on a rotating Teflon disk coated with a PDMS film on one of its surfaces. In this way, the disk, which has a high surface area, contacts only the liquid sample, which can be stirred at higher velocity than with the stir bar used in stir-bar sorptive extraction (SBSE), without damaging the phase while at the same time facilitating analyte mass transfer to the PDMS surface. We refer to the procedure as rotating-disk sorptive extraction (RDSE). Extraction variables such as disk rotational velocity, extraction time, and surface area of PDMS film were studied to establish the best conditions for extraction. With increasing rotational velocity, the amount of extracted analyte significantly increases because the stagnant layer concomitantly decreases. On the other hand, the extracted amount concomitantly increases with extraction time, reaching equilibrium at approximately 20 min, which can be reduced to 10 min when the surface area of PDMS increases from 1.74 to 6.97 cm². Precision of the method was determined by using the same disk (n = 6) and different disks (n = 3), showing relative standard deviations for the analyte of 3.7% and 10%, respectively. The detection limit of the method was 0.09 μg/L NP, defined at a signal to noise ratio of 3. The method was applied to a real sample, achieving quantitative recovery. The PDMS phase on the disk could be used for at least 50 experiments. In any case, replacement of the PDMS film on the disk is very easy and inexpensive, as compared to the commercial alternative SBSE.     

First direct fluorescence polarization assay for the detection and quantification of spirolides in mussel samples

In 2009, we achieve the first inhibition FP assay to detect imine cyclic toxins. In the present paper we propose a new FP assay for direct quantify spirolides. This new method has resulted in significant improvement of sensitivity, rapidity and accessibility. In the method design, nicotinic acetylcholine receptor from Torpedo marmorata membranes labelled with a derivative of fluorescein was used. Spirolides, 13-desmethyl spirolide C (13-desMeC) and 13,19-didesmethyl spirolide C (13,19-didesMeC) were extracted and purified from cultures of the Alexandrium ostenfeldii dinoflagellate. Data showed the decrease of FP when toxin concentration was increased. Thus, a relationship between the FP units and the spirolides amount present in a sample was obtained. This direct assay is a reproducible, simple and very sensitive method with a detection limit about 25 nM for 13-desMeC and 150 nM for 13,19-didesMeC. The procedure was used to measure spirolides in mussel samples using an extraction and clean up protocol suitable for the FP assay. Results obtained show that this method is able to quantify 13-desMeC in the range of 50–350 μg kg⁻¹ meat. Other liposoluble toxins did not interfere with the assay, proving a specific method. Moreover, the matrix do not affect in the range of toxin concentrations that involving risk of spirolides intoxication.     

Immunoenzyme assay of nonylphenol: study of selectivity and detection of alkylphenolic non-ionic surfactants in water samples

Immunoenzyme assay (ELISA) is proposed and characterized for determination of alkylphenol ethoxylates, a primary class of manufactured non-ionic surfactants. The assay is based on the obtained polyclonal antibodies against nonylphenol (NP), the main stable intermediate of the decomposition of nonylphenol ethoxylates. A mixture of non-modified branched isomers of NP was applied as hapten coupled to protein carriers by Mannich reaction with the use of formaldehyde. The proposed ELISA format is based on immobilized NP-(soybean trypsin inhibitor) conjugate as a competitor of antigen molecules contained in the tested sample for binding with specific antibodies indirectly labeled via an anti-species immunoperoxidase conjugate. The developed ELISA allows to reveal NP with the limit of detection about 10 ng ml⁻¹ and NP-related compounds such as octylphenol, alkylphenoletoxylates, alkylphenolcarboxylates and their halogenated derivatives. The ELISA was applied for assaying polluted water samples, namely influents and effluents from different wastewater treatment plants (WWTP) and tap water. ELISA and chromatographic data demonstrate good correlation (r = 0.94), while ELISA gives higher values. Due to endocrine disrupting and other toxic activities of some metabolites of alkylphenolic non-ionic surfactants, the developed assay may be effectively used in ecological monitoring and sanitary control.     

Part-per-trillion level detection of estradiol by competitive fluorescence immunoassay using DNA/dye conjugate as antibody multiple labels

Fluorescent organic dyes are currently the standard signal-generating labels used in microarray quantification. However, new labeling strategies are needed to meet the demand for high sensitivity in the detection of low-abundance proteins and small molecules. In this report, a long-chain DNA/dye conjugate was used to attach multiple fluorescence labels on antibodies to improve signal intensity and immunoassay sensitivity. Compared with the 30 base-pair (bp) oligonucleotide used in our previous work [Q. Zhang, L.-H. Guo, Bioconjugate Chem. 18 (2007) 1668-1672], conjugation of a 219 bp DNA in solution with a fluorescent DNA binder SYBR Green I resulted in more than sixfold increase in signal intensity, consistent with the increase in bp number. In a direct immunoassay for the detection of goat anti-mouse IgG in a mouse IgG-coated 96-well plate, the long DNA conjugate label also produced higher fluorescence than the short one, accompanied by about 15-fold improvement in the detection limit. To demonstrate its advantage in real applications, the DNA/dye conjugate was employed in the competitive immunoassay of 17β-estradiol, a clinically and environmentally important analyte. The biotin-terminated DNA was attached to biotinylated anti-estradiol antibody through the biotin/streptavidin/biotin bridge after the immuno-reaction was completed, followed by conjugation with SYBR Green I. The limit of detection for 17β-estradiol is 1.9 pg mL⁻¹, which is 200-fold lower than the assay using fluorescein-labeled antibodies. The new multiple labeling strategy uses readily available reagents, and is also compatible with current biochip platform. It has great potential in the sensitive detection of protein and antibody microarrays.     

量子点标记链霉亲和素荧光免疫分析测定雌三醇

以生物素化羊抗兔免疫球蛋白和量子点标记链霉亲和素为荧光探针,采用竞争免疫分析模式,建立了一种灵敏度高、选择性好的检测雌三醇的荧光免疫分析方法.对几种测量参数如温育时间和温度、缓冲溶液pH、试剂浓度等进行了优化;方法的线性范围为0.01～1000ng/mL,检出限0.0029 ng/mL,血清样品加标回收率在91%～117%之间.     

Preparation of pH-sensitive polymer by thermal initiation polymerization and its application in fluorescence immunoassay

A fluorescence immunoassay for human IgG (Ag) was developed using a pH-sensitive polymer prepared by thermal initiation or redox initiation polymerization as a carrier. In the competitive immunoassay, appropriate quantity of Ag was immobilized on the polymer and the standard Ag (or sample) solution, and a constant amount of fluorescein isothiocyanate labeled goat anti-human IgG antibody (Ab-FITC) was added. Immobilized Ag and the standard (or sample) Ag competed for binding to the Ab-FITC in 37 °C in homogeneous format. After changing the pH to separate the polymer-immune complex precipitate, it was re-dissolved and determined by fluorescence method. The results showed that the immobilization efficiency, immunological reaction activities of immobilized Ag and phase transition pH range were improved as Ag was immobilized by thermal initiation instead of redox initiation polymerization. Under optimum conditions, the calibration graphs for the Ag in both methods, thermal initiation and redox initiation, were linear over the concentration range of 0.0-1000 ng mL⁻¹, with detection limits 8 (thermal initiation) and 12 ng mL⁻¹ (redox initiation), respectively. Moreover, some pH-sensitive polymer prepared only in organic solvent or under high temperature could also be used as an immunoreaction carrier by thermal initiation polymerization. Thermal initiation polymerization was a better immobilization mode.     

Preliminary evaluation of new polymer matrix for solid-phase extraction of nonylphenol from water samples

Molecularly imprinted (MIP) and blank polymers with affinity for nonylphenol were designed using computational modelling. Chromatographic tests demonstrated higher affinity of imprinted polymers towards the template nonylphenol as compared with blank polymers. The performance of both polymers in solid-phase extraction was however very similar. Both blank and imprinted polymers appeared to be suitable for the removal and pre-concentration of nonylphenol from contaminated water samples with 99% efficiency of the recovery. The commercial resins PH(EC) (Biotage) and C18 (Varian) tested in the same conditions used for comparative purposes had recovery rate <84%. The polymer capacity for nonylphenol was 231 mg g⁻¹ for blank and 228 mg g⁻¹ for MIP. The synthesised materials can have significance for sample pre-concentration and environmental analysis of this class of compounds.     

Comparative study of three immunoassays based on monoclonal antibodies for detection of the pesticide parathion-methyl in real samples

Three immunoassay systems: indirect, direct competitive enzyme-linked immunosorbent assay (IC-ELISA and DC-ELISA), fluorescence polarization immunoassay (FPIA) based on monoclonal antibodies for the detection of parathion-methyl (PM) were developed and optimized. Several PM derivatives (haptens) were conjugated to proteins and fluoresceinthiocarbamyl ethylenediamine (EDF) to obtain immunogens and competitors. The influence of immunogen and competitor structures on the assay performance was investigated. IC-ELISA was the most sensitive of all techniques developed, with a detection limit of 0.08 ng ml⁻¹, but assay time was the longest (3.5 h per 96-well microtitre plate). DC-ELISA was easier to perform and quicker (1.5 h per 96-well microtitre plate) but less sensitive than IC-ELISA (detection limit was 0.5 ng ml⁻¹). FPIA was the fastest and simplest (7 min per 10 samples) but the least sensitive (detection limit was 15 ng ml⁻¹) technique. The methods were characterized by high specificity and reproducibility. The cross-reactivity for parathion-ethyl was around 30-40% for IC-ELISA and FPIA, but significantly higher (125%) for DC-ELISA. The immunoassays were applied to the analysis of PM residues in different food and environmental matrices. Methanol extracts of vegetable, fruit and soil samples were used for the analysis. Recoveries for most spiked samples averaged between 85 and 110%. The methods developed can be used for screening of food and environmental samples for PM residues without complicated clean-up.     

Specific liquid DNA hybridization kinetics measured by fluorescence polarization

The kinetics of specific DNA hybrid formation were monitored directly in solution. Detection was based on fluorescence polarization measurements of labelled oligonucleotides at different stages during the hybridization. The effect of mismatched base pairs on the kinetics was measured. Significant differences could be observed in the kinetics when as few as three mismatches were introduced by the polymerase chain reaction technique in the target DNA sequences.     

固相萃取-高效液相色谱-荧光检测法同时测定水中的壬基酚和双酚A

利用C18固相萃取柱富集和净化水样,采用高效液相色谱分离、荧光检测建立了水中的壬基酚（NP）和双酚A（BPA）的测定方法,并成功用于污水处理厂各处理单元出水的测定。不同加标水平的NP和BPA的回收率为95．3％～98．6％．其相对标准偏差为2、5％～3．7％。     

A microfluidic protease activity assay based on the detection of fluorescence polarization

This article describes a fluorescence polarization (FP)-based protease assay on a microfluidic device that is compatible with fast and reproducible analyses of protease activities. The optical systems were arranged for simultaneously measuring fluorescence intensities of vertical and horizontal polarization planes, and the binding of tetramethylrhodamine (TMR) labeled-biotin with streptavidin was utilized for optimizing FP detection in continuously flowing solutions within 74-μm wide, 12-μm deep microchannels of a glass chip. In developing off-chip FP-based assays for proteinase K, trypsin, papain and elastase, TMR conjugated-casein protein (TMR-α-casein) was employed as a universal substrate. After optimization of the hydrodynamic flow control to allow complete mixing of TMR-α-casein and short proteolysis time as possible, and of buffer composition to minimize protein sticking problems, the developed assay was transferred to the microfluidic chip by monitoring FP changes of TMR-α-casein in the main microchannel. The results indicate that the proposed device would serve as an integrated microfluidic platform with automated injection of reacting species, diffusion-controlled mixing, reaction and detection for protease activities without the need to separate the products.     

A homogeneous assay for protein analysis in droplets by fluorescence polarization

We present a novel homogeneous (“mix‐incubate‐read”) droplet microfluidic assay for specific protein detection in picoliter volumes by fluorescence polarization (FP), for the first time demonstrating the use of FP in a droplet microfluidic assay. Using an FP‐based assay we detect streptavidin concentrations as low as 500 nM and demonstrate that an FP assay allows us to distinguish droplets containing 5 μM rabbit IgG from droplets without IgG with an accuracy of 95%, levels relevant for hybridoma screening. This adds to the repertoire of droplet assay techniques a direct protein detection method which can be performed entirely inside droplets without the need for labeling of the analyte molecules.     

Homogeneous indirect fluorescence quenching immunoassay for the determination of low molecular weight substances

This paper describes the principle of a homogeneous indirect fluorescence quenching immunoassay that uses monoclonal antibodies. It is a carrier-free assay system that is performed completely in solution. The assay system was established for the determination of a low molecular weight substance (hapten), the herbicide diuron, used as a model analyte. A fluorescein–monuron conjugate together with a fluorescence-quenching monoclonal anti-fluorescein antibody and an anti-analyte antibody (here an anti-diuron/monuron monoclonal antibody) were used as central components of the assay. The fluorescein–monuron conjugate can be bound either by the anti-fluorescein monoclonal antibody or by the anti-diuron/monuron monoclonal antibody. Due to steric hindrance, binding of both antibodies to the conjugate was not possible at the same time. By selecting the antibody concentrations appropriately, a dynamic equilibrium can be established that permits the preferential binding of the anti-diuron/monuron antibody to the conjugate, which allows the fluorescein in the conjugate to fluoresce. This equilibrium can be easily altered by adding free analyte (diuron), which competes with the conjugate to bind to the anti-diuron/monuron antibody. A reduction of anti-diuron/monuron antibody binding to the conjugate results in an increase in the binding of the anti-fluorescein antibody, which leads to a decrease in the fluorescence of the conjugate. The fluorescence is therefore a direct indicator of the state of equilibrium of the system and thus also the presence of free unconjugated analyte. The determination of an analyte based on this test principle does not require any washing steps. After the test components are mixed, the dynamic equilibrium is rapidly reached and the results can be obtained in less than 5 min by measuring the fluorescence of the fluorescein. We used this test principle for the determination of diuron, which was demonstrated for concentrations of ∼5 nM.     

Fluorescence polarization immunoassays and related methods for simple, high-throughput screening of small molecules

Fluorescence polarization immunoassay (FPIA) is a homogeneous (without separation) competitive immunoassay method based on the increase in fluorescence polarization (FP) of fluorescent-labeled small antigens when bound by specific antibody. The minimum detectable quantity of FPIAs with fluorescein label (about 0.1 ng analyte) is comparable with chromatography and ELISA methods, although this may be limited by sample matrix interference. Because of its simplicity and speed, FPIA is readily automated and therefore suitable for high-throughput screening (HTS) in a variety of application areas. Systems that involve binding of ligands to receptor proteins are also susceptible to analysis by analogous FP methods employing fluorescent-labeled ligand and HTS applications have been developed, notably for use in candidate drug screening.     

An ultrasensitive quantum dots fluorescent polarization immunoassay based on the antibody modified Au nanoparticles amplifying for the detection of adenosine triphosphate

In this work, an ultrasensitive fluorescent polarization immunoassay (FPIA) method based on the quantum dot/aptamer/antibody/gold nanoparticles ensemble has been developed for the detection of adenosine triphosphate (ATP). DNA hybridization is formed when ATP is present in the PBS solution containing the DNA-conjugated quantum dots (QDs) and antibody-AuNPs. The substantial sensitivity improvement of the antibody-AuNPs-enhanced method is mainly attributed to the slower rotation of fluorescent unit when QDs-labeled oligonucleotides hybridize with antibody modified the gold nanoparticle. As a result, the fluorescent polarization (FP) values of the system increase significantly. Under the optimal conditions, a linear response with ATP concentration is ranged from 8 × 10⁻¹² M to 2.40 × 10⁻⁴ M. The detection limit reached as low as 1.8 pM. The developed work provides a sensitive and selective immunoassay protocol for ATP detection, which could be applied in more bioanalytical systems.     

A simple, rapid and high-throughput fluorescence polarization immunoassay for simultaneous detection of organophosphorus pesticides in vegetable and environmental water samples

A simple, rapid and high-throughput fluorescent polarization immunoassay (FPIA) for simultaneous determination of organophosphorus pesticides (OPs) using a broad-specificity monoclonal antibody was developed. The effects of tracer structure, tracer concentration, antibody dilution, methanol content and matrix effect on FPIA performance were studied. The FPIA can detect 5 OPs simultaneously with a limit of detection below 10 ng mL(-1). The time required for the equilibrium of antibody-antigen interaction was less than 10 min. The recovery from spiked vegetable and environmental samples ranged from 71.3% to 126.8%, with the coefficient of variations ranging from 3.5% to 14.5%. The developed FPIA was applied to samples, followed by confirmation with high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis. The developed FPIA demonstrated good accuracy and reproducibility, and is suitable for rapid and high-throughput screening for OP contamination with high-efficiency and low cost.