Recent progress in nanomaterial-enhanced fluorescence polarization/anisotropy sensors

As a promising signaling transduction approach, fluorescence polarization (FP)/fluorescence anisotropy (FA), provides a powerful quantitative tool for the rotational motion of fluorescently labeled molecules in chemical or biological homogeneous systems. Unlike fluorescence intensity, FP/FA is almost independent the concentration or quantum of fluorophores, but they are highly dependent on the size or molecular weight of the molecules or materials attached to fluorophores. Recently, significant progress in FP/FA was made, due to the introduction of some nanomaterials as FP/FA enhancers. The detection sensitivity is thus greatly improved by using nanomaterials as FP/FA enhancers, and nanomaterial-based FP/FA is currently used successfully in immunoassay, and analysis of protein, nucleic acid, small molecule and metal ion. Nanomaterial-based FP/FA provides a new kind of strategy to design fluorescent sensors and establishes innovative analytical methods. In this review, we summarize the scientific publications in the field of FP/FA sensor in recent five years, and first introduce the recent progress of FP/FA sensor based on nanomaterial. Subsequently, the various analytical applications of FP/FA based on nanomaterial are discussed. Finally, we provide perspectives on the current challenges and future prospects in this promising field.     

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 tetramethylrhodamine (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 interaction and restricted 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     

Recent progress in fluorescent and colorimetric sensors for the detection of ions and biomolecules

Optical sensors are widely used in the field of analytical sensing and optical imaging because of their high sensitivity, fast response time, and technical simplicity. This review focuses on recent contributions concerning the ions, neutral molecules and especially tumor micro-environment-related parameters based fluorescent or colorimetric sensors and is organized according to their target classifications.     

毛细管电泳-电致化学发光联用技术应用进展

介绍了毛细管电泳-电致化学发光联用技术(CE-ECL)的检测原理,重点评述了1983-2010年间CE-ECL在药物制品、生物样品、环境和食品、农产品分析中的应用(引用文献66篇)。     

Simultaneous determination of glycols based on fluorescence anisotropy

Simultaneous determination of non-fluorescent glycols in mixtures without separation or chemical transformation steps is described. Two methods based in the measure of fluorescence anisotropy of a probe such as fluorescein dissolved in the analyte or analyte mixtures are described. In the first method, the anisotropy spectra of pure and mixtures of analytes are used to quantitative determination (if the fluorophor concentration is in a range where fluorescence intensity is proportional to concentration). In the second method, a calibration curve anisotropy-concentration based on the application of the Perrin equation is established. The methods presented here are capable of directly resolving binary mixtures of non-fluorescent glycols on the basis of differences on the fluorescence anisotropy of a fluorescence tracer. Best analytical performances were obtained by application of the method based on Perrin equation. This method is simple, rapid and allows the determination of mixtures of glycols with reasonable accuracy and precision. Detection limits are limited by the quantum yield and anisotropy values of the tracer in the solvents. Recovery values are related to the differences in anisotropy values of the tracer in the pure solvents. Mixtures of glycerine/ethylene glycol (GL/EG), ethylene glycol/1,2-propane diol (EG/1,2-PPD) and polyethylene glycol 400/1,2-propane diol (PEG 400/1,2-PPD) were analysed and recovery values are within 95-120% in the Perrin method. Relative standard deviation are in the range 1.3-2.9% and detection limits in the range 3.9-8.9%.     

A data treatment method for detecting fluorescence anisotropy peaks in capillary electropherograms

A data treatment method is presented to detect fluorescence anisotropy (FA) peaks in capillary electrophoresis electropherograms. The data treatment method converts plots of fluorescence anisotropy vs. time that contain no peaks that are distinguishable from the noise of the anisotropy background into plots that show distinct fluorescence anisotropy peaks. The method was demonstrated using laser-induced fluorescence anisotropy data from individual Aβ (1–42) aggregates separated using capillary electrophoresis. Applying this data treatment method enabled the detection of anisotropy peaks for individual Aβ aggregate fluorescence peaks that were not observed prior to the data treatment method. The data treatment method is not specifically designed for Aβ aggregate analysis or capillary electrophoresis, and it should be applicable to other applications and other separation methods with FA detection.     

A new optical platform for biosensing based on fluorescence anisotropy

A novel fluorescence-based optical platform for the interrogation of an optical biochip was designed and developed. The optical biochip was made of poly(methyl methacrylate) (PMMA) formed by two pieces of PMMA appropriately shaped in order to obtain four microchannels that are 500-μm wide and 400-μm high. The lower part includes the microchannels and the inlet and outlet for the fluidics, while the sensing biolayer was immobilized on the upper part. The optical signal comprised the fluorescence emitted by the biolayer, which was anisotropically coupled to the PMMA cover and suitably guided by the PMMA chip. The potentiality of the optical chip as a biosensor was investigated by means of a direct IgG/anti-IgG interaction carried out inside the flow channels. The mouse-IgG was covalently immobilized on the internal wall of the PMMA cover, and the Cy5-labelled anti-mouse IgG was used for the specific interaction. Several chemical treatments of the PMMA surface were investigated, poly(L-lactic acid), Eudragit L100 and NaOH, in order to obtain the most effective distribution of carboxylic groups useful for the covalent immobilisation of the mouse-IgG. The treatment with Eudragit L100 was found to be the most successful. Limits of detection and quantification of 0.05 μg mL⁻¹ and 0.2 μg mL⁻¹, respectively, were obtained with the configuration described.     

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.     

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 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.     

Express detection of nonylphenol in water samples by fluorescence polarization immunoassay

The development of express method for detection of endocrine-disrupting chemicals (EDC) such as alkylphenols is required for ecological monitoring. Several attempts have been made to produce antibodies against 4-nonylphenol (NP) in recent years. This work describes the production of new antibodies against NP and also summarizes the characterization of antibodies obtained earlier. Three approaches used to produce alkylphenol-specific antibodies are compared; these are based on: 1. omega-(4-hydroxyphenyl)nonanoic or omega-(4-hydroxyphenyl)heptanoic acid NP derivatives designed to mimic the linear NP isomer; 2. 4-aminophenol, which potentially mimics various substituted phenolic compounds with different side-chain structures at position 4 of the benzene ring; and 3. a mixture of branched NP isomers, conjugated to the carrier protein via a benzene ring by the Mannich reaction, and expected to be the closest mimic of NP structure by preserving its natural alkyl moiety.Fluorescence polarization immunoassays based on different combinations of antibody and labeled antigen for screening detection of NP were developed and structural aspects of assay sensitivity and specificity were investigated. The assays based on the antisera raised against omega-(4-hydroxyphenyl)nonanoic acid and NP conjugate via Mannich reaction are capable of express detection of NP with detection limit of 7 microg mL(-1 )and assay dynamic range of 18-300 microg mL(-1).     

Recent advances in fluorescence sensor for the detection of peroxide explosives

The detection of peroxide explosives (PEs) has attracted considerable attention all over the world in global security owing to their simple preparation, poor chemical stability and easy decomposition. In recent years, great efforts have been devoted to developing organic fluorescence sensors for detecting the PEs because of their fast response, high sensitivity and high selectivity. In this short review, we firstly discuss the sensing mechanisms for fluorescence based the PEs detection. Next, we reviewed recent progress of PE probes in the nearly 5 years and the design strategies of the material structures to enhance the sensitivity or selectivity, such as conjugated polymers and assembled nanoparticles.     

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.     

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.     

A sensitive fluorescence anisotropy method for detection of lead (II) ion by a G-quadruplex-inducible DNA aptamer

Sensitive and selective detection of Pb²⁺ is of great importance to both human health and environmental protection. Here we propose a novel fluorescence anisotropy (FA) approach for sensing Pb²⁺ in homogeneous solution by a G-rich thrombin binding aptamer (TBA). The TBA labeled with 6-carboxytetramethylrhodamine (TMR) at the seventh thymine nucleotide was used as a fluorescent probe for signaling Pb²⁺. It was found that the aptamer probe had a high FA in the absence of Pb²⁺. This is because the rotation of TMR is restricted by intramolecular interaction with the adjacent guanine bases, which results in photoinduced electron transfer (PET). When the aptamer probe binds to Pb²⁺ to form G-quadruplex, the intramolecular interaction should be eliminated, resulting in faster rotation of the fluorophore TMR in solution. Therefore, FA of aptamer probe is expected to decrease significantly upon binding to Pb²⁺. Indeed, we observed a decrease in FA of aptamer probe upon Pb²⁺ binding. Circular dichroism, fluorescence spectra, and fluorescence lifetime measurement were used to verify the reliability and reasonability of the sensing mechanism. By monitoring the FA change of the aptamer probe, we were able to real-time detect binding between the TBA probe and Pb²⁺. Moreover, the aptamer probe was exploited as a recognition element for quantification of Pb²⁺ in homogeneous solution. The change in FA showed a linear response to Pb²⁺ from 10 nM to 2.0 μM, with 1.0 nM limit of detection. In addition, this sensing system exhibited good selectivity for Pb²⁺ over other metal ions. The method is simple, quick and inherits the advantages of aptamer and FA.     

双波长分光光度法的研究进展

文中对双波长分光光度法在近十年来的研究进展作了评述。内容分为4部分：①常规双波长分光光度法;②标准加入法-双波长分光光度法;③计算双波长分光光度法及④双峰双波长分光光度法,引述文献23篇。     

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.     

Biosensing in microfluidic channels using fluorescence polarization

Microfabricated microfluidic devices provide useful platforms for sensing and conducting immunoassays for high throughput screening and drug discovery. In this paper, fluorescence polarization (FP) has been used as a technique for probing binding events within 500 μm and smaller microfluidic channels fabricated in polydimethylsiloxane. The binding of concanavalin A to a lectin-dextran and a glycoprotein-acetylcholinesterase has been used to demonstrate the homogeneous, ratioing format of fluorescence polarization for the quick and accurate determination of extremely low concentrations. Concentrations of concanavalin A in the 0.2-1.0 nmole range were detected within 500 μm channels. Polarization has also been used to sense for a polyaromatic hydrocarbon (PAH) within a microfluidic channel using binding to a TRITC-labeled antibody. Specifically, concentrations of pyrene in a 10-40 nmole range were sensed in 500 μm microfluidic channels. We have also demonstrated a simple pH sensor based on the change in anisotropy of a pH sensitive fluorophore-SNAFL. The ease of fabrication and measurement using such polarization-based devices make them extremely suitable for micro-sized sensors, assays and total analysis systems.     

Enzymatic cleavage and mass amplification strategy for small molecule detection using aptamer-based fluorescence polarization biosensor

Fluorescence polarization (FP) assays incorporated with fluorophore-labeled aptamers have attracted great interest in recent years. However, detecting small molecules through the use of FP assays still remains a challenge because small-molecule binding only results in negligible changes in the molecular weight of the fluorophore-labeled aptamer. To address this issue, we herein report a fluorescence polarization (FP) aptamer assay that incorporates a novel signal amplification strategy for highly sensitive detection of small molecules. In the absence of adenosine, our model target, free FAM-labeled aptamer can be digested by nuclease, resulting in the release of FAM-labeled nucleotide segments from the dT-biotin/streptavidin complex with weak background signal. However, in the presence of target, the FAM-labeled aptamer–target complex protects the FAM-labeled aptamer from nuclease cleavage, allowing streptavidin to act as a molar mass amplifier. The resulting increase in molecular mass and FP intensity of the aptamer–target complex provides improved sensitivity for concentration measurement. The probe could detect adenosine from 0.5 μM to 1000 μM, with a detection limit of 500 nM, showing that the sensitivity of the probe is superior to aptamer-based FP approaches previously reported for adenosine. Importantly, FP could resist environmental interferences, making it useful for complex biological samples without any tedious sample pretreatments. Our results demonstrate that this dual-amplified, aptamer-based strategy can be used to design fluorescence polarization probes for rapid, sensitive, and selective measurement of small molecules in complicated biological environment.     

Fluorescence anisotropy of acridinedione dyes in glycerol: Prolate model of ellipsoid

Time-dependent reorientations of resorcinol-based acridinidione (ADR) dyes in glycerol were studied using steady-state and time-resolved fluorescence studies. The difference between fluorescence anisotropy decays recorded at 460 nm when exciting at 250 nm and those obtained when exciting at 394 nm are reported. When exciting at 394 nm, the fluorescence anisotropy decay is bi-exponential, while on exciting at 250 nm a mono-exponential fluorescence anisotropy decay is observed. We interpret this in terms of different directions of the absorption dipole at 394 and 250 nm with the emission dipole respectively, which is experimentally validated and further analysed as a prolate model of ellipsoid.