Modulating fluorescence anisotropy of terminally labeled double-stranded DNA via the interaction between dye and nucleotides for rational design of DNA recognition based applications.

Effective signal enhancement for fluorescence anisotropy in a simple manner is most desirable for fluorescence anisotropy method development. This work aimed to provide insights into the fluorescence anisotropy of terminally labeled double-stranded DNA (dsDNA) to facilitate a facile and universal design strategy for DNA recognition based applications. We demonstrated that fluorescence anisotropy of dsDNA could be regulated by the nature of dyes, the molecular volume, and the end structure of dsDNA. Fluorescence anisotropy ascended with the increased number of base pairs up to 18 bp and leveled off thereafter, indicating the molecular volume was not the only factor responsible for fluorescence anisotropy. By choosing dyes with the positively charged center, high fluorescence anisotropy signal was obtained due to the confinement of the segmental motion of dyes through the electrostatic interaction. By properly designing the end structure of dsDNA, fluorescence anisotropy could be further improved by enlarging the effective overall rotational volume, as supported by two-dimensional (2D) (1)H-(1)H nuclear Overhauser enhancement spectroscopy (NOESY). With the successful enhancement of the fluorescence anisotropy for terminally labeled dsDNA, simple and universal designs were demonstrated by sensing of major classes of analytes from macromolecules (DNA and protein) to small molecules (cocaine).

The electron-transfer based interaction between transition metal ions and photoluminescent graphene quantum dots (GQDs): a platform for metal ion sensing.

The electron-transfer based quenching effect of commonly encountered transition metal ions on the photoluminescence of grapheme quantum dots (GQDs) was for the first time investigated, and was found to be associated with electron configuration of the individual metal ion. Ethylene diamine tetraacetic acid (EDTA), the metal ion chelator, can competitively interact with metal ions to recover the quenched photoluminescence of GQDs. Basically, metal ions with empty or completely filled d orbits could not quench the photoluminescence of GQDs, but this quenching effect was observed for the metal ions with partly filled d orbits. Based on the quenching-recovering strategy, a simple optical metal sensing platform was established by taking Ni(2+) as an example. Using the nickel ion-specific chelating reagent, dimethylglyoxime (DMG), to replace EDTA, a detection limit of 4.1 µM was obtained in standard solution. This proposed strategy does not need further functionalization of GQDs, facilitating the application for simple, fast and cost-effective screening of metal ions.

Gold nanoparticle enhanced fluorescence anisotropy for the assay of single nucleotide polymorphisms (SNPs) based on toehold-mediated strand-displacement reaction.

We developed a highly differentiating, homogeneous gold nanoparticle (AuNP) enhanced fluorescence anisotropic method for single nucleotide polymorphism (SNP) detection at nanomolar level using toehold-mediated strand-displacement reaction. The template strand, containing a toehold domain with an allele-specific site, was immobilized on the surface of AuNPs, and the solution fluorescence anisotropy was markedly enhanced when the fluorescein-labeled blocking DNA was attached to the AuNP via hybridization. Strand-displacement by the target ssDNA strand resulted in detachment of fluorescein-labeled DNA from AuNPs, and thus decreased fluorescence anisotropy. The drastic kinetic difference in strand-displacement from toehold design was used to distinguish between the perfectly matched and the single-base mismatched strands. Free energy changes were calculated to elucidate the dependence of the differentiation ability on the mutation site in the toehold region. A solid negative signal change can be obtained for single-base mismatched strand in the dynamic range of the calibration curve, and a more than 10-fold signal difference can still be observed in a mixed solution containing 100 times the single-base mismatched strand, indicating the good specificity of the method. This proposed method can be performed with a standard spectrofluorimeter in a homogeneous and cost-effective manner, and has the potential to be extended to the application of fluorescence anisotropy method of SNP detection.

The homogeneous fluorescence anisotropic sensing of salivary lysozyme using the 6-carboxyfluorescein-labeled DNA aptamer.

A simple and sensitive fluorescence anisotropy method was developed for lysozyme, employing the coupling of fluorophore, 6-carboxyfluorescein (FAM), with lysozyme upon recognition between the target molecule and its DNA aptamer. It was found in this study that the rotational dynamic of the detecting system is crucial to obtain a high anisotropy signal that cannot always be achieved by simply increasing the molecular volume, because molecular volume increase may not be able to efficiently retard the rotational movement of the fluorophore. FAM was selected as the label of the ssDNA aptamer to effectively facilitate the change of the fluorophore from a primarily independent segmental movement to slow global rotation. The time-resolved measurements, including lifetime and dynamic fluorescence anisotropy, were conducted to study the recognition interaction and to better understand the methodology. The proposed method had a wide linear dynamic range of 12.5-300 nM and a high sensitivity with the limit of detection of 4.9 nM (3S/N). This proposed method was successfully applied to assay of human salivary lysozyme. The results based on the standard addition recovery and comparison with enzyme-linked immunosorbent assay (ELISA) demonstrated the feasibility of this method for biological samples. Using coupling between the fluorophore and the analyte can be one of the approaches working toward expanding the application of fluorescence anisotropy based on aptamer-target and antibody-antigen recognitions.

Determination of human urinary kanamycin in one step using urea-enhanced surface plasmon resonance light-scattering of gold nanoparticles.

The purpose of this study was to establish a simple, sensitive analytical method for kanamycin (KANA) in human urine. Enhancement of the plasmon resonance light-scattering (PRLS) of gold nanoparticles (AuNPs) by KANA provided the basis for this analytical method. At pH 6.7, KANA induced AuNPs aggregation with enhanced PRLS. The PRLS of the AuNPs-KANA system was further enhanced by addition of urea. The linear range and detection limit for KANA were from 20-800 nmol L(-1) and 2 nmol L(-1), respectively. Potential interfering substances present in urine had a negligible effect on the determination, thus preliminary sample separations were not necessary. Recovery of KANA from spiked human urine was 94-104%. This simple, sensitive method, using urea to enhance the PRLS of the AuNPs-KANA system, may provide a new approach for determination of compounds rich in OH groups.

Novel method to measure enamel surface porosity with hydrogen peroxide bleaching.

PURPOSE: To establish methods to quantitate the physical surface change, not the chemical color bleaching change, of enamel occurring with hydrogen peroxide treatment in solution and commercially available plastic strips. METHODS: Bovine enamel was polished to a sound, uniform, optical flat, white subsurface that was used as the initial substrate for all substrate modification, treatment and instrumental measurement using digital photography-image analysis, SEM and profilometry. Sound enamel was treated with 10, 20 and 30% solutions of hydrogen peroxide. Plastic strips were used to treat both sound and acid modified enamel surfaces. Etched enamel, similar to a 10-second exposure to lemon juice, was treated with 0.5, 1, 3, 5, 10, and 15% hydrogen peroxide for 24-hour exposure at 37 degrees C to obtain a dose response curve to this modified enamel. RESULTS: The digital photography-image analysis system and scanning electron microscopy (SEM) were effective in detecting enamel surface porosity and structure disruptive changes, respectively. Plastic strip treatment of both sound and etched enamel produced little surface change. Measurable surface change of etched enamel was detected with as low as 1% hydrogen peroxide in solution. The surface change with 15% hydrogen peroxide was statistically significant. Dye uptake as measured by image analysis indicated an increase in surface porosity that was more evident with the acid modified surface. SEM studies were consistent with this observation.