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Concept: Fluorescence spectroscopy

165

Chlorophyll a fluorometry has long been used as a method to study phytoplankton in the ocean. In situ fluorometry is used frequently in oceanography to provide depth-resolved estimates of phytoplankton biomass. However, the high price of commercially manufactured in situ fluorometers has made them unavailable to some individuals and institutions. Presented here is an investigation into building an in situ fluorometer using low cost electronics. The goal was to construct an easily reproducible in situ fluorometer from simple and widely available electronic components. The simplicity and modest cost of the sensor makes it valuable to students and professionals alike. Open source sharing of architecture and software will allow students to reconstruct and customize the sensor on a small budget. Research applications that require numerous in situ fluorometers or expendable fluorometers can also benefit from this study. The sensor costs US$150.00 and can be constructed with little to no previous experience. The sensor uses a blue LED to excite chlorophyll a and measures fluorescence using a silicon photodiode. The sensor is controlled by an Arduino microcontroller that also serves as a data logger.

Concepts: Fluorescence, Fluorescence spectroscopy, Diode, Ocean, Transducer, Infrared, Pulse oximeter, Fluorometer

28

A solvent switchable rotaxane equipped with a pyrene stopper and with two ferrocenyl units on the macrocycle is reported, in which three different states, two nondegenerate and one degenerate, can be obtained in different solvents at room temperature. This is accompanied by high contrast changes in fluorescence intensity of the pyrene stopper by the presence of the ferrocenyl moieties on the macrocycle, which quench the emission of pyrene more efficiently with proximity.

Concepts: Fluorescence, Oxygen, Light, Fluorescence spectroscopy, Solubility, Solvent, Rotaxane, Degeneracy

28

The formation of a thin antibody film on a glass surface using pneumatic spray was investigated as a potential immobilization technique for capturing pathogenic targets. Goat-Escherichia coli O157:H7 IgG films were made by pneumatic spray and compared against the avidin-biotin bridge immobilized films by assaying with green fluorescent protein (GFP) transformed E. coli O157:H7 cells and fluorescent reporter antibodies. Functionality, stability, and immobilization of the films were tested. The pneumatic spray films had lower fluorescence intensity values than the avidin-biotin bridge films but resulted in similar detection for E. coli O157:H7 at 10(5)-10(7)cells/ml sample concentrations with no detection of non-E. coli O157:H7 strains. Both methods also resulted in similar percent capture efficiencies. The results demonstrated that immobilization of antibody via pneumatic spray did not render the antibody non-functional and produced stable antibody films. The amount of time necessary for immobilization of the antibody was reduced significantly from 24h for the avidin-biotin bridge to 7 min using the pneumatic spray technique, with additional benefits of greatly reduced use of materials and chemicals. The pneumatic spray technique promises to be an alternative for the immobilization of antibodies on glass slides for capturing pathogenic targets and use in biosensor type devices.

Concepts: Immune system, Protein, Bacteria, Fluorescence spectroscopy, Green fluorescent protein, Escherichia coli, Escherichia coli O157:H7, Shiga toxin

28

A differential scanning fluorimetry (DSF) based high-throughput screening assay with the fluorescent molecular rotor CCVJ (9-(2-carboxy-2-cyanovinyl)julolidine) was developed. CCVJ is mainly sensitive to viscosity and less to polarity in comparison to polarity-sensitive dyes like SYPRO Orange, which was commonly used in DSF measurements. Therefore DSF with CCVJ is a suitable approach for high-throughput screening and stability testing of surfactant-containing protein formulations. Due to the different detection principles of CCVJ and SYPRO Orange, the midpoint of the fluorescence curve of CCVJ, defined as temperature of aggregation (Tagg), was obtained at a higher temperature than the midpoint of the SYPRO Orange fluorescence curve, defined as temperature of hydrophobic exposure (Th). Granulocyte colony stimulating factor (G-CSF) was used as model protein for all measurements. Commonly used surfactants in therapeutic protein formulations (polysorbate 20, polysorbate 80 and poloxamer 188) were investigated by DSF with CCVJ and SYPRO Orange. The fluorescence properties of CCVJ were minimally affected by investigated surfactants at concentrations typically used in pharmaceutical protein formulations. SYPRO Orange however, showed high background fluorescence as it also interacts with hydrophobic groups of surfactants. CCVJ was also capable of detecting thermally induced aggregation in the commercial polysorbate 80-containing product Neupogen®.

Concepts: Protein, Fluorescence, Oxygen, Molecular biology, Fluorescence spectroscopy, Polysorbate 80, Polysorbate, Polysorbate 20

28

Amantadine hydrochloride (AMA) and rimantadine hydrochloride (RIM) are non-fluorescent in aqueous solutions. This property makes their determination through direct fluorescent method difficult. The competing reactions and the supramolecular interaction mechanisms between the two drugs and coptisine (COP) as they fight for occupancy of the cucurbit[7]uril (CB[7]) cavity, were studied using spectrofluorimetry, (1)H NMR, and molecular modeling calculations. Based on the significant quenching of the supramolecular complex fluorescence intensity, a fluorescent probe method of high sensitivity and selectivity was developed to determine AMA or RIM in their pharmaceutical dosage forms and in urine samples with good precision and accuracy. The linear range of the method was from 0.0040 to 1.0μgmL(-1) with a detection limit ranging from 0.0012 to 0.0013μgmL(-1). This shows that the proposed method has promising potential for therapeutic monitoring and pharmacokinetics and for clinical application.

Concepts: Fluorescence, Pharmacology, Fluorescence spectroscopy, Adamantane, Rimantadine, Amantadine

27

Herein, fluorescent DNA-templated gold/silver nanoclusters (DNA-Au/Ag NCs) are presented as a novel probe for sensitive detection of deoxyribonuclease I (DNase I). The procedure is based on quenching fluorescence of DNA-Au/Ag NCs by DNase I digestion of the DNA (5'-CCCTTAATCCCC-3') template. This decrease in fluorescence intensity permitted sensitive detection of DNase I in a linear range of 0.013-60μgmL(-1), with a detection limit of 3ngmL(-1) at a signal-to-noise ratio of 3. Furthermore, the practicality of this probe for detection of DNase I in human serum and saliva samples was validated, demonstrating its advantages of simplicity, selectivity, sensitivity and low cost. Importantly, satisfactory agreement between results obtained by the fluorescent method described here and high performance liquid chromatography (HPLC) further confirmed the reliability and accuracy of this approach.

Concepts: Fluorescence, Fluorescence spectroscopy, Sensitivity and specificity, Chromatography, High performance liquid chromatography, Deoxyribonuclease, Deoxyribonuclease I, EC 3.1.21

27

A novel enzyme-free and label-free fluorescence aptasensor based on target-catalyzed hairpin self-assembly is developed for amplified detection of adenosine. This aptasensor contains four DNA strands termed as aptamer-catalysis strand, inhibit strand, hairpin structures H1 and H2 which are partially complementary. Meanwhile, a sequence that can form DNA G-quadruplex is partly hidden in the stem of H2. In the absence of adenosine, aptamer-catalysis strand is inhibited, and cannot trigger the self-assembly between H1 and H2. Upon the addition of adenosine, the binding event of aptamer and adenosine triggers the self-assembly between H1 and H2, resulting in the formation of G-quadruplex at the end of H1-H2 complex. The addition of N-methyl mesoporphyrin IX, which has a pronounced structural selectivity for G-quadruplex, generates label-free fluorescence signal. In the optimum conditions, we could detect adenosine as low as 6 μM by monitoring the change in fluorescence intensity. Furthermore, this amplified aptasensor shows high selectivity toward adenosine against its analogs due to the specific recognition ability of the aptamer for the target. Thus, the proposed aptasensor could be used as a simple and selective platform for target detection.

Concepts: DNA, Signal transduction, Structure, Fluorescence spectroscopy, The Target, American films, Trigger finger, Trigger

27

The first fluorescent sensor for HF2 (-) anion, N(1), N(3)-di(naphthalene-1-yl)isophthalamide (L) has been derived from α-Napthylamine and isopthaloyl chloride. In 1:1 (v/v) DMSO:H2O, L exhibits high selectivity towards HF2 (-) anion with a 4-fold enhancement in fluorescent intensity. Very little enhancement in fluorescence intensity is observed for F(-), Cl(-), Br(-), I(-), SCN(-), PO4 (3-), SO4 (2-), and CH3COO(-) anions. The stoichiometry interaction between L and HF2 (-) is found to be 1:1 from fluorescence and UV/Visible spectral data. DFT calculation shows that binding between HF2 (-) and L is 1:1 and increases the relative planarity between the two naphthyl rings causing fluorescence enhancement. A shift of 0.080 V in oxidation potential of L is observed on interaction with HF2 (-) by cyclic voltammetry and square wave voltammetry.

Concepts: Electron, Light, Hydrogen, Fluorescence spectroscopy, Electrochemistry, Atom, Ion, Squarewave voltammetry

27

A novel dual-emission fluorescence probe has been developed for specific and sensitive detection of hypochlorite (ClO(-)). Upon addition of ClO(-), significant changes in fluorescence emission intensity at two discrete wavelengths were observed. Meanwhile OONO(-) led to only a single-channel fluorescence enhancement. This feature makes it a clear advantage in distinguishing ClO(-), RNS from other ROS.

Concepts: Fluorescence, Spectroscopy, Optics, Light, Fluorescence spectroscopy, Stokes shift

26

In this study, we report here a general protocol for making core-shell magnetic Fe3O4/SiO2-MPS/MIPs (MPS = 3-(methacryloxyl) propyl trimethoxysilane, MIPs = molecularly imprinted polymers, Fe3O4/SiO2-MPS as core, MIPs as shell) via a surface molecular imprinting technique for optical detection of trace λ-cyhalothrin. The fluorescent molecularly imprinted polymer shell was first prepared by copolymerization of acrylamide with a small quantity of allyl fluorescein in the presence of λ-cyhalothrin to form recognition sites without doping. The magnetic Fe3O4/SiO2-MPS/MIPs exhibited paramagnetism, high fluorescence intensity, and highly selective recognition. Using fluorescence quenching as a detecting tool, Fe3O4/SiO2-MPS/MIPs were successfully applied to selectively and sensitively detect λ-cyhalothrin, and a linear relationship could be obtained covering a wide concentration range of 0-50 nM with a correlation coefficient of 0.9962 described by the Stern-Volmer equation. The experimental results of practical detection revealed that magnetic Fe3O4/SiO2-MPS/MIPs as an attractive recognition element was satisfactory for determination of trace λ-cyhalothrin in honey samples. This study, therefore, demonstrated the potential of MIPs for detection of λ-cyhalothrin in food.

Concepts: Fluorescence, Oxygen, Ultraviolet, Light, Fluorescence spectroscopy, Polymer, Quenching, Polymers