Fluorescent and Electrochemical Sensing of Polyphosphate Nucleotides by Ferrocene Functionalised with Two ZnII(TACN)(pyrene) Complexes

The [Fc? bis{Zn^II(TACN)(Py)}] complex, comprising two Zn^II(TACN) ligands (Fc=ferrocene; Py=pyrene; TACN=1,4,7‐triazacyclononane) bearing fluorescent pyrene chromophores linked by an electrochemically active ferrocene molecule has been synthesised in high yield through a multistep procedure. In the absence of the polyphosphate guest molecules, very weak excimer emission was observed, indicating that the two pyrene‐bearing Zn^II(TACN) units are arranged in a trans‐like configuration with respect to the ferrocene bridging unit. Binding of a variety of polyphosphate anionic guests (PPi and nucleotides di‐ and triphosphate) promotes the interaction between pyrene units and results in an enhancement in excimer emission. Investigations of phosphate binding by ³¹P NMR spectroscopy, fluorescence and electrochemical techniques confirmed a 1:1 stoichiometry for the binding of PPi and nucleotide polyphosphate anions to the bis(Zn^II(TACN)) moiety of [Fc? bis{Zn^II(TACN)(Py)}] and indicated that binding induces a trans to cis configuration rearrangement of the bis(Zn^II(TACN)) complexes that is responsible for the enhancement of the pyrene excimer emission. Pyrophosphate was concluded to have the strongest affinity to [Fc? bis{Zn^II(TACN)(Py)}] among the anions tested based on a six‐fold fluorescence enhancement and 0.1 V negative shift in the potential of the ferrocene/ferrocenium couple. The binding constant for a variety of polyphosphate anions was determined from the change in the intensity of pyrene excimer emission with polyphosphate concentration, measured at 475 nm in CH₃CN/Tris‐HCl (1:9) buffer solution (10.0 mM , pH 7.4). These measurements confirmed that pyrophosphate binds more strongly (K_b=(4.45±0.41)×10⁶ M ^?1) than the other nucleotide di‐ and triphosphates (K_b=1–50×10⁵ M ^?1) tested.     

Aggregation‐Controlled Excimer Emission from Anthracene‐Containing Polyamidoamine Dendrimers

Lower generations of polyamidoamine (PAMAM) dendrimers were peripherally modified with anthracene moieties, and excimer emission from anthracene chromophores was investigated in an acetonitrile–water mixture at acidic and basic pH values. Results from fluorescence spectroscopic experiments suggest that 1) the propensity of anthracene‐modified PAMAM dendrimers to aggregate in acetonitrile is substantial in the presence of 15–20 vol % of water, and 2) aggregate formation in anthracene‐modified PAMAM dendrimers leads to unique morphologies in the ground state, where the anthracene units are pre‐arranged to form stable excimers upon photoexcitation. Three types of anthracene excimers are generated in the system, with face‐to‐face, angular, and T‐shaped geometry. The formation of different types of anthracene excimers was confirmed by steady‐state and time‐resolved fluorescence spectroscopic experiments. Experimental results further suggest that it is feasible to alter the type of excimer formed by anthracene units attached to the PAMAM dendrimers through altering the propensity for ground‐state aggregation. Most excitingly, increased π conjugation in the molecular framework of anthracene‐substituted PAMAM dendrimers leads to intense and exclusive excimer emission from anthracene at room temperature.     

Cross‐Reactive Sensor Arrays for the Detection of Peptides in Aqueous Solution by Fluorescence Spectroscopy

A simple but powerful method for the sensing of peptides in aqueous solution has been developed. The transition‐metal complexes [PdCl₂(en)], [{RhCl₂Cp*}₂], and [{RuCl₂(p‐cymene)}₂] were combined with six different fluorescent dyes to build a cross‐reactive sensor array. The fluorescence response of the individual sensor units was based on competitive complexation reactions between the peptide analytes and the fluorescent dyes. The collective response of the sensor array in a time‐resolved fashion was used as an input for multivariate analyses. A sensor array comprised of only six metal–dye combinations was able to differentiate ten different dipeptides in buffered aqueous solution at a concentration of 50 μM . Furthermore, the cross‐reactive sensor could be used to obtain information about the identity and the quantity of the pharmacologically interesting dipeptides carnosine and homocarnosine in a complex biological matrix, such as deproteinized human blood serum. The sensor array was also able to sense longer peptides, which was demonstrated by differentiating mixtures of the nonapeptide bradykinin and the decapeptide kallidin.     

Intensely Fluorescent Azobenzenes: Synthesis,Crystal Structures,Effects of Substituents,and Application to Fluorescent Vital Stain

2‐[Bis(pentafluorophenyl)boryl]azobenzenes bearing hydrogen, methoxy, dimethylamino, trifluoromethyl, fluoro, n‐butyl, and tert‐butyldimethylsiloxy groups at the 4′‐position or methoxy and bromo groups at the 4‐position have been synthesized. The 4‐bromo group of the 2‐boryl‐4‐bromoazobenzene derivative was converted to phenyl and diphenylamino groups by palladium‐catalyzed reactions. The absorption and fluorescence properties have been investigated using UV/Vis and fluorescence spectroscopy. The 2‐borylazobenzenes emitted an intense green, yellow, and orange fluorescence, in marked contrast to the usual azobenzene fluorescence. The 4′‐siloxy derivative showed the highest fluorescence quantum yield (0.90) among those reported for azobenzenes to date. The correlation between the substituent and the fluorescence properties was elucidated by studying the effect of the substituent on the relaxation process and from DFT and TD‐DFT calculations. An electron‐donating group at the 4′‐position was found to be important for an intense emission. Application of fluorescent azobenzenes as a fluorescent vital stain for the visualization of living tissues was also investigated by microinjection into Xenopus embryos, suggesting these compounds are nontoxic towards embryos.     

Systematic Investigation of Photoinduced Electron Transfer Controlled by Internal Charge Transfer and Its Consequences for Selective PdCl2 Coordination

Fluoroionophores of fluorophore–spacer–receptor format were prepared for detection of PdCl₂ by fluorescence enhancement. The fluorescent probes 1 – 13 consist of a fluorophore group, an alkyl spacer and a dithiomaleonitrile PdCl₂ receptor. First, varying the length of the alkylene spacer (compounds 1 – 3 ) revealed a dominant through‐space pathway for oxidative photoinduced electron transfer (PET) in CH₂‐bridged dithiomaleonitrile fluoroionophores. Second, fluorescent probes 4 – 9 containing two anthracene or pyrene fragments connected through CH₂ bridges to the dithiomaleonitrile unit were synthesized. Modulation of the oxidation potential (E_Ox) through electron‐withdrawing or ‐donating groups on the anthracene moiety regulates the thermodynamic driving force for oxidative PET (ΔG_PET) in bis(anthrylmethylthio)maleonitriles and therefore the fluorescence quantum yields (Φ_f), too. The new concept was confirmed and transferred to pyrenyl ligands, and fluorescence enhancements (FE) greater than 3.2 in the presence of PdCl₂ were achieved by 7 and 8 (FE=5.4 and 5.2). Finally, for comparison, monofluorophore ligands 10 – 13 were synthesized.     

Evaluation of the effects of Candidatus Liberibacter asiaticus on inoculated citrus plants using laser-induced breakdown spectroscopy (LIBS) and chemometrics tools

This study investigated the organic and inorganic constituents of healthy leaves and Candidatus Liberibacter asiaticus (CLas)-inoculated leaves of citrus plants. The bacteria CLas are one of the causal agents of citrus greening (or Huanglongbing) and its effect on citrus leaves was investigated using laser-induced breakdown spectroscopy (LIBS) combined with chemometrics. The information obtained from the LIBS spectra profiles with chemometrics analysis was promising for the construction of predictive models to identify healthy and infected plants. The major, macro- and microconstituents were relevant for differentiation of the sample conditions. The models were then applied to different inoculation times (from 1 to 8 months). The models were effective in the classification of 82-97% of the diseased samples with a 95% significance level. The novelty of this method was in the fingerprinting of healthy and diseased plants based on their organic and inorganic contents.     

Microemulsion electrokinetic chromatography: an application for the simultaneous determination of suspected fragrance allergens in rinse-off products

The feasibility of microwave-accelerated derivatization for capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection was evaluated. The derivatization reaction was performed in a domestic microwave oven. Histidine (His), 1-methylhistidine (1-MH) and 3-methylhistidine (3-MH) were selected as test analytes and fluorescein isothiocyanate (FITC) was chosen as a fluorescent derivatizing reagent. Parameters that may affect the derivatization reaction and/or subsequent CE separation were systematically investigated. Under optimized conditions, the microwave-accelerated derivatization reaction was successfully completed within 150 s, compared to 4-24 h in a conventional water-bath derivatization process. This will remarkably reduce the overall analysis time and increase sample throughput of CE-LIF. The detection limits of this method were found to be 0.023 ng/mL for His, 0.023 ng/mL for 1-MH, and 0.034 ng/mL for 3-MH, respectively, comparable to those obtained using traditional derivatization protocols. The proposed method was characterized in terms of precision, linearity, accuracy and successfully applied for rapid and sensitive determination of these analytes in human urine.     

Synchronous fluorescence measurement of enrofloxacin in the pharmaceutical formulation and its residue in milks based on the yttrium (III)-perturbed luminescence

A simple, rapid and sensitive synchronous fluorescence method is put forward for the determination of enrofloxacin (ENRO) in the pharmaceutical formulation and its residue in milk based on the yttrium (III)-perturbed luminescence. When Y³⁺ is added into the ENRO solution, the fluorescence of ENRO is significantly enhanced. The synchronous fluorescence technology is employed in the method to determine trace amount of ENRO residue in milks. The synchronous fluorescence intensity of the system is measured in a 1-cm quartz cell with excitation wavelength of 328 nm, Δλ = 80 nm. A good linear relationship between the fluorescence intensity and the ENRO concentration is obtained in the range of 1.0 × 10⁻⁹ to 2.0 × 10⁻⁶ mol L⁻¹ (r² = 0.9992). The limit of detection (LOD) of this method attains as low as 3.0 × 10⁻¹⁰ mol L⁻¹ (S/N = 3). The selectivity of this method is also very good. Common metal ions, rare-earth ions and some pharmaceuticals, which are usually used together with ENRO, do not interfere with the determination of ENRO under the actual conditions. The proposed method can be applied to determine ENRO residue in milks, and limit of quantification (LOQ) determined in the spiked milk is estimated to be 2.8 × 10⁻⁸ mol L⁻¹ (10 μg L⁻¹). Moreover, this method can be used as a rapid screening for judging whether the ENRO residues in milks exceed Minimal Risk Levels (MRLs) or not. In addition, the mechanism of the fluorescence enhancement is also discussed in detail.     

Two different spectrofluorimetric methods for simultaneous determination of gemfibrozil and rosiglitazone in human plasma

Two accurate, reliable, and highly sensitive spectrofluorimetric methods were developed for simultaneous determination of binary mixture gemfibrozil and rosiglitazone in human plasma without prior separation steps. The first method is based on synchronous fluorescence spectrometry using double scans. At Δλ = 27 nm, gemfibrozil yields detectable signal that is independent of the presence of rosiglitazone. Similarly, at Δλ = 120 nm the signal of rosiglitazone is not influenced by the presence of gemfibrozil. Signals at two wavelengths, 301 (Δλ = 27 nm) and 368 nm (Δλ = 120 nm) vary linearly with gemfibrozil and rosiglitazone concentrations over the range 100-700 ng mL⁻¹ (for gemfibrozil) and 20-140 ng mL⁻¹ (for rosiglitazone), respectively. The limits of detection (LOD) were 2.3 and 2.72 ng mL⁻¹ for gemfibrozil and rosiglitazone, respectively. The second method is based on the technique of simultaneous equations (Vierodt's method), in which 258 nm was selected as the excitation wavelength. Two equations are constructed based on the fact that at (λEm₂=302 nm of gemfibrozil) and (λEm₂=369 nm of rosiglitazone) the fluorescence of the mixture is the sum of the individual fluorescence of gemfibrozil and rosiglitazone. The limits of detection (LOD) were 28.1 and 23.63 ng mL⁻¹ for gemfibrozil and rosiglitazone, respectively. The proposed methods were successfully applied for the determination of the two compounds in synthetic mixtures and in human plasma with a good recovery.     

Development and characterization of a magnetic bead-quantum dot nanoparticles based assay capable of Escherichia coli O157:H7 quantification

The development and characterization of a magnetic bead (MB)-quantum dot (QD) nanoparticles based assay capable of quantifying pathogenic bacteria is presented here. The MB-QD assay operates by having a capturing probe DNA selectively linked to the signaling probe DNA via the target genomic DNA (gDNA) during DNA hybridization. The signaling probe DNA is labeled with fluorescent QD₅₆₅ which serves as a reporter. The capturing probe DNA is conjugated simultaneously to a MB and another QD₆₅₅, which serve as a carrier and an internal standard, respectively. Successfully captured target gDNA is separated using a magnetic field and is quantified via a spectrofluorometer. The use of QDs (i.e., QD₅₆₅/QD₆₅₅) as both a fluorescence label and an internal standard increased the sensitivity of the assay. The passivation effect and the molar ratio between QD and DNA were optimized. The MB-QD assay demonstrated a detection limit of 890 zeptomolar (i.e., 10⁻²¹ mol L⁻¹) concentration for the linear single stranded DNA (ssDNA). It also demonstrated a detection limit of 87 gene copies for double stranded DNA (dsDNA) eaeA gene extracted from pure Escherichia coli (E. coli) O157:H7 culture. Its corresponding dynamic range, sensitivity, and selectivity were also presented. Finally, the bacterial gDNA of E. coli O157:H7 was used to highlight the MB-QD assay's ability to detect below the minimum infective dose (i.e., 100 organisms) of E. coli O157:H7 in water environment.