A nanogold-quenched fluorescence duplex probe for homogeneous DNA detection based on strand displacement

A nanogold-quenched fluorescence duplex probe has been developed for lighting up homogenous hybridization assays. This novel probe is constructed from two strands of different lengths, and labeled by nanogold and a fluorophore at the long-strand 5′-end and the short-strand 3′-end, respectively. The two tags are in close contact, resulting in complete quenching of the probe fluorescence. If perfectly complemented to the nanogold-labeled strand, a long target oligonucleotide would displace the short fluorophore-labeled strand, and as a result, restore the fluorescence. By using nanogold in the probe, an extremely high quenching efficiency (99.1%) and removal of free fluorophore-labeled strand is achieved. The signal-to-noise ratio and the detection limit (50 pmol L⁻¹) of homogenous assays are therefore improved significantly, in comparison with similar probes using organic acceptors. Moreover, the probe has a great inhibition effect on hybridization to a mismatched oligonucleotide. This effect provides the assay with a high specificity, and particularly the assay has great potential in applications for discriminating variations in sequences. The assay sensitivity could be markedly enhanced by using fluorescent materials in the signal strand that are brighter and not quenched by nucleobases.     

Non-destructive assessment of parchment deterioration by optical methods

A non-destructive and non-invasive method for quantitative characterization of parchment deterioration, based on spectral measurements, is proposed. Deterioration due to both natural aging (ancient parchments) and artificial aging (achieved by means of controlled UV irradiation and temperature treatment) was investigated. The effect of aging on parchment native fluorescence was correlated with its deterioration condition. Aging causes fluorescence intensity drop, spectral shift of the main peak, and an overall change in the fluorescence spectral features. Digital color imaging analysis based on visible reflectance from the parchment surface was also applied, and the correspondent color components (RGB) were successively correlated with the state of parchment deterioration/aging. The fluorescence and color imaging data were validated by analysis of historical parchments, aged between 50 and 2000 years and covering a large variety of states of deterioration. The samples were independently assessed by traditional microscopy methods. We conclude that the proposed optical method qualifies well as a non-destructive tool for rapid assessment of the stage of parchment deterioration.     

Fluorescence sensor for water in organic solvents prepared from covalent immobilization of 4-morpholinyl-1, 8-naphthalimide

A new fluorescent dye, N-allyl-4-morpholinyl-1,8-naphthalimide (AMN), was synthesized as a fluorescence indicator in the fabrication of a sensor for determining water content in organic solvents. To prevent leakage of the fluorophore, AMN was photo-copolymerized with acrylamide, (2-hydroxyethyl)methacrylate, and triethylene glycol dimethacrylate on a glass surface treated with a silanizing agent. The sensing mechanism is based on the solvatochromic feature of the covalently immobilized AMN. The fluorescence intensity of AMN decreased with increasing water contents when it was excited at 400 nm. In the range of ca. 0.00–4.40% (v/v), the fluorescence intensity of AMN changed as a linear function of water content. The sensor exhibited satisfactory reproducibility, reversibility, and a response time (t ₉₉) of the order of 50 s. The detection limit was solvent-dependent, when acetonitrile was used as the solvent, and the detection limit could be as low as 0.006% (v/v) of water. Additionally, the prepared sensor is pH-insensitive and possesses a relatively long lifetime of at least one month.     

Studies on DMSO-containing carbanilation mixtures: chemistry,oxidations and cellulose integrity

The oxidative effect of carbanilation mixtures containing dimethylsulfoxide (DMSO) was demonstrated by means of alcohol model substances in which competitive carbanilation was prevented due to steric hindrance of the hydroxyl function, rendering those compounds specific probes for oxidation effects. Dimethylsulfonium ions and derived ylide species were shown to be the actually oxidizing species according to trapping methodology using lipophilic olefins which were converted into the corresponding cyclopropane and epoxide derivatives. The experimental data were in good agreement with DFT computations carried out on the B3LYP/6-311+G(d,p) level of theory. The direct interaction of cellulose and sulfoxide solvent was proven by means of methyl-(2-naphthyl)sulfoxide (MNSO) as a model for DMSO, which caused introduction of UV-detectable methylthionaphthyl ether moieties into the cellulose, formed in Pummerer-type side reaction paralleling the chemical behavior of DMSO. A facile color test—responding to sulfoxide-derived oxidizing species—was developed to assess the suitability of carbanilation conditions with regard to cellulose oxidation and degradation. DMSO-based carbanilation systems have to be used with great caution for determination of molecular weight parameters and for similar purposes which require complete maintenance of the cellulose integrity. Cellulose oxidation/degradation by DMSO-derived intermediates upon carbanilation can be minimized but cannot be avoided completely. Thus, if cellulose integrity is an issue as it is in cellulose analytics, it is recommended to replace DMSO by solvent components of similar solution behavior but without the inherent danger of generating oxidants, such as pyridine or DMAc, whenever possible.

Spectroscopic characterization and theoretical simulation of 1,4-diallylquinoxaline-2,3-dione self dimer

Room temperature UV-vis absorption and emission spectra of the 1,4-diallylquinoxaline-2,3-dione (DAQX) are measured in solution at different concentrations. Even at very low concentration (approximately 10(-7)M), DAQX is shown to form ground state van der Waals dimers and excited dimers. These later species do not seem to rearrange into an excimer geometry. The theoretical simulation of the dimer, performed using the analytical atom-atom pair potential described below, predicts a non sandwich face-to-reverse slipped structure with head-to-tail orientation. The allowed absorption transitions, calculated using ZINDO/S package, reproduce satisfactory the experimental spectrum for both the monomer and the simulated dimer.     

Selective recognition iodide in aqueous solution based on fluorescence enhancement chemosensor

A novel and simple fluorescence enhancement method for selectively sensing iodide was proposed based on metal complex formation between mercuric(II) ion with fluorophore (p-((dimethylamino)benzylidene)thiosemicarbazide, 1) and with anion in aqueous solution. The 1-Hg complex was found to show selectively and sensitively fluorescence enhancement response toward iodide over than S(2-), EDTA, SCN-, CH(3)CO(2-), Br-, Cl-, F-, H2PO4- and SO4(2-), which was attributed to the higher stability of inorganic complex between iodide and mercuric(II).     

Synthesis and investigation on the interaction with calf thymus deoxyribonucleic acid of a novel fluorescent probe 7-oxobenzo[b][1,10]phenanthroline-12(7H)-sulfonic acid

In this paper, the synthetic route of a potential antitumor reagent, benzo[b][1,10] phenanthrolin-7(12H)-one (BPO), was improved. A sulfonic group was introduced to BPO to form a new compound, 7-oxobenzo[b][1,10]phenan-throline-12(7H)-sulfonic acid (OPSA), in order to enhance its water-solubility. The molecular structure of OPSA has been confirmed by IR, UV, MS, ¹H NMR and elements analysis. It was proved in our experiments that DNA could quench the fluorescence of OPSA and the maximum quenched intensity appeared at 408 nm (λ_ex = 284 nm). The quenched fluorescence intensity was proportional to the concentration of DNA. Based on this phenomenon, OPSA had been used as the fluorescent probe for detection of calf thymus DNA (ct-DNA) and the corresponding linear response range was from 1.0 to 150.0 μg mL⁻¹ and the limit of detection (LOD) was 3.8 ng mL⁻¹. Its interaction with ct-DNA was investigated by fluorescence, absorption and viscosity measurements. When binding to ct-DNA, OPSA showed obvious fluorescence quenching and the quenched intensity was stable with the presence and absence of NaCl. The absorption spectra of OPSA had no evidence of increasing or decreasing when ct-DNA was added. The viscosity of OPSA and ct-DNA mixture showed no obvious change comparing with the viscosity of ct-DNA along. The results suggested that the interaction between OPSA and ct-DNA was groove binding in nature. Scatchard plots constructed from fluorescence titration data gave a binding constant of 8.9 × 10⁵ L mol⁻¹ and a binding site size of 0.35 base pairs per bound drug molecule.     

Development of a peptide inhibitor-based cantilever sensor assay for cyclic adenosine monophosphate-dependent protein kinase

A highly sensitive nanomechanical cantilever sensor assay based on an electrical measurement has been developed for detecting activated cyclic adenosine monophosphate (cyclic AMP)-dependent protein kinase (PKA). Employing a peptide derived from the heat-stable protein kinase inhibitor (PKI), a magnetic bead system was first selected as a vehicle to immobilize the PKI-(5-24) peptide for capturing PKA catalytic subunit and the activity assay was applied for indirectly assessing the binding. Synergistic interactions of adenosine triphosphate (ATP) and the peptide inhibitor with the kinase were then investigated by a solution phase capillary electrophoretic assay, and by surface plasmon resonance technology which involved immobilization of the peptide inhibitor. After systemically evaluated by a homogeneous direct binding assay, the ATP-dependent recognition of the catalytic subunit of PKA by PKI-(5-24) was successfully transferred on to the nanomechanical cantilevers at protein concentrations of 6.6 pM-66 nM, exhibiting much higher sensitivity and wider dynamic range than the conventional activity assay. Thus, direct assessment of activated kinases using the cantilever sensor system functionalized with specific peptide inhibitors holds great promise in analytical applications and clinical medicine.     

Optical sensing systems for microfluidic devices: a review

This review deals with the application of optical sensing systems for microfluidic devices. In the “off-chip approach” macro-scale optical infrastructure is coupled, while the “on-chip approach” comprises the integration of micro-optical functions into microfluidic devices. The current progress of the use of both optical sensing approaches in microfluidic devices, as well as its applications is described. In all cases, sensor size and shape profoundly affect the detection limits, due to analyte transport limitation, not to signal transduction limitation. The micro- or nanoscale sensors are limited to picomolar-order detection for practical time scales. The review concludes with an assessment of future directions of optical sensing systems for integrated microfluidic devices.     

Fluorimetric determination of phytic acid in urine based on replacement reaction

A sensitive fluorimetric method for determination of phytic acid in human urine samples was described. The method was based on a fluorimetric replacement reaction, in which the added phytic acid replaced the Cu²⁺ ion from Cu²⁺-gelatin complex, liberating the fluorescent gelatin molecule. The fluorescence of the solution was accordingly recovered proportionally to the amount of the foreign phytic acid. The excitation wavelength was 273.5 nm and the characteristic emission wavelength was 305.0 nm, respectively. The calibration graph was obtained by plotting the recovered fluorescent intensity at maximum 305.0 nm against the added standard phytic acid, and was divided into two sections. One section was linear over the range of 0.40-2.40 mg L⁻¹ with a linear regression equation of I_f = −0.895 + 15.146c (R² > 0.9993), and the other over the range of 2.40-9.20 mg L⁻¹ with a linear regression equation of I_f = −29.526 + 26.113c (R² > 0.9996), respectively. The relative standard deviation (R.S.D.) at 95% confidence degree for a 2.0 mg L⁻¹ of standard phytic acid within 1 month was less than 1.26% (n = 5), indicating the procedure is reproducible. The detection and the quantification limits of phytic acid were estimated to be 0.23 and 0.40 mg L⁻¹, respectively. The proposed method was applied to the determination of phytic acid in urine samples and the found concentrations of phytic acid in urine were in the range of 0.49-0.75 mg L⁻¹ with recoveries of 96.2-108.8%. Comparison of the obtained results with the reported HPLC was performed, indicating the proposed method was reliable.