Real-time observation of single-molecule fluorescence in microdroplet streams

We report real-time observation of fluorescence bursts from individual Rhodamine 6G molecules in streams of microdroplets (peak signal-to-noise ratios, approximately 30) whose trajectories are constrained with a linear electric quadrupole. This approach offers a reasonable dynamic range in droplet size (3- 12-microm diameter) with <1% shot-to-shot size fluctuations and sensitivity comparable with that of droplet levitation techniques with at least 10(3) higher analysis rates. Applications to the study of single-molecule microcavity effects and stimulated emission are discussed.     

Appropriate rather than representative sampling, based on acceptable levels of uncertainty

Appropriate sampling, that includes the estimation of measurement uncertainty, is proposed in preference to representative sampling without estimation of overall measurement quality. To fulfil this purpose the uncertainty estimate must include contribution from all sources, including the primary sampling, sample preparation and chemical analysis. It must also include contributions from systematic errors, such as sampling bias, rather than from random errors alone. Case studies are used to illustrate the feasibility of this approach and to show its advantages for improved reliability of interpretation of the measurements. Measurements with a high level of uncertainty (e.g. 50%) can be shown to be fit for some specified purposes using this approach. Once reliable estimates of the uncertainty are available, then a probabilistic interpretation of results can be made. This allows financial aspects to be considered in deciding upon what constitutes an acceptable level of uncertainty. In many practical situations ”representative” sampling is never fully achieved. This approach recognises this and instead, provides reliable estimates of the uncertainty around the concentration values that imperfect appropriate sampling causes. Received: 28 December 2001 Accepted: 25 April 2002     

Quantification of deoxynivalenol in wheat using an immunoaffinity column and liquid chromatography

A simple and accurate method to quantify the mycotoxin deoxynivalenol (DON) in wheat is described. The method uses immunoaffinity chromatography for DON isolation and liquid chromatography (LC) for toxin detection and quantification. Wheat samples are extracted in water, filtered twice and applied to an immunoaffinity column. Following a water wash, DON is eluted from the column with methanol and injected onto an LC system with a UV detector for quantification. Test performance was evaluated in terms of antibody specificity, limit of detection, percentage recovery, precision, column capacity, assay linearity and comparison with the GC-electron-capture detection (ECD) method of Tacke and Casper. Specificity of the immunoaffinity column cleanup procedure was confirmed with only DON (>80%) and its 15-C derivatives (40-50%) being recognized by the antibody while 3-C DON derivatives, nivalenol, T-2 and fusarenon-X did not bind. The limit of detection is at least 0.10 microg/g. Percentage recovery for the entire assay range averages 90% with an average relative standard deviation of 8.3%. Naturally contaminated samples showed comparable precision. Column capacity was determined to be 3.3 microg. The assay showed a high degree of linearity (r2=0.999) and an optimum assay range of 0.10 to 10.0 microg/g. Comparative analysis of 28 naturally or artificially contaminated wheat samples using DONtest-HPLC and the GC-ECD method of Tacke and Casper showed that DONtest-HPLC is a statistically significant predictor of the GC-ECD method (r2=0.982).     

Disodium guanosine 5'-monophosphate self-associates into nanoscale cylinders at pH 8: a combined diffusion NMR spectroscopy and dynamic light scattering study

We report a combined NMR and dynamic light scattering (DLS) study on the size of supramolecular structures formed by disodium guanosine 5'-monophosphate, Na(2)(5'-GMP), at pH 8. In general, two distinct types of aggregate species are present in an aqueous solution of Na(2)(5'-GMP). One type consists of stacking 5'-GMP monomers, and the other contains stacking G-quartets. Both types of aggregates can be modeled as rodlike cylinders. The cylinder diameter is 10 and 26 A for monomer aggregates and quartet aggregates, respectively. For Na(2)(5'-GMP) concentrations between 18 and 34 wt %, the cylinders formed by stacking G-quartets have an average length between 8 and 30 nm, corresponding to a stack of approximately 24-87 G-quartets. These nanoscale aggregates are significantly larger than what had previously been believed for Na(2)(5'-GMP) self-association at pH 8. The length of both types of 5'-GMP aggregates was found to increase with Na(2)(5'-GMP) concentration but was insensitive to the added NaCl in solution. While the aggregate size for monomer aggregates increases with a decrease in temperature, the size of G-quartet aggregates is essentially independent of temperature. We found that the size of G-quartet aggregates is slightly larger in D(2)O than in H(2)O, whereas the size of monomer aggregates remains the same in D(2)O and in H(2)O. We observed a linear relationship between the axial ratio of the 5'-GMP cylinders and the Na(2)(5'-GMP) concentration for both types of 5'-GMP aggregates, which suggests a common stacking mechanism for monomers and G-quartets.     

Semiempirical molecular orbital calculations on boranes

Traditional Pariser-Parr-Pople and variable electronegativity calculations have been carried out on C₆H₅B(OR)₂ and p-CH₃OC₆H₄B(OR)₂, and the results compared with calculations for C₆H₅BR₂. It is concluded that the VE-SCF method offers a real advantage over the simple PPP method for predicting percent charge transfer and transition intensity in cases where excited states possess substantial C.T. character. The restriction that empirically chosen parameters fit the observed transition energies and intensities of both triarylboranes and ArB(OR)₂ requires the choice of a boron VSIP greater than 2.0 eV in the fixed parameter procedure of the usual PPP-SCF-CI method for these molecules. Observed transitions in C₆H₅B(OR)₂ correlate with ¹ L _b, ¹ L _a, ¹ B _b, whereas the first absorption maximum of (C₆H₅)₃B is assigned to C.T. (¹ A ₁¹ A ₁) local C _2v symmetry.

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Zusammenfassung PPP- und VE SCF-Rechnungen wurden für C₆H₅B(OR)₂ und p-CH₃OC₆H₄(OR)₂ durch- geführt, und die Ergebnisse wurden mit denjenigen für C₆H₅BR₂ verglichen. Es kann der Schluß gezogen werden, daß die VE SCF-Methode einen Vorteil gegenüber der einfachen PPP-Methode bietet, um den prozentualen Charge Transfer und Übergangsintensitäten in Fällen zu bestimmen, in denen die angeregten Zustände einen wesentlichen C.T.-Charakter besitzen. Die Bedingung, daß die empirisch gewählten Parameter den beobachteten Übergangsenergien und -intensitäten von sowohl Triarylboranen als auch ArB(OR)₂ angepaßt sein sollen, erfordert die Wahl eines Bor-VSIP größer als 2,0 eV im Rahmen der üblichen Parametrisierung der PPP-SCF-CI-Methode. Beobachtete Über-gänge in C₆H₅B(OR)₂ korrelieren mit ¹ L _b, ¹ L _a, ¹ B _b, wogegen das erste Absorptionsmaximum des (C₆H₅)₃B einem C.T.-Übergang (¹ A ₁¹ A ₁) lokaler C_2v -Symmetrie zugeordnet wird.

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Résumé Des calculs traditionnels Pariser-Parr-Pople et des calculs d'électronégativité variable ont été effectués sur C₆H₅B(OR)₂ et p-CH₃OC₆H₄B(OR)₂ avec comparison aux résultats obtenus pour C₆H₅BR₂. La conclusion est que la méthode VE-SCF offre un réel avantage sur la méthode PPP simple en ce qui concerne la prédiction du transfert de charge et de l'intensité de transition pour les états excités possédant un net caractère de transfert de charge. La restriction selon laquelle les paramètres empiriques doivent permettre de reproduire les énergies de transition et les intensités des deux triarylboranes et de ArB(OR)₂, nécessite le choix d'un potential d'ionisation de l'état de valence du bore supérieur de 2 eV à celui employé dans les méthodes ordinaires. Les transitions observées dans C₆H₅B(OR)₂ sont reliées à ¹ L _b, ¹ L _a, ¹ B _b, tandis que la première absorption de (C₆H₅)₃B est attribuée à un transfert de charge (¹ A ₁¹ A ₁) de symétrie locale C_2v.