Adequacy and Inadequacy in the Metrology of Chemical Analysis

The adequacy (inadequacy) of samples to each other and reference materials to test samples is an important notion in the metrology of chemical analysis. However, the literature gives no strict definition for this notion. In this paper, it was proposed to determine adequacy based on the measure of inadequacy. For the case of two samples, the measure of inadequacy is the absolute value of the difference between the systematic errors of the results of analyses of the samples; it depends on the difference in the composition and properties of the samples. The measure of inadequacy of a standard reference material to a series of test samples is the maximum distance between the systematic error of the reference material and one of the quantiles of the systematic error distribution of the test samples Q(0.025); Q(0.975); the errors in this case are due to the composition and the properties of the test samples. The samples are considered adequate if the measure of inadequacy can be neglected compared to the normalized error of the analysis. Using the determination of total cholesterol in the blood serum of humans as an example, it was shown that the standard reference material in the analysis of samples of complex composition is often inadequate to test samples.     

Calibration lines passing through the origin with errors in both axes

Linear regression of calibration lines passing through the origin was investigated for three models of y-direction random errors: normally distributed errors with an invariable standard deviation (SD) and log normally and normally distributed errors with an invariable relative standard deviation (RSD). The weighted (weighting factor is x ² _i ), geometric and arithmetic means of the ratios y _i /x _i estimate the calibration slope for these models, respectively. Regression of the calibration lines with errors in both directions was also studied. The x-direction errors were assumed to be normally distributed random errors with either an invariable SD or invariable RSD, both combined with a constant relative systematic error. The random errors disperse the true, unknown x-values about the plotted, demanded x-values, which are shifted by the constant relative systematic error. The systematic error biases the slope estimate while the random errors do not. They only increase automatically the slope estimate uncertainty, in which the uncertainty component reflecting the range of the possible values of the systematic error must be additionally included. Received: 9 May 2000 Accepted: 7 March 2001     

Voltammetric Antioxidant Analysis in Mineral Oil Samples Immobilized into Boron‐Doped Diamond Micropore Array Electrodes

Mineral oil microdroplets containing the model antioxidant N,N‐didodecyl‐N′,N′‐diethyl‐phenylene‐diamine (DDPD) are immobilized into a 100×100 pore‐array (ca. 10 μm individual pore diameter, 100 μm pitch) in a boron‐doped diamond electrode surface. The robust diamond surface allows pore filling, cleaning, and reuse without damage to the electrode surface. The electrode is immersed into aqueous electrolyte media, and voltammetric responses for the oxidation of DDPD are obtained. In order to further improve the current responses, 20 wt% of carbon nanofibers are co‐deposited with the oil into the pore array. Voltammetric signals are consistent with the oxidation of DDPD and the associated transfer of perchlorate anions (in aqueous 0.1 M NaClO₄) or the transfer of protons (in aqueous 0.1 M HClO₄). From the magnitude of the current response, the DDPD content in the mineral oil can be determined down to less than 1 wt% levels. Perhaps surprisingly, the reversible (or midpoint) potential for the DDPD oxidation in mineral oil (when immersed in 0.1 NaClO₄) is shown to be concentration‐dependent and to shift to more positive potential values for more dilute DDPD in mineral oil solutions. An extraction mechanism and the formation of a separate organic product phase are proposed to explain this behavior.     

Isotope pattern vector based tandem mass spectral data calibration for improved peptide and protein identification

Tandem mass spectra contain noisy peaks which make peak picking for peptide identification difficult. Moreover, all spectral peaks can be shifted due to systematic measurement errors. In this paper, a novel use of an isotope pattern vector (IPV) is proposed for denoising and systematic measurement error prediction. By matching the experimental IPVs with the theoretical IPVs of candidate fragment ions, true ionic peaks can be identified. Furthermore, these identified experimental IPVs and their corresponding theoretical IPVs are used in an optimization process to predict the systematic measurement error associated with the target spectrum. In return, the subsequent spectral data calibration based on the predicted systematic measurement error enhances the data quality. We show that such an integrated denoising and calibration process leads to significantly improved peptide and protein identification. Different from the commonly employed chemical calibration methods, our IPV‐based method is a purely computational method for individual spectra analysis and globally optimizes the use of spectral data. Copyright © 2009 John Wiley & Sons, Ltd.     

Robust Chemical Preservation of Digital Information on DNA in Silica with Error‐Correcting Codes

Information, such as text printed on paper or images projected onto microfilm, can survive for over 500 years. However, the storage of digital information for time frames exceeding 50 years is challenging. Here we show that digital information can be stored on DNA and recovered without errors for considerably longer time frames. To allow for the perfect recovery of the information, we encapsulate the DNA in an inorganic matrix, and employ error‐correcting codes to correct storage‐related errors. Specifically, we translated 83 kB of information to 4991 DNA segments, each 158 nucleotides long, which were encapsulated in silica. Accelerated aging experiments were performed to measure DNA decay kinetics, which show that data can be archived on DNA for millennia under a wide range of conditions. The original information could be recovered error free, even after treating the DNA in silica at 70 °C for one week. This is thermally equivalent to storing information on DNA in central Europe for 2000 years.     

Identification of methylated regions with peak search based on Poisson model from massively parallel methylated DNA immunoprecipitation-sequencing data

DNA methylation is one of the most important epigenetic modification types, which plays a critical role in gene expression. High efficient surveying of whole genome DNA methylation has been aims of many researchers for long. Recently, the rapidly developed massively parallel DNA‐sequencing technologies open the floodgates to vast volumes of sequence data, enabling a paradigm shift in profiling the whole genome methylation. Here, we describe a strategy, combining methylated DNA immunoprecipitation sequencing with peak search to identify methylated regions on a whole‐genome scale. Massively parallel methylated DNA immunoprecipitation sequencing combined with methylation DNA immunoprecipitation was adopted to obtain methylated DNA sequence data from human leukemia cell line K562, and the methylated regions were identified by peak search based on Poison model. From our result, 140 958 non‐overlapping methylated regions have been identified in the whole genome. Also, the credibility of result has been proved by its strong correlation with bisulfite‐sequencing data (Pearson R²=0.92). It suggests that this method provides a reliable and high‐throughput strategy for whole genome methylation identification.     

Alpha‐Helical Fragaceatoxin C Nanopore Engineered for Double‐Stranded and Single‐Stranded Nucleic Acid Analysis

Nanopores are used in single‐molecule DNA analysis and sequencing. Herein, we show that Fragaceatoxin C (FraC), an α‐helical pore‐forming toxin from an actinoporin protein family, can be reconstituted in sphingomyelin‐free standard planar lipid bilayers. We engineered FraC for DNA analysis and show that the funnel‐shaped geometry allows tight wrapping around single‐stranded DNA (ssDNA), resolving between homopolymeric C, T, and A polynucleotide stretches. Remarkably, despite the 1.2 nm internal constriction of FraC, double‐stranded DNA (dsDNA) can translocate through the nanopore at high applied potentials, presumably through the deformation of the α‐helical transmembrane region of the pore. Therefore, FraC nanopores might be used in DNA sequencing and dsDNA analysis.     

Tracking chemical kinetics in high-throughput systems

Combinatorial chemistry and high‐throughput experimentation (HTE) have revolutionized the pharmaceutical industry—but can chemists truly repeat this success in the fields of catalysis and materials science? We propose to bridge the traditional “discovery” and “optimization” stages in HTE by enabling parallel kinetic analysis of an array of chemical reactions. We present here the theoretical basis to extract concentration profiles from reaction arrays and derive the optimal criteria to follow (pseudo)first‐order reactions in time in parallel systems. We use the information vector f and introduce in this context the information gain ratio, χ_r, to quantify the amount of useful information that can be obtained by measuring the extent of a specified reaction r in the array at any given time. Our method is general and independent of the analysis technique, but it is more effective if the analysis is performed on‐line. The feasibility of this new approach is demonstrated in the fast kinetic analysis of the carbon–sulfur coupling between 3‐chlorophenylhydrazonopropane dinitrile and β‐mercaptoethanol. The theory agrees well with the results obtained from 31 repeated C? S coupling experiments.     

Fitting multiple datasets in kinetics: n‐butane + OH → products

Offsets due to systematic calibration errors are a common feature of the literature on temperature‐dependent rate constants. We present a formalism for dealing with these offsets within the context of least‐squares fitting, using a priori parameter constraints based on the estimated accuracy of individual studies. This methodology not only eliminates biases caused by calibration errors, it also ensures that the metric used to compare different studies is their accuracy, not their precision. Consequently, studies with single measurements at room temperature can be meaningfully compared with studies comprising dozens of measurements spanning a wide temperature range. We apply this procedure to the complete literature dataset for two reactions: OH + propane and OH + n‐butane, after first presenting new data for OH + n‐butane spanning the temperature range 180–300 K and extending the low‐temperature limit of the literature by 50 K. There is outstanding agreement among a very large set of studies, including relative measurements of the propane:n‐butane rate constant ratio. We present new reduced transition state theory fits for each reaction that accurately reproduce the observed rate constants between 180 and 1000 K, and argue that these two reactions are the optimal reference reactions for many relative rate studies. © 2004 Wiley Periodicals, Inc. Int J Chem Kinet 36: 259–272, 2004     

Contaminant-induced current decline in capillary array electrophoresis

High-throughput capillary array electrophoresis (CAE) instruments for DNA sequencing suffer to varying degrees from read length degradation associated with electrophoretic current decline and inhibition or delay in the arrival of fragments at the detector. This effect is known to be associated with residual amounts of large, slow-moving fragments of template or genomic DNA carried through from sample preparation and sequencing reactions. Here, we investigate the creation and expansion of an ionic depletion region induced by overloading the capillary with low-mobility DNA fragments, and the effect of growth of this region on electrophoresis run failure. Slow-moving fragments are analytically and experimentally shown to reduce the ionic concentration of the downstream electrolyte. With injection of large fragments beyond a threshold quantity, the anode-side boundary of the nascent depletion region begins to propagate toward the anode at a rate faster than the contaminant DNA migration. Under such conditions, the depletion region expands, the capillary current declines dramatically, and the electrophoresis run yields a short read length or fails completely.     

SYSTEMATIC ERRORS DUE TO EXCITATION PULSE WIDTH IN FAST KINETIC TECHNIQUES

Abstract— Convolution of the decay function with the excitation pulse function leads to systematic error in both kinetic and spectral analysis of transients. The error in rate constant determination for a first order decay can be kept to about 1% if the decay half-life is equal to the pulse half-width and to well below 1% for half-lives equal to or longer than twice the half-width. The errors in transient absorbance measurements are much greater. Only if the half-life approaches 200 times the pulse half-width is the error reduced to 1%. It is suggested that spectral measurements should be corrected for this error.     

毛细管阵列电泳与规模化DNA测序

 根据 10 0 80份基因组DNA测序的结果 ,讨论了毛细管阵列电泳测序方法的技术特点 ,并对影响测序结果的一些因素进行了分析。在此基础上与平板凝胶电泳方法进行比较 ,显示了毛细管阵列电泳的优点。同时也对大规模测序技术环节之间的协调进行了探讨 。     

Simultaneously Sizing and Quantitating Zeptomole‐Level DNA at High Throughput in Free Solution

Determining the sizes and measuring the quantities of DNA molecules are fundamental tasks in molecular biology. DNA sizes are usually evaluated by gel electrophoresis, but this method cannot simultaneously size and quantitate a DNA at low zeptomole (zmol) levels of concentration. We have recently developed a new technique, called bare‐narrow‐capillary/hydrodynamic‐chromatography or BaNC‐HDC, for resolving DNA based on their sizes without using any sieving matrices. In this report, we utilize BaNC‐HDC for measuring the sizes and quantities of DNA fragments at zmol to several‐molecule levels of concentration. DNA ranging from a few base pairs to dozens of kilo base pairs are accurately sized and quantitated at a throughput of 15 samples per hour, and each sample contains dozens of DNA strands of different lengths. BaNC‐HDC can be a cost‐effective means and an excellent tool for high‐throughput DNA sizing and quantitation at extremely low quantity level.     

Translocation of biomolecules through solid‐state nanopores: Theory meets experiments

The interest in polynucleotide translocation through nanopores has moved from purely biological to the need of realizing nanobiotechnological applications related to personalized genome sequencing. Polynucleotide translocation is a process in which biomolecules, like DNA or RNA, are electrophoretically driven through a narrow pore and their passage can be monitored by the change in the ionic current through the pore. Such a translocation process, which will be described here offers a very promising technology aiming at ultra‐fast low‐cost sequencing of DNA, though its realization is still confronted with challenges and drawbacks. In this review, we present the main aspects involved in the polynucleotide translocation through solid‐state nanopores by discussing the most relevant experimental, theoretical, and computational approaches and the way these can supplement each other. The discussion will expose the goals that have been reached so far, the open questions, and contains an outlook to the future of nanopore sequencing. © 2011 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 49: 985–1011, 2011     

DNA sequencing with titanium nitride electrodes

We construct a hydrogen‐bond based metal–molecule–metal junction, which contains two identical “reader” molecules, one single DNA base as a bridged molecule, and two titanium nitride electrodes. Hydrogen bonds are formed between “reader” molecules and DNA base, whereas titanium–sulfur bonds are formed between “reader” molecules and titanium nitride electrodes. We perform electronic structure calculations for both the bare bridged molecule and the full metal–molecule–metal system. The projected density of states shows that when the molecule is connected to the titanium nitride electrode, the energy levels of the bridged molecule are shifted, with an indirect effect on the hydrogen bonds. This is similar to the case for a gold electrode but with a more pronounced effect. We also calculate the current–voltage characteristics for the molecular junctions containing each DNA base. Results show that titanium nitride as an electrode can generate distinct conductance for each DNA base, providing an alternative electrode for DNA sequencing. © 2013 Wiley Periodicals, Inc.     

Screening and isolation of cyclooxygenase‐2 inhibitors from Trifolium pratense L. via ultrafiltration,enzyme‐immobilized magnetic beads,semi‐preparative high‐performance liquid chromatography and high‐speed counter‐current chromatography

Nonsteroidal anti‐inflammatory drugs reportedly reduce the risk of developing cancer. One mechanism by which they reduce carcinogenesis involves the inhibition of the activity of cyclooxygenase‐2, an enzyme that is overexpressed in various cancer tissues. Its overexpression increases cell proliferation and inhibits apoptosis. However, selected cyclooxygenase‐2 inhibitors can also act through cyclooxygenase‐independent mechanisms. In this study, using ultrafiltration, enzyme‐immobilized magnetic beads, high‐performance liquid chromatography, and electrospray‐ionization mass spectrometry, several isoflavonoids in Trifolium pratense L. extracts were screened and identified. Semi‐preparative high‐performance liquid chromatography and high‐speed counter‐current chromatography were then applied to separate the active constituents. Using these methods, seven major compounds were identified in Trifolium pratense L. As cyclooxygenase‐2 inhibitors: rothindin, ononin, daidzein, trifoside, pseudobaptigenin, formononetin, and biochanin A, which were then isolated with >92% purity. This is the first report of the presence of potent cyclooxygenase‐2 inhibitors in Trifolium pratense L. extracts. The results of this study demonstrate that the systematic isolation of bioactive components from Trifolium pratense L., by using ultrafiltration, enzyme‐immobilized magnetic beads, semi‐preparative high‐performance liquid chromatography, and high‐speed counter‐current chromatography, represents a feasible and efficient technique that could be extended for the identification and isolation of other enzyme inhibitors.     

A Quadruplex‐Based,Label‐Free,and Real‐Time Fluorescence Assay for RNase H Activity and Inhibition

We demonstrate a unique quadruplex‐based fluorescence assay for sensitive, facile, real‐time, and label‐free detection of RNase H activity and inhibition by using a G‐quadruplex formation strategy. In our approach, a RNA–DNA substrate was prepared, with the DNA strand designed as a quadruplex‐forming oligomer. Upon cleavage of the RNA strand by RNase H, the released G‐rich DNA strand folds into a quadruplex in the presence of monovalent ions and interacts with a specific G‐quadruplex binder, N‐methyl mesoporphyrin IX (NMM); this gives a dramatic increase in fluorescence and serves as a reporter of the reaction. This novel assay is simple in design, fast in operation, and is more convenient and promising than other methods. It takes less than 30 min to finish and the detection limit is much better or at least comparable to previous reports. No sophisticated experimental techniques or chemical modification for either RNA or DNA are required. The assay can be accomplished by using a common spectrophotometer and obviates possible interference with the kinetic behavior of the catalysts. Our approach offers an ideal system for high‐throughput screening of enzyme inhibitors and demonstrates that the structure of the G‐quadruplex can be used as a functional tool in specific fields in the future.     

Formation of the Compounds with an Epoxychromene Framework: Role of the Methoxy Groups

Earlier, it was found that the reaction of para‐mentha‐6,8‐diene‐2,3‐diol with 2,4,5‐trimethoxybenzaldehyde in the presence of Montmorillonite K 10 clay led to the formation of a compound with an unusual epoxychromene framework among other products. In the current work, systematic studies of the effect of the number and position of MeO groups in the aromatic ring of the aldehyde on the reaction route were performed to reveal the structural parameters, which favor the formation of compounds with an epoxychromene framework. Compounds with an epoxychromene framework were shown to be formed if the benzaldehyde contained MeO substituents in o‐ and p‐position. The highest yield was achieved in the case of 2,4,5‐trimethoxybenzaldehyde.     

Screening and investigation of triplex DNA binders from Stephania tetrandra S. Moore by a combination of peak area‐fading ultra high‐performance liquid chromatography with orbitrap mass spectrometry and optical spectroscopies

The identification and screening of triplex DNA binders are important because these compounds, in many cases, are potential anticancer agents as well as promising drug candidates. Therefore, the ability to screen for these compounds in a high‐throughput mode could dramatically improve the drug screening process. A method involving a combination of 96‐well plate format and peak area‐fading ultra high‐performance liquid chromatography coupled with Orbitrap mass spectrometry was employed for screening bioactive compounds binding to the triplex DNA from the extracts of Stephania tetrandra S. Moore. Two compounds were screened out and identified as fangchinoline and tetrandrine based on the comparison of retention time and tandem mass spectrometry data with those of standards. The binding mechanisms of fangchinoline and tetrandrine at the molecular level were explored using tandem mass spectrometry, fluorescence spectroscopy, ultraviolet‐visible spectroscopy, and circular dichroism. Collision‐induced dissociation experiments showed that the complexes with fangchinoline and tetrandrine were dissociated by ligand elimination. According to these measurements, an intercalating binding is the most appropriate binding mode of these two alkaloids to the triplex DNA. The current work provides not only deep insight into alkaloid‐triplex DNA complexes but also useful guidelines for the design of efficient anticancer agents.