Machine Learning Color Feature Analysis of a High Throughput Nanoparticle Conjugate Sensing Assay.

Plasmonic nanoparticles are finding applications within the single molecule sensing field in a "dimer" format, where interaction of the target with hairpin DNA causes a decrease in the interparticle distance, leading to a localized surface plasmon resonance shift. While this shift may be detected using spectroscopy, achieving statistical relevance requires the measurement of thousands of nanoparticle dimers and the timescales required for spectroscopic analysis are incompatible with point-of-care devices. However, using dark-field imaging of the dimer structures, simultaneous digital analysis of the plasmonic resonance shift after target interaction of thousands of dimer structures may be achieved in minutes. The main challenge of this digital analysis on the single-molecule scale was the occurrence of false signals caused by non-specifically bound clusters of nanoparticles. This effect may be reduced by digitally separating dimers from other nanoconjugate types. Variation in image intensity was observed to have a discernible impact on the color analysis of the nanoconjugate constructs and thus the accuracy of the digital separation. Color spaces wherein intensity may be uncoupled from the color information (hue, saturation, and value (HSV) and luminance, a* vector, and b* vector (LAB) were contrasted to a color space which cannot uncouple intensity (RGB) to train a classifier algorithm. Each classifier algorithm was validated to determine which color space produced the most accurate digital separation of the nanoconjugate types. The LAB-based learning classifier demonstrated the highest accuracy for digitally separating nanoparticles. Using this classifier, nanoparticle conjugates were monitored for their plasmonic color shift after interaction with a synthetic RNA target, resulting in a platform with a highly accurate yes/no response with a true positive rate of 88% and a true negative rate of 100%. The sensor response of tested single stranded RNA (ssRNA) samples was well above control responses for target concentrations in the range of 10 aM-1 pM.

Chemical data evaluation: general considerations and approaches for IUPAC projects and the chemistry community (IUPAC Technical Report).

The International Union of Pure and Applied Chemistry (IUPAC) has a long tradition of supporting the compilation of chemical data and their evaluation through direct projects, nomenclature and terminology work, and partnerships with international scientific bodies, government agencies and other organizations. The IUPAC Interdivisional Subcommittee on Critical Evaluation of Data (ISCED) has been established to provide guidance on issues related to the evaluation of chemical data. In this first report we define the general principles of the evaluation of scientific data and describe best practices and approaches to data evaluation in chemistry.

Interlaboratory comparison of the intensity of drinking water odor and taste by two-way ordinal analysis of variation without replication.

A case study of ordinal data from human organoleptic examination (sensory analysis) of drinking water obtained in an interlaboratory comparison of 49 ecological laboratories is described. The recently developed two-way ordinal analysis of variation (ORDANOVA) is applied for the first time for the treatment of responses on the intensity of chlorine and sulfurous odor of water at 20 and 60 °C, which is classified into the six categories from 'imperceptible' to 'very strong'. The one-way ORDANOVA is used for the analysis of the 'salty taste' intensity of the water. A decomposition of the total variation of the ordinal data and simulation of the multinomial distribution of the data-relative frequencies in different categories allowed the determination of the statistical significance of the difference between laboratories in classifying chlorine or sulfurous odor intensity by categories, while the effect of temperature was not significant. No statistical difference was found between laboratories on salty taste intensity. The capabilities of experts to identify different categories of the intensity of the odor and taste are also evaluated. A comparison of the results obtained with ORDANOVA and ANOVA showed that ORDANOVA is a more useful and reliable tool for understanding categorical data such as the intensity of drinking water odor and taste.

Redox Recycling Amplification Using an Interdigitated Microelectrode Array for Ionic Liquid-Based Oxygen Sensors.

A new design for a membrane-free gas sensor modified with a thin layer of ionic liquid is described. The new approach uses miniaturized interdigitated microelectrodes for detecting gases having reversible electrochemistry, for example, dioxygen. Analyte molecules are reduced on the first working electrode, creating an intermediate species (e.g., superoxide, O2•-, from dioxygen) that can be reoxidized back to the original molecule at the second working electrode. The loop of redox reactions enhances the measured current, leading to high sensitivity (3.29 ± 0.06 nA cm-2 ppm-1) and low detection limit (LOD = 174 ppm). The gas sensor design was demonstrated to monitor typical concentrations of oxygen with good accuracy and precision. The enhancement in the current is characteristic only of gas molecules with reversible electrochemistry, which indicates that the proposed gas sensor can analyze these molecules with greater sensitivity over those with irreversible electrochemistry.

Dual Signaling DNA Electrochemistry: An Approach To Understand DNA Interfaces.

Electrochemical DNA biosensors composed of a redox marker modified nucleic acid probe tethered to a solid electrode is a common experimental construct for detecting DNA and RNA targets, proteins, inorganic ions, and even small molecules. This class of biosensors generally relies on the binding-induced conformational changes in the distance of the redox marker relative to the electrode surface such that the charge transfer is altered. The conventional design is to attach the redox species to the distal end of a surface-bound nucleic acid strand. Here we show the impact of the position of the redox marker, whether on the distal or proximal end of the DNA monolayer, on the DNA interface electrochemistry. Somewhat unexpectedly, greater currents were obtained when the redox molecules were located on the distal end of the surface-bound DNA monolayer, notionally furthest away from the electrode, compared with currents when the redox species were located on the proximal end, close to the electrode. Our results suggest that a limitation in ion accessibility is the reason why smaller currents were obtained for the redox markers located at the bottom of the DNA monolayer. This understanding shows that to allow the quantification of the amount of redox labeled target DNA strand that hybridizes to probe DNA immobilized on the electrode surface, the redox species must be on the distal end of the surface-bound duplex.

Investigating the interfacial properties of electrochemically roughened platinum electrodes for neural stimulation.

Platinum electrodes have been electrochemically roughened (roughness factors up to 430) and evaluated for use as neural stimulation electrodes. The roughened electrodes show superior interfacial properties with increasing surface roughness. The roughened electrode (fR = 250) has a charge injection limit of 1.0 mC cm(-2) (400 µs pulse width), which is superior to that of titanium nitride (0.87 mC cm(-2)) but comparable to that of carbon nanotubes (1.0-1.6 mC cm(-2)). The surface roughness can also be optimized for different neural stimulation applications based on the available charge density at a particular pulse width of stimulation. The roughened platinum electrodes demonstrated good mechanical stability under harsh ultrasonication and electrochemical stability under continuous biphasic stimulation, indicating the potential of this biological interface to be safe and stable.

Gas chromatography with parallel hard and soft ionization mass spectrometry.

RATIONALE: Mass spectrometric identification of compounds in chromatography can be obtained from molecular masses from soft ionization mass spectrometry techniques such as field ionization (FI) and fragmentation patterns from hard ionization techniques such as electron ionization (EI). Simultaneous detection by EI and FI mass spectrometry allows alignment of the different information from each method. METHODS: We report the construction and characteristics of a combined instrument consisting of a gas chromatograph and two parallel mass spectrometry ionization sources, EI and FI. When considering both ion yield and signal-to-noise it was postulated that good-quality EI and FI mass spectra could be obtained simultaneously using a post-column splitter with a split fraction of 1:10 for EI/FI. This has been realised and we report its application for the analysis of several complex mixtures. RESULTS: The differences between the full width at half maximum (FWHM) of the EI and FI chromatograms were statistically insignificant, and the retention times of the chromatograms were highly correlated (r(2) =0.9999) with no detectable bias. The applicability and significance of this combined instrument and the attendant methodology are illustrated by the analysis of standard samples of 13 compounds with diverse structures, and the analysis of mixtures of fatty acids, fish oil, hydrocarbons and yeast metabolites. CONCLUSIONS: This combined dual-source instrument saves time and resources, and more importantly generates equivalent chromatograms aligned in time, in EI and FI (i.e. peaks with similar shapes and identical positions). The identical FWHMs and retention times of the EI and FI chromatograms in this combined instrument enable the accurate assignment of fragment ions from EI to their corresponding molecular ions in FI.

Formation constants of copper(II) complexes with tripeptides containing Glu, Gly, and His: potentiometric measurements and modeling by generalized multiplicative analysis of variance.

We report a systematic study of the effects of types and positions of amino acid residues of tripeptides on the formation constants logß, acid dissociation constants pKa, and the copper coordination modes of the copper(II) complexes with 27 tripeptides formed from the amino acids glutamic acid, glycine, and histidine. logß values were calculated from pH titrations with l mmol L(-1):1 mmol L(-1) solutions of the metal and ligand and previously reported ligand pKa values. Generalized multiplicative analysis of variance (GEMANOVA) was used to model the logß values of the saturated, most protonated, monoprotonated, logß(CuL) - logß(HL), and pKa of the amide group. The resulting model of the saturated copper species has a two-term model describing an interaction between the central and the C-terminal residues plus a smaller, main effect of the N-terminal residue. The model supports the conclusion that two copper coordination modes exist depending on the absence or presence of His at the central position, giving species in which copper is coordinated via two or three fused chelate rings, respectively. The GEMANOVA model for pKamide, which is the same as that for the saturated complex, showed that Gly-Gly-His has the lowest pKamide values among the 27 tripeptides. Visible spectroscopy indicated the formation of metal-ligand dimers for tripeptides His-His-Gly and His-His-Glu, but not for His-His-His, and the formation of multiple ligand bis compexes CuL2 and Cu(HL)2 for tripeptides (Glu/Gly)-His-(Glu/Gly) and His-(Glu/Gly)-(Glu/Gly), respectively.

Confirmation of ethanol administration in racing greyhounds by LC-MS-MS.

Ethanol is a prohibited substance in professional animal racing as its administration causes physiological effects such as depression of the central nervous system. Regulation of potential doping agents, including those that inhibit performance, is critical to ensure integrity and animal welfare in greyhound racing, but the detection of ethanol is complicated by dietary and/or environmental exposure. In response, a reliable analytical method capable of detecting recent ethanol administration in greyhound urine samples was validated and implemented. Liquid chromatography-tandem mass spectrometry (LC-MS-MS) was used to investigate the variation in urinary ethanol metabolites; ethyl-ß-D glucuronide (EG; γ ¯ EG $$ {\overline{\gamma}}_{\mathrm{EG}} $$ = 1.0 µg/ml, s EG $$ {s}_{\mathrm{EG}} $$ = 3.3 µg/ml) and ethyl sulfate (ES; γ ¯ ES $$ {\overline{\gamma}}_{\mathrm{ES}} $$ = 0.9 µg/ml, s ES $$ {s}_{\mathrm{ES}} $$ = 1.9 µg/ml) levels from a reference population of 202 racing greyhounds. These were compared to urine samples collected following administration of ethanol to one male and one female greyhound. Results were used to establish a threshold within the national rules of greyhound racing: γ ¯ EG $$ {\overline{\gamma}}_{\mathrm{EG}} $$ and γ ¯ ES $$ {\overline{\gamma}}_{\mathrm{ES}} $$ > 20 µg/ml in urine are defensible criteria to confirm ethanol administration to greyhounds. Case studies of competition samples are provided to demonstrate the forensic translation of this work.

Measurement of stable isotope ratios in methylamphetamine: a link to its precursor source.

The illicit drug methylamphetamine is often prepared from the precursor ephedrine or pseudoephedrine, which in turn are obtained by three processes: extraction from the Ephedra plant ("natural"), via fermentation of sugars ("semi-synthetic"), and by a "fully synthetic" route from propiophenone. We report the first method to differentiate between the three industrial routes used to produce the precursors ephedrine and pseudoephedrine by measurement of stable isotope ratios of nitrogen (δ(15)N), hydrogen (δ(2)H), and carbon (δ(13)C). Analysis of 782 samples of seized methylamphetamine allowed classification into three groups using k-means clustering or the expectation-maximization algorithm applied to a Gaussian mixture model. By preparation of 30 samples of ephedrine by the "fully synthetic" industrial process and measuring their δ(15)N, δ(2)H, and δ(13)C values, we observed that (15)N becomes significantly depleted compared to the methylamine starting material. Conversion of ten ephedrine samples to methylamphetamine showed that this depletion is maintained in the final drug product, of which the δ(15)N, δ(13)C, and δ(2)H values were distinct from those of ephedrine and methylamphetamine samples of a semi-synthetic (fermentation pathway) origin. Combining modeling analysis with the new experiments and published information on the values of δ(2)H gave a definitive assignment of the three model groups, and equations to obtain probabilities for the precursor origin of any new sample. A simple rule of thumb is also presented. Making an assignment using delta values is particularly useful when no other chemical profiling information is available.

A study of the conditions of measurement required to evaluate bias in analytical results illustrated by the use of data from a multi-round, blind-duplicated, proficiency test.

Measurement uncertainty estimated under repeatability conditions in batch chemical analysis using calibrated instruments may be considered to be composed of contributions from two major effects: (i) precision of the analysis that encompasses sufficient variability of the measurement and (ii) the assessment of trueness by quantifying and if necessary correcting for bias. This paper considers under what conditions of measurement to assess bias, and from the results of a six-round blind-duplicated interlaboratory proficiency program for creatinine in urine shows that bias is present in each individual run with components from that batch and from the laboratory over the rounds of the program. We conclude that bias should be determined in each batch run under repeatability conditions. Measurement of laboratory bias alone is not sufficient to account for effects in each batch run.

Enhancing the usefulness of cross dehydrogenative coupling reactions with a removable protecting group.

A removable protecting group has been identified that allows the products of widely-used cross dehydrogenative couplings to be synthetically elaborated. The method can be used with enantiopure amines with no loss of enantiomeric excess. The methodology is exemplified by a new synthesis of enantiopure praziquantel, the drug used in the treatment of millions of people suffering from the neglected tropical disease, schistosomiasis.

Systematic comparison of δ13C measurements of testosterone and derivative steroids in a freeze-dried urine candidate reference material for sports drug testing by gas chromatography/combustion/isotope ratio mass spectrometry and uncertainty evaluation using four different metrological approaches.

An alternative calibration procedure for use when performing carbon isotope ratio measurements by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) has been developed. This calibration procedure does not rely on the corrections in-built in the instrument software, as the carbon isotope ratios of a sample are calculated from the measured raw peak areas. The method was developed for the certification of a urine reference material for sports drug testing, as the estimation of measurement uncertainty is greatly simplified. To ensure that the method is free from bias arising from the choice of calibration material and instrument, the carbon isotope ratios of steroids in urine extracts were measured using two different instruments in different laboratories, and three different reference materials (CU/USADA steroid standards from Brenna Laboratory, Cornell University; NIST RM8539 mineral oil; methane calibrated against NIST RM8560 natural gas). The measurements were performed at LGC and the Australian National Measurement Institute (NMI). It was found that there was no significant difference in measurement results when different instruments and reference materials were used to measure the carbon isotope ratio of the major testosterone metabolites androsterone and etiocholanolone, or the endogenous reference compounds pregnanediol, 11- ketoetiocholanolone and 11ß-hydroxyandrosterone. Expanded measurement uncertainties at the 95% coverage probability ranged from 0.21‰ to 1.4‰, depending on analyte, instrument and reference material. The measurement results of this comparison were used to estimate a measurement uncertainty of δ(13)C for the certification of the urine reference material being performed on a single instrument using a single reference material at NMI.

Alternative instrumentation for the analysis of total carbon dioxide (TCO(2)) in equine plasma.

The analysis of total carbon dioxide (TCO(2)) in equine plasma is conventionally done in Australia and elsewhere using Beckman Synchron EL-ISE(R) analysers. This instrument is no longer being manufactured and has not been supported by the supplier since December 2008. For testing for TCO(2) to continue, it is necessary to evaluate and commission alternative instrumentation. In this paper, we compare the Beckman Synchron EL-ISE, the Beckman Synchron CX5, the Beckman UniCel DxC 600 and the Randox Daytona instruments. Results indicate that all four instruments perform in accordance with the manufacturer's specifications. The Beckman CX 5, DxC 600 and Randox Daytona instruments are therefore all suitable alternatives for routine screening in a laboratory environment. Only the Randox Daytona instrument is sufficiently 'portable', i.e. it can be readily transported and used on-site at a racecourse (typically in a purpose-built modest-size laboratory vehicle). The three Beckman instruments are suitable for 'confirmatory analysis' using the quality-accredited method (Racing Science Centre), but the principle of operation of the Randox Daytona instrument may preclude its use in confirmatory analysis. Instrument costs may affect purchase decisions.

A probabilistic approach to heroin signatures.

The probability density functions of amount ratios of compounds (total codeine/total morphine, 6-monoacetylemorphine/total morphine, papaverine/total morphine, and noscapine/total morphine) from the analysis of seized heroin, originating from known world regions (South East Asia, South West Asia, South America, Mexico) allows calculation of likelihood ratios for 'unknown' samples. Application of Bayes Theorem with a suitable prior probability, for example the frequency of a particular region in the database, leads to the probability that a particular profile comes from a given target region. Data from 2549 seizures of heroin at Australia's border illustrates the method, and results are compared with simple HS1 ratio approaches for assigning geographical origin. The method can be implemented in a spreadsheet and gives more refined intelligence of the origins of seized drugs than simple ranges.

Siderophore-Assisted Dissolution of Iron(III) Hydroxide Oxides from Iron-Rich Fossil Matrices.

Current paleontological techniques to separate vertebrate fossils from encasing iron-rich cements by chemical means are limited by the low solubility of common iron(III) hydroxide oxides such as hematite and goethite. This study examines novel geochemical extractions capable of selectively dissolving iron(III) hydroxide oxides, in aqueous solutions of pH 9-11, without damaging fossilised bones or teeth (hydroxidecarbonate-apatite). This involves the siderophore ligands pyridoxal isonicotinoyl hydrazone (PIH), salicylaldehyde isonicotinoyl hydrazone (SIH), and acetohydroxamic acid (aHA), whose coordination complexes with iron(III) show exceptionally high formation stability constants. The methods have been tested on natural hematite and fossil containing samples from the Riversleigh World Heritage Area in Australia. Both 0.01 mol dm-3 aHA and 0.001 mol dm-3 PIH at pH 9.7 were able to dissolve over 0.1 mmol dm-3 of the goethite coating bone fragments.

Determination of the composition of fatty acid mixtures using GC x FI-MS: a comprehensive two-dimensional separation approach.

Gas chromatography using a highly polar column combined with field ionization mass spectrometry (FI-MS) is used as a comprehensive two-dimensional (2D) separation approach to analyze mixtures of fatty acid methyl esters (FAMEs). A unique ordered pattern and classification of FAMEs is obtained in a 2D GC x FI-MS separation plot based on the number of carbons, the degree of unsaturation, and a combination of both by which the geometrical, positional, and structural isomers group together. FAMEs with different chain length but identical geometry, position, and degree of unsaturation follow linear patterns. These subclassifications (linear functions) can provide information about the geometry, position, and structure of unsaturation of an unknown FAME. Non-FAMEs and FAMEs with different functional groups are identified using the ordered separation pattern of the FAMEs in the GC x FI-MS plot and the exact mass data from the FI-MS mode. Measurement of exact mass also acts as a high-resolution separation technique to separate overlapping peaks. The method is illustrated by application to samples of fish, canola, and biodiesel oils and standard mixtures of 37 FAMEs and of alpha-linolenic acid methyl ester geometrical isomers. A great wealth of information is achieved in a single run.

Compatibility of electron ionization and soft ionization methods in gas chromatography/orthogonal time-of-flight mass spectrometry.

Orthogonal acceleration time-of-flight (oa-TOF) mass spectrometry (MS) was coupled to gas chromatography (GC) to measure ion yields (ratio of ion counts to number of neutrals entering the ion source) and signal-to-noise (S/N) in the electron ionization (EI) mode (hard ionization) as well as in the soft ionization modes of chemical ionization (CI), electron capture negative ion chemical ionization (NICI) and field ionization (FI). Mass accuracies of the EI and FI modes were also investigated. Sixteen structurally diverse volatile organic compounds were chosen for this study. The oa-TOF mass analyzer is highly suited for FI MS and provided an opportunity to compare the sensitivity of this ionization method to the more conventional ionization methods. Compared to the widely used quadrupole mass filter, the oa-TOF platform offers significantly greater mass accuracy and therefore the possibility of determining the empirical formula of analytes. The findings of this study showed that, for the instrument used, EI generated the most ions with the exception of compounds able to form negative ions readily. Lower ion yields in the FI mode were generally observed but the chromatograms displayed greater S/N and in many cases gave spectra dominated by a molecular ion. Ion counts in CI are limited by the very small apertures required to maintain sufficiently high pressures in the ionization chamber. Mass accuracy for molecular and fragment ions was attainable at close to manufacturer's specifications, thus providing useful information on molecular ions and neutral losses. The data presented also suggests a potentially useful instrumental combination would result if EI and FI spectra could be collected simultaneously or in alternate scans during GC/MS.

High throughput screening arrays of rhodium and iridium complexes as catalysts for intramolecular hydroamination using parallel factor analysis.

Parallel factor analysis (PARAFAC) was used to analyze data from the high throughput screening of an array of organometallic rhodium and iridium complexes as catalysts for the intramolecular hydroamination of 2-(2-phenylethynyl)aniline to give 2-phenylindole. The progress of the hydroamination reactions was monitored using UV-visible spectroscopy. The overlapped UV-visible spectra of the mixture of starting material, product and solvent in the samples taken at different times were deconvoluted using PARAFAC. Unique PARAFAC models led to close approximations of the actual UV-visible spectra of the compounds in the mixture. The performance of the catalysts was then compared by estimating the final concentration of the starting material and product using PARAFAC loadings. A library of 63 complexes generated in situ was examined in a single experiment using this methodology. The complexes were generated from combinations of seven ligands (bis(N-methyl2-imidazolyl)methane, bis(1-pyrazolyl)methane, 1,10-phenanthroline, N,N'-bis(p-tolyl)diazabutadiene, N,N'-bis(p-tolyl)1,2-dimethyldiazabutadiene, N,N'-bis(mesityl)1,2-dimethyldiazabutadiene and bis(2,4,6-trimethylphenylimino)acenapthene) and nine metal precursors ([Ir(COD)Cl](2) (COD = 1,5-cyclooctadiene), [Ir(CO)(2)Cl](n), [Ir(COE)(2)Cl](2), [IrCp*Cl(2)](2) (Cp* = 1,2,3,4,5-pentamethylcyclopentadiene), [Rh(COD)Cl](2), [Rh(CO)(2)Cl](2), [Rh(COE)(2)Cl](2), [RhCp*Cl(2)](2) and [RhCpCl(2)](2)) (Cp = cyclopentadiene)). The proposed method can be used for the fast screening of arrays of metal complexes for identifying effective catalysts, providing information that can augment traditional methods used for the analysis of catalyzed reactions.