Showing papers in "Analytical Chemistry in 2009"
TL;DR: CR-GO with the nature of a single sheet showing favorable electrochemical activity should be a kind of more robust and advanced carbon electrode material which may hold great promise for electrochemical sensors and biosensors design.
Abstract: In this paper, the characterization and application of a chemically reduced graphene oxide modified glassy carbon (CR-GO/GC) electrode, a novel electrode system, for the preparation of electrochemical sensing and biosensing platform are proposed. Different kinds of important inorganic and organic electroactive compounds (i.e., probe molecule (potassium ferricyanide), free bases of DNA (guanine (G), adenine (A), thymine (T), and cytosine (C)), oxidase/dehydrogenase-related molecules (hydrogen peroxide (H2O2)/β-nicotinamide adenine dinucleotide (NADH)), neurotransmitters (dopamine (DA)), and other biological molecules (ascorbic acid (AA), uric acid (UA), and acetaminophen (APAP)) were employed to study their electrochemical responses at the CR-GO/GC electrode, which shows more favorable electron transfer kinetics than graphite modified glassy carbon (graphite/GC) and glassy carbon (GC) electrodes. The greatly enhanced electrochemical reactivity of the four free bases of DNA at the CR-GO/GC electrode compare...
1,587 citations
TL;DR: A detailed study on wax printing, a simple and inexpensive method for fabricating microfluidic devices in paper using a commercially available printer and hot plate, which creates complete hydrophobic barriers in paper that define hydrophilic channels, fluid reservoirs, and reaction zones.
Abstract: This technical note describes a detailed study on wax printing, a simple and inexpensive method for fabricating microfluidic devices in paper using a commercially available printer and hot plate. The printer prints patterns of solid wax on the surface of the paper, and the hot plate melts the wax so that it penetrates the full thickness of the paper. This process creates complete hydrophobic barriers in paper that define hydrophilic channels, fluid reservoirs, and reaction zones. The design of each device was based on a simple equation that accounts for the spreading of molten wax in paper.
1,403 citations
TL;DR: A novel polyvinylpyrrolidone-protected graphene/polyethylenimine-functionalized ionic liquid/GOD electrochemical biosensor is constructed, which achieved the direct electron transfer of GOD, maintained its bioactivity and showed potential application for the fabrication of novel glucose biosensors with linear glucose response up to 14 mM.
Abstract: We first reported that polyvinylpyrrolidone-protected graphene was dispersed well in water and had good electrochemical reduction toward O2 and H2O2. With glucose oxidase (GOD) as an enzyme model, we constructed a novel polyvinylpyrrolidone-protected graphene/polyethylenimine-functionalized ionic liquid/GOD electrochemical biosensor, which achieved the direct electron transfer of GOD, maintained its bioactivity and showed potential application for the fabrication of novel glucose biosensors with linear glucose response up to 14 mM.
1,253 citations
TL;DR: The FiehnLib libraries can be used in conjunction with GC/MS software but also support compound identification in the public BinBase metabolomic database that currently comprises 5598 unique mass spectra generated from 19,032 samples covering 279 studies of 47 species.
Abstract: At least two independent parameters are necessary for compound identification in metabolomics. We have compiled 2 212 electron impact mass spectra and retention indices for quadrupole and time-of-flight gas chromatography/mass spectrometry (GC/MS) for over 1 000 primary metabolites below 550 Da, covering lipids, amino acids, fatty acids, amines, alcohols, sugars, amino-sugars, sugar alcohols, sugar acids, organic phosphates, hydroxyl acids, aromatics, purines, and sterols as methoximated and trimethylsilylated mass spectra under electron impact ionization. Compounds were selected from different metabolic pathway databases. The structural diversity of the libraries was found to be highly overlapping with metabolites represented in the BioMeta/KEGG pathway database using chemical fingerprints and calculations using Instant-JChem. In total, the FiehnLib libraries comprised 68% more compounds and twice as many spectra with higher spectral diversity than the public Golm Metabolite Database. A range of unique c...
1,153 citations
TL;DR: A platform that integrated the chemical analysis, including identification and relative quantification, data reduction, and quality assurance components of the process enabled the high-throughput collection and relative quantitative analysis of analytical data and identified a large number and broad spectrum of molecules with a high degree of confidence.
Abstract: To address the challenges associated with metabolomics analyses, such as identification of chemical structures and elimination of experimental artifacts, we developed a platform that integrated the chemical analysis, including identification and relative quantification, data reduction, and quality assurance components of the process. The analytical platform incorporated two separate ultrahigh performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS2) injections; one injection was optimized for basic species, and the other was optimized for acidic species. This approach permitted the detection of 339 small molecules, a total instrument analysis time of 24 min (two injections at 12 min each), while maintaining a median process variability of 9%. The resulting MS/MS2 data were searched against an in-house generated authentic standard library that included retention time, molecular weight (m/z), preferred adducts, and in-source fragments as well as their associated MS/MS spectra for all molecul...
1,139 citations
TL;DR: A novel instrument for real time analysis of individual biological cells or other microparticles is described and real-time simultaneous detection of multiple isotopes from individual 1.8 microm polystyrene beads labeled with lanthanides is shown.
Abstract: A novel instrument for real time analysis of individual biological cells or other microparticles is described. The instrument is based on inductively coupled plasma time-of-flight mass spectrometry and comprises a three-aperture plasma−vacuum interface, a dc quadrupole turning optics for decoupling ions from neutral components, an rf quadrupole ion guide discriminating against low-mass dominant plasma ions, a point-to-parallel focusing dc quadrupole doublet, an orthogonal acceleration reflectron analyzer, a discrete dynode fast ion detector, and an 8-bit 1 GHz digitizer. A high spectrum generation frequency of 76.8 kHz provides capability for collecting multiple spectra from each particle-induced transient ion cloud, typically of 200−300 μs duration. It is shown that the transients can be resolved and characterized individually at a peak frequency of 1100 particles per second. Design considerations and optimization data are presented. The figures of merit of the instrument are measured under standard indu...
1,072 citations
TL;DR: This study shows the successful integration of paper-based microfluidics and electrochemical detection as an easy-to-use, inexpensive, and portable alternative for point of care monitoring.
Abstract: We report the first demonstration of electrochemical detection for paper-based microfluidic devices. Photolithography was used to make microfluidic channels on filter paper, and screen-printing technology was used to fabricate electrodes on the paper-based microfluidic devices. Screen-printed electrodes on paper were characterized using cyclic voltammetry to demonstrate the basic electrochemical performance of the system. The utility of our devices was then demonstrated with the determination of glucose, lactate, and uric acid in biological samples using oxidase enzyme (glucose oxidase, lactate oxidase, and uricase, respectively) reactions. Oxidase enzyme reactions produce H2O2 while decomposing their respective substrates, and therefore a single electrode type is needed for detection of multiple species. Selectivity of the working electrode for H2O2 was improved using Prussian Blue as a redox mediator. The determination of glucose, lactate, and uric acid in control serum samples was performed using chron...
1,037 citations
TL;DR: MetAlign software is capable of automatic format conversions, accurate mass calculations, baseline corrections, peak-picking, saturation and mass-peak artifact filtering, as well as alignment of up to 1000 data sets, and is compatible with most multivariate statistics programs.
Abstract: Hyphenated full-scan MS technology creates large amounts of data. A versatile easy to handle automation tool aiding in the data analysis is very important in handling such a data stream. MetAlign software—as described in this manuscript—handles a broad range of accurate mass and nominal mass GC/MS and LC/MS data. It is capable of automatic format conversions, accurate mass calculations, baseline corrections, peak-picking, saturation and mass-peak artifact filtering, as well as alignment of up to 1000 data sets. A 100 to 1000-fold data reduction is achieved. MetAlign software output is compatible with most multivariate statistics programs.
661 citations
TL;DR: In this article, the authors investigated achievable uncertainties for the determination of fluorescence quantum yields of these chromophores and illustrate common pitfalls exemplarily for differently sized water-soluble CdTe QDs.
Abstract: Despite the increasing use of semiconductor nanocrystals (quantum dots, QDs) with unique size-controlled optical and chemical properties in (bio)analytical detection, biosensing and fluorescence imaging and the obvious relevance of reliable values of fluorescence quantum yields for these applications, evaluated procedures for the determination of the fluorescence quantum yields (Φf) of these materials are still missing. This limits the value of literature data of QDs in comparison to common organic dyes and hampers the comparability of the performance of QDs from different sources or manufacturers. This encouraged us to investigate achievable uncertainties for the determination of Φf values of these chromophores and to illustrate common pitfalls exemplarily for differently sized water-soluble CdTe QDs. Special attention is dedicated to the colloidal nature and complicated surface chemistry of QDs thereby deriving procedures to minimize uncertainties related to these features.
566 citations
TL;DR: Through this approach, aqueous Hg(2+) can be detected at 50 nM (10 ppb) with colorimetry in a facile way, with high selectivity against other metal ions.
Abstract: Mercury ion (Hg2+) is able to specifically bind to the thymine−thymine (T−T) base pair in a DNA duplex, thus providing a rationale for DNA-based selective detection of Hg2+ with various means. In this work, we for the first time utilize the Hg2+-mediated T−T base pair to modulate the proper folding of G-quadruplex DNAs and inhibit the DNAzyme activity, thereby pioneering a facile approach to sense Hg2+ with colorimetry. Two bimolecular DNA G-quadruplexes containing many T residues are adopted here, which function well in low- and high-salt conditions, respectively. These G-quadruplex DNAs are able to bind hemin to form the peroxidase-like DNAzymes in the folded state. Upon addition of Hg2+, the proper folding of G-quadruplex DNAs is inhibited due to the formation of T−Hg2+−T complex. This is reflected by the notable change of the Soret band of hemin when investigated by using UV−vis absorption spectroscopy. As a result of Hg2+ inhibition, a sharp decrease in the catalytic activity toward the H2O2-mediated...
454 citations
TL;DR: A novel approach has been developed for the quantitative determination of circulating drug concentrations in clinical studies using dried blood spots (DBS) on paper, rather than conventional plasma samples, and a quantitative bioanalytical HPLC-MS/MS assay requiring small blood volumes has been validated using acetaminophen as a tool compound.
Abstract: A novel approach has been developed for the quantitative determination of circulating drug concentrations in clinical studies using dried blood spots (DBS) on paper, rather than conventional plasma samples. A quantitative bioanalytical HPLC-MS/MS assay requiring small blood volumes (15 microL) has been validated using acetaminophen as a tool compound (range 25 to 5000 ng/mL human blood). The assay employed simple solvent extraction of a punch taken from the DBS sample, followed by reversed phase HPLC separation, combined with selected reaction monitoring mass spectrometric detection. In addition to performing routine experiments to establish the validity of the assay to internationally accepted criteria (precision, accuracy, linearity, sensitivity, selectivity), a number of experiments were performed to specifically demonstrate the quality of the quantitative data generated using this novel sample format, namely, stability of the analyte and metabolites in whole human blood and in DBS samples; effect of the volume of blood spotted, the device used to spot the blood, or the temperature of blood spotted. The validated DBS approach was successfully applied to a clinical study (single oral dose of 500 mg or 1 g acetaminophen).
TL;DR: Both the color and fluorescence changes of the chemosensor are remarkably specific for Cu(2+) in the presence of other heavy and transition metal ions (even those that exist in high concentration), which meet the selective requirements for biomedical and environmental monitoring application.
Abstract: The design and synthesis of a novel rhodamine spirolactam derivative and its application in fluorescent detections of Cu(2+) in aqueous solution and living cells are reported. The signal change of the chemosensor is based on a specific metal ion induced reversible ring-opening mechanism of the rhodamine spirolactam. It exhibits a highly sensitive "turn-on" fluorescent response toward Cu(2+) in aqueous solution with an 80-fold fluorescence intensity enhancement under 10 equiv of Cu(2+) added. This indicates that the synthesized chemosensor effectively avoided the fluorescence quenching for the paramagnetic nature of Cu(2+) via its strong binding capability toward Cu(2+). With the experimental conditions optimized, the probe exhibits a dynamic response range for Cu(2+) from 8.0 x 10(-7) to 1.0 x 10(-5) M, with a detection limit of 3.0 x 10(-7) M. The response of the chemosensor for Cu(2+) is instantaneous and reversible. Most importantly, both the color and fluorescence changes of the chemosensor are remarkably specific for Cu(2+) in the presence of other heavy and transition metal ions (even those that exist in high concentration), which meet the selective requirements for biomedical and environmental monitoring application. The proposed chemosensor has been used for direct measurement of Cu(2+) content in river water samples and imaging of Cu(2+) in living cells with satisfying results, which further demonstrates its value of practical applications in environmental and biological systems.
TL;DR: The observed drift in instrumental performance over time and its improvement with adjustment of the length of analytical block are described and allowed the authors to prepare SOPs for "fit for purpose" long-term UPLC-MS metabolomic studies, such as are being employed in the HUSERMET project.
Abstract: A method for performing untargeted metabolomic analysis of human serum has been developed based on protein precipitation followed by Ultra Performance Liquid Chromatography and Time-of-Flight mass spectrometry (UPLC−TOF-MS). This method was specifically designed to fulfill the requirements of a long-term metabolomic study, spanning more than 3 years, and it was subsequently thoroughly evaluated for robustness and repeatability. We describe here the observed drift in instrumental performance over time and its improvement with adjustment of the length of analytical block. The optimal setup for our purpose was further validated against a set of serum samples from 30 healthy individuals. We also assessed the reproducibility of chromatographic columns with the same chemistry of stationary phase from the same manufacturer but from different production batches. The results have allowed the authors to prepare SOPs for “fit for purpose” long-term UPLC−MS metabolomic studies, such as are being employed in the HUSER...
TL;DR: It is expected this Au NPs-based Hg(2+) sensor will be a promising candidate for field detection of environmentally toxic mercury and exhibits excellent selectivity over a spectrum of interference metal ions.
Abstract: We report a highly sensitive electrochemical sensor for the detection of Hg2+ ions in aqueous solution by using a thymine (T)-rich, mercury-specific oligonucleotide (MSO) probe and gold nanoparticles (Au NPs)-based signal amplification. The MSO probe contains seven thymine bases at both ends and a “mute” spacer in the middle, which, in the presence of Hg2+, forms a hairpin structure via the Hg2+-mediated coordination of T−Hg2+−T base pairs. The thiolated MSO probe is immobilized on Au electrodes to capture free Hg2+ in aqueous media, and the MSO-bound Hg2+ can be electrochemically reduced to Hg+, which provides a readout signal for quantitative detection of Hg2+. This direct immobilization strategy leads to a detection limit of 1 μM. In order to improve the sensitivity, MSO probe-modified Au NPs are employed to amplify the electrochemical signals. Au NPs are comodified with the MSO probe and a linking probe that is complementary to a capture DNA probe immobilized on gold electrodes. We demonstrated that t...
TL;DR: Measuring transmittance through paper represents a new method of quantitative detection that expands the potential functionality of micro-PADs and is potentially attractive for use in resource-limited environments and developing countries.
Abstract: This article describes a point-of-care (POC) system—comprising a microfluidic, paper-based analytical device (μ-PAD) and a hand-held optical colorimeter—for quantifying the concentration of analytes in biological fluids. The μ-PAD runs colorimetric assays, and consists of paper that has been (i) patterned to expose isolated regions of hydrophilic zones and (ii) wet with an index-matching fluid (e.g., vegetable oil) that is applied using a disposable, plastic sleeve encasement. Measuring transmittance through paper represents a new method of quantitative detection that expands the potential functionality of μ-PADs. This prototype transmittance colorimeter is inexpensive, rugged, and fully self-contained, and thus potentially attractive for use in resource-limited environments and developing countries.
TL;DR: The new MIP-based RTP sensing protocol was applied to detect trace pentachlorophenol (PCP) in water samples without the interference of autofluorescence and scattering light of matrixes.
Abstract: A new type of molecularly imprinted polymer (MIP)-based room-temperature phosphorescence (RTP) optosensor was developed by anchoring the MIP layer on the surface of Mn-doped ZnS quantum dots (QDs) via a surface molecular imprinting process. The synergetic combination of the RTP property of the Mn-doped ZnS QDs and the merits of the surface imprinting polymers not only improves the RTP selectivity of the Mn-doped ZnS QDs but also makes the MIP-based RTP optosensor also applicable to selective detecting of those nonphosphorescent analytes without the need for any inducers and derivatization. The new MIP-based RTP sensing protocol was applied to detect trace pentachlorophenol (PCP) in water samples without the interference of autofluorescence and scattering light of matrixes. The detection limit for PCP was 86 nM, and the precision for five replicate detections of 0.4 μM PCP was 2.8% (relative standard deviation). The recovery of spiked PCP in river water samples ranged from 93% to 106%.
TL;DR: A nonenzymatic amperometric glucose sensor was developed with the use of the Pd-SWNT nanostructure as an electrocatalyst and had good electrocatalytic activity toward oxidation of glucose and exhibited a rapid response.
Abstract: A new electrocatalyst, palladium nanoparticle-single-walled carbon nanotube (Pd-SWNTs) hybrid nanostructure, for the nonenzymatic oxidation of glucose was developed and characterized by X-ray diffraction (XRD) and the transmission electron microscope (TEM). The hybrid nanostructures were prepared by depositing palladium nanoparticles with average diameters of 4-5 nm on the surface of single-walled carbon nanotubes (SWNTs) via chemical reduction of the precursor (Pd(2+)). The electrocatalyst showed good electrocatalytic activity toward the oxidation of glucose in the neutral phosphate buffer solution (PBS, pH 7.4) even in the presence of a high concentration of chloride ions. A nonenzymatic amperometric glucose sensor was developed with the use of the Pd-SWNT nanostructure as an electrocatalyst. The sensor had good electrocatalytic activity toward oxidation of glucose and exhibited a rapid response (ca.3 s), a low detection limit (0.2 +/- 0.05 microM), a wide and useful linear range (0.5-17 mM), and high sensitivity (approximately 160 microA mM(-1) cm(-2)) as well as good stability and repeatability. In addition, the common interfering species, such as ascorbic acid, uric acid, 4-acetamidophenol, 3,4-dihydroxyphenylacetic acid, and so forth did not cause any interference due to the use of a low detection potential (-0.35 V vs SCE). The sensor can also be used for quantification of the concentration of glucose in real clinical samples. Therefore, this work has demonstrated a simple and effective sensing platform for nonenzymatic detection of glucose.
TL;DR: A graphene oxide amplified electrogenerated chemiluminescence (ECL) of quantum dots (QDs) platform and its efficient selective sensing for antioxidants is reported and its detection limit was 8.3 microM for glutathione.
Abstract: Here we report a graphene oxide amplified electrogenerated chemiluminescence (ECL) of quantum dots (QDs) platform and its efficient selective sensing for antioxidants. Graphene oxide facilitated the CdTe QDs•+ production and triggered O2•− generation. Then, a high yield of CdTe QDs* was formed due to the combination of CdTe QDs•+ and O2•−, leading to an ∼5-fold ECL amplification. Glutathione is the most abundant cellular thiol-containing peptide, but its selective sensing is an intractable issue in analytical and biochemical communities because its detection is interfered with by some thiol-containing compounds. This platform showed a detection limit of 8.3 μM (S/N = 3) for glutathione and a selective detection linear dependence from 24 to 214 μM in the presence of 120 μM cysteine and glutathione disulfide. This platform was also successfully used for real sample (eye drug containing glutathione) detection without any pretreatment with a wide linear range from 0.04 to 0.29 μg mL−1.
TL;DR: Results demonstrate that the UCL imaging technique appears particularly suited for applications in tracking and labeling components of complex biological systems.
Abstract: Fluorescence targeted imaging in vivo has proven useful in tumor recognition and drug delivery. In the process of in vivo imaging, however, a high autofluorescence background could mask the signals from the fluorescent probes. Herein, a high contrast upconversion luminescence (UCL) imaging protocol was developed for targeted imaging of tumors based on RGD-labeled upconversion nanophosphors (UCNPs) as luminescent labels. Confocal Z-scan imaging of tissue slices revealed that UCL imaging showed no autofluorescence signal even at high penetration depth (∼600 μm). More importantly, region of interest (ROI) analysis of the UCL signal in vivo showed that UCL imaging achieved a high signal-to-noise ratio (∼24) between the tumor and the background. These results demonstrate that the UCL imaging technique appears particularly suited for applications in tracking and labeling components of complex biological systems.
TL;DR: Demonstration of quantitative colorimetric correlations using a scanner or camera to image the zones and to measure the intensity of color, makes it possible to conduct assays without a microplate reader.
Abstract: This paper describes 96- and 384-microzone plates fabricated in paper as alternatives to conventional multiwell plates fabricated in molded polymers. Paper-based plates are functionally related to plastic well plates, but they offer new capabilities. For example, paper-microzone plates are thin (∼180 μm), require small volumes of sample (5 μL per zone), and can be manufactured from inexpensive materials ($0.05 per plate). The paper-based plates are fabricated by patterning sheets of paper, using photolithography, into hydrophilic zones surrounded by hydrophobic polymeric barriers. This photolithography used an inexpensive formulation photoresist that allows rapid (∼15 min) prototyping of paper-based plates. These plates are compatible with conventional microplate readers for quantitative absorbance and fluorescence measurements. The limit of detection per zone loaded for fluorescence was 125 fmol for fluorescein isothiocyanate-labeled bovine serum albumin, and this level corresponds to 0.02 the quantity o...
TL;DR: The intuitive graphical user interface of MetaboliteDetector additionally allows for a detailed examination of a single GC/MS chromatogram including single ion chromatograms, recorded mass spectra, and identified metabolite spectra in combination with the corresponding reference spectra obtained from a reference library.
Abstract: We have developed a new software, MetaboliteDetector, for the efficient and automatic analysis of GC/MS-based metabolomics data. Starting with raw MS data, the program detects and subsequently identifies potential metabolites. Moreover, a comparative analysis of a large number of chromatograms can be performed in either a targeted or nontargeted approach. MetaboliteDetector automatically determines appropriate quantification ions and performs an integration of single ion peaks. The analysis results can directly be visualized with a principal component analysis. Since the manual input is limited to absolutely necessary parameters, the program is also usable for the analysis of high-throughput data. However, the intuitive graphical user interface of MetaboliteDetector additionally allows for a detailed examination of a single GC/MS chromatogram including single ion chromatograms, recorded mass spectra, and identified metabolite spectra in combination with the corresponding reference spectra obtained from a ...
TL;DR: The strategies underlying the automated identification and quantification of individual lipid molecular species through array analysis of multidimensional mass spectrometry-based shotgun lipidomics (MDMS-SL) data, which are acquired directly from lipid extracts after direct infusion and intrasource separation are presented.
Abstract: This article presents the strategies underlying the automated identification and quantification of individual lipid molecular species through array analysis of multidimensional mass spectrometry-based shotgun lipidomics (MDMS-SL) data, which are acquired directly from lipid extracts after direct infusion and intrasource separation. The automated analyses of individual lipid molecular species in the program employ a strategy in which MDMS-SL data from building block analyses using precursor ion scans, neutral loss scans, or both are used to identify individual molecular species, followed by quantitation. Through this strategy, the program screens and identifies species in a high-throughput fashion from a built-in database of over 36 000 potential lipid molecular species constructed employing known building blocks. The program then uses a two-step procedure for quantitation of the identified species possessing a linear dynamic range over 3 orders of magnitude and reverifies the results when necessary throug...
TL;DR: The first example of the utilization of MOFs for solid-phase microextraction (SPME) is reported, with MOF-199 with unique pores and open metal sites employed as the coating for SPME fiber to extract volatile and harmful benzene homologues.
Abstract: Metal−organic frameworks (MOFs) have received great attention due to their fascinating structures and intriguing potential applications in various fields. Herein, we report the first example of the utilization of MOFs for solid-phase microextraction (SPME). MOF-199 with unique pores and open metal sites (Lewis acid sites) was employed as the coating for SPME fiber to extract volatile and harmful benzene homologues. The SPME fiber was fabricated by in situ hydrothermal growth of thin MOF-199 films on etched stainless steel wire. The MOF-199-coated fiber not only offered large enhancement factors from 19 613 (benzene) to 110 860 (p-xylene), but also exhibited wide linearity with 3 orders of magnitude for the tested benzene homologues. The limits of detection for the benzene homologues were 8.3−23.3 ng L−1. The relative standard deviation (RSD) for six replicate extractions using one SPME fiber ranged from 2.0% to 7.7%. The fiber-to-fiber reproducibility for three parallel prepared fibers was 3.5%−9.4% (RSD)...
TL;DR: It is shown that dansylation labeling and fast LC/FTICR MS can be a powerful technique for quantitative profiling of at least 672 metabolites in urine samples in 12 min, and it is demonstrated that a linear response of over 2 orders of magnitude is achieved for relative metabolite quantification.
Abstract: We report a new quantitative metabolome profiling technique based on differential 12C-/13C-isotope dansylation labeling of metabolites, fast liquid chromatography (LC) separation and electrospray ionization Fourier-transform ion cyclotron resonance mass spectrometry (ESI-FTICR MS) detection. An isotope reagent, 13C-dansyl chloride, can be readily synthesized. This reagent, along with 12C-dansyl chloride, provides a simple and robust means of labeling metabolites containing primary amine, secondary amine, or phenolic hydroxyl group(s). It is shown that dansylation labeling offers 1−3 orders of magnitude ESI signal enhancement over the underivatized counterparts. Dansylation alters the chromatographic behaviors of polar and ionic metabolites normally not retainable on a reversed phase (RP) column to an extent that they can be retained and separated by RPLC with high efficiency. There is no isotopic effect on RPLC separation of the differential isotope labeled metabolites, and 12C-/13C-labeled isoforms of me...
TL;DR: Understanding as much as possible about proteins in the shortest amount of time has long been a goal of hydrogen exchange (HX) MS, but has this goal yet been achieved?
Abstract: Understanding as much as possible about proteins in the shortest amount of time has long been a goal of hydrogen exchange (HX) MS. Recent technological advances have led to improvements in the technique, but has this goal yet been achieved? (To listen to a podcast about this Feature, please go to the Analytical Chemistry Web site at pubs.acs.org/journal/ancham.)
TL;DR: The preparation and characterization of novel hybrid materials combining a catalytic MIP, capable of oxidizing the template, catechol, with an electrically conducting polymer, and the catalytic properties of the sensor were characterized by chronoamperometry and were found to be consistent with Michaelis-Menten kinetics.
Abstract: One of the difficulties with using molecularly imprinted polymers (MIPs) and other electrically insulating materials as the recognition element in electrochemical sensors is the lack of a direct path for the conduction of electrons from the active sites to the electrode. We have sought to address this problem through the preparation and characterization of novel hybrid materials combining a catalytic MIP, capable of oxidizing the template, catechol, with an electrically conducting polymer. In this way a network of "molecular wires" assists in the conduction of electrons from the active sites within the MIP to the electrode surface. This was made possible by the design of a new monomer that combines orthogonal polymerizable functionality; comprising an aniline group and a methacrylamide. Conducting films were prepared on the surface of electrodes (Au on glass) by electropolymerization of the aniline moiety. A layer of MIP was photochemically grafted over the polyaniline, via N,N'-diethyldithiocarbamic acid benzyl ester (iniferter) activation of the methacrylamide groups. Detection of catechol by the hybrid-MIP sensor was found to be specific, and catechol oxidation was detected by cyclic voltammetry at the optimized operating conditions: potential range -0.6 V to +0.8 V (vs Ag/AgCl), scan rate 50 mV/s, PBS pH 7.4. The calibration curve for catechol was found to be linear to 144 microM, with a limit of detection of 228 nM. Catechol and dopamine were detected by the sensor, whereas analogues and potentially interfering compounds, including phenol, resorcinol, hydroquinone, serotonin, and ascorbic acid, had minimal effect (< or = 3%) on the detection of either analyte. Non-imprinted hybrid electrodes and bare gold electrodes failed to give any response to catechol at concentrations below 0.5 mM. Finally, the catalytic properties of the sensor were characterized by chronoamperometry and were found to be consistent with Michaelis-Menten kinetics.
TL;DR: It is found that the choice of extraction method can drastically affect measured metabolite levels, to an extent that for some metabolites even the direction of changes between growth conditions can be inverted.
Abstract: Accurate determination of intracellular metabolite levels requires well-validated procedures for sampling and sample treatment. Several methods exist for metabolite extraction, but the literature is contradictory regarding the adequacy and performance of each technique. Using a strictly quantitative approach, we have re-evaluated five methods (hot water, HW; boiling ethanol, BE; chloroform−methanol, CM; freezing-thawing in methanol, FTM; acidic acetonitrile−methanol, AANM) for the extraction of 44 intracellular metabolites (phosphorylated intermediates, amino acids, organic acids, nucleotides) from S. cerevisiae cells. Two culture modes were investigated (batch and chemostat) to check for growth condition dependency, and three targeted platforms were employed (two LC-MS and one GC/MS) to exclude analytical bias. Additionally, for the determination of metabolite recoveries, we applied a novel approach based on addition of 13C-labeled internal standards at different stages of sample processing. We found tha...
TL;DR: To the best of the knowledge, this is the first example of a single DNA-based sensor that allows the detection of both Hg(2+) and Pb( 2+) ions.
Abstract: We have developed a technique for the highly selective and sensitive detection of Pb(2+) and Hg(2+) using a thrombin-binding aptamer (TBA) probe labeled with the donor carboxyfluorescein (FAM) and the quencher 4-([4-(dimethylamino)phenyl]azo)benzoic acid (DABCYL) at its 5' and 3' termini, respectively. The TBA has a random coil structure that changes into a G-quartet structure and a hairpin-like structure upon binding Pb(2+) and Hg(2+) ions, respectively. As a result, the fluorescence decreases through fluorescence resonance energy transfer (FRET) between the fluorophore and quencher. These changes in fluorescence intensity allow the selective detection of Pb(2+) and Hg(2+) ions at concentrations as low as 300 pM and 5.0 nM using this TBA probe in the presence of phytic acid and a random DNA/NaCN mixture, respectively. The linear correlation existed between the fluorescence intensity and the concentration of Pb(2+) and Hg(2+) over the range of 0.5-30 nM (R(2) = 0.98) and 10-200 nM (R(2) = 0.98), respectively. To the best of our knowledge, this is the first example of a single DNA-based sensor that allows the detection of both Hg(2+) and Pb(2+) ions. This simple and cost-effective probe was also applied to separately determine Pb(2+) in soil samples and spiked Hg(2+) in pond samples.
TL;DR: A method for high-throughput isothermal amplification of single DNA molecules in a droplet-based microfluidic system and fast and accurate "digital" quantification of the template DNA based on the Poisson distribution ofDNA molecules in droplets is developed.
Abstract: We have developed a method for high-throughput isothermal amplification of single DNA molecules in a droplet-based microfluidic system. DNA amplification in droplets was analyzed using an intercalating fluorochrome, allowing fast and accurate “digital” quantification of the template DNA based on the Poisson distribution of DNA molecules in droplets. The clonal amplified DNA in each 2 pL droplet was further analyzed by measuring the enzymatic activity of the encoded proteins after fusion with a 15 pL droplet containing an in vitro translation system.
TL;DR: For the first time, DLS is able to directly and quantitatively measure the binding stoichiometry between a protein-conjugated GNP probe and a target analyte protein in solution.
Abstract: Dynamic light scattering (DLS) is an analytical tool used routinely for measuring the hydrodynamic size of nanoparticles and colloids in a liquid environment. Gold nanoparticles (GNPs) are extraordinary light scatterers at or near their surface plasmon resonance wavelength. In this study, we demonstrate that DLS can be used as a very convenient and powerful tool for gold nanoparticle bioconjugation and biomolecular binding studies. The conjugation process between protein A and gold nanoparticles under different experimental conditions and the quality as well as the stability of the prepared conjugates were monitored and characterized systematically by DLS. Furthermore, the specific interactions between protein A-conjugated gold nanoparticles and a target protein, human IgG, can be detected and monitored in situ by measuring the average particle size change of the assay solution. For the first time, we demonstrate that DLS is able to directly and quantitatively measure the binding stoichiometry between a protein-conjugated GNP probe and a target analyte protein in solution.