Showing papers in "Analytical Chemistry in 2001"
TL;DR: An automated method for shotgun proteomics named MudPIT, which combines multidimensional liquid chromatography with electrospray ionization tandem mass spectrometry, improves the overall analysis of proteomes by identifying proteins of all functional and physical classes.
Abstract: We describe an automated method for shotgun proteomics named multidimensional protein identification technology (MudPIT), which combines multidimensional liquid chromatography with electrospray ionization tandem mass spectrometry. The multidimensional liquid chromatography method integrates a strong cation-exchange (SCX) resin and reversed-phase resin in a biphasic column. We detail the improvements over a system described by Link et al. (Link, A. J.; Eng, J.; Schieltz, D. M.; Carmack, E.; Mize, G. J.; Morris, D. R.; Garvik, B. M.; Yates, J. R., III. Nat. Biotechnol. 1999, 17, 676−682) that separates and acquires tandem mass spectra for thousands of peptides. Peptides elute off the SCX phase by increasing pI, and elution off the SCX material is evenly distributed across an analysis. In addition, we describe the chromatographic benchmarks of MudPIT. MudPIT was reproducible within 0.5% between two analyses. Furthermore, a dynamic range of 10 000 to 1 between the most abundant and least abundant proteins/pep...
1,804 citations
TL;DR: The precision of the method is better than 0.2/1000 (1 SD) at concentrations of nitrate down to 1 microM, and the nitrogen isotopic differences among various standards and samples are accurately reproduced.
Abstract: We report a new method for measurement of the isotopic composition of nitrate (NO3-) at the natural-abundance level in both seawater and freshwater. The method is based on the isotopic analysis of nitrous oxide (N2O) generated from nitrate by denitrifying bacteria that lack N2O-reductase activity. The isotopic composition of both nitrogen and oxygen from nitrate are accessible in this way. In this first of two companion manuscripts, we describe the basic protocol and results for the nitrogen isotopes. The precision of the method is better than 0.2‰ (1 SD) at concentrations of nitrate down to 1 μM, and the nitrogen isotopic differences among various standards and samples are accurately reproduced. For samples with 1 μM nitrate or more, the blank of the method is less than 10% of the signal size, and various approaches may reduce it further.
1,562 citations
TL;DR: It is shown how quantitative data about the thickness, shear elastic modulus, and shear viscosity of the protein film can be obtained with the QCM-D technique, even beyond the Sauerbrey regime, if frequency (f) and energy dissipation (D) measurements measured at multiple harmonics are combined with theoretical simulations using a Voight-based viscoelastic model.
Abstract: We have measured the time-resolved adsorption kinetics of the mussel adhesive protein (Mefp-1) on a nonpolar, methyl-terminated (thiolated) gold surface, using three independent techniques: quartz crystal microbalance with dissipation monitoring (QCM-D), surface plasmon resonance, and ellipsometry. The QCM-D and ellipsometry data shows that, after adsorption to saturation of Mefp-1, cross-linking of the protein layer using NaIO4 transforms it from an extended (∼20 nm), water-rich, and hydrogel-like state to a much thinner (∼5 nm), compact, and less water-rich state. Furthermore, we show how quantitative data about the thickness, shear elastic modulus, and shear viscosity of the protein film can be obtained with the QCM-D technique, even beyond the Sauerbrey regime, if frequency (f) and energy dissipation (D) measurements measured at multiple harmonics are combined with theoretical simulations using a Voight-based viscoelastic model. The modeling result was confirmed by substituting H2O for D2O. As expect...
1,126 citations
TL;DR: This electrode shows high selectivity for the response to cysteine, as compared with many common inorganic anions, salicylate, and other kinds of amino acids, and has a fast response time, micromolar detection limit, and good long-term stability.
Abstract: A novel cysteine-selective electrode based on lead phthalocyanine (PbPc) as ionophore is described. The electrode was prepared by incorporating PbPc into a plasticized poly(vinyl chloride) (PVC) membrane, which was directly coated on the surface of a graphite electrode. This electrode shows high selectivity for the response to cysteine, as compared with many common inorganic anions, salicylate, and other kinds of amino acids. The influence of membrane composition, pH, and the effect of lipophilic cationic and anionic additives on the response characteristics of the electrode were investigated. The resulting sensor demonstrates nernstian response over a wide linear range of cysteine concentration (1 × 10-6 to 5 × 10-2 M). The electrode has a fast response time, micromolar detection limit (∼1 × 10-6 M), and good long-term stability (more than 1 month). The prepared electrode was used for determination of cysteine in a synthetic human serum sample, and very good recovery results were obtained over a wide con...
958 citations
TL;DR: In this paper, the authors describe the generation of gradients having complex shapes in solution using microfluidic networks, which can be useful in both biological and nonbiological research.
Abstract: This paper describes the generation of gradients having complex shapes in solution using microfluidic networks. Flowing multiple streams of fluid each carrying different concentrations of substances laminarly and side-by-side generated step concentration gradients perpendicular to the direction of the flow. Appropriately designed networks of microchannels for controlled diffusive mixing of substances generated a range of shapes for the gradients, including linear, parabolic, and periodic. The lateral dimensions of the gradients ranged from 900 to 2200 μm. This paper also demonstrates the generation of overlapping gradients composed of different species. Since solutions in the microfluidic network exist as steady states and are continuously renewed, the gradients established in the capillaries are spatially and temporally constant and can be maintained easily for periods of hours. Using laminar flow to generate gradients should be useful in both biological and nonbiological research.
839 citations
TL;DR: Proteolytic 18O labeling enables a shotgun approach for proteomic studies with quantitation capability and is proposed as a useful tool for comparative proteomics studies of very complex protein mixtures.
Abstract: A new method for proteolytic stable isotope labeling is introduced to provide quantitative and concurrent comparisons between individual proteins from two entire proteome pools or their subfractions. Two 18O atoms are incorporated universally into the carboxyl termini of all tryptic peptides during the proteolytic cleavage of all proteins in the first pool. Proteins in the second pool are cleaved analogously with the carboxyl termini of the resulting peptides containing two 16O atoms (i.e., no labeling). The two peptide mixtures are pooled for fractionation and separation, and the masses and isotope ratios of each peptide pair (differing by 4 Da) are measured by high-resolution mass spectrometry. Short sequences and/or accurate mass measurements combined with proteomics software tools allow the peptides to be related to the precursor proteins from which they are derived. Relative signal intensities of paired peptides quantify the expression levels of their precursor proteins from proteome pools to be comp...
790 citations
TL;DR: The electrochemical behavior of a film of single-wall carbon nanotubes functionalized with carboxylic acid groups was studied extensively on a glassy carbon (GC) electrode and showed favorable electrocatalytic behavior toward the oxidation of biomolecules such as dopamine, epinephrine, and ascorbic acid.
Abstract: The electrochemical behavior of a film of single-wall carbon nanotubes (SWNTs) functionalized with carboxylic acid groups was studied extensively on a glassy carbon (GC) electrode. One stable couple corresponding to the redox of the carboxylic acid group, which was supported by XPS and IR experiments, was observed. The electrode process involved four electrons, while the rate-determining step was a one-electron reduction. The SWNT film-modified electrode showed favorable electrocatalytic behavior toward the oxidation of biomolecules such as dopamine, epinephrine, and ascorbic acid.
783 citations
TL;DR: A new molecular conjugation method has been developed to label biomolecules with optically stable metalorganic luminophores, such as tris(2,2'-bipyridyl)dichlororuthenium(II) hexahydrate (Rubpy), which are otherwise not possible for direct linking with the biomolecule.
Abstract: A new molecular conjugation method has been developed to label biomolecules with optically stable metalorganic luminophores, such as tris(2,2‘-bipyridyl)dichlororuthenium(II) hexahydrate (Rubpy), which are otherwise not possible for direct linking with the biomolecules. Unique biochemical properties of the biomolecule can, thus, be associated with photostable luminophores. This opens a general way to conjugate desired biomolecules using a sensitive signal transduction method. It also promotes the application of excellent luminescent materials, especially those based on photostable metalorganic luminophores, in biochemical analysis and biomolecular interaction studies. The conjugation method is based on uniform luminophore-doped silica (LDS) nanoparticles (63 ± 4 nm). These nanoparticles have been prepared using a water-in-oil (W/O) microemulsion method. The controlled hydrolysis of tetraethyl orthosilicate (TEOS) in W/O microemulsion leads to the formation of monodisperse LDS nanoparticles. The luminophor...
758 citations
TL;DR: A method has been developed for the trace analysis of two classes of antimicrobials consisting of six sulfonamides and five tetracyclines, which commonly are used for veterinary purposes and agricultural feed additives and are suspected to leach into ground and surface water.
Abstract: A method has been developed for the trace analysis of two classes of antimicrobials consisting of six sulfonamides (SAs) and five tetracyclines (TCs), which commonly are used for veterinary purposes and agricultural feed additives and are suspected to leach into ground and surface water. The method used solid-phase extraction and liquid chromatography/mass spectrometry (LC/MS) with positive ion electrospray. The unique combination of a metal chelation agent (Na2EDTA) with a macroporous copolymer resulted in quantitative recoveries by solid-phase extraction (mean recovery, 98 ± 12%) at submicrogram-per-liter concentrations. An ammonium formate/formic acid buffer with a methanol/water gradient was used to separate the antimicrobials and to optimize the signal intensity. Mass spectral fragmentation and ionization characteristics were determined for each class of compounds for unequivocal identification. For all SAs, a characteristic m/z 156 ion representing the sulfanilyl fragment was identified. TCs exhibit...
721 citations
TL;DR: It is possible to break a broadband mass spectrum into 1-Da segments, rotate each segment by 90 degrees, scale each segment according to its mass defect, and compress the spacing between the segments to yield a compact display, illustrated for experimental electrospray ionization FTICR ultrahigh-resolution mass spectra of a petroleum crude oil.
Abstract: At currently achievable Fourier transform ion cyclotron resonance broadband mass spectrometry resolving power (m/Δm50% > 350 000 for 200 < m/z < 1000), it would be necessary to spread out a conventional mass spectrum over ∼200 m in order to provide visual resolution of the most closely resolved peaks. Fortunately, there are natural gaps in a typical mass spectrum, spaced 1 Da apart, because virtually no commonly encountered elemental compositions yield masses at those values. Thus, it is possible to break a broadband mass spectrum into 1-Da segments, rotate each segment by 90°, scale each segment according to its mass defect (i.e., difference between exact and nominal mass), and then compress the spacing between the segments to yield a compact display. For hydrocarbon systems, conversion from IUPAC mass to “Kendrick” mass (i.e., multiplying each mass by 14.00000/14.01565) further simplifies the display by rectilinearizing the peak patterns. The resulting display preserves not only the “coarse” spacings (e...
705 citations
TL;DR: Surface plasmon resonance imaging is used to quantitatively detect the hybridization adsorption of short (18-base) unlabeled DNA oligonucleotides at low concentration, as well as, for the first time, the hybridized adsorbed RNA oligon nucleotides and larger 16S ribosomal RNA isolated from the microbe Escherichia coli onto a DNA array.
Abstract: Surface plasmon resonance (SPR) imaging is a surface-sensitive spectroscopic technique for measuring interactions between unlabeled biological molecules with arrays of surface-bound species. In this paper, SPR imaging is used to quantitatively detect the hybridization adsorption of short (18-base) unlabeled DNA oligonucleotides at low concentration, as well as, for the first time, the hybridization adsorption of unlabeled RNA oligonucleotides and larger 16S ribosomal RNA (rRNA) isolated from the microbe Escherichia coli onto a DNA array. For the hybridization adsorption of both DNA and RNA oligonucleotides, a detection limit of 10 nM is reported; for large (1500-base) 16S rRNA molecules, concentrations as low as 2 nM are detected. The covalent attachment of thiol-DNA probes to the gold surface leads to high surface probe density (1012 molecules/cm2) and excellent probe stability that enables more than 25 cycles of hybridization and denaturing without loss in signal or specificity. Fresnel calculations are...
TL;DR: This device demonstrates the most sensitive PCR possible in a microfabricated device and will also facilitate single-cell and single-molecule studies to expose the genetic variation underlying ensemble sequence and expression averages.
Abstract: Stochastic PCR amplification of single DNA template molecules followed by capillary electrophoretic (CE) analysis of the products is demonstrated in an integrated microfluidic device. The microdevice consists of submicroliter PCR chambers etched into a glass substrate that are directly connected to a microfabricated CE system. Valves and hydrophobic vents provide controlled and sensorless loading of the 280-nL PCR chambers; the low volume reactor, the low thermal mass, and the use of thin-film heaters permit cycle times as fast as 30 s. The amplified product, labeled with an intercalating fluorescent dye, is directly injected into the gel-filled capillary channel for electrophoretic analysis. Repetitive PCR analyses at the single DNA template molecule level exhibit quantized product peak areas; a histogram of the normalized peak areas reveals clusters of events caused by 0, 1, 2, and 3 viable template copies in the reactor and these event clusters are shown to fit a Poisson distribution. This device demon...
TL;DR: The efficacy of the method is demonstrated by measuring temperature distributions resulting from Joule heating in a variety of microfluidic circuits that are electrokinetically pumped.
Abstract: A technique is described for the measurement of fluid temperatures in microfluidic systems based on temperature-dependent fluorescence. The technique is easy to implement with a standard fluorescence microscope and CCD camera. In addition, the method can be used to measure fluid temperatures with micrometer spatial resolution and millisecond time resolution. The efficacy of the method is demonstrated by measuring temperature distributions resulting from Joule heating in a variety of microfluidic circuits that are electrokinetically pumped. With the equipment used for these measurements, fluid temperatures ranging from room temperature to 90 °C were measured with a precision ranging from 0.03 to 3.5 °Cdependent on the amount of signal averaging done. The spatial and temporal resolutions achieved were 1 μm and 33 ms, respectively.
TL;DR: A "MS BLAST" homology searching protocol was developed to overcome specific limitations imposed by mass spectrometric data, such as the limited accuracy of de novo sequence predictions.
Abstract: MALDI-quadrupole time-of-flight mass spectrometry was applied to identify proteins from organisms whose genomes are still unknown. The identification was carried out by successively searching a sequence database-first with a peptide mass fingerprint, then with a packet of noninterpreted MS/MS spectra, and finally with peptide sequences obtained by automated interpretation of the MS/MS spectra. A "MS BLAST" homology searching protocol was developed to overcome specific limitations imposed by mass spectrometric data, such as the limited accuracy of de novo sequence predictions. This approach was tested in a small-scale proteomic project involving the identification of 15 bands of gel-separated proteins from the methylotrophic yeast Pichia pastoris, whose genome has not yet been sequenced and which is only distantly related to other fungi.
TL;DR: The new electrochemical stripping metallogenomagnetic protocol couples the inherent signal amplification of stripping metal analysis with discrimination against nonhybridized DNA, the use of microliter sample volumes, and disposable transducers and, hence, offers great promise for decentralized genetic testing.
Abstract: A new nanoparticle-based electrical detection of DNA hybridization, based on electrochemical stripping detection of the colloidal gold tag, is described. In this protocol, the hybridization of a target oligonucleotide to magnetic bead-linked oligonucleotide probes is followed by binding of the streptavidin-coated metal nanoparticles to the captured DNA, dissolution of the nanometer-sized gold tag, and potentiometric stripping measurements of the dissolved metal tag at single-use thick-film carbon electrodes. An advanced magnetic processing technique is used to isolate the DNA duplex and to provide low-volume mixing. The influence of relevant experimental variables, including the amounts of the gold nanoparticles and the magnetic beads, the duration of the hybridization and gold dissolution steps, and the parameters of the potentiometric stripping operation upon the hybridization signal, is examined and optimized. Transmission electron microscopy micrographs indicate that the hybridization event leads to t...
TL;DR: For DC18C6 in 1-octyl-3-methylimidazolium hexafluorophosphate, the alkali metal cation extraction selectivity and efficiency were unaffected by variation of the aqueous-phase anion from chloride to nitrate to sulfate.
Abstract: An improved method for the preparation of 1-alkyl-3-methylimidazolium hexafluorophosphates provides a series of room-temperature ionic liquids (RTILs) in which the 1-alkyl group is varied systematically from butyl to nonyl. For competitive solvent extraction of aqueous solutions of alkali metal chlorides with solutions of dicyclohexano-18-crown-6 (DC18C6) in these RTILs, the extraction efficiency generally diminished as the length of the 1-alkyl group was increased. Under the same conditions, extraction of alkali metal chlorides into solutions of DC18C6 in chloroform, nitrobenzene, and 1-octanol was undetectable. The extraction selectivity order for DC18C6 in the RTILs was K+ > Rb+ > Cs+ > Na+ > Li+. As the alkyl group in the RTIL was elongated, the K+/Rb+ and K+/Cs+ selectivities exhibited general increases with the larger enhancement for the latter. For DC18C6 in 1-octyl-3-methylimidazolium hexafluorophosphate, the alkali metal cation extraction selectivity and efficiency were unaffected by variation of...
TL;DR: Whether their high solubalizing power, negligible vapor pressure, and broad liquid temperature range are advantageous if they are used as matrixes for UV-MALDI is examined.
Abstract: Room-temperature ionic liquids are useful as solvents for organic synthesis, electrochemical studies, and separations. We wished to examine whether their high solubalizing power, negligible vapor pressure, and broad liquid temperature range are advantageous if they are used as matrixes for UV-MALDI. Several different ionic matrixes were synthesized and tested, using peptides, proteins, and poly(ethylene glycol) (PEG-2000). All ionic liquids tested have excellent solubilizing properties and vacuum stability compared to other commonly used liquid and solid matrixes. However, they varied widely in their ability to produce analyte gas-phase ions. Certain ionic matrixes, however, produce homogeneous solutions of greater vacuum stability, higher ion peak intensity, and equivalent or lower detection limits than currently used solid matrixes. Clearly, ionic liquids and their more amorphous solid analogues merit further investigation as MALDI matrixes.
TL;DR: The ability of the LF isotherm to model MIPs suggests that a unimodal heterogeneous distribution is an accurate approximation of the distribution found in homogeneous and heterogeneous MIPS.
Abstract: The majority of binding models that have been applied to molecularly imprinted polymers (MIPs) have been homogeneous models. MIPs, on the other hand, are heterogeneous materials containing binding sites with a wide array of binding affinities and selectivities. Demonstrated is that the binding behavior of MIPs can be accurately modeled by the heterogeneous Langmuir-Freundlich (LF) isotherm. The applicability of the LF isotherm to MIPs was demonstrated using five representative MIPs from the literature, including both homogeneous and heterogeneous MIPs. Previously, such comparisons required the use of several different binding models and analyses, including the Langmuir model, the Freundlich model, and numerical approximation techniques. In contrast, the LF model enabled direct comparisons of the binding characteristics of MIPs that have very different underlying distributions and were measured under different conditions. The binding parameters can be calculated directly using the LF fitting coefficients that yield a measure of the total number of binding sites, mean binding affinity, and heterogeneity. Alternatively, solution of the Langmuir adsorption integral for the LF model enabled direct calculation of the corresponding affinity spectrum from the LF fitting coefficients from a simple algebraic expression, yielding a measure of the number of binding sites with respect to association constant Finally, the ability of the LF isotherm to model MIPs suggests that a unimodal heterogeneous distribution is an accurate approximation of the distribution found in homogeneous and heterogeneous MIPs.
TL;DR: The device was used to detect the separation of a mixture of proteins and small molecules by capillary electrophoresis; the separation illustrated the suitability of this integrated fluorescence detection system for bioanalytical applications.
Abstract: This paper describes a prototype of an integrated fluorescence detection system that uses a microavalanche photodiode (μAPD) as the photodetector for microfluidic devices fabricated in poly(dimethylsiloxane) (PDMS). The prototype device consisted of a reusable detection system and a disposable microfluidic system that was fabricated using rapid prototyping. The first step of the procedure was the fabrication of microfluidic channels in PDMS and the encapsulation of a multimode optical fiber (100-μm core diameter) in the PDMS; the tip of the fiber was placed next to the side wall of one of the channels. The optical fiber was used to couple light into the microchannel for the excitation of fluorescent analytes. The photodetector, a prototype solid-state μAPD array, was embedded in a thick slab (1 cm) of PDMS. A thin (80 μm) colored polycarbonate filter was placed on the top of the embedded μAPD to absorb scattered excitation light before it reached the detector. The μAPD was placed below the microchannel an...
TL;DR: Results indicate that conventional environmental risk assessment overestimates FQ concentrations in surface waters by 1 to 2 orders of magnitude.
Abstract: Fluoroquinolones (FQs) are among the most important antibacterial agents (synthetic antibiotics) used in human and veterinary medicine. An analytical method based on reversed-phase liquid chromatography with fluorescence detection was developed and validated for the simultaneous determination of nine FQs and the quinolone pipemidic acid in urban wastewater. Aqueous samples were extracted using mixed-phase cation-exchange disk cartridges that were subsequently eluted by ammonia solution in methanol. Recoveries were above 80% at an overall precision of better than 10%. Instrumental quantification limits varied between 150 and 450 pg injected. The presented method was successfully applied to quantify FQs in effluents of urban wastewater treatment plants. The two most abundant human-use FQs, ciprofloxacin and norfloxacin, occurred in primary and tertiary wastewater effluents at concentrations between 249 and 405 ng/L and from 45 to 120 ng/L, respectively. The identity of FQs in urban wastewater was confirmed ...
TL;DR: The discrimination of DNA mismatches is demonstrated using an elegantly simple microcantilever-based optical deflection assay, without the need for external labeling, that is readily adaptable to a high-throughput array format and provides a distinct positive/negative signal for easy interpretation of oligonucleotide hybridization.
Abstract: Characterization of single-nucleotide polymorphisms is a major focus of current genomics research. We demonstrate the discrimination of DNA mismatches using an elegantly simple microcantilever-based optical deflection assay, without the need for external labeling. Gold-coated silicon AFM cantilevers were functionalized with thiolated 20- or 25-mer probe DNA oligonucleotides and exposed to target oligonucleotides of varying sequence in static and flow conditions. Hybridization of 10-mer complementary target oligonucleotides resulted in net positive deflection, while hybridization with targets containing one or two internal mismatches resulted in net negative deflection. Mismatched targets produced a stable and measurable signal when only a four-base pair stretch was complementary to the probe sequence. This technique is readily adaptable to a high-throughput array format and provides a distinct positive/negative signal for easy interpretation of oligonucleotide hybridization.
TL;DR: The function of the monolithic concentration device was first demonstrated using very dilute solutions of Coumarin 519 and the performance was then demonstrated with the enrichment of a hydrophobic tetrapeptide and also of green fluorescent protein for which an increase in concentration by a factor as high as 10(3) was achieved.
Abstract: Monolithic porous polymers have been prepared by photoinitiated polymerization within the channels of a microfluidic device and used for on-chip solid-phase extraction and preconcentration. The preparation of the monolithic material with hydrophobic and ionizable surface chemistries is easily achieved by copolymerization of butyl methacrylate with ethylene dimethacrylate, or 2-hydroxyethyl methacrylate and [2-(methacryloyloxy)ethyl]trimethylammonium chloride with ethylene dimethacrylate, respectively. The porous properties, and consequently the flow resistance, of the monolithic device are controlled by the use of a mixture of hexane and methanol as a porogenic mixture. This mixture was designed to meet the specific requirements for pore formation within macroporous monoliths useful in the microfluidic formats. The low flow resistance enables high flow rates of up to 10 μL/min, which corresponds to a linear flow velocity of 50 mm/s and far exceeds the flow velocities typical of the common analytical micro...
TL;DR: The applicability of ECD for glycopeptide analysis to N-glycosylated peptides and to peptides containing branched, highly substituted glycans is extended and the two types of spectra, obtained with the same instrument, provide complementary structural information about the glycopePTide.
Abstract: Glycoproteins are a functionally important class of biomolecules for which structural elucidation presents a challenge. Fragmentation of N-glycosylated peptides, employing collisionally activated dissociation, typically yields product ions that result from dissociation at glycosidic bonds, with little occurrence of dissociation at peptide backbone sites. We have applied two dissociation techniques, electron capture dissociation (ECD) and infrared multiphoton dissociation (IRMPD), in a 7-T Fourier transform ion cyclotron resonance mass spectrometer, in the investigation of an N-glycosylated peptide from an unfractionated tryptic digest of the lectin of the coral tree, Erythrina corallodendron. ECD provided c and z• ions derived from the peptide backbone, with no observed loss of sugars. Cleavage at 11 of 15 backbone amine bonds was observed. The lack of cleavage at sites located close to the glycosylated asparagine residue may result from steric blocking by the glycan. IRMPD provided abundant fragment ions...
TL;DR: Easy preparation of the MIPs, their high stability, and their ability to recognize small and large proteins, as well as to discriminate molecules with small variations in charge, make this approach attractive and broadly applicable in biotechnology, assays and sensors.
Abstract: A technique for coating microplate wells with molecularly imprinted polymers (MIPs) specific for proteins is presented. 3-Aminophenylboronic acid was polymerized in the presence of the following templates: microperoxidase, horseradish peroxidase, lactoperoxidase, and hemoglobin, via oxidation of the monomer by ammonium persulfate. This process resulted in the grafting of a thin polymer layer to the polystyrene surface of the microplates. Imprinting resulted in an increased affinity of the polymer toward the corresponding templates. The influence of the washing procedure, template concentration, and buffer pH on the polymer affinity was analyzed. It was shown that the stabilizing function of the support and spatial orientation of the polymer chains and template functional groups are the major factors affecting the imprint formation and template recognition. Easy preparation of the MIPs, their high stability, and their ability to recognize small and large proteins, as well as to discriminate molecules with small variations in charge, make this approach attractive and broadly applicable in biotechnology, assays and sensors.
TL;DR: This microchip-based diagnosis system is the first micro chip-based system that is practically useful for clinical diagnoses with short analysis time, high sensitivity, and easy procedures.
Abstract: A bead-bed immunoassay system was structured on a microchip and applied to determine carcinoembryonic antigen (CEA), which is a commonly used marker of colon cancer. Polystyrene beads precoated with anti-CEA antibody were introduced into a microchannel, and then a serum sample containing CEA, the first antibody, and the second antibody conjugated with colloidal gold were reacted successively. The resulting antigen−antibodies complex, fixed on the bead surface, was detected using a thermal lens microscope (TLM). A highly selective and sensitive determination of an ultratrace amount of CEA in human sera was made possible by a sandwich immunoassay system that needs three antibodies for an assay. A detection limit dozens of times lower than the conventional ELISA was achieved. Moreover, when serum samples for 13 patients were assayed with this system, there was a high correlation (r = 0.917) with the conventional ELISA. The integration reduced the time necessary for the antigen−antibody reaction to ∼1%, thus ...
TL;DR: The firstsol-gel-based, ratiometric, optical nanosensors, or sol-gel probes encapsulated by biologically localized embedding (PEBBLEs), are made and demonstrated here to enable reliable, real-time measurements of subcellular molecular oxygen.
Abstract: The first sol−gel-based, ratiometric, optical nanosensors, or sol−gel probes encapsulated by biologically localized embedding (PEBBLEs), are made and demonstrated here to enable reliable, real-time measurements of subcellular molecular oxygen. Sensors were made using a modified Stober method, with poly(ethylene glycol) as a steric stabilizer. The radii of these spherical PEBBLE sensors range from about 50 to 300 nm. These sensors incorporate an oxygen-sensitive fluorescent indicator, Ru(II)−tris(4,7-diphenyl-1,10-phenanthroline) chloride ([Ru(dpp)3]2+), and an oxygen-insensitive fluorescent dye, Oregon Green 488-dextran, as a reference for the purpose of ratiometric intensity measurements. The PEBBLE sensors have excellent reversibility, dynamic range, and stability to leaching and photobleaching. The small size and inert matrix of these sensors allow them to be inserted into living cells with minimal physical and chemical perturbations to their biological functions. Applications of sol−gel PEBBLEs insert...
TL;DR: This study characterizes various features of the proteins that are detected in MALDI mass spectra when whole bacteria cells are analyzed, in an effort to understand why some proteins are successfully detected and many others are not.
Abstract: This study characterizes various features of the proteins that are detected in MALDI mass spectra when whole bacteria cells are analyzed, in an effort to understand why some proteins are successfully detected and many others are not. Forty peaks observed in the mass range 4,000-20,000 Da in the spectra of Escherichia coli K-12 and 11775 are tentatively assigned to proteins in a protein database, and these proteins are characterized by cell location, copy number, pI, and hydropathicity. Those detected originate in the cytosol and generally share the traits of high abundance within the cell, strong bacisity, and medium hydrophilicity.
TL;DR: The PhIAT labeling technique provides a reliable method for making quantitative measurements of differences in the O-phosphorylation state of proteins.
Abstract: A method has been developed that utilizes phosphoprotein isotope-coded affinity tags (PhIAT) that combines stable isotope and biotin labeling to enrich and quantitatively measure differences in the O-phosphorylation states of proteins. The PhIAT labeling approach involves hydroxide ion-mediated β-elimination of the O-phosphate moiety and the addition of 1,2-ethanedithiol containing either four alkyl hydrogens (EDT-D0) or four alkyl deuteriums (EDT-D4) followed by biotinylation of the EDT-D0/D4 moiety using (+)-biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine. The PhIAT reagent, which contains the nucleophilic sulfhydryl and isotopic label covalently linked to a biotin moiety, was synthesized and has the potential utility to reduce the O-phosphorylation derivatization into a one-step process. The PhIAT labeling approach was initially demonstrated using the model phosphoprotein β-casein. After proteolytic digestion, the PhIAT-labeled peptides were affinity isolated using immobilized avidin and analyzed using ...
TL;DR: Porous silicon surfaces optimized for DIOS response were examined for their applicability to quantitative analysis, organic reaction monitoring, post-source decay mass spectrometry, and chromatography.
Abstract: Desorption/ionization on porous silicon mass spectrometry (DIOS-MS) is a novel method for generating and analyzing gas-phase ions that employs direct laser vaporization. The structure and physicochemical properties of the porous silicon surfaces are crucial to DIOS-MS performance and are controlled by the selection of silicon and the electrochemical etching conditions. Porous silicon generation and DIOS signals were examined as a function of silicon crystal orientation, resistivity, etching solution, etching current density, etching time, and irradiation. Pre- and postetching conditions were also examined for their effect on DIOS signal as were chemical modifications to examine stability with respect to surface oxidation. Pore size and other physical characteristics were examined by scanning electron microscopy and Fourier transform infrared spectroscopy, and correlated with DIOS-MS signal. Porous silicon surfaces optimized for DIOS response were examined for their applicability to quantitative analysis, ...