Picomole Amounts Research Articles

Highly sensitive salicylic fluorophore for visual detection of picomole amounts of Cu2+

Four new fluorophores containing multiple fluorogenic branches of phenylene-ethynylene and salicylic acid Cu2+ binding sites are synthesized and evaluated as Cu2+ sensors in aqueous media. The fluorophore, F4, having a triphenylamine core and three fluorogenic branches, possesses superior sensitivity, selectivity and emission properties for fluorescence quenching detection of Cu2+. This fluorophore exhibits the highest Cu2+ quenching sensitivity in aqueous solution; Ksv = 5.79 × 106 M−1. The relatively high fluorescence emission of this fluorophore provides excellent visual contrast in a solid state paper-based sensor that enables naked eye detection of Cu2+ at the picomole level.

Label-free colorimetric detection of picomolar amounts of hydrazine using a gold nanoparticle-based assay

We report the development of a simple and rapid colorimetric detection method for hydrazine in boiler feed water using label-free aggregation-based gold nanoparticles as probe. This assay relies upon the distance-dependent optical properties of gold nanoparticles. Hydrazine could rapidly induce the aggregation of gold nanoparticles, thereby resulting in visual color change from red to blue (or purple). The concentration of hydrazine can be determined by the naked eye or by a UV–vis spectrometer. Calibration curve was linear ranging from 1.0 × 10−11 to 1.0 × 10−7 M of hydrazine. The present limit of detection for hydrazine was 1.0 × 10−12 M. The method is rather simple, and the whole process including sample pretreatment takes only 15 min at room temperature. The merits (such as simplicity, rapidity, low cost and visual colorimetry) make the proposed method especially useful for on-site screening of hydrazine levels well in boiler feed water.

Multiplexed Enrichment and Detection of Malarial Biomarkers Using a Stimuli-Responsive Iron Oxide and Gold Nanoparticle Reagent System

There is a need for simple yet robust biomarker and antigen purification and enrichment strategies that are compatible with current rapid diagnostic modalities. Here, a stimuli-responsive nanoparticle system is presented for multiplexed magneto-enrichment and non-instrumented lateral flow strip detection of model antigens from spiked pooled plasma. The integrated reagent system allows purification and enrichment of the gold-labeled biomarker half-sandwich that can be applied directly to lateral flow test strips. A linear diblock copolymer with a thermally responsive poly(N-isopropylacrylamide) (pNIPAm) segment and a gold-binding block composed of NIPAm-co-N,N-dimethylaminoethylacrylamide was prepared by reversible addition-fragmentation chain transfer polymerization. The diblock copolymer was used to functionalize gold nanoparticles (AuNPs), with subsequent bioconjugation to yield thermally responsive pNIPAm-AuNPs that were co-decorated with streptavidin. These AuNPs efficiently complexed biotinylated capture antibody reagents that were bound to picomolar quantities of pan-aldolase and Plasmodium falciparum histidine-rich protein 2 (PfHRP2) in spiked pooled plasma samples. The gold-labeled biomarker half-sandwich was then purified and enriched using 10 nm thermally responsive magnetic nanoparticles that were similarly decorated with pNIPAm. When a thermal stimulus was applied in conjunction with a magnetic field, coaggregation of the AuNP half-sandwiches with the pNIPAm-coated iron oxide nanoparticles created large aggregates that were efficiently magnetophoresed and separated from bulk serum. The purified biomarkers from a spiked pooled plasma sample could be concentrated 50-fold into a small volume and applied directly to a commercial multiplexed lateral flow strip to dramatically improve the signal-to-noise ratio and test sensitivity.

Positron emission tomography detection of human endothelial cell and fibroblast monolayers: effect of pretreament and cell density on 18FDG uptake

BackgroundThe non-destructive assessment and characterization of tridimensional (3D) cell and tissue constructs in bioreactors represents a challenge in tissue engineering. Medical imaging modalities, which can provide information on the structure and function of internal organs and tissues in living organisms, have the potential of allowing repetitive monitoring of these 3D cultures in vitro. Positron emission tomography (PET) is the most sensitive non-invasive imaging modality, capable of measuring picomolar amounts of radiolabeled molecules. However, since PET imaging protocols have been designed almost exclusively for in vivo investigations, suitable methods must be devised to enable imaging of cells or tissue substitutes. As a prior step to imaging 3D cultures, cell radiotracer uptake conditions must first be optimized.MethodsIn this study, human umbilical vein endothelial cells (HUVEC) and human fibroblasts were cultured at different densities and PET was used to non-destructively monitor their glycolytic activity by measuring 18F-fluorodeoxyglucose (18FDG) uptake. Various cell preconditioning protocols were investigated by adjusting the following parameters to optimize 18FDG uptake: glucose starvation, insulin stimulation, glucose concentration, 18FDG incubation time, cell density and radiotracer efflux prevention.ResultsThe conditions yielding optimal 18FDG uptake, and hence best detection sensitivity by PET, were as follows: 2-hour cell preconditioning by glucose deprivation with 1-hour insulin stimulation, followed by 1-hour 18FDG incubation and 15-minute stabilization in standard culture medium, prior to rinsing and PET scanning.ConclusionsA step-wise dependence of 18FDG uptake on glucose concentration was found, but a linear correlation between PET signal and cell density was observed. Detection thresholds of 36 ± 7 and 21 ± 4 cells were estimated for endothelial cells and fibroblasts, respectively.

Platelet Biomarkers in Tumor Growth

As the clinical use of biologic response modifiers continues to increase, the old way of estimating effective doses in oncology, i.e. reaching dose-limiting toxicities, is becoming obsolete. Biologic response modifiers and targeted therapies are less toxic and their effective dose is often left shifted on the dose response curve. This is why the majority of pharmaceutical companies have opened new programs seeking biomarker of therapeutic response, and why numerous research programs have been announcing Request for Applications in order to find and evaluate biomarkers of disease. Because of the ease of procurement of the clinical specimen, plasma and serum, remain the favorite clinical analytes. However, the sheer numbers of different plasma proteins, the many thousand fold differences in the amounts of the potential protein biomarkers, and the lack of specificity of plasma proteins have hindered the search. The ability to detect changes in the levels of proteins expressed in picomolar quantities are mired by the presence of more abundant nonspecific proteins such as albumin or immunoglobulins. Most meaningful changes, those that occur in the amounts of free proteins, remain very difficult to evaluate. The recent discovery that angiogenesis regulators are actively and selectively sequestered in platelets early in cancer, has led to renewed hopes of finding circulating biomarkers that would help in early disease detection, and improve our ability to detect an early therapeutic response. There are early indicators that this is the case in other diseases as well, and that the focus may be shifting from analyzing plasma and serum, to analyzing platelets.

In Vitro Detection and Quantification of Botulinum Neurotoxin Type E Activity in Avian Blood

Botulinum neurotoxin serotype E (BoNT/E) outbreaks in the Great Lakes region cause large annual avian mortality events, with an estimated 17,000 bird deaths reported in 2007 alone. During an outbreak investigation, blood collected from bird carcasses is tested for the presence of BoNT/E using the mouse lethality assay. While sensitive, this method is labor-intensive and low throughput and can take up to 7 days to complete. We developed a rapid and sensitive in vitro assay, the BoTest Matrix E assay, that combines immunoprecipitation with high-affinity endopeptidase activity detection by Förster resonance energy transfer (FRET) to rapidly quantify BoNT/E activity in avian blood with detection limits comparable to those of the mouse lethality assay. On the basis of the analysis of archived blood samples (n = 87) collected from bird carcasses during avian mortality investigations, BoTest Matrix E detected picomolar quantities of BoNT/E following a 2-h incubation and femtomolar quantities of BoNT/E following extended incubation (24 h) with 100% diagnostic specificity and 91% diagnostic sensitivity.

A targeted mass spectrometry-based metabolomics strategy of human blastocoele fluid: a promising tool in fertility research

We describe a fast HPLC-MS-based metabolomic approach which enables detection, validation and quantitation of metabolites of biological interest for embryo development from blastocoele fluid down to the picomole quantities, in order to determine the correlation between the metabolic levels and blastocyst inplantation. We show how the sensitivity of the proposed mass spectrometry MS-based approach allows for direct measurement in blastocoele fluid of a handful of metabolites relevant for embryo development, as reported in the literature, with no harm to the blastocyst and without disrupting the clinical pipeline prior to vitrification. Indeed, samples as low as 0.5nl, which routinely represent a waste product of the vitrification process of blastocyst prior to cryostorage, can be processed for metabolite detection through rapid resolution reversed phase RR-RP high performance liquid chromatography HPLC-MS. 1 Blastocoele fluid collection 2 Metabolite extraction 3 Rapid resolution reversed-phase HPLC 4 ESI mass spectrometry From sample volumes as low as 0.5 nl we could detect and quantify against external standards a group of metabolites, whose roles in blastocyst development and embryo metabolism have long been postulated. The list included ATP, glucose-6-phosphate; lactate; NAD+, NADH and NADPH; 6-phosphogluconic acid; glutamic acid and α-ketoglutarate. Our method allowed detection and quantitation of glutamate and ketoglutarate down to picomole quantities 1.35and13.70 injected pmol, respectively. The high sensitivity of the MS analysis on the blastocoele fluid enables to gather direct information on embryo metabolism, in contrast with the approaches adopted so far which aim at indirectly determining metabolite levels from the culture media or rather directly through disruptive analysis on the whole blastocyst.

HPLC with In-Capillary Optical Fiber Laser-Induced Fluorescence Detection of Picomolar Amounts of Amino Acids by Precolumn Fluorescence Derivatization with Fluorescein Isothiocyanate

In this paper, a fluorescein isothiocyanate (FITC) precolumn derivatization technique in conjunction with an HPLC-in-capillary optical fiber laser-induced fluorescence (HPLC-ICOF-LIF) detection method has been developed for determination of amino acids. The HPLC separation of FITC-labeled amino acids and the ICOF-LIF detection system are studied and optimized. Optimum separation conditions were obtained with a gradient elution program of acetonitrile and phosphate buffer (10 mM, pH 6.8). The ICOF-LIF detection system comprises a 530-μm capillary and a 380-μm optical fiber. The analyses of amino acids display excellent linear relationship between peak area and concentration with correlation coefficients greater than 0.999 and the method also provides good repeatability with RSD < 3%. The detection limits for FITC-tagged amino acids are very low and the lowest LOD for tyrosine is 51 pM. The proposed method has been successfully applied to determination of amino acids in human serum. Our developed HPLC-ICOF-LIF system is cheap, simple, stable, and sensitive which is potentially useful for the formulation analysis and bioanalysis.

Bioanalysis of picomole amounts of acetylcholine by ion-pair partition chromatography applied to rat sciatic nerve

A method for determination of acetylcholine in small, discrete biological objects by use of ion-pair technique has been developed. Acetylcholine is extracted as an ion pair with 3,5-di-t-butyl-2-hydroxybenzenesulphonate and separated from co-extracted components by ion-pair partition chromatography with picrate as the counter ion and porous cellulose as support. The quantitative evaluation is made from the acetylcholine peak in the chromatogram obtained by ultraviolet detection. Acetylcholine has been analysed in 1 cm large pieces of rat sciatic nerve containing about 60 pmol (10 ng). The overall recovery of the method is 100 +/- 10% at the 120 pmol level of acetylcholine in a sample.

Ultrasensitive characterization of site-specific glycosylation of affinity-purified haptoglobin from lung cancer patient plasma using 10 μm i.d. porous layer open tubular liquid chromatography-linear ion trap collision-induced dissociation/electron transfer dissociation mass spectrometry.

Site-specific analysis of protein glycosylation is important for biochemical and clinical research efforts. Glycopeptide analysis using liquid chromatography-collision-induced dissociation/electron transfer dissociation mass spectrometry (LC-CID/ETD-MS) allows simultaneous characterization of the glycan structure and attached peptide site. However, due to the low ionization efficiency of glycopeptides during electrospray ionization, 200-500 fmol of sample per injection is needed for a single LC-MS run, which makes it challenging for the analysis of limited amounts of glycoprotein purified from biological matrixes. To improve the sensitivity of LC-MS analysis for glycopeptides, an ultranarrow porous layer open tubular (PLOT) LC column (2.5 m × 10 μm i.d.) was coupled to a linear ion trap (LTQ) collision-induced dissociation/electron transfer dissociation mass spectrometer to provide sensitive analysis of N-linked protein glycosylation heterogeneity. The potential of the developed method is demonstrated by the characterization of site-specific glycosylation using haptoglobin (Hpt) as a model protein. To limit the amount of haptoglobin to low picomole amounts of protein, we affinity purified it from 1 μL of pooled lung cancer patient plasma. A total of 26 glycoforms/glycan compositions on three Hpt tryptic glycopeptides were identified and quantified from 10 LC-MS runs with a consumption of 100 fmol of Hpt digest (13 ng of protein, 10 fmol per injection). Included in this analysis was the determination of the glycan occupancy level. At this sample consumption level, the high sensitivity of the PLOT LC-LTQ-CID/ETD-MS system allowed glycopeptide identification and structure determination, along with relative quantitation of glycans presented on the same peptide backbone, even for low abundant glycopeptides at the ∼100 amol level. The PLOT LC-MS system is shown to have sufficient sensitivity to allow characterization of site-specific protein glycosylation from trace levels of glycosylated proteins.

Spontaneous Waveguide Raman Spectroscopy of Self-Assembled Monolayers in Silica Micropores

Advances in nanoscience are critically dependent on the ability to control and probe chemical and physical phenomena in confined geometries. A key challenge is to identify confinement structures with high surface area to volume ratios and controlled surface boundaries that can be probed quantitatively at the molecular level. Herein we report an approach for probing molecular structures within nano- to microscale pores by the application of spontaneous Raman spectroscopy. We demonstrate the method by characterization of the structural features of picomole quantities of well-organized octadecyltrichlorosilane (OTS) monolayers self-assembled on the interior pore surfaces of high aspect ratio (1 μm diameter × 1-10 cm length), near-atomically smooth silica microstructured optical fibers (MOFs). The simple Raman backscattering collection geometry employed is well suited for a wide variety of diagnostic applications as demonstrated by tracking the combustion of the hydrocarbon chains of the OTS self-assembled monolayer (SAM) and spectral confirmation of the formation of an adsorbed monolayer of human serum albumin (HSA) protein. Using this MOF Raman approach, molecular processes in precisely defined, highly confined geometries can be probed at high pressures and temperatures, with a wide range of excitation wavelengths from the visible to the near-IR, and under other external perturbations such as electric and magnetic fields.

A label-free biosensor assay for botulinum neurotoxin B in food and human serum

Botulinum neurotoxins (BoNTs) are among the most toxic substances known. Surveillance and diagnostics require methods for rapid detection of BoNTs in complex media such as foodstuffs and human serum. We have developed in vitro assays to specifically detect the protease activity of botulinum neurotoxin B (BoNT/B) on a time scale of minutes. Cleavage of the BoNT/B substrate VAMP2, a membrane SNARE protein associated with synaptic vesicles, was monitored using real-time surface plasmon resonance to measure vesicle capture by specific antibodies coupled to microchips. The assay is functional in low-ionic-strength buffers and stable over a wide range of pH values (5.5–9.0). Endoproteolytic cleavage of VAMP2 was detected in 10min with 2pM native BoNT/B holotoxin. Contamination of liquid food products such as carrot juice, apple juice, and milk with low picomolar amounts of BoNT/B was revealed within 3h. BoNT/B activity was detected in sera from patients with type B botulism but not in healthy controls or patients with other neurological diseases. This robust, sensitive, and rapid protein chip assay is appropriate for monitoring BoNT/B in food products and diagnostic tests for type B botulism and could replace the current in vivo mouse bioassay.

Quantifying the Labeling and the Levels of Plant Cell Wall Precursors Using Ion Chromatography Tandem Mass Spectrometry

The biosynthesis of cell wall polymers involves enormous fluxes through central metabolism that are not fully delineated and whose regulation is poorly understood. We have established and validated a liquid chromatography tandem mass spectrometry method using multiple reaction monitoring mode to separate and quantify the levels of plant cell wall precursors. Target analytes were identified by their parent/daughter ions and retention times. The method allows the quantification of precursors at low picomole quantities with linear responses up to the nanomole quantity range. When applying the technique to Arabidopsis (Arabidopsis thaliana) T87 cell cultures, 16 hexose-phosphates (hexose-Ps) and nucleotide-sugars (NDP-sugars) involved in cell wall biosynthesis were separately quantified. Using hexose-P and NDP-sugar standards, we have shown that hot water extraction allows good recovery of the target metabolites (over 86%). This method is applicable to quantifying the levels of hexose-Ps and NDP-sugars in different plant tissues, such as Arabidopsis T87 cells in culture and fenugreek (Trigonella foenum-graecum) endosperm tissue, showing higher levels of galacto-mannan precursors in fenugreek endosperm. In Arabidopsis cells incubated with [U-(13)C(Fru)]sucrose, the method was used to track the labeling pattern in cell wall precursors. As the fragmentation of hexose-Ps and NDP-sugars results in high yields of [PO(3)](-)/or [H(2)PO(4)](-) ions, mass isotopomers can be quantified directly from the intensity of selected tandem mass spectrometry transitions. The ability to directly measure (13)C labeling in cell wall precursors makes possible metabolic flux analysis of cell wall biosynthesis based on dynamic labeling experiments.

Characterization of Picomole Amounts of Oligosaccharides from Glycoproteins by 1H NMR Spectroscopy

Can you see it? NMR spectroscopy is indispensible for structure analysis. It is believed that its use is limited if less than several nanomoles are available. The characterization of oligosaccharides as components of glycoproteins by NMR spectroscopy utilizing significantly less material is highly desirable. Optimization of the sample preparation, water suppression, and instrument setup has enabled only 15 picomoles of oligosaccharides to be analyzed by NMR spectroscopy.

Picomole-scale characterization of protein stability and function by quantitative cysteine reactivity

The Gibbs free energy difference between native and unfolded states ("stability") is one of the fundamental characteristics of a protein. By exploiting the thermodynamic linkage between ligand binding and stability, interactions of a protein with small molecules, nucleic acids, or other proteins can be detected and quantified. Determination of protein stability can therefore provide a universal monitor of biochemical function. Yet, the use of stability measurements as a functional probe is underutilized, because such experiments traditionally require large amounts of protein and special instrumentation. Here we present the quantitative cysteine reactivity (QCR) technique to determine protein stabilities rapidly and accurately using only picomole quantities of material and readily accessible laboratory equipment. We demonstrate that QCR-derived stabilities can be used to measure ligand binding over a wide range of ligand concentrations and affinities. We anticipate that this technique will have broad applications in high-throughput protein engineering experiments and functional genomics.

Measurement of Human Surfactant Protein-B Turnover in Vivo from Tracheal Aspirates Using Targeted Proteomics

We describe a method to measure protein synthesis and catabolism in humans without prior purification and use the method to measure the turnover of surfactant protein-B (SP-B). SP-B, a lung-specific, hydrophobic protein essential for fetal-neonatal respiratory transition, is present in only picomolar quantities in tracheal aspirate samples and difficult to isolate for dynamic turnover studies using traditional in vivo tracer techniques. Using infusion of [5,5,5-(2)H(3)] leucine and a targeted proteomics method, we measured both the quantity and kinetics of SP-B tryptic peptides in tracheal aspirate samples of symptomatic newborn infants. The fractional synthetic rate (FSR) of SP-B measured using the most abundant proteolytic fragment, a 10 amino acid peptide from the carboxy-terminus of proSP-B (SPTGEWLPR), from the circulating leucine pool was 0.035 +/- 0.005 h(-1), and the fractional catabolic rate was 0.044 +/- 0.003 h(-1). This technique permits high-throughput and sensitive measurement of turnover of low abundance proteins with minimal sample preparation.

Method for the Affinity Purification of Covalently Linked Peptides Following Cyanogen Bromide Cleavage of Proteins

The low resolution structure of a protein can sometimes be inferred from information about existing disulfide bridges or experimentally introduced chemical crosslinks. Frequently, this task involves enzymatic digestion of a protein followed by mass spectrometry-based identification of covalently linked peptides. To facilitate this task, we developed a method for the enrichment of covalently linked peptides following the chemical cleavage of a protein. The method capitalizes on the availability of homoserine lactone moieties at the C-termini of cyanogen bromide cleavage products which support selective conjugation of affinity tags. The availability of two C-termini within covalently linked peptides allows for the conjugation of two distinct affinity tags and thereby enables subsequent removal of unmodified peptides by tandem affinity chromatography. Here, we demonstrate the stepwise implementation of this method using a polyhistidine tag and a biotin tag for the selective two-step purification of covalently linked cyanogen bromide fragments from increasingly complex protein samples. The method is independent of the nature of the covalent bond, is adaptable to fully denaturing conditions, and requires only low picomole quantities of starting material.

Determination of linear aliphatic aldehydes in heavy metal containing waters by high-performance liquid chromatography using 2,4-dinitrophenylhydrazine derivatization

A simple and sensitive method is described for the determination of picomolar amounts of C 1–C 9 linear aliphatic aldehydes in waters containing heavy metal ions. In this method, aldehydes were first derivatized with 2,4-dinitrophenylhydrazine (DNPH) at optimized pH 1.8 for 30 min and analyzed by HPLC with UV detector at 365 nm. Factors affecting the derivatization reaction of aldehydes and DNPH were investigated. Cupric ion, an example of heavy metals, is a common oxidative reagent, which may oxidize DNPH and greatly interfere with the determination of aldehydes. EDTA was used to effectively mask the interferences by heavy metal ions. The method detection limits for direct injection of derivatized most aldehydes except formaldehyde were of the order of 7–28 nM. The detection limit can be further lowered by using off-line C 18 adsorption cartridge enrichment. The recoveries of C 1–C 9 aldehydes were 93–115% with a relative standard deviation of 3.6–8.1% at the 0.1 μM level for aldehydes. The HPLC–DNPH method has been applied for determining aldehyde photoproducts from Cu(II)–amino acid complex systems.

Generation of Highly Specific Aptamers via Micromagnetic Selection

Aptamers are nucleic acid-based reagents that bind to target molecules with high affinity and specificity. However, methods for generating aptamers from random combinatorial libraries (e.g., systematic evolution of ligands by exponential enrichment (SELEX)) are often labor-intensive and time-consuming. Recent studies suggest that microfluidic SELEX (M-SELEX) technology can accelerate aptamer isolation by enabling highly stringent selection conditions through the use of very small amounts of target molecules. We present here an alternative M-SELEX method, which employs a disposable microfluidic chip to rapidly generate aptamers with high affinity and specificity. The micromagnetic separation (MMS) chip integrates microfabricated ferromagnetic structures to reproducibly generate large magnetic field gradients within its microchannel that efficiently trap magnetic bead-bound aptamers. Operation of the MMS device is facile and robust and demonstrates high recovery of the beads (99.5%), such that picomolar amounts of target molecule can be used. Importantly, the device demonstrates exceptional separation efficiency in removing weakly bound and unbound ssDNA to rapidly enrich target-specific aptamers. As a model, we demonstrate here the generation of DNA aptamers against streptavidin in three rounds of positive selection. We further enhanced the specificity of the selected aptamers via a round of negative selection in the same device against bovine serum albumin (BSA). The resulting aptamers displayed dissociation constants ranging from 25 to 65 nM for streptavidin and negligible affinity for BSA. Since a wide spectrum of molecular targets can be readily conjugated to magnetic beads, MMS-based SELEX provides a general platform for rapid generation of specific aptamers.

Analysis of Semivolatile Pharmaceuticals and Pollutants in Organic Micro Extracts Using Hot Cell Membrane Inlet Mass Spectrometry

This paper presents the first membrane inlet method that can be used together with field portable mass spectrometers for the analysis of semivolatile pharmaceuticals (pethidine, benzophenone, and cocaine) and environmental pollutants (terbutryne and butylated hydroxyl toluene (BHT)) dissolved in organic micro extracts. A microliter of the organic extract is simply injected into a closed hot cell membrane inlet (hc-MIMS), and an electron ionization mass spectrum of the vaporized semivolatile sample molecules can be recorded shortly thereafter. Detection limits at low picomole quantities or low/sub ng/microL concentrations in the extract are demonstrated for solutes in methanol, ethanol, acetone, and toluene. A linear correlation between analyte concentration and signal was found in the range of 1-100 ng/microL, and the relative standard deviation (RSD) was approximately 10%. As a practical example we demonstrate the detection of cocaine in extracts from dried coca leaves. The analysis of organic micro extracts using hc-MIMS represents a considerable extension of the type and complexity of analytes that can be measured using a field portable MIMS system, since it does not require special and field tedious modifications to the standard MIMS system.