Common Reagents Research Articles

Quantification of miRNAs by a simple and specific qPCR method.

MicroRNAs (miRNAs) are powerful regulators of gene expression at posttranscriptional level and play important roles in many biological processes and in disease. The rapid pace of the emerging field of miRNAs has opened new avenues for development of techniques to quantitatively determine miRNA expression levels in different systems. In this chapter we describe a PCR method for quantification of miRNAs based on a single reverse transcription reaction for all miRNAs combined with real-time PCR with two miRNA-specific DNA primers. This method quantifies synthetic templates over eight orders of magnitude and successfully discriminates miRNAs that differ by one single nucleotide. Due to the usage of DNA primers this method allows higher amplification efficiencies than a similar method based on locked nucleic acid-spiked primers. The high efficiency translates into higher sensitivity and precision in miRNA quantification. Furthermore, the method is easy to perform with common laboratory reagents, which allows miRNA quantification at low cost.

Assessment of apixaban plasma levels by laboratory tests: suitability of three anti-Xa assays

While laboratory monitoring is not required in patients treated with apixaban, a direct factor-Xa inhibitor, assessment of its concentration is useful in some critical situations. However, few data are available on its effect on coagulation tests and on the suitability of anti-Xa assays for its quantification. It was the objective of this study to identify laboratory tests suitable for apixaban concentration assessment. Coagulation tests - PT and aPTT- and anti-Xa assays were performed in apixaban-spiked plasma samples. To evaluate the sensitivity of PT and aPTT to apixaban, we conducted a first monocenter part, with a wide range of concentrations (50-1,000 ng/ml), a large panel of reagents (20 reagents), and two coagulometers (STAR®, Stago and ACL TOP®, IL), and a second multicenter part involving 13 laboratories using either a common PT reagent (RecombiPlastin2G®) or the local PT and aPTT reagents. In the multicentre part, five blinded apixaban-spiked plasma samples (0/100/200/400/800 ng/ml - checked by HPLC-MS/MS) were used; apixaban concentrations were measured with three anti-Xa assays, apixaban calibrators and controls (Stago). PT and aPTT tests using a large panel of reagents displayed a low sensitivity to a wide range of apixaban concentrations. The concentrations to double PT ranged from 400 to >1,000 ng/ml with the 10 reagents. With the three anti-Xa assays, inter-laboratory precision and accuracy were below 11% and 12%, respectively. In conclusion, whereas PT and aPTT tests were not sensitive enough to detect apixaban, the three anti-Xa assays tested using lyophilised apixaban calibrators and controls allowed to reliably quantify a wide range of apixaban concentrations.

Colorimetric Detection of Trace Arsenic(III) in Aqueous Solution Using Arsenic Aptamer and Gold Nanoparticles

In this study, trace arsenic(iii) (AsIII) in aqueous solution was detected by applying a classical aptamer-based gold nanoparticles colorimetric sensing strategy. An arsenic aptamer was used as a sensing probe and gold nanoparticles as a colorimetric indicator. In the absence of AsIII, the gold nanoparticles were stabilised by the arsenic aptamer and remained dispersed at high NaCl concentrations, displaying a red solution. Contrarily, in the presence of AsIII, the gold nanoparticles were prone to aggregation, owing to the formation of aptamer–AsIII complex between the arsenic aptamer and AsIII, and thus exhibited a blue solution. By monitoring the colour change, a simple and fast colorimetric assay for AsIII was established with a detection range of 1.26–200 ppb and a detection limit of 1.26 ppb. Because this colorimetric assay only involves common reagents and can be assessed visually, it holds great potential for arsenic(iii) monitoring in environment-related and other applications.

Ионохроматографическое определение перхлоратов в питьевой воде с предварительным микроэкстракционным концентрированием

PURPOSE OF THE ARTICLE. Ion chromatography method with microextraction preconcentration and conductometric detection basis of domestic equipment and common reagents for determination of perchlorate ions low concentrations in different water samples was developed. METHODOLOGY. Reducing the influence of drinking water matrix components on the analytical signal was achieved by microextraction preconcentration perchlorate-ions using selective ion-pair reagent - tetraethylammonium chloride. The proposed methodological approach does not require a costly mass-spectrometric method, application of high-performance commercial columns and special cartridges. SCIENTIFIC PURPOSE. Different nature sorbents, including volume-porous anionite «Oka-1» were studied. Decreasing perchlorate-ions retention time to 12-13 minutes was estimated using «Oka-1» sorbent. The method of liquid-phase microextraction perchlorates concentration was developed. Concentrate was injected into the flow of amino acid eluent. RESULTS. The detection limit (3s- criterion) was 5∙10 −4 mg/l. Perchlorate-ions were determined in the concentrations range of was 2∙10 −3 - 2∙10 −1 mg/l. Relative error was not exceed 20%. The content of matrix ions Cl − , SO 4 2− , HCO 3− up to 5 g/l was not interfere perchlorate-ions determination. Recommended method had been successfully tested in the determination of perchlorate-ions in different water samples and calcium hypochlorite disinfecting solutions. CONCLUSIONS. Microextraction concentration of perchlorate ions from aqueous solvents using drip extraction method can improve the selectivity determination of perchlorate-ions low concentrations by ion chromatography in isocratic elution with conductometric detection. Recommended simple and accessible method allows to determine perchlorate-ions in different water samples. Keywords: perchlorates, ion chromatography, determination, microextraction concentration. (Russian) DOI: http://dx.doi.org/10.15826/analitika.2014.18.1.008 P.N. Kulikov, E.V. Nayanova, E.V. Elipasheva, G.M. Sergeev Lobachevsky State University, Nizhny Novgorod, Russian Federation

Versatile and scalable synthesis of graphene nanoribbons

The inability to readily upscale nanofabrication of carbon nanomaterials often restricts their application, despite outstanding performances reported in both the research laboratory and prototype stages. Here we report the direct chemical synthesis of graphene nanoribbons by a bottom-up approach based on the common laboratory reagents sodium and propanol; these are solvothermally reacted to give an intermediate precursor that is then rapidly pyrolized yielding single- and few-layer graphene nanoribbons. Our results show that confinement of the lateral dimensions of graphene can be achieved simply by varying the alcohol feedstock. The ability to produce bulk quantities of graphene nanoribbons by a low cost and scalable approach is anticipated to enable a wider range of affordable real-world graphene applications.

High-resolution chemical ionization mass spectrometry (ToF-CIMS): application to study SOA composition and processing

Abstract. This paper demonstrates the capabilities of chemical ionization mass spectrometry (CIMS) to study secondary organic aerosol (SOA) composition with a high-resolution (HR) time-of-flight mass analyzer (aerosol-ToF-CIMS). In particular, by studying aqueous oxidation of water-soluble organic compounds (WSOC) extracted from α-pinene ozonolysis SOA, we assess the capabilities of three common CIMS reagent ions: (a) protonated water clusters (H2O)nH+, (b) acetate CH3C(O)O− and (c) iodide water clusters I(H2O)n− to monitor SOA composition. Furthermore, we report the relative sensitivity of these reagent ions to a wide range of common organic aerosol constituents. We find that (H2O)nH+ is more selective to the detection of less oxidized species, so that the range of O / C and OSC (carbon oxidation state) in the SOA spectra is considerably lower than those measured using CH3C(O)O− and I(H2O)n−. Specifically, (H2O)nH+ ionizes organic compounds with OSC ≤ 1.3, whereas CH3C(O)O− and I(H2O)n− both ionize highly oxygenated organics with OSC up to 4 with I(H2O)n− being more selective towards multi-functional organic compounds. In the bulk O / C and H / C space (in a Van Krevelen plot), there is a remarkable agreement in both absolute magnitude and oxidation trajectory between ToF-CIMS data and those from a high-resolution aerosol mass spectrometer (HR-AMS). Despite not using a sensitivity-weighted response for the ToF-CIMS data, the CIMS approach appears to capture much of the chemical change occurring. As demonstrated by the calibration experiments with standards, this is likely because there is not a large variability in sensitivities from one highly oxygenated species to another, particularly for the CH3C(O)O− and I(H2O)n− reagent ions. Finally, the data illustrate the capability of aerosol-ToF-CIMS to monitor specific chemical change, including the fragmentation and functionalization reactions that occur during organic oxidation, and the oxidative conversion of dimeric SOA species into monomers. Overall, aerosol-ToF-CIMS is a valuable, selective complement to some common SOA characterization methods, such as AMS and spectroscopic techniques. Both laboratory and ambient SOA samples can be analyzed using the techniques illustrated in the paper.

Ammonium Tetrathiomolybdate: A Versatile Catalyst for Hydrogen Evolution Reaction from Water under Ambient and Hostile Conditions

The lack of catalysts that can selectively reduce protons to produce hydrogen from water in the presence of oxygen and other conventional inhibitors of hydrogen evolution reaction (HER) has been a fundamental problem stalling the development of a practical hydrogen economy. Ammonium tetrathiomolybdate (ATM), a common laboratory reagent, spontaneously assembles on Au electrodes. Atomic force microscopy, scanning electron microscopy, and X-ray photoelectron spectroscopy data indicate formation of multiple layers of ATM which are stable over a wide pH range for days. These assemblies can produce hydrogen with very low onset potentials. It shows a turnover rate of 1.4 s(-1) and turnover number >5 × 10(4) in pH 7 at 180 mV overpotential. The pH dependence of the peak potential suggests that the generation of H2 from water proceeds likely via a ligand based proton coupled electron transfer process which precludes inhibition by O2. The ATM functionalized Au electrodes are found to efficiently catalyze HER in saline rich, CO saturated, and sulfide rich water sources with minimal inhibition of catalytic activity.

Highly-Efficient Preparation of Key Intermediate of Huperzine A

Huperzine A was approved for curing Alzheimers disease in the early 1990s in China. We explored a highly-efficient synthetic route for the key intermediate in the total synthesis of this natural product. Only common reagents and conditions were involved. So our new route has potential application in large-scale preparation of the key intermediate.

Bioanalytical platform comparison using a generic human IgG PK assay format

A comparison of four different ligand-binding assay technology platforms (ELISA, Meso Scale Discovery®, Gyros® and AlphaLISA®) was conducted using quantitative assays for the measurement of a human IgG1 monoclonal antibody (MAb) in rat serum. The assays used common reagents for Fc-specific measurement to determine total levels of a human IgG MAb drug analyte, and all were fully optimized for use on each platform. Mock MAb study samples were prepared and analyzed using all platforms to assess assay performance. Assay parameters such as sensitivity, dynamic range, minimum required dilution and sample volume as well as other considerations such as per-run cost, technology availability, requisite equipment and necessary reagent modifications were evaluated toward the determination of a default go-to assay platform for monoclonal antibody biotherapeutics in this laboratory. Based primarily on superior assay performance, Meso Scale Discovery and Gyros were selected from the four technologies evaluated as our default platforms for non-regulated (discovery) study support. As an adjunct, immunoaffinity LC-MS/MS was explored as an alternate platform for generic Fc quantitation and was found to perform similarly to the ligand-binding assays.

Polymeric pseudo-crown ether for cation recognition via cation template-assisted cyclopolymerization

Cyclopolymerization is a chain polymerization of bifunctional monomers via alternating processes of intramolecular cyclization and intermolecular addition, to give soluble linear polymers consisting of in-chain cyclic structures. Though cyclopolymers comprising in-chain multiple large rings potentially show unique functionality, they generally require the elaborate design of bifunctional monomers. Here we report cation template-assisted cyclopolymerization of poly(ethylene glycol) dimethacrylates as an efficient strategy directly yielding polymeric pseudo-crown ethers with large in-chain cavities (up to 30-membered rings) for selective molecular recognition. The key is to select a size-fit metal cation for the spacer unit of the divinyl monomers to form a pseudo-cyclic conformation, where the two vinyl groups are suitably positioned for intramolecular cyclization. The marriage of supramolecular chemistry and polymer chemistry affords efficient, one-pot chemical transformation from common chemical reagents with simple templates to functional cyclopolymers.

Preprogrammed, Parallel On-Chip Immunoassay Using System-Level Capillarity Control

Fully manual use of conventional multiwell plates makes enzyme-linked immunosorbent assay (ELISA)-based immunoassays highly time-consuming and labor-intensive. Here, we present a capillarity-driven on-chip immunoassay that greatly saves time and labor with an inexpensive setup. Our immunoassay process starts with pipetting multiple solutions into multiwells constructed on a microfluidic device chip. Subsequently, capillarity spontaneously transports multiple sample solutions and common reagent solutions into assigned detection channels on the chip in a purely passive and preprogrammed manner. Our device implements capillarity-driven immunoassays involving four sample and six reagent solutions within 30 min by orchestrating the functions of on-chip passive components. Notably, our immunoassay technique reduces the total number of pipetting processes by ~5 times, as compared to assays on multiwell plates (48 vs 10). This assay technique allows us to quantify the concentrations of C-reactive protein and suppressor of tumorigenicity 2 with a detection limit of 8 and 90 pM, respectively. This device should be useful for sophisticated, parallel biochemical microfluidic processing in point-of-care settings under limited resources.

Comprehensive Assessment and Standardization of Solid Phase Multiplex-Bead Arrays for the Detection of Antibodies to HLA

Solid phase multiplex-bead arrays for the detection and characterization of HLA antibodies provide increased sensitivity and specificity compared to conventional lymphocyte-based assays. Assay variability due to inconsistencies in commercial kits and differences in standard operating procedures (SOP) hamper comparison of results between laboratories. The Clinical Trials in Organ Transplantation Antibody Core Laboratories investigated sources of assay variation and determined if reproducibility improved through utilization of SOP, common reagents and normalization algorithms. Ten commercial kits from two manufacturers were assessed in each of seven laboratories using 20 HLA reference sera. Implementation of a standardized (vs. a nonstandardized) operating procedure greatly reduced MFI variation from 62% to 25%. Although laboratory agreements exceeded 90% (R2), small systematic differences were observed suggesting center specific factors still contribute to variation. MFI varied according to manufacturer, kit, bead type and lot. ROC analyses showed excellent consistency in antibody assignments between manufacturers (AUC > 0.9) and suggested optimal cutoffs from 1000 to 1500 MFI. Global normalization further reduced MFI variation to levels near 20%. Standardization and normalization of solid phase HLA antibody tests will enable comparison of data across laboratories for clinical trials and diagnostic testing.

Standardization and Cross Validation of Alloreactive IFNγ ELISPOT Assays Within the Clinical Trials in Organ Transplantation Consortium

Emerging evidence indicates memory donor-reactive T cells are detrimental to transplant outcome and that quantifying the frequency of IFNγ-producing, donor-reactive PBMCs by ELISPOT has potential utility as an immune monitoring tool. Nonetheless, differences in assay performance among laboratories limit the ability to compare results. In an effort to standardize assays, we prepared a panel of common cellular reagent standards, developed and cross validated a standard operating procedure (SOP) for alloreactive IFNγ ELISPOT assays in several research laboratories supported by the NIH-funded Clinical Trials in Organ Transplantation (CTOT) Consortium. We demonstrate that strict adherence to the SOP and centralized data analysis results in high reproducibility with a coefficient of variance (CV) of ∼30%. This standardization of IFNγ ELISPOTassay will facilitate interpretation of data from multicenter transplantation research studies and provide the foundation for developing clinical laboratory testing strategies to guide therapeutic decision-making in transplant patients.

Direct catalytic cross-coupling of organolithium compounds

Catalytic carbon-carbon bond formation based on cross-coupling reactions plays a central role in the production of natural products, pharmaceuticals, agrochemicals and organic materials. Coupling reactions of a variety of organometallic reagents and organic halides have changed the face of modern synthetic chemistry. However, the high reactivity and poor selectivity of common organolithium reagents have largely prohibited their use as a viable partner in direct catalytic cross-coupling. Here we report that in the presence of a Pd-phosphine catalyst, a wide range of alkyl-, aryl- and heteroaryl-lithium reagents undergo selective cross-coupling with aryl- and alkenyl-bromides. The process proceeds quickly under mild conditions (room temperature) and avoids the notorious lithium halogen exchange and homocoupling. The preparation of key alkyl-, aryl- and heterobiaryl intermediates reported here highlights the potential of these cross-coupling reactions for medicinal chemistry and material science.

Delivery of small interfering RNAs in human cervical cancer cells by polyethylenimine-functionalized carbon nanotubes

Carbon nanotubes are capable of penetrating the cell membrane and are widely considered as potential carriers for gene or drug delivery. Because the C-C and C=C bonds in carbon nanotubes are nonpolar, functionalization is required for carbon nanotubes to interact with genes or drugs as well as to improve their biocompatibility. In this study, polyethylenimine (PEI)-functionalized single-wall (PEI-NH-SWNTs) and multiwall carbon nanotubes (PEI-NH-MWNTs) were produced by direct amination method. PEI functionalization increased the positive charge on the surface of SWNTs and MWNTs, allowing carbon nanotubes to interact electrostatically with the negatively charged small interfering RNAs (siRNAs) and to serve as nonviral gene delivery reagents. PEI-NH-MWNTs and PEI-NH-SWNTs had a better solubility in water than pristine carbon nanotubes, and further removal of large aggregates by centrifugation produced a stable suspension of reduced particle size and improved homogeneity and dispersity. The amount of grafted PEI estimated by thermogravimetric analysis was 5.08% (w/w) and 5.28% (w/w) for PEI-NH-SWNTs and PEI-NH-MWNTs, respectively. For the assessment of cytotoxicity, various concentrations of PEI-NH-SWNTs and PEI-NH-MWNTs were incubated with human cervical cancer cells, HeLa-S3, for 48 h. PEI-NH-SWNTs and PEI-NH-MWNTs induced cell deaths in a dose-dependent manner but were less cytotoxic compared to pure PEI. As determined by electrophoretic mobility shift assay, siRNAs directed against glyceraldehyde-3-phosphate dehydrogenase (siGAPDH) were completely associated with PEI-NH-SWNTs or PEI-NH-MWNTs at a PEI-NH-SWNT/siGAPDH or PEI-NH-MWNT/siGAPDH mass ratio of 80:1 or 160:1, respectively. Furthermore, PEI-NH-SWNTs and PEI-NH-MWNTs successfully delivered siGAPDH into HeLa-S3 cells at PEI-NH-SWNT/siGAPDH and PEI-NH-MWNT/siGAPDH mass ratios of 1:1 to 20:1, resulting in suppression of the mRNA level of GAPDH to an extent similar to that of DharmaFECT, a common transfection reagent for siRNAs. Our results indicate that the PEI-NH-SWNTs and PEI-NH-MWNTs produced in this study are capable of delivering siRNAs into HeLa-S3 cells to suppress gene expression and may therefore be considered as novel nonviral gene delivery reagents.

Texas 3-Step decellularization protocol: Looking at the cardiac extracellular matrix

The extracellular matrix (ECM) is a critical tissue component, providing structural support as well as important regulatory signaling cues to govern cellular growth, metabolism, and differentiation. The study of ECM proteins, however, is hampered by the low solubility of ECM components in common solubilizing reagents. ECM proteins are often not detected during proteomics analyses using unbiased approaches due to solubility issues and relatively low abundance compared to highly abundant cytoplasmic and mitochondrial proteins. Decellularization has become a common technique for ECM protein-enrichment and is frequently used in engineering studies. Solubilizing the ECM after decellularization for further proteomic examination has not been previously explored in depth. In this study, we describe testing of a series of protocols that enabled us to develop a novel optimized strategy for the enrichment and solubilization of ECM components. Following tissue decellularization, we use acid extraction and enzymatic deglycosylation to facilitate re-solubilization. The end result is the generation of three fractions for each sample: soluble components, cellular components, and an insoluble ECM fraction. These fractions, developed in mass spectrometry-compatible buffers, are amenable to proteomics analysis. The developed protocol allows identification (by mass spectrometry) and quantification (by mass spectrometry or immunoblotting) of ECM components in tissue samples. The study of extracellular matrix (ECM) proteins in pathological and non-pathological conditions is often hampered by the low solubility of ECM components in common solubilizing reagents. Additionally, ECM proteins are often not detected during global proteomic analyses due to their relatively low abundance compared to highly abundant cytoplasmic and mitochondrial proteins. In this manuscript we describe testing of a series of protocols that enabled us to develop a final novel optimized strategy for the enrichment and solubilization of ECM components. The end result is the generation of three fractions for each sample: soluble components, cellular components, and an insoluble ECM fraction. By analysis of each independent fraction, differences in protein levels can be detected that in normal conditions would be masked. These fractions are amenable to mass spectrometry analysis to identify and quantify ECM components in tissue samples. The manuscript places a strong emphasis on the immediate practical relevance of the method, particularly when using mass spectrometry approaches; additionally, the optimized method was validated and compared to other methodologies described in the literature.

ChemInform Abstract: Photochemical Reactions of Aromatic Compounds and the Concept of the Photon as a Traceless Reagent

AbstractReview: 263 refs.

A physical sciences network characterization of non-tumorigenic and metastatic cells

To investigate the transition from non-cancerous to metastatic from a physical sciences perspective, the Physical Sciences–Oncology Centers (PS-OC) Network performed molecular and biophysical comparative studies of the non-tumorigenic MCF-10A and metastatic MDA-MB-231 breast epithelial cell lines, commonly used as models of cancer metastasis. Experiments were performed in 20 laboratories from 12 PS-OCs. Each laboratory was supplied with identical aliquots and common reagents and culture protocols. Analyses of these measurements revealed dramatic differences in their mechanics, migration, adhesion, oxygen response, and proteomic profiles. Model-based multi-omics approaches identified key differences between these cells' regulatory networks involved in morphology and survival. These results provide a multifaceted description of cellular parameters of two widely used cell lines and demonstrate the value of the PS-OC Network approach for integration of diverse experimental observations to elucidate the phenotypes associated with cancer metastasis.

Picoinjection enables digital detection of RNA with droplet rt-PCR.

The ability to add reagents to drops in a sequential fashion is necessary for numerous applications of microfluidics in biology. An important method for accomplishing this is picoinjection, a technique in which reagents are injected into aqueous drops using an electric field. While picoinjection has been shown to allow the precise addition of reagents to drops, its compatibility with biological reactions is yet to be thoroughly demonstrated. Here, we investigate the compatibility of picoinjection with digital RT-PCR Taqman assays, reactions that incorporate nucleic acids, enzymes, and other common biological reagents. We find that picoinjection is compatible with this assay and enables the detection of RNA transcripts at rates comparable to workflows not incorporating picoinjection. We also find that picoinjection results in negligible transfer of material between drops and that the drops faithfully retain their compartmentalization.

Ionic liquids as surfactants in micellar liquid chromatography

This paper is devoted to application of ionic liquids as surfactants in LC of organic compounds, derivatives of 1,4-thiosemicarbazides. According to HPLC requirements the most advantageous conditions such as transparency for ultraviolet light, low CMC, additional inorganic salt additives, and appropriate organic solvent were established. The CMC was determined using conductivity measurements. Suitability of two different stationary phases: RP-C18 and cyanopropyl bonded phase was examined under micellar conditions. Chosen ionic liquid surfactant was compared to common traditional amphiphilic reagent - SDS. Elaborated on chromatographic micellar conditions were tested as a pilot technique for prediction of distribution coefficients of organic analytes in ionic liquid-based aqueous two-phase system.