Minimal Reagent Research Articles

How To Shrink Paper Money: A Macroscopic Demonstration of the Malaprade Reaction

Shrinking paper money is a captivating and memorable demonstration of chemistry in action that can be carried out using minimal equipment and reagents. Paper bills can be shrunk down to ∼25% of their initial surface area by treating them with an aqueous solution of sodium periodate. The cellulose in the paper bill is oxidized to dialdehyde cellulose via the Malaprade reaction, and the resulting change in size of the paper money provides macroscopic evidence of the reaction. The chemical change that occurs in the cellulose can be confirmed by a Tollen’s test.

Development and validation of probe-based multiplex real-time PCR assays for the rapid and accurate detection of freshwater fish species.

Reliable species identification methods are important for industrial environmental monitoring programs. Probe based real-time quantitative polymerase chain reaction (qPCR) provides an accurate, cost-effective and high-throughput method for species identification. Here we present the development and validation of species-specific primers and probes for the cytochrome c oxidase (COI) gene for the identification of eight ecologically and economically important freshwater fish species: lake whitefish (Coregonus clupeaformis), yellow perch (Perca flavescens), rainbow smelt (Osmerus mordax), brook trout (Salvelinus fontinalis), smallmouth bass (Micropterus dolomieu), round whitefish (Prosopium cylindraceum), spottail shiner (Notropis hudsonius) and deepwater sculpin (Myoxocephalus thompsonii). In order to identify novel primer-probe sets with maximum species-specificity, two separate primer-probe design criteria were employed. Highest ranked primer-probe sets from both methods were assayed to identify sequences that demonstrated highest specificity. Specificity was determined using control species from same genus and non-target species from different genus. Selected primer-probe sets were optimized for annealing temperature and primer-probe concentrations to identify minimum reagent parameters. The selected primer-probe sets were highly sensitive, with DNA concentrations as low as 1 ng adequate for positive species identification. A decoder algorithm was developed based on the cumulative qPCR results that allowed for full automation of species identification. Blinded experiments revealed that the combination of the species-specific primer/probes sets with the automated species decoder resulted in target species identification with 100% accuracy. We also conducted a cost/time comparison analysis between the qPCR assays established in this study with other species identification methods. The qPCR technique was the most cost-effective and least time consuming method of species identification. In summary, probe-based multiplex qPCR assays provide a rapid and accurate method for freshwater fish species identification, and the methodology established in this study can be utilized for various other species identification initiatives.

Rapid prototyping of proteins: Mail order gene fragments to assayable proteins within 24 hours.

In this study, we present a minimal template design and accompanying methods to produce assayable quantities of custom sequence proteins within 24 hr from receipt of inexpensive gene fragments from a DNA synthesis vendor. This is done without the conventional steps of plasmid cloning or cell-based amplification and expression. Instead the linear template is PCR amplified, circularized, and isothermally amplified using a rolling circle polymerase. The resulting template can be used directly with cost-optimized, scalably-manufactured Escherichia coli extract and minimal supplement reagents to perform cell-free protein synthesis (CFPS) of the template protein. We demonstrate the utility of this template design and 24 hr process with seven fluorescent proteins (sfGFP, mVenus, mCherry, and four GFP variants), three enzymes (chloramphenicol acetyltransferase, a chitinase catalytic domain, and native subtilisin), a capture protein (anti-GFP nanobody), and 2 antimicrobial peptides (BP100 and CA(1-7)M(2-9)). We detected each of these directly from the CFPS reaction using colorimetric, fluorogenic, and growth assays. Of especial note, the GFP variant sequences were found from genomic screening data and had not been expressed or characterized before, thus demonstrating the utility of this approach for phenotype characterization of sequenced libraries. We also demonstrate that the rolling circle amplified version of the linear template exhibits expression similar to that of a complete plasmid when expressing sfGFP in the CFPS reaction. We evaluate the cost of this approach to be $61/mg sfGFP for a 4 hr reaction. We also detail limitations of this approach and strategies to overcome these, namely proteins with posttranslational modifications.

An integrated microfluidic system with field-effect-transistor sensor arrays for detecting multiple cardiovascular biomarkers from clinical samples

Certain blood-borne biomarkers offer a potent methodology for understanding the risk of cardiovascular diseases (CVDs) with clinicians generally advocating the use of multiple biomarkers for proper risk assessment of CVDs. Herein four such CVDs biomarkers- C-reactive protein (CRP), N-terminal pro b-type natriuretic peptide (NT-proBNP), cardiac troponin I (cTnI), and fibrinogen- were rapidly (5 min) analyzed from clinical samples (~ 4 µL) on an integrated microfluidic platform equipped with 1) immobilized highly specific aptamer probes and 2) field-effect transistor (FET)-based sensor arrays. The calibration curve from the FET sensor arrays showed good agreement in the physiological concentration ranges for CRP (0.1–50 mg/L), NT-proBNP (50–10,000 pg/mL), cTnI (1–10,000 pg/mL), and fibrinogen (0.1–5 mg/mL). The developed prototype of this fully automated portable device requires minimal reagent and sample inputs and consequently shows great promise for next-generation point-of-care devices assaying multiple CVDs biomarkers in clinical samples.

A rink-amide soluble support: high purity conotoxins and other peptides accessed with minimal reagents

The use of peptides as therapeutics has been growing due to their biocompatibility. Solid phase peptide synthesis typically used to access these peptides requires excess reagents and/or microwave irradiation to drive reactions to completion because the reaction medium is heterogeneous. Reported herein is a soluble polynorbornene support containing rink amide attached sites for synthesizing oligopeptides and conotoxins in high purity using only 1.2 to 2 equivalents of coupling reagents. The support can be isolated as a precipitate from the reaction medium by adding ether. The loading capacity of the support can be easily determined by spectroscopy and can also be tuned by varying the monomer ratio. This support is promising for accessing peptides as the methodology uses minimal reagents and by-products can be easily separated.

Disposable Microfluidic Sensor Based on Nanocellulose for Glucose Detection.

Point‐of‐care devices that are inexpensive, disposable, and environmentally friendly are becoming increasingly predominant in the field of biosensing and biodiagnostics. Here, microfluidics is a suitable option to endow portability and minimal reagent and material consumption. Nanocellulose is introduced to manufacture microfluidic channels and as a storage and immobilization compartment of glucose oxidase. Improved enzymatic activity retention is demonstrated in a simple and disposable point‐of‐care diagnostic unit that is able to detect glucose from fluid matrices at 0.1 × 10−3 m concentration and in less than 10 min. It is concluded that the patterning and fluidic technologies that are possible with nanocellulose enable easily scalable multianalyte designs.

An Integrated Lab‐on‐a‐chip Approach to Study Heterogeneous Enantioselective Catalysts at the Microscale

AbstractAn integrated lab‐on‐a‐chip enables the rapid analysis of heterogenized enantioselective organocatalysis at the microscale. A packed‐bed microreactor was seamlessly integrated with a downstream chiral high pressure liquid chromatography (HPLC) functionality to study enantioselective transformations on a single microfluidic glass chip. Hyphenation to mass spectrometry allows for the rapid investigation of the selectivity and the substrate scope of microgram amounts of catalyst beads. Optimization of reaction conditions is possible with minimal reagent consumption and instant analytical feedback.

Efficient One-Step PEG-Silane Passivation of Glass Surfaces for Single-Molecule Fluorescence Studies.

Surface passivation to inhibit nonspecific interactions is a key requirement for in vitro single-molecule fluorescent studies. Although the standard passivation methods involve the covalent attachment of poly(ethylene glycol) (PEG) in two steps preferably over quartz surfaces, this protocol and improvements thereon require extensive labor and chemicals. Herein, we report an efficient one-step surface grafting of PEG-silane that yields enhanced passivation, as evidenced by reduced nonspecific interactions, over the conventional method at a minimal time and reagent cost and on glass surfaces. Our method is rooted in a mechanistic understanding of the silane reaction with the silanol groups on the glass surface. Single-molecule fluorescence studies with fluorescently tagged proteins and DNA on PEG-silane-functionalized glass surfaces validate the enhanced performance of the method. Combined with atomic force microscopy surface characterization, our study further illustrates that few remaining pinhole defects, plausibly from defects on the glass, on PEG-silane glass-coated surfaces account for the minimal background, where typically no more than one molecule is nonspecifically attached in a given diffraction-limited spot on the surface.

Fluorescence polarization immunoassay for rapid screening of the pesticides thiabendazole and tetraconazole in wheat.

Fluorescence polarization immunoassays (FPIAs) for thiabendazole and tetraconazole were first developed. Tracers for FPIAs of thiabendazole and tetraconazole were synthesized and the tracers' structures were confirmed by HPLC-MS/MS. The 4-aminomethylfluorescein-labeled tracers allowed achieving the best assay sensitivity and minimum reagent consumption in comparison with aminofluorescein-labeled and alkyldiaminefluoresceinthiocarbamyl-labeled tracers. Measurements of fluorescence polarization were performed using a portable device. The developed FPIA methods were applied for the analysis of wheat. Fast and simple sample preparation technique earlier developed by authors for pesticides was adapted for thiabendazole and tetraconazole. The limits of detection of thiabendazole and tetraconazole in wheat were 20 and 200μg/kg, and the lower limits of quantification were 40 and 600μg/kg, respectively. The recovery test was performed by two methods-FPIA and HPLC-MS/MS. The results obtained by FPIA correlated well with those obtained by HPLC-MS/MS (r2 = 0.9985 for thiabendazole, r2 = 0.9952 for tetraconazole). Average recoveries of thiabendazole and tetraconazole were 74 ± 4% and 72 ± 3% by FPIA, and average recoveries of thiabendazole and tetraconazole were 86 ± 2% and 74 ± 1% by HPLC-MS/MS (n = 15). Graphical abstract ᅟ.

A Self-Digitization Dielectrophoretic (SD-DEP) Chip for High-Efficiency Single-Cell Capture, On-Demand Compartmentalization, and Downstream Nucleic Acid Analysis.

The design and fabrication of a self-digitization dielectrophoretic (SD-DEP) chip with simple components for single-cell manipulation and downstream nucleic acid analysis is presented. The device employed the traditional DEP and insulator DEP to create the local electric field that is tailored to approximately the size of single cells, enabling highly efficient single-cell capture. The multistep procedures of cell manipulation, compartmentalization, lysis, and analysis were performed in the integrated microdevice, consuming minimal reagents, minimizing contamination, decreasing lysate dilution, and increasing assay sensitivity. The platform developed here could be a promising and powerful tool in single-cell research for precise medicine.

A Self‐Digitization Dielectrophoretic (SD‐DEP) Chip for High‐Efficiency Single‐Cell Capture, On‐Demand Compartmentalization, and Downstream Nucleic Acid Analysis

AbstractThe design and fabrication of a self‐digitization dielectrophoretic (SD‐DEP) chip with simple components for single‐cell manipulation and downstream nucleic acid analysis is presented. The device employed the traditional DEP and insulator DEP to create the local electric field that is tailored to approximately the size of single cells, enabling highly efficient single‐cell capture. The multistep procedures of cell manipulation, compartmentalization, lysis, and analysis were performed in the integrated microdevice, consuming minimal reagents, minimizing contamination, decreasing lysate dilution, and increasing assay sensitivity. The platform developed here could be a promising and powerful tool in single‐cell research for precise medicine.

Microscale radiosynthesis, preclinical imaging and dosimetry study of [18F]AMBF3-TATE: A potential PET tracer for clinical imaging of somatostatin receptors

BackgroundPeptides labeled with positron-emitting isotopes are emerging as a versatile class of compounds for the development of highly specific, targeted imaging agents for diagnostic imaging via positron-emission tomography (PET) and for precision medicine via theranostic applications. Despite the success of peptides labeled with gallium-68 (for imaging) or lutetium-177 (for therapy) in the clinical management of patients with neuroendocrine tumors or prostate cancer, there are significant advantages of using fluorine-18 for imaging. Recent developments have greatly simplified such labeling: in particular, labeling of organotrifluoroborates via isotopic exchange can readily be performed in a single-step under aqueous conditions and without the need for HPLC purification. Though an automated synthesis has not yet been explored, microfluidic approaches have emerged for 18F-labeling with high speed, minimal reagents, and high molar activity compared to conventional approaches. As a proof-of-concept, we performed microfluidic labeling of an octreotate analog ([18F]AMBF3-TATE), a promising 18F-labeled analog that could compete with [68Ga]Ga-DOTATATE with the advantage of providing a greater number of patient doses per batch produced. MethodsBoth [18F]AMBF3-TATE and [68Ga]Ga-DOTATATE were labeled, the former by microscale methods adapted from manual labeling, and were imaged in mice bearing human SSTR2-overexpressing, rat SSTR2 wildtype, and SSTR2-negative xenografts. Furthermore, a dosimetry analysis was performed for [18F]AMBF3-TATE. ResultsThe micro-synthesis exhibited highly-repeatable performance with radiochemical conversion of 50 ± 6% (n = 15), overall decay-corrected radiochemical yield of 16 ± 1% (n = 5) in ~40 min, radiochemical purity >99%, and high molar activity. Preclinical imaging with [18F]AMBF3-TATE in SSTR2 tumor models correlated well with [68Ga]Ga-DOTATATE. The favorable biodistribution, with the highest tracer accumulation in the bladder followed distantly by gastrointestinal tissues, resulted in 1.26 × 10−2 mSv/MBq maximal estimated effective dose in human, a value lower than that reported for current clinical 18F- and 68Ga-labeled compounds. ConclusionsThe combination of novel chemical approaches to 18F-labeling and microdroplet radiochemistry have the potential to serve as a platform for greatly simplified development and production of 18F-labeled peptide tracers. Favorable preclinical imaging and dosimetry of [18F]AMBF3-TATE, combined with a convenient synthesis, validate this assertion and suggest strong potential for clinical translation.

Microfluidic Print-to-Synthesis Platform for Efficient Preparation and Screening of Combinatorial Peptide Microarrays.

In this paper, we introduce a novel microfluidic combinatorial synthesis platform, referred to as Microfluidic Print-to-Synthesis (MPS), for custom high-throughput and automated synthesis of a large number of unique peptides in a microarray format. The MPS method utilizes standard Fmoc chemistry to link amino acids on a polyethylene glycol (PEG)-functionalized microdisc array. The resulting peptide microarrays permit rapid screening for interactions with molecular targets or live cells, with low nonspecific binding. Such combinatorial peptide microarrays can be reliably prepared at a spot size of 200 μm with 1 mm center-to-center distance, dimensions that require only minimal reagent consumption (less than 30 nL per spot per coupling reaction). The MPS platform has a scalable design for extended multiplexibility, allowing for 12 different building blocks and coupling reagents to be dispensed in one microfluidic cartridge in the current format, and could be further scaled up. As proof of concept for the MPS platform, we designed and constructed a focused tetrapeptide library featuring 2560 synthetic peptide sequences, capped at the N-terminus with 4-[( N'-2-methylphenyl)ureido]phenylacetic acid. We then used live human T lymphocyte Jurkat cells as a probe to screen the peptide microarrays for their interaction with α4β1 integrin overexpressed and activated on these cells. Unlike the one-bead-one-compound approach that requires subsequent decoding of positive beads, each spot in the MPS array is spatially addressable. Therefore, this platform is an ideal tool for rapid optimization of lead compounds found in nature or discovered from diverse combinatorial libraries, using either biochemical or cell-based assays.

Optimizing NBE PK/PD assays using the Gyrolab Affinity Software; conveniently within the bioanalyst's existing workflow.

The fully automated microfluidics-based Gyrolab is a popular instrument for the bioanalysis of protein therapeutics; requiring minimal sample and reagent volumes. Gyros offers affinity software for determining binding affinity in solution using a high-throughput method and miniaturized reactions. Using this affinity software, multiple CTGF-targeting reagents were characterized on the Gyrolab after <100% target coverage was seen in a cynomolgus pharmacokinetic/PD study dosed with anti-CTGF antibodies. The results uncovered magnitude differences in binding affinities between the dosed antibody, target and assay reagents. The binding affinity values were used to investigate reduced target coverage and results highlight potential of the affinity software for incorporation into the bioanalyst's existing Gyrolab workflow for characterizing reagents and optimizing pharmacokinetic/PD bioanalytical assays.

Selenium analysis by an integrated microwave digestion-needle trap device with hydride sorption on carbon nanotubes and electrothermal atomic absorption spectrometry determination

An integrated microwave assisted digestion (MW-AD) – needle trap device (NTD) for selenium determination in grape pomace samples is presented. The NTD was filled with oxidized multiwall carbon nanotubes (oxMWCNTS) where Se hydrides were preconcentrated. Determination was carried out by flow injection-electrothermal atomic absorption spectrometry (FI-ETAAS). The variables affecting the system were established by a multivariate design (Plackett Burman), indicating that the following variables significantly affect the system: sample amount, HNO3 digestion solution concentration, NaBH4 volume and elution volume. A Box-Behnken design was implemented to determine the optimized values of these variables. The system improved Se atomization in the graphite furnace, since only trapped hydrides reached the graphite furnace, and the pyrolysis stage was eliminated according to the aqueous matrix of the eluate. Under optimized conditions the system reached a limit of quantification of 0.11μgkg−1, a detection limit of 0.032μgkg−1, a relative standard deviation of 4% and a preconcentration factor (PF) of 100, reaching a throughput sample of 5samples per hour. Sample analysis show Se concentrations between 0.34±0.03μgkg−1 to 0.48±0.03μgkg−1 in grape pomace. This system provides minimal reagents and sample consumption, eliminates discontinuous stages between samples processing reaching a simpler and faster Se analysis.

ACTIVITY OF THE EXPERT CENTER ON PRODUCTION PROCESSES CHEMISTRY IN RN-UFANIPINEFT LLC

Background. On the basis of RN-UfaNIPIneft LLC an expert center for the production processes chemicalization has been established, the area of responsibility of which are conducting laboratory and pilot testing of chemical reagents at the Company's facilities; examination of the programs conducted by third parties; development of recommendations and technological instructions for the use of chemical reagents; providing expert and methodological support to the Company's extracting Companies. The process control system for the production chemization includes traditional meth- ods of optimization in accordance with the principle of achieving maximum technological effect with minimal chemical reagent use taking into account their chemical and technological compatibility at all stages of production from the formation to the commodity park. At the same time, the system for the chemicalization of technological processes that is being implemented can respond quickly to challenges associated with oil production in complicated conditions (high water cut, low-permeability collectors, high-viscosity emulsions, heavy oil production, high water salinity, corrosiveness, etc.). The presented approach is in good agreement with the trends in the development of the world's main mining and service companies in this area. Aims and Objectives. Conduct an analysis of the activities of the expert center for the chemicalization of production processes aimed at reducing the costs of Rosneft for chemical treatment without loss of technological efficiency of the chemical reagents used. Results. The main objective of the works carried out by the expert center of RN-UfaNIPIneft LLC is to reduce the costs of Rosneft for chemical treatment without loss of technological efficiency of the chemical reagents used. For the system solution of the problem of accumulation, storage and processing of information on the reagents used, a corporate product was developed - an electronic reference book «Rosneft Chemical Reagent Line», which allows to carry out on-line monitoring of the experimental and field trials, selection of reagents for new facilities taking into account geological and/or technical specifications of objects, physical and chemical properties of the reagent, experience of its application under similar conditions, economic feasibility. Oil Company Rosneft, a major consumer of chemical products, has formed its own corporate Uniform technical requirements for chemical reagents that establish uniform requirements for chemical reagents used at the Company's facilities, as well as methods for determining their properties; requirements for the organiza- tion of laboratory and pilot testing of chemical reagents, input/output and current monitoring of chemical reagents, the requirements for the process of monitoring the efficiency of industrial application of chemical reagents. On a regular basis, methodical support is provided to the mining companies of Rosneft the development and justification of the technology for the use of chemical reagents for use in complex geological conditions of the Company's fields are being developed, and normative local documents in chemicalization is being developed and updated.

A novel flow injection chemiluminescence method for automated and miniaturized determination of phenols in smoked food samples

An easily performed fully automated and miniaturized flow injection chemiluminescence (CL) method for determination of phenols in smoked food samples has been proposed. This method includes the ultrasound assisted solid-liquid extraction coupled with gas-diffusion separation of phenols from smoked food sample and analytes absorption into a NaOH solution in a specially designed gas-diffusion cell. The flow system was designed to focus on automation and miniaturization with minimal sample and reagent consumption by inexpensive instrumentation. The luminol – N-bromosuccinimide system in an alkaline medium was used for the CL determination of phenols. The limit of detection of the proposed procedure was 3·10−8·molL−1 (0.01mgkg−1) in terms of phenol. The presented method demonstrated to be a good tool for easy, rapid and cost-effective point-of-need screening phenols in smoked food samples.

Miniaturised sample preparation method for the multiresidual determination of regulated organohalogenated pollutants and related compounds in wild bird eggs.

A simplified, miniaturised matrix solid-phase dispersion (MSPD)-based method allowing the simultaneous extraction and purification of contaminants belonging to selected families of regulated persistent organic pollutants (POPs), i.e. polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs) and penta- to octa-polybrominated diphenyl ethers (PBDEs), and related contaminants of emerging concern, such as deca-BDE and Dechlorane Plus (DP), has been proposed. Wild bird eggs were used as biotic fat-containing model matrices. Once optimised, the procedure allowed sample preparation to be accomplished within 30min, in a single step, and with minimal sample and reagent consumption and waste generation. These features contributed to speeding up and greening the analytical process as compared to the large-scale multistep procedures for these types of analyses. The method was combined with gas chromatography-quadrupole mass spectrometry (GC-qMS) for PCB and OCP analysis and with GC-negative ion chemical ionisation-quadrupole mass spectrometry (NCI-qMS) for PBDE and DP determination. The complete analytical method provided satisfactory recoveries of the target compounds (above 83% for all analytes, except for PBDE 209, 63%), although as smallan amount of sample as 0.300g was used. The repeatability of the complete procedure was less than 16% (with the only exception for PCB 153, which looked to be affected by an interference). The limits of detection were in all cases lower than 34pgg-1 dry weight (as calculated for real samples), demonstrating the feasibility of the proposed procedure for accurate determination of the target compounds in biological samples. The proposed procedure was applied to the analysis of the target POPs in unhatched eggs of wild bird species. Graphical abstract Scheme of the miniaturised methodology proposed for environmental monitoring of POPs and related compounds in wild bird eggs.

Analytical Protein Microarrays: Advancements Towards Clinical Applications.

Protein microarrays represent a powerful technology with the potential to serve as tools for the detection of a broad range of analytes in numerous applications such as diagnostics, drug development, food safety, and environmental monitoring. Key features of analytical protein microarrays include high throughput and relatively low costs due to minimal reagent consumption, multiplexing, fast kinetics and hence measurements, and the possibility of functional integration. So far, especially fundamental studies in molecular and cell biology have been conducted using protein microarrays, while the potential for clinical, notably point-of-care applications is not yet fully utilized. The question arises what features have to be implemented and what improvements have to be made in order to fully exploit the technology. In the past we have identified various obstacles that have to be overcome in order to promote protein microarray technology in the diagnostic field. Issues that need significant improvement to make the technology more attractive for the diagnostic market are for instance: too low sensitivity and deficiency in reproducibility, inadequate analysis time, lack of high-quality antibodies and validated reagents, lack of automation and portable instruments, and cost of instruments necessary for chip production and read-out. The scope of the paper at hand is to review approaches to solve these problems.

Human Induced Pluripotent Stem (hiPS) Cells from Urine Samples: A Non-Integrative and Feeder-Free Reprogramming Strategy.

Human induced pluripotent stem (hiPS) cell technology has already revolutionized some aspects of fundamental and applied research such as study of disease mechanisms and pharmacology screening. The first clinical trial using hiPS cell-derived cells began in Japan, only 10 years after the publication of the proof-of concept article. In this exciting context, strategies to generate hiPS cells have evolved quickly, tending towards non-invasive protocols to sample somatic cells combined with "safer" reprogramming strategies. In this unit, we describe a protocol combining both of these advantages to generate hiPS cells with episomal plasmid transfection from urine samples of individuals carrying the desired genotype. Based on previous published works, this simplified protocol requires minimal equipment and reagents, and is suitable both for scientists familiar with the hiPS cells technology and neophytes. HiPS cells displaying classical features of pluripotency and suitable for all desired downstream applications are generated rapidly (<10 weeks) and with high efficiency. © 2017 by John Wiley & Sons, Inc.