Amounts Of Reagents Research Articles

Release of protein N-glycans by effectors of a Hofmann carboxamide rearrangement.

Background: Chemical methods for glycan release have gained traction because of their cost efficiency, accelerated reaction time and ability to release glycans not amenable to enzymatic cleavage. Oxidative chemical glycan release via hypochlorite treatment has been shown to be a convenient and efficient method that yields N-glycans similar to classical PNGase F digestion. We observed that the initial steps of the suggested mechanism for the oxidative release of glycans from glycoproteins by hypohalites showed similarities to the initiating steps of the classical Hofmann rearrangement of carboxamides. Therefore, we investigated the ability of different stable effectors of a Hofmann-type carboxamide rearrangement to efficiently and selectively release N-glycans from glycoproteins. Methods: Released glycans obtained from different experimental chemical release approaches were analyzed by HILIC-FLD, BHZ-FACE and ESI-MS and evaluated with respect to electrophoretic mobility, retention time and integrated peak area for resolved glycans. Results: We show that the known Hoffmann catalysts 1,3-dichloro-5,5-dimethylhydantoin, the hypervalent organoiodine (III) compound diacetoxy-iodobenzene as well as in-situ hypobromite generation using Oxone® and potassium bromide are all capable of releasing protein-bound N-glycans in good yield. Among the compounds investigated, diacetoxy-iodobenzene was capable of releasing glycans in the absence of alkali. Detailed investigations of the bromide/Oxone® method revealed a dependence of N-glycan release efficiency from the temporal order of bromide addition to the reaction mix as well as from a molar excess of bromide over Oxone®. Conclusions. These findings suggest that the oxidative release of N-glycans occurs via the initiating steps of a Hofmann carboxamide rearrangement. Hypervalent organoiodine compounds hold the promise of releasing glycans in the absence of alkali. The in-situ generation of hypobromite by bromide/Oxone® produces a consistent defined amount of reagent for rapid N-glycan release for both analytical and preparative purposes.

Pretreatment Process on Reverse Osmosis Brine as Electrodialysis Feed

Reverse Osmosis (RO) Brine is waste generated from the desalination process using the RO method. RO Brine is generally directly thrown back into the sea, even though it has the potential to be reprocessed because it still contains a variety of ions in it. The best method in RO Brine processing is Electrodialysis. But it has a problem of decreased membrane performance caused by the formation of fouling. The fouling problem can be overcome by doing a pretreatment process to eliminate impurities contained in RO Brine, one of which is Ca2+. The existence of Ca2+ can trigger the formation of CaSO4 deposits. Therefore, it needs excess reagent Na2CO3 with a certain amount to eliminate the whole Ca2+. Currently, it isn’t yet known the best pretreatment conditions that can eliminate impurities ions and produce high concentrations of NaCl. Pretreatment trials are needed in various variations of reagents amount to reduce impurities. The purpose of this study is to find out the best RO Brine pretreatment process that will later be used for the electrodialysis process to produce high NaCl recovery. The best results were obtained in the pretreatment process with variations NaOH excesses by 15% and Na2CO3 by 30% from the ideal stoichiometry.

Successful synthesis of a glial-specific blood-brain barrier shuttle peptide following a fragment condensation approach on a solid-phase resin.

Successful manual synthesis of the TD2.2 peptide acting as a blood-brain barrier shuttle was achieved. TD2.2 was successfully synthesised by sequential condensation of four protected peptide fragments on solid-phase settings, after several unsuccessful attempts using the stepwise approach. These fragments were chosen to minimise the number of demanding amino acids (in terms of coupling, Fmoc removal) in each fragment that are expected to hamper the overall synthetic process. Thus, the hydrophobic amino acids as well as Arg(Pbf) were strategically spread over multiple fragments rather than having them congested in one fragment. This study shows how a peptide that shows big challenges in the synthesis using the common stepwise elongation methodology can be synthesised with an acceptable purity. It also emphasises that choosing the right fragment with certain amino acid constituents is key for a successful synthesis. It is worth highlighting that lower amounts of reagents were required to synthesise the final peptide with an identical purity to that obtained by the automatic synthesiser.

Thread-Based Analytical Device for Determination of Phosphorus Content in Fertilizers

A simple microfluidic thread-based analytical device (mTAD) for determination of phosphorus content in fertilizers based on the formation of phosphomolybdenum blue (PMB) complex was proposed. The proposed mTAD was simply fabricated by stitching the 100 % cotton thread to the polyethylene (PE) mesh canvas. A color developing reagent, which are the mixture of sulfuric acid, ammonium molybdate, potassium antimony tartrate, and ascorbic acid, was firstly loaded into the middle of the thread. A sample solution was subsequently introduced into the same position to form PMB. A digital camera and image processing program were employed for evaluating color intensity of PMB. Variable parameters affecting the mTAD efficiencies were optimized i.e., solution volumes, chemical concentrations, and reaction time. The applicability of the proposed mTAD method was evaluated by analyzing fertilizer samples. The accuracy as expressed by percentages of recovery ranged between 83 and 96 %. The method was highly reproducible with the relative standard deviation (RSD) of 0.84 % (1 mgP/L), 0.92 % (3 mgP/L) and 1.04 % (10 mgP/L) for 10 replicates. The main features of the system are the used of commonly available materials, simple and rapid fabrication, small scale, reduced chemical waste, less sample consumption, and low-cost.
 HIGHLIGHTS
 
 Simple materials such as 100 % cotton thread and PE mesh canvas can be used for fabricating µTAD
 The proposed µTAD method was fast analysis and good sensitivity (as low as nmol P level within 6 minutes) by using small amount of reagent and sample solutions
 The method was successfully determining the phosphorus content in fertilizers
 
 GRAPHICAL ABSTRACT

Superfast desulfurization for protein chemical synthesis and modification

Superfast desulfurization for protein chemical synthesis and modification

Purification and characterization of inorganic pyrophosphatase for in vitro RNA transcription.

Inorganic pyrophosphatase (iPPase) is an enzyme that cleaves pyrophosphate into two phosphate molecules. This enzyme is an essential component of in vitro transcription (IVT) reactions for RNA preparation as it prevents pyrophosphate from precipitating with magnesium, ultimately increasing the rate of the IVT reaction. Large-scale RNA production is often required for biochemical and biophysical characterization studies of RNA, therefore requiring large amounts of IVT reagents. Commercially purchased iPPase is often the most expensive component of any IVT reaction. In this paper, we demonstrate that iPPase can be produced in large quantities and high quality using a reasonably generic laboratory facility and that laboratory-purified iPPase is as effective as commercially available iPPase. Furthermore, using size exclusion chromatography coupled with multi-angle light scattering and dynamic light scattering, analytical ultracentrifugation, and small-angle X-ray scattering, we demonstrate that yeast iPPase can form tetramers and hexamers in solution as well as the enzymatically active dimer. Our work provides a robust protocol for laboratories involved with RNA in vitro transcription to efficiently produce active iPPase, significantly reducing the financial strain of large-scale RNA production.

Intensification of the Processes of Extraction of Humic Acids from Biosubstrates under the Action of Electric Discharges

The article deals with the study of factors participating in the extraction of humic acids from plant substrates under the action of electric discharges. Using the example of the electric discharge treat-ment of the peat-water suspension, it was demonstrated that the main factor affecting the intensity of extraction is the degree of the solid phase of the biosubstrate-water suspension grinding, which depends on the pressure amplitude at a distance of the inner radius of the chamber and the number of discharge pulses. Experimental studies have shown that the amount of chemical reagents (alkalis) commonly used in the extraction of humic acids from peat can be significantly reduced due to the appearance of radicals and peroxide compounds in the peat-water suspension during the ac-tion of an electric discharge. The perspectives of a non-thermal electric discharge meth-od for intensifying the extraction of humic acids from biosubstrates are determined.

Working electrode geometry effect: A new concept for fabrication of patterned polymer brushes via SI-seATRP at ambient conditions

First reports concerning the use of atom transfer radical polymerization (ATRP) to prepare patterned hybrid materials appeared more than 20 years ago. However, the development of new methods of preparation of patterned materials is still at the forefront of scientific interest. In this paper, we describe surface-initiated simplified electrochemically mediated ATRP (SI- se ATRP) under constant current conditions, used for the fabrication of patterned polymer brushes under ambient conditions at the microliter scale. It is shown that appropriate selection of surface and shape of the working electrode (WE) allows handling the polymerization of (meth)acrylates, acrylates and acrylamides directly on the laboratory bench. The complete procedure includes a minimum amount of reagents and an optimal amount of a catalytic complex equal to 300 ppm. The use of a platinum wire mesh electrode guarantees obtaining polymers forming unique patterns. The observed patterning phenomenon could be explained by the mechanism of electrochemically mediated ATRP ( e ATRP) and is directly related to the working electrode geometry, and diffusion of the catalyst Cu I Br/TPMA (where TPMA: tris(2-pyridylmethyl)amine). • Diffusion-controlled polymerization for the preparation of hybrid materials. • Patterned polymer brushes fabricated via SI- se ATRP at ambient conditions. • Two-electrode system applied under constant current conditions for μL-scale ATRP. • Dual activator (re)generation during SI- se ATRP of DMAEMA in (in)organic medium. • XPS and EDS analysis of patterned polymer brushes.

Fit-for-Purpose Assessment of QuEChERS LC-MS/MS Methods for Environmental Monitoring of Organotin Compounds in the Bottom Sediments of the Odra River Estuary.

Organotin compounds (OTCs) are among the most hazardous substances found in the marine environment and can be determined by either the ISO 23161 method based on extraction with non-polar organic solvents and gas chromatography analysis or by the recently developed QuEChERS method coupled to liquid chromatography-mass spectrometry (LC-MS/MS). To date, the QuEChERS LC/MS and ISO 23161 methods have not been compared in terms of their fit-for-purpose and reliability in the determination of OTCs in bottom sediments. In the case of ISO 23161, due to a large number of interferences gas chromatography-mass spectrometry was not suitable for the determination of OTCs contrary to more selective determination by gas chromatography with an atomic emission detector. Moreover, it has been found that the derivatization of OTCs to volatile compounds, which required prior gas chromatography determination, was strongly affected by the sediments’ matrices. As a result, a large amount of reagent was needed for the complete derivatization of the compounds. Contrary to ISO 23161, the QuEChERS LC-MS/MS method did not require the derivatization of OTC and is less prone to interferences. Highly volatile and toxic solvents were not used in the QuEChERS LC-MS/MS method. This makes the method more environmentally friendly according to the principles of green analytical chemistry. QuEChERS LC-MS/MS is suitable for fast and reliable environmental monitoring of OTCs in bottom sediments from the Odra River estuary. However, determination of di- and monobutyltin by the QuEChERS LC-MS/MS method was not possible due to the constraints of the chromatographic system. Hence, further development of this method is needed for monitoring di- and monobutyltin in bottom sediments.

Magnetic solid-phase extraction based on benzalkonium chloride-coated Fe3O4@SiO2 nanoparticles for spectrophotometric determination of ritodrine hydrochloride and salbutamol sulfate in water and urine samples

A simple, fast, and cost-efficient magnetic solid-phase extraction (MSPE) based on benzalkonium chloride-coated Fe3O4@SiO2 magnetic nanoparticles (Fe3O4@SiO2@BZC MNPs) was developed for extraction and pre-concentration of ritodrine hydrochloride (RET) and salbutamol sulfate (SAL) after derivatization with a diazotized drug as a reagent. RET was coupled with diazotized metoclopramide (DMC) under an oxidative coupling reaction in alkaline media and an orange azo dye (λmax = 470 nm) was produced. Another orange azo dye (λmax = 457 nm) was produced from coupling of SAL with diazotized sulfadiazine (DSZ). The extraction conditions such as pH of the solution, amount of surfactant, reagent, and sorbent, sample volume, and extraction time were optimized. The developed extraction technique provided a pre-concentration factor of 25 and 50 for RET and SAL, respectively. The extraction process was followed by spectrophotometric determination of the target analytes. The linear ranges were achieved as 0.1–6 and 0.05–5 µg/mL, and detection limits were obtained as 0.04 and 0.02 µg/mL for RET and SAL, respectively. This method was successfully employed for the pre-concentration and detection of RET and SAL in water and urine samples.

Photocatalytic Degradation of Some Typical Antibiotics: Recent Advances and Future Outlooks.

The existence of antibiotics in the environment can trigger a number of issues by fostering the widespread development of antimicrobial resistance. Currently, the most popular techniques for removing antibiotic pollutants from water include physical adsorption, flocculation, and chemical oxidation, however, these processes usually leave a significant quantity of chemical reagents and polymer electrolytes in the water, which can lead to difficulty post-treating unmanageable deposits. Furthermore, though cost-effectiveness, efficiency, reaction conditions, and nontoxicity during the degradation of antibiotics are hurdles to overcome, a variety of photocatalysts can be used to degrade pollutant residuals, allowing for a number of potential solutions to these issues. Thus, the urgent need for effective and rapid processes for photocatalytic degradation leads to an increased interest in finding more sustainable catalysts for antibiotic degradation. In this review, we provide an overview of the removal of pharmaceutical antibiotics through photocatalysis, and detail recent progress using different nanostructure-based photocatalysts. We also review the possible sources of antibiotic pollutants released through the ecological chain and the consequences and damages caused by antibiotics in wastewater on the environment and human health. The fundamental dynamic processes of nanomaterials and the degradation mechanisms of antibiotics are then discussed, and recent studies regarding different photocatalytic materials for the degradation of some typical and commonly used antibiotics are comprehensively summarized. Finally, major challenges and future opportunities for the photocatalytic degradation of commonly used antibiotics are highlighted.

Sustainable Synthesis of Aryl and Heteroaryl Aldehydes

AbstractAromatic aldehydes are important intermediates for the synthesis of valuable fine and specialty chemicals and many procedures for their preparation have been developed but, nowadays, most of these synthetic methods require large amounts of reagents and multiple steps, with production of wastes. In this paper, we focused on the synthesis of some (hetero)aromatic aldehydes, used either as industrial flavor/fragrances of commercial interest, such as Heliotropine®, or as key intermediates for the preparation of some APIs and of other fragrances such as Lioral® and Helional®, by reductive carbonylation of halo derivative precursors. In order to carry out an easier and more applicable process, hydrogen donors, instead of gaseous flammable and explosive hydrogen, and recyclable phosphine free low metal content catalysts, were employed.

Design of a versatile microfluidic device for imaging precision-cut-tissue slices

Precision-cut-tissues (PCTs), which preserve many aspects of a tissue’s microenvironment, are typically imaged using conventional sample dishes and chambers. These can require large amounts of reagent and, when used for flow-through experiments, the shear forces applied on the tissues are often ill-defined. Their physical design also makes it difficult to image large volumes and repetitively image smaller regions of interest in the living slice. We report here on the design of a versatile microfluidic device capable of holding mouse or human pancreas PCTs for 3D fluorescence imaging using confocal and selective plane illumination microscopy (SPIM). Our design positions PCTs within a 5 × 5 mm × 140 µm deep chamber fitted with 150 µm tall channels to facilitate media exchange. Shear stress in the device is localized to small regions on the surface of the tissue and can be easily controlled. This design allows for media exchange at flowrates ∼10-fold lower than those required for conventional chambers. Finally, this design allows for imaging the same immunofluorescently labeled PCT with high resolution on a confocal and with large field of view on a SPIM, without adversely affecting image quality.

Development of a Label-Free Immunosensor for Quantitative, Electrical Detection of Target Antigens

Point-of-care immunoassays require rapid, sensitive, and user-friendly operation. The most common example is an at-home pregnancy test, which falls under a class of diagnostics called a lateral flow assay (LFA). However, LFAs present many disadvantages such as poor sensitivity and a difficulty for users to interpret test results accurately. The gold standard immunoassay, ELISA, exhibits excellent limits of detection (LOD) and limits of quantitation (LOQ). However, barriers to the implementation of ELISA must be considered, such as the consumption of large amounts of expensive reagents, the need for trained personnel to perform the assay, and the possibility of false positives or negatives due to improper washing or procedure. A clear demand exists for a label-free immunological sensor with high sensitivity and specificity, requiring only a small amount of sample and with fully automated operation. Previous work in our group has demonstrated the successful label-free detection of nucleic acids in a microfluidic device with an embedded 3D porous electrode by their hybridization to a bed of bioconjugated microbeads. An increase in conductivity, observed by a current-voltage curve, was attributed to the negative charge added to the beads upon hybridization of the target nucleic acids. In this presentation, we demonstrate label-free sensing of antibody targets by an analogous method despite their low net charge under the conditions employed. In this system, biotinylated goat anti-rabbit IgG immobilized on streptavidin-modified beads serves as the capture antibody. The analyte, Rabbit IgG, is introduced into the channel and incubated for 30 min. A current-voltage curve (CVC) is obtained before and after incubation to detect and quantify binding of Rabbit IgG to the bead surface. We evaluated the sensitivity of this method and found that the LOD is approximately 5 nM. Following validation of this method, we examined binding kinetics by obtaining CVCs at several time points during incubation. Next, to improve the LOD in this device, faradaic ion concentration polarization (fICP) was employed to electrokinetically enrich the negatively charged IgG over the bead bed test line. After an enrichment period of 30 min, the antigen was preconcentrated over 100-fold, which resulted in greater sensitivity. Finally, we characterize the impact of ionic strength on the sensor and evaluated its performance in a simulated biofluid, saliva, under several conditions: neat, spiked with Tris, and diluted. Ongoing research in our group will translate this method to biomarkers relevant to viral diagnostics. The authors gratefully acknowledge the Roy J. Carver Charitable Trust for the funding of this project.

Fabrication of anti-fouling and photocleaning PVDF microfiltration membranes embedded with N-TiO2 photocatalysts

Polyvinylidene fluoride (PVDF) membranes are promising materials for purifying and recycling wastewater in cold regions due to their high mechanical strength and low temperature stability. However, in practical wastewater treatment services, PVDF is easily fouled by organic pollutants because of its hydrophobic characteristics. In addition, because of the large amount of reagent utilized in the conventional cleaning process, a new hydrophilic membrane with simple cleaning characteristics needs to be developed. In this work, a novel anti-fouling membrane (N-TiO2@PVDF) was obtained by implanting a N-TiO2 photocatalyst into a PVDF membrane. N-TiO2@PVDFshows a more homogeneous pore size distribution and a larger average pore diameter, which results in a higher water flux (1017.8 L/m2•h compared to 217.4 L/m2•h PVDF), higher model pollutant bovine serum albumin (BSA) solution flux and higher BSA rejection rate (92.3% compared to 81.4% for PVDF). Furthermore, N-TiO2@PVDF shows an outstanding hydrophilicity performance compared to the PVDF membrane. The water contact angle of N-TiO2@PVDF 14 decreases sharply from 93.7° (PVDF) to 53.5°, and the BSA adsorption amount decreases significantly from 1.41 mg/cm2 (PVDF) to 0.05 mg/cm2. The total fouling ratio of N-TiO2@PVDF is decreased to a value of 25.5% compared to a value of 70.3% for PVDF, and the irreversible BSA fouling ratio is even decreased to 5.8% compared to 57.2% for PVDF. This result indicates that N-TiO2@PVDF shows outstanding antifouling performance. The N-TiO2@PVDF membrane also has a photocleaning function, and the BSA flux recovery ratio reaches up to 94.7% under simulated sunlight irradiation for 2 h without additional treatment. The results obtained from hydroxyl radical (OH) detection indicate that OH degradation of organic pollutants is the dominant mechanism for the N-TiO2@PVDF photocleaning. This anti-fouling and photocleaning N-TiO2@PVDF membrane offers a solution to wastewater treatment problems of membrane brittleness and easy fouling in cold places. It can also be a good choice for the drinking water purification process.

Advances in chiral separation and analysis by capillary electrophoresis-mass spectrometry

目前使用的绝大多数药物为手性化合物,它们具有相似的物理和化学性质,但药理活性不同,且常以外消旋混合物的形式存在,因此对手性化合物的分离在生物、环境、食品和医药等领域一直备受关注。与广泛使用的液相色谱-质谱(LC-MS)相比,毛细管电泳-质谱(CE-MS)作为一种新型分离分析技术,具有分离效率高、样品和试剂消耗量低、选择性高和分离模式多样化等诸多优势,已经发展成为手性分析领域中有广阔应用前景的分析方法之一。CE-MS结合了CE的高分离效率和低样品消耗以及MS的高灵敏度和强结构解析能力,在蛋白质组学和代谢组学等领域发挥了重要作用。CE杰出的手性拆分能力与MS优势的结合,亦使CE-MS成为实现手性化合物高效分离分析的完美组合。在过去的十几年里,基于不同CE-MS分离模式的高性能手性分析体系层出不穷,如电动色谱-质谱(EKC-MS)、胶束电动色谱-质谱(MEKC-MS)和毛细管电色谱-质谱(CEC-MS)等,并成功应用于医药、生物、食品和环境科学等领域的手性化合物分析。该文主要综述了2011~2021年,CE-MS在手性化合物分析领域的技术、手性选择剂(如改性环糊精和聚合物表面活性剂等)的使用以及在医药等领域应用方面的研究进展,并讨论了不同手性分析模式的局限性,为未来的CE-MS手性分离分析技术发展及应用提供借鉴。

Environmentally Friendly Method of Assembly of Cardanol and Cholesterol into Nanostructures Using a Continuous Flow Microfluidic Device

This study shows a viable and straightforward microfluidic method of assembly of cardanol (CA) and cholesterol (CH) into amphiphile nanostructures obtained through a hydrodynamic focusing approach according to which an alcoholic solution of CA and CH is constrained within a two-dimensional lamina shape by two lateral streams of borate buffer solution. The process is performed within glass-made cross-shaped micro-sized fluidic chips specially designed to achieve a laminar regime. CA, distilled from the cashew nut shell liquid, is demonstrated as a surface-active molecule in borate buffer basic medium and when mixed with CH it produces versatile nanovesicles through an in-batch solvent-free process. Compared to this conventional method, the microfluidic route allows operating under continuous flows, with a reduced amount of reagents and at lower experimental temperatures, ensuring no waste formation and the achievement of size-monodisperse amphiphile nanostructures that do not need further steps of purification. Electron microscopy analyses demonstrate that upon increasing CH in the lipid mixture, a switchover from spherical CA micelles to CA/CH mixed closed vesicles occurs. Differential scanning microcalorimetry confirms the formation of vesicular structures and evidences the primary role of CH, which increasingly lowers the temperature of transition depending on its concentration.

The beneficiation of the Pütürge pyrophyllite ore by flotation: mineralogical and chemical evaluation

AbstractSamples from the pyrophyllite reserves in the Malatya–Pütürge region, used in the production of the whitest cement in Europe, were beneficiated using flotation. The mineralogical composition of the natural pyrophyllite, as determined using X-ray diffraction, includes pyrophyllite, kaolinite, quartz, illite–mica and feldspar. The chemical composition of pyrophyllite contains 69.75% SiO2 and 23.04% Al2O3. The pyrophyllite percentage (40–45 wt.%) of the natural sample increased to 60–80 wt.% after flotation. In flotation experiments, the effects of reagent amounts, types and their mixtures were investigated. Methyl isobutyl carbinol (MIBC) and pine oil as frothers and kerosene as a collector were used in the flotation studies. The use of reagents as mixtures has a positive effect on the beneficiation compared to use on their own. The best result was obtained for a mixture of MIBC with kerosene, which is a non-ionic hydrocarbon oil, yielding a concentrate containing 26.63% Al2O3. Improved results were also observed for kerosene plus frother mixtures after flotation cleaning circuits. The bubbles formed during flotation were photographed and the bubble diameters were measured using the ImageJ program. The Al2O3 content was evaluated by correlating the bubble diameters. In general, selectivity decreased during experiments in which bubble diameters were reduced.

Advances in Mixer Design and Detection Methods for Kinetics Studies of Macromolecular Folding and Binding on the Microsecond Time Scale.

Many important biological processes such as protein folding and ligand binding are too fast to be fully resolved using conventional stopped-flow techniques. Although advances in mixer design and detection methods have provided access to the microsecond time regime, there is room for improvement in terms of temporal resolution and sensitivity. To address this need, we developed a continuous-flow mixing instrument with a dead time of 12 to 27 µs (depending on solution viscosity) and enhanced sensitivity, sufficient for monitoring tryptophan or tyrosine fluorescence changes at fluorophore concentrations as low as 1 µM. Relying on commercially available laser microfabrication services, we obtained an integrated mixer/flow-cell assembly on a quartz chip, based on a cross-channel configuration with channel dimensions and geometry designed to minimize backpressure. By gradually increasing the width of the observation channel downstream from the mixing region, we are able to monitor a reaction progress time window ranging from ~10 µs out to ~3 ms. By combining a solid-state UV laser with a Galvano-mirror scanning strategy, we achieved highly efficient and uniform fluorescence excitation along the flow channel. Examples of applications, including refolding of acid-denatured cytochrome c triggered by a pH jump and binding of a peptide ligand to a PDZ domain, demonstrate the capability of the technique to resolve fluorescence changes down to the 10 µs time regime on modest amounts of reagents.

Multiplexed-Based Assessment of DNA Damage Response to Chemotherapies Using Cell Imaging Cytometry

The current methods for measuring the DNA damage response (DDR) are relatively labor-intensive and usually based on Western blotting, flow cytometry, and/or confocal immunofluorescence analyses. They require many cells and are often limited to the assessment of a single or few proteins. Here, we used the Celigo® image cytometer to evaluate the cell response to DNA-damaging agents based on a panel of biomarkers associated with the main DDR signaling pathways. We investigated the cytostatic or/and the cytotoxic effects of these drugs using simultaneous propidium iodide and calcein-AM staining. We also describe new dedicated multiplexed protocols to investigate the qualitative (phosphorylation) or the quantitative changes of eleven DDR markers (H2AX, DNA-PKcs, ATR, ATM, CHK1, CHK2, 53BP1, NBS1, RAD51, P53, P21). The results of our study clearly show the advantage of using this methodology because the multiplexed-based evaluation of these markers can be performed in a single experiment using the standard 384-well plate format. The analyses of multiple DDR markers together with the cell cycle status provide valuable insights into the mechanism of action of investigational drugs that induce DNA damage in a time- and cost-effective manner due to the low amounts of antibodies and reagents required.