Streamlined Data Processing for Determination of Equilibrium Dissociation Constants with Accurate Constant via Transient Incomplete Separation (ACTIS).

The determination of accurate equilibrium dissociation constants, Kd, of protein-small molecule complexes is important but challenging as all established methods have inherent sources of inaccuracy. Accurate Constant via Transient Incomplete Separation (ACTIS) is a new method for Kd determination using transient incomplete separation of the complex from the unbound small molecule in a pressure-driven flow inside a capillary. ACTIS is accurate, and its accuracy is invariant to variations in geometries of both the fluidic system and the flow. Furthermore, ACTIS is implemented using a simple fluidic system supporting its accuracy and providing a simple-to-follow/copy template for instrumentation. Despite the simple and robust instrumentation/acquisition, the current data processing workflow is cumbersome, time consuming, and prone to hard-to-trace human errors therefore hindering ACTIS' ability to become a practical reference method for Kd determination. This technical note describes a streamlined workflow for processing ACTIS data; the workflow is implemented as a set of open-source software tools called prACTISed (https://github.com/prACTISedProgram/prACTISed). These tools allow all steps of data processing to be performed in a fast and straightforward fashion. These practical software tools complement the simple instrumentation serving both developers and users of ACTIS.

Circular Geometry in Molecular Stream Separation to Facilitate Nonorthogonal Field-to-Flow Orientation.

Molecular stream separation (MSS) is a promising complement for continuous-flow synthesis. MSS is driven by forces exerted on molecules by a field applied at an angle to the stream-carrying flow. MSS has only been performed with a 90° field-to-flow angle because of a rectangular geometry of canonic MSS; the second-order rotational symmetry of a rectangle prevents any other angle. Here, we propose a noncanonic circular geometry for MSS, which better aligns with the polar nature of MSS and allows changing the field-to-flow. We conducted in silico and experimental studies of circular geometry for continuous-flow electrophoresis (CFE, an MSS method). We proved two advantages of circular CFE over its rectangular counterpart. First, circular CFE can support better flow and electric-field uniformity than rectangular CFE. Second, the nonorthogonal field-to-flow orientation, achievable in circular CFE, can result in a higher stream resolution than the orthogonal one. Considering that circular CFE devices are not more complex in fabrication than rectangular ones, we foresee that circular CFE will serve as a new standard and a testbed for the investigation and creation of new CFE modalities.

Template Instrumentation for "Accurate Constant via Transient Incomplete Separation".

Accurate Constant via Transient Incomplete Separation (ACTIS) is a new method for finding the equilibrium dissociation constant Kd of a protein-small molecule complex based on transient incomplete separation of the complex from the unbound small molecule in a capillary. This separation is caused by differential transverse diffusion of the complex and the small molecule in a pressure-driven flow. The advection-diffusion processes underlying ACTIS can be described by a system of partial differential equations allowing for a virtual ACTIS instrument to be built and ACTIS to be studied in silico. The previous in silico studies show that large variations in the fluidic system geometry do not affect the accuracy of Kd determination, thus, proving that ACTIS is conceptually accurate. The conceptual accuracy does not preclude, however, instrumental inaccuracy caused by run-to-run signal drifts. Here we report on assembling a physical ACTIS instrument with a fluidic system that mimics the virtual one and proving the absence of signal drifts. Furthermore, we confirmed method ruggedness by assembling a second ACTIS instrument and comparing the results of experiments performed with both instruments in parallel. Despite some unintentional differences between the instruments (caused by tolerances in sizes, positions, etc.) and noticeable differences in their respective separagrams, we found that the Kd values determined for identical samples with these instruments were equal. Conclusively, the fluidic system presented here can serve as a template for reliable ACTIS instrumentation.

Topino: A Graphical Tool for Quantitative Assessment of Molecular Stream Separations.

In molecular-stream separation (MSS), a stream of a multicomponent mixture is separated into multiple streams of individual components. Quantitative evaluation of MSS data has been a bottleneck in MSS for decades as there was no conventional way to present the data in a reproducible and uniform fashion. The roots of the problem were in the multidimensional nature of MSS data; even in the ideal case of steady-state separation, the data is three-dimensional: intensity and two spatial coordinates. We recently found a way to reduce the dimensionality via presenting the MSS data in a polar coordinate system and convoluting the data via integration of intensity along the radius axis. The result of this convolution is an angulagram, a simple 2D plot presenting integrated intensity vs angle. Not only does an angulagram simplify the visual assessment, but it also allows the determination of three quantitative parameters characterizing the quality of MSS: stream width, stream linearity, and stream deflection. Reliably converting an MSS image into an angulagram and accurately determining the stream parameters requires an advanced and user-friendly software tool. In this technical note, we introduce such a tool: the open-source software Topino available at https://github.com/Schallaven/topino. Topino is a stand-alone program with a modern graphical user interface that allows processing an MSS image in a fast (<2 min) and straightforward way. The robustness and ruggedness of Topino were confirmed by comparing the results obtained by three users. Topino removes the analytical bottleneck in MSS and will be an indispensable tool for MSS users with varying levels of experience.

Assessing Accuracy of an Analytical Method In Silico: Application to "Accurate Constant via Transient Incomplete Separation" (ACTIS).

Analytical methods may not have reference standards required for testing their accuracy. We postulate that the accuracy of an analytical method can be assessed in the absence of reference standards in silico if the method is built upon deterministic processes. A deterministic process can be precisely computer-simulated, thus allowing virtual experiments with virtual reference standards. Here, we apply this in silico approach to study "Accurate Constant via Transient Incomplete Separation" (ACTIS), a method for finding the equilibrium dissociation constant (Kd) of protein-small-molecule complexes. ACTIS is based on a deterministic process: molecular diffusion of the interacting protein-small-molecule pair in a laminar pipe flow. We used COMSOL software to construct a virtual ACTIS setup with a fluidic system mimicking that of a physical ACTIS instrument. Virtual ACTIS experiments performed with virtual samples-mixtures of a protein and a small molecule with defined rate constants and, thus, Kd of their interaction-allowed us to assess ACTIS accuracy by comparing the determined Kd value to the input Kd value. Further, the influence of multiple system parameters on ACTIS accuracy was investigated. Within multifold ranges of parameter values, the values of Kd did not deviate from the input Kd values by more than a factor of 1.25, strongly suggesting that ACTIS is intrinsically accurate and that its accuracy is robust. Accordingly, further development of ACTIS can focus on achieving high reproducibility and precision. We foresee that in silico accuracy assessment, demonstrated here with ACTIS, will be applicable to other analytical methods built upon deterministic processes.

Visualization of Streams of Small Organic Molecules in Continuous-Flow Electrophoresis.

Continuous-flow electrophoresis (CFE) separates a stream of a multicomponent mixture into multiple streams of individual components inside a thin rectangular chamber. CFE will be able to benefit flow chemistry when it is both compatible with nonaqueous solvents utilized in organic synthesis and capable of generically detecting streams of small organic molecules. While stable nonaqueous CFE has been demonstrated, generically detecting molecular streams has not been achieved yet. Here we propose a general approach for molecular stream visualization in CFE via analyte-caused obstruction of excitation of a fluorescent layer underneath the separation chamber-fluorescent sublayer-based visualization (FSV). The concept of FSC-based visualization has been adapted from visualization of small organic molecules on fluorescent plates in thin-layer chromatography. We designed and fabricated a CFE device with one side made of quartz and another side made of UV-absorbing visibly fluorescent, chemically inert, machinable plastic. This device was demonstrated to support nonaqueous CFE of small organic molecules and quantitative detection of their streams in real-time with a limit of detection below 100 µM. Thus, CFE may satisfy conditions required for its seamless integration with continuous flow organic synthesis in flow chemistry.

Non-aqueous continuous-flow electrophoresis (NACFE): potential separation complement for continuous-flow organic synthesis.

We introduce non-aqueous continuous-flow electrophoresis (NACFE) in which the electrolyte is a solution of an organic salt in an aprotic organic solvent. NACFE can maintain steady-state separation of multiple hydrophobic organic species into individual molecular streams. It is a potential separation complement for continuous-flow organic synthesis. This proof-of-concept work will serve as a justification for efforts towards making NACFE a practical tool in flow chemistry.

Transient Incomplete Separation Facilitates Finding Accurate Equilibrium Dissociation Constant of Protein-Small Molecule Complex.

Current practical methods for finding the equilibrium dissociation constant, Kd , of protein-small molecule complexes have inherent sources of inaccuracy. Introduced here is "accurate constant via transient incomplete separation" (ACTIS), which appears to be free of inherent sources of inaccuracy. Conceptually, a short plug of the pre-equilibrated protein-small molecule mixture is pressure-propagated in a capillary, causing fast transient incomplete separation of the complex from the unbound small molecule. A superposition of signals from these two components is measured near the capillary exit and used to calculate a fraction of unbound small molecule, which, in turn, is used to calculate Kd . Herein the validity of ACTIS is proven theoretically, its accuracy is verified by computer simulation, and its practical use is demonstrated. ACTIS has the potential to become a reference-standard method for determining Kd  values of protein-small molecule complexes.

Cytometry of Reaction Rate Constant: Measuring Reaction Rate Constant in Individual Cells To Facilitate Robust and Accurate Analysis of Cell-Population Heterogeneity.

Robust and accurate analysis of cell-population heterogeneity is challenging but required in many areas of biology and medicine. In particular, it is pivotal to the development of reliable cancer biomarkers. Here, we prove that cytometry of reaction rate constant (CRRC) can facilitate such analysis when the kinetic mechanism of a reaction associated with the heterogeneity is known. In CRRC, the cells are loaded with a reaction substrate, and its conversion into a product is followed by time-lapse fluorescence microscopy at the single-cell level. A reaction rate constant is determined for every cell, and a kinetic histogram "number of cells versus the rate constant" is used to determine quantitative parameters of reaction-based cell-population heterogeneity. Such parameters include, for example, the number and sizes of subpopulations. In this work, we applied CRRC to a reaction of substrate extrusion from cells by ATP-binding cassette (ABC) transporters. This reaction is viewed as a potential basis for predictive biomarkers of chemoresistance in cancer. CRRC proved to be robust (insensitive to variations in experimental settings) and accurate for finding quantitative parameters of cell-population heterogeneity. In contrast, a typical nonkinetic analysis, performed on the same data sets, proved to be both nonrobust and inaccurate. Our results suggest that CRRC can potentially facilitate the development of reliable cancer biomarkers on the basis of quantitative parameters of cell-population heterogeneity. A plausible implementation scenario of CRRC-based development, validation, and clinical use of a predictor of ovarian cancer chemoresistance to its frontline therapy is presented.

Predicting efficiency of NECEEM-based partitioning of protein binders from nonbinders in DNA-encoded libraries.

Nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) is an affinity method for separating binder-target complexes from nonbinders by gel-free CE. NECEEM is a promising high-efficiency method for partitioning protein binders from nonbinders in DNA-encoded libraries (DEL), such binders are used as "hits" in drug development. It is important to be able to predict the efficiency of NECEEM-based partitioning, which is the efficiency of collecting binders while removing nonbinders for a specific protein and a specific DEL with a minimum of empirical information. Here, we derive and study the dependence of efficiency of NECEEM-based partitioning on electrophoretic mobilities of the protein and the DNA moiety in DEL compounds. Our derivation is based upon a previously found relation between the electrophoretic mobility of protein-binder complex and measured electrophoretic mobilities of the protein and unbound DEL and their estimated sizes. The derivation utilizes the assumption of Gaussian shapes of electrophoretic peaks and the approximation of the efficiency of partitioning by the background of nonbinders - a fraction of nonbinders, which elutes along with protein-binder complexes. Our results will serve as a guiding tool for planning the NECEEM-based partitioning of protein binders from non-binders in DELs. In particular, it can be used to estimate a minimum number of rounds of partitioning required for the desired level of DEL enrichment.

Quantitative Characterization of Molecular-Stream Separation.

Molecular-stream separation (MSS), for example, by free flow electrophoresis or continuous annular chromatography, has great potential for applications that require continuous downstream separation such continuous flow synthesis. Despite its potential, MSS still needs to be greatly advanced, which requires currently lacking tools for quantitative characterization of streams in MSS. We developed and introduce here an analytical toolbox for this task. The first tool is a method to convolute 3D raw MSS data into a 2D "angulagram" via signal integration over the whole separation zone using a polar coordinate system. The second tool is three quantitative parameters characterizing stream width, linearity, and deflection, which are determined from an angulagram. The third tool is the analysis of the three parameters in relation to physicochemical characteristics of MSS which reveals deficiencies and guides improvements in MSS devices and methods. Examples of toolbox application to validation of previously published MSS data are provided.

Image processing and analysis system for development and use of free flow electrophoresis chips.

We present an image processing and analysis system to facilitate detailed performance analysis of free flow electrophoresis (FFE) chips. It consists of a cost-effective self-built imaging setup and a comprehensive customizable software suite. Both components were designed modularly to be accessible, adaptable, versatile, and automatable. The system provides tools for i) automated identification of chip features (e.g. separation zone and flow markers), ii) extraction and analysis of stream trajectories, and iii) evaluation of flow profiles and separation quality (e.g. determination of resolution). Equipped with these tools, the presented image processing and analysis system will enable faster development of FFE chips and applications. It will also serve as a robust detector for fluorescence-based analytical applications of FFE.

Hyphenation of Production-Scale Free-Flow Electrophoresis to Electrospray Ionization Mass Spectrometry Using a Highly Conductive Background Electrolyte.

In this technical note, we demonstrate the hyphenation of production-scale free-flow electrophoresis (FFE) and sheathless electrospray ionization mass spectrometry (ESI-MS). In contrast to previous hyphenation approaches, we used a highly conductive background electrolyte (BGE) required for production-scale FFE. We found that this kind of BGE as well as a production-scale setup leads to significant electric interference between FFE and MS. This interference prevents steady-state FFE operation. We examine this interference in detail and discuss possible solutions to this issue. We demonstrate that the straightforward grounding of the transfer line removes the influence of ESI-MS on FFE, but creates a current leak from the ESI interface, which adversely affects the ESI spray. Furthermore, we show that only the electrical disconnection of the ESI probe from the FFE-MS transfer line suppresses this undesirable current. In order to facilitate the electrical disconnection we used a low conductivity, silica-based ESI probe with withdrawn inner capillary. This approach allowed the interference-free hyphenation of production-scale FFE (using a highly conductive BGE) with ESI-MS.

Two-dimensional separation of ionic species by hyphenation of capillary ion chromatography × capillary electrophoresis-mass spectrometry.

The separation of complex mixtures such as biological or environmental samples requires high peak capacities, which cannot be established with a single separation technique. Therefore, multidimensional systems are in demand. In this work, we present the hyphenation of the two most important (orthogonal) techniques in ion analysis, namely, ion chromatography (IC) and capillary electrophoresis (CE), in combination with mass spectrometry. A modulator was developed ensuring a well-controlled coupling of IC and CE separations. Proof-of-concept measurements were performed using a model system consisting of nucleotides and cyclic nucleotides. The data are presented in a multidimensional contour plot. Analyte stacking in the CE separation could be exploited on the basis of the fact that the suppressed IC effluent is pure water.

Simultaneous separation and detection of anions and thiophilic cations using capillary-size anion exchange chromatography with suppressed conductivity detection.

In this fundamental study, the simultaneous separation and detection of anions and thiophilic cations in anion exchange chromatography with suppressed conductivity detection is investigated. Mercury(II) and cadmium(II) served as model analytes. Separation and detection was performed by introducing 2-mercaptoethanesulfonate, which forms complexes with both mercury and cadmium with a strong metal-sulfur bond, into the KOH eluent. Additional to the separation on the column, these complexes were able to pass the suppressor. Subsequently, they could be detected as negative peaks. A simple model for the separation mechanism was developed based on these results. Furthermore, the effect of the eluent concentration on the retention factors of both cation complexes and standard anions was examined and quantified. It revealed that the concentration of 2-mercaptoethanesulfonate has more influence on the cations than the KOH concentration. Also, 2.0 mM of 2-mercaptoethanesulfonate had about the same effect on the anion separation as 60 mM KOH. Finally, selectivity and detection limits were investigated. The detection limits were 4.9 µM for mercury and 2.2 µM for cadmium.

Hyphenation of capillary high-performance ion-exchange chromatography with mass spectrometry using sheath-flow electrospray ionization.

RATIONALE: Mass spectrometry (MS) is an attractive method for extending capillary-size ion chromatography (cHPIC) to create a valuable technique for speciation analysis. For hyphenation, the aqueous effluent of cHPIC has to be transformed into a volatile mixture for MS while preserving analytical concentrations as well as peak shapes during transfer from cHPIC to MS. Finally, the approach should technically be flexible and easy-to-use. A combination of cHPIC and sheath-flow electrospray ionization (ESI)-MS offers to solve all these challenges. METHODS: cHPIC/sheath-flow-ESI-TOFMS was used in this study for the speciation analysis of various arsenic model compounds. These model compounds were analyzed with different hyphenation setups and configurations of cHPIC/MS and their respective assets and drawbacks were examined and discussed. The parameters (flow rate and composition of sheath liquid) of sheath-flow ESI and their influence on the performance of the spray and the sensitivity of the detector were investigated and compared with those of sheathless ESI. RESULTS: Using an injection valve to couple cHPIC and MS was found to be the best method for hyphenation, since it constitutes a flexible and dead-volume-free approach. The investigation of sheath-flow ESI revealed that the flow rate of the sheath liquid has to resemble the flow rate of the IC effluent to ensure a stable spray and that a composition of 2-propanol/water/ammonia at 50:50:0.2 (v/v/v) suits most applications without unilaterally promoting the sensitivity for either organic or inorganic compounds. The optimized setup and conditions were successfully applied to the analysis of a mixture of important arsenic species and used to determine limits of detection of organic and inorganic arsenic species (3.7 µg L(-1) elemental arsenic). CONCLUSIONS: A method for cHPIC/sheath-flow-ESI-MS was developed. The method was shown to be a valuable tool for speciation and trace analysis. It features no dead volume, fast transfer from IC to MS, only minimal peak-widening, high reproducibility, and the ability to fine-tune the ESI spray for higher sensitivity and stability by adjusting the composition of the sheath-liquid.

Beryllium10: a free and simple tool for creating and managing group safety data sheets.

BACKGROUND: Countless chemicals and mixtures are used in laboratories today, which all possess their own properties and dangers. Therefore, it is important to brief oneself about possible risks and hazards before doing any experiments. However, this task is laborious and time consuming. SUMMARY: Beryllium10 is a program, which supports users by carrying out a large part of the work such as collecting/importing data sets from different providers and compiling most of the information into a single group safety data sheet, which is suitable for having all necessary information at hand while an experiment is in progress. We present here the features of Beryllium10, their implementation, and their design and development criteria and ideas. CONCLUSION: A program for creating and managing of group safety data sheets was developed and released as open source under GPL. The program provides a fast and clear user-interface, and well-conceived design for collecting and managing safety data. It is available for download from the web page http://beryllium.keksecks.de.

Sensing and imaging of oxygen with parts per billion limits of detection and based on the quenching of the delayed fluorescence of (13)C70 fullerene in polymer hosts.

We report on a new method for sensing trace oxygen in the gas phase. It is based on the extreme efficiency of the quenching of the thermally activated delayed fluorescence of isotopically enriched carbon-13 fullerene C(70) ((13)C(70)). This fullerene was dissolved in polymer matrixes of varying oxygen permeability, viz., polystyrene (PS), ethyl cellulose (EC) and an organically modified silica gel ("ormosil"; OS). The sensor films (5-10 µm thick), on photoexcitation at 470 nm, display a strong delayed photoluminescence with peaks between 670 and 700 nm. Its quenching by molecular oxygen was studied at 25 and 60 °C and at concentrations from zero up to 150 ppmv of oxygen in nitrogen. The rapid lifetime determination (RLD) method was applied to determine oxygen-dependent lifetimes and for fluorescence lifetime imaging of oxygen. The lower limits of detection (at 1% quenching) vary with the polymer used (EC ∼250 ppbv, OS ∼320 ppbv, PS ∼530 ppbv at 25 °C) and with temperature. The oxygen sensors reported here are the most sensitive ones described so far.

The pH dependence of the total fluorescence of graphite oxide.

Graphite oxide was characterized by pH dependent excitation-emission matrices from 300 to 500 nm in excitation and from 320 to 600 nm in emission which reveal the presence of two pH steps. These are assigned to the presence of carboxy groups and phenolic hydroxy groups, respectively. Fluorescence is strongest at 470 nm excitation and 555 nm emission. The fluorescence intensity is a function of pH but not of temperature, and is not quenched by oxygen.