Nonsteady-State Electric Circuit in Electrophoresis on Paper: Thermal Consideration of Electrophoretic Lateral-Flow Assays.

Nonsteady-state behaviors are not expected in electric circuits that lack significant capacitance, inductivity, and/or active feedback. Here, we report that electrophoresis on paper─used, e.g., to electrophoretically driven lateral-flow immunoassays (LFIA)─can create a nonsteady-state electric circuit. We studied electrophoresis on 50 × 4 mm nitrocellulose membrane strips utilized in LFIA. The voltage was applied to strip termini immersed in reservoirs with a running buffer. If the electric power of this circuit exceeded approximately 0.5 W, neither the electric current nor the temperature map reached their steady states on a multiminute time scale. The current grew slowly to its maximum and then slowly decreased. The temperature map evolved slowly, with one or more hot spots appearing and disappearing gradually in different positions on the strip. The slow evolution of a temperature map led to the occurrence of a terminal hot spot in which the strip burned. No chaotic behavior was observed, i.e., time dependences of both the current and temperature map were reproducible. We analyzed major processes involved in paper-based electrophoresis and explained the nonsteady-state behavior. Unlike ordinary electric circuits with metal conductors, paper-based electrophoresis involves two slow processes: (i) intense buffer evaporation from hot spots and (ii) buffer supply from the reservoirs by an interplay of the capillary penetration and the electroosmotic flow. These processes affect heat generation and/or dissipation on the strip and, accordingly, the resistivity profile. The slow evolution of the resistivity profile is responsible for the nonsteady-state behavior. The results of our computer modeling support this explanation. The hot spots may have a destructive effect on electrophoretically driven LFIA. To avoid denaturation of immunoreagents, experimentalists should empirically confirm that spatiotemporal temperature maps are compatible with the developed assay.

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.

Fundamental Determinants of the Accuracy of Equilibrium Constants for Affinity Complexes.

The equilibrium constant of a chemical reaction is arguably the key thermodynamic parameter in chemistry; we naturally expect that equilibrium constants are determined accurately. The majority of equilibrium constants determined today are those of binding reactions that form affinity complexes, such as protein-protein, protein-DNA, and protein-small molecule. There is growing awareness that the determination of equilibrium constants for highly stable affinity complexes may be very inaccurate. However, fundamental (i.e., method-independent) determinants of accuracy are poorly understood. Here, we present a study that explicitly shows what the accuracy of equilibrium constants of affinity complexes depends on. This study reveals the critical importance of the choice of concentration of interacting components and creates a theoretical foundation for improving the accuracy of the equilibrium constants. The predicted influence of concentrations on accuracy was confirmed experimentally. The results of this fundamental study provide instructive guidance for experimentalists independently on the method they use.

Electrophoresis-Assisted Multilayer Assembly of Nanoparticles for Sensitive Lateral Flow Immunoassay.

Lateral flow immunoassay (LFIA) is a rapid, simple, and inexpensive point-of-need method. A major limitation of LFIA is a high limit of detection (LOD), which impacts its diagnostic sensitivity. To overcome this limitation, we introduce a signal-enhancement procedure that is performed after completing LFIA and involves controllably moving biotin- and streptavidin-functionalized gold nanoparticles by electrophoresis. The nanoparticles link to immunocomplexes forming multilayer aggregates on the test strip, thus, enhancing the signal. Here, we demonstrate lowering the LOD of hepatitis B surface antigen from approximately 8 to 0.12 ng mL-1 , making it clinically acceptable. Testing 118 clinical samples for hepatitis B showed that signal enhancement increased the diagnostic sensitivity of LFIA from 73 % to 98 % while not affecting its 95 % specificity. Electrophoresis-driven enhancement of LFIA is universal (antigen-independent), takes two minutes, and can be performed by an untrained person.

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.

Quantitative Characterization of Partitioning in Selection of DNA Aptamers for Protein Targets by Capillary Electrophoresis.

Partitioning of protein-DNA complexes from protein-unbound DNA is a key step in selection of DNA aptamers. Conceptually, the partitioning step is characterized by two parameters: transmittance for protein-bound DNA (binders) and transmittance for unbound DNA (nonbinders). Here, we present the first study to reveal how these transmittances depend on experimental conditions; such studies are pivotal to the effective planning and control of selection. Our focus was capillary electrophoresis (CE), which is a partitioning approach of high efficiency. By combining a theoretical model and experimental data, we evaluated the dependence of transmittances of binders and nonbinders on the molecular weight of the protein target in two modes of CE-based partitioning: nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) and ideal-filter capillary electrophoresis (IFCE). Our data suggest that as the molecular weight of the protein target decreases: (i) the transmittance for binders remains close to unity in NECEEM but decreases drastically in IFCE and (ii) the transmittance for nonbinders increases orders of magnitude in NECEEM but remains relatively stable at a very low level in IFCE. To determine the optimal CE conditions for a given size of protein target, a balance between transmittances of binders and nonbinders must be reached; such a balance would ensure the collection of binders of sufficient purity and quantity. We conclude that, as a rule of thumb, IFCE is preferable for large-size protein targets while NECEEM should be the method of choice for small-size protein targets.

Bulk Affinity Assays in Aptamer Selection: Challenges, Theory, and Workflow.

Selection of oligonucleotide aptamers involves consecutive rounds of affinity isolation of target-binding oligonucleotides from a random-sequence oligonucleotide library. Every next round produces an aptamer-enriched library with progressively higher fitness for tight binding to the target. The progress of enrichment can only be accurately assessed with bulk affinity assays in which a library is mixed with the target and one of two quantitative parameters, the fraction of the unbound library (R) or the equilibrium dissociation constant (Kd), is determined. These quantitative parameters are used to help researchers make a key decision of either continuing or stopping the selection. Despite the importance of this decision, the suitability of R and Kd for bulk affinity assays has never been studied theoretically, and researchers rely on intuition when choosing between them. Different approaches used for bulk affinity assays expectedly hinder comparative analyses of selections. Our current work has two goals: to give bulk affinity assays a thorough theoretical consideration and to propose a scientifically justified and practical bulk-affinity-assay approach. We postulate a formal criterion of suitability: a quantitative parameter must satisfy the principle of superposition. R satisfies this principle, while Kd does not, suggesting R as a theoretically preferable parameter. Further, we propose a solution for two limitations of R: its dependence on target concentration and narrow dynamic range. Finally, we demonstrate the use of this algorithm in both computer-simulated and experimental aptamer selection. This study sets a cornerstone in the theory of bulk affinity assays, and it provides researchers with a scientifically sound and instructive approach for conducting bulk affinity assays.

Transient Incomplete Separation of Species with Close Diffusivity to Study the Stability of Affinity Complexes.

Large molecules can be generically separated from small ones, though partially and temporarily, in a pressure-driven flow inside a capillary. This transient incomplete separation has been only applied to species with diffusion coefficients different by at least an order of magnitude. Here, we demonstrate, for the first time, the analytical utility of transient incomplete separation for species with close diffusion coefficients. First, we prove in silico that even a small difference in diffusivity can lead to detectable transient incomplete separation of species. Second, we use computer simulation to prove that such a separation can be used for the reliable determination of equilibrium dissociation constant (Kd) of complexes composed of similar-sized molecules. Finally, we demonstrate experimentally the use of this separation for the accurate determination of Kd value for a protein-aptamer complex. We conclude that "accurate constant via transient incomplete separation" (ACTIS) can serve as a reference method for affinity characterization of protein-aptamer binding in solution.

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.

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.

How to Develop and Prove High-Efficiency Selection of Ligands from Oligonucleotide Libraries: A Universal Framework for Aptamers and DNA-Encoded Small-Molecule Ligands.

Screening molecular libraries for ligands capable of binding proteins is widely used for hit identification in the early drug discovery process. Oligonucleotide libraries provide a very high diversity of compounds, while the combination of the polymerase chain reaction and DNA sequencing allow the identification of ligands in low copy numbers selected from such libraries. Ligand selection from oligonucleotide libraries requires mixing the library with the target followed by the physical separation of the ligand-target complexes from the unbound library. Cumulatively, the low abundance of ligands in the library and the low efficiency of available separation methods necessitate multiple consecutive rounds of partitioning. Multiple rounds of inefficient partitioning make the selection process ineffective and prone to failures. There are continuing efforts to develop a separation method capable of reliably generating a pure pool of ligands in a single round of partitioning; however, none of the proposed methods for single-round selection have been universally adopted. Our analysis revealed that the developers' efforts are disconnected from each other and hindered by the lack of quantitative criteria of selection quality assessment. Here, we present a formalism that describes single-round selection mathematically and provides parameters for quantitative characterization of selection quality. We use this formalism to define a universal strategy for development and validation of single-round selection methods. Finally, we analyze the existing partitioning methods, the published single-round selection reports, and some pertinent practical considerations through the prism of this formalism. This formalism is not an experimental protocol but a framework for correct development of experimental protocols. While single-round selection is not a goal by itself and may not always suffice selection of good-quality ligands, our work will help developers of highly efficient selection approaches to consolidate their efforts under an umbrella of universal quantitative criteria of method development and assessment.

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.

Analytical Challenges in Development of Chemoresistance Predictors for Precision Oncology.

Chemoresistance, i.e., tumor insensitivity to chemotherapy, shortens life expectancy of cancer patients. Despite the availability of new treatment options, initial systemic regimens for solid tumors are dominated by a set of standard chemotherapy drugs, and alternative therapies are used only when a patient has demonstrated chemoresistance clinically. Chemoresistance predictors use laboratory parameters measured on tissue samples to predict the patient's response to chemotherapy and help to avoid application of chemotherapy to chemoresistant patients. Despite thousands of publications on putative chemoresistance predictors, there are only about a dozen predictors that are sufficiently accurate for precision oncology. One of the major reasons for inaccuracy of predictors is inaccuracy of analytical methods utilized to measure their laboratory parameters: an inaccurate method leads to an inaccurate predictor. The goal of this study was to identify analytical challenges in chemoresistance-predictor development and suggest ways to overcome them. Here we describe principles of chemoresistance predictor development via correlating a clinical parameter, which manifests disease state, with a laboratory parameter. We further classify predictors based on the nature of laboratory parameters and analyze advantages and limitations of different predictors using the reliability of analytical methods utilized for measuring laboratory parameters as a criterion. Our eventual focus is on predictors with known mechanisms of reactions involved in drug resistance (drug extrusion, drug degradation, and DNA damage repair) and using rate constants of these reactions to establish accurate and robust laboratory parameters. Many aspects and conclusions of our analysis are applicable to all types of disease biomarkers built upon the correlation of clinical and laboratory parameters.

Necessity and Challenges of Sample Preconcentration in Analysis of Multiple MicroRNAs by Capillary Electrophoresis.

Thousands of putative microRNA (miRNA)-based cancer biomarkers have been reported, but none has been validated for approval by the Food and Drug Administration. One of the reasons for this alarming discrepancy is the lack of a method that is sufficiently robust for carrying out validation studies, which may require analysis of samples from hundreds of patients across multiple institutions and pooling the results together. The capillary electrophoresis (CE)-based hybridization assay proved to be more robust than reversed transcription polymerase chain reaction (the current standard), but its limit of quantification (LOQ) exceeds 10 pM while miRNA concentrations in cell lysates are below 1 pM. Thus, CE-based separation must be preceded by on-column sample preconcentration. Here, we explain the challenges of sample preconcentration for CE-based miRNA analyses and introduce a preconcentration method that can suit CE-based miRNA analysis utilizing peptide nucleic acid (PNA) hybridization probes. The method combines field-amplified sample stacking (FASS) with isotachophoresis (ITP). We proved that FASS-ITP could retain and concentrate both near-neutral PNA with highly negatively charged PNA-miRNA hybrids. We demonstrated that preconcentration by FASS-ITP could be combined with the CE-based separation of the unreacted PNA probes from the PNA-miRNA hybrids and facilitate improvement in LOQ by a factor of 140, down to 0.1 pM. Finally, we applied FASS-ITP-CE for the simultaneous detection of two miRNAs in crude cell lysates and proved that the method was robust when used in complex biological matrices. The 140-fold improvement in LOQ and the robustness to biological matrices will significantly expand the applicability of CE-based miRNA analysis, bringing it closer to becoming a practical tool for validation of miRNA biomarkers.

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.

Spheroid-Based Approach to Assess the Tissue Relevance of Analysis of Dispersed-Settled Tissue Cells by Cytometry of the Reaction Rate Constant.

Cytometry of Reaction Rate Constant (CRRC) uses time-lapse fluorescence microscopy to measure a rate constant of a catalytic reaction in individual cells and, thus, facilitate accurate size determination for cell subpopulations with distinct efficiencies of this reaction. Reliable CRRC requires uniform exposure of cells to the reaction substrate followed by their uniform imaging, which in turn, requires that a tissue sample be disintegrated into a suspension of dispersed cells, and these cells settle on the support surface before being analyzed by CRRC. We call such cells "dispersed-settled" to distinguish them from cells cultured as a monolayer. Studies of the dispersed-settled cells can be tissue-relevant only if the cells maintain their 3D tissue state during the multi-hour CRRC procedure. Here, we propose an approach for assessing tissue relevance of the CRRC-based analysis of the dispersed-settled cells. Our approach utilizes cultured multicellular spheroids as a 3D cell model and cultured cell monolayers as a 2D cell model. The CRRC results of the dispersed-settled cells derived from spheroids are compared to those of spheroids and monolayers in order to find if the dispersed-settled cells are representative of the spheroids. To demonstrate its practical use, we applied this approach to a cellular reaction of multidrug resistance (MDR) transport, which was followed by extrusion of a fluorescent substrate from the cells. The approach proved to be reliable and revealed long-term maintenance of MDR transport in the dispersed-settled cells obtained from cultured ovarian cancer spheroids. Accordingly, CRRC can be used to determine accurately the size of a cell subpopulation with an elevated level of MDR transport in tumor samples, which makes CRRC a suitable method for the development of MDR-based predictors of chemoresistance. The proposed spheroid-based approach for validation of CRRC is applicable to other types of cellular reactions and, thus, will be an indispensable tool for transforming CRRC from an experimental technique into a practical analytical tool.

Empirical predictor of conditions that support ideal-filter capillary electrophoresis.

Ideal-filter CE (IFCE) is a method for the selection of affinity binders for protein targets from oligonucleotide libraries, for example, random-sequence oligonucleotide libraries and DNA-encoded libraries, in a single step of partitioning. In IFCE, protein-oligonucleotide complexes and unbound oligonucleotides move in the opposite directions, facilitating very high efficiency of their partitioning. For any given protein target and oligonucleotide library, protein-oligonucleotide complexes and unbound oligonucleotides move in the opposite directions only for a limited range of EOF mobilities, which, in turn, corresponds to a limited range of pH and ionic strength values of the running buffer. Rational design of IFCE-based partitioning requires a priori knowledge of this range of pH and ionic strength values, and here we introduce an approach to predict this range for a given type of the running buffer. The approach involves measuring EOF mobilities for a relatively wide range of pH and ionic strength (I) values and finding an empirical predictor function that related the EOF mobility with pH and ionic strength. In this work, we developed a predictor function for a running buffer (Tris-HCl) that is commonly used in CE-based partitioning of affinity binders for protein targets. This predictor function can be immediately used for the rational design of IFCE-based partitioning in this running buffer, while the described approach will be used to develop predictor functions for other types of running buffer if needed.

Spherical-Shape Assumption for Protein-Aptamer Complexes Facilitates Prediction of Their Electrophoretic Mobility.

DNA aptamers are single-strand DNA (ssDNA) capable of selectively and tightly binding a target molecule. Capillary electrophoresis-based selection of aptamers for protein targets requires the knowledge of electrophoretic mobilities of protein-aptamer complexes, while measuring these mobilities requires having the aptamers. Here, we report on breaking this vicious circle. We introduce a mathematical model that allows prediction of protein-aptamer complex mobility, while requiring only three easy-to-determine input parameters: the number N of nucleotides in the aptamer, electrophoretic mobility of N-nucleotide-long ssDNA, and a sum molecular weight of the protein-aptamer complex. The model was derived upon simplifying assumptions of a spherical shape of the protein-aptamer complex. According to this model, the protein-aptamer complex mobility is a linear function of a combination of the three input parameters with empirically determined line's intercept and slope. The intercept and slope were determined using experimental data for seven complexes. The model was then cross-validated with the leave-one-out approach revealing only 2% residual standard deviations for both the slope and the intercept. Such a precise determination of these constants allowed accurate mobility prediction for the excluded complexes with only a 3% maximum deviation from the experimentally determined mobilities. The model was tested by applying it to three protein-aptamer complexes that were not a part of the training/cross-validation set; deviations of the predicted mobilities from the experimentally determined ones were within 5% of the latter. To complete this study, the model was fine-tuned using the 10 complexes. Our results strongly suggest the validity of the spherical-shape assumption for the protein-aptamer complexes when considering complex mobility. The developed model will make it possible to rationally design capillary electrophoresis-based selection of DNA aptamers for protein targets.

Determination of the Equilibrium Constant and Rate Constant of Protein-Oligonucleotide Complex Dissociation under the Conditions of Ideal-Filter Capillary Electrophoresis.

Ideal-filter capillary electrophoresis (IFCE) allows selection of protein binders from oligonucleotide libraries in a single step of partitioning in which protein-bound and unbound oligonucleotides move in the opposite directions. In IFCE, the unbound oligonucleotide does not reach the detector, imposing a problem for finding the equilibrium constant ( Kd) and rate constant ( koff) of protein-oligonucleotide complex dissociation. We report a double-passage approach that allows finding Kd and koff under the IFCE conditions, i.e. near-physiological pH and ionic strength. First, a plug of the protein-oligonucleotide equilibrium mixture passes to the detector in a pressure-driven flow, allowing for both the complex and free oligonucleotide to be detected as a single first peak. Second, the pressure is turned off and the voltage is applied to reverse the migration of only the complex which is detected as the second peak. The experiment is repeated with a lower voltage consequently resulting in longer travel time of the complex to the detector, greater extent of complex dissociation, and the decreased area of the second peak. Finally, the peak areas are used to calculate the values of Kd and koff. Here we explain theoretical and practical aspects of the double-passage approach, prove its validity quantitatively, and, demonstrate its application to determine Kd and koff for an affinity complex between a protein and its DNA aptamer. The double-passage approach for finding Kd and koff of protein-oligonucleotide complexes under the IFCE conditions is a perfect complement for IFCE-based selection of protein binders from oligonucleotide libraries.

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.