Diffusion Of Laminar Flow Profiles Research Articles

Human protein kinase inhibitor screening by capillary electrophoresis using transverse diffusion of laminar flow profiles for reactant mixing

A capillary electrophoresis (CE)-based enzyme assay method has been developed to screen protein kinase inhibitors. Four human kinases GSK3β, DYRK1A, CDK5/p25 and CDK1/cyclin B were chosen to test this novel method. These enzymes have been identified as very promising targets to develop treatments against cancer and neurodegenerative diseases. The efficiency of drugs against these relevant biological targets has never been carried out by CE. For this proposal, the capillary was used as a nanoreactor in which four reactants (the enzyme, its two substrates and its potential inhibitor) were successively injected, mixed by using transverse diffusion of laminar flow profiles and incubated. The adenosine 5′-diphosphate (ADP) formed during the enzymatic reaction was detected by UV and quantified. The efficiency of the developed CE method was validated by determining the IC50 values of a wide variety of inhibitors covering a large domain of affinity toward kinases and containing representative and chemically divergent skeletons. Excellent agreement was found between the results obtained by CE and those reported in the literature when using conventional radiometric enzyme assays. Moreover, CE was successfully used to determine the inhibitory effect of several potential inhibitors that was not yet assessed by conventional methods and is crucial for structure activity relation studies. This novel CE method is simple, rapid, very economic (few tens of nanoliters per IC50) and eco-friendly since no radioactivity was required. It could be extended to high-throughput screening of kinase inhibitors, which is of great interest for biomedical and pharmaceutical research fields.

Transverse diffusion of laminar flow profiles – a generic method for mixing reactants in capillary microreactor

The capillary is an attractive format for integrated microanalyses, which start with the injection of separate reactants into the capillary and their mixing inside the capillary. Due to the nonturbulent nature of flow inside the capillary, mixing reactants in a generic way is a challenging task. Three approaches have been suggested as a solution: mixing by electrophoresis, mixing by longitudinal diffusion, and, most recently, mixing by transverse diffusion of laminar flow profiles (TDLFP). This is the first review on TDLFP, describing: (i) the physical basis of the method, (ii) its theory, (iii) analytical and numerical solutions for the calculation of concentration profiles of mixed reactants, (iv) up-to-date applications, and (v) problems to be solved and future directions.

Kinetic capillary electrophoresis-based affinity screening of aptamer clones

DNA aptamers are single stranded DNA (ssDNA) molecules artificially selected from random-sequence DNA libraries for their specific binding to a certain target. DNA aptamers have a number of advantages over antibodies and promise to replace them in both diagnostic and therapeutic applications. The development of DNA aptamers involves three major stages: library enrichment, obtaining individual DNA clones, and the affinity screening of the clones. The purpose of the screening is to obtain the nucleotide sequences of aptamers and the binding parameters of their interaction with the target. Highly efficient approaches have been recently developed for the first two stages, while the third stage remained the rate-limiting one. Here, we introduce a new method for affinity screening of individual DNA aptamer clones. The proposed method amalgamates: (i) aptamer amplification by asymmetric PCR (PCR with a primer ratio different from unity), (ii) analysis of aptamer–target interaction, combining in-capillary mixing of reactants by transverse diffusion of laminar flow profiles (TDLFP) and affinity analysis using kinetic capillary electrophoresis (KCE), and (iii) sequencing of only aptamers with satisfying binding parameters. For the first time we showed that aptamer clones can be directly used in TDLFP/KCE-based affinity analysis without an additional purification step after asymmetric PCR amplification. We also demonstrated that mathematical modeling of TDLFP-based mixing allows for the determination of K d values for the in-capillary reaction of an aptamer and a target and that the obtained K d values can be used for the accurate affinity ranking of aptamers. The proposed method does not require the knowledge of aptamer sequences before screening, avoids lengthy (3–5 h) purification steps of aptamer clones, and minimizes reagent consumption to nanoliters.

Mathematical Model for Mixing Reactants in a Capillary Microreactor by Transverse Diffusion of Laminar Flow Profiles

Transverse diffusion of laminar flow profiles (TDLFP) was recently suggested as a generic approach for mixing reactants inside a capillary microreactor. Conceptually, solutions of reactants are injected inside the capillary by high pressure as a series of consecutive plugs. Because of the laminar nature of the flow inside the capillary, the nondiffused plugs have parabolic profiles with predominantly longitudinal interfaces between the plugs. After the injection, the reactants are mixed by transverse diffusion across the longitudinal interfaces. TDLFP-based mixing is still in its infancy as only the principle was proved. Here, we develop the theory of TDLFP and introduce a dimensionless parameter, York number, which can be used in predicting the quality of TDLFP-based mixing. The theory uses a single simplifying assumption that the longitudinal diffusion is negligible; this assumption is readily satisfied. We then develop a numerical model of TDLFP and use it to simulate the concentration profiles of three reactants mixed by TDLFP in the capillary. The correlation between the York number and quality of mixing is analyzed. Two ways of improving the quality of TDLFP-based mixing are suggested and studied: (i) increasing the longitudinal interface between the plugs by a long last plug of a solvent and (ii) "shaking" the injected reactants by a series of alternating negative and positive pressure pulses. The developed theory and computational simulation of TDLFP will stimulate the practical use of capillary microreactors.

“Inject-Mix-React-Separate-and-Quantitate” (IMReSQ) Method for Screening Enzyme Inhibitors

Many regulatory enzymes are considered attractive therapeutic targets, and their inhibitors are potential drug candidates. Screening combinatorial libraries for enzyme inhibitors is pivotal to identifying hit compounds for the development of drugs targeting regulatory enzymes. Here, we introduce the first inhibitor screening method that consumes only nanoliters of the reactant solutions and is applicable to regulatory enzymes. The method is termed inject-mix-react-separate-and-quantitate (IMReSQ) and includes five steps. First, nanoliter volumes of substrate, candidate inhibitor, and enzyme solutions are injected by pressure into a capillary as separate plugs. Second, the plugs are mixed inside this capillary microreactor by transverse diffusion of laminar flow profiles. Third, the reaction mixture is incubated to form the enzymatic product. Fourth, the product is separated from the substrate inside the capillary by electrophoresis. Fifth, the amounts of the product and substrate are quantitated. In this proof-of-principle work, we applied IMReSQ to study inhibition of recently cloned protein farnesyltransferase from parasite Entamoeba histolytica. This enzyme is a potential therapeutic target for antiparasitic drugs. We identified three previously unknown inhibitors of this enzyme and proved that IMReSQ could be used for quantitatively ranking the potencies of inhibitors.

Cell lysis inside the capillary facilitated by transverse diffusion of laminar flow profiles (TDLFP)

Chemical cytometry studies the molecular composition of individual cells by means of capillary electrophoresis or capillary chromatography. In one of its realizations an intact cell is injected inside the capillary, the plasma membrane is disrupted to release the cellular contents into the separation buffer, and, finally, the molecules of interest are separated and detected. The solubilization of the plasma membrane with a surfactant is a simple and efficient way of achieving cell lysis inside the capillary. To facilitate cell lysis by a surfactant the cell has to be contacted with the surfactant inside the capillary. We recently introduced a generic method for mixing solutions inside the capillary termed transverse diffusion of laminar flow profiles (TDLFP). In this work, we propose that TDLFP can facilitate efficient cell lysis inside the capillary. Conceptually, a short plug of the surfactant is injected by pressure prior to cell injection. The cell is then injected by pressure within a plug of the physiological buffer. Due to the parabolic profiles of pressure-driven laminar flows the interface between the plug of the surfactant and that of the physiological buffer is predominantly longitudinal. Transverse diffusion mixes the surfactant with the physiological buffer, which leads to surfactant's contact with the cell and subsequent cell lysis. Here, we demonstrate that the proposed concept is valid. TDLFP-facilitated cell lysis by a short plug of the surfactant allows us to exclude the surfactant from the run buffer, and, hence, facilitates modes of separation, which are incompatible with the surfactant's presence in the run buffer. In addition to cell lysis, TDLFP will be used to mix the cellular components with labeling reactants, affinity probes, inhibitors, etc. We foresee that the generic nature and enabling capabilities of TDLFP will speed up the maturation of chemical cytometry into a practical bioanalytical tool.

Nonequilibrium Capillary Electrophoresis of Equilibrium Mixtures (NECEEM): A Novel Method for Biomolecular Screening

Nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) is a new separation-based affinity method. It has kinetic capabilities exceeding those of surface plasmon resonance (SPR) and does not require immobilization of molecules on the surface. Another distinctive feature of NECEEM is that--if it is combined with an advanced method for the mixing solutions inside a capillary, termed transverse diffusion of laminar flow profiles (TDLFP)--it requires only nanoliter volumes of solutions. The proven applications of NECEEM to biomolecular screening include 1) measuring kinetic and thermodynamic parameters of protein-ligand interactions, 2) quantitative affinity analyses of proteins and hybridization analyses of DNA and RNA, and 3) selection of binding ligands from combinatorial libraries. NECEEM is easy to automate and parallelize. Because of its simplicity and analytical power, NECEEM has the potential to become a workhorse in studies of biomolecular interactions. The author reviews theoretical bases of NECEEM and its applications to biomolecular screening.

Transverse Diffusion of Laminar Flow Profiles To Produce Capillary Nanoreactors

We introduce transverse diffusion of laminar flow profiles (TDLFP), the first generic method for mixing two or more reactants inside capillaries. Conceptually, solutions of reactants are injected inside the capillary by pressure as a series of consecutive plugs. Due to the laminar nature of flow inside the capillary, the nondiffused plugs have parabolic profiles with predominantly longitudinal interfaces between them. After injection, the plugs are mixed by transverse diffusion; longitudinal diffusion does not contribute to mixing. To prove the principle, we used TDLFP to mix two reactants-an enzyme and its substrate. After mixing the reactants by TDLFP, we incubated reaction mixtures for different periods of time and measured the reaction kinetics. We found that the reaction proceeded in time- and concentration-dependent fashion, thus confirming that the reactants were mixed by TDLFP. Remarkably, the experimental reaction kinetics were not only in qualitative agreement but also in good quantitative agreement with theoretically predicted ones. TDLFP has a number of enabling features. By facilitating the preparation of reaction mixtures inside the capillary, TDLFP lowers reagent consumption to nanoliters (microliters are required for conventionally mixing reagents in a vial). The reaction products can be then analyzed "on-line" by capillary separation coupled with optical, electrochemical, or mass spectrometric detection. The combination of TDLFP with capillary separation will be an indispensable tool in screening large combinatorial libraries for affinity probes and drug candidates: a few microliters of a target protein will be sufficient to screen thousands of compounds. The new method paves the road to a wide use of capillary nanoreactors in different areas of physical and life sciences.