Electrokinetic mixing in electrode-embedded multiwell plates to improve the diffusion limited kinetics of biosensing platforms.

Rapid and accurate biosensing with low concentrations of the analytes is usually challenged by the diffusion limited reaction kinetics. Thus, as a remedy, long incubation times or excess amounts of the reagents are employed to ensure the reactions to go to completion. Therefore, mixing becomes both a serious problem and necessity to overcome that diffusion limitation and homogenize the samples, especially for the biochemical reactions that take place in multiwell plates. Because the current mixing platforms such as shakers/vortexers, sonicators, magnetic stirrers and acoustic mixers have disadvantages including, but not limited to, being invasive/harfmul to the samples, causing the samples to splash out or stick to the walls of the wells and allowing foreign compartments to enter the solutions in the wells. Here we propose a noninvasive and safer (considering the risk of sample loss) technology that provides electrokinetic-mixing (EKM) of the reagents placed in electrode-embedded multiwell plates where the incubation times, or in other words, the time required for the desired molecules to meet in stationary solutions, can be reduced substantially. In order to demonstrate the power of this innovation, in this specific case, a simple Förster resonance energy transfer (FRET) based quenching bioplatform was adopted, where a molecular beacon DNA (MB) modified with sulfhydryl (-SH) and fluorescein (FITC) dye at opposite terminals was incubated with 10 nm sized gold nanoparticles (AuNPs) in the wells of an electrode-embedded multiwell plate, in which a printed circuit board (PCB) was attached at the bottom to control the liquid flows by EKM. When the MB binds to AuNPs through thiolate chemistry in the solution, FITC dye comes in close proximity to the AuNP surface and the emission is quenched via FRET principle. Thus, this quenching percentage over time was our comparison parameter for the mixing and no mixing cases to demonstrate the impact of mixing on the quenching kinetics. This reaction was conducted with different concentrations of AuNPs to observe the impact of mixing on MB quenching kinetics when the concentrations of the AuNPs were increased. Total quenching efficiency could go up to 90% in the presence of the AuNPs and it took about 60 min to reach stability. When the EKM was involved, fluorescence quenching time for the MBs could be reduced by up to 4.1 times. Thus, it was demonstrated that this technology may improve the kinetics of the diffusion limited biological reactions take place in multiwell plates substantially so that it may be adopted in various different sensing platforms for rapid measurements.

DNA Antenna Tile-Associated Deoxyribozyme Sensor with Improved Sensitivity.

Some natural enzymes increase the rate of diffusion-limited reactions by facilitating substrate flow to their active sites. Inspired by this natural phenomenon, we developed a strategy for efficient substrate delivery to a deoxyribozyme (DZ) catalytic sensor. This resulted in a three- to fourfold increase in sensitivity and up to a ninefold improvement in the detection limit. The reported strategy can be used to enhance catalytic efficiency of diffusion-limited enzymes and to improve sensitivity of enzyme-based biosensors.

Monte Carlo simulations of enzymatic reactions in crowded media. Effect of the enzyme-obstacle relative size.

We perform Monte Carlo simulations in three-dimensional (3D) lattice in order to study diffusion-controlled and mixed activation-diffusion reactions following an irreversible Michaelis-Menten scheme in crowded media. The simulation data reveal the rate coefficient dependence on time for diffusion-controlled bimolecular reactions developing in three-dimensional media with obstacles, as predicted by fractal kinetics approach. For the cases of mixed activation-diffusion reactions, the fractality of the reaction decreases as the activation control increases. We propose a modified form of the Zipf-Mandelbrot equation to describe the time dependence of the rate coefficient, k(t)=k0(1+t/τ)(-)(h). This equation provides a good description of the fractal regime and it may be split into two terms: one that corresponds to the initial rate constant (k0) and the other one correlated with the kinetics fractality. Additionally, the proposed equation contains and links two limit expressions corresponding to short and large periods of time: k1=k0 (for t≪τ) that relates to classical kinetics and the well-known Kopelman's equation k∼t(-)(h) (for t≫τ) associated to fractal kinetics. The τ parameter has the meaning of a crossover time between these two limiting behaviours. The value of k0 is mainly dependent on the excluded volume and the enzyme-obstacle relative size. This dependence can be explained in terms of the radius of an average confined volume that every enzyme molecule feels, and correlates very well with the crossover length obtained in previous studies of enzyme diffusion in crowding media.

Crowding, diffusion, and biochemical reactions.

Diffusion is the basic mode of transport for molecules in living cells. Diffusion leads to dispersion of individual molecules, but it is also the driving force behind biochemical reactions and pattern formation as diffusional motion mediates reactant encounters. Owing to macromolecular crowding in all cellular fluids and biomembranes, diffusion of molecules in cells is quite different from the motion observed in dilute solutions in a test tube. Hindered and anomalous diffusion are seen in cells, and biochemical reactions are affected by these. This review is intended to give an introduction and a brief overview about causes and consequences of crowding-induced diffusion anomalies and their impact on biochemical reactions.