Nuclemeter: a reaction-diffusion based method for quantifying nucleic acids undergoing enzymatic amplification.

Real-time amplification and quantification of specific nucleic acid sequences plays a major role in medical and biotechnological applications. In the case of infectious diseases, such as HIV, quantification of the pathogen-load in patient specimens is critical to assess disease progression and effectiveness of drug therapy. Typically, nucleic acid quantification requires expensive instruments, such as real-time PCR machines, which are not appropriate for on-site use and for low-resource settings. This paper describes a simple, low-cost, reaction-diffusion based method for end-point quantification of target nucleic acids undergoing enzymatic amplification. The number of target molecules is inferred from the position of the reaction-diffusion front, analogous to reading temperature in a mercury thermometer. The method was tested for HIV viral load monitoring and performed on par with conventional benchtop methods. The proposed method is suitable for nucleic acid quantification at point of care, compatible with multiplexing and high-throughput processing, and can function instrument-free.

Scaling relationship and optimization of double-pulse electroporation.

The efficacy of electroporation is known to vary significantly across a wide variety of biological research and clinical applications, but as of this writing, a generalized approach to simultaneously improve efficiency and maintain viability has not been available in the literature. To address that discrepancy, we here outline an approach that is based on the mapping of the scaling relationships among electroporation-mediated molecular delivery, cellular viability, and electric pulse parameters. The delivery of Fluorescein-Dextran into 3T3 mouse fibroblast cells was used as a model system. The pulse was rationally split into two sequential phases: a first precursor for permeabilization, followed by a second one for molecular delivery. Extensive data in the parameter space of the second pulse strength and duration were collected and analyzed with flow cytometry. The fluorescence intensity correlated linearly with the second pulse duration, confirming the dominant role of electrophoresis in delivery. The delivery efficiency exhibited a characteristic sigmoidal dependence on the field strength. An examination of short-term cell death using 7-Aminoactinomycin D demonstrated a convincing linear correlation with respect to the electrical energy. Based on these scaling relationships, an optimal field strength becomes identifiable. A model study was also performed, and the results were compared with the experimental data to elucidate underlying mechanisms. The comparison reveals the existence of a critical transmembrane potential above which delivery with the second pulse becomes effective. Together, these efforts establish a general route to enhance the functionality of electroporation.

Quantification of propidium iodide delivery using millisecond electric pulses: experiments.

The transport mechanisms in electroporation-mediated molecular delivery are experimentally investigated and quantified. In particular, the uptake of propidium iodide (PI) into single 3T3 fibroblasts is investigated with time- and space-resolved fluorescence microscopy, and as a function of extracellular buffer conductivity. During the pulse, both the peak and the total integrated fluorescence intensity exhibit an inverse correlation with extracellular conductivity. This behavior can be explained by an electrokinetic phenomenon known as Field-Amplified Sample Stacking (FASS). Furthermore, the respective contributions from electrophoresis and diffusion have been quantified; the former is shown to be consistently higher than the latter for the experimental conditions considered. The results are compared with a compact model to predict electrophoresis-mediated transport, and good agreement is found between the two. The combination of the experimental and modeling efforts provides an effective means for the quantitative diagnosis of electroporation.

Vesicle deformation and poration under strong dc electric fields.

When subject to applied electric pulses, a lipid membrane exhibits complex responses including electrodeformation and electroporation. In this work, the electrodeformation of giant unilamellar vesicles under strong dc electric fields was investigated. Specifically, the degree of deformation was quantified as a function of the applied field strength and the electrical conductivity ratio of the fluids inside and outside of the vesicles. The vesicles were made from L-α-phosphatidylcholine with diameters ranging from 14 to 30 µm. Experiments were performed with field strengths ranging from 0.9 to 2.0 kV/cm, and intra-to-extra-vesicular conductivity ratios varying between 1.92 and 53.0. With these parametric configurations, the vesicles exhibited prolate elongations along the direction of the electric field. The degree of deformation was, in general, significant. In some cases, the aspect ratio of a deformed vesicle exceeded 10, representing a strong-deformation regime previously not explored. The aspect ratio scaled quadratically with the field strength, and increased asymptotically to a maximum value at high conductivity ratios. Appreciable area and volumetric changes were observed both during and after pulsation, indicating the concurrence of electroporation. A theoretical model is developed to predict these large deformations in the strongly permeabilized limit, and the results are compared with the experimental data. Both agreements and discrepancies are found, and the model limitations and possible extensions are discussed.