An efficient and high-throughput electroporation microchip applicable for siRNA delivery

Here we report a novel electroporation microchip with great performance and compatibility with the standard multi-well plate used in biological research. The novel annular interdigitated electrode design makes it possible to achieve efficient cell transfection as high as 90% under low-strength electrical pulses, thereby circumventing the many adverse effects of conventional cuvette-type and previously reported microchip-based electroporation devices. Using this system, we demonstrated substantially improved cell transfection efficacy and viability in cultured and primary cells, for both plasmid and synthetic siRNA. Improvements of this system open new opportunities for high-throughput applications of siRNA technology in basic and biomedical research.     

Gene delivery by electroporation after dielectrophoretic positioning of cells in a non-uniform electric field

We report the use of dielectrophoresis (DEP) to position U-937 monocytes within a non-uniform electric field, prior to electroporation (EP) for gene delivery. DEP positioning and EP pulsing were both accomplished using a common set of inert planar electrodes, micro-fabricated on a glass substrate. A single-shell model of the cell's dielectric properties and finite-element modeling of the electric field distribution permitted us to predict the major features of cell positioning. The extent to which electric pulses increased the permeability of the cell membranes to fluorescent molecules and to pEGFPLuc DNA plasmids were found to depend on prior positioning. For a given set of pulse parameters, EP was either irreversible (resulting in cytolysis), reversible (leading to gene delivery), or not detectable, depending on where cells were positioned. Our results clearly demonstrate that position-dependent EP of cells in a non-uniform electric field can be controlled by DEP.     

Effects of the electric field distribution on microchip valving performance

Valving characteristics on microfluidic devices were controlled through manipulation of the electric field strengths during both the sample loading and dispensing steps. Three sample loading profiles for the constant volume valve (pinched injection) in conjunction with four dispensing schemes were investigated to study valving performance. The sample confinement profiles for the sample loading step consisted of a weakly pinched sample, a medium pinched sample, and a strongly pinched sample. Four dispensing schemes varied the electric field strengths in the sample and sample waste channels relative to the analysis channel to control the volume of the sample dispensed from the valve. The axial extent of the sample plug decreased as the electric field strengths in the sample and sample waste channels were raised relative to the analysis channel. In addition, a trade-off existed between sample plug length and sensitivity.     

Enhancement of an electroporation system for gene delivery using electrophoresis with a planar electrode

In this paper a new electroporation (EP) system is developed, which includes an EP microchip and a logic circuit, which combined with electrophoresis (ES), can provide site-specific enhancement of gene concentration. In this ES-EP microchip, an arc planar electrode provides the ES function for DNA attraction, and interdigitated array electrodes provide appropriate electric fields for the EP on the chip surface. In addition, the adherent cells can be manipulated in situ without detachment of the ES-EP microchip, which performs the "Lab on a chip". Experimental results have shown that the efficiency of gene transfection with an attracting-electric field (35.89%) becomes much higher than that without an attracting-electric field (16.62%). Cell numbers as low as 10(4) cells, and DNA as little as 4 microg are sufficient for evaluating the phenotypic effects following the over-expression of the introduced genes on the ES-EP microchip. The proposed system has the advantages of portability, cost-effectiveness, a high transfection rate and ease of operation.     

Flow splitting at the inlet electrode as a method for decreasing the electric current in electric field assisted liquid chromatography

The combination of pressurized flow and electric field offers, with the use of capillary columns, several options for retention control. However, it has been shown that the utility of this technique is strongly limited by the high electric current that is generated at the high electric field strengths that are needed. We have earlier shown that the high current is a result of locally increased mobile phase ion concentration in the electric field, particularly around the inlet electrode. In this paper, we report that by splitting the mobile phase flow around the inlet electrode a relatively constant ion concentration around the electrode can be obtained and the high currents are there by reduced.     

Determination of the electric field and anomalous heating caused by exponential pulses with aluminum electrodes in electroporation experiments

Electroporation is well known to depend non-linearly on the magnitude and duration of the change ΔU(t) in transmembrane voltage. In the case of cell suspension experiments, an electric field E_e(t) within the electrolyte causes ΔU(t), which is governed by both the size and shape of a cell, and also by E_e(t). It is therefore important to determine the magnitude and time dependence of the electric field to which cells are actually exposed in electroporation experiments. This can be significantly different from the nominal field E_n, which is calculated by using electrode voltages and geometries alone. Throughout we used single, nominally exponential pulses with time constants τ_pulse ranging from about 0.6 to 5 ms and found that E_e was always less than E_n. In order to determine the actual electric field pulse, we measured the voltage across the electrodes, the current through the cuvette, the temperature rise of the pulsing medium, and the voltage across two special electrodes placed within the cuvette. From these measurements we calculated the field strength inside the cuvette using two different methods. In addition, we compared the measured temperature rise with that expected from the electrical power dissipation. In some cases there was much larger (“anomalous”) heating, due to interfacial electrochemical heat production; for one pulsing solution T_e(t) was about 30 K larger than expected. These effects are important for experiments aimed at elucidating the electroporation mechanism, comparing results obtained under different conditions, and guiding applications.     

Effect of sample and substrate electric properties on the electric field enhancement at the apex of SPM nanotips

Finite element (FE) models were built to define the optimal experimental conditions for tip-enhanced Raman spectroscopy (TERS) of thin samples. TERS experimental conditions were mimicked by including in the FE models dielectric or metallic substrates with thin dielectric samples and by considering the wavelength dependence of the dielectric properties for the metallic materials. Electromagnetic coupling between the substrate/sample and the SPM tips led to dramatic changes of both the spatial distribution and magnitude of the scattered electric field which depended on the substrate dielectric permittivity and excitation wavelength. Raman scattering as high as 10(8) with a spatial resolution of approximately 8 nm was estimated for gold SPM tips and gold substrate when excitation is performed at 532 nm (near-resonance wavelength). For dielectric samples (approximately 4 nm thick), the enhancement of Raman scattering intensity is estimated at approximately 10(5); this does not depend significantly on the sample dielectric permittivity for dielectric samples. These results suggest that TERS experimental conditions should be estimated and optimized for every individual application considering the geometric factors and electric properties of the materials involved. Such optimizations could enlarge the range of applications for TERS to samples eliciting weaker intrinsic Raman scattering, such as biological samples.     

Advances in electric field and atomic surface derived properties from experimental electron densities

The present study is devoted to a general use of the Gauss law. This is applied to the atomic surfaces derived from the topological analysis of the electron density. The method proposed here is entirely numerical, robust and does not necessitate any specific parametrization of the atomic surfaces. We focus on two fundamental properties: the atomic charges and the electrostatic forces acting on atoms in molecules. Application is made on experimental electron densities modelized by the Hansen-Coppens model from which the electric field is derived for a heterogenic set of compounds: water molecule, NO(3) anion, bis-triazine molecule and MgO cluster. Charges and electrostatic forces are estimated by the atomic surface flux of the electric field and the Maxwell stress tensor, respectively. The charges obtained from the present method are in good agreement with those issued from the conventional volume integration. Both Feynman and Ehrenfest forces as well as the electrostatic potential at the nuclei (EPN) are here estimated from the experimental electron densities. The values found for the molecular compounds are presented and discussed in the scope of the mechanics of atomic interactions.     

环糊精超分子聚合物在药物/基因递送载体领域的研究进展

超分子聚合物通常以非共价键作为构筑驱动力,其结构具有动态可逆的特点,在新型响应性聚合物材料中具有突出优势。环糊精可通过主客体识别作用与客体分子如二茂铁、偶氮苯、金刚烷、苯环等形成包合,以此构筑的超分子组装体展现出丰富的自组装-解组装特性、刺激响应性、较低的细胞毒性和较好的生物相容性,有望在药物/基因载体领域得到应用。本文从环糊精超分子聚合物的生物医用出发,着重对近年来环糊精超分子聚合物载体在药物控制释放、基因转染以及药物/基因共递送三方面的研究进展进行了总结和评述,并在此基础上展望了环糊精超分子聚合物的研究方向和发展趋势。     

Deoxyribonucleic acid modified poly(dimethylsiloxane) microfluidic channels for the enhancement of microchip electrophoresis

In this paper, deoxyribonucleic acid (DNA) was employed to construct a functional film on the PDMS microfluidic channel surface and apply to perform electrophoresis coupled with electrochemical detection. The functional film was formed by sequentially immobilizing chitosan and DNA to the PDMS microfluidic channel surface using the layer-by-layer assembly. The polysaccharide backbone of chitosan can be strongly adsorbed onto the hydrophobic PDMS surface through electrostatic interaction in the acidic media, meanwhile, chitosan contains one protonatable functional moiety resulting in a strong electrostatic interactions between the surface amine group of chitosan and the charged phosphate backbone of DNA at low pH, which generates a hydrophilic microchannel surface and reveals perfect resistance to nonspecific adsorption of analytes. Aminophenol isomers (p-, o-, and m-aminophenol) served as a separation model to evaluate the effect of the functional PDMS microfluidic chips. The results clearly showed that these analytes were efficiently separated within 60 s in a 3.7 cm long separation channel and successfully detected on the modified microchip coupled with in-channel amperometric detection mode at a single carbon fiber electrode. The theoretical plate numbers were 74,021, 92,658 and 60,552 N m^?1 at the separation voltage of 900 V with the detection limits of 1.6, 4.7 and 2.5 μM (S/N = 3) for p-, o-, and m-aminophenol, respectively. In addition, this report offered an effective means for preparing hydrophilic and biocompatible PDMS microchannel surface, which would facilitate the use of microfluidic devices for more widespread applications.     

Magnetically separable template assisted iron nanoparticle for the enhancement of latent fingerprints

The article focuses on the cost-effective and high-quality non-destructive template-assisted magnetic mesoporous iron nanomaterial for latent Fingerprint examination (LFPs). The iron nanomaterial is synthesized using the CTAB template by the co-precipitation method. The synthesized nanomaterial is characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), N₂ sorption- Brunauer-Emmett-Teller (BET), Fourier transform infrared spectroscopy (FTIR), Field Emission Scanning Electron Microscopy (FE-SEM), EDX elemental mapping, Vibrating sample magnetometer (VSM), UV–Visible spectroscopy, and XPS. The N2 sorption showed that the synthesized material is mesoporous with an H1 hysteresis loop. The TEM images showed the spherical shape and particle size to be around 55–65 nm. The VSM analysis successfully established its ferromagnetic nature. The synthesized nanomaterial showed excellent magnetic recovery after its utilization and retained fingerprints for 2–3 months with the same resolution. We collected the fingerprint on both non-porous and non-porous surfaces with better-developed ridges of fingerprints compared to conventional fingerprinting dyes (charcoal).     

Degradable self-assembling dendrons for gene delivery: experimental and theoretical insights into the barriers to cellular uptake

This paper uses a combined experimental and theoretical approach to gain unique insight into gene delivery. We report the synthesis and investigation of a new family of second-generation dendrons with four triamine surface ligands capable of binding to DNA, degradable aliphatic-ester dendritic scaffolds, and hydrophobic units at their focal points. Dendron self-assembly significantly enhances DNA binding as monitored by a range of experimental methods and confirmed by multiscale modeling. Cellular uptake studies indicate that some of these dendrons are highly effective at transporting DNA into cells (ca. 10 times better than poly(ethyleneimine), PEI). However, levels of transgene expression are relatively low (ca. 10% of PEI). This indicates that these dendrons cannot navigate all of the intracellular barriers to gene delivery. The addition of chloroquine indicates that endosomal escape is not the limiting factor in this case, and it is shown, both experimentally and theoretically, that gene delivery can be correlated with the ability of the dendron assemblies to release DNA. Mass spectrometric assays demonstrate that the dendrons, as intended, do degrade under biologically relevant conditions over a period of hours. Multiscale modeling of degraded dendron structures suggests that complete dendron degradation would be required for DNA release. Importantly, in the presence of the lower pH associated with endosomes, or when bound to DNA, complete degradation of these dendrons becomes ineffective on the transfection time scale-we propose this explains the poor transfection performance of these dendrons. As such, this paper demonstrates that taking this kind of multidisciplinary approach can yield a fundamental insight into the way in which dendrons can navigate barriers to cellular uptake. Lessons learned from this work will inform future dendron design for enhanced gene delivery.     

A theoretical and experimental study of the electric field and magnetic anisotropy effects on proton chemical shifts

Proton magnetic shielding constants are divided into different contributions using the IPPP technique (inner projections of the polarization propagator). Total magnetic shielding constants are calculated within the CHF-INDO-GIAO approach (coupled-Hartree-Fock-INDO-gauge-invariant atomic orbitals). In order to compare the electric field and magnetic anisotropy effects of neighbouring groups, two model compounds were chosen, namely, ethyl cyanoformate, I, and ethylformate, II, which show to frozen and unequally populated rotamers each at room temperature. Their proton spectra were measured and the difference in shielding of methylene protons in each pair of rotamers was theoretically analysed with the abovementioned technique. The experimental difference in chemical shifts is quantitatively reproduced with the present analysis.     

In vitro demonstration of the heavy-atom effect for photodynamic therapy

Photodynamic therapy (PDT) is an emerging treatment modality for a range of disease classes, both cancerous and noncancerous. This has brought about an active pursuit of new PDT agents that can be optimized for the unique set of photophysical characteristics that are required for a successful clinical agent. We now describe a totally new class of PDT agent, the BF2-chelated 3,5-diaryl-1H-pyrrol-2-yl-3,5-diarylpyrrol-2-ylideneamines (tetraarylazadipyrromethenes). Optimized synthetic procedures have been developed to facilitate the generation of an array of specifically substituted derivatives to demonstrate how control of key therapeutic parameters such as wavelength of maximum absorbance and singlet-oxygen generation can be achieved. Photosensitizer absorption maxima can be varied within the body's therapeutic window between 650 and 700 nm, with high extinction coefficients ranging from 75,000 to 85,000 M(-1) cm(-1). Photosensitizer singlet-oxygen generation level was modulated by the exploitation of the heavy-atom effect. An array of photosensitizers with and without bromine atom substituents gave rise to a series of compounds with varying singlet-oxygen generation profiles. X-ray structural evidence indicates that the substitution of the bromine atoms has not caused a planarity distortion of the photosensitizer. Comparative singlet-oxygen production levels of each photosensitizer versus two standards demonstrated a modulating effect on singlet-oxygen generation depending upon substituent patterns about the photosensitizer. Confocal laser scanning microscopy imaging of 18a in HeLa cervical carcinoma cells proved that the photosensitizer was exclusively localized to the cellular cytoplasm. In vitro light-induced toxicity assays in HeLa cervical carcinoma and MRC5-SV40 transformed fibroblast cancer cell lines confirmed that the heavy-atom effect is viable in a live cellular system and that it can be exploited to modulate assay efficacy. Direct comparison of the efficacy of the photosensitizers 18b and 19b, which only differ in molecular structure by the presence of two bromine atoms, illustrated an increase in efficacy of more than a 1000-fold in both cell lines. All photosensitizers have very low to nondeterminable dark toxicity in our assay system.     

Real time electroporation control for accurate and safe in vivo non-viral gene therapy

In vivo cell electroporation is the basis of DNA electrotransfer, an efficient method for non-viral gene therapy using naked DNA. The electric pulses have two roles, to permeabilize the target cell plasma membrane and to transport the DNA towards or across the permeabilized membrane by electrophoresis. For efficient electrotransfer, reversible undamaging target cell permeabilization is mandatory. We report the possibility to monitor in vivo cell electroporation during pulse delivery, and to adjust the electric field strength on real time, within a few microseconds after the beginning of the pulse, to ensure efficacy and safety of the procedure. A control algorithm was elaborated, implemented in a prototype device and tested in luciferase gene electrotransfer to mice muscles. Controlled pulses resulted in protection of the tissue and high levels of luciferase in gene transfer experiments where uncorrected excessive applied voltages lead to intense muscle damage and consecutive loss of luciferase gene expression.     

Numerical evaluation of matrix elements of the electric field and electric field gradient operators in a slater basis set

A method is proposed for evaluating all matrix elements of the electric field and electric field gradient operators in a Slater basis set fo systems with an arbitrary number of nuclei and geometry. These integrals are evaluated using a numerical quadrature after having modified the integrand to remove all infinities. The integration ranges are broken to avoid integrating through cusps in the integrand. With reasonable grids these procedures are adequate for numerical evaluation with an accuracy of five to six decimal places (in a.u.). Particular cases are briefly discussed.     

On the enhancement of the electric dipole moment in molecular complexes

In the formation of molecular complexes an enhancement Δμ of the molecular dipole moment with respect to the vectorial sum of the moments of the monomers is often observed. The charge distribution provided by infrared intensity studies successfully predicts, in several cases, Δμ as due to polarization effects.     

Poly(beta-aminosulfonamides) as gene delivery vectors: synthesis and in vitro screening

A series of poly(beta-aminosulfonamides) was synthesized and demonstrated to be efficient in vitro transfection reagents.     

Precise determination of nonlinear function of ion mobility for explosives and drugs at high electric fields for microchip FAIMS

High‐field asymmetric waveform ion mobility spectrometry (FAIMS) separates ions by utilizing the characteristics of nonlinear ion mobility at high and low electric fields. Accurate ion discrimination depends on the precise solution of nonlinear relationships and is essential for accurate identification of ion species for applications. So far, all the nonlinear relationships of ion mobility obtained are based at low electric fields (E/N <65 Td). Microchip FAIMS (μ‐FAIMS) with small dimensions has high electric field up to E/N = 250 Td, making the approximation methods and conclusions for nonlinear relationships inappropriate for these systems. In this paper, we deduced nonlinear functions based on the first principle and a general model. Furthermore we considered the hydrodynamics of gas flow through microchannels. We then calculated the specific alpha coefficients for cocaine, morphine, HMX, TNT and RDX, respectively, based on their FAIMS spectra measured by μ‐FAIMS system at ultra‐high fields up to 250 Td. The results show that there is no difference in nonlinear alpha functions obtained by the approximation and new method at low field (<120 Td), but the error induced by using approximation method increases monotonically with the increase in field, and could be as much as 30% at a field of 250 Td. Copyright © 2015 John Wiley & Sons, Ltd.