PDMS-based Microfluidic Chip Research Articles

A standalone and portable imaging detection system with embedded computing for automated defect inspection of microfluidic devices

The existing methods for defect detection in PDMS microfluidic chips typically involve complex image recognition algorithms or manual inspection and still lack efficiency and reliability. Although some automatic defect detection methods have been proposed in recent years, most of them still rely on external computation systems to deploy. To address these challenges, we propose an independent portable defect detection system with embedded computing for microfluidic devices. This portable system is completely self-contained, integrating an image acquisition module, a control panel module, a power module, and an embedded computing control module to realize chip detection, processing, and result display functions. Experimental results show that the system can effectively detect most of the commonly seen defects in PDMS-based microfluidic chips, proving to be more efficient and reliable than manual inspection. With the control of the embedded system, two detection methods: template matching (based on comparison with standard samples) and automatic defect detection (based on surface defect recognition) were used to identify defects in PDMS-based microfluidic chips. The proposed system can automatically inspect and analyze chips without the need for external laboratory support and can provide a promising solution for future microfluidic chip manufacturing and operation.

Time resolution in cryo-EM using a PDMS-based microfluidic chip assembly and its application to the study of HflX-mediated ribosome recycling

The rapid kinetics of biological processes and associated short-lived conformational changes pose a significant challenge in attempts to structurally visualize biomolecules during a reaction in real time. Conventionally, on-pathway intermediates have been trapped using chemical modifications or reduced temperature, giving limited insights. Here, we introduce a time-resolved cryo-EM method using a reusable PDMS-based microfluidic chip assembly with high reactant mixing efficiency. Coating of PDMS walls with SiO2 virtually eliminates non-specific sample adsorption and ensures maintenance of the stoichiometry of the reaction, rendering it highly reproducible. In an operating range from 10 to 1,000ms, the device allows us to follow invitro reactions of biological molecules at resolution levels in the range of 3Å. By employing this method, we show the mechanism of progressive HflX-mediated splitting of the 70S E.coli ribosome in the presence of the GTP via capture of three high-resolution reaction intermediates within 140ms.

A fully automatic dPCR microfluidic system for detection of large-volume and low-abundance bacteria sample

Digital polymerase chain reaction (dPCR) has become a key technology for bacteria detection with superior sensitivity and accuracy, enabling in reducing the spread of waterborne disease. However, the current systems lack a complete dPCR detection workflow for low-abundance bacteria water sample in large-volume, including bacteria enrichment, nucleic acid (NA) extraction and digital arrays generation. In this work, a fully automated workflow system has been developed for dPCR detection. A preprocessing cartridge designed for single-use has been fabricated to realize the bacterial capture (∼ 98%), NA absorption (∼ 81%), mixing and heating. A PDMS-based microfluidic chip and a vacuum cover are employed for the generation of digital arrays without the pre-degas of PDMS. The proposed system exhibits a good linear relationship of Escherichia coli (E. coli) detection within the range from 2.5 × 101 CFU/mL to 2.5 × 104 CFU/mL, with a limit of detection ∼ 2.5 CFU/mL when enrich 5 mL sample. Furthermore, the dPCR detection results of four actual water samples are comparable to those obtained by the gold standard method plate counting, demonstrating its potential as a point-of-care testing (POCT) tool for accurate quantification of large-volume and low-abundance bacteria samples.

Embedded 3D Printing of PDMS-Based Microfluidic Chips for Biomedical Applications

Abstract Microfluidic devices made from polydimethylsiloxane (PDMS) have diverse biomedical applications. However, due to the poor printability of PDMS, current 3D printing techniques are rarely used to fabricate microfluidic devices. This study aims to investigate a fumed silica-PDMS suspension that can function as a matrix bath for embedded 3D printing (e-3DP) purposes, making it technically feasible to print microfluidic chips with complex embedded channels via low-cost extrusion 3D printing. The rheological properties, mechanical properties, transparency, and filament fidelity of the fumed silica-PDMS suspension have been systematically studied. It is found that the addition of fumed silica particles can effectively change PDMS from a viscous solution to a yield-stress suspension with suitable rheological properties for e-3DP. Also, the mechanical properties of the crosslinked fumed silica-PDMS are enhanced with an increased concentration of fumed silica. Although the transparency of PDMS has been lessened by mixing it with fumed silica particles, the visibility of the printed microfluidic chips is still acceptable. The filament fidelity has been studied by embedded printing filaments using a sacrificial ink in the fumed silica-PDMS suspension. Finally, two representative microfluidic chips for biomedical applications have been successfully printed to validate the effectiveness of the proposed fumed silica-PDMS suspension-enabled e-3DP method.

Validation of Easy Fabrication Methods for PDMS-Based Microfluidic (Bio)Reactors

The common method for producing casting molds for the fabrication of polydimethylsiloxane (PDMS) chips is standard photolithography. This technique offers high resolution from hundreds of nanometers to a few micrometers. However, this mold fabrication method is costly, time-consuming, and might require clean room facilities. Additionally, there is a need for non-micromechanics experts, who do not have specialized equipment to easily and quickly prototype chips themselves. Simple, so-called, makerspace technologies are increasingly being explored as alternatives that have potential to enable anyone to fabricate microfluidic structures. We therefore tested simple fabrication methods for a PDMS-based microfluidic device. On the one hand, channels were replicated from capillaries and tape. On the other hand, different mold fabrication methods, namely laser cutting, fused layer 3D printing, stereolithographic 3D printing, and computer numerical control (CNC) milling, were validated in terms of machine accuracy and tightness. Most of these methods are already known, but the incorporation and retention of particles with sizes in the micrometer range have been less investigated. We therefore tested two different types of particles, which are actually common carriers for the immobilization of enzymes, so that the resulting reactor could ultimately be used as a microfluidic bioreactor. Furthermore, CNC milling provide the most reliable casting mold fabrication method. After some optimization steps with regard to manufacturing settings and post-processing polishing, the chips were tested for the retention of two different particle types (spherical and non-spherical particles). In this way, we successfully tested the obtained PDMS-based microfluidic chips for their potential applicability as (bio)reactors with enzyme immobilization carrier beads.

Rapid Prototyping of Moulds for PDMS-based Microfluidic Chips

Abstract To shorten the production time for PDMS-moulds by additive manufacturing (AM) several 3D printers have been investigated in comparison to standard micro-milling by producing benchmark structures. These are evaluated regarding their shape accuracy, the transparency of the casted PDMS which is linked to the surface quality of the mould, and the production time until the moulds are ready to use. Even though the additively manufactured moulds showed significantly better surface quality and shorter production time, the necessary shape accuracy for non-square-structures or structures with < 250 μm edge length could not be achieved due to limiting factors like nozzle diameter, size of the digital micromirror device or spot size of the LCD-panel.

Light-based 3D printing of functional polydimethylsiloxane- based microfluidic chips

The objective of the work is to fabricate and functionalize 3D printed PDMS-based microfluidic chips through digital light processing DLP-3D printing.

PDMS leaching and its implications for on-chip studies focusing on bone regeneration applications

Polydimethylsiloxane (PDMS) is among the most widely used materials for organ-on-chip systems. Despite its multiple beneficial characteristics from an engineering point of view, there is a concern about the effect of PDMS on the cells cultured in such devices. The aim of this study was to enhance the understanding of the effect of PDMS on cellular behavior in a context relevant for on-chip studies. The focus was put on an indirect effect of PDMS, namely leaching of uncrosslinked oligomers, particularly for bone regeneration applications. PDMS-based chips were prepared and analyzed for the potential release of PDMS oligomers within the microfluidic channel when kept at different flow rates. Leaching of uncrosslinked oligomers from PDMS was quantified as silicon concentration by inductively coupled plasma - optical emission spectrometry and further confirmed by mass spectrometry. Subsequently, PDMS-leached media, with a silicon concentration matching the on-chip experiment, were prepared to study cell proliferation and osteogenic differentiation of MC3T3-E1 pre-osteoblasts and human mesenchymal stem cells. The silicon concentration initially detected in the media was inversely proportional to the tested flow rates and decreased to control levels within 52 h. In addition, by curing the material overnight instead of 2 h, regardless of the curing temperature (65 and 80 °C), a large reduction in silicon concentration was found, indicating the importance of the PDMS curing parameters. Furthermore, it was shown that PDMS oligomers enhanced the differentiation of MC3T3-E1 pre-osteoblasts, this being a cell type dependent effect as no changes in cell differentiation were observed for human mesenchymal stem cells. Overall, this study illustrates the importance of optimization steps when using PDMS devices for biological studies, in particular PDMS curing conditions and extensive washing steps prior to an experiment. • PDMS oligomers leach out from PDMS-based microfluidic chips. • The amount of PDMS oligomers is reduced by longer curing times or washing. • MC3T3-E1 pre-osteoblasts show enhanced differentiation after indirect PDMS exposure. • The effect of PDMS oligomers on cellular behavior is cell type specific.

A flexible precise volume sensor based on metal-on-polyimide electrodes sandwiched by PDMS channel for microfluidic systems

This paper reports a flexible precise volume sensor with metal-on-polyimide (PI) electrodes to substitute for the peripheral ration pump of a microfluidic system, thus beneficial for integration and miniaturization. This in-channel volume sensor consists of multi-electrode pairs, and it can perform volume measurement of the fluid flowing through microchannels by testing the resistance variation of the electrode pairs, which makes the device possible to help automatically control the sample volume in the mixing and reacting processes inside a microfluidic chip without peripheral ration pumps. The electrode pairs of the sensor are fabricated on flexible PI surface directly by inkjet printing. Then, the electrodes with the PI substrate are transferred and sandwiched by a polydimethylsiloxane (PDMS) substrate layer and a PDMS channel layer to form the flexible precise volume sensor. This method overcomes the challenge of patterning metals on PDMS and the sandwiched PDMS–PI–PDMS structure is beneficial for integration with other PDMS-based microfluidic chips. The effects of electrode-tip shapes and numbers of the electrode pairs are also investigated. A novel calculating method is proposed to obtain more precise results when different numbers of electrode pairs are used in different situations. According to the experimental results, the more electrode pairs are used in the same spacing, the better measurement precision can be obtained. The volume sensor with optimized electrode-tip and multi-electrode pairs can detect the fluid volume in nanolitre scales with the relative error of < 0.8%. This work exhibits the potential to form a total lab-on-a-chip without peripheral ration pumps.

Abstract 39: Microvessel-on-a-chip for investigating glioma-vascular interactions

Abstract Introduction: Gliomas, which are heterogeneous tumors made up of malignant glial and stromal cells, can often grow and progress without angiogenesis and thus escape anti-angiogenic therapies. One of the alternative mechanisms of tumor blood supply is vessel co-option, where cancer cells migrate along the pre-existing vessel of the host organ, preserving the blood-brain barrier (BBB). However, studying this phenomenon is currently limited mainly to animal models. With the push to reduce in vivo approaches, developing an experimental, organ-on-a-chip model to encompass one or more tractable microenvironmental factors, will enable us to better understand their mechanistic roles in brain tumor progression. Methods and Results: An extracellular matrix-integrated PDMS-based microfluidic chip with a rounded microvessel, mimicking the BBB, was generated using a human microvascular cell line (hCMEC/D3), in the presence of flow. In the chip, a vessel of ~100µm diameter was interfaced with a 3D brain cancer cell culture (either U87, glioma neural stem or normal neural stem cell, embedded in a collagen-based ECM). The system was coupled with live-cell imaging and image analysis, which enabled tracking of cell-cell and cell-microenvironment interactions. Changes in gene expression and protein distribution in endothelial cells were successfully quantified, thus enabling the characterization of the influence of cancer cells population on the microvessel. Conclusions: Development and optimization of the novel device has given us the opportunity to study the influence of glioma cells on normal brain endothelium, when agiogenesis does not occur. Crucially, this can be done in controlled, user-defined environment (i.e. choice of ECM components and stiffness, microvessel size and flow rate) unlike in animal models. Note: This abstract was not presented at the meeting. Citation Format: Magda Gerigk, Harry J. Bulstrode, Yan Yan Shery Huang. Microvessel-on-a-chip for investigating glioma-vascular interactions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 39.

On-chip light-sheet fluorescence imaging flow cytometry at a high flow speed of 1 m/s.

We present on-chip fluorescence imaging flow cytometry by light-sheet excitation on a mirror-embedded microfluidic chip. The method allows us to obtain microscopy-grade fluorescence images of cells flowing at a high speed of 1 m/s, which is comparable to the flow speed of conventional non-imaging flow cytometers. To implement the light-sheet excitation of flowing cells in a microchannel, we designed and fabricated a mirror-embedded PDMS-based microfluidic chip. To show its broad utility, we used the method to classify large populations of microalgal cells (Euglena gracilis) and human cancer cells (human adenocarcinoma cells). Our method holds promise for large-scale single-cell analysis.

Proizvodnja mikrokanala u laboratoriji i primena mikro PIV-a

We present a method for microfluidic channel fabrication that has the following advantages compared to conventionally-used methods: technical simplicity, dramatically lower fabrication costs, and fabrication time. The method entails printing channel designs on a thermoplastic film from a LaserJet printer. Exposure to high temperatures elicits isotropic shrinkage of the film (in the x-y plane), in addition to thickening (in the z-direction), resulting in a positive relief mold. The microfluidic channel design of the mold is then transferred to a polydimethyl siloxane (PDMS) chip through soft lithography, resulting in a ready-to-use microfluidic chip. Through this approach, chips with complex channel geometries can be generated with low cost equipment and in as little as a couple hours. Flow visualizations from several chips from the 'Do-it-yourself Microfluidics Workshop', held at the Faculty of Mechanical Engineering Univeristy of Belgrade, are presented in this paper. We also discuss possibilities for adapting micro particle image velocimetry (PIV) measurements to channel designs on PDMS-based microfluidic chips using the fabrication method delineated here.

An Ultraviolet-Visible (UV) Photometry System Based on the PDMS-based Microfluidic Chip

In order to avoid a problem remains with the low accuracy and poor portability of the photometry system, the system based on the photometry method and microfluidic chip technology was built. As the characteristics of cheap, solid and good transmission, Polydimethylsiloxane (PDMS) was chosen as the material of the designed chip in the paper. To the designed UV photometry system, the light-emitting diode with wavelength of 580m is chosen as the light source. The experimental result indicates that there is no significant deviation between the designed UV photometry system and the conventional immuneturbidimetric assay, the correlation coefficient is 0.95 obtained by adopting the linear regression analysis. The linearity of the designed UV photometry system based on the PDMS-based microfluidic chip has increased by 17.3% in comparison with the system based on the silicon-based microfluidic chip.

Optofluidic analysis system for amplification-free, direct detection of Ebola infection.

The massive outbreak of highly lethal Ebola hemorrhagic fever in West Africa illustrates the urgent need for diagnostic instruments that can identify and quantify infections rapidly, accurately, and with low complexity. Here, we report on-chip sample preparation, amplification-free detection and quantification of Ebola virus on clinical samples using hybrid optofluidic integration. Sample preparation and target preconcentration are implemented on a PDMS-based microfluidic chip (automaton), followed by single nucleic acid fluorescence detection in liquid-core optical waveguides on a silicon chip in under ten minutes. We demonstrate excellent specificity, a limit of detection of 0.2 pfu/mL and a dynamic range of thirteen orders of magnitude, far outperforming other amplification-free methods. This chip-scale approach and reduced complexity compared to gold standard RT-PCR methods is ideal for portable instruments that can provide immediate diagnosis and continued monitoring of infectious diseases at the point-of-care.

(Invited) Observation of Stress Responses of Bacteria Confined in a MEMS Microfluidic Chip

This work describes the new and simple method for observing bacterial behavior of stress response such as to light irradiation in a polydimethylsiloxane (PDMS) -based MEMS microfluidic chip. The fluorescence was observed from Legionella cells confined in small spaces among microbeads. Ultraviolet (UV) light irradiation was found to be a trigger for producing fluorescent material for Legionella.Confining bacteria in the small spaces made by the MEMS technique is an effective way for investigating their stress response. Sensing bacteria is attracting a great deal of interest to avoid an outbreak of bacterial infection. We have been developing sensors for bacteria, Legionella pneumophila (L. pneumophila), which is widely known as one of the pathogenic bacteria [1]. The sensors used for this purpose have small cavity structures for trapping the bacteria. During the course of the study for trapping bacteria in the small cavities, we found that the cells emit fluorescence by the UV excitation just when they are trapped and restricted their motion in the cavities or narrow spaces [2]. This fact implies that such bacteria might show previously-unknown stress responses in a small space. In this work, applying MEMS technique, we made the cells of L. pneumophila and Legionella dumoffii (L. dumoffii) confined in small spaces and investigated their stress responses to UV light irradiation. To confine Legionella cells in small spaces, we used a glass microbeads suspension and a PDMS-made microfluidic chip. We previously made a mixed suspension of Legionella cells and the beads. Then we introduced them into a PDMS-made MEMS microfluidic chip that has protrusions which work as stoppers both for the cells and the beads. Thus Legionella cells are trapped in small spaces among the microbeads. The blue fluorescence from the trapped L. pneumophila cells was observed, indicating that the cells were restricted their motion among the beads. Spectroscopic analyses then compared the fluorescence with and without the beads. Both L. pneumophila and L. dumoffii showed their fluorescence for the case with the beads, meaning that they emit fluorescence when they are restricted their motion and not when they swim freely in water. The UV light irradiation time dependence of the fluorescence spectra for L. pneumophila and L. dumoffii in the case with the beads was further investigated. The spectra for L. pneumophila had a main and a shoulder peaks at around 450 nm and 550 nm respectively at the initial stages of the irradiation. Then the main peak grew with the time, showing the photo irradiation triggers to produce fluorescent materials. The spectra for L. dumoffii, on the other hand, drastically varied depending on the irradiation time. The spectral analysis showed that the fluorescent material which L. dumoffii originally had in their cells as reported in ref. 3 is photodissociated and then the production of the different fluorescent materials begins to occur. Thus our proposed method successfully revealed the previously-unknown characteristics of Legionella confined in the small spaces. In summary, the fluorescent characteristics of Legionella were investigated by using the PDMS-made MEMS microfluidic chip with the combination of microbeads suspension. Our developed MEMS-based method for observing bacterial behavior paves the way for obtaining new knowledge about the stress response of bacteria by controlling their surrounded conditions in a MEMS microfluidic chip.

Surface Modification of Poly(dimethylsiloxane) Using Ionic Complementary Peptides to Minimize Nonspecific Protein Adsorption.

Poly(dimethylsiloxane) (PDMS) has become a widely used material for microfluidic and biological applications. However, PDMS has unacceptably high levels of nonspecific protein adsorption, which significantly lowers the performance of PDMS-based microfluidic chips. Most existing methods to reduce protein fouling of PDMS are to make the surface more hydrophilic by surface oxidization, polymer grafting, and physisorbed coatings. These methods suffer from the relatively short-term stability, the multistep complex treatment procedure, or the insufficient adsorption reduction. Herein, we developed a novel and facile modification method based on self-assembled peptides with well-tailored amino acid composition and sequence, which can also interact strongly with the PDMS surface in the same way as proteins, for suppressing the nonspecific protein fouling and improving the biocompatibility of PDMS-based microfluidic chips. We first demonstrated that an ionic complementary peptide, EAR16-II with a sequence of [(Ala-Glu-Ala-Glu-Ala-Arg-Ala-Arg)2], can readily self-assemble into an amphipathic film predominantly composed of tightly packed β-sheets on the native hydrophobic and plasma-oxidized hydrophilic PDMS surfaces upon low concentrations of carbohydrates. The self-assembled EAR16-II amphipathic film exposed its hydrophobic side to the solution and thus rendered the PDMS surface hydrophobic with water contact angles (WCAs) of around 110.0°. However, the self-assembled EAR16-II amphipathic film exhibited excellent protein-repelling and blood compatibility properties comparable to or better than those obtained with previously reported methods. A schematic model has been proposed to explain the interactions of EAR16-II with the PDMS surface and the antifouling capability of EAR16-II coatings at a molecular level. The current work will pave the way to the development of novel coating materials to address the nonspecific protein adsorption on PDMS, thereby broadening the potential uses of PDMS-based microfluidic chips in complex biological analysis.

Prototyping chips in minutes: Direct Laser Plotting (DLP) of functional microfluidic structures

We report a fast and simple prototyping method to fabricate polymer-based microfluidic chips using Direct Laser Plotting (DLP) technique, by which various functional micro-structures can be realized within minutes, in a mask-free and out-of-cleanroom fashion. A 2D Computer-Aid-Design (CAD) software was employed to layout the required micro-structures and micro-channels, a CO2 laser plotter was then used to construct the microstructures. The desired patterns can be plotted directly on PDMS substrates and bio-compatible polymer films by manipulating the strength and density of laser pulses. With the DLP technique, chip-embedded micro-electrodes, micro-mixers and 3D microfluidic chips with 5 layers, which normally require several days of work in a cleanroom facility, can be fabricated in minutes in common laboratory. This novel method can produce microfluidic channels with average feature size of 100μm, while feature size of 50μm or smaller is achievable by making use of the interference effect from laser impulsion. In this report, we present the optimized parameters for successful fabrication of 3D microchannels, micro-mixers and microfluidic chips for protein concentration measurements (Bovine Serum Albumine (BSA) test), and a novel procedure to pattern flexible embedding electrodes on PDMS-based microfluidic chips. DLP offers a convenient and low cost alternative to conventional microfluidic channel fabrication technique which relies on complicated and hazardous soft lithography process.

The Specific Hybridization of &lt;I&gt;p53&lt;/I&gt; Gene on Bead-Quantum Dot Complex in Microfluidic Chip

Recently, nanobiosensors using nanoparticles, such as gold, silver, and quantum dots, have been studied extensively. Among them, fluorescence resonance energy transfer (FRET)-based DNA sensor is prominent device, especially for the medical diagnosis and biomolecular investigations. FRET is a phenomenon of the emitted energy transfer from one fluorescent dye to another dye through a convoluted wavelength for the excitation. PDMS-based microfluidic chips with pillar structure were prepared for the detection of exon 7 of p53 gene by using QD-DNA probe attached to polystyrene micro beads. The specific hybridization was investigated with 4 different target oligonucleotides. Fluorescence quenching was observed only from the target oligonucleotide for exon 7 with proper sequence for the hybridization. The fluorescence intensity from QDs decreased rapidly due to hybridization and FRET between QDs and intercalating dyes.

Vision-Based Automated Single-Cell Loading and Supply System

Automated continuous individual cell transfer is a critical step in single-cell applications using microfluidic devices. Cells must be aspirated gently from a buffer before transferring to operation zone so as not to artificially perturb their biostructures. Vision-based manipulation is a key technique for allowing nondestructive cell transportation. In this paper, we presented a design for an automated single-cell loading and supply system that can be integrated with complex microfluidic applications for examining or processing one cell at a time such as the current nuclear transplantation method. The aim of the system is to automatically transfer mammalian donor ( approximately 15 microm) or oocyte ( approximately 100 microm) cells one by one from a container to a polydimethylsiloxane (PDMS) microchannel and then transport them to other modules. The system consists of two main parts: a single-cell suction module, and a PDMS-based microfluidic chip controlled by an external pump. The desired number of vacuumed cells can be directed into the microfluidic chip and stored in a docking area. From the batch, they can be moved to next module by activating pneumatic pressure valves located on two sides of the chip. The entire mechanism is combined with monitoring systems that perform detection/tracking and control.

Fabrication of UV epoxy resin masters for the replication of PDMS-based microchips

This paper presents two methods for the fabrication of UV epoxy resin masters for the replication of PDMS-based microfluidic chips. In the first method, the epoxy resin master is fabricated from a negative glass template manufactured using conventional lithography and wet etching techniques. However, in the second method, the master is produced simply by exposing a layer of UV epoxy resin coated on a glass substrate. Although the first method enables the replication of multiple PDMS structures from a single master, the latter method avoids the requirement for a wet chemical etching process and enables the epoxy master to be produced in 40 min or less. The experimental results show that the epoxy resin masters enable the mass production of PDMS replicas with highly precise geometrical tolerances. A series of electrokinetic focusing experiments are performed using PDMS microchips replicated from the current epoxy resin masters. The experimental results obtained for the width of the electrokinetically-focused sample stream under different focusing ratios are found to be in good agreement with the theoretical predictions. The sample handling characteristics of the microfluidic chips are also investigated. It is shown that the sample flow can be electrokinetically pre-focused into a narrow stream and then guided to the desired outlet port by applying a simple voltage control model. Finally, it is demonstrated that through an appropriate alignment of the sample flow and the conductivity gradient, the electrokinetic instability phenomenon can be induced at a relatively low electrical field strength of 0.35 kV/cm.