An ultra-low-cost electroporator with microneedle electrodes (ePatch) for SARS-CoV-2 vaccination.

Vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other pathogens with pandemic potential requires safe, protective, inexpensive, and easily accessible vaccines that can be developed and manufactured rapidly at a large scale. DNA vaccines can achieve these criteria, but induction of strong immune responses has often required bulky, expensive electroporation devices. Here, we report an ultra-low-cost (<1 USD), handheld (<50 g) electroporation system utilizing a microneedle electrode array ("ePatch") for DNA vaccination against SARS-CoV-2. The low cost and small size are achieved by combining a thumb-operated piezoelectric pulser derived from a common household stove lighter that emits microsecond, bipolar, oscillatory electric pulses and a microneedle electrode array that targets delivery of high electric field strength pulses to the skin's epidermis. Antibody responses against SARS-CoV-2 induced by this electroporation system in mice were strong and enabled at least 10-fold dose sparing compared to conventional intramuscular or intradermal injection of the DNA vaccine. Vaccination was well tolerated with mild, transient effects on the skin. This ePatch system is easily portable, without any battery or other power source supply, offering an attractive, inexpensive approach for rapid and accessible DNA vaccination to combat COVID-19, as well as other epidemics.

A low-cost smartphone controlled portable system with accurately confined on-chip 3D electrodes for flow-through cell electroporation.

Microscale flow-through electroporation at DC voltage has advantages in delivering small molecules. Yet, electroporation based on constant voltage are liable to generate electrolysis products which limits the voltage-operating window. Parallel on-chip 3D electrodes with close and uniform spacing are required to cut down voltage as well as provide enough electric field for electroporation. Here we present a simple electrode fabrication method based on capillary restriction valves in Z-axis to achieve parallel 3D electrodes with controllable electrode spacing in PDMS chips. With electrodes accurately placed in close range, a low voltage of only 1.5 V can generate enough electric field (>400 V/cm) to make cell membrane permeable. Squeeze flow is introduced to produce higher electric field (>800 V/cm) at a fixed voltage for more efficient electroporation. Benefit from the electrode fabrication method and application of squeeze flow, we develop a smartphone controlled microfluidic electroporation system which integrate functions of sample injection, pressure regulating, real-time observation and constant DC power supply. The system is used to electroporate two cell lines, showing a permeabilization percentage of 63% for HEK-293 cells and 58% for CHO-K1 cells with optimal parameters. Thus, the portable microfluidic system provides a cost-effective and user-friendly flow-through cell electroporation platform.

ElectroPen: An ultra-low-cost, electricity-free, portable electroporator.

Electroporation is a basic yet powerful method for delivering small molecules (RNA, DNA, drugs) across cell membranes by application of an electrical field. It is used for many diverse applications, from genetically engineering cells to drug- and DNA-based vaccine delivery. Despite this broad utility, the high cost of electroporators can keep this approach out of reach for many budget-conscious laboratories. To address this need, we develop a simple, inexpensive, and handheld electroporator inspired by and derived from a common household piezoelectric stove lighter. The proposed "ElectroPen" device can cost as little as 23 cents (US dollars) to manufacture, is portable (weighs 13 g and requires no electricity), can be easily fabricated using 3D printing, and delivers repeatable exponentially decaying pulses of about 2,000 V in 5 ms. We provide a proof-of-concept demonstration by genetically transforming plasmids into Escherichia coli cells, showing transformation efficiency comparable to commercial devices, but at a fraction of the cost. We also demonstrate the potential for rapid dissemination of this approach, with multiple research groups across the globe validating the ease of construction and functionality of our device, supporting the potential for democratization of science through frugal tools. Thus, the simplicity, accessibility, and affordability of our device holds potential for making modern synthetic biology accessible in high school, community, and resource-poor laboratories.

Lyse-Reseal Erythrocytes for Transfection of Plasmodium falciparum.

Simple and efficient transfection methods for genetic manipulation of Plasmodium falciparum are desirable to identify, characterize and validate the genes with therapeutic potential and better understand parasite biology. Among the available transfection techniques for P. falciparum, electroporation-based methods, particularly electroporation of ring-infected RBCs is routinely used. Nonetheless, transfection of P. falciparum remains a resource-intensive procedure. Here, we report a simple and economic transfection method for P. falciparum, which is termed as the lyse-reseal erythrocytes for transfection (LyRET). It involved lysis of erythrocytes with a hypotonic RBC lysis buffer containing the desired plasmid DNA, followed by resealing by adding a high salt buffer. These DNA-encapsulated lyse-reseal erythrocytes were mixed with P. falciparum trophozoite/schizont stages and subjected to selection for the plasmid-encoded drug resistance. In parallel, transfections were also done by the methods utilizing electroporation of DNA into uninfected RBCs and parasite-infected RBCs. The LyRET method successfully transfected 3D7 and D10 strains with different plasmids in 63 of the 65 attempts, with success rate similar to transfection by electroporation of DNA into infected RBCs. The cost effectiveness and comparable efficiency of LyRET method makes it an alternative to the existing transfection methods for P. falciparum, particularly in resource-limited settings.

An optimized electroporation approach for efficient CRISPR/Cas9 genome editing in murine zygotes.

Electroporation of zygotes represents a rapid alternative to the elaborate pronuclear injection procedure for CRISPR/Cas9-mediated genome editing in mice. However, current protocols for electroporation either require the investment in specialized electroporators or corrosive pre-treatment of zygotes which compromises embryo viability. Here, we describe an easily adaptable approach for the introduction of specific mutations in C57BL/6 mice by electroporation of intact zygotes using a common electroporator with synthetic CRISPR/Cas9 components and minimal technical requirement. Direct comparison to conventional pronuclear injection demonstrates significantly reduced physical damage and thus improved embryo development with successful genome editing in up to 100% of living offspring. Hence, our novel approach for Easy Electroporation of Zygotes (EEZy) allows highly efficient generation of CRISPR/Cas9 transgenic mice while reducing the numbers of animals required.

Cell electroporation with a three-dimensional microelectrode array on a printed circuit board.

Electroporation is a commonly used approach to rapidly introduce exogenous molecules into cells without permanent damage. Compared to classical electroporation protocols, microchip-based electroporation approaches have the advantages of high transfection efficiency and low consumption, but they also commonly rely on costly and tedious microfabrication technology. Hence, it is desirable to develop a novel, more affordable, and effective approach to facilitate cell electroporation. In this study, we utilized a standard printed circuit board (PCB) technology to fabricate a chip with an interdigitated array of electrodes for electroporation of suspended cells. The electrodes (thickness ~35 µm) fabricated by PCB technology are much thicker than the two-dimensional (2D) planar electrodes (thickness < 1 µm) fabricated by conventional microfabrication techniques and possess a smooth corner edge. Numerical simulations showed that the three-dimensional (3D) electrodes fabricated by PCB technology can provide a more uniformly distributed electric field compared to 2D planar electrodes, which is beneficial for reducing the electrolysis of water and improving cell transfection efficiency. The chip constructed here is composed of 18 individually addressable wells for high throughput cell electroporation. HeLa, MCF7, COS7, Jurkat, and 3T3-L1 cells were efficiently transfected with the pEGFP-N1 plasmid using individually optimal electroporation parameters. This work provides a novel method for convenient and rapid cell transfection and thus holds promise for use as a low-cost disposable device in biomedical research.

A cost-effective approach to microporate mammalian cells with the Neon Transfection System.

Electroporation is one of the most efficient nonviral methods for transferring exogenous DNA into mammalian cells. However, the relatively high costs of electroporation kits and reagents temper the routine use of this fast and easy to perform technique in many laboratories. Several years ago, a new flexible and easy to operate electroporation device was launched under the name Neon Transfection System. This device uses specialized pipette tips containing gold-plated electrodes as electroporation chamber. Here we report a protocol to regenerate these expensive tips as well as some other Neon kit accessories, thereby reducing the cost of electroporation at least 10-fold.

In vivo electroporation of adult mouse sensory neurons for studying peripheral axon regeneration.

Electroporation has been a widely used tool to introduce DNA plasmids or RNA oligos into cultured cells and recently in vivo into chick or mouse embryos. Here we report a rapid and efficient approach to transfect adult mouse dorsal root ganglion neurons in vivo with precise spatiotemporal control via electroporation. This approach will allow both gain- and loss-of-function experiments in vivo to study the function of adult sensory neurons, such as sensory axon regeneration.

A detailed description of an economical setup for electroporation of chick embryos in ovo.

One of the challenges of the postgenomic era is characterizing the function and regulation of specific genes. For various reasons, the early chick embryo can easily be adopted as an in vivo assay of gene function and regulation. The embryos are robust, accessible, easily manipulated, and maintained in the laboratory. Genomic resources centered on vertebrate organisms increase daily. As a consequence of optimization of gene transfer protocols by electroporation, the chick embryo will probably become increasingly popular for reverse genetic analysis. The challenge of establishing chick embryonic electroporation might seem insurmountable to those who are unfamiliar with experimental embryological methods. To minimize the cost, time, and effort required to establish a chick electroporation assay method, we describe and illustrate in great detail the procedures involved in building a low-cost electroporation setup and the basic steps of electroporation.

A detailed description of an economical setup for electroporation of chick embryos in ovo

One of the challenges of the postgenomic era is characterizing the function and regulation of specific genes. For various reasons, the early chick embryo can easily be adopted as an in vivo assay of gene function and regulation. The embryos are robust, accessible, easily manipulated, and maintained in the laboratory. Genomic resources centered on vertebrate organisms increase daily. As a consequence of optimization of gene transfer protocols by electroporation, the chick embryo will probably become increasingly popular for reverse genetic analysis. The challenge of establishing chick embryonic electroporation might seem insurmountable to those who are unfamiliar with experimental embryological methods. To minimize the cost, time, and effort required to establish a chick electroporation assay method, we describe and illustrate in great detail the procedures involved in building a low-cost electroporation setup and the basic steps of electroporation.

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.

Efficient transfection of endothelial cells by a double-pulse electroporation method.

Primary endothelial cells are largely recognized as hard-to-transfect cells. We have been using a double-pulse electroporation technique to efficiently insert genetic material into human umbilical vein endothelial cell (HUVEC). Previously, this technique has been successfully used on hard-to-transfect monocytic cells. Using a conventional electroporation device, we have tested this protocol on HUVECs and compared it with conventional transfection techniques. The average transfection efficiency was up to 68% as measured by the ability of the cells to efficiently express the red fluorophore of the tdTomato gene. Similar results were obtained in human aortic endothelial cells and human microvascular endothelial cells. This technique does not require any particular expensive device, specific medium, or reagent, and the results we obtained so far exceed those of any other previous protocol. This is therefore an affordable and efficient transfection technique that opens new avenues in vascular endothelial research.