Cheap, portable, electricity-free electroporator for gene transfer in cells

Abstract

The invention is a cheap, portable, electricity-free electroporator for gene transfer in single cells. In order to transfer substances such as the gene DNA in cells, it generates electricity in during 20 microseconds in the range of about 200 volts to 5 kv. This causes a temporary opening of the cell pore in bacteria / cells and the targeted substance enters. The first part of the product: Cuvette holder: It is designed to hold the current electroporation cuvettes in a suitable size. The 2 copper connection cables from the piezoelectricity supply electricity to the cuvette. The second part of the product: Main case: It contains the 1st part of the product and also contains the piezoelectric system, cables, and button. It weighs approximately 30 grams and is a 3D printed single piece. Its dimensions are preferably 27 mm x 62 mm x 35 mm. The third part of the product: Button: Thanks to this button, electricity through piezo system is transferred to the cuvette with a single press.

Description

[0001] DESCRIPTION

[0002] CHEAP, PORTABLE, ELECTRICITY-FREE ELECTROPORATOR FOR GENE TRANSFER IN CELLS

[0003] TECHNICAL FIELD

[0004] Electroporation is a biophysical phenomenon in which an external electric field generated around a cell disrupts the physical structure of the cell membrane, increasing cell permeability. This physical principle has been applied in a wide range of applications, including molecular biology for mammalian and bacterial genes. One of the uses of electroporation is electroporation-based gene therapy, non-viral gene transfer, and many studies have been and will be conducted on this field. In some studies, electroporation has been extensively tested for gene transfer to tumours. In molecular biology and genetic engineering, electroporation is used to transform bacteria, yeast, mammalian cells, plant cells and other microorganisms into genetically modified organisms for various purposes, including bioproduction with recombinant DNA, and for gene-based therapies. In the field of gene editing, electroporation can facilitate the delivery of gene editing tools such as CRISPR-Cas9 components, siRNA, miRNA, mRNA, non-coding long / short RNA into cells and enable precise gene editing for therapeutic or research purposes. Electroporation as a route of drug delivery can be used to enhance the delivery of drugs, including chemotherapeutic agents, to target cells, tissues or tumours. This can improve the efficacy of drug therapies. In vaccine research, electroporation can be used to deliver antigens or genetic material encoding antigens into cells to stimulate an immune response. This can increase the effectiveness of the vaccines. Some biotechnological and cellular therapy processes require the expression of recombinant proteins in cells. Electroporation can be used to deliver plasmids encoding these proteins into host cells. It can be used to make plant lines more qualified and resistant to various external / internal factors, to produce more crops, and to increase their commercial value. This system can also be used in biotechnology-oriented programmes such as molecular biology and genetics, bioengineering, pharmacy, medicine, veterinary medicine, biomedical engineering, agriculture, food engineering, biomedical engineering, and in educational systems as course material in laboratories. BACKGROUND

[0005] Two noteworthy scientific papers on this subject in the state of the art have demonstrated the feasibility of piezoelectric-driven electroporation: In a comprehensive analysis of literature and patents, [1] successfully used a piezoelectric actuator to induce electroporation in microfluidic channels. In another study, a piezoelectric device for trans-dermal drug delivery via electroporation was introduced, as documented by [2], Despite an extensive patent search using keywords such as ‘piezoelectric’, ‘electroporation’ and ‘cell delivery’, no relevant patents directly related to piezoelectric powered electroporation devices could be identified. Innovation of the Innovative Electroporation Device This innovation attempts to overcome the limitations of existing technologies by introducing a pioneering electroporation device that combines a piezoelectric energy source and a cuvette holder in a single unit. This integrated design provides several advantages: 1. Cost effectiveness: By eliminating the need for an external power supply, production costs are reduced and device portability is increased. 2. Usability: Combining the power supply and cuvette holder increases operational efficiency and simplifies setup and manipulation. 3. Compatibility with Existing Cuvettes: Compatibility with standard cuvette sizes ensures compatibility with existing laboratory applications, minimising reliance on special consumables. In summary, [Reference 2] previously pioneered a piezoelectric device for transdermal drug delivery via electroporation. A rigorous patent search using keywords such as ‘piezoelectric’, ‘electroporation’ and ‘cell delivery’ yielded no existing patents directly related to piezoelectric powered electroporation devices. The unique integration of piezoelectric energy source and cuvette holder in the present invention differentiates it from others as a forwardthinking solution that addresses the limitations of existing technologies. [1] G. Byagathvalli, S. Sinha, Y. Zhang, M. P. 30 Styczynski,J. Standeven, and M. S. Bhamla, “ElectroPen: An ultra- low-cost, electricity-free, portable electroporator,” PLoS Biol, vol. 18, no. 1, p.e3000589, Jan. 2020, doi: 10.1371 / journal.pbio.3000589. [2] D. Xia etal., “An ultra-low-cost electroporator with microneedle electrodes(ePatch) for SARS-CoV-2 vaccination,” Proceedings of the NationalAcademy of Sciences, vol. 118, no. 45, Nov. 2021, doi: 10.1073 / pnas.2110817118.

[0006] The Canadian application number CA3178545A1 can also be cited as a relevant patent application. The invention provides devices and methods for administering a drug to biological tissue in a patient, for example by intracellular and / or dermal administration. The device comprises a piezoelectric pulse generator; and an array of microneedle electrodes electrically coupled to the piezoelectric pulse generator, wherein the device is configured to generate and transmit one or more electrical pulses through the microneedle electrodes effective to electroporation of cells following insertion of the microneedle electrodes into biological tissue.

[0007] The main disadvantages of all the mentioned applications are: they are extremely expensive, they are not domestically produced and cause continuous foreign dependency, their current use is difficult, they require training and experience, they are not compatible with existing electroporation cuvettes, they are large in size, they are not portable, they require wired electrical power from outside, and the risk of arc.

[0008] DESCRIPTION OF THE INVENTION

[0009] The invention aims to eliminate the disadvantages mentioned in the prior art.

[0010] Other advantages of the invention are that it provides high voltage in a short time, creates a functional drop without using electronic components and can be easily produced with 3D printing.

[0011] It is an invention that will benefit researchers working in biotechnology laboratories, the field of food engineering, agricultural faculties, bioengineering, molecular biology and genetics, as well as companies involved in cellular therapy, RNA and DNA vaccine production, and gene therapy.

[0012] Description of the Figures

[0013] Figure 1. Exploded view of the electroporator

[0014] Figure 2. Perspective view of the electroporator

[0015] Figure 3. Right perspective view of the electroporator

[0016] Figure 4. Top view of the electroporator

[0017] Figure 5. Left perspective view of the electroporator Reference List

[0018] 1 Main part

[0019] 2 Voltage regulator cover

[0020] 3 Easy volt device

[0021] 4 Voltage rod

[0022] 5 Piezoelectric holder

[0023] 6 Cuvette holder

[0024] 7 Press button acceptor

[0025] 8 Press button

[0026] 9 Cover

[0027] 10 Cuvette holder cover

[0028] DETAILED DESCRIPTION OF THE INVENTION

[0029] In this detailed description, the preferred alternatives of the cheap, portable, electricity -free electroporator for gene transfer in the cells of the invention are described only for a better understanding of the subject matter and without any limitations.

[0030] The main part (1) is the central assembly covering all components. Its design is compact and can cover all components of the device. The main box also serves as a protective coating to ensure the safety of the internal components and their longevity. In addition, it provides structural support that maintains the durability and stability of the device during use.

[0031] The voltage regulator cover (2) encloses the voltage regulating part and allows easy adjustment of the switches. This cover provides safety by protecting the user from intentional collisions or exposure to high voltages. In addition, it helps to maintain the cleanliness of the device, preventing dust or rubbish from entering and interfering with the functionality of the voltage device.

[0032] Easyvolt devices (3) are equipped with a voltage regulator. This is a next generation feature and allows them to optimise the electroporation system for various cell types in order to adjust the voltage. The voltage device also helps to prevent possible damage to the electroporation system by extending the lifetime of the device. This saves researchers time and resources by reducing the need for frequent replacements or repairs.

[0033] The voltage adjusting cover (4) for the voltage regulating component maintains the stability of the voltage regulator and prevents its fluctuations. The-voltage adjusting cover provides a secure seal to protect the voltage regulator from external factors that can affect its performance. This helps to ensure consistent and accurate voltage settings for reliable electroporation results.

[0034] The piezoelectric holder (5), the core of the easyvolt device is a piezoelectric component. It is compatible with a push button receptor, allowing the receptor to move smoothly without any problems. The piezoelectric component is known for its durability and efficiency in converting mechanical energy into electrical energy, making it a reliable choice for long-term use. In addition, compatibility with the push button receptor ensures smooth operation and a user- friendly experience.

[0035] The cuvette holder (6) is used to hold electroporation cuvettes where the electroporation process takes place. This section has a copper wall on two sides, which acts as a conduit for the electrical energy generated from the piezoelectric component. This component is compatible with readily available electroporation cuvettes for ease of purchase. The copper wall efficiently conducts electrical energy through the cuvette, optimising the electroporation process. Furthermore, the device is designed for convenient insertion and removal of the cuvette, making it user-friendly for researchers.

[0036] The push button receptor (7) is a component that allows energy to be radiated from the-push button under similar conditions. If the positioning is compatible with the piezoelectric source and has the necessary cleaning with the piezoelectric holder, this facilitates the transfer of energy from the pressure button receptor to the piezoelectric system leading to an efficient and reliable operation. Overall, the design of the cabinet and its compatibility with the push buttons make this device durable and user-friendly for long-term use.

[0037] The push button (8) assists in activating the piezoelectric component. This is a user-friendly feature that enables easy activation of the component that generates the energy required for the operation of the device. This design ensures that users can easily and efficiently use the device without any complications.

[0038] The cover (9) is designed to protect the piezoelectric system from external elements and potential damage to ensure long life and performance. In addition, its compatibility with push buttons and print receivers ensures seamless integration and operation of the system within various devices.

[0039] The cuvette holder cover (10) helps to maintain stability and ensures accurate and consistent results during trials or measurements.

[0040] The product is an electroporator. It generates electricity in the range of about 200 volts to 5 kv in during 20 microseconds to transfer substances such as the gene DNA in cells. This leads to a temporary opening of the cell pore in bacteria and the targeted substance enters. First part of the product: Cuvette holder part: It is designed to hold the current electroporation cuvettes in a suitable size. The 2 copper connection cables from the piezoelectricity supply electricity to the cuvette. Second part of the product: Main case: It contains the first part of the product and also contains the piezoelectric system, cables and button. It weighs approximately 30 grams and is a 3D printed single piece. Its dimensions are preferably 27 mm x 62 mm x 35 mm. Third part of the product: Button: Thanks to this button, electricity is transferred to the cuvette with a single press.

[0041] First part of the product is the newest feature. The cuvette holder does not need any additional part. Since there is no additional part to hold the cuvette, the media should be added on the cells immediately after electroporation, which is one of the most critical times. This is a feature that makes our system stand out compared to current systems. As mentioned before, the biggest benefit is that it is portable and lightweight. The fact that it does not need a wired power for electricity is the most effective and environmentally friendly feature. It can be used for years by getting power from piezoelectricity in a free and unlimited way. It is easy enough to work with a single button and even students can easily use it in their own projects.

[0042] When the button is pressed, piezoelectricity is generated and transmitted by cables to the cuvette holder section and from there to the cuvette. Thanks to the electrically conductive structure on the edges of the cuvette, electricity reaches the cells in the cuvette evenly. This causes a short and temporary shock in the cells and the cell membrane becomes semi-permeable in bacterial mammalian cells. It allows the target molecule we want to introduce into the cell to be taken in. This process was used to transform the experimental Muc-1 gene in E.coli bacteria and its operability was proven.

[0043] The applied voltage is an important factor in electroporation. The efficiency of transfection depends on the precise voltage setting as each cell type requires different voltage levels. Our advanced piezoelectric device has a sophisticated ability to adjust the voltage providing precise customisation to meet the specific needs of different cell types. This innovative feature makes the transfection process better while remaining cost-effective. Switches on the front panel of the instrument provide a user-friendly interface for voltage customisation that is convenient for users to change the desired voltage. This tool simplifies the process of optimising transfection settings for different cell types and consequently increases the overall experimental results.

Claims

CLAIMS1. The invention relates to a cheap, portable, electricity-free electroporator for gene transfer in cells, characterised in that it comprises- The main part (1) that protects the device parts and ensures durability,- voltage regulator cover (2), which protects the device from deliberate collisions or high voltage and helps to keep the device clean and prevent dust etc. from reaching the device,- voltage adjuster easyvolt devices (3), which enable voltage adjustment,- voltage adjusting cover (4), which acts as a secure seal to protect the voltage regulator from external factors that maintain its stability and prevent fluctuations,- piezoelectric holder (5),- cuvette holder (6) for holding electroporation cuvettes where the electroporation process takes place, optimising the electroporation process and ensuring ease of use,- the push button receptor (7), which ensures the transfer of energy from the pressure button receptor to the piezoelectric system leading to an efficient and reliable operation,- push button (8) to activate the piezoelectric component,- cuvette holder cover (10), which ensures accurate and consistent results during trials or measurements.

2. The invention according to claim 1 is a cheap, portable, electricity -free electroporator for gene transfer in cells, characterised in that it comprises an easyvolt device for increasing the efficiency and sensitivity of electroporation experiments.

3. The invention according to claim 1 is a cheap, portable, electricity -free electroporator for gene transfer in cells, characterised in that it comprises a cover (9) which protects the piezoelectric system from external elements and potential damage to ensure long life and performance.

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