This application claims the benefit under 35 U.S.C. §119 of Taiwanese Patent Application No. 101139140, filed Oct. 23, 2012, which is hereby incorporated by reference in its entirety.
1. Field of the Invention
The present invention relates to the field of a nano-electrode based transparent chip, in particular to apply an intense electric field for localized electroporation toward the individual single cell by using nano-gap with nano-electrodes, which are very smaller than the size of the single cell to be tested for achieving the goal, to deliver drugs/medicine through a nano region of single cell without destroying the single cell membrane structure.
2. Description of the Related Art
The delivery of drugs into cell is an important phenomenon for biological studies and therapeutic applications. The gene delivery technique which is related to it includes a plurality of different methods and means. Although viral transfection being one of the traditional method which is widely acceptable and effective method for drug delivery, but it may cause some problems such as response of immunity, low controlling rate and so on. These reasons resulting, this technique has some chances to be improved.
In addition, the methods of the non-viral transfection used for gene delivery technique including, jet injection, lipid mediated entry into cells, sonoporation, or electroporation. The conventional electroporation may cause some problems such as fusing or collapsing of the cell membrane structure easily, so that genes or drugs could not be effectively and fast, delivered inside of the single cell. Furthermore, the conventional method is difficult to be controlled in the polar direction. Thus, it is an ineffective to control the injecting position of genes or drugs. Due to affected of whole cell membrane by electric field, it is also impossible to control the doses of drugs delivery inside the cell by using conventional electroporation technique.
Therefore, a precisely drug delivery systems of the non-viral transfection through a specific region of the single cell is a main topic in the field of single cell research and development in recent years. Wherein it includes the electroporation by utilizing a high intense electric field. Many conventional techniques generate an AC/DC electric field by using two large electrodes to permeabilize the cell membrane structure cause to form membrane nano-pores. However, these methods usually cause some problems such as large electrodes area can generate more hydroxyl and hydrogen ion causes more toxicity of the cell environment resulting to reduce the cell viability. Also these methods have some problem for the cell membrane resealing, for example, its reversibility being disappeared and its structure being collapsed, finally cell death.
Therefore, it is urgent and necessary to provide a method to apply an intense electric field through a specific region (submicron to nano region) of single cell membrane to deliver drugs inside the individual single cell to achieve the goal of upgrading the transmittance of medicine without destroying the whole membrane structure of the single cell.
Based on the aforementioned problems in the prior art technique, one objective of the present invention is to provide a nano-electrode based transparent chip with nano-gap between two electrodes to overcome the defect of inaccurate delivery of drugs into the single cell. This nano-gap with nano-electrodes resulting fully controllable drug delivery through a specific region of single cell with membrane reversibility to deliver specific doses of drugs into the single cell and avoided to collapse of the cell membrane structure with negligible cell toxicity, when the cell is electroporated.
The present invention comprises a silicon-based layer, a structural layer, an insulating layer, and a micro fluidic layer. The structural layer is deposited on the silicon-based layer, and a plurality of nano-electrodes is deposited on the structural layer. The insulating layer is deposited on the top of the plurality of nano-electrodes. Then final nano-gap with nano-hole is form, in between two nano-electrodes throughout the insulating and structural layer. This nano-gap is form in all of plurality of nano-electrodes. As nano-gap form throughout the insulation and structural layer which is nothing but nano-hole form in between two nano-electrodes. When the individual single cell is disposed on the nano-gap between the plurality of nano-electrodes, which is able to generate at least an intense electric field on specific region of single cell membrane, then localized single cell membrane can deform to permeabilize the drugs or medicine through the nano-hole in each plurality of nano-gap electrodes. The drugs diffuse through the nano-hole by microfluidic nano-channel and it is enter into the localized single cell through the specific region of membrane nano-pores.
The size of the nano-gap between the plurality of nano-electrodes is in the range from 25 nanometers to 500 nanometers. As the 25 nanometer gap enhance much more an intense electric field to compare with 500 nm nano-gap between plurality of nano-electrodes. To enhance much more intense electric field on local area (25 nm), which affect the localized cell membrane area is less with compare 500 nm nano-gap between the plurality of nano-electrodes. As results the drugs or medicine can enter with more controllable doses for 25 nanometer gap between the plurality of nano-electrodes.
Preferably, the micro fluid flows within the plurality of nano-holes through the nano-channel and the nano-gap between two nano-electrodes.
Preferably, the plurality of nano-electrodes are respectively applied with a single positive square wave pulse to generate at least an intense electric field.
Preferably, the plurality of nano-electrodes is indium tin oxide (ITO) nano-electrodes.
Preferably, the plurality of nano-electrodes is nano-electrodes with triangular shape nano-tips, which form an intense electric field at specific point.
Preferably, the insulating layer is a silicon oxide (SiO2) layer to inhibit hydroxyl and hydrogen ion generation which can avoid toxic issue.
Preferably, the micro fluid circulates through at least one of the plurality of nano-hole with nano-channel through a plurality of nano-gap with nano-electrodes.
Preferably the structural layer, insulating layer and a transparent layer are further deposited on a portion of a bottom of the silicon-based layer.
Preferably, the silicon-based layer, the structural layer, insulating layer and the transparent layer define a micro fluidic chamber, where to allow the micro fluid flow in and out of the micro fluidic chamber through the microfluidic channel.
In summarization of the description above, the nano-electrode based transparent chip of the present invention includes one or more advantages as follows:
(1) The nano-electrode based transparent chip of the present invention is applicable to improve the localize drugs delivery into the single cell without destroying much more area of the single cell membrane structure which enhance the transfection efficiency and viability of the single cell.
(2) The nano-gap with nano-electrodes reduces the hydrogen and hydroxyl ion generation during electroporation experiment. Moreover the insulation layer protect the ion generation from the nano-electrodes resulting to reduces the toxicity effect on the cell membrane and enhance the cell viability.
(3) Through controlling the strength of the electric field, electroporation gets a better control to form the nano-pores on the membrane. The electric field affects such a small region of the single cell membrane, which successfully generate a reversible electroporation with fully controllable drug delivery inside the single cell and transparent chip provides a clear optical path to trace the drugs or DNAs deliver into the cell.
(4) To control electric pulse length, number of pulses and time between two pulses, as results the number of nano-pores opening, the nano-pores size and the density of the nano-pores should be well controlle for localized single cell electroporation.
The technical contents and characteristics of the present invention will be apparent with the detailed description of a preferred embodiment accompanied with related drawings as follows. For simplicity, the same numerals are used for the same respective elements in the description of the following preferred embodiments and the illustration of the drawings.
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In addition, in the present embodiment, when the individual single cell 80 is disposed on the nano-gap 30 in between the plurality of nano-electrodes 30 which is able to generate at least an intense electric field, then the electric field acts on the individual single cell membrane 80 to cause the localize electroporation on a specific position of cell membrane of the individual single cell 80 results to form nano-pores 800. Therefore, a portion of the micro fluid 90 flows from the plurality of the microfluidic channel 100 and passing through the nano-hole with nano-channel 40 between the plurality of nano-gap electrodes 30 and then later flows into the individual single cell 80 through the localize area 800 after passing through the portion of nano-channel 70. More specifically, when the micro fluid flows through the nano-channel 40 disposed between the plurality of nano-electrodes 30 from the portion 70 and then passes through a plurality of nano-pores on the cell membrane 800 of the individual single cell 80, one or the combination of a biomolecular material with nano size, a molecular drug and a molecular probe contained in the micro fluid 90 could be delivered into the individual single cell 80 for further observation.
It is noteworthy that because the size of the nano-hole with nano-channel 40 disposed between the plurality of nano-gap electrodes 30 is smaller than that of the individual single cell 80, the electric field intense by the nano-gap electrodes 30 works merely a range such as it acts on a small portion of the cell membrane of the individual cell 80 to cause the localize single cell electroporation. Therefore, a plurality of nano-pores 800 generated on the single cell membrane by using nano-electrode based transparent chip of the present invention for successful reversible electroporation. That is, those nano-pores would not produce any effect on the main structure of the whole body of the individual single cell 80. More specifically, when the micro fluid 90 passes though the plurality of nano-pores 800 on the cell membrane of the individual single cell 80 with electroporation technique, after some time of successful localize electroporation, the individual single cell 80 where the plurality of nano-pores 800 on the cell membrane are disappeared and the cell membrane recovers back to its original condition. Therefore, this is called a reversible electroporation. After reversible electroporation, the intensity inside the single cell 80 should be constant, because no more drugs can enter inside the single cell 80.
In addition, the electric field intense by applying the positive voltage (+V) and negative voltage (−V) on the plurality of nano-electrodes can be in the form of electric pulse. Preferably, it can be a square wave electric pulse with much more preferably, it can be a single positive square wave electric pulse. The user can control pulse length, number of pulses, time between two pulses, as results the number of nano-pores opening, the nano-pores size and the density of the nano-pores should be controlled on nanopores 800 of the individual single cell 80 for corresponding to the size of the molecule, the drug, the probe or the biomedical material included in the micro fluid 90. The user wants to deliver drugs into an individual single cell 80 through adjusting the frequency, the field strength and the duration of the electric field. Furthermore, in the nano-hole with nano-channel 40, the pulse of electric field generated by the plurality of nano-electrodes 30 with nano-gap (25 nm) is highly intense, and the effective area of the pulse of electric field is minimized because of nano-gap between two nano-electrodes 30 which extremely lowering the damage toward the individual single cell 80.
In addition, the bottom layer of the micro fluidic layer 60, structural layer 20, nano-electrodes layer 30 and oxide layer 50 of the nano-electrode based transparent chip 1 of the present invention comprises a fully light transparent layer. The advantage of adopting the light transparent layer 110 is that, when the molecular drug or material included in the micro fluid 90 flows from the micro fluidic layer 60 to moves toward the individual single cell 80, the sensor connected from the outside, senses and receives variable signals, particularly the optical signals, through the light transparent layer 110 when the nano-electrode based transparent chip 1 is in operation mode. For example, if the micro fluid 90 comprises the drug emission fluorescence like propidium iodide (PI)/calcein/quantum dot (QD) or GFP flow into the individual single cell 80, the biomolecules insert into the individual single cell 80 and then gradually accumulate inside the individual single cell 80. When the plurality of nano-pores 800 on the cell membrane of the individual single cell 80 is resealed and the cell membrane recovers back to its original condition, then biomolecules are left inside the individual single cell 80. At the same time, the sensor connected from outer place immediately detects the variation of the fluorescent signals coming from the biomolecules when it flows like the process mentioned above and then obtains the usage effect of the nano-electrode based transparent chip 1 of the present invention Also the variation of fluorescence intensity of the single cell can confirm the resealing mode of nano-pores 800, where the invertor used Inverted fluorescence microscope for which the chip with all layers should be needed to be transparent. This nano-electrodes based transparent chip should be comfortable for electroporation experiment in both inverted and non-inverted fluorescence microscope.
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Number | Date | Country | Kind |
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101139140 | Oct 2012 | TW | national |