Apparatus and method for tip alignment in multiwell plates

Automatic screening and manipulation of biomaterials in multiwells using chip assemblies.

Ribonucleic acid interference (RNAi) is one of the most exciting discoveries of modern biology and represents a revolution in the analysis of gene function. So far, genome-scale screening has become an increasingly important part of the target discovery process. However, there is a lack of devices and methods for efficient transfection of bio-related cell types with sufficient processing capacity. For example, lipid-based methods can provide processing power but are unable to effectively transfect most biorelated cell types. Conventional electroporation-based methods can transfect primary and difficult-to-transfect cell types, but not at the cost, efficiency, and processing power required.

Electroporation increases the conductivity and permeability of cell membranes caused by an externally applied electric field. In molecular biology, electroporation is used to introduce substances into cells. For example, nucleic acids can be introduced into cells to alter their function. Electroporation is generally useful for introducing nucleic acids or chemicals or physical substances into cultured tissue cells, including mammalian cells and target organs in vivo. The application of electroporation includes cancer treatment, gene therapy, cell-based treatment, and drug discovery.

In conventional electroporation techniques, the electroporator generates an electric current that allows it to pass through, for example, a cell solution in a cuvette containing two metal electrodes on its sides. The cell suspension contained in the cuvette is mixed with the plasmid and introduced into the cells. The cuvette is inserted into the electroporator, and the electroporator applies a voltage (eg, 240 volts) to the electrodes to apply an electric field to the cell solution to put the plasmid into the cell. After electroporating a cell solution, the cells must be treated carefully until they have the potential for division to produce new cells containing the regenerated plasmid.

In many of the current electroporation procedures, cells are detached from cell culture vessels, placed in suspensions and transferred to cuvettes for electroporation as described above. This process requires a large amount of labor and limits processing capacity and effectiveness. In addition, the electroporation process itself, and in combination with complex cell operations such as exfoliation, digestion, transfer, pipetting, etc., can cause significant stress on the cells. As a result, high rates of cell morbidity and mortality are often observed.

<p num="0006"> Objectives and advantages of the present invention will be described and will become apparent in the detailed description below. Further advantages of the present invention will be realized and achieved by the devices and methods particularly noted in the detailed description and claims of the present specification, as well as apparent from the drawings. Many embodiments of the present invention align and lower the tip manifold and multi-well plate aligner, as well as the chips of the tip manifold, and polarize on the surface of adherent cells cultured on the floor of the wells in the multi-well plate. Provide a method that can be entered in close proximity. In the application of electroporation, the subject technology can focus on the electric field between the bottom of the well and the hollow tip electrode. In this way, adherent (immobilized) cells are directly electroporated in their natural state.</p><p num="0007"> Another important advantage of the present invention is that the alignment devices and methods can be extended to a number of research processes that facilitate high-throughput screening and enable genome-wide RNAi screening for organism-related cell types. is there. Other high-throughput / large-scale applications include cDNA screening, characterization of intracellular targets, interrogation of biological systems in signaling pathways, and administration of intracellular drugs. Further, the devices of various embodiments of the present invention can be manufactured relatively easily and inexpensively.</p>

<p num="0008"> In order to achieve these and other advantages according to the purposes of the present invention, as embodied herein, the present invention provides a method of aligning at least one tip of a tip manifold with the wells of a multi-well plate. Including. This method comprises providing at least one alignment hole. The preferred method has two alignment holes that are spaced apart enough to give sufficient angular accuracy.</p><p num="0009"> For example, two alignment holes can be formed at both ends of the multiwell plate. The method further comprises providing at least one alignment pin, which is located alongside at least one alignment hole. The method further comprises inserting at least one alignment pin into at least one alignment hole to guide at least one tip into at least one of the plurality of wells. The number, size and arrangement of alignment holes / pins are assumed to be flexible given that they favor the purpose of aligning the tip axis with respect to the wells.</p><p num="0010"> According to a further aspect of the invention, the at least one chip is an electroporation chip.</p><p num="0011"> According to another aspect of the invention, the at least one chip is equal to or plural as the number of wells in a multi-well plate.</p><p num="0012"> According to a further aspect of the invention, the at least one chip is equal to and plural as some of the many wells of a multi-well plate.</p><p num="0013"> According to another aspect of the invention, the at least one chip is a collection of a large number of chips, the plurarity is the number of wells, and the number of wells is the number of chips. It is a multiple. Preferably, the method further comprises repeating the induction step, in which the total number of times the induction step is performed can be up to a tip so that the tip can be inserted into all or part of the well. Equal to a multiple of the number of wells relative to the number of.</p><p num="0014"> The present invention also provides a multi-well plate for receiving at least one tip of the manifold. The multi-well plate includes a plurality of wells for holding biological material, a body that defines a first alignment hole and a second alignment hole, and the second alignment hole faces the first alignment hole. Generally, the first and second alignment holes are asymmetrically formed on the multiwell plate. Preferably, the wells of the multi-well plate are non-perforated.</p><p num="0015"> According to a further aspect of the invention, the multi-well plate further forms a first alignment slot and a second alignment slot. The first and second alignment slots can be formed adjacent to the first and second alignment holes, respectively.</p><p num="0016"> In a further aspect of the invention, the body has a rectangular shape. The first alignment hole and the first alignment slot can be formed on the first short side of the body, and the second alignment hole and the second alignment slot can be formed on the second short side of the body. It is preferred that the body has sides, the first alignment slot is formed closer to the side surface than the first alignment hole, and the second alignment slot is formed closer to the side surface than the second alignment hole.</p><p num="0017"> In a further aspect of the invention, the alignment holes and alignment slots are formed as circles with diameters within the exemplary range of 0.2-10.0 mm, and each alignment slot is formed within the center of the alignment slot. It has a slot center point, each alignment hole has a hole center point formed within the center of the alignment hole, and each slot center point is 0.2-10. From the center point of each adjacent hole. It is formed within or more of the exemplary range of 0 mm.</p><p num="0018"> According to a further aspect of the invention, the body is processed from a material selected from the group consisting of metals, ceramics, plastics, rubbers, glasses and combinations thereof. The number of wells may be 6, 12, 24, 48, 96, 384, 1536, or 3456 wells.</p><p num="0019"> The present invention also provides an apparatus containing a multi-well plate. The multi-well plate includes a plurality of wells for holding biomaterials, a body that defines at least one alignment hole. In some embodiments, the second alignment hole faces the first alignment hole. The device includes a table and a robot member for aligning the multi-well plate located on the table with the chip manifold. It is preferred that the chip manifold contains at least one chip and the robot member aligns the multiwell plates perpendicular to the table.</p><p num="0020"> According to a further embodiment of the invention, the multiwell plate further forms at least one alignment slot. Preferably, the at least one alignment slot is a first and second alignment slot for securing the position of the multiwell plate on the table.</p><p num="0021"> The present invention also provides an apparatus that includes a chip manifold. The chip manifold includes a plate, at least one chip dependent on the plate, and at least one alignment pin dependent on the plate. The second chip alignment pin may face the first chip alignment pin.</p><p num="0022"> According to a further embodiment of the present invention, the at least one chip includes an electrode, a light guiding member, a disposable plastic chip for dispensing, and the like.</p><p num="0023"> According to another embodiment of the present invention, the chip manifold is an electroporation chip manifold.</p><p num="0024"> According to a further embodiment of the invention, the device further comprises a multi-well plate. The multi-well plate includes a body that defines a plurality of wells (eg, non-perforated wells), first alignment holes, and second alignment holes that hold biological material. Preferably, the second alignment hole faces the first alignment hole. In a further embodiment of the invention, at least one tip comprises a capillary electrode filled with at least one electrolyte having a non-conductive capillary wall, where at least one tip is in each well. It has been lowered. Each well has a surface defined by the bottom of the well, and at least one tip can be lowered a predetermined distance from the surface of each well. In some embodiments, the predetermined distance is 75 micrometers, but variations from one or a few millimeters to micrometer or submicrometer are also conceivable.</p><p num="0025"> According to a further embodiment of the invention, the alignment holes form a circle, and the alignment pins have a circular cross section and are made to fit snugly into the alignment holes. When a single alignment hole is used with a single alignment pin, the pin and receiving hole have a geometric shape that allows alignment in the xy direction. For example, the receiving holes can be star-shaped, cross-shaped, and triangular, and the cross-sections of the pins can be star-shaped, cross-shaped, and triangular, respectively. Preferably, the first and second alignment pins have a pin length, and at least one tip has a chip length. The length of the pins is longer than the length of the tips, and the tip manifold is configured so that the alignment pins can be inserted into the respective alignment holes before inserting at least one tip into each well for precise alignment. There is.</p><p num="0026"> According to another embodiment of the invention, the alignment pins (s) have rounded tips pointing towards the alignment holes (s) so that the alignment pins (s) can be inserted into the alignment holes (s). It has and the multi-well plate slides laterally until the alignment pins (s) are inserted into the alignment holes (s).</p><p num="0027"> In a further embodiment of the invention, when the tip comes into contact with the plate, the tip is spring-loaded for vertical compliance. The well further defines the bottom surface at the bottom of the well, and the bias body normally extends to the end by the force from the spring, but when inserted into the well and comes into contact with the bottom surface, it moves proximally to the multi-well plate body. Brings compliance in the direction perpendicular to the plane of. Preferably, the tip is an electroporation tip.</p><p num="0028"> According to a further embodiment of the present invention, the robot member is connected to the chip manifold. The robot member assists in aligning the alignment pins with the alignment holes and lowering at least one tip of the tip manifold into their respective wells.</p><p num="0029"> According to another embodiment of the invention, at least one chip is an array of many chips, and the chips in the array are arranged in at least one row containing at least one chip. The number of chips may be equal to the number of wells in the multi-well plate. The number of wells may be 6, 12, 24, 48, 96, 384, 1536, or 3456 wells.</p><p num="0030"> According to a further embodiment of the invention, the number of wells is equal to a multiple of the number of chips so that at least one chip is inserted into each well portion in alignment with each well portion. It is composed.</p><p num="0031"> According to another embodiment of the invention, the chips are aligned in a matrix of at least one chip containing a large number of chips. The pluralities of the wells form a well matrix containing a large number of wells. The number of wells is a multiple of the number of chips. The well matrix is divided into at least one group of wells. The total number of alignment holes may be equal to a multiple of the chips relative to the wells. Half of the alignment holes can be formed on one opposite side of the multi-well plate and the other half of the alignment holes can be formed on the other opposite side of the multi-well plate. The alignment pins are configured to align with each alignment hole and be inserted into the hole, and the number of dips is equal to the number of wells for the tip.</p><p num="0032"> As a matter of course, the above general description and the following description are exemplary and are intended to provide further description of the claimed invention.</p><p num="0033"> The accompanying drawings, which are incorporated herein by reference and are in part thereof, are provided to illustrate the devices and methods of the present invention and provide further understanding. Along with the description, the drawings serve to explain the principles of the invention. All relative statements, such as left, right, up, down, anterior, and posterior, are in reference to the drawings and are not meant to be limiting.</p>

Although cited as an example, the following statements, which are not intended to limit the invention to the particular embodiments described, are understood in the context of the accompanying drawings incorporated herein by reference. Will be done. Many preferred embodiments of the invention are described as non-limiting examples and with reference to the accompanying drawings.<figref num="1">FIG. 1 illustrates a system in which the present invention can be implemented.</figref><figref num="2">FIG. 2 is a schematic view of a partial top surface of the environment described in FIG.</figref><figref num="3">FIG. 3 illustrates embodiments of the subject art showing an electroporation tip manifold containing alignment pins and electroporation tips, and a multi-well plate containing eight pairs of alignment holes and slots. ing.</figref><figref num="4">FIG. 4 illustrates a perspective view of a multi-well plate of an embodiment of the subject art shown in FIG.</figref><figref num="5A">FIG. 5A is a detailed top view or plan view of the multi-well plate according to the embodiment of the present invention shown in FIG.</figref><figref num="5B">FIG. 5B is a detailed partial view of the circle B showing some alignment holes and alignment slots shown in FIG. 5A.</figref><figref num="5C">FIG. 5C is a side view of the multi-well plate shown in FIG. 5A.</figref><figref num="6">FIG. 6 is a perspective view of the electroporation chip manifold of the embodiment of the present invention shown in FIG.</figref><figref num="7A">FIG. 7A illustrates an embodiment of an alignment pin of a chip manifold having a rounded tip adjacent to an alignment hole in a multi-well plate.</figref><figref num="7B">FIG. 7B illustrates a pin lowered into the alignment hole of FIG. 7A.</figref><figref num="8">FIG. 8 illustrates one possible insertion sequence for covering a 384 multiwell plate with the 48 chip manifold shown in FIG.</figref><figref num="9A">FIG. 9A is an exploded view of another electroporation tip manifold that includes an alignment pin and 96 electroporation tips.</figref><figref num="9B">FIG. 9B is a front view of the electroporation tip manifold of FIG. 9A with the cover removed to show the components therein.</figref><figref num="9C">9C is a side view of the electroporation tip manifold of FIG. 9A.</figref><figref num="9D">9D is a top view of the electroporation tip manifold of FIG. 9A.</figref><figref num="9E">9E is a cross-sectional view of the electroporation tip manifold along line EE of FIG. 9D.</figref><figref num="9F">FIG. 9F is an exploded view of the electroporation chip assembly.</figref><figref num="9G">FIG. 9G is a perspective view of the electroporation chip assembly of FIG. 9F.</figref><figref num="9H">9H is a top view of the electroporation chip assembly of FIG. 9F.</figref><figref num="9I">FIG. 9I is a cross-sectional view of the electroporation tip assembly along line I-I of FIG. 9H.</figref><figref num="9J">FIG. 9J illustrates a spring-loaded tip of FIG. 9A inserted into the well of a multi-well plate.</figref><figref num="10">FIG. 10 is a flowchart relating to a method for carrying out an embodiment of the present invention.</figref><figref num="11">FIG. 11 shows the results of a plasmid transfection assay using the subject technology. And</figref><figref num="12">FIG. 12 shows the results of a siRNA transfection assay according to the disclosure herein.</figref>

I. Definition The term "multi-well plate" is intended to include structures that define any number of wells for retaining biological, chemical, or physical material for the screening process.

The term "non-perforated" is intended to characterize the body material for retaining the material placed in the well without the material leaking through the body material. For example, a plastic body material can be said to be non-perforated because it can retain the biological material without leaking through the plastic body material.

The term "chip manifold" is used to hold any number of chips that are structured to hold and / or dispense biomaterials, including liquids, and / or electroporate biological samples. Is intended to include the structure of. The tips in the tip manifold are generally configured and arranged to align with the wells.

The term "electroporation" is intended to include the application of significant voltages that penetrate a cell double membrane, such as a cell membrane, due to the applied electric field. Electroporation, among other applications, in molecular biology, for example, is a method of introducing a substrate (eg, DNA, RNA, siRNA, small molecule, peptide, protein, antibody) into a cell, eg, a cell. It is used to incorporate molecular probes, drugs that can alter cell function, or nucleic acids into cells.

The term "biomaterial" is intended to include any substance formed or recently formed from an organism. For example, biomaterials can include organically formed substances such as compounds occurring in cell tissues, plants, living cells, processed biomaterials, viable substances, and soil and other organic substances. ..

The term "screening" is intended to include exploring a large number of things (eg, biological samples) to find out what has a particular problem or characteristic. Screening can be performed in a number of areas where the present invention can be practiced, including, but not limited to, pharmacology, medicine and the like. For example, in pharmacology, screening can be performed to search for pharmacological activity during drug discovery (eg, to detect biological activity of a chemical compound on cells (eg cell proliferation)).

The term "nucleic acid" is intended to include macromolecules consisting of nucleotide chains. For example, a molecule can carry genetic information or form a structure within a cell. Common nucleic acids include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

II. System and method Automatic screening and manipulation of biological material in multi-well plates requires alignment of the chip assembly with respect to the plate wells. For example, the systems and methods of the invention are suitable for performing electroporation-based complementary DNA (cDNA) high-throughput screening transfections using RNA interference (RNAi) and electrolyte-filled capillaries (EFCs).

The multi-well plate of the present invention can be used for many screening applications. One common use is to dispense a liquid into a multi-well plate so that each well in the plate receives a controlled amount of the agent. Examples of such agents include genetic material, proteins, peptides, drugs, enhancers, bioactive compounds in general, inhibitors, and dyes. Dispensing of liquid into a multi-well plate can be performed using a pipette tip manifold, where each pipette tip corresponds to a given well in the well plate.

For example, all 96 wells in a 96-well multi-well plate simultaneously receive up to 96 different solutions from 96 different pipette tips in the tip manifold so that each additional solution is distributed to each well. Chips can be arranged in the chip manifold so that the arrangement of each chip (eg, the spacing between the chips) corresponds to the arrangement of the wells in the multi-well plate.

Wells can contain, for example, cells grown on the bottom, cells in suspension, or chemical species such as reagents or enzymes. The solution added to the wells can contain the drug so that information on binding affinity for cells can be obtained by adding the drug to the wells at different concentrations. The solution can contain the substrate so that information about the reaction rate can be obtained by adding the substrate to the wells containing the enzyme.

The multi-well plates of the present invention are of great importance to experimental results for many robot screening applications, including capillary electroporation, and for example, accurate placement of chips on cell layers grown on the bottom of each well. It can be used in several electrochemical and optical applications.

FIG. 1 shows a system (100) capable of performing automatic screening and manipulation of biological material according to the present invention. The system (100) can be provided with a sealed, temperature and humidity controlled, filtered space to facilitate parameters suitable for the methods described herein. The system (100) is provided with a tip manifold (200) and a multi-well plate (300). The robot member (110) is for general positioning of the chip manifold (200). The robot member (110) has a lower end (111) adapted and configured to selectively combine various chip manifolds. When not in use, the tip manifold can be placed at the park station (113).

The multi-well plate (300) can be placed on the table (120). The tip manifold (200) can be attached to the robot member (110) and moved in various directions with respect to the multi-well plate (300). For example, with respect to the axes (112), (114), (116) shown in FIG. 1, the tip manifold (200) is forward along the axis (112) in a plane parallel to the plane of the multiwell plate. And rearward, left and right along an axis (114) parallel to the surface of the multiwell plate, and an axis (116) perpendicular to the surface of the multiwell plate (200) or table (120). ) Can be moved upwards and downwards. Also, the robot member (110) can be attached to a multi-well plate (200) or table (120) for general positioning of the tip manifold (200).

Here, with reference to FIG. 2, a schematic top view of the system (100) is shown. System (100) provides stations (132), (134), (136), (138), (140) to hold various components and substances for screening. For example, stations (132), (134), (136), (138), (140) are buffer compound (132), control compound (134), various media (136), source (138), and Cellular material (140) can be retained. The other stations (142), (144), (146) can hold the chip block (142), the cleaning station (144), and the chip manifold (146). The robot member (110) attached to the chip manifold (200) is loaded with one or more substances for screening, such as buffer (132), control (134), and cell culture (140). Can be operated to combine. The robot member (110) can be programmed to capture the chip block (142) and clean it at the cleaning station (144) as needed during the screening process.

With reference to FIG. 3, the present invention provides a multi-well plate (300) for receiving by a plurality of chips (202) of the chip manifold (200). The multi-well plate (300) is also shown in FIGS. 4, 5A, 5B, and 5C and is provided with a body (304) that defines a plurality of non-perforated wells (302) for holding biological material. .. The biological material can contain cells grown on the bottom of the well (302) or cells in suspension.

The multi-well plate (300) can have one or more banking surfaces (315) for the initial positioning of the multi-well plate (300) on the table (120). In order to accurately fit the tip (202) into the well (302), the tip manifold (200) initially fits the alignment opening (310a-h), (312a-h) into the multi-well plate (300). It has pins (210) and (212).

Within the multi-well plate (300) are eight pairs of aligned openings (310a-h), (312a-h). In each pair, the alignment slots (310a-h) face the circular alignment holes (312a-h). A pair of alignment slots and holes (310a-h), (312a-h) can be asymmetrically formed on the multi-well plate (300). For example, alignment holes (310a) and (312a) can be formed on both sides of the body (304). Separately, a pair of alignment openings is formed on the adjacent side of the body (304), either close to each other, or in a structure suitable for the screening process that allows accurate alignment with the chip manifold (200). can do. In the structure shown, the alignment openings are arranged in four groups, each group having two slots (310) near the perimeter and two holes (312) located internally from adjacent slots (310). have.

The body (304) of the multi-well plate (300) has 16 or more or less aligned openings (310), depending on the structure of the multi-well plate (300), the insert manifold (200), and the needs of the screening application. (312), for example, 1, 2, 3, 4, 5 devices and the like can be specified.

As shown in FIGS. 3, 4, 5A-C, and 6, the multi-well plate (300) has 16 alignment openings, and the chip manifold (200) has 4-12 arrays of chips. doing. Therefore, in order to cover 384 wells with the 48 tips of the chip manifold (200), the body (304) of the multi-well plate (300) was replaced by a total of 8 different chips manifold (200). It can be configured to define 16 openings (310a-h), (312a-h) (eg, 8 pairs) for the dip. In this way, each dip utilizes a different pair of aligned openings (310), (312). The tip manifold (200) is an electroporation tip manifold having 4 × 12 rows of tips (202) with a 9 mm spacing with respect to the 4.5 mm spacing of the wells (302) in the multi-well plate (300). Is preferable.

According to a further embodiment of the invention, the multi-well plate (300) is characterized by a bottom surface or an aligned opening (310) for placing the multi-well plate (300) on a table (120). (312) can be used completely, partially or temporarily. In addition, another portion of the banking surface (315) or multi-well plate (300) should be readily adjacent to the complementary surface on the table (120) for rough positioning of the multi-well plate (300). Can be done. The multi-well plate (300) is a flat portion, on the bottom side, for the multi-well plate (300) to be placed in six axes (axis 112, 114, 116 and coaxial rotation) in a highly precise manner. It may have 3 points (not shown) to interact with 3 points such as the jagged part and the semi-dome part. It is also preferred that the bottom is provided with a support beam to increase structural stability. In one embodiment, it has a plurality of long support beams extending parallel to the edge and a plurality of shorter support beams extending vertically to the edge. The number and structure of support beams can be changed as desired.

Preferably, alignment openings (310), (312) can be formed in another portion of the multi-well plate (300), eg, at the corners of the multi-well plate. The multi-well plate (300) can further form three, four, or any number of aligned openings of various sizes and shapes, depending on the needs of the application. For example, larger mechanical parts will require more than one slot to increase stability. In another example, one or more slots are "" so that only a single hole can be combined with a "+" or "-" alignment pin to place the multiwell plate (300) laterally and rotationally. It can be in the shape of "+" or "-". Many other shapes of alignment pin and hole combinations can provide tertiary adjustment (shaft 112, shaft 114 and rotation shaft 116 with respect to FIG. 1), such as shapes such as triangles, keyholes and the like.

As best seen in FIG. 5A, the body 304 has a rectangular shape. Alignment openings (310), (312) can be formed on the first short side of the body (304) by forming alignment slots (310) closer to the outer circumference (336) than alignment holes (312). .. The body (304) is not limited to a rectangular shape for screening applications and may have any suitable shape of square, circular, polygonal, elliptical, or any other, or a combination thereof. .. Further, it is not necessary to form the alignment slot (310) near the outer circumference, for example, to form the alignment slot (310) at a portion of the body (310) substantially at the center of the body (304), for example, away from the outer circumference than the hole (312). Can be done.

Alignment openings (310), (312) can be formed in distant portions of the multiwell plate body (304). The table (120) can also have an upright protrusion or similar structure to help receive the alignment pin (210) by first guiding the multi-well plate (300). Therefore, the multi-well plate (300) will not require a aligned opening. In yet another embodiment, the table (120) may include a moving pin that first aligns the multi-well plate (300) through the alignment openings (310), (312), which is then the moving pin. The tip manifold (200) can be aligned by retracting and using the alignment openings (310), (312).

In a further embodiment of the invention, both the aligned openings (310) and (312) are formed as circular. Further, each of the aligned openings (310) and (312) has a formed hole center point. The alignment holes (310) and (312) can be formed in other shapes, such as squares and shapes with four or more sides, such as hexagons. In addition, the diameter of the alignment holes and the distance between the alignment hole center points (325) can be varied as needed for screening applications. In some embodiments, the body (304) is manufactured by injection molding. During such a manufacturing process, a discharge pin (not shown) is used to remove the body (304) from the mold (not shown). As a result, traces of discharge pins may appear on the body (304) as multiple circles (319). This circle (319) does not pass through the hole or even sink.

The body (304) can be made of metal, ceramic, plastic, rubber, glass, etc., and even combinations thereof. The number of wells includes 6, 12, 24, 48, 96, 384, 1536 or 3456 wells. The number of wells may be even or odd.

The system (100) also includes a chip manifold (200), the embodiment of which is shown in FIG. The chip manifold includes a plate (204) and a plurality of chips (202) subordinate to the plate (204). The first and second chip alignment pins (210), (212) are also dependent on the plate (204). The second chip alignment pin (212) faces the first chip alignment pin (210) so that a pair of opposing alignment openings (310), (312) can be used for alignment.

The tip (202) includes an electrode or light guide member, or a dispensing tip such as a disposable plastic pipette tip. The electrodes can be used for electroporation of biological material and the tips can be capillaries or tips filled with electrolytes. The electrode may be a solid, eg, a cylindrical electrode, for measuring the oxidation or reduction process. The light guide member is used to expose the biological material to light, and the chip may be an optical fiber lumen for guiding light or for measuring light emitted from wells and / or cells, such as fluorescence or luminescence. .. Disposable tips can be used in many applications for adding and recovering liquids that require a high degree of accuracy and cleanliness.

Here, with reference to FIGS. 7A and 7B, an alignment for aligning the chips (202) and wells (302) is partially shown to illustrate the process. In particular, FIG. 7A illustrates an alignment pin (210) of a rounded tip manifold that is close to the alignment slot (310) of the multiwell (300). As described above, the chip manifold (200) is selectively connected to the robot member (110). By moving along the axes (112), (114), (116), the robot member (110) positions the alignment pins (210) and (212) in the alignment holes (310) and (312), respectively. For simplicity, only the pins (210) and alignment slots (310) are shown in FIGS. 7A and 7B.

The alignment pin has a rounded tip pointing towards the alignment openings (310), (312). The multi-well plate (300) can be placed on the table (120) and positioned first, but the multi-well plate (300) can be freely moved as described above. The multi-well plate (300) is initially arranged so that the alignment pins (210), (212) of the chip manifold (200) are at least partially aligned with the alignment openings (310), (312). The final alignment of the well plate (300) to the tip manifold (200), and thereby the tip (202) to the well (302), aligns the alignment pins (210), (212) with the opening (310). Achieved by inserting into (312).

Here, referring to FIG. 7B, the pin (210) is fully inserted into the alignment hole (310). The axes (112), (114), (116) of FIG. 1 are reproduced for the reference direction. The tip manifold (200) is lowered along the axis (116) towards the multiwell plate (300) to insert the pins (210), (212). As the pins (210), (212) enter the alignment openings (310), (312), the rounded tips (280) of the alignment pins (210), (212) are axially (112) the multiwell plate (300). , (114) to move laterally in the horizontal plane. Since there are two aligned openings (310), (312) that are moved by the two pins (210), (212), the multiwell plate (300) can also be adjusted for how it rotates around the axis 116, eg. 3-axis adjustment. Since the chips (210), (212) are relatively long and closely depend on the multi-well plate (300), alignment occurs before the chips (202) reach the wells (302). In this way, all chips (202) and wells (302) are fully positioned and aligned in preparation for the chips (202) being inserted into the wells (302). Further, a triangular pin and one triangular hole can be used for triaxial adjustment and the like. As another example, the multi-well plate is held in precise alignment by a wall extending vertically from the table (120), and the multi-well plate (300) contacts the banking surface (315) or another location. If so, rotational alignment can be prevented. Therefore, only side adjustments can be required by inserting the pin into the hole.

With reference to FIG. 8, the 48-chip manifold (200) shown in FIG. 1 illustrates one possible insertion pattern for covering every well of the 384-well plate (300). The multi-well plate (300) has 384 wells (302) arranged in 16 rows of 24 wells / row. The chip manifold (200) has four rows of 12 chips / row, spaced twice as much as the wells (302). Therefore, eight alignment dips of the chip manifold (200) are required to access each well (302). Eight pairs of facing alignment holes (310a-h) and (312a-h) are formed facing the eight dips. The subject technology is not limited to this structure, for example, chip rows, well rows every 3 rows, well rows every 4 rows, etc. for each signal well row, based on the needs of the screening process. Can be spaced.

More specifically, in order to carry out the screening, the robot member (110) moves the tip manifold (200) between the eight pairs of alignment holes (310a-h), (312a-h). (202) is inserted into every well (302). For example, when the pins (210) and (212) of the chip manifold (200) are aligned in the alignment holes (310a) and (312a) as described above, the tip (202) is labeled "a" in the well (302). ) Are aligned and inserted. When the pins (210) and (212) of the tip manifold (200) are aligned in the alignment holes (310b) and (312b) as described above, the tip (202) is in the well (302) labeled "b". It is aligned and inserted in. After the fourth dip is completed by aligning with the holes (310d) and 312 (d), the robot member (110) is placed on the pair (310e-h), (312e-h) below the alignment hole. Jump down and continue. As described above, the robot member (110) moves the chip manifold in the two-step boustrophedonic pattern indicated by the wells (302) labeled with a to h.

In an embodiment, the tip (202) has a 9 mm row spacing and a 9 mm column spacing, and the well (302) has a 4.5 mm row spacing and a 4.5 mm column spacing. Therefore, the chips (202) of the chip manifold (200) are aligned in a row that is vacant by a row of wells and in a column that is vacant by a row of wells.

In certain embodiments, the subject art comprises a device that includes a multi-well plate (300). The multi-well plate comprises a plurality of non-perforated wells (302) for holding biological material, a first alignment hole (310), and a body (304) defining a second alignment hole (312). The two alignment holes (312) face the first alignment hole (310). The device includes a table (120) and a robot member (110) for aligning the multi-well plate (300) located on the table (120) with the chip manifold (200). The chip manifold (200) contains at least one chip (202), and the robot member (110) further aligns the multiwell plate perpendicular (116) (vertically) to the plane of the table (120). Is preferable.

According to a further embodiment of the invention, the multi-well plate further secures the position of the multi-well plate on the table (120) to help align the chip manifolds, a pair of alignment holes (310), (. 312) is formed.

In certain embodiments, the subject art comprises aligning at least one chip (202) of the chip manifold (200) with a plurality of wells (302) of a multi-well plate (300). In one method, at least one alignment hole is formed on one side of the multi-well plate (300) and at least one alignment hole is formed on the opposite side of the multi-well plate (300), at least two alignments. Includes providing holes (310), (312). The method is that at least one of the alignment pins is connected to one side of the chip manifold (200) and at least one of the alignment pins is connected to the other side of the chip manifold (200), at least two alignment pins (210). , (212). The method is to insert at least one alignment pin connected to one side of the chip manifold into at least one of the alignment holes, and to align at least one alignment pin connected to the other side of the chip manifold to the other alignment. It involves guiding at least one tip (202) into at least one of the plurality of wells (302) by inserting it into at least one of the holes. According to a further aspect of the invention, at least one chip is an electroporation chip.

According to another embodiment of the invention, the chip manifold (200) has an array of a large number of chips (202). The chips (202) in the array are arranged in at least one row containing at least one chip (see FIG. 6 having 4 × 12 rows of chips (202)). The number of chips (202) may be equal to the number of wells (302). The plurality of wells (302) includes any number of wells such as 6, 12, 24, 48, 96, 384, 1536 or 3456. In a further embodiment of the invention, the number of wells is such that at least one tip is aligned with one portion of each well and can be inserted into one portion of each well. Equal to a multiple of the number of. Suitable electroporation chips and methods of use are known in the art. For example, in U.S. Pat. Nos. 6,521,430 and U.S. Patent Application Publication Nos. 2005/0048651 and 2005/0036283, all of which are incorporated herein by reference in their entirety. The electroporation chips described can be applied to the use of the apparatus of the present invention.

Referring to FIG. 9A, an exploded view of another electroporation tip manifold (400) containing an alignment pin and 96 spring-loaded electroporation tips is shown. For further clarity, the following description also refers to FIGS. 9B-9E, which show a front view, a side view, a top view and a cross-sectional view of the electroporation tip manifold (400). The manifold has 96 spring-loaded electroporation chip assemblies (402) arranged in 8x12 rows. Also, the distance between the electroporation tip assemblies (402) is twice that of the wells. Therefore, the 348 well plate is covered after the manifold (400) has passed four times. Having a spring-loaded electroporation tip results in a quaternary adjustment of the tip (402) (ie, adjustment along axis 116 in FIG. 1).

The manifold (400) includes a cover (404) that forms an opening (406) for the tip guide plate (408). The tip guide plate (408) provides an opening (410) for each electroporation assembly (402). The tip guide plate (408) holds two first alignment pins (412) to align the multiwell plate with the electroporation tip assembly (402). The tip guide plate (408) also holds any second alignment pin (414) for shallow fitting into the alignment holes on the multiwell plate to provide additional stability and positioning.

The chip guide plate (408) is aligned with the interconnected printed circuit boards (pcb) (416) by alignment pins (418). The pcb (416), along with the tip guide plate (408), is coupled with the respective electroporation tip assembly (402) to provide electrical interconnection and mechanical spring loading to the electroporation tip assembly (402). PCBs (416) have bifurcated pin assemblies (420) that depend on them. The pcb (416) defines a hole holding a bias element, such as a spring (not shown), to provide downward energy for each bifurcated pin assembly.

Here, with reference to FIGS. 9F-9I, various diagrams of the electroporation chip assembly (402) are shown. Each electroporation chip assembly (402) has an external electrode (422) with a lower portion (424) that is relatively narrower than the upper portion (426). The external electrode (422) has a constricted portion (428) between the upper portion and the lower portions (424) and (426). The external electrode (422) defines an interior (430) for receiving the electrode spacer (432) at substantially the upper portion (426). The external electrodes (422) and spacers (432) each have a complementary rectangular ring (434), (436) to establish a relationship between them. The ring (434) of the external electrode also forms the banking surface (438) (best visible in FIG. 9I), which allows the electroporation tip assembly (402) to pass through the respective holes in the tip guide plate (408). Prevent that. Thus, the electroporation tip assembly (402) is simply stationary within the tip guide plate (408) and can move upwards. There is also an electrode contact (440) adjacent to the ring (434) on the external electrode (422).

The lower portion (424) of the external electrode (422) substantially houses the chip base (442). Both the spacer (432) and the tip base (442) extend within the constriction region (428) so that each can be securely connected to the external electrode by fastening, welding, gluing, etc. Since the tip base (442) has an end (444) of a predetermined size, when the end (444) is adjacent to the bottom of the well, the substance there in the well and the manipulation site of the chip electrode You can set the interval between them. The end portion (444) forms a shoulder (445) that is the end of the lower portion (424). The spacer (432) also defines the interior (446) that receives the internal electrode (448). The internal electrode (448) also has an internal electrode contact portion (450) located within the spacer ring. As the internal electrode (448) extends deeper into the external electrode interior (430), the internal electrode (448) is also anchored therein at the constriction (428).

Referring again to FIGS. 9A-E, when assembled, a bifurcated pin assembly (420) subordinate to the pcb (416) connects the electroporation tip assembly (402). In particular, one of the bifurcated protrusions is configured to be in electrical contact with the external electrode contact (440), whereas the other of the protrusions is configured to be in electrical contact with the internal electrode contact (450). , This completes the electrical circuit via the connector (453). Further, since the bifurcated pin assembly (420) is spring-loaded, the electroporation tip assembly (402) may move upward when upward energy acts on the electroporation tip assembly (402). Contact is retained.

For example, if the well plate has irregular well depths, the electroporation tip assembly (402) can be inserted regardless of the depth. The ability to move upwards and the inclusion of spacers (442) allow each electroporation tip assembly (402) to be conveniently located at the same distance from the bottom of the well. With reference to FIG. 9J, an exemplary electroporation chip assembly (402) is shown arranged in an exemplary well (302). A spring-loaded electroporation tip assembly (402) is inserted into the wells (302) of the multi-well plate (300). The electroporation tip assemble (402) can be applied to perform suction and / or electroporation.

The movement of the robot component (110) along the axis (116) shown in FIG. 1 lowers the electroporation chip assembly (402) into each well (302). Preferably, each well (302) has a substantially flat surface at the bottom of the well (302). The electroporation tip assembly (402) is lowered into each well (302) and beyond the point where the spacer (442) contacts the bottom. As a result, the spring load is used to set a predetermined distance between the bottom (360) of each well (302) and the operating site of the electroporation tip assembly (402). In some embodiments, this predetermined distance is about 75 micrometers.

It is preferred that the electroporation solution be treated in the wells (302) and the biomaterial to be electroporated (eg, cells) be treated in the bottom of the wells (302). The biological material may be mammalian cells, but may include other suitable substrates (eg, fatty vesicles). The biomaterial may be at the bottom of the well (302) or may be attached to the bottom. The well (302) defined in the multi-well plate body (304) is usually a square that fits snugly into the shape of the spacer (442).

Referring again to FIGS. 9A-9E, the manifold (400) also includes a tube (452) for establishing a liquid flow path between the electroporation tip assembly (402) and the liquid connection plate assembly (454). .. The liquid connection plate assembly (454) has an outer frame (456) that supports the liquid distribution plate (458). Two carrier support assemblies (460) are provided on the outer frame (456) of the liquid connection plate assembly (454) so that the manifold (400) can be connected to another component. Multiple fasteners (462) and washers (464) together secure the manifold components (only some of them are shown for convenience).

FIG. 10 illustrates a flowchart having the steps of the method of the invention. In particular, this flowchart illustrates electroporation using electroporation steps S102-S116, which are defined by being surrounded by a dotted frame. However, this method is not limited to the electroporation method and can be used for other screening experiments. Steps S150-S160 can be performed to prepare the medium in the chip (202) before starting the electroporation method.

First, a multi-well plate must be prepared. In step S150, the robot member (110) can capture the liquid processing chips to recover the liquid sources stored at stations (132), (134), (136), (138), (140).

For illustration, FIG. 10 describes transfection of HeLa cells with siRNA specific for polo-like kinase 1 (PLK1). Here, successful transfection results in a complete loss of viability compared to controls 72 hours after post-transfection culture. First, 1000 HeLa-S3 cells (ATCC No. CCL-2.2) were supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin in each well of the multi-well plate (300) in DMEM medium (DMEM medium. Seed in 40 μl (available from Invitrogen of Carlsbad, California under trade number 32430-027). After sowing, 5% CO on multi-well plate (300)_２Incubate at 37 ° C. for 24 hours.

After culturing, the multiwell plate (300) is ready for transfection. In step S152, the robot member (110) is ready to use a suitable liquid processing chip on the chip manifold. Remove the cell culture medium (usually leaving 10 μl of residual medium).

In step S154, the electroporation medium is added to the wells (302). In step S156, nucleic acid (eg, siRNA specific for PLK1) can be added from the source plate with control from the control plate. For example, siRNA and electroporation buffer are added. In one embodiment, siRNA and electroporation buffer add a total of 27 μl, for a total of 37 μl in each well (302). To clean the intermediates in steps S154 and S156, the robot member (110) can move the liquid processing chip to the cleaning station. In step S158, the robot member (110) prepares to separate the liquid processing chip and connect it to the electroporation chip manifold (ETM) (200).

In step S160, the ETM (200) is captured by the robot member (110) and is ready for electroporation operation. In step S102, the robot member (110) moves the ETM (200) to a position above the multi-well plate (MWP) (300) located on the table (120). At the end of step S102, the ETM (200) is roughly aligned on top of the MWP (300) for accurate alignment in the next step. In step S104, the ETM pins (210) and (212) are more accurately aligned with the MWP alignment holes (310a) and (312a).

In step S160, the ETM pins (210) and (212) are inserted into the alignment holes (310a) and (312a) to align the MWP (300) exactly with the ETM (200) as described above.

In step S108, the liquid is sucked to create an electric current or an electrical circuit. For example, the chip (202) collects a total of 15 μl of liquid, leaving 22 μl in each well (302). The tip (202) is then lowered into the well (302) and stopped 2 mm above the bottom of the well (360). Having 15 μl of liquid sucked into the chip (202) creates electrical contact between the respective internal and external electrodes of the electroporation chip. For example, the electrical circuit of the chip is closed. The electrodes of the chip (202) are connected to a square wave pulse generator (not shown) capable of transmitting high voltage pulses to the chip.

In step S110, the tip (202) is moved to contact the bottom (360) of each well. The tip (202) is preferably spring-loaded as described above. In step S112, a pulse protocol is added. The pulse protocol can be significantly varied depending on the cell type. For PLK1, a suitable pulse protocol is 25 pulses with an applied voltage of 130 V and a pulse length of 25 ms at 0.1 second intervals.

In step S114, the tip (202) can be lifted onto the bottom surface (360) of the well (302) to distribute most of the liquid. Alternatively, the chip (202) can be advanced directly to a cleaning station that distributes and disposes of the liquid.

In step S116, the ETM chip (202) moves out of the MWP well (302). The electroporation operation may be repeated to cover another portion of the well with a chip (120), or may be aborted and cultured in step (122). In the 384-well (302) and 48-chip (202) embodiments, a pair of alignment holes (310b-h), (312b-h) to perform an electroporation operation on all 384 wells (302). ), The processing steps S106 to S116 will be carried out seven more times. It is also possible to wash and replace the chips during processing to collect new biomaterials so that some of the wells can contain different screening material.

Once the electroporation procedure is complete, the electroporated cells can be cultured in the presence of the transfected molecule. For example, in the PLK-1 assay, 28 μl of medium is added after electroporation. Additional medium was supplemented with 15% fetal bovine serum and 1.5% penicillin / streptomycin to a final volume of 50 μl with 10% fetal bovine serum and 1% penicillin / streptomycin, followed by 5% CO._２Incubate at 37 ° C. for 72 hours.

After culturing, transfection efficiency and viability can be evaluated. This system (100) can facilitate the evaluation or the evaluation can be performed outside the system. In the example of PLK1, the system can remove some medium from MWP (300). Using a liquid processing manifold (not shown) connected to robotic member (110), system (100) added 40 μl of 10% Alamar Blue reagent to DMEM medium supplemented with 2% fetal bovine serum. Can be added. After culturing for 2 hours in the dark at room temperature, transfection efficiency and viability were obtained from SAFIRE available from Tecan Trading Group AG in Lausanne, Switzerland.^２Evaluate using a (registered trademark) plate reader according to the manufacturer's instructions.

11 and 12 show the results of automatic screening using the apparatus and method of the present invention. FIG. 11 shows the results of viability and efficiency by plasmid transfection. Percentages of efficiency and viability have been shown for cell type DRG, Schwann cells, PC-12, SH-SY5Y, endothelium (human), A549, and Neuro-2a. FIG. 12 shows the percentage of viability and efficiency of siRNA transfections of HeLa, HeLa-S3, and HEK239 cell types.

One advantage of this subject technology is to provide an electroporation chip manifold with alignment pins that provide precise placement.

Another advantage of this subject technology is that it provides an electroporation chip manifold and multi-well plate alignment device, as well as a method that allows the chips to be aligned, lowered, and placed in close proximity to the surface of the cell culture wells. That is. During electroporation, the electric field can be focused between the bottom of the well and the tip capillary electrode, thus creating a substantial electroporation cuvette. This method allows cells to be electroporated directly in their intrinsic state, with improved viability.

Another advantage is that alignment devices and methods facilitate high-throughput screening. It can be extended to a number of research processes that enable applications such as genome-wide RNAi screening for biorelated cell types. Other high-throughput / large-scale applications include cDNA screening, characterization of intracellular targets, interrogation of biological systems in signaling pathways, and administration of intracellular drugs. Furthermore, the devices of various embodiments of the present invention can be manufactured relatively easily and inexpensively.

Of course, both the general description above and the description below are intended to provide further explanation of the invention as exemplary and claimed.

The accompanying drawings incorporated in and in part thereof are included to illustrate the devices and methods of the present invention and provide further understanding. Along with the description, the drawings serve to explain the principles of the invention.

The specification describing the ideas, aspects, and embodiments of the present invention, as well as specific examples thereof, are intended to include both their structural and functional equivalents. Also, such equivalents include both currently known equivalents and further improved equivalents in the future, i.e., any improved component that achieves the same function regardless of structure. Intended to do.

Although the above invention has been described in some detail as a means of description and examples for clarity and understanding, it will be apparent to those skilled in the art that some modifications and modifications can be made. Therefore, the description and examples should not be construed to limit the scope of the invention, which is described in detail in the appended claims.