Technical Field The present invention relates to a technology for pipetting by accurately regulating infinitesimal amount of samples for a single cell for suction, extration, dispensation, or injection to the single cell.

Background Art

The technique for sucking and dispensing an infinitesimal amount of substance has been developed for operations for removing or transplanting a nucleus from a single cell, such as a fertilized ovum. . In order to achieve a flow of fluid through a pipet tip performing such pipet operation, the prior mechnical pumping devices applied air pressure, oil pressure (a positive pressure or a negative pressure) , or used piezoelectric elements . In both kinds of the devices, a conduit between the pressure generating unit and the tip is filled with a fluid in advance, such as silicon oil, to effectively transmit the generated pressure to the pipet tip. A pressure generating unit, having electrical control units to control the strength and time for the pressure generation, are added to control the operation of the

pressure generating unit The ultra-micro pipet tip is produced to have an inner diameter of several micrometers

(μm) or less by melting a glass tube or a fused silica tube through electrical heating or laser heating and then pulling the melted tube.

However, in the device in which an air pressure is used as the driving force, due to a large difference in the size between the driving force generating unit and the pipet capillary, there is a large dead volume at the connecting section between the driving force generating unit and the pipet capillary ; and it is easy to capture air. In this case, a delay of a pipet operation is generated by a cushion effect in this area, and so the regulation of the amount of the infinitesimal sample becomes unreliable. In the device adopting the mechanical pumping method, the conduit between the inside of the piston to the pipet tip should be filled entirely with a fluid, such as silicon oil. However the problem is that it is very troublesome to fill the conduit with a fluid and it is not an easy operation to perform for unskilled users.

Disclosed in Korean Patent Laid Open Publication No.

10-2004-0004448, there is a electrical pipet device, which can be associated to an infinitesimal amount fluid system, which transmitsobject materials by electroosmotic pressure. However, the above device is driven in a

continuous micro channel of a chip, and so there is a limitation for a continuous and stable suction and dispensation of the fluid.

Disclosure of the Invention

The embodiment of the invention relates to a new type pipet for infinitesimal amount of sample, which the manufacture and the theoretically accurate pipetting operation performance can be simplified compared with the conventional manner. By applying the ultra-micro pipet device according to the present invention, the single cell experiment and the bio-chip experiment, requiring an accurate handle of infinitesimal amount of sample can be performed conveniently. The present invention is related to an electroosmotic pressure driven ultra-micro pipet device using an electroconductive tube and a method for manufacturing the same .

The pipet device according to the present invention uses an electroosmotic pressure for sucking and dispensing the fluid, and a brief description thereon is as follow.

When a suitable fluid is flowed on a channel with a surface having a functional group with a polarity, the functional group can be ionized. When a suface of the channel has a hydroxyl functional group with polarity, for

example, proton leaves the suface of channel and enters in the fluid. In this condition, the surface of channel has net-negative-charge, while the fluid especially adjacent on the interface between the surface of the channel and the fluid has excessive proton or positive charge locally. By applying an electric field along the entire length of the channel, positive ion flows toward the chathode. In the fluid adjacent to the suface of the inner wall of the channel, a movement of positive-charged particles attracts the fluid solution. In general, a velocity of such fluid movement in a normal state is decided by the equation below.

V= 1&

4πη

Wherein, "V" is the flow rate of an electroosmotic flow of fluid, "ε" is the dielectric constant of fluid, "ξ" is the zeta electric potential of surface, "E" is the strength of electric field, and "η" is the viscosity of the solution. As known from the above equation, the flow rate of electroosmotic flow of fluid is directly in proportion to the zeta electric potential and the applied electric field.

The generation of a hydromechanical fluid meets with a bigger resistance as the inner diameter of the fluid conduit becomes smaller, due to a resistance against a

flow of fluid on a surface of an inner wall of the capillary. On the contrary, the electroosmotic flow generates fluid almost without hydromechanical resistance since the electroosmotic flow is formed on a surface of an inner wall of the capillary. Accordingly, in the pipet device using as ultra-micro capillary tube as the pipet tip, the electroosmotic driven method may be significantly advantageous. In the above method, the flow rate at the pipet tip section is encountered with a resistance. However, within the driving force generating device there is no resistance generated, so all the generated pressure is applied to the pipet tip, making a effective pipet operation. This fact is the fundamental basis allowing the ultra-micro pipe employing this driving method, to be precisely operated.

In the present invention, the electroosmotic flow is used as a driving force of the pipet device. However, an electric field for generating the electroosmotic flow is isolated at the part where a pipetting operation is performed, preventing discriminative suction/dispension caused by an electrical characteristic of each substance in the sample. An isolation of the electric field also prevents the object for a pipetting operation, such as a cell, from being electrically impacted. Also, The embodiment of the invention is to simplyfy

the electroosmotic flow driven ultra-micro pipet. The technical point of the present invention is to directly connect the capillary 11 (a second capillary) for generating the electroosmotic flow and the capillary 17 (a first capillary) for a pipeting operation to the electroconductive tube 16. (see Fig. 1) .

Structural elements of the ultra-micro pipet device according to the present invention are as follow. The ultra-micro pipet device of the present invention comprises a first capillary 17 provided at the front, having a taper section formed at the front part ; a second capillary 11 provided at the rear front side disposed coaxially with the first capillary 17; an electroconductive tube 16 forming a predetermined space 19 between the first and second capillaries and wrapping end sections of the two capillaries ; a supporting porous membrane 14 communicated in fluid with the electroconductive tube placed in the space through penetrating holes formed theron, wrapping the electroconductive tube and supporting the electroconductive tube to the first and second capillaries,-a buffer solution receiving unit 12 receiving the supporting porous membrane, wraping each of a portion of the first and second capillaries, and receiving the buffer solution by forming a receiving section spaced apart from the supporting porous membrane; a buffer solution

reservoir 20 connected to the end of the second capillary so that the buffer solution flows into the second capillary or to receive the buffer solution from the second capillary; electrodes 15 and 18 respectively provided in the buffer solution receiving unit and the buffer solution reservoir, ; and a power supplying unit 30 connected to each electrode to supply electric power to the electrodes. A glass tube or a fused silica tube is used for the first and second capillaries. The inner diameter of the second capillary is 100 μm or less, and a front side of the first capillary through, where the fluid is entered or discharged, has an inner diameter of 20 /zm or less and an outer diameter of 50 /mi or less.

The electroconductive tube 16 has a tubular shape and serves as a passage for electric current, which generates the electroosmotic flow, as well asa minimized transmitting space for transmitting a hydromechanical driving force of the electroosmotic flow to the capillary (the first capillary) for a pipetting operation with minimum influence of retention volume. As shown in Fig. 1, an electric circuit is connected to an outer electrode 15, by the membrane of the electroconductive tube, at a portionwhere the two capillaries are coupled to each other.. Accordingly, the difficulty to provide the electrode in the fluid conduit section (the first capillary or the

second capillary) is removed. Not only providing the electrode in the fluid conduit section is structurally difficult, but also (the first capillary or the second capillary) and due to the gas generated from the electrode by electrolysis, problems regarding in the transmission of the hydromechanical driving force can be caused. In the present invention, the problem mentioned above is fundamentally solved: the electrode is provided at the outside of the fluid conduit section (the first capillary or the second capillary) so the generated gas is radiated to the atmosphere . . The driving force generated by the electroosmotic flow can not escape outward by the wall of the electroconductive tube, transmitting the driving force to the capillary for a pipetting operation (the first capillary) , helping the pipet operation to be performed.

In the present invention, the method for connecting the first and second capillary is by using an electroconductive tube having an excellent flexibility. When the two capillaries are simply yet strongly connected to each other without using a bonding agentby an electroconductive tube with excellent flexibility: the electroconductive tube is swollen by wetting the electroconductive tube with a suitable solvent (if the electroconductive tube is the Nafion tube, the solvent is alcohol) ; next the two capillaries are inserted in the

increased inner diameter of the electroconductive tube; finally the solvent is dried for contracting the electroconductive tube, (see the embodiment) .

In the above coupling process, the supporting porous membrane is used to prevent the deformation of the electroconductive tube andan element to cover and support the electroconductive tube. Among the electroconductive tubes, a tube havinig flexibility, such as the Nafion tube, can be deformed when the electroosmotic flow is faces resistance by the capillary for a pipetting operation. Such deformation causes a delay of pressure transmission, as a result, the accurate regulation of the pipetting operation difficult. In the present invention, the electroconductive tube is coverd and supported by a porous tube having a high hardness in order to prevent the electroconductive tube from being deformed by the pressure (see Fig. 1) . When the porous tube is not used, fine openings are formed on the suporoting tube to prevent a flow of electric current from being interrupted. The supporting porous membrane 14 prevents the electroconductive tube from being damaged and helps to prevent the outside leakage of the fluid.

The above buffer solution is a solution having electroconductivity and the high/low ion strength are selected as buffer solution. Specific ions introducing

electroconductivity to solution are derived from mineral salt (for example, NaCl, KI, CaCl ₂ , FeF ₃ , (NH ₄ ) ₂ SO ₄ , Na ₃ PO ₄ and the like) , organic salt (for example, pyridinium benzoate, benzalkonium laurate) , or mixture of mineral salt/organic salt (for example, sodium benzoate, sodium deoxysulfate, benzylaminehydrochloride) .

The buffer solution receiving unit and the buffer solution reservoir are made of the materials which are chemically stable against buffer solution since they receive buffer solution. And, the buffer solution receiving unit and the buffer solution reservoir receive buffer solution in the liquid state or receive a supporting object, which the buffer solution is absorbed, such as sponge or cotton. As described above, the electroosmotic flow generates a flow having a flow rate proportation to the magnitude of the applied voltage. Accordingly, by properly regulating the magnitude of the applied voltage, the suction or dispensation velocity of fluid can be regulated at the pipet tip. At this time, a tube having an inner wall which is charge-treated with alkaline aqueous solution, is used as the second capillary in order to reinforce electroosmotic flow, and the the second capillary is operated in a state when the buffer solution receiving unit, the buffer solution reservoir, the first capillary, the

second capillary and the space are filled with the buffer solution. If the inside of the capillary or the space is filled with air, the pipetting operation is delayed, and so a regulation of infinitesimal amount of sample can be unreliable. In general, if a voltalge of several kV to several tens kV is applied to the electrode, and inner wall of the capillary , where the osmotic pressure is generated, has negative charge, the electroosmotic flow flows from the posite voltage side to the negative side (see Fig. 3) . An inner wall of the fused silica capillary used in the present invention is charged with silicate negative ion, and so the present invention obeys the above directivity. Accordingly, whenthe electrode 15 of the buffer solution receiving unit is grounded and a positive voltage is applied to the opposite electrode 18, the electroosmotic flow flows toward the pipet, and dispensation occurs at the pipet tip (see Fig. 4) . On the contraray, when a negative voltage is applied to the opposite electrode 18, the electroosmotic flow flows toward the operating electrode, and suction occurs at the pipet tip. By the regulation of the magnitude of the voltage along with the time for suppling the voltage , the total amount of suction of dispensation of the sample can be regulated (see Fig. 5 and Fig. 6) .

Brief Description of the Drawings

Fig. 1 is a diagram showing the essential section of an ultra-micro pipet device driven by an electroosmotic flow, which passes through a electroconductive tube and forms an electrical circuit, according to the present invention;

Fig. 2 is a diagram showing the entire structure of the ultra-micro pipet device according to the present device; Fig. 3 and Fig. 4 are diagrams showing the electroosmotic flow according to the present invention;

Fig. 5 is a diagram showing the sequential process of a high molecular solution (polyethylene oxide, PEO 2%) with high visicosity being sucked into the pipet by means of the device according to the present invention; and

Fig. 6 is a diagram showing the sequential process of a sucked substance being dispensed by means of the device according to the present invention.

10 : Main part of the ultra-micro pipet device

11 : Second capillary

12 : Buffer solution receiving unit

13 : Buffer solution

14 : Supporting porous membrane 15 : Electrode of the buffer solution receiving unit

16 : Electroconductive tube

17 : First capillary

18 : Electrode provided on the buffer solution reservoir 19 : Space between capillaries 20 : Buffer solution reservoir 30 : Power supplying unit

Best Mode for Carrying Out the Invention An experiment of suction and dispension of high molecular solution with high viscosiy (polyethylene oxide, PEO 2%) using the device of the present invention

The ultra-micro pipet device was manufactured using the material described below and operated under the conditions as follow according to the experimental method mentioned below to prove the effectiveness thereof.

The fused silica capillary (commercially available from Polymicor Technologies Company, U.S.A.) having an inner diameter of 75μm and a length of 1 m was used as the capillary for generating the electroosmotic flow, was used after it was filled with NaOH of 0. IN for 1 hour or more to sufficiently alkalify the surface of the inner wall of the capillary.

The Nafion tubing (commercially available from Permapure Company, U.S.A.) was used for the

electroconducitve tube for coupling the capillaries, and for the capillary for pipet operation, a glass tube from Sutter Company, U.S.A. was manufactured to have outer dimeter of 35/mi and an inner diameter of 5μm for the operating part, using a capillary pulling apparatus of Narishige Company, Japan.

The supporting porous membrane, supporting the electroconductive tube was manufactured by previously preparing penetraing holes on a heat-contracted tube for providing passages for the electric current flowed, and afterwards, this was placed on the electroconductiive tube, and heat was applied, so that the electroconductiive tube strongly covered the contracted tube (porous membrane) . Sodium phosphate (pH 7.5) of 15 mM was used as the buffer solution for generating the electroosmotic flow, and a mixture with 2% PEO (polyethylene oxide) solution and fluorescence dyes of 0.01M was used as suction/dispensation sample solution. For the high voltage generating apparatus (30 kV, 300 M), an apparatus manufactured by this applicant, was used and voltage of 5,000 -10,000 V was used for generatging the electroosmotic flow. And, the 488 ran Ar ion laser, a 60Ox optical microscope, and a CCD camera were used as the apparatus for verifying the suction/dispensation process .

After the above manufactured device was contacted with the above sample solution, the voltage of 10 kV was then applied to the electrode 18 provided on the buffer solution reservoir, acting as the cathode electrode, for 12 seconds. In this state, a process for suction of solution was photographed every three seconds . The results are shown in Fig. 4. Fig. 6 is a diagram showing the state of dispensention of the sample sucked in Fig. 5, The dispensing method was the same as the above method, however, the voltage was applied to the electrode provided on the buffer solution receiving unit acting as the cathode electrode.

As the experimental results, as shown in Fig. 5 and Fig, 6, by determining a flow direction of electric current and applying the voltage, a desired amout of PEO solution having a high viscosity can be sucked or dispensed.

Industrial Applicability

As compared with the conventional air pressure type pump device or mechanical pumping device, the ultra-micro pipet device driven by the electroosmotic flow according to the present invention can be adjusted conveniently and accurately for extracting and injecting the ultra-mintue sample since the high voltage generating device is used as a source of the drving force. And, since the

electroosmotic flow acting as the driving force is generated in the ultra-micro capillary which is similar to the capillary for a pipetting operation in a size, a dimension of the dead-volume formed in the space through which the driving force is transmitted is very small, and so a delay of transmission of a pipet operation is decreased so that it is possible to accurately regulate the volume .

Also, by changing a polarity of the voltage for generating the electroosmotic flow, a suction/dispension operation can be easily converted into a dispension/suction operation, and by exchanging the fluid via the capillary for a pipetting operation using syringe, it is possible to exchange easily the solution in the capillary for a pipetting operating

In the process for manufacturing the device according to the present invention, in case of Nafion electroconductive tube which is expandable by alcohol, an inner diameter of the Nafion tube is increased by wetting with alcohol, and so the Nafion tube couples conveniently to the capillary for a pipetting operating with the driving capillary, and the capillaries are sealed strongly without using any bonding agent by the evaporation of alcohol. If the heat-contracted tube is used as the outer tube for supporting the electroconductive tube, the

electroconductive tube can be covered and supported by the heat-contracted tube by using an electric heat device (a heat gun) . Accordingly, the ultra-micro pipet device according to the present invention can be employed in operations requiring an accurate adjustment of ultra-micro volumefor extracting and injecting a sample for single cell and for sucking and dispensing the sample in a bio chip.

Those skilled in the art will appreciate the conceptions and specific embodiments disclosed in the foregoing description and may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims.