JP5581236B2 - Dispensing chip and nucleic acid analyzer - Google Patents

Description

  The present invention relates to a dispensing chip and a nucleic acid analyzer used when determining sequence information of nucleic acid fragments.

  New techniques for determining the base sequences of DNA and RNA have been developed.

  In the currently used method using electrophoresis, a cDNA fragment sample synthesized in advance by reverse transcription reaction from a DNA fragment or RNA sample for sequencing is prepared, and a dideoxy reaction is performed by a well-known Sanger method. After that, electrophoresis is performed, and the molecular weight separation development pattern is measured and analyzed.

  On the other hand, in recent years, a method has been proposed in which a large number of DNA fragments as samples are fixed to a substrate and the sequence information of many fragments is determined in parallel.

  In Non-Patent Document 1, PCR is performed on microparticles using microparticles as a medium carrying DNA fragments. Thereafter, fine particles carrying PCR-amplified DNA fragments are put on a plate having a large number of holes in which the hole diameter is adjusted to the size of the fine particles, and read by a pyrosequencing method.

  In Non-Patent Document 2, PCR is performed on microparticles using microparticles as a medium carrying DNA fragments. Thereafter, the microparticles are dispersed and fixed on the glass substrate, an enzyme reaction (ligation) is performed on the glass substrate, a fluorescent dye is incorporated, and the sequence information of each fragment is obtained.

  Furthermore, in Non-Patent Document 3, a large number of DNA probes having the same sequence are fixed on a smooth substrate. In addition, after cutting the DNA sample, a DNA probe sequence and an adapter sequence of a complementary strand are added to the end of each DNA sample fragment. By hybridizing these on the substrate, the sample DNA fragments are immobilized on the substrate randomly one molecule at a time. In this case, the DNA elongation reaction is performed on the substrate, the substrate with the fluorescent dye is taken in, the unreacted substrate is washed and the fluorescence is detected, and the sequence information of the sample DNA is obtained.

  As described above, a method for determining the sequence information of a large number of fragments in parallel by fixing a large number of nucleic acid fragment samples on a smooth substrate has been developed and put into practical use.

  As a nucleic acid analysis reaction cell used in these systems, it is desirable that the detection region to which the sample DNA or the fine particles carrying the sample DNA are fixed is wider. Further, in order to reduce the amount of sample DNA and the amount of reaction reagent necessary for the sequence reaction, it is desirable that the capacity in the flow cell is smaller. Furthermore, in order to reduce the dead volume outside the detection region, it is necessary to narrow the reagent inlet.

In addition, it is desirable that the nucleic acid analysis reaction cell is thin in order to transmit temperature and for detection.
The chemical reaction associated with these methods generally consists of a number of steps using different reagents, and it is necessary to supply solutions containing different reagents for each step. The reagent used in the previous step often causes a reaction inhibition factor or a false detection in the next step, and it is necessary to perform a washing process between steps and completely remove the reagent used in the previous step.

  In addition, the solution containing the reagent used is often an expensive reagent or a solution containing a valuable DNA sample, and it is not desirable to replace each reaction step by using a large amount. . Then, since the replacement efficiency is generally better when the gas is replaced with the liquid than when the liquid is replaced with the liquid, when filling the flow cell with these solutions, the step of sucking or flushing with the gas is performed. Then, it is necessary to completely remove the reagent and the washing solution used in the previous step.

  Furthermore, in order to correctly perform the assumed chemical reaction, it is necessary that the reagent and the sample solution used in the reaction step are supplied to all detection regions without containing bubbles.

  In order to realize these, it is possible to supply a solution containing a reagent and a sample to the flow cell without leakage, and it does not contain bubbles, and also has the function of pushing away gas and sucking liquid. A supply and removal system is required.

  However, since the solution containing the sample is often a different sample every time depending on the intention of the user, it is not always efficient to automate these operations. Therefore, it is desirable that the system to be realized should be compatible with both automation and manual work, and the system to be realized should be based on the fact that manual injection of trace solutions using a quantitative pipette is common. It is also desirable to assume the use of a quantitative pipette.

  Here, Patent Document 1 discloses a structure of a pipette tip that enables dispensing of an accurate amount of liquid.

Special table 2010-510488 gazette

Nature 2005, Vol. 437, pp. 376-380 Science 2005, Vol. 309, pp. 1728-1732 Science 2008, Vol. 320, pp. 106-109 P.N.A.S. 2006, Vol. 103, pp. 19635-19640

  A general pipette tip is designed to aspirate and inject liquid from a container with a deep bottom such as a reaction tube. An accurate amount can be dropped on a flat surface such as a slide glass, or all of this can be aspirated. It is not adapted to work.

  Patent Document 1 is a structure that can accurately distribute the amount of extremely minute liquid dropped from a pipette tip. When this structure is used, it is not possible to supply reagents and solutions in a planar shape by an accurate amount. it can.

  However, it does not realize that the liquid is injected into a narrow channel structure having a high channel resistance such as a flow cell without leaking and sucked.

  For example, if the tip of the tip is smaller than the hole of the flow cell, the liquid cannot be stopped from overflowing, and the gas cannot be swept away or the liquid cannot be sucked.

  If the tip of the chip is larger than the hole of the flow cell, the hole of the flow cell can be sealed by the bottom of the chip, but the work of pressing the bottom of the chip to seal the hole accurately is extremely difficult. Moreover, the groove on the bottom surface of the pipette tip, which is a feature of the structure, does not exhibit the effect sufficiently. As a result, its precise dispensing ability is lost and the performance is no different from a general pipette tip.

  The present invention solves the above-described problems, and can inject liquid without leaking without bubbles into a narrow channel structure having high channel resistance like a flow chip, and can suck it without leaking. An object of the present invention is to provide an inflow tip or an inflow tip attachment that assumes use of manual or automatic quantitative pipettes.

  The dispensing tip of the present invention has a space for accommodating a liquid or a gas inside, and is a flow channel through which liquid or gas is introduced or sucked in a dispensing tip that flows in or out of the liquid or gas from the tip. A flat portion for sealing around the hole is provided.

  By providing a flat portion that seals the periphery of the hole of the flow path, the liquid or gas injected from the dispensing tip is injected into the flow path without leaking. When the quantitative pipette performs suction, the liquid or gas in the flow path is sucked by the quantitative pipette. These effects can be provided by a simple structure.

It is a figure for demonstrating an example of a structure of this invention. It is a figure for demonstrating an example of a structure of this invention different from FIG. It is a figure for demonstrating an example of a structure of this invention different from FIG. 1, FIG. It is a figure for demonstrating an example of a structure of this invention different from FIG.1, FIG.2, FIG.3. It is a disassembled perspective view explaining the structure of FIG. It is a disassembled perspective view explaining the one part structure of FIG.

[First Embodiment]
An embodiment of the present invention will be described with reference to FIG. In the configuration example of FIG. 1, the quantitative pipette tip (dispensing tip 101) of the present invention has a flow path 102 inside and a sealing portion 103 having a flat tip and a hole (outflow) continuous with the flow path 102. A distal end portion 105 having an inlet 106 is integrally formed. As shown in FIG. 1, the sealing portion 103 has a circular cross section, and the diameter of the circle decreases as the tip portion 105 is approached. Moreover, it has the collar part spread on the outer side at the dispensing tip 101 side. The tip portion 105 is disposed so as to protrude from the sealing portion 103.

  The tip 105 is anastomosed to the inlet 201 of the flow cell 204 shown in FIG. 4, and the liquid or gas flows into the flow path 202 of the flow cell 204 through the outflow port 106 or is sucked from the flow path (see FIG. 5). ).

  Again, the sealing part 103 is flat on the side close to the outflow inlet 106. When the tip 105 is anastomosed to the inlet 201 of the flow cell 204, the planar portion seals the periphery of the inlet 201 of the flow cell 204, so that liquid or gas flows into the flow path 202 of the flow cell 204 without leaking. Alternatively, suction is performed from the flow path 202 of the flow cell 204.

[Second Embodiment]
FIG. 2 is a diagram for explaining an example of the configuration of the present invention which is different from FIG. In the configuration example of FIG. 2, the present invention takes the form of a tip attachment 111 that is anastomosed to a tip 110 for a quantitative pipette.

  The tip attachment 111 is provided with a through hole 107, and the dispensing tip 101 passes through the through hole 107 from the distal end portion 105 side.

  The tip attachment 111 has a flat tip as in the sealing portion 103 shown in FIG. 1, has a circular cross section, and the diameter of the circle decreases as the tip portion 105 is approached. Moreover, it has the collar part extended on the outer side on the opposite side to the front-end | tip part 105. FIG.

  As shown in FIG. 2, the tip portion 105 of the chip 110 protrudes from the chip attachment 111 with the chip attachment 111 mounted on the chip 110.

  By adopting such a configuration, when the distal end portion 105 anastomoses with the inlet 201 of the flow cell 204, the flat portion seals the periphery of the inlet 201 of the flow cell 204, so that liquid or gas does not leak. It flows into the flow path 202 of the flow cell 204 or is sucked from the flow path 202 of the flow cell 204.

[Third Embodiment]
FIG. 3 is a diagram for explaining an example of the configuration of the present invention different from those in FIGS. In the configuration example of FIG. 3, the present invention takes the form of a chip attachment as in FIG. 2, but may be integrally formed as shown in FIG. 1. However, the difference from FIG. 2 is that the tip portion 105 does not protrude from the chip attachment 111.

  Therefore, although it cannot be anastomosed to the injection port 201 of the flow cell 204, a flow path is secured by aligning the outflow inlet 106, which is a hole of the chip attachment 111, with the injection port 201 of the flow cell 204, and the planar portion of the chip attachment 111 is By sealing, liquid or gas flows into the flow cell channel or is sucked from the flow cell channel without leaking. Compared to the configuration example of FIGS. 1 and 2, the tip end portion 105 of the chip does not enter the inlet 201 of the flow cell 204, so that it is difficult to damage the vicinity of the inlet 201 of the flow cell 204.

  Here, in the configuration example similar to FIGS. 2 and 3, a soft elastomer is suitable as a material for forming the chip attachment 111. Specifically, nitrile rubber (NBR) with excellent oil resistance, wear resistance, and aging resistance, hydrogenated nitrile rubber (HNBR) with improved NBR heat resistance and weather resistance, high heat resistance and chemical resistance Fluorine rubber (FKM), Silicone rubber (VMQ) with high heat resistance and cold resistance, Ethylene propylene rubber (EPDM) with excellent aging resistance, ozone resistance, weather resistance, chemical resistance, and abrasion resistance ), Chloroprene rubber (CR) excellent in weather resistance, ozone resistance, heat resistance, chemical resistance, etc., acrylic rubber (ACM) excellent in oil resistance at high temperature, weather resistance, ozone resistance, and gas permeability Butyl rubber (IIR) that resists polar solvents well, Urethane rubber (U) with excellent mechanical strength, Chlorsulfonated polyethylene (CSM) with excellent aging resistance, ozone resistance, chemical resistance, and abrasion resistance, high temperature In Oil well, gas permeation resistance, aging resistance is good epichlorohydrin rubber (CO, ECO), mechanical strength such as abrasion resistance and the like large elastic high natural rubber (NR).

  Dispensing tip 101 and tip portion 105 are made of fluororesin (PTFE) excellent in heat resistance and chemical resistance, metals such as aluminum from the viewpoint of mechanical strength, and members such as glass excellent in chemical resistance. It is done.

  In the configuration example similar to FIG. 1, since all are integrated, it is desirable to form all with the soft elastomer mentioned as the constituent material of the chip attachment 111 in FIGS.

[Fourth Embodiment]
FIG. 4 is a diagram for explaining an example of the configuration of the present invention different from those in FIGS.

  In the first to third embodiments, the chip side is characterized to prevent liquid leakage and bubble injection. In the present embodiment, the present invention is not limited to this, and even a normal chip (for example, a chip without the attachment 111 in FIGS. 2A and 2B) is provided with characteristics on the flow cell side and has the same effect. It is what you play.

  The configuration example of FIG. 4 is an example of a nucleic acid analyzer that sandwiches the flow cell 204 in which liquid or gas flows into the flow cell or suction from the flow cell is performed through the inlet 201 and the outlet 205.

  FIG. 5 is an exploded perspective view illustrating the configuration of FIG. In the configuration of FIG. 4, as shown in FIG. 5, the flow cell 204 is placed on the base 401, and the cover 206 is covered, and is fixed using a set screw 207. When it is desired to dispose the flow cell 204 at a specific position without displacement, positioning is performed using means such as the positioning protrusion 402.

  FIG. 6 is an exploded perspective view illustrating the configuration of the cover 206 in FIG. 5 in detail. The cover 206 incorporates a packing (sealing member) 210 between the cover upper plate 208 and the cover lower plate 209, and each has a hole shape that becomes the inlet 201 and the outlet 205 in FIG. The cover upper plate 208 and the cover lower plate 209 are fixed by a cover set screw 211.

  In order for the liquid or gas to flow through the flow path of the flow cell 204, the packing 210 is pressed around the hole of the flow cell 204, and the periphery is sealed by the flat portion of the packing 210. As a result, the hole of the packing 210 and the hole of the flow cell 204 are connected, and liquid or gas is introduced or sucked from the inlet 201 or the outlet 205 in FIG. By adopting such a structure, liquid leakage and bubble injection can be prevented even when a normal chip is used.

  When the inlet 201 or the outlet 205 is tapered, various types of pipette tips can be anastomosed.

  4, 5, and 6, the packing 210 is preferably the above-described soft elastomer. The base 203, the cover upper plate 208, and the cover lower plate 209 can be made of PTFE, resin such as acrylic, metal such as aluminum, glass, or the like.

  Fig. 1, Fig. 2, Fig. 3 and Fig. 4 describe the configuration example to use a quantitative pipette and pipette tip on the premise of manual operation used as power for injection and suction, but use the pipette tip for automation. Any infusion or suction device that can be used.

  In the configuration examples of FIGS. 2 and 3, if the attachment can be anastomosed, not only a pipette tip but also a nozzle can be used.

  In the configuration example of FIG. 4, not only the pipette tip but also a nozzle can be used as long as it can be anastomosed to the inlet 201 or the outlet 205.

  It will be readily appreciated by those skilled in the art that the present invention is not limited to the above-described embodiments, and various modifications can be made within the scope of the invention described in the claims.

101 Dispensing tip 102 Channel 103 Sealing portion 105 Tip portion 106 Outflow inlet 107 Through hole 111 Tip attachment 201 Inlet 202 Channel 204 Flow cell 205 Outlet 206 Cover 207 Set screw 208 Cover upper plate 209 Cover lower plate 210 Packing 211 Cover set screw 401 Base 402 Positioning protrusion

Claims (1)

In a dispensing tip that has a space for containing liquid or gas inside, and that flows in or out of the liquid or gas from the tip,
Dispensing tip main body part, having a flow path inside, a sealing part having a flat tip, and a tip part having a hole continuous with the flow path are integrally formed,
The tip portion is arranged to protrude from the sealing portion,
The sealing part has a circular cross section and the diameter of the circle decreases as it approaches the tip.
A dispensing tip, wherein the sealing part is formed of a soft elastomer.

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