JP2009058288A - Liquid sample dispensation method, heating device, and automatic dispensation method and automatic dispensing device of liquid sample - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a liquid sample dispensation method capable of performing simply and highly accurately analysis of an organism-related substance, such as detection of nucleic acid, and a device used therefor. <P>SOLUTION: The liquid sample dispensation method has a process for warming a liquid sample to be provided to dispensation in a pipette chip by warming the pipette chip for liquid sample dispensation. A warming device used for the dispensation method comprises a warming means for warming the pipette chip for liquid sample dispensation to 40-100°C; and a holding means for holding the pipette chip, with its suction/discharge port out of contact with the warming means. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a liquid sample dispensing method and an automatic dispensing method that are suitable for performing analysis of biological materials with high accuracy and ease, and a liquid sample heating device and an automatic dispensing device used therefor.

In recent years, as a technique for detecting gene expression frequency, a probe nucleic acid having a base sequence complementary to a target nucleic acid to be detected is immobilized on a substrate, and the target nucleic acid is hybridized to the probe nucleic acid and detected. An analysis method using a so-called probe array is employed.
Conventionally, a nylon membrane is used as a substrate, and the probe nucleic acid is immobilized on the nylon membrane by utilizing the positive charge in the molecule.
Furthermore, in recent years, efficient analysis methods have been proposed due to advances in microarray technology. For example, using a standard slide glass (approximately 7.5 cm x 2.5 cm), a method of immobilizing many types of probe nucleic acid on a substrate at high density, or binding a probe nucleic acid to a semiconductor substrate using photolithography technology The method of making it known is known. By adopting these microarray technologies, when a nylon film is conventionally used as a substrate, the spot diameter per one was 3 to 5 mm, while using a glass substrate, the spot diameter was 100 to 100 mm. It has become possible to make it as extremely small as 300 μm, and the probe immobilized on the substrate has been greatly densified. Specifically, thousands to tens of thousands of probes can be immobilized on a single microarray, which is a useful means for large-scale gene expression frequency analysis. In the future, further dramatic progress is expected by improving detection sensitivity, reducing sample usage by microfabrication, and simplifying data acquisition and processing.

On the other hand, detection of a target nucleic acid by hybridization is conventionally performed by detection of chemiluminescence or radioisotope. The detection accuracy such as the detection sensitivity and the reproducibility of the detection result greatly depends on the accuracy of the hybridization itself as well as the accuracy of the microarray.
For example, in hybridization, the results greatly depend on the salt concentration and temperature of the liquid sample used and the procedure. Therefore, suppressing these fluctuations is essential for improving detection accuracy.
For example, a method of obtaining a highly accurate detection result by managing the temperature of a liquid sample under analysis has been disclosed (see Patent Document 1). This method is more accurate by automating the hybridization process, which takes the longest time for detecting nucleic acids and requires a large experimental space, using magnetic particles on which nucleic acids are immobilized. It is intended to obtain a correct detection result. In particular, since the magnetic separation stage and the annealing stage are integrated, it is not necessary to move the container into which the liquid sample is injected, and the temperature change from the magnetic separation process to the hybridization process is minimized. In addition, by heating the liquid sample for analysis to 95 ° C., the nucleic acid in the sample is single-stranded (thermally denatured), thereby suppressing nonspecific adsorption during hybridization, It is intended to detect with high accuracy.
JP 2003-274925 A

  However, in the method described in Patent Document 1, the liquid sample is held at 95 ° C. for 5 minutes, and further maintained at 95 ° C. during operation and standby state of the magnetic control device. There is a problem that the solvent evaporates, the salt concentration of the liquid sample fluctuates, and in the worst case, the solvent disappears. Such problems are particularly noticeable as the number of specimens increases because the liquid sample dispensing time becomes longer during hybridization. There is a problem that the difference in salt concentration of the sample becomes large, it is difficult to perform hybridization under certain conditions, and a highly accurate detection result cannot be obtained. There is also a problem that the detection device is large and complicated. The actual situation is that no method or apparatus for solving such problems has been proposed.

  The present invention has been made in view of the above circumstances, and provides a liquid sample dispensing method suitable for highly accurate and simple analysis of biological materials such as nucleic acid detection and an apparatus used therefor. Let it be an issue.

To solve the above problem,
The invention according to claim 1 includes a step of heating a pipette tip for dispensing a liquid sample and heating the liquid sample to be dispensed in the pipette tip. This is a sample dispensing method.
The invention according to claim 2 is characterized in that the liquid sample is an aqueous solution containing a nucleic acid or an aqueous solution containing a water-soluble substance having a solubility in water equal to or lower than that of sodium dodecyl sulfate. The liquid sample dispensing method described in 1.
The invention according to claim 3 is the liquid sample dispensing method according to claim 1 or 2, wherein the liquid sample is used for nucleic acid hybridization.

Invention of Claim 4 is a heating apparatus used for the liquid sample dispensing method as described in any one of Claims 1-3, Comprising: The pipette tip for liquid sample dispensing is 40-100 degreeC. A heating apparatus comprising: a heating means for heating; and a holding means for holding the pipette tip without bringing the suction / discharge port into contact with the heating means.
According to a fifth aspect of the present invention, the heating device is box-shaped, and the holding means holds the pipette tip with its suction / discharge port protruding into the heating device, and further the heating device The heating apparatus according to claim 4, further comprising gas circulation means for circulating an internal gas.
The invention according to claim 6 is characterized in that the heating device is in the form of a pipette, and further comprises operating means for operating suction and discharge of a liquid sample, and the heating means is integrated with the holding means. The heating apparatus according to claim 4.
The invention according to claim 7 is characterized in that the heating means is made of one or more materials selected from the group consisting of metal, glass, engineering plastic and super engineering plastic. It is a heating apparatus as described in any one of these.
In the invention according to claim 8, the heating device is a metal block, and the holding means is an opening portion of an insertion hole formed in the metal block for inserting the pipette tip into the metal block. It is a heating apparatus of Claim 4 characterized by these.
The invention according to claim 9 is provided with a plurality of the holding means, and the distance between the centers of the adjacent pipette tips is 8 to 10 mm. It is a temperature device.
The invention according to claim 10 is characterized in that the heating means is electrically connected to a temperature control means for automatically controlling the temperature during heating. The heating device according to item.

  The invention according to claim 11 includes a step of automatically sucking a liquid sample using a pipette tip for dispensing a liquid sample, the pipette tip is heated, and the liquid sample is automatically added in the pipette tip. A method for automatically dispensing a liquid sample, comprising a step of performing a temperature treatment and a step of automatically discharging the liquid sample in the pipette tip.

The invention according to claim 12 is an automatic liquid sample dispensing device used in the automatic liquid sample dispensing method according to claim 11, the operation unit operating the suction and discharge of the liquid sample, and the pipette A heating device comprising heating means for heating the tip to 40 to 100 ° C., and holding means for holding the pipette tip without contacting the suction / discharge port to the heating means, An automatic dispensing device for a liquid sample, wherein the operation unit and the heating means are electrically connected to a control means for automatically controlling the suction, discharge and heating of the liquid sample.
According to a thirteenth aspect of the present invention, the heating device is a metal block, and the holding means is an opening of an insertion hole formed in the metal block for inserting the pipette tip into the metal block. The automatic dispensing apparatus for liquid samples according to claim 12, wherein:
According to a fourteenth aspect of the present invention, the heating device section is box-shaped, and the holding means holds the pipette tip with its suction discharge port projecting into the heating device section, and further the heating device. The automatic dispensing device for a liquid sample according to claim 12, further comprising a gas circulation means for circulating the gas inside the temperature device section.
The invention according to claim 15 is the automatic dispensing of a liquid sample according to claim 12, characterized in that the heating device section is in the form of a pipette, and the heating means is integrated with the holding means. Device.

  According to the present invention, a biological substance can be analyzed with high accuracy and simplicity, and the present invention is particularly suitable for detecting a nucleic acid accompanied by hybridization.

Hereinafter, the present invention will be described in detail.
<Liquid sample dispensing method>
The liquid sample dispensing method of the present invention includes a step of heating a pipette tip for dispensing a liquid sample and heating the liquid sample to be dispensed in the pipette tip.
And when dispensing the liquid sample which needs heating, this invention suppresses quality changes, such as a density | concentration change, by suppressing the evaporation of the solvent. Specifically, the liquid sample sucked and held in the pipette tip is heated through the pipette tip. At this time, the pipette tip has its suction and discharge opening opened to the atmosphere, but since the opening area is small, the evaporation amount of the solvent is small. Furthermore, the evaporation amount of the solvent can be extremely reduced by setting the holding time of the liquid sample after heating to a certain time within about 1 to 5 minutes.

The liquid sample to be dispensed may be appropriately selected depending on the purpose, and is not particularly limited. The dispensing method of the present invention is particularly suitable when the amount of detection target substance is very small, and any sample that requires analysis of trace components can be used. For example, a sample containing a biological substance is suitable.
Here, the biological substance refers to a substance extracted or isolated from a living body, or a substance obtained by chemical treatment or chemical modification thereof. Specific examples include, for example, antigens, antibodies, enzymes, abzymes, cytokines, tumor markers, other proteins, or DNA, cDNA, RNA, other nucleic acids, hormones, haptens, and the like. Examples include chemical treatment or chemical modification.

  In particular, when a liquid sample containing a nucleic acid, preferably a nucleic acid having a non-specific double-stranded structure, is used as the liquid sample, the present invention has a more excellent effect. For detecting a nucleic acid, a technique of hybridizing a probe nucleic acid to the nucleic acid to be detected is widely used. In order to perform hybridization with high accuracy, it is necessary to denature the nucleic acid to form a single strand by heating, but it contains nucleic acid because it needs to be kept in a denatured state until hybridization. The time for heating the sample to be heated tends to be long. In particular, the lower the concentration of the sample used for nucleic acid hybridization, the more susceptible to changes in concentration due to solvent evaporation.

In particular, when a sample containing a biological substance such as nucleic acid is used as the liquid sample, a dispensing operation of the cleaning liquid is often required. Some cleaning liquids contain components that are easily crystallized even at room temperature. Examples of such a washing solution include sodium dodecyl sulfate (hereinafter abbreviated as SDS) aqueous solution, phosphate buffered saline (PBS) and citrate buffered saline (SSC), and a mixture thereof. It is done. In particular, SDS is a component that is easily crystallized, and its solubility is about 10 g / 100 ml. In the case of using a cleaning liquid, there are various adverse effects if some of the components are crystallized. For example, not only the cleaning ability is lowered, but also crystals deposited on the microarray used for detection are recognized as impurities, which may reduce the analysis accuracy. In addition, when a microarray with a narrow channel is used, the channel may be clogged with precipitated crystals, and analysis may not be performed.
However, according to the present invention, even when an aqueous solution containing a water-soluble substance having a solubility in water equal to or less than that of SDS is used as a liquid sample to be dispensed, crystal precipitation is suppressed by heating. Dispensing of the cleaning liquid improves the cleaning efficiency and cleaning accuracy, and enables analysis with high accuracy.

The pipette tip for dispensing the liquid sample is not particularly limited as long as it has heat resistance when heated, and may be a disposable one or a reusable one, and a commercially available product that is usually used. Commercially available pipette tips used in the biochemical field are usually autoclavable and can be used in the present invention.
Most commercially available pipette tips are made of polypropylene with a low thermal conductivity of 0.14 W / mK or polycarbonate made of 0.19 W / mK, but from the viewpoint of efficiently heating a liquid sample. For example, a pipette tip manufactured by WATOSON containing carbon having a high thermal conductivity (thermal conductivity: 129 W / mK) is more preferable. A pipette tip made of a resin material having a high carbon filling rate such as PC or ABS may be used. The increase in thermal conductivity when carbon fiber is added at 10% by mass to PC is approximately 0.2 W / mK (JIS R2616, probe method).

  The liquid sample may be heated by, for example, sucking and holding the liquid sample using a pre-heated pipette tip and heating the liquid sample via the pipette tip. You may carry out by heating the pipette tip of the state attracted | sucked and hold | maintained. The temperature of the liquid sample at this time may be appropriately selected according to the purpose, and is not particularly limited. However, in consideration of the material of the pipette tip, the temperature of the liquid sample normally required, etc., it is preferable to heat the pipette tip so that the temperature of the pipette tip is 40 to 100 ° C.

<Heating device>
Next, the heating apparatus used in the liquid sample dispensing method of the present invention will be described.
The heating device includes a heating means for heating the pipette tip to 40 to 100 ° C., and a holding means for holding the pipette tip without bringing the suction / discharge port into contact with the heating means. It is.

  The heating means transmits heat generated from a heat source to the pipette tip directly or indirectly via gas or a suitable wall material. The material is not particularly limited as long as it has heat resistance. Examples thereof include various metals such as aluminum, iron and copper, various alloys such as stainless steel, and various heat conductive resins. Of these, aluminum is preferred because of its excellent stability and thermal conductivity.

  The holding means holds the pipette tip as long as the suction / discharge port of the pipette tip is not in contact with the heating means. For example, a part of the holding means may be fitted and held in a pipette tip mounting opening, or a hole having a smaller diameter than the pipette tip outer diameter may be formed in a part of the holding means. Then, a pipette tip may be inserted here, and a part other than the suction outlet may be held in contact with the hole. Or you may hold | maintain so that site | parts other than the suction discharge port of a pipette tip may be hold | gripped.

  In the present invention, since the suction / discharge port of the pipette tip is not brought into contact with the heating means, for example, even if an impurity is attached to the heating means, this impurity does not adhere to the suction / discharge port of the pipette tip, It does not hinder analysis of liquid samples. Here, the impurities refer to, for example, chemicals or microorganisms that inhibit the analysis when a liquid sample is subjected to the analysis.

The temperature of the heating means rises to warm the pipette tip during the dispensing operation. Therefore, the higher the heating temperature, the more the growth of microorganisms on the surface of the heating means is suppressed, and the microorganisms in the liquid sample Mixing is suppressed.
Furthermore, the heating means is preferably made of a material that can be sterilized or washed. Before subjecting the heating device to a dispensing operation, for example, sterilization or washing can be performed with higher accuracy by sterilization or washing by autoclave, ultraviolet irradiation, boiling sterilization, ultrasonic washing, or the like. Specific examples of the material that can be sterilized or cleaned include one or more selected from the group consisting of metal, glass, engineering plastic, and super engineering plastic. When two or more kinds of materials are used in combination, the combination may be appropriately selected according to the purpose.

Hereinafter, specific embodiments will be described with reference to the drawings, and the liquid sample dispensing method will be described in more detail.
(First embodiment)
FIG. 1 is a schematic view illustrating a heating device according to a first embodiment of the present invention, in which (a) is a plan view and (b) is a longitudinal sectional view taken along line AA of (a). .
The heating device 1 has a box shape, and circular insertion holes 12a, 12a,... For inserting the pipette tips 10 are formed on the upper surface 12. The hole diameters of the insertion holes 12a, 12a,... Are set slightly smaller than the outer diameter of the mounting end of the mounting port 10a to the pipette (not shown) of the pipette tip 10. The number of insertion holes 12a, 12a,... Is three per row, for a total of three rows, that is, nine.
And by inserting the pipette tip 10 into the heating device 1 from the suction / discharge port 10b through the insertion hole 12a, the outer surface in the vicinity of the mounting port 10a is in contact with the peripheral edge of the insertion hole 12a, The pipette tip 10 is detachably held. That is, the insertion hole 12a functions as a holding means.

Pipette tip 10 is, while being held by such insertion hole 12a, the length H _L from the holding portion to suction and discharge ports 10b are in size than the height H ₁ of the heating device 1 inside the space small. That is, the pipette tip 10 is in contact with any part of the heating device 1 with the suction / discharge port 10b protruding and held inside the heating device 1.

  Inside the warming device 1, a heater 11 is disposed on the upper surface 12 as a warming unit, and a fan 13 is disposed on the side surface as a gas circulation unit that circulates gas inside the warming device 1. And these are arrange | positioned so that the rotating shaft of the fan 13 and the center axis | shaft of the helical site | part which is a heat generation source of the heater 11 may be located on a substantially straight line, 13 is circulated efficiently. Although illustration is omitted here, for example, in order to efficiently circulate the warmed gas, for example, in FIG. 1, the fan 13 is on the side surface near the bottom of the heating device 1. The extension line of the rotation axis of the fan 13 may intersect with the central axis of the spiral portion of the heater 11. In FIG. 1, the heater 11 may be arranged so that the central axis of the spiral portion is orthogonal to the extension line of the rotation axis of the fan 13. However, even if the heater 11 and the fan 13 are not arranged in the above positional relationship, the gas can be sufficiently circulated inside the heating device 1.

The heater 11 only needs to be able to heat the pipette tip to 40 to 100 ° C. For that purpose, it is necessary that the gas in the heating device 1 can be heated to at least 40 ° C. or more. Any known device such as a nichrome wire heater may be used.
In addition, the fan 13 may be any known one including those having a rotor blade as shown here as long as it can generate an air flow.

  Of the heating device 1, the material of the six wall surfaces including the upper surface 12 is preferably a material having high heat insulation. For example, from the viewpoint of increasing the strength of the heating device 1, the six wall surfaces are preferably made of metal. However, since the metal wall surfaces have high thermal conductivity, the heat of the gas in the heating device 1 is high. Is easy to escape to the outside. Therefore, in order to achieve both strength and heat insulation, for example, a structure in which a heat insulating material and a metal plate are laminated one by one as a wall surface, a structure in which a metal plate is sandwiched between heat insulating materials, and a heat insulating material is sandwiched between metal plates. It is good to use the thing of the structure. Or you may just cover the outer surface of the heating apparatus 1 which consists of metal wall surfaces with a heat insulating material. Here, examples of the heat insulating material include known materials such as glass wool and polystyrene foam. Or you may heat directly the heating apparatus 1 by well-known means, such as a water jacket, without using a heat insulating material in this way.

  The upper surface 12 is preferably detachable or openable / closable with respect to the heating device 1 main body, and more preferably detachable. By making it attachable / detachable or openable / closable, for example, workability such as maintenance management of the heating device 1 is improved. In particular, if a plurality of upper surfaces 12 holding the pipette tip 10 in advance are prepared separately by being detachable, these can be exchanged instantaneously, and the dispensing operation can be performed more efficiently. Can do.

  The gas inside the heating device 1 may be any gas as long as it is inert and has no problem such as explosion, and examples thereof include air, nitrogen, helium, and argon. Of these, air is most preferable in consideration of versatility and economy.

In this embodiment, the pipette tip 10 can be held without bringing the suction / discharge port 10b into contact with the heating device 1 by inserting the pipette tip 10 into the heating device 1 from the insertion hole 12a through the suction / discharge port 10b. . And by operating the heater 11 and the fan 13 in this state, the heated gas can be circulated inside the heating device 1, and the temperature inside the heating device 1 is kept almost constant regardless of the place. It is.
Then, the pipette tip 10 is heated through the heated gas, and when the pipette tip 10 thus heated is attached to the pipette (not shown) and then the liquid sample is sucked, the liquid sample Is heated using the pipette tip 10 as a heat source.

Alternatively, the pipette tip 10 may be attached to the pipette, the liquid sample may be sucked and held in advance, and the pipette tip 10 may be held in the insertion hole 12a while being attached to the pipette. At this time, it is advisable to use a stand separately in order to prevent the pipette from falling.
In this case, the liquid sample in the pipette tip 10 is heated through the heated gas and the pipette tip 10. As long as the pipette tip 10 is held in the insertion hole 12a, the liquid sample is continuously heated.

  Here, an example in which the number of insertion holes 12a, 12a,... Drilled in the upper surface 12 is nine has been described, but the present invention is not limited to this, and is appropriately adjusted according to the purpose. Just do it. For example, in the biochemical field, those equipped with a plurality of pipette tip attachment sites, such as an eight-pipe pipette capable of attaching eight pipette tips in series, are commercially available and widely used. Considering the arrangement state, for example, it is preferable to provide the insertion holes 12a by a multiple of eight per row. Furthermore, since the center-to-center distance between adjacent pipette tip mounting sites such as the above eight pipettes is about 9 mm, the center-to-center distance between the adjacent insertion holes 12a is 8 to 10 mm also in the present invention. Is preferable, and 9 mm is particularly preferable. By doing in this way, dispensing operation can be performed still more efficiently.

  The number of heaters 11 and fans 13 provided inside the heating device 1 is one here, but is not limited to this in the present invention, and may be appropriately adjusted according to the purpose.

  Furthermore, although illustration is abbreviate | omitted here, it is preferable that the heater 11 is electrically connected to the temperature control means which controls the temperature at the time of heating automatically. By doing in this way, the heating process of the pipette tip 10 can be performed simply and stably.

(Second embodiment)
FIG. 2 is a front view illustrating a heating device according to the second embodiment of the invention.
The heating device 2 shown here is in the form of a pipette, a main body portion 23 that can be gripped with one hand, a mounting portion 21 that is a holding means that holds the pipette tip 10 by fitting the mounting port 10a, and the mounting portion 21. An operation unit 22 is provided for operating the suction of the liquid sample into the pipette tip 10 attached to and the discharge of the liquid sample outside the pipette tip 10. The mounting portion 21 includes a heater (not shown) inside, and can be heated by the heater. That is, in the present embodiment, the heating means is integrated with the holding means, and in other respects, it has the same structure as a commonly used pipette.

  A heater will not be specifically limited if it can arrange | position in the mounting part 21 and can heat a pipette tip to 40-100 degreeC. For example, an electrothermal type such as a nichrome wire heater or a ceramic heater may be used, or a type in which a combustible gas is combusted, or a known one may be selected as appropriate.

  The material of the mounting part 21 may be the same as that mentioned above as the material of the heating means.

In addition, although the thing with the number of the attachment parts 21 is shown as the heating apparatus 2 here, in this invention, it is not limited to this, The eight-series pipette etc. which were described in 1st embodiment As described above, a plurality of mounting portions 21 may be provided.
As in the first embodiment, it is preferable that the heater in the mounting portion 21 is electrically connected to a temperature control unit that automatically controls the temperature during heating.

In the present embodiment, the pipette tip 10 is attached to the heating device 2 and the pipette tip 10 is heated, so that the liquid sample held therein is heated through the pipette tip 10. As long as the liquid sample is held in the pipette tip 10, the liquid sample is continuously heated. Of course, the heating of the pipette tip 10 may be started either before or after the liquid sample is aspirated.
Needless to say, in this embodiment, the pipette tip 10 is held without the suction / discharge port 10b coming into contact with the heating device 2.

  According to the heating device and the liquid sample dispensing method of the present invention, it is possible to analyze biologically related substances with high accuracy and simplicity, and the effect becomes more remarkable as the number of liquid samples used for the analysis increases. It becomes.

<Automatic dispensing method of liquid sample>
The liquid sample automatic dispensing method of the present invention includes a step of automatically sucking a liquid sample using a pipette tip for dispensing a liquid sample, heating the pipette tip, and placing the liquid sample in the pipette tip. It has a process of automatically heating and a process of automatically discharging the liquid sample in the pipette tip.
In order to automatically suck and discharge a liquid sample, a pipette tip is attached to this pipette using a pipette that is electrically connected to a suction / discharge control means that automatically controls the suction and discharge operation of the liquid sample. And use it. The pipette used here may be the same as a known pipette except that it is electrically connected to the suction / discharge control means. Furthermore, for example, in the heating device 2 according to the second embodiment, the operation unit 22 may be electrically connected to the suction / discharge control means.

  In order to automatically heat the liquid sample in the pipette tip, for example, in the heating device described above, the heating means automatically controls the temperature at the time of heating and the start and stop of the heating. What is electrically connected to the temperature control means may be used. Specifically, the heater in the first or second embodiment electrically connected to the temperature control means can be exemplified.

<Automatic dispensing device for liquid samples>
The liquid sample automatic dispensing apparatus of the present invention is used in the above-described liquid sample automatic dispensing method of the present invention, and includes an operation unit for operating suction and discharge of the liquid sample, and the pipette tip between 40 and 100. The heating device includes a heating unit that heats the pipette tip and a holding unit that holds the pipette tip without bringing the suction / discharge port into contact with the heating unit.
The operation unit and the heating unit are electrically connected to a control unit that automatically controls suction, discharge, and heating of the liquid sample. Here, automatically controlling the heating of the liquid sample means automatically controlling the temperature at the time of heating the liquid sample and the start and stop of the heating.
Hereinafter, specific embodiments will be described with reference to the drawings, and the automatic dispensing method will be described in more detail.

(Third embodiment)
FIG. 3 is a diagram illustrating a liquid sample automatic dispensing apparatus according to a third embodiment of the present invention, in which (a) is a schematic configuration diagram and (b) is an enlarged longitudinal sectional view of a heating means. .
The automatic dispensing device 3 includes an operation unit 33 and a heating device unit 30.
The operation unit 33 includes a pipette unit 331 that performs suction and discharge of a liquid sample, a first position adjustment unit 332 that adjusts a position in the Y direction among the arrangement positions of the pipette unit 331, and positions in the X direction and the Z direction. A second position adjusting unit 333 is provided. The first position adjustment unit 332 is provided integrally with the pipette unit 331, and is movable on the second position adjustment unit 333 in the longitudinal direction (Y direction) of the adjustment unit 333. The adjustment unit 333 is movable in a direction (X direction and Z direction) perpendicular to the longitudinal direction by a drive unit (not shown). Furthermore, the operation unit 33 is electrically connected to the control means 34, and the arrangement position of the operation unit 33 and the suction and discharge of the liquid sample can be automatically controlled.

The heating device unit 30 is a metal block, and its main body 31 functions as a heating means, and is installed at the lower part of the operation unit 33. A plurality of insertion holes 32 for inserting the pipette tip 10 into the metal block are formed on the surface of the main body 31 facing the operation portion 33.
The insertion hole 32 has a substantially conical shape, and the opening 32a has a diameter slightly smaller than the outer diameter of the mounting end of the mounting port 10a to the pipette portion 331 of the pipette tip 10. The diameter of the part other than the opening 32 a is set larger than the outer diameter of the part other than the mounting end of the pipette tip 10.
The pipette tip 10 is detachably held by being inserted into the insertion hole 32 through the suction / discharge port 10b, with the outer surface near the mounting port 10a being in contact with the opening 32a.

Pipette tip 10 is, while being held in this way by the opening 32a, the length H _M from the holding portion to suction and discharge ports 10b is smaller in size than the height H ₃₂ of the insertion hole 32. That is, the pipette tip 10 is inserted and held in the insertion hole 32, and the suction / discharge port 10 b is not in contact with any part of the warming device main body 31.

  Any known method may be used as the method for heating the heating device unit 30 as long as it is a method capable of conducting heat using electricity, such as a method of heating using a Peltier element or the like.

  The heating device main body 31 is electrically connected to the control means 34 so that the temperature during heating can be automatically controlled.

  In addition, the heating apparatus part 30 can be used as a heating apparatus at the time of liquid sample dispensing for the same purpose as the heating apparatus shown in the first and second embodiments. In this case, the heating device may be heated by, for example, immersing in a liquid whose temperature is adjusted to an appropriate temperature according to the purpose, instead of automatically controlling the temperature at the time of heating by the control means.

  Further, a sample holding unit 37 that holds a liquid sample to be dispensed and a warmed sample holding unit 35 that dispenses a heated liquid sample are provided in the vicinity of the heating device unit 30. Further, for example, when using a plurality of warmed sample holders 35, it is preferable to use a sample arrangement means 36 for arranging and arranging them.

  The sample holder 37 may have any material and shape as long as it can stably hold the filled liquid sample. For example, the sample holder 37 has a well-known sample bottle shape made of various materials such as resins and glasses. A thing, a thing provided with this, a well plate, etc. may be used.

The heated sample holder 35 may be made of any material as long as it has the same heat resistance as the pipette tip. The shape can be appropriately selected according to the purpose. For example, when it is necessary to store a dispensed liquid sample, the shape may be the same as that of the sample holding unit 37. On the other hand, when only spotting a liquid sample, it may be in the form of a plate or sheet. For example, a microarray in which a supplementary substance for capturing molecules and atoms in the liquid sample is immobilized is suitable. In particular, a microarray for detecting nucleic acids by hybridization is particularly suitable.
And the sample arrangement | positioning means 36 may be anything as long as this warm sample holding | maintenance part 35 can be arrange | positioned so that attachment or detachment is possible.

In the present embodiment, the heating device unit 30 may have a different form, and FIG. 3C is an enlarged cross-sectional view showing another example of such a heating unit.
In the heating device section 30 ′ shown here, a plurality of substantially cylindrical through holes 32 ′ into which the pipette tip 10 is inserted are provided in the heating device section main body 31 ′. Thus, it is the same as that of the heating device unit 30 except that a through hole is provided.

  Further, the number and arrangement of the insertion holes 32 and the through-holes 32 ′ are not limited to those shown here. For example, as with the insertion hole 12a described in the first embodiment, pipette tips are attached. You may adjust suitably so that the commercially available pipette provided with multiple site | parts can be used.

  In the present embodiment, the amount of liquid sample suction and discharge, the time from suction to discharge, the amount dispensed per time, the number of times dispensed, the arrangement position of the sample holder 37 and the heated sample holder 35, and heating By inputting necessary information such as the temperature at the time of heating of the device unit 30 and the start and stop times of the heating to the control means 34 in advance, the liquid sample can be dispensed automatically.

  Specifically, after holding the pipette tip 10 in the heating device body 31, the control means 34 is operated to perform a heating process for the pipette tip 10 based on the input information. After completion of the heating process, the pipette unit 331 is moved to a predetermined location based on the input information, and after the pipette tip 10 that has been heated is attached, the pipette unit 331 is further moved to the predetermined location based on the input information. The liquid sample in the sample holding unit 37 is aspirated and held in the pipette tip 10 for a predetermined time, whereby the liquid sample is heated. Next, the pipette unit 331 is moved to a predetermined location based on the input information, and the heated liquid sample is discharged into the heated sample holding unit 35. At this time, the heating process of the liquid sample and the movement of the pipette unit 331 in the direction of the heated sample holding unit 35 may be performed in parallel.

  Here, an example in which the operation of holding the pipette tip 10 on the heating device main body 31 is not automatically performed is shown. However, as another example, the pipetting unit 331 is not heated based on input information. After the pipette tip 10 is attached, the pipette portion 331 may be moved to a predetermined location, and the heating device portion main body 31 may hold the pipette tip 10.

As yet another example, the pipette tip 10 may be heated after the liquid sample is aspirated.
That is, based on the input information, after the pipette tip 331 that has not been heated is attached to the pipette section 331, the pipette section 331 is moved to a predetermined location, and the liquid sample in the sample holding section 37 is sucked. Subsequently, the pipette unit 331 is moved to a predetermined location to hold the pipette tip 10 on the heating device main body 31, and this state is maintained for a predetermined time, and the liquid sample is heated in the pipette tip 10. Next, the pipette unit 331 may be moved to a predetermined location based on the input information, and the heated liquid sample may be discharged into the heated sample holding unit 35.

(Fourth embodiment)
The automatic dispensing apparatus for liquid samples according to the fourth embodiment of the present invention is the automatic dispensing apparatus according to the third embodiment, wherein the heating device section 30 is heated according to the first embodiment. It has been replaced by the device 1. However, here, the heater 11 provided in the heating device 1 is electrically connected to the control means 34 so that the heating of the liquid sample can be automatically controlled. Further, the fan 13 may be electrically connected to the control means 34 as necessary, and its operation may be automatically controlled.
Also in this embodiment, a liquid sample can be automatically dispensed as in the third embodiment.

(Fifth embodiment)
The automatic dispensing device for a liquid sample according to the fifth embodiment of the present invention is provided in the operation unit 33 while the heating device 30 is removed in the automatic dispensing device according to the third embodiment. The pipette unit 331 is replaced with the heating device 2 according to the second embodiment. However, here, the heater in the mounting portion 21 of the heating device 2 is electrically connected to the control means 34 so that the heating of the liquid sample can be automatically controlled.
In the present embodiment, instead of holding the pipette tip 10 in the heating device main body 31 and heating it, the pipette tip 10 is attached to and held in the mounting portion 21 of the heating device 2 except for heating. As in the third embodiment, a liquid sample can be automatically dispensed.

  According to the automatic dispensing apparatus and the automatic dispensing method of the liquid sample of the present invention, it is possible to analyze biologically related substances with higher accuracy and ease, and to rapidly analyze a large amount of liquid sample.

Hereinafter, the present invention will be described in more detail with reference to specific examples. However, the present invention is not limited to the following examples.
(Example)
Nucleic acid hybridization was performed using the automatic dispensing apparatus 3 according to the third embodiment.
That is, the heating device part 30 was heated to 90 ° C., and the pipette tip was held in the warming device part main body 31 and allowed to stand for 5 minutes to sufficiently warm the pipette tip.
Then, an aqueous solution containing a fluorescently labeled nucleic acid that has not been denatured (single-stranded) is held for about 1 to 5 minutes using the heated pipette tip, and the heated sample holding unit 35 is obtained. Were dispensed onto a microarray manufactured by NGK.
After dispensing, hybridization was performed at 38 ° C. for 30 minutes under light-shielding conditions, and then the solution on the microarray was removed to obtain 1 × SSC, 0.1% SDS as in the case of the aqueous solution containing the nucleic acid. The washing solution was dispensed to the corresponding locations on the microarray using a pipette tip that had been heated.
After washing, the washing solution on the microarray was removed, the microarray was rinsed with milliQ, and centrifuged at 2000 rpm for 1 minute to dry the microarray.
And the fluorescence signal on the dried microarray was detected with the laser scanner, and data were analyzed (n = 4).

(Comparative example)
Except that the aqueous solution containing the fluorescently labeled nucleic acid and the 1 × SSC, 0.1% SDS washing solution were dispensed onto the microarray using a non-warmed pipette tip. Hybridization was performed in the same manner, and a fluorescent signal was detected, and data was analyzed (n = 4).

以上の結果から、本発明により、ハイブリダイゼーションを伴う核酸の検出を、高精度かつ簡便に行えることが確認された。 When the analysis results of the example and the comparative example were compared, in the example, the detection luminance of the fluorescent signal was about 10% higher than that of the comparative example, and the reproducibility was also high.
On the other hand, in the comparative example, the impurities deposited on the microarray are attached, and it becomes difficult to distinguish this impurity from the fluorescence signal obtained as a result of the hybridization. It took time to analyze the work and the work efficiency deteriorated. Table 1 shows specific data of detected luminance in the examples and comparative examples.

From the above results, it was confirmed that the detection of nucleic acids accompanied by hybridization can be carried out with high accuracy and simplicity according to the present invention.

  The present invention can be used for analysis of biologically relevant substances in the medical field and biochemical research.

It is the schematic which illustrates the heating apparatus which concerns on 1st embodiment of this invention, (a) is a top view, (b) is a longitudinal cross-sectional view in the AA of (a). It is a front view which illustrates the heating apparatus which concerns on 2nd embodiment of this invention. It is a figure which illustrates the automatic dispensing apparatus of the liquid sample which concerns on 3rd embodiment of this invention, (a) is a schematic block diagram, (b) is an expanded longitudinal cross-sectional view of a heating means, (c) is heating. It is an enlarged longitudinal cross-sectional view which shows the other example of a temperature means.

Explanation of symbols

1, 2 ... Heating device, 3 ... Automatic dispensing device, 10 ... Pipette tip, 10a ... Mounting port, 10b ... Suction / discharge port, 11 ... Heater, 12 ... -Upper surface, 12a, 32 ... insertion hole, 13 ... fan, 21 ... mounting part, 22, 33 ... operating part, 30, 30 '... heating device part, 32' ...・ Through hole, 34 ... control means, 331 ... pipette part

Claims (15)

  A liquid sample dispensing method comprising a step of heating a pipette tip for dispensing a liquid sample and heating the liquid sample to be dispensed in the pipette tip.   The liquid sample dispensing method according to claim 1, wherein the liquid sample is an aqueous solution containing a nucleic acid or an aqueous solution containing a water-soluble substance having a solubility in water equal to or lower than that of sodium dodecyl sulfate.   3. The liquid sample dispensing method according to claim 1, wherein the liquid sample is used for nucleic acid hybridization. A heating apparatus used in the liquid sample dispensing method according to any one of claims 1 to 3,
A heating means for heating a pipette tip for dispensing a liquid sample to 40 to 100 ° C., and a holding means for holding the pipette tip without bringing its suction / discharge port into contact with the heating means. Characteristic heating device. The heating device is box-shaped,
5. The holding means includes a gas circulation means for holding the pipette tip with a suction / discharge port projecting inside the heating device, and further circulating a gas inside the heating device. The heating device according to 1. The heating device is in the form of a pipette;
5. The heating apparatus according to claim 4, further comprising operating means for operating suction and discharge of the liquid sample, wherein the heating means is integrated with the holding means.   7. The heating according to claim 4, wherein the heating means is made of one or more materials selected from the group consisting of metal, glass, engineering plastic, and super engineering plastic. Temperature device. The heating device is a metal block;
The heating device according to claim 4, wherein the holding means is an opening portion of an insertion hole formed in the metal block for inserting the pipette tip into the metal block.   The heating apparatus according to any one of claims 4 to 8, wherein a plurality of the holding means are provided, and a distance between centers of the adjacent pipette tips is 8 to 10 mm.   The heating apparatus according to any one of claims 4 to 9, wherein the heating means is electrically connected to a temperature control means for automatically controlling a temperature during heating.   A step of automatically sucking a liquid sample using a pipette tip for dispensing a liquid sample, a step of heating the pipette tip and automatically heating the liquid sample in the pipette tip, and the step of automatically An automatic liquid sample dispensing method comprising a step of discharging a liquid sample in a pipette tip. An automatic liquid sample dispensing apparatus for use in the automatic liquid sample dispensing method according to claim 11,
An operation unit for operating suction and discharge of a liquid sample, a heating means for heating the pipette tip to 40 to 100 ° C., and the pipette tip are held without contacting the suction / discharge port to the heating means. A heating device comprising a holding means for
The liquid sample automatic dispensing apparatus, wherein the operation section and the heating means are electrically connected to a control means for automatically controlling suction, discharge and heating of the liquid sample.   13. The heating device section is a metal block, and the holding means is an opening portion of an insertion hole formed in the metal block for inserting the pipette tip into the metal block. Automatic dispensing apparatus for liquid samples as described.   The warming device portion is box-shaped, and the holding means holds the pipette tip with its suction / discharge port projecting into the warming device portion, and further circulates gas inside the warming device portion. 13. The automatic liquid sample dispensing apparatus according to claim 12, further comprising a gas circulation means.   The automatic dispensing device for a liquid sample according to claim 12, wherein the warming device section has a pipette shape, and the warming means is integrated with the holding means.

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