JP2007212310A - Trace liquid sample dispenser - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To solve the problem in a conventional analysis technique for trace liquid specimens, wherein it is difficult to enhance the throughput by simplifying the system as a whole, since the analysis technique involves a complex and large-sized detection device. <P>SOLUTION: This trace liquid specimen dispenser comprises a multitude of trace liquid control parts disposed in an array form, with the control parts each made up of a pipet part, and is equipped with a mechanism for automatically controlling them. Each pipet part is equipped with a trace liquid holding chip, comprising a structure for sucking/discharging trace liquid samples and holding the liquid samples in the inside, and an opening/closing control mechanism performing opening/tight-closing internal space. Providing the sample dispenser enables a rapid and simple device to be provided, making it possible to analyze trace samples and to control movement thereof, while dispensing with complex detection device. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a technique related to an automatic liquid sample dispensing apparatus, and more particularly to a technique that enables control of movement according to an analysis processing result for a very large number of trace liquid samples, and a technique related to an apparatus using the same. is there.

  In recent years, automatic dispensing devices widely used in each field of life science have been progressing in high throughput, small amount, and multi-function. In the automatic dispensing apparatus, multi-well plates such as 96-well, 384-well, and 1536-well are used, and liquid samples accommodated in the same multi-well plate are simultaneously subjected to analysis processing. However, it is rare that the analysis result is constant for each plate, and there are more cases where the analysis result is not uniform. Therefore, it is desirable to selectively dispense into different containers depending on the analysis result.

  In the conventional technology, after a part of the liquid sample is transferred to another multiwell plate, analysis processing using colorimetry, fluorescence, etc. is performed, and the analysis result is collated with the original multiwell plate. Means are provided for dispensing each liquid sample.

For example, Patent Documents 1 to 3 describe examples of trace liquid sample dispensing apparatuses.
JP 2004-325117 A JP 2001-299388 A JP-A-11-201976

However, such means have the following problems. That is,
(1) The number of liquid sample dispensing increases, and the number of steps increases.
(2) Complex and large analyzers that use colorimetric, fluorescent, etc. of trace liquid samples are common, and when these are incorporated into a system, it becomes difficult to simplify and downsize the entire system. End up.

  An object of the present invention has been made in consideration of the above-described circumstances, and uses a simple alternative method that enables analysis of a small amount of liquid sample and control of movement while eliminating the need for a detection apparatus itself, and the same. Is to provide a device.

As a result of intensive studies to solve the above problems, the present inventor has reached the present invention. Specifically, it is a minute liquid sample dispensing apparatus that focuses on surface tension, which is a dominant characteristic when the amount of the sample is small.
A minute liquid sample dispensing apparatus of the present invention is a pipette provided with a minute liquid holding chip having a structure for sucking and discharging a minute liquid sample and holding the minute liquid sample therein, and an opening / closing control mechanism for opening and closing the inner space. A plurality of trace liquid samples having different surface tensions between samples are dispensed into different containers according to the difference in surface tension.

  In the present invention, since the influence of gravity is reduced and the trace liquid sample in which the influence of the surface tension becomes dominant is a target, specifically, a trace liquid sample of about several μl to 500 μl or less is desirable.

  Further, the kind of the trace liquid sample is not particularly limited except for a liquid sample having an extremely high viscosity. Specifically, various aqueous solutions, organic compounds, chemical reaction solutions, bacterial culture solutions, or cell culture solutions may be used.

Therefore, according to the present invention, it is possible to provide a quick and simple device that enables analysis of a small amount of sample and control of movement while eliminating a complicated detection device.
INDUSTRIAL APPLICABILITY The present invention can be applied to a method for analyzing a trace liquid sample and a device using the same, which are in great demand for simultaneous multi-sample high-speed processing.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings.

  FIG. 1 is a perspective view showing the overall structure of a multi-well plate automatic dispensing apparatus 1 to which Example 1 of a trace liquid sample dispensing apparatus according to the present invention is applied. A multi-well plate automatic dispensing apparatus 1 as a micro-liquid sample dispensing apparatus shown in FIG. 1 is configured to apply a micro-liquid sample having different surface tension into each well 2 of a multi-well plate A3 according to the difference in surface tension. The apparatus dispenses into the multiwell plate B4 and the multiwell plate C5. The multi-well plate automatic dispensing apparatus 1 includes a plate holder 6 for fixing three multi-well plates, a plate holder driving mechanism 7 for moving the plate holder 6 in the XY direction, a liquid control unit 8 for sucking and discharging a trace liquid sample, The liquid control unit drive mechanism 9 moves the liquid control unit 8 in the YZ direction. Further, the liquid control unit 8 includes a pipette 10, a pipette holder 11, a pipette control unit 12, and a trace liquid holding tip 13.

  FIG. 2 is a plan view showing the liquid control unit 8 in cross section. Hereinafter, the configuration of the liquid control unit 8 will be described in detail with reference to FIG. The same number of pipettes 10 as the number of wells of the multi-well plate are arranged in an array in the pipette holder 11 so as to correspond to each well of the multi-well plate, and the trace liquid holding tips 13 are attached to all the pipettes. All the pipettes 10 are automatically controlled by the pipette controller 12 at the same time.

  Each pipette 10 includes a cylindrical barrel 14, a plunger 15 that moves up and down in the barrel, and an open / close valve 16 that switches between an open and closed state inside the barrel. The up / down operation of the plunger 15 and the opening / closing operation of the open / close valve 16 are automatically controlled by the plunger control unit 17 and the valve control unit 18 in the pipette control unit 12, respectively.

  The trace liquid holding chip 13 has a conical shape in which the inner diameter becomes smaller toward the lower end, and the maximum part of the inner diameter is 5 mm and the minimum part is 0.5 mm. The material is polypropylene. In addition, since the trace liquid holding | maintenance chip | tip 13 is inserted and connected with the pipette 10, it is exchangeable.

  Next, based on FIG. 3 and FIG. 4, the operation | movement mechanism of the multiwell plate automatic dispensing apparatus 1 as a trace liquid sample dispensing apparatus is demonstrated. FIG. 3 is an operation diagram of the liquid control unit 8. Among the trace liquid samples in each well of the plate A, the trace liquid sample with a small surface tension is placed in the well of the plate B, and the trace liquid sample with a large surface tension is placed in the plate. The mechanism of dispensing into each well of C is shown.

  The trace liquid sample having a low surface tension described here is, for example, an alcohol solution such as ethanol or isopropanol, an organic compound such as hexane or octane, or an aqueous solution of a surfactant such as sodium dodecyl sulfate. On the other hand, the trace liquid sample having a large surface tension is, for example, water, an aqueous solution having a low concentration, or an aqueous solution of a metal salt such as sodium chloride.

  FIG. 3A shows one well 19 of the multiwell plate A in which a trace liquid sample 24 having a small surface tension is accommodated, and one well 20 of the multiwell plate B at the same coordinate position as the well. A single well 21 of the multiwell plate C, a pipette 22 corresponding to these wells, and a liquid holding tip 23 attached to the pipette are shown.

  Hereinafter, based on FIG. 3 (A), an operation of dispensing a small amount of liquid sample having a small surface tension in one well of the multi-well plate A to one well of the multi-well plate B will be described. First, the multi-well plate A moves so as to be positioned below the liquid control unit, and the plunger is pulled up with the open / close valve closed, whereby a trace liquid sample is inhaled, and the upper portion inside the liquid holding chip (FIG. 3A-1).

  Second, the multiwell plate B moves so as to be positioned below the liquid control unit, and the open / close valve is opened. At this time, although the trace liquid sample is subjected to the action of atmospheric pressure, since the surface tension is small, the trace liquid sample slides downward, falls from the liquid holding chip, and enters the well of the multiwell plate B (FIG. 3 ( A-2)).

  Third, the multiwell plate C moves so as to be positioned below the liquid control unit, and the plunger is pulled down with the open / close valve closed, but there is nothing in the liquid holding chip, Nothing is dispensed into the wells of plate C (FIG. 3 (A-3)).

  On the other hand, FIG. 4B shows one well 25 of the multiwell plate A in which the trace liquid sample 30 having a large surface tension is accommodated, and one well of the multiwell plate B at the same coordinate position as this well. 26, one well 27 of the multiwell plate C, a pipette 28 corresponding to these wells, and a liquid holding tip 29 attached to the pipette.

  Hereinafter, based on FIG. 4 (B), an operation of dispensing a trace amount liquid sample having a large surface tension in one well of the multiwell plate A into one well of the multiwell plate C will be described. First, the multiwell plate A moves so as to be positioned below the liquid control unit, the plunger is pulled up with the open / close valve closed, and the trace liquid sample in one well of the multiwell plate A is inhaled. And held in the upper part inside the liquid holding chip (FIG. 4B-1).

  Second, the multiwell plate B moves so as to be positioned below the liquid control unit, and the open / close valve is opened. At this time, although the trace liquid sample is subjected to the action of atmospheric pressure, since the surface tension is large, the trace liquid sample slides down to a portion having a small inner diameter, stops halfway, and does not fall from the liquid holding chip (FIG. 4 (B- 2)).

  Third, the multi-well plate C moves so as to be positioned below the liquid control unit, and the plunger is pulled down in a state where the open / close valve is closed. Enter the well of plate C (FIG. 4 (B-3)).

  Therefore, as described above, in a single operation of the multi-well plate automatic dispensing apparatus 1, among the trace liquid samples contained in the multi-well plate A, the sample having a small surface tension is the multi-well plate B. In addition, a sample having a large surface tension is dispensed into the multiwell plate C. Moreover, the next multiwell plate can be dispensed by exchanging the trace liquid holding chip 13.

  As described above, according to Example 1 of the present invention, a large number of trace liquid samples having different surface tensions can be measured in a very simple and quick manner according to the difference in surface tension without any need for surface tension measurement. It is possible to dispense.

  Example 2 of the present invention is the same as the trace liquid sample dispensing apparatus of Example 1 described above, except that different trace liquid holding tips are used. FIG. 5 is a cross-sectional view of a liquid control unit to which a trace liquid holding chip used characteristically in the second embodiment is attached. The trace liquid holding chip 13 in the second embodiment has a cylindrical shape with an inner diameter of about 1 mm and is made of polypropylene. Moreover, it has the hydrophobic surface part 31 above an inner wall, and has the hydrophilic surface part 32 in inner wall parts other than a hydrophobic surface part.

It is assumed that the hydrophobic surface portion 31 is surface-treated with a hydrophobic material such as a fluorine resin, a silicon resin, or polypropylene. The hydrophilic surface portion 32 is surface-treated with a hydrophilic material such as nylon or polyethylene terephthalate.
In addition, since the trace liquid holding | maintenance chip | tip 13 is inserted and connected with the pipette 10, it is exchangeable.

  Next, based on FIG.6 and FIG.7, the operation | movement mechanism of the multiwell plate automatic dispensing apparatus 1 as a trace liquid sample dispensing apparatus is demonstrated. FIG. 6 is an operation diagram of the liquid control unit 8. Among the trace liquid samples in each well of the plate A, the trace liquid sample with a small surface tension is placed in the well of the plate B, and the trace liquid sample with a large surface tension is placed in the plate. The mechanism of dispensing into each well of C is shown.

  The trace liquid sample having a small surface tension described here is, for example, an alcohol solution such as ethanol or isopropanol, an organic compound such as hexane or octane, or an aqueous solution of a surfactant such as sodium dodecyl sulfate. Any liquid sample that has high wettability with respect to the hydrophilic surface portion may be used. On the other hand, a trace liquid sample having a large surface tension is, for example, water, a low concentration aqueous solution, or an aqueous solution of a metal salt such as sodium chloride. Any liquid sample having high wettability may be used.

  FIG. 6A shows one well 19 of the multiwell plate A in which a small amount of liquid sample 24 having a low surface tension is accommodated, and one well 20 of the multiwell plate B at the same coordinate position as this well. A single well 21 of the multiwell plate C, a pipette 22 corresponding to these wells, and a liquid holding tip 23 attached to the pipette are shown.

  Hereinafter, based on FIG. 6 (A), an operation in which a small amount of liquid sample having a small surface tension in one well of multiwell plate A is dispensed into one well of multiwell plate B will be described. First, the multi-well plate A moves so as to be positioned below the liquid control unit, and the plunger is pulled up with the open / close valve closed, whereby a trace liquid sample is inhaled, and the hydrophobic surface inside the liquid holding chip (Fig. 6 (A-1)).

  Second, the multiwell plate B moves so as to be positioned below the liquid control unit, and the open / close valve is opened. At this time, the trace liquid sample is subjected to the action of atmospheric pressure, but moves downward due to the low surface tension. When moving slightly downward, a slight driving force is applied to the trace liquid sample due to the difference in wettability of the trace liquid sample with respect to the hydrophobic surface portion and the hydrophilic surface portion. Move to. Since the trace liquid sample has high wettability at the hydrophilic surface portion, the trace liquid sample slides downward, falls from the liquid holding chip, and enters the well of the multi-well plate B (FIG. 6 (A-2)).

  Third, the multiwell plate C moves so as to be positioned below the liquid control unit, and the plunger is pulled down with the open / close valve closed, but there is nothing in the liquid holding chip, Nothing is dispensed into the wells of plate C (FIG. 6 (A-3)).

  On the other hand, FIG. 7B shows one well 25 of the multiwell plate A in which the trace liquid sample 30 having a large surface tension is accommodated, and one well of the multiwell plate B at the same coordinate position as this well. 26, one well 27 of the multiwell plate C, a pipette 28 corresponding to these wells, and a liquid holding tip 29 attached to the pipette.

  Hereinafter, based on FIG. 7B, an operation in which a trace amount liquid sample having a large surface tension in one well of the multi-well plate A is dispensed into one well of the multi-well plate C will be described. First, the multi-well plate A moves so as to be positioned below the liquid control unit, the plunger is pulled up with the open / close valve closed, and the trace liquid sample that has entered one well 21 of the multi-well plate A Inhaled and held on the hydrophobic surface portion inside the liquid holding chip (FIG. 7B-1).

  Second, the multiwell plate B moves so as to be positioned below the liquid control unit, and the open / close valve is opened. At this time, the trace liquid sample is subjected to the action of atmospheric pressure. However, since the surface tension is large, the trace liquid sample does not move from the hydrophobic surface portion inside the liquid holding chip and does not fall from the liquid holding chip (FIG. 7B -2)).

  Third, the multi-well plate C moves so as to be positioned below the liquid control unit, and the plunger is pulled down in a state where the open / close valve is closed. It enters the well of plate C (FIG. 7 (B-3)).

  Therefore, as described above, in a single operation of the multi-well plate automatic dispensing apparatus 1, among the trace liquid samples contained in the multi-well plate A, the sample having a small surface tension is the multi-well plate B. In addition, a sample having a large surface tension is dispensed into the multiwell plate C. Moreover, the next multiwell plate can be dispensed by exchanging the trace liquid holding chip 13.

  As described above, according to the second embodiment of the present invention, a large number of micro liquid samples having different surface tensions can be measured very easily and quickly without any surface tension measurement. It is possible to dispense according to.

  In addition, since no detection device or measurement device is used in the present apparatus, even a minute liquid sample can be dispensed sufficiently even with about 1 μl, and even a minute liquid sample can be realized. Since it was difficult to reduce the size of the detection device or the measurement device in the conventional device, the entire device system could not be reduced in size, but the size reduction of the device can be achieved very easily.

  Example 3 of the present invention utilizes the above-described trace liquid sample dispensing apparatus of Example 1 or Example 2 as an analysis system that selectively and automatically dispenses a reaction solution according to the success or failure of a nucleic acid amplification reaction. It is. In the present example, the minute liquid sample to be dispensed is a nucleic acid amplification reaction solution, specifically, a liquid sample after completion of a PCR reaction that is widely used in many fields.

  FIG. 8 is a conceptual diagram showing DNA, primers, and probes used in the nucleic acid amplification reaction that can be used in the present invention. A general nucleic acid amplification reaction consists of at least one DNA 33 to be amplified, primers 34 and 35, four different types of deoxynucleotide triphosphates (not shown) (adenine, thymine, cytosine, guanine), and a nucleic acid polymerase. In addition to the above, in the nucleic acid amplification reaction that can be used in the present invention, it contains a DNA polymerase (not shown) and other reagents and solvents necessary for the PCR reaction. It contains a probe 38 in which OH is changed to -H and the lipophilic chain 37 is modified at the 3 'end. The lipophilic chain 37 described here is, for example, a linear alkyl group having 8 to 18 carbon atoms, and may have a part of a highly hydrophobic functional group such as a branched alkyl group or alkylnaphthalene. Good (FIG. 8A).

  The DNA 33 is configured by forming single strands 33A and 33B having different base sequences in a complementary manner and forming a helical double strand. The primers 34 and 35 are nucleotides that act as starting points for the synthesis of the DNA 33 to be amplified in the presence of deoxynucleotide triphosphate and DNA polymerase, and are complementary to the DNA 33. It is a base sequence that binds.

  The probe 38 is a reagent characteristically used in the present embodiment, and has a base sequence that binds complementarily to the DNA at a position between the primers 34 and 35. Therefore, when the DNA to be amplified is synthesized, this probe is decomposed by 1 base dNTP39 from the 5 ′ side by the exo activity of the DNA polymerase, and finally the hydrophilic part and the lipophilic part are separated. It acts as a surfactant 40 having a molecular structure. That is, when one molecule of DNA 33 to be amplified is amplified, one molecule of surfactant 40 is generated (FIG. 8B). The above-described reagents are mixed, and the temperature cycle including the heat denaturation step, the annealing step, and the extension step is repeated a plurality of times for the mixed solution.

  Here, in the heat denaturation step, by setting the reaction solution to a heat denaturation temperature (usually about 94 ° C.) for several seconds, the double strands of DNA 33 in the reaction solution are dissociated to form single strands 33A and 33B. It is a process to do. In the annealing step, the reaction solution is set at an annealing temperature (usually about 55 ° C.) for several seconds, whereby the single strand 33A or 33B (template DNA) generated in the heat denaturation step is added to the primer 34. Alternatively, 35 is a step of complementary bonding (annealing).

  Further, in the extension step, the reaction solution is set to the extension temperature (usually about 72 ° C.) for several minutes, whereby the single strand 33A or 33B (template DNA) of the DNA 33 in which the primer 34 or 35 is annealed in the annealing step. , Which is a step of synthesizing double-stranded DNA 33 by complementary binding of deoxynucleotide triphosphates by the action of DNA polymerase to synthesize primer 34 or 35, and at the same time, probe 15 is decomposed and surfactant 16 is It is a process of generating.

  By repeating the above temperature cycle a plurality of times (n times), the DNA 33 to be amplified is amplified 2n times, and a surfactant having the same number of molecules as the amplified DNA is generated at the same time. Here, since the surfactant greatly reduces the surface tension, the surface tension of the nucleic acid amplification reaction solution is greatly reduced as compared with that before the reaction. On the other hand, in the nucleic acid amplification reaction described above, DNA 33 to be amplified is sufficiently amplified due to low primer specificity, inappropriate temperature cycle, inappropriate salt concentration, or failure to prepare reaction solution. Otherwise, the surfactant is not generated sufficiently, so that the surface tension of the nucleic acid amplification reaction solution is hardly changed compared with that before the reaction, and remains the same as water.

  Using the multi-well plate, the nucleic acid amplification reaction described above is performed and used for the trace liquid sample dispensing apparatus described in the first embodiment or the second embodiment. The multiwell plate containing the nucleic acid amplification reaction solution after completion of the reaction corresponds to the multiwell plate A in Example 1 or Example 2.

  The nucleic acid amplification reaction solution in which the nucleic acid amplification reaction was successful, that is, the nucleic acid amplification reaction solution in which the DNA 33 to be amplified was sufficiently amplified and the surface tension was low was the same as in Example 1 or Example 2 in the same manner as in the multiwell plate. Dispense into B.

  On the other hand, the nucleic acid amplification reaction solution in which the nucleic acid amplification reaction has failed, that is, the nucleic acid amplification reaction solution in which DNA 33 to be amplified is hardly amplified and has a surface tension as high as that of water is the same as in Example 1 or Example 2. Similarly, it is dispensed into the multiwell plate C.

  As described above, according to Example 3 of the present invention, the successful nucleic acid amplification reaction solution and the failed nucleic acid amplification reaction solution can be dispensed easily and quickly simultaneously with the determination of the success or failure of the nucleic acid amplification reaction. it can.

  In the conventional method, a part of the nucleic acid amplification reaction solution is collected, and the collected solution is subjected to a nucleic acid analysis method such as agarose electrophoresis, and the result of the analysis is collated to determine the success or failure of the nucleic acid amplification reaction. It was. For this reason, in such a method, labor and time are required for analysis, and there has always been a risk of an ID error due to a mistake in the sample after verification. However, according to the present invention, analysis of the sample becomes unnecessary, and determination of success / failure and dispensing can be performed at the same time.

It is a perspective view which shows the whole structure of the trace amount liquid sample dispensing apparatus of Example 1 of this invention. It is a top view which shows a trace liquid control part in a cross section. It is an operation | movement figure (A1-A3) of a trace amount liquid control part. It is an operation | movement figure (B1-B4) of a trace amount liquid control part. It is a top view which shows the trace liquid control part of Example 2 of this invention in a cross section. It is an operation | movement figure (A1-A3) of a trace amount liquid control part. It is an operation | movement figure (B1-B4) of a trace amount liquid control part. It is a conceptual diagram which shows each DNA, primer, and probe which are used for PCR reaction of Example 3 of this invention.

Explanation of symbols

1 Multiwell plate automatic dispensing device 2 Well 3 Multiwell plate A
4 Multiwell plate B
5 Multiwell plate C
6 Plate Holder 7 Plate Holder Drive Mechanism 8 Liquid Control Unit 9 Liquid Control Unit Drive Mechanism 10 Pipette 11 Pipette Holder 12 Pipette Control Unit 13 Trace Liquid Holding Tip 14 Barrel 15 Plunger 16 Open / Close Valve 17 Plunger Control Unit 18 Valve Control Unit 19 Multiwell One well of plate A 20 One well of multiwell plate B 21 One well of multiwell plate C 22 Pipette corresponding to well 23 Liquid holding chip corresponding to well 24 Micro liquid sample with low surface tension 25 Multiwell plate One well of A 26 One well of multi-well plate B 27 One well of multi-well plate C 28 Pipette corresponding to well 29 Liquid holding tip corresponding to well 30 Fine surface tension of fine Liquid sample 31 Hydrophobic surface part 32 Hydrophilic surface part 33 DNA
33A, 33B single-stranded DNA
34 Primer 35 Primer 36 Oligonucleotide 37 Lipophilic chain 38 Probe 39 dNTP
40 Surfactant

Claims (7)

  A trace liquid sample dispensing apparatus having a trace liquid holding tip having a structure for sucking and discharging a trace liquid sample and holding the trace liquid sample inside, and a pipette having an open / close control mechanism for opening and closing the internal space. And dispensing a plurality of the trace liquid samples having different surface tensions between the samples into different containers according to the difference in the surface tension. In the trace liquid sample dispensing apparatus of Claim 1,
A trace liquid sample dispensing apparatus, wherein the trace liquid holding tip has a conical shape with an inner diameter decreasing toward a lower end. In the trace liquid sample dispensing apparatus of Claim 1,
A trace liquid sample dispensing apparatus comprising a hydrophobic surface portion and a hydrophilic surface portion on an inner wall of the trace liquid holding chip. In the trace liquid sample dispensing apparatus of Claim 1,
A trace liquid sample dispensing apparatus comprising a rough surface portion and a smooth flat surface portion on a surface of the trace liquid holding chip. In the trace amount liquid sample dispensing apparatus in any one of Claims 1 thru | or 4,
A trace liquid sample dispensing apparatus, wherein a large number of trace liquid control units including the trace liquid holding tip and the pipette unit are arranged in an array, and a large number of the trace liquid samples are dispensed simultaneously. In the trace amount liquid sample dispensing apparatus of Claim 5,
A trace liquid sample dispensing apparatus comprising a mechanism for automatically operating the liquid control unit and the trace liquid sample container. In the trace liquid sample dispensing apparatus of Claim 1,
A trace liquid sample dispensing apparatus, wherein the trace liquid sample is a nucleic acid amplification reaction solution, and the nucleic acid amplification reaction solution is automatically dispensed into different containers according to the success or failure of the nucleic acid amplification reaction.

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