WO2004104584A1 - Procede pour tester des substances apparentees a la biologie, appareil et procede de transfert fluidique associes - Google Patents

Procede pour tester des substances apparentees a la biologie, appareil et procede de transfert fluidique associes Download PDF

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Publication number
WO2004104584A1
WO2004104584A1 PCT/JP2004/007450 JP2004007450W WO2004104584A1 WO 2004104584 A1 WO2004104584 A1 WO 2004104584A1 JP 2004007450 W JP2004007450 W JP 2004007450W WO 2004104584 A1 WO2004104584 A1 WO 2004104584A1
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WO
WIPO (PCT)
Prior art keywords
fluid
substrate
flow
pump
reaction
Prior art date
Application number
PCT/JP2004/007450
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English (en)
Japanese (ja)
Inventor
Shuzo Mishima
Morinao Fukuoka
Kenji Takahashi
Takami Shibazaki
Original Assignee
Olympus Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Olympus Corporation filed Critical Olympus Corporation
Priority to JP2005506439A priority Critical patent/JPWO2004104584A1/ja
Publication of WO2004104584A1 publication Critical patent/WO2004104584A1/fr

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/02—Burettes; Pipettes
    • B01L3/0289—Apparatus for withdrawing or distributing predetermined quantities of fluid
    • B01L3/0293—Apparatus for withdrawing or distributing predetermined quantities of fluid for liquids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
    • G01N35/1009—Characterised by arrangements for controlling the aspiration or dispense of liquids
    • G01N35/1016—Control of the volume dispensed or introduced

Definitions

  • the present invention relates to a method for testing a biological substance.
  • the invention also provides for controlling fluid transfer for that purpose.
  • Human genome analysis has shifted from systematic sequencing to t> or systematic functional analysis, and the horoscope has been shifted.
  • the method of analyzing genetic information is mainly used. One is to analyze “what the genes and the mRNAs and proteins expressed from the genes themselves are”. The other is to analyze under what conditions the mRNA or protein is synthesized.
  • the methods belonging to the former include the Southern blot method, the Northern blot method, and the Eastern blot method. These methods are mainly used for DNA, RNA, or protein of interest. It is for analyzing quality. Therefore, it is not suitable for comprehensive analysis of all DNA, RNA, or protein extracted from cells.
  • DNA thisop is obtained by dividing a silicon chip into a number of compartments by means of photolithography technology, and directly synthesizing single-stranded DNA with a specific base sequence on each compartment.
  • the DNA micro-lay is designed to reduce the size of the DNA micro-lay that was conventionally plotted on the membrane to about 300 m or more.
  • Is 3 ⁇ 4 and connected to the computing unit system, for example, fluorescently labeled D supplied on the chip or on the microphone array
  • Japanese Patent Application Laid-Open Publication No. 2000-501520 and U.S. Patent Application Publication No. 200 / 200,533 A1 describe specific examples of such a DNA chip.
  • a pull-up DNA is placed on a porous substrate made of aluminum oxide. Is immobilized.
  • the solution containing the fluorescently labeled DNA is transported inside and out of the porous substrate at a predetermined constant speed so that the solution is transported. y, high efficiency and fast speed 5 ⁇ .
  • Japanese Patent Application Laid-Open No. 2000-501-5251 discloses a porous substrate.
  • a method of detecting a biological substance by immobilizing a solid phase and flowing a sample liquid by a pressure difference is disclosed.
  • U.S. Patent Application Publication No. 200/002 / 530533A1 describes that the detection is performed after the substrate is washed.However, the flow of the solution at the time of washing and at the time of detection is described. Is not shown. This means that only one flow condition is always applied in one process.
  • the present invention is directed, in part, to a method for testing a biological substance using a test chip including a substrate capable of detecting a biological substance ft, which is not limited to a gene. That is, the above-mentioned D N
  • test chips composed of a functional substrate for testing genes and biological substances other than genes are used. Is directed to the test method used o
  • the inspection method of the present invention is a method for inspecting a biological substance using a functional substrate, comprising a plurality of steps of flowing a fluid and contacting the fluid with a general-purpose substance.
  • the fluid flows at the kinetic speed i.
  • Another inspection method of the present invention is a method for inspecting a biological substance using a functional substrate, comprising a step of causing a fluid to reciprocate and repeatedly contact the fluid with the functional substrate. The fluid is caused to flow at different flow speeds at and after the return.
  • Another inspection method of the present invention is a method of inspecting a biological substance using a functional substrate, comprising a step of flowing a fluid to contact the fluid with the functional substrate, and causing the fluid to flow at an irregular flow velocity.
  • Another inspection method includes a plurality of steps of causing a fluid to flow using a volume pump to contact the fluid with the functional substrate, and the volume of the volume pump changing the volume of the fluid is changed.
  • test method of the present invention is a test method for a bio-related substance in which a test for a bio-related substance is performed while a functional and flowable substrate is held in a fluid in a plurality of steps. In each step, the substrate is flowed at a flow rate suitable for the process.
  • the present invention is also directed, in part, to a transfer device used in the above-described method for inspecting a biological substance.
  • a transfer device includes: transfer means for transferring a fluid; holding means for holding a functional substrate capable of continuously contacting the transfer of the fluid; means for controlling the transfer means; And a control condition setting means for selectively setting at least one control condition. Further, the present invention is directed, in part, to a transfer method applied to the above-described method for testing a biological substance.
  • the transfer method of the present invention includes a step of continuously contacting a substrate that functions with a fluid with the fluid, a step of transferring the fluid under at least two control conditions, and a step of controlling the control conditions according to characteristics of the fluid. Setting step.
  • FIG. 1 shows the D N commonly used in the embodiment of the present invention.
  • FIG. 1 shows a schematic configuration of an A-thisop reading device.
  • FIG. 2 is a perspective view schematically showing the configuration of the reaction vessel shown in FIG.
  • Figure 3 shows a cross section of the reaction vessel shown in Figures 1 and 2.o
  • FIG. 4 shows a general drive pattern of a syringe pump in connection with the first embodiment of the present invention.
  • FIG. 5 shows a driving pattern of a syringe pump according to the second embodiment of the present invention.
  • FIG. 6 shows a driving pattern of a syringe pump according to the third embodiment of the present invention.
  • FIG. 7 shows a driving pattern in a comparative example of the driving pattern of FIG.
  • Fig. 8 shows a measurement example of the temporal change of the luminance of a certain probe obtained by driving according to the driving pattern shown in Fig. 6.o
  • Fig. 9 is obtained by driving according to the driving pattern of Fig. 7.
  • FIG. 6 shows a measurement example of the temporal change in the luminance of the fluorescence of the same pump.
  • FIG. 10 shows a driving pattern of the reaction process in the fourth embodiment with a lightness of 3%.
  • FIG. 11 shows a driving pattern of a cleaning step in the fourth embodiment of the present invention.
  • FIG. 12 is a view showing an apparatus for implementing the fifth embodiment of the present invention.
  • FIG. 13 is a diagram showing a garment implementing the 9th / 9th embodiments of the present invention.
  • the country 14 shows the drive of a syringe pump capable of using the invention in the invention o
  • FIG. 15 shows a more preferable drive pattern of a syringe pump that can be used in the present invention.
  • the DNA chip reading device has a stage 1 on which a reaction container 2 is placed.
  • the stage 1 has a fixed plate 1a, a Y movable plate 1b movable on the fixed plate 1a in the Y-axis direction, and an X movable plate movable on the Y movable plate 1b in the X-axis direction.
  • the reaction vessel 2 can be moved in the X-Y axis directions by the Y moving plate 1b and the X moving plate 1c.
  • Reaction vessel 2 has a rectangular parallelepiped upper plate as shown in Fig. 2. It is constructed by superimposing 210 and the lower plate 202.
  • the upper plate 201 includes, but is not limited to, four cylindrical holes 200, for example.
  • the lower plate 202 has a hole 201 a
  • Cylindrical holes 202 a, 202 b, 202 c, and 202 d at positions corresponding to 201 b, 201 c, and 201 d in a row along the longitudinal direction Is formed in.
  • the upper plate 201 and the lower plate 202 are superimposed to form four reaction chambers 3a, 3b, 3c and 3d.
  • the reaction chambers 3a, 3b, 3c, and 3d have the DNA chips containing the reaction sections 4a, 4b4c, and 4d of the DNA chip 4 as the functional base, respectively.
  • 4 is the reaction chamber 3a, 3b,
  • the A chip 4 has, for example, four reaction portions 4a, 4b, 4c, and 4d having a diameter of about 6 mm formed on a substrate of about 25 mm ⁇ 75 mm at a pitch of 9 mm.
  • the reaction sections 4a, 4b, 4c, and 4d become reaction chambers.
  • 3a, 3b, 3c, and 3d are accommodated at positions slightly above the respective bottom surfaces.
  • the reaction progress of the reaction sections 4a, 4b, and 4c4d is detected by the fluorescence detection unit.
  • the openings 301a, 301b, 301c, and 30Id are opened so that the sample solution and the reagent solution required for the reaction can be supplied sequentially, simultaneously, or in a mixed state. I have.
  • the openings 301a, 301b, 301c, and 30Id may contain liquids other than samples and reagents, such as diluents, washing solutions, and assay reagents (substrates). Enzymes etc.) are also supplied.
  • the openings 301a, 301b, 301c, and 301d may be covered with an appropriate lid member at a time when liquid is not supplied.
  • connection holes 3 leading out to the side surfaces of the reaction vessel 2 are provided on the side surfaces of the holes 202 of the lower plate 202, 202 a 200 b, 202 c and 202 d.
  • the reaction vessel 2 includes reaction chambers 3a and 3b.
  • stage 1 X-moving plate 1 c Placed on stage 1 X-moving plate 1 c so that 3 c and 3 d are arranged in a line along the X-axis direction
  • a switching valve 6 as a channel switching means is connected to 2 d via distribution lines 5 a, 5 b 5 c, and 5 d.
  • a pump 8 that functions as a flow source of the transfer means is connected to the switching valve 6 via a pipe 7.
  • the pump 8 is used to give a flow to the pump solution in each of the reaction chambers 3 a, 3 b 3 c, and 3 d.
  • a pump such as a silicon pump (for example, MIC510B, an eight-mil-ton company) It is composed of Switching par, lube 6 is, for example, a rotary 4-way selection valve (example For example, Hamilton HVDP 4_5).
  • the operating shaft a has a pinion 9 attached to it.
  • the pini 9 attached to the switching valve 6 is a rack
  • the pinion 9 is rotated via the rack 10 by the movement of the stay 1 in the X-axis direction.
  • the rotation of the pinion 9 rotates the operating shaft 6a, whereby one of the arrangements 5a, 5b, 5c, 5d can be selectively connected to the pump 8 via the pipe 7 .
  • the pinion 9 has a size corresponding to a pitch (9 mm) of the reaction portion 4 a 4 b, 4 c, and 4 d of the DNA chip 4 in a quarter rotation.
  • It functions as a flow condition setting means for setting a preferable speed condition from among a plurality of flow speeds, while keeping in mind the characteristics of the fluid bath fluid flowing through the DNA chip 4.
  • the necessary solutions are prepared for each step.
  • the required solution is a sample liquid containing the biological substance to be inspected that reacts with the probe, and the remaining solution on the test chip
  • the washing water is used.
  • the necessary solutions prepared in each step are summed up. Simply called a solution
  • biological substance J includes not only cells such as animals, plants, and microorganisms, but also substances derived from viruses, bacteria, and the like that cannot multiply on their own unless they infest them.
  • Bio-related substances include not only those in the natural form directly extracted and isolated from these cells and the like, but also those produced using genetic techniques and those chemically modified. More specifically, it includes hormones, enzymes, antibodies, antigens, Abzymes, and other nucleic acids (for example, DNA, RNA, PNA), and the like.
  • a substance that specifically binds to a biological substance refers to a substance that specifically binds to a biological substance.
  • a ligand such as a hormone and its receptor, an enzyme and its substrate, an antigen and its antibody, a nucleic acid having a specific sequence and a sequence complementary thereto.
  • nucleic acids having Re or are included.
  • ⁇ DNA chip J refers to the above-mentioned “functional substrate having a reactivity to perform a biological reaction using probe J as a reagent.” “Functional substrate J responds to the above-described biological substance.
  • the functional substrate of the present invention is a functional substrate having a functional property that causes a measurable change. Supports of any shape with various reagent components o
  • fluid refers to any fluid containing a substance that comes into contact with a functional substrate and causes the substrate to function. ⁇
  • the fluid is a solution, it contains a gas that is inert with respect to the above-mentioned biological substance. Even Good.
  • the fluid is innumerable fine particles, the fluid may be contained in a body that is inert to the biological substance.
  • Flow J is a movement in which the liquid changes the contact area with the substrate.
  • O Flow is actively moved by means for controlling the movement of the liquid. Controlling the movement of the liquid
  • the means includes a dynamic parameter of adding-damping 'maintaining and stopping the flow velocity in an appropriate combination, and has a function of distributing an appropriate time distribution to each selected dynamic parameter.
  • the solution is flowed by the syringe pump 8 and moved back and forth to one of the reaction sections 4 a 4 b 4 c and 4 d of the DNA chip 4. That is, the solution is reciprocally flowed and repeatedly passed through one of the reaction sections 4a4b4c4d.
  • a preferred embodiment of the substrate that can be applied to the present invention has a hole through which a fluid can pass through the substrate, and more preferably at least a part of the functional portion made of a porous material.
  • a substrate having another shape may be used as long as the substrate has a function and a fluid can contact the functional surface in a flowing state.
  • FIG. 4 shows a general driving pattern of the silicon pump 8.
  • the X-axis indicates the elapsed time
  • the Y-axis indicates the position of the piston of the syringe pump 8.
  • the downward-sloping section corresponds to the “bow 1” action
  • the upward-sloping section corresponds to the “push” action.o
  • the magnitude of the inclination indicates the piston's movement speed. 2004/007450
  • the piston of the syringe pump 8 is pushed at a constant speed, stopped for a fixed time, and then pulled at the same speed as the pushing operation. This series of operations is repeated at regular intervals.
  • the present embodiment is directed to flowing a solution at a flow rate suitable for each of a plurality of steps in a test of a biological substance. That is, in each process, the syringe pump 8 is driven according to a drive pattern in which the movement speed of the piston is different. In other words, the movement speed of the piston of the syringe pump 8 is changed in each step of the method for examining a biological substance.
  • the piston of the syringe pump 8 was drawn at a constant speed giving a flow velocity of 10 IZ seconds to the solution and stopped for a certain period of time according to the driving pattern in FIG. The solution is then pushed at a rate that gives the solution a flow rate of 10 tI / s. This series of operations is repeated at a certain interval and repeated as many times as necessary.
  • the piston of the syringe pump 8 is drawn at a constant speed that gives the solution a flow speed of 20 IZ seconds according to the driving pattern in FIG. After being stopped for a while, the solution is pressed at a speed that gives a flow rate of 20 nIZ seconds. This series of operations is repeated at a certain time interval and repeated as many times as necessary.
  • Optimal flow rate and washing of the solution (sample liquid) in the reaction process The optimal solution (washing water) in the four processes is different. Therefore, by allowing the solution to flow at a flow rate suitable for the process in each process, it is possible to complete each process in a short time. Each process can be performed efficiently.As a result, the time required for the inspection of biological substances can be reduced.
  • the present embodiment is directed to a process in which a solution is caused to flow forward and a test chip is repeatedly passed through the solution, and the solution is caused to flow at different flow speeds at the time of going and returning in the process. Has been.
  • the movement of the piston of the silicon pump 8 is changed between the “pull” operation and the ⁇ push J operation.
  • the flow rate of the solution is different between the time when the J pump of the syringe pump 8 is pulled and the time that it is pressed.
  • Fig. 5 shows a drive pattern in which the piston moves at different speeds during the "pull” operation and the “push” operation.o
  • the piston of the syringe pump 8 for example, is drawn at a constant rate that gives the solution a flow rate of 10 At 1 / s, and 5 ⁇ m is applied to the solution stopped for a certain period of time.
  • the reaction parts 4 a, 4 b, 4 c, and 4 d of the D ⁇ chip 4 have different withstand pressure characteristics with respect to positive pressure and withstand pressure with respect to negative pressure depending on the material and the shape of the peripheral holding part.
  • the positive pressure is the pressure received by the reaction part 4 d during the “push” operation of the piston of the syringe pump 8
  • the negative pressure is the pressure received during the “pull” operation of the piston of the syringe pump 8. It is.
  • the reaction parts 4 a, 4 b, 4 c, and 4 d have particularly low withstand pressure characteristics against positive pressure, so they tend to be broken in the worst case during the “push” operation of the piston of the syringe pump 8.
  • the movement speed of the piston during the "push” operation is kept low, so the reaction sections 4a, 4b, 4c, 4 during the "push” operation
  • the pressure on d is kept low.
  • the flow velocities of the solution at the time of going and returning are set in accordance with the pressure resistance characteristics at the time of going and returning of the reaction sections 4a, 4b, 4c and 4d.
  • the solution may be flowed at a speed suitable for the process. As a result, the process can be completed in a short time, and as a result, it is possible to reduce the time required for examining a biological substance.
  • the present embodiment is directed to flowing the solution at a non-constant flow rate in one step of flowing the solution and passing the test chip through the solution.
  • the solution is intermittently flowed at a constant one-step motion.
  • the biston of the syringe pump 8 is intermittently moved at a constant speed. Pressing ”indicates a drive pattern in which the piston moves intermittently during operation.
  • reaction sections 4a, 4b, 4c, and 4d of 4 Excessive rise in the pressure of the reaction sections 4a, 4b, 4c, and 4d of 4 is suppressed. As a result, the reaction sections 4a, 4b, 4c,
  • FIG. 7 shows a drive pattern in a comparative example.
  • FIG. 8 shows a measurement example of a temporal change in the luminance of the fluorescence of a certain probe obtained by driving according to the driving pattern of FIG.
  • FIG. 9 shows a measurement example of a temporal change in the luminance of the fluorescence of the same pump obtained by driving according to the driving pattern shown in FIG. 04 007450
  • the driving pattern that suppresses the generation of laminar flow is the same as when
  • the piston is moved intermittently--not limited to a drive pattern that moves at a constant is degree.
  • the drive unit moves the piston so that the solution flows in a pulsating manner during the operation.
  • the drive unit moves in a sinusoidal waveform based on a constant angle. It may be a drive pattern for moving the. Alternatively, the driving pattern may be such that the piston moves at an irregularly changing speed during the “pull” operation and the “push” operation.o
  • the solution may be caused to flow at the same flow rate. ⁇ This allows the process to be completed in a short time. 0 As a result, it is possible to reduce the time required for testing biomaterials. ⁇ Fourth embodiment
  • FIG. 11 shows the driving pattern of Reaction I in this embodiment
  • FIG. 11 shows the driving pattern of the cleaning step in this embodiment.
  • the horizontal axis indicates elapsed time
  • the vertical axis indicates the position of the piston of the syringe pump 8.
  • the downward sloping section corresponds to the "pull" action
  • the upward sloping section corresponds to
  • Substantially horizontal portion indicates the stop position of the piston in each drive pattern, and corresponds to the amount of liquid to be driven in each process.
  • the stop position of the piston of the syringe pump 8 is lower in the washing process than in the center of fe. ⁇ (There is «-nX At S. Accordingly, after the sample solution is reacted, By supplying a larger amount of the cleaning liquid than the reaction liquid, the cleaning step drives a larger amount of the cleaning liquid.
  • the present embodiment is directed to flowing a solution at a drive position suitable for each of a plurality of steps in the inspection of a biological substance. That is, in each process, the syringe pump 8 is driven according to a drive pattern in which the drive position of the piston is different. O In other words, the drive region of the piston of the syringe pump 8 is ⁇ IO for each of the related substances will change. The changed drive amount of the piston Supply the solution together ⁇ M t Changed 0
  • the piston of the syringe pump 8 is drawn for 5 seconds at a constant speed that gives the solution a flow rate of 10 t Iz seconds.
  • the solution connected to the pipes connected to the connection ports 302a to 302d is sucked according to the amount of the liquid to be flown.
  • the inside diameter of the solution is 1 mm
  • the length of the solution to be sucked is about 64 mm, and due to the repeated flow, a solution residue occurs near the solution tip of 64 mm.
  • the transfer control of the present invention focuses on the point that the characteristics of the fluid affect the reactivity depending on the contact conditions with the functional substrate, and changes the control conditions of the transfer of the fluid according to the characteristics of the fluid.
  • the control conditions are appropriately selected from one or a combination of one or more of a flow velocity, a moving time, a number of transfers, a transfer direction, and the like.
  • Fluids have physical and chemical properties.
  • the “physical properties” include, for example, viscosity, volume, steam / soil, light refractive index, magnetic property, and diffusion constant.
  • “Chemical properties J include, for example, Tm value, reactive force, and functional components (nucleic acid, in, body, enzyme, etc.).
  • the transfer pressure to the DNA tip is changed in accordance with the viscosity of the sample, the formula 3 ⁇ 4, or both.
  • Samples applied to a DNA chip as a functional substrate have different viscosities depending on the individual from which they are derived. Since this viscosity is related to the various components in the sample, it is usually difficult to adjust the viscosity to a constant value. The same component present in separate samples will come into contact with the substrate under different viscosity conditions. Different liquids move with the same surface with different frictional resistances. Therefore, in the present embodiment, the m of the substrate is changed by changing the flow state of the sample in accordance with the viscosity level. The syringe pump 8 is not driven so as to exert the same effect. For the sake of convenience, in the present embodiment, variations in the fluidity due to breakage of the base or failure due to clogging are not considered.
  • Fig. 12 shows the schematic configuration of a DNA chip reading and reading apparatus for implementing the Hondeido form.
  • the same reference numerals as those shown in Fig. ⁇ indicate "C”.
  • the members shown are the same members, and detailed descriptions are omitted. 7450
  • each reaction chamber 3a can be connected through the piping 5a, 5b, 5c, 5d corresponding to each switched connection flow path.
  • the flow resistance at 4 d is selectively detected by a common pressure sensor 17.
  • the value of the pressure sensor 17 is fed back to the pump control unit 14 of the thin pump 8 via the arithmetic unit 15.
  • the feedback control to the syringe pump 8 by the pressure sensor 17 is not suitable for abnormal pressure changes related to abnormal quality of the DNA chip 4 (for example, cracks, breakage, clogging).
  • the flow resistance value resulting from the standard quality abnormality of the substrate shows a similar pressure fluctuation pattern even with different fluid solutions, so that it can be clearly distinguished from the response increase / decrease pattern due to viscosity.
  • a porous material made of alumina having a pore size of m to 0.6 ⁇ m is formed into a thickness of 0 • 0.5 cm to 0.5 cm.
  • the speed of the standard viscosity sample is set to 35 At I / sec for a sample with a standard viscosity, 50 n I sec for a sample of a relatively high viscosity group It is preferable to use a flow rate of 20 t 1 Z seconds for a sample of a relatively low viscosity group, which makes it possible to maximize the function of the functional substrate.
  • the reagent information is sent to the operation unit 15 based on the input data of the test item, and the characteristics of the reagent included in the reagent information or the characteristics of the components in the sample on which the reagent acts or the characteristics thereof are described.
  • the sample applied to the DNA chip as a functional substrate or the reagent for reacting with this sample contains nucleic acid which is a temperature-dependent component.
  • the temperature dependence of nucleic acids is related to the temperature at which the DNA duplex dissociates into single strands and the temperature at which it returns to double strand.
  • the Tm value which is the temperature at which a single strand dissociates, differs depending on the nucleotide sequence of the nucleic acid. Since different temperature conditions affect the flow of samples and reagents, it is preferable to control the flow rate to correspond to the difference in Tm values. By adjusting the flow conditions so that the thermodynamic conditions of the sample and / or reagent are kept constant for each temperature corresponding to each Tm value, a large number of nucleic acid priming reactions can be performed under constant reaction conditions. Run on 3 ⁇ 4 ⁇
  • FIG. 13 shows a schematic configuration of a DNA chip ⁇ -pickup apparatus for carrying out the present embodiment.
  • the members indicated by the same reference numerals as those shown in FIG. 1 are the same members, and the detailed description thereof is omitted. 4 007450
  • the 24 sampling device is provided with an input device 18 for causing the arithmetic unit 15 to recognize the Tm value of the sample on the DNA chip 4.
  • the input device 18 allows the user to input information related to the Tm value using, for example, a keyboard, a touch panel, or a mouse.
  • the entered information is confirmed by the user on the display 19.
  • Another embodiment of the input device 18 is a code reader that decodes an information medium such as a bar code (consisting of a one-dimensional or two-dimensional pattern) by a decoding process.
  • Known code readers may be operated by a user or automatically accessed on a substrate mounted and held within an inspection system.
  • the operation unit 15 changes the intensity of stirring or the time according to the difference in the Tm value.
  • excess dissociation is reduced by controlling the flow rate to be slower. Stable dissociation conditions in the nucleic acid hybridization reaction contribute to the reliability of the reaction result.
  • the reagent is for PCR (polymerase chain reaction)
  • change the agitation intensity according to the reaction curve in the thermal cycle consists of different temperature conditions applied to the dissociation and reversion of the nucleic acid duplex. Even for the same sample, the fluid properties at high and low temperatures are different, so the contact conditions for the same DNA chip are different.
  • the comparison ⁇ ! Strong i dynamic condition is suitable. Conversely, relatively good flow conditions are appropriate for returning from a single strand.
  • the flow is degree is relatively high compared to the low temperature process. If the cycle of the nucleic acid hybridization reaction always proceeds under the same contact conditions, the R concentration as the reaction result will be the highest.
  • the reagent information is sent to the operation unit 15 based on the input data of the test items sent from the input unit 18, and the pump flow rate is calculated every thermal cycle related to the characteristics of the PCR reagent. Sends control signal to control unit
  • the strength of the flow of the washing liquid is changed in accordance with the reaction sensitivity of the ⁇ chemical, and an extra measure that causes a decrease in accuracy in the measurement of J5Z. Need to suppress the reaction
  • the reagent information is sent to the tray 15 based on the input data of the test item by the input device 18.
  • a control signal is sent to the pump control unit 14 that controls the flow rate of the pump for each reaction sensitivity related to the strength of the washing liquid as a chemical.
  • the pump control unit 14 controls the flow rate of the pump for each reaction sensitivity related to the strength of the washing liquid as a chemical.
  • the flow is changed to the fastest flow in which no bubbles are generated according to the water level on the tip when the pump is driven.
  • the liquid level changes during the movement of liquid l3 ⁇ 4j.
  • the lower the water level on the DNA tip the slower the speed.
  • This embodiment can be carried out by the same apparatus as that shown in FIG. 1; however, the arithmetic unit 15 is provided with a detector for detecting light absorption or light reflection of the excitation beam of the fluorescence detection unit 12. On the basis of the,
  • the water level is calculated by analyzing the water surface image on the DNA chip.
  • the fluorescence detection unit 2 is used to measure the reaction progress and results of the 3d anti-) L, part 4d of the ventricle. Is moved above the reaction part 4 d by moving the stain 1 to the illustrated 2 cm in the X-axis direction. ⁇ 0
  • the rack 10 is moved along with the movement of the X moving plate 1 c.
  • the operation unit 1 is moved while the pin 9 rotates and the operation shaft 6 a of the switching valve 6 rotates, and the reaction 3 c and the flow path of the pump 8 are connected via the switching valve 6.
  • the solution in the reaction chamber 3C can be made to flow.
  • the objective lens 13 of the fluorescence detection unit 12 can be moved up and down, and the water level on the DNA chip can be changed from the light source by changing the TU occupation by nj ′. Continuously detects light absorption or light reflection near the water surface due to the output beam of.
  • an optical system that changes the focal position without moving the objective lens Two technologies are also available.
  • the data detected by the light detection unit is used to monitor the flow state of the solution in the reaction chamber 3c. In this way, the light absorption (or light reflection) data obtained by the CCD of the fluorescence detection unit 12 is transmitted to the arithmetic unit 15 and fed back to the flow velocity control unit of the pump.
  • the flow velocity on the substrate is increased or decreased in accordance with the measurement timing of the D ⁇ chip.
  • the fluid state should be as static as possible, and at the time of non-measurement, the fluid state should be as dynamic as possible.
  • This embodiment can be implemented by the same device as in FIG. First, the reaction part 4 d of the reaction chamber 3 d is moved to the focal position of the objective lens 13 of the fluorescence detection unit 12. Next, Stage 1
  • the rack 10 moves with the movement of the X moving plate 1c, the pini pin 9 rotates, and the operation shaft 6a of the switching valve 6 also rotates, as described above.
  • the flow path of the pump 8 is connected to the flow path of the pump 8 via the switching valve 6 ⁇
  • the operation unit 5 controls the pump 8 via the pump control unit 14 via the pump control unit 14.
  • the solution in the reaction chamber 3c can be made to flow.
  • the reaction on the DNA chip by the fluorescence detection unit 12 is continuously measured while the driving by the pump 8 is performed intermittently.
  • a constant low-speed condition for example, 5 At 1 / s
  • the fluorescence data obtained at this low speed is used as the measured value.
  • the constant low speed condition means that the speed is reduced to a flow rate that suppresses the turbulence of the liquid surface, or that the speed is accelerated or decelerated according to the liquid surface so as to perform measurement in a uniform convection state.
  • the fluorescence detection unit 12 can also be used to monitor the dynamic and static state of a liquid using detection data on light absorption or light reflection of the excitation beam.
  • Table 1 shows the relationship between the suitable piston drive speed for each sample or reagent property.
  • the syringe pump can be driven as follows.
  • Figure 8 shows two types of driving conditions that are cfc in the general driving pattern.
  • the X-axis indicates the elapsed time
  • the Y-axis indicates the position of the piston of the syringe pump 8.
  • the section corresponds to a “push” action.
  • the magnitude of the inclination indicates the moving speed of the piston.
  • the solid line indicates a relatively high-speed drive pattern
  • the dotted line indicates a relatively low-speed ( ⁇ half the speed of the solid line) drive pattern.
  • the piston of the syringe pump 8 is continuously pushed at a constant speed, stopped for a fixed time, and then continuously pulled at the same speed as the pushing operation. .
  • This series of operations is repeated at regular intervals.
  • the drive pattern of the non-linear pump 8 is not limited to the above-described example, and various modifications and changes may be made without departing from the essentials of the present invention.
  • the piston of the inline pump 8 is pulled at a constant speed for 1 second during the pulling J operation, stopped for 1 second, and this series of operations is repeated four times. After that, during a “push” operation, it is pressed at a constant speed for 1 second and stopped for 1 second, and this series of operations is repeated four times.
  • the piston is intermittently moved during the subtraction J operation and the “push” operation, so that laminar flow is effectively suppressed. Is controlled.
  • the drive pattern that suppresses the generation of laminar flow is not limited to a drive pattern that moves the piston intermittently at a constant speed during the “pull” operation and the “push” operation.
  • a drive pattern that moves the piston to pulsate the solution // it i.e., a speed that changes sinusoidally with reference to a constant speed
  • a driving pattern for moving the piston with the button may be used.
  • the driving pattern may be such that the button is moved at an is degree that changes irregularly during the “pull” operation and the “push” operation.
  • two types of flow speed conditions are selectively set according to the characteristics of the fluid.
  • three or more types of flow speed conditions may be provided. For example, for characteristics where there is an unspecified large number of variations for each sample or reagent (viscosity, volume, etc.), standard conditions corresponding to intermediate flow rates are set, and for characteristics within the standard level, use standard conditions.
  • the fluid may be transferred.
  • the present invention comprises a functional substrate for testing a biologically-related substance other than a gene. Testing for other bio-related substances using other test chips
  • the present invention may be applied to examination of an immune reaction, a biochemical reaction, an electrochemical reaction, and the like.
  • various forms can be applied. For example, two-dimensional substrates such as silicon wafers and glass, various fine particles, various porous substrates, etc. Gel Various carrier-type arrays (Japanese Patent Laid-Open No. 11-7
  • a functional substrate and a functional substrate can be used. It may be a combination with a substrate that does not have it.
  • a necessary amount of a functional reagent may be immobilized on the inner wall of the microchannel, and the functional reagent may be used.
  • the beads or fine particles having the required number of solid phases immobilized thereon may be positioned in the flow channel.
  • a plurality of substrates can be arranged in the same area, or can be conveyed so as to be positioned in a perfume with respect to a common position.
  • the inspection by a body having a plurality of functionalities may be executed by a control system that sorts and processes each substrate for each set / operating condition.
  • the non-permeable one-dimensional body can be formed into a flat medium vessel that surrounds the entire substrate according to the opening used. In a flat hollow cell, branch passages having partially different widths are provided, and a mechanism for electrochemically or mechanically controlling the flow direction or flow is flow rate of the fluid or both is appropriately provided.
  • the present invention also includes a method and an apparatus for magnetically controlling the mi-motion condition of a fluid having a component containing a magnetic substance.
  • the control of magnetic force can be automated by changing the power of the electromagnet or increasing or decreasing the distance from the permanent magnet.
  • the capillary type array is installed on the inner surface of the capillary or inside the capillary.
  • fluorescence detection using a reagent that emits fluorescence has been described as an example. However, a measurable signal other than fluorescence is emitted.
  • Another detection unit using a generated reagent may be used. Further, detection of the detection unit is not limited to a wide field of view using a CCD for imaging, but may be a measurement relating to a narrow area such as a confocal laser microscope. Multiple scanning points can be measured efficiently by scanning a narrow area o
  • the preferred type of pump is a displacement pump.
  • a displacement pump for example, a reciprocating pump, a diaphragm pump, and a rotary pump can be used.
  • O for a reciprocating pump, for example, a piston pump.
  • Brancher pumps are available
  • Rotary pumps are, for example, m-table pumps, partition plate pumps, screw screw pumps, and ⁇ -port pumps. It is possible to easily control the pressure change because it sucks and discharges the liquid by changing it.
  • Preferred is a reciprocating pump when the liquid is brought into and out of contact with the solid-phased substrate in a reciprocating manner.
  • the amount and speed of driving of the solution can be controlled by the reciprocating distance and the mobility of the piston-blanker, so that the precise movement of the solution can be easily performed.
  • the syringe is dropped to the position B corresponding to the volume corresponding to the added amount of the solution, and the solution is aspirated. Further, the cylinder pump is lowered to the position C corresponding to the offset volume (suction), and if necessary, is held for a certain time T1, and then the syringe pump is initialized via the positions D and E. Position F corresponding to position
  • the present invention can be summarized as follows. ⁇
  • different kinds of fluids corresponding to different processes are produced under different flow conditions for each process. Contacts the functional substrate.
  • the same type of fluid may come in contact with a plurality of functional substrates at the same time or at different times.
  • the fluid that contacts the substrate in each step is different Since it has a purpose, it is possible to enhance the quality of inspection for bio-related substances by setting flow conditions suitable for achieving the purpose for each process.
  • the substrate When the substrate is a porous filter, it is caused to flow in a direction or in both directions so as to pass through the filter.
  • a DNA chip having a three-dimensional structure of a through-hole type in which a plurality of types of probes as reagents can be arranged on the same surface as in the embodiment is used as a base, a DNA chip as shown in the drawing is used.
  • the liquid level as a fluid is raised (pump is positive pressure) or lowered (pump is negative pressure) by leveling the surface.
  • the substrate is a groove or a tubular microchannel, the fluid flows in one or both directions along the channel.
  • the flow direction is arbitrary and may be, for example, unidirectional or bidirectional in a circular, helical or linear flow path. Different beads can be immobilized on multiple beads or microparticles.
  • a variety of flow conditions can be set by stopping or moving without depending on the flow of the fluid. Regardless of the shape of the substrate, the laminar flow due to the constant continuous pump pressure is used for M as described above, and the pulsating flow is applied by applying the intermittent or intermittent pump pressure. However, it has a remarkable effect that it is possible to bring more bio-related substances in the fluid into contact with the substrate. Testing with constant flow conditions will reduce the quality of testing for any combination of reaction, washing and measurement.
  • the present invention can be used for all functional substrates, but a liquid bio-related substance which is constantly and uniformly present in a biological fluid.
  • Non-stationary or heterogeneous insoluble biomaterials e.g., nucleic acids or cells
  • insoluble biomaterials e.g., nucleic acids or cells
  • functional substrates rather than (e.g., electrolyte components and enzyme components) And the tumor marker protein
  • the substrate immobilized with an insoluble bio-related substance as a specialty I- to ⁇ drug has an extremely fine protruding structure composed of a bio-related substance
  • Controlling the cC wit dynamic conditions so as to induce the protrusions dynamically contributes to good contact with the entire substrate surface.
  • it is a place where it is difficult to make contact with the fluid and a place where the ratio or probability of contact with the fluid is low.
  • the immobilization step of immobilizing a bio-related substance as a sample and / or a reagent to a predetermined region of the substrate is included as a pre-stage of the reaction step, It is preferable to provide a period of suspension of the flow sufficient to achieve immobilization.
  • the immobilization process is not limited to permanent immobilization, in which suspended biological substances are directly immobilized electrochemically or organically on a substrate, but beads or fine particles that have previously been immobilized with biological substances are immobilized. Includes a reversible immobilization process that immobilizes electrically or magnetically at an addressless location on the substrate.
  • the present invention provides a method and apparatus for maximizing the functionality of a functional substrate in contact with any different fluids at a high rate and with a high probability. Also, in a multi-item measurement in which a functional substrate has a plurality of different functionalized components (for example, reagents), it is effective to ensure that each function is performed at the required timing.
  • a functional substrate has a plurality of different functionalized components (for example, reagents)

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  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Clinical Laboratory Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé pour tester des substances apparentées à la biologie, ce procédé comprenant plusieurs étapes pour créer un flux de fluide et mettre ce fluide en contact avec une substance de base fonctionnelle. Dans chacune des étapes, le flux de fluide est réalisé à un débit adapté à l'étape concernée.
PCT/JP2004/007450 2003-05-26 2004-05-25 Procede pour tester des substances apparentees a la biologie, appareil et procede de transfert fluidique associes WO2004104584A1 (fr)

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JP2005506439A JPWO2004104584A1 (ja) 2003-05-26 2004-05-25 生体関連物質の検査方法と、そのための流体移送装置と流体移送方法

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JP2003147685 2003-05-26
JP2004-070813 2004-03-12
JP2004070813 2004-03-12

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007132805A (ja) * 2005-11-10 2007-05-31 Canon Inc 反応装置
JP2008253227A (ja) * 2007-04-09 2008-10-23 Hitachi Software Eng Co Ltd 反応装置及び反応チップ
JP2009042104A (ja) * 2007-08-09 2009-02-26 Canon Inc 物質固定装置、物質検出装置および物質固定方法
JP2009521924A (ja) * 2005-12-29 2009-06-11 アイ−スタット コーポレイション 分子診断用増幅システムおよび方法
US8716008B2 (en) 2010-10-18 2014-05-06 Fujifilm Corporation Detection method and detection system

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007132805A (ja) * 2005-11-10 2007-05-31 Canon Inc 反応装置
JP2009521924A (ja) * 2005-12-29 2009-06-11 アイ−スタット コーポレイション 分子診断用増幅システムおよび方法
JP2008253227A (ja) * 2007-04-09 2008-10-23 Hitachi Software Eng Co Ltd 反応装置及び反応チップ
JP2009042104A (ja) * 2007-08-09 2009-02-26 Canon Inc 物質固定装置、物質検出装置および物質固定方法
US8716008B2 (en) 2010-10-18 2014-05-06 Fujifilm Corporation Detection method and detection system

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