Patent Application
20060275892
1. Field of the Invention
The present invention relates to a reaction vessel provided for the detection of biorelated substances, and a reaction apparatus and a detection apparatus using the reaction vessel.
2. Description of the Related Art
Analysis has recently been performed on the genes of many creatures including human beings and many plants including rice. In the recent years, there has been developed a new examination method using a DNA chip having DNAs regularly sequenced on a semiconductor or the like or a DNA microarray. According to this new examination method, a plurality of genes can be simultaneously examined.
The present applicant has proposed a gene examination apparatus which uses a three-dimensional DNA microarray and can easily examine genes in a short period of time in International Publication WO03/027673. In this case, a reaction solution is placed in a reaction vessel for a three-dimensional DNA microarray using a porous filter as a carrier, and is caused to repeatedly pass through a reaction portion by being pressurized and depressurized by a syringe pump. Therefore, gene examination can be performed with high repeatability within a short period of time. In addition, International Publication WO03/005013 has proposed a microarray having a structure which uses a porous substrate and causes a reaction solution to repeatedly pass through a reaction portion by a syringe piston pump.
The apparatus disclosed in International Publication WO03/027673 or International Publication WO03/005013 is configured so that a reaction solution is dispensed into a reaction vessel from above a microarray substrate, a reaction state is observed from above, and pressurization and depressurization by a syringe pump are performed from below.
According to an aspect, the present invention is directed to a reaction vessel for detecting biorelated substances. A reaction vessel of the present invention comprises a three-dimensional substrate on which a probe for detecting a biorelated substance is immobilized, a coupling portion allowed to be coupled to liquid driving means in an airtight fashion and to be coupled to detection means so as to prevent leakage of light, and a solution sucking portion allowed to suck a solution. The coupling portion and the solution sucking portion are arranged with a three-dimensional substrate held between them.
According to another aspect, the present invention is directed to a biorelated substance reaction apparatus using the above reaction vessel. A reaction apparatus of the present invention comprises a fitting portion allowed to be fitted on the coupling portion of the reaction vessel, pressure control means for transferring a pressure for sucking and discharging a sample solution to the reaction vessel through the fitting portion, and temperature regulating means for regulating a temperature of the sample solution in the reaction vessel.
According to still another aspect, the present invention is directed to a biorelated substance detection apparatus using the above reaction vessel. A detection apparatus of the present invention comprises a fitting portion allowed to be fitted on a coupling portion of a reaction vessel and detection means for detecting a reaction of a biorelated substance on the three-dimensional substrate in the reaction vessel.
Advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Advantages of the invention may be realized and obtained by means of the instrumentalities and combinations particularly pointed out hereinafter.
The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate presently preferred embodiments of the invention, and together with the general description given above and the detailed description of the preferred embodiments given below, serve to explain the principles of the invention.
The embodiments of the present invention will be described below with reference to the views of the accompanying drawing.
The present embodiment is directed to a new reaction vessel for the detection of biorelated substances and a biorelated substance examination apparatus (including a reaction apparatus and a detection apparatus) using the reaction vessel.
In this specification, “biorelated substances” include not only cells of animals, plants, microorganisms, and the like but also substances originating from viruses which cannot proliferate by themselves unless parasitizing such cells. Biorelated substances include substances in natural forms which are directly extracted/isolated from these cells, substances produced by using a gene engineering technique, and chemically modified substances. More specifically, biorelated substances include hormones, enzymes, antibodies, antigens, abzymes, other proteins, nucleic acids, and the like.
In addition, a “probe” means a substance which specifically binds with the above biorelated substance, and includes any one of substances in the following relationships: a ligand such as a hormone and its acceptor, an enzyme and its substrate, an antigen and its antibody, a nucleic acid having a specific sequence and a nucleic acid having a sequence complementary thereto, and the like.
As shown in
In this specification, a “three-dimensional substrate” is a substrate on which probes are immobilized. As this substrate, a substrate made of any material and having any structure can be used as long as it has a structure which has a spread in the three-dimensional direction and allows a liquid to pass therethrough. For example, various kinds of filters and porous members can be used. As this substrate, a porous member obtained by etching an Si wafer, hollow fiber, metal oxide film, beads, or capillary is especially preferable.
The three-dimensional substrate 102 includes a reaction region in which a plurality of probe molecules which trap a target substance are immobilized in the form of a circle having a diameter of 120 μm. The coupling portion 103 and the solution sucking portion 104 are coupled to each other with the three-dimensional substrate 102 held between them. The coupling portion 103 and the solution sucking portion 104 comprise a non-optical-transparent material.
The examination apparatus includes a fitting portion 111 which is allowed to be fitted on the coupling portion 103 of the reaction vessel 101, a pressure controller 112 which controls the pressure of the reaction vessel 101 through the fitting portion 111, and a biaxial robot 113 which movably supports the fitting portion 111. The pressure controller 112 preferably includes a means for generating a pressure and a means for detecting a pressure.
The examination apparatus also includes a sample vessel 114 which stores a sample solution and a cleaning fluid vessel 115 which stores a cleaning fluid. The sample vessel 114 comprises, for example, a micro-titer plate. The cleaning fluid vessel 115 comprises, for example, a micro-titer plate or a bottle.
The coupling portion 103 of the reaction vessel 101 has an opening end. Fitting the fitting portion 111 in the opening end of the coupling portion 103 allows the reaction vessel 101 to be detachably mounted on the fitting portion 111. The reaction vessel 101 mounted on the fitting portion 111 is coupled to the pressure controller 112 in an airtight fashion. The biaxial robot 113 can move the reaction vessel 101 mounted on the fitting portion 111.
The pressure controller 112 transfers a pressure to the reaction vessel 101 mounted on the fitting portion 111, causing a sample solution or cleaning fluid to be sucked from the sample vessel 114 or cleaning fluid vessel 115 into the reaction vessel 101 or a sample solution or cleaning fluid to be discharged from the reaction vessel 101 through the solution sucking portion 104.
The examination apparatus further includes a heater 116 for controlling a sample temperature during a hybridization reaction or a cleaning fluid temperature at the time of cleaning operation, a temperature measuring resistor 117 for measuring the temperature of the heater 116, a temperature controller 118 which controls the temperature of the heater 116 on the basis of the information obtained by the temperature measuring resistor 117.
The heater 116 has a space for housing the reaction vessel 101. The reaction vessel 101 mounted on the fitting portion 111 is moved into the space of the heater 116 by the biaxial robot 113, as needed.
The heater 116, temperature measuring resistor 117, and temperature controller 118 constitute a temperature regulating means for regulating the temperature of a solution in the reaction vessel 101. This temperature regulating means cooperates with the fitting portion 111 and pressure controller 112 to constitute a reaction apparatus which promotes a hybridization reaction.
The examination apparatus also includes an observation optical system 120 for optically observing the three-dimensional substrate 102 in the reaction vessel 101. The observation optical system 120 is, for example, a fluorescence observation optical system. The observation optical system 120 includes a light source 122 which emits light with a visible wavelength, an excitation filter 123 which selectively transmits a wavelength for exciting a fluorescent material bound to a sample molecule from the light emitted by the light source 122, a dichroic mirror 124 which selectively reflects light transmitted through the excitation filter 123 and transmits fluorescence emitted from the fluorescent material, and a fluorescence filter 125 which selectively transmits fluorescence emitted from the fluorescent material and transmitted through the dichroic mirror 124.
The observation optical system 120 also includes an objective lens 126 for optically detecting the trapping of sample molecules in probe molecules on the three-dimensional substrate 102, an illumination optical system 127 which brings light from the light source 122 to the three-dimensional substrate 102 through the excitation filter 123 and the dichroic mirror 124, an imaging optical system 128 for imaging the light from the three-dimensional substrate 102 captured by the objective lens 126, and a CCD camera 129 which converts the optical image imaged by the imaging optical system 128 into an electrical signal.
The coupling portion 103 has a tapered inner surface which gradually increases in diameter with an increase in distance from the three-dimensional substrate 102. The coupling portion 103 has an opening end in which the fitting portion 111 is to be inserted. The taper angle between this opening end and the three-dimensional substrate 102 is larger than the NA of the objective lens 126. Accordingly, light from the reaction region on the three-dimensional substrate 102 is focused without vignetting.
The operation of the examination apparatus of this embodiment will be described below in accordance with a general examination process.
(1) Preparation
A nucleic acid is extracted from a biological sample, labeled with a fluorescent material such as FITC, and then dissolved in a buffer solution.
The dissolved sample solution is dispensed into the sample vessel 114, which is then placed at a sample vessel set position.
In addition, a cleaning fluid is dispensed into the cleaning fluid vessel 115, which is then placed at a cleaning fluid vessel set position.
The reaction vessel 101 is set at a predetermined position.
(2) Suction of Sample Solution
The reaction vessel 101 is mounted on the fitting portion 111.
The interior of the reaction vessel 101 is depressurized by the pressure controller 112 through the fitting portion 111, causing a sample solution is sucked from the sample vessel 114. A portion of the solution sucking portion 104 of the reaction vessel which is positioned below the solid support is preferably tapered as shown in the figure, when a sample solution, in particular, is sucked from a micro-titer plate or the like, because of decreasing the remaining amount of solution. After the suction of a sample solution, an air layer is preferably sucked, which prevents the sample solution from dripping from the reaction vessel when it moves to the temperature controller, and also the sample solution from dripping from the reaction vessel when the sample solution is moved up and down through the three-dimensional substrate.
(3) Transportation of Reaction Vessel to Temperature Controller (Heater)
The reaction vessel 101 is attached in the recess portion of the heater 116 in tight contact.
(4) Hybridization
The heater 116 is controlled to a desired temperature.
The interior of the reaction vessel 101 is repeatedly pressurized and depressurized by the pressure controller 112, which moves the sample solution up and down through the three-dimensional substrate 102 to hybridize the nucleic acid contained in the probes and sample solution.
(5) Cleaning
After the end of a hybridization reaction, the interior of the reaction vessel 101 is pressurized by the pressure controller 112 through the fitting portion 111, discharging the sample solution into the sample vessel 114.
the interior of the reaction vessel 101 is depressurized by the pressure controller 112 through the fitting portion 111, causing a cleaning fluid is sucked from the cleaning fluid vessel 115.
The interior of the reaction vessel 101 is repeatedly pressurized and depressurized by the pressure controller 112, which cleans the three-dimensional substrate 102 with the cleaning fluid, washing off the nucleic acid contained in the sample solution which is not bound to any probe molecule.
(6) Decoupling
The reaction vessel 101 is removed from the fitting portion 111 to expose the three-dimensional substrate 102.
(7) Reading of Reaction Result
The observation optical system 120 is placed above the reaction vessel 101. Exciting light is applied to the three-dimensional substrate 102, light from probe spots on the three-dimensional substrate 102 is imaged by the CCD camera 129 as electronic image data, and the data of the image or the quantity of light from the probe spots is stored.
(8) Examination Result
This stored data is analyzed to examine the expression state, mutation, polymorphism, and the like of the nucleic acid contained in the sample solution.
According to this embodiment, by the single pressure controller 112, solutions necessary for the examination of a biorelated substance such as a sample solution and a cleaning fluid can be sucked from the respective vessels, and the sample solution can be moved to efficiently come into contact with a porous member. That is, the dispensing mechanism for a sample solution and cleaning fluid and the driving mechanism for the sample solution are integrated. This simplifies the apparatus arrangement.
The present embodiment is preferably configured so that a solution necessary for the examination of a biorelated substance such as a sample solution or a cleaning fluid is placed in micro-titer plates. That is, the sample vessel 114 and the cleaning fluid vessel 115 are preferably constituted from micro-titer plates. In this case, many samples can be examined at high speed.
The reaction vessel 101 does not substantially transmit light, which preferably eliminates the need for preparing any specific light-shielding means in the case of performing examination with light such as fluorescence or chemiluminescence. When a target substance to be detected is to be labeled with fluorescence and detected, since light shielding must be provided even during a reaction with a probe, the reaction vessel 101 preferably has a light-shielding function. Although no specific limitations are imposed on materials for the reaction vessel 101 as long as they can substantially block light, a crystalline resin is preferably used in consideration of chemical resistance and heat resistance against a solution to be handled. Crystalline resins include, for example, polyproprene, polyacetal, polyamide-based synthetic resins, polymethylpentene, polyethyleneterephthalate, and polyethylene. These materials are inexpensive, can be easily made to contain carbon particles, and is easily moldable. Polyproprene and polyacetal containing carbon particles can easily provide a light shielding function, and hence is more preferable. Furthermore, polyacetal is high in transferability from a mold for molding, and allows easy manufacture of a reaction vessel with high dimensional accuracy.
In addition, the inner surface of the coupling portion 103 of the reaction vessel 101 is tapered, and the taper angle between the opening end of the coupling portion 103 and the three-dimensional substrate 102 is preferably larger than the NA of the objective lens 126 because all signals from spots formed on the three-dimensional substrate 102 as a support for probes can be received. In order to efficiently receive all signals from spots without excessively increasing the size of an optical system, it is especially preferable that the NA of the observation optical system 120 be substantially equal to the taper angle of the inner surface of the coupling portion 103 of the reaction vessel 101.
Reaction vessels 101 having such tapered portions can be mass-produced by molding with a resin, and a vessel having sufficient strength can be manufactured even if the thickness of the resin is about 0.1 to 0.2 mm or less. Therefore, the manufactured vessel has a small thermal capacity and high temperature responsibility, and hence a biorelated substance can be examined with high accuracy.
A portion of the reaction vessel 101 on which the three-dimensional substrate 102 is placed preferably has a diameter of 1 to 6 mm. In clinical examination, it can be thought from the number of examination items that the number of spots is about 10 to 250. If, for example, single nucleotide polymorphisms in about 30 regions are to be examined, eight types of probes may be required for a single nucleotide polymorphism in one region. Therefore, 240 types of probes are required. When such a probe is to be immobilized on a three-dimensional substrate, therefore, a portion of the examination vessel in which the three-dimensional substrate is placed preferably has a diameter of 1 to 6 mm because about 10 to 250 spots can be efficiently arranged.
In addition, a biorelated substance as a target substance to be examined is sampled and extracted from blood or tissue. Therefore, the amount of solution from which sampling can be performed is limited. It is desirable that examination can be performed with a minimum amount of solution. If, however, the volume of sample solution is small, since the solution often is set at a predetermined temperature of about 30° C. to 60° C. suitable for a reaction at a reaction stage, the evaporation of the sample solution may influence an examination result. In addition, in order to perform reaction and detection of a biorelated substance with high repeatability, a certain volume of solution is required in consideration of the movement of the solution, the retention of its temperature, and the evaporation of the solution. The volume of 10 to 100 μL allows performing reaction and detection of a biorelated substance with high repeatability. Accordingly, the volume of solution finally regulated is preferably 10 to 100 μL. The substantial volume of the reaction vessel 101 is preferably 10 to 100 μL in order to store this sample solution, provide sufficient contact between the three-dimensional substrate 102 and the sample solution, and realize an efficient reaction with a probe.
A sample solution stored in a micro-titer plate enables reaction and detection to be performed within a very short period of time. Examination can be performed with a high throughput.
The present embodiment is directed to another examination apparatus using the reaction vessel according to the first embodiment.
A fitting portion 130 in this embodiment includes an observation optical system for optically observing a three-dimensional substrate 102. That is, the fitting portion 130 comprises an imaging optical system 131 for forming an optical image of the three-dimensional substrate 102, a CCD 132 which converts an optical image of the three-dimensional substrate 102 formed by the imaging optical system 131 into an electrical signal, and an LED 133 which emits light having a wavelength which can excite fluorescent molecules bound to sample molecules.
Although not shown in particular, the fitting portion 130 is coupled to a pressure controller 112 (see
In the examination apparatus of this embodiment, since an observation optical system is provided in the fitting portion 130, the three-dimensional substrate 102 in the reaction vessel 101 can be optically observed without detaching the reaction vessel 101 from the fitting portion 130.
In addition, since the fitting portion 130 can transfer a pressure from the pressure controller 112 to the reaction vessel 101 like the fitting portion 111 in the first embodiment, a change in reaction state can be observed while a sampling solution is driven. In order to perform measurement for each probe under an optimal condition, measurement is performed first under a given condition, and then measurement is performed under another condition. The present embodiment is especially suitable for a case wherein measurement is performed while a temperature is changed or a reaction condition is changed, e.g., a case wherein measurement is performed with a given probe at 40° C., and measurement is performed with another probe at 60° C. In addition, a proper reaction time can also be obtained by measuring a change over time.
The present embodiment is directed to another observation optical system which can be used in place of the observation optical system according to the first embodiment.
An observation optical system 120A of this embodiment includes optical elements functionally equivalent to those of the observation optical system 120 of the first embodiment. The observation optical system 120A also includes a sleeve 160 which is allowed to be fitted in a coupling portion 103 of a reaction vessel 101. The sleeve 160 is made of a non-optical-transparent material. An objective lens 126 is housed and held in the sleeve 160.
For this reason, each optical element of the observation optical system 120A is functionally equivalent to that of the observation optical system 120 of the first embodiment, but is much smaller than that of the observation optical system 120 of the first embodiment.
In this embodiment, a three-dimensional substrate 102 in the reaction vessel 101 is observed while the sleeve 160 housing the objective lens 126 is fitted in the coupling portion 103 of the reaction vessel 101.
In this embodiment, since the sleeve 160 housing the objective lens 126 is fitted in the coupling portion 103 of the reaction vessel 101, the space between the three-dimensional substrate 102 and the objective lens 126 is properly shielded from light. Accordingly, disturbance light from entering the observation optical system 120A is properly prevented. Therefore, the measurement accuracy improves.
The present embodiment is directed to another examination apparatus using the reaction vessel according to the first embodiment.
As shown in
It suffices if the rack 141 can at least house the reaction vessel 101. More preferably, the rack 141 can house a plurality of reaction vessels 101.
Although not shown in particular, the fitting portion 111 is coupled to a pressure controller 112 (see
As shown in
As shown in
The operation of the examination apparatus according to this embodiment will be described below in accordance with a normal examination process:
(1) The reaction vessel 101 as an examination target is placed on the rack 141.
(2) A prepared sample solution and cleaning fluid are dispensed into the microplate 142.
(3) The rack 141 on which the reaction vessel 101 is mounted is placed in the rack housing portion 143.
(4) The microplate 142 in which the solutions are dispensed is placed in the microplate housing portion 144.
(5) The XYZ robot 146 is caused to fit the fitting portion 111 in the target reaction vessel 101 placed on the rack 141.
(6) The fitting portion 111 on which the reaction vessel 101 is mounted is moved by the XYZ robot 146 to position the reaction vessel 101 at a position where a solution can be sucked from a predetermined well of the microplate 142.
(7) The interior of the reaction vessel 101 is depressurized by the pressure controller 112 (see
(8) The fitting portion 111 on which the reaction vessel 101 is mounted is moved by the XYZ robot 146, placing the reaction vessel 101 in the recess portion 150 of the temperature regulating unit 147. The temperature regulator 151 regulates the temperature of the temperature regulating unit 147 to a temperature suitable for a hybridization reaction.
(9) The interior of the reaction vessel 101 is repeatedly pressurized and depressurized by the pressure controller 112, which moves the solution up and down through the three-dimensional substrate 102, causing a hybridization reaction between the sample and the probes in the reaction region.
(10) After the end of the reaction, the fitting portion 111 on which the reaction vessel 101 is mounted is moved by the XYZ robot 146 to position the reaction vessel 101 to the original well of the microplate 142. The interior of the reaction vessel 101 is then pressurized by the pressure controller 112, discharging the sample solution into the original well.
(11) The reaction vessel 101 is positioned to the well in which the cleaning fluid is dispensed by the XYZ robot 146.
(12) The interior of the reaction vessel 101 is depressurized, sucking the cleaning fluid into the reaction vessel 101.
(13) The interior of the reaction vessel 101 is repeatedly pressurized and depressurized, driving the cleaning fluid by a proper number of times.
(14) The interior of the reaction vessel 101 is pressurized, discharging the cleaning fluid into the original well.
(15) The reaction vessel 101 in which a reaction has been terminated is returned to the rack 141 by the XYZ robot 146.
(16) The operation from (5) to (15) is repeatedly executed for all the reaction vessels 101 as examination targets housed in the rack 141.
(17) The observation optical system 120A is moved by the XYZ robot 156 to be fitted in the processed reaction vessel 101 from above, detecting the fluorescence intensity in the reaction region on a three-dimensional substrate 102.
(18) The operation in (17) is repeatedly executed with respect to all the processed reaction vessels 101 as examination targets. The operation in (17) is preferably performed every time the operation from (5) to (15) is terminated with respect to one reaction vessel 101.
According to this embodiment, since a hybridization reaction and detection can be separately performed and sequentially processed, experiment and detection concerning a large amount of sample can be performed at high speed.
The present embodiment is directed to a new reaction vessel for detecting biorelated substances and a biorelated substance examination apparatus using the reaction vessel.
As shown in
The examination apparatus includes a fitting portion 181 on which the reaction vessel 171 is to be mounted and an XYZ robot for moving the fitting portion 181. The fitting portion 181 is conductive and has a voltage measurement electrode. The XYZ robot includes an arm 182 which supports the fitting portion 181, a direct-acting guide 183 which supports the arm 182 so as to allow it to move in the Z direction, a ball screw 186 engaged with the arm 182, and a control motor 185 which drives the ball screw 186. The ball screw 186 converts the rotational motion of the control motor 185 into rectilinear motion in the Z direction of the arm 182.
The examination apparatus further includes a vessel 191 which stores a solution necessary for examination such as a sample solution or a cleaning fluid and a rack 193 which houses the vessel 191. Both the vessel 191 and the rack 193 are conductive, and the rack 193 has a voltage measurement electrode.
The examination apparatus further includes an AC power supply 201 for applying an AC voltage between the fitting portion 181 and the rack 193, a voltmeter 202 for measuring the voltage between the two terminals of the AC power supply 201, an ammeter 205 for measuring a current flowing between the fitting portion 181 and the rack 193, a motor driver 204 which drives the control motor 185, and a control device 203 for controlling the motor driver 204. The control device 203 obtains an impedance from the voltage value measured by the voltmeter 202 and the current value measured by the ammeter 205, and performs feedback control on the motor driver 204 on the basis of the impedance.
The operation of the examination apparatus according to this embodiment will be described below. Assume that in the following description, a sample solution is stored in the vessel 191.
The reaction vessel 171 is lowered toward the solution level of the sample solution stored in the vessel 191 while the impedance between the fitting portion 181 and the rack 193 is measured.
The impedance gradually decreases as the reaction vessel 171 approaches the solution level of the sample solution. When the impedance decreases below a threshold, the lowering speed of the reaction vessel 171 is decreased.
When the reaction vessel 171 keeps moving down and comes into contact with the sample solution, the impedance abruptly changes. This position is therefore set as a solution level position. The reaction vessel 171 is further lowered from this solution level position by a distance by which a predetermined amount of sample solution can be sucked, and is stopped there. After the reaction vessel 171 reaches this position, sucking of a sample solution is started by a pressure controller (not shown). Monitoring an impedance informs that the suction of a solution is reliably performed. When the impedance abruptly changes, a suction failure has occurred. In such case, therefore, the reaction vessel is lowered to continue suction.
According to another method, when the reaction vessel 171 comes into contact with a sample solution, the impedance abruptly changes, and hence this position is set as a solution level position. The reaction vessel 171 is further lowered from this solution level by a predetermined distance, and is stopped there. After the vessel reaches this position, suction of a sample solution is started by the pressure controller (not shown). Furthermore, while the sample solution is sucked, the reaction vessel 171 is lowered in accordance with the solution level movement amount which can be calculated from the sucked volume and the volume of the reaction vessel. At this time, an impedance is measured to check whether the reaction vessel is in contact with the solution level.
In this embodiment, detecting a solution level makes it possible to properly control the velocity at which the reaction vessel 171 enters the liquid level. This can prevent a solution as a suction target from scattering. In addition, since suction can be performed while a solution level is checked, the solution can be reliably sucked.
Since the coupling portion 173 and the solution sucking portion 174 contain black carbon, light can be blocked.
The present embodiment is directed to another examination apparatus using the reaction vessel according to the first embodiment.
As shown on the left side of
The pressure controller 213 applies a negative pressure to the conduit 212, and a change in the pressure of the conduit 212 is measured by a pressure gauge while a sample solution is sucked from a sample vessel 114. As shown on the middle of
According to this embodiment, since a sample solution can be driven (sucked and moved) by the pressure controller 213 comprising only a negative pressure source, the apparatus arrangement can be simplified. In addition, since there is no pressure difference between the two sides of the three-dimensional substrate 102 in a steady state, the three-dimensional substrate 102 is hard to crack.
The present embodiment is directed to another reaction vessel which can be used in place of the reaction vessel according to the first embodiment.
As shown in
The three-dimensional substrate 222 is equivalent to the three-dimensional substrate 102 in the first embodiment, and the fitting portion 224 and 223 are respectively equivalent to the coupling portion 103 and the solution sucking portion 104 in the first embodiment. A detailed description of them will be omitted. The filter 225 has a function of filtering a solution passing therethrough.
When a solution is sucked into the reaction vessel 221, the solution passes through the filter 225 to be filtered.
In this embodiment, since the reaction vessel 221 comprises the filter 225, there is no need to filter a solution to remove dust before it is used.
The filter 225 preferably has a hole having a diameter almost equal to that of the three-dimensional substrate 222, and is more preferably made of the same material as that of the three-dimensional substrate 222. In this case, adhesion of dust to the three-dimensional substrate 222 can be prevented more properly.
The present embodiment is directed to another examination apparatus using the reaction vessel according to the first embodiment.
As shown in
A sample vessel 114 storing a sample solution is set at a predetermined position. While the valve 237 is closed, a reaction vessel 101 is mounted on the fitting portion 231. The reaction vessel 101 is lowered toward the sample vessel 114 by a direct-acting mechanism (not shown), and the distal end of the reaction vessel 101 is stopped at a position where the solution stored in the sample vessel 114 can be sucked. A negative pressure is transferred from the pressure controller 234 to the solution through the pressure transfer conduit 232 to suck a predetermined amount of solution into the reaction vessel 101. The reaction vessel 101 is then pulled upward from the sample vessel 114 by the direct-acting mechanism (not shown) to separate the reaction vessel 101 from the solution level. The sample solution is moved up and down through a three-dimensional substrate 102 by a predetermined number of times by interacting positive and negative pressures using the pressure controller 234.
A positive pressure is applied to the sample solution by using the pressure controller 234 to move the sample solution below the three-dimensional substrate 102. After the valve 233 is opened to be released to atmospheric pressure, a cleaning fluid from the cleaning fluid tank 236 is supplied from above the three-dimensional substrate 102 by opening the valve 237 to pressurize the cleaning fluid tank 236 by using the pump 239. The valve 237 is closed, and a positive pressure is applied to the reaction vessel 101 by using the pressure controller 234, moving the sample solution below the three-dimensional substrate 102, together with the cleaning fluid. When cleaning is to be repeated, the discharge of the solution and the supply of the cleaning fluid are alternately executed.
As described in the first embodiment, in order to wash off the nucleic acid which has not been used for a hybridization reaction, a sample solution must be discharged from the reaction vessel. Assume however that the surface tension of each kind of solution (sample solution or cleaning fluid) with respect to a three-dimensional substrate is large. In this case, when the solution is to be discharged by applying a positive pressure from the fitting portion, the solution does not move due to the surface tension as an end face of the three-dimensional substrate coincides with the solution level of the solution. For this reason, if the surface tension is large, since the sample solution cannot be discharged from the reaction vessel by a pressure from above, the nucleic acid cannot be washed off. According to this embodiment, however, since a cleaning fluid is supplied from above, the sample solution can be washed off while being sequentially diluted with the cleaning fluid.
The present embodiment is directed to driving of the reaction apparatus according to the first embodiment. The apparatus arrangement of this embodiment is therefore the same as that of the first embodiment.
In the first embodiment, the reaction apparatus and detection apparatus which constitute the examination apparatus are formed as discrete apparatuses. For this reason, after the end of a reaction, the reaction vessel 101 needs to be detached from the fitting portion 111 of the reaction apparatus.
In this embodiment, when a reaction vessel 101 is detached from a fitting portion 111 of the reaction apparatus, the pressure above a three-dimensional substrate 102 is made positive by a pressure controller 112. That is, the pressure is set to be equal to or more than the pressure given by P1=(V0+V1)/V1*P0 where V1 is a change in volume until the pressure is released to atmospheric pressure. Thereafter, the reaction vessel 101 is detached, and imaging is performed by the detection apparatus.
According to this embodiment, when the reaction vessel 101 is detached, a solution does not move above the three-dimensional substrate 102. For this reason, imaging is performed by the detection apparatus while the solution is held below the reaction vessel 101, and hence an imaging state is not disturbed by the solution level. As a consequence, a signal with less noise can be acquired.
The present embodiment is directed to another examination apparatus using the reaction vessel according to the first embodiment.
As shown in
As shown in
The reaction vessel transfer guide 247 includes a rack 261 attached on the arm 182, a pinion 262 engaged with the rack 261, a rack 263 engaged with the pinion 262, a solution receiver 265 which is coupled to the lower end portion of the rack 263 through a hinge 264, and a pin 266 for guiding the solution receiver 265.
The reaction vessel 101 mounted on the fitting portion 251 is moved up and down by the control motor 185 as in the fifth embodiment. Upon the vertical movement of the fitting portion 251, the rack 261 attached on the arm 182 which supports the fitting portion 251 moves up and down. Upon the vertical movement of the rack 261, the rack 263 moves up and down in the reverse direction to the rack 261. In addition, upon the vertical movement of the rack 263, a portion of the solution receiver 265 which is coupled to the hinge 264 moves up and down while being guided by the pin 266.
As shown in
As the reaction vessel 101 is lowered, the rack 261 moves down, the rack 263 moves up, and the solution receiver 265 moves up while tilting. As shown in
The solution receiver 265 preferably has a shape that prevents a stored solution from dripping off in the state shown in
According to this embodiment, since a solution which drips off from the reaction vessel 101 is stored in the solution receiver 265, the examination apparatus is not contaminated with the solution.
The present embodiment is directed to another examination apparatus using the reaction vessel according to the first embodiment.
The present embodiment is basically similar to the second embodiment. A fitting portion 271 on which a reaction vessel 101 is to be mounted includes an observation optical system for optically observing a three-dimensional substrate 102. That is, as shown in
As shown in
The reaction vessel 101 is mounted on the fitting portion 271. A negative pressure is generated by the pressure controller 279, and the valve 280 is opened to suck a sample solution from a sample vessel (not shown) into the reaction vessel 101. The sample solution is moved up and down through the three-dimensional substrate 102 by interacting positive and negative pressures using the pressure controller 279. After the end of the solution driving operation, the valve 280 is opened to release the reaction vessel 101 to atmospheric pressure.
Subsequently, in order to clean the lower end portion of the fitting portion 271, the valve 283 is opened, and the cleaning fluid tank 282 is pressurized by the pump 285. A cleaning fluid is then fed from the cleaning fluid tank 282 to the fitting portion 271. The cleaning fluid fed to the fitting portion 271 is sprayed against the lower end portion of the fitting portion 271 by the cleaning nozzle 286, cleaning the lower end portion of the fitting portion 271.
After the cleaning operation, the valve 283 is closed. The valve 280 is then opened, and a positive pressure is generated by the pressure controller 279 to move the cleaning fluid below the three-dimensional substrate 102. Thereafter, an image is acquired by the CCD 273.
According to this embodiment, even if the imaging lens 272 becomes fogged or stained, imaging can be stably performed by cleaning the imaging lens 272.
The present embodiment is directed to another examination apparatus using the reaction vessel according to the first embodiment.
As shown in
In this embodiment, the housing of the reaction vessel 101, i.e., a coupling portion 103 and a solution sucking portion 104, are made of PP, whose linear expansion coefficient is about 6×10−5. The fitting portion 111 is made of high-density PE, whose linear expansion coefficient is about 1×10−4. That is, the linear expansion coefficient of the fitting portion 111 is larger than that of the coupling portion 103 of the reaction vessel 101.
When the reaction vessel 101 is heated by the heater 291, the fitting portion 111 is also heated. With this heating, the coupling portion 103 of the reaction vessel 101 expands, and the diameter of its opening end increases. However, since the linear expansion coefficient of the fitting portion 111 is larger than that of the coupling portion 103, the fitting portion 111 expands more than the opening end.
According to this embodiment, therefore, while the temperature of the reaction vessel 101 is regulated, the reaction vessel 101 and the fitting portion 111 are kept in an airtight fashion.
The present embodiment is directed to another examination apparatus using the reaction vessel according to the first embodiment.
As shown in
In this embodiment, as in the 12th embodiment, the housing of the reaction vessel 101, i.e., a coupling portion 103 and a solution sucking portion 104, are made of PP, whose linear expansion coefficient is about 6×10−5. The fitting portion 301 is made of stainless steel, whose linear expansion coefficient is about 7×10−6. Therefore, the linear expansion coefficient of the fitting portion 301 is smaller than that of the coupling portion 103 of the reaction vessel 101.
In this embodiment, the temperature of the fitting portion 301 is controlled by the temperature control device 304 so as to be always higher than the temperature of the reaction vessel 101. Preferably, the temperatures of the fitting portion 301 and the coupling portion 103 of the reaction vessel 101 are controlled so that their linear expansion amounts are almost equal to each other. For example, in order to control the temperature of the reaction vessel 101 to a temperature T optimal for a hybridization reaction of a gene, the temperature of the reaction vessel 101 is controlled by the temperature control device 304. In addition, the temperature of the fitting portion 301 is controlled to be higher than that of the reaction vessel 101 by 10° C. by the temperature control device 304.
According to this embodiment, since the temperatures of the fitting portion 301 and a reaction vessel 101 are controlled by the temperature control device 304 so that their linear expansion amounts become almost equal to each other, the reaction vessel 101 and the fitting portion 301 of the coupling portion 103 are properly kept in an airtight fashion.
The present embodiment is directed to an examination apparatus using the reaction vessel according to the first embodiment.
As shown in
In this embodiment, the housing of the reaction vessel 101, i.e., the coupling portion 103 and the solution sucking portion 104, are made of PP, and the fitting portion 311 is made of stainless steel. The fitting portion 311 has an inner fitting surface facing the inner surface of the coupling portion 103 of the reaction vessel 101 and an outer fitting surface facing the outer surface of the coupling portion 103 of the reaction vessel 101.
A case wherein the temperature of a sample solution is controlled to be higher than a reference temperature will be described below.
The reaction vessel 101 in which a sample solution is sucked by a pressure controller (not shown) is set in the heater 291. A target temperature is set in the temperature control device 293. The temperature control device 293 energizes the heater 291 until the set temperature is reached on the basis of temperature information from the temperature sensor 292. The temperatures of the reaction vessel 101, sample solution, and fitting portion 311 are simultaneously regulated by heat conduction from the housing of the reaction vessel 101, i.e., the coupling portion 103 and the solution sucking portion 104.
With this temperature regulating, the coupling portion 103 of the reaction vessel 101 expands. Owing to this expansion, the wall of the coupling portion 103 increases in thickness. In addition, owing to the difference in linear expansion coefficient between the coupling portion 103 and the fitting portion 311, the inner surface of the coupling portion 103 tries to separate from the inner fitting surface of the fitting portion 311. However, before the inner surface of the coupling portion 103 separates from the inner fitting surface of the fitting portion 311, the outer surface of the coupling portion 103 comes into contact with the outer fitting surface of the fitting portion 311 because of increases in the diameter of the opening end of the coupling portion 103 and the thickness of the wall.
In other words, the coupling portion 103 and the fitting portion 311 are designed such that the outer surface of the coupling portion 103 comes into contact with the outer fitting surface of the fitting portion 311 before the inner surface of the coupling portion 103 separates from the inner fitting-surface of the fitting portion 311.
For this reason, according to this embodiment, the coupling portion 103 of the reaction vessel 101 and the fitting portion 311 are always kept in an airtight fashion.
The present embodiment is directed to another examination apparatus using the reaction vessel according to the first embodiment.
As shown in
The sample supply unit 322 sequentially supplies, for examination, a rack 321 housing a plurality of sample vessels in which sample solutions are respectively dispensed. As shown in
As shown in
Each reaction vessel housing portion 337 has a housing space for housing the reaction vessel 101. The housing space is in fluid communication, through a conduit, with a pressure source (not shown) placed below the base 331. The reaction vessel housing portion 337 includes a lid 338 which is openable and closable. The lid 338 includes an optical window 339 which allows optical observation from above using the detection apparatus 330.
The turntable 324 further comprises lid opening/closing mechanisms 341 for opening and closing the lids 338 of the reaction vessel housing portions 337 and a fixed disk 335 which supports the lid opening/closing mechanisms 341. The fixed disk 335 is fixed to the base 331 and does not rotate. As shown in
As shown in
Although not shown, as in the embodiments described above, the fitting portion 351 is in fluid communication with a pressure controller through a conduit, and the sampling device 325 can supply positive and negative pressures to the reaction vessel 101 mounted on the fitting portion 351.
With this arrangement, in
The first solution rack 326 and the second solution rack 328 are substantially identical structures, and store solutions necessary for examination. For example, in each rack, a vessel in which a reagent necessary for examination is dispensed is stored at a proper temperature and humidity. The vessel for the necessary reagent is placed at a suction position.
The first dispenser 327 and the second dispenser 329, which are substantially identical structures, can dispense solutions from the first solution rack 326 and the second solution rack 328 into the reaction vessel 101, respectively.
The detection apparatus 330 includes, for example, a fluorescence microscope, and can sense a fluorescent image of the three-dimensional substrate in the reaction vessel 101.
The examination device 320 of this embodiment operates as follows.
First of all, the reaction vessel 101 supplied from the reaction vessel supply device 323 is mounted on the fitting portion 351 of the sampling device 325. The sampling device 325 sucks a sample solution from the sample rack 321 of the sample supply unit 322 into the reaction vessel 101, and places the reaction vessel 101, in which the sample solution is sucked, in the reaction vessel housing portion 337 of the turntable 324. The lid 338 of the reaction vessel housing portion 337 is then closed by the lid opening/closing mechanism 341.
The reaction vessel 101 housed in the reaction vessel housing portion 337 sequentially passes through the first dispenser 327, second dispenser 329, and detection apparatus 330 upon rotation of the turntable 324, and finally returns to the sampling device 325.
While the reaction vessel 101 is transferred from the sampling device 325 to the first dispenser 327, the sample solution in the reaction vessel 101 is driven by repeatedly applying positive and negative pressures to the reaction vessel housing portion 337, executing a complement fixation reaction.
When the reaction vessel 101 reaches the first dispenser 327, the lid 338 of the reaction vessel housing portion 337 is opened by the lid opening/closing mechanism 341. A cleaning fluid is dispensed into the reaction vessel 101 by using the first dispenser 327, and the sample solution is washed away by repeatedly applying positive and negative pressures to the reaction vessel housing portion 337. Thereafter, the lid 338 of the reaction vessel housing portion 337 is closed by the lid opening/closing mechanism 341.
When the reaction vessel 101 reaches the second dispenser 329, the lid 338 of the reaction vessel housing portion 337 is opened by the lid opening/closing mechanism 341. A chemiluminescent substrate is dispensed from the second dispenser 329 into the reaction vessel 101, and is promoted by repeatedly applying positive and negative pressures to the reaction vessel housing portion 337. Thereafter, the chemiluminescent substrate is washed away with a cleaning fluid, and the lid 338 of the reaction vessel housing portion 337 is closed by the lid opening/closing mechanism 341.
When the reaction vessel 101 reaches the detection apparatus 330 afterward, an image of a three-dimensional substrate in the reaction vessel 101 is sensed by the detection apparatus 330, and a luminance at each spot on the three-dimensional substrate is measured by image analysis software.
When the reaction vessel 101 having undergone the measurement reaches the sampling device 325, the sampling device 325 extracts the reaction vessel 101 from the reaction vessel housing portion 337 of the turntable 324, and discards the reaction vessel 101 into a discard hole 363 shown in
According to this embodiment, since no apparatus for handling the reaction vessel 101 is required, the examination apparatus can be reduced in size.
The present embodiment is directed to a method of manufacturing a new reaction vessel for detecting biorelated substances appearing in the embodiments described above. Assume that in the embodiment described below, a nylon filter is applied to a three-dimensional substrate.
First of all, as shown in
As shown in
Subsequently, as shown in
As shown in
In addition, an acrylic adhesive 451 (see
As shown in
An acrylic adhesive 452 (see
Finally, as shown in
Referring to
When the coupling portion 430, three-dimensional substrate 440, and solution sucking portion body 410 are manufactured such that when they are properly abutted against the abutment jig 463, the center of the through hole 433, the center of the spot region 444, and the center of the through hole 413 are aligned with each other. Since the probe spots are stably placed at the same positions, the arrangement of the probe spots can be easily checked.
The present embodiment is directed to another method of manufacturing a reaction vessel. The manufacturing method of this embodiment will be described below with reference to FIGS. 29 to 31. The same reference numerals as in the 16th embodiment denote the same members in FIGS. 29 to 31, and a detailed description thereof will be omitted.
First of all, as shown in
As shown in
Subsequently, for example, 100 kinds of nucleic acid probes are spotted at predetermined positions on the nylon filter 441 by the inkjet printer to form a spot region. With this process, a three-dimensional substrate 440 on which probes are immobilized is manufactured. For example, the spot diameter and the spot intervals are adjusted to 100 μm and 200 μm, respectively.
As shown in
Finally, as in the 16th embodiment, a tapered cylindrical member 420 is bonded to a truncated cone portion 412 of the solution sucking portion body 410, or fixed thereto by fitting if the cylindrical member 420 is to be detachable, completing a reaction vessel similar to the reaction vessel shown in
The reaction vessel manufactured in this embodiment is the same as that manufactured in the 16th embodiment except that no mark is formed on the nylon filter 441.
The present embodiment is directed to another method of manufacturing a reaction vessel. The manufacturing method of this embodiment will be described with reference to FIGS. 32 to 34. The same reference numerals as in the 16th embodiment denote the same members in these drawings, and a detailed description thereof will be omitted.
First of all, as shown in
As shown in
Subsequently, for example, 100 kinds of nucleic acid probes are spotted at predetermined positions on the nylon filter 441 by the inkjet printer to form a spot region. With this process, a three-dimensional substrate 440 on which probes are immobilized is manufactured. For example, the spot diameter and the spot intervals are adjusted to 100 μm and 200 μm, respectively.
As shown in
Finally, as in the 16th embodiment, a tapered cylindrical member 420 is bonded to a truncated cone portion 412 of the solution sucking portion body 410, or fixed thereto by fitting if the cylindrical member 420 is to be detachable, completing a reaction vessel similar to the reaction vessel shown in
The reaction vessel manufactured in this embodiment is the same as that manufactured in the 16th embodiment except that no mark is formed on the nylon filter 441.
In the 16th, 17th, and 18th embodiments, an acrylic adhesive is used to join the nylon filter 441 to the solution sucking portion body 410 and join the nylon filter 441 to the coupling portion 430. However, a means other than an acrylic adhesive, e.g., ultrasonic welding, heat welding, or laser welding, may be used.
The present embodiment is directed to another method of manufacturing a reaction vessel. The manufacturing method of this embodiment will be described below with reference to FIGS. 32 to 34.
First of all, as shown in
Referring to
As shown in
Subsequently, for example, 100 kinds of nucleic acid probes are spotted at predetermined positions on the nylon filter 541 by the inkjet printer to form a spot region 544 (see
The three-dimensional substrate chip 540 is then attached on a solution sucking portion body 510 shown in
Attachment of the three-dimensional substrate chip 540 on the solution sucking portion body 510 is performed by placing an O-ring 551 on the bottom surface of the recess portion 514 of the solution sucking portion body 510 and placing the three-dimensional substrate chip 540 on the O-ring.
Subsequently, the coupling portion 530 shown in
The coupling portion 530 is attached on the solution sucking portion body 510 as follows. An O-ring 552 (see
As shown in
Finally, as shown in
Referring to
The coupling portion 530, three-dimensional substrate chip 540, and solution sucking portion body 510 are manufactured so that when they are assembled in accordance with a proper positional relationship, the center of the through hole 533, the center of the spot region 544, and the center of the through hole 513 are aligned with each other.
According to the relationships of R1>R2 and R5>R4, the three-dimensional substrate chip 540 and the solution sucking portion body 510 are kept in an airtight fashion by the O-ring 551, and the three-dimensional substrate chip 540 and the coupling portion 530 are kept in an airtight fashion by the O-ring 552.
In addition, according to the relationships of R4>R2 and R5>R4, when the spots on the three-dimensional substrate chip 540 are observed, the protective member 545, the adhesive 549 applied to the protective member 545, and the solution sucking portion body 510 are not observed.
The reaction vessel manufactured in this embodiment is substantially the same as that manufactured in the 16th embodiment.
In the embodiments described above, the members constituting the reaction vessel are fixed by an adhesive, fitting, and screws. However, fixing methods to be used are not limited to them as long as the reaction vessel can be kept in an airtight fashion in which a solution can be sucked and discharged. For example, the members constituting the reaction vessel can be fixed by using heat welding or ultrasound welding as another method.
When a tapered cylindrical member is fitted on the truncated cone portion of the solution sucking portion body or fixed thereto with screws so as to be detachable, detection can be performed after the tapered cylindrical member is detached upon completion of a reaction or a cleaning process. This arrangement is especially effective when the reaction apparatus and the detection apparatus are discrete apparatuses, allowing the size of the detection apparatus to be reduced. In addition, when the coupling portion is detached from the plate-like portion, there is no part which interferes with the three-dimensional substrate, so that scanning with plate-like portions aligned can detect a reaction result at higher speed.
Although the embodiments of the present invention have been described with reference to the accompanying drawings, the present invention is not limited to these embodiments. The embodiments can be variously modified and changed within the spirit and scope of the invention.
The first embodiment has exemplified the case wherein a biorelated substance is labeled by using a fluorescent dye. However, various detection methods and labels can be applied to the present invention. When detection is to be performed by a chemiluminescence method, since luminescence is caused by a reaction between an enzyme and a substrate, it is not necessary to use any light source which illuminates a sample with light. In addition, even when detection is to be performed with fluorescence, various kinds of fluorescent materials can be used as labels. In addition to various kinds of fluorescent dyes, fluorescent glass particles and the like can be used. Furthermore, when detection is to be performed with scattered light or reflected light, metal particles or dielectric particles are used as a label. For example, fine gold, silver, platinum, and silicon particles and latex particles can be used. Metal fine particles such as fine gold, silver, or platinum particles with a particle size of 10 to 100 nm are especially preferable because the velocity of a particle in motion becomes optimal. Likewise, latex particles with a particle size of 0.1 to 1 μm are especially preferable because the velocity of a particle in motion becomes optimal. A suitable particle diameter is determined by the specific gravity of a particle and the velocity of Brownian motion. In this case, the motion state of particles includes is, for example, Brownian motion or vibration.
Additional advantages and modifications will readily occur to those skilled in the art. Therefore, the invention in its broader aspects is not limited to the specific details and representative embodiments shown and described herein. Accordingly, various modifications may be made without departing from the spirit or scope of the general inventive concept as defined by the appended claims and their equivalents.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2003-405789 | Dec 2003 | JP | national |
This is a Continuation Application of PCT Application No. PCT/JP2004/018063, filed Dec. 3, 2004, which was published under PCT Article 21(2) in Japanese. This application is based upon and claims the benefit of priority from prior Japanese Patent Application No. 2003-405789, filed Dec. 4, 2003, the entire contents of which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/JP04/18063 | Dec 2004 | US |
| Child | 11445999 | Jun 2006 | US |
