Automatic analysis method and apparatus for enzyme reaction

• US 4,835,707 A
• Filed: 07/22/1987
• Issued: 05/30/1989
• Est. Priority Date: 07/23/1986
• Status: Expired due to Fees

• Current Assignee: Takeda Chemical Industries, Ltd. (Takeda Pharmaceutical Company Limited)
• Original Assignee: Takeda Chemical Industries, Ltd. (Takeda Pharmaceutical Company Limited)
• Inventors: Amano, Toshio, Imada, Isuke
• Primary Examiner(s): Lall, Parshotam S.
• Assistant Examiner(s): Teska, Kevin J.
• Application Number:
  US07/076,720
• Time in Patent Office: 678 Days
• Field of Search: 364/496, 364/497, 364/500, 364/513, 364/413, 73/863.01, 73/863.25, 73/863.32, 73/864.81, 422/62, 422/67
• US Class Current: 700/266
• CPC Class Codes: C12Q 1/00 Measuring or testing processes involving enzymes, nucleic acids or microorganisms measuring or testing apparatus with condition measuring or sensing means, e.g. colony counters, C12M1/34; Compositions therefor; Processes of preparing such compositions
• CPC Class Codes: G01N 2035/00217 involving measurement of weight
• CPC Class Codes: G01N 2035/00316 Detecting door closure
• CPC Class Codes: G01N 2035/00326 Analysers with modular structure
• CPC Class Codes: G01N 2035/00396 where the fluid is a liquid
• CPC Class Codes: G01N 2035/0097 monitoring reactions as a function of time
• CPC Class Codes: G01N 2035/1032 Dilution or aliquotting
• CPC Class Codes: G01N 30/06 Preparation
• CPC Class Codes: G01N 30/24 Automatic injection systems
• CPC Class Codes: G01N 35/0099 comprising robots or similar manipulators robots per se B25J

Abstract

An automatic analysis method of and apparatus for the full automation of an enzyme reaction analysis from the pretreatment step of the reaction to the data processing step, wherein the operation of the pretreatment step is arranged to be sequentially performed on many samples with full automation by the use of robots and computers to improve savings efficiency and measurement accuracy. Also, the pretreatment steps of weighing samples in many sample tubes, adding the given amount of solvent corresponding to the weighing value and placing the samples into the dissolution vessel to dissolve the sample in the solvent are adapted to be sequentially performed with full automation with the use of the robot, computer and electronic balance. Furthermore, the filtration, concentration and the injecting operations into the HPLC are automatically performed. The automatic apparatuses are coupled to each other so that the filtration, concentration, injecting operations are allowed to be sequentially performed with full automation using an on-line system.

Claims

1. An apparatus for automatically analyzing the effect of a sample on an enzyme, said apparatus comprising:

weighing means for weighing a sample;

dissolving solvent adding means for adding solvent to the sample;

dissolving means for facilitating the dissolution of the sample in the solvent to produce a sample solution;

dilution solvent adding means for adding solvent to the sample solution to produce a diluted sample solution;

diluted sample solution pipetting means for injecting at least a portion of the diluted sample solution into a reaction tube;

enzyme adding means for adding an enzyme into the reaction tube into which the diluted sample solution is injected by said diluted sample solution pipetting means;

substrate adding means for adding a substrate of the enzyme into the reaction tube to obtain a reaction mixture in which an enzyme reaction takes place;

a preservation vessel for preserving the reaction mixture;

moving means for moving the reaction tube into said preservation vessel to preserve the reaction mixture;

filtering means for filtering the reaction mixture as preserved by the preservation vessel to produce a filtrate of the reaction mixture;

concentrating means for concentrating the filtrate to produce a concentrated filtrate of the reaction mixture;

analysis means for producing data based on an analysis of the concentrated filtrate of the reaction mixture;

filtrate injecting means for injecting the concentrated filtrate of the reaction mixture into the analysis means to allow the analysis means to produce data based on the analysis of the concentrated filtrate;

data processing means for processing the data based on the analysis of the concentrated filtrate into information indicative of the effect of the sample on the enzyme reaction; and

control means operatively connected to said dissolving and said dilution adding means, said sample solution pipetting means, said enzyme adding means, said substrate adding means, said moving means and said filtrate injecting means for automatically controlling the adding of the solvent by said dissolving solvent adding means and by said dilution solvent adding means, the injection of the diluted sample solution by said diluted sample solution pipetting means, the adding of the enzyme by said enzyme adding means, the adding of the substrate by said substrate adding means, the movement of said moving means, and the injection of the concentrated filtrate by said filtrate injecting means.

2. An apparatus according to claim 1,

wherein said weighing means is an electronic balance,

said dissolving solvent adding means is a solvent dispenser connectable to a source of solvent for dispensing solvent,

said dissolving means is a dissolution vessel having an ultrasonicator,

and further comprising a sample rack for supporting sample test tubes containing the sample, and a first robot having a movable arm equipped with a chuck,

said control means operatively connected to said first robot for controlling said robot to grasp a sample tube supported by said sample rack with said chuck, and to sequentially move the grasped sample tube, via said movable arm, onto said electronic balance, to said solvent dispenser and to said dissolution vessel;

wherein said dilution solvent adding means is a dilution dispenser including a dilution nozzle connectable to a source of solvent for dispensing solvent therefrom,

said diluted sample solution pipetting means is a sample pipetter for pipetting diluted sample solution,

said enzyme adding means is an enzyme dispenser including an enzyme injection nozzle connectable to a source of enzyme for dispensing enzyme therefrom,

said substrate adding means is a substrate dispenser including a substrate injection nozzle connectable to a source of substrate for dispensing substrate therefrom,

said preservation vessel includes a preservation rack for supporting a plurality of said reaction tubes,

said moving means is a second robot including a chuck,

and further comprising a dilution rack for supporting a plurality of sample tubes, and an incubator including an incubation rack for supporting a plurality of reaction tubes,

said control means operatively connected to said second robot for controlling said second robot to grasp said dilution nozzle with the chuck thereof and move the same to said dilution rack, to grasp said sample pipetter with the chuck thereof and move the same between said dilution rack and said incubator rack, to grasp said enzyme injection nozzle with the chuck thereof and move the same to said incubator rack, to grasp said substrate injection nozzle with the chuck thereof and move the same to said incubator rack, and to grasp the reaction tubes supported by said incubator rack and move the same to said preservation rack; and

wherein said filtering means includes a web of filter material, a supply means from which said filter material is supplied, a take-up reel for receiving the filter material from said supply means and means for continuously winding said filter material around said take-up reel,

said concentrating means is a concentration column for absorbing the filtrate, said concentration column connected to said analysis means, and

said filtrate injecting means comprises a valve operatively connected between said concentration column and an eluting source of solvent, said valve movable to a position at which said eluting source of solvent is open to said concentration column for allowing the solvent, from said eluting source, to pass through said concentration column to elute the filtrate absorbed thereby and to force the eluted filtrate into said analysis means connected to said concentration column.

3. An automatic weighing and dissolution apparatus for automatically weighing and dissolving a sample with a solvent, said apparatus comprising:

a sample rack for supporting a plurality of sample tubes containing a sample;

an electronic balance;

solvent adding means for dispensing solvent;

a dissolution vessel for facilitating the dissolution of the sample in the solvent dispensed by said solvent adding means;

a robot having a movable arm including a chuck; and

a computer operatively connected to said electronic balance, said solvent adding means, said dissolution vessel and said robot for controlling said robot to move a sample tube supported by said sample rack to said electronic balance and to the solvent adding apparatus once weighed by said electronic balance, for controlling said solvent adding means to dispense an amount of solvent based on the weight of the sample contained in the sample tube, for controlling the robot to transfer the sample tube to said dissolution vessel once the solvent is added by said solvent adding means, and for controlling the operation of said dissolution vessel once the sample tube has been transferred thereto by said robot.

4. An apparatus according to claim 3, wherein the movable arm of said robot is displaceable along a horizontal plane and has a plurality of points about which portions of said movable arm are pivotable.

5. An apparatus according to claim 3, wherein said solvent adding means includes a plurality of dispensers for dispensing a plurality of different types of solvents, respectively.

6. An apparatus according to the claim 3, wherein said dissolution vessel includes a heater comprising an ultrasonicator, said computer operatively connected to said heater for controlling the operation of said heater.

7. An automatic dilution and reacting apparatus for diluting a sample solution and facilitating an enzyme reaction within said sample solution, said apparatus comprising:

a dilution rack for supporting a plurality of sample tubes containing a sample solution;

a dilution dispenser including a dilution nozzle mounted on flexible piping and connectable to a source of solvent for dispensing solvent therefrom, and a dilution nozzle stand for supporting the dilution nozzle;

an incubator, said incubator having support means for supporting a plurality of reaction tubes therein;

a sample pipetter having a pipetter nozzle and a pipetter stand for supporting said pipetter nozzle;

a plurality of different reaction mixture dispensers for dispensing different components of a reaction mixture;

each of said reaction mixture dispensers including a reaction nozzle mounted on flexible piping and connectable to a respective source of the components, and reaction nozzle stands for supporting said reaction nozzles;

at least one washing vessel for rinsing at least one of said nozzles;

a robot having a movable arm including a chuck; and

a controller operatively connected to said robot for controlling said robot to grasp said nozzles with said chuck and move the same between said stands and said racks, respectively, and to said at least one washing vessel, in a manner in which the sample solution is diluted in the sample tube supported in said dilution rack by solvent dispensed by said dilution dispenser via said dilution nozzle grasped by said robot and moved to said sample rack to produce diluted sample solutin, the diluted sample solution is withdrawn from the sample tubes by said sample pipetter and is dispensed thereby into the reaction tubes supported by said support means of said incubator, and the components of the reaction are dispensed into said reaction tubes via said reaction nozzles grasped by said robot and moved to said support means of said incubator.

8. An apparatus according to claim 7, and further comprising a preservation vessel for preserving the reaction mixture after the reaction components are introduced into the reaction tubes,

said controller further controlling said robot to grasp said reaction tubes supported by said support means of said incubator, once the reaction components are introduced therein, and to move the reaction tube to said preservation vessel.

9. An apparatus according to claim 7, wherein the movable arm of said robot is displaceable along a horizontal plane and has a plurality of joints about which portions of said movable arm are pivotable.

10. An apparatus according to claim 7, wherein each of said dispensers comprises a digital diluter pipetter,

each said flexible piping comprises a coil-shaped teflon tube,

and said nozzles are integral with said teflon tubes at tip ends thereof, respectively.

11. An apparatus according to claim 7, wherein said incubator includes a magnetic stirrer for stirring the reaction mixture contained in reaction tubes supported thereon by said support means.

12. An apparatus according to claim 7,

wherein said reaction mixture dispenser comprises a plurality of independent dispensers each having a said reaction nozzle and each connectable to a respective one of the sources of the components, and

said controller further controls said robot to sequentially grasp and move the nozzles of said independent dispensers to said support means of said incubator during predetermined time intervals.

13. An apparatus according to claim 7, wherein said reaction mixture dispenser is a reaction mixture dispenser for dispensing at least enzyme solution.

14. An automatic filtration/concentration/injection (FCI) apparatus for filtering a solution to produce a filtrate, concentrating the filtrate to produce a concentrated filtrate, and injecting the concentrated filtrate to facilitate the analyis thereof, said apparatus comprising:

a sampling nozzle, and a syringe pump operatively connected to said sampling nozzle for drawing at least a portion of a reaction mixture through said sampling nozzle;

a filter unit disposed in the apparatus downstream of said sampling nozzle for filtering the reaction mixture drawn through said sampling nozzle;

said filter unit including filter paper, a supply means from which said filter paper is supplied, a take-up reel for receiving the filter from said supply means and means for continuously winding said filter paper around said take-up reel;

a first valve connected to a sampling loop and disposed in the apparatus downstream of said filter unit for receiving a predetermined volume of filtrate passing through said filter paper;

a concentration column for adsorbing the filtrate and disposed in the apparatus downstream of said first valve and said sampling loop;

a water pump operatively connected in the apparatus for pumping water and the filtrate in said sampling loop through said concentration column;

an analytical column for analyzing the reaction mixture and connected to said concentration column; and

eluting means for eluting the filtrate adsorbed in said concentration column and allowing the eluted filtrate to pass into said analytical column.

15. A method for automatically analyzing the effect of a sample on an enzyme, comprising the steps of:

weighing a sample,

adding a solvent to the sample,

dissolving the sample in the solvent to produce a sample solution,

dispensing further solvent to at least a portion of the sample solution to produce a diluted sample solution,

pipetting at least a portion of the diluted sample solution into a reaction tube,

dispensing enzyme and substrate of the enzyme into the reaction tube containing diluted sample solution to produce a reaction mixture,

moving the reaction tube into a preservation vessel capable of preserving a reaction to preserve the reaction mixture,

filtering the reaction mixture as preserved by the preservation vessel to produce a filtrate,

concentrating the filtrate to produce a concentrated filtrate of the reaction mixture,

injecting the concentrated filtrate into an analysis apparatus capable of analyzing solution to produce data based on the analysis of the concentrated filtrate, and

processing the data based on the analysis of the concentrated filtrate into information indicative of the effect of the sample on the enzyme,

wherein all the steps are performed automatically.

16. A method according to claim 15, wherein the steps of weighing the sample, adding solvent to the sample, and dissolving the sample in the solvent are automatically performed with a weighing and dissolution apparatus by

grasping a sample tube supported in a sample rack with a chuck provided on a movable arm of a first robot,

moving the sample tube to an electronic balance,

moving the sample tube to a solvent adding apparatus,

dispensing an amount of solvent into the sample tube from the solvent adding means based on the weight of the sample contained in the sample tube,

moving the sample tube to a dissolution vessel capable of facilitating dissolution of the sample in the solvent after the addition solvent to the sample tube to produce a sample solution, and

moving the sample tube containing the sample solution to a dilution rack in a dilution and weighing apparatus;

wherein the steps of dispensing further solvent to at least a portion of the sample solution, pipetting at least a portion of the diluted sample solution into a reaction tube, dispensing enzyme and substrate into the reaction tube, and moving the reaction tube into a preservation vessel are automatically performed with a dilution and reacting apparatus by

grasping a nozzle of a dilution dispenser with a chuck of a second robot,

moving the nozzle of the dilution dispenser to the sample tube supported in the dilution rack,

dispensing solvent from the dilution dispenser through the nozzle thereof to at least a portion of the sample solution to produce a diluted sample solution,

grasping a sampling pipetter with the chuck of the second robot,

pipetting at least a portion of the diluted sample solution with said sampling pipetter into a reaction tube supported in a reaction tube rack in an incubator,

grasping each respective nozzle of a plurality of reaction mixture dispensers for dispensing enzyme and substrate necessary for an enzyme reaction with the chuck of the second robot,

moving sequentially each of the nozzles of the reaction mixture dispensers to the reaction tube containing the diluted sample solution,

dispensing enzyme and substrate into the reaction tube from the reaction mixture dispensers and through the nozzle thereof at predetermined time intervals to produce a reaction mixture,

grasping the reaction tube with the chuck of the second robot,

moving the reaction tube into a preservation vessel capable of preserving the reaction mixture,

grasping the reaction tube in the preservation vessel, and

moving the reaction tube, once the reaction mixture is preserved, to a sampling nozzle of a filtration/concentration/injection apparatus capable of withdrawing at least a portion of the reaction mixture from the reaction tube;

wherein the steps of filtering the reaction mixture, concentrating the filtrate, and injecting the concentrated filtrate into an analysis apparatus are automatically performed with the filtration/concentration/injection apparatus by

drawing from the reaction tube at least a portion of the reaction mixture through the sampling nozzle into the filtration/concentration/injection apparatus,

filtering the reaction mixture drawn through the sampling nozzle with a filter unit disposed in the apparatus downstream of the sampling nozzle to obtain a filtrate,

concentrating the filtrate by adsorbing at least a portion of the filtrate on a concentration column disposed in the apparatus downstream of the filter unit,

eluting the filtrate adsorbed on the concentration column from said column with an eluting solvent to obtain a concentrated filtrate,

injecting the concentrated filtrate eluted from the concentration column into an anaylsis column of a high pressure liquid chromatograph disposed in the apparatus downstream of the concentration column to produce data based on the analysis of the concentrated filtrate,

and wherein the steps performed by the first and second robots are automatically controlled by a computer.

17. A method according to claim 15, wherein said step of dispensing enzyme comprises dispensing lipoxygenase.

18. A method according to claim 15, wherein said step of dispensing enzyme comprises dispensing cyclooxygenase.

19. A method according to claim 15, wherein said step of dispensing enzyme comprises dispensing lipoxygenase or cyclooxygenase, and said step of dispensing substrate comprises dispensing arachiodonic acid, and further comprising a step of dispensing ethanol.

20. A method of automatically weighing and dissolving samples, comprising grasping a sample tube retained in a sample rack with a chuck provided on a movable arm of a first robot,

moving the sample tube with said robot to an electronic balance,

moving the sample tube with said robot to a solvent adding apparatus,

dispensing an amount of solvent into the sample tube based on the weight of the sample contained in the sample tube,

moving the sample tube with said robot to a dissolution vessel capable of facilitating the dissolution of the samplein the solvent after the addition of solvent to the sample tube to produce a sample solution, and

moving the sample tube containing the sample solution with said robot to a dilution rack in a dilution and weighing apparatus,

wherein the operation of said robot, the determining of the amount of solvent to add to the sample based upon the weight of the sample, the adding the solvent into the sample tube, the operation of the electronic balance, the operation of the solvent adding apparatuses, and in the operation of the dissolution vessel are automatically controlled by a control means.

21. A method of diluting and reacting a sample, comprising grasping with a chuck of a robot a dilution nozzle connectable to a dilution dispenser with a flexible tube,

moving the dilution nozzle with said robot to a sample tube containing a sample solution supported in a dilution rack,

dispensing solvent into the sample solution in a manner which produces a diluted sample solution,

moving the dilution nozzle with said robot into a nozzle washing vessel capable of rinsing the nozzle to rinse the dilution nozzle,

grasping with the chuck of the robot a pipetter nozzle connectable to a sampling pipetter with a flexible tube,

moving the pipetter nozzle to the sample tube containing the diluted sample solution,

pipetting at least a portion of the diluted sample solution with said sampling pipetter into a reaction tube supported in a rack in an incubator,

moving the pipetter nozzle into the nozzle washing vessel to rinse the nozzle,

grasping with the chuck of the robot a plurality of a reaction mixture nozzles one by one, each nozzle connectable to a reaction mixture dispenser for dispensing a different one of reaction mixture components,

moving sequentially with said robot the respective reaction mixture nozzles to the reaction tube, and

injecting sequentially the respective reaction mixture components from said reaction mixture dispenser into the reaction tube to produce a reaction mixture,

wherein all the steps performed by the robot are automatically controlled by a computer.

22. A method of automatically filtering a reaction mixture, concentrating the filtrate, and injecting the concentrated filtrate into an analysis apparatus, said method comprising:

drawing a reaction mixture contained in a reaction tube through a sampling nozzle operatively connected to a syringe pump;

filtering the reaction mixture in a filtering unit disposed downstream of the sampling nozzle,

controlling a first valve connected to a sampling loop and disposed downstream of the filter unit to cause a predetermined volume of filtrate to be received therein from said filtering unit,

feeding the filtrate into a concentration column capable of adsorbing the filtrate,

feeding an eluting solvent into the concentration column to elute the filtrate adsorbed on the concentration column, and

injecting the eluted filtrate into an analytical column capable of analyzing the filtrate,

wherein the steps are performed automatically.