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Description  |
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Background of the Invention
The present invention relates generally to an enzymatic cycling technique
for analyzing a small or extremely small amount of a substance contained
in a sample and more particularly to an automatic enzymatic cycling
reaction apparatus and an automatic analyzing apparatus for automatically
analyzing a very small or extremely small amount of a substance in a
sample with the aid of the automatic enzymatic cycling reaction apparatus.
For instance, in the field of biochemistry, a small amount of a substance
contained in a sample is usually detected by a radio isotope method in
which the substance to be analyzed is marked by a radio isotope and then
is detected by a scintillation counter, a mass spectrometric method in
which a substance is labeled with a stable isotope and is detected by a
mass spectrometer and an immunological method in which a substance is
analyzed by utilizing antigen-antibody reaction for labeled substance.
In the radio isotopic method, since the radio isotope is used, it is
necessary to provide an apparatus which satisfies the safety standards for
the isotope and further in order to avoid the radioactive contamination
the treatment of wasted materials is very cumbersome. Further, the
operation might be subjected to the radioactivity. In the mass
spectrometric method using the stable isotope, since substances which can
be marked with the stable isotopes are limited, the number of items to be
tested is small. Further, since use is made of a mass spectrometer, it is
very cumbersome to evaporate the marked substance. In the immunological
method, there are a radio immuno assay using radio isotope markers, an
enzyme immuno assay using enzyme markers, and a fluoroimmuno assay using
fluorescent markers. In these methods it is necessary to mark antibody or
antigen effecting the antigen-antibody reaction. In the radio immuno assay
the same problems as those in the radio isotope method occur.
Recently, there has been proposed an enzymatic cycling method by means of
which a very small amount of a substance in a sample can be analyzed
without causing the above mentioned problems of the radioactive
contamination, the restriction of the number of test items, etc. In the
enzymatic cycling method, a substance is measured in a multiplying manner
by combining two enzyme reactions. Nowadays, the following three kinds of
cycling reactions have been performed as routine work.
__________________________________________________________________________
Multiplying Maximum
substrate multiplying
Name (coenzyme)
Cycling reaction enzyme
Excess substrate
Multiplied product
rate per
__________________________________________________________________________
hour
NAD cycling
NAD.sup.+
NADH
##STR1##
##STR2##
acetaldehyde
malate*
60,000
NADP cycling
NADP.sup.+
NADPH
##STR3##
##STR4##
6-P-gluconate*
glutamate
20,000
CoA cycling
CoASH
acetyl- CoA
##STR5##
##STR6##
phosphate
citrate*
37,500
__________________________________________________________________________
Substances marked by * are reacted with an indicator and then produced
fluorescent substances such as NADH and NADPH are measured.
##STR7##
##STR8##
##STR9##
##STR10##
- Now the principle of the cycling reaction will be explained with
reference to typical AND cycling. In the AND cycling, malate and
acetaldehyde are produced in a multiplying manner by the following
reaction.
##STR11##
At first, to a mixture of excess amounts of ethanol and oxalacetate are
added two kinds of enzymes, i.e. alcohol dehydrogenase and malate
dehydrogenase of given concentrations to form a cycling mixture. To the
cycling mixture thus formed is added a very small amount of NAD.sup.+
(nicotiramide adenine dinucleotide oxidation type) which is a kind of a
coenzyme.
Then one molecule of NAD.sup.+ is reduced by the catalytic action of the
alcohol dehydrogenase using the ethanol as substrate to produce one
molecule of acetaldehyde and one molecule of NADH. Then, one molecule of
NADH thus produced is oxidized by the catalytic action of the malate
dehydrogenase using the oxalacetate as substrate to produce one molecule
of NAD.sup.+ and one molecule of malate. Therefore, when the cycle
reaction is repeated by 1,000 times, there are produced 1,000 molecules of
acetaldehyde and malate although the initial liquid contains only one
molecule of NAD.sup.30.
To the mixture containing the acetaldehyde and malate produced in the
multiplying mode are added an excess amount of NAD.sup.+ and a given
amount of malate dehydrogenase to effect the following indicator reaction.
##STR12##
In this manner, the accumulated or multiplied malate is transferred
quantitatively into fluorescent NADH. Therefore, by measuring the
intensity of fluorescent light emitted from the excited NADH, it is
possible to measure a very small amount of NAD.sup.+ in a sample with the
aid of a calibration curve relating the fluorescent light intensity to the
concentration of NAD.sup.+. In the NAD cycling, NADH is simultaneously
produced in the multiplying manner. Therefore, NADH may be measured in the
same manner as that explained above.
In the NADP cycling and CoA cycling, the multiplying reaction is carried
out in the similar manner to that explained above.
By means of the enzymatic cycling method, it is also possible to measure
substances which can be transferred into the multiplying substrate such as
NAD.sup.+, NADH, NADP and CoA. For instance, a very small amount of
ethanol contained in a blood serum may be measured in the following
manner. At first, the ethanol in the serum sample is transferred
quantitatively into NADH under the existence of an excess amount of
NAD.sup.+ in accordance with the following transfer reaction, while
alcohol dehydrogenase is used as a catalyst.
##STR13##
Next, the solution is heated to, for instance 70.degree. C., while a pH
value of the solution is adjusted to 11 to 12. During this treatment,
NAD.sup.+ remained in the solution is destroyed. Then, the above explained
NAD cycling reaction is carried out, while NADH remained in the solution
is used as multiplying substrate. In this manner, a very small amount of
ethanol may be measured accurately by the enzymatic cycling method.
Most of substances of living bodies or substances produced by enzyme
reactions in the living bodies may be transferred into multiplying
substrates in the cycling reactions, and therefore the enzymatic cycling
method is very effective for measuring various substances by utilizing
specificities of various enzymatic reactions.
Nowadays the enzymatic cycling method has been used to analyze various
substances such as glucides and their intermediary metabolites, amino
acids and their relating substances, some kinds of lipids (glucide
phospholipid) and substances relating to nucleotide, and to effect the
enzyme assay for various kinds of enzymes relating to metabolism. For
instance, in case of analyzing an amniotic fluid extracted from a pregnant
woman, it is possible to diagnose congenital metabolisms of fetus such as
Krabbe's disease, galactosemia, G.sub.Ml -gangliosidosis and Fabry's
disease.
As explained above, by utilizing the transfer reactions for transferring
substances to be analyzed into the multiplying substrates (coenzymes), the
enzymatic cycling method can afford the measurement of extremely small
amounts of substances in the multiplying mode. Therefore, the enzymatic
cycling method can be applied not only to biochemistry and medicine, but
also to a broader sense biology including biochemistry, physiology and
cell biology, pharmacology, agricultural chemistry and chemical analysis.
In the medical field, since a sample amount is extremely small, it is
possible to diagnose not only various diseases of fetus, but also various
diseases of newborn and infant. Further, the enzymatic cycling method may
be applied to forensic medicine and pathology. In the application to the
biology, pharmacology and agricultural chemistry, since given substances
may be analyzed quantitatively and qualitatively, various cells such as
microorganism, cultured cell and living tissue may be analyzed one by one
and thus quality of particular cells can be investigated in detail. In the
field of analytical chemistry, extremely small amounts of samples in the
organic chemistry may be analyzed accurately.
Heretofore, the above explained enzymatic cycling method has been carried
out manually. That is to say, at first a given amount of a sample
(multiplying substrate) and an aliquot of a cycling reaction enzyme and an
excess amount of a substrate are poured into a reaction vessel such as a
test tube. Until a given number of samples have been delivered into
reaction vessels, the reaction vessels are immersed into a first
thermostat which is held at a temperature such as -30.degree. C. at which
the cycling reaction does not proceed. After a given number of samples
have been delivered into the reaction vessels, the reaction vessels are
transferred into a second thermostat held at a temperature such as
25.degree. C. at which the cycling reaction occurs. Times at which
particular reaction vessels are transferred into the second thermostat are
recorded manually. When a given cycling reaction period has been elapsed
for a reaction vessel, the relevant reaction vessel is immersed for two or
three minutes into a third thermostat held at a temperature such as
100.degree. C. at which the cycling reaction is stopped due to the
alternation of the enzymes. Then, the reaction vessel is transferred into
a fourth thermostat held at a temperature such as 38-40.degree. C. at
which the indicator reaction takes place and an indicator reagent is
delivered into the reaction vessel. After the indicator reaction has been
performed for a predetermined period, the liquid contained in the reaction
vessel is introduced into a fluorometer and is excited by radiation of a
given wavelength to emit fluorescent light. Then the intensity of
fluorescent light thus emitted is measured. It should be noted that in the
CoA cycling, after the lapse of the predetermined indicator reaction
period, but prior to the fluorometry a given amount of a buffer solution
is delivered into the reaction vessel.
In the enzymatic cycling method, the temperature and period of the cycling
reaction are important factors which determine an amount of an accumulated
substance such as malate. For instance, in the NAD cycling, the following
relation is generally obtained.
P=k.sub.c Ct
wherein C is a sum of concentrations of NAD.sup.+ and NADH, t is the
reaction period, P is the concentration of accumulated malate and k.sub.c
is the cycling rate. It is apparent that the amount of malate is
proportional to the reaction period t. Further, the cycling rate k.sub.c
is expressed as follows.
##EQU1##
wherein k.sub.a is a primary reaction coefficient of alcohol dehydrogenase
with respect to NAD.sup.+ and k.sub.b is a primary reaction coefficient of
malate dehydrogenase with respect to NADH. Since k.sub.a and k.sub.b are
proportional to concentrations of alcohol dehydrogenase and malate
dehydrogenase, respectively, the cycling rate k.sub.c is also proportional
to the concentration of these enzymes. However, when the enzyme
concentrations in the cycling mixture become higher, the cycling reaction
does not proceed, because NAD.sup.+ and NADH are bound to the enzymes. In
NAD cycling, the cycling reaction proceeds at a temperature range of
4.degree. to 25.degree. C., in NADP cycling the cycling reaction takes
place at a temperature range of 4' to 38.degree. C., and in CoA cycling
the cycling reaction is carried out at a temperature range of 4.degree. to
30.degree. C. The maximum multiplying rates per hour of 60,000, 20,000 and
37,500 in these cyclings are obtained at 25.degree. C., 38.degree. C. and
30.degree. C., respectively. However, when the cycling reactions are
continued for more than three hours at these temperatures, the activity of
enzymes is lost and thus the multiplying rates are gradually decreased.
For instance, in NAD cycling the multiplying rate per hour at 4.degree. C.
is decreased to 17% of the maximum multiplying rate at 25.degree. C., but
since at 4.degree. C. the enzymes do not loose the activity, given the
cycling reaction is continued for more than three hours, for example,
twenty hours, the amount of malate can be increased by 200,000. Therefore,
in the cycling reaction, the reaction temperature and period are very
important factors for increasing the amount of accumulated substance by
any desired multiplier.
As explained above, in the enzymatic cycling, the multiplying factor of the
accumulated substance is predominantly determined by the reaction
temperature and period. Therefore, in the known manual method times of
immersion of particular reaction vessels into the second thermostat have
to be recorded accurately and after a given reaction period has elapsed,
the reaction vessel has to be immediately transferred into the third
thermostat held at 100.degree. C. to stop the cycling reaction. This
requires a lot of labor of an operator and might introduce inevitable
human errors. Therefore, it is difficult to obtain highly accurate and
reliable analytic results.
In order to avoid the above mentioned drawbacks, it has been desired to
develop an apparatus for easily carrying out the enzymatic cycling method.
In such an apparatus, it is necessary to control the cycling reaction
liquids contained in the reaction vessels at various temperatures and
further the reaction temperature and/or period has to be varied in order
to obtain a desired multiplying rate. It is considered that a conventional
biochemical analyzer is altered so as to carry out the enzymatic cycling.
The conventional analyzer comprises only one thermostat usually held at
37.degree. C. and reaction vessels are successively fed through the
thermostat at a given pitch. Therefore, by merely making the feeding pitch
variable and providing a plurality of thermostats held at different
temperatures, there might occur various problems, because in the enzymatic
cycling the multiplying rate has to be varied over a very wide range. Due
to the above reason, there have not been proposed an automatic cycling
reaction apparatus which can perform automatically the enzymatic cycling
method in a simple and reliable manner and an automatic analyzing
apparatus utilizing the enzymatic cycling reaction.
SUMMARY OF THE INVENTION
The present invention has for its object to provide an automatic cycling
reaction apparatus in which an enzymatic cycling reaction can be carried
out automatically in a simple and precise manner.
It is another object of the invention to provide an automatic cycling
reaction apparatus in which the amplifying rate, i e. the cycling rate can
be easily adjusted over a wide range.
It is still another object of the invention to provide an automatic
analyzing apparatus in which an extremely small amount of a substance can
be measured in a highly accurate and reliable manner by using the
enzymatic cycling reaction.
According to the invention, an automatic cycling reaction apparatus
comprises
first means for supporting a plurality of reaction vessels each containing
given amounts of a sample and a cycling mixture including enzymes; and
second means for holding simultaneously liquids contained in all the
reaction vessels at a given cycling reaction temperature for a given
period, holding all the liquids simultaneously at a first temperature at
which a cycling reaction is stopped due to loss of activity of enzymes,
and then keeping all the liquids simultaneously at a second temperature
lower than the first temperature.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic view showing an embodiment of the automatic analyzing
apparatus according to the invention;
FIG. 2 is a perspective view illustrating an outer appearance of the
apparatus shown in FIG. 1;
FIG. 3 is a graph showing the variation of the temperature during the
analysis;
FIGS. 4, 5 and 6 are flow charts explaining the operation of the apparatus
shown in FIG. 1;
FIG. 7 is a perspective view depicting another embodiment of the automatic
analyzing apparatus according to the invention;
FIG. 8 is a plan view showing a reaction unit of the apparatus shown in
FIG. 7;
FIG. 9 is a perspective view illustrating a mechanism for driving reaction
vessels;
FIG. 10 is a perspective view showing delivery nozzles; and
FIG. 11 is a schematic view illustrating still another embodiment of the
automatic analyzing apparatus according to the invention.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
FIG. 1 is a schematic view showing an embodiment of the automatic analyzing
apparatus according to the invention. In the present embodiment, the
apparatus comprises only one reaction tank 1 in which a plurality of
reaction vessels 2 are contained. A thermostatic medium of the reaction
tank 1 is controlled to have various temperatures so as to keep
simultaneously a plurality of liquids in the reaction vessels at given
temperatures. In the reaction tank 1 is rotatably arranged a turntable 3
which can hold removably a hundred reaction vessels 2 in the form of test
tube arranged equidistantly along a periphery thereof. The turntable 3
comprises an upper disc 3-1 and a lower disc 3-2, these discs being
coupled with a driving shaft of a motor 4. In the upper disc 3-1 there are
formed a hundred holes through which the reaction vessels are inserted
until their bottoms are brought into contact with the lower disc 3-2. The
rotational angle of the driving shaft of motor 4 is detected by a rotary
encoder 5. Under the control of the detected rotational angle, the
turntable 3 is rotated in a stepwise manner in a direction shown by an
arrow at a pitch equal to a pitch of the array of holes formed in the
upper disc 3-1. The reaction tank 1 is filled with a thermostatic medium
such as an antifreeze liquid which is circulated through the reaction tank
by means of pipe 6, circulating pump 7, switching valve 8, heater 9 or
refrigerator 10. The pipe 6 is covered with heat insulating material and
its inlet 6-1 is connected to a side wall of the reaction tank 1 and its
outlet 6-2 is coupled with a bottom of the tank so that the thermostatic
fluid can circulate effectively within the reaction tank 1. Further,
inside the reaction tank 1 is arranged a temperature sensor 11 for
detecting a temperature of the antifreeze liquid. It should be noted that
the antifreeze liquid is contained in the reaction tank 1 to such a level
that portions of reaction vessels containing liquids are sufficiently
immersed in the antifreeze liquid.
Besides the reaction tank 1 is arranged an arm 17 which is moved up and
down by a mechanism 15 as well as is rotated by a rotating mechanism 16.
To a front end of the arm 17 are secured three nozzles 18, 19 and 20 which
may be inserted into a reaction vessel indexed at a liquid delivery
position.
At a position outside the reaction tank 1 there is further arranged a
washing tank 21. By rotating the arm 17 above the washing tank 21 and then
descending the arm, it is possible to immerse the nozzles 18 to 20 into
the washing tank 21. The washing tank 21 is connected to a waste liquid
tank 23 via a valve 22. Above the washing tank 21 are arranged two nozzles
24 and 25, the nozzle 24 being communicated with a washing liquid tank 27
by means of a pump 26 so as to eject a washing liquid into the washing
tank 21. The other nozzle 25 is coupled with an air pump 28 to jet an air
stream.
The nozzle 18 secured to the arm 17 communicates with an indicator reagent
tank 34 via valve 31, delivery syringe 32 and valve 33. By driving the
valves 31, 32 and a syringe driving mechanism 35, it is possible to
deliver a given amount of an indicator reagent into a reaction vessel 2.
It should be noted that a conduit extending from the indicator reagent
tank 34 to a tip of the nozzle 18 is always filled with the indicator
reagent. The nozzle 19 is coupled with an air pump 36 so as to eject an
air stream from the nozzle tip. Further, the nozzle 20 is extended to a
waste liquid tank 39 by means of a pump 37 and a fluorometer 38 to supply
a reaction liquid in a reaction vessel 2 into the fluorometer 38 after the
indicator reaction. The fluorometer 38 comprises a flowcell 38-1 in which
the reaction liquid is introduced, a light source 38-2, a filter 38-3 for
projecting a light flux having a given wavelength into the flowcell, a
filter 38-4 for transmitting fluorescent light and a photoelectric
detector 38-5 for detecting the fluorescent light.
In the present embodiment, in order to control the operation of various
units, there are arranged a main computer 41 and two sub computers 42 and
43 connected to the main computer 41. Under the command from the main
computer 41, the sub computer 42 controls the temperature of the
thermostatic medium, i.e. antifreeze liquid in the reaction tank 1 and the
sub computer 43 controls the rotational movement of the turntable 3 and
other various movements related thereto. To this end, the output of the
temperature sensor 11 is supplied to the sub computer 42 and then the sub
computer 42 controls the circulating pump 7, switching valve 8, heater 9
and refrigerator 10. The output of the rotary encoder 5 is supplied to the
sub computer 43 which then controls the motor 5, up and down mechanism 15
and rotating mechanism 16 for the arm 17, valve 22, pump 26, air pump 28,
valves 31, 33, syringe driving mechanism 35, air pump 36 and pump 37. The
output of the photoelectric detector 38-5 of the fluorometer 38 is
supplied to the main computer 41 and the main computer 41 performs given
calculations on the basis of the received output to identify and measure a
kind and an amount of a substance to be analyzed. To the main computer 42
are connected a keyboard 44 for entering various kinds of information, a
floppy disc device 45 for storing the entered information relating to the
analytic operation and for reading out the stored information, a printer
46 for printing out analytic results and a monitor 47 for displaying
various kinds of information such as the entered information and analytic
results.
FIG. 2 is a perspective view illustrating an outer appearance of the
automatic analyzing apparatus shown in FIG. 1. A main apparatus 51
comprises reaction unit 52, printing and displaying unit 53, fluorometry
unit 54, control unit 55 and pump unit 56. The reaction unit 52 comprises
the reaction tank 1 and its temperature controlling system, turntable 3
and its driving system, arm 17 and its driving system, washing tank 21 and
thermostat 57 which is kept at 4.degree. C. so as to prevent the indicator
reagent contained in the indicator reagent tank 34 from being altered. The
temperature of the thermostat 57 is controlled by the refrigerator 10
which is used to control the temperature of the reaction tank 1. It should
be noted that the opening of the reaction tank 1 is covered with a
removable lid 58 except for a portion through which the nozzles 18 to 20
are moved. Similarly, the thermostat 57 is covered with a removable lid
59. The printing and displaying unit 53 comprises the printer 46 and
monitor 47 shown in FIG. 1, and the fluorometry unit 54 comprises the pump
37 and fluorometer 38. The control unit 55 comprises the main and sub
computers 41 and 42, 43, keyboard 44, and floppy disc device 45. The
control unit 55 further comprises a start button 60 for initiating the
analysis. The pump unit 56 comprises the nozzle washing pump 26 and air
pump 28, indicator reagent delivery valves 31, 33 and syringe 32, syringe
driving mechanism 35, and air pump 36 connected to the nozzle for mixing
the contents in a reaction vessel.
Now, the operation of the automatic analyzing apparatus will be explained
by taking NAD cycling by way of example.
First, the circulating pump 7 is operated and the switching valve 8 is
switched on the side of the refrigerator 10. Then the refrigerator 10 is
controlled in an on-off manner in accordance with the output of the
temperature sensor 11 so as to keep the antifreeze liquid at -30.degree.
C. Then a given number of reaction vessels 2, i.e. a hundred reaction
vessels each containing 1 .mu.l of a sample and 50 .mu.l of a cycling
mixture are set on the turntable 3. This may be performed in the following
manner. Prior to the delivery of samples, 50 .mu.l of cycling mixture is
delivered into all reaction vessels which are kept cold by ice, and a
hundred samples which contain NAD.sup.+ transferred from substance to be
analyzed by means of a transfer reaction are delivered into a hundred
sample cups. Then, 1 .mu.l of each samples in respective sample cups are
delivered into respective reaction vessels one by one and the reaction
vessels are successively set on the turntable 3.
After a hundred reaction vessels each containing given aliquots of sample
and cycling mixture have been set on the turntable 3, the reaction tank 1
is covered with the lid 58 and the start button 60 is depressed. Then the
switching valve 8 is changed onto the side of the heater 9 so as to heat
the antifreeze liquid. Under the control of the output of the temperature
sensor 11, the switching valve 8, heater 9 and refrigerator 10 are so
controlled that the temperature of the antifreeze liquid is maintained at
25.degree. C. for one hour.
FIG. 3 is a graph showing a temperature variation of the reaction tank 1.
After the cycling reaction period of one hour has elapsed, the switching
valve 8 is maintained to be switched on the side of heater 9 and the
antifreeze liquid is rapidly heated and the temperature of reaction tank 1
is kept at 100.degree. C. for three minutes by controlling the valve 8,
heater 9 and refrigerator 10 in accordance with the output of the
temperature sensor 11. By heating the cycling reaction liquid up to
100.degree. C., the enzymes contained in the liquid loose their activity
and therefore the cycling reaction is stopped.
Then, the antifreeze liquid is cooled and the reaction tank 1 is kept at
38.degree. C. as illustrated in FIG. 3. Then 1.0 ml of the indicator
reagent is delivered into successive reaction vessels in the following
manner.
The arm 17 is moved downward by means of the up and down mechanism 15 and
the tips of nozzles 18 to 20 are immersed into a liquid contained in a
reaction vessel which is just indexed at the delivery position. Then after
the valve 31 has been closed and the valve 33 has been opened, the syringe
driving mechanism 35 is operated to suck 1.0 ml of the indicator reagent
into the syringe 32. Then, after the valve 31 has been opened and the
valve 33 has been closed, the mechanism 35 is driven again to discharge
the 1.0 ml of the indicator reagent from the nozzle 18 into the liquid
contained in the reaction vessel 2. At the same time, the air pump 36 is
driven to eject the air stream from the nozzle 19 into the liquid to
agitate or mix the cycling reaction liquid and indicator reagent in the
reaction vessel 2. Next, the arm 17 is moved upward by the up and down
mechanism 15 so that the nozzles 18 to 20 are removed from the reaction
vessel 2. Then the rotating mechanism 16 is driven to rotate the arm 17
into the position just above the washing tank 21, and the arm 17 is moved
downward to immerse the nozzles 18 to 20 into the washing tank 21. Then
the pump 26 is operated to deliver a given amount of the washing liquid
contained in the tank 27 by means of the nozzle 24 into the washing tank
21. During this delivery of the washing liquid, the valve 22 is closed so
that parts of nozzles 18 to 20 which have been brought into contact with
the liquid in the reaction vessel are immersed into the washing liquid
remained in the washing tank 21. Then the valve 22 is opened to discharge
the washing liquid in the tank 21 into the waste liquid tank 23. Then the
air pump 28 is driven to jet the air stream from the nozzle 25 against the
nozzles 18 to 20 to remove any washing liquid adhered to the outer walls
of nozzles 18 to 20. After that, the arm driving mechanisms 15 and 16 are
operated to ascend and rotate the arm 17 and the nozzles 18 to 20 are
indexed at the delivery position above the turntable 3. During the above
indicator reagent delivery operation, the turntable 3 is rotated by one
pitch in the given direction. By repeating the above operation, 1.0 ml of
indicator reagent is delivered into successive reaction vessels 2. It
should be noted that during the delivery of the indicator reagent, the
pump 37 connected to the nozzle 20 is remained inoperative.
In each of the reaction vessels 2, the indicator reaction is carried out
for one hour and then the liquids contained in successive reaction vessels
2 are introduced into the fluorometer 38 to measure the intensity of
fluorescent light. For this purpose, the arm 17 is moved in the same
manner as that explained for the indicator reagent delivery, and the
nozzles 18 to 20 are first immersed into a liquid in a reaction vessel 2
just situating at the delivery position. Then the pump 37 is operated to
introduce a given amount of the liquid (0.3 ml) from the nozzle 20 into
the flowcell 38-1. Then the arm 17 is moved upward, then is rotated into
the washing position above the washing tank 21, and is moved downward.
During this movement the liquid introduced into the flowcell is measured
and then discharged into the tank 39 by driving the pump 37. Now the
nozzles 18 to 20 are washed by operating the pumps 26 and 28 in the same
manner as that explained above. During the fluorometry period, the valves
31, 33 connected to the nozzle 18, syringe 32 and the air pump 36
connected to the nozzle 19 are remained inoperative. Further, in order to
avoid contamination between successive liquids in the conduit coupled with
the fluorometer 38, the conduit is washed by flowing the washing liquid
therethrough. This may be done as follows. After the outer walls of
nozzles 18 to 20 have been washed and the wasted washing liquid has been
discharged into the waste liquid tank 23, the fresh washing liquid is
again introduced in the washing tank 21. Then the pump 37 is driven again
to flow the washing liquid through the nozzle 20 and fluorometer 38. It
should be noted that the conduit connected to the fluorometer 38 may be
washed by passing the air stream therethrough.
The output from the photoelectric detector 38-5 is supplied to the main
computer 42 and the analytic result obtained by effecting the calculation
based on the output is printed out by the printer 46 as well as displayed
on the monitor 47.
After the liquids contained in all the reaction vessels 2 have been
successively measured by the fluorometer 38, the operation of the
apparatus is stopped. It should be noted that the turntable 3 may be
always rotated intermittently at a given period, or may be rotated
intermittently only during the indicator reagent delivery period and
fluorometry period.
In the present embodiment, the various units are controlled by the main
computer 41 via the sub computers 42 and 43 in accordance with the program
recorded in the floppy disc device 45. FIGS. 4, 5 and 6 are flow charts
showing the operations controlled by the main computer 41, and sub
computers 42 and 43, respectively. The operations represen | | |
