Analysis by sensor placement in recprocating flow

A method and apparatus for analyzing a sample to determine an analyte content has a sensor for sensing the analyte positioned between a first and second transducer. The analyte or derivatives thereof are reciprocated between the first and second transducer to reciprocate at the sensor and allow instantaneous sensing of an indication of the analyte's presence at a point between the first and second transducer. Rapid analysis can be carried out in many different systems including for example where the transducers are enzyme reactors and the analytes are body metabolites.

BACKGROUND OF THE INVENTION 
A large number of analyzing means and methods for various analytes are 
known in the medical and other processing arts. In many cases, immobilized 
enzyme membranes and enzyme reactors are coupled with electrochemical or 
other detectors to obtain indications of analyte concentration in various 
media such as body fluids and the like. T he chemistry for analyzing y 
means of O.sub.2 sensors, pH sensors and the like, of materials such as 
glucose, creatinine and gases in blood such as CO.sub.2 are known. 
British Patent 1,485,123 published Sept. 8, 1977 to National Research 
Development Corporation discloses the utility of enzymes and other 
biologically active material in biochemical reactions which can be 
attached to solid supports and used to determine the analyte contents of 
body fluids. A tubular reactor having a solid support and carrying an 
enzyme is specifically described for use in a blood glucose determination 
as well as other determinations. 
In such known methods, a nylon tube having a solid support on which is 
mounted glucose oxidase is used as a reactor. A sample of body fluid or 
diluted body fluid, for example, passed through the tube and reacted with 
the glucose oxidase and the oxygen consumed is monitored with a standard 
oxygen sensor such as described in U.S. Pat. No. 3,539,455 of Leland C. 
Clark. Such enzyme reactions have long been known for use. In some cases, 
reactions carried out in this manner are limited in speed and do not 
maximize the efficiency possible with automated equipment. 
SUMMARY OF THE INVENTION 
It is an object of this invention to provide a means and method for 
analyzing a sample to determine an analyte content thereof rapidly and 
efficiently. 
It is still another object of this invention to provide a means and method 
in accordance with the preceding object wherein a fluid is passed back and 
forth between two transducers with an intermediate sensor for sensing and 
determining analyte content of an unknown sample rapidly with good 
accuracy. 
Still another object of this invention is to provide an enzyme reactor 
useful in carrying out the methods of the present invention. 
Still another object of this invention is to provide means particularly 
adopted for us in connection with analytical determinations of an analyte 
in a rapid and efficient manner. 
According to the invention, a method of analyzing a sample to determine an 
analyte content thereof is carried out by passing the sample or a 
derivative thereof to a first transducer and a second transducer and 
reciprocating between said first and second transducer while sensing an 
indicator of said analyte's presence at a point between said first and 
second transducer. 
Preferably the apparatus for carrying out the method in the case of an 
immobilized enzyme based test comprises a first modular tubular reactor 
and second modular tubular reactor which are mounted in a block or housing 
and removable therefrom. A conduit between the reactors extends to a 
sample chamber interconnected with an electrode sensor such as an O.sub.2 
sensor. 
In the preferred embodiment, the method is carried out for use in testing 
biological fluids for metabolites, and an electrochemical sensor in the 
form of an electrode is positioned between two reactors used. A 
determination of the concentration of metabolite is preferably obtained in 
short time periods of for example 60 seconds or less. Preferably, the 
space within which fluid is exposed to the sensor for test is dimensioned 
to be such that the reactor chamber areas are at least two times said 
space and more preferably at least four times the test space to allow 
rapid reciprocation of liquids between the two reactors and rapid readout 
of results. 
It is a feature of the method and apparatus used that the testing can be 
carried out rapidly and efficiently at high fluid throughputs. Long life 
reactors can be used which can be interchanged in various systems. 
Maintenance can be minimized and small sample size can be used. Since 
automated equipment can easily be designed, unskilled operation is 
possible. The method and apparatus can be used with various transducers 
including immobilized enzyme reactors and dialysis arrangements.

PREFERRED EMBODIMENTS OF THE INVENTION 
A testing apparatus and method in accordance with the present invention is 
diagrammatically shown at FIG. 1 in a system for testing a sample 
material. Basically, the method and apparatus of this invention can allow 
the carrying out of clinical diagnostic testing and analytical 
measurements of dissolved organic and inorganic molecules and liquids 
using optical, gas, ion or other sensors. The key feature of the invention 
is the use of a sensor mounted between two transducers for reciprocation 
of a test fluid therebetween whereas the prior art has often mounted the 
sensor before or after a sensing surface or transducer. 
The means and method of the present invention can be used in clinical or 
process testing of various metabolites such as BUN, glucose, creatinine, 
uric acid, total bilirubin, total cholesterol, triglycerides and the like; 
blood gases such as oxygen, carbon dioxide; enzymes such as CPK amylase, 
SGPT, LDH, SGOT, alkaline phosphatase; therapeutic drugs such as 
theophyline; hormones such as thyroxin, T4; proteins such as albumin, 
alpha feto, IgG, IgA, IgE and other materials wherever rapid testing is 
desired in the range of preferably less than five minutes and more 
preferably in less than one minute. For example, glucose testing can be 
carried out in less then 45 seconds. 
The sensors used in the present invention can be any conventional 
electrochemical or other sensor arrangements as known in the art. Such 
sensors include standard CO.sub.2 electrodes, pH electrodes, optical 
curvettes positioned between two transducers where photo multiplier tubes 
and interference filters can be used to obtain optical readouts with the 
use of fiber optic light sources or the like or gas dialysis blocks for 
sensing gases generated from the analytes indicated above. 
As used in this specification and claims, the analytes can be any of the 
materials to be tested utilizing the system and apparatus of this 
invention and include the material generated from such analytes in the 
testing process. For example, O.sub.2 is actually sensed in the preferred 
process of testing to determine glucose content of a sample. Similarly, 
O.sub.2 is tested at a O.sub.2 electrode when testing for cholesterol 
using immobilized enzymes reactors. Similarly, blood gas determinations 
require the use of gas permeable membranes in dialysis blocks but should 
be considered within the scope of the analyte meaning as used in this 
application. 
Any sensor can be used including optical amperometeric, conductimeteric, 
thermal or potentiometric type sensors. In some cases, multiple sensors 
can be positioned between the transducers. 
The transducer used on either side of the sensor in the present invention 
can be of various structures. In the preferred embodiment, immobilized 
enzyme reactors are used. Such reactors can comprise tubular members 
having solid supports for enzymes which react with sample material during 
flow through the tubular members. The transducers can be reactors of other 
types which allow flow therethrough or can be gas dialysis blocks. 
Heaters, irradiators, catalyst chambers or the like can be the transducers 
used. 
In each case, the dead volume or space where sensing occurs is small with 
relation to the transducer or reactor on either side of the sensor. 
Preferably, the reactor space of each reactor on either side of the sensor 
is at least four times, preferably eight or more times the dead space in 
which sensing occurs. While the two reactors are preferably identical, 
they can be different in size, content or other value. 
In all cases, a reciprocal motion of a fluid between the sensor and 
transducers surrounding it is used with sensing carried out preferably 
during the reciprocal motion of the fluid to determination rate of 
reaction. In some cases, end point or steady state is useful to obtain 
rapid readout of results to determine concentration of the analyte being 
tested for in the process. In the preferred embodiments bulk conversion of 
analytes in mobilized enzyme reactors is carried out with simultaneous 
sensing of the reaction as it progresses. 
It is best shown in FIG. 1, the system 10 provides for fluid passageways or 
conduits between a mixing chamber 11, first transducer 12, sensor 13, 
second transducer 14, peristaltic pump 15 and discard area 16. Fluid 
supply means are shown at 17 interlinked with the mixing chamber through 
diluter 21. A probe 20 is connected to the fluid diluter 21 and is capable 
of moving to a sample chamber area 22 as shown in the dotted position to 
pick up a sample, which can be diluted prior to the probe being positioned 
within the mixing chamber 11 to discharge the diluted sample. Alternately, 
as preferred the sample is diluted with the fluid supply 17 directly 
through line 78 to the mixing chamber 11. 
The diluter 21 can be a conventional syringe-type diluter or more 
preferably is a multi-mode differential dilution pump arrangement as 
described in applicants' copending application filed simultaneously 
herewith entitled: Multi-Mode Differential Fluid Displacement Pump the 
entire specification and drawings of which are incorporated by reference 
herein. Such a dilution system comprises a defined volume chamber having 
at least two different diameter pistons mounted for movement therein 
whereupon fluid displaced by one or both of said pistons comprises a first 
volume and fluid dispensed by the second of said pistons comprises a 
second volume enabling dilution of a sample as described therein. 
The sample fluids are preferably liquids and may comprise blood, serum, 
plasma, spinal fluid, urine, cell culture media, industrial liquids 
containing analytes or other samples to be tested. The liquid supply 17 
and diluter 21 can provide for cleaning or flushing materials to be 
entered into the mixing chamber and system or alternately, for reactants 
or carriers such as buffer systems or inert carrier liquids when testing 
samples of various types. 
The peristaltic pump 15 can be of conventional design and is designed to 
move the liquid within the system reciprocally in the directions of arrows 
23, 24 during testing or sensing. 
In the preferred embodiment of this invention, transducers 12 and 14 are 
enzyme reactors having enzyme attached to solid supports to form a 
biologically active matrix within a tubular reactor as best shown in FIGS. 
2 and 3. Tubular reactors per se are known as is their use in testing for 
glucose or other analytes. Glucose determination in immobilized enzyme 
reactors are described in British Patent 1,485,123, Published Sept. 8, 
1977. In such reactors, there is usually a hollow tube of an organic 
polymer suitable for use as a support for biologically active material. 
The tube can comprise a water insoluble polymer having a wall carrying 
primary or secondary amido groups, amino substituted aromatic groups or 
nitrile groups to convert at least some of the amido, amino or nitrile 
groups on the inner surface of the tube to imidate groups capable of 
linkage reaction with a biologically active material. Such conversion is 
affected without substantial cleavage of the polymer backbone. 
The organic polymer which may comprise primary or secondary amido groups, 
amino-substituted aromatic groups, or nitrile groups may be a synthetic 
polymer such as for example polyacrylamide or homologues thereof (primary 
amido groups), a nylon, a polyurethane or a urea-formaldehyde polymer 
(secondary amido groups), polyaminostyrene (amino groups), or 
polyacrylonitrile (nitrile groups). Excellent results are obtained using 
nylon and this is the preferred polymer. 
The organic polymer is preferably in the shape of a hollow circular tube 
although other shapes can be used. Primary or secondary amido groups may 
be converted to imidate groups using an alkylation reagent, for example a 
dialkyl sulfate such as dimethyl sulphate or diethyl sulphate, ethyl 
chloroformate, an alkyl halide such as iodomethane or iodoethane, or a 
triethyloxonium salt. The reaction is preferably carried out at room 
temperature but can be at temperatures for example from 30.degree. to 
100.degree. C. The alkylation reagent may be dissolved in a suitable 
organic solvent, for example methylene chloride or toluene, if necessary. 
Amino groups substituted in aromatic nuclei may be converted to imidate 
groups by reaction with ortho esters. Nitrile groups may be converted to 
imidate groups by condensation with an alcohol in the presence of an acid. 
A wide range of biologically active materials may be linked to the organic 
polymers to produce biologically active matrices. These include enzymes 
present in or isolated from animal, plant or micro-biological tissue such 
as, for example, proteolytic enzymes such as trypsin, chrymotrypsin and 
pepsin; hydrolases such as galactosidase, ribonuclease, alkaline 
phosphate, amyloglucosidase, dextranase; cholesterol esterase, urease, 
penicillinase and invertase; dehydrogenases such as lactic dehydrogenase, 
liver alcohol dehydrogenase, yeast alcohol dehydrogenase and 
glucose-6-phosphate dehydrogenase; kinases such as creatinine 
phosphokinase, pyruvate kinase and hexokinase; oxidases such as glucose 
oxidase, cholesterol oxidase and catalase; transaminases such as 
glutamate-pyruvate transaminase and glutamate-oxalacetate transaminase; 
and amidases such as amidase and penicillin amidase. Alternatively, the 
biologically active material may be a co-factor such as, for example, 
nicotinamide adenine dinucleotide (AND), nicotinamide adenine dinucleotide 
phosphate (NADP) and their reduced forms, adenosine diphosphate ribose 
(ADP-Ribose), adenosine triphosphate (ATP), adenosine diphosphate (ADP), 
adenosine monophosphate (AMP), pyridoxamine phosphate, pyridoxal 
phosphate, a pterin, a flavin, or co-enzyme A; an inhibitor such as, for 
example, an organophosphorus compound; or an antigen or antibody. The 
linking reaction in which the biologically active material is covalently 
bound to the organic polymer is preferably carried out under very mild 
conditions. Alkaline conditions are preferred, and the most favourable pH 
at which to bind the biologically active material to the organic polymer 
is the highest pH which the biologically active material in solution can 
tolerate without losing its activity. Usually this pH is within the range 
of from 7 to 10, and preferably from 7.5 to 9. 
A further description of nylon tube glucose oxidase derivatives and their 
use in the automated analysis of glucose is further described in 
Biochimica Et Biophysica Acta, 384 (1975), page 307-316, Campbell, Hornby 
and Morris, The Preparation of Several New Nylon Tube Glucose Oxidazed 
Derivatives and Their Incorporation Into the Reagentless Automated 
Analysis of Glucose. 
With reference now to FIGS. 2 and 3, an enzyme reactor is shown which is 
suitable for use as a removable or disposable module in a system of the 
type shown in FIG. 1. In this case, a enzyme module 40 can replace the 
transducers 12 and 14 in that two reactors are contained within the module 
40. The first reactor 42 is substantially identical to reactor 43 and only 
one will be fully described. The reactors comprise tubular members of 
nylon having a wall thickness of 0.015 inch and each having a length of 
7.0 inch. The tubes are coiled intermediate their ends as best shown in 
FIGS. 2 and 3. The ends of the tube 41 of each reactor is mounted in a 
header 44 at circular recesses 45 and potted therein by an epoxy sealant 
not shown. The header 44 has through holes for the ends of the tubular 
reactors which can interengage with O-ring liquid seals (not shown) of a 
mixing chamber block or housing 11, and carries an outer notch 46. Notch 
46 and bore 80 act together as a locating notch and bore when the module 
is plugged into an operating block of a testing device in accordance with 
the present invention. 
Preferably the inside diameter of the tubes can be from 0.020 to 0.088 inch 
and the tubes can have lengths from 1.5 inches to 12 inches or more when 
uncoiled. The reactors are encased within a cylindrical shell 50 of hard 
plastic with an end closure plate 51 sealed at an edge as known in the 
art. The internal volume 52 can be filled with a heat dissipating material 
such as a filled epoxy resin as known in the art. 
Reactors of this type can be used as replaceable modules in automated 
equipment as when used in a reactor and mixing block of the type 
diagrammatically shown in FIG. 6. 
An operating block 70 for the system 10 is diagrammatically shown at FIG. 
6. Note that FIG. 4 shows a mixing chamber 11, first reactor 42, second 
reactor 43, O.sub.2 electrode 48 with a peristaltic pump 15 and a waste 
area 16. The peristaltic pump is capable of moving the fluid in the 
direction of arrows 23, 24 as desired. 
In the embodiment of FIGS. 4 and 6, the reactor shell 50 fits within a 
tubular bore 60 in a aluminum block 70 of an automated test apparatus. A 
spring loaded cap 61 retains the reactor in the block with the O-ring 
seals pressed against corresponding passageways within the block. 
The chamber 11 can be an insert block of plexiglass. In a typical setup, 
one end of a reactor tube as at 71 can be connected to an outlet 72 from 
the mixing chamber 11, outlet 73 can lead to a passageway 74 of an O.sub.2 
sensor 48. Reactor 42 has one end O-ring sealed tube and interconnected 
with a passageway 75 of the electrode 48 as at 76 and a second end as at 
77 interconnected with a fluid passageway to the peristaltic pump 15. 
Preferably, the mixing chamber is in a slide-in block having the fluid 
passageways noted between the reactor shell 50 and the electrode 48 as 
diagrammatically illustrated in FIG. 6. 
In another embodiment of the invention where gases are sensed, the 
transducers can be in the form of gas dialysis blocks as diagrammatically 
shown in FIG. 5. In transducers of this type, a pH sensor 80 of known 
design can be used having a face adjacent a first conduit or passageway 81 
which can carry a flow of buffer on one side of a gas permeable membrane 
82 with a flow of sample or diluted sample passing through passageway 83 
on another side of the gas permeable membrane. Conventional gas dialysis 
blocks 84, 85 can be used, however, the electrode 80 is mounted in the 
center of the gas dialyzer with a minimum of dead space volume between the 
active dialyzer surface and the electrode surface. In this case, gas from 
the sample flowing in passageway 83 passes through the membrane which has 
a portion on either side of the sensor and thus lies on either side 
thereof. Reciprocal movement of the buffer or any other fluid for 
capturing the gas in conduit 81 is shown at arrows 23, 24. This enables a 
sensing of the gas through pH detection of the pH electrode 80 or other 
sensors as known in the art. The CO.sub.2 gas generated changes the pH of 
the buffer. 
In the case of glucose determinations as well as cholesterol 
determinations, the rate of of decrease of O.sub.2 is measured as an 
electrode current as the reaction progresses and the liquid reciprocates 
between the reactors. This rate in the case of glucose and the steady 
state current in the case of cholesterol is an indication of glucose, or 
cholesterol concentration respectively. The rate or steady state current 
is read as an electrical readout through the electrode. 
The electrode used is preferably in a module form similar to the module 
form of the reactors. This allows plugging into the block 70 previously 
described. Thus, an electrode in the form of sensor 13 can be a tubular 
electrode having a membrane face which membrane is directly exposed to the 
fluid flow in the fluid passageway between the immobilized enzyme reactors 
42 and 43 as best seen in FIG. 4. 
In FIG. 4, the sample dead space area is within passageways 74, 75 and 
facing the O.sub.2 electrode. The area is bound by the ends of the 
reactors 42, 43 contacting 74 and 75. A diluted sample is pulled into the 
sensor area with the peristaltic pump and passed by the sensor at least 4 
times the dead volume of the sensor area in the tubes 74, 75. The pump can 
them be halted and rotated forward the number of steps required to move a 
volume of liquid eguivalent to approximately 5 times the dead volume of 
the sensor space. This sensor space is preferably in the form of a bore 
through a cuvette or block 70. In FIG. 4 the cuvette is diagrammatically 
indicated at 100. The pump direction can be reversed and the head rotated 
the same number of steps in the backward direction. The forward and 
backward rotation can continue for a specified number of cycles for each 
species to be analyzed. The data can be taken during the cycling, the 
sample then flushed with diluent or cleaning fluid and the cycle stopped 
with diluent left in the sensor area. Thus, the process is ready for 
immediate repeat. 
In the preferred embodiment for measuring glucose, immobilized glucose 
oxidase is used on nylon tubing having an inside diameter of 0.86 
millimeters. The sensor is an O.sub.2 electrode having a membrane of 0.75 
mil polyethylene. The serum sample is human serum diluted 1:65, pulled 
into the reactor electrode dead space area with a conventional peristaltic 
pump. The pump is then rotated a given number of steps forward and the 
same number of steps in a reverse direction. The number of steps 
correspond to a particular volume of fluid which in the preferred 
embodiment is 4 times the dead space volume in the electrode cuvette 
providing the passageways 74, 75. For example, when the dead space volume 
and the cuvette is 10 microliters, the rocking volume should be 
approximately 40 microliters. The reactor volume in each reactor should be 
approximately 2 times the rocking volume (one back and forth movement). 
The number of rocking cycles for glucose can be 7 with the start of data 
being taken at the end of the second cycle. Data is then taken for five 
cycles. The rate of decrease in O.sub.2 concentration is then computed and 
compared to the rate of the standard. From this, a value for the unknown 
concentration of glucose in the sample can be computed. Preferably, the 
temperature is maintained at 33.degree. C. and the total cycle time is 
less than 50 seconds. Ten microliters of sample can be used in an 
extremely short time to give desirable results. 
The dead space around the sensor 48 starts at the interface to each of the 
reactors on either side of the sensor and comprises the fluid conduit 
through which liquid flow passes from the first to second reactor or 
transducer. In most cases, the reactors are fully lined with enzyme and 
reaction occurs at the very start. In the preferred embodiment, the dead 
space maintained is small in the order of from 2 to 20 microliters but 
could be 100 microliters or more. The volume in each reactor is at least 
four times the dead volume but preferably at least eight times or more as 
for example in the preferred embodiment each reactor has a space for fluid 
in its tubular conduit of 90 microliters. With normal peristaltic pump 
action, reciprocation of the fluid over the sensor can be carried out 
rapidly and results can be obtained in time periods of less than one 
minute and preferably 45 seconds. The specific reactors used can have long 
lives although they are replaceable as modules as shown in FIGS. 2 and 3. 
Preferably, they have life spans of at least two months which in normal 
usage would permit testing of at least 10,000 samples. Reaction 
temperatures within the reactors are preferably maintained at 32.degree. 
C. while suitable temperature control means not shown. The reactors are 
preferably surrounded by material having high thermal conductivity to 
allow temperature regulation of the reactors. While spring-clip or 
bayonet-type mounting as shown in FIGS. 2 and 3 is preferred, various 
constructions for a replaceable module can be used. 
The mixing chamber is preferably in the form of a clear see through plastic 
block in module form as shown in FIG. 6. The block automatically 
interconnects its side passage 78 with a fluid supply for cleaning and 
diluting if required and a bottom passage 72 with one of the two reactors 
used. The mixing chamber module is positioned adjacent the reactors in the 
mounting block as well as in proximity to the O.sub.2 probe when glucose 
is to be determined. The chamber 11 is in effect a fluid module. 
Preferably it has a 1/2 sphere bottom. The reagent is introduced from the 
line 78 through a hole offset from the center of the axially elongated 
chamber 11 and at least 1/2 of the distance from the start of the 
spherical bottom to the bottom end of the chamber. This creates a swirling 
vortex within the well which is useful for cleaning, mixing and 
maintaining uniformity of the sample to be tested. The sample to be tested 
can be diluted prior to entrance to the chamber 11. 
In the preferred embodiment, where glucose is to be tested, the probe moves 
from the sample cup 24 to the chamber or well 11 after picking up the 
sample and positions the diluted or full sample as the case may be in the 
chamber 11. Buffer which can be introduced through line 78 cleans the 
outside of the probe. A peristaltic pump can be used to evacuate the 
chamber and part of the reactors leaving liquid around the electrode or 
perhaps evacuates the entire fluid system. Diluent can be introduced from 
line 78 in an amount of for example 150 microliters into the chamber 11. 
With the probe 20 tip below the liquid level in chamber 11, the sample 
which can be 10 microliters, is dispensed within the chamber 11 and then 
the probe is withdrawn from the chamber. An additional quantity of buffer 
as for example 450 microliters can be introduced to chamber 11 through 
line 78 in the form of a swirling vortex at a speed of 20 milliliters per 
minute into the sample chamber to form a volume of 650 ml. About 650 
microliters of total liquid is preferably used in the test of this 
invention. The mixture from the chamber 11 is then drained into the 
reactors 42 and 43 as well as the dead space within the O.sub.2 sensor. 
Additional buffer from supply 17, as for example 700 microliters, cleans 
the walls and the outside of the probe. Everything within the chamber 11 
can then be drained to a waste area. The chamber 11 is then filled with 
air. 300 microliters of buffer can then be entered through line 78 into 
the chamber 11 and to fill the reactors and electrode after the test. It 
should be noted that the reactors have a coiled section in order to 
compact the size of the reactors. 
Because the probe can be introduced into the chamber with fluid flow as 
described, the chamber 11 can be used for both cleaning the probe and 
mixing or holding the sample for delivery to the reactors. Because the 
entrance port 78 is offset from the central axis of the elongated chamber 
and is positioned part way up the spherical curve of the bottom, force of 
the diluent or other fluid through line 78 creates a swirling vortex for 
mixing and cleaning. Draining at the bottom of the spherical bottom allows 
complete draining from the chamber 11. Minimized cleaning fluids can be 
used and small modules can be used since the fluid use is maximized. The 
outlet 78 is preferably at least half a way to the bottom of the spherical 
curved bottom portion of the chamber 11. This portion is a half sphere. 
Access must be provided at the top of the chamber for the probe so that 
the chamber 11 in effect has an upper access port, a lower drain port and 
an intermediate fill port. 
It should be understood that the use of dialysis membranes as the 
transducers can be in the form of two separate transducers just as there 
are two reactors in FIG. 4. Alternately, a single membrane arrangement of 
FIG. 5, effectively is a split transducer by locating the electrode 80 at 
the center point thereof. 
The sensors used in connection with this invention can vary as stated 
above. When an electrode sensor is used, it is preferred to use a 
cartridge type electrode sensor as known in the art. For example as shown 
in FIGS. 7 and 8, the sensor can have a cartridge body 90 of tubular cross 
section for fitting within the block 70 by a bayonet type arrangement as 
previously described (not shown). The electrode shown at 91 has a screw 
thread end 92 through which a platinum wire cathode 93 extends. The 
cathode 93 is covered by a membrane cap 94 screw threaded at 95. The cap 
94 has a membrane 96 retained in place by an O-ring 97. 
Cartridge-type replaceable membranes of this type can be made to fit within 
cuvette section 100 diagrammatically shown in FIG. 4. This replaceable 
cuvette forms a portion of the block 70. It carries the fluid passageways 
74, 75 and an entrance bore for positioning the electrode 91 or 48, as for 
example shown in FIG. 4. 
The membranes used with the O.sub.2 electrodes of this invention can be any 
of several types. Clysar EH, a trademark product of Polymer Products 
Department of Dupont, Wilmington, Del. made of biaxially oriented 
polyethylene film having thicknesses of from 0.6 to 1.0 mil (nominal) can 
easily be used with a preferred thickness of 0.75 mil. Clysar EHC, a 
biaxially oriented polyethylene/polypropylene copolymer film also 
available from Dupont is useful. Other useful membranes for the electrodes 
of this invention include Cryovac MPD 2055, a trademark product of W. R. 
Grace & Co. of Duncan, S.C. comprising a polyolefin multi-layered film in 
thicknesses of from 0.5 to 1.0 mil and Cryovac D-955, a polyolefin 
cross-linked multi-layered film. TEFLON film sold under the tradename 
NEN-FLO having a thickness of from 0.5 to 1.0 mil and manufactured by 
Dupont is preferred for certain glucose uses. 
The O.sub.2 electrode is a conventional O.sub.2 electrode having a 
conventional electrical readout through known systems as known in the art. 
The Beckman O.sub.2 electrode manufactured by Beckman Instruments, Inc. of 
Brea, Calif. can be used. This electrode is about 1.5 inches long with a 
rhodium cathode 5 millimeters in diameter and a silver anode. The 
electrolyte maintaining electrical contact between the anode and cathode 
is a highly viscus gel containing potassium chloride. The electrolyte is 
held in position by a Teflon membrane 1 millimeter thick which is pressed 
tightly against the cathode. Because of this pressure, only a thin layer 
of electrolyte is trapped between the cathode and teflon membrane. The 
anode is held at 0.55 volts versus the cathode. Only the TEFLON membrane 
is in contact with the solution in the passageways 74 and 75 in the 
preferred embodiment of this invention. 
In a first specific example of this invention, the glucose content of a 
human serum sample is determined using a system of the type illustrated 
diagrammatically in FIGS. 1, 4 and 6. The electrode used is substantially 
similar to Beckman O.sub.2 electrode as previously described, however, it 
has a small platinum cathode with a potential between the electrodes of 
650 millivolts. Each of the two reactors 42 and 43 have a volume of 80 
microliters and each are formed of a nylon tube having an inside diameter 
of 0.86 millimeters with a length of 17.5 centimeters coiled as shown and 
coated with glucose oxidase by activating nylon with triethyloxonium 
tetrafluoroborate (10% in methylene chloride). This is carried out by flow 
through for 20 minutes at 3.degree. C. A flush with methylene chloride is 
used. 10% hexane diamine spacer in methanol is added and rapidly flowed 
through for one minute. The tube is then allowed to sit in hexane diamine 
for one hour after which it is flushed with distilled water. Reactivation 
is carried out with 5% gluteraldehyde pH 8.5 in a buffer. Washing at a pH 
of 7.8 with phosphate buffer is then carried out. Enzyme glucose oxidase 
is added in 900 units per/ml solution in a pH 7.2 phosphate buffer. This 
solution is allowed to remain at 4.degree. C. for six hours. Tris buffer 
is then used to flush the tube of excess unattached enzyme. 
The O.sub.2 sensor is a platinum cathode electrode with silver anode as 
shown in FIG. 7 with the potential between the electrodes of 650 
millivolts which can cause reduction of O.sub.2 in the sample after 
passing through the membrane. The membrane is a 0.75 mil thick 
polyethylene sold by Dupont, Wilmington, Del. under the tradename CLYSARE 
H. 
The sample is human serum but can, of course, be plasma, urine, spinal 
fluid, cell growth media or the like. The serum is diluted 1:65 in Tris 
buffer pH 7.2 in the sample chamber 11. The peristaltic pump is used to 
deliver and pull through the reactor the serum dilution and to move the 
solution back and forth. 400 microliters is pulled into the system shown 
in FIG. 1 with a reciprocation of about 40 microliters in six complete 
back and forth cycles. 
The electrode output is read after the second reciprocation at eight spaced 
intervals to have two readings on each reciprocation Each back and forth 
reciprocation takes 1.6 seconds for a total analysis time of 9.6 seconds. 
Time and picoamp output are measured at specific intervals and then a 
linear regression analysis is carried out to determine the best straight 
line through the points to test for the presence of glucose, thus 
assigning a slope to the line. This procedure is carried out for a known 
concentration of glucose and from which a calibration curve is determined 
and the slope of the unknown sample is then compared to determine glucose 
concentration. The reaction is carried out at 33.degree. C. 
Table 1 shows the target value of four control sera samples tested in the 
system. 
TABLE 1 
__________________________________________________________________________ 
C.V. Within Run 
TARGET VALUE 
SAMPLE X (unknown 
(% coefficient 
C.V. 
(mg/deciliter) 
(control/serum) 
measured value) 
of variation) 
(Day to Day) 
__________________________________________________________________________ 
76 MONI I 72.5 1.13% 1.80% 
258 MONI II 260.1 1.14% 1.46% 
68 SER CHEM I 
65.5 1.52% 2.26% 
257 SER CHEM II 
256.3 0.92% 1.30% 
__________________________________________________________________________ 
Note that three percent is considered a good C.V (percent coefficient of 
variation) in test instruments of this type. The above test data clearly 
indicates that accuracy and repeatability is good in the system of the 
present invention. 
The above example involves the use of known glucose testing chemistry where 
the reactions occur as described below: 
EQU H.sub.2 O+Glucose+O.sub.2 .fwdarw.Gluconolactone+H.sub.2 O.sub.2 
In a second example using the system of this invention to determine 
cholesterol in a blood serum sample of a human, the first example is 
repeated with the following changes. The reactors used are identical 
except that the enzyme immobilized in the reactor is cholesterol esterase 
and cholesterol oxidase formed in the same manner as previously described 
in the first example with 3 milligrams per/ml of each used. The Tris 
buffer is repeated but added to it is 0.4% of Triton X100 produced by Rhom 
and Hass. Sodium chlorate is added to the buffer at a value of 0.2 
millimolar along with magnesium chloride and ethylene diamine tetra acidic 
acid (EDTA) in an amount of 5 and 2.5 millimolar respectively. The 
reaction temperature varies between 20.degree. to 40.degree. C. and is 
preferably maintained at about 39.degree. C. 
The reaction of cholesterol in the blood with the enzyme immobilized in the 
reactor produces O.sub.2 and is detected by the sensor. 
Here again readout is carried out during the reciprocation of the diluted 
sample as the reaction approaches steady state as previously described. 
Extremely accurate results can be obtained 
The chemistry of this reaction is as follows: 
EQU Cholesterol Ester+H.sub.2 O.fwdarw.Cholesterol+Free Fatty Acid 
EQU Cholesterol+O.sub.2 .fwdarw.Cholesterol+H.sub.2 O.sub.2 
In a third example of this invention, a 20 microliter serum sample is 
aspirated out of the probe and dispensed into the well 11 containing 20 
millimoles of H.sub.2 SO.sub.4 (100 microliters). The sample is mixed by 
rapid addition of 400 microliters of 20 millimole H.sub.2 SO.sub.4. The 
diluted sample is then pulled into the gas dialyzer shown in FIG. 5 where 
the buffer flow is substituted by 4.0 millimoles sodium bicarbonate at a 
pH of 8.9. The sulfuric acid in the diluent causes the release of CO.sub.2 
gas from the sample as illustrated below: 
EQU NaHCO.sub.3 +H.sub.2 SO.sub.4 .fwdarw.H.sub.2 CO.sub.3 +NaHSO.sub.4 
EQU H.sub.2 CO.sub.3 .fwdarw.H.sub.2 O+CO.sub.2 (l) 
EQU CO.sub.2 (l).fwdarw.CO.sub.2 (g) 
The CO.sub.2 gas dissolves in the membrane which is 1 mil thick and then 
passes through to the bicarbonate buffer side and undergoes the following 
reactions: 
EQU CO.sub.2 (g).fwdarw.CO.sub.2 (l) 
EQU CO.sub.2 (l)+H.sub.2 O.fwdarw.H.sub.2 CO.sub.3 
EQU Na.sub.2 CO.sub.3 +H.sub.2 CO.sub.3 .fwdarw.2NaHCO.sub.3 
This causes a decrease in pH of the bicarbonate buffer due to the CO.sub.2 
going from one side of the membrane to the other. The peristaltic pump 
controlling the bicarbonate is then rotated back and forth to bring the 
acidified buffer to the electrode. 
This results in the rate of CO.sub.2 evolution being sensed by the pH 
electrode, which rate is proportional to the bicarbonate concentration in 
blood in the range of 5-45 millimoles per liter. The total cycle time is 
less than one minute and an accurate readout of CO.sub.2 content is 
obtained. 
After the readout the buffer and the diluted sample flow on either side of 
the dialyzer membrane is flushed with fresh solutions which speeds the 
return to base line of the electrode. 
While specific embodiments of this invention have been shown and described, 
it should be understood that many variations are possible. 
Generally, it is preferred to have the system as described which reacts 
very rapidly. It is almost an instantaneous reaction and thus, is faster 
than a normal membrane electrode method, because the enzyme surface and 
the electrode surface are exposed to diluted sample which is constantly 
mixed because of the reciprocal motion of the fluid. Having the enzyme 
layer on the electrode creates a barrier to stirring which can only be 
traversed by diffusion of molecules through the layer. Thus, the response 
of the sensor slows down in some cases in particular during the recovery 
stage. In the case of an enzyme coil reactor which may have been used 
before the sensor, the reactor is slower because a rate can only be taken 
by pumping diluted sample very slowly through the reactor and monitoring 
the rate of reaction. Essentially no mixing takes place in the coiled 
tubing, and diffusion must be relied upon to bring analyte from the center 
of the tubing to the wall. In the present method, the analysis is faster 
than having a coil reactor before the sensor and faster than straight flow 
through. 
Temperature control is easier than when carried out in a single coil 
reactor or gas dialyzer. Because the same segment of liquid is 
reciprocated back and forth in the reaction chamber during the entire 
analysis time, temperature control can be more concentrated and more 
precise. Equilibrium of the reciprocated liquid can be rapidly brought to 
thermal equilibrium by heating or cooling of the cuvette, reactors or the 
like. 
Using a split coil or two reactor system of the present type allows great 
flexibility because miniaturizable sensors such as optical, amperometric, 
conductimetric, thermal or potentiometric can be utilized. In addition, 
multiple sensors between the coils can also be used. 
While the block or housing 70 has been described as an aluminum mounting 
for a mixing chamber 11, reactor module and electrode, the form can bary 
greatly. Preferably the three components are removably mounted in the 
housing by spring caps or detents as is known. Supplemental heaters can be 
mounted in the housing to heat the components through the aluminum block. 
Other good heat transfer materials are also preferred for use as the 
housing material.