Note: Descriptions are shown in the official language in which they were submitted.
This invention relates to a method and apparatus for
the determination and analysis of urea. More particularly, the
present invention relates to the analysis of urea in physiological
fluids such as blood and urine, and other aqueous specimens of
medical and industrial interest.
There is a great need in the medical field today for a
rapid and accurate analytical technique for determining blood
urea nitrogen (B~). In the past, such analyses have been
performed by cumbersome wet chemical methods, conductivity methods
or spectrophotometric and colormetric techniques. While these
methods are generally accurate, they are somewhat time consuming
and can require careful interpretation.
7'~,
1039163
More recently there has been research directed
toward the development of the so called "enzyme electrode" for
determining urea. Enzyme electrodes are made by immobilizing
urease on the surface of a cation sensitive electrode. The
enzyme electrode is then contacted with the urea specimen and
the urea is converted to ammonium ions which are sensed by the
electrode. While this system is suitable for some applications,
the presence of other monovalent cations such as are present in
physiological fluids, interferes with the electrode response.
Accordingly, the present invention overcomes these
disadvantages of the prior art by providing an apparatus and
method for analysis of urea by hydrolysis thereof with
immobilized urease to ammonium ions, conversion of the resulting
ammonium ions to ammonia by reaction with a base, permeation of
the resulting ammonia through a hydrophobic membrane and into an
electrolyte for potentiometric detection with a pH sensitive
electrode.
Thus, in accordance with the present teachings, a
method is provided for determining urea in an aqueous specimen
which contains urea. The method comprises passing the specimen
into a hydrolysis zone containing immobilized urease and retaining
the specimen in the hydrolysis zone for a time sufficient to
hydrolyze the urea to ammonium ions. The resulting hydrolysis
mixture is removed from the hydrolysis zone and the pH of the
mixture is raised to at least about 11 to convert substantially
all of the ammonium ions to an aqueous ammonia solution. The
aqueous ammonia solution is contacted with a hydrophobic,
ammonia permeable membrane for a time sufficient to allow
gaseous ammonia to permeate the membrane. The gaseous ammonia
permeating the membrane is determined by dissolving the ammonia
in an electrolyte and potentiometrically determining the resulting
increase in pH of the electrolyte and converting the ammonia
,~
--2--
~B
10391fà3
determination to the urea equivalent of the specimen.
In accordance with a further embodiment of the
present teachings, an apparatus is provided for determining
urea in an aqueous specimen which contains urea. The
apparatus comprises an hydrolysis chamber which contains
immobilized urease with the chamber having a specimen inlet
and a hydrolysis product outlet. A mixing chamber is inter-
connected with the hydrolysis produ,ct outlet with the chamber
including an inlet for base, a mixture positioned for mixing
the hydrolysis product with the base and an effluent outlet
for the resulting mixture. An hydrophobic, ammonia gas permeable
membrane is positioned to have one surface for intimate physical
contact with any effluent flowing through the effluent outlet
and the opposing surface of the membrane defining a portion of
the reservoir of liquid electrolyte in intimate physical contact
with the opposing surface of the membrane. The membrane is
positioned to separate the effluent from the liquid electrolyte.
A pH sensitive electrode is provided immersed in the electrolyte
with the electrode being electrically connected to a pH cell for
response to an increase in pH in the electrolyte.
A primary feature of the present invention is
that the immobilized urease is physically separated from the
--2~-
, . . .
,~, .
1039163 G-12~41
potentiometric electrode. This allows the ammonium ions
generated by the urease hydrolysis of urea to be removed from
the immobilized urease (where the pH must be maintained in the
range of 5 to 9 for efficient urease hydrolysis) and mixed with
a base to raise the pH to at least about 11 for conversion of
the ammonium ion to soluble gaseous ammonia The soluble ammonia
gas is then permeated through a hydrophobic membrane for detec-
tion with a pH sensitive electrode.
By this technique, the ammonia gas is solely responsible
for any change in pH and interference of any cation which may
be present in the specimen is prevented. Thus, the present
invention is capable of analyzing urea in a wide variety of
aqueous specimen with or without the presence of monovalent
cations such as sodium, potassium, or lithium. This represents
a marked improvement over the "enzyme electrode" types discussed
above where the electrode comes in contact with any extraneous
cations in the specimen which can interfere with the test
results. Furthermore, an enzyme electrode constructed with an
ammonia electrode would be inoperative because the pH sufficient
to convert ammonium ions to ammonia would deactivate the enzyme
immobilized on the electrode.
The present invention will be described with reference to
the drawings wherein Fig. 1 is a schematic process flow diagram
for practicing the present invention, and Figs. 2 and 3 are
cross sectional illustrations of one type of pH electrode
cell containing a hydrophobic ammonia permeable membrane for
10;~9163 . G-1244l
¦ practicing the present invention.
¦ Referring now to Fig. 1, an aqueous specimen containing
¦ urea flows into a bed of immobilized urease which functions
¦ as a hydrolysis zone where the specimen is maintained for a
¦ time and at a temperature sufficient to hydrolyze urea to
¦ ammonium ions. Preferably the specimen is maintained in
¦ contact with the immobilized urease for a time sufficient to
¦ hydrolyze substantially all of the urea to ammonium ions.
¦ Typically, this hydrolysis is completed within a few seconds
¦ to 30 minutes or longer at temperatures ranging from 0C to
¦ about 50C and higher. The hydrolysis reaction is believed
¦ to proceed according to the reaction:
2H+ + H20 + H2N-C-NH2 Urease)2~H4 + HC03 and/or C02
The urease is believed to be most efficient in hydrolyzing
urea at a pH of about 5 to 9. Because urease is most
efficient in hydrolyzing urea in the 5 to 9 pH range, the urea
specimen, prior to contact with the urease, is usually mixed
with an aqueous diluent which is buffered to pH 5 to 9~
The ratio of dilution of the urea specimen in the buffered
diluent varies with the concentration of urea in the specimen.
For physicological fluids such as blood or urine having unknown
concentration within the expected concentration range, a ratio
of 1 part of volume by specimen to 25 to 50 parts of diluent is
suitable for an acceptable electrode response. Usually,
for efficiency and economy, a small specimen (e.g~ about 10
to 50 microliters)is injected into a stream of buffered diluent
flowing at the rate of 0.1 to 10 ml per minute for introduction
-4-
¦ G-124~1 j
1039163
into the bed of immobilized urease. Suitable buffered diluents
include O.OlM sodium citrate (pH 6.0); O.OlM sodium maleate
(pH 6.2) and O.OlM tris (hydroxymethyl) aminomethane adjusted to
pH 7 with HCl.
Additional reagents can be incorporated into the buffered
diluent for the purpose of retarding deactivation of the im-
mobilized urease and deterioration of the support material.
These include: salts of ethylene diamine tetraacetic acid, to
prevent heavy metal ion poisoning of the enzyme; beta-mercapto
ethanol, to protect the urease from oxidation; and sodium azide,
a bacterial inhibitor.
Any of the known methods for immobilizing urease on an
insoluble support can be used in prac~icing the present invention.
For instance, urease can be covalently coupled to a porous glass
support with an amino-functional silane coupling agent as
disclosed in the article entitled, "Urease Covalently Coupled to
Porous Glass," by H. H. Weetall and L. S. Hersh; Biochim.
Biophys. Acta, 185 (1969) 464-465, and U. S. Patent 3,519,538,
urease can be coupled to water insoluble diazonium salts as in
the article entitled, "Preparation and Properties of Water-
insoluble Derivatives of Urease," by E. Riesel and E. Katchalski;
Journal of Biological Chemistry, Vol. 239, No. 5 (1964) 1521;
urease covalently coupled to nylon by the method in the article
entitled, "The Immobilization of Enzymes on Nylon Structures and
their Use in Automated Analysis," by D. J. Inman and W. E. Hornby;
Biochem. J. (1972) 129, 255-262; urease immobilized on a
polyacrylamide gel by the method in the article "A Urea-Specific
Enzyme Electrode," by G. G. Guilbault and J. G. Montalvo, Jr.;
G-124~1 ~
10391W
Journal of the l~merican Chemical Society, 91, (1969) 2164-5;
urease can be adsorbed on the surface of kaolinite as in the
article, "Preparation and Properties of Solid-Supported Urease,"
by P. V. Sundaram and E. M. Crook; Canadian Journal of Biochemistr~
Vol. 49 (1971) 1388-94; and urease can be immobilized with
cyanogen bromide according to the method of Patent 3,645,852
entitled, "Method of Binding Water-soluble Proteins and Water-
soluble Peptides to Water-insoluble Polymers Using Cyanogen
Halide," by R. Axen, J. Porath, and E. Ernbach; and "The
Preparation and Characterization of Lyophilized Polyacrylamide
Enzyme Gels for Chemical Analysis" by G. P. Hicks and S. J.
Updike appearing in Analytical Chemistry, Vol. 38, No. 6, May 1966
at page 726. Thus, in forming the bed of i~unobilized urease
the selection of the support from materials such as porous
15 glass, clay, water insoluble polymers and immobilizing the
urease thereon by chemical or physical means is well known
in the art and forms no part of the present
invention.
After hydrolysis of the urea, the resulting hydrolysis mix-
20 ture containing ammonium ions flows from the bed of immobilized
urease and is mixed with sufficient base in a suitable mixing
chamber to adjust the pH of the mixture to at least about 11.
At this pH and above substantially all of the ammonium ions are
converted to an aqueous ammonia solution. The mixing chamber has
25 an inlet for the hydrolyzed urea, an inlet for base, and an outlet
for the resulting reaction mixture. Any type of mixer such as
an impeller or blade type mixer can be used in the mixing chamber
to mix the base with the hydrolyzed urea, although a small
magnetically operated mixing bar has been found to be quite
30 satisfactory.
-6-
,.,~,,,,"
1039163 G-l244l
Any type of base ~hich does not contain ammonia or ammonium
ion can be used to adjust the pH to at least about 11. Suitable
bases include the alkali metal hydroxides (e.g.-Ca(OH)2 or ~g
(OH)2] although dilute aqueous solutions of alkali metal
hydroxides, particularly NaOH, having concentrations in the range
of about 0.01 to about lN are preferred for efficiency and
economy in pH adjustment.
After adjustment of the pH to at least 11, the resulting
aqueous ammonia solution is contacted ~ith a hydrophobic, ammonia
permeable membrane for a time sufficient to allo~ gaseous ammonia
to permeate throuyh the membrane. Such hydrophobic membranes
permit the passage of gaseous ammonia while retaining aqueous
solutions and can be in the form of hydrophobic porous and micro-
porous plastic films having a thickness of about 0.1 to about 10
mils, a porosity of about 10 to 85% and a pore size diameter of
about 0.05 to 10 microns. Preferably such microporous plastic
films have a thickness of about 0.5 to 5 mils, a porosity of
about 25~o to 80% and anaverage pore size diameter of about 0. 05 to
5 microns. Suitable plastic membranes are commercially available
in the form of porous copolymers of acrylonitrile and vinyl
chloride on nylon support (AcroporT sold by Gelman Instrument
ompany) porous hydrophobic cellulose acetate, porous polytetra-
fluoroethylene (Teflon sold by DuPont), microporous polypropy-
lene (Celgard sold by Celanese Corporation~, porous polyvinyli-
dene fluoride and other membrane materials as disclosed in U.S.Patent 3,649,505. These membranes permit diffusion of gaseous
ammonia while monovalent ions such as Na , K , or Li , remain
in the aqueous solution
G-124~1 ~
1039~63
which does not diffuse through the membrane.
The gaseous ammonia permcating the membrane is then passed
to a pH electrode cell which contains an aqueous electrolyte
solution. The gaseous ammonia dissolves in this electrolyte
solution to increase the pH of the electrolyte solution. This
increase in pH is potentiometrically measured with a pH sensitive
electrode.
The electrolyte solution is usually a dilute solution of
an ammonium salt (e.g. - O.lM ~H4Cl) to provide baseline
ammonium ion concentration from which an increase in pH is readily
measurable. This increase in pH is a function of the amount of
ammonia gas permeating through the membrane and the corresponding
potentiometric reading on the pH electrode can be readily
converted to the urea equivalent of the original specimen. The
urea equivalent of the original specimen is usually reported
in mg blood urea nitrogen 'i.e. BUN)/lO0 ml specimen. These units
are conventional in clinical applications.
Fig. 2 is a cross sectional illustration of a pH cell for
use in the pres~nt invention. Figure 3 is a broken away enlarge-
ent of Figure 2 showing the membrane and electrode in detail.In Fig. 2 and 3 pH cell lO comprises an electrode chamber lOa to
which membrane housing lOb is engaged by means of screw threads
lOc. Chamber lOa contains a pH sensitive electrode ll which can
e a conventional glass electrode referenced against a suitable
conventional reference standard electrode 12 such as a platinum
ire coated with silver/silver chloride. Both of those
1039~63
electrodes are held in position by electrode support 20 equipped
with gasket 21. Electrodes 11 and 12 are- electrically connected
to a conventional potentiometric pH meter which is not shown.
The sensing tips of electrodes 11 and 12 extend into
electrolyte cavity 13 which contains an aqueous 0.lM NH4Cl
solution. The bottom of electrolyte cavity 13 is defined by
hydrophobic, ammonia permeable membrane 14 and the sensing tip
of electrode 11 is positioned adjacent thereto. Membrane 14 is
held in contact with electrolyte cavity 13 by membrane housing
10b and membrane holder 22. A'liquid seal is maintained by means
of gasket 16. Membrane housing 10b is also provided with a
narrow passageway 17 through which the sample containing the ~
ammonia flows in permeation chamber 23. The passageway 17 and
permeation chamber 23 are of such dimensions to assure turbulent
flow therein for maximum exposure of the sample to membrane 14
to allow effic~ient ammonia permeation. After contact with
membrane 14 the specimen residue which is depleted in ammonia
leaves through passageway 18. The potentiometric measurement
which results from the increase in pH is converted to the urea
concentration of the original urea specimen by conventi~nal
potentiometric calibration techniques.
In the above technique, a conventional ammonia gas
sensing electrode such as a Model 95-10 gas sensing electrode sold
by Orion Research Incorporated or an ammonia electrode which
electrodes incoporate the hydrophobic ammonia permeable membrane
into the pH electrode cell can be employed.
_ f_~
1~9163 G-12441
In the most efficient practice for the clinical
laboratory the buffered diluent and the base are pumped
continously through the system sho~Jn in Figure l at the rate
of about O.l to about lO ml/minutes and usually about l ml/minute.
About 10-25 microliter "shot" of urea specimen is rapidly
injected with a syringe directly into the buffered diluent
stream at the inlet to the bed of immobilized urease. In
,the bed of immobilized urease, the urea is hydrolyzed to
ammonium ions and bicarbonate ions. Upon leaving the bed
of immobilized urease the reaction product is mixed with
the base to raise the pH to at least about ll to convert
the ammonium ions to soluble ammonia gas. The bicarbonate ions
are converted to carbonate ions at this increased pH.
.
G-12441
1039163
The buffered diluent stream containing the dissolved ammonia
then contacts the hydrophobiG ammonia permeable membrane and
ammonia permeation begins. Before equilibrium on both sides of
the membrane is reached, however, the concentration of the
dissolved ammonia in the buffered diluent has decreased to the
point where ammonia diffuses back from the electrode electrolyte
solution into the buffered diluent stream.
Because the urea specimen has been injected in a relatively
high localized concentration in the buffered diluent stream,
this reaction occurs quickly, (e.g. within about 2 or 3 minutes)
and produces a rapid increase in the pH which results in a sharp
peak in the potentiometric electrode response. The rate of
change of pH is a function of the concentration, i.e. the
higher the concentration of NH3 in the micro-environrnent of
the electrode the greater the slope of the pH curve. The
sharpness of the peak is also a function of the rate of change
of ~H3 concentration, i.e. how rapidly the pH decreases depends
on how rapidly the ~H3 back-diffuses into the diluent buffer
stream. Small samples should be removed faster than large
samples. The height of this sharp peak is a measure of the
concentration of urea. The next urea specimen can then be
injected when the potentiometric reading has returned to the base
line or a point near enough to the base line such that the next
urea determination is not detrimentally affected.
Another method of operation employsthe slow introduction of
urea specimen over longer periods of time until equilibrium in
ammonia diffusion is reached across the membrane. For instance,
a 10-25 microliter urea specimen is slowly and continuously
introduced with comp~ete and instantaneous miY~ing over a 10
minute period into a buffered diluent stream flowing at
I iO39163 G-12441
1 ml/minute. l'his reaches equilibrium with a constant pH in
the electrode electrolyte and a coxrespondingly constant
potentiometric response. After the constant reading has been
taken, the introduction of urea specimen is stopped and the
potentiometric reading returns to the base line. This
technique is less preferred because of the longer time period
required for each determination.
The invention will be further illustrated in the examples
that follow wherein all parts are parts by weight, all
percentages are weight percentages, and all temperatures are
in C unless stated otherwise.
10~9 1~ 3 G-12441
EXAMPLE 1
In Example 1 the buffered diluent is an aqueous O.OlM
solution of tris (hydroxymethyl) aminomethane which has been
adjusted to pH 7.0 (with HCl) containing disodium ethylene
diamine tetraacetic acid, beta-mercapto ethanol and sodium
azide in a concentration of O.OOlM with respect to each of
these chemicals.
The base used to adjust the pH is a 0.03N sodium
hydroxide solution.
The urease enzyme is obtained from Worthington Biochemical
Corporation and has an activity of 139 International Units per
milligram.
The support for the immobilization of the urease enzyme
is agarose gel (a highly porous polydextran) obtained fro~
Pharmacia Fine Chemicals Inc. under their trademark Sepharose
4B. The electrolyte in cavity 13 is O.lM NH4Cl.
The pH cell is a conventional glass pH electrode employing
a silver-silver chloride element in HCl electrolyte referenced
against a silver-silver chloride electrode.
The ammonia permeable hydrophobic membrane is a microporous
polypropylene film having a thickness of 1 mil, porosity of
35%, an average pore diameter of less than 0.1 microns obtained
from Celanese Corporation under the trademark of "Celgard 2400.
Part A
Forty-four ml of a 4~ by weight aqueous dispersion of the
agarose gel described above is poured onto a Buchner funnel
and washed with distilled water. The agarose on the filter is
~''`'
G-1244L
1039163
transferred to a beaker and distilled water is added with
agitation to yield a dispersion of 40 ml which is then
centrifuged in centrifuge tubes at about 1000 rpm for five
minutes. After decantation of the supernatant, the agarose
in the bottom of the centrifuge tubes is transferred again to
a beaker and distilled water is added with stirring to produce
a uniform gel having a volume of 40 ml.
Part B
Four hundred mg of the urease described above is dissolved
in 20 ml of a 0.05M sodium borate aqueous solution which has
been adjusted to pH 9.5 with 6N sodium hydroxide. The resulting
solution is stored in an ice bath until ready for use in Part C.
Part C
Four grams of reagent grade cyanogen bromide crystals are
added to the agarose gel of Part A and the pH is quickly
adjusted to about 11 with 6.0~ sodium hydroxide while stirring
continuously. The pH of the mixture is maintained at or near
this value by addition of the 6.0N sodium hydroxide as
required, during which time the cyanogen bromide crystals
slowly dissolve and react. This dissolution reaction requires
about 15 minutes during which time crushed ice is added to
maintain the temperature below 20C. A total of about 40 ml
of 6.ON sodium hydroxide are required over this 15 minute
period to maintain the pH at 11.
After the cyanogen bromide has completely dissolved and re-
acted, the resulting gel is washed on a sintered glass funnel
with 300 ml ofcold 0.05M sodium borate solution adjusted to pH
-13
.
G-12441
~039163
9.5 with NaOH.
The resulting agarose gel is transferred to a 100 ml beaker
and the cold urease solution prepared in Part B is added
immediately while stirring. The urease/agarose gel is then
S frozen quickly and kept frozen for 5 minutes. It is then
maintained at 0C for 20 hours ~.~hile stirring gently with a
magnetic stirrer.
The resulting immobilized urease/agarose composite gel
is filtered on a sintered glass funnel with suction and ~ashed
first with a 0.5M sodium chloride solution and then with
distilled, deionized water until the filtrate washings are free
of urease as shown by the absence of any absorption at the
characteristic wavelength 272 nm. The immobilized urease/
agarose composite gel is stored in 0.05M tris (hydroxymethyl)
aminomethane buffer solution (pH 7.5) at 0-5C.
Part D
The activity of the immobilized urease/agarose composite
gel of Part C is determined by mixing a kno~n quantity of the
gel-in a 0.15M solution of urea which is 0.005 molar in tris
(hydroxymethyl) aminomethane and 1.0 x 10 3 molar in disodium
ethylenediaminetetraacetic acid and measuring the change in pH
with time. The rate of change of pH is converted to enzyme
activity by the method of L. Jaco~sen, K. Lindstrom~Lang,
M. Ottesen and D. Glick Ed., in "'~'ethods o~ Biochemical
Analysis," Vol. IV, Interscience Publishers, N~ Y., 1957, p. 171
,
. -14-
.,,"; . "
1 ~ 9 16 3 G-12441
This analytical technique gives an activity for urease
of lO00 I~u~/ml of gel which decreases to about 400 I.U./ml
of gel after storage for 2 months at 0-4C in 0.05M tris
(hydroxymethyl) aminomethane.
Part E
A glass column is prepared from a 75 mm borosilicate glass
capillary tube with an inside diameter of 2.8 mm and an outside
diameter of 6 mm. A 400 mesh nylon disc is attached to one
end of the column. The immobilized urease/agarose composite
gel of Part C is charged thereto to fill the tube. The urease/
agarose gel packs into the column by gravity and the other end
of the column is also fitted with a 400 mesh nylon disc after
the column is filled with the urease/agarose gel.
The column ends are then fitted with a plastic tubing
fittings one of which is in the form of a "tee" for sample
injection. The injection tee is provided with a rubber membrane
for sample injection with a hypodermic needle.
Part F
The buffered diluent and base solutions described above are
pumped through the apparatus described in Fig. l at a rate of
l.0 ml/min. for each stream. Duplicate lO microliter samples
of each of the a~ueous urea specimen concentration describedkelCw
are quickly injected with a hypodermic needle through the injection
"tee" into the immobilized enzyme column as shown in Figure l.
The analysis takes place as described above in conjunction
with the drawing. Correspondins millivolt readings obtained are:
. -15-
lOJ916;~ G-1244 1
¦Aq~_ous Urea Concentration EMF Change ~in Millivolts) On pH Meter
¦ 0.10M 194.5
- - 194.0
l 0.01M 138.5
1 137.5
0.001M 78.0
78.0
A calibration graph is prepared by plotting the millivolt change
against the logarithm of the urea concentration. This graph is
essentially a straight line which indicates Nernstian behavior.
A blood serum specimen of unknown urea concentration is
analyzed by injecting 10 microliter specimens in the column
using the procedure described above. A maximum millivolt reading
is obtained about one minute after specimen injection. Successive
samples are injected into the column at approximately oAe minute
intervals. A total of 50 specimens of the serum provides an EMF
change of 130~1.0 millivolts This change in EMF correspond to
a concentration of 7.50 x 10 molar urea from the above
calibration graph. The concentration corresponds to a blood urea
nitrogen (BUN) value of 21.0- 0.8 mg nitrogen/100 ml of serum.
Similar results are obtained in the above procedure for BUN
analysis when the immobilized enzyme bed is prepared from a -
cross-linked polydextran obtained from Pharmacia Fine Chemicals
Inc. under the trademark of Sephadex G-200. Thus, one gram
of the cross-linked polydextran, 4 grams of cyanogen bromide,
6.5 ml of 6N sodium hydroxide and 200 mg of urease are reacted
by the above procedures to yield an immobilized urease/polydextran
composite gel having an activity of about 400 I.U./ml of gel.
ll G-12441
10391~i3
i Similar results are obtained when the hydrophobic ammonia permeable
membrane is a copolymer of acrylonitrile and vinyl chloride sold
by Gelman Instr~lent Company under the trademark of Acropor ANH-
3000 instead of the microporous polypropylene membrane.-
EXAMPLE 2
Thirteen 20 microliter specimens of a serum sample having
been chemically analyzed to contain 15.9 mg BUN/l00 ml by
conventional clinical spectrophotometric techniques are analyzed
by the procedures of Example l. The results indicate a BUN value
;l0 of 16.l mg/l00 ml with a standard deviation of 0.2 mg/l00 ml.
Similarly precise results are obtained when the analysis is
repeated with chemically analyzed specimens containing 49 mg BUN/
l00 ml.
EXAMPLE 3 .
lS '` Part A
~wenty ml of distilled water is mixed with l0 ml (about l
gram) of agarose used in Example l to form a suspension of
agarose. Ten ml of a 10% by weight solution of cyanogen bromide
is added to the above agarose suspension and the pH adjusted to
ll.0 by addition of 3.0N NaOH while the temperature of the
agarose suspension is maintained at about 22 to 27C by the
addition of ice as necessary.
After pH adjustment, the agarose is quickly washed by
vacuum filtration with approximately one liter of cold aqueous
0.2M sodium borate buffer at pH 8.5. One half of the resulting
cyanogen bromide "activated" agarose is added to a solution of
urease prepared by dissolving 20 mg of urease (92 International
Units/mg) in 5 ml of the above 0.2M sodium borate buffer
-17-
~.~, .
1~9163 G-12441
which has been cooled to 0C. The resulting urease/agarose
suspension is stirred overnight at 0-3C to complete the im-
mobilization reactions.
The activity of the resulting immobilized urease is
determined by a laboratory pH meter (Model pHR sold by Sargent-
Welch)and a calibrated monovalent cation electrode (Beckman Model
39137) referenced to a standard calomel electrode. The activity
of the immobilized urease is determined to be approximately 500
International Units per gram of urease/agarose composite gel.
1.5 ml of the immobilized urease/agarose composite gel
is placed into a 4 mm inside diameter glass tube and the glass
tube is fitted at each end with a disc of 400 mesh nylon to form
a small column filled with immobilized urease. Distilled water
is passed through the column at the rate of 1 ml/minute to
hydraulically pack the immobilized urease as a bed.
The tube containing immobilized urease/agarose composite
gel is connected as the immobilized urease bed in Example l. A
calibrated ammonia electrode containing a hydrophobic ammonia
permeable membrane and pH electrode cell (available from Orion
Corporation as electrode Model 95-10) is employed.
Six blood serum specimens from six different human patients
are chemically analyzed in a hospital laboratory. Three of these
samples are analyzed to have a BUN value of 14.0 mg/100 ml and
three specimens are analyzed to have a BUN value of 17.0 mg/100
ml. These serum specimens are diluted in 0.01 M tris
(hydroxymethyl) aminomethane buffer in the
10~9163 G-1~441
ratio 1 to 25 and the diluted serum specimens are introduced
into the column of immobilized urease of this example at the
~low rate of about 1 ml/minute. About 1 ml/minute of 0.2M
sodium hydroxide is used as the base to adjust the pH to 13.
Under these conditions, chemical equilibrium is attained
and change in EMF (in millivolts) is measured. The corresponding
BUN value is determined from the calibration graph. The results
are set forth below.
Serum BUN Value by Chemical Analysis BU~ Value by Invention
Sample (mq BUN/100 ml serum sample) (mq BU~/100 ml serum)
1 14 13.8
2 14 14.0
3 14 13.0
4 17 17.0
17 16.1
6 17 16.8
EXAMPLE 4
Urease is immobilized on a particulate porous alumina
support by mixing 100 mg urease and 1.0 g of particulate alumina
in 200 ml of O.OlM tris (hydroxymethyl) aminomethane (adjusted
to pH 8.2 with HCl) at 40C and stirring for one hour. The partic-
ulate alumina has a particle size in range of from -50 to +100
mesh (U.S. sieve screen) and an average pore size diameter of
G-12441
1039163
about 0.1 to 0.2 microns, The immobilized urease/alumina
reaction product is allowed to stand overnight at 0.
The immobilized urease reaction product is then vacuum
filtered on a scintered glass funnel and washed first with
500 ml of 0.5 M NaCl, followed by washing with 1 to 2 liters of
distilled water. The washed immobilized urease reaction
product is stored in 10-20 ml of 0.01 M tris (hydroxymethyl)
aminomethane buffer until ready for use. The activity of the
immobilized urease/alumina product is analyzed to be 1500 I.U./
cm3.
This activity decreases sharply upon use in urea hydrolysis
. due to leaching of the urease from the alumina support, and
has a relatively short service life when used for urea
analysis according to Example 1. .
~ EXAMPLE 5
A solution of 1,2-dibromoethane is prepared by diluting
0.25 ml of 1,2-dibromoethane in 20.0 ml of methanol. This
dibromoethane solution is added to 200 ml of a tris
(hydroxymethyl) aminomethane at pH 8.2 buffered solution. -~
The pH of the resulting solution is adjusted to and maintained
at 8.2 by the dropwise addition of 1.0 M HCl.
100 mg of urease and 1.0 g porous alumina powder
(the same alumina powder used in Example 4) are slowly added
to the buffered dibromoethane solution with stirring while
keeping the temperature at 40C. This reaction mixture is
stirred for one hour at 40C and allowed to stand overnight
at 0. After filtering and washing the immobilized urease/
G-124~1
1 10391~;3
alumina composite is assayed and determined to have an
¦ activity of 618 I.U./cm . The activity of this composite
¦ decreases very slowly in use and has prolonged service life
¦ when used for urea analysis according to Example 1. The
¦ dibromoethane apparently functions as a cross-linking agent
¦ in immobilizing the urease on the porous alumina.
EXAMPLE 6
l _ .
¦ Part A
Urease is immobilized on porous alumina as in Example
5 except that 0.25 ml of 1,3-dibromopropane is used as
the cross-linking agent instead of the 1,2-dibromoethane
The immobilized urease/alumina composite has an activity of
approximateiy 1,000 I.U./cm .
This immobilized urease/alumina composite is pacXed
in a column and used to analyze urea samples following the
procedure of Example 1. A calibration graph i5 prepared by
plotting the change in EMF (in millivolts) against urea
concentration of three serum samples having 14, 28, and 70
mg BUN/100 ml serum. These serum samples produce average
millivolt change of 70, 90, and 111 respectively.
Part B
Two blood serum specimens having been analyzed in a
hospital laboratory to contain 12.2 and 30.7 mg BUN/100 ml
of serum are analyzed using the method and procedures of Part A
of this Example. Based on the calibration graph, the
potentiometric response indicates that the serum samples have
12.2l 0 4 and 30.8~ 0.6 mg BUN/100 ml of serum, respectively.
G-12441
1 1039163
¦ Similar results are obtained in the above proccdure for
¦ BUN analysis when the immobiliæed enzyme bed is a urease/
¦ acrylamide composite gel prepared by the method of the Hicks
¦ and Updike article discussed above. Such a composite gel
¦ can be prepared by reacting 1.0 ml of a 0.1 M phosphate buffer
solution (pH 7.4) containing 400 mg of acrylamide, 4.0 ml of
a solution of the same buffer containing 23 mg of N,N
methylenebis (acrylamide); 1.0 ml of the same buffer solution
containing 10 mg of urease; together with 0.03 mg of riboflavin
and 0.03 mg of potassium persulfate to catalyze a photo-
polymerization reaction. The reaction mixture isstirred
in an ice bath while photolyzing with a flood lamp. Gellation
occurs in about 10 minutes. The gel is mechanically dispersed
and washed with 0.1 M phosphate buffer before use in the
procedure of Example 1.
Similar results are obtained in the above procedure for
BUN analysis when the immobilized enzyme bed is prepared from
a ureas~ kaolinite composite prepared by the method of the articl~
of Sundaram and Crook described above.Such a composite can
be prepared by reacting 200 g powdered kaolinite (average
particle size less than 0.1 micron) suspended in 8 ml of tris
(hydroxymethyl) aminomethane buffer with 12 mg of urease
in a stirred reactor for 20 minutes at 30C. The resulting
immobilized urease/kaolinite composite is washed to remove
residual soluble urease before use in the procedures of Example 1.
G-124~1
10391bi3
Similar results are obtained in the above procedure for
BUN analysis when the immobilized enzyme bed is prepared
by the method of U. S. Patent 3,519,538 discussed above. Such
a composite can be prepared by chemically coupling urease to
96~ silica glass powder (about 100 mesh) having an average pore
size of approximately 0.1 micron. Thus, 1.0 g of porous glass
is combined with 50 ml of a 10~ solution of ~-aminopropyltrietho-
silane in toluene. This mixture is stirred overnight with
continuous refluxing, filtered and washed with acetone. After
further reaction with p-nitrobenzoic acid, reduction of the
incorporated pendant nitro group and its subsequent diazotization
the thus activated porous glass is reacted with 10 mg of
urease in 10 ml of 0.05 M tris (hydroxymethyl) aminomethane
l buffer solution (pH 7.5). The mixture is stirred overnight
¦ at 5C, filtered and washed with buffer before use in the
l procedures of E~ample 1.
¦ Similar results are obtained in the above procedure for
BUN analysis when the immobilized enzyme bed is a urease nylon
l composite prepared by the method of the Inman and Hornby
¦ article discussed above. The nylon is a low molecular weight
type 6 polymer in powder form (120-150 mesh). Pre-treatment
of the nylon with glutaraldehyde is carried out by suspending
250 mg of the powdered nylon in 10.5 ml of 12.5% (weight per
volume) glutaraldehyde~ The latter reagent is dissolved in
0.10 M sodium borate buffer adjusted to a pH of 8.5. The nylon-
glutaraldehyde mixture is stir~ed rapidly at 0 for 20 minutes
an~ then filtered on a scintered glass funnel and washed with
1039163 G-12441
¦ 0.2 M sodium.boratc buffer. The washed activated nylon powder
¦ is suspended in 5 ml of a urease solution containing 10 mg
¦ urease, 25 micromoles of ethylene diamine tetraacetic acid and
¦ 5 micromole of mercaptoethanol in 0.05 M KH2P04 buffer adjusted tc
¦ pH 7.0 with dilute sodium hydroxide. The urease nylon mixture
¦ is stirred for 16 hours at about 1C. The suspension of
¦ immobilized urease nylon composite is washed free of unreacted
¦ urease with a 0.2 M sodium chloride solution before use in the
¦ procedures of Exampl.e 1.
