U.S. patent number 3,926,734 [Application Number 05/427,322] was granted by the patent office on 1975-12-16 for urea analysis.
This patent grant is currently assigned to Owens-Illinois, Inc.. Invention is credited to Don N. Gray, Melvin H. Keyes, Frank E. Semersky.
United States Patent |
3,926,734 |
Gray , et al. |
December 16, 1975 |
**Please see images for:
( Certificate of Correction ) ** |
Urea analysis
Abstract
Disclosed is a method and apparatus for the determination of
urea in an aqueous specimen such as blood or urine. The specimen is
passed through a bed of immobilized urease to hydrolyze the urea to
ammonium ion. The ammonium ion is then converted to ammonia by
reaction with a base. The resulting ammonia is then selectively
passed as a gas through a hydrophobic, ammonia permeable membrane
for potentiometric detection with a pH sensitive electrode.
Inventors: |
Gray; Don N. (Sylvania, OH),
Keyes; Melvin H. (Sylvania, OH), Semersky; Frank E.
(Toledo, OH) |
Assignee: |
Owens-Illinois, Inc. (Toledo,
OH)
|
Family
ID: |
23694363 |
Appl.
No.: |
05/427,322 |
Filed: |
December 21, 1973 |
Current U.S.
Class: |
435/12;
435/287.9; 435/176; 435/181; 435/182; 435/807 |
Current CPC
Class: |
C12Q
1/58 (20130101); G01N 33/48742 (20130101); C12Q
1/005 (20130101); Y10S 435/807 (20130101) |
Current International
Class: |
C12Q
1/58 (20060101); C12Q 1/00 (20060101); G01N
27/416 (20060101); G01N 33/487 (20060101); C12K
001/04 () |
Field of
Search: |
;195/13.5R ;23/23B
;55/16,158 ;204/195B,195P |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
D J. Inman and W. E. Hornby, "The Immobilization of Enzymes on
Nylon Structures and Their Use in Automated Analysis" BioChem. J.
(1972) 129, p. 255-262..
|
Primary Examiner: Monacell; A. Louis
Assistant Examiner: Fan; C. A.
Attorney, Agent or Firm: Bruss, Jr.; Howard G. Holler; E.
J.
Claims
Having thus described the invention, what is claimed is:
1. A method for determining urea in an aqueous specimen containing
urea, comprising the sequential steps of:
passing said specimen into a hydrolysis zone containing immobilized
urease,
retaining said specimen in said hydrolysis zone for a time
sufficient to hydrolyze said urea to ammonium ions,
removing the resulting hydrolysis mixture from said hydrolysis
zone,
raising the pH of said hydrolysis mixture to at least about 11 to
convert substantially all of said ammonium ions to an aqueous
ammonia solution,
contacting said aqueous ammonia solution with a hydrophobic,
ammonia permeable membrane for a time sufficient to allow gaseous
ammonia to permeate said membrane,
determining the gaseous ammonia permeating said membrane by
dissolving said ammonia in an electrolyte and potentiometrically
determining the resulting increase in pH of said electrolyte,
and
converting said ammonia determination to the urea equivalent of
said specimen.
2. The method of claim 1 when substantially all of said urea is
hydrolyzed to ammonium ions.
3. The method of claim 1 wherein said hydrolysis zone comprises a
bed of urease immobilized on a solid support.
4. The method of claim 1 wherein said aqueous specimen containing
urea is an aqueous solution buffered to a pH in the range of about
5 to 9.
5. The method of claim 1 wherein said membrane comprises a thin
sheet of porous plastic having a thickness of about 0.1 to about 10
mils, a porosity of about 10% to about 85% with an average pore
size diameter of about 0.05 to about 5 micron.
6. The method of claim 5 wherein said membrane comprises a thin
sheet of porous plastic having a thickness of about 0.5 to about 5
mils, a porosity of about 25% to about 80% with an average pore
size diameter of about 0.05 to about 5 micron.
7. The method of claim 1 wherein said liquid electrolyte comprises
a dilute aqueous solution of ammonium chloride.
8. Apparatus for determining urea in an aqueous specimen containing
urea, comprising in combination,
an hydrolysis chamber containing immobilized urease, said
hydrolysis chamber having a specimen inlet and a hydrolysis product
outlet,
a mixing chamber interconnected with said hydrolysis product
outlet, said mixing chamber including an inlet for base, an mixer
positioned for mixing said hydrolysis product with base, an
effluent outlet for the resulting mixture,
an hydrophobic, ammonia gas permeable membrane having one surface
positioned for intimate physical contact with any effluent flowing
through said effluent outlet, said membrane having its opposing
surface defining a portion of a reservoir of liquid electrolyte in
intimate physical contact with said opposing surface of said
membrane, said membrane being positioned to separate said effluent
from the liquid electrolyte, and
a pH sensitive electrode immersed in said electrolyte, said
electrode being electrically connected to a pH cell for response to
an increase in pH in said electrolyte.
9. The apparatus of claim 8 wherein said hydrolysis chamber
contains a bed of urease immobilized on a solid support.
10. The apparatus of claim 8 wherein said mixer is magnetically
activated.
11. The apparatus of claim 8 wherein said membrane comprises a thin
sheet of porous plastic having a thickness of about 0.1 to about 10
mils, a porosity of about 10% to about 85% with an average pore
size diameter of about 0.05 to about 10 micron.
12. The apparatus of claim 11 wherein said membrane comprises a
thin sheet of porous plastic having a thickness of about 0.5 to
about 5 mils, a porosity of about 25% to about 80% with an average
pore size diameter of about 0.05 to about 5 micron.
13. The apparatus of claim 8 wherein said liquid electrolyte
comprises a dilute aqueous solution of ammonium chloride.
Description
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
(BUN). In the past, such analyses have been performed by cumbersome
wet chemical methods, conductivity methods or spectrophotometric
and colormetric techniques as described in the article entitled,
"The Use of Immobilised Derivatives of Urease and Urate Oxidase in
Automated Analysis" by H. Filippusson, W. E. Hornby, and A.
McDonald, FEBS Letters, Vol. 20, p. 291 (1972). While these methods
are generally accurate, they are somewhat time consuming and can
require careful interpretation.
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. Such enzyme electrodes are discussed
in the article entitled, "An Enzyme Electrode for the Substrate
Urea" by G. G. Guilbault and J. G. Montalvo appearing in the Apr.
22, 1970 issue of J.A.C.S. at page 2533. This article is
representative of the state of the art although many other
publications in this field are appearing in the recent
literature.
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.
A primary feature of the present invention is that the immobilized
urease is physically separated from the 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 detection 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 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 0.degree. to about 50.degree.C and
higher. The hydrolysis reaction is believed to proceed according to
the reaction: ##EQU1## 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 into the bed of
immobilized urease. Suitable buffered diluents include 0.01M sodium
citrate (pH 6.0); 0.01M sodium maleate (pH 6.2) and 0.01M 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 immobilized 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 practicing 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. Pat. No. 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.; Journal of the American
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 Biochemistry Vol. 49 (1971) 1388-94; and urease
can be immobilized with cyanogen bromide according to the method of
U.S. Pat. No. 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. The disclosures of these publications are incorporated herein
by references. Thus, in forming the bed of immobilized urease the
selection of the support from materials such as porous 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 mixture
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 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 satisfactory.
Any type of base which 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).sub.2 or Mg
(OH).sub.2 ] although dilute aqueous solutions of alkali metal
hydroxides, particularly NaOH, having concentrations in the range
of about 0.01 to about 1N 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 with a hydrophobic, ammonia permeable
membrane for a time sufficient to allow gaseous ammonia to permeate
through 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 microporous 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% to 80% and an average 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
(Acropor sold by Gelman Instrument Company) porous hydrophobic
cellulose acetate, porous polytetrafluoroethylene (Teflon sold by
DuPont), microporous polypropylene (Celgard sold by Celanese
Corporation), porous polyvinylidene fluoride and other membrane
materials as disclosed in U.S. Pat. No. 3,649,505 the disclosure of
which is incorporated by reference. These membranes permit
diffusion of gaseous ammonia while monovalent ions such as
Na.sup.+, K.sup.+, or Li.sup.+, remain in the aqueous solution
which does not diffuse through the membrane.
The gaseous ammonia permeating 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. -- 0.1M NH.sub.4 Cl) 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)/100 ml specimen. These units are
conventional in clinical applications.
FIG. 2 is a cross sectional illustration of a pH cell for use in
the present invention. FIG. 3 is a broken away enlargement of FIG.
2 showing the membrane and electrode in detail. In FIG. 2 and 3 pH
cell 10 comprises an electrode chamber 10a to which membrane
housing 10b is engaged by means of screw threads 10c. Chamber 10a
contains a pH sensitive electrode 11 which can be a conventional
glass electrode referenced against a suitable conventional
reference standard electrode 12 such as a platinum wire coated with
silver/silver chloride. Both of these 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 0.1M NH.sub.4 Cl 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 efficient 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 conventional 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 as shown in U.S. Pat.
No. 3,649,505 which electrodes incorporate the hydrophobic ammonia
permeable membrane into the pH electrode cell can be employed.
In the most efficient practice for the clinical laboratory the
buffered diluent and the base are pumped continously through the
system shown in FIG. 1 at the rate of about 0.1 to about 10
ml/minutes and usually about 1 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 11 to convert the ammonium
ions to soluble ammonia gas. The bicarbonate ions are converted to
carbonate ions at this increased pH.
The buffered diluent stream containing the dissolved ammonia then
contacts the hydrophobic 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 NH.sub.3 in the micro-environment 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 NH.sub.3 concentration,
i.e., how rapidly the pH decreases depends on how rapidly the
NH.sub.3 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 than 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 employs the 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
complete and instantaneous mixing over a 10 minute period into a
buffered diluent stream flowing at 1 ml/minute. This reaches
equilibrium with a constant pH in the electrode electrolyte and a
correspondingly 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 .degree.C unless
stated otherwise.
EXAMPLE 1
In Example 1 the bufferred diluent is an aqueous 0.01M 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 0.001M
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 from Pharmacia Fine
Chemicals Inc. under their trade name Sepharose 4B. The electrolyte
in cavity 13 is 0.1M NH.sub.4 Cl.
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 trade name 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 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 5 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 6.0N 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 20.degree.C.
A total of about 40 ml of 6.0N sodium hydroxide are required over
this 15 minute period to maintain the pH at 11.
After the cyanogen bromide has completely dissolved and reacted,
the resulting gel is washed on a sintered glass funnel with 300 ml
of cold 0.05M sodium borate solution adjusted to pH 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 frozen quickly and kept
frozen for 5 minutes. It is then maintained at 0.degree.C for 20
hours while stirring gently with a magnetic stirrer.
The resulting immobilized urease/agarose composite gel is filtered
on a sintered glass funnel with suction and washed 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.degree.-5.degree.C.
Part D
The activity of the immobilized urease/agarose composite gel of
Part C is determined by mixing a known quantity of the gel in a
0.15M solution of urea which is 0.005 molar in tris (hydroxymethyl)
aminomethane and 1.0 .times. 10.sup.-.sup.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. Jacobsen, K. Lindstrom-Lang, M. Ottesen and D.
Glick Ed., in "Methods of Biochemical Analysis," Vol. IV,
Interscience Publishers, N.Y., 1957, p 171, the disclosure of which
is incorporated by reference.
This analytical technique gives an activity for urease of 1000
I.U./ml of gel which decreases to about 400 I.U./ml of gel after
storage for 2 months at 0.degree.-4.degree.C 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 bufferred diluent and base solutions described above are pumped
through the apparatus described in FIG. 1 at a rate of 1.0 ml/min.
for each stream. Duplicate 10 microliter samples of each of the
aqueous urea specimen concentration described below are quickly
injected with a hypodermic needle through the injection "tee" into
the immobilized enzyme column as shown in FIG. 1. The analysis
takes place as described above in conjunction with the drawing.
Corresponding millivolt readings obtained are:
Aqueous EMF Change Urea (in Millivolts) Concentration On pH Meter
______________________________________ 0.10M 194.5 194.0 0.01M
138.5 137.5 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 1
minute after specimen injection. Successive samples are injected
into the column at approximately 1 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 .times. 10.sup.-.sup.3 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 trade name of Sephadex G-200. Thus, 1 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. Similar results are
obtained when the hydrophobic ammonia permeable membrane is a
copolymer of acrylonitrile and vinyl chloride sold by Gelman
Instrument Company under the trade name 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/100 ml by conventional
clinical spectrophotometric techniques are analyzed by the
procedures of Example 1. The results indicate a BUN value of 16.1
mg/100 ml with a standard deviation of 0.2 mg/100 ml. Similarly
precise results are obtained when the analysis is repeated with
chemically analyzed specimens containing 49 mg BUN/100 ml.
EXAMPLE 3
Part A
Twenty ml of distilled water is mixed with 10 ml (about 1 gram) of
agarose used in Example 1 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 11.0 by addition of
3.0N NaOH while the temperature of the agarose suspension is
maintained at about 22.degree. to 27.degree.C by the addition of
ice as necessary.
After pH adjustment, the agarose is quickly washed by vacuum
filtration with approximately 1 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 which has been cooled to
0.degree.C. The resulting urease/agarose suspension is stirred
overnight at 0.degree.-3.degree.C to complete the immobilization
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 1. 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 ratio 1 to 25 and the diluted serum
specimens are introduced into the column of immobilized urease of
this example at the flow 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 BUN Value by Invention Sample
(mg BUN/100 ml serum sample) (mg BUN/100 ml serum)
__________________________________________________________________________
1 14 13.8 2 14 14.0 3 14 13.0 4 17 17.0 5 17 16.1 6 17 16.8
__________________________________________________________________________
EXAMPLE 4
Urease is immobilized on a particulate porous alumina support by
mixing 100 mg unrease and 1.0 g of particulate alumina in 200 ml of
0.01M tris (hydroxymethyl) aminomethane (adjusted to pH 8.2 with
HCl) at 40.degree.C and stirring for 1 hour. The particulate
alumina has a particle size in range of from -50 to +100 mesh (U.S.
sieve screen) and an average pore size diameter of about 0.1 to 0.2
microns. The immobilized urease/alumina reaction product is allowed
to stand overnight at 0.degree..
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./cm.sup.3.
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
a 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 40.degree.C. This reaction mixture is stirred for 1 hour at
40.degree.C and allowed to stand overnight at 0.degree.. After
filtering and washing the immobilized urease/alumina composite is
assayed and determined to have an activity of 618 I.U./cm.sup.3.
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
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 approximately 1,000 I.U./cm.sup.3.
This immobilized urease/alumina composite is packed in a column and
used to analyze urea samples following the procedure of Example 1.
A calibration graph is 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 12.2.+-. 0.4 and 30.8.+-. 0.6
mg BUN/100 ml of serum, respectively.
Similar results are obtained in the above procedure for BUN
analysis when the immobilized 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 photopolymerization reaction. The reaction mixture is
stirred 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
urease/kaolinite composite prepared by the method of the article 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 30.degree.C. The resulting
immobilized urease/kaolinite composite is washed to remove residual
soluble urease before use in the procedures of Example 1.
Similar results are obtained in the above procedure for BUN
analysis when the immobilized enzyme bed is prepared by the method
of U.S. Pat. No. 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 .alpha.-aminopropyltriethosilane 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 buffer solution (pH 7.5). The mixture is stirred
overnight at 5.degree.C, filtered and washed with buffer before use
in the procedures of Example 1.
Similar results are obtained in the above procedure for BUN
analysis when the immobilized enzyme bed is a urease nylon
composite prepared by the method of the Inman and Hornby article
discussed above. The nylon is 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 stirred rapidly
at 0.degree. for 20 minutes and then filtered on a scintered glass
funnel and washed with 0.2 M sodium borate 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
KH.sub.2 PO.sub.4 buffer adjusted to pH 7.0 with dilute sodium
hydroxide. The urease nylon mixture is stirred for 16 hours at
about 1.degree.C. 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 Example 1.
* * * * *