Note: Descriptions are shown in the official language in which they were submitted.
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METHOD FOR PREPARING LACTATE BIOSENSING STRIP
Field of the invention
The invention relates to a lactate biosensing strip for the measurement of
lactate
solution. The present invention also relates to a method for the manufacture
of a novel lactate
biosensing strip and to the use thereof for lactate sensing.
Background of the invention
Physicians rely on personal examination and clinical laboratory results to
determine
the presence and concentration of biological analytes in critical care
patients. Clinical
laboratories offer a wide range of automated systems for high-volume testing
and analytical
support in a well controlled, high quality environment. However, clinical
laboratories can not
provide the immediate results needed to properly treat trauma and multi organ
dysfunction/failure patients. To meet the clinical need for immediate test
results, several
technologies are emerging for testing using reliable, automated analyzers at
the patient's
bedside including electrochemical biosensors, optical fluorescence sensors,
paramagnetic
particles for coagulation test systems, and micromachined devices for both
chemical and
immunochemical testing. These technologies have allowed mufti-analyte
chemistry panels to
be performed rapidly and addressed previous obstacles such as calibration of
test devices.
These tests can be classified as: 1) in vitro, which is performed at the
bedside; 2) ex
vivo or para vivo, which is performed at wrist-side; and 3) in vivo, which is
performed inside
the patient. Such tests offer indirect cost efficiencies and savings such as
reduced labor costs,
decreased blood identification and transport errors, and reduced patient
complications. In
vitro or bedside devices are used typically in several departments of the
hospital including
intensive care units; operating rooms; emergency departments (ER);
interventional
departments; general patient care departments; and outpatient surgery and
ambulatory care
units. In vitro diagnostic tests offer a wide range of diagnostic tests,
similar to the clinical
laboratory. In vitro diagnostic test systems typically are not connected on-
line to the patient
and require an operator for blood sampling.
Key categories of diagnostic test in the diagnostic market include arterial
blood gases,
blood chemistries, blood glucose, coagulation, drugs-of abuse testing,
hemoglobin,
hematocrit, infectious diseases, and therapeutic drug monitoring: Other
categories include
cancer markers, cardiac markers, cholesterol detection, immunodiagnostics,
infectious
disease detection, lactate, and thrombolytic monitoring.
Ex vivo diagnostics use external sensors for on-line real-time testing with
little to no
blood loss. Typically, sampled blood flows through a closed system to minimize
blood
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contact. Ex vivo systems minimize problems associated with in vivo sensors,
including
clotting, inaccuracy, calibration drift, and an inability to recalibrate once
in the patient. U.S.
Pat. No. 5,505,828 discloses an exemplary ex vivo system.
In vivo diagnostics offer considerable potential in the treatment of most
critical and
unstable patients. Although many companies are developing in vivo sensors,
technical
hurdles have thus far kept in vivo sensors from common commercial use.
Ex vivo and in vivo diagnostics, since they are on-line systems, can reduce
quality
control and information integration errors that occur with clinical or in
vitro tests. Quality
control errors are commonly due to operator errors, not instrument errors or
device failures.
Exemplary errors include inappropriate specimen volume, inaccurate
calibration, use of
deteriorated test strips, inadequate validation, insufficient instrument
maintenance, bad timing
of the test procedure, and use of the wrong materials. Clinical information
system integration
allows test data collected at the bedside to be put directly into the patient
record. This
improves the efficiency of the patient management process, allowing the
integration of the
laboratory's information system and clinical information systems, providing a
"seamless"
flow of all types of patient information..
Lactate is the byproduct of carbohydrate metabolism and product of glycolysis
(pyrovate) is converted into lactate under an aerobic condition i.e.
deficiency of oxygen in
cells. Lactate estimations are therefore important in respiratory disorder,
heart ailment, labor
deseases etc. normal concentration of lactate in human blood is in the range
of 1.2 to 2.7mM.
Procedure for lactate determination for example, has employed a variety of
chemical
and physical technique. Traditional assay involves chemical treatment of
lactate in human
blood and thereby converting it into colour products which can be measured
spectrophotometrically, the methods consists in reacting the blood under test
with enzyme
namely lactate dehydrogenise (LDH). In such process absorbance at 340nm is
measured due
to the NADH formation, it becomes a measurement of lactate originally present
in blood.
US Patent 6,117,290 discloses an on-line lactate sensor arrangement. The
sensor
arrangement includes a lactate sensor, a catheter for withdrawing a test
sample, and a first
fluid flow line provided fluid communication between the lactate sensor and
the catheter. The
sensor arrangement also includes a source of sensor calibration and
anticoagulant solution,.
and second fluid flow line providing fluid communication between the source of
sensor
calibration and anticoagulant solution and the lactate sensor.
In practice there are some difficulties in adopting such a detection procedure
for use
with blood sample. The disadvantage of such methods, include, lack of
specificity, difficulty
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of standardization, requirement of large amount of blood and use of unstable
and corrosive
regents. Such methods also involve optical detection and are therefore
expensive and time
consuming. Additionally, the samples must be prepared. Another disadvantage is
that the
measurement of lactate level by prior art methods need to be done in
laboratory by qualified
personnel.
Asha Chaubey et al disclose in Electrochimica Acta Vol 46, 723 - 729 (2000)
the
immobilization of lactate dehydrogenase on electrochemically prepared
polypyrrole
polyvinyl sulphonate composite films. The response time reported is about 40
seconds and a
shelf life of about 2 weeks under refrigerated conditions. In another
disclosure (Asha
Chaubey et al, Analyticla Chimica Acta Vol 49, 98 - 103, 2000), the
immobilization of
lactate dehydrogenase on conducting polyaniline films is disclosed. The
linearity of response
is shown from 0.lmM to 1mM lactate concentration with a shelf life of about 3
weeks under
refrigerated conditions.' It is preferable to obtain sensors with longer shelf
life and shorter
response time.
Accordingly, it is important to provide a lactate biosensing strip that can
overcome the
disadvantages of the prior art without losing out on efficiency and accuracy
of measurement.
Objects of the invention
The main object of this invention is to provide a novel lactate biosensing
strip for the
measurement of lactate in aqueous medium.
It is another object of the invention to provide a lactate biosensing strip
which
performs rapidly and accurately the estimation of lactate in an aqueous
medium.
It is yet another object of the invention to provide a lactate biosensing
strip which is
low cost and is capable of being used by even non-medical persons.
A further object of this invention is an assay, which can be performed without
the
need for elaborate preliminary treatment of blood sample.
Another object of this invention is to proposed a lactate biosensing strip,
which has a
high activity of 75%.
Still another object of this invention is to proposed a lactate-sensing strip,
which is
capable for providing a reading at site.
- Surinnary of the invention
Lactate biosensing strip have many advantages over traditional methods, such
as fast
response, small size convenience, specificity of response, lack of need of any
sample
preparation, low cost and high sensitivity of measurement. The main advantage
of this sensor
over the traditional method is sample operation it can be done by ordinary
person.
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The present invention provides a lactate biosensing strip for use in the assay
of lactate
in a sample, said sensor comprising a dry strip sensor of an electrically
conducting material
having at least:
i. an external surface.
ii. a screen printed reference electrode and
iii. a screen-printed working electrode.
Accordingly, the present invention provides a lactate sensor comprising an
electrically
insulated base support (1), a pair of isolated first and second silver layers
deposited thereon
(2), a pair of graphite layers, each one of said pair of graphite layers being
deposited on one
respective silver layer and electrically connected to said respective silver
layer (2), the first
silver layer being covered fully by the respective graphite layer, the second
silver layer being
covered partly in the middle thereof with the respective graphite layer
leaving the connecting
and working zone area of the said layer uncovered , a Ag/AgCI electrode (4)
provided on top
of the working zone area of said second silver electrode layer, lactate
oxidase deposited with
a mediator on the working zone area of graphite layer covering the first
silver layer, the said
silver/silver chloride reference electrode (4) and enzyme with mediator
working electrode
being supported on said support (1), the entire working areas of referene and
working
electrode being covered with a hydrophilic membrane.
In one embodiment of the invention, the electrically insulated base support
used is
made of polyvinyl chloride.
In one embodiment of the invention the distance between the silver layers is
in the
range of 0.5 to 1mm
In another embodiment of the invention, the thickness of each silver layer is
in the
range of 15 to 25 microns.
In another embodiment of the invention, the electron mediator layer comprises
a layer
of potassium ferricyanide or ferrocene.
In another embodiment of the invention, the hydrophilic membrane is made of
nylon
or polyester.
In another embodiment of the invention, the working zone are of electrode is a
target
area used for=dispensing the analyte sample
In another embodiment of the invention, the connecting terminal zone area of
electrode is an area used for the connectivity of electrode to an electrometer
The lactate biosensing strip of the invention shows an activity of 75% and a
response
time for lactate detection is in the range of 30 to 40 seconds. The shelf life
of the strip of the
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invention is about 4 months under refrigerated conditions. Under ambient
conditions (25 to
30°C) the shelf life of the biosensing strip is seen to be about 2
months. The strip of the
invention is disposable.
The invention also relates to a method for the manufacture of a lactate
biosensing strip
said strip comprising an electrically insulated base support (1), a pair of
isolated first and
second silver layers deposited thereon (2), a pair of graphite layers, each
one of said pair of
graphite layers being deposited on one respective silver layer and
electrically connected to
said respective silver layer (2), the fast silver layer being covered fully by
the respective
graphite layer, the second silver layer being covered partly in the middle
thereof with the
respective graphite layer leaving the connecting and working zone area of the
said layer
uncovered, a AglAgCl electrode (4) provided on top of the working area of said
second silver
electrode layer, lactate oxidase deposited with a mediator on the working area
of graphite
layer covering the first silver layer (5), the said silver/silver chloride
electrode (4) and
enzyme with mediator working electrode being supported on said support (I),
said process
1 S comprising
(a) depositing a pair of silver layers on an electrically insulated base
support by any
conventional method;
(b) depositing a pair of graphite layers on said silver layers by any
conventional method,
each of said silver layers being deposited with one graphite layer, a first
graphite
layer completely covering the first silver layer, and the second graphite
layer covering
the second silver layer only in part of the surface thereof that is away from
the surface
facing the base support;
(c) depositing a silver chloride layer on the second silver layer on the part
thereof that is
not deposited with a graphite layer to obtain a silverlsilver chloride
electrode;
(d) adsorbing physically lactate oxidase enzyme with an electron mediator on
the first
graphite deposited silver layer to obtain a working electrode; and
(e) applying an outer hydrophilic membrane on the above said first reference
electrode
and second working electrode to obtain the desired pair of electrodes on an
electrically insulated base support in a single assembly.
In one eriibodiment of the-invention the electrically insulated base support
comprises
of polyvinyl chloride.
In another embodiment of the invention, said silver layer used is applied by
the step of
screen-printing.
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In another embodiment of the invention, said graphite layer used is applied by
the step
of screen-printing.
In another embodiment of the invention, the sample being tested is an aqueous
Lactate
solution or blood sample in an amount of 25 to 30~,L.
In another embodiment of the invention, the electron mediator used is selected
from
potassium ferricyanide and ferrocene.
In another embodiment of the invention, connecting terminal zone area of
electrode is
an area used for the connectivity of electrode to an electrometer
In another embodiment of the invention, the hydrophilic membrane is made of
nylon
or polyester.
Brief description of the accompanying drawings
Figure 1 is a schematic representation of the biosensing strip of the
invention.
Figure 2 is the response curve of the lactate biosensing strip of the
invention for
standard lactate test samples.
Figure 3 shows the calibration curve for the sensor against standard lactate
test
samples prepared in a laboratory.
Figure 4 shows the shelf life stability characteristics of the lactate strip
of the
invention.
Detailed description of the invention
As shown in figure 1, the invention comprises an electrically insulated base
support
(1) for supporting an electrode assembly (2), (3), (4) and (5). The electrode
assembly
comprises two electrode systems, a working electrode system (2), (3) and (5)
consisting of a
silver layer with a graphite layer deposited thereon and an enzyme and
mediator layer
adsorbed in the inorganic matrix. The other electrode assembly comprises a
reference
electrode comprising a silver layer partly deposited with a graphite layer and
a silver/silver
chloride layer thereon. Figure 1 shows the PVC sheet (i) which comprises the
supporting
substrate for the electrode. Conducting silver tracking (ii) is the screen-
printed conducting
graphite layer onto the surface of conducting silver tracking (iii) for the
connection of the
sensor to read out apparatus.
30. The target area consists of-the working electrode (iv). and the reference
electrode (v)
applies to the end of tracking by screen printing. An insulated layer is
applied over the
printed electrode to give them protection; the xna,ss can be coated with one
or more legends.
The conducting graphite track (ii) does not extend to the complete length of
the silver track
and the reference electrode.
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To achieve calibration of the biosensing strip, the strip was used to detect
currents
when the lactate solutions were used in concentrations of 1 to BxnM. The
current measured
for each of the concentrations was measured and plotted in Figure 2. In Figure
2, curve (1) is
the response curve for 1mM lactate solution, curve (2) is for 2mM solution,
curve (3) is for
4mlVI solution, curve (4) is for 6mM solution and curve (5) is for 8mM
solution. This shows
that the biosensing strip of the invention can be used to measure lactate in a
blood sample if
the range lies in the region of 1 to 8 mM in a subject. The sensitivity of the
system in terms of
the response time to attain a stable current value was determined by analyzing
the strip time
variation of current. This comprised initiating current measurement from the
time fo putting
the drop of standard test solution on the strip to the time when the current
asymptotically
reaches a stable value. It was observed (Figure 3) that the current attains
the stable value in
30 to 40 seconds. Shelf life characteristics were determined by measuring the
current due to a
known lactate concentration on strips stored for different periods of time.
The data is given in
Figure 4. In Figure 4, curve (1) is for strips stored under refrigerated
conditions (at 4°C)
while curve (2) is for strips stored at 25 - 30°C.
The invention also provides a process for producing a lactate sensor strip
which
comprise in forming a first and second electrode on a substrate by applying a
layer of silver
for each of said electrodes in said electrode, applying a layer graphite on
the handling zone of
said second electrode to silver chloride, applying a mediator and enzyme on
the graphite
layer of the working zone of the first electrode. An outer hydrophilic
membrane is applied
zone of said first electrode. The silver layers and the graphite layers are
preferably applied by
the step of screen-printing.
The main feature of this invention is that the sensor is a dry strip sensor.
It is found
that a similax mix of reagents employed in a wet sensor system did not give
good result
across a desired range of detectable lactate concentration. This invention
comprises a
substrate for supporting an electrode assembly said electrode assembly
comprising two
electrode systems, one working electrode and another one as a reference
electrode supported
on said substrate and disposed in a spaced relationship to each other. The
lactate sensing strip
comprising of'a substrate for supporting a first or working electrode and
second or reference
electrode, said electrode disposed in a spacedrelationship to each other: The
first electrode is
a working electrode and has a terminal extending into a working zone through a
handling
zone. The second electrode is a reference electrode and has a terminal
extending into a
working zone through a handling zone. In both cases, the respective terminals
are of a
material different to the base conducting layer of said first and second
electrodes.
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Commercially obtained lactate oxidase is mixed in a phosphate bufFer, then
proper
amount of this solution is injected onto a preprinted working electrode. This
solution is
allowed to dry in allow temperature, followed by
i. printing of conducting tracking
ii. printing of reference electrode
iii. printing of working electrode
iv. fixing of membrane onto electrode.
The working and reference electrode each comprise a base conducting layer of
silver
material along the handling and working zone. A graphite layer is deposited on
the silver
layer of the working electrode and extends to the terminal; the graphite layer
is applied on the
handling zone of the reference electrode and extends to the terminal. Ag/AgCI
is deposited on
the target area of the reference electrode. Working electrode comprising
conducting surface
carrying mediator compound and lactate oxidase enzyme. Mediator compound
transfer
electrodes from the enzyme to the electrode, when such catalytic activity
takes place. A
hydrophilic membrane must be provided on the working zone of said electrode.
It appears
that the surfactant serves to break up the lipoprotein complex of blood and
lactate is then
oxidized to the pyruvate by the lactate oxidase. The mediator compound is
electrochemically
reduced at the electrode producing a current measurable at the electrode,
which current is
relative to the activity of the lactate oxidize and hence the amount of
lactate present in the
sample this current is generated through a serious of coupled reactions
L-Lactate+LOD ~°X) --------------- Pyruvate +LOD (red)
LOD t~d)+Me ~oX) __________________- LQD ~oX)+ Me (re)
The redox mediator is oxidized at the base electrode and the current
proportional to
the lactate concentration. Current can be measured by any conventional
electronic system.
The following examples are given by the way of illustration and therefore
should not
be construed as limiting the scope of the invention
Example 1: Preparation of graphite paste with mediator
100mg of graphite powder and polyvinyl pyrrolidon (binder) was mixed with
O.O1M
Potassium ferricyanide (mediator) in ethylene glycol monobutyl ether to
prepare screen
printable working electrode graphite paste:
Example 2: Preparation of Dry strip
Commercially obtained lactate oxidase solution (2~.L) containing 2U of lactate
oxidase was physically adsorbed on the mediator mixed graphite electrode strip
and was kept
over night to dry at 25°C. The dry strip electrode was covered with a
hydrophilic nylon
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membrane. Before the membrane was applied, it was placed in 10% surfactant
(Tween 80)
solution in distilled water for some time the dried membrane was then fixed
over the strip.
Example 3: Preparation of lactate standard lactate solutions
Stock lactate solution 10 mM was prepared in 0,1M phosphate buffer. Standard
solutions of 2 mM, 4 mM, 6 mM and 8 mM were prepared by diluting the stock
solution with
phosphate buffer.
Example 4: Preparation of enzyme stock solution
mg of enzyme lactate oxidase was dissolved in 100.1 of 0.1M phosphate buffer
to
get the concentration SU/~..1 to get the working enzyme solution, the stock
solution was
10 further diluted to lU/p,l.
Example 5: Immobilization of enzyme on the mediator mixed graphite dry strip
2 ~.1 of enzyme solution containing 2 U of lactate oxidase was physically
adsorbed on
the mediator mixed graphite electrode strip and was kept over night to dry at
25°C. The said
dry strip electrode was covered by a hydrophilic nylon membrane. Before
applying the
15 membrane, it was placed in 10% surfactant (Tween 80) solution in distilled
water for some
time and then dried membrane was fixed over the strip.
Example 6: Enzyme activity
Sigma protocol for activity of lactate oxidase was used to estimate the
lactate oxidase
activity. The basic principle is that lactate oxidase converts 1-lactate to
pyruvate and H20~.
H20~ is subsequently converted into a colored dye by peroxidase in the
presence of 4-amino
antipyrine (4AAP) and dirnethylaniline(DMA).
LOD
L-lactate + 02 --~ Pyruvate + H20a
LOD
2 H2O2 + 4-AAP + DMA ---~ Quinonediimine dye + HBO
In the optimum conditions of temperature= 37°C and pH = 6.5, the dye
absorbs at 565
nm at the light path of 1 cm.
The activity of the immobilized enzyme was calculated according to the
following
formula:
U cni 2 = AV/ s t s
Where A is the change in absorbance before and after incubation
V is the total volume (3ml)
s is the milimolar extinction coefficient of Quinonediimine dye at 565 nm
(35.33)
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t is the reaction time (10 min)
s is the surface area of the enzyme electrode
The enzyme activity of immobilized LOD on the working graphite strip was found
to
be 75%.
Egample7: Amperometric Response Studies
The lactate biosensing strip comprising enzyme(LOD) immobilized on graphite as
working electrode and Ag/AgCI reference electrode is connected to the input of
the
electrometer was polarized at a bias voltage of 0.4V for the measurement of
amperometric
calibration response to lactate (1-8 mlVn(Figure 2). A maximum current of 60
p.A was
obtained for 8 mM lactate solution above which no significant change in
current could be
observed. The response time for lactate solution (1-8 mM) was found to be 40
seconds for
each concentration of lactate (Figure 3). Results were found to be
reproducible to within 5%.
Following principle was involved in the amperometric measutrements.
Lactate + LOD (ox) Pyruvate + LOD (red)
LOD (red) + Fe3+ -1 LOD (ox) + Fea+
0.4V
Fe2+ --1 Fe3+
Advantages of the invention
1. The lactate biosensing strip provides a quick estimation of lactate in a
sample
2, the shelf life of the sample is 4 months under refrigerated conditions.
3, the strip has a linear response in a lactate concentration of 1 to 8 mM.
4, the strip is disposable without causing any environmental hazard.
5, the strip is easily used even by people without any formal medical
training.
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