Note: Descriptions are shown in the official language in which they were submitted.
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BIOSENSOR INCORPORATING A SURFACTANT
The present invention relates to a sensor and more particularl~~ to a
biosensor.
Biosensors are used for determining the presence and/or amount of a selected
analyte in a sample. The particular type of biosensor to v-hich the present
invention
relates comprises an enzyme which is specific for the analyte to be determined
and
which interacts therewith to produce a chemical change indicative of the
presence
and/or amount of the analyte. The change may be detected by any suitable
means, e.g.
an electrode arrangement. Thus. for example. an enzyme electrode system for
the
determination of glucose comprises an enzyme layer incorporatin~~ ''lucose
oxidase
which catalyses the oxidation of ~~lucose by molecular oxygen to produce
gluconic
acid and hydrogen peroxide, either of which may be determined by an
amperometric
electrode.
In use of such biosensor, it is necessary that the enzyme be provided with any
substance essential to its activity for effecting the chemical change which
forms the
basis of the detection procedure. Examples ol~ SllCh SLlbStal7CC;S 111Clllde
ellZfll7e Co-
factors and electron transfer mediators. Such substances are <~enerallv
provided in the
sample being analysed and, in the case where the biosensor includes a
diffusion
limiting membrane between the enzyme and sample, diffuse through the
Illelllbl'alle SO
as to be available for the enzyme.
The need to provide such substances in the sample is however a considerable
disadvantage for several reasons. Firstly. the need to add the substances to
the analyte
is an extra step in the detection procedure. Secondly. it is necessary to use
substantially more of the substances than actually required by the. enzyme.
This is
particularly the case if measurements on the sample are being made under
continuous
flow conditions.
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It is therefore an object of a first aspect of the present invention to
obviate or
mitigate the abovementioned disadvanta~~es.
According to a first aspect oi~ the present invention there is provided a
biosensor for the determination of a selected analyte in a sample, the bio-
sensor
comprising an enzyme layer incorporating an enzyme which is capable of
interacting
with said analyte to provide a detectable change. detecting means on one side
of said
layer for detecting said change, and an outer diffusion limiting barrier
membrane
which is provided on the opposite side of said layer to the detecting means.
said
membrane incorporating a surfactant so as to render it permeable to said
analyte
wherein said enzyme layer incorporates at least one substance essential to
enzyme
activity and the nature and amount of said surfactant is such that the
membrane
inhibits release of said substance into the sample whilst retainin~~
permeability to the
analyte.
We have found, and this fOr111S the basis of the present invention, that
surfactant incorporating membranes may be produced which are capable of
permitting
sufficient diffusion of analyte species whilst nevertheless inhibiting
diffusion of
substances essential to enzyme activity (e.g. enzyme co-factors. electron
transfer
mediators, coenzymes and activators). Using 511C11 Il7ell1bra11eS It is
possible to
produce biosensors in which substances required for enzyme activity are
provided in
the enzyme layer rather than in the Salllple llledllllll COlltallllllg the
analyte.
Non-limiting examples of enzymes which may be used in the sensor of the
invention are given in Table 1 below together with examples of co-factors etc.
essential for their activity which are incorporated, together with the enzyme
in the
enzyme layer.
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TABLE 1
ENZYME CO-FACTORS ETA.
(i) Pyruvate Oxidase Cocarboxylase (thiamine pyrophosphate
chloride), Mg2*) flavin adenine
clinucleotide (FAD)
(ii) Lactate DehydrogenaseDiaphorase. potassium ferricyanide,
NAD+
(iii) Malate DehydrogenaseDiaphorase, pOtasSll1177 leI'1'lCya171de)
NAD+
The combinations (i), (ii) and (iii) may be used in sensors for determination
of
pyruvate, lactate and malate respectively.
Other cofactors may be additionally or alternatively used wlthln the enzymes
described in Table 1. These include:-
for (ii) NADH
for (iii) NADH, NADPI-I and/or NADP+
Many other enzymes recluirin~ COfaCt01'S/171ed1at01'S Illay be Sllltably used
according to the invention. Further examples include a variety of other
dehydrogenase
enzymes (e.g. glucose, alcohol, fructose or glutamate dehydrogenases) which
require
cofactors similar to (ii) or (iii) in Table 1 above. Kinases (such as pyruvate
kinase or
protein kinase) may also be used. Such kinases usually require cofactors such
as
Mg2+ ADP and/or ATP. I11 fact, n7en7branes incolporating a surfactant
accordn7g t0
the invention greatly expand the range of enzymes that may be used 111 5uCh
5e17 SOTS.
The surfactant may be a non-ionic, cationic, anionic or zwitter ionic
surfactant
and will generally_ be present in the lllelllbralle 111 a17 aiTlOLlilt of 10%
to 60% based on
the total weight of the membrane.
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A preferred cationic surfactant is a methyl mixed triallcyl quaternary
ammonium salt (e.g. the chloride) in which the said alkyl groups each have up
to l00,
preferably 10 to l00 carbon atoms. One example of such a surfactant which we
have
found to be useful is that available under the name Adogen 464.
A further surfactant which may be used is Aliquat 336.
A preferred non-ionic surfactant is a polyoxyethylene sorbitan monooleate
having for example 15 to 25 oxyethylene units in the chain, e.g.
polyoxyethylene 20
sorbitan monooleate. A suitable example of such a surfactant is available
under the
name Tween-80.
We have found that certain poly mer/surfactant combinations are to be
preferred as membranes for particular types of sensor. Thus, in the case of
pyruvate
and lactate sensors of the type given by (i) and (ii) in Table 1, it is
preferred that the
membrane is cellulose acetate modified by a methyl triall<yl quaternary
ammonium
chloride (e.g. Adogen 464), most preferably SLICI7 that tl7e sLlrfaCtailt
provides about
SO% by weight of the total weight of the membrane. However in the case of a
malate
sensor as represented by (iii) in Table 1. it is preferred that the membrane
comprises
cellulose acetate modified with a polyoxyethylene sorbitan monooleate (e.g.
Tween
80), most preferably SLICK that the surfactant provides about 50% by weight of
the
total weight of the membrane. This leads to a further aspect of the present
invention
in that we have ascertained that a membrane incorporatin~~ a cationic
surfactant has
the ability to select between similar sized organic molecules such as malate
and
pyruvate, possibly on the basis of the extent of ionisation at a given pH
(malic acid
being diabasic and pyruvic acid being monobasic).
Therefore according to a second aspect of the present invention there is
provided a biosensor for the determination of a selected analytc in a sample,
the bio-
SUBSTITUTE SHEET (RULE 2fi)
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sensor comprising an enzyme liver incorporating an enzyme which is capable of
interacting with said analyte to larovi(ie a detectable chan~~e. (letectin~'
means on one
side of said layer for detecting said change, and an outer diffusion limiting
barrier
membrane which is provided on the opposite side of said layer to tile
detecting means,
said membrane incorporating a surfactant so as to render it permeable to said
analyte
wherein said surfactant is a cationic surfactant.
The enzyme layer of the sensor of the second aspect of the invention
preferably (but not necessarily) i11C01'pOrateS SllbStaIlCes eSSelltlal ~Ol'
the activity of the
enzyme, i.e. as described for the first aspect of the invention. If such
substances are
not incorporated in the enzyme layer they play be provided in the sample to be
analysed.
The preferred surfactant for use in the sensor of the second aspect of the
invention is a methyl triall:yl quaternary a1111110111L1111 Salt of the type
discussed above.
Therefore according to a third aspect of the present invention there is
provided
a membrane for use in a biosensor, said membrane incorporating a methyl
trialkyl
quaternary ammonium salt.
The preferred base material for the membrane of any aspect of the invention is
a cellulosic material, e.g. cellulose. cellulose nitrate or a cellulose ester
such as
cellulose acetate or cellulose butyrate. The preferred material is cellulose
acetate,
preferably having an acetyl content of about 40%. The membrane preferably
contains
10% to 60% by weight of the surfactant based on the total weight of the
membrane.
Membranes for any aspect of the present invention may be produced by
conventional C3Stlllg techniques in which a sOllltlOll ~lll a vOlatlle
SOlVellt)Ol' the base
material of the membrane (e.g. cellulose acetate) and the requisite amount of
surfactant is cast onto a flat surface and the solvent evaporated.
Alternatively it is
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possible to employ a "spin casting'" technidue in which the membrane is
produced by
applying a solution (of the type defined in the previous sentence) to a flat
surface
which is then rotated (usually about a vertical axis) at a speed which causes
the
solution to be evenly distributed and the solvent to be evaporated so as to
produce a
membrane of uniform thickness.
Typically the membrane Ior any aspect of the invention will have a thickness
of 0.1 to 200 microns, preferably 4 to 50 microns.
The enzyme layer may be produced by immobilisation of the enzyme (and any
substances necessary for the activity thereof) using conventional techniques,
e.g. by
incorporation in a cross-linked gllltal'aldehyde Illatl'1X.
If desired the enzyme layer may be laminated to at least one highly permeable
support layer, e.g. a dialysis membrane.
Furthermore, the sensor of the invention may incorporate an inner membrane
between the enzyme layer and the detection means for selectively preventing
the
passage to the detector of interferant species.
The detecting means may be an electrochemical means, most preferably of the
non-potentiometric type. An amperometric detection is preferred.
The invention will be fiuthcr illustrated by the following 11O11-lllllltlllg
Examples and the accompanying drawings which illustrate the results of the
Example.
As described membranes were prepared from lml of a 5% w/v cellulose
acetate in acetone containing varying amounts (%v/v) Adogen 404. The following
"Conversion Table" gives the amount by weight of the surfactant in the final
membrane (based on the total weight of the membrane).
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Casting Solution COiIIpOSItlOr1/, Adogen 464 by w~eI'~ht
in membrane
(a) 5% Cellulase Acetate/_5%50%
Adogen
(b) 5% Cellulase Acetate/4% 44%
Adogen
(c) 5% Cellulase Acetate/3% 38%
Adogen
(d) S% Cellulase Acetate/2% 29%
Adogen
(e) 5% Cellulase Acetate/I% 17%
Adogen
All subsequent references to the Adogcn (also referred to as MTAC)
concentrations are to the composition of the castily~ solution. Thus for
example
reference in Fig. I to 5'% C:A/5°/« MTAC is to a membrane corresponding
to (a)
above.
E am 1
cuter Membrane for Pyruvate Biosensor
EXPERIMENTAL
Chemicals
Pyruvate oxidase (EC 1.2.3.3) from Pediococca~.s species (75% protein, 80
U.mg-~ protein), albumin (Bovine, Fraction V powder. 98-99% albun-lln),
pyruvic acid
(sodium salt, 99+%}, cocarboxylase (thiamine pyrophosphate chloride, 98%),
flavin
adenine dinucleotide {FAD) (= ~)4'%>). were obtained ti~om Si~~ma (Poole, UK).
Hydrochloric acid, cellulose acetate (39.8% acetyl content). acetone (99.9+%,
HPLC
grade) and Adogen 464 were from Aldrich (Poole, UK). SOdllllll dihydrogen-
phosphate, disodium hydrogen-phosphate. magnesium chloride, sodium hydroxide,
glutaraldehyde (25% solution, EM grade), alL1T111111L111~ OXld2 Wel'e from BDH
(Poole,
UK).
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Buffer
A buffer comprising I 8.4 nnnoles 1 ~ NaH,PO;,.H,O. 8 I .6 mmoles 1-~ MgCI
was prepared in distilled water and adjusted to pH 7.0 with I-IC1 or NaOH. All
solutions were made up in this buffer.
Membranes
Spin Cast Outer Membrane
The outer membrane was formed by spin-coating 1 ml of 5% w/v cellulose
acetate in acetone solution containing 1-5% v/v Adogen 464 onto a 1 cm' piece
of
Cuprophan dialysis membrane {Gambro. Lurid, Sweden} at 1000 rpm for 60s using
a
photo-resist spinner (E/C I O 1 D-R485. Headway Research Inc., Garland. Texas,
USA).
Enzyme Laminate Fubriccrtinn
A composite solution of pyruvate oxidase (POD) (200 U ml-~ }. cocarboxylase
(5 mmoles 1-~), Fad (5 mmole I-~). MgCI, (1 nnnole 1-~j and albLlllllll {0.1 g
ml-~) was
prepared in buffer solution. I OyL of POD-albumin solution and ~yl of
glutaraldehyde
(5% v/v in buffer) were mixed rapidly and placed on a 1 cm' portion of
dialysis
membrane. A further I ellh pOrt1011 Ot dlalySIS Il1e111bralle Was then placed
on top, and
glass plates were used to compress the enzyme film so that it was evenly
distributed
between the membranes. The crosslinlced enzyme ! membrane laminate was allowed
to air-dry for 10 min then washed in buffer to remove excess glutaraldehyde.
The
laminate was used in all experiments) with the additional modified cellulose
acetate-
coated membrane placed on the upperside.
Apparatus and Electrode ~Is.semhly
The amperometric cell (Rank Brothers, Bottisham, UK} consisted of a central
2 mm diameter platinum working electrode with an outer concentric 12 mm
diameter
and 1 mm wide silver ring (Ag/Ag C.'1) as a counter / reference electrode.
Before use,
electrodes were polished with wet and tl~len dry aluminium oxide powder. The
electrodes were then covered with a small volume of buffer ad the enzyme
laminate
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c)
plus the additional outer membrane was placed over the electrodes. The working
electrode was polarised at +650 mV (vs. AgJAgC:I) for the oxidation of
enzymatically
generated H2O2, using a potentiostat (Chemistry Workshops. University of
Newcastle,
UK) with an output to a chart recorder (Lloyd Instruments. Fareham, UK) for
recording of the current/time response.
Pyruvate Response
Baseline current in buffer (~~5 nA) was attained before measurement. 1 ml of 5
mmoles I-~ of buffered pyruvate solution was added to the sample chamber and
the
current/time response was monitored. Between successive exposures the sample
chamber was rinsed three times with buffer and left to recondition 1-cw s0
min.
Resul
Fig. 1 shows the ability of the CA/Adogen (MTAC) membrane to retain the
essential cofactors (cocarboxylase, FAD, Mg'+) allowing a reagentless,
reusable
pyruvate sensor. With a dialysis outer membrane only. the sensor rapidly loses
activity as the cofactors are lost. Fi~~. ? SIIOVVS 1 Sllllllal' effect for a
range Of Adogen
concentrations and contrasts with unmodified cellulose acetate where cofactors
are
retained but the membrane is very impermeable to pyruvate.
x 2
Outer Membrane of Lactate Sensor
The most effective membrane for a reagentless lactate sensor was f ound to be
5%CA/5% Adogen. The method of fabricating the membrane/sensor was the same as
for pyruvate except:
Enzyme Laminate Fabrication
4p1 of lactate dehydrogenase (LDH) ( 1000 U ml~ ~ ) in buffer was mixed with
11p1 of diaphorase (181.5U ml-~) in buffer containing 0.75 mmoles 1-~
potassiLUn
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ferricyanide and 0.75 mmoles 1-~ NAD~~-. lSyL of the composite solution were
dropped onto a lcmZ piece of 0.45yin nixed cellulose ester HA lllelllbl'alle
(Millipore)
and air-dried for 30 min. A further 1 cm~ portion of modified cellulose
acetate-coated
dialysis membrane was then placed on top (see pyruvate), and the laminate was
positioned on the amperometric cell as above.
xam le 3
Outer Membrane for Malate Sensor
The optimum membrane for a reagentless malate sensor was found to be
5%CA/5% Tween-80.
Methods as above, except:
Enzyme Laminate Fahricotion
15p.1 of a composite solution of malatc dehydrogenase (MDH) (2S0 U mI-~),
diaphorase (l25 ml-~), potassium ferricyanide (50 U ml-~ ) and NAD+ (50 U
ml~~) were
dropped into a 1 cm2 piece of 0.451.m mixed cellulose ester HA membrane
(Millipore)
and air-dried for 30 min. A further 1 cm' portion of modif ed cellulose
acetate-coated
dialysis membrane was then placed on top (see pyruvate). and the laminate was
positioned on the amperometric cell as above.
Results
Fig. 3 shows how NAD and ferricyanide are rapidly lost fi~on~ a sensor when a
dialysis membrane is used as an outer membrane. Note that the sensor is
regenerated
by the addition of NAD/ferricyanide in solution. Fig. 4 shows the effect of
varying
Tween content. If the Tween content is too high the membrane is too permeable
and
the cofactor/mediator are lost, and if the Tween content is too low the
membrane is
impermeable to malate.
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