Determining Glucose Content of a Sample
Introduction
A number of metals are known to oxidise carbohydrates under alkaline conditions, and this concept
has been used in commercial applications, such as for example in flow-through detectors used for
monitoring of separation of carbohydrates by HPLC. The literature contains several references that
describe detection of carbohydrates, including glucose, using metals such as platinum , gold, silver
and copper; often involving complex treatments and preparation to modify the metal surface prior to
measurement [Luo et al, Journal of Electroanalytical Chemistry, 1995, v387, pp87-94,
Characterisation of carbohydrate oxidation at copper electrodes; Marioli et al, Electrochim . Acta 1992,
v37(7), pp1 187-1 197, Electrochemical characterisation of carbohydrate oxidation at copper
electrodes; Rahman et al, Sensors, 201 0 , 10 , pp4855-4886, A Comprehensive Review of Glucose
Biosensors Based on Nanostructured Metal-Oxides; Toghill et al, Int. J. Electrochem. Sc , 201 0 , v5,
pp1 246-1 301 , Electrochemical Non-enzymatic Glucose Sensors: A Perspective and an Evaluation ;
Sivasankari et al, International Journal of Pharmacy and Biological Sciences, 201 2 , v2(1 ) , pp1 88-1 95,
NON-ENZYMATIC AMPEROMETRIC GLUCOSE BIOSENSOR BASED ON COPPER
HEXACYANOFERRATE-FILM MODI FIED-GNP-GRAPHITE COMPOSITE ELECTRODE; the
contents of which are incorporated herein]. However to date, there has been no disclosure in the
literature or commercial application or exploitation of the use of unmodified copper metal electrode
technology in a point of care test for the non-enzymatic measurement of glucose in finger prick blood.
Summary of the invention
Relevant paragraphs:
1. A method for determining the glucose content of a sample comprising causing complete
ionisation of the glucose and determining the ionised glucose electrochemically.
2 . A method for determining the glucose content of a sample comprising ionising the glucose
while the sample is in contact with an un-modified copper electrode and determining the quantity of
ionised glucose by detecting changes of current at one or more pre-determined voltage settings.
3 . The method of paragraph 1 or 2 where in the conditions causing ionisation of glucose
comprises alkalisation of the sample.
4 . The method of paragraph 3 wherein the alkalisation comprises increasing the pH of the
sample to at least pH1 4 .
5 . The method of paragraph 3 wherein the alkalisation is caused by mixing the sample with a
strong base.
6 . The method of paragraph 5 wherein the strong base is sodium hydroxide, potassium
hydroxide, barium hydroxide, ammonium, ammonium hydroxide or methylammonium.
7 . The method of any one of paragraphs 1 to 6 wherein the electrochemical detection comprises
electro-catalysis
8 . The method of paragraph 7 wherein the electro-catalysis comprises oxidation of copper.
9 . The method of paragraph 8 wherein the oxidation of copper comprises oxidation of copper
2+ to copper 3+.
10 . The method of any one of paragraphs 1 to 9 wherein the determination is by voltammetry.
11. The method of paragraph 9 wherein the voltammetry is sweeping voltammetry.
12 . The method of paragraph 9 wherein the voltammetry is cyclic voltammetry.
13 . The method of paragraphs 10 or 11 wherein the voltammetry sweeps across a range of 500
to 1200mV.
14 . The method of paragraph 11 or 13 wherein the sweeping voltammetry is forward and/or
reverse sweeping.
15 . The method of any one of paragraphs 1- 14 where in the sample is blood, plasma, serum ,
urine tears, saliva, or CSF.
16 . The method of any one of paragraphs 1 to 15 which further comprises mixing the sample with
a polyion.
17 . The method of paragraph 16 wherein the polyion is a polyanion .
18 . The method of paragraph 16 wherein the polyion is a polycation.
19 . The method of paragraphs 6 where in thee polyion is a polyzwitterion.
20. The method of paragraph 16 wherein the polyion is EDTA and /or, polyethyeleneimine.
2 1. The method of any one of paragraphs 1 to 20 further comprising mixing the sample with a
surfactant.
22. The method of paragraph 2 1 wherein the surfactant is sorbate.
23. A device for determining the glucose content of a sample comprising a sample analysis area
wherein the sample analysis area comprises electrodes and pre-deposited reagent for alkalisation of
the sample.
24. The device of paragraph 23 wherein the electrodes comprise metals or conducting polymers.
25. The device of paragraph 23 or 24 wherein the electrodes comprise copper working electrode,
a silver/silver chloride reference electrode and a platinum counter electrode.
26. The device of paragraph 23 or 24 wherein the working, counter and reference electrodes are
all gold.
27. The device of paragraph 23 or 24 wherein the working and counter electrodes are gold and
the reference electrode is silver/silver chloride.
28. The device of paragraph 23 or 24 wherein the electrodes comprise gold working electrode, a
silver/silver chloride reference electrode and a platinum counter electrode.
29. The device of paragraph 23 or 24 wherein the working, counter and reference electrodes are
all copper.
30. The device of paragraph 23 or 24 wherein the working and counter electrodes are copper and
the reference electrode is silver/silver chloride.
3 1. The device of any one of paragraphs 23 to 30 wherein the copper and platinum electrodes
comprise evaporated film electrodes.
32. The device of any one of paragraphs 23 to 3 1 wherein the reagent for alkalisation of glucose
comprises a strong base.
33. The device of paragraph 32 wherein the strong base comprises sodium hydroxide, potassium
hydroxide, Barium hydroxide, ammonium , ammonium hydroxide or methylammonium .
34. The device of any one of paragraphs 23 to 33 wherein the reagent for alkalisation of glucose
further comprises a polyion.
35. The device of paragraph 34 wherein the polyion comprises EDTA and or polyethyleneimine.
36. The device of any one of paragraphs 23 to 35 wherein the reagent for alkalisation for the
sample further comprises a surfactant.
37. The device of any one of paragraphs 23 to 36 wherein the electrodes and reagent for
alkalisation of the sample are physically separate but fluidically connected.
38. The device of any one of paragraphs 23 to 37 where the electrodes are capable of electrocatalysis
of ionised glucose.
39. The device of paragraph 25 wherein the electrodes comprise alternative electrode
arrangements.
40. The device of any one of paragraphs 23 to 29 wherein glucose is determined
electrochemically following ionisation and electrocatalysis of glucose.
4 1. The device of any one of paragraphs 23 to 40 wherein the glucose can be determined at
more than one electrode potential.
42. A biosensor, comprising ;
a base layer having disposed thereon at least one conductive track extending from a first end
to a second end, wherein the conductive track comprises copper;
an assay zone at the first end of the base layer, comprising a reagent capable of increasing
the pH of a sample applied to the assay zone;
a terminal at the second end of the base for connection of the at least one conductive track to
a processor.
43. The biosensor of paragraph 42 further comprising a capillary chamber at the first end for
receiving a sample of body fluid, wherein the capillary chamber is disposed over the assay zone such
that a portion of the at least one conductive track is exposed within the capillary chamber.
44. The biosensor of paragraph 42 or 43, wherein the base layer has disposed thereon at least
three conductive tracks, each conductive track being electrically insulated from the other.
45. The biosensor of paragraph 44 wherein the at least three conductive tracks comprise copper
and wherein a portion of the at least three conductive tracks is exposed within the capillary chamber,
and further wherein the capillary chamber contains the pH altering reagent.
46. The biosensor of any one of paragraphs 43-45 wherein the pH altering reagent is disposed on
an inner surface of the capillary chamber.
47. The biosensor of any one of paragraphs 44-46 wherein the pH altering reagent is disposed on
the base layer, but not in contact with the at least three conductive tracks within the capillary
chamber.
48. The biosensor of any one of paragraphs 43-45 wherein the pH altering reagent is disposed
within the capillary chamber.
49. The biosensor of any one of paragraphs 42-48 wherein the at least three conductive tracks
define at least one measurement electrode, at least one reference electrode and at least one counter
electrode, and wherein the measurement electrode, counter electrode and reference electrode are
located within the capillary chamber in the assay zone.
50. A method, comprising :
ionizing glucose present in whole blood ; and
electrochemically determining the presence of the ionized glucose in the whole blood.
5 1 . The method of paragraph 50, wherein ionizing the glucose comprises combining the whole
blood with a dried reagent.
52. The method of paragraph 5 1, wherein the dried reagent is present in an amount sufficient to
increase the pH of the whole blood by an amount sufficient to ionize the glucose.
53. The method of any one of paragraphs 50-52, wherein the electrochemically determining is
performed in a chamber having a total volume of less than about 5 microliters.
54. The method of any one of paragraphs 50-53, wherein the electrochemically determining
comprises electrochemically determining the ionized glucose via an electrochemical circuit comprising
at least one copper electrode in contact with the whole blood .
55. The method of any one of paragraphs 50-54, wherein the method is performed in the absence
of enzymes/mediators.
56. A test strip for determining the presence of glucose, comprising :
a capillary chamber defining a total volume of less than about 2.5 microliters;
at least one copper electrode in electrochemical communication with the capillary chamber; and
a dried reagent present in an amount sufficient to increase a pH of a whole blood sample introduced
into the capillary chamber and filling the volume of the capillary chamber by an amount sufficient to
ionize glucose present in the whole blood.
57. The device of paragraph 56 wherein the test strip comprises three copper electrodes
configured as:
i) a working electrode at which measurement of glucose oxidation occurs;
ii) a counter electrode, which supplies or consumes electrons in response to the reaction at
the working electrode; and
iii) a reference electrode, which acts to monitor and maintain the potential applied between
the working electrode and counter electrode.
58. The device of paragraphs 56 or 57 wherein the capillary chamber defines a volume of less
than about 2 microlitres.
59. The device of paragraphs 56 or 57 wherein the capillary chamber defines a volume of less
than about 1 microlitre.
60. The device of paragraphs 56 or 57 wherein the capillary chamber defines a volume of less
than about 0.5 microlitres.
6 1 . The device of any one of paragraphs 56 to 60 wherein the dried reagent is disposed on a
surface of the capillary chamber not in direct contact with the one or more copper electrodes.
62. The device of any one of paragraphs 56 to 6 1 wherein the dried reagent comprises base and
a surfactant.
63. The device of paragraph 62 wherein the surfactant is polyvinyl alcohol and the base is sodium
hydroxide.
64. A method of determining the quantity of glucose in a sample of blood obtained from a finger
prick or alternate site using a device of paragraphs 56 to 63, comprising ;
removing the test strip from a storage compartment;
inserting the test strip into a meter and following the instructions presented on the display of
the meter;
pricking a finger or alternate site to release a drop of blood ;
contacting the drop of blood with the sample port on the test strip;
removing the test strip from the drop of blood when the meter indicates sufficient sample has
been acquired on the test strip;
allowing the blood to react in the test strip for at least 1 second ; and
displaying a blood glucose concentration on the display of the meter.
65. The method of paragraph 64, wherein the blood reacts in the test strip for at least 3 seconds
before a glucose concentration is displayed.
66. The method of paragraph 64, wherein the blood reacts in the test strip for at least 5 seconds
before a glucose concentration is displayed.
67. The method of paragraph 64, wherein the blood reacts in the test strip for at least 7 seconds
before a glucose concentration is displayed.
68. The method of paragraph 64, wherein the blood reacts in the test strip for at least 10 second
before a glucose concentration is displayed.
69. The method of any one of paragraphs 64 to 68 wherein no more than 2.5 microlitres of blood
has been acquired on the test strip.
70. The method of any one of paragraphs 64 to 68 wherein no more than 1.5 microlitres of blood
has been acquired on the test strip.
7 1 . The method of any one of paragraph 64 to 68 wherein no more than 1 microlitre of blood has
been acquired on the test strip.
72. The method of any one of paragraphs 64 to 68 wherein no more than 0.5 microlitres of blood
has been acquired on the test strip.
Description of the Figures
Fig 1 shows an example of a general 3-electrode design according to the invention.
Fig 2 shows an expanded area of Fig. 1 showing the electrode design which will be exposed to the
sample for testing.
Fig 3 : diagram to show the position of the block mask to leave an enlarged exposed electrode area.
Fig 4 : diagram to show the position of a typical capillary chamber located over the 3-electrode design.
Fig 5 : current response for low range of glucose in whole sheep blood using 3x copper electrodes
(WE.CE. RE).
Fig 6 : current response for high range of glucose in 0.5M NaOH using 3x copper electrodes
(WE.CE. RE).
Fig 7 : current response from fast chrono method showing the high range glucose response.
Fig. 8 : Mean ACuTEGA signals in whole sheep blood, spiked with various glucose concentrations,
showing SD and CofV for each value (n = 5 for each point).
Fig. 9 : Current/time curves of repeat ACuTEGA glucose assays in glucose-spiked sheep blood to
show speed of response and precision (repeatability).
Fig 10 : Mean ACuTEGA signals in whole sheep blood , spiked with glucose at 1, 3 and 5mM to prove
adequate performance of the system in the clinically important range (n = 5 for each point).
Fig 11: Comparative ACuTEGA signal responses from glucose and maltose under identical
conditions. Note that 15mM maltose gives the same signal as 1mM glucose.
Fig 12 : Dose response profiles of the ACuTEGA system across the most clinically relevant range of
0-1 OmM
Fig 13 : Dose response profiles of the ACuTEGA system up to 30mM
Detailed Description
A new non-enzymatic approach to measuring glucose has been developed and is disclosed herein.
The non-enzymatic measurement of glucose is based on the direct oxidation of glucose using
unmodified copper metal electrodes. A potential is applied to a copper measurement/working
electrode, which potential is monitored by a separate reference electrode and the current within the
system is balanced with a counter electrode. The presence of the ionized glucose in the sample can
then be determined electrochemically. Disclosed herein are methods, devices, and test systems
using this novel approach.
Several exemplary embodiments of copper based measurement systems are described in Table 1. In
a first aspect a copper working electrode is used in combination with a silver/silver chloride reference
electrode and a platinum counter electrode. In a second embodiment, a copper working electrode is
used in combination with a silver/silver chloride counter/reference electrode. In a third aspect a
copper working electrode is used in combination with a copper counter/reference electrode. And, in a
fourth aspect a copper working electrode is used in combination with a copper reference electrode
and a copper counter electrode.
Table . Copper-based measurement systems
An exemplary copper-based measurement system is based on the All Copper Triple Electrode
Glucose Assay (ACuTEGA) technology. Without wishing to be bound by any theory, ACuTEGA may
work by directly oxidising glucose which has been converted into an anionic state at a pH sufficient to
ionize the glucose. For example, at a pH of about 13 to 14 , glucose is subject to electrocatalytic
oxidation, peaking at a potential around 900mV (vs copper reference), yielding 6 formate molecules
and 12 electrons for each glucose molecule oxidised. Such an oxidation process yields three or six
times the number of electrons per glucose molecule oxidised when compared with more traditional
enzyme based self-monitoring blood glucose sensors. Consequently it is expected the measurement
of glucose using an ACuTEGA device may allow for more sensitive determination of glucose at lower
concentration than might be achieved using more traditional measurement modalities, leading to
improved measurement performance.
Under conditions sufficient to ionize glucose in a sample using the novel approach described herein,
electrochemical determination of the ionized glucose is not impaired by factors known to interfere with
traditional glucose measurements. For example, at pH values in the order of 13 to 14 there is no
apparent response detected on the copper electrode from species such as ascorbate, paracetamol,
urate, dopamine, etc., which are known to interfere with measurement of glucose at pH close to
neutral. Furthermore measurements made using copper electrodes at pH in the region of 14 appears
to be unaffected by the haematocrit of the blood under test; which is another factor known to
compromise measurement of glucose in traditional enzymic sensor devices. An apparent increase in
viscosity of blood that occurs when the pH of the sample is raised to at least 14 , appears to cause the
blood to be held tightly in the reaction chamber of the test strip. This apparent increase in viscosity
appears to negate any effect that haematocrit may otherwise have on the resultant signal measured
by the electrode during the oxidation of glucose to formate.
In one aspect a method for determining the glucose content of a sample comprising causing complete
ionisation of the glucose and determining the ionised glucose electrochemically, is described. The
glucose content of the sample is typically determined by completely ionising the glucose in the sample
while it is in contact with an un-modified copper electrode; the quantity of ionised glucose is
determined by detecting changes of current at one or more pre-determined voltage settings. The
conditions causing ionisation of glucose typically involve alkalisation of the sample; and the pH of the
sample is often increased to at least 13 or 14 through the mixing of a strong base, such as for
example sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, barium
hydroxide, ammonium, ammonium hydroxide or methylammonium.
The electrochemical detection of glucose oxidation in alkaline solution may be achieved using cyclic
voltammetry, chronoamperometry or like techniques which monitor the flow of current when a
potential is applied to a working or measurement electrode at which oxidation of the glucose occurs.
In one aspect the oxidation of glucose on a copper electrode may follow a process where the copper
is changed from copper 2+ to copper 3+. Typically an applied potential in the range of +500 to
+ 1200mV may be used, depending on the reference electrode being utilised. For example a
silver/silver chloride reference electrode may require a different potential be applied compared with
using a copper reference electrode.
The strong alkali may be formulated additional additives that may aid drying and resuspension of the
dry reagent upon sample addition ; such agents may include a polyion, such as a polyanion, a
polycation, or a polyzwitterion. In some formulations the polyion may be either EDTA and /or,
polyethyeleneimine. The formulation may further include a surfactant, such as for example sorbate,
polyvinyl alcohol, saponin.
In another aspect a device for determining the glucose content of a sample that includes a sample
analysis area, which includes one or more electrodes and pre-deposited dried reagent for alkalisation
of the sample is disclosed . The electrodes may be formed using metals or conducting polymers,
including for example, platinum , gold, silver, copper, zinc, ruthenium, palladium , poly(3,4-
ethylenedioxythiophene), polypyrrole, polyaniline, polythiophene. In some embodiments the
electrodes may include a copper working electrode, a silver/silver chloride reference electrode and a
platinum counter electrode; or the working, counter and reference electrodes may all be formed from
gold. In other embodiments the working and counter electrodes may be formed from gold and the
reference electrode may be of silver/silver chloride; or the electrodes may include a gold working
electrode, a silver/silver chloride reference electrode and a platinum counter electrode. In an
exemplary embodiment the working, counter and reference electrodes are all formed from copper; or
the working and counter electrodes may be formed from copper and the reference electrode from
silver/silver chloride. In some embodiments the electrodes and reagent used for alkalisation of the
sample are physically separate but fluidically connected ; in other cases the reagents are deposited
directly over the electrodes. In general, the materials from which the electrodes are made will be
capable of direct measurement of any ionised glucose in the sample, leading to a signal that is
proportional to a concentration of glucose present.
In an exemplary embodiment, disclosed is a device for the quantitative determination of blood glucose
in a sample. For example, the device can be used for determination of glucose in a sample of whole
blood. The device may also be used to determine the presence of glucose in plasma, serum , urine
and other fluid samples. Whole blood can be readily obtained from a finger prick or other alternate
site that is readily accessible, using a lancing device available for personal use. Blood may also be
obtained by a suitably qualified phlebotomist using venipuncture. The device utilizes copper
electrodes to determine glucose within the sample with no requirement for enzymes or mediator
compounds. The device may be a test strip including a capillary chamber, at least one copper
electrode, and a dried reagent. In some embodiments, the capillary chamber is in electrochemical
communication with the at least one copper electrode. In some embodiments, the dried reagent is
present in the capillary chamber. The dried reagent may be present in an amount sufficient to
increase the pH of the sample, for example whole blood sample, introduced into the capillary chamber
to at least 13 and more preferably to at least 14 . The capillary chamber may define a total volume of
less than 5 ul, less than 4 ul, less than 3 ul, less than 2.5 ul, less than 1.5ul, less than 1ul, less than
0.5ul.
A device such as a test strip can be stored individually or as a package of strips. A test strip can be
used with a meter. For example a test strip can be removed from its packaging or storage
compartment and then inserted into a meter. A user would typically use a test strip to determine the
quantity of glucose in a sample of blood obtained from a finger prick. The user would first remove the
test strip from a storage compartment, which may be an individual foil pouch or similar containment
means designed to keep the strip "dry", or which may be a vial that holds several test strips, which
contains a desiccant material to maintain the strips in a "dry" atmosphere. Once removed from the
protective container, the user would insert the test strip into a meter and following the instructions
presented on the display of the meter. Such instructions will typically indicate the following : prick a
finger or alternate site to release a drop of blood ; discard the first one or two droplets of blood ; contact
the drop of blood with the sample port on the test strip; remove the test strip from the drop of blood
when the meter indicates sufficient sample has been acquired ; wait for the blood to react within the
test strip; read the glucose concentration on the display of the meter. The time taken for the blood
sample to react within the test strip before the meter displays a glucose reading to the user is typically
less than 10 seconds, and more often less than 7 seconds, generally less than 5 seconds and may
even be less than 3 seconds and may even be less than 1 second . The technology is thus well suited
to providing rapid measurement results, which may be critical in certain circumstances.
Also disclosed herein are biosensors comprising a base layer, an assay zone, and a terminal. The
biosensor, can include a base layer having disposed thereon at least one conductive track which
extends from one end to the other end of the base layer. The conductive track may be formed using
copper. The biosensor also includes an assay zone at one end of the base layer, which may include a
dried reagent that is capable of increasing the pH of a sample applied to the assay zone. A terminal at
the other end of the base layer is used for making a connection of the at least one conductive track to
a microprocessor in an analysis device or meter with which the biosensor is intended to be used.
Typically the biosensor will have a capillary chamber at the one end for receiving a sample of body
fluid ; the capillary chamber is frequently located over the assay zone such that a portion of the at least
one conductive track is exposed within the capillary chamber. Therefore when a sample is applied to
the biosensor, the sample will be collected within the capillary chamber, where it will make contact
with the conductive track. In some cases the biosensor can have at least three conductive tracks one
the base layer, with each of the conductive tracks being electrically insulated from the other. In a
particular embodiment the biosensor includes at least three conductive tracks that are formed using
copper metal, with at least a portion of the three separate conductive tracks being exposed within the
capillary chamber and thus accessible for direct contact with a sample applied to the biosensor.
Frequently the capillary chamber will include a dried reagent that can alter the pH of a sample applied
to the biosensor. The pH altering reagent is typically dried on an inner surface of the capillary
chamber; however the pH altering reagent can also be dried down on the base layer, but not in direct
contact with the at least three conductive tracks within the capillary chamber. The conductive tracks
will generally represent at least one working or measurement electrode, at least one reference
electrode and at least one counter electrode, and each of these will exist within the confines of the
capillary chamber in the assay zone.
The disclosure further defines a method of measuring glucose that might be present in a sample of
whole blood. The method, generally involves completely ionizing any glucose that may be present in a
sample of whole blood and then electrochemically determining the presence of the ionized glucose in
the whole blood . The process of ionizing the glucose includes combining the whole blood with a dried
reagent, which dried reagent is present in an amount sufficient to increase the pH of the whole blood
by an amount sufficient to ionize the glucose. The process of electrochemically determining the
quantity of ionised glucose is performed in a chamber having a total volume of less than about 5
microliters, more often than not the chamber has a volume of less than 2.5ul, and in many cases a
volume less than 1ul. The electrochemical determination of the ionized glucose can be achieved
using an electrochemical circuit that includes at least one copper electrode which will be in contact
with the whole blood. One aspect of the disclosed method is that it does not require the presence of
either enzymes or mediators that are utilised in many commercial systems for self-monitoring blood
glucose.
The disclosure also includes description of a test strip for determining the presence of glucose in a
fluid sample obtained from a human subject. The test strip includes a capillary chamber which defines
a total volume of typically less than about 2.5 microliters, and more frequently less than 1 microliter
and in some cases less than 0.5 microliters. The test strip also includes at least one copper electrode
in electrochemical communication with the capillary chamber; along with a dried reagent present in an
amount sufficient to increase a pH of a whole blood sample introduced into the capillary chamber and
filling the volume of the capillary chamber by an amount sufficient to ionize glucose present in the
whole blood. The test strip will often include at least three copper electrodes that are arranged as: i) a
working electrode at which measurement of glucose oxidation occurs; ii) a counter electrode, which
supplies or consumes electrons in response to the reaction at the working electrode; and iii) a
reference electrode, which acts to monitor and maintain the potential applied between the working
electrode and counter electrode. The dried reagent is generally present on a surface of the capillary
chamber not in direct contact with the one or more copper electrodes, and it may contain an alkali or
base and a surfactant. The base can include sodium hydroxide, potassium hydroxide, calcium
hydroxide, magnesium hydroxide, barium hydroxide, ammonium, ammonium hydroxide or
methylammonium, and the surfactant can include sorbate, polyvinyl alcohol, or saponin.
Examples
Test method :
Two different electrochemical tests, cyclic voltammetry (CV) and chronoamperometry (Chrono) were
used to characterise the performance of copper working electrodes for direct measurement of glucose
under alkali conditions. CV conducts a 3V potential sweep while Chrono applies a single, fixed
potential. Both methods have given good detection of glucose in both buffer and blood environments.
Electrode Preparation:
Copper coated polyester was supplied from Vacuum Depositing Inc. (VDI LLC (Louisville, Kentucky,
USA)). A polyester (polyethylene terephthalate (PET)) sheet was used (Lumirror T62, 750gauge
nominal (~190microns)) as the base layer. A tie layer of Chromium and Nickel was sputter coated to
act as a bonding layer to improve the adherence of the copper layer to the PET. Following this,
copper was sputter coated onto the Cr/Ni tie layer. The tie layer was approximately 3-5nm in thick, the
copper layer was used with a maximum thickness of about 40nm . No treatment or modification of the
pure copper metal surface was performed. The stock copper metal coated polyester supplied by VDI
LLC was delivered as a real of material, from which devices for testing were prepared.
In an exemplary embodiment, test sensors were prepared by first removing a section of material
approximately 16 cm x 16 cm from the master real, being careful not to contaminate the surface.
Articles were ultimately cut into strips approximately 5mm wide by about 35mm long. The strips of
copper coated polyester were pattered using laser etching to define two or more individual electrically
insulated tracks; one end of which was used to make electrical connection with a potentiostat or meter
that supplied the required voltage polarisation to perform CV or Chrono, as well as acquiring the
resultant current corresponding to the oxidation of glucose.
Three separate electrodes (WE, RE and CE) were defined by laser etching, using a Ulyxe laser
etching system (Datalogic Automation (supplied by Laserlines Ltd (UK)) was used. The Ulyxe has a
6w YAG laser, operated at a wavelength of 1064nm which was demonstrated to cleanly remove both
the copper and Cr/Ni tie layer from the PET backing , thus revealing the PET in regions exposed to
laser energy. The laser system was typically operated used with the following settings: power (80%),
frequency (20,000Hz), scan speed (500mm/s), dot delay (5 is), shot time (I . m ) , with only a single
pass. The lens used was an F254. The Ulyxe was used in-conjunction with a filter extraction system,
which removes the vapour debris emitted by the ablation steps.
Several designs of electrodes were investigated , each with slight variation in the area of copper metal
exposed for each electrode surface. An exemplary design is shown in Figure 1.
The configuration of the individual electrodes is shown in more detail in Figure 2 . The RE is positioned
at the centre of the array, which is in turn surrounded by the WE which itself is surrounded by the CE.
Depending on how the electrode was used, different masking was applied. Under some
circumstances a capillary chamber having a volume of no more than 2.5 microlitres was adhered
directly over the electrodes. On other occasions a capillary chamber having a volume of no more than
1 microlitre was applied over the electrodes. In general, the end of the electrode is masked off with
the use of a non-conductive adhesive tape, or a dielectric insulating ink. Figures 3 and 4 depict
different approaches to masking off portion of the copper metal as a way of controlling the surface
area of metal that may be contacted by a sample.
Once a series of electrodes have been defined on the PET substrate, they were masked with
insulating material as shown in Figures 3 and 4 , and cut from the master sheet to give a sensor with
typical dimensions of 35 x 5.5mm.
Hardware:
The following equipment was used.
• Potentiostat:
o Supplied by Whistonbrook Technologies. Product name is Ezescan. The model typically used
is the Ezescan 4 . It is a single test potentiostat, with inputs for WE, RE and CE. Software is supplied
with the instrument, which allows CV and Chrono methods to be performed. A user interface allows
parameters to be determined by the user.
• Sensor connection :
o A 9-pin D-sub type connector was used for connection to the Ezescan 4 potentiostat. 7-strand
copper core wire (conductor area = 0.22mm 2) was used for all wiring. A pcb vertical slide connection
socket, with 1.27mm pitch between the pins was used for connection to the copper electrode.
Materials:
Sodium hydroxide: any high quality, low impurity grade can be used. For example, Sigma-Aldrich
Code S5881 , >98% purity.
Potassium hydroxide: any high quality, low impurity grade can be used. For example, Sigma-Aldrich
Code 48401 6 , >90% purity
Analytical water: < 15MOhm.
Glucose: any high quality, low impurity grade can be used. For example, Sigma-Aldrich Code G8270,
>99.5% purity.
General purpose microtitre plate (or any equivalent small volume container).
Measurement method for glucose in buffer:
The following procedure was performed when measuring glucose in aqueous buffer samples. The
example describes testing with a masked electrode as shown in Fig 3 .
1. Individual electrodes are prepared as described under the electrode preparation section.
2 . Hydroxide solution is prepared by dissolving pellets in analytical water to give 4M
concentration . Preferred cation is potassium , although sodium may also be used.
3 . Glucose solution is prepared by dissolving powder in analytical water to give 1M
concentration .
4 . To an individual microtitre plate well, volumes are dispensed to give a final volume of 200m I.
This volume is sufficient to cover the exposed area of the electrodes when it is submerged to the
masked area. The volume is not critical , but there should be sufficient to cover the exposed
electrodes.
a . Add hydroxide solution to give the required concentration, for example 0.5M. For example,
25m I of 4M stock solution in 200m I final volume.
b. Add glucose solution to the well to give the required concentration , for example, 12m I of 1M
stock in 200m I final volume to give 30mM final concentration. Further volumes of glucose are added to
wells to give differing glucose concentrations.
c . Make the volume up to 200m I with analytical water. Aspirate the well to ensure all solutions
are mixed well.
5 . Take the connection lead, and plug into the potentiostat.
6 . Take a single, masked electrode and slide into the connector block, ensuring the electrodes
are lined up correctly with the connector pins.
7 . Using the user interface with the potentiostat software, choose the method to be used for the
test, for example, cyclic voltammetry. Ensure the settings are correct, for example the following
settings are typically used :
a . Potential sweep range: - 1500mV forward sweep to + 1500mV with reverse sweep back to -
1500mV.
b. Step interval = 10ms
c . Potential step = 10mV
d . Scan rate equivalent to 1v/s.
8 . Dip the end of the electrode into the test solution, ensuring the exposed area of the sensor is
submerged in the test solution. Only submerge the electrodes when the test is ready to be performed.
Ensure no air bubbles are trapped or attached to the surface of the electrode.
9 . Start the scan, holding the electrode as still as possible to prevent movement of the test
sample across the surface of the electrode. The aim is to conduct the test under static conditions.
10 . After the scan has been completed, remove the electrode from the test solution and
connector and discard.
11. Save the data file.
12 . The data is typically imported into a graphics package such as Microsoft Excel. The data is
plotted as potential (mv, x-axis) vs current (mA, y-axis). Multiple graphs may be plotted to examine
trends throughout the sweep profiles. In addition, specific data (current) can be extracted from the
data set which relate to specific peaks which correspond to responses from changes in the presence
of glucose.
Measurement method for glucose in whole blood:
If blood is to be tested, the analytical water used as described above is replaced with 200 m I whole
blood. Typically the blood is collected into citrate-only tubes. Sodium citrate is used as the ant i
coagulant, with a final concentration of approximately 0.3%. The whole blood is stored cooled at 4-
8°C, until used. If a zero glucose baseline is required, the blood is placed in a 37° C incubator and
monitored with a commercial glucose detection device until the reading is too low to read (typically
< 1mM glucose). Glucose may then be spiked back into the depleted blood to give known
concentrations of soluble glucose. Differences in the volume of glucose added to the blood sample
are compensated for by additional water.
The following procedure is performed when measuring glucose in whole blood samples. The example
describes testing with a masked electrode as shown in Fig 3 .
1. Individual electrodes are prepared as described under the electrode preparation section.
2 . Hydroxide solution is prepared by dissolving pellets in analytical water to give 4M
concentration . Preferred cation is potassium , although sodium may also be used.
3 . Glucose solution is prepared by dissolving powder in analytical water to give 1M
concentration .
4 . To an individual microtitre plate well, volumes are dispensed to give a final volume of 200 m I.
This volume is sufficient to cover the exposed area of the electrodes when it is submerged to the
masked area. The volume is not critical , but there should be sufficient to cover the exposed
electrodes.
a . Add the blood sample to the well.
b. Add glucose solution to the well to give the desired concentration, for example, 12m I of 1M
stock in 200 m I final volume to give 30mM final concentration. Further volumes of glucose are added to
wells to give differing glucose concentrations.
c . Aspirate the well to ensure all solutions are mixed well.
5 . Take the connection lead, and plug into the potentiostat.
6 . Take a single, masked electrode and slide into the connector block, ensuring the electrodes
are lined up correctly with the connector pins.
7 . Using the user interface with the potentiostat software, choose the method to be used for the
test, for example, cyclic voltammetry. Ensure the settings are correct, for example the following
settings are typically used :
a . Potential sweep range: - 1500mV forward sweep to + 1500mV with reverse sweep back to -
1500mV.
b. Step interval = 10ms
c . Potential step = 10mV
d . Scan rate equivalent to 1v/s.
8 . Just prior to testing, add hydroxide solution to the blood to give the desired concentration, for
example 0.5M. To achieve this, add 25m I of 4M stock solution in 200m I final volume. Mix quickly,
because the effect of the sharp rise in pH in the blood is that the blood becomes very viscous and
gelatinous.
9 . Dip the end of the electrode into the test solution, ensuring the exposed area of the sensor is
submerged in the test solution. Only submerge the electrodes when the test is ready to be performed.
Ensure no air bubbles are trapped or attached to the surface of the electrode.
10 . Start the scan, holding the electrode as still as possible to prevent movement of the test
sample across the surface of the electrode. The aim is to conduct the test under static conditions.
11. After the scan has been completed, remove the electrode from the test solution and
connector and discard.
12 . Save the data file.
13 . The data is typically imported into a graphics package such as Microsoft Excel. The data is
plotted as potential (mv, x-axis) vs current (mA, y-axis). Multiple graphs may be plotted to examine
trends throughout the sweep profiles. In addition, specific data (current) can be extracted from the
data set which relate to specific peaks which correspond to responses from changes in the presence
of glucose.
Chronoamperommetry measurement of glucose:
A fast chrono method may be used for fixed potential interrogation of the sample. Typically this fixed
applied potential is +900mV, although this should be optimised to reflect the format of the electrode
array.
The basic method of sample preparation is the same as described for the cyclic voltammetry
methods.
The method used is Fast Chrono with the following parameters:
• Potential : +900mV
• Step 10ms
• Time to complete the test: 5 seconds.
TYPICAL RESPONSES:
Cyclic voltammetry data:
Fig 5 shows an example of the glucose response, using a laser ablated electrode array, in the
presence of whole sheep blood in 0.5M NaOH . The range tested was 0-1 OmM to demonstrate the
differentiation that was possible with this format.
Fig 6 shows an example of the glucose response using a laser ablated electrode array, in 0.5M NaOH
only. The range tested was 0-30mM to demonstrate the high range linearity of the format.
Chronoamperometry data:
Fig 7 : fast chrono method was used with an applied potential of +900mV. In this example, individual
electrode strips were used rather than a laser ablated array. The result demonstrates the linearity of
the glucose response using the chrono single potential method.
The graphs above demonstrate a typical response to the addition of glucose to both just the 0.5M
NaOH and to whole sheep blood with 0.5M NaOH.
ACuTEGA in general operation: For general testing of the devices depicted in Figure 3 , the fast
chrono mode is used, with the potential poised at around +900mV vs copper reference. A strip is
connected to a reader using a push fit connector, after which typically less than 1m I_ of finger-stick
blood is applied to the end of the strip. As the blood flows into the capillary chamber, it meets and
rehydrates the dried sodium hydroxide en-route to the electrode array. An exemplary design of the
electrode array as shown in figures 3 and 4 , was used. Rehydration of the hydroxide reagent is near
instantaneous, causing rapid ionisation of glucose, which typically permit a glucose measurement in
less than 5 seconds, frequently less than 3 seconds, and regularly requires less than 1 second from
the time of sample introduction to determine a glucose concentration within the sample. The data
shown in Figure 8 represent a dose response curve when glucose was spiked into glucose depleted
sheep blood. The chrono time-course profiles for each measurement signal was captured over 5
seconds. The time/current curves are shown in figure 9 , which clearly show both the rapid response
and the reproducibility of the signal in ACuTEGA. In particular it can be seen that stable responses
are achieved after just 1 second ; allowing determination of the glucose content of the sample to be
determined at such time point.
For routine glucose testing by diabetic subjects, it will be essential to gain good discrimination and
linearity at glucose levels below 10mM and ideally below 5mM - the recommended target level for
blood glucose; in this context a series of blood samples spiked with 1mM, 3mM and 5mM glucose
were prepared and assayed. The data are shown in figure 10 .
The ACuTEGA system has been shown to be unaffected by interference from the usual interfering
substances that cause problems for enzyme driven tests (paracetamol, ascorbate and urea etc. , data
not shown), but market forces now requires that glucose tests should discriminate between glucose
and maltose. Maltose is a 1,6-linked glucose dimer, and it can sometimes be found in patients who
are receiving peritoneal dialysis (who are given intra-peritoneal maltodextrin solutions as "osmotic
agents", known as "lcodextrin") and very ill cancer patients (who receive oncology medication in which
maltodextrin is present as an excipient). There have been rare but high-profile cases in which PQQglucose
dehydrogenase based enzyme sensors have given falsely elevated readings for glucose,
leading to excessive insulin dosing. This is due to the lack of specificity of PQQ-GDH, which will utilise
maltose as a substrate in place of glucose. It is reported that maltose levels as high as 3mM can be
found. To the best of our knowledge, higher maltose levels are not encountered.
To demonstrate that ACuTEGA has adequate discrimination against maltose, calibration solutions for
each sugar were prepared with concentrations between 1mM and 30mM. These were assayed by
ACuTEGA under identical conditions, giving the results shown in Figure 11.
The results in figure 11 indicate that at the high pH necessary for the operation of the ACuTEGA
system, maltose at clinically relevant concentrations shows a much lower electrochemical response
compared with glucose. With such a difference in response, one can be confident that an ACuTEGA
glucose value in a patient with maltose as high as 30mM ( 10 times the maximum reported clinical
level) would at most be compromised by about 1mM, which would not lead to a miss-reporting of
blood glucose that would either wrongly deny glucose in a hypoglycaemic state, or wrongly identify a
hyperglycaemic state, resulting in an overdose with insulin.
Creation of 1 I volume capillary chambers that reliably fill with whole blood from a finger-prick
o Capillary chamber tops (self-adhesive) are standard units, and reels of suitable materials
have been acquired on a research scale.
o Approximately 1m I_void volumes are created, using hydrophilic capillaries.
o Dried reagents are placed within the capillary chamber, which in turn are rehydrated when the
test sample enters the capillary space.
Reliable deposition of solid sodium hydroxide within the chamber without corroding the ultrathin
copper film and without impeding capillary filling.
o Pre-dosing the electrode chamber with correct volume and concentration of sodium hydroxide
is critical to test operation.
o The pH of the whole blood sample has to be raised above the ionisation point of glucose,
higher than pH 13 , before a measurement is taken (less than 5 seconds).
o To achieve stability, hydroxide has to be present as a dry reagent presenting several issues:
■ Hydroxide contact with copper initiates a destructive process, so dry hydroxide cannot be
stored in direct contact with the electrode surface.
■ Dry hydroxide has been used in submarines and spacecraft as a C02 scrubber, in which the
hydroxide reacts rapidly with carbon dioxide to form sodium carbonate. This reaction also occurs in
ACuTEGA chambers when are open to the atmosphere. If the storage atmosphere is uncontrolled,
over time, the pH of the dry reagent drops. If substantial conversion occurs, the blood pH is not
raised high enough to ionise glucose.
o Drying hydroxide pure from simple aqueous solution results in crystal formation that are too
large to dissolve quickly (within seconds) to allow a measurement within the target timescale.
o It was discovered that a carrier, or a "dispersing agent", was required . A detergent, Proteric-
JS, is used to allow the hydroxide to dry as far smaller crystals, thus increasing the surface area such
that when the blood is applied the hydroxide can quickly dissolve.
Dosing of hydroxide without loss of potency in storage (through reaction with carbon dioxide)
with instant, uniform alkalinisation of the blood.
o The surface area to volume (of the dry film) ratio is very large. For this reason, even though
C02 concentration in air is low (-0.04%) enough is absorbed to force a pH drop. To overcome this,
the pre-dosed sensors are packaged in the presence of molecular sieve. This material reduces the
moisture content of the air within the packaging to almost complete dryness and simultaneously
absorbs C02.
o The dried reagent is located on the capillary chamber surface, rather than directly on the
copper electrode surface. Direct deposition of the hydroxide reagent onto copper is not effective due
to the corrosive nature of the hydroxide.
o In practice, the pre-dosed dry hydroxide almost instantly dissolves into the blood, raising the
pH sufficiently to allow the copper oxidation chemistry to work.
o
Performance of the dried, pre-dosed system with 1uL chambers
The dried system operating with capillary chambers manufactured by hand on small-scale is
vulnerable to some variation compared to electrodes of similar dimensions that are operated with wet
reagents and larger sample volumes. Thus, the capillary chamber versions were subjected to
rigorous performance testing to understand impact of manufacturing parameters on the resuspension
of the dried reagents within the capillary chambers. The following data were obtained using fully dried
and miniaturised devices.
Linearity: Excellent linearity is observed when testing either 0 - 10mM (short range) and 0 - 30mM
(long range) in whole blood, as shown in figures 12 and 13 respectively.
Correlation of ACuTEGA with a reference device:
The ACuTEGA device is used to measure glucose in blood during a non-fasting glucose tolerance
test. A non-diabetic volunteer consumes a glucose containing drink. A finger-prick blood sample is
tested by ACuTEGA, the YSI STAT Plus analyser, and a commercial self test blood glucose systems,
the Bayer Contour XT.
Capillary blood is drawn via lancet puncture of a finger. A 1m I_ drop of blood is applied to the
ACuTEGA capillary chamber. Electrochemical measurements are made by the "fast chrono" method,
as previously described. Another sample of blood from the same puncture is also measured by the
YSI analyser and the Contour XT device. Blood glucose levels are measured every 30 minutes
following consumption of the glucose containing drink over a 2 hour period using each device. The
level of glucose within a first blood sample represents a baseline level ; the level of glucose within a
second blood sample will increase above the baseline; the level of glucose in a third and subsequent
blood samples is similar to the baseline. Signals from each technology correspond to the expected
glucose levels and the changes exhibited by the signals measured using the copper electrode are
correlated to the changes in glucose levels determined using the classic technologies.

Claims
1. A method for determining the glucose content of a sample comprising causing complete
ionisation of the glucose and determining the ionised glucose electrochemically.
2 . A method for determining the glucose content of a sample comprising ionising the glucose
while the sample is in contact with an un-modified copper electrode and determining the quantity of
ionised glucose by detecting changes of current at one or more pre-determined voltage settings.
3 . The method of claim 1 or 2 wherein the conditions causing ionisation of glucose comprises
alkalisation of the sample, optionally wherein the alkalisation comprises increasing the pH of the
sample to at least pH1 4 , optionally wherein the alkalisation is caused by mixing the sample with a
strong base, optionally wherein the strong base is sodium hydroxide, potassium hydroxide, barium
hydroxide, ammonium, ammonium hydroxide or methylammonium .
4 . The method of any one of claims 1 to 3 wherein the electrochemical detection comprises
electro-catalysis, optionally wherein the electro-catalysis comprises oxidation of copper, optionally
wherein the oxidation of copper comprises oxidation of copper 2+ to copper 3+.
5 . The method of any one of claims 1 to 4 wherein the determination is by voltammetry,
optionally wherein the voltammetry is sweeping voltammetry or cyclic voltammetry.
6 . The method of claim 5 wherein the voltammetry sweeps across a range of 500 to 1200mV.
7 . The method of claim 5 or 6 wherein the sweeping voltammetry is forward and/or reverse
sweeping.
8 . The method of any one of claims 1-7 wherein the sample is blood, plasma, serum, urine
tears, saliva, or CSF.
9 . The method of any one of claims 1 to 8 which further comprises mixing the sample with a
polyion. , optionally wherein the polyion is a polyanion, polycation, polyzwitterion , EDTA and /or,
polyethyeleneimine.
10 . The method of claim any one of claims 1 to 9 further comprising mixing the sample with a
surfactant, optionally wherein the surfactant is sorbate.
11. A device for determining the glucose content of a sample comprising a sample analysis area
wherein the sample analysis area comprises electrodes and pre-deposited reagent for alkalisation of
the sample.
12 . The device of claim 11 wherein the electrodes comprise metals or conducting polymers.
13 . The device of claim 11 or 12 wherein :
a . the electrodes comprise copper working electrode, a silver/silver chloride reference
electrode and a platinum counter electrode
b. the working, counter and reference electrodes are all gold
c . the working and counter electrodes are gold and the reference electrode is
silver/silver chloride
d . the electrodes comprise gold working electrode, a silver/silver chloride reference
electrode and a platinum counter electrode
e. the working, counter and reference electrodes are all copper; or
f . the working and counter electrodes are copper and the reference electrode is
silver/silver chloride.
14 . The device of any one of claims 11 to 13 wherein the (copper and platinum) electrodes
comprise evaporated film electrodes.
15 . The device of any one of claims 11 to 14 wherein the reagent for alkalisation of glucose
comprises a strong base, optionally wherein the strong base comprises sodium hydroxide,
potassium hydroxide, Barium hydroxide, ammonium , ammonium hydroxide or methylammonium .
16 . The device of any one of claims 11 to 15 wherein the reagent for alkalisation of glucose
further comprises a polyion, optionally wherein the polyion comprises EDTA and or
polyethyleneimine.
17 . The device of any one of claims 11 to 16 wherein the reagent for alkalisation for the sample
further comprises a surfactant.
18 . The device of any one of claims 11 to 17 wherein the electrodes and reagent for alkalisation
of the sample are physically separate but fluidically connected.
19 . The device of any one of claims 11 to 18 where the electrodes are capable of electrocatalysis
of ionised glucose.
20. The device of claim 13 wherein the electrodes comprise alternative electrode arrangements.
2 1. The device of any one of claims 11 to 20 wherein glucose is determined electrochemically
following ionisation and electrocatalysis of glucose.
22. The device of any one of claims 11 to 2 1 wherein the glucose can be determined at more
than one electrode potential.
23. A biosensor, comprising ;
a base layer having disposed thereon at least one conductive track extending from a first
end to a second end, wherein the conductive track comprises copper;
an assay zone at the first end of the base layer, comprising a reagent capable of increasing
the pH of a sample applied to the assay zone;
a terminal at the second end of the base for connection of the at least one conductive track
to a processor.
24. The biosensor of claim 23 further comprising a capillary chamber at the first end for
receiving a sample of body fluid, wherein the capillary chamber is disposed over the assay zone
such that a portion of the at least one conductive track is exposed within the capillary chamber.
25. The biosensor of claim 23 or 24, wherein the base layer has disposed thereon at least three
conductive tracks, each conductive track being electrically insulated from the other, optionally
wherein the at least three conductive tracks comprise copper and wherein a portion of the at least
three conductive tracks is exposed within the capillary chamber, and further wherein the capillary
chamber contains the pH altering reagent.
26. The biosensor of any one of claims 23-25 wherein the pH altering reagent is disposed :
a . on an inner surface of the capillary chamber;
b. on the base layer, but not in contact with the at least three conductive tracks within
the capillary chamber; and/or
c . within the capillary chamber.
27. The biosensor of any one of claims 23-26 wherein the at least three conductive tracks define
at least one measurement electrode, at least one reference electrode and at least one counter
electrode, and wherein the measurement electrode, counter electrode and reference electrode are
located within the capillary chamber in the assay zone.
28. A method, comprising :
ionizing glucose present in whole blood ; and
electrochemically determining the presence of the ionized glucose in the whole blood.
29. The method of claim 28, wherein ionizing the glucose comprises combining the whole blood
with a dried reagent, optionally wherein the dried reagent is present in an amount sufficient to
increase the pH of the whole blood by an amount sufficient to ionize the glucose.
30. The method of claim 28 or 29, wherein the electrochemically determining is performed in a
chamber having a total volume of less than about 5 microliters.
3 1. The method of any one of claims 28-30, wherein the electrochemically determining
comprises electrochemically determining the ionized glucose via an electrochemical circuit
comprising at least one copper electrode in contact with the whole blood.
32. The method of any one of claims 28-31 , wherein the method is performed in the absence of
enzymes/mediators.
33. A test strip for determining the presence of glucose, comprising :
a capillary chamber defining a total volume of less than about 2.5 microliters;
at least one copper electrode in electrochemical communication with the capillary chamber; and
a dried reagent present in an amount sufficient to increase a pH of a whole blood sample introduced
into the capillary chamber and filling the volume of the capillary chamber by an amount sufficient to
ionize glucose present in the whole blood.
34. The device of claim 33 wherein the test strip comprises three copper electrodes configured
as:
i) a working electrode at which measurement of glucose oxidation occurs;
ii) a counter electrode, which supplies or consumes electrons in response to the reaction at
the working electrode; and
iii) a reference electrode, which acts to monitor and maintain the potential applied between
the working electrode and counter electrode.
35. The device of claim 33 or 34 wherein the capillary chamber defines a volume of less than
about 2 microlitres, less than about 1 microlitre or less than about 0.5 microlitres.
36. The device of any one of claims 33 to 35 wherein the dried reagent is disposed on a surface
of the capillary chamber not in direct contact with the one or more copper electrodes.
37. The device of any one of claims 33 to 36 wherein the dried reagent comprises a base and a
surfactant, optionally wherein the surfactant is polyvinyl alcohol and the base is sodium hydroxide.
38. A method of determining the quantity of glucose in a sample of blood obtained from a finger
prick or alternate site using a device of claims 33 to 37, comprising ;
removing the test strip from a storage compartment;
inserting the test strip into a meter and following the instructions presented on the display of
the meter;
pricking a finger or alternate site to release a drop of blood ;
contacting the drop of blood with the sample port on the test strip;
removing the test strip from the drop of blood when the meter indicates sufficient sample
has been acquired on the test strip;
allowing the blood to react in the test strip for at least 1 second ; and
displaying a blood glucose concentration on the display of the meter.
39. The method of claim 38, wherein the blood reacts in the test strip for at least 3 , 5 , 7 or 10
seconds before a glucose concentration is displayed.
40. The method of claim 38 or 39 wherein no more than 2.5, 1.5, 1 or 0.5 microlitres of blood
has been acquired on the test strip.