Anal. Chem. 1995, 67, 4594-4599
`
`Catalytic Materials, Membranes, and Fabrication
`Technologies Suitable for the Construction of
`Amperometric Biosensors
`
`Jeffrey D. Newman,* Stephen F. White, lbtlsam E. Tothill, and Anthony P. F. Tumer
`
`
`
`Cranfield Biotechnology Centre, Cranfield University, Cranfield, Beds MK43 OAL, U.K.
`
`
`
`
`
`A selection of recently available catalytic carbon powders
`
`the electrical double layer.7 In this instance, the difference in the
`
`were assessed and compared with the more convention­
`electronic work functions of platinum and carbon result in an
`
`
`ally used platinized material. Their suitability for incor­
`increase in the electron density on the platinum.
`Several methods for the incorporation of the metal into the
`
`
`poration in amperometric biosensors is discussed. In
`carbon matrix have been reported. These include electrochemical
`conjunction
`with this study, methods of applying mem­
`deposition using, for example, cyclic voltammetry,& sputtering9 and
`
`
`were investigated. branes to the surfaces of these devices
`straightforward mixing of the metal in a carbon paste.10 The
`
`
`Advanced fabrication technologies, potentially suitable for
`conventionally preferred working electrode material, platinized
`
`
`scale-up of sensor production, such as screen printing and
`carbon, is able to reduce greatly the oxidation potential of
`
`
`
`ink·jet printing, were used for manufacture of the com­
`hydrogen peroxide. Unfortunately, this material is also highly
`
`
`plete sensor structure. Hydrogen peroxide-sensing elec­
`electrocatalytic for a large number of other substances, such as
`
`trodes and glucose biosensors were produced as model
`glucose and other reducing sugars. In complex samples, the
`
`
`systems, demonstrating the advantages of these ap­
`presence of such interfering compounds will clearly compromise
`
`
`proaches. The commercially available rbodinized carbon
`specificity. To overcome this drawback, selectivity can be
`
`
`
`density at low potentials MCA4 produced a high current
`enhanced by the use of membranes, typically constructed from
`over a plateau region (300-400 mV vs SCE). In addition,
`polymers such as cellulose acetate, polyurethane, Nafion, and a
`
`
`direct oxidation of glucose (seen with platinized carbon)
`variety of others. !I Membranes are also a convenient way of
`
`
`of +350 mV. was not observed at the chosen potential
`extending the linear range of a biosensor. They form a diffusion
`
`
`Further interference studies using fermentation media
`barrier, and substrate/product concentration profiles within this
`
`
`for use highlighted its suitability as an electrode material
`layer are, therefore, a function of both diffusion and reaction. One
`
`in complex samples. Ink-jet printing proved to be a
`parameter limiting the linear range of such an electrode is the
`
`
`successful method for the deposition of Nafion mem­
`Km of the enzyme, which exhibits saturation kinetics, and a
`
`
`branes of defined and reproducible geometry.
`nonlinear signal is anticipated. If the diffusional effects are
`dominant, the limitation on the linear range, due to a low Km value,
`becomes negligible. In such situations, "apparent" kinetic pa­
`rameters are often used.12
`A drawback in the use of membrane-modified electrodes arises
`from the time-consuming and inconsistent manual fabrication
`methods typically employed. Automated methods, such as screen
`printing13 and ink-jet printing, 14 can be used to accelerate and
`regulate production. This is an area which is often overlooked
`when considering the commercialization of such devices.1;
`This paper reports on the suitability of some recently available
`catalytic carbon powders and examines methods of depositing
`reproducible membrane structures on screen-printed electrodes
`constructed of such materials. In addition, this manufacturing
`process enables biological components such as enzymes to be
`printed, since it involves a room temperature curing process.
`
`A number of approaches to the construction of amperometric
`biosensors have been adopted since the conception of the first
`device by Clark in 1962.1 The first biosensors were based on the
`Clark oxygen electrode, detecting the depletion of oxygen result­
`ing from the action of an oxidase enzyme.2 Alternatively,
`hydrogen peroxide, generated as a result of the enzymatic
`reaction, can be detected and related to the analyte concentration.3
`Further refinements involved the use of mediators to circumvent
`the reliance of the sensor on the local oxygen concentration, to
`reduce the operating potential and hence enhance selectivity.4 In
`addition, the use of metals such as platinum and rhodium, coated
`onto carbon electrodes as a fine dispersion, has been widely
`adopted.5·6 This results in a highly catalytic surface as long as
`the particle size of the deposited metal is comparable to that of
`
`(1) Clark, L C .. Jr.: Lyons, C. Ann. N. Y. Acad. Sci. 1962, 102, 29.
`(2) Updike. S. J.; Hicks. G. P. Nature 1967. 214, 986.
`(3) Clark, L C .. Jr. U.S. Patent 3539455, 1970.
`(4) Cass. A E.G.: Davis. G.; Francis, G.D.: Hill, H. AO.: Aston. W. J.: Higgins.
`l. J.: Plotkin. E. V.: Scott. L. D. L.: Turner. AP. F. Anal. Chem. 1984, 56,
`667.
`(5) Bennetto, H. P.; DeKeyser, D. R.: Delaney, G. M.; Koshy, A; Mason. J. R;
`Mourla, G.: Razack, L. A; Sterling, J. L: Thurston. C. F.: Anderson. 0. G.:
`Mullen. W. H. bit. Ind. Biotechnol. 1985. 8, 5.
`(6) White , S. F.; Turner, A P. F.; Bilitewski. U.: Schmid. R. D.; Bradley. J.
`Electroanalysis 1994, 6. 625.
`
`(7) Mukerjee. S. ]. Appl . Electrochem. 1990. 20, 537.
`
`(8) Gunasingham. H.: Tan. C.·H. Electroanalysis 1989, J. 223.
`{9) Gorton. L: Svensson. T.]. Mo/. Cata/. 1986, 38, 49.
`(10) Wang, J.; N aser. N.: Angnes, l.: Wu. H.: Chen. L A11al. Chem. 1992. 64.
`1285.
`(11) McDonnell. M. B.; Vadgama, P. M. Ion-Se/. Electrode Reu. 1989. 11. 17.
`leypoldt, J. K: Gough. D. A. Anal. Chem. 1984. 56, 2896.
`(12)
`1991. 6, 109.
`(13) Kulys. J.: D'Costa. E. ). Biosens.
`Bioelectrqn,
`(14) Newman. J. D.: Turner. A. P. F.: Marrazza. G. Anal Chim. Acta 1992,
`262. 13.
`
`
`(15) Alvarez.Icaza. M.; Bilitewski, t.:. Anal. Cltem. 1993 , 65. 525A.
`
`4594 Analytical Chemistry, Vol. 67, No. 24, December 15, 1995
`
`0003·2700/9510367·4594$9.00/0 � 1995 American Chemical Society
`
`AGAMATRIX, INC. EXHIBIT NO. 1018
`Page 1 of 6
`
`

`

`EXPERIMl!NTAL SECTION
`Electrode Construction and Test Procedure. Screen·
`printed electrodes were used throughout these investigations and
`were constructed in planar arrays of eight electrodes. These were
`fabricated using a DEK 247 screen-printer (DEK Printing Ma­
`chines, Weymouth, UK) in a multistage printing process whereby
`a silver conducting track (Electrodag 477SS RFU, Acheson
`Colloids, Plymouth, UK) was placed on a PVC substrate (Geno­
`therm, Sericol, Surrey, UK). A graphite pad (Electrodag 423 SS,
`Acheson Colloids) was printed at one end of the silver track, and
`an insulation shroud (Matt Vinyl White M.V. 27, Apollo Colours,
`London, UK) was deposited over these layers, such that only a
`contact pad at one end and a working electrode (8 mm x 2 mm)
`at the other end were exposed. In each case, the printed layer
`was cured for 1 h at 40 °C.
`A range of commercially available metalized carbon powders
`(fable 1) were obtained from ETEC and MCA Services (Royston,
`UK) and investigated. The MCA powders are proprietary materi·
`als consisting of highly dispersed precious metals and containing
`promoters. For studies of the response of the catalytic materials
`to hydrogen peroxide, the powders were mixed with a 2.5% (w/
`v) hydrox:yethyl cellulose (HEC; Fluka, Gillingham, UK) solution
`dissolved in 0.1 M, pH 7.0 phosphate buffer. The metalized carbon
`powder was incorporated into the HEC solution in the ratio 1:2
`(w/w). The ink was mixed using a rotary stirrer until a uniform
`consistency was achieved (-1 h at25 °C). Finally, the resultant
`ink was screen-printed onto the graphite pad. Despite this layer
`being slowly soluble in the test solution. it remained bound to
`the working electrode surface for sufficient time to enable the
`analysis to be carried out.
`Experiments were carried out in a batch mode using a three­
`electrode amperometric system consisting of a saturated calomel
`reference electrode (SCE; Russell, Auchtennuchty, Fife, UK), a
`2 mm diameter platinum wire counter electrode, and the working
`electrode as described above. All experiments were performed
`under stirred conditions at 25 •c using 0.1 M sodium phosphate
`buffer containing 0.1 M potassium chloride at pH 7.0. Aliquots
`of hydrogen peroxide were added to the buffer solution, and the
`dynamic current response was monitored and recorded using an
`Autolab electrochemical analyzer (EcoChemie, Utrecht, Nether­
`lands), which was controlled by the software package GPES3.
`Enzyme Electrode Construction. Having determined the
`preferred composition of the electrode base material, the next
`stage was to incorporate the enzyme into the structure. Glucose
`ox.idase (Glucox PS, ABM. Stockport, UK) was chosen as a model
`system. Two sensor configurations were investigated. First, the
`enzyme was mixed with the catalytic powder to form a printable
`ink (electrode types A). Second, the enzyme was printed over
`the catalytic powder in a separate layer (electrode types B).
`The following ink formulations were used: type A, 1 g of 2.5%
`(w/v) HEC in phosphate buffer (pH 7.0) solution, 100 mg of
`glucose oxidase, and 400 mg of catalytic graphite powder; type
`B, the catalytic graphite layer deposited as described earlier and
`containing no glucose oxidase. The enzyme was subsequently
`incorporated into an ink consisting of 1 g of 3% HEC, 100 mg of
`glucose oxidase, and 400 mg of TIO graphite (Acheson Colloids).
`After printing, all electrodes were left to dry for 2 h at room
`temperature. Because the organic binder is soluble in aqueous
`solution, an outer protective membrane was required. We
`investigated two such materials, Nafion (Aldrich, Gillingham, UK),
`
`a persulfionic charge exclusion material, and cellulose acetate
`(Aldrich), a size exclusion barrier. Three techniques were used
`ink-jet printing, diixoating, and spin­
`to apply these membranes:
`coating. Ink-jet printing requires a charged solution for deposition
`and is therefore suitable for applying the Nafion layer. Cellulose
`acetate solutions, which are nonconducting, require the presence
`of a charged species, which can be provided by the inclusion of
`an organic salt such as tetrabutylammonium toluenesulfonate
`(I'BATS). Diixoating and spllK:oating are not subject to this
`constraint and were suitable for the application of both membrane
`materials.
`Nafion was supplied as a 5.0% (w/v) solution, dissolved in a
`mixture of lower aliphatic alcohols and water. It was necessary
`to dilute this to reduce the viscosity for printing and coating. Water
`was added to make a final concentration of 1% (w/v) for ink-jet
`printing and 0.5% (w/v) for spin<oating and diixoating. Cellulose
`acetate was prepared as a 2.0% solution in acetone.
`Ink-Jet Printing. A Biodot printer (Biodot, Diddington, UK)
`was used for all ink·jet experiments. Such a printer operates by
`forcing tluid under pressure through a small nozzle (75 µm). As
`the tluid passes through the print head, a drive rod actuated by
`an oscillating piezoelectric crystal produces a shock wave which
`breaks the jet into a series of regular droplets. This occurs at a
`frequency of -64 000 Hz, dependent on the modulation voltage.
`Printing is achieved by charging individual droplets via a charge
`electrode which is controlled by a microprocessor. The printed
`pattern is constructed in a dot matrix format.
`An array of 16 x 21 droplets of membrane solution (total
`printed volume, 0.4 7 µL) was printed onto the electrode surface
`such that it completely covered the active area and overlapped
`the insulation shroud by -2 mm. The amount of solution was
`varied by repeatedly passing the electrode array under the print
`head.
`Spin·Coating. The electrodes (Individually) were mounted
`on a purpose-built spigot and rotated at 2000 rpm. An aliquot (10
`µL) was pipetted onto the surface of the working electrode, which
`was left to rotate for 15 min, allowing the solvent to evaporate,
`leaving a thin membrane deposit. All spin<oated electrodes were
`prepared using this approach.
`Dip·Coating. The enzyme electrode was immersed in the
`membrane solution and immediately removed. Following immer­
`sion, the electrodes were suspended vertically and left to dry for
`30 min (allowing the solvent to evaporate).
`Glucose Biosensor Test Procedure. Glucose stock solu·
`tions (0.1 M) were all prepared in 0.1 M, pH 7.0 phosphate buffer
`containing 0.1 M KC!. These solutions were allowed to mutarotate
`overnight prior to use. All glucose experiments were carried out
`under the conditions described earlier for hydrogen peroxide
`determination. pH optimization was carried out using five
`electrodes which were tested at 25 •c in the appropriate buffer
`under stirred conditions, with the working electrode potential
`poised at -350 mV (vs SCE). The following buffers were used:
`
`pH 5-5.5
`
`0.1 M sodium acetate
`
`p H 6-7.5
`
`0.1 M sodium phosphate
`
`p H 8-8.9
`0.1 M Tris-HCI
`When the electrode had reached a steady state, a 5 mM
`concentration of glucose (prepared in the appropriate buffer) was
`added to the buffer, and the electrode response was monitored.
`Three measurements were made at each pH.
`
`Analytical Chemistry, Vol. 67, No. 24, December 15, 1995 4595
`
`AGAMATRIX, INC. EXHIBIT NO. 1018
`Page 2 of 6
`
`

`

`Table 1, Catalytic Graphite Analysis: Electrochemlcal Response to Hydrogen Peroxide In the Range 0-2.5 mM at
`25 °C and pH 7.0
`
`material
`
`ETEC (10% Pt)
`MCAl (5% Ni, 5% Cr)
`MCA2 (5% Pt)
`MCA3 (0.1% Pt)
`MCA4 (5% Rh)
`MCA5 (0.1% Rh)
`MCA6 (5%Ru)
`MCA7 (0.1% Ru)
`MCA8 (10% Pd)
`MCA9 (5% Pt, 5% Pd)
`MCAlO (5% Pd, 0.5% Ru)
`MCAll (5% Ni, 596 Cr, 596 Pd)
`MCA12 (10% Ag)
`
`lOOmV
`
`-23.4 (-0.9975)
`-0.1 (-0.9165)
`-22.3 (-0.9992)
`-2.0 (-0.9943)
`-10.9 (-0.9961)
`no response
`-13.1 (-0.9971)
`no resp0nse
`-14.3 (-0.9994)
`-30.4 (-0.9993)
`-22.8 (-0.9990)
`-1.8 (-0.9948)
`no response
`
`hydrogen peroxide resp0nse (uA/mM) and correlation coefficient
`(r, in parentheses) at the indicated applied potentials
`300 mV
`400 mV
`200mV
`
`13.9 (0.9995)
`0.1 (0.9165)
`8.2 (0.9997)
`4.6 (0.9991)
`15.0 (0.9973)
`no response
`-11.2 (-0.9985)
`no response
`7.9 (0.9984)
`4.4 (0.9898)
`2.0 (0.9999)
`0.9 (0.9853)
`no response
`
`29.1 (0.9998)
`0.8 (0.9820)
`33.l (0.9937)
`14.8 (0.9987)
`27.3 (0.9925)
`no response
`-9.l (-0.9991)
`no response
`23.5 (0.9998)
`28.9 (0.9982)
`27.4 (0.9998)
`3.7 (0.9935)
`no response
`
`29.3 (0.9997)
`2.7 (0.9750)
`41.7 (0.9934)
`22.9 (0.9989)
`26.8 (0.9982)
`no response
`-0.3 (-0.9846)
`no resp0nse
`31.0 (0.9998)
`44.5 (0.9985)
`36.6 (0.9995)
`7.4 (0.9988)
`no response
`
`500mV
`
`5.1 (0.9979)
`
`0.8 (0.9586)
`52.8 (0.9976)
`7.6 (0.9952)
`
`no response
`
`Interference Studies. To validate the potential use of the
`selected catalytic material as a suitable base for practical biosen·
`sors, interference studies were carried out by studying the direct
`oxidation of glucose and the electrode response in fermentation
`media. For these experiments, the non-enzyme electrodes were
`used. All experiments were carried out under the conditions
`described above, with the potential interferent solutions being
`added in place of hydrogen peroxide. The first six media
`investigated were sterile samples in which no cells had been
`grown. Following this, tests were carried out in cell-free extracts;,­
`obtained during two bacterial fermentations and one yeast
`fermentation. The media were prepared according to the follow·
`ing (amounts given in grams per liter):
`
`medium 1 (medium for yeast)
`KH2P04
`12
`5
`MgS04·7H20
`0.2
`FeCl3·6H20
`yeast extract
`20
`hycase
`20
`
`medium 3 (LB medium)
`
`bactotryptone
`yeast extract
`NaCl
`
`10
`5
`10
`
`medium 2 (medium for bacteria)
`CaClr4H20
`0.1
`MgCh·6H20
`2
`5
`KiS04
`2
`NaCl
`10
`KC!
`20
`yeast extract
`20
`hycase
`KiHP04
`2
`
`medium 4
`(brain-heart infusion medium)
`1.25
`calf brain
`5
`beef heart infusion solids
`proteose pep tone
`10
`5
`NaCl
`2
`dextrose
`2.5
`Na2HP04
`
`medium 5
`(reinforced clostridial medium)
`
`yeast extract
`lablemco powder
`peptone
`soluble starch
`dextrose
`cysteine hydrochloride
`NaCl
`sodium acetate
`agar
`
`3
`10
`10
`1
`5
`0.5
`5
`3
`0.5
`
`medium 6 (MRS broth)
`
`bacteriological peptone
`beef extract
`yeast extract
`Na2HP04
`sodium acetate
`triammonium citrate
`Mg$Q4·4H20
`MnS04·4H20
`Tween 80
`
`10
`8
`4
`2
`5
`2
`0.2
`0.05
`l
`
`E. coli was grown with shaking at 37 °C in nutrient broth
`consisting of 10 g/L beef extract, 10 g/L peptone no. 1, and 5
`g/L NaCl. Samples were taken at regular intervals over a period
`of 7.5 h.
`was grown in a stationary mode at 30 °C in
`L piantarum
`medium 6 (outlined above). Once more, samples were taken over
`a 7.5 h period.
`was grown in a stationary mode at 30 °C in a
`S. cerevisiae
`medium consisting of 7 g/L yeast extract, 10 g/L mycological
`peptone, and 20 g/L glucose. Aliquots were removed at intervals
`over a 24 h period.
`
`RESULTS AND DISCUSSION
`Selection of Electrocatalytic Material. The criteria used to
`select the catalytic material were based on (i) high current density,
`thus maximizing the signal; (ii) low operating potential, with a
`response plateau, such that small fluctuations in the applied
`potential have a negligible effect on precision; (ill) low interference
`from other electroactive compounds, a problem highlighted earlier
`for metal/carbon electrodes; and (iv) linear response in the
`working range.
`Electrochemical Response to H202. The response to
`hydrogen peroxide over a range of concentrations between 0 and
`2.5 mM was determined amperometrically for each of the
`electrode types over a series of applied potentials between 100
`and 500 m V. Results of these measurements are summarized in
`Table 1. From this, it can be seen that several catalytic carbons
`show significant electrocatalytic activity. This is characterized by
`a high positive slope value at low potentials. From these data, it
`is apparent that the following materials exhibited the highest
`current densities in this range: ETEC Pt-C, MCA2, MCA3,
`MCA4, MCA8, MCA9 and MCAlO.
`The electrodes which exhibited the best plateau responses
`were ETEC Pt-C and MCA4, which both displayed this feature
`between 300 and 400 m V. Electrodes which produced a significant
`signal were all linear over the tested hydrogen peroxide concen·
`tration range.
`Direct electrooxidation of glucose has been previously de­
`scribed.16 Generally, the anodic oxidation was carried out using
`
`Fennentation Broths. Fermentations were canied out in 250
`mL shake flasks, containing 50 mL of inoculated media. The
`(16) Giner. J.: Malachesk-y, P. Proceedings or the Artificial Heart Conference.
`organisms grown were Escherichia
`
`coli, Lactobacillus plantarum
`Washington. June 1969, U.S. Dept. of Health. Education and Welfare; 1969:
`p 839.
`LPCO·lO, and Saccharomyces
`cerevisiae.
`
`4596 Analytical Chemistry. Vol. 67, No. 24, December 15, 1995
`
`AGAMATRIX, INC. EXHIBIT NO. 1018
`Page 3 of 6
`
`

`

`Table 2. llectrocle Materlal AesponH to Hydrogen
`Peroxide and Glucose
`
`1.8£-85
`ti/A
`
`(&)
`
`
`
`material hydrogen peroxide glucose
`
`ETEC
`MCAl
`MCA2
`MCA3
`MCA4
`MCA5
`MCA6
`MCA7
`MCAS
`MCA9
`MCAlO
`MCAll
`MCA12
`
`+++
`+
`++
`+
`+++
`+
`+++
`++
`++
`++
`++
`++
`+
`
`++
`+++
`
`+
`
`+
`++
`+
`+
`
`+
`
`'l.BE-85
`
`0 -, no noticeable response; +, slight response; ++, significant
`
`
`
`
`
`response; +++, strong response.
`
`&.8£-85
`f l/A
`
`•£/\I
`
`e.1ee
`
`(c)
`
`either pure (Au, Pt) or adatom (Pb, TI, Bi)-modified
`noble metal
`
`at platinized electrodes. Similar behavior has been reported
`
`
`carbon electrodes. The effects of direct glucose oxidation may
`
`
`
`cause problems due to electrode fouling and may complicate the
`
`biosensor kinetics, leading to a poorly defined relationship
`
`
`
`between the glucose concentration and the recorded signal. It is
`
`also likely that if glucose is electrochemically active at these
`
`
`electrode surfaces, then other organic compounds, such as
`
`
`
`alcohols and carbohydrates, will also exhibit this characteristic.6
`-8.BE-85
`
`
`Hence, the electrodes were tested for their sensitivity to direct
`
`
`glucose oxidation using cyclic voltammetry. The potential was
`1.BE-85
`!!IA
`0 and 700 mV at a scan rate of 50 mV/s in the
`cycled between
`
`
`buffer solution described above, in buffer containing 1.0 mM
`
`hydrogen peroxide, and finally in buffer containing 25.0 mM
`
`
`
`glucose. The magnitude of the response in each case, which
`
`
`represents a qualitative interpretation of the cyclic voltammo­
`grams, is shown in Table 2.
`that MCA4 offers the best It is apparent from these results
`
`
`
`
`
`response to hydrogen peroxide with no noticeable interference
`
`from the direct oxidation of glucose. The cyclic voltammograms
`of the MCA4 material in buffer, buffer+ hydrogen
`
`peroxide, and
`buffer + glucose are shown in Figure 1. As can be seen, the
`scans for the buffer and the buffer + glucose are virtually identical.
`Figure 1. Cyclic voltammograms of MCA4-based electrodes. (A)
`
`
`
`
`The scan when hydrogen peroxide is present, however, clearly
`
`buffer. to 0.1 M, pH 7.0 phosphate (B) Response to 1.0 M
`Response
`
`
`
`shows a large electrocatalytic oxidation of hydrogen peroxide. As
`h yd rogen peroxide in phosphate buffer (as In A). (C) Response to
`
`
`a result of these experiments, it was concluded that MCA4 is the
`
`(as in A).
`25.0 mM glucose In phosphate buffer
`
`
`best of the materials tested. It offers a significant advantage, when
`
`
`compared with the commonly used platinized carbon material, in
`
`that it does not suffer interference from direct oxidation of
`This suggests that this electrode material may be well suited to
`
`
`
`reducing sugars, and hence it was used for all subsequent
`operation in complex samples, where potential electrochemical
`experiments.
`
`
`interferences may be present, such as in fennenters.
`Results of Interference Studies. A three-electrode ampero­
`Enzyme Electrodes: Initial Membrane Test Results.
`
`
`metric cell, as described earlier, was poised with an applied
`
`
`Table 3 illustrates the mean responses from five of each type of
`
`
`
`were immersed in the electrodes potential of +350 mV. Initially,
`
`
`electrode to a 5 mM addition of glucose. Electrode types A and
`
`
`
`20 mL of phosphate buffer (pH 7.0) under stirred conditions at
`B are as defined earlier. All of the electrodes
`
`fabricated using
`
`
`had reached a steady current 25 °C. When the electrode response
`
`
`
`dip-coating as the method of membrane application produced very
`
`(-60 s), buffer was removed and replaced with the same volume
`
`small currents and exhibited poor reproducibility.
`It can be seen that type A electrodes
`
`sample over a dilution of the appropriate media or fermentation
`
`(enzyme and catalytic
`
`
`the addition of the media range between 1:20 and 1:1. Following
`
`:powder in the same layer) produced significantly higher currents
`
`sample, hydrogen peroxide was added to ensure that the opera­
`.and were found to be more stable than those of type B.
`
`
`
`tional characteristics of the electrode were unimpaired. For all
`
`
`
`
`Furthermore, producing electrodes via this approach eliminates
`
`
`
`of the samples tested, no significant interference was observed.
`
`
`the need for a separate step during manufacture. Nafion-coated
`
`I
`
`Analytical Chemistry, Vol. 67, No. 24, December 15, 1995 4597
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`AGAMATRIX, INC. EXHIBIT NO. 1018
`Page 4 of 6
`
`

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`[GI ucoseJ (mM)
`pH
`
`
`glucose Figure 3. Typical calibration plot for a screen-printed
`response at a glucose Figure 2. pH profile of glucose biosensor
`
`
`
`
`biosensor with an ink-jet-printed Nafion membrane.
`
`
`conditions. concentration of 5 mM at 25 °C under stirred
`
`Table 3. Results of Membrane Fabrication Studies
`
`response to 5 mM
`glucose (µA)
`electrode and membrane type mean
`SD
`
`type Nink-jet Nafion (8 passes) 15.8
`type Blink-jet Nafion (8 passes)
`4.8
`
`type Nspin-coated cellulose
`10.7
`acetate
`type B/spin-coated cellulose
`acetate
`type Nspin-coated Nafion
`type B/spin-coated Nafion
`
`17.7
`3.5
`
`1.8
`
`�
`<I:
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`comments
`
`low noise
`noisy
`noisy
`
`very noisy
`
`noisy
`very noisy
`
`1.2
`0.4
`1.0
`
`0.6
`
`1.2
`1.0
`
`6
`Time (days)
`over an 8 day period. Sensor stored
`Figure 4. Biosensor stability
`
`electrodes produced higher currents with less noise than those
`
`
`
`
`
`response dry at 4 °c between measurements. Plot depicts biosensor
`
`
`
`coated with ceUulose acetate. Ink-jet printing proved to be the
`to a glucose concentration
`
`of 5 mM at 25 °C under stirred conditions.
`
`
`most controllable method of applying the membranes. Spin­
`
`
`coated, Nafion-covered electrodes produced marginally hlgher
`by the number of printed droplets) which was ink-jet-printed onto
`
`
`
`
`
`currents, but the response was significantly noisier. From these
`
`
`the sensor surface, were tested for their response to a range of
`
`
`would be results, it was decided that all future electrodes
`from 0 to 15 mM. Generally,
`in the type A fonnat, with initial work
`glucose concentrations
`linear
`constructed
`being carried
`
`out using an ink-jet-printed Nafion membrane.
`
`
`
`responses were obtained at the lower glucose concentrations, with
`Investigation of the pH Profile. The graph shown in Figure
`
`
`the linear range extending as the amount ofNafion deposited was
`2 depicts the electrode response
`over a pH range between 5.0
`
`increased. Above 22.09 µL, the current response declined
`
`
`
`and 9.0. Obviously, the optimum applied potential for hydrogen
`
`
`significantly, indicating that the memb1ane thickness restricted
`peroxide detection and the activity of the enzyme will be
`
`the flux of glucose to the immobilized enzyme. A representative
`
`
`influenced by the pH. However, for simplicity of associated
`
`calibration graph is shown in Figure 3.
`
`
`instrumentation of an analytical device based on this technology,
`Stability. Using the same operating conditions
`as those
`
`it is useful to operate at a fixed potential. Glucose oxidase operates
`
`
`described above (phosphate buffer, pH 7), an electrode was tested
`
`over a wide pH range (2.7-8.5) and the optimum is usually
`over 8 days. Three electrodes
`for stability
`
`were tested against a
`
`reported to lie between 4.8 and 6.0 for the free enzyme utilizing
`
`5 mM glucose concentration daily over this time. When not in
`17 Numerous publications
`
`dioxygen as the electron acceptor.
`stored in buffer at 4 °C. Figure 4, which
`use, the electrodes were
`
`
`glucose biosensors have described a shift in the pH
`describing
`
`
`
`
`depicts the response of a typical enzyme electrode, indicates that
`
`optimum exhibited due to the use of artificial electron acceptors.18
`
`
`
`
`by a more gradual is followed an initial sharp decline in activity
`
`Immobilization is another factor which may alter the operating
`and that after 8 days, the sensor was still producing
`decrease
`a
`
`
`that the of the enzyme.19 Hence, the observation
`characteristics
`significant response.
`
`optimum value is 7.0 is a function of the electrode and its mode
`Nafion is a perfluorosulfonic ion-exchange membrane material
`
`
`
`
`of operation at a constant voltage and is not necessarily an
`
`
`
`materials which differs from conventional ion-exchange in that it
`
`
`indication that the behavior of the enzyme has been altered.
`
`
`
`is not a cross-linked polyelectrolyte, but a thermoplastic polymer
`Calibration Curves. A series of enzyme electrodes
`(five of
`
`
`neutral­with pendant sulfonic acid groups partially or completely
`2.35
`
`
`each). prepared using different amounts of N afion (between
`
`
`ized to fonn salts. This also provides a negatively charged surface.
`and 22.09 µL, calculated
`
`
`by multiplying the mean droplet volume
`
`It is believed that the membrane, by virtue of the electrostatic
`
`
`
`interaction between enzyme and membrane, is helping to retain
`
`
`the enzyme electrostatically as well as physically. Furthermore,
`
`
`this immobilization procedure may lead to a greater
`rigidity,
`
`
`
`may enhancing physical resilience of the enzyme. Another effect
`
`(17) Uhlig. H. Jn Enr.ymts in lnd�try. Gerhartz, W., Ed.; VCH: Weinheim, 1990:
`p 77.
`(18) Wilson, R: Turner, AP. F. Biosens. Bioelectron. 1992. 7. 165.
`(19) Gorton. L: Csoregi. E.: Dominguez. E.: Emneus, J.: Jonsson-Pettersson,
`G.: Markc>-Varga. G.; Persson, B. Anal. Chim. Acta 1991. 250. 203.
`
`4598 Analytical Chemistry, Vol. 67, No. 24, December 15, 1995
`
`AGAMATRIX, INC. EXHIBIT NO. 1018
`Page 5 of 6
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`

`

`be that the use of a membrane of this type may stabilise the local
`microenvironment.
`
`CONCLUSIONS
`Of the catalytic powders investigated, MCA4 proved to have a
`response comparable to the best of the other materials, coupled
`with reduced interference effects. Experiments to determine the
`best way of depositing the enzyme and catalytic graphite dem­
`onstrated that it is preferable that both components are screen­
`printed in the same layer as an water-based ink using hydroxyethyl
`cellulose as the polymeric binder.
`Na:fion proved to be amenable to deposition via several different
`methods. Compared to cellulose acetate, Nafion-coated sensors
`produced higher currents and were more stable. Of the methods
`investigated, ink-jet printing proved to be more controllable and
`reproducible. Furthermore, sensors fabricated by this method
`exhibited the highest signal-to-noise ratios.
`
`Using these materials and fabrication methods, biosensors can
`be produced via an automated process, with its inherent advan­
`tages of mass production capability and reproducibility.
`
`ACKNOWLEDGMENT
`The authors thank Gilson Medical Electronics (France) SA for
`funding this project, Fred Jones of MCA Services, Melbourn,
`Cambs, UK, for supply of the catalytic carbon powders, and Philip
`Shaw of Biodot Ltd., Diddington, Cambs, UK, for providing the
`ink-jet printer.
`
`review January 19, 1995. Accepted
`for
`Received
`September 26, 1995.0
`
`AC950062L
`
`•Abstract published in Advance ACS Abstracts, November l, 1995.
`
`Anslytical Chemistry, Vol. 67, No. 24, December 15, 1995 4599
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`AGAMATRIX, INC. EXHIBIT NO. 1018
`Page 6 of 6
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