Gated Voltammetry

The invention may be better understood with reference to ths following drawings and description. The components in the figures are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention. Moreover, in the figures, like referenced numerals designate corresponding parts throughout the different views.

[0076] FIG, 1A is a perspective representation of an assembled sensor strip.

[0077] , FIG. IB is a top-view diagram of a sensor strip, with the Kd removed.

[0078] FIG. 2 depicts an end-view diagram of the sensor strip of HG. IB, [0079] FIGs. 3A and 3E depict a working electrode having a surface conductor and a

DBL during the application of long and short read pulses, [0080] FIGs, 4A and 4B are graphs illustrating the improvement in measurement accuracy when, a DBL is combined with a short excitation.

2016200959 15 Feb 2016 [0081] FIG, 5 represents an elselroehsmical analytic method of determining the presence and concentration of an analyte in a sample, [0082] FIGs. 6A-6F represent six examples of pulse sequences where multiple duty cycles were applied io the sensor strip after introduction of the sample.

[0083] FIG. 7A is a graph showing a cyclic voKarnmogram fiom a sensor system, [0084] FIG. 7B compares a cyclic scan to an acyclic scan, where the forward excitation of the acyclic scan was started near the formal potential E^<ss for the redox pair, [0085] FIG. 7C shows an acyclic scan, where the reverse scan is terminated the reverse carrentpeali.

[0086] FIG. 7D shows a cyclic scan with an acyclic scan superimposed in the DLC region.

[0087] FIGs. 8A-8D shows the output currents plotted as voltamnrograms from the pulse sequence represented, in FIG. 6C for 40% hematocrit WB samples containing 50> 100_;and 400 mg/dL glucose [0088] FIGs, 9A-9C shew contour profiles of the voitenraograrns of FIGs. 3A-SC.

[0089] FIG, IDA is a graph of the semi-integral corresponding to the cyclic voltammogram of FIG. 7A.

[0090] FIG. 10B presents the serai-integral of the acyclic data corresponding to the acyclic votfomniogram of FIG. 7C, [0091] FIG. 10C presents the semi-integrals of the cyclic and acyclic excitations of FIG, ?B.

[0092] * ' FIG. Ι0Ρ shows the* semi-integral and recorded current values for the acyclic excitation of FIG. 7D.

[0093] FIG. Π shows contour profiles prepared by semi-integrating voltammograms from, a seven excitation pulse sequence for WB samples containing varying amounts of glucose, [0094] FIG. 12A depicts the cyclic voltammogram, semi-integral., and semi-derivative of 16 nfrd ferrocyanide in a 20% hematocrit WB sample.

[0095] FIG. 123 is an enlargement of ths semi-derivative carve of FIG. 12A.

[GO96] FIGs; 13A-13C depict the derivatives of cyclic voltarnmogratns.

[0097] FIG. 14 plots the semi-integral currents recorded as a function of time for the contour profiles of PIG. 11.

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I [0098] FIG. 15 depicts the cyclic voltammograras obtained from an under-filled < ::- , i sensor strip.

. [0099] FIGs. 16A depicts semi-integral plots of cyclic voliammograras obtained from five sensor strips wife 1 V/sec seas rates for a sample including 100 mg/dt glucose and 40% hematocrit in WB.

⁵ [00100] FIG. 16B plots the ratio of the forward and reverse scan current values taken > at the 0.1S potential as a function of enzyme concentration.

>

[00101] FIG. 16C depicts a typical response of the slope of the linear response )

< calihr alien of the sensor strip as a function of the GO content (%-dry weight).

>

[00102] HG. 17 is a schematic representation of a measuring device.

P&TAIbto> RggCWl'tOtt [00103] .An electrochemical analytic system determines the concentration of analytes in a sample, such as the glucose concentration of whole blood. The system includes at least one device, that applies gated voltammetric pulse sequences including multiple duly cycles to the sample. Each duty' cycle includes a linear, cyclic, or acyclic excitation during which currents (amperage) are measured from a sensor strip while a potential (voltage) applied to the strip is varied linearly with time. Each duty cycle also includes a relaxation that may be provided by a® open circuit, The system may compare the resulting current data to determine the concentration of the analyte in the sample, white correcting the results for variations in uon-aualyts responsive factors. The system also may apply one or more data treatments, including those based on semi-integration, derivatives, arid semi-derivatives to analyse the voltamsnetric data.

[00104] The gated voltammetric pulse sequences may provide improved accuracy and , precision to the analysis, white reducing the completion time of the analysis. Accuracy errors introduced by the hematocrit effect and precision errors introduced by varying cap-gap volume may be reduced through the combination of a diffusion barrier layer wath the gated pulse sequences. Errors otherwise resulting fiom a noa-steady-state sensor condition, and/or mediator background also maybe reduced. The time required for analysis may be reduced by eliminating the need for additional delays and pukes, such as “incubation delays to provide reagent rehydration, ’ ‘bum-off’ pulses to renew the electrodes, and mediator regeneration pulses to renew the oxidation state of the mediator. The gated pulse sequences also may allow the determination of dynamic current and contour profiles that provide multiple

2016200959 15 Feb 2016 calibration points, under-fiH detection, and the ability to apply temperature compensation to the analysis. Because the gated pulse sequences may generate useful data rapidly, the tong wait times of co.nveniional coulometry and the inaccuracy of non-steady-state measurements in convendonal amperometry may be avoided, [00105} HGs.l A-IB depict a sensor strip 100, which, may be used in the present sensor system, FIG. 1A is a perspective representation. of an assembled sensor strip 100 including a sensor base 110, at least partially covered by a lid 1X0 that includes a vent 130, a concave area 140, and an input end opening ISO. A partially-enclosed volume IdQ (the cap-gap) is formed between the base 110 and the lid 120, Other sensor strip designs compatible with the present invention also may be used, such as those described in U.S. Pat. Nos. 5,120,420 and 5,798,031.

[00106j A liquid sample for analysis may bs transferred into the cap-gap IdO by introducing the liquid to the opening 150. The liquid fills the cap-gap 160 while expelling the previously contained ah through the vent 130. The cap-gap 150 may contain a composition (not shown) that assists in retaining Bis liquid sample in the cap-gap. Examples of such compositions include water-swellable polymers, such as carboxymethyi cellulose and polyethyleno glycol·, and porous polymer matrices, such as dextran and polyacrylamide. [00107] FIG. IB depicts a top-view of the sensor strip 100, with the lid 120 removed. Conductors 170 and 180 may run under a dielectric layer 190 from the opening ISO to a working electrode 175 and a counter electrode 185, respectively. In one aspect, the working and counter electrodes 175, 185 may be in substantially tbs same plane, as depicted in the figure, to another aspect, the electrodes 175, 185 may be facing, such as described in U.S. Pat. App. 2004/0054267.

[00108] While the working and counter electrodes 175, 185 may be closer, in one aspect the electrodes 175,185 may be separated by greater than 200 or 250 pm. Similarly, while at least one of the electrodes 175, 185 may be closer, in one aspect at toast one electrode may ho separated ftom an upper portion of the lid 120 by at least 100 pm. In one aspect, the working and counter electrodes 175, '185 may have surface areas of approximately I mm“ and 12 renti. respectively. The dielectric layer 190 may partially cover the electrodes 175, 185 and may be made from any suitable dielectric material, such as an insulating polymer.

[00109] The counter electrode 185 balances the potential at the working electrode 175 of the sensor strip 100, In one aspect, this potential may be a reference potential achieved by

2016200959 15 Feb ί

>

ί forming Λ© counter electrode 18$ from a redox pair, such as Ag/AgCl, to provide a <

combined refeence-counter electrode. In another aspect, the potential may be provided to the sensor system by forming fee counter electrode 185 from an inert material,, such as carbon, and including a soluble redox species, sc eh as ferricyanide, within. the cap-gap 160, [00110] Alternatively, the sensor strip 100 may he provided with a third conductor and > electrode (not shown) to provide a reference potential to the sensor system. Tins third > electrode may be configured as a toe reference electrode or as an. inert material that relies on >

I a soluble redox species to provide the reference potential. The third electrode also may allow !

t · the measuring device to determine the insertion of a sensor strip and/or if the cap-gap 160 has ϊ

S filled with sample. Additional conductors and/or electrodes also may he provided on the strip

100 to provide these and other functions.

[00111] > FIG. 2 depicts an end-view diagram of the sensor strip depicted in FIG. IB showing the layer structure of the working electrode 175 and the counter electrode 185. The conductors 170 and 180 may he directly on the base 110. Surface conductor layers 270 and 280 optionally may be deposited on the conductors 170 and ISO. respectively. The surface conductor layers 270,280 may be made from the same or from different materials.

[OOH 2] The material or materials used to form the conductors 170,180 and the surface conductor layers 270, 280 may include any electrical conductor. Preferable electrical conductors are non-ioniriug, such that the material does not undergo a net oxidation or a net reduction during analysis of the sample. The conductors 170,180 preferably Include a thin layer of a metal pasts or metal, such as gold, silver, platinum, palladium, copper, or tungsten. The surface conductor layers 270, 280 preferably include carbon, gold, platinum, palladium, or combinations thereof. If a surface conductor layer is not present on a conductor, the conductor is preferably made from anon-ionizing material.

[00113] The surface conductor material may be deposited on the conductors 170, 180 by any conventional means compatible with the operation of the sensor strip, including foil deposition, chemical vapor deposition, slurry deposition, metallization, and the like. In the case of slurry deposition, the mixture may be applied as an ink to the conductors 170, 18Q, as . described in U.S, Pat. No. $,798,031.

[00114] The reagent layers 275 and 285 maybe deposited on the conductors 170 and 180, respectively, and include reagents and optionally a binder. The binder material is • preferably a polymeric material that is at least partially water-soluble, Suitable partially water-soluble polymeric materials for use as the binder may include polyfethylene oxide)

2016200959 15 Feb 2016 ί >

I (PBO), carboxy methyl cellulose (CMC), polvvmyl alcohol (PVA), hydroxycthylene

I cellulose (EEC), hydroxypropyl cellulose (HFC), methyl cellulose, ethyl cellulose, ethvl >

hydroxyethyl cellulose, oarboxymethyl ethyl cellulose, polyvinyl pynolldone (PVT), poiysssmo acids such as polylysine, polystyrene sulfonate, gelatin, acrylic acid, methacrylic acid, starch, maleic anhydride, salts thereof, derivatives thereof, and combinations thereof.

>

j Among the above binder materials, PBO, PVA, CMC, and PVA are preferred, with CMC and > PBO being more preferred at present.

>

| (0011-5], In addition to the binder, the reagent layers 27S and 283 may include the same ί

< or different reagents., In one aspect, fee reagents present in the first layer 275 may be selected >

I for use wife the working electrode 173, while fee reagents present in fee second layer 285 may be selected for use with the counter electrode 185. For example, the reagents In feelayer 285 may facilitate the free flow of electrons between the sample and the conductor 180. Similarly, the reagents in fee layer 27S may facilitate fee reaction of fee analyte, (00116] The reagent layer 275 may include an oxidoreductase specific to the analyte feat may facilitate the reaction of tire analyte while enhancing the specificity of fee sensor system to the analyte, especially in complex, biological samples. Examples of some specific oxidoreductases and corresponding analytes are given below in Table Π.

<td> Oxidoreductase (reagent layer)</td><td colspan="2"> Analyte</td>

<td> Glucose dehydrogenase</td><td> β-glucose</td><td></td>

<td> Glucose oxidase</td><td> β-glucose</td><td> &lt;</td>

<td> ί Cholesterol esterase; cholesterol oxidase</td><td> Cholesterol</td><td> Ϊ</td>

<td> Lipoprotein lipase; glycerol kinase; glycerol-3phosphate oxidase</td><td> Triglycerides</td><td> i i : i</td>

<td> i Lactate oxidase;igctate dehydrogenase; diaphorase • . .. ..... -. ............................</td><td> Lactate .....</td><td></td>

<td> Pyruvate oxidase</td><td> Pyruvate</td><td> I</td>

<td> ; Alcohol oxidase</td><td> Alcohol</td><td></td>

<td> ; Bilirubin oxidase</td><td colspan="2"> Bilirubin</td>

<td> i XMcass</td><td colspan="2"> Uric acid</td>

<td> Glutathione reductase</td><td colspan="2"> NAD(P)H</td>

<td> Ϊ Carbon. monoxide oxidoreductase</td><td colspan="2"> Carbon monoxide</td>

<td colspan="3"> Table H</td>

At present, especially preferred oxidoreduntases for glucose analysis include glucose oxidase, glucose dehydrogenase, derivatives thereof, or combinations thereof,

2016200959 15 Feb 2016 < 20 >

(00117] Tire reagent layer 275 also may include a mediator to more effectively «ί communicate the results of the analyte reaction to the surface conductor 270 and/or the s

• conductor 170. Examples of mediators include QTM complexes, coordination complexes, and electro-active organic molecules. Specific examples indude ferrocene compounds, ferrocyanide, ferricyanide, coenzymes of substituted or unsubstituted pyrroloquinoKne quinones (PQQ), substituted or unsubstitoted S-phsuyliminoGH-phsnothiaztires (ΡΪΡΤ), 34 > phenylimino-SH-phenoxazine (FIFO), substituted or unsubstituted benzoqumoncs, >

substituted or nssubstituted naphthoquinones, N oxides, nitroso compounds, hydroxylamines, oxmes, flavins, phenazine®, phenazine derivatives, phesothiarincs, indophenols, and >

mdammes. Those,· and other mediators that may be included in the reagent layer may he found in U.S. Fat. Nos. 5,653,863; 5,520,786; 4,746,607; 3,791,988; and in BP Fat. Nos. 0 · 354 441 andO 330 517.

' (00118] At present, especially preferred mediators for glucose analysis include ferricyanide, ruthenium hexaamine, P1PT, FIFO, or combinations thereof. A review of useful electrochemical mediators for biological redox systems may be found in Analytics Clinics Ada. 140 (1982)·, pages 1-18.

[00379] The reagent layers 275, 285 may be deposited by arty convenient means, such as printing, liquid deposition, or ink-jet deposition, in one aspect, the layers are deposited by printing. With other factors being equal, the angle of the printing blade may inversely affect the thickness of the reagent layera. For example, when the blade is moved at an approximately 82° angle to the base 710, the layer may have a thickness of approximately 10 pm. Similarly, when a blade angle of approximately 62° to the base 170 is used, a thicker 30 pm layer may be produced. Thus, lower blade angles may provide thicker reagent layers, hr addition to blade angle, other factors, such as the viscosity of the material being applied as well as the screen-size and emulsion combination, may affect the resulting thickness of the reagent layers 275,235.

[00120] The working electrode 175 also may include a diffusion barrier layer (DEL) that is integral to a reagent layer 275 or that is a distinct layer 240, such as depicted iu FIG, 2. Thus, the DEL may he formed as a combination reageatZDBL on the conductor, as a distinct layer on the conductor, or as a distinct layer os the reagent layer, Vtireu the working electrode 175 includes the distinct DBL 290, the reagent layer 275 may or may not reside on the DBL 290. Instead of residing on the DBL 290, fts reagent layer 275 may reside on any

2016200959 15 Feb 2016

Ί >

s portion of the sensor strip 100 that allows the reagent to solubilize in the sample. For

S example, the reagent layer 275 may reside on the base 110 or on tbs lid 120, [00121] The DBL provides a porous space having an internal volume where a

Ί measurable species may reside. The pores of the DBL may be selected so that the ₃ measurable species may difihse into the DBL, while physically larger sample constituents,

J such as RB cells, are substantially excluded. Although conventional sensor ships have used > various materials to filter RB cells from the surface of the working electrode, a DBL provides

I an internal volume to contain and isolate a portion of the measurable species from the sample.

&lt; [00122] . When the reagent layer 275 includes a water-soluble binder, any portion of the binder that does not solubilize into the sample prior to the application of an excitation may function as an integral DBL. The average initial thickness of a combination DBI/reagent layer is preferably less than 30 or 23 micrometers (pm) and more preferably less than 16 pm.

At present, an· especially preferred average initial thickness of a combination DBLAeagsuf layer is from 1 to 30 nm or from 3 to 12 pan. The desired average initial thickness of a combination DBL/reagent layer may be selected for a specific excitation length on the basis of when the- diffusion rate of the measurable species from the DBL to a conductor surface, such as the surface of the conductor 170 or the surface of the surface conductor 270 from FIG. 2, becomes relatively constant [00123] Furthermore, using too thick of a DBL with a short excitation length may delay when tbs diffusion rate of the measurable species from the DBL to the conductor surface becomes relatively constant. For example, when duty cycles including sequential 1 second excitations separated by 0.5 second relaxations are applied to a working electrode • using a combination DBL/reagent layer having an average initial thickness of 30 μηχ, a preferred measurable species diffusion rate from the DBL to the conductor surface may not bo reached until at least 6 duty cycles have been applied (>-10 seconds). Conversely, when the same duty cycles are applied to a working electrode using a combination DB.Vreage.oi layer having an average initial thickness of 11 pm, a relatively constant diffusion rate may be reached after the second excitation (-25 seconds). Thus, there is an upper limit &r the preferred average initial thickness of the DBL for a given duty cycle. A more in-depth treatment of the correlation between DBL thickness, excitation length, and time to reach a relatively constant diffusion rate may be found in WO 2006/042304, filed October 12, 2005, entitled 'Concentration Determination in a Diffusion Barrier Laysrf.

2016200959 15 Feb 2016 ί f )

ι [00124] The distinct DBL 290 may include any material that provides the desired pore ί

I space, while being partially or slowly soluble to the sample. to one aspect, tire distinct DBL

290 may include a reagent binder material lacking reagents, The distinct DBL 290 may have i

an average initial thickness of at least 1 μια, preferably, from 5 to 25 pm, and more ₍ preferably from Stole pm.

J [00125] FIGs, 3A and 3B depict a working electrode 300 having a surface coadnetor ’ 330 and a distinct DBL 305 during the application of long and short read pulses. When a WB

I sample is applied to the working electrode '300, RB cells 320 cover the DBL 305. Anaiyis !

¹ present to the sample forms external measurable species 318 external to the DBL 305. A i · portion of the external measurable species 310 diffuses into the distinct DBL 305 to give internal measurable species 315.

(00126] · As-shown to FIG. 3A, when a. continuous 10 second rend pulse is applied to the working electrode 300, both the external and internal measurable species 310 and 315 are excited at the surface conductor 33Ο by a change to oxidation state. During the long read pukre, the external measurable species 3X0 diffuses through the sample region where the RB cells 320 reside and through the DBL 305 to the surface conductor 330, Diffusion of the external measurable species 310 through the RB cells 320 during the read pulse introduces the hematocrit effect to the analysis. Because a substantial portion of toe measurable species excited at the surface conductor 330 originates from outside the DBL 320, a long read pulse applied to a sensor strip having a DBL may perform similarly with regards to the hematocrit afreet to a short read pulse applied to a strip lacking a DBL.

[00127] Conversely, FIG. 3B represents the situation where a short excitation is applied to the DBL equipped sensor strip. 300 to excite the internal measurable species 315, while substantially excluding from excitation toe measurable species 310 external to toe DBL 305. During the short excitation, fee measurable species 310 either remains external to the DBL 305 or doss not substantially diffuse through, the DBL to reach the surface conductor 330. to tins manner, the short excitation may provide a substantial reduction in the influence of the hematocrit effect on the analysis. By reducing the hematocrit effect, analysis errors . (bias) introduced by the sample constitaeats, including RB cells, maybe reduced.

[00.128] Another advantage of selectively analyzing the measurable species internal to the DBL with a short excitation is a reduction of measurement imprecision from sensor strips having varying sap-gap volumes. Variances in toe cap-gap volume between sensor ships may lead to imprecision because too electronics in conventional measuring devices apply the

2016200959 15 Feb 2016 ₍ 23 >

δ same electric potential and perform the same calculations for each analysis. If a read puke &lt;

I contmues past the time when substantially all of the measurable species present in the capgap has been analyzed, the analysis no longer represents the concentration of measurable species in fee sample, bal instead represents the amount of measurable species in the cap-gap; a very different measurement. Thus, a sensor strip having a larger cap-gap volume will show > a higher analyte concentration than a sensor strip having a smaller cap-gap volume, i independent of the analyte concentration of the sample. By substantially limiting analysis to )

! the measurable species present in the DBL, the imprecision otherwise introduced by i

i - manufacturing variability between sensor strips may h e reduced.

j

I [00129] FIGs, 4A and 4B are graphs illustrating the Improvement in measurement accuracy when a DBL was combined with ‘a short excitation. FIG. 4A shows a large inaccuracy represented as the difference between the 16% and 48% calibration lines (the total .hematocrit bias span) resulting from a sensor strip lacking a DBL after a 1 second excitation, Conversely, FIG. 4B shows a smeller difference between the calibration lines representing a more accurate result when a DBL was combined with a 1 second excitation, The total bias hematocrit span for fee DBL combined, with a short excitation was nearly two-thirds less than the total bias span without fee DBL, [00130] As described above and in further detail in WG 2006/042304, a short read, pulse or excitation may provide an improvement in the accuracy and/or precision of an analysis. However, if a single short excitation is used for the analysis, a relatively constant diffusion rate of the measurable species from, the DBL to the conductor surface may not be reached during the analysis.. This condition also may result in measurement inaccuracy because the concentration of the measurable species within fee DBL does not accurately represent that,in fee sample, Furthermore, the single excitation may not effectively reduce fee background signal from the mediator.

[00131] FIG. 5 represents an electrochemical analysis SCO for determining the presence and optionally fee concentration of an analyte 522 in a sample 512 that may overcome the disadvantages associated with short excitations. In one aspect, the analysis 500 may reduce bias from mediator backcyound while providing a shorter analysis time wife or without a DBL, In a preferred aspect, the analysis 500 may be completed In less than 3 or less than 1 minute. In a more preferred aspect, the analysis 500 may be completed in from 2 to 50 or from 4 to 32 seconds.

2016200959 15 Feb 2016 > f >

* [00132) In 51.0, the sample 512 is introduced to a sensor strip 514, such as the sensor

S atrip depicted in FIGs. 1 A-IB and 2. The reagent layers, such as 275 and/or 285 front FIG. 2, i

begin to solubilize into the sample 512, thus allowing reaction. At this point in the analysis, cn initial time delay, or “incubation period,” optionally may be provided tor trie reagents to react with the sample 512. Preferably, the optional time delay may be from 1 to 10 seconds.

ί ’ A more ίη-deptb treatment of initial time delays may be found in U.S. Pat. Nos. 5.620,579 ι

> and 5,653,863. In one aspect, the analysis 500 may reduce the need for an incubation period.

>

I [90133} During the reaction, a portion of the analyte 522 present in the sample 512 is i

i chemically or biochemically oxidized or reduced in 520, snob as by an oxidorsduefese. Upon ί

j oxidation or reduction, electrons. optionally may he transferred between the analyte 522 and a.

mediator 532 in 530.

[00134} In 540, a measurable species 542, which may bs the charged analyte 522 from 520 or the charged mediator 533 from 530, is electrochemicaFy excited (oxidized or reduced). For example, when the sample 512 is whole blood containing glucose oxidized by glucose oxidase in 520 and transferring an electron to reduce a ferricyanide (III) mediator to ferrocyanide (Π) in 530, the excitation of 540 oxidizes ferrocyanide (H) to ferricyanide (HI) at the working electrode. In this manner, an electron is selectively transferred from the glucose analyte to the working electrode of the sensor strip where it may be detected by a measuring device (not shown).

[00135] The excitation 540 includes vollammefrie scanning where a varying potential or “scan” is applied across the electrodes of the sensor strip 514 at a substantially Sxed rate (V/sec). The scan rate may be· stow or fast; however, fast scans are preferred due to dis nature of the gated pulse sequences. In one aspect, the rate at which the potential is scanned is at least 2 mV/sec, preferably from 20 to 5000 mV/sec, more preferably from 200 to 2000 mV/sec. At present, an especially preferred scan rate is from 500 to 1500 mV/sec.

[00136} The duration of the excitation S40 is at most 4 or 5 seconds, and preferably less than 3, 2,1.5, or 1 second, hi another aspect, the duration of the excitation 540 is from 0.1 to 3 seconds, from 0.1 to 2 seconds, or from. 0.1 to 1.5 seconds. More preferably, the duration of the excitation 540 is from 0.4 to 1.2 seconds.

[00137j frj 550, fire currents resulting from the scanning excitation 540 may he monitored and recorded as a function of the applied potential (voltage). Tins contrasts with conventional amperomefry and coulometry where a constant voltage is applied while tire current is measured, as a function, of time. In one aspect, tbs current is monitored and

2016200959 15 Feb 2016 >

$ recorded daring the excitation S48. In another aspect, the current is not monitored during tire s· s relaxation 560 or at least during a portion of the relaxation 560. Tn another aspect, the current s

and the potential at the working electrode may be monitored during at least a portion of the relaxation 560, but the: values are not used in determining the concentration of the analyte 522.

>

) [00138} In 560, the sample undergoes relaxation, where the measuring device may

Ί > open the circuit through the sensor strip 514, thus allowing the system to relax. During the relaxation 560, the current applied during the excitation 540 is substantially reduced by at * least one-half, preferably by an order of magnitude, and more preferably to zero, Preferablv, )

j a zero current state is provided by an open circuit. In one aspect, the relaxation 560 is at least

10, 5, 3,2,1,5,I, or 0.5 seconds in duration. In another aspect, the relaxation 560 is from 0,1 to 3 seconds, from 0.1 to 2 seconds, or from 0.1 to 1.5 seconds.in duration. More preferably, the relaxation 360 is from 0,2 to 1.5 seconds in duration and provided by an open circuit. [00339] During the relaxation 560, the ionizing agent may react with the analyte to generate additional measurable species without the effects of an electric potential. Thus, for a glucose sensor system including glucose oxidase and a ferricyanide mediator as reagents, additional ferrocyanide (reduced mediator) responsive to the analyte concentration of the sample may be produced without interference from an electric potential dicing the relaxation 560.

[8014b] The excitation 540, the recordation 550, and the relaxation 560 constitute a single duty cycle. In 578, the duty cycle is repeated at least once for a total of at least two duty cycles. In one aspect, fee duty cycle is repeated at least twice for a total of at least three duty cycles within 188 seconds, 90 seconds, or less. In another aspect, the pulse sequence of the analysis 580 includes at least 4, 6, 8, 1.0, 14, 18, or 22 duty cycles applied during an independently selected 120, 90, 60, 30, 15, 10, or 5 second time period. In another aspect, the duty cycles are applied during a 5 to 60 second time period. In another aspect, from 3 to 18 or fe?·¹. 3 to 10 duty cycles may be applied within 30 seconds or less. In another aspect, from 4 to 8 duty cycles may be applied within 3 to 16 seconds.

[80141} Tire repetitive “on^SJ and ‘riff’ nature of the duty cycles of the analysis 500 directly contrast with conventional methods where voltage is continuously applied to and current is continuously drawn from a sensor strip tor from 5 to 10 seconds during the duration of fee rend pulse. For these conventional methods, fee applied voltage may have a fixed potential or may have a potential that is swept from a positive to a negative potential or from

2016200959 15 Feb 2016 >

# a positive or a negative potential to a zero potential relative to a reference potential. Even at a zero relative potential, these methods continuously draw current fe© the sensor strip during fee read pulse, which permits the electrochemical reaction to continue throughout the read pulse, Thus, in these conventional methods the reaction that produces measurable species responsive to the analyte concentration cud the diffusion of the measurable species to the working electrode are both affected by current during the zero potential portion of a > conventional read pulse. The analysis 500 pulse sequences also are. markedly different tiom

J* conventional methods that use a single long duration pulse with multiple measurements, such as, those disclosed in U.S. Pat. No, 5,243,516, due to the multiple relaxations 560.

Ϊ V . [80142] In 580, the recorded current and voltage values may be transformed with one , or more data treatments. The transformed values may be used to determine the presence and/or concentration of the analyte 522 in the sample 512. The transformed values also may be used to determine other characteristics of the analysis 500, including the hematocrit concentration of the sample, multiple calibration sets, under-fill, and the active ionizing agent content of the sensor strip, s&amp; outlined below, [00143] FIGs. 6A-6F depict six examples of gated voltammstric pulse sequences that may he used with the method 500. In each pulse sequence, multiple duty cycles were applied to the sensor strip after introduction of the sample. The voltammetric excitation portion of each duty cycle may be applied in a linear {FIG. 6A), cyclic (FIG. 63), or acyclic manner .· (PIGs, 6C-6F), hr these examples, tilted (linear) or triangular-wave (cyclic or acyclic) excitation pulses were used; however, other wave types compatible with the sense? system and ills sample also may be used.

[00144] FIG. 6A depicts multiple tilted excitations where the voltage increased linearly with time to sn endpoint. FIG. 6B depicts multiple triangular-wave excitations providing , cyclic data that includes the complete potential rang® of the ferricyanide mediator. FIG. 6C . depicts six duty cycles including six triangular-wave excitations providing acyclic data that starts and ends at substantially the same voltage. Because the last excitation of FIG. 6C. a terminal read pulse 640, lacks a relaxation, only six duty cycles are shown. FIG. SD depicts seven duty cycles including seven triangular-wave excitations providing acyclic data. The fust duty cycle is preceded by an initial incubation period. FIG. 63 depicts multiple triangular-wave excitations providing acyclic data that starts and ends at different voltages. FIG, 6F depicts multiple triangular-wave excitations resulting in acyclic data that

2016200959 15 Feb 2016

Ί s

ί )

)

J i

substantially exclude the oxidation and reduction peaks of the fenicyanide/ferrosyanide [00145) The terminal read pulse 640 may have the same duration and scan rate as the excitations of the prior duty cycles, as depicted in FIG. 6C, or the terminal read pulse 640 may have a different duration or rate. In· one aspect, the terminal road pulse 648 may be of longer duration and increased voltage in relation to die excitations of the prior duty cycles. The increased voltage may provide the ability to detect a species having a Higher oxidation potential, such as a control solution, A more complete discussion regarding terminal read pulses may be found in U.S. Provisional· App, No. 60/669,729, filed April 8, 2005, entitled ‘Oxidisable Species as an Menial Reference in Control Solutions for Biosensors,” [00146) Control solutions containing known amounts of glucose may be used to verify thatthe analysis system is operating properly. Specific formulations for control solutions maybe found in U.S. Pat. Nos. 3,920,580; 4,572,899; 4/729,959; 5,028,542; 5,605, 83?;‘md

PCT publications WO 93/21928; WO 95/13535; and WO 95/13536. If the measurement device cannot distinguish between a signal from a control solution versus a sample, control .solution readings may be stored as analyte values. Thus, the history of &amp; patent’s glucose readings, for example, may be inaccurate regarding diabetic condition,

100147] If the control solutions csrniot be identified and their responses separated from those of the blood samples by the test meter, glucose readings of the control solutions will be included in the history of the glucose measurements, which could lead to wrong interpretation of a patient’s diabetic condition.

[09148) Bach of the duty cycles for the pulse sequences depicted in BIGs. 6A-6F provide excitation times of shorter duration than the folk-wing open circuitrelaxstion times;

however, this is not required. In BIG, 6C the duration of the excitations is 0.8 seconds at a rate of 1 V/'sec white the duration of each relaxation is about 3.2 seconds. Thus, each duty cycle has a duration of about 4 seconds and the pulse sequence tests for about 24.8 seconds, including a terminal read pulse to provide a redox intensity (RI) of 0.226 (>.6/24,8). The pulse sequence of FIG. 6D provides & lower RI of 0.2 (5.6/28), atrribntabts to the incubation period before the first duty cycle.

ί [00149) The higher the RI for a pulse sequence, the less background, inaccuracy introduced into the analysis by the mediator. The pulse sequences represented in FIGs, 6Α» 6B are oxidative pulses, designed to excite (e.g. oxidize) a reduced mediator, which is the measurable species. Thus, the greater the oxidative current applied to the sensor strip in a

2016200959 15 Feb 2016 ι 28 >

* given fens period, the less chance feat mediator reduced by pathways other than oxidation of

S the analyte contributes to the recorded current values. In combination, the multiple excitations of the gated voltsmmetric pulse sequence may eliminate fee need for an initial pulse to renew the oxidation state of the mediator. For ferricyanide, pulse sequences having RI values of at least 0.01, 0.3, 0.6, or 1 are preferred, with RI values of irons 0,1 to 0,8, from )

> 0.2 to 0.7, or from 0.4 to 0.6 being more preferred at present, > [00750] During a linear excitation, such as forward excitation 610 depicted in FIG. 6A, >

ί the current at the working electrode is measured while the potential at the working electrode >

: changes linearly with time at a constant rate. The excitation range, such as from -0.5 V to ί +0.5 V, may cover the reduced and oxidised states of a redox pah so that a transition from a first state to a second state occurs. The current measured at the working elsctrode may be thought of as having three components: the equilibrium current, the diffusion current, and the surface current. The surface current which may derive from any species, adsorbed on the electrode, is generally small. The equilibrium and diffusion currents are the primary components represented in the resulting voltammogram.

[00151] A ‘linear voltammogram (a plot of current verses voltage) may be characterized by a plot that starts at an initial current, reaches apeak, current, and decays to &amp; lower diffusion-limited current (DLC) level during the excitation. The initial current is substantially dependent on the applied potential, while the DLC is not. If the scan is slow' enough, the DLC may be seen as a plateau region in a voltammogrem.

[00152] The DLC region represents a state where the oxidation or reduction of the .measurable species at the conductor surface reaches a maximum rate substantially limited by diffusion. The diffusion may be limited by the rate at which the measurable species travels from the sample to the conductor surface. Alternatively, when die working electrode of the sensor strip includes a D3L, the diffusion may be limited by fee rate at which the measurable species travels from the DBL to the conductor surface.

[00153] DLC values recorded at a relatively constant diffusion rate after rehydration of • the reagent layer may minimize inaccuracies that would otherwise be Introduced by variations in the rehvdration and diffusion rates of the reagents. Thus, once a relatively constant diffusion rate is reached, the recorded DLC values may more accurately correspond to the concentration of the measurable species, and thus fee analyte.

[00754] After completion of fee forward excitation 61G, for a cyclic or acyclic excitation, such as. those depicted in FIGs. 6B and 6C, respectively, a reversed potential linear

2016200959 15 Feb 2016 ι

( ϊ * excitation 620 is applied. Tire reversed potential linear scan of fee excitation 620 may be i

applied at substantially fee same rate as the forward scan 610, Thus, fee excitation range is scanned from a first lower value to a higher value and back to a second lower value, where i

the first arjd second lower values may or may not be the same for cyclic or acyclic scans, ₍ respectively. Cyclic, and in some instances acyclic, excitations may examine the transition of

J a redox species from, a reduced state to an oxidized state (and vice versa) in relation to the ¹ applied, potential or in relation to the diffusion rate of the redox species to fee conductor [00155] In relation to a linear excitation, cyclic and acyclic excitations may provide a better representation of the DLC region of the excitation. The advantage of cyclic and acyclic -excitations may be especially advantageous for quantifying the DLC from quasireversible redox pairs at fast scan rates. Additional information about linear and cyclic scan voltammetry may he found in “Etectrochenncal Methods: Fundamentals and Applications” by A. J. Bard and L.R. Faulkner, 1980.

[00156] FIG, 7A presents the data from a 25 mV/sec cyclic excitation of a fenicyanide/ferrccyamde redox pair as a cyclic voltammogram. The voltasunogram is characterized by a forward current peak during the forward portion of the scan from --0 3 V to +0.6 V indicating ferrocyanide oxidation and a reverse current peak during the reverse voltage scan from 9-0,6 V back to -0.3 V indicating ferricyanide reduction. The forward and reverse current peaks center around the formal potential E^0, of fee fen'oeyanide/femeyanido redox pair, when referenced to the counter electrode. In this aspect, the potential of the counter electrode is substantially determined by fee reduction potential of ferricyanide, the major redox species present on the counter electrode.

[00157] While the potentials where the forward and reverse scans begin, (the excitation range) may be selected to include the reduced and oxidized states of the redox pair, the excitation range may be reduced to shorten the analysis time. However, the excitatkm range preferably includes the DLC region for the redox pair. For example, at a scan rate of 25 mVZsec, the concentration of fee reduced [R.&amp;d1 and oxidized [Ox] species of the fenOcyanideVforricyamde reversible redox pair and the resulting electrode potential are described by fee Nernst equation as follows.

<img file="AU2016200959B2_D0001.tif" />

+0.059Sog

<img file="AU2016200959B2_D0002.tif" />

CO

2016200959 15 Feb 2016 )

) >

I [00158} In the Nemst equation, R is the gas. constant of 8.314 Joui/QmdeHC), F is the Faraday constant of 96,5000 CouLtoquiv., a is fee number of equivaleats per mole, and T is ths temperature in degrees Kelvin. When the potential at the woridng electrode is referenced to its own redox potential, ths formal potential S⁵’ will become substantially £ero and the equation collapses to:

: 0.0591oe ’[Red] _: 0,0.50 tog [Fe(CN)^] (2)

From equation (2), when the ratio of the oxidized mediator to the reduced mediator changes by 10» tire potential at the working electrode changes by about 60 mV. The reverse is also true; Thus, for ferricyanide [Ox] to ferrocyanide [Red] concentration ratios of 10:1, 100:1, -1000:1 and 10,000:1, the potential at the working electrode will be approximately 60, 120, 180, and 240 mV away .from the zero potential» respectively.

[00159] Thus, when die ratio of ferricyanide to ferrocyanide is —IQOG;1, a scan range of -180 mV to H8G mV would provide substantially complete oxidation of the reduced species at the working electrode. At ISO mV, the oxidation rats is limited by how fast the reduced form of fee mediator can diffuse to the conductor surface, and from this potential forward, there exists, a DLC region. Thus, if the reversing point is set -400 mV from the zero potential, -200 mV of DLC region may be provided.

[00160] For· reversible systems, it may be preferable to provide an excitation range of from- 400 to 600 mV, feus exciting from 200 to 300 mV on each side of the formal potential ft

Ή ’ of the redox paw. For quasi-rev ersihle systems, it may be preferable to provide an excitation range of from 600 to lOOOmV, thus exciting from 300 to 500 mV on each side of ft the formal potential E ’ of the redox pair.

[00103} The larger excitation range may be preferred for qnasi-reversible systems because the DLC region may be smaller. In addition to redox pairs feat are inherently quasireversibto, fast scan excitation may cause a redox pair that is reversible at slow excitation rates to demonstrate quasi-reversibte behavior. Thus, it maybe preferable to provide a larger quasi-reversibte excitation range for a reversible redox pair at fast excitation rates.

[00162] Preferably, at toast 25, 50, 100. 150, or 300 mV of DLC region is provided byfee selected excitation range. Is another aspect, the reversing point for a cyclic or acyclic excitation is selected so that from 25 to 400 mV, from 50 to 350 mV, from 1Q0 to 300 mV, or from 175 tc 225 mV of DLC region is provided. For reversible systems, the reversing point for a cyclic or acyclic excitation may be selected so that from 180 to 260 mV or from 200 to

2016200959 15 Feb ⁵ 240 mV of DLC region is provided. For qnasi-reversibie systems, the reversing point for &amp;

I cyclic or acyclic excitation may be selected so that 6cm 180 to 400 mV or from 200 to s

260 mV of DLC region is provided.

[00163} Once the reversing, point to selected to provide the desired DLC region, tbs > duration of the revsme scan may be selected for an acyclic scan. As can be seen in FIG. 7B, > starting the forward scan and terminating the reverse scan at approximately -0.025 mV > resulted in an acyclic scan that included more of the forward current peak than the reverse >

I current peak. From the FIG. 7B comparison, while the peak enrrente obtained for the cyclic i

&lt; (a) and acyclic (b) scans differ, the DIG region of the scans were nearly the same, especially s

| with regard to foe reverse scan.

[00164] In another aspect, the reverse excitation may be terminated before the reverse current peak is reached, as depicted in FIG. 7C. When the forward excitation was started at a potential sufficiently negative, such as at -0.3 mV in FIG. 7C, to die middle of the potential range of the redox pair, such as -0.Q5 mV in FIG. 7C, the forward excitation included the fell range of the redox potential of the redox pair. Thus, by terminating the reverse excitation at a potential from 50 to 500 mV, from 150 to 450, or from 300 fo 400 mV negative from fee reversing point, for example, the reverse current peak may be excluded for the ferrioyanids/ferrocyanide redox pair.

[001G5] Similarly, the reverse excitation also may be terminated before the reverse .current peak is reached by. terminating the excitation when the reverse excitation current deviates in value from the DLC. A change in the reverse excitation current of at least 2%, 5%, 10%, or 2.5% may he used to indicate the beginning of the reverse excitation current peak.

[00166] FIG. 7D compares a 1 V/sec cyclic Yoltammograrn including the forward and reverse oxidation peaks of the redox pair with a 1 V/sec acyclic voRamuwgram that excludes the forward and reverse oxidation peaks of a redox pair. The acyclic excitation had starting and ending points of 200 mV and a reversing pofrit of 300 mV. Preferable excitation ranges for acyclic excitations within the- DLC region of the ferricyamde/ferrocy&amp;mde redox part·, which exclude the forward and reverse oxidation and reduction peaks, are from 10 to 200 mV, more preferably from 50 to 100 mV. While the cyclic voltajnmogram including the complete scan range significantly decayed after reaching the current peak, the acyclic voltammogram provided a substantially flat current region aver the scan range. This current region maybe directly correlated with the analyte concentration of the sample.

2016200959 15 Feb 2016 <ί >

[00167] As seen in FIG, 7D, the current values recorded for the acyclic excitation are

I numerically smaller than those of the cyclic excitation, while the background current is lower for the acyclic excitation. This beneficial reduction m background current was unexpectedly ’j ....

obtained without having to initiate the acyclic excitation in the reduction peak portion of the cyclic excitation. Thus, a fast and short acyclic excitation within the DLC region of a redox pair may increase the accuracy of analyte determination due to a reduction in the background > current, which may provide an increase in the signaMo-baclrgronnd ratio.

[60168] Cyclic and acyclic excitations may provide multiple benefits in relation to &lt; linear excitations, fit one aspect, the portion of the reverse scan from the reversing point to i the point where the reverse current peak begins may be a better representation of the true

DLC values than the DLC region of the forward scan. The DLC region of the reverse excitation may be a more accurate representation of analyte concentration for quasi-rev srsihle redox systems or at fast excitation rates because the forward excitation may not show a distinct DLC region.

[00169] Acyclic excitations may have multiple advantages over cyclic excitations including a shorter excitation time and a substantial decrease in the amount of mediator elecfeochemically converted to the measurable state. Thus, if the mediator is reduced in response to the analyte and elsctrochemically oxidised during measurement, terminating the reverse excitation before the oxidised mediator is electiochemically reduced decreases the amount·of reduced mediator in the sample not responsive to the analyte. Similarly, starting the forward excitation at a potential above that at which the measurable species is reduced also may decrease, the amount of reduced mediator in the sample not responsive to the analyte, Both acyclic excitations may allow for a shorter analysis time, a significant benefit for the user.

[00.170] FIGs, SA-SD show the output dynamic currents plotted as a function of potential from the pulse sequence of FIG. &lt;$€ using 7 triangular waveform excitations for WB samples containing 40% hematocrit and 0, 50, 100» and 400 mg/dL of glucose. The scan rate was '1 V/sec. Instead of a conventional long duration read pulse resulting is extensive oxidation of the measurable species, each, triangular excitation was followed by a relaxation to provide a break in the current profile. The currents from each successive excitation were plotted as a different ‘hep*³ line, thus providing rep 1 through rep? for each Figure.

[60171] The current values from each of the multiple excitations (each rep) in the voltammograms of FIGs. 3 A--8D were converted to a single data point and connected to give

2016200959 15 Feb 2016 ths contour profiles of FIGs. 9A-9C. For FIGs. 9 A and 9B, the conversion was accomplished

I by selecting a current value at the same potential in ths DLC region of each successive s

excitation, such as 300 mV, In FIG, 9A, the current values from FIGs, SA.-8D were directly £

plotted as a function of time from the ending of the pulse sequence. In FIG. 9B, a semiintegral data treatment was applied to the current values before plotting. For FIG. 9C, the > multiple excitations were converted to single data points by selecting the peak current value ΐ

> of each rep and using a semi-derivative data treatment. In this manner, the X-axis of the >

I contour profiles are expressed in terms of time, thus mimicking the data obtained from a t conventional system at steady-state, where the current change with time is substantially »

constant. While the recorded voltenmogram. currents may be treated in multiple ways to extract useful information, semi-integral, semi-derivative, and derivative data treatments are presently preferred.

[00172] Hie dynamic current profiles obtained from gated voltammetric pulse sequences are fundumentelly different from the current profiles obtained from a conventional analysis using a single read pulse. While currents recorded from a single read pub® derive .from a single relaxatiew'diffusion, each time point in ths contour profile of the dynamic currents originates from an excitation after an independent relaxation/difibsion process. Furthermore, as the length of an excitation increases, the correlation between the current and the analyte concentration may decrease, often due to the- hematocrit effect. Thus, the accuracy of an analysis using multiple, short excitations may be increased in comparison to an analysis using a longer read pulse having, ths duration of tire multiple excitations combined, [00173} Ths application of these data treatments to glucose analysis is described below. However, a more in-depth discussion of data treatments for transforming electrochemical currents and the related digital implementations may be found in Bard, A.J., Faulkner, L,R_&lt;S “Electrochemical Methods: Fundamentals snd Applications, 1980; Oldham, K.B.; A Si^ial-Independent Blecfroanalyfeal Method,” Anal, C%em. 1972. 44_s 196; Goto, M._s Oldham, K.B., “Semi-integral Eleotroaualysis: Shapes of Neopolarogranisrircaf Ch&amp;n, . 1973, 4J, 2043; Dalrymple-Alford, P._t Goto, M-, Oldham, K.B., Peak Shapes in Semidifferential Electroaualysis,” AaaL Cfem, 1977, 49. 1390; Oldham, R.B., “Convolution: A General Electrochemical Procedure Implemented by a Universal Algorithm,” dwti. Cheat. 1286,, 58, 2296; Pedrosa, LM.., Martin, M.T., Ruiz, J.J., Camacho, L.. “Application of the Cyclic Sssni-totegral Voltammetry and Cyclic Semi-Differential Voltammetry to the

2016200959 15 Feb 2016 ί

Ί >

I j

a )

>

>

Determination of the Reduction Mechanism of a bli-Porphyrin,” J. Etectroanai Cheat, 2002, 523_s 160; Klicka, R, Adsorption in Semi-Differential Voltammetry,” J. Electraanah Cheat. m8, 455., 253, [00174] Semi-mtegratiou of a voltamnrograra may separate the DLC from the hematocrit affected equilibrium current (initial peak) because separate signals may he observed for the hematocrit-affected equilibrium si current and the hematocrit. This is especially true at slow scan rates. The serai-integral of the experimentally obtained vottammehic current ifri has the following mathematical form:

dr*¹² f(0/(0

<img file="AU2016200959B2_D0003.tif" />

f /(«) /(r = «)* clu (3) where i(t) is the time function of fee voltammeirie current obtained during the scan; l(t) Is a transformation and the semi-integral of u is a transformation parameter; and is the semi-integration operator, [00175] At a sufficiently high oxidation potential, the steady-state semi-integral current is given by:

/«,, - ftFriD^C (coni/set?²) (4) where /&amp;,, is the DLC under fee condition of fee surface concentration of the oxidteable species being zero. Note that tire unit of semi-integral current is coul/sec^, which is not the traditional unit for expressing electrical current, which is coul/sec, [00176] For simplicity, is refared to as the serai-integration. DLC (SI) with a unit of coul/sec^ia. Tins SI current (eonl/serf'D is only a half-step integration from current (coul/sec). The half-step iutegrarion is fendamentally different from coulometry where s full integral is applied to the f-ί curve to proride the total charge passing through the electrodes. [00177] Although equation (3) gives a theoretical definition of the semi-integral, for digital processing the ri? data may be divided into H equally spaced time intervals between t ~ 0 and ; = Arii, One such digital processing algorithm is given by equation (5) where t ^;Arif and u -yrit, and I Is determined at the midpoint of each interval.

7(W) /(/At 1/ 2Δί)Δί^,i3! /b-Jil/’s (S)

A preferred algori-bra for digital processing is given by:

2016200959 15 Feb 2016 *

ΐ i

ΐ ί

ί

J ϊ

i ϊ

ϊ !

ί j

7(/εΔί)^:

1/?.)

Λν ²ί&amp;'ώ/} where Γ(χ) is the gamma function of x, where Γ{1/2) ™ π^ν2, Γ(3/2) ™ l/fefe², and Γ(5/2) ™ 3/2*1/2π’^β, etc.

[00778] From equation (4) it may be seen that the SI current lacks the time-dependence factor of conventional amperometdc methods. Thus, the SI current response may be considered a series of plateau currents, instead of the continuously changing amperemeteic currents obtained from conventional amperemetvy. Because the semi-mtegrarion allows for quantification of the DLC, a faster scan rate may be used than when peak currents. are quantified. Thus, linear, cyclic, or acyclic voltammetry hi combination with, semi-integration may rapidly generate a DLC in response to glucose concentrations, In this manner, fee disadvantages of the long wait rimes of coulometry and the nom-sleady-siate nature of tire current in conventional amperometry may be reduced, [00179] Equation (4) also shows that reversible or quasi-reversihle redox pairs are preferred for use with semi-integration. This is because the semi-integral from a reversible or quasi-reversible redox pair can exhibit a sharp transition from the reduced state to the oxidized stats (and vice versa) and a wide DLC region, thus making the transition easier to determine. Fsmcyanide/ferrocyanide and fee +3 and 42 states of rufeemum hexaamine are examples of redox pairs demonstrating preferred reversible (slow scan) or quasi-reversihle (fast scan) behaviors, [00180] Poorly activated electrodes may not provide an acceptable DLC condition even with reversible or quasi-reversible redox pairs. Thus, electrode activation procedures, such as those described in U.S. Pat. No, §,429,735, may be used to achieve the preferred electrode activity.

[00187] in addition to semi-integrals, semi-derivatives of a vcltsmmogram also may be used to quantity the analyte by measuring fee peak of the semi-derivative. The semiderivative of fee experimentally obtained, voltammetric current i(t) has fes following mathematical forms:

di \n

KO ri^!?i ... ri [' 1 V Ife) riw i, (cred / sec%³) (8)

J

2016200959 15 Feb 2016 's

J

,.. . _a where I{t) is the semi-integral of fee time function iff). The equations used for the scanJ integral., semi-dsrivativs, and the derivative data treatment described below, were ‘ implemented with the Electrochemical Workstation software package, version 4.07, revised.

April 26, 2004, which accompanies &amp;e CH Installments Electrochemical Workstation, model x CHIS6QA, \ ......

' [00182] FIG, 10A presents the semi-integral plot of the cyclic voltammogram from ’ BIG. 7A. Similarly, PIG. 1QB presents tbs semi-integral plot of the acyclic voltammogram

J from FIG. 7C_S where the reverse excitation terminated before initiation of the reverse current * . peak. FIG. IOC establishes that when the semi-integral of the cyclic and acyclic excitations of BIG. 7B ar e plotted, the DLC region of the return, scan was readily established, permitting an accurate current reading in as little as 50 mV from, the reversing point. Furthermore, the peak portion of Ge semi-integral plot was responsive to the hematocrit content of the sample and the magnitude of the peak may be quantitatively related to the hematocrit level, [00183] FIG. If© shows the semi-integrals for the cyclic and 200 to 300 mV acyclic excitations of HG. ?D. The shape of the si voltammogram from the short acyclic excitation differs from the voltammogram of the cyclic excitation because the region of oxidationreduction transition ia missing from the acyclic excitation. By starting the acyclic excitation in the DLC region, the background si current decreased at a faster rate in comparison to that observed for Ge cyclic voltammogram, thus improving the si-gnal-to-backgroimd ratio for the acyclic excitation. Furthermore, the reverse ,« current from the acyclic excitation shows a _s plateau more accurately describing the analyte concentration of the sample than the forward ri current, frt this manner, fee acyclic scan of the DLC region provided an increase in accuracy for fee analysis when compared to the cyclic excitation.

[00184] FIG. 11 shows contour profiles prepared by semi-integrating voltammograms from a seven excitation pulse sequence for WB samples containing 0, 5-6, 111, 221.75, 455.25, and 712,5 mg/d’L of plasma glucose. For each of fee glucose concentrations, equilibrium with regards to DEL rehydration was reached at the highest current value in the contour profile for each glace se. concentration. Thus, readings Π10 (highest) and 1120 (lower) establish that equilibrium was reached regarding DEL rehydration at about four seconds for fee 455 mg/dL glucose ccEce.nbu.tion, [00185] Current valn.es recorded at a relatively constant diffusion rate may minimise inaccuracies that would otherwise be introduced by variations In fee rehyfeation and diffusion rates of the reagents. Thus, once a relatively constant diffusion rate is reached, the

2016200959 15 Feb 2016

3?

recorded current values may more accurately correspond ώ the concentration of the

I measurable species, asfo thus the analyte. Furthermore, for FIG. 11, the complete analysis &amp;

may be completed io as few as seven seconds because once the highest current value 1110 of ή

the contour profile is known, its value may be directly correlated to the analyte concentration, Additional data points may be obtained to reduce background error attributable to the mediator, as previously discussed with regard to RL [00186] Another form of data treatment that may be used to generate a contour profile

I is semi-derivatizatiott. Os® implementation of a semi-derivative is to take a foil step s

&lt; derivative of the semi-integral, as previously described in relation to equation (S). Unlike the

I plateau region representing the vcdtammetric scan to semi-integral plots, semi-derivative plots convert the voltammstric scan data into a peak centered at the transition of the redox pair. · [00187] FIG. I2A depicts the cyclic voltammogram (a), semi-integral (b), and semiderivative (c) of 16 mM ferrocyanide to a 20% hematocrit WB sample. In (his instance, the writing electrode of the sensor strip lacked enzyme and oxidized mediator. FIG. 12B is an enlargement of the semi-derivative curve of FIG, 12A showing tbs peak height for the forward scan. The value of the forward or reverse scan peak height may be correlated with the analyte concentration of the sample. Furthermore, the semi-derivative data treatment may inherently provide hematocrit compensation for glucose determination, especially for samples including less than 40% hematocrit, A more detailed description of the application of semiderivative data treatment to glucose analysis may be found in WO 2005/114164, filed May 16, 2005, entitled ''Woltammetric Systems for Assaying Biological Analytes,” [00188] In addition to semi-integral and semi-derivative data treatments, a derivative data treatment also may he used to generate a contour profile, and thus determine the concentration of the analyte in the sample. FIGs, 13A-13C depict the derivatives of cyclic voltammograms for samples having 20, 40. and 60 % hematocrit. These derivative plots show an initial increase in current as voltage increases, followed by a decrease, and finally a DEC region. The hematocrit effect may be seen to the negative peak located at about 0,1 volts in FIGs. 12A-12C, with higher RB cell concentrations reflected as more negative peak values.

[00189] While the values of the positive and negative derivative peaks, such as those depicted in the derivative plot of FIG. 13B. arc concentration dependent, toe ratio of tire negative peak to toe positive peak cancels cut the concentration dependence, thus being

2016200959 15 Feb 2016

4 >

hematocrit dependent. Because this ratio (HI-DBR) is concentration independent and ί hematocrit dependent, the ratio indicates the percent hematocrit in the sample. Thus, this ratio of the derivative peaks may bo used to determine a hematocrit compensation equation ί

for analyte determination, A more detailed description of the application of derivative data ϊ treatment to glucose analysis may he found in WO 2005/114164.

' [00190] In addition to the ability of the gated pulse sequences to reduce inaccuracy

J from the hematocrit effect and from the mediator background signal, the combination of the i dynamic current profile of each excitation and the resulting contour profiles may be used to * provide multiple sets of calibration constants to the sensor system, thus increasing the accuracy of the analysis. Each sst of calibration constants obtained may be used to correlate a specific current reading io a specific concentration of measurable species in the sample. Thus, in one aspect, an increase in accuracy may be obtained by averaging the glucose values obtained using multiple sets of calibration constants.

[00191] Conventional electrochemical sensor systems generally use one set of calibration constants, such as slope and intercept, to convert current readings into a corresponding concentration of the analyte in the sample. However, a single set of calibration constants may result in inaccuracies in the analyte concentration determined from the recorded current values because random noise is included in the measurement.

[00192] By taking the current value or the transformed current value after data treatment at a fixed time within, each duty cycle of a gated voltasometric pulse sequence, multiple sets of calibration constants may be- established, FIG. 14 plots the semi-integral currents recorded at 7.4, 10.65, 13.9, and 17.15 seconds for tire contour profiles of FIG. 11. Bach of these four calibration fines are independent of the other and may be used in at least two ways, [00193] First, the multiple sets of calibration constants may be used io determine the number of duty cycles that should be applied during the pulse sequence to obtain the desired accuracy and precision. For example, if the current values obtained from the first throe excitations indicate a high glucose concentration, such as >150 or 200 mg/dL, the sensor system may terminate the analysis early, such as after the 4* excitation depicted in FIG. 11. to this manner, foe time required for the analysis may be substantially shortened. Such a shortening may be possible because imprecision at high glucose concentrations Is typically less than at lower glucose concentrations. Conversely, if the current values obtained from the first three excitations indicate a low glucose concentration, such as <150 or 100 mg/dL, the

2016200959 15 Feb 2016 i

i ® sensor system may extend the analysis to greater than 5 or 7 excitations. Thus, the accuracy > and/or precision of tire analysis may be increased by including 5 or more duty cycles.

• (00194] Second, the multiple sets of calibration constants may be used to increase fee t

accuracy and/or precision of the analysis by averaging. For example, if fee target glucose ₍ measurement time is 17.15 seconds, the currents at 10.65, 13,9, and 17,15 seconds can be ‘ utilised to calculate the glucose concentrations using the slopes and intercepts from the ’ corresponding calibration lines; therefore, G_!0,$s fO&gj Gn._? ® >

I tei/xgl/b’lppejj.s, sod Gnus - (iitjs ~ Ed}7j^/Siopsirji. Theoretically, these three glucose

I i values should be equivalent, differing only by random variations, Thus, the glucose values

J i G/suro Gti-s, and G/zjj may he averaged and fee final glucose value of (Grass T G/o ⁺

G/z/i)/3 may be calculated, Averaging the values from fee calibration lines may provide a reduction in. noise at fee rate of 1/ife).

[00195] An unexpected benefit of gated voltammetric pulse sequences including relatively short excitations and relatively long relaxations, such as that depicted in FIG. 6C, is the ability to simplify calibration. While fee multiple sets of calibration constants feat may be obtained from the dynamic and contour profiles may provide an advantage to fee accuracy of the analysis, a pulse sequence such as depicted in FIG. 6C, may provide similar accuracy to feat obtained using multiple sets of calibration constants from a single set of calibration constants. This effect may be observed in the contour profiles of FIG. 11 and fee resulting calibration lines-in FIG. 14.

[00106] This unexpected increase in accuracy may be. attributable to fee relatively long relaxation times in comparison to fee short relaxations. In one aspect, excitation/relaxation time (BRT) ratios from 0.3 to 0.2 are preferred, wife BRT ratios, from 0.27 to 0.22 being more preferred. For example, a gated voltammetric pulse sequence having an ERT ratio of 0,25 (0,8 seconds/3.2 seconds), such as depicted in FIG, SC, may be preferred to a pulse having an ERT ratio of greater than 0.3, such as fee FIG. -SB pulse sequence having an ERT ratio of 0.56 (1,4 seconds /25 seconds). While not intending to be bound by any particular theory, tire relatively long relaxation times may provide a state where fee average consumption rate of measurable species during the excitation- is balanced by the supply rate of measurable species diffusing into the DBL ,·&amp; this manner, the multiple sets of calibration constants may collapse into a single set and fee conversion of the recorded data into an analyte concentration may be simplified by carrying out fee averaging process on fee recorded current date before determining the analyte concentration.

2016200959 15 Feb 2016

I used to determine if the sensor strip has been under-filled with sample, thus allowing , the user to add additional sample to the sensor strip. In addition to working and counter ’ electrodes, conventional sensor systems may determine an underfill condition through the use of a third electrode or electrode pair; however, the third electrode or electrode ’ pair adds complexity and cost to the sensor system.

* (00198] Conventional two electrode systems, may be able to recognize that an ► analysis is “had,” but may not determine if the reason for the failed, analysis was caused j by underfill or a defective sensor strip. The ability to determine if under-fill caused the

J failure of the analysis is beneficial because it may be corrected by adding additional

I sample to the same sensor strip, and repeating the analysis, thus preventing a good strip from being discarded, (00199] PIG. 15 depicts the cyclic vo Mammograms obtained from an under-filled sensor strip, while FIG. 8A depicts a series of seven cyclic vo Mammograms obtained with a gated voltammetric pulse sequence from a normal-filled sensor strip. In both instances, the scan rate was 1 V/sec, Even though the PIG. 8A sample lacked, any glucose and. the sample used for FIG. 15 included 400 mg/dL of glucose, the current values obtained from the underfilled strip having the 400 nsg/dL glucose concentration were much lower than those from the normal-filled 3trip having no glucose. Thus, if may be determined by the second duty cycle of the pulse sequence that the currents obtained are lower than a previously selected value and that the sensor strip is underfilled, For example, for the system of FIG. 15, initial current values less than 0 signify that the sensor strip is under-filled, [00200] In this manner, the gated voltammetric pulse sequences of embodiments of the present invention allowed for undsr-fiU defection in a two-electrode sensor strip, a function typically requiring a third electrode for conventional sensor systems. Furthermore, the under-fill determination may be made in less than 5 seconds, providing time for the measuring device to signal the user, such as by sending a signal to a light emitting device or a display, to add more sample to the strip.

[00201] A common problem for the accuracy of strip based analysis methods is that the reagents, especially the enzyme, degrade over time. One of the effects of enzyme degradation is a change in the calibration values, and thus the precision and/or accuracy of the analysis.

[00202] The dynamic current profiles provided, by the multiple duty cycles of embodiments of the present invention may be used to determine the active ionising agent content of aged sensor lillfii&lt;3 ΡΠΰϊΦ,AL», i ΐίϊ3

2016200959 15 Feb 2016 > j ’ strips, where the ionising species may have degraded. Knowing the amount of ionising agent ί

available to react with the analyte may allow for the identification of defective sensor strips ί

' and for the correction of the analyte concentration value to provide the desired accuracy and

I precision to the analysis. In this manner, the accuracy and/or precision of the analysis obtained from sensor strips having varying amounts of active ionizing agent due to > manufacturing variability or reagent degradation may be obtained.

’ (00203] FIG, 16A depicts seafi-iategral plots of cyclic voltsmmograms obtained fora ! five sensor strips with 1 V/sec sons rates for a sample metading 100 mg/dL glucose and 40% }

¹ hematocrit in WB·. While HG. ISA presents acyclic voltammograms, the method also may >

i be applied to cyclic scans. The ionizing agent used in the reagent layer for the sensor strips was the glucose oxidase (GO) enzyme. Bach sensor strip included a dry weight percentage of 1.7, 3.5, 5.3, 7, or 10 percent (weight/weight) GO in relation to the total dry weight of the material fenning the regent layer. As seen in the figure, the current values for the forward scans increase in relation to those for foe reverse scans as the percentage of ionizing agent increases, Thus, the difference between the forward, and reverse scan current values may be used to determine foe percent of active ionising agent present in the reagent layer of the sensor strip, [00204] FIG. ItiB plots the ratio of the forward and reverse scan current values taken at foe 0.1,5 potential as a taction of percent GO. Once the correlation between the forward and reverse current ratios and the percent active GO is determined, the amount of active GO present in a reagent layer may be determined from tire current values measured for a strip. The ratio of foe forward and reverse scans may be determined before or during the analyte analysis portion of the pulse sequence, thus allowing the user to be notified if the strip is defective.

[00205] The actual active ionizing agent content of the atrip may then be used to alter fee calibration slope, through a relationship such as shown in FIG. 16C. FIG. I6C depicts a typical response of the slope of the linear response calibration of foe sensor strip as a taction of the GO content (%~fey weight). This plot shows that as the GO content increases, foe calibration slope decreases. Thus, if foe actual GO content of fee reagent layer is calculated from HG, 16B, the affected slope of fee GO-based sensor strip way be calculated from foe 2^Ώί5 order polynomial of PIG. 16C using the GO content as the input. The output slope then may be used to correct the glucose concentration value in response to differing amounts of active ionizing agent present in the reagent layer of fee sensor strip, In this manner,

2016200959 15 Feb 2016 >ί inaccuracy and/or imprecision that would otherwise result from enzyme degradation may be reduced.

ί [00206] FIG. 17 is a schematic representation of a measuring device 1700 including

Ί contacts 1720 in electrical communication with electrical circuitry 1710 and a display 1730.

3h one aspect, Ore measuring device 1700 is portable and is adapted to be handheld and to receive a sensor strip, such as the ship 100 from FIG. 1A. In another aspect, tire measuring > device 170G is a handheld measuring device adapted to receive a sensor strip and implement gated voltammetric onlse seouenees.

[00207} Toe contacts 1720 are adapted to provide electrical communication with the

I electrical circuitry 1710 and fee contacts of a sensor strip, such as the contacts. 170 and 180 of the sensor strip 100 depicted in FIG. IB. The electrical circuitry 1.710 may include an electric charger 1750, a processor 1740, and a computer readable storage medium 1745. The electrical charger 17S0 may be a potentiostat, signal generator, or the like. Thus, the charger 1750 may apply a voltage to the contacts 1720 while recording the resulting current to function as a charger-recorder, [00208] The processor 1740 may be in electrical communication with the charger 1750, the computer readable storage medium 1745, and the display 1730. If the charger is not adapted to record current, the processor 1740 may he adapted to record the current at the contacts 1720, [00209] The computer readable storage medium 1745 may be any storage medium, such as magnetic, optical, semiconductor memory, and fee like. The computer readable storage medium 1745. nray be a fixed memory device or a removable memory device, such as a removable memory card. The display 1730 may be analog or digital, in one aspect a LCD display adapted to displaying a numerical reading.

[00210] When the contacts of a sensor strip containing a sample are in electrical communication with the contacts 1720, the processor 1740 may direct the charger 1750 to apply a gated voltammetric pulse sequence to fee sample, thus starting the analysis. The processor 1740 may start the analysis m response to the insertion of a sensor strip, the application of a sample to a previously inserted sensor ship, or in response to a user input, for example.

[00211] Instructions regarding implementation of fee gated voltammetric pulse sequence may be provided by computer readable software code stored in fee computer readable storage medium 1745. The cods may be object code or any other code describing or

2016200959 15 Feb 2016 «ί s , controlling the functionality described in this application. The data feat results from fee

J gated voitamiRetric pulse sequence may be subjected to one or more date treatments, including the determination of decay rates, K constants, slopes, intercepts, and/or sample ’J temperature In the processor 1740 and the results, such as a corrected analyte concentration, output to the display 1730. As wife the instructions regarding the pulse sequence, fee data .

&lt; treatment may be implemented by the processor 1740 from computer readable software code ⁵ . stored in the computer readable storage medium 1745.

[ EXAMPLES * [Θ0212] Example 1: Collection of Voltarometric Date.

[00213] Tbs cyclic voltammogram of FIG. 7A was obtained from a CH

Electrochemical Work Station by applying a potential between fee working and counter electrodes of a sensor strip that varied linearly by 1 V/see at a scan rate of 0,025 V/sec. The current generated at fee working electrode during fee application of fee potential was recorded and plotted as a function of the applied potential. After the initial 0.3 second excitation, fee potentiosfet opened the circuit to provide a 3,2 second relaxation. Six additional excitations were applied to the strip using the pulse sequence of FIG. 6C. Γη this manner, seven acyclic voltammogr&amp;ms for glucose concentrations of 0, SO, 100, and 400 mg/dL, as shown in FIGs. 8A.-8D, respectively, were obtained.

[00214] Example 2: Establishing Contour Plots for Multiple Data Treatments [00275] FIGs, 9A_S 9B, and 9C are contour plots from unprocessed voltammetrfc currents, semi-integral, and semi-derivative data treatments, respectively. In TIG. 9A, unprocessed current values at 0.3 V were taken from each forward scan to provide seven data points. The resulting contour plot presents tire unprocessed current values as a tonction of time since each duty cycle included a 0.S second excitation followed by a 3,2 second relaxation, [0021S] FIG. 9B presents a contour plot of fee same voltammetric data converted wife semi-integral date processing according to equation (3) and implemented with equations (5) and (6). The implemented semi-integral data processing was feat present in the CH Electrochemical Work Station software package, version 4.07, revised April 26', 2004, which accompanies the CH Instruments Blecfrochemical Workstation, model CHI 660,4.. After semi-integral processing, fee semi-integral currents at 0,3 V were taken from fee reverse portion of each scan and plotted as function of time, as previously described wife regard to FIG. 9A.

2016200959 15 Feb 2016 [00217] FIG. 90 presents a contour plot of the same voltammetric data converted . processing used was that present in the CH Electrochemical Work Station software * package, version 4.07, revised April 28, 2004, which accompanies the CH Instruments

Electrochemical Workstation, model CHI 860A. After semi-derivative processing, the ’ peak currant value was taken from each scan and plotted as function of time, as < previously described with regard to FJGs. 0A and 9B. Thus, the Y-axis of FIG. 9C has > the unit of uCoul/seeS 2 for the semi-derivative currents.

[00218] Example 3: Constructing Calibration Plots and Determining Analyte

Concentration [00219] As shown in FIG, .14, a calibration plot for the semi-integral data processing method was formed by taking the semi-integral currents from the four different glucose concentration at 8,8, 12.8, 18,8, and 20.8 seconds from FIG. 9B and plotting the currents as a function of YSI plasma glucose concentration. Glucose sample concentrations were determined from the calibration plot by plugging in the semi-integral processed current from a sample measurement at a specific time into the slope and intercept of the calibration line,

00220] Calibration plots for the unprocessed and semi-derivative processed data were generated similarly. The calibration plots were then used to determine glucose sample concentrations from unprocessed and semi-derivative processed measured current values taken at a specific time.

[00221] Example 4’ Determining Analyte Concentration from. Multiple Calibration [00222] FIG, 4 depicts at least four calibration, lines for times up to 20,8 seconds. For an analysis time of 18.8 seconds, the calibration points at 8,8 and 12.8 seconds were used to calibrate the glucose values. The three glucose values calculated from the 8,8, 12.8 and 18.8 second calibration points were the result of independent oxidations separated by the relaxation time before the 8.8, 12.8 and 18.8 second excitation. While representing the same sample glucose concentration, the concentration values differ by the experimental, noise. Thus, by averaging, G ~ (Gs,s ± Gl2.s ± G 16,8)/3, these values, the signaHo·noise ratio of the final glucose concentration value was increased.

[00223] While various embodiments of the invention have been described, it will be apparent to those of ordinary skill in the art that other embodiments and implementations are possible within the scope of the invention.

[00224] In the claims which fellow and in the preceding description of the

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2016200959 15 Feb 2016 • 44a “ί $ invention, except where the context requires otherwise due to express language or j necessary implication, the word “comprise” or variations such as “comprises or „ comprising” is used in an inclusive sense, i.e. to specify the presence of the stated ·* features but not to preclude the presence or addition of further features in various embodiments of the invention.

> [00225] It ie to be understood that, if any prior art publication is referred to herein, ! such reference does not constitute an admission that the publication forms a part of the > common general knowledge in the art, m Australia or any other country.

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35?mV iC2iMSi!«?iPW87i·Λϋ.5 *5>.Ί3

2016200959 15 Feb 2016