CN101297196B - Gated amperometry - Google Patents

Abstract

A sensor system, device, and methods for determining the concentration of an analyte in a sample is described. Gated amperometric pulse sequences including multiple duty cycles of sequential excitations and relaxations may provide a shorter analysis time and/or improve the accuracy and/or precision of the analysis. The disclosed gated amperometric pulse sequences may reduce analysis errors arising from the hematocrit effect, variance in cap-gap volumes, non-steady-state conditions, mediator background, under-fill, temperature changes in the sample, and a single set of calibration constants.

Description

Gated amperometry

The reference of related application

That the application requires to submit on July 20th, 2005, exercise question is the U.S. Provisional Application No.60/700 of " Gated Amperometry (gated amperometry) ", submitted in 787 and 2006 years 05 month 08 day, exercise question is the U.S. Provisional Application No.60/746 of " Abnormal Output Detection system for a biosensor (the unusual output detection system of biology sensor) ", 771 right of priority, and the content of these two provisional application is incorporated herein as a reference.

Background technology

Analyte in the quantitative measurement biofluid is for the diagnosis and treatment of physically different.For example, measure such as the glucose level in the biological fluid, such as blood, for blood sugar level that must running check oneself so that the diabetic of adjusting diet and/or medication is very important.

Electro-chemical systems has been used for such analysis.In analytic process, the redox reaction with enzyme or similar substance (species) has occured in analyte, thereby produces electric current that can measure and that be associated with analyte concentration.Analyze the required time by shortening, desired accuracy and precision is provided simultaneously, can allow the user fully benefited.

An example of electrochemical sensor system that is used for the analyte of analyzing biologic fluids comprises measurement mechanism and sensing zone.Sensing zone comprises reagent and electrode, and this reagent reacts with analyte during analyzing and the electronics of analyte is shifted, and this electrode transmits above-mentioned electronics by the conductor that sensing zone and measurement mechanism are coupled together.Measurement mechanism has from sensing zone and receives the contact of electronics and apply the ability of voltage difference between each contact.This device can record the electric current that passes sensor and the assessed value that this current value is changed into the analyte content of sample.These sensing systems can be analyzed a whole blood (WB), and for example volume is the whole blood of 1～15 microlitre (μ L).

The example of desk-top (bench-top) measurement mechanism comprises: the BAS 100B analyser of being sold by the BAS Instruments company of the western Lafayette of Indiana, USA (Indiana) (West Lafayette), the CH instrumental analysis instrument of being sold by Texas, USA (Texas) Austin's (Austin) CH Instruments company, the Cypress electrochemical workstation of being sold by the CypressSystems company of Kan. (Kansas) Lao Lunsi (Lawrence) and the EG﹠amp that is sold by the Princeton Research Instruments company of N.J. (New Jersey) Princeton (Princeton); The G electrochemical apparatus.The example of Portable type measurement unit comprises the AscensiaBreeze of Bayer Corporation company

And Elite

Instrument.

Sensing zone can comprise that analyte at the working electrode place a kind of electrochemical reaction occurs to electrode for a working electrode and one, and the electrode place is being occured relative (oppsite) electrochemical reaction, thereby electric current can be flow through between these two electrodes.Therefore, if at the working electrode place oxidation occurs, then the electrode place is being reduced.For example, referring to " Fundamentals Of Analytical Chemistry, 4 ^ThEdition, D.A.Skoog and D.M.West; Philadelphia:Saunders College Publishing (1982), pp304-341 " (" the analytical chemistry basis " of D.A.Skoog and D.M.West works, the 4th edition, Philadelphia; Saunders institute publishes (nineteen eighty-two), 304-341 page or leaf).

Sensing zone can also comprise true (true) contrast electrode, thereby provides constant reference potential to measurement mechanism.Although multiple contrast electrode material all is known, the potpourri of silver (Ag) and silver chloride (AgCl) is the typical case relatively, because this potpourri is insoluble in the aqueous environment of analytical solution.Contrast electrode also can be with doing electrode.U.S. Patent No. 5,820 has been described in 551 and has been used this reference-to the sensing zone of electrode combination.

Thereby can form sensing zone on the dielectric base by using multiple technologies that electrode is printed on, above-mentioned technology for example is U.S. Patent No. 6,531,040, No.5,798,031 and No.5, and those technology described in 120,420.Can by the one or more electrodes of coating, such as working electrode and/or to electrode, thereby form one or more reagent layers.In one aspect, can be covered by identical reagent layer more than one above-mentioned electrode, for example when working electrode with to electrode on when being coated with identical component.In yet another aspect, can use the U.S. Provisional Patent Application No.60/513 that submitted on October 24th, 2003, the method described in 817 will have the reagent layer printing of different component or microdeposit at working electrode with on to electrode.Therefore, the reagent layer on the working electrode can comprise enzyme, amboceptor and bonding agent, and the reagent layer on the electrode is comprised bonding agent and solubility redox materials that can be identical or different with above-mentioned amboceptor.

Reagent layer can comprise for the oxidation that promotes analyte or the ionization preparation of reduction, and helps any amboceptor or other materials that allow electronics shift between analyte and conductor.The ionization preparation can be the analyte enzyme-specific, and for example glucose oxidase or glucose dehydrogenase are with the oxidation of the glucose in the catalysis WB sample.Reagent layer also can comprise the bonding agent that enzyme and amboceptor are kept together.Below Table I provide the enzyme that uses for specific analyte and the routine of amboceptor to make up.

Table I

Bonding agent can comprise the polymkeric substance of all kinds such as CMC (carboxymethyl cellulose) and/or PEO (polyethylene oxide) and molecular weight.Bonding agent also can assist to filter red blood cell except each reagent is bonded together, prevent that them from covering on the electrode surface.

The example of conventional electrochemical sensor system that is used for the analyte of analyzing biologic fluids comprises: the precision of being sold by the Abbott company in Illinois, America (Illinois) Albert park (Abbott Park)

Biology sensor, the Accucheck that is sold by the Roche company of Indiana, USA (Indiana) Indianapolis (Indianapolis)

Biology sensor and the One Touch Ultra that is sold by the Lifescan company of California, USA (California) Mil's Pitta this (Milpitas)

Biology sensor.

A kind of electrochemical method that has been used for the analyte of quantification biofluid is coulometry.For example, Heller etc. are in U.S. Patent No. 6,120, have described in 676 to be used for the coulometry that full blood glucose is measured.In coulometry, with the analyte exhaustive oxidation of small size very, and on oxidization time, electric current is quadratured, thereby produce the electric charge that represents analyte concentration, with this quantitative analysis substrate concentration.In other words, the glucose total amount in the sensing zone has been obtained in coulometry.

An importance of coulometry is, towards the end of electric charge to the integrated curve of time, electric current is the rate substantially constant that becomes over time, thereby stable situation occurs.This stable state of coulometry curve partly forms relatively straight flat region, thereby just can measure corresponding electric current.Yet coulometry requires the analyte of whole volume to change to reach stable situation fully.As a result, this method is consuming time, and the required fast results of user such as electrochemical appliances such as glucose monitoring products can not be provided.Another problem of coulometry is, for accurately result is provided, must controls sensor pond (sensor cell) and make its volume little, and this may be difficult to realize for full-scale plant.

The another kind of electrochemical method that has been used for the analyte of quantification biofluid is amperometry.In amperometry, when constant potential (voltage) is applied to the working electrode of sensing zone and to electrode between the time, during read pulse, measure electric current.Measured electric current out is used for quantizing the analyte of sample.Amperometry is measured near electroactive substance (thereby measuring analyte) oxidized or speed of being reduced working electrode.For example U.S. Patent No. 5,620, described many variations of biology sensor amperometry in 579, No.5,653,863, No.6,153,069 and No.6,413,411.

The shortcoming of conventional current analytic approach is the unstable state character of electric current after applying current potential.At first, electric current is very fast with respect to the rate of change of time, and then along with the carrying out of analyzing, this rate of change is slack-off owing to the qualitative change of basic (underlying) diffusion process.Until the amboceptor that is reduced before the wear rate at electrode surface place equals rate of diffusion, can not obtain steady-state current.Therefore, for amperometry, the transient state before reaching stable situation is measured electric current in the cycle, can cause than more inaccurate when stable state was measured in period.

" hematocrit effect " hindered the concentration of glucose of analyzing exactly in the WB sample.The WB sample contains red blood (RB) cell and blood plasma.The blood plasma major part is water, but contains some protein and glucose.Hematocrit be the RB cellular component with respect to the long-pending shared volume of WB population of samples, and be typically expressed as number percent.Whole blood sample generally has 20%～60% hematocrit number percent, and its average level is～40%.

At the conventional sensing zone that is used for measuring concentration of glucose, glucose can be by oxydasis, and then enzyme is transferred to amboceptor with electronics.So this amboceptor that is reduced moves to working electrode also there by electrochemical oxidation.The amount of oxidized amboceptor can with the working electrode of sensing zone with to electrode between the electric current that flows be associated.Aspect quantitative, the electric current of measuring at the working electrode place directly is proportional to the coefficient of diffusion of amboceptor.The hematocrit effect has hindered this process, spreads to working electrode because the RB cell hinders amboceptor.Thereupon, the hematocrit effects amount of the electric current measured at the working electrode place, and with sample in the amount of glucose without any relation.

Inaccuracy during the WB sample of the vicissitudinous RB cell concentration of tool may cause measuring is because sensor can not distinguish that the RB cell occurring stops amboceptor to spread the higher mediator concentration of this situation and low mediator concentration to working electrode.For example, when analysis contains same glucose level and hematocrit when being 20%, 40% and 60% WB sample, can report out three different glucose readings based on the conventional sensors system of one group of calibration constants (for example, slope and intercept).Even concentration of glucose is identical, but because the RB cell has hindered amboceptor to spread to working electrode, system will report that 20% hematocrit sample comprises more glucose than 60% hematocrit sample.

Human normal plasma cell specific volume ranges (RBC concentration) is 20%～60%, and its intermediate value is about 40%.The hematocrit deviation refers to, for the sample that comprises different hematocrit levels, from the reference concentration of glucose of reference instrument acquisition and from the difference between the experiment glucose readings of portable sensor system acquisition.Above-mentioned reference instrument for example is the YSI 2300 STAT PLUS that sold by the YSI Inc. company in yellow hot spring city, Ohio (Ohio) (Yellow Springs) ^TMDifference between reference concentration and the experiment reading derives from the hematocrit levels of the variation between each specific whole blood sample.

Except the hematocrit effect, but when measurement of species concentrations and analyte concentration onrelevant, also may cause the measurement inaccuracy.For example, when sensing system was measured the concentration of the amboceptor that is reduced that produces because of the analyte oxidation, any was not all can cause sensing system owing to the amboceptor back end provides as judging because of the amboceptor that is reduced that the analyte oxidation produces: have in sample than the more analyte of correct option.

Except hematocrit effect and amboceptor back end effect, other factors also may cause the ability of analyte concentration in the working sample of conventional electrochemical sensor system inaccuracy to occur.In one aspect, because the sensing zone of splendid attire sample part may be different on volume, therefore can introduce above-mentioned these inaccuracies.When not providing enough samples to fill up cap crack volume fully, this is called the situation of not filling up, and also may introduce inaccuracy.In other respects, at random " noise " and when sensing system lacks the ability that the Accurate Determining sample temperature changes may be incorporated into inaccuracy in the measurement.

In order to overcome the one or more shortcomings in these shortcomings, the conventional sensors system has attempted multiple technologies, and these technology are not only selected about Machine Design and the reagent of sensing zone, but also applies the mode of current potential to sensing zone about measurement mechanism.For example, comprise for reducing the conventional method of the hematocrit effect of amperometric sensor: use filtrator, such as U.S. Patent No. 5,708, in 247 and No.5,951,836 disclosed like that; The polarity of the counter-rotating electric current that applies is as disclosed among the WO01/57510; And utilize the maximized method of intrinsic impedance make sample, such as U.S. Patent No. 5,628, in 890 disclosed like that.

Used multiplely to apply the method for current potential to sensing zone, be commonly referred to pulse method, pulse train or pulse cycle, solved the inaccuracy problem in the determined analyte concentration.For example, in U.S. Patent No. 4,897, in 162, pulse method comprises and applies continuously the lifting current potential, obtains the triangular form ripple thereby they are mixed.In addition, the pulse method of describing in WO2004/053476 and U.S. patent documents 2003/0178322 and 2003/0113933 comprises applying continuously current potential lifting and that change polarity.

Other conventional methods are combined the specific electrode structure with the pulse train that is suitable for this structure.For example, U.S. Patent No. 5,942,102 specific electrode structure and continuous impulses that provided by sheet cell combine, and can arrive working electrode so that come to the Electrode reaction product.This combination is used to driving a reaction, until that electric current becomes over time is constant, thereby mobile amboceptor reaches a real stable situation in applying the step of current potential (potential step) so that at working electrode and between to electrode.Although each method in these methods has all been weighed various merits and demerits, is desirable without any a kind of method.

Can find out from above-mentioned explanation, still need improved electrochemical sensor system, especially those can be in shorter time the electrochemical sensor system of determination and analysis substrate concentration more exactly.System of the present invention, apparatus and method have overcome at least one shortcoming relevant with conventional system.

Summary of the invention

A kind of method of the analyte concentration for working sample is provided, and described method comprises to sample and applies pulse train that described pulse train comprised at least 3 working cycle (DutyCycle) in 180 seconds.Each working cycle can be included in exciting under the set potential, and comprises relaxation, can record current at duration of exciting.Described pulse train can comprise the terminal count pulse, and can be applied to the sensing zone of the diffusion barrier layer (DBL) that comprises on the working electrode.The deviation owing to the amboceptor back end that may comprise in the analyte concentration of measuring is less than according to there not be use to comprise the Same Way of pulse train of at least 3 working cycle or the deviation in the analyte concentration that additive method is measured in 180 seconds.By using the momentary current data, when during the excitation portion of the working cycle of pulse train, not arriving stable situation, just can the determination and analysis substrate concentration.Can carry out data to measured electric current and process, thus the analyte concentration in the working sample.

A kind of hand-held analysis measurement device of the analyte concentration for working sample is provided.Described device comprises the gating current analysis measuring device that is suitable for admitting sensing zone.Described gating current analysis measuring device comprises at least two by the device contact of circuit and the mutual telecommunication of display.Described sensing zone comprises the first and second sensing zone contacts at least.Described the first sensing zone contact is by conductor and the mutual telecommunication of working electrode, and described the second sensing zone contact is by conductor and to the mutual telecommunication of electrode.At at least one described electrode the first reagent layer is arranged, described the first reagent layer comprises oxidoreducing enzyme and at least a redox couple material.

A kind of handheld measuring device that is suitable for admitting sensing zone is provided, is used for the analyte concentration of working sample.Described device comprises: contact; At least one display; And circuit, be used for setting up the telecommunication between described contact and the described display.Described circuit comprises charger and processor, the mutual telecommunication of wherein said processor and computer-readable recording medium.Described storage medium comprises the computer-readable software code, when described processor is carried out described computer-readable software code, so that described charger is applied to the pulse train that comprises at least 3 working cycle in 180 seconds between described contact.

A kind of method for reducing in the analyte concentration of determined sample owing to the deviation of amboceptor back end is provided, and described method comprises to sample and applies pulse train that described pulse train comprised at least 3 working cycle in 180 seconds.

The method of a kind of duration of the pulse train of determining to comprise at least 3 working cycle in 180 seconds is provided, the analyte concentration that is used for working sample, described method comprises: according to the electric current that records during described at least 3 working cycle, determine many group calibration constants; And in response to the analyte concentration in the sample of measuring, determine the duration of described pulse train

The method that provides a kind of signaling user to add extra sample to sensing zone, described method comprises: measure disintegration constant according to analyzing the electric current that records during the pulse train at gating current, judge thus whether described sensing zone does not fill up; And if described sensing zone do not fill up, then the signaling user adds extra sample to described sensing zone.

A kind of method of temperature of the sample of measuring sensing zone institute splendid attire is provided, and described method comprises: measure disintegration constant according to analyzing the electric current that records during the pulse train at gating current; And described disintegration constant and temperature value associated.

The method of a kind of duration of definite pulse train is provided, the analyte concentration that is used for working sample, described method comprises: measure the temperature of the sample of sensing zone institute splendid attire according to disintegration constant, described disintegration constant is to measure out according to analyzing the electric current that records during the pulse train at gating current.

The below provides some definition, so as clear, verily understand instructions and claims.

Term " analyte " is defined as being present in one or more materials in the sample.The existence of analyte and/or the concentration of existing analyte in the described analysis working sample.

Term " sample " is defined as a kind of composition that may contain the unknown quantity analyte.Usually, the sample of electrochemical analysis is liquid form, and preferably, sample is aqueous mixture.Sample can be biological samples such as blood, urine or saliva.Sample also can be the derivant of biological sample, for example sediment of extract, dilution, filtrate or rehydration.

Term " but measurement of species " is defined as being at the working electrode of electrochemical sensing band any electroactive substance oxidized under the suitable potential or that be reduced.But the example of measurement of species comprises analyte, oxidoreducing enzyme and amboceptor.

Term " amperometry " is defined as a kind of analytical approach, wherein, and by oxidation or the rate of reduction of analyte under a certain current potential carried out electrochemical measurement, the analyte concentration in the working sample.

Term " system " or " sensing system " are defined as a kind of sensing zone, and it is by oneself conductor and the mutual telecommunication of measurement mechanism, make it possible to quantize the analyte in the sample.

Term " sensing zone " be defined as a kind of during analyzing the device of the telecommunication between splendid attire sample and sampling and the measurement mechanism.The sensing zone of splendid attire sample partly is commonly referred to " cap crack (cap-gap) ".

Term " conductor " is defined as a kind of changeless conductive materials that keeps during electrochemical analysis.

Term " measurement mechanism " is defined as one or more electronic installations, and it can impose on current potential the conductor of sensing zone and measure resulting electric current.Measurement mechanism also can have measures the existence of one or more analytes and/or the processing power of concentration in response to the current value that records.

Term " accuracy " is defined as the degree of closeness of the substantial amount of the amount of the analyte measured by sensing zone and the analyte in the sample.In one aspect, accuracy can be expressed as the form of deviation.

Term " precision " is defined as the degree of closeness to the repeatedly analysis measurement of same sample.In one aspect, precision can be expressed as repeatedly the difference (spread) between the measurement or the form of difference.

Term " redox reaction " is defined between two kinds of materials, relate to the chemical reaction of at least one electronics from the first substance transfer to the second material.Therefore, redox reaction comprises oxidation and reduction.Oxidation half cell reaction relates to the first material and loses at least one electronics, and the reduction half-cell reaction relates to the second material and increases at least one electronics.The electron number that the ionic charge increase value of oxidized material equals to lose.Equally, be reduced the electron number that the ionic charge reduction value of material equals to obtain.

Term " amboceptor " be defined as a kind of can be oxidized or be reduced and can shift the material of one or more electronics.Amboceptor is the reagent in the electrochemical analysis, and it is not target analytes, and provides the indirect measurement to analyte.In the system that oversimplifies, redox reaction occurs in response to the oxidation of analyte or reduction in amboceptor.Then, relative reaction occurs at the working electrode place of sensing zone in amboceptor oxidized or that be reduced, and returns to its initial oxidation number.

Term " bonding agent " is defined as a kind of material, and it provides physical support and hold reagent to reagent, has simultaneously the chemical compatibility with reagent.

Term " amboceptor back end " is defined as being incorporated into the deviation in the measured analyte concentration, but it can be owing in response to the measurement of species of fundamental analysis substrate concentration.

Term " does not fill up " and is defined as when the sample of deficiency being introduced in the sensing zone when obtaining accurate analysis.

Term " redox couple " is defined as having two kinds of pairing materials of the chemical substance of different oxidation numbers.The material reduction that will have the higher oxygen number can produce the material that has than the suboxides number.The substance oxidation that will have than the suboxides number alternatively, can produce the material with higher oxygen number.

Term " oxidation number " is defined as the form ionic charge such as chemical substances such as atoms.Oxidation number is higher, and for example (III), then electropositivity is larger; Oxidation number is lower, and for example (II), then electropositivity is less.

Term " solubility redox materials " be defined as occuring oxidation or reduction and can be in water (the pH value is 7,25 ℃) with the material of the magnitude dissolving of at least 1.0 grams per liters.The solubility redox materials comprises electroactive organic molecule, organic transition metal complex compound and transition metal coordination complexes.Term " solubility redox materials " does not comprise elemental metals and isolated metallic ion, does not especially comprise those that are insoluble to or are insoluble in water.

Term " oxidoreducing enzyme " is defined as promoting any enzyme of oxidation or the reduction of analyte.Oxidoreducing enzyme is a kind of reagent.Term " oxidoreducing enzyme " comprising: " oxidase " of energy accelerating oxidation reaction, and in this oxidation reaction, molecular oxygen is electron accepter; Can promote " reductase " of reduction reaction, in this reduction reaction, analyte is reduced, and molecular oxygen is not analyte; And " dehydrogenasa " of the reaction of energy accelerating oxidation, in this oxidation reaction, molecular oxygen is not electron accepter.For example, referring to " OxfordDictionary of Biochemistry and Molecular Biology, Revised Edition; A.D.Smith, Ed., New York:Oxford University Press (1997) pp.161; 476; 477, and 560 " (" biological chemistry and the molecular biology oxford dictionary " that A.D.Smith, Ed. write, revised edition, New York, Oxford University Press (1997), the 161st, 476,477 and 560 page).

Term " electroactive organic molecule " is defined as containing metal not and the organic molecule of oxidation or reduction reaction can occur.Electroactive organic molecule can be used as amboceptor.

Term " organic transition metal complex compound ", be also referred to as " OTM complex compound ", be defined as a kind of complex compound, wherein, transition metal by σ key and at least one carbon atom bonding (with the formal charge of transition metal on the carbon atom of σ key bonding as-1) or by π key and at least one carbon atom bonding (with the formal charge of transition metal on the carbon atom of π key bonding as 0).For example, ferrocene is a kind of OTM complex compounds with two luxuriant dialkylenes of ring (Cp) ring, each ring by oneself five carbon atoms by two π keys and σ key and iron center bonding.Another example of OTM complex compound is ferrocyanide (II) counter pair through reduction of the ferricyanide (III) and it, wherein six cyano group ligands (formal charge on each in six ligands is-1) by carbon atom and iron center with σ key bonding.

Term " co-ordination complex " is defined as a kind of complex compound with clearly defined coordination geometry, for example octahedral complex or square plane (shape) complex compound.From different by the defined OTM complex compound of the bonding of self, co-ordination complex is by their geometric configuration definition.Therefore, co-ordination complex can be OTM complex compound (for example previously mentioned ferricyanide), or a kind of like this complex compound, wherein, non-metallic atom except carbon, the heteroatom that for example comprises nitrogen, sulphur, oxygen and phosphorus is to give mode (datively) and transition metal center bonding.For example, it is a kind of co-ordination complex that hexamethylene tetramine closes ruthenium, and it has clearly defined octahedral geometry, wherein six NH ₃Ligand (formal charge on each of 6 ligands is 0) is to give mode and ruthenium center bonding.The more comprehensively discussion of organic transition metal complex compound, co-ordination complex and transition metal bonding can be referring to " Collman etal., Principles and Applications of Organotransition Metal Chemistry (1987) " and " Miessler﹠amp; Tarr, Inorganic Chemistry (1991) " (" principle of organotransition metal chemistry and application " (1987) of the works such as Collman and " inorganic chemistry " (1991) of Miessler and Tarr works ".

Term " stable state " is defined as when electrochemical signals (electric current) with respect to the variation substantial constant of its independent input variable (voltage or time) time, for example should change ± 10% or ± 5% within.

Term " instantaneous point " but be defined as when measurement of species is transformed into relatively constant rate of diffusion to the cumulative rate of diffusion of conductive surface the current value as the function of time that obtains.Before instantaneous point, electric current changes rapidly in time.Similarly, after instantaneous point, it is relatively constant that the electric current decay rate becomes, thereby but reflected the relatively constant rate of diffusion of measurement of species to conductive surface.

Term " relatively constant " be defined as when the variation of current value or rate of diffusion ± 20%, ± 10% or ± 5% within.

Term " average original depth " refers to the average height of the layer before introducing fluid sample.Using term " on average " is because the end face of layer is uneven, has projection and depression.

Term " redox intensity (RI) " is defined as total firing time of pulse train divided by total firing time and total relaxation time sum.

Term " handheld apparatus " is defined as being held in the staff and portable device.An example of handheld apparatus is Ascensia

The measurement mechanism that the Elite blood glucose monitoring system is equipped with, by the Bayer HealthCare of the state of Indiana (IN) Ai Er Elkhart (Elkhart), LLC company sells.

Term " ... upper (on) " be defined as " ... top " and for described direction.For example, if the first element is deposited at least a portion of the second element, is then write as the first element and be deposited on the second element.In another example, if the first element is positioned at above at least a portion of the second element, then write as the first element on the second element.Use term " ... on " time and be not precluded within and also exist material between described upper element and the lower element.For example, the first element can have coating at its end face, and the second element above at least a portion of the first element and Topcoating thereof can be write as " on the first element ".Therefore, use term " ... on " can represent that two relevant elements carry out physical contact or do not carry out physical contact.

Description of drawings

The present invention may be better understood with explanation in conjunction with following accompanying drawing.Ingredient in the accompanying drawing needn't be according to ratio, but focuses on explaining principle of the present invention.In addition, in the accompanying drawings, the counterpart in all different views is represented by similar Reference numeral.

Figure 1A is the stereographic map of the sensing zone of assembling.

Figure 1B is the vertical view that removes the later sensing zone of lid.

Fig. 2 shows the end-view of sensing zone among Figure 1B.

Fig. 3 shows for the existence of working sample analyte and the electrochemical analysis method of concentration.

Fig. 4 A and Fig. 4 B show apply long read pulse and short read pulse during, have the working electrode of surface conductor and DBL.

Fig. 5 A～Fig. 5 E shows five examples of pulse train, is used for after introducing sample a plurality of working cycle being put on sensing zone.

Fig. 6 A shows for comprising 50,100,200,400 and 40% hematocrit WB sample of 600mg/dL glucose, the instantaneous output current of the pulse train shown in Fig. 5 B.

Fig. 6 B show by draw and connection layout 6A shown in the current profile curve that obtains of last current value of each momentary current curve.

Fig. 6 C shows the current profile curve that obtains from the momentary current curve that is produced by the pulse train shown in Fig. 5 E.

Fig. 6 D has explained and has used gating current to analyze the output signal that is associated with input signal in the electro-chemical systems of pulse train.

Fig. 7 A and Fig. 7 B explained when DBL when short read pulse is combined, the raising of accuracy of measurement.

Fig. 7 C and Fig. 7 D have explained when gating current and have analyzed pulse train when DBL is combined, the reduction of hematocrit deviation.

Fig. 8 has drawn when the pulse train with Fig. 5 B imposes on the WB sample that comprises various concentration of glucose, at the end points electric current of a plurality of working cycle place record.

Fig. 9 A shows when 2.0 μ L samples being introduced in 10 different sensing zones, the momentary current curve that obtains from the pulse train shown in Fig. 5 B.

Fig. 9 B shows from Fig. 9 A and changes the decay rate of each pulse train that obtains as the curve of the function of time.

Figure 10 has drawn for 50,100 and the 400mg/dL concentration of glucose, and the K constant of measuring out from pulse train is as the function of temperature.

Figure 11 is the synoptic diagram of measurement mechanism.

Embodiment

The present invention has utilized such discovery, that is, the gating current that contains a plurality of working cycle is analyzed pulse train and can improved accuracy and precision is provided, shorten deadline of above-mentioned analysis simultaneously for analysis.Exciting of once can providing under relatively constant voltage is provided each working cycle.The relaxation that can be provided by open circuit (open circuit) also is provided once each working cycle.Pulse train of the present invention needn't need the delay and the pulse that add, for example be used for providing " (incubation) hides " delay of reagent rehydration, the amboceptor that is used for upgrading " fusing (the burn-off) " pulse of electrode and being used for renewal amboceptor oxidation state to recover pulse, reduce analysis time with this, thereby just can shorten the required time of above-mentioned analysis.

Even have short analysis time, gating current analysis pulse train of the present invention is compared still with conventional method and can be improved accuracy and/or precision.In one aspect, can be by the combination of diffusion barrier layer and pulse train of the present invention, reduce accuracy error and because the trueness error that the cap crack volume of variation is introduced introduced by the hematocrit effect.In yet another aspect, can reduce the other error that is caused by unstable state sensor situation and/or amboceptor back end.The contour curve that gating pulse sequence of the present invention also makes it possible to measure momentary current and is used for the simulation stable situation.The momentary current curve can be used to provide many group calibration constants, does not fill up the ability of detection and working sample temperature, rather than depends on the temperature from measurement mechanism.

Figure 1A and Figure 1B show and can be used for sensing zone 100 of the present invention.Figure 1A is the stereographic map of the sensing zone 100 of assembling, and sensing zone 100 comprises sensor base 110, and it is covered by the lid 120 that comprises discharge orifice 130, recessed district 140 and input end opening 150 at least in part.Between pedestal 110 and lid 120, be formed with partially enclosed space 160 (that is, cap crack).Also can use other sensing zones design that conforms to the present invention, for example U.S. Patent No. 5,120, and 420 and No.5, those sensing zones described in 798,031.

Can will be transported to cap crack 160 for the fluid sample of analyzing by inserting the liquid into opening 150.Liquid filling cap crack 160, the air that will before comprise is simultaneously discharged by discharge orifice 130.Cap crack 160 can be contained a kind of helping fluid sample is remained on component (not shown) in this cap crack.The example of this component comprises the polymkeric substance such as the water-swellable such as carboxymethyl cellulose and polyglycol, and the matrix such as the porous polymer such as glucosan and polyacrylamide.

Figure 1B shows the vertical view that removes the later sensing zone 100 of lid 120. Conductor 170 and 180 can be below dielectric layer 190 extends to working electrode 175 and to electrode 185 from opening 150 respectively.In one aspect, as shown in the figure, working electrode 175 and can be located substantially on same plane to electrode 185.In related fields, working electrode 175 and can be separated by greater than the distance of 200 or 250 μ m to electrode 185, and at least 100 μ m of can being separated by with the top of lid 120.Dielectric layer 190 is coated electrode 175,185 partly, and can be by making such as any suitable dielectric materials such as insulating polymers.

Sensing zone 100 electrode 185 is equilibrated at the current potential at working electrode 175 places.In one aspect, this current potential can be the reference potential that obtains in the following way: by forming electrode 185 such as redox couples such as Ag/AgCl, so that reference-to electrode combination to be provided.In yet another aspect, this current potential can be provided for sensing system in the following way: formed electrode 185 by the material such as inertia such as carbon, and comprise such as solubility redox materials such as the ferricyanides in cap crack 160.Alternatively, sensing zone 100 can be provided with the 3rd conductor and electrode (not shown), to provide reference potential to sensing system.

Fig. 2 shows the end-view of the sensing zone shown in Figure 1B, is used for explanation working electrode 175 and to the layer structure of electrode 185. Conductor 170 and 180 can directly place on the pedestal 110.Optionally, can be at conductor 170 and 180 difference deposition surface conductor layers 270 and 280.Surface conductor layer 270,280 can be made by identical or different material.

Be used to form conductor 170,180 and surface conductor layer 270,280 material (or each material) can comprise any electric conductor.Preferred electric conductor is non-ionic (non-ionizing's), thereby clean oxidation (net oxidation) or clean reduction (net reduction) can not occur during sample analysis this material.Conductor 170,180 preferably includes a metal film or thin metal layer, for example Au Ag Pt Pd, copper or tungsten.Surface conductor layer 270,280 preferably includes carbon, gold, platinum, palladium or their combination.If conductor does not have the surface conductor layer, then this conductor is preferably made by non-ionic material.

Described surface conductor material can be deposited on by any usual manner that conforms to the work of sensing zone on the conductor 170,180, and these modes comprise the paper tinsel deposition, chemical vapor deposition, and the slurry deposition, etc.In the situation of slurry deposition, potpourri can be put on conductor 170,180 as China ink, such as U.S. Patent No. 5,798, described in 031 like that.

Reagent layer 275 and 285 can be deposited on respectively on conductor 170 and 180, and comprises reagent, optionally also comprises bonding agent.Adhesive material is preferably polymeric material, and it is water-soluble that it has part at least.Suitable part is water miscible, polymeric material as bonding agent can comprise polyethylene oxide (PEO), carboxymethyl cellulose (CMC), polyvinyl alcohol (PVA) (PVA), hydroxy ethylene cellulose (HEC), hydroxypropyl cellulose (HPC), methylcellulose, ethyl cellulose, ethylhydroxyethylcellulose, carboxymethylethylcellulose, polyvinylpyrrolidone (PVP), such as polyaminoacid such as polylysines, Polystyrene Sulronate, gelatin, acrylic acid, methacrylic acid, starch, their maleic anhydride salt, their derivant and their combination.At present, in above-mentioned adhesive material, PEO, PVA, CMC and PVA are preferred, and wherein CMC and PEO are preferred.

Reagent layer 275 and 285 can also comprise identical or different reagent except comprising above-mentioned bonding agent.In one aspect, reagent in the first reagent layer 275 can be selected to be present in using with working electrode 175, and reagent in the second reagent layer 285 can be selected to be present in with electrode 185 is used.For example, the reagent in the layer 285 can promote electronics moving freely between sample and conductor 180.Similarly, the reagent in the layer 275 can promote the reaction of analyte.

Reagent layer 275 can comprise has specific oxidoreducing enzyme to analyte, and it can promote the reaction of analyte, strengthens simultaneously sensing system to the specificity of analyte, especially to the specificity of the analyte in the complex compound biological sample.The below provides the example of some specific oxidation reductases and respective analyte in Table II.

Table II

At present, particularly preferred oxidoreducing enzyme for glucose analysis comprises glucose oxidase, glucose dehydrogenase, their derivant or their combination.

Reagent layer 275 can also comprise amboceptor, with effectively with the readout of analyte response to surface conductor 270 and/or conductor 170.The example of amboceptor comprises OTM complex compound, co-ordination complex and electroactive organic molecule.Concrete example comprises: ferrocene-containing compound, ferrocyanide, the ferricyanide, the PQQ that is substituted or is unsubstituted (PQQ) coenzyme, the 3-that is substituted or is unsubstituted phenylimino-3H-phenothiazine (PIPT), 3-phenylimino-3H-phenoxazine (PIPO), the benzoquinones that is substituted or is unsubstituted, the naphthoquinones that is substituted or is unsubstituted, nitrogen (N) oxide, nitroso compound, azanol, hydroxyquinoline, flavine, azophenlyene, the azophenlyene derivant, phenothiazine, indophenols, and indamines.These and other amboceptors that can comprise in the reagent layer can be with reference to U.S. Patent No. 5,653, and 863, No.5,520,786, No.4,746,607 and No.3,791,988 and European patent No.0 354 441 and No.0 330 517.

At present, particularly preferred amboceptor for glucose analysis comprises that the ferricyanide, hexamethylene tetramine close ruthenium, PIPT, PIPO or their combination.The summary that can be used for the galvanochemistry amboceptor of biological oxidation system can be referring to Analytica ClinicaActa.140 (1982), 1-18 page or leaf.

Reagent layer 275,285 can be by depositing such as printing, liquid deposition or any easily mode such as inkjet deposited.In one aspect, each layer deposits by printing.In the identical situation of other factors, the angle of printing cutter can affect the thickness of reagent layer conversely.For example, when moving when printing 110 one-tenth about 82 ° of angles of cutter and pedestal, bed thickness can be about 10 μ m.Similarly, when adopting when spending with the printing nose angle of 110 one-tenth about 62 ° of pedestals, can produce 30 μ m than thick-layer.Therefore, printing nose angle degree is less, and the reagent layer that then can provide is thicker.Except printing nose angle degree, also can affect resulting reagent layer 275,285 thickness such as the viscosity of material therefor and other factors such as step sizing (screen-size) and emulsion fluid compound.

Working electrode 175 can also comprise diffusion barrier layer (DBL), and this DBL and reagent layer 275 are integrally formed, or independently the layer 290, as shown in Figure 2.Therefore, DBL can be formed independent stratum on reagent on the conductor/DBL combination layer, the conductor or the independent stratum on the reagent layer.When working electrode 175 comprised independently DBL290, reagent layer 275 can be positioned at or not be positioned on the DBL290.Reagent layer 275 can not be positioned on the DBL290, but be arranged in can so that agent dissolves on the arbitrary portion of the sensing zone 100 of sample.For example, reagent layer 175 can be positioned on pedestal 110 or the lid 120.

DBL provides between a porous air with internal capacity, but measurement of species can place this space.Can select the hole of DBL, but so that measurement of species can be spread among the DBL, simultaneously so that basically all foreclose such as the larger sample component on volume such as RB cell.Although conventional sensing zone has used various materials with the surperficial filtering RB cell from working electrode, but DBL provide for admit and the inside porous air of the part measurement of species of isolation sample between.

When reagent layer 275 comprised water-soluble binder, any adhesive portion that is insoluble in the sample can play the effect of a complete DBL before exciting.The average original depth of DBL/ agent combination layer preferably less than 30 or 23 microns (μ m), is more preferably less than 16 μ m.At present, the average original depth of particularly preferred DBL/ agent combination layer is 1～30 μ m or 3～12 μ m.For specific energised length, but can when become relatively constant based on measurement of species rate of diffusion on (for example surface of the surface of conductor 170 or surface conductor 270 among Fig. 2) from DBL to the conductive surface, select the average original depth of required DBL/ agent combination layer.

In addition, for short energised length, but use too thick DBL may postpone the moment that the rate of diffusion of measurement of species from DBL to the conductive surface becomes relatively constant.For example, the working cycle that excited in 1 second continuous when comprising, that separated by 0.5 second relaxation is applied to and uses average original depth is on the working electrode of DBL/ agent combination layer of 30 μ m the time, apply (＞～10 seconds) at least 6 working cycle and before, may not reach preferred rate of diffusion.Conversely, to use average original depth be on the working electrode of DBL/ agent combination layer of 11 μ m the time when identical working cycle is applied to, and just can reach relatively constant rate of diffusion after exciting (～2.5 seconds) for the second time.Therefore, for predetermined working cycle, the preferred average original depth of DBL has a upper limit.About DBL thickness, energised length with reach more thoroughly discussing of association between time of relatively constant rate of diffusion, can with reference to submitted on February 22nd, 2005, exercise question is the U.S. Provisional Application No.60/655 of " Concentration Determination in a Diffusion BarrierLayer (concentration determination in the diffusion barrier layer) ", 180.

Independently DBL290 can comprise can provide required space with holes, partly or lentamente be dissolved in any material in the sample simultaneously.In one aspect, independently DBL290 can comprise the reagent adhesive material that does not contain reagent.Independently the average original depth of DBL290 can be at least 5 μ m, preferred 8～25 μ m, more preferably 8～15 μ m.

Fig. 3 shows electrochemical analysis method 300, is used for the existence of working sample 312 analytes 322, the concentration of going back according to circumstances analyte 322 in the working sample 312.In step 310, sample 312 is incorporated into sensing zone 314, for example Figure 1A～Figure 1B and sensing zone shown in Figure 2.Reagent layers such as the reagent layer 275 among Fig. 2 and/or 285 begins to be dissolved in the sample 312, thereby makes it possible to react.About this point in analyzing, the initial time that provides reagent and sample 312 to react postpones or " latent period " may be useful.Preferably, the initial time delay can be 1 second～10 seconds.Initial time postpones, and more thoroughly discuss can be with reference to U.S. Patent No. 5,620, and 579 and No.5,653,863.

During reaction, in step 320, the part of the analyte 322 that contains in the sample 312 is such as by oxidoreducing enzyme etc. chemistry or biochemical oxidation or reduction occuring.Utilize oxidation or reduction, electronics can shift between analyte 322 and amboceptor 332 in step 330 according to circumstances.

In step 340, excite (oxidation or reduction) but measurement of species 342 is carried out galvanochemistry, but should measurement of species can be from the charged analyte 322 of step 320 or from the charged amboceptor 332 of step 330.For example, thereby when sample 312 be contain in step 320 by the glucose oxidase oxydasis, when then in step 330, shifting an electronics and the ferricyanide (III) amboceptor being reduced into the whole blood of glucose of ferrocyanide (II), exciting of step 340 is oxidized to the ferricyanide (III) with ferrocyanide (II) at the working electrode place.In this way, the working electrode of sensing zone is transferred to from the glucose analysis thing in electronic selection ground, and it can detect by measured device there.

The electric current that is caused by exciting step 340 during exciting step 340 can be used as the function of time, records in step 350.In step 360, sample has experienced relaxation.Preferably, record current not between the relaxation period of step 360.

In step 370, according to total total at least three this modes of working cycle in 180 seconds or shorter time section, repeat exciting step 340, recording step 350 and relaxation step 360 at least twice.In step 380, can analyze the electric current and the time value that record, thus the existence of analyte 322 and/or concentration in the working sample 312.

Thereby the amperometric sensor system applies a current potential (voltage) to sensing zone but excites measurement of species, while monitoring current (amperage).Conventional amperometric sensor system can keep this current potential, measures simultaneously for example electric current of 5～10 seconds continuous-reading pulse lengths.Compare with conventional method, employed working cycle excites with relaxation with a plurality of short duration and has replaced continuous long duration read pulse in the electrochemical analysis method 300.

But because be present in from measurement of species differently in the cap crack of sensing zone be, but the measurement of species that in 540, excites at the working electrode place basically from DBL inside, therefore, analytical approach 300 can improve analyte determination result's accuracy and/or precision.Fig. 4 A and Fig. 4 B show apply long read pulse and short excite during, have the working electrode 400 of surface conductor 430 and independent DBL405.When the WB sample placed on the working electrode 400, RB cell 420 covered DBL405.But the analyte that exists in the sample forms outside measurement of species 410 in the outside of DBL405.But the part of outside measurement of species 410 is diffused among the independent DBL405, thereby but obtains inside measurement of species 415.

Shown in Fig. 4 A, when read pulse imposed on working electrode 400 in continuous 10 seconds with one, but outside and inside measurement of species 410 and 415 all change by oxidation state and are excited at surface conductor 430 places.During long read pulse, but outside measurement of species 410 diffuses through the sample area of RB cell 420 and passes DBL405 arrival surface conductor 430.But outside measurement of species 410 diffuses through RB cell 420 during read pulse, and this just is incorporated into the hematocrit effect in the analysis.Because but the major part of the measurement of species that excites at surface conductor 430 places derives from the outside of DBL420, therefore, aspect the hematocrit effect, the possibility of result that is applied to the long read pulse on the sensing zone with DBL is similar with the result who is applied to the short read pulse on the sensing zone that does not have DBL.

On the contrary, Fig. 4 B shows short exciting is imposed on the sensing zone 400 of being furnished with DBL, thereby but but excites inside measurement of species 415 basically not excite the situation of the measurement of species 410 of DBL405 outside simultaneously.At short duration of exciting, but measurement of species 410 or remain on the DBL405 outside perhaps basically can not diffuse through DBL and arrive surface conductor 430.By this way, short exciting just can obviously be reduced the impact of hematocrit effect on analyzing.

By being controlled at the energised length at working electrode place, but can analyze the measurement of species of DBL inside, but basically not analyze the measurement of species of DBL outside simultaneously.But with respect to the rate of diffusion of outside measurement of species 410, but believe that the thickness of DBL405 and internal capacity should be able to change inside measurement of species 415 to the rate of diffusion of working electrode surface conductor 430.

Because but but the measurement of species of DBL inside can diffuse to according to the speed of the measurement of species that is different from the DBL outside conductor of working electrode, but the length that therefore excites at the working electrode place can be selected the measurement of species of precedence parse.From the viewpoint of molecule equally as can be known, but the different rate of diffusion of the inside and outside measurement of species of DBL can allow difference.

Although be not the restriction of wanting to be subject to any particular theory, think at present, but the rate of diffusion of measurement of species in from the DBL outside to DBL change, but and the rate of diffusion of measurement of species from the DBL inner space to conductor is relatively constant.But the rate of diffusion of the variation of DBL outside measurement of species may be to be caused by the RB cell that exists in the sample and other components, and may cause the hematocrit effect.Therefore, but can analyze having to the measurement of species of the relative constant diffusion rate of conductor diffusion by abundant restriction, reduce the analytical error (deviation) of being introduced by the sample sets branch that comprises the RB cell.

But another advantage of optionally analyzing the measurement of species of DBL inside is, has reduced the measurement inexactness that causes owing to the sensing zone with different caps crack volume.But surpass all measurement of species of existing in the cap crack analyzed used time all basically if read pulse continues, but the so above-mentioned analysis measurement of species concentrations in the representative sample no longer just, but but determine the amount of the measurement of species in the cap crack; This is a very different measurement result.Because energised length is longer with respect to cap crack volume, so current measurement result will depend on cap crack volume, rather than basic analyte concentration.Therefore, when pulse length " overshoot is in (overshoot) " but in the cap crack during existing measurement of species, long read pulse may cause the very inaccurate measurement result about analyte concentration.

As submitted on October 12nd, 2004, exercise question is the U.S. Provisional Application No.60/617 of " Concentration Determination ina Diffusion Barrier Layer (concentration determination in the diffusion barrier layer) ", described in 889 like that, thereby but can select single short read pulse or excite the measurement of species that limits fully DBL to excite.When using single-shot, the thickness of preferably selective excitation length and DBL, but so that realize the relatively constant rate of diffusion of measurement of species from DBL to the conductive surface at duration of exciting.If do not have to realize relatively constant rate of diffusion at duration of exciting, but but the measurement of species concentrations in the representative sample exactly not just of the measurement of species concentrations in the DBL then, thus analysis is had a negative impact.In addition, single-shot can not reduce the back end signal from amboceptor effectively.

With reference to Fig. 3, exciting step 340, recording step 350 and relaxation step 360 consist of a working cycle, and it can put on sensing zone at least three times in the cycle at 180 seconds or shorter time.More preferably, within independent 120 seconds, 90 seconds, 60 seconds, 30 seconds, 15 seconds, 10 seconds of selecting or 5 second time cycle, apply at least 4,6,8,10,14,18 or 22 working cycle.In one aspect, within 5～60 second time cycle, applied above-mentioned working cycle.In yet another aspect, can in 30 seconds or shorter time, apply 3～18 or 3～10 working cycle.In yet another aspect, can within 3～16 second time, apply 4～8 working cycle.

The current potential that applies during exciting step 340 parts of working cycle preferably applies according to substantially invariable voltage and polarity on the whole duration.This changes with voltage during record data or the conventional read pulse of " going through (swept) " a plurality of voltage potentials and/or polarity forms direct contrast.In one aspect, the duration of exciting step 340 is at most 4 seconds or 5 seconds, preferably less than 3 seconds, 2 seconds, 1.5 seconds or 1 second.In yet another aspect, the duration of exciting step 340 is 0.01～3 second, 0.01～2 second or 0.01～1.5 second.More preferably, the duration of exciting step 340 is 0.1～1.2 second.

After exciting step 340, in step 360, measurement mechanism can be opened circuit by sensing zone 314, thus so that system's relaxation.During relaxation step 360, the electric current that occurs during exciting step 340 reduces at least half basically, preferably reduces a larger magnitude, more preferably is decreased to zero.Preferably, zero current condition by open circuit or known to persons of ordinary skill in the art, be used for providing the additive method of the electric current that is substantially zero to provide.Relaxation can be provided 3 times during the working cycle of pulse train at least.

In one aspect, the duration of relaxation step 360 is at least 10 seconds, 5 seconds, 3 seconds, 2 seconds, 1.5 seconds, 1 second or 0.5 second.In yet another aspect, the duration of relaxation step 360 is 0.1～3 second, 0.1～2 second or 0.1～1.5 second.More preferably, the duration of relaxation step 360 is for 0.2～1.5 second and provided by open circuit.

During relaxation step 360, thereby but the ionization preparation can generate additional measurement of species with analyte response under the effect that does not have current potential.Therefore, for comprising glucose oxidase and the ferricyanide amboceptor glucose sensor system as reagent, can not be subjected to the interference of the current potential during the relaxation step 360, just can produce the additional ferrocyanide (amboceptor that is reduced) in response to the analyte concentration of sample.

Many conventionals method of analysis apply voltage in during the read pulse duration continuously.The voltage that applies can have set potential, perhaps can have following current potential, and it changes to negative potential or change to zero potential with respect to a current potential from positive potential or negative potential from positive potential.Even zero relatively during current potential, these methods also continue to obtain electric current from sensing zone during read pulse, this so that electrochemical reaction in whole read pulse process, recur.Therefore, but but produce corresponding to the reaction of the measurement of species of analyte concentration and measurement of species and all can be subject to the influence of peak current during the zero potential part of conventional read pulse to the diffusion of working electrode.

Even with respect to the zero potential of a current potential time, also apply voltage and obtain the conventional method of electric current from sensing zone to sensing zone continuously, with relaxation fundamental difference of the present invention.Because a plurality of relaxation of the present invention, a plurality of working cycle of the present invention also are obviously to be different from the conventional method of using single long duration pulse to take multiple measurements, and for example U.S. Patent No. 5,243, those disclosed conventional method in 516.Opposite with these conventional methods, each working cycle of pulse train of the present invention provides independent diffusion and the analyte response time between relaxation period.

Fig. 5 A～Fig. 5 E has shown five examples of gating current analysis pulse train, and wherein after introducing sample, a plurality of working cycle are applied to sensing zone.In these examples, user's wave impulse; But, other wave modes that also can use conform to testing sample with sensing system.Fig. 5 C～Fig. 5 D has shown the pulse train that comprises a plurality of working cycle that have identical firing time and open a way time delay.

Fig. 5 A～Fig. 5 B has shown the pulse train that comprises 9 working cycle, and except the terminal count pulse 510 than long duration and voltage increase was arranged, these working cycle had identical firing time and open a way time delay.The increase voltage of this terminal count pulse provides the ability that the amboceptor with higher oxygen current potential (oxidation potential) is detected.Can submit to referring on April 8th, 2005 about discussing more comprehensively of terminal count pulse, exercise question is the U.S. Provisional Application No.60/669 of " Oxidizable Species as anInternal Reference in Control Solutions for Biosensors ", 729.

Fig. 5 A shows 9 working cycle pulse trains, and wherein each excited by 1 second open circuit and postpones to be spaced from each other in 0.5 second, was 0.357 (5/14) thereby obtain redox intensity (RI).Like this, in Fig. 5 A, second working cycle has excitation portion 520 and relaxation part 530.Fig. 5 B has shown 9 working cycle pulse trains, and wherein each excited by 0.5 second open circuit and postpones to be spaced from each other in 1 second, was 0.69 (10/14.5) thereby obtain RI.Fig. 5 C has shown 7 working cycle pulse trains, and wherein each excited by 1 second open circuit and postpones to be spaced from each other in 1 second, was 0.53 (8/15) thereby obtain RI.Duration and voltage used during the duration of terminal count pulse 540 and voltage and 7 working cycle are identical.Fig. 5 D has shown 6 working cycle pulse trains, wherein excited in those 1.5 seconds by 1 second open circuit to postpone to be spaced from each other, and be 0.636 (10.5/16.5) thereby obtain RI.As among Fig. 5 C, the duration of terminal count pulse 540 is identical with duration and the voltage of front working cycle pulse with voltage.Fig. 5 E has shown 7 working cycle pulse trains, wherein relatively excites by 1.5 seconds relatively long relaxation in short those 0.25 seconds to be spaced from each other.The pulse train of Fig. 5 E begins with 1 initial pulse per second (PPS) 550, and finishes with terminal count pulse in 1.25 seconds 540, is 0.25 (4/16) thereby RI is provided.

The RI of pulse train is higher, and the back end that is incorporated in the analysis by amboceptor will be fewer.Pulse train shown in Fig. 5 A～Fig. 5 E is the oxidisability pulse, is designed to the amboceptor of the also conduct measurement of species that excites (that is, oxidation) but be reduced.Therefore, the oxidation current that is applied on the sensing zone within cycle predetermined time is larger, and the amboceptor that then is reduced because of the approach outside the analyte oxidation is less to the contribution possibility of institute's record current value.

Below Table III the ratio of slope, intercept and intercept and slope of the contour curve of pulse train (a) and last four working cycle (b) is provided.For pulse train (a):

9 * (unlatching in 0.5 second was interrupted in+1.0 seconds)+0.5 second=14 seconds, RI=5/14=0.357.

For pulse train (b):

9 * (unlatching in 1.0 seconds was interrupted in+0.375 second)+1.0 seconds=13.375 seconds, RI=10/13.375=0.748.

Table III

The ratio of intercept and slope provides the assessment owing to the amount of the back end signal of amboceptor, and wherein, the major part of higher ratio value representation institute tracer signal is owing to the amboceptor back end.Therefore, similar when the pulsed frequency of sequence (a) and (b) (excite number of times/always chemically examine the time (second)) and be approximately 0.7sec ^-1The time, the increase of the RI that is provided by pulse train (b) so that owing to the signal section of amboceptor back end reduced more than half.When making up, repeatedly the exciting of pulse train can be eliminated the needs to the inceptive impulse that is used for upgrading the amboceptor oxidation state.Because the back end electric current may be subjected to the impact of amboceptor, therefore for the ferricyanide, preferred RI value is at least 0.01,0.3,0.6 or 1 pulse train, and more preferably the RI value is 0.1～0.8,0.2～0.7 or 0.4～0.6 pulse train.

Refer again to Fig. 3, in step 350, can for each working cycle of pulse train, the electric current that passes the conductor of sensing zone 314 be recorded as the function of time.Fig. 6 A has shown that the output current of the pulse train shown in Fig. 5 B is as the function of time for containing 50,100,200,400 and 40% hematocrit WB sample of 600mg/dL glucose.But different from the conventional long duration read pulse that causes a large amount of oxidations of measurement of species is in current curve, to excite the back that an interruption is arranged at every turn.

In Fig. 6 A, when output current is drawn out as the function of time, excite to cause a momentary current curve at every turn, it has the initial higher current value along with the time decay.Preferably, that working cycle comprises is short, independently excite and the prevention system arrives slowly the decay relaxation of situation of stable state or electric current at each duration of exciting, as required during the read pulse of conventional system.Different from electric current conventional stable state or that slowly decay is, analyze pulse train from gating current and obtained instantaneous (fast decay) current value, but but because measurement of species is faster than the speed that the measurement of species diffusion is supplied to working electrode in the electrochemical reaction at working electrode place.

Fig. 6 B has shown by last current value (that is, last current value that excites) with every momentary current curve shown in Fig. 6 A at every turn and has coupled together, and the contour curve figure that obtains.This contour curve can be used for simulating the data that obtain from conventional system under stable state, electric current is constant over time basically under this stable state.

The momentary current curve and the resulting profile current value that obtain from gating current analysis pulse train are fundamentally different than the current curve that uses single read pulse to obtain from conventional analysis.The electric current that records from single read pulse is from single relaxation/diffusion, and each time point in the contour curve of momentary current derives from exciting after relaxation/diffusion process independently.In addition, when energised length lengthened, the correlativity between electric current and the analyte concentration may reduce owing to the hematocrit effect usually.Therefore, compare with the analysis of the read pulse of using the duration that repeatedly excites that is long, that have combination, can improve the repeatedly accuracy of the short analysis that excites of use.

Refer again to Fig. 6 A, when from exciting the last moment current value that obtains to represent that last moment from the maximum that excites arbitrarily acquisition during current value, just reaches the instantaneous point 605 in the current curve.Therefore, according to Fig. 6 A, when approximate 5 second, reach instantaneous point.For every kind of concentration of glucose, can the maximum current value place in the contour curve of every kind of concentration of glucose reach the balance about DBL rehydration.Therefore, when the momentary current of Fig. 6 A is transformed into profile electric current among Fig. 6 B, for the 600mg/dL concentration of glucose, reading 610 (the highest) and 620 (lower) but so that when about 5 second, reach about measurement of species to the diffusion of DBL and the balance of DBL rehydration.

The current value that records under relatively constant rate of diffusion makes inaccuracy reduce to minimum, and on the contrary, the rate of diffusion of reagent and the variation of rehydration will be introduced inaccuracy.Therefore, in case reach relatively constant rate of diffusion, but the current value that records so will be more accurately corresponding to the concentration of measurement of species, and thereby more accurately corresponding to the concentration of analyte.In addition, according to Fig. 6 B, within 7 short seconds, just can finish whole analysis, because if the maximum current value 610 of known profile curve, its value can directly be associated with analyte concentration.As previously mentioned, can obtain extra data point, thereby reduce the back end error owing to amboceptor.

Fig. 6 C has shown the current profile curve that the momentary current curve that produces according to the pulse train among Fig. 5 E obtains.At 0.25 second each duration of exciting, in the centre constantly (～0.125 second) and the finish time (～0.25 second) record current value, it can be used for judging disintegration constant.Utilize the long short inceptive impulse that excites and relatively grow relaxation that has, can in about 4 seconds, finish analysis.

Fig. 6 D has shown the output signal relevant with input signal in the electro-chemical systems that uses gating current analysis pulse train.Input signal is the current potential that imposes on biologicfluid sample.Input signal comprises Polling (polling) input signal and chemical examination input signal.Output signal is the electric current that produces from sample.Output signal comprises Polling output signal and chemical examination output signal.Sample response produces the chemical examination output signal in the redox reaction of the glucose of chemical examination input signal from whole blood.Input and output signal can be for having working electrode and to the biology sensor of electrode.Can use other biology sensor, comprise that those have the biology sensor of supplemantary electrode and different structure.Can measure other analyte concentration, comprise those analyte concentrations in the other biological fluid.Other output signal be can produce, those signals of initial decay and those signals of in whole pulses, decaying comprised.

During use, biologicfluid sample is placed biology sensor.Biology sensor applies approximately-1.25 second～0 second Polling signal to sample.Pulse has about 5～10 milliseconds pulse width and about 125 milliseconds recurrent interval.Biosensor response produces the Polling output signal in the Polling input signal.Biology sensor is measured the Polling output signal.Biology sensor can have provides the Polling output signal to the voltage stabilizer of the input end of analog comparator.

When the Polling output signal was equal to or greater than the Polling threshold value, biology sensor applied from about 0 second to about 7 seconds chemical examination input signal to electrode.The Polling threshold value can be about 250nA.Comparer can compare Polling output signal and Polling threshold value.When the Polling output signal surpassed the Polling threshold value, the output signal of comparer can trigger the transmission of chemical examination input signal.

During the chemical examination input signal, biology sensor is to working electrode and electrode is applied the working cycle of the first pulse of the about 400mV current potential with about 1 second time.Ensue 0.5 second relaxation after the first pulse, in fact this relaxation can be open circuit etc.Measure chemical examination output signal or the electric current in the first pulse and be stored in the memory storage.Biology sensor can and apply the second pulse of about 200mV current potential of about 1 second time to working electrode to electrode.Measure chemical examination output signal or the electric current in the second pulse and be stored in the memory storage.Biology sensor applies pulse to working electrode with to electrode continuously by the chemical examination input signal, until chemically examine end cycle or reach the required time of biology sensor.The chemical examination cycle can be about 7 seconds.The chemical examination output signal in each pulse can be measured and store to biology sensor.

The Polling input signal is a kind of electric signal such as electric current or current potential, and it is according to the interrupted appearance of the frequency of setting or interval or unlatching and interruption.Sample response produces the Polling output signal in the Polling input signal.The Polling output signal is a kind of electric signal such as electric current or current potential.Biology sensor can be presented at the Polling output signal on the display and/or the chemical examination output signal can be stored in the memory storage.Thereby biology sensor can apply the Polling signal and detect when sample is connected with electrode.Biology sensor can detect when want analytic sample with additive method and device.

The Polling input signal is the working cycle that a kind of wherein Polling pulse train is separated by the Polling relaxation.At the Polling impulse duration, electric signal is opened.Between the Polling relaxation period, electric signal interrupts.Time cycle when unlatching can include electric signal and exists.Interruption can comprise the time cycle when not having electric signal to exist.Interrupt should not including the electric signal existence the time cycle when but it there is no much amplitudes.Electric signal can be changed in unlatching and between interrupting by closing and open circuit respectively.Circuit can open and close by modes such as machinery, electric power.

The Polling input signal can have one or more Polling recurrent intervals.A Polling recurrent interval is a Polling pulse and a Polling relaxation sum.Each Polling pulse has amplitude and Polling pulse width.Amplitude represents the intensity of current potential, electric current of electric signal etc.Amplitude can change or individual constant at the Polling impulse duration.The Polling pulse width is the duration of Polling pulse.Polling pulse width in the Polling input signal can change or be substantially the same.Each Polling relaxation has Polling relaxation width, and it is the duration of Polling relaxation.Polling relaxation width in the Polling input signal can change or be substantially the same.

The Polling input signal can have less than the Polling pulse width of about 300 milliseconds (ms) with less than about 1 second Polling recurrent interval.The Polling input signal can have less than about 100 milliseconds Polling pulse width with less than about 500 milliseconds Polling recurrent interval.The Polling input signal can have about 0.5 millisecond～75 milliseconds Polling pulse width and about 5 milliseconds～300 milliseconds Polling recurrent interval.The Polling input signal can have about 1 millisecond～50 milliseconds Polling pulse width and about 10 milliseconds～250 milliseconds Polling recurrent interval.The Polling input signal can have about 5 milliseconds Polling pulse width and about 125 milliseconds Polling recurrent interval.The Polling input signal can have other pulse width and recurrent interval.

Biology sensor can apply the Polling input signal to sample during the Polling cycle.The Polling cycle can be less than about 15 minutes, 5 minutes, 2 minutes or 1 minute.Depend on the user and how to use biology sensor, the Polling cycle can be longer.The Polling cycle can be about 0.5 second (sec)～15 minute.The Polling cycle can be about 5 seconds～5 minutes.The Polling cycle can be about 10 seconds～2 minutes.The Polling cycle can be about 20 seconds～60 seconds.The Polling cycle can be about 30 seconds～40 seconds.The recurrent interval in Polling cycle can be less than about 200,100,50 or 25.The recurrent interval in Polling cycle can be about 2～150.The recurrent interval in Polling cycle can be about 5～50.The recurrent interval in Polling cycle can be about 5～15 recurrent intervals.The recurrent interval in Polling cycle can be about 10.Can use other Polling cycle.

When the Polling output signal was equal to or greater than the Polling threshold value, biology sensor applied the chemical examination input signal.When the Polling threshold value can begin greater than the first pulse about 5% of desired chemical examination input signal.When the Polling threshold value can begin greater than the first pulse about 15% of desired chemical examination input signal.The Polling threshold value can be the first pulse when beginning desired chemical examination input signal about 5%～50%.Can use other Polling threshold value.Biology sensor can be presented at the Polling output signal that is equal to or greater than the Polling threshold value on the display.

Chemically examining input signal is a kind of electric signal, for example is electric current or current potential, and it is according to the interrupted appearance of the frequency of setting or interval or unlatching and interruption.Sample response produces the chemical examination output signal in the chemical examination input signal.The chemical examination output signal is a kind of electric signal, for example is electric current or current potential.

The chemical examination input signal is the chemical examination pulse train of being separated by the chemical examination relaxation.At the chemical examination impulse duration, electric signal is opened.Between the chemical examination relaxation period, electric signal interrupts.Time cycle when unlatching includes electric signal and exists.Interruption comprises the time cycle when not having electric signal to exist, and does not include the electric signal existence the time cycle when but it there is no much amplitudes.Electric signal can be changed in unlatching and between interrupting by closing and open circuit respectively.Circuit can open and close by modes such as machinery, electric power.

The chemical examination input signal can have one or more chemical examination recurrent intervals.A chemical examination recurrent interval is a chemical examination pulse and a chemical examination relaxation sum.Each chemical examination pulse has amplitude and chemical examination pulse width.Amplitude represents the intensity of current potential, electric current of electric signal etc.Amplitude can change or individual constant at the chemical examination impulse duration.The chemical examination pulse width is the duration of chemical examination pulse.Chemical examination pulse width in the chemical examination input signal can change or be substantially the same.Each chemical examination relaxation has chemical examination relaxation width, and it is the duration of chemical examination relaxation.Chemical examination relaxation width in the chemical examination input signal can change or be substantially the same.

The chemical examination input signal can have less than about 5 seconds chemical examination pulse width with less than about 15 seconds chemical examination recurrent interval.The chemical examination input signal can have less than the chemical examination pulse width of about 3 seconds, 2 seconds, 1.5 seconds or 1 second with less than chemical examination recurrent interval of about 13 seconds, 7 seconds, 4 seconds, 3 seconds, 2.5 seconds or 1.5 seconds.The chemical examination input signal can have about 0.1 second～3 seconds chemical examination pulse width and about 0.2 second～6 seconds chemical examination recurrent interval.The chemical examination input signal can have about 0.1 second～2 seconds chemical examination pulse width and about 0.2 second～4 seconds chemical examination recurrent interval.The chemical examination input signal can have about 0.1 second～1.5 seconds chemical examination pulse width and about 0.2 second～3.5 seconds chemical examination recurrent interval.The chemical examination input signal can have about 0.4 second～1.2 seconds chemical examination pulse width and about 0.6 second～3.7 seconds chemical examination recurrent interval.The chemical examination input signal can have about 0.5 second～1.5 seconds chemical examination pulse width and about 0.75 second～2.0 seconds chemical examination recurrent interval.The chemical examination input signal can have about 1 second chemical examination pulse width and about 1.5 seconds chemical examination recurrent interval.The chemical examination input signal can have other pulse width and recurrent interval.

Biology sensor applies the chemical examination input signal to sample during the chemical examination cycle.The chemical examination cycle can have the duration identical or different with the Polling cycle.The chemical examination cycle of chemical examination input signal can be less than about 180 seconds, 120 seconds, 90 seconds, 60 seconds, 30 seconds, 15 seconds, 10 seconds or 5 seconds.The chemical examination cycle can be about 1 second～100 seconds.The chemical examination cycle can be about 1 second～25 seconds.The chemical examination cycle can be about 1 second～10 seconds.The chemical examination cycle can be about 2 seconds～3 seconds.The chemical examination cycle can be about 2.5 seconds.The chemical examination recurrent interval in chemical examination cycle can be less than about 50,25,20,15,10,8,6 or 4.The chemical examination recurrent interval in chemical examination cycle can be about 2～50.The chemical examination recurrent interval in chemical examination cycle can be about 2～25.The chemical examination recurrent interval in chemical examination cycle can be about 2～15.The chemical examination recurrent interval in chemical examination cycle can be about 10.Can use other chemical examination cycle.

Fig. 7 A and Fig. 7 B shown when DBL when short read pulse is combined, the raising of accuracy of measurement.With whole blood sample and ferrocyanide according to 1: thereby 5 dilution ratios mix as basic concentration of glucose, then measure with read pulse in 1 second.Therefore, become 16%, 32% and 48% hematocrit (three hematocrite value have all descended 20%) with 60% hematocrit WB Sample Dilution with initial 20%, 40%.20%, 40% and 60% line represents respectively the electric current of measuring from the blood sample sample that contains 16%, 32% and 48% hematocrit.

Fig. 7 A has shown the inaccuracy of introducing by hematocrit with owing to caused other effects of the bare exposed conductor sensing zone that does not contain DBL.Inaccuracy is expressed as poor (total hematocrit deviation spacing) between the 20% and 60% hematocrit line, and has represented owing to the maximum of hematocrit effect and measure inaccuracy.Less deviate represents more accurately result.As reference Fig. 4 A discusses, when DBL uses with longer read pulse, observe similar effect in the above.

On the contrary, Fig. 7 B shown when DBL when read pulse was combined in 1 second, spacing significantly reduces between 20% and 60% lubber-line.As above-mentioned Fig. 7 A is employed, the independent DBL that is made of PEO polymkeric substance and 10%KCl (not having reagent) is printed on the conductor.Total departure hematocrit spacing when the short read pulse of DBL/ is arranged the almost total departure spacing when not having DBL is little by 2/3rds.Therefore, the pulse train that comprise a plurality of working cycle, combines with DBL can improve significantly accuracy of measurement and reduce the amboceptor back end with achieving one's goal.

Fig. 7 C and Fig. 7 D have shown when gating current and have analyzed pulse train when DBL is combined, and can reduce the hematocrit deviation.Fig. 7 C demonstrates as DBL and is combined with the pulse train of Fig. 5 E and at 14.875 seconds or when being separated by 0.125 second record current value with final pulse, owing to the measured deviation of hematocrit effect within ± 5%.For relatively, Fig. 7 D shown when the current value that uses the moment 16 seconds (being separated by 1.25 seconds with final pulse) comes the concentration of glucose of working sample, deviation increases to ± and 15%.Therefore, the duration that excites is longer, and the hematocrit deviation that then observes is larger.

Except the present invention can reduce ability from the inaccuracy of hematocrit effect and amboceptor back end signal, can use combination and the resulting contour curve of the momentary current curve that at every turn excites, organize calibration constants to provide to sensing system more, thereby improve the accuracy of analyzing.Thereby can use the every group of calibration constants that obtains that but the measurement of species certain concentration in specific current indication and the sample is associated.Therefore, in one aspect, can be by the dextrose equivalent that obtains with many group calibration constants be averaged to improve accuracy.

Conventional electrochemical sensor system uses one group of calibration constants such as slope and intercept usually, and current indication is transformed into analyte concentration in the corresponding sample.Yet, because in measurement, comprise random noise disturbance, so single group calibration constants may cause the analyte concentration measuring out according to the current value that records inaccurate.

Obtain current value by the set time in each working cycle of pulse train of the present invention, can set up many group calibration constants.Fig. 8 has drawn when the pulse train shown in Fig. 5 B is imposed on the WB sample that comprises various concentration of glucose, at the end points electric current of 8.5 seconds, 10 seconds, 11.5 seconds, 13 seconds and 14.5 seconds (first of working cycle 6～9 and terminal count pulse) record.Each bar in these five lubber-lines is separate with other all, and can use according at least two approach.

At first, can be determined at many groups calibration constants the quantity of the working cycle that should apply during the pulse train, thus accuracy, precision and chemical examination time that acquisition is wanted.For example, if from beginning excite the current value of acquisition to show high glucose concentration three times, for example＞150 or 200mg/dL, then sensing system can be in the time of about 5.5 seconds termination analysis, thereby shorten significantly the required time of analyzing.This shortening is feasible, because the common inexactness less than than low glucose concentrations time of the inexactness during high glucose concentration.On the contrary, if from beginning excite the current value of acquisition to show low glucose concentrations three times, for example≤150 or 100mg/dL, then sensing system can extend to analysis greater than 7 seconds, for example greater than 8 seconds or 10 seconds, thereby improve accuracy and/or the precision of analyzing.

Next, thus can be with many groups calibration constants by being averaged to improve accuracy and/or the precision of analysis.For example, if the target glucose Measuring Time is 11.5 seconds, the electric current in the time of can utilizing 8.5 seconds, 10 seconds and 11.5 seconds uses the slope of corresponding lubber-line and intercept to calculate concentration of glucose; Therefore G is just arranged _8.5=(i _8.5-Int _8.5)/Slope _8.5, G ₁₀=(i ₁₀-Int ₁₀)/Slope ₁₀, and G _11.5=(i _11.5-Int _11.5)/Slope _11.5In theory, these three dextrose equivalents should be equal to, and difference only is random deviation (random variation).Therefore, can be to dextrose equivalent G _8.5, G ₁₀And G _11.5Be averaged, and calculate final dextrose equivalent (G _8.5+ G ₁₀+ G _11.5)/3.Average according to lubber-line and noise can be reduced by 1/ √ 3.

The gating current that comprises the relatively short relaxation that excites and relatively grow shown in Fig. 5 E is analyzed pulse train, and its unforeseeable effect is to simplify the ability of calibration.Although the accuracy that can be conducive to analyze from many groups calibration constants that instantaneous and contour curve obtains, but the pulse train shown in Fig. 5 E also can provide the accuracy that obtains to using many group calibration constants similar accuracy, and it is obtained by single group calibration constants.Although be not to be to want to limit with any particular theory, this possibility of result is owing to the relatively long relaxation time relative with short relaxation.The long relaxation time can provide a kind of like this state, and in this state, but but the mean speed that measurement of species changes at duration of exciting and measurement of species diffuse to the rate equation among the DBL.In this manner, organize more calibration constants can collapse (collapse) be single group, and can be by before the determination and analysis substrate concentration, the current data that records being averaged process, and simplify record data to the transformation of analyte concentration.

Also can judge whether sensing zone is not filled up by sample with combination and the resulting contour curve of the momentary current curve that excites at every turn, thereby so that the user can add extra sample to sensing zone.The conventional sensors system can also be by judging the situation of not filling up with third electrode or electrode pair except the use working electrode with to the electrode; Yet third electrode or electrode pair have increased complicacy and the cost of sensing system.

Two conventional electrode systems can identify whether an analysis result is " bad ", but whether reason that can not determine this bad analysis result is owing to not filling up or defective sensing zone causes.Determine whether that owing to not filling up the ability that has caused bad analysis result be useful, can be by adding extra sample to same sensing zone and replicate analysis is proofreaied and correct because do not fill up, thus prevented from good sensing zone is discarded.

Fig. 9 A has shown the momentary current curve that obtains from the pulse train shown in Fig. 5 B, has wherein carried out 10 times and has analyzed, and has all used different sensing zones, and 2.0 μ L samples are incorporated in the sensing zone at every turn.According to filling speed and the cap crack volume of specific sensing zone, 2.0 μ L samples may enough or be not enough to fill sensing zone.

In Fig. 9 B, become decay rate as the contour curve of the function of time the momentary current Curve transform of Fig. 9 A.In one aspect, decay rate can be expressed as from following arbitrary equation and determine next K constant:

${\mathrm{<figure-callout\; id="1"\; label="K"\; filenames="S2006800263462E00041.png,S2006800263462E00071.png"\; state="\{\{state\}\}">K</figure-callout>}}_1=\frac{\ln \left(i_{0.125}\right)-\ln \left(i_{1.0}\right)}{\ln \left(t_{0.125}\right)-\ln \left(t_{1.0}\right)}$

${\mathrm{<figure-callout\; id="2"\; label="K"\; filenames="S2006800263462E00041.png,S2006800263462E00051.png"\; state="\{\{state\}\}">K</figure-callout>}}_2=\frac{\ln \left(i_{0.5}\right)-\ln \left(i_{1.0}\right)}{\ln \left(t_{0.5}\right)-\ln \left(t_{1.0}\right)}$

Wherein, the unit of value 0.125,0.5 and 1.0 is second.Therefore, use the K constant of decay process, the current curve of Fig. 9 A can be converted to the disintegration constant curve of Fig. 9 B.

Fig. 9 B shown the decay curve of the sensor that does not fill up and the decay curve of the sensor that normally fills up between significant difference, the especially difference in 3 seconds～7 seconds time ranges.Can by the difference between actual disintegration constant and the previous value of selecting, determine not fill up according to the disintegration constant curve.For example, if select-0.1 upper limit as the sensor that normally fills up among Fig. 9 B, during 3～5 second time cycle excite mensuration out be lower than any K of-0.1 ₁Constant value can be thought normally to fill up.Similarly, K ₁Value is higher than that any sensor of-0.1 can think not fill up.In this manner, just can not fill up in response to judging from the decay rate of momentary current curve acquisition.

Therefore, the sensing zone by series 3 and series 8 expressions among Fig. 9 B fully fills up, and is not filled up by eight sensing zones of series 1～2, series 4～7 and series 9～10 expressions.In this manner, gating current analysis pulse train of the present invention allows the detection of not filling up in the two electrode sensing bands, and this is the function that the conventional sensors system needs third electrode to realize usually.In addition, just can not fill up judgement within less than 10 seconds time, Multi-example provides the time for measurement mechanism signaling user adds more to sensing zone, and above-mentioned signalling for example is to send a signal to light-emitting display device or display.

Because can judge from the momentary current curve and do not fill up, can determine whether that with the same electrical flow valuve of the existence that is used for the determination and analysis thing and/or concentration existence do not fill up situation.Therefore, duration that will electrochemical analysis extends to the time required above concentration determination, just can judge during a plurality of working cycle of pulse train and not fill up.

Also can determine sample temperature changes whether analysis is had adverse effect with combination and the resulting contour curve of the momentary current curve that excites at every turn.The conventional sensors system comprise in the measurement mechanism or sensing zone on thermistor, thereby the temperature of measurement mechanism or sensing zone is provided respectively.Usually, although this temperature near sample temperature, the temperature of measurement mechanism or sensing zone is different from sample temperature.The temperature difference of measurement mechanism or sensing zone and sample may be incorporated into deviation in the analysis.

For example by determining decay rate with K constant previously discussed, just can the working sample temperature.Figure 10 has shown the relation curve of K constant and temperature, these K constants are to be 50,100 and 400mg/dL for concentration of glucose, excites from the 5th of pulse train to obtain.This relation curve presents the decay rate that absolute value increases along with the rising of temperature.Although be not to be to want to limit with any particular theory, this phenomenon may be owing to so that the lower temperature that the rate of diffusion of the various components in the cap crack reduces.In this manner, can come the working sample temperature in response to the decay rate that obtains from the momentary current curve.

Because sample temperature can be measured out according to the momentary current curve, therefore, can come the working sample temperature with the existence that is used for the determination and analysis thing and/or the same electrical flow valuve of concentration.So, can during a plurality of working cycle of pulse train, determine sample temperature, and extend to and exceed the required time of concentration determination duration that will electrochemical analysis.

In one aspect, the temperature of sample can be measured by solving K according to following Solving Equations:

$K=\frac{\ln i_{0.125}-\ln i_{0.375}}{\ln \left(0.125\right)-\ln \left(0.375\right)}$

I wherein _0.125And i _0.375Be to temperature variation the most responsive excite beginning 0.125 second and 0.375 second the time electric current, this excites for example is to produce exciting that the electric current the most responsive with respect to temperature variation decay.Ln (0.125) and ln (0.375) are respectively 0.125 second and 0.375 second natural logarithm form constantly.As shown in figure 10, according to the relation curve of these K constants and temperature, can come from the related function of relation curve the working sample temperature.Related function can be the fitting of a polynomial of curve.The temperature of measuring from this relation curve may be different from the temperature of device, and may reflect more accurately sample temperature.

Relative with said apparatus is, the advantage that sample temperature is measured is, can regulate the time length of analysis, thereby so that there is time enough to reach balance by DBL rehydration, thereby improve the accuracy of analyzing.For example, if the sample temperature of measuring then can make pulse train elongated for being lower than at least 5 ℃ of environment temperatures or 10 ℃, for example has the extra work circulation during pulse train.

Figure 11 is the synoptic diagram of measurement mechanism 1100, comprises the contact 1120 with circuit 1110 and display 1130 mutual telecommunications.In one aspect, measurement mechanism 1100 be portable and be suitable for hand-held and admit shown in Figure 1A with the sensing zone 100.In yet another aspect, measurement mechanism 1100 is to be suitable for admitting sensing zone and to implement the handheld measuring device that gating current is analyzed pulse train.

Contact 1120 is suitable for providing the telecommunication with the contact (for example contact 170 and 180 of the sensing zone shown in Figure 1B 100) of circuit 1110 and sensing zone.Circuit 1110 can comprise charger 1150, processor 1140 and computer-readable recording medium 1145.Charger 1150 can be voltage stabilizer, signal generator, etc.Therefore, charger 1150 can apply voltage to contact 1120, thereby records simultaneously the effect that resulting electric current plays charger-register.

Processor 1140 can with charger 1150, computer-readable recording medium 1145 and display 1130 mutual telecommunications.If charger is not suitable for record current, then processor 1140 can be suitable for recording the electric current at contact 1120 places.

Computer-readable recording medium 1145 can be such as any storage medium such as magnetic store, optical memory, semiconductor memory.Computer-readable recording medium 1145 can be fixed memory device or such as movable memory equipments such as mobile memory cards.Display 1130 can be simulation or digital, can be the LCD display that is suitable for showing the numerical value reading in one aspect.

When the contact of the sensing zone of splendid attire sample and contact 1120 mutual telecommunication, processor 1140 can be indicated charger 1150 to apply gating current to sample and be analyzed pulse train, thereby begins to analyze.Processor 1140 can for example be inputted in the sensing zone of previous insertion or in response to the user in response to insertion, the sample application of sensing zone, begins to analyze.

The instruction of analyzing pulse train about implementing gating current can be provided by the computer-readable software code that is stored in the computer-readable recording medium 1145.Described code can be any other codes that object code maybe can illustrate or control function described in the application.Can carry out the one or many data to the data that obtain from gating current analysis pulse train and process, be included in and measure decay rate, K constant, slope, intercept and/or sample temperature in the processor 1140 and will output to display 1130 such as the results such as analyte concentration after proofreading and correct.Utilization is about the instruction of pulse train, and data are processed and can be carried out according to the computer-readable software code that is stored in the computer-readable recording medium 1145 by processor 1140.

Be not that aforesaid method and system can be realized with following steps under the prerequisite for limited range, application or enforcement:

Step 1: start the biology sensor power supply

Step 2: carry out the biology sensor self check

Step 3: arrange, the Polling sample application is in sensor

Be v with ASIC Polling potential setting _Poll

Be i with the ASIC set of threshold levels _Trigger

The Polling cycle timer is set as at int _PollPlace's expiration

Step 4: be used for the setting of the sensor current of chemical examination

Wait for the expiration of Polling cycle timer

Start ASIC charge pump (charge pump)

Make ASIC threshold dector (i _Trigger) work

Make Polling current potential (v _Poll) work

Selection applies the sensor channel of current potential to sensor

Wait for t Time Created _Poll

Step 5: whether the detecting sensor electric current surpasses threshold value

Step 6: postpone also again detecting sensor electric current

Step 7: based on the detection of sample application

The beginning timing

Send pulse train

Step 8: pulse 1-survey sensor current i _1,1And i _1,8

At t _P1Constantly, pulse 1 beginning

1 duration of pulse is set as d _P1

Pulse 1 sensor current potential is set as v _P1

Selection applies the sensor channel of current potential to sensor

At t _1,1Constantly, the survey sensor signal is stored as AD with value _S11

At t _1,8Constantly, the survey sensor signal is stored as AD with value _S18

Step 9: postpone again standardized electronic device (electronics) of 1-

At AD ₂When reading finishes, postpone 1 beginning, disconnect the sensor channel

When pulse 2 beginning, postpone 1 and finish

Be V with potential setting _Standardize

At t _C1Constantly, select then measuring-signal of reference resistor channel, value is stored as AD _R1

At t _C2Constantly, select then measuring-signal of compensate for channel, value is stored as AD _O1

Attention: the sensor current that begins at pulse 1 place is from AD _R1And AD _O1Measurement result is calculated

Step 10: pulse 2-survey sensor current i _2,1And i _2,8

At t _P2Constantly, pulse 2 beginnings

2 duration of pulse are set as d _P2

Pulse 2 sensor current potentials are set as v _P2

Selection applies the sensor channel of current potential to sensor

At t _2,1Constantly, the survey sensor signal is stored as AD with value _S21

At t _2,8Constantly, the survey sensor signal is stored as AD with value _S28

Step 11: postpone 2-

At AD _S3When reading finishes, postpone 2 beginnings, disconnect the sensor channel

When pulse 3 beginning, postpone 2 and finish

Select compensation (offset) channel, to disconnect sensor

Step 12: pulse 3-survey sensor electric current: i _3,1And i _3,8

At t _P3Constantly, pulse 3 beginnings

3 duration of pulse are set as d _P3

Pulse 3 sensor current potentials are set as v _P3

Selection applies the sensor channel of current potential to sensor

At t _3,1Constantly, the survey sensor signal is stored as AD with value _S31

At t _3,8Constantly, the survey sensor signal is stored as AD with value _S38

Step 13: postpone 3-T ₁And i _Wet

At AD _S38When reading finishes, postpone 3 beginnings, disconnect the sensor channel

When pulse 4 beginning, postpone 3 and finish

Be V with potential setting _Standardize

At t _C3Constantly, select then measuring-signal of thermistor channel, value is stored as AD _T1

At t _WetConstantly, select then measuring-signal of compensate for channel, value is stored as AD _Wet

Step 14: pulse 4-survey sensor electric current: i _4,1, i _4,4And i _4,8

At t _P4Constantly, pulse 4 beginnings

4 duration of pulse are set as d _P4

Pulse 4 sensor current potentials are set as v _P4

Select the sensor channel, to apply current potential to sensor

At t _4,1Constantly, the survey sensor signal is stored as AD with value _S41

At t _4,4Constantly, the survey sensor signal is stored as AD with value _S44

At t _4,8Constantly, the survey sensor signal is stored as AD with value _S48

Step 15: postpone 4-

At AD _S48When reading finishes, postpone 4 beginnings, disconnect the sensor channel

When pulse 5 beginning, postpone 4 and finish

Select compensate for channel, to disconnect sensor

Step 16: pulse 5-survey sensor electric current: i _5,1, i _5,4And i _5,8

At t _P5Constantly, pulse 5 beginnings

5 duration of pulse are set as d _P5

Pulse 5 sensor current potentials are set as v _P5

Select the sensor channel, to apply current potential to sensor

At t _5,1Constantly, the survey sensor signal is stored as AD with value _S51

At t _5,4Constantly, the survey sensor signal is stored as AD with value _S54

At t _5,8Constantly, the survey sensor signal is stored as AD with value _S58

Make the ASIC analog functuion ineffective

Step 17: slope and the intercept of searching one group of amount of calibration

The slope value of current this group amount of calibration of S=

The values of intercept of current this group amount of calibration of Int=

Step 18: for temperature effect, regulate slope and intercept

Step 19: the concentration of glucose under calculating 25 ℃

[01991 step 20: convert target reference (blood plasma and WB reference) to

Step 21: check and do not fill up

Step 22: check " abnormal behaviour "

Step 23:, reexamine " abnormal behaviour " if glucose is low

Step 25: check limit glucose level

Step 26: show the result

Above-mentioned algorithm can have other subroutine, comprises that those are used to check such as sample temperature and the subroutine of not filling up the situation equal error.Listed the constant that can be used for above-mentioned algorithm in the lower Table III.Also can use other constants.

Table III

Although described various embodiment of the present invention, to it will be evident to one of ordinary skill in the art that within the scope of the invention, also other embodiment and embodiment can have been arranged.

Claims (26)

1. amperometry that is used for the analyte concentration of working sample comprises:

Apply pulse train to sample, described pulse train comprised at least 3 working cycle in 180 seconds, each described working cycle comprises that amperometry excites and relaxation, wherein, the described relaxation of each of described at least 3 working cycle provides independent diffusion and analyte response time, but analyte generates measurement of species at described analyte response time durations;

Excite from a described amperometry with duration of 0.01～1.5 second and to measure output signal, but described output signal in response to described measurement of species, but the analyte concentration of the concentration-response of described measurement of species in sample; And

Analyte concentration in response to measured output signal in the working sample, but the analyte concentration of wherein measuring is in response to described measurement of species oxidized or speed of being reduced under the effect of described pulse train.

2. the method for claim 1, wherein, the included deviation owing to the amboceptor back end in the analyte concentration of measuring according to described method, less than according in the analyte concentration of other method mensuration that not have to use the pulse train that in 180 seconds, comprises at least 3 working cycle owing to the deviation of amboceptor back end.

3. the method for claim 1, wherein, the included deviation owing to the amboceptor back end in the analyte concentration of measuring according to described method, less than according in the analyte concentration of the Same Way mensuration that not have to use the pulse train that in 180 seconds, comprises at least 3 working cycle owing to the deviation of amboceptor back end.

4. the method for claim 1, wherein said sample is the liquid that comprises biofluid.

5. the method for claim 1, wherein said analyte is glucose.

6. the method for claim 1, wherein each working cycle is included in exciting and relaxation under the set potential.

7. method as claimed in claim 6 is wherein in each duration of exciting record current value.

8. the method for claim 1, wherein said pulse train comprises the terminal count pulse.

9. the method for claim 1 wherein imposes on the sensing zone that comprises the diffusion barrier layer on the working electrode with described pulse train.

10. the method for claim 1 also comprises and measures resulting electric current and determine a plurality of calibration points from described electric current.

11. method as claimed in claim 10 is wherein determined duration of described pulse train from described a plurality of calibration points.

12. method as claimed in claim 10, the mensuration of wherein said analyte concentration comprise that the signal to using described a plurality of calibration point to obtain is averaged.

13. the method for claim 1 comprises also whether the sensing zone of judging the splendid attire sample is not filled up by sample.

14. method as claimed in claim 13, whether the sensing zone of wherein said judgement splendid attire sample does not fill up comprises the mensuration decay rate.

15. the method for claim 1 also comprises the temperature of the sample of measuring sensing zone institute splendid attire.

16. method as claimed in claim 15, the temperature of the sample of wherein said mensuration sensing zone institute splendid attire also comprises the mensuration decay rate.

17. the method for claim 1, the described relaxation of wherein said at least 3 working cycle is in response to open circuit.

18. the method for claim 1, the described relaxation of wherein said at least 3 working cycle is at least 0.5 second.

19. the method for claim 1, wherein measured output signal is the momentary current curve.

20. method as claimed in claim 17, wherein the electric current between described relaxation period is zero.

21. the method for claim 1, wherein said pulse train comprises that square wave excites.

22. the method for claim 1, but also comprise when described measurement of species reaches relatively constant rate of diffusion the determination and analysis substrate concentration in response to the current curve of this moment.

23. method as claimed in claim 22 also comprises in response to described current curve setting up to organize calibration constants more.

24. method as claimed in claim 23 also comprises:

In response to every group of calibration constants, the analyte concentration in the working sample; And

To be averaged from the analyte concentration that described many group calibration constants obtain.

25. the method for claim 1 determines also when sensing zone did not fill up when decay rate when described current curve was less than set point value.

26. method as claimed in claim 25, wherein said set point value is-0.1.

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