The application is to be that on January 24th, 2011, denomination of invention are for " improving the degree of accuracy applying dateDehumidizer " the divisional application of the application number patent application that is 201180006867.2.
That the application requires is that on January 22nd, 2010 submits to, denomination of invention is for " improving the degree of accuracyDehumidizer (AccuracyImprovingDesiccants) " U.S. Provisional Application No.61/297,515 priority, is incorporated herein its content whole by reference.
Background technology
Biology sensor provide to biofluid (as whole blood, serum, blood plasma, urine, saliva,Interstitial fluid or intracellular fluid) analysis. Conventionally, biology sensor has being positioned at test sensingThe measurement mechanism that sample in device is analyzed. Described sample is generally liquid form, and canThat the derivative of biofluid or biofluid is (as extract, dilution, filtrate or redissolution(reconstituted) sediment). In the Analysis deterrmination biofluid being undertaken by biology sensorExistence and/or the concentration of one or more analytes. The example of analyte comprise alcohol, glucose,Uric acid, lactate/ester, cholesterol, bilirubin, free fatty, triglycerides, protein,Ketone, phenylalanine or enzyme. Described analysis can be applied in physically different diagnosis and treatment. ExampleAs, can measure the glucose level in diabetic individual whole blood with biology sensor, and thisInformation can be used to regulate this individual diet and/or medication.
Biology sensor can be designed to be analyzed and can use one or more analytesDifferent sample volumes. Some biology sensor can analysis list drips (as by volume for 0.25-15 is micro-Rise (μ L)) whole blood. Can use biology with desk-top, portable and similar measurement mechanismSensor. Portable type measurement unit can be portable, and allow a kind of or many in sampleKind of analyte is identified and/or quantitatively. The example of Portable type measurement unit comprises BayerHealthCare's (Ta Lidun, New York)WithMeasuring instrument, and platformThe example of formula measurement mechanism comprises and can locate from CHInstruments (Austin, Texas)The ElectrochemicalWorkstation arriving.
In electrochemica biological sensor, in the time that input signal is applied to sample, by analyteThe signal of telecommunication that oxidation/reduction reaction or redox reaction generate or the thing that analyte is respondedDefinite point of the signal of telecommunication that the oxidation/reduction reaction of matter kind (species) or redox reaction generateAnalyse substrate concentration. Described input signal can be used as that single electric pulse applies or with multiple-pulse, sequence or circulationMode applies. Can be by redox materials (as amboceptor (mediator), enzyme or similar substance kind)Add described sample to strengthen during redox reaction from the first substance classes to the second material kindThe electronics of class shifts. Described redox materials can react with single analyte, provides thusThe specificity of the output signal that generates for a part.
Electrochemica biological sensor comprises the measurement with electrical contact (electricalcontacts) conventionallyDevice, the electric conductor in described electrical contact connecting test sensor. Described testing sensor can be suitableIn the outside at live organism, inside or partial interior use. When in the outside use of live organism,The sample of biofluid is introduced in the sample bomb in described testing sensor. Use can introducedBefore the sample of analyzing, after introducing is used for the sample of analyzing or at the sample of introducing for analyzingDuring this time described testing sensor is placed in measurement mechanism. When or part inner at live organism existWhen the inner use of live organism, described testing sensor can be immersed continuously maybe can be by sample in sampleProduct are introduced into described testing sensor off and on. Described testing sensor can comprise that part isolation is certainThe holder of volume sample, or described testing sensor can be open to sample. Similarly, described inSample flows through serially described testing sensor or is interrupted to analyze.
By being equipped with on insulated substrate or printing electrode (by being equipped with on one or more conductorsOne or more reagent compositions) can form described testing sensor. For example, when working electrode and rightWhen electrode is applied by same composition, can utilize multiple in described conductor of reagent composition of the same raceConductor applies. Multiple technologies known to persons of ordinary skill in the art can be used for described reagentComposition is provided on testing sensor. Described reagent composition can be provided in as reagent fluidOn conductor, be then dried. In the time that sample is introduced into described testing sensor, described reagentComposition starts rehydration.
The reagent composition being equipped with on each conductor can be identical or different. Therefore, working electrodeReagent composition can comprise enzyme, amboceptor and adhesive, and can only wrap the reagent composition of electrodeContaining amboceptor and adhesive, this amboceptor can be identical or different with the amboceptor of described working electrode. Reagent setCompound can comprise ion agent (promoting oxidation or the reduction of analyte, as oxidoreducing enzyme) andAmboceptor or other material (assisting the electronics between analyte and working electrode to shift) arbitrarily.
Before the described testing sensor of use, one or more components of reagent composition can experienceLearn and transform. Especially, it is believed that, under certain condition, the oxidation state of amboceptor can be sent out in timeChanging. In the situation that water exists, as the amboceptors such as the iron cyanide and organic quinone and quinhydrones can be throughGo through reduction. The existence of the amboceptor through reducing in reagent composition can cause the background current liter of sensorHeight, especially, for the low sample of analyte concentration, this can produce inaccurate analysis result.
Conventionally,, by testing sensor is stored in and is adjacent to dehumidizer place, suppress agent combinationThe chemical conversion of less desirable and/or too early generation in thing. Dehumidizer is applied to testing sensor conventionallyIn primary package (as bottle or paper tinsel bag), to prevent the degraded of reagent composition, thereby maintain testThe working life (shelflife) of the expectation of sensor. Tradition for testing sensor stocking system is removedHumectant can absorb the moisture that may bleed in the packaging that comprises testing sensor soon. For the protection ofThe example of the dehumidizer of testing sensor comprises molecular sieve, and described molecular sieve is even at low-humidity environmentUnder also can promptly absorb moisture.
Protecting the shortcoming of testing sensor with dehumidizer is one or more components of reagent compositionMay need the moisture of threshold level to keep their functions in described composition. For example, FADDependent glucose dehydrogenase (FAD-GDH) is considered to need some residual moistures to maintain its daySo active configuration. Moisture in reduction reagent composition, to lower than threshold level, can cause enzyme conformationChange and inactivation.
Conventionally in the following way to the loss of enzyme activity occurring because testing sensor is over-dryingProcess: in reagent composition, comprise excessive enzyme or recognized to adding in reagent compositionFor making the material of enzyme stabilization. Can make the thing of the enzyme stabilization in test sensor reagent compositionThe example of matter comprises: sugar, as trehalose or sucrose; And sugar alcohol, as sweet mellow wine, maltitolOr sorbierite. Can in freeze-dry process, preserve enzymatic activity with these materials. Referring to, for example EP1785483A1. But the high amount of fill of enzyme or other solid (as stabilizing agent) can produce otherDifficulty. Because enzyme component is conventionally very expensive, improving enzyme amount of fill is not conform to exceeding analysis desired levelDemand. In addition, especially at lower temperature, enzyme or other solid reagent set that can slow downThe rehydration that compound is undertaken by sample, causes analysis time longer. Exceed with analyte and mutually doWith excessive other one-tenth in the excessive enzyme in the testing sensor outside aequum and/or reagent compositionPoint (as amboceptor) also can reduce as described in the degree of accuracy of sensor.
Therefore, there is the lasting demand of improving bio-sensor system, especially can provide sampleIn analyte concentration carry out more accurately and/or bio-sensor system and/or the energy of micrometric measurementThe bio-sensor system of the analysis time of shortening is provided. In addition, exist and improve biology sensor systemThe following demand of system: providing the degree of accuracy of expectation, precision and/or in analysis time,Under the storage condition of relative broad range, there is the bio-sensor system of the working life of prolongation. Of the present inventionSystem, apparatus and method have overcome at least one shortcoming relevant with conventional biosensor system.
Detailed description of the invention
A kind of bio-sensor system, described bio-sensor system comprises the survey being encapsulated in containerExamination sensor, described container has the dehumidizer that keeps the residual moisture level in this container. LowIn humidity environment, described dehumidizer can promptly not absorb moisture, and this can make testing sensorReagent composition maintains moisture to contribute to make enzyme to maintain the level of its active configuration. Be stored inComprise traditional dehumidizer or compare without the comparable testing sensor in the container of dehumidizer, being stored inTesting sensor in the container that comprises this class dehumidizer can provide to analyte concentration more accurately and/Or more accurate mensuration. Therefore, even in the time that described testing sensor is long under non-optimum conditionBetween while storing, described testing sensor also can provide has the accurate consistently of rapid analysis timeTrue analysis.
A kind of bio-sensor system, described bio-sensor system comprises multiple testing sensors,Each testing sensor comprises: at least two conductors, wherein, one in described conductor is work electricityThe utmost point; And reagent composition, described reagent composition is provided on described working electrode or contiguous instituteState working electrode. Described bio-sensor system further comprises container, and described container comprises dehumidifyingAgent. Described multiple testing sensors are encapsulated in described container.
When at 40 DEG C during with the environmental exposure of 10%-20% relative humidity (RH), in described containerDehumidizer preferably at most absorb the water of its weight 15%. When at 40 DEG C of rings with 10%-20%RHWhen border contacts, more preferably described dehumidizer absorbs at most the water of its weight 10%. When 40 DEG C withWhen the environmental exposure of 10%-20%RH, more preferably described dehumidizer absorbs its weight 5%-10%'sWater.
When during with the environmental exposure of 10%-20%RH, absorbing the water of its weight 5%-10% at 40 DEG CThe example of dehumidizer comprise silica gel. For 0% to about 60% RH value, silica gel can be with greatlyCause to the proportional level of relative humidity of surrounding environment and absorb moisture. By contrast, use traditionallyMolecular sieve dehumidizer in testing sensor container can be promptly from having the ring of 10%-20%RHIn border, absorb large quantity of moisture. When at 40 DEG C during with the environmental exposure of 5%RH, molecular sieve can absorb itThe water of weight 15%-20%, then, along with relative humidity increases, absorbable extra water is few.
When at 40 DEG C during with the environmental exposure of 10%-20%RH, can absorb at most its weight 15%The example of the dehumidizer of water comprises that blend has the composition of the molecular sieve of polymer. Can be by dehumidifyingAgent and polyblend reduce the water suction effect of dehumidizer. Due to the dehumidizer in polymerOnly partly be exposed to environment, moisture absorption can be with the speed slower than the infiltration rate of pure dehumidizerDegree occurs. When during with the environmental exposure of 10%-20%RH, absorbing at most its weight 15% at 40 DEG CAnother example of dehumidizer of water comprise the blend of molecular sieve and silica gel. In described blendMolecular sieve and type and the relative quantity of silica gel select, make it possible to design this blend compositionThe total moisture absorbing under low relative humidity.
It is 400 milligrams/deciliter (mg/dL) and blood that Figure 1A-Fig. 1 C shows from concentration of glucoseSpecific volume of cell amount is the testing sensor output signal of 40% whole blood sample. By described test sensingDevice is sealed in following container: described container has traditional dehumidizer of 22.5mg/ testing sensor" 13x zeolite " (Figure 1A), the silica gel (Figure 1B) of 30mg/ testing sensor or without dehumidizer(Fig. 1 C). For all types of containers, the described container of half is stored to two weeks at 50 DEG C, withTime by the described container of other half-20 DEG C store two weeks. 50 DEG C of heat stress environment of two weeks beBe usually used to evaluate the acceleration stressed condition of biology sensor performance in latter stage working life. In described storageDeposit after date, testing sensor is used for carrying out to the electrochemical analysis of described whole blood sample.
As disclosed in 2009/0145779 with United States Patent (USP) in United States Patent (USP) discloses 2008/0173552Described, the signal that inputs to testing sensor by measurement mechanism is gate ampere pulse train,And one or more output current values are relevant to the analyte concentration of sample. By reference willThese patent applications are relevant about gate ampere pulse train and output current value and analyte concentrationProperty disclosure be incorporated to herein. Be used for the packet of pulses of the figure that generates Figure 1A-Fig. 1 C containing relaxing by 7 timesHenan excites for separated 8 times. Being energized into for the second time the duration exciting for the 8th time is about 0.4s,The duration that relaxes towards for the second time the 7th relaxation is about 1s. Swash being energized into for the second time the 8th timeDuring sending out, three output current values are recorded.
Analyzing by drawing for the concentration known analyte in a series of liquid storages that comprise analyteIn special time under output current, can obtain one or more output current values and sample pointAnalyse the correlation of substrate concentration. Dense for making from the output current value of output signal and the analyte of sampleDegree is relevant, from the described initial current value exciting preferably than in decay (decay) subsequentlyCurrent value is higher. Preferably, the output current value relevant to the analyte concentration of sample is from as followsDecay: the current data that described decay comprises reflection testing sensor maximum power performance. FormThe dynamics of oxidation-reduction on output current basis is subject to the impact of multiple factors. These factors can be wrappedDraw together: reagent composition rehydration speed, enzyme system and analyte response speed, enzyme system are by electronicsBe transferred to the speed that the speed of amboceptor and amboceptor are transferred to electronics at electrode.
In the time thering is the initial current value exciting of decay current value and be the maximum in repeatedly exciting,Can reach the maximum power performance of testing sensor at gate ampere pulse train duration of exciting. ExcellentSelection of land, when having the electric current final value (lastintimecurrent that exciting of decay current value is obtainedValue) be while repeatedly exciting obtained maximum current final value, the maximum that reaches testing sensor is movingMechanical property. More preferably, when the initial current value exciting with decay current value is repeatedly to swashMaximum in sending out, and same excite obtained electric current final value be repeatedly excite obtainedWhen large electric current final value, reach the maximum power performance of testing sensor. Decay current can be there isWhen the exciting for the first time of value, reach maximum power performance, or can (as have exciting subsequentlyThe exciting for the second time, excite for the third time or exciting more below of decay current value) time reach maximum movingMechanical property.
Can describe maximum power performance with regard to parameter " time to peak ", described " time to peak " isAfter the sample that comprises analyte contacts with testing sensor, electrochemical test sensor obtains itThe time of large output current value. Maximum output current value is preferred for the analyte concentration of sampleCorrelation. The time to peak of testing sensor is preferably introducing sample in described testing sensorAfter be less than about 7s, be more preferably less than about 5s. Preferably, described time to peak is to described surveyExamination is introduced after sample about 0.4s in sensor to about 7s, more preferably about 0.6s is to about 6.4sIn, more preferably about 1s to about 1.1s in about 5s and more preferably to about 3.5s.
With reference to Figure 1A, for the testing sensor being encapsulated in the container with traditional dehumidizer,Store after two weeks compared with thering is longer time to peak at-20 DEG C after storing two weeks at 50 DEG C. Compare itUnder, for the sensor (Figure 1B) of silica gel dehumidizer sealing or not with the biography of dehumidizer sealingSensor (Fig. 1 C), the time to peak 50 DEG C of storages after two weeks is compared with storing after two weeks at-20 DEG CTime to peak does not increase.
Because the glucose result of testing sensor stems from the electricity of measuring at set time point conventionallyStream, any variation of testing sensor current curve (currentprofile) can produce inconsistentGlucose analysis result. For the analysis of carrying out under the short period (below 10s),Inaccuracy increases especially obvious. For the testing sensor detecting for Figure 1A-Fig. 1 C, with biographyThe variation of the current curve of the testing sensor of system dehumidizer sealing has caused at described biology sensorLess desirable the increasing in bias aspect.
Limit at the measurement performance to biology sensor aspect the degree of accuracy and/or precision. AccurateExactness and/or precision increase the improvement that biology sensor measurement performance aspect is provided. The degree of accuracyCan be expressed as the bias of the analyte reading of the biology sensor compared with reference analyte reading,The larger lower degree of accuracy of bias value representation. Precision can be expressed as multiple analyte readingsBias is with respect to decentralization (spread) or the variance of mean value. Bias is by biosensor assayOne or more values and biofluid in one or more reference values of adopting of analyte concentration(acceptedreferencevalues) difference between. Therefore, in Measurement and analysis or manyIndividual error has caused the bias of the analyte concentration of being measured by bio-sensor system. Per sampleAnalyte concentration, bias can be expressed as to " definitely bias " or " percentage bias ". DefinitelyBias can represent as mg/dL with measurement unit, and can be used for analyte concentration and be less than 100mg/dLSituation. Percentage bias can be expressed as the percentage of absolute bias value with respect to reference value, and canBe at least the situation of 100mg/dL for analyte concentration. The reference value of adopting can be by calibration instrumentDevice is (as the YSI2300STAT that can obtain from YSI Inc. (YellowSprings, Ohio)PLUSTMGlucose analyser) obtain.
Fig. 2 A and Fig. 2 B described hematocrit amount be 40% and concentration of glucose be 50The glucose analysis of the whole blood sample of mg/dL, 100mg/dL, 400mg/dL or 600mg/dLThe figure of bias. The testing sensor using in analyzing is encapsulated into and comprises 0-22.5mg/ test sensingIn the container of traditional dehumidizer 13x zeolite of device (Fig. 2 A) or comprise 0-30mg/ test and passIn the container of the silica gel of sensor (Fig. 2 B), and store two weeks at 50 DEG C.
When without dehumidizer (0mg dehumidizer/testing sensor), for comprising low concentration glucose(50mg/dL) sample, the analysis of blood sugar after testing sensor heat stress has 15mg/dLPositive skewness; For thering is the sample of 100mg/dL and 400mg/dL concentration of glucose,Described analysis of blood sugar has the bias of 7%-10%; For comprising high concentration glucose (600mg/dL)Sample, described analysis of blood sugar is almost without bias. Testing sensor and traditional molecular sieve are removedHumectant seals together, proofreaied and correct there is low concentration glucose and normal concentration glucose sample justBias; But, along with dehumidizer level increases, have 600mg/dL glucose sample partiallyLean on and increase to-10% and-15% (Fig. 2 A). By contrast, for thering is the 100mg/dL of being less than PortugalThe sample of grape sugar, the bias of the sensor storing with the silica gel of 30mg/ sensor is 5In mg/dL bias, for thering is the sample of 100mg/dL-600mg/dL glucose, described inBias is (Fig. 2 B) in ± 5% bias.
Than not with dehumidizer or with the carrying out of weak silica gel dehumidizer sealing similar processingThe result of testing sensor, in the situation that traditional dehumidizer exists, for sealing two weeks at 50 DEG CTesting sensor, analyzing, time to peak and analysis increasing aspect bias is astonishing.Conventionally, dehumidizer has been used to prevent that the component (comprising amboceptor) of reagent layer from making at testing sensorWith front conversion. Therefore, unexpectedly, with respect to not with dehumidizer or with weak dehumidifyingThe comparable testing sensor that agent stores, especially in the time that analytic sample has high concentration of glucose,The testing sensor storing with traditional dehumidizer by weaken described testing sensor the degree of accuracy and/orIts working life.
For comprising the bio-sensing that is encapsulated into the multiple testing sensors in the container with dehumidizerDevice system, can evaluate this system: use test sensor is measured in the following mannerThere is analyte content in the sample of concentration known (crossing over certain concentration range) analyte, soRear calculating measured value is with respect to the bias of actual concentrations. In an example, by multiple test sensingsDevice enters to comprise in the container of dehumidizer two weeks at the temperature lower seal of 50 DEG C, wherein, respectively tests sensingDevice comprises: at least two conductors, and one in described conductor is working electrode; And agent combinationThing, described reagent composition is provided on described working electrode or contiguous described working electrode. Then,Described testing sensor is shifted out from container, and each testing sensor is passed through to described at least twoConductor is connected to measurement mechanism. Once connect, each testing sensor is contacted with in sample one,And for the analyte concentration of working sample. In this example, cross over for analyte concentrationThe sample of 10mg/dL-600mg/dL scope: be less than 100mg/dL for analyte concentrationSample, preferably the bias of each analyte concentration is after measured in ± 10mg/dL; For dividingAnalyse substrate concentration and be at least the sample of 100mg/dL, preferably each analyte concentration after measuredBias is in ± 10%. The meaning of phrase " analyte concentration is crossed over 10mg/dL-600mg/dL scope "Think of is that at least one the analyte concentration in sample is 10mg/dL, at least one in other sampleIndividual analyte concentration is 600mg/dL. If any, remaining sample can have 10mg/dLAnd analyte concentration between 600mg/dL.
In above-mentioned example, for the sample that is less than 100mg/dL for analyte concentration, preferablyThe bias of each analyte concentration is after measured in ± 7mg/dL; Be at least 100 for analyte concentrationThe sample of mg/dL, the bias of preferred each analyte concentration is after measured in ± 7%. More excellentSelection of land, for the sample that is less than 100mg/dL for analyte concentration, preferably after measured each pointThe bias of analysing substrate concentration is in ± 5mg/dL; Be at least the sample of 100mg/dL for analyte concentrationProduct, the bias of preferred each analyte concentration is after measured in ± 5%. Preferably, at thisIn example, the quantity of described multiple testing sensors is at least 10, be preferably at least 25,At least 50 or at least 100. Preferably, in this example, described sample has crosses over 50The analyte concentration of mg/dL-600mg/dL scope.
For comprising the bio-sensing that is encapsulated into the multiple testing sensors in the container with dehumidizerDevice system, can evaluate this system: use test sensor is measured to be had in the following wayKnow the analyte content of the sample of concentration analysis thing, then calculate the coefficient of variation of this measured value(CV%). In above-mentioned example, the CV% of each analyte concentration is after measured at the most 2.5%.In above-mentioned example, the CV% of analyte concentration after measured more preferably at the most 2%.
Table 1 listed hematocrit amount be 42% and concentration of glucose be 50mg/dL, 100The CV% of the glucose analysis of the whole blood sample of mg/dL, 400mg/dL or 600mg/dL. ShouldThe testing sensor using in analysis is encapsulated in following container: described container has 0-22.5mg/Traditional dehumidizer 13x zeolite of testing sensor or there is the silicon of 0-30mg/ testing sensorGlue dehumidizer, and store two weeks at 50 DEG C. Listed each result is based on using 10 tests to passThe measured value of sensor.
Table 1 is at the analytical precision of 50 DEG C of heat stresses testing sensor of 2 weeks
Table 2 has been listed the CV% of glucose analysis as described in Table 1, and still, test wherein passesSensor is to store two weeks at-20 DEG C. Listed each result is based on using 10 testing sensorsMeasured value.
Table 2 is at the analytical precision of-20 DEG C of storages testing sensor of 2 weeks
When without dehumidizer (0mg dehumidizer/testing sensor), cross over 50 for analyte concentrationThe sample of mg/dL-600mg/dL scope, at testing sensor in 50 DEG C of heat stresses after 2 weeksThe analysis of blood sugar carrying out has the coefficient of variation of 1.3%-2.4%. Make testing sensor and traditional molecular sieveDehumidizer (7.5mg/ testing sensor or 22.5mg/ testing sensor) or pass with 10.0mg/ testThe silica gel sealing of sensor does not reduce the upper limit of the CV% of analysis of blood sugar. But, make to test sensingThe silica gel sealing of device and 30.0mg/ testing sensor is reduced to the upper limit of the CV% of analysis of blood sugar1.5%. Testing sensor, is also being measured for analysis of blood sugar after 2 weeks in-20 DEG C of sealingsSimilar trend. For the 50mg/dL-600mg/dL scope of sample cross over to(for) analyte concentration,The analysis of blood sugar tool that the testing sensor of the silica gel sealing of use and 30.0mg/ testing sensor carries outThere is the CV% value lower than 2.1%.
Fig. 3 A and Fig. 3 B have described the background electricity that does not contain the glucose analysis of the whole blood sample of glucoseThe figure of stream. The testing sensor using in analyzing is encapsulated in container, and described container comprisesTraditional dehumidizer 13x zeolite (Fig. 3 A) of 0-22.5mg/ testing sensor or comprise 0-30The silica gel (Fig. 3 B) of mg/ testing sensor, and by described testing sensor-20 DEG C, room temperature (RT,25 DEG C) or 50 DEG C store two weeks. Because described sample is not containing glucose, the background current measuringOwing in through reduction oxidation state material (as reduction amboceptor) existence.
When without dehumidizer, being stored in testing sensor in container demonstrates at heat stress artifact and passesLarge the increasing of sensor background current aspect. This increases consistent with following traditional theory: dehumidizerTo maintaining low background current (may be the autoreduction by preventing amboceptor) in testing sensor veryImportant. Increasing of sensor background current may be to produce in having compared with the sample of low glucose concentrationsThe reason of the positive analysis bias shown in raw Fig. 2 A and Fig. 2 B. Compared with in the situation that silica gel existsThe testing sensor (Fig. 3 B) storing stores in the situation that traditional molecular sieve dehumidizer existsTesting sensor (Fig. 3 A) needs less dehumidizer to maintain low background current. Therefore, traditionDehumidizer has seemed to reach the predictive role that suppresses the too early generation of amboceptor reduction.
Amboceptor in the reagent composition of the testing sensor using in Fig. 1-Fig. 6 is bielectronTransfer amboceptor 3-(2 ', 5 '-disulfophenyl imino group)-3H-phenthazine double sodium salt. At testing sensorThe effect of the moisture of observing between the storage life is considered to be applicable to other bielectron and shifts amboceptor, as itIts organic quinone and quinhydrones. The example of this amboceptoid comprises: phenanthroline quinone (phenathrolineQuinone); Phenthazine is with phenoxazine derivative, as: 3-phenylimino-3H-phenthazine (PIPT)With 3-phenylimino-3H-phenoxazine (PIPO); 3-(phenyl amino)-3H-phenoxazine; Phenthazine;With 7-hydroxyl-9,9-dimethyl-9H-acridine-2-ketone and derivative thereof. Between the testing sensor storage lifeThe effect of the moisture of observing is also considered to be applicable to single electron transfer amboceptor, as 1,1 '-dimethyl twoLuxuriant iron, ferrocyanide and the iron cyanide, six ammino rutheniums (III) and six ammino rutheniums (II).
One to these about the surprising result of time to peak, bias and/or precision canCan explanation be that weak dehumidizer can be enzyme unforeseeable high-caliber protection is provided. Compared with biographySystem dehumidizer, weak dehumidizer (as silica gel) seems more compatible with FAD-GDH enzyme,And described weak dehumidizer also provides enough protections to amboceptor. Especially for high grapeSugar sample, may underestimate loss of enzyme activity before this on analyzing the impact of bias.
Fig. 4 has described in the container of dehumidizer with dissimilar and level in-20 DEG C of (rhombusesSymbol), 50 DEG C (triangle symbol) or room temperature (square symbol) the sealing test sensing of two weeksThe figure of FAD-GDH enzymatic activity in the sensor of device. Filled symbols dehumidifies corresponding to traditional molecular sieveAgent, open symbols is corresponding to silica gel dehumidizer. These two kinds of dehumidizers seem can not make enzyme to be livedProperty is-20 DEG C of losses. Store after two weeks, at the sensor (0mg without packing under dehumidizer at 50 DEG CDehumidizer/sensor) sensor in the loss of enzymatic activity be approximately 10%. (solid with molecular sieveTriangle symbol) together the enzymatic activity of sensor of packaging be reduced to about 60%, or even in phaseAlso like this during to low-level 7mg dehumidizer/sensor. By contrast, pack together with silica gelThe enzymatic activity of sensor exceeded approximately 25%, kept 75%-80% (the hollow triangle of enzymatic activityShape symbol). Even, for room temperature storage, the testing sensor storing with molecular sieve is (just solidSquare symbols) demonstrate than the enzyme of the testing sensor (open squares symbol) storing with silica gelActive low about 5% enzymatic activity.
The result of Fig. 4 is consistent with following analysis in conjunction with the result of Fig. 1-Fig. 3: FAD-GDH enzyme needsWant the moisture of threshold level to maintain its natural structure and activity. For the glucose of 600mg/dLConcentration, with molecular sieve dehumidizer increase and the negative bias that increases lean on (Fig. 2 A) and use molecular sieve dehumidizerAbout 40% Loss Correlation (Fig. 4) of the FAD-GDH enzymatic activity of the testing sensor storing. PhaseThan under, for the concentration of glucose of 600mg/dL, increase and relatively constant with silica gel dehumidizerAnd the FAD-GDH of the testing sensor that nearly zero bias (Fig. 2 B) and use silica gel dehumidizer storeThe only 20-25% Loss Correlation (Fig. 4) of enzymatic activity.
Fig. 5 has described for being encapsulated into and having dissimilar dehumidizer and have or tool not at 50 DEG CHave in the container of reagent composition of enzyme stabilizers sorbierite in the sensor of testing sensor of two weeksFAD-GDH enzymatic activity (" enzyme reclaims % "). The dehumidizer using is silica gel (SG), molecular sieve13x (MS-13x), the bottle cover cylinder (Bottle-MS) that comprises molecular sieve 4A and two kinds differentBlend has the dehumidizer of polymer (be coated with the polypropylene screen (SLF/MS) of molecular sieve and be coated withThe polypropylene screen (SLF/SG) of silica gel). From MultisorbTechnologies (Buffalo, NY)Place obtains described blend the dehumidizer of polymer.
Be formed for being marked with by reagent fluid is deposited and is dried " PD18-contrast " andThe reagent composition of the testing sensor of " PD16-contrast ", described reagent fluid comprises water, 80MM (mM) 3-(2 ', 5 '-disulfophenyl imino group)-3H-phenthazine double sodium salt amboceptor, 3.750.2% (w/w) that enzyme unit FAD-GDH/ μ L, weight average molecular weight (Mw) are 300,0000.362% (w/w) HEC that hydroxyethylcellulose (HEC) adhesive, Mw are 90,000 is stickyMixture, 112.5mMNa2HPO4Buffer salt, 0.225% (w/w) N-caprylyl-N-methyl D-Gucosamine (MEGA-8) and 0.01% (w/w) sodium methyl cocoyl taurate (GeroponTC-42). As the reagent composition of the sensor for being marked with " PD18-contrast ", prepare markNote has the reagent composition for testing sensor of " PD18 adds 0.4% sorbierite ", differenceBe in the sorbierite that also comprises 0.4% (w/w) in this reagent fluid.
With pure molecular sieve dehumidizer (MS-13x) or with bottle a dehumidizer sleeve (Bottle-MS) storeTesting sensor aspect enzymatic activity, reduced approximately 30%, and store up with silica gel dehumidizer (SG)The testing sensor of depositing has only reduced by 15% aspect enzymatic activity. The enzyme stabilization of 0.4% sorbierite is doneWith having reduced loss of enzyme activity; But, with molecular sieve dehumidizer store testing sensor again makeThe degree that obtains enzyme deactivation doubles. The PD18-storing with pure molecular sieve dehumidizer or with silica gel dehumidizerThe difference of the enzyme recovery aspect between contrast testing sensor and PD16-contrast testing sensor is considered toWithin the scope of experimental error.
Molecular sieve dehumidizer and polyacrylic blend (SLF/MS) provide can with by silica gel dehumidizerThe enzymatic activity providing retains the enzymatic activity of comparing and retains. Therefore, the dehumidifying effect of Inhibitory molecules sieve makesEnzyme is kept its activity during heat stress. Also suppress the dehumidifying effect of silica gel. Analyze accuratelyThe reduction of degree may make other reagent composition composition avoid the anti-of moisture with lacking during heat stressProtect relevant.
For comprising the bio-sensing that is encapsulated into the multiple testing sensors in the container with dehumidizerDevice system, can come this system to evaluate: by described testing sensor in the following wayAfter storing, measure the redox in the reagent composition of described testing sensor under different conditionThe reservation of enzymatic activity. In an example, the temperature lower seal at 50 DEG C by multiple testing sensorsIn the container that comprises dehumidizer two weeks, wherein, each testing sensor comprised: at least two conductors,One in described conductor is working electrode; And reagent composition, described reagent composition is equipped withOn described working electrode or contiguous described working electrode, and described reagent composition comprises oxidationReductase. Then from described container, shift out testing sensor, to the reagent set of each testing sensorThe activity of the oxidoreducing enzyme in compound is measured. In this example, each testing sensorReagent composition preferably retains at least 75% oxidoreductase activity. More preferably, real at thisIn example, the reagent composition of each testing sensor preferably retain at least 80% oxidoreductase activity,And more preferably retain at least 85% oxidoreductase activity. Preferably, in this example,The quantity of the testing sensor in described multiple testing sensors is at least 10, is preferably at least25, at least 50 or at least 100.
Adjustable one or more output current value is (as the output electricity described at Figure 1A-Fig. 1 CFlow valuve) and the analyte concentration of sample between correlation in measuring with compensation (accountfor)Error. A kind of approach of proofreading and correct the error relevant with biosensor analysis is to utilize by output currentThe index function (indexfunction) that the intermediate current value of value is extracted regulates for electric by outputThe correlation of the analyte concentration in flow valuve working sample. Index function can compensate for electric by outputOne or more errors of the correlation of flow valuve determination and analysis substrate concentration, described error can cause through surveyingThe bias of fixed analyte concentration. Index function is corresponding to the one or more errors by analyzingBias in correlation between analyte concentration and the output current value causing.
Glucose analysis bias % can be one or more by what obtain from one or more error parametersΔ S value represents. Described Δ S value represent the analyte concentration measured by one or more error parameters andThe slope deviation (slopedeviation) of the correlation between output current value. Described correlation tiltedlyRate corresponding to the given variation for sample concentration of glucose in the variation aspect output current. CanBy the index function normalization corresponding to slope or slope variation, with reduce output current value changeStatistics impact, improve difference (differentiationinvariations) that output current value changes,Make measurement standard and their combination etc. of output current value. Can will be used for through the correlation regulatingMeasure the analyte concentration in biological sample by output current value, and and conventional biosensorCompare and there is the improved degree of accuracy and/or precision. In as Publication about Document, describe and made index of reference functionThe error correction of carrying out with Δ S value: for example, United States Patent (USP) discloses 2009/0177406; And 2009The denomination of invention that submit on December 8, in is the international monopoly of " ComplexIndexFunctions "Application No.PCT/US2009/067150. By reference, by these patent applications about useDuring the disclosure that index function and Δ S value are carried out error correction is incorporated herein.
Therefore, make the Δ S/S of index of reference function representation, can be by the output of response sample concentration of glucoseCurrent value converts the calibrated concentration of glucose of sample to. Or, make index of reference function and equation asGcorr=Graw/ (1+f (Index)), can determine calibrated concentration of glucose by corrected glucose concentration value notValue, wherein, GcorrFor the calibrated concentration of glucose of sample, GrawWhen uncompensated after measuredThe analyte concentration of sample, f (Index) is index function.
Index function can comprise by output signal (as the output signal of describing in Figure 1A-Fig. 1 C)The ratio extracting. For example, output signal value can compare in individual pulse-signal attenuation circulation,As ratio R 3=i3,3/i3,1Deng, wherein, i3,3Represent the 3rd electricity recording for the 3rd signal attenuationFlow valuve, and i3,1Represent the first current value recording for the 3rd signal attenuation. In another example,Output signal value between more independently pulse-signal attenuation circulation, as ratio R 4/3=i4,3/i3,3Deng, wherein, i4,3Represent the 3rd current value recording for the 4th signal attenuation. Index function canThe combination that comprises the ratio extracting from output signal. In an example, index function can comprise ratioThe simple rate of rate, as Ratio3/2=R3/R2. In another example, index function can compriseThe more complicated combination of simple index function. For example, index function Index-1 can be expressed asIndex-1=R4/3-Ratio3/2. In another example, index function Index-2 can be expressed asIndex-2=(R4/3)p-(Ratio3/2)q, wherein, p and q are positive number independently.
Preferably, the index function correction error relevant with the variation of hematocrit amount. For example,Can conventional biosensor system be set to report and be assumed to 40% (v/v) hematocrit amountThe concentration of glucose of whole blood sample, and do not consider the actual hematocrit amount of sample. At these beIn system, to comprising any glucose measurement carrying out below or above the blood sample of 40% hematocritTo comprise error, and therefore there is the bias that is attributable to hematocrit impact.
Produce the testing sensor of the output signal changing with hematocrit amount by use, canContribute to the calculating of the index function that the error relevant with hematocrit quantitative changeization proofreaied and correct.For some biology sensors, R5/4 ratio parameter has been served as the instruction of the hematocrit in sample,And be used to the analyte concentration of adjusting through measuring to compensate the hematocrit amount of sample. R5/4Ratio parameter has represented the 4th of response gate ampere pulse train (as the sequence of Figure 1A-Fig. 1 C)Pulse and the 5th pulse and relation between the electric current that generated by analyte.
Fig. 6 has described with respect to the R5/4 ratio parameter at-20 DEG C of storages testing sensor of two weeks,At 50 DEG C of figure of variation that store the R5/4 ratio parameter of the testing sensor of two weeks, wherein, described inTesting sensor has the enzyme density of various levels on the working electrode of described testing sensor. TwoType data point represented two kinds of different anion surfactant PhospholanCS131(polyoxyethylene nonylphenol phosphate (nonylphenolethoxylatephosphate)) and GeroponTC-42。
Under higher enzyme concentration, be stored in the testing sensor of 50 DEG C and be stored in the test of-20 DEG CDifference between the R5/4 ratio parameter of sensor is less. This trend is to for reagent compositionThe anion surfactant of two types all clearly. Owing to R5/4 ratio parameter can being usedMake the variable in index function that calibration analyte measures, need to this parameter because environmental factor is drawnThe variation rising is less. Therefore the enzymatic activity increasing, being provided by weak dehumidizer retains can be providedReduce the additional benefit of correction factor changeability (variability).
Enzyme in the reagent composition of the testing sensor using in Fig. 1-Fig. 6 is FAD-GDHEnzyme. The effect of the residual moisture of observing between the testing sensor storage life is considered to be applicable to otherEnzyme, described enzyme for example comprises following enzyme: alcohol dehydrogenase, lactic dehydrogenase, beta-hydroxy-butanoic acid dehydrogenationEnzyme, glucose-6-phosphate dehydrogenase (G6PD), glucose oxidase (GOx), GDH, formaldehydeDehydrogenase, malic dehydrogenase and the dehydrogenation of 3-hydroxy steroid.
Preferred enzyme system is oxygen dependent/non-dependent, does not therefore substantially pass through dioxygen oxidation. A kind of thisClass oxygen dependent/non-dependent enzyme family is GDH (GDH). Use different coenzyme or co-factor,Can mediate GDH in a different manner by different amboceptors. Depend on itself and GDHIn conjunction with situation, co-factor (as flavin adenine dinucleotide (FAD) (FAD)) can be tightly by apoenzyme (hostEnzyme) maintain, as the situation for FAD-GDH; Or co-factor is (as PQQ(PQQ)) can be covalently linked to described apoenzyme, as the situation of PQQ-GDH. These enzyme systemsCo-factor in each of system can for good and all be maintained by described apoenzyme, or can flow to reagentIn body, add before described enzyme system coenzyme and pheron described in reconstruct. Also coenzyme can be added independentlyBe added in the apoenzyme part in reagent fluid the catalysis to assist apoenzyme, for example nicotinoyl amine gland is fastNicotinamide adenine dinucleotide NAD/NADH+Or nicotinamide-adenine dinucleotide phosphate NADP/NADPH+The situation of combining with the dependent GDH of NAD (NAD-GDH).
For the composition of the reagent composition of testing sensor be used to form described reagent compositionThe composition of reagent fluid is for example recorded in: United States Patent (USP) discloses 2009/0178936; With 2009The denomination of invention that December 7 submitted to is " LowTotalSaltReagentCompositionsAndSystemsForBiosensors " international patent application No.PCT/US2009/066963. To drawWith mode by these patent applications about reagent composition composition be used to form reagent compositionThe disclosure of fluid is incorporated to herein.
Enzymatic activity in testing sensor and the analytical performance of testing sensor seem to be all subject to instituteState the impact of the dehumidizer type using in the container of sensor. When at 40 DEG C and 10%-20%When the environmental exposure of RH, absorb at most the water of its weight 15% or preferably absorb at most its weight10% or the dehumidizer of the water of its weight 5%-10% residual moisture water can be provided in reagent compositionFlat, make enzyme maintain its activated state. By contrast, by powerful dehumidizer (as molecular sieve)Make the over-drying enzyme deactivation that causes of described reagent composition. Weak dehumidizer is by only working asWhen humidity level in packaging exceedes 20%RH, just from environment, absorb water, can balance be used for testThe demand that amboceptor in the container of sensor and enzyme are contrary to moisture. Therefore, weak dehumidizer canProtection amboceptor avoids high-moisture and can not produce adverse effect to enzymatic activity.
Fig. 7 has described and has represented that the biology of the analyte concentration in use test sensor working sample passesThe schematic diagram of sensor 700. Described bio-sensor system 700 comprises measurement mechanism 702 and testSensor 704, described measurement mechanism 702 can any analytical instrument (comprise desktop apparatus, portableFormula or hand held device) form use. Can utilize described biology sensor 700 to carry out determination and analysisThe concentration of thing (comprising glucose, uric acid, lactate/ester, cholesterol, bilirubin etc.). AlthoughShow a kind of concrete structure, but biology sensor 700 can have other structure, comprises and havingThe biology sensor 700 of additional components.
Described testing sensor 704 has pedestal 706, and described pedestal 706 forms has opening 712Passage 710 and holder 708. Described holder 708 and described passage 710 can be by having apertureLid hide. Described holder 708 limits partially enclosed cavity (volume). Described storageDevice 708 can comprise composition (as water-swelling polymer or porous polymer matrix), described compositionAssist liquid hold-up sample. Reagent can be deposited in described holder 708 and/or passage 710. ?The reagent composition at working electrode 707 places comprises the low reagent composition of total salt amount, and can comprise oneThe substance classes such as kind or plurality of enzymes system and amboceptor. Can use identical or different reagent composition (excellentChoosing lacks the reagent composition of enzyme system) carry out shape paired electrode 705. Described testing sensor 704Also can have sample interface 714, described sample interface 714 is provided to contiguous described holder708 places. Described sample interface 714 can part around or Perfect Ring around described holder 708. InstituteState testing sensor 704 and can there is other structure.
Described sample interface 714 has and is connected to described working electrode 707 and described to electrode 705Conductor 709. Described electrode can be substantially in same plane or in more than one plane. DescribedElectrode 705,707 can be provided on pedestal 706 surfaces that form described holder 708. Described electricityThe utmost point 705,707 is extensible or stretch into (projectinto) described holder 708. Dielectric layer canPartly cover described conductor 709 and/or described electrode 705,707. Described sample interface 714Can there is other electrode and conductor.
Described measurement mechanism 702 comprises the electricity that is connected to sensor interface 718 and display 720Road 716. Described circuit 716 comprises and is connected to signal generator 724, optional temperature sensor 726Processor 722 with storage medium 728.
As the response to described processor 722, described signal generator 724 provides electricity input letterNumber to described sensor interface 718. Can be by electrical input signal by described sensor interface 718Transfer to sample interface 714, thereby electrical input signal is applied to biologicfluid sample. Electricity inputSignal can be electromotive force or electric current, and can apply with multiple-pulse, sequence or endless form. Described letterNumber generator 724 also can record the signal being received by described sensor interface place, thereby as sending outRaw device-logger.
Described optional temperature sensor 726 has been measured the sample in the holder of testing sensor 704Product temperature. Can draw in the following manner sample temperature: measure, calculate or suppose by output signalFor being equal to or being similar to the environment temperature of measurement or using the temperature of the device of described biology sensor.Can use thermistor, thermometer, infrared sensor, thermoelectric pile or other temperature sensing device to surveyMeasure described temperature. Can be by other technology for working sample temperature.
Described storage medium 728 can be magnetic storage, optical memory or semiconductor memory,Other storage device etc. Described storage medium 728 can be remote access mobile storage means(as storage card), fixed-storage device etc.
Described processor 722 uses computer readable software code and is stored in described storage mediumData in 728 are implemented analyte analyzation and data processing. Described processor 722 can respond described biographyThe existence of testing sensor 704 on sensor interface 718, the sample carrying out to testing sensor 704Product apply and user inputs etc. and initial analyte analyzation. Described processor 722 instructs described letterNumber generator 724 provides electrical input signal to described sensor interface 718. Described processor 722Can receive the sample temperature from optional temperature sensor 726. Described processor 722 receivesFrom the output signal of described sensor interface 718. At the analyte in described holder 708The response of redox reaction in generate described output signal.
Described processor 722 is preferably measured output signal to obtain self-excitation (this rising in excitingBeginning current value is higher than the current value in decay subsequently), in described testing sensor 704Introduce the current value in the time that is less than about 3s after sample. More preferably, described processor 722Measure output signal and introduce sample in described testing sensor 704 after, be less than about 3s to obtainTime in current value; And obtain the first current value from instigated recordings, wherein, continue firstCurrent value after current value constantly reduces. Even more preferably, described processor 722 is measured defeatedGoing out signal is less than in the time of about 3s introduce sample in described testing sensor 704 after to obtainCurrent value; Obtain the first current value from instigated recordings, wherein, the electricity after the first current valueFlow valuve constantly reduces; And obtain the electricity during the maximum power performance of described testing sensorFlow valuve.
Use the one or more correlation equations in described processor 722 to make one or more obtainingCurrent value relevant to the analyte concentration of sample. The result of analyte analyzation can be output to demonstrationDevice 720 also can be stored in described storage medium 728. Preferably, the result of analyte analyzationIntroduce sample in testing sensor after, in 5s or shorter time, be output to described display720; More preferably, described result 3s or shorter time introduce sample in testing sensor afterInside be output to described display 720.
The correlation equation that relates to analyte concentration and output current value can be by graphical method, mathematics sideThe expressions such as method or its combination. Described correlation equation can be by being stored in described storage medium 728The expressions such as program number (PNA) table or other question blank. Can be by being stored in described storage medium 728In computer readable software code provide about implementing the description of analyte analyzation. Described codeCan be object identification code or description or other arbitrary code of controlling function as herein described. From dividingAnalyse the data of thing analysis and can accept one or more data processings, be included in described processor 722Measure decay rate, K constant, ratio etc.
Described sensor interface 718 has the sample interface 714 with described testing sensor 704In conductor 709 connect or the contact of electric connection. Described sensor interface 718 touches by describedHead transfers to the electric excitation signal from described signal generator 724 in sample interface 714Conductor 709. Described sensor interface 718 also by described contact by the output signal from sampleTransfer to described processor 722 and/or described signal generator 724.
Described display 720 can be simulation or digital. Described display can be LCD,LED, OLED, TFT or other are suitable for showing the display of reading.
In use, by introduce sample in described opening 712, will turn for the sample of analyzingMove in described holder 708. Described sample flows through passage 710, fills described holder 708Discharge contained before this air simultaneously. Described sample be deposited on described passage 710 and/or holderReagent generation chemical reaction in 708. Preferably, described sample is fluid, more preferably liquid.
Described testing sensor 704 is provided as and is adjacent to described measurement mechanism 702. In abutting connection with comprising instituteState the position of sample interface 714 and described sensor interface 718 electric connections. Electric connection comprisesConductor in contact in described sensor interface 718 and described sample interface 714 709 itBetween input and/or the wired or wireless transfer of output signal.
Fig. 8 has described the bio-sensor system 800 that comprises container 810, and described container 810 comprisesDehumidizer and multiple testing sensor 830. Described container 810 comprises closure member 812, described sealingPart 812 can be sealed in described testing sensor 830 in container 810. Described container 810 can wrapContaining the dehumidizer 820 in the independent packaging in this container. Described container 810 can comprise in instituteState the dehumidizer 822 in closure member 812. Described container 810 can comprise the dehumidifying in chamber wallAgent 824. Described container 810 can comprise the dehumidizer 826 in this container base. Described container810 can be made of a variety of materials, and comprise plastics, metal forming and/or glass. Can select in describedThe amount of the dehumidizer in container 810 and type, thus predetermined moisture level is provided in this container.
Although described numerous embodiments of the present invention, those of ordinary skill in the art comeSay and it is evident that, other embodiment and occupation mode also may be within the scope of the invention.Therefore, the present invention is only limited by appending claims and equivalent thereof.