CN106053585A - Detection method for judging sample distribution status - Google Patents

Abstract

The invention relates to a detection method. The method is characterized in that a working voltage is provided between the first electrode and the second electrode of an electrochemical sensor to obtain a first current value and a second current value, wherein a ratio of the first current value to the second current value can represent the full distribution rate of samples fully distributed on the surface of the first electrode and the surface of the second electrode, and is used to judge the validity of test.

Description

A kind of method for detecting of judgement sample coverage status

The application is the Application No. 200910141438.0 submitted on May 12nd, 2009 and invention entitled " one is sentenced The method for detecting of disconnected sample coverage status " the divisional application of patent application.

Technical field

Present disclosure generally relates to a kind of for judging sensor test effectiveness method, espespecially a kind of through a series of current values Acquired ratio, uses understanding one sample to be tested distribution scenario on the electrode of an electrochemical sensor, and judges this electricity The method of the test validity of chemical sensor.

Background technology

Electrochemical sensing system (Electrochemical Sensor Systems) has been widely used in life at present In the analysis detection of thing sample analyte, such as, detect the concentration of glucose in blood, cholesterol concentration etc..In general, This electrochemical sensing system comprises a sensing test piece and a measuring instrument, particularly this sensing test piece and is designed to single time make With, jettisonable, use for common people's life at home is convenient.

Electrochemical sensor uses ferment current methods (enzymatic amperometric methods) to be the most general Time, this kind of sensor has the electrode covering the reagent containing ferment, this reagent and analyte response and produce an electrochemical source of current, And can be sensed by this electrode.The ferment tool unicity used, and have good special with the specific analyte in sample to be tested One property reaction, the reaction of this species specificity can reduce the interference of other analyte.During such as cholesterol concentration in test sample, can Use the reagent containing the ferment to cholesterol tool specificity, and when measuring the concentration of glucose in blood, then can use containing Fructus Vitis viniferae The reagent of carbohydrate oxidase (glucose oxidase).Glucoseoxidase will not with cholesterol effect, also will not with in blood Other saccharide effect.In general, glucoseoxidase to the glucose single selective in sample up to 99%, the most this The measurement system utilizing ferment method can produce a measurement result the most accurately.

Measuring in sample in the method for analyte concentration in the way of measuring sensing electric current, this sensing electric current is referred to as Ke Te Leier electric current (Cottrell current), and can draw by below equation:

I (t)=K.n.F.A.C.D0.5.t-0.5；

Wherein, i is the instant value of sensing electric current；

K is a constant；

N is the quantity of electron transmission；

F is Faraday constant；

A is the surface area of working electrode；

C is the concentration of test analyte in sample；

D is the diffusion coefficient of reagent；

T is the special time that a predeterminated voltage applies to electrode.

It is said that in general, on the structure and measuring program of known discard type electrochemical sensing test piece, comprise as follows:

1. a base sheet (Base sheet) is with being the mechanism carrying this test piece；

The most at least two separate conductive electrodes be placed on this base sheet, wherein the first end of the first conductive electrode as One " working electrode (Working Electrode) ", and the second end of the first conductive electrode is as an extraction of this working electrode End, the exit of this working electrode for doing contacting of electricity with a scale (measuring meter): another second conductive electrode First end is as one " to electrode (Counter Electrode) ", and the second end of the second conductive electrode is as pair of electrodes Exit, this is used for doing contacting of electricity with this scale to the exit of electrode.Electrode is joined in mechanism by this working electrode with this It is placed in the region closed on, to form an electrode test district；

3. this electrode test district coated by a chemical reagent containing ferment, for treating that with one certain analyte of fluid measured produces One chemical reaction；

4. by this scale apply a running voltage (Working voltage) in working electrode and to electrode between, this work For allowing this chemical reaction work in oxidation, (running voltage now putting on working electrode is with polarity of voltage to make voltage swing Just) or the duty of reduction (running voltage now putting on working electrode be negative), reaction of oxidation (or reduce) at this Under operating voltage state, measuring the electrochemical source of current of this chemical reaction, it is a Ke Teleier electric current (Cottrell current)；

5. by the electrochemical source of current of this measurement, further in accordance with Ke Teleier current formula (i (t)=K.n.F.A.C.D0.5.t- 0.5), can be calculated this and treat the concentration of certain analyte in fluid measured.

Wherein, " working electrode " should above be coated with the chemical reagent containing ferment, be used for and certain analyte treated in fluid measured Produce chemical reaction, and in chemical reaction process, apply the running voltage (containing voltage swing and polarity of voltage) of a reaction in this On the surface of electrode, this chemical reaction is made to work in zoneofoxidation (or reducing zone) in order to the electrochemistry electricity measuring in this chemical reaction Stream, the electrochemical source of current of this zoneofoxidation (or reducing zone), it is Ke Teleier electric current (Cottrell current)；And this one " To electrode " for producing opposing current loop when measuring electrochemical source of current.

The selection of the running voltage of this reaction, can be by cyclic voltammetry curve (cyclic known in electrochemistry Voltammograms) learn appropriate oxidation or reduction potential, describe in detail as follows:

1. change and circulate the size of voltage of this working electrode, to measure electric current during different operating voltage, can obtain Shown in the I point of one oxidation wave peak current such as Fig. 1 (A), the voltage swing when this oxidation wave peak current, it is one the sensitiveest " Oxidation running voltage " VI, optimal by obtaining in this peak current point " signal noise ratio (S/N Ratio) ", this operating point is then There is optimum oxidation reaction operating potential, one of optimal oxidation Ke Teleier electric current (Cottrell current) II can be obtained, And this signal noise ratio is above or equal to one.Otherwise, if shown in the II point of Fig. 1 (A), just cannot have optimum signal noise One of ratio oxidation Ke Teleier electric current (Cottrell current).

The most identical in the peak current point gone back in virgin curve (the III point with reference to Fig. 1 (A) is shown), one can be obtained the cleverest Quick " reduction running voltage " VIII, this operating point is optimum response operating potential, can obtain one of tool optimum signal noise ratio Reduction Ke Teleier electric current (Cottrell current) IIII, and this signal noise ratio is also above or equal to one.

Select the working electrode voltage of appropriate voltage polarity and voltage swing the most from the above mentioned, to measure certain point in body to be measured The Ke Teleier electric current (Cottrell current) of analysis thing and the chemical reagent electrochemistry in time aoxidizing (or reduction).

From Ke Teleier electric current (Cottrell current) formula, the concentration C of determinand just becomes with sensing electric current i Ratio, therefore the concentration C of a test analyte can be calculated by the size of measure electrochemical current i (t).Because this measurement electricity Stream i (t) also surface area A with working electrode is directly proportional, therefore this concentration computing formula system is fixing with the area of working electrode Premise is for assuming.So, the more precise definition of working electrode surface area A should be " in measuring this electrochemical source of current " at that time " work Electrode surface area A is fixing ", therefore condition of further explaining is that " the surface area A of working electrode, need to be by this in measuring at that time Treat that fluid measured is covered with completely, to guarantee that this working electrode surface area A is fixing ", if working electrode surface area A is because of the most tested Examination fluid is covered with, then this test analyte calculated by above-mentioned Ke Teleier electric current (Cottrell current) formula Concentration C can be wrong.

Thereby, the concentration C of determinand can be detected and obtain, and this concentration C is directly proportional to sensing electric current i.It addition, because Sensing electric current is also directly proportional with working electrode surface area A, therefore for one accurately measuring instrument, and explication in sensing test piece Working electrode surface area be also a key factor.

Judge object under test accumulated amount in electrochemical sensor reaction zone the most enough, be then that another is to be measured with accurately measurement The key factor that substrate concentration is relevant.When there are enough object under test accumulated amounts the electrochemical sensor reaction zone covering reagent, Sensing electric current can be measured according to Ke Teleier electric current (Cottrell current) formula, use and extrapolate testing concentration；But It is when object under test accumulated amount deficiency, will be the most incorrect according to the extrapolated testing concentration of sensing electric current of formula gained. Therefore, when being precisely controlled working electrode surface area, determinand volume is enough in reaction zone, then become epochmaking One of key factor.

Sensor like this and measuring instrument in patent document such as: US 5,266,179, US 5,366,609 or EP12728331 is disclosed for.

Measuring instrument operational approach disclosed in above-mentioned file is roughly the same.First, a sensing test piece is inserted to measuring In instrument, sensing test piece the most suitably inserts among measuring instrument, is to be come by mechanical type and/or the switch of electronic type and/or contact Detecting.After sensing test piece is suitably inserted, user is required to provide sample, and typically one bleeds.Blood sample is then Entering a reaction zone of sensing test piece, this reaction zone has at least two electrodes, and this electrode is covered by suitable reagent place.

In order to whether inspecting samples occurs in this reaction zone, when sensing test piece is suitable upon insertion, i.e. apply a voltage to being somebody's turn to do Electrode, between electrode, the resistance of reagent is the highest when not having sample to occur, but if sample haptoreaction district at the beginning, between electrode Resistance will decline.One electric current can be detected in the decline of this resistance, to be present in a finger of reaction zone as sample Show,

In order to be explained in more detail the detection method of known techniques, refer to Fig. 1 (B) to Fig. 1 (C).

The method that the measuring instrument of Fig. 1 (B) display known techniques is used, is also United States Patent (USP) US 5, disclosed by 366,609 Content.Fig. 1 (C) is then shown in sample and detects the interim electric current produced because applying a voltage and the enlarged drawing of scope S.

According to Fig. 1 (B) Suo Shi, when sensing test piece was inserted in measuring instrument in the time 100, there is the detecting phase 101 in sample Apply a fixed voltage 102, to detect whether a sample to be tested is present in reaction zone.A droplet sample is added in sense in the time 108 On test sheet.

Please refer to Fig. 1 (C), when electric current reaches a sample detecting door 112, and namely a sample is in time 116 quilt In the presence of detecting, start sample size retardation period 114.Enough in order to continue confirmatory sample amount, measuring instrument may proceed to Apply this fixed voltage 102 to electrode, until a time point 103 (referring to Fig. 1 (B)).

Then, the magnitude of current 109 contrasts with a sample size door 113 of time point 115, defines this sample size retardation period The terminal of 114.If current intensity is less than this sample size door 113, measuring instrument will send in warning expression sensing test piece and deposit Sample size not enough, then measuring process will stop.

If there being enough sample sizes to be present in reaction zone, namely at time point 115 magnitude of current more than this sample Amount door 113, measuring instrument will continue to next step, namely stands the beginning of phase 105.In the standing phase 105, measuring instrument closes Close this fixed voltage 102 and change into no-voltage 104.During standing the phase 105, it is special that needs one are dissolved in the mixing of sample and reagent Time fixed, that preset.After completing to stand, measuring instrument will start a measurement phase, measures purpose for this, detects the side phase in one 106 apply predeterminated voltage 107 a to electrode, and the interelectrode magnitude of current 110 (referring to Fig. 1 (B) lower section) is by measured.

The decision of test analyte concentration, is according to aforesaid Ke Teleier electric current (Cottrell current), is surveying The concentration value that the amount phase 106 calculates according to Ke Teleier current formula can be shown on the display of measuring instrument.

Therefore the size definition of sample detecting door 112 is the most particularly important for judgment sample amount.

Duty is event, and applicant, in view of the disappearance produced in known techniques, is through concentrated test and research, and one is carved And the spirit do not given up, visualize this case " method for detecting of a kind of judgement sample coverage status " eventually, below for this case briefly Bright.

Summary of the invention

The purpose system of this case is for providing a kind of electrode table being covered in electrochemical sensor for detecting fluid testing sample Area be covered with rate, its can in formal measure testing sample concentration before or after measuring testing sample concentration, do this with the present invention It is covered with the judgement of rate, in order to judge the effectiveness of this test.Detecting phase applying one reaction DC voltage is there is to extremely at sample Having the first electrode and the electrochemical sensor of the second electrode less, this applying response voltage size system defines according to cyclic voltammetry Go out to make sample generation electrochemical action to produce oxidation (reduction) voltage of optimum oxidation (reduction).

For reaching above-mentioned purpose, the present invention provides a kind of method for detecting judging testing sample one coverage status, is application In at least having one of one first electrode and one second electrode sensor, its step includes: (a) provides this testing sample so that it is This second electrode is flowed to from this first electrode；(b) in one first set time, apply one first reaction DC voltage in this first Between electrode and this second electrode, make the current potential of this first electrode current potential higher than this second electrode, and record this first when fixing Between one of produced a Cottrell current value；C () removes this first reaction DC voltage and stagnates a first period；(d) In one second set time, apply one second reaction DC voltage between this first electrode and this second electrode, this second reaction The magnitude of voltage of DC voltage is equal with the magnitude of voltage of this first reaction DC voltage, and make the current potential of this second electrode higher than this The current potential of one electrode, and record this second set time and one of produced the 2nd Cottrell current value；(e) remove this second Reaction DC voltage also stagnates a second phase；(f) repeat step (b) to step (e) at least twice, and distinctly add up this first Cottrell current value and the 2nd Cottrell current value；And (g) calculates this Cottrell current value that adds up and tires out with this Adding one of the 2nd Cottrell current value ratio, wherein this ratio reflects that this testing sample is at this first electrode and this second electrode This coverage status.

The present invention repeats the first reaction DC voltage and the second reaction DC voltage applied, and will make the first electrode electricity respectively Position is more than the first electrode potential more than the second electrode potential and the second electrode potential, and by its produced Cottrell electricity Stream and the 2nd Cottrell electric current calculate its ratio after adding up respectively, and enough in order to judgment sample, the method can overcome Known techniques is with the shortcoming setting a sample detecting door electric current.

According to said method, wherein this first reaction DC voltage and the second reaction DC voltage system are bent through cyclic voltammetric Line (cyclic voltammograms) determines, and this first reaction DC voltage is made an uproar with this second signal reacting DC voltage Acoustic ratio (S/N Ratio) is more than or equal to 1.

According to said method, wherein this first electrode and this second electrode are positioned at same base version.

According to said method, wherein on this first electrode and this second electrode, there is a ferment and an electron transmission intermediary Thing, wherein this ferment carries out oxidation reaction to this testing sample.

According to said method, wherein on this first electrode and this second electrode, there is a ferment and an electron transmission intermediary Thing, wherein this ferment carries out reduction reaction to this testing sample.

According to said method, wherein this first set time is 3ms to 2s.

According to said method, wherein this second set time is 3ms to 2s.

According to said method, wherein this first set time is identical with this second set time.

According to said method, wherein this first set time and this second set time are 20ms.

According to said method, wherein this first period is 0ms to 50ms.

According to said method, wherein this second phase is 0ms to 50ms.

According to said method, wherein this first period and this time second phase are identical.

According to said method, wherein this first period and this second phase are 20ms.

According to said method, wherein this first electrode is identical with the electrochemical reaction area of this second electrode.

According to said method, wherein when this ratio is 1, represent that this testing sample is covered with this first electrode and this second electricity Pole.

According to said method, wherein an electrochemical reaction area of this first electrode is more than an electrochemistry of this second electrode Response area.

According to said method, wherein an electrochemical reaction area of this first electrode is less than an electrochemistry of this second electrode Response area.

According to said method, wherein this sensor is an electrochemical sensor.

According to said method, wherein the method is in order to judge the effectiveness that one of this sensor is tested.

According to said method, wherein can there is the detecting phase in a sample in the method, a sample stands the phase or a sample is measured Interim arbitrary time point is carried out.

According to said method, when wherein this ratio is 0.3 to 3.0, this test representing this sensor is effective.

The present invention also provides a kind of method for detecting judging testing sample one coverage status, is to be applied at least have one One of first electrode and one second electrode sensor, its step includes: (a) provides a testing sample so that it is from this first electrode Flow to this second electrode；B (), in one first set time, applies one first DC voltage in this first electrode and this second electrode Between, make the current potential of this first electrode current potential higher than this second electrode, and record this first set time and one of produced first Cottrell current value；C () removes this first DC voltage and stagnates a first period；D (), in one second set time, applies One second DC voltage between this first electrode and this second electrode, the magnitude of voltage of this second DC voltage and this first unidirectional current The magnitude of voltage of pressure is equal, and makes the current potential current potential higher than this first electrode of this second electrode, and records this second set time One of produced the 2nd Cottrell current value；And (e) calculates a Cottrell current value and the 2nd Cottrell electricity One of flow valuve ratio, wherein this ratio reflects that this testing sample is at this first electrode and this coverage status of this second electrode.

According to said method, wherein this first set time is 3ms to 2s.

According to said method, wherein this second set time is 3ms to 2s.

According to said method, wherein this first set time is identical with this second set time.

According to said method, wherein this first set time and this second set time are 20ms.

According to said method, wherein this first period is 0ms to 50ms.

According to said method, wherein this first period is 20ms.

According to said method, wherein this sensor is an electrochemical sensor.

According to said method, wherein the method is in order to judge the effectiveness that one of this sensor is tested.

According to said method, when wherein this ratio is 0.3 to 3.0, this test representing this sensor is effective.

According to said method, wherein the method can be to be measured in a testing sample detecting phase, a testing sample standing phase or one Sample is measured interim arbitrary time point and is carried out.

The present invention must by the following example and illustrate, in order to must deeper into understanding.

Accompanying drawing explanation

Fig. 1 (A) is a cyclic voltammetry curve；Fig. 1 (B) is the schematic diagram of known measuring instrument institute using method；Fig. 1 (C) is then Enlarged drawing for Fig. 1 (B)；

Fig. 2 (A) is the schematic appearance of the measuring instrument 10 of the present invention；Fig. 2 (B) is then after electrochemical sensing test piece 20 amplifies Front view and rear view；Fig. 2 (C) is the inside circuit diagram of known measuring instrument；

Fig. 3 (A) is the electrochemical sensing test piece 20 cross section view along A-A ' section line；Fig. 3 (B) to Fig. 3 (E) is then sample 29 at the schematic diagram of capillary tube 23 flowing；

Fig. 4 (A) to Fig. 4 (C) is the internal circuit schematic diagram of the measuring instrument 40 of the present invention；

Fig. 5 (A) to Fig. 5 (G) is that sample 29 flows and is covered with the schematic diagram of electrode；

Fig. 6 (A) to Fig. 6 (D) is the present invention at sample 29 to flow and is covered with measured electric current and electricity during electrode Pressure schematic diagram；The circulation volt that Fig. 6 (E) to Fig. 6 (I) is measured during being then covered with electrode for the present invention flows at sample 29 Peace curve；

Fig. 7 (A) and another internal circuit schematic diagram of measuring instrument 40 that Fig. 7 (B) is the present invention；

Fig. 8 is another internal circuit schematic diagram of the measuring instrument 40 of the present invention；

Fig. 9 (A) is another embodiment of electrochemical sensing test piece of the present invention；Fig. 9 (B) is then the electrochemical sensing of Fig. 9 (A) Test piece regards along the section of B-B ' section line；

Figure 10 (A) is another embodiment of electrochemical sensing test piece of the present invention；Figure 10 (B) is the electrochemistry sense of Figure 10 (A) The exploded view of test sheet；Figure 10 (C) is that the electrochemical sensing test piece of Figure 10 (A) regards along the section of C-C ' section line；Figure 10 (D) is then Enter for sample the electrochemical sensing test piece of Figure 10 (A) schematic diagram and

Figure 11 (A) is another embodiment of electrochemical sensing test piece of the present invention；Figure 11 (B) is the electrochemistry sense of Figure 11 (A) The exploded view of test sheet；Figure 11 (C) then electrochemical sensing test piece for Figure 11 (A) regards along the section of D-D ' section line.

Primary clustering symbol description

100, there is the detecting phase in 108,116 time 101 samples

102 fixed voltage 103,115 time points

104 no-voltages 105 stand the phase

106 detect side phase 107 default voltage

109,110 magnitude of current 112 sample detecting door

113 sample size door 114 sample size retardation periods

10 measuring instrument 11 slots

12 display 20 electrochemical sensing test pieces

21,22,91,92 electrode 23 capillary tube

24,25 contact 26,1009 sample entrance port

27 cover plate 28 pore

29,1008 sample 210,1010 groove

211,212 electrode top 213 perforation

214 reagent 41,701 microprocessors

42 display 43 power-supply units

44 current measuring unit 45 electric currents

46 current-to-voltage convertor 47 analog-digital converters

48 current buffer 49 voltage regulators

410 temperature measurement units 411, Vwc electrode running voltage

Detecting unit SW, S1, S2, S3, S4 switch is inserted in 412 test pieces

415,715,815 voltage switching cell arrangement

413,414, Vc1, Vc2 contact

416,716 control point 417 reverser

420 switching switches set Vo analog voltages

Vw, Vc, Vx, Vy, Vr, Vref current potential

Vxy voltage difference R1, R2 resistance

X, Y output point t0～t8 time

Ixa, Iya, Ixb, Iyb, Ixc, Iyc, Ixd, Iyd current value

93 the 3rd electrode 1104 the 3rd membrane electrodes

1001,1101 membrane electrode sensing test piece

1002,1003,1102,1103,1104 membrane electrode

A-A ', B-B ', C-C ', D-D ' section line

Detailed description of the invention

The offer one of this case is used for detecting fluid testing sample and is covered in electrochemical sensor electrodes surface area and is covered with rate With the method judging test validity, can be illustrated by below example and be fully understood, and make to be familiar with this skill Personage can complete it according to this, but the enforcement kenel of this case is not restricted in the following example.

Referring to Fig. 2 (A), it is the schematic appearance of a measuring instrument 10 of use electrochemical sensing test piece of the present invention. Measuring instrument 10 comprises a shell with a display 12, in order to show measurement result, and includes a slot 11, in order to insert one Electrochemical sensing test piece 20.Fig. 2 (B) then regards for the front view (Fig. 2 (B) is left) after electrochemical sensing test piece 20 amplification and the back side The schematic diagram of figure (Fig. 2 (B) is right), wherein electrochemical sensing test piece 20 further includes electrode 21 and 22.

Fig. 2 (C) is the schematic diagram of known measuring instrument, and this measuring instrument 10 comprises microprocessor 13, display 14, electricity Source feeding unit 15, one current measuring unit 16, electric current 17, current-to-voltage convertor 18, analog-digital converter 19, One current buffer 120, voltage regulator 121, temperature measurement unit 122 and have one of switch SW test piece insert Entering detecting unit 124, wherein current-to-voltage convertor 18 is contained among current measurement unit 16, in order to by electrode 21,22 it Between electric current 17 be converted into an analog voltage Vo (wherein Vo=I × Rf), then through analog-digital converter 19 by this simulation electricity Pressure Vo changes into the digital equivalents of voltage, calculates for microprocessor 13.And voltage regulator 121 and resistance R1, R2 institute Constitute potentiometer, in order to apply voltages to contact Vc1, current buffer 120 then has a high current drive capability, in order to The current potential that contact Vc2 output is identical with contact Vc1.Now the current potential of contact 125 is Vw, and the current potential of contact 126 is Vc, one Electrode running voltage 123 is Vwc, that is equal to the potential difference between Vw and Vc.This electrode running voltage 123 is applied in contact 125, between 126, and the output contact 24 and 25 of sensing test piece 20 can be connected to.

Please continue refering to Fig. 3 (A) to (E), it is that sample to be tested flows into electrochemical sensing test piece 20, and be covered with in electrode 21 and The schematic diagram of 22 processes.

Fig. 3 (A) is the electrochemical sensing test piece 20 cross section view along A-A ', and electrochemical sensing test piece 20 comprises a capillary tube 23, output contact 24,25, sample entrance port 26, covers table on plate 27, pore 28, sample 29, groove 210, electrode Face 211 and 212, perforation 213 and reagent 214.Wherein electrode 21,22 is arranged in the groove 210 of electrochemical sensing test piece 20 Perforation 213 in.The surrounding of electrode 21,22 by perforation 213 close around and be formed without any breach.The diameter of perforation 213 It is designed to the diameter of slightly smaller than electrode 21,22, makes electrode 21,22 mechanically can be caught in perforation 213.

The upper surface 211,212 of electrode 21,22 forms the work area of electrode, and the size of upper surface 211,212 is permissible Being same to each other or different to each other, the lower end of electrode then forms the output contact 24,25 of sensing test piece 20, and these output contacts can be distinguished It is connected with the contact 125,126 of measuring instrument 10 in Fig. 2 (C).And hydrophilic covers plate 27 and has the pore 28 communicated with the external world, and Covering groove 210 is to form capillary tube 23, and this capillary tube 23 defines a reaction zone, it is provided that reagent 214 is coated on groove 210 In, and cover the electrode top 211,212 of electrode 21,22.Reagent 214 comprises known oxidation or a reduction enzyme such as Glucoseoxidase, an electron transmission mediator such as potassium ferrocyanide (Fe (CN) 63-), and some hydrophilic chemicals Matter.The emphasis of composition part non-invention for known techniques of reagent.In addition this sensing test piece 20 more provides sample entrance port 26, uses To insert sample 29, such as one bleeds.

Refer to Fig. 3 (B), when sample 29 is put the opening of sample entrance port 26, because capillarity or hydrophilic work With, this drop of blood can be inhaled into capillary tube 23 automatically.Fig. 3 (B) to Fig. 3 (E) then shows the sample 29 flowing in capillary tube 23 Situation.When enough samples 29 are dropped into sample entrance port 26, will start along capillary tube 23 as shown in Fig. 3 (C) and (D) Flowing, until electrode being completely covered as shown in Fig. 3 (E), now the air in capillary tube 23 then can be discharged via opening 28.? In Fig. 3 (B), owing to sample 29 not yet flows to electrode 22, though therefore having applied voltages to electricity when sample exists the detecting phase 101 Extremely go up, but carry on as before and do not turn on so not yet producing electric current.At Fig. 3 (C), sample 29 has been completely covered electrode 21 and part covers electricity Pole 22, now applies a voltage on electrode, it will have electric current to produce, and measuring instrument 10 i.e. needs to judge that this electric current has arrived sample the most Detecting door 112, therefore the definition of sample detecting door 112 is particularly important.Can substantially be found by Fig. 3 (C), now sample 29 is also Electrode 22 is not completely covered, if sample detecting door 112 defines too small, then measuring instrument 10 may be made to judge by accident, and start to stand the phase 105 to measuring the step of phase 106, and the concentration of specimens of such gained is the most incorrect；Otherwise, if sample detecting door 312 defined Greatly, there is different component contents, such as hematocrit ratio (HCT), oxygen content, concentration of glucose or fat in blood because of different samples The difference of fat content, such as Fig. 3 (D) or (E), though sample 29 has been almost completely filled with in groove 210, but there is the detecting phase in sample The electric current that 101 are produced possibly cannot exceed sample detecting door 112 so that measuring instrument 10 cannot be carried out standing phase 105 to survey The step of amount phase 106.Therefore it provides one can correctly judge that whether enough object under test accumulated amount in reaction zone, with obtain effectively it The method of sensing electric current, particularly important for this type of measuring instrument.

Referring to Fig. 4 (A), it is the schematic diagram of measuring instrument 40 of use electrochemical sensing test piece 20 of the present invention, needs spy Not it is emphasised that, although Fig. 4 (A) and measuring instrument 40 described thereafter are identical with known person with electrochemical sensing test piece 20 outward appearance, Only the present invention is on the internal circuit and method for measurement thereof of measuring instrument 40, compared to the progress the most significantly of front case.As for Fig. 4 (A) electrochemical sensing test piece 20 in, describes in detail in Fig. 2 (A) to (E) and corresponding explanation thereof, therefore will be the heaviest in this Multiple.

Please continue refering to Fig. 4 (A), this measuring instrument 40 comprises microprocessor 41, display 42, power-supply unit 43, current measuring unit 44, temperature measurement unit 410, have one of switch SW test piece insert detecting unit 412, with And a voltage regulator 49, current measurement unit 44 comprises a current-to-voltage convertor 46, in order to by between contact 413,414 one Electric current 45 is converted into an analog voltage Vo (wherein Vo=I × Rf), then through an analog-digital converter 47 by this analog voltage Vo changes into the digital equivalents of voltage and calculates for microprocessor 41.Voltage regulator 49 and resistance R1, R2 are constituted Potentiometer, in order to apply voltages to contact Vc1, a current buffer 48, there is a high current drive capability, in order at contact Vc2, exports a current potential identical with contact Vc1.Now the current potential of contact 413 is Vw, and the current potential of contact 414 is Vc, an electrode Running voltage 411 is that Vwc (potential difference equal between Vw and Vc) is therefore applied between contact 413,414.

Measuring instrument schematic diagram used in the present invention shown in Fig. 4 (A), in addition to voltage switching cell arrangement 415, its Its part is designed as known techniques with circuit.

Voltage switching cell arrangement 415 comprises the switching switch being made up of S1, S2, S3 and S4 tetra-switch (Switches) Group 420, these four switches can be by known mechanical relay (Relay) or the analog switch (Analog of electronic type IC type Switch) bridge switch or with electronic type transistor (MOSFET or Bipolar transistor) formed is to reach handoff functionality. And voltage switching cell arrangement 415 further includes a control point 416, in order to transmit the digital control news spread out of by microprocessor 41 Number, it is controlled by switching Guan Bi or the open circuit of S1 and the S4 switch in switches set 420；Wherein, when the signal at control point 416 it is When 1, then S1 and S4 switch is closed at connecting；Otherwise, when the signal at control point 416 is 0, then S1 and S4 switch is opened a way simultaneously Isolation.Voltage switching cell arrangement 415 further includes a reverser 417, and this is the basic composition of digital circuit (logic circuit) One, in order to by reverse for input signal, for binary logic, when inputting 0, it is output as 1, otherwise, when input is 1, then Being output as 0, it is for controlling Guan Bi or the open circuit of S2 and the S3 switch in this switches set 420, and allows closing moment of S2 and S3 Just contrary with the moment of S1 and S4, these two groups switches can only select one group to be closure state in synchronization.This voltage switching The control of cell arrangement 415 is described as follows:

Such as Fig. 4 (B), when microprocessor 41 gives control point 416 1 digital signal 1,

Now output point X connects with contact 413 via the Guan Bi of S1 and current potential is identical, the current potential of output point X and contact 413 It is respectively Vx and Vw；

And output point Y connects with contact 414 via the Guan Bi of S4 and current potential is identical, output point Y divides with the current potential of contact 414 Wei Vy and Vc；

Now Vx=Vw and Vy=Vc, therefore potential difference between two output point X and Y, be the potential difference of contact 413 and 414 411 (Vwc),

Because of Vx > Vy, so time the electrode 21 that is connected with output point X be working electrode.

For another Fig. 4 (C), when microprocessor 41 gives control point 416 1 digital signal 0,

Now output point X connects with 414 via the Guan Bi of S2 and current potential is identical, and the current potential of output point X and contact 414 is respectively For Vx and Vc；

And output point Y connects with 413 via the Guan Bi of S3 and voltage is identical, the current potential of output point Y and contact 413 is respectively Vy and Vw；

Now Vx=Vc and Vy=Vw, and between two output point X and Y, potential difference 411 is still Vwc,

So because of Vx < Vy, so time the electrode 22 that is connected with output point Y be working electrode.

Fig. 5, Fig. 6 are one of the measuring method according to present invention embodiment, and wherein Fig. 5 is the enlarged partial top of Fig. 2 (A) Figure, and carried out measurement instrument operates in respect of with purgation step (please refer to Fig. 2 (A) and Fig. 4 (A) to (C)).

(1) insert the slot 11 of electrochemical sensing test piece 20 to measurement instrument 10, by activate switch 412, make microprocessor 41 Start the cycle over sends the signal of 1 and 0 to control point 416.Now output point X and output point Y voltage are respectively such as Fig. 6 (A) and Fig. 6 (C)。

(2) then display 12 by display request supply sample 29, be typically a drop of blood sample, be also in each figure of Fig. 5 it Sample 29.

(3) (referred to Fig. 2 (A) or Fig. 5 (A)) when sample 29 puts sample entrance port 26 because capillarity or Hydrophilic interaction, this sample 29 can be automatically drawn into capillary tube 23.Fig. 5 (B) to Fig. 5 (G) display drop of blood sample 29 is at capillary tube 23 Interior flow situation.

(4) now microprocessor 41 initially receives kinetic current:

A (), when the time was 0～to time, sample 29 flow condition such as Fig. 5 (B) is to Fig. 5 (C), now from output point institute The electric current received is respectively as shown in Fig. 6 (B), (D).Can clearly show that, now sample 29 not yet flows to electrode surface 212, Therefore produce without any electric current；Now utilize the cyclic voltammetry in the method for testing of electrochemistry available as shown in Fig. 6 (E) Result；

B (), when the time is to～t2, such as Fig. 5 (D), now blood sample 29 part covers electrode surface 212, therefore Fig. 6 (D) clearly shows that existing electric current produces, and is now Ixa from current value received by output point X, and receives from output point Y Current value is Iya；The most therefore time, sample 29 has been completely covered electrode top 211, but only part covers electrode top 212, Therefore Ixa is much larger than Iya；When time point is to～t1, electrode running voltage is Vwc, and now working electrode is electrode 21, work Electrode surface area is the electrode top 211 of electrode 21；When time point is t1～t2, electrode running voltage is still Vwc, but now Working electrode is electrode 22, and working electrode surface area is the part electrode top 212 of electrode 22；Public from Ke Teleier electric current The conclusion that formula gained electric current is directly proportional to work area, therefore will obtain the result of this smaller current Iya；Now utilize electrification The available result as shown in Fig. 6 (F) of cyclic voltammetry in method of testing；

C (), when the time is t2～t4, such as Fig. 5 (E), electric current received by output point X, Y is respectively Ixb and Iyb, wherein Ixb is about roughly equal to Ixa, and its reason is after a bit of time, the consumption of existing fraction electric current, but its value is the least negligible Disregarding, and Iyb is more than Iya, time therefore, sample 29 covers to electrode top 212 area of electrode 22 relatively big caused；Now profit By the available result as shown in Fig. 6 (G) of the cyclic voltammetry in the method for testing of electrochemistry；

(D) when the time is t4～t6, such as Fig. 5 (F), electric current received by output point X, Y is respectively Ixc, Iyc, wherein Ixc is about roughly equal to Ixb, and Iyc is more than Iyb, time therefore sample 29 cover to electrode 22 electrode top 212 area the most earlier above One time point is bigger caused；Now utilize the available knot as shown in Fig. 6 (H) of the cyclic voltammetry in the method for testing of electrochemistry Really；

(E) when the time is t6～t8, such as Fig. 5 (G), electric current received by output point X, Y is respectively Ixd, Iyd, wherein Ixd is about roughly equal to Ixc, Iyd and is more than Iyc, and Iyd is almost equal with Ixd, because now blood sample 29 has been completely covered to electricity The electrode top 212 of pole 22, and electrode top 211,212 area is identical；Now utilize in the method for testing of electrochemistry The available result as shown in Fig. 6 (I) of cyclic voltammetry.

(5), after microprocessor 41 receives electric current, i.e. start computing judgment sample 29 enough in reaction zone, have Multiple judgment mode, such as: set a time range, microprocessor 41 constantly compares from the electricity received by output point X, Y Flow valuve, (also or when Ix with Iy ratio (Ix/Iy) is less than when Ix with Iy ratio (Iy/Ix) is more than or equal to a certain predetermined ratio Or during equal to a certain predetermined ratio), next step can be carried out, be the standing phase 305 that initially enters, otherwise i.e. from display 12 Display sample 29 volume (i.e. blood volume) is not enough；Also can set a time range, all Ix with Iy values are tired out by microprocessor 41 respectively Add, and cumulative Ix with Iy value calculated, the ratio after this accumulated value is computed be more than or equal to (also or be less than or etc. In) a certain predetermined ratio time, next step can be carried out, otherwise i.e. show sample 29 volume (i.e. blood volume) no from display 12 Foot.

(6) when through microprocessor 41 judgment sample 29 in reaction zone enough, the standing phase can be carried out to the phase of measurement Standard step, and obtain a correct measurement electric current, through microprocessor 41, gained is measured in the sample after electric current is computed 29 It is intended to analyte concentration be shown on display 12.

How to determine that the ratio range of the preferable coverage status of side sample is treated in judgement, be utilize different sample volumes go into Result obtained by row experiment.It is described as follows:

Offer is applicable to one of measuring instrument sensing test piece, and this sensing test piece volume required being covered with is 0.7 μ l, and this sensing There is in test piece one first electrode and one second electrode, and this first electrode is less than this second electrode.

Making sample be flowed to this second electrode by this first electrode, this sample volume is 0.3 μ l to 0.8 μ l and carries out repeatedly real Test.

In one first set time 20ms, apply one first DC voltage 0.1V in this first electrode and this second electrode Between, make the current potential of this first electrode current potential higher than this second electrode, and record this first set time and one of produced first Cottrell current value.

Remove this first DC voltage and stagnate a first period 20ms.

In one second set time 20ms, apply one second DC voltage 0.1V in this first electrode and this second electrode Between, make the current potential of this second electrode current potential higher than this first electrode, and record this second set time and one of produced second Cottrell current value.

Calculate a Cottrell current value and one of the 2nd Cottrell current value ratio.

Each sample volume above-mentioned is all verified more than above step 10 time repeatedly, and adds up this ratio range, And calculate the degree of accuracy of a Cottrell current value, obtain a statistical table (table one), as follows:

Table one:

From upper table one, when this sample volume is too small, such as 0.3 μ l, a Cottrell current value cannot measure, Because now sample volume is not enough to flow to this second electrode from this first electrode.When sample volume is 0.4 μ l, though can measure Oneth Cottrell current value, but its current accuracy the poorest (CV > 10%).Increase sample volume to 0.45 μ l～0.8 μ l Time, the degree of accuracy of a Cottrell current value is preferable (CV < 5%), now (first/second) Cottrell current ratio model Enclosing is 0.3～1.7, otherwise (the second/the first) Cottrell current ratio scope is 0.6～3.3, therefore falls when current ratio 0.3～during 3.3 scope, sample to be tested coverage status in this sensing test piece can be represented good.

Fig. 7 (A) and (B) are another embodiment of this case, and the method used in voltage switching cell arrangement 715 is electric with Fig. 4 Pressure switching unit device 415 is different, and the voltage switching cell arrangement 715 in Fig. 7 is received by microprocessor by control point 716 701 sent orders carry out the switching between S1, S2, S3, and it is described as follows.

When S1 with S2 is connected, as shown in Fig. 7 (A), now:

Vx=Vref=V1=Vr

Vy=V2=[(R2+R3)/(R1+R2+R3)] Vr,

Vxy=Vx-Vy=Vr-[(R2+R3)/(R1+R2+R3)] Vr

=[R1/ (R1+R2+R3)] Vr；

Therefore Vx > Vy, the electrode 21 that now output point X is connected is working electrode.

And when S1 with S3 is connected, as shown in Fig. 7 (B), then:

Vx=V3=[R3/ (R1+R2+R3)] Vr,

Vy=V2=[(R2+R3)/(R1+R2+R3)] Vr,

Vxy=Vx-Vy=[R3/ (R1+R2+R3)] Vr-[(R2+R3)/(R1+R2+R3)] Vr；

=[-R2/ (R1+R2+R3)] Vr；

Therefore Vy > Vx, the electrode 22 that now output point Y is connected is working electrode.

In circuit design, when selecting R1=R2, then can make the Vxy voltage difference when S1 with S2 is connected and S1 Yu S3 phase Voltage value during connection is identical, and simply its polarity of voltage is contrary.

Mutually switch between S1 and S2, S3 according to this embodiment, it is possible to reach as shown in Figure 6, obtain the number being intended to calculate Value, and then judge that blood volume is enough.

Fig. 8 is this case another embodiment, in voltage switching cell arrangement 815 and preceding embodiment the most difference, in In this embodiment, Vx=Vr is a fixed voltage value, and voltage switching cell arrangement 815 is according to by the sent life of microprocessor 801 Order carries out digital signal and is converted into analog voltage (Digital to Analoy Voltage Converter) output to Vc1, warp Strengthened the output driving force of its electric current by current buffer OP2, now voltage is constant still for making Vy=Vc1, i.e. by microprocessor 801 sent orders carry out digital signal to adjust the voltage swing of Vy, to reach the function of voltage switching.It controls program such as Under:

The absolute value being pre-designed electrode running voltage Vxy between output point X to be put on and output point Y is Q；

In the very first time, the sent order of microprocessor 801 carry out digital signal to adjust Vc1, allow

Vc1=Vy=Vx Q；

Then Vxy=Vx Vy=Vx (Vx Q)=Q；

Now Vx > Vy, therefore the electrode 21 that output point X is connected is working electrode.

In the second time, the sent order of microprocessor 801 carry out digital signal to adjust Vc1, allow

Vc1=Vy=Vx+Q；

Then Vxy=Vx Vy=Vx (Vx+Q)=-Q；

Now Vx < Vy, therefore the electrode 22 that output point Y is connected is working electrode.

According to this embodiment, carried out digital signal in the very first time and the second time by the sent order of microprocessor 801 Change the mutual switching adjusting Vc1 magnitude of voltage, it is possible to reach as shown in Figure 6, obtain the numerical value being intended to calculate, and then judge blood Measure enough.

Through the present invention, more can work as that sample 29 enters sample entrance port 26 and microprocessor 41,701,801 receives an electricity During stream, set a sample detecting door 112 according to known techniques, after this electric current arrives this door, i.e. enter standing phase 105 to survey The standard step of amount phase 106, and in standing the phase 105 or measuring a certain time point in the phase 106, carry out above example of the present invention it Voltage switching, to obtain Ix and Iy for microprocessor 41,701,801 calculating, then carries out the calculating of above example of the present invention With the step of judgement, the most correct in order to determine the result of this measurement after date gained.

In other words, method of the present invention, in there is detecting phase 101, standing phase 105 or measurement phase 106 in sample A certain time point all can calculate, in order to judge the result of calculation effectiveness of this measurement after date.

Refer to Fig. 9 (A) and (B), for another embodiment of Fig. 2 electrochemical sensing test piece.This structure comprise electrode 91, 92, as it was previously stated, electrode 91,92 when voltage switching device start, and can become working electrode in a certain time point and produce institute Need to calculate the Cottrell electric current judged, this embodiment is more preferably containing one the 3rd electrode 93, and the 3rd electrode 93 can be One reference electrode (reference electorde).When the measuring instrument of this electrochemical sensing test piece confirms blood volume sample enough, Entering the measurement phase 106 after standing the phase 105, this reference electrode 93 can assist in the predeterminated voltage 107 of measurement phase 106 required applying More stable and a most accurate sensing electric current.

Membrane electrode sensing test piece 1001 and 1101, its molding it is respectively shown in Figure 10 (A) to (D) and Figure 11 (A) to (C) Refer to such as U.S. Patent No. 5,997,817, U.S. Patent No. 5,985,116, European EP 1,098,000 with structure Number patents etc., this membrane electrode 1002,1003,1102,1103 and 1104 etc., the available method such as screen painting, metal evaporation becomes Type, and the present invention is in application such as Figure 10 of this membrane electrode sensing test piece.As shown in Figure 10 (C), when in providing a blood volume sample 1008 when being entered to this sensing test piece 1001 by sample entrance port 1009, described in its similar Fig. 2, when blood volume sample only flow to thin-film electro During pole 1002, now also no current produces, when blood volume sample flow to membrane electrode 1003 such as Figure 10 (D), i.e. available such as Fig. 6 Result and obtain one and required calculate in order to the electric current judged, with judge blood volume sample 1008 in groove 1010 enough. This embodiment, more preferably such as Figure 11, comprises one the 3rd membrane electrode 1104, can be a thin film reference electrode.

Even if the present invention is described in detail by above-mentioned embodiment, and craftsman can be executed by appointing at this field usual skill of tool Think and be all as modify, the most de-claim such as is intended to protector.

Claims (14)

1. judge a method for detecting for testing sample coverage status, be the sensor of the electrode being applied to comprise separation, described The electrode separated is made up of the first electrode and the second electrode, and described first electrode is covered by reagent layer with described second electrode, its Being characterised by, step includes:

A () provides described testing sample so that it is flow to described second electrode from described first electrode；

B () provides microprocessor and has the voltage switching cell arrangement of switches set, when described microprocessor provides signal to make institute When stating the first circuit ON in switches set and second circuit disconnection, in the first set time, apply the first reaction DC voltage In between described first electrode and described second electrode, make the current potential current potential higher than described second electrode of described first electrode, and Record a Cottrell current value produced by described first set time, wherein said first electrode as working electrode, And a described Cottrell current value is directly proportional to the surface area of described first electrode covered by described testing sample, described The first set time be 3ms to 20ms；

C () removes described first reaction DC voltage and stagnates first period, described first period is 0ms to 20ms；

D () described microprocessor provides signal to switch described first electricity in the described switches set of described voltage switching cell arrangement Road is connection for disconnection and described second circuit, and in the second set time, applies the second reaction DC voltage in described first Between electrode and described second electrode, the magnitude of voltage of described second reaction DC voltage and the described first voltage reacting DC voltage Be worth equal, and make the current potential of the described second electrode current potential higher than described first electrode, and record described second the set time institute The 2nd Cottrell current value produced, wherein said second electrode is as described working electrode, and described 2nd Cottrell Current value is directly proportional to the surface area of described second electrode covered by described testing sample, and the second described set time is 3ms to 20ms；

E () removes described second reaction DC voltage and stagnates the second phase, the described second phase is 0ms to 20ms；

F repetition step (b) that () is circulated to step (e) at least twice, and distinctly add up a described Cottrell current value with Described 2nd Cottrell current value；

G () calculates the ratio of a described cumulative Cottrell current value and described cumulative 2nd Cottrell current value, wherein Described ratio is 0.3 to 3.3, in order to reflect that described testing sample is covered with shape at described first electrode and described second electrode Condition；And

H () is when sample volume is more than 0.45 μ l, it is judged that described testing sample is enough, the most then represent described sensor Test to the concentration being intended to analyte in described testing sample is effective.

2. judge a method for detecting for testing sample coverage status, be applied to that at least there is the first electrode and the second electrode Sensor, it is characterised in that step includes:

A () provides described testing sample so that it is flow to described second electrode from described first electrode；

B () between described first electrode and described second electrode, makes institute in the first set time, applying the first reaction DC voltage State the current potential of the first electrode current potential higher than described second electrode, and record produced by described first set time first Cottrell current value；

C () removes described first reaction DC voltage and stagnates first period；

D (), in the second set time, applying the second reaction DC voltage is between described first electrode and described second electrode, described The magnitude of voltage of the second reaction DC voltage is equal with the magnitude of voltage of described first reaction DC voltage, and makes described second electrode Current potential is higher than the current potential of described first electrode, and records the 2nd Cottrell current value produced by described second set time；

E () removes described second reaction DC voltage and stagnates the second phase；

F () repeats step (b) to step (e) at least twice, and the described Cottrell current value and described that distinctly adds up Two Cottrell current values；And

G () calculates the ratio of a described cumulative Cottrell current value and described cumulative 2nd Cottrell current value, wherein Described ratio reflects that described testing sample is at described first electrode and the coverage status of described second electrode.

3. method as claimed in claim 2, it is characterised in that the first described reaction DC voltage and the second reaction unidirectional current Pressure is to determine through cyclic voltammetry curve (cyclic voltammograms), and described first reaction DC voltage is with described the The signal noise ratio (S/N Ratio) of two reaction DC voltages is more than or equal to 1.

4. method as claimed in claim 2, it is characterised in that the first described electrode and described second electrode are positioned at same base Version.

5. method as claimed in claim 2, it is characterised in that

Having ferment and electron transmission mediator on the first described electrode and described second electrode, wherein said ferment is to described Testing sample carries out oxidation reaction；Or

Having ferment and electron transmission mediator on the first described electrode and described second electrode, wherein said ferment is to described Testing sample carries out reduction reaction.

6. method as claimed in claim 2, it is characterised in that

The first described set time is 3ms to 2s；And/or

The second described set time is 3ms to 2s.

7. method as claimed in claim 2, it is characterised in that

The first described set time is identical with described second set time；And/or

The first described set time and described second set time are 20ms.

8. method as claimed in claim 2, it is characterised in that

Described first period is 0ms to 50ms；Or

The described second phase is 0ms to 50ms.

9. method as claimed in claim 2, it is characterised in that

Described first period and the described time second phase are identical；And/or

Described first period and the described second phase are 20ms.

10. method as claimed in claim 2, it is characterised in that the first described electrode and the electrochemistry of described second electrode Response area is identical, wherein when described ratio is 1, represents that described testing sample is covered with described first electrode and described second electricity Pole.

11. methods as claimed in claim 2, it is characterised in that

The electrochemical reaction area of the first described electrode is more than the electrochemical reaction area of described second electrode；Or

The electrochemical reaction area of the first described electrode is less than the electrochemical reaction area of described second electrode.

12. methods as claimed in claim 2, it is characterised in that described sensor is electrochemical sensor.

13. methods as claimed in claim 2, it is characterised in that described method is in order to judge the test of described sensor Effectiveness, when wherein said ratio is 0.3 to 3.3, the described test that can represent described sensor is effective.

14. 1 kinds of method for detecting judging testing sample coverage status, are to be applied at least have the first electrode and the second electrode Sensor, it is characterised in that step includes:

A () provides described testing sample so that it is flow to described second electrode from described first electrode；

B () provides microprocessor and has the voltage switching cell arrangement of switches set, when described microprocessor provides signal to make institute When stating the first circuit ON in switches set and second circuit disconnection, in the first set time, apply the first reaction DC voltage In between described first electrode and described second electrode, make the current potential current potential higher than described second electrode of described first electrode, and Record a Cottrell current value produced by described first set time, wherein said first electrode as working electrode, And a described Cottrell current value is directly proportional to the surface area of described first electrode covered by described testing sample, described The first set time be 20ms；

C () removes described first reaction DC voltage and stagnates first period, described first period is 20ms；

D () described microprocessor provides signal to switch described first electricity in the described switches set of described voltage switching cell arrangement Road is connection for disconnection and described second circuit, and in the second set time, applies the second reaction DC voltage in described first Between electrode and described second electrode, the magnitude of voltage of described second reaction DC voltage and the described first voltage reacting DC voltage Be worth equal, and make the current potential of the described second electrode current potential higher than described first electrode, and record described second the set time institute The 2nd Cottrell current value produced, wherein said second electrode is as described working electrode, and described 2nd Cottrell Current value is directly proportional to the surface area of described second electrode covered by described testing sample, and the second described set time is 20ms；

E () removes described second reaction DC voltage and stagnates the second phase, the described second phase is 20ms；

F repetition step (b) that () is circulated to step (e) at least twice, and distinctly add up a described Cottrell current value with Described 2nd Cottrell current value；

G () calculates the ratio of a described cumulative Cottrell current value and described cumulative 2nd Cottrell current value, wherein Described ratio is 0.3 to 3.3, in order to reflect that described testing sample is covered with shape at described first electrode and described second electrode Condition；And

H () is when sample volume is more than 0.45 μ l, it is judged that described testing sample is enough, the most then represent described sensor Test to the concentration being intended to analyte in described testing sample is effective.