CN101495838A - Reversible electrochemical sensors for polysions - Google Patents

Abstract

The present invention is directed to a reversible electrochemical sensor for polyions. The sensor uses active extraction and ion stripping, which are controlled electrochemically. Spontaneous polyion extraction is suppressed by using membranes containing highly lipophilic electrolytes that possess no ion-exchange properties. Reversible extraction of polyions is induced by constant current pulse of fixed duration applied across the membrane. Subsequently, polyions are removed by applying a constant stripping potential. The sensors provide excellent stability and reversibility and allow for measurements of heparin concentration in whole blood samples via protamine titration. The sensors can also monitor a polyion concentration and an enzyme activity, wherein the polyion decomposition is directly proportional to the enzyme activity in a sample. Additionally, the sensors can monitor an enzyme inhibitor activity. Also, an immunoassay can be used to detect analytes by employing one of a polyion and an enzyme as markers.

Description

The reversible electrochemical sensors that is used for polyion

Related application

Present patent application is that the name of application on July 8th, 2004 is called the Application No. 10/887 of " reversible electrochemical sensors that is used for polyion ", 251 continuation-in-part application, it requires the U.S. Provisional Application number 60/485 of on July 9th, 2003 application, 856 right of priority, Application No. 10/887 with application on July 8th, 2004, both all are incorporated herein by reference the U.S. Provisional Application of on July 9th, 251 and 2003 application number 60/485,856 at this.

According to 35U.S.C. § 119 (e), the application also requires application time on August 5th, 2005 U.S. Provisional Patent Application (sequence number 60/706 co-pending, 117 and name be called " utilizing enzymatic activity Electrochemical Detection and the polyion selectivity reversible electrochemical sensors of polyion " as matrix) right of priority, it all is incorporated herein by reference at this.

The research or the exploitation of federal government's patronage

The part support of research of the present invention is the fund from National Institutes of Health (NIH) grant number GM071623 and EB002189.U.S. government enjoys interests to theme of the present invention.

Invention field

The present invention relates to the polyion sensor.The invention further relates to the film that is used to detect polyion (for example nucleoprotamine and heparin), and by this film being introduced this detection method in the electrochemical cell.Particularly the present invention relates to by making the polyion positive motion pass the method that this film detects polyion, wherein the motion of polyion is reversible, for use in re-using of this film.

Background of invention

In the past decade, along with the polymer film that adopts plasticising is used for the exploitation of the potentiometric sensor that the polyion macromolecule detects, new direction has appearred in the ion-selective electrode field.Early stage work in this field has advised containing the polymer membrane electrode of lipophilicity anionite, and it can detect the polyanion heparin.Referring to Ma, S.C, Yang, V.C, and Meyerhoff, M.E.Anal.Chem.1992,64,694.Heparin selective polymerisation thing membrane electrode further is described in U.S. Patent number 5,236,570 and U.S. Patent number 5,453,171 in.

Heparin be have mean charge for-70 and mean molecular weight be the Sulfated polysaccharide of 15,000 daltonian height.The molecular formula of a unit of heparin compound is provided below.

Heparin is at main operation and manipulation in vitro, as being used as anticoagulant in cardiac operation under direct vision, shunt operation and the dialysis.Yet in medical procedure, use excessive heparin to be harmful to, need be to the precise monitoring of heparin administration.The real-time monitoring of heparin concentration is useful especially for risk that prevents out hyperhematosis in surgical procedure and minimizing postoperative complication in blood.Activated clotting time is measured the commonsense method that (ACT) is heparin concentration in the estimation whole blood.Though this method is widely-used, it is non-specific and indirect, and the result can be subjected to the influence of many variablees.Compare with ACT, the heparin electrodes selective can directly detect heparin concentration in whole blood or plasma sample.

Similarly, also advised being used for the electrode of sensing polycation nucleoprotamine.Referring to Yun, J.H., Yun, J.H., Meyerhoff, M.E. and Yang, V.C.Anal Biochem.1995,224,212.During the polypeptide nucleoprotamine is generally used for and heparin activity (that is, promote solidify).Below illustrational nucleoprotamine be have mean charge for+20 polycation and its be rich in arginine residues.

The sulfonation base static of the alkaline guanidine radicals of nucleoprotamine and heparin is compound, makes that the anticoagulant active of heparin is invalid.Yet the excessive use of nucleoprotamine also is harmful to.For example, the use of nucleoprotamine usually causes disadvantageous Hemodynamics and hematology spinoff, has lung's blood platelet concurrently as oxygen consumption, the thrombopenia of hypertension, attenuating and separates and leukopenia.Therefore the nucleoprotamine concentration that can accurately detect and measure in biofluid such as blood is useful.

The reliable detection of nucleoprotamine can realize the careful administration of this reagent, has therefore avoided above-indicated relevant issues.In addition, also might be owing to detecting nucleoprotamine by come the heparin concentration in the working sample with nucleoprotamine titration sample by ion-selective electrode.This may be because above-mentioned specific Heparin-Protamine interacts.This type of effect also by people such as Ramamurthy at Clin.Chem.1998, be described in 606.

The viewed response of the specific membrane electrode of heparin well known in the prior art can not be explained according to traditional balance method.Because the high electric charge of these ions, for heparin and nucleoprotamine, nernst equation should obtain electrode function slope respectively for being lower than 1mV/ 10 years and 2mV/ 10 years.The quasi steady state model of explaining this unusual mechanism has been described subsequently.Referring to Fu, people such as B., Anal Chem.1994,66,2250.The response of measuring the polyion sensor of current potential is dynamic (dynamical) in nature.The powerful circulation of polyion has all appearred in mutually at aqueous solution and film, this be since polyion spontaneous be extracted in the polymer film and incident its with exchange from the water wettability ion in the film, this can cause the potential change in the presence of polyion.

Because when using the heparin specific membrane electrode of prior art, the extraction of polyion is an irreversible process, so observe the drift of forceful electric power position usually.After the relative short time with the contact of polyion solution, sensor begins to lose its response.The polyion of extraction must come by the reparation (as in concentrated sodium chloride solution) of sensor to remove mutually from film.Advised in the prior art that several different methods overcomes because the caused response loss of the lip-deep polyion concentration of film.The someone advises the mutual responsive current potential heparin sensor of pH, and wherein this sensor contains ion exchanger and charged H+ ionophore.According to this method, heparin is stripped and can be realized by regulating sample pH value.The another kind of method that overcomes sensor response loss is to use disposable sensor.

Therefore, although there is the selective extraction principle, still can not design reversible polyion sensor so far.Therefore, though the polyion sensor is very useful in critical nursing is used, their use is subjected to the sensor response restriction of loss fast.Disposable sensor can cause increasing cost, and need be by dividing other method taking-up sensor and readjust this sensor the validity that is quite time-consuming and has limited sensor unfriendly.Therefore, the sensor that has the detection polyion of completely reversibility is useful, wherein this reverse can be promptly, repeatedly and under need not to take out the situation of sensor in the independent solution carry out.

Summary of the invention

The invention provides the reversible electrochemical sensors that is used for polyion.This sensor comprises that potential response mechanism measures the concentration of polyion analyte, but the process that extraction and ion are stripped is subjected to galvanochemistry control.Spontaneous polyion extraction can contain the electrolytical film of the highly lipophilic with ion-exchange performance by use and suppress.If apply the fixedly continuous current pulse of duration at membrane electrode of the present invention two ends, but then can cause the counterextraction of polyion.Subsequently, remove polyion by applying constant reextraction current potential.The ability of this reextraction polyion makes sensor regeneration effectively, solved the problem that previously presented polyion sensor is faced, it has the tendency of drift and needed with the dense saline solution Long contact time so that strip polyion from sensing membrane before can carrying out another time measurement.

Containing the electrolytical film of lipophilicity can use with electrochemical cell, so that the polyion concentration in the continuous coverage sample solution and need not to take out electrode and readjust or change this electrode.Therefore, the titration of polyion such as heparin is possible.For example, be possible by the heparin concentration that adopts the nucleoprotamine titrimetry to measure in the whole blood sample.

According to an aspect of the present invention, provide a kind of polyion selective membrane that is used for electrochemical cell, wherein this film comprises the lipophilicity electrolyte with lipophilic cation component and lipophilicity anionic group.Preferentially, a kind of selective in lipophilic cation component and the lipophilicity anionic group for specific polyion.The desirable especially polyion that is used to detect is heparin and nucleoprotamine according to the present invention.Comprise DNA (deoxyribonucleic acid) (DNA), ribonucleic (RNA), humic acid, carrageenan and other polyion macromolecule with detectable other polyion of film of the present invention.

As mentioned above, a kind of selective in electrolytical lipophilic cation component of this lipophilicity and the lipophilicity anionic group for special polyion.Therefore, in an embodiment of the invention, this lipophilicity electrolyte comprises nucleoprotamine lipophilicity anionic group selectively.Preferentially, in this embodiment, this lipophilicity electrolyte is four (dodecyl) ammonium 1,3-dinonyl naphthalene-4-sulfonate (TDDA-DNNS).Similarly, in another embodiment, this lipophilicity electrolyte comprises heparin lipophilic cation component selectively.Preferentially, in this embodiment, this lipophilicity electrolyte is dodine four (right-chlorphenyl) borate (DDG-TCIPB).

In yet another embodiment of the present invention, a kind of polyion selective membrane is provided, it comprises polymer film-forming material, plastifier and has the lipophilic cation component and the lipophilicity electrolyte of lipophilicity anionic group, wherein a kind of selective for specific polyion in this lipophilic cation and the lipophilicity anionic group.Preferentially, this polymer film-forming material is that Polyvinylchloride and this plastifier are 2-nitrobenzophenone Octyl Ether.

In according to another implementation of the invention, a kind of polyion selective membrane that is used for electrochemical cell is provided, wherein this film comprises that having a kind of potpourri is scattered in wherein many microporous hydrophobics property matrix, and this potpourri comprises plastifier and has the lipophilicity electrolyte of lipophilic cation component and lipophilicity anionic group.Preferably, selective for specific polyion one of in this lipophilic cation component and the lipophilicity anionic group.

In another aspect of the present invention, provide a kind of polyion selective membrane electrode that is used for electrochemical cell.In one embodiment, this polyion selective membrane electrode comprises shell, comprise reference solution in the enclosure and operationally place in the enclosure electrode, makes electrode contact with reference solution.In addition, according to this embodiment, the polyion selective membrane is set at an end of shell.Reference solution in this film and the shell contacts, and operationally places this film so that it touches the sample solution outside shell.This film comprises the lipophilicity electrolyte with lipophilic cation component and lipophilicity anionic group, wherein at least a selective for specific polyion in lipophilicity negative ion and the lipophilic cation component.

In another aspect of this invention, provide the method for polyion material concentration in a kind of sample solution.This method on the one hand can controllably, reversibly transport this polyion material and pass film aspect galvanochemistry according to the present invention.Therefore, this method can be used for the METHOD FOR CONTINUOUS DETERMINATION of the polyion material concentration in the sample solution (as biological sample).

An embodiment according to this method provides a kind of sample solution that wherein contains the polyion material.Preferably, this sample solution also comprises supporting electrolyte.This solution contacts with membrane electrode with the contrast electrode of electrical connection.The film of membrane electrode is made up of the lipophilicity electrolyte with lipophilic cation component and lipophilicity anionic group, at least a selective for the polyion material in this lipophilicity negative ion and the lipophilic cation component.When sample solution contacts with electrode, the circuit that comprises membrane electrode and sample solution is applied the extrinsic current pulse, the electric current that is applied orders about the polyion material and is transported to the film from sample solution.Preferably, this extrinsic current pulse has fixed duration.The potential response of measuring between membrane electrode and the contrast electrode can carry out during this current impulse.The concentration of polyion material can be calculated as the function of potential response then.

In this another embodiment on the one hand of the present invention, this method also comprises the external electrode current potential is put on membrane electrode and the contrast electrode, transports out from film thereby order about the polyion material.In this embodiment, this method can be used for reversible sensor, and wherein polyion is stripped, so this film is repaired for further use.

In according to this another embodiment of the present invention on the one hand, provide the method for the polyion material concentration in a kind of sample solution.This method may further comprise the steps:

A) provide a kind of sample solution that comprises polyion material and supporting electrolyte;

B) provide a kind of fuel cell arrangement, it comprises i) comprise the polyion selective membrane electrode of film, this film comprises the lipophilicity electrolyte with lipophilic cation component and lipophilicity anionic group, at least a selective in this lipophilicity anionic group and the lipophilic cation component wherein for specific polyion, ii) be electrically connected to the contrast electrode on this membrane electrode, iii) be electrically connected to the counter electrode on this membrane electrode, the control device that iv) is operably connected to the electrochemical apparatus on this electrode and v) is communicated with this electrochemical apparatus;

C) sample solution is contacted with the electrode of fuel cell arrangement;

D) fixedly the extrinsic current pulse of duration is applied to the circuit that comprises membrane electrode, counter electrode and sample solution;

E) measure potential response during this current impulse;

F) calculating is as the polyion material concentration of the function of this potential response; And

G) the external electrode current potential is applied to membrane electrode and contrast electrode, from film, transports out thereby order about the polyion material.One preferred embodiment in, repeating step d) to g) to obtain one or more additional measurements of polyion material concentration.

In according to a further aspect in the invention, a kind of fuel cell arrangement is provided, this device can be used for the polyion concentration in the sample solution.Device according to an embodiment comprises: the polyion selective membrane electrode that comprises a kind of film, this film comprises the lipophilicity electrolyte with lipophilic cation component and lipophilicity anionic group, wherein at least a selective for specific polyion in this lipophilicity anionic group and the lipophilic cation component; Be electrically connected to the contrast electrode on the membrane electrode; And be operably connected to electrochemical apparatus on membrane electrode and the contrast electrode.

In another embodiment of this respect according to the present invention, this fuel cell arrangement further comprises the counter electrode that is electrically connected on the membrane electrode.

In another embodiment of this respect according to the present invention, this fuel cell arrangement also comprises the controller equiment that is communicated with electrochemical apparatus.One preferred embodiment in, this controller equiment is computerized controller.Sort controller is convenient to the part or all of robotization of this fuel cell arrangement.

Another aspect of the present invention discloses a kind of sensor.This sensor comprises the electrode that is positioned at shell.A kind of film is disposed in this shell one end and contacts with shell sample solution outward, the polyion concentration in this film test sample solution, and the enzymatic activity that reduces in speed and this sample solution of this polyion concentration is in direct ratio.

This electrode can be the Ag/AgCl electrode.This film can have the 10mm of being about ²-100mm ²Surface area.On the other hand, this film can have the 20mm of being about ²-50mm ²Surface area.The average thickness that this film can have is about 10 μ m-1000 μ m.On the other hand, this film average thickness that can have is about 20 μ m-300 μ m.Polyion concentration can be the concentration of nucleoprotamine.The activity of enzyme can be tryptic activity.

In another aspect of this invention, disclose the method for polyion concentration in a kind of working sample solution, this method comprises the step that a kind of sample solution is provided.This method is further comprising the steps of: this sample solution is contacted with film, and wherein said film detects the polyion concentration in this sample solution, and the enzymatic activity that reduces in speed and the described sample solution of wherein said polyion concentration is in direct ratio.This sample solution can comprise biotic component, and this sample solution can comprise blood.

In another aspect of this invention, a kind of reversible fuel cell arrangement is disclosed.This cell apparatus comprises the polyion selective membrane electrode that contains film, and this film can be measured the polyion concentration in this sample solution, and the enzymatic activity that reduces in speed and the sample solution of this polyion concentration is in direct ratio.This cell apparatus also comprises and is used to apply the device that current potential cleans this film.Being used to apply the device that current potential cleans this film can be the external electrode current potential that applies between contrast electrode and polyion selective membrane electrode.

In another aspect of this invention, a kind of reversible electrochemical cell apparatus is disclosed.This cell apparatus comprises the polyion selective membrane electrode that contains film, and this film is measured the polyion concentration that solution has; This cell apparatus also is included between contrast electrode and the polyion selective membrane electrode and applies current potential to clean the device of this film.

In another aspect of this invention, a kind of sensor is disclosed, this sensor comprises the electrode that is positioned at shell, this sensor also comprise be arranged in this shell one end and with the outer contacted film of sample solution of shell, this film can detect enzymatic activity and the corresponding inhibitor activity in this sample solution.

This corresponding inhibitor activity can be a kind of activity in activity, Aprotinin and the soybean inhibitor of α 1-antiprotease inhibitor, alpha2-macroglobulin.Current potential among the present invention reduces can be depending on corresponding enzyme inhibitor concentration in the sample solution.

In another aspect of this invention, a kind of immunoassays are disclosed.These immunoassays can be measured analyte by adopting a kind of thing that serves as a mark in polyion and the enzyme.

Brief description of drawings

Fig. 1 is the synoptic diagram according to the fuel cell arrangement that comprises polyion selective membrane electrode of a preferred embodiment of the invention;

Fig. 2 be illustrate (A) according to polyion selective membrane electrode of the present invention and (B) ion-selective electrode of prior art at 0.1M NaCl with contain the figure of the electrode repeatability when being used alternatingly between the 0.1MNaCl of 10mg/L nucleoprotamine;

Fig. 3 illustrates to adopt a kind of measuring method of having introduced polyion selective membrane electrode of the present invention, and the solution of 0.1M NaCl solution and 0.1M NaCl and 10mg/L nucleoprotamine is carried out the electric current/time locus of pulse continuous current measurement and the figure of current potential/time locus;

Fig. 4 illustrates to adopt a kind of measuring method of having introduced polyion selective membrane electrode of the present invention, and the solution of 0.1M NaCl and the solution of 0.1M NaCl and 50mg/L nucleoprotamine are carried out the electric current/time locus of pulse continuous current measurement and the figure of current potential/time locus;

Fig. 5 utilizes the pulse continuous current to measure and adopts (A) according to polyion selective membrane electrode of the present invention and (B) correction graph of nucleoprotamine among the illustrated 0.1M NaCl of ion-selective electrode of prior art;

Fig. 6 shows the blank solution of 0.1M NaCl and when having under the 10mg/L nucleoprotamine cathode current that applies-2 μ A, stirs the figure for the influence of the response of polyion selective membrane electrode according to the present invention;

Fig. 7 shows does not have nucleoprotamine (lower curve) and when having added 25mg/L nucleoprotamine (upper curve), and under the cathode current of-2 μ A, sample pH value is for the figure of the influence of the response of polyion selective membrane electrode of the present invention;

Fig. 8 shows at NaCl, KCl, MgCl ₂, CaCl ₂Pure solution in and the solution of nucleoprotamine in 0.1M NaCl supporting electrolyte in, the correction graph of polyion selective membrane electrode according to the present invention;

Fig. 9 provides two figure, show respectively (A) in the presence of the supporting electrolyte (0.01M NaCl, 0.03M NaCl and 0.1M NaCl) of variable concentrations nucleoprotamine calibration curve and (B) be with or without under the situation of 0.1M KCl, KCl concentration is to the influence of nucleoprotamine calibration curve among the 0.1M NaCl;

Figure 10 shows the amplitude-time response figure of use polyion selective membrane electrode according to the present invention for the current potential of nucleoprotamine titration in the whole blood;

Figure 11 is a curve map, it shows (A) and adopts according to polyion selective membrane electrode of the present invention, utilize the titration of 1g/L nucleoprotamine solution to contain the whole blood sample of concentration for the heparin of 0mM, 0.25mM, 0.5mM, 1mM and 2mM, (B) adopt according to polyion selective membrane electrode of the present invention, for the corresponding calibration curve of Heparin-Protamine titration in the whole blood;

Figure 12 a shows the reaction rate (Δ E/ Δ t) of just adding potential change after the trypsase figure as the function of trypsinase concentration;

Figure 12 b shows the reaction rate (Δ C/ Δ t) of just adding nucleoprotamine concentration change after the trypsase figure as the function of trypsinase concentration;

Figure 13 a shows the reaction rate (Δ E/ Δ t) of just adding potential change after trypsase and the SI inhibitor figure as the function of SI inhibitor concentration; And

Figure 13 b shows the reaction rate (Δ E/ Δ t) of just adding nucleoprotamine concentration change after trypsase and the SI inhibitor figure as the function of SI inhibitor concentration.

The detailed description of embodiment

Now the present invention will be described more fully hereinafter.Yet the present invention can be embodied in many different forms and not will be understood that and only limit to the embodiment of illustrating at this; Or rather, provide these embodiments so that present disclosure satisfies the legal requiremnt that is suitable for.Employed singulative in instructions and claims " one ", " a kind of " and " being somebody's turn to do " comprise the indicated thing of plural number, unless clearly indicate in addition in context.

The invention provides a kind of reversible polyion sensor, it will combine with the follow-up reextraction that is used for handling again under instrument is controlled continuously by the polyion extraction process of mass transport limitation during measuring.The present invention uses and comprises the electrolytical polyion selective membrane of lipophilicity.The instrument control that film is passed in ion circulation make can be repeatedly with ion extractuin to the film neutralization ion of from film, stripping, obtain the high sensor response of reproducibility.In addition, promptly recover the ability of sensor film, in test process, it is moved serially so that the actual value of polyion concentration in the solution to be provided.

The current potential polyion selective sensor of this area previously known is a passsive sensor.This sensor has the lipophilic cation of containing exchanger molecule and (generally is expressed as general formula R ^-Na ⁺) and the film of electrolyte aqueous solution.Phase boundary potential between the film in sample and this sensor can be determined according to equation (1):

$E_{\mathrm{PB}}={\mathrm{<figure-callout\; id="0"\; label="E"\; filenames="A20068002870100381.png,A20068002870100411.png"\; state="\{\{state\}\}">E</figure-callout>}}^0+\frac{\mathrm{RT}}{F}\ln \frac{a_{\mathrm{Na}}}{{\left[{\mathrm{Na}}^{+}\right]}_{\mathrm{pb}}}---\left(1\right)$

α wherein _NaBe the activity of Na in Solution ion, [Na ⁺] be so-called on the phase boundary of film phase the free concentration and the E of sodion ⁰Expression is transferred to the free energy of film in mutually with sodium from water.Term R and T are respectively gas law constant and absolute temperature.In sample, do not have under the situation of nucleoprotamine (or other polycation), by ignoring ion pairing, determine Na ion concentration during film mutually, i.e. R by the total concentration of lipophilic cation exchanger _T, it can calculate according to equation (2).

[Na ⁺] _pb＝R _T (2)

Therefore, the behavior of this film is similar to based on the sodium electrode of ion exchanger and can expect can this special response slope.

If nucleoprotamine is present in the aqueous solution, then will exist the nucleoprotamine kation logical with the high current of film in mutually on the surface simultaneously, form two stagnant diffusion layers.Because diffusion is a rate-limiting step in the layer that should stagnate of water, on the interface, can be observed the metastable state diffusion.The nucleoprotamine kation displaces sodium cation from the film phase boundary.This ion exchange process reduced film in mutually Na ion concentration and improved the current potential that is observed, as in equation (1), calculating.It is because the kation total concentration in the system must satisfy according to the described electroneutrality condition of equation (3) that this raising of the current potential that is observed may be interpreted as.

[Na ⁺] _pb＝R _T-z[PA ^z+] _pb (3)

[PA wherein ^Z+] _PbBe the concentration that has the nucleoprotamine of electric charge z in the film phase boundary.Calculate according to equation (4) on the basis of considering the circulation of pseudostationary attitude, this concentration can be expressed as the function of nucleoprotamine volumetric concentration:

${\left[{\mathrm{PA}}^{Z+}\right]}_{\mathrm{pb}}=\frac{D_{\mathrm{aq},\mathrm{PA}}{\mathrm{\&delta;}}_m}{D_{m,\mathrm{PA}}{\mathrm{\&delta;}}_{\mathrm{aq}}}{c}_{\mathrm{PA},\mathrm{bulk}}---\left(4\right)$

D wherein _m, D _Aq, δ _mAnd δ _AqBe respectively film mutually in, the nucleoprotamine coefficient of diffusion in the aqueous solution and in the gained thickness of diffusion layer.Equation (4) can be inserted into now in equation (3) and the equation (1), to obtain the nucleoprotamine response under the low concentration.The equation (5) of gained is provided below.

$E_{\mathrm{PB}}={\mathrm{<figure-callout\; id="0"\; label="E"\; filenames="A20068002870100381.png,A20068002870100411.png"\; state="\{\{state\}\}">E</figure-callout>}}^0+\frac{\mathrm{RT}}{F}\ln \frac{a_{\mathrm{Na}}}{R_T-\frac{z{D}_{\mathrm{aq},\mathrm{PA}}{\mathrm{\&delta;}}_m}{D_{m,\mathrm{PA}}{\mathrm{\&delta;}}_{\mathrm{aq}}}{c}_{\mathrm{PA},\mathrm{bulk}}}---\left(5\right)$

As confirmable in aforementioned calculation, if α _NaFix, then the phase boundary potential of this film demonstrates the direct response to nucleoprotamine.Therefore, under high nucleoprotamine concentration, near-this special response slope of energy to nucleoprotamine is replaced and be contemplated to sodion quantitatively from film.This quantitative displacement also can take place under rare nucleoprotamine solution, but needs time exposure (about 24 hours).The response slope of gained is too little, so that analytically of no use.

This spontaneous ion extractuin sensor can be undermined because of foregoing problems, is limited to disposable problem such as signal drift and most of sensor after long-time the use.Therefore, up to the present invention, just occurred a kind ofly being used to repair sensor so that continuous simple and easy, the reliable method of using.

With depend on the ion extractuin process in the above-mentioned passive type potentiometric sensor of spontaneous ion-exchange opposite, the present invention brings out ion extractuin by applying continuous current pulse electrochemical ground.In order to prevent spontaneous extraction, this film contains highly lipophilic electrolyte, and it generally can be according to general formula R ⁺R ^-Define and do not have an intrinsic ion-exchange performance.Therefore, the initial concentration of nucleoprotamine or sodium cation is assumed to be zero in membrane body.The cathode current i that is applied has brought out the net flux of kation J on film phase direction.In order to simplify resulting equation, only can suppose sodium and nucleoprotamine ion can be extracted to film mutually in.Therefore, at current i and sodium flux J _NaAnd nucleoprotamine flux J _PABetween relation can calculate according to equation (6).

i＝FAJ _Na+zFAJ _PA (6)

Wherein A is the membrane area that exposes.For the sake of simplicity, suppose linear concentration gradient and think that the na concn in the membrane body is zero that the sodium flux can be relevant with the concentration gradient across organic phase boundary, shown in the following equation (7).

$J_{\mathrm{Na}}=-\frac{D_{m,\mathrm{Na}}}{{\mathrm{\&delta;}}_m}{\left[{\mathrm{Na}}^{+}\right]}_{\mathrm{pb}}---\left(7\right)$

If there is no nucleoprotamine, equation (6) and equation (7) can be inserted in the equation (1) and obtain equation as follows (8).

$E_{\mathrm{PB}}={\mathrm{<figure-callout\; id="0"\; label="E"\; filenames="A20068002870100381.png,A20068002870100411.png"\; state="\{\{state\}\}">E</figure-callout>}}^0+\frac{\mathrm{RT}}{F}\ln \frac{{\mathrm{FAD}}_{m,\mathrm{Na}}}{{\mathrm{i\&delta;}}_m}{a}_{\mathrm{Na}}---\left(8\right)$

Fixedly the cathode current pulse of duration and intensity and constant potential reextraction pulse subsequently do not have sodion to keep membrane body, will obtain near-energy nernst electrode slope.If nucleoprotamine is present in the sample solution, then nucleoprotamine will be competed with sodion effectively in extraction process.Can be to be similar to the mode rewrite equation formula (6) of equation (7), shown in following equation (9).

$i=-\mathrm{FA}\frac{D_{m,\mathrm{Na}}}{{\mathrm{\&delta;}}_m}{\left[{\mathrm{Na}}^{+}\right]}_{\mathrm{pb}}-\mathrm{zFA}\frac{D_{m,\mathrm{PA}}}{{\mathrm{\&delta;}}_m}{\left[{\mathrm{PA}}^{Z+}\right]}_{\mathrm{pb}}---\left(9\right)$

Always greater than the flux of being kept alone by the polycation diffusion, equation (4) still effectively also can be inserted in the equation (9) flux that the electric current that present hypothesis is applied is forced.Therefore, sodium flux J _NaReduced, it has improved current potential according to equation (1).Equation (4) is inserted in the equation (9), solves [Na ⁺] _Pb, and insert in the equation (1), therefore obtained the nucleoprotamine response of the expectation under oligomeric ion concentration, it is provided in the following equation (10).

$E_{\mathrm{PB}}={\mathrm{<figure-callout\; id="0"\; label="E"\; filenames="A20068002870100381.png,A20068002870100411.png"\; state="\{\{state\}\}">E</figure-callout>}}^0+\frac{\mathrm{RT}}{F}\ln \frac{a_{\mathrm{Na}}}{\frac{{\mathrm{\&delta;}}_m}{D_{m,\mathrm{Na}}}(\frac{-i}{\mathrm{FA}}-z\frac{D_{\mathrm{aq},\mathrm{PA}}}{{\mathrm{\&delta;}}_{\mathrm{aq}}}{c}_{\mathrm{PA},\mathrm{bulk}})}---\left(10\right)$

For the known potentiometric sensor of prior art, between the response of the nucleoprotamine shown in equation (10) and the equation (5), there are differences.Importantly, existing with the thickness of diffusion layer of continuous current mode controlling diaphragm in mutually, and also the constant potential film between pulse upgrades the repeatably δ that has guaranteed from a pulse to another pulse _mValue.Though embodiment provided by the present invention adopts the electric current of at first selecting to obtain maximum potential range, the present invention does not plan to be so limited.Therefore, the current impulse intensity that is applied can be used for regulating the measurement range of polyion response.Because be the electric current that is applied, rather than ion exchanger, define sodion and be extracted in the film that pulse timing controls in the current potential mode, the coefficient of diffusion of film in mutually do not influence the responding range of nucleoprotamine.This forms contrast with the prior art potentiometric sensor that is subjected to equation (5) domination, wherein known at sodium-nucleoprotamine the competitiveness extraction and the film coefficient of diffusion between have direct dependence.

Suitably utilize above background theories, now can easily understand advantage provided by the present invention.Particularly the invention provides a kind of polyion selective membrane that is used for electrochemical cell.In addition, this polyion selective membrane an integral part that can be the electrochemical cell electrode.But this polyion selective membrane and membrane electrode can be used for the inverse approach of the polyion material concentration in the sample solution.

Polyion selective membrane of the present invention is characterised in that and comprises the lipophilicity electrolyte with lipophilic cation component and lipophilicity anionic group, wherein at least a selective for specific polyion in this lipophilicity anionic group and the lipophilic cation component.Term " lipophilicity " generally is understood that to be used for describing the material that fat is had affinity and has high lipid solubility.Lipophilicity is a kind of physicochemical property of describing the partition equilibrium of predetermined substance between water and immiscible organism.When water also existed, lipophilicity can further be described to a kind of material and be dissolved in the ability of lipid in mutually.This relation (that is partition factor) can be defined as the equilibrium constant of material concentration in the two-phase.Comparative standard generally is a 1-octanol/water partition factor.Can come calculation of distribution coefficient according to equation shown below (11).

According to equation (11), can expect that showing high oil loving molecule preferentially demonstrates dissolubility in lipid rather than in the water.

Being used for measuring a lipophilic functional test is the container that compound to be tested is placed on the potpourri that has held 50% water and 50% lipid (as the 1-octanol).The compound of being concerned about can be placed in the container, and can apply mixing force and be distributed in the two-phase to force this compound.Container is left standstill so that this compound reaches concentration balance between each phase.Can measure this compound concentration in each phase then, and this concentration can be used for equation (11) to determine lipophilicity.

Computer software also can be used to determine the lipophilicity of material.Determine that a lipophilic computer program example is at http: the last online ALOGPS program that gets of // 146.107.217.178/lab/alogps.Relevant lipophilic principle is also by Bakker, E.and Pretsch, E, at " Lipophilicity of tetraphenylborate derivatives as anionic sites in neutralcarrier-based solvent polymeric membranes and the lifetime ofcorresponding ion-selective electrochemical and optical sensors " AnalyticaChimica Acta, 1995,309,7-17, in discuss, at this its full content is incorporated herein by reference.

Usually, have compound greater than the P value of 100,000 calculating and be considered to highly lipophilic, and be useful therefore according to the present invention.Yet, can estimate usually to use to have even the compound of higher P value can cause sensor life-time to increase.Therefore, the P value of preferred lipophilic compound used according to the invention is greater than 100,000, more preferably greater than 1,000,000, and most preferably greater than 10,000,000.

The known polyion selective membrane of prior art comprises lipophilicity electrolyte and anti-lotus kation (that is R, of water wettability ^-Na ⁺).According to the present invention, the water wettability gegenion is replaced by the lipophilicity gegenion.Therefore, polyion selective membrane of the present invention comprises lipophilicity electrolyte (that is R, with lipophilic cation component and lipophilicity anionic group ^-R ⁺).By using two kinds of lipophilicity electrolyte, the lipophilicity gegenion no longer spontaneously with sample in measured polyion mass exchange.Preferably, selective for specific polyion one of in this lipophilicity anionic group and the lipophilic cation component, make this polyion detected easily.Want the non-limitative example of the specific polyion that detects to comprise nucleoprotamine, heparin, humic acid, carrageenan, DNA, ribonucleic and other polyion macromolecule.

In an embodiment of the invention, the electrolytical lipophilicity anionic group of lipophilicity is selective for nucleoprotamine.The nucleoprotamine selectivity of this lipophilicity anionic group depends on this anionic functional group.Nucleoprotamine contains alkaline guanidine radicals (that is arginine residues).Therefore, for selective to nucleoprotamine, this lipophilicity negative ion must comprise the functional group that can form ion pair with the guanidine radicals of nucleoprotamine.One preferred embodiment in, adopt to have carboxyl (COOH), sulfo group (SO ₃H) or sulfate (OSO ₃H) lipophilicity negative ion is used for the selectivity of nucleoprotamine.In a particularly preferred embodiment, the electrolytical lipophilicity anionic group of lipophilicity is selected from 1,3-dinonyl naphthalene-4-sulfonate, 2,6 dinonyl naphthalene-4-sulfonate, dodecyl benzene sulfonate and 3,9-diethyl-6-tridecyl sulfate.The chemical formula of these compounds is as follows.

1,3-dinonyl naphthalene-4-sulfonate

2,6 dinonyl naphthalene-4-sulfonate

Dodecyl benzene sulfonate

3,9-diethyl-6-tridecyl sulfate

As mentioned above, when the water wettability gegenion of known membrane electrolyte is replaced by the second lipophilicity electrolyte, prevented that ion is extracted into the polyion selective membrane from sample is spontaneous.Usually, the water wettability gegenion is a sodium, because sodium is the gegenion of the maximum that exists in the sample solution.Replace sodion by chemosynthesis with the lipophilicity gegenion.When the polyion chosen anion is selective for nucleoprotamine, any lipophilicity quaternary ammonium cation that can estimate to have about 4 to about 16 alkyl side arm chain length will be suitable gegenion.

One preferred embodiment in, with the water wettability gegenion of nucleoprotamine selectivity lipophilicity negative ion pairing be the kation that is selected from four (dodecyl) ammonium, three (dodecyl) ammonium methyls and dodecyl trimethyl ammonium.These cationic chemical formulas are as follows.

Four (dodecyl) ammonium

Three (dodecyl) ammonium methyl

Dodecyl trimethyl ammonium

According to above description, might select lipophilicity negative ion and the cationic combination of the anti-lotus of lipophilicity selectively to suppress spontaneous ion-exchange with sample solution to nucleoprotamine about the lipophilicity ion.Therefore, above nucleoprotamine chosen anion that provides and cationic any may the combination of anti-lotus can be provided spendable nucleoprotamine-selectivity lipophilicity electrolyte according to the present invention.According to a preferred implementation, being used for optionally from the lipophilicity electrolyte of solution extraction nucleoprotamine is four (dodecyl) ammonium 1,3-dinonyl naphthalene-4-sulfonate (TDDA-DNNS).

In according to another implementation of the invention, the lipophilicity electrolyte comprises heparin lipophilic cation component selectively.The heparin selectivity of cationic components depends on the functional group of this compound.Heparin contains sulfo group and carboxyl.Therefore, for selective to heparin, suitable kation must contain one or more groups that can form ion pair with the sulfo group and the carboxyl of heparin.Being particularly conducive to heparin is provided optionally is guanidine radicals.In addition, long aliphatic lateral chain or the aromatic ring of the heparin (as film according to the present invention) by neighboring cation piles up and stabilized from sample extraction to organic sensing phase.Therefore, the kation with highly lipophilic can have chain length and is about on the aliphatic chain of 4-18 carbon atom and/or suitable aromatic functionality and prepares by one or more guanidine radicals are connected to.One preferred embodiment in, the electrolytical lipophilic cation component of lipophilicity is selected from dodine and N, N '-1,10-decane two base two (guanidine).These cationic chemical formulas are as follows.

Dodine

N, N '-1,10-decane two bases two (guanidine)

In addition, when the water wettability gegenion is replaced by the second lipophilicity electrolyte, prevented spontaneous extraction ion from sample.Usually, the negative ion of maximum in testing liquid, chlorion is replaced by the lipophilicity negative ion through chemosynthesis.The one group of negative ion that can be used as gegenion according to this embodiment is the tetraphenyl borate salts derivant, as three kinds of borates that provide below.

Tetraphenyl borate salts (TPB)

Four (right-chlorphenyl) borates (CIPB)

Four (3, two (trifluoromethyl) phenyl of 5-) borate (TFPB)

The suitable negative ion of another combination is lipophilicity (fully halogenated or alkylating) 12 carboranes.12 carborane radicals are in the icosahedron carborane anion, have chemical formula CB when it is in complete unsubstituted form ₁₁H ₁₂ ^-12 carboranes of halogenation are as the 1-H-CB according to the present invention ₁₁Cl ₁₁, 1-H-CB ₁₁Br ₁₁And 1-H-CB ₁₁I ₁₁Be be particularly useful and more fully by Peper, S. wait the people, be described in " Ion-pairing Ability, Chemical Stability, and SelectivityBehavior of Halogenated Dodecacarborane Cation Exchangers in NeutralCarrier-Based Ion-Selective Electrodes; " Analytical Chemistry, (2003) 75 (9), 2131-2139 all is incorporated herein by reference it.Also useful according to the present invention is alkylating 12 carboranes, and wherein aforesaid halogen group is replaced by various alkyl.One preferred embodiment in, with heparin optionally the water wettability gegenion of lipophilic cation pairing be four (right-chlorphenyl) borate anion.

According to above description, might select lipophilic cation and the anionic combination of the anti-lotus of lipophilicity selectively to suppress spontaneous ion-exchange with sample solution to heparin about the lipophilicity ion.Therefore, above heparin selectivity kation that provides and anionic any may the combination of anti-lotus can be provided spendable heparin-selectivity lipophilicity electrolyte according to the present invention.According to a preferred implementation, being used for from the lipophilicity electrolyte of solution selective extraction heparin is dodine four (right-chlorphenyl) borate (DDG-TCIPB).

Use the principle of above-mentioned outline, also might determine the specific polyion except that nucleoprotamine or heparin is selectively had the lipophilicity electrolyte of lipophilic cation component or lipophilicity anionic group.Therefore, containing the electrolytical film of this type of lipophilicity also is included in the present invention.

The electrolytical amount of lipophilicity that exists in film according to the present invention can change according to the physical property of this film, and it can limit the solubleness of salt in film.Preferably, the electrolytical amount of lipophilicity is based on about 1-15wt% of film general assembly (TW).More preferably, the electrolytical amount of lipophilicity is the about 5-12wt% that accounts for the film general assembly (TW).One preferred embodiment in, the electrolytical amount of lipophilicity is the about 10wt% that accounts for the film general assembly (TW).

Except that the lipophilicity electrolyte, film according to the present invention may further include one or more plastifier.Plastifier has promoted mixing uniformity and has helped to control the flux of polyion from sample solution to film surface and membrane body.Various plastifier can be used in the film of the present invention, it includes but not limited to be selected from following plastifier: 2-nitrobenzophenone Octyl Ether, dioctyl phthalate, di-n-octyl sebacate, dioctyl adipate, dibutyl sebacate, dibutyl phthalate, 1-decyl alcohol, 5-phenyl-1-amylalcohol, benzohydrol 3,3 ', 4,4 '-tetrabasic carboxylic acid four (undecyl) ester, benzylic ether, phosphonic acids dioctyl phenylester, tri-2-ethylhexyl phosphate and 2-nitrobenzophenone Octyl Ether.In according to a preferred embodiment of the present invention, the plastifier that adopts in the film is a 2-nitrobenzophenone Octyl Ether (NPOE).

Usually preferably, film of the present invention except that lipophilicity electrolyte and plastifier, also comprises the host material that forms material as the body of film.The multiple matrix that is used to form permeable membrane is well known by persons skilled in the art, and the present invention is intended to comprise all this type of matrix.

In one embodiment, host material is a polymer film-forming material.Polymer film-forming material according to this embodiment can be any polymeric material compatible with the plastifier chemistry with the lipophilicity electrolyte.In addition, this polymeric material should be able to be shaped as film, as casting by solvent streams.As nonrestrictive example, according to the present invention can with polymeric material comprise Polyvinylchloride, polyurethane, cellulose triacetate, polyvinyl alcohol (PVA), silicon rubber, with and multipolymer and terpolymer.One preferred embodiment in, this polymer film-forming material is a Polyvinylchloride.

In an embodiment of the invention, the polyion selective membrane comprises the lipophilicity electrolyte of about 1-15wt%.Further comprise the polymer film-forming material of about 28-49.5wt% and the plastifier of about 42.5-66wt% (total weight is that the general assembly (TW) with film is a benchmark) according to the film of this embodiment.Preferably, the ratio of polymer film-forming material and plastifier existence is about 1: 1-1: 2 (by weight).

One preferred embodiment in, the polyion selective membrane comprises four (dodecyl) ammonium 1 of about 10wt%, the 2-nitrobenzophenone Octyl Ether of the Polyvinylchloride of 3-dinonyl naphthalene-4-sulfonate, about 30wt% and about 60wt% (total weight is that the general assembly (TW) with film is a benchmark).

Can be suitable for curtain coating by employing according to the polyion selective membrane of one of above embodiment becomes the organic solvent of film such as tetrahydrofuran (THF) to carry out solvent streams to cast and prepare.Preferably, with polymer film-forming material, plastifier and lipophilicity preparation of electrolyte be homogeneous solution in the solvent.Solution casting can be become film then.In case be prepared as film, this film can be cut into the size of any regulation so that be used for the polyion sensor subsequently.Not to be configured as film, but this coating solution can be applied on base material such as the electrode, and it is dry on electrode, thus directly on electrode, form film.

According to an embodiment of the invention, at least a in lipophilicity anionic group and the lipophilic cation component can be covalently bound in the backbone structure of polymer film-forming material.For example utilize copolyreaction or some other suitable chemical reaction form of vinyl bonds anionic group can be connected on the polymer chain.In addition, the lipophilic cation or the anionic group that can be connected on the polymer architecture can be polyion optional ingredient or gegenion component.

In another embodiment, host material is the hydrophobic base of porous.According to this embodiment, plastifier and lipophilicity electrolyte form potpourri, are distributed to then on many microporous hydrophobics property matrix, and wherein the electrolytical potpourri of plastifier and lipophilicity is inhaled among the hole of matrix, and makes its curing.Process for use in the polyion sensor wherein being dispersed with the electrolytical many microporous hydrophobics property matrix of plastifier and lipophilicity then.According to many microporous hydrophobics property matrix of an embodiment of the present invention can be selected from tygon, polypropylene, nylon, polyvinylidene fluoride, polycarbonate, teflon, acrylic copolymer, polyethersulfone, with and multipolymer and terpolymer.According to a preferred implementation, the hydrophobic base of this porous is a tygon.What be preferably used as many microporous hydrophobics property matrix especially is can be from Celgard, Inc., the Celgard that Charlotte, N.C obtain (R) film.Celgard (R) film is the polyethylene based film that can be used as flat sheet membrane and hollow-fibre membrane.

In a preferred embodiment of the present invention, the polyion selective membrane comprises the porous hydrophobic matrix that has contacted with the potpourri that comprises about 1-15wt% lipophilicity electrolyte and about 85-99wt% plastifier, described lipophilicity electrolyte comprises lipophilic cation component and lipophilicity anionic group, and percentage by weight is based on the general assembly (TW) of potpourri.

The present invention further provides a kind of polyion selective membrane electrode that can be used for electrochemical cell.In an embodiment of the invention, polyion selective membrane electrode comprises shell, comprise in the enclosure reference solution and location electrode in the enclosure operationally, and electrode is contacted with reference solution.In addition, according to this embodiment, the polyion selective membrane is arranged on an end of shell.Reference solution in this film and the shell contacts, and this film is operationally located so that it touches the sample solution outside shell.As mentioned above, this film comprises the lipophilicity electrolyte with lipophilic cation component and lipophilicity anionic group, wherein at least a selective for specific polyion in lipophilicity anionic group and the lipophilic cation component.

This embodiment according to the present invention can adopt any normal electrode, as long as this electrode can be introduced aforesaid polyion selective membrane.In a particularly preferred embodiment of the present invention, membrane electrode comprises and is introduced in normal electrode such as Philips electrode body (IS-561, GlasblsereiMoller, Zurich, Switzerland) the polyion selective membrane in.

The reference solution that adopts in the electrode shell can be those skilled in the art's known spendable any electrolyte solutions usually.One preferred embodiment in, this electrolyte solution is a sodium chloride solution, particularly 1M NaCl solution.In addition, electrode itself can be any kind electrode that can be used in the electrochemical cell with current potential as described below and current value.Useful especially is the Ag/AgCl electrode.

According to a preferred implementation, the polyion selective membrane of introducing in the membrane electrode is selective to nucleoprotamine.Preferably, according to this embodiment, the lipophilicity electrolyte that is used for the polyion selective membrane is TDDA-DNNS.

When using with membrane electrode, preferably the surface area of polyion selective membrane is about 10mm ²-100mm ²More preferably surface area is about 20mm ²-50mm ²In order to reach such surface area, film preparation can be as mentioned above, and film is cut into required size (for example using cork borer), so that match with electrode.More preferably, the average thickness of polyion selective membrane is about 10 μ m-1000 μ m, is more preferably 20 μ m-300 μ m.

The invention further relates to a kind of fuel cell arrangement.In one embodiment, this fuel cell arrangement comprise foregoing polyion selective membrane electrode, be electrically connected to the contrast electrode on the membrane electrode and be operably connected to membrane electrode and contrast electrode on electrochemical apparatus.

An embodiment according to fuel cell arrangement of the present invention is provided among Fig. 1, the figure shows the fuel cell arrangement 5 that can be used for polyion material in the measuring samples.Fig. 1 shows polyion selective membrane electrode 10, contrast electrode 30 and operationally is positioned over counter electrode 50 in the test sample vessel 60 that sample solution 65 has been housed.Membrane electrode 10 comprises electrode shell 15, reference solution 17 and reference electrode wire 21.What be arranged in electrode shell 15 1 ends is according to polyion selective membrane 25 of the present invention.Contrast electrode 30 as shown in Figure 1 is binode electrodes, though do not deviating under the present invention, the contrast electrode of other type also can be used.Contrast electrode 30 comprises shell 33, inner casing 36, shell reference solution 39, inner casing reference solution 41 and reference electrode wire 43.

As shown in Figure 1, polyion selective membrane electrode 10, contrast electrode 30 and counter electrode 50 are operably connected on the electrochemical apparatus 75 separately, and it further is communicated with control device 90.Electrochemical apparatus 75 is preferably continuous current-potentiostat.Therefore, electrochemical apparatus can be with the Current Control by electrochemical cell in predetermined value and also can be with the control of Electric potentials between working electrode (for example, polyion selective membrane electrode 10) and the contrast electrode 30 in predetermined value.During a function, electrochemical apparatus 75 can be forced necessary any electric current to keep required current potential between working electrode (for example, polyion selective membrane electrode 10) and counter electrode 50 after execution.In a particularly preferred embodiment, electrochemical apparatus 75 is two constant potentials, as from PineInstruments (Grove City, Pa.) available AFCBPl Bipotentiostat.

Control device 90 as shown in fig. 1 is preferably the computing machine that can carry out a kind of algorithm, and this algorithm is designed to control automatically the function of electrochemical apparatus 75 aspect control required electric current, current potential or electrochemical activity.Control device 90 also preferably can be collected from the data of electrochemical apparatus 75 and is shown as the observable data of user and/or store this data.Certainly, should be understood that electrochemical apparatus 75 among Fig. 1 and control device 90 boths can be connected on the power supply (not shown).

The invention further relates to the method for polyion material concentration in a kind of sample solution.This method generally includes following steps: the sample solution that comprises polyion material and supporting electrolyte a) is provided; B) sample solution is contacted with the polyion selective membrane electrode with film, this film comprises the lipophilicity electrolyte with lipophilic cation component and lipophilicity anionic group, wherein at least a selective for this polyion material in this lipophilicity anionic group and the lipophilic cation component; C) sample is contacted with contrast electrode, polyion selective membrane electrode and contrast electrode are electrically connected; D) circuit that comprises polyion selective membrane electrode and sample solution is applied the fixedly extrinsic current pulse of duration, transfer to this film from sample solution thereby order about the polyion material; E) measure the potential response between polyion selective membrane electrode and contrast electrode during this current impulse; And f) calculating is as this polyion material concentration of the function of potential response.

The preferred fixedly extrinsic current pulse of duration has applied about 0.1 second to about 2 seconds duration.The general potential response that does not need to measure the full duration of the current impulse that applies.But preferably measure potential response for the only part in applying extrinsic current duration of pulse.In a particularly preferred embodiment, potential response is current impulse outside and fixedly measures during last about 100 milliseconds of duration.

The potential value of measuring in the above method depends on the polyion type that exists in the sample.For example, if there is kation,, then cathode current (bearing) is put on this battery as nucleoprotamine.When applying electric current, the current potential that records becomes bigger negative value.When having negative ion such as heparin, anode current (just) is put on this battery.When applying electric current, the current potential that records become bigger on the occasion of.

Generally include polyion selective membrane electrode and sample solution although be applied in the circuit of extrinsic current, this circuit also can comprise one or more other assemblies of electrochemical cell.For example, in one embodiment according to the present invention, this circuit also comprises counter electrode.This embodiment will comprise as usual the electro-chemical systems that is called " three electrodes " electrochemical cell.In addition, in another embodiment, this circuit also comprises contrast electrode.This embodiment comprises as usual the electro-chemical systems that is called " two electrodes " electrochemical cell.Usually preferred three-electrode system is to avoid the occurring contrast electrode deterioration when extrinsic current is applied on these electrodes.

Usually can estimate that the current potential absolute value of measuring in the above method will reduce as time passes, this stably increases owing to thickness of diffusion layer in the film.As mentioned above, when the existing of test polycation such as nucleoprotamine, apply cathode current and observe negative potential.When nucleoprotamine (or another kind of polycation) when being present in the sample, measured current potential is than the situation that does not have polycation, obviously be bigger on the occasion of.On the contrary, when test during the existing of polyanion, apply anode current and observed current potential is positive.If heparin (or another kind of polyanion) can be estimated measured current potential than the situation that does not have polyanion when being present in the sample, obviously is bigger negative value.In two kinds of situations, to the corrigendum or more the mobile of negative charge be that polyion is extracted into the indication in the film from sample solution.Behind time enough, because polyion gathering in film, measurement begins to interrupt.

In a preferred embodiment of the present invention, this film is restored.According to this embodiment, above method further comprises the external electrode current potential is put on polyion selective membrane electrode and the contrast electrode, is shifted out from the film transfer thereby order about the polyion material.In case this film is by the polyion of having stripped effectively, this polyion selective membrane electrode can be used for the polyion of sample solution once more.By applying the pulse train that comprises extrinsic current pulse and volta potential pulse subsequently repeatedly, make continuous, the reversible detection of polyion material become possibility.

Preferably, the external electrode current potential that is applied for reextraction polyion from film is a baseline potential.The baseline potential value can change along with the symmetry of electrochemical cell.For example, in one embodiment, membrane electrode adopts identical electrode and has to sample solution with contrast electrode forms similar interior reference solution.In this preferred implementation, baseline potential is 0V.Also imagined other embodiment, wherein electrode is demonstrating lower symmetry in varying degrees.In these other embodiments, can estimate that baseline potential will begin to change from 0V.By disconnecting electrochemical apparatus (referring to Fig. 1), and with high-impedance voltmeter it is replaced with the zero current current potential of measuring between membrane electrode and the contrast electrode and can measure best baseline potential.

For the polyion of stripping from film effectively, the fixedly duration of the duration that preferably applies volta potential than extrinsic current pulse is about 10-20 doubly.

According to following experimental example the more embodiment of the present invention can be described more clearly.

The present invention is the polyion in the test sample solution on the other hand, nucleoprotamine for example, and this detects based on itself and for example tryptic direct relation of enzymatic activity.If endonuclease capable makes polyion be divided into short section, then the activity of this enzyme can be recorded, because these short sections can not be detected well by the polyion sensor usually.The Electrochemical Detection that is similar to a kind of detection disclosed by the invention has the advantage that is better than the spectrophotometric method of enzymatic activity in conventional opaque, the coloured or muddy sample of detection.

A kind of embodiment of this respect of the present invention is the purposes that trypsase is used for nucleoprotamine.Trypsase is the enzyme in basic amino acid lysine and arginic carboxyl side place scinderin matter.Because nucleoprotamine is rich in arginine residues, trypsase will be at these contact place cutting nucleoprotamine.When adopting electrochemical appliance disclosed by the invention and nucleoprotamine sensor, can find that the initial rate and the tryptic activity of nucleoprotamine decomposition is in direct ratio.Sensor signal potential change speed as the function of time is owing to the digestion of trypsase to nucleoprotamine.This relation can expand to various polyions and corresponding enzyme thereof.

Another embodiment of the present invention is to apply current potential to clean film it to be reused in enzymatic activity, polyion concentration and inhibitor activity detect.Should be noted that and utilize the present invention can detect other biology and chemical activity.

In addition, the present invention can be used for the inhibitor activity in the test sample solution.Enzyme inhibitor combines with enzyme and has reduced enzyme reaction speed effectively.The concentration of enzyme inhibitor in the sample solution is depended in the reduction of initial potential after the potpourri that adds enzyme and enzyme inhibitor.

A kind of embodiment of this respect of the present invention is the inhibitor that is used for enzyme trypsase and following a kind of corresponding enzyme: α 1-antiprotease inhibitor, alpha2-macroglobulin, Aprotinin and soybean inhibitor.

In another aspect of the invention, potentiometric titration polyion sensitive electrode can find application in no separating immune is measured.These immunoassays can adopt the polyion of mark or the relevant enzyme thing that serves as a mark to detect analyte, and this analyte can be used as label through free analyte with have in the competitive bidding of mark analyte of antibody.

Experiment

Can illustrate the present invention more fully by following embodiment, listing these embodiment is to think to limit the present invention in order to demonstrate the invention and not.Except as otherwise noted, all percentages is meant that with polyion selective membrane general assembly (TW) be the percentage by weight of benchmark.

Embodiment 1

The preparation of nucleoprotamine-selective membrane

Test comprising the sensor capability that is ready to use in the polyion selective membrane of the present invention in the electrochemical cell.The polycation selective membrane is mixed with for the polycation nucleoprotamine selective especially.The film that forms is for to have 10wt% TDDA-DNNS in 2: 1 weight ratio potpourris of 2-nitrobenzophenone Octyl Ether and Polyvinylchloride.By adopting THF to prepare this film as the solvent streams casting of solvent.Make the potpourri drying and forming-film, made nucleoprotamine-selective membrane that thickness is about 200 μ m.With diameter be the cork borer of 6mm cut this film with preparation for the film that is incorporated in the electrode.

Embodiment 2

The preparation of nucleoprotamine-selective membrane electrode

Nucleoprotamine-the selective membrane that will prepare in embodiment 1 is incorporated in the electrode.Electrode comprises interior reference solution and the Ag/AgCl electrode wires of Philips electrode body (IS-561), 0.1M NaCl.Nucleoprotamine-selective membrane electrode before using, is regulated whole night in the solution identical with interior reference solution in experiment.

Prepare one group of 10 identical electrode as mentioned above, and before the experiment of reality is used, in 0.1M NaCl solution, test its denseness.Test be presented between given electric current bottom electrode in 0 to-10 μ A scope changeability for+/-7mV (standard deviation).

The also tested reversibility of estimating of membrane electrode.Film is exposed in two kinds of independent solution repeatedly a kind of 0.1M NaCl that contains, a kind of 0.1M NaCl and 10mg/L nucleoprotamine of containing.Also adopt the ion-selective electrode of prior art to carry out identical test.The results are shown among Fig. 2 of test, nucleoprotamine wherein of the present invention-electrodes selective film is shown in the curve A, and the electrode of prior art is shown in the curve B.In two curves, as seen, when having nucleoprotamine, observe higher current potential.In curve A, repeatably potential measurement have+/-variation of 1mV.It changes greater than 50mV in 5 cycles less yet but be presented in curve B.

Embodiment 3

There is and do not have the time-measuring electric potential response of the sample of nucleoprotamine

Be produced on and do not have a chronopotentiogram among the 0.1M NaCl of nucleoprotamine.Utilize nucleoprotamine-selective membrane electrode of describing among the embodiment 2 to set up electrochemical cell as shown in fig. 1.Contrast electrode is to have the electrolytical binode Ag/AgCl of 1M LiOAc bridge electrode.Counter electrode is a platinum filament.

The voltammetry experiment is AFCBP1Bipotentiostat (the Pine Inst. that utilizes by the control of PCI-MIO-16E4 interface board, Grove City, Pa.) and 5.0 softwares of the LabVIEW on the macintosh computer (National Instruments, Austin Tex.) carry out.Before experiment, convert the first electrode output function of double potentiostat (K1) under the control of second working electrode (K2) output constant potential Current Control.In order to apply current impulse, the analog switch that working electrode is controlled via external software is connected to K1 output.When between current impulse, applying the baseline current potential, working electrode is connected to K2 output.

In the chronopotentiometric titration experiment, the continuous current pulse (1 second duration) that is at every turn applying-3 μ A is the constant potential pulse (10 seconds duration) under 0V afterwards.The sampling current potential of gained (its expression sensor response) is the mean value during the last 100ms of each current impulse.All experiment is carried out under laboratory environment temperature (21.5 ± 0.5 ℃).The fiducial interval level of calculating is 95%.

Experiment is carried out in two samples.First sample only contains 0.1M NaCl, and second sample contains 0.1M NaCl and concentration is the nucleoprotamine (PA) of 10mg/L.-3 μ A cathode currents that applied cause nucleoprotamine to be extracted in the film, compare with the sample that does not have nucleoprotamine, and are significantly different for the viewed current potential of the sample that nucleoprotamine is arranged.Electric current-the time locus and the current potential-time locus of chronopotentiometric titration experiment are provided among Fig. 3.

Embodiment 4

There is and do not have the more chronopotentiometric titration response of the sample of the nucleoprotamine of high-load

Adopt and to be manufactured with the identical test unit that in embodiment 2, is provided and not have second chronopotentiogram among the 0.1M NaCl of nucleoprotamine.In the chronopotentiometric titration experiment, the continuous current pulse (1 second duration) that at every turn applies-2 μ A is the constant potential pulse (15 seconds duration) under 0V afterwards.The sampling current potential of gained (its expression sensor response) is the mean value during the last 100ms of each current impulse.All experiment is carried out under laboratory environment temperature (21.5 ± 0.5 ℃).The fiducial interval level of calculating is 95%.

In two samples, experimentize once more.First sample only contains 0.1M NaCl, and second sample contains 0.1M NaCl and concentration is the nucleoprotamine of 50mg/L..-3 μ A cathode currents that applied cause nucleoprotamine is extracted in the film, compare with the sample that does not have nucleoprotamine, and are significantly different for the viewed current potential of the sample that nucleoprotamine is arranged.Electric current-the time locus and the current potential-time locus of chronopotentiometric titration experiment are provided among Fig. 4.

During the constant potential rest pulse, can be observed ion antidiffusion from film.When nucleoprotamine was present in the sample, this diffusion was more slowly, had shown the difference on dispersal behavior between sodium and nucleoprotamine ion.When asking current integration on the whole rest pulse at 15 seconds, the electric charge that calculates is corresponding to 90% of the electric charge that applies during this current impulse.

Embodiment 5

The calibration curve that compares nucleoprotamine of the present invention-selective membrane electrode and prior art polyion-selective membrane for nucleoprotamine

Take a sample by the electrogram that applies repeatedly when finishing, make continuous, the reversible detection of nucleoprotamine become possibility in the pulse train as shown in Fig. 3 and Fig. 4 and to each current impulse.Therefore, might obtain the nucleoprotamine calibration curve.

Utilize said method in embodiment 3 and 4 can be in 0.1M NaCl the time locus of nucleoprotamine calibration curve.Utilize the ion-selective electrode of the nucleoprotamine described in the embodiment 2-selective membrane electrode and prior art can obtain some curves.The contrast of two kinds of curves is provided among Fig. 5, wherein adopt the curve of nucleoprotamine of the present invention-selective membrane electrode gained to be shown in the curve A, and the curve of prior art electrode is shown in the curve B.It is poor that observed forceful electric power position drift comes from the film side thickness of diffusion layer control in curve B.The nucleoprotamine concentration (mg/L) of logarithm is indicated on the track.

Embodiment 6

Stirring is for the influence of sensor response

For the potentiometric titration polyion sensor of prior art, known observed current potential can be subjected to the strong influence of sample stir speed (S.S.), and it has changed moisture diffusion layer and has therefore changed the polyion flux that enters into film.In fact, recent achievement the is verified definite relation between measurement range and rotating speed in the electrode assembly of rotation.To stir the response that can how to influence in order investigating in the continuous current pulse test, in agitating solution not and under the stir speed (S.S.) at 100rpm, to measure current potential according to polyion selective membrane electrode of the present invention.Both contrasts are provided among Fig. 6.

Opposite with the potentiometric titration result who adopts heparin response film (wherein sample stirs the potential change that stops to have caused about 20mV suddenly), the response of pulse continuous current sensor of the present invention does not have to show the appreciable impact that is subjected to stirring rate.Stir and not the potential difference (PD) between the stirred sample surpass 2-3mV.

Embodiment 7

The pH value is for the influence of sensor response

Though it is to work in 7.4 the whole blood that this nucleoprotamine sensor is intended to the physiological pH value, also investigated of the influence of pH value for the sensor response.Fig. 7 provides current potential observed under the cathode current of-2 μ A.Following track is for the blank solution that comprises 0.1M NaCl, 6.6mmol citric acid, 11mmol boric acid and 10mmol phosphoric acid, viewed current potential under the pH value that adopts 1M NaOH to regulate.Last track is for the viewed current potential of the same solution that contains the 25mg/L nucleoprotamine in sample.Because nucleoprotamine concentration height, the difference of two kinds of current potentials can be thought maximum sensor response or electrochemical window in 0.1M NaCl.

Embodiment 8

The film selectivity

The selectivity of film is to measure by the independent calibration curve of the chloride salt of record sodium, potassium, calcium and magnesium pH 7.4 times.The curve of current potential-salinity logarithm is provided among Fig. 8.The gained selectivity factor with reported about the DNNS base ISE film that does not have the additional ions carrier in the past those are very consistent.The whole slopes of discovery in the 0.001M-0.1M concentration range are super slightly (slightlysuper)-Nemstian (70-72mV), and it departs from selectivity factor to a certain extent.This slope might explain that by the contribution of ion migration on the membrane interface this equation also was not studied according to the Nernst-Plank equation in the theoretical model of simplifying.About 10 ⁴The sudden change current potential of M rises to and comes from the lip-deep consumption process of film.Nucleoprotamine calibration curve in 0.1M NaCl also is shown among Fig. 8.Higher electrogram shows, compares with whole other kations of testing, has greatly preferred property for this film of nucleoprotamine.

Embodiment 9

The influence of supporting electrolyte concentration

Can estimate that supporting electrolyte concentration can influence the nucleoprotamine response curve, because this response theory is based on the competitiveness extraction between polyion and the sodion.For example, can haggle over low sodium background concentration in advance and can produce bigger potential range (referring to equation 10), and also may cause this response to be transformed into lower nucleoprotamine concentration (equation 10) for the nucleoprotamine response.Fig. 9 A has shown the experiment nucleoprotamine calibration curve under three kinds of sodium chloride concentrations (10mM, 30mM and 100mM).The nucleoprotamine potential range descends along with increasing of NaCl concentration.

Potassium is shown among Fig. 9 B for the trickle influence of nucleoprotamine response, and wherein showing is having and do not having two kinds of nucleoprotamine calibration curves among the 0.1M NaCl of 10mM KCl.In fact observed response maximum deviation is no more than 5mV under low nucleoprotamine concentration.

Embodiment 10

The calibration curve of nucleoprotamine in whole blood

Figure 10 for example understand in the whole blood the nucleoprotamine calibration curve and under the cathode current of-2 μ A the corresponding current potential-time locus of this calibration curve.Discovery potential response scope in whole blood is about 60mV, and this practical measurement for nucleoprotamine in whole blood sample is very gratifying.The standard deviation of current potential is increased to 1.5mV, observes 0.7mV by contrast in the NaCl solution of buffering.The result shows, utilizes the current impulse chronopotentiometric titration, and sensor can be measured the nucleoprotamine concentration that is low to moderate 0.5mg/L.

Embodiment 11

The titration of whole blood sample

This embodiment can be used for measuring heparin in the blood via the end point determination of nucleoprotamine titration, its with the previous class of operation that adopts the potentiometric titration sensor seemingly.Little aliquot (2 * 10 with the heparin stock solution ^-5M 1.5g/L) adds in the whole blood sample, so that obtain different typical heparin concentration in 0.25-2 μ M (0.6-4.5kU/L) scope, and carries out titration with the 1g/L nucleoprotamine.The gained titration curve is shown among Figure 11 A.

The each point that calculates is the mean value of 10 continuous electrograms, obtains being not more than the standard deviation of 1.5mV.Estimate repeatability by each titration is repeated 4 times, the deviation that has obtained the take-off potential from a sample to another sample and finished current potential is below 7mV, and total variation of current potential keeps identical in titration process.Because each receiving tube contains the sylvite of 7.2mg EDTA and the blood flow volume collected in each pipe is to change between 2-4mL, most of deviation is attributable to the variation (referring to Fig. 9 B) of potassium concn.

The observed terminal point that is plotted among Figure 11 B is the function of whole blood heparin concentration, and has found the linear relationship (related coefficient 0.995) of expection.The linear regression of gained calibration curve is measured as C _Heparin=V (6.6 ± 0.4) * 10 ^-3M/L-0.6 μ M.

Embodiment 12

Life-span and sensor stability

The life-span of sensor and stability are important parameters, are to carry out in Physiological Medium if especially measure.3 hours the continuous impulse chronopotentiometric titration measurement (measuring intervals of TIME is 1 minute) in containing the pH buffering 0.1M NaCl of 10mg/L nucleoprotamine does not produce significant potential drifting and maximum potential is changed to 2mV.For whole blood sample, the titration curve that is shown in Figure 11 B is to utilize identical sensor to obtain, and the overall measurement time in blood surpasses 2.5 hours (for each point, having collected 10 potential measurement values).After in being exposed to blood, sensor is positioned among the 0.1M NaCl of buffering, finds that the baseline current potential turns back to initial value (for each sensor ± 5mV).The life-span of sensor (being defined as the time that the baseline potential change is no more than 20mV) was at least 2 weeks, and the T.T. that is exposed in the undiluted whole blood sample is 10 hours.

Embodiment 13

Preparation nucleoprotamine selective membrane electrode and mensuration tryptic activity and inhibitor activity

It is 1: 2 PVC and the lipophilicity salt TDDA-DNNS of o-NPOE and 5wt% that ion selective membrane (thickness is 200 μ m) contains weight ratio.THF is as solvent in employing, casts by solvent streams to prepare this film.Be cut into this film by female film and attach it in the Philips electrode body (S-561) with cork borer (diameter is 6-mm).Interior filling solution is made up of in 10mMTris-HCl buffering agent (pH=7.4) 0.1M NaCl and is contacted with inner Ag/AgCl electrode.With one night of this electrode regulating, the binode Ag/AgCl electrode with 1MLiOAc bridge electrolytic solution is used as outer contrast electrode before experimentizing in the solution identical with interior filling solution.

The Pulstrode measurement is carried out in the three-electrode battery system, and wherein Philips body electrode (as working electrode), outer contrast electrode and counter electrode (platinum line) are dipped in this sample.Utilize pulse continuous current/constant potential technology to control ion selective membrane.At experimental session, the current density that at every turn applies is 0.5 μ A/cm ²Be another zero continuous current pulse (duration is 0.5 second) after the continuous current pulse of (duration is 0.5 second), add constant potential pulse (duration is 15 seconds) then.The mean value of the sampling current potential that can represent sensor response during as the last 50ms of first current impulse.

Nucleoprotamine has the basic amino acid of high-load, and wherein about 50% has arginine residues.Therefore, it is the good zymolyte that is used for the trypsinization reaction.Shown in Figure 12 a and Figure 12 b, when adding high concentration proteases trypsin enzyme, the response of nucleoprotamine significantly reduces, and proteolysis reaction has appearred in this expression.Adopt identical electrode all to test, and between the different measurement of trypsinase concentration, clean this film as background solution with identical buffering agent.The baseline total displacement is about 7mV, and it represents the efficient recovery that has of sensing membrane.

The slope that initial potential descends during by interpolation trypsase can be assessed the reaction rate of trypsinization nucleoprotamine.The slope of Xia Jianging means that when trypsinase concentration increases reaction rate strengthens gradually, therefore, shown in nucleoprotamine digestion and the sample tryptic activity in direct ratio.

Be in the past utilize potentiometric titration nucleoprotamine sensor directly to detect the active of protease inhibitors and successfully measured in the pre-service plasma sample like the trypsin inhibitor Aprotinin.Utilize Pulstrode nucleoprotamine sensor can assess the activity of tryptic soy inhibitor.By under the changeless trypsinase concentration of every milliliter 50 unit, inhibitory reaction speed is estimated in the variation of current potential and nucleoprotamine concentration during initial 96 seconds after the potpourri that adds trypsase and soybean inhibitor.Result with respect to the soybean inhibitor concentration has been shown in Figure 13 a and 13b.Figure 13 a shows the potential change with respect to the soybean inhibitor concentration, and Figure 13 b shows the nucleoprotamine concentration change with respect to the soybean inhibitor concentration.When the soybean inhibitor concentration increases, shown in reaction rate reduce.

The instruction that the technician who belongs to field of the present invention provides in having benefited from narrating in front can be learnt many modification of the present invention described herein and other embodiment.Therefore, should be understood that the invention is not restricted to disclosed specific embodiment and these modification and other embodiment is believed to comprise within the scope of the appended claims.Though used particular term here, they only are used for the meaning described with generality, and purpose without limits.

Claims (42)

1. sensor, it comprises:

Be positioned at the electrode of shell, and

Be arranged in this shell an end and with the outer contacted film of sample solution of shell, the polyion concentration in the wherein said film test sample solution, and wherein said polyion concentration to reduce the interior enzymatic activity of speed and described sample solution in direct ratio.

2. sensor according to claim 1, wherein said electrode are the Ag/AgCl electrodes.

3. sensor according to claim 1, the surface area of wherein said film is about 10mm ²-100mm ²

4. sensor according to claim 3, the surface area of wherein said film is about 20mm ²-50mm ²

5. sensor according to claim 1, the average thickness of wherein said film are about 10 μ m-1000 μ m.

6. sensor according to claim 5, the average thickness of wherein said film are about 20 μ m-300 μ m.

7. sensor according to claim 1, the concentration of wherein said polyion are the concentration of nucleoprotamine.

8. sensor according to claim 1, the activity of wherein said enzyme are tryptic activity.

9. the method for polyion concentration in the test sample solution, this method comprises:

A kind of sample solution is provided; And

This sample solution is contacted with film, and wherein said film detects the polyion concentration in this sample solution, and the enzymatic activity that reduces in speed and the described sample solution of wherein said polyion concentration is in direct ratio.

10. method according to claim 9, the concentration of wherein said polyion are the concentration of nucleoprotamine.

11. method according to claim 9, the activity of wherein said enzyme are tryptic activity.

12. method according to claim 9, wherein said sample solution comprises biotic component.

13. method according to claim 9, wherein said sample solution is a blood.

14. a reversible fuel cell arrangement, it comprises:

The polyion selective membrane electrode that contains film, wherein said film detect the polyion concentration in this sample solution, and the enzymatic activity that reduces in speed and the described sample solution of wherein said polyion concentration is in direct ratio; And

Be used to apply the device that current potential cleans described film.

15. reversible fuel cell arrangement according to claim 14, wherein said polyion concentration are the concentration of nucleoprotamine.

16. reversible fuel cell arrangement according to claim 14, the activity of wherein said enzyme are tryptic activity.

17. reversible fuel cell arrangement according to claim 14, wherein said to be used to apply the device that current potential cleans described film be the external electrode current potential that applies between contrast electrode and polyion selective membrane electrode.

18. a reversible fuel cell arrangement, it comprises:

The polyion selective membrane electrode that contains film, wherein said film detect the polyion concentration in the described sample solution; And

Be used between contrast electrode and polyion selective membrane electrode, applying current potential to clean the device of described film.

19. a reversible fuel cell arrangement, it comprises:

Polyion concentration in the polyion selective membrane electrode that contains film, wherein said film test sample solution, and the enzymatic activity that reduces in speed and the described sample solution of wherein said polyion concentration is in direct ratio; And

Be used between contrast electrode and polyion selective membrane electrode, applying current potential to clean the device of this film.

20. reversible fuel cell arrangement according to claim 19, the concentration of wherein said polyion are the concentration of nucleoprotamine.

21. reversible fuel cell arrangement according to claim 19, the activity of wherein said enzyme are tryptic activity.

22. one kind makes the fuel cell arrangement reversal method, this method comprises:

A kind of sample solution is provided;

This sample solution is contacted with the polyion selective membrane electrode that contains film, and wherein said film detects the polyion concentration in this sample solution, and the enzymatic activity that reduces in speed and the described sample solution of wherein said polyion concentration is in direct ratio, and

Apply current potential to clean this film.

23. method according to claim 22, the concentration of wherein said polyion are the concentration of nucleoprotamine.

24. method according to claim 22, the activity of wherein said enzyme are tryptic activity.

25. method according to claim 22, wherein said sample solution comprises biotic component.

26. method according to claim 22, wherein said sample solution is a blood.

27. method according to claim 22, the wherein said current potential that is used to clean described film is applied between contrast electrode and the polyion selective membrane electrode.

28. a sensor, it comprises:

Be positioned at the electrode of shell, and

Be arranged in this shell an end and with the outer contacted film of sample solution of shell, wherein said film detects enzymatic activity and the corresponding inhibitor activity in the described sample solution.

29. sensor according to claim 28, wherein said electrode are the Ag/AgCl electrodes.

30. sensor according to claim 28, the surface area of wherein said film is about 10mm ²-100mm ²

31. sensor according to claim 30, the surface area of wherein said film is about 20mm ²-50mm ²

32. sensor according to claim 28, the average thickness of wherein said film are about 10 μ m-1000 μ m.

33. sensor according to claim 32, the average thickness of wherein said film are about 20 μ m-300 μ m.

34. sensor according to claim 28, the activity of wherein said enzyme are tryptic activity.

35. sensor according to claim 28, the activity of wherein said corresponding enzyme inhibitor are the activity of α 1-antiprotease inhibitor.

36. sensor according to claim 28, the activity of wherein said corresponding enzyme inhibitor are the activity of alpha2-macroglobulin.

37. sensor according to claim 28, the activity of wherein said corresponding enzyme inhibitor are the activity of α 1-Aprotinin.

38. sensor according to claim 28, the activity of wherein said corresponding enzyme inhibitor are the activity of soybean inhibitor.

39. sensor according to claim 28, wherein current potential reduces to depend on the concentration of corresponding enzyme inhibitor in the described sample solution.

40. sensor according to claim 28 also comprises being used to detect the immunoassays of deciding by the analyte of label mark.

41. according to the described sensor of claim 40, wherein said label is a polyion.

42. according to the described sensor of claim 40, wherein said label is an enzyme.

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