AU706362B2 - Improvement in ionic reservoir through application of an electrical potential - Google Patents
Improvement in ionic reservoir through application of an electrical potential Download PDFInfo
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- AU706362B2 AU706362B2 AU56403/96A AU5640396A AU706362B2 AU 706362 B2 AU706362 B2 AU 706362B2 AU 56403/96 A AU56403/96 A AU 56403/96A AU 5640396 A AU5640396 A AU 5640396A AU 706362 B2 AU706362 B2 AU 706362B2
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Description
WO 96/36871 PCT/AU96/00304 1 IMPROVEMENT IN IONIC RESERVOIR THROUGH APPLICATION
OFAN
ELECTRICAL POTENTIAL The present invention relates to an improved membrane based biosensor and to a method of improving the performance of membrane based biosensors.
Biosensors based on ion channels or ionophores contained within lipid membranes that are deposited onto metal electrodes, where the ion channels are switched in the presence of analyte molecules have been described in International Patent Application Nos WO 92/17788, WO 93/21528, WO 94/07593 and US 5,204,239 (the disclosures of which are incorporated herein by reference). As these biosensors rely on changes in ion conduction through the membrane, usually mediated by an ionophore, it is important that there exists an ionic reservoir between the electrode and the lipid membrane. Ideally this ionic reservoir between the electrode and the lipid reservoir is not totally depleted or filled, by conduction through the ionophore, during the course of the measurement cycle. The usual method of measuring the conductance changes is the use of alternating current (AC) impedance spectroscopy. The abovementioned disclosures have shown that good reservoirs can be produced using special linker lipid compounds.
The present inventors have now found that the application of a direct current (dc) potential offset superimposed onto the AC impedance signal can influence the apparent conduction of ions by the ionophore through the membrane. Without wishing to be bound by scientific theory it is believed that this modification of the ionophore conduction occurs through the modulation of the reservoir capacity and improvement in the reservoir homogeneity. This improvement in conduction of ions by the ionophore therefore allows the use of less ionophore which may be useful in producing more sensitive sensor membranes as less analyte is required to switch the ionophore on/off. A negative dc potential applied to the metal
I
WO 96/36871 PCT/AU96/00304 2 electrode has been shown to improve the ion conduction by ionophores, whereas a positive dc potential applied to the metal electrode has been shown to decrease and even negate the apparent conduction of the ionophores through the membrane. This effect is especially noticeable when membranes are formed containing phosphatidyl choline based lipids. The inventor has found that by controlling the dc offset, the reproducibility of the ionophore conduction is greatly improved.
Accordingly, in a first aspect the present invention consists in an improved membrane based biosensor comprising a lipid membrane incorporating ionophores the conductivity of the membrane being dependent on the presence or absence of an analyte, a reference electrode, a sensing electrode onto which is deposited the lipid membrane such that a functional reservoir exists between the lipid membrane and the sensing electrode, the improvement comprising including in the biosensor means to apply a dc electrical potential offset to the sensing electrode relative to the reference electrode.
In a second aspect the present invention consists in an improved method of detecting the presence or absence of an analyte in a sample using a membrane based biosensor comprising a lipid membrane incorporating ionophores the conductivity of the membrane being dependent on the presence or absence of the analyte, a reference electrode, a sensing electrode on to which is deposited the lipid membrane such that a functional reservoir exists between the lipid membrane and the sensing electrode, the improvement comprising applying a dc electrical potential offset to the sensing electrode relative to the reference electrode.
In a third aspect by incorporating ionisable, polarisable, dipolar or otherwise electroactive species within the membrane based biosensor comprising a lipid membrane incorporating ionophores the conductivity of the membrane being dependent on the presence or absence of an analyte, a reference electrode, a sensing electrode onto which is deposited the lipid WO 96/36871 PCT/AU96/00304 3 membrane such that a functional reservoir exists between the lipid membrane and the sensing electrode, the appropriate dc potential can be induced between the sensor electrode and the analyte solution.
Although it is envisaged that generally it is preferred to apply a negative potential onto the metal sensor electrode in order to improve the ionophore conduction, it may be useful in some circumstances to apply a positive potential onto the metal sensor electrode thus reducing or negating the apparent ionophore conduction through the membrane.
In a preferred embodiment of the present invention a dc potential of between +500mV to -500mV is applied to the sensing electrode.
In a further preferred embodiment the dc offset is produced through the use of a counter electrode where the electrochemical potential between the counter electrode and the sensing electrode produces an electrical potential of between 0 to -500mV, with the sensing electrode being at the negative potential.
In a preferred embodiment the counter electrode is made from stainless steel.
In a further preferred embodiment the counter electrode is made from titanium.
In a further preferred embodiment the counter electrode is made from silver, gold, platinum, palladium, copper, chromium or molybdenum.
In another preferred embodiment the counter electrode is made from metals that are capable of being deposited in a thin film onto a plastic, glass or silicon substrate, said metals being stable for at least 30 minutes in aqueous solution and sets up the appropriate electrochemical potential relative to the sensing electrode on addition of an aqueous solution.
In a further preferred embodiment of the present invention the counter electrode is an electrochemically neutral metal relative to the sensing electrode and the dc electrical potential of between +500 to -500mV is created by electronic means.
WO 96/36871 PCT/AU96/00304 4 In a further preferred embodiment of the present invention the counter electrode produces an electrochemical potential relative to the sensing electrode which is enhanced or negated or reversed using a dc electrical potential created by electronic means to give a potential of between +500 to -500mV.
In yet another preferred embodiment of the present invention, the dc offset potential at the sensing electrode, onto which is deposited the lipid membrane, is controlled using a three terminal measurement, where the impedance measurement is made between the counter electrode and the working electrode which is the sensing electrode and where the dc offset potential is controlled by a reference electrode to be between +500 to 500mV as required.
The metals used for the counter electrode and the reference electrode in the three terminal measurement may be any of the commonly used metals and electrode combinations commonly used in these measurements as known to those skilled in the art.
In a further preferred embodiment of the present invention the metal used for the sensing electrode is a layer of freshly evaporated or sputtered gold. Alternatively, a freshly cleaned gold surface, which can be produced using plasma etching or ion-beam milling, can be used.
It is further preferred that the first layer of the lipid membrane is produced using the linker lipid shown in figure 1, the disulfide of mercaptoacetic acid, linker gramicidin shown in figure 2, the membrane spanning lipid and the membrane spanning lipid both shown in figure 3.
It is further preferred that the second layer of the lipid membrane is produced from diphytanyl phosphatidyl choline, glycerol diphytanyl ether, shown in figure 7, and biotinylated gramicidin shown in figure 4.
WO 96/36871 PCT/AU96/00304 In a further preferred embodiment the second layer lipid contains at least a proportion of a phosphatidyl choline, or phophatidyl ethanolamine or phosphatidic acid lipid.
In a further preferred embodiment the second layer lipid contains at least a proportion of a charged lipid.
In a further preferred embodiment the lipid membrane is a monolayer.
As will be appreciated by those skilled in the art, if the sensing of an analyte occurs through the switching off or on of an ionophore contained within the lipid sensing membrane on addition of analyte, then it is possible to monitor this change in conduction by measuring the amount of electrical potential required in order to maintain the membrane conduction value at the initial ungated membrane conduction value. The magnitude and sign of the electrical potential is then related to the amount of analyte present in the sample.
By increasing the signal spectral inhomogeneity the information content in the signal can be increased with the consequent possibility of improved signal to noise. One mechanism for achieving this is to take advantage of the system voltage dependence by applying a non sinusoidal excitation and then analysing the results by fourier transform in which case the signal information content will be increased due to the cross modulation products in the output.
By automatically selecting a dc potential the sensitivity can be optimised. This may sometimes require the use of a calibrating dose of analyte for each measurement. (See Example 2 as a means of minimising drift.) The present invention also provides an improved method for detecting response to an analyte in which a signal may derived by altering and monitoring dcbias potential, while analyte is binding to the channels during the biosensor gating event, either to maintain the admittance WO 96/36871 PCT/AU96/00304 6 constant preferably at the frequency for minimum phase or similarly to maintain the phase constant preferably at the frequency for minimum phase.
The present invention further provides an improved method for detecting the electrode response to analyte in which the signal response is optimised by automatically altering the dc bias potential to obtain maximum sensitivity or minimum drift.
In order that the nature of the present invention may be more clearly understood the invention will now be described by way of non-limiting example.
Example 1 On a clean glass or plastic slide, an adhesion layer of chromium angstrom) followed by a gold layer (200-2000 angstroms) is evaporated. The freshly evaporated gold coated electrode is taken and immediately immersed in an ethanolic solution of linker lipid (Fig 1) (300ul of 10mM), the disulfide of mercaptoacetic acid (150ul of 10mM), linker gramicidin (Fig 2) (150ul of 0.01 mg/ml), membrane spanning lipid C (Fig 3)(2.25 ul of 0.1 mM) and membrane spanning lipid D (Fig 3) (45ul of 1 mM) in ethanol (50 ml). The gold coated electrode is left immersed in the solution for 5-60 minutes, rinsed with ethanol and assembled into a teflon slide assembly holder such that an electrode surface is defined by a circular teflon well pressed onto the gold electrode. The teflon well forms a tight, water impermeable seal at the electrode perimeter. This procedure forms the first layer of the bilayer sensor membrane and may be stored in ethanol, glycerol, ethylene glycol or other alcohol for several months. Formation of the second layer of the bilayer membrane is carried out by addition of 5ul of a solution containing 14mM of diphytanyl phosphatidyl choline/glyceryl diphytanyl ether (7:3 ratio), biotinylated gramicidin (Fig 4) in a ratio of 100,000:1 (total lipid):gramicidin. The well assembly was then rinsed twice with phosphate buffered saline (PBS) resulting the formation of the second lipid layer of the bilayer sensing membrane. The well assembly holds approximately 150ul of WO 96/36871 PCT/AU96/00304 7 PBS. Into this 150 ul of PBS in the well is placed a counter electrode, a connection is made between this counter electrode and the impedance bridge measuring apparatus. To complete the electrical circuit, the other connection is made between the gold electrode and the impedance bridge.
In order to control the dc potential offset a reference electrode is inserted into the well also contacting the PBS solution and the potential is controlled such that the gold electrode potential may be varied. The apparatus needed to make such three terminal measurements are known to those skilled in the art. Alternatively, the dc offset may be varied by changing the metal type which makes up the counter electrode. This sets up electrochemical potential between the counter electrode and the gold electrode. A dc offset may also be produced electronically in a two terminal measurement. Using the impedance bridge the conduction of the membrane may then be determined. Standard Bode plots are shown in figure 5. The effect of changing the counter electrode material, thus changing the potential, on gramicidin induced membrane conduction is shown. As can be seen stainless steel and titanium counter electrodes produce more conductive membranes than silver or gold counter electrodes when equivalent membrane sensor electrodes are measured.
Using a three terminal measurement it was found that the gramicidin induced membrane conduction increases as a negative potential is applied to the sensor membrane in the range of between OmV to -500mV. Figure 6 shows the effect of varying the potential on gramicidin containing membranes. As an indication of conduction the frequency at phase minimum is used. The higher the frequency at phase minimum, the more conductive the membrane. However, on application of a positive potential (OmV to +500mV) relative to the gold electrode the gramicidin induced membrane conduction decreased, such that at +200mV the membrane was ionically insulating.
WO 96/36871 PCT/AU96/00304 8 It is believed that using counter electrode metals such as stainless steel or titanium places a dc offset of between -150mV to -400mV on the gold electrode relative to the counter electrode. It has been further found that the reproducibility in terms of conduction for a particular concentration of ionophore in the membrane has been improved from coefficients of variation (cv's) of 30-60% using silver counter electrodes to cv's of 10-15% using stainless steel electrodes.
Similar effects were found if ionophores such as valinomycin were used instead of the gramicidin derivatives.
Example 2 A membrane was formed as described above in Example 1. A dc offset across the biosensor membrane was established using a three terminal voltage clamp with a platinum counter electrode, a silver chloride reference electrode and a gold sensing electrode.
Figure 10 shows the effect of varying the dc offset on the drift in the biosensor output. The output signal was the frequency at minimum phase.
The graph Y axis shows the rate of the frequency at minimum phase divided by the initial frequency at minimum phase.
The RC network in Figure 10 is a represntation of the passive electrical properties of the sensor membrane. This model consists of two capacitors connected in series, one with a value of 0.1 microFarad the second with a value of 0.01 microFarad and a resistor of about 300 kilOhm connected in parallel with the 0.01 microFarad capacitor. When this network was connected to the measuring apparatus the intrinsic drift in the apparatus was found to be negligible as is indicated in Fig It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the WO 96/36871 PCT/AU96/00304 9 invention as broadly described, the present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.
Claims (27)
1. An improved membrane based biosensor including a lipid membrane incorporating ionophores the conductivity of the membrane being dependent on the presence or absence of an analyte, a reference electrode, a sensing electrode on to which is deposited the lipid membrane such that a functional reservoir exists between the lipid membrane and the sensing electrode, the improvement comprising including in the biosensor means to apply a dc electrical potential offset to the sensing electrode relative to the reference electrode.
2. An improved membrane based biosensor according to claim 1, wherein the means to apply a dc electrical potential is a means capable of applying a dc electrical potential of between +500mV to -500mV to the sensing 15 electrode.
3. An improved membrane based biosensor according to claim 1 or 2, wherein the dc offset is produced by a counter electrode. 20 4. An improved membrane based biosensor according to claim 3, wherein the electrochemical potential between the counter electrode and the sensing electrode produces an electrical potential of between 0 to -500mV, with the sensing electrode being at the negative potential. C
5. An improved membrane based biosensor according to claim 3 or 4, wherein the counter electrode is made from stainless steel.
6. An improved membrane based biosensor according to claim 3 or 4, wherein the counter electrode is made from titanium. WO 96/36871 PCT/AU96/00304 11
7. An improved membrane based biosensor according to claim 3 or 4, wherein the counter electrode is made from silver, gold, platinum, palladium, copper, chromium or molybdenum.
8. An improved membrane based biosensor according to claim 3 or 4, wherein the counter electrode is made from a metal that is capable of being deposited in a thin film on to a plastic, glass or silicon substrate, said metal being stable for at least 30 minutes in aqueous solution and sets up the appropriate electrochemical potential relative to the sensing electrode on addition of an aqueous solution.
9. An improved membrane based biosensor according to any one of claims 3 to 8, wherein the counter electrode is an electrochemically neutral metal relative to the sensing electrode and the dc electrical potential of between +500mV to -500mV is created by electronic means. An improved membrane based biosensor according to any one of claims 3 to 8, wherein the counter electrode produces an electrochemical potential relative to the sensing electrode which is enhanced or negated or reversed using a dc electrical potential created by electronic means to give a potential of between +500mV to -500mV.
11. An improved membrane based biosensor according to any one of claims 3 to 10, wherein the dc offset potential at the sensing electrode, onto which is deposited a lipid membrane, is controlled using a three terminal measurement, wherein the impedance measurement is made between the counter electrode and the working electrode which is the sensing electrode and where the dc offset potential is controlled by a reference electrode to be between +500mV to -500mV as required.
12. An improved membrane based biosensor according to anyone of claims 3 to 11, wherein the sensing electrode comprises a metal.
13. An improved membrane based biosensor according to claim 12, wherein the metal used for the sensing electrode is a layer of freshly evaporated, sputtered, plasma etched or ion beam milled gold.
14. An improved membrane based biosensor according to any one of claims 3 to 13, wherein the lipid membrane includes a first layer of linker lipid (Fig the disulfide of mercaptoacetic acid, linker gramicidin (Fig 2), membrane spanning lipid C (Fig 3) and membrane spanning lipid D (Fig 3). An improved membrane based biosensor according to claim 14, wherein the lipid membrane includes a second layer of diphytanyl phosphatidyl choline, glycerol diphytanyl ether, and biotinylated gramicidin i (Fig 4).
16. An improved membrane based biosensor according to claim wherein the said second layer contains at least a proportion of a phosphatidyl 20 choline, or phosphatidyl ethanolamine or phosphatidic acid lipid.
17. An improved membrane based biosensor according to claims 15 or 16, wherein the said second layer contains at least a proportion of a charged lipid.
18. An improved membrane based biosensor according to any one of claims 1 to 13, wherein the lipid membrane is a monolayer.
19. An improved method of detecting the presence or absence of an analyte in a sample using a membrane based biosensor including a lipid membrane incorporating ionophores the conductivity of the membrane being dependent on the presence or absence of the analyte, a reference electrode, a sensing electrode on to which is deposited the lipid membrane such that a functional reservoir exists between the lipid membrane and the sensing electrode, the improvement including applying a dc electrical potential offset to the sensing electrode relative to the reference electrode. An improved method according to claim 19, wherein a dc electrical potential of between +500mV to -500mV is applied to the sensing electrode.
21. An improved method according to claim 19 or 20, wherein the dc offset is produced by a counter electrode.
22. An improved method according to claim 21, wherein the electrochemical potential between the counter electrode and the sensing electrode produces an electrical potential of between 0 to -500mV, with the sensing electrode being at the negative potential.
23. An improved method according to claim 21 or 22, wherein the counter electrode is made from stainless steel. S. *W S 24. An improved method according to claim 21 or 22, wherein the counter electrode is made from titanium.
25. An improved method according to claim 21 or 22, wherein the counter electrode is made from silver, gold, platinum, palladium, copper, chromium or molybdenum.
26. An improved method according to claim 21 or 22, wherein the counter electrode is made from a metal that is capable of being deposited in a thin 25 film on to a plastic, glass or silicon substrate, said metal being stable for at least 30 minutes in aqueous solution and sets up the appropriate electrode chemical potential relative to the sensing electrode on addition of an aqueous solution.
27. An improved method according to any one of claims 21 to 26, wherein the counter electrode is an electrochemically neutral metal relative to the sensing electrode and the dc electrical potential of between +500mV to -500mV is created by electronic means.
28. An improved method according to any one of claims 21 to 26, wherein the counter electrode produces an electrochemical potential relative to the sensing electrode which is enhanced or negated or reversed using a dc electrical potential created by electronic means to give a potential of between +500mV to -500mV.
29. An improved method according to any one of claims 21 to 28, wherein the dc offset potential at the sensing electrode, onto which is deposited a lipid membrane, is controlled using a three terminal measurement, wherein the impedance measurement is made between the counter electrode and the working electrode which is the sensing electrode and where the dc offset potential is controlled by a reference electrode to be between +500mV to -500mV as required. An improved method according to any of claims 21 to 29, wherein the sensing electrode comprises metal.
31. An improved method according to claim 30, wherein the metal used for the sensing electrode is a layer of freshly evaporated, sputtered, plasma etched or ion beam milled gold. 20 32. An improved method according to any one of claims 21 to 31, wherein the lipid membrane includes a first layer of linker lipid (Fig the disulfide of mercaptoacetic acid, linker gramicidin (Fig membrane spanning lipid C (Fig. 3) and membrane spanning lipid D (Fig 3). 25 33. An improved method according to claim 32, wherein the lipid membrane includes a second layer diphytanyl phosphatidyl choline, glycerol diphytanyl ether, and biotinylated gramicidin (Fig 4). a. a a
34. An improved method according to claim 33, wherein the said second layer contains at least a proportion of a phosphatidyl choline, or phosphatidyl ethanolamine or phosphatidic acid lipid. An improved method according to claim 33 or 34, wherein the said second layer contains at least a proportion of a charged lipid.
36. An improved method according to any one of claims 19 to 31, wherein the sensing membrane is a monolayer. Dated this twentieth day of April 1999, AUSTRALIAN MEMBRANE AND BIOTECHNOLOGY RESEARCH INSTITUTE and THE UNIVERSITY OF SYDNEY Patent Attorneys for the Applicant: F B RICE CO a a e too& 0:60 *o e e* ee
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU56403/96A AU706362B2 (en) | 1995-05-17 | 1996-05-17 | Improvement in ionic reservoir through application of an electrical potential |
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AUPN303195 | 1995-05-17 | ||
| AUPN3031 | 1995-05-17 | ||
| AU56403/96A AU706362B2 (en) | 1995-05-17 | 1996-05-17 | Improvement in ionic reservoir through application of an electrical potential |
| PCT/AU1996/000304 WO1996036871A1 (en) | 1995-05-17 | 1996-05-17 | Improvement in ionic reservoir through application of an electrical potential |
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| Publication Number | Publication Date |
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| AU5640396A AU5640396A (en) | 1996-11-29 |
| AU706362B2 true AU706362B2 (en) | 1999-06-17 |
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| Application Number | Title | Priority Date | Filing Date |
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| AU56403/96A Ceased AU706362B2 (en) | 1995-05-17 | 1996-05-17 | Improvement in ionic reservoir through application of an electrical potential |
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Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997025616A1 (en) * | 1996-01-11 | 1997-07-17 | Australian Membrane And Biotechnology Research Institute | Ion channel sensor typing |
| JP3775799B2 (en) * | 1996-02-08 | 2006-05-17 | アンブリ・リミテッド | Enzyme detection biosensor |
| AUPN980796A0 (en) * | 1996-05-13 | 1996-06-06 | Australian Membrane And Biotechnology Research Institute | Improved reservoir components |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1987000168A2 (en) * | 1985-06-27 | 1987-01-15 | Willis John P | Ionophores and ion-selective membranes containing the same |
| WO1992017788A1 (en) * | 1991-03-27 | 1992-10-15 | Australian Membrane And Biotechnology Research Institute | Ionic reservoir at electrode surface |
| US5368712A (en) * | 1989-11-02 | 1994-11-29 | Synporin Technologies, Inc. | Biologically mimetic synthetic ion channel transducers |
-
1996
- 1996-05-17 AU AU56403/96A patent/AU706362B2/en not_active Ceased
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1987000168A2 (en) * | 1985-06-27 | 1987-01-15 | Willis John P | Ionophores and ion-selective membranes containing the same |
| US5368712A (en) * | 1989-11-02 | 1994-11-29 | Synporin Technologies, Inc. | Biologically mimetic synthetic ion channel transducers |
| WO1992017788A1 (en) * | 1991-03-27 | 1992-10-15 | Australian Membrane And Biotechnology Research Institute | Ionic reservoir at electrode surface |
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| AU5640396A (en) | 1996-11-29 |
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