CA2245664A1 - Enzyme detection biosensors - Google Patents
Enzyme detection biosensors Download PDFInfo
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- CA2245664A1 CA2245664A1 CA002245664A CA2245664A CA2245664A1 CA 2245664 A1 CA2245664 A1 CA 2245664A1 CA 002245664 A CA002245664 A CA 002245664A CA 2245664 A CA2245664 A CA 2245664A CA 2245664 A1 CA2245664 A1 CA 2245664A1
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- membrane
- biosensor
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- enzyme
- ionophores
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
- G01N33/5438—Electrodes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/001—Enzyme electrodes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/001—Enzyme electrodes
- C12Q1/002—Electrode membranes
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/817—Enzyme or microbe electrode
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/806—Electrical property or magnetic property
Abstract
The present invention provides a biosensor for use in detecting the presence of an enzyme or enzymes in a sample. The biosensor comprises a membrane and means for determining the impedance of the membrane. The membrane includes ionophores therein to which are attached linkers. The linkers are cleavable by the enzyme or enzymes to be detected, with the cleavage of the linker causing a change in the ability of ions to pass through the membrane via the ionophores.
Description
CA 0224~664 l998-08-06 Enzyme Detec~on Biosensors The present invention relates to biosensors and methods involving the use of these biosensors in detecting the presence of enzymes by 5 detecting their enzymatic activity.
A number of proteins which are useful as immunodiagnostic analytes and disease markers have the additional property of enzymatic activitv~ in particular protease activity. In addition. other classes of proteins exhihibit nuclease activity.
Prostate Specific Antigen (PSA~, a dia~nostic marker for prostate cancer. is an example of a protein which exhibits protease activity, and belongs to the class of proteins known as the serine proteases. Examples of other proteases which are important immunodiagnostic markers include blood coagulation enzymes, elastase. cathepsin B.
There are also a number of important industrial enzvmes such as subtilisin, papain and a-amylase.
Examples of important nucleases are restriction enzvmes, e.g., ~3amH1. Hind III, polymerases which can act as nucleases under certain conditions! e.g., T4 l~NA polymerase, reverse transcriptase. ~hich acts as an 2 0 Rnase under certain conditions, e.g.. Rnase H, and exo- and endo-nucleases, e.g., S1 nuclease.
Current diagnostic tests employ immunoassays for the detection of PSA (e.g a number of analytical instruments such as Abbott's AXsym, Boehringer ~Iannheim's Elecsys, and CI~A-Corning's ACS-180. all have 25 ELISA-based PSA tests). These tests use antibodies raised against the PSA
molecule which recognise the specific epitope sites within the protein molecule.
A variation on these approaches is disclosed in International Patent application No. PCT/AU95/00536. In this reference there is disclosed a range 30 of substrates specifically cleaved by PSA. There is also disclosure in this reference of an assay system for proteases such as PSA which malce use of the activity of the protease. This assay system involves the use of a ligand to capture ~he PSA and the subsequent use of a substrate for the PSA.
The present inventors have developed devices and methods for the 35 detection of enzvmes which make use of the protein's protease activity.
- These devices and methods involve the use of membrane based biosensors.
CA 0224~664 1998-08-06 ?
Information regarding such biosensors can be found in International Patent Application Nos PCT/AU88/OOZ73, PCT/AU89/00352, PCT/AU90/00025, PCT/AIJ92/00132, PCT/AU93/00~09, PGT/AU93/00620, PCT/AU94/00202 and PCT/AU95/00763. The disclosure of each of these applications is included herein by reference.
The present invention involves providing a substrate for the enzyme to be detected and then sensing the digestion of the substrate by ~he enzyme.
This may be achieved in a number of ways, for example the digestion of the substrate may rernove a group from the ionophore thereby releasing the ionophore so that it diffuses laterally within the membrane or may result in an increase in the ability of ions to pass through the ionophore simply by a reduction in "steric" hindrance. Alternatively the digestion of the substrate vvhen attached to a membrane spanning component may result in the release of the ionophore such that it rmay diffuse laterally within the membrane.
Clearly this could also be achieved by digestion of substrates attached to both the ionophore and membrsne spanning component.
In another arrangement the digestion of the substrate results in the release of ionophore including probe which then inserts itself into the membrane.
Accordingly, in a first aspect the present invention consists in a biosensor for use in detecting the presence of an enzyme in a sample, the biosensor comprising a membrane and means for determining the impedance of the nlembralle. the membrane ha~Ting ionophores therein to which are attached linkers, the linkers being cleavable by the enzyme to be detected, the cleavage of the linker causing a change in the ability of iOIlS topass through the membrane via the ionophores.
In a preferred embodiment of the present invention the linker is attached to the membrane such that the ionophore is prevented from diffusing laterally within the membrane. It is preferred that the linker is attached to membrane spanning components provided in the membrane.
This attachment may be achieved in a number of ways such as covalent attachment, however. it is presently preferred that the attachment is achieved by providing on each of the linker and membrane sp~nn;ng component one member of a ligand binding pair. A preferred ligand binding pair is biotin streptavidin. In another preferred arran8ement both the - membrane spalming component alld the linker are provided with moieties -CA 0224~664 1998-08-06 which are both bound to the same molecule, for example biotin is provided on both the membrane spanning component and the linker and there is cross-linking via streptavidin.
The moiety on the membrane spRnning component may also be attached via a linker. This may be the same linker as that provided on the ionophore or may be different.
In a further preferred embodiment the membrane comprises a first and second layer of a closely packed array of amphiphilic molecules, a plurality of ionophores and a plurality of membrane-sp~nning lipids prevented from lateral diffusion in the membrane, the ionophores comprising first and second half membrane spRnning monomers. the first half membrane spanning monomers being provided in the first layer and the second half membrane spRnning monomers being provided iIl the second layer. the first half membrane sp~nning monomers being prevented from lateral diffusion in the first layer, the second half membrane sp~nning monomers being linked to the membrane sp~nning lipids via the linker.
Following cleavage of the linker by the enzyme the second half membrane spanning monomers can diffuse laterally within the second layer independent of the first half membrane spRnn;ng monomers.
II1 a second aspect the present invention consists in a biosensor for use in detecting the presence of an enzyme in a sample! the biosensor comprising a membrane and means for determining the impedance of the membrane, the membrane having a plurality of ionophores and a plurality of membrane-spaIlning components therein, the membralle-spR n n i ng components having attached thereto linker molecules to which are connected the ionophores, the linker molecules being cleavable by the enzyme to be detected, the cleavage of the linker molecules causing a change iIl the abilitv of ions to pass through the membrane via the lonophores.
A number of proteins which are useful as immunodiagnostic analytes and disease markers have the additional property of enzymatic activitv~ in particular protease activity. In addition. other classes of proteins exhihibit nuclease activity.
Prostate Specific Antigen (PSA~, a dia~nostic marker for prostate cancer. is an example of a protein which exhibits protease activity, and belongs to the class of proteins known as the serine proteases. Examples of other proteases which are important immunodiagnostic markers include blood coagulation enzymes, elastase. cathepsin B.
There are also a number of important industrial enzvmes such as subtilisin, papain and a-amylase.
Examples of important nucleases are restriction enzvmes, e.g., ~3amH1. Hind III, polymerases which can act as nucleases under certain conditions! e.g., T4 l~NA polymerase, reverse transcriptase. ~hich acts as an 2 0 Rnase under certain conditions, e.g.. Rnase H, and exo- and endo-nucleases, e.g., S1 nuclease.
Current diagnostic tests employ immunoassays for the detection of PSA (e.g a number of analytical instruments such as Abbott's AXsym, Boehringer ~Iannheim's Elecsys, and CI~A-Corning's ACS-180. all have 25 ELISA-based PSA tests). These tests use antibodies raised against the PSA
molecule which recognise the specific epitope sites within the protein molecule.
A variation on these approaches is disclosed in International Patent application No. PCT/AU95/00536. In this reference there is disclosed a range 30 of substrates specifically cleaved by PSA. There is also disclosure in this reference of an assay system for proteases such as PSA which malce use of the activity of the protease. This assay system involves the use of a ligand to capture ~he PSA and the subsequent use of a substrate for the PSA.
The present inventors have developed devices and methods for the 35 detection of enzvmes which make use of the protein's protease activity.
- These devices and methods involve the use of membrane based biosensors.
CA 0224~664 1998-08-06 ?
Information regarding such biosensors can be found in International Patent Application Nos PCT/AU88/OOZ73, PCT/AU89/00352, PCT/AU90/00025, PCT/AIJ92/00132, PCT/AU93/00~09, PGT/AU93/00620, PCT/AU94/00202 and PCT/AU95/00763. The disclosure of each of these applications is included herein by reference.
The present invention involves providing a substrate for the enzyme to be detected and then sensing the digestion of the substrate by ~he enzyme.
This may be achieved in a number of ways, for example the digestion of the substrate may rernove a group from the ionophore thereby releasing the ionophore so that it diffuses laterally within the membrane or may result in an increase in the ability of ions to pass through the ionophore simply by a reduction in "steric" hindrance. Alternatively the digestion of the substrate vvhen attached to a membrane spanning component may result in the release of the ionophore such that it rmay diffuse laterally within the membrane.
Clearly this could also be achieved by digestion of substrates attached to both the ionophore and membrsne spanning component.
In another arrangement the digestion of the substrate results in the release of ionophore including probe which then inserts itself into the membrane.
Accordingly, in a first aspect the present invention consists in a biosensor for use in detecting the presence of an enzyme in a sample, the biosensor comprising a membrane and means for determining the impedance of the nlembralle. the membrane ha~Ting ionophores therein to which are attached linkers, the linkers being cleavable by the enzyme to be detected, the cleavage of the linker causing a change in the ability of iOIlS topass through the membrane via the ionophores.
In a preferred embodiment of the present invention the linker is attached to the membrane such that the ionophore is prevented from diffusing laterally within the membrane. It is preferred that the linker is attached to membrane spanning components provided in the membrane.
This attachment may be achieved in a number of ways such as covalent attachment, however. it is presently preferred that the attachment is achieved by providing on each of the linker and membrane sp~nn;ng component one member of a ligand binding pair. A preferred ligand binding pair is biotin streptavidin. In another preferred arran8ement both the - membrane spalming component alld the linker are provided with moieties -CA 0224~664 1998-08-06 which are both bound to the same molecule, for example biotin is provided on both the membrane spanning component and the linker and there is cross-linking via streptavidin.
The moiety on the membrane spRnning component may also be attached via a linker. This may be the same linker as that provided on the ionophore or may be different.
In a further preferred embodiment the membrane comprises a first and second layer of a closely packed array of amphiphilic molecules, a plurality of ionophores and a plurality of membrane-sp~nning lipids prevented from lateral diffusion in the membrane, the ionophores comprising first and second half membrane spRnning monomers. the first half membrane spanning monomers being provided in the first layer and the second half membrane spRnning monomers being provided iIl the second layer. the first half membrane sp~nning monomers being prevented from lateral diffusion in the first layer, the second half membrane sp~nning monomers being linked to the membrane sp~nning lipids via the linker.
Following cleavage of the linker by the enzyme the second half membrane spanning monomers can diffuse laterally within the second layer independent of the first half membrane spRnn;ng monomers.
II1 a second aspect the present invention consists in a biosensor for use in detecting the presence of an enzyme in a sample! the biosensor comprising a membrane and means for determining the impedance of the membrane, the membrane having a plurality of ionophores and a plurality of membrane-spaIlning components therein, the membralle-spR n n i ng components having attached thereto linker molecules to which are connected the ionophores, the linker molecules being cleavable by the enzyme to be detected, the cleavage of the linker molecules causing a change iIl the abilitv of ions to pass through the membrane via the lonophores.
3 0 In a preferred embodiment the membrane comprises a first and second layer of a closely packed array of amphiphilic molecules and the membrane-spanning components are prevented from lateral diffusion in the membrane. Preferably the ionophores comprise first and second half membrane spannillg moIlomers. the first half membrane sp~ntling monomers being provided in the first layer and the second half membrane sp~nning - monomers being provided in the second layer with the first half membrane CA 0224~664 1998-08-06 W O 97/29366 PCT/AUg7/00071 SpZ~nning monomers being prevented from lateral diffusion in the first layer.
The second half membrane sp~nning monomers are connected to the membrane-spRnning components via the linker molecule.
The ionophores in both these aspects are preferably gramicidin or 5 analogues thereof.
While a range of enzymes can be detected using the biosensor or the present invention the biosensor is particularly useful in the detection of proteases. in particular those of clinical importance such as PSA, fibrinogen etc.
II1 a third aspect the present invention consists in a biosensor for the detection of enzymes comprisin~g first and second zones! means to allow addition of a sample suspected to contain an enzyme to the first zone, the first zone containing a probe linked to a carrier via a linker cleavable by the enzvme and means to allow passage of n~ lk~d probe from the first zone to 15 the second zone; the second zone including a membrane the impedance of which is dependent on the presence or absence of the probe and means to measure the impedance of the membrane.
In a preferred embodiment of this aspect of the present invention the membrane comprises a first and a second layer of a closely packed array of 20 amphiphilic molecules and a plurality of ionophores comprising a first and second half membrane sp~nning monomers. the first half membrane spanning monomers being provided in the first layer and the second half membralle spanniIlg monomers being provided in the second layer, the second half membrane sp~nning monomers being capable of lateral diffusion 25 within the second laver independent of the first half membrane sp~nning monomers. the first half membrane sp~nning monomers being prevented from lateral diffusion in the first layer, and a ligand provided on at least thesecond half membrane sp~nning monomers, said ligand being reactive with the probe or a portion thereof, the binding of the probe to the ligand causing 30 a change in the relationship between the first half membrane sp~nning monomers and the second half me~nbrane sp~llning monomers such that the flow of iOllS across the membrane via the ionophores is allowed or prevented.
II1 a preferred embodiment the probe includes streptavidin and the 35 li,gand includes biotin.
CA 0224~664 1998-08-06 W O 97/29366 PCT/AU97/OOn71 In yet another preferred embodiment the probe includes an ionophore such that when the probe comes into contact with the membrane the ionophore inserts itself into the membrane r:hflnging the impedance of the membrane. As an example of such an arrangement the probe may 5 include valinomycin which inserts itself into the membrane.
In a preferred embodiment of the present invention the enzyme to be detected is a protease in particular Prostate Specific Antigen. In this case it is preferred that the linker or linker molecule includes the sequence Ala-Val-Tyr.
As will be recognised by those skilled in the art the actual linker used will depend on the enzyme to be detected. Examples of some enzymes and their corresponding substrates are set out in VVhittaker et al. Analytical Biochemish~y: Z20, 238-243 (1994). the disclosure of which is incorporated by cross-reference In a further aspect the present invention consists in a method of detecting the presence of an enzyme in a sample comprising adding the sample to the biosensor of the first or second or third aspect of the present invention and measuring the change in impedance of the membrane.
As ~rill be readily apparent the biosensors and methods of the 2 0 present inventiol1 do not detect total enzyme. they detect only active enzyme. This is important as in a number of situations it is the amount of active enzyme present which is of importance not simply the total amount of enzyme present as would be measured in a standard sandwich ~:LISA.
It t~Till also be apparent that the sensors of the present invention can be used to detect a wide range of enzymes. These enzymes include nucleases, protease amylases etc. The sensors are adapted to the particular enzyme to be detected by adjusting the make-up of the linker. For example to detect proteases the linker will typically include a peptide portion which is cleaved by the enzyme. Information re,garding peptide sequences cleaved by specific proteases is provided in Whittaker et al referred to above. Where the eIlzyme to be detected is a nuclease the linker will typically include a nucleic acid sequence. Information regarding specific sequences cleaved by specific enzvmes can be found in "Current Protocols in Molecular Biology"
Ausebel et al (1987) John Wiley & SOI1S, NY.
The sensors of the present invention may also find use in drug - de~elopment for determinin~ DNA-dru~g binding sites The sensors could CA 0224~664 1998-08-06 W O 97/29366 PCT/AU97/~0071 also be used in determ~ning DNA-protein binding sites. The sensors may also find use iIl diagnosing infection. For example the sensors could be used to detect enzyme activity specifically associated with a pathogen.
Industrially and clinically relevant proteases and substrates include thrombin and serine proteases including PSA. A list of lysis enzymes is found in "Specificity of Proteolysis" Borivoj Keil (1992) Springer Verlag NY
pp. 283-323. Useful ones are the serine and cysteine proteases. See also "Proteolyhc Enzymes": a Prachcal Approach" R.J. Benyon & J.S. Bond (eds) 1989 Oxford University Press NY p232, pp. 241-z49. Commercially significant proteases and protease inhibitors for which the present technology is relevant are available in serine, cysteine, aspartic and metallo types. The serine proteases incIude the endoproteinase-Arg-C, -Glu-C, Lys-C, factor Xa, proteinase K. subtilisin and trypsin. and the exopeptidases acylamino-acid-releasing enzyme, carboxypeptidase P, and carboxypeptidase Y. The cysteine proteases include the endopeptidases bromelain, cathepsin B! closhipain~ papain, and the exopeptidases cathepsin C and pyroglutamate aminopeptidase. The aspartic proteases include the endopeptidases cathepsin D and pepsin. The metallo proteases include the endopeptidase thermolysin and the exopeptidases aminopeptidase M. carboxypeptidase-A, -B and leucine aminopeptidase. The listing is not intended to be exclusive and indicates the broad utility of the present invention. Other commercially useful proteases are listed in the publications cited above, which are included herein by reference. For example it aIso includes the endopeptide endoproteinase-Asp-N of unknown type.
In order that the nature of the present invention may be more clearly understood preferred forms thereof will now be described by reference to the following E7camples and accompanying Figures.
Figure 1 shows a schematic representation of an embodiment of the 3 0 device of the third aspect of the present invention. As can be seen from this Figure the device 10 includes a first zone 11 and a second zone 12. First zone 11 is provided with polymer ~eads 13 (carrier) linked to streptavidin 14 (probe) via a peptide linker 15. The peptide linker 15 is cleavable by the protease 16.
As shown in this Figure upon addition of the protease (or a nuclease) - 16 the sheptavidin 14 is released and passes to the second zone 12. Second CA 0224~664 1998-08-06 ?
zone 1~ includes a biosensor membrane 17 which detects the presence of sheptavidin 14. Streptavidin 14 reaching biosensor membrane 17 causes a change in the impedance of the membrane.
Figure 2 shows an embodiment of the first and/or second aspect of 5 the invention. As shown in Figure 2 the biosensor membrane 20 includes a membrane 21 and electrode 22. The membrane 21 has a first layer 23 and second layer 24 of arrays of amphiphilic molecules. Included in layer 24 is a first half men1brane-sp~nnitlg monomer 25 which is prevented from lateral diffusion within the membrane. Layer 23 includes a second half membrane-10 sp;~nning monomer 26. The membrane also includes a membrane-spRnning lipid 27 which is also prevented from diffusing laterally within the membrane. The second half membrane-sp~Tlnillg monomer 26 is linked to the membralle-spanning lipid Z7 via a peptide 28. The peptide 28 is cleavable by protease 29. Upon cleavage of the peptide 28 by protease 29 the 15 half membrane-spal1ning monomer 26 is free to diffuse laterallv within the membrane. This results in a change in impedance of the membrane.
Examples 20 Example 1:
Protease cleavage of streptavidin-gramicidil1 linkage 1st layer: 9.3nM Linker Gramicidin B ~Fig 3) ~ M Membrane Spanner Lipid D (Fig 4) 3 711~I MAAD ~Fig 5) 75~ Linker Lipid ~ ~Fig 6) 2nd layer: 10n~I (DPE-PC (Fig 7):GDPE (Fig 8~ = 7:3): Biotinylated Gramicidin F (Fig 9) = 6~i,677:1 in ethanol.
Elechodes with freshly evaporated gold (1000A) on a chrome adhesion layer (200A on glass microscope slides) were dipped into an ethanolic solution of the first layer components for 1 hour at room 35 temperature. ril1sed with ethanol. then stored at 4~C under ethanol until - used for impedance meas-lrements. The slide was clamped into a block .
containIng teflon coated wells which defined the area of the working electrode as approximately l6mm2.
5~L of the second layer was added to the working electrode before addition of a 150,uL volume of phosphate buffered saline (6.26mM NaCI, 59.4mM NaH~PO4.ZH2O, Z.53mM Na~HPO4. 12H2O, 50mM EDTA at pH 7.4;
PBS). The electrode was then washed 4 times using PBS and raised to 60~C
over a 30 minute period. Streptavidin was added to the sensor wells ~5,uL
0.01mg/ml in PBS) and incubated. The bindin8 Of streptavidin to the biotinylated gramicidin E gave a decrease in the admittance at minimum phase (Figure 10~. After 15 minutes the excess streptavidin was washed out with PBS. Wells with no added streptavidin were run as controls.
Proteinase K was added to sensing and control wells to give end well concentration at 12.5mglml (Boehringer Mannheim D-682~8 made in PBS).
Addition of Proteinase K to control wells caused no significant change in membrane admittance characteristic. Sensor membranes to which streptavidin was bound exhibited an increase in admittance at minimum phase (Figure 11). The amount and rate of increase of admittance at minimum phase is related to the amount of proteinase K present in the test solution and therefore can be used to determine enzymatic activity in test 2 0 solutiolls.
Example 2:
Dnase 1 cleavage of DNA-bound channels 2s 1st layer: 9.3nM Linker Gramicidin B
LM Membrane Spanner Lipid D
27.5nM Membrane Spanner Lipid C (Fig 123 37~M MAAD
75~I Linker Lipid A
Znd layer: 14n~I (DPE-PC:GDPE = 7:3): Biotinylated Grarmicidin E
=50.000:1 in ethanol.
Electrodes ~ith freshly evaporated gold (loooA) OIl a chrome - adhesioll layer (200A) on glass microscope slides) were dipped into an CA 0224~664 1998-08-06 ethanolic solution of the first layer components for 1 hour at room temperature. rinsed with ethanol, then stored at 4~C under ethanol until used for impedance measurements. The slide was clamped into a block containing teflon coated wells which defined the area of the working electrode as approximately 16mm2.
5~1L of the second layer was added to the working electrode before addition of a 180~L volume of phosphate buffered saline (lOmM NaHzPO4, lm~f KH2PO4, 137mM NaCl, 2.7mM KCl: PBS). The electrode was washed 4 times using PBS. These steps were carried out at room temperature. All the subseguent steps were carried out at 30~C. Streptavidin was added to all the wells (5~1L O.Olmg/ml in PBS) and allowed to react with biotinylated gramicidin E for 10-15 minutes before washing out excess unbound streptavidin with PBS, 5~L of a 1:1 mixture of DNA probe F (200n~): DNA
probe G (200nM in PBS) was added to the sensor wells. A DNA non-specific binding probe H (5,uL 400 nM in PBS) was added to control wells. Binding probe H is non-complementary to the target DNA of interest and hence target DNA should not bind. The probes were allowed to react with streptavidin for 10-15 minutes then excess unbound probes were washed out with PBS.
100 uL of DNA target I (10nM) in PBS was added to each well. The binding of DNA target I to the sensor wells gave a decrease in the admittance at minimum phase, but no significant change in meml~rane admittance in control wells (Figure 13). After 15 mil1utes unbound DNA target I was washed out with DNase 1 activation buf~er. DNase 1 activation buf~er consists of 50nM Tris. ~ICl, pH 7.6. 5QnM NaCl. 10n~ MgClz, 10nM MnClz, 0.2 mg/mL BSA. DNase 1 was added (2~lL lmg/mL in a 50%w/v glycerol solution of 2~mM Tris.HCl, p~ 7.~. lmM MgClz) to sensor and control wells.
Addition of DNase 1 gave an increase in admittance at minimum phase for sensor wells. but no significant change for control wells (Figure 14). The amount and rate of increase of admittance at minimum phase is related to the amount of DNase 1 present in the test solution and therefore can be used to determine enZymatiC activity in test solutions.
DNA probe F:
5'biotinylated listeria probe DNA with a 31-atom phosphoramidite linker group between the biotin and DNA.
5 !-bio-L-M-ATA~ l 11 lATGGGATTAGC-3' DNA probe G:
5'biotinylated cholera toxin probe DNA with a 13-atom phosphoramidite linker group between the biotin and DNA.
5 '-bio-L-CTCCGGAGCATAGAGCTTGGAGG-3 ' DNA non-specific binding probe H:
5'biotinvlated 15-mer oligonucleotide with a 31-atom phosphoramidite linker group between the biotin and DNA, which is non-complementary to al~ parts o~ the target DNA sequence.
5 '-bio-L-M-ATTGCTACGTATACG-3 ' ~ 0 DNA target I:
5Z base DNA seguence containing the 1~-base listeria sequence, a 10 I}ase 'spacer and the 23 base cholera toxin sequence.
5~-CCTl~ATCCCAT~CTATGCI~TGCI~'I ~TCC T CCA~GCTCTl~ l GCTCCGG~G-3-CA 02245664 l998-08-06 where.
bio = biotin NH ~ o - DMT
o--11--O
O
M=
O ~ 0~~~O ~o~
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 invention as broadly described. The present embodiments are. therefore. to be considered in all respects as illustrative and not restrictive.
The second half membrane sp~nning monomers are connected to the membrane-spRnning components via the linker molecule.
The ionophores in both these aspects are preferably gramicidin or 5 analogues thereof.
While a range of enzymes can be detected using the biosensor or the present invention the biosensor is particularly useful in the detection of proteases. in particular those of clinical importance such as PSA, fibrinogen etc.
II1 a third aspect the present invention consists in a biosensor for the detection of enzymes comprisin~g first and second zones! means to allow addition of a sample suspected to contain an enzyme to the first zone, the first zone containing a probe linked to a carrier via a linker cleavable by the enzvme and means to allow passage of n~ lk~d probe from the first zone to 15 the second zone; the second zone including a membrane the impedance of which is dependent on the presence or absence of the probe and means to measure the impedance of the membrane.
In a preferred embodiment of this aspect of the present invention the membrane comprises a first and a second layer of a closely packed array of 20 amphiphilic molecules and a plurality of ionophores comprising a first and second half membrane sp~nning monomers. the first half membrane spanning monomers being provided in the first layer and the second half membralle spanniIlg monomers being provided in the second layer, the second half membrane sp~nning monomers being capable of lateral diffusion 25 within the second laver independent of the first half membrane sp~nning monomers. the first half membrane sp~nning monomers being prevented from lateral diffusion in the first layer, and a ligand provided on at least thesecond half membrane sp~nning monomers, said ligand being reactive with the probe or a portion thereof, the binding of the probe to the ligand causing 30 a change in the relationship between the first half membrane sp~nning monomers and the second half me~nbrane sp~llning monomers such that the flow of iOllS across the membrane via the ionophores is allowed or prevented.
II1 a preferred embodiment the probe includes streptavidin and the 35 li,gand includes biotin.
CA 0224~664 1998-08-06 W O 97/29366 PCT/AU97/OOn71 In yet another preferred embodiment the probe includes an ionophore such that when the probe comes into contact with the membrane the ionophore inserts itself into the membrane r:hflnging the impedance of the membrane. As an example of such an arrangement the probe may 5 include valinomycin which inserts itself into the membrane.
In a preferred embodiment of the present invention the enzyme to be detected is a protease in particular Prostate Specific Antigen. In this case it is preferred that the linker or linker molecule includes the sequence Ala-Val-Tyr.
As will be recognised by those skilled in the art the actual linker used will depend on the enzyme to be detected. Examples of some enzymes and their corresponding substrates are set out in VVhittaker et al. Analytical Biochemish~y: Z20, 238-243 (1994). the disclosure of which is incorporated by cross-reference In a further aspect the present invention consists in a method of detecting the presence of an enzyme in a sample comprising adding the sample to the biosensor of the first or second or third aspect of the present invention and measuring the change in impedance of the membrane.
As ~rill be readily apparent the biosensors and methods of the 2 0 present inventiol1 do not detect total enzyme. they detect only active enzyme. This is important as in a number of situations it is the amount of active enzyme present which is of importance not simply the total amount of enzyme present as would be measured in a standard sandwich ~:LISA.
It t~Till also be apparent that the sensors of the present invention can be used to detect a wide range of enzymes. These enzymes include nucleases, protease amylases etc. The sensors are adapted to the particular enzyme to be detected by adjusting the make-up of the linker. For example to detect proteases the linker will typically include a peptide portion which is cleaved by the enzyme. Information re,garding peptide sequences cleaved by specific proteases is provided in Whittaker et al referred to above. Where the eIlzyme to be detected is a nuclease the linker will typically include a nucleic acid sequence. Information regarding specific sequences cleaved by specific enzvmes can be found in "Current Protocols in Molecular Biology"
Ausebel et al (1987) John Wiley & SOI1S, NY.
The sensors of the present invention may also find use in drug - de~elopment for determinin~ DNA-dru~g binding sites The sensors could CA 0224~664 1998-08-06 W O 97/29366 PCT/AU97/~0071 also be used in determ~ning DNA-protein binding sites. The sensors may also find use iIl diagnosing infection. For example the sensors could be used to detect enzyme activity specifically associated with a pathogen.
Industrially and clinically relevant proteases and substrates include thrombin and serine proteases including PSA. A list of lysis enzymes is found in "Specificity of Proteolysis" Borivoj Keil (1992) Springer Verlag NY
pp. 283-323. Useful ones are the serine and cysteine proteases. See also "Proteolyhc Enzymes": a Prachcal Approach" R.J. Benyon & J.S. Bond (eds) 1989 Oxford University Press NY p232, pp. 241-z49. Commercially significant proteases and protease inhibitors for which the present technology is relevant are available in serine, cysteine, aspartic and metallo types. The serine proteases incIude the endoproteinase-Arg-C, -Glu-C, Lys-C, factor Xa, proteinase K. subtilisin and trypsin. and the exopeptidases acylamino-acid-releasing enzyme, carboxypeptidase P, and carboxypeptidase Y. The cysteine proteases include the endopeptidases bromelain, cathepsin B! closhipain~ papain, and the exopeptidases cathepsin C and pyroglutamate aminopeptidase. The aspartic proteases include the endopeptidases cathepsin D and pepsin. The metallo proteases include the endopeptidase thermolysin and the exopeptidases aminopeptidase M. carboxypeptidase-A, -B and leucine aminopeptidase. The listing is not intended to be exclusive and indicates the broad utility of the present invention. Other commercially useful proteases are listed in the publications cited above, which are included herein by reference. For example it aIso includes the endopeptide endoproteinase-Asp-N of unknown type.
In order that the nature of the present invention may be more clearly understood preferred forms thereof will now be described by reference to the following E7camples and accompanying Figures.
Figure 1 shows a schematic representation of an embodiment of the 3 0 device of the third aspect of the present invention. As can be seen from this Figure the device 10 includes a first zone 11 and a second zone 12. First zone 11 is provided with polymer ~eads 13 (carrier) linked to streptavidin 14 (probe) via a peptide linker 15. The peptide linker 15 is cleavable by the protease 16.
As shown in this Figure upon addition of the protease (or a nuclease) - 16 the sheptavidin 14 is released and passes to the second zone 12. Second CA 0224~664 1998-08-06 ?
zone 1~ includes a biosensor membrane 17 which detects the presence of sheptavidin 14. Streptavidin 14 reaching biosensor membrane 17 causes a change in the impedance of the membrane.
Figure 2 shows an embodiment of the first and/or second aspect of 5 the invention. As shown in Figure 2 the biosensor membrane 20 includes a membrane 21 and electrode 22. The membrane 21 has a first layer 23 and second layer 24 of arrays of amphiphilic molecules. Included in layer 24 is a first half men1brane-sp~nnitlg monomer 25 which is prevented from lateral diffusion within the membrane. Layer 23 includes a second half membrane-10 sp;~nning monomer 26. The membrane also includes a membrane-spRnning lipid 27 which is also prevented from diffusing laterally within the membrane. The second half membrane-sp~Tlnillg monomer 26 is linked to the membralle-spanning lipid Z7 via a peptide 28. The peptide 28 is cleavable by protease 29. Upon cleavage of the peptide 28 by protease 29 the 15 half membrane-spal1ning monomer 26 is free to diffuse laterallv within the membrane. This results in a change in impedance of the membrane.
Examples 20 Example 1:
Protease cleavage of streptavidin-gramicidil1 linkage 1st layer: 9.3nM Linker Gramicidin B ~Fig 3) ~ M Membrane Spanner Lipid D (Fig 4) 3 711~I MAAD ~Fig 5) 75~ Linker Lipid ~ ~Fig 6) 2nd layer: 10n~I (DPE-PC (Fig 7):GDPE (Fig 8~ = 7:3): Biotinylated Gramicidin F (Fig 9) = 6~i,677:1 in ethanol.
Elechodes with freshly evaporated gold (1000A) on a chrome adhesion layer (200A on glass microscope slides) were dipped into an ethanolic solution of the first layer components for 1 hour at room 35 temperature. ril1sed with ethanol. then stored at 4~C under ethanol until - used for impedance meas-lrements. The slide was clamped into a block .
containIng teflon coated wells which defined the area of the working electrode as approximately l6mm2.
5~L of the second layer was added to the working electrode before addition of a 150,uL volume of phosphate buffered saline (6.26mM NaCI, 59.4mM NaH~PO4.ZH2O, Z.53mM Na~HPO4. 12H2O, 50mM EDTA at pH 7.4;
PBS). The electrode was then washed 4 times using PBS and raised to 60~C
over a 30 minute period. Streptavidin was added to the sensor wells ~5,uL
0.01mg/ml in PBS) and incubated. The bindin8 Of streptavidin to the biotinylated gramicidin E gave a decrease in the admittance at minimum phase (Figure 10~. After 15 minutes the excess streptavidin was washed out with PBS. Wells with no added streptavidin were run as controls.
Proteinase K was added to sensing and control wells to give end well concentration at 12.5mglml (Boehringer Mannheim D-682~8 made in PBS).
Addition of Proteinase K to control wells caused no significant change in membrane admittance characteristic. Sensor membranes to which streptavidin was bound exhibited an increase in admittance at minimum phase (Figure 11). The amount and rate of increase of admittance at minimum phase is related to the amount of proteinase K present in the test solution and therefore can be used to determine enzymatic activity in test 2 0 solutiolls.
Example 2:
Dnase 1 cleavage of DNA-bound channels 2s 1st layer: 9.3nM Linker Gramicidin B
LM Membrane Spanner Lipid D
27.5nM Membrane Spanner Lipid C (Fig 123 37~M MAAD
75~I Linker Lipid A
Znd layer: 14n~I (DPE-PC:GDPE = 7:3): Biotinylated Grarmicidin E
=50.000:1 in ethanol.
Electrodes ~ith freshly evaporated gold (loooA) OIl a chrome - adhesioll layer (200A) on glass microscope slides) were dipped into an CA 0224~664 1998-08-06 ethanolic solution of the first layer components for 1 hour at room temperature. rinsed with ethanol, then stored at 4~C under ethanol until used for impedance measurements. The slide was clamped into a block containing teflon coated wells which defined the area of the working electrode as approximately 16mm2.
5~1L of the second layer was added to the working electrode before addition of a 180~L volume of phosphate buffered saline (lOmM NaHzPO4, lm~f KH2PO4, 137mM NaCl, 2.7mM KCl: PBS). The electrode was washed 4 times using PBS. These steps were carried out at room temperature. All the subseguent steps were carried out at 30~C. Streptavidin was added to all the wells (5~1L O.Olmg/ml in PBS) and allowed to react with biotinylated gramicidin E for 10-15 minutes before washing out excess unbound streptavidin with PBS, 5~L of a 1:1 mixture of DNA probe F (200n~): DNA
probe G (200nM in PBS) was added to the sensor wells. A DNA non-specific binding probe H (5,uL 400 nM in PBS) was added to control wells. Binding probe H is non-complementary to the target DNA of interest and hence target DNA should not bind. The probes were allowed to react with streptavidin for 10-15 minutes then excess unbound probes were washed out with PBS.
100 uL of DNA target I (10nM) in PBS was added to each well. The binding of DNA target I to the sensor wells gave a decrease in the admittance at minimum phase, but no significant change in meml~rane admittance in control wells (Figure 13). After 15 mil1utes unbound DNA target I was washed out with DNase 1 activation buf~er. DNase 1 activation buf~er consists of 50nM Tris. ~ICl, pH 7.6. 5QnM NaCl. 10n~ MgClz, 10nM MnClz, 0.2 mg/mL BSA. DNase 1 was added (2~lL lmg/mL in a 50%w/v glycerol solution of 2~mM Tris.HCl, p~ 7.~. lmM MgClz) to sensor and control wells.
Addition of DNase 1 gave an increase in admittance at minimum phase for sensor wells. but no significant change for control wells (Figure 14). The amount and rate of increase of admittance at minimum phase is related to the amount of DNase 1 present in the test solution and therefore can be used to determine enZymatiC activity in test solutions.
DNA probe F:
5'biotinylated listeria probe DNA with a 31-atom phosphoramidite linker group between the biotin and DNA.
5 !-bio-L-M-ATA~ l 11 lATGGGATTAGC-3' DNA probe G:
5'biotinylated cholera toxin probe DNA with a 13-atom phosphoramidite linker group between the biotin and DNA.
5 '-bio-L-CTCCGGAGCATAGAGCTTGGAGG-3 ' DNA non-specific binding probe H:
5'biotinvlated 15-mer oligonucleotide with a 31-atom phosphoramidite linker group between the biotin and DNA, which is non-complementary to al~ parts o~ the target DNA sequence.
5 '-bio-L-M-ATTGCTACGTATACG-3 ' ~ 0 DNA target I:
5Z base DNA seguence containing the 1~-base listeria sequence, a 10 I}ase 'spacer and the 23 base cholera toxin sequence.
5~-CCTl~ATCCCAT~CTATGCI~TGCI~'I ~TCC T CCA~GCTCTl~ l GCTCCGG~G-3-CA 02245664 l998-08-06 where.
bio = biotin NH ~ o - DMT
o--11--O
O
M=
O ~ 0~~~O ~o~
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 invention as broadly described. The present embodiments are. therefore. to be considered in all respects as illustrative and not restrictive.
Claims (26)
1. A biosensor for use in detecting the presence of an enzyme or enzymes in a sample, the biosensor comprising a membrane and means for determining the impedance of the membrane, the membrane having ionophores therein to which are attached linkers, the linkers being cleavable by the enzyme or enzymes to be detected, the cleavage of the linker causing a change in the ability of ions to pass through the membrane via the ionophores.
2. A biosensor as claimed in claim 1 in which the linker is attached to the membrane such that the ionophore is prevented from diffusing laterally within the membrane.
3. A biosensor as claimed in claim 2 in which the linker is attached to membrane spanning components provided in the membrane.
4. A biosensor as claimed in claim 3 in which the linker is attached to the membrane spanning component via a ligand binding pair.
5. A biosensor as claimed in any one of claims 1 to 4 in which the membrane comprises a first and second layer of a closely packed array of amphiphilic molecules, a plurality of ionophores and a plurality of membrane-spanning lipids prevented from lateral diffusion in the membrane. the ionophores comprising first and second half membrane spanning monomers. the first half membrane spanning monomers being provided in the first layer and the second half membrane spanning monomers being provided in the second layer, the first half membrane spanning monomers being prevented from lateral diffusion in the first layer, the second half membrane spanning monomers being linked to the membrane spanning lipids via the linker.
6. A biosensor as claimed in any one of claims 1 to 5 in which the ionophores are gramicidin or analogues thereof.
7. A biosensor as claimed in any one of claims 1 to 6 in which the enzyme to be detected is a protease.
8. A biosensor as claimed in claim 7 ill which the protease is PSA.
9. A biosensor as claimed in any one of claims 1 to 6 in which the enzyme to be detected is a nuclease.
10. A biosensor for use in detecting the presence of all enzyme in a sample, the biosensor comprising a membrane and means for determining the impedance of the membrane, the membrane having a plurality of ionophores and a plurality of membrane-spanning components therein, the membrane-spanning components having attached thereto linker molecules to which are connected the ionophores, the linker molecules being cleavable by the enzyme to be detected, the cleavage of the linker molecules causing a change in the ability of ions to pass through the membrane via the ionophores.
11. A biosensor as claimed in claim 10 in which the membrane comprises a first and second layer of a closely packed array of amphiphilic molecules and the membrane-spanning components are prevented from lateral diffusion in the membrane.
12. A biosensor as claimed in claim 10 or claim 11 in which the ionophores comprise first and second half membrane spanning monomers.
the first half membrane spanning monomers being provided in the first layer and the second half membrane spanning monomers being provided in the second layer with the first half membrane spanning monomers being prevented from lateral diffusion in the first layer.
the first half membrane spanning monomers being provided in the first layer and the second half membrane spanning monomers being provided in the second layer with the first half membrane spanning monomers being prevented from lateral diffusion in the first layer.
13. A biosensor as claimed in any one of claims 10 to 12 in which the ionophores are gramicidin or analogues thereof.
14. A biosensor as claimed in any one of claims 10 to 13 in which the enzyme to be detected is a protease.
15. A biosensor as claimed in claim 14 in which the protease is PSA.
16. A biosensor as claimed in any one of claims 10 to 13 in which the enzyme to be detected is a protease.
17. A biosensor for the detection of enzymes comprising first and second zones, means to allow addition of a sample suspected to contain a protease to the first zone, the first zone containing a probe linked to a carrier via a linker cleavable by the enzyme and means to allow passage of unlinked probe from the first zone to the second zone; the second zone including a membrane the impedance of which is dependent on the presence or absence of the probe and means to measure the impedance of the membrane.
18. A biosensor as claimed in claim 17 in which the membrane comprises a first and second layer of a closely packed array of amphiphilic molecules and a plurality of ionophores comprising first and second half membrane spanning monomers, the first half membrane spanning monomers being provided in the first layer and the second half membrane spanning monomers being provided in the second layer. the second half membrane spanning monomers being capable of lateral diffusion within the second layer independent of the first half membrane spanning monomers, the first half membrane spanning monomers being prevented from lateral diffusion in the first layer, and a ligand provided on at least the second half membrane spanning monomers, said ligand being reactive with the probe or a portion thereof, the binding of the probe to the ligand causing a change in the relationship between the first half membrane spanning monomers and the second half membrane spanning monomers such that the flow of ions across the membrane via the ionophores is allowed or prevented.
19. A biosensor as claimed in claim 17 or claim 18 in which the enzymes to be detected are proteases.
20. A biosensor as claimed in claim 19 in which the protease is PSA.
21. A biosensor as claimed in claim 17 or claim 18 in which the enzyme to be detected is a nuclease.
22. A biosensor as claimed in any one of claims 17 to 21 in which the half membrane spanning monomers are gramicidin or analogues thereof.
23. A biosensor as claimed in claim 17 in which the probe includes an ionphore.
24. A method of detecting the presence of an enzyme in a sample comprising adding the sample to the biosensor as claimed in any one of claims 1 to 23 and measuring the change in impedance of the membrane.
25. A method as claimed in claim 24 in which the enzymes to be detected are proteases.
26. A method as claimed in claim 25 in which the protease is PSA.
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|---|---|---|---|
| US1131496P | 1996-02-08 | 1996-02-08 | |
| US60/011,314 | 1996-02-08 |
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| CA2245664A1 true CA2245664A1 (en) | 1997-08-14 |
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| CA002245664A Abandoned CA2245664A1 (en) | 1996-02-08 | 1997-02-10 | Enzyme detection biosensors |
Country Status (6)
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| US (1) | US6348319B1 (en) |
| EP (1) | EP0879412A4 (en) |
| JP (1) | JP3775799B2 (en) |
| AU (1) | AU708021B2 (en) |
| CA (1) | CA2245664A1 (en) |
| WO (1) | WO1997029366A1 (en) |
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| CN107389758A (en) * | 2017-07-31 | 2017-11-24 | 重庆微奥云生物技术有限公司 | A kind of enzyme detecting system and detection method of quality control |
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| CA2609720C (en) | 2005-07-20 | 2015-06-30 | Bayer Healthcare Llc | Gated amperometry |
| US7504235B2 (en) * | 2005-08-31 | 2009-03-17 | Kimberly-Clark Worldwide, Inc. | Enzyme detection technique |
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| US8758989B2 (en) * | 2006-04-06 | 2014-06-24 | Kimberly-Clark Worldwide, Inc. | Enzymatic detection techniques |
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1997
- 1997-02-10 AU AU15851/97A patent/AU708021B2/en not_active Ceased
- 1997-02-10 CA CA002245664A patent/CA2245664A1/en not_active Abandoned
- 1997-02-10 US US09/125,068 patent/US6348319B1/en not_active Expired - Fee Related
- 1997-02-10 JP JP52799597A patent/JP3775799B2/en not_active Expired - Fee Related
- 1997-02-10 WO PCT/AU1997/000071 patent/WO1997029366A1/en not_active Application Discontinuation
- 1997-02-10 EP EP97902105A patent/EP0879412A4/en not_active Withdrawn
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107389758A (en) * | 2017-07-31 | 2017-11-24 | 重庆微奥云生物技术有限公司 | A kind of enzyme detecting system and detection method of quality control |
Also Published As
| Publication number | Publication date |
|---|---|
| JP3775799B2 (en) | 2006-05-17 |
| AU708021B2 (en) | 1999-07-29 |
| JP2000509140A (en) | 2000-07-18 |
| EP0879412A1 (en) | 1998-11-25 |
| WO1997029366A1 (en) | 1997-08-14 |
| AU1585197A (en) | 1997-08-28 |
| EP0879412A4 (en) | 2004-08-11 |
| US6348319B1 (en) | 2002-02-19 |
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