JP4929427B2 - Immunological sensor - Google Patents

Immunological sensor Download PDF

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Publication number
JP4929427B2
JP4929427B2 JP2002514406A JP2002514406A JP4929427B2 JP 4929427 B2 JP4929427 B2 JP 4929427B2 JP 2002514406 A JP2002514406 A JP 2002514406A JP 2002514406 A JP2002514406 A JP 2002514406A JP 4929427 B2 JP4929427 B2 JP 4929427B2
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Prior art keywords
reaction chamber
chamber
hole
sheet
sample
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JP2004505246A (en
Inventor
シャテリアー・ロン
ホッジズ・アラステア
Original Assignee
ユニバーサル バイオセンサーズ ピーティーワイ リミテッドUniversal Biosensors Pty Limited
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Priority to US09/615,691 priority Critical
Priority to US09/616,433 priority
Priority to US61643300A priority
Priority to US09/615,691 priority patent/US6638415B1/en
Priority to US09/616,556 priority
Priority to US09/616,512 priority patent/US6632349B1/en
Priority to US09/616,512 priority
Priority to US09/616,556 priority patent/US6444115B1/en
Priority to PCT/US2001/022202 priority patent/WO2002008763A2/en
Application filed by ユニバーサル バイオセンサーズ ピーティーワイ リミテッドUniversal Biosensors Pty Limited filed Critical ユニバーサル バイオセンサーズ ピーティーワイ リミテッドUniversal Biosensors Pty Limited
Publication of JP2004505246A publication Critical patent/JP2004505246A/en
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001Enzyme electrodes
    • C12Q1/004Enzyme electrodes mediator-assisted
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001Enzyme electrodes
    • C12Q1/005Enzyme electrodes involving specific analytes or enzymes
    • C12Q1/006Enzyme electrodes involving specific analytes or enzymes for glucose
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00Investigating or analysing materials by the use of electric, electro-chemical, or magnetic means
    • G01N27/26Investigating or analysing materials by the use of electric, electro-chemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28Electrolytic cell components
    • G01N27/30Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327Biochemical electrodes electrical and mechanical details of in vitro measurements
    • G01N27/3271Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/84Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving inorganic compounds or pH

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to instruments and methods for performing immunological assays. The instrument has a disposable, immunological sensor.
[0002]
[Prior art]
Biomedical sensors are used to record the presence and / or concentration of a wide variety of analytes. When the analyte is a protein, the detection element used is usually an antibody. This is because the interaction between the antibody and the protein (antigen) is very specific. Such immunoassays are typically divided into two categories: “yes / no answers” obtained by the concentration of antigen determined, for example, by simple visual detection or quantification methods. Most quantification methods include expensive equipment such as scintillation counters (for monitoring radioactivity), spectrophotometers, spectrofluorometers (see, eg, US Pat. No. 5,156,972), surfaces A plasmon resonance instrument (see, for example, US Pat. No. 5,965,456) is required. Therefore, it would be advantageous to develop a quantitative immunoassay that is inexpensive enough to be suitable for home or field use and simple to use.
[0003]
Conventional immunoassays fall into two categories: competitive assays and sandwich assays. In a competition assay, the antigen in the test sample is mixed with the antigen-probe complex, and the mixture then competes for binding to the antibody. The probe may be an enzyme, a phosphor or a chromophore (color group). Second, in a sandwich immunoassay, the antigen in the test sample binds to the antibody, and then the second antibody-probe complex binds to the antigen. These conventional assays usually require one or more washing steps. The washing step adds complexity to the assay procedure and can result in liquid waste that is harmful to life. Therefore, it would be advantageous to develop an instrument for performing an immunoassay that does not require a washing step. Naturally, such devices are designed to be single use disposable devices.
[0004]
Summary of the Invention
An inexpensive quantitative disposable immunological sensor is provided that does not require a washing step and thus does not produce liquid waste. Furthermore, the preferred embodiment sensor does not require a time adjustment step on the part of the user and the sensor can easily adapt to antigen-antibody reactions over a wide reaction rate range.
[0005]
  In one embodiment, a disposable instrument used in detecting a target antigen in a fluid sample having a pH, the reaction chamber having an inner surface, a proximal end, and a distal end, and secured within the reaction chamber An immobilized antibody capable of binding to the target antigen, a reporter complex that is present in the reaction chamber, forms a probe, and can be mixed with the sample, a wall, an inner surface, A detection chamber with a distal end and a proximal end; and a reaction chamberproximalAn instrument is provided having a sample inlet provided at the end and a sample passage provided between the distal end of the reaction chamber and the proximal end of the detection chamber.
[0006]
In one aspect of this embodiment, an agent is provided that is housed in the reaction chamber and that can prevent non-specific binding of proteins to the inner surface of the reaction chamber. The drug is selected from the group consisting of a surfactant and a blocking protein, such as BSA (bovine serum albumin).
[0007]
In another feature of this embodiment, the reporter complex further comprises a second antigen capable of binding to the target antigen or capable of binding to the target antigen so that it can bind to the immobilized antibody.
[0008]
In another feature of this embodiment, the probe is selected from the group consisting of a chromophore and a phosphor. The probe may consist of an enzyme such as glucose oxidase or glucose dehydrogenase. In addition, an enzyme substrate such as an oxidizable substrate such as galactose, acetic acid or glucose may be further provided.
[0009]
In another feature of this embodiment, the detection chamber further comprises a mediator. The mediator may comprise dichlorophenolindphenol, a complex of a transition metal and a nitrogen-containing heteroatomic species, or ferricyanide.
[0010]
In another feature of this embodiment, the device further comprises a buffer that can adjust the pH of the sample, such as a phosphate or citrate.
[0011]
In another feature of this embodiment, the immobilized antibody and / or reporter complex is supported on the inner surface of the reaction chamber. The reporter complex should be separated from the immobilized antibody by about 1 mm or less.
[0012]
In another feature of this embodiment, the device further comprises a stabilizing agent that stabilizes one or more of the antigen, enzyme and antibody.
[0013]
In another feature of this embodiment, the enzyme substrate is supported on the inner surface of the detection chamber.
[0014]
In another feature of this embodiment, the instrument further comprises a support material. Support material may be placed in the detection chamber, and one or more substances, such as enzyme substrates, mediators and buffers, may be supported on or within the support material. A support material is placed in the reaction chamber, and one or more substances, such as an immobilized antibody, a reporter complex, and an agent capable of preventing nonspecific binding of proteins to the inner surface of the reaction chamber are supported on or within the support material You may put in. The support material may be a mesh or fiber-filled material comprising a polymer selected from the group consisting of polyolefin, polyester, nylon, cellulose, polystyrene, polycarbonate, polysulfone and mixtures thereof, a porous material such as polysulfone, polyvinylidene difluoride It may have a macroporous membrane comprising a polymeric material selected from the group consisting of nylon, cellulose acetate, polymethacrylate, polyacrylate and mixtures thereof, or a sintered powder.
[0015]
In another feature of this embodiment, the detection chamber has at least two electrodes. The electrode may be composed of a material selected from the group consisting of palladium, platinum, gold, iridium, carbon, a mixture of carbon and binder, indium oxide, tin oxide, and mixtures thereof.
[0016]
In another feature of this embodiment, the walls of the detection chamber are transparent to the radiation emitted or absorbed by the probe, the radiation indicating the presence or absence of a reporter complex in the detection chamber.
[0017]
In another feature of this embodiment, a detector is further provided that can detect when the reaction chamber is substantially full. Further, it is preferable to further provide a perforation means capable of forming a detection chamber degassing at the distal end of the detection chamber. A reaction chamber vent may also be provided at the distal end of the reaction chamber.
[0018]
In a second embodiment, a method for manufacturing a disposable instrument used to detect a target antigen in a fluid sample having a pH, comprising: a first material sheet having a proximal end and a distal end; Passing through the reaction chamber side wall, the detection chamber side wall and a first hole defining a first sample passage between the reaction chamber distal end and the detection chamber proximal end; Attaching the first layer to the first surface of the first sheet in a covered state to form the first reaction chamber end wall and the first detection chamber end wall; and A second reaction chamber end wall and a second detection chamber end wall in a substantially overlapping relationship with the first layer, wherein the sheet and the layer comprise: Forming a strip with a plurality of outer surfaces; Penetrating the outer surface of the strip and extending into the reaction chamber at the distal end of the reaction chamber to form a second passage defining the reaction chamber vent, passing through the outer surface of the strip, Extending into the reaction chamber at the proximal end of the reaction chamber to form a third passage defining a sample inlet, immobilizing the antibody in the reaction chamber, and configuring a probe Placing a reporter complex in the reaction chamber.
[0019]
In one aspect of this embodiment, the hole penetrates the proximal end of the first sheet to form a third passage.
[0020]
In another feature of this embodiment, the first sheet, the first layer, and the second layer are composed of an electrically resistive material, and the first layer faces the first surface of the first sheet. Having a first electrode, the second layer has a second electrode facing the second surface of the first sheet. At least one of the electrodes is composed of a material selected from the group consisting of palladium, platinum, gold, iridium, carbon, a mixture of carbon and binder, indium oxide, tin oxide, and mixtures thereof. The first electrode substantially covers the first detection chamber end wall, and the second electrode substantially covers the second detection chamber end wall. At least one of the electrodes may be a sputter coated metal deposit. The second electrode is in a face-to-face relationship with a distance of about 500 microns or less from the first electrode, about 150 microns or less from the first electrode, or about 150 microns or less and about 50 microns or more from the first electrode. Is provided.
[0021]
In another feature of this embodiment, the layer is sheeted with an adhesive, for example, a heat activated adhesive, a pressure sensitive adhesive, a thermosetting adhesive, a chemical curing adhesive, a hot melt adhesive, and a hot flow adhesive. It is attached to. At least one of the sheets or layers may consist of a polymeric material such as polyester, polystyrene, polycarbonate, polyolefin and mixtures thereof or polyethylene terephthalate. At least one of the layers may be transparent to a wavelength of radiation selected from the group consisting of infrared light, visible light and ultraviolet light.
[0022]
In another feature of this embodiment, the method may further comprise providing an enzyme substrate and a mediator, wherein the enzyme substrate and mediator are placed in a detection chamber, the probe is an enzyme, and the mediator is The reaction of the enzyme electrode can be mediated to direct the occurrence of an electrochemical reaction.
[0023]
In another feature of this embodiment, the method further comprises providing a buffer that can adjust the pH of the sample.
[0024]
In 3rd Embodiment, it is a manufacturing method of the disposable instrument used for detecting the target antigen in the fluid sample which has pH, Comprising: The 1st electric resistance material sheet provided with a proximal end part and a distal end part Forming a first hole through the first hole, the first hole having a reaction chamber portion of the first hole and a detection chamber portion of the first hole, and a first portion of the reaction chamber side wall; A second passage through the second electrically resistive material sheet comprising the detection chamber side wall and the sample passage between the reaction chamber distal end and the detection chamber proximal end and comprising the proximal end and the distal end; Forming a second hole, wherein the second hole constitutes a second portion of the reaction chamber side wall and extends through a third sheet of electrically resistive material comprising a proximal end and a distal end. 3 holes are formed, and the third hole is formed on the reaction chamber side. Forming a third portion of the first sheet and attaching the first surface of the second sheet to the first surface of the first sheet, wherein the second sheet includes a detection chamber portion of the first hole. Extending over to form a first detection chamber end wall, wherein the second portion of the reaction chamber sidewall is in a substantially overlapping relationship with the first portion of the reaction chamber sidewall, Attaching the first surface to the second surface of the first sheet, the third sheet extending over the detection chamber portion of the first hole to form a second detection chamber end wall and reacting The third portion of the chamber side wall is in a substantially overlapping relationship with the first portion of the reaction chamber side wall and covers the second layer with the first layer on the second surface of the second sheet. To form a first reaction chamber end wall and cover the second hole with the second hole covered To the second surface of the third sheet to configure the second reaction chamber end wall in a substantially overlapping relationship with the first thin layer, the sheet and layer comprising a plurality of outer surfaces. Forming a second strip extending through the exterior of the strip and extending into the reaction chamber at the distal end of the reaction chamber, the second passage comprising venting the reaction chamber Forming a third passage extending through the exterior of the strip and into the reaction chamber at the proximal end of the reaction chamber, the third passage constituting a sample inlet and an antibody There is provided a method comprising the step of immobilizing a reporter complex in a reaction chamber and disposing a reporter complex constituting a probe in the reaction chamber, wherein the reporter complex constitutes a probe. The
[0025]
  In a fourth embodiment, a method for determining the presence or absence of a target antigen in a fluid sample, which is a disposable instrument used to detect a target antigen in a fluid sample having a pH, comprising an inner surface, a proximal end A reaction chamber with a portion and a distal end; an immobilized antibody immobilized within the reaction chamber and capable of binding to a target antigen; and present within the reaction chamber to constitute a probe and to constitute a sample A reporter complex that can be mixed with a detection chamber with a wall, an inner surface, a distal end and a proximal end, and a reaction chamberproximalA sample inlet provided at the end and a sample passage provided between the distal end of the reaction chamber and the proximal end of the detection chamber so that the reporter complex binds to the immobilized antibody Providing a device further comprising a second antigen capable of competing with the target antigen or a second antibody capable of binding to the target antigen, the method comprising contacting the fluid sample with the sample inlet, Substantially filling the reaction chamber with a fluid sample by allowing it to flow from the sample inlet to the reaction chamber, so that substantially all of the reporter complex is immobilized in the absence of antigen in the sample. Allowing the sample to flow from the reaction chamber through the sample passage toward the detection chamber, with a stage sufficient to allow the antibody to bind. Substantially filling the detection chamber with a fluid sample, and detecting the presence or absence of an antigen-probe complex in the detection chamber, wherein the presence or absence of the antigen-probe complex A method is provided that represents presence or absence.
[0026]
In a first feature of this embodiment, the method further comprises drilling a wall of the detection chamber to form a detection chamber degassing at the distal end of the detection chamber, the drilling step being a predetermined time. Implemented as soon as it has passed.
[0027]
In 5th Embodiment, it is a manufacturing method of the disposable instrument used when detecting the target antigen in the fluid sample which has pH, Comprising: The process of forming the 1st hole which penetrated the 1st electrical resistance material sheet | seat And the first hole has a detection chamber portion and constitutes a detection chamber side wall, the detection chamber has a proximal end and a distal end, and the first layer covers the hole. Is attached to the first surface of the first sheet to form the first detection chamber end wall, and the second layer is attached to the second surface of the first sheet in a state of covering the hole. Forming a second detection chamber end wall in a substantially overlapping relationship with the first layer, the sheet and the layer forming a strip and forming a second hole extending through the strip. The strip has a proximal end and a distal end; The hole has a reaction chamber portion, the reaction chamber has a distal end and a proximal end, and the second hole is adjacent to the reaction chamber side wall and the distal end of the reaction chamber and the detection chamber. Forming a sample passage between the first end of the strip and attaching the first surface of the third layer to the first surface of the strip, the third layer comprising the second pore reaction chamber portion. Extending over to form a first reaction chamber end wall and attaching the first surface of the fourth layer to the second surface of the strip, the fourth layer reacting to the second hole Extending over the chamber portion and configuring the second reaction chamber end wall in a substantially overlapping relationship with the first reaction chamber end wall and through the surface of the instrument at the reaction chamber distal end Forming a third hole extending therein, wherein the third hole vents the reaction chamber. Forming a fourth hole through the surface of the instrument and extending into the reaction chamber at the proximal end of the reaction chamber, the fourth hole forming a sample inlet and reacting the antibody There is provided a method comprising the step of immobilizing in a chamber, the step of placing a reporter complex in a reaction chamber, the reporter complex having a probe.
[0028]
In one aspect of this embodiment, the first layer and the second layer are composed of an electrically resistive material, and the first layer has a first electrode facing the first surface of the first sheet. The second layer has a second electrode facing the second surface of the first sheet.
[0029]
DETAILED DESCRIPTION OF THE INVENTION
In the following, preferred embodiments of the present invention will be described in detail. Those skilled in the art will envision many variations and modifications of the present invention that fall within the scope of the invention. Accordingly, the description of preferred embodiments does not limit the scope of the invention.
[0030]
A single stage non-cleaning immunological sensor is disclosed. This sensor is a single use disposable instrument that utilizes two adjacent chambers, a reaction chamber and a detection chamber. Antigen-antibody reactions occur in the reaction chamber, and the results of these reactions are detected in the detection chamber, and the presence or absence of the antigen in the sample is estimated.
[0031]
Any suitable detection method can be utilized. Suitable detection methods include, for example, visual detection where color development is observed or spectroscopic detection in which changes in light absorbance are measured using reflected or transmitted light. In a preferred embodiment, the detection method is an electrochemical method that measures the current or potential difference indirectly caused by the product of the antigen-antibody reaction.
[0032]
A method and apparatus for obtaining electrochemical measurements of fluid samples is disclosed in co-pending US patent application Ser. No. 09 / 615,691, filed Jul. 14, 2000 (Title of Invention: ANTIOXIDANT SENSOR), 2000. No. 09 / 616,512 filed Jul. 14 (Title of Invention: HEMOGLOBIN SENSROR) and No. 09/616, filed Jul. 14, 2000 No. 556 (Title of Invention: ELECTROCHEMICAL METHOD FOR MEASURING CHEMICAL REACTION RATES), the entire disclosure of each of these US patent specifications is hereby incorporated by reference as forming part of this specification. To do.
[0033]
Various test phases, i.e. the reaction phase and the detection phase, can be timed in a manual manner. As a variant, the time adjustment may be made automatically in response to a trigger signal that occurs when the reaction chamber is charged.
An embodiment of a sensor suitable for use in electrochemical detection is shown in FIGS. FIG. 1 is a plan view of a sensor strip, and FIG. 2 is a cross-sectional view showing details of a reaction chamber and a detection chamber.
[0034]
Sensor
The immunosensors of preferred embodiments can be prepared using well-known thin film device fabrication methods used in preparing electrochemical glucose detection devices (see, eg, US Pat. No. 5,942,102). Note that the entire contents of such US patent specifications are hereby incorporated by reference as forming part of this specification.) Such techniques are also used to prepare immunological sensors that utilize non-electrochemical detection methods, with some design changes.
[0035]
In the preferred embodiment of the immunological sensor shown in FIGS. 1 and 2, the detection chamber 28 constitutes an electrochemical cell. To prepare the reaction chamber 22 and the detection chamber 28, first a hole is formed that extends through the sheet 36 of electrically resistive material. This hole is shaped to form a sample passage 38 between the side walls of both the reaction chamber 22 and the detection chamber 28 and between the two chambers 22, 28. A sample inlet 24 is also formed by extending the hole from the proximal end 24 of the reaction chamber 22 through the edge of the sheet 37. In one embodiment, the thickness of the sheet 36 defines the total height of the reaction chamber 22 and the detection chamber 28, which are the same height. In another embodiment, the height of the reaction chamber 22 is greater than the height of the detection chamber 28. The reaction chamber 22 having a height higher than that of the detection chamber 28 is prepared by stacking a large number of sheets 32, 34, 36 on each other. The intermediate sheet 36 of the layers has holes that define the side walls 74, 76 of both the reaction chamber 22 and the detection chamber 28 as described above. The intermediate layer 36 is then sandwiched between two or more additional layers 32, 34 that have holes that form the side walls 74 of the reaction chamber 22 only, and the layers 32, 34 thereby constitutes the end walls 60, 62 of the detection chamber 28. In this embodiment, the end walls 60, 62 of the detection chamber constitute electrodes 52, 54 that can be prepared as described below.
[0036]
After the side walls 74 and 76 of the reaction chamber 22 and the detection chamber 28 are formed, a first thin electrode layer 52 is deposited on one side 70 of the sheet 36 of electrical resistance material with the holes covered, and the detection chamber 28 is And the end wall 60 is formed. The layer 52 may be adhered to the sheet 36 by an adhesive, for example. Suitable adhesives include, for example, heat activated adhesives, pressure sensitive adhesives, thermosetting adhesives, chemical curing adhesives, hot melt adhesives, hot flow adhesives, and the like. To prepare the electrode layer 52, a sheet of electrically resistive material 32 (eg, by sputter coating) suitable metal, such as platinum, palladium, carbon, indium oxide, tin oxide, a mixture of indium and tin oxide, gold, silver, Cover with iridium, a mixture of these, and the like. Materials suitable for use as the electrodes 52, 54 are compatible with the reagents present in the sensor 20, i.e., these materials do not chemically react with the reagents.
[0037]
Next, a second thin electrode layer 54 is also applied to the opposite surface 71 of the sheet of electrical resistance material 36, covering the hole, forming the detection chamber 28 and the second end wall 62. Form. In a preferred embodiment, the electrode layers 52, 54 are provided in an opposing relationship at a distance of less than about 500 microns, more preferably less than 150 microns, and most preferably 50 microns to 150 microns. Even if the sample inlet 24 is not already formed, it is provided, for example, by forming a notch in the edge 37 of the instrument 20 that intersects the proximal end 23 of the reaction chamber 22.
[0038]
The electrode layers 52 and 54 are provided with connection means that can arrange the sensor 20 in the measurement circuit. At least one of the electrodes 52 or 54 in the cell 28 is a detection electrode, that is, a predetermined amount of a reduced redox agent in the case of an antioxidant and a predetermined amount of an oxidized redox agent in the case of an oxidant. The affected electrode. In the case of a potentiometric sensor 20 in which the potential difference of the detection electrode 52 or 54 represents the presence level of the analyte, a second electrode 54 or 52 acting as a reference electrode is present that serves to provide a reference potential. In the case of an amperometric sensor 20 in which the detection electrode current represents the level of the analyte in the sample, there is at least one other electrode 54 or 52 that serves as the counter electrode that constitutes the electrical circuit. The second electrode 54 or 52 preferably functions as a reference electrode. Alternatively, a separate electrode (not shown) may perform the function of the reference electrode.
[0039]
When the immunological sensor 20 is operated as an electrochemical cell 28, the sheets 32, 34, 36 having holes constituting the reaction chamber 22 and / or the detection chamber 28 need to be made of an electrically resistive material. . Suitable electrical resistance materials include, for example, polyester, polystyrene, polycarbonate, polyolefin, and mixtures thereof. A preferred polyester is polyethylene terephthalate. When the immunological sensor 20 is operated using a detection method other than the electrochemical detection method, the constituent material need not be an electrical resistance material. However, the polymer materials described above are preferred for use in constructing the immunological sensor of the preferred embodiment. This is because they are easy to process, inexpensive, and not reactive to reagents and samples. For detection methods that include absorbing, transmitting, or emitting light of a specific frequency, the end walls 60 and / or 62 and the layers 32, 46, and / or layers located on the end walls of the detection chamber 28 34 and 42 need to be transparent to the frequency of the light.
[0040]
Reagents used in the cell 28, such as immobilized antibodies, probe-linked antigens, buffers, mediators, etc., on the walls 44, 48 and / or 74 of the reaction chamber 22 and on the wall 60 of the detection chamber 28. , 62 and / or 76 on a separate and independent support housed in the chamber, may be supported in the matrix, or may be self-supporting. When the reagents are supported on the chamber walls or electrodes 52, 54, the chemicals may be applied using printing methods well known in the art, such as inkjet printing, screen printing, lithography, and the like. In a preferred embodiment, a solution containing the reagent is applied to the surface in the chamber and dried.
[0041]
The antibody 44, probe-bound antigen 50 or other chemical is immobilized on the surface 40, 48, 60, 62, 74 and / or 76 of the reaction chamber 22 or detection chamber 28, or they are not allowed to dry adhere to them. It may be advantageous to support on these separate and independent supports or place them in the chamber and then place them in the chamber. Suitable separate independent supports include, but are not limited to, mesh materials, nonwoven materials, fiber-filled materials, macroporous membranes, or sintered powders. Advantages of using separate and independent supports include increased surface area, thus allowing larger amounts of antibody and probe-bound antigen to be placed in reaction chamber 28 if desired. In such an embodiment, the antibody is immobilized on one piece of porous material, placed in a first reaction chamber, and the probe-bound antigen is dry deposited on another piece of porous material, which is then attached to the reaction chamber. Place in. Alternatively, either the antibody or the probe-bound antigen is deposited on the porous material and the other component is supported on the reaction chamber wall as described above. In yet another embodiment, the reaction chamber walls themselves are porous, and antibodies and / or probe-bound antigens are contained within these walls. In this embodiment, the liquid can penetrate into the porous wall, but does not leak out of the limited area. This is accomplished by using a macroporous membrane to form the reaction chamber wall and compressing the membrane around the reaction chamber to prevent sample leakage from the desired area.
[0042]
Suitable separate and independent supports, such as mesh materials, nonwoven materials and fiber filler materials, include polyolefins, polyesters, nylons, celluloses, polystyrenes, polycarbonates, polysulfones, mixtures thereof, and the like. Suitable macroporous membranes are preferably prepared from polymeric materials, such polymeric materials include polysulfone, polyvinylidene diflurolide, nylon, cellulose acetate, polymethacrylate, polyacrylate, mixtures thereof, and the like.
[0043]
The protein or antibody may be contained in a matrix such as polyvinyl acetate. By changing the solubility of the matrix in the sample, controlled release of the protein or antibody into the sample can be achieved.
[0044]
In all cases, the material used in the sensor is in a form suitable for mass production, and the cell itself is designed for use in a single experiment and can then be disposed of.
[0045]
A preferred embodiment of an immunological sensor fabricated as described above is shown in FIGS. In this preferred embodiment, layers 32 and 34 are substrates coated with conductive materials 52 and 54. The conductive material 52, 54 on the surface 60 or 62 faces the detection chamber 28 and the adhesive layer (not shown) coated on the surface 33 or 35 faces the layer 42 or 46, respectively.
[0046]
How to use a sensor to determine the presence or absence of an antigen
In a preferred embodiment, the sensor 20 is shown in FIG. 1, i.e. a plan view of such a sensor 20, and as shown in FIG. An electrochemical cell 28 utilizing glucose oxidase or glucose dehuman logase. In this embodiment, the presence or absence of the analyte is estimated as follows.
[0047]
Initially, the user introduces the sample from the sample inlet 24 into the reaction chamber 22 of the sensor 20. The sample is drawn into the reaction chamber 22 under the influence of capillary action or wicking action. During filling, the reaction chamber vent (vent) 26 is open to the atmosphere, thus allowing air displaced by the sample to escape. The sample is drawn into the reaction chamber 22 until it is full up to the reaction chamber vent 26 and filling stops when it is full. The volume of the reaction chamber 22 is selected to be at least equal to, and preferably greater than, the volume of the detection chamber 28.
[0048]
A circle indicated by a broken line in FIG. 1 indicates the hole 30 provided in the layers 32, 34, and 36 instead of the layers 42 and 46, and the hole of the layer 34 opens into the detection chamber 28. Since layers 42 and 46 are not initially perforated, it is only the sample passageway 38 that opens into the reaction chamber 22 that the detection chamber 28 is open to the atmosphere. Thus, when the reaction chamber 22 is full of sample, the reaction chamber blocks the passage 38 leading to the detection chamber 28. This traps air into the detection chamber 28 and substantially prevents the detection chamber from becoming full of sample. A small amount of sample enters the detection chamber 28 during the period between when the sample first contacts the opening 38 leading to the detection chamber 28 and when the sample contacts the far side of the opening 38. become. However, once the sample is completely wet across the opening 38 to the detection chamber 28, no further filling of the detection chamber 28 occurs.
[0049]
The inner surface 40 of the substrate 42 forming the base of the reaction chamber 22 is coated with an antibody 44 corresponding to the antigen to be detected. The antibody 44 is adsorbed or immobilized on the surface 40 of the substrate 42 so that it is not removed from the substrate 42 during the test. After application of the antibody 44 to the inner surface 40 of the substrate 42, an agent (not shown) prepared to prevent nonspecific binding of proteins to the surface may be applied. It is optional. One such example well known in the art is BSA (bovine serum albumin). Nonionic surfactants such as Triton X100 ™ from Rohm and Haas, Philadelphia, Pennsylvania, or Tween ™ from Icy Americas, Wilmington, Delaware are used as such agents. May be. The selected nonionic surfactant does not denature the protein. The coating 44 on the inner surface 40 of the substrate 42 is in a dry state when ready for use in inspection.
[0050]
Another substrate 46 constitutes the top surface 48 of the reaction chamber 22. An enzyme (ENZ) bound to the antigen 50 to be detected is applied on the inner surface 48 of the substrate 46. Examples of suitable enzymes include, but are not limited to, glucose, oxidase and glucose dehydrogenase. When the inner surface 48 of the substrate 46 is wetted by the sample, the enzyme-linked antigen 50 is dry deposited on the inner surface 48 so that the enzyme-linked antigen can be released into the sample. Therefore, the coating method of the inner surface 48 of the substrate 46 and the enzyme-bound antigen 50 is selected so that only a weak bond is generated between the enzyme-bound antigen 50 and the inner surface 48 of the substrate 46. The rate of dissolution of enzyme-linked antigen 50 from surface 48 is selected such that little lysis occurs during the time required for the sample to fill reaction chamber 22. In this way, enzyme-bound antigen 50 will be uniformly distributed throughout the region of reaction chamber 22 after filling.
[0051]
The relative amounts of enzyme-linked antigen 50 and antibody 44 are selected so that there is only slightly more antibody 44 than enzyme-linked antigen 50. In this connection, a slight excess is defined such that it is small compared to the number of antigen molecules to be detected in the sample.
[0052]
Thus, as the sample fills the reaction chamber 22, the enzyme-bound antigen 50 enters and mixes with the sample. Next, the enzyme-bound antigen 50 is brought into contact with the antibody 44 for a sufficient time. Due to the presence of excess antibody 44, substantially all of the enzyme-bound antigen 50 binds to antibody 44 and is effectively immobilized, even if no antibody is present in the sample. If antigen is present in the sample, less than enzyme-bound antigen 50, antigen already present throughout the sample contacts and binds to antibody 44, after which enzyme-bound antigen 50 is antibody 44 is contacted. Accordingly, the antibody 44 is blocked from binding to the enzyme-bound antigen 50. Thus, if the antigen is initially present in the sample, at the end of the reaction phase, the enzyme linked antigen 50 will remain mobile in the sample. If the antigen is not initially present in the sample, enzyme-linked antigen 50 will be immobilized on the inner surface 48 of the substrate 46 at the end of the reaction phase.
[0053]
The end of the reaction phase is a predetermined position after the sample is introduced into the reaction chamber 22. The predetermined time point is set such that there is sufficient time for substantially all of the enzyme-bound antigen 50 to bind to antibody 44 under the test conditions if the antigen is not initially present in the sample.
[0054]
The time for introducing the sample into the reaction chamber 22 can be indicated by the user, for example, by pressing a button on an instrument connected to the sensor 20. This action is used to trigger the time adjustment device. In the case of the visual detection method, no instrument is required. In such embodiments, the user manually times the reaction period.
[0055]
When detecting the result of an antigen-antibody reaction using an electrochemical detection method, an indication that the sample has been introduced into the reaction chamber 22 should be automated. As described above, when the sample fills the reaction chamber 22, a small portion of the detection chamber 28 at the opening 38 leading to the reaction chamber 22 will be wetted by the sample. When using electrochemical detection methods, at least two electrodes 52, 54 will be provided in the detection chamber 28. If these electrodes 52, 54 are placed in the detection chamber 28 so that at least a portion of each electrode 52, 54 is in contact with the sample during filling of the reaction chamber 22, the electrode 52 is present if a sample is present. , 54 are bridged to generate an electrical signal that can be used to trigger the time adjustment device.
[0056]
After triggering the time adjustment device by the user or automatically, at a given point in time, the antigen-antibody reaction phase of the test is considered complete. When the antigen-antibody reaction phase of the test is complete, a vent 56 to the atmosphere is opened. For example, a solenoid operated needle in the instrument may be used to pierce layer 42 or 46, or both layers 42 and 46, thus opening the distal end 58 of the detection chamber 28 to the atmosphere. The drilling may be performed automatically by the instrument as in the above example, or manually by the user in the case of a visual detection scheme where the instrument is not used, for example, the user may The layers 42 and 46 are pierced, thereby forming a vent 56.
[0057]
By opening the vent 56 to the atmosphere, air trapped within the detection chamber 28 can escape, thereby filling the detection chamber 28 with the reacted sample from the reaction chamber 22. The reacted sample will be drawn into the detection chamber 28 because the capillary action force in the detection chamber 28 is greater than the capillary action force present in the reaction chamber 22. In a preferred embodiment, the increase in capillary action force is achieved by appropriately coating the surfaces 60, 62 of the detection chamber 28, or more preferably, the capillary action distance for the detection chamber 28, which is the capillary action of the reaction chamber 22. By choosing to be shorter than the distance. In this embodiment, the capillary working distance is defined to be the smallest dimension of the chamber.
[0058]
A dry reagent 64 is optionally provided in the detection chamber 28. The dry reagent consists of an enzyme substrate and a mediator, and reacts with the enzyme portion of the enzyme-bound antigen 50 to produce a detectable signal. Can be output. The enzyme substrate and mediator (if present) should be of an amount sufficient for the reaction rate of any enzyme present with the enzyme substrate 64 to be determined by the amount of enzyme present. For example, if the enzyme is glucose oxidase or glucose dehydrogenase, a suitable enzyme mediator 64 and glucose (if not already present in the sample) will be provided in the detection chamber 28. A buffer that helps to adjust the pH of the sample in the detection chamber 28 may also be used. In embodiments using an electrochemical detection scheme, ferricyanide is a suitable mediator. Other suitable mediators include dichlorophenolindphenol and complexes of transition metals with nitrogen-containing heteroatomic species. Glucose, mediator and buffer 64 are present in sufficient amounts such that the reaction rate of the enzyme and enzyme substrate 64 is limited by the concentration of enzyme present.
[0059]
When the detection chamber 28 is filled, the reagent 64 dissolves into the sample. The enzyme component of the reagent 64 reacts with glucose and the mediator to generate a reduced mediator. This reduced mediator is electrochemically oxidized at the electrode 52 or 54 acting as an anode in the detection chamber 28 to generate an electric current. In one embodiment, this rate of change of current over time is used as an indicator of the presence and abundance of the enzyme in the reacted sample. If the rate of change of current is less than a predetermined threshold, this indicates that an effective amount of enzyme-bound antigen 50 is not present in the reacted sample, and thus there is no antigen present in the original sample. Have not instructed. If the rate of current change is higher than the threshold, this also indicates that enzyme-bound antigen 50 is present in the reacted sample and thus the antigen is present in the initial sample. In one embodiment, the rate of change of current is used to provide a measure of the relative amount of antigen initially present in the sample.
[0060]
In a preferred embodiment of the electrochemical detection system, the electrodes 52, 54 in the detection chamber 28 are sputtered, for example, by sputtering as described in WO 97/18464. It is formed as a conductive layer coated on 62. These conductive layers 52, 54 are of a material that does not chemically react with existing reagents and serve as electrodes 52, 54 as a possibility of selection. Examples of suitable materials include, but are not limited to, palladium, platinum, gold, iridium, carbon, a mixture of carbon and binder, indium oxide, tin oxide, and a mixture of indium and tin oxide. .
[0061]
In this embodiment, an inert electrical insulating layer 36 separates the electrode support bases 32 and 34 from each other. Preferably, the insulating layer 36 functions to keep the layers 32, 34 in a predetermined spaced state. If this separation is sufficiently small, for example less than 500 microns, more preferably 50 microns to 150 microns, the current flowing between the electrodes 52, 54 will have a reasonably short time relative to the detection time employed. And the concentration of the reduced mediator. In this embodiment, the rate of increase in current is directly proportional to the reaction rate of the enzyme and thus the amount of enzyme present.
[0062]
A connection end 66 is shown in FIG. The electrodes 54 and 56 in the detection chamber 28 may be placed in electrical connection with a meter (not shown) via a connection end 66. The connecting means (not shown) is in electrical contact with the electrodes 54, 56 in the detection chamber 28 by a conductive track (not shown). In the preferred embodiment shown in FIG. 1, these consist of an extension of the film of conductors 52, 54 coated on the inner surfaces of the layers 32, 34. The instrument in connection with the connection region 66 applies a potential difference between the electrodes 52, 54 in the detection chamber 28, analyzes the resulting electrical signal, displays the response result, and optionally displays the response result. It can be stored in a storage device.
[0063]
Another embodiment utilizing an electrochemical detection scheme uses a stripe of conductive material deposited on one or both of the inner surfaces of the detection chamber, in which case at least two electrodes, i.e., a detection electrode and A counter / reference electrode is provided. A third electrode is provided that optionally serves as a separate reference electrode.
[0064]
For embodiments where the detection method uses visual detection or reflection spectroscopy, at least layers 32, 46 or layers 34, 42 are transparent to the wavelength of the radiation to be observed. In the case of a visual detection scheme, a simple color change in the detection chamber 28 is observed. In the case of reflection spectroscopy, the detection radiation is illuminated through layers 32, 46 or layers 34, 42 and the radiation reflected from the solution in detection chamber 28 is analyzed. In the case of transmission spectroscopy used as a detection method, at least the layers 32, 46, 34, 42 are transparent to radiation of a selected wavelength. The radiation is illuminated through the sample in the detection chamber 28 and measures the beam attenuation.
[0065]
In a preferred embodiment of the method for constructing the sensor, layer 36 is a substrate having an adhesive layer (not shown) applied to the upper surface 70 and lower surface 72 of the substrate. Examples of materials suitable for the substrate of layer 36 include polyester, polystyrene, polycarbonate, polyolefin, preferably polyethylene terephthalate. Examples of suitable adhesives are pressure sensitive adhesives, heat and chemical cure adhesives, hot melt and hot flow adhesives.
[0066]
Use of melittin as a probe
A conventional ELISA binds the antigen to the enzyme. However, it is also possible to bind the antigen to melittin, a polypeptide found in bee venom. In this embodiment, the probe-bound antigen comprising the antigen-melittin complex may be dry attached to the reaction chamber wall as described above. The detection chamber may contain a mediator consisting of ferrocyanide in liposomes or lipid vesicles. When the antigen-melittin complex reaches the liposome, the liposome ruptures and releases ferrocyanide. This results in a rapid amplification of the signal, ie a small amount of free antigen competes with the antigen-melittin complex for binding sites on the antibody, resulting in a high concentration of ferrocyanide.
[0067]
Use of horseradish peroxidase and alkaline phosphatase in electrochemical assays
Conventional ELISA uses horseradish peroxidase (HRP) or alkaline phosphatase (AP) as the enzyme in the colorimetric assay. However, substrates have been developed that allow both of these enzymes to be used in electrochemical assays. In this embodiment, AP may be used with p-aminophenyl phosphate and HRP may be used with tetrathiafulvalene.
[0068]
Method for obtaining electrochemical measurements using an immunological sensor
In some embodiments, a sensor is used to localize a chemical reaction at a location that is remote from an electrode used to electrochemically react an electroactive product. , Information on the rate of chemical reactions that produce at least one electroactive product can be obtained.
[0069]
The location of the chemical reaction is sufficiently offset from the electrode so that the mass transfer of the electroactive species from the location of the chemical reaction to the electrode effectively controls the current flowing at the electrode at any given time. With this configuration, the concentration gradient of electroactive species between the chemical reaction site and the electrode is substantially linear. The concentration of the electroactive species is effectively kept at zero at the electrode by the electrochemical reaction taking place here. Therefore, the temporal transition of the magnitude of the concentration gradient is substantially determined only by the temporal transition of the concentration of the electroactive chemical species at the chemical reaction site and the diffusion coefficient of the electroactive reaction product in the liquid medium. Since the current flowing at the electrode is proportional to the concentration gradient of the electroactive species at the electrode, the time course of this current will reflect the time course of the chemical reaction occurring at this remote location. . This allows the current measured at the electrode (or the charge passed if the current is integrated) to be used as a convenient measure of the rate and extent of chemical reaction.
[0070]
An example of a suitable method for causing a chemical reaction to occur at a location remote from the working electrode is to immobilize (immobilize) one or more of the reaction components on a solid surface located far from the electrode. It is in. The reaction component may be immobilized by drying it on a solid surface or incorporating it into a polymer matrix attached thereto. Alternatively, the reaction components may be directly bonded to the solid surface by chemical bonding or physical bonding. As a variant, one or more of the reaction components may be simply dried on the solid surface and attached to it without using any special immobilization means. In this situation, one or more of the reaction components are sufficiently low in mobility in a liquid matrix that fills the electrochemical cell, so that the electrochemical current can be effectively monitored and the required measurement can be performed. Does not move substantially from the dry position. In this case, substantially moving means that the slowest moving component required for the chemical reaction is sufficient to cause the Cottrell type reduced kinetic state to begin to achieve a time course of current flowing at the electrode. It means getting closer.
[0071]
In a preferred embodiment, the range of the separation distance between the chemical reaction site and the working electrode is desirably about 1 cm, preferably less than 5 mm, more preferably 5,10,50,100,200,500 microns to 5 mm, more preferably. Is 5,10,50,100,200 microns to 500 microns, most preferably 5,10,50,100 microns to 200 microns.
[0072]
In addition to the working electrode, at least a counter electrode in contact with the liquid sample is provided to constitute the electrochemical circuit. The counter electrode may function as a combination of the counter electrode and the reference electrode, or may be provided with a separate reference electrode, but this is optional. In a preferred embodiment, the working electrode and counter electrode are desirably at a distance of about 300 microns or more, preferably a distance of about 500 microns or more, more preferably a distance of about 500 microns to 10 mm, more preferably about 500 microns. They are provided at a distance of microns to 1,2,5 mm, most preferably at a distance of 1 mm to 2,5,10 mm.
[0073]
The working electrode is composed of a material that, during use, does not chemically react with any component that contacts the electrode's current response to the extent that it interferes. When the working electrode is used as an anode, examples of suitable materials are platinum, palladium, carbon, carbon combined with an inert binder, iridium, indium oxide, tin oxide, a mixture of indium and tin oxide. When the working electrode is used as a cathode, in addition to the materials described above, other suitable materials include steel, stainless steel, copper, nickel, silver and chromium.
[0074]
Examples of suitable materials for the counter electrode are platinum, palladium, carbon, a combination of carbon and inert binder, iridium, indium oxide, tin oxide, a mixture of indium and tin oxide, steel, stainless steel, copper, nickel, chromium Silver, a substantially insoluble silver salt, such as silver chloride, silver bromide, silver iodide, silver ferrocyanide, silver coated with ferricyanide.
[0075]
The chemical reaction site may be localized on a bare wall or counter electrode located far from the working electrode. The chemical reaction site may be located in the same plane as the working electrode, more preferably in a plane that faces the working electrode and is substantially parallel thereto.
[0076]
A sensor suitable for use in certain embodiments has a working electrode and a counter electrode provided on an electrically insulating substrate. A layer of chemical reactant is deposited on the second substrate, and at least one of the reactants is substantially solidified on the substrate. In use, the space between the sensor walls is filled with a liquid containing a substance that can react with the reagent to produce at least one electroactive species. The product of the chemical reaction diffuses toward the working electrode where it reacts electrically with the electroactive species to produce an electric current. Using the magnitude of the current or the charge passed at a specific time, or the time course of the current or charge passed, a measure of the rate or extent of the chemical reaction occurring at the reactant layer can be obtained.
[0077]
In another embodiment of the sensor, the reactant is deposited on a counter electrode provided on the substrate with electrical resistance. In this embodiment, the constituent material of the counter electrode does not react with any of the reactant components deposited on the electrode.
[0078]
The method of obtaining electrochemical measurements described above can be applied to any suitable electrochemical system, including an electrochemical immunoassay system. An example of a method that is a non-immunological assay but is adapted to a typical electrochemical system is the measurement of glucose in whole blood using the enzyme PQQ-dependent glucose dehydrogenase (GDHpqq) and a redox mediator. . In this reaction, glucose in the blood reacts with GDHpqq to produce gluconic acid. In this process, PQQ in the enzyme is reduced. In this case, a mediator such as potassium ferricyanide oxidizes PQQ in the enzyme to produce a ferrocyanide. The oxidized form of the enzyme can then react further with glucose. The net effect of this reaction is to generate two ferrocyanide molecules for each reacted glucose molecule. Ferrocyanide is an electroactive species and therefore can be oxidized at the electrode to produce a current. Other enzymes suitable for this reaction are glucose oxidase (GOD) or NAD-dependent glucose dehydrogenase. For other reactions, lactate dehydrogenase and alcohol dehydrogenase can be used. Other suitable redox mediators include ferrocinium, osmium complexes with bipyridine, and benzophenone.
[0079]
The reaction of glucose and enzymes in the whole blood can be slow and takes several minutes to complete. Also, the higher the hematocrit of the blood sample, the slower the reaction. Blood hematocrit is the volume fraction of red blood cells in a whole blood sample. For example, a solution containing 50 mg / ml GDHpqq, 0.9 M potassium ferricyanide and 50 mM buffer at pH 6.5 was attached to the counter electrode and a dry reactant layer was formed after removing the water. Within this layer, GDHpqq is large enough to be effectively immobilized on the counter electrode, whereas ferricyanide can mix more uniformly throughout the liquid in the electrochemical cell. A blood sample was introduced into the cell and immediately a potential difference of +300 mV was generated between the working electrode and the counter electrode. A potential difference of +300 mV is most preferred for oxidizing the ferrocyanide, but the potential difference is desirably +40 mV to +600 mV, preferably +50 mV to +500 mV, more preferably +200 mV to +400 mV. Within the cell, the working electrode consisted of a gold layer sputtered on a polyester substrate and the counter electrode consisted of a palladium layer sputtered on the polyester substrate.
[0080]
Current traces were recorded for different hematocrit blood samples showing a fast reaction rate in blood with low hematocrit (ie, 20%, 42% and 65% hematocrit in the blood). The glucose level in each blood sample is approximately the same, ie, 5.4 mM for the 65% hematocrit sample, 5.5 mM for the 42% hematocrit sample, and 6.0 mM for the 20% hematocrit sample.
[0081]
The current measurement can be approximated by the following equation:
i = −FADC / L
In the above equation, i is the current, F is the Faraday constant (964486.7 C / mol), A is the electrode area, D is the diffusion coefficient of ferrocyanide in the sample, and C is the ferrocyanide at the reaction site. The concentration of the compound, L, is the distance between the reaction site and the electrode. Therefore, the reaction rate represented by the rate of change of C with respect to time is given by:
[0082]
dC / dt =-(L / FAD) di / dt
For the above reactions, the average di / dt for the 6 to 8 seconds for the 20%, 42% and 65% hematocrit samples is 3.82 microamperes / second, 2.14 microamperes / second and 1.32 microseconds, respectively. Amps / second. The ferrocyanide diffusion coefficients for these samples were 2.0 × 10 2 for the 20%, 42% and 65% hematocrit samples, respectively.-6cm2 / Second, 1.7 × 10-6cm2 Per second and 1.4 × 10-6cm2 / Sec. The electrode area is 0.1238cm2 L was 125 microns. These values gave reaction rates of 2.0 mM / second, 1.3 mM / second and 0.99 mM / second for the 20%, 42% and 65% hematocrit samples, respectively.
[0083]
The above-described method for measuring glucose reactivity in blood is suitable for other electrochemical systems, including immunoassay formats, as will be appreciated by those skilled in the art, for example, as appropriate for the measurement of antigens. The design can be changed.
[0084]
  The above description discloses several methods and materials of the present invention. The present invention allows design changes of such methods and materials, as well as fabrication methods and equipment modifications. Such modifications will become apparent to those skilled in the art from consideration of the specification or practice of the invention disclosed herein. Accordingly, the invention is not limited to the specific embodiments disclosed herein, but the invention covers all design modifications and spirits that fall within the true scope and spirit of the invention as set forth in the claims. Modification examples are included.
The preferred embodiments of the present invention are as follows.
(1) A disposable instrument used for detecting a target antigen in a fluid sample having a pH, which is a reaction chamber having an inner surface, a proximal end, and a distal end, and is fixed in the reaction chamber. An immobilized antibody capable of binding to the target antigen, a reporter complex that is present in the reaction chamber and constitutes the probe and can be mixed with the sample, the wall, the inner surface, the distal end and the vicinity. A detection chamber with a distal end, a sample inlet provided at the proximal end of the reaction chamber, and a sample provided between the distal end of the reaction chamber and the proximal end of the detection chamber A device having a passage.
(2) The device according to embodiment 1, further comprising an agent housed in the reaction chamber and capable of preventing nonspecific binding of proteins to the inner surface of the reaction chamber.
(3) The device according to embodiment 2, wherein the drug is selected from the group consisting of a surfactant and a blocking protein.
(4) The device according to Embodiment 3, wherein the blocking protein is BSA (bovine serum albumin).
(5) The device according to embodiment 1, wherein the reporter complex further comprises a second antigen capable of competing with the target antigen so that it can bind to the immobilized antibody, or a second antibody capable of binding to the target antigen.
(6) The instrument according to embodiment 1, wherein the probe is selected from the group consisting of a chromophore and a phosphor.
(7) The instrument according to embodiment 1, wherein the probe comprises an enzyme.
(8) The instrument according to embodiment 7, wherein the enzyme comprises glucose oxidase.
(9) The device according to embodiment 7, wherein the enzyme comprises glucose dehydrogenase.
(10) The instrument according to embodiment 7, wherein the detection chamber further comprises an enzyme substrate.
(11) The instrument according to embodiment 7, wherein the enzyme substrate is an oxidizable substrate.
12. The device of embodiment 11, wherein the oxidizable substrate is selected from the group consisting of galactose and acetic acid.
(13) The device according to embodiment 11, wherein the enzyme substrate comprises glucose.
(14) The instrument according to embodiment 7, wherein the detection chamber further comprises a mediator.
15. The instrument of embodiment 14, wherein the mediator is selected from the group consisting of dichlorophenolindphenol and a complex of a transition metal and a nitrogen-containing heteroatomic species.
(16) The device according to embodiment 14, wherein the mediator comprises ferricyanide.
(17) The instrument according to embodiment 7, further comprising a buffer capable of adjusting the pH of the sample.
(18) The device according to embodiment 17, wherein the buffer is a substance selected from the group consisting of phosphate and citrate.
(19) The instrument according to embodiment 1, wherein the detection chamber has at least two electrodes.
(20) The electrode is made of a material selected from the group consisting of palladium, platinum, gold, iridium, carbon, a mixture of carbon and a binder, indium oxide, tin oxide, and a mixture thereof. Embodiment 20. The instrument of embodiment 19.
21. The embodiment of claim 1, wherein the walls of the detection chamber are transparent to radiation emitted or absorbed by the probe, said radiation indicating the presence or absence of a reporter complex in the detection chamber. Appliances.
(22) The apparatus according to embodiment 1, further comprising a detector capable of detecting a state in which the reaction chamber is substantially filled.
23. The instrument of embodiment 1, further comprising perforation means capable of forming a detection chamber degasser at a distal end of the detection chamber.
24. The apparatus of embodiment 1 further comprising a reaction chamber vent at the distal end of the reaction chamber.
(25) A method for producing a disposable device used to detect a target antigen in a fluid sample having a pH,
A first material sheet having a proximal end and a distal end passes through the first material sheet, the reaction chamber sidewall, the detection chamber sidewall, and a first between the reaction chamber distal end and the detection chamber proximal end. Forming a first hole that constitutes the sample passage;
Attaching the first layer to the first surface of the first sheet in a state of covering the holes to form the first reaction chamber end wall and the first detection chamber end wall;
A second layer is attached to the second surface of the first sheet with the hole covered, and the second reaction chamber end wall and the second detection chamber end wall are substantially overlapped with the first layer. Forming a strip with a plurality of outer surfaces, wherein the sheet and layer comprise a relationship;
Penetrating the outer surface of the strip and extending into the reaction chamber at the distal end of the reaction chamber to form a second passage defining the reaction chamber venting;
Passing through the outer surface of the strip and extending into the reaction chamber at the proximal end of the reaction chamber to form a third passage defining a sample inlet;
Immobilizing the antibody in the reaction chamber;
Placing the reporter complex constituting the probe in a reaction chamber.
(26) The first sheet, the first layer, and the second layer are made of an electric resistance material, and the first layer constitutes a first electrode facing the first surface of the first sheet. 26. The method of embodiment 25, wherein the second layer constitutes a second electrode facing the second surface of the first sheet.
27. The method of embodiment 26, wherein the second electrode is provided in a face-to-face relationship at a distance of about 500 microns or less from the first electrode.
28. The method of embodiment 26, wherein the second electrode is provided in a face-to-face relationship at a distance of about 150 microns or less from the first electrode.
29. The method of embodiment 26, wherein the second electrode is provided in a face-to-face relationship at a distance of about 150 microns or less and about 50 microns or more from the first electrode.
30. The method of embodiment 25, wherein at least one of the layers is transparent to a wavelength of radiation selected from the group consisting of infrared light, visible light, and ultraviolet light.
(31) The method further comprises the steps of preparing an enzyme substrate and a mediator, wherein the enzyme substrate and the mediator are placed in a detection chamber, the probe is an enzyme, and the mediator directs the occurrence of an electrochemical reaction to the enzyme electrode 26. The method of embodiment 25, wherein the reaction of
(32) A method for producing a disposable device used to detect a target antigen in a fluid sample having a pH,
Forming a first hole through the first sheet of electrical resistance material comprising a proximal end and a distal end, wherein the first hole comprises a reaction chamber portion of the first hole and a first hole. And a sample passage between the first portion of the reaction chamber side wall, the detection chamber side wall and the reaction chamber distal end and the detection chamber proximal end,
Forming a second hole through a second sheet of electrically resistive material having a proximal end and a distal end, the second hole forming a second portion of the reaction chamber sidewall. ,
Forming a third hole through a third sheet of electrically resistive material having a proximal end and a distal end, the third hole constituting a third portion of the reaction chamber sidewall. ,
Attaching the first surface of the second sheet to the first surface of the first sheet, the second sheet extending over the detection chamber portion of the first hole and extending to the first detection chamber; Constituting an end wall, wherein the second portion of the reaction chamber sidewall is in a substantially overlapping relationship with the first portion of the reaction chamber sidewall;
Attaching the first surface of the third sheet to the second surface of the first sheet, the third sheet extending over the detection chamber portion of the first hole and extending to the second detection chamber; Constituting an end wall, and the third portion of the reaction chamber sidewall is in a substantially overlapping relationship with the first portion of the reaction chamber sidewall;
Attaching the first layer to the second surface of the second sheet in a state of covering the second hole to form the first reaction chamber end wall;
A second layer is attached to the second surface of the third sheet with the third hole covered, and the second reaction chamber end wall is configured to substantially overlap the first thin layer. The sheet and the layer form a strip with a plurality of outer surfaces;
Forming a second passage through the exterior of the strip and extending into the reaction chamber at the distal end of the reaction chamber, the second passage comprising a reaction chamber venting;
Forming a third passage extending through the exterior of the strip and into the reaction chamber at the proximal end of the reaction chamber, the third passage constituting a sample inlet;
Immobilizing the antibody in the reaction chamber;
A method comprising disposing a reporter complex constituting a probe in a reaction chamber, wherein the reporter complex constitutes a probe.
(33) A method for determining the presence or absence of a target antigen in a fluid sample, comprising the step of preparing the instrument according to embodiment 1, wherein the reporter complex is capable of competing with the target antigen for binding to the immobilized antibody. Further comprising two antigens,
Contacting the fluid sample with the sample inlet;
Substantially filling the reaction chamber with a fluid sample by allowing the sample to flow from the sample inlet toward the reaction chamber;
Having a predetermined time sufficient for substantially all reporter complexes to bind to the immobilized antibody in the absence of antigen in the sample;
Substantially filling the detection chamber with a fluid sample by allowing the sample to flow from the reaction chamber through the sample passage toward the detection chamber;
A method comprising detecting the presence or absence of an antigen-probe complex in a detection chamber, wherein the presence or absence of an antigen-probe complex represents the presence or absence of an antigen in a sample.
(34) A method for producing a disposable device used when detecting a target antigen in a fluid sample having a pH,
Forming a first hole through the first sheet of electrical resistance material, the first hole having a detection chamber portion and forming a detection chamber sidewall, the detection chamber comprising a proximal end and Having a distal end,
Attaching the first layer to the first surface of the first sheet in a state of covering the hole to form the first detection chamber end wall;
Attaching the second layer to the second surface of the first sheet in a state of covering the hole, and configuring the second detection chamber end wall in a substantially overlapping relationship with the first layer. Sheet and layer form a strip;
Forming a second hole extending through the strip, the strip having a proximal end and a distal end, the second hole having a reaction chamber portion, the reaction chamber comprising: Having a distal end and a proximal end, the second hole defining a reaction chamber side wall and a sample passageway between the reaction chamber distal end and the detection chamber proximal end;
Attaching the first side of the third layer to the first side of the strip, the third layer extending over the second hole reaction chamber portion to constitute a first reaction chamber end wall; And
Attaching the first side of the fourth layer to the second side of the strip, the fourth layer extending over the reaction chamber portion of the second hole to connect the second reaction chamber end wall; A substantially overlapping relationship with the first reaction chamber end wall;
Forming a third hole through the surface of the instrument and extending into the reaction chamber at the reaction chamber distal end, wherein the third hole constitutes a reaction chamber vent.
Forming a fourth hole extending through the surface of the instrument into the reaction chamber at the reaction chamber proximal end, the fourth hole defining a sample inlet;
Immobilizing the antibody in the reaction chamber;
Placing the reporter complex in a reaction chamber, wherein the reporter complex constitutes a probe.
[Brief description of the drawings]
FIG. 1 is a plan view (not drawn to scale) of an immunological sensor incorporating an electrochemical cell.
2 is a cross-sectional view (not drawn to scale) taken along the line A-A ′ of the embodiment of the immunological sensor of FIG. 1;

Claims (34)

  1. In a disposable instrument used for detecting a target antigen in a fluid sample having a pH, a reaction chamber having an inner surface, a proximal end and a distal end, and a reaction chamber fixed in the reaction chamber An immobilized antibody capable of binding; a reporter complex that is present in the reaction chamber and comprises a probe and can be mixed with the sample; and a wall, an inner surface, a distal end and a proximal end A detection chamber; a sample inlet provided at the proximal end of the reaction chamber; and a sample passage provided between the distal end of the reaction chamber and the proximal end of the detection chamber;
    Before SL instrument, by capillary action forces, it is adapted to move fluid to the detection chamber from the reaction chamber,
    The reporter complex further comprises a second antigen capable of competing with the target antigen for binding to the immobilized antibody or a second antibody capable of binding to the target antigen;
    The reporter complex is initially separated from the immobilized antibody,
    A device characterized by that.
  2.   The instrument of claim 1, wherein the distal end of the detection chamber has no outlet.
  3.   The instrument according to claim 1, further comprising an agent housed in the reaction chamber and capable of preventing non-specific binding of proteins to the inner surface of the reaction chamber.
  4.   4. A device according to claim 3, wherein the drug is selected from the group consisting of a surfactant and a blocking protein.
  5. The medical device of claim 4, wherein the blocking protein, characterized by being a BSA (bovine serum albumin), the instrument.
  6.   The instrument of claim 1, wherein the probe is selected from the group consisting of a chromophore and a phosphor.
  7.   The instrument according to claim 1, wherein the probe comprises an enzyme.
  8. 8. A device according to claim 7 , characterized in that the enzyme comprises glucose oxidase.
  9. 8. A device according to claim 7 , wherein the enzyme comprises glucose dehydrogenase.
  10. 8. An instrument according to claim 7 , wherein the detection chamber further comprises an enzyme substrate .
  11. 8. A device according to claim 7 , wherein the enzyme substrate is an oxidizable substrate .
  12. 12. The device according to claim 11 , wherein the oxidizable substrate is selected from the group consisting of galactose and acetic acid.
  13. 12. A device according to claim 11 , characterized in that the enzyme substrate comprises glucose.
  14. The instrument of claim 7 , wherein the detection chamber further comprises a mediator.
  15. 15. A device according to claim 14 , characterized in that the mediator is selected from the group consisting of dichlorophenolindphenol and a complex of a transition metal and a nitrogen-containing heteroatomic species.
  16. 15. A device according to claim 14 , wherein the mediator comprises ferricyanide.
  17. 8. The instrument according to claim 7 , further comprising a buffer capable of adjusting the pH of the sample.
  18. 18. A device according to claim 17 , characterized in that the buffering agent is a substance selected from the group consisting of phosphate and citrate.
  19.   The instrument of claim 1, wherein the detection chamber has at least two electrodes.
  20. 20. The device of claim 19 , wherein the electrode is composed of a material selected from the group consisting of palladium, platinum, gold, iridium, carbon, a mixture of carbon and binder, indium oxide, tin oxide, and mixtures thereof. A device characterized by that.
  21.   The instrument of claim 1, wherein the wall of the detection chamber is transparent to radiation emitted or absorbed by the probe, the radiation indicating the presence or absence of a reporter complex in the detection chamber. To do the instrument.
  22.   The instrument of claim 1, further comprising a detector capable of detecting a state in which the reaction chamber is substantially full.
  23.   The instrument of claim 1, further comprising perforation means capable of forming a detection chamber degasser at a distal end of the detection chamber.
  24.   The instrument of claim 1 further comprising a reaction chamber vent at the distal end of the reaction chamber.
  25. The method for producing a disposable device according to claim 1, which is used for detecting a target antigen in a fluid sample having a pH.
    A first material sheet having a proximal end and a distal end passes through the first material sheet, the reaction chamber sidewall, the detection chamber sidewall, and a first between the reaction chamber distal end and the detection chamber proximal end. Forming a first hole defining a sample passageway of:
    Attaching a first layer to the first surface of the first sheet over the hole to define a first reaction chamber end wall and a first detection chamber end wall;
    A second layer is attached to the second surface of the first sheet with the hole covered, and the second reaction chamber end wall and the second detection chamber end wall are substantially overlapped with the first layer. Defining the relationship so that the sheet and the layer form a strip with a plurality of outer surfaces;
    Passing through the outer surface of the strip and extending into the reaction chamber at the distal end of the reaction chamber to form a second passage defining reaction chamber degassing;
    Passing through the outer surface of the strip and extending into the reaction chamber at the proximal end of the reaction chamber to form a third passage defining a sample inlet;
    Immobilizing the antibody in the reaction chamber;
    Placing a reporter complex comprising a probe in a reaction chamber.
  26. 26. The method of claim 25 , wherein the first sheet, the first layer, and the second layer are composed of an electrically resistive material, the first layer facing the first surface of the first sheet. A method comprising: one electrode, wherein the second layer comprises a second electrode facing the second surface of the first sheet.
  27. 27. The method of claim 26 , wherein the second electrode is provided in a face-to-face relationship at a distance of about 500 microns or less from the first electrode.
  28. 27. The method of claim 26 , wherein the second electrode is provided in a face-to-face relationship at a distance of about 150 microns or less from the first electrode.
  29. 27. The method of claim 26 , wherein the second electrode is provided in a face-to-face relationship at a distance of about 150 microns or less and about 50 microns or more from the first electrode.
  30. 26. The method of claim 25 , wherein at least one of the layers is transparent to a wavelength of radiation selected from the group consisting of infrared light, visible light and ultraviolet light.
  31. The method of claim 25, further comprising the step of providing an enzyme substrate and mediator, enzyme substrate and mediator are placed in the detection chamber, the probe is an enzyme, the mediator, the generation of electrochemical reaction A method characterized in that it can mediate the reaction between the enzyme and the electrode to indicate.
  32. The method for producing a disposable device according to claim 1, which is used for detecting a target antigen in a fluid sample having a pH.
    Forming a first hole through the first sheet of electrical resistance material comprising a proximal end and a distal end, wherein the first hole comprises a reaction chamber portion of the first hole and a first hole. And defining a sample passage between the first portion of the reaction chamber side wall, the detection chamber side wall and the reaction chamber distal end and the detection chamber proximal end,
    Forming a second hole through a second sheet of electrically resistive material comprising a proximal end and a distal end, the second hole defining a second portion of the reaction chamber sidewall. ,
    Forming a third hole through a third sheet of electrically resistive material having a proximal end and a distal end, the third hole defining a third portion of the reaction chamber sidewall. ,
    Attaching the first surface of the second sheet to the first surface of the first sheet, the second sheet extending over the detection chamber portion of the first hole and extending to the first detection chamber; Defining an end wall, the second portion of the reaction chamber sidewall being in a substantially overlapping relationship with the first portion of the reaction chamber sidewall;
    Attaching the first surface of the third sheet to the second surface of the first sheet, the third sheet extending over the detection chamber portion of the first hole and extending to the second detection chamber; Defining an end wall and the third portion of the reaction chamber sidewall is in a substantially overlapping relationship with the first portion of the reaction chamber sidewall;
    Attaching a first layer to a second surface of a second sheet with the second hole covered to define a first reaction chamber end wall;
    A second layer is attached to the second surface of the third sheet with the third hole covered to define a second reaction chamber end wall in a substantially overlapping relationship with the first thin layer. The sheet and the layer form a strip with a plurality of outer surfaces;
    Forming a second passage through the exterior of the strip and extending into the reaction chamber at the distal end of the reaction chamber, the second passage defining a reaction chamber degassing;
    Forming a third passage through the exterior of the strip and extending into the reaction chamber at the proximal end of the reaction chamber, the third passage defining a sample inlet;
    Immobilizing the antibody in the reaction chamber;
    Placing the reporter complex in a reaction chamber, the reporter complex comprising a probe.
  33. A method for determining the presence or absence of a target antigen in a fluid sample, comprising the step of providing the instrument of claim 1, wherein the reporter complex further comprises a second antigen capable of competing with the target antigen for binding to the immobilized antibody. Including
    Contacting the fluid sample with the sample inlet;
    Substantially filling the reaction chamber with a fluid sample by allowing the sample to flow from the sample inlet toward the reaction chamber;
    Having a predetermined time sufficient for substantially all reporter complexes to bind to the immobilized antibody in the absence of antigen in the sample;
    Substantially filling the detection chamber with a fluid sample by allowing the sample to flow from the reaction chamber through the sample passage toward the detection chamber;
    A method comprising detecting the presence or absence of an antigen-probe complex in a detection chamber, wherein the presence or absence of an antigen-probe complex represents the presence or absence of an antigen in a sample.
  34. The method for producing a disposable device according to claim 1, which is used when detecting a target antigen in a fluid sample having a pH, wherein the device comprises a plurality of outer surfaces, and the method comprises:
    Forming a first hole through the first sheet of electrical resistance material, the first hole having a detection chamber portion and defining a detection chamber sidewall, the detection chamber comprising a proximal end and Having a distal end,
    Attaching a first layer to the first surface of the first sheet with the hole covered to define a first detection chamber end wall;
    Attaching a second layer to the second surface of the first sheet over the hole to define a second detection chamber end wall in a substantially overlapping relationship with the first layer. Sheet and layer form a strip;
    Forming a second hole extending through the strip, the strip having a proximal end and a distal end, the second hole having a reaction chamber portion, the reaction chamber comprising: A distal end and a proximal end, the second hole defining a reaction chamber side wall and a sample passageway between the distal end of the reaction chamber and the proximal end of the detection chamber;
    Attaching the first side of the third layer to the first side of the strip, the third layer extending over the second hole reaction chamber portion and defining a first reaction chamber end wall; And
    Attaching the first side of the fourth layer to the second side of the strip, the fourth layer extending over the reaction chamber portion of the second hole to connect the second reaction chamber end wall; Defining in a substantially overlapping relationship with the first reaction chamber end wall;
    Forming a third hole through the surface of the instrument and extending into the reaction chamber at the distal end of the reaction chamber, the third hole defining a reaction chamber venting;
    Forming a fourth hole extending through the surface of the instrument and into the reaction chamber at the reaction chamber proximal end, the fourth hole defining a sample inlet;
    Immobilizing the antibody in the reaction chamber;
    Placing the reporter complex in a reaction chamber, the reporter complex comprising a probe.
JP2002514406A 1995-11-16 2001-07-13 Immunological sensor Active JP4929427B2 (en)

Priority Applications (9)

Application Number Priority Date Filing Date Title
US61643300A true 2000-07-14 2000-07-14
US09/615,691 US6638415B1 (en) 1995-11-16 2000-07-14 Antioxidant sensor
US09/616,556 2000-07-14
US09/616,512 US6632349B1 (en) 1996-11-15 2000-07-14 Hemoglobin sensor
US09/616,512 2000-07-14
US09/616,433 2000-07-14
US09/616,556 US6444115B1 (en) 2000-07-14 2000-07-14 Electrochemical method for measuring chemical reaction rates
US09/615,691 2000-07-14
PCT/US2001/022202 WO2002008763A2 (en) 2000-07-14 2001-07-13 Immunosensor

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JP2004505246A JP2004505246A (en) 2004-02-19
JP4929427B2 true JP4929427B2 (en) 2012-05-09

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IL (3) IL153584A (en)
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US20060134713A1 (en) * 2002-03-21 2006-06-22 Lifescan, Inc. Biosensor apparatus and methods of use
NZ590411A (en) * 2008-07-11 2013-07-26 Universal Biosensors Pty Ltd Enhanced immunoassay sensor

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WO1998043074A1 (en) * 1997-03-25 1998-10-01 Usf Filtration And Separations Group Inc. Improved electrochemical cell
JP2000500572A (en) * 1995-11-16 2000-01-18 メムテック・アメリカ・コーポレイション Electrochemical cell

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AUPP238898A0 (en) * 1998-03-12 1998-04-09 Usf Filtration And Separations Group Inc. Heated electrochemical cell
WO1999053312A1 (en) * 1998-04-15 1999-10-21 Biofutura S.R.L. Apparatus adapted to perform a plurality of determinations on wine samples or the like
EP1084133B1 (en) * 1998-06-01 2002-08-28 Roche Diagnostics Corporation Redox reversible bipyridyl osmium complex conjugates

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US4859583A (en) * 1985-02-25 1989-08-22 Amoco Corporation Chemiluminescent immunochemical technique for low molecular weight antigens
JP2000500572A (en) * 1995-11-16 2000-01-18 メムテック・アメリカ・コーポレイション Electrochemical cell
WO1998043074A1 (en) * 1997-03-25 1998-10-01 Usf Filtration And Separations Group Inc. Improved electrochemical cell

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IL153582A (en) 2008-03-20
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IL153584A (en) 2008-03-20
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IL153583A (en) 2007-03-08
SG143056A1 (en) 2008-06-27
CA2733852A1 (en) 2002-01-24

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