WO1997002487A1 - Electrochemical biosensor test strip - Google Patents
Electrochemical biosensor test strip Download PDFInfo
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- WO1997002487A1 WO1997002487A1 PCT/US1996/011240 US9611240W WO9702487A1 WO 1997002487 A1 WO1997002487 A1 WO 1997002487A1 US 9611240 W US9611240 W US 9611240W WO 9702487 A1 WO9702487 A1 WO 9702487A1
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- WIPO (PCT)
- Prior art keywords
- reagent
- cutout portion
- electrodes
- millimeters
- working
- Prior art date
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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
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
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- 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
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- 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
- C12Q1/005—Enzyme electrodes involving specific analytes or enzymes
- C12Q1/006—Enzyme electrodes involving specific analytes or enzymes for glucose
Definitions
- This invention relates generally to the determination of the concentration of analytes in fluids and more specifically to an amperometric biosensor for use in such determination.
- Biosensors are not new. Their use in the determination of concentrations of various analytes in fluids is also known.
- Nankai et al. discloses a method in which the reaction of glucose and ferricyanide may run to completion prior to the application of an electrical potential, this method is referred to as the "end-point" method of amperometric determination.
- Nankai et al. discloses a system, wherein the glucose oxidase and potassium ferricyanide are held on a nonwoven nylon mesh.
- the mesh is positioned so that it is in contact with a working electrode, a counter electrode and a reference electrode.
- the total surface area of the counter and reference electrodes is twice that of the working electrode.
- the anode is formed from an anode material, such as platinum, and the cathode is formed from a cathode material, such as silver.
- the anode is coated with an enzyme.
- the coated electrode is covered with an elastomer that is permeable to glucose.
- Pottgen et al. WO 89/08713, published Sept. 21, 1989, discloses the use of a two electrode biosensor, wherein the electrodes are made of the same noble metal, but one of the electrodes (referred to as a pseudoreference electrode) is larger than the other (working) electrode.
- the Pollmann et al. test strip includes a reagent well that will accommodate a testing sample of human whole blood from about 10 to about 70 microliters.
- errors in the measurement of an analyte, such as glucose, from a whole blood sample may result (low dosing errors).
- the low dosing error is manifested as an understated measurement of the analyte, or no measurement of the analyte by the meter used in conjunction with the test strip.
- Low dosing errors are a particular concern for infants and elderly persons who often have difficulty in expressing a reasonably sized blood drop for testing upon pricking their finger with a lancet.
- test strip that requires a minimum volume of blood for the testing of an analyte, such as blood glucose.
- the invention is an electrochemical biosensor test strip that has a lower minimum volume blood sample requirement than prior art strips of similar construction.
- the present inventive test strip has a smaller reagent well and smaller spreading mesh than similar prior art strips. Further, the reagent well is positioned differently than in similar prior art test strips.
- the minimum blood volume sample requirement for the new strip is about 9 microliters.
- the smaller sample volume requirement means fewer low sample volume dosing errors result when measuring an analyte, such as glucose, from a whole blood sample. This result is especially important for those persons, such as infants and the elderly, who have difficulty expressing a reasonably sized drop of blood by pricking their finger with a lancet. Also, with the present inventive strip it is easier for the meter, which collects current measurements and correlates those measurements to a concentration of analyte from a sample, to discriminate low sample volume dosing errors. Further, the smaller reagent well requires less reagent per biosensor strip, thereby increasing the production volume for mass production of biosensor test strips.
- the tape when the spreading mesh is affixed to the test strip by an adhesive tape, the tape includes a hole that exposes the reagent well and spreading mesh, and further includes air vents on opposing sides of the hole. These air vents reduce the occurrence of air bubbles trapped in the reagent well when a sample is being tested. Air bubbles can produce testing errors.
- FIG. 1 is an exploded view of the present inventive biosensor test strip.
- FIG. 2 is a top view of the biosensor test strip without the reagent, spreading mesh, and adhesive tape with air vents.
- FIG. 3 is a top view of the fully constructed, preferred biosensor test strip.
- FIG. 4 is a cross-sectional view of the biosensor of FIG. 3 along lines 21-21.
- FIG. 5 illustrates hypothetical calibration curves for different lots of biosensor test strips.
- the present inventive biosensor test strip is similar to the preferred embodiment of the test strip described in Pollmann et al., U.S. Patent No. 5,288,636, issued Feb. 22, 1994, the disclosure of which is hereby incorporated by reference.
- the Pollmann et al. strip has a construction such that too many low dosing errors result when whole blood samples below about 13 microliters are tested for blood glucose.
- reagent well 9. (Fig. 4) has been reduced in size over the Pollmann et al. reagent well and repositioned so that a smaller surface area of the counter electrode 5_ than the working electrode 4 is exposed by cutout portion 8_, which forms reagent well 9_.
- Mesh 12 which is a spreading mesh, is also reduced in size over the Pollmann et al. mesh. (Figs. 1, 3, 4)
- FIGs. 1 through 4 there is shown the presently preferred embodiment of the inventive biosensor test strip.
- Test strip I comprises first and second electrically insulating layers 2 and 2, respectively. Any useful insulating material will be suitable. Typically, plastics, such as vinyl polymers and polyimides provide the electrical and structural properties which are desired. Preferably, these layers are Melinex 329, 7 mil.
- the biosensor test strip shown in Figs. 1 through 4 is intended to be mass produced from rolls of material, necessitating the selection of a material which is sufficiently flexible for roll processing and at the same time sufficiently stiff to give a useful stiffness to the finished biosensor test strip.
- Layers 2 and 2 may be of any useful thickness. In a preferred embodiment, layers 2 and 2 are about 7 mil thick.
- Working electrode 4. and counter electrode 5_ are preferably deposited on a backing of insulator material 7, such as polyimide, to reduce the possibility of tearing the electrode before it is affixed to layer 2-
- Working electrode 4 and counter electrode 5_ are substantially the same size and are made of the same electrically conducting material.
- electrically conducting materials that may be used are palladium, platinum, gold, silver, carbon, titanium, and copper.
- Noble metals are preferred because they provide a more constant, reproducible electrode surface area. Palladium is particularly preferred because it is one of the more difficult noble metals to oxidize and because it is a relatively inexpensive noble metal. Silver is not preferred because it is more readily oxidized by air than the other noble metals listed above.
- electrodes 4 and 5_ are about 0.1 micron thick and backing 7 is about 25 microns thick (commercially available from Courtaulds Performance Films in California and Southwall Technologies, Inc.).
- Electrodes 4 and 5_ must be sufficiently separated so that the electrochemical events at one electrode do not interfere with the electrochemical events at the other electrode.
- the preferred distance between electrodes 4 and 5_ is about 1.2 millimeters.
- electrodes 4 and 5_ affixed to backing 7, are unspooled from reels and attached to layer 2 by the use of hot melt adhesive (not shown). Electrodes 4 and 5_ also preferably extend from one end of layer 2 to the other end in parallel configuration.
- Insulating layer 2 is fixed on top of layer 2 and electrodes 4 and 5_ by the use of hot melt adhesive (not shown).
- Layer 2 includes cutout portion 8_, which defines reagent well 2- Both the size and the position of cutout portion 8 are critical to the invention. Cutout portion £ must be sufficiently small and must be sufficiently positioned such that in combination with the spreading mesh, described below, a minimum whole blood sample volume of about 9 microliters may be accurately analyzed by the test strip.
- the preferred size of cutout portion S is 4 millimeters by 4.2 millimeters.
- the 4 mm side of cutout portion 8. runs parallel to the long side of the test strip shown in Figs. 1-4.
- cutout portion 8 . is positioned over electrodes 4 and 5_ such that a smaller surface area of counter electrode 5_ than working electrode 4 is exposed.
- the exposed surface area of working electrode 4 is twice as large as the exposed surface area of counter electrode 5_.
- offsetting cutout portion 8. to expose a smaller surface area for the counter electrode than the working electrode does not adversely affect measurement of an analyte from a sample being measured.
- electrodes 4 and 5_ are 1.5 mm in width.
- Biosensor test strip 1 may be accompanied by a power source (not shown) in a electrical connection with the working and counter electrodes and a current measuring meter (not shown) which is also in a electrical connection with the working and counter electrodes.
- Biosensor reagent H (Fig. 4) is placed in well 2 so that it covers substantially all of exposed surfaces IQ and 2Q of working electrode 4 and counter 5_, respectively.
- a reagent that may be used in the biosensor test strip of the present invention is a reagent for measuring glucose from a whole blood sample.
- a protocol for making a glucose reagent utilizing the enzyme glucose oxidase and ferricyanide as the oxidized form of the redox mediator is as follows:
- Step 1- Prepare 1 liter (in a volumetric flask) of a buffer/NATROSOL mixture by adding 1.2000 grams (g) NATROSOL-250 M to 0.740 M aqueous potassium phosphate buffer (including 80.062 g monobasic potassium phosphate and 26.423 g dibasic potassium phosphate) at pH 6.25. Allow the NATROSOL to stir and swell for 3 hours.
- Step 2- Prepare an AVICEL mixture by stirring 14.0000 g AVICEL RC-591 F and 504.7750 g water for 20 minutes.
- Step 3- Prepare a TRITON mixture by adding 0.5000 g TRITON X-100 to 514.6000 g of the buffer/NATROSOL mixture and stir for 15 minutes.
- Step 4- While stirring, add the total TRITON mixture dropwise with an addition funnel or buret to the total AVICEL mixture. Once addition is complete, continue stirring overnight.
- Step 5- To the mixmre resulting from Step 4, add, while stirring, 98.7750 g potassium ferricyanide. (Add a little potassium ferricyanide at a time to allow the potassium ferricyanide to dissolve as added.)
- Step 6- Stir the resulting mixmre of Step 5 for 20 minutes.
- Step 7- Adjust the pH of the mixmre resulting from Step 6 to 6.25 by adding potassium hydroxide.
- Step 8- To the resulting mixmre of Step 7, add 9.1533 g glucose oxidase (218.50 tetramethyl benzidine units per milligram (mg) from Biozyme) and stir at least 20 minutes.
- Step 9- To the resulting mixmre of Step 8, add 20 g potassium glutamate and stir at least 20 minutes.
- Step 10- Filter the resulting mixmre of Step 9 through a 100 micron sieve bag to remove any AVICEL clumping.
- the filtrate is the resulting reagent composition o (reagent H), which is added to reagent well 2 and is then dried at about 50 C° for about 3 minutes.
- reagent H the reagent composition
- 4 microliters of reagent made by the above-stated protocol is added to well 2 formed by cutout 8_. This amount of reagent 11 will substantially cover surface areas IQ and 2 ⁇ of the electrodes 4 and 5.
- Another glucose reagent that may be formulated includes 300 millimolar potassium ferricyanide, 250 millimolar potassium phosphate buffer, 14 grams microcrystalline cellulose (AVICEL RC-591 F) per liter of reagent, 0.6 grams hydroxyethy lcellulose (NATROSOL-250 M) per liter of reagent, 0.5 grams Triton X- 100 surfactant per liter of reagent, 37 millimolar sodium succinate, and 1.57 million tetramethyl benzidine units of glucose oxidase per liter of reagent.
- Sodium hydroxide (6 Normal solution) is used to titrate this reagent to a pH of 6.6.
- This reagent may be formulated by the same protocol described above, but amounts of components should be adjusted and components substimted (sodium succinate for potassium glutamate and sodium hydroxide for potassium hydroxide) to achieve the component concentrations stated above. Drying of this reagent in reagent well 2 typically results in a loss of enzyme activity of about 30-35 % .
- a spreading mesh 12 which has been impregnated with a surfactant, is placed over cutout portion 8. and is affixed to second electrical insulator 2- Speading mesh 12 is preferably a polyester monofilament mesh from ZBF (Zurich Bolting Cloth Mfg. Co. Ltd., R ⁇ schlikon, Switzerland).
- the spreading mesh is preferably dipped in a solution of 0.8% (wt.:vol.) dioctylsodium sulfosuccinate (DONS) in a solution of 50:50 (vol. : vol.) methanol: water, and then dried.
- DONS dioctylsodium sulfosuccinate
- Spreading mesh H must be small enough such that in combination with the size of cutout portion £ and placement of cutout portion £ the biosensor strip will accurately measure analyte from a minimum whole blood sample of about 9 microliters.
- the preferable dimensions of spreading mesh 12 are 6 mm x 5.8 mm. In the most preferred biosensor strip, the 6 mm side of the mesh is parallel to the long side of the strip shown in Figs. 1-4.
- spreading mesh 12 is affixed to adhesive tape 14, which includes hole 15_.
- Adhesive tape 14 is preferably made of polyester with an adhesive backing. (Available from Tapemark, Medical Products Division, 223 E. Marie Ave., St. Paul, Minnesota 55118) Adhesive tape 14 is preferably dyed maroon and hole 15_ provides a target area for application of a sample to be analyzed by the biosensor. Hole 15 exposes at least a portion of spreading mesh 12 and cutout portion 8, and preferably exposes substantially all of cutout portion £.
- Tape 14 preferably includes slits 16, as shown in Figs. 1 and 3, located on opposing sides of hole 15_.
- Slits 16 constitute air vents, which reduce the occurrence of air bubbles trapped in the reagent well upon the addition of a sample such whole blood to the reagent well. Reducing the occurrence of air bubbles trapped in reagent well 2 results in fewer testing errors.
- the roll-formed biosensors are separated by die punching to form discrete biosensors, which are used in conjunction with 1) a power source in electrical connection with the working and counter electrodes and capable of supplying an electrical potential difference between the working and counter electrodes sufficient to cause diffusion limited electrooxidation of the reduced form of the redox mediator at the surface of the working electrode, and 2) a meter in electrical connection with the working and counter electrodes and capable of measuring the diffusion limited current produced by oxidation of the reduced form of the redox mediator when the above-stated electrical potential difference is applied.
- the meter described above will normally be adapted to apply an algorithm (discussed below) to the current measurement, whereby an analyte concentration is provided and visually displayed.
- additional cutout portion 12 (Figs. 1 through 4), exposing portions of the working and counter electrodes, are preferably provided in the biosensor device.
- the biosensor device described above may be used to determine the concentration of an analyte in a fluid sample by performing the following steps:
- reaction completion is defined as sufficient reaction between the analyte and the oxidized form of the redox mediator to correlate analyte concentration to diffusion limited current generated by oxidation of the reduced form of the redox mediator at the surface of the working electrode.
- analyte-containing fluids may be analyzed. For example, analytes in human body fluids such as whole blood, blood serum, urine and cerebrospinal fluid may be measured. Also, analytes found in fermentation products and in environmental substances, which potentially contain environmental contaminants, may be measured.
- the potential difference applied between the electrodes is preferably no more than about 500 millivolts.
- a potential difference above about 500 millivolts is applied between the electrodes, oxidation of the working electrode surface (for palladium) and of some blood components may become intolerable, thereby preventing an accurate and precise correlation of current to analyte concentration.
- a potential difference from about 150 millivolts to about 500 millivolts may be applied between the electrodes to achieve diffusion limited electrooxidation of the reduced form of the redox mediator at the surface of the working electrode.
- about 300 millivolts potential difference is applied between the electrodes.
- Current generated from the oxidation of the reduced form of the redox mediator may be measured at any time from about 0.5 seconds to about 30 seconds after the potential difference is applied between the electrodes. At less than about 0.5 seconds, diffusion limited current is difficult to measure due to the charging current. After about 30 seconds, convection becomes significant, thereby interfering with the measurement of a diffusion limited current.
- the current measured during the assay of an analyte from a fluid sample may be correlated to concentration of the analyte in the sample by application of an algorithm by the current measuring meter.
- the algorithm may be a simple one, as illustrated by the following example:
- [Analyte] represents the concentration of the analyte in the sample (see Fig. 5), i 7 5 is the current (in microamps) measured at 7.5 seconds after application of the potential difference applied between the electrodes, C is the slope of line 22 (Fig. 5), and d is the axis intercept (Fig. 5).
- calibration curve 22 (Fig. 5) may be constructed. This calibration will be stored in the Read Only Memory (ROM) key of the meter and will be applicable to a particular lot of biosensor test strips. Lines 24 and 26 in Fig. 5 represent other hypothetical calibration curves for two other different lots of biosensor test strips. Calibration for these biosensor lots would generate slightly different values for C and d in the above algorithm.
- ROM Read Only Memory
- glucose and ferricyanide are preferably added to the above-stated glucose reagent.
- the reaction of glucose and ferricyanide is allowed to go to completion, thereby forming gluconic acid and ferrocyanide.
- This reaction normally requires a short time, preferably less than about 20 seconds, to go to completion.
- a potential difference of about 300 millivolts is applied between the electrodes, thereby oxidizing ferrocyanide to ferricyanide at the surface of the working electrode.
- Current measurements are made at 0.5 second intervals from 1 second to 7.5 seconds after the potential difference is applied between the electrodes. These current measurements are correlated to the concentration of glucose in the blood sample.
- [Glucose] Ci ii + C2 12 + C3 -3 + ...C n i n + d, wherein ii is the current measured at the first measurement time (1 second after application of the 300 millivolt potential difference), i2 is the current measured at the second measurement time (1.5 seconds after application of the 300 millivolt potential difference), i3 is the current measured at the third measurement time (2 seconds after application of the 300 millivolt potential difference), i n is the current measured at the n m measurement time (in this example, at the 14 m measurement time or 7.5 seconds after application of the 300 millivolt potential difference), C ⁇ , C2, C3, and C n are coefficients derived from a multivariate regression analysis technique, such as Principle Components Analysis or Partial Least Squares, and d is the regression intercept (in glucose concentration units). (A modification of this procedure may be used in the event that calibration curves illustrated by Fig. 5 have considerable curvature.)
- the concentration of glucose in the sample being measured may be determined by integrating the curve generated by plotting current, i, versus measurement time over some time interval (for example, from 1 second to 7.5 seconds after application of the 300 millivolt potential difference), thereby obtaining the total charge transferred during the measurement period.
- the total charge transferred is directly proportional to the concentration of glucose in the sample being measured.
- the glucose concentration measurement may be corrected for differences between environmental temperamre at the time of acmal measurement and the environmental temperamre at the time calibration was performed. For example, if the calibration curve for glucose measurement was constructed at an environmental temperamre of 23 °C, the glucose measurement is corrected by using the following equation:
- each of a multiplicity of glucose concentrations is measured by the meter at various temperatures, T, and at 23°C (the base case).
- T various temperatures
- 23°C the base case
- the glucose concentration of a sample may be accurately and precisely measured by the present inventive method utilizing the present inventive biosensor. Further, when a sample of human whole blood is measured, error due to hematocrit effect is insignificant in the range of 30-55 % hematocrit.
- At least one additional enzyme is used as a reaction catalyst.
- some of the examples shown in Table 1 may utilize an additional mediator, which facilitates electron transfer to the oxidized form of the redox mediator.
- the additional mediator may be provided to the reagent in lesser amount than the oxidized form of the redox mediator.
- the present inventive biosensor When compared to the preferred embodiment of the closest prior art biosensor test strip, disclosed in Pollmann et al., the present inventive biosensor has the following distinguishing feamres:
- reagent well 9 is 30% smaller
- the exposed surface area of the counter electrode in the reagent well is less than the exposed surface area of the working electrode in the reagent well;
- air vents are included on opposing sides of the reagent well.
- a smaller sample volume requirement to properly dose the test strip means fewer underdosing errors will result. This result is especially important for those persons, such as infants and the elderly who have difficulty in obtaining a reasonably sized blood drop after pricking their finger with a lancet.
- the present inventive strip makes it easier for a current measuring meter to discriminate low sample volume dosing errors. Also, using less reagent per sensor increases production volume for mass producing sensors. Further, providing side air vents near the reagent well reduces the occurrence of air bubbles trapped in the reagent well, which results in fewer testing errors.
Abstract
Description
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Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP96922651A EP0874984B1 (en) | 1995-06-30 | 1996-06-28 | Electrochemical biosensor test strip |
DE0874984T DE874984T1 (en) | 1995-06-30 | 1996-06-28 | TEST STRIP FOR AN ELECTOCHEMICAL BIOSENSOR |
CA002224308A CA2224308C (en) | 1995-06-30 | 1996-06-28 | Electrochemical biosensor test strip |
JP50529097A JP3819936B2 (en) | 1995-06-30 | 1996-06-28 | Electrochemical biosensor test strip |
AU63451/96A AU712527B2 (en) | 1995-06-30 | 1996-06-28 | Electrochemical biosensor test strip |
DE69617464T DE69617464T2 (en) | 1995-06-30 | 1996-06-28 | TEST STRIP FOR AN ELECTOCHEMICAL BIOSENSOR |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US08/496,939 US5762770A (en) | 1994-02-21 | 1995-06-30 | Electrochemical biosensor test strip |
US08/496,939 | 1995-06-30 |
Publications (1)
Publication Number | Publication Date |
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WO1997002487A1 true WO1997002487A1 (en) | 1997-01-23 |
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ID=23974813
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1996/011240 WO1997002487A1 (en) | 1995-06-30 | 1996-06-28 | Electrochemical biosensor test strip |
Country Status (9)
Country | Link |
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US (1) | US5762770A (en) |
EP (1) | EP0874984B1 (en) |
JP (1) | JP3819936B2 (en) |
KR (1) | KR100344740B1 (en) |
AU (1) | AU712527B2 (en) |
CA (1) | CA2224308C (en) |
DE (2) | DE874984T1 (en) |
ES (1) | ES2154250T3 (en) |
WO (1) | WO1997002487A1 (en) |
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US9795747B2 (en) | 2010-06-02 | 2017-10-24 | Sanofi-Aventis Deutschland Gmbh | Methods and apparatus for lancet actuation |
Also Published As
Publication number | Publication date |
---|---|
CA2224308A1 (en) | 1997-01-23 |
DE69617464T2 (en) | 2002-08-29 |
AU712527B2 (en) | 1999-11-11 |
ES2154250T1 (en) | 2001-04-01 |
US5762770A (en) | 1998-06-09 |
MX9710374A (en) | 1998-07-31 |
EP0874984A4 (en) | 2000-05-10 |
KR100344740B1 (en) | 2003-03-15 |
EP0874984A1 (en) | 1998-11-04 |
JP3819936B2 (en) | 2006-09-13 |
CA2224308C (en) | 2008-05-06 |
KR19990028415A (en) | 1999-04-15 |
ES2154250T3 (en) | 2002-06-16 |
DE69617464D1 (en) | 2002-01-10 |
AU6345196A (en) | 1997-02-05 |
DE874984T1 (en) | 2000-10-05 |
JP2001511881A (en) | 2001-08-14 |
EP0874984B1 (en) | 2001-11-28 |
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