WO1994018559A1 - Asymmetric membrane sensor - Google Patents

Asymmetric membrane sensor Download PDF

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Publication number
WO1994018559A1
WO1994018559A1 PCT/DK1994/000058 DK9400058W WO9418559A1 WO 1994018559 A1 WO1994018559 A1 WO 1994018559A1 DK 9400058 W DK9400058 W DK 9400058W WO 9418559 A1 WO9418559 A1 WO 9418559A1
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WO
WIPO (PCT)
Prior art keywords
sensor
test cell
chemical species
enzyme
chemical
Prior art date
Application number
PCT/DK1994/000058
Other languages
French (fr)
Inventor
John P. Willis
Rayvenne L. Pivato
Original Assignee
Radiometer Medical A/S
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Publication date
Application filed by Radiometer Medical A/S filed Critical Radiometer Medical A/S
Publication of WO1994018559A1 publication Critical patent/WO1994018559A1/en

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • G01N33/521—Single-layer analytical elements

Definitions

  • the invention relates to sensors and apparatus for detecting one or more chemical species in a sample fluid.
  • One aspect of the invention is particularly suited to measurements performed on fluids requiring solids removal prior to the measurement, a particular example of which is whole blood.
  • Chemical species or parameters of particular interest are, for example: pH,
  • concentrations of electrolytes such as Li+, Na+, K+, Ca 2 + , Mg 2 + , Cl “ , HC0 3 " , and NH 3 (NH 4 + ) ,
  • concentrations of dissolved gases notably oxygen and carbon dioxide (conventionally reported in the form of partial pressures, e.g. p0 2 , pC0 2 ) ,
  • metabolic factors such as glucose, creatinine, urea (BUN) , uric acid, lactic acid, pyruvic acid, ascorbic acid, phosphate, protein, bilirubin, cholesterol, triglycerides, phenylalanine and tyrosine,
  • enzymes such as lactic acid dehydro- genase (LDH) , lipase, amylase, choline esterase, alka ⁇ line phosphatase, acid phosphatase, alanine a ino
  • ligands such as antibodies and nucleotide fragments
  • ethyl alcohol for example, to determine the state of intoxication
  • therapeutic drugs e.g. anesthetics
  • illicit drugs e.g. anesthetics
  • SUBSTITUTESHEET and increased staff flexibility in clinical chemical analysis have provided an incentive for the development of easy-to-use, easy-to- aintain, reliable, relatively cheap, compact and, if possible, portable equipment, based in part on discardable components, for the bed ⁇ side measurement of those characteristics of chemical species which constitute fundamental clinical chemical parameters of body fluids.
  • Equipment based in part on disposable components may also be of great value in numerous non-medical ana ⁇ lytical applications where the ability to carry out decentralized or field analyses is of importance. Examples of such applications are the determination of pH, color and concentrations of chemical species such as chloride, nitrite, nitrate, sulfate and phosphate in relation to control of the quality of bodies of water used for domestic supplies, and on-site analyses of the contents of process vessels, e.g. in fermentation pro- Deads such as the production of beers and wines, in sugar refining and in industrial syntheses.
  • chemical species such as chloride, nitrite, nitrate, sulfate and phosphate
  • This disposable part is designed for introduction into a reading appa ⁇ ratus which contains whatever meters and associated electronic components may be required.
  • the system is designed so that the sample fluid only comes into contact with the disposable part. See, e.g., parent
  • EP 0012031 discloses a method and apparatus for measuring a chemical characteristic of a liquid, in particular for measuring the pH of a blood sample.
  • the preferred embodiments of the measuring devices disclosed in the first five of the above mentioned sources are intended for discardment after a single use, whereas a preferred embodiment disclosed in EP 0189316 and embodiments dis ⁇ closed in EP 0012031 are intended to be disposed of after repeated use.
  • Disposable sensing devices for measuring a component concentration in a sample fluid are also described in PCT Application WO90/02938 and EPO 0354895.
  • the device described in PCT WO90/02938 consists of a housing with- in which various chambers are provided for reagent and the sample fluid.
  • the housing also contains a sample collection mechanism which includes an orifice for drawing the sample into the sample chamber.
  • the housing further contains a sensor for the component concen- tration to be measured. Under control of a reading apparatus within which the housing is inserted, the reagent and the sample fluid are caused to flow through conduits provided within the housing which connect the chambers to the sensor. Sample displacement is enabled
  • SUBSTITUTE SHEET by means disposed within the housing and activated by the reading apparatus.
  • a clinical application of particular interest is the measurement of glucose concentration in whole blood.
  • the determination of clucose concentration in a body fluid sample is described, for example, in U.S. Patent No. 4,273,868.
  • the described technique utilizes a com ⁇ position which includes glucose oxidase, peroxidase, a buffer effective to maintain a pH of from 4 to 7.5, a stabilizing agent and 3,3', 5,5'-tetramethylbenzidene.
  • the 3,3', 5,5'-tetramethylbenzidene is in a concen ⁇ tration of at least about 2.6 millimoles per thousand International Units of glucose oxidase activity.
  • U.S. Patent No. 4,380,585 describes the use of a branched or cyclic hexanol as an enhancer compound for such benzidene type indicators.
  • an absorbent carrier is impregnated first with, e.g., a peroxidase or peroxidatively active substance dissolved in a water-containing solvent, and then with a solution of a chromogen dissolved in an organic liquid containing solvent.
  • the sensing device described in U.S. Patent No. 4,816,224 is specifically designed for blood analysis in cases in which it is necessary to separate plasma or serum from whole blood.
  • the device includes a glass fiber layer having a density of from 0.1 to 5 g/cm 3 wherein the glass fibers have an average diameter from 0.2 to 5 microns.
  • the device further includes a reaction layer in fluid communication with the glass fiber layer, at least a portion of the glass fiber layer being free from direct attachment to the reaction layer.
  • U.S. 4,477,575 likewise describes a method for separat ⁇ ing plasma or serum from whole blood using a glass fiber layer, wherein the glass fibers have an average diameter of 0.2 to 5 microns and a density of 0.1 to 0.5 g/cm 3 .
  • the total volume of plasma or serum to be separated is at most 50% of the absorption volume of the glass fiber layer. Separation is carried out by slowly trickling whole blood on one side of the glass fiber layer and collecting separated plasma or serum from the other side.
  • Particular advancements include an improved sensor which solves the bleed through problem without compro ⁇ mising reliability and ease of construction and use. This is achieved using an asymmetric membrane having a chromogen indicator coating located downstream of and adjacent to the downstream side side of the asymmetric membrane, i.e. the side having the smaller porosity. In this way it has been found that bleed through is not a problem and precise measurement of various blood param ⁇ eters is thereby achieved.
  • the sensor may be used for measurement of a variety of analytes, including glu- cose, lactate, creatinine, urea (BUN) , uric acid, pyruvic acid, ascorbic acid and cholesterol. Techniques for constructing the sensor and operating same are also disclosed herein.
  • the invention relates to sensors employ ⁇ ing asymmetrjc membranes having a porosity gradient from the upstream side to the downstream side.
  • An in ⁇ dicator layer is included on the downstream side only and is preferably applied by spray coating.
  • the up ⁇ stream side of the membrane has a porosity in the range of 10-20 ⁇ m.
  • the indicator layer is selected for a particular chemi- cal species, examples of which are:
  • enzyme substrates such as glucose, lactate, tri ⁇ glycerides, total cholesterol, high-density lipo protein, urea, creatinine, ascorbic acid, uric acid or ethanol;
  • - ions such as potassium, sodium, hydrogen, bicarbon ⁇ ate, lithium, phosphate, magnesium, calcium or am ⁇ monium;
  • enzymes such as amylase, creatinine-kinase, creatinine-kinase-MB or gamma-glutamy1-transferase;
  • proteins such as antibody, nucleotide fragments, albumin or a ino acid
  • - antibodies such as an HIV antibody
  • hemoglobin such as hemoglobin, bilirubin and antigens, including viruses, drugs, drug metabolites, native sub-units of viruses and haptens.
  • the sensors exhibit excellent sensitivity while exhib ⁇ iting a strong linear response.
  • Fig. 1 is a cross-sectional view of an asymmetric membrane
  • Fig. 2a is a top plan view of a sensor housing
  • Fig. 2b is a side cross-sectional view of the sensor housing of Fig. 2a;
  • Figs. 2c and d are partial side cross-sectional views of the sensor housing showing, respectively, placement of the asymmetric membrane therein and fixation of the asymmetric membrane;
  • Fig. 3a is an exploded view of another embodiment of a sensor in accordance with the invention.
  • Fig . 3b is a side cross-sectional view of the sensor of Fig . 3a taken along line 3b-3b;
  • FIGS. 3c and 3d are perspective views of the sensor of Figs. 3a and b, showing alternative embodiment s of a sample fluid inlet;
  • Figs. 4 and 5 are graphs of glucose cencentration vs. reflectance (as represented by a Kubelka-Munk Trans ⁇ form) ;
  • Figs. 6-10 are top plan views of a disposable analyti- cal cassette in accordance with the invention.
  • Figs. 11-14 are top plan views of an alternative em ⁇ bodiment of a diposable analytical cassette in accor ⁇ dance with the invention.
  • the present invention comprises a method for measuring a characteristic which is a function of the concentra- tion of one or more chemical species in a sample fluid.
  • fluid as used here denotes either a liquid phase or a gaseous phase.
  • Liquid phases notably aque ⁇ ous solutions, are the more important in connection with clinical chemical applications of the present in- vention, for which reason preferred embodiments of measuring devices for use according to the present in ⁇ vention are adapted for the measurement of character ⁇ istics of chemical species in aqueous media.
  • SUBSTITUTE SH Sensors generally perform a conversion function to convert the energy form associated with the change occurring at the sensing surface part to electrical energy or electromagnetic radiant energy, the sensor response thereby being registerable in the form of an electrical or optical signal.
  • a more detailed descrip ⁇ tion of non-limiting examples of conversion principles which are relevant in connection with sensors is given by Middelhoek & Noorlag (S. Middelhoek & D.J.W. Noorlag, "Three-Dimensional Representation of Input and Output Transducers", Sensors and Actuators 2, 1981/1982, pp. 29-41).
  • a characteristic for example the concentration of a chemical species
  • concentration of a chemical species the relationship between the magnitude of the response of the sensor in question and the magnitude of the characteristic, under the condi ⁇ tions pertaining during the measurements.
  • Certain types of sensors can be manufactured with such a high degree of reproducibility that the relationship between response and characteristic (as generally de ⁇ termined by sensor sensitivity and sensor response at least one value of the characteristic) for all the members of a production batch of the same type of sensor is substantially identical and thus predetermin- able.
  • Certain other types of sensors may be manufac ⁇ tured with such a degree of reproducibility that at least one of, but not all the parameters determining the relationship between response and characteristic will be predeterminable.
  • Other aspects of sensor re ⁇ sponse for example drift and variation with tempera ⁇ ture, may also be predetermined. It is therefore possi ⁇ ble for the manufacturer to equip a measuring device, incorporating such a sensor, with appropriate sensor
  • SUBSTITUTE SHEET data together with any other desirable information, e.g. date of manufacture, expiry date, measurement cycle control information, etc. , in the form of a code, such as a bar-code which can be read by an optical reading device, or a magnetic code which can be read by a magnetic reading device.
  • Reading means for decod ⁇ ing the information contained in the code can advanta ⁇ geously be incorporated in an analyzer.
  • ana- lyzer designates an apparatus adapted to removably accommodate the measuring device and provided with means for bringing about movement of the sensor and the sample fluid chamber relative to each other when the measuring device is accommodated in the analyzer, sensor response transmission means, and means for registering the response generated by the sensor, said sensor response transmission means facilitating commu ⁇ nication between the sensor response output means of the sensor and the sensor response registering means of the analyzer.
  • two-point or multiple-point cali ⁇ bration of sensor response may be carried out; for example, by a procedure involving the successive expo-
  • the sensor is based on an asymmetric polysulphone membrane of the type available from Filterite Corp. , Timonium, Maryland under the trademark BTS TM .
  • the pore size decreases gradually, e.g. from an average of 10-20 um on the upstream side to 0.1 um on the downstream side. This gradient enables the membrane to separate plasma from whole blood.
  • Fig. 1 illustrates a preferred asymmetric membrane sensor 5.
  • the asymmetric membrane sensor 5 has an upstream side 200 with a pore size of about 10-20 microns, a downstream side 210 with a pore size of about 0.1 to 1.0 microns and a thickness L of about 125 microns.
  • An enzyme coating layer 220 approximately 10 to 20 microns in thickness is applied to the downstream side 210.
  • Blood 250 applied to the upstream side 200 is separated whereby the red cells 230 are filtered out by the mem ⁇ brane and only plasma 240 reaches the enzyme coating layer 220.
  • an analyte in the plasma interacts with the enzyme coating layer to bring about a detactable change indicative of the analyte's concen ⁇ tration, e.g. a change in color intensity.
  • the asymmetric membrane sensor is a two layer device incorporating blood separation and color development in separate layers. Two coating procedures have proven effective.
  • a buffer stock solution is prepared as follows:
  • the solution is spray coated on the down stream side of the membrane and dried in a 45 C oven. Disks are then punched and placed in a cassette device for measure ⁇ ment.
  • Solution 1 is spray coated on the downstream side of the membrane and dried in a 45 C oven.
  • Solution 2 is then spray coated over Solution 1 and dried in a 45 C oven.
  • Disks are then punched and inserted into a cas ⁇ sette for measurement.
  • the preferred coating technique is to spray the solu ⁇ tion onto the downstream side of the asymmetric mem ⁇ brane using a spray valve available from EFD, Inc. , 977 Waterman Avenue, East Buffalo, RI 02914, Model No. 780S.
  • the spray valve is stationary and is mounted in a coating machine beneath the membrane to be sprayed.
  • the membrane is fed through the coating machine above the spray path.
  • the operating parameters are as follows:
  • Figs. 2a-d illustrate a glucose cassette 100 incorpor ⁇ ating an asymmetric membrane 5 as described above.
  • the cassette 100 includes a chamber 110 in which the asy - metric membrane 5 is held by ultrasonic weld.
  • An air chamber 120 is provided adjacent the indicator layer 220.
  • Figs. 3a-c illustrate a flow-cell sensor 300.
  • the sensor 300 comprises a top half 320 and a bottom half
  • the top half 320 includes a chamber 310 which houses the asymmetric membrane 5.
  • the tcp half and bottom half together define a sample fluid conduit 350, that includes a receiving end 360 and hydrophobic vent 370.
  • Fig. 3c also shows a transfer vessel 400.
  • Fig. 3d illustrates an alternative embodiment of a receiving end 360' for receiving sample either directly from a patient or from an intermediate transfer vessel.
  • the asymmetric membrane in the measuring device it is preferable to orient the asymmetric membrane in the measuring device so that, when in use, the sensor will be substantially above the fluid sample forcing the sample to flow upwards against gravity by capillary action.
  • This is an especially advantageous arrangement in the case of blood samples, because red blood cells migrate slower than plasma which, therefore, reaches the topmost indicator layer first. When plasma (which contains glucose, for example) reaches and wets the indicator layer, flow essentially stops. As a result, red blood cells are substantially excluded from the indicator layer where they could otherwise interfere with measurement of the color change which is indica- tive of glucose concentration.
  • Glucose sensors made in accordance with Example 1 were tested at five separate glucose concentrations between 10 mg/dL (0.5 mmol/L) and 600 mg/dL (30 mmol/L) .
  • the test method was as follows.
  • a fluid sample of predetermined concentration was applied to the upstream side of the membrane.
  • Whole blood enters the cassette under the upstream side (larger pore size 10-20 um) .
  • the red cells separate as
  • the intensity of the color is proportional to glucose concentration and the color produced may be measured by reflectance spectro- scopy.
  • Wetting of the enzyme coating layer was detected by a change in the reflectance of light at a wavelength of 500 nm. This point is taken as time 0. The intensity of light reflected at 650 nm was measured at time 0 and again 6 seconds later.
  • glucose sensors employing asymmetric membranes were prepared by dip coating using the same indicator solution prepared in Example 1.
  • the weight gain of indi ⁇ cator for the spray-coating and dip-coating methods is set forth below:
  • m and b represent constants in the calibration curve
  • Y m X + b
  • Y glucose cone, in mg/DL
  • X Kubelka-Munk transform
  • r 2 the correlation coeffi ⁇ cient
  • Figs. 4 and 5 The results are illustrated graphically in Figs. 4 and 5. It can be readily seen that the slope of the cali- bration curves for the sprayed membranes are much less steep than those for the dipped membranes. Consequent ⁇ ly, the sprayed membranes will have a correspondingly greater sensitivity to glucose concentration changes. Additionally, the 0.1 ⁇ m sprayed membrane has a corre- lation coefficient approaching unity which indicates purely linear behavior as well as a high degree of precision.
  • the senor is mounted in a disposeable cassette which is designed for automatic operation.
  • a cassette is shown in Figures 6-10.
  • Fig. 6 shows the housing sensor 500.
  • the sensor housing includes a sample tube 511 connected to a major flow path 512 which, in turn, is connected to various sen ⁇ sors 521-526.
  • An air tube 513 surrounds the sample tube 511.
  • the sensor housing 500 also includes a guide tube 530, the function of which is described below.
  • the sensor housing 500 further includes an air inlet 540, a reference fluid reservoir 550, a vent path 560, a
  • SUBSTITUTESHEET hydrophobic vent 570 an overflow chamber 580 and a luer collar 590.
  • Fig. 7 shows the sensor housing 500 after an intermedi- ate vessel or sampler device 600 has been inserted into the guide tube 530 and locked in place.
  • the sampler device 600 has a septum 610 which is punctured by the sample tube 511 and air tube 513. Both the sample tube 511 and air tube 513 project into the sampler device 600.
  • Fig. 8 is an enlarged view of the interface between the sensor housing 500 and the sampler device 600.
  • air is forced into the sampler device 600 through the air tube 513. This forces the sample through the sample tube 511 and into the major flow path 512 whereupon it contacts the various sensors within the housing 500.
  • Fig. 9 shows the sensor housing 500 and sampler device
  • Fig. 10 shows the complete sensor housing 500 and sam ⁇ pler device 600 immediately after air A is introduced to force the sample B from the sampler device 600 into the major flow path 512 and into contact with the sensors. The sample eventually reaches the hydrophobic vent 560 whereupon it stops flowing at point C.
  • Figs. 11-14 shows an alternative cassette design which is configured to apply the sample to the center of the asymmetric membrane.
  • Figs. 11 and 12 show? a top plate 800 with recessed flow channels covered by cover tape 810.
  • sample enters through flow hole 811 and flows to tapered inlet hole 812.
  • the sample will wet the asymmetric membrane sensor 5 that will be positioned directly below.
  • Excess sample flows out though flow hole 813, and vent channel 814 until it wets vent plug 815.
  • the vent plug 815 can be a hydrophyobic vent, for example.
  • Figs. 13 and 14 show a bottom plate which, combined with the top plate of Figs. 11 and 12, forms a complete cassette.
  • the bottom plate 900 includes the asymmetric membrane 5 and has a reflectance reading window 911 underneath the asymmetric membrane.
  • a number of variations of the basic concepts described above are possible. For example, it may be desirable to incorporate two or more membranes from the same manu- facturing batch in each sensor. One or more membranes may be tested as controls using reagents of known analyte concentration prior to testing the sample of unknown analyte concentration. The control membranes can thus be used to confirm that the test membrane is within a predetermined specification or may be used for a single or multipoint calibration.

Abstract

The invention relates to an asymmetric membrane having a chromogen indicator coating located downstream of and adjacent to the downstream (smaller porosity) side of the asymmetric membrane. Bleed-through is not a problem and precise measurement of various blood parameters is thereby achieved. The sensor may be used for measurement of a variety of analytes, including glucose, lactate, creatinine, urea (BUN), uric acid, pyruvic acid, ascorbic acid and cholesterol. Additionally, the invention relates to disposable cassettes employing the sensor and analytical systems employing same. The invention further relates to techniques for constructing and operating the sensor.

Description

Asymmetric membrane sensor.
Field of Invention
The invention relates to sensors and apparatus for detecting one or more chemical species in a sample fluid. One aspect of the invention is particularly suited to measurements performed on fluids requiring solids removal prior to the measurement, a particular example of which is whole blood.
Background of the Invention
Chemical species or parameters of particular interest are, for example: pH,
concentrations of electrolytes, such as Li+, Na+, K+, Ca2 +, Mg2 +, Cl", HC03 ", and NH3 (NH4 +) ,
concentrations of dissolved gases, notably oxygen and carbon dioxide (conventionally reported in the form of partial pressures, e.g. p02, pC02) ,
haemoglobin concentration,
concentrations of metabolic factors, such as glucose, creatinine, urea (BUN) , uric acid, lactic acid, pyruvic acid, ascorbic acid, phosphate, protein, bilirubin, cholesterol, triglycerides, phenylalanine and tyrosine,
concentrations of enzymes, such as lactic acid dehydro- genase (LDH) , lipase, amylase, choline esterase, alka¬ line phosphatase, acid phosphatase, alanine a ino
SUBSTITUTESHE transferase (ALAT) , aspartate amino transferase (ASAT) and creatinine kinase (CK) ,
concentrations of ligands, such as antibodies and nucleotide fragments,
and concentrations of ethyl alcohol (for example, to determine the state of intoxication) , therapeutic drugs (e.g. anesthetics) and illicit drugs.
In the past, clinical chemical analysis systems have tended to be large in size, expensive and complex to operate, and in general only relatively large medical institutions have been able to afford the purchase, operation and maintenance of such systems. Smaller hospitals, clinics, general practitioners etc. usually have had to employ centralized commercial or hospital laboratories for clinical chemical analyses, leading to unavoidable delays in the procedure.
Since abnormal values of certain clinical chemical parameters are indicative of serious danger to health, the rapid and reliable determination of clinical chemi¬ cal parameters in general is of crucial importance for proper and effective medical treatment. This is like¬ wise of importance in determining the individuals state of intoxication, for example, or impairment due to in- gestion of illicit drugs in relation to legally defined limits. Furthermore, quite apart from the acute aspects of medical treatment, it is clearly an advantage, both for patients from a psychological viewpoint and for medical staff from an administrative viewpoint, that clinical analysis results are accessible as quickly as possible. Thus, increasing demands for reduction in costs, more rapid turnover, greater decentralization
SUBSTITUTESHEET and increased staff flexibility in clinical chemical analysis have provided an incentive for the development of easy-to-use, easy-to- aintain, reliable, relatively cheap, compact and, if possible, portable equipment, based in part on discardable components, for the bed¬ side measurement of those characteristics of chemical species which constitute fundamental clinical chemical parameters of body fluids.
Equipment based in part on disposable components may also be of great value in numerous non-medical ana¬ lytical applications where the ability to carry out decentralized or field analyses is of importance. Examples of such applications are the determination of pH, color and concentrations of chemical species such as chloride, nitrite, nitrate, sulfate and phosphate in relation to control of the quality of bodies of water used for domestic supplies, and on-site analyses of the contents of process vessels, e.g. in fermentation pro- cesses such as the production of beers and wines, in sugar refining and in industrial syntheses.
In clinical applications, reliability and ease of use are obviously important criteria. Disposability and safety are also important considerations. Cost is like¬ wise a factor.
The need for disposability led to the introduction of disposable test devices with self-contained fluid re- servoirs for the sample and reagents. This disposable part is designed for introduction into a reading appa¬ ratus which contains whatever meters and associated electronic components may be required. The system is designed so that the sample fluid only comes into contact with the disposable part. See, e.g., parent
SUBSTITUTESHEET application Serial No. 689,727 filed April 22, 1991, now U.S. Patent No. 5,114,859, which describes such a system.
PCT applications WO 85/02257, WO 85/04719 and WO
86/05590, US patent 4,436,610, US patent 4,225,410 and European application EP 0189316, disclose apparatuses, all of which comprise a disposable measuring device and an analyzer, suitable for bedside clinical chemical analyses, notably of blood samples. European patent EP 0012031 discloses a method and apparatus for measuring a chemical characteristic of a liquid, in particular for measuring the pH of a blood sample. The preferred embodiments of the measuring devices disclosed in the first five of the above mentioned sources are intended for discardment after a single use, whereas a preferred embodiment disclosed in EP 0189316 and embodiments dis¬ closed in EP 0012031 are intended to be disposed of after repeated use.
Disposable sensing devices for measuring a component concentration in a sample fluid are also described in PCT Application WO90/02938 and EPO 0354895. The device described in PCT WO90/02938 consists of a housing with- in which various chambers are provided for reagent and the sample fluid. The housing also contains a sample collection mechanism which includes an orifice for drawing the sample into the sample chamber. The housing further contains a sensor for the component concen- tration to be measured. Under control of a reading apparatus within which the housing is inserted, the reagent and the sample fluid are caused to flow through conduits provided within the housing which connect the chambers to the sensor. Sample displacement is enabled
SUBSTITUTE SHEET by means disposed within the housing and activated by the reading apparatus.
A clinical application of particular interest is the measurement of glucose concentration in whole blood. The determination of clucose concentration in a body fluid sample is described, for example, in U.S. Patent No. 4,273,868. The described technique utilizes a com¬ position which includes glucose oxidase, peroxidase, a buffer effective to maintain a pH of from 4 to 7.5, a stabilizing agent and 3,3', 5,5'-tetramethylbenzidene. The 3,3', 5,5'-tetramethylbenzidene is in a concen¬ tration of at least about 2.6 millimoles per thousand International Units of glucose oxidase activity. U.S. Patent No. 4,380,585, describes the use of a branched or cyclic hexanol as an enhancer compound for such benzidene type indicators.
The construction of sensing devices using such indica- tors is described for example in U.S. Patent No. 4,385,114. According to this patent, an absorbent carrier is impregnated first with, e.g., a peroxidase or peroxidatively active substance dissolved in a water-containing solvent, and then with a solution of a chromogen dissolved in an organic liquid containing solvent.
The use of such chromogen type indicators in connection with blood analysis presents special problems in view of the capacity for solids in the whole blood to inter¬ fere with the color change being measured. U.S. Pat¬ ents Nos. 4,774,192, 4,987,085 and 4,994,238 describe the use of asymmetric membranes to separate solids, e.g. red blood cells, from the test sample. In each of these documents, a chromogen type indicator is distrib-
SUBST1TUTE SHEET uted throughout the membrane. The porosity gradient cf the asymmetric membrane described in U.S. Patent No. 4,987,085 is from 20 to 0.45 microns. However, as recognized in this latter patent, certain blood compo- nents may nevertheless interfere with the measurement of reflectance of the chromogen indicator. This problem is sometimes referred to as "bleed through".
In this regard, the sensing device described in U.S. Patent No. 4,816,224 is specifically designed for blood analysis in cases in which it is necessary to separate plasma or serum from whole blood. The device includes a glass fiber layer having a density of from 0.1 to 5 g/cm3 wherein the glass fibers have an average diameter from 0.2 to 5 microns. The device further includes a reaction layer in fluid communication with the glass fiber layer, at least a portion of the glass fiber layer being free from direct attachment to the reaction layer.
U.S. 4,477,575 likewise describes a method for separat¬ ing plasma or serum from whole blood using a glass fiber layer, wherein the glass fibers have an average diameter of 0.2 to 5 microns and a density of 0.1 to 0.5 g/cm3. The total volume of plasma or serum to be separated is at most 50% of the absorption volume of the glass fiber layer. Separation is carried out by slowly trickling whole blood on one side of the glass fiber layer and collecting separated plasma or serum from the other side.
The use, however of multi-layer membranes has not heretofore resulted in an entirely satisfactory solu¬ tion to the "bleed through" problem referenced herein- above.
SUBSTITUTE SHEET As a result of the aforementioned problems, chromogen indicators used in reflectance spectroscopy systems have been found to demonstrate a non-linear response to analyte concentration. See, e.g., U.S. Patent No.
5,049,487, issued September 17, 1991. This has hither- tofore necessitated that complicated methods be em¬ ployed for calibrating such indicators, including the use of third order (and higher) polynomial curve fit- ting or other methods designed to account for the interfering contributions from unwanted blood compo¬ nents.
Summary of the Function
Various improvements in disposable test devices made in accordance with the invention are disclosed herein.
Particular advancements include an improved sensor which solves the bleed through problem without compro¬ mising reliability and ease of construction and use. This is achieved using an asymmetric membrane having a chromogen indicator coating located downstream of and adjacent to the downstream side side of the asymmetric membrane, i.e. the side having the smaller porosity. In this way it has been found that bleed through is not a problem and precise measurement of various blood param¬ eters is thereby achieved. The sensor may be used for measurement of a variety of analytes, including glu- cose, lactate, creatinine, urea (BUN) , uric acid, pyruvic acid, ascorbic acid and cholesterol. Techniques for constructing the sensor and operating same are also disclosed herein.
SUBSTITUTE SHEET In particular., the invention relates to sensors employ¬ ing asymmetrjc membranes having a porosity gradient from the upstream side to the downstream side. An in¬ dicator layer is included on the downstream side only and is preferably applied by spray coating. The up¬ stream side of the membrane has a porosity in the range of 10-20 μm.
The indicator layer is selected for a particular chemi- cal species, examples of which are:
- enzyme substrates, such as glucose, lactate, tri¬ glycerides, total cholesterol, high-density lipo protein, urea, creatinine, ascorbic acid, uric acid or ethanol;
- ions, such as potassium, sodium, hydrogen, bicarbon¬ ate, lithium, phosphate, magnesium, calcium or am¬ monium;
- gases, such as chlorine, oxygen or carbon dioxide;
- enzymes, such as amylase, creatinine-kinase, creatinine-kinase-MB or gamma-glutamy1-transferase;
- proteins, such as antibody, nucleotide fragments, albumin or a ino acid;
- antibodies, such as an HIV antibody;
- amino acids, such as phenylalinine or tyrosine;
- chemical species, such as hemoglobin, bilirubin and antigens, including viruses, drugs, drug metabolites, native sub-units of viruses and haptens.
SUBSTITUTE SHEET Suitable indicators are known in the art. See, e.g., Trocken chemie : Analytik mit tragergebundenen Reagenzien Oswald Sonntag. - Stuttgart; New York: Thieme, 1988.
The sensors exhibit excellent sensitivity while exhib¬ iting a strong linear response.
Description of the Drawings
Preferred embodiments of the invention will now be described with reference to the following drawings, wherein:
Fig. 1 is a cross-sectional view of an asymmetric membrane;
Fig. 2a is a top plan view of a sensor housing;
Fig. 2b is a side cross-sectional view of the sensor housing of Fig. 2a;
Figs. 2c and d are partial side cross-sectional views of the sensor housing showing, respectively, placement of the asymmetric membrane therein and fixation of the asymmetric membrane;
Fig. 3a is an exploded view of another embodiment of a sensor in accordance with the invention;
Fig . 3b is a side cross-sectional view of the sensor of Fig . 3a taken along line 3b-3b;
SUBSTITUT Figs. 3c and 3d are perspective views of the sensor of Figs. 3a and b, showing alternative embodiment s of a sample fluid inlet;
Figs. 4 and 5 are graphs of glucose cencentration vs. reflectance (as represented by a Kubelka-Munk Trans¬ form) ;
Figs. 6-10 are top plan views of a disposable analyti- cal cassette in accordance with the invention; and
Figs. 11-14 are top plan views of an alternative em¬ bodiment of a diposable analytical cassette in accor¬ dance with the invention.
Detailed Description of the Preferred Embodiments
The present invention comprises a method for measuring a characteristic which is a function of the concentra- tion of one or more chemical species in a sample fluid. The term "fluid" as used here denotes either a liquid phase or a gaseous phase. Liquid phases, notably aque¬ ous solutions, are the more important in connection with clinical chemical applications of the present in- vention, for which reason preferred embodiments of measuring devices for use according to the present in¬ vention are adapted for the measurement of character¬ istics of chemical species in aqueous media. These embodiments will be described and exemplified in detail in the following, although it will be evident to a person skilled in the art that the principles of the invention as disclosed herein may equally well be adapted for use in measurements on nonaqueous fluids and fluids which are gases.
SUBSTITUTE SH Sensors generally perform a conversion function to convert the energy form associated with the change occurring at the sensing surface part to electrical energy or electromagnetic radiant energy, the sensor response thereby being registerable in the form of an electrical or optical signal. A more detailed descrip¬ tion of non-limiting examples of conversion principles which are relevant in connection with sensors is given by Middelhoek & Noorlag (S. Middelhoek & D.J.W. Noorlag, "Three-Dimensional Representation of Input and Output Transducers", Sensors and Actuators 2, 1981/1982, pp. 29-41).
The derivation of a characteristic, for example the concentration of a chemical species, from the response of a sensor requires that the relationship between the magnitude of the response of the sensor in question and the magnitude of the characteristic, under the condi¬ tions pertaining during the measurements, is known. Certain types of sensors can be manufactured with such a high degree of reproducibility that the relationship between response and characteristic (as generally de¬ termined by sensor sensitivity and sensor response at least one value of the characteristic) for all the members of a production batch of the same type of sensor is substantially identical and thus predetermin- able. Certain other types of sensors may be manufac¬ tured with such a degree of reproducibility that at least one of, but not all the parameters determining the relationship between response and characteristic will be predeterminable. Other aspects of sensor re¬ sponse, for example drift and variation with tempera¬ ture, may also be predetermined. It is therefore possi¬ ble for the manufacturer to equip a measuring device, incorporating such a sensor, with appropriate sensor
SUBSTITUTE SHEET data, together with any other desirable information, e.g. date of manufacture, expiry date, measurement cycle control information, etc. , in the form of a code, such as a bar-code which can be read by an optical reading device, or a magnetic code which can be read by a magnetic reading device. Reading means for decod¬ ing the information contained in the code can advanta¬ geously be incorporated in an analyzer. As used in connection with the present invention, the term "ana- lyzer" designates an apparatus adapted to removably accommodate the measuring device and provided with means for bringing about movement of the sensor and the sample fluid chamber relative to each other when the measuring device is accommodated in the analyzer, sensor response transmission means, and means for registering the response generated by the sensor, said sensor response transmission means facilitating commu¬ nication between the sensor response output means of the sensor and the sensor response registering means of the analyzer. Various embodiments of such an analyzer for use in an analysis system according to the present invention are described in greater detail below.
It is thus possible, using such an analyzer, to cali- brate the response of the sensor in relation to the magnitude of the characteristic simply by carrying out a single measurement of the response generated by the sensor upon exposure to a calibration fluid for which the magnitude of the characteristic in question is known; calibration in this matter is referred to here¬ after as "single-point calibration".
However, if desired, two-point or multiple-point cali¬ bration of sensor response may be carried out; for example, by a procedure involving the successive expo-
SUBSTITUTE SHEET sure of the sensing surface part of the sensor to an appropriate number of calibration fluids of different known concentration.
The sensor is based on an asymmetric polysulphone membrane of the type available from Filterite Corp. , Timonium, Maryland under the trademark BTS™. In an asymmetric membrane, the pore size decreases gradually, e.g. from an average of 10-20 um on the upstream side to 0.1 um on the downstream side. This gradient enables the membrane to separate plasma from whole blood.
Fig. 1 illustrates a preferred asymmetric membrane sensor 5. The asymmetric membrane sensor 5 has an upstream side 200 with a pore size of about 10-20 microns, a downstream side 210 with a pore size of about 0.1 to 1.0 microns and a thickness L of about 125 microns. An enzyme coating layer 220 approximately 10 to 20 microns in thickness is applied to the downstream side 210.
Blood 250 applied to the upstream side 200 is separated whereby the red cells 230 are filtered out by the mem¬ brane and only plasma 240 reaches the enzyme coating layer 220. At this point, an analyte in the plasma interacts with the enzyme coating layer to bring about a detactable change indicative of the analyte's concen¬ tration, e.g. a change in color intensity.
The asymmetric membrane sensor is a two layer device incorporating blood separation and color development in separate layers. Two coating procedures have proven effective.
SUBSTITUTE SHEET Both methods involve coating on only one side of the membrane; the side with the smaller diameter pore size (downstream side) . Conversely, if the entire membrane is dip-coated as described below, thereby covering both sides, some bleed-through occurs resulting in uneven color development. For optimum precision, sensitivity and accuracy, only the downstream side of the membrane should be coated.
Procedure 1;
A buffer stock solution is prepared as follows:
8 g Sodium Dioctylsulfosuccinate (DOSS) and 13.8 g sodium alginate are dissolved in 1000 mL of 0.05M sodium citrate buffer, pH 5.7.
To 25 L of the above buffer stock solution is added with mixing 0.3125g TMB dissolved in 3.0 mL acetone, followed by 5.5 mL of 400 U/mL horseradish peroxidase (2200 units) dissolved in buffer stock plus 1.1 mL of 400 U/mL Glucose oxidase (440 units) dissolved in buffer stock.
The solution is spray coated on the down stream side of the membrane and dried in a 45 C oven. Disks are then punched and placed in a cassette device for measure¬ ment.
Procedure 2:
(Two solution coating procedure.)
Solution 1: 1.25% TMB in Acetone.
Solution 2: To 25 mL of the buffer stock solution of procedure 1 is added, with mixing, 5.5 mL of 400 U/mL horseradish peroxidase (2200 units) dissolved in buffer
SUBSTITUTE SHEET stock plus 1.1 mL of 400 U/mL Glucose oxidase (440 units) dissolved in buffer stock.
Solution 1 is spray coated on the downstream side of the membrane and dried in a 45 C oven. Solution 2 is then spray coated over Solution 1 and dried in a 45 C oven. Disks are then punched and inserted into a cas¬ sette for measurement.
The preferred coating technique is to spray the solu¬ tion onto the downstream side of the asymmetric mem¬ brane using a spray valve available from EFD, Inc. , 977 Waterman Avenue, East Providence, RI 02914, Model No. 780S. The spray valve is stationary and is mounted in a coating machine beneath the membrane to be sprayed. The membrane is fed through the coating machine above the spray path. The operating parameters are as follows:
Operating pressure 65 psi Needle stroke 180°
Liquid pressure 2 psi
Spray distance 4 inch
Spray pattern Fan
Membrane Delivery speed 0.29 inch/min. Liquid delivery rate 2.5 l/min.
Figs. 2a-d illustrate a glucose cassette 100 incorpor¬ ating an asymmetric membrane 5 as described above. The cassette 100 includes a chamber 110 in which the asy - metric membrane 5 is held by ultrasonic weld. An air chamber 120 is provided adjacent the indicator layer 220.
Figs. 3a-c illustrate a flow-cell sensor 300. The sensor 300 comprises a top half 320 and a bottom half
SUBSTITUTESHEET 330. The top half 320 includes a chamber 310 which houses the asymmetric membrane 5. The tcp half and bottom half together define a sample fluid conduit 350, that includes a receiving end 360 and hydrophobic vent 370.
Fig. 3c also shows a transfer vessel 400. Fig. 3d illustrates an alternative embodiment of a receiving end 360' for receiving sample either directly from a patient or from an intermediate transfer vessel.
It is preferable to orient the asymmetric membrane in the measuring device so that, when in use, the sensor will be substantially above the fluid sample forcing the sample to flow upwards against gravity by capillary action. This is an especially advantageous arrangement in the case of blood samples, because red blood cells migrate slower than plasma which, therefore, reaches the topmost indicator layer first. When plasma (which contains glucose, for example) reaches and wets the indicator layer, flow essentially stops. As a result, red blood cells are substantially excluded from the indicator layer where they could otherwise interfere with measurement of the color change which is indica- tive of glucose concentration.
Glucose sensors made in accordance with Example 1 were tested at five separate glucose concentrations between 10 mg/dL (0.5 mmol/L) and 600 mg/dL (30 mmol/L) . The test method was as follows.
A fluid sample of predetermined concentration was applied to the upstream side of the membrane. Whole blood enters the cassette under the upstream side (larger pore size 10-20 um) . The red cells separate as
SUBSTITUTE SHEET the blood diffuses up through the membrane such that only plasma reaches the side containing the enzymes and indicator producing a blue color. The intensity of the color is proportional to glucose concentration and the color produced may be measured by reflectance spectro- scopy.
Wetting of the enzyme coating layer was detected by a change in the reflectance of light at a wavelength of 500 nm. This point is taken as time 0. The intensity of light reflected at 650 nm was measured at time 0 and again 6 seconds later.
For comparison, glucose sensors employing asymmetric membranes were prepared by dip coating using the same indicator solution prepared in Example 1. In the case of the 0.1 μm cutoff membrane the weight gain of indi¬ cator for the spray-coating and dip-coating methods is set forth below:
0.1 um Membrane
Untreated Sprayed Dipped
Average (mg) 1.05 1.24 1.21 Std. dev. (mg) 0.02 0.03 0.02
Max. (mg) 1.08 1.28 1.25
Min. (mg) 1.01 1.19 1.16
Calibration curves were prepared using the least squares method. The results were as follows:
SUBSTITUTE SHEET 0.1 μm 0.45 um
spraved dipped sprayed dipped m 6566.5 17075 5385.2 21684 b 15.02 11.03 13.25 35.66 r2 0.9937 0.9720 0.9813 0.9816
Wherein m and b represent constants in the calibration curve, Y = m X + b (Y = glucose cone, in mg/DL, X = Kubelka-Munk transform and r2 = the correlation coeffi¬ cient) .
The results are illustrated graphically in Figs. 4 and 5. It can be readily seen that the slope of the cali- bration curves for the sprayed membranes are much less steep than those for the dipped membranes. Consequent¬ ly, the sprayed membranes will have a correspondingly greater sensitivity to glucose concentration changes. Additionally, the 0.1 μm sprayed membrane has a corre- lation coefficient approaching unity which indicates purely linear behavior as well as a high degree of precision.
Preferably the sensor is mounted in a disposeable cassette which is designed for automatic operation. Such a cassette is shown in Figures 6-10.
Fig. 6 shows the housing sensor 500. The sensor housing includes a sample tube 511 connected to a major flow path 512 which, in turn, is connected to various sen¬ sors 521-526. An air tube 513 surrounds the sample tube 511. The sensor housing 500 also includes a guide tube 530, the function of which is described below. The sensor housing 500 further includes an air inlet 540, a reference fluid reservoir 550, a vent path 560, a
SUBSTITUTESHEET hydrophobic vent 570, an overflow chamber 580 and a luer collar 590.
Fig. 7 shows the sensor housing 500 after an intermedi- ate vessel or sampler device 600 has been inserted into the guide tube 530 and locked in place. The sampler device 600 has a septum 610 which is punctured by the sample tube 511 and air tube 513. Both the sample tube 511 and air tube 513 project into the sampler device 600.
Fig. 8 is an enlarged view of the interface between the sensor housing 500 and the sampler device 600. During operation, air is forced into the sampler device 600 through the air tube 513. This forces the sample through the sample tube 511 and into the major flow path 512 whereupon it contacts the various sensors within the housing 500.
Fig. 9 shows the sensor housing 500 and sampler device
600 just prior to introduction of a sample. As shown in this figure, a calibration fluid has been introduced near the left end of the major flow path 512.
Fig. 10 shows the complete sensor housing 500 and sam¬ pler device 600 immediately after air A is introduced to force the sample B from the sampler device 600 into the major flow path 512 and into contact with the sensors. The sample eventually reaches the hydrophobic vent 560 whereupon it stops flowing at point C.
Figs. 11-14 shows an alternative cassette design which is configured to apply the sample to the center of the asymmetric membrane.
SUBSTITUTE SHEET Figs. 11 and 12 show? a top plate 800 with recessed flow channels covered by cover tape 810. As shown in th detailed underside view of Fig. 12, sample enters through flow hole 811 and flows to tapered inlet hole 812. At this point, the sample will wet the asymmetric membrane sensor 5 that will be positioned directly below. Excess sample flows out though flow hole 813, and vent channel 814 until it wets vent plug 815. The vent plug 815 can be a hydrophyobic vent, for example.
Figs. 13 and 14 show a bottom plate which, combined with the top plate of Figs. 11 and 12, forms a complete cassette. The bottom plate 900 includes the asymmetric membrane 5 and has a reflectance reading window 911 underneath the asymmetric membrane.
A number of variations of the basic concepts described above are possible. For example, it may be desirable to incorporate two or more membranes from the same manu- facturing batch in each sensor. One or more membranes may be tested as controls using reagents of known analyte concentration prior to testing the sample of unknown analyte concentration. The control membranes can thus be used to confirm that the test membrane is within a predetermined specification or may be used for a single or multipoint calibration.
SUBSTITUTE SHEET

Claims

e claim:
1. A sensor for a chemical species comprising:
a. an asymmetric membrane having an upstream side, a downstream side and a porosity gradient from the upstream side to the downstream side; and
b. indicator means for generating a measurable re- sponse indicative of the presence of the chemical species, said indicator means being provided as a coating located on and being in fluid communica¬ tion with the downstream side of said asymmetric membrane.
2. The sensor of claim 1, wherein the porosity gradi¬ ent decreases from the upstream side to the down¬ stream side of the asymmetric membrane.
3. The sensor of claim 1, wherein the upstream side has a porosity in the range of about 10-20 um.
4. The sensor of claim 1, wherein the downstream side has a porosity in the range of about 0.1-1.0 um.
5. The sensor of claim 1, wherein the chemical spe¬ cies is an enzyme substrate, ion, gas, enzyme, protein, hemoglobin, bilirubin or antigen.
6. The sensor of claim 1, wherein the chemical spe¬ cies is an enzyme substrate.
7. The sensor of claim 6, wherein the enzyme sub¬ strate is glucose, lactate, triglycerides, total cholesterol, high-density lipoprotein, low-density
SUBSTITUTE SHEET lipoprotein, urea, creatinine, ascorbic acid, uric acid or ethanol.
8. The sensor of claim 7, wherein the enzyme sub- strate is glucose, lactate or urea.
9. The sensor of claim 8, wherein the enzyme substrate is glucose.
10. The sensor of claim 8, wherein the enzyme sub¬ strate is lactate.
11. The sensor of claim 8, wherein the enzyme sub¬ strate is urea.
12. The sensor of claim 1, wherein the chemical spe¬ cies is ethanol.
13. The sensor of claim 1, wherein the chemical spe- cies is an ion.
14. The sensor of claim 13, wherein the ion is potass¬ ium, sodium, hydrogen, bicarbonate, lithium, phos¬ phate, magnesium, calcium or ammonium.
15. The sensor of claim 1, wherein the chemical spe¬ cies is a gas.
16. The sensor of claim 15, wherein the gas is chlo- rine, oxygen or carbon dioxide.
17. The sensor of claim 1, wherein the chemical spe¬ cies is an enzyme.
SUBSTITUTE SHEET
18. The sensor of cl=ιim 17, wherein the enzyme is amylase, creatiniπe-kinase, creatinine-kinase-MB or gamma-glutamyl-transferase.
19. The sensor of claim 1, wherein the chemical spe¬ cies is a protein.
20. The sensor of claim 19, wherein the protein is an antibody, albumin or amino acid.
21. The sensor of claim 20, wherein the antibody is an HIV antibody.
22. The sensor of claim 20, wherein the amino acid is phenylalinine or tyrosine.
23. The sensor of claim 1, wherein the chemical spe¬ cies is hemoglobin.
24. The sensor of claim 1, wherein the chemical spe¬ cies is bilirubin.
25. The sensor of claim 1, wherein the chemical spe¬ cies is an antigen.
26. The sensor of claim 25, wherein the antigen is a virus.
27. The sensor of claim 25, wherein the antigen is a drug.
28. The sensor of claim 25, wherein the antigen is a drug metabolite.
SUBSTITUTE SHEET
29. The sensor of claim 25, wherein the antigen is a native sub-unit of a virus.
30. A test cell comprising:
a. a sensor for a chemical species, the sensor in¬ cluding an asymmetric membrane and indicator means for generating a measurable response indicative of the presence of the chemical species, the asym et- ric membrane having an upstream side, a downstream side and a porosity gradient from the upstream side to the downstream side, the indicator means being provided as a coating located on the down¬ stream side of and being in fluid communication with the downstream side of said asymmetric mem¬ brane; and
b. means for receiving a sample containing the chemi¬ cal species and delivering same to the upstream side of the asymmetric membrane.
31. The test cell of claim 30, wherein the porosity gradient decreases from the upstream side to the downstream side of the asymmetric membrane.
32. The test cell of claim 30, wherein the upstream side has a porosity in the range of about 10-20 um.
33. The test cell of claim 30, wherein the downstream side has a porosity in the range of about 0.1-1.0 um.
βuBβπruTHβHsa
34. The test cell of claim 30, wherein the chemical species is an enzyme substrate, ion, gat, enzyme, protein, hemoglobin, bilirubin or antigen.
35. The test cell of claim 30, wherein the chemical species is an enzyme substrate.
36. The test cell of claim 35, wherein the enzyme substrate is glucose, lactate, triglycerides, total cholesterol, high-density lipoprotein, low- density lipoprotein, urea, creatinine, ascorbic acid, uric acid or ethanol.
37. The test cell of claim 36, wherein the enzyme substrate is glucose, lactate or urea.
38. The test cell of claim 37, wherein the enzyme substrate is glucose.
39. The test cell of claim 37, wherein the enzyme substrate is lactate.
40. The test cell of claim 37, wherein the enzyme substrate is urea.
41. The test cell of claim 30, wherein the chemical species is ethanol.
42. The test cell of claim 30, wherein the chemical species is an ion.
43. The test cell of claim 42, wherein the ion is potassium, sodium, hydrogen, bicarbonate, lithium, phosphate, magnesium, calcium or ammonium.
SUBSTITUTESHEET
44. The test cell of claim 30, wherein the chemical species is a gas.
45. The test well of claim 44, wherein the gas is chlorine, oxygen or carbon dioxide.
46. The test cell of claim 30, wherein the chemical species is an enzyme.
47. The test cell of claim 46, wherein the enzyme is a ylose, creatinine-kinase, creatinine-kinase-MB or gamma-glutamyl-transferase.
48. The test cell of claim 30, wherein the chemical species is a protein.
49. The test cell of claim 48, wherein the protein is an antibody, albumin or amino acid.
50. The test cell of claim 49, wherein the antibody is an HIV antibody.
51. The test cell of claim 49, wherein the amino acid is phenylalinine or tyrosine.
52. The test cell of claim 30, wherein the chemical species is hemoglobin.
53. The test cell of claim 30, wherein the chemical species is bilirubin.
54. The test cell of claim 30, wherein the chemical species is an antigen.
SUBSTITUTESHEET
55. The test cell of claim 54, wherein the antigen is a virus.
56. The test cell of claim 54, wherein the antigen is a drug.
57. The test cell of claim 54, wherein the antigen is a drug metabolite.
58. The test cell of claim 54, wherein the antigen is a native sub-unit of a virus.
59. An analyzer comprising:
a. a test cell having,
i) a sensor for a chemical species, the sensor in¬ cluding an asymmetric membrane and indicator means for generating a measurable response indicative of the presence of the chemical species, the asymmet¬ ric membrane having an upstream side, a downstream side and a porosity gradient from the upstream side to the downstream side, the indicator means being provided as a coating located on the down- stream side of and being in fluid communication with the downstream side of said asymmetric mem¬ brane, and
ii) sample transfer means for receiving a sample con- taining the chemical species and delivering same to the upstream side of the asymmetric membrane; and
SUBSTITUTE SHEET b. detection means for measuring a change .in a characacteristic of the indicator means responsive to the presence of the chemical species.
60. The analyzer of claim 59, wherein the test cell is a disposable test cell.
61. The analyzer of claim 59 wherein the sample trans¬ fer means comprises a sample inlet, a sample out- let, a first conduit connecting the sample inlet to the test cell and a second conduit connecting the test cell to the sample outlet.
SUBSTITUTE SHEET
PCT/DK1994/000058 1993-02-11 1994-02-09 Asymmetric membrane sensor WO1994018559A1 (en)

Applications Claiming Priority (2)

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US08/016,524 1993-02-11

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US5526120A (en) * 1994-09-08 1996-06-11 Lifescan, Inc. Test strip with an asymmetrical end insuring correct insertion for measuring
US5563031A (en) * 1994-09-08 1996-10-08 Lifescan, Inc. Highly stable oxidative coupling dye for spectrophotometric determination of analytes
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US5780304A (en) * 1994-09-08 1998-07-14 Lifescan, Inc. Method and apparatus for analyte detection having on-strip standard
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Cited By (21)

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US5526120A (en) * 1994-09-08 1996-06-11 Lifescan, Inc. Test strip with an asymmetrical end insuring correct insertion for measuring
US5563031A (en) * 1994-09-08 1996-10-08 Lifescan, Inc. Highly stable oxidative coupling dye for spectrophotometric determination of analytes
US5780304A (en) * 1994-09-08 1998-07-14 Lifescan, Inc. Method and apparatus for analyte detection having on-strip standard
US6335203B1 (en) 1994-09-08 2002-01-01 Lifescan, Inc. Optically readable strip for analyte detection having on-strip orientation index
US6491870B2 (en) 1994-09-08 2002-12-10 Lifescan, Inc. Optically readable strip for analyte detection having on-strip orientation index
EP0832430A1 (en) * 1995-05-09 1998-04-01 Smithkline Diagnostics, Inc. Devices and methods for separating cellular components of blood from liquid portion of blood
EP0832430A4 (en) * 1995-05-09 2000-12-06 Beckman Coulter Inc Devices and methods for separating cellular components of blood from liquid portion of blood
EP0926484A2 (en) * 1997-12-24 1999-06-30 Terumo Kabushiki Kaisha Test paper and analyte collecting head
EP0926484A3 (en) * 1997-12-24 1999-10-20 Terumo Kabushiki Kaisha Test paper and analyte collecting head
WO2008131769A2 (en) * 2007-04-27 2008-11-06 Radiometer Medical Aps A sensor board
EP1985994A1 (en) * 2007-04-27 2008-10-29 Radiometer Medical ApS An optical sensor system
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EP2227269A1 (en) * 2007-12-12 2010-09-15 Micropoint Bioscience Inc. Rapid and efficient filtering whole blood in a capillary flow device
EP2227269A4 (en) * 2007-12-12 2014-01-15 Micropoint Bioscience Inc Rapid and efficient filtering whole blood in a capillary flow device
US9968931B2 (en) 2007-12-12 2018-05-15 Nan Zhang Rapid and efficient filtering whole blood in capillary flow device
EP2238444A2 (en) * 2008-01-09 2010-10-13 Orono Spectral Solutions, Inc. Apparatus and method for determining analyte content in a fluid
EP2238444A4 (en) * 2008-01-09 2011-12-21 Orono Spectral Solutions Inc Apparatus and method for determining analyte content in a fluid
US8393198B2 (en) * 2008-01-09 2013-03-12 OronoSpectral Solutions, Inc. Apparatus and method for determining analyte content in a fluid
US8613214B2 (en) 2008-01-09 2013-12-24 Orono Spectral Solutions, Inc. Apparatus and method for determining analyte content in a fluid
WO2014024157A1 (en) * 2012-08-08 2014-02-13 Koninklijke Philips N.V. Plasma separation using a drop of blood

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Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase