AU7996798A - Automatic analysing apparatus - Google Patents

Description

AUSTRALIA

PATENT$ ACT 1990 COMPLETE

SPECIFICATION

FOR A STANDARD

PATENT

ORIGINAL

Name of Applicant: USF FILTRATiON AND SEPARATIONS GROUP

INC.

A..a.Ivetos Ian Andrew MAXWELL, Thomas William BECK and Alastair Mclndoe

HODGES

Address of Service* B3ALDWIN SHELSTON

WATERS

MARGARET

STREET

SYDNEY NSW 2000 Invention Title: "AUTOMATIC ANALYSING

APPARATUS"

Details of Associated Provisional Application No. P0 8558 dated 13th August, 1997 The following statement is a full description of this invention, including the best method of performring it known to us:- -2- FIELD OF THE INVENTION This invention relates to automatic analysing apparatus. The invention will be described herein with particular reference to apparatus for measuring the concentration of glucose or other analytes in blood but is not limited to that use.

BACKGROUND

ART

Currently glucose in blood samples is measured in pathology laboratories and the like by means of apparatus such the YSI blood analyser in which successive samples are i analysed by means of a hollow cylindrical probe in which is mounted a silver and a platinum electrode. The face of the probe is fitted with a three layer membrane. The middle layer contains an immobilised enzyme which is sandwiched between a cellulose Sacetate and a polycarbonate membrane. The face of the probe, covered by the membrane, is situated in a buffer filled sample chamber into which successive samples are injected.

Some of the sample diffuses through the membrane. When it contacts the immobilised oxidase enzyme it is rapidly oxidised producing hydrogen peroxide, the glucose forming a glucono-delta-lactone.

The hydrogen peroxide is in turn oxidised at the platinum anode producing electrons.

A dynamic equilibrium is achieved when the rate of peroxide production and removal reach a steady state. The electron flow is linearly proportioned to the steady state peroxide concentration and therefore to the concentration of the glucose.

The platinum electrode is held at an anodic potential and is capable of oxidising many substances other than hydrogen peroxide. To prevent these reducing agents from Scontribution to sensor current, the membrane contains an inner layer consisting of a very thin film of cellulose acetate. This film readily passes hydrogen peroxide but excludes IH chemical compounds with molecular weights above approximately 200. The acetate film fc. -3also protects the platinum surface from proteins, detergents, and other substances that could foul it. However the cellulose acetate film can be penetrated by compounds such as hydrogen sulphide, low molecular weight mercaptans, hydroxylamines, hydrozines, phenols and analytes.

In use, the sample (or a calibration standard) is dispensed in to the chamber, diluted into 600 microlitres of buffer, and then a measurement is made by the probe. The sensor response increases and then reaches a plateau when a steady state is reached. After several seconds a buffer pump flushes the chamber and the sensor response decreases.

The apparatus monitors the base line current. If it is unstable a buffer pump will continue to flush the sample chamber with buffer. When a stable base line is established S .an automatic calibration is initiated. The apparatus calibrates itself for example after every .I .five samples or 15 minutes. If a difference of more than 2% occurs between the present Sand previous calibration, the apparatus repeats the calibration. Recalibration also occurs if the sample chamber temperature drifts by more than 1 °C.

The apparatus described suffers from a number of disadvantages. Firstly, a high proportion of its time in use is spent in performing calibrations rather than analysis.

Furthermore the consumption of buffer and calibrating solutions is a substantial cost.

Another disadvantage is that as the enzyme membrane ages, a graph of reading versus concentration becomes non-linear. It would be highly desirable to provide apparatus which is able to make measurements of the kind described with improved speed, efficiency, and at lower running cost.

-4- OBJECT OF THE INVENTION An object of the present invention is an improved apparatus for automatically analysing samples which avoids or ameliorates at least some of the disadvantages of prior art. An object of the preferred embodiment of the invention is an automatic apparatus for estimating the concentration of glucose in samples of blood.

DISCLOSURE OF THE INVENTION According to one aspect the present invention consists in automatic apparatus comprising: a plurality of cells, each cell comprising a first electrode spaced a predetermined 10 distance from a second electrode, each cell containing a predetermined quantity of one or more reagents, S means for moving the cells successively through a filling station said filling station comprising means for delivering successive samples to respective successive cells, means for applying a potential between the electrodes of a cell and measuring the current as a function of time, the electrodes being in sufficiently close proximity that reaction products from one electrode arrive at the other electrode while the current is being measured.

BRIEF DESCRIPTION OF DRAWINGS The invention will now be more particularly described with reference to the drawings wherein figure 1 is a schematic diagram of a cross-section of apparatus according to the invention in elevation.

Figure 2 shows a band of cells in plan view.

Figure 3 is a schematic diagram of measuring apparatus according to the invention.

e DESCRIPTION OF PREFERRED EMBODIMENTS Our earlier applications PCTiAU/00723; PCT/AU/00724 and PCT/AU/00365 (the description of each of which is incorporated herein by reference) describe methods for sensitive measurement of concentration of an analyte in a sample. Those methods require a thin layer cell having electrodes of predetermined area and predetermined separation (less than 500 microns and typically less than 200 micron spacing).

We have now found that the use of such cells provides a superior automatic analysing apparatus having unexpected advantages.

s In one preferred embodiment of the present invention, a succession of cells are 1 disposed as a continuous band which is driven through the filling station at which successive cells are filled for example by an automatic pipetting device and is then driven through a measuring station at which a potential is applied between the electrodes and current measured. For preference the band is flexible and can be wound or coiled.

According to a second aspect the invention consists in: apparatus comprising means for forming a cell (or pseudo cell), each cell or (pseudo cell) comprising a first electrode spaced a predetermined distance from a second electrode and containing a predetermined quantity of one or more reagents; means for delivering a sample to fill one of said cells or (pseudo cells) before or after its formation; means for applying a potential between the electrodes of the cell or (pseudo cell) and measuring the current as a function of time, the electrodes being in sufficiently close J proximity that reaction products from one electrode arrive at the other electrode while the S current is being measured.

-6- In preferred embodiments pseudo cells are formed successively in situ at or in advance of a sample filling station by bringing two spaced apart continuous strips of electrode into close proximity.

The invention will now be more particularly described by way of example only with reference to specific embodiments.

SApparatus according to a first embodiment of the invention will now be described by way of example only with reference to figure 1.

SA continuous separator sheet I of Melinex (a chemically inert, and electrically resistive Polyethylene Terephthalate approximatel; 100 micron thick is fed to a j 10 punch2 atwhich circular holes 3 (each 3.4 mmn in diameter) are punched out at eg at 5 mm centres. The punched separator sheet then passes through a rotogravure printing station 4 at which the lower side 4 of separator sheet I is coated with a water based heat activated adhesive 5 (ICI Novacoat system) to a thickness of 12 microns wet (approximately microns dry). Water is then evaporated by means of a hot air dryer 6 leaving a contact adhesive surface 7 on the lower surface of separaIr sheet 1. Asheet 8ofMylar

PET

bearing a sputter coating 9 ofpalladium at a thickness of 100-1000 angstroms is then brought into engagement with adhesive coating 7 and the adhesive is activated whereby the palladium layer is bonded to the lower surface of separator sheet I covering aperture SThe assembly then passes to a station where the well formed by apertures 3 closed with palladium coating 9 may be dosed with one or more chemical reagents eg.by a pipette and drying tunnel I I. The assembly then passes to a second rotogravure station 12 at which a further layer of adhesive 13 is applied to the upper side of separator sheet I and a further eet 14 of Mylar PET carrying a coating ofpalladium 15 of a thickness of 100- 1 -7- 1000 angstroms is brought into contact with, and then adhered to, the upper surface of separator sheet 1 enclosing punched holes 5 between the two palladium layers. If preferred, an adhesive may be applied to a spacer layer prior to punching and prior to laminating with the sputter coated layers.

Strip 1 is then provided with notches 16 at opposite edges as shown in Figure 2 so as to provide access from each side to the cell defined by apertures 5 together with palladium coatings 15 and 9.

At station 11 each cell is provided with a predetermined quantity of chemicals for example an enzyme and a ferro/ferricyanide redox mediator. Suitable enzymes and S 10 mediators may be selected (without limitation) from those described in Table 1. The dosages may be metered into the well of the cell prior to closure with the upper metallised layer 15 and may be dried in an oven or may be freeze dried in situ. The cell may also contain buffers, surfactants etc.

In use according to the first embodiment of the invention a continuous length 20 of PET defining spaced apart cells 3 is fed to a sample filling station 21 at which a predetermined volume of successive samples are admitted to successive cells by means of a micro pipette 22. The band then passes through a measuring station where a potential is applied to the upper and lower electrodes via contacting means 23 and the current is measured by measuring circuit 24. As described in our co-pending applications PCT/AU96/00723 and PCT/AU96/00724, the potential between the electrodes is set such that the rate of electro-oxidation of the reduced form of the species (or of electro-reduction S- of the oxidised form) is diffusion controlled. Because the working and counter electrodes are placed in very close proximity (about 0.5 mm apart or less) ferrocyanide that is generated at the counter electrode has time to reach the working electrode and contribute to i ;i ii ic~.i r- i; i.ri.~a -8the current at the working electrode. That is, a ferricyanide molecule can be reduced at the counter electrode to ferrocyanide, and can then diffuse to the working electrode, where it will be re-oxidised to ferricyanide. This situation results in a decreasing current at short times that steadies to reach a constant value at longer times (the steady state current). This steadying of the current occurs because a constant stream of ferrocyanide is being supplied to the working electrode from the counter electrode. This mechanism is quite distinct from that which occurs in a Cottrell device in which the electrodes are separated so that ferrocyanide that results from the reduction of ferricyanide at the counter electrode does not influence the observed current 10 In the present cell the steady state current is givenby 2DFAC o (1)

L

wherein i is the steady state current, D is the diffusion coefficient, F is the Faraday constant, A is the area of the electrode, Co is the concentration of the analyte (ferrocyanide) and L is the separation of the electrodes.

15 The current I at time t is given by the equation: zD i =i(l+2 e) t where p is pi.

At longer times the higher exponential terms in equation 2 can be ignored. Therefore equation 2 can be approximated by equation 3 for times greater than a certain value S-4p D~t~p2 i i,(1+2exp S' L I -9- If it is assumed that equation 2 can be approximated by equation 3 when the second exponential term in equation 2 is 1% of the first exponential term, equation 3 is valid for 0.0389L 2 times greater than t=-

D

It will be understood that Equation 3 can be transformed to give: I l D 'iss

L

So a plot of the left hand side of equation versus time will give a straight line ea a with new slope=4p2 Combining equations and gives C. 2p 2 iss (6 FAL slope SA and L are predetermined by the thickeners of separator sheet 1 and the cross sectional area of apertures 3 since parameters slope" and "iss are measured in the test and p and F are universal constants, the concentration of the analyte derived from the test (Co) determined very accurately by calibration using a small number of the cells.

SUsed cells are disposable and can be cut from the roll for example by a guillotine and if desired can be isolated in a sealed container thereby eliminating biohazardous liquid waste and contact of liquid samples with the user.

The cells can be assembled in situ or manufactured inexpensively for supply eg on a Ii roll ready for use and since, in the latter case, they may contain all necessary chemicals r r i; i. DC1_. i l i- i I there is no need to maintain separate supplies of reagents or to provide pumps and dispensing means for the reagents. If desired the chemistry of successive cells could be different one from another so that a multiplicity of different tests could be performed on successive pipetted volumes of sample in successive cells. Because the chemical reagents are used only once they can be better protected against deterioration prior to use. Because the cells are very much smaller than the sample cell of for example the YSI device smaller samples are required and each test can be performed more rapidly and at lower materials cost.

In the embodiment described above the electrodes are spaced apart by means of a 10 PET spacer layer which is punched to define a cell volume contained between the electrode surfaces.

In an alternative construction a porous layer is interposed between the electrode layers. This may simply be a porous absorbent substance such as a paper or may be a porous membrane such as described in our copending application PCT/AU96/00210.

When a porous layer is used to define a cell volume it may be divided into cells for example by compression of the porous layer at predetermined locations as also described in said copending application The porous layer may have a metallised layer adhered on one or both sides or may have the metal layers deposited directly thereupon for example by sputter coating.

Although the first embodiment has been described with reference to a continuous band of cells. severed separated cells could be fed successively to a filling station and a S- measuring station by a suitable feeding and conveying mechanism. The cells may be manufactured by other means and may be of a different form than those exemplified.

kr 4 -11- Although in the first embodiment described a single column of cells is used, the cells could also be punched out in rows, or in some other arrangement. It will also be apparent from the teaching hereof that the filling and/or measuring stations can move relative to the cells rather than the cells move relative to the stations.

A second embodiment of the invention (not illustrated) will now be described in which a cell or pseudo cell is formed in situ.

In a second embodiment of the invention two metallised tapes are fed to a filling station. One tape consists of a spacer sheet 1 of PET having holes 2 punched out, eg at Smm centres and having a sheet 8 of Mylar bearing a sputter coating 9 of palladium Sto; laminated on one side of the spacer sheet so that the palladium coating 9 surface seals the "lower opening of holes 2 to form a well. The well may contain reagents relevant to the test to be conducted. The reagents may be in the form of solids dried on the conducting surface or the walls of the well or may be a solution dispensed into the well priorto addition of a *sample. The reagents may be added to the well at the filling station or at a separate preceding station or stations.

At the filling station a sample to be analysed is pipetted into the well and then a second tape comprising, for example. a Mylar strip 14 bearing a sputtered layer of palladium 15 on its lower surface is brought into contact with the top of the volume of Ssample which desirably extends above the upper surface of spacer sheet 1 such that excess sample volume is squeezed out as the upper metal layer 15 comes into contact with the top surface of the spacer sheet 1.

The volume of sample ipetted is such that the height of the drop is equal to or t preferably slightly greater than the thickness of the spacer layer I.

's r -12- It will also be understood that the spacer layer can be replaced with a porous layer for example a porous paper, non-woven mesh, or felt, or a porous membrane, which acts to immobilise the sample spatially with respect to the electrode layers and to hold the reagents.

A spacer layer 1 need not be adhered to metal layer 9. A temporary seal can be formed at the filling station eg by vacuum or pressure between an electrode strip and a spacer strip to form a temporary floored well which becomes a pseudo cell.

It will also be understood that the metal layer tapes or bands need not be travelling in *i the same direction. For example, one metallised electrode layer may be proceeding I; 10 transversely of the other, each tape being advanced after each measurement to expose a fresh lower and fresh upper electrode surface and fresh reagent at the sample filling station.

In each case the resulting current is measured as a function of time while the electrodes are in contact with a sample drop of predetermined volume.

In another embodiment of the invention the current can be followed with time after a potential has been applied between the electrodes until a predetermined time or state has Sbeen reached. The sign of the applied potential would then be reversed and analysis performed similar to that given above except with equations and being replaced with 2 Dt it i=iss(l+4exp(-4p 2 (7) I In( 4 4 p (8) This protocol has the advantage of being able to allow for slow processes occurring in the test. This can be done by: i r J; j -13a) waiting for the current to change by less than a predetermined amount per second before reversing the potential, such that any slow processes which effect the measurement are substantially complete, or b) using the change in the current with time before the potential is reversed to compensate for the slow processes occurring (as has been described in our earlier patent applications).

t r 0S 9 00*S 1 99 9 I S ft *4*

V.

TABLE I -7

'I

~1 rANALYTE I c o se I NAD d e p endet Cliolesterol GDI-pqq G lucose dehydrogenase and diaphcrase cholesterol esterase and cholesterol oxidase

RDXMEDIATORI

(OXIDISED)

FORM)I

rerricyanide Ferricyanide ADDITIONAL

MEDIATOR

erricyariide I IVl- elmlesverol Cholesterol es terase and cholesterol Ferricyanide idasea..i) 2.6-dinsethyl-l .4-them'.ocjiiinolic2.

dicitloro- 1,4-henzoqu inone or pheinazinc ethosulfate 2,6-dim ethyl- 1,4-benzoqu inoile dicliloro- I ,4lbenzoquinoflC or phenaine ethosul fatc L enazine mcthosulfaic 2,6-diehioro- It,4-bueazoquinone Friel'. cerides Lactate L~actate Lipoprotein lipase, glycerol kinase, and glycerol-3-pliosphate oxidase Lactate oxidasc Lactate dehydrogenase and diaphorase Ferricyanide or pitenazine ethosulphate1 FFerricyanide, hnzn toufto Ferricyanide, phenazine ethosulfate, or phenazine methosulfate IFerricyanide IPhenylenediamine l.actnte dehydrogenase Diaphorase Pyrivate Alcohol Hilinmhtn 1. ric acid Pyrsivate oxidase Alcohol oxidase Hilirubin oxidase Uricase I -toethoxy-phenazifle methosulfate Ferricyanide I 1

I

Claims (13)

1. Automatic apparatus comprising: a plurality of cells, each cell comprising a first electrode spaced a predetermined distance from a second electrode, each cell containing a predetermined quantity of one or more reagents, means for moving the cells successively relative to a filling station said filling station comprising means for delivering a predetermined volume of successive samples to respective successive cells, and means for applying a potential between the electrodes of a cell and measuring the current as a function of time, the electrodes being in sufficiently close proximity that reaction products from one electrode arrive at the other lo electrode while the current is being measured.

2. Apparatus according to claim I wherein a plurality of cells are disposed in a strip. S:

3. Apparatus according to claim 2 wherein the strip is sufficiently flexible to be wound Sor coiled. S4. Apparatus according to any one of the preceding claims wherein the plurality of cells comprises a continuous strip offirst electrode spaced from one or more second electrodes by spacer means, said spacer means together with the first and second electrode defining a cell volume bounded by said electrodes and said spacer. Apparatus according to any one of the preceding claims wherein a cell of the W plurality comprises a continuous strip bearing a discrete first electrode spaced from a second electrode by spacer means, the spacer means being apertured to define in use a cell volume between the first and second electrode.

6. Apparatus according to any one of the preceding claims wherein adjacent cells of the plurality contain an identical reagent or reagents. -16-

7. Apparatus according to any one of claims 1 to 5 wherein a cell of the plurality contains a different reagent from an adjacent cell.

8. Apparatus comprising means for forming a cell (or pseudocell) each cell (or pseudocell) comprising a first electrode spaced a predetermined distance from a second electrode and containing a predetermined quantity of one or more reagents, and means for delivering a predetermined volume of a sample to each cell or pseudocell before or after the formation of that cell.

9. Apparatus according to claim 8 wherein the means for forming a cell comprises means for bringing a continuous strip of first electrode into close proximity with, and a i. predetermined distance from, a second electrode. Apparatus according to claim9 wherein the first and second electrode are spaced apart by a spacer of predetermined thickness.

11. Apparatus according to claim 8 wherein the spacer comprises an aperture which C1, Aapara*us ac 1 1 i together with an overlying and underlying electrode defines an effective cell volume.

12. Apparatus according to claim 8 wherein a pseudocell is formed in situ at or in Sadvance of a sample filling station by bringing two continuous strips of electrode into close proximity but spaced one from the other at a predetermined distance in combination with Smeans for admitting a predetermined volume of a sample into the space between the electrodes so that the sample is in contact with both electrodes and the sample defines an effective cell volume.

13. Apparatusaccording to claim 8 wherein-a pseudocell is formed by providing a first and second electrode spaced each from the other by a thin porous spacer arranged so that in use predetermined volume of sample can be absorbed betwen the electrodes and in Oka r -17- contact with both electrodes, the sample volume defining an effective volume of the pseudocell.

14. Apparatus according to any one of the preceding claims wherein the first electrode is a working electrode and the second electrode is an counter electrode and said electrodes are spaced apart by less than about 0.5 mm. Apparatus substantially as herein described with reference to any one of the Examples.

16. A method comprising the steps of: forming a cell or pseudo cell, each cell having a first electrode and a second electrode 10 spaced apart by a predetermined distance and containing a predetermined quantity of one or more reagents, delivery a sample to fill one of said cells (or pseudo cells) before or after its formation, °c 0" applying a potential between the electrodes, and measuring the current as a function of time, the electrodes being in sufficiently close proximity that reaction products from one electrode arrive at the other electrode while the current is being measured.

17. A method substantially as herein described with reference to any one of the Examples. DATEDthis13th Day of August, 1998 USF FILTRATION AND SEPARATIONS GROUP INC. Attorney: IAN T. ERNST Fellow Institute of Patent Attorneys of Australia of BALDWIN SHELSTON WATERS