CA2189994A1 - Flow-through electrochemical biosensor - Google Patents

Abstract

A flow-through solid phase immunoassay system or device with electrochemical detection capabilities is described wherein the solid phase is in close connection with a porous working electrode and a suitable counter electrode is present in the system. The novel device provides very fast, sensitive diagnostic capabilities.

Description

WO 95131725 ~18 9 9 9 4 r~ C`~ Y
. ;,.

r ; ~ ~iosensor Background of the Invention The present invention relates to a flow-throuch solid phase ~ y system or device with :' . ' ' detection capabilities.
~ diagnostics are well established as the routine method of diagnosis for a wide ranae of substances in serum, plasma or other fluids and the principle of these assays can be extended to other fields such as L .u~ ll monitoring or DNA analysis.
The classical methods for ~ use either ~i) a capture antibody on a solid phase, such as a plastic microtitre piate, exposure to the biological sample to attach the antigen of interest, washing and then exposure to a second labelled antibody. The label on the antibody can be a radioactive tracer or enzyme for example. Further washing is followed by detection of the label ~and hence the amount of antigen in the original sample). This is known as a sandwich assay or two-site assay or liil a capture antibody on the solid phase followed by exposure to the biological sample containing antigen and an added amount of labelled antigen. Labelled and unlabelled antigen compete on the solid phase for the antibody sites. The amount of label revealed after washing is inversely proportional to the amount of true antigen in the biological sample. This is known as a competitive assay.
The revelation of label is achieved by various means. For enzyme labels the choices are ul~u~û5~ , fluorescent, ~ "G"L or ul~_l,u.,l.~ .l detection. Chromogenic s, where the enzyme label produces a coloured product uPOn addition of a suitable substrate, are well established under the generic term ELISA, using ~ principlesto detect the colour change in the substrate solution. They are relatively easy to perform but suffer from long incubation times and difficulty in achieving low limits of detection.
L~ _u,.~ and fluorescent products from a substrate solution can also be achieved via enzyme labelling and take the limits of detection lower by a factor of 10 for sandwich assays IPortsmann, T. and Kiessi9, S.T., Enzyme Immunoassay Techniques-, J. Immuno. Meth., 150, pp 5-21, 1992~ when compared to cl--u,--u~ , methods and significantly reduce the sample volumes required. Difficulties arise in stabilisinq the substrates for commercial distribution and SUBSTITllTE SHEE~ (PJJLE 26) WO95/31725 218999~ r~ L IIIY ~
~ _ he expense of the I ' 'r;uu.c~cc..I detectors in the instrument costs. '' . ' ' / is considered to lie within the field of biosensors and essentially seeks to combine the detection of an ' . ' ' 'Iy active species Iwhich is the product of the enzyme-substrate reaction~ by oxidation or reduction of the said species on an electrode in close proximity to the solid phase. There are many exampies in the literature of such biosensors ~see for examples Heineman, W. R. and Halsall, H. B., ~Strategies for ~
Anal. Chem., 57,2, pp 1321-1331, 1985~ but the majority have faiied to achieve the required sensitivity and detecion iimits in any manner that could be used to fabricate a practical device for commercial use.
Another aspect of practical Inn~-~~y is the ,' ' of assays on various solid phases of large surface areas in order to shorten the incubation times for the assay. Solid phases with large surface areas include polymer beads, magnetic particles and polymeric membranes.
Uquid flow technology in various forms has been appiied in conjunction with a selection of solid phases to shorten assay times and enhance sensitivity. US-A-5006309 describes a device with a membrane as a solid phase for ' , ...~ y with optical detection. JP-A-60188839 describes the use of a selecbve electrode which is projected into the stream of flow of a sample for detection.
EP-a1-0033188, EP-B1-0060082 and EP-A1-0469830 all describe the construction of porous electrodes that may be used to improve Ll~_~lu~l-_.,.;~al detection in analytical systems such as HPLC. EP-31-0122009 is an electrode intended for the detection of some biological molecules which are Ll~_.., ' ' "~ active provided that the samples are ~1". ~ filtered prior to ' The electrodes described in these patents are intended to be used as semi-permanent, re-usable detectors within ~ _, ' ' systems.
EP-~2-0352138 and EP-A2-0525723 both describe particular devices which incorporate membranes as solid phases and use the standard solid electrode formats normally employed in biosensors as Ll~_L~I ' ' ' detectors. The devices are intended for use as disposables with a suitable instrumentation system.
Summary of the Invention The present invention is based on the finding that the detection capabilities of a llu.. ~,.uu~ll solid phase ' ~ system or device can be significantly augmented by havina the solid SUBSTITUTE SHEET IhiJLE ~bl 21~9994 phase in close connection with a porous working electrode. It appears that this effoct is achieved by the increased surface area of the electrode in contact with the ~ ~l . ' e species and the enhanced diffusion kinetics at the ~I~_L.u i~. s"_~ interface. The large surface areas provided by porous electrodes apparently lead to extrcmely high oxidation and reduction efficiency of :' u~ ;.e species under the proper flow and voltage conditions. The act of flowing the liquid through the electrode appears to instant~y replenish the species for detection in the diffusion layer at the _I,,_L,u~i~ liqu;J interface as it is oxidised or reduced. This interface has been the limiting factor in many ~k,_l~ c~nenrS and other biosensors since the species as it is oxidised or reduced is depleted from the double layer and its cul~.-' ' has to be controlled, usually by stirring the solution above a solid detection electrode or flowinu liquid over a solid detection electrode. The application of the electrode in this invention therefore covers ;.,,~,.u.. to two aspects of critical importance for electrodes to be used in LII ' ' ' jmrr-lnn~n~nrS or other biosensors, the need for high efficiency in oxidation and reduction Isurface area~ and the need to quickly replenish the double layer under controlled conditions (flow-through . . The electrode can be any suitable porous conductingmaterial such as pre-formed graphite sheets, fused carbon powders, metallic-based porous plugs or conductinu inks printed on suitable porous bases.
The invention therefore relates to a flow-through solid phase ; system or devicewith Ll~_Llu~ al detection capabilities, .I~ Lc.i,~d in that the solid phase is in close connection with a porous working eiectrode and a suitable counter electrode is present in the SYstem.
Further subject matter is evident from the claims.
he sensitivity of the electrode according to this invention to a particular species can be by controlling flow speed and voltage at the electrode. Voltage control can be by the normal, means to provide a current or charue reading in proportion to the amount of species in solution which is flowed through the electrode. Fu~ voltagecontrol can be of any of the normal vulL~ y or ~ u_.u~ll_l.y formats for _~ ,u~,l-_...:_.,l analysis but would preferably use the application of a dc-potentiai suiuble for oxidation or reduction of the species and current at the electrode would be monitored by suitable instrumentation .
The el~_~-u~ active species lalso referred to as Ll~._LIU.,.,L;.3 species) can be generated in the course of a sandwich or competitive or other ' , type by the use of any suiuble enzyme-substrate pair which produces a readily oxidised or reduced species and the SUBSTITUTE SHEEt (RULE 26) WO 9'i/31725 ; ~ 1 / /Y ~

2.18g994 enzymo nonmally forms the assay label or pan of the assay. The assay preferably uses the enzyme alkaline ~ and a phosphate substrate but could use any other suitable enzyme-substrate pair where the substrate generates an ~.' u"l, '~ detectable species on conuct with the enzyme. For example the use of naphthyl phosphate in solution as the substrate in an alkaline ,' ,' labelled immunoassay yields the Ll~,_3. . speciesnaphthol under appropriate buffer and substrate conditions. This can be readily oxidised under ' control on the porous electrode to yield one electron for every molecule of naphthol produced. Similarly 1,' ,I phosphate can be used as a substrate with alkaline ul,u~ul,clL~l:u:~ Other examples of enzyme substrate pairs that could be employed are ~1-u ~ 9 with p . , ' jl ~ D-galactoside to produce the ~ ,_I..,a.,t;.r, species , ' "~1, horseradish peroxidase with glucûse to produce the L~ ' . e species hydrogen peroxide and lactate ~' ' ,d~._ with lactate in the presence of NAD~ to produce the ;.e species NADH.
The electrode is either ;.,~.u-~ : ' in a suitable flow cell or device which allows the proper r~ control of the _Iu_L~ud~ Ik~ukl ell. .UIlll~ by the use of a three electrode system comprising a working Idetection) e~ectrode, counter and reference electrodes or in a device which requires only the presence of two electrodes, namely a working and a counter electrode to properly function with an applied potential between the electrodes. The Ll~._l.l ' ' ' detection can be carried out by means of peak current or total charge measurement and this can be calibrated against analyte cull~e..L,~.iu-, for each panicular parameter subjected to L'~_LIU~ l '' "''..J-`7-~ in the system.
The second aspect of this invention is to incorporate the said porous electrode and the solid phase in a complete device to perform an ;~ u.~ .a~, in sequential flow steps or continuous flow, with high surface area and enhanced diffusion kinetics for both the antibody-antigen interactions on the solid phase and for the Ll~._l.U.,l._.l.;....l detection phase, creating a device which is practicable and economic to fabricate requiring less su~,l, ' electronic control than either fluorescence or lu~ _el~,e detection and providing very fast, sensitive diagnostic capabilities. The solid phase can be any suitable protein-binding material which ' .
high surface area to volume ratios and good porosity such as polymer membranes, polymer beads or panicle ~-Icr~n~innc Since the solid phase is preferably located close to the porous electrode and included in a complete , device the preferred solid phase is a polymer membrano.
A fuller description of possible configurations of the invention is now given and illustrated by the way of diagrams.
SUBSTITUTE SHEET (P,ULE 26~

' ~W095/3~725 ~,~ 8999 4 r~ /Y
- 5 - ~ , List of diagrams Fiuure 1 shows a schematic cross section of a flow-through cell design i.lr,Ul~uula~ g a porous membrane to act as the solid phase for ~ and porous working and counter electrodes for -u"I, ' detection.
Figure Z shows a schematic cross section of a flow-through cell with wicking action.
Fiuure 3 is a diagram showinu the results of the assay described in Example 3.
Figuro 4 is a diagram showinu the results of the assay described in Example 4.
Description of the device Figure 1 illustrates a cross section through one possible ~on';D~ ;nn for a flow-through device or cell according to this invention for attachment to a pumped system for liquid flow control allowinvo the passage of the sample, reauem and wash solutions in a suitable sequence of steps through the cell to the final vlv_LII ' ' detection step. The cell body 1 can be of any suitable pre-formed or machined chemically inert material but is preferably formed from a polymeric material and is illustrated in this case as being formed from two separable halves to allow the ~vu~ vu~ , of the device to be replaced as necessary. All reauents enter the cell through the circular aperture 2 and are pulled through in the direction of the arrow 3 by a pump 14 attached below the cell with suitable tubing 15. The cell in this case is configured with a three electrode alla.,_ for full _ control where the porous electrode 5 is the workinu electrode, the porous electrode ~ is the counter electrode and a solid Ag/AgCI reference electrode 7 is exposed to contact with the reagent flow at the inner wall of the cell. The porous electrodes are fixed in position and sealed auainst the cell body by the aid of gaskets 10, 11 and 12 and 'o'-ring 13 as is the porous membrane 4 which provides the solid phase for the immunoassay reactions. The membrane 4 can be any suitable membrane for protein ' but would preferably be made of one of the following materials; -;~" " ' poly~ ' difluoride ~PVDF), nylon or ulass fibres. Pore diameter and thickness of the - membrane can be tailored to the ~ e.. ~ of the particular ;~--- u~o~ y and required fluidic behaviour. The membrane 4 would be prepared according to the usual aspects of il~l...ullua:~vay known to those skilled in the art with capture antibody or antigen and inserted into the cell. The inner bore diameter, d, of the cell can be varied but preferred dimensions are in the range 2 -SUBSTITUTE SHEET ~P,UL' 2b) WO 9~il3172~ r~
1. ;` '.~ : ~ 6; ~
10 mm to limit the sample Yolume required for propcr detection of product. The thickness of the working elecuode 5 should be dictated by the : , range of the product to be detected to allow high oxidation or reduction efficiency and therefore high sensitivity or by the need to svoid rapid fouling of the electrode. Fouling describes the, ' whereby the oxidation or reduction of some .' uc~.;;.L species can create substances which are insoluble and which will gradually deposit on the electrode, blocking the surface snd reducing its cfficiency.
Enhancing the available surface area of the electrode by selecting thicker porous electrodes increases the amount of time the electrode can be used before it must be replaced or the -~Lc.-l-a~;U~- of species that can be detected before significant fouling occurs. Electrical connections are made through the cell to the electrodes and are connected externally to a potentiostat or other electroanalytical device with via contacts 7, a and 9.
The cell then forms the complete immunoassay .,.. ~ :., for competitive or two-site u~ l.Jllua:.~.ay as illustrated by Examples 3 and 4. The device ~u.,, ' ' in Figure 1 could analyse plasma, serum or another fluid suitable for analysis by I J.
The concept of the ,' .. L'~._LI~ ' ' ' Inn~ y device can also be .' . ' in a device adapted for wicking of solutions by capillary action in order that no pumping of fluids throuoh the device need uke place. The concept diagram of this type of device derived from the basic principles of the invention is illustrated by a cross section view thouoh the device in Figure 2. Sample and reaoents in the sequence required for a two-site or competitlve ~ua~:~ay according to the parameter being tested enter the device at the aperture 14 and wick vertically throu,oh the solid phase membrane 15, spacer membrane 16a, porous working electrode 21, spacor membrane 16b, porous /lerc-c-,,~c electrode 22 and into absorbent material 19.
Contacts are made to the working and counter electrodes via conducting materials 17 and 18 which can be suitably connected to a potentiostat or other ~_LIl '~L;I,dl device. The porous counter Ireference eiectrode 22 is of a stable material but preferably of Ag/AgCI ink printed on a suitable porous support. The final rcagent to bo passed through the device is the substrate solution for the en2yme label which is instantly converted to Ll~ ua~Li~_ product which is in turn detected by oxidation or reduction at the working electrode. The device is llnr~r~ll' ' in container 20 and is disposed of after one sample measurement. Plasma, serum or other fluids which can be analysed by ;,. ~ _y can be used on this device. In addition the device could be adapted for whole blood analysis by the addition of a blood/plasma separation membrane at or adjacent to the test sample entrance point.
Devices can be realised from the conceptual drawings of Figures 1 and 2 by many different routes of fabrication and preparation. The porous electrode may be il)cul~uu.a~d in the device SUBSTITUTE SHEET (RULE 2d~

WO 95/31?25 2 1 8 9 9 9 4 PCTIEP95101779 directly as a prc-formed material or may be printed using a porous ink onto a suitable porous surface. The device may be constructed with a working, reference and counUr electrode or simply as a two electrode system accordin~ to the demands of the analytical , ' of the device. To reduce generation of waste materials the device, of a type described in concept in Fi~ure 1 could be constructed in two separable halves, one to contain the membrane or other ' ~yl...,li~_., capture phase and the other to contain the detection electrode system.
The electrode half of the device would be re-usable for a ~ . ;' ' ' number of tests and only that part of the device containing the solid phase would have to be removed and replaced for every new sample tested.

SUBSTITIJTE SHEET !RU~ 20 WO 9!i/3172~i 218 9 9 9 l PCTIEP95/~11779 Examplcs ExDmplc 1 disc of porous oraphite, diameter 5 mm, thickness 0.35 mm, was cut from a sheet of available material IToray Industries, Japan).
As a first test it was placed in a l,u...~ lal planar elecuode cell where the only liquid contact with the disc was at the upper surface of the graphite as in nonmal solid electrodes for biosensors. The cell for "~ ..c contained an Ag/AgCI reference electrode and a titanium counter elecuode and could be stirred to provide diffusion layer control in the fashion. This provided a model for the . .l of :' ~,a~;;.r, species from ~ IJI,llcll~;~,al j on normal solid phase electrodes. The measurement of 10~mol/1 naphthol solutions in dk.lllallula~ c DEA buffer (DEA 50 mmol/l, 100 mmol/l NaCI, pH 9.6~ with the working electrode poised at a voltage of + 300 mV against the reference electrode oave an average peak current response of 100 nA.
In a second test another disc electrode was cut from the graphite sheet and placed in a flow cell of the type illustrated in Figure 1. The contact diameter of the electrode with the liquid was 3 mm. Samples of naphthol, at various cu,.~c., ,:. in DEA buffer,were flowed through the cell at 300 ~I/min with the electrode poised at +300 mV against the Ag/AgCI reference and the resulting current peaks for each 300 ~l sample volume were recorded. Peak currents of -100 nA were recorded for naphthol cu,, of 100 nmol/l makinq the porous electrode 100 times more sensitive than the ,.u....: ' solid electrode under these voltage and flow conditions. This sensitivity is achieved because the ~",r~ :", of the cell allows the total oxidation or reduction of the e~ t~Jal.;i._ species as it passes through the electrode at appropriate flow rate and applied potential. The peak current response can be increased by increasing the velocity of '' .. ~l~uuul~ the cell.
In this mannbr it is possible to distinguish naphthol l.ull~cllLla~iull~ to at least 0.5 nmol/l a~ainst background currents provided by the carner solutions (biological buffers~. The limits to this are set in practico by the fluidics of the system. In particular, velocities which are high enough to generate air bubbles within an open system should be avoided.
Example 2 A Cu.l._.lL;ullal ELISA sandwich assay was used to compare the standard 1.1lll _ assay SUBSTITUTE SHEET ~'UL~ 2B) ~w095~ 1 8 9 9 9 4 with an . _ flow detection system in an assay to determine levels of thyroid stimulatin~ hormone ~TSH) in serum standard solutions.
The assay was performed on a 96 well microtitre plate coated with a capture antibody for TSH.
TSH standard solutions, 100 Ill/well, of different cuncc~ were incubated for 1 hour on the plate, the wells were then washed and a solution of the anti-TSH secondary antlbody. 100 ~I/well, labelled with alkaline ~ (ALP), was incubated for 1 hour on the plate. The plate was again washed with buffer solution to remove any unbound secondary antibody.
Half of the wclls in the microplate were then exposed to a 2.8 mmol/l solution of a .
substrate for ALP, p-nitro-phenyl phosphate, 300 ~I/well. The other half were exposed to one of the _ . substrates for ALP, naphthyl phosphate at a Cull~el~ iull of tmmol/l.
300 ~llwell. After an incubation of 20 min at 37C the solutions in the wells were either measured for colour uk.:. , in a plate reader 1~ substratel or extracted from the wells and measured I, . - I~ by the flow method described in Exampie 1 u~ll substrate~. The normal "I". ~ assay typically could not measure reliably below levels of TSH of 1 mU/I while the ~ ,u~,ll flow detection method continued to measure to 0.01 mU/I.
Example 3 A competitive immunoassay was prepared for thyroxin (T4~ in a flow-throuGh system.
A T4-alkaline ~ T4-ALP~ conjugate was prepared from L-Thyroxin sodium salt and linked to alkaline ul~u:~ull~lL~-~e ~Bio~yme~ usin~ di-C~ ~I suberate ~DSS~.
The anti-T4 antibody was loaded at 2 ~g/ml in carbonate buffer pH 9.6 on to 0.45 ~rn ' uu~.lI.Jlo ~e membrane ~Schleicher and Schuell~ using an ELIFA membrane assay system ~Pierce). The membrane was blocked with blocking solution.
Disks of membrane ~6 mm diameter~ were cut from the prepared mombrane and loaded into a flow cell of the type illustrated in Figure 1. A new piece of membrane was used for every sample tested. For the antigen incubation step 1 ~/glml T4-ALP conjugatc in Tris buffered saline, pH 8.6 was mixed with T4 standard solutions in a 1: 5 ratio to provide the competitive assay at a total sample volume of 200 ~1. The assay steps in the cell which were carried out . :~ without interruption were as follows:

WO 95/3172~ 8 g 9 9 ~ , 19 (i) Passage of 1 ml of buffer solution through the cell.
(ii) Passa~e o~ the 200 ~JI of , ' '~ 1~ mix, liii) Passa~e of 1 ml of buffer throu~h CDIl as a wash step.
Iiv) Passage of 300 11l of substrate solution ~1 mmol/l naphthyl phosphate in buffer).
The :' J..~.t;..l product of this incubation, naphthol, was detected directly at the electrode in She cell IToray, 3 mm diameter in contact with fluid) which was poised at +300 mV vs the A~/A~CI reference electrode. The results, in agreement with the form obtained from classical competitive assay are shown in Figure 3. The total assay time was 27 minutes at room ...
Examplo 4 A sandwich ~or two-sitel ~ y for TSH was performed in continuous flow in the following manner:
t: u~,~l ulus~ membrane ~Millipore1 was loaded with a primary antibody against TSH at a ~u~ iull of 10 ~g/ml in carbonate buffer using the ELIFA system ~Pierce). The membrane was blocked using blocking solution.
Discs of 6 mm diameter were cut from the membrane and placed in the flow cell of the "_ shown in Fi~ure 1. Human serum standards containing TSH were passed through the membranes at a sample volume of 300 ~1, a new membrane and electrode were employed for each . 1~ : .. For the assay a sample was flowed through the system and immediately followed by a secondary ~...il,od, _" ' ul~.e~ul~ conjugate in solution and then 1.5 ml of wash solution. Finally, 300 ~1 of 1 mmol/l naphthyl phosphate solution in DEA buffer was passed through the device. The cell was polarised only from the wash step onwards to reduce the risk of fouling the electrode with oxidation or reduction products from the serum sample.
Naphthol was detected immediately at the porous electrode by its oxidation current with the electrode poised at +300 mV vs a Ag/AgCI reference electrode. Total incubation time for the assay from sample addition to results was 29 minutes at room temperature. Results are shown in Flgure 4 and conform to the expected response from a sandwich or two-site i, ~ y.
SUBSTITUTE SHEET (RULE 2~)

Claims (7)

Claims

1. A flow-through solid phase immunoassay system or device with electrochemical detection capabilities, characterised in that the solid phase is in close connection with a porous working electrode (5, 21) and a suitable counter electrode (6, 22) is present in the system.

2. A flow-through solid phase immunoassay system or device with electrochemical detection capabilities according to claim 1, characterised in that the solid phase is at least one porous membrane (4, 15) in close connection with the porous working electrode (5, 21).

3. A flow-through solid phase immunoassay system or device with electrochemical detection capabilities according to claim 1, characterised in that a flow control system is present.

4. A flow-through solid phase immunoassay system or device with electrochemical detection capabilities according to claim 3, characterised in that the flow control system is a pump producing a pressure difference.

5. A flow-through solid phase immunoassay system or device with electrochemical detection capabilities according to claim 3, characterised in that the flow control system is represented by a wicking material (19).

6. A flow-through solid phase immunoassay system or device with electrochemical detection capabilities according to claim 1, characterised in that a reference electrode (7, 22) is also present.

7. A flow-through solid phase immunoassay system or device with electrochemical detection capabilities according to claim 1, characterised in that the porous working electrode (5, 21) is a graphite or carbon paper electrode or a printed electrode on porous material.