GB2337332A

GB2337332A - Affinity electrode for electrochemical analysis

Info

Publication number: GB2337332A
Application number: GB9810103A
Authority: GB
Inventors: Silke Kroeger; Anthony Peter Francis Turner; Karsten Olaf Haupt; Klaus Mosbach
Original assignee: Cranfield University
Current assignee: Cranfield University
Priority date: 1998-05-13
Filing date: 1998-05-13
Publication date: 1999-11-17
Anticipated expiration: 2018-05-13
Also published as: GB2337332B; GB2337332A8; GB9810103D0

Abstract

The electrode, has a surface modified with a synthetic polymer which is functionalised to enable the polymer to specifically recognise, bind and concentrate, in close proximity to the electrode surface, an analyte or group of analytes. Functional groups in the polymer can be randomly distributed, or the polymer can be molecularly imprinted. The polymer can be attached to the electrode through various methods such as adsorption, entrapment within a membrane or macroporous polymer or direct polymerisation at the electrode surface. The electrodes may be screen-printed. The polymer concentrates analyte from the sample solution at the electrode increasing its concentration and lowering the detection limit of the analyte. Either the bound analyte itself or an electrochemically active derivative thereof preincubated with the electrode or added to the sample (competition/displacement assay) is quantified electrochemically (for example by differential pulse or square wave voltammetry) directly at the electrode surface. The device can be used in the detection of analytes of medical, environmental or nutritional relevance, detection of substituted phenols, morphine and phenoxyacid herbicides being specified. The device may be used in harsh environments such as organic solvents and may be disposable.

Description

1 r 1 Title: DISPOSABLE AFFINITY SENSOR

Background of the invention

2337332 In recent years, the number of samples that have to be analysed for medical or environmental reasons has increased manifold. Conventional analytical methods are time consuming and expensive and therefore alternative techniques, particularly those suitable for decentralised analysis, are required. Biosensors have proven very useful in this context. In this group, electrochemical sensors based on disposable screen-printed electrodes have the advantage of being versatile, low-cost and easily mass-produced [1,2]. Screen-printed electrodes in combination with enzymes as biological detection element have found application in numerous amperometric biosensors [3-11] and form the basis for some very successful commercial biosensors such as the ExacTech glucose pen for diabetes monitoring. Immunosensors based on the same style of electrodes but utilising the affinity interaction between antigen and antibody in theii detection step are also being investigated [12-13], particularly in the light of the increasing acceptance of immunochemical methods.

The three main problem areas for analysis of medical and environmental samples, especially when dealing with complex sample matrices such as blood, serum, natural water or soil, are selectivity, detection limit and robustness of the sensing element. In order to lower the detection limit and reduce the number of interferences, analytical methods are often combined with solid phase extraction (SPE) [141. In SPE the sample is passed through a column filled with adsorptive resins containing functional groups to which the analyte binds. After washing of the SPE matrix to remove interferences, the analyte is eIuted with a suitable solvent. This eluate can either be analysed directly or treated further before analysis (for example dilution, evaporation of the solvent). While the use of SPE allows pre-concentration and purification of the analyte, it is a process involving a number of different sample preparation steps and solvents. Furthermore, it generally does not yield the analyte in a solution that is very compatible with biosensor analysis (high concentration of organic solvents, often non-physiological pH).

To facilitate measurements under these conditions, solvent-resistant screen-printed electrodes have been developed [151. These electrodes formed the basis for some enzymatic studies [16,171 and have been employed as transducers in the development of an immunological method for the detection of 2,4-D [ 18]. The natural recognition elements utilised in these studies limit the applicability of the devices in terms of stability and robustness.

Molecular imprinting is increasingly becoming recognised as a technique for the preparation of synthetic polymers containing tailor-made recognition sites for certain target molecules [ 19]. This is achieved by co-polymerising functional and cross-linking monomers in presence of the target analyte acting as a molecular template. After elution of the template, complementary binding sites are obtained that allow rebinding of the template with a sometimes very high specificity comparable to that of antibodies [20, 211. The so obtained artificial receptors have been used in different applications that require specific ligand binding, such as separation of closely related compounds [22] and immunoassay-type binding assays [20, 231. Another publication has been as recognition element in sensors of different formats [24-26]. Molecularly imprinted polymers have been produced that specifically recognise herbicides e.g. atrazine [27] and 2,4-D [28], drugs e.g. theophylline [201 or the beta- blocker propranolol [2-')], hormones [29, 211 and many other compounds including proteins '(19]. Polymers can be imprinted with substances for which natural receptors do not exist or.are difficult to obtain. Moreover, imprinted polymers can be used in organic solvents and at high temperatures, and because of their great chemical and mechanical stability they retain theirmolecular memory over long time periods. They therefore have advantages over biomolecules as recognition elements in many sensor applications.

1 Description:

The invention consists in disposable screen-printed electrodes derivatised with synthetic polymers capable of binding target analytes for the production of electrochemical affinity sensors. The polymers can be a) polymers containing randomly distributed functional groups, or b) polymers in which the functional groups have been pre-arranged by molecular imprinting to form specific binding sites for the selected analyte.

The functional polymer can for example be attached to the working electrode surface by physical adsorption of small polymer particles, direct polymerisation at the electrode surface, covalent linkage of a thin polymer layer to the electrode surface, entrapment of functional polymer particles in a support polymer covalently or non-covalently linked to the electrode surface, or entrapment at the electrode surface through a membrane. Non-limiting examples of techniques that can be employed to directly deposit the support and/or the functional polymer at the electrode surface are dip-coating, spray-coating, spin-coating, screen-printing and jetprinting.

Affinity sensors produced in this way can serve as combined analyte enrichment and detection systems. The working principle is an in situ solid phase extraction with subsequent electrochemical detection of the adsorbed analyte at the electrode surface. Advantages are the decreased detection limit and a more selective electrochemical detection of the target analyte in complex samples. Analytes that are not electroactive can be measured in a competitive format using a derivative of the analyte that carries an electrochernical label, or a non-related electroactive probe that can bind to the binding sites in the polymer. The advantages of such sensor systems are the reduction in sample handling steps and thus analysis time, reduced solvent consumption, and compared to immunosensors, the superior robustness of the artificial receptor element combined with low production cost.

Target analytes for detection are substances of interest in environmental and food analysis, process monitoring, biomedical analysis and in other areas.

Example 1

Detection of substituted phenols. A linear poly-4-vinylpyridine-polymer was prepared by radically polymerising 4-vinylpyridine (4-VP) with heat initiation using 2,2'-azobis(2,4-dimethyl valeronitrile) as initiator, in the presence of excess methanol resulting in a highly viscous solution. This solution was flirther diluted with methanol and aliquots of 2-4 gI were pipetted onto the working electrode surface of the screen-printed electrodes produced as outlined in Ref. 15 (Figure 1). The methanol was left to evaporate and the remaining 4-VP formed a smooth, porous film covering the carbon electrode (Fi IgUe 2a). Electrodes modified in this way were incubated in solutions containing substituted phenols such as 2,4-dichlorophenol. The phenols were attracted to the electrode surface were they bound to the polymer layer through interaction with the pyridine groups. After this process had resulted in an enrichment of the phenolic substances from the sample at the electrode surface, they were detected directly using different pulse voltammetry (dpv). The bound phenols were hereby oxidised during a single potential scan and the height or surface area of the resulting oxidation-peak could be correlated to the analyte concentration. Figure 3 shows a dpv scan obtained with 4-VP modified electrodes when detecting 2, 4-dichlorophenol. Such electrodes could be used either in "dip-stick-mode " to accumulate analytes from quiescent or stirred solutions for single- point measurements or in G'continuos-flow mode" inserted into an analyte stream (for example wastewater) were repeated 3 scans at discrete intervals would allow semi-continous monitoring. The short time required for a dpv scan (approximately 12 s) facilitates high data density if required. The lifetime of the disposable affinity sensor would depend on the analyte concentration and the composition of the polymer film.

Example 2

Detection of morphine. Morphine can be detected electrochemically by dpv on screen-printed carbon electrodes. Figure 4 shows dpv scans of a buffer solution containing increasing morphine concentrations (0-1 mg/1). To selectively enrich morphine at the electrode surface, a molecularly imprinted polymer specific for morphine was used as adsorbent. The morphine-molecularly imprinted polymer is a co-polymer of divinylbenzene and methacrylic acid. It was prepared by dissolving 20 mmol of divinylbenzene, 4 mmol of methacrylic acid, 0.5 mmol. of morphine and 0.3 mmol of 2,2'-azobis-isobutyronitrile (polymerisation initiator) in chloroform. Polymerisation was initiated by UV-irradiation at 366 nm and allowed to proceed for 6 h at 4 C. The polymer was ground to a particle size of Igm and the template eluted by incubation with methanolacetic acid (7:3) followed by methanol.

The particles were then mixed with a diluted solution (10' mg/ml) of poly(methylmethacrylate) in methanol at a particle concentration of 100 mg/ml. 5 gI of the suspension was deposited and evenly distributed on the working electrode surface of a screenprinted solvent-resistant carbon electrode (produced as outlined in Ref. 15) and allowed to dry. The poly(methylmethacrylate) acting as a binder immobilised the polymer particles at the electrode surface (Figure 2b). Alternatively, polymer particles were suspended only in methanol (100 mglml) and deposited onto the working electrode jurface. Subsequently the particles were covered with a round piece of a porous PVD17 membrane with a diameter 3 mm larger than the working electrode, which was glued to the electrode substrate (Eligure 2c).

Incubation of the electrode in a stirred solution of morphine (10 ppb) resulted in an accumulation of morphine close to the electrode surface by binding to the imprinted polymer, increasing its concentration to above the detection limit, and thus allowing subsequent quantification of the morphine by dpv.

Example 3

Detection of phenoxyacid herbicides. A molecularly imprinted polymer specific for 2,4dichlorophenoxyacetic acid (2,4-D) was used as adsorbent. The 2,4-D-molecularly imprinted polymer is a co-polymer of ethyleneglycol dimethacrylat ' e. and 4-vinylpyridine, and was prepared and processed as outlined in Ref. 28. In that way, polymer particles with a diameter of =1 gm were obtained. Solvent-resistant screen-printed carbon electrodes prepared as outlined in Ref. 15. Since 2,4-D is not electroactive, a competitive system was used based on homogentisic acid (HGA) as a nonrelated electroactive probe that can bind to the imprinted polymer. HGA can be measured by differential pulse voltammetry on screen-printed carbon electrodes. Figure 5 shows typical scans of HGA in 20 % ethanol/buffer using unmodified electrodes. To modify the electrodes with imprinted polymer, they were coveredwith adhesive masks leaving only the working electrode surface free. The electrodes were then submerged in a stirred suspension of the 2,4-D-molecularly imprinted polymer in 20 mM phosphate buffer pH 6. After 30 min the surfaces of the working electrodes were covered with a thin layer of physically adsorbed polymer particles (Figure 2d). The electrodes were rinsed once with the same buffer and then the adhesive masks were removed. Into a series of test tubes was added 2,4-D at varying concentration and 4 HGA at a constant concentration of 6 iM. One electrode was placed in each test tube so that the working electrode surface was submerged in the solution. After 5 min incubation under stirring, the amount of homogentisic acid bound to the electrode was determined by differential pulse voltammetry in 20 mM phosphate buffer pH 6 containing 0. 1 M KCl. The current peak obtained after incubation of the polymer-modified electrode in a solution of 100 iM 2,4-D was smaller than after incubation in pure buffer, which proves that some of the HGA had been displaced from the polymer by the analyte 2,4-1).

References 2.

5.

1.

1. Beraveld, P. & Turner, A. P. F. (1993). Fabrication and Mass Production. In: Advances in Biosensors, Supplement 1, pp. 165-186. Edited by A. P. F. Turner. London: JAI Press Ltd. Cardosi, M. F. & Turner, A. P. F. (1990). Recent Advances in Enzyme-Based Electrochemical Glucose Sensors. The Diabetes Annual 5: 254-272.

3. Bilitewski, U., RUger, P., & Schmid, R. D. (199 1). Glucose Biosensors Based on Thick Film Technology. Biosensors & Bioelectronics 6: 369-373. Cagnini, A., Palchetti, L, Lionti, L, Mascini, M., & Turner, A. P. F. (1995). Disposable Ruthenized Screen-Printed Biosensorsfor Pesticides Monitoring. Sensors and Actuators B 24-25: 85-89. Gilmartin, M. A. T. & Hart, J. P. (1994). Fabrication and Characterization of a Screen Printed Disposable, A mperometric Cholesterol Biosensor. Analyst 119: 23 3 1-233 6. Hampp, N., Eppelsheim, C., & Silber, A. (1994). Thick-Film Biosensors. In: Thick Film Sensors, p. 341-356. Edited by M. Prudenziati. London: Elsevier Science. Hart, A. L., Turner, A. P. R, & Hopcroft, D. (1996).- On the Use of Screen- and Ink-Jet Printing to Produce Amperometric Enzyme Electrodesfor Lactate. Biosensors & Bioelectronics 11: 263-270.

8. Newman, J. D., White, S. R, Tothill, I. E., & Turner, A. P. F. (1995). Catalytic Materials, Membranes, and Fabrication Technologies Suitablefor the Construction ofAmperometric Biosensors. Anal. Chern 67: 4594-4599.

9. Schmidt, A., Rohm, L, RUger, P., Weise, W., & Bilitewski, U. (1994). Application ofScreen Printed Electrodes in Biochemical Analysis. Fresenius J. Anal. Chem. 349: 607-612.

10. Wang, L, Chen, Q, Pedrero, M., & Pingarron, J. M. (1995). ScreenPrintedAmperometric Biosensorsfor Glucose and Alcohols based on RutheniumDispersed Carbon Inks. Anal. Chim. Acta 300: 111-116.

11. White, S. R, Turner, A. P. F., Schmid, R. D., Bilitewski, U., & Bradley, J. (1994).

Investigations of Platinized and Rhodinized Carbon Electrodesfor Use in Glucose Sensors.

Electroanalysis 6: 625-63)2.

KalAb, T. & SkIddal, P. (1995). A Disposable Amperometric Immunosensorfor 2,4 Dichlorophenoxyacetic Acid. Analytica Chimica Acta 304: 3 61 -3 68.

SkIddal, P. & Kalfib, T. (1995). A Multichannel Immunochemical Sensorfor Determination of 2,4-Dichlorophenoxyacetic Acid. Anal. Chim. Acta 316: 73-78.

14. Pic6, Y., Molt6, J. C., Mafles, J., & Font, G. (1994). Solid Phase Techniques in the Extraction'of Pesticides and Related Compoundsfrom Foods and Soils. J. Microcol. Sep. 6: 3311-359. - 15. Kr6ger, S. and Turner, A.P.F (1997) Solvent-Resistant Carbon Electrodes Screen Printed onto Plasticfor Use in Biosensors. Anal. Chim. Acta 347: 9-18.

16. Kr6c-,er, S., Setford, S.J. and Turner, A.P.F (1998) Electrochemical Assay Methodfor the Rapid Determination of Oxidase Enzyme Activities. Biotechnology Techniques 12: 123- 127.

17. Krd-er S Setford S.J. and Turner A P F (1998) Assessment ()f oxidase ew e behaviour -,. - ' 9..

in alcoholic solutions using disposable e ctrodes. Anal. Chim. Acta. In press.

18. Krd-er, S., Setford, S.J. and Turner, A.P.F (submitted) An Immunosensorfor 2,4- Dichlorophenoxyacetic Acid in AqueouslOrganic Solvent Soil extracts. Anal. Chem.

19. Mosbach. K. and Ramstrdm, 0. (1996) The emerging technique of molecular imprintina and C> g itsjuture impact on Biotechnology. B io/Technolo a 14: 16 3) - 170..y 20. V1ataki s, G., Andersson, L1, Mtiller, R. and Mosbach, K. (1993)) Drug assay using antibody mimics made by molecular imprinting. Nature 361: 645-647.

21. Ramstr6m, 0., Ye, L. and Mosbach, K. (1996) Artificial antibodies to corticosteroids prepared by molecular imprinting. Chemistry & Biology 3: 471-477.

22. Fischer, L., Miffier, R., Ekberg, B., and Mosbach, K. (199 1) Direct enantioseparation offladrenergic blockers using a chiral stationary phase prepared by molecular imprinting. J. Am. Chem. Soc. 113: 9358-9360.

2 3. Andersson, L.I. (1996) Application of molecular imprinting to the development of aqueous buffer and organic solvent based radioligand binding assaysfor (S)-propanolol. Anal. Chem. 68: 111-117.

24. Hedborg, E, Winquist, F., Lundstr6m, L, Andersson, L.I. and Mosbach, K. (199-3) Some studies of molecularly imprintedpolymer membrane in combination with field-effect devices. Sensors Actuators A 37-38: 796-799.

25. Kriz, D., Ramsti-6m, 0., Svensson, A. and Mosbach, K. (1995) Introducing biomimetic sensors based on molecularly imprintedpolymers as recognition elements. Anal. Chem. 67: 2142-2144.

26. Kriz, D., Kempe, M., Mosbach, K. (1996) Introduction of molecularly imprintedpolymers as recognition elements in conductometric chemical sensors. Sensors Actuators B 33: 178181.

27. Muldoon, M.T. and Stanker, L.H. (1995) Polymer synthesis and characterization ofa molecularly imprinted sorbent assayfor atrazine. J. Agric. Food Chem. 43: 1424-1427.

28. Haupt, K., Dzgoev, A. and Mosbach, K. (1998) Assay system for the herbicide 2,4dichlorophenoxyacetic acid using a molecularly imprinted polymer as the artificial recognition element. Anal. Chem. 70: 628-63 1.

29. Andersson, L1, MUller, R. and Mosbach, K. (1996) Molecular imprinting of the endogenous neuropeptide Leu5-enkephalin and some derivatives thereof. Macromol. Rapid Commun. 17: 657 1.

r^

Claims

Claims

1. An electrode intended for controlled-potential electrochemical analysis, characterised in that the electrode surface is modified with a synthetic polymer functionalised to enable the polymer to specifically recognise, bind and concentrate, in close proximity C1 to the electrode surface, an analyte or group of analytes.

2. An electrode according to claim 1 where the polymer is randomly functionalised.

to claim 2 where the analyte is an organic compound.

An electrode according 4. An electrode accordina to claim 1 characterised in that the polymer contains specific 0 binding sites generated by molecular imprinting for a target analyte.

C -- 5. An electrode according to claim 1 characterised in that the electrode is produced by screen-printing.

6. An electrode according to claim 5 characterised in that the electrode is a screen- C> printed solvent-resistant carbon electrode.

7.

An electrode according to claim 1 characterised in that the polymer is attached to the electrode surface by physical adhesion.

8. An electrode according to claim 7 characterised in that the polymer is in the form of a thin film.

9. An electrode according to claim 7 characterised in that the polymer is in the form of small particles.

10. An electrode according to claim 1 characterised in that the polymer is attached to the electrode surface by covalent linkage.

11. An electrode according to claim 1 characterised in that the polymer is attached to the 1 electrode surface through entrapment of polymer particles in a binder covalently or noncovalently attached to the electrode surface.

12. An electrode according to claim 1 characterised in that the polymer is attached to the z> electrode surface by entrapment at the electrode surface using. a membrane.

An electrode according to claim 1 characterised in that the polymerisation mixture, the polymer particle suspension or the polymer particle suspension containing a 1 1 binder, are deposited onto the electrode surface by dip-coating, spray- coatina. spincoating, screen-printing, ink-jet printing. deposition with for example a pipette or by other means.

-1 14. Application of electrodes according to claims 1 - 1 3), whereby the analyte is detected or quantified in a competitive format, using an electrochemically active derivative of the analyte, or a non-related electrochemic ally active compound, as a probe which can adsorb to the binding sites in the polymer.

15. Application of electrodes according to claims 1, 2, 3), 5, 6, 7, 8, 13), where the analytes are substituted pheno-15.

16. Application of electrodes according to claims 1, 2, 5, 6, 7, 8, 1 where the polymer is poly(vinylpyridine).

17. Application of electrodes according to claims 1, 4, 5, 6, 9, 11, 12, 13), where the analyte is morphine.

18. Application of electrodes according to claims 1, 4, 5, 6, 9, 11, 12, 13), where the polymer is molecularly imprinted with morphine.

19. Application of electrodes according to claims 1, 4, 5, 6, 7, 9, 13), 14, where the analytes are phenoxyacid herbicides.

20. Application of electrodes accordinC, to claims 1, 4, 5, 6, 7, 9, 13, 14, where the polymer is molecularly imprinted with 2,4dichlorophenoxyacetic acid.

21. Application of electrodes according to claims 1, 5, 6, where detection is by ---1 differential pulse voltammetry. square wave voltammetry or linear sweep voltarnmetry.