C o m m u n i c a t i o nAn enzyme flow immunoassay using alkaline phosphatase asthe label and a tyrosinase biosensor as the label detector

Catalin Nistor and Jenny Emnéus*

Department of Analytical Chemistry, Lund University, P. O. Box 124, S-221 00 Lund,Sweden. E-mail: jenny.emneus@analykem.lu.se; Fax: +46-46-2224820 or 2220104

Received 13th October 1998, Accepted 13th November 1998

The present work demonstrates the possibility of employinga tyrosinase biosensor as a label detector for a continuousheterogeneous flow immunoassay. The tyrosinase biosensormonitors the phenolic product liberated by an alkalinephosphatase label in a non-competitive flow immunoassayfor digoxin.

Introduction

Biological affinity interactions (BAIs) such as antigen–anti-body, receptor–ligand, and enzyme–substrate reactions havefound valuable use as analytical tools for the determination oftrace levels of analytes in complex biological matrices, e.g., inserum and blood samples and in environmental samples such assurface and waste water.127 In particular, immunoassays havefound widespread use due to the increasing availability ofantibodies against small haptens. The detection is performed viaa specific marker molecule such as a radioactive, fluorescent orenzymatic label. Immunoassays have intrinsically a highselectivity as well as a good sensitivity and limit of detection,which is determined primarily by the affinity properties of theantibody, but also by the mode of label detection.

Immunoassays performed in the flow mode have attractedgreat interest in recent years8–12 and offer some advantages overconventional microtiter plate immunoassays with regards tospeed, precision, and the potential for simple and cost effectiveautomation. The detection of the label is most commonlyperformed by colourimetric, fluorescence or electrochemicaldetection. Recently, a highly sensitive electrochemical micro-titer plate ELISA was developed for 2,4-dichlorophenoxyaceticacid13 using a biosensor based on glucose dehydrogenase co-immobilised with tyrosinase onto the surface of a Clark sensor(GDH–TYR–Clark) for the detection of the phenol liberated byan alkaline phosphatase (AP) label. The same group developeda cocaine flow immunoassay with an AP label which wasmonitored off-line by the GDH–TYR–Clark biosensor.14

The present work demonstrates the possibility of employinga tyrosinase biosensor as a label detector in a continuous flowimmunoassay. The tyrosinase sensor monitors the phenolproduct liberated by an alkaline phosphatase label in a non-competitive flow immunoassay for the determination of themodel compound digoxin. The system is based on theseparation of bound and free label by an immunoaffinitysupport with immobilised digoxin.11,15 The detected amount ofphenol was found to be proportional to the analyte concentra-tion in the sample.

Materials and methods

Chemicals

Stock solutions (0.1 mM) of digoxin from Sigma (St. Louis,USA) were prepared by first dissolving the powder in 800 mL of

dimethyl sulfoxide (DMSO) (Merck, Darmstadt, Germany),and then diluting with 99.5% ethanol (Kemetyl, Stockholm,Sweden) to a final volume of 100 mL. Polyclonal anti-digoxigenin Fab-fragments labelled with alkaline phosphatase(Fab-AP, 150 U per 0.2 mL) were obtained from Boehringer-Mannheim (Mannheim, Germany). Glycine (Sigma), tris(hy-droxymethyl)aminomethane (TRIS, Merck), Tween 20(Sigma), magnesium chloride (Merck), sodium chloride(Merck), zinc chloride (Merck), disodiumphenylphosphate(Merck) and sodium dihydrogen phosphate (Merck) were usedto prepare different buffers in water. All buffer solutions wereprepared using water purified with a Milli-Q (Millipore,Bedford, USA) system.

Lyophilised mushroom tyrosinase (E.C. 1.14.18.1) powderwas purchased from Sigma (LOT 24H9524, 4400 U mg21),One unit of tyrosinase will cause a decrease in A280nm of 0.001per min at pH 6.5 and 25 °C in a 3 ml reaction mixturecontaining l-tyrosine. Eastman AQ Polymer 29 D, a 30% (w/v)water dispersion, was purchased from Eastman Kodak (King-sport, TN, USA ). Graphite powder and paraffin oil werepurchased from Fluka (Fluka Chemie AG, Buchs, Switzer-land).

Label detecting biosensor

Unmodified carbon paste (CP) was prepared as follows: 1 g ofgraphite powder and 400 mL of paraffin oil were mortered for 20min. The unmodified CP was packed into 1 ml plastic syringes(ONCE, ASIK, Denmark). To obtain electrical contact, a silverwire was inserted around the tip of the piston, which waspressed into the syringe containing unmodified CP. Theexposed geometric surface area of the electrodes was 0.051 cm2.The biosensor was then prepared according as follows: a stocksolution of tyrosinase–Eastman AQ mixture was prepared bymixing tyrosinase powder directly with a 1.0 % Eastman AQsolution prepared in 0.1 M phosphate buffer at pH 6.5, giving afinal tyrosinase concentration of 38500 U ml21 (declaredactivity) in the immobilisation mixtures. 15 ml of this enzyme–polymer mixture was then applied on top of the syringeelectrode prepared above and, after drying, the modifiedelectrode was kept for 20 h at 4 °C before use.

The biosensor was fitted into a Teflon holder and insertedinto a flow-through wall jet amperometric cell. The tyrosinasebiosensor was used as the working electrode, an Ag/AgCl (0.1M KCl) electrode as the reference electrode, and a platinumwire served as the auxiliary electrode. The electrodes wereconnected to a three electrode potentiostat (Zäta Elektronik,Lund, Sweden) and the current was recorded on a printer (Kipp& Zonen, The Netherlands, mod. BD111). All measurementswere performed at an applied potential of 250 mV vs. Ag/AgCl.

Non-competitive flow immunoassay

The digoxin immunoaffinity support was prepared by im-mobilising digoxin to Poros-NH support (PerSeptive Bio-

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systems, USA) according to a previously published proce-dure.11 The immobilised digoxin was then packed into a 10 30.2 mm, volume 31.4 mm3 column and connected as describedin the flow immunoassay system presented in Fig. 1. Digoxinwas first pre-incubated with excess of Fab-AP for 10 min andthen injected via a six port injection valve (Rheodyne model7010, LabPRO, CA, USA) with a 20 mL injection loop into aTRIS-buffered saline (TBS) carrier, containing 25 mM tris(hydroxymethyl)aminomethane, 0.15 M NaCl and 1 mMMgCl2 at pH 7.5. The effluent from the affinity column was thenmixed on-line with the substrate solution for Fab-AP containing0.5 mM phenylphosphate in 0.1 M glycine and 1 mM MgCl2 atpH 10.4. Before reaching the label detecting biosensor, the pHof the substrate solution was adjusted on-line to pH 6.5 by 0.1M NaH2PO4. All carriers contained 0.025% Tween 20 toprevent non-specific binding of Fab fragments to the affinitycolumn or to the connecting tubing in the system. All threecarriers were pumped with a four channel peristaltic pump(Alitea, Sweden) at a flow rate of 0.25 ml min21, resulting in afinal flow of 0.75 ml min21 through the label detectingbiosensor. The different flows were connected using mixingT’s, followed by knitted mixing coils of Teflon tubing. Allcarriers were degassed and filtered through a 0.45 mm filter(Millex HA, Millipore) before use.

Results and discussion

The analyte digoxin is incubated off-line for 10 min with excessof alkaline phosphatase (AP) labelled Fab-fragments (Fab–AP).20 ml of this mixture is then injected into the flow immunoassaydepicted in Fig. 1 and the excess non-bound Fab–AP is trappedinside the immobilised digoxin column. The Fab–AP–digoxincomplex is eluted from the digoxin column and mixed on-linewith the AP substrate phenyl phosphate, leading to phenol as thereaction product. The generated phenol is detected by thetyrosinase biosensor, according to the principle presented inFig. 2.

The reaction catalysed by alkaline phosphatase takes place athigh pH, i.e., glycine buffer at pH 10.4, a pH not compatiblewith the tyrosinase biosensor (pH optimum at around 6.5). Anon-line pH corrector that shifts the pH to 6.5 is thus introducedand the enzymatically generated phenol is monitored down-stream by the tyrosinase biosensor.

Fig. 3 shows a typical response of the system for 20 mLinjections of 15 mU L21 Fab–AP fragments in the absence (A)and in the presence of the digoxin column (B). The residualpeak seen in Fig. 3B is probably due to the presence of free APin the original Fab–AP solution or the presence of inactiveantibodies not able to bind to the digoxin column. The peak inFig. 3B does not decrease with an increase in the incubationtime in the column (decrease in flow rate).11 Fig. 3C shows theresponse for injection of 10 nM digoxin incubated with 15mU L21 Fab–AP. Fig. 4 presents the calibration graph for

digoxin with a limit of detection of about 2.5 nM digoxin,calculated from the slope as three times the signal to noiseratio.

Conclusions and further directions

The integration of biosensors as label detectors in flow-basedimmunoassays is a new approach, which combines the high

Fig. 1 The flow immunoassay system. Injection volume 20 mL. Buffer 1:TRIS-buffered saline (TBS) carrier, containing 25 mM tris(hydroxy-methyl)aminomethane, 0.15 M NaCl and 1 mM MgCl2 at pH 7.5. APsubstrate buffer: 0.5 mM phenylphosphate in 0.1 M glycine also containing1 mM MgCl2 at pH 10.4. pH-corrector: 0.1 M NaH2PO4. Each flow waspumped at 0.25 ml min21.

Fig. 2 Detection principle of a tyrosinase-modified electrode. Thephenolic substrate is converted by the enzyme via catechol to o-quinone thatis reduced on the electrode surface back to catechol, leading to anamplification cycle.

Fig. 3 Typical response for the flow immunoassay system using atyrosinase biosensor as the label detector. Injections of 15 mU L21 Fab–AP(A) in the absence and (B) in the presence of the digoxin column. (C) 10 nMdigoxin incubated with 15 mU L21 Fab–AP in the presence of the digoxincolumn. All incubation times = 10 min. Conditions as in Fig. 1.

Fig. 4 Calibration plot for digoxin. Different concentrations of digoxinincubated with 15 mU L21 Fab–AP in the presence of the digoxin column.Conditions as in Fig. 1.

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selectivity of the antigen–antibody reactions with the goodsensitivity and short response time of enzyme-based biosensors.The present work demonstrates that a tyrosinase biosensor canbe used as a label detector in a continuous flow immunoassayfor the detection of the phenol product generated by the enzymelabel AP. The advantage is that the actual phenol measurementis performed at –50 mV vs. Ag/AgCl, i.e., the optimal potentialrange for electrochemical measurements where noise andbackground currents due to interferences in real samples arelow. The system is at present not optimised with regards to theflow rates, substrate concentration or the length of the reactioncoil used for incubation of the substrate with the label. Suchoptimisations are currently performed and will improve theoverall sensitivity, detection limit and sample throughput of thesystem. The use of a suppresser column–membrane for pHcorrection will also make possible the elimination of the secondmake-up flow and thus most likely lead to improvements of thesystem performance. Currently several different biosensorsbased on different enzymes, e.g., tyrosinase, NADH independ-ent dehydrogenases (glucose dehydrogenase, cellobiose dehy-drogenase, oligosaccharaide dehydrogenase) are tested for theiruse as detectors for monitoring different phenolic products fromenzyme labels such as alkaline phosphatase and b-galactosidasein flow immunoassays.

Acknowledgements

The authors kindly acknowledge financial support from theEuropean Community, EC Contracts No. ENV4-CT97-0476and No. S-JEP-09227-95.

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