ELSEVIER A D V A N C E D TECHNOI ( ~ ; Y

Biosensors & Bioelectronics Vol. 11, No. 8, pp. 805--810, 1996 © 1996 Elsevier Science Limited

Printed in Great Britain. All rights reserved 0956--5663/96/$15.00

Electrochemiluminescence detection of glucose oxidase as a model for flow

injection immunoassays

Robert Wilson,* Jens Kremesk6tter & David J. Schiffrin

Department of Chemistry, University of Liverpool, PO Box 147, Liverpool, L69 3BX, UK Tel: [44] (151) 794 3573 Fax: [44] (151) 794 3588

James S. Wilkinson

Optoelectronics Research Centre, University of Southampton, Southampton, SO9 5NH, UK

(Received 10 May 1995; accepted 28 November 1995)

Abstract: Glucose oxidase was covalently attached to aminosilanized indium tin oxide coated glass wafers and these were used as working electrodes in an electrochemiluminescence flow injection analyser as a model for enzyme immunoassays. Light from the chemiluminescent reaction between enzy- matically produced hydrogen peroxide and electrochemically oxidized luminol was detected and the effect of pH and luminol concentration on this reaction was determined. Repetitive assays for glucose were carried out to investigate the surface chemistry. Results suggested that electrochemiluminescence enzyme immunoassays with glucose oxidase as the antibody label could be carried out. © 1996 Elsevier Science Limited

Keywords: electrochemiluminescence, indium tin oxide electrode, flow injec- tion analysis, glucose oxidase

INTRODUCTION

Chemiluminescence is a sensitive technique used to detect a wide range of analytes in commercially available immunoassays. In many of these, hydrogen peroxide is used as an oxidant to detect trace amounts of a label such as horseradish peroxidase (Whitehead et al., 1983), isoluminol (De Boever et al., 1984), or an acfidinium ester (Richardson et a/., 1985). Hydrogen peroxide can be generated

* To whom correspondence should be addressed.

enzymatically by glucose oxidase (Wilson & Turner, 1992) and therefore this enzyme can be detected chemiluminescently (Arakawa et al., 1982), but it is not often used in immunoassays because it has a low turnover compared with commonly used alternatives like horseradish peroxidase, alkaline phosphatase and /3-galactosidase (Porstmann et al., 1985). The aim of work described here was to examine the possibility of using this enzyme in electrochemiluminescence immunoassays, based on the reaction between hydrogen peroxide and electro- chemically oxidized luminol (Haapakka & Kankare, 1982; Kremesk6tter, 1995), that combine the semi-

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R. Wilson et al. Biosensors & Bioelectronics

tivity of chemiluminescence with the spatial organiza- tion of electrochemistry. As a model for this kind of immunoassay glucose oxidase was covalently attached to indium tin oxide (ITO) coated glass wafers that were used as working electrodes in an electrochemiluminescence flow injection analyser. Electrochemiluminescence detection of the enzyme was demonstrated and the effect of pH and luminol concentration on the chemiluminescent reaction was investigated. Repetitive assays for glucose were carried out to show that the surface chemistry was suitable for flow injection immunoassays.

EXPERIMENTAL

Apparatus

1TO glass from Balzer Ltd. (Buckinghamshire, UK) was cut into wafers that measured 36 x 20 × 1 mm thick. The ITO layer was 20 um thick and had a resistance of 200 D,~]. The flow injection analyser pump, six-way valve and injection loop were part of a single unit supplied by Ismatec UK Ltd. (Surrey, UK). Solutions were pumped at a flow rate of 0.475 ml/min through an electrochemical flow cell connected to a potentiostat, both made in-house. Light generated in the flow cell was detected with a type 9558QA photomultiplier tube (PMT) (Thorn EMI, Middlesex, UK) connected to a model 475R power supply (Brandenburg, UK). The entire system was controlled by a 486¢33 PC programmed with Viewdac data acquisition software (Keithley Data Acquisition, Taunton, MA, USA) and fitted with two DAS1602 Data Acquisition Boards (Keithley). All potentials are relative to a SCE.

Reagents and solutions

All solutions were prepared in water from a Still Plus purification unit fitted with a deionizer and carbon filter from Purite Ltd (Oxfordshire, UK). Reagents of the highest purity available were used. Glucose oxidase (GOD) (EC 1.1.3.4) Type X-S, ovalbumin (grade V), 3-(2-pyridyldithio) propionic acid N-hydroxysuccinimide ester (SPDP), glucose (HPLC grade); 4-acetamidophenol, uric acid (99%) and L-ascorbic acid (ACS reagent) (vitamin C) were from Sigma. Luminol was from Fluka Ltd (UK). Glucose solutions were allowed to stand for 48 h before use to allow mutarotafion to take place.

806

Covalent attachment of protehls to ITO ctmted wafers

ITO was aminosilanized as described previously (Wilson & Schiffrin, 1995). GOD (50 mg) was dissolved in 4.35 ml of 0.1 M phosphate buffer, pH 7.5, that contained 0.1 M NaC1, and to this was added (dropwise with stirring) 0.65 ml of 12 mg/ml SPDP solution in ethanol. The solution was allowed to stand for 1 h at room temperature, loaded onto a column packed with Sephadex G-25, and eluted with the same buffer. The absorbance of the eluate at 450 am was monitored and fractions with a high optical density were pooled and stored at -20°C. Dithiol content was determined by the method of Carlsson (Carlsson e ta / . , 1978), and GOD concentration from the absorbance at 450 nm using an extinction coeffcient of 1.41 x 104 mo1-1 cm -1 (Swoboda & Massey, 1%5). The same concentration of ovalbumin was treated with SPDP in the same way except that eluate was monitored at 280 nm, and protein concentration determined with Bio-Rad Protein Assay (Bio-Rad, Miinchen, Germany) by the method of Bradford (Bradford, 1976; Spector, 1978). Proteins were attached to 1TO coated glass wafers as described for horseradish peroxidase (Wilson & Schiffrin, 1995).

Eleetroehemiluminescence mea~area~ts

The flow injection analyser is shown in Fig. 1. Derivatized ITO wafers were damped into the flow cell as shown in Fig. 2. The electrochemiluminescence reaction takes place under alkaline conditions, but prolonged exposure to these results in deterioration of the silanized surface (Plueddemann, 1982) and loss of GOD activity (Wilson & Turner, 1992). To avoid this 10 mM KC1 running solution with pH close to neutral and no buffering capacity was pumped through the flow cell in between assays. The initial composition of the assay solution, chosen from a knowledge of the enzymatic and electrochemil- uminescence reactions, was 10 mM KCI, 10 mM glucose and 100 ~VI luminol in 10 mM Tris buffer, pH 8.0. It was injected into the running solution in 110/zl amounts and light and current were recorded as it passed through the flow cell.

~ t i o n of pH and luminol concentration

The pH was optimized by measuring the change in light intensity as nmning solution in the pH range 7.2-9.4 was pumped through the flow cell. The

Biosensors & Bioelectronics Detection of glucose oxidase

PMT

PMT Power Supply

Fountain Cell

I I i

g

Potentiostat

t

!

Asia Flow Injection Pump

Fig. 1. Electrochemiluminescence flow injection ana- lyser. The ASIA flow injection pump, potentiostat and photomultiplier tube (PMT) were all controlled by the computer. Solutions were manipulated with the ASIA pump, and light and current were measured simul- taneously as they passed through the fountain cell. Data was fed back to the computer where integrals for light and current were calculated. The reference electrode

(R.E.) was located in the effluent solution.

procedure was repeated with ITO wafers derivafized with ovalbumin as a control. The luminol concen- tration was op "tunized in the same way except that pH was fixed at 9.0 and the luminol concentration varied between 0 and 100 #M. In between all measurements glucose was reassayed at pH 8-0 and 100 #aM luminol to monitor the reproducibility of the electrochemiluminescence reaction.

Detection of glucose

Light and current were recorded as 0-10 mM glucose in pH 9.0 assay solution that contained 100 pM

Light

i ~ ITO Coated Glass : /

-x, ,,

/ J

Working Electrode Contact

J

W E l l l ~ ~ ~ / Stainless Steel Counter Electrode / / " /"

/ oP,~g /

C.E.

Liquid In

Fig. 2. Exploded half section through centre line of the fountain cell. Light from the chemiluminescence reaction was transmitted through the ITO coated glass electrode and detected by a PMT positioned above the cell. Both cell and PMT were enclosed in a light tight box. C.E.,

counter electrode; W.E., working electrode.

luminol was pumped through the flow cell. The effect of 4-acetamidophenol (200 wM), uric acid (0.5 mM) and vitamin C (100/aM) on electrochemilu- minescence was determined under the same con- ditions.

RESULTS AND DISCUSSION

Previous work has shown that the immobilization technique gives a monolayer of electroactive ferro- ceneacetic acid covalently attached to the surface of ITO coated glass wafers (Wilson & SchiffiJn, 1995). GOD and ovalbumin treated with SPDP contained, respectively, nine and 10 blocked dithiols per mole of protein and it is assumed they were also attached as a monolayer. On pumping assay solution through the flow cell, light and current varied as shown in Fig. 3, demonstrating that monolayer amounts of GOD could be detected by electrochemiluminescence as shown schematically in Fig. 4. In the absence of an applied potential, there was no light. No previous knowledge about the stability of 1TO coated glass

807

R. Wilson et al. Biosensors & Bioelectronics

6O

4o

20

80

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/

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i l , I , I ,

40 60 80

Time (s)

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100

1.2

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~ 0.6

0.4

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0.2 , I , I ~ I , i ~ l 0 20 40 60 80 100

Time (s)

Fig. 3. Variation in light and current as a pH 8.0 solution of glucose (10 m) and luminol (100 izM) was pumped through the fountain cell.

derivatized with proteins under these conditions was available, and therefore glucose was redetermined under fixed conditions at the end of each change in pH or luminol concentration. Light intensity under these conditions remained constant throughout the experiment, demonstrating that the surface was suitable for repetitive flow injection assays. The effect of pH and luminol concentration on light intensity for GOD and ovalbumin derivatized wafers is shown in Figs. 5 and 6, and from these results pH 9.0 and 100 ~ luminol were chosen for sub- sequent glucose assays. When glucose solutions were

pumped through the flow cell, light intensity increased linearly from 0 to 10 mM as shown in Fig. 7. The limit of detection for glucose (defined as 2 x SD of 21 zero calibrators) was 0.419 mM and the correlation coefficient was 0.9962 for 42 assays. It was not possible to detect glucose in the presence of paraceta- mol, vitamin C or uric acid because they suppressed chemiluminescence at concentrations likely to be found in blood.

In a competitive enzyme immunoassay, in which analogues of the antigen are covalenfly attached to an electrode, GOD labelled antibodies would bind

808

Biosensors & Bioelectronics Detection of glucose oxidase

~ Glucose + Oxygen

~.o ~-~ ~'~".o ~ Hydrogen Peroxide ~_~i~ 'i"' 'ci"' ~~ 'x id ized Luminol ~ - - ~ - - ...... ~ A ~

Si Si

i t I I I I

ITO Electrode

Fig. 4. The electrochemiluminescence reactions. Luminol is oxidized at the ITO electrode and reacts chemilumines- cently with hydrogen peroxide generated by glucose oxidase.

7 -

6-

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2-

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Fig. 5. Graph showing how light varied with pH for electrodes derivatized with GOD (1) or ovalbumin (e). The glucose concentration was 10 mM and the

luminol concentration 100 IxM.

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/ /

/ /

,/ /

2

/ 0 • • / •

o o ~o l'oo 1'~ 2bo 2~o

[Luminol]/pM Fig. 6. Graph showing how light varied with luminol concentration for electrodes derivatized with GOD (1) or ovalbumin (e). The pH was 9.0 and the concentration

of glucose 10 mM.

8 0 9

R. Wilson et al. Biosensors & Bioelectronics

2 0 - REFERENCES

15

,,-:,

"--" 10

0 0

[GlucoseJ/mM

Fig. 7. Graph showing how light varied with glucose concentration at pH 9.0 and 100 I~M luminol.

to the surface in monolayer amounts, and this work suggests it may be possible to detect them by electrochemiluminescence. This kind of immunoassay would be attractive because light is generated only when a potential is applied and therefore more than one analyte could be detected by interrogating individually derivatized units in an array of electrodes. There was a gradual increase in current during glucose assays and investigation showed the 1TO electrode became more active with time, but light intensity remained proportional to the glucose concen- tration. This was because luminol (100 tabl) was present in excess, as shown in Fig. 6, and it illustrates another advantage of combining electrochemistry with chemiluminescence. In an electrochemical assay change in electrode activity would make it necessary to recalibrate the system, but it had no effect on the electrochemiluminescence flow injection analyser described here. Electromagnetic noise does not interfere with light intensity for the same reason. In contrast to its potential for use in multianalyte immunoassays, interference from common blood components and a modest limit of detection for glucose means that the electrochemiluminescence system described~ here is unsuitable for detecting glucose in blood.

ACKNOWLEDGEMENTS

The authors are grateful to Andreas Brecht, Gerd Lang and Jacob Piehler of Tfibingen University, Germany, for helpful advice, and Jack Dutton and Charles Clavering of Liverpool University, UK, for technical assistance.

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