Avidin—biotin complexation for enzyme sensor applications

Avidin—biotin complexation for enzyme sensor applications

205 trends in analytical chemistry, vol. 13, no. 5, 1994 R.J. Gettens and G.L. Stout, Painting Materials: A Dover, New York, 1966. 0 R. Klockenkampe...

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trends in analytical chemistry, vol. 13, no. 5, 1994

R.J. Gettens and G.L. Stout, Painting Materials: A Dover, New York, 1966. 0 R. Klockenkamper, A. von Bohlen, L. Moens and W. Devos, Spectrochim Actu, 48B (1993) 239. 10 M. Mantler, M. Schreiner, F. Weber, R. Ebner and F. Maininger, Adv. X-Ray Anal., 35 (1992) 987. 11 G. Gigante, C. Maltese, S. Rinaldi and S. Sciuti, in E. Pernicka and G. Wagner (Editors), Archeometry 8

Short Encyclopaedia,

‘90, Proc. 27th Symp. Archeometry, Heidelberg, 1990, Birkhauser Verlag, Basel, 1991, p. 255. 12 S.P. Best, R.J.H. Clark and R. Withnall, Endeavour, 16 (1992) 66. 13 J. Dufilho and C. Coupry, Analusis, 20 (1992) 15. 14 B. Guineau, Stud. Conserv., 29 (1984) 35. 15 J.-C. Merlin, in B. Guineau (Editor), Pigments et Colorants de l’tintiquite’ et du Moyen, editions du

Centre National de la Recherche Scientitique, Paris, 1990, p. 41. 16 P Dharnlincourt, J. Barbillat and M. Delhaye, Spectrosc. Eur, 5 (1993) 16. 17 M. Schreiner and M. Grasserbauer, Fresenius’ Z. Anal. Chem., 322 (1985) 181.

18 J.P.W. Houtman and J. Turkstra, in Radiochem. Methods Anal., Proc. Symp. Salzburg, 1964, IAEA, Vienna, 1965, Vol. I, p. 85. 19 F. Lux, R. Zeisler and J. Reher, Radiochem. Ra-

dioanal. Lett., 42 (1980) 341. 20 J. Plesters, Stud. Conserv., 11 (1966) 62. 21 B. Mtihlethaler and J. Thissen, Stud. Conserv., 14 (1969) 47. 22 J. Plesters, Stud. Conserv. 14 (1969) 62. 23 H.-P. Schranun and B. Hering, Historische Mulmaterialien und ihre Identifizierung, Akademische

Druck- u. Verlagsanstalt, Graz, 1988, p. 55. 24 H. Ki.ihn,Stud. Conserv, 13 (1968) 7. Luc Moens is a professor in the Department of Analytical Chemistry of the University of Ghent, Proeftuinstraa t 86, B-9000 Ghent, Belgium. His research topics are archaeometry and a tom spectrometric methods of analysis. Wim Devos is in the same group, preparing a Ph.D. thesis on the analytical characterization of inorganicpigments and colorants used in artifacts. Reinhold Klockenkamper is head of the Physical Analysis Research Group at the Dortmund Institute and a professor at the University of Dortmund. Alex von Bohlen is in the lnstitut fiir Spectrochemie und angewandte Spectroskopie in Dortmund where his research interests include micro-, trace- and local analysis using X-ray methods.

Avidin-biotin complexation for enzyme sensor applications Jun-ichi Anzai, Tomonori Hoshi and Tetsuo Osa*


Sendai, Japan

Enzyme sensors are usually fabricated by immobilizing enzymes on the surface of electrodes, optrodes, or other transducers [ 11. Therefore, the technique of enzyme immobilization is of crucial importance for the development of high-performance enzyme sensors. Recently, much attention has been devoted to the molecular-level modification of electrode surfaces with enzymes by techniques including covalent bonding [2], monolayer deposition [3] and simple adsorption [4]. One of the advantages of the molecular-level modification techniques is that rapid-response sensors can be prepared by removing the conventional type of thick membranes from the surface of transducers. In this context, we and other groups have recently developed a new class of technique for enzyme immobilization using an avidin-biotin system.

The use of an avidin-biotin system in the preparation of enzyme sensors is described. The biotin-labelled enzymes can be immobilized on the avidin-modified electrode surface through avidin-biotin complexation. The various techniques for the surface derivatization with biotin and avidin and for the coupling with enzymes are cited. The possibility of constructing a protein architecture on the electrode is also discussed: this is based on the non-covalent interaction of avidin and biotin. *To whom correspondence should be addressed.


0 1994 Elsevier Science B.V. All rights reserved


trends in analyTica/ chemistry, vol. 73, no. 5, 1994

that each subunit contains a binding site to biotin and forms a highly stable complex with biotin or its derivatives, the association constant being reported to be cu. 1015 M-l [5] (Fig. 1). Becallse of the highly specific and strong binding interaction, the avidin-biotin system has been widely used in a variety of fields such as affinity chromatography [6], binding assay [7], and immunochemical stains [8]. For this purpose, many kinds of biotin- or avidin-labelled reagents, including fluorophores, lectines, binding proteins, DNA and RNA, and enzymes, have been developed and are now commercially available.

Biotin Avidin

Fig. 1. Complex formation from avidin and biotin.

This article is an overview of recent work on the use of this system in enzyme immobilization for biosensor applications. Avidin-biotin


Avidin is a highly stable glycoprotein (molecular mass 67 000) found in egg-white. It is isolated as a tetramer of identical 12%residue polypeptide chains. The most characteristic feature of avidin is


: -a

Enzyme immobilization biotin complexation



Two different routes are avialable for immobilizing biotin-labelled enzymes on the electrode surface through avidin-biotin complexation. The two procedures are schematically depicted in Fig. 2. The first procedure employs the biotin-modified surface, on which biotin-labelled enzymes are im-

, B-GOD :

, Avidin : *

Fig. 2. Schematic illustration of enzyme immobilization via avidin-biotin


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mobilized through avidin as binder protein. Using this strategy, Kuhr and co-workers have anchored biotin residues on the surface of carbon electrodes through a covalent linkage [9,10]. They treated the biotin-modified electrode with avidin and biotinlabelled glutamate dehydrogenase to prepare carbon-fibre microsensors for the measurement of neurotransmitter dynamics. The carbon-fibre microsensors showed a rapid response time (0.3 s). An alternative way to modify the electrode surface with biotin was reported by Snejdarkova et al., using a biotin-modified phospholipid bilayer [ 111. They prepared a biotinylated phospholipid from the N-hydroxysuccinimide ester of biotin and the phospholipid for this purpose. The phospholipidcoated electrode was used to construct a glucose sensor by coupling it with a biotin-labelled glucose oxidase (B-GOD). Biotin-modified synthetic polymers were also used for the preparation of fibre-optic enzyme sensors [ 121. We have tried to immobilize avidin directly on the electrode surface in order to circumvent the cumbersome procedure of surface-derivatization with biotin. If avidin could be immobilized on the electrode surface directly, without loss of the binding acivity to


biotin, biotin-labelled enzymes could be loaded more easily on the electrode surface. Adsorption

of avidin on LB film surface

Avidin is known to be adsorbed strongly on the hydrophobic surface through hydrophobic interactions [ 13 3. Therefore, we first prepared the hydrophobic surface by coating a metal oxide electrode with a Langmuir-Blodgett (LB) monolayer composed of stearic acid. The B-GOD was immobilized on the LB film-coated electrode via two different routes [ 141 (Fig. 3, routes a and b). Both electrodes exhibited an amperometric response to glucose, showing that the B-GOD was immobilized successfully on the electrode surface. Typical calibration curves are shown in Fig. 4. It should be noted that the electrodes prepared by route (b) showed a higher response than those prepared by route (a). This shows that more B-GOD is loaded onto the electrode by route (b), in which a multiple complex of avidin and B-GOD was adsorbed directly onto the hydrophobic surface. In any case, these results clearly demonstrate the value of the avidin-biotin system for the immobilization of

. ::.:4 0







Fig. 3. Immobilization of glucose oxidase on LB film-coated electrodes by routes (a) and (b).


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250 route b)

200 Y 5 P 1 d


1.50 100 50

route a)


0 0






[Glucose]/mM Fig. 4. Calibration curves for glucose sensors prepared by the routes (a) and (b).

enzymes. Unfortunately, the long-term stability of the LB film-based glucose sensors was not satisfactory. The current-response of the sensors decreased in a few days after preparation, even if the probes were stored in buffer at low temperature. Weak adhesion of the LB film to the electrode surface may be responsible for this short life. Electrodeposition

of avidin

In order to enhance the surface-adhesion of avidin, the electrodeposition of avidin films was studied [ 15,161. Fig. 5 shows the time-course of the electrodeposition of avidin on a platinum (Pt) electrode, monitored with a quartz-crystal microbalance (QCM). The decrease in the resonance

frequency (AF) means that avidin is deposited on the Pt electrode surface. The QCM data clearly show that the deposition of avidin is accelerated by applying an alternating potential, from -0.5 to + 2.1 V vs. Ag/AgCl, to the Pt electrode in an avidin solution. An advantage of the present technique is that the loading of avidin protein can be controlled accurately, because the AF values of the QCM depend linearly on the weight of avidin deposited on the electrode (-1 Hz in AF corresponds to cu. 1 ng of avidin deposited, in our QCM equipment). The B-GOD is immobilized by immersing the avidin-deposited electrode in a B-GOD solution (50 pg/ml) for 30 min. A typical response curve of the glucose sensor thus prepared is shown in Fig. 6a. The sensor showed a high and rapid response to glucose, confirming that avidin retains its ability to bind to biotin even after the electrochemical treatment. This rapid response of the sensor suggests that there is smooth transport of glucose or H202 through the avidin film. The possibility of non-specific adsorption of enzyme to the avidin layer can be excluded because little or no response was observed for the electrode treated with a native GOD in place of B-GOD. The long-term stability of the sensor compared to the LB film-based sensors was improved by using the electrodeposition technique. The sensor can be used for the determination of glucose for more than half a year.

Enzyme architecture face

on the electrode sur-

It may be possible to build up a protein architecture, using avidin and biotin-labelled proteins, in which the proteins can be compared to “molecu-

Time / s Fig. 5. Monitoring of electrodeposition of avidin on a Pt electrode with a quartz-crystal microbalance. The electrolysis was started at the time indicated by the arrow.


Fig. 6. Typical response curves of the glucose sensors prepared with B-GOD (a) and native GOD (b).

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1) avidin 2) B-GOD

1) avidin 2) B-GOD


sensor 1



sensor 2

sensor 3

: Avidin,



Fig. 7. Deposition of B-GOD multilayers on electrode surface.

lar Lego” [17]. The two-or three-dimensional architectures may be constructed using proteins which are tagged with more than two biotin residues, because avidin contains four biotin-binding sites per molecule. Along this line we have undertaken some preliminary experiments to prepare enzyme multilayers on the electrode surface [ 181. An alternating deposition of avidin and B-GOD was repeated up to three times to prepare the B-GOD multilayers (Fig. 7). The deposition can be carried out simply by immersing the electrode in the avidin and B-GOD solutions, alternately. Jnterestingly, the size of the output current from the sensors 1, 2 and 3 varies nearly linearly with the number of B-GOD layers deposited. For example, the output current from the sensors 1,2, and 3 is 45, 90, and 140 nA/cm2, respectively, in the presence of 5 mM glucose, suggesting that the loading of B-GOD is dependent on the number of depositions. In other words, B-GOD seems to build up in the deposited multilayers as schematically illustrated in Fig. 7. Although further study is needed to establish the detailed structure of the multilayers, we can conclude at least that the sensitivity (or size of the output current) of the sensor is controllable, to some extent, by repeated deposition of avidin and B-GOD. These results imply that the avidin-biotin system can be used to construct protein architectures on the electrode surface. The protein architecture may be useful not only for biosensor applications but also for the development of other protein-based electronic devices [ 191.

Miscellaneous systems using avidin-biotin complexation Immunosensors can be prepared by immobilizing biotin-labelled antibodies to the avidinmodified surface of transducers. Optical immunosensors which are sensitive to prolactin [20] and sex-hormone-binding globulin [2 l] were devised, using their antibodies tagged with biotin. A biotinlabelled oligonucleotide was used to modify the surface of a quartz-crystal microbalance for use in a DNA hybridization assay [22].

Conclusion Biotin-labelled enzymes can be immobilized on the biotin- or avidin-modified surface through avidin-biotin complexation for the preparation of biosensors. For this purpose, various techniques have been developed to immobilize avidin or biotin on the surface of electrodes and optrodes. The avidin-biotin system may be also applicable to the construction of protein architectures,

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Jun-ichi Anzai is Associate Professor of Pharmaceutical Science at the Pharmaceutical Institute, Tohoku University, Aobayama, Sendai 980, Japan. His current research interests include functional artificial membranes and chemical sensors. Tomonori Hoshi is a graduate course student at the Pharmaceutical Institute, Tohoku University He is current/y studying enzyme sensors based on avidin-biotin complexation. Tetsuo Osa is Professor of Physical Chemistry at the Pharmaceutical Institute, Tohoku University His current interests are in electro-organic chemistry and host-guest chemistry

Multivariate sensor arrays as industrial and environmental monitoring systems W. Patrick Carey Seattle, WA, USA


The advances in the development of chemical sensors from individual probes to oneand two-dimensional sensor arrays will play an important role in the remote sensing of complex samples in the future. However, special attention must be given to the selectivity and response characteristics of each sensor and to determining the best chemometric calibration technique to use. Examples of chemical sensor arrays are presented by examining the role of chemometrics and the applications to field problems in analytical science.

One of the biggest challenges to analytical chemists today is the monitoring of the environment and industrial processes. There is a large arsenal of techniques and instrumentation to characterize environmental samples using laboratory based methods such as gas chromatography and mass spectrometry. To continually monitor waste sites, or industrial processes that might emit hazardous compounds, analytical tools based on sensors are a feasible approach. Chemical sensors have the potential to be low cost, long term monitoring devices, especially when they are dispersed in large numbers across a waste site or industrial plant.

0 1994 Elsevier Science B.V. All rights reserved