Sensors and Actuators B, 3 (1991) 295-304 295

A concanavalin A-coated piezoelectric crystal biosensor

Christopher Barnes, Claudius D’Silva, John P. Jones and T. John J_.ewis Institute of Molecular and Biomolecular Electronics, VnivemIy of Wales, Bangor, Dean Street, Gwynedd LL57 IVT (U.K.)

(Received January 30, 1991; in revised form July 8,, 1991; accepted July 12, 1991)

Abstract

The lectin concanavalin A (Con A) has been immobilized on the surface of AT-cut quartz crystals to produce piezoelectric sensors for sugars in solution. Immobilization has been achieved by treating the crystals with 3-aminopropyltriethoxysilane followed by terephthaldicarboxaldehyde to which the lectin is then bound. The change in resonance frequency of the coated crystal, when immersed in glucose solutions of millimolar concentrations, confirms the efficacy of Con A as a detector of glucose having a sensitivity of approximately 100 IWmillimole at concentrations below 2 millimole. By analysing the viscoelastic response of the system to shear waves, we demonstrate that the measured changes in resonance frequency cannot be interpreted in the conventional way as simple changes in mass of the active layers. Instead, it is probable that they arise because reaction with the anaiyte alters the conformation of the sensing Con A layer and so changes the viscoelastic response of the system to the transmission of shear waves. The ability to detect changes in the viscoelastic response of a molecular layer produced by a reaction offers a means of examining liquid-phase reactions at the monolayer level, and has significant implications for the design of sensing systems based on coated quartz.

Keywords: concanavalin A, microbalance, AT-cut quartz crystal, biosensor, piezoelectric, lectin, glucose sensor, shear wave.

Introduction

AT-cut piezoelectric crystals have been widely used as environmental monitoring de- vices since the application was first described by King et al. [l]. In air or vacuum these crystals can act effectively as microbalances because a small mass change per unit area,Am, at the crystal surface produces a proportional shift in the resonance frequency Af which may be described by the Sauerbrey [2] equa- tion

Af = - V&p,) - ‘h = -2.26x IO-“p&n (1)

where f. is the resonance frequency of the crystal in fundamental shear mode and ps, V, are respectively the density and shear wave velocity for quartz.

Subsequent studies have shown that mod- ification of the crystal surface by organic compounds or enzymes can enhance the spec- ificity of the device for a wide variety of vapours, such as SOZ [3], NH, [4], organo-

phosphorous compounds and pesticides [5], HzS [63, HCl [71, aromatic hydrocarbons [8], and mercury vapour [9]. Recently, AT-crystals immersed in water and organic solvents have found applications in the determination of electrodeposited metal ions [lo], electro- chemical analysis [ll], evaluation of chemo- receptive membranes [12] and as biosensors for the determination of Candida albicam [13], galactosyltransferase [14] and immu- noglobulin G [15]. When used in liquids to monitor large biomolecules there is clearly an expected advantage due to the large mass [13, 141, but more recently it has been sug- gested that the response of a crystal is not simply related to it 1161. Shear waves are generated at the oscillating crystal surface and propagate out into the surrounding liquid, generally becoming adsorbed within a few microns of the surface [16]. Changes in the resonance frequency of the system may then be brought about not by the addition of mass per se but by the viscoelastic response of the layer adjacent to the crystal surface into which

09254005/91/$3.50 0 1991 - Elsevier Sequoia, Lausanne

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the shear waves propagate. The success of an immersed AT-crystal sensor in which the surface has been chemically modified to make it selectively sensitive to an analyte in solution thus depends on detecting changes in the nature of the viscoelastic interactions between the crystal surface and the liquid brought about by the addition of the analyte. In the present work we have sought to develop a sensor for glucose based on AT-cut crystals, the electrode surfaces of which have been modified by the covalent attachment of the lectin concanavalin A (Con A). Although not a true antibody, this plant lectin, isolated from jack beans, exhibits antibody-antigen type reactions with carbohydrates and is a metalloprotein of molecular weight 26 000. At pH>5.6 it exists as a tetramer with a requirement for Mn*+ and Ca*+ ions for agglutination activity with carbohydrates. Con A binds a variety of carbohydrates [17], in- cluding sugars such as glucose, fructose and galactose, dissaccharides such as maltose, su- crose and gentiobiose, and polysaccharides such as dextrans, glycogen, amylopectin, tei- choic acids, D-fructans and mannans.

A potentiometric Con A immunoelectrode was previously reported by Janata [I81 but was insensitive to oligosaccharides, respond- ing only to large ionically charged biomole- cules. A fibre optic probe for glucose using Con A in a competitive binding assay with glucose and fluorescence-labelled dextran was reported by Schultz et al. [19].

Glucose sensing has recently been suc- cessfully achieved using a gel-entrapped en- zyme, namely hexokinase, at the surface of a 5 MHz crystal [20]. It is believed, however, that the study described here is one of the few in which a fully immersed crystal has been used for small molecule determination via an antibody-antigen type reaction, being possibly the only example to date where true covalent immobilization has been employed for glucose sensing and where any form of theoretical interpretation of the anomalously sensitive response to the aqueous analyte has been advanced. Also, the broad specificity of Con A suggests that is might be a suitable sensing agent for use in a variety of bio- technological applications.

Materials and methods

Materials Planar AT-cut crystals of nominal reso-

nance frequency 14.33 MHz and with silver electrode areas of 20 mm2 were obtained from the HY-Q Crystal Company (U.K.). 3-Ami- nopropyltriethoxysilane, concanavalin A, glucose, tris(tris(hydroxymethyl)aminometh- ane) and sodium borohydride were obtained from the Sigma Chemical Co (U.K.). Calcium chloride and manganese sulphate were from BDH (U.K.) and terephthaldicarboxaldehyde and anhydrous dichloromethane from Aldrich Chemical Co (U.K.).

Immobilization of Con A Methods for the immobilization of proteins

on quartz crystals have generally involved the chemical attachment of a functionalized silane to the hydroxylic groups on the crystal surface. The amine [13,14] or diol [21] functionalized surface is then reacted with a spacer, for example, glutaraldehyde [13, 141 or sodium periodate [21], to produce a terminal aldehyde which is coupled to the protein. The presence of a spacer between the surface and protein can enhance the reactivity of the latter to solution species [22].

In the present case Con A had to be immobilized on the silver electrode surfaces of the crystal. Silver has a stable hydroxide layer which makes it a suitable surface for attachment of silanes such as 3-aminopro- pyltriethoxysilane (APTES), which have a use- ful reactivity with surface hydroxylic groups P31.

After experimenting with several methods, the following coating procedure was adopted. Both sides of the electroded quartz crystals were treated with 5% APTES in ACS dry chloroform for 2 h at 20 “C. The crystals were then washed with chloroform, air dried and completely covered with a solution of 10% terephthaldicarboxaldehyde (TPDCA) in anhydrous dichloromethane. After reacting for 2 h at 20 “C, the quartz crystals were removed from solution, washed with dich- loromethane, acetone and water, and air dried. The use of TPDCA instead of glu- taraldehyde as a spacer was prompted by the poor reproducibility of the immobilization in terms of area1 uptake obtained in studies on

297

quartz slices (unpublished data) as well as on the AT-cut crystals using this latter ma- terial. Glutaraldehyde is quite reactive and commercial preparations quickly deteriorate to unsuitable polymeric products, and this combined with its complex reaction with amines [24] occasionally resulted in undesir- able surface loading of crystals. The dialde- hyde TPDCA was a natural replacement due to its greater stability (being a crystalline compound) and proved to be sufficiently re- active to complex with the 3-amino group of APTES. Con A was then immobilized via the surface aldehyde by immersing the crystal in a solution of Con A (1 mg/ml, 0.1 M Tris-HCL at pH 7.5) for 16-20 h at 4 “C. To increase the stability of the immobilized layer, the imines formed by reaction with the 3-amino group of APTES and the lysine residues on Con A were reduced in sihc by reacting with sodium borohydride (0.6 mg/ml at 4 “C for 45 min) [25] to give the corresponding sec- ondary amine, thus preventing dissociation of the starting materials. The coated crystals were then washed in water and stored in Tris-HCl solution pH 7.5, at 4 “C. The overall structure of the Con A attachment to the silver electrode is depicted in Fig. 1.

Determination of crystal resonance The resonance frequencies of the Con A

crystals were determined using the system shown schematically in Fig. 2. The crystal was

P t ’

0--SinNH-CH KHz-NH-Concanavalin A

0

Fig. 1. Generalized structure of a Con A reaction site produced by chemical modification of the siker electrode

surface.

Fig. 2. Schematic diagram of system used for frequency measurements on Con A-coated ATcut crystals immersed in liquids.

mounted in a standard type HC25 socket and connected to a high gain Pierce-type oscillator designed to drive the quartz crystal at its parallel resonance frequency with both faces rather than one [26] immersed. The oscillator had sufficient gain to overcome the losses incurred when the crystal operated fully im- mersed, but it was necessary to reduce stray parallel capacitance to a minimum. This was achieved by keeping the leads short and en- closing the oscillator circuit in a screened box with the crystal in its holder protruding out on a short insulating column into the liquid. It was also necessary to maintain a fairly low liquid conductivity. The oscillator frequency was monitored using a frequency counter (Marconi model 2432A). In order to display the frequency changes of the oscillator cor- responding to changes in crystal resonance frequency, the frequency was down-converted to the range 4-14 kHz by subtractive mixing with a reference frequency derived from a frequency synthesizer (Farnell model SSG 1000). The difference frequency was moni- tored by a separate frequency counter. Changes in frequency, Af, were displayed on a chart recorder after frequency/voltage con- version.

Measurements were made with the Con A-treated crystal immersed in 70 ml of deion- ized water containing trace amounts (200 PM) of Ca’+ and Mn” ions necessary to maintain Con A activity. To this was added a measured volume of glucose solution of known con- centration (Fig. 2). The changes in resonance frequency resulting from the step-wise ad- ditions of glucose were recorded.

Con A-modified crystals, operated in air without special screening, exhibited a drift in resonance frequency of about 1 Hz/h, which increased typically to 320 Hz/h when they were immersed in water containing the above trace ions. Slow ion absorption and changes in surface conditions and temperature are likely to be responsible for the drift. Special precautions to control temperature were not taken, because the relatively large thermal inertia of the 70 ml of water prevented rapid fluctuations in temperature. Some long-term drift occurred.

298

Results

Crystal characteristics As expected, the effect of Con A layers

was to reduce the resonance frequencies of crystals. Operated in air, the average reduc- tion was about 8 kHz, but could be between 6 and 9 kHz depending on the degree of Con A layer coverage. Subsequent immersion in deionized water caused a further reduction by about 21 kHz. If the coating is considered simply to give an increased mass on the crystal surface, the Sauerbrey eqn. (1) may be used to estimate Af. The combined molecular weight of the silane and TPDCA part of the coating (Fig. 1) is 253 and assuming a close- packed structure, the area1 mass is about 8 x low6 kg m-‘. The Con A molecule, with a molecular weight of 26 000, is likely to have a hydration sheath which, if typical of proteins, will be about two monolayers of water thick [27, 281. If the hydrated Con A groups are also considered to form a close-packed layer to give the maximum density, then the area1 mass is also about 8X 10m6 kg m-*. With the combined mass of 16x low6 kg m-*, the Sauerbrey equation predicts Af to be about - 1 kHz for the crystal in air, which is eight times less than the experimental value. Grande et al. [14] reported a similar discre- pency following the covalent attachment of glucosamine to quartz surfaces via silanes. The difference may reflect the fact that the true surface area is greater than the geometric one due to roughness of the electroded sur- faces and, also, because multiple layers of silane have been deposited. It may also mean, as will be discussed below, that the Sauerbrey equation is not appropriate and does not take proper account of the viscoelastic properties of the coating.

The change in resonance frequency when an uncoated crystal is immersed in a liquid has been considered by a number of authors [29-321. The equation derived by Kanazawa and Gordon [29] for the situation when only one face is immersed can be written in the form

Af = -finhl~)‘Rh,~,) -’ (2) Where p, 7 are the density and absolute viscosity of the liquid. This equation, which should be compared with eqn. (l), is derived

on the basis of a strongly attenuated shear wave in the liquid coupled to the shear wave generated in the quartz. The predicted Af for immersion in water is -3.5 kHz, which is very much less than the observed value (- 21 kHz), even allowing for both faces of the crystal being immersed. In the treatment given by Bruckenstein and Shay [32] the liquid is assumed to provide a viscous boundary layer at the crystal surface of a thickness determined by the kinematic viscosity and frequency. The mass of this boundary layer (PrllfoY is then considered as an additional mass load on the crystal as in the Sauerbrey treatment (eqn. (1)) to give

Af = - W-?*(P~‘“(P,~,) - ’ where n is 1 or 2 depending on whether one or two faces of the crystal are in contact with the liquid. This equation differs from eqn. (2) only by a factor 2n(n-)lE and the predicted Af on immersion in water is -24.8 kHz, which is much nearer to the experimental value. None of these treatments provides for the situation in which the crystal is coated with layers having different viscoelastic properties from those of the bulk of the immersion liquid. We shall consider this situation below.

The typical time-dependent response of a coated crystal immersed in 70 ml of deionized water at 20 “C to step-wise increases in glucose concentration is shown in Fig. 3. In response to each glucose injection, the resonance fre- quency falls immediately and then fluctuates, probably because of the created turbulence, before setting down within a period of about 20 s to a steady value which finally saturates when sufficient glucose has been added, as

Fig. 3. Change of resonance frequency, 4, of sensor following step-wise increases in glucose concentration. The initial volume of water was 70 ml and at the times indicated the stated quantity of a 200 mglml solution of glucose was injected.

299

shown in Fig. 4. At this stage all available Con A sites are likely to have been complexed with glucose. Although Fig. 4 represents a typical response, quite large differences from crystal to crystal are found in the saturation values of the frequency shift, AfmaX, reflecting the difficulty in controlling the evenness and chemical stability of the Con A coatings. The former is a function of the crystal electrode surface as supplied [15]. Coated crystals were stored dry at 4 “C and showed a steady decline in activity, as indicated in Fig. 5. The useful lifetime was about three days and was probably determined by bacterial attack, but it may also have been influenced by the migration of the silver ions from the underlying electrode causing the replacement of the Mn2+ ions in the Con A complex by Ag’ . Rigorous exclusion of bacterial contamination and the use of alternative electrodes may make it possible to extend the lifetime of these sensors.

Go0

i

0 5 10 15 20

gl”cose/mM

Fig. 4. Magnitude of shift in steady-stat: resonance fre- quency, IAft, as a function of glucose concentration.

Fig. 5. Decline in crystal activity with time. The saturation value of resonance frequency shift, 4,..! decreases with the age of the Con A coating on the crystal.

Analysis As mentioned above, it is necessary to

recognize that the environment, especially when glucose is bound, is one with complex viscoelastic properties affecting the resonance frequency of the crystal. The multilayered structure of the coated crystal is illustrated in Fig. 6. The silver electrode layers, E, extend over only the central portions of the faces of the crystal, Q, but the silane layers, S, and dialdehyde-Con A layers, C, extend over the whole of the quartz and silver faces, being bonded to them at the silane interface. The immersion medium, I, may be air or the aqueous glucose solution. A characteristic complex shear-wave impedance may fined for each layer of thickness 1 familiar form [33]:

Z=Apu(l -b)-’

be de- in the

(4)

where A and p are the area and density of the layer and r= ctvlo, where a is the at- tenuation coefficient and u is the velocity for shear waves propagating at angular frequency o across the layer. Since 1 will be small for each layer, it is more convenient to write eqn. (4) in the form

Z=Ao-u*(l -ir)-’ (5)

where (+ is the area1 density of the layers, p!, v*=vII and r=a*v*/o where cy*=cyI is the attenuation exponent for the layer.

The characteristic complex impedance pre- sented by the combined Con A layer C and immersion medium I is then

z ,_z Z+Z,tanh 4 cl- c

2, + Zi tanh &

where Z,, Zi are the impedances for the Con A and immersion medium, respectively (eqn.

(5)), and

Fig. 6. Idealized multilayer structure on the quartz crystal.

300

& = q* + iwlv,* (6)

We shall assume, without proof, that Zi tanh +=-K 2, for both real and imaginary com- ponents. This condition is certainly true when the crystal is in air since Zi=O, but it is also likely to hold when the crystal is in water because of the factor tanh & With this as- sumption

Z,i=Zi i-Z, tanh 4,

In the same way the characteristic impedance presented by the silane and Con A layers and immersion medium combined is

z.=z &+Ztanh 4, 51

’ Z, + Z.-i tanh &

assuming that

Z,-i tanh & e Z,

Z,i = Z, tanh &5 + Z.-i

Finally, including the silver electrode, the combined impedance of the layers presented to the crystal becomes

Zei=Ze tanh &+Z, tanh &+Z, tanh &+Zi

(7)

Following Mason [33] and Crane and Fischer [34], the change in crystal resonance frequency caused by the layers is

Af= - 2fo x. ~PqVsA, e’

where f. is the resonance frequency of the quartz crystal. A, is the active area of the crystal, pq, v, are the density and shear-wave velocity for quartz and Xei is the imaginary part of Zci (eqn. (7)). The factor 2 allows for both sides of the crystal being coated. For the crystals used in the present exper- iments we find

Af = - 1.04X,JA, (9)

For the crystal with only silver electrode layers and operating in air, the impedance (eqn.

(7)) b ecomes

Zci =Z, tanh &

Now o/v,* will be small and using and (6) it is easy to show that

eqns. (5)

x = Aeueve* Cl l+T-,2

(‘k tanh (.y,* + o/v,* sech ‘a,*)

(10) If (ye* is small, the hyperbolic functions may be reduced to give

Xci =A,u~w (11)

and if the active area of the crystal A,., is equal to A, then, from eqn. (9),

Afe = - 1.04~~24 (12)

Usually Af= efo so that f-f0 and eqn. (12) is then the Sauerbrey equation.

When the crystal is covered with silane and Con A and operated in air, we have from

eqn. (7)

Xei =X,’ +X,‘+Xc (13)

where X,’ is the imaginary part of Z, tanh 4e etc. (see eqn. (10)). The change in resonance frequency due to the additional layers is

Afsc = - 1.04(X,’ +Xe’) (14)

At this point it is necessary to note that the silane (and Con A) layer is bonded to the whole crystal and not just to the electrode area. As a consequence the effective area A, of the silane layer will be greater than A, by an amount depending on the coupling of the crystal-metal electrode-silane interface and by the likelihood of mode conversions and the generation of other wave patterns at the crystal surface [35]. Without more detailed knowledge of the interface, we can take ac- count of these effects only by introducing a simple factor p so that eqn. (14) becomes

Afs, = - l.O4p(x,’ +xc’) (15)

where x:, x,’ are now impedances per unit area.

When the system is immersed in water, an impedance Xi =X, has to be included in eqn. (13). If we assume that the silane layer is hydrophobic, and will therefore not hydrate upon immersion, and that the Con A layer is already fully hydrated in normal laboratory air, thenX,’ and X,l will not alter on immersion and the frequency shift will be

Afw = - 1.04/3x, (16)

The addition of glucose to the water phase will changex,’ because of the Con A sensitivity

301

to glucose, but it may also change X,. The change in the latter should be negligible because at the maximum glucose concentra- tions used (20 mM) the reported change in the viscosity of the solution is less than 0.8% [36]. Consequently the frequency shift on adding glucose becomes

A& = - l.O4p(x,,’ -x&) (17)

where x,, ’ is the imaginary component of the impedance of the Con A layer as a result of binding glucose at a given concentration.

There is no way of determining a priori the viscoelastic properties of the Con A layer, but if the attenuation parameter q* were small, then an approximation similar to that used in deriving eqn. (11) will transform eqn. (17) into a simple mass relationship

A& = - l.O4Pa,o[(o&,) - 11 (18)

We have argued that the Con A layer in water will have much the same properties as in air and consequently o, N 8 x 1O-6 kg m-‘. At the saturation limit .we find Afg= -575 Hz and thus, from eqn. (18), there is a predicted density gain in the layer due to glucose complexation such that 0~~1 o-,, = 1+0.77/p. The molecular weight of Con A is 26 000 and that of glucose less than 200, so that ~,,/a, when the layer is fully com- plexed is less than 1.01 and p would have to be >77.

Returning to eqn. (16), since A&= -21 kI-Iz, & would have the value 2.6 X 10’ kg rnwz s-‘. From eqn. (5) x, may be written as ~,v,*r,(l +?$)-l where r, = ~*v,*/o. Es- timates of the various parameters at a fre- quency of 1 MHz have been given by Matheson [37] from which x, is deduced to be 1.75 X lo3 kg me2 s-’ at 1 MHz. From the work of Madsen et al. [38] on a number of water- based materials, it would appear likely that r,,, would be a weak function of frequency, while v,* could increase with frequency. At 14.3 MHz, therefore, we can expect x, to be greater than 1.75X lo3 kg mm2 s-‘, i.e., very much greater than the value we have deduced from the argument based on the increased mass of the glucose-complexed Con A system. We conclude that an argument for A’g based on a simple glucose-induced mass increase in the Con A layer is not justified.

The alternative is to consider that com- plexation with glucose alters the attenuation of the Con A layer. For an arbitrary level of attenuation the analysis becomes intract- able, but in the limit of strong attenuation it is easy to show, using an equation similar to eqn. (lo), that xcg’ will be of the form o~~cQ~*-‘o. Because A& is negative, the Con A layer must become less attenuating when glucose is complexed with it. Equation (17) becomes

= - l.o4pwa,(a,,*-’ -a,*-‘) (19)

If the complexed Con A layer becomes only weakly attenuating, then LY,~* may be replaced by unity in eqn. (19).

The binding of glucose (G) to Con A may be considered as a simple bimolecular reaction

Con A + glucose t----,

Con A-glucose complex

with a dissociation constant K= [Con A][G]/ [Con A-G]. If each Con A site contributes a specific absorption, acw when uncomplexed and acg when complexed with glucose, and if 8 is the proportion of Con A sites complexed at a given glucose concentration, then

CY cg* =lv[hcg + (I- @,I

where N is the concentration of Con A sites in the layer. Similarly ~~,*=iva,. Thus

% *-1 -%w *-I= e(a, - acg) Na,(fbg + Cl- @a&

(20) which, as e-4, reaches the maximum (a, -a,)/(Na~,). Thus from eqn. (19)

Afgmax = 1 + Cl- Ww Af, bcis =l+S (21)

which is an equation of the Eadie type. A plot of Afg against AfJ[G] should be linear with a slope - K(a,/a,). Figure 7 shows such a plot from the data of Fig. 4. The plot is linear except at the lowest glucose concen- tration and yields a slope Ka,/a,=3.1 mM.

So and Goldstein [39] report a dissociation constant K equal to 1.7 mM and Schultz et al. [19], using a fluorescent technique found 2.5 mM, giving a mean value of 2.1 mM.

302

when the Con A layer becomes fully com- plexed are about 1.8 x lo-’ and 0.67.

Fig. 7. Eadie-type plot according to eqn. (21).

Thus there is excellent agreement with the present result if the ratio of specific absorp- tions aou/acg is N 1.5. Using this result and eqns. (19) and (20)

Afgmax = - l.O4@~J(2c~,*) (22)

Returning to eqn. (15) and assuming that the silane layer is only weakly attenuating, i.e., cy,* is small, so that x,’ = 00, and writing x,’ as OO,/(Y,*, we find

Af= = - 1.04&( cr, + o,_,,/(Y,*) (23)

Now the silane layer is likely to be more than a monolayer thick and we can account for this by writing o,= yu, where y is greater than unity. Taking the ratio AfgmaxlAfssc from eqns. (22) and (23) and substituting exper- imental values, we find (Y,* =5.95/r and (Y,~* would be about 4.0/y. Returning to eqn. (22) and using the estimate 8X 10e6 kg m-* for a,, then /3r= 9.2 and, from eqn. (16), x, = ~(2.4 x 103)kg me2 s-‘. Bearing in mind that the parameters given by Matheson for water at a frequency of 1 MHz give x,= 1.75 x lo3 kg m-’ s-l and that x, may be expected to increase with frequency, the present estimate of x, is very satisfactory. If, as is unlikely, only a monolayer of silane existed so that y= 1, x, would be 2.4 X lo3 kg me2 s-l; otherwise it will have a value greater than this.

The attenuation factor for the wave in crossing the uncomplexed Con A layers, exp (-(Ye*), has a value 2.6X 10e3 if y= 1 but would be larger than this if several monolayers exist. For 10 layers, for example, where y H 10, the value is 0.55. The corresponding values

Conclusions

It has been shown that appropriately coated AT-cut crystals can detect glucose at low concentration. The sensitivity to glucose de- pends on concentration and is of the order 100 Hz/millimole over the concentration range 0 to 2 millimole (Fig. 4). Other lectins, immobilized in the same way, could provide sensors with selective response to particular carbohydrates. Reproducibility and lifetime can undoubtedly be improved by rigorous exclusion of bacterial contamination, and pos- sibly by using a less poisoning metal than silver in proximity to Con A.

By careful interpretation of the observed frequency shifts, the present experiments have demonstrated the usefulness of standard AT- cut quartz crystals, with appropriately mod- ified surfaces, as fully immersed sensors of liquid-phase reaction.

Particularly significant is the demonstration that sensing action depends on detecting changes in the viscoelastic properties of the Con A layer, and not on a simple mass change due to glucose binding. Shear wave absorption by the large molecular group of Con A will be sensitive to the structure and the various permitted vibrational modes. The action of binding glucose might be expected to modify the structure and the vrbrational modes, and, since absorption is apparently reduced by binding glucose, would seem to stiffen the Con A system. This type of effect may be expected for a variety of enzyme and related binding systems and the ability to detect viscoelastic changes opens up the possibility of new classes of sensors based on optimi- zation of the shear-wave system, with atten- uation of the wave rather than mass change in mind.

More work is required to understand the underlying determining processes. In the pres- ent experiments p could be as large as 9.2 if y= 1, which is unlikely; but, if y were say 3, B would be only 3.1 and might then be attributed merely to the area change across the quartz-electrode&lane boundary. By suitable design of electrode geometry and

control of the silane deposition, if might be possible to optimize this parameter and hence the sensitivity of the sensor.

The characteristic complex impedance functions involved in the analysis are cum- bersome to use without simplifying assump- tions. We are currently developing a pro- gramme to compute Af without approximations, thus permitting the deter- mination of the sensitivity of Af to changes in the essential parameters, a, V* and (Y*, of the various layers.

Acknowledgments

The authors acknowledge support through the Specially Promoted Programme on Med- ical Engineering of the U.K. Science and Engineering Research Council. C. D’S. also thanks GEC, Plc and the Fellowship of En- gineering for the award of a Senior Fellowship.

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Biographies

Christopher Barnes obtained a B.S.c. (Hons) degree from the University of Wales, Bangor, in 1977, an M.S.c. in electronic materials and devices in 1979 and a Ph.D. in 1982 in elec- tronic materials science from the University of Wales, Bangor. He is currently employed as a research fellow at the University of Wales, Bangor engaged in studies in liquid biosensing, utilizing piezo-devices.

Claudius D’Silva obtained a B.A. (Hons) and Ph.D. in chemistry from the Universities of Lancaster and Birmingham respectively. He has been a research fellow in the De- partment of Chemistry, University of Essex and senior fellow in the Department of Bi- ological Chemistry, University of Michigan, Ann Arbor, U.S.A., until 1986. He returned to the U.K. as a research fellow in the De- partment of Biotechnology and Biophysics, University of Leeds and was appointed in 1988 as the Fellowship of Engineering/GEC senior fellow in molecular and biomolecular electronics, University of Wales, Bangor. He currently heads an interdisciplinary group consisting of chemists, physicists and elec- tronic engineers working on chemical sensors and molecular electronics.

John P. Jones graduated in chemistry from the University of Wales, and obtained Ph.D and D.Sc. degrees from the University of London. Following an appointment at the National Physical Laboratory, he joined the University of Wales, Bangor as a lecturer and is now a senior lecturer in electrical material science. His interests are studying the be- haviour of metallic surfaces and interfaces in atomic detail by high field microscopy, the development of molecular electronic systems, and the development of chemical sensors based on shear wave absorption in the quartz/ liquid boundary region.

T. John Lewis is Professor Emeritus in the University of Wales. He was formerly pro- fessor of electrical materials science at the University of Wales, Bangor. His interests are in the electronic and electrokinetic prop- erties of molecular materials, ultrasonics and the development of sensors based on these properties.