Chern. Anal. (l*lrsaw), 41, 715 (1996)

Itnmunosensors in Analytical Chemistry

by Wojciech Dzwolak, Robert Koncki and Stanislaw GJ~b

Department ofChemistry, University of Warsaw Pasfeura 1, 02-093 Warsaw, Poland

REVIEW

Key words: biosensors, immunosensors, potentiometry, amperometry, piezoelectric

sensors, optodes

This article is a review of literature devoted to immunological biosensors. The descrip­tion of the principles of their action and comparison of basic types of immunosensorsand their applications are presented. Electrochemical, piezoelectrical and optical immu­nbsensors are characterized.

Artykul stanowi przeglc\d literatury dotyczC\cej bioczujnikow immunologicznych. Za­wiera omowienie zasad dzialania i por6wnanie podstawowych typ6w immunoczujni­k6w i zakresu zastosowall. W pracy scharakteryzowano i om6wiono immunoczujnikielcktrochemiczne, piezoclektryczlle i optyczllc.

Introduction

The increasing expectations of the modern clinical and environmental analysiscause enormous development of all analytical methods which allow to estimate smallamounts of biologically active substances. In the field of molecular biology andmedicine, especially important seem to be the problem of identification and quanti­tative analysis of macromolecular biopolimers. This problem could not be solved byusing only classical analytical methods. Almost each published method employs anclement of the so called molecular recognition of a given sequence of nucleotides ina DNA chain, or aminoacids in a protein chain. In the first case the role of themolecular recognition clement is played by restriction enzymes or radioisotope­labeled short oligonucleotides which show affinity to the complementary site at theDNA chain. In the case of protein analysis, only specific antibodies - gammaglobu­lins arc applicable. This approach utilizes the ability of gammaglobulins (antibodies)for selective binding to the defined sites of an other protein - an31yte (antigens).

716 W Dzwolak, R. Koncki and S. Glqb

Specific reactions bctween antibody and antigen arc basis for all immunologicalmethods like enzyme linked immunosorbent assay (ELISA), radioinullunoassay(RIA),immunoanalysis with antibodies labelled with a fluorochrome [1-3].

Combination of certain substrates of the immunological reaction with a trans­ducer leads to a special group of biosensors called immunosensors. These analyticaldevices allow to detcrmine specifically various biologically active compoundS, as itcan be done with traditional inllllunoanalytical methods, and give a possibility ofminiaturization of the analytical instrumcntation. Another advantage of ihis methodis the fact that automatization of particular analytical procedures becomes easier. Itis also very important that immunosensors can be used repeatedly, what lowers theconsumption of very expensive immunochemical compounds. Many types manykinds of immunosensors based on different physical and chemical transducers andvarious methods of protein immobilization on the surface of a sensor have beendescribed in the literature. Recently several monographs and review articles devotedto immunosensors have beenpublished [4-9]. Despite intensive work on immunosen­sors they have not reached until now a commercial success. This phenomenon isanalyzed in article [10]. The present article reviews various kinds of immunosensor,compares them, and evaluates their likely applications in the future.

The immunological complex

Formation of thc immunological complcx is a consequcnce of formation of thespecific bonds between antibody and antigen. From the chemical point of viewantigens arc mostly proteins and glicoproteins. There is one more characteristicfcature of all types of antigens beyond the ability to form an immunological complexwith antibody: its ability of inducing specific immunological response ill vivo againstthis antigen. Thcre is a large group of chemical compounds of low molecular weightcalled htlptens which, like antigens, can react with antibody. However, in this caseno immunogenic propcrties are observed. The structure of immunoglobulin molecule(antibody IgG) is shown in Figure 1. IgG molecular mass is about 160.000 daltons.It consists of four protein chains of two differcnt kinds: Hand L which arc connectedby disulfide bonds. It is known that only the Fab fragment of gauullaglobulin(Fragmcnt antigen binding) is involved in the process of immunological recognition.Another fragment, Fc (Fragment crystallizable), is not engaged in recognition ofantigen, and is utilized in many immunological methods [2] because of its selectiveand firm binding with proteins A or G. The structure of Fab fragment involves bothkinds (H and L) of chains. The molecular recognition process betwecn antibody andantigen leads to gencration of an immunological complex (Figure 2). This proccssconsists in connecting the antibody with the antigcn by the so-called immunologicalbinding at special sites of both molecules. The site at the Fab fragment that isresponsible for this binding is called paratope and the complementary site on thesurface of antigen is called epitope. Very high complementarity between the paratopeand the epitope is responsible for very specific immunological interactions betweenthe antibody and the antigen. The nature of immunological binding is non-covalent

Immunosensors in analytical chemistry 717

and it consists in electrostatic and dispersive interactions between the side groups ofboth the protein chains. Dissociation of the immunological complex can be reversiblyachieved in the solution of very high ionic strength and low pH [1,7,11].

Disulfide Bonds

COOH

Figure 1. The basic immunoglobulin structure

Fe

Figure 2. Formation of immunological complex

Paratope

~Antibody

In the case of enzymatic biosensors, the geometrical orientation of the proteinmolecule on the surface of the sensor is not very important unless a decline in theenzyme activity is caused. This is because of usualy lowlllOlecular Inass and small

718 lY. Dm-'olak, R. Koncki and S. Glqb

dimensions of the molecule of the analyte. Owing to that, the analyte can penetratethe enzymes structure freely, and finally, despite any sterical obstacle, it can reachthe active center of the enzyme. The situation seems to be different in the case ofimmunosensors being applied to assays ofbiopolimers of very high molecular mass,when the role of the sterical factor is much more significant. Random immobilizationof the antibodies makes many of them turn towards the sensors surface with the onlyone biologically active fragment Fab. Then a big molecule of the analyte cannot reachthe compiementary molecular receptor, what resuits in disfunction of the antibody.An obvious solution to this problem is using the fragment Fc for forming a properlybuilt antibodies monolayer [7].

Generation and transduction of the analytical signal

Unfortunately it is a characteristic feature of the immunological complex that itsformation is not accompanied by generation of any chemical signal. That is whyphysical transducers which can be sensitive to formation of immunological complex(e.g. quartz microbalance) arc widely utilized in the construction of immunosensors.Another approach employs antibodies or antigens labelled with substances capableof generating a chemical signal (e.g. enzymes), and chemical transducers as the innersensor (e.g. potentiometric and amperometric electrodes). The only one exceptionfrom this rule is a group of immunosensors employing a special kind of antibodies ­so called catalytic antibodies (abzymes) [12-14]. There are no structural differencesbetween abzymes and ordinary antibodies, however, their specific properties arcrelated to the fact that the abzymes have affinity towards metastable state of certainreactions rather than towards typical antigens. This property of abzymes makes ametastable state of a reaction more stable which results in a considerable increase ofthe rate of this reaction. The potential advantage of abzymes over enzymes is thesusceptibility of forming antibodies catalyzing any needed chemical reaction. Ac­tually catalytic abilities of abzymes are several orders of magnitude lower than thoseof enzymes catalyzing the same reactions. Special characterof molecular recognitionprocess between abzyme and analyte suggests that the biosensors employing abzymcsshould be classified among enzymatic biosensors rather thiHl immunosensors. So faronly abzymes cati)lyzing hydrolysis of esters and short-chain peptides has beenobtained [12-14]. This makes the abzyme based biosensors even less promising forthe future. These disadvantages and a rather small number of articles devoted to theabzyme biosensors (e.g. [15]) point how weak is the hope that this group of sensorswill ever make a substantial progress in the immunological analysis.

A very useful division of immunosensors into homogenic and heterogenic sensorsis often given in the literature [4,7]. Homogenic immunosensors are these whichduring the analytical procedure demand only one (in the sample solution) incubation.Heterogenic immunosensors (based on competition or sandwich systems) demandseveral incubations in different solutions. Instead of this division we will laterintroduce another classification considering the kind of transducer that is applied forcooperation with immunological element of molecular recognition.

Immunosensors in analytical chemistry

Electrochemical immunosensors

719

Many different immunosensors based on electrochemical transducers like ionselective electrodes, ISFETs, Clarks electrode and other amperometric sensors havebeen already introduced. Their basic advantages are their availability and a low costof all necessary equipment. Some typical solutions among electrochemical immu­nosensors were reviewed in article [16].

Potentiometry has accelerated considerably its progress in the field of enzymaticbiosensors [17], and found application also in the construction of immunosensors.The simplest potentiometric immunosensorwas described by Aizawa in the seventies[18]. In that paper approach a membrane electrode was covered with an antibodyagainst another kind of antibody (Wassermann's antibody) existing in the blood ofpeople infected with syphilis. The consequence of binding between the antibodyimmobilized on the membrane and the antigen from the sample solution was theaccumulation of electrically charged protein particles on the electrode, what wasaccompanied by a chclllge in the electrode potential. This change was not caused bythe selective transport ofa certain ion through the membrane. Avery similar principlewas applied in an immunosensor described by Li and co-workers [19]. Their purposewas to work out an inullunosensor for detection of the hepatitis surface antigen type B.This sensor allowed only qualitative detection of the antigen in human plasma. ThecompctillOn bCtWCCllthc antigen immobilized on the surface ofsensor and thc antigenfrom the sample solution (analyte) in formation of the immunological complex withantibody was utilized in the construction of a heterogenic potentiometrici1l11l1unosen­sor for estimation of digoxin [20). Theimmunosensor was made of an ion selectiveelectrode with a modi fied ionophore. The applied ionophore was a crown etherconjugated with hapten - digoxin. The ionophore modified in this way is still able totransport the potassium ion through the membrane that results in a change of thepotential of the electrode. The immunosensor was incubated in a solutio!l containinganalyte - digoxin and a constant concentration of antibody against digoxin. In thecase of low concentration of the analyte in the sample thc antibody is mostly boundby an antigcn conjugatcd to the ionophore from thc surface of the sensor. Hydrophi!­icily and large dimensions prevent the antibody particles to get inside of the hydro­phobic mcmbrane. The freshly formed immunological complex between the antibodyand the digoxin concentrates at the membrane-sample solution interphase. Then theionophore linked to digoxin, which is present in the membrane, becomes immobilizedand ruled out from the ionic transport through this membrane. This latter fact causeschange in the potential of the electrode. If the concentration of the analyte is higherthe formation of immunological complex wilh the antigen in the solution is favoured,more molecules of the ionophore are still able to carry ions through the membraneand the changes of the electrodes potential are smaller. That is why the potential shiftis inverscly proportional to the analyte concentration. This immunoelectrode can bealso used as homogenic inll11UnOSensor for antibody against digoxin.

A similclr principle is utilized in the immunosensor for determination of prosta­glandin PGEz [21]. It seems that the major disadvantage of the described methods isthe need of preparing conjugatc ionophores with haptens, what results in a dramatical

720 W DntJolak, R. Koncki and S. Glqb

decline of the ionophore mobility in the membrane and in increase in the assay time.There are different ways to eliminate these difficulties: one of them refers to a veryfew systems where the antigen and the ionophore is the same compound, or theantigen is capable of selective complexation of a particular ion. The potentiometricimll1unosensor with the membrane sensitized with hapten-dinitrophenol which is anionophore for potassium ions can be recalled here as an example [22]. The potentialof this electrode is proportional to the concentration of potassiUIu ions. The immu­nosensor is incubaied in a solution containing a constant amount of potassium ionsand the analyte - antibody for dinitrophenol. In this situation thei potential of theinuuunosensor depends on the concentration of the latter substance. This phenome­non can be easily explained by the blocking influence of the antibody on theionophore, like it was in the immunosensor for digoxin.

It must be stressed here that most of potentiometric imluunosensors employantibodies lclbelled with enzymes (so called conjugates). This group of itmuunosen­sors can be divided into the immunosensors adapting a competition system (Figure 3)and the immunosensors adapting a sandwich systems (Figure 4). There is an interes­ting example of the first group - the immunosensor made of a gold electrode coveredwith the antigen-pig insulin [23]. The sensor was incubated in a solution containinginsulin as analyte and a constant, known amount of the conjugate of the antibodyagainst insulin with laccase, the enzyme catalyzing reduction of oxygen. In theincubating solution the antigen from the surface of the electrode and the antigen(analyte) from the solution compete to form the immunological complex with theantibody-enzyme conjugate. The lack or low concentration of analyte in the samplesolution allowes the process of billding the enzyme to the surface ofelectrode throughthe "immunological bridge". The higher concentration of analyte leads to immuno-

T\

sensor atter incubation

~ Washing step )II~I~ (Q ~

~Immunological Complex

in Solution

Conjugate of antibody -, ([)with enzyme ~

~(J)"'0o.1=o(J)

w

""hUn

LY'

Sensor

~II'%1"dn I

Analyte

)<'igllre 3. Competitive immunoassay of insulin

Immunosensors in analytical chemi~try

After Second IncubationI

second Antibody

• Annbody

'"Protein A

Figure 4. Sandwich immunoassay of human IgG

721

logical saturation of the conjugate in the bulk solution and therefore to low surfaceactivity of the enzyme. After incubation this immobilized activity is inverselyproportional to the concentration of insulin in the sample (see Fig. 3). The fact thatlaccase catalyses some redox reactions allows to use the potentiometric detectionwith a particular redox electrode. The concentration of oxygen in water is usuallyhigher than the Michaelis constant of laccase for oxygen, therefore this reaction isnot limited by oxygen concentration.

The competition and the antibody labeled with enzyme were also applied toconstruct an immunosensor sensitive for human IgG [24]. In this case the humangammaglobulin was the estimated analyte and the source of antibody was the goatplasma. The antibody was labeled with urease which catalyses the hydrolysis of urea.A pH-ISFET with crosslinked IgG was adapted as an interior sensor. The followinganalytical procedure was similar to the previous example (Fig. 3). The amount ofurease immunologically bound to ISFETs surface was inversely proportional to theconcentration of human IgG. After biosensor incubation in the sample solution, itwas placed in the urea solution. Urea cannot be found as an electroactive agent,however the products of its hydrolysis (ammonia and carbon dioxide) can be, sincethey arc in equilibrium with hydrogen ions. The pH shift caused by the enzymaticreaction in the transistor sensitizing layer was detected by ISFET. The authors of thiswork treated as analytical signals both the change of the potential and the initial rateof potential change. They both arc inversely proportional to concentration of IgG inthe sample. Application of the FETtechnology to construction of immunosellsors canlead to miniaturization of the sensors. The quoted example of an inUllunosensor isalso characterized with a good stability and a possibility of muHiple use (up to 30times) The detection limit equal to 10 ~g ml-1 of human IgG is in accordance withthe expectations of clinical analysis.

722 l¥. Dzwolak, R. Koncki and S. Glqb

Another example of heterogenic competitive immunosensor is a modified ISFETfor determination of a herbicide, atrazine [25,26]. In this case protein Ais covalentlyimmobilized on the sensors surface. The origin of protein A are cellular walls ofStaphylococcus. The protein has a great affinity towards fragment Fc of gammaglo­bulin [2]. For this reason the antibody against atrazine is coupled to sensor on theprotein A matrix. Application of protein A provides an inllllunosensor with a highlyordered arrangement of antibody layer. This layer is made of molecules of IgGoriented to the solution with the Fab fragments. The advantage of this solution wasexplained earlier. The biosensor was incubated in a solution containing atrazine andthe conjugate of ametryn-glucose oxidase at a known concentration. Ametryn is acompound having the structure very similar to atrazine but much more prone tochemical linkage with proteins. Glucose oxidase is an enzyme which catalyzes thereaction of oxydation of glucose to gluconic acid. During incubation a competitionbetween atrazille and the labelled ametryn takes places. This concerns the bindingwith antiboHy immobilized on the sensors surface. The direct source of the analyticalsignal is acidification of the biocatalyticallayer due to appearing of gluconic acid asan effect of enzymatic reaction in glucose solution. The immunosensor allowed toachieve a limit of detection at the ppb level. The principle of functioning of theimmunosensor is presented in Fig. 3.

Another application of protein A was that in the construction ofan immunosellsorsensitive for human IgG [25,26]. This time instead of a competitive method asandwich system was applied (see Fig. 4). The process of immobilization of theantibody on the sensor surface was performed like in the previous case with protein A.Then the modified immunoFETwas incubated in the solution containing the analyte.After this the next incubation in conjugate of glucose oxidase with antibody againsthuman IgG solution took place. It is important to stress here that the antibody usedin the second incubation had an affinity to another epitope in the IgG molecule. Asa consequence of such a procedure a proportional dependence between the concen­tration of the analyte during the first incubation and the surface activity of boundenzyme was observed by potentiometric measurements during incubation in glucosesolution - like in the previous case. The detection limit was found to be at the levelof 0.1 ~g ml-t . Again it should be stressed that the sandwich systems cannot supportanalysis of the antigens having only one antigenic determinant (haptens like atrazine)because binding the antigen with the first antibody (bond to protein A) results inblocking this site for another immunological binding. Since this moment the antigenwould be immunologically irrecognizable for another antibody. Except for these twoapproaches, which are quoted after articles [25,26], there are many other sandwichand competitive methods. The major origin of the plenty of different designs in thisfield is a possibility of immobilization of different immunocompounds labelled withvarious enzymes. Sensors with firmly, covalcnlly bound protein A were reported tobe universal bases for the cOlistruction of immunosensors for various antigens andantybodies.

Another application of the pH-ISFET is the construction of an iUllllunosensorsensitive to herbicide 2,4-dichlorophcnoxyacetic acid [27]. In this work an antibodyagainst the analyte was immobilized on porous photo-activated cellulose membranes

Immunosensors in analytical chemistry 723

which then were attached to the gate of the FET. During the incubation step thecompetition between the analyte from sample solution and the conjugate of herbicideand horseradish peroxidase was taking place.

An example of rare homogenic ImmunoFET was presented by Schasfoort et a1.[28]. In this case the process of generating the analytical signal concerns the directpotentiometric sensing of protein charges.

The most obvious disadvantage of the most of mentioned types of immunosen­sors is their heterogenic character resulting in an inevitable need to perform severalincubations step by step. One of the trys to overcome this problenl counted onabzymes utilization. Another project for the construction of an immunosensor wasreported in article [29]. This immunosensor for human IgG employs a complexbienzymatic system where the product of one enzyme - catalyzed reaction is asubstrate of the next one. The biosensor was based on a potentiometric electrodesensitive for the ammonium ions with the surface covered with two proteins: humanIgG and enzyme - adenosine desaminase (E1), which catalyses the following reaction:

Adenosine + H20 ---(El)~ Inosine + NH3

The immunosensor was incubated in a solution containing adenosinemonophosphate(AMP), the analyte - human IgG, and the conjugate of the molecular receptor for theanalyte - protein A, with enzyme - alkali phosphatase (E2), which catalyzes thereaction:

5'-AMP +H20 ---(E2)~Adenosine + PO~-

Protein A fulfills the function of the antibody against IgG. Under the conditions ofthe incubation the competition process between IgG from the surface of the sensorand IgG from solution (analyte) in the formation complex with protein A takes place.The higher concentration of analyte results in more saturated protein A and lessactivity of alkaline phosphatase at the electrode surface. Finally the amount ofdirectly bound (through the bridge protein A-IgG) molecules of enzyme (E2) isinversely proportional to the analyte concentration. In this way the direct neighbor..hood of molecules of two enzymes was favoured. Then the diffusion of continuouslygenerated adenosine to dcsaminase is fast and the resulting potential change of theelectrode (as a consequence the increase of the concentration of ammonium ions) isalso fast. As the analyte concentration is higher, "free" IgG competes with IgGbonded to adenine desaminase in binding with protein A. The distance to be coveredby diffusing adenosine to desalllinase (El) layer is effectively longer and this resultsin a less rapid potential change of the electrode. The obtained limit of detection ofhuman IgG by this method was 5 Ilg llll-l.

Amperometry was found not to be less promising in applications of imlllunosen­sors than potentiollletry is. The first paper devoted to amperollletric immunosensorsappeared in seventies [30]. Amperometric immunosensors, just like potentiometricinununosensors, demand only quite inexpensive and widely available equipment. The1113illadvantage of amperometricimmullosensors over potentiometric are theirbetter

724 W Dnvolak, R. Koncki and S. Glqb

sensitivity, shorter response times and greater prospects for miniaturization (ultra­microelectrodes). Most of amperometric imlUunosensors employ the antibodieslabeled with enzymes producing electroactive substances. Various competitive andsandwich systems were used to construct such immunosensors.

The amperometric immunosensor for the cancer marker, a-fetoprotein (AFP),determination is a typical example of this kind [31]. In this work a Clark electrodewas applied. On the electrode surface an antibody against AFP was immobilized. Thesensor was incubated in a solution containing the analyte and the conjugate catalase­AFP, and then dipped into a solution of hydrogen peroxide. Catalase decomposeshydrogen peroxide into oxygen and water. The measured current was proportional tothe concentration of oxygen and activity of bound catalase. Thus the amount ofcatalase bound immunologically to the Clark electrode directly influences the analy­tical signal. Considering the conditions of the competition, the analytical signal isinversely proportional to AFP concentration in the sample. Other kinds of amperome­tric iUllllunosensors employing both a Clark electrode and the immunoconjugateswith catalase were introduced [5].

An inullunosensor employing a Clark electrode covered with the antibody againstglicoprotein GP41 of the HIV virus was decribed [32]. The analytical procedure wasbased on immunological binding to this antibody of the peptide EGIEE labelled withcatalase. The antigenic properties of EGIEE arc very similar to glicoprotein GP41,however, the immunological affinity towards corresponding antibody is slightlysmaller. The immunosensor was incubated in a solution containing glicoproteinGP41. Then labelled EGIEE was replaced by GP 41 from the immunological bindingwith a Clark electrode. In this way, after the incubation in the sample solution, theenzyme activity was inversely proportional to the analyte concentration. The higherconcentration of ana lyte the lower the activity of enzyme, the higher concentrationof oxygen in the sensing layer of electrode, and finally the higher current measured.

An amperometric immunosensor for the determination of mouse IgG employdthe labelling with another enzyme [33]. For the assembly of this immunosensor asandwich system with galactosidase was adapted. The enzyme is capable of catalyz­ing the reaction of p-aminophenyl-f3-D-galactopyranoside hydrolysis which givesp-aminopbenol and galactopiranoside. The first product is electroactive and can bemeasured voltametrically at a glassy carbon electrode. The application of cyclicvoltametry with the described sensor, used as a working electrode, leads to a verylow (0.1 ppb) detection limit.

The competition was found to be applicable in the construction of the amperome­tric inullunosensor for detection of herbicide - 2,4-dichlorophenoxyacetic acid [34].This antigen was immobilized on the surface o.f the working electrode - in this casea glassy carbon or a gold electrode. Then the sensor was incubated in a solutioncontaining the conjugate of an antibody against 2,4-dichlorophenoxyacetic acid withperoxidase and analyte. The competition between the immobilized and free antigenscaused the inverse dependence of the analyte concentration on the surface activity ofthe enzyme. After this step the sensor was placed in a solution containing twosubstrates of the enzymatic reaction: hydroquinone and hydrogen peroxide. Theproduct of this reaction-quinone was then reduced electrochemically and that resulted

Immunosensors in analytical chemistry 725

in the generation of the analytical signal. The obtained detection limit was 0.1 ppb.In the same paper [34] a possibility of using different substrates for the enzyme wasalso examined.

A very similar approach was adapted for the assembling of an immunosensorsensitive for mainllletabolite of coumarin in human body -7-hydroxycoumarin [35].Small molecules of 7-hydroxycoumarinwere immobilized on a glassy carbon elec­trode with the help of a supporting protein (thyroglobulin). In this work the compe­tition and the antibody labelled with peroxidase were applied. TIle substrates for theenzyme were hydrogen peroxide and hydroquinone. The limit of detection was24/.tmoll-1•

The aim of the authors of the article [36] was to work out an amperometricimmunosensor sensitive for apolipoprotein E, the level of which in serum is anindicator of the condition of lipid metabolism in the human system. A sandwichsystem and an antibody labelled with alkaline phosphatase were applied. The sub­strate for the enzylnatic reaction was p-aminophenol phosphate and p-aminophenolwas itselectroactive product. The latter compound was then oxydized at a glassycarbon electrode. The measured current was proportional to the activity of the boundenzyme and the concentration of the analyte. The immunosensor based on thisprinciple gave a linear response in the analyte concentration range frot» 50 to 1000ng ml-1 of apolipoprotein E.

The first amperometric immunosensor for simultaneous estimation of two mac­romolecular analytes - hormones FSH (follicle stimulating hormone) and LH (lutei­nising hormone) has been described [37]. Both hormones are responsible for thepubescence. In wonlen, the cyclic changes of the level of each of these hormonesdepend on the current stage of the menstruation cycle. That is the origin for theimportance of these hormones in clinical analysis. The authors applied. a sandwichsystem with special "light addressable" method of immobilization of antibodiesagainst FSH and LH on the surface of a gold electrode. A biosensor with sites sensitiveto two different antigens was received. The immunosensorwas incubated in solutionscontaining the analytes, which were bound to the corresponding antibodies. The nextincubation was done in the presence of the conjugates of the antibodies to bothantigens with peroxidase. The substrates for enzymatic reaction were hydrogenperoxide and ferrocenic acid. Duringchronoamperometric measurements the currentreferring to the sites with immobilized antibody against FSH or LH was proportionalto the concentration of the corresponding antigen (hormone). The detection limitswere 2.1 V ml-1 and 1.8 V mr1 for FSH and LH, respectively ("V" is a unit ofhormone activity).

One of the most important problems in all immunoenzymatic methods is the needto apply very expensive and unstable conjugates of enzymes with antibodies (sand­wich systems) or with antigens (competitive assays). This problem can be overcomepartly in the immunological methods with amperometric detection and adaptation ofthe antibodies labelled with elcctroactive agents. However, among all quoted solu­tions it is hard to point even one example which could to be called "immunosensor"[38,39], but rather an amperomctric detector in immunochemical, analytical configu­ration.

726 lv. Dzwolak, R. Koncki and S. Glqb

Barnett atai. [40] introduced an antibody against p-cresol into the structure ofthe conducting polymer (polypyrrole). The polimer was electrochemically depositedon a platinum disc. The immunosensor obtained in this way was used as a workingelectrode for determination ofp-cresol by pulse voltametry in flow analysis system.The measured current was proportional to the concentration of the analyte in thesample solution. Although the details of the mechanism of functioning of thisimmunosensor are still not quite understood it must already be admitted that thisdevice can detect p-cresol down to 0.1 ppm.

A rare example of conductometric immunosensor was described [41]. It wasbased on the dependence between the amount of electrically charged protein particlesdeposited on the metal surface and the impedance of the system metal/proteinlayer/solution. The authors of this work managed to achieve a success in thisenterprise by using a very thin platinum film of the thickness 25 l.tIn. On this film,an antibody against Staphylococcus enterotoxin type B was immobilized. The de­pendence between the logarithm from the impedance changes and the concentrationof enterotoxin in the sample solution was linear in the analyte concentration rangefrom 0.4 to 10 ppb. It seems that the most obvious advantages of this immunosensorare its homogenic character and the ability of miniaturization. The main shortcoming,typical for all conductometric methods is their unspecifity.

Piezoelectric immunosensors

Piezoelectric immunosensors do not require, like the already mentioned abzymeusing biosensors and the optical sensors that will be discussed later, any antibody andthe antigen labelling. The basic physical transducer used to build a piezoelectricimmunosensor is a quartz crystal microbalance. Piezocrystals have already found awide range of applications in analytical chemistry long before their use for theimmunosensors building [42]. The idea of the measurement utilises the change of theoscillation frequency of the crystal caused by adsorption of a mass on its surface.

The first applications of the piezoelectric phenomenon in analytical chemistryappeared in early sixties. The first piezoelectric immunosensor has been presentedin 1972 [43]. In this paper only initial research has been presented on the possibilityof applying piezoelectric immunosensor for determining the Bovine Serum Albuminantibody activity. Aquartz crystal has been coated with polymer to which the proteinmolecules (in this case Bovine Serum Albumin antibody) adhere easily. Binding ofthe antibody (the analyte) with the antigen immobilized on the piezocrystal surfacetakes place during the biosensor incubation in the solution of the antibody. Analyticalmeasurement is conducted after washing and drying the sensor. In this way theinstability of the crystal oscillation, caused by its contact with the solution can beavoided. This phase of the analytical procedure is typical for all of the early designedpiezoimmunosensors. Recently some papers appeared, in which the piezoelectricimmunosensor is reported to remain in the sample of the examined liquid during thewhole time of the analysis. These problems are more thoroughly discussed in a reviewpaper dedicated to piezoelectric sensors [42,44,45]. In all previously discussed types

Immunosensors in analytical chemistry 727

of immunosensors the requirement of water in which the measurement is carried outwas necessary for three major reasons: stabilization of the proteill molecule, trans­portation of the electroactive agent to the sensor, and fixing the electrochemicalequilibria at the membrane-solution interface.

Because of a completely different method of the measurement, the piezoelectricimmunosensors gave new possibilities, what resulted in some elaborations on thepiezoelectric immunosensors for the estimation of such heptens like toluene, nitro­toluene or parathion in the gas phase [46,47]. As an example serves an immunosensorfor detection of ricin, a strong toxin of potential military use [48]. In this work twomethods of immobilization of the antibody on the piezocrystal surface has beentested: with protein A, and through the adhesion methods. The latter method gave anabsolute detection limit of 0.5 f.lg of the toxin, which is twenty times lower than thatobtained by using the immunosensor with the antibody immobilized by protein A.Such regularity should be explained by a large and passive damping mass of proteinA itself, which decreases the sensitivity of the sensor. A similar comparison of themethods of the antibody immobilization was performed with an immunopiezoelectricsensor for cortisol [49]. The metl:od with protein A is more stable compared to theadsorption and the crosslinking (esing glutaral) of antibody. The working range ofthis immunosensor was 40-35CC ppb. The biosensor was successfully tested forclinical use. Other steroids did no~ interfere.

The immunoscnsor for dctection of virions of the hepatitis type A and B viruswas described in [50]. Protein A was noncovalently immobilized on the sensorsurface. Through this protein antibody against the virus thc virions surface glicopro­tcins were bonded to the scnsor. Absolute detection limit achieved with this methodwas 10000 virions.

The piezoimmunosensor for IgM [51] is worth of noticing due to utilized thereimmobilization technique of molecular recognition element on the surface of thesensor. This method allows to· achieve a high superficial concentration of the proteinon the sensor surface, what results in a better sensitivity of the analytical method anda better durability of the protcin layer, thus expanding thc sensor lifetime. It consistsin the silanizcd crystal surface. Adiffercnt mcthod ofcovalcnt protein immobilizationfor piezoelectric immunosensors on thc quartz surface with cyjanogcn bromide wasalso described [52]. It gavc an antibody layer capable ofantigcn binding for 3 months.A piezoelectric sensor for the atriazine estimation at the level of 0.03 ppb was alsopresented [53]. In this case a typical method of immobilization using the protein Ahas been utilized. To eliminate the influence of the nonspecific adsorption differentialmeasurements have been made. The reference crystal was coated with the antibodyagainst human IgG showing no immunological affinity to atriazine but having thesame, as the antibody against atriazine, ability to bind nonspecificaUy with othersubstances.

A more sensitive method of detcction of atriazine which is based on a compilationof conventional piezoelectric immunosensor and the competition as discussed earlieris described in [54]. An antibody against atriazine is bound to the surface of the scnsorthrough protein A. Thc sensor is then incubated in a solution containing analyte, theatriazine, and the conjugate of atriazine and much bigger protein molecules (perox-

728 lY. Dzwolak, R. Koncki and S. Glqb

idase - in this case the enzyme plays the role of the mass marker). In the case of lowanalyte concentration, mainly atriazine labelled with the protein binds with thesensor, which is followed by a significant growth of mass and a large change ofoscillation frequency. The higher is the analyte concentration the stronger it bindswith the sensor, and the lower is the change of the oscillation frequency. The limit ofthe atriazine detection given by the authors is approximately 0.001 ppb.

An immunosensor with gold electrodes of piezoelectric system coated with theantigen, the gammagiobulin, oniy one of which is immersed in the solution contain­ing the analyte, an antibody against gammaglobulin [55]. This concept is important,since it makes possible measurements of oscillation frequency changes withoutremoving the·sensor from the solution.

Piezoelectric immunosensors have a potential allow of detection of even largerbiological objects such as microorganisms, analytical detection of which by electro­chemical immunosensors seems to be impossible. A prototype of piezoelectric sensorcoated with antybodies against antigens located on the surfaces of the Escherichiacoli bacteria cells was already reported [56]. The immunological binding of wholecells drastically changes the analytical signal of sensor. The most important advant­age of this method, in comparison with traditional coli estimation, is its rate.Biosensor that has been described here produced a linear answer to the analyte, thebacterial cells, in the range from 10 to 106 in 1 ml.

Piezoelectric immunosensors constitute a reasonable alternative for electro­chemical immunosensors. They do not require labelling antibodies with enzymes,and allow a very fast measurement without any additional incubations. Although thelevel of noise registered during the measurement utilizing piezoelectric immunosen­sors is significant, their detection limits can be much lower than those of theeiectrochemical immunosensors. The latest papers in this field aim at further increas­ing of their sensitivity through binding masses much larger than the analyte itself tothe sensor surface during the analysis (sandwich and competitive methods).

Optical immunosensors

Optical immunosensors undoubtedly constitute the most technologically ad­vanced and rapidly developing group of immunosensors. The most common opticaltechniques used are: ellipsometry, Internal Reflection Spectroscopy (IRS), and mole­cular spectrophotometry (absorption sensors) [7,57,58]. A specially detailed discus­sion of the physical principles of the optical phenomena utilized in these methodscan be found in monographs [57,59]. The first of the mentioned methods utilizes theelliptical depolarization of the polarized light interacting with the given environment.The second method includes a whole group of different solutions that have a commonthing: the transport of light by the optical fiber, which is based on the principle of thetotal internal reflection; the beam of light cannot leave the optical fiber due to theratio of the refractive indexes of the external environment and the material of thefiber [59]. Whereas the absorption methods utilize the Lambert-Beer law for the caseof light propagation through a thin layer of receptor at the end of the optical sensor

Immunosensors in analytical chemistry 729

••II

"Antigen -Analyte

~~~~ Porous Membrane

.·1/•• IDS

a......-..I-----I.-......&.....-..I.

[57,59]. An extensive discussion of many optical methods applied in building opticalsensors, considering the specific cases of immunosensors has been published else­where [57].

Ellipsometry had already been used in analytical chemistry before its applicationin immunosensors [57]. A competitive biosensor for y-interferon and human serumalbumin (HSA) is a typical example ofan ellipsometrical immunosensor [60]. On thesurface of the sensor, the proper antigen has been immobilized (e.g. 'Y-interfero~l) andthen the sensor has been incubated in a solution containing an unknown concentrationof the analyte (the same antigen) and the antibody against it. The surface concentra­tion of the antibody immunologically bound to the sensor surface was inverselyproportional to the analyte concentration in the incubation solution. Surface concen­tration of this antibody has been ellipsometrically traced by the measurement of theprotein layer refractive index changes. The sensor showed the detection limit fory-interferon at the level of 15 nmoll-1and for the HSA at 2.5 nmoll-1.A significantdisadvantage of the ellipsometric method is the necessity of using complicatedapparatus. The methods utilizing IRS include the following techniques: Total InternalReflection Fluorescence (TIRF), Attenuated Total Reflection (ATR), Surface Plas­mon resonance (SPR), Fluorescence Capillary Fill Device (FCFD).

TIRF is a miniaturized version of the Fluorescence Molecular Photometry. Atypical experimental design in such the case is shown in Fig. 5. An optical fibertransmits the excitation light beam into a small cell on its end equipped withsemipermeable membranes. Inside the cell there are molecular receptors for theantibody against the given antigen (the analyte) immobilized on its walls, and anumber of non-immobilized antigens labeled with fluorochrome. The end of thesensor is immersed in the examined solution so that the analyte can penetrate throughthe semipenneable membrane into the cell. Under such conditions a competitionoccurs between the fluorescently labelled and non-labelled antigens. In the case of

AntibQdy

Figure S. Scheme of typical TIRF device

730 lY. Dzwolak, R. Koncki and S. Glqb

highanalyte concentration only a small number of antigen molecules labeled with.fluorochrome can immunologicaly bind with the cell facets, because many of themremain in the area of the cell which is "enlighted" by the exciting laser beam. Thesemolecules of labelled antigen become the source of the secondary fluorescentradiation. Final intensity of the fluorescence is inversely proportional to the analyteconcentration and is measured via the optical fiber by a spectrophotometer. Adiscussion of some instrumental details and a theoretical model of such immunosen­sor work are included in paper [61].

ATR in opposition to TIRF does not require fluorochromes as labels. This methodis based on the light propagation through the optical fiber partly outside of the coreat a depth of 10 to 40 nm. The intensity of this beam becomes lower with the distancefrom the core of the fiber. This method is called the Evanescent Wave Spectroscopy(EWS) [59]. Han antibody molecule is placed in the vicinity of the fiber the lightwill partly diffuse on it. H the antibody binds with the antigen the diffusing obstaclewill become larger and the light intensity will become proportionally lower. Thus theevanescent wave spectroscopy is a method similar to turbidimetry. A typical use ofthis method is preselltedin paper [62]. ThCfimmunosensor for Botulinum toxin - Bpresented here utilizes an antibody against this analyte immobilized on the surfaceofthe optical fiber.

/ The sensors based on the SPR (a specific form of ATR), similarly to thosepreviously discussed, do not use fluorescent labels. Their construction is presentedin Figure 6 and is interesting. The optical fiber is coated with a thin film of conductingmetal on the surface of which an antibody or antigen is immobilized. At a specificangle of incidence, the evanescent wave striking the metallic film causes the reson­ance oscillation of the metal electrons to diminish. This manifests itself in theabsorption of the evanescent wave when striking at this angle. At the same time theenergy caused by it to propagate is dissipated. This is very sensitive to refractiveindex of the substance adjoining the metal layer. The binding of a proper immuno­logical component to the metallized surface of the SenfJr changes sharply therefraction index of the layer deposited on the metal and, because of it, the angle atwhich the evanescent wave is absorbed. Thespectra showing the dependence of therefracted beam intensity minimum point to the angle at which the beam strikes themetal for three superfjcial cases are shown in Fig. 7. Paper [63] presents an SPRimmunosensof sensitive to the specific antibodies present in the serum of syphiliticpatients. A glass slide coated with 50 nm gold film has been utilized to build thesensor. The surface of the sensor was coated with an analyte receptor, a TmpA protein.The TmpA protein is an antigen against which the syphilitic patients produceantibodies. In order to increase sensitivity a sandwich system has been applied - thesensor with immunologically deposited antibody against. The TmpA protein has beenincubated in a solution containing the rabbit antibody against human IgG. Therefractive index shift caused by the presence of the analyte increases. Nowdays theSPR sensors are the largest group of immunooptodes. A review presents manydifferent problems important to SPR at the background of historical development ofthis optical method [64]. The paper [65] deals with the theoretical analysis of thedependencies in the SPR technique with special emphasis given to the problems.

Immunosensors in analytical chemistry 731

~Fiber Op11c

Bound Antibody

~•

~tigen

rThin Metal Film

Incident Beam Reflected Beam

Figure 6. Surface Plasmon Resonance (SPR)

f ':~':'·'::·:':'·'::·:':'·:·"i-$···;":':'·"::"·':'·"::"·':'·"';.! . . \

iQ) .....~\ \nf",~ 7:': ~:~ Melotwtth

adsorbed antigen Melol covel9d withiI'nnuloIogIcal complex

Angle of Reflected Beam

Figure 7. SPR spectra

Immunosensors described as FCFD sensors generate, likethe SPR sensors, a lightbeam at some angle. The angle is measured. Construction of a sensor of this type isshown in Figure 8. A fluorescence inactive beam of light strikes at the right angle,the glass cell on the interior facets of. which the antibody is immobilized. Thesubstance being inducted for this fluorescence is the fluorochrome labelled antigenof known concentration which is present together with the non-labelled analyte.Under such conditions a competition in forming an immunological complex occursbetween the labelled antigen and the analyte, non-labelled antigen. In the case of lowanalyte concentration the equilibrium of this process is shifted towards the immuno­logical binding of the fluorochrome to the glass facet. Each fluorochrome moleculebecomes a source of the secondary radiation emitted from the solution or from theimmunological complex monolayer. They reach the other glass slide playing the roleof the second optical fiber at different angle. Finally the immunological fluorochromebinds, through the antigen labelled with it, to the capillary, and this increases theintensity of the wave leaving the capillary at the lower angle. It should be mentionedthat, for a given analyte concentration, the angle changes are much larger than thosemeasured in SPR. The first detailed description of the FCFD immunosensor forhuman gammaglobulin detection is given in [66].

732 w: Dzwolak, R. Koncki and S. Glqb

Incident Light

I

-.Emitted Ught

Figure 8. Fluorescence Capillary Fill Device

Absorption immunosensors are based on the molecular spectrophotometry [57-59].An absorption immunosensor for human IgG detection is presented in paper [67]. Atthe end of the optical fiber a membrane containing the dye - pH indicator (BrilliantYellow) has been placed. The sensor has been equipped with an additional selectivegas permeable membrane, on the surface of which the antigen - human IgG has beenimmobilized. This is an ammonium gas optosensor senstitized immunologically. Thebiosensor has been then incubated in a solution containing an unknown concentrationof the analyte (human IgG) and a constant concentration of the antibody against IgGas well as the urease enzyme conjugate. Under such conditions a competition inbinding with the antibody between the IgG from the sensor surface and the IgG, theanalyte from the solution, occu.rred. The activity of the enzyme immunologicallybound to the ammonia optode surface was inversely proportional to the analyteconcentration in the first incubation. The sensor with the bound urease has beenmoved to the urea solution. Ammonia formed as a result of the enzymaticalycatalyzed hydrolysis penetrates through the semipermeable membrane to the dyelayer. The increase of ammonia concentration in the pH indicator layer results in achange of the pH indicator colour.

Table 1 summarized this review of immunooptical biosensors.

Table 1. Immunooptodes

Type of sensor Analyte Marker Ref.

Absorption human IgG urease 67SPR hCG hormone - 68SPR SHBG protein - 69SPR hCG hormone fluorochrome 70SPR GP41-HIV - 71

Immunosensors in analytical chemistry 733

Table 1 (continued)

SPR P24-HIV 1 - 72SPR mioglobine - 73SPR teofilin - 74SPR Ab(GP-HSV1) - 75

Ellipsometry humanIgG - 76Ellipsometry interferon - 60

TIRFlEllipsometry prolaktin HRP 77FCFD IVRVAg fluorochrome 78EWS rabbitIgG fluorochrome 79EWS H-CK-MB fluorochrome 80EWS botulinum toxin fluorochrome 81

Interferometry IgG - 82Interferometry HBsAg - 83

Immunosensors - comparison and trends

Present publications in the field of immunosensors lead to. a conclusion thatpiezoelectric and optical immunosensorsare the most rapidly developing sensors.The review of the optical immunosensors [84] pays special attention to their capa­bility to fulfill the market demamJ.. A specially important factor is that the progressbeing constantly made in construction of optoelectronic immunosc.nsors is muchfaster than the parallel development of electrochemical or piezoelectric immunosen­sors. It seems that optical immunosensors fulfill the requirements of an ideal immu­nosensor most easily: most of them do notrequire any additional incubation duringthe analytical measurement, they allow carrying out a remote measurements -due toa possibility of shaping the optkal fiber into a long and thin fiber, the fiber opticsmaterial allows applications ofa full range of immobilization methods ofthe antibodyon the sensor surface, application of sandwich and competition systems allows adramatic increase in the sensitivity of such sensors.

Consistent development of optoelectronics detennines that the final limitationsof the optical immunosensors abilities, effectiveness and price are unknown. But italso gives a hope that optical immunosensor will playa significant role in futureanalytical chemistry. Fundamental disadvantages of optical immunosensors are theirhigh price and necessity of a very complicated and often unique instrumentation.

A valuable comparison of the SPR method and the quartz crystal microbalanceas two possible attempts for immunosensors under the flow conditions has beenpresented [61].

Piezoelectric immunosensors are a reasonabie alternative for the electrochemicalimmunosensors: they do not require any antibody either antigen labelling withenzymes, they allow very quick measurement without any additional incubations.Although the level of the noise registered during the measurements with the pie­zoelectric immunosensors is significant, their detection limits can be far lower thanthe ones of the electrochemical immunosensors. The latest papers in the field ofanalytical chemistry aim at further increase of their sensitivity by binding to thesensor surface a mass much larger than the mass of the analyte (sandwich andcompetitive methods).

734 lY. Dzwolak, R. Koncki and S. Glqb

Interesting for analysts comparison of features of different types of immunosen­sors can be made by studying the results quoted in various papers on the differenttypes of immunosensors. for the same analyte detection. And so the previously quotedpaper using ISFET as an external sensor [25,26], an article dedicated to similar useof piezocrystals [53,54], and described in paper [85] the optical immunosensorutilizing RIFS allow a comparison of the detection limits achieved with differentimmunosensors for the same analyte, which was the atriazine. Previously presentedimillUnOenzymatic method [25,26] allowed detecting atriazine at the level of a fewppb, whereas a typical piezoelectric immunosensor described in work [53] can stilldetect this herbicide at the level of concentration of 0.03 ppb. But the most sensitivemethod of the atriazine detection is the one presented in paper [54] the limit ofdetection here was only 0.001 ppb. The detection limit for atriazine in the case ofoptosensor measurements, achieved by authors ofpaper [85] was approximately 0.1 ppb.

Immunosensors are a very attractive analytical tool that allows for detection ofsmall molecule herbicides as well as large molecule proteins. In each of these cases,the application of a immunosensor is followed by high sensitivity, low detection limit,short analysis time and the possibility of its further automatization. Those advantagesare partly compensated for a short lifetime of the sensors, and not excellent precisionof these methods. Decrease of the noise can be expected in the future by eliminationof the nonspecific molecular receptor-analyte interactions and reducing the apparatusnoise by still better electronics. A lot of attention is already paid to the problem ofnonspecific interactions. In turn this leads to highly specific monoclonal antibodiesor to the highly ordered molecular recep~or layers, for example with the Langmuir­Blodgett method [77,86-88], or with the application of the proteins selectivelybinding the gammaglobulin molecules, for example the protein A [25,26], or thebiotin-avidin system [37,69]. Paper [88] is devoted to the thermodynamic studies onthe Langmuir-Blodgett films with HIgG in aspect of their application for TIRFimmunosensors.

Opposite to other analytical methods, all types of immunosensors have veryimportant advantages in the case of application on the field of the environmentalanalytical chemistry. Those rely on the fact that methods ofmaking antibodies againstany analyte are known very well. The only need for preparation of a new immunosen­sor for detection of a new analyte is to obtain the proper antibody through standardimmunological procedures without any change of instrumentation. In addition,immunosensors, as all biosensors, can be used for remote measurements.

Specially important is the low cost of analysis with immunosensors due tomultiple use of the same costly antibodies in the form of a monolayer immobilizedon the sensor surface. Immunosensors are likely to be the only variant of immuno­logical methods possible for application in t~e on line measurements. Thus immu­nosensors, with their further development, can become a valuable tool, especially inmedical and environmental analytical chemistry.

Acknowledgments

The autlwrs are grateful to Prof. Adam Hulanicki for valuable discussions. One of the autlwrs (RK.)acknowledges a scholarship from the Foundation for Polish Science. This work was supported by a grantBW1301/13/95 from the University of WarsaMl.

lmmunosensors in ~nalytical chemistry

REFERENCES

735

1. Immunology (M. Jak6bisiak, Ed), PWN, Warszawa 1995 (in Polish).2. Immunocytochemistry (M. zabel, Ed.), PWN, Warszawa 1990 (in Polish).3.BiomedicalApplications ofImmobilizedEnzymes andProteins (T. Ming Swi Chang, Ed.), Plenum, New

York 1977.4.Analytical Uses ofImmobilized Compoundsfor DeteCtion, Medical andIndustrial Uses (G.G.Guilbault

and M. Mascini, Eds.), D. Reidel Publishing Company, Dordrecht 1988.5.Electrochemical Sensors in ImmunoiogicalAnalysis (T. T. Ngo, Ed.), Plenium Press, New York 1987.6.BiosensorsApplications in Medicine, Enviromental Protection and Process Control (RD. Schmid and F.

Scheller, Eds.), VCH Publishers, New York 1989.7. Campbell AM., Monoclonal Antibody andImmunosensor Technology,· Elsevier, New York 1991.8. Aizawa M., Phil. Trans. R. Soc. Lond., B 316, 121 (1987).9. Marco M.P., Gee S. and Hammock B.D., TrendsAnal.Chem., 14,341 (1995).

10. Manning B. and Maley T., Biosens. Bioelectron., 7, 391 (1992).11. Boitieux J., Biron M.P., Desmet G. and Thomas G;, Clin. Chem., 35 , 1026 (1989).12. Gallacher G., Biochem. Soc. Trans., 21, 1087 (1993).13. I7Atdyar 1..., Friboulet A, Remy H., Roseto A and Thomas D., Proc. Natl Acad. Sci., 90, 8876 (1993).14. Tramontano A, Janda K.D. and Lerner RA, Science, Z34, 1586 (1986).15. Blackburn G.F., Talley D.B., Booth P.M., Durfor C.N., Martin M.T., Napper AD. and Rees AR,Anal.

Chem., 62, 2211 (1990).16. Green M., Phil. Trans. R. Soc. Lond., B 316, 135 (1987).17. Koncki R. and GIl\b S., Chem.Anal. (Warsaw), 36,423 (1991).18.Aizawa M., Kato S. and Suzuki S.,J. Membr. Sci., 2,125 (1977).19. Li Y., Chen K., Wei S. and He J., Sens.Actuators,B12,49 (1993).ZO.KeatingM.Y and Rechnitz G.A,Anal. Chem., 56,801 (1984).21. Connell G.R., Williams R.1... and Sanders K.M., Biophys. J., 44, 123 (1983).22. Tran-Minh C., Pandey P.C. and Chavanne D., Biosens. Bioelectron., 7, 147(1992).23. Ghindilis AL., Skorobogatko O.~, Gavrilova~P. and YaropolovAP.,Biosens. Bioelectron., 7, 301 (1992).24. Sakai H., Kaneki N., Hara H. and ltoK., Anal. Chim.Acta, 230, 189 (1990).25. Colapicchioni C., Barbaro A, Porcelli F. and Giannini!., Sens.Actuators, B4, 24S (1991).26. Barbaro A, Colapichioni C., Davini E., Mazzamurro G., Piotto Piotto A and Porcelli F., Adv. Mater.,

4,402 (1992).27. Khomutov S.M., Zherdev A V:,Dzantiev B.B., Reshetilov A.N., Anal. Lett., 27, 2983(1994).28.Schasfoort RB.M., Bergveld P., Kooyman RP.H. and GreveJ.,Anal. Chim.Acta, 238,323 (1990).29. Brown D.~ and Meyerhoff M.E., Biosens.Bioelectron., 6, 615 (1991).30. Aizawa M., Marioka A, Matsuoka H., Suzuki S., Nagamura Y, Shinohara Rand Ishiguro I., J.Solid

Phase Biochem., 1,319 (1976).31. Aizawa M., Morioka A and Suzuki S.,Anal. Chim. Acta, US, 61 (1980).32. Uda T., Hifumi E., Kobayashi T.and Shimizu K., Biosens. Bioelectron., 10, 477 (1995).33. Masson M., LiuZ,Haruyama T., KohltakeE., Ikariyama Y andAizawa M.,Anal. Chim.Acta, 304, 353(1995).34. Kalab T. and Skladal P.,Anal. Chim. Acta, 304, 361 (1995).35. Deasy B., Dempsey E., Smyth M.R., Egan D., Bogan D. and OKeooedyR., Anal. Chim.Acta, 294,291 (1994).36. Meusel M., Renneberg R, Spener F. and Schmitz G., Biosens.Bioelectron., .10, 577 (1995).37. Pritchard D.J., Morgan H. and Cooper J.M.,Anai. Chim. Acta, 310, 111 (1995).38. Weber S.G. and Purdy W.C., Anal. Lett., 12,·1 (1979).39. Doyle M.J., HalsallH.B. and Heineman W.R., Anal. Chem., 54, 2318 (1982).40. Barnett D., Laing D.G., Skopec S., Sadik O. and Wallace G.G., Anal. Lett., 27,2417(1994).41. DeSilva M.S., Zhang Y, Hesketh P.J., Maclay G.J., Gendel S.M. and Stetter J.R., Biosens. Bioelectron.,

10,675 (1995).42. Guilbault G.G. and Jordan J.M., CRC Crit. Rev. Anal. Chem., 19, 1 (1988).43.ShonsA., Dorman F. and NajarianJ.,J.Biomed.Mater.Res., 6, 565 (1972).44. Suleiman A.A.and Guilbault G.G., Anal. Lett., 24, 1283 (1991).45. Minunni M.,Mascini M., Guilbault G.G. and Hock B., Anal. Lett., 28, 749 (1995).46. Ngeh-Ngwainbi J., Foley J., Kuan P.H. and GuilbaultG.G.,J.Am.Chem. Soc., 108, 5444 (1986).47. Rajakovic L., Ghaemmaghami ~ and Thompson M.,Anal. Chim.Acta, 217,111 (1989).48. Carter RM., Jacobs M.B., Lubrano G.J. and Guilbault G.G., Anal. Lett., 28, 1379 (1995).49.Attili B.S. and Suleiman AA,Anal. Lett, 28,2149 (1995).SO. Konig B. and GratzeIM.,Anal. Chim.Acta, 309, 19 (1995).

736 If. Dzwolak, R. Koncki and S. Glqb

51. Suri C.R., Raje M. and Mishra G.C., Biosens. Bioelectron., 9, 325 (1994).52. Krapivinskaya L.D., Krapivinsky G.B. and Ratner V:L, Biosens. Bioelectron., 7, 509 (1992).53. Guilbault G.G., Hock R and Schmid R,Biosens. Bioelectron., 7,411 (1992).54. Yokoyama K, Ikebukuro K, Tamiya E., ~arube I., Ichiki N. and, Arikawa Y.,Anal. Chim. Acta, 304,

139 (1995).55. Geddes NJ., Paschinger E.M., Furlong D.N., Ebara ~, Okahata ~, Than KA and Edgar J.A, Sens.

Actuators, B17, 125 (1994).56. He E, Geng Q., Zhu W., Nie L., Yao S. and Meifeng C., Anal. Chim. Acta, 289,313 (1994).57. Seitz W.R CRC Crit. Rev. Anal. Chem., 19, 135 (1988).58. Proceedings ofthe 1stEuropean Conference on Optical Chemical Sensors andBiosensorsEuropt(r)ade

1 (O.s.Wolfbeis, Ed.), Sens. Actuators, 811, 1993.59. Fiber Optic Chemical Sensors andBiosensors (O.S.Wolfbeis, Ed.), CRC Press, Boca Raton 1991.60. Ruzgas T.A, Razumas V:J. and Kulys J.J., Biosens. Bioelectron., 7,305 (1992).61. Domenici C.,SchironeA, CelebreM., Ahluwalia A and De Rossi D.,Biosens.Bioelectron., 10,371 (1995).62. Kumar P.,Colston IT.,Chambers J.P., Rael E.D. and Valdes J.J. Biosens. Bioelectron., 9, 57 (1994).63. Severs AH., Schasfoort RRM. and Salden M.H.L., Sens. Actuators, 824-25,98 (1995).64. Liedberg B., Nylander C. and Lundstrom I. Biosens. Bioelectron., 10, i (1995).65. Lukosz W., Biosens. Bioelectron., 6,215 (1991).66. Badley RA, Drake R.AL, Shanks I.A, Smith A.M. and P.R Stephenson Phil. Trans. R. Soc. Lond.,

B316, 143 (1987).67. Sansubrino A. and Mascini M., Biosens. Bioelectron., 9, 207 (1994).68. Severs AH. and Schasfoort R.RM., Biosens. Bioelectron., S, 365 (1993).69. Morgan H. and Taylor D.M., Biosens. Bioelectron., 7,405 (1992).70. Attridge J.W., Daniels P.R, Deacon J.K, Robinson G.A. and Davidson G.P., Biosens. Bioelectron., 6,

201 (1991).71. Kolinger C., Vttenthaler E., Drost S., Aberl E, Wolf H., Brink G., Stanglmaier A. and Sackmann E.,

Sens. Actuators, 824-25, 107 (1995).72. Fagerstam LG., Frostell A, Karlsson R, Kullman M., Larsson A., Malmqvist M. and Butt H., J. Mol.

Recognit., 3,208 (199O).73. Johne B., Gandnell M. and Hansen K.,J. Immunol. Methods, 160,191 (1993).74. Jonsson V., Fagerstam L, Ivarsson B., Johnsson R, Karlsson R, Lundh K., Llfas S., Persson B., Roos H.

and Ronnberg I., Biotechniques, 11, 620 (1991).75. Van den Heuvel D.J., Kooyman RP.H., Drijtbout J.w. and Welling G.w., AnaL Biochem., 215,233 (1993).76. Lu R, Xie J., Lu C., Wu C. and Wei Y.,Anal. Chem., 67,83 (1995).77. Ahluwalia A, De Rossi D., Ristori C., Schirone A and Serra G., Biosens. Bioelectron., 7, 207 (1991).78. Parry RP., Love C. and Robinson C.,J. Virol. Methods, 27, 39 (199O).79. Feldman S.E, Vzgiris E.E., Murray Penney C., Gui J.Y., Shu E.~ and Stokes E.S.,Biosens. Bioelectron.,

10, 423 (1995).80. Walczak I.M., Love W.E, Cook 'f.A and Slovacek RE., Biosens. Bioelectron., 7, 39 (1992).81. OgertRA,Brown J.E.,Singh RR, Shriver-Lake LC. and Lingler ES.,AnaI.Biochem., 205,306 (1992).82. Brecht A, Gauglitz G. and Polster J., Biosens. Bioelectron., S, 387 (1993).83. Schlatter D., Barner R., Fattinger Ch., Huber w., Hubscher J., Hurst J., Koller H., Mangold C. and Muller H,

Biosens. Bioelectron., S, 109 (1993).84. Robinson G.A, Biosens. Bioelectron., 6, 183 (1991).85. Brecht A, Piehler J., Lang G. and Gauglitz G., Anal. Chim. Acta, 311, 289 (1995).86. Dubrovsky 'f.B., Demcheva M.V:, Savitsky AP., Mantova E.Yu., Yaropolov Al, Savransky V.v: and

Belovolova V:L., Biosens. Bioelectron., S, 377 (1993).87. Dubrovsky T., Vakula S. and Nicolini c., Sens. Actuators, B22, 69 (1994).88. Ahluwalia A, De Rossi D., Monici M., Schirone A, Biosens. Bioelectron., 6, 133 (1991).

Received January 1996AcceptedApril 1996