Detection and quantification of biomolecular interactions with optical biosensors

trends in analytical chemistry,

[ 81 T.C. O’Haver and G.L. Green, Int. Lab., 5 (1975) [9] :.k. Cahill.Am.

49

vol. 14, no. 2, 1995

Lab., 11 (1979) 79.

Bernhard W. Glombitza and Peter C. Schmidt are at the Department of Pharmaceutical Technology, Eberhard-Karls-University Ttibingen, Auf der Morgenstelle 8, 72076 Tiibingen, Germany.

Detection and quantification of biomolecular interactions with optical biosensors D. Yeung, A. Gill, C.H. Made, R.J. Davies * Cambridge,

UK

Optical biosensors have the potential for rapid detection and quantification of the interactions between a wide range of biomolecules and macromolecular complexes. A new biosensor system which incorporates a resonant mirror (RM) as the optical sensor is described, and the results obtained with it from a number of biomolecular interaction experiments are compared with those found using another system in which the sensor is based on the surface plasmon resonance (SPR) effect. Both sets of results are compared with those obtained using classical procedures. It is concluded that optical biosensor systems will in future probably provide the method of choice for studying biomolecular interactions.

1. Introduction In the 1990s the discovery of new biological molecules continues apace. Many biomolecules work with others in concert as functional systems: the contractile proteins of muscle, G-protein coupled receptors, DNA transcription complexes, and cytoplasmic vesicle targeting proteins are typical examples. In some cases once a protein has been incorporated into the complex, new surfaces are created which allow other molecules to attach. * Corresponding author.

0 199.5 Elrevier Science B.V. All rights reserved

Much emphasis has been placed on obtaining structural detail on single macromolecules, facilitated by advances in NMR, crystal formation, X-ray and synchrotron radiation techniques. However, to understand how these biomolecules function requires that the interactions of their components are monitored as they occur. Perturbation of the environment in which they operate, either before, during or after their assembly, may also give physiological insight. Following the assembly of simple bimolecular complexes in real time is difficult, but now with the advent of new types of optical biosensors this may be simply and rapidly performed. Uses for such instruments range from real-time assays on high value biological materials, such as recombinant interferon or monoclonal antibodies in bioreactor broths, to the rapid determination of biomolecular association and dissociation rate constants. Such rate constants are of academic and practical value, for instance, the suitability of an antibody for an ELISA type assay will often be determined by its dissociation rate. An optical biosensor can follow binding interactions between biological macromolecules directly as they occur. Because it can collect data rapidly, rate constants characterising the binding event can also be determined. Dedicated software packages can facilitate the evaluation of these kinetic constants. Such information can be obtained with Pharmacia’s robotic system, BIAcoreTM (launched in 1990), or their more recent manual system, BIAliteTM. The Pharmacia systems utilise the optical phenomenon of surface plasmon resonance (SPR), but since this instrument is well described in the literature [ 1,2] it will be discussed but not described further here. A newly formed Swiss com-

0165-9936/95/$09.50

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trends in analytical chemistry, vol. 14, no. 2, 1995

pany, A.S.I., also have a sensor, Bios 1, based upon a planar waveguide [ 31. However, it does not use the sophisticated biorecognition matrix of the other sensors, and as literature is scarce, because of its recent commercialisation, it will not be discussed here. IAsysTM, a new commercial product of Fisons, utilises the principle of optical resonance [ 41, which is described in more detail below.

to within a few hundred nanometres of the surface of the waveguide. Changes in the refractive index at the surface, caused for example by the binding of a macromolecule from the sample to a site near the surface, will alter the resonance angle which can be tracked by a suitable optical detection system. The instrument and RMs are constructed so that only TE resonances are utilised. SPR and RM devices are similar in that both produce an intense, surface confined optical wave at a discrete resonance angle. While the SPR device has over twice the resonance angle shift of the RM device (360 vs. 163 arc set ng- ’ mmm2, respectively), its resonance width is broader. The ultimate sensitivity (i.e., the lower limit of detection in a given bioassay) will, however, be determined by instrumental noise and in practice we have found that both instruments have equivalent lower detection limits. Both instruments will determine association rate constants (k,,,) in the range 103lo6 M-‘s-l and dissociation constants (kdiss) in the range 1O- 5- lop2 s- ‘. Differences between the two instruments are, therefore, related to instrumental and configurational details.

2. The origin of the sensor optical

3. The bioselective layer

.

Ligate (Analyte)

Carboxymethyldenran matrix 1

I&and (Antibody)

Fig. 1. The optical and biological components of the RM-based system IAsysTM.

resonance and its measurement The sensing element is an integrated optical chip, called the resonant mirror (RM). The RM comprises a glass prism upon which are deposited in turn a low refractive index silica spacer layer and a thinner layer of a high refractive index monomode waveguide of titania, hafnia or silicon nitride (Fig. 1) . Laser light (A = 670 nm) passes through a polariser to produce equal intensities of transverse electric and magnetic (TE and TM) components of light which is directed at the prism at an angle that is swept repeatedly. Some of the light entering the prism will tunnel through the spacer where, at an angle unique for each polarisation (their respective resonant angles), it propagates by multiple internal reflections along the monomode waveguide. The light also tunnels back across the spacer to leave the prism. At resonance an intense element of the light (the evanescent wave) is generated at the waveguide surface which decays exponentially into the sample. For the RM its intensity drops to 1/eth of that at the sensor surface at about 100 nm into the sample. The resonant angle is therefore particularly sensitive to the refractive index of the sample in immediate contact with and

It might perhaps be considered that the bioselective layer could be formed by direct physical adsorption of the biomolecules of interest from their solution on the top surface of the resonant mirror (RM, Fig. 1) . However, direct adsorption of protein to surfaces impairs their function, either because of partial denaturation, or simply because of steric interference of the adsorbent. Hence to preserve the biological activity of the immobilised binding partner (the ligand) , a matrix of carboxymethylated dextran is attached to the top surface of the RM. The original dextran has a number average degree of polymerisation of about 3000 and is carboxylated in solution to about 0.5 carboxylic acid groups per glucose residue. The matrix is activated by an aqueous solution of carbodiimide and Nhydroxysuccinimide, in the course of which it becomes partially cross-linked, presumably the result of p-alanine formation [ 51. Based on the radius of gyration of dextran in solution [ 61, the electrostatic expansion due to the carboxylic acid moieties, and the observation that adsorbed polyelectrolytes form layers substantially thicker than expected [7] the matrix thickness is estimated to be 200400 nm. This matrix appears equally acces-

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sible to proteins spanning the mass range 14 000 (lysozyme) to 640 000 (thyroglobulin), however, bacteria and larger cells cannot penetrate it. For intact microbial cell studies aminosilanised RM surfaces are available to allow ligands to be chemically immobilised directly to the sensor surface

5. Quantitative determination of the relative binding strengths and rate constants of Staphylococcal Protein A to different immunoglobulins 5.1. Relative binding strengths

m. A further benefit of the highly charged matrix is that many proteins may be greatly concentrated into it during immobilisation. By presenting the protein in a low ionic strength solution at a pH between its pl and pH 3.5 (below which the matrix loses its charge) the complementary charges of matrix and protein ensure electrostatic concentration. Thus with immunoglobulin G (IgG) the matrix becomes saturated with protein when applied at concentrations > 20 mg ml- ‘; combining this with the minimum cuvette volume of 50 ~1 results in a ligand requirement of only 1 pg. The cuvette can hold 50-250 ~1 of sample which is stirred by a vibro-stirrer (patent pending) to minimise mass transport of sample molecules to the matrix.

4. Biological applications: comparative

studies

Two examples will now be given to illustrate and, where possible, to compare the types of information provided by the RM-based system with other techniques. Here we summarise its application to two biological systems, further detailed information may be found in the literature [9-l I 1. Table 1 Relative binding strengths of immunoglobulins

Protein A (SPA), isolated from the cell wall of Staphylococcus aureus, binds a range of antibodies of the IgG class. The molecular details of the interaction are well known [ 121; SpA has four binding sites for the Fc region of IgG, although in solution it can bind at most two IgGs because of steric hindrance. SpA is of great bio-technological utility, being used in diagnostic tests and affinity purification of monoclonal antibodies. Information on the binding of SpA to a range of IgG molecules has, until the advent of direct measuring technologies like those described here, been of a qualitative nature. SpA (1M, 42 000; pl 5.1) was first covalently immobilised to the matrix by standard carbodiimide-succinimide coupling chemistry [ 131. Residual ester groups were then deactivated with ethanolamine, the cuvette loaded with an IgG solution and binding followed for about 40 min. Dissociation could also be followed, after a brief but thorough wash with buffer. Finally, complete removal of the IgG was effected by a brief wash in 20 mMHC1. This does not impair the binding activity of SpA, so further IgG solutions at the same concentration of 1 mg ml ~ ’ could be tested. In this way the relative binding strengths for different IgGs were obtained, using one cuvette, simply by taking the change in resonance angle after a constant contact time and the results tabulated in column 2 of Table 1. A comparison of these

to protein A

Antibody

IAsys response _+SD. (arc seconds)

Precipitin test”

Human IgG, Human IgG, Human IgG, Rabbit Goat Mouse Chicken Sheep

171&12 190+6 Ok0 142+9 75+2 64k3 0 58+22

++++ ++++ -

a-t + -I- + indicates a strong binding interaction, - indicates no precipitate formation [22]. S.D. =standard deviation of 3 to 4 replicate measurements.

++++ +I++ +Iformation and +/-

indicates weak or no precipitate

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trends in analytical chemistry, vol. 14, no. 2, 1995

1250

The instrument can also be used to determine equilibrium binding values, which may then be subjected to a Scatchard-type analysis to determine the binding constant (K,,) . However, because the vibro-stirrer component ensures solution homogeneity up to the matrix, analysis of the real-time binding profile also allows extraction of the association and dissociation rate constants and hence KD = kdiss/kass. Thus Fig. 2 shows raw data for the binding of human IgGI at different concentrations to immobilised SpA. The kinetic parameters that characterise the interaction, viz. association (k,,,) and dissociation (kdiss), are readily obtained from this data by the following treatment. The association of immobilised SpA with IgG in solution can be represented by the equilibrium:

15 nM

1.5 nM

250

4 nM 2nM

Oi0.0

ti 20.0

10.0

M.0

50.0

40.0

Time / minutes Fig. 2. The binding of human IgG, at various concentrations to immobilised SpA. The arrow indicates the

point at which the IgG was added to the cuvette. quantitative results with those obtained by

the usual

precipitin type assays, shows that they are far more discriminating.

8

Intercept: k(j= 1.83x10-4 Slope: k,=

s-l

1.12x10~M-1s-l

6

kas SpA + IgG + SpA:IgG k&s with the equilibrium (dissociation) constant KD being related to the two rate constants thus: K,=

hiss. _ [SPAI *l&G1 k ass

[ SpA:IgG]

The concentration of bimolecular complex at a particular time is directly proportional to the sensor signal response (R) at that time. As a corollary the concentration of free binding sites on the SpA will vary as W,,, -R] , where R,,, is the maximum response that will be seen at high concentrations of IgG that saturate the SpA sites. To extract rate constants from the binding profiles in Fig. 2 the following differential rate equation for complex formation applies: dR z=

k,,s[R,,

-RI *[I@1 -kc&

= kdgWLax

-R(kass[IgGl +bss)

Since the measurements are carried out under pseudo-first order conditions (i.e., the concentration of IgG does not change during the binding process) further differentiation with respect to response eliminates the requirement of determining R ,,,=, thus: I

1

40

20

(

60

hIgGl / ti

Fig. 3. Evaluation of the rate constants for human IgG, binding to immobilised SpA.

dRldt dR

-=

-

(kss[I@l +biss)

The raw data is therefore first transformed to a plot of dR/dt versus R and the gradient of the initial

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(low R) linear part is then taken and plotted against [ IgG] as shown in Fig. 3. Consideration of the last equation shows that the slope of the plot in Fig. 3 gives the association rate constant (kass,, and the intercept the dissociation constant (kdiss). Kinetic analysis of data by the above method has two drawbacks. The first is that in practice the dR/ dt vs. R plots are often curved; nonetheless attempts to fit a straight line to such data are evident in the literature [ 141. The second is that such multiple data transformations are time consuming and contrary to the goal of a rapid measurement. To overcome these problems a rapid data analysis package, FASTfi tTM, has been developed. This applies exponential curve fitting algorithms to both the binding and dissociation phases, to abstract the best values of k,,, and kdlsn.With these values in hand there is no need for the more usual, protracted methods (e.g., dialysis, fluorescence titration, radioisotopic assay, and ELISA) for determining the equilibrium constant. Frontal [ 151 and zonal [ 161 analytical affinity chromatography can also rapidly give values for K,, but require substantial effort to optimise the column; eluate detection may also be difficult with some systems. For the HIV protein gp 120 and its T-cell receptor CD4 frontal analysis ( 18 nA4) and optical biosensor ( 11 M) gave similar values for& [ 171. Obtaining rate constants, even in solution, is rareIy as straightforward as discussed above. Fluorescent techniques (particularly stopped-flow) can achieve this, but often will require attachment of a Auorophore to one of the interactants. Such labels cannot be attached uniformly. Even proteins with intrinsic fluorophores (tryptophan and NADH) are heterogeneous because they possess a large number of different conformational microstates, many of which are likely to alter the properties of the Huorophore. Substantial knowledge of the binding mechanism may therefore be needed before true rate constants can be assigned with confidence [ IS]. Kinetic values determined by optical biosensors may not in all circumstances precisely mirror solution measurements. Discrepancies may arise if movement of material through the matrix is retarded compared to the bulk phase; the close spacing of immobilised ligand (approximately 20 nm at a loading of 1000 arc seconds of a protein of M, 100 000 distributed uniformly throughout a matrix of thickness 300 nm) may favour multiple interactions during transit to (and from) the surface. Some of these factors are discussed in a forth-

coming paper [ 191. More work needs to be done to establish the exact relationship between rate constants determined in free solution and with these biosensors. However, in those situations where one wishes to select, for example an antibody with the quickest ‘on rate’ from a given population, then the systems discussed here offer a rapid and easy analysis. 6. Binding of native and bioengineered antibody fragments to lysozyme Specificity, affinity and rate of binding are factors that determine the suitability of an antibody for a particular application. Antibody design is currently an area of intensive research which aims to increase the affinity for the target molecule and to genetically graft binding sites from other species onto human antibodies (chimeric antibodies) thereby preventing immuno-recognition and removal. More recently small antibody fragments have been prepared with acceptable affinities and they are expected to have greater tissue penetrative power and hence enhanced therapeutic or diagnostic efficacy. In this second example, hen egg lysozyme (M, 14 000; pl 10.5) was immobilised on the matrix. Its interaction with either a whole, bivalent, antilysozyme antibody (D 1.3; A4,. 1.50 000), or just a fragment (D1.3 Fv; M, 25 000) having univalent binding capacity, have been studied using optical biosensors or by classical techniques. The kinetic constants, derived as shown in Section 5, from measurements made on the RM- and SPR-based systems under the same conditions, together with other SPR system data, as well as with those obtained using standard solution techniques, are given in Table 2. A study of the data listed shows that, in agreement with earlier findings from the SPR system, the RM system affinity values (Kb) are also substantially lower than those obtained from solution measurements such as intrinsic (tryptophan) fluorescence titration or equilibrium sedimentation. ELBA, a frequently used technique, is unable to give a precise value for Kb in this case. However, both optical biosensing systems gave kinetic parameters, k,,, and kdiss, (and hence Kb) that are in good agreement, particularly with measurements conducted on the same materials under similar conditions (see data marked with superscripts a and b). The validity of the biosensor association rates (k,,,) is established by their similarity to the fluo-

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trends in analytical chemistry, vol. 74, no. 2, 1995

Table 2 Equilibrium and rate constants solution measurements

for anti-lysozyme

k,,, (AK

Antibody Fv Fragment Di .3

Whole Antibody D1.3

3.0 0.17 0.4 1.0 6.3 2.0

0.63 < 0.01
antibodies

s-‘)

and fragments

determined

k,,,ss(s-l)

1.9.106 1.0.106 1.2.106 5.1 105 2.8.1 O5 1.4.105 3.0.105 1.8.105 1.7.105

1.7.10-4 5.2.10-4 5.1 .10-4 <1.10-6
carried out with the same materials, in the same laboratory

rescence quench measurements. A recent study comparing association rate constants of whole D I .3 and four humanised antibodies to lysozyme indicated that k,,, (free solution) was about fivefold higher than k,,, (optical biosensor) [ 201. The lack of dissociation data from solution measurements prevents comparison with the new optical technologies. Dissociation measurements on macromolecules in solution are difficult to perform, which may account for the lack of such data on this model system. This is a serious limitation of some solution methods as it is largely through changes in kdiss, rather than k,,,, that an antibody’s affinity is controlled. Variability in the measured values may be apportioned out between the variable nature of biological materials and different measurement techniques. Even the binding affinity of radioiodinated antibodies to suspended erythrocytes, a straightforward measurement, has recently been shown to vary by almost an order of magnitude simpIy by varying the equilibration volume [ 171. Across the literature, where comparisons can be made, substantial variation will usually be found. For the practical biochemist/biotechnologist what is required is a consistent, reproducible measure of affinity and rate constants such as these optical biosensors provide. 7. Biological applications: spectrum

the broader

It may be confidently stated that any interaction that increases the refractive index within the eva-

by IAsysTM, BIAcoreTM and conventional

Method

Ref.

Fluorescence: quench and titration BIAcoreTM IAsysTM a BIAcoreTM a Calorimetry Fluorescence quench BIAcoreTM Fluorescence quench Sedimentation equilibrium. BIAcoreTM BIAcoreTM b lAsysTM b ELISA

[231 [241

[I 91 [I 91 [251 I231

PO1 PO1

[261 [241

c c

[271

by the same user.

nescent field may be followed by the optical biosensors discussed here. Such sensors are ideally suited to work with the vast range of available monoclonal antibodies; an anti-murine antibody may be simply coupled to the matrix to capture the selected monoclonal. After binding their target analyte they may be removed from the matrix to allow attachment of another monoclonal. Topological interference maps (epitope maps) are readily constructed by determining whether one of a pair of antibodies can prevent binding of the other. Such information is useful when designing assays that involve the binding of two antibodies to a target analyte (as in ELISA). Small molecules (haptens), themselves incapable of eliciting an immune response and so generating an antibody binding partner, may do so by being conjugated to a carrier molecule, such as a protein. By using these antibodies, and the appropriate hapten-conjugate, binding assays may be set up on the sensor. Since the conjugate usually competes with free hapten for sites in the matrix, this opens up the large area of drug assay and screening for possible therapeutic agents. DNA binding to either polynucleotides or DNA binding proteins may also be investigated. Membrane receptors binding to immobilised ligand may be readily followed if the receptors are in vesicular form. As indicated previously, whole cells - bacterial or mammalian - have been bound to RMs via specific ligands and binding prevented by the appropriate inhibitor. Consideration of this range of applications of optical biosensors leads us to predict that the scientific literature will

trends in analyticalchemistry,vol. 14,no. 2,

1995

become rich with reports of such studies. Already optical biosensors have been applied to the apparently intractable problem of protein complex formation. Thus it is gratifying to note that the assembly of the membrane-bound chemotactic response complex of E. coli from its four protein components has been tracked with an evanescent wave optical biosensor, and the effect of phosphorylation determined [ 211. The ability to investigate such multicomponent complexes represents a major advance over the more traditional methods.

8. Conclusions Optical biosensors that utilise evanescent fields can quickly provide information on the interaction of many biological macromolecules and biomolecular assemblies. Affinity and kinetic constants, which are not always measured by traditional methods, are readily obtained. It is considered likely that they will complement and may largely replace radioassay, fluorescence spectroscopy, ELISA, equilibrium dialysis, stopped-flow photometry and some chromatographic methods as affinity-assessing methods. Their versatility, ease of use, and ability to use impure samples containing small amounts of analyte that requires no labelling should make them the first choice of bioanalysts in tackling such problems.

References and R. Reed, Bioflechnology, 10 ( 1992) 390. [ 21 L.G. Fagerstam, Techniques in Protein Chemistry, II (1991) 65. [ 31 J. J. Ramsden, J. Phys. Chem., 97 ( 1993) 4479. [4] R.Cush, J.M. Cronin, W.J. Stewart, C.H. Maule, J. Molloy and N.J. Goddard, Biosensors and Bioelectronics, 8 ( 1993) 347. [ 51 M. Wilchek and T. Miron, Makromol. Chem. Macromol. Symp. 17 ( 1988) 22 1. [ 61 D.B. Sellen, Polymer, 16 ( 1975) 561. [ 71 R.J.Davies, L.R. Dix and C. Toprakcioglu, J. Colloid. Inte$ace Sci., 129 (1988) 145. [ 81 H.J. Watts, C.R. Lowe and D.V. Pollard-Knight, Anal. Chem., 66 ( 1994) 2465. [9] P.E. Buckle, R.J. Davies, T. Kinning, D. Yeung, P.R. Edwards, D. Pollard-Knight and CR. Lowe, Biosensors and Bioelectronics, 8 ( 1993) 355. [ I ] R. Granzow

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[101 R.J.Davies, P.R. Edwards, H.J. Watts, CR. Lowe. P.E. Buckle, D. Yeung, T.M. Kinning and D.V. Pollard-Knight, Techniques in Protein Chemistry, V (1994) 285. Am. 1111 R.J. Davies and D. Pollard-Knight, Biotechnol. Lab., July (1993), p.52. [I21 J.J. Langone, Adv. Immunol., 32 ( 1982) 157. [I31 J.V. Staros, R.W. Wright and D.M. Swingle,Anal. Biochem., 156 (1986) 220. K.K. [14 D. J. O’Shannessy, M. Brigham-Burke, Soneson, P. Hensley and I. Brooks, Anal. Biochem., 212 (1993) 457. [15 D.J. Winzor, in P.D.G. Dean, W.S. Johnson and F.A. Middle (Editors), Afinity Chromatography, A Practical Approach, TRL Press, Oxford, 1987, Ch. 6 1161D.M. Abercrombie and I.M. Chaiken, in P.D.G. Dean, W.S. Johnson and F.A. Middle (Editors), Afinity Chromatography, A Practical Approach, IRL Press, Oxford, 1987, Ch.7 1171 G.L. Ong and M.J. Mattes, Mol. lmmunof., 30 ( 1993) 1455. [I81 M.P. Jackman, M.A.A. Parry, J. Hofsteenge and S.R. Stone, J. Biol. Chem., 267 ( 1992) 15375. A. Gill, R.J. Leatherbarrow, M. Hoare, D.V. I191 Pollard-Knight and D.H. Fortune, Anal. Biochem., submitted for publication. [20] I. Chaiken, S. Ros& and R. Karlsson, Anal. Biochem., 201 (1992) 197. [21] S.C. Schuster, R.V. Swanson, L.A. Alex, R.B. Bourret and M.I. Simon, Nature, 365 ( 1993) 343. [221 E. Harlow and D. Lane, Antibodies, A Laboraton, Manuat, Cold Spring Harbor Laboratory, 1988, p. 616. ~31 E.S. Ward, D. Giissow, A.D. Griffiths, P.T. Jones and G. Winter, Nature, 341 ( 1989) 544. Borrebaeck, A-C. Malmborg, C. r241 C.A.K. Furebring, A. Michaelsson, S. Ward, L. Danielsson and M. Ohlin, Bioflechnofogy, 10 ( 1992) 679. ~51 W. Ito and Y. Kurosawa, J. Biol. Chem., 268 ( 1993) 20668. [26] T.L. McInemey, G.J. Hewlett, L.C. Gruen and D.C. Jackson, Mol. Immunol., 30 ( 1993) 47. [27] M. Verhoeven, C. Milstein and G. Winter, Science. 239 ( 1988) 1534.

Miss Debra Yeung graduated in microbiology at King’s College, London University in 1986. For the past four years she has been working on SPR and RM based biosensors at Fisons Applied Sensor Technology (Saxon Way, Bar Hill, Cambridge, CB3 8SL, UK). Mr Andrew Gill graduated in applied biology at Coventry Polytechnic in 1992. He is now studying for a Ph.D. on monitoring fermentation products in

56

real time with lAsysTM at Fisons Applied Sensor Technology. Dr. Colin Hugh Maule graduated in physics at Bristol University in 1984. Since 1989 he has been working on optical biosensors at Fisons Applied Sensor Technology.

trends in analytical chemistry, vol. 14, no. 2, 7995

Dr. Robert John Davies graduated in biochemistry at Birmingham University in 1979. Since 1989 he has been working on protein attachment to optical biosensor surfaces at Fisons Applied Sensor Technology.

Recent developments in the analysis and environmental chemistry of toxaphene with emphasis on the marine environment Derek C.G. Muir * Winnipeg, Canada

Jacob de Boer IJmuiden, The Netherlands Toxaphene (camphechlor) is a pesticide consisting primarily of chlorinated bornanes (CHBs) which was widely used until the mid 1980s. Toxaphene continues to be a major contaminant in marine and freshwater biota, with levels in marine fish exceeding some regulatory guideline limits. Methods of analysis for CHBs include gas chromatography with detection by electron-capture negative ion and electron ionization mass spectrometry, as well as electron-capture detection. Until recently quantification of toxaphene was limited by lackof suitableanalytical standards and by limited information on thestrucof CHB residues in identity tural environmental samples. This article reviews recent developments, such as the identification and synthesis of CHB congeners, and new information on spatial and temporal trends.

Environmental Protection Agency [ 11, toxaphene was the most extensively used pesticide in the USA and many other parts of the world. Global production has been estimated to be 1.33 megatons [ 31, although accurate data are only available for the USA ( 1946 to 1982) where about 0.45 megatons was produced [ 1,3]. Although no longer manufactured in the USA, toxaphene and similar products are still being used in Central and South America, Africa, Eastern Europe, the Indian sub-continent, and in regions of the former USSR [ 1,3,4]. Despite the ban on its use in the USA, Canada and Western Europe, interest in the environmental levels and biological effects of toxaphene has continued because of the presence of elevated levels in soils, air, and especially in fish and marine mammals. There have recently been major advances in the analytical chemistry and measurements of the environmental distribution of toxaphene resulting from the identification and synthesis of environmentally significant congeners [ 5,6] and the determination and partial characterization of CHB residues in a variety of matrices ranging from air [ 71 to marine mammal blubber [ 8-101. In this article we review these developments, with emphasis on the analysis, distribution, and possible effects of toxaphene in the marine environment.

1. Background 2. Analytical methods Toxaphene is a complex mixture consisting primarily of chlorinated bornanes (CHBs) with an average elemental composition of CloHloCls [ 1,2]. Prior to its banning in 1982 by the U.S. * Corresponding author.

2.7. Synthesis, structural considerations nomenclature

and

Toxaphene is synthesized by controlled chlorination of camphene (an isomerization product of