Optical-fibre sensors by silylation techniques

Sensorsand Acfuators zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA B, I1 (1993)353-360 353 Optical-fibre sensors by silylation techniques* F. Baldini and S. Bracci Istituto di Ricerca mile On& Eleftromagneiiche, via Panciatichi 44, 50127 Florence (&a&) Abstract The detection of chemical parameters by means of optical fibres has had a decisive boost in recent years; the realization of these devices is related to the development of a great number of techniques. The former are characterized by a reagent fixed to an optical fibre, a procedure which is not in itself unique. The silylation technique is one of the most widkly used for optrode realization. It generally involves an organosilicon compound that reacts with an external support or directly on the surface of the fibres with one or more functional groups and gives the supports a new functionality. Obviously the activation of the surface must be followed by other appropriate reactions so as to be able to immobilize the optically sensitive reagent. Silylation can be performed either on external glass or quartz supports or directly on the fibre itself. From a chemical point of view, there is not a big difference on where the chemical reaction takes place, but the same cannot be stated from the optical point of view. By changing where the silylation is performed, it is possible to change the optical path and consequently the signal-to-noise ratio, which is strictly related to the performance of the sensor. In fact, if the optical-fibre sensor is to be launched on the market, it must be competitive, in terns of cost and performance, when comparedwith other instruments that measure the same chemicalspecies.Some of the most significantsilylated optrodes are reported here, together with the associated optoelectronic instrumentation. Moreover, the advantages and drawbacks of the different procedures are taken into account. Introduction Optical-fibre chemical sensors have undergone exceptional development in recent years. Such increased interest is perfectly justified in view of the typical advantages that optical fibres offer. The possibility of remote detection is very important, and often essential for industry as far as process control and detection in hostile environments are concerned. Miniaturization is another important aspect that is of great interest for clinical and biomedical applications, since it enables in uiuo performance, which is often ‘unique’ to take place. In environmental analysis, the possibility of performing continuous in situ controls without the need for drawing samples is often a winning characteristic. In the design of an optical-fibre sensor many aspects must be taken into account for the choice of the optical and electronic units and for the construction of the optrode. The latter aspect has fundamental importance in the functionality of the sensor, as it can condition the choice of the optoelectronic components. Therefore, the immobilization of a chemical that is sensitive to the investigated parameters plays an essential role in the final performance of the sensor. In this work our attention will be given to optrodes realized by means of silylation techniques. *Invitedpaper. 0925-4005/93/$6.00 Organosilicon compounds have been known for a long time, and the related chemistry has undergone rapid growth in the past 20 years. This type of reagent is widely used in both analytical and synthetic chemistry. In synthetic organic chemistry, silyl reagents are employed to protect functional groups and to control the stereochemistry of chemical reactions. In analytical chemistry, silylation is used in gas chromatography and mass spectrometry for the modification of a variety of inorganic substrates and functional groups in order to enhance the performance of the analytical techniques. Silylation is also utilized in the realization of optrodes, since it can provide the supports (in some cases, the fibre itself) with a new functionality, so as to make possible the detection of chemical parameters using optical methods; it also makes it possible to fix the chromophore to the support using a covalent bond, thereby almost completely avoiding its leakage. In this paper, a general view on silylation techniques for optical-fibre sensors is considered, paying particular attention to the optoelectronic implications in relation to the construction of the optrode. Geuersl aspects The term silylatiun is defined as the substitution of a hydrogen atom, bound to a hetero atom of an inorganic substrate, with a silyl group, forming a silicon-hetero @ 1993- ElsevierSequoia.All rights reserved 354 atom bond without any further alteration of the molecule. The silylation is generally realized with an organosilane having the general formula R, Si1<(4_Z) (0 f z 6 4), where X is a hydrolysable group, typically alkoxy or chlorine, and R is a non-hydrolysable organic radical possessing a functionality that imparts particular characteristics to the substrates or enables further reactions to occur for bonding different reagents. The most common alkoxy groups are methoxy (-OCH,) and ethoxy (-OCH,CH,), which give methanol and ethanol as by-products during coupling reactions, They are generally utilized more than chlorosilane because the latter generates hydrogen chloride, which may be an undesirable by-product in some reactions, After the reaction, the bond between X and the silicon atom in the coupling agent is replaced by a bond between the inorganic substrate and the silicon atom. Generally, thanks to the characteristics previously described, the reactions involving silylating agents can be performed on various supports having reactive sites on their surface, The most connion substrates are silica-based oxides such as quartz, glasses of different shapes, or optical fibres themselves. As a rule, siiylation is performed when it is necessary to change the characteristics of the supports: for instance, in order to increase the hydrophobicity or even to introduce a reactive group on the surface. The introduction of a new functional group makes it possible to perform other chemical reactions in order to fix the proper reagent. As stated previously, silylation can be performed either on glass/quartz supports or directly on the fibre itself. From a chemical point of view, it is not of major importance where the chemical reaction takes place, but there are important considerations from the optical point of view. If the final and main goal in realizing the sensor is the extreme miniaturization of the optrode, which is often a fundamental requirement in the biomedical field, silylation directly on the fibre is preferable. On the other hand, if this aim is pursued, the signal-to-noise ratio can be a critical point, especially if modulation of the light comes from a monolayer located on the fibre surface with a consequent very low dynamic range of the detected signal. In such a case, it is necessary to utilize powerful sources (lasers, halogen lamps with an optical system providing for a good coupling of the light with the fibre) and a highly sensitive detecting system (i.e., lock-in, amplifier, photomultiplier, etc.) Altogether this makes the sensor very expensive and sometimes not very easy to produce on an industrial scale, especially if it is to be portable. To overcome this problem, silylation can be utilized to fix a polymer, with the chromophore entrapped inside, to increase the optical path, or it can be performed along the fibre core, exploiting the modulation coming from the evanescent field. It is apparent, in this case, that the longer the piece of fibre silylated, the higher the signal-to-noise ratio; therefore light-emitting diodes (LEDs) and specifically developed electronic circuits can be used as sources and detecting systems, respectively, with a consequent strong decrease in the cost of the overall system. On the other hand, if the treated fibre is too long, the fragility of the fibre itself increases and must be taken into account. Hence, a good compromise must be chosen. If silylation is performed on an external support (glass/quartz slides or platelets, controlled-pore glasses, etc.), it is possible both to immobilize a larger quantity of the chromophore and also to work with a longer optical path; therefore, better modulation of the light and, consequently, a good electrical dynamic range are obtained. In this case, the optrode can be properly planned so as to guarantee a satisfactory optical path for the light coming from the fibre; but highly miniaturized optrodes are obtained with difhculty, because they require a suitable envelope for attaching the support to the tip-end of the fibre. Moreover, even if it makes it possible to obtain a good signal-to-noise ratio, the presence of a layer (no longer limited to a monolayer) has the disadvantage that the diffusion of the chemical species under study occurs along a thicker path and this mass transfer can give rise to a very slow response time. As appears clear from the above considerations, optoelectronic requirements must also be taken into account before performing the chemical sequence used to 6x the reagent, together with the performance required of the optical-fibre sensor on the basis of its application field. In fact, if the optical-fibre chemical sensor is to be launched on the market, it must be competitive, in terms of cost and performance, when compared with other instruments that measure the same chemical species. From this point of view, the realization of simple optoelectronic systems coupled to the optrode, making use of LEDs and simple photodiodes and without any traditional optical systems (lenses, mirrors, dichronic filters), may be a winning solution. zyxwvutsrqponmlkjihgfedcb Examples So far many optical-fibre sensors have been realized by using the silylation technique. Table 1 summarizes some of the chemical species investigated up to now, along with the silylating agents utilized and references to the literature. zyxwvutsrqponmlkjihgfedcbaZYXWVUTS Silylation on the optical fibre One of the first examples of silylated fibre is a pH sensor exploiting the fluorescence of a modilied fluorescein [l]. In this case, the surface of glass/glass fibre 355 TABLE 1. Summary of fibre-optic sensors developed up to zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLK no w by using silylation techniques. (I = chemical reaction on the fibre; s = chemical reaction on an external support). APTS = 3-aminopropyltriethoxysihme; OOPS = 3-glycidoxytrimethoxysilane; OTES = octadecyltriethoxysilaae; OTCS = octadecyltrichlorosilane; MAPS = y-methacryloxypropyltrimethoxysilane); DMECS = dimethyldichlorosilane Investigated parameters APTS GOPS f Antibodies Bile acids c&se-s Glucose Haptens High acidity Hydrocarbons Ionic strength Metal ions Penicillin Pesticides PI-I f S DMECS f f S S [61 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA I191 [lOI WI L711 [3,221 [121 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA I231 u41 L 7-51 [7-9.11, 131 [51 laser beam Fluoreecw~ee return Spectrometer entrance at b fibre Fig. rin. MAPS WI (core diameter = 100/14Opm) was treated with 3aminopropyltriethoxysilane ( APTS) and, successively, with acryloyl chloride; this permits a polymerizable vinyl group to be obtained, which reacts successively with acrylamide, NJ-methylenebis(acrylamide) and acryloylfluorescein. In this way, a polymer that is sensitive to pH was obtained. In its first version, the optoelectronic system coupled with the optrode makes use of an Ar-ion laser as source and of a spectrometer coupled to a photon-counting system for the detection. Only one fibre is used, and the discrimination between the excitation and emission light is obtained with the optical arrangement shown in Fig. 1. A mirror with a small hole allows the passage of the light coming from the laser, while the light coming from the fibre is reflected by the surface of the mirror and sent to the detecting system. Alternatively, a dichroic mirror can be used. Good results have also been obtained by replacing the laser with an incandescent light source coupled with a mechanical chopper. The optoelectronic system with the laser was also coupled with another i @put S OTES/OTCS 1. View of the optical arrangement for the pH sensor (from PI optrode (always for pH detection [2]), which exploits the energy transfer between eosin and phenol red. In this case, surface silylation of a glass/glass fibre (200/250 pm) was performed with (y-methacryloxy-propyl)-trimethoxysilane (MAPS). The polymer was obtained in the way described previously, and the pa-sensitive reagents were introduced by using phenol red and a polymerizable compound of eosin: N-(eosinyl)-acrylamide. The performance of the two sensors is quite good: the response time is less than 9 s and the precision for the sensor, based on energy transfer, is 0.008 pH units. Even if the chemical treatment of the fibre increases the surface area available for the attachment of the chromophore, the signal levels obtainable from this optrode are too low to permit the use of LEDs and simple photodiodes in the optoelectronics system. Recently a good many optrodes, able to recognize small amounts of specific antigen by means of immunochemical techniques, have been realized by silylation. One of the silylating agents most utilized for fixing the reagents on the chosen support is 3-glycidoxytrimethoxysilane (GOPS), which has an aliphatic chain terminating with a very reactive epoxy group. Vo-Dinh et al. [ 31used this silylating agent to realize a sensor for the detection of benzo(a)pyrene (BaP), a well-known carcinogen. After stripping a quartz fibre (600 p core) of its cladding for a length of about 7 mm and activating it with GOPS, the surface was oxidized with periodic acid in order to obtain aldehydic groups capable of reacting with BaP antibodies (BaP molecules coupled to bovine serum albumin) to form Schiff bases. A gentle reduction of these Schiff bases was achieved with NaBH,. What is important to emphasize is that BaP is a fluorescent substance, so that there is no need for fluorescent tagged labels. 356 The optoelectronic system coupled with such an optrode is a laboratory system: excitation at 325 run from a helium cadmium laser was coupled to the 600 p fibre used both to transmit the excitation light and to collect the fluorescence emission. A beam splitter makes it possible to send the fluorescence radiation to the detection system, which is constituted by a monochromator and a photomultiplier connected with a picoammeter. The measurement was performed by dipping the fibre tip with the immobilized antibody into a 5 d Fig. 2. Covalent binding on the fibre tip of poly-(N-vinylimidasample of a BaP solution; after a 10 miu incubation and zole) in view of the immobiliition of acid-base indicators (from a 10 s rinse with a phosphate buffer saline solution, the [51F response of the optrode was analysed by transmitting the excitation radiation and recording the fluorescence important to emphasize that a red emitting diode and emission. The detection limit was of the order of 10m9mol/l. an infrared emitting diode were also used. In these cases, even if the output end of the fibre is lower by a As outlined above, in order to increase the electronic factor of 100 in comparison with the use of the laser dynamic range, it can be useful to treat the fibre along source, this shows the feasibility of realizing sensors the core for a longer length. Kawahara zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA et al. [4] developed based on the evanescent wave coupling with electronia sensor for hydrocarbons by silylating a piece of fibre cally modulated cheap and light optoelectronics compo80 cm in length (core diameter = 140 pm). The chemical nents, since the dynamic range of the parameter under treatment was done in order to prepare the organophilic study is strictly connected to the length of the treated surface so that the hydrocarbon substances to be meafibre. sured would be adsorbed onto the surface. The refractive The only industrial prototype of an optical-fibre senindex of the silica used is n = 1.4585 at 589.3 nm; when sor with an optrode obtained by using silylation of the the fibre is immersed in pure water, a total reflection fibre surface is a pH sensor realized by Boisde and his occurs. If hydrocarbons are dispersed in the aqueous collaborators [5] for the detection of pH in the physiosolution, they are adsorbed on the core of the fibre, giving logical range, for medical applications. The silylating rise to an increase of the refractive index in the immediate agent used is GOPS, which permits bonding a polymer environment of the fibre core. Due to the properties (poly-(N-vinylimidazole), PVI) on a PCS fibre core of the evanescent field, a great quantity of light comes (core diameter = 1000 run), for a length of 1 cm by out from the fibre core, and consequently the amount covalent binding, as reported in Fig. 2. Subsequently, of reduced intensity may be related to the amount of after the reaction with a cross-linking reagent such as contaminant. Different chemical reagents were used to epichlorhydrin, and the quatemization of un-crossrealize the organopliilic layer on the fibre surface, in linked sites with iodomethane, the fixation of the dye particular two silylating agents having the same R was achieved, via ionic interactions, by immersing the group (R = -CL8H3,) but different leaving group, treated fibre in a 80% methanol-20% water (v/v) solu(X = OCH,CH,,X = Cl), namely, octadecyltriethoxysition of the dye. The depth of the layer obtained on the lane (OTES) and octadecyltrichlorosilane (OTCS), refibre is of the order of a few microns. Different indicaspectively. Other reagents used have the same leaving tors were fixed, in particular, 3,4,5,6+etrabromophenolgroup (X = Cl) but different R groups (R = Ph or R = sulphonephtalein (TBPSP), which presents a linear n-C&HZ,), diphenyldichlorosilane (DDCS) and n-decylrange between pH 6.8 and 7.5. The optoelectronic unit trichlorosilane (DTCS), respectively. The different coatutilizes two LEDs emitting at 580 and 850~1 for the ings were tested and the results obtained indicated that signal and the reference, respectively. In Fig. 3, a schethe coating reagent and the method of applying it affected matic representation of the fibre-optic link is shown. the capability of absorbing hydrocarbons. The detection With this system, modulation of the two light-emitting limit is variable, and seems to be related to the solubility diodes and a synchronous demodulation (f = 1 kHz) of the pollutants in water. Problems related to the are realized. A logarithmic tiplifier makes it possible discrimination of different hydrocarbons still remain, but to obtain a voltage proportional to the absorbance the physical and chemical approaches are interesting. In difference at 580 and 850nm. A bundle of 400 mixed this case, a 5 mW He-Ne laser (632.8 nm) is used as a fibres is used to connec,t the optrode with the optoelecsource, and the detector is constituted by appropriate tronic unit. Four different branches are connected with electronic circuitry with a simple pin photodiode. Other the two sources and two solid-state Si(pin) photodisources were tested and the signals obtained were odes. The diameter of the bundle is 1 mm; the latter can compared with the ones obtained with the laser. It is 357 tentional. As emphasized before, this is due to the utilization of very inexpensive sources, which can be electronically modulated and easily connected to optical fibres without any optical system of mirrors or lenses; this is also due to the use of appropriate electronic circuits connected with cheap solid-state photodiodes. Of course, easy optical connections and manageable equipment play a fundamental role in the development of industrial devices. zyxwvutsrqponmlkjihgfedcbaZYX Silylationon exfernul support Fig. 3. Multifibre link of the industrialized prototype and exploded view of the pH optrode. be connected with the treated fibre by means of a commercial SMA connector. In this way, a disposable optrode is obtained and can be easily changed. An industrialized version of this instrument is produced by Photonetics (Marly le Roi, France). A modification of the above optoelectronic device has been effected to make possible the use of an optrode with more than one immobilized chromophore in order to increase the pH range to be measured. The related chemistry is analogous to that described for the previous optrode, the only difference being the fixation of the chromophores, which was achieved with a specially prepared mixture taking into account the extinction molar coefficient and the molecular weight of the chosen reagents. For example, a mixture of four reagents (bromophenol blue:chlorophenol red:TBPSP: thymol blue; molar ratio 1:1.9:1.2:3) was immobilized on the grafted PVI optrode. The results showed two dynamic ranges, the former between pH 1 and 3, and the latter between pH 3 and 9. In the above case, three light-emitting diodes were utilized (2, = 590 nm, 1, = 620 run, I, = 850 nm). Each LED is associated with three fibres (200 pm core), and the three LED-fibre combinations are joined to the optrode using a standard connector. One central fibre (400 pm core) carries the modulated light to the photodetector. The electronic unit, also equipped with an internal C language software, allows the modulation of the three lightemitting diodes and a synchronous demodulation (f = 1 kHz) of the collected signal to be achieved. These examples show how, with a good knowledge of chemistry and the use of a very effective optoelectronic system, it is possible to realize prototypes of opticalfibre chemical sensors with the chromophore fixed on the fibre, making use of LEDs and photodiodes as sources and detectors, respectively. The fact that the above two sensors are the only industrialized prototypes of optical-fibre sensors connected with a silylated optrode, and in general two of the very few optical-fibre chemical sensors, is not unin- As mentioned above, if silylation is not realized on the surface of the fibre, an external support, which may differ according to the successive realizations of the optrode, is required. Silylating agents have been used on waveguides to immobilize antibodies [6], and on quartz powder [7] or controlled-pore glasses (CPG) to immobilize a variety of reagents, for example, pH indicators [8,9] or enzymes [lo]. An interesting support was used by Wolfbeis et al. [ 11, 121. In this case, in order to increase the surface of the chosen support (a glass slide), a small layer of CPG (IO-17OA) was sintered on the slide itself. Subsequently, these supports were placed in a solution containing the product of the reaction between APTS and l-hydroxypyrene-3,6,8trisulphonate (HPTS). Alternatively, the sintered glass platelets were treated with APTS and a different indicator, 7-hydroxycoumarin-3-carboxylic acid (HCC), was coupled to the amino surface by using a peptide coupling reagent I-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. A spectrofluorimeter, coupled with a bifurcated fibre-optic bundle (common branch diameter = 6 mm), was used to characterize the layers obtained. At constant ionic strength, an accuracy of 0.01 pH units was obtained. Another pH sensor, which makes use of an external support, was developed in our laboratory [13]. In this sensor, acid&base indicators were covalently bound on CPG (120-200 mesh, pore size 700 A) by using APTS and successive reactions as reported in Fig. 4. The optrode (Fig. 5) is realized with a stainless-steel capillary (external diameter 1 mm), coupled with two optical fibres filled with treated CPG and closed with a stainless-steel cap. Exchange with the external environment is assured by 70 pm holes on the lateral surface of the capillary, made with a laser beam. The size of these holes prevents the exit of the CPG, the dimensions of which are between 70 and 130 pm. The optoelectronic unit (Fig. 5) makes use of two LEDs emitting at 595 and 805nm for the signal and the reference, respectively. An optical-fibre coupler (Gould, core diameter of the fibres = 200 pm) makes it possible to send both wavelengths to the probe using the same fibre. A second optical fibre (core diameter = 200 pm) collects the modulated light and sends it to a photodetector. A suitable 358 !E +(E10~SilCH2’& - loluene/lldC/ZZ h IAfT I ucCCti qNo2 CHCl3 / N(EI)3 rellux I24 h I NaN02 I HCI 2M O’Cl30min wpling with indkalors Fig. 4. Reactions followed for fixing acid-base indicators on CPG. Fig. 5. Block diagram of the optical-fibre system and exploded view of the optrode for pH measurement. detecting system, consisting of a pin photodiode connected with an appropriate electronic circuit [7], processes the signals and supplies the readout of the measurement. The overall instrument is very compact (30 x 30 x 10 cm”) and easily transportable. At present the interest of the authors has been focused on the characteristics of the supports, which may be relevant in a practical realization of the sensor. In fact, when the chemical reactions (reported in Fig. 4) are performed on CPG with a different pore diameter, the results are quite different. The supports used are characterized by the same dimensions (70- 130 pm), but have different pore sizes and, consequently, different surface areas. We have used four different supports: CPG lo-75 8, (mean pore diameter = 77 A, surface area = 182 m*/g), CPG lo-350 A (mean pore diameter = 313 A, surface area = 66.7 m*/g), CPG lo-700 A (mean pore diameter = 810 A, surface area = 27.6 m’/g) and CPG 10-2000 A (mean pore diameter = 2059 A, surface area = 11.1 m’/g). In Fig. 6 the relationship between absorbance and pH is shown for the four different supports; for simplicity of comparison, the lowest absorbance value was set equal to 0. In Fig. 7 the slope of the linear part of these curves is related to the pore size of the CPG: as the pore size decreases and, consequently, the available surface for the chemical bond increases, the value of AA/ApH does not increase monotonically, as might be expected, but is characterized by a maximum value. The reason for this behaviour is under study and could be ascribed to a steric hindrance. Another example of an optrode realized with silylation of an external support is the optrode developed by Bright et zyxwvutsrqponmlkjihgfedcbaZYXWVUTS al. for the detection of haptens [ 14,151, by using different antibody fragments Fcab’)as the recognition element. Human serum albumin, digoxin and theophylline were measured by immobilizing the corresponding antibody fragment and following a quite similar procedure: the chosen support was quartz platelets, cut into 0.5 cm squares whose surfaces were treated with GOPS; after hydrolysis’of the epoxy groups, the resultant hydroxyl groups were reacted with tresyl chloride. The resulting surface was then reacted with Fcab’) fragments, which after immobilization were fluorescently labelled with dansyl chloride. When the analyte is present, it binds to the sensor, thus producing a significant increase in the fluorescence. This is due to 359 A fibre, filtered by a 420 mn long-pass filter and detected with a photomultiplier tube. In this case even if an external support is used, a not very high signal-to-noise ratio can be achieved, since the light is modulated only by a monolayer immobilized on the quartz platelets. As stated before, silylating agents have been used not only to introduce reactive chemical groups on the surfaces of the supports, but also to make the surface hydrophobic in order to allow the formation of Langmuir-Blodgett (L-B) 6lms. Here, after the treatment of the surfaces, the supports were covered with L-B films which are generally composed of a chemical suitable for the formation of fihns and by a fluorophore. Wolfheis et al. have described optical chemical sensors for ions obtained by using glass platelets treated with dimethyldichlorosilane (DMECS) on which L-B fihns selective to sodium [ 161 and to calcium [ 17] were formed. Krull et al. [ 181demonstrated that L-B multilayers obtained on surfaces treated with OTCS can be sensitive to different vapours, such as hexane, chloroform, ethanol and N,N-dimethylaniline. I Conclusions Fig. 6. Relationshipbetweenabsorbanceand pH for bromopheno1 blue immobilizedon CFG .tiwithdifferentpore sizes (0, 2oooA; q, 700A; l , 350A; A, 75Aj. d 0 I. ” ” 500 ” ” 1OfM ” ” 1500 Silylation is a good technique for obtaining compact and effective optrodes having no problems of leakage of the immobilized chromophore. Particular attention must be devoted to the optoelectronics to be coupled to the optrode, since certain requirements must be satisfied so as to realize an optical-fibre sensor that is easy to industrialize and is competitive on the market. LEDs and simple photodiodes connected with an appropriate circuitry seem to be the best solution from this point of view, but this solution implies some restrictions. Therefore, the optrode and the associated optoelectronics must be designed simultaneously in order to build up a ‘real’ optical-fibre sensor, and not simply a laboratory ” zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGF ,269Q system. poresizeA Fig. 7. Relationship between pore size of treated CPG and AAIApH. the fact that dansyl is an environment-sensitive probe. In a polar environment, a bathochromic shift and a decrease in emission yield are observed, while in nonpolar media, a hypsochromic shift and an increase in fluorescence yield are observed. Accordingly, when the antibody-antigen complex is formed on the support, the dansyl label is located in a more hydrophobic environment, so an increase in fluorescence occurs. As far as the optoelectronics are concerned, light from a He-Cd laser (325 nm) was focused on a quartz fibre (200 m core); the fluorescence coming from the treated substrate was collected by a 600 pm quartz Acknowledgements The authors wish to thank Professor A. M. Scheggi (Istituto di Ricerca sulle Onde Elettromagnetiche) for her continuing interest and Dr M. Bacci (Istituto di Ricerca sulle Onde Elettromagnetiche) for his helpful suggestions during the course of this work. References 1 C. Munkholm, D. R. Walt, F. 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Anal. Chem., 334 (1989) 162-165. of pesticides, Sensors and Acruarors B, 7 (1992) 509-512. 9 M. S. Fuh, L. W. Burgess, T. Hirschfeld and G. D. Christian, 25 Y. Kawabata, T. Imasaka and N. Ishibashi, Fiber optic pH Single fibre optic tluorescence pH probe, Analyst, 112 (1987) sensor based on laser fluorimetry, Proc. OFS’ 86, 4rh Inm. 1159-1163. Coni. ‘Optical Fiber Sensors’, Tokyo, Japan, 1986, p. 39. 10 J. Ruzicka and J. Flossdorf. Characterization of immobilized enzymes by flow-injection techniques with variable forward flow, Anal. Chim. Acta, 218 (1989) 291-301. Biographies 11 H. Hoffenbacher, 0. S. Wolf%eis and E. Fiirlinger, Fluorescence optical sensors for continuous determination of nearneutral pH values, Sensors and Actuators, 9 (1986) 73-84. Francesco Baldini was born in Florence in 1961. He 12 0. S. Wolbeis and H. HolTenbacher, Fluorescence sensor for received a degree in physics from the University of monitoring ionic strength and physiological pH values, zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLK SenFlorence in 1986. In 1989 he became a researcher at the sors and Actuators, 9 (1986) 85-91. Institute of Electromagnetic Wave of CNR. His research 13 F. Baldini, S. Bracci and M. Bacci, A fiber-optic pH sensor using acid-base indicators covalently bound on controlled activity is principally devoted to optical-fibre sensors for pore glasses, Proc. SPIE, Chemical, Biochemical and Environphysical and chemical parameters and to optical methmental Fiber Sensors III, Boston, MA, USA, Sept. 3-6, 1991, ods used for the restoration of paintings and frescoes. pp. 67-73. 14 F. V. Bright, T. A. Betts and K. S. Litwiler, Regenerable fiber-optic-based immunosensor, Anal. Chem., 62 (1990) Susanna Bracci was born in Florence in 1961. She 1065-1069. graduated in chemistry from the University of Florence 15 T. A. Bet& G. C. Catena, J. Huang, K. S. Litwiler, J. Zhang, in 1988. Her research activity is mainly concerned with J. Zagrobelny and F. V. Bright, Fiber-optic-based imorganic synthesis. Since 1989 she has been involved in munosensors for haptens, Anal. Chim. Acra, 246 (1991) 55research concerning optical-fibre chemical sensors. 63.