ANALYST, JUNE 1993, VOL. 118 593 Flow-through (6io)Chemical Sensors* Plenary Lecture Miguel Valcarcel and Maria Dolores Luque de Castro Department of Analytical Chemistry, Faculty of Sciences, University of Cordoba, E- 14004 Cordoba, Spain The basic features and most salient examples of flow-through chemical and biochemical sensors based on the integration of derivative analytical reactions, separation processes (dialysis, gas diffusion, sorption, liquid-liquid extraction) and detection (optical, electroanalytical, mass, thermal) are presented and discussed, and critically compared with those of probe-type sensors and conventional continuous-flow configurations, where such processes take place sequentially in separate modules. Keywords: flow-through sensor; chemical and biochemical sensors; continuous flow Devices providing direct, immediate analytical information on a given system under study are still a future goal, the accomplishment of which relies heavily on automation and miniaturization.' The main trends in the science and tech- nology of the late 20th century, in response to the basic objectives of today's and tomorrow's analytical chemistry, are the acquisition of more chemical information of higher quality by expending less material, time, effort and economic resources .2,3 What is a sensor? Defining this word is far from easy.Because sensors are the magic keys to many doors, their name is often used improperly. Ideally, a (bio)chemical sensor is an analytical device that responds in a direct, reversible, contin- uous, rapid and accurate (precise) manner to changes in the concentration of chemical or biochemical species in an untreated sample (Fig.1). It may consist of a sensing microzone where a chemical or biochemical reaction (and, occasionally, a separation process) takes place, which is connected or integrated with an optical, electrical, thermal or mass transducer. In short, the principal use of an ideal sensor is for integrating two of the three general steps of the analytical process (preliminary operations and measurement and transduction of the analytical signal). In principle, sampling, addition of reagents, detection, separation tech- niques, etc., need not be included. However, many of the sensors developed in the last few years fail to meet one or more of the defining criteria. Interestingly, many books on this topic provide no description of sensors or begin with a stringent definition, yet they subsequently deal with devices that do not operate in a direct, reversible, rapid, continuous or accurate fashion. This is also so with many papers that include the word sensor in their title, yet are concerned with devices that only meet a few if any of the basic requirements for the ideal (bio)chemical sensor, even in the absence of a sensing microzone containing an immobilized (bio)chemical species.The large number and variety of sensors reported so far make it necessary to establish classifications according to a host of different criteria: the monitored parameter (chemical or biochemical) , their nature (reversible, irreversible, dispos- able or reusable), their external shape (planar, probes or flow cells), the relationship between the sensitive microzone and the transducer (connected or integrated), the operational mode (batch or continuous), the occurrence or not of a (bio)chemical process (whether active or passive), the inclu- sion or not of an additional separation process, the type of transducer (optical, electrical, thermal, mass or otherwise), and the number of monitored species (single parameter or multi-parameter; individual or integrated).A detailed de- * Presented at SAC '92, an International Conference on Analytical Chemistry, Reading, UK. September 20-26, 1992. scription of each classification is beyond the scope of this paper. In any event, such classifications provide a good idea of how varied sensors are and show how difficult it is to establish general rules and describe their behaviour in broad terms.However, defining the generic properties of a (bio)chemical sensor is easy. Some of them coincide with basic analytical features (accuracy, precision, sensitivity and selectivity), whereas others are related to appropriate performance (reversibility , reusability for irreversible, regenerable systems and suitability for a single use for irreversible, non-regener- able systems). Time-related features such as (near) real-time response, rapidity in the reversible and regeneration processes involved and stability (long shelf and operational lifetimes) are crucial. One other set of features are related to reliability, viz., simplicity of construction and operation, ruggedness, low cost, usability with complex samples, suitability for evolving systems (portable manifolds) and the need for no interpreta- tion by the operator.Some of these features are indispensable, others only desirable. This paper reviews the state-of-the-art and trends of (bio)chemical sensors based on the integration of detection and reaction (and/or separation) in a flow cell that can be integrated with or connected to an optical, electroanalytical, thermal or mass detector for direct determination of one or more (bio)chemical analyte(s) in gas or liquid samples. Far from being exhaustive, it summarizes the fundamental prin- ciples and most significant alternatives of this type of sensor, with special emphasis on optical systems.Critical comparisons with matching probe-type and continuous-flow [flow injection (FI)] configurations are also made throughout. Generic Features of Flow-through (Bio)Chemical Sensors In conventional continuous-flow analytical systems,4.5 separa- tions involving mass transfer between two phases, (bio)chem- ical reactions and continuous detection in a conventional flow Reliable Analytical instrument Sensor '-' '-' Complex sample 1 -A---- I- Accurate (precise) I Real-time I - - - - - - - I t I - Dirkt- -I I '1 Reversible ' Basic features -,,,,I Fig. 1 Features of the ideal sensor594 ANALYST, JUNE 1993, VOL. 118 cell take place in different modules that are isolated in space, i.e., in a sequential manner [Fig. 2(a)]. There are four generic ways of integrating these steps [Fig.2(b)], namely, by combining: (1) continuous separation(s) and analytical reac- tion(s); (2) (bio)chemical reaction(s) and detection; (3) continuous separation(s) and detection; and (4) all three types of process. A device that performs detection almost simul- taneously with reaction (type 2) and separation (type 4 or 3) in the flow cell can be considered to be a sensor as it meets the main requirements described above. There are three basic types of flow-through (bio)chemical sensors (Fig. 3) according to the location of the sensitive microzone where the bio(chemica1) reaction and/or separation take place and its the relationship to the detector. Two of them [(a) and ( b ) ] rely on the use of probes connected to the instrument; the scnsitive microzone can be attached to the end of the probe (a) or incorporated into the flow cell (b).The third type of sensor involves a conventional instrument in which the sensitive microzone is incorporated into a special flow cell ( c ) . They differ from ‘probe sensors’ because in the latter the probe, either containing (active) or not containing (passive) the sensing (bio)chemical microzone, is plunged into the gas or liquid sample rather than being introduced (by aspiration or injection) into the flow cell. There is a tendency to use the word probe instead of sensor in describing disposable (single-use) sensors;6 in any event, the borderline between the two is blurred. For this reason, we prefer to use the word probe to denote rod-shaped sensors that are connected to transducers.The sensitive microzone is a single flow cell, with no reactive element in the so-called passive sensors, which makes it difficult to distinguish between a flow-through sensor and a conventional continuous-flow configuration (e.g. , an FI mani- fold). In active flow-through (bio)chemical sensors, a reactive microzone is included in the flow cell. The physico-chemical interaction between such a microzone and the components of the gas or liquid streams flowing through the system can be in the form of a simple (bio)chemical reaction, a separation process or a combination of both. As a rule, these processes are based on the immobilization of one of the ingredients of a (bio)chemical reaction, whether this be the analyte, reagent, catalyst or reaction product.7 Immobilization must be per- manent if the active component (reagent or catalyst) is to be used for a large number of determinations.If the reagent is consumed in the process, the flow-through sensor can only be used a limited number of times. On the other hand, when the analyte or its reaction product is to be immobilized, their residence in the flow cell will be temporary, so the sensor must be regenerated (i. e., the temporarily immobilized species must be removed) after each determination. Integrated separation can be accomplished by means of a membrane (dialysis and gas-diffusion processes) or a solid support in the form of a film, a porous solid or particles (sorption).X.9 The integrated (bio)chemical reaction can take place on the support (when the analyte or reagent is the immobilized (a) Conventional Detector (b) Integrated Detector Fig.2 figurations ( a ) Conventional and (b) Separations (BioIChemical 00 reactions Detection 0 integrated continuous-flow con- species), in the solution held in the cell (when the catalyst is supported on the solid) or in a reaction coil (when the reaction product is the temporarily immobilized species). Flow-through sensors are available in a variety of shapes suited to the type of detection required, their relationship (connected or integrated) to the (bio)chemical microzone and the inclusion or not of a simultaneous or sequential separation process. The most frequently used configurations are depicted in Fig. 4. In the optical flow-through cell, the light beam can reach and leave the cell directly or through optical fibres for absorption (Al) and (A2), fluorescence (Al), reflectance (A3) or (bio)chemiluminescence measurements (A4).The use of a potentiometric or voltammetric electrode in an active flow cell allows the implementation of a variety of flow-through sensors (B). Most mass and thermal flow-through (bio)sensors rely on differential measurements; they use two identical cells (one of them containing the sensitive microzone) that are arranged either in series (C) or in parallel (D). A gas-diffusion or dialysis membrane can be incorporated into the active flow- through cell by passing one (El) and (E3) or two streams (E2) through it. One of the most salient features of flow-through sensors is their compatibility with unsegmented-flow configurations,4~5 which allows the main advantages of continuous-flow mani- folds [automation, flexibility, ease of sample conditioning and calibration, development of previous (bio)chemical reactions, etc.] to be extended to this type of sensors, thereby also allowing for use on real samples. These configurations, therefore, make excellent links between real samples and flow-through sensors.The large variety of configurations of this type is described and classified according to the immobi- lized species elsewhere.7 Probably, one of their greatest assets is their suitability for performing the regeneration step in irreversible, reusable sensors in a very simple, automatic fashion compared with probe-type sensors [Fig. 5 (A)]. The three principal regeneration modes of flow-through (bio)- chemical sensors are shown in Fig. 5 (B).The first made, the simplest (Bl) , involves injecting rather a large sample volume Instrument - Sample I I rSMZ Sample -4 I Sample I Instrument ‘1‘ Fig. 3 Generic types of flow-through (bio)chemical sensors. Sensitive (bio)chemicai microzone (for details, see text) SMZ:ANALYST, JUNE 1993, VOL. 118 "1 S l ( E l ) lS * /K- SMZ I I I C I Fig. 4 Examples of flow-through cell sensors in which a sensing (bio)chemical microzone (SMZ) (type A, B, C and D) and a membrane (M) (type E) arc integratcd with an optical (type A, E l and E2). electroanalytical (type B and E3), mass (type D) and thermal (type D) detector. S, samples; RP, reflection plate; PMT, photomultiplier tubc; E, potentiometric or voltammetric electrode; PC, piezoelectric crystal; T, thermistor; FO, fibre optic; and AS, accepting stream (0.2-2 ml) into a carrier containing the (bio)chemical reac- tants for regeneration via an injection valve; as soon as the tailing end of the sample plug reaches the sensor, the regeneration process starts.The second mode (B2) involves sequential aspiration of the sample and regeneration streams via a switching valve. Finally, the third mode (B3) is based on injection of the sample volume into a (conditioning) carrier and aspiration or injection of the regenerating solution. In all instances, the signal increases as the sample plug passes through the sensor. When the tailing end of the sample plug reaches the sensor (first type), the selecting valve is switched (second-type) or the regeneration solution is introduced (third type) and the baseline, which is established by passing the regenerating (types 1 and 2) or conditioning carriers (type 3) through the system, is restored.Thus, the temporary signal obtained can be a peak (type 1 and 2) or plateau (type 3). Analytical information can be drawn from the peak or plateau height or the slope of the rising portion of the signal (kinetic method). Various examples of flow-through (bio)chemical sensors are described below, classified according to the processes that take place in the flow-through cell. All of them must meet the following basic requirements for appropriate performance: (i) the system should be reversible (or easily regenerable if irreversible); (ii) the kinetics of the processes involved (chemical and biochemical reactions, separation process, etc.) should be fast unless the catalyst is the immobilized species; (iii) the immobilization linkage should be very stable when the reagent or catalyst is to be immobilized in the flow cell; and ( i v ) the sensitive (bio)chemical microzone and the detection system should be fully compatible.Integration of Reaction and Detection Flow-through (bio)chemical sensors based on the integration of reaction and detection in a suitable flow cell rely on the permanent immobilization of one or several reagents and/or the catalyst on an appropriate support. The difference between conventional continuous-flow configurations, in which a mini-reactor is placed before the detector flow cell (occasionally called sensor systems, as in ref.lo), and true sensors is illustrated in Fig. 6. This type of sensor involves no separation process unless the immobilized reagent is partly consumed in each determination. They differ from those in which the immobilized reagent retains the analyte temporarily and a detectable change (colour, fluorescence) occurs simul- taneously. A single immobilized reagent located in a flow-through cell can be used for the determination of a variety of species by different detection principles. Surface-modified electrodes inserted into flowing systems are representative examples. 11 One such system is the carbon electrode modified with immobilized FeVFelII sites that respond amperometrically to various nitrogen oxides.12 Many of these flow-through sensors are based on immobilized, non-regenerable ( e . g . , luminol13) or regenerable [e.g., tris-(2,2'-bypiridyl)ruthenium(1i)] com- plexes14 that act as chemiluminescence reagents, as well as on fluorophores15 and phosphors. 16 Frei and co-workers used a flow-through cell containing two reagents, viz., a solid [bis(2,4,5-trichlorophenyl) oxalate (TCPO)] and a fluoro- phore (3-aminofluoranthene), immobilized on different types of supports for the determination of hydrogen peroxide,17 glucosels and anilines. Ic) By immobilizing pyrenebutyric acid596 ANALYST, JUNE 1993, VOL. 118 B ,&, S I RC I S 1 Flow-th roug h sensor / C I Fig. 5 Regeneration modes in the use of irreversible-reusable or probe-typc sensors (A) and flow-through sensors (B).S, samples; RD, regenerating dissolution; C, carrier; RC, regenerating carrier; IV, injection valve; and SV, switching valve in a silicone membrane or on a glass support placed in a flow- through cell, molecular oxygen can be determined in gases by its quenching effect on the native fluorescence of the reagent .20 The same principle was used by immobilizing benzo[ghi]pyrene in a silicone-rubber membrane that was placed on a planar glass plate, separation of excitation light and fluorescence emission being accomplished by total inter- nal reflection.21 A flow-through cell containing a palladium wire as sensing element attached to a monomode optical fibre was used to determine hydrogen in gases by formation of palladium hydrides, which resulted in longitudinal strain of the optical fibre that was transduced to a phase retardance in a light beam guided by the fibre and detected by inter- ferometry.22 Catalysts (enzymes) immobilized in flow-through cells can be used to develop biosensors based on various detection principles that can be coupled on-line to continuous-flow configurations. The main alternatives in this context are: (i) passing the sample-reaction plug through the sensitive micro- zone once (conventional) or several times (iterative reversal of the flow direction and open-closed configurations); and (ii) stopping the flow as the plug reaches the sensor. The choice is dictated by the rate of the (bio)chemical reaction. Chemi(bio)luminescence reactions are generally fast enough for implementation in straightforward continuous- flow configurations with a catalyst immobilized in the flow cell.Thus, haemin, haemoglobin and horseradish peroxidase were used to determine hydrogen peroxide by the luminol reac- tion,23 co-immobilized bacterial luciferase and oxidoreductase were employed to determine nicotinamide adenine dinucleo- tide (reduced form) (NADH), nicotinamide adenine dinu- cleotide phosphate (reduced form) (NADPH), flavin mono- nucleotide (FMN) and FMNH2 (FMN, reduced form),24 and firefly luciferase was utilized to determine adenosine triphos- phate (ATP), creatine phosphate and phosphokinase.25 A set- up consisting of a flow-through cell housing an enzyme-coated optical fibre and a combined optical-enthalpimetric detector was reported by Dessy et aZ.26 The ability of FI configurations to implement stopped-flow methodologies can be exploited to improve sensitivity and selectivity (reaction-rate measure- ments) by means of (bio)chemical sensors [Fig.7(a)]. In this way, ethanol in real samples can be determined spectrophoto- metrically using alcohol dehydrogenase immobilized on the R and/or C (a) s D ( b ) , R and/or C Fig. 6 Difference between (a) a sensor system and ( h ) sensor in which the reagent (R) and/or the catalyst (C) is immobilized on a suitable support and placed in a reactor or a flow-through cell that is integrated with the detector (D). rcspectively. S, sample flow-cell walls.27 Optical glucose sensors based on glucose oxidase immobilized on a germanium crystal28 and an As-Se- Te fibre29 accommodated in a flow-through cell and combined with Fourier-transform infrared-attenuated total reflection detection were recently reported. Measurements were made under stopped-flow conditions.Multi-peak recordings can be obtained by passing the sample plug through the (bio)chemical sensor containing the immobilized catalyst several times. This can be accomplished by iteratively reversing the flow direction [Fig. 7(b)]3" or by using an open-closed configuration31 [Fig. 7(c)]. Thus, by using immobilized P-D-glucuronidase in a photometric flow cell, the hydrolysis of 4-nitrophenol-~-glucoronide was moni- tored with five devices: three of them were true biosensors (an enzyme was immobilized in the flow cell) that differed in the way that reaction rates were measured (by the stopped-flow mode or with iterative passage of the sample plug)32 (see Fig.7), whereas the other two were biosensor systems [Fig. 6(a)] relying on the iterative, sequential passage of the sample plug through the enzyme reactor and the flow cell.33 The use of parallel dual biosensors (see Fig. 4, E) for implementing calorimetric measurements makes an interest- ing alternative to immobilized enzymes integrated in trans- ducing elements (two thermistors) .34-38 They have been used to determine a variety of substrates. Integration in flow-through cells of an enzyme (glucose oxidase39.4" or lactate oxidase41) and an indicator reagent whose fluorescence is dynamically quenched by molecular oxygen has facilitated the development of a new series of biosensors. Inverted video imaging microscopes have been used to develop receptor-based flow-through biosensors using cray- fish antennae, the nerves of which are connected to a physiological amplifier via ground and reference wire poten- tiometric electrodes.One such sensor was successfully applied to the determination of pyrazinamide in water.42 Integration of Separation and Detection This type of (bio)chemical sensor involves reversible mass transfer between two phases (solid-liquid, liquid-liquid or gas-liquid) in a flow cell that is integrated with or connected to the detector.8 The species involved in this process can be analyte(s) (when their physico-chemical features are directly usable for detection) or previously formed reaction products.They differ from the other types of sensor in that no (bio)chemical reaction takes place in the flow-through cell (see Fig. 8). There are several examples of flow-through sensors that rely on a separation process through a membrane without (bio)chemical reaction; such is the case with an integrated gas-diffusion-atomic absorption cell for the deter- mination of mercury,43 a variety of film-coated voltammetric and potentiometric electrodes used in continuous-flow systems that allow matrix effects to be minimized11 and a gas- diffusion membrane coating an iridium-metal oxide thermal detector.44ANALYST, JUNE 1993, VOL. 118 597 stop Time - 4 Change - D t w II C U A Time - Time ---t Fig. 7 Alternatives to reaction-rate measurements by use of immobilized enzymes ( C ) in the flow-through cell of a photometric detector (D).(a) By stopping the flow; ( h ) by iterative reversal of the flow direction; and ( c ) by using an open-closed configuration. SV, Selecting valve; W, waste; and P, pump The most frequently employed flow-through sensors of this type are based on the use of sorbent material (e.g., ion- exchange beads, C18 bonded phase silica beads) that is packed in the flow cell of a non-destructive detector (e.g., a photometer or fluorimeter), where the analytes or their reaction products are temporarily immobilized. These are irreversible, reusable sensors inasmuch as two steps (retention and elution as implemented in the configurations depicted in Fig. 5 ) take place sequentially but simultaneously with detection in each determination.If the analyte(s) can be directly and temporarily retained by the support, they must lend themselves to continuous detection. Such is true for the direct spectrophotometric determination of ionic copper based on its native colour; the ions are temporarily retained in a flow cell packed with a cation-exchange resin and elution is effected by injecting a nitric acid solution.45 Flow-through sensors based on the temporary immobiliza- tion, on a solid support, of reaction products formed previously in the continuous manifold offer a wide range of applications. For example, bismuth can be determined by forming iodide complexes and retaining them in a flow- through cell containing Sephadex anion exchanger as sup- port.46 In addition, the temporary retention of the product formed between CrVI and diphenylcarbazide on a cation- exchange resin was exploited for the determination of this ion.47 Cyanide was determined at the ng ml-I level using a Fig.8 Flow-through sensors based on the integration of a reversible separation process, through a membrane (M) or on the surface of a solid support (SP), and detection. S , sample containing the analyte; RP, reaction product; and DZ, detection zone merging-zones FI manifold into which two plugs of sample and reagent (pyridoxal-5-phosphate) were simultaneously in- jected; fluorimetric flow-through cell packed with an anion exchanger allowed retention and detection of the reaction product to be integrated.48 Phosphate was determined by forming an ion associate between molybdophosphate and Malachite Green that was temporarily retained on Sephadex LH-20 packed in a photometric flow-through cell.49 The classical Molybdenum Blue reaction with ascorbic acid has also been used in this context .SO Ammonia can be determined at the pg ml-l level by temporarily immobilizing its product (Berthelot reaction) on Sephadex QAE packed in a photo- metric flow ce11.51 A fluorimetric flow-through sensor for the determination of very low concentrations of fluoride based on the temporary immobilization of the ternary complex zirco- nium(1v)-Calcium Blue-fluoride showed improved analytical features compared with its matching probe-type sensor.52 Aluminium can be determined using a room-temperature phosphorescence chemical sensor based on the formation in a coil of a complex between aluminium and 8-hydroxy-7-iodo- quinoline-5-sulfonic acid (ferron) , which is temporarily immo- bilized on an anion-exchange resin packed in a flow ce11.53 The determination of p-aminobenzoic acid was accomplished by using a customized flow cell accommodating a paper filter in a straightforward FI configuration through which a silver hydrosol was circulated; detection was effected by surface- enhanced Raman spectrometry.54 A film of Prussian Blue coating a quartz crystal microbalance was used as a flow- through mass sensor to determine dissolved electroinactive ions .55 One of the most promising aspects of this type of sensor is the ability to take advantage of photometric diode-array detectors to monitor simultaneously absorbances on a support (with or without an immobilized species) at various wave- lengths.The joint use of this principle and classical deconvolu- tion chemometric approaches allows the development of multi-parameter sensors. Mixtures of amines can be deter- mined on the basis of their intrinsic absorbances by injecting a large sample volume into a methanol-water carrier in order to drive the plug to a flow cell packed with CIS bonded phase silica beads for temporary retention.56 A determination of carbamate pesticide mixtures at the ng ml-l level based on the temporary retention of their reaction products on CI8 bonded phase silica beads placed in the flow cell of a diode-array spectrophotometer was recently reported.57 The same flow- through sensor was successfully used as a post-column system in high-performance liquid chromatography (HPLC) .58359 Other spectroscopic approaches can be used to develop multi-parameter flow-through (bio)chemical sensors.An example is the simultaneous determination of the different forms of vitamin B6 (i. e., pyridoxal, pyridoxal-5-phosphate and pyridoxic acid) based on the formation of fluorescent complexes with Be" in ammonia solution and their temporary retention on CIS bonded phase silica beds packed in a conventional flow cell. Discrimination relied on derivative synchronous fluorescence measurements .60598 ANALYST, JUNE 1993, VOL. 118 Integration of Reaction, Separation and Detection With flow-through (bio)chemical sensors based on triply integrated continuous systems, separation processes and reactions take place either sequentially or simultaneously.On the other hand, detection occurs simultaneously with one or the two other processes (Fig. 9). This type of sensor involves permanent immobilization of the reagent and/or the catalyst. Occasionally, however, no active ingredient of the (bio)chem- ical reaction is immobilized, i.e., the reaction takes place in the solution held in the flow cell. Separation processes can be enacted through membranes (dialysis, gas diffusion) or solid supports packed with beads or coated with a film. The immobilized reagent can play a single- or two-fold role: acting as ingredient of the derivatizing reaction and/or faciliting the separation processes. Ionophores immobilized in a flow-through cell can tempor- arily retain ionic species from the flowing sample solution.The derivatizing reagent can be either dissolved or immobilized [Fig. 9(a)]. Werner el al. 61 developed various fluorimetric flow-through sensors based on the temporary retention of cationic analytes (K+, NH4+) on controlled-pore glass beads, where different ionophores (dibenzo-18-crown-6, valinomy- cin, nonacin) were non-covalently immobilized; a dissolved fluorescence probe (8-anilinonaphthalene- 1-sulfonic acid) previously mixed with the sample yielded a fluorescent ion pair on the solid surface. A calcium ion selective flow-through optrode based on the fluorescence quenching of a dye (the C- 18 ester of Rhodamine B) incorporated into a lipid membrane containing a calcium ion selective ionophore was recently reported.62 A flow-through photometric sensor with a plasti- cized poly(viny1 chloride) (PVC) membrane containing an NH4+-selective macrotetrolide ionophore, an H+-selective neutral chromatographic and a lipophilic anion was success- fully used for the determination of ammonium ions.63 Also, a PVC membrane placed in a fluorimetric flow-through cell containing Hexadecylacridine Orange and valinomycin was employed for the determination of potassium .64 Permanent retention of a colorimetric or fluorimetric pH indicator dye in a flow cell was first accomplished by Kirkbright et al.in 1984.65 Temporary retention of H+, OH- or other species is the operational principle of this type of sensor [Fig. 9 (C)]. Sulfide ions can be determined by using various reagents immobilized on porous organic polymers and fibre optics.66 Commercially available indicator papers placed in a flow cell provided a reflecting plate that was used in conjunction with two optical fibres for pH measurements by reflectance spectroscopy.67 They were applied to the determi- nation of rainwater pH68 and the sequential determination of acids and b a ~ e s .6 ~ If an immobilized enzyme is incorporated into cellulose acid-base pads, the resulting sensor can be used for the determination of urea70 and penicillin.71 The use of reagents immobilized on solid supports (either as films or beads) that are in turn placed in a flow cell allows temporary retention (separation) and derivatization to take place simultaneously [Fig. 9(c)]. This approach has been applied to the spectrophotometric and spectrofluorimetric determination of metal ions and other chemical species. Thus, copper was determined at the ng ml-l level by immobilizing 4- (2-pyridy1azo)resorcinol on a cation-exchange resin packed in a spectrophotometric flow cell; the system was made reusable by including an eluting ligand (2-mercaptoacetic acid) in the carrier.72 By using a similar arrangement, beryllium was determined spectrofluorimetrically with morin immobilized on an anion-exchange resin .73 Chlortetracycline immobilized on an anion-exchange membrane placed in a flow cell to which a branched optical fibre was attached was used for the spectrofluorimetric determination of calcium by formation of a chelate.74 Aluminium was determined in dialysis fluids and concentrates with a flow-through spectroiluorimetric fibre- optic sensor containing the 8-hydroxyquinoline derivative Kelex-100 immobilized on Amberlite XAD-7.75 Also, low Fig.9 Generic types of (bio)chcmical flow-through sensors based on the triple integration of separation, reaction and detection. Differ- ences lie in whether the integrated rocesses take place sequentially [ ( a ) and (b)] or simultaneously (cf S, sample; and K, additional reagent (for details, see text) concentrations of iron in waters and wines were determined by means of a spectrophotometric flow-through sensor contain- ing an anion-exchange resin in the thiocyanate form.76 A flow- through spectrophotometric flow cell accommodating a PVC membrane on to which a lipophilized chromogenic ligand, 3 - octadecyloxy-4-(2-pyridylazo)resorcinol, was immobilized was successfully used to determine zinc.77 An optical wave- guide coated with a copper-organophosphine complex placed in a flow-through cell was employed as a reversible chemical sensor for sulfur dioxide.78 Water and ethanol can be determined in a spectrophotometric flow-through sensor based on the immobilization of lipophilic trifluoroaceto- phenone derivatives on a PVC membrane that extract the analytes to form hydrates and hemiacetals, respectively, and change their absorption spectra.79 These triply integrated (bio)chemical sensors have been demonstrated to be suitable for implementing immunoassays based on the immobilization of one of the components of the biochemical interaction.Thus, antibodies [anti-mouse immu- noglobulin G (IgG)] covalently immobilized on to a rigid beaded support placed in a bioluminescence flow-through cell was used for the determination of antigens (mouse TgG); a two-site immunoassay was accomplished by consecutive injection of the sample, acridinium ester-labelled antibodies and alkaline hydrogen peroxide to initiate luminescence.80 An immunomembrane containing the antigen bovine IgG mounted in a bioluminescence sandwich flow cell was used in an FI system for the determination of mouse anti-bovine IgG; four sequential injections (analyte, goat anti-mouse IgG- horseradish peroxidase conjugate, buffer and substrates plus luminol reagents) into an assay carrier containing bovine serum albumin and p-iodophenol were required.81 A capaci-ANALYST, JUNE 1993, VOL.118 599 tance flow-through cell using antibody or antigen immobilized on a tantalium oxide surface for implementing real-time immunoassays was recently reported.** Sensors based on integration of dialysis, reaction and detection rely on the use of a membrane located in a flow cell to deliver one of the ingredients of the (bio)chemical reaction directly. Determination of metal ions was accomplished using a pressurized membrane through which a spectrofluorimetric reagent was forced into the flow ce11.83 Sucrose was deter- mined after conversion into glucose (using an enzyme reactor containing invertase and mutarotase) and development of the classical luminol chemiluminescence reaction of glucose oxidase, which was delivered directly through a membrane accommodated in the chemiluminescence flow ce11.84 Perm- selective film-coated enzyme electrodes are another example of this type of triply integrated sensor.” There are several examples of integrated gas-diffusion- chemical reaction-detection (bio)chemical systems in the literature.Most of them are ammonia chemical sensors. In these, the analyte or its reaction product is the species involved in the separation process. A universal sandwich membrane flow-through cell integrated with absorption, reflectance or chemiluminescence detection for determination of ammonia and hypochlorite was reported recently .X5 Ammonia can also be determined using a gas-diffusion membrane placed in a flow cell and fibre-optic reflectance measurements; detection is based on an acid-base indicator that can be either immobilized67 or not.86 A flow cell shaped like that in Fig.4 (Fl), was developed for the determination of ammonia,s7 the sensitive microzone consisting of a PVC optrode membrane containing the same ingredients as used in ref. 63. A flow-through gas chemiluminescence sensor for the determination of molecular oxygen based on the diffusion of the analyte through a membrane and its subsequent reaction with 1,1’,3,3’-tetraethyl-A-bis(imidazolidine) dissolved in hexane was reported by Freeman and Seitz.88 Conclusion A critical comparison between flow-through (bio)chemical sensors based on integrated separation, reaction and detection with their matching probe-type sensors and conventional continuous-flow configurations (in which all three steps take place sequentially in separate modules) allows the advantages and disadvantages of using the former in analytical chemistry to be clearly established.7.8 The most outstanding advantages of integrating flow- through (bio)chemical sensors are: (i) the increased sensitiv- ity89 resulting from miniaturization, which reduces dispersion of the inserted sample and concentration of the analyte or its reaction product, a typical feature of integrated separation techniques; (ii) the selectivity being indirectly enhanced by the removal of interfering species and the avoidance of parasitic signals by use of kinetic approaches based on differential rather than absolute measurements; (iii) the implementation of the regeneration step, which can be carried out readily, quickly and conveniently, is dramatically facilitated; and (iv) the use of FI configurations to implement (bio)chemical sensors offers interesting, practical assets such as high versatility and sample throughput, ready calibration, reduced sample and reagent consumption and easy adaptation to on- line process monitoring.On the other hand, the most serious drawbacks of this type of flow-through sensors are: (i) the lack of compatibility between the sensitive microzone (support, membrane, etc.) and the detection system required to ensure appropriate performance of the analytical method concerned; (ii) prob- lems arising from the kinetics of the separation and reaction steps involved; (iii) difficulties in remote sensing and micro- zone monitoring; and ( i ~ ) the scarcity of appropriate chemical and biochemical systems meeting the essential requirements for integration in a flow-through cell.We believe that it would be interesting to start a new age in the development of (bio)chemical sensors by systematically applying them to real analytical problems ( e . g . , environmen- tal, food, pharmaceutical and industrial samples) to exploit the myriad of academic developments achieved so far. The implementation of validated analytical methods based on sensors and their subsequent use by routine laboratories is no doubt going to bc the crucial test for analytical sensors in the 1990s. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 References Luque de Castro, M.D., and Valcarcel, M., Lab. Robot. Aurom., 1991, 3, 199. Valcarccl, M., Quim. Anal., 1990, 9, 225. Valcarcel, M., FreJeniuJ ’ J . Anul. Chem., 1992, 343. 814. RGiiEka, J., and Hansen, E., Flow Injection Analysis, Wiley, New York, 2nd edn., 1988. Valcarcel, M., and Luque dc Castro, M. D., Flow Injection Analysis. Principles and Applications, Ellis Horwood, Chiches- tcr, 1987. Wolfbeis, 0. S . , Fiber Optic Chemicul Sensors, CRC Press. Boca Raton. FL, vol. I , 1991. Valcarcel, M., and Luque de Castro, M. D., Analysf. 1990, 115, 699. Luque de Castro, M. D., and Valcarccl, M., TrAC Trends Anal. Chem., 1991, 10, 114. RGiiEka, J., and Christian, G. D., Anal. Chim. Acta, 1990, 234, 31. Zhu, C.. and Hieftje, G. M., Anal. Chim. Acta, 1992,256,97. Wang, J., Anal.Chim. Actu, 1990, 234, 41. Bonakdar, M.. Yu, Y . , and Mottola, H. A., Taluntu, 1989, 36, 219. Hool, K., and Nieman, T. A., Anal. Chem.. 1987, 59. 869. Downey, T. M.. and Nieman, T. A., A n d . Chem., 1992, 64, 261. Gubitz. G.. van Zoonen, P., Gooijer, C., Velthorst, N. H . , and Frci, R. W., Anal. Chem., 1985, 57, 2071. Baumann, R. A., Gooijer, C., Velthorst, N. H . , Frci, R. W., Aichinger, I . . and Gubitz, G., Anal. Chem.. 1988, 60, 1237. van Zoonen, P., Kamminga, D. A., Gooijer, C., Velthorst, N. H., Frei, R. W., and Gubitz, G., Anal. Chim. Acfa, 1985, 174, 151. van Zoonen, P., Kerder, I., Gooijer, C., Vclthorst, N. H.. Frei, R . W., Kuntzberg, E., and Gubitz, G., Anal. Lett., 1986, 19, 1949. van Zoonen, P., Kamminga, D. A . , Gooijcr, C., Velthorst, N. H., Frei, R.W., and Gubitz, G., Anal. Chem., 1986, 58, 1248. Tai, H., Tanaka, H., and Yoshino, T., Opt, Lett.. 1987,12,437. Kroneis, H. W., unpublished results, 1983; from ref. 6, vol. 11. Farahi, F., Akhavan-Leilabady, P., Jones, J. D. C . , and Jackson, D. A.. J . Phys. E , 1987, 20, 432. Hool, K., and Nieman, T. A., Anal. Chem.. 1988, 60, 834. Nabi, A., and Worsfold, P. J., Analyst, 1986, 111, 1321. Worsfold, P. J., and Nabi, A., Anal. Chim. Actu, 1986, 179, 307. Dessy, R. E., Burgess, L., Arney, L., and Pctersen, J., in Chemical Sensors and Microinstrumentation, eds. Murray, R. W., Dessey, R. E.. Heineman, W. R., Janata. J.. and Seitz, W. R., ACS Symposium Series, Vol. 403. American Chemical Society, Washington, DC, 1989, ch. 10. Linares, P., Luque de Castro, M.D., and Valcarcel, M., Anal. Chim. Acta, 1990, 230, 199. Weigel, Ch., and Kellner. R., SPIE, 1989,1145. 134 (Proceed- ings of 7th Fourier Transform Spectroscopic Conference. Fairfax, 1989). Kellner, R., andTaga. K., SPIE. 1991, 1510, 232. Rios, A., Luquc de Castro, M. D., and Valcarcel, M., Anal. Chem., 1988, 60, 1540. Rios, A., Luquc de Castro, M. D., and Valcarcel, M., Anal. Chem., 1985, 57, 1803. Fernandez-Romero. J . M . , Luque de Castro, M. D., and Valchrcel, M., Sens. Actuators, in the press. Fernandez-Romero, J. M., Luque de Castro, M. D., and Valchrcel, M., Anal. Chim. Acfa, in the press. Mosbach, K., and Danielsson, B., Anal. Chem., 1981,53,83A. pp. 277-279.600 ANALYST, JUNE 1993, VOL. 118 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 Danielsson.B., Mattiasson, B., and Mosbach, K., Appl. Biochem. Bioeng., 1981, 3, 97. Danielsson, B. Appl. Biochem. Biotechnol., 1982, 7, 127. Danielsson, B., and Mosbach, K., in Biosensors, ed. Turner, A. P. F., Karube, I., and Wilson, G. E. S . , Oxford Science Publications, Oxford, 1987, pp. 575-595. Satdh, I., and Ishii, T.. Anal. Chim. Acta, 1988, 214, 409. Trettnad, W., Leiner, M. J . P., and Wolfbeis, 0. S . , Analyst, 1988, 113, 1519. Dremel, B. A., Schaffar, B. P. H . , and Schmid, R. D., Anal. Chim. Actu, 1989, 225, 293. Dremel, B. A., Yang. W., and Schmid, R. D., Anal. Chim. Acta, 1990. 234, 107. Wijesuriya, D.. and Rechnitz, G. A., Anal. Chim. Acta, 1992, 256, 39. Andrade, J . C., Pasquini. C., Baccan, N.. and van Loon, J.C., Spectrochim. Acta, Part B, 1983, 38, 1329. Winquist, F., Lundstrom, I., and Danielsson, B., Anal. Chem., 1986, 58, 145. Yoshimura. K., Anal. Chem., 1987, 59, 2924. Yoshimura, K., Bunseki Kagaku. 1987, 36, 656. Yoshimura, K., Analyst, 1988, 113, 471. Chen, D.. Luque de Castro, M. D., and Valcarcel, M., Talanta, 1990,37, 1049. Yoshimura, K., Nawata, S . , and Kura, G., Analyst, 1990, 115, 843. Lancy, N., Christian, G. D., and RfiiiEka, J . , Anal. Chem., 1990, 62, 1482. de la Torre. M., Luque de Castro, M. D., and Valchrcel, M., Talanta, 1992,39, 869. Chen, D., Luque de Castro, M. D., and Valcarcel, M., Anal. Chim. Acta, 1990, 234, 345. Pereiro Garcia, R., Liu, Y. M., Diaz Garcia, M. E., and Sanz- Medel, A . , Anal. Chem.. 1991, 63, 1759. Berthod, A.. Laserna, J.J., and Winefordner, J. D., J. Pharm. Biomed. Anal., 1988, 6, 599. Deakin, M. R., and Byrd. H., Anal. Chem.. 1989, 61,290. Fcrnandcz-Band, B., Lazaro, F., Luque de Castro, M. D., and Valcarcel, M., Anal. Chim. Acta, 1990. 229, 177. Fernandez-Band, B., Luque dc Castro, M. D., and Valcarcel, M., Anal. Chem., 1991 ~ 63, 1672. Tcna, M. T., Linares, P., Luque de Castro, M. D., and Valcarcel, M., Chromatographia, 1992, 33, 449. Tena, M. T., Luque de Castro, M. D., and Valcarcel, M., J. Chromatogr. Sci., 1992, 30, 276. Chen, D., Luque de Castro, M. D., and Valcarcel, M., Anal. Chim. Acta, 1992. 261, 269. Werner, T. C., Commings. J. G., and Seitz, W. R., Anal. Chem.. 1989, 61, 211. Schaffar, B. P. H . . and Wolfbeis, 0. S . , Anal. Chim. Actu, 1989, 217. 1 . Seiler, K.. Morf, W. E . , Rusterholz, B., and Simon, W., Anal. Sci., 1989, 5 , 557. 64 Kanabata. Y., Imasama, T., and Ishibashi, N., Anal. Chim. Acta, 1991, 255,97. 65 Kirkbright, G. F., Narayanaswamy, R., and Welti, N. A., Analyst, 1984, 109, 15. 66 Narayanaswamy, R., and Sevilla, F . , 111, Analyst, 1986, 111, 1085. 67 RfiiiEka, J . , and Hansen, E. H., Anal. Chim. Acta, 1985, 173, 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 3. Woods, B. A., RfiiiCka, J., Christian, G. D., Rose, N. J . , and Charlson, R. J . , Analyst, 1988, 113, 301. Woods, B. A,, RfiiiEka, J . , and Christian, G. D., Anal. Chem., 1987, 59,5767. Yerian, T. D., Christian, G. D., and RfiiiEka, J . , Anal. Chim. Acta, 1988, 204, 7. Yerian, T. D., Christian, G. D., and RfiiiEka, J . , Anal. Chem., 1988, 60, 12.50. Lazaro, F., Luque de Castro, M. D., and Valchrcel, M., Anal. Chim. Acta, 1988, 214,217. de la Torre, M., Fernandez-Gamez, F., Lazaro, F., Luque de Castro, M. D., and Valcarcel, M., Analyst, 1991, 116, 81. Kanabata, Y., Tahara, R., Imasaka, T., and Ishibashi, N., Anal. Chim. Acta, 1988, 212, 267. Pereiro Garcia, M. R., Diaz Garcia, M. E . , and Sanz-Medel, A., Analyst, 1990, 115, 575. Lazaro, F., Luque de Castro, M. D., and Valcarcel, M., Anal. Chim. Acta, 1989, 219, 238. Wang, K., Seiler, K., Rusterholz, B., and Simon, W., Analyst, 1992, 117, 57. Cook, R. L., Macduff, R. C., and Sammells, A. F., Anal. Chim. Acta, 1989, 226, 153. Seiler, K., Wang, K., Kuratli, M., and Simon, W., Anal. Chim. Acta, 1991, 244, 151. Shellum, C., and Gubitz. G., Anal. Chim. Acta, 1989, 227, 97. Liu, H., Yu, J . C., Bindra, D. S., Givens, R. S . , and Wilson, G. S., Anal. Chem., 1991, 63, 666. Gebbert, A., Alvarez-Icaza, M., Stocklein, W., and Schmid, R. D., Anal. Chem., 1992,64,997. Inman, S. M., Stromvall, E. S., and Lieberman, S. H., Anal. Chim. Acta, 1989, 217,249. Koerer, C. A., and Nieman, T. A. , Anal. Chem., 1986,58,116. Perez-Pavon, J . L., Rodriguez-Gonzalo, E., Christian, G. D., and RfiiiCka, J . , Anal. Chem., 1992, 64, 923. Petersson, B. A., Andersen, H. B., and Hansen, E. H . , Anal. Lett., 1987, 20, 1977. Ozana, S., Hanser, P. C., Seiler, K., Tan, S. S. S . , Morf. W. E., and Simon, W., Anal. Chem., 1991, 63, 640. Freeman, T. M., and Seitz, W. R., Anal. Chem., 1981,53,98. Valcarcel, M., and Luque de Castro, M. D., Microchem. J., 1992, 45, 189. Paper 21051 58C Received September 25, 1992