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Flow injection analysis with immobilized reagents
Flow injection analysis with immobilized reagents Elizabeth A.H. Hall University of Cambridge, Cambridge, UK Immobilized reagent phase flow injection analysis can be configured as discrete reagent cells upstream of the sensor element or as an integral reagent/transduction system (flow injection analysis-biosensor). The former approach has attracted greater attention because several assays can be assembled with greater versatility in reagent column units employing a single sensor, than can be co-immobilized on the surface of a transducer. Current Opinion in Biotechnology 1991, 2:9-16
Introduction Since its advent in the 1970s more than 2000 publications have been concerned with flow injection analysis (FIA). In the early days of its application, there was frequent criticism that it compared unfavourably with other automated methods of analysis, because it was claimed that only a few simultaneous determinations could be carried out, whereas many other methods allowed up to 20 parameters to be measured simultaneously per sample. Since then, the versatility of the principle of FIA has proved favourable for a vast spectrum of assays requiring both single and multiple reaction pathways on route to the detector. FIA is a mode of continuous flow analysis (CFA) where the flow is not segmented by air bubbles. In fact, it is often described as a hybrid between CFA and high-pressure liquid chromatography. By definition, FIA involves sample determination under non-steady--state conditions, where a quantity of dissolved sample is injected or introduced into a carrier stream, which flows through the system, upstream of the detector. Additional processes, such as chemical reaction or extraction, may also occur between the sample and the carder in order to generate the detected species. As the analyte or reaction product passes a continuous detector, a transient signal is produced. The measurement is performed under non-equilibrium conditions, as neither physical homogeneity nor chemical equilibrium have been attained by the time of the detection. The system, therefore, involves kinetic methods of analysis in the fixed-time mode. The evolution of the FIA biosensor can be followed through three distinct generations (Fig. 1.). In the first generation system, the sample and reagents, present in single or multiple reagent channels, react in solution before passing the detector and going to waste (Fig. la). In the second generation system, an immobilized reagent column is situated upstream of the detector (Fig. lb).
The sample passes through the column in the carrier stream and the product from the reaction in the column is monitored either direcdy or via a further reagent introduced into the carrier stream. The third generation system integrates the chemical reaction with the detector, so that the detected species is produced in situ (Fig. lc). Third generation FIA systems include biosensots and have been reported since the early 1980s, but there still remain only a handful of publications involving these integrated FIA biosensors. Indeed, the attention remains on second generation reagent columns rather than on the achievement of the complete integrated sensor. This review will consider the possible applications of the biosensor configuration in the latest developments in immobilized reagent Fib_together with the features which have been evaluated in designing an 'optimum' system. FIA in fermentation monitoring is discussed by Danielsson (this issue, pp17-22), and a more detailed discussion on biosensors can be found in the review by Gulibauilt (this issue, pp 3-8).
Design of the immobilized reagent phase The use of immobilized biomolecules in analytical procedures can offer important features, such as high selectivity together with the capacity of regeneration in a catalytic cycle. Particularly relevant is the use of biomolecules in a continuous flow system, because this permits recirculation of the reagents. One of the earliest reports of the use of enzymes in FIA corresponds with the first report of a closed loop assay [1 ]. Nevertheless, the long term success of such an immobilized reagent phase (even in applications other than FIA) requires the efficient irreversible attachment of the molecule to the support, without loss of activity. In discrete reagent systems, immobilization of the bioreagent via glutaraldehyde still appears to be the most
Abbreviations CFA-~continuous flow analysis; FIA~-flow injection analysis; GOl~glucose oxidase; ISFET--ion selective field-effect transistor; 1-1"F--tetrathiafulvalene. ~) Current Biology Ltd ISSN 0958-1669
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favoured method, because it is considered generally applicable and it produces good mechanical properties in a flowing stream. Indeed, a prerequisite of an enzyme sensor to be used in a flowing system is that the 'membrane' should be strong and, particularly when in use as a method in process control, the fouling by clogging of the membrane by macromolecular substances may be influenced by membrane and solute properties and processing parameters. Failure to eliminate these effects can lead t o a drastic decline in the flux to the sensor with time. Even for a single immobilization procedure, the conditions are critical. Ukeda et al. [2.] for example, found that for a complex multienzyme reactor involving alcohol dehydrogenase, diaphorase and NAD + immobilization, temperature and time influenced the stability and activity of the enzymes, respectively. Recently, Narinesingh et aL [3 °] used a novel method of immobilization, which involved attaching urease on 2-fluoro- 1-methylpyridinium toluene sulphonate-activated Fractogel (Merck). This was chosen as a support because of its good mechanical, chemical and thermal stabilities. Ammonia produced by the reaction of urease with urea in serum was partitioned under alkaline conditions, across a microporous P'ITE membrane into a carrier stream containing the flurogenic reagent. Thus, the detection circuit was separated from the sample carrier stream.
Fig. 1. The three generations of flow injection analysis: (a) the first generation system involving soluble reagents; (b) the second generation system in which immobilized reagent columns are employed; (c) the integral reagent cell/transducer of the third generation system. Kfinnecke and Schmid [4"] have reported a similar configuration of parallel 'sample' and 'detector' flows, for the determination of ethanol in beverages but, in this case, the function of the membrane separating the two carrier streams is primarily to cause sample dilution in order to increase the detection range. Sample dilution between donor and acceptor streams is proportional t o membrane thickness. PrinzJng et aL [5"] have also developed a pervapoura-
tion method to transfer ethanol samples from the complex media of a fermentation line into the parallel analytical circuit across a temperature gradient and through a hydrophobic membrane. The analytes of interest, in this case, were not only ethanol but also diacetyl which is formed during the production of beer. These products could be monitored via alcohol dehydogenase and diacetyt reductase, respectively. The enzymes were immobiliTed on alkylated porous glass beads in small tubes. The ethanol and diacetyl are constantly extracted into the detection stream across a porous hydrophobic membrane separating the fermentation broth from the acceptor stream, and a pulse of NADH/H + injected into the system induces a transient analytical signal at the fluorimeter, downstream of the enzyme columns. Although this paper discusses a biosensor, in fact no integral reagent phase/detector is proposed. Such a third generation development would have to unite the immobilized
Flow injection analysiswith immobilized reagents Hall 11 reagent tube with the fluorimeter. This modification will probably not be straightforward if the enzyme has to remain immobilized on porous glass beads packed in a tube, in order to retain the flow characteristics conducive to maximal efficiency of the enzyme reaction. It has been reported [6] that open-tube wall immobilization offers better flow properties than the packed beds used in this system because a dead volume develops in the latter case. However, immobilization on beads packed in tubing gives a higher and sharper peak than seen with an open tube. It is suggested that this is due to changes in axial and radial dispersion properties. In open tube immobilization, the reagent is immobilized within the 'stagnant' layer at the wall of the tube, thus causing greater dispersion of the product as it enters the fluid stream. In packed tube columns, with reagent also in the high velocity flow in the centre of the tube, dispersion is reduced, but the diameter of the particle support and its packing influence dispersion.
Multiple enzyme pathways HA has very often taken advantage of coupling multipleenzyme pathways in order to achieve the desired analysis. Very complex pathways can be devised in this way, particularly when the enzymes are immobilized in a series of short columns, rather than circulating in solution. On the other hand, the greater the number of enzymes required, the more difficult it is to apply the system to the integral FIA biosensor. Inorganic phosphate, for example, has been determined via hypoxanthine produced in an immobiliTed enzyme column containing purine nucleoside phosphorytase [7°]. The hypoxanthine was then estimated via HzO 2 generated in a second column containing xanthine oxidase and urate oxidase. The final detection of peroxide was performed optically through its reaction with chemfluminescent reagents. Although a suitable reagent column might be expected to achieve this final step, nevertheless, the report describes chemiluminescent reagents injected in a second carrier stream, so that the final system configuration is comparable with the previous example [5°], in that it still requires non-immobiliTed reagents in the carrier stream. The enzymes in the reaction columns were immobilized on aminopropyl-controlled pore glass beads via glutaraldehyde so that, in principle, the conversion of this assay system into an integrated sensor would require co-immobiliT~tion of all the ermymes, together with the chemiluminescent reagents (rather than in different discrete columns) on a support which could also be interrogated optically. Peterson et aL [8] have described an optical pH sensor, accurate to 0.01pH units, which involves a pH reagent immobilized to light-scattering beads, packed into a hollow dialysis tube at the end of a bifurcated fibre optic. A similar design might be appropriate for use in this inorganic phosphate assay but, in general, such a totally self-contained system is probably less versatile than
the enzyme column 'units', which can be assembled in various combinations to perform different assays. Nevertheless, Ruzicka and Christian [9] have considered the model of an optical HA biosensor composed of a voltune packed with spherical partially light-reflecting particles, modified with reagent. The amount of transmitted light decreases with particle size and increases in scattering coefficient. Ideally, transluscent materials should be used, although various polymer materials have been found to transmit sufficient light in the visible region. KurkijLrvi et aL [10.] also report the rudiments of an integral HA biosensor system with immobilized bacterial bioluminescence enzymes on a packed bed in the measuring chamber of a luminometer. The optical biosensor consisted of glutamate dehydrogenase co-immobilized with bioluminescent enzymes onto cyanogen bromideactivated agarose to monitor glutamate. The reactor was placed directly in the luminometer. This could represent a good compromise approach as, in conjunction with separate reactor columns, other analytes could be selected. For example, if this column was to be combined with an aspartate aminotransferase and malate dehydrogenase column it could be employed for aspartate estimation (Fig. 2.) [10-].
Influence of a flowing system on electrochemical biosensors Immobilized enzyme-linked assays suitable for HA are by no means confined to optical transduction methods. Indeed, much of the evolutionary pathway of the steadystate biosensor can be seen mirrored in developments in FIA. Nevertheless, the flow parameters and the kinetic nature of the assay impose rather different constraints on the design and success of the system. In the first instance, the precision, sensitivity, selectivity and dynamic range of a FIA sensor can be related loosely to the same sensor in operation in the steady-state mode. However, for any system with a dispersion coefficient greater than one, the concentration-maximum detected will be less than the concentration of analyte introduced into the system. In designing a FIA, therefore, it is necessary to consider such features as the dispersion of the sample in the carrier stream in order to achieve maximum response. It has been noted already that the dispersion characteristics are apparently more favourable for an enzyme system immobilized on beads packed into a tube than for open tube-wall immobilizations. The velocity profile of a sample zone injected in a laminar flowing system is parabolic [6] because under these conditions the layer of liquid in contact with the tube surface is practically stationary while in the centre of the tube the velocity is twice the mean velocity of the liquid. Theoretically this would imply that by the time the sample reached the detector it would have an infinitely long 'taft' and that the system would become permanently polluted with 'carry-over' from the sample. However, lateral diffusion of molecules perpendicular to the direction of flow causes mixing between sample and carrier which modifies this effect.
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Analytical bio~echnology
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When a solute that does not undergo on-line/in-carrierstream chemical reaction is injected into a FIA system, physical dispersion is the only band-broadening process involved. In this case, changes in one or more of the mean velocity, tube diameter, monitoring distance, or diffusion coefficients, will alter the dispersion of the sample. On the other hand, when an on-line reaction takes place (for example, a reaction with the immobilized biomolecule column), two kinetic processes occur simultaneously: chemical reaction and physical dispersion. Dispersion is measured as a ratio of the signal from the nondispersed analyte to that of the anatyte after transport through the FIA system. For soluble reagents injected into the carrier stream, the design is optimised so that the chemical reaction generally has no major effect on the dispersion profile at the flow rates normally employed. Nevertheless split peaks can be observed at large injection volumes, due to reagent depletion in the middle of the sample zone. Other factors influencing the kinetics of the reaction and positions of equilibrium can also influence the size and shape of the signal. Chandler et al. [11..] have considered the application of an enzyme field-effect transistor
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Fig. 2. The glutamate bioluminescent flow injection analysis (FIA) biosensor (bottom), which measures glutamate in terms of a luminescence signal, could, in principle, be coupled to a discrete reagent column (above) and so be used to measure aspartate. The reagent column would generate NAD in proportion to the aspartate present, and the NAD would be measured in terms of a luminescent signal from the biosensot. (O), immobilized aspartate amino transferase;(C)), immobilized malate dehydrogenase; (C)) glutamate dehydrogenase co-immobilized with bioluminescent enzymes ((~)).
(EnFET) to FIA for assay of urea, but have concluded that fabrication of a discrete enzyme column is easier than production of the integrated device. Urease was immobilized on silanised controlled pore glass via glutaraldehyde and ion selective field-effect transistors (ISFETs) placed before and after the reaction column to monitor the change in pH caused by the enzymecatalysed breakdown of urea (Fig. 3.). The calibration of this system depends on several factors such as the position of equilibrium on the column, the sample and carrier pH, and dispersion. For a given system, the dispersion can control the output signal and, even in a controUed pH buffer carrier stream, the unknown pH of the sample can seriously alter the position of the equilibrium and thus the response. If the enzyme reaction goes to completion, that is all the urea is hydrolysed in the column, then the reaction can be considered in three stages: initial mixing of sample and carrier; total urea hydrolysis; and pH equilibration with the buffer and sample zone. In certain cases, for example where the sample pH is greater than the buffer pH, the dispersion may result in a decrease in the monitored pH between the first and second ISFETs. In principle, ff the dispersion is known, these changes
Flow injection analysis with immobilized reagents Hall could be computed and the calibration adjusted according to the pH monitored by the ISFET upstream of the column. The carrier buffer can be chosen to give the minimum interference from the sample buffer and obviously, in this instance, the greater the dispersion the less the influence of the pH of the sample. On the other hand, the signal peak height decreases with dispersion so that, inpractice, a compromise between signal size, linearity and effects of buffer-interference must be made.
Solute focusing Unlike the soluble reagent present in the carrier stream, where the length of the coil between the port and detector can be designed to optimise sample-reagent reaction time, the dispersion of the sample flowing past a stationery reagent phase will be strongly influenced by the geometry and activity of that phase; even the nature of the enzyme catalysis itself may influence the dispersion if it is 'slow' to release the product from its binding site. In these cases, a separate product zone distinct from the reaction zone may even be created, and will be detected downstream. Indeed, the dispersion of the sample can be considerably altered by a stationery phase, even when no chemical reaction is involved with that phase. Johnson and Dorsey [12] have described a technique of solute focusing where the sample injected into a solvent stream passes through a 'focusing column' which acts as an adsorbant for the sample, focusing it into a concentrated band, which is totally retained on the column. In this technique, the column does not serve a second function of immobilized reagent: instead, the sample is eluted from the column with a suitable carrier solvent, to encounter chemical reaction down-stream, prior to the detector. In contrast to solute focusing before chemical reaction, enhancement of the sensitivity of the more conventional FIA chemical methods for phosphate determination has also been accomplished via a post-reaction
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concentration-extraction technique. Lacy et al, [13] have reported the use of a column for a sorbent extraction method involving a standard reaction based on the formation of a complex of phosphate with molybdenum blue. The reaction was allowed to occur in the carrier stream before passing into a microcolumn of silica-based C-18, which was placed in the light-path of a spectrophotometer. The column 'collects' the complex, and the signal that is generated when the complex is reduced with ascorbic acid is proportional to the phosphate concentration. With this technique, the dispersion characteristics of the system are important only in respect to the mixing of sample with reagent, and do not otherwise effect the signal response. A 200-fold enhancement of sensitivity is claimed to be achieved with the concentration column. This corresponds to a lower limit of detection of 6ppb for a lml sample and compares favourably with the value of 500 fmol Pi for the enzyme method of phosphate determination discussed above [7°].
Selectivity in oxidase-linked assays The sensitivity and selectivity of the FIA signal is strongly influenced by the transduction method, in the same way as in steady-state assays, as well as by other features more unique to flow analysis. Sulphite can be estimated via H202 produced in the sulphite oxidase catalysed reaction: sulphite SO32- + O 2+ H20
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where peroxide is measured on a platinum electrode. Matsumoto et al. [14o] used sulphite oxidase immobilized on cyanogen bromide-activated Sepharose in the flow injection manifold for sulphite determination in wine. The study investigated the effect on the sig-
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hal of the following factors: enzyme loading (increase in signal with loading to 150 U/g dry Sephamse); column length (increase in signal with length); flow rate (Increase with flow to maximum at 0.45ml rain -1, then decrease to plateau); and the elimination of interference from other sample species. This enabled optimal conditions to be established. Red wine tannins (polyphenols) are particularly bad interferents of amperometric assay techniques, and they may also inactirate the enzyme. A dialysis membrane on the platinum electrode was able to decrease interference of the electrode signal to 12 % of its original value, but this is insufficient for performing an adequate wine assay. In this study the authors found that pre-treatment of white wine with gelatine (1%), together with the introduction of an on-line filter into the flow, allowed the sulphite to be measured. After similar treatment of red wine, however, the high level of tannins still caused erratic behaviour of the dectrode signal. Because this enzyme-linked assay requires sample pretreatment, it is far from being described as a simple integral self-contained biosensor assay system. A sinailar problem had been encountered by Yao [15] in the choline oxidase-linked amperometric assay of cholinesterase. In this case, the author suggested the use of a copper (II) dithiocarbamate-modified silica gel column placed upstream to remove interfering electroactive species normally present in human sera, such as ascotbate, urate, tyrosIne, cysteine, glutathione, and bilimbin. Naturally, the very nature of FIA allows the introduction of as many such 'dean up' columns as are required. However, in view of the preceding discussion on the effects of dispersion on the size and shape of the detected signal, it should be clear that multiple columns cannot be inserted without due consideration for their effect on the analyte dispersion. In steady-state non-flowing systems this option of multiple column insertion does not exist, and so other means have been sought to by-pass the problem, particularly for the amperometric redox enzyme-linked assays. Low molecular weight mediators have been employed in place of oxygen to shuttle electrons between the enzyme redox centre and the electrode, with favourable results. With a view to opamizing the conditions for efliciency and stability, attempts have been made to design mediators with a redox potential close to 0V. (This should be compared with oxidation of H202 at 0.65V versus saturated calomel electrode). By achieving a lower oxidation potential, it is possible to avoid the interference from many other sample species. Gunasingham and Tan [16-] have used tetrathiafulvalene (TIT) as a mediator in a glucose oxidase (GOD)/TrF-carbon paste electrode in a wall-iet cell to measure glucose. In conventional walliet electrodes, the electrode is positioned so that the inlet lies just at the edge of the wall-jet boundary layer. In this electrode, with a reaction layer on the surface, the inlet-electrode separation could be significantly increased without any loss in peak current response. Not only does this study address the problem of sample interferents through the use of mediators, it also demonstrates a totally integrated reagent phase/detector. This
glucose assay system requires no further modification to be classified as a third generation FIA-biosensor.
The polypyrrole/GOD electrode has also undergone considerable investigation as a steady-state electrode, so it is hardly surprising to lind that it has received attention in relation to FIA. In flowing systems the membrane or polymer thickness for an amperometric electrode is an essential parameter because partitioning at the interface and within the membrane layer will effect the sample dispersion and the depletion layer from the electrode. In the mediated GOD electrode above, the distance between the electrode and the wall-jet boundary layer was less critical than for an unmodified electrode. Trojanowicz et at [17 °°] have considered the effect of the polymer thickness on the stability and response of the polypyrrole/GOD electrode and found that very thick membranes gave poor stability and reproducibility. This was attributed to transport limitations of H20 z produced by the enzyme reaction on the surface of the electrode.
The use of an oxygen optrode with immobilized lactate oxidase has demonstrated an alternative means of circumventing the interference of the amperometric H202 signal, inherent in the reaction:
lactate + 02
lactate oxidase
) pyruvate + H202
This biosensor was designed to monitor L-lactate concentration during kefir fermentation [18o], but as the linear range was only 0.02 to 0.5 mM, zone sampling to give dilution had to be applied to extend the linear range sufficiently (to 60 mM). This dilution technique, although very accurate, reduces the sampling frequency.
Assays involving immobilized binding proteins Most of the effort directed at the use of immobilized biomolecules in FIA has been for enzyme-linked systems. Mattiasson etal. [19.o], on the other hand, have directed their attention to the use of an immobilized binding protein. Labelled target molecules (in this case an enzyme label) were added to the sample containing the target molecules to be determined, and the sample was injected into the carrier flow. The labelled and un/abelled target molecules competed for binding to immobilized conconavalin A. The unbound labelled target molecules downstream of the reagent column were assayed after adding a substrate of the labelling enzyme. This is more complex than conventional FIA as it involves two simultaneous processes: the binding reaction between soluble and immobilized reactants, which is far from equilibrium because the system is flowing, and the enzyme reaction
Flow injection analysis with immobilized reagents Hall giving the signal. After the assay, bound protein must be eluted from the colunm. Dissociation can be followed by iniecting pulses of substrate, and this can also be used as an alternative means of analyte determination. Unlike the immobilized enzyme columns, calibration does not vary with lifetime, but capacity decreases. Capacity can be evaluated by injecting a fixed amount of label and observing the binding efficiency.
Conclusion In conclusion, attention towards the use of immobilized bioreagents in FIA remains focused on reagent columns. Although integral enzyme FIA biosensors have been reported, it seems that greater versatility is possible with discrete immobilized reagent columns, which can be linked together, as required, to perform different analyses. The majority of reports have been concerned with enzyme systems and, like their stationery system counterparts, have investigated a variety of transduction techniques. Some effort has also been directed towards the use of binding proteins as the bioactive reagent, but because the system is flowing, the reversible complex-formation between immobilized reagent and soluble analyte is far from equilibrium. Optimisation of this system is, therefore, likely to be more complex than for 'conventional' FIA.
ages without Sample Pretreatment. Anal Chim Acta 1990, 234,213-220. The influence of membrane modification on the linear range of analyses is described for a gas diffusion unit separating donor (sample) and acceptor streams, upstream of an enzyme reactor. The application of the system in the determination of ethanol in samples of beverages and medicine is considered.
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PRINZINGU, OGBOMO I, LEHN C, SCHMn3T H-L: Fermentation Control with Biosensors ill FlOW Injection Systems: Problems and Progress. Sens Actua 1990, B1:542-545. A pervapouration module is described which transfers volatile substances from the sample cartier stream to the acceptor detector stream, upstream of the enzyme reactor. Membrane fouling is avoided by introducing an air space between the process fluid and the membrane. Ethanol and diacetyt are determined via an enzyme reactor by fluorimetry. 6.
KAWASAKIH, SATO K, OGAWAJ, HASEGAWAY', YUKI H: Deterruination of Inorganic Phosphate by Flow Injection Method with Immobilised Enzymes and Chemiluminescence Detection. Anal Biochem 1989, 182:366-370. inorganic phosphate determination is linked to an enzyme pathway generating H202, which is determined via chemiluminescent reagents. The enzymes are immobilized in enzyme reactor columns but, because the luminescent reagents are added to the carrier stream, the system is not fully solid-state. An impressive limit of detection of 500 tool Pi is reported. In fact for phosphate in DNA, measured as a test of applicability to biological samples, the method is claimed to be 160 t~nes more sensitive than conventional spectrophotometric techniques. 7. .
8.
PETERSONJI, GOLDSTEIN SR, FITZGERALD RV, BUCKHOLD DK: Fibre Optic pH Probe for Physiological Use. Anal Cbem 1980, 52:864--868.
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RUZlCKAJ, CHRISTIANGD: Reversible Optosensing in Packed Flow-through Detectors: Flow Injection or Chromatography? Anal Cbim Acta, 1990, 234:31-40.
References and recommended reading Papers of special interest, published within the annual period of review, have been highlighted as: • of interest .• of outstanding interest 1. 2.
BERGMEYERHU, HAGENA: New Principle o f Enzymatic Analysis. Fresenius Z Anal Cbem 1972, 261:333-337. UKEDAH, IMABAYASHIM, MATSUMOTO K, OSAJIMAY: Coimmo-
•
bilization of Alcohol Deydrogenase, Diaphorase and NAD and its Application to Flow Injection Analytical System for Ethanol. dgrm Biol t ~ e m 1989, 53(11):2909-2915. Alcohol dehydrogenase, diaphorase and NAD were co-immobilized on Sepharose modified with hexamethylenediamine and glutaraldehyde. In contrast with many other NAD-linked systems, the advantage of this technique is that the NAD is directly coupled to the support. The effect of immobilization parameters on stability, activity and reusability was considered. The ratio of ethanol to 02 consumed was monitored via the Clark electrode. NARINESINGHD, MUNGAL R, NGO T'I': Flow Injection Analysis of Serum Urea Using Urease Covalently Immobflised on 2-Fluro-1-methylpyridinium Salt Activated Fractogel and Fluorescence Detection. Anal Bfbcbem 1990, 188:325-329. A novel method in which urea was determined via the ammonia product of the urease reaction. The isoxoindole product with typhthaldehyde was assayed fluorometrically at kex 340nm and Zero 455nm. Partitioning was required to separate the ammonia from other interfering substances present in serum. Urea detection limit was reported as 0.1mg per litre urea nitrogen at 0.3ml m i n - 1 carrier flow rate, and this decreased as the flow rate was lowered. Linearity to 140mg litre- l could 3. .
be achieved. 4. •
KONNECKEW, SCHMID RD: Gas-diffusion Dilution Flow-injection Method for the Determination o f Ethanol in Bever-
ROCKSB, ~ C: Flow Injection Analysis: A New Approach to Quantitative Measurements in Clinical Chemistry. CUn Chem 1982, 28:409-421.
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KURKIJARWK, Vmmjom T, KORPELAT: FlOW Injection Analysis of Amino Acids and their Metabolites by Immobilised Vitamin B6-dependent Enzymes. A n n N e w York Acad Sci 1989, 585:394-403. Aspartate aminotransferase together with malate dehydrogenase was immobilized onto cyanogen bromide-activated agarose in one column and glutamate dehydrogenase with bioluminescent enzymes was immobilized onto another cyanogen bromide-activated agarose column. The latter reactor was placed directly in the luminometer and could be employed alone to detect glutamate in the range 10-100 nmol. For detection of L-aspartate it was necessary to use the first column as well, in the presence of excess oxaloacetate, and a detection range of 0.1-5 nmol was achieved. The immobilized enzymes were stable for several months and could be used for 600-4300 analyses. Thus, this study suggests that, at least for glutamate, an integrated device could be developed. 11. ••
CHANDLERGK, DODGSON JR, EDDOWES MJ: An ISFET-based Flow Injection Analysis System for Determination of Urea: Experiment and Theory. Sens Actua 1990, B1:433-437. An ISFET pH-sensitive scheme is described with the pH change across a urease reactor column related to sample urea concentration. The influence of sample and buffer pH on the output is discussed. 12.
JOHNSONBF, DORSEYJG: Solute FocusIng in Flow Injection Analysis: Limits of Detection and Linear Dynamic Range. Anal ~ 1990, 62:1392-1397.
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LACY N, CHRISTIANGD, RUZlCKAJ: Enhancement of Flow Injection Optosensing by Sorbent Extraction and Reaction Rate Measurement. Anal C~em 1990, 62:1482-1490.
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MATSUMOTOK, MATSUBARAH, UKEDA H, OSAJIMAY: Determination of Sulfite in White Wine by Amperometric Flow Injection Analysis with an Immobilised Sulfite Oxidase Reactor. Agric Biol t ~ e m 1989, 53(9):2347-2353.
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Analytical biotechnology This study investigates the optimal conditions for sulphite determination in wine. The authors find that pre-treatment of the wine with gelaOne (1%) and introduction of an on-line filter into the flow, allows the sulphite to be measured in white wine over the range 20-200 ppm. 15.
YAO T: Flow Injection Analysis for Cholinesterase in Blood Serum by use of a Choline.Sensitive Electrode as Amperometric Detector. Anal (Thim Acta 1983, 153:169-174.
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GUNASINGHAMH, TAN C-H: Carbon Paste-tetrathiafulvalene Amperometic Enzyme Electrode for Determination of Glucose in Flowing Systems. Analyst 1990, 115:35--39. A carbon-paste-TTF electrode with glucose oxidase immobilized on the surface via glutaraldehyde cross-linking with bovine serum albumin is employed in a wall-jet configuration to measure glucose. The electrode can be operated continuously for 2000 injections of 10 mM glucose with only 8% decline in current. In general, the standard deviation of the response in 100 samples was 0.6%. In whole blood, the electrode was orientated vertically to decrease deposition of cells and other blood particles. 17. •6
TROJANOWICZM, MATUSZEWSK~W, PODSIADLAM: Enzyme Entrapped Polypyrrole Modified Electrode for Flow Injection Determination o f Glucose. Biosens Bioelectron 1990, 5:149-156. A report of an integrated biosensor. Electrochemically polymerised pyrrole as the immobilization matrix for GOD was evaluated for FIA glucose determination. Polymer thickness and flow rate were optimised. After five days use, the electrode output had reduced to 20% of its original value. This is comparable with similar electrodes in use in non-flowing systems.
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DREMELBAA, YANG W, SCHMID RD: On-line Determination of Lactic Acid During Kelir Fermentation Based on a Fibreoptic Lactic Acid Biosensor and Flow Injection Analysis. Anal Chim Acta 1990, 234:107-112. A lactate biosensor based on an oxygen optrode is described, with lacrate oxidase immobilized via glutaraldehyde. The assay is based on the estimation o f dissolved oxygen via its dynamic quenching of the fluorescence from an indicator. 19. oo
MATI'IASSONB, BERDEN P, LING TGI: Flow Injection Binding Assays: a Way to increase the Speed in Binding Analyses. Anal Biocbem~ 1989, 181:379-382. This report investigates the use of immobilized biomolecules for the determination of proteins which are not enzymes. A competitive binding assay is described with immobilized binding protein and the target molecule (sample) mixed with enzyme-labelled target molecule injected as pulses. Concanavalin A immobilized to Sepharose via a tresyl chloride method, was employed as binding protein and horseradish peroxidase as the competitive label (competing with samples of other glucosides or mannosides). The enzyme's substrate was included in the buffer, and the signal was measured with respect to a baseline established after a fixed time from a reference point.
EAH Hall, Institute of Biotechnology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QT, UK,
Introduction Since its advent in the 1970s more than 2000 publications have been concerned with flow injection analysis (FIA). In the early days of its application, there was frequent criticism that it compared unfavourably with other automated methods of analysis, because it was claimed that only a few simultaneous determinations could be carried out, whereas many other methods allowed up to 20 parameters to be measured simultaneously per sample. Since then, the versatility of the principle of FIA has proved favourable for a vast spectrum of assays requiring both single and multiple reaction pathways on route to the detector. FIA is a mode of continuous flow analysis (CFA) where the flow is not segmented by air bubbles. In fact, it is often described as a hybrid between CFA and high-pressure liquid chromatography. By definition, FIA involves sample determination under non-steady--state conditions, where a quantity of dissolved sample is injected or introduced into a carrier stream, which flows through the system, upstream of the detector. Additional processes, such as chemical reaction or extraction, may also occur between the sample and the carder in order to generate the detected species. As the analyte or reaction product passes a continuous detector, a transient signal is produced. The measurement is performed under non-equilibrium conditions, as neither physical homogeneity nor chemical equilibrium have been attained by the time of the detection. The system, therefore, involves kinetic methods of analysis in the fixed-time mode. The evolution of the FIA biosensor can be followed through three distinct generations (Fig. 1.). In the first generation system, the sample and reagents, present in single or multiple reagent channels, react in solution before passing the detector and going to waste (Fig. la). In the second generation system, an immobilized reagent column is situated upstream of the detector (Fig. lb).
The sample passes through the column in the carrier stream and the product from the reaction in the column is monitored either direcdy or via a further reagent introduced into the carrier stream. The third generation system integrates the chemical reaction with the detector, so that the detected species is produced in situ (Fig. lc). Third generation FIA systems include biosensots and have been reported since the early 1980s, but there still remain only a handful of publications involving these integrated FIA biosensors. Indeed, the attention remains on second generation reagent columns rather than on the achievement of the complete integrated sensor. This review will consider the possible applications of the biosensor configuration in the latest developments in immobilized reagent Fib_together with the features which have been evaluated in designing an 'optimum' system. FIA in fermentation monitoring is discussed by Danielsson (this issue, pp17-22), and a more detailed discussion on biosensors can be found in the review by Gulibauilt (this issue, pp 3-8).
Design of the immobilized reagent phase The use of immobilized biomolecules in analytical procedures can offer important features, such as high selectivity together with the capacity of regeneration in a catalytic cycle. Particularly relevant is the use of biomolecules in a continuous flow system, because this permits recirculation of the reagents. One of the earliest reports of the use of enzymes in FIA corresponds with the first report of a closed loop assay [1 ]. Nevertheless, the long term success of such an immobilized reagent phase (even in applications other than FIA) requires the efficient irreversible attachment of the molecule to the support, without loss of activity. In discrete reagent systems, immobilization of the bioreagent via glutaraldehyde still appears to be the most
Abbreviations CFA-~continuous flow analysis; FIA~-flow injection analysis; GOl~glucose oxidase; ISFET--ion selective field-effect transistor; 1-1"F--tetrathiafulvalene. ~) Current Biology Ltd ISSN 0958-1669
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Analytical biotechnolosy
I Reagent2~=~S>' ~
I Reagent3 Pump (b)
carrier Pump
(c)
m~mm Immobilized reagent column
Intergrated reagents and sensor Buffer/ carrier
Biosen~ Pump
favoured method, because it is considered generally applicable and it produces good mechanical properties in a flowing stream. Indeed, a prerequisite of an enzyme sensor to be used in a flowing system is that the 'membrane' should be strong and, particularly when in use as a method in process control, the fouling by clogging of the membrane by macromolecular substances may be influenced by membrane and solute properties and processing parameters. Failure to eliminate these effects can lead t o a drastic decline in the flux to the sensor with time. Even for a single immobilization procedure, the conditions are critical. Ukeda et al. [2.] for example, found that for a complex multienzyme reactor involving alcohol dehydrogenase, diaphorase and NAD + immobilization, temperature and time influenced the stability and activity of the enzymes, respectively. Recently, Narinesingh et aL [3 °] used a novel method of immobilization, which involved attaching urease on 2-fluoro- 1-methylpyridinium toluene sulphonate-activated Fractogel (Merck). This was chosen as a support because of its good mechanical, chemical and thermal stabilities. Ammonia produced by the reaction of urease with urea in serum was partitioned under alkaline conditions, across a microporous P'ITE membrane into a carrier stream containing the flurogenic reagent. Thus, the detection circuit was separated from the sample carrier stream.
Fig. 1. The three generations of flow injection analysis: (a) the first generation system involving soluble reagents; (b) the second generation system in which immobilized reagent columns are employed; (c) the integral reagent cell/transducer of the third generation system. Kfinnecke and Schmid [4"] have reported a similar configuration of parallel 'sample' and 'detector' flows, for the determination of ethanol in beverages but, in this case, the function of the membrane separating the two carrier streams is primarily to cause sample dilution in order to increase the detection range. Sample dilution between donor and acceptor streams is proportional t o membrane thickness. PrinzJng et aL [5"] have also developed a pervapoura-
tion method to transfer ethanol samples from the complex media of a fermentation line into the parallel analytical circuit across a temperature gradient and through a hydrophobic membrane. The analytes of interest, in this case, were not only ethanol but also diacetyl which is formed during the production of beer. These products could be monitored via alcohol dehydogenase and diacetyt reductase, respectively. The enzymes were immobiliTed on alkylated porous glass beads in small tubes. The ethanol and diacetyl are constantly extracted into the detection stream across a porous hydrophobic membrane separating the fermentation broth from the acceptor stream, and a pulse of NADH/H + injected into the system induces a transient analytical signal at the fluorimeter, downstream of the enzyme columns. Although this paper discusses a biosensor, in fact no integral reagent phase/detector is proposed. Such a third generation development would have to unite the immobilized
Flow injection analysiswith immobilized reagents Hall 11 reagent tube with the fluorimeter. This modification will probably not be straightforward if the enzyme has to remain immobilized on porous glass beads packed in a tube, in order to retain the flow characteristics conducive to maximal efficiency of the enzyme reaction. It has been reported [6] that open-tube wall immobilization offers better flow properties than the packed beds used in this system because a dead volume develops in the latter case. However, immobilization on beads packed in tubing gives a higher and sharper peak than seen with an open tube. It is suggested that this is due to changes in axial and radial dispersion properties. In open tube immobilization, the reagent is immobilized within the 'stagnant' layer at the wall of the tube, thus causing greater dispersion of the product as it enters the fluid stream. In packed tube columns, with reagent also in the high velocity flow in the centre of the tube, dispersion is reduced, but the diameter of the particle support and its packing influence dispersion.
Multiple enzyme pathways HA has very often taken advantage of coupling multipleenzyme pathways in order to achieve the desired analysis. Very complex pathways can be devised in this way, particularly when the enzymes are immobilized in a series of short columns, rather than circulating in solution. On the other hand, the greater the number of enzymes required, the more difficult it is to apply the system to the integral FIA biosensor. Inorganic phosphate, for example, has been determined via hypoxanthine produced in an immobiliTed enzyme column containing purine nucleoside phosphorytase [7°]. The hypoxanthine was then estimated via HzO 2 generated in a second column containing xanthine oxidase and urate oxidase. The final detection of peroxide was performed optically through its reaction with chemfluminescent reagents. Although a suitable reagent column might be expected to achieve this final step, nevertheless, the report describes chemiluminescent reagents injected in a second carrier stream, so that the final system configuration is comparable with the previous example [5°], in that it still requires non-immobiliTed reagents in the carrier stream. The enzymes in the reaction columns were immobilized on aminopropyl-controlled pore glass beads via glutaraldehyde so that, in principle, the conversion of this assay system into an integrated sensor would require co-immobiliT~tion of all the ermymes, together with the chemiluminescent reagents (rather than in different discrete columns) on a support which could also be interrogated optically. Peterson et aL [8] have described an optical pH sensor, accurate to 0.01pH units, which involves a pH reagent immobilized to light-scattering beads, packed into a hollow dialysis tube at the end of a bifurcated fibre optic. A similar design might be appropriate for use in this inorganic phosphate assay but, in general, such a totally self-contained system is probably less versatile than
the enzyme column 'units', which can be assembled in various combinations to perform different assays. Nevertheless, Ruzicka and Christian [9] have considered the model of an optical HA biosensor composed of a voltune packed with spherical partially light-reflecting particles, modified with reagent. The amount of transmitted light decreases with particle size and increases in scattering coefficient. Ideally, transluscent materials should be used, although various polymer materials have been found to transmit sufficient light in the visible region. KurkijLrvi et aL [10.] also report the rudiments of an integral HA biosensor system with immobilized bacterial bioluminescence enzymes on a packed bed in the measuring chamber of a luminometer. The optical biosensor consisted of glutamate dehydrogenase co-immobilized with bioluminescent enzymes onto cyanogen bromideactivated agarose to monitor glutamate. The reactor was placed directly in the luminometer. This could represent a good compromise approach as, in conjunction with separate reactor columns, other analytes could be selected. For example, if this column was to be combined with an aspartate aminotransferase and malate dehydrogenase column it could be employed for aspartate estimation (Fig. 2.) [10-].
Influence of a flowing system on electrochemical biosensors Immobilized enzyme-linked assays suitable for HA are by no means confined to optical transduction methods. Indeed, much of the evolutionary pathway of the steadystate biosensor can be seen mirrored in developments in FIA. Nevertheless, the flow parameters and the kinetic nature of the assay impose rather different constraints on the design and success of the system. In the first instance, the precision, sensitivity, selectivity and dynamic range of a FIA sensor can be related loosely to the same sensor in operation in the steady-state mode. However, for any system with a dispersion coefficient greater than one, the concentration-maximum detected will be less than the concentration of analyte introduced into the system. In designing a FIA, therefore, it is necessary to consider such features as the dispersion of the sample in the carrier stream in order to achieve maximum response. It has been noted already that the dispersion characteristics are apparently more favourable for an enzyme system immobilized on beads packed into a tube than for open tube-wall immobilizations. The velocity profile of a sample zone injected in a laminar flowing system is parabolic [6] because under these conditions the layer of liquid in contact with the tube surface is practically stationary while in the centre of the tube the velocity is twice the mean velocity of the liquid. Theoretically this would imply that by the time the sample reached the detector it would have an infinitely long 'taft' and that the system would become permanently polluted with 'carry-over' from the sample. However, lateral diffusion of molecules perpendicular to the direction of flow causes mixing between sample and carrier which modifies this effect.
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Analytical bio~echnology
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When a solute that does not undergo on-line/in-carrierstream chemical reaction is injected into a FIA system, physical dispersion is the only band-broadening process involved. In this case, changes in one or more of the mean velocity, tube diameter, monitoring distance, or diffusion coefficients, will alter the dispersion of the sample. On the other hand, when an on-line reaction takes place (for example, a reaction with the immobilized biomolecule column), two kinetic processes occur simultaneously: chemical reaction and physical dispersion. Dispersion is measured as a ratio of the signal from the nondispersed analyte to that of the anatyte after transport through the FIA system. For soluble reagents injected into the carrier stream, the design is optimised so that the chemical reaction generally has no major effect on the dispersion profile at the flow rates normally employed. Nevertheless split peaks can be observed at large injection volumes, due to reagent depletion in the middle of the sample zone. Other factors influencing the kinetics of the reaction and positions of equilibrium can also influence the size and shape of the signal. Chandler et al. [11..] have considered the application of an enzyme field-effect transistor
I
Fig. 2. The glutamate bioluminescent flow injection analysis (FIA) biosensor (bottom), which measures glutamate in terms of a luminescence signal, could, in principle, be coupled to a discrete reagent column (above) and so be used to measure aspartate. The reagent column would generate NAD in proportion to the aspartate present, and the NAD would be measured in terms of a luminescent signal from the biosensot. (O), immobilized aspartate amino transferase;(C)), immobilized malate dehydrogenase; (C)) glutamate dehydrogenase co-immobilized with bioluminescent enzymes ((~)).
(EnFET) to FIA for assay of urea, but have concluded that fabrication of a discrete enzyme column is easier than production of the integrated device. Urease was immobilized on silanised controlled pore glass via glutaraldehyde and ion selective field-effect transistors (ISFETs) placed before and after the reaction column to monitor the change in pH caused by the enzymecatalysed breakdown of urea (Fig. 3.). The calibration of this system depends on several factors such as the position of equilibrium on the column, the sample and carrier pH, and dispersion. For a given system, the dispersion can control the output signal and, even in a controUed pH buffer carrier stream, the unknown pH of the sample can seriously alter the position of the equilibrium and thus the response. If the enzyme reaction goes to completion, that is all the urea is hydrolysed in the column, then the reaction can be considered in three stages: initial mixing of sample and carrier; total urea hydrolysis; and pH equilibration with the buffer and sample zone. In certain cases, for example where the sample pH is greater than the buffer pH, the dispersion may result in a decrease in the monitored pH between the first and second ISFETs. In principle, ff the dispersion is known, these changes
Flow injection analysis with immobilized reagents Hall could be computed and the calibration adjusted according to the pH monitored by the ISFET upstream of the column. The carrier buffer can be chosen to give the minimum interference from the sample buffer and obviously, in this instance, the greater the dispersion the less the influence of the pH of the sample. On the other hand, the signal peak height decreases with dispersion so that, inpractice, a compromise between signal size, linearity and effects of buffer-interference must be made.
Solute focusing Unlike the soluble reagent present in the carrier stream, where the length of the coil between the port and detector can be designed to optimise sample-reagent reaction time, the dispersion of the sample flowing past a stationery reagent phase will be strongly influenced by the geometry and activity of that phase; even the nature of the enzyme catalysis itself may influence the dispersion if it is 'slow' to release the product from its binding site. In these cases, a separate product zone distinct from the reaction zone may even be created, and will be detected downstream. Indeed, the dispersion of the sample can be considerably altered by a stationery phase, even when no chemical reaction is involved with that phase. Johnson and Dorsey [12] have described a technique of solute focusing where the sample injected into a solvent stream passes through a 'focusing column' which acts as an adsorbant for the sample, focusing it into a concentrated band, which is totally retained on the column. In this technique, the column does not serve a second function of immobilized reagent: instead, the sample is eluted from the column with a suitable carrier solvent, to encounter chemical reaction down-stream, prior to the detector. In contrast to solute focusing before chemical reaction, enhancement of the sensitivity of the more conventional FIA chemical methods for phosphate determination has also been accomplished via a post-reaction
(NH2)2CO + 2H20 ~
concentration-extraction technique. Lacy et al, [13] have reported the use of a column for a sorbent extraction method involving a standard reaction based on the formation of a complex of phosphate with molybdenum blue. The reaction was allowed to occur in the carrier stream before passing into a microcolumn of silica-based C-18, which was placed in the light-path of a spectrophotometer. The column 'collects' the complex, and the signal that is generated when the complex is reduced with ascorbic acid is proportional to the phosphate concentration. With this technique, the dispersion characteristics of the system are important only in respect to the mixing of sample with reagent, and do not otherwise effect the signal response. A 200-fold enhancement of sensitivity is claimed to be achieved with the concentration column. This corresponds to a lower limit of detection of 6ppb for a lml sample and compares favourably with the value of 500 fmol Pi for the enzyme method of phosphate determination discussed above [7°].
Selectivity in oxidase-linked assays The sensitivity and selectivity of the FIA signal is strongly influenced by the transduction method, in the same way as in steady-state assays, as well as by other features more unique to flow analysis. Sulphite can be estimated via H202 produced in the sulphite oxidase catalysed reaction: sulphite SO32- + O 2+ H20
5042- -I- H202 oxidase
where peroxide is measured on a platinum electrode. Matsumoto et al. [14o] used sulphite oxidase immobilized on cyanogen bromide-activated Sepharose in the flow injection manifold for sulphite determination in wine. The study investigated the effect on the sig-
CO32-+ 2NH3 + 2H +
NH4+
pKa=9.25
I-tl20 3-
pK2=10.25
H2CO3
pK1=6.35
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H20
pKw=14
H++ HPO4 2- ~
H2PO4-
pKo=7.20
Fig. 3. The enzyme-catalysedhydrolysis of urea showing the pK values of the pH-dependent equilibria in phosphate buffer.
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Analytical
biotechnology
hal of the following factors: enzyme loading (increase in signal with loading to 150 U/g dry Sephamse); column length (increase in signal with length); flow rate (Increase with flow to maximum at 0.45ml rain -1, then decrease to plateau); and the elimination of interference from other sample species. This enabled optimal conditions to be established. Red wine tannins (polyphenols) are particularly bad interferents of amperometric assay techniques, and they may also inactirate the enzyme. A dialysis membrane on the platinum electrode was able to decrease interference of the electrode signal to 12 % of its original value, but this is insufficient for performing an adequate wine assay. In this study the authors found that pre-treatment of white wine with gelatine (1%), together with the introduction of an on-line filter into the flow, allowed the sulphite to be measured. After similar treatment of red wine, however, the high level of tannins still caused erratic behaviour of the dectrode signal. Because this enzyme-linked assay requires sample pretreatment, it is far from being described as a simple integral self-contained biosensor assay system. A sinailar problem had been encountered by Yao [15] in the choline oxidase-linked amperometric assay of cholinesterase. In this case, the author suggested the use of a copper (II) dithiocarbamate-modified silica gel column placed upstream to remove interfering electroactive species normally present in human sera, such as ascotbate, urate, tyrosIne, cysteine, glutathione, and bilimbin. Naturally, the very nature of FIA allows the introduction of as many such 'dean up' columns as are required. However, in view of the preceding discussion on the effects of dispersion on the size and shape of the detected signal, it should be clear that multiple columns cannot be inserted without due consideration for their effect on the analyte dispersion. In steady-state non-flowing systems this option of multiple column insertion does not exist, and so other means have been sought to by-pass the problem, particularly for the amperometric redox enzyme-linked assays. Low molecular weight mediators have been employed in place of oxygen to shuttle electrons between the enzyme redox centre and the electrode, with favourable results. With a view to opamizing the conditions for efliciency and stability, attempts have been made to design mediators with a redox potential close to 0V. (This should be compared with oxidation of H202 at 0.65V versus saturated calomel electrode). By achieving a lower oxidation potential, it is possible to avoid the interference from many other sample species. Gunasingham and Tan [16-] have used tetrathiafulvalene (TIT) as a mediator in a glucose oxidase (GOD)/TrF-carbon paste electrode in a wall-iet cell to measure glucose. In conventional walliet electrodes, the electrode is positioned so that the inlet lies just at the edge of the wall-jet boundary layer. In this electrode, with a reaction layer on the surface, the inlet-electrode separation could be significantly increased without any loss in peak current response. Not only does this study address the problem of sample interferents through the use of mediators, it also demonstrates a totally integrated reagent phase/detector. This
glucose assay system requires no further modification to be classified as a third generation FIA-biosensor.
The polypyrrole/GOD electrode has also undergone considerable investigation as a steady-state electrode, so it is hardly surprising to lind that it has received attention in relation to FIA. In flowing systems the membrane or polymer thickness for an amperometric electrode is an essential parameter because partitioning at the interface and within the membrane layer will effect the sample dispersion and the depletion layer from the electrode. In the mediated GOD electrode above, the distance between the electrode and the wall-jet boundary layer was less critical than for an unmodified electrode. Trojanowicz et at [17 °°] have considered the effect of the polymer thickness on the stability and response of the polypyrrole/GOD electrode and found that very thick membranes gave poor stability and reproducibility. This was attributed to transport limitations of H20 z produced by the enzyme reaction on the surface of the electrode.
The use of an oxygen optrode with immobilized lactate oxidase has demonstrated an alternative means of circumventing the interference of the amperometric H202 signal, inherent in the reaction:
lactate + 02
lactate oxidase
) pyruvate + H202
This biosensor was designed to monitor L-lactate concentration during kefir fermentation [18o], but as the linear range was only 0.02 to 0.5 mM, zone sampling to give dilution had to be applied to extend the linear range sufficiently (to 60 mM). This dilution technique, although very accurate, reduces the sampling frequency.
Assays involving immobilized binding proteins Most of the effort directed at the use of immobilized biomolecules in FIA has been for enzyme-linked systems. Mattiasson etal. [19.o], on the other hand, have directed their attention to the use of an immobilized binding protein. Labelled target molecules (in this case an enzyme label) were added to the sample containing the target molecules to be determined, and the sample was injected into the carrier flow. The labelled and un/abelled target molecules competed for binding to immobilized conconavalin A. The unbound labelled target molecules downstream of the reagent column were assayed after adding a substrate of the labelling enzyme. This is more complex than conventional FIA as it involves two simultaneous processes: the binding reaction between soluble and immobilized reactants, which is far from equilibrium because the system is flowing, and the enzyme reaction
Flow injection analysis with immobilized reagents Hall giving the signal. After the assay, bound protein must be eluted from the colunm. Dissociation can be followed by iniecting pulses of substrate, and this can also be used as an alternative means of analyte determination. Unlike the immobilized enzyme columns, calibration does not vary with lifetime, but capacity decreases. Capacity can be evaluated by injecting a fixed amount of label and observing the binding efficiency.
Conclusion In conclusion, attention towards the use of immobilized bioreagents in FIA remains focused on reagent columns. Although integral enzyme FIA biosensors have been reported, it seems that greater versatility is possible with discrete immobilized reagent columns, which can be linked together, as required, to perform different analyses. The majority of reports have been concerned with enzyme systems and, like their stationery system counterparts, have investigated a variety of transduction techniques. Some effort has also been directed towards the use of binding proteins as the bioactive reagent, but because the system is flowing, the reversible complex-formation between immobilized reagent and soluble analyte is far from equilibrium. Optimisation of this system is, therefore, likely to be more complex than for 'conventional' FIA.
ages without Sample Pretreatment. Anal Chim Acta 1990, 234,213-220. The influence of membrane modification on the linear range of analyses is described for a gas diffusion unit separating donor (sample) and acceptor streams, upstream of an enzyme reactor. The application of the system in the determination of ethanol in samples of beverages and medicine is considered.
5. •
PRINZINGU, OGBOMO I, LEHN C, SCHMn3T H-L: Fermentation Control with Biosensors ill FlOW Injection Systems: Problems and Progress. Sens Actua 1990, B1:542-545. A pervapouration module is described which transfers volatile substances from the sample cartier stream to the acceptor detector stream, upstream of the enzyme reactor. Membrane fouling is avoided by introducing an air space between the process fluid and the membrane. Ethanol and diacetyt are determined via an enzyme reactor by fluorimetry. 6.
KAWASAKIH, SATO K, OGAWAJ, HASEGAWAY', YUKI H: Deterruination of Inorganic Phosphate by Flow Injection Method with Immobilised Enzymes and Chemiluminescence Detection. Anal Biochem 1989, 182:366-370. inorganic phosphate determination is linked to an enzyme pathway generating H202, which is determined via chemiluminescent reagents. The enzymes are immobilized in enzyme reactor columns but, because the luminescent reagents are added to the carrier stream, the system is not fully solid-state. An impressive limit of detection of 500 tool Pi is reported. In fact for phosphate in DNA, measured as a test of applicability to biological samples, the method is claimed to be 160 t~nes more sensitive than conventional spectrophotometric techniques. 7. .
8.
PETERSONJI, GOLDSTEIN SR, FITZGERALD RV, BUCKHOLD DK: Fibre Optic pH Probe for Physiological Use. Anal Cbem 1980, 52:864--868.
9.
RUZlCKAJ, CHRISTIANGD: Reversible Optosensing in Packed Flow-through Detectors: Flow Injection or Chromatography? Anal Cbim Acta, 1990, 234:31-40.
References and recommended reading Papers of special interest, published within the annual period of review, have been highlighted as: • of interest .• of outstanding interest 1. 2.
BERGMEYERHU, HAGENA: New Principle o f Enzymatic Analysis. Fresenius Z Anal Cbem 1972, 261:333-337. UKEDAH, IMABAYASHIM, MATSUMOTO K, OSAJIMAY: Coimmo-
•
bilization of Alcohol Deydrogenase, Diaphorase and NAD and its Application to Flow Injection Analytical System for Ethanol. dgrm Biol t ~ e m 1989, 53(11):2909-2915. Alcohol dehydrogenase, diaphorase and NAD were co-immobilized on Sepharose modified with hexamethylenediamine and glutaraldehyde. In contrast with many other NAD-linked systems, the advantage of this technique is that the NAD is directly coupled to the support. The effect of immobilization parameters on stability, activity and reusability was considered. The ratio of ethanol to 02 consumed was monitored via the Clark electrode. NARINESINGHD, MUNGAL R, NGO T'I': Flow Injection Analysis of Serum Urea Using Urease Covalently Immobflised on 2-Fluro-1-methylpyridinium Salt Activated Fractogel and Fluorescence Detection. Anal Bfbcbem 1990, 188:325-329. A novel method in which urea was determined via the ammonia product of the urease reaction. The isoxoindole product with typhthaldehyde was assayed fluorometrically at kex 340nm and Zero 455nm. Partitioning was required to separate the ammonia from other interfering substances present in serum. Urea detection limit was reported as 0.1mg per litre urea nitrogen at 0.3ml m i n - 1 carrier flow rate, and this decreased as the flow rate was lowered. Linearity to 140mg litre- l could 3. .
be achieved. 4. •
KONNECKEW, SCHMID RD: Gas-diffusion Dilution Flow-injection Method for the Determination o f Ethanol in Bever-
ROCKSB, ~ C: Flow Injection Analysis: A New Approach to Quantitative Measurements in Clinical Chemistry. CUn Chem 1982, 28:409-421.
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KURKIJARWK, Vmmjom T, KORPELAT: FlOW Injection Analysis of Amino Acids and their Metabolites by Immobilised Vitamin B6-dependent Enzymes. A n n N e w York Acad Sci 1989, 585:394-403. Aspartate aminotransferase together with malate dehydrogenase was immobilized onto cyanogen bromide-activated agarose in one column and glutamate dehydrogenase with bioluminescent enzymes was immobilized onto another cyanogen bromide-activated agarose column. The latter reactor was placed directly in the luminometer and could be employed alone to detect glutamate in the range 10-100 nmol. For detection of L-aspartate it was necessary to use the first column as well, in the presence of excess oxaloacetate, and a detection range of 0.1-5 nmol was achieved. The immobilized enzymes were stable for several months and could be used for 600-4300 analyses. Thus, this study suggests that, at least for glutamate, an integrated device could be developed. 11. ••
CHANDLERGK, DODGSON JR, EDDOWES MJ: An ISFET-based Flow Injection Analysis System for Determination of Urea: Experiment and Theory. Sens Actua 1990, B1:433-437. An ISFET pH-sensitive scheme is described with the pH change across a urease reactor column related to sample urea concentration. The influence of sample and buffer pH on the output is discussed. 12.
JOHNSONBF, DORSEYJG: Solute FocusIng in Flow Injection Analysis: Limits of Detection and Linear Dynamic Range. Anal ~ 1990, 62:1392-1397.
13.
LACY N, CHRISTIANGD, RUZlCKAJ: Enhancement of Flow Injection Optosensing by Sorbent Extraction and Reaction Rate Measurement. Anal C~em 1990, 62:1482-1490.
14. .
MATSUMOTOK, MATSUBARAH, UKEDA H, OSAJIMAY: Determination of Sulfite in White Wine by Amperometric Flow Injection Analysis with an Immobilised Sulfite Oxidase Reactor. Agric Biol t ~ e m 1989, 53(9):2347-2353.
15
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Analytical biotechnology This study investigates the optimal conditions for sulphite determination in wine. The authors find that pre-treatment of the wine with gelaOne (1%) and introduction of an on-line filter into the flow, allows the sulphite to be measured in white wine over the range 20-200 ppm. 15.
YAO T: Flow Injection Analysis for Cholinesterase in Blood Serum by use of a Choline.Sensitive Electrode as Amperometric Detector. Anal (Thim Acta 1983, 153:169-174.
16. •~
GUNASINGHAMH, TAN C-H: Carbon Paste-tetrathiafulvalene Amperometic Enzyme Electrode for Determination of Glucose in Flowing Systems. Analyst 1990, 115:35--39. A carbon-paste-TTF electrode with glucose oxidase immobilized on the surface via glutaraldehyde cross-linking with bovine serum albumin is employed in a wall-jet configuration to measure glucose. The electrode can be operated continuously for 2000 injections of 10 mM glucose with only 8% decline in current. In general, the standard deviation of the response in 100 samples was 0.6%. In whole blood, the electrode was orientated vertically to decrease deposition of cells and other blood particles. 17. •6
TROJANOWICZM, MATUSZEWSK~W, PODSIADLAM: Enzyme Entrapped Polypyrrole Modified Electrode for Flow Injection Determination o f Glucose. Biosens Bioelectron 1990, 5:149-156. A report of an integrated biosensor. Electrochemically polymerised pyrrole as the immobilization matrix for GOD was evaluated for FIA glucose determination. Polymer thickness and flow rate were optimised. After five days use, the electrode output had reduced to 20% of its original value. This is comparable with similar electrodes in use in non-flowing systems.
18. •
DREMELBAA, YANG W, SCHMID RD: On-line Determination of Lactic Acid During Kelir Fermentation Based on a Fibreoptic Lactic Acid Biosensor and Flow Injection Analysis. Anal Chim Acta 1990, 234:107-112. A lactate biosensor based on an oxygen optrode is described, with lacrate oxidase immobilized via glutaraldehyde. The assay is based on the estimation o f dissolved oxygen via its dynamic quenching of the fluorescence from an indicator. 19. oo
MATI'IASSONB, BERDEN P, LING TGI: Flow Injection Binding Assays: a Way to increase the Speed in Binding Analyses. Anal Biocbem~ 1989, 181:379-382. This report investigates the use of immobilized biomolecules for the determination of proteins which are not enzymes. A competitive binding assay is described with immobilized binding protein and the target molecule (sample) mixed with enzyme-labelled target molecule injected as pulses. Concanavalin A immobilized to Sepharose via a tresyl chloride method, was employed as binding protein and horseradish peroxidase as the competitive label (competing with samples of other glucosides or mannosides). The enzyme's substrate was included in the buffer, and the signal was measured with respect to a baseline established after a fixed time from a reference point.
EAH Hall, Institute of Biotechnology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QT, UK,