Journal of Biotechnology, 14 (1990) 3-31
3
Elsevier BIOTEC 00479
Minireview
Flow injection analysis (FIA) based on enzymes or antibodies - applications in the life sciences Rolf D. Schmid and Wolf gang Kiinnecke Gesellschaft fiir Biotechnologische Forschung (GBF), Division of Enzyme Technology and Chemistry of Natural Substances, Braunschweig, F.R.G.
Flow injection analysis; FIA; Enzymes; Antibodies
Introduction
Flow injection analysis (FIA) - a relatively new technique described only in 1975 by Ruzicka and Hansen - has proven to be applicable to a wide variety of analytical problems. In fact, the 1989 handbook of commercial instruments sold since 1982 counts 120 applications (Tecator Co., HSganas, Sweden), and in 1988 alone Chemical Abstracts registered 372 new entries on FIA-related techniques. The astounding success of this method is due to several features, the most important of which are: - its extremely high flexibility in adapting most chemical and biochemical reaction procedures, - its compatibility with virtually any detection method, and its reliability in low volume, rapid experiments, allowing applications in on-line monitoring of chemical processes. It is not surprising that these advantages have also led to a large number of applications of F I A in the field of life sciences. Surprisingly, however, most of these methods feature the detection of analytes in biological samples by standard chemical procedures. Although the first application of an enzyme in F I A was reported by Ruzicka and Hansen in 1979, the concept of enzyme- or antibody-supported flow injection systems received relatively little attention, although some notable examples
Correspondence to." R.D. Schmid, Gesellschaft ftir Biotechnologische Forschung (GBF), Division of Enzyme Technology and Chemistry of Natural Substances, Mascheroder Weg, D-3300, Braunschweig, F.R.G.
0168-1656/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
do exist and will be discussed later in this mini review. Only recently, the advantage of the specificity inherent in most enzyme and immuno reactions began to be exploited in FIA procedures, and the first measurement of DNA hybridization in FIA still remains to be published. Against this background, an assessment of these procedures appears to be warranted. The major textbooks on FIA (Ruzicka and Hansen, 1988; Valcarcel and Luque de Castro, 1987) contain chapters on this topic, and reviews on FIA in food (Osborne and Tyson, 1988), clinical (Linares et al., 1985) and pharmaceutical analysis (Calatayud, 1986) have been published recently. In this paper, emphasis is put on the compilation of all FIA procedures involving enzymes and other biological recognition molecules. This will be further illustrated by the original papers in this issue. Since the number of publications in this area is quite large already, but no compilation appears to have been published to date, this mini review is followed by several tables which try to summarize the present state of the art.
Procedures
While biological components such as enzymes and antibodies usually increase the selectivity of an analytical assay, their kinetics have to be reconciled with the conditions of FIA. This implies that (a) special FIA modes have to be applied in order to prolong the contact time of the analyte solution and the biochemical reagent (enzyme, antibody), and (b) FIA configurations have to be adapted to the limited linear range of the biological reaction and the detection units. Condition (a) is fulfilled by the concept of "stopped-flow" (Ruzicka and Hansen, 1979), which is of paramount importance especially in the case of enzyme activity determination. Advantage is taken of the fact that the dispersion of the sample zone does not change significantly even if its residence time is increased by stopping the carrier stream. The stopped-fow technique can be used for the measurement of enzymatic reaction rates (kinetic stopped-flow) as well as for the measurement of analytes (non-kinetic stopped-flow). Condition (b) is accomplished by gradient techniques such as "electronic dilution" (Olsen et al., 1982) as illustrated in Fig. 1, and zone sampling (Reis et al., 1981) demonstrated in Fig. 2. By coupling two manifolds in line, the ratio of the analyte and a buffer stream can be controlled by zone sampling over a dilution range of 104 and more (Fig. 2). Thus, for example, the limited dynamic range of 102 of an enzyme-supported optode system can be significantly expanded (Dremel et al., 1989). A large number of detection systems have been described in the context of these analytical procedures. Optical assays (photometric, fluorimetric and luminometric) prevail but many electrochemical determinations have also been investigated. FIA procedures relevant for this minireview can conveniently be classified in three sections: (a) the measurement of enzyme activity, (b) the use of enzymes as biocatalysts in FIA assays, and (c) flow injection immuno analysis (FIIA).
D1
D2
D3
Fig. 1. Electronic dilution of alcohol oxidase (AOD) using the concentration gradient of the FIA peak. The kinetic stopped-flow technique was used to demonstrate the determination of alcohol oxidase activity via spectrophotometric monitoring of H202. The dispersed sample zone, shown on the left, contains a range of substrate/enzyme ratios, resulting in decreasing slopes of the individual reaction rate curves as measured at increasing delay times because of increasing dispersion (D1 < D2 < D3). To the right is shown a series of recorded reaction-rate curves obtained by injecting identical concentrations of AOD but stopping at different delay times.
Measurement of enzyme activity by FIA procedures F I A techniques can b e used to analyze e n z y m e activity in s a m p l e s of b i o l o g i c a l origin. T h e F I A configurations for this p u r p o s e have b e e n reviewed r e c e n t l y b y F e r n a n d e z - R o m e r o a n d L u q u e de C a s t r o (1988). A c o m p i l a t i o n of p u b l i s h e d literature c a n b e f o u n d in A p p e n d i x 1. G i v e n the speed, reliability a n d a u t o m a t i o n of F I A , it is clear t h a t this m e t h o d is very useful for a variety of applications. Thus, F I A m i g h t e v e n t u a l l y c o m p e t e with p r e s e n t clinical l a b o r a t o r y a u t o m a t s in the m o n i t o r i n g of i n d i c a t o r enzymes such as lactate d e h y d r o g e n a s e ( L D H ) ( T o y o d a et al., 1985) a n d a l a n i n e a m i n o t r a n s f e r a s e ( A L T ) (Sugaya et al., 1988). Several e x a m p l e s for this will b e discussed in the section on Medicine. A n o t h e r i m p o r t a n t a p p l i c a t i o n is in b i o p r o c e s s o p t i m i z a t i o n a n d d o w n s t r e a m processing as far as processes for e n z y m e p r o d u c t i o n are involved.
$1 C
R1
1
'W
Fig. 2. The zone-sampling manifold. Sample is injected at S1 and disperses in the manifold components which include the loop of injection valve $2. At a controlled time after the initial injection, $2 injects a subsample of the original solution into the reagent stream R1 for transport to the detector D and then to waste W.
Thus, already in 1984 a FIA system has been described for the on-line monitoring of alkaline protease production (Kroner and Kula, 1984). A presently unexploited potential application is the detection of the presence or absence of enzyme activity in food which may contain important information on storage stability, off-flavour development, etc. Use of enzymes as biocatalysts in FIA assays
The use of enzymes has greatly expanded the scope of biochemical analysis (Bergmeyer, 1985). Since the late 1960's, immobilized enzyme reagents became available as multi-use, inexpensive analytical reagents (Carr and Bowers, 1980; Guilbault, 1984). In the framework of FIA, the use of both soluble and immobilized enzymes has been described in several hundred publications (Ruz et al., 1988a). A compilation is given in Appendix 2. Glucose is an analyte of central importance in medicine, food and bioprocess analysis. Of the three enzyme systems available, only glucose oxidase and NADHdependent glucose dehydrogenase appear to have been investigated due to the easy detection of their reaction products, whereas there seems to be yet no comparison with PQQ-dependent glucose dehydrogenase (D'Costa et al., 1986). Optical and electrochemical detection systems have been extensively studied, as indicated in Table 1. Usually, an immobilized enzyme column is positioned after the mixing coil for the analyte solution and the reagents. The enzymatically formed indicator reagent, e.g. N A D H or H202, is then transported to and assayed in the detection unit. If the immobilized enzyme and the detector are in sufficiently close contact, the detector becomes a "biosensor" where a transducer (e.g. amperometric electrode, oxygen optode) is in direct proximity to the enzyme membrane. In most enzyme-supported FIA designs, immobilized enzyme and detector unit are kept separate. The reason for this is a 20-fold higher operational stability of enzyme reactors compared with enzyme electrodes (Yao, 1989). Another obvious advantage is that both modules can be serviced separately. Recently, post-column detectors in HPLC have stimulated the incorporation of real "biosensors". An example in this issue is the paper of Yao et al. (1990) where guanase or phosphorylase a/xanthine oxidase are directly immobilized at an amperometric electrode for the post-column HPLC detection of nucleosides. If several enzymes have to be used in one assay, or if multichannel assays are to be performed, the kinetics and stability of the single enzymes may vary considerably. As a result, it can prove to be advantageous to immobilize only one enzyme and add the other enzyme(s) in the carrier solution (Fernandez-Romero et al., 1987). Alternatively, immobilized enzyme columns may be used in parallel and detection of one indicator reagent is controlled via delay coils (Masoom, 1988a). Multichannel detectors fed from independent lines and plural immobilized enzyme reactors are another option discussed in this issue by Matsumoto et al. (1990) for simultaneous FIA of glucose, ethanol and lactate.
H 202
NAD
NADPH NADH
Glucose oxidase/ mutarotase
Glucose dehydrogenase
Hexokinase/ glucose-6-P-dehydrogenase
amperometry
02/hexacyanoferrate
02/hexacyanoferrate
Glucose oxidase/ catalase
photometry
H202/dye
Glucose oxidase/ peroxidase
photometry fluorimetry
(a) photometry (b) amperometry
(a) amperometry (b) photometry
amperometry
100 h l
1
30 h -1
4 0 h -1
120 h -1 300 h -1
8h
lOOh 1
Ruz et al., 1988b Linares et al., 1987
Roehrig et al., 1983 Marko-Varga et al., 1986
Yang, 1989 Toei, 1988
Yao et al., 1984
Yao et al., 1984
Stults et al., 1987
Wieck et al., 1984
75 h -1 6h-1
(a) potentiometry (b) optode
Reference Marko-Varga et al., 1986 Petersson, 1989 Petersson et al., 1986
Sampling frequency 300 h -1 90 h -1 120 h - l
(a) amperometry chemiluminescence
Glucose oxidase
Detection principle
Detected species
H202 (a) H202/luminol (b) H 202/luminol / hexacyanoferrate H+
Enzyme
Glucose assay by enzyme-supported FIA
TABLE 1
fluorimetry chemiluminescence
fluorimetry ion-selective electrode photometry
precipitation competitive immunoassay reversible absorption homogeneous immunoassay competitive liposome enhanced immunoassay (f) antigen-conjugated liposomes
energy transfer immunoassay
competitive immunoassay
competitive immunoassay, HPLC
competitive immunoassay secondary antibodies
competitive immunoassay
Human serum albumin
a-Feto-protein, insulin 17-a-hydroxy-progesterone
Theophylline, valproic acid
Theophylline, insuline
Human transferrin
fluorimetry, amperometry etc.
turbidimetry amperometry ellipsometry fluorimetry fluorimetry
(a) (b) (c) (d) (e)
Human IgG
Detection principle
Assay principle
Analyte
Immunological assays by FIA
TABLE 2
15 h - 1
4h 1
10 h - 1
1
h- 1 h 1 h- 1 h-1
60 h -
-
40 5 10 60
Sampling frequency
Larsson et al., 1987
Lee and Meyerhoff, 1988
Allain et al., 1989
Maeda and Tsuji, 1985
Lim et al., 1980
Durst et al., 1988
Worsfold et al., 1985 De Alwis and Wilson, 1987 JSnsson et al., 1985 Kelly and Christian, 1982 Plant et al., 1988
Reference
Flow injection immuno analysis (FIIA) Obvious difficulties in applying FIA techniques to immunoanalysis are (a) the relatively slow progression of immunological reactions, and (b) quantitative detection. A survey of the methods proposed to date is indicated in Table 2. The first example of FIIA is probably contained in a paper by Lim et al. (1980) who proposed a homogeneous fluorescence energy-transfer immunoassay for serum albumin. They labelled antigen and antibody with different fluorescent groups suitable for energy transfer after immunological binding. Using merging-zone and stopped-flow principles, the detection limit was in the order of 10 -7 M, and the sampling frequency was 10 h -1. Kelly and Christian (1982) proposed a homogeneous immunoassay for serum IgG using peroxidase as enzyme label and H202/peroxidase-catalyzed oxidation of leuco-diacetyldichlorofluorescein as indicator reaction. Serum IgG concentrations from 1.4 to 25 mg ml-1 could be determined with a sampling frequency of 60 h -1. Other detection procedures for homogeneous immunoassays include ellipsometry (J~Snsson et al., 1985) and turbidity (Hughes and Worsfold, 1985). Most other systems proposed to date are based on heterogeneous immunoassays where a separation of labelled from unlabelled antibody or antigen is required before quantification. Secondary antibodies (Lee and Meyerhoff, 1988), liquid chromatography (Heineman and Halsall, 1987), HPLC (Allain et al., 1989) or antigen-containing reporter liposomes (Plant et al., 1988) have been suggested for this purpose. The latter principle is based on the concept that antigen-sensitized liposomes compete with sample analyte molecules for binding to immobilized antibodies in a immunoreactor column. For every liposome which does not bind to the column due to the presence of an analyte molecule, approximately l0 s fluorescent molecules entrapped in the liposome are released and detected. In the model experiments described so far with antigen-binding IgG Fab', the sensitivity and sampling frequency seem to be excellent although no detailed data are provided.
Application of FIA based on enzymes or antibodies in the life sciences
Medicine FIA systems have largely been investigated in the field of medicine, and a commercial FIA system for the clinical laboratory is being marketed in Japan by Shimadzu. FIA being a technique for either the automatic analysis of a large number of samples or for continuous monitoring, emphasis has been placed on the accelerated assay of clinical parameters. These include the measurement in blood, serum or urine of either enzyme activity or of metabolites via enzymatic assay, as indicated in Table 3. For practical application in the clinical laboratory, precision, reliability and ease of operation are major requirements. An ingenious system which allows the determination of six clinical parameters in a "one-shot FIA" (Murachi et al., 1987) has been commercialized (Fig. 3). It is based on the use of a sequence of enzyme
Creatinine
Cholesterol serum serum serum serum
blood blood blood blood blood/urine/ saliva saliva brain tissue serum serum
Alcohol
Ascorbic acid Choline Cholesterol (total)
blood
creatinine iminohydrolase
cholesterol oxidase
cholesterol oxidase/POD
ADH ADH ascorbate oxidase
acetylchohneesterase/choline oxidase AOD ADH ADH ADH
Enzyme system
Body fluid
photometry
amperometry chemiluminescence chemiluminescence
photometry
fluorimetry photometry/fluorimetry amperometry chemiluminescence
amperometry amperometry amperometry photometry photometry
Jeppesen and Hansen, 1988
Fernandez-Romero et al., 1987 Yao and Wasa, 1988 Yao, 1983 Malavolti et al., 1985
G o m e z et al., 1985 Linares et al., 1987 Bradberry and Adams, 1983 Yao, 1983
Masoom, 1988a Yao and Wasa, 1985 Fernandez-Romero et al., 1987 Ruz et al., 1987 Worsfold et al., 1981
Reference
Lain6-Cessac et al., 1989 Yao, 1983 Hayashi et al., 1987a Riley et al., 1983 Hayashi et al., 1987b
photometry amperometry fluorimetry photometry fluorimetry
choline xanthine pyruvate/NADH hypoxanthine Detection principle
Sugaya et al., 1988 Sugaya et al., 1988
Reference
photometry photometry
Detection principle
pyruvate/NADH oxalacetate/NADH
Assay principle
serum serum plasma/ erythrocytes blood/serum blood serum erythrocytes
Body fluid
Acetylcholine/choline
2. Analytes
Analyte
Guanase Lactate dehydrogenase Purine nucleoside phosphorylase
Alanine-aminotransferase (ALT) Aspar tate-aminotrans ferase (AST) Cholinesterase
1. Enzymes
Enzyme activity
FIA assays for enzyme activity or metabolites in body fluid
TABLE 3
Uric acid
Protein Theophylline/triglyceride Urea
Lactate
Glutathione ox.
Glutathione
Galactose Glucose
serum serum/urine serum
serum serum blood
blood serum plasma bodyfuids serum
blood
plasma/urine serum plasma serum serum serum serum blood blood blood blood biological material biological material blood
urease urease uficase
GSSG reductase lactate dehydrogenase lactate dehydrogenase/glutamate pyruvate transaminase lactate oxidase Lowry EMIT-kit urease urease/glutamate-DH/ glutamate oxidase urease urease urease
GOD/POD GOD/POD GOD GOD GOD GOD GOD GOD/mutarotase hexokinase/G-6P-DH glutathione reductase
Tabata and Murachi, 1988 Ruzicka et al., 1979 Yerian et al., 1986
chemiluminescence pH-electrode pH-optrode N H 3"selective electrode pH-electrode conductivity amperometry
Petersson, 1988 Ruzicka et al., 1979 Taylor and Nieman, 1986 Iob and Mottola, 1980
Bergqvist et al., 1988 Zaitsu et al., 1987 Salerno et al., 1985 Rocks et al., 1984 Solich et al., 1989
Karlsson et al., 1983
Redegeld et al., 1988
Winquist et al., 1986 Yao, 1983 H w a n g and Dasgupta, 1987 Sanghera, 1988 Yao, 1983 Ridder et al., 1982 Tabata et al., 1984 M a s o o m and Townshend, 1984 Petersson, 1989 Toei, 1988 Gizurarson, 1989 van Opstal et al., 1988
fluorimetry fuorimetry photometry photometry photometry
fluorimetry
photometry
chemiluminescence photometry photometry photometry
photometry chemiluminescence fuorescence amperometry amperometry chemiluminescence chemiluminescence
12
Fig. 3. S h i m a d z u FIA.
reactions which all eventually lead to H202 which in turn is determined by a luminometer (Fig. 4). In this issue, Murachi et al. (1990) have elegantly applied the same concept to the sensitive luminometric determination of N A D H , the cofactor of a wide range of substrates which can be determined by means of dehydrogenase, by using immobilized N A D H oxidase in the presence of luminol and potassium ferricyanide.
Urea
Urease
Creatinine
CD
Glutamicacid
GOT
Asparticacid
GPT
Glucose
Hh
3
Elsevier BIOTEC 00479
Minireview
Flow injection analysis (FIA) based on enzymes or antibodies - applications in the life sciences Rolf D. Schmid and Wolf gang Kiinnecke Gesellschaft fiir Biotechnologische Forschung (GBF), Division of Enzyme Technology and Chemistry of Natural Substances, Braunschweig, F.R.G.
Flow injection analysis; FIA; Enzymes; Antibodies
Introduction
Flow injection analysis (FIA) - a relatively new technique described only in 1975 by Ruzicka and Hansen - has proven to be applicable to a wide variety of analytical problems. In fact, the 1989 handbook of commercial instruments sold since 1982 counts 120 applications (Tecator Co., HSganas, Sweden), and in 1988 alone Chemical Abstracts registered 372 new entries on FIA-related techniques. The astounding success of this method is due to several features, the most important of which are: - its extremely high flexibility in adapting most chemical and biochemical reaction procedures, - its compatibility with virtually any detection method, and its reliability in low volume, rapid experiments, allowing applications in on-line monitoring of chemical processes. It is not surprising that these advantages have also led to a large number of applications of F I A in the field of life sciences. Surprisingly, however, most of these methods feature the detection of analytes in biological samples by standard chemical procedures. Although the first application of an enzyme in F I A was reported by Ruzicka and Hansen in 1979, the concept of enzyme- or antibody-supported flow injection systems received relatively little attention, although some notable examples
Correspondence to." R.D. Schmid, Gesellschaft ftir Biotechnologische Forschung (GBF), Division of Enzyme Technology and Chemistry of Natural Substances, Mascheroder Weg, D-3300, Braunschweig, F.R.G.
0168-1656/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
do exist and will be discussed later in this mini review. Only recently, the advantage of the specificity inherent in most enzyme and immuno reactions began to be exploited in FIA procedures, and the first measurement of DNA hybridization in FIA still remains to be published. Against this background, an assessment of these procedures appears to be warranted. The major textbooks on FIA (Ruzicka and Hansen, 1988; Valcarcel and Luque de Castro, 1987) contain chapters on this topic, and reviews on FIA in food (Osborne and Tyson, 1988), clinical (Linares et al., 1985) and pharmaceutical analysis (Calatayud, 1986) have been published recently. In this paper, emphasis is put on the compilation of all FIA procedures involving enzymes and other biological recognition molecules. This will be further illustrated by the original papers in this issue. Since the number of publications in this area is quite large already, but no compilation appears to have been published to date, this mini review is followed by several tables which try to summarize the present state of the art.
Procedures
While biological components such as enzymes and antibodies usually increase the selectivity of an analytical assay, their kinetics have to be reconciled with the conditions of FIA. This implies that (a) special FIA modes have to be applied in order to prolong the contact time of the analyte solution and the biochemical reagent (enzyme, antibody), and (b) FIA configurations have to be adapted to the limited linear range of the biological reaction and the detection units. Condition (a) is fulfilled by the concept of "stopped-flow" (Ruzicka and Hansen, 1979), which is of paramount importance especially in the case of enzyme activity determination. Advantage is taken of the fact that the dispersion of the sample zone does not change significantly even if its residence time is increased by stopping the carrier stream. The stopped-fow technique can be used for the measurement of enzymatic reaction rates (kinetic stopped-flow) as well as for the measurement of analytes (non-kinetic stopped-flow). Condition (b) is accomplished by gradient techniques such as "electronic dilution" (Olsen et al., 1982) as illustrated in Fig. 1, and zone sampling (Reis et al., 1981) demonstrated in Fig. 2. By coupling two manifolds in line, the ratio of the analyte and a buffer stream can be controlled by zone sampling over a dilution range of 104 and more (Fig. 2). Thus, for example, the limited dynamic range of 102 of an enzyme-supported optode system can be significantly expanded (Dremel et al., 1989). A large number of detection systems have been described in the context of these analytical procedures. Optical assays (photometric, fluorimetric and luminometric) prevail but many electrochemical determinations have also been investigated. FIA procedures relevant for this minireview can conveniently be classified in three sections: (a) the measurement of enzyme activity, (b) the use of enzymes as biocatalysts in FIA assays, and (c) flow injection immuno analysis (FIIA).
D1
D2
D3
Fig. 1. Electronic dilution of alcohol oxidase (AOD) using the concentration gradient of the FIA peak. The kinetic stopped-flow technique was used to demonstrate the determination of alcohol oxidase activity via spectrophotometric monitoring of H202. The dispersed sample zone, shown on the left, contains a range of substrate/enzyme ratios, resulting in decreasing slopes of the individual reaction rate curves as measured at increasing delay times because of increasing dispersion (D1 < D2 < D3). To the right is shown a series of recorded reaction-rate curves obtained by injecting identical concentrations of AOD but stopping at different delay times.
Measurement of enzyme activity by FIA procedures F I A techniques can b e used to analyze e n z y m e activity in s a m p l e s of b i o l o g i c a l origin. T h e F I A configurations for this p u r p o s e have b e e n reviewed r e c e n t l y b y F e r n a n d e z - R o m e r o a n d L u q u e de C a s t r o (1988). A c o m p i l a t i o n of p u b l i s h e d literature c a n b e f o u n d in A p p e n d i x 1. G i v e n the speed, reliability a n d a u t o m a t i o n of F I A , it is clear t h a t this m e t h o d is very useful for a variety of applications. Thus, F I A m i g h t e v e n t u a l l y c o m p e t e with p r e s e n t clinical l a b o r a t o r y a u t o m a t s in the m o n i t o r i n g of i n d i c a t o r enzymes such as lactate d e h y d r o g e n a s e ( L D H ) ( T o y o d a et al., 1985) a n d a l a n i n e a m i n o t r a n s f e r a s e ( A L T ) (Sugaya et al., 1988). Several e x a m p l e s for this will b e discussed in the section on Medicine. A n o t h e r i m p o r t a n t a p p l i c a t i o n is in b i o p r o c e s s o p t i m i z a t i o n a n d d o w n s t r e a m processing as far as processes for e n z y m e p r o d u c t i o n are involved.
$1 C
R1
1
'W
Fig. 2. The zone-sampling manifold. Sample is injected at S1 and disperses in the manifold components which include the loop of injection valve $2. At a controlled time after the initial injection, $2 injects a subsample of the original solution into the reagent stream R1 for transport to the detector D and then to waste W.
Thus, already in 1984 a FIA system has been described for the on-line monitoring of alkaline protease production (Kroner and Kula, 1984). A presently unexploited potential application is the detection of the presence or absence of enzyme activity in food which may contain important information on storage stability, off-flavour development, etc. Use of enzymes as biocatalysts in FIA assays
The use of enzymes has greatly expanded the scope of biochemical analysis (Bergmeyer, 1985). Since the late 1960's, immobilized enzyme reagents became available as multi-use, inexpensive analytical reagents (Carr and Bowers, 1980; Guilbault, 1984). In the framework of FIA, the use of both soluble and immobilized enzymes has been described in several hundred publications (Ruz et al., 1988a). A compilation is given in Appendix 2. Glucose is an analyte of central importance in medicine, food and bioprocess analysis. Of the three enzyme systems available, only glucose oxidase and NADHdependent glucose dehydrogenase appear to have been investigated due to the easy detection of their reaction products, whereas there seems to be yet no comparison with PQQ-dependent glucose dehydrogenase (D'Costa et al., 1986). Optical and electrochemical detection systems have been extensively studied, as indicated in Table 1. Usually, an immobilized enzyme column is positioned after the mixing coil for the analyte solution and the reagents. The enzymatically formed indicator reagent, e.g. N A D H or H202, is then transported to and assayed in the detection unit. If the immobilized enzyme and the detector are in sufficiently close contact, the detector becomes a "biosensor" where a transducer (e.g. amperometric electrode, oxygen optode) is in direct proximity to the enzyme membrane. In most enzyme-supported FIA designs, immobilized enzyme and detector unit are kept separate. The reason for this is a 20-fold higher operational stability of enzyme reactors compared with enzyme electrodes (Yao, 1989). Another obvious advantage is that both modules can be serviced separately. Recently, post-column detectors in HPLC have stimulated the incorporation of real "biosensors". An example in this issue is the paper of Yao et al. (1990) where guanase or phosphorylase a/xanthine oxidase are directly immobilized at an amperometric electrode for the post-column HPLC detection of nucleosides. If several enzymes have to be used in one assay, or if multichannel assays are to be performed, the kinetics and stability of the single enzymes may vary considerably. As a result, it can prove to be advantageous to immobilize only one enzyme and add the other enzyme(s) in the carrier solution (Fernandez-Romero et al., 1987). Alternatively, immobilized enzyme columns may be used in parallel and detection of one indicator reagent is controlled via delay coils (Masoom, 1988a). Multichannel detectors fed from independent lines and plural immobilized enzyme reactors are another option discussed in this issue by Matsumoto et al. (1990) for simultaneous FIA of glucose, ethanol and lactate.
H 202
NAD
NADPH NADH
Glucose oxidase/ mutarotase
Glucose dehydrogenase
Hexokinase/ glucose-6-P-dehydrogenase
amperometry
02/hexacyanoferrate
02/hexacyanoferrate
Glucose oxidase/ catalase
photometry
H202/dye
Glucose oxidase/ peroxidase
photometry fluorimetry
(a) photometry (b) amperometry
(a) amperometry (b) photometry
amperometry
100 h l
1
30 h -1
4 0 h -1
120 h -1 300 h -1
8h
lOOh 1
Ruz et al., 1988b Linares et al., 1987
Roehrig et al., 1983 Marko-Varga et al., 1986
Yang, 1989 Toei, 1988
Yao et al., 1984
Yao et al., 1984
Stults et al., 1987
Wieck et al., 1984
75 h -1 6h-1
(a) potentiometry (b) optode
Reference Marko-Varga et al., 1986 Petersson, 1989 Petersson et al., 1986
Sampling frequency 300 h -1 90 h -1 120 h - l
(a) amperometry chemiluminescence
Glucose oxidase
Detection principle
Detected species
H202 (a) H202/luminol (b) H 202/luminol / hexacyanoferrate H+
Enzyme
Glucose assay by enzyme-supported FIA
TABLE 1
fluorimetry chemiluminescence
fluorimetry ion-selective electrode photometry
precipitation competitive immunoassay reversible absorption homogeneous immunoassay competitive liposome enhanced immunoassay (f) antigen-conjugated liposomes
energy transfer immunoassay
competitive immunoassay
competitive immunoassay, HPLC
competitive immunoassay secondary antibodies
competitive immunoassay
Human serum albumin
a-Feto-protein, insulin 17-a-hydroxy-progesterone
Theophylline, valproic acid
Theophylline, insuline
Human transferrin
fluorimetry, amperometry etc.
turbidimetry amperometry ellipsometry fluorimetry fluorimetry
(a) (b) (c) (d) (e)
Human IgG
Detection principle
Assay principle
Analyte
Immunological assays by FIA
TABLE 2
15 h - 1
4h 1
10 h - 1
1
h- 1 h 1 h- 1 h-1
60 h -
-
40 5 10 60
Sampling frequency
Larsson et al., 1987
Lee and Meyerhoff, 1988
Allain et al., 1989
Maeda and Tsuji, 1985
Lim et al., 1980
Durst et al., 1988
Worsfold et al., 1985 De Alwis and Wilson, 1987 JSnsson et al., 1985 Kelly and Christian, 1982 Plant et al., 1988
Reference
Flow injection immuno analysis (FIIA) Obvious difficulties in applying FIA techniques to immunoanalysis are (a) the relatively slow progression of immunological reactions, and (b) quantitative detection. A survey of the methods proposed to date is indicated in Table 2. The first example of FIIA is probably contained in a paper by Lim et al. (1980) who proposed a homogeneous fluorescence energy-transfer immunoassay for serum albumin. They labelled antigen and antibody with different fluorescent groups suitable for energy transfer after immunological binding. Using merging-zone and stopped-flow principles, the detection limit was in the order of 10 -7 M, and the sampling frequency was 10 h -1. Kelly and Christian (1982) proposed a homogeneous immunoassay for serum IgG using peroxidase as enzyme label and H202/peroxidase-catalyzed oxidation of leuco-diacetyldichlorofluorescein as indicator reaction. Serum IgG concentrations from 1.4 to 25 mg ml-1 could be determined with a sampling frequency of 60 h -1. Other detection procedures for homogeneous immunoassays include ellipsometry (J~Snsson et al., 1985) and turbidity (Hughes and Worsfold, 1985). Most other systems proposed to date are based on heterogeneous immunoassays where a separation of labelled from unlabelled antibody or antigen is required before quantification. Secondary antibodies (Lee and Meyerhoff, 1988), liquid chromatography (Heineman and Halsall, 1987), HPLC (Allain et al., 1989) or antigen-containing reporter liposomes (Plant et al., 1988) have been suggested for this purpose. The latter principle is based on the concept that antigen-sensitized liposomes compete with sample analyte molecules for binding to immobilized antibodies in a immunoreactor column. For every liposome which does not bind to the column due to the presence of an analyte molecule, approximately l0 s fluorescent molecules entrapped in the liposome are released and detected. In the model experiments described so far with antigen-binding IgG Fab', the sensitivity and sampling frequency seem to be excellent although no detailed data are provided.
Application of FIA based on enzymes or antibodies in the life sciences
Medicine FIA systems have largely been investigated in the field of medicine, and a commercial FIA system for the clinical laboratory is being marketed in Japan by Shimadzu. FIA being a technique for either the automatic analysis of a large number of samples or for continuous monitoring, emphasis has been placed on the accelerated assay of clinical parameters. These include the measurement in blood, serum or urine of either enzyme activity or of metabolites via enzymatic assay, as indicated in Table 3. For practical application in the clinical laboratory, precision, reliability and ease of operation are major requirements. An ingenious system which allows the determination of six clinical parameters in a "one-shot FIA" (Murachi et al., 1987) has been commercialized (Fig. 3). It is based on the use of a sequence of enzyme
Creatinine
Cholesterol serum serum serum serum
blood blood blood blood blood/urine/ saliva saliva brain tissue serum serum
Alcohol
Ascorbic acid Choline Cholesterol (total)
blood
creatinine iminohydrolase
cholesterol oxidase
cholesterol oxidase/POD
ADH ADH ascorbate oxidase
acetylchohneesterase/choline oxidase AOD ADH ADH ADH
Enzyme system
Body fluid
photometry
amperometry chemiluminescence chemiluminescence
photometry
fluorimetry photometry/fluorimetry amperometry chemiluminescence
amperometry amperometry amperometry photometry photometry
Jeppesen and Hansen, 1988
Fernandez-Romero et al., 1987 Yao and Wasa, 1988 Yao, 1983 Malavolti et al., 1985
G o m e z et al., 1985 Linares et al., 1987 Bradberry and Adams, 1983 Yao, 1983
Masoom, 1988a Yao and Wasa, 1985 Fernandez-Romero et al., 1987 Ruz et al., 1987 Worsfold et al., 1981
Reference
Lain6-Cessac et al., 1989 Yao, 1983 Hayashi et al., 1987a Riley et al., 1983 Hayashi et al., 1987b
photometry amperometry fluorimetry photometry fluorimetry
choline xanthine pyruvate/NADH hypoxanthine Detection principle
Sugaya et al., 1988 Sugaya et al., 1988
Reference
photometry photometry
Detection principle
pyruvate/NADH oxalacetate/NADH
Assay principle
serum serum plasma/ erythrocytes blood/serum blood serum erythrocytes
Body fluid
Acetylcholine/choline
2. Analytes
Analyte
Guanase Lactate dehydrogenase Purine nucleoside phosphorylase
Alanine-aminotransferase (ALT) Aspar tate-aminotrans ferase (AST) Cholinesterase
1. Enzymes
Enzyme activity
FIA assays for enzyme activity or metabolites in body fluid
TABLE 3
Uric acid
Protein Theophylline/triglyceride Urea
Lactate
Glutathione ox.
Glutathione
Galactose Glucose
serum serum/urine serum
serum serum blood
blood serum plasma bodyfuids serum
blood
plasma/urine serum plasma serum serum serum serum blood blood blood blood biological material biological material blood
urease urease uficase
GSSG reductase lactate dehydrogenase lactate dehydrogenase/glutamate pyruvate transaminase lactate oxidase Lowry EMIT-kit urease urease/glutamate-DH/ glutamate oxidase urease urease urease
GOD/POD GOD/POD GOD GOD GOD GOD GOD GOD/mutarotase hexokinase/G-6P-DH glutathione reductase
Tabata and Murachi, 1988 Ruzicka et al., 1979 Yerian et al., 1986
chemiluminescence pH-electrode pH-optrode N H 3"selective electrode pH-electrode conductivity amperometry
Petersson, 1988 Ruzicka et al., 1979 Taylor and Nieman, 1986 Iob and Mottola, 1980
Bergqvist et al., 1988 Zaitsu et al., 1987 Salerno et al., 1985 Rocks et al., 1984 Solich et al., 1989
Karlsson et al., 1983
Redegeld et al., 1988
Winquist et al., 1986 Yao, 1983 H w a n g and Dasgupta, 1987 Sanghera, 1988 Yao, 1983 Ridder et al., 1982 Tabata et al., 1984 M a s o o m and Townshend, 1984 Petersson, 1989 Toei, 1988 Gizurarson, 1989 van Opstal et al., 1988
fluorimetry fuorimetry photometry photometry photometry
fluorimetry
photometry
chemiluminescence photometry photometry photometry
photometry chemiluminescence fuorescence amperometry amperometry chemiluminescence chemiluminescence
12
Fig. 3. S h i m a d z u FIA.
reactions which all eventually lead to H202 which in turn is determined by a luminometer (Fig. 4). In this issue, Murachi et al. (1990) have elegantly applied the same concept to the sensitive luminometric determination of N A D H , the cofactor of a wide range of substrates which can be determined by means of dehydrogenase, by using immobilized N A D H oxidase in the presence of luminol and potassium ferricyanide.
Urea
Urease
Creatinine
CD
Glutamicacid
GOT
Asparticacid
GPT
Glucose
Hh
