Biosensors and flow injection analysis Chien-Yuan Chen and Isao Karube

National Taiwan University, Tapei, Taiwan and University of Tokyo, Tokyo, Japan

Combining flow injection analysis with a biosensor is a novel biosensing process which has allowed speedy and accurate analysis. Diagnostic analysis is the most important application for biosensing flow injection analysis, but other applications include bioprocess monitoring, analysis of food and agricultural products, as well as environmental analysis. In addition, the analysis of compounds, such as explosives and abused drugs, and monitoring of Salmonella, the microorganism that causes food poisoning, have been reported.

Current Opinion in Biotechnology 1992, 3:31-39

Introduction into electrical signals; and an element for recording these electrical signals. It is difficult to give an exact definition of a biosensor. Widely speaking, a biosensor can be considered as an The sensing elements used in biosensors can be gener- analytical device that responds to biological substances ally classified into four groups: proteins, organelles, cells selectively and reversibly. According to this definition, any and tissues. Biosensors themselves can be divided into system that can be used to analyse a biological material several groups according to the biological materials used can be classified as a biosensor. This definition, however, or the reaction type involved, and a list is shown in Fig. 2. would include almost all physical and chemical sensors On the other hand, conversion elements used in biosen- and is therefore obviously too wide. Thus, a more gener- sors employ most methods used in the fields of physical ally acceptable definition would be analytical devices that and chemical analysis. Spectrophotometry, amperometry, use a biological material as the sensing element. potentiometry, thermometry, fluorimetry and resonance luminescence are widely adopted for this purpose. Three elements are required for the construction of a biosensor (shown in Fig. 1): a recognition element com- The use of biosensors started in 1962 when Clark and posed of biological materials that react selectively with Lyons [1] combined an oxygen probe with glucose oxi- the specified substrate; a conversion element for convert- dase to determine glucose levels. The fact that biosensors hag the related information from the bio-catalytic reaction still attract a lot of interest three decades after they were

Bio-fu nctional membrane

O :)-- Chemical Electrode )- substance semiconductor

Heat = Thermistor OD.I-I )- Photon Electrical [] © Light signals )- counter Sound Sound ©E3 detector Electrical o,. )-'J-- signal'

Molecular . Conversion recognition

Fi&l. General biosensor construction and function. Chemical substances, heat,light, sound and electrical signals are produced durmg bio-catalyzed reactions in the bio-functional mwmbrane. These are converted into electrical signal which are then detected by electronic devices.

Abbreviations FIA--flow injection analysis; G-~P--glucose-6-phosphate; TNT--trinitrotoluene.

© Current Biology Ltd ISSN 0958-1669 31 32 Analytical biotechnology

Molecular recognition element Biosensors

Proteins Antibodies Transducer Immuno sensors

Receptors Receptor sensors

......

Organelles Organelles Organelle sensors

...... !1 Micr°°rganisms ~ ? ~ ~a Micr°bial sensors C Is and Animaland plant cells [ 1 Cell sensors tissues ~ Fig. 2. Classification of blosensors ac- cording to the sensing elements used. Animal and plant tissues Tissue sensors Proteins, organelles, cells and tissues are major biomaterials used for construct- ing biosensors.

first developed demonstrates their importance in many reason, however, is the applicability of glucose sensors fields of analysis, to many fields, especially clinical analysis. Although the technique of flow injection analysis (FIA) is Glucose sensors have been studied for some decades and not new, the combination of FIA with a detection device over this time have been much improved. Amperometric is novel in many analytical systems. There are still many glucose enzyme sensors can be divided into three gen- studies concerned with the theory or operation of FIA. erations according to the electrodes and mediators used. Some have tried to devise good methods for calibrating The first generation sensors (Fig. 3) detect hydrogen per- data and reducing interference [2]. Others have tried to oxide formation or oxygen consumption in the reaction. establish mathematical models [3,4], and still others have The second generation sensors use a mediator to trans- carried out error analysis of signals and then tried to fer electrons between the enzyme and the electrode and establish a more reasonable operation process [5]. All the third have an electrode made from a special material of these have improved FIA to some extent. A biosensing which allows direct transfer of electrons to it, from the FIA system is constructed by combining a biosensor with enzyme. Conductive organic complex salts, for example an FIA system. Most of the biological materials used as Fulvadene-tetracyanoquinodimethane, have been used as recognition elements in biosensors have the disadvantage electrode materials for this purpose. A recent study com- of losing their activity when sterilized by heat or chemi- pared these three generations of sensor combined with cal treatment. However, a biosensor does not need to FIA in determining glucose concentration in undiluted be sterilized if the sample is not returned to its source. whole blood [6°°]. The second generation sensors seem Another problem is the decrease in response intensity to be better than the others, although the problem of if the biological material being used is kept in contact mediator leakage from the electrode must be taken into with its substrate for a long period of time. This can be consideration because most of the mediators used are overcome by using an FIA system to minimize the contact small molecules that are difficult to immobilize together period between a biosensor and its substrate and to rinse with enzyme. the biological material with the carrying solution. In ad- Glucose sensors might also be improved by altering the dition, it is possible to obtain almost-real-time data even materials and methods used for enzyme immobilization. though the sensor is not directly connected with the sys- Nylon has been used as the insoluble carrier for glucose tem under investigation, for example a fermenter. Other oxidase immobilization and the effects of various spacers merits of a biosensing FIA system include the possibility have been investigated in order to improve the efficiency of on-line calibration of the sensor and the manipulation of enzyme immobilization [7]. A nylon membrane with of the sample before contact with the sensor. enzymes immobilized using spacers was combined with an FIA system and used to determine the concentration of glucose in blood serum [8]. Immobilization of enzymes in electropolymerized polypyrrole film on the surface of Glucose determination for clinical analysis a platinum electrode was reported to provide a conve- nient sensor for flow injection glucose analysis [9°]. The Glucose determination is the most widely studied appli- technique of electropolymerization has also been applied cation of biosensors. This is partly because the high sta- to prevent interference and electrode fouling [10]. If, bility and satisfactory specificity of the glucose-related en- however, whole blood instead of serum is used as the zymes, including glucose oxidase and glucose dehydro- sample, the influence of the blood cells should be taken genase, make it quite easy to get good results. The major into account. This problem can be solved by improv- Biosensors and flow injection analysis Chen and Karube 33

Hydrogen peroxide Immobilized glucose Reaction electrode oxidase membrane chamber Anode 1/202 Buffer solution

2e- 3 Glucose + 02

Corpuscle Fig. 3. A first generation enzymatic glu- 21-1++11202 Protein cose sensor showing sensing and con- version elements. Hydrogen peroxide L i Bilirubin is generated from glucose and oxy- gen through the action of glucose oxi- Ascorbic dase immobilized to a membrane. This -~2e- y, 20 acid hydrogen peroxide reacts at the anode • ", ., Glutathione to produce two hydrogen ions, one oxy- gen atom and two electrons. The elec- trons are measured by an ammeter (A).

ing the operation of the FIA system, for example, by xide poisoning. This enzyme catalyzes a reaction that equipping it with on-line dialysis and segmented sample transfers the phosphate group of creaflne phosphate to injection [11o]. A similar study used segmented sample ADP. The ATP produced in this reaction can be used injection, membrane-deposited reagent and membranes in the conversion of glucose into glucose-6-phosphate of low permeability. This system was used to determine (G-6-P), catalyzed by hexokinase. The G-6-P is further L-lactate and crea~_ine in addition to glucose in whole converted to gluconolactone-6-P by G-6-P dehydroge- blood samples [12]. Other studies have tried to elimi- nase and the coenzyme NADP. The reduced form of nate interference [13] or to evaluate the amperometric NADP (NADPH) produced in this reaction can then be determination of glucose in blood serum or whole blood determined spectrophotometrically or spectrofluorimet- [141. rically. In accordance with this reaction sequence, an im- As a result of these studies, application of biosensing FIA mobiliT.ed enzyme column containing hexokinase and G- systems in the determination of glucose concentration 6-P dehydrogenase, in conjunction with an FIA system, for clinical purposes seems very attractive. The enzyme was used to determine the activity of creatine kinase in system can be immobiliTed, packed as a column and serum samples [16o]. The activity of two other enzymes connected to the FIA system at a point before the sample that are valuable indicators of health, glutamate pyruvate passes through the detector, or immobiliTed directly onto aminotransferase [ 17] and lactate dehydrogenase [ 18], the working surface of an electrode. In both cases, the can also be determined using procedures that incorpo- reusability of the immobiliTed enzyme system effectively rate enzymatic reactions in series. reduces operation costs by decreasing the consumption In most cases, the quantification of FIA operation is usu- of enzyme. Other merits are the fast response time and ally calculated by a univadate calibration based on the the ability to alter the sample solution to meet the con- height or the area of the peak. For very complex bio- ditions optimal for the enzyme system so that longer op- logical samples with many interfering materials, however, eration periods are possible. a multivariate calibration system is more convenient. A photodiode array detector was applied to produce such a multivariate signal and this system was used to determine teniposide, an antitumor agent, in blood serum [19]. Other clinical applications The components of urine can also be determined by a Analysis systems composed of a biosensor and an FIA biosensing FIA system. Oxalate, a marker of calculosis, system have many other applications in clinical analysis. has been determined by a kinetic-enzymatic procedure The measurement of serum cholesterol, believed to be an [20]. The enzymes used in this analysis include oxalate important factor in coronary heart disease, has become a decarboxylase and formate dehydrogenase; the former routine diagnostic analysis. An FIA system for determin- catalyzes the decarboxylation reaction of oxalate to pro- ing serum cholesterol was designed using immobilized duce formic acid, which is then converted to carboxylic cholesterol esterase and cholesterol oxidase [15]. An acid by the latter. NADH is formed in this reaction and anion exchange resin was used to remove materials caus- monitored spectrophotometrically at 340 nm. ing interference prior to analysis in order to increase ac- Although NADH itself is detectable by photometric or curacy and precision. The accuracy of the results con- fluorimetric methods, the application of NADH oxidase formed to the guidelines of the national cholesterol ed- to convert NADH into NAD + and produce hydrogen per- ucation program of Canada. oxide in the same reaction has been reported as a more An increase in the activity of serum creatine kinase is sensitive way of determining NADH [21 ]. Higher sensitiv- a valuable indicator for some health disorders includ- ity was obtained by mixing the hydrogen peroxide with ing myocardial infarction, cerebral diseases, Duchenne's luminol and potassium ferricyanide and measuring the muscular dystrophy, Reye's syndrome and carbon mono- emitted chemiluminescence with a photon counter. Us- 34 Analyticalbiotechnology

ing this method, the detection limit of NADH is 10pmol. cephalosporinase onto the gate of a pH-sensitive field- It is possible to link this system to any NAD-dependent effect transistor. These bio-field-effect transistors for peni- reaction and it has been used to determine magnesium cillin G, penicillin V and cephalosporin C are usable for in human serum by linking it to hexokinase and G-6-P up to 3 months, 4 months and I-2 days, respectively. dehydrogenase catalyzed reactions. Other drugs including tetracycline, chlortetracycline, Techniques for determining several substrates simultane- oxytetracycline, doxycycline and methacycline have been ously have also been investigated by using an arrange- determined by FIA with a spectrophotometric detector ment of several immobilized enzyme columns in an FIA [28] or a flow-through sensor [29]. The operation rate system. Glucose, ethanol and lactate in serum samples of the latter was emphasized as one hundred samples were measured simultaneously by a paraUel configura- per hour. Berberine and benzethonium in drugs have tion of three columns containing immobilized glucose been determined spectrophotometricaUy by batchwise oxidase, alcohol oxidase and lactate oxidase [22..]. A and flow injection methods after a solvent extraction pro- 16-way switching valve was designed and adopted as the cess [30]. sample injection port for this system. An ascorbate oxi- dase column was used to pretreat the sample and a uric- ase column was connected before the immobilized en- zyme columns to eliminate interference from ascorbic Applications in fermentation processes, the acid and uric acid. A schematic diagram of the multichan- food industry and agricultural products nel FIA system is shown in Fig. 4. Other clinically interesting analyses have been performed Fermentation monitoring, as discussed further by Raju such as the determination of the concentration of bile and Cooney (this issue, pp 40--44), is an essential part of acids in serum, which has been measured by a fluori- bioprocess control. A schematic diagram for connecting metric enzymatic FIA system [23] and a combination of fermenters with an FIA system is illustrated in Fig. 5. In FIA and liquid chromatographic processes [24]. Amino this diagram, a recycling sampling system composed of acids in serum have also been determined: L-alanine was a pump and a filter is shown. This design is suitable for measured using a packed-bed enzyme reactor compris- a small jar-fermenter only. For a big fermenter for large- ing alanine dehydrogenase immobilized on poly(vinyl al- scale production (e.g. 300 M3 ton capacity) a sampling cohol) beads [25]. Trace amounts of thiocyanate in saliva rate of a few milliliters every minute makes no difference samples were measured by a kinetics spectrophotometric to the final volume. Therefore, there is no reason to take assay of a flow injection system [26]. the risk of contamination by recycling the sample solu- tion. The dialyzer may be unnecessary if the flow system is designed to flush the surface of the sensor effectively or ff the surface of the sensor is covered by materials Determination of drug components that effectively obviate the adhesion of components of the medium. Bio-field-effect transistors combined with an FIA system Glucose is still the major compound of interest in the have been used to determine penicillin G, penicillin V field of fermentation. A chemically modified graphite elec- and cephalosporin C concentrations during a fermenta- trode with immobilized glucose dehydrogenase was used tion process [27] and gave results that matched well with as the detector in an FIA system to determine glucose off-line liquid chromatographic data. This was achieved concentration in wine production [31o]. This system was by immobilizing penicillin G amidase, peniciUinase and reportedly used for 3 days of continuous operation with

Micro-tubular Air pump damper I I Bifer t i i I ~j Potentiostat Sample--] ecH~order

16-way Fig. 4. Schematic diagram of a mul- sWjta~hing ~Jy~ Waste tichannel flow injection analysis sys- tem. I, lactate reactor; II, glucose reac- tor; III, ethanol reactor; IV, urate-elim- ~ Flow-cell mating reactors; V, ascorbate-eliminat- ing reactor. Buffer A, phosphate (0.1M, Waste pH8.0); buffers B and C, phosphate (0.1 M, pH 7.0). Biosensors and flow injection analysis Chen and Karube 35

Pump ' ~ ~

,,er l I

Fermenter

=- Waste ste.~~~Dia Sample Wa lyzer t!~ ..... I_t Pump

Buffer Injector Ca I ---~ Waste Liquid- Glucose l chromatography sensor pump

Waste Fig. 5. Incorporationof a fermenterwith a flow injection analysissystem. no interference from alcohol observed. Another FIA sys- pyruvate aminotransferase. Using this method, sensitivity tem using a double injection process to measure glucose is increased 20--30-fold and concentrations of L-glutamate in a fermentation broth has also been reported [32]. The as low as 1 picomolar could be detected. L-alanine in sample solution and enzyme solution were injected in- serum and beverages can be determined by FIA with a dividually into a carrier stream. The hydrogen peroxide fluorimetric detector [25]. Some other amino acids in produced after passing a reaction coil was measured am- cheese have been determined chemiluminescently by FIA perometrically. [36]. Amino acids are another important class of compounds Aspartame is an important artificial sweetener composed in fermentation. L-lysine is an important essential amino of aspartic acid and phenyialanine. The'concentration of acid. Commercially, it is mainly produced by fermentation this dipeptide can be determined by FIA using two en- and used as an additive in fodder. The concentration zyme columns containing peptidase and aspartate amino- of L-lysine in fermentation broths and fodder concen- transferase, respectively, and an enzyme sensor with im- trate was determined with high specificity by a combi- mobilized L-glutamate oxidase [37]. Aspartame is hydro- nation of an immobilized L-lysine monooxygenase col- tyzed into aspartic acid and phenylalanine by the pepti- umn, a dissolved oxygen probe and an FIA system [33]. dase. Aspartic acid is then converted into glutamic acid L-lysine was also measured using the same FIA and im- by aspartate aminotransferase and the product, L-glutamic mobilized enzyme column, but with a different detection acid, can then be determined by the glutamate sensor. system: the hydrogen peroxide produced reacted with L-ascorbic acid is also an analyte that has attracted much peroxidase, phenol and 4-amino antipyrine to produce attention. L-ascorbate oxidase converts ascorbate into de- a red quinoneimine dye which was detected spectropho- hydroascorbate. The hydrogen peroxide produced at the tometfically at 500 nm [34]. t-glutamic acid, an important same time is detectable using a hydrogen-peroxide-sens- amino acid used to prepare monosodium L-glutamate, ing electrode. Ascorbate itself, however, is electroactive at which is widely used in the food industry was measured, the potential used to detect hydrogen peroxide. A system was by an amplification system based on substrate recy- for ascorbate determination using FIA, an immobilized cling [35"']. L-glutamate was converted to 2- oxoglutarate L-ascorbate oxidase column and a blank blank column by L-glutamate oxidase. The glutarate produced was sub- with no enzyme was designed. On passing through the sequently converted back to L-glutamate by glutamate enzyme column some of the L-ascorbic acid was con- 36 Analyticalbiotechnology

vet'ted into the dehydro form and the decrease in re- phosphorylase and alkaline phosphatase is co-immobi- sponse (measured against the blank column) was used lized along with xanthine oxidase. This amplification sys- to calculate the concentration of ascorbate [38]. Another tem increases sensitivity up to 12-fold. system using cucumber juice as the carrier solution has also been used to determine ascorbate [39]. In still an- other system, measurement of the total ascorbic acid con- centration has been reported using kinetic fluorimetric Applications to microbiological and enzymatic FIA and two serial injection valves [40]. reactions Many carbohydrates have also been determined by FIA coupled to a biosensing system. In addition to fermen- The activities of many enzymes have been determined us- tation broths (described above, [31"]), systems for mea- ing biosensing FIA systems, often by incorporating addi- suring the concentration of glucose in milk [41] and al- tional enzymes to convert the product formed by analyte coholic beverages [22..] have also been described. In catalysis into a detectable material. Two examples of this, contrast with glucose oxidase, which oxidizes the C-1 as mentioned above, are glutamate pyruvate transaminase hydroxyl group of [3-D-glucose, pyranose oxidase oxi- (an indicative enzyme of liver function) [17] and lactate dizes the C-2 hydroxyl group of the pyranose ring in dehydrogenase. The activity of lactate dehydrogenase can hexoses and pentoses. This enzyme has been used in be determined using an immobilized pyruvate oxidase the determination of monosaccharides including glucose, membrane on an oxygen probe in an FIA system [47]. xytose and galactose produced during the hydrolysis of Another system that can detemline the activities of lac- cellulosic materials [42°]. This system can give a relative tate dehydrogenase and glutamate pyruvate transaminase measurement of the total pyranose concentration in a simultaneously was developed by the same group and is sample of crude broth or an absolute value of an individ- based on measuring the pyruvate produced with pyruvate ual substrate in a sample without competing analytes. In oxidase immobilized on an oxygen probe [48]. Guanase another study, the determination of starch by FIA and a activity has been determined by measuring the hydrogen multi-enzyme system was reported [43°]. Amyloglucosi- peroxide produced in the analyte-catalyzed reaction in dase, mutarotase and glucose oxidase were co-immobi- conjunction with xanthineoxidase and uricase [49]. The lized on a nylon membzmae and bound to a platinum FIA system used consisted of series-injection, stopped- electrode. The concentration of starch in solution, when flow and merging-zone methods. Another multi-enzyme pretreated with at-amylase at room temperature for one FIA system, for determining the activity of creatine kinase hour, was determined by measuring the hydrogen per- in serum samples by photometric and fluorimetric detec- oxide produced. In one recent report, an immobilized tion has also been reported [50]. mannitol dehydrogenase column combined with FIA was Microorganisms, bioreactions of microorganisms and cell used to detect n-mannitol (e.g. in celery and chewing components can also be monitored using biosensing gum) [44] and the NADH produced in this enzymatic FIA systems. The presence of Salmonella in food prepa- reaction was measured fluorimetrically. rations causes severe food poisoning. To check for Salmonella in food products by the traditional method is time consuming, cosdy and often ditticuk because it is only present at very low concentrations. An FIA immuno- Applications in environmental protection assay system has been developed [51 °° ] for the purpose of detecting microorganisms belonging to this genus. A Environmental protection has become the focus of much sandwich system composed of antibody, microorganism attention in recent years. FIA systems equipped with and glucose oxidase labeled antibody was constructed biosensors have been used to measure indicators of en- by immobilizing the antibody onto the inner wall of a Ty- vironmental pollution, such as pesticides and herbicides. gon tube through which the sample solution and enzyme Paraoxon, an organophosphorus insecticide with a wide labeled antibody solution were pumped in succession. range of activity that is very toxic to mammals, was de- Finally, a glucose solution was pumped through the tube termined by both continuous flow and stopped flow sys- and hydrogen peroxide determined electrochemically. tems in combination with an immobilized cholinesterase Another system, combining FIA with a membrane intro- column. As an organophosphorus compound, paraoxon duction tandem mass spectrometric system, was used to can form a stable complex with cholinesterase and in- monitor liquid and gas components during the produc- hibit its activity. Paraoxon determinations were made at tion of 2,3-butanediol by Bacillus polymyxa or Kletr a rate of 60 per hour [45"] on the basis of its ability siella oxytocdn these microorganisms [52°°]. Although to inhibit enzymatic hydrolysis of ~-naphthyl acetate and this study did not use a biosensor for measuring the an- the subsequent reaction of the product, 0t-naphthol, with alytes, we have included it because it was used to monitor p-nitro-benzenediazonium fluoroborate. a fermentation process. The concentration of phosphate in water is considered to An FIA system incorporating a fibre-optic biosensor to be another important indicator of pollution. An amper- determine ATP and NADH has been designed [53°°]. ometric FIA system with an immobilized enzyme reactor Three enzymes, firefly lucfferase, bacterial oxidoreduc- has been designed for determining phosphate in water tase and bacterial lucfferase are co-immobilized onto a samples [46"]. In the enzyme reactor, an amplification polyamide membrane. The first enzyme is used to meas- system for substrate recycling using purine nucleoside ure ATP and the other two enzymes to measure NADH. Biosensors and flow injection analysis Chen and Karube 37

By changing the reagents, ATP or NADH can be deter- References and recommended reading mined with the same sensing element. The activity of enzymes that produce ATP or NADH in their catalyzing Papers of particular interest, published within the annual period of re reactions, for example kinase and dehydrogenase, can be vaew, have been highlighted as: detemained by this system. • of special interest e. of outstanding interest 1. CLARK LC, LYONS C: Electrode Systems for Continuous Moo- itoring in Cardiovascular Surgery. Ann NY Acad Sci 1962, Other applications 102:29-45 2 FAN S, FANG Z: Compensation of Calibration Graph Curva- ture and Interference in Flow-injection Spectrophotome- A flow-injection biosensing system has been constructed try Using Gradient Ratio Calibration. Anal Cbim Acts 1990, using an immobilized alkaline phosphatase column, a 241:15-22 planar pH-sensitive field effect transistor and a flow in- 3 KOLEV SP, NAGY G, PUNGOR E: Mathematical Modeling of jection system. This system was used to determine the Single-layer Flow-injection Analysis System with Single-layer concentration of zinc(II) ions by activation of the immo- Enzyme Electrode Detection. Part 1. Development of the bilized cofactor-free apoenzyme in the column [54o.]. A Mathematical Model. Anal Chim Acta 1990, 241:43-53. chelating agent, 2,6-pyridine dicarboxyiate, was injected 4. KOLEVSP, NAGY G, PUNGOR E: Mathematical Modeling of between successive samples to regenerate the column. Single-layer Flow-injection Analysis System with Single-layer Enzyme Electrode Detection. Part II. Stimulation of the Two immuno-FIA systems have been developed for de- Mathematical Model. Anal Chim Acta 1990, 241:55-69. termining methamphetamine [55"], a drug that is often abused, and ttinitrotoluene (TNT) [56], an organic ex- 5. CHEN D, ZENG Y: Processing and Error Analysis of Signals in Flow-injection Analysis. Anal Chim Acta 1990, 235:337-342. plosive. The latter involves mixing TNT with anti-TNT and determining the decrease of fluorescence of anti-TNT 6. GUNASINGHAMH, TAN CH, AW TC: Comparative Study of e. lst-Generation, 2rid-Generation and 3rd-Generation Am- after mixing. The former incorporates immobilized al- perometric Glucose Enzyme Electrode in Continuous-flow bumin conjugated to methamphetamine on the surface Analysis of Undiluted Whole Blood. Anal Chim Acta 1990, of an AT-cut quartz crystal. The decrease of the reso- 234:321-330. nant frequency of the crystal after contacting a solution The three generations of electrodes are described, and the advantages of methamphetamine and monoclonal antibody against and disadvantages of each in the analysis of biological samples are dis- cussed. Second generation sensors seem best. methamphetamine is measured, with the frequency de- crease correlating with the concentration of anti-metham- 7. INMANDJ, HORNLY WE:The Immobilization of Enzymes on phetamine in the flowing solution. At a fixed concentra- Nylon Structures and Their use in Automated Analysis. BiocJxrm J 1972,129.255-262 tion of anti-methamphetamine, the frequency decrease was found to be attenuated by the concentration of 8 ALVES DA SILVA M, HELENA GIL M, REDINI-1AJS, OLIVEIRABRETr AM, COSTA PEREIRAJL: Immobilization of Glucose Oxidase methamphetamine that was present in the solution. Con- on Nylon Membranes and its Application in a Flow-through centrations of methamphetamine as low as 0.02 ppm can Glucose Reactor. J Poly Sci [A] 1991, 29:275-279. be detected in about 5 min. 9 TRojANow]cz M, MATSUZEWSK~W, PODStADLA M: Enzyme En- • trapped Polypyrrole Modified Electrode for Flow-injection Determination of Glucose. Biosensors Bioelectronics 1990, 5:149-156. Conclusion Using electropolymerized polypyrrole films, enzymes can be immobi- lized onto the surface of electrodes of various shapes. A very thin mem- brane with a very fast response time can be created. The papers cited in this review demonstrate the wide ap- plicability of a combined biosensing FIA system to many 10. GEISERJ, ADAMSJM, BARONE NJ, YACYNYCH AM: Electropoly- merized Films to Prevent Interferences and Electrode Foul- fields including clinical diagnosis, the food industry, agri- ing in Biosensors. Biosensors Bioelectronics 1991, 6:151-160. cultural products and environmental control. The range and number of these systems is expected to increase as 11 BUCH-RASMUSSENT: Determination of D-Glucose in Undiluted • Whole Blood Using Chemically Modified Electrodes and immobilization, flow injection and detection techniques Segmented Sample Injection in a Flow System. Anal Cbzm improve. To analyze a biomaterial using traditional meth- Acta 1990, 237:405--411. ods may be time consuming and even then the data may When analyzing whole blood samples, mterference and fouling by the be of little value because the result is obtained hours or blood cells needs to be taken into consideration. This paper describes an FIA system with segmented sample injection and on-line dialysis to perhaps days after the sample was obtained. The acqui- decrease dependence on the volume of the red blood cell fraction. sition of real-time, or almost-real-time, data is the most important advantage of biosensing FIA systems. Increas- 12. BUCH-RASMUSSENT: Flow System for Direct Determination of Enzyme Substrate in Undiluted Whole Blood. Anal Chem ing use of this kind of system may be expected in the 1990, 62:932-936. future. 13. MATSUZEWSmW, TROJANOWICZ M, LEWENSTAM A: Elin~tion of Interference in Flow-Injection Amperometric Determi- nation of Glucose in Blood Serum Using Immobilized Glu- cose Oxidase. Electroanalysis 1990, 2:607-610 Acknowledgement 14 GUNASINGHAMH, TAN CH, AW TC. Clinical Evaluation of Am- perometric Enzyme Electrodes in Continuous-flow Analysis The authors would like to express their appreciation to Dr Robert for Glucose in Undiluted XVhole Blood. Clin CJmm 1990, W'dson for helpful discussions and assistance with the English. 36:1657-1661. 38 Analytical biotechnology

15. CARPENTERA, PURDYWC: The Determination of Total Serum 30 SAKAI T: Solvent Extracfion-Spectrophotometric Determina- Cholesterol by Flow-injection Analysis with Amperometric tion of Berberine and Benzethonium in Drugs with Tetra- bromophenolphthalein Ethyl Ester by Batchwise and Flow Detection. Anal Lett 1990, 23:425--435. Injection Methods. Analyst 1991, 116:187-190. 16. FERNANDEZ-ROMEROJM, LUQUE DE CASTRO MD: Determination • of Creatine Kinase Activity UsIng a Co-immobiilzed Auxil- 31. APPELQVIST R, HANSEN EH: Determination of Glucose in iary Enzyme Reactor Coupled On-line with a Flow-injection . Fermentation Processes by Means of an On-line Cou- System. Analyst 1991, 116:167-170. pled Flow-injection System Using Enzyme Sensors Based The operation of multi-enzyme systems can be simplified by choosing on ChemicaRy Modified Electrodes. Anal Chim Acta 1990, enzymes that function in similar reaction conditions and co-tmmobihz- 235:265-271. ing them in the same reactor. In this study an example is described for This paper outlines the standard model of a biosensing FIA system for determining creatine kinase activity. monitoring fermentation. 17. FERNANDEZ-ROMEROJM, LUQUE DE CASTRO MD, VALCARCEL 32. WEN XW, KIPTON H, POWELL J, CHRISTIAN GD, RUZlCKA J: M: Determination of Alanine Aminotransferase in Human Double Injection Enzymatic Flow Analysis of Glucose Using Serum in an Open--Closed Flow Injection Configuration. J Amperometric Detection and an Oxygen-permeable Reac- Biotech 1990, 14:43-52. tion Coil. Anal CAn'm Acta 1991, 249:451-460. 18. TABATAM, TOTANI M, MURACm T: Determination of Lac- 33. SIMONIANAL, KHACHATRIAN GE, TATIK1AN SSH, AVAKIAN TSM, tate and Lactate Dehydrogenase Activity In Serum with the BADALIAN IE: A Flow-through Enzyme Analyzer for Determi- Flow Injection Analysis System Involving Immobilized En- nation of L-Lysine Concentration. Biosensors Bioelectronics zyme Column and Chemiluminescence. Anal Bic~em 1991, 1991, 6:93-99. 193:112-117. 34. POHI~ANNA, STAMM WW, KUSAKABEH, KULA MR: Enzymatic 19. GERR1TSENMJP, KATEMANG. Flow-Injection System with Pho- Determination of L-Lysine by Flow-Injection Techniques. todiode Array Detection and Multivariate Data Evaluation Anal Chim Acta 1990, 235:329-335. for Quantifying Teniposide in Blood Plasma. Anal Chim Acta I990, 241:23-50. 35. YAO T, KOBAYASH1N, WASA T: Amperometric Flow-Injection ., Analysis of L-Glutamate Using an Immobilized Enzyme Re- 20. INFANTESJA, LUQUE DE CASTRO MD, VAtC~CEL M: Kinetic-en- action Amplification by Substrate Recycling. Electroanalysis zymatic Determination of Oxalate in Urine by Flow-injec- 1990, 2:563-566. tion Analysis with Double-stopped Flow. Anal Ooim Acta The response intensity of an analysis system could be amplified up to 1991, 242:179-183. 30-fold by incorporate an enzyme that can convert the product of the reaction back to the substrate. This paper describes an example of this 21. MURACHIT, TOTANI M, IKEMODO M, TABATA M: A Flow In- whereby glutarate is converted back to L-glutamate by glutamate pyru- jection Analysis System Involving Immobilized NADH Oxi- vate aminotransferase. dase in Column Form for Clinical Analysis. Or Biotech 1990, 14:33-42. 36. PUCHADES1L LEMIEUX L, S~t~O RE: Determination of Free Amino Acids in Cheese by Flow Injection Analysis with an 22. MATSUMOTOK, MATSUBARAH, HAMADAM, UKEDAH, OSAJIMAY: Enzymic Reactor and Chemiluminescence Detector. J Food • . Simulmeous Determination of Glucose, Ethanol and Lac- Sci 1991, 55:1555-1558. tate in Alcoholic Beverages and Serum by Amperometric Flow Injection Analysis with Immobilized Enzyme Reactors. 37. MALEKB, LUONG JHT, MULCHANDAN] A: Determination of J Biotech 1990, 14:115-126. Aspartame in Dietary Food Products by a FIA Biosensor. A parallel configuration of immobilized enzyme columns was used to Biosensors Bioelectronics 1991, 6:117-123. determine several components in the same sample simultaneously This procedure, though not a new one, is still a useful method for simulta- 38. GREENWAYGM, ONGOMO P: Determination of L-ASCorbicAcid neous determination of multiple analytes. in Fruit and Vegetable Juices by Flow Injection with Im- mobilized Ascorbate Oxidase. Analyst 1990, 115:1297-1299. 23. MEMBmLAA, LAZARO F, LUQUE DE CASTRO MD, VALCARCEL M: Fluorimetric Enzymatic Flow-Injection Determination of 39. UCHIYAMAS, SUZUK~S: Flow-injection Determination of Total Bile Acids in Human Serum. Fresenius J Anal Chem 1990, Vitamin C Using Cucmber Juice Carrier. Bunseki Kagaku 338:749-751. 1990, 39:793-797. 24. MEMBIELAA, LAZARO F, LUQUE DE CASTRO MD, VALCARCELM: 40. CHUNGHK, INGLEJD JR: Kinetic Fluorometric FIA Determi- Alternative Use of Flow-injection Analysis and the Combi- nation of Total Aseorbic Acid, Based on Use of Two Serial nation of Liquid Chromatography and Flow-injection Anal- Injection Valves. Talanta 1991, 38:355-357. ysis for the Determination of Total and Individual Bile Acid 41 PILLOTONP,, MASCINIM: Flow Analysis of Lactose and Glucose Concentration In Serum. Anal Chim Acta 1991, 249:461-467 in Milk with an Improved Electrochemical Biosensor. Food 25. KIBA N, TAGAMI H, FURUSAWA M: Determination of L-Alanhle O0em 1990, 36:213-222. in a Flow-injection System with an Immobilized Enzyme 42. OLSSONI, MANDENIUS CF, VOLC J: Determination of Mono- Reactor. Anal CAnm Acta 1990, 239:307-310. • saccharides in Cellulosic HydrolyzateS Using Immobilized 26. ANDERSBROE B, ELO ~ H: Spectrophotometric Flow Pyranose Oxidase in a Continuous Amperometric Analyzer. Injection Determination of Trace Amounts of Thiocyanate Anal Chem 1990, 62:2688-2691. Based on Reaction with 2-(5-bromo-2-pyridylazo)-5Miethyl- Cellulosic hydrolyzates contain several kinds of fermentable sugars. aminophenol and Dichromate: Assay of the Thiocyatmte Le- Pyranose oxidase, the enzyme used m this case, can be used to de- vel in Saliva from Smokers and Non-smokers. Analyst 1991, termine a variety of monosaccharides in addition to glucose. 116:647-651. 43. HAMIDJA, MOODY GJ, THOMAS JDR: Multi-enzyme Electrodes 27. BRANDU, REINHARDT B, RUTHER F, SCHEPER T, SCHUGERL K' . for the Determination of Starch by Flow Injection. Analyst Bio-fieid-Effect Transistors as Detectors In Flow Injection 1990, 115:1289-1295. Analysis. Anal Cbim Acta 1990, 238:201-210. Enzymic biosensing systems are rarely used to determine complex molecules such as starch. This paper describes one such study in 28. ALWARTHANAA, AL-TAMRAH SA, SULTAN SM: Spectrophotomet- which pre-hydrolysis was used to obtain a measurable response from a ric Determination of Oxytetracycline by Flow Injection. An- multienzyme system. alyst 1991, 116:183-186. 44. KIBA N, 1NOUE Y, FURUSAWA M: Flow-injection Determina- 29. LIu WZ, J1ANG H, DU AZ: Determination of Tetracyclines by tion of D-Manitol with Immobilized Mannitol Dehydroge- Flow Injection Analysis. Yaoxue Xuebao 1991, 26:391-394 nase. Anal Chim Acta 1991, 244:105-107. Biosensors and flow injection analysis Chen and Karube 39

45. LEON-GONTALEZME, TOWNSHENI) A: Flow-injection Determi- 52. HAYWARDMJ, KOTIAHO T, LISTER AK, COOKS RG, AUSTIN GD, • nation of Paraoxon by Inhibition of Immobilized Acetyl- • o NARAYNR, TSAO GT: On-line Monitoring of Bioreactions of cholinesterase. Anal Cbim Acta 1990, 236:267-272. Bacillus polymyxa and Klebsiella oxytoca by Membrane Toxic materials can be determined by virtue of their ability to inhibit Introduction Tandem Mass Spectrometry with Flow Injec- the activity of biomaterials. In this case paraoxon was determined by its tion Analysis Sampling. Anal Chem 1990, 62:1798-1804. inhibition of cholinesterase catalyzed hydrolysis of at-naphthyt acetate. An outstanding method that can monitor major products and volatile This method has potential to measure toxic materials using living or- metabolites during a fermentation process. gai~$ms. 53. GAUTIERSM, BLUM LJ, COULET PR: Multi-function Fibre-optic 46. YAO T, KOBAYASHIN, WASA T: Amperometric Flow-injection ,. Sensor and the Bioluminescent Flow Determination of ATP • System with an Immobilized Enzyme Factor for the Highly or NADH. Anal Cbim Acta 1990, 235:243-253. Selective Detection of Phosphate and On-line Amplification Two enzymatic systems for measuring different analytes were by Substrate Recycling. Anal OMm Acta 1990, 238:339-343. co-immobilized on the same membrane to construct the sensing unit. A system for detecting phosphate in water samples and for increasing By changing the reagents and reaction conditions, analytes can be se- sensitivity to it by substrate amplification is described. lectively determined. 47. OKUMAH, SEK~CdKAI S, HOSHI M, TOYAMA K, WATANABE E: 54. SATOH I, Aom Y' Biosensing of Zinc (I1) Ions Using Biosensor System for Continuous Flow Determination of o• an Apoenzyme Reactor and an ISFET Detector in Flow Enzyme Activities. I. Determination of Glucose Oxidase Streams. Denki Kagaku 1990, 58:1114-1118. and Lactic Dehydrogenase Activities. Enzyme Microb Tec2r A very clever idea for measuring the concentration of metallic ions is nol 1989, 11:824-829 described in which determination relies on activation of an apoenzyme 48. OKUMAH, TAKAHASm H, SEKIMUK~d S, WATANABE E: Biosen- by binding the analyte as cofactor. sor System for Continuous Flow Determination of Enzyme Activities. II. Simultaneous Determination of Plural Enzyme 55. MIURAN, NIGOBASHIH, TAKEYASUA, UDA T, YAMAZOEN: Piezo- Activities. Enzyme Microb T~ol 1991, 13:134-138. • electric Crystal Immunosensor for Sensitive Detection of Methamphetamine. Chemical Sensor 1991, 7:53-56. 49. TOMODAM, UCHIDA K, HIGUCH1 N, SAIl K, SAITO S: Flow In- This study is a good example of applying immuno-FIA to the detertrn- jection Analysis of Guanase Activity. J Flow Injection Anal nation of drugs of abuse. 1990, 7:113-129. 56 NAKAYAMAH, SUGIHARAH, TAKAHASHIK, MIYAZAKIJ, MITSUMATA 50. LUQUE DE CASTRO MD, FERNANDEZ-ROMEROJM: Photomet- T: The Development of a Sensor for Explosives Using an tic and Fluorimetric Determination of Creatine Kinase Immune Reaction. ~ical Sensor 1991, 7:57--60. Activity by Using Co-immobilized AuTiliary Enzymes and an Open/Close Flow Injection Manifold. Anal Lett 1991, 24:749-765. 51. LUONGJHT, PRUSAK-SOCHACza~WSmE: Development of a Flow C-Y Chert, Research Center for Advanced Science and Technology, Uni- • . Immunoassay System for the Detection of Salmonella ty versity of Tokyo, 4-6-1, Komaba, Meguro-Ku, Tokyo, Japan and Depart- phtmurlum. Anal Lett 1990, 23:1809-1826. A sandwich type immunoassay system set up by immobilizing antibod- ment of Agricultural Chemistry, National Taiwan University, 1, Sec. 4, ies raised against S. Ophimurium on the wall of a Tygon tube was used Roosevelt Road, Taipei, Taiwan. to detect microorganisms of this species. This immuno FIA system was I Karube, Research Center for Advanced Science and Technology, Uni- reused more than 50 tLmes. versity of Tokyo, 4-6-1, Komaba, Meguro-Ku, Tokyo, Japan.