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Biosensors and Flow Injection Analysis Chien-Yuan Chen and Isao Karube

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Biosensors and Flow Injection Analysis Chien-Yuan Chen and Isao Karube 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,
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