02-02-2013, 09:39 AM
GENERAL REQUIREMENTS FOR THE PRACTICAL USE OF ENZYME BIOSENSORS IN FOOD ANALYSIS
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INTRODUCTION
Over the last decade, a variety of biosensors based on elegant concepts were discovered .In most cases, these highly attractive analytical tools result from the intimate combination of a transducer and an immobilized enzyme layer. Electrochemical sensors for oxygen or hydrogen peroxide detection or involving a mediator are the most common transducers used for the design so called enzyme electrode. Practically, B-D-glucose and L-lactate are the two main analytes for which instruments based on this technology are available on the world market and which are routinely used in bioprocesses. The special attention was given to the preparation of the enzyme layer. Electrochemical could be achieved using pre-activated polyamide membranes enabling enzyme immobilization in a few minutes. The electrochemical based sensors then developed allowing the determination of glucose, lactate, phosphate etc. with the suitable enzymes. Assays could be performed in various foodstuffs including wine, honey, dairy products etc. more recently, a fibre optic-based enzyme sensor with enzymes producing luminescence from fireflies for adenosine triphosphate (ATP) or from luminescent bacteria for the reduced form of nicotinamide adenine dinucleotide phosphate (NAD(P)H) was designed. Auxiliary enzymes, namely dehydrogenases co-immobilized with the bacterial luminescent system allowing different metabolites to be determined, like sorbitol, ethanol or oxaloacetate. Enzyme activity could also be assayed with lactate dehydrogenase as model enzyme in cell culture processes. Both type of transduction lead to very low detection limits and wide dynamic ranges. The fact that generally no tedious pre-treatment of the sample is required makes such system particularly attractive for food analysis.
ENZYME IMMOBILIZATION
The properties and characteristics of the bio sensing layer are extremely important in the design of biosensors, especially for the reliability versus time since they are repeatedly in contact with the sample. Two main categories of layers can be distinguished: pre-formed artificial membranes bearing functional groups, which can be activated and further coupled to selected enzyme or polymer matrices in which the enzyme molecules are trapped and which can be used for the direct coating of the transducer. Chemically activated collagen membrane were first used as pre formed membrane for enzyme immobilization.4 The activation was based on acyl –azide formation through a three step treatment of the carboxyl group of lateral chains of glutamate and aspartate residues available in collagen. After washing to remove the entire reagent which could be damaging for the enzymes, spontaneous coupling was achieved by simple immersion of the activated membrane in a freshly prepared enzyme solution. A variety of enzymes including, oxidases, transferases, hydrolases, or lyases were immobilized on such membranes. Despite good stability of the bound enzymes, the main drawback was the time required, more than three days, for the activation process which should be kept mild with regards to the proteinic nature of the support itself. ImmunodyneTM membrane from Pall, Glencove, USA provide in a preactivated form were selected for their handiness and good reproducibility of the immobilization procedure.5 these polyamide membrane are provided in a dry state in moisture proof bags. In briefly, enzyme immobilization can be obtained ass follows by 0.1M phosphate buffer, pH 7 is prepared and 10ul applied to both faces of the membrane. The reaction is completed within one minute. The enzymic membrane is rinsed in buffer and bioactive discs of a suitable size adapted to the electrode tip are cut of the membrane. The main advantage of the membrane is the extremely simple and fast procedure which enables enzymes to be immobilized in a few minutes without tedious and hazardous steps. The bio-active membrane can be stored either in buffer or in a dry form.
ENZYME ELECTRODES
This paper manly focused on oxidase based electrode or on oxidase –containing multi enzyme system when such sequence were needed if no oxidase could be found and used directly for the target analyte to be assayed . Two approaches can be considered either oxygen consumption is followed with a clark-type electrode, or hydrogen peroxide production is monitored with platinum electrode, both poised at the adequate potential. Among the two possibilities, monitoring the appearance of hydrogen peroxide was chosen (Figure 1).
SELECTIVITY
Selectivity is one key parameter to consider when thinking of the practical use of biosensors. Indeed, a high selectivity is needed for direct analysis. The selectivity of the sensor depends on both the specificity of the enzyme and on the presence of electroactive species in the sample. With glucose oxidase, the specificity of the enzyme is good and the case foe other enzymes, especially galactose oxidase and alcohol oxidase which accept several substrates or with D-amino acid oxidases active on various amino acids.
In complex media various contaminants which are electroactive at the poised pontential may exit in the sample; this is not only restricted to food analysis but also affects biochemical analysis or environmental control. An example of this is ascorbic acid which can be readily oxidized together with hydrogen at the above mentioned potential. Several ways have been considered by different authors to avoid such a drawback. In the approach developed by clark and his group, 7 the bioactive membranes used were obtained by sandwiching a glutaraldehyde-treated enzyme layer between a cellulose acetate membranes in contact with the platinum electrode is to act as a barrier for small molecules and to prevent oxidation of interfering species with hydrogen peroxide leading to an overestimated measurement.
RESPONSE TIME
For the self –contained enzyme electrode Gluc 1, the steady – state response time varies between 3 and 5 minutes. This time was considered too long for practical application of an analyzer and in this instrument the derivative of the current was monitored instead of the direct current. The result was then obtained on a digital display only 30 seconds after manual injection of the glucose – containing sample, but a 90 second interval between asssays was necessary to allow washing and filling of the measurement cell.
USE IN FOOD ANALYSIS
In the laboratory, tests were performed with either the self-contained electrode Gluc 1 or the semi-automatic analyzer GlucoprocesseurR equipped with collagen or polyamide membranes. Either single or, when necessary, multi-enzyme systems were tested. Results obtained with different target analytes are summarized below.
GLUCOSE: Determination of glucose in honey and jam could be performed with the enzyme electrode analyzer. With a series of 10 assays, excellent values of the coefficient of variation ranging from 0.7-1.7% obtained.5
GALACTOSE: A galactose electrode10 could be made with galactose oxidase but the enzyme is not specific for galactose and oxidizes other galactosides like lactose or raffinose. This restricts, in principle, the use of a galactose electrode to medium where such interferent sugars are not present.
LACTOSE: B-galactosidase hydrolyses lactose to produce glucose and galactose. The performance of this sensor were poor with a rather high detection limit and a narrow linear range.10
SUCROSE: Three enzymes were involved: invertase which hydrolyses sucrose into fructose and a-D-glucose, mutarotase converting a-D-glucose into B-D-glucose, and glucose oxidase. The linear range was narrow compared to glucose and the sensitivity low.10
LUMINESCENCE PHOTOBIOSENSOR
Based on experience in the development of enzyme electrodes, a fibre optic sensor was designed,14 based on luminescence enzyme from either the firefly for ATP or from marine bacteria for NAD(P)H detection. The enzymes were bound to pre-activated polyamide membranes associated through a screw –cap to one end of a fibre optic bundle connected to a luminometer (Figure 2). The bioactive tip was inserted in a light-proof measurement cell in which samples can be injected through a septum. The same set-up can be used whatever the light emitting system on the membrane.
CONCLUSION:
Attempts were made by research peoples to overcome identified bottlenecks impairing their use for practical application. Enzyme electrodes and luminescence photobiosensors were developed and several analytes important in food analysis could be reliably determined with such sensors in real samples. From the exprement, the constraints existing in the different domains where biosensors could be used as valuable tools, explain the difficulties encountered for their acceptance. The main problem may be the selectivity of the system and thus the confidence the user has in the values obtained. A second point concerns the lifetime of the bioactive membrane in complex media. Other points have to be carefully addressed when the design of a new instrument is planned.
[attachment=49307]
INTRODUCTION
Over the last decade, a variety of biosensors based on elegant concepts were discovered .In most cases, these highly attractive analytical tools result from the intimate combination of a transducer and an immobilized enzyme layer. Electrochemical sensors for oxygen or hydrogen peroxide detection or involving a mediator are the most common transducers used for the design so called enzyme electrode. Practically, B-D-glucose and L-lactate are the two main analytes for which instruments based on this technology are available on the world market and which are routinely used in bioprocesses. The special attention was given to the preparation of the enzyme layer. Electrochemical could be achieved using pre-activated polyamide membranes enabling enzyme immobilization in a few minutes. The electrochemical based sensors then developed allowing the determination of glucose, lactate, phosphate etc. with the suitable enzymes. Assays could be performed in various foodstuffs including wine, honey, dairy products etc. more recently, a fibre optic-based enzyme sensor with enzymes producing luminescence from fireflies for adenosine triphosphate (ATP) or from luminescent bacteria for the reduced form of nicotinamide adenine dinucleotide phosphate (NAD(P)H) was designed. Auxiliary enzymes, namely dehydrogenases co-immobilized with the bacterial luminescent system allowing different metabolites to be determined, like sorbitol, ethanol or oxaloacetate. Enzyme activity could also be assayed with lactate dehydrogenase as model enzyme in cell culture processes. Both type of transduction lead to very low detection limits and wide dynamic ranges. The fact that generally no tedious pre-treatment of the sample is required makes such system particularly attractive for food analysis.
ENZYME IMMOBILIZATION
The properties and characteristics of the bio sensing layer are extremely important in the design of biosensors, especially for the reliability versus time since they are repeatedly in contact with the sample. Two main categories of layers can be distinguished: pre-formed artificial membranes bearing functional groups, which can be activated and further coupled to selected enzyme or polymer matrices in which the enzyme molecules are trapped and which can be used for the direct coating of the transducer. Chemically activated collagen membrane were first used as pre formed membrane for enzyme immobilization.4 The activation was based on acyl –azide formation through a three step treatment of the carboxyl group of lateral chains of glutamate and aspartate residues available in collagen. After washing to remove the entire reagent which could be damaging for the enzymes, spontaneous coupling was achieved by simple immersion of the activated membrane in a freshly prepared enzyme solution. A variety of enzymes including, oxidases, transferases, hydrolases, or lyases were immobilized on such membranes. Despite good stability of the bound enzymes, the main drawback was the time required, more than three days, for the activation process which should be kept mild with regards to the proteinic nature of the support itself. ImmunodyneTM membrane from Pall, Glencove, USA provide in a preactivated form were selected for their handiness and good reproducibility of the immobilization procedure.5 these polyamide membrane are provided in a dry state in moisture proof bags. In briefly, enzyme immobilization can be obtained ass follows by 0.1M phosphate buffer, pH 7 is prepared and 10ul applied to both faces of the membrane. The reaction is completed within one minute. The enzymic membrane is rinsed in buffer and bioactive discs of a suitable size adapted to the electrode tip are cut of the membrane. The main advantage of the membrane is the extremely simple and fast procedure which enables enzymes to be immobilized in a few minutes without tedious and hazardous steps. The bio-active membrane can be stored either in buffer or in a dry form.
ENZYME ELECTRODES
This paper manly focused on oxidase based electrode or on oxidase –containing multi enzyme system when such sequence were needed if no oxidase could be found and used directly for the target analyte to be assayed . Two approaches can be considered either oxygen consumption is followed with a clark-type electrode, or hydrogen peroxide production is monitored with platinum electrode, both poised at the adequate potential. Among the two possibilities, monitoring the appearance of hydrogen peroxide was chosen (Figure 1).
SELECTIVITY
Selectivity is one key parameter to consider when thinking of the practical use of biosensors. Indeed, a high selectivity is needed for direct analysis. The selectivity of the sensor depends on both the specificity of the enzyme and on the presence of electroactive species in the sample. With glucose oxidase, the specificity of the enzyme is good and the case foe other enzymes, especially galactose oxidase and alcohol oxidase which accept several substrates or with D-amino acid oxidases active on various amino acids.
In complex media various contaminants which are electroactive at the poised pontential may exit in the sample; this is not only restricted to food analysis but also affects biochemical analysis or environmental control. An example of this is ascorbic acid which can be readily oxidized together with hydrogen at the above mentioned potential. Several ways have been considered by different authors to avoid such a drawback. In the approach developed by clark and his group, 7 the bioactive membranes used were obtained by sandwiching a glutaraldehyde-treated enzyme layer between a cellulose acetate membranes in contact with the platinum electrode is to act as a barrier for small molecules and to prevent oxidation of interfering species with hydrogen peroxide leading to an overestimated measurement.
RESPONSE TIME
For the self –contained enzyme electrode Gluc 1, the steady – state response time varies between 3 and 5 minutes. This time was considered too long for practical application of an analyzer and in this instrument the derivative of the current was monitored instead of the direct current. The result was then obtained on a digital display only 30 seconds after manual injection of the glucose – containing sample, but a 90 second interval between asssays was necessary to allow washing and filling of the measurement cell.
USE IN FOOD ANALYSIS
In the laboratory, tests were performed with either the self-contained electrode Gluc 1 or the semi-automatic analyzer GlucoprocesseurR equipped with collagen or polyamide membranes. Either single or, when necessary, multi-enzyme systems were tested. Results obtained with different target analytes are summarized below.
GLUCOSE: Determination of glucose in honey and jam could be performed with the enzyme electrode analyzer. With a series of 10 assays, excellent values of the coefficient of variation ranging from 0.7-1.7% obtained.5
GALACTOSE: A galactose electrode10 could be made with galactose oxidase but the enzyme is not specific for galactose and oxidizes other galactosides like lactose or raffinose. This restricts, in principle, the use of a galactose electrode to medium where such interferent sugars are not present.
LACTOSE: B-galactosidase hydrolyses lactose to produce glucose and galactose. The performance of this sensor were poor with a rather high detection limit and a narrow linear range.10
SUCROSE: Three enzymes were involved: invertase which hydrolyses sucrose into fructose and a-D-glucose, mutarotase converting a-D-glucose into B-D-glucose, and glucose oxidase. The linear range was narrow compared to glucose and the sensitivity low.10
LUMINESCENCE PHOTOBIOSENSOR
Based on experience in the development of enzyme electrodes, a fibre optic sensor was designed,14 based on luminescence enzyme from either the firefly for ATP or from marine bacteria for NAD(P)H detection. The enzymes were bound to pre-activated polyamide membranes associated through a screw –cap to one end of a fibre optic bundle connected to a luminometer (Figure 2). The bioactive tip was inserted in a light-proof measurement cell in which samples can be injected through a septum. The same set-up can be used whatever the light emitting system on the membrane.
CONCLUSION:
Attempts were made by research peoples to overcome identified bottlenecks impairing their use for practical application. Enzyme electrodes and luminescence photobiosensors were developed and several analytes important in food analysis could be reliably determined with such sensors in real samples. From the exprement, the constraints existing in the different domains where biosensors could be used as valuable tools, explain the difficulties encountered for their acceptance. The main problem may be the selectivity of the system and thus the confidence the user has in the values obtained. A second point concerns the lifetime of the bioactive membrane in complex media. Other points have to be carefully addressed when the design of a new instrument is planned.