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The use of immobilized enzyme-membrane sandwich reactors in automated analysis
ANALYTICAL
BIOCHEMISTRY
83, 330-335
(1977)
The Use of Immobilized Enzyme-Membrane Reactors in Automated Analysis
Sandwich
Glucose oxidase from A. niger (EC 1.1.3.4) was physically entrapped between two dialysis membranes. The resultant enzyme-membrane sandwich reactors were employed in a standard Technicon dialyzer unit at 37°C and were found to perform efficiently in the automated analysis of aqueous glucose standards in the range 2.5-50 mg%. Over a lo-day period with intermittent usage, slight increases in the activities of the reactors were observed. The potential of such reactors in automated analysis is discussed.
The use of immobilized enzyme derivatives in automated analysis has been clearly established (l-3). Immobilized glucose oxidase, for example, has been utilized in the form of packed bed reactors (4,5), membrane reactors (5), and open tubular reactors (3,5) for the analysis of both aqueous glucose standards and serum glucose levels. In the case of the former, immobilized enzyme configuration, new types of analytical systems had to be evolved in order to utilize the insoluble catalyst efficiently (4). On the other hand, in the case of open tubular reactors and membrane reactors, standard Technicon AutoAnalyser equipment could be readily employed (l-3,5). Using the fairly sophisticated chemistries of immobilization available for the attachment of enzymes to the inside surface of nylon tube (6,7), it is usually possible to obtain adducts with practically significant activities. However, in the case of enzymes with low turnover numbers, the intrinsic low surface area available for the covalent attachment of protein may be a problem. Semipermeable microcapsule preparations (8,9) containing enzymes have a large surface area to volume relationship, and this type of immobilized enzyme has been shown to work efficiently in packed bed reactors. However, this would disturb the bubble segmented flow pattern used in standard Technicon AutoAnalysers to maintain sample integrity. In this work, a new approach using membrane sandwich reactors in automated analysis is described. EXPERIMENTAL
Type V glucose oxidase (Sigma Chemical Co.) with 1400 units/ml was used without further purification. Type C dialysis membranes and all automated analysis equipment were obtained from Technicon Corp., Tarrytown, New York. KI solutions were prepared immediately prior to 330 Copyright All rights
0 1977 by Academic Press. Inc. of reproduction in any form reserved.
ISSN 0003.2697
SHORT COMMUNICATIONS Dialyser
Donor
331
Top Plate
Stream
Enzyme
Layer
Recipient
Stream
Dialyser
Bottom
Plate
FIG. 1. Schematic representation of the glucose oxidase-membrane (not drawn to scale); GO, glucose oxidase.
sandwich reactors
use and were protected from light. Aqueous glucose standards were prepared 24 hr prior to use to ensure complete mutarotation. Preparation of glucose oxidase-membrane reactors. Typically, two dialysis membranes were soaked in distilled water for 15 min prior to use. The first membrane was placed over the lower grooved plate of a standard Technicon dialyzer unit. Surplus water was removed, and 2 ml of glucose oxidase solution was applied evenly over the grooved portion of the membrane-covered dialyzer plate. Excess water was removed from the second membrane while held in an applicator frame, and this membrane was placed carefully over the enzyme layer. Then, the top plate of the dialyzer unit was lowered into place and clamped firmly. The complete unit was placed in the dialyzer water bath at 37°C for at least 1 hr prior to use. The assembled glucose oxidase-membrane sandwich reactor is shown schematically in Fig. 1. Automated analysis using glucose oxidase-membrane sandwich reac#or. The assembled glucose oxidase-membrane sandwich reactors were inserted in a continuous-flow automated analysis system shown schematically in Fig. 2. The sample lines, reagent concentrations, etc., were as shown in Fig. 2. The enzyme sandwich reactors were treated basically the same as were normal dialysis membranes. In the flow system shown, small metabolites (such as glucose) which appear in the donor stream diffuse into the sandwich reactor. In the case of glucose, the glucose oxidase catalyzes the conversion of glucose and dissolved oxygen into &gluconolactone and hydrogen peroxide. The presence of hydrogen peroxide in the recipient stream was detected by a modification of the HCVKI method described elsewhere (5), using the flow system shown in Fig. 2. RESULTS AND DISCUSSION
Glucose standards in the range of 2.5-50 mg% were sampled, and the change in absorbance at 349 nm was continuously monitored using a spectrophotometer (Beckman DB) fitted with a l-cm light path flow cell.
332
SHORT COMMUNICATIONS
2 1
_
2
_
D.U. SW uuuo 1
_
3
4
-
DONOR ,E.MSR RECIPIENT
-WASTE :
37°C
SMC “0””
6
FIG. 2. Flow system for the automated determination of glucose using the glucose oxidase-membrane sandwich reactors. The sampler (S), pump (P). dialyzer unit (D.U.; 25ml volume), small mixing coils (SMC), and debubbler (DB) were standard Technicon AA1 equipment. SPEC was a Beckman DB spectrophotometer fitted with a l-cm light path flow cell. The pump tubing lines l-8 gave flow rates of 1.2, 0.8, 0.23, 1.4, 0.8, 1.2, 1.2, and 2.5 ml/mm. Glucose standards were sampled through line 1, and air segmentation was introduced through line 2. Line 3 carried 0.5 M sodium acetate buffer, pH 5.5. Line 4 carried 0.1 M sodium acetate buffer, pH 5.5, and line 5 introduced the air segmentation. Line 6 carried 1.25 M HCl, and line 7 carried 0.25 M KI. IE-MSR: immobilized enzyme-membrane sandwich reactor.
The traces thus generated exhibited satisfactory flow characteristics (Fig. or cross-contamination was not a 3, inset) in that sample “carryover” problem. A standard curve derived from such traces is also shown in Fig. 3. The standard curve is slightly concave downward, but this was not seen as a difficulty here, as individual glucose concentrations were easily distinguishable. The glucose oxidase-sandwich reactors were retained in position in the dialyzer water bath (37°C) and were assayed intermittently. After each run, the lines were washed and filled with water. Figure 4 shows that no decrease in activity was observed over the IO-day period studied. Indeed a slight increase in activity was observed, presumably due to an increase in the membrane permeability with time. In any event, Fig. 4 suggests that it is unlikely that there was any leaching of the enzyme from the membrane sandwich reactor. The observed increase in activity with time would not be a drawback to the incorporation of such reactors in the automated analysis of unknown glucose concentrations, since standard curves are normally generated prior to analysis of the unknowns. Physical entrapment of enzymes, in the fashion described here, appears to offer an extremely facile and flexible method of economically employ-
333
SHORT COMMUNICATIONS
0
so*
204
0/
15.0
0I
10.0
7.5,
I
50, I
2.5
J
0
10
20
30
GLUCOSE
40
50
Cmg %I
FIG. 3. Standard curve for the automated determination of glucose using the glucose oxidase-membrane sandwich reactor (20 samples/hr). Inset shows typical set of traces obtained, exhibiting satisfactory flow characteristics. The flow system shown in Fig. 2 was used.
ing the catalytic power of enzymes in automated analysis. There are several advantages inherent in this method of immobilizing enzymes. First, the use of immobilized enzyme-sandwich reactors does not introduce any new component into the continuous-flow system. Dialysis
334
SHORT COMMUNICATIONS 1.0 -
Glucose
1
0' 0
2
1 4
I 6
8
mg %
I 10
T I ME (days)
FIG. 4. Stability of glucose oxidase-membrane sandwich reactor at 37°C with intermittent usage. The reactors were maintained in the dialyzer unit between assays and were perfused with water before and after each run. Reactors were kept filled with water during this study.
is a routine technique employed in automated analysis, and, in fact, the enzyme sandwich reactor still performs this function. Second, the method of preparing the reactors is extremely easy, and, as no complex chemistries are required, this method could be applied to all enzymes. In addition, there is virtually no limit to the amount of enzyme which could be entrapped by this method or to the variety of different enzymes which could be co-immobilized. Consequently, multienzyme systems could be entrapped simultaneously, and, if coenzymes were required, then macromolecular coenzyme analogs (10) plus coenzyme-regenerating multienzyme systems could also be incorporated, thus broadening the horizons of automated analysis. Furthermore, plasma proteins or fixed elements of blood do not come into contact with the entrapped enzyme. Glucose oxidase is intrinsically rather a stable enzyme. However, if a more labile enzyme were to be entrapped, then a more stable immobilized enzyme module could be obtained by the co-entrapment of a high concentration of an inert protein. If still further stabilization were necessary, then the enzyme and inert protein (while entrapped) could be subjected to a cross-linking procedure, which has been found to induce stability in certain cases (11,12). In conclusion, the immobilized enzyme-membrane sandwich reactors described in this work functioned well in the automated analysis of glucose. The membranes used were commercially available and exhibited satisfactory permeability and flow characteristics. However, by generating
SHORT COMMUNICATIONS
335
membranes specifically for the purpose described here, it ought to be possible to maximize the activity expressed by these reactors. ACKNOWLEDGMENTS This research is supported by Grants MRC-MT-2100 and MRC-SP-4 to T.M.S.C. from the Medical Research Council of Canada. J.C. is a postdoctoral fellow supported by Grant MRC-MT-2100. A.S.C. is an MRC Scholar.
REFERENCES 1. Homby, W. E., Inman, D. J., and McDonald, A. (1972) FEES Letr. 23, 114. 2. Inman, D. J., and Homby, W. E. (1973) Biochem. J. 129, 255. 3. Campbell, J., Homby, W. E., and Morris, D. L. (1975) Biochim. Biophys. Ac?a 384, 307. 4. Weibel, M. K., Dritschilo, W., Bright, H. J., and Humphrey, A. E. (1973) Anal. Biochem. 52, 402. 5. Inman, D. J., and Homby, W. E. (1972) Biochem. J. 129, 255. 6. Homby, W. E., and Morris, D. L. (1975) in Immobilized Enzymes, Antigens, Antibodies, and Peptides (Weetall, H. H., eds.), pp. 141-169, Marcel Dekker, New York. 7. Morris, D. L., Campbell, J., and Homby, W. E. (1975) Biochem. J. 147, 593. 8. Chang, T. M. S. (1964) Science 146, 524. 9. Chang, T. M. S. (1972) Artificial Cells, Charles C Thomas, Springfield, Ill. 10. Larsson, P.-O., and Mosbach, K. (1971) Biotechnol. Bioeng. 13, 393. 11. Chang, T. M. S. (1971) Biochem. Biophys. Res. Commun. 44, 1531. 12. Broun, G., Thomas, D., Gellf, G., Domurado, D., Bejonneau, A. M., and Guillon, C. (1973) Biotech. Bioeng. 15, 359.
J. CAMPBELL A. S. CHAWLA T. M. S. CHANG Physiology Department Artijcial Organs Research Unit McGill University Montreal, Quebec, Canada Received June 8, 1976; accepted July 18, 1971
BIOCHEMISTRY
83, 330-335
(1977)
The Use of Immobilized Enzyme-Membrane Reactors in Automated Analysis
Sandwich
Glucose oxidase from A. niger (EC 1.1.3.4) was physically entrapped between two dialysis membranes. The resultant enzyme-membrane sandwich reactors were employed in a standard Technicon dialyzer unit at 37°C and were found to perform efficiently in the automated analysis of aqueous glucose standards in the range 2.5-50 mg%. Over a lo-day period with intermittent usage, slight increases in the activities of the reactors were observed. The potential of such reactors in automated analysis is discussed.
The use of immobilized enzyme derivatives in automated analysis has been clearly established (l-3). Immobilized glucose oxidase, for example, has been utilized in the form of packed bed reactors (4,5), membrane reactors (5), and open tubular reactors (3,5) for the analysis of both aqueous glucose standards and serum glucose levels. In the case of the former, immobilized enzyme configuration, new types of analytical systems had to be evolved in order to utilize the insoluble catalyst efficiently (4). On the other hand, in the case of open tubular reactors and membrane reactors, standard Technicon AutoAnalyser equipment could be readily employed (l-3,5). Using the fairly sophisticated chemistries of immobilization available for the attachment of enzymes to the inside surface of nylon tube (6,7), it is usually possible to obtain adducts with practically significant activities. However, in the case of enzymes with low turnover numbers, the intrinsic low surface area available for the covalent attachment of protein may be a problem. Semipermeable microcapsule preparations (8,9) containing enzymes have a large surface area to volume relationship, and this type of immobilized enzyme has been shown to work efficiently in packed bed reactors. However, this would disturb the bubble segmented flow pattern used in standard Technicon AutoAnalysers to maintain sample integrity. In this work, a new approach using membrane sandwich reactors in automated analysis is described. EXPERIMENTAL
Type V glucose oxidase (Sigma Chemical Co.) with 1400 units/ml was used without further purification. Type C dialysis membranes and all automated analysis equipment were obtained from Technicon Corp., Tarrytown, New York. KI solutions were prepared immediately prior to 330 Copyright All rights
0 1977 by Academic Press. Inc. of reproduction in any form reserved.
ISSN 0003.2697
SHORT COMMUNICATIONS Dialyser
Donor
331
Top Plate
Stream
Enzyme
Layer
Recipient
Stream
Dialyser
Bottom
Plate
FIG. 1. Schematic representation of the glucose oxidase-membrane (not drawn to scale); GO, glucose oxidase.
sandwich reactors
use and were protected from light. Aqueous glucose standards were prepared 24 hr prior to use to ensure complete mutarotation. Preparation of glucose oxidase-membrane reactors. Typically, two dialysis membranes were soaked in distilled water for 15 min prior to use. The first membrane was placed over the lower grooved plate of a standard Technicon dialyzer unit. Surplus water was removed, and 2 ml of glucose oxidase solution was applied evenly over the grooved portion of the membrane-covered dialyzer plate. Excess water was removed from the second membrane while held in an applicator frame, and this membrane was placed carefully over the enzyme layer. Then, the top plate of the dialyzer unit was lowered into place and clamped firmly. The complete unit was placed in the dialyzer water bath at 37°C for at least 1 hr prior to use. The assembled glucose oxidase-membrane sandwich reactor is shown schematically in Fig. 1. Automated analysis using glucose oxidase-membrane sandwich reac#or. The assembled glucose oxidase-membrane sandwich reactors were inserted in a continuous-flow automated analysis system shown schematically in Fig. 2. The sample lines, reagent concentrations, etc., were as shown in Fig. 2. The enzyme sandwich reactors were treated basically the same as were normal dialysis membranes. In the flow system shown, small metabolites (such as glucose) which appear in the donor stream diffuse into the sandwich reactor. In the case of glucose, the glucose oxidase catalyzes the conversion of glucose and dissolved oxygen into &gluconolactone and hydrogen peroxide. The presence of hydrogen peroxide in the recipient stream was detected by a modification of the HCVKI method described elsewhere (5), using the flow system shown in Fig. 2. RESULTS AND DISCUSSION
Glucose standards in the range of 2.5-50 mg% were sampled, and the change in absorbance at 349 nm was continuously monitored using a spectrophotometer (Beckman DB) fitted with a l-cm light path flow cell.
332
SHORT COMMUNICATIONS
2 1
_
2
_
D.U. SW uuuo 1
_
3
4
-
DONOR ,E.MSR RECIPIENT
-WASTE :
37°C
SMC “0””
6
FIG. 2. Flow system for the automated determination of glucose using the glucose oxidase-membrane sandwich reactors. The sampler (S), pump (P). dialyzer unit (D.U.; 25ml volume), small mixing coils (SMC), and debubbler (DB) were standard Technicon AA1 equipment. SPEC was a Beckman DB spectrophotometer fitted with a l-cm light path flow cell. The pump tubing lines l-8 gave flow rates of 1.2, 0.8, 0.23, 1.4, 0.8, 1.2, 1.2, and 2.5 ml/mm. Glucose standards were sampled through line 1, and air segmentation was introduced through line 2. Line 3 carried 0.5 M sodium acetate buffer, pH 5.5. Line 4 carried 0.1 M sodium acetate buffer, pH 5.5, and line 5 introduced the air segmentation. Line 6 carried 1.25 M HCl, and line 7 carried 0.25 M KI. IE-MSR: immobilized enzyme-membrane sandwich reactor.
The traces thus generated exhibited satisfactory flow characteristics (Fig. or cross-contamination was not a 3, inset) in that sample “carryover” problem. A standard curve derived from such traces is also shown in Fig. 3. The standard curve is slightly concave downward, but this was not seen as a difficulty here, as individual glucose concentrations were easily distinguishable. The glucose oxidase-sandwich reactors were retained in position in the dialyzer water bath (37°C) and were assayed intermittently. After each run, the lines were washed and filled with water. Figure 4 shows that no decrease in activity was observed over the IO-day period studied. Indeed a slight increase in activity was observed, presumably due to an increase in the membrane permeability with time. In any event, Fig. 4 suggests that it is unlikely that there was any leaching of the enzyme from the membrane sandwich reactor. The observed increase in activity with time would not be a drawback to the incorporation of such reactors in the automated analysis of unknown glucose concentrations, since standard curves are normally generated prior to analysis of the unknowns. Physical entrapment of enzymes, in the fashion described here, appears to offer an extremely facile and flexible method of economically employ-
333
SHORT COMMUNICATIONS
0
so*
204
0/
15.0
0I
10.0
7.5,
I
50, I
2.5
J
0
10
20
30
GLUCOSE
40
50
Cmg %I
FIG. 3. Standard curve for the automated determination of glucose using the glucose oxidase-membrane sandwich reactor (20 samples/hr). Inset shows typical set of traces obtained, exhibiting satisfactory flow characteristics. The flow system shown in Fig. 2 was used.
ing the catalytic power of enzymes in automated analysis. There are several advantages inherent in this method of immobilizing enzymes. First, the use of immobilized enzyme-sandwich reactors does not introduce any new component into the continuous-flow system. Dialysis
334
SHORT COMMUNICATIONS 1.0 -
Glucose
1
0' 0
2
1 4
I 6
8
mg %
I 10
T I ME (days)
FIG. 4. Stability of glucose oxidase-membrane sandwich reactor at 37°C with intermittent usage. The reactors were maintained in the dialyzer unit between assays and were perfused with water before and after each run. Reactors were kept filled with water during this study.
is a routine technique employed in automated analysis, and, in fact, the enzyme sandwich reactor still performs this function. Second, the method of preparing the reactors is extremely easy, and, as no complex chemistries are required, this method could be applied to all enzymes. In addition, there is virtually no limit to the amount of enzyme which could be entrapped by this method or to the variety of different enzymes which could be co-immobilized. Consequently, multienzyme systems could be entrapped simultaneously, and, if coenzymes were required, then macromolecular coenzyme analogs (10) plus coenzyme-regenerating multienzyme systems could also be incorporated, thus broadening the horizons of automated analysis. Furthermore, plasma proteins or fixed elements of blood do not come into contact with the entrapped enzyme. Glucose oxidase is intrinsically rather a stable enzyme. However, if a more labile enzyme were to be entrapped, then a more stable immobilized enzyme module could be obtained by the co-entrapment of a high concentration of an inert protein. If still further stabilization were necessary, then the enzyme and inert protein (while entrapped) could be subjected to a cross-linking procedure, which has been found to induce stability in certain cases (11,12). In conclusion, the immobilized enzyme-membrane sandwich reactors described in this work functioned well in the automated analysis of glucose. The membranes used were commercially available and exhibited satisfactory permeability and flow characteristics. However, by generating
SHORT COMMUNICATIONS
335
membranes specifically for the purpose described here, it ought to be possible to maximize the activity expressed by these reactors. ACKNOWLEDGMENTS This research is supported by Grants MRC-MT-2100 and MRC-SP-4 to T.M.S.C. from the Medical Research Council of Canada. J.C. is a postdoctoral fellow supported by Grant MRC-MT-2100. A.S.C. is an MRC Scholar.
REFERENCES 1. Homby, W. E., Inman, D. J., and McDonald, A. (1972) FEES Letr. 23, 114. 2. Inman, D. J., and Homby, W. E. (1973) Biochem. J. 129, 255. 3. Campbell, J., Homby, W. E., and Morris, D. L. (1975) Biochim. Biophys. Ac?a 384, 307. 4. Weibel, M. K., Dritschilo, W., Bright, H. J., and Humphrey, A. E. (1973) Anal. Biochem. 52, 402. 5. Inman, D. J., and Homby, W. E. (1972) Biochem. J. 129, 255. 6. Homby, W. E., and Morris, D. L. (1975) in Immobilized Enzymes, Antigens, Antibodies, and Peptides (Weetall, H. H., eds.), pp. 141-169, Marcel Dekker, New York. 7. Morris, D. L., Campbell, J., and Homby, W. E. (1975) Biochem. J. 147, 593. 8. Chang, T. M. S. (1964) Science 146, 524. 9. Chang, T. M. S. (1972) Artificial Cells, Charles C Thomas, Springfield, Ill. 10. Larsson, P.-O., and Mosbach, K. (1971) Biotechnol. Bioeng. 13, 393. 11. Chang, T. M. S. (1971) Biochem. Biophys. Res. Commun. 44, 1531. 12. Broun, G., Thomas, D., Gellf, G., Domurado, D., Bejonneau, A. M., and Guillon, C. (1973) Biotech. Bioeng. 15, 359.
J. CAMPBELL A. S. CHAWLA T. M. S. CHANG Physiology Department Artijcial Organs Research Unit McGill University Montreal, Quebec, Canada Received June 8, 1976; accepted July 18, 1971