- Email: [email protected]
[25] Routine analysis with immobilized enzyme nylon tube reactors
288
ANALYTICAL APPLICATIONS
[25]
[25] R o u t i n e A n a l y s i s w i t h I m m o b i l i z e d E n z y m e N y l o n Tube Reactors B y P. V. SUNDARAM
Immobilized enzymes offer some distinct advantages including cost reduction in the various ingenious applications that they lend themselves to by virtue of a variety of insoluble polymer supports of different chemical structures and physical forms that are available for immobilization. In particular, new analytical devices such as enzyme electrodes, enzyme immunoassay (EIA), and enzyme reactors have become popular for this reason. In 1970 Sundaram and Hornby I showed that enzymes bound to the inside of nylon tubes may be used in flow-through analysis. Then Sundaram et al. 2 developed a variety of these "immobilized enzyme nylon tube reactors" (a generic name given by us), for assaying most of the commonly required and clinically relevant analytes such as blood urea, 3 uric acid, 4 glucose, 5 pyruvate and lactate, 6 creatinine and creatine, 7 triglycerides, 8 and cholesterol. 9 Extensive clinical trials including stability tests, cost analysis, and problem solving acceptable in laboratories for routine use were undertaken in the development of these tests. 2 Principle of Operation A typical reactor consists of a l-m-long nylon tube (i.d. 1 mm) wound to form a coil 1 cm in diameter. The enzymes are covalently attached to the inside walls of the tube, and analysis is accomplished by perfusion of the samples separated by air bubbles, i.e., a segmented flow. In routine analysis the reactor is incorporated into the flow system of a Technicon i p. V. Sundaram and W. E. Hornby, FEBS Lett. 10, 325 (1970). 2 p. V. Sundaram, Enzyme Microb. Technol. 4, 290 (1982). 3 p. V. Sundaram, M. P. Igloi, R. Wassermann, W. Hinsch, and K.-J. Knoke, Clin. Chem. 24, 234 (1978). 4 p. V. Sundaram, M. P. Igloi, R. Wassermann, and W. Hinsch, Clin. Chem. 24, 1813 (1978). 5 p. V. Sundaram, B. Blumberg, and W. Hinsch, Clin. Chem. 25, 1436 (1979). 6 p. V. Sundaram and W. Hinsch, Clin. Chem. 25, 285 (1979). 7 p. V. Sundaram and M. P. Igloi, Clin. Chim. Acta 94, 295 (1979). 8 W. Hinsch, W.-D. Ebersbach, and P. V. Sundaram, Clin. Chim. Acta 104, 95 (1980). 9 W. Hinsch, A. Antonijevic, and P. V. Sundaram, J. Clin. Chem. Clin. Biochem. 19, 307 (1981).
METHODS IN ENZYMOLOGY, VOL. 137
Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
[25]
NYLON TUBE REACTORS
289
AutoAnalyzer AA I or AA II. Dialyzed samples flow through the reactor at a predetermined flow rate, and the effluent is analyzed either colorimetrically or, if a cofactor is involved, by absorbance measurements at 340 nm. Reactors are washed, filled with a suitable buffer, and stored at 4° when not in use. Reactor coil length, enzyme concentration in the reactor, specific activity of the immobilized enzyme, flow rate, and in turn the residence time of the substrate in the reactor are factors that influence the turnover of a substrate in addition to pH, temperature, and buffer composition. Testing and Optimization of Reactor Performance Individual reactor performance is tested thoroughly before optimization of conditions since very often the kinetic properties such as the pH optima, 4,8,1° apparent Kin, and specific activity of enzymes change on immobilization. Sometimes these changes can be dramatic so that diffusive mass transfer can perturb the system and produce phase changes in the (v) versus (s) progress curve thus leading to a biphasic or triphasic curve. 1° It is critical to ensure that the standard curve for a given method of analysis falls entirely within one of these phases or segments or else routine analysis cannot be automated to give reliably reproducible results. Routine Analysis Normally 50-60 assays per hour are carried out in routine automated analysis. Individual flow diagrams for the analysis of the different analytes such as urea, glucose, and cholesterol using reactors containing the respective enzymes are different as shown in the publications. TM Methods of Immobilization Nylon tubing (i.d. 1 mm) purchased from Portex Ltd. (Hythe, Kent, UK) is used to make reactors. Unless otherwise specified, reactors are made of 1-m-long tubes cut from the bulk supply, wound around a plastic rod 1 cm in diameter, and fixed in position with adhesive tape. Once the 10 W. Hinsch and P. V. Sundaram, Clin. Chim. Acta 104, 87 (1980). u p. V. Sundaram, J. Solid-Phase Biochem. 3, 185 (1978). 12 W. Hinsch, A. Antonijevic, and P. V. Sundaram, Z. Lebensm.-Unters. Forsch. 171, 449 (1980). 13 W. Hinsch, A. Antonijevic, and P. V. Sundaram, Clin. Chem. 26, 1652 (1980). 14 W. Hinsch, A. Antonijevic, and P. V. Sundaram, Fresenius Z. Anal. Chem. 309, 25 (1981).
290
ANALYTICALAPPLICATIONS
[25]
immobilization procedure is complete the coiled tubing may be removed from the plastic rod, which retains its coiled structure. This coiling is essential to ensure turbulence during perfusion of substrates and thus enable a proper mixing of the substrate which thereby facilitates maximum contact of substrate with the immobilized enzyme molecules on the matrix. Basically two approaches are used in immobilizing enzymes. Either the tube is first partially hydrolyzed before the COOH and NH2 groups released are further activated to couple the enzyme, or nylon is directly O-alkylated by treatment with dimethyl sulfate (DMS) or triethyloxonium tetrafluoroborate (TTFB). This alkylation produces an imidate derivative of nylon which is very reactive and can be amidinated by reaction with NH2-bearing compounds. Thus either an enzyme can be directly coupled to the matrix or a spacer may first be coupled followed by an enzyme (Fig. 1).
Enzyme Coupling to Hydrolyzed Nylon Coiled tubing is filled with 3.6 M HC1, ends sealed after connecting with a piece of soft Tygon tubing, and covered with Parafilm. The tube is incubated in a water bath at 70 ° for 4-6 min depending on the extent of hydrolysis desired. It is then thoroughly washed starting with warm water and followed by cold water. Cross-Linking with Glutaraldehyde. Hydrolyzed tube is washed with a NaHCO3 or borate buffer (0.1 M), pH 9.4, and filled or perfused with a freshly prepared 1.25% (v/v) glutaraldehyde solution made up in either of these buffers of choice and allowed to react for 40 min, The tube is then washed with water followed by coupling buffer, usually phosphate buffer (0.1 M), pH 7-8, and filled with the enzyme solution. The average maximum coupling capacity being about 0.1 mg/m tubing by this method, a 2 mg/ml solution of enzyme protein is sufficient provided that the specific activity is around 20 U/mg. 1,j5-17 Cross-Linking with Bisimidates. Hydrolyzed tube is washed with borate buffer (0.1 M), pH 9.4, and then filled with a 4 mg/ml solution of dimethyl adipimidate or suberimidate made fresh. After reaction at room temperature for 2 hr the tube is washed once again with coupling buffer and filled with a 2 mg/ml enzyme solution made in a buffer at pH 6-9. Tris t5 p. V. Sundaram and D. K. Apps, Biochem. J. 161, 441 (1977). 16p. V. Sundaram, in "BiomedicalApplications of Immobilized Enzymes and Proteins" (T. M. S. Chang, ed.), Vol. 1, p. 317. Plenum, New York, 1977. ~7p. V. Sundaram, in "Enzyme Labelled Immunoassays of Hormones and Drugs" (S. B. Pal, ed.), p. 107. de Gruyter, Berlin, 1978.
[25]
NYLON TUBE REACTORS -C=NH I
291
-
O O
~ (C2H5) 30BF4 O
- C= NH -
03 Q C=NHI
Enzy me
I
v
OC2H 5
HN - Enzyme
NH2 I
•.(.CH2-CH- ,CH- CH-)-n I
I
NH2 O
-C=NH -
l +~
PE|
HN- CH 1 HCI
HC -NH 2 I
CH2
Nylon- PE! copolymcr
I OHC (CH2)3CHO
Q O
- C=NH -
Enzyme
HN - CH
NN-CH I NCI
I
HC H
HC-N = C (CH2)3CHO I
CH2
oc
-C=NH-
I
H
H
HC-N=C(CH2)3C=N-Enzyme I
CH2
"4"
Nylon - PEI -Enzyrn¢ FIG. 1. Chemical rection schemes for attaching an enzyme to O-alkylated nylon (directly) and to nylon-PEI copolymer (indirectly).
buffer is avoided. Above pH 7 coupling is faster but so is the hydrolysis of the imidate. Thus a judicious choice of pH is made. After standing overnight at 4° the tube is washed well with a 0.1 M NaC1 solution and then water. The reactor is filled with a suitable buffer and stored. Coupling to COOH Groups Activated with Soluble Carbodiimides. Hydrolyzed tube is washed well with dry dimethylformamide (DMF) and perfused with 40 mM NHS made in dry DMF for a few minutes after which enough 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide(EDAC) or
292
ANALYTICALAPPLICATIONS
[25]
l-cyclohexyl-3[2-morpholinoethyl]-carbodiimide metho-p-toulene sulfonate (CMC) weighed out to give a final concentration of 20 mM is added in over a few minutes to the NHS solution ensuring good stirring simultaneously. The mixture is stirred well and perfused through the tube in a closed circuit for 90 min. The tube is then washed quickly with dry DMF and once with coupling buffer before filling with the enzyme solution made up in phosphate buffer, pH 7-8, and left overnight at 4°. The activation is conducted at room temperature. The next day the reactor is washed as usual and stored.
Coupling to Nylon after Alkylation Nylon yields an imidate derivative on alkylation to which enzymes may be coupled directly or through a spacer such as a diamine, polylysine, or polyethylenimine (PEI). Enzymes are crosslinked to the NHz groups of the spacer with dialdehydes or bisimidates. Alkylation with TTFB 18 requires milder conditions than with DMS 19 and is thus preferred to the latter. A 1-m-long coiled tube is filled with a 0.1 M solution of TTFB (supplied by Aldrich Chemical Co., Milwaukee, WI) in dichloromethane and after sealing the ends is allowed to react for 4 min at room temperature. The tube is then emptied into a safety flask by suction and flushed with 50 ml ice-cold methanol followed by ice-cold water. The tube which is now ready for coupling may be filled with an enzyme solution at 2 mg/ml in phosphate buffer (0.1 M), pH 7-9, and allowed to react overnight at 4 °, or a spacer molecule is attached. However, kinetics of the coupling process shows that more than 60% couples in 30 min at room temperature, z° Spacers are coupled by filling the tube with either 0. l M hexamethylenediamine or a 4 mg/ml solution of polylysine or a 0.6 M PEI solution (Serva GmbH., FRG, 10,000 MW) made up in NaHCO3 buffer (0.1 M), pH 9.2. After coupling, the tube is washed well with 0.1 M NaC1 followed by water and is now ready for coupling enzymes by cross-linking, the same conditions being used as before.15.z° Performance Characteristics The performance characteristics and the statistical parameters of the performance of the various methods evaluated from clinical trials are given in Tables I and II, respectively. Exhaustive information is available is D. L. Morris, J. Campbell, and W. E. Hornby, Biochem. J. 147, 593 (1975). 19 p. V. Sundaram, Nucleic Acids Res. 1, 1587 (1974). 2o p. V. Sundaram, Biochem. J. 183, 445 (1979).
[9-5]
NYLON
TUBE
REACTORS
293
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294
ANALYTICALAPPLICATIONS
[25]
in these tables and only some special features of some of the tests are discussed below. As already pointed out, z reduction in cost of testing is a feature common to all the methods using immobilized enzyme nylon tube reactors. We have employed single enzyme systems for the estimation of urea, uric acid, glucose, and pyruvate using urease, 3 uricase, 4 glucose dehydrogenase (Gluc-DH), 5 and lactate dehydrogenase (LDH) 6 reactors, respectively. Creatinine and creatine 7 may be estimated using a creatininase-creatine kinase reactor connected to a pyruvate kinase (PK)-lactate dehydrogenase (LDH) reactor. Although all four enzymes may be immobilized in a single reactor, sufficiently high activity of the four enzymes for analysis at the rate of 50-60 per hour was not easily achieved with the enzymes available. Hitherto triglycerides have been estimated by a chemical method, and now a method using glycerol kinase is commerically available. Our method uses glycerol dehydrogenase which acts on glycerol released from sera after the lipolytic enzymes lipase and esterase have acted on them. The method is very efficient and specific) Immobilization shifts the pH optimum of the enzyme to pH 10.0 and also stabilizes it. 1° This high pH and excess NAD ÷ are required for the enzyme to act on glycerol. Approximately 3500 tests are possible with each reactor. Heterogeneous Multienzyme Systems A novel approach to analysis is used in a method where yeast aldehyde dehydrogenase (ALDH) is used along with glucose oxidase (GOD), uricase, or cholesterol oxidase and catalase, the last mentioned in solution form, that together make heterogeneous multienzyme systems that are employed in the estimation of glucose, 13 uric acid, 14 or cholesterol.19 Since these heterogeneous multienzyme systems comprise immobilized enzymes that are separated by an enzyme in solution, transport of substrate and products to and from the polymer surface is involved (Fig. 2), and this could limit the efficient functioning of the systems. However, use of a large excess of catalase and ethanol, which in the presence of H202 produces CH3CHO for ALDH to act on, guarantees proper functioning of the systems. In the case of cholesterol, since cholesterol oxidase and cholesterol esterase are unstable on immobilization, the sera are exposed to these enzymes in solution before being passed through an ALDH reactor. In the same way glucose or uric acid may be assayed with an ALDH reactor provided the sample sera are first treated with GOD or
[25]
NYLONTUBEREACTORS
;
Cholesterol esterase a n d oxidose
Esterified ond free cholesterol
~
295
~~k~celaIde"hyde~'~ H202/- - • \NAD÷ t ~.ototo$¢ ~ NADH + H+
Glucose J i or
Ethanol
acetate
uric acid Ambient medium
FIG. 2. Reactionschemesof heterogeneousmultienzymesystems containingimmobilized aldehydedehydrogenaseand other enzymes. uricase prior to passage through the dialyzer in the AutoAnalyzer. An ALDH reactor is stable for 5000 tests and gives excellent performance (Table II).
Glucose Estimation with the G O D - A L D H Reactor
Having established that the optimum performance of the G O D ALDH reactor is at pH 7.8,13 the reactor is assembled as part of the flow circuit of a Technicon AutoAnalyzer II (Fig. 3). Only three different reagent solutions are used in routine analysis of glucose by this system. Buffer A is a potassium phosphate solution (0.1 M), pH 7.8, containing 0.3 g Brij-35 per liter, and Buffer B is the same buffer which in addition contains 1.5 M ethanol, 1 mM EDTA, and 1 mM mercaptoethanol. NAD + catalase solution is made in phosphate buffer (0.1 M), pH 7.5, containing 5 mM NAD and 1.5 × 106 U of catalase. This method is linear up to 4500 mg/liter glucose. Uric Acid Estimation with the Uricase-ALDH Reactor
Using similar principles uric acid may be determined with a uricaseALDH reactor, 14 analysis being carried out in a Tris-HC1 buffer, pH 8.6. The flow diagram is slightly modified, and the NAD+/catalase solution contains 10 mM NAD + and 6 × 106 U of catalase. The method is linear up to 120 rag/liter of uric acid.
296
ANALYTICAL APPLICATIONS
r~
I
od
I
I
--
rq I
I I I
II
r~-2~ I
[25]
I
I
I
,d ,~ oi -2 I
I
rq I
0 f<
+ + +
~++~ +~l
,~
~
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o
+
Z
0
~ e q ~
(Ir~
E 1..,
•" ~
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t.,
-6
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[25]
NVLOr~ TUBE REACTORS
297
samplerIV
ml/rnin Waste
t
5 turns 12"dialyzer
I
I ~
i
GOD/ALDH reactor(25")
romp
10 turns
Waste l 340 nm
0.32
air
O.&2 0.16
buffer A sample
0,32
air
0,42
buffer B
0.16
NAD+lcatalas¢ NAD+lcat,
J 0.80 ~ 0,32
I H20/Brij
FIG. 3. Flow diagram for the determination of glucose using a GOD-ALDH reactor connected to a Technicon AutoAnalyzer AA II. Summary T h e basic strategy i n v o l v e d in the design, d e v e l o p m e n t , and application o f immobilized e n z y m e n y l o n tube r e a c t o r s for routine analysis is d e s c r i b e d in this c h a p t e r , t o u c h i n g on s o m e o f the attractive features o f these m e t h o d s . E x t e n s i v e data (Tables I and II) and the references provide details w h i c h m a y be n e e d e d b a s e d on specific m e t h o d s . Acknowledgment The work described herein was supported by grants from DFVLR and Deutsche Forschungsgemeinschaft. This article was written when the author was a visiting Professor at The Voluntary Health Services Centre, Adyar, Madras, India.
ANALYTICAL APPLICATIONS
[25]
[25] R o u t i n e A n a l y s i s w i t h I m m o b i l i z e d E n z y m e N y l o n Tube Reactors B y P. V. SUNDARAM
Immobilized enzymes offer some distinct advantages including cost reduction in the various ingenious applications that they lend themselves to by virtue of a variety of insoluble polymer supports of different chemical structures and physical forms that are available for immobilization. In particular, new analytical devices such as enzyme electrodes, enzyme immunoassay (EIA), and enzyme reactors have become popular for this reason. In 1970 Sundaram and Hornby I showed that enzymes bound to the inside of nylon tubes may be used in flow-through analysis. Then Sundaram et al. 2 developed a variety of these "immobilized enzyme nylon tube reactors" (a generic name given by us), for assaying most of the commonly required and clinically relevant analytes such as blood urea, 3 uric acid, 4 glucose, 5 pyruvate and lactate, 6 creatinine and creatine, 7 triglycerides, 8 and cholesterol. 9 Extensive clinical trials including stability tests, cost analysis, and problem solving acceptable in laboratories for routine use were undertaken in the development of these tests. 2 Principle of Operation A typical reactor consists of a l-m-long nylon tube (i.d. 1 mm) wound to form a coil 1 cm in diameter. The enzymes are covalently attached to the inside walls of the tube, and analysis is accomplished by perfusion of the samples separated by air bubbles, i.e., a segmented flow. In routine analysis the reactor is incorporated into the flow system of a Technicon i p. V. Sundaram and W. E. Hornby, FEBS Lett. 10, 325 (1970). 2 p. V. Sundaram, Enzyme Microb. Technol. 4, 290 (1982). 3 p. V. Sundaram, M. P. Igloi, R. Wassermann, W. Hinsch, and K.-J. Knoke, Clin. Chem. 24, 234 (1978). 4 p. V. Sundaram, M. P. Igloi, R. Wassermann, and W. Hinsch, Clin. Chem. 24, 1813 (1978). 5 p. V. Sundaram, B. Blumberg, and W. Hinsch, Clin. Chem. 25, 1436 (1979). 6 p. V. Sundaram and W. Hinsch, Clin. Chem. 25, 285 (1979). 7 p. V. Sundaram and M. P. Igloi, Clin. Chim. Acta 94, 295 (1979). 8 W. Hinsch, W.-D. Ebersbach, and P. V. Sundaram, Clin. Chim. Acta 104, 95 (1980). 9 W. Hinsch, A. Antonijevic, and P. V. Sundaram, J. Clin. Chem. Clin. Biochem. 19, 307 (1981).
METHODS IN ENZYMOLOGY, VOL. 137
Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
[25]
NYLON TUBE REACTORS
289
AutoAnalyzer AA I or AA II. Dialyzed samples flow through the reactor at a predetermined flow rate, and the effluent is analyzed either colorimetrically or, if a cofactor is involved, by absorbance measurements at 340 nm. Reactors are washed, filled with a suitable buffer, and stored at 4° when not in use. Reactor coil length, enzyme concentration in the reactor, specific activity of the immobilized enzyme, flow rate, and in turn the residence time of the substrate in the reactor are factors that influence the turnover of a substrate in addition to pH, temperature, and buffer composition. Testing and Optimization of Reactor Performance Individual reactor performance is tested thoroughly before optimization of conditions since very often the kinetic properties such as the pH optima, 4,8,1° apparent Kin, and specific activity of enzymes change on immobilization. Sometimes these changes can be dramatic so that diffusive mass transfer can perturb the system and produce phase changes in the (v) versus (s) progress curve thus leading to a biphasic or triphasic curve. 1° It is critical to ensure that the standard curve for a given method of analysis falls entirely within one of these phases or segments or else routine analysis cannot be automated to give reliably reproducible results. Routine Analysis Normally 50-60 assays per hour are carried out in routine automated analysis. Individual flow diagrams for the analysis of the different analytes such as urea, glucose, and cholesterol using reactors containing the respective enzymes are different as shown in the publications. TM Methods of Immobilization Nylon tubing (i.d. 1 mm) purchased from Portex Ltd. (Hythe, Kent, UK) is used to make reactors. Unless otherwise specified, reactors are made of 1-m-long tubes cut from the bulk supply, wound around a plastic rod 1 cm in diameter, and fixed in position with adhesive tape. Once the 10 W. Hinsch and P. V. Sundaram, Clin. Chim. Acta 104, 87 (1980). u p. V. Sundaram, J. Solid-Phase Biochem. 3, 185 (1978). 12 W. Hinsch, A. Antonijevic, and P. V. Sundaram, Z. Lebensm.-Unters. Forsch. 171, 449 (1980). 13 W. Hinsch, A. Antonijevic, and P. V. Sundaram, Clin. Chem. 26, 1652 (1980). 14 W. Hinsch, A. Antonijevic, and P. V. Sundaram, Fresenius Z. Anal. Chem. 309, 25 (1981).
290
ANALYTICALAPPLICATIONS
[25]
immobilization procedure is complete the coiled tubing may be removed from the plastic rod, which retains its coiled structure. This coiling is essential to ensure turbulence during perfusion of substrates and thus enable a proper mixing of the substrate which thereby facilitates maximum contact of substrate with the immobilized enzyme molecules on the matrix. Basically two approaches are used in immobilizing enzymes. Either the tube is first partially hydrolyzed before the COOH and NH2 groups released are further activated to couple the enzyme, or nylon is directly O-alkylated by treatment with dimethyl sulfate (DMS) or triethyloxonium tetrafluoroborate (TTFB). This alkylation produces an imidate derivative of nylon which is very reactive and can be amidinated by reaction with NH2-bearing compounds. Thus either an enzyme can be directly coupled to the matrix or a spacer may first be coupled followed by an enzyme (Fig. 1).
Enzyme Coupling to Hydrolyzed Nylon Coiled tubing is filled with 3.6 M HC1, ends sealed after connecting with a piece of soft Tygon tubing, and covered with Parafilm. The tube is incubated in a water bath at 70 ° for 4-6 min depending on the extent of hydrolysis desired. It is then thoroughly washed starting with warm water and followed by cold water. Cross-Linking with Glutaraldehyde. Hydrolyzed tube is washed with a NaHCO3 or borate buffer (0.1 M), pH 9.4, and filled or perfused with a freshly prepared 1.25% (v/v) glutaraldehyde solution made up in either of these buffers of choice and allowed to react for 40 min, The tube is then washed with water followed by coupling buffer, usually phosphate buffer (0.1 M), pH 7-8, and filled with the enzyme solution. The average maximum coupling capacity being about 0.1 mg/m tubing by this method, a 2 mg/ml solution of enzyme protein is sufficient provided that the specific activity is around 20 U/mg. 1,j5-17 Cross-Linking with Bisimidates. Hydrolyzed tube is washed with borate buffer (0.1 M), pH 9.4, and then filled with a 4 mg/ml solution of dimethyl adipimidate or suberimidate made fresh. After reaction at room temperature for 2 hr the tube is washed once again with coupling buffer and filled with a 2 mg/ml enzyme solution made in a buffer at pH 6-9. Tris t5 p. V. Sundaram and D. K. Apps, Biochem. J. 161, 441 (1977). 16p. V. Sundaram, in "BiomedicalApplications of Immobilized Enzymes and Proteins" (T. M. S. Chang, ed.), Vol. 1, p. 317. Plenum, New York, 1977. ~7p. V. Sundaram, in "Enzyme Labelled Immunoassays of Hormones and Drugs" (S. B. Pal, ed.), p. 107. de Gruyter, Berlin, 1978.
[25]
NYLON TUBE REACTORS -C=NH I
291
-
O O
~ (C2H5) 30BF4 O
- C= NH -
03 Q C=NHI
Enzy me
I
v
OC2H 5
HN - Enzyme
NH2 I
•.(.CH2-CH- ,CH- CH-)-n I
I
NH2 O
-C=NH -
l +~
PE|
HN- CH 1 HCI
HC -NH 2 I
CH2
Nylon- PE! copolymcr
I OHC (CH2)3CHO
Q O
- C=NH -
Enzyme
HN - CH
NN-CH I NCI
I
HC H
HC-N = C (CH2)3CHO I
CH2
oc
-C=NH-
I
H
H
HC-N=C(CH2)3C=N-Enzyme I
CH2
"4"
Nylon - PEI -Enzyrn¢ FIG. 1. Chemical rection schemes for attaching an enzyme to O-alkylated nylon (directly) and to nylon-PEI copolymer (indirectly).
buffer is avoided. Above pH 7 coupling is faster but so is the hydrolysis of the imidate. Thus a judicious choice of pH is made. After standing overnight at 4° the tube is washed well with a 0.1 M NaC1 solution and then water. The reactor is filled with a suitable buffer and stored. Coupling to COOH Groups Activated with Soluble Carbodiimides. Hydrolyzed tube is washed well with dry dimethylformamide (DMF) and perfused with 40 mM NHS made in dry DMF for a few minutes after which enough 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide(EDAC) or
292
ANALYTICALAPPLICATIONS
[25]
l-cyclohexyl-3[2-morpholinoethyl]-carbodiimide metho-p-toulene sulfonate (CMC) weighed out to give a final concentration of 20 mM is added in over a few minutes to the NHS solution ensuring good stirring simultaneously. The mixture is stirred well and perfused through the tube in a closed circuit for 90 min. The tube is then washed quickly with dry DMF and once with coupling buffer before filling with the enzyme solution made up in phosphate buffer, pH 7-8, and left overnight at 4°. The activation is conducted at room temperature. The next day the reactor is washed as usual and stored.
Coupling to Nylon after Alkylation Nylon yields an imidate derivative on alkylation to which enzymes may be coupled directly or through a spacer such as a diamine, polylysine, or polyethylenimine (PEI). Enzymes are crosslinked to the NHz groups of the spacer with dialdehydes or bisimidates. Alkylation with TTFB 18 requires milder conditions than with DMS 19 and is thus preferred to the latter. A 1-m-long coiled tube is filled with a 0.1 M solution of TTFB (supplied by Aldrich Chemical Co., Milwaukee, WI) in dichloromethane and after sealing the ends is allowed to react for 4 min at room temperature. The tube is then emptied into a safety flask by suction and flushed with 50 ml ice-cold methanol followed by ice-cold water. The tube which is now ready for coupling may be filled with an enzyme solution at 2 mg/ml in phosphate buffer (0.1 M), pH 7-9, and allowed to react overnight at 4 °, or a spacer molecule is attached. However, kinetics of the coupling process shows that more than 60% couples in 30 min at room temperature, z° Spacers are coupled by filling the tube with either 0. l M hexamethylenediamine or a 4 mg/ml solution of polylysine or a 0.6 M PEI solution (Serva GmbH., FRG, 10,000 MW) made up in NaHCO3 buffer (0.1 M), pH 9.2. After coupling, the tube is washed well with 0.1 M NaC1 followed by water and is now ready for coupling enzymes by cross-linking, the same conditions being used as before.15.z° Performance Characteristics The performance characteristics and the statistical parameters of the performance of the various methods evaluated from clinical trials are given in Tables I and II, respectively. Exhaustive information is available is D. L. Morris, J. Campbell, and W. E. Hornby, Biochem. J. 147, 593 (1975). 19 p. V. Sundaram, Nucleic Acids Res. 1, 1587 (1974). 2o p. V. Sundaram, Biochem. J. 183, 445 (1979).
[9-5]
NYLON
TUBE
REACTORS
293
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._= N
II
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294
ANALYTICALAPPLICATIONS
[25]
in these tables and only some special features of some of the tests are discussed below. As already pointed out, z reduction in cost of testing is a feature common to all the methods using immobilized enzyme nylon tube reactors. We have employed single enzyme systems for the estimation of urea, uric acid, glucose, and pyruvate using urease, 3 uricase, 4 glucose dehydrogenase (Gluc-DH), 5 and lactate dehydrogenase (LDH) 6 reactors, respectively. Creatinine and creatine 7 may be estimated using a creatininase-creatine kinase reactor connected to a pyruvate kinase (PK)-lactate dehydrogenase (LDH) reactor. Although all four enzymes may be immobilized in a single reactor, sufficiently high activity of the four enzymes for analysis at the rate of 50-60 per hour was not easily achieved with the enzymes available. Hitherto triglycerides have been estimated by a chemical method, and now a method using glycerol kinase is commerically available. Our method uses glycerol dehydrogenase which acts on glycerol released from sera after the lipolytic enzymes lipase and esterase have acted on them. The method is very efficient and specific) Immobilization shifts the pH optimum of the enzyme to pH 10.0 and also stabilizes it. 1° This high pH and excess NAD ÷ are required for the enzyme to act on glycerol. Approximately 3500 tests are possible with each reactor. Heterogeneous Multienzyme Systems A novel approach to analysis is used in a method where yeast aldehyde dehydrogenase (ALDH) is used along with glucose oxidase (GOD), uricase, or cholesterol oxidase and catalase, the last mentioned in solution form, that together make heterogeneous multienzyme systems that are employed in the estimation of glucose, 13 uric acid, 14 or cholesterol.19 Since these heterogeneous multienzyme systems comprise immobilized enzymes that are separated by an enzyme in solution, transport of substrate and products to and from the polymer surface is involved (Fig. 2), and this could limit the efficient functioning of the systems. However, use of a large excess of catalase and ethanol, which in the presence of H202 produces CH3CHO for ALDH to act on, guarantees proper functioning of the systems. In the case of cholesterol, since cholesterol oxidase and cholesterol esterase are unstable on immobilization, the sera are exposed to these enzymes in solution before being passed through an ALDH reactor. In the same way glucose or uric acid may be assayed with an ALDH reactor provided the sample sera are first treated with GOD or
[25]
NYLONTUBEREACTORS
;
Cholesterol esterase a n d oxidose
Esterified ond free cholesterol
~
295
~~k~celaIde"hyde~'~ H202/- - • \NAD÷ t ~.ototo$¢ ~ NADH + H+
Glucose J i or
Ethanol
acetate
uric acid Ambient medium
FIG. 2. Reactionschemesof heterogeneousmultienzymesystems containingimmobilized aldehydedehydrogenaseand other enzymes. uricase prior to passage through the dialyzer in the AutoAnalyzer. An ALDH reactor is stable for 5000 tests and gives excellent performance (Table II).
Glucose Estimation with the G O D - A L D H Reactor
Having established that the optimum performance of the G O D ALDH reactor is at pH 7.8,13 the reactor is assembled as part of the flow circuit of a Technicon AutoAnalyzer II (Fig. 3). Only three different reagent solutions are used in routine analysis of glucose by this system. Buffer A is a potassium phosphate solution (0.1 M), pH 7.8, containing 0.3 g Brij-35 per liter, and Buffer B is the same buffer which in addition contains 1.5 M ethanol, 1 mM EDTA, and 1 mM mercaptoethanol. NAD + catalase solution is made in phosphate buffer (0.1 M), pH 7.5, containing 5 mM NAD and 1.5 × 106 U of catalase. This method is linear up to 4500 mg/liter glucose. Uric Acid Estimation with the Uricase-ALDH Reactor
Using similar principles uric acid may be determined with a uricaseALDH reactor, 14 analysis being carried out in a Tris-HC1 buffer, pH 8.6. The flow diagram is slightly modified, and the NAD+/catalase solution contains 10 mM NAD + and 6 × 106 U of catalase. The method is linear up to 120 rag/liter of uric acid.
296
ANALYTICAL APPLICATIONS
r~
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od
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,~
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+
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NVLOr~ TUBE REACTORS
297
samplerIV
ml/rnin Waste
t
5 turns 12"dialyzer
I
I ~
i
GOD/ALDH reactor(25")
romp
10 turns
Waste l 340 nm
0.32
air
O.&2 0.16
buffer A sample
0,32
air
0,42
buffer B
0.16
NAD+lcatalas¢ NAD+lcat,
J 0.80 ~ 0,32
I H20/Brij
FIG. 3. Flow diagram for the determination of glucose using a GOD-ALDH reactor connected to a Technicon AutoAnalyzer AA II. Summary T h e basic strategy i n v o l v e d in the design, d e v e l o p m e n t , and application o f immobilized e n z y m e n y l o n tube r e a c t o r s for routine analysis is d e s c r i b e d in this c h a p t e r , t o u c h i n g on s o m e o f the attractive features o f these m e t h o d s . E x t e n s i v e data (Tables I and II) and the references provide details w h i c h m a y be n e e d e d b a s e d on specific m e t h o d s . Acknowledgment The work described herein was supported by grants from DFVLR and Deutsche Forschungsgemeinschaft. This article was written when the author was a visiting Professor at The Voluntary Health Services Centre, Adyar, Madras, India.