Lower Critical Solution Temperatures of Aqueous Copolymers of

Chapter 18

Lower Critical Solution Temperatures of Aqueous Copolymers of N-Isopropylacrylamide and OtherN-SubstitutedAcrylamides 1

1

1

2

John H.Priest ,Sheryl L. Murray , R. JohnNelson ,and Allan S. Hoffman 1

Genetic Systems Corporation, 3005 1st Avenue, Seattle, WA 98121 Chemical Engineering Department, Center for Bioengineering, FL-20, University of Washington, Seattle, WA 98195

Downloaded by UNIV LAVAL on July 10, 2014 | http://pubs.acs.org Publication Date: September 14, 1987 | doi: 10.1021/bk-1987-0350.ch018

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High polymers of N-isopropyl acrylamide (NIPAAM) exhibit a lower critical solution temperature (LCST) i n phosphate buffered s a l i n e : above 31°C p r e c i p i t a t i o n occurs, with minimal concentration dependence. Copolymers of NIPAAM with acrylamide (AAM), N-methyl acrylamide (NMAAM) and N-ethyl acrylamide (NEAAM) exhibited LCSTs elevated i n proportion to t h e i r comonomer content. As expected acrylamide was the most e f f e c t i v e at elevating the LCST of the copolymer formed. However, NEAAM was more e f f e c t i v e than NMAAM. The reason for this i s not c l e a r . In contrast, copolymers of NIPAAM with N-t-butyl acrylamide (NTBAAM) exhibited LCSTs that were lowered as a l i n e a r function of the comonomer input r a t i o . Copolymers with N-n-butyl acrylamide (NNBAAM) displayed s i m i l a r behavior up to 40% NNBAAM, but then the LCST dropped abruptly fron 17°C to below zero. The use of these copolymers i n diagnostics and bioseparations i s discussed. The water s o l u b i l i t y at room temperature of polymers of N-substituted acrylamides changes abruptly as the N-alkyl substituent i s changed from ethyl to b u t y l . PolyAAM,* polyNMAAM and polyNEAAM are a l l soluble i n water, while polyNNBAAM and polyNTBAAM are insoluble i n water under ambient conditions. It i s i n t e r e s t i n g to note that polyNIPAAM i s positioned i n this series at the border between the very soluble and the very insoluble, and i t exhibits a lower c r i t i c a l solution temperature (LCST) i n water at 31-33°C (1-3). Below the LCST, a 20% solution of polyNIPAAM can be prepared. Above the LCST the s o l u b i l i t y of polyNIPAAM i s less than 10" %. The dramatic s o l u b i l i t y difference below and above the LCST allows the quantitative removal from solution of the polymer and any material s p e c i f i c a l l y bound to i t . This phenomenon has been exploited for a number of uses, among them the delivery or separation of biomolecules to or from aqueous solutions (4, and references therein) and i n an immunoassay (5,6). 3

0097-6156/87/0350-0255S06.00/0 © 1987 American Chemical Society

In Reversible Polymeric Gels and Related Systems; Russo, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

REVERSIBLE POLYMERIC GELS AND RELATED SYSTEMS


256

Assays based s o l e l y on polyNIPAAM to cause quantitative separations lack the f l e x i b i l i t y for choosing the assay temperature, since the polymer precipitates at or near 31°C. For many immunoassays i t i s desirable to incubate the antibody with the sample at 37°C or above (7). For others i t may be desirable to incubate at low temperatures, such as overnight at 4°C, followed by p r e c i p i t a t i o n at room temperature. The l a t t e r avoids the necessity of heating a centrifuge above 22°C. A t h i r d application of the LCST phenomenon to diagnostics i s i n assays based upon the binding of a DNA probe to i t s complementary sequence i n a sample to detect, for example, v i r a l or b a c t e r i a l nucleic acids, or hereditary diseases such as s i c k l e c e l l anemia or β thallasemia (8). However, these hybridization assays require incubation at 50-60°C i n high s a l t (9). The a b i l i t y to fine-tune the LCST of polyNIPAAM-based copolymers extends the potential of this technology. The purpose of this study was to prepare a series of random copolymers of NIPAAM with predictable and well defined temperatures of p r e c i p i t a t i o n covering the range of 0 to 45°C, as well as some that p r e c i p i t a t e above 55°C under the conditions of high s a l t used i n DNA hybridization assays. The N-substituted acrylamides o f f e r the greatest chemical s i m i l a r i t y to NIPAAM and therefore should copolymerize randomly with the l a t t e r (10). Thus, copolymers of NIPAAM with AAM, NMAAM, NEAAM, NNBAAM and NTBAAM were prepared at selected monomer ratios and their aqueous solution behavior was evaluated. EXPERIMENTAL Materials NMAAM, (Pfaltz and Bauer), NEAAM, NNBAAM, NTBAAM (Monomer-Polymer and Dajac Laboratories, Inc., Trevose, PA), NIPAAM (Eastman Kodak Company, Rochester, NY), AAM, ammonium persulfate (Bio-Rad Laboratories, Richmond, CA) and TEMED (Sigma Chemical Co., St. Louis, MO) were used as received. NEAAM was also obtained from Polysciences. Sephacryl S-400 was obtained from Sigma. A l l other chemicals were reagent grade or better. Polymerizations Where possible, polymers and copolymers were prepared i n normal saline buffered with 10 mM sodium phosphate (PBS), pH 7.4, by room temperature i n i t i a t i o n of 1% monomer solutions using 40 mM TEMED (HC1) and 5 mM ammonium persulfate. However, due to poor s o l u b i l i t y , 0.2% solutions of NTBAAM and proporationately lower i n i t i a t o r concentrations were used. PolyNEAAM was also synthesized by polymerization i n tetrahydrofuran as described by Cole et a l . (5). Lower C r i t i c a l Solution Temperatures LCSTs were determined from plots of o p t i c a l density at 600 nm versus temperature for 0.03% solutions of each polymer i n PBS and were defined as the temperature at which A oo = 0.1. Temperatures were raised at less than 0.3°C per minute and were measured with a thermometer that had been calibrated against an NBS primary standard thermometer. LCSTs for Figure 6 were determined from the cloud points of 0.01% solutions. 6


18.

PRIEST ET AL.

Critical Solution Temperatures of Aqueous Copolymers 257

Gel Permeation Chromatography The r e l a t i v e molecular weights of the copolymers were compared by chromatography on a 1.0 χ 116 cm Sephacryl S-400 column with detection by absorbance at 214 nm. This also enabled the determination of the r e l a t i v e e f f i c i e n c y of polymerization reactions v i a integration of the t o t a l column volume (V ) peak. This was possible because the monomers are the predominant species of small molecules that absorb at 214 nm.


t

Analysis of the Copolymerizabilities of Monomers The composition of the copolymers formed was determined by measuring the r e l a t i v e amounts of each monomer, NIPAAM and AAM, that remained i n solution a f t e r a copolymerization. Copolymerizations were terminated by addition of 1 ml of reaction mix to 9 ml of 0.1% phosphoric acid at 50°C, followed by centrifugation of a 0.4 ml aliquot at 6,500 χ g for 5 minutes i n an Eppendorf microfuge. After 100 fold d i l u t i o n of an aliquot of the supernate, 200 μΐ of this was injected onto an IBM reversed phase C i HPLC column pre-equilibrated with 2% a c e t o n i t r i l e i n 0.1% aqueous phosphoric acid and the eluent monitored at 214 nm. The monomers were eluted using a 0.1% aqueous phosphoric acid (solvent A): a c e t o n i t r i l e (solvent B) gradient as follows: for 5 minutes the solvent was 98% solvent A and 2% solvent B, followed by a linear gradient to 80% A and 20% Β over 10 minutes. After 5 more minutes at 80% A and 20% B, the solvent was returned to 98% A and 2% B. 8

RESULTS The LCSTs of copolymers of NIPAAM with AAM, NMAAM or MEA were elevated as a regular function of their comonomer input r a t i o (Figure 1.) As expected, acrylamide was the most e f f e c t i v e at r a i s i n g the LCST of the copolymer formed, but s u r p r i s i n g l y NEAAM was more e f f i c i e n t than NMAAM. Since substitution of isopropyl groups with methyl groups has less effect than substitution with ethyl groups, one might expect polyNMAAM to exhibit an LCST. Below 95°C i t did not, but polyNEAAM exhibited an LCST between 85°C and 90°C. This was true whether the NEAAM i s polymerized using TEMED-persulfate or AIBN, ruling out an i n i t i a t o r e f f e c t on the LCST. Copolymers of NIPAAM with AAM, NMAAM and NEAAM were also analyzed by gel permeation chromatography both for molecular weight of the copolymers and for polymerization e f f i c i e n c y . A representative chromatogram i s i l l u s t r a t e d in Figure 2. The results of the analysis indicated no s i g n i f i c a n t differences i n the polymerization reactions which would explain the greater effectiveness of NEAAM over NMAAM at r a i s i n g the LCST of the copolymer formed (Table I ) . The gel permeation chromatogram of polyNEAAM was also not s i g n i f i c a n t l y d i f f e r e n t from that of polyNIPAAM. Additional tests of the randomness of the copolymers were conducted. Plots of absorbance at 600nm versus temperature for the polymer and the copolymers did not reveal any differences i n d i c a t i v e




258

\ 100

I 90

I 80

I 70

I 60

1 50

percent N-isopropyl acrylamide in polymerization mix Figure 1. Lower c r i t i c a l solution temperatures of copolymers of N-isopropyl acrylamide with other N-alkyl acrylamides as a function of monomer input r a t i o s .

Figure 2. Sephacryl S-400 Chromatogram of Acrylamide/N-isopropyl Acrylamide (10/90) Copolymer.


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PRIEST ET AL. Table I.


Polymer

Critical Solution Temperatures of Aqueous Copolymers 259 Results of Gel Permeation Chromatographic Analysis of Copolymers of NIPAAM

Kav

Residual Monomer (%)

polyNIPAAM

0.42

5.3

polyNIPAAM-AAM

0.45

7.9

polyNIPAAM-NMAAM

0.40

5.6

po1yNIΡAAM-NEAAM

0.40

5.1

l

K

a v

= V V t

e

- Vo - Vo

where V = e l u t i o n volume of the polymer Vo = column void volume V = t o t a l column volume e

t

of heterogeneity (Figure 3). However, the randomness of the copolymers of NIAPAAM and AAM was best supported by measurement of the copolymerization r e a c t i v i t y ratios for the two monomers. Figure 4 shows the results for a 10 minute reaction, a f t e r which at least 87% of the monomers had polymerized. These results indicated that the copolymerization was almost perfectly random (ri=r2=1.0). This high extent of polymerization usually accentuates deviations from random copolymerization using this protocol, but none was seen even after 3 hours (data not shown). Copolymerization of NIPAAM with NTBAAM proved to be an e f f e c t i v e method for producing material with an LCST that was lowered i n direct and apparently linear proportions to the amount of NTBAAM added (Figure 5). The poor water s o l u b i l i t y of NTBAAM was apparently not a problem. Likewise NNBAAM demonstrated a s i m i l a r dependence of LCST on co-monomer input r a t i o up to a point. Copolymers containing 40% or more NNBAAM, however, would not redissolve i n PBS at any of several temperatures including -2 and -4°C. Thus i t was not possible to produce copolymers of NIPAAM and NNBAAM that precipitate between 0 and 17°C. Beyond a c r i t i c a l number of η-butyl side chains per polymer molecule water s o l u b i l i t y v i r t u a l l y disappears. As observed for other copolymers that exhibit an LCST (3), sodium chloride depresses the LCST of NIPAAM copolymers (Figure 6). The difference appears to be very large for NIPAAM copolymers i n 0.9 M sodium chloride versus PBS (15 to over 20°C). These studies indicated that the NIPAAM-AAM copolymer that precipitated most e f f i c i e n t l y above 55°C and below 65°C i n 0.9M NaCl contained 67% NIPAAM and 33% AAM.


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0.7Γ

OL_ud 30

ΙΛ h Πι 40

50

60

Temperature, °C

Figure 3. Turbidity versus Temperature Curves f o r poly-N-isopropyl acrylamide and i t s copolymers.

1.0

0.8

0.6

mole fraction N I P A A M in polymerization mix

Figure 4. Copolymerization plot f o r acrylamide and N-isopropyl acrylamide (NIPAAM).



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PRIEST ET AL.


100

90

80

70

60

50

40

30

% N-isopropyl Acrylamide in Polymerization Mix

Figure 5· Lower C r i t i c a l Solution Temperatures of Copolymers of N-isopropyl Acrylamide and N-n- and N-t-butyl Acrylamide as a Function of Monomer Input Ratios.

Figure 6. Lower C r i t i c a l Solution Temperatures of Copolymers of NIPAAM and ΝΕΑ or AAM i n 0.15 M and 0.9 M NaCl.


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DISCUSSION Using N-isopropyl acrylamide and acrylamide or other N-substituted acrylamides i t i s possible to design copolymers that w i l l precipitate at any desired temperature between 0 and 65°C. I t i s not surprising that acrylamide i s the most e f f e c t i v e comonomer i n r a i s i n g the LCST. However, the greater effectiveness of NEAAM over that of NMAAM at r a i s i n g the LCST of the copolymer i s d i f f i c u l t to explain. If the NEAAM had contained a larger amount of polymerization i n h i b i t o r than the other monomers, which would have lowered the molecular weight of the polymer formed, the LCST might have been higher. This would also have lowered the extent of polymerization. Less than 1% of this kind of impurity could have these effects and s t i l l meet the purity s p e c i f i c a t i o n s of the manufacturer. This was found not to be the case by the two c r i t e r i a of polymer molecular weight and extent of polymerization. In addition, while the discrepancy between the l i t e r a t u r e values for the LCST for polyNEAAM (74°C) (3) and those determined i n our laboratories (85-90°C) also points to the NEAAM as the cause, the r e l a t i v e l y poor e f f i c i e n c y by which NMAAM raises the LCST of i t s NIPAAM copolymers indicates that the l a t t e r i s the abnormal monomer (Figure 1). For our purposes acrylamide appeared to be the best choice for a comonomer for the synthesis of high LCST copolymers, especially since i t can be obtained i n very high purity. For these copolymers to be used i n immunoassays, an antibody s p e c i f i c for the analyte of interest must be covalently bonded to them. This can be accomplished by the synthesis of an activated polymer followed by conjugation to an antibody (5). However, one may also accomplish this v i a the aqueous copolymerization (or terpolymerization) of an antibody-monomer conjugate with the free monomer (or monomers) by methods previously described (11-13). Due to the necessity to e f f i c i e n t l y incorporate antibody into these copolymers, a high extent of polymerization was desirable. This does not s i g n i f i c a n t l y change the LCSTs of copolymers of NIPAAM and AAM (3). However the determination of copolymerizability of the two monomers by the c l a s s i c a l method of analysis of the polymer composition i s usually not v a l i d at high extents of polymerization. While deviations from random copolymerization are obscured by measurements of the polymer composition at high conversion, they are amplified by measurement of the residual monomers. The finding of almost perfectly random behavior by these two monomers indicated to us that they had passed a c r i t i c a l and s u f f i c i e n t test. While our copolymerization r e a c t i v i t y ratios for NIPAAM and AAM disagree with those of C h i k l i s and Grasshoff (10), the extent of the disagreement i s r e l a t i v e l y small. Similar disagreements are often found i n the l i t e r a t u r e for other pairs of monomers (14). Thus the discrepancies could e a s i l y be due to the differences i n polymerization conditions, such as pH, i n i t i a t o r s and concentrations, monomer concentrations, etc. Many immunoassays are performed at 37°C or above (7). An advantage of the novel immunoassay methodology disclosed i n the previous paper i s that i t avoids a s i g n i f i c a n t problem encountered with many heterogeneous immunoassays, that of slow k i n e t i c s when binding of a molecule i n solution to a s o l i d surface i s involved.



18.

PRIEST ET AL.


The a b i l i t y to incubate a AAM-NIPAAM copolymer-antibody conjugate with the sample at 37°C should further improve this advantage. The effect of the butyl side chains on the LCST's of copolymers of NIPAAM i s interesting, e s p e c i a l l y i n the case of the n-butyl copolymer. One hypothesis i s that the η-butyl groups associate and precipitate more readily than the less f l e x i b l e t-butyl groups do. This i s consistent with the observation that poly-N-n-propyl acrylamide i s insoluble at room temperature i n water (3). For use i n immunoassays, copolymers that p r e c i p i t a t e at 0 to 17°C are d i f f i c u l t to synthesize by this method, especially when considering a third monomer component such as an antibody-monomer conjugate. Perhaps a better method i n this case i s the conjugation with activated copolymers (5), or the use of copolymers of AAM and NNBAAM. Another application of this methodology i s to use several d i f f e r e n t copolymers to detect multiple analytes i n a single sample. Each polymer would have a d i f f e r e n t antibody attached and be precipitated at a d i f f e r e n t temperature. This appears to be possible since one copolymer that precipitates at a lower temperature does not appear to remove a copolymer with a higher LCST from solution. ( J . H. P r i e s t , unpublished observations). It i s also conceivable that a DNA probe-based assay could be included with a panel of immunoassays. After removal of the various antibody-copolymer conjugates at lower temperature sodium chloride would be added, the hybridization would be performed at 55°C and the DNA-copolymer conjugates precipitated at 65°C. Acknowledgments We wish to thank Dr. Niels H. Andersen and associates of the Department of Chemistry, University of Washington, for many NMR, infrared and mass spectral analyses. Abbreviations AAM, acrylamide; NMAAM, N-methyl acrylamide; NEAAM, N-ethyl acrylamide; NNBAAM, N--n-butyl acrylamide; NTBAAM, N-t-butyl acrylamide; NIPAAM, N-isopropyl acrylamide; LCST, lower c r i t i c a l solution temperature; TEMED, Tetramethylethylenediamine; PBS, phosphate-buffered s a l i n e ; AIBN, 2,2' a z o b i s ( i s o b u t y r o n i t r i l e ) . Literature Cited 1. Heskins, M.; G u i l l e t , J . E. J . Macromol. S c i . Chem. 1968, A2 (8), 1441. 2. Haas, H. C.; MacDonald, R. L.; Schuler, A. N. J . Polymer S c i . 1970, 8 (part A-1), 3405. 3. Taylor, L. D.; Cerankowski, L. D. J . Polymer S c i . 1975, 13, 2551. 4. Hoffman, A. S.; A f r a s s i a b i , A.,; Dong, L. C., this symposium. 5. Cole, C-A.; Schreiner, S. M.; P r i e s t , J . H.; Monji, N.; Hoffman, A. S.,this symposium. 6. Jones, T.; Houghton, R. L. C l i n . Chem. 1986, 32, 1067.



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7. Tsang, V. C. W.; Wilson, B. C.; Peralta, J . M. In Immunochemical Techniques; Longone, J . J . ; Van Vunakis, H., Eds.; Methods i n Enzymology Vol. 9, part Ε Academic Press, Orlando, F l o r i d a , 1983. 8. Meinkoth, J . ; Wahl, G. Anal. Biochem. 1984, 138 267-284. 9. Young, B. D.; Anderson, M. L. M. In Nucleic Acid Hybridization, A P r a c t i c a l Approach; Hames, B. D.;Higgins S. J . , Eds.; IRL Press, Washington D.C., 1985. 10. C h i k l i s , C. K.; Grasshoff, J . M. J . Polymer S c i . 1970, 8, 1617. 11. Pollak, Α.; Blumenfeld, H.; Wax, M.; Baughn, R. L.; Whitesides, G. M. J . Am. Chem. Soc. 1980, 56, 6324-6336. 12. Plate, Ν. Α.; Valuev, L. I.; Chupov, V. V. Pure and Appl. Chem. 1984, 56(10), 1351-1370. 13. Nowinski, R. C.; Hoffman, A. S. U.S. Patent 4 511 478, 1985. 14. Young, L.J. In Polymer Handbook, (Brandrup, J . ; Immergut, E. H., Eds.) p.II-105. 2nd Edition, John Wiley əSons, New York, 1974; 11-105. RECEIVED April 24, 1987


Lower Critical Solution Temperatures of Aqueous Copolymers of

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