Anal. Chem. IQQ2,64, 694-697 Coupland, R. E.; Pyper, A. S.; Hopwood, D. Nature 1984, 207, 124-242. Benedeczky, A.; Puppi, A.; Tlgyl, A.; Lissak, K. Nature 1964, 204, 591-592. Benedeczky, I.; Puppl, A.; Tigyi, A.; Lissak, K. Nature 1988, 209, 592-594. Hagen, P.; Bannet, R. J. Adrenergic Mechanisms; Little, Brown and Co.: Boston, 1960; pp 83-99. MlcheCBechet. M.; Cotte, 0.; Haon, A.-M. J. Mlcroscop. 1963, 2 , 449-460. Kennedy, R. T.; Jorgenson, J. W. Anal. Chem. 1988, 67, 436-441. Oates, M. D.; Cooper. B. R.; Jorgenson, J. W. Anal. Chem. 1990, 62, 1573-1577. Kennedy. R. T.: Oates, M. D.; COODW,B. R.; Nickerson, B.; Jorgenson, J. w. sc/ence 1089,.?46,57-63: Okflrowicz. T. M.;Ewing, A. G. Anal. Chem. 1890, 62, 1872-1876. McCaman, R. M.; Welnrlch, D.; Borys, H. J. Neurochem. 1973, 21, 473-476. McAdoo, D. J. Blochemstry of Characterized Neurons; Pergamon: Oxford, U.K., 1978; pp 19-45.
(14) Lent, C. M.; Meuiler, R. L.; Haycock, D. A. J. Neurochem. 1089, 47, 481-490. (15) Kennedy, R. T.; Jorgenson, J. W. Anal. Chem. 1980, 67, 1128-1135. (16) Knecht, L. A.; Guthrie, E. J.; Jorgenson, J. W. Anal. Chem. 1984, 56, 479-482. (17) St. 186-191. Claire, R. L.; Jorgenson, J. W. J. Chromatogr. Scl. 1985, 23, (18) Wilson, S. P.; Viveros, 0. H. €xp. Cell Res. 1861, 733. 159-169. (19) Leszczyszyn, D. J.: Jankowskl, J. A.; Vlveros, 0. H.; Dlliberto, E. J.; Near, J. A.; Wightman, R. M. J. Neufochem. 1001, 56, 1855-1863. (20) Moro, M. A.; Lopez, M. G.; Ganda, L.; Michebna, P.; Garcia, A. G. Anal. Blochem. 1890, 785, 243-248. (21) Kennedy, R. T.; Jorgenson, J. W. Anal. Chem. 1988, 60, 1521-1524. (22) White. J. G.; St. Claire, R. L.; Jorgenson, J. W. Anal. Chem. 1086, 58, 293-298. (23) Phillips, J. H. Neurosclence 1082, 7, 1595-1609.
RECEIVED for review June 24,1991. Revised manuscript received November 5, 1991. Accepted November 11, 1991.
CORRESPONDENCE Heparin-Responsive Electrochemical Sensor: A Preliminary Study Sir: Polymer membrane type ion-selective electrodes (ISEs) are now used routinely within biomedical instruments to measure clinically important ions (e.g., Na+, Li+, K+, H+,C1-, etc.) in undiluted whole Efforts to develop similar sensors (including immune-based biosensors) suitable for the direct detection of larger biomolecules, such as drugs and specific proteins, have been less successful3 owing to the difficulty in identifving appropriate membrane chemistries that will yield a significant and specific electrochemical response toward the desired analytes. Toward this goal, we describe herein a polymer membrane-based electrode that exhibits significant potentiometric response to heparin in the clinically relevant concentration range. Heparin is an anionic rodlike polysaccharide (copolymer of uronic/iduronic acids alternating with sulfated glucosamine residues; Figure 1)with an average molecular weight of approximately 15000 Daa4 It is the anticoagulant drug used universally during surgical procedures and extracorporeal therapies?" The anticoagulant activity of thisdrug is believed to be due to its ionic interaction with antithrombin I11 (ATIII, a serine protease inhibitor), causing the formation of heparin-ATIII complexes which potentiate the inhibition of ATIII on enzymes involved in the coagulation cascade.' Because of the potential bleeding risks associated with its use? accurate monitoring of heparin is critical. At present, there is no method suitable for direct and rapid determination of physiological heparin levels. Currently available heparin assays such as the Activated Clotting Time (ACT) are all based on the measurements of blood clotting time. Despite wide clinical use for many years, these clotting time tests are not specific for heparin and lack speed and accuracy, as well as a defiied bio~hemistry.~ We were interested therefore in applying conventional ISE polymer membrane technology to devise a membrane electrode that is capable of detecting directly the concentrations of heparin in blood or plasma samples. Initial studies focused on the use of a quaternary ammonium salt, tridodecylmethylammonium chloride (TDMAC),as the "heparin carrier" incorporated within the polymer membrane phase. TDMAC has structural similarity to polybrene, a highly potent heparin antagonist,l0and is known to possess strong ion-association with heparin." Indeed, TDMAC-heparin complexes have
been incorporated into polymeric materials previously for preparing biocompatible devices,12J3from which heparin is released slowly via an ion-exchangeprocess. In addition, PVC membranes doped with TDMAC or other quaternary ammonium salts have been suggested for fabricating conventional ISEs for small anions including ~ h l o r i d e . ~Nevertheless, ~J~ none of these quaternary ammonium salt-based polymer membrane electrodes have ever been examined in detail with respect to heparin response. Surprisingly, we now find that membranes doped with TDMAC do in fact exhibit significant potentiometric response toward heparin in the presence of normal physiological levels of NaCl. The purpose of this correspondence is to report our initial results aimed at characterizing this response in terms of its dependence on membrane composition and its potential analytical utility for detecting heparin in plasma or whole blood samples. EXPERIMENTAL SECTION Reagents. Poly(viny1 chloride) (PVC) was obtained from Polyscience, Inc. (Warrington,PA). Dioctylsebacate (DOS) and tridodecylmethylammonium chloride (TDMAC) were purchased from Fluka Chemika-Biochemika (Ronkonkoma, NY). Sodium heparin was from Hepar Industries, Inc. (Franklin, OH) and poly(viny1sulfate), chondroitin sulfate A, chondroitin sulfate B, D-glUCOSamine, D-glUCOSamine 2,3-disulfate, D-glUCOSamine 2sulfate,D-gh"ine 3-sulfate, and D-glucosamhe &sulfate were products of Sigma Chemical Co. (St. Louis, MO). Citrated fresh frozen human plasma was obtained from American Red Cross (Southfield, MI). All other reagents were of analytical grade. Solutions were prepared with double-distilled deionized water. Preparation of Polymer Membranes and Apparatus. Polymer membranes were cast using the conventional method for ISE membrane preparation.I6 Small disks (-5 mm i.d.) of polymer membranes were cut and incorporated into Phillips electrode bodies (IS-561, Glasblaserei Moller, Zurich). A 15 mmol/L NaCl solution was used as the internal filling solution throughout the study. Electrodes were soaked in the same NaCl solution overnight prior to their use. The potentiometric response of the membrane electrode was measured relative to an external double junction Ag/AgCl reference electrode at room temperature (-22 "C), with the sample solution being stirred constantly (see Figure 2).
RESULTS AND DISCUSSION Although it was known that tridodecylmethylammonium
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 6, MARCH 15, 1992
695
Table I. Potentiometric Heparin Response of TDMAC-Based PVC Membranes with Various Membrane Compositions
AE,"mV OH
OR
R
R = H or SO;
R'/
x
membrane
R * =~ ~ j COCH, o r
Flgure 1. Oligosaccharide sequence of carbohydrate residues in heparin. f?
h 1
j
k
AdAgCI-
4moVLKCI
a
.O15 moVL NaCl
0.01 5 moVL NaCl
stir bar
~
1 I
heparin
Figure 2. 1set up of the heparinsensing electrode system and an expanded view of the process occurring on TDMAWPVC membrane/sample interface.
ions bind strongly with heparin,12J3preliminary studies focused on examining a wide range of quaternary ammonium salts as membrane-active components for heparin sensing using membrane formulations similar to those applied in conventional polymer membrane ISEs (i.e., -33 w t YO PVC, -65 wt 90plasticizer, and -1-3 wt 940 quaternary ammonium salt.I6 Among 14 quaternary ammonium salts examined, however, only membranes doped with TDMAC or Aliquat 336 (tricaprylmethylammonium chloride) exhibited significant potentiometric anion response when heparin was added to a 0.15 mol/L NaCl solution. It is interesting to note that membranes doped with the tetraalkyl analogues of these two species (i.e., tetradodecylammonium and tetraoctylammonium) exhibited negligible heparin response, suggesting that some accessibility to the positively charged nitrogen atom is required for heparin interaction within the membrane. On the other hand, a much smaller potentiometric response to heparin was observed when only one or two of the alkyl chains of the quaternary ammonium species were 2 C8 and the rest were small, less lipophilic methyl groups (e.g., trimethylhexadecylammonium, dioctadecyldimethylammonium,etc.). These early studies indicated that the specific structure of the quaternary ammonium species doped within the polymeric
(0-1.4 units/mL heparin in 0.12
PVC content,
DOS content,
TDMAC content,
mol/L NaCl)
wt 7'%
wt 90
wt%
-11.6 -10.4 -2.1 -7.0 -5.0 -30.7 -41.8 -38.0 -44.4 -3 1.O -45.0
33.0 32.6 32.7 32.4 32.4 48.9 65.8 73.1 65.5 66.4 65.5
66.0 65.9 65.3 65.1 64.6 50.5 33.3 26.0 33.1 33.1 32.5
1.0 1.6 2.0 2.5 3.0 1.1 0.9 0.9 1.4 0.5 2.0
Averane of duplicates obtained from three electrodes.
membrane was a very importat factor in determining the magnitude of the potentiometric response to heparin. Subsequent studies with Aliquat 336-doped membranes showed significant nonspecific response to components in blood and plasma samples (without heparin added), and further examination of such Aliquat-based membranes was therefore discontinued. To further optimize heparin response, various TDMACdoped PVC membranes with different formulations of plasticizers and/or TDMAC were prepared and tested. As shown in Table I, membranes prepared with a lower wt % plasticizer relative to the PVC content exhibit the greatest response to heparin. Indeed, the TDMAC-doped membranes formulated with normal (high) levels of plasticizer (i.e., -65 wt 9%) display significant response to heparin only at high heparin levels, whereas membranes formulated with lower wt YO plasticizer exhibit much greater total heparin response and at lower concentrations of heparin. The results shown in Table I indicate that membranes formulated with 65 wt % PVC, 33 wt % plasticizer, and 1.4-2.0 wt 940 TDMAC have optimum response to heparin (i.e., membranes i and k), and these were used in all further studies. As shown in Figure 3A, the electrodecontaining membrane i (see Table I) is able to detect low levels of heparin (0.1-1.0 units/mL) in solution even in the presence of 0.12 mol/L chloride. Figure 3A also shows that the response to chloride over its normal/and narrow physiological concentrationrange is much less than that for heparin. Indeed, for a 1 unit/mL heparin sample, changing the concentration of background chloride from 0.10 mol/L to 0.12 mol/L, alters the membrane electrode's potential by 0.6 mV. This would correspond to an apparent change in heparin concentration of only 2.9% (based on calibration curve data). It should be noted that no response to added heparin is observed when a dialysis membrane (MW cutoff: 12000 Da) is placed over the surface of the sensing membrane to block the heparin-TDMAC interaction (see Figure 4). Furthermore, when protamine (a clinically used heparin antagonist)1° is added to the heparin sample, the potential of the electrode immediately shifts toward a more positive value, as the activity of free heparin in solution decreases (data are not shown). These observations indicate that the electrode is responding directly to the macromolecular heparin and not to smaller ionic impurities within the heparin preparation used in these studies. Linear potentiometric response was also observed in undiluted human plasma samples a t heparin concentrations between 1.0 and 9.8 units/mL (Figure 3B). The somewhat reduced response in plasma versus saline solution may be due to the binding of heparin by endogenous proteins (e.g., an-
696
ANALYTICAL CHEMISTRY, VOL. 64, NO. 6, MARCH 15, 1992 Lop [CU, M -1.05 10
-1.00
-0.95
-0.90
Table 11. Potentiometric Response of Heparin Sensor toward Various Compoundsa sulfate content,
0
.10
tested compounds* (12 pg/mL)
> E
-20
3
-30
glycosaminoglycans heparin dermatan sulfate chondroitin sulfate A hyaluronic acid poly(viny1 sulfate)e glucosamine residues glucosamine g1ucosamin e 2-sulfate g1ucosamin e 3-sulfate (free acid) glucosamine 6-sulfate (free base) glucosamine 2,3-disulfate
-40
.so .LO
1.0
0.0
Log [heparin], Uiml
-15
%
-25
w' a
-3s
0.2
0.0
0.4
0.6
0.8
1.0
Log [heparin], U/ml
Figure 3. Response characteristics of heparin sensor: (A) response toward CI- In the concentration range of 0.09 mol/L and 0.12 moi/L (O),and toward heparln In background of 0.12 mol/L NaCi (0); (B) response toward heparin In cltrated fresh human plasma sample. A€ represents the potential change from the original cell potential (wlthout added heparln or chloride) and is plotted against logarithm of concentration of the analytes. The results plotted In A and B are the mean f sd for dupilcates of two different heparin electrodes. Response slopes are 49.2 mV/dec and 28.2 mV/dec separately for linear ranges In 0.12 moi/L NaCi (heparln concentration between 0.2 and 1.0 unit/mL) and undiluted human plasma (heparin concentration between 1.0 and 9.8 units/mL) samples, respectively.
I
10 min.
Flgurs 4. Chart recording of the potentiometric response toward the addition of 1.4 units/mL heparin (final concentration) In the presence of 0.12 mol/L NaCi: (A) of TDMAC membrane; (B) of TDMAC membrane covered with a piece of dialysis membrane.
tithrombin-111), and/or the inhibition of heparin extraction into the membrane phase by surface adsorbed plasma proteins. Nonetheless, given that heparin levels encountered in most surgical procedures are within 2-8 unita/mL,5 the sensitivity of the electrode appears to be more than adequate to meet this clinical need. Preliminary studies using heparinized sheep whole blood samples reveal similar electrode response characteristics.
AE,'mVd
wt%
-50 -25 -10 0 0
13 9 7 0 62
0 0
0 25
0
27
0
27
0
42
"All compounds were prepared in 0.12 mol/L NaCl solution at the same concentration (Le., 12 pg/mL). For heparin, this concentration is equivalent to 2 units/mL activity. Unless otherwise specified, all compounds are in their sodium salt form. Average of duplicates obtained from three electrodes. dChange in cell potential compared to solution of 0.12 mol/L NaC1. eThe [poly(vinyl sulfate)] may be different from the others since only the soluble part present in the supernatant is used, the nonsoluble part is filtered off.
Among many glycosaminoglycanstested (see Table 11),the electrode was found to yield increased potentiometric response correlating to the degree of sulfate content of these compounds. Interestingly, the electrode displays no measurable response to poly(viny1 sulfate) (PVS), a highly-sulfated polymer, nor to sulfated or nonsulfated glucosamine residues which are the major monosaccharide building blocks of heparin. These results indicate that heparin's high sulfate content and ita specific polymeric structure are both essential for ita interaction with the TDMAC-doped membrane. A two-phase extraction experiment conducted by mixing a TDMAC/DOS phase with a fluorescein-labeledheparin/H,O phase shows that heparin is in fact extracted into the organic DOS layer, suggesting that the response of the electrode is not due solely to simple adsorption of highly anionic heparin on the surface of the TDMAC-PVC membrane. Preliminary electrodialysis studies, performed as described by Thoma et al.,17also reveal the permeation of fluorescein-labeled heparin into the bulk phase of the TDMAC/DOS/PVC membrane. These results indicate that the response mechanism of the sensor may resemble that observed with charged-carrier based polymer membrane electrodes,'* which involves solvent extraction of the analyte into the organic membrane phase and concomitant ion complexation of analyte with the carrier. The preferred selectivity of heparin to ita analogues and the sulfated glucosamine residues may thus be due to the ability of heparin's many sulfate groups to interact simultaneously with a large number of relatively immobile, positively charged tridodecylmethylammonium sites in the hydrophobic membrane (see Figure 2). Lack of response to PVS may be attributed to the rather hydrophilic nature of PVS that diminishes ita initial extraction into the hydrophobic organic membrane. Notably, as with other polymer membrane electrodes prepared with quaternary ammonium ion-exchange species, the heparin sensor does exhibit substantial response to more lipophilic anions such as salicylate and bromide; however, these species would normally not be present at interfering levels in the blood of systemically heparinized patients.
Anal. Chem. 1992. 64, 697-700
We have demonstrated that by impregnating a suitable "carrier" into a polymer membrane with adequate composition, it is feasible to devise an electrochemical sensor that responds direcly and rather selectively to a polyionic macromolecule. The response time of the electrode to heparin is sufficiently rapid to yield stable signals in 2-5 min at therapeutic heparin doses (1-10 unita/mL) (see Figure 3). Response to decreasing levels of heparin is considerably slower, although the rate of this reverse response can be greatly enhanced by rinsing the electrode with protamine or a high ionic strength saline solution (to extract and/or dissociate the heparin from the membrane). Nonetheless, given the importance and critical need for monitoring heparin ievels, the sensor described here may ultimately offer attractive advantages over existing approaches based on clotting time measurements. Indeed, it is envisioned that proposed membrane chemistry could be implemented in a disposable single-use electrode format (similar to the Kodak's Ektachem d e ~ i g n ' ~for , ~ )rapid estimation of free heparin levels in undiluted blood. Work on demonstrating the clinical utility of this approach is currently in progress as are efforts to understand more fully the exact mechanism of heparin response.
ACKNOWLEDGMENT This work is partially supported by grants from the National Institute of Health, R29-HL 38353 and GM-28882. REFERENCES (1) Oesch, U.; Ammann, D.; Simon, W. Clln. Chem. 1988, 32, 1448. (2) Byrne, T. P. S e k t i v e E k t . Rev. 1988, 10, 107. (3) Arold, M. A.; Meyerhoff, M. E. CRC Crlt. Rev. Anal. Chem. 1988, 20 (a), 149. (4) Danishefsky, I.; Rosenfeld, L.; Kuhn. L.; Lahiri, B.; Whyzmuzis, C. I n Heperln and Related pdysecchark%s I Structure and Actfvltles, Ann. New York Acad. Scl. 556; Ofosu, F. A., et al., Eds.; The New York Academy of Sciences: New York, 1989, p 29. (5) Jaques, L. 8. pharmacal. Rev. 1980, 31, 99. (6) Lindhardt. R. J. Chem. Ind. 1991, 45.
607
(7) O'Reiily, R. A. In OOODMAN and OILMAN'S the mmcdoglcel Besls of TherepeutlcS, 7th ed.; Oliman. A. G., et ai., Eds.; Macmiilan Publishing Co.: New York, 1985, p 1338. (8) Hirsh, J. Now. Rev. Fr. Hematol. 1984. 2 6 , 261. (9) Walenga, J. M.; Fareed, J.; Hoppensteadt, D.; Emanuele, R. M. CRC Crlt. Rev. Clln. Lab. Scl. 1985, 22, 361. (10) -1, H. C. Scandlnav. J . (%. Lab. Invest. 1960. 12. 446. (11) &ode, 0. A.; Faib. R. D.; Crowley. J. P. J . 8 M . Meter. Res. Symp. 1972, 3 , 77. (12) NalJar, F. B.; Gott, V. L. Swg. 1970, 66,1053. (13) Gott, V. L. Ann. Thcfacic Surg. 1972, 14, 219. (14) Hartman, K.; Luterotti, S.; Osswald, H. F.; Oehme, M.; Meler, P. C.; Ammann, D.; Simon, W. Mikrochlm. Acta 1978, 11, 235. (15) Wegmann, D.; Weiss. H.;Ammann, D.; Morf. W. E.; Pretsch, Sugahara, E. K.; Simon, W. Mikrochim. Acta 1984, 111, 1. (16) Craggs, A.; Moody. 0. J.; Thomas, J. D. R. J . Chem. Ed. 1974, 51. 541. (17) Thoma, A. P.; VivlanCNauer, A.; S. Arvanltis. Mod. W. E.; Simon, W. AMI. Chem. 1977. 49(11), 1567. (18) Mod, W. E. In The Mnclples of Ion-Sekcthre E&ctro&s and of Membrane Transport. Studies In Anal. Chem. 2 ; Pungor, E., et ai., Eds.; Elsevier Sci. Publishing Co.: New York, 1981. p 211. (19) Curme, H. G.; Babaogiu. K.; Babb, B. E.; Battagila, C. J.; Beavers, D. J.; Bogdenowicz, M. J.; Chang. J. C.; Daniel, D. S.; Kim, S. H.;Klssel, T. R.; Sandifer, J. R.; Schnipelsky, P. N.; Searie, R.; Secord. D. S.; Spayd, R. W. Clln. Chem. 1979, 2 5 , 1115. (20) Walter, B. Anal. Chem. 1983, 55 (4), 498A. To whom correspondence and reprint requests should be addressed.
Shu-Ching Ma Victor C. Yang* College of Pharmacy The University of Michigan Ann Arbor, Michigan 48109
Mark E. Meyerhoff Department of Chemistry The University of Michigan Ann Arbor, Michigan 48109 RECEIVED for review September 26,1991. Accepted December 30, 1991.
Trace Analysis in Solution Using Zeolite-Modified Electrodes Sir: The quest for devices and procedures to quantitatively determine specific solution-phase moieties has led analytical chemists into many cross disciplinary ventures. The need for rapid detection of various analytes in both living and nonliving systems has indeed provoked a massive research contributi~n.'-~A particularly attractive goal has been the pursuit of selective chemical sensors that will reproducibly detect a target analyte without the attendant interference problems associated with the analysis of multicomponent systems. The selectivity problems associated with chemical systems that have been devised or proposed as sensors has therefore been a recurrent theme in this area,, Many elegant schemes have been proposed and demonstrated to circumvent selectivity problems. This correspondence reports the implementation of zeolite-modified electrodes in solution-phase, amperometric analysis. The principal advantage of amperometric techniques over potentiometric determir'ations being one of sensitivity. For example, differential pulse anodic stripping methods offer sub-ppb detection limits for many inorganic ions. One challenge in applying amperometric detection methods is to build Lhemical selectivity into the device. Although unmodified electrodes have shown little promise in this area, chemically modified electrodes offer some hope in the fruition of 0003-2700/92/0364-0697$03.00/0
this goal. Indeed size-selective phenomena have been demonstrated for chemically modified electrodes on a number of
occasion^.^^^ The zeolite molecular sieves are interesting materials for application in electroanalyticalchemistry, and a review of this area has recently appeared.6 The size and shape selectivity of zeolites is well documented'* and has been exploited in several industrially important processes.1° In this paper we show that the properties of ion-exchange and size-selectivity of zeolite molecular sieves play important roles in their use as electroanalyticaldevices. The following discwion will focus on two applications; (i) determination of solution-phase cations at and below ppm concentrations; (ii) selective determinations of trace quantities of water in organic liquids."
EXPERIMENTAL SECTION The zeolites used in this study were donated by Union Carbide. They were received in the sodium form. The samples were first washed in 0.1 M NaCl solutions prior to further exchange with electroactive species (vide infra). In this study zeolites Linde Type A and Y (LZY 72) were used. These were ion-exchanged with
copper and silver ions in aqueous solution at room temperature using 5-10 mM solutions of the acetate or nitrate of the cation. Typically, 1g of zeolite was exchanged overnight in 250 mL of HzO. Following this, the zeolite was washed with Barnstead0 1992 American Chemical Society
