Anal. Chem. 1980, 6 0 , 1455-1458
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Elimination of the Asymmetry in Neutral-Carrier-Based Solvent Polymeric Membranes Induced by Proteins Lucas F. J. Durselen, Dorothke W e g m a n n , Kurt May, Urs O e s c h , and Wilhelm Simon* Department of Organic Chemistry, Swiss Federal Institute of Technology (ETH),CH-8092 Zurich, Switzerland
Ion-selective electrode cell assemblles based on solvent polymeric membranes suffer from a shMt In the standard potential of the cell when changlng the sample from an aqueous electrolyte solutlon to protein-contalnlng solutlons (e.g. blood serum). This shift Is due to the Induction of a membrane asymmetry potential In inherently symmetric membranes through a contamlnatlon of the membrane surface by proteins. Membranes prepared with vinyl chloride-vlnyl alcohol copolymers do not suffer from such shifts and exhibit a similar electromotive behavior (selectlvlty, slope of electrode response) as the parent poly(vinyl chlorlde) membranes.
Among t h e ion-selective potentiometric sensors, t h e glass and the solvent polymeric membrane electrodes have become a routine tool in clinical chemistry and neurophysiological laboratories (for reviews see ref 1-4). It has, however, repeatedly been observed that such potentiometric sensors may suffer from a shift in the standard cell potential Eo when t h e ion-selective membrane is contacted by sample solutions containing proteins after conditioning t h e cell assembly in aqueous standards (5,6). A solution t o this problem is normally attempted by aqueous recalibration after t h e sample contact (5). If the protein-induced Eo shift remained constant and sample independent, this could be accepted as a reliable calibration procedure. I n some solvent polymeric membranes, poor reproducible E, shifts of u p t o 2 mV (4-6)have been observed; they were reduced by coating t h e sensor with a cellulose acetate membrane (5) at the cost of prolonged response time. Since these shifts may be large as compared with t h e emf change due t o varying t h e activity of the primary ion over the physiological range (Ca2+, 2.4 mV; Na', 2.4 mV ( 4 , 7)), t h e procedure is questionable. Because of t h e sample dependence of t h e E, shift (5) even cdibrations with standards containing proteins (5, 8 ) are unreliable. T h e present study was undertaken t o quantify t h e shift in the standard potential of neutral-carrier-based solvent polymeric membranes induced by a protein contact and t o eliminate this effect. EXPERIMENTAL SECTION Electrode Systems. For all measurements of the asymmetry potential E, of the membranes, a cell of the following type was used: Hg;Hg2C12,KC1(satd)lO.l M KCllelectrolyte solution11 membrane11electrolyte so1ution)O.l M KClJKC1(satd),Hg,Cl,;Hg. The electrolyte solution was a 0.1 M chloride solution of the corresponding primary ion, i.e. KCl for the K+-selective membranes (valinomycin), NaCl for the Na+-selective membranes (ETH 157 or E T H 21201, and CaClz for the Ca2+-selective membranes (ETH 1001). Each half-cell contained about 15 mL of solution, which was not stirred. The body of the cell shown in Figure 1 was made of Teflon. It contained two reference electrode half-cells with free-flowing free-diffusion liquid junctions as described earlier (9). For the emf measurements with ion-selective electrodes, cells of the type Hg;Hg,Cl,,KCl (satd)(bridge electrolytelsample solution(lmembrane)linnerfilling solution,AgCl;Ag were used. The 0003-2700/88/0360-1455$01 S O / O
membranes were incorporated into Philips IS 560 electrode bodies (Philips N.V. Gloeilampenfabriken, Eindhoven, The Netherlands), the inner filling solution being a 0.01 M chloride solution of the respective primary ion. The reference electrode was as described above (9), with 1 M LiOAc as bridge electrolyte for the measurements with valinomycin membranes and 3 M KCl for the remaining ones. Reagents. All electrolyte solutions for the potentiometric measurements were prepared with doubly quartz distilled water and salts of p.a. grade (E. Merck, Darmstadt, FRG, and Fluka AG, Buchs, Switzerland). The membranes listed in Table I were prepared according to ref 9 and ref 14 in ref 9. Poly(viny1chloride) (PVC hochmolekular), the ionophores valinomycin, ETH 157, ETH 2120, and ETH 1001, the plasticizer BBPA [(bis(l-butylpentyl) adipate] as well as potassium tetrakis(pchloropheny1)borate (KTpClF'B; purum pa.), and tetrahydrofuran (THF, puriss. p.a., distilled under N2before use) were obtained from Fluka AG, CH-9470 Buchs. For the protein-conditioning experiments bovine serum albumin (BSA; Fluka) as a 7 wt % solution in double quartz distilled water or human serum albumin (HSA; Central Laboratory of the Swiss Red Cross Blood Transfusion Service) as a 6 wt % solution in a physiological NaCl solution (0.9 wt % ) were used. Measurement of the Asymmetry Potential. The asymmetry potentials were measured at 21 f 1 "C. The offset of the reference electrodes was first obtained by immersing them into a glass beaker containing the electrolyte solution. After about 1 2 h the cell with the membrane was assembled, both electrolyte compartments were filled with the same solutions as used for the offset measurements, and both reference electrodes were connected to a Digital Multimeter Type 7150 (Solartron Schlumberger Instruments, Farnborough, UK). For the remote control and data storage and handling, a personal computer Apple IIe was used with an IEEE 488 interface, an extended RAM memory (Apple Computer, Inc., Cupertino, CA), a real-time clock (Thunderware, Inc., Oakland, CA), a Model RX 80 matrix printer (Epson Corp., Nagano, Japan), and a Graphic Plotter Color Pro (HewlettPackard, San Diego, CA), managed by a high-level language program (UCSD Pascal 1.3) written for this use. Values for E, were taken every 60 s or 5 min over a period of 24-60 h. emf Measurements w i t h Ion-Selective Electrodes. The measurements were performed with the equipment described earlier (ref 17 in ref 9). The electrode response functions, selectivities, and liquid-junction potentials were calculated off-line with a H P 85 calculator system as mentioned elsewhere (9). Conditioning Experiments. The membranes were mounted into the measuring cell (Figure l), one half-cell being filled with the corresponding electrolyte solution and the other with a 7 wt % BSA solution. The conditioning time was 24 h. Then both half-cells were washed several times with the same electrolyte solutions used afterward for the measurement of E,. The time delay between the protein contact and the beginning of the E,, measurements was typically about 1min. For the conditioning of both membrane surfaces with BSA, the same procedure was applied to the other half-cell after measuring E,, during 24 h. P r e p a r a t i o n of the Vinyl Chloride-Vinyl Alcohol Copolymer (OH-PVC). The educt for the preparation of the polymer (OH-PVC) was a copolymer of 81 wt % vinyl chloride, 17 wt % vinyl acetate, and 2 w t % maleic acid, which was obtained from Scientific Polymer Products, Inc., Ontario, NY. The copolymer educt for the membranes la, lb, 5a, and 5b consisted of 82.9 wt % vinyl chloride and 17.1 wt % vinyl acetate prepared on a small scale (charge 916D) by I.C.I. (Switzerland) AG, Kunstoffwerk, CH-5643 Sins. The poly(viny1 chloride)-poly(viny1 1988 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 14, JULY 15, 1988
Table I. Composition of the Membranes Studied
type of membrane
primary ion
polymer
la Ib 2a
K+ K+ Na+ Na+ Na+ Na+ Ca2+ Ca2+ Ca2+ Ca2+
33 PVC 33 OH-PVC 33 PVC 33 OH-PVC 33 PVC 33 OH-PVC 33 PVC 33 OH-PVC 30.7 PVC 30.7 OH-PVC
2b
3a 3b
4a 4b
5a 5b
composition in wt '30 plasticizer ionophore
additive
1 valinomycin 1 valinomycin 1 ETH 157 1 ETH 157 1 ETH 2120 1 ETH 2120 1 ETH 1001 1 ETH 1001
66 BBPA 66 BBPA 66 BBPA 66 BBPA 66 BBPA 66 BBPA 66 BBPA 66 BBPA 63.9 BBPA 63.9 BBPA
3.3 ETH 1001 3.3 ETH 1001
2.1 KTpClPB 2.1 KTDClPB
1 cm H
C
15
30
0
15
30
, 0
15
30
T'ME [ m ~ n ]
Figure 2. Asymmetry potentials E, of the reference electrodes and
a membrane before and after conditioning.
7
L
3
5 6
3
L
7
Figure 1. Cell used for the measurement of asymmetry potentials: 1, membrane; 2, reference electmdes (SCE); 3, electrolyte compartments; 4, cell body (Teflon);5, membrane holder (poly(methy1 methacrylate) ring); 6, membrane support (poly(methyi methacrylate) ring); 7, cell support (aluminum).
acetate) copolymer (5 g) was dissolved in 100 mL of T H F and, during a period of 10 min, added dropwise to a solution of 1wt % NaOH (puriss., Siegfried AG, CH-4800 Zofingen) in 30 mL of methanol (MeOH; Fluka, puriss. p.a.) at 50-60 "C. The solution was stirred during 1.5 h at 63 "C, reduced to half of the volume, and then passed through filter paper into 1.5 L of water (25 "C) under stirring. After 10 min, the precipitate was filtered off, suspended in 300 mL of MeOH, and filtered again. The residue was suspended once more in 200 mL of MeOH and poured into 0.8 L of water (25 OC). After the product was filtered and dried (60"C, vacuum), 1.2 g of product was obtained. The IR spectrum of a membrane cast with 50 mg of OH-PVC in 1.5 mL of T H F showed a broad OH signal a t 3400 cm-l, the sharp signal at 1732 cm-' due to the CO stretching vibration of the educt being absent. P r e p a r a t i o n of Radiotracer Solutions. HSA was labeled with I3lI (IA4, Swiss Federal Institute for Reactor Research, CH-5303 Wurenlingen) according to the following method: To 37 MBq l 3 I I as NaI in a septum vial, 500 pL of buffer solution (0.1 M TRIS buffer), 500 fiL of a HSA solution (250 mg/mL in 0.9 wt % NaCl solution), and 200 p L of chloramine T solution (2 mg/mL, Fluka) were added through syringes. The mixture was shaken during 5 min, after which the reaction was quenched by adding 200 WLof a sodium disulfite solution (3 mg/mL). Purification of the labeled material was achieved by quick column chromatography (DEAE Sephadex A 25-120, Sigma Chemicals, St. Louis, MO) and elution with HSA solution. The labeling efficiency and purity were determined by TLC (Polygram CEL 300, Macherey Nagel, Hamburg, FRG; mobile phase MeOH/water 85:15 (v/v)). The purified labeled HSA solution was made up with inactive HSA to 50 mL of a 6 wt '30 solution. The labeled BSA solution was prepared by diluting 185 kBq of l4C-methylated BSA (CFA 621, Amersham International plc, GB) with inactive
BSA (Fluka) to give 10 mL of a 6 wt % protein solution. Membrane Preparation f o r Tracer Experiments. Membranes were prepared by pouring a solution of the components in THF into a glass ring of 24 mm diameter (ref 14 in ref 9),which rested on a glass plate covered with fluoroethylene propylene foil, and letting the T H F evaporate overnight. The resulting membrane tightly adhered to the glass ring. Into this vessel the radiotracer solution was filled and allowed to stand for 24 h. The active solution was withdrawn and the membrane rinsed several times with electrolyte solution and finally with water. After the membrane was dried, the center part of the membrane with a diameter of 18 mm was cut out and assayed for radioactivity. Radioactivity Measurements. For y counting the membrane segments as well as the reference solutions were placed into Eppendorf vials to assure constant geometry and the activity was determined with a Gammascint BF 5300 counter (Fa. Berthold, Wildbad, FRG). p counting was performed with a liquid scintillation spectrometer Mark I1 (Searl, USA) by dissolving the membrane segments in 1mL of THF and adding 15 mL of toluene (Merck) which contained 105 mg of butyl-PBD-scintillator (obtained from Ciba-Geigy AG, Basle, Switzerland). The amount of adsorbed protein was calculated according to the respective reference activities.
RESULTS AND DISCUSSION All analytically relevant, freshly prepared neutral-carrierbased ion-selective solvent polymeric membranes studied so far are electrically symmetrical after equilibration (conditioning) on both sides with aqueous sample solutions. This means that t h e asymmetry potential E , of membranes conditioned with the same aqueous solution on both sides (Figure 2, right) is zero within 30 fiV. A similar time curve is also obtained when the cell emf is measured without inserting a membrane between t h e two half-cells (Figure 2 , left). This is probably due to thermal disturbances induced by the sample transfer. In contrast, a freshly prepared membrane has a considerable asymmetry which does not disappear within 45 min of contact with aqueous solutions (Figure 2 , middle). If, however, a membrane is conditioned with aqueous electrolyte solutions on both sides, it exhibits symmetric behavior even after drying.
ANALYTICAL CHEMISTRY, VOL. 60, NO. 14, JULY 15, 1988 ASVMME TRY
-.
EMF
[mVI
ETH 2120iPVC
+ .
3
i?
TIME
ETH 2120iOH-PVC
Figure 3. Asymmetry potentials of PVC- and OH-PVC-based solvent polymeric membranes (type 2, 3, and 4, Table I).
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f-
A3
AI
5-1
r-
$1
60
'
120
'
180 '
'
240
300 TIME
[min]
Flgure 5. Response of Na+-selectIve PVC- and OH-PVC-based membranes (type 3, Table I) to alternating samples: A, aqueous 5
10
20
15
TIME
[min]
Figure 4. Asymmetry potential of a K+-selective membrane (valinomycin, type l a , Table I) after a 20-min treatment with hexane.
At least in some membranes a contact with a 7 wt % solution of bovine serum albumin (BSA) on one side induces surprisingly high asymmetries (Figure 3, upper left). These asymmetries decrease somewhat but reach a fairly constant value which persists over several hours. If the other membrane side is also brought into contact with the same BSA solution, E, finally becomes approximately zero (Figure 3, upper right). Since aqueous BSA solutions are much more lipophilic than aqueous electrolyte solutions (4, 6, 7),we speculated that E, shifts might occur because of a n enhanced extraction of membrane components on the sample side ( 4 , 6). A very efficient extraction by contacting the membrane with hexane (Figure 4),however, leads to a n asymmetry potential which decreases much faster than those presented in Figure 3. Although the membrane becomes quite brittle, E,, tends toward zero in about 5 min after extracting it on one side with hexane. T h e induction of E , by BSA therefore is due to an adsorption on the membrane surface and/or migration into the boundary region of BSA components as assumed in ref 5. T o study the relationship between the asymmetry induced by proteins and a protein deposition on the membrane surface, radiotracer techniques were used. For this purpose, membranes were treated with BSA or human serum albumin (HSA), which were radiolabeled with 1311and 14C,respectively. Irreversible adsorption took place and the adsorbed proteins could not completely be removed even by thoroughly rinsing the membranes with water or aqueous electrolyte solutions. Moreover, the tools which came in contact with the treated membranes were not significantly contaminated by the radiolabels. Experiments with high concentrations of inactive
solutions (4.0 mM KCI, 1.1 mM CaCI,, 0.6 mM MgCI,) with A1 50 mM NaCI, A2 140 mM NaCI, and A3 400 mM NaCI; S, serum samples containing S1 141 mM Na+, S2 171 mM Na', and S3 211 mM Na'. For S2 and S3, NaCl was added to S1.
iodide corroborated that the adsorption of proteins is not mimicked by the presence of small amounts of 1311-. Nevertheless, a quantification of the amount of proteins adsorbed was not possible, which might be due to differences in the adsorption behavior between labeled and unlabeled proteins. Stimulated by a series of efforts to increase the biocompatibility of artificial polymers (for a review see ref lo), we tried to increase the polarity of the membrane surface by different methods. One was the introduction of hydroxy groups into the PVC matrix. T o this end, a vinyl chloridevinyl acetate copolymer was hydrolyzed (see Experimental Section). Whereas a copolymer with about 2% poly(viny1 alcohol) showed no effect, a material with 17% hydroxy functionality (OH-PVC) had a surprising influence on the induction of asymmetry in membranes by proteins (see Figure 3, upper left). All PVC membranes showing large asymmetries after a protein contact completely lost their asymmetric behavior when substituting the PVC by OH-PVC (Figure 3). As a consequence, the emf of ion-selective cell assemblies with neutral-carrier-based OH-PVC membranes became drastically more reproducible when alternating between aqueous electrolyte solutions (calibration solutions) and serum samples (see Figures 5 and 6). The relevant selectivities and the slope of the electrode response were comparable to those of the classical PVC-based membrane electrodes (Table 11). These findings are in agreement with the observation that the electrochemical characteristics of K+-selective electrodes prepared with a OH-PVC (0.75% OH functionality) are very similar to those of the parent PVC-based K+ electrodes (11). In this reference, the OH functionality was introduced in view of a high adhesion of the membranes to semiconductor substrates (11). Indeed, we also observed that the OH-PVC-based
ANALYTICAL CHEMISTRY, VOL. 60, NO. 14, JULY 15, 1988
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ETH 1001 / PVC
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