Anal. Chem. 2005, 77, 5221-5228
Response Characteristics of a Reversible Electrochemical Sensor for the Polyion Protamine Alexey Shvarev and Eric Bakker*
Department of Chemistry and Biochemistry, Auburn University, Auburn, Alabama 36849
We describe here in detail the first reversible electrochemical sensors for the polyion protamine. Potentiometric sensors were proposed in recent years, mainly for the determination of the polyions heparin and protamine. Such potentiometric polyion sensors functioned on the nonequilibrium extraction of polyions into a hydrophobic membrane phase via ion pairing with lipophilic ion exchangers. This made it difficult to design sensors that operate in a truly reversible fashion. The reversible sensors described here utilize the same basic response mechanism as their potentiometric counterparts, but the processes of extraction and ion stripping are now fully controlled electrochemically. Spontaneous polyion extraction is avoided by using membranes containing highly lipophilic electrolytes that possess no ion-exchange properties. Reversible extraction of polyions is induced if a constant current pulse of fixed duration is applied across the membrane, followed by a baseline potential pulse. The key theoretical response principles of this new class of polyion sensors are discussed here and compared to those of its classical potentiometric counterpart. The electrochemical sensing system is characterized in terms of optimal working conditions, membrane composition, selectivity, and influence of sample stirring and organicphase diffusion coefficient on the response characteristics. Excellent potential stability and reversibility of the sensors are observed, and measurements of heparin concentration in whole blood samples via protamine titration are demonstrated. In the past decade a new direction in the field of ion-selective electrodes has emerged with the development of potentiometric sensors with plasticized polymeric membranes for the detection of polyionic macromolecules.1-4 In the first work that started this research,5 Ma et al. proposed a polymer membrane electrode containing a lipophilic anion exchanger, which was capable of detecting the polyanion heparin. Heparin is a highly sulfated * To whom correspondence should be addressed. E-mail: bakkere@ purdue.edu. (1) Fu, B.; Bakker, E.; Wang, E.; Yun, J. H.; Yang, V.; Meyerhoff, M. E. Electroanalysis 1995, 7, 823. (2) Yun, J. H.; Han, I. S.; Chang, L.; Ramamurthy, N.; Meyerhoff, M. E.; Yang, V. C. Pharm. Sci. Technol. 1999, 2, 102. (3) Dai, S.; Esson, J. M.; Lutze, O.; Ramamurthy, N.; Yang, V. C.; Meyerhoff, M. E. J. Pharm. Biomed. Anal. 1999, 19, 1. (4) Fu, B.; Bakker, E.; Yun, J. H.; Yang, V. C.; Meyerhoff, M. E. Anal. Chem. 1994, 66, 2250. (5) Ma, S.-C.; Yang, V. C.; Meyerhoff, M. E. Anal. Chem. 1992, 64, 694. 10.1021/ac050101l CCC: $30.25 Published on Web 07/12/2005
© 2005 American Chemical Society
polysaccharide with an average charge -70 and average molecular mass of 15 000 Da. It is used as an anticoagulant in major surgical and extracorporeal procedures, such as open-heart surgery, bypass surgery, and dialysis.6 Real-time monitoring of heparin concentration in blood is important to prevent the risk of possible bleeding and reduce postoperative complications.7 The activated clotting time measurement (ACT) is a common method for estimating the heparin concentration in whole blood. Although this method is widely used, it is nonspecific and indirect, and the results can be affected by many variables.8 In contrast to ACT, the heparinselective electrode is able to detect heparin concentration directly in whole blood or plasma samples. At around the same time, an electrode for sensing the polycation protamine was proposed.9 This polypeptide is usually administered in order to neutralize heparin activity.10 Protamine is a polycation with an average charge of +20 and is rich in arginine residues.11 Detection of protamine via ion-selective electrodes created the possibility of determining the heparin concentration via titration with protamine, utilizing very specific heparin-protamine interactions.12 The observed response of the heparin electrode could not be explained in terms of the classic equilibrium approach. The Nernst equation should yield a slope of the electrode function of about 1 and 2 mV/decade for heparin and protamine, respectively, because of the high charge of these ions. A quasi-steady-state model to explain this unusual mechanism was subsequently described.4 The potentiometric polyion sensor response is kinetic in nature. A strong flux of polyions occurs both in the aqueous solution and in the membrane phase due to the spontaneous extraction of polyions into the polymeric membrane and concomitant exchange with hydrophilic ions from the membrane, which results in a nonNernstian potential change in the presence of polyions. Unfortunately, because the extraction of polyions is an irreversible process, a strong potential drift is normally observed. In fact, after a relatively short time in contact with a polyion solution, the sensor starts to lose its response.4 Extracted polyions (6) Hirsh, J.; Dalen, J.; Deykin, D.; Poller, L. Chest 1992, 102, 337S. (7) Despotis, G. J.; Joist, J. H.; Goodnough, L. T. Clin. Chem. 1997, 43, 16841696. (8) Culliford, A. T.; Gitel, N. S.; Starr, N. Ann. Surg. 1981, 193, 105. (9) Yun, J. H.; Meyerhoff, M. E.; Yang, V. C. Anal. Biochem. 1995, 224, 212. (10) Metz, S.; Horrow, J. C. Protamine and Newer Heparin Antagonists. In Pharmacology & Physiology in Anesthetic Practice; Stoelting, R. K., Lippincott: Philadephia, 1994; p 1. (11) Ando, T.; Yamasaki, M.; Suzuki, K. Protamines; Springer-Verlag: New York, 1973; p 116. (12) Ramamurthy, N.; Baliga, N.; Wahr, J.; Schaller, U.; Yang, V. C.; Meyerhoff, M. E. Clin. Chem. 1998, 44, 606.
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may be removed from the membrane phase by reconditioning of the sensor in concentrated sodium chloride. More recently, a pH cross-sensitive potentiometric heparin sensor was proposed, which contained the ion exchanger and a charged H+ ionophore. Here, heparin stripping could be accomplished by adjusting the pH of the sample.13 Another approach, which has been employed by Meyerhoff, requires disposable sensors.12 A new technique utilizing electrochemically controlled, reversible ion extraction into polymeric membranes in an alternating galvanostatic/potentiostatic mode was recently described.14,15 This approach has interesting applications for small ion detection and tremendous promise in the area of polyion sensing. The instrumental control of ion fluxes across the membrane allows one to repeatedly extract and strip ions into and from the membrane, yielding highly reproducible sensor responses. A preliminary study of a chronopotentiometric protamine sensor was recently reported.16 Alternatively, cyclic voltammetry was recently introduced by others to detect the polyions heparin and protamine at liquid-liquid interfaces.17,18 Here, a detailed description of the response mechanism of this new pulsed galvanostatic polyion detection approach, further improvement of the sensing technology, and first applications toward polyion sensing in undiluted whole blood samples are presented. EXPERIMENTAL SECTION Reagents. High molecular weight poly(vinyl chloride) (PVC), 2-nitrophenyl octyl ether (o-NPOE), dinonylnaphthalenesulfonic acid (DNNS) as a 50% solution in heptane, tetradodecylammonium chloride (TDDACl), tetradodecylammonium tetrakis(4-chlorophenyl)borate (ETH 500), tetrahydrofuran (THF), and all salts were purchased from Fluka Chemical Corp. (Milwaukee, WI). Heparin, sodium salt (from bovine intestinal mucosa, 151 units/mg) and protamine sulfate (from herring) were purchased from Sigma (St. Louis, MO). Aqueous solutions were prepared with Nanopure deionized water (18.2 MΩ‚cm). The lipophilic salt DNNS-TDDA was prepared by metathesis in benzene of stoichiometric quantities of dinonylnaphthalenesulfonic acid and tetradodecylammonium chloride. The solution of DNNS-TDDA in benzene was washed several times with distilled water. After evaporation of the solvent, the product was dissolved in THF and used. Membrane Preparation. Ion-selective membranes (∼200 µm thick) for voltammetric experiments contained PVC and o-NPOE, 1:2 by weight. The membranes were prepared by solvent casting, with THF as a solvent. Membranes containing 10 wt % lipophilic salts ETH 500 or DNNS-TDDA without ion exchanger were prepared. Electrodes. The ion-selective membranes were cut with a cork borer (6-mm diameter) from the parent membrane and incorporated into a Philips electrode body (IS-561, Glasbla¨serei Mo¨ller, Zu¨rich, Switzerland). The inner solution consisted of 0.1 M NaCl and was contacted with an internal Ag/AgCl electrode. The electrodes were conditioned overnight before experiments in a (13) Mathison, S.; Bakker, E. Anal. Chem. 1999, 71, 4614. (14) Shvarev, A.; Bakker, E. Anal. Chem. 2003, 75, 4541. (15) Shvarev, A.; Bakker, E. Talanta 2004, 63, 195. (16) Shvarev, A.; Bakker, E. J. Am. Chem. Soc. 2003, 125, 11192. (17) Samec, Z.; Trojanek, A.; Langmaier, J.; Samcova, E. Electrochem. Commun. 2003, 5, 867. (18) Yuan, Y.; Amemiya, S. Anal. Chem. 2004, 76, 6877.
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solution identical to the inner filling solution. A double-junction Ag/AgCl electrode with a 1 M LiOAc bridge electrolyte was used as an external reference electrode both for potentiometric and for voltammetric measurements. Experimental Setup. Voltammetric measurement were conducted in a three-electrode cell system where the internal Ag/ AgCl electrode acted as a working electrode and the external reference electrode and counter electrode (platinum wire) were immersed into the sample. For the experiments with whole blood samples, a small (10 mL) cell was used. The galvanostatic control of the ion-selective membrane utilized the pulse galvanostatic/potentiostatic technique, described in detail elsewhere.14-16 The voltammetric experiments were performed with an AFCBP1 bipotentiostat (Pine Inst., Grove City, PA) controlled by a PCI-MIO-16E4 interface board and LabVIEW 5.0 Software (National Instruments, Austin, TX) on a Macintosh computer. Prior to the experiment, the operation of the first electrode output of the bipotentiostat (K1) was switched to current control with potentiostatic control of output of second working electrode (K2). To apply the current pulse, the working electrode was connected to the K1 output via an analog switch controlled by external software. When the baseline potential between current pulses was applied, the working electrode was connected to the K2 output. During the chronopotentiometric experiments, each applied constant current pulse (of 1-s duration) was followed by a constant potential pulse (of 15-s duration). Sampled potentials, which represent the sensor response, were obtained as the average value during the last 100 ms of each current pulse. The observed amplitude-time behavior of current and potential has been described.14 Titration of protamine was performed by addition of 1.0 g L-1 stock solution; titration of heparin was conducted by addition of 1.5 g L-1 (2 × 10-5 M) stock solution. Unless mentioned otherwise, all solutions were buffered to pH 7.4 with 10 mM Tris-HCl buffer. To study the pH influence on the sensor response, the sample solution was composed of 0.1 M NaCl, 6.6 mM citric acid, 11 mM boric acid, and 10 mM phosphoric acid. The desired pH was adjusted with 1 M NaOH. The solutions were stirred during measurements. Heparin and protamine concentrations were calculated by assuming an average molecular mass of 15 000 and 5000 Da, respectively. An AR-20 pH meter and an Accutron combination glass electrode (Fisher Scientific, Pittsburgh, PA) were used for pH measurement in aqueous solutions and blood samples. All experiments were conducted at laboratory ambient temperature (21.5 ( 0.5 °C). Confidence intervals were computed at the 95% level. Blood Collection. Whole blood samples were collected from a healthy human donor into 4-mL collecting tubes (Fisher Scientific, Pittsburgh, PA), each containing 7.2 mg of K2EDTA. The blood samples (40-50 mL) were collected in the morning, placed in the refrigerator, and used the same day. The blood pH was found to be 7.5 with a calibrated glass pH electrode. Roomtemperature blood samples were homogenized prior to measurement by slowly inverting the tube several times. THEORY It is useful to compare the pulsed chronopotentiometric polyion sensors described here to their passive potentiometric counter-
parts.4 The model is illustrated here for a sensor selective for the polycationic protamine. Classical Potentiometric Polyion-Selective Sensor. It is assumed that the polymeric membrane contains a lipophilic cationexchanger R-Na+ in contact with a NaCl sample solution. The sample-membrane phase boundary potential for this case may be formulated as follows:
EPB ) E0 +
aNa RT ln F [Na+]
(1) pb
where aNa is the activity of sodium ions in the aqueous solution, [Na+]pb is the so-called free concentration of sodium ions at the organic side of the sample-membrane phase boundary, and E° incorporates among other constant terms the free energy of transfer for sodium from water to the membrane phase. Symbols aspecies or cspecies refer to activities and concentrations of the corresponding species in the aqueous phase and brackets ([species]) to concentrations in the organic membrane phase. In the absence of protamine in the sample, and by neglecting ion pairing, the concentration of sodium ions in the membrane phase is determined by the total concentration of lipophilic cation exchanger, RT:
[Na+]pb ) RT
(3)
where [PAz+]pb is the concentration of protamine cations with charge z in the membrane-phase boundary. That concentration can be formulated as a function of the bulk sample protamine concentration on the basis of a pseudo-steady-state flux consideration:4 z+
[PA ]pb ) (Daq,PAδm/Dm,PAδaq)cPA,bulk
aNa RT ln (5) F RT -(zDaq,PAδm/Dm,PAδaq)cPA,bulk
Consequently, if aNa is fixed, the phase boundary potential shows a direct response for protamine. At high protamine concentration, the sodium ions are quantitatively replaced from the membrane and a near-Nernstian response slope for protamine is expected.4 Such a quantitative replacement also occurs upon prolonged exposure (∼24 h) to dilute protamine solutions. The resulting response slope is too small to be analytically useful.4 Normal Pulse Chronopotentiometric Sensor. In contrast to potentiometric polyion-selective sensors the ion-extraction process is here induced electrochemically by applying a constant current pulse.14-16 To prevent spontaneous extraction, the membrane contains a highly lipophilic electrolyte R+R- and does not possess intrinsic ion-exchange properties. The initial concentration of protamine or sodium cations in the membrane is therefore close to zero. An applied cathodic current i induces a net flux J of cations in the direction of the membrane phase. Assuming for simplicity that only sodium and protamine ions may be extracted into the membrane phase, the relationship between current i and the fluxes of sodium, JNa, and protamine, JPA, can be formulated as
i ) FAJNa + zFAJPA
(6)
(2)
Consequently, the membrane behaves as an ion-exchanger-based sodium electrode and a Nernstian response slope is expected. If protamine is present in the aqueous solution, a strong flux of protamine cations occurs both to the surface and into the membrane phase, forming two stagnant diffusion layers. Because the diffusion in the stagnant layer of the aqueous phase is the rate-limiting step, a quasi-steady-state diffusion may be observed at the interface.4 Protamine cations displace the sodium cations from the membrane-phase boundary. This ion-exchange process reduces the concentration of sodium ions in membrane phase and increases the observed potential (eq 1) because the total concentration of cations must satisfy the electroneutrality condition:
[Na+]pb ) RT - z[PAz+]pb
EPB ) E0 +
(4)
where Dm,PA, Daq,PA, δm, and δaq are the diffusion coefficients of protamine in the membrane phase and aqueous solution and the resulting diffusion layer thickness, respectively. This eq 4 may now be inserted into eqs 3 and 1 to obtain the protamine response at low concentrations:
where A is the exposed membrane area. Assuming linear concentration gradients and recalling that the sodium concentration in the membrane bulk is zero, the sodium flux can be related to the concentration gradient across the organic phase boundary as follows:
JNa ) - (Dm,Na/δm)[Na+]pb
(7)
If no protamine is present, eqs 7 and 6 may be inserted into eq 1 to give
EPB ) E0 +
RT FADm,Na ln aNa F iδm
(8)
As established earlier,14 a cathodic current pulse of fixed duration and magnitude, followed by a potentiostatic baseline pulse to keep the membrane bulk void of sodium ions, will give a near-Nernstian electrode slope. As above, protamine will compete with sodiums in the extraction process if it is present in the sample solution. Equation 6 can be rewritten in analogy to eq 7 as follows:
Dm,Na Dm,PA i ) -FA [Na+]pb - zFA [PAz+]pb δm δm
(9)
Assuming that the applied current imposes a flux that is always larger than the flux that can be sustained by polycation diffusion alone and that electromigration is neglected, eq 4 is still valid and may be inserted into eq 9. As a result, the sodium flux, JNa, is decreased, which increases the potential according to eq 1. Inserting eq 4 into eq 9, solving for [Na+]pb, and substituting into Analytical Chemistry, Vol. 77, No. 16, August 15, 2005
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eq 1 therefore yields the predicted protamine response at low polyion concentrations:
EPB ) E0 +
aNa RT ln Daq,PA F δm -i c -z Dm,Na FA δaq PA,bulk
(
)
(10)
If the protamine concentration in the aqueous phase increases, it will eventually be able to sustain the imposed ion flux alone. In this case, the phase boundary concentration of protamine in the sample differs from zero and the phase boundary potential will be dictated by protamine with a slope of ∼3 mV/decade due to the high charge of protamine. This critical point at which the sensor response becomes independent of protamine concentration can be estimated15 for the cathodic current of 2 µA, a net charge of 20, and an aqueous diffusion coefficient of 10-6 cm2/s. The calculated value of 3 × 10-5 mol L-1 is in good agreement with the experimental results described below. Evidently, there are differences between eq 10 and the protamine response for a classical potentiometric sensor shown in eq 5. Importantly, the diffusion layer thickness in the membrane phase is now dictated galvanostatically, and potentiostatic membrane renewal between pulses ensures repeatable δm values from pulse to pulse. Also, the magnitude of the applied current pulse may, in principle, be used to adjust the measuring range for polyion response to some extent, although in this work, the current was primarily chosen to give a maximum potential change. Interestingly, since the applied current and not an ion exchanger dictates the extraction of sodium ions into a pulsed chronopotentiometrically controlled membrane, the diffusion coefficient in the membrane phase does not appear to influence the protamine response range. This is in contrast with the potentiometric sensor (eq 5), where a direct dependence between competitive extraction of sodium by protamine and the membrane diffusion coefficients is known to exist. This has historically been the main reason to use polymeric membranes with a much lower plasticizer content than traditionally used for other types of potentiometric sensors, which now appears to be unnecessary. RESULTS AND DISCUSSION Initial studies used membranes containing the lipophilic electrolyte ETH 500 because both its cation and anion bind to polyanions and polycations such as heparin and protamine, respectively, albeit with less than optimal selectivity.5,12,19 It was therefore anticipated that in a NaCl background applied cathodic current pulses would extract polycations from the sample and changing the sign of the current would allow one to extract polyanions. A sequence of current pulses changing from -20 to +20 µA was applied in a 0.1 M NaCl background, and the potential, measured according to the protocol described above, was plotted as a function of current. The measurements were repeated in the presence of the relevant concentration of protamine or heparin in the sample. The difference between two potentials readings, each measured at the same current without and with polyion in the sample, represents the sensor response at the given current. However, in 0.1 M NaCl, the membrane containing 10 wt % ETH (19) Ma, S.-C.; Yang, V. C.; Fu, B.; Meyerhoff, M. E. Anal. Chem. 1993, 65, 2078.
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500 did not show any polyion response. The registered potential differences were less than 2 mV despite high concentrations of heparin (10 mM) and protamine (100 mg L-1). As anticipated, the selectivity of this membrane was judged to be insufficient for the chosen NaCl background. Heparin or protamine sensing was, however, demonstrated in a more dilute and less lipophilic electrolyte (0.01 M Na2SO4), which encouraged us to optimize the membrane composition further (see Supporting Information for details). DNNS was proposed in earlier work as a cation exchanger with a strong affinity to protamine.12,20 To suppress any spontaneous ion exchange, the hydrophilic counterion was here replaced by the tetradodecylammonium cation. Initial results obtained with this new protamine-selective membrane formulation were recently reported.16 The improved polycation-selective membranes were formulated with 10 wt % TDDA-DNNS in o-NPOE-PVC (2:1 by mass). Preliminary experiments indicated that this membrane composition is optimal. A reduction of the TDDA-DNNS concentration or an increase of the PVC content did not give advantages in terms of low detection limit or lifetime but significantly increased the membrane resistance, which added a large iR drop to the measured potential. A set of 10 electrodes prepared for these experiments demonstrated good reproducibility of the baseline potential (in 0.1 M NaCl), with an interelectrode variability of (7 mV (standard deviation) at a given current in the range of 0 to -10 µA. A number of basic experiments were performed to characterize the sensing properties of the galvanostically controlled protamineselective membranes. A chronopotentiogram in 0.1 M NaCl with and without 10 mg L-1 protamine revealed a maximum potential change of 50 mV at a cathodic current of -2 µA (membrane area, 8 mm2; giving a current density of 25 µA/cm2), consistent with earlier results.16 This current was used for all further experiments. Figure 1 shows the current-time (Figure 1A) and potentialtime (Figure 1B) profiles for a pulse sequence applied to a fresh, newly prepared membrane. As reported before,16 the potential readings during the current pulse are more positive if protamine is present in the sample (recall that the last 100 ms of each pulse are here used as the final sensor readout). During the potentiostatic pulse, the back-diffusion of the ions from the membrane can be observed. Obviously, the current decay is slower when protamine is present in the sample, indicating a difference in the diffusion behavior between sodium and protamine ions. When the current was integrated over the entire resting pulse of 15 s, the calculated charge corresponded to more than 90% of the applied charge during the current pulse. The back-diffusion currents were found to be increasingly similar for samples with and without protamine after a few dozen pulses, perhaps indicating that a residual, constant amount of protamine remains extracted in the membrane phase. The corresponding potential difference without and with protamine (50 mg L-1) tended to be surprisingly large for fresh membranes (100-130 mV) but quickly stabilized over the next 1-2 pulses to ∼60 mV. The potential readings during subsequent uptake pulses remained highly reproducible. To evaluate the influence of baseline potentials on the sensor behavior, we monitored the potential under applied current as a (20) Han, I. S.; Ramamurthy, N.; Yun, J. H.; Schaller, U.; Meyerhoff, M. E.; Yang, V. C. FASEB J. 1996, 10, 1621.
Figure 1. Current-time and potential-time profiles for a pulse sequence applied to a fresh, newly prepared membrane without protamine in 0.1 M NaCl solution (10 mM TRIS, pH 7.40) and in the presence of 50 mg L-1 protamine. Subsequent potentials tend to be smaller (see text).
function of time in 0.1 M NaCl at pH 7.4. Prior to the experiment, the open-circuit potential of the sensor was measured with respect to the same reference electrode that was used in galvanostatic pulse experiments. The observed value was found to be stable (-30 ( 2 mV) for one sensor in a series of 10 consecutive measurements. Figure 2 illustrates the sensor potentials at a cathodic current of -2 µA for three different values of baseline potentials applied between current pulses. One of the baseline potentials was equal to the open-circuit potential value (Figure 2B), one was 50 mV more negative (Figure 2A), and one was 50 mV more positive (Figure 2C). If the baseline potential significantly deviated from open-circuit potential, an instability of the sensor response (6-7 mV/h) was observed. This was likely due to the continuous extraction of the ions at both sides of the membrane that takes place if the baseline potential is not equal to the equilibrium (open-circuit) value. Shifting of the baseline potential within (20 mV from the open-circuit value did not lead to a visible drift of more than 1.5 mV/h. In a separate experiment, we measured the back-diffusion currents at the end of the stripping process for different baseline potentials under the same conditions. It was found that the absolute value of the back-diffusion current is minimal (3 nA) if the baseline potential is equal to the opencircuit potential (Figure 2D). These experiments confirmed our previous assumptions,14,15 and now allow one to experimentally determine the optimal value of the baseline potential. With classical potentiometric polyion sensors, the observed potentials are known to be strongly influenced by the rate of sample stirring, which alters the aqueous diffusion layer and hence the polyion flux to the membrane.4 Indeed, more recent work has confirmed a clear relationship between measuring range and rotation speed in a rotating electrode setup.21 To examine how stirring may affect the sensor response in a galvanostatic pulse experiment, the potential was measured in an unstirred solution
Figure 2. Sensor response in blank solution (0.1 M NaCl, 10 mM Tris, pH 7.40) as a function of time at different baseline potentials: (A) -80, (B) -30, and (C) +20 mV. (D) Back-diffusion currents measured at the end of the potentiostatic interval plotted as a function of the baseline potential (1-s current pulses at a cathodic current of -2 µA).
Figure 3. Effect of stirring on the sensor response at a cathodic current of -2 µA in blank solution (0.1 M NaCl, 10 mM Tris, pH 7.40) and in the presence of 10 mg L-1 protamine.
and at a stirring rate of 100 rpm (see Figure 3). In contrast to potentiometric results with a heparin-responsive membrane, where sudden stoppage of sample stirring caused the potential to change by ∼20 mV,1 the response of the pulse galvanostatic sensor did not show significant influence on the stirring rate. The potential difference between a stirred and unstirred sample did not exceed 2-3 mV. The limiting current calculated according to the wellknown Levich equation using concentration of protamine of 10-6 mol L-1, a net charge of 20, a stirring rate of 100 rpm, and an aqueous diffusion coefficient of 10-6 cm2/s yields the value 0.5 µA. This is lower than currents applied to the electrode. Most (21) Qin, W.; Zhang, W.; K. P., X.; Meyerhoff, M. E. Anal. Bioanal. Chem. 2003, 377, 929.
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likely, therefore, the constant applied current pulse dictates, via electromigration, to some extent the thickness of the Nernst diffusion layer and is now less affected by sample stirring. Previous work with classical potentiometric sensors also showed that the response range is strongly influenced by the diffusion coefficient of the polyion in the organic membrane phase.1 In that case, the extraction process is driven by an ionexchange reaction in the membrane phase. An increase in the plasticizer content slows the polyion diffusion in the membrane and shifts the measuring range to lower concentrations because of a more effective enrichment of the polyion in the organic-phase boundary region, while the concentration of the exchanging counterion sodium is dictated by the ion exchanger and is therefore constant (see eq 5).4,19 With pulsed galvanostatically controlled membranes, polyion extraction does not occur based on an ion-exchange reaction but via assisted ion transfer. The difference between diffusion of protamine and competing sodium ions in the membrane phase does not play a major role in this case. A membrane with reduced plasticizer content will accumulate not only polyions to a larger extent but the competing sodium ions as well. As also predicted by eq 10, a much smaller influence of the plasticizer content on the measuring range is expected. Indeed, a decrease in the o-NPOE/PVC ratio from 2:1 to 1:1 gave a shift of 0.3 logarithmic unit of protamine concentration to lower concentrations, which is significantly smaller than observed with classical zero current potentiometry.4 A notable drawback with formulations containing less plasticizer was the increased membrane resistance, leading to an undesired iR drop across the membrane. For this reason, membranes containing the 2:1 plasticizer-to-polymer ratio were preferred here. While the protamine sensor is intended to work in whole blood at the physiological pH of 7.4, the influence of pH on the sensor response was examined. The potentials were measured without and with 25 mg L-1 protamine in the sample, respectively, at a cathodic current of -2 µA. Owing to the high protamine concentration, the difference between the two potentials may be regarded as the maximum sensor response, or potential window, in 0.1 M NaCl. Under acidic conditions, the sensor response remains independent of pH, but higher pH values clearly reduce the potential difference (see Supporting Information). Fortunately, pH 7.4 is still adequate for practical use. The observed pH effect may be best explained on the basis of the dissociation equilibria of protamine in the sample. The selectivity of the membrane was determined at pH 7.4 by recording separate calibration curves for the chloride salts of sodium, potassium, calcium, and magnesium (Figure 4). The resulting selectivity coefficients are in good agreement with those reported previously for DNNS-based ISE membranes without additional ionophore.22 All slopes in the concentration range of 0.001-0.1 M were found to be slightly super-Nernstian (70-72 mV), which biases the selectivity coefficients to some extent.23 The slopes may perhaps be explained by the contribution of ion migration at the membrane interface on the basis of the NernstPlank equation, which has not yet been considered in the simplified theoretical model. The abrupt potential jump around (22) Rosatzin, T. Optische and potentiometrischen Sensoren auf der Basis von Fluessigmembranen mit immobilisierten Komponenten. Thesis No. 9829, ETH Zurich, 1992. (23) Bakker, E.; Pretsch, E.; Buhlmann, P. Anal. Chem. 2000, 72, 1127.
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Figure 4. Calibration curves in pure solutions of NaCl, KCl, MgCl2, CaCl2 and protamine in the presence of a 0.1 M NaCl background (10 mM Tris, pH 7.40).
Figure 5. (A) Calibration curves of protamine in the presence of 0.01, 0.03, and 0.1 M supporting electrolyte NaCl at pH 7.40 and (B) protamine calibration curves in 0.1 M NaCl (10 mM Tris, pH 7.40) without and with 0.01 M KCl.
10-4 M originates from depletion processes at the membrane surface.14,15 A protamine calibration curve in 0.1 M NaCl is also shown in Figure 4. The higher potential readings demonstrate a strong preference of this membrane for protamine over all other tested cations. The background electrolyte concentration is expected to influence the protamine response curve because the response principle is based on a competitive extraction between the polyion and sodium ions. A lower sodium background concentration, for instance, is expected to give a larger potential range for the protamine response and may also lead to a shift of the response to lower protamine concentrations (see eq 10). Figure 5A shows experimental protamine calibration curves in the presence of three sodium chloride concentrations, 10, 30, and 100 mM. Indeed, the protamine potential range decreases with increasing NaCl concentrations. This decrease is consistent with the separately measured slope for sodium observed in Figure 5 of ∼70 mV for
Figure 6. Amplitude-time behavior of the potential for a protamine calibration in a whole blood sample. The membrane contained 10 wt % DNNS-TDDA. The numbers above the plot represent log cProtamine (mg L-1).
a 10-fold sodium activity change. The concentration range of sodium in whole blood is very narrow (136-145 mM), 24 so that this type of interference may not be very important in practice. Fortunately, the concentration of potassium also varies in a relatively small range in whole blood (from 3 to 5 mM), and Figure 4 suggests an approximately equal discrimination of sodium and potassium. The fact that the more lipophilic potassium ion is not preferred more substantially may be explained by the ion-pairing characteristics of DNNS.22 The small influence of potassium on the protamine response is confirmed in Figure 5B, where two protamine calibration curves in 0.1 M NaCl with and without 10 mM KCl are shown. The maximum deviation of the response observed at low protamine concentration does indeed not exceed 5 mV. Figure 6 illustrates a potential-time trace for a protamine calibration curve at the cathodic current of -2 µA. In whole blood, the potential response range was found as ∼60 mV, acceptably large for a practical determination of protamine in whole blood samples. Standard deviations of the potentials (n ) 10) increased to 1.5 mV compared to 0.7 mV in buffered NaCl solutions.16 The results indicate that protamine concentrations as low as 0.5 mg L-1 may be determined with the current pulsed chronopotentiometric sensor. The experimental protocol can be used for determining heparin in blood via end point detection of a protamine titration, in analogy to previous work with potentiometric sensors.12 Small aliquots of heparin stock solution were added to whole blood samples in order to obtain different model concentrations of heparin in the range of 0.25-2 µM (0.6-4.5 kU L-1) and titrated with protamine stock solution. The resulting titration curves are represented in Figure 7A. Each point was calculated as an average of 10 consecutive potential readings. Reproducibility was evaluated by repeating each titration four times, giving deviations of starting and ending potentials of at most 7 mV from sample to sample, while the total change of potential during titrations remained the same. Since each collection tube contained 7.2 mg of the potassium salt of EDTA and the amount of blood collected in each tube varied from 2 to 4 mL, much of the deviation may be attributed to variations in the potassium concentration (see Figure 5B). The observed end points are plotted in Figure 7B as a function of the whole blood heparin concentration, and an expected linear relationship (24) Oesch, U.; Ammann, D.; Simon, W. Clin. Chem. 1986, 32, 1448.
Figure 7. (A) Titration of whole blood samples containing 0, 0.25, 0.5, 1, and 2 mM heparin with 1 g L-1 protamine solution. (B) Resulting calibration curve for the heparin-protamine titration in whole blood samples. Dashed line represents the linear regression of the data.
was found (correlation coefficient 0.995). The linear regression of this calibration curve yielded a confidence interval for the determination of heparin concentration of (0.14 µM or (0.31 kU L-1 (n ) 5). The established Hepcon HMS assay system (Medtronics Blood Management Corp.) based on ACT measurements has a resolution between 0.4 and 0.7 kU L-1 depending on the chosen cartridge range. The lifetime and stability of the sensor are important parameters, especially if measurements are conducted in physiological media. Continuous pulsed chronopotentiomeric measurement in pH buffered 0.1 M NaCl containing 10 mg L-1 protamine for 3 h, with 1-min measurement intervals, gave no visible potential drift and a maximum potential variation of 2 mV (see Supporting Information). For whole blood samples, the titration curves shown in Figure 7B were obtained with the same sensor and the total time of measurements in the blood exceeded 2.5 h (for each point, 10 potential measurements were collected). After exposure to blood, the sensors were placed in buffered 0.1 M NaCl and the baseline potential was found to return to the initial value ((5 mV for each sensor). The lifetime of the sensors, arbitrarily defined here as the time where baseline potential shifts did not exceed 20 mV, was at least 2 weeks, after a 10 h total exposure time to undiluted whole blood samples. CONCLUSIONS Potentiometric polyion electrodes have, so far, been irreversible systems because of the intrinsic thermodynamics of the polyion Analytical Chemistry, Vol. 77, No. 16, August 15, 2005
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extraction process. This limitation may be overcome by operating with membranes where polyion extraction does not occur spontaneously but is placed under galvanostatic control. Here, a simplified theoretical model points to differences and similarities of the two types of polyion sensors. Conveniently, both sensing systems yield response signals that are comparable to each other and are essentially indistinguishable for a practical user in terms of calibration procedure. In addition to reversible sensing characteristics, pulsed galvanostatic systems are not expected to show a significant influence of the diffusion behavior in the membrane phase (e.g., plasticizer content), which contrasts to that found in zero current potentiometry. These expectations are largerly confirmed in practice. Interestingly, the response of instrumentally controlled polyion sensors are found to be largely independent of sample stirring, very different from the behavior of corresponding potentiometric polyion sensors. The selectivity over other small cations and the influence of the polyion measuring range (potential window) are found to be comparable in both sensing principles. The pulsed galvanostatic protamine sensor is successfully applied to the measurement in undiluted whole blood samples, with excellent stability, reproducibility, and selectivity. The successful
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heparin determination in whole blood via protamine titration demonstrates that this is an attractive sensor principle for the continuous monitoring of polyions in clinical samples. ACKNOWLEDGMENT The authors are grateful to the National Institutes of Health (GM071623) for financial support of this research. The authors acknowledge Kathryn Milly West, Coordinator of Medical and Laboratory Technology in the Department of Chemistry and Biochemistry at Auburn University, for drawing the blood samples. SUPPORTING INFORMATION AVAILABLE Additional figures: response of a membrane containing ETH 500 to heparin and protamine in 10 mM Na2SO4, effect of the pH on the protamine sensor response, and long-term stability of the protamine sensor potentials in 0.1 M NaCl. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review January 26, 2005. Accepted June 13, 2005. AC050101L
