Use of Ionomsr Membranes To Enhance the Seiectivity of Electrode-Based Biosensors in Flow- Injection Analysis Sara A. Rosario, Geun Sig Cha, and Mark E.Meyerhofp
Department of chemistry, The University of Michigan, Ann Arbor, Michigan 48109-1055 Marek Trojanowicz
Department of Chemistry, University of Warsaw, Pasteura I, 02-093 Warsaw, Poland
The use of lonomcH membranes toenhance the de4cfMty of potmtkmtrk enzyme electrodes Infbw-ln)ectkn measure ment ar-s io examined. The lonomer membranes employed are permeable to an8lyte subsbates but relatively knpenneabb to detectable bns that would nonnelly krtertere wlth the measurement of the subetrates H the enzyme electrodes were In d M contact with the sample. As a model system, the selectivtty of enzyme electrodes prepared with n o n a c t k r - b a s e depolynmlcnmnkanesls ~ evaluated. I n the preferred configuratkn, a thln hydropMllc anion-exchsnge membrane Is Incorporated wlthln a flowthrough tlialysls unit upstream from the enzyme-eledrode detector. As the sample pa8ses through the dlalysls unit, neutral or eRkncc andyte mdecdes (urea 0rQMamIne)move through the membrane while the permestbn of endogenous ammonium ions and other cations In the sample is retarded. A flowlng rbclplent buffer on the other M e ot the membrane carrles the analyte substrate to the enzyme-electrode detector. Enhancements In selectivity for analyte substrates over endogenwo ammonkm and potarskm ions me greater than or equat to 9-fdd when compared to enzymeelectrode flow-lsl)ectlonanalyrls ( H A ) sydems assmbkd without the ionomermembraneudl. Theanalytlcalutmydtheproposed system Is demonstrated by the accurate measurements of urea In wood serum and L-glutamlne in hyakloma Moteactor media.
Although a wide variety of enzyme electrodes based on potentiometric ion- and gas-selective membrane electrode transducers have been described in the literature (1-5),relatively few have been incorporated into commercial instruments for routine measurements of biomolecules in complex samples (e.g., blood, urine, bioreactor media, etc.). In practice, the main limitation of such sensors in these applications is the lack of adequate selectivity. This selectivity problem does not arise from the immobilized biocatalytic reaction but rather from the innate response of the base ion or gas transducer to endogenous ionic and gaseous species in the sample. For example, a large number of bioprobes have, been fabricated by using enzymes that liberate ammonia. The liberated ammonia can be detected with either a gas-selective sensor or an ion-selective electrode (6-12). In the former case, large background levels of endogenous ammonia-nitrogen (total ammonia and ammonium) in the sample can cause positive errors in the measurement of a given substrate. When the ammonium-selective electrode is used as a transducer (based on nonactin as the membrane ionophore (13)),interference from both endogenous ammonium and potassium can occur (14). We now describe how the use of an anion-exchange
* To whom correspondence should be addressed. 0003-2700/90/0362-2418$02.50/0
perfluorinated membrane in conjunction with a nonequilibrium flow-injection measurement arrangement can be used to greatly reduce endogenous ionic interferences, allowingsuch biosensors to be employed more readily as flow-through detectors for direct measurement of analyte substrates in complex samples. Various methods of eliminating endogenous ionic and gas interferences have been proposed previously in conjunction with using enzyme-based electrode systems. Tedious and time-consuming manual sample pretreatment steps such as ion exchange (11, 15) have been reported. Simultaneous measurement of the interferent species with additional sensors (e.g. ammonium/potassium) and subsequent subtraction of these values from the enzyme-electrode signals has also been suggested, particularly for the determination of urea in blood (14).Immobilized glutamate dehydrogenase (GLDH) has been employed to remove endogenous ammonia enzymatically (7, 1618). For automated flow-through arrangements involving enzyme reactions, Fraticelli reported the use of an on-lime gas dialyzer to remove endogenous ammonia in plasma samples for the determination of L-asparagine (19).C o h o n et al. (m), through a temperature modification of the Fraticelli method, were able to remove up to 99.8% of endogenous ammonia in a continuous-flow system suitable for selective ammoniaelectrode-based enzymatic determination of creatinine in serum and urine samples. Selectivity and sensitivity enhancement has been reported previously in electroanalytical chemistry by using ionomerfilm-modified voltammetric electrodes (21-23). Indeed, perfluorinated ion exchangers have been used in voltammetry, as an alternate supporting electrolyte medium (24,25),as a means of preconcentrating cationic analytes at the electrode surface (21,26),as well as for reducing interference from electroactive anionic interferences (e.g., ascorbate, urate, etc.) (27). In the latter case, such coated electrodes have proven useful for the more selective detection of primary neurotransmitters in rat brain (28)and as outer discriminating membranes in the amperometric determination of glucose in whole blood (29). In the present work, we describe how a suitable anion-exchange membrane coupled to a FIA potentiometric enzymeelectrode-based detection system can also be used to enhance analytical selectivity toward a particular analyte substrate. Figure 1illustrates the two configurations examined. In one case (Figure la), the anion-exchange membrane is placed directly over the surface of the potentiometric enzyme electrode and this biosensor unit is utilized as the FIA detector. In the second and more practical measurement arrangement (Figure lb), the ionomer membrane is placed within a flowthrough dialysis unit upstream from the enzyme-electrode detector. As shown in Figure l b , neutral and anionic analyte species present in the injected sample can permeate the membrane while positively charged interferent species such as endogenous ammonium and potassium ions are, for the 0 1990 American Chemlcal Soclety
ANALYTICAL CHEMISTRY, VOL. 62, NO. 22, NOVEMBER 15. 1990
BUFFER
It:
DDH20
I I
71'-a
most part, repelled and thus never reach the enzymeelectmde detector. Measurements are based on the potentiometric detection of ammonium ions formed as the substrate molecules entering in the recipient buffer stream flow by the surface of the enzyme electrode. For the purpose of this study, the enzymes urease and glutaminase are employed for the selective determinations of urea and 1.-glutamine, respectively, in accordance with the following reactions:
"IC-
COP + 2NH,*
gl"faml"mc
glutamine
n S
w
w
/
DDH20
Flpure 1. Membrane arrangements used to enhance selectivlly of potentbmetric enzyme eIectr&s. (Part a) Membrane placed over surtace 01 me enzyme electrode: (a) nonactin CTA rnemorane; ( 0 ) immobilized enzyme layer: (cJanion-8xcMnge memorane: (d) Sample stream. (Part bl Membrane lined in a dialysis unit with an expanded view of the chemical processes occurring within.
urea
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glutamate
+ NH,+
Both enzymes are immobilized on the surface of hydroxylated asymmetric ammonium-selective cellulose triacetate (CTA) membranes (30)to form the required biosensor detectors. The practical analytical application of the proposed FIA arrangements is demonstrated by the measurements of urea in blood serum and glutamine in hybridoma hioreactor media. EXPERIMENTAL SECTION Apparatus. The various flow manifolds uwd for flow-injection measurements are shown in Figure 2. In all cases, solution delivery was accomplished with a low-pulsation multichannel peristaltic pump (Hainin Inc.. Wohurn, MA). A rotary valve (Teflon fluorocarhon resin) with variable.volume sample loups (Rheodyne. Cotati. CA) was used for sample introduction. The anion-exchange membrane, Type R-1030, was obtained from RAI Research Cow.(Hauppauge. NY). A piece of this memhrane WBS mounted iotu a homemade dialysis block (22.8 cm) containing a high surface area to volume ratio. The exart geometry of this flow-through dialysis unit has been reported previously (31). Flow-through potentiometric measurements were carried out by utilizing the enzyme electrodes in a large-volume wall-jet style membrane electrode detection arrangement as described in previous work (321. All potentiometric measurements were made with a digital pH/mV meter (Altex. Model 45001 in conjunction with a Fisher Recordall 5000 potentiometric recorder. A saturated calomel tlectrode was used as the reference electrode. Measurements were made at room temperature. Reagents. Cellulose triacetate (CTAJ,1 ,I'-rarbonyldiimidazole (CDII. and nonactin were obtained from Fluka (Ronkonkoma. NYI.and dipentyl phthalate was purchad from Eastman Kodak (Rochester, SYJ. Ureaw (EC3.5.1.5.Type C.3 from lark kansi, glutaminase (EC0.5.1.2,grade I1 and grade V from Eacherirhia coli), Iscove's Modified Dulbecco's Medium (IMDM,. and Controlled Process Serum Replacement (CPSR-01 were purrhased from Sigma Chemical Co.(St. huir. MOJ. Normal human control serum was a product of Fisher Scientifir (Orangeburg, NY). All other chemicals used were analytical-reagent grade. Standard solutions and hufferswere prepared with distiUed-.ieionized water (DD-H,Oi.
.. H D B
u
a
.. = iniection valve
Flgure 2. Three different FIA manifolds used in these stdes: P, peristaltic pump: S, Sample injection; DB, dialysis block; D. enzymeelectrode detector; W, waste; IM, ionomer membrane. Preparation of the Enzyme Electrodes. The enzymes urease and glutaminase were immobilized on the surface of hydroxylated asymmetric ammonium ion selective CTA membranes, which were mounted in Phillips electrode bodies, 1.5-561 (Glasbliiserei Moller, Zurich). These membranes were prepared according to a method described by Cha and Meyerhoff (301,in which hydroxyl groups on the surface of asymmetric ion selective membranes are either aminated or activated with CDI. After the membranes were mounted in the electrode bodies, the following procedures were employed. For the urea electrodes, 10 pL of urease solution (1M) mg/mL; 615 pmol units/mg in 0.05 mol/L sodium phoshate buffer, pH 7.0) and 4.5 pL of 2.5% glutaraldehyde solution in the same buffer were sequentially applied to the aminated or CDIactivated surface of the ion-selective membrane. For the glutamine electrodes, the same protocol was followed except that 6 pL of glutaminase solution (loo0 units/mL in 0.05 mol/L sodium phosphate buffer, pH 7.0) was added to the CDI-activated surface of the ammonium-selective membrane. In both cases, after 12 h at 4 "C, the membranes were rinsed thoroughly. Urea sensors were rinsed with a 0.05 mol/L tris(hydroxymethy1)aminomethanehydrochloric acid buffer (Tris-HCI), pH 7.2, while the glutamine sensors were washed with a 0.01 mol/L lithium acetate buffer, pH 4.9. Both types of enzyme electrodes were stored at 4 OC when not in use. Typically, with daily use within the various FIA systems, both enzyme electrodes exhibited analytically useful responses for at least 2 weeks Evaluation of Ionomer Membrane for Enhancing Seleetivity. Static Measurements. To assess the relative permeahilities of substrates and cation interferents through the anionexchange membrane, initial evaluation of the membrane was conducted by using the sensor configuration shown in Figure l a with and without the outer ionomer membrane in place (for urea electrode only). For these static measurements, 0.5 mL of 1mol/L aqueous standard solutions of urea, NH,C1, and KCI were added to a well-stirred volume (50 mL) of Tri-HCI buffer, pH 7.2. The potentiometric responses as a function of time of the urea enzyme electrode toward each of the species were recorded. Flow-Injection Measurements. The manifold arrangement shown in Figure 2a was used to evaluate the ionomer-membrane-covered urea enzyme electrode (Figure lb) as a direct detector in FIA measurements. For these studies, 20 pL of urea standard solutions or control serum WBS injected into a distilled water stream (2.3 mL/min), and this stream was merged with a stream (1.1mL/min) of 0.05 mol/L Tri-HCl buffer, pH 7.2. Peak
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 22, NOVEMBER 15, 1990
potentials (AE, mV, from baseline values) were recorded and used to quantitate urea concentrations in control serum samples. Both the urea and glutamine enzyme electrodes were evaluated in arrangements in which the anion-exchange membrane was placed in a dialysis unit upstream from the enzyme-electrode detector. For the urea system, 100 pL of urea standards and/or urea standards containing varying levels of KCl and NH4Clwas injected into the system using the manifold arrangement shown in Figure 2b. The flow rate of the distilled water carrier stream was 5.4mL/min. The recipient buffer was a 0.05 mol/L Tris-HC1 buffer, pH 7.2, flowing at 2.7 mL/min. Selectivity of the urea FIA system was assessed by comparing the peak heights (in millivolts) for injected urea standards to those observed when the same standards where spiked with varying amounts of KC1 and NH4Cl. A more quantitative evaluation of the improvement in selectivity was carried out with the glutamine-enzyme-electrode FIA system. Apparent selectivity coefficients, kb[j, for glutamine over ammonium and potassium ions with and without the anion-exchange membrane unit incorporated into the system were determined in the following manner. Initially, the dialysis unit containing the ion-exchange membrane was removed from the system (Figure 2a) and 100 WLof aqueous KCl, NH4Cl, and glutamine standards ranging between 0.1 and 10 mmol/L were injected into the system. Distilled water was used as the sample carrier stream (flow rate = 1.7 mL/min), and a lithium acetate buffer, pH 4.9,as the diluent stream (flow rate = 1.1 mL/min). The ionomer membrane unit was then reincorporated into the system (Figure 2 4 . In this case, the donor stream consisted of distilled water (1.1mL/min) merged with a 0.005 mol/L MOPS buffer [3-(N-morpholino)propanesulfonicacid] pH 7.3 (1.1 mL/min), while a 0.01 mol/L lithium acetate buffer, pH 4.9 (at 1.1mL/min), was employed B the recipient stream. Peak heights in millivolts were recorded in both cases for ammonium, potassium, and glutamine, and these values were plotted vs the logarithm of the concentration of each species. The fixed potential-separate solution method was then utilized to determine the apparent selectivity coefficients at two different glutamine levels (33). Correlation Studies. Urea. Thirty serum specimens were obtained from the Clinical Chemistry Laboratory at the University of Michigan Hospital. These samples had been previously assayed for urea by using the Kodak Ektachem 700 system (a urease-based colorimetric method). The specimens were then redetermined for urea by using the ionomer-membrane-based FIA system described above (with ionomer membrane dialysis unit), and the urea content of each sample was determined from a prior urea calibration curve. Glutamine. Hybridoma bioreactor medium samples were provided by the Cell Culture Laboratory in the Chemical Engineering Department at the University of Michigan. A calibration curve for fresh glutamine standards prepared in cell culture medium was recorded prior to sample introduction. Values for glutamine in the bioreactor samples were determined by using a second-order equation of the glutamine calibration curve data. Results were compared to a standard HPLC method (34).
RESULTS AND DISCUSSION The concept of using ionomer membranes to enhance the selectivity of biosensors originates from the fact that the permeability of such membranes to species that are neutral, or possess a charge that is opposite to that of the fixed membrane sites, is significantly greater than that of ionic species with the same charge as the fixed ionic sites of the membrane. Thus, when using anion-exchange membranes containing quaternary ammonium sites, the flux of small cationic species should be reduced relative to neutral or anionic species. Figure 3 illustrates this point for the anion-exchange membrane used in this work. This figure shows the effect of covering a potentiometric urea enzyme electrode (based on detection of NH4+) with a perfluorinated anion-exchange membrane on the time response of the enzyme electrode to additions of urea, NH4Cl, and KCl (at 10 mmol/L). In the absence of the membrane (Figure 3a), the enzyme electrode
.5c
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-100
b-
zy+ 0 P Y
0 O K
t 2 W
- 1 50
-200
. 5 min
Figure 3. Strip-chart recordlng showing the response of urea electrodes without (a)and with (b) an outer anion-exchange membrane to 10 rnmol/L urea (l), NH,CI (2), and KCI (3).
responds rapidly to all three species (in well-stirred sample solution). Although two ammonium ions are liberated per urea molecule, the response toward both species is about the same. This is because the response for urea is dependent on a steady-state level of ammonium ions formed in the layer adjacent to the membrane, and this is governed by the amount of immobilized enzyme activity, thickness of immobilized enzyme layers, etc. Equilibrium response to potassium is somewhat less than that to urea and ammonium. This is due to the fact that the nonactin-based polymer membrane electrode exhibits 10-fold selectivity for ammonium over potassium ions (35). Nonetheless, use of such a biosensor in samples containing high levels of endogenous potassium and/or ammonium would obviously yield a significant error in the measurement of urea. When an anion-exchange membrane is placed over the surface of the enzyme electrode, the rate of response to all three species decreases substantially (Figure 3b; note: the baseline is shifted due to the additional membrane potential generated by the anion-exchange membrane). As expected, however, the rate of response to neutral urea is enhanced relative to that observed for the two inorganic cations. Advantage can be taken of this fact to improve the practical selectivity of the enzyme electrode. That is, by employing the ionomer-membrane-based sensor in a nonequilibrium flow-through detection arrangement, enzymatic response toward analyte substrate can be observed before significant levels of endogenous interferent ions permeate the membrane and contribute to the observed potentiometric signal. To this end, preliminary experiments involved using the ionomer-membrane-covered urea sensor as a direct detector in a conventional FIA measurement arrangement (Figure 2a). A typical strip-chart recording illustrating responses to injections of urea standards (1-10 mmol/L) and control serum is shown in Figure 4. When the membrane-covered enzyme electrode is employed directly in a wall-jet detection configuration, a rather asymmetric response to urea is observed. The initial negative potential spikes appear to be due to a small transient change in the potential across the outer anion-exchange membrane owing to differences in the electrolyte composition of the samples (and standards) and the carrier
ANALYTICAL CHEMISTRY, VOL. 62, NO. 22, NOVEMBER 15, 1990
10mM Urn8
I
I
2421
10 mM Urea
10 mM Urea
50mV Control serum
1L 1:l dlluted serum
Potentiometric flow-injection urea response obtained with manifold b of Figure 2: (a) 1 mmol/L urea + 2 mmoi/L K'; (b) 5 mmoilL urea 5 mmol/L K+; (c) 5 mmol/L urea + 1 mM NH,'; (s) control serum. Figure 5.
+
10 mln
The
45
Potentiometric flow-injection response obtained with manifold a of Figure 2 using a urea enzyme electrode covered with anion-exchange membrane. Figwe 4.
stream. The subsequent rapid increase in potential as the dispersed sample initially comes in contact with the detector originates from the relatively rapid influx of neutral urea through the outer anion-exchange membrane to the immobilized urease, and concomitant conversion of this urea to detectable ammonium ions. The slow return to baseline arises from the relatively slow rate at which the enzyme-generated ammonium ions can permeate back through the outer anion-exchange membrane once the sample slug completely passes the detector. This slow reversal in response represents the major disadvantage of using the membrane-covered enzyme electrode as the FIA detector. Despite this shortcoming, such an FIA arrangement does in fact yield analytically useful selectivity enhancements and responses. Indeed, separate injections of KC1 and NH4C1into the FIA system yielded little potentiometric response until the concentrations of the cations approached 10 mmol/L levels (not shown). In addition, based on a calibration curve (not shown) for the urea responses in Figure 4, urea concentration values determined for the undiluted and 1:l diluted control serum samples were in good agreement with the values supplied by the manufacturer of this product (undiluted = 5.15 mmol/L; diluted 1:l = 5.60 mmol/L; average value reported by manufacturer = 5.24 mmol/L). Given that such serum normally contains very high levels of endogenous ammonia (approximately 1 mmol/L) (36, 37) and physiological levels of potassium (3.6-5.0 mmol/L), these preliminary results further supported the notion that a selectivity enhancement in real samples can be achieved by using the proposed approach. On the basis of the results shown in Figures 3 and 4, it is clear that the anion-exchange membranes do not reject cationic species completely. Indeed, electrochemical anion transference numbers of these membranes are reported to be on the order of 0.81 (38). Thus, some leakage of cations through the membrane is expected. Further, in the case of ammonium ions, apparent rates of permeability can be even larger than expected due to the fact that this cation is always in a pH-dependent equilibrium with gaseous dissolved ammonia and the neutral gas form will permeate the membrane more rapidly than ammonium ions. Given the recovery problems encountered when the ionomer-membrane-covered enzyme electrode is utilized as the FIA detector, an alternate FIA configuration involving the use of the ion-exchange membrane in a flow-through dialysis unit upstream from the enzyme-electrode detector was examined. Figure 1b illustrates an expanded view of the anion-exchange membrane unit and the overall chemical processes that take
> E
c I
30
P w
I Y
a w
n
15
a CONCENTRATION, mrnollL
Typical urea ( 0 )and glutamine (0)calibration curves for FIA-biosensor detection systems assembled with an upstream ionomer membrane unit. Figure 6.
place within this assembly. To demonstrate the performance of the membrane unit in reducing cationic interferences, several urea standards with and without background levels of interferent cations were injected into the FIA arrangement (together with control serum). The strip-chart recording for this experiment is shown in Figure 5. As can be seen, the presence of background ammonium and potassium ions at levels comparable to the level of urea present do not significantly alter the observed potentiometric responses to urea, even when the urea concentrations were as low as 1 mmol/L (physiological normal range = 1.3-4.3 mmol/L). Subsequent experiments in which separate solutions of NH&l and KCl standards were injected into the system yielded minimal response ( 1 5 mV) for concentrations of K+ 550 mmol/L and for NH,+ at concentrations 1 5 mmol/L. When Figure 5 is compared with Figure 4, these results clearly show the improvement in sample throughput (24 vs 6 sample/h) that can be achieved by using the membrane dialysis unit rather than the system where the membrane is placed directly over the enzyme electrode. Figure 6 shows the typical calibration curve for urea obtained with this FIA-biosensor detection system. Because the enzyme-electrode detector is a potentiometric device, when plotted on linear coordinates, a nonlinear response is observed. Over a wider and higher concentration of injected urea standards (e.g., 5-50 mmol/L), plots of peak height in millivolts vs the logarithm of urea yield a nearly Nernstian re-
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 22, NOVEMBER 15, 1990 4s
1
i
10.0 mM
IS -I
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10 min
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Flgurs 7. Fbw-injectkm response obtained with manifold c of Figure 2 for glutamine standards.
sponse (e.g., 50-55 mV/decade; not shown). It should be noted that since the system operates in a nonequilibrium mode, only a small fraction (measured to be approximately 1 % ) of the urea present in the sample stream actually enters the recipient stream as the sample flows through the ion-exchange membrane unit. Thus, for low-level injected urea standards, the actual concentrations of urea in the recipient stream, as detected by the downstream enzyme electrode, are below the concentration range where this type of detector will display classical Nernstian response (Le., in nonlinear range of peak height vs log [urea] plots). Moreover, the detection limits and throughput of this system can be altered by changing the flow rates of the sample and recipient streams that pass through the membrane unit. The flow rates used for the urea measurements were optimized for measurements of urea in the physiological range normally found in human serum. It should also be noted that two possible FIA manifold arrangements can be utilized for urea detection (Figure 2b or c). Since urea is uncharged regardless of the sample pH, it is not essential to dilute the sample slug with buffer before passing it through the ionomer membrane unit (e.g., Figure 2c). That is, unlike glutamine permeability (see below), urea permeability through the membrane is pH independent (i.e., not based on an ion-exchange mechanism). Thus, a simplified manifold (Figure 2b), in which the sample is carried to the membrane dialyzer unit in a distilled water stream, is adequate for most analytical applications. However, as mentioned above, selectivity enhancement over ammonium ions will in fact be pH dependent, and thus samples that would normally have pH values approaching the pK, of the ammonia-ammonium equilibrium should be run on a Figure 2c type manifold (with a buffer less than pH 7.5 as sample diluent stream) to ensure optimal selectivity enhancement over endogenous ammonium-ammonia present (see below). A more quantitative evaluation of the selectivity enhancement possible with the proposed FIA-biosensor system incorporating the ionomer membrane dialyzer unit was carried out with the glutamine sensing arrangement. Figure 7 shows a stripchart recording for detection of Gglutamine standards in the range of 0.2-10 mmol/L while Figure 6 illustrates the typical calibration plot for such an experiment. In this case, the sample stream must be diluted to a fixed pH (manifold c in Figure 2) to ensure that the fraction of glutamine in anionic form is constant. Indeed, given that the pHI (isoelectric pH) value for L-glutamine is 5.65, rates of glutamine permeability through the ionomer membrane (and thus larger potentiometric signals) were enhanced when the pH of the donor stream was increased. Therefore, a 5 mmol/L MOPS buffer, pH 7.3, was used as the diluent for the sample stream. However, since the pH optimum for L-glutaminase enzyme
150
-
100
-
50
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.
-2.0
.
,
-1.0
.
I
.
,
0.0
1.0
.
,
2.0
LOG CONCENTRATION, mrnol/L
Flgure 8. Calibration curves corresponding to flow-injectlan measurements of glutamine (O), NH,+ (A),and K+ (0) obtained using
manifold c (a) and manifold a (b) of Figure 2. Table I. Apparent Selectivity Coefficients for Glutamine over Interferent Ions, krlnli,for the Glutamine Enzyme Electrode in an FIA Measurement Systema
[glutamine], mM
without anion-exchange membrane
with
anion-exchange membraneb
kghlK+
kgh/NHd+
kgln/K+
kghINHdt
1
0.91
0.72
4.55 5.26
0.06
10
0.10 0.26
0.08
Calculated on the basis of fixed potential-separate solution method. Perfluorinated anion-exchange membrane Type R-1030 from RAI Research Corp. (Hauppauge,NY). activity is approximately pH 4.9, a lithium acetate buffer, pH 4.9, was chosen as the recipient stream solution that flows by the enzyme electrode. Consequently, some of the glutamine is transported through the membrane via an ion-exchange mechanism. This, in combination with the larger size of glutamine (relative to urea), results in significantly lower rate of membrane permeability for this species. Therefore, a lower total sample-stream flow rate was used for glutamine detection, and this leads to somewhat broader peaks and decreased sample throughput when results are compared to the urea system described above. For assessing the glutamine selectivity, responses were obtained for injections of NH,Cl, KC1, and glutamine standards with (manifold c of Figure 2) and without (manifold a of Figure 2) the ionomer membrane incorporated into the FIA system. The resulting peak heights plotted vs the logarithm of the injected sample concentrations are shown in Figure 8 for both cases. Apparent potentiometric selectivity coefficients, kgh,K+ and kghpH4+,were determined for two different glutamine standards (1and 10 mmol/L; see Table I) from these curves. As indicated in Table I, the selectivity enhancements for a 1 mmol/L glutamine solution over endogenous ammonium and potassium ions were 15- and &-fold when compared to the enzyme-electrode FIA systems assem-
ANALYTICAL CHEMISTRY, VOL. 62, NO. 22, NOVEMBER 15, 1990 50 1
I
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A
EE
Y
4 Y
V
.
0
10
20
30
40
50
Ektachem (mmollL)
T
2423
prior pretreatment. Results for 21 samples measured by both a standard HPLC method (34) and the FIA-biosensor system correlated well (see Figure 9) given the extremely narrow concentration range for glutamine present in the samples assayed (1.5-4 mmol/L) (FIA = 0.931 (HPLC) - 0.105; R2= 0.875). In summary, we have reported here that the selectivity of potentiometric enzyme electrodes based on ammonium ion detection can be enhanced significantly through the use of an appropriate anion-exchange membrane within a nonequilibrium flow-injection measurement system. Given the large number of enzyme electrodes already reported that incorporate enzymes that liberate ammonium ions (1-5) and given the further improvements in the quality of the anionexchange membranes available (e.g. improved rejection of cations, thinner membranes to enhance substrate fluxes, etc.), it is likely that this approach will offer a relatively simple route to promote the practical and more routine analytical application of such biosensor devices.
ACKNOWLEDGMENT
EE
We thank Don Giachero, Director of the Clinical Chemistry Laboratory at The University of Michigan Hospital, and Gyun Min Lee and Craig R. Halberstadt of the Department of Chemical Engineering (University of Michigan), for providing the serum and hybridoma media samples used in these studies. Registry No. Urea, 57-13-6; glutamine, 56-85-9.
Y
a
ii:
0
1
2
3
4
5
HPLC (mmol/L)
Figure 9. Correlation plots for determination o f (a) urea in human serum samples and (b) glutamlne in hybridoma reactor media by the new FIA-bbsensw method vs commercial Kodak Ektachem analyzer and conventlonal HPLC methods, respectively.
bled without the ion-exchange membrane unit. At 10 mmol/L glutamine, the enhancement factors were 9 and 20, respectively. These values clearly illustrate the selectivity advantage offered by using the ionomer membrane in the FIA-potentiometric-biosensor system. Practical Analytical Applications. Finally, the ionomer-membrane-based FIA-biosensor systems described above were tested for their ability to quantitate urea in human blood serum and L-glutamine in cell culture media. The latter measurement is becoming increasingly important in biotechnology since L-glutamine and glucose serve as the sole energy sources for growth and monoclonal antibody production by hybridoma cells (39). Both serum and hybridoma media samples normally contain levels of endogenous potassium and/or ammonium that would cause positive errors in the measurement of these species by the nonactin-membranebased urea and glutamine enzyme electrodes if the additional ionomer membrane were not used. For evaluation of the urea system, unidentifiable serum samples were obtained from the University of Michigan Hospital and assayed with the ionomer membrane FIA-biosensor system. Results correlated well with the values measured in the clinical laboratory with a commercial Ektachem 700 clinical analyzer (see Figure 9) (FIA = 0.873 (Ektachem) + 1.02; R2 = 0.987). For glutamine measurements, in preliminary experiments with fresh cell culture medium, some matrix problems were encountered due to the high concentration of inorganic salts and buffer molecules present in the sample medium. This caused a competition between the endogenous anionic species and glutamine for ion-exchange sites in the membrane, thereby yielding in a decrease in the transport of glutamine across the membrane. To compensate for this matrix effect, glutamine standards were prepared in fresh cell culture medium instead of DD-H20. Subsequently, hybridoma culture medium samples were injected directly into the FIA system without any
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Anal. Chem. 1990, 62, 2424-2429
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(34)Seavy, S. S. In Rapid I.llgh Perfwmenoe LlquM chromaiop~phy Technques for &#onA m h Acids in C W e F W ; Sewer, S. S.. Ed.: Mafcel Dekker: New York. 1967: na 315-318. (35) kyerhotf, M. E. Atmi. chem. ioio. 5z,'i52-1534. (36) mprhotf, M. E.; RoMns, R. ti. AIM!. chem.igoo. 52, 2383-2387. (37)Fratldi, Y. M.; m f f , M. E. Anal. Chem. 1981, 53, 992-997. (38) ProductendDete &&?; R A I Research Corp.: Hauppauge. NY, 1987; n A
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REC-
for review June 18,1990. Accepted August 7,1990. We gratefully acknowledge the National Science Foundation (Grant EET-8712756) and Mallinckrodt Sensor Systems for the support of these studies.
Detailed in Situ Scanning Tunneling Microscopy of Single Crystal Planes of Gold(111) in Aqueous Solutions Hidetoshi Honbo, Shizuo Sugawara, and Kingo Itaya* Department of Engineering Science, Faculty of Engineering, Tohoku University, Sendai 980, J a p a n
An In situ eledrochmlcal swmning tunnelkrg mlcrorcope (ESTM) was applied to a goM(ll1) surface with atomically flat terraces In aqueous perchloric acid Odutkns. The pits, a dngkr layer deep, were formed durlng the reductlon of OXW tayers with higher owldatlon states. The surface dmudon d gokl atoms In a pure^" HCIO, SdUHon was qulte slow and comparable to that observed In ultrahigh vacuum (UHV) reported In the prevlous literature. An electrochemical dlsgolution of gold was investigated In a HCIO, solution In the presence of chkrkle Ion. Strongly adsorbed CMOridb ionr, on gold( 111) remarkably enhanced the surface dlffuslon of gold.
INTRODUCTION One of the most exciting fields in electrochemistry seems to be the understanding of the relationship between the structures and properties of the solid-electrolyte interface at an atomic level (1-4). Although electrochemical techniques such as cyclic voltammetry have provided remarkably sensitive tools for characterization of various monolayer processes a t electrode surfaces (1-3),there are not many in situ methods for the determination of the structure of electrode surfaces a t the atomic level within electrochemical environments (4). However, recent efforts using in situ scanning tunneling microscopy (STM) have provided persuasive evidence that the in situ STM is a powerful new method for samples (metals and semiconductors), immersed in aqueous solutions (5-13). Real space imaging a t an atomic level has recently been achieved with graphite (5-7),Au(ll1) (8-101, and Pt(ll1) (11) surfaces in aqueous electrolyte solutions. Wiechers et al. first demonstrated resolution of monatomic Au(ll1) layers using a bulk single crystal (8). However, more recently, Trevor et al. reported the roughening and dissolution accompanying the oxidation and rereduction of the Au(ll1) surface (9). An underpotential deposition of a monolayer of lead has also been investigated on Au(ll1) by Green e t al. (10). The Au substrates used for the latter two studies cited above (9,10) were Au(ll1) films prepared by vacuum evaporation onto freshly cleaved mica. A critical question might be raised whether the surface can be transferred from the vacuum to the electrolyte solution without chemical changes and contaminations, which has been discussed in previous literature (1-41, even though individual clase-packed Au atoms could be imaged on these surfaces in an air environment (14, 15). In our previous study for Pt(ll1)surface, the Pt electrode 0003-2700/90/0362-2424$02.50/0
was annealed in a hydrogen-oxygen flame near loo0 "C for 1 min and then quickly brought into contact with ultrapure water saturated with hydrogen for a final preparation of atomically clean surfaces (11). Based on both electrochemical and in situ STM measurements, we concluded that Pt surfam have been kept clean during the in situ STM observation for a t least several hours (11). In this paper, therefore, we report our in situ STM study of clean Au(ll1) surfaces of single crystal spheres in aqueous perchloric acid solutions. Surface diffusion, pit formation, and electrochemical anodic dissolution are discusaed on Au(111) surfaces. EXPERIMENTAL SECTION Experimental conditions were similar to those described in our previous papers (7,11,15). Single crystals of Au spheres were prepared by the method of Clavilier et al. (16). A molten ball of gold at the end of a gold wire was slowly solidified in a flame of hydrogen and oxygen. Carefully prepared spheres, 2-3 mm in diameter, always consisted of eight facets of Au(ll1) in an octahedral confiiation. The crystallographic axe8 of the spheres were determined by both X-ray diffraction (11)and laser beam reflection from a Au(ll1) facet as described by Furuya et al. (17, 18). Mechanically exposed Au(ll1) surfaces (denoted by polished Au) with successively finer grades of alumina were annealed in a hydrogen-oxygen flame at ca. 600 OC for 10 min. Both facet and polished Au(ll1) surfaces were investigated. As a final pretreatment for all measurements, the Au electrode was again annealed in a hydrogen-oxygen flame near 600 "C for 30 s and then quickly brought into contact with ultrapure water saturated with hydrogen. The electrochemical cell for STM measurements made by poly(chlorotrifluoroethy1ene)was the same as described previously (11). The Au(ll1) electrode prepared above was transferred to the electrochemical cell. During the transfer, the Au(ll1) surface was protected from contamination by a droplet of ultrapure water on it. Bright Pt wires were used as counter and quasireference electrodes, respectively. All electrochemical potentials are reported with respect to a reversible hydrogen electrode (RHE) in the same electrolyte. Perchloric acid used was Kanto Chem. (super grade) whose content of chloride ion is less than 5 ppm. It is well known that trace chloride ion can substantiallyincrease the amount of soluble Au ions at anodic potential regions (19,20). Therefore, a special effort has been made to prevent contamination of chloride ions in our procedure of the preparation of solutions. The STM unit used for the present study was similar to that described by Hansma et al. (21). A fine mechanical approach is achieved by a 101 reducing lever. We employed a combined STM-bipotentiostat system for the present study in which the electrode potentials of both the tip and the substrate can be independently controlled with respect to a reference electrode 0 1990 American Chemlcal Society
