Anal. Chem. 1998, 70, 3137-3145
Analytical Applications of Cooperative Interactions Associated with Charge Transfer in Cyanometalate Electrodes: Analysis of Sodium and Potassium in Human Whole Blood David R. Coon,† Linda J. Amos, and Andrew B. Bocarsly*
Department of Chemistry, Frick Laboratory, Princeton University, Princeton, New Jersey 08544 Patricia A. Fitzgerald Bocarsly
Department of Pathology, University of Medicine & Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey 07103
Nickel electrodes chemically modified with an interfacial layer of nickel ferrocyanide are shown to be of analytical utility for simultaneously sensing sodium and potassium ions in aqueous solutions, human whole blood serum, and human whole blood. By controlling the chargetransfer characteristics of this versatile interface, interfering blood proteins and potential interferences associated with other alkali cations can be avoided. A solid-state model which explains the excellent simultaneous selectivity and sensitivity of the nickel ferricyanide interface is proposed. Historically, electroanalytical chemistry has played a central role in the detection and determination of species having biomedical importance.1-3 Typically, detection schemes have been based on potentiometric techniques. Although well-established electroanalytical approaches are associated with membrane-based ionselective electrodes, recent developments in biochemistry and interfacial electrochemistry have allowed for the generation of electrode interfaces which couple biological materials with more classical electrochemical sensing techniques to introduce highly selective electrode responses.4 For example, surface confinement of specific enzymes,5 immunological reagents,6 or biological tissues7,8 have been used to interface with specific biomedical components thereby generating chemical species that can be detected amperometrically or potentiometrically. An alternate approach to the selective detection of biomedical analytes is the † Current address: Hawaii Biosensor Laboratory University of Hawaii Department of Chemistry Honolulu, HI 96826. (1) Cosofret, V. V.; Buck, R. P. Crit. Rev. Anal. Chem. 1993, 24, 1-58. (2) Koryta, J.; Brezina, M. Methods For Electroanalysis in Vivo; Marcel Dekker: New York, 1979. (3) Patriarche, G. J.; Chateau-Gosselin, M.; Vandenbalck, J. L. Polarography and Related Electroanalytical Techniques in Pharmacy and Pharmacology; Marcel Dekker: New York, 1979. (4) Wang, A.-J.; Rechnitz, G. Anal. Chem. 1993, 65, 3067-3070. (5) Heller, A. J. Phys. Chem. 1992, 96, 3579-3587. (6) Duan, C.; Meyerhoff, M. Anal. Chem. 1994, 66, 1369-1377. (7) Babb, C.; Coon, D.; Rechnitz, G. Anal. Chem. 1995, 34, 763-769. (8) Coon, D.; Babb, C.; Rechnitz, G. Anal. Chem. 1994, 66, 3193-3197.
S0003-2700(97)00975-X CCC: $15.00 Published on Web 06/20/1998
© 1998 American Chemical Society
rational and systematic design of interfacial transducer elements based on known chemistry and physics. Unlike electrode systems based on naturally occurring biological components, such “designed” systems offer the advantage of being amenable to finetuning thereby allowing for the optimization of electrode response with respect to sensitivity and sensor lifetime. We report here on a new electrochemical approach for the detection of nonelectroactive metal cations of biomedical importance which takes advantage of the well-defined solid-state properties of chemically modified interfaces containing ∼1000 Å of polycrystalline nickel ferricyanide. Unlike prior analytical schemes, the present approach is voltammetric in nature and relies on the electroanalytical detection of solid-state structural changes in the nickel ferricyanide lattice upon intercalation of different alkali cations. The basis of the technique involves well-defined structure-current-potential relationships in the described system. The nickel ferricyanide chemically modified electrode (CME) is composed of an electroactive layer of nickel ferricyanide that can be electrochemically cycled between the oxidized and reduced states of the iron ions (FeII/III). This electroactive layer exchanges cations between itself and the electrolyte during charge transfer in order to maintain charge neutrality. During oxidation, half of the cations inside of the film are expelled and the same number of cations are taken back into the lattice during the subsequent reduction.9 The voltammetric characteristics of the redox process including the redox potential of the FeII/III surface couple, the voltammetric wave shape, and the peak currents are dependent on the cations being incorporated inside the lattice.10 These variations correlate with changes in the nickel ferricyanide solidstate lattice structure induced by the intercalation/ion exchange of the charge-balancing cations.11 This has been verified by X-ray diffraction results.12,13 These results show that, for bulk powder samples of nickel ferrocyanide, the lattice expands going from (9) Humphrey, B.; Sinha, S.; Bocarsly, A. J. Phys. Chem. 1984, 88, 736-743. (10) Sinha, S.; Humphry, B.; Bocarsly, A. Inorg. Chem. 1984, 23, 203-212. (11) Chun, J. Ph.D. Thesis, Princeton University, 1993. (12) Luangdilok, C.; Arent, D.; Bocarsly, A. Langmuir 1992, 8, 650-657. (13) Amos, L. Ph.D. Thesis, Princeton University, 1988.
Analytical Chemistry, Vol. 70, No. 15, August 1, 1998 3137
sodium down the alkali metals to cesium. This is also seen when the lattice is confined to an electrode surface.11,13 The changes in the voltammetric characteristics of the electrode as the type and concentrations of ions in the solution are varied can be used to quantify these ions. As such, the analytic technique reported here is fundamentally different from other electrochemical techniques previously employed to detect alkali cations. In contrast to classic ion-selective electrode techniques, which utilize measured shifts in potential across an ion-selective interface to detect specific ion concentrations, the technique explored here uses voltammetric response (i.e., current vs controlled potential) to determine the type(s) and concentration(s) of fundamentally electroinactive species. The observed current is a secondary parameter. This current directly reflects the electrochemistry of the surface-confined FeII/III couple. Since the solid-state environment of these sites is perturbed by the alkali cations in solution in an electrochemically reversible manner, the voltammetric response of the system becomes analytically useful in the determination of alkali cation concentrations. The reversible nickel ferricyanide modified electrode functions over a broad pH range and under both aerobic and anaerobic conditions. Further, the system can be miniaturized. The zeolitic nature of this interface makes it an ideal way to sense ions of interest while excluding other species. Moreover, the exclusivity brought about by the size of the ion channels in the lattice allows the interface to function in matrixes that contain large, neutral, or anionic interferences such as proteins. We have chosen to demonstrate the electrode’s ion-sensing ability in untreated, human whole blood. Untreated human whole blood is difficult to analyze since it contains many chemical species that tend to cause fouling when existing electrochemical and photometric techniques are employed.14,15 Human blood begins to clot and degrade after it is exposed to air. The degradation of human whole blood produces ammonium as well as increases in the electrolyte concentration due to lysing of blood cells. In aqueous solution, NH4+ is the same size and charge as K+; thus, it is indistinguishable from K+ at the nickel ferricyanide interface. This species is the major interference in the analysis under discussion. In the currently employed clinical analysis of blood, it is centrifuged to separate it into two fractions, the lighter serum and the heavier red blood cells.14 White blood cells form the principal component of the serum, which is easily separated and collected from the heavier red cells. This serum is then analyzed for the desired substances. Ion-selective electrodes are currently used by many laboratories for inorganic ion measurements in the serum. This limitation precludes the adaptation of this technology to in vivo monitoring of blood electrolytes. In this report, human whole blood is voltammetrically analyzed for blood electrolyte concentration (i.e., sodium and potassium ion concentrations) using the nickel ferricyanide electrode. The voltammetric behavior of this electrode in aqueous solutions is used as a paradigm for understanding the simultaneous interactions of these two ions with the modifying layer in a biological medium. (14) Altman, P. L.; Katz, D. D. In Biological Handbooks II; Federation of American Societies for Experimental Biology: Bethesda, MD, 1977. (15) Eastman Kodak Co. KODAK EKTACHEM Clinical Chemistry Products; product literature, 1993.
3138 Analytical Chemistry, Vol. 70, No. 15, August 1, 1998
EXPERIMENTAL SECTION Electrode Fabrication. A polyethylene beaker (Dynalab Corp.) containing 10 mL of a solution of 5 mM K3Fe(CN)6 and 0.1 mM NaNO3 was employed as the electrosynthetic cell. Nickel wire (Alpha; 99.7%, diameter 0.63 mm) was abraded with sand paper, washed with distilled water, and wiped clean. A Tygon tube was fastened to the inside of the cell through which the wire could be placed to ensure it would remain stationary throughout the derivatization process. The cell was set at 1.0 V vs SCE and the nickel wire inserted for 60 s. The wire was then removed and washed by gently running deionized water down its sides. Blood Donation. Blood was collected from nine volunteers into heparinized and unheparinized Venoject blood collection tubes (Terumo Medical Corp.). The blood from the unheparinized tubes was centrifuged so the serums could be collected. The serums were submitted to Roche Biomedical Laboratories for analysis. The second tube contained lithium heparin to minimize the sodium contamination of the whole blood. Various unused tubes were sampled at random and their contents analyzed by ICP spectroscopy to ensure that there was no sodium or potassium contamination from the heparin salt. All analysis on the whole blood was done within 24 h of sampling to minimize NH4+ contamination and elevated cation levels resulting from deterioration of the blood samples. Electrochemistry. A three-electrode cell was used with a platinum auxiliary electrode. The potential was controlled with a PAR 173 connected to a PAR 175 universal programmer with data output to a Houston Instruments XY recorder. An SCE reference was used for all experiments. The scan rate was 100 mV/s for all experiments. Numerical Methods. As the cathodic and anodic peaks are symmetric about the potential axis, only the anodic portion of the voltammogram was used for numerical analysis. The anodic wave of the voltammograms of interest were first traced into an Apple IIe computer using Vidichart software. They were then transferred, by Kermit software, to a VAX 11/780 computer. Separate in-house FORTRAN programs were written to add the potential axis and scale the data to the proper current values and to evaluate the reproduced voltammogram. The program was used to calculate the fit of the desired model to the digitized voltammogram using the nonlinear least-squares subroutine DUNSLF (double precision). All the programs and their updates were tested with a computer-generated ideal cyclic voltammetric curve. Determination of Alkali Cations in the Modifying Layer. Large-area plate electrodes were used for the experiments in order to optimize the amount of nickel ferricyanide present. The derivatized area was monitored carefully by marking a spot on the electrode’s surface above which there would be no derivatization. The electrodes were cycled five times from 0 to 0.8 V vs SCE in various solutions of differing sodium-to-potassium ratio. In all experiments, the cycle was stopped at 0 V vs SCE to ensure the iron ions in the lattice were all in the reduced state. It was found that soaking the electrode for 10 min in 1 M LiOH stripped the electroactive layer off the nickel surface. High-purity LiOH (99%, Na+ 91 mV, indicates repulsive site-site interactions. Table 3 shows the calculated interaction parameters for various mole fractions of sodium ions using eq 2. The magnitude of the interaction parameters are comparable to those found in the sodium/cesium case; however, there is not a functional variation with variations in solution cation composition. Thus, the fit appears arbitary. The inability to fit the data in a physically meaningful manner using eq 2 along with the compositional data reported in Figure 4 points to a phase change in the structure of the derivatizing layer when the sodium mole fraction in solution goes below ∼0.33. This is the region where the directly determined internal and external lattice ion concentrations show a minimum, i.e., a preference for potassium over sodium ions. The reversal of the partitioning of Na+ and K+ at this cation ratio is best explained by a reorganization of the nickel ferrocyanide lattice. Similar behavior in the electrochemical response of ionic solid-state systems has previously been used to infer a phase transition.24,25 Directly observed structural changes due to alkali ion interaction have been reported for electrodes chemically modified with CdFe(CN)62-/-, a nickel ferricyanide analogue.12 The voltammetric characteristics of the CdFe(CN)62-/- film were found to be determined in large part by the structural changes, supporting the conclusion that the nickel ferrocyanide lattice undergoes a phase transition as the Na+ to K+ ratio is varied. The similar shape of the experimental voltammograms (Table 2) despite the mole fraction of sodium in solution leads to the conclusion that the phase change occurring in the mole fraction region around 0.33 is independent of the potential of the electrode. When an electrode with its modifying layer is placed in a solution of sodium and potassium ions, the lattice structure changes due to redox-induced ion exchange into the lattice framework. The equilibrium ratio of Na+ to K+ and the consequential structural changes in the nickel ferricyanide lattice are established regardless of the potential of the electrode. The electrode potential serves only to dictate the ion loading of the lattice, not the ratio of Na+ to K+ within the lattice. (24) Dahn, D.; Haering, R. Solid State Commun. 1982, 44, 29-32. (25) Dahn, J.; Dahn, S.; Haering, R. Solid State Commun. 1982, 42, 179-183.
Analytical Chemistry, Vol. 70, No. 15, August 1, 1998
3143
Table 2. Summary of Cyclic Voltammetric Results Obtained as a Function of Sodium and Potassium Mole Ratio at Constant Ionic Strengtha
c
mole fraction of sodium
sodium concn (M)
potassium concn (M)
anodic peak potential (V vs SCE)
∆Epb (mV)
fwhm (mV)
anodic peak current (ipa) (mA)
ipa/ipcc
1.0 0.971 0.882 0.706 0.588 0.412 0.294 0.118 0.059 0
0.170 0.165 0.150 0.120 0.100 0.070 0.050 0.020 0.010 0
0 0.005 0.020 0.050 0.070 0.100 0.120 0.150 0.160 0.170
0.370 0.380 0.410 0.437 0.445 0.453 0.450 0.460 0.465 0.470
115 80 85 87 90 83 75 85 95 95
135 140 145 140 140 140 140 135 135 130
0.366 0.248 0.296 0.322 0.308 0.345 0.312 0.384 0.334 0.373
1.08 1.08 1.04 1.04 1.04 1.03 1.03 1.01 1.04 1.04
a All data were obtained at a scan rate of 100 mv/s. An ionic strength of 0.170 M was employed. b Anodic peak to cathodic peak separation. Ratio of anodic peak current to cathodic peak current after baseline correction.
Table 3. Fitting Parameters for the Voltammetric Behavior of the Nickel Ferricyanide Electrode Using a Nearest-Neighbor Interaction Model mole fraction of sodium
Rr/107a
Ro/107b
R/107c
surface coverage/10-8d
sum of squarese
1.0 0.971 0.882 0.706 0.588 0.412 0.294 0.118 0.059 0.0
-3.01 -4.22 -3.85 -3.78 -4.26 -3.76 -3.76 -3.06 -3.54 -2.79
-2.93 -4.55 -3.62 -2.65 -2.60 -2.42 -3.02 -1.97 -2.53 -1.98
-5.94 -8.77 -7.47 -6.43 -6.84 -6.18 -6.78 -5.04 -6.07 -4.77
4.63 3.41 3.65 3.77 3.60 4.07 3.66 4.39 3.77 4.05
0.146 0.0803 0.0586 0.0848 0.0699 0.0993 0.712 0.131 0.0735 0.0908
a R is the interaction parameter for the effect of a reduced species r on a reduced species minus the effect of an oxidized species on a reduced species. b Ro is the interaction parameter for the effect of an oxidized species on another oxidized species minus the effect of reduced species on an oxidized species. c R is the algebraic sum of Ro and Rr. d Surface coverage is in mol/cm2. e The sum of squares is the algebraic sum of the squares of the differences between the fitted and experimental curves.
The observed redox potential of the modifying layer must therefore be composed of at least two independent terms, one term based purely on the idealized Nernstian potential (eq 3a)
θ)
[
]
[ ]
Fe(II) nF ) exp (E°′ - E) RT R Fe(III)
(3a)
2 3 4 E°′ R ) E° K - 0.10χNa + 0.22χNa - 0.25χNa + 0.19χNa -
0.16χ5Na + ... (3b) and the other based on the Na+ mole fraction (eq 3b), where (E°K) is the standard redox potential of the nickel ferrocyanide lattice in a pure potassium nitrate supporting electrolyte. Substitution of eq 3a into eq 2a,b provides the current-voltage response as a function of χNa in the electrolyte. The Nernstian term is chosen in the idealized form, based on the observation that the fwhm values of the cyclic voltammetric waves approach the idea one-electron value, independent of the χNa value. The form of the formal potential term (E°R) in eq 3b is provided as an arbitary fit to a power series in χNa based on the data provided in Figure 4. 3144 Analytical Chemistry, Vol. 70, No. 15, August 1, 1998
This functional form is not intended to be suggestive of a specific physical process. Rather, it is an empirical fit, designed to account for the observed solid-state phase transition of the nickel ferricyanide lattice upon sodium/potassium cation exchange. The above ideas nicely explain the experimentally observed shift in E1/2 with cation ratio, but the question as to why there is only one wave that moves for the sodium/potassium system yet two fixed waves for the sodium/cesium system still remains. It has been reported that only one type of ion may dominate the process of maintaining the electroneutrality of redox films that act as ion exchangers.26 We believe that this may be the case for the nickel ferricyanide electrode. The difference between the ionic radii of Na+ and K+ is 26% while the difference in the sizes of a Na+ and Cs+ ion is 62%.27 In the Na+/K+ ion case, the ions are so similar in size that they can dynamically undergo site exchange within the nickel ferricyanide lattice. If the exchange is rapid, then the electrochemical response should appear as a single redox event. The small difference in the ionic sizes, however, would still be large enough to cause structural changes in the lattice as the concentration of the dominating internal ion shifts with solution concentrations. The large difference in sodium and cesium ion radii should preclude rapid exchange between the two types of cation lattice sites, leading to two separate charge-transfer events in that system. Although we have focused on a solid-state mechanism to explain the observed electrochemistry, one might also examine mechanistic sources related to interfacial charge characteristics such as a shift in Donan potentials, ion-induced variations in double-layer capacitance, or the point of zero charge. However, none of these phenomena can explain the presence of two voltammetric events for the surface-confined iron systems in the presence of a Na+/Cs+ electrolyte and only a single well-defined voltammetric feature when a Na+/K+ electrolyte is employed. Likewise, no process related to interfacial electric field can provide a satisfactory explanation of the relationship between χIn Na and χNa. Blood Work. Since the mole fraction range of interest for human whole blood and serum is above 0.9, the phase change in (26) Redepennig, J.; Miller, B. R.; Burnham, S. Anal. Chem. 1994, 66, 15601565. (27) Huheey, J. E.; Keiter, E. A.; Keiter, R. L. Inorganic Chemistry: Principles of Structure and Reactivity, 4th ed.; HarperColllins: New York, 1993.
the low-sodium mole fraction region does not affect the sensor’s performance. The relatively quiescent and stable region above a sodium mole fraction of 0.9 allows the nickel ferricyanide electrode to perform as a stable sensor. As noted in the results section, the human whole blood analyses, which were conducted on freshly obtained samples, yielded results that were consistent with the clinically obtained values. These data showed a random error (based on standard deviation) which was well within the clinically accepted range and lower than the error associated with the clinically accepted ion-selective electrode techniques. Similarly, analyses carried out on human blood serum samples were found to produce acceptable values for both Na+ and K+ cations. However, while stored human whole blood serum yielded reasonable analytical values for sodium concentration, the observed potassium values were inconsistent with the clinical laboratory values and highly variable. We believe this was due to decomposition of the blood samples, a process known to produce ammonium ions, the only observed interference for K+ using the nickel ferricyanide electrode detection system. Presumably, the observed interference could be overcome by placing a strong base, cation-exchange resin membrane over the nickel ferricyanide modification layer. However, given the pragmatic fact that under typical operating conditions blood analysis is not carried out on “old blood” the observed interference does not represent a practical problem. Further, since the nickel ferricyanide electrode demonstrates an intrinsic protection from protein fouling, a relatively unusual situation, addition of overlayers composed of other materials may destabilize the system. We speculate that the lack of protein fouling is due to the net anionic charge of the nickel ferricyanide system interacting with the overall negative charge of most proteins. CONCLUSIONS The nickel ferrocyanide blood electrolyte detector is easily, rapidly, and inexpensively fabricated and calibrated. In addition,
the use of cyclic voltammetry provides for rapid data collection and reduction. As such this system, unlike presently available techniques, could form the basis of a disposable technology. Consistent with this suggestion is the ability of the electrode to hold its calibration for 8-10 h. The relatively short lifetime of the electrode (∼8 assays) would not be a limiting factor if utilized in a disposable system. Alternatively, further development of this system can be expected to improve the electrode stability and sensor lifetime. The system proves an added advantage in terms of its intrinsic antifouling- and interference-immune nature in human whole blood (properties not currently available in presently utilized diagnostic sensors). Freedom from interferences includes the alkali earth dications studied in the present work and a range of biologically prevalent anions (chloride, carbonate, nitrate, sulfate, phosphate) as previously reported.20 The proposed detection system also provides a response that is independent of oxygen partial pressure, a problem that has limited other bioelectrochemical detection schemes.10 Because of these properties, the nickel ferrocyanide system presents interesting possibilities with respect to an in vivo blood electrolyte sensor. Future work will be necessary to address this possibility. ACKNOWLEDGMENT Support from NSF under Grant CHE-9631380 is gratefully acknowledged. The authors thank Dr. Gene Knight for helpful discussions and computer assistance. We also extend many thanks to volunteers who donated blood for this work.
Received for review September 4, 1997. Accepted May 11, 1998. AC970975A
Analytical Chemistry, Vol. 70, No. 15, August 1, 1998
3145
