Anal. Chem. 7007, 59, 2049-2053
2049
Polymer Immobilized Enzyme Optrodes for the Detection of Penicillin Thomas J. Kulp,* Irene Camins, and Stanley M. Angel Environmental Sciences Division, Lawrence Liuermore National Laboratory, Liuermore, California 94550 Christiane Munkholm and David R. Walt* Max Tishler Laboratory for Organic Chemistry, Department of Chemistry, Tufts University, Medford, Massachusetts 02155
The preparation and performance of two enzymebased fiber-optic sensors (optrodes) capable of detecting penicillin are described. Each sensor consists of a polymer membrane that is covalently attached to the tip of a glass optical fiber. The membrane contains the enzyme peniclilnase and a pHsensitive fluorescent dye. A signal Is produced when the enzyme catalyzes the cleavage of the &lactam ring of penicillin to produce penicilloic acid and, consequently, a pH change in the microenvironmmt of the membrane. The sensors differ in the way the polymer membrane is constructed and in the type of pH indicator dye used. Both optrodes exhibit response times (40-60 8 ) significantly lower than those of the corresponding enzyme electrodes (2 min). Each gives a linear response over the concentration range of 0.000 25 to 0.01 M penicillin 0, when measured in a 0.005 M phosphate butler. The data indicate that these ImmoMilzation strategies produce similar results and may be considered complementary alternatives in future enzyme optrode appiications.
Given the established success of enzyme electrodes (1-3) and the recent advances in the production of usable fiber-optic chemical sensors (optrodes) (4-61, the development of enzyme optrodes is a logical area of interest. In general, the pursuit of integrated sensors consisting of a naturally occurring bioreceptor linked to a physical sensor holds great promise. These sensors exploit the inherent ability of the biomolecule to selectively and sensitively recognize a particular chemical species in a complex mixture of other chemicals. In the case of enzyme sensors, the signal is produced as a result of a selective enzyme-catalyzed chemical reaction of the analyte resulting in a product that is transduced by the sensor. The reaction, and thus the analyte concentration, is monitored through either the rate of formation of products or the steady-state concentration of products. Because the enzyme is catalytic, the response is inherently reversible. The first optical enzyme biosensor was presented by Lubbers and Opitz for the measurement of glucose (7). The feasibility of using an enzyme in an optrode was demonstrated by Arnold (8). More recently, Burgess et al. have presented an enzyme optrode for penicillin (9). In this paper, the preparation and performance of two alternative types of penicillin enzyme optrodes are described. All of these penicillin optrodes operate by the same mechanism. The enzyme, penicillinase, catalyzes the cleavage of the p-lactam ring of penicillin to form penicilloic acid, which dissociates into penicilloate and a proton, thus producing a pH change in the sensor medium. The enzyme reaction is monitored by a pHdependent fluorescent dye. The sensors described here consist of a surface coating of dye and enzyme incorporated into a polymer attached directly to the optical fiber. In one, the 0003-2700/87/0359-2849$01.50/0
enzyme is entrapped in polyacrylamide that has fluorescein covalently incorporated into it and is bonded directly on the activated fiber tip. The other sensor is constructed of penicillinase, a derivatized form of the pH-sensitive dye hydroxypyrenetrisulfonate (HPTS), and bovine serum albumin (BSA), which are intermolecularly cross-linked with glutaraldehyde on the tip of an amino silanized optical fiber. In each polymer, the pH-sensitive dye is intermixed with the enzyme, thus eliminating the diffusional barrier between the membrane and the dye. The data demonstrate that such measurements can be made on a microscale (200-pm sensor diameter) and, moreover, that the fibers retain the rapid response times of earlier sensors based on surface amplification (10)-a method that involves increasing the number of binding sites on the distal end of the fiber. The response times noted here are considerably faster than those for the corresponding enzyme electrodes (11).
EXPERIMENTAL SECTION Materials. Penicillinase (Type 1, from Bacillus cereus), penicillin G (potassiumsalt), and bovine serum albumin (A-7906) were purchased from Sigma Chemical Co. Potassium persulfate, riboflavin, acrylamide, and N,”-methylenebis(acry1amide) (BIS) were acquired from Bio-Rad Laboratories. The trisodium salt acid was obtained from Moof 8-acetoxy-l,3,6-pyrenetrisulfonic lecular Probes and y-(methacry1oxy)propyltrimethoxysilanefrom Pharmacia. Fluoresceinamineisomer I and acryloyl chloride were from Aldrich Chemical Co. All reagents were used without further purification. Optical fibers (glass/glass) of nominally 200/240 pm core/ cladding diameter were purchased from Diaguide (Fort Lee, NJ). The distal end of the fiber was stripped of the protective plastic buffer and polished prior to attachment of the sensor material. In all cases, the fiber lengths were approximately 1 m. Apparatus. All measurements for the polyacrylamide fluorescein penicillin optrode were performed with a portable fiber-optic fluorometer built by Douglas Instruments (Palo Alto, CA). This instrument was described in detail elsewhere (12). Its features include tungsten-halogen lamp illumination chopped at 30 Hz by a tuning fork and fitered with a narrow band-pass filter centered at the peak of the fluorescein absorption maximum. The collimated excitation light is transmitted by a 4 5 O dichroic beam splitter into a lens that focuses it onto the terminated end of the fiber. The numerical aperture (NA) of the Diaguide fibers used to make the optrodes is 0.2 and that of the coupling optics is 0.5. Thus, the fibers were overfilled with respect to the exciting light. Fluorescence returning from the distal end of the fiber is reflected by the beam splitter, passed through a narrow band-pass filter to remove any stray excitationlight, and focused onto a photodiode detector. The signal from the photodiode is amplified by a phase-sensitive amplifier tuned to the chopping frequency of 30 Hz. The apparatus used for testing the BSA-HPTS penicillin optrode has been described previously (10). OPTRODE CONSTRUCTION Polyacrylamide Gel Penicillin Optrode. The synthesis and attachment of the polyacrylamide gel proceeded in a 0 1987 American Chemical Society
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ANALYTICAL CHEMISTRY. VOL. 59, NO. 24. DECEMBER 15, 1987
manner similar to that used in the construction of polyacrylamide pH optrodes (described in Ref lo). The major steps are as follows: Surface Silanization. After the polished ends of the fibers were cleaned in chromic acid, the glass was silanized by soaking for 30 min in a 2% aqueous solution of y-(methacry1oxy)propyltrimethoxysilane adjusted to pH 3.5 with glacial acetic acid. Polymerization Reagents. Aqueous solutions of acrylamide and N,N'-methylenehis(acry1amide) (BIS) were prepared according to the procedure of Hicks and Updike (23). Acryloylfluoresceinwaa prepared hy mixing 3 mL of distilled THF, acryloyl chloride (90 pL, 1.1 mmol), and fluoresceinamine (isomer I, 90 mg, 0.26 mmol) and allowing the mixture to sit in the dark overnight. Typically, stock solutions of the acrylamide monomer and a mixture of the BIS and acryloylfluorescein solutions (401v/v ratio) were stored at 4 "C until required for a polymerization run. Polymerization and Enzyme Entrapment. An 8-mL volume of the acrylamide stock solution was mixed thoroughly with 2 mL of the BIS/acryloylfluorescein stock solution. To this mixture were added the initiators, riboflavin (1.5 mg) and potassium persulfate (3.0 mg). The solution was deoxygenated with nitrogen for 15-30 s. Then 3 mL of this mixture was transferred to a small beaker and mixed with penicillinase (2 mg, 5000 units). Next, the silanized ends of the fibers were inserted into the monomer-enzyme solution and polymerization was initiated with a Ken-Rad no. 2 photoflood lamp. To equally illuminate the solution from all angles, the position of the lamp was manually changed during the reaction. In addition, light was sent through the fibers to promote polymerization on the fiber-tip surface. The fibers were allowed to remain in the solution until there was a considerable buildup of gel on the walls of the beaker. At this point, they were withdrawn and illuminated for an additional 10 min to complete the polymerization of any adhering monomer solution. The fibers were then soaked in distilled water, placed in a plastic bag containing a small amount of pH 7 buffer, and stored a t 4 "C. BSA-HPTS Penicillin Optrode. Surface Silanization. The glass fiber tip was silanized by submerging it in a 10% solution (v/v) of (aminopropy1)triethoxysilane(adjusted to pH 3.75 with 4 N HCI) and then waa heated a t 75 "C for 3 h. Fibers were rinsed in water and dried overnight in a 100 "C oven. Polymerization and Cross-Linking of Enzyme. The amide linkage between hydroxypyrenetrisulfonate (HPTS) and hexanediamine was formed according to the procedure of Wolfbeis (14). An alhumin-dye solution was prepared by adding 700 pL of the derivatized dye to 5 mL of a 15% albumin solution (25) (w/v, 0.02 M phosphate buffer, pH 7.0). This stock solution was stored a t 4 OC. To prepare the enzyme sensor, pencillinase (1 mg, 2500 units) was dissolved in the albumin-dye solution (200 pL). Glutaraldehyde (16) (20 pL, 12.5%, v/v, 0.02 M phosphate buffer, pH 6.8) was then added and stirred 2 min. This mixture was pipetted onto the fiber tips, which were secured on a glass plate. After 30 min, the fibers were removed from the gelatinous membrane material, after carefully cutting with a razor blade. The glutaraldehyde cross-linking reaction can result in loss of the pH sensitivity of the dye if it proceeds too long. Cross-linking times of 30 min or less were found to he optimal. The sensors were soaked in 0.005 M phosphate buffer Overnight to rinse noncovalently attached dye molecules from the sensor. Measurements. A stock solution of 0.1 M penicillin G was prepared daily for the optrode measurements. Aliquots of the
+
"Py
+
C. "lllll,"...
+
1
yyq-
"0"
P.~I.W~...
Figure 1. Intermolecular Cross-linkingof penicillinase,BSA, and HPTS glass fiber with glutaraldehyde.
onto a
stock solution were diluted with a pH 7 phosphate buffer to obtain the requisite concentration solutions for the measurements. The buffer concentration was 0.005 M, although 0.003 M and 0.0005 M buffers were used to test the effect of buffering strength on response. Each diluted solution was adjusted to 0.1 M in KCI to maintain constant ionic strength. Prior to the substrate measurements, the optrode was soaked in a phosphate buffer of a concentration and ionic strength equal to that of the penicillin solutions. Upon reaching a stable fluorescence signal, the optrde was assumed to be ready for use. To make a measurement, the optrde was transferred from the starting buffer to the penicillin solution (15 mL). Upon reaching a steady state, the optrode was rinsed with the starting buffer solution to remove penicilloic acid. Once the original fluorescence value was restored, the next measurement was taken. Data were collected with a computer that read and plotted the output of the lock-in amplifer at prescribed time intervals. In all experiments, the illumination source was shuttered except during the measurement period in order to minimize photohleaching of the dye. Although the measurements were made every 20 s, the shutter was actually opened 2 s prior to the measurement to allow for the stabilization of the detection circuit (0.5 s time constant). RESULTS AND DISCUSSION The two penicillin optrodes are prepared by entrapping or binding the enzyme in a polymer-dye matrix bonded to the fiber tip. One preparation is based on the technique of cross-linked gel entrapment, using a fluorescein-derivatized polyacrylamide gel developed for use in a pH optrode (10). The second optrode is constructed of a polymer matrix formed by glutaraldehyde cross-linking of BSA, derivatized HPTS, and penicillinase. The chemistly of the latter optrde is shown in Figure 1. To incorporate the dye into the polymer, acetyl HPTS was converted into an amide (1 in Figure 1) by consecutive treatment with PCI, and hexanediamine. The dye was deacetylated leaving HPTS with amine residues available for reaction with glutaraldehyde. The amino silanized fiber is placed into a solution of derivatized HPTS, penicillinase, and BSA, which are all cross-linked with glutaraldehyde to produce a cross-linked matrix (2 in Figure 1). The responses of the penicillin optrodes to various concentrations of penicillin G as a function of time are displayed in Figures 2 and 3. The signal (plotted in units of either volts or thousand photon counts per second, Kcps) represents the net fluorescence intensity from the fiber a t a given penicillin concentration. The measurements were made in unstirred solutions a t pH 7.0 (acrylamide-fluorescein) and pH 7.12 (BSA-HPTS) maintained with 5 X M phosphate buffer and 0.1 M KCI. Prior to the optrode tests, measurements made with a conventional pH electrode indicated that the pH
ANALYTICAL CHEMISTRY, VOL. 59, NO. 24, DECEMBER 15, 1987 Penlclllln 0 Concentration(mM) 2.0 4.0 6.0 8.0 1 0 . 0
0.0 0.7
Penicillin G Concentration
0.6
-z E
rn iii
Q
.00025M
0.8 "O
+ .00050 M 4 9
0.5
+
0.4
4
0
20
40
60
EO
100
,00075M .00250M .00500M .01000 M
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i
120
nrne ( 0 ) 7.0
Flgure 2. Time response of acrylamide-fluorescein penicillin optrode. Buffer Is 0.005 M phosphate, 0.1 M KCI.
220
B Y
Penicillin G Concentration
200
Q
-c
180
8 rn
160
6.0
Flgure 4. Substrate response and pH response of acrylamide-fluorescein penicillin optrode. Open squares denote response in pH buffers (with no penicillin) as labeled on lower axis. Solid squares indicate response In penicillin solutions labeled on upper axis. Penicillin measurements were made in 0.005 M phosphate, 0.1 M KCI.
0.00025M
+ 0.00250M 9 9
+
5.0
PH
Penlclllln G Concentration (mM)
0.00625M 0.0125M 0.02500M
0
5
10
140
--
, 0
20
40
60 nms (8)
EO
100
120
Flgure 3. Time response of HPTS-BSA penicillin optrode. Buffer is 0.005 M phosphate, 0.1 M KCI.
of the solutions were equal to within 0.1 pH unit. After the run, the pH values of the solutions were measured again to confirm that the bulk pH of each solution had not changed. In addition, a fluorescein fiber (with no enzyme) was used to measure each solution to ensure that there were no artifacts peculiar to the fluorescein-acrylamide configuration that might lead to the observed signal. In all cases, the signal from the plain fluorescein fiber indicated only minor pH differences between the solutions and bore no resemblance to the signal produced by the enzyme optrode. For each optrode, the time required to reach a 95% steady-state signal was 40-60 s, as can be seen in Figures 2 and 3. The recovery time of the optrode following its return to the buffer solution was about 2 min. This could, however, be decreased by stirring. The optrode response time is significantly faster than those (2-3 min) reported for penicillin electrodes (1I ) . The difference can be attributed to the very small diameter of the fiber and the thinness of the polymer layer attached to it. Furthermore, the response is greatly aided by the fact that the pH detector (fluorescein or HPTS) is intimately mixed with the active enzyme membrane. The enzyme electrode requires the crossing of two diffusional barriers to produce a signal (substrate to enzyme, products to electrode), whereas the enzyme optrode requires only one. The decrease in the fluorescence signal with increased penicillin concentration is due to the decrease in pH that occurs in the microenvironment of the sensing layer when penicillin is converted to penicilloic acid. As the penicillin concentration is increased, more acid is produced and, thus, a lower pH and lower fluorescence signal results. A steady state is reached when the production and loss rates of hydrogen ions are balanced. To more fully understand the characteristics of the optrode, we examined the relationship between the fluorescenceintensity (microenvironmental pH) and the penicillin concentration. In unbuffered solutions, it is expected that the local pH produced by the enzymatic reaction will be linear with respect to the logarithm of the penicillin concentration (17,18). For buffered solutions, it has also been observed that a direct linear
z O
o'2 0.0
1 7.0
5.0
6.0
4.0
PH
Figure 5. Substrate response and pH response of BSA-HPTS penicillin optrode. Open squares denote response in pH buffers (with no penicillin) as labeled on lower axis. Solid squares indicate response in penicillin solutions labeled on upper axis. Penicillin measurements were made in 0.005 M phosphate, 0.1 M KCI.
relationship exists between pH and the molarity of the substrate (17,19); however the buffering strength determines the concentration range of the linear response and the sensitivity of the sensor. It has been observed that pH-based enzyme electrodes exhibit the highest sensitivity with low buffer strengths. However, the response under those conditions becomes very sensitive to changes in that variable. At high buffer strengths, the buffer concentrationis less critical. Thus, one must balance the need for a sensitive measurement against the necessity to carefully control the buffering strength of the solution. To evaluate the pH produced in the sensor, it is useful to f i s t measure the response to the sensor in solutions of varying pH, that contain no substrate. This provides a calibration of the pH response of the immobilized dye. A plot of the fluorescence signal of the acrylamide penicillin optrode in a variety of 0.1 M phosphate buffers of pH ranging from 4 to 7 is shown in Figure 4. A similar plot for the HPTS-BSA penicillin optrode is given in Figure 5. The normalized signal (S,) was derived according to the equation where S p H x is the fluorescence signal at a given pH and SpH and S p ~are 7 the signals observed at pH 4 and pH 7, respectively (for the BSA-HPTS sensor, SpH7 was replaced by SpH It may be noted that the fluorescence intensity of both dyes is nonlinear with respect to pH, although the HPTS curve is somewhat more linear than that of fluorescein. Although not shown in the figure, each dye is capable of responding to pH values above 7, the starting point for the
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 24, DECEMBER 15, 1987
shelf life was not investigated for the acrylamide optrode, it was noted that the BSA-HPTS sensor responded (with a reduced signal) to 2.5 X M penicillin after 3 months of storage at 5 “C immersed in phosphate buffer. +
0.0005M 0.0030M
0
0.0050 M
Q
0 000
0 002 0 004 0.006 0 0 0 8 Penicillin G Concentration (M)
0.010
Figure 8. Effect of buffer on optrode response for acrylamidefluorescein optrode. Microenvironmental pH was calculated by using the polynomial fit of the fluorescein pH response given in the text.
penicillin measurements. That value was selected as the initial pH because the optimal activity of penicillinase extends from pH 5.8 to 6.8 (20). Also plotted in Figures 4 and 5 are the steady-state signals (normalized) of each optrode at various penicillin concentrations. To our knowledge, these are the first direct measurements of the microenvironmental pH of an immobilized enzyme under working conditions. Each set of measurements was performed in solutions buffered a t pH 7 (acrylamidefluorescein) or 7.12 (BSA-HPTS) with 0.005 M phosphate and 0.1 M KCl. S, was calculated by using the optrode responses in the pH 4 and 7 (or 7.12) buffers (with no penicillin). It may be noted that the shapes of the pH and the penicillin response curves are in each case very similar. Thus, at this buffering strength, there is a linear relationship between the analyte concentration and the resulting microenvironmental pH. The solid curve in Figures 4 and 5 represents a polynomial fit of the pH response of each dye, defined by the following equations: acrylamide-fluorescein p H = 4.00
+ 14.15Sn - 42.75Sn2+ 75.55S2 -
BSA-HPTS p~ = 4.01
67.61Sn4+ 23.66Sn5
+ 5.73 s, - 5 . 8 3 ~ ~+23.26 s:
Using these equations, one can accurately relate the fluorescence signal of the optrode in a given penicillin concentration to the microenvironmental pH produced in the membrane. Figure 6 shows the acrylamidefluorescein optrode response converted to units of pH. The most linear data are those measured in 0.005 M phosphate buffer. Also plotted is the response of the optrode in two other buffer concentrations. As predicted, at these relatively low buffering capacities, the sensitivity is increased. For the lowest concentration buffer (5.0 X M), a response is obtained with a penicillin G concentration of 7.5 X M. The response appears to saturate, however, at relatively low analyte concentrations (perhaps due to diminished enzyme activity at these low pH values). The use of higher concentration buffers suppresses the pH change to a greater extent producing a linear response curve over a wider range of analyte concentrations. All of these measurements were performed under unstirred conditions to allow a maximum steady-state change. In tests in which the solutions were stirred, the steady-state signal was unaffected a t low concentrations and reduced by about 25-30% at moderate and high concentrations. Stirring the solution increases the transport of protons out of the polymer layer and in doing so increases the local pH. Under conditions of constant use, both optrodes retained their activity for an average of 2-3 weeks. While the long-term
CONCLUSION Our results demonstrate that this construction of fiber-optic sensors can produce improved performance, while maintaining miniature dimensions. The two enzyme entrapment strategies used to manufacture these optrodes should be considered as complementary methods. An immobilization technique that proves successful for one enzyme does not necessarily work for another. Furthermore, the diffusional properties of each membrane may favor certain analytes. Although apparently successful, the penicillin optrode does have certain drawbacks. As noted above, to obtain high sensitivities it is necessary to use low buffer concentrations and to maintain strict control over the buffering strength of the solution. At high buffer concentrations sensitivity will be sacrificed; however stringent control over the buffer strength is not critical. These problems mainly arise because pH is used as the indicator of the enzymatic reaction-a choice that was made due to the ease of optically monitoring that parameter. Nonetheless, applications of penicillinase electrodes in measuring fermentation broths ( I 7) have been demonstrated and such measurementsshould prove successful with these optrodes as well. It is likely that certain applications, such as clinical and process control measurements, can advantageously utilize the small sample volumes and rapid response times of these sensors. The use of enzymes in fiber-optic sensors has great potential due to their diverse, selective, sensitive, and reversible nature. In the future, it is certain that other optical “transducersn besides fluorescein and HPTS will be used to make enzyme optrodes. Some enzymes produce products that cannot be detected by optical means. These products may then participate in other enzyme reactions that can give rise to an optical signal. Such a scheme may further extend the scope of this optrode technology. ACKNOWLEDGMENT The authors thank Drs. Paul F. Daley and Fred Milanovich for technical discussions. Registry No. CH,=CHCOCl, 814-68-6;penicillin G, 61-33-6; penicillin, 1406-05-9;penicillinase, 9001-74-5;fluoresceinamine, 27599-63-9; acryloylfluorescein,53413-37-9. LITERATURE CITED Guilbault, G. G. In Immobilized Enzymes, Antigens, Antibodies, and PeptMes: Weetall, H. H., Ed.; Dekker: New York, 1975; p 293. Vadgama, P. I n Ion-Selective Nechode Methodology; Covington, A. K., Ed.; CRC Press: Boca Raton, FL, 1979; Vol. 2, p 23. Carr, P. W.; Bowers, L. D. I n Chemical Analyses: A Series of’Moflographs on Analytical Chemistry and lfs Applicatlons; Elving, P. J., Winefordner, J. D., Kolthoff, I. M., Eds.; Wlley: New York, 1980; Vol. 56, pp 95-147. Seltz, W. R. Anal. Chem. 1984, 56, 16A. Wolfbeis, 0. S. TrAC, Trends Anal. Chem. (Pers. Ed.) 1985, 4 , 184. Angel, S.M. Spectroscopy (SprjflgfieM, Oreg.) 1987, 2 , 38. Lubbers, D. W.; Opltz, N. Sens. Actuators 1983, 4 , 641. Arnold, M. A. Anal. Chem. 1985, 5 7 , 565. Fuh, M. S.; Burgess, L. W.; Christian, G. D. Presented at the Pittsburgh Conference, Atlantic City, NJ, 1987; paper 601. Munkholm, C.; Wan, D. R.; Milanovlch, F. P.; Klainer, S. M. Anal. Chem. 1986, 58, 1427. Tor, R.; Freeman, A. Anal. Chem. 1986, 58, 1042. Mllanovlch, F.; Daley, P. F.; Klainer, S.M.; Eccles, L. Anal. Instrum. ( N . Y . ) 1986, 75, 347. Hicks, G. P.; Updike, S . J. Anal. Chem. 1966, 38, 762. Offenbacher, H.; Wolfbeis, 0. S.; Furlanger, E. Sens. Actuators 1986, 9 . 73. Whlte, C. W.; Gulibault, G. G. Anal. Chem. 1978, 5 0 , 1481. Gullbault, G. G.; Danielsson, 6.; Mandenlus, C. F.; Mosbach, K. Anal. Chem. 1983, 55, 1582. Nilsson. H.; Mosbach. K.; Enfors, S.;Molin, N. Biotechnol. Bioeng. 1978, 20, 527. CUllen, L. F.; Rusling, J. F.; Schleifer. A,; Papariello, G. J.; Anal. Chem. 1974, 4 6 , 1955.
Anal. Chem. 1987, 59, 2853-2860
Hansen, E. H.; Ghose, A. K.; Mottola, H. A.; Anal. Chem. 1979, 57, 199. (20) Hou, J. P.; Poole, J. W. J . phefm. Scl. 1972, 67, 1594. (19) Ruzlcke, J.;
RECEIVED for review May 11,1987. Accepted August 17,1987. The work at Lawrence Livermore Laboratory was performed under the auspices of the US Department of Energy under
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Contract W-7405-Eng-48. The group at Lawrence Livermore Laboratory would like to express thanks to Gerald Goldstein of the Office of Health and Environmental Research for supporting their work under Contract No. RPIS-003906. The work at Tufts University was supported by the Environmental Protection Agency through the Tufts Center for Environmental Management.
Assessment of Conditions under Which the Oxidation of Ferrocene Can Be Used as a Standard Voltammetric Reference Process in Aqueous Media A. M. Bond,* E. A. McLennan,l R. S. Stojanovic, and F. G. Thomas2 Division of Chemical and Physical Sciences, Deakin University, Waurn Ponds 321 7, Victoria, Australia
The one-electron owldatlon process for ferrocene (Fc), Fc e-, has been studied extensively by cycllc, normal Fc+ pulse, and dlfferentlal pulse voltammetry and chronocoulometry to determine the condltlons under whlch this reactlon can be used as a voltammetrlc standard In aqueous medla. I n water, the oxldatbn of ferrocene Is not a shrple revenlMe one-electron process as Is the case In organlc solvents. Rather, weak reactant adsorptlon Is exhlblted, whlch Is electrode and electrolyte dependent. The oxldatlon of a saturated solutlon corresponds to conslderably less than a monolayer of coverage. The relatlve order of adsorption wlth respect to the electrode materlal follows the trend Hg > glassy carbon > Au > Pt, whlle wlth electrolyte lt Is NaCIO, > LI,SO, > NaF. Desplte the presence of weak reactant adsorptlon, essentially electrode, electrolyte, and technlque Independent voltammetric data can be obtalned at sufflclently slow scan rates or long pulse widths. The E,,, value calculated under these condltkns is 0.400 0.005 V vs NHE whkh agrees very well wlth the standard redox potential reported from potentkmetrlc measurements under genuine equlllbrlum condltlons. The data suggest that ferrocene can be used as a voltammetrlc standard under carefully chosen condltlons where the Influence of adsorptlon Is mlnlmal.
+
*
The standard or formal reduction potential (Eo or E3 is often the basic thermodynamic quantity used to characterize a redox system. In aqueous media, redox potentials are usually measured relative to reliable and universally accepted reference electrodes such as the standard hydrogen eledrode (SHE) or the saturated calomel electrode (SCE) (1,2). Unfortunately, no universally accepted reference electrode exists for work in the majority of nonaqueous solvents (3, 4): Frequently electrode potentials obtained in nonaqueous solvents have been reported versus aqueous reference electrodes such as the SCE, the silver-silver chloride electrode, or the SHE. The problem with using these reference electrodes in nonaqueous Present address: Division of Biological and Health Sciences, Deakin University. *On leave from James Cook University, Townsville 4811, Queensland, Australia.
solvents is that an unknown liquid junction potential is introduced into the measurements. In view of the solvent-dependent liquid junction potential problem, there has been considerable interest in finding solvent independent redox couples that can be used as reference redox systems in both organic and aqueous solvents (5-13). Unfortunately, the standard redox potential of a given redox system in solution invariably depends to some degree on the nature of the solvent in which it is measured (14). Some redox couples are very strongly solvent dependent (electrode potentials are a function of the coordinating ability of the solvent) and are clearly not suitable as reference redox systems (14-18). A systematic search for a solvent-independent reference redox system has been based on theory involving various extrathermodynamic assumptions. One conclusion reached from these assumptions is that the redox potentials of large ions, molecules, or complexes might be essentially independent of the nature of the solvent (6, 10, 11, 19). More recently, it has been proposed that large symmetrical ions in which the charge is deeply buried should have the same activity as an uncharged molecule of the same size and structure in all solvents. Such redox couples involving a one-electron transfer between the ionic and neutral forms will include only minor contributions from the solvent. Strehlow (6) used this concept to propose that the redox systems ferricenium ion-ferrocene (Fc+/Fc) (ferrocene is bis(q5-cyclopentadienyl)iron(II) and cobalticenium ion/cobaltocene (Cc+/Cc) (cobaltocene is bis(q5-cyclopentadienyl)cobalt(II)) should be solvent independent. Since then, several reviews have appeared in the literature (20-25) that suggest that the Fc+/Fc assumption (6)may not be perfect. In view of the lack of complete agreement of the use of ferrocene, other reference redox systems (7,10-13,26-28) have been suggested. These include bis(biphenyl)chromium(l) / bis(biphenyl)chromium(O) (Bcr+/Bcr) ( 1 0 , I I ) and redox systems based on polynuclear aromatic hydrocarbons and the respective radical ions (13). Despite some questions being raised with respect to the Fc+/Fc redox couple, extensive research into reference redox systems has led to the IUPAC Commission on Electrochemistry recommending that either of the redox couples ferrocene/ferricenium ion or bis(q-biphenyl)chromium(I)/bis(qbiphenyl)chromium(O)be used tw reference redox systems (29).
0003-2700/87/035Q-2853$01.50/0 0 1987 American Chemical Society
