Anal. Chem. 1995, 67,1546-1552
Oxygen Transport Characteristics of Refunctionalized Fluoropolymeric Membranes and Their Application in the Design of Biosensors Based upon the Clark-Type Oxygen Probe D. J. Tamowski, E. J. Bekos,t and C. Kotzeniewski* Department of Chemistty, University of Michigan, Ann Arbor, Michigan 481 09
Surface refunctionalized fluoropolymer membranes were applied in the design of whole cell and enzyme biosensors based on the Clark-type oxygen sensor. Fluoropolymer membranes (poly(hexatluoropropylene-co-teMuoroethylene) (FEP)) were treated using a recently developed procedure that employs a hydrogedmethanol vapor radio frequency glow discharge plasma to introduce hydroxyl functionality into the polymer backbone in a controlled fashion. Hydroxylated materials were aminated by treatment with (y-aminopropy1)triethoxysilane(AF'TES). The surface amine groups served as attachment sites for whole cells and enzymes. Initial work measured the permeability and diffusion coefficients for oxygen in hydroxylated, aminated, and base (nonmodified) FEP membranes. Refunctionalizedmembranes retained the oxygen permeability and diffusion characteristics of the base fluoropolymer. Subsequent experiments investigated the response of biosensors constructed using aminated FEP as the gas-permeable membrane of a Clark-type oxygen sensor. The respiration of NB2a neuroblastoma cells was recorded following cell attachment to the membrane through natural growth processes. In a quiet solution, the response of the oxygen sensor decreased by -40% in the presence of a monolayer of respiring cells. Sensor response slowly returned to baseline after the cells were exposed to millimolar levels of sodium azide. The response of an enzyme electrode, constructed by linking glucose oxidase and albumin to free amine sites of aminated FEP, is also demonstrated. The calibration curve for glucose was linear over a concentration range between 0.1 and 6.5 mM, and the sensor response reached a steady state within about 60 s of exposure to glucose.
Clark-type amperometric oxygen sensor^^-^ are frequently employed in biosensing schemes that involve detecting substrates through their degradation by oxygen consuming enzymes or
' Permanent address: Department of Chemistry, Natural Sciences & Mathematics Complex, State University of New York, Buffalo, NY 1426@3000. (1) Clark, L. C. Trans. Am. SOC.Ad. Intem. Organs 1956,2, 41. (2) Hitchman, M. L. Measurement ofDissolued Ckygen;Wiley: New York, 1978. (3) Hale, J. M. In Polarographic Ozygen Sensors, Aquatic and Physiological Applications; Gnaiger, E., Forstner, H., Eds.; Springer-Verlag: New York, 1983. (4) Short, G. L.; Shell, G. S. G. 1.Phys. E.:Sci. Instrum. 1984,17, 1085. 1546 Analytical Chemistty, Vol. 67,No. 9,May 7, 7995
whole cell~.~-lO The steady-statecurrent of the Clark-type sensor is directly proportional to the oxygen partial pressure in the test solution, which is altered by enzymatic conversion of substrate. Through careful design of the biosensing system, a linear relationship between probe current and substrate concentration can be achieved over a useful range of substrate concentrations. An important component of the Clark-type oxygen sensor is the gas-permeable membrane, which separates the anode, cathode, and internal electrolyte solution from the analyte matrix. The gas-permeable membrane prevents interference from nonvolatile electroactive components in the sample, which facilitates probe use in complex environments. The membrane also confines the oxygen diffusion layer to a finite region outside the cathode, making the response of the sensor relatively time independent and table.^-^ Membranes are chosen on the basis of their permselective properties, mechanical strength, and resistance to chemical attack. Perfluorinated membrane materials, such as poly (hexafluoropropylene-cc-tetrafluoroethylene) PEP) and poly(tetrafluoroethylene) W E ) , are frequently e m p l ~ y e d . ~ , ~ In using Clark-type oxygen sensors for biosensor applications, a common probe design locates the biologically active component immediately adjacent to the gas-permeable membrane. A variety of strategies have been used to contain the biocatalyst so that the active layer can be secured to the probe tip.5,6J1,12 Common containment procedures involve entrapping the biocatalyst in a polymer gel or on a filter pad or covalently linking the biocatalyst to a porous support. The semirigid layer is then gently secured to the gas-permeable membrane, often with a porous dialysis membrane. Biocatalyst attachment schemes that provide facile oxygen transport to the biocatalyst and that maintain high catalyst activity are d e ~ c r i b e d . ~ . ~ J - ~ ~ (5) Karube, I.; Suzuki, M. In Biosensors: A Practical Approach; Cass, A E. G., Ed.: Oxford University Press: New York, 1990. (6) Wilson, G. S.; Thevenot, D. R In Biosensors: A Practical Approach; Cass, A E. G., Ed.; Oxford University Press: New York, 1990. (7) Goldblum, D. K; Holodnick, S. E.; Mancy, K H.; Briggs, D. E. Enuiron. Prog. 1991,10, 24. (8) Janata, J. Principles of Chemical Sensors; Plenum: New York, 1989. (9) Amold, M. A; Meyerhoff, M. E. In CRC Critical Reviews in Analytical Chemistty; CRC Press, Inc.: Boca Raton, FL, 1988 Vol. 20, p 149. (10) Karube, I. In Biosenson: Fundamentals and Applications; Turner, A P. F., Karube, I., Wilson, G. S., Eds.: Oxford: New York, 1987. (11) Barker, S. A. In Biosensors: Fundamentals and Applications; Turner, A P. F., Karube, I., Wilson, G. S., Eds.; Oxford: New York, 1987. (12) Guilbault, G. G.; de Olivera Neto, G. In Immobilised Cells and Enzymes; Woodward, J., Ed.; IRL Press: Oxford, 1985 p 55. (13) Rosevear, A; Kennedy, J. F.; Cabral, J. M. S. Immobilized Enzymes and Cells; Adam Hilger: Philadelphia, PA, 1987. 0003-2700/95/0367-1546$9.00/0 0 1995 American Chemical Society
The present study tests a new class of surface refunctionalized fluoropolymer membranes for their application in the design of biosensing probes based on the Clark-type oxygen sensor. In contrast to conventional oxygen electrode membranes, the fluoropolymers used here were surface modified to permit the direct, covalent attachment of biocatalysts, circumventing the need for a secondary biocatalyst support matrix, such as a porous gel or filter pad. Specifically,these experiments employ FEP membranes that have been exposed to a hydrogen/methanol radio frequency glow discharge plasma to introduce hydroxyl functionality into the fluoropolymer backbone in a controlled fashion (FEP-OH).14-19 Compared to other plasma-based surface refunctionalization methods, the process used here minimizespolymer morphological damage; the HZin the plasma scavenges ejected fluorine species, and the solvent vapor attacks defluorinated (radical) sites on the surface, creating new functionality on the polymer backbone.14-19 Studies that employ a battery of surface analytical techniqueshave shown that this process incorporates hydroxyl functionality at sites adjacent to -CF2 groups and that treated materials retain their original morphological characteristics and have surface hydrophobicities similar to that of the base p ~ l y m e r . ' ~ - ' ~ JFurther~J~ more, the surface OH functionality has been shown to be extremely reactive toward a variety of organosilane coupling agents, providing a means to introduce additional surface chemical functi~nality.~~-~~~~~-~~ For example, reacting hydroxylated fluoropolymer membranes with (y-aminopropyl)triethoxysilane (APES) creates a surface that is rich in primary amine functionality,which can also serve as sites for further derivatization. The surface properties of these plasma-treated fluoropolymers have led to their use as artificial supports for the immobilization of biological species.16J8,20.21 Whole cells,16J8-20 immunoglobins,21 and minimal peptide ~equences'~J~ have been linked to these materials using a variety of chemical and physical approaches. The -CF2 functionality surrounding the chemically derivatized sites provides a unique microenvironment for immobilized biological species, and the extent to which this environment improves the stability of attached agents has been of interest.21 An important application of these materials has been in studies of cell adhesion.16J8-20 Early work showed that plasma-treated and APTESmodified fluoropolymers containing preadsorbed protein support the growth of adherent cell populations."QO Cell attachment to the modified fluoropolymer surface is thought to occur through intermediate protein linkages,23and a more refined molecular level view of this process is emerging from experiments that probe the attachment of neural cells to immobilized minimal peptide sequence~.'~J~ (14) Vargo, T.G.; Gardella, J. A, Jr.; Meyer, A E.; Baier, R E. J. Polym. Sci. Part A : Polym. Chem. 1991,29, 555. (15) Hook, D.J.; Vargo, T.G.; Gardella, J. A, Jr.; Litwiler, K S.; Bright, F.V. Lungmuir 1991,7,142. (16) Vargo, T.G.; 'Thompson, P. M.; Gerenser, L. J.; Valentini, R F.;Aebischer, P.; Hook, D.J.; Gardella, J. A, Jr. Lungmuir 1992,8,130. (17) Vargo, T.G.; Gardella, J. A, Jr.; Calvert, J. M.; Chen, M.-S. Science 1993, 262,1711. (18) Ranieri, J. P.; Bellamkonda, R; Bekos, E. J.; Vargo, T.G.; Gardella, J. A, Jr.;Aebischer, P. J. Biomed. Mater. Res.,submitted. (19) Ranieri,J. P.; Bellamkonda, R; Bekos, E. J.; Gardella,J. A, Jr.; Mathiew, H. J.; Ruiz, L.; Aebischer, P. J. Neurosci., submitted. (20) Ranieri, J. P.; Bellamkonda, R; Jacob, J.; Vargo, T.G.; Gardella, J. A, Jr.; Aebischer, P. J. Eiomed. Mater. Res. 1993,27, 917. (21) Bright, F.V.; Litwiler, K S.;Vargo, T.G.; Gardella, J. A, Jr. Anal. Chim. Acta 1992,262, 323. (22) Li, M.; Pacholski, M. L.; Bright, F.V. Appl. Spectrosc. 1994,48, 630. (23) Pierschbacher, M. D.;Ruoslahti, E. Nature 1984,309, 30.
Observing the natural adhesion of cell populations at these modified fluoropolymer surfaces stimulated our interest in biosensor applications.2l Our aim was to detect the respiration of surface immobilized cells by using a modified fluoropolymer membrane in place of the standard gas-permeable membrane on a Clark-type oxygen sensor. We were interested in exploring this approach as a means to monitor cellular metabolic processes without the damaging effects of artificial immobilization. The present paper reports on experiments that employed these plasma-treated and APTESmodified fluoropolymers in whole cell and enzyme biosensors based on the Clark-type oxygen sensor. Initial work explored the effect of surface modification on fluoropolymer oxygen transport properties through experiments that measured the permeability and diffusion coefficients for oxygen in the base material and the surface hydroxylated and aminated forms. Corresponding oxygen transport parameters were the same for each material, withii experimental error. Subsequent experiments measured the respiratory response of surface immobilized neuroblastoma cells and demonstrated the response of an enzyme electrode constructed by covalently linking glucose oxidase to the free amine sites on the modified FEP.
EXPERIMENTAL SECTION
Oxygen Detection and Membrane Transport Parameters. Clark-type oxygen sensors were obtained from Yellow Springs Instruments (Yellow Springs, OH). Two probe types were used: the YSI Model 5357 (measured cathode radius = 127 pm) and the YSI Model 5331 (measured cathode radius = 591 pm). For both sensors, the anode (silver/silver chloride) and cathode (platinum) are located in a coplanar geometry at the probe tip. In preparing the sensors for oxygen quantification, a drop of electrolyte solution (2 M KC1) was placed in contact with the anode/cathode assembly, and then a membrane was positioned over the probe tip and held in place with an O-ring. A YSI 5300 Biological Oxygen Monitor was used to apply the polarizing voltage (-0.8 V at the cathode) and to monitor the current response. A limitation of the YSI 5300 is that it does not provide a calibrated current output. For permeability measurements, the current output was calibrated manually for a particular gain setting using a known resistance in the range of 30-63 MQ and applied voltages of 0.7 and 0.8 V. The oxygen monitor was interfaced to a 28Gbased microcomputer via a 12-bitA/D converter (ComputerBoards, CIO-ADO8). Oxygen transport measurements were made within thermostated cells. The YSI 5357 oxygen probe was used in a waterjacketed chamber with an internal volume of about 0.5 mL and a special port to accommodate the YSI 5357 probe (Instech Laboratories, Plymouth Meeting, PA). The YSI 5331 oxygen probe was used in a water-jacketed glass cell with an internal volume of about 30 mL. Both cells allowed for variable speed magnetic stirring and were thermally controlled by using a constant temperature water bath circulator (VWR Scienac, Model 1131), which provided temperature control and stability of better than *0.2 "C. The oxygen partial pressure of a test solution was determined from the solution temperature and atmospheric pressure, according to Analytical Chemistry, Vol. 67, No. 9,May 1, 1995
1547
where Pc is the atmospheric pressure, P H ~isOthe partial pressure of water vapor in the gas above the solution,24and 0.2095 is the volume fraction of oxygen in air.25 The atmospheric pressure was monitored in the laboratory using a mercury barometer. For membrane permeability studies, cathode areas were determined in separate experiments by using chronocoulometry. For these measurements, the anode/cathode assembly was placed in an electrochemical cell containing an aqueous 0.5 M KNO3 electrolyte solution and 6.7 mM Ru(NH3)6(N03)3 (Johnson Matthey). The platinum cathode was connected to the working electrode contact of a three-electrode potentiostat (FAR Model 273/96, Princeton, NJ) that was controlled via an IEEE-488 interface bus by a 386 microcomputer running MSDOS. A platinum ring electrode and a saturated calomel electrode (SCE) served as the counter and reference electrodes, respectively. The current transient measured upon stepping the potential from -0.15 to -0.35 V (vs SCE) was digitized and numerically integrated. Plots of charge vs t1/2were constructed, and the cathode area was determined from the slope of the chronocoulometry plot, cm/s as the diffusion coefficient of Ru(NH3)6(N03)3 using 9 x in 0.5 M KN0326at 25 "C. The measured cathode areas were 0.3 f 0.1 mm2 and (1.4 f 0.1) x mm2for the YSI Models 5331 and 5357 probes, respectively. Just prior to area determinations, and periodically before oxygen measurements, the cathode/anode assembly was polished using 0.1 pm alumina (Buehler) and sonicated. Subsequently, the Ag/AgCl layer was re-formed by anodization in 2 M KCl. Reagent grade KCl was obtained from Aldrich and used as received. The sensor response measured in oxygen purged solutions was ~ 1of% the value measured in air-saturated solution. Values for the thickness of the electrolyte layer between the cathode and the membrane (ze values) were confirmed using an infinity corrected confocal microscope (Olympus BH-2, Overland Park, KS). Membrane Preparation. FEP membranes were obtained from American DuraFilm (Holliston, MA) in 25.4 and 50.8 pm (1and 2-mil) thickness. Plasma-modified membranes were prepared in a radio frequency glow discharge plasma in the presence of a flowing Hz/methanol vapor, as described in ref 16. Plasma treatment removes surface fluorine atoms and replaces them with OH groups that covalently attach to the polymer backbone. Except where noted, aminated materials were prepared by dipping the plasma-treatedmembranes into a solution of ARES in hexane (1 mL of APTES to 40 mL of hexane), as described in refs 15,16. APTES was obtained from Sigma Chemical Co. (St. Louis, MO) and used as received. Cell Culture and Whole Cell Biosensor Preparation. Mouse neuroblastoma subclone NB2a (American Type Culture Collection, Catalog No. CCL 131) was cultured in 25 cmz disposable culture flasks (Coming 2510&25) in a sterile medium containing Dulbecco's Modified Eagle Medium @MEW (Gibco Laboratories, Grand Island, NY), 5% (v/v) fetal bovine serum (24) Forstner, H.; Gnaiger, E. In Polarographic Oxygen Sensors; Gnaiger, E., Forstner, H., Eds.; Springer-Verlag: Berlin, 1983; p 321. (25) CRC Handbook of Chemistry and Physics, 72nd ed.; CRC Press, Inc.: Ann Arbor, MI, 1991; p 14. (26) Morita, M.; Longmire, M. L.; Murray, R W. Anal. Chem. 1988, 60,2770.
1548 Analytical Chemistry, Vol. 67, No. 9, May 1, 1995
Outer Sleeve 0-Ili n g
Inner Sleeve
Gas Perm e a b le Mem h ran e
Vln n i t 0 r
I r
Figure 1. Schematic drawing of the chamber assembly and oxygen probe used for cell growth and cellular respiration experiments.
(FBS) (Gibco),penicillin G (100 units/mL) ,and streptomycin (100 pg/mL). Adherent cells were detached from the culture flask surface by using a Hanks balanced salt solution (HBSS) (Gibco) containing trypsin-EDTA (0.5%(w/v) trypsin, 5.3 mM EDTA*4Na, Gibco) and replated every 96 h. Spent DMEM medium was removed from the culture flask using a sterile pipet, followed by a 2 s rinse with 1mL of the trypsin-EDTA solution. Cells were then covered with 0.3 mL of the trypsin-EDTA solution, and the flask was gently rocked until the cells detached. Released cells were dispersed in 6 mL of DMEM medium, and 1mL of this cell mixture was added to 7 mL of fresh DMEM in a new, sterile flask for continued growth. This procedure was also used to detach cells immediately before biosensor preparation, except that released cells were brought to a final volume in DMEM medium of 3 mL, and a fraction of this mixture was used for attachment to modified membranes. Cells were maintained at 37 "C in a water-jacketed incubator (VWR Scientific, Model 2250) under a 5%C02 atmosphere. All procedures were performed under sterile conditions in a laminar flow biological safety cabinet (Baker). Cell immobilization onto APTESmodified FEP (FEP-APS) was performed with the modified membrane secured to an oxygen probe. The YSI 5357 oxygen probe was employed for all cellular respiration measurements. A special chamber was constructed that allowed for cell growth on the portion of the membrane covering the probe tip. A schematic drawing of the chamber and oxygen probe is shown in Figure 1. The probe was mounted in a threaded plastic sleeve (inner sleeve, Figure 1) and positioned such that the top half of the sensor protruded through the end. A gas-permeable membrane (FEP or FEP-APS) was then secured to the probe tip with an O-ring,which sat in the groove just below the top surface of the probe. Once the gas-permeable membrane was secured, the electrode assembly was inserted into a plastic fitting (outer sleeve, Figure l), which was closed at one end, except for a small hole through which the probe tip could extend. The electrode assembly was pushed forward until the O-ring made contact with the lip at the top of the fitting. The inner sleeve was
then screwed into the fitting until it contacted the O-ring with sufficient pressure to form a water-tight seal. In an earlier step, a piece of Tygon tubing was cut to a length of about 2 cm and stretched over the end of the outer sleeve. This short piece of tubing formed a compartment that held the cell suspension during the period of cell attachment and growth. Just before an FEP-APS membrane was attached to the probe tip, the membrane was rinsed in a water cascade (Barnstead Nanopure II) for -5 min and then dipped in 95%ethanol and left to dry on a tissue in the laminar flow hood. M e r the membrane was attached to the oxygen electrode (with the modified side facing the solution) and the probe mounted in the cell growth chamber, the membrane was rinsed with 95%ethanol a second time, along with the entire chamber assembly, and left to dry in the laminar flow hood under ultraviolet illumination. The assembly was flushed with sterile phosphatebuffered saline (PBS) (Gibco, pH 7.1) and sterile DMEM before the cell suspension was added to the chamber. Cell growth was initiated by covering the membrane with 0.25 mL of the cell suspension, loosely capping the top of the chamber, and placing the assembly in an incubator for 12-48 h. Enzyme Electrode Preparation. Hydroxylated FEP (25.4 pm thickness) was aminated by using a freshly prepared 5%(v/ v) AFTES solution in 20%ethanol/water. The solution was mixed at ambient temperature and allowed to stand for 30 min. The mixture was then spread over the hydroxylated FEP surface and allowed to cure for 24 h at room temperature. Compared to FEPAPS membranes prepared using the m S / h e x a n e solution described earlier, membranes silanized under aqueous solution conditions provided for greater enzyme loading, due to more extensive surface amination. Glucose oxidase from Aspergillus niger (Type VII, 177 units/mg, Sigma) was coupled to the aminated surface using a glutaraldehyde/albuminmixture. Glucose oxidase was added to a 0.1 M carbonate/bicarbonate buffer (PH 9.0) that contained 5% (v/v) glutaraldehyde (25%,Grade 1 (G5882), Sigma) and 60 mg/mL albumin (Albumax 11, Sigma). The h a 1 glucose oxidase concentrationwas 400 units/mL. This mixture was spread over the surface of the animated FEP and allowed to react overnight at 3 "C. Following coupling, the membranes were washed with PBS (PH 7.1) and stored dry at 3 "C. RESULTS AND DISCUSSION
Oxygen Transport Properties of Modified FEP. In using the plasma-treated and chemically modified membranes in an oxygen detection system, it was important to assess the iduence of fluoropolymer surface modification on the permeability and diffusion coefficients for oxygen in the various materials. These transport parameters were measured by using standard Clarktype oxygen sensors. The membranecovered Clark-type oxygen sensor employed in the present work responds to oxygen through its four-electron reduction at a platinum cathode: 0,
+ 2H,O + 4e- - 4 0 H -
(1)
Under conditions of planar diffusion, the mass transport limited current (9 is directly proportional to the partial pressure of oxygen in the test solution (PpOz), and it also depends upon the magnitude of the membrane oxygen permeability coefficient (Pd and the membrane thickness (zm) according to the following relationship:
where A is the area of the cathode and F is the Faraday constant. Since probe sensitivity is directly proportional to membrane permeability, the approximate value of P, for various materials that are employed in Clark-type oxygen sensors is of interest. P, is the product of the membrane oxygen diffusion coefficient (D,,,) and the oxygen solubility in the membrane phase (SA, as shown below:
P, = D,S,
(3)
In initial experiments, oxygen P, values were determined for various membranes by measuring the steady-state current of the probe when the tip was submerged in a thermostated cell containing airequilibrated deionized water. Steady-state current was recorded under well-stirred conditions, under which the flux of oxygen to the membrane was sufficient to maintain the 02 concentration at the outer surface at bulk levels (i.e., when the probe current remained constant with increases in the stirring rate). These experimentalconditions confine the diffusion layer to the membrane, so that the steady-state current can be directly related to the oxygen transport properties of the membrane.2 Computing the oxygen permeability coefficients from the steadystate probe current required accurate knowledge of the cathode area, which was determined in independent experimentsby using chronocoulometry (see Experimental Section). Oxygen P, values were determined for base (nonmodified) FEP membranes and for FEP membranes that had been plasmatreated (FEP-OH) and subsequentlyAFTESmodified (FEP-APS). Figure 2 reports the oxygen permeability coefficients measured using both the YSI 5357 and 5331 oxygen probes, which have different cathode sizes. Figures 2a shows the oxygen P, values computed from measured steady-state currents using eq 2. For similar materials, substantial differences in the P, values were determined using the two different probes. While the P, values measured using the larger probe are in close agreement with typical literature P, values for untreated FEP (i.e., 1.5 x 10-l2 mol.m-l.s-l-kPa-l 27,28),considerably larger values were obtained using the smaller probe. This discrepancy was traced to nonplanar diffusion in the electrolyte layer behind the membrane.B.30 Subsequently,P, values were corrected for nonplanar diffusion effects using the model described by Jensen et aLB (Figure 2b). Specifically, the permeability coefficients reported in Figure 2b were computed using eq 2 with values for the steady-state current arising from planar diffusion (&,lanu), derived from the measured steady-state current (imeasd)using eq 4:
In eq 4, KlhR) and K&R) are modified Bessel functions of fist and zero order, respectively, and XR is a dimensionless radius given (27) Polymer Handbook, 3rd ed.; Pauly, S.,Ed.; Wlley-Interscience: New York, 1991; p 435. (28) Pastemak, R A; Burns,G.L.;Heller, J. Macromolenrles 1971,4, 470. (29)Jensen, 0.J.; Jacobsen,T.; Thomsen, K.J. Electrochem. SOC.1978,87,203. (30) Garavaghan,D.J.; Rollett, J. S.;Hahn,C. E.W.J.Electroanal. Chem. 1992, 325.23.
Analytical Chemistty, Vol. 67, No. 9, May 1, 1995
1549
5.0
Table 1. Fluoropolymer Oxygen Transport Parametersa
.a FEP
4.0
FEP-APS
FEP-OH
FEP FEP-OH FEP-APS FEPb
P, (mol*m-l*s-l*kPa-l) x 10l2
D, (cm2/s) x lo7
1.24 f 0.04 (N = 25) 1.23 f 0.02 ( N = 12) 1.19 f 0.05 ( N = 13) 1.5
1.2 f 0.2 ( N = 6) 1.2 f 0.5 ( N = 3) 1.2 f 0.1 ( N = 5) 1.7
a Parameters measured at 25 f 0.2 "C. Error bars represent the 95%confidence level. Literature values from refs 27, 28.
I
I
1 .o
0.5
0.0
Figure 2. Membrane permeability coefficients measured for FEP and modified FEP using the following YSI oxygen probes: the Model 5357 b),which has a measured cathode radius of 127 pm, and the Model 5331 (M), which has a measured cathode radius of 591 pm. (a) Permeability values computed from the measured steady-state probe current. (b) Permeabilityvalues computed from the measured steady-state probe current after correction for nonplanar diffu ~ i o n . * ~
where R is the cathode radius, Pe is the oxygen permeability coefficient in the electrolyte, and Ze is the thickness of the electrolyte layer between the cathode and the membrane. The Pe value used was for 02 in aqueous 2.0 M KC1 electrolyte (1.13 x 10-l' mol*m-1*s-1kPa-1).2 The ze value is more difficult to determine, and typical values are reported to be in the range of 3-7 pm.29 A ze value of 5 pm was used here; however, since the FEP membranes were transparent, we verified that 5 pm was a reasonable estimate using a confocal microscope (see Experimental Section). Measurements were made at the probe center by focusing first on the cathode surface and then on the inside surface of the membrane; this procedure gave Ze values of 5-10 pm. Corrected f" values were computed from corresponding measurements made with the two different oxygen electrodes by using the iterative approach described by Jensen et al.B to determine optimal values for XR and iPlanar for each probe. Maple (v 5.0) was used in the evaluation of the modified Bessel functions. For similar materials, the correction for nonplanar diffusion brings the permeability coefficients measured using the two probes into closer agreement. Average values for the permeability coefficients, computed by pooling the measurements made using the two dif€erentprobes, are listed in Table 1. For the base FEP material, the measured value is slightly lower than the literature value,27*28 but the correspondence is very good considering the wide variability in permeability coefficient values reported for well1550 Analytical Chemistry, Vol. 67, No. 9,May 1, 1995
studied fluoropolymers, such as PTFE (cf. ref 31). Figure 2 also indicates that oxygen permeability coefficients for the surface hydroxylated and surface aminated materials are not significantly different from the value measured for the base FEP material. Diffusion coefficients for oxygen in the membrane (03were determined from the current transient recorded in response to a step change in oxygen partial pressure at the membrane's outer surface? The transient current response (iJ is given by
+
it = is, (1 2C(-l)"e-n24)
where q = (+'D,t)/zm and is, is the electrode steady-state current response.2 As described by Hitchman: restricting the sum to the first-order term provides an expression for the diffusion coefficient for times greater than 1s. Using this approximation,eq 6 reduces to
Diffusion coefficients for oxygen in the FEP membranes were determined from the current transients by plotting the logarithmic term on the left side of eq 7 as a function of time, for t > 1s, and then computing D m from the slope of the line (typical current transients decay over a period longer than 7 s) . A step change in oxygen partial pressure at the outer membrane surface was brought about by changing the solution stir rate. Specifically, after equilibrating the electrode in a quiescent solution, under conditions where the oxygen diffusion layer extends beyond the membrane and into solution, the stir bar was switched on to initiate stirring at a rate that was sufficient to keep the oxygen partial pressure at the membrane's outer surface at bulk levels. A current transient recorded in a typical experiment using an FEP membrane is shown in Figure 3a, and the corresponding log plot is shown Figure 3b. Values for the diffusion coefficients for the base FEP material are listed in Table 1. The values are the same, within experimental error, for all materials. Electrode Response to a Respiring Adherent Cell Population and Cross-LinkedEnzyme. To test the application of these materials in biosensors based upon the Clark-type oxygen electrode, two approaches were taken. In the first, the oxygen electrode was used to record changes in the respiration rate of an adherent cell population that was plated on the outer surface of the FEP-APS gas-permeable membrane. In the second a p proach, the electrode recorded the consumption of oxygen by glucose oxidase, which was immobilized at the outer surface of (31) Myland, J. C.; Oldham, K B. J. Electrochem. SOC.1984, 131,1815.
f -3.2
Pm
-Blank
-k
30 minutes
I
120
-3.5
-3.8 -4.2
-4.5 -4.0
.
-5.2
IO 20 30 40 50 60
-5.5
Time (seconds)
12
90
13
15
16
17
19
,
20
Time (seconds)
420
k
-10
o
0
2
4
6
8
1012
[Glucose] mM 80
Figure 3. Current transient and log plot used to determine the diffusion coefficient for oxygen in an FEP membrane. (a) Transient probe response recorded during a step change in oxygen partial pressure at the membrane's outer surface. Measurements were made using a YSI 5357 oxygen sensor. (Using the factor 0.2 n N response unit converts the sensor output to current.) (b) Log plot used to determine the oxygen diffusion coefficient in the FEP membrane (after Hitchman, ref 2). The slope of this plot is equal to -(~?&f)lz,,,. The dashed line shows the least-squares fit to the data. 120
a .
~ o o % o o ~ ~ ~
.....................
100
E ..... t *--.
....*a*
U
a
20 80 2
2 60
5
&
40
m
20
0
Time Figure 4. Experiments run in parallel with two YSI 5357 oxygen sensors using the experimental arrangement shown in Figure 1. (U) Response of probe with an FEP gas-permeable membrane. (0) Response of probe with an FEP-APS gas-permeable membrane. See text for details. (See Figure 3 and ExperimentalSection for current axis conversion factor.)
the FEP-APS gas-permeablemembrme through glutaraldehyde/ albumin cross-linking.6332 Figure 4 shows the response of the oxygen probe in experiments with mouse neuroblastoma (NB2a) cells. In addition to studies with FEP-AFS, parallel control experiments were performed with a separate oxygen probe using base FEP as the gaspermeable membrane. Just before cell growth was initiated, both probes were equilibrated in PBS at 26 "C to establish a baseline reading that could be used for reference. The first 30 min trace in Figure 4 shows the response of both probes during equilibration in PBS. At the end of this 30 min period, the PBS solution was removed with a sterile syringe, and the membranes were covered with 0.25 mL of a suspension of freshly released NB2a cells ~
~~
~~~
(32) Mascini, M.; Guilbault, G. S. Anal. Chem. 1977,49, 795.
Figure 5. Response of glucose oxidase enzyme-basedbiosensor to repeated injections of glucose in a stirred PBS solution at pH 7.1 and 26 "C. Glucose oxidase was immobilized onto an FEP-APS membrane and then secured to the tip of a YSI 5357 oxygen probe. The glucose concentrationsfollowing each injection were (a) 0.1, (b) 0.8, (c) 1.5, and (d-e) 2.5-10.5 mM, in 1.O mM increments. Inset: Calibration curve showing least-squares fit to data for glucose concentrations in the range 0.1-6.5 mM. (See Figure 3 and Experimental Section for current axis conversion factor.)
(-104-105 cells/mL). The oxygen probes were disconnected from the power supply at this point, and the probe assemblies were allowed to incubate for 12-15 h to promote cell attachment. At the end of the incubation period, the probe assemblies were reconnected to the power supply and equilibrated in a sterile hood for a period of about 1 h. The second 30 min trace in Figure 4, which starts at point A, shows the response of the oxygen probes immediately following the incubation and subsequent equilibration period. For both probes, the oxygen levels in the test solution drop substantially compared to the reference trace recorded in PBS, owing to the respiring cell population in the solution above each membrane. To measure the response of the adherent cell population, nonadherent cells were removed by washing the probe assemblies with aliquots of sterile DMEM medium. At this point, the Tygon tubing and the outer plastic sleeve were gently removed from each assembly and replaced with new, freshly sterilized pieces. This latter step was required to eliminate interferences from respiring cells that attached to the assembly walls during incubation. Immediately after being washed, the membranes were covered with 0.25 mL of air-equilibrated DMEM medium at 26 "C, and the response of each probe was recorded for a 30 min period, beginning at the point marked B in Figure 4. The probe with the FEP-APS membrane, which supports the growth of adherent cells, gives an attenuated response, while the probe with the base FEP membrane stabilizes near the baseline response. Subsequent experiments tested the response of the sensor after cellular respiration was inhibited by the addition of an aqueous sodium azide solution. In each cell, 10 pL of a 0.1 M sodium azide solution was added to the solution layer above each membrane. The trace which begins at point C marks the probe response 1.5 h after the addition of azide, and the trace which begins at point D marks the probe response 1.5 h after an additional 5 pL injection of 0.1 M sodium azide solution. In each case, the probe with the FEP-APS membrane supporting a layer of adherent cells gives an attenuated response that shifts toward Analytical Chemistry, Vol. 67,No. 9,May I, 1995
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baseline with the addition of azide. The response of the probe with the base FEP membrane is not affected by the addition of azide. To complete the experiments, each sensor system was washed with 95% ethanol to remove adherent cells and then flushed with PBS. Each chamber was filled with a 0.25 mL aliquot of air-equilibrated PBS and allowed to stand in a sterile hood at ambient temperature for 1 h. The trace marked E in Figure 4 records the response of the sensors immediately after the 1 h stabilization period. The response of both probes is nearly , constant over the 30 min test period and is near the level recorded before cell growth was initiated. The data in Figure 4 demonstrate that the Clark-type oxygen electrode responds to a layer of respiring adherent neuroblastoma cells following their attachment and growth at the outer surface of an FEP-APS gas-permeable membrane. Experiments that tested the utility of using FEP-APS in the design of enzyme electrodes based upon the Clark-type oxygen sensor are summarized in Figures 5 and 6. Figure 5 shows the response of an oxygen probe with the attached glucose oxidasemodified FEP-APS membrane (see Experimental Section) to repeated injections of glucose when the probe was immersed in a stirred PBS solution @H 7.1) at 26 "C. The response of the probe is linear out to about 6.5 mM glucose (Figure 5, inset). Following an injection of glucose, the sensor response reaches a steady state after about 60 s (Figure 6). Experiments are underway to determine the long-term stability of the sensor, and alternative coupling strategies33 are being explored. SUMMARY FEP membranes retained their original oxygen permeability and diffusion characteristics after exposure to a hydrogen/ methanol vapor radio frequency glow discharge plasma and after subsequent treatment with APTES. These modified membranes were used as the gas-permeable membrane of a Clark-typeoxygen probe. The oxygen sensor, fitted with an FEP-APS membrane, responded to changes in the respiration of an adherent cell population that was confined to the outer surface of the membrane through natural attachment mechanisms. The application of FEP(33) Kallury. IC M. R; Lee, W. E.; Thompson, M. Anal. Chem. 1993,65,2459.
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Add Glucose
0&, 1 1 2
2
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104
ACKNOWLEDGMENT We thank S. Wilson for assistance during the initial stages of this work with the support of the UM-REU program. We are grateful to A. Govil and M. D. Morris for the use of their confocal microscope and for providing assistance with the measurements. This work was supported by American Cancer Society Grant JFRA-321 awarded to C.K. E.J.B. acknowledges a grant from the National Science Foundation @MR-9303032). Received for review September 19, 1994. February 24, 1995.@ AC9409290 Abstract published in Advance ACS Abstracts, April 1, 1995.
Accepted
