Anal. Chem. 1995,67, 936-944
Bilayer Lipid Membranes for Flow Injection Monitoring of Acetylcholine, Urea, and Penicillin Dimitrios P. Nikolelis* and Christina 0. Siontorou
Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, Panepistimiopolis-Kouponia, 15771-Athens, Greece
This work describes a technique for the rapid and sensitive determination of acetylcholine, urea, and penicillin in flowing solution streams using stabilized systems of solventless bilayer lipid membranes ( B u s ) . This method of monitoring substrates of hydrolytic enzyme reactions made use of BLMs which were supported on ultra6ltration membranes such as polycarbonate and glass microfiber; these flter membraneswere found to enhance the stability of BLMs for uses in flow injection experiments. The enzymes were immobilized on BLMs by incorporatingthe protein solution into the lipid matrix at the aidelectrolyte interface before the BLM formation, followed by injections of the substrates into flowing streams of a carrier electrolyte solution. Hydronium ions produced by the enzymatic reaction at the BLM surface caused dynamic alterations of the electrostatic fields and phase structure of BLMs, and as a result ion current transients were obtained; the magnitude of these signals was correlated to the substrate concentration,which could be determined at the micromolar level. The response times were ca. 10 s, and acetylcholine, urea, and penicillin could be determined in continuous flowing systems with a maximum rate of 220 samples/h. It is expected that this analytical utility of stabilizedBLMs for flow stream uses will provide new opportunities in this strategy of chemical sensing. Biological recognition of molecules in living cells is controlled by the perception, identification, transduction, and amplification of chemical signals. In nature this molecular recognition is generally accompanied by a conformational change of the membrane-embedded “receptor” (protein). This can lead to an immediate opening of an “ion channel” and a depolarization of the membrane potential or can activate an enzyme system, catalyzing the formation of a “second messenger”, which may initiate a cascade of enzyme modulations with the effect of amplification and distribution of the original signal. The adaptation of natural sensory and molecular recognition systems for biosensor construction is currently being focused in the use of ion channel opening of artificial lipid membranes.’q2 Biosensors based on these artijicial biomimetic systems do not have the complexity of the biological living cells and provide a route of generic transduction of an analytical signal with advantages of high sensitivity and selectivity and fast response times; lipid membranes can also be excellent host matrices for the maintenance of the activity of many biochemically selective species, such as enzymes, antibodies, and (1) Nikolelis, D. P.; Krull, U.J. Electroanalysis 1993,5, 539-545. ( 2 ) Sugao, N.; Sugawara, M.; Minami, H.; Uto, M.; Umezawa, Y. Anal. Chem. 1993,65,363-369.
936 Analytical Chemisfiy, Vol. 67,No. 5, March 7, 7995
receptors. Recent advances in the construction of biosensors based on lipid membranes include devices, e.g., for possible uses in the determination of odorous and taste and eyeinitant substance^.^*^ Also, a large number of bilayer lipid membrane (BLM)-based biosensors for the determination of a wide range of biochemicals have been recently reported in the literature.1,215-9 These studies have shown that these ultrathin films have provided the basis for the construction of devices with rapid response times and high sensitivity and selectivity; compounds such as glutamic acid and glucose can be selectively determined at nanomolar concentration level^.^^^ Of greater importance, perhaps, is the use of BLMs for the rapid (‘2 min) measurement of antigensa and the direct selective and sensitive monitoring of some insecticides, such as monocrotofos and ~arbofuran.~ Considerable progress has been made in the immobilization of active proteins in close proximity to electrochemical devices to improve the response time of an enzyme electrode for uses in flow-through bioreactors.lOJ1 The best response times were achieved by immobilizing very thin layers of enzymes (1-2 pm thickness) on glass electrodes;10the response times of these enzyme electrodes (which were the fastest ever realized) were in the range of 10 s, but sensitivity was compromised, and these fast response times were only observed for substrate concentrations in the millimolar range. Previous studies have investigated the interactions of hydrolytic enzyme-substrates at a BLM surface; the representative enzymatic reactions were between membraneassociated acetylcholinesterase (AchE), urease, and penicillinase with acetylcholine (Ach), urea, and penicillin, respectively, and the results have shown that these BLMs can be used as generic transducers to monitor these reaction~.~J~ Some major limitations, though, have been imposed in these investigations, such as the inherent fragility of BLMs, which makes them unpractical, Le., for flow-through injection applications;in addition, diffusioncontrolledprocesses resulted in timedependent transient signals which appeared in periods of minutes at micromolar (3) Okahata, Y.; En-na, G.4.; Ebato, H. Anal. Chem. 1990,62,1431-1438. (4) Okahata, Y.; Ebato, H. Anal. Chem. 1991,63,203-207. (5) Minami, H.; Sugawara, M.; Odashima, IC: Umezawa, Y.; Uto, M.; Michaelis, E. K; Kuwana, T. Anal. Chem. 1991,63, 2787-2795. (6)Snejdarkova, M.; Rehak, M.; Otto, M. Anal. Chem. 1993,65,665-668. (7) Nikolelis, D. P.; Tzanelis, M. G.; Krull, U. J. Anal. Chim. Acta 1993,281, 569-576. (8) Nikolelis, D. P.; Tzanelis, M. G.; Krull, U. J. Anal. Chim. Acta 1993,282, 527-534. (9) Nikolelis, D. P.; Krull, U. J. Anal. Chim.Acta 1994,288,187-192. (10) Kumaran, S.; Meier, H.; Danna, A. M.; Tran-Minh, C. Anal. Chem. 1991, 63, 1914-1918. (11) Meier, H.;Kumaran, S.; Danna, A. M.; Tran-Minh, C. Anal. Chim. Acta 1991,249, 405-411. (12) Nikolelis, D. P.; Tzanelis, M. G.; Krull, U. J. Biosens. Bioelectron. 1994,9, 179-188. 0003-2700/95/0367-0936$9.00/0 0 1995 American Chemical Society
concentrationsof analytes and extended the time of analysis. The reliance on diffusion also implies that strict attention must be given to stirring if calibration curves are to be reproducible. The aim of the present work was to prepare stabilized BLMs for use as practical biosensors and to exhibit their potentiality for applications in flow injection analysis. This paper exploits the use of filter-supportedBLMs for the rapid and sensitive determination of substrates of hydrolytic enzymes such as Ach, urea, and penicillin in flowing solution streams. Ultrafiltration membranes such as polycarbonate and glass microfiber were used as supports to prepare stabilized BLM assemblies for flow-through injection experiments. Injection of substrates into the carrier electrolyte solution followed the immobilization of the hydrolytic enzymes in these filter supported BLMs; as a result, current transients were obtained and the magnitude of these signals was correlated to the substrate concentration which could be determined at micromolar concentration levels. The response times were ca. 10 s, faster than any previously reported values at these concentration levels of analytes. These transient signals were attributed to the dynamic pH alterations at the BLM surface owing to the enzymatic reaction, which caused artilicial “ion gating” events and conductivity alterations. The present use of BLMs in flowing streams suggests the possibility of adaptation of the method for the continuous analysis of protein-free samples in flow-throughbioreactors or batch fermentors. EXPERIMENTAL SECTION
Materials and Equipment. The lipids used throughout this study were egg phosphatidylcholine (PC, lyophilized; Avanti Biochemicals, Birmingham,AL) and dipalmitoylphosphatidicacid (DPPA; Sigma, St. Louis, MO). Other chemicals supplied by Sigma included HEPES (4(-2-hydroxyethyl)-l-piperazineethanesulfonic acid), gramicidin D, AchE (EC 3.1.1.7, type VI-S from electric eel, lyophilized powder with an activity of 275 units/mg of protein and 225 units/mg of solid), acetylcholinechloride, urea, penicillinase (EC 3.5.2.6, type I from Bacillus cereus, with an activity of 2450 units/mg of protein toward benzylpenicillin as substrate and containing 14.9 mg of protein/mg of solid, balance phosphate and citrate buffer salts), and penicillin G and V (potassium salts). Urease (EC 3.5.1.5, from jack beans, highly purified lyophilized powder with an activity of 269 units/mg) was supplied from Serva Feinbiochemica GmbH & Co. (Heidelberg, Germany). Enzyme activities were estimated according to s u p plier. The filters and (nominal) pore sizes used were GF/F glass microfiber (0.7 pm; Whatman Scientific Ltd., Kent, U.K.) and UniPore polycarbonate (1.0 pm; Bio-Rad Laboratories, Mississauga, Canada). Water was purified by passage through a Milli-Q cartridge filtering system (Millipore, El Paso, TX) and had a minimum resistivity of 18 MB cm. All other chemicals were of analytical reagent grade. The apparatus for the formation of stabilized BLMs consisted of two Plexiglas chambers separated by a Saran Wrap (PVC; Dow Chemical Co., Mindland, MI) partition of a thickness of ca. 10 pm (Figure 1). This plastic sheet was cut to more than twice the size of the contact area of the faces of the chambers with a paper cutter and folded in half; an orifice of 0.32 mm diameter was made through the double layer of the plastic film by punching it with a perforation tool as previously described.13 A microporous glass fiber or polycarbonate filter disk (diameter of about 9 mm) was (13) Nikolelis, D. P.; Krull, U. J. Talanta 1992, 39, 1045-1049.
Reference Electrode to Electrometer Reference Electrode to Power Supply source Sample Injection
II
fWaste III)
’
Partition
Ultrafiltration Membrane Filter -Supported BLMs
Figure I. Simplified setup of the apparatus used for the formation of the filter-supported BLMs for flow-through experiments. The ultrafiltration membrane and the aperture for BLM formation are not drawn to scale (in reality they are smaller than shown).
placed in this aperture between the two plastic layers, with the filter centered on the 0.32 mm orifice. The partition with the filter in place was then clamped tightly between the Plexiglas chambers. One of the Plexiglas chambers was machined to contain an electrochemical cell connected with a plastic tubing for the flow of the carrier electrolyte solution (Figure 2A); a Ag/AgCl reference electrode was immersed in the waste of the carrier electrolyte solution. The second Plexiglas chamber was machined to contain a cell with a cylindrical shape having its longitudinal axis perpendicular to the flow of the carrier electrolyte solution of the opposing cell (Figure 2B). The upper circular hole of this cell was at a distance of 1.5 mm from the front and 2.5 mm from the back of the Plexiglas chamber; the lower elliptical hole faced the circular hole of the opposing cell chamber, and the 0.32 mm circular aperture was placed approximately at the center of the assembly. A Ag/AgCl reference electrode was placed into the cylindrical cell, and an external 25 mV dc voltage was applied across the lipid membrane between the two reference electrodes. A digital electrometer (Model 614, Keithley Instruments, Cleveland, OH) was used as a current-to-voltageconverter. A peristaltic pump (Masterflexwith SRC Model 7020 speed controller and 7014 pump head) was used to carry the electrolyte solution from the reservoir. Injections of substrate samples were made in close proximity to the detector system with a Hamilton repeating dispenser with a disposable tip (Hamilton Co., Reno, NV). The electrochemical cell and electronic equipment were isolated in a grounded Faraday cage. Procedures. The dilute lipid solution used for the formation of the solventless stabilized BLMs contained 0.04 mg mL-l total lipid and was composed of 15, 35, and 60% (w/w) DPPA; these solutions were prepared daily from stock solutions of PC (2.5 mg mL-’) and DPPA (2.5 mg mL-’) in n-hexane-absolute ethanol (80:20). The stock lipid solutions were stored in the dark in a nitrogen atmosphere at -4 “C. The BLMs were supported in a 0.1 M KC1 electrolyte solution buffered with HEPES having a pH value of 8.0 for the Ach/AchE enzymatic system, a pH 6.0 for the urea/urease and a pH 7.0 for the penicillin/penicillinase reaction. Stock solutions of AchE in 10 mM Tris-HC1 buffer (PH 7.4) containing 0.4 mg of solid/mL and urease in 50%glycerol and penicillinase (each containing 0.25 mg of solid/mL) were prepared and stored in the freezer. The stock solutions of substrates (0.1 Analytical Chemistry, Vol. 67, No. 5, March I, 1995
937
A Z
Cltcular Hob ( d i a “
0.5
)
Y
Elliplid Hole
6 Figure 2. Dimensional version of the Plexiglas chambers showing the opposing electrochemical cells separated by the Saran Wrap partition. (A) Chamber for the flow of the carrier solution. (B) Chamber used for lipid-protein codeposition and casting of the lipid bilayer on the Saran Wrap partition.
M) in water were prepared daily just before use, and the injected dilute substrate solutions were buffered and had the same ionic strength as the carrier electrolyte solution. Lipid solution (10 pL) was added dropwise from a microliter syringe to the water surface in the cylindrical cell (Figure 2B) near the partition. A volume of 3 pL of the protein solution was applied to the same air/electrolyte interface subsequent to the deposition of lipid. The level of the electrolyte solution was dropped below the aperture and then raised again within a few seconds. The formation of a membrane was verified by the magnitude of the ion current obtained and by electrochemical characterization using gramicidin D. Over 95%of attempts (with a freshly prepared dilute lipid solution) for BLM formation were successful, and the obtained membranes were stable for periods of more than 8 h. The repetitive injections of 75 pL of substrate solutions followed the ion current stabilization, which occurred within 5-10 min after BLM formation. All experiments were made at 25 f 1 “C. 938 Analytical Chemisfry, Vol. 67, No. 5, March I , 7995
RESULTS AND DISCUSSION Formation of Stabilized BLMs for Flow Injection Uses. Ultrafiltration systems such as polycarbonate have previously been used for the stabilization of planar BLMs prepared with the “brush” technique retaining hydrocarbon solvent (“black lipid film^").'^-'^ To achieve reproducibility, a large number of microBLMs with the same characteristics have to be simultaneously formed. In addition, a variable substantial proportion of n-decane used to carry the lipid to the filter may become trapped in BLMs, causing irreproducibility in thinning and the formation of an environment which is not conducive to the function of ion channels. Accordingly, recent methods of BLM formation are focused toward “solventless”or “solvent-free”BLMs (a very low level of residual solvent may be still retained in the BLM torus, and therefore these membranes are nominally solvent-free).1s13J7J* The use of the filter-supported lipid membranes prepared by the brush technique has not though been explored in continuous flow experiments. Hydrostatic stabilization of solvent-fi-ee BLMs has been achieved by the transfer of two lipid monolayers upon the aperture of a closed however, the construction of the hydrostatically closed chamber and the concurrent use of automated digital control of the transmembrane pressure differentials is not an easy task for practical routine analysis. Recent approaches for the construction of stabilized BLM-based biosensors are focused on the direct deposition of lipids on metal electrode^.^^^^^ The construction of a glucose minisensor has exhibited the potentiality of these self-assembledlipid bilayers for uses in human serum matrix.6 In the present work, the monolayer folding technique13 was combined with the use of microfilter supports for the construction of stabilized BLMs for uses in flow-through experiments. Solventfree BLMs were used throughout this study because the retention of solvent reduces the precision of quantitative experimental results (vide supra), and the physical phenomena associated with effects of membrane phase structure and electrostatics can be related to signal generation of BLM-based transducers with greater confidence; however, the design of the present flowthrough apparatus prohibits the formation of black lipid films (owing to the small size of the cell chamber used for lipid membrane casting), so a comparison of results with respect to precision and limit of detection using both techniques of BLM preparation cannot be made. Accordingly, an amount of 10 pL of the lipid solution (0.04 mg mL-’) was found adequate for the formation of these filter-supported BLMs. The accompanying solvent (n-hexane) was allowed su€ficient time to completely evaporate (ca. 5 min) before the process of monolayer casting, and the formation of BLMs took place with the first attempt without it being necessary to redeposit more solvent at the air/ electrolyte interface. The ultr<ration membranes were found to prevent the extensive leakage of the carrier electrolyte solution through the 0.32 mm aperture of the plastic fih such a leakage would have hindered formation of BLMs. The closed chamber (14) Mountz, J. M.; Tien, H. T. Photochem. Photobiol. 1978,28, 395-400. (15) Thompson, M.; Lennox, R B.; McClelland, R. A. Anal. Chem. 1982,54, 76-81.
(16)Yoshikawa, K; Hayashi, H.; Shimooka, T.; Terada, H.; Ishii, T. Bzochem. Biophys. Res. Commun. 1987,145, 1092-1097. (17) Vodyanoy. V.; Halverson, P.; Murphy R B. j . Colloid Interface Sci. 1982, 88, 247-257. (18) Vodyanoy, V.;Murphy, R. B. Biochim. Eiophys. Acta 1982,687, 189-194. (19) Ti Tien, H.; Salamon, 2. Bioelectrochem. Eioenerg. 1989,22, 211-218. (20) Otto, M.; Snejdarkova, M.; Rehak, M. Anal. Lett. 1992,25, 653-662.
of the flow ~ell,’~J* as combined in the present technique with the small pore size of the filters, resulted in increased stability of BLMs,I5which therefore can be used in flowing streams. Special care was exercised to avoid contamination of the WC partition and filter support^.'^ Polycarbonate filters were selected for BLM supports as they contain insignificant quantities of humectants and wetting agents present in other types of ultrafiltration supports, e.g., cellulose ester filters;I5both types of contaminants can potentially interact with the lipid bilayer membrane. In addition, reagents added to one side of the polycarbonate filters cannot percolate through the filter skeleton and simultaneously interact with the opposing side of the lipid membrane.15 It was also observed that the electrolyte level at the side used for membrane casting and BLM formation can be brought below the level of the elliptical aperture for short periods of time (ca. 6 min) and then raised without inducing membrane collapse. In addition, the filter-supported BLMs have exhibited enhanced mechanical stability at high flow rates of the carrier electrolyte solution (i.e., 20.4 mL min-I). Pretreatment of the filters with octadecyltrichlorosilane or any other hydrophobic material was unnecessary to facilitate the formation of BLMs. The aperture thickness contributes considerably to the stability of BLMs, and conventional methods for formation of solventless lipid bilayers cannot successfully be applied when partitions thicker than 25 pm are used.13 In our studies, the thickness of the filters used can vary from 15 (for polycarbonate filters) to 420 pm (for the glass microfiber filters), This is a direct result of using filters as supports of BLMs with reduced pore size;the ratio of aperture thickness to diameter for the filters used was ca. 35 for polycarbonate filters, which is larger than previously reported values, and an increase of this value results in BLM formation with pronounced stability.15 Typical values for the specific resistance of the stabilized BLMs used in our studies were about lo7 R cm2. The electrical capacitance of membranes was measured to test whether a single lipid membrane occupies the total area of a flkr or whether microBLMs cover the pores of the filter paper. The capacitance of BLMs was measured as previously described13and was found to be 0.7 nF for the polycarbonate filters and 0.85 nF for the glass microfiber filters. The membrane capacitance approaches the typical value for solventless BLMs (ca. 1.0 p F ~ m - 9 only ’ ~ if the lipid membrane would cover the total area of the aperture with a diameter of 0.32 mm in the Saran-Wrap partition. The bimolecular thickness of the filter-supported BLMs was confirmed by conductance alterations that were induced when the channel-forming agent gramicidin D (at concentration levels of 1.0 pM) was added to either the carrier electrolyte solution or the solution at the side where the lipid membrane was formed. It is well known that gramicidin does not induce conductance changes if the lipid membrane is thicker than one bi1a~er.I~ The experimental technique for the formation of filter-sup ported BLMs developed in the present studies offers a new route to the uses of these membranes as practical biosensors since BLM-based systems have not been used for flow-through applications. The present method also provides physical tolerance in BLMs so that an exchange of solutions (carrier electrolyte or injected sample) is feasible during experiments. Selection of the Enzymatic Systems. Preliminary results have shown that for mod$ers, such as hydrolytic enzymes that are immobilized onto a BLM, a selective transient charging signal can be obtained based on the rate and extent of the biochemical
Table 1. Results of Optlmlzed Amounts of Enzymes Immobilized in BLMs without Inducing Transient or Permanent Ion Current Changes
enzyme reaction
AchE/Ach
urease/urea penicillinase/penicillin
optimum acid or base 1ipid:protein enzyme produced ratio activity (units) (nmol/min) 141:l 3861 1101
4.0 10-4 3.0 10-4 4.3 x 10-4
0.40 0.54 0.43
interaction with substrate; the AchE/Ach, urease/urea, and penicillinase/penicillin enzymatic reactions were used in these inve~tigations.~J~ The results have shown that it is possible to construct “switchable”sensors in terms of conductivity states at submicromolar concentrations of substrate without implementation of ion channel proteins from natural sources. As the reactions proceed, the ionizable groups in the membrane are effectively dynamically titrated through an equivalencepoint with consequent rapid reorganization of the double layer and the BLM structure; in addition, enzyme conformational changes during substrate turnover can disrupt the local membrane lipid packing, which may also cause the current leakage. These investigations were offered to be adapted by the use of stabilized filter-supported BLMs for flow-through injection experiments; the ultimate purpose was to develop a technique for the rapid and sensitive repetitive determination of substrates of hydrolytic enzymes such as Ach, urea, and penicillin. Our approach begins with the immobilization of enzymes in BLMs by directly codepositing the active protein onto a carefully defined lipid mixture at an air/electrolyte interface, followed by a film casting technique to give filter-supported BLMs containing protein. Subsequent experiments with these filtersupported BLMs including repetitive injections of substrates into the carrier electrolyte solution would result in an electrochemical ion current signal related to analyte concentration. Immobilization of Enzymes at BLMs. The proteins used in our studies were deposited directly onto the lipid mixtures at the air/electrolyte interface to maximize the loading of these biological species in the BLMs. However, incorporation of a protein in a lipid membrane may induce ion channels even in the absence of a “stimulant”, 8~12 and experiments were done to determine the maximum amount of the hydrolytic enzymes that could be codeposited with the lipid mixture without inducing permeability alterations. Table 1 summarizes the final lipid-toprotein ratio (as used in the Experimental Section), provided that the protein is homogeneously distributed and that there is no loss of protein from the air/electrolyte interface. Smaller lipid-toprotein ratios than those reported in Table 1 resulted in the appearance of transient signals reminiscent of ion gating events21 or permanent conductivity increases that prohibit the further use of these BLMs for analytical purposes: e.g., a codeposition of 3 pL of AchE containing 1.0 mg of solid/mL (lipid-to-protein ratio corresponding to 561) resulted in the appearance of transients of increasing magnitude after 15 min of membrane formation. Instability of ion current with time and permanent permeability changes similar to those previously reported12were also noticed for volumes larger than 3 pL of urease solution: e.g., the ion current value was initially as expected when a volume of 6 pL of (21) Hille, B. Ionic Channek; ofhkcitable Membranes, 2nd ed.; Sinauer: Sunderland, MA, 1992; Chapter 1.
Analytical Chemistry, Vol. 67, No. 5, March 1, 1995
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enzyme (lipid-to-protein ratio corresponding to 1939) was used, but it increased over a period of minutes to 22 PA, and finally the BLM was ruptured in a total time of 20 min after its formation. Some models of the association of enzymes with BLMs have been p r o p o ~ e d . ~The ~ - ~structure ~ of the enzymes is such that there are hydrophobic portions of the molecule which permit incorporation into BLMs, leaving the active site at the aqueous interface.22The design of the present apparatusfor BLM casting to prepare stabilized BLMs in flowing streams was based on the modified procedure for BLM formation described previ0us1y.l~It is likely that the enzymes may be on both sides of the BLM, owing to the casting procedure for membrane formation. The enzyme orientation may affect detector sensitivity or accuracy as was previously reported in methods for BLM preparation by raising synchronously the water levels in both cell compartments, and the active protein was introduced in the form of proteoliposomes at one side of a BLM;2 deviations of the initial ion conductivity between different membrane preparationswere noticed owing to a varying number of proteins incorporated with each BLM which affected the biosensor response.2 Aggregative events associated with increasing amounts of enzyme in a BLM could readily cause disruption of a double layer, and the rapid reorganization of the double layer would be observed as a transient current signal. The aggregation of charged protein molecules and the interactions with the charged lipid component of the BLMs @rotein binding to hydrogen bond accepting sites of DPPA or electrostatic interactions between DPPA and the enzymes25) can induce electrostatic field gradients at a BLM surface26which would result in restructuring of the BLM double layer. The destabilizing effects which are noticed when larger amounts of proteins are used (vide supra) and which cause a membrane leakage leading to a BLM rupture are likely due to concurrent protein-lipid and protein-protein interactions which tend to disorganize the lipid membrane. Presently, little is known about the molecular reorganization during BLM rupture,27 but these electrochemical results do indicate that a critical protein:lipid ratio (Table 1) exists, beyond which BLM destabilization and failure occur. Substrate Concentration Dependence. The AchE/Ach, ureasehrea, and penicillinase/penicillin interactions were examined at pH values of 8.0,6.0, and 7.0, respectively, in the presence of calcium ions as a compromise between optimum enzyme activity and enhancement of signal sensitivity (vide infra). Figure 3 shows recordings of the signals obtained at pH 8.0 for different concentrations of Ach. It can be seen that transient responses appear as singular events (no further transients were observed over periods of 10 min) as a result of the hydrolytic enzymatic reaction. The magnitude of these transient responses is in direct proportion to the Ach concentration in the carrier electrolyte solution 01 = 1.419~+ 2.064,P = 0.9994, Figure 4). The variability of response of the BLMs to repetitive substrate injections and the ability to reproducibly incorporate active protein is also indicated in Figure 3 (relative standard deviation, 5.1%). Similar transient signals were obtained for the penicillinase-catalyzed hydrolysis (22) Wiedmer, T.; Brodbeck. U.; Zahler, P.; Fulpius. B. W. Biochim. Biophys. Acta 1978,506, 161-172. (23) Dutta-Choudhury,T. A; Rosenbeny, T. L. J. Biol. Chem. 1984,259,56535660. (24) The Structure of Biological Membranes: Yeagle, P., Ed.; CRC Press, Inc.: Boca Raton, FL, 1992. (25) Boggs, J. M. Biochem. Cell Biol. 1986,64, 50-57. (26) Lundstrom, I. Febs Lett. 1977,83,7-10. (27) Dimitrov, D.S.; Jain, R. K. Biochim. Biophys. Acta 1984,779,437-468.
940 Analytical Chemistry, Vol. 67,No. 5, March 1, 1995
A
-
B
C
F
D
f
lOpA
1
-zosec
Figure 3. Experimental results obtained for the AchVAch reaction at pH 8.0 (0.1 M KCI, 10 mM HEPES, and 1 mM Ca2+) with BLMs composed of 35% DPPA and supported in glass microfiber filters. 5.00; (C) 10.0; (D)30.0. The Ach concentrations (pM): (A) 2.00; (6) injection of each sample was made at the beginning of each recording. The recordings shown in (B) were selected randomly from a large number of injections made. 501
I
40
Y
01 0
8
16
24
32
40
W h I (W Figure 4. Calibration of the analytical signal from experiments as shown in Figure 3. 30
!5
Figure 5. Calibration of the analytical signal obtained for the urea/ urease system using glass microfiber filters.
of penicillin, whereas the signals for the urea/urease system were opposite in direction. Figures 5 and 6 provide calibration of the analytical signal for urea and penicillin G, respectively. Statistical treatment of results of Figures 5 and 6 gave regression equations: y = 0.231x 3.868, P = 0.990, for urea and y = 42.33~1.377, P = 0.9970, and y = 46.2~ 3.914, P = 0.9971, for penicillin
+
+
16-
45 -
ze E s” I
8E f
30-
12-
/’
a 15-
0.25
0.5
0.75
1
1
[Ponicillin 01 (mM)
Figure 6. Calibration of the analytical signal obtained for the penicillin/penicillinase system. Ultrafiltration membranes used were (A) glass microfiber filters, and (8)polycarbonate filters.
using glass microfiber and polycarbonate filters, respectively. The use of either glass microfiber or polycarbonate filters provided similar results, as also shown in Figure 6, though a difference in their structures exists, as was previously found by using scanning electron micrography?8 A difference in sensitivity was observed when penicillin V was used as substrate, and the peak heights were reduced (to about 54%) as compared to penicillin G. This is expected, owing to the varying susceptibility of the particular penicillin species to penicillinase-catalyzed hydr0lysis.2~In general, a good linear correlation was achieved, as observed from the obtained values of 12, and replicate analyses of substrate samples indicated that the reproducibility is on the order of ca. f5%,which is better than previously reported primarily on the basis of diffusion effe~ts.~J* A number of control experiments were completed to demonstrate that the current signals were due to the enzymatic reactions. Signals were not evident when substrate (buffered identically to the carrier electrolyte solution) was injected in flowing stream in the absence of enzyme or in the presence of denaturated enzymes (e.g., by heat) in the BLM structure. Effect of pH. The selection of the experimental conditions, such as pH values, bu€fercapacity, etc., was studied in our previous work, in which BLMs were used as generic transducers of hydrolytic enzyme reaction~.~J~ However, the pH effect with respect to enhancement of detector response and optimum enzyme activity was presently examined. The pH and the presence of calcium ions could atfect BLM response to pH alterations of the carrier electrolyte solution, and the pH also sets the enzymatic activity. BLM response to pH changes of the electrolyte solution was studied thoroughly in a previous paper, and it was found that the signal magnitude increased when the degree of ionization and amount of DPPA are large (as, for example, at pH 8.0 and for BLMs composed of 35%DPPA) and also in the presence of calcium ions;28e.g., the signal observed with membranes consisting of 35%w/w DPPA to pH alterations from 8.0 to 5.5 (made with injections of a buffered solution into the carrier electrolyte solution, both having the same ionic (28) Nikolelis, D. P.: Siontorou, C. G.; Andreou, V. G.; Krull, U. J. Electroanalysis,
in press. (29) Fuh, M.-R S.; Burgess, L. W.; Christian, G. D. Anal. Chem. 1988,60,433435.
/
OJ
I
5.5
8
7
6.5
7.5
8
PH Figure 7. Effect of pH on signal magnitude for the AchE/Ach system using ELMS composed of 35% (w/w) DPPA in the presence of 1.0 mM calcium ions (Ach concentration used was 10 pM).
strength) had about a 2-fold increase in magnitude as compared with signals obtained with BLMs composed of 15% DPPA and altering the pH from 5.5 to 3.0?8 Figure 7 shows the effect of pH for the AchE/Ach system. It can be seen in this figure that the optimum pH for Ach determination is in the range of 7.5-8.0; this is in agreement with values given in literature as the optimum pH with respect to AchE activity is between 7 and 8.3O The optimum pH for the urease/ urea system has been given in literature between 6.0 and 7.5,31-34 and the number of moles of base produced is 1.8 at pH 6.0 and 1.4 at pH 6.5 if 1 mol of urea is consumed in bulk solution, resulting in an increase of the pH of the acidic solution.33A study of the pH effect on the signal magnitude for this enzymatic system using BLMs composed of 35% (w/w) DPPA and in the presence of 1.0 mM calcium ions has exhibited that the signal decreased to about 60%when the pH was 6.5 instead of 6.0. A slightly smaller pH value was used in our present studies as compared to the previous12for the penicillinase/penicillin reaction, as it was found that there is a 2-fold increase in the signal magnitude at a pH 7.0 instead of 7.5, which is in agreement with the optimum pH for this enzymatic system given in literature.35~36 Effect of Amount of Enzyme on the Sensitivity of the Calibration Graphs. Table 1also provides the number of units of enzyme activity immobilized at either BLM surface (provided that the activity of the enzyme remained unchanged after incorporation of the protein into the BLMs),and the related number of nanomoles of acid or base produced per minute during the hydrolytic enzyme reaction at the BLM surface. All enzyme solutions were first assayed for both enzyme activity and protein content before use. The deposition of smaller amounts of enzymes to provide lipid-to-proteinratios larger than those reported in Table 1 resulted in a decrease of the signal magnitude. For instance, (30) Blake, C.; Gould, B. J. Analyst 1 9 8 4 , 109,533-547. (31) Fishbein, W. N.; Carbone, P. P.J Bid. Chem 1 9 6 5 , 240,2407-2414. (32) Guilbault, G. G. Handbook ofEnzymatic Methods ofAnalysis; Marcel Defier, Inc.: New York, 1976 pp 164-166. (33) Adams, R. E.; Carr, P. W. Anal. Chem. 1 9 7 8 , 5 0 , 944-950. (34) Van Der Schoot, B. H.; Bergveld, P. Anal. Chim. Acta 1990,233,49-57. (35) Citri, N.; Pollock, M. R. In Advances in Entymology and Related Subjects of Biochemisty; Nord, F. F., Ed.: Interscience Publishers: New York, 1966; pp 237-323. (36) Waley, S. G. Eiochem. J. 1975, 149, 547-551.
Analytical Chemistry, Vol. 67,No. 5,March 1, 1995
941
I
2.51
1
1.5-
8
p
1-
0.54
01
1.7
2.4
3.2
4.2
5.4
I
Flow Rate (mUmln)
Figure 8. Effect of flow rate on noise levels when glass microfiber filters were used at ambient temperature.
the use of 3 pL of AchE solution containing 0.2 mg of solid/mL provided a signal of 7.2 PA for 10 pM of Ach; a deposition of half the optimum amount of urease resulted also in a decrease of sensitivity to about 20%as compared to Figure 5. These results exhibit that the use of enzymes of high specific activity (number of units/mg of protein) is necessary to obtain a practical transduction scheme and to optimize the sensitivity of the method. The enzyme preparations used in our present studies for the urease/ urea system had higher specific activity than those previously used.I2 As a result, an improvement of the detection limit for urea was noticed, and the obtained detection limit was about 5-fold smaller than the previous value.12 The AchE preparations used were selected as a compromize between cost and high purity (ca. 82%), because the protein loading for optimum results may vary depending on the enzyme purity; penicillinase was chosen on the basis of using a highly specific activity preparation at low cost though enzyme purity was low, at approximately 15%. Effect of Flow Rate. The filter-supported BLMs described in this work are more suitable for practical biosensor implementation, such as for flow-through applications, than are the conventional solventless BLMs.13 However, noise level increases with an increase of the flow rate used. Typical flow rates that can be used are up to 3.2 mL min-I, which result in noise levels of less than 1 PA (Figure 8). These stabilized BLM systems exhibit stability in flow rates of up to about 20 mL min-', but the resulting noise level and problems associated with the mechanical stability of the ultra&-ationfilter supports make detection unpractical at such high flow rates. On the other hand, the signal magnitude and the delay time of signal appearance decreases as the flow rate increases; the delay time was decreased from about 6 to 3 s when the flow rate increased from 1.7 to 3.2 mL min-', but the signal magnitude was decreased to 60%as it was observed with injections of Ach. Therefore, the flow rate was chosen on the basis of optimization of signal enhancement and delay time of signal appearance. Principle of Signal Generation for the Ach, Urea, and Penicillin Sensors. The ion conductivities of BLMs composed of mixtures of egg PC and DPPA have been previously studied, and the results have shown that the ion permeation through these BLMs is sensitive to pH levels of the electrolyte solution, the composition of these membranes, and the calcium concentration in bulk electrolyte sol~tion.3~,~* Recent work provided pH alter942 Analytical Chemistry, Vol. 67,No. 5,March 1 , 1995
ations in one cell compartment? and as a result, transient current signals were obtained. These signals were the result of an interfacial charging phenomenon, owing to the pH changes which control surface charge and phase structure of these BLMs. The time delay for observation of the transient was directly and reproducibly related to the concentration of the substrate. The quantitative signal of these BLMs-based transducerswas primarily based on diffusion effects, which controlled the time delay of the appearance of the current transients. Transients of similar direction and amplified magnitude were observed during experiments involving the response of BLMs to gradual pH changes; these studies included pH titrations of the acidic lipid constituent of membrane on one side of a BLM.39The mechanism of ion current signal was exploited, and the results have shown that a rearrangement of the electrical double layer is not the only phenomenon responsible for the transient signals that were observed. A structural rearrangement must occur in the head group region of the lipid membranes (and not a phase reorganization of the hydrocarbon interior of BLMs) to influence the electrochemical properties of BLMs, and this hypothesis was supported by fluorescence microscopy studies of monolayers of DPPMPC. A detailed comparison of the profiles and magnitudes of the current transients obtained in the present work with the transients observed in recent investigations7J2indicates important similarities and substantial differences. Of greatest significance is the appearance of well-defined transient currents of the same direction in both types of experiment as the hydronium ion concentration at the BLM surface is altered during the enzymatic reactions. The time of appearance of the transients in the previous studies depended on the substrate concentration: their magnitudes had a maximum amplitude of about 10 PA and they lasted for a period of a few seconds; the transients of the present work have a maximum magnitude of about 50 PA and have a duration of about 10 s. The time delay of signal appearance in our present studies depends on the flow rate of the carrier solution and the distance from the injection point to the detection zone. This time delay can be theoretically calculated and is in agreement with the movement of an indicator (methyl orange) from the injection point to the detector. Recent differential scanning calorimetric studies with egg PC/ DPPA vesicles have exhibited that the phase transition temperature for the gel to liquid crystalline phase of such vesicles is pH dependentF8 The results have shown that at pH 8.0 and at 25 "C, there is a coexistence of these two phases induced by the presence of calcium ions.40s41Such a phase separation results in an increase of defects in the phase structure of a lipid membrane and therefore of ion permeation through membrane~~2Each surface of a BLM is in equilibrium with its adjacent bulk solution, and any charging of either of the membrane surfaces will result in a reorganization of the BLM double layer. The appearance of structural defects at the same time (Le., artificial ion channel (37) Nikolelis, D. P.; Brennan, J. D.; Brown R S.; Krull, U . J . Anal. Chim. Acta 1992,257, 49-57. (38) Nikolelis, D. P.; Krull, U. J. Anal. Chim. Acta 1992,257,239-245. (39) Krull, U. J.; Seethaler, S. L.; Brennan, J. D.; Nikolelis, D. P. Thin SolidFilms 1994,244,917-922. (40) Galla, H.-J.; Sackmann, E. Biochim. Biophys. Acta 1975,401,509-529. (41) Van Dijck, P. W. M.; De Kruijff, B.; Verkleij, A J.; Van Deenen, L. L. M.: De Gier, J. Biochim. Biophys. Acta 1978,512,84-96. (42) Nagle, J. F.; Scott, H. L. Biochim. Biophys. Acta 1978,513,236-243.
Tablo 2. Summary of Rosponre for Dlfferent Comporltlons of BLMs and Ll~ctrolytoSolution for 1x Ach Concentratlon
DPPA in BLMs (%w/w)
PH
15 15 35 35 60
5.5 8.0 8.0 8.0 8.0
[Ca2+1
Signal
(mM)
magnitude (PA)
none none none
4.2 7.4 3.6
1.0 0.5
15.8 21.0
formation) will lead to ion diffusion through the defect sites.43The fraction of ions that permeate through the BLMs depends on the speed of channel opening and the diffusion of ions from the surface to the bulk solution.43A slow channel formation implies that the majority of ions will be dissipated into the bulk solution; on the other hand, a fast channel opening will result in the passage of an appreciable portion of the ions through the lipid membrane.43 The former phenomena were observed in previous diffusioncontrolled alterations of the pH at one side of a BLM (hydrolytic enzyme reactions) with concurrent observations of charging current transient~,~J~ while the latter phenomena were observed in our present flow experiments, in which the number of defects dynamicallyvaries as the pH of the carrier electrolyte solution is altered. These results indicate how amplification of analytical signal of BLM-based transducers may be achieved. Intrinsic Signal AmplXcation. The response characteristics of the micro-BLMs located in the pores of the filter media were found to differ in magnitude (still observed transients of current) from those of the conventional solventless planar BLMS.~J~ The filter-supportedBLMs used in our flow injection experiments can also serve as generic transducers of pH alterations at the BLM surface caused by a hydrolytic enzyme reaction, but the magnitude of the signals obtained was correlated to the substrate concentration. For the filter-supported BLMs, these transient signals are on the order of tens of picoamperes (factors of 5-10-fold larger than for planar BLMs) . The signal profile provides this interesting opportunity for development of a sensor which can act as a modulator or switching device. The signal magnitude depended on the extent of ionization of DPPA. This acid behaves as a diprotic acid in lipid membranes having interfacial pKa values of the acidic phosphate groups of about 4 and 8 (in the absence of calcium ions) or 4 and 7 (in the presence of calcium ions) DPPA dissociation constants also depend on the surface charge density and lipid composition of membranes and ionic strength of the electrolyte s o l ~ t i o n the ; ~ ~local , ~ ~hydronium activity at the surface of a BLM may be different than that of bulk solution and depends on the surface potential of membranes and ultimately on the surface charge density.1~37~38~46,47 The signal magnitude is increased when the degree of ionization of DPPA is large, as for example at pH 8.0 (Table 2). The signal is also increased with larger quantities of DPPA in BLMs and in the presence of calcium ions, as shown by the results in Table 2 for membranes composed of 15, 35, and 60%DPPA .37338,44,45
(43) Blank, M. Biochim. Biophys. Acta 1987,906, 277-294. (44) Papahadjopoulos, D. Biochim. Biophys. Acta 1968,163, 240-254. (45) Boggs, J. M. Biochim. Biophys. Acta 1987,906, 353-404. (46) Clementz, T.; Christiansson, A; Weislander, A In Adoances in Membrane Fluidify;Aloia, R C., Curtain, C. C., Gordon, L. M., Eds.; Alan R. Liss,Inc.: New York, 1988 Vol. 3, pp 41-74. (47) Davies, J. T.; Rideal, E. K Interfacial Phenomena, 2nd ed.; Academic Press: New York, 1963; pp 94-95.
The applications reported here were performed at ambient temperature (25 O C ) since the sensitivity is sufficient without introducing the complicating factor of temperature control of the flow-through apparatus system, Le., by thermostating the room where experiments are performed, which is d ~ c u ltot achieve. It would be expected that for reasons of increasing the rate of the enzymatic reactions, a temperature of 37 "C should be employed in the assay; however, previous DSC studies have shown that the phase structure of lipid membranes is fluid (liquid crystalline) at this temperature, which imposes a limiting factor in signal generation of BLM-based devices.1*28 Comparison with Other Ach, Urea, and Penicillin Sensors. Different sensors for the determination of substrates of hydrolytic enzymatic reactions, such as Ach, urea, and penicillin, have been described in the literature, including optical and electrochemical detection scheme^.^^^^^@ The best achieved response times are on the order of about 10 s, attributed to the extremely thin films of the immobilized enzyme layer.IOHowever, sensitivity was compromised in these systems, and substrates were determined at millimolar concentration levels. The present method offers response times on the order of 10 s without losing sensitivity; the obtained response times for these low concentrations of analytes, i.e., at micromolar concentrations,are the fastest achieved up to now as compared to other similar detectors; however, this could possibly not be adequate and fast enough for uses of the present BLM-based detector, i.e., in high-performance liquid chromatographic systems. The detection limits in the present studies are on the order of 1, 10, and 100 pM for Ach, urea, and penicillin, respectively, which are similar to those obtained by fluorescence method^.^^^^^ It is interesting also to notice the large detection range of Ach without saturation and that the analytical signal is linearly related to substrate in the concentration range between 2 and 30 pM Ach (Figure 4); it was found that the signal remains practically constant for Ach concentrations higher than 60 pM. AchE is known to be one of the most efficient enzymes and has a turnover number within 3-6 x lo4 s-1.49,50 Very good reversibility is also observed with repetitive determinations of substrates in our system with no sample carryover or membrane memory effects ( F i r e 3). The return to the base line after each measurement was almost instantaneous, and this permits repetitive substrate determinations with a rate of 220 samples/h; this measurement rate is based on theoretical calculations, as the number of injections performed was practically limited by the syringe capacity used in the Hamilton repeating dispenser (Le., 2.5 mL). However, repetitive cycles of injections of substrates were performed to exploit the retention of the analytical signal, and no reduction of the signal magnitude was observed during each cycle. Therefore, it is anticipated that the analytical utility of stabilized BLMs for flow stream uses will provide new opportunities for biosensing in practical applications, which demand rapid response at a micromolar concentration level. This provides a scope for application of this technique in flow-through systems for the analysis of fermentation broths, pharmaceutical preparations, or monitoring of industrial processes. (48) Wolfbeis, 0. S. Anal. Chim. Acta 1991,250, 181-201. (49) Taylor, P. In The Pharmacological Basis of Therapeutics, 6th ed.; Goodman Gilman, A, Goodman, L. S., Gilman, A, Eds.; MacMillan Publishing Co., Inc.: New York, 1980; Chapter 6. (50) Stryer, L. Biochemisty, 3rd ed.; W. H. Freeman and Co.: New York, 1988 Chapter 8.
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In conclusion, the results from applications of the present thin lipid film technology,which is a continuation of our previous work, highlight the concept that microfabricated stabilized BLM-based biosensors for flow injection analysis provide fast response times (on the order of a few seconds), high sensitivity, micromolar detection limits, reversibility, and capability of analyzing small volumes and can now be reliably fabricated with simplicity and low cost. It is obvious that an improvement of the characteristics of lipid membrane transducers has been achieved in terms of ruggedness,time of analysis, and precision as compared to related techniques developed previously by us7J2or other groups.2~~ Our technique has significant advantages over the existing methods of analysis, such as liquid chromatographic (LC) procedures (Le., (51) Analytical Currents. Anal. Chem. 1994,66, 569-570A (52) Analytical Currents. Anal. Chem. 1994, 66, 81-8%
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analysis times, size, and cost of LC instrumentation limit the use of this technology for screening applications in the field)51and chromogenic immunoassays (which are highly sensitive and selective, but they take from many minutes to hours to complete and usually require multiple steps, both of which hamper their adaptation to biosensor format).52 Work is in progress to extend the versatility of choice of a wide range of chemically selective reagents and is focusing on the use of these stabilized filtersupported BLMs as electrochemical detectors in flow immunoassays with concurrent regeneration of antibody binding activity. Received for review August 2, 1994. Accepted December 2, 1994.@ AC940769S @Abstractpublished in Adoance ACS Abstracts, January 15, 1995.
