Anal. Chem. 1998, 70, 2366-2371
Flow Injection Monitoring of Aflatoxin M1 in Milk and Milk Preparations Using Filter-Supported Bilayer Lipid Membranes Vangelis G. Andreou and Dimitrios P. Nikolelis*
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 electrochemical flow injection monitoring of aflatoxin M1 (AFM1) using stabilized systems of filter-supported bilayer lipid membranes (BLMs). Injections of AFM1 were made into flowing streams of a carrier electrolyte solution, and a transient current signal with a duration of seconds reproducibly appeared less than 10 s after exposure of the lipid membranes to the toxin. The magnitude of this signal was linearly related to the concentration of AFM1, with detection limits at the subnanomolar level. The mechanism of signal generation was investigated by differential scanning calorimetric experiments. The technique was applied for the rapid flow injection determination of AFM1 in milk and milk preparations. The effect of potent interferences such as proteins and lipids was investigated, and the results show that interferences from these milk constituents can be eliminated by modulation of the flow rate of the carrier solution so as not to allow adsorption of these compounds in BLMs. AFM1 could be determined in continuous flowing systems with a rate of at least 4 samples min-1. Repetitive cycles of injection of AFM1 showed no signal degradation during each cycle. Aflatoxins, produced by certain fungi, occur naturally in a wide variety of foods. The most abundant of the group, aflatoxin B1, is a recognized carcinogen. When aflatoxin B1 is ingested by cows, it is secreted as its hydroxylated metabolite, aflatoxin M1 (AFM1) (Figure 1, inset). Due to the potential carcinogenicity of AFM1, detection and determination in milk is of increasing interest.1 An action level of 0.5 ppb (i.e., 1.5 nM) for AFM1 in milk has been established by the U.S. Food and Drug Administration; however, this level may be legislated lower in some European countries (i.e., 0.15 nM).2 To date, most of the procedures for AFM1 determination are based on liquid chromatography, thinlayer chromatography, immunoassays, and fluorescence emission and usually require cleanup steps using immunoaffinity chromatography.1,3-5 Investigations of interactions of toxins with (1) De Boevere, C.; Van Peteghem, C. Anal. Chim. Acta 1993, 275, 341-345. (2) Qian, G.-S.; Yasei, P.; Yang, G. C. Anal. Chem. 1984, 56, 2079-2080. (3) Ioannou-Kakouri, E.; Christodoulidou, M.; Christou, E.; Constandinidou, E. Food Agric. Immun. 1995, 7, 131-137. (4) Hansen, T. J. J. Food Prot. 1990, 53, 75-77.
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bilayer lipid membranes (BLMs) that have been reported in the literature to date are limited only to peptidic toxins,6 which form ionic channels by aggregation and induce constant or transient ion current increases.7,8 Studies that have investigated the interactions of AFM1 with BLMs were reported recently in the literature by our group.9 BLM-based biosensors and related thin-film technology have been described as an attractive route for the construction of devices useful for monitoring or rapidly screening (in a single format) a large number of compounds of biomedical, pharmaceutical, environmental, and agricultural interest.10-13 Recent research targeted on the preparation of a practical biosensor (in terms of mechanical stability, simplicity of use, etc.).12,14 However, no analytical applications of BLM-based biosensors in real samples have been reported in the literature to date. The aim of the present work was to use stabilized filtersupported BLMs as practical electrochemical biosensors to continuously monitor a species such as AFM1 in real samples. Injection of AFM1-spiked milk into the carrier electrolyte solution provided ionic current transients with a magnitude linearly related to the toxin concentration (nanomolar level). The present adaptation of the selective BLM/toxin interactions using filter-supported BLMs provides a simple and inexpensive technique for continuous flow monitoring (i.e., repetitive cycles of injection of the toxin could be performed, with no signal degradation during each cycle). The maximum number of injections (about 30) performed was limited only by the capacity of the injector used. EXPERIMENTAL SECTION Materials and Apparatus. The lipids used throughout this study were lyophilized egg phosphatidylcholine (egg PC; Avanti (5) Diaz, S.; Moreno, M. A.; Dominguez, L.; Suarez, G.; Blanco, J. L. J. Dairy Sci. 1993, 76, 1845-1849. (6) Jain, M. K.; Zakim, D. Biochim. Biophys. Acta 1987, 906, 33-68. (7) Mellor, I. R.; Thomas, D. H.; Sansom, M. S. P. Biochim. Biophys. Acta 1988, 942, 280-294. (8) Krasilnikov, O. V.; Muratkhodjaev, J. N.; Voronov, S. E.; Yezepchuk, Y. V. Biochim. Biophys. Acta 1991, 1067, 166-170. (9) Andreou, V. G.; Nikolelis, D. P.; Tarus, B. Anal. Chim. Acta 1997, 350, 121-127. (10) Nikolelis, D. P.; Krull, U. J. Electroanalysis 1993, 5, 539-545. (11) Nikolelis, D. P.; Krull, U. J. Anal. Chim. Acta 1994, 288, 187-192. (12) Nikolelis, D. P.; Siontorou, C. G. Anal. Chem. 1995, 67, 936-944. (13) Nikolelis, D. P.; Siontorou, C. G. J. Autom. Chem. 1997, 19 (1), 1-8. (14) Nikolelis, D. P.; Siontorou, C. G.; Krull, U. J.; Katrivanos, P. L. Anal. Chem. 1996, 68, 1735-1741. S0003-2700(97)01209-2 CCC: $15.00
© 1998 American Chemical Society Published on Web 04/28/1998
Figure 1. Experimental setup used for the flow injection monitoring of aflatoxin M1 in milk. Inset: the chemical structure of AFM1.
Polar Lipids, Birmingham, AL) and dipalmitoylphosphatidic acid (DPPA; Sigma Chemical Co., St. Louis, MO). HEPES (4-(2hydroxyethyl)-1-piperazineethanesulfonic acid) was used for the preparation of buffer and was supplied by Sigma. Aflatoxin M1 was purchased from Serva (Serva Feinbiochemica GmbH & Co. KG, Hidelberg, Germany). The filters and (nominal) pore size used were GF/F glass microfiber, 0.7 µm (Whatman Scientific Ltd., Kent, U.K.). Water was purified by passage through a Milli-Q cartridge filtering system (Millipore, El Paso, TX) and had a minimum resistivity of 18 MΩ‚cm. All other chemicals were of analytical reagent grade. The apparatus for the formation of stabilized BLMs has been described elsewhere (Figure 1).12,15 The apparatus consisted of two Plexiglas chambers separated by a Saran Wrap (PVDC; DowBrands L.P., Indianapolis, IN) partition of ∼10-µm thickness. This plastic sheet was cut to more than twice the size of the contact area of the faces of the chambers and folded in half; a hole of 0.32-mm diameter was made through the double layer of the plastic film by punching with a perforation tool.16 A microporous glass fiber disk (diameter ∼0.9 cm) was placed between the two plastic layers and centered on the 0.32-mm orifice. The partition with the filter membrane in place was then clamped between the Plexiglas chambers. One of the chambers was machined to contain an electrochemical cell with a circular shape (diameter 1.0 cm and depth 0.5 cm), connected with plastic tubing for the flow of the carrier solution; a Ag/AgCl reference electrode was immersed in the waste of the carrier electrolyte solution. The second chamber was machined to contain a cylindrical cell, having (15) Nikolelis, D. P.; Siontorou, C. G.; Andreou, V. G.; Krull, U. J. Electroanalysis 1995, 7 (6), 531-536. (16) Nikolelis, D. P.; Krull, U. J. Talanta 1992, 39 (8), 1045-1049.
its longitudinal axis perpendicular to the flow of the carrier solution. The upper hole of this cell was circular (surface area ∼0.2 cm2), and the lower was elliptical (with diameters 0.5 and 1.4 cm parallel and vertical to the flow of the carrier electrolyte solution, respectively), facing the opposing cell. The microporous glass fiber filter was positioned approximately at the center of the cells. A Ag/AgCl reference electrode was placed into the cylindrical cell, and an external 25-mV dc voltage was applied across the filter membrane between the two reference electrodes. A digital electrometer (model 614, Keithley Instruments, Cleveland, OH) was used as a current-to-voltage converter. A peristaltic pump (Gilson Minipuls 3) was used for the flow of the carrier electrolyte. Injections of the AFM1 samples were made 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. A Perkin-Elmer differential scanning calorimeter (model DSC4) was used for the DSC experiments; the thermograms were processed by means of the thermal analysis data station (TADS) of the DSC-4. Procedures. A stock solution of 2.5 mg mL-1 phosphatidylcholine in a mixture of n-hexane and absolute ethanol (80:20 v/v) was used for the preparation of lipid solutions of 0.04 mg mL-1 lipid in the same solvent that was used for BLM casting in the electrochemical experiments. These dilute lipid solutions were prepared daily, just before the commencement of experiments. The stock lipid solution was stored in the dark in a nitrogen atmosphere at -4 °C. The BLMs were supported in a 0.1 M KCl electrolyte solution, buffered with 10 mM HEPES at pH 7.4. A stock solution of 6.09 µM AFM1 was prepared by using the above electrolyte solution to dissolve solid AFM1. This stock solution Analytical Chemistry, Vol. 70, No. 11, June 1, 1998
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was used for the preparation of the injected AFM1 standard solutions, contamination of milk samples, and DSC experiments. The ionic strength and pH of the injected standards and the carrier electrolyte solution were adjusted to match the ionic strength and pH values of the milk samples. The process of formation of stabilized BLM for flow injection experiments was described recently.12,15 Lipid solution (10 µL) was added dropwise from a microliter syringe to the water surface in the cylindrical cell near the partition. The level of the electrolyte solution was then dropped below the aperture and then raised again within a few seconds. The formation of “solventless” BLMs was verified by the magnitude of the transmembrane ion current obtained and by electrochemical characterization using gramicidin D.15 Repetitive injections of 75 µL of AFM1 standard solutions and AFM1-spiked milk samples were made following the ion current stabilization, which occurred about 5 min after BLM formation. All experiments were carried out at 21 ( 1 °C. Lipid vesicles composed of mixtures of DPPA and PC (2.5 mg mL-1 of total lipid) and containing 10%, 15%, and 35% (w/w) of DPPA were used for the DSC experiments. These vesicles were prepared from stock PC (2.5 mg ml-1) and DPPA (2.5 mg ml-1) solutions in a solvent system of n-hexane and absolute ethanol (80:20 v/v). The organic solvent of the lipid solution was evaporated under a stream of nitrogen gas. The lipid was resuspended by sonication with a 0.1 M KCl electrolyte buffered solution, at a temperature above the estimated phase transition temperature, Tm, of the vesicles (i.e., 60 °C in our experiments). The suspensions had a concentration of 3.0 mg mL-1 total lipid and were left refrigerated overnight. An amount of 20 µL of lipid suspension was withdrawn using a calibrated microsyringe and mixed into an aluminum pan with 10 µL of identically buffered AFM1 solution (0.914 or 3.66 µM) or electrolyte solution. The pan was hermetically sealed. Vesicles were scanned between 10 and 60 °C with a scanning rate of 2 and/or 4 °C min-1 using a Perkin-Elmer DSC-4 differential scanning calorimeter (buffer solution was used as the control). Scanning was initiated 5 min after mixing of the lipid suspension with AFM1 solution to ascertain adsorption of AFM1 in BLMs. RESULTS AND DISCUSSION Adaptation of AFM1-BLM Interactions Using FilterSupported BLMs for the Determination of AFM1. The potential of BLMs as devices for the construction of one-shot biosensors for direct monitoring of AFM1 has recently been suggested.9 These studies have made use of “freely suspended” BLMs prepared in an aperture of a thin plastic film separating two identical Plexiglas chambers. The results have shown that a transient current signal appeared as a single event at a relatively constant time after exposure of the membrane to AFM1 (6.6 ( 1.34 s, N ) 5). The magnitude of the transient of current increased with an increase in the bulk AFM1 concentration. This magnitude could be used to quantify the concentration of the toxin as the peak heights of the transients were related to the concentration of AFM1 in bulk solution. The application of the above interactions, when using these systems of freely suspended BLMs, to the direct determination of AFM1 in milk is not possible, since the proteins that are present in milk (i.e., casein, lactalbumin, and lactoglobulin) were found to interfere with the BLM irreversibly and nonselectively when 2368 Analytical Chemistry, Vol. 70, No. 11, June 1, 1998
Figure 2. Experimental results obtained for AFM1 using as carrier electrolyte 0.1 M KCl with 10 mM HEPES at a pH of 7.4 with BLMs composed phosphatidylcholine and supported in glass microfiber filters. The flow rate value used was 4.0 mL min-1. AFM1 concentrations of the injected samples (75 µL) in the carrier electrolyte (nM): (A) 0.609, (B) 1.22, (C) 3.05, (D) 6.09, (E) 9.13, (F) 15.2, and (G) 21.2. The injection of each sample was made at the beginning of each recording. The recordings shown were chosen randomly from a number of injections made.
present in the bulk electrolyte solution, even at very low concentrations. However, the previous electrochemical studies of interactions of AFM1 with BLMs were considered to be adapted in the flow injection monitoring of AFM1. Figure 2 shows recordings of the signals obtained with injections of AFM1 in continuous flowing streams of a carrier electrolyte solution (0.1 M KCl, 10 mM HEPES, pH adjusted to 7.4). Experiments were done using a flow rate of 4.0 mL min-1 (vide infra). It can be seen in this figure that a transient current signal as a single event is obtained by the interactions of AFM1 with the filter-supported BLMs; a constant time delay for the appearance of the transient currents of 12.3 ( 0.93 s (N ) 5) is observed in Figure 2. This time is in agreement with the time delay of the transient signals obtained with the use of freely suspended BLMs of ∼7 s (vide supra), given that a period of ∼5 s is expected for the centroid of the dispersed zone of the injected solution to reach the BLM (i.e., observed with injections of a solution indicator such as phenolphthalein using a flow rate of 4.0 mL min-1). The magnitude of these transient responses is directly proportional to the concentration of the injected AFM1 solution in the carrier electrolyte solution (Figure 3):
∆I (pA) ) 3.82[AFM1] (nM) - 0.732, r2 ) 0.998
A detection limit of 0.585 nM can be achieved as determined by a noise level of 0.5 pA and a S/N ratio equal to 3. The variability of response of the BLMs to repetitive sample injections, as expressed by the relative standard deviation, is on the order of (4-7%. Effect of Flow Rate. Figure 4 shows the effect of the flow rate of the carrier electrolyte solution on the magnitude of the
Figure 3. Calibration graph obtained from the results of AFM1 determination shown in Figure 2.
Figure 4. Effect of flow rate on signal magnitude. AFM1 concentration used was 3.05 nM. The dashed line below flow rate 1.2 mL min-1 indicates the region were the determination is not possible due to milk protein interference.
transient signal. The results of Figure 4 show that the adsorption of AFM1 in BLMs is fast enough to provide a signal of constant magnitude when using flow rates up to ∼4 mL min-1, and the signal magnitude decreases for flow rates larger than 4 mL min-1. Flow rates larger than ∼5 mL min-1 cannot be analytically useful, as they can result in increased noise levels (i.e., more than 2 pA).12 To adapt the above electrochemical interactions of AFM1 with BLMs, using the stabilized systems of filter-supported BLMs composed of PC, to the direct determination of this toxin in fresh milk, matrix effects due to milk constituents have to be eliminated. Control experiments using a buffered casein solution of concentration similar to that found in undiluted milk were performed, and the results showed that no interference is observed when using flow rates larger than 1.2 mL min-1. For larger flow rates, the protein interference is practically eliminated due to the insufficient time of interaction of protein with the lipid membrane. The same results were observed when using skimmed milk instead of buffered casein solutions. For flow rates smaller than 1.2 mL min-1, interference from casein in the form of random ion current transients of no discrete pulse height, due to casein adsorption in BLMs, occurred. These interferences were also observed in previous investigations.17 These transients occur with delay times shorter than the times of appearance of the AFM1 signals, which results an interference for these flow rates. (17) Nikolelis, D. P.; Siontorou, C. G.; Andreou, V. G.; Viras, K. G.; Krull, U. J. Electroanalysis 1995, 7 (11), 1082-1089.
Lipids that are present in milk may also interfere with the lipid molecules that constitute the BLM. However, when non-AFM1contaminated full cream milk (3.5% w/v in fat) was injected in the flow of the carrier electrolyte solution (at flow rates above 1.2 mL min-1), no alterations of the background membrane conductivity occurred. The milk lipids are probably not able to fuse with the BLM due to the protective effect exerted by milk proteins, since those two milk constituents are known to form lipoproteins,18 with the proteins protruding into the aqueous phase. Therefore, a flow rate between 1.2 and 4 mL min-1 should be used to obtain a signal with minimal matrix effects, maximized signal magnitude, and decreased noise levels. Investigation of Aflatoxin Interactions with BLMs and Mechanism of Signal Generation. As shown in a recent study,9 the magnitude and sensitivity of the signal for AFM1 determination depend on the quantity and degree of ionization of DPPA in the BLM structure and the presence of calcium ions in bulk solution. It was previously suggested that addition of DPPA in BLMs composed of PC and the presence of calcium ions in the bulk electrolyte solution can substantially alter the electrostatic fields and/or phase structure of BLMs.19,20 DPPA was presently selected among other negatively charged lipids (i.e., dipalmitoylphosphatidyl serine (DPPS) or dipalmitoylphosphatidyl glycerol (DPPG) because this acidic lipid has extensively been studied experimentally and it is well-known that this lipid may provide phase structure changes that may be used in biosensors based on BLMs.19-21 The apparent pKa values of DPPA when in a mixture with egg PC are about 4.0 and 8-9;19 therefore, a substantial electrical double layer would form at the surface of a BLM if it contained negatively charged phospholipid headgroups, and the surface charge density and surface potential would increase with an increase of DPPA concentration in membranes.19 At pH values around 7, for DPPA concentrations less than 25% (w/w) and in the absence of calcium ions, a phase separation of PC and DPPA occurred in the membrane structure, and the ion permeation took place through defect sites that were associated with a heterogeneous mixed-phase domain system.19 For higher than 25% (w/w) acid concentrations, the predominant factor that determined the magnitude of the ion current was the homogeneous distribution of DPPA and egg PC. At pH values of about 3.5 and in the absence of calcium ions, less than 25% of the acid is ionized, which results in a substantial decrease of the surface charge and in homogeneous distribution of those two lipids in membranes.19 At concentrations of DPPA >25% at neutral pH values and in the presence of calcium ions in bulk electrolyte solution, the interaction of BLMs with the divalent cations and the subsequent phosphate-calcium complex formation resulted in a phase separation of the two lipids in membranes.20 In our recent investigations on BLM/AFM1 interactions,9 it was found that at neutral pH values in the absence of calcium ions and for BLMs containing 0-20% (w/w) DPPA, the signal magnitude decreased with an increase of DPPA concentration in BLMs (i.e., increased surface charge density in membranes and hetero(18) Encyclopedia of Analytical Chemistry; Alan Townshend, Ed.; Academic Press Limited: London, 1995; Vol. 4, p 2520. (19) Nikolelis, D. P.; Brennan, J. D.; Brown, R. S.; Krull, U. J. Anal. Chim. Acta 1995, 257, 49-57. (20) Nikolelis, D. P.; Krull, U. J. Anal. Chim. Acta 1995, 257, 239-245. (21) Boggs, J. M. Biochim. Biophys. Acta 1987, 906, 353-404.
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geneous structure19). No significant transient responses were observed when using BLMs composed of 35% or 60% (w/w) DPPA and in the presence of calcium ions (i.e., heterogeneous structures20). BLMs containing between 30 and 60% w/w DPPA and in the absence of calcium ions have increased surface charge density, but their phase structure is homogeneous,19 and the signal magnitude obtained when using such BLMs was found to be increased as compared to those obtained when using heterogeneous structures. The results also indicated that the signal magnitude is increased as the pH value is decreased, i.e., decreased surface potential and homogeneous structure in membranes.19 As a result of all the above, it was concluded that the signal magnitude seemed to depend on both surface charge density and surface potential and phase structure of BLMs (i.e., signal magnitude decreased with an increase of surface charge and potential and for homogeneous BLMs). Also, an increase of DPPA concentration in BLMs between 0 and 20% (w/w) (i.e., increase of surface charge and surface charge density) provided the largest trend of increase of signal magnitude upon AFM1 concentration increases, as compared to the other experimental conditions. In the presence of calcium ions (i.e., complete neutralization of surface charge), there were no measurable signals. The formation of homogeneous and uniformly charged membranes resulted in an apparent decrease of the trend of increase of signal magnitude with toxin concentration, as compared to a heterogeneous distribution of the two lipids in the membrane structure. Such a phase alteration of the membrane structure from heterogeneous to homogeneous results in reduced surface charge in areas that were enriched in DPPA for an heterogeneous distribution of the two lipids in BLMs.19 This also leads to alterations of the molecular packing, i.e., decrease of average area per molecule, in this mixed-phase lipid system.19 However, presently, BLMs composed of egg PC and containing no DPPA were selected for our experiments as a compromize between signal magnitude and trend of increase of signal magnitude with toxin concentration. To further investigate the previous electrochemical results,9 DSC experiments that simulate the interactions of these freely suspended BLMs with AFM1 were performed. Vesicles composed of pure egg PC were not examined, as this lipid is known to have a phase transition temperature, Tm, of -10 °C, and the fluidity of BLMs composed of egg PC is not significantly altered above this temperature.22 The Tm of vesicles composed of 35% DPPA (pH 8.0 and in the presence of 1.0 mM Ca2+) was found to be 23.6 °C and did not practically change in the presence of AFM1, thus indicating no adsorption of the latter onto the surface of BLMs. When vesicles containing 10% DPPA at pH 7.0 or 15% DPPA at pH 3.5 (in the absence of calcium ions) were investigated a shift in Tm occurred. In the former case, the melting temperature, 53.9 °C in the absence of AFM1, decreased to 47.9 °C in the presence of 0.305 µM AFM1 and further to 45.4 °C when using 1.22 µM AFM1. In the latter, the transition temperature, Tm, decreased from 57.9 to 54.4 °C in the presence of 0.305 µM AFM1 and further to 42.6 °C for 1.22 µM AFM1. These changes may be attributed to the fact that AFM1 destabilizes the phase structure of BLMs by forming (22) Fleischer, S.; Packer, L. In Methods in Enzymology (Biomembranes, Part B); Colowick, S. P., Kaplan, N. O., Eds; Academic Press: New York, 1974; Vol 32, pp 485-554.
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hydrogen bonds with the lipid molecules and, therefore, disrupting their bonding network and the lipid-lipid continuation.21 All the transition temperatures are higher than the temperature used in our electrochemical experiments (i.e., 21 ( 1 °C). Therefore, the phase structure of BLMs during these electrochemical experiments does not change upon injection of AFM1, and the electrochemical signals observed using these BLMs are the smallest. In the case of vesicles composed of 35% DPPA (pH 7.0 and in the absence of Ca2+), a transition temperature, Tm, of 22.1 °C was observed in the absence of AFM1. This temperature was decreased to 18.7 °C in the presence of 0.305 µM AFM1 and further to 15.3 °C when using 1.22 µM AFM1. These results show that a phase transition from gel (solid) to liquid crystalline (fluid) occurs during AFM1/ BLM interactions, in addition to the changes of the electrostatic fields of BLM that occur during these interactions. This may explain why larger transient electrochemical signals are observed with BLMs composed of 35% DPPA in the absence of calcium ions. The transient signals obtained from membranes supported in filters look similar to those obtained using planar BLMs,9 and they take about the same time to appear after exposure of AFM1 to BLMs, but their magnitudes are directly proportional to AFM1 concentration; the signal magnitudes for the same AFM1 concentrations are larger when using filter-supported rather than planar BLMs, probably due to the dynamic changes of AFM1 concentration experienced by the BLM during the passage of the injected zone. The data in Figure 2 indicate that the time required for signal appearance is independent of AFM1 concentration, for all tested concentrations. The relatively invariable delay time suggests a mechanism of response that may occur in two steps: adsorption of AFM1 molecules onto the BLM surface, which may then associate as aggregates to provide electrostatic perturbation of the lipid membrane. Aggregation should be the slow and ratedetermining step, as based on movement across the surface of membranes. At a given temperature (our data have been collected only at room temperature), the process of aggregation should be relatively constant and independent of AFM1 concentration. The aggregative process could be responsible for the delay time in the sense that a threshold amount of aggregates may be necessary to induce changes of BLM electrostatics. The extent of the process could lead to a subsequent rapid reorganization of electrostatic fields at the surface of a membrane. At higher concentrations of AFM1, aggregates may be larger and cause a greater effect, as evidenced by the increases of signal magnitudes obtained with increasing toxin concentrations; therefore, the signal would depend on AFM1 concentration. The return of the current to residual values indicates that the AFM1 aggregates that had been formed onto the membrane surface are unstable and easily detach from the BLM. The destruction of these aggregates for freely suspended BLMs is probably due to lateral diffusion of AFM1 on the BLM surface. According to our DSC experiments, at least some AFM1 molecules remain associated with the BLM, as indicated by the alteration of the phase transition temperature, Tm, of liposomes in the presence of AFM1. Note that temperature scanning in DSC experiments was initiated about 5 min after mixing of AFM1 with the liposomes. No additional electrical transients during the electrochemical experiments were observed for at least 10 min after the appearance
Table 1. Dependence of Applied Potential on Signal Magnitude for AFM1 Concentration of 8.10 nM (Average of Five Determinations ( 1SD) applied potential (mV)
signal magnitude (pA)
25 50 75 100
30.6 ( 1.28 49.1 ( 2.61 63.5 ( 3.44 75.1 ( 4.27
of the transient ion current signal. These attached AFM1 molecules may serve as aggregation nuclei, after the injection of another sample aliquot. For filter-supported BLMs, the breakdown of toxin aggregates is further facilitated by mass action (infinite dilution). The effect of the flow rate on peak height shown in Figure 4 (i.e., decrease of signal magnitude for flow rates above 4 mL min-1) supports the above mechanism of signal generation due to aggregation of the toxin in BLMs. It is possible that an increase of the flow rate from 4 to 5 mL min-1 caused AFM1 to move through the chamber at just enough speed for aggregation to occur. This also would have a high value for studing membrane aggregation rates. Monitoring membrane aggregation rates is difficult for most compounds. The present method may offer a route to monitor the aggregation of numerous proteins-peptides and biologically relevant solutes in membranes. The response of the BLM to alterations of AFM1 concentration, observed as rapid changes of BLM ion permeability, is indicative of the presence of charging events associated with alterations of the surface or dipolar potential of the membrane. As can be seen in Table 1, there is a trend of linearly increasing signal magnitude upon increasing applied potential for a given AFM1 concentration, for potential values up to 100 mV. Repetitive Monitoring of AFM1 Using Filter-Supported BLMs. The mechanism proposed above of signal generation has allowed good signal reversibility, as observed with repetitive determinations of AFM1 in our system, with no sample carryover or membrane memory effects (the relative standard deviation was ∼4-7%). Figure 1 shows that there is an immediate return to the baseline after each measurement (vide supra). This has permitted repetitive AFM1 determinations, with a rate of at least 4 samples/min. A number of cycles of repetitive injections were performed to exploit the retention of the analytical signal, and no reduction of the signal magnitude was observed during each cycle (Figure 5). The number of injections performed in each cycle was about 30; the number of injections was limited only by the syringe capacity (2.5 mL) used in the Hamilton repeating dispenser. Application of the Method to the Direct Determination of AFM1 in Commercial Milk Products. As shown in Table 2, a number of commercial products have been artificially contaminated with AFM1 at various concentrations, and the AFM1 content has been assayed using the above method and a calibration equation. Recoveries within the range of ∼90-110% have been obtained. In conclusion, the results from the applications reported herein that use a technology which is an extension of our previous work highlight the concept that AFM1 in whole milk can be monitored
Figure 5. Recordings showing the variability of response of the BLMs to repetitive AFM1-spiked milk samples injections at a flow rate value of 4.0 mL min-1. AFM1 concentrations were (A) 6.09 and (B) 3.05 nM. The injection of each sample was made at the beginning of each recording. Table 2. Results of Quantification of AFM1 Added in Commercial Milk Preparations (Numbers in Parentheses are the Spike Amounts of AFM1)a sample ID FAGE Dairy Products, S.A., skimmed milk Delta Dairy Products S.A., half cream milk Delta Dairy Products S.A., full cream milk Carnation instant nonfat dry milk, Societe des Produits Nestle S.A. (reconstituted) Noulac, Fiesland Dairy Foods a
AFM1 content (nM) (7.15) 6.61 ( 0.30 (3.81) 4.14 ( 0.21 (14.6) 14.7 ( 0.97
(11.1) 10.2 ( 0.58 (11.1) 11.6 ( 0.70
Results presented are the average of five determinations ( 1SD.
by using stabilized BLM-based sensors for flow injection analysis with very fast response times (on the order of 15 s or less). The detection limits of the present technique are approximately the same as those obtained by immunoassays or chromatographic methods1-3 and within the action limits set by FDA. Moreover, it has significant advantages over the immunoassays or chromatographic procedures in terms of analysis times, sample volumes, and preparation, size, and cost of instrumentation. The applications presented herein show that a BLM-based biosensor can provide an attractive alternative to currently used chromatographic and imunosensor devices. ACKNOWLEDGMENT This work was carried out in the framework of “Copernicus” Contract No. CIPA CT94-0231 and Inco-Copernicus Contract No. IC15CT96-0804 with financial contribution by the European Commission. Received for review November 3, 1997. February 24, 1998.
Accepted
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