Anal. Chem. 1998, 70, 3674-3678
Capacitive Approach To Determine Phospholipase A2 Activity toward Artificial and Natural Substrates Vladimir M. Mirsky,* Markus Mass, Christian Krause, and Otto S. Wolfbeis
Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, 93040 Regensburg, Germany
A capacitive approach has been employed to develop a novel method to determine phospholipase activity. The sensing electrodes have a structure like Au/S(CH2)17CH3/ substrate/electrolyte. Hydrolysis of the substrate, mediated by phospholipase A2, leads to the formation of watersoluble products from the insoluble substrate. This results in desorption of these products into aqueous phase and corresponding increase of the electrode capacitance. The requirement of high water solubility of the reaction products can be achieved in two ways. In the first, short-chain phospholipids are used as the substrate, in which case, water-soluble products are formed and no additional reagents are required to promote desorption of these products. The sensors prepared by this strategy provide sensitive qualitative detection of phospholipases. The second way is based on the use of a water-soluble acceptor (for example, β-cyclodextrin) to solubilize the products of hydrolysis. It allows semiquantitative detection of phospholipase activity toward long-chain natural substrates. The reaction kinetics for this case was found to be monoexponential and linearly dependent on the phospholipase concentration. The detection limit of this method, as tested with phospholipase A2 from bee venom and soy bean lecithin as the substrate, is ∼0.5 ng/mL (500 µunits/mL). The Langmuir technique is one of the most widely used methods to study lipolytic processes.1-6 It is based on the measurement of the decrease of the monolayer area under fixed surface pressure resulting from the formation of water-soluble products and their subsequent desorption from the monolayer into the aqueous phase. This approach was successfully used to study different aspects of phospholipase hydrolysis such as kinetics,1,2 effect of inhibitors or activators,3,5 and electrically coupled autocatalytic phenomena.4 However, its application for medical and industrial purposes is rather limited for several a Corresponding author: (e-mail)
[email protected]. (tel) +(49-941)9434911; (fax) +(49-941)9434064. (1) Zografi, G.; Verger, R.; deHaas, G.H. Chem. Phys. Lipids 1971, 7, 185206. (2) Verger, R. Methods Enzymol. 1980, 64, 340-392. (3) Thuren, T.; Tulkki, A.-P.; Virtanen, J. A.; Kinnunen, P. K. J. Biochemistry 1987, 26, 4907-4910. (4) Mirsky, V. M. Chem. Phys. Lipids 1994, 70, 75-81. (5) Pieroni, G.; Gargouri, Y.; Sarda, L.; Verger, R. Adv. Colloid Interface Sci. 1990, 32, 341-378. (6) Laurent, S.; Ivanova, M. G.; Pioch, D.; Graille, J.; Verger, R. Chem. Phys. Lett. 1994, 70, 35-42.
3674 Analytical Chemistry, Vol. 70, No. 17, September 1, 1998
Figure 1. Principle of the capacitive method for studying phospholipase activity. The capacitance of a hydrophobized electrode, covered with a layer of phospholipids, is monitored. The reaction products desorb into the aqueous phase resulting in the corresponding increase in the electrode capacitance: ∆C ) C02(S0 - Sads)/(C0 + Cads), where C0 is the specific capacitance of the bare electrode, Cads is the specific capacitance of the adsorbed (in this work - lipid) layer, Sads is the area of the covered surface of the electrode, S0 is a total electrode area. β-Cyclodextrin forms a water-soluble complex with insoluble reaction products thus increasing their desorption rate; it allows applications of the method not only for short-chain substrates (arrow up) forming water-soluble reaction products but also for natural (longchain) substrates (arrow down).
reasons: (i) expensive instrumentation (Langmuir trough) is required, (ii) the assay is time-consuming, and certain steps can hardly be automated, and (iii) the method cannot be miniaturized. Recent studies7-11 have revealed that a capacitive method can be applied to analyze adsorption and desorption of different species on/from electrodes. We now report a novel scheme for the detection of lipolytic enzymes based on capacitive registration, which was realized in two ways. The first one (Figure 1, top) can be applied for short-chain phospholipids. It allows a highly sensitive qualitative indication of phospholipase A2 without the use of any additional reagents. The second way was applied for (7) Mirsky, V. M.; Krause, C.; Heckmann, K. D. Thin Solid Films 1996, 284/ 285, 939-941. (8) Krause, C.; Mirsky, V. M.; Heckmann, K. D. Langmuir 1996, 12, 60596064. (9) Mirsky, V. M.; Riepl, M.; Wolfbeis, O. S. Biosens. Bioelectron. 1997, 12, 977-989. (10) Rickert, J., Go¨pel, W., Beck, W., Jung, G.; Heiduschka, P. Biosens. Bioelectron. 1996, 11, 757-768. (11) Berggren, C.; Johansson, G. Anal. Chem. 1997, 69, 3651-3657. S0003-2700(98)00102-4 CCC: $15.00
© 1998 American Chemical Society Published on Web 07/24/1998
long-chain phospholipids; in this case, a water-soluble acceptor of hydrolysis products (β-cyclodextrin) must be added. This latter approach is less sensitive than the former one but allows a semiquantitative detection of phospholipase activity (Figure 1). EXPERIMENTAL SECTION All measurements were performed with a two-electrode system, the reference electrode being an Ag/AgCl electrode with a surface of ∼10 cm2. The working (sensitive) electrodes were prepared from a gold wire by first cleaning it with a hot “piranha” solution (3 mL of concentrated H2SO4 and 1 mL of 30% H2O2), rinsing with water, and drying. Caution: piranha solution reacts violently with most organic materials and must be handled with extreme care. The electrodes were then placed in a 10 mM solution of octadecanethiol in chloroform for at least 12 h and washed shortly with chloroform. The macroscopic surface area of the sensitive electrodes was ∼6 mm2. The electrode capacitance was measured as a 90° component of the capacitive current by means of a lock-in amplifier (PAR, Model 121) at 20 Hz, the amplitude of the sine voltage on the electrode being ∼10 mV. A homemade current-voltage converter (10 V‚mA-1) was used. The analog capacitance signal was digitized with a 16-bit AD card (PC-20 NextView, from BMC Systeme GmbH). All measurements were performed at the electrode potential of +300 mV (reference Ag/AgCl, 100 mM KCl) at room temperature. To prevent a formation of air bubbles, the cell with electrolyte was degassed under vacuum prior to the experiment. The cell volume was 17 mL. To prepare a liposome suspension, 8 mg of the phospholipid (L-R-phosphatidylcholine from soybeans) was dissolved in a small quantity of chloroform and dried under nitrogen in order to deposit a layer on the walls of the flask. A 4-mL aliquot of electrolyte was then added, and after a 30-min incubation, the mixture was ultrasonicated for 20 min with a sonicator (Bandelin Sonorex RK31) in a water bath at room temperature. L-R-Dilauroylphosphatidylcholine and L-R-dilauroylphosphatidylethanolamine were used as short-chain phospholipids, and L-R-phosphatidylcholine from soy beans was used as a long-chain phospholipid. All the lipids were from Sigma. Deionized water was additionally purified by passing it through a Milli-Q-Plus system (Millipore). Gold wire (purity 99.998%) was from Alfa Johnson Matthey. All solvents, inorganic compounds, and octadecanethiol were from Merck. Phospholipase A2 (from bee venom, specific activity ∼1000 units/mg) was purchased from Boehringer Mannheim. Octadecanethiol (98%) was recrystallized from ethanol. The other chemicals (p.a.) were used without additional purification. RESULTS Phospholipid Deposition. Several methods were tested in order to deposit short-chain phospholipids on octadecanethiolcoated electrodes. With the use of either the Langmuir-Blodgett technique or liposome method, no decrease of electrodes capacitance as well as no phospholipase activity toward these electrodes occurred. However, both effects were observed if the electrodes were covered with the short-chain phospholipids by several subsequent immersions into a highly concentrated lipid solution in chloroform (300 mg/mL) typically 5 times for 2 s each, interrupted by pauses of 10 s to evaporate the chloroform. The
Figure 2. Formation of the phospholipid monolayer by liposome fusion with a hydrophobized electrode (a) and the changes in the capacitance of the electrode due to this process (b): phospholipid: L-R-phosphatidylcholine from soy beans; electrolyte, 100 mM NaCl, 2 mM imidazole, 1.5 mM CaCl2, pH 7.22.
Figure 3. Effect of calcium and magnesium ions on the rate of the liposome fusion (capacitance changes) with hydrophobized electrodes: phospholipid, L-R-phosphatidylcholine from soy beans; electrolyte, 100 mM NaCl, 2 mM imidazole, pH 7.22.
specific electrical capacitance of the electrodes decreased during this procedure from 1.2 µF‚cm-2 to typically 1.0-1.1 µF‚cm-2. Long-chain natural phospholipids were deposited on the hydrophobic surface of the alkanethiol-covered gold electrodes by the liposome fusion technique (Figure 2a). The process can be monitored by capacitance measurements (Figure 2b). The total changes in the capacitance were in the range of 15-20%. The kinetics of the decrease of the capacitance after addition of liposomes can be described by a biexponential function. The presence of calcium ions accelerated this process, while magnesium had no effect (Figure 3). The acceleration by Ca2+ was reversible, and a complexing of Ca2+ by addition of EDTA led to inhibition of the capacitance decrease. Phospholipase Action toward Short-Chain Phospholipids. Addition of phospholipase A2 in the presence of calcium leads to Analytical Chemistry, Vol. 70, No. 17, September 1, 1998
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Figure 4. Detection of the phospholipase activity with a short-chain substrate (L-R-dilauroylphosphatidylcholine). Changes in the capacitance of the sensor after subsequent phospholipase additions (a) and the corresponding dependence of the rate of the capacitance increase on the phospholipase A2 concentrations (b). Capacitance effect due to deposition of phospholipid layer on the electrode was considered as 100%: electrolyte, 200 mM NaCl, 10 mM imidazole, 0.25 mM CaCl2, pH 7.0.
an increase in the capacitance of the hydrophobized gold electrodes covered with short-chain phospholipid (Figure 4a). Subsequent addition of an excess of EDTA completely inhibits the capacitance increase. Such cycles of calcium additions followed by additions of an excess of EDTA can be repeated several times. Since it is well-known that phospholipase A2 is a calcium-dependent enzyme, the experiment proves that the capacitance increase after the phospholipase addition is indeed caused by the phospholipase action. The rate of capacitance rise increases with the enzyme concentration (Figure 4). The results allow an indication of the phospholipase activity at levels as low as ∼50 pg/mL (i.e., about 50 µunits/mL). To exclude any effects of decreasing substrate concentration on the rate of the capacitance increase, the concentration dependence was measured when only a small part (
