Biotechnol. Prog, 1995, 11, 699-703
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NOTES Chemiluminescence-Based Inhibition Kinetics of Alkaline Phosphatase in the Development of a Pesticide Biosensor Madhu S. AyyagariJto Sanjay Kamtekar? Rajiv PandeJ Kenneth A. Man,*,? Jayant Kumar,*Sukant K. Tripathy? Joseph Akkara,o and David L. Kaplano Centers for Advanced Materials and Intelligent Biomaterials, Departments of Chemistry and Physics, University of Massachusetts Lowell, Lowell, Massachusetts 01854, and Biotechnology Division, US. Army Natick RD&E Center, Natick, Massachusetts 01760
The use and application of the enzyme alkaline phosphatase in a chemiluminescence assay are discussed. The enzyme catalyzes the hydrolysis of a macrocyclic phosphate compound generating a chemiluminescence signal. On the basis of inhibition of this signal, a methodology for the detection and quantitation of organophosphorus-based pesticides has been developed. The methodology is studied with alkaline phosphatase in the bulk aqueous phase, and detection of the signal is accomplished by a simple optical setup. Parts per billion level detection of paraoxon and methyl parathion in bulk solutions is achieved. The technique is rapid and sensitive and is applicable to the detection of most organophosphorus-based pesticides. The results from kinetic studies indicate a mixed type of inhibition of the enzyme by paraoxon and methyl parathion. The detection methodology forms a n integral part of a biosensor under development and is adaptable to incorporating optical fibers for remote detection of pesticides.
Introduction The persistence of chlorinated hydrocarbon pesticides such as DDT, endrin, and lindane in the biosphere and the resultant ecological impact have turned the attention of chemists to the development of environmentally compatible pesticides. These chlorinated compounds are not easily degraded, and their environmental mobility in soil makes them ubiquitous (Coats, 1993). As an alternative, organophosphorus (0P)-based pesticides have been widely used since the 1950s. Typically, OP-based pesticides are more soluble in water in comparison to the chlorinated pesticides and pose a threat to aquatic life. It is therefore essential to monitor the levels of these pesticides in various environments to determine compliance with EPA regulations as well as efficacy of treatments (EPA report EP 1.29/2,1980). Among a number of methods employed to separate and/or detect the pesticides (Martinez et al., 1992; Sanchez et al., 1991; Bier et al., 1992; Palleschi et al., 1992), biological methods can afford rapid, specific, and sensitive detection as well as compatibility with miniaturized and portable devices. In this article, we discuss the action of the enzyme alkaline phosphatase on a 1,2-dioxetane phenyl phosphate compound. These compounds release chemical energy without the addition of oxidants. Dephosphorylation produces an unstable phenolate anion (Tizard et al., 1990). The weak 0-0 bonds in the highly strained + Department of Chemistry, University of Massachusetts
Lowell. Department of Physics, University of Massachusetts Lowell. U.S. Army Natick RD&E Center. * Address correspondence to Prof. Kenneth A. Marx, Center for Intelligent Biomaterials, Department of Chemistry, University of Massachusetts Lowell, Lowell, MA 01854. Tel: (508)934 3658. Fax: (508) 458 9571.
ring break, and the decomposition of the phenolate anion emits light [Ayyagari et al., 1994al. The half-life of the anion varies from a few minutes to a few hours depending on its surrounding environment. In the presence of OPbased pesticides, the enzyme activity is inhibited, which leads to the generation of a weaker chemiluminescence signal. Thus the signal intensity, which is inversely proportional to the inhibitor concentration, is related to the amount of the pesticide in solution. The in-situ generation of light in the reaction mixture is easily detected and processed using a simple optical setup. Unlike the case of a fluorescence signal, there is no need for an external excitation light source and a monochromator for the detection of a chemiluminescence signal. Although this article discusses the inhibition kinetics of alkaline phosphatase, we underscore the importance of the technique for biosensor applications in the detection of OP-based pesticides. A number of approaches based on amperometry,chromatography,fluorescence, or a simple color change for the detection of pesticides have been reported in the literature (Martinez et al., 1992; Sanchez et al., 1991; Bier et al., 1992; Palleschi et al., 1992; Trettnak et al., 1993). Although sensitive, some of these techniques involve either extensive sample preparation or lengthy analytical procedures or both. In this article, we describe a methodology by which a parts per billion level detection is possible within a few seconds, and at least 2 orders of magnitude faster than the literature values. This detection methodology, in conjunction with a generic molecular assembly of enzymes and conjugated polymers developed for optical fibers (Ayyagari et al., 19951, forms the sensing element of a biosensor under study for the detection of environmental pollutants.
8756-7938/95/3011-0699$09.00/0 0 1995 American Chemical Society and American Institute of Chemical Engineers
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700
[I] = 1.0 mM
10
Photon counter
Amplifier
12
sy,, *
High voltage
Sample holder
Dark room
Figure 1. Experimental setup for chemiluminescence detection.
Materials and Methods An aqueous preparation of streptavidin-conjugated alkaline phosphatase was supplied as a part of SouthernLight Chemiluminescent Detection System by Tropix, Inc. (Bedford,MA). Diethylamine (DEA) and 3-(5'-chloro4-methoxyspiro[1,2-dioxetane-3,2'-tricycl0[3.3.1.1397]-decan]-4-y1)phenyl phosphate (CSPD) were also a part of this system. CSPD was available as a 25 mM aqueous solution. Sapphire, a luminescence amplifying material (referred to as an enhancer), was also supplied by Tropix, Inc. Magnesium chloride was purchased from Fisher Scientific (Fair Lawn, NJ). Paraoxon was supplied by Sigma Chemicals Co. (St. Louis, MO), and malathion, methyl parathion, and diazinon were purchased from PolyScience (Nile, IL). Deionized and distilled water was used in all preparations. The assay buffer (0.1 M DEA, 1 mM MgC12, pH 10) was prepared once every week, as recommended. The pH was adjusted with 0.1 M HC1. Stock solutions of pesticides were prepared just before the experimentation, and necessary dilutions were carried out with assay buffer. CSPD stock solution was prepared by first making a 10% solution of enhancer in DEA buffer. The 25 mM CSPD solution was diluted with this solution to the extent necessary. The resultant solution was stable at room temperature for weeks. The enzyme stock solution (0.5 nM)was always freshly prepared just before the reaction. The stock solutions were stored at 4 "C until the time the reaction mixture was to be prepared. A typical reaction mixture was prepared by first adding predetermined volumes of substrate and buffer or pesticide solutions in a glass test tube. After bringing the mixture to room temperature, the reaction was initiated by adding a known volume of enzyme solution. Wherever required, buffer was used to make up the volumes. The composition of the final reaction mixture was arrived at after extensive experimentation to minimize the amounts of enzyme and CSPD and maximize the signal to noise ratio. A photomultiplier tube (PMT), a photon counter, an amplifier, and a personal computer were used to collect and process the data. Figure 1illustrates the schematic of this simple experimental setup. Less than 10 s elapsed between the initiation of the reaction and the start of data acquisition. The photon counter was set up to count
c
/
5
10
/
7
15
=O
20 25
L
1/s,mM-' Figure 2. Lineweaver-Burk plots for (a) paraoxon-mediated and (b) methyl parathion-mediated inhibition of alkaline phosphatase.
photons detected during a 1 s interval. The raw data are therefore a plot of the first derivative of photon counts (with respect to time) versus time. Hence, the first few points were integrated with time, and a counts-versustime plot was generated. Initial velocities of the reaction were computed from the slopes calculated on the basis of the first 20 data points collected in as many seconds.
Results and Discussion Alkaline phosphatase is catalytically active at relatively high pH values. The enzyme is most active at pH 10, and accordingly, all reactions were conducted at that value in 0.1 M DEA buffer. The following text discusses the results from the enzyme kinetics studies and the detection and quantitation of OP-based pesticides. Kinetics. The amount of light generated in the hydrolysis of CSPD, catalyzed by alkaline phosphatase, is proportional to the amount of product generated. Thus the rate of light generation is proportional to the rate of product formation which is the velocity, u , of the reaction. In the first set of experiments, the reactions were conducted by varying the CSPD concentration (0.05-0.3 mM) in the reaction mixture. In the second set of experiments, CSPD concentration was varied again in the same range, but in the presence of 1.0 mM paraoxon. The third set of experiments was a repeat of the second set, but in the presence of 1.5 mM paraoxon. The concentration of the enzyme was 125 pM in all reaction mixtures in kinetic studies. A Lineweaver-Burk plot was prepared for these three sets of data as shown in Figure 2a. Typically the lines in a Lineweaver-Burk plot meet either on the x-axis or on the y-axis for noncompetitive or competitive inhibition, respectively. However, it is interesting to note that the lines do not intersect on either of the axes in the present case (Figure 2). This behavior suggests a mixed type of inhibition in which the inhibitor not only binds to the enzyme but affects the association of the substrate with the enzyme (Webb, 1963; Segel, 1975). Another OP-based pesticide, methyl parathion, also exhibited a similar behavior (Figure 2b). The scheme in Figure 3 illustrates the mixed inhibition of the
701
Biotechnol. frog., 1995,Vol. 11, No. 6 Table 1. Kinetic Parameters for Paraoxon and Methyl Parathion Inhibition
+
+
I
I
iT
K1
E1
inhibitor none paraoxon methyl parathion
Ks
+ S
itu
Ki
ESI
P2 44, P3 t P* PP4+hv Figure 3. Schematic of (a)the reaction equilibria and (b) the kinetic scheme.
enzyme by the pesticide. A theoretical analysis of this observation is presented in the following text. Figure 3b portrays the reaction in three steps written to derive a kinetic expression. The expression was derived for the rate of generation of chemiluminescence or, from an experimental point of view, the rate of photons countedldetected. As noted earlier, typical postexperimental data processing involved conversion of a real time curve of photon count rate-versus-time to a counts-versus-time curve by data integration. The slope of the curve in its linear portion was then computed and equated to the initial velocity of the reaction. Assuming steady state conditions at very small times into the reaction, the rate expression, after simple mathematical manipulations, reduces to the standard MichaelisMenten expression (see Appendix). The final rate expression is
u = d(n)/dt = [(k,)(ET)(S)MK, + SI
(1)
where n is the number of photons emitted, ET is the total enzyme concentration in the reaction mixture, Kmis the Michaelis-Menten constant, and S is the initial concentration of CSPD. The enzyme behavior, however, in the presence of pesticides is quite different. For a multicomponent reaction, the total amount of enzyme in the reaction system can be written as ET
=E
+ EX1 + EX2 + ... +EX, + ES + ESX1+ ESX, + ... + ESX,
(2)
where S is the substrate and X l , X z , ...,X , are cofactors, inhibitors, or activators for the enzyme E. If the X's are inhibitors, they compete with the substrate for binding sites on the enzyme. Substituting for the corresponding terms, the above expression can be rearranged into the Lineweaver-Burk form as shown below. The rationale for adopting this kinetic scheme becomes clear in the following arguments. Writing eq 2 in terms of velocity (Webb, 1963)
where u is the velocity of the reaction which is the rate of photon emission in the present case. K1, Kz, ...,K,, are the dissociation constants for binding of the enzyme with corresponding substrates. a, /3, ..., v represent the
umax (5-l) 3 104 -
K,(mM) 0.81 -
a
Ki(mM) -
-
1.7 0.06
0.334 0.380
interaction constants. The physical significance of the interaction constants is as follows. The parameter a takes an infinitely large value if its corresponding inhibitor does not prevent the substrate from binding to the enzyme. On the other hand, a takes a value of 5 if the inhibitor causes a 5-fold increase in the dissociation constant between the enzyme and the substrate. For a specific case where there is one substrate and one inhibitor, as is the case under discussion, the above expression reduces to
where I is the inhibitor concentration and Ki is the dissociation constant for the inhibitor. The equation reduces to the normal Michaelis-Menten expression arranged in Lineweaver-Burk form
l/v = l/U"
+ K,/(V,&s)
(5)
in the absence of inhibitor (i.e., I = 0). The point of intersection of the lines defined by eqs 4 and 5 is (-l/ [aKm] , [a - 1Ua~maJ).Once this point of intersection is obtained graphically, the interaction parameter, a, can be easily computed. The inhibitor dissociation constant, Ki,is calculated from the slope, [l 1/KJKm/Umax,of the line represented by eq 4. According to this model, E1 has a higher affinity for S than E (for a .e 1and the point of intersection falling in the third quadrant), and the ESI complex is nonproductive (i.e., no product formation is possible). At very high 1,all enzyme can be driven to E1 and ESI forms, and since the ESI complex is nonproductive, the velocity can be driven to zero by increasing I. Similarly, as a approaches zero, the inhibition may be complete since all the enzyme is in ESI form. The behavior shown in Figure 2 is not unexpected especially in view of the fact that both the substrate and the inhibitor are capable of binding to the enzyme. The data obtained from the experiments fit eqs 1and 4, as is evident from the sets of straight lines in Figure 2. The kinetic constants were calculated from the slopes and intercepts of the resulting curves and their points of intersection and are listed in Table 1. It is also interesting to note that the magnitude of the interaction constant, a, is about the same for both types of pesticides, presumably due to the structural similarity of these OPbased pesticides. The K,value for paraoxon was found to be higher than that for methyl parathion, implying that paraoxon is less strongly bound to the enzyme than methyl parathion. This is in accordance with the experimental observation that, to achieve the same level of inhibition, the amount of paraoxon required was greater than that of methyl parathion (see Figure 2). Detection and Quantitation of Pesticides. The extent of inhibition of alkaline phosphatase activity by a range of pesticide concentrations was studied at a fmed CSPD concentration. A stock solution of paraoxon or methyl parathion of a known concentration was prepared. As before, the reaction mixture was prepared by mixing predetermined volumes of CSPD, pesticide, and enzyme solutions. The concentrations of CSPD and the enzyme
+
6iofechnol. Prog., 1995, Vol. 11, No. 6
702 Table 2. Paraoxon Detection Limit Versus Molar Ratio of CSPD to Paraoxon [CSPDI @M)
paraoxon detn limit (ppm)
[CSPDl/[paraoxonl in reactn mixture
290 1.82 0.874 0.290
50 0.4 0.11 0.05
1.6 1.3 2.2 1.6
in the reaction mixture were fixed at 0.29 pM and 0.14 pM, respectively, and the pesticide concentration was varied either by adding a given volume of diluted pesticide solutions or by adding different volumes of the concentrated stock pesticide solution and making up the reaction mixture volume with buffer. In all cases, the reaction was initiated by adding the enzyme solution just before the data collection, which was continued well past the linear portion of the count rate-versus-time curve. The initial slopes of these curves (in their linear portions) are proportional to the enzyme activity. Unlike the treatment in kinetics, the enzyme activity here is expressed directly as slopes of count rate-versus-time curves for simplicity. This sequence of steps was carried out at each successively lower pesticide concentration until the detection limit was reached. In practice, the data points required for computing slopes could be collected within the first 10-20 s. Interestingly, during the course of establishing detection limits, the difference in the initial slopes for a sufficiently low concentration of pesticide and the corresponding control (without pesticide) disappeared when the molar ratio of CSPD to the pesticide approached a value in the range of 1-2 (Table 2). For example, at 0.14 nM enzyme concentration, 1.82pM CSPD concentration, and 100 ppm paraoxon concentration, which was sufficient to completely inhibit the enzyme activity, the The corresponding slope molar ratio would be 5 x of a count rate-versus-time curve would be negligible or very small compared to that of the control. As the pesticide concentration was reduced from 100 ppm at constant CSPD and enzyme concentrations, the molar ratio and the curve initial slope would increase and approach the control value. As the ratio approached the above range, the difference between the initial slopes of the curves for pesticide and the control would diminish and the detection limit with respect to that particular CSPD concentration used would have been reached (0.4 ppm in this example, see Table 2). In other words, at a concentration above 1.26 CSPD molecules per every pesticide molecule in this example, the enzyme inhibition due to the pesticide was negligible. Figure 4 illustrates the dependence of the lowest detectable concentration of paraoxon on CSPD concentration. In principle, any concentration of paraoxon could be detected in solution, provided the concentration of CSPD would not exceed the corresponding value from the plot. Thus the CSPD concentration range could be adjusted in a dynamic fashion to detect any concentration of a given OP-based pesticide. The above relation could be used to advantage in developing an automated process for the remote detection of toxic compounds present in unknown quantities. The amount of CSPD could be adjusted by a feedback controller which compares the inhibited chemiluminescence signal to the corresponding control signal. The data presented in Figure 4 clearly indicates that the detection limit of paraoxon could be further lowered by reducing the CSPD concentration, but only at the cost of the instrument signal to noise ratio (S/N) and the intensity of photon emission. Without a significant
4 3
-1
-2 -2
0
-1
2
1
log,, ( [Paraoxon], ppm )
Figure 4. CSPD-dependent detection limit of paraoxon (0.14 nM enzyme).
8
L 0
0 Paraoxon 0 Methyl parathion
50
100
150
200
250
300
Pesticide conc., ppb
Figure 5. Calibration curves for paraoxon and methyl parathion inhibitions (0.14 nM enzyme and 0.29 pM CSPD).
sacrifice in S/N ratio, we were able to reduce the CSPD concentration to 0.29 pM to enable the detection of 50 ppb of paraoxon and 80 ppb of methyl parathion. We believe that, by enhancing the efficiency of collection optics, lower CSPD concentrations could be used that would permit sub-parts per billion level detection. Figure 5 illustrates the calibration curves for both paraoxon and methyl parathion. Other OP-based pesticides that showed a similar behavior (data not shown) were malathion and diazinon. Malathion was not particularly stable at the working pH 10 due to spontaneous hydrolysis (Merck Index, 1983). This problem could be overcome by conducting the reactions at a lower pH and using acid phosphatase. The technique described so far can play a potentially important role in the development of fiber-optic biosensors for pesticide detection. For example, alkaline phosphatase could be immobilized on an optical fiber and the chemiluminescence signal generated on the fiber surface could be transduced via fiber to the detector. We have already established a novel molecular cassette approach to attach a biomolecule to an optical fiber surface through biotin-streptavidin interactions (Ayyagari et al., 1995). Three different methodologies have been studied in our laboratory to immobilize enzymes on a glass surface. These methodologies have been shown to be successful in conjunction with optical fibers and glass capillaries. In addition to the self-assembly, a brushlike polymer was grown on a glass surface and the conjugated polymer functionalized with biotin for subsequent streptavidinconjugated protein binding. The third approach was a
Biofechnol. Prog., 1995, Vol. 11, No. 6
covalent attachment of biomolecules to glass surfaces (Ayyagari et al., 1994a,b).
703
From Figure 3b, the rate of light emission is d(hv)/dt = k,[P*l
Conclusions A novel technique to detect and quantitate organophosphorus-based pesticides is described. Alkaline phosphatase-catalyzed chemiluminescence signal generation and its inhibition are used to establish the reaction kinetics and calibration curves for the detection of paraoxon and methyl parathion. A mixed type (i.e., a mixture of competitive and noncompetitive types of inhibition) of inhibition is exhibited by the enzyme in the presence of the pesticides at sufficiently high concentrations. The reaction kinetics show that the levels of inhibition of the enzyme by paraoxon and methyl parathion are about the same. A 50 ppb detection level for paraoxon and 80 ppb detection level for methyl parathion are achieved at a 0.29 pM concentration of CSPD. These detection levels could be lowered by reducing the CSPD concentration. Besides rapid and sensitive detection, the technique only requires a simplified optical setup. Experiments with other OP-based pesticides show that the technique is general to all OP-based pesticides studied. This technique forms an integral part of a biosensor system under development for the detection of various environmental pollutants. Future studies are focused on lowering the detection limits, optimizing the immobilization strategy, and expanding the utility of the device to cover a variety of pesticides.
Acknowledgment The authors acknowledge support from ARO Grant DAAL03-91-G-0064 and Contract DM60-93-K-0012 and ARO Coop Grant DAAH07-94-2-0003.
Appendix Figure 3b depicts the hydrolysis of the substrate CSPD
(S)catalyzed by the enzyme alkaline phosphatase (E). One of the two products formed is an unstable phenolate anion, P2, which breaks down into a stable product, P3, and a second product in its excited state, P*. During the de-excitation step, P* emits light to form a stable compound, P4. The rate constants for different steps are represented by k1, kz, k3, k4,and ks.
Under steady state conditions d[P*Ydt = d[P2l/dt = d[ESYdt = 0 The resultant equations were solved for [P*l, [P21, and [ESI to arrive at eq 1. Velocity, u , is the rate of product (P4) formation and is proportional to the rate of photon emission.
Literature Cited Ayyagari, M.; Kamtekar. S.; Pande, R.; Marx, K.; Kumar, J.; Tripathy, S.; Kaplan, D. Proc. Znt. Conf: Zntell. Mater. 1994a, 2, 85-96. Ayyagari, M.; Gao, H.; Chittibabu, K. G.; Bihari, B.; Marx, K.; Kumar, J.; Tripathy, S.; Kaplan, D. SPZE Proc. 1994b, 2068, 168-177. Ayyagari, M. S.; Pande, R.; Kamtekar, S.; Gao, H.; Marx, K. A.; Kumar, J.; Tripathy, S. K.; Akkara, J. A,; Kaplan, D. L. Biotechnol. Bioeng. 1995, 45, 116-121. Bier, F. F.; Stocklein, W.; Bocher, M.; Bilitewski, U.; Schmid, R. D. Sens. Actuators, B 1992, 7, 509-512. Coats, J. R. CHEMTECH 1993,23 (2), 25-29. Martinez, R. C.; Gonzalo, E. R.; Moran, M. J.; Mendez, J. H. J . Chromatogr. 1992,607,37-45. Palleschi, G.; Bernabie, M.; Cremisini, C.; Mascini, M. Sens. Actuators, B 1992, 7, 513-517. Sanchez, F. G.; Lopez, M. H.; Pareja, A. G. Anal. Chim. Acta 1991,255, 311-316. Segel, I. H. In Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady State Enzyme Systems; John Wiley & Sons: New York, 1975. Tizard, R.; Cate, R. L.; Ramachandran, K. L.; Wysk, M.; Voyta, J. C.; Murphy, 0.J.; Bronstein, I. Proc. Natl. Acad. Sci. U S A . 1990,87,4514-4518. Trettnak, W.; Reininger, F.; Zintrel, E.; Wolfbeis, 0. S. Sens. Actuators, B 1993, 11, 87-93. Webb, J. L. In Enzyme and Metabolic Inhibitors; Academic Press: New York, 1963; Vol. 1. Accepted March 31, 1995.@
BP950019J
* Abstract published in Advance ACS Abstracts, June 1, 1995.
