A Biotinylated Undecylthiophene Copolymer Bioconjugate for Surface

Dec 1, 1995 - of Massachusetts, Lowell, Massachusetts 01854, and Biotechnology Division, U.S. Army Natick RD&E Center,. Natick, Massachusetts 01760...
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Bioconjugate Chem. 1996, 7, 159−164

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TECHNICAL NOTES A Biotinylated Undecylthiophene Copolymer Bioconjugate for Surface Immobilization: Creating an Alkaline Phosphatase Chemiluminescence-Based Biosensor Rajiv Pande,† Sanjay Kamtekar,† Madhu S. Ayyagari,†,§ Manjunath Kamath,† Kenneth A. Marx,*,† Jayant Kumar,‡ Sukant K. Tripathy,† and David L. Kaplan§ Centers for Intelligent Biomaterials and Advanced Materials, Departments of Chemistry and Physics, University of Massachusetts, Lowell, Massachusetts 01854, and Biotechnology Division, U.S. Army Natick RD&E Center, Natick, Massachusetts 01760. Received April 11, 1995X

Methodology is described for the creation of a molecular assembly consisting of the enzyme alkaline phosphatase immobilized onto a glass surface using a biotinylated conjugated copolymer, poly(3undecylthiophene-co-3-thiophenecarboxaldehyde) 6-biotinamidohexanohydrazone. The biotinylated polymer is attached to the inside walls of a silanized glass capillary via hydrophobic interactions, and a streptavidin-conjugated alkaline phosphatase is interfaced with the polymer through the classical biotin-streptavidin interaction. Utilizing a simple optical setup, we can detect the activity of as little as approximately 0.1 fmol of alkaline phosphatase with this molecular assembly. The assembly is mechanically robust and retains the majority of bound enzyme activity for up to 30 days. We have utilized this molecular assembly for the detection of organophosphorus-based pesticides. Both paraoxon and methyl parathion inhibit the enzyme-mediated generation of chemiluminescence signal. We are able to detect paraoxon and methyl parathion concentrations down to 500-700 ppb.

INTRODUCTION

We are pursuing the creation of monolayer and thin film materials that incorporate biological macromolecules and conjugated polymer for applications in optoelectronics and biosensors (1). The development of such practical devices is dependent upon devising appropriate procedures for the immobilization and stabilization of the biological component. Over the last few years, our aim has been to interface biomolecules with conducting polymers which possess unusual optical and electronic properties. Besides being potentially useful in signal transduction, these polymers offer ruggedness and stability. Streptavidin, a tetrameric protein, has the ability to bind four molecules of biotin and derivatized forms of biotin with exceptionally high affinity (2, 3). This property has led to its wide application in bioanalytical and biomedical research (4, 5). Previous studies undertaken by our group have involved creation of molecular assemblies using streptavidin as a coupling component between a polymer support and biotinylated macromolecules. These assemblies included a biotinylated lipid monolayer cassette system (6) and biotinylated derivatives of conjugated polymers (7) for the creation of a more robust cassette system. Derivatives of polythiophenes are a widely investigated * Corresponding author: Kenneth A. Marx, Center for Intelligent Biomaterials, Department of Chemistry, University of Massachusetts, Lowell, MA 01854. † Department of Chemistry, University of Massachusetts, Lowell. ‡ Department of Physics, University of Massachusetts, Lowell. § U.S. Army Natich RD&E Center. X Abstract published in Advance ACS Abstracts, December 1, 1995.

1043-1802/96/2907-0159$12.00/0

class of conjugated polymers because of their stability and processability (8-10). We recently reported the synthesis of a biotinylated copolymer of a 3-alkylthiophene and 3-(hydroxymethyl)thiophene. The copolymer was used to orient and spatially organize protein molecular assemblies utilizing the Langmuir technique (11). In the present report, we modify this system by employing a longer spacer arm between the polymer and the biotin group. The copolymer synthesized was poly(3-undecylthiophene-co-3-thiophenecarboxaldehyde) 6-biotinamidohexanohydrazone (B-PUHT). Longer spacers are known to enhance biotin binding to the deep set biotin binding cleft of the streptavidin subunits (12). In addition to enhancing thermal stability, the alkyl chain side groups on B-PUHT impart hydrophobicity for van der Waals interactions with hydrophobic surfaces. We describe the development of a molecular assembly created by the attachment of B-PUHT to the inside walls of a silanized glass capillary via hydrophobic interactions. Streptavidin-conjugated alkaline phosphatase was immobilized on the polymer matrix. Alkaline phosphatase catalyzes the dephosphorylation of organophosphorus (OP) compounds. An interesting substrate for this enzyme is the chemiluminescent 3-(4-methoxyspiro[1,2dioxetane-3,2′-tricyclo[3.3.1.1]decan]-4-yl)phenyl chlorophosphate (CSPD) (13). The utilization of chemiluminescence, the production of light as a result of chemical reactions, has gained favor as a method of choice for molecule detection. Its major advantages over fluorescence-based techniques are the lack of the requirement of an external light source for excitation energy and the avoidance of background buildup as is the case for fluorescent substrates. Dephosphorylation of CSPD results in the formation of a product in an excited state. Upon de-excitation, a chemiluminescence signal is spontaneously generated. Detection is based on chemilumi© 1996 American Chemical Society

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nescence measurements by a simple optical setup. We also describe the utilization of the molecular assembly for the detection of OP-based pesticides. These pesticides act as inhibitors to the alkaline phosphatase-catalyzed dephosphorylation of CSPD. Without optimization of this system, we can detect paraoxon and methyl parathion around 500-700 ppb. We discuss this immobilization strategy as a generic approach for a number of different sensing applications. EXPERIMENTAL METHODS

Materials. 3-Undecylthiophene was purchased from TCI America (Portland, OR). 3-Thiophenecarboxaldehyde, ferric chloride, and acetic acid were obtained from Aldrich Chemicals (Milwaukee, WI). Chloroform, dichloromethane, and methanol were also obtained from Aldrich Chemicals but distilled under nitrogen prior to use. 6-Biotinamidohexanohydrazide (or biotin-LC-hydrazide) was purchased from Pierce (Rockford, IL), and all reagents were used without further purification. Streptavidin-conjugated alkaline phosphatase, supplied as an aqueous preparation, was purchased as a part of the Southern-Light Chemiluminescent Detection System from Tropix, Inc. (Bedford, MA). Diethylamine (DEA), blocking buffer (0.2% casein in phosphate-buffered saline), and CSPD were among other components of the system. CSPD was received as a 25 mM aqueous solution. Sapphire, a luminescence-amplifying material (referred to as enhancer), was also obtained from Tropix, Inc. Magnesium chloride was purchased from Fisher Scientific (Fair Lawn, NJ). Paraoxon and methyl parathion were supplied by Sigma Chemicals Co. (St. Louis, MO). All other solvents were supplied by Aldrich Chemicals (Milwaukee, WI). Deionized and distilled water was used in all preparations. Instrumentation. Characterization of the polymer was facilitated via IR and UV-vis spectroscopy. The IR spectra were recorded on a Perkin-Elmer 1760X FTIR spectrophotometer. UV-vis spectra were recorded on a Perkin-Elmer Lambda 9 spectrophotometer. Number and weight average molecular weights (Mn and Mw) of the polymers were determined with a Waters 410 differential refractometer using chloroform as an eluent. The gel permeation chromatography (GPC) calibration was based on polystyrene standards. The column set consisted of Waters styragel 100, 500, 1000, and 10 000 Å. Silanization. Glass capillaries (accupette, 100 mL, Dade Diagnostics, Miami, FL) were cleaned with Chemsolv detergent (10% v/v in water) in an ultrasonic bath and rinsed thoroughly in water. The cleaned capillaries were dried under nitrogen and subsequently immersed in a 2% (w/v) solution of chlorodimethyloctadecylsilane (CDOS) in petroleum ether for 30 min. After silanization, the capillaries were washed with petroleum ether to remove any unbound silane compound, air dried, and stored in a dust-free environment at room temperature. Polymer Synthesis. Synthesis of B-PUHT was carried out as follows. 3-Undecylthiophene and 3-thiophenecarboxaldehyde were copolymerized in a molar ratio of 1:1 by the chemical dehydrogenation method using anhydrous FeCl3 (14). A 10 mmol solution of the mixture in chloroform was polymerized using 40 mmol of ferric chloride at room temperature under nitrogen. After being stirred for 5 h, this copolymer, PUAT, was precipitated in methanol, filtered, and washed with an acetonemethanol mixture. This was followed by a Soxhlet extraction with methanol for 3 days to remove oligomeric and iron impurities. The copolymer was finally washed with aqueous ammonia solution to remove any trace

Pande et al.

amounts of iron impurities and dried under vacuum. The yield was 93%, the Mn ) 17 000, and the Mw ) 27 800 by GPC analyses. 6-Biotinamidohexanohydrazide (0.024 g, 0.064 mmol) and PUAT (50 mg, 0.125 mmol equiv of CHO) in dichloromethane (50 mL) was stirred at room temperature until the Schiff’s base was formed. The reaction mixture was then washed with water (3 × 10 mL) and dried (MgSO4). The B-PUHT solution was concentrated and reprecipitated in methanol. The precipitated polymer was repeatedly washed with acetone and dried overnight under vacuum, at room temperature. Mn ) 20 000 and Mw ) 32 700 for the B-PUHT. Polymer Attachment. The silanized capillaries were immersed in a 10 mg/mL solution of B-PUHT in chloroform for 16 h at room temperature. The adsorption of the polymer on the silanized glass surface was attributed to van der Waals interactions between the extended octadecyl alkyl group of CDOS and the alkyl side chain of the biotinylated polymer. The control involved immersion of the silanized capillaries in a 10 mg/mL solution of PUAT (the nonbiotinylated polymer) for 16 h at room temperature. These capillaries were treated in a manner identical to that of those coated with B-PUHT in further experimental procedures. Immobilization and Detection of Bound Streptavidin-Conjugated Alkaline Phosphatase. The capillaries were removed from the B-PUHT-containing solution, and the polymer solution was drained. These capillaries were immersed in blocking buffer for 30 min to minimize nonspecific interactions. Streptavidinconjugated alkaline phosphatase solution (25 nM in blocking buffer) was drawn into a capillary. The tube was sealed and placed in a water bath at 45 °C for 90 min. The optimal incubation temperature was derived from a temperature dependence study. Following the 90 min incubation, excess conjugate solution was blotted out. The enzyme-bound capillaries were immersed in blocking buffer for 10 min before being washed for 30 min (3 × 10 minutes) in wash buffer (1X phosphate-buffered saline solution, pH 7.2). The capillaries were equilibrated in a freshly prepared assay buffer (0.1 M DEA, 1 mM MgCl2, pH 10) for 10 min prior to the addition of the substrate. Typically, 100 mL of 0.4 mM CSPD (in assay buffer, 10% enhancer) was drawn into the capillary to initiate the reaction. The capillary was wrapped in aluminum foil and placed on end, in front of the photomultiplier tube (PMT) window for chemiluminescence signal detection. Kinetic Measurements. Different substrate concentrations were used to initiate the chemiluminescent reaction by aliquoting or diluting appropriate amounts of CSPD from the stock solution. The substrate solution was always made up with assay buffer containing 10% enhancer. The substrate concentration was varied from 0.05 to 0.6 mM. Higher CSPD concentrations were not achievable due to solubility constraints. The corresponding changes in reaction rates as a function of substrate concentration were computed from the changes in the rates of chemiluminescence signal generation. Pesticide Detection. Stock solutions of paraoxon (2400 ppm) and methyl parathion (50 ppm) were prepared by dissolving them separately in assay buffer (15). Appropriate fractions from the stock solution were aliquoted into a microfuge tube containing 90 mL of 0.4 mM CSPD. The final volume was always adjusted to 100 mL. This reaction mixture was drawn into the capillary to initiate the chemiluminescence signal generation. This procedure was repeated for each successive lower paraoxon and methyl parathion concentration until the detection limit was reached.

Technical Notes

Figure 1. Schematic of the experimental setup for the detection of the chemiluminescence signal.

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Figure 3. Hypothetical representation of biotinylated copolymer immobilization by hydrophobic interactions between the protruding octadecyl alkyl chain from a silanized glass surface and the extended undecyl alkyl side chain of the biotinylated thiophene polymer, B-PUHT.

Figure 4. Typical chemiluminescence spectra of streptavidinconjugated alkaline phosphatase immobilized on the inside walls of a glass capillary (0.4 mM CSPD, 10% enhancer): (O) enzyme conjugate interfaced with B-PUHT and (b) control-enzyme conjugate reacted with nonbiotinylated polymer. Figure 2. Schematic of the synthesis of B-PUHT. (A) Synthesis of the copolymer of 3-undecylthiophene and 3-thiophenecarboxaldehyde (PUAT) and (B) attachment of 6-biotinamidohexanohydrazide through Schiff’s base formation.

Signal Detection and Data Calibration. A photomultiplier tube (PMT, Hamamatsu R 943-02), a photon counter (Stanford Research Systems Inc., SR 400 2 channel-gated photon counter), and a microcomputer were used to collect and process the data. Figure 1 is a schematic of this simple experimental setup. The capillary was placed in front of the PMT glass window in a dark room. A photon counter connected to the PMT via an amplifier was placed in an anteroom. A personal computer hooked up to the photon counter was used to acquire and process the data. A typical experiment involved obtaining a real time curve of total photon counts per second versus time, as the photon counter was set up to count photons detected during 1 s intervals. Thus, enzymatic activity was the rate of chemiluminescence signal generation, which was the slope of the initial linear portion of the photon counts versus time plot. The final counts were corrected for background noise level. For kinetic studies, the raw data (real time curve) had to be integrated with time to generate a counts versus time plot. Initial rates of the reaction were then com-

puted from the slopes, calculated on the basis of initial data points collected. The signal profiles were highly reproducible. We observed minor batch variation effects with respect to the conjugate activity, and the data were normalized to account for these variations. RESULTS AND DISCUSSION

The schematic for the synthesis of the conjugated polymer B-PUHT is shown in Figure 2 and involved two steps: (A) the synthesis of the copolymer of 3-undecylthiophene and 3-thiophenecarboxaldehyde (PUAT) and (B) subsequent attachment of 6-biotinamidohexanohydrazone through Schiff base formation. The presence of the biotinamidohexanohydrazide moiety was confirmed from FTIR results (data not shown). The Schiff’s base formation was confirmed from the peak at 1627 cm-1, the peak being characteristic of CdN stretching vibrations. Figure 3 is a schematic of interactive forces involved in the creation of the molecular assembly. B-PUHT was physisorbed onto the silanized glass capillary through hydrophobic interactions between the alkyl chain of the silane compound and the undecyl alkyl side chain of the polymer. Streptavidin-conjugated alkaline phosphatase was immobilized onto the polymer matrix via biotinstreptavidin interactions.

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Figure 6. Lineweaver-Burk plot for paraoxon-mediated inhibition of alkaline phosphatase: (O) [I] ) 0 mM, (4) [I] ) 0.18 mM, and (b) [I] ) 0.36 mM. Figure 5. Effect of temperature on the conjugate binding step. The effect is also shown for the control-enzyme conjugate reacted with the nonbiotinylated polymer. Table 1. Comparative Study of the Complete Molecular Assembly and Different Control Assemblies controls dark counts unsilanized capillaries silanized capillaries (no polymer) enzyme complexed with free biotin absence of the conjugated enzyme absence of the substrate nonbiotinylated polymer experimental molecular assembly

slope, arbitrary units 0.0 0.01 0.06 0.10 0.06 0.05 0.15 4.0

Figure 4 illustrates a set of chemiluminescence data obtained with 100 µL of 0.4 mM CSPD drawn into the glass capillary containing the immobilized enzyme. Typically, the signal increased steadily for 10 min and leveled off over a period of at least 50 min, as shown in the inset. The control consisted of silanized glass capillaries coated with PUAT (polythiophene copolymer lacking the biotin group of the side chain), and the resultant chemiluminescence signal observed was at background level as shown in the figure. Additional controls, shown in Table 1, were performed, including unsilanized capillaries, the absence of the enzyme conjugate, and the absence of CSPD. As is evident, the controls showed only background intensity levels. These results implied that the assembly successfully formed via specific biotin-streptavidin interactions and hydrophobic surface interactions with the polymer and retained enzymatic function. The dependence of the conjugate binding step on temperature was investigated. Figure 5 represents the relative reaction rates computed from the generated chemiluminescence profiles of enzyme-immobilized capillaries, with the enzyme immobilization step being facilitated at the temperatures shown. The reaction rate at 45 °C was almost 3 times that observed at 25 °C. It is apparent from the figure that enzyme immobilization was maximized at 45 °C. This could be due to an increase in temperature, enhancing binding of the streptavidin conjugate by virtue of increased mobility of the biotinylated copolymer chain segments and the streptavidin binding site. This result led us to routinely process our conjugate binding step at 45 °C. In an attempt to determine the amount of enzyme bound in the molecular assembly on the capillary surface, this activity was compared to known solution concentrations of the enzyme. First, the enzyme conjugate was dissolved in assay buffer at various concentrations. With

Table 2. Relative Activity of Immobilized Alkaline Phosphatase as a Function of Storage Time at 4 °C days elapsed (after enzyme immobilization)

slope (normalized), arbitrary units

0 5 10 17 24 30

1.0 0.80 0.86 0.78 0.83 0.76

a 0.4 mM CSPD concentration, the reaction rates were computed for each enzyme concentration in the buffer. Next, 0.4 mM CSPD was drawn into the capillary containing the immobilized enzyme. The reaction rates were computed, and the enzyme concentration was determined from the solution calibration curve. This indicated the presence of 10-15 mol of the conjugate. Thus, the number of molecules immobilized was approximately 109. Monolayer coverage involving the available surface area of the capillary and the size of the biomolecule [110 Å × 70 Å cross-section dimensions (16)] indicated approximately 1% surface coverage. We also estimated that 10% of the available enzyme was immobilized. The molecular assembly is mechanically robust and unaffected by vigorous washing. The immobilized enzyme retains up to 80% of its activity for a period of 30 days, when stored in 1X PBS at pH 7.2 at 4 °C as shown in Table 2. As mentioned earlier, OP-based pesticides act as inhibitors during the process of alkaline phosphatasecatalyzed dephosphorylation of CSPD. Therefore, the kinetics of the immobilized enzyme in the molecular assembly were studied by a series of substrate dependent studies with the CSPD concentration varied from 0.05 to 0.6 mM, at a constant enzyme concentration. A plot of the reaction velocity, [V], as a function of the substrate concentration, [S], showed a classical [S] versus [V] dependence of an enzyme obeying Michaelis-Menten kinetics. A Lineweaver-Burk plot was generated for this set of data (Figure 6). The Km for the immobilized enzyme was 0.58 mM-1. This was close to the Km measured for the free solution enzyme, 0.81 mM-1 (18). An investigation of paraoxon-mediated inhibition kinetics of capillary-immobilized alkaline phosphatase was carried out by varying the CSPD concentration while keeping the inhibitor (paraoxon) concentration at a constant value of either 0.36 or 0.18 mM. It can be observed in Figure 6 that the linear fits to these data do not intersect either on the x axis or on the y axis as would be the case for noncompetitive or competitive inhibition, respectively. This behavior, indicative of a mixed type of inhibition,

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Technical Notes

concentrations. This system is capable of detecting 700 ppb methyl parathion. Similar calibration curves for paraoxon and methyl parathion can be accounted for, presumably due to the structural similarity of these inhibitors. We are currently investigating methods for enhancing the signal to noise ratio in the immobilized alkaline phosphatase assembly. Use of a conjugate with a higher specific activity and the utilization of a new enhancer (Sapphire-II), which affords a 3-fold signal amplification (19), are being pursued. Studies to investigate potential electrochemical signal transduction capabilities of the biotinylated thiophene copolymers and to create a more rugged molecular assembly are in progress. The biotinylated thiophene copolymer immobilization strategy holds promise for the development of a rapid and reasonably sensitive biosensor for OP-based pesticides. CONCLUSIONS Figure 7. Calibration curve for paraoxon (0.4 mM CSPD, 10% enhancer). The inset shows the effect at higher paraoxon concentrations and the structure of the inhibitor.

A new methodology for the immobilization of biomolecules has been described. This generic approach was used to create a bioconjugate assembly for the detection of organophosphorus-based pesticides, paraoxon and methyl parathion. The enzyme alkaline phosphatase (conjugated with streptavidin in this case) was the sensing element in the detection of OP-based pesticides. The enzyme kinetics were studied and were in agreement with our previous demonstration of a mixed type of inhibition. In a simplified optical setup, 500 ppb paraoxon and 700 ppb methyl parathion were detectable and the molecular assembly was found to be reasonably stable and reusable. We have established the prototype of a conjugate alkaline phosphatase-based biosensor for organophosphorus compound detection in this study. ACKNOWLEDGMENT

Support from the ARO Grant DAAL 03-91-G-0064 and from the Joint U.S. Army Natick/USDA Advanced Materials Program Contract DAAK60-93-K-0012 is gratefully acknowledged. LITERATURE CITED Figure 8. Calibration curve for methyl parathion (0.4 mM CSPD, 10% enhancer). The inset shows the profile for higher inhibitor concentrations and the structure of methyl parathion.

was similar to that observed and reported in solutionbased studies of alkaline phosphatase (18). Figure 7 demonstrated the extent of inhibition of the chemiluminescence signal in the presence of different paraoxon concentrations at fixed enzyme (10-15 mol) and substrate (0.4 mM CSPD) concentrations. The inset illustrated the chemiluminescence profile at higher paraoxon concentrations. The nonlinear behavior seen in the inset was commonly observed and may be attributed to high inhibitor concentrations altering the mechanism of inhibition. This system is capable of detecting 500 ppb paraoxon. Previously, we reported a 1.2 ppm detection limit utilizing a similar molecular assembly (17), but the conjugate binding step was performed at 25 °C. The improvement in the sensitivity of detection reported in this study is attributed to the higher levels of bound enzyme resulting from the change in the incubation temperature of the conjugate binding step to 45 °C. Figure 8 illustrates the extent of chemiluminescence signal inhibition for another organophosphorus pesticide, methyl parathion. The inset illustrates an increased chemiluminescence inhibition at higher methyl parathion

(1) Marx, K. A., Samuelson, L. A., Kamath, M., Sengupta, S., Kaplan, D. L., Kumar, J., and Tripathy, S. K. (1994) Intelligent Biomaterials Based on LB Monolayer Films. In Molecular and Biomolecular Electronics (R. R. Birge, Ed.) pp 395-412, Advances in Chemistry Series 240, American Chemical Society Books, Washington, DC. (2) Chaiet, L., and Wolf, F. J. (1964) The properties of Streptavidin, a biotin binding protein produced by Streptomycetes. Arch. Biochem. Biophys. 106, 1. (3) Green, N. M. (1975) Adv. Protein Chem. 29, 85. (4) Langer, P. R., Waldrop, A. A., and Ward, D. C. (1981) Enzymatic synthesis of biotin-labeled polynucleotides; Novel nucleic acid affinity probes. Proc. Natl. Acad. Sci. U.S.A. 78, 6633. (5) Wilchek, M., and Bayer, E. A. (1988) The Avidin-Biotin Complex in Bioanalytical Applications. Anal. Biochem. 171, 1. (6) Samuelson, L. A., Miller, P., Galotti, D. M., Marx, K. A., Kumar, J., Tripathy, S. K., and Kaplan, D. L. (1991) Oriented Fluorescent Streptavidin Conjugated Phycoerythrin Protein on Biotinylated Lipid L-B Monolayer Films. In Proteins Structure, Dynamics and Design (V. Renugopalakrishnan, P. R. Carey, I. C. P. Smith, S. G. Huang, and A. C. Storer, Eds.) pp 160-164, Escom Science Publishers BV, Leiden, The Netherlands. (7) Samuelson, L. A., Kaplan, D. L., Lim, J. O., Kamath, M. N., Marx, K. A., and Tripathy, S. K. (1994) Molecular

164 Bioconjugate Chem., Vol. 7, No. 1, 1996 Recognition Between a Biotinylated Polythiophene Copolymer and Phycoerythrin Utilizing the Biotin-Steptavidin Interaction. Thin Solid Films 242, 796. (8) Burroughs, J. H., Jones, C. A., and Friend, R. H. (1988) New Semiconductor device physics in polymer diodes and transistors. Nature 335, 137. (9) Naarman, H. (1990) The Development of Electrically Conducting Polymers. Adv. Mater. 2, 8, 345. (10) Elsenbaumer, R. L., Jen, K. Y., Miller, G. G., and Shacklette, L. W. (1987) Processable Environmentally stable, Highly Conductive forms of Polythiophene. Synth. Met. 18, 277. (11) Kamath, M., Lim, J. O., Chittibabu, K. G., Sarma, R., Samuelson, L. A., Akkara, J. A., Kaplan, D. L., Marx, K. A., Kumar, J., and Tripathy, S. K. (1993) Biotinylated Poly(3-Hexylthiophene-co-3-Methanolthiophene): A Langmuir Monolayer-Forming Copolymer. J. M. S. Pure Appl. Chem. A 30 (8), 493. (12) Green, N. M., Konieczny, L., Toms, E. J., and Valentine, R. C. (1971) The use of biotinyl compounds to determine the arrangement of subunits in avidin. Biochem. J. 125, 781. (13) Martin, C., Bresnick, L., Juo, R. R., Voyta, J. C., and Bronstein, I. (1991) Improved Chemiluminescent DNA Se-

Pande et al. quencing. BioTechniques 11, 1, 110. (14) Sugimoto, R., Takeda, S., Gu, H. B., and Yoshino, K. (1986) Preparation of soluble polythiophene derivatives transition metal halides as catalysts and their property. Chem. Express 1, 635. (15) The Merck Index (1989) p 1112, Merck & Co. Ltd., Rahway, NJ. (16) Coleman, J. E. (1992) Structure and Mechanism of Alkaline Phosphatase. Annu. Rev. Biophys. Biomol. Struct., 441. (17) Ayyagari, M. S., Pande, R., Kamtekar, S., Marx, K. A., Kumar, J., Tripathy, S. K., Akkara, J. A., and Kaplan, D. L. (1995) Molecular Assembly of Proteins and Conjugated Polymers: Toward Development of Biosensors. Biotechnol. Bioeng. 45, 116. (18) Ayyagari, M. S., Kamtekar, S., Pande, R., Marx, K. A., Kumar, J., Tripathy, S. K., Akkara, J. A., and Kaplan, D. L. (1995) Biotechnol. Prog 11, 699. (19) Tropix catalog (1994) p 8, Tropix Inc., Bedford MA (Sapphire, Sapphire-II, Emerald, Emerald-II, Nitro BlocLuminescence Enhancers.

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