Synthesis and Characterization of a Biotin-Alginate Conjugate and

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Synthesis and Characterization of a Biotin-Alginate Conjugate and Its Application in a Biosensor Construction† Boris Polyak, Shimona Geresh, and Robert S. Marks* The Institute for Applied Biosciences and Department of Biotechnology Engineering, Ben-Gurion University of the Negev, P.O. Box 653, Beer Sheva 84105, Israel Received November 5, 2003

Biotin was covalently coupled with alginate in an aqueous-phase reaction by means of carbodiimide-mediated activation chemistry to provide a biotin-alginate conjugate for subsequent use in biosensor applications. The synthetic procedure was optimized with respect to pH of the reaction medium (pH 6.0), the degree of uronic acid activation (20%), and the order of addition of the reagents. The biotin-alginate conjugate was characterized by titration with 2-anilinonaphthalene-6-sulfonic acid (2,6-ANS), 4-hydroxyazobene-2′carboxylic acid (HABA) and by an HPSEC-MALLS analytical method as well as by FTIR and 13C NMR spectroscopy. As a compromise between the need for a high percent of molar modification of the alginate, on one hand, and sufficient gelling capability, on the other hand, an optimal modification of 10-13% of biotin-alginate was used. The new biotin-alginate conjugate was used for the encapsulation of bioluminescent reporter cells into microspheres. A biosensor was prepared by conjugating these biotinylated alginate microspheres to the surface of a streptavidin-coated optical fiber, and the performance of the biosensor was demonstrated in the determination of the antibiotic, mitomycin C as a model toxin. Introduction Alginates are unbranched polysaccharides produced by marine brown algae and by some bacteria. They consist of 1,4-linked β-D-mannuronic (M) and R-L-guluronic (G) acid residues in different sequences and proportions.1 The physical properties of alginates depend on the sequence of M and G residues as well as on the average molecular weights and the molecular weight distribution of the polymer.2 In the presence of divalent ions, such as calcium, alginates form gels spontaneously in a single-step process. The technical success of using alginates for entrapment and encapsulation may be attributed to the gentle environment provided by the gels for the entrapped material as well as the high porosity provided by the open lattice structure of the gel. Chemical modifications of alginates for different purposes have recently been the focus of a number of studies. In particular, the modification of carboxylic groups has been used for the following purposes: (1) to couple the alginate to a short peptide (GRGDY) with the aim of creating an adhesive hydrogel substrate for cultivation of anchoragedependent mammalian cells (myoblasts) and for the expression of a differentiated phenotype;3 (2) to couple galactose moieties, such as ASGP-R ligands, to the alginate to improve anchorage and the interaction of hepatocytes with the alginate, with the aim of enhancing the functions of the encapsulated hepatocytes in three-dimensional culture;4 and (3) to covalently cross-link alginate chains with poly(ethylene glycol) diamines and to study the mechanical properties of † This work was performed in partial fulfillment of the requirements for the Ph.D. degree of B. P. * To whom correspondence should be addressed. Phone: (972) 8 6477182. Fax: (972) 8 647-2857. E-mail: [email protected].

newly modified alginates.5 Alginates have also been used in the preparation of model cells or tissue-specific drug delivery systems6 in which lectins were attached by means of an avidin-biotin interaction to the surface of spermine alginate microcapsules. In the first step of the latter twostep process, avidin was covalently bound to the capsule surface via carbodiimide chemistry. In the second step, avidin-coated capsules were bound to different biotinylated lectins. In the present work, we linked biotin to the carboxylic residues of alginate to provide the alginate with a new conjugation property. Coupling of biotin to the alginate was achieved by using aqueous-phase carbodiimide activation chemistry, followed by the binding of biotin hydrazide. We have previously reported the use of genetically engineered bioluminescent bacteria immobilized within an alginate matrix on an optical fiber as a biosensor for the detection of genotoxicants in water.7,8 In that type of biosensor, physical adsorption was used to entrap reporter cells into cross-linked calcium alginate by forming adlayers of the bacterial-polymer matrix on the surface of the optical fiber. The use of biotin-alginate will enable us to entrap reporter cells in a spherical geometry and to couple those microspheres onto the optical transducer surface. The microsphere-based method has a variety of advantages over the previously reported layered configuration: well-defined geometrical shape and uniformity of the beads; simplicity of preparation; improvement of diffusion properties; ability to store beads separately from the optical fibers; and, finally, the possibility of placing a mixture of microspheres containing different biospecificities onto the same fiber, thus facilitating the creation of nonspecific probes with a triggering system sensitive to a wider range of toxicants. The

10.1021/bm034454a CCC: $27.50 © 2004 American Chemical Society Published on Web 01/07/2004

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biotinylated alginate microspheres may be coupled to the transducer surface with the aid of the widely used avidinbiotin affinity system. The avidin-biotin interaction is an extremely specific and strong noncovalent binding method (association constants, Ka, of the glycoprotein avidin for biotin in solution or immobilized on the surface are ∼1015 or 1010 mol-1 L, respectively).9 The bond between biotin and avidin is established very rapidly and, once formed, is essentially undisturbed by extreme pH values or exposure to organic solvents or other denaturating agents.10 In this study, avidin/streptavidin-coated optical fibers were shown to successfully bind biotin-coated calcium alginate microspheres. The covalent attachment of streptavidin to the silica surface was carried out by means of previously described silanization methods.11,12 In the current paper, we present a new system for use in the construction of optical fiber sensors. This new immobilization material combines the advantages of the alginate’s gelling properties to entrap cells, providing them with a gentle hydrated and highly porous environment, and the high affinity interaction of the avidin/ streptavidin-biotin system. Experimental Section Materials. Chemicals obtained from commercial sources were of analytical grade and were used without further purification. Sodium alginate Manugel DMB (from Laminaria hyperborea, 70% G content, average molecular weight of 180 kDa) was supplied by Kelco International (U.S.A.). Biotin hydrazide (B-7639), 1-ethyl-[3-(dimethylamino)propyl]-3-ethylcarbodiimide HCl (EDAC; E-1769), 2-[N-morpholino]ethanesulfonic acid (MES) buffer (M-8250), phosphate-buffered saline (PBS) (P-3813), avidin affinity-purified (A-9275), 3-aminopropyltrimethoxysilane, 97% (28,177-8), glutaraldehyde, 50% aqueous solution (G-7651), calcium chloride (C-5426), and sodium hydrogen carbonate (23,6527) were purchased from Sigma and Aldrich Co. (U.S.A.). d-Biotin (29129), N-hydroxysulfosuccinimide (NHSS; 24510), and 4′-hydroxyazobenzene-2-carboxylic acid (HABA; 28010), streptavidin affinity-purified (21125) were acquired from Pierce (Rockford, U.S.A.). 2-anilinonaphthalene-6-sulfonic acid (2,6-ANS) was obtained from Molecular Probes (U.S.A.). Preparation of Biotin-Conjugated Alginate. The biotinalginate conjugate was prepared as follows: an amount of 0.052 g (0.2 mmol) of biotin hydrazide was added to a 1% (w/v) solution of alginate (20 mL solution, 1.00 mmol alginate monomer) in 0.1 M MES buffer, pH 6.0. The reaction mixture was stirred at room temperature for 60 min to facilitate a homogeneous dispersion of the biotinylating reagent in the reaction solution. Then, 0.0216 g (0.1 mmol) of NHSS and 0.0384 g (0.2 mmol) of EDAC were added (ratios of reagents were calculated for a theoretical 20% molar modification of the number of carboxylic groups of alginate). After 3 h at room temperature, the resulting polymer was dialyzed against doubly deionized water using a 10,000 MWCO membrane (66410, Rockford, U.S.A.). The water was changed twice a day for 3 days, after which time the modified alginate was lyophilized. Spectroscopic Analysis. For FTIR spectroscopy, polymer samples were prepared as thin films as follows: 4 mg/mL

Polyak et al.

of the modified alginate was dissolved in doubly deionized water. The resulting solution was poured into a polystyrene Petri dish and dried in an oven at 50 °C for 24 h to produce a thin transparent polymer film.13 Infrared measurements were performed in transmission mode on a Bruker Equinox 55 infrared spectrometer. The FTIR spectra were averaged over 128 scans at a resolution of 4 cm-1. For 13C NMR spectroscopy, samples of modified alginate were dissolved in D2O. 13C NMR measurements were performed on a Bruker Advance DNX instrument (500 MHz) utilizing standard Pals programs. Quantitative Assay of the Extent of Biotinylation of the Alginate. The fluorescence-based assay was adapted to a 96-well microtitration plate format. Volumes of 1 µL of a 2,6-ANS (6 mg/mL in DMSO) and 66 µL of avidin (2 mg/ mL) were added to different volumes of biotin (0.1 mM) from 0 to 120 µL (in increments of 10 µL) in the wells of a microtitration plate. Each well was brought up to a total volume of 200 µL with PBS, pH 6.0. Biotin-alginate samples (1 mg/mL) in volumes from 2 to 20 µL (in increments of 2 µL) were then assayed to determine the amounts of biotin on the alginate available for complexing with avidin. The fluorescence was monitored at 320 nm (excitation) and 405 nm (emission) by a POLARStar Galaxy fluorimeter (BMGlabtechnologies GmbH, Germany). All solutions, except 2,6ANS, were prepared in PBS at pH 6.0. When the absorbance-based HABA titration method was used for quantification of the extent of biotinylation of the alginate, precipitates formed, probably due to the formation of a conjugate-avidin-conjugate cross-linked network. In this assay, a 20-µL sample of 10 mM HABA in 10 mM NaOH was added to 0.4 mL of 0.5 mg/mL avidin solution in 50 mM phosphate buffer, pH 6.0, and NaCl, 0.9% (w/v). Biotinalginate or nonmodified alginate at a concentration of 1 mg/ mL in doubly deionized water was then added to the HABAavidin mixture. The mixture was kept at 4 °C overnight to allow the final precipitate to form. Viscosity Measurements. The viscosity of various biotinalginate and nonmodified alginate solutions [1.5-3.5% (w/v) in doubly deionized water] was determined using a Carrimed CLS 50 controlled-stress rheometer (TA Instruments, UK). The measurements were performed using coneplate geometry (4 cm/4°). HPSEC-MALLS Analysis. Molecular weight distributions of the polymers were determined with a multi-angle laser light scattering (MALLS) photometer (DAWN DSP, Wyatt Technology Inc., Santa Barbara, U.S.A.), fitted with a K5 flow cell and a He-Ne laser (633 nm). Polymer samples, 3 mg/L, were prepared in a buffer containing 0.02% (w/v) sodium azide and 0.1 M sodium nitrate in 10 mM imidazol solution at pH 7.0. Fractional separations were performed on 100-, 300-, and 1000-Å PSS Suprema separation columns (Polymer Standard Service, Germany). The mobile phase was delivered at the ambient temperature at a nominal flow rate of 0.7 mL/min. MALLS and differential refractive index (DRI) detectors were calibrated with filtered HPLC-grade toluene and NaCl solutions, respectively. The MALLS instrument was normalized using standard pululan P-23 (isotropic light scattering). The dn/dc value (specific

Synthesis and Characterization of a Biotin-Alginate

refractive index increment) for the studied polymers was estimated at 0.155 [mL/g], according to the value reported in the literature for sodium alginate in the presence of NaCl.14 Strain Description. The organism chosen for the demonstration of the feasibility of the proposed system was Escherichia coli strain DPD1718.15,16 This organism contains a chromosomally integrated fusion of the E. coli recA promoter region to the Photorhabdus luminescens lux CDABE reporter operon. These five promotorless structural genes are responsible for the heterodimeric luciferase units (lux A and B) and the synthesis of the luciferase substrate, tetradecanal, by an ATP- and NADPH-dependent multienzyme complex composed of fatty acid reductase, transferase, and a synthetase (lux C, D and E).17 Media and Growth Conditions. Prior to immobilization, the transformant cells were grown with shaking overnight in Luria-Bertani (LB) medium18 in a rotary thermoshaker (Gerhardt, Germany) at 140 rpm. The cultures were maintained at 37 °C, in the presence of chloramphenicol (30 mg/ l) for positive selection of the lux transposon. Cultures were then diluted to approximately 107 cells/mL and regrown under the same conditions, but without chloramphenicol, to an early exponential growth phase of a measured optical density of 50 Klett units (Klett-Summerson colorimeter, 800-3, U.S.A.), which was determined to be about 1.52.0 × 108 cells per milliliter (OD660 of 0.37-0.38). Encapsulation of Bioreporter Cells into Biotin-Alginate Microspheres. Biotin-alginate beads we prepared as described below using the gas shear method, which employs a concentric air stream to shear the drop from the needle tip controlled essentially by the gas flow.19-22 The harvested cells were mixed 1:1.6 with a 4% (w/v) biotin-alginate solution. Biotin-alginate at a final concentration of 2.5% (w/v) containing reporter cells was loaded into a 5-mL Becton Dickinson (U.S.A.) plastic syringe. The syringe was then attached to a syringe pump (KD Scientific model 100, U.S.A.). Beads 900-1000 µm in diameter were prepared by using a 0.61-mm outside diameter needle at a flow rate of 20 mL/h. The biotin-alginate beads so produced were allowed to stand in a 0.1 M calcium chloride solution for 20-30 min for further hardening. Optical Fiber Coating with Streptavidin and Microsphere Conjugation Multimode optical fibers (PUV 1000 BN, CeramOptec GmbH, Germany), 1000 µm in diameter, were used for microsphere conjugation experiments. The black nylon jackets of the fibers were stripped away, leaving a 1-cm long optical fiber tip, which was then used for the conjugation of biotin-alginate beads. The stripped ends of the optical fibers were cleaned successively with ethanol and piranha solution (H2SO4:H2O2, 4:1 (v/v)). Cleaned fibers were silanized for 2 h in a vapor atmosphere of 3-aminopropyltrimethoxysilane at 80 °C in a vacuum oven (Vacucell 22, MMM Medcenter GmbH, Germany) at a pressure of 50 mbars. The amine-conjugated fibers were washed three times with 95% (v/v) ethanol to remove the excess un-reacted aminosilane and then washed once with doubly deionized water. Thereafter, the fibers were immersed for 30 min at 4 °C in a 2.5% (v/v) glutaraldehyde solution prepared in a 0.2 M bicarbonate buffer (pH 9.7). Excess glutaraldehyde

Biomacromolecules, Vol. 5, No. 2, 2004 391

was rinsed off with doubly deionized water, and the fibers were immersed for 30 min in a PBS-buffered streptavidin solution at a concentration of 2 mg/mL.23 After incubation with streptavidin, fibers were washed gently with doubly deionized water to remove any remaining phosphates from the fiber surface (phosphates are chelators of calcium ions, and their presence can lead to the disruption of the alginate gel matrix). Finally, streptavidin coated fibers were brought into physical contact with biotin-alginate beads (previously washed with doubly deionized water to remove any remaining calcium chloride), and the beads were conjugated to the optic fiber solid surface. Measurement Procedure and Data Analysis. The optical fiber with its biological probe was placed into a 0.5-mL sample tube (Jonplast, Italy) containing the analyte, and bioluminescence readings were integrated for one second. The far end of the optical fiber was connected to a Hamamatsu HC135-01 PMT Sensor Module. To receive and treat data, a specific driver was developed using LabWindows/CVI (version 6.0), which allowed the combined monitoring of the bioluminescent signal and data handling in real time. Results and Discussion Optimization of Biotin Incorporation. Biotin-alginate was prepared in an aqueous phase via carbodiimide chemistry according to the scheme shown in Figure 1. The aqueousphase carbodiimide chemistry approach uses a water-soluble carbodiimide (EDAC) that catalyzes the formation of amide bonds between carboxylic acids and amines by activating the carboxylate to form an O-acylisourea intermediate.24,25 This intermediate is unstable in aqueous solution and undergoes fast hydrolysis. For the procedure to be successful, the active form of alginate carboxyls would have to be more stable than the O-acylisourea derivative. Such a condition could be fulfilled by the so-called “active esters”, such as N-hydroxysuccinimidyl esters (NHS) developed for peptide synthesis,26 or their sulfonated derivatives.27 NHS and sulfoNHS active esters have previously been used for the covalent attachment of small molecules to carboxyl groups of proteins28-33 or to carboxylic groups on gold surfaces.34 The chemistry for biotin conjugation to alginate was optimized with respect to various parameters. The crucial parameter for the biotin coupling reaction was activation of the carboxylic group. The amount of EDAC present in the reaction dramatically affected the efficiency of biotin incorporation. A common side reaction that occurs when EDAC is used in large amounts is the internal rearrangement of the O-acylurea activated ester of EDAC to a N-acylurea. The latter compound becomes irreversibly incorporated onto the substrate, in this case the alginate polymer backbone.35 Thus, the EDAC content was varied in the optimization experiments as a percentage of the available carboxylic acid groups on the alginate. Biotin incorporation increased with increasing carbodiimide concentration and reached a maximum at 40% of the theoretical molar ratio of uronic acids (Figure 2). To minimize side reactions associated with carbodiimide chemistry, 20% uronic acid activation was chosen in the

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Figure 1. Biotin coupling to alginate via carbodiimide chemistry.

Figure 2. Variation of the theoretical uronic acid activation by altering the concentration of EDAC/NHSS added to the reaction medium.

Figure 3. Biotin incorporation into alginate at different constant pH values for the activation and coupling stages (20% uronic acid activation).

subsequent optimization experiments. This level of activation offered relatively good reaction efficiencies (∼60%) with minimal N-acylurea rearrangement. An additional parameter that affects the reaction efficiency is the pH of the reaction medium. The EDAC activation step is most effective at mild acidic pH (i.e., pH 4.5).36 The second step of biotin attachment is more effective at higher pH values. Higher pH values are required to prevent protonation of amine (hydrazide), as required in the reaction with the NHSS esters.33,34 We examined two approaches for the optimization of the pH; in the first approach, the pH was kept constant for both the activation and the coupling stages, and in the second approach, the optimal pH range was sought for each stage. With the first approach, an optimal pH of 6.0 was found to offer the highest biotin incorporation, with a reaction efficiency of approximately 75% (Figure 3). This pH facilitated a balance in the reactivities between the carbodiimide and the biotin hydrazide. A decrease in the efficiency of biotin incorporation at higher pH values was also related to an increase in the hydrolysis of the NHS ester intermediate. NHS esters are most stable in solution between pH 5 and 6.33 In the second approach of modulating the pH in each stage, lower incorporation efficiencies for all pH intervals were obtained. The finding could be a consequence of the

hydrolysis of the NHS ester intermediate as a result of local pH changes during the gradual pH increase (by addition of 0.1 M NaOH). It was also observed that the order of introduction of the reagents was important. Because the solubility of biotin hydrazide in water is rather low (10 mg/mL),37 much better incorporation of biotin was observed when biotin hydrazide was dispersed in the reaction medium before the addition of EDAC and NHSS. Control reactions without the addition of EDAC for activation were run for each experiment. After dialysis, less than 1.5% of biotin was detected, suggesting that the incorporation of biotin is not a result of nonspecific interactions or physical entrapment of biotin hydrazide by the polysaccharide. Spectroscopic Characterization of Biotin Alginate. The biotin-alginate product was characterized by FTIR and 13C NMR spectroscopy. The FTIR spectra of biotin-alginate and nonmodified sodium alginate are shown in Figure 4. The characteristic features of the biotin-alginate spectrum are a strong sharp peak at 1666 cm-1 (amide I band, CdO stretching vibration), with a medium shoulder at 1565 cm-1 (amide II band, N-H bending vibration), and a mediumsharp peak at 1240 cm-1 (amide II band, interaction between


Figure 4. FTIR spectra of (a) biotin-alginate, (b) original sodium alginate, and (c) subtraction spectrum of (a) and (b). Polymer samples were prepared as thin transparent films.13

the N-H bending and C-N stretching vibrations).38 The subtraction spectrum of sodium alginate and biotin-alginate (Figure 4c) clearly shows the amide II band at 1560 cm-1. The degree of biotinylation was correlated with the appearance of the amide I band. The 13C NMR spectrum confirmed the conjugation of biotin to alginate via the formation of an amide bond. In contrast to the chemical shift of the carboxyl group at 175.5 ppm present in the nonmodified alginate, the biotin-alginate product showed two new chemical shifts, that of 1-amide carbonyl (CONH) at 174 ppm and that of a carbonyl on the ureido ring of biotin (CO(NH)2) at 160.5 ppm. Quantification of the Biotin Content in the Modified Alginate. To quantify the degree of biotinylation, we initially tried to apply the HABA titration method,39 in which HABA binds to avidin to give an absorption maximum at 500 nm. When biotin or a biotinylated entity are added, biotin displaces HABA from the complex with avidin, and the absorbance at 500 nm decreases (the Kd for HABA-avidin complex is 6 × 10-6 at pH 4.7 vs that of approximately 10-15 M for the avidin-biotin complex).10 The decrease in absorption is then used to determine the extent of biotinylation. In our case, this method was not satisfactory, because precipitation occurred when biotin-alginate was added to the HABA-avidin complex. In qualitative terms, however, the HABA method confirmed that biotin was covalently coupled to alginate, a finding that enabled us to exclude nonspecific interactions or physical entrapment of the biotinylating reagent by the polysaccharide. The precipitation that occurred was probably due to two types of interactions: (1) sandwichtype cross-linking between the biotinylated polysaccharide chains through avidin bridges, which created a polymer network that was insoluble in the aqueous medium; and (2) electrostatic interactions between the negatively charged alginate and the positively charged avidin (IP ∼ 10) at pH 6.0 (reaction conditions). The degree of precipitation was greater for biotin-alginate than for the nonmodified alginate. Raising the pH from 6.0 to 11 resulted in the total dissolution of the slight precipitate obtained from the nonmodified alginate, but it almost did not affect the precipitate from the biotin alginate conjugate. In the former case, the dissolution


Figure 5. Demonstration of 4:1 stoichiometries in saturated complexes of biotin-alginate conjugates with avidin. Titrations of known concentrations of d-biotin against: (9) avidin with 99% functional purity; (2) biotin-alginate 13% modification; (b) biotin-alginate 10% modification. The later two curves are displaced by +1000 and +2000 fluorescence units, respectively, to improve the clarity of the plot.

of the precipitates at the higher pH was due to electrostatic repulsion between the now negatively charged avidin and the alginate macromolecules. For both the biotin-alginate and HABA-avidin complexes, the stability of precipitates was due to the strength of the avidin-biotin interaction that preserves the cross-linking between the alginate chains under basic conditions, i.e., at pH 11. Because the HABA method could not be used quantitatively, a fluorescence-based method was used to determine the available biotin content in the modified alginate. In the presence of avidin, the fluorescence of 2,6-ANS is blue shifted (from 463 to 422 nm) with a large increase in quantum yield. Biotin binding causes complete displacement of the bound fluorophore, with a concomitant quenching of the fluorescence.40,41 This method is both sensitive and accurate over a wide range of probe and avidin concentrations. The optimal molar ratio of avidin to 2,6-ANS was found to be 1:10, a value that depends on the instrument sensitivity. A 4-fold molar excess of 2,6-ANS over avidin was required to ensure that all of the biotin binding sites were indeed bound to 2,6-ANS. The biotin content in modified alginate was interpolated from the calibration curve plotted by using standard biotin solutions. Then, the biotin/ avidin ratio was obtained by dividing the previously calculated biotin content in the modified alginate by the known amount of avidin added to the sample. Biotin-alginate products were synthesized with 10-13% molar modification. Figure 5 shows titrations of avidin (99% functionally pure) against two biotin-alginate samples and titration of avidin against known solutions of d-biotin. In the latter case, we observed the sharp breakpoint that unequivocally corresponds to the saturation of avidin with exactly 4 biotin molecules per functional avidin tetramer (Figure 5). However, for the biotin-alginate conjugates, no sharp breakpoint was observed. In the breakpoint region, additions of biotin-alginate to the ANS-avidin complex had almost no influence on the fluorescence. The maximal molar biotin-alginate/avidin ratio was found to be about 3.7-3.8. These titration results are probably a consequence of the

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Polyak et al. Table 1. Viscosity of 13% Modified Biotin-Alginate and Original Alginate Measured at a Shear Rate of 100 s-1 viscosity (cp)a

polymer concentration (% w/v)

biotin-alginate

original alginate

1.5 2.0 2.5 3.0 3.5

68 139 252 414 731

126 278 482 822 1230

a Data represent the means of three independent experiments with maximal SD of 5%.

Figure 6. (A) Cumulative and (B) differential molecular weight distributions for the (O) nonmodified alginate and (9) biotin-alginate (13% of modification) as determined by SEC.

steric hindrance of alginate chains, which prevent free biotin residues on the alginate from replacing the remaining ANS molecules in the complex with avidin. As a result of this limitation, about 5-7% of biotin is not accessible for quantification at the stage of full saturation of all the avidin binding sites. Characterization of Molecular Parameters and Viscosity. Figure 6 shows the cumulative (A) and differential (B) molecular weight distributions for biotin-alginate (13% of molar modification) and for nonmodified alginate. The differential molar mass distribution shows how much material (differential weight fraction) is present in any molecular weight interval. The cumulative distribution gives, for each molar mass, the weight fractions of material having molar mass less than the given weight. Thus, the cumulative distribution approaches zero at low molecular weights and unity at high molecular weights. The cumulative distribution is thus particularly useful in determining which molecular weight fractions are contained in the high and low molecular tails of the sample. The biotin-alginate product shows a clear shift to higher molecular weights in both distribution presentations. This result implies that biotin is probably distributed homogeneously on the alginate backbone. The weight-average molecular weight (Mw) for biotin-alginate (13% modification) was measured as 307 kDa (Mw/Mn ) 1.82) by means of SEC. This value represents an increase from the initial Mw of alginate (Mw ) 180 kDa, Mw/Mn ) 1.42). The ratio Mw/Mn is a measure of the dispersivity of the polymer chains. The meaning of the narrow ratio of Mw/ Mn for biotin-alginate and alginate is that we have nearly mono dispersed polymers. The viscosity of the alginate solutions to be used in bead preparation is regarded as an important parameter in predicting the geometry of the beads.42 Uniform spherical beads, defined as populations with a standard deviation below 10%, could be obtained only with alginate preparations exhibiting viscosities greater than 125 cp. Our measurements of the viscosity of biotin-alginate solutions at various concentrations showed that the viscosities were lower than that of the original alginate solution (Table 1). This finding reflects the increase of steric hindrance between the polymer molecules

in the biotin-alginate solution, which reduces the shearing forces between the polymer chains. Biotin-alginate in the concentration range of 2.0-3.5% (w/v) gave microspheres of uniform geometry. An optimal concentration of biotinalginate of 2.5% (w/v) was used to demonstrate the conjugation of microspheres to the optical fiber surface. Higher concentrations of polymer solutions were too viscous for further use. Biotin-alginate (5-10% degree of modification according to the company specifications) has recently become commercially available (CarboMer Inc., Westborough, MA). The commercial product has already been used for the immobilization and conjugation of biotin-labeled glucose oxidase on the surface of Pt electrodes for the construction of an amperometric biosensor. Cosnier and co-workers reported that a biosensor based on a biotin-alginate immobilized enzyme and conjugated through the avidin-biotin bridges to the matrix gave better performance characteristics and stability over a longer time than the biosensor constructed with nonmodified alginate.43 We compared our biotinalginate product with the biotin-alginate from CarboMer. The degree of biotinylation for the commercial biotin-alginate was found to be about 2-3% of molar modification (using fluorescence competitive assay). This low biotin content on the alginate was not sufficient to bind microspheres prepared from this product to a solid surface. The viscosity of the commercial biotin-alginate (3.0% w/v) was 63 cp at 100 s-1 shear rate, whereas a viscosity of 414 cp was found for our biotin-alginate under same conditions and concentration. The low viscosity of commercial biotin-alginate requires a preparation of more concentrated solutions of this polymer so as to obtain a sufficient degree of cross-linking. The molecular parameters of CarboMer’s product were also determined. The Mw of the CarboMer biotin-alginate was about 210 kDa (Mw/Mn ) 5.1). The relatively high Mw/Mn ratio indicates a poly-dispersed polymer. The viscosity and polydispersivity of the CarboMer product, together with the low biotin content, are the main reasons why this material was not suitable for the production of our biosensor. Biosensor Construction. One of the potential applications of biotin-modified alginate is its use as an immobilization matrix for genetically engineered bioluminescent cells that respond to the presence of toxic substances by the production of light. These sensor cell elements can be entrapped in a biotin-alginate immobilization matrix in the microsphere geometry and then conjugated to the optical fiber that serves as a transducer of captured light. This conjugation approach



streptavidin, the nonglycosylated and hydrophobic bacterial analogue of the egg-white glycoprotein avidin, produced more satisfactory results in the conjugation process. Figure 8 shows the bioluminescent response of E. coli strain DPD1718 entrapped within biotin-alginate microspheres that had been conjugated to the optical fibers and exposed to various concentrations of mitomycin C. Previous studies have clearly demonstrated that in both liquid culture and an adlayer-alginate immobilized form this strain responds to DNA-damaging agents such as mitomycin C, nalidixic acid or UV radiation, by a dose-dependent increase in bioluminescence of blue-green radiation at 490 nm.8,44 In the present studies, mitomycin C was used as a representative model chemical in an experiment that demonstrated the performance of the constructed biosensor. The results presented in Figure 8 were obtained using the lone-bead configuration. An approximately 1 h lag phase was followed by a dose-dependent increase in bioluminescence. A calibration curve obtained at 180 min of incubation with the analyte showed very good linear behavior (see inset in Figure 8, R2 ) 0.99, slope ) 6.58 photon-counts s-1 µg-1 L, and intercept ) 120 photon-counts) in the range of mitomycin C concentrations from 800 to 100 µg L-1. Summary Figure 7. Biotin-alginate microspheres conjugated to an optical fiber via avidin-biotin affinity interactions: (a) attachment of a lone bead to the end face of the fiber and (b) coating of the fiber with a number of microspheres. Diameter of the optical fibers is in both cases 1000 µm.

is presented in Figure 7. We attached biotinylated alginate microspheres with previously entrapped reporter cells to the fiber in different configurations. As shown in Figure 7a, it was possible to attach a lone bead to the end face of the fiber or, alternatively, to coat the fiber with a number of microspheres (Figure 7b). Conjugation of beads was achieved using either avidin or streptavidin. However, the use of

The goal of this study was to prepare and characterize biotin-modified alginate as a new material that combines the advantages of the gelling capability of alginates (to entrap cells) with the high-affinity interaction of the avidin/ streptavidin-biotin system, with the ultimate aim of constructing optical fiber sensors. The biotinylation chemistry (based on the carbodiimide approach) was optimized with respect to pH of the reaction medium (pH 6.0), degree of uronic acid activation (20%), and order of addition of the reagents. The conjugation of biotin to alginate was confirmed by FTIR, 13C NMR, HABA titration, and HPSEC-MALLS

Figure 8. Bioluminescence response curves obtained from reporter cells encapsulated within biotin-alginate beads and conjugated to the optical fiber surface using the streptavidin/biotin affinity interaction. The main figure shows the response at a mitomycin C concentration range of 800-100 µg/L; the inset shows the linear range of the calibration curve based on the light signal measured at 180 min of incubation with the mitomycin C. (9) 800 µg/L, (b) 400 µg/L, (2) 200 µg/L, (1) 100 µg/L, ([) blank (water).

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analytical methods. The biotin-alginate conjugate was prepared at a degree of 10-13% of molar modification, as determined by means of a fluorescence competitive assay. A fluorescence-based analytical procedure for the unequivocal quantification of biotin residues on the alginate backbone was established. The attachment of biotin-modified alginate microspheres as conjugates of streptavidinylated optical fiber surfaces was confirmed experimentally. The applicability of the new conjugate was demonstrated in a new biosensor configuration by measuring the optical response of the encapsulated bioluminescent reporter cells onto optical fibers using biotin-alginate as an immobilization matrix. Acknowledgment. This work was funded by the Israel Ministry of Science and the Arts, infrastructure Grant No. 13139-1-98; Ben-Gurion University Infrastructure grant to the Institute for Applied Biosciences; and the Commission of the European Communities Research Directorate, MENDOS project Contract No QLRT-2001-02323. The authors thank Prof. Smadar Cohen, Ben-Gurion University of the Negev, Beer-Sheva, for the generous gift of sodium alginate and Dr. Mark Karpasas, Interdisciplinary Equipment Center, Institute for Applied Biosciences, Ben-Gurion University, Beer-Sheva, Israel for the help in performing viscosity and SEC measurements. The generous gift of strain DPD1718 by Mr. R. A. LaRossa and Mr. T. K. Van Dyk of DuPont, Wilmington, DE, U.S.A. and Prof. Shimshon Belkin, The Hebrew University of Jerusalem are gratefully acknowledged. Abbreviations 2,6-ANS, 2-anilinonaphthalene-6-sulfonic acid EDAC, 1-ethyl-[3-(dimethylamino)propyl]-3-ethylcarbodiimide HCl G, R-L-guluronic acid M, β-D-mannuronic acid HABA, 4′-hydroxyazobenzene-2-carboxylic acid MES, 2-[N-morpholino]ethanesulfonic acid PBS, phosphate-buffered saline NHSS, N-hydroxysulfosuccinimide RT, room temperature BA, biotin-alginate DMSO, dimethyl sulfoxide HPSEC, high-performance size exclusion chromatography MALLS, multi-angle laser light scattering

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Synthesis and Characterization of a Biotin-Alginate Conjugate and

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