ng C 27 (2007) 787–793www.elsevier.com/locate/msec

Materials Science and Engineeri

Immobilization of the enzyme β-lactamase by self-assembly on thin filmsof a poly(phenyleneethynylene) sequenced with flexible

segments containing sulfur atoms

Erika Vázquez a, Abdieel Esquivel Aguilar a,b, Ivana Moggio a,⁎, Eduardo Arias a, Jorge Romero a,Héctor Barrientos a, José Roman Torres a, Maria de la Luz Reyes Vega b

a Centro de Investigación en Química Aplicada (CIQA), Blvd. Enrique Reyna 140, 25253, Saltillo, Mexicob Facultad de Ciencias Químicas, Universidad Autónoma de Coahuila, Blvd. V. Carranza and Ing. J. Cardenas, 25000 Saltillo, Mexico

Received 20 April 2006; received in revised form 7 August 2006; accepted 27 August 2006Available online 18 October 2006

Abstract

Anovel poly(phenyleneethynylene) sequenced in themain conjugated chain with flexible groups containing sulfur atoms has been synthesized byHeck–Sonogashira coupling reaction. Layer-by-layer films of the polymer have been prepared with a linear growth in thickness up to four layers asevidenced by UV–Vis spectroscopy and perfilometry. On the top of these multilayers, the enzyme β-lactamase was deposited by self-assembly. Theenzymatic activity was measured by a modified spectrophotometric standard assay method for penicillin G, ampicillin and amoxicillin. A higher andfaster activity was obtained for penicillin G and thus preliminary study of the biosensor response by fluorescence was carried out for this antibioticrevealing a decrease in the polymer fluorescence as function of the penicillin G concentration.© 2006 Elsevier B.V. All rights reserved.

Keywords: Poly(phenyleneethynylene); β-Lactamase; Immobilization; Self-assembly; Atomic force microscopy; Biosensors

1. Introduction

Since the discovery of penicillin, β-lactamic antibiotics arethe most common drugs used in bacterial therapy. Theirefficiency consists in the possibility to inhibit an importantreaction involved in the formation of the cell wall [1]. Despite ofthe enormous quantity of antibiotics now available in the phar-maceutical industry, bacteria usually develop defense mechan-isms. One of these is producing special enzymes, β-lactamases,that can hydrolyze theβ-lactamic ring [2]. For this reason, it is ofincreasing interest to develop a method which permits theanalysis of new antibiotics and the efficiency of the attack ofthese enzymes to them. Biosensors emerged as a quick, simpleand low cost technology alternative to classical analytical me-thods [3]. Most of the commercial prototypes use enzymes aselement of molecular recognition (receptor) because of theirspecificity to defined analytes. As a main problem with enzymes

⁎ Corresponding author. Tel.: +52 844 438 98 30; fax: +52 844 4389839.E-mail address: imoggio@ciqa.mx (I. Moggio).

0928-4931/$ - see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.msec.2006.08.022

is their stability, they are usually immobilized on the detectionelement (transducer). Bibliography on biosensors in general hasgrown exponentially in the last years; specifically references onβ-lactamase immobilized biosensors mainly deal with twobiosensor categories. (1) Potentiometric biosensors. Since thefirst papers by Papariello et al. [4] and then by Caras and Janata[5], a great work has been done to immobilize β-lactamase onsilicon substrates or gates to obtain sensors based on semi-conductor devices such as Field-Effect Transistors [6–8]. (2)Optical biosensors have been also proposed based mainly in theuse of fluorescein [9–12]. The fluorescence of this molecule ispH sensitive and thus it can be exploited to detect the change inpH which occurs as a consequence of the enzymatic reaction.The immobilization in these cases is obtained through avidin–biotin coupling [13]. The covalent union between an enzyme andthe transducer surface is one of the most commonly used me-thods for immobilization together with adsorption, microencap-sulation, cross-linking and entrapment in a matrix [2]. Although,other immobilization techniques have been proposed based onsol–gel [14], plasma assisted polymerization [15], the use of

788 E. Vázquez et al. / Materials Science and Engineering C 27 (2007) 787–793

bilayer lipid membranes [16] or bacterial magnetic particles [14]and self-assembly [17]. The latter technique presents someadvantage with respect to the others, (1) it is simple and low cost;(2) it can permit simultaneous detection of various analytes byimmobilizing different enzymes in separated layers of the samedevice, (3) it allows the preparation of devices at nanoscale bythe deposition of nanofilms, (4) as it is induced by no covalentinteractions between specific parts of the enzyme and functionalgroups of the transducer, a defined spatial distribution of theenzyme on the surface can be usually obtained.

In a previous paper, we demonstrated the possibility toconstruct alternated multilayers by self-assembly of a glycolester sequenced poly(phenyleneethynylene) with poly(aniline)salt through electrostatic interactions between the positivecharge of the poly(aniline) salt and the partial negative chargeoriginating from the resonance of the carbonyl group of thepolymer [18]. On the basis of this previous work, we report herethe elaboration of films of a poly(phenyleneethynylene) bearingflexible sequences containing sulfur atoms (Fig. 1) and theimmobilization of the enzyme β-lactamase on them by self-assembly as well as the results of biosensoring to penicillin G byfluorescence. It is to be mentioned that phenyleneethynyleneoligomers and polymers (PEs) are a class of conjugated mate-rials with excellent photoluminescence properties and their usein chemical and biological sensors has been already reported[19–22]. In literature, the pPEs are substituted with carbohy-drate groups which work as receptors. Here we propose toconstruct the biosensor by immobilizing the recognizing ele-ment (an enzyme) on the transducer (the polymer films) by self-assembly. This strategy is aimed to (i) allow the possibility torecover the receptor after the assay, (ii) avoid difficult syntheticroutes to insert enzymes in a conjugated backbone and (iii) openthe way for the future to widen the range of enzymes which canbe immobilized on the pPE films by using protocols similar tothat here reported.

2. Experimental

Analytical grade reagents from Sigma, Fluka and JT Bakerwere used for the synthesis without further purification. Trie-thylamine and THF were dried on KOH and sodium ben-zophenone complex, respectively and distilled before use. β-Lactamase (Type II from Bacillus cereus), antibiotics andstandard kits for enzymatic activity tests were from SigmaChemical Company. 1H and 13C NMR data were obtained atroom temperature with a Jeol Eclipse 300 instrument at300 MHz (1H) and 75.4 MHz (13C) using CDCl3 as solvent.UV–Vis absorption spectra were carried out with a Shimadzu

Fig. 1. Chemical structure of the poly(phenyleneethynylene) studied in thiswork.

2401 spectrophotometer. The emission spectra were recordedwith a Perkin-Elmer LS 50B spectrofluorimeter and the exci-tation wavelength was chosen to be 10 nm under the lowerenergy peak in the absorption spectrum. Quantum yield (q.y.)was obtained as reported in [23] and by using sulfate quinine asstandard. The molecular weight was obtained by GPC on an HP1100 HPLC, using PS as standard, refractive index as detector,in THF at 40 °C and 1 ml/min. Glass and quartz slides werefrom Corning and SPI. Inc, respectively. For the films pre-paration, the dipper of a KSV 5000 Langmuir-Blodgett equip-ment was used for the deposition at a speed of 68.55 mm/minand in a closed atmosphere. The film morphologies were stu-died at ambient conditions with an AFM microscope Nano-scope III Dimension™ 3100 from Digital Instruments. Filmswere analyzed in tapping mode at scanning rates of 0.3–0.5 Hz.The morphology of the multilayers was studied after eachdeposition step in scanning areas of 50×50, 10×10, 5×5 and1×1 μm2. The roughness of the surface topography (Rq) isgiven by the root mean square average (RMS) of height devia-tion and is taken from the mean data plane, and expressed as:Rq= [(1/N)∑(Zi)

2]1/2, where Zi is the current Z value, and N isthe number of points within the box. The thickness of the layerswas obtained indirectly by performing perfilometry on a thicksample prepared by spin-coating a 2 g/l CHCl3 solution at1500 rpm on a quartz slide by using a modified Clay Adamscentrifuge. The perfilometer was a Dektak Stylus with a12.5 μm tip and a force of 5 mg was applied. The value of thethickness found for the spun film (850 nm) was introduced in amodified Lambert-Beer law A=εt where A is the absorbance, εis the absorption coefficient in film (nm−1) and t is the thicknessin nm. The value of ε (3.62×10−3 nm−1) was then introducedin the same formula but using the absorbance values measuredfor the different depositions of the multilayer to give the cor-responding thickness.

2.1. Synthesis

pPET3OC12-sqS was obtained by polycondensation cata-lyzed by Pd/Cu of (2-S-thiodiethyl)4,4′-bis(yodobenzencar-bothiolate) and 1,4-diethynyl-2,5-bis(dodecanoxy)benzene byHeck–Sonogashira coupling [24 25]. This latter monomer wassynthesized from 1,4-hydroquinone as reported in Ref. [26]while the yodophenyl monomer was prepared by esterificationof yodobenzoic acid with mercaptoethyl sulfide as follows.

2.1.1. (2-S-thiodiethyl)4,4′-bis(yodobenzencarbothiolate)In a two neck round flask, 24.19 mmol (6 g) of 4-yodobenzoic

acid, 12.1 mmol (1.476 g, d=1.183 g/ml, 1.38 ml) of 2-mercap-toethyl sulfide, 25.4 mmol (5.24 g) of dicyclohexylcarbodiimide(DCC) and 3.63 mmol (0.44 g) of dimethylaminopyridine in150 ml of CH2Cl2 were introduced. The mixture was stirred for18 h, then filtered to eliminate the urea formed as by-product. Tothe filtrated portion, acidified water (20% HCl) was added tocompletely precipitate any residual quantity of unreacted DCC.After further filtration, the organic phase was dried on Na2SO4.The product was purified by column chromatography (SiO2,CH2Cl2). 5.4 g (72.3% yield). Pf 140–144 °C. 1HNMR (CDCl3):

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δ=7.8 (d, 4H, –PhH), 7.65 (d, 4H, –PhH), 3.35 (t, 4H, –COSCH2–), 2.85 (t, 4H, –CH2S–).

13CNMR (CDCl3): δ=190.8,138.0, 136.2, 128.64, 101.4, 31.8, 29.3.

2.1.2. pPET3OC12-sqSThe procedure to synthesize the polymer was adapted from

Ref. [18]. In brief, in a two neck round flask, 0.814 mmol (0.5 g)of 2-(S-thiodiethyl)4,4′-bis(yodobenzencarbothiolate),0.814 mmol (0.4 g) of 1,4-diethynyl-2,5-bis(dodecanoxy)ben-zene [26], 0.0244 mmol (0.017 g) of PdCl2(PPh3)2,0.0081 mmol (1.5 mg) of CuI and 50 ml of triethylaminewere introduced under nitrogen. The mixture was heated ataround 50 °C for 20 h. After cooling, the mixture was filteredoff to eliminate the ammonium salt and the organic phase waspartially evaporated and precipitated in methanol (saturatedwith sodium diethyldithiocarbamate) to eliminate any trace ofPd and Cu. The precipitate was centrifuged, dissolved andprecipitated twice in pure methanol. The final product wasrecuperated in 10 ml of chloroform yielding 82%. 1H NMR(CDCl3): δ=7.95 (d, –COPhH), 7.55 (d, –CgC–PhH–), 7.0 (s, –OPhH–), 4.0 (t, –CH2O–Ph–), 3.3 (t, –COSCH2–), 2.9 (t, –CH2S–), 1.85 (q, –CH2–β-O–), 1.5 (q, –CH2–γ-O–), 1.25 (s, –CH2–), 0.85 (t, –CH3).

13C NMR (CDCl3): δ=192, 153.9, 135.9,131.7, 128.7, 127.3, 116.9, 114, 94.3, 89.7, 69.7, 32, 31.9, 29.4,26.1, 22.7, 14.2. UV–Vis (CHCl3): ε (327 nm)=46.78 g/l cm; ε(394 nm)=50.71 g/l cm. Fluorescence (CHCl3): λmax=458 nm,q.y=0.59. Mn=5350, Mw=16,885.

2.2. Films preparation

Thin films were deposited on glass or quartz slides pre-viously treated with a sulfochromic solution for at least 1 day,washed with distilled water, sonicated in H2O and then dried inoven at 60 °C. For the polymer deposition, the slide was im-mersed in 1 g/l CHCl3 solution for 10 min at room temperatureand dried in air. This process was repeated to obtain four layers.For the enzyme deposition, a fresh 100 mM (1.56 mg/25 mlcorresponding to 10 units/ml) solution in phosphate buffer at pH7 (adjusted with 1 M KOH) was prepared. The polymer multi-layer was immersed in the enzyme solution for 20 min with thesame procedure used for the polymer deposition. Then the filmwas rinsed with water at pH 7 and dried with a gentle flux of air.After this we obtain a film with a configuration of (pPE-T3OC12-sqS)4/β-lactamase.

2.3. Enzymatic activity

The enzymatic activity of β-lactamase in solution to peni-cillin G is usually measured by using a spectroscopy method[27]. According to the enzymatic reaction of hydrolysis of theantibiotic by the β-lactamase, penicillin G is converted topenicilloic acid. Consequently, the absorbance at the wavelengthcorresponding to the electronic transitions of the β-lactamic ringof penicillin decreases with the time of contact of the antibioticwith the enzyme. The enzymatic activity is then obtained inμmol of antibiotic hydrolyzed per minute (μmol/min) with thefollowing formula considering that a unit is defined as the

quantity of enzyme that can hydrolyze 1 μmol of penicillin perminute.

Units=Ag ¼ ½jAmin test−jAmin blank� � VjeE

where

– ΔAmin test is the change in the absorbance read at the maxi-mum wavelength of each antibiotic in a lapse of a minuteafter the contact with the enzyme.

− ΔAmin blank is the change in the absorbance read at themaximum wavelength of each antibiotic in a lapse of aminute before the contact with the enzyme.

– V is the volume analyzed in the experiment, i.e. 3.01 ml. Inthe reaction mix, the final concentrations are 100 mM ofpotassium phosphate, 0.1 mM of antibiotic and 0.05–0.1cephaloridine unit of penicillinase.

– Δε is the change in the millimolar absorption coefficient ofeach antibiotic at the end of the hydrolysis and before thecontact with the enzyme.

– E is the volume (in milliliter) of enzyme used.

For the β-lactamase immobilized in the films, the enzymaticactivity to penicillin G, ampicillin and amoxicillin was studiedby slightly modifying the standard assay reported for solutions.In this case, the control solution does not contain the enzyme asit is immobilized on the polymer film. The experiment wasrealized in a UV–Vis spectroscopic cell with temperature controlat 25 °C. First, the absorbance at the maximum wavelengthcorresponding to the β-lactamic electronic transitions of thethree different antibiotics (206 nm for penicillin G, 205 nm foramoxicillin and 207 nm for ampicillin) was measured for thecontrol solution. Then, the film was immersed in the controlsolution for 10 s, lifted out and the absorbance was measured.This procedure was repeated in intervals of 10 s until a constantvalue of absorbance was obtained (complete hydrolysis). Akinetic of the enzymatic activity was constructed by plotting theabsorbance vs. immersion time. A comparative value was ob-tained for the other two antibiotics by calculating the quantity ofthe substrate hydrolyzed in μmol/min.

2.4. Determination of the deposited polymer

To calculate the quantity of polymer deposited in the multi-layer, two samples with four polymer layers have been preparedas aforementioned. After having performed the UV–Vis spec-trum, the films were immersed in a known volume of chloroformuntil completely dissolved. From the absorbance of the obtainedsolution and using the absorption coefficient in CHCl3 ofpPET3OC12-sqS, the μg of the deposited polymer was obtained.

2.5. Determination of the immobilized enzyme

The quantity of enzyme immobilized in the films wasdetermined by using a Biorad method [28]. This method isbased on the observation that the absorbance maximum

Fig. 2. 1H NMR spectrum of pPET3OC12-sqS in CDCl3.

790 E. Vázquez et al. / Materials Science and Engineering C 27 (2007) 787–793

wavelength for an acidic solution of the dye Coomassie BrilliantBlue G-250 shifts from 450 nm to 595 nm when binding toprotein occurs. Over a broader range of protein concentrations,the dye binding method gives an accurate response with asensitivity of 1 μg. Different solutions of the enzyme wereprepared in a concentration range of 1–25 μg/ml and a standardcurve was constructed.

In the assay, 1.6 ml of the protein dilution and 0.4 ml of thedye were placed in a UV spectrophotometric cell, mixed bygentle inversion and after 5 min the absorbance at 595 nm(A595 nm) was measured. The standard curve was obtained byplotting A595 nm vs. the concentration of standards. The enzymedeposited was measured as the difference of the amount ofenzyme present in the solution after and before the immersion ofthe polymer film in the β-lactamase solution.

2.6. Biosensor assay

Penicillin G solutions with concentrations equal to 2.1, 4.2, 7and 9 μM have been prepared in buffer and used in the testtogether with pure buffer as control sample. One biosensor, i.e.(pPET3OC12-sqS)4+β-lactamase film, has been used for eachconcentration. For each sample, the fluorescence has beenmeasured before the immersion into the penicillin solution.Then it was introduced in the cell containing the penicillinsolution and the fluorescence was measured again. The valuesof fluorescence reported in Fig. 6 have been obtained byintegrating the area under the emission spectra.

3. Results and discussion

The synthesis of pPET3OC12-sqS was realized by polycon-densation catalyzed by Pd/Cu of the iodophenyl and phenyla-cetylene derivative according to the Heck–Sonogashiracoupling reaction [24,25]. The iodophenyl monomer (2-S-thiodiethyl)4,4′-bis(iodobenzencarbothiolate) was prepared byesterification of iodobenzoic acid with mercaptoethyl sulfide byusing carbodiimide as activating agent for the nucleophilicsubstitution of the 2,2′-thioethanethiol. This monomer gives theflexible sequences containing sulfur atoms, which are expectedto promote the assembly with the enzyme. To confirm thispoint, the same procedure used for the immobilization of theenzyme on pPET3OC12-sqS was carried out on multilayers ofpoly[2,5-bis(dodecanoxy)phenyleneethynylene] [26]. No activ-ity was detected supporting the hypothesis that the polarsequences are necessary to immobilize the β-lactamase,probably through electrostatic interactions between the dipolecreated by resonance in the carbonyl of the polymer and thecharges present in the enzyme. The sulfur atoms are also likelyinvolved through no covalent S–S bonds with sulfur containingproteins of the enzyme. The optical properties of thephenyleneethynylene are due to the rigid conjugated chainthat is obtained by using, as the acetylene derivative, the 1,4-diethynyl-2,5-bis(dodecanoxy)benzene where the long aliphaticchains are inserted to promote the solubility of the polymer.

The polymer pPET3OC12-sqS is soluble in CHCl3, THF,CH2Cl2, toluene and it precipitates in methanol and acetone. Its

molecular structure was corroborated by 1H NMR spectroscopy(Fig. 2). All the resonance peaks are broader than those of thecorresponding monomers. The peaks due to the acetylenicproton at δ=3.32 ppm disappear, indicating that afterpurification all traces of unreacted monomer or short oligomerswith terminal acetylenic groups were eliminated and that thereare no H termini groups in the polymer chains. From theanalysis of the aromatic region, the presence of small peaks inthe 7.6–7.9 ppm region indicates that the polymer chainsterminate more likely with iodine substituted rings according towhat was reported by other authors [29]. The 13C NMR spectraare also in agreement with the expected structure.

The growth of the multilayer was followed by UV–Visspectroscopy after each deposition. Fig. 3 shows the absorptionspectra for a (pPET3OC12-sqS)4+β-lactamase multilayer, wheretwo peaks at 333 and 405 nm are observed. The higher energypeak is due to the electronic transitions of the dodecanoxysubstituted benzene,while the lower energy peak is ascribed to theπ–π⁎ electronic transitions of the conjugated chains. Thesespectral features are retained layer-by-layer and the value of theabsorbance at the maximum increases linearly as function of thelayers (inset). This behavior indicates that the same quantity ofmatter is transferred to the substrate in each deposition suggestingthat the film thickness increases regularly layer-by-layer. After thedeposition of the enzyme on the fourth polymer layer (dot line),the absorbance decreases due to partial dissolution of the polymermultilayer in water, which cannot be avoided even with a longdrying time. These data are confirmed by the measurements ofthickness after each deposition (Table 1). The decrease in thick-ness after the enzyme immobilization likely affects the supramo-lecular organization of the multilayer. Further study by X-rayreflectivity is currently in progress to elucidate this point. Never-theless, investigating by spectroscopy the residual enzyme solu-tion after the immobilization, no trace of the polymer bands isdetected by UV–Vis and only by fluorescence could a lowemission of the polymer be observed. The quantity of polymerdissolved during the process is thus estimated on the basis of theexperiment carried out to determine the amount of polymer de-posited in the multilayers (see Experimental). 10.18 μg ofpPET3OC12-sqS was obtained from this measurement and cor-responded to a multilayer with absorbance of 0.096. Thus fromthe difference in the absorbance of the multilayer before and afterthe immobilization of the enzyme, we deduced that a 20% of

Fig. 3. UV–Vis spectra of (pPET3OC12-sqS)4+β-lactamase multilayer. Solidlines correspond to the polymer layers while dash lines correspond to thespectrum of the film after the β-lactamase deposition. Inset figure: absorbance at405 nm normalized to 600 nm vs. number of immersion.

791E. Vázquez et al. / Materials Science and Engineering C 27 (2007) 787–793

material is lost. This is indeed a non-negligible amount and futureworks might be addressed to change slightly the chemical struc-ture of the polymer by decreasing the polarity of the sequencesand consequently its solubility in water. Nevertheless, severalimportant remarks are to be considered. The decrease in theabsorbance of the multilayers is the same for all the samples andidentical fluorescence intensities were measured for the β-lac-tamase solutions after the immobilization in different experi-ments. These results indicate the reproducibility of thephenomenon. On the other hand, the most relevant point for thepossible application is that this aspect does not affect the activityvalue of the deposited enzyme or the response of the biosensor tothe antibiotic. Moreover, once the enzyme is immobilized, theabsorption spectra of the films do not change with time revealingthat the polymer is not further lost. The enzymatic studies alsodemonstrated that neither the enzyme is liberated after the immo-bilization as it will be discussed later.

The morphology of the biosensor was studied by AFM aftereach deposition (Fig. 4). For the polymer layers, a granularmorphology was observed since the first deposition (Fig. 4a)and along with the multilayer formation (Fig. 4b), analogouslyto what was already reported for the glycol ester polymer [18].This topography seems to be favored by the co-existence of stiffand flexible parts in the same structure which promotes phaseseparation or agglomerate formation. The Rq and the grain

Table 1Thickness of the biosensor layer-by-layer and the corresponding absorbance

Deposition Absorbance normalizedto 600 nm

Thickness(nm)

One layer of polymer 0.034 9.4Two layers of polymer 0.057 15.7Three layers of polymer 0.075 20.7Four layers of polymer 0.096 26.5β-Lactamase immobilized on

four layers of polymer0.077 21.2

diameter, however, decrease with the number of polymer layers.Rq for the first polymer layer is equal to 6.241 nm and decreasesto 5.421 nm for the fourth deposition. The grain diameter is inthe range of 900 (for the biggest ones) to 100–200 nm (for thetiniest grains) for the first polymer layer and it is 100–300 nm inthe last deposition. After the enzyme deposition (Fig. 3c), the

Fig. 4. 10×10 μm2 bidimensional AFM images of (a) one deposition ofpPET3OC12-sqS, (b) 4 depositions of pPET3OC12-sqS, (c) enzyme depositionon 4 layers of pPET3OC12-sqS.

Table 2μmol of antibiotic hydrolyzed per minute by the β-lactamase immobilized onpPET3OC12-sqS films for the three antibiotics of this study

Antibiotic μmol/min

Amoxicillin 0.0144Ampicillin 0.0136Penicillin G 0.0502

792 E. Vázquez et al. / Materials Science and Engineering C 27 (2007) 787–793

same morphology is observed with a good homogeneity interms of rugosity (Rq=6.600 nm). No appreciable changes thatcould indicate the presence of the β-lactamase are evident. Asthe enzyme presents a globular conformation, it is presumablethat it is immobilized on the polymer film in form of grains thatcannot be distinguished from the polymer aggregates. On theother hand, as the self-assembly is promoted by the interactionsbetween the amino acids of β-lactamase and the polar sequencesof the polymer, it is likely deposited where these groups are

Fig. 5. Kinetic of the hydrolysis of ampicillin (a), amoxicillin (b) and penicillinG (c) catalyzed by β-lactamase immobilized on the pPET3OC12-sqS films asfunction of the contact time of the biosensor with the antibiotic.

available in the polymer layer, i.e. the enzyme deposition doesnot correspond reasonably to a continuous layer but rather toisolated clusters in defined position of the polymer layer.

The amount of enzyme immobilized in the films evaluatedby the Biorad method was 3.1±0.9 μg. In order to discard thatthe immobilization process could affect the enzymatic activityof β-lactamase as well as for making a comparative study of itscatalytic properties towards the three antibiotics (penicillin G,amoxicillin and ampicillin), the kinetic of the hydrolysis of theantibiotics by β-lactamase immobilized in the films was studiedby using a modified method based in Ref. [27] (see Expe-rimental). In Fig. 5, the kinetic for the three antibiotics isreported. The absorbance decreases as a function of the im-mersion time of the film in the solution in agreement with thedecreasing quantity of hydrolyzed antibiotic and indicating thatthe immobilization process does not affect the catalyzer pro-perties of the enzyme. A first induction time can be observed,more marked for penicillin G and amoxicillin with respect toampicillin. This behavior can be ascribed to diffusion of theantibiotic molecules towards the enzyme film, i.e. a molecularrearrangement of the enzyme is necessary before it can interactwith the antibiotic. From the comparison of the three curves, itis evident that the hydrolysis of penicillin G and amoxicillin bythe β-lactamase occurs in less time with respect to ampicillin.This different behavior must be related to the different chemicalstructure of the three antibiotics and not to the immobilization asit was observed also for the enzyme in solution (data notreported). The values of the degradation of the antibiotic by theenzyme are reported in Table 2 and follow the tendency peni-cillinNamoxicillinNampicillin. This is related to the different

Fig. 6. The change in the fluorescence of the biosensor after and before thecontact with the penicillin solution vs. the antibiotic concentration.

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chemical structure of the antibiotics and the different affinity ofthe β-lactamase to them. It is to be pointed out that after havinglift out the film, the absorbance of the control solution wasmonitored in a minute to check if a liberation of β-lactamaseoccurred. As the absorbance remains constant, we deduced thatβ-lactamase stays efficiently immobilized in the film. In thecase that the enzyme was released into the solution during the10 s of contact, after lifting out the film, the enzymatic reactionwould continue producing a decrease in the absorbance. On thebasis of these results and with the aim to corroborate the hypo-thesis that it was possible to construct an optical biosensor basedon the emitting properties of the polymer, we measured thefluorescence of five (pPET3OC12-sqS)4+β-lactamase films topenicillin G solutions of different concentrations and to buffer.Fig. 6 reports the fluorescence integration area of the biosensorafter the contact with the antibiotic solution with respect to theinitial value (A−A0) vs. the concentration of penicillin G. Thefluorescence decreases linearly with the concentration of peni-cillin G. This effect cannot be ascribed to the partial polymerdissolution in water as the emission of the penicillin solutionchecked after each assay was constant as already mentioned. Twoother possible explanations are likely: (1) it is well known that theenzymes change their conformation as a consequence of thebiomolecular interaction with their substrate [30]. Thus the con-formational change in the β-lactamase immobilized on the poly-mer layers could cause a change in the pPET3OC12-sqSconformation, affecting the optical emission properties of thepolymer. (2) The formation of water and heteroatom containingmolecules (penicillinoic acid) during the reaction could quenchthe polymer fluorescence. In this case, the linear dependence of thefluorescence could be explained on the basis of the “SternVolmer”relationship as shown in the inset where the data are reportedaccording to that equation. Further studies are actually in progress.

4. Conclusions

In this work we report on the immobilization of β-lactamaseon thin films of poly(phenyleneethynylene) bearing flexiblepolar sequences containing sulfur atoms. The enzymatic activityassay reveals that the enzyme is effectively immobilized in thefilms and that presents a higher and faster response to penicillinG with respect to its synthetic analogues, ampicillin and amo-xicillin. Studies by fluorescence of the biosensor to penicillin Gindicate that there is a decrease in the polymer fluorescence as afunction of the antibiotic concentration. Further studies arenecessary to clearly understand the origin of this behavior.Nevertheless, the most relevant result is that the catalyticreaction causes a change in the fluorescence of pPET3OC12-Swhich can be easily detected. This opens the possibility to use

this biosensor for the analysis of new β-lactamic antibiotics andthat similar protocols could be exploited to immobilize otherenzymes on pPE films by self-assembly.

Acknowledgments

This work was financially supported by CONACYT throughthe Project 43166-R, and Grant No. 170338 of Erika Vázquez.The authors also acknowledge CIQA (Internal Project F70623).

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