sensitive biosensors based on (dendrimer encapsulated pt nanoparticles)/enzyme multilayers

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Sensitive Biosensors Based on (Dendrimer Encapsulated PtNanoparticles)/Enzyme Multilayers

Yihua Zhu,* Huiyu Zhu, Xiaoling Yang, Lihuan Xu, Chunzhong Li

Key Laboratory for Ultrafine Materials of Ministry of Education, School of Materials Science and Engineering, East ChinaUniversity of Science and Technology, Shanghai 200237, P. R. China*e-mail address: [email protected]

Received: November 2, 2006Accepted: January 8, 2007

AbstractA sensitive enzymed-based biosensor for glucose has been obtained by introducing dendrimer encapsulated Ptnanoparticles via a layer-by-layer assembling method. The free amine groups located on each poly(amidoamine)dendrimer molecule were exploited to covalently attach enzyme to the dendrimer chains using carbodiimide coupling.The resultant enzyme electrodes are shown to have excellent sensitivity (as high as 30.33 mA mM�1 cm�2) and a limitof detection (about 0.1 mmol L�1), depending on metal nanoparticles within dendrimers and the biocompatibility ofdendrimers, the linear response range to glucose (from 5 mM to 1.0 mM), a fast response time (within 5 s), and goodreproducibility (<8% relative standard deviation between electrodes at low substrate concentration). Thesensitivities, and stabilities determined experimentally have demonstrated the potential of dendrimer encapsulatedPt nanoparticles as a novel candidate for enzymatic glucose biosensors.

Keywords: Biosensor, Dendrimer, Pt nanoparticles, Layer-by-layer, Glucose oxidase, Nanocomposite

DOI: 10.1002/elan.200603802

1. Introduction

Biosensor systems that detect biological and chemicalagents have important medical, environmental, publicsafety, and defense applications. An ideal biosensor wouldbe sensitive, rapid, reliable, robust, and inexpensive [1].Development of newmethods and implementation of novelmaterials for the construction of desirable biocompositenanostructures have been the subject of intensive research,for example application of carbon nanotubes towardelectrochemical sensors and biosensors [2, 3], and ultrathinfilms containing enzymes used to achieve direct electronexchange between redox cofactor sites and electrodes [4],avoiding the complications of mediators in devices such asbiosensors and bioreactors.In recent years, much attention has been paid to the

construction of the third generation biosensor, which isbased on the direct electron transfer between enzyme andelectrode [5, 6]. As a building unit for themultilayer films inbiosensors, highly branched dendritic macromolecules areof great interest [7]. Theyposses a unique surface ofmultiplechain ends. The high concentration of functional end groupsof dendrimers enables synthetic modifications for themolecularly ordered nanostructures, which include deposi-tion of dendritic multilayers via electrostatic interaction [8],reaction with grafted copolymer [9, 10]. Meanwhile, noblemetal nanoparticles are of fundamental interest and tech-nological importance because of their high catalytic activ-ities for many chemical reactions, nanoparticles can also

facilitate the electron transfer and can be easily modifiedwith a wide range of biomolecules. Therefore, dendrimer-encapsulated Pt nanoparticles of uniform size can undoubt-edly be assumed as the novel electroanalytical sensingnanobiocomposite material, because it can not only provideclose contact between a transducer and the enzyme, but alsoimmobilize the enzyme in an active state due to the excellentbiocompatibility of dendrimer [11].The use of layer-by-layer (LbL) assemblymethod is being

employed for the immobilization of molecules to obtainchemically modified electrodes [12]. This approach, whichenables the formation of a monolayer of the chosenmolecules on the electrode surface is very simple and isdriven affinity between the electrode material and themolecules to be immobilized. Very recently, the self-assembly technique is being exploited for construction ofamperometric enzyme biosensors [13 – 17]. The analyticaladvantages associatedwith self-assembledmultilayer-basedconfiguration are as follows: this approach is simple andelegant, which enables easy covalent binding of the enzymeand bioconjugation onto the electrode. It is also possible tocovalently bind enzyme onto the electrode surfaces andtherefore membrane free biosensors can be developed, alsoleads to rapid response time, free from diffusion problem ofsubstrates or products through the enzyme membrane.Combining the merits of metal nanoparticles to enhance

sensitivity of biosensors and dendrimer to improve stabilityof biosensors, this work is mainly focused on developing anew type of biosensor based on dendrimer encapsulated Pt

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nanoparticles (Pt-DENs). In this paper poly(amidoamine)(PAMAM) dendrimers are recognized as the promisingcandidate for immobilizing glucose oxidase (GOx) in thelayer-by-layer (LbL) films owing to multiple homogeneouschain ends of dendrimer valuable for providing multipleconjugation sites with enzyme [18]. Meanwhile, in themultilayer films, the encapsulated Pt nanoparticles can actas efficient conduits for electrons to facilitate them trans-ferring in the enzyme active center, and poly(amidoamine)dendrimer, one kind of biomacromolecule, ensuring desiredenzymatic and electron catalytic reactions over the wholerange of multilayers. Highly sensitive and reproducibleenzyme electrodes can be fabricated using dendrimerencapsulated Pt nanoparticles where glucose oxidase wasused as a model enzyme. Analytical performance of theresulting glucose biosensor was evaluated in terms ofsensitivity and stability. Details are reported herein.

2. Experimental

2.1. Chemicals and Materials

Fourth-generation poly(amidoamine) (PAMAM) dendrim-ers having amine terminal groups (G4-NH2) were synthe-sized according to the previous literature [24, 25]. Thefollowing chemicals were used as received: glycidyltrime-thylammonium chloride (ca. 90%, Fluka Chemie A G), K2

PtCl4 (45%Pt, Sino-Chemical. Co.), NaBH4 (90%, Sino-Chemical.Co.), methanol, glucose oxidase (GOx)(EC.1.1.3.4, TYPEVII, 150 Umg�1, Fluka), (3-aminoprop-yl) triethoxysilane (APTES) (90%, Fluka). The otherchemicals were of analytical grade. Doubly distilled anddeionized water was used through this work.Carbohydrate groups on the peripheral surface of the

glucose oxidase molecule were oxidized with periodate tocarbaldehydes. For this reaction, we used the periodate-oxidized glucose oxidase (IO�

4 -glucose oxidase) synthesizedcomplied with the literature [18, 21], a 20 mmol glucoseoxidase solution in 5 mL of 0.1 mol L�1 phosphate buffersolution (pH 6.8) was slowly stirred with 30 mg of sodiumperiodate for 1 h in 4 8C, and the product was purified andconcentrated with ultrafiltration.

2.2. Preparation of Dendrimer-Encapsulated PtNanoparticles (Pt-DENs)

The preparation of amine terminated PAMAM dendrimerencapsulated Pt nanoparticles followed previously proce-dures reported for preparing metal nanoparticles encapsu-lated within hydroxyl-terminated PAMAM dendrimers(G4-OH) [25, 26]. Briefly, to 10 mL of water was added1 mL of a 1 mM poly(amidoamine) dendrimer aqueoussolution and, subsequently, 0.25 mL of a 0.1 M K2PtCl4aqueous solution. After the mixture was stirred for 48 h,1.67 mLof a 0.3 MNaBH4 aqueous solution was added. Theresulting dark brown solution was purified by dialysis

against water for 24 h to give dendrimer encapsulated Ptnanoparticles.

2.3. Preparation of Pt-DENs/GOx Modified PlatinumElectrode

The bare platinum electrode was polished with aluminausing different grades of emery paper, sonicated in distilledwater and cleaned with piranha solution before modifica-tion. The electrode was then transferred to the electro-chemical cell for cleaning by cyclic voltammetry between�0.5 V to 1.2 V versus Ag/AgCl at 100 mV/s in phosphatebuffer solution (PBS), until a stable CV profile wasobtained. For the activation of the electrodeKs surface aswell as the improvement of Pt-DENs and GOx adhesion toPt electrode, the cycling was terminated by stepping thepotential to þ1.2 V for 2 min. Afterwards, the electrodeethanol and treated with an aqueous poly(allylamine)hydrochloride (PAH) (3 mg/mL) solution for 15 min, andthen rinsed with anGOx solution (5 mgmL�1 in 0.1 MPBS)for 10 min. Afterwards the electrode was treated with Pt-DENs solution and IO�

4 -glucose oxidase solution alterna-tively for obtaining layer-by-layer configuration. Theamount of enzyme activity presented on the biosensor canbe determined from the voltammetric curves.

2.4. Characterization

Transmission electron microscopy (TEM) images wereobtained using a JEOL-2100 TEM. The samples of the Pt-DENs/GOx multilayer growth for the UV-visible adsorp-tion measurements (Unic-UV-2101-Infrared spectropho-tometer) on the quartz slides. Atomic force microscopy(AFM) observations were performed with an atomic forcemicroscope (Park Scientific. CA) operated in the contactmode using a standard silicon nitride cantilever in ambientair. All electrochemical experiments in this work wereperformed using an Autolab Electrochemical Analyzer. APt working electrode, a platinum wire counter electrode,and an Ag/AgCl reference electrode were all purchasedfrom Sino-Pharm Co. Ltd.

3. Results and Discussion

3.1. Fabrication of Pt-DENs/GOx NanocompositeMultilayers

The resulting Pt-DENs were examined by HR-TEM, whichshowed that the mean particle size is about 3 nm (Fig. 1).The inset of the Figure 1 shows the well defined, almostspherical Pt nanoparticles are prepared using the dendrimeras the template. The well defined, multiply charged surfacesof the dendrimer nanocomposite, due to the terminal aminogroups of the dendrimer, is utilized for the multilayerassembly. The nanocomposite has been deposited onto the

699(Dendrimer Encapsulated Pt Nanoparticles)/Enzyme Multilayers

Electroanalysis 19, 2007, No. 6, 698 – 703 www.electroanalysis.wiley-vch.de E 2007 WILEY-VCH Verlag GmbH&Co. KGaA, Weinheim

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negatively charged GOx with terminal groups of aldehydefilm surface, as shown in Figure 2, as a result the uniformmultiplayer of Pt-DENs/GOx has been obtained. For UV-vis spectra (Fig. 3) of the Pt-DENs/GOx nanocompositemultilayers, the adsorption peak at 210 – 220 nm character-istic of Pt nanoparticles appears and the absorbanceincreases with the number of layers, indicating that in eachdeposition step the amount of Pt-DENs and enzymeimmobilized is regular.AFM studies of the Pt-DENs/GOx nanocomposite film

can provide the surfacemorphology and the ordered degreeof the nanocomposites in three-dimensional direction. TheAFM images of blank (a), Pt-DENs (b) and Pt-DENs/GOxmultilayers (c) coating on the slides (Fig. 4) show that thehomogenization of the Pt-DENs and Pt-DENs/GOx nano-biocomposite films are arrayed in a uniform spatial ordermanner. In Figure 4c, the GOx is miscible in the matrix ofthe composite film. This corresponds to the fact that thebiocatalytic activity and the conductivity of the material areuniform throughout the sensing material. A good homog-enization implies a higher degree of intimate contact of thephases that favors a more stable and reproducible manner.The film of nanobiocomposite can be seen clearly, formingin a densely packed and structurally stable architecture,which attributed to strong electrostatic interaction andcovalent bonding between the multiply charged amineterminated dendrimer and the IO�

4 -GOx, as well asstructural homogeneity, controllable composition, compa-rable size to the participating biomolecules of dendrimer.

3.2. Performance of Biosensors Based on the Pt-DENs/GOx Multilayers

The nanobiocomposite film can be classified as conductivematerials, support materials, and biomolecule materials

Fig. 1. HR-TEM image of dendrimer-encapsulated Pt nano-particles (Pt-DENs). The inset shows a lattice image of the singlePt nanoparticle. The well defined, almost spherical particle withthe size of 3 nm is clearly seen.

Fig. 2. Schematic representation of the multilayered Pt-DENs/GOx network construction on the electrode using layer-by-layerapproach.

Fig. 3. UV-vis absorption spectra of Pt-DENs/GOx nanocompo-site multilayers on the quartz slide. The absorbance increases withthe number of layers, indicating that in each deposition step theamount of Pt-DENs and enzyme immobilized is regular.

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according to the nature of Pt-DENs and GOx. The mostimportant phase in a nanobiocomposite is the biologicalreceptor, GOx, as it is responsible for the selectivity of thedevice to a given analyte.However, the other two phases arealso very important, as they define the electrical conductiv-ity needed for the conduction of electrical signals. Becauseof the Pt nanoparticles as the conductive materials encap-sulated in dendrimersK internal porosity, the metal-glucoseoxidase interactions are avoided, replaced bybiocompatiblepolymer-glucose oxidase interactions, allowing for greaterflexibility in choosing the deposition and dispersion ofmetalnanoparticles in the film to exhibit special nanometer effectswith the possibility of minimizing enzyme denaturing.Cyclic voltammetry (CV) is a valuable and convenient

tool to monitor the barrier of the modified electrode,therefore it was chosen as a marker to investigate thechanges of electrode behavior after each assembly step. The

electrodes (2 mm� 6 mm) were treated in 0.1 M PBS(pH 6.8), added different concentration of glucose. Figure 5shows cyclic voltammograms of differently modified elec-trode in 5.0 mmol L�1 glucose. Comparing the magnitude ofthe catalytic current obtained with two kinds of enzymeimmobilization manner, respectively using dendrimer, anddendrimer encapsulated Pt nanoparticles, the results allowus to state that most of the immobilized enzyme is activethrough the Pt-DENs. The anodic currents from Pt-DENselectrodes were significantly enhanced in contrast toelectrodes without inclusion Pt nanoparticles.The effect of pH value on the performance of the Pt-

DENs/GOx biosensor is studied. Figure 6 shows that theinfluence on pH as the activity of the immobilized GOx ispH dependent. To avoid the interference from otherelectroactive species, obtain themaximum current responseandkeep the goodactivity, a pH 6.8wereused in the flowing.

Fig. 4. AFM images of blank (a), Pt-DENs (b), and Pt-DENs/GOx multilayers (c) on the silicon wafer showing that thehomogenization of the Pt-DENs and Pt-DENs/GOx nanobiocomposite films are arrayed in a uniform spatial order manner.




Figure 7 (curve a) shows the amperometric response for abiosensor builtwith 5-bilayer operating at�0.2 Vin abuffersolution at pH 6.8. The feasibility of the method forbiosensing is demonstrated by the increase in oxidativecurrent upon addition of successive aliquots of glucose.Each addition corresponds to an increase of 15 mmol L�1 inthe glucose concentration. The linear range spans theconcentration of glucose from 5 mmol L�1 to 1.0 mmol L�1.A low detection limit of 0.1 mmol L�1 glucose was estimatedat signal-to-noise ratio of 3. The biosensor sensitivity of 5-bilayer (determined from the slope of the calibration) was30.33 mA mM�1 cm�2 (Fig. 7, curve b). Such an increase insensitivity is attributed to the Pt nanoparticles dispersed inthe thin film. These results are promising because thesensitivity achieved here is higher than inmost similarworks[2, 14, 19, 20] involving glucose biosensors based ondendrimer and GOx [21], for the same 5-bilayer biosensor,the sensitivity from their electrodes modified by ferrocenyl-tethered dendrimers is 7.38 mA mM�1 cm�2, detection limitof their electrodes is 1 mmol, which tested that the methodwe adapted combined the advantages of dendrimer and Ptnanoparticles for biosensing purpose, dendrimer as abuilding unit in the network, and Pt nanoparticles encapsu-lated in dendrimers playing a role as conductor throughGOx with electrodes [22 – 24].From Figure 7a, the amperometric response implies that

the multilayered GOx provides a reproducible high perfor-mance, depending on the filmKs compact and dense struc-ture. In addition, the response time of biosensors with 4, 6,and 10 bilayers is short (within 5 s). Based on the config-uration as shown in Figure 2, the film is ultrathin and couldgenerate a fast signal transduction due to the catalyticproperties of Pt nanoparticles [15].The reproducibility of bioelectrode construction was

estimated by the response to 2 mM glucose at eight electro-des. The results revealed that the bioelectrode has satisfac-tory reproducibility with a mean current response of 28 mAmmol L�1 cm�2 and a relative standard deviation of 8%.Good reproducibility can be ascribed to the controllableLbL process of the sensor construction.The operational stability of the Pt-DENs/GOx electrode

was investigated by consecutive measurements of itsresponse to 0.5 mmolL�1 glucose.About 86% initial activityhad been remained after 100 measurements. When westored the enzyme electrode under 4 8C at dry conditionsandmeasured daily in 30 days, the response to 0.5 mmol L�1

glucose maintained over 80% of the first day response. Thestability is probably due to the structural integrity ofmultilayered network, which result from the multiplecovalent linkages formed between enzyme molecules anddendrimers, and also the physical adsorption driven by ionicattraction between GOx and dendrimer encapsulated Ptnanoparticles.

Fig. 5. Cyclic voltammograms (second run recorded) for modi-fied electrodes in 0.1 M PBS (pH 6.8) in the presence of 5 mMglucose. a) 5-Bilayer G4-NH2 dendrimer/GOx modified electrode(2 mm� 6 mm), b) 3-bilayer and 5-bilayer Pt-DENs/GOx modi-fied electrode (2 mm� 6 mm).

Fig. 6. The response current as a function of the pH value. Themaximum current response and the good activity exhibit atpH 6.8.

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4. Conclusion

We have demonstrated that dendrimer encapsulated Ptnanoparticles can be effectively employed for the construc-tion of biosensor by simple and direct layer-by-layerdeposition of Pt-DENs and glucose oxidase on Pt electro-des. The technology combined the advantages of dendrimer,stem from its unique characters, and metal nanoparticles inbiosensor construction. The response of the enzyme bio-electrode was significantly amplified with Pt nanoparticlesexisted, which indicated that the technology is promising forthe efficient fabrication of microelectrode. We believe that

the multilayered Pt-DENs/GOx network will find applica-tions in the field of biosensors, bioelectronic devises, andseparative and catalytic biomembranes.

5. Acknowledgement

The authors gratefully acknowledge National NaturalScience Foundation of China (20236020, 20276019,20676038), Nano-Science and Technology Foundation ofShanghai, Shanghai Basic Research Major Program(04DZ14002), and Development Project of Shanghai Prior-ity Academic Discipline for financial support.

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Fig. 7. Amperometric responses for detecting increasing glucose(a) and calibration curve for glucose concentrations ranging from5 mM and 7.5 mM (b). The high sensitivity (30.33 mA mM�1 cm�2)and the linear response range to glucose (from 5 mM to 1.0 mM)were achieved.




sensitive biosensors based on (dendrimer encapsulated pt nanoparticles)/enzyme multilayers

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