Biotemplated Synthesis of Gold Nanoparticle-Bacteria Cellulose Nanofiber Nanocomposites and Their Application in Biosensing

Biotemplated Synthesis of Gold Nanoparticle-Bacteria Cellulose Nanofiber...

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Biotemplated Synthesis of Gold Nanoparticle–Bacteria Cellulose Nanofiber Nanocomposites and Their Application in Biosensing

By Taiji Zhang, Wei Wang, Dayong Zhang, Xinxiang Zhang, Yurong Ma,* Yinglin Zhou,* and Limin Qi*

The bioinorganic hybrid nanostructures combine the optical, electronic, and mechanical properties of inorganic nanomaterials with the good biocompatibility and low cost of natural biomaterials, which may find potential applications in optics,[1,2] electronics,[3–5] magnetics,[6,7] mechanics,[8] catalysts,[9] and battery materials.[10] While 1D inorganic composite nanostructures have been realized by functionalizing various 1D inorganic nanomaterials such as nanotubes,[1–14] nanowires (NWs),[15–17] or nanofibers[18] with nanoparticles (NPs), quite a few 1D biological materials such as tissues,[19] live fungus[9,20] and bacteria,[3–5,21] virus,[10,2–24] and biomolecules[25–27] have been used to produce

1D bioinorganic hybrid materials. For example, Sastry and co-workers synthesized Au–spider silk bioconjugate, which showed excellent efficiency in vapor sensing.[19] Dutta et al.[20] and Eychmuller et al.[9] used live fungus to assemble with Au NPs forming fungal–Au hybrid NWs, whichcanbeusedincatalysis.Sarafandcoworkers reported the assembly of Au nanorods on individual live bacterium to obtain highly conductive hybrid system[3,4] or to fabricate electronic devices.[5] However,mostofthereportedbiotemplates such as virus and biomolecules often have low chemical and mechanical stability, which limits the applications of these kinds of hybrid materials.

Cellulose is an attractive natural material because of its good stability and abundance innature.Asaspecialkindofcellulose,bacteriacellulose(BC)isan interesting biological material produced by acetic acid bacteria Acetobacter xylinum using D-glucose as the carbon source, which usually forms netlike pellicles made up of ribbon-shaped ultrafine nanofibers with widths less than 100nm.[25] Quite different from plantcelluloses,BChasavarietyofnotablepropertiessuchasgood biocompatibility, high tensile strength, ultrafine nanofiber network structure, high water retention capability, high hydrophilicity,and high crystallinity.[25,26] Due to these special properties, BC has been applied as constituents of food, medical materials, and paper, and in optical, electronic, and optoelectronic devices and acoustic diaphragms.[25,27–30] Yano et al.[1,28] combined BC membrane and polymer to produce transparent films with a low coefficient of thermal expansion. Bismarck et al.[31,32] attached BC to the surface of sisal fibers by in situ fermentation and obtained a new class of completely renewable and biodegradable hierarchical composites. In our previous work, mesoporous titania networks weresynthesizedbyusingBCmembranesasbiotemplatesandthe obtained titania networks show enhanced photocatalytic activity.[3]

Noble metal nanoparticles such as Au NPs exhibit rich optical and electronic properties in addition to their pronounced biocompatibility and chemical inertness, which therefore can be used as biorelated functional materials in nanomedicine,[34] such as cell imaging,[35] photothermal therapy,[36] and gene delivery.[37] Furthermore, Au NPs are a very promising source for electrochemicalbiosensors,inparticulartoconnectenzymestoelectrode w.afm-journal.de w.MaterialsViews.com

[*] Prof. Y. Ma, Dr. Y. Zhou, Prof. L. Qi, T. Zhang, W. Wang, D. Zhang,

Prof. X. Zhang Beijing National laboratory for Molecular Sciences (BNLMS) State Key Laboratory for Structural Chemistry of Unstable and Stable Species Key Laboratory of Biochemistry and Molecular Engineering College of Chemistry, Peking University Beijing 100871 (China) E-mail: yurong.ma@pku.edu.cn; zhouyl@pku.edu.cn; liminqi@pku.edu.cn

DOI: 10.1002/adfm.200902104

Bacteria cellulose (BC) nanofibers are used as robust biotemplates for the facile fabrication of novel gold nanoparticle (NP)–bacteria cellulose nanofiber (Au–BC) nanocomposites via a one-step method. The BC nanofibers are uniformly coated with Au NPs in aqueous suspension using poly(ethyleneimine) (PEI) as the reducing and linking agent. With the addition of different halides, Au–BC nanocomposites with different Au shell thicknesses are formed, and a possible formation mechanism is proposed by taking into account the special role played by PEI. A novel H2O2 biosensor is constructed using the obtained Au–BC nanocomposites as excellent support for horseradish peroxidase (HRP) immobilization, which allows the detection of H2O2 with a detection limit lower than 1mM. The Au–BC nanocomposites could be further used for the immobilization of many other enzymes, and thus, may find potential applications in bioelectroanalysis and bioelectrocatalysis.

surfaces, mediateelectrochemical reactionsas redoxcatalysts, and amplifyrecognitionsignalsforbiologicalprocesses.[38]AuNPsare often combined with other materials, such as sol–gel matrices, polymers, and other nanomaterials, which can provide a network structure or a basal matrix that immobilizes Au NPs onto the electrode surface. A novel horseradish peroxidase (HRP) biosensor has been developed by adsorbing HRP onto the surface of the Au-NP-modified sol–gel network, which exhibited fast amperometricresponse,highsensitivity,goodreproducibility,and long-term stability to H2O2.[39] Nanocomposites of polymeric dopamine–enzyme–metallic (Au or Pt) NPs via one-pot prepara- tion showed high detection sensitivity for glucose in comparison with those based on conventional multistep procedures.[40] Other nanocomposites prepared recently such as Au NPs–carbon nanotubes[41] and Au NPs–carbon nanospheres[42] also showed improvedbiosensingpropertiesforproteinsorenzymes.However materialssuchassol–gelmatrices,polymers,andcarbonmaterials may restrain the bioactivity of HRP to some extent because these supports are not really biocompatible when HRP is encapsulated. Therefore an excellent biocompatible support composed of biomaterials would improve the electrochemical activities of the Au-NP-based biosensors. In this regard, BC with a ultrafine nanofiber network structure, high stability, and excellent biocompatibility would be a very promising support for Au-NP-based biosensors.

Inthiswork,novel1DnanocompositesofAu

NP–BC nanofibers (Au–BC) with adjustable shell thickness were prepared using BC nanofibers as biotemplates via a facile one-step method. The obtained nanocomposites were characterized in detail by scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), and UV–visible spectroscopy. A plausible formation mechanism for the Au–BC nanocomposites with different Au thicknesses was proposed. HRP was successfully immobilized on the surface of Au–BC nanocompositemodified glassy carbon electrode (GCE), and displayed excellent bioactivity in the reduction

ofH2O2.OnemicromolarofH2O2caneasilybe detected via this HRP/Au–BC biosensor. The excellent performance of the HRP/Au–BC nanocomposites in electrocatalysis can be attributed to the synergistic effect of gold nanoparticles and BC in the Au–BC nanocomposites.

2. Results and Discussion

2.1. Formation of Au–BC Nanocomposites

TheBCnanofibersproducedbybacteriausually form pellicles about several millimeters thick. An optical photograph of a BC pellicle ( 1mm in thickness) is shown in Figure S1a (see

Supporting Information (SI)). The BC pellicle is semitransparent, soft,andelastic.BeforeassemblingAuNPsonto thesenanofibers, the BC pellicle was cut into pieces. After smashing the BC pellicle by ultrasonication, fiberlikestructures were formed and dispersed inwater(Fig.S1b,SI).FigureS2(seeSI)showsthetypicalSEMand TEM images of the obtained BC nanofibers, which suggests that thefibersareribbon-shaped,withanaveragediameterof 40nm. It may be noted that the contrast grade of BC nanofibers in TEM image is pretty low without dyeing.

Poly(ethyleneimine) (PEI), an amine-rich cationic polyelectrolyte,workedbothasalinkingmoleculeandareducingagentfor the synthesis of Au–BC nanocomposites, similar to the synthesis of Au-related composites in recent years.[43,4] During the reduction of HAuCl4 by PEI in the solution, the BC nanofibers turnedgraduallyfromcolorlesstopurple,indicatingtheformation ofAuNPsonthefibers.TheSEMimageinFigure1ashowsthatthe product obtained after 1h of reaction at 608C remained a netlike structure and the nanofibers were separated from each other, in contrasttotheaggregatedpureBCnanofibers(Fig.S2b(SI)).From the high-magnification image (Fig. 1b), it can be seen that the BC w.MaterialsViews.com w.afm-journal.de

Figure 1. a,b) SEM, c–e) TEM, and f) HRTEM images of Au–BC nanocomposites obtained after 1h of reaction at pH 8.2. Inset shows the related ED pattern. [PEI]¼1g L 1, [HAuCl4]¼1mM.

nanofibers were capped with Au NPs uniformly. The contrast grade of the obtained Au–BC nanocomposites became much higher compared with BC nanofibers and the networklike structure was also much clearer (Fig. 1c). These Au–BC nanocomposites looked like necklaces (Fig. 1d), which suggests that Au NPs did not enwrap BC nanofibers completely and covered about 80% of the surface area of BC nanofibers. The electron diffraction (ED) pattern of the Au–BC nanocomposites is shown in Figure 1c, which exhibits sharp rings indexed to the (1), (200), (220) and (311) planes of the face-centered cubic (fcc) Au.Thehigh-magnificationTEMimageinFigure1eindicatesthat the Au NPs on BC nanofibers are nearly spherical and discrete, with relatively uniform size ( 9nm). The diameter of the Au–BC nanocomposites is about 60nm (Fig. 1e), slightly larger than the diameter of BC nanofibers (40nm) due to the attachment of Au NPs. Most of the Au NPs are directly attached to the surface of the BCnanofibers.Thehigh-resolution TEM(HRTEM)imageshown in Figure 1f shows clear lattice fringes 0.24nm, attributed to the (1) plane of fcc Au, indicating that each Au particle is a single crystal.

Figure 2a shows the XRD patterns of BC and Au–BC nanocomposites. The XRD pattern of pure BC nanofibers shows three peaks at 14.18, 16.98, 2.68, indexed to (110), (110), and (200) reflections of cellulose I, respectively.[45] Besides these three peaks of BC, the XRD pattern of the Au–BC nanocomposites exhibits additional four peaks indexed to the (1), (200), (220), and (311) planes of fcc Au. The XRD patterns confirm the existence of Au NPs on the BC nanofibers in the nanocomposites.

Figure 2b gives the UV–vis absorption spectra of Au–BC nanocomposites and monodisperse Au NPs ( 9nm) synthesized by PEI and HAuCl4 without BC nanofibers. There is no absorption peak from 400 to 800nm in the absorption spectrum of pure BC nanofibers. Theabsorption spectrumofmonodisperse Au NPsshowsapeakat520nm,duetothesurface plasmonresonanceabsorptionofAuNPs.Au– BC nanocomposites have a broader peak from 520 to 580nm. The absorption peak of the Au NPs in the Au–BC nanocomposites became broader and slightly red-shifted because of the surface plasmon coupling between closely spaced nanoparticles,[46] similar to the absorption of silica nanofibers–Au NPs hybrid nanostructure.[15]

2.2. Effects of Reaction Conditions

The Au–BC nanocomposites synthesized with different reaction times are shown in Figure 3. A very small amount of Au NPs formed on the surface of BC nanofibers after 5min of reaction in theaqueoussolutionat608C(Fig.3a).After15min,moreAuNPs formed on the surface of BC nanofibers (Fig. 3b). However, the amount of Au NPs is still too low to wrap the surface of the BC nanofibers. As the reaction time increased, more Au NPs attached to the surface of BC nanofibers. After 30min, most of the BC nanofibers were wrapped by the Au NPs, and fiberlike nanostructures can be seen clearly (Fig. 3c). When the reaction timewasextendedto1h,BCnanofibersbecamewellcoatedbyAu NPs as shown in Figure 1. Thermogravimetric (TGA) curves showed that the weight percent of Au in Au–BC nanocomposites increased from 65.4% to 70.7% when the reaction time was increased from 30 to 60min (Fig. S4 (SI)), confirming an increase in the amount of Au NPs on the surface of BC nanofibers with increasing time. The time-dependent experiment shows that the

AuCl4 ions can be reduced by PEI quickly at mild conditions in aqueous solution and thewell-coated Au–BCnanocomposites can be formed after 1h.

To better understand the influence of PEI, Au–BC nanocomposites were synthesized in aqueous solutions with different concentrations of PEI (Fig. S3 (SI)). The amount of Au NPs on the w.afm-journal.de w.MaterialsViews.com a) b)

∗ 311∗ 220∗ 200

BC-Au BC

Intensity (a.u.) 2θ (degree)

- BC ∗ Au

Au NPs

BC-Au Absorbance

Wavelength(nm)

Figure 2. a) XRD patterns and b) UV–vis absorption spectra of BC, Au NPs, and Au–BC nanocomposites after 1h of reaction at pH 8.2. [PEI]¼1g L 1, [HAuCl4]¼1mM.

Figure 3. TEM images of Au–BC nanocomposites obtained at pH 8.2 with different reaction times: a) 5, b) 15, and c) 30min. [PEI]¼1g L 1, [HAuCl4]¼1mM.

surface of the BC nanofibers was much lower at a high concentrationofPEI(10gL 1)(Fig.S3a(SI)).Astheconcentration of PEI was decreased to 1gL 1, Au NPs with a size about 9nm grew on the surface of the BC nanofibers (Fig. 1). When the concentration of PEI was decreased to 0.2gL 1, Au NPs formed aggregated in the solution, and much less Au NPs attached to the surface of the BC nanofibers (Fig. S3b (SI)). Triangular and hexangular plates, micrometers in size, appeared as the concentration of PEI was further decreased to 0.05gL 1 (Fig. S3c (SI)). Since PEI functions both as reducing agent and linking molecule,[43,4] the protonation degree of the NH3 group of PEI or the pH of the solution would considerably influence the preparation of the Au–BC nanocomposites. The effect of the pH on the synthesis of Au–BC nanocomposites was investigated by adjusting the pH of the reaction solution with concentrated NaOH or HCl solution. The pH of the solution for the synthesis of the Au–BC nanocomposites under standard condition is 8.2 before reaction. Some of the Au NPs were badly aggregated while most of the BC nanofibers were still covered by Au NPs when the pH of the reaction solution was decreased to 2.5 (Fig. 4a,b). The Au NP aggregates are markedbyblackarrowsinFigure4a.Comparedwiththestandard Au–BC composite nanofibers shown in Figure 1, the diameter of Au NPs coated onto the surface of the BC nanofibers increased to 20nm; meanwhile, the number density of the Au NPs coated on each BC nanofiber was considerably decreased, namely from 40 to 15 particles per 100nm distance (Fig. 4b). In contrast, only monodisperse Au NPs were obtained and no evident attachment of Au NPs onto the BC nanofibers can be seen from the sample obtained at a pH of 1.5 (Fig. 4c,d). It is interesting that the diameter of the Au NPs obtained under basic condition is very uniform (Fig. 4c, inset). Obviously, many of the BC nanofibers are naked without coating particles (Fig. 4d). This result is consistent with the synthesis of the monodisperse pyridine-capping Au NPs synthesized under basic conditions,[47] which indicates that the PEI may act both as a stabilizing and reducing agent, but not as linking molecule under basic conditions. It is worth noting that the color of the solution after reaction waslight pinkunder basic conditions, verydifferent from the color of the reaction solution under acidic and neutral conditions,i.e.,darkpurple.Thecolordifferenceindicatesthatless Au NPs were obtained in the basic solution than those in the neutral or acidic solutions. In other words, the reduction rate of

AuCl4 wasmuchslowerunderbasicconditionsthanthoseunder neutral and acidic conditions.

The pH of the solution can change the steric structure of polymers with amine groups.[48] The amine groups of PEI are protonated at low pH values or neutral conditions, and PEI chains are fully stretched due to the electrostatic repulsion between the protonated amine groups. Streched PEI molecules may attach to the surface of BC nanofibers easily to act as linking molecules. Therefore, Au–BC nanocomposites can be fabricated both under neutral and acidic conditions. At high pH, the polymer chains are uncharged, resulting in a compact cluster conformation which inhibits PEI wrapping of BC nanofibers. The compact PEI molecules with less protonated free amine groups inhibited the reduction and attachment of Au NPs to the surface of BC nanofibers. As a result, almost no Au NPs deposited on the BC nanofibers under basic condition.

2.3. Halide-Assisted Synthesis of Au–BC Nanocomposites with Thick Shells

The influence of various chloride salts, such as

NaCl,KCl,MgCl2,onthepreparationofAu–BC nanocompositeswasstudied.When0.1MNaCl

was added to the reaction solution, a uniform netlikeAu–BCcompositewithasmoothsurface can be formed (Fig. 5a,c). The high-magnification SEM and TEM images indicate that the surfaceoftheBCnanofibersarewrappedbyAu NPs uniformly, forming a dense Au NPs shell (Fig.5b,d).ThediameteroftheobtainedAu–BC nanocomposites was about 60–100nm with an average diameter of 80nm, larger than the diameter of Au–BC obtained without the addition of salts shown in Figure 1 (60nm). The Au NPs were still spherical and singlecrystalline,withadiameterof 9nm(Fig.5d).It was supposed that the thick coating of Au NPs was formed due to the reduction of the z potentials of Au NPs in the presence of the Cl ions. Chloride ion has a significant affinity totheAusurfaceandmayreplacesomeligands such as citrate and pyridine on the Au surface.[49,50] In this work, Au NPs are positively charged, as they are coordinated and stabilized w.MaterialsViews.com w.afm-journal.de

Figure 4. TEM images of Au–BC nanocomposites synthesized with different pH values for 1h:

a,b)pH¼2.5andc,d)pH¼11.5.[PEI]¼1g L 1,[HAuCl4]¼1mM.Thearrowsinaanddindicate Au NPs aggregates and naked BC nanofibers, respectively. The inset in c shows the high- magnification image of dispersed Au NPs.

by PEI, which has protonated free amine groups. After adding NaCl, chloride ions may be absorbed to the surface of Au NPs and reduce the surface charge, leading to the reduction of electrostatic repulsionbetweenAuNPs,somoreAuNPsarecoatedontotheBC nanofibers to form thickly coated Au–BC nanocomposites. The above proposition is approved by the characterization of the surface charge. The Au NPs synthesized without any salts have a z potential about 53mV. With the addition of 0.1M NaCl, the z potential of the Au NPswasreducedto31mV.Theeffect ofKClor

MgCl2 is essentially the same as NaCl for the preparation of Au–BC nanocomposites.

The influence of other halide ions such as

F ,B r , and I was also investigated for the synthesis of Au–BC nanocomposites since different halide ions have different affinity with gold (Au–I>Au–Br>Au–Cl>Au–F).[50] When fluoride was added, the amount of Au NPs coating BC nanofibers did not obviously increase as was observed with the addition of chloride.ThezpotentialofAuNPssynthesized with the existence of 0.1M F was measured to be 49mV, close to the z potential of Au NPs synthesized without any salts ( 53mV). As F has the weakest affinity with Au among halide ions, there are less F ions adsorbed onto Au NPs, and the reduction of the z potential of Au NPs caused by absorption of F can be neglected. Thus F barely has the ability to promote the attachment of Au NPs to BC nanofibers.

In the presence of bromide, Au NPs were aggregated and large Au NPs agglomerates appeared on the surface of the Au–BC nanocomposites(Fig.6a,b).MoreAuNPswere coated on BC nanofibers compared with the sample obtained with NaCl. The nanocomposites has nodule-like structures with diameters from50to130nm(Fig.6b),verydifferentfrom theAu–BCcompositeswithanalmostuniform diameterasshowninFigure5.Weproposethat more bromide adsorbed on the surface of Au NPs than chloride because of the stronger affinity of the bromide ion with gold.[49,50] The z potential of the Au NPs obtained with the addition of 0.1M NaBr is 20mV, much lower than the samples with NaCl (31mV). Thus the electrostatic repulsion among Au NPs decreased and more Au NPs coated on the surface of BC nanofibers with nodule-like structure.

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