Copper-Glucosamine Microcubes: Synthesis, Characterization, and C

Jun 7, 2011 - Furthermore, primitive electrochemical (cyclic voltammetry) properties of these MC's were evaluated for the detection of C-reactive prot...
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Copper-Glucosamine Microcubes: Synthesis, Characterization, and C-Reactive Protein Detection Murugan Veerapandian,†,|| Ramesh Subbiah,† Guei-Sam Lim,‡ Sung-Ha Park,§ KyuSik Yun,*,† and Min-Ho Lee*,|| †

Department of Bionanotechnology, Kyungwon University, Gyeonggi 461-701, Republic of Korea Healthcare Group, Emerging Technology Lab, LG Electronics Advanced Research Institute and Department of Biomicrosystem Technology, Korea University § Sungkyunkwan Advanced Institute of Nanotechnology (SAINT) and Department of Physics, Sungkyunkwan University Korea Electronics Technology Institute, Medical IT Technology, Gyeonggi 463-816, Republic of Korea

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bS Supporting Information ABSTRACT: Cubelike microstructures of glucosamine-functionalized copper (GlcN-CuMC’s) have been fabricated by the integration of injection pump and ultrasonochemistry. Although bulk microstructures and the nanostructure of metallic copper exhibit distinct applications, the amino sugar surface-functionalized copper is almost biocompatible and exhibits advanced features such as more crystallinity, high thermal stability, and electrochemical feasibility toward biomolecule (C-reactive protein, CRP) detection. An electrochemical test of this GlcN-CuMC’s was demonstrated by immobilization on a conventional gold-PCB (Au-PCB) electrode. The combination of a biointerface membrane, from glucosamine functionalization, and electroactive sites of metallic copper provides a very efficient electrochemical response against various concentration of CRP. A perfect scaling of steady-state currents with r2 values of 0.9862 (Ipa) and 0.9972 (Ipc) indicate the promise of this kind of biofunctionalized microstructure electrode for many surface and interface applications.

’ INTRODUCTION The hybridization of new class nano/microstructures consisting of metal and organic/composite materials is always of great importance because of their remarkable applications in science and technology.1,2 Copper is economical and has good electrical and thermal conductivity, making it a widely used materials for machining and manufacturing processes. Metal copper particles and its composites provide versatile interface activity in biosensing and biological properties.3 Biocompatibility, hydrophilic character, high stability against aggregation, and versatility in surface modification are the key features required for biological and biomedical applications.4 Several approaches have been proposed for the synthesis of metal copper particles, such as thermal reduction and sonochemical reduction,1 surface-protecting chemical reduction,5,6 a radiation method,7 and a laser ablation method.8 Nevertheless, these synthesis schemes and obtained metal copper structures have some limitations such as the use of toxic chemical reactants, precipitation, and inappropriateness for large-scale production. To overcome these issues, biofunctionalization or several surface chemical modifications are required. Certainly, scientists have largely been involved in the development of the large-scale manufacturing of nanomaterials (metal particles) through biosynthetic approaches. For instance, prokaryotic (bacterial species) and eukaryotic (fungal and plant species) organisms are utilized for the production of metal r 2011 American Chemical Society

nanoparticles of different compositions and sizes.9 11 The stabilizing properties of proteins and other biocompatible materials from the bioreactor (i.e., bioextracts used for synthesis) provide green chemistry to the synthesis process and synthesized nano/microstructures.9 11 Limitations persist, such as the long period of time required to grow the cultures, incubation, and synthesis. Therefore, it is of fundamental importance to establish an alternative, robust, environmentally benign synthesis strategy for the preparation of biocompatible metal particles with the above features as observed from other chemical processes.1,5 8 Here, we have devised an integrated approach (injection pump and ultrasonochemistry) to synthesize gram-scale biocompatible metal copper microstructures (cube-shaped) that are surface functionalized with glucosamine. Glucosamine (GlcN) is a naturally occurring amino sugar resulting from the amidation of fructose-6 phosphate.12 It is well known as an alternative cure for osteoarthritis. Recently, we have functionalized the surfaces of silver nanoparticles with GlcN because of its glyconanoparticles13 and antibacterial application.14 The ability to functionalizing nano/microstructures with naturally occurring substances is one of the most significant and interesting challenges in interdisciplinary nanobiotechnology.15 It is Received: March 14, 2011 Revised: June 3, 2011 Published: June 07, 2011 8934

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Langmuir widely accepted that these materials provides excellent physicochemical and biological properties and are expected to broaden its application in different fields, including biosensing, imaging, catalysis, electronics, and other biomedical applications.16 Furthermore, the functionalization of the surfaces of metal/metal oxide nano/ microstructures with biocompatible substances significantly reduces the toxicity toward human health and the environment. An ultrasonochemical system is obviously not new for the preparation of nano/microstructures.1 Recently, a wide variety of metal and alloy particles with significant surface modifications were obtained from simple ultrasonochemistry.17 This includes our previous research on the generation of hetero/hybrid nanostructures composed of organic polymers and inorganic metalloids.18,19 However, the integration of injection pump and ultrasonochemistry for synthesis is a fairly new synergistic approach to obtaining flow regulation between a metal precursor and other chemical reactants. This will significantly provide the harmonized flow of reactants and the reaction environment. GlcN-CuMC’s were fabricated by using copper(II) nitrate hemi(pentahydrate) (as a metal precursor), hexamethylenetetramine (HMTA, as chelating ligand), and Dglucosamine (as surface-functionalizing agent). The physicochemical and morphological properties of as-fabricated GlcN-CuMC’s were characterized. Furthermore, primitive electrochemical (cyclic voltammetry) properties of these MC’s were evaluated for the detection of C-reactive protein (CRP) with respect to various concentrations in comparison with the commercial Au-PCB electrode. The purpose of selecting CRP immunodetection for this experiment is its normal presence in plasma, synthesized by hepatocytes. In case of injury, infection or acute inflammation CRP concentrations can increase 104-fold. Several clinical investigations have used CRP levels for diagnostic purposes in cardiovascular disease20 or as a prognostic indicator of gastresophageal cancer.21 However, several other tumor markers such as carcinoembryonic antigen (CEA), carbohydrate antigen (CA19-9), and R-fetoprotein (AFP) are clinically significant for prognosis.22 Because of their lowgrade sensitivity, these markers possess limited applications in the early prognostic evaluation of gastric cancer patients. In like manner, positive correlations between CRP and chronic inflammatory lead to a diagnosis of recurrent inflammation after gastrectomy.23 Therefore, CRP has been selected as a model biomolecule to elucidate the electrochemical properties of GlcN-CuMC’s. Moreover, the in situ biofunctionalization of metal copper particles positively enhances its crystalline and thermal stability, which will play a promising role in interdisciplinary fields.

’ EXPERIMENTAL SECTION Materials. Copper(II) nitrate hemi(pentahydrate) (Cu(NO3)2 3 2.5 H2O), hexamethylenetetramine (HMTA), D-glucosamine (GlcN), 3-mercaptopropionic acid (3-MPA), 3-aminopropyltriethoxysilane (3-APTES), glutaraldehyde (GTA), anhydrous ethanol, and phosphate-buffered saline (pH 7.4) were commercially obtained from Sigma-Aldrich. Milli-Q water with a resistance greater than 18 MΩ was used in all of our experiments. Recombinant human CRP and antibody to human CRP were purchased from R&D Systems (Minneapolis, MN). All of the chemicals and protein samples were used as received (without further purification). Protein samples were diluted in phosphate-buffered saline (PBS) at pH 7.4. The gold-PCB (Au-PCB) working electrode used in the cyclic voltammetry study was made from a conventional Au-PCB chip. Instrumentation. The ultrasonic system and injection pump used in the current experiment were a VCX 505 Sonics and a KDS101 syringe pump (KD Scientific). Morphological characteristics were studied from

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a conventional field-emission scanning electron microscope (FE-SEM, JEOL JSM-7500F), a high-resolution transmission electron microscope (HR-TEM, FEI Titan 80-300), and a bioatomic force microscope (BioAFM; Nanowizard II, JPK) in intermittent air mode. Ultraviolet visible spectra were analyzed in Optizen 3220. The identity and crystallinity of GlcN-CuMC’s were investigated with an X-ray diffractometer (XRD, Scintag-SDS 2000) at 40 kV/20 mA using continuous-scanning 2θ mode. Fourier transform infrared (FT-IR) spectra of the samples were recorded at room temperature using a TENSOR 27 instrument (Bruker) in the 600 4000 cm 1 region. Thermal gravimetric analysis (TGA) was carried out using Perkin-Elmer instrument under a nitrogen atmosphere at a heating rate of 15 °C/min from 0 to 800 °C. Water contact angle measurements for the thin solid films of GlcN-CuMC’s were performed using a Kr€uss DSA10-MK2 contact angle measuring system (Kr€uss GmbH) and were analyzed with drop shape analysis software. Five measurements were made and averaged. Cyclic voltammetry (CV) measurements were recorded using VersaSTAT 3 (Versa studio software) in a three-electrode configuration. Synthesis and Thin Solid Film Growth of GlcN-CuMC’s. In a typical synthesis of GlcN-CuMC’s, an aqueous solution of the Cu(NO3)2 (25 μM) metal precursor was taken in a 10 mL syringe. It was injected into a reaction vessel containing 10 mL of HMTA (25 μM) at a flow rate of 0.166 mL/min and allowed to stirring vigorously for 15 min at a heating temperature of 80 110 °C. Then an appropriate amount (10 mL) of GlcN (20 μM) was injected into the reaction mixture and ultrasonicated for about 30 min with controlled parameters of probe temperature (60 °C), amplitude (35%), and a pulsed on off (5 10) cycle. As-obtained colloidal dispersions of GlcN-CuMC’s were separated by centrifugation. The separated microstructures were washed twice with anhydrous ethanol in an ultrasonication bath and dried in an electronically heated oven at 85 °C for 4 h. An ethanolic dispersion of GlcN-CuMC’s was then drop coated onto an oxygen plasma-treated platinum substrate (2.0  2.0 cm2) and evaporated to obtain thin solid film growth.

Electrode Pretreatment and Immobilization of GlcNCuMC’s. Electrochemical measurements were performed using a three-electrode cell with a Au-PCB working electrode (electrode area ∼1 mm), a Pt wire auxiliary electrode, and a Ag/AgCl reference electrode. The surface of the working Au-PCB electrode were cleaned by soaking the electrode under ethanol and acetone for 5 min separately and rinsing thoroughly with deionized water. Prior to surface-functionalization procedures, the surface of the Au-PCB electrode was made hydrophilic by oxygen plasma treatment for 60 s. After plasma cleaning, the electrode surface was drop coated with 4 μL of 3-MPA (50 mM) and kept under room temperature for 2 h. The 3-MPA-activated Au-PCB surface were then drop coated with 4 μL of an aqueous colloidal dispersion of GlcN-CuMC’s and allowed to dry for 1 h. Electrode Preparation for the Detection of CRP. Electrochemical characterization for the detection of CRP was done by functionalizing the surface of the Au-PCB electrode with anti-CRP and CRP. After following the same pretreatment (oxygen plasma, 3-MPA activation) and GlcN-CuMC’s immobilization procedures, the antibodies were attached to the surface of the GlcN-CuMC’s electrode surface by modifying the reported procedure for CRP to silica nanowires.23 In the present study, the Au-PCB/GlcN-CuMC’s surface was again oxygen plasma treated for 60 s, which activates the hydroxyl groups on the Au-PCB/GlcN-CuMC’s electrode surface. Then, silanization was achieved by coating 4 μL of 3-APTES (30 μL in 1 mL of C2H5OH) and allowing the samples to rest for 2 h. The resulting amine groups were then reacted with 4 μL of GTA (1.25% in PBS), resulting in an aldehyde functional group at the surface. The terminal aldehyde surface was then coupled to antibodies to CRP (100 μg/mL in PBS, pH 7.4). All of the electrochemical experiments were recorded in 10 mM PBS at pH 7.4 in the potential range of 0.25 to +0.45 V. A reproducible 8935

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Scheme 1. Mechanism of the Chemical Reaction and a Reasonable Structure of the Copper-Glucosamine Microstructure

voltammogram can be obtained under steady-state conditions after about eight cycles.

’ RESULTS AND DISCUSSION Synthesis of GlcN-CuMC’s. An integrated approach devised by the injection-pump-assisted flow of reactants was carried out and in situ ultrasonochemistry-derived colloidal dispersions of copper-glucosamine microcubes was successfully carried out. Unlike other chemical processes, the utilization of the injection pump regulates the controlled flow volume at a rational interval, which brings about an effective reaction environment. The importance of ultrasonochemistry in the generation of a wide range of metal particles and heteronanostructures is described in the literature.1,17 19 The ultrasonochemical reaction for the generation of copper microstructures is initiated from the decomposition of a chelating ligand (HMTA) in the reaction vessel, which was reported to decompose into formaldehyde (HCHO) and ammonia (NH3) at 80 °C.24 Generated NH3 forms several types of complexes such as copper hexamethylene tetrammonium, ammonium ions (NH4+), and a copper ammonia complex [Cu(NH3)4)2+], which results in the formation of copper(II) ions (Cu2+) and hydroxyl ions in the reaction mixture, followed by the introduction of aqueous glucosamine to react with Cu2+ to form the copper-glucosamine complex. Here, in situ acoustic microstreaming and the cavitation force resulting from the pressurized waves of an ultrasonic probe mediate the growth of GlcN-CuMC’s. The reaction mechanisms for the generation of GlcN-CuMC’s are schematically drawn in Scheme 1. The specific geometry of the copper-glucosamine complex was drawn on the basis of previous reports related to the interaction between copper(II) and chitin/chitosan.25 27 It was demonstrated that the nitrogen atom containing a free electron doublet may be the active site for binding metal cations; oxygen atoms of hydroxyl groups, especially those in the C-3 position, may be assigned to metal sorption. From that study, the possible geometries formed

Figure 1. X-ray diffractograms: (a) copper(II) nitrate hemi(pentahydrate), (b) D-glucosamine hydrochloride, and (c) copper-glucosamine (GlcNCuMC’s). Functionalization of the copper surface with glucosamine results in the occurrence of new characteristic peaks at 12.66, 25.58, 36.26 (where * denotes Cu2O), 43.42, 53.36, and 61.18°.

between Cu2+ and the nitrogen of the amine group and between Cu2+ and a nearby oxygen of the hydroxyl groups were specified. Chitosan is a linear polysaccharide consisting of randomly distributed β-(1,4)-linked D-glucosamine and N-acetyl-D-glucosamine. In general, the geometries between Cu2+ and chitosan/ chitin residues were explained in the form of pendant and bridge models. Similarly, Wang et al. studied the three different possible structures (chitosan-Zn complex) corresponding to different chelating ratios and discussed the coordination mechanisms based on Lewis acid base theory in which Zn2+ ions acting as the acid are the acceptor of a pair of electrons given by chitosan acting as the base, resulting in complex formation (chitosan-Zn)2+.28 Powder X-ray Characterization. The formation of the copper microstructure is ensured from the powder X-ray diffraction (XRD). Figure 1 displays the XRD profile of copper nitrate salt, glucosamine hydrochloride, and the copper-glucosamine microstructures. The XRD peaks of (Figure 1a) the copper nitrate salt correlate to the earlier diffractograms.29 To compare the crystalline 8936

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Figure 2. UV vis spectrum of D-glucosamine (black trace) and copperglucosamine microcubes (red trace).

properties of as-synthesized copper-glucosamine microstructures, raw glucosamine hydrochloride was also included. As seen in Figure 1c (blue trace), the major peaks are located at 12.66, 25.58, 36.26, 43.42, 53.36, and 61.18°. The peaks in the 43.42 (111) and 53.36 (200) planes belong to fcc metallic Cu (JCPDS, PDF, file no. 00-001-1241). As discussed in the synthesis part (Scheme 1(7), (8)), the copper ammonium complex and copper hydroxide ions are decomposed to form copper oxide structures. This is evinced by the peak located at 36.26° belonging to Cu2O (JCPDS, PDF, file no. 01-701-3645), denoting that Cu2O also partially exists with copper microstructures. In addition, the possible pathway to the generation of Cu2O was also demonstrated in the sonochemical synthesis of Cu nanoparticles.1,30 The partial oxidation of Cu0 by the secondary species was formed by the recombination of H• and OH• radicals. Thus, the generated H2O2 from these radicals initiates the oxidation of Cu clusters into Cu2O. Furthermore, GlcN-CuMC’s has two major peaks at 12.66 and 25.58, which could possibly be due to surface-functionalized glucosamine, revealing that functionalization of glucosamine on the surfaces of copper structures results in the formation of new crystalline phase. Similarly, polysaccharide chitosan chelated with Cu and Zn metal substances is reported to have improved crystalline properties.27,28 UV Visible Spectral Study. Figure 2 shows the UV vis absorption spectra of an aqueous solution of raw D-glucosamine and a colloidal solution of GlcN-CuMC’s. As observed in Figure 2 (black trace), D-glucosamine has no adsorption peak in the selected region. In the case of GlcN-CuMC’s, the wavelength of maximum absorbance was found to be 545 nm (red trace) with a broad absorption peak. This peak can be assigned to the absorption of surface-modified copper microstructures. Copper nanoparticles and submicrometer particles synthesized by other methods display absorption bands at ∼570 and ∼600 nm, respectively.1,31 Factors such as the size, shape, solvent, capping agent, and reducing agent used in the synthesis process are the key features in determining the absorption band.6 Larger copper particles are reported to have a shifted absorbance peak at a higher wavelength.32 However, the λmax for the current GlcN-CuMC’s is located at a lower wavelength compared to that of other copper colloids; this is probably due to the surface modification of glucosamine on the copper microcubes. Furthermore, the partial coexistence of oxide content as observed from XRD patterns could also be related to the low intensity of the band and its broadness. The interaction between the surface-modified glucosamine and shape-controlled copper microcubes results in the blue shift of GlcN-CuMC’s rather than other copper microstructures.

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Morphological Characterization. Figure 3 shows typical (a, b) FE-SEM, (c, d) HR-TEM, and (e, f) AFM images of prepared copper-glucosamine microstructures. The micrograph of injection pump and ultrasonochemistry-derived copper-glucosamine (Figure 3a) shows the well-dispersed microstructures. The microstructures have a regular shape of a cubic surface with an average size distribution of about 1.0 ( 0.1 μm (Figure 3a, inset histogram). At a higher magnification (Figure 3b,d), the micrograph shows that the microstructures have welldefined sharp faces and cubelike surfaces that are formed by regular arrangements of sheetlike structures (Figure 3b). This also reveals the existence of smaller particles within the cubical surface. This implies the crystallinity of cubic copperglucosamine microstructures. Figure S1 shows the EDS profile for the obtained GlcN-CuMC’s. Apart from the regular cubelike structures observed from the FE-SEM and AFM, HR-TEM image reveal that there are sheetlike copperglucosamine microstructures that also exist (Figure 3c). This is probably due to the acoustic microstreaming and cavitation force obtained from the ultrasonic probe, which determines the surface structures of the final product. It was demonstrated that the sonochemical cavitation effect generates the interparticle collision that results in the formation of larger network of (Cu0 )n structures.1,33 The formation of such a larger network because of interparticle collision/ coalescence has also been observed for other systems. 34 36 In another report, the growth of larger particles is discussed on the basis of the desorption of encapsulating ligands at higher temperatures (∼150 °C), which allows the metal atom or neighboring particle to participate in interparticle aggregation. 32 However, in the current study the temperature conditions used for the chelation (80 110 °C) and ultrasonication (65 °C) are comparatively lower. Furthermore, other microscopic images seem to have well-dispersed structures with no aggregation. Therefore, the physical agglomeration seen in the HR-TEM image possibly occurs because of the drying process. The 2D and 3D surface topographies of the copper-glucosamine microstructures are shown in Figure 3e,f. Direct observation of the image showed that the maximum size of GlcN-CuMC’s was found to be ∼1 μm. The size distribution of the GlcN-CuMC’s along with the line profiles drawn at a scan size of 5.0  5.0 μm 2 is shown in Figure S2. It clearly indicates that the observed GlcN-CuMC’s were falling in the range of 0.9 1.0 μm. The data obtained from AFM correlates well with the other two microscopy results. The thin solid film growth of well-dispersed GlcN-CuMC’s was obtained by drop coating the ethanolic solution of copper-glucosamine on a freshly plasma-treated Pt substrate. The mechanism behind the thin solid film growth was evaporation-induced self-assembly. 37,18 As discussed in these reports, the evaporation of solvent allowed the reorganization of the microstructure and formed a continuous film with MC’s in a large network. Several studies were performed on this drying process toward the development of a bulk film consisting of nanoparticles, as reviewed elsewhere.37 The successful formation of thin solid film structures can be observed from the FE-SEM image shown in Supporting Information Figure S3a c. The average thickness of the obtained film was measured to be 41.23 μm (three measurements were made at different places and averaged). The surface properties of the as-fabricated copperglucosamine thin solid film were determined by water contact 8937

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Figure 3. (a, b) FE-SEM, (c, d) HR-TEM, and (e, f) AFM images of GlcN-CuMC’s. (a, c) Scale bar = 1 μm, (b, d) scale bar = 0.1 μm, and (e, f) scale bar = 5.0  5.0 μm2.

angle measurements. It has been demonstrated that hierarchical structures organized by nanostructures on microstructures offer a novel strategy for constructing superhydrophobic surfaces.38 Li et al. synthesized the macroporous copper films by utilizing the hydrogen bubble template method, from which phenomena such as the pore size (or a reduction in pore size) and wall thickness are highlighted to be tunable factors for obtaining a superhydrophobic surface.38 Likewise, in the present study the tightly packed microstructures of the copper-glucosamine film have a reduced pore size that results in an increased contact angle of about 130 ( 8° (Supporting Information Figure S2d). Although the surface of copper is functionalized with the glucosamine moiety, the densely organized microstructures impart a reasonable hydrophobicity to the fabricated film surface. Such biocompatible hydrophobic surfaces are important in technological applications. FT-IR Characterization. An FT-IR spectral study was performed to ensure the successful functionalization of glucosamine

on the surface of the copper microstructure. Powder samples of D-glucosamine (a, black trace) and copper-glucosamine (b, red trace) were utilized for comparative FT-IR analysis (Figure 4). Thereby, structural changes such as bending and stretching vibrations can be distinguished from raw glucosamine and the copper-glucosamine complex. The significant sets of vibration bands observed for D-glucosamine were assigned as follows (cm 1). There is a characteristic short out-of-plane bend at 698 (OH), an intense peak located at 773 assigned as a C C vibration; and peak signals between 856 and 912 associated with the C H out-of-plane bend. The C O stretching of C6 of GlcN is observed at 1029. The shoulder peak stretchings located at 1089 and 1093 were obtained from the C O bond of the C3 (containing a secondary OH) group. The asymmetric stretching located at 1139 is associated with C O C (νCOC). The other peaks located at 1423 correspond to CH2 scissoring, 1618 1537 relates to N H bending, and shifts identified between 2850 and 8938

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Figure 4. FT-IR spectra of (a) D-glucosamine and (b) copper-glucosamine microstructures.

2945 were ascribed to symmetric and asymmetric modes of CH2 group vibrations, respectively. A sharp peak observed at 3282 is assigned to be from N H and hydrogen-bonded OH stretching vibrations.27,28 The FT-IR spectrum of GlcN-CuMC’s (Figure 4b, red trace) exhibits many alterations from that of raw D-glucosamine. Such a change suggests that significant complex formation occurred between the glucosamine and the copper microstructures. The major differences are observed to be the appearance of new sharp peaks at 669 (Cu OH) and 1311 (C H in plane bend). The frequency of vibrational modes attributed to Cu OH functional groups depends on the degree of hydrogen bonding and is found to be located at 669. That was in good agreement with those reported Cu OH frequencies by John et al. and Henrist et al. (676 and 673 cm 1).29,39 Other notable increased intensities were observed at 773 (C C), 881 (C H out-of-plane bend), 1045 (C O stretch), 1118 (C O C), and 1415 (CH2 stretching band). A very weak signal centered at 2893 denotes that the shift and loss in intensities of the band (between 2750 and 3000) are caused by the environmental changes in the protonated amino groups (NH3+).27 Those influenced the symmetrical and asymmetrical modes of CH2 group vibrations of glucosamine. The stretching bands of N H and OH from GlcN-CuMC’s are observed to be broader and more unfolded (with two centered peaks at 3411 and 3541) than those of raw glucosamine. This could possibly be due to the interaction between metallic copper ions and glucosamine. This evinces the event of intra- and intermolecular hydrogen bonding in the coordination chemistry between Cu and glucosamine. Thus, the distinguished FT-IR spectral information from copper-glucosamine and raw D-glucosamine provides us with structural evidence for the successful surface functionalization of the final microstructure. Thermal Gravimetric Analysis (TGA). Figure 5 shows the TGA thermograms of prepared GlcN-CuMC’s. The initial weight loss was observed to occur at 254.32 °C and was attributed to the

Figure 5. Thermal analysis of copper-glucosamine microcubes (GlcNCuMC’s).

condensation of Cu OH and other hydroxyl groups from the surfaces of GlcN-CuMC’s. The weight loss observed for this region is 2.168% (0.4535 mg). The concurrent losses at 388.30 °C and the third-stage weight loss at ∼800 °C were ascribed to the loss of water molecule via the condensation of Cu OH and other hydroxyl groups. Here, the thermal weight losses were observed to be nearly 23.98% (5.017 mg) and 0.8877% (0.1857 mg), respectively. A lower total weight percentile loss of about 27.035% obtained from the thermal analysis suggests that fabricated GlcN-CuMC’s could withstand high temperatures. It has been illustrated that the incorporation of metal and metalloid nano/ microstructures into organic matrices has feasibly enhanced the decomposition of the hybrid component.18,19 The above trace indicates the improved stability of obtained GlcN-CuMC’s compared to that of other copper microcomposites,40 which could also be an additive material in the fabrication of other heterostructures. Design and Electrochemical Behavior of the Au-PCB/GlcNCuMC’s Biosensor Platform. Scheme 2 illustrates the procedure for the surface immobilization of GlcN-CuMC’s, anti-CRP, and 8939

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Scheme 2. Illustration of the Preparation of the Au-PCB/GlcN-CuMC’s Biosensor Platform

Figure 6. Cyclic voltammograms of (a) Au-PCB/GlcN-CuMC’s and (c) Au-PCB/GlcN-CuMC’s/anti-CRP-CRP at different scan rates in 10 mM PBS (pH 7.4) and corresponding plots of peak currents against the square root of scan rates for (b) Au-PCB/GlcN-CuMC’s and (d) Au-PCB/GlcNCuMC’s/anti-CRP-CRP. The concentration ranges for anti-CRP and CRP were 100 μg/mL and 10 ng/mL, respectively.

CRP on Au-PCB. The working electrode (Au-PCB) utilized in the current experiment has 16 gold patterns. Depending on the CV experiment, an appropriate number of electrodes were selectively modified with the prepared samples. Optical microscope images of bare Au-PCB, GlcN-CuMC’s, and GlcN-CuMC’s/anti-CRP-CRPmodified Au-PCB electrodes after CV experiments were morphologically observed and are depicted in Supporting Information Figure S4. Generally, the metal-particle-based detection of biomolecules

such as proteins, DNA, and other small molecules is developed by optical methods such as colorimetry, light scattering, fluorescence, and surface-enhanced Raman scattering (SERS).4,41 44 In addition, the electrochemical properties of metal nanoparticles also play a vital role in several biosensing studies.45 Compared to conventional metallic electrode substrates, chemically modified electrodes containing surface-entrapped catalytic species have shown renowned advantages. The feasibility of modifying metallic particles with an 8940

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Figure 7. (A) Cyclic voltammograms of (two segments after eight cycles are represented) (a) bare Au-PCB, (b) Au-PCB/anti-CRP, (c) Au-PCB/antiCRP-CRP, (d) Au-PCB/GlcN-CuMC’s, (e) Au-PCB/GlcN-CuMC’s/anti-CRP, and (f) Au-PCB/GlcN-CuMC’s/anti-CRP-CRP. (B) Cyclic voltammograms of AuPCB-GlcN-CuMC’s/anti-CRP at various CRP concentrations in 10 mM PBS (pH 7.4).

organic polymer or an inert surface provides a good physical dispersion leading to highly efficient electrocatalysis.46 48 The electrochemical activity of GlcN-CuMC’s was evaluated to assess its applicability as a (transducer material) platform for the electrochemical biosensor. Figure 6a presents the CV curves of GlcN-CuMC’s in PBS solution at scan rates of 0.20, 0.25, 0.30, 0.35, and 0.40 V/s. The current signals and peak-to-peak potential separation (ΔEp) were increased as a function of the scan rate. In particular, GlcN-CuMC’s ensured the linear relationship of Ip versus ν1/2. As shown in Figure 6b, a perfect scaling of steady-state currents in GlcN-CuMC’s (r2 is 0.9703 and 0.9173 for Ipa and Ipc, respectively) suggests that the interfacial properties of the modified electrode governed the necessary conduction pathways and electron transfer between the electroactive components. The changes in the peak current signals by the immobilization of anti-CRP-CRP should be clarified to demonstrate the detection range of GlcN-CuMC’s on a Au-PCB-based biosensor. Figure 6c shows the CV curves of Au-PCB/GlcN-CuMC’s after the immobilization of anti-CRP-CRP at various scan rates (0.20 to 0.40 V/s). The significant enhancement in the peak current was in relation to the scan rate, and the linear relationship for these CV curves was found to be 0.9862 (Ipa) and 0.9972 (Ipc). To investigate the effect of GlcN-CuMC’s on the function for the detection of CRP, CV curves of bare Au-PCB, Au-PCB/anti-CRP, and Au-PCB/anti-CRP-CRP were obtained in the presence and absence of GlcN-CuMC’s modification, as shown in Figure 7A. The bare Au-PCB, anti-CRP, and CRP-modified Au-PCB did not show any characteristic voltammograms for the immobilization of anti-CRP and CRP in the potential region used in the investigation. However, the GlcN-CuMC’s modified Au-PCB electrode exhibited more characteristic voltammetric responses to the current signals and ΔEp compared to bare Au-PCB because of the high surface area to volume ratio and facile accessibility to surface-modified metallic microstructures. The biocompatible layer provided by hybrid GlcN-CuMC’s advanced the specific immobilization of chemical linkers (APTES and GTA), anti-CRP and CRP, respectively. The favorable interfacial characteristics from the hybrid GlcN-CuMC’s possess a strong electrochemical response toward the adsorption of CRP at the anti-CRP-modified electrode. This results in the increased peak current and a shift in the cathodic peak (Figure 7A(f)). Moreover, four different concentrations of CRP were prepared (10, 3.33, 1.11, and 0.37 ng/mL) and utilized for sequential modification on an appropriate electrode

surface. As shown in Figure 7B, the peak current increases with increasing concentration of CRP. This further confirms that GlcNCuMC’s modification can enhance the signal response in a concentration-dependent manner. The electrochemical response (redox signal) obtained from the GlcN-CuMC’s on the electrode surface can be explained by its chemical structures. As discussed before regarding the surface functionalization of glucosamine on Cu microcubes, a chemically modified metallic species allows electrical contact between the redox centers in adsorbing biomolecules and the electrode surface, thereby providing an important sensing interface for electroanalysis. We consider that the GlcN-CuMC’s modified Au-PCB electrode was more apparent than that of the bare Au-PCB electrode and it acts as a very efficient transducer material for the immobilization of CRP. The detection limit of Au-PCB/GlcN-CuMC’s for CRP by the current devised protocol was found to be 0.37 ng/mL, which is comparatively more sensitive than other CV-based biosensors reported for the detection of CRP (Supporting Information Table S1). An extended study is in progress to explore its further biosensing properties and cytotoxicity, and we hope to report the results in the near future.

’ CONCLUSIONS Shape-controlled microstructures of glucosamine-surfacemodified copper have been obtained by the integration of the injection pump and ultrasonochemistry. A possible mechanism involving the cooperation of chelation, the in situ coordination of glucosamine, and copper microstructures has been proposed for the integrated strategy-assisted synthesis of Cu microcubes. In addition to metallic Cu peaks, the powder XRD shows the formation of new crystalline phase and the Cu2O phase that are formed by glucosamine and radicals from (water) ultrasonication. The size, shape, and distribution of the copper-glucosamine microcubes are very dependent on the reaction temperature and ultrasonication parameters. Morphological studies revealed that the derived microcubes are uniform in size, with sharp faces. As-obtained copper-glucosamine ensures its high-temperature stability. The surface immobilization of copper-glucosamine microcubes on the Au-PCB electrode is capable of enhancing the electrochemical activity and exhibits a characteristic voltammetric response against anti-CRP/CRP interaction. The results obtained from the primitive electrochemical detection of CRP are superior to that of the conventional Au-PCB electrode. The biocompatible synthesis strategy, shape-controlled microstructures, thermal 8941

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Langmuir stability, and electrochemical properties of the obtained hybrid structure will direct us to several applications and developments in micro/nanobiotechnology. More experiments are underway to determine the interdisciplinary applications of these microstructures.

’ ASSOCIATED CONTENT

bS

Supporting Information. EDS spectrum, 2D AFM amplitude image and line profile of GlcN-CuMC’s, FE-SEM images and water contact angle of a GlcN-CuMC’s thin solid film, optical microscope image of bare Au-PCB and the Au-PCB electrode after the sequential deposition of GlcN-CuMC’s and anti-CRPCRP, calibration plot, and a comparison of the detection performance. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: (K.Y.) [email protected] and (M.-H.L.)mhlee@ keti.re.kr.

’ ACKNOWLEDGMENT This research was supported by the Kyungwon University Research Fund in 2011, the Regional Technology Innovation Program of the Ministry of Knowledge Economy (grant no. 10032112 to K.-S.Y.), and the International Collaborative R&D Project for Semiconductor from the Ministry of Knowledge Economy (grant no. 10030694 to M.-H.L.). ’ REFERENCES (1) Arul Dhas, N.; Paul Raj, C.; Gedanken, A. Chem. Mater. 1998, 10, 1446–1452. (2) Huang, H. H.; Yan, F. Q.; Kek, Y. M.; Chew, C. H.; Xu, G. Q.; Ji, W.; Oh, P. S.; Tang, S. H. Langmuir 1997, 13, 172–175. (3) Cai, H.; Zhu, N.; Jiang, Y.; He, P.; Fang, Y. Biosens. Bioelectron. 2003, 18, 1311–1319. (4) Subramanian, T.; Timothy Thatt, Y. T.; Dong Kee, Y.; Nikhil, R. J. Langmuir 2010, 26, 11631–11641. (5) Wu, S. H.; Chen, D. H. J. Colloid Interface Sci. 2004, 273, 165–169. (6) Abdulla-Al-Mamun, M.; Kusumoto, Y.; Muruganandham, M. Mater. Lett. 2009, 63, 2007–2009. (7) Joshi, S. S.; Patil, S. F.; Iyer, V.; Mahumuni, S. Nanostruct. Mater. 1998, 10, 1135–1144. (8) Yeh, M. S.; Yang, Y. S.; Lee, Y. P.; Lee, H. F.; Yeh, Y. H.; Yeh, C. S. J. Phys. Chem. 1999, 103, 6851–6857. (9) Narayanan, K. B.; Sakthivel, N. Adv. Colloid Interface Sci. 2010, 156, 1–13. (10) Thakkar, K. N.; Mhatre, S. S.; Parikh, R. Y. Nanomed. NBM 2010, 6, 257–262. (11) Absar, A.; Satyajyoti, S.; Islam Khan, M.; Rajiv, K.; Murali, S. Langmuir 2003, 19, 3550–3553. (12) Ju, H. W.; Koh, E. J.; Kim, S. H.; Kim, K. I.; Lee, H.; Hong, S. W. J. Plant. Physiol. 2009, 166, 203–212. (13) Murugan, V.; Kyusik, Y. Synth. React. Inorg., Met.-Org., NanoMet. Chem. 2010, 40, 56–64. (14) Murugan, V.; Suk-Kyung, L.; Hyang-Mi, N.; Gobianand, K.; Kyusik, Y. Anal. Bioanal. Chem. 2010, 398, 867–876. (15) Jiang, W.; Papa, E.; Fischer, H.; Mardyani, S.; Chan, W. C. W. Trends Biotechnol. 2004, 22, 607–609. (16) Tseng, R. J.; Tsai, C.; Ma, L.; Ouyang, J.; Ozkan, C. S.; Yang, Y. Nat. Nanotechnol. 2006, 1, 72–77.

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