Polymer-Functionalized Silica Nanosphere Labels for Ultrasensitive

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Polymer-Functionalized Silica Nanosphere Labels for Ultrasensitive Detection of Tumor Necrosis Factor-alpha Liang Yuan, Xin Hua, Yafeng Wu, Xiaohu Pan, and Songqin Liu* State Key Laboratory of Bioelectronics, School of Chemistry and Chemical Engineering, Jiangning District, Southeast University, Nanjing, 211189, People’s Republic of China

bS Supporting Information ABSTRACT: A signal amplification strategy for sensitive detection of tumor necrosis factor-alpha (TNF-R) using quantum dots (QDs)-polymer-functionalized silica nanosphere as the label was proposed. In this approach, silica nanospheres with good monodispersity and uniform structure were employed as carriers for surface-initiated atom transfer radical polymerization of glycidyl methacrylate, which is readily available functional monomer that possessing easily transformable epoxy groups for subsequent CdTe QDs binding through ring-open reaction. Then, human anti rabbit TNF-R antibody (anti-TNF-R, Ab2, served as a model protein) was bonded to CdTe QDsmodified silica nanospheres coated with polymer to obtain QDs-polymer-functionalized silica nanosphere labels (Si/PGMA/QD/ Ab2). The Si/PGMA/QD/Ab2 labels were attached onto a gold electrode surface through a subsequent “sandwich” immunoreaction. This reaction was confirmed by scanning electron microscopy (SEM) and fluorescence microscopic images. Enhanced sensitivity could be achieved by an increase of CdTe QD loading per immunoassay event, because of a large number of surface functional epoxy groups offered by the PGMA. As a result, the electrochemiluminescence (ECL) and square-wave voltammetry (SWV) measurements showed 10.0- and 5.5-fold increases in detection signals, respectively, in comparison with the unamplified method. The detection limits of 7.0 pg mL
1 and 3.0 pg mL
1 for TNF-R antibodies by ECL and SWV measurements, respectively, were achieved. The proposed strategy successfully demonstrated a simple, reproducible, specific, and potent method that can be expanded to detect other proteins and DNA.

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NF-R is an extremely potent inflammatory peptide cytokine produced by cells of the immune system. It is involved in a wide range of pathological and physiological processes.1,2 Generally, TNF-R is present at very low concentrations in the subpM range in healthy human blood, whereas a several-fold increase is observed in septic patients. When overproduced, the TNF-R plays a major role in chronic inflammatory diseases such as rheumatoid arthritis, psoriasis, and Crohn’s disease.4
7 It has also been linked to conditions such as septic shock syndrome, diabetes, and preeclampsia.8
10 This allows TNF-R to be an early stage indicator of an inflammatory reaction, in response to infection or cancer.3 Therefore, the development of sensitive methods for the detection of TNF-R is very important for the understanding of biological infection processes, inherent mechanisms, and drug discovery. Some methods, such as enzymelinked immunosorbent assays (ELISA),11,12 radioimmunoassay,13 immuno-PCR assay,14 chemiluminescence imaging,15 fluorescence immunoassays,16
18 chemiluminescence assays,19,20 electrophoretic immunoassays,21 and electrochemical immunoassays22 have been developed for TNF-R detection. Graham et al.12 proposed a novel method by replacing the traditional colorimetric detection with resonance Raman spectroscopy for sensitive detection of TNF-R. In their work, horseradish peroxidase (HRP) bounded to the detection antibody catalyzed the r 2011 American Chemical Society

oxidation of 3,30 ,5,50 -tetramethylbenzidine (TMB) by hydrogen peroxide, which tuned the Raman wavelength in resonance with an electronic excited state of the oxidized TMB. Consequently, the relative intensity of the enhanced Raman band was proportional to the amount of TMB. Via this approach, the sensitivity can be improved 50-fold, compared to that of the traditional colorimetric detection by a conventional ELISA kit. Niwa and co-workers reported a highly sensitive approach for TNF-R detection.20 The ELISA method was performed in a 96-well microtiter plate, which was modified with an anti TNF-R antibody for capture. After a sandwiched immunoassay procedure, the biotinylated anti TNF-R antibody was captured through the antigen
antibody interaction. The avidin-acetylcholinesterase conjugate then was immobilized in the well of the microtiter plate through biotin
avidin interaction, which converted acetylthiocholine to thiocholine. The thiocholine was subsequently collected on the surface of a gold electrode through the Au
S bond, which produced a bright ECL emission as a coreactant in the presence of Ru(bpy)32+. Very recently, some nanoparticles have also been used for signal amplification in the TNF-R assay. Lin and co-workers Received: June 20, 2011 Accepted: August 1, 2011 Published: August 01, 2011 6800

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Analytical Chemistry reported an electrochemical immunosensor for the detection of TNF-R based on poly-(guanine)-functionalized silica nanoparticle (NPs) labels on a dual-signal amplification.23 Through the sandwich immunoreactions, the TNF-R antibody coupled with the poly(guanine)- and avidin-functionalized silica NP labels was introduced onto the electrode surface. As a result, a significant enhancement of anodic current produced by Ru(bpy)32+ inducing catalytic oxidation of guanine was observed. This was due to a large amount of guanine residues that had assembled onto the electrode surface. Zhu and co-workers22 described a novel immunoassay method using functionalized gold nanoparticles/ poly(styrene
acrylic acid) (GNPs/PSA) as the label for the sensitively detection of TNF-R. The multienzyme functionalized GNPs/PSA label was constructed by conjugation of alkaline phosphatase (ALP) to the colloidal gold-coated PSA nanospheres. Biocompatible polyaniline doped with poly(acrylic acid) was electropolymerized at the glass carbon electrode for the immobilization of TNF-R antibody. After the sandwich immunoreactions, the attached ALP enzyme on electrode catalyzed the hydrolysis of R-naphthyl phosphate to produce the corresponding electroactive R-naphthol, which allowed monitoring of the quantity of antigen by the electrochemical signals of R-naphthol. On the other hand, polymer nanocomposites represented an attractive family of composite materials in which the nanometersize reinforcing fillers were uniformly dispersed in the polymer, at a nanometer scale, compared to conventional phase-separated macrocomposites. Because of the interesting mechanical, electronic, optical, catalytic, and magnetic characteristics of the polymer nanocomposites, surface-initiated polymerization has also been developed into a powerful strategy for modifying and tailoring the surface properties of metal and semiconductor nanospheres, oxide nanospheres, oxide nanorods, and carbon nanotubes toward nanostructured functional materials.24 The grafted fibrous polymer chains increased the adsorption capacity of the support by allowing multilayer protein binding.25
28 Surfaceinitiated atom transfer radical polymerization (SI-ATRP) is one of the most investigated of controlled graft polymerization methods, because it can be performed with a variety of functional monomers at mild temperatures, and in aqueous or organic solvents.29
31 Among the various chemical functional groups that could be integrated into polymers, the epoxy group is one of the most attractive, because it can be readily modified using various chemical reactions to introduce various functional moieties.32,33 Epoxy groups undergo a ring-opening reaction with various compounds that possess hydroxyl, amine, or activated methylene groups.34,35 For this reason, polymers with epoxy groups offer numerous modification possibilities under mild reaction conditions.36,37 Meanwhile, long chain polymeric materials with numerous chemically modifiable functional groups were capable of providing extra redox tags in the same fashion. Indeed, the use of polymer films to increase the loading of capture probes has been employed routinely.38
41 The growth of long-chain polymeric materials provided numerous sites for subsequent aminoferrocene coupling, which, in turn, significantly enhanced signal output. Finally, the detection limits of 15 pM and 0.07 ng mL
1, for DNA and ovalbumin, respectively, were obtained.42 The present work was also motivated by very promising applications of the nanostructured materials for biosensing. To verify the signal amplification, a CdTe QDs and polymerfunctionalized silica nanosphere label was first fabricated according to the steps of SI-ATRP of glycidyl methacrylate (GMA). Other options included covalent binding with CdTe QDs to

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PGMA-coated silica microspheres through ring-open reaction in aqueous phase, and covalent binding of the TNF-R antibody to CdTe QDs. Enhanced sensitivity could be achieved by increasing the CdTe QDs loading per immunoassay event.

’ EXPERIMENTAL SECTION Materials. Gold substrates (50 Å of chrome, followed by 1000 Å of gold on float glass) were purchased from Evaporated Metal Films (Ithaca, NY). Rabbit tumor necrosis factor-alpha antigen (TNF-R, Ag, 2 μg mL
1) and human anti rabbit TNF-R antibody (anti-TNF-R, Ab, 200 μg mL
1, polyclonal) were obtained from Boster Biological Technology Co., Ltd. (Wuhan, PRC). Human R-fetoprotein antigen (AFP) and carcinoembryonic antigen (CEA) were received from Boson Biotech Co. Ltd. (Xiamen, PRC). Bovine serum albumin (BSA) was obtained from SunShine BIO (Nanjing, PRC). 2-Bromoisobutyryl bromide (BriBuBr, 98%), glycidyl methacrylate (GMA), 1-ethyl-3-(3dimethylaminopropyl) carbodiimide hydrochloride (EDC), Nhydroxysuccinimide (NHS), (3-aminopropyl)-triethoxysilane (APTES), and copper(I) bromide (CuBr, 98%) were purchased from Sigma
Aldrich (Shanghai, PRC). Tetraethoxysilane (TEOS) was obtained from Zhang-Jiagang Guotai-Huagong New Chemical Materials Co., Ltd. (Zhang-Jiagang, PRC). o-Aminobenzoic acid was a gift from Xingshengchem (Yancheng, PRC). CuBr was purified by stirring in acetic acid, washing with acetone, and drying under vacuum. Glycidyl methacrylate (GMA) was purified inhome and passed through a column with activated Al2O3 (Aldrich, neutral, Brockmann I, standard grade, ∼150 mesh, 58 Å) to remove the inhibitor before polymerization. All other chemicals were of analytical grade and were used as received. 0.1 M phosphate buffer solutions (PBS) were prepared by mixing 0.1 M NaH2PO4 and Na2HPO4. Twice-distilled water was used throughout the study. Apparatus. Electrochemiluminescence (ECL) measurements were carried out on a MPI-E multifunctional electrochemiluminescent analytical system (Xi’an Remex Analyze Instrument Co., Ltd., PRC). All ECL measurements were performed in a 5-mL glass cell comprised of a platinum wire auxiliary electrode, a saturated calomel electrode (SCE) reference electrode, and a modified Au working electrode. Cyclic voltammetry (CV) and square-wave voltammetry (SWV) measurements were conducted with a CHI Instruments Model 830C electrochemical workstation (Shanghai Chenhua Instrument Co., Ltd., PRC). A conventional three-electrode system for SWV detection consisted of a modified bismuth film modified glassy carbon electrode (BFE), a platinum wire, and a saturated calomel electrode (SCE) as working, auxiliary, and reference electrodes, respectively. Fourier transform infrared spectroscopy (FTIR) was performed on a Bruker Tensor 27 instrument using dry KBr pellets. The morphology and sizes of various nanospheres were analyzed using a transmission electron microscopy (TEM) system (Model S-2400N, Hitachi, Japan) and a scanning electron microscopy (SEM) system (Model 1530 VP, LEO, Germany) with an acceleration voltage of 10 kV. The fluorescence microscopy images were obtained from an Olympus Model IX71 inverted optical microscope (Olympus, Japan). A Hewlett
Packard Model HP 8453E ultraviolet
visible light (UV-Vis) spectrophotometer was used to monitor QD-functionalized silica nanoparticles. Photoluminescence (PL) spectra were obtained on a Model FluoroMax-4 spectrophotometer (Horiba, Japan). X-ray 6801

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Analytical Chemistry photoelectron spectroscopy (XPS) (Model PHI5000 VersaProbe, ULVAC-PHI, Japan) was used to probe the binding nature of Ab2 with CdTe QD-capped silica nanoparticles. Synthesis of Antibody/QDs/PGMA-Coated SiO2 Nanosphere Labels (Si/PGMA/QD/Ab2). Synthesis of Amino-Functionalized Silica Particles (SiO2
NH2). Monodispersed SiO2 nanoparticles were synthesized according to our previously reported seed-growth method.48
50 The diameters of the assynthesized silica nanoparticles were 110 ( 3.0 nm, determined by TEM microscopy. As-synthesized silica nanoparticles (0.4 g), toluene (5 mL), and 3-aminopropyltriethoxysilane (APTES) (1 mL) were placed in a 10-mL glass tube. After the solutions were degassed, by purging with N2 for 20 min, the reaction tube was immersed in an oil bath at 95 °C. The mixture was stirred overnight; then, the solution was cooled and exposed to air, washed with abundant toluene and acetone in turn, and, finally, the resulting product (SiO2
NH2) was dried under vacuum at room temperature for 12 h. Immobilization of Initiators on SiO2 Nanoparticles (SiO2
Br). The as-synthesized amino-functionalized silica particles were then dispersed in a solution of triethylamine (4 mL) and freshly distilled toluene (10 mL) while under magnetic stirring. After the mixture was cooled to 0 °C in an ice
water bath, a solution of 2-bromoisobutyryl bromide (2 mL) and toluene (4 mL) was added drop by drop into the solution. After 4 h of reaction, the solution was thoroughly washed by ultrasound with an abundant amount of acetone
water solution (v/v = 1/1). It was then washed by toluene, then acetone, and dried under vacuum at room temperature; the SiO2
Br was thus obtained for the subsequent polymerization. Surface-Initiated ATRP Polymerization on SiO2 Nanoparticles (Si/PGMA). An amount of 0.2 g of SiO2
Br was resuspended in a mixture solution of 4 mL of dimethyl formamide (DMF) and 2 mL of glycidyl methacrylate (GMA). After bubbling the mixture with high-purity nitrogen (99.99%) for 30 min, 20 mg of CuBr and 64 mg of bpy (CuBr/bpy =1:2, molar ratio) were added to trigger the ATRP polymerization. The polymerization was performed under a nitrogen atmosphere while being continuously stirred at room temperature for 2 h. The solution became progressively viscous, which indicated the onset of polymerization. The polymerization was terminated by an instant injection of a large amount of DMF in air. The PGMAcoated silica microspheres were then washed with five cycles of centrifugation and then redispersion in DMF and absolute ethanol. Finally, the sample was dried under vacuum to obtain PGMA-coated silica microspheres (Si/PGMA). Binding of CdTe QDs to PGMA-Coated Silica Nanoparticles (Si/PGMA/QD). Aqueous CdTe QDs capped with mercaptocarboxylic acid (MPA) was prepared using procedures that have been described previously.51 For the preparation of CdTe QDmodified PGMA-coated silica nanoparticles, 1 mL of CdTe QDs (5 mg mL
1) was added dropwise to an aqueous suspension of PGMA-coated silica microspheres (5 mg mL
1) under continuous stirring. The reaction occurred at room temperature over 2 h. The carboxylic groups on the surface of CdTe QDs could react with the epoxy groups in PGMA through ring-open reaction, which led to the binding of CdTe QDs to PGMAcoated silica nanoparticles. The resultant silica nanoparticles were collected by centrifugation and washed with water several times. Simultaneously, unbound CdTe QDs were removed. Finally, the as-prepared Si/PGMA/QD nanospheres, which had the same orange color as the CdTe QDs themselves, were

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Scheme 1. Schematic Representation of the Preparation of the Si/PGMA/QD/Ab2 Label

Scheme 2. Schematic Representation of the Sandwich Immunoassay with Si/PGMA/QD/Ab2 as the Label

obtained and dispersed in water to a final volume of 1 mL for the next use. Synthesis of Nanoimmunological Label (Si/PGMA/QD/Ab2). To generate nanoimmunological labels, 1 mL of the above Si/ PGMA/QD suspension was mixed with 1 mL of Ab2 solution (anti-TNF-R, 10 μg mL
1 in 0.01 M pH 7.4 PBS). Then, 100 μL of freshly prepared EDC (10 mg mL
1 in 0.1 M pH 7.4 PBS) and 100 μL of NHS (10 mg mL
1 in 0.1 M pH 7.4 PBS) were added. The incubation was conducted at room temperature for 2 h. Subsequently freed antibodies were removed by centrifugation and were washed with 0.01 M PBS several times to obtain the Ab2-modified Si/PGMA/QD nanoparticles (Si/PGMA/QD/Ab2). Finally, Si/ PGMA/QD/Ab2 nanoparticles were redispersed by stirring in 5 mL of 1% BSA solution over 2 h, to block the excess amino groups and nonspecific binding sites of the Si/PGMA/QD/Ab2 nanospheres. The resultant Si/PGMA/QD/Ab2 nanoparticles were acquired by centrifugation and washed with PBS. They were then dispersed with 0.01 M of pH 7.4 PBS to a final volume of 2 mL and stored at 4 °C for later use. The entire process for construction of the Si/PGMA/ QD/Ab2 label is illustrated in Scheme 1. For control experiments, CdTe QD-coated silica nanoparticles (Si/QD) were prepared via the dispersion of 0.02 g of silica nanospheres in 2 mL of ethanol, and then treated with 0.4 mL of APTES. After stirring for 6 h, the suspension was centrifuged and washed with ethanol repeatedly for four times. The as-prepared amino-functionalized nanoparticles then were dispersed in a mixture of 1 mL of CdTe QDs (5 mg mL
1) and 100 μL of EDC (20 mg mL
1). The mixed suspension was stirred at 4 °C for 12 h. After unbound QDs were removed by successive centrifugation and several washes with water, the as-prepared Si/QD nanospheres were obtained and dispersed in water to a final volume of 1 mL. 6802

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Analytical Chemistry Preparation of the Anti-TNF-r-Modified Au Substrates. Scheme 2 illustrates the preparation of the anti-TNF-R-modified Au substrates. For the anti-TNF-R immobilization, a poly(o-ABA) (PAB) film was electropolymerized on the gold electrode surface. Briefly, a clean electrode was dipped in a 1 M H2SO4 solution containing 50 mM o-ABA and scanned in a potential range of 0
1.0 V for 10 cycles at a scan rate of 40 mV s
1.38 After being removed from solution, the PAB-modified electrode was rinsed with water three times, immersed in water for 30 min to remove any physically adsorbed materials, and then dipped in 200 μL of EDC/NHS solution (20 mg mL
1 EDC and 10 mg mL
1 NHS in 0.1 M pH 7.4 PBS) for another 30 min. After being washed thoroughly with water, 10 μL of anti-TNF-R (10 μg mL
1, in 0.01 M pH 7.4 PBS, denoted Ab1) was dropped onto the surface of PAB-modified Au electrode. After 4 h of incubation at 4 °C, the electrode was washed thoroughly with buffer (0.05 M phosphate buffer containing 0.1% v/v Tween, pH 7.4). The electrode was soaked in 1% BSA solution at 4 °C for 30 min to block excess active groups and nonspecific binding sites on the electrode surface. The anti-TNF-R-modified Au electrode (Ab1-PAB-Au) was then stored at 4 °C for subsequent use. Sandwich Immunoassay with Si/PGMA/QD/Ab2 as the Label. The Ab1-PAB-Au was incubated with the desired concentration of TNF-R solution (Ag, 100 μL) at 37 °C for 45 min to capture Ag with the first immunoreaction (Ag-Ab1-PAB-Au). After being thoroughly washed with PBS, the Ag-Ab1-PAB-Au was exposed to 0.5 mL of Si/PGMA/QD/Ab2 suspension for another 45 min at room temperature. The exposure introduced the Si/PGMA/QD/Ab2 label onto the electrode surface through the second immunoreaction. The electrode was thoroughly rinsed again and was gently shaken in PBS to remove physically adsorbed Si/PGMA/QD/Ab2 nanoparticles. Finally, the sandwich immunoassay was completed, and the Si/PGMA/QD/Ab2modified Au electrode (Si/PGMA/QD/Ab2-Ag-Ab1-PAB-Au) was thus obtained. With the use of the same procedure, Ab1-PAB-Au was directly incubated in Si/PGMA/QD/Ab2 suspension without prior exposure to the TNF-R antigen solution for the control experiment. ECL Detection. The electrolyte was a solution of 0.05 M pH 7.4 PBS that contained 0.05 M KCl and 0.05 M K2S2O8. The potential range applied to the Au working electrode in the CV measurement was from 0 V to
1.7 V, at a scan rate of 100 mV s
1. The emission window was placed in front of the photomultiplier tube, which was biased at 1000 V. Standard curves were plotted as ECL intensity versus logarithm of TNF-R concentration. From the standard curves, we can obtain the 50% inhibition value (IC50). Fabrication of Bismuth Film Modified Glassy Carbon Electrode (BFE). Bismuth film was electrodeposited on the surface of clean glassy carbon electrode for 120 s by a deposition potential of
1.0 V (versus SCE) under stirring. The electrolyte was a solution of bismuth nitrate in acetate with a final pH of 2.0 and Bi(III) ion concentration of 1.25 mg mL
1. Stripping Voltammetric Analysis. Si/PGMA/QD/Ab2-AgAb1-PAB-Au was immersed in 200 μL of 0.1 M glycine-HCl (pH 2.2) solution for 10 min. It then was washed by 0.1 M PBS three times and diluted with 0.1 M pH 7.0 PBS to a final volume of 3 mL. The analytical procedure involved three rounds of 120 s electrodeposition at
1.2 V. After that, it was stripped by scanning from
1.3 V to
0.5 V, using SWV measurements with potential steps of 4 mV, a frequency of 15 Hz, and an amplitude of 25 mV.

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Figure 1. High-magnification TEM images of (A) silica microspheres and (B) CdTe QDs
polymer-modified silica microspheres Si/PGMA/QD.

’ RESULTS AND DISCUSSION Characterization of the Si/PGMA/QD/Ab2 Label. In the present work, SiO2 nanoparticles were employed as carriers for ATRP initiators, subsequent polymerization, and CdTe QDs immobilization. Thus, silica nanoparticles with good monodispersion and similar surface morphology were vital for consistent loading of the same amount of initiators on each microsphere. This, in turn, influenced the loading of the QDs and TNF-R antibody, and the sensitivity, reproducibility, and analytical performance of the resultant immunosensor. Here, the silica nanospheres were synthesized with our previously reported seed-growth method.43,44 The resultant silica nanospheres were chemically clean and displayed the homogenized structure of a nanosphere. The diameter of the nanospheres was ∼110 ( 3.0 nm, which was demonstrated by TEM images (see Figure 1A). To achieve a polymer-coated shell, a uniform and densely packed monolayer of APTES was first generated on the surface of silica nanospheres. It was then reacted with 2-bromoisobutyryl bromide to immobilize the polymerization center on the surface of the silica nanosphere. Subsequently, surface-initiated ATRP of GMA was performed on these microspheres to form PGMA-coated shells, using CuBr/bpy system in DMF at room temperature. The successful ATRP process of PGMA-coated silica nanospheres was verified by FTIR (see Figures S1 and S2 in the Supporting Information). The coating of CdTe QDs on the Si/ PGMA nanospheres could also be demonstrated by the color change under sunlight and UV illumination (λex = 365 nm), as well as UV
vis and PL spectra (see Figures S3 and S4 in the Supporting Information). When PGMA coating shells were formed on the SiO2 surface, no obvious color change was observed between the Si/PGMA and bare SiO2 nanoparticles. However, after being coated with CdTe QDs, the color changed, and Si/PGMA/QD showed the same color as the CdTe QDs in water. In addition, the resultant nanospheres displayed an absorption peak at 480 nm in UV
vis spectra and a strong PL emission peak at 516 nm (the λex value was 350 nm), which were also consistent with the CdTe QDs themselves. It was indicated that the Si/PGMA/QDs retained the properties of the CdTe QDs. In addition, the TEM images could be used to confirm the successful binding of the CdTe QDs to the silica nanospheres (see Figure 1B). After coating with QDs, the attached small nanoparticles with a probable uniform distribution were clearly observed on the Si/PGMA surface. Meanwhile, compared to the clean surface of SiO2 shown in Figure 1A, partial adhesion between particles and polymer brush generated on the surface of silica nanospheres was observed after the PGMA coating. This 6803

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Analytical Chemistry

Figure 2. (A) SEM and (B) fluorescence microscopic images of an Ab1PAB-modified Au substrate exposed to 10 ng mL
1 TNF-R solution, followed by Si/PGMA/QD/Ab2 suspension. The excited wavelength was 488 nm for fluorescence microscopic images.

suggested that a successful polymerization reaction occurred. All these results confirmed that CdTe QDs had been successfully implanted onto the surface of Si/PGMA with good dispersivity, which avoided a common caveat of small nanoparticles as biological labels where particles formed agglutinations.45 The unreacted carboxyl groups located on CdTe QDs surface could be further coupled to the amino groups of TNF-R antibody through acrylamide binding in the presence of EDC and NHS as activating reagents. This caused the TNF-R secondary antibody to be loaded on Si/PGMA/QD. Attachment of the antibody did not affect PL emission of the QDs coated on the Si/PGMA/QD (see Figure S4-B in the Supporting Information). The binding of Ab2 to Si/PGMA/QD was demonstrated by XPS spectra. The XPS spectrum of the Si/PGMA/QD exhibited the Cd 3d2/5 peak signal at the binding energy of 405.1 and 411.8 eV (see Figure S5 in the Supporting Information). A weaker N 1s peak was also displayed at 399.8 eV, because of the N
H group in the APTES. After further coupling with the Ab2, the XPS spectra showed two smaller Cd 3d2/5 peaks and one enhanced N 1s peak at the same binding energy as Si/PGMA/QD. This was due to the coating of antibodies on the surface of the QDs. In addition, the change of elemental compositions for several elements before and after being Ab2-coated (without BSA blocking process) could be used to support this conclusion. The elemental compositions for carbon (C 1s), nitrogen (N 1s) and cadmium (Cd 3d2/5) were 95.42%, 2.4%, and 2.18% before being Ab2-coated and 95.06%, 4.01%, and 0.93% after being Ab2-coated. It was suggested that coupling of Ab2 on Si/PGMA/QD resulted in an increase in the

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relative elemental compositions of nitrogen, and a decrease in the irrelative elemental compositions of cadmium. All these facts confirmed that the Ab2 and CdTe QDs had been successfully attached on the surface of silica nanospheres. Sandwich Immunoassay Using Si/PGMA/QD/Ab2 as the Label. The process of the sandwiched immunoassay using Si/ PGMA/QD/Ab2 as the label was outlined in Scheme 2. Specifically, PAB film with abundant carboxyl groups was electrodeposited on the gold surface for antibody coupling. The antibody-coated electrode then was used to capture TNF-R from the TNF-R-containing solution through the first immunoreaction. The sandwiched immunoassay process was demonstrated by its electrochemical response to redox active species, Fe(CN)63
/4
, and the electrochemical impedance spectroscopy measurements (see Figures S6 and S7 in the Supporting Information). The sandwiched immunoassay with Si/PGMA/ QD/Ab2 labels could be further confirmed by SEM and fluorescence images (excited at a blue-light excitation corresponding to 488 nm), as shown in Figure 2. Several particles, singly or mildly aggregated, were observed in Figure 2A for Si/PGMA/ QD/Ab2-Ag-Ab1-PAB-Au, at a TNF-R concentration of 10 ng mL
1. The diameter of an individual nanosphere was ∼110 nm. Corresponding to SEM images, some small green fluorescence spheres were viewed in fluorescence microscopy (see Figure 2B). While the SEM images for both bare Au electrode and PAB-Au displayed plane uniform structures (see Figure S8 in the Supporting Information), no bright spots were observed from the fluorescence microscopy of Ag-Ab1-PABAu (see Figure S9 in the Supporting Information). In control experiments, the Ag/Ab1/PAB/Au was incubated in a Si/PGMA/ QD suspension without coupling with Ab2, the Ab1/PAB/Au was incubated in Si/PGMA/QD/Ab2 suspension without prior exposure to the TNF-R-containing solution, and Ab1/PAB/Au was incubated in 20 ng mL
1 AFP and 20 ng mL
1 CEA, respectively. This was followed by soaking in the Si/PGMA/QD/Ab2 suspension. No particles were observed in either case in the SEM or fluorescence microscopic images (data not shown). All of the above confirmed that the immobilized particles were from a highly specific immunoassay, and the CdTe QDs kept their fluorescence properties during the immunoreaction. More quantitative measurements could be carried out in the following electrochemical and ECL analyses. Signal Amplification in ECL Detection Using Si/PGMA/QD/ Ab2 as the Label. The sandwiched immunassay used Si/ PGMA/QD/Ab2 as label was also demonstrated by ECL measurements. Figure 3A showed the ECL signal (curve “e” in the figure) of Si/PGMA/QD/Ab2-Ag-Ab1-PAB-Au in air-saturated 0.05 M pH 7.4 PBS that contained 0.05 M KCl and 0.05 M K2S2O8. The corresponding CVs also are shown (see Figure 3B). A large increase of ECL emission intensity at
1.68 V was highlighted from Si/PGMA/QD/Ab2-Ag-Ab1-PAB-Au (Figure 3A, curve “e”), whereas no obvious increase of ECL emission could be observed from bare gold (Figure 3A, curve “a”), PAB-Au (data not shown), Ab1-PAB-Au (data not shown), and Ag-Ab1-PABAu at a TNF-R concentration of 1 ng mL
1 (Figure 3A, curve “b”). Therefore, the enhanced ECL emission intensity was attributed to the attached Si/PGMA/QD/Ab2 on the electrode surface, which reacted with S2O82
. To support this opinion, a series of control experiments were conducted. No discernible difference could be discriminated after incubation of Ag-Ab1-PABAu in 1 ng mL
1 of Ab2 solution without QDs (Figure 3C, curve “a”); Ab1-PAB-Au in 0 ng mL
1 TNF-R (Figure 3C, curve “b”) 6804

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Figure 3. (A) ECL intensity of CdTe QDs versus potential obtained at bare gold (curve a), Ab1-PAB-Au incubated in 1 ng mL
1 TNF-R solution (curves b
e), followed by QD/Ab2 suspension (curve c), Si/QD/Ab2 suspension (curve d), and Si/PGMA/QD/Ab2 suspension (curve e). (B) CV of Si/PGMA/QD/Ab2-Ag-Ab1-PAB-Au at a TNF-R concentration of 1 ng mL
1 at 50 mV s
1. (C) ECL response of Ag-Ab1-PAB-Au at a TNF-R concentration of 1 ng mL
1 (curve a) and Ab1-PAB-Au incubated in 0 ng mL
1 TNF-R (curve b), 20 ng mL
1 AFP (curve c), 20 ng mL
1 CEA (curve d), 1 ng mL
1 TNF-R (curve e) for 45 min, followed by Si/PGMA/QD/Ab2 suspension for 45 min. (D) ECL response of bare gold (curve a) and Si/PGMA/QD/Ab2-Ag-Ab1-PAB-Au in air-saturated 0.05 M pH 7.4 PBS-containing 0.05 M KCl with 0.05 M K2S2O8 (curve c) or without the presence of 0.05 M K2S2O8 (curve b); curve “d” shows data for Si/PGMA/QD/Ab2-Ag-Ab1-PAB-Au in 0.05 M pH 7.4 PBS-containing 0.05 M KCl and 0.05 M K2S2O8 after the removal of oxygen with pure nitrogen for 30 min.

for 45 min followed in Si/PGMA/QD/Ab2 suspension; Ab1PAB-Au in 20 ng mL
1 AFP (Figure 3C, curve “c”) or 20 ng mL
1 CEA (Figure 3C, curve “d”) followed in Si/ PGMA/QD/Ab2 suspension for 45 min. The ECL responses in these control experiments showed slight increases in curve “a”. However, when Ab1-PAB-Au was incubated in 1 ng mL
1 TNF-R solution, followed by Si/PGMA/QD/Ab2 suspension (Figure 3C, curve “e”), the ECL signal increased significantly. All results confirmed that the QDs label could be loaded onto the electrode surface through highly specific sandwich immunoreactions, and they were not generated from nonspecific physical absorption or some cross-reaction; the attached Si/PGMA/QD/ Ab2 effectively retained its ECL properties. All these advantages allowed it to be used as a biological label for ECL immunoassay. Moreover, the ECL intensity of Si/PGMA/QD/Ab2-Ag-Ab1PAB-Au increased 9.98-fold more than that of QD/Ab2-Ag-Ab1PAB-Au (Figure 3A, curve “c”, CdTe QDs bound directly with Ab2 and used as the label). It also increased 2.03-fold more than that of Si/QD/Ab2-Ag-Ab1-PAB-Au (Figure 3A, curve “d”), at the same TNF-R concentration. The IC50 value of TNF-R for Si/ PGMA/QD/Ab2 labels was 0.046 μg mL
1, while IC50 values for QD/Ab2 labels and Si/QD/Ab2 labels were 0.330 μg mL
1 and 0.132 μg mL
1, respectively (see Figure S10 in the Supporting Information). Therefore, the presence of polymer was

demonstrated to further amplify the ECL signal of CdTe QDs by attaching Si/PGMA/QD/Ab2 on the electrode surface. The signal amplification was due to the increase of CdTe QDs loading per immunological event. A series of new experiments had been executed in order to gain a better understanding of the ECL generation. A clear ECL emission peak was observed from Ab2-Ag-Ab1-PAB-Au in a 0.05 M pH 7.4 air-saturated PBS solution containing 0.05 M K2S2O8 and 0.05 M KCl (Figure 3D, curve “a”). Once in the presence of Si/PGMA/QD/Ab2, this ECL emission could be greatly enhanced (Figure 3D, curve “c”), while no ECL emission was observed for Si/PGMA/QD/Ab2-Ag-Ab1-PAB-Au in a 0.05 M pH 7.4, air-saturated, PBS-containing 0.05 M KCl, without K2S2O8 (Figure 3D, curve “b”). This proved that S2O82
played a crucial role in the cathodic ECL. In Figure 3B, two cathodic peaks were observed at
1.22 V and
0.64 V, respectively. This was in good agreement with the observations for CdS QDs bound directly with protein labels.46 Thus, the peak at
1.22 V was attributed to the reduction of attached CdTe QDs on the electrode surface, which produced negatively charged radicals of CdTe
•. While the other peak at
0.64 V was attributed to the reduction of S2O82
to anion sulfate radical SO4
•. According to the report, the electrochemically reduced and oxidized CdSe QDs could react with the 6805

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Figure 4. (A) ECL profiles of the immunosensor in air-saturated 0.05 M pH 7.4 PBS-containing 0.05 M KCl and 0.05 M K2S2O8, at TNF-R concentrations of (a) 0.1, (b) 0.5, (c) 1, (d) 10, (e) 100, (f) 1000, (g) 1500, and (h) 2000 ng mL
1. (B) Plot of ECL intensity versus TNF-R concentration in incubation solution. Inset: linear plot for TNF-R determination.

coreactants to produce ECL.47 In this case, upon the potential scan with an initial negative direction, the CdTe QDs immobilized on the electrode were reduced to CdTe
• species by charge injection. The coreactant S2O82
was reduced to the strong oxidant, SO4
•, and then reacted with by injection into a hole with the highest occupied molecular orbital of CdTe
• to obtain an excited state (CdTe*). This state emitted light in the aqueous solution to produce an ECL signal. According to the literature, the coreactant could also be dissolved oxygen or H2O2.47,48 When dissolved oxygen was removed from the solution by bubbling with high-purity nitrogen, the ECL emission was slightly lower than that of the air-saturated solution (Figure 3D, curve “d”). These results agreed with the result that oxygen can be reduced and used to catalyze ECL emission of QDs. The corresponding ECL processes were as follows:47,49 CdTe þ e
f CdTe
•

ð1Þ

S2 O8 2
þ e
f SO4 2
þ SO4
•

ð2Þ

O2 þ H2 O þ 2e
f OOH
þ OH


ð3Þ

SO4
• þ CdTe
• f CdTe þ SO4 2


ð4Þ

2CdTe
• þ OOH
þ H2 O f 3OH
þ 2CdTe

ð5Þ

CdTe f CdTe þ hν

ð6Þ

The ECL Detection of TNF-r. Based on the sandwich immunoassay with Si/PGMA/QD/Ab2 as the label, an ECL immunosensor was developed for sensitive detection of the target TNF-R. Figure 4A showed the ECL intensity of the biosensor in the absence (peak a), and presence (peaks b
g), of different concentrations of TNF-R. During the dose
response curve for different TNF-R concentration, the ECL intensity increased gradually with increased TNF-R concentration, because morefrequent binding of Ag with QDs-coated Ab2 greatly increased the amount of CdTe QDs on the electrode, thus enhancing the ECL intensity, which suggested that the TNF-R concentration could be determined by the ECL measurement of the biosensor. Figure 4B showed the calibration curve of the Si/QD/Ab2-AgAb1-PAB-Au electrode after the deduction of the background signal. The calibration range of TNF-R was from 0.1 ng mL
1 to

2.0 μg mL
1. The linear equation was IECL ¼ 1748:819 þ 811:48 logðc=ng ML
1 Þ where IECL is the ECL intensity and c is the TNF-R concentration in the incubation solution. The correlation coefficient (R2) of 0.9766. The linear response range of the immunosensor to TNFR concentration (inset in Figure 4B) was from 0.1 ng mL
1 to 1 μg mL
1, with a detection limit of 7 pg mL
1 at a signal-to-noise ratio of 3. The low detectable concentration of 7 pg mL
1 in the present work was similar to other current immunoassays, such as 0.01 ng mL
1 by Zhu et al.22 According to the linear equation, we could detect TNF-R concentration quantitatively. ECL emission from the immunosensor upon continuous potential scanning for nine cycles is shown in Figure S11 in the Supporting Information. The coincident signals indicated the reliability and stability of the immunosensor, which could be applied to the ECL detection. Signal Amplification Using Si/PGMA/QD/Ab2 as the Label in SWV Assay. The captured Si/PGMA/QD/Ab2 on Au substrates was well-detected by a bismuth film modified glassy carbon electrode (BFE), which was fabricated by electrodeposition in Bi(III) ion solution. Figure 5A showed the SWV curve of Ab1-PAB-Au electrode incubated in 1 ng mL
1 TNF-R, followed by incubation in a Si/PGMA/QD/Ab2 suspension. A welldefined peak for the oxidation of Cd was observed at around
0.9 V (see Figure 5A, curve “d”). No peak was observed for Ab1-modified gold electrode incubation in Si/PGMA/QD/Ab2 suspension without prior exposure to the TNF-R solution (Figure 5A, curve “a”). Control experiments were done by incubation of Ag-Ab1-PAB-Au electrode in a suspension of QDs, coated in Si/PGMA nanospheres, without further coupling with Ab2 or in a Si/Ab2 suspension in the absence of QDs. No detectable signal could be observed in the same potential scan, which suggested that the stripping signal was attributed to the oxidation of the deposited Cd after it was dissolved and captured by CdTe QDs in glycine
HCl. On the other hand, the oxidation current of 30.31 μA by the Si/PGMA/QD/Ab2 label was 5.5 times larger than that of 5.528 μA by the QD/Ab2 label (Figure 5A, curve “b”). In addition, it was 2.3 times larger than that of 13.37 μA by the Si/QD/Ab2 label (Figure 5A, curve “c”). The IC50 value of TNF-R for Si/PGMA/QD/Ab2 label was 0.009 μg mL
1, while IC50 values for QD/Ab2 and Si/QD/Ab2 labels were 0.050 μg mL
1 and 0.025 μg mL
1, respectively (see Figure S12 in the Supporting Information). These result confirmed the signal amplification by Si/PGMA/QD/Ab2 label. 6806

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Figure 5. (A) SWV curves recorded after the Ab1-PAB-Au was incubated in 0 ng mL
1 TNF-R solution (curve a) and 1 ng mL
1 TNF-R solution (curves b
d) for 45 min, followed by QD/Ab2 (curve b), Si/QD/Ab2 (curve c), and Si/PGMA/QD/Ab2 (curves a and d) for 45 min. It was then dissolved in 0.1 M glycine
HCl for 10 min. (B) SWV curves of Si/PGMA/QD/Ab2-Ag-Ab1-PAB-Au at TNF-R concentrations of (a) 0.01, (b) 0.05, (c) 0.1, (d) 1, (e) 10, (f) 100, (g) 500, and (h) 1000 ng mL
1, respectively. (C) Plot of peak current obtained by dissolved Si/PGMA/QD/Ab2-Ag-Ab1PAB-Au vs TNF-R concentration in the incubation solution. (D) Linear plot for TNF-R determination.

The SWV Detection of TNF-r. The oxidation current of Cd2+

was found to be proportional to the concentration of TNF-R in the incubation solution, within a calibration range from 0.01 ng mL
1 to 1 μg mL
1 (Figure 5B, curves a
h). The standard calibration curve for the detection of TNF-R was shown in Figure 5D. The SWV peak intensity increased linearly with the TNF-R concentration from 10 pg mL
1 to 100 ng mL
1 with a detection limit of 3 pg mL
1 (see Figure 5D). The linear equation was IP ¼ 27:24 þ 10:90 logðc=ng mL
1 Þ μA where IP was the oxidation current and c was the TNF-R concentration in the incubating solution. The correlation coefficient was R2 = 0.998. The lowest detectable concentration of 3.0 pg mL
1, at a signal-to-noise ratio of 3 in the present work, was lower than that of the traditional sandwich immunoassay for TNF-R.23,50 According to the detection limit and broad linear equation, the proposed methods were demonstrated to detect TNF-R concentration sensitively. Reproducibility, Regeneration, and Stability of the Immunosensor. The reproducibility of the biosensor for TNF-R was investigated with intra-asay and interassay precision. The intra-assay precision of the biosensor was evaluated by the assay of one TNF-R level for four replicate measurements. The

interassay precision was estimated by determining one TNF-R level, with four biosensors made at the same electrode. The intraassay and interassay variation coefficients (CVs) obtained from 1 ng mL
1 TNF-R were 4.2% and 8.6% for ECL assay, 5.1% and 6.7% for SWV assay, which indicated good electrode-to-electrode reproducibility of the fabrication protocol described above. The regeneration of the proposed immunsensor was performed using 0.1 mol L
1 glycine
HCl (pH 2.2) to interrupt the antigen
antibody immunocomplex. Accordingly, after each sandwiched immunoassay, the electrode was immersed in pH 2.2 glycine
HCl for 10 min. No SWV peak current could be detected at this moment. The same immunosensor was reacted again with 1 ng mL
1 TNF-R and Si/PGMA/QD/Ab2. Similar SWV responses to their original values were obtained. In repetition of these steps, SWV intensity recovered 95.7% of the initial value after six assay runs (see Figure S13 in the Supporting Information). This observation also demonstrated that the assynthesized Si/PGMA/QD/Ab2 possessed good reproducibility, because of good monodispersion and extreme uniformity. The as-synthesized Si/PGMA/QD/Ab2 could be used as the label for sandwiched immunoassay. Storage stability of the Si/PGMA/QD/Ab2-Ag-Ab1-PAB-Au was also investigated by detection of SWV after a sandwich immunoreaction at 1 ng mL
1 TNF-R. No evident decrease in intensity was observed after 2 weeks of storage in 0.01 M PBS at 4 °C, which indicated that the prepared immunosensor 6807

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Analytical Chemistry possessed good storage stability and potential for practical application.

’ CONCLUSION In this work, two versatile immunosensors based on electrochemiluminescence (ECL) and square-wave voltammetry (SWV) assays for detection of tumor necrosis factor-alpha (TNF-R) were developed, using the Si/PGMA/QD nanospheres as immunological labels for signal amplification. The PGMA shell was formed through surface-initiated atom transfer radical polymerization (SI-ATRP), thanks to easily transformable epoxy groups for subsequent quantum dot (QD) coupling. After the sandwich immunoreactions, the CdTe QDs and PGMA-coated silica nanoparticles labels were introduced to the surface of a gold substrate. Enhanced sensitivity was achieved by a large number of surface functional groups offered by the polymer shell, which permitted the fabrication of high QDs coverage per immunoassay event. The ECL and SWV measurement confirmed effective signal amplification with a 10.0- and 5.5-fold increase of detection signal, respectively, compared with the unamplified method. The presented strategy was demonstrated to be simple, specific and could be extended to study other biological interactions, containing protein
protein, peptide
protein, and DNA
proteins. ’ ASSOCIATED CONTENT

bS

Supporting Information. Synthesis of Si/PGMA/QD/ Ab2 labels, characterization of SiO2, Si/PGMA, Si/PGMA/QD, and Si/PGMA/QD/Ab2, EIS curves, XPS spectra, SEM images and fluorescence microscopic images of the modified Au substrate, optimization of immunoassay conditions, and stability and regeneration of the resultant immunosensor. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Phone: +86-25-52090613. Fax: +86-25-52090618. E-mail: liusq@ seu.edu.cn.

’ ACKNOWLEDGMENT The project is supported by the National Basic Research Program of China (No. 2010CB732400), the Key Program (21035002) from the National Natural Science Foundation of China, National Natural Science Foundation of China (Grant Nos. 20875013), and the Key Program (BK2010059) from the Natural Science Foundation of Jiangsu Province. ’ REFERENCES (1) Old, L. J. Science 1985, 230, 630–632. (2) Jones, E. Y.; Stuart, D. I.; Walker, N. P. C. Nature 1989, 338, 225–228. (3) Old, L. J. Nature 1987, 326, 330–331. (4) Tetta, C.; Camussi, G.; Modena, V.; Di Vittorio, C.; Baglioni, C. Ann. Rheum. Dis. 1990, 49, 665–667. (5) Ettehadi, P.; Greaves, M. W.; Wallach, D.; Aderka, D.; Camp, R. D. R. Clin. Exp. Immunol. 1994, 96, 146–151. (6) D’Haens, G. Curr. Pharm. Des. 2003, 9, 289–294. (7) DeKossodo, S.; Houba, V.; Grau, G. E.; Group., W. C. S. J. Immunol. Methods 1995, 182, 107–114.

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