Fluorescent Artificial Enzyme-Linked Immunoassay System Based on

Oct 28, 2013 - School of Electronic Science and Engineering, Southeast University, Nanjing, Jiangsu 210096, China. •S Supporting Information. ABSTRA...
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Fluorescent Artificial Enzyme-Linked Immunoassay System Based on Pd/C Nanocatalyst and Fluorescent Chemodosimeter Zhifei Wang,*,† Shuang Zheng,† Jin Cai,† Peng Wang,† Jie Feng,† Xia Yang,† Liming Zhang,‡ Min Ji,‡ Fugen Wu,‡ Nongyue He,*,‡ and Neng Wan§ †

School of Chemistry and Chemical Engineering, Southeast University, Nanjing, Jiangsu 211189, China School of Biological Science and Medical Engineering, Southeast University, Nanjing, Jiangsu 210096, China § School of Electronic Science and Engineering, Southeast University, Nanjing, Jiangsu 210096, China ‡

S Supporting Information *

ABSTRACT: Artificial enzyme mimics have recently attracted considerable interest because they possess many advantages compared with natural enzymes, such as low cost of preparation and high stability. Herein, we present a novel fluorescent artificial enzyme-linked immunoassay strategy by utilizing Pd/C nanocatalyst as the enzyme mimic and bisallyloxycarbonyl rhodamine 110 (BI-Rho 110) as the substrate, and the amplification procedure is based on the palladiumcatalyzed Tsuji-Trost reaction. Pd/C nanocatalyst with the average size of 150 nm was prepared by the impregnation− reduction method, and high resolution transmission electron microscopy (HRTEM), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS) analyses reveal that Pd clusters with an average size of about 1 nm are dispersed uniformly on each carbon nanosphere’s surface. Kinetic studies show that this reaction follows Michaelis−Menten kinetics and the fluorescence intensity is proportional to the concentration of Pd/C nanocatalyst under certain conditions. The turnover number of Pd/C nanocatalyst reaches up to 3.3 × 107 (h−1). The analytical performance of this system in detecting hCG shows that after a 24 h incubation the sensitivity limit can reach 0.1 ng/mL and the dynamic linear working range is 1−10 ng/mL. Our findings pave the way to use Pd-catalyzed reaction for design and development of novel analytical methods.

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enzymatic signal amplification and develop an alternative to the above biological assays, it is necessary to construct both novel artificial enzyme labeling and the corresponding signal amplification system by combining chemical, material, and biological approaches. Meanwhile, rapid development of nanotechnology over the past decades has allowed us to view conventional heterogeneous catalysts with a new perspective.9 Catalytic materials, such as noble metals which often serve as active catalytic components, can now be prepared with greater precision via nanotech-enabled processes, which offers great opportunities for developing nanomaterial-based signal amplification strategy in conjunction with the special catalytic system.1,10−15 For example, one favorable characteristic of Au nanoparticle (NP) as label is its signal amplification capability offered by the catalytic silver deposition,11 usually termed as silver enhancement. In this process, silver ions are reduced chemically by hydroquinone to silver metals at the surfaces of the Au NPs,

he development of novel analytical methods for the simple and sensitive detection of disease-related proteins or other biomarkers has been attracting considerable attention in biological studies, clinic diagnosis, and prevention.1,2 Among the conventional techniques, enzyme-linked immunosorbent assay (ELISA) has been the most commonly used analytical strategy for measuring trace biomarkers since its introduction in 1971.3,4 In this method, enzyme molecules including horseradish peroxidase (HRP) and alkaline phosphatase (AP) are employed as labels and the detection signals are greatly amplified by the conversion of substrates to the colored5 or fluorescent compounds6 or chemiluminescence7 under the catalysis of the formed antibody−target−enzyme complex. For fluorescence-based ELISA in particular, since the fluorescence generated during the enzymatic reaction can be accumulated,6 the detection limit is expected to be further improved by prolonging the incubation time to increase the fluorescence intensity. However, despite the advantages listed above, the enzymatic labeling always involves the time-consuming preparation and sophisticated purification processes.7,8 Additionally, as a kind of proteins, enzyme also tends to denature under environmental changes,8 which further limits its application. Hence, in order to overcome the limitations of © 2013 American Chemical Society

Received: September 20, 2013 Accepted: October 28, 2013 Published: October 28, 2013 11602

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carbon nanospheres were dispersed in water with the concentration of 0.08 mg/mL. Surface Modification of Carbon Nanosphere with Lysine. Lysine (1.46 g) was dissolved in 10 mL of carbon nansphere aqueous solution, and the pH of the resulting mixture was adjusted to 10 using 0.1 M NaOH aqueous solution. The mixture was then incubated at 37 °C for 12 h. After that, the resulting carbon nanospheres were washed five times with water to remove free lysine and finally resuspended in 10 mL of water. Preparation of Pd/C Nanocatalyst. Pd/C nanocatalyst was synthesized by the impregnation−reduction method. First, 2 mL of lysine-coated carbon nanosphere suspension was added to 5 mL of H2PdCl4 (2 mM) solution, and the resulting mixture was stirred gently for 30 min at the room temperature. After impregnation, the resulting carbon nanospheres were washed with water for three times to remove the remaining H2PdCl4 and then resuspended in 2 mL of water. The reduction of PdCl42− ion on C nanospheres was performed by the addition of 0.2 mL of NaBH4 aqueous solution (3 mM), and the reaction lasted for 5 min. Finally, the resulting Pd/C nanocatalyst was washed for three times and resuspended in 20 mL of water with the concentration of 8 μg/mL. General Procedure for the Catalytic Tests. The transformation of BI-Rho110 to rhodamine 110 (abbreviated as Rho110) over Pd/C nanocatalyst was tested in H2O/DMSO (v/v = 49/1) solution at 37 °C. In a typical procedure, a 20 mM solution of BI-Rho110 in DMSO was first prepared by dissolving 1 mg of BI-Rho110 in 1 mL of DMSO. Then, 10 μL of the above solution was diluted with 490 μL of water to give a final concentration of 0.4 mM. Various amounts of Pd/C nanocatalyst aqueous solution depending on the final concentration of Pd/C nanocatalyst in the mixture (for example, 0.75 μL for 12 ng/mL) were added, and the reaction was kept for 36 h at 37 °C. Reaction progress was monitored by detecting the fluorescence intensity of solution (Ex/Em = 498/ 525 nm) with a RF-5301PC spectrofluorophotometer (Shimadzu Scientific Instrument). Linkage of hCG Ab2 to Pd/C Nanocatalysts’ Surface. To a dispersion of Pd/C nanocatalyst (0.2 mL, MES buffer, pH 6.0, 4 μg/mL), 2 mg of EDC and 2 mg of NHS were added separately under room temperature, and the activation reaction lasted for 20 min. Then, the activated Pd/C nanocatalysts were collected by centrifugation at 15 000 rpm for 4 min under 4 °C and dispersed in 0.2 mL of icy PB buffer (pH 7.4). Next, 50 μL of hCG Ab2 (1 mg/mL) was added to the above solution, and the reaction was kept for 24 h under 4 °C. To block remaining active NHS sites on Pd/C nanocatalysts’ surface, 100 μL of aqueous BSA solution (1 mg/mL) was successively added. The reaction was kept for another 24 h under 4 °C. After thorough washing, hCG Ab2-coated Pd/C nanocatalysts were finally dispersed in PBS buffer (pH 7.2) with the concentration of 4 μg/mL. Linkage of hCG Ab1 to Magnetic Beads’ Surface. The procedure for the functionalization of magnetic beads with hCG Ab1 was similar to that above. Carboxyl group-coated magnetic beads (100 μL, 10 mg/mL) and 50 μL of hCG Ab1 (1 mg/mL) were used in this procedure, and hCG Ab1-coated magnetic beads were finally resuspended in PBS buffer (pH 7.2) with the concentration of 1 mg/mL. Sandwich-Based Assay for hCG. hCG Ab1 coated-magnetic beads (50 μL, 1 mg/mL) were first transferred into Eppendorf tube (1.5 mL), and then, 200 μL of hCG solution with different

resulting in a 100-fold reduction in the detection limits for both optical and electrochemical methods. In addition, the merging of nanotechnology with biology has also ignited research efforts for designing functional materials with mimetic enzymatic activity.10−15 For instance, Au NPs,11,12 Pt NPs,13 FeS NPs,14 and magnetite (Fe3O4) NPs15 have been reported separately to possess an intrinsic peroxidase-like activity, which can be utilized to catalyze chemiluminescent reactions, and have potential to replace the enzyme used in ELISA. In contrast, although Pd-catalyzed reactions such as the Heck, SuzukiMiyaura, and Tsuji-Trost reaction play an important role in the formation of a C−C bond16 or cleavage of an ally-O bond,17 up to now, the exceptional ability of palladium to catalyze a wide variety of chemical transformations has been hardly utilized in nanocatalyst-based signal amplification. Very recently, chemodosimeter-based optical molecular probes for the qualitative and quantitative analysis of palladium species have been attracting much attention.17−19 As an abiotic molecule, a chemodosimeter can form a new fluorescent product and further provides an observable signal through a usually irreversible chemical reaction between the dosimeter molecule and palladium. In most cases, such interaction is based on a Pd-catalyzed reaction such as the Tsuji-Trost reaction.17,19 For example, Koide et al.17 developed a fluorescein-based Pd probe where the allylic C−O bond of an allylic ether can be catalytically cleaved by allylic oxidative insertion under the presence of Pd (0), and high sensitivity down to the 3 nM Pd level is achieved with this chemodosimeter. Inspired by these works, herein, we propose a conceptually novel fluorescent artificial enzyme-linked immunoassay system by replacing enzyme with Pd nanocatalyst to label the second antibody (Ab2). Bis-allyloxycarbonyl Rhodamine 110 (abbreviated as BI-Rho110 later, Supporting Information, Figures S1 and S2) was chosen as the corresponding substrate according to the literature.20

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EXPERIMENTAL SECTION Chemicals. 1-Ethyl-3-[3-dimethylaminopropyl] carbodimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), and dimethyl sulfoxide (DMSO) were purchased from Aldrich. Glucose (C6H12O6), palladium dichloride (PdCl2), hydrochloric acid (HCl, ≥37%), lysine (C6H14N2O2), sodium hydroxide (NaOH), and sodium borohydride (NaBH4) were obtained from Shanghai Chemical Reagent Corporation. All chemicals were used as received. Magnetic beads (Dynabeads, 1 μm) were purchased from Invitrogen. Anti-alfa-human chorionic gonadotropin (hCG) (mouse, hCG Ab1), antiBata-hCG (mouse, hCG Ab2), and hCG antigen (human) were purchased from Shanghai Linc-Bio Science Co., Ltd. Water used in this experiment was purified by distillation of deionized water. Experimental Procedures. Preparation of Carbon Nanospheres. Carbon nanospheres were synthesized by the hydrothermal method as reported in the literature.21 Glucose (4 g) was first dissolved in 40 mL of water to form a clear solution. Before the reaction, the reactant mixture placed in a 50 mL Teflon-sealed autoclave was deoxygenated under the flowing nitrogen. Then, the mixture was heated to 170 °C and maintained at this temperature for 4 h. Next, the carbon nanospheres isolated from the above mixture by centrifugation were cleaned by three cycles of centrifugation/washing/ redispersion in water and in alcohol. Finally, the obtained 11603

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Figure 1. (A) Schematic diagram of fluorescent artificial enzyme-linked immunoassay system based on Pd/C nanocatalyst and fluorescent chemodosimeter. (B) Fluorescence emission spectra under 498 nm excitation and corresponding picture (inset) of Bi-Rho110 (a) and Rho110 (b) in H2O/DMSO (v/v = 49/1) solution. (C) Mechanism for the palladium-catalyzed cleavage of allyl carbamates.

concentrations (from 100 to 0.1 ng/mL) was added. The resultant mixture was incubated for 2 h at 37 °C with gentle mixing. After that, the magnetic beads isolated from the mixture by the external magnetic field were rinsed with 100 μL of PBS− 0.05% Tween 20 buffer (PBSTB) three times for 5 min each. Next, 200 μL of hCG Ab2-coated Pd/C nanocatalysts were added and incubated at 37 °C for 1 h. Following this step, the magnetic beads were rinsed again with 100 μL of PBSTB 3 times (100 μL for 5 min each), followed by a PBS wash for 5 min. After carefully removing the rinsing solution, 200 μL of PBS/DMSO (49:1) solution containing 0.4 mM bis-allyoxycarbonyl rhodamine 110 was added into the EP tube, and the resultant mixture was incubated at 37 °C for 24 h under mild stirring. The fluorescence intensity of the supernatant was measured (Ex/Em = 498/525 nm) with a RF-5301PC spectrofluorophotometer. Characterization. The morphology and size of the samples were analyzed by transmission electron microscopy (TEM) JEOL-3010, and the elemental analysis of Pd/C nanocatalyst was conducted by its equipped energy dispersive X-ray spectroscopy analyzer (EDX). Before the characterization, the samples for TEM were prepared by placing a drop of the colloidal dispersion of carbon sphere or Pd/C nanocatalyst onto a carbon-coated copper grid followed by naturally evaporating the solvent. The surface of Pd/C nanocatalyst was detected by the X-ray photoelectron spectra recorded on

an ESCALAB MK II, using a nonmonochromatized Mg Kα Xray as the excitation source and choosing C (1s) (284.6 eV) as the reference line. Before the analysis, the obtained Pd/C nanocatalysts were freeze-dried to remove water completely and kept under air.

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RESULTS AND DISCUSSION As a proof of concept, the detection of hCG was employed as a model system in the experiment considering its diagnostic significance. Figure 1A presents a schematic representation of this assay, and it can be seen that, in the presence of hCG (target antigen), Pd/C nanocatalyst captured by the formation of sandwich structure can catalytically convert the substrate BiRho 110 to Rh110, which further generates the strong fluorescent signal (centered at 525 nm) under 498 nm excitation, by the cleavage of the allyloxycarbonyl protecting group. Before the allylcarbamate deprotection, the fluorophore in the BI-Rho110 is nonfluorescent owing to the steric hindrance caused by allylcarbamate group (Figure 1B), and a plausible mechanism for the palladium-catalyzed cleavage of allylcarbamates is presumably associated with the formation of the π−allyl palladium complex (2, in Figure 1C) which induces the transfer of the allyl unit to allyl acceptor (H2O, herein).20,22 The successive decarboxylation of intermediate (3, in Figure 1C) endows Rho 110 with the green fluorescence. 11604

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Scheme 1. (A) Procedure for the Preparation of Pd/C Nanocatalyst by the Impregnation−Reduction Method; (B) Picture of 0.02 mg/mL of Pd/C Nanocatalyst Aqueous Solution

Figure 2. TEM images of C nanospheres (A) and Pd/C nanocatalysts (B); HRTEM image of Pd/C nanocatalysts (C); EDX pattern of Pd/C nanocatalysts deposited on a carbon-coated copper grid (D).

by the impregnation−reduction method using carbon nanospheres as the support (Scheme 1). Carbon nanospheres with an average size of about 150 nm are first synthesized by hydrothermal carbonization of glucose (Figure 2A).21 FT-IR spectroscopic characteristic reveals that there are large numbers of functional groups such as −OH and CO groups present on their surface (Supporting Information, Figure S3A). As shown in Scheme 1, before loading Pd species onto their

It should be pointed out that in the pre-experiment pure Pd NPs stabilized by 11-mercaptoundecanoic acid (MUA)23 were also tried as the label to catalyze the conversion of BI-Rho110 to Rho110, but no obvious activity was observed even after a 72 h incubation. This decline in catalytic activity can be attributed to the effect of the MUA ligands which passivate Pd NPs’ surface. To address this problem and get Pd nanocatalyst with a much higher activity, a novel Pd/C nanocatalyst was prepared 11605

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Figure 3. XPS spectra of Pd/C nanocatalysts. (A) XPS survey spectrum; (B) XPS pattern of C1s; (C) XPS pattern of N1s; (D) XPS pattern of Pd 3d.

species attached to the surface of lysine-modified carbon nanosphere exist in the form of Pd0. To be applicable to immunoassays, the Pd/C nanocatalystbased signal amplification system must meet the following criteria: First, the substrate BI-Rho110 has good stability under a wide range of reaction conditions. Second, the rate of fluorescence development is proportional, over a certain range, to the amount of Pd/C nanocatalysts captured by the formation of sandwich structure. Third, Pd/C nanocatalysts possess a high turnover number. We first studied the stability of BI-Rho110 through a set of control experiments, where the effect of solvent, carbon nanospheres modified with lysine, and protein molecule (represented by BSA) involved in the reaction were especially investigated with an aim to rule out the possibility of any fluorescence seen being the result of allylcarbamate cleavage in the absence of Pd/C nanocatalyst. Considering the poor aqueous solubility of BI-Rho110, all evaluations were carried out in DMSO/H2O (1/49) solution at 37 °C according to Mark Bradley et al.20 As shown in Table S1 (Supporting Information), after a 24 h incubation, no obvious change in fluorescence intensity was observed in the above negative control. In contrast, strong green fluorescence is clearly observed from the reaction containing Pd/C nanocatalysts (3 ng/mL), indicating that the allylcarbamate cleavage only occurs in the presence of Pd (0). To investigate the kinetic behavior of this Pd/C NPscatalyzed reaction, the effect of the various reaction parameters, including pH, the concentration of BI-Rho110, and the concentration of Pd/C nanocatalyst, on the formation of

surfaces, carbon nanospheres’ surfaces were further modified with lysine through the formation of Schiff base between amino group from lysine and carbonyl group from carbon nanosphere. The purpose of lysine modification is to provide the coordination environment for the formation of Pd clusters as well as to facilitate covalent immobilization of hCG Ab2 on Pd/ C nanocatalyst surface through carboxyl group (For more details, see Figure S5, Supporting Information.). The successful modification of lysine is confirmed by the FT-IR spectrum in Figure S3B (Supporting Information). TEM, high resolution transmission electron microscopy (HRTEM), and EDX analyses in Figure 2B−D, respectively, reveal that Pd clusters with an average size of about 1 nm are dispersed uniformly on each carbon nanosphere’s surface, which is also supported by the characteristic peaks in the X-ray diffraction (XRD) spectrum (Supporting Information, Figure S4). The palladium content of Pd/C nanocatalyst is about 1.2% according to ICPMS analysis. Figure 3 further illustrates the X-ray photoelectron spectroscopy (XPS) spectra of the resulting Pd/C nanocatalysts. The survey spectrum shows the presence of C, O, N, and Pd in the product (Figure 3A). As shown in Figure 3B, the main C1s peak centered at 284.8 eV can be assigned to C−C or CC, which agrees with the aromatization and carbonization process of glucose. The N1s signals (Figure 3C), associated with amino acid, are evidenced at 399.9 eV, further confirming the existence of lysine on nanosphere.24 For the Pd 3d spectrum, the peaks (336.0, 341.15 eV) can be attributed to Pd0 species (Figure 3D).25 All these data taken together offer clear evidence that Pd 11606

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rate of formation of Rho110 within the first 4 h against the concentration of BI-Rho110 ranging from 0.5 to 3.5 μM, and a typical Michaelis−Menten curve with Km = 1.2 μM and Vmax = 0.15 μmol·h/L was observed (Supporting Information, Figure S7), indicating that this reaction follows Michaelis−Menten kinetics.26 According to the Michaelis−Menten theory, when the concentration of BI-Rho110 is much higher than Km, Pd/C nanocatalysts will be saturated with BI-Rho110 and the formation rate of Rho110 will be hardly affected by the variation of BI-Rho110 concentration. This is why the fluorescence intensity is similar at the initial 12 h when the concentration of BI-Rho110 changes from 5 μM to 0.4 mM. However, once most of BI-Rho110 is consumed or the concentration of BI-Rho110 is close to or lower than Km, the diffusion velocity of BI-Rho110 from the bulk liquid to the external catalyst surface will control the formation rate of Rho110. To be sure that the concentration of BI-Rho110 is in vast excess and much higher than Km during the whole incubation, 0.4 mM of BI-Rho110 is adopted in the following experiments. Figure 4B shows the evolution of fluorescence intensity as a function of incubation time under the various concentrations of Pd/C nanocatalyst (0.4 mM of BI-Rho110). The resulting data were further linearly fitted, and the parameters of the fitted curve were listed in the inset table. It can be seen that no matter what concentration of Pd/C nanocatalysts used, the fluorescence signal increases nearly linearly with the incubation time, indicating that Rho110 is formed at a constant rate. Meanwhile, from the slope of a line, which represents the formation rate of Rho110, it can be found that with the increase of the concentration of Pd/C nanocatalysts (0.75, 1.5, 3, 6, and 12 ng/mL) the corresponding slope varies (4.8, 9.8, 22.9, 40.6, and 81.6), indicating that the formation rate of Rho110 is proportional to the concentration of Pd/C nanocatalysts. This lays a good foundation for the future quantitative analysis. Moreover, it can be further induced that the turnover number of Pd/C nanocatalysts reaches up to 3.3 × 107 (h−1) (see the detailed calculation in the Supporting Information), indicating that the resulting Pd/C nanocatalyst performs a high catalytic efficiency for the conversion of BI-Rho-110 to Rho110. According to the above results, in the following experiments, incubation times of 12 and 24 h are selected representatively as a result of the compromise between the detectable fluorescence intensity and the appropriate incubation time required in practical clinical diagnosis. To further explore the practical application of this artificial enzyme-based signal amplification system, we evaluated the potential of this system to detect hCG using a sandwich-based approach (as shown in Figure 1A), where magnetic beads with an average size of 1 μm were used as the solid support for the immobilization of hCG Ab1 instead of 96-well plates in conventional ELISA considering their convenience in wash steps. hCG Ab2 was covalently attached to the Pd/C nanocatalysts’ surface using EDC coupling chemistry.27 The resulting hCG Ab2-coated Pd/C nanocatalysts were characterized by UV−visible absorption spectra and dynamic light scattering. As illustrated in Figures S9 and S10 (Supporting Information), after the linkage of hCG Ab2, the typical absorption signal (a broad absorption peak between 260 and 280 nm) from protein is observed and the mean hydrodynamic diameters of Pd/C nanocatalysts shifts from 288.7 to 550.0 nm, indicating the successful attachment of hCG Ab2 to Pd/C nanocatalysts’ surface. Similarly, hCG Ab1 was also covalently

Rho110 were separately studied. Results show that the pH variation between 6 and 9 has no obvious effects on the formation of Rho110 (Supporting Information, Figure S6). Thus, we adopted PBS with pH 7.2 as reaction solvent for subsequent analysis. Figure 4A shows the typical evolution of

Figure 4. (A) The evolution of fluorescence intensity of PBS/DMSO (v/v = 49/1) solution containing various concentrations of Bi-Rho110, catalyzed by 6 ng/mL of Pd/C nanocatalysts, as a function of incubation time. (B) The evolution of fluorescence intensity of PBS/ DMSO (v/v = 49/1) solution containing 0.4 mM of Bi-Rho110, which is catalyzed by the different concentrations of Pd/C nanocatalysts (ng/ mL): 12 (a), 6 (b), 3 (c), 1.5 (d), and 0.75 (e), as a function of incubation time.

fluorescence intensity as a function of incubation time under the various concentration of BI-Rho110 (6 ng/mL of Pd/C nanocatalysts), and it can be seen that with the increase of BIRho110 concentration from 0.5 to 5 to 15 μM the time taken for the fluorescence intensity to reach a plateau increases from 8 to 14 to 32 h. For 0.4 mM of BI-Rho110, the fluorescence signal continues to increase even after a 36 h incubation. Additionally, during the initial 12 h incubation, despite the difference in the concentration of BI-Rho110 between 5 μM and 0.4 mM, there are no significant variations in their fluorescence intensities. However, they are obviously higher than that generated for 0.5 μM of BI-Rho110. To understand better the above phenomena, we further plotted the average 11607

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sandwich-layered structure. Meanwhile, upon the formation of “sandwich” structure, the intensity of the background fluorescence is obviously high, which is due to the nonspecific binding of Pd/C nanocatalysts modified with hCG Ab2 to hCG Ab1-functionalized magnetic beads. After a 12 h incubation, there is only a slight difference in signals between a hCG concentration of 0.l ng/mL (143) and the control (130) but the signal can be differentiated from the control for a hCG concentration of 1 ng/mL (240). When the incubation time is extended to 24 h, such a signal difference between a hCG concentration of 0.l ng/mL (309) and the control (241) can be further amplified through the accumulated Rho110 constantly generated during the incubation. If we refer to sensitivity as the difference in signal caused by the difference in antigen concentration, the sensitivity limit as low as 0.1 ng/mL is obtained after a 24 h incubation. Moreover, from the inset in Figure 5B, we can see that the increase of the fluorescence intensity is nearly linear in the range of 1 to 10 ng/mL after a 24 h incubation. One of the reasons why a higher concentration of hCG such as 100 ng/mL is beyond the dynamic linear range is presumably related to concentration quenching of Rho110.28 Therefore, it can be concluded that, in comparison with other artificial enzyme-based signal amplification strategies (Fe3O4 NPs, the sensitivity limit of 1 ng/mL for hCG),8 this assay provides a much stronger signal by the accumulated fluorescent molecule and thus increases the sensitivity.

attached to the surface of magnetic beads coated by carboxylic acid groups. Figure 5A shows the fluorescence intensity of the

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CONCLUSION

In conclusion, we have preliminarily established the novel fluorescent artificial enzyme-linked immunoassay system by utilizing Pd/C nanocatalyst as the enzyme mimic and BI-Rho 110 as the substrate. To the best of our knowledge, this is the first attempt to explore Pd-catalyzed reactions on signal amplification as an alternative for luminescence ELISA. The results show that the resulting Pd/C nanocatalyst performs the high catalytic efficiency for the conversion of substrate BI-Rho 110 to product Rho110 and its turnover number reaches up to 3.3 × 107 (h−1). More importantly, the formation rate of Rho110 is proportional to the amount of Pd/C nanocatalysts. Meanwhile, the substrate BI-Rho 110 also exhibits good stability under the reaction condition and is only transformed to the product in the presence of Pd/C nanocatalysts. The analytical performance of this system in detecting hCG shows that after a 24 h incubation the sensitivity limit can reach 0.1 ng/mL and dynamic linear working range is 1−10 ng/mL. When compared with traditional ELISA or other artificial enzyme-linked immunoassay, this system shows several unprecedented advantages. First of all, Pd/C nanocatalyst is low-cost, easy to prepare, more resistant to biodegradation, and less vulnerable to denaturation. Second, the fluorescence generated during the reaction can be accumulated by prolonging the incubation time, which can be used to improve the detection limit. Third, the versatility of Pd-catalyzed reactions provides more opportunities for designing new nanocatalysts and corresponding substrates in future studies. Future research will focus on how to shorten the reaction time by designing the novel substrate that needs a lower activation energy for Pd-catalyzed transformation as the current incubation time (such as 24 h) is too long to be applicable to practical clinical diagnosis.

Figure 5. (A) Effect of different samples (Pd/C nanocatalysts, BSAcoated Pd/C nanocatalysts, and BSA-coated magnetic beads) on the transformation of Bi-Rho110 to Rho110 after a 12 and 24 h incubation. (B) Pd/C nanocatalyst-linked immunofluorescence assay of hCG.

solution incubated with Pd/C nanocatalysts, protein-coated Pd/C nanocatalysts, and protein-coated magnetic beads for both 12 and 24 h, respectively. It can be seen that after the linkage of hCG Ab2 the increment of fluorescence intensity is slightly lower compared with that for blank Pd/C nanocatalysts, presumably due to the steric hindrance of outer protein which decreases the conversion efficiency of BI-Rho110 to Rho110 over Pd/C nanocatalysts. As a control experiment, no obvious catalytic activity was observed over protein-coated magnetic beads. The analytical performance of the above system was investigated by measuring various known concentrations of hCG between 0 and 100 ng/mL in PBS. In the control experiment, the background fluorescence level of the system was measured by using 10 ng/mL of BSA solution instead of hCG to complete the whole procedure. As shown in Figure 5B, the fluorescence intensity increases with the increase of concentration of hCG. This result was expected, as an increasing concentration of hCG translates into an increasing amount of Pd/C nanocatalysts captured by the formation of 11608

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (Z.W.). *E-mail: [email protected]. Phone/Fax: +86-25-83790885 (N.H.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the State key Basic Research Program of the PRC (2014CB744501, 2010CB933903) and the NSF of China (61271056)

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dx.doi.org/10.1021/ac403001y | Anal. Chem. 2013, 85, 11602−11609