Polydopamine-Functionalization of Graphene Oxide to Enable Dual

Apr 8, 2014 - Surface plasmon resonance imaging (SPRi) is one of the powerful tools for immunoassays with advantages of label-free, real-time, and ...
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Polydopamine-Functionalization of Graphene Oxide to Enable Dual Signal Amplification for Sensitive Surface Plasmon Resonance Imaging Detection of Biomarker Weihua Hu,*,†,‡,§,⊥ Guangli He,†,‡,§ Huanhuan Zhang,†,‡,§ Xiaoshuai Wu,†,‡,§ Jialin Li,†,‡,§ Zhiliang Zhao,†,‡,§ Yan Qiao,†,‡,§ Zhisong Lu,†,‡,§ Yang Liu,∥ and Chang Ming Li*,†,‡,§ †

Institute for Clean Energy & Advanced Materials, Southwest University, Chongqing 400715, China Chongqing Key Laboratory for Advanced Materials and Technologies of Clean Energies, Southwest University, Chongqing 400715, China § Faculty of Materials and Energy, Southwest University, Chongqing 400715, China ∥ Institute of Agro-products Processing Science and Technology Chinese Academy of Agricultural Sciences, Key Laboratory of Agro-products Processing, Ministry of Agriculture, Beijing 100193, China ⊥ Key Laboratory of Analytical Chemistry for Biology and Medicine, Wuhan University, Ministry of Education, 430072, China ‡

S Supporting Information *

ABSTRACT: Surface plasmon resonance imaging (SPRi) is one of the powerful tools for immunoassays with advantages of label-free, real-time, and high-throughput; however, it often suffers from limited sensitivity. Herein we report a dual signal amplification strategy utilizing polydopamine (PDA) functionalization of reduced graphene oxide (PDA-rGO) nanosheets for sensitive SPRi immunoassay in serum. The PDA-rGO nanosheet is synthesized by oxidative polymerization of dopamine in a gentle alkaline solution in the presence of graphene oxide (GO) sheets and then is antibody-conjugated via a spontaneous reaction between the protein and the PDA component. In the dual amplification mode, the first signal comes from capture of the antibody-conjugated PDA-rGO to form sandwiched immunocomplexes on the SPRi chip, followed by a PDA-induced spontaneous gold reductive deposition on PDA-rGO to further enhance the SPRi signal. The detection limit as low as 500 pg mL−1 is achieved on a nonfouling SPRi chip with high specificity and a wide dynamic range for a model biomarker, carcinoembryonic antigen (CEA) in 10% human serum.

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chips. A chemical approach has also been investigated to amplify the weak SPRi signal through biomolecular interactions for specific bioconjugates leading to local dielectric constant change for more sensitive SPRi detections. Antibody-conjugated nanomaterials including gold nanoparticles (AuNPs), gold nanorods (AuNRs), inorganic quantum dots, magnetic particles, and silica nanoparticles have been reported as amplification tags to improve the SPR/SPRi sensitivity in sandwich immunoassays.16−21 The enzyme-catalyzed precipitation reaction and surface-initiated atom transfer radical polymerization (SI-ATRP) have also been utilized to amplify the SPR signals in immunoassays.7,20,22,23 These approaches have advanced SPRi immunoassay technology; however, it remains a great challenge for SPRi in immunoassays to achieve not only high sensitivity but also broad detection range and excellent specificity for practical applications such as early diagnosis of tumors.

urface plasmon resonance imaging (SPRi) offers a unique tool for the study of biomolecular interaction, analysis of interfacial behaviors of (bio)molecules, and particularly, immunoassay of target biomolecules with prominent advantages of label-free, real-time, and high-throughput.1−10 It has demonstrated great potentials in a wide variety of research field such as drug screening, clinic diagnosis, and environment monitoring among others. Its limited sensitivity, however, is a major impediment for various practical applications.1−4,10 In particular, quantification of specific proteins (biomarkers) in human serum is essentially important for early diagnosis of tumors but generally requires cutoff levels of the detected biomarkers at ng mL−1,11,12 which is much lower than the detection limit of a SPRi chip. Therefore, it is critical but very challenging to boost the sensitivity of the SPRi immunoassay. One strategy to improve SPRi sensitivity is to use precision instrumental setups such as phase-sensitive SPRi, differential SPRi, and long-range SPR and/or specifically designed SPRi chip including etched/patterned SPRi chip and gold/silver bimetallic with improved sensitivity due to the enhancement of the signal and/or suppression of noise.1,3,8,9,13−15 However, this strategy requires sophisticated instruments and/or expensive © 2014 American Chemical Society

Received: January 27, 2014 Accepted: April 8, 2014 Published: April 8, 2014 4488

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Figure 1. Cartoon representations of (a) synthesis of PDA-rGO and antibody-conjugated PDA-rGO and (b) dual signal amplification strategy for SPRi detection. Step I, direct detection of target in 10% human serum; step II, first signal amplification with Anti-Mouse IgG conjugated PDA-rGO to form immunocomplexes on the sensing surface; step III, second signal amplification by flowing HAuCl4 solution to grow gold nanostructures on PDA-rGO.

were put into a 250 mL flask, and 38 mL of concentrated sulfuric acid (H2SO4, 98%) was added with stirring for 30 min in an ice bath. Then 4.5 g of potassium permanganate (KMnO4) was added slowly under vigorous stirring, and the mixture was continuously stirred at room temperature for 12 h. A volume of 36 mL of deionized water was slowly poured into the solution under vigorous stirring. After stirring for 24 h, 12 mL of 30 wt % H2O2 was added to the mixture and reacted for 3 h. Finally, the mixture was washed by centrifugation and rinsed with 5 wt % HCl and deionized water several times. The yellow-brown aqueous suspension of GO was obtained by ultrasonication. PDA-rGO was prepared by oxidative polymerization of dopamine in the presence of GO (Figure 1a). Typically, 20 mg of dopamine was dissolved in 10 mL of TrisHCl buffer (10 mM, pH 8.5) containing 1.0 mg mL−1 GO, and the solution was magnetically stirred at room temperature for 24 h. The PDA-rGO product was collected after centrifugation and washed with deionized water three times. For the synthesis of antibody conjugated PDA-rGO, 100 μg of PDA-rGO in 50 μL of water was added dropwise into 1 mL of phosphate buffered saline (PBS, 0.01 M, pH 7.4) containing 100 μg mL−1 anti-Mouse IgG with energetic stirring, and the solution was further stirred for 24 h at room temperature. Excessive antibody was removed by multiple centrifugations and washing with 0.01 M PBS buffer containing 1 mg mL−1 bovine serum albumin (BSA). The product was finally resuspended in 1 mL of 0.01 M PBS buffer containing 1 mg mL−1 BSA. Preparation of SPRi Antibody Chip. The SPRi antibody chip was prepared according to our previous work.5,30 Briefly, the bare SPRi gold chip (SPRi SpotReady chip with 4 × 4 spot array) was first functionalized with a nonfouling POEGMA-coGMA polymer brush by surface-initiated atom transfer radical polymerization (SI-ATRP), followed by manually spotting monoclonal antibody solution (anti-CEA mAb, 250 μg mL−1 in 0.01 M PBS, developed in Rabbit) onto each gold spot. The chip with spotted antibody solution was stored in a dry cabinet for 8 h, followed by intensive washing with 0.01 M PBS. The POEGMA-co-GMA polymer brush was prepared by immersing the SPRi gold chip into a cysteamine ethanol solution (2 mg mL−1) for 2 h, followed by incubating in 10 mL of tetrahydrofuran (THF) solution containing 77 μL of triethylamine (TEA) and 64 μL of 2-bromoisobutyryl bromide (BIB) for 2 h. The initiator-attached gold chip was put into a SIATRP growth solution containing 4.5 mL of DI water, 4.5 mL of methanol (HPLC grade), 1 mL of OEGMA (typical Mn = 360), 50 μL of GMA (97%), 2,2′-bipyridyl (Bipy, 44.5 mg), and copper(II) bromide (CuBr2, 33.5 mg). After bubbling the

Polydopamine (PDA), a mussel-inspired polymer, as a versatile functional material has sparked considerable research interest in bio- and energy-related areas.21,24−26 It has been discovered that PDA is able to firmly adhere to almost all surfaces by facile oxidative polymerization of dopamine under mild alkaline conditions in the presence of dissolved O2.24,27 Besides facile preparation and excellent adhesive stability, PDA also enables a wide variety of chemical reactions for subsequent functionalization due to its reactive catechol/quinone groups. Via Michael addition or Schiff base reaction, PDA is capable of reacting with thiols and amines, thus enabling protein conjugation/attachment and surface modification without need of any activation process.24 Various immunoassay devices have been fabricated based on PDA functional materials.21,28 PDA also allows spontaneous deposition of noble metal (electroless plating) via reduction of metal ions without any reductant assistance.24 Adherent and uniform metal deposit could be grown on the PDA film due to the reducing ability and metal binding ability of its catechol groups, which facilitates its application in immunoassay as a versatile platform, for example, for signal readout and signal amplification. In this study, by utilizing the intriguing properties of PDA, we developed a novel dual signal enhancement strategy based on PDA-functionalization of reduced graphene oxide (PDArGO) nanosheets to significantly improve the performance of SPRi immunoassay. Although PDA modified materials have been applied for immunoassay, it is the first time to combine PDA modified graphene and subsequent PDA-mediated metal deposit for dual signal amplification on the SPRi platform. PDA-rGO nanosheets were synthesized via oxidative polymerization of dopamine in the presence of graphene oxide (GO), and antibody was then conjugated with the PDA-rGO (Figure 1a). The resulting antibody-conjugated PDA-rGO is used to form a sandwich immunocomplex on the SPRi sensing surface for the first signal amplification, and PDA mediates gold deposition in HAuCl4 solution to further enhance the SPRi signal, as shown in Figure 1b. With this dual signal amplification, a detection limit of as low as 500 pg mL−1 with a broad detection range for a model biomarker, carcinoembryonic antigen (CEA) in 10% human serum, is achieved on a SPRi platform.

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EXPERIMENTAL SECTION

Synthesis of PDA-rGO and Antibody Conjugated PDA-rGO. Graphene oxides (GO) was synthesized from graphite powder using the modified Hummers method.29 Briefly, 1.0 g of graphite and 0.6 g of sodium nitrate (NaNO3) 4489

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solution with pure nitrogen for 15 min, 0.5 mL of 60 mg mL−1 ascorbic acid solution was rapidly added into the solution to start the polymerization. After 8 h growth in an inert atmosphere, the chip was pulled out and rinsed with ethanol and DI water, then dried under gentle nitrogen flow for antibody spotting. SPRi Detection. SPRi measurements were performed on a GWC imager II system (GWC Technologies Inc., WI), and the in situ SPR responses on each gold spot were collected by the V ++ software. During the detection, 0.01 M PBS was first flowed through the sensing surface at a constant rate of 300 μL min−1 to obtain a stable baseline, followed by the sample (CEA spiked 10% serum solution) for 1 h and 0.01 M PBS for 5 min at the same flowing rate (step I in Figure 1b). To enhance the SPRi signal, 5 μg mL−1 anti-CEA polyclonal antibody (anti-CEA pAb, developed in Mouse) solution was flowed on the sensing surface for 15 min, followed by anti-Mouse IgG functionalized PDA-rGO solution at 10 μL min−1 for 1 h (step II in Figure 1b). The gold deposit enhancement was further carried out by flowing 0.01% (w/w) HAuCl4 solution for 20 min, followed by rinsing with 0.01 M PBS buffer (step III in Figure 1b). Characterizations. Fourier transform-infrared (FT-IR) spectra were recorded using the Nicolet FTIR 6700 spectrophotometer (Thermo Nicolet). Scanning electron microscopy (SEM) images were performed on a JSM-6510LV instrument (JEOL, Tokyo, Japan). Transmission electron microscopy (TEM) images were obtained on a JEM-2100 TEM system (JEOL, Tokyo Japan). UV−vis spectra were collected with a Shimadzu UV-2550 UV−vis spectrophotometer. The atomic force microscopy (AFM) image was obtained in tapping mode using a Nanoman AFM (Veeco Metrology Group). Thermogravimetric analysis (TGA) was performed on a TA Instruments high resolution TGA Q50 from 0 to 700 °C at a heating rate of 5 °C min−1 in an oxygen atmosphere.

some grains at the edges, and their average height of PDA-rGO dramatically increases to 2.3 nm, as shown in Figure 2b, confirming the successful functionalization of GO with PDA coating. It is also unveiled that the whole surface of GO sheets are fully covered by the PDA film, and there are no other PDA nanostructures scattered on the AFM substrate (mica), suggesting that the growth of PDA film exclusively occurs on the GO surface but not in bulk solution. On the FT-IR spectrum (Figure S1 in the Supporting Information), the GO demonstrates characteristic peaks located at 1731, 1623, 1226, and 1056 cm−1, contributed from the stretching vibrations of CO (carboxyl), CC of the graphitic domains, O−H (hydroxyl), and C−O (epoxy or alkoxy)) groups, respectively.29,32 For PDA-rGO, the peak at 1610 cm−1 represents the overlap absorption band of the stretching vibrations of CC in aromatic rings of both rGO and PDA. The new peaks at 1510 cm−1, 1353 cm−1, and 1271 cm−1 are characteristic peaks of PDA, which could be assigned to the scissoring vibration of N−H, bending vibration of O−H in catechol groups, and stretching vibration of phenolic C−O.25 Notably, no signal from CO stretching vibration around 1731 cm−1 can be observed, indicating the carboxyl groups of the GO were eliminated and the GO was partially reduced during the PDA deposition process.32 It has been discovered that dopamine shows fair reducing ability in a weak alkaline solution and could be easily oxidized by dissolved oxygen and other oxidants.24 Therefore, it is not surprising that the GO sheets, with abundant oxidative groups in its surface, are reduced by dopamine. The GO sheets, as oxidizing reagents, facilitate the nucleation and growth of PDA film, thus enabling them to be fully coated by PDA film, which is consistent with the AFM observation shown in Figure 2b. Anti-Mouse IgG was further conjugated to the PDA-rGO by simply mixing PDA-rGO with the antibody for 24 h of reaction as PDA is able to spontaneously react with amine groups of proteins, which is also confirmed by the FT-IR spectrum shown in Figure S1 in the Supporting Information. After the antibody conjugation, the amide I (1648 cm−1, CO stretching) and amide II (1536 cm−1, overlap of N−H bending and C−N stretching) bands of the bound antibody are observed on the FT-IR spectrum,29 which is strongly suggestive that the antibody is successfully attached on the PDA-rGO nanosheets and the secondary structures of the immobilized antibody are fully retained. Also, as-prepared antibody-conjugated PDA-rGO can stably disperse in PBS buffer for days without observable precipitation, as shown in the inset of Figure S1 in the Supporting Information, which enables its specific bioaffinity binding onto the SPRi sensing surface for the first signal amplification, as demonstrated below. After the capture of antibody-conjugated PDA-rGO, spontaneous reductive deposition of elemental gold onto PDA-rGO from the HAuCl4 solution is employed for the second signal amplification. The reduction process of HAuCl4 on PDA-rGO is in situ monitored by UV−vis, as shown in Figure S2a in the Supporting Information. A characteristic peak at around 520 nm, which is the localized surface plasmon resonance (LSPR) adsorption of AuNPs, appears upon the addition of HAuCl4. Its intensity gradually increases with time during 30 min reaction, suggesting that the gold deposition is progressing with a controllable manner. For GO sheets without PDA functionalization, it is observed in Figure S2b in the Supporting Information that no any absorbance peak appears in the presence of HAuCl4, indicating the indispensable role of

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RESULTS AND DISCUSSION Dispersing GO in a slightly alkaline dopamine solution and stirring in ambient atmosphere at room temperature enable the attachment of PDA on the surface of GO via the oxidative polymerization of dopamine.31,32 The AFM image shown in Figure 2a reveals that the spatial dimensions of the GO sheets

Figure 2. AFM images and the height profiles of (a) GO and (b) PDA-rGO nanosheets.

are around or less than 1 μm while their thickness is around 0.6 nm, which is consistent with previous observations, 31 suggesting the complete exfoliation of the graphite oxide to form single-layered GO sheets. After the coating of PDA for 24 h polymerization, in sharp contrast, the roughness of the basal planes of the resulted PDA-rGO is evidently increased with 4490

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PDA-rGO is estimated to be 40 wt %, which is much higher than that of GO and PDA-rGO, illustrating the successful grown of the gold nanostructures onto the PDA-rGO nanosheets. To demonstrate the potential of the dual amplification strategy, CEA is chosen as a model biomarker for the SPRi immunoassay. When 10 ng mL−1 CEA in 10% human serum was flowed onto the chip surface for 1 h incubation, as shown in Figure 4a, the in situ SPRi signal on the sensing spot (with anti-CEA mAb attached, as indicated with the blue rectangle) and control spot (with nonspecific BSA attached) remain stable and flat without an evident increase. Consistently, the SPRi differential image and its line profile (Figure 4b) show negligible noise signals on all sensing spots as well as control spots, suggesting that the SPRi chip possesses a nonfouling nature to suprress the nonspecific protein adsorption from 10% human serum containing abundant nonspecific proteins, and as low as 10 ng mL−1 CEA cannot arouse detectable signal using direct SPRi detection, which is in line with our previous results,5 further highlighting the importance of signal amplification for sensitive SPRi immunoassay. After the capture of CEA target from the sample solution, polyclonal antibody (anti-CEA pAb) is introduced to form sandwich structure on the sensing surface, followed by antiMouse IgG conjugated PDA-rGO nanosheets to generate the first signal amplification. As shown in Figure 4c, the anti-Mouse IgG conjugated PDA-rGO nanosheets is directed to the sensing surface driven by the affinity binding, resulting in a significant increase of the SPRi signal (2.63 ± 0.35 pixels) on the sensing spots preincubated with 10 ng mL−1 CEA, suggesting that the PDA-rGO sheet with a conjugated huge amount of antibodies is able to specifically bind on the sensing surface and efficiently intensify the SPRi signal as an amplification label. The in situ SPRi responses of anti-Mouse IgG conjugated PDA-rGO nanosheets in Figure S4 in the Supporting Information demonstrate a dependent relation of the SPRi signal vs CEA-

PDA as a reducing agent for gold deposition. On the SEM and TEM images shown in Figure 3, there are a lot of gold

Figure 3. SEM and TEM images of PDA-rGO after reacting with HAuCl4 for 30 min.

nanostructures, mainly AuNPs and some gold nanorods homogeneously distributed on the PDA-rGO nanosheets, and no isolated/free-standing gold nanostructures could be observed, suggesting that AuNPs grow specifically on the PDA-rGO but cannot in the solution, which guarantees the specificity of secondary signal amplification with gold deposition. TGA curves in Figure S3 in the Supporting Information exhibit that there are two sharp slopes of mass loss at 180 °C and 450 °C for GO sheets, assigning to the destruction of oxygen-containing functional groups and carbon oxidation, respectively.32 The first slope disappears for PDArGO, further proving the successful reduction of GO during the oxidative polymerization of dopamine on the GO surface.32 Interestingly, the residue content of the HAuCl4-incubated

Figure 4. In situ SPRi sensing curves, differential images, and line profiles for detection of 10 ng mL−1 CEA in human serum. (a,b) Direct detection, (c,d) signal amplification with antibody-conjugated PDA-rGO, and (e,f) sequential gold deposit enhancement. The red lines in parts a, c, and e represent the responses on the sensing spot, and the black lines are collected from the control spot. The blue rectangles in parts b, d, and f indicate the four sensing spots while other spots are control spots on the SPRi chip. 4491

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concentration. PDA-rGO sheets are clearly observed on the SEM images of the sensing surface after the signal amplification, as shown in Figure S5 in the Supporting Information. On the control spots, in sharp contrast, the signal remains at noise level (0.37 + 0.09 pixels), indicating the efficient supression of nonspecific adsorption of the nanosheets. On the differential image and line profile shown in Figure 4d, it is observed that after the incubation with anti-Mouse IgG conjugated PDA-rGO nanosheets, all the sensing spots demonstrate considerable signals and can be easily differentiated from the control spots, which further confirms the efficiency and specificity of the first signal amplification with antibody conjugated PDA-rGO nanosheets. When HAuCl4 solution was flowed through the chip surface for second signal amplification, the SPRi signals on sensing spots dramatically increase (24.41 ± 1.05 pixels) due to the growth of gold nanostructures on the captured PDA-rGO, while on the control spots where no PDA-rGO was captured, the SPRi signal changes remain negligible (0.76 ± 0.25 pixels), as shown in Figure 4e,f and Figure S6 in the Supporting Information. It suggests that flowing HAuCl4 solution on the polymer brush-modified SPRi gold chip without precaptured PDA-rGO sheets does not result in significant SPRi response, possibly because the densely packed polymer chains are able to efficiently prevent the HAuCl4 from accessing the gold chip and thus the deposit does not occur to interfer the signal amplification. The captured PDA-rGO sheets on the sensing sruface mediate the deposition of gold nanostructures when HAuCl4 solution was flowed (see the SEM images in Figure S7 in the Supporting Information) and induces a striking signal enhancement due to the high mass-density and dielectric constant of the gold nanostructures and the electromagnetic coupling between the gold nanostructures and the underlying gold thin film. As the gold deposition exclusively occurs on PDA-rGO sheets, the SPRi signals originating from the immunoassay is further efficiently and specifically enhanced by this second signal amplification and the signal-to-noise ratio for the immunoassay is significantly elevated for improved performance. The SPR signal increases are plotted as a function of CEA concentration, as shown in Figure 5. Each value is obtained by averaging the data from four parallel spots with standard deviation shown on the point. It is unveiled that the signal intensities are positively related to the CEA concentrations. Using PDA-rGO alone for signal amplification, the detection limit is as low as 2.5 ng mL−1 for CEA in 10% human serum according to the Mb + 3Sb definition. When the dual signal amplification is applied, the detection limit can be further decreased to 500 pg mL−1. The whole dynamic range covers a wide range from 0.5−50 ng mL−1 and moreover, higher concentrations above 50 ng mL−1 could be directly quantified by the SPRi chip without the need for signal amplification, as demonstrated previously.5 Considering that the sample matrix is 10% human serum, this dual signal amplification also possesses excellent specificty and high reliability. CEA is one of the valuable indicators closely related to various tumors, and its serum concentration in healthy individuals is several ng mL−1 while significantly elevated upon the onset or recurrence of a tumor.11,12 Therefore, the dual signal amplification reported here provides adequate sensitivity for the SPRi immunoassay of CEA and other biomarkers in serum.

Figure 5. Calibration curves for SPRi detection of CEA using (a) PDA-rGO alone for signal amplification and (b) using the dual signal amplification.

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CONCLUSIONS We have demonstrated the use of PDA-rGO for dual signal amplification of SPRi immunoassay, in which antibodyconjugated PDA-rGO functions as a signal-enhancing label for the first signal amplification, while as a unique reductant and matrix to facilitate the metal deposit for second signal amplification, resulting in a detection limit of 500 pg mL−1 for the model CEA in human serum. Most importantly, the accomplished dynamic range covers all concentrations above 500 pg mL−1. Considering the immunoassay is carried out in 10% human serum, this dual signal amplification indeed provides high specificity and sensitivity for potential practical applications.

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ASSOCIATED CONTENT

S Supporting Information *

FT-IR spectra of (a) GO, (b) PDA-rGO, and (c) antibody conjugated PDA-rGO; time-course UV−vis spectra of (a) PDA-rGO and (b) GO solution in the presence of HAuCl4; TGA curves of GO, PDA-rGO, and HAuCl4-incubated PDArGO in oxygen atmosphere; representative in situ SPRi responses for flowing antibody conjugated PDA-rGO to the 4492

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sensing spots which are preincubated with CEA target and antiCEA polyclonal antibody; SEM images of the SPRi sensing chip after 10 ng mL−1 CEA detection and subsequent signal amplification by antibody conjugated PDA-rGO; representative in situ SPRi responses for flowing HAuCl4 solution to the sensing spots which are preincubated with CEA target, antiCEA polyclonal antibody, and antibody conjugated PDA-rGO; and SEM images of the SPRi sensing chip after 10 ng mL−1 CEA detection and subsequent dual signal amplification by antibody conjugated PDA-rGO and gold deposition. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*Fax: 08623-68254969. Phone: 08623-68254969. E-mail: [email protected]. *Phone: 08623-68254727. E-mail: [email protected]. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by National Natural Science Foundation of China (Grant No. 21205098, 21273173), National Program on Key Basic Research Project of China (973 Program Grant No. 2013CB127804), Natural Science Foundation Project of CQ CSTC (Grant cstc2012jjA10099), Key Laboratory of Analytical Chemistry for Biology and Medicine (Wuhan University), Ministry of Education (Grant ACBM2012006), Institute for Clean Energy & Advanced Materials (Southwest University), Chongqing Key Laboratory for Advanced Materials and Technologies of Clean Energies, Start-up grant under Grant SWU111071 from Southwest University, Chongqing Engineering Research Center for Rapid Diagnosis of Dread Disease, and Chongqing Development and Reform Commission.

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