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May 6, 2016 - Alkaline Phosphatase Tagged Antibodies on Gold Nanoparticles/TiO2 Nanotubes Electrode: A Plasmonic Strategy for Label-Free and Amplified...
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Alkaline Phosphatase Tagged Antibodies on Gold Nanoparticles/TiO2 Nanotubes Electrode: A Plasmonic Strategy for Label-Free and Amplified Photoelectrochemical Immunoassay Yuan-Cheng Zhu,† Nan Zhang,† Yi-Fan Ruan, Wei-Wei Zhao,* Jing-Juan Xu,* and Hong-Yuan Chen State Key Laboratory of Analytical Chemistry for Life Science and Collaborative Innovation Center of Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, Jiangsu 210023, China S Supporting Information *

ABSTRACT: This work reports a plasmonic strategy capable of label-free yet amplified photoelectrochemical (PEC) immunoassay for the sensitive and specific detection of model protein p53, an important transcription factor that regulates the cell cycle and functions as a tumor suppressor. Specifically, on the basis of Au nanoparticles (NPs) deposited on hierarchically ordered TiO2 nanotubes (NTs), a protein G molecular membrane was used for immobilization of alkaline phosphatase (ALP) conjugated anti-p53 (ALP-a-p53). Due to the immunological recognition between the receptor and target, the plasmonic charge separation from Au NPs to the conduction band of TiO2 NTs could be influenced greatly that originated from multiple factors. The degree of signal suppression is directly associated with the target concentration, so by monitoring the changes of the plasmonic photocurrent responding after the specific binding, a new plasmonic PEC immunoassay could be tailored for label-free and amplified detection. The operating principle of this study could be extended as a general protocol for numerous other targets of interest. rotein detection is essential in numerous fields such as clinical diagnosis, biomedical research, and environmental monitoring.1−3 Using the newly developed photoelectrochemical (PEC) bioanalytical technique, the PEC immunoassay has been increasingly recognized as an important methodology for sensitive protein assays.4−23 Recently, explosive PEC immunoassays have been proposed, and these works could be classified into two main categories: the label-free6,15,21 and labelbased16−18 methods. On the basis of the direct antibody (Ab)−antigen (Ag) affinity for obvious simplicity, the label-free works suffer from their own drawbacks of low protein abundance and inability for amplification, and sensitivity needs to be improved. In contrast, albeit with higher sensitivity, the label-based methods necessitate the additional labeling step toward the target protein involving complicated and tedious assay processes, and hence, increased time and cost. Obviously, there appears to be a dilemma between the simplicity for practical application and the sensitivity for trace amount analysis. Inspired by this, one question is naturally raised: whether the amplified PEC immunoassay could be ingeniously achieved with a simple label-free protocol? On the basis of the unique localized surface plasmon resonance (LSPR) feature, the plasomonic photoelectrochemistry of noble metal nanoparticles (NPs) deposited on various semiconductors has been an extremely hot topic drawing substantial research interest from many fields.24−27 Recent activities have demonstrated its unprecedented applications in,

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© XXXX American Chemical Society

e.g., photovoltaics and photocatalysts.28−30 In fact, given the metallic NPs can concentrate light near a semiconductor/liquid junction to produce more photocarriers that can reach the interface, the appropriate integration of such LSPR enhanced photoelectrochemistry with the judiciously designed biological reactions may offer great opportunities to novel biomolecular detection. In this regard, we previously have exploited the LSPR-based photoelectrochemistry and catalytic growth reaction of Au NPs on the TiO2 film to develop a plasmonic PEC enzymatic biosensor.26 Then, Zheng et al. proposed a novel SPR enhanced PEC protein assay by decorating Au NPs on TiO2 nanowires (NWs).19 More recently, using Au NPs and TiO2 NPs, Liu and co-workers reported another dye-sensitized and SPR enhanced PEC biosensor for sensitive analysis of protein kinase activity.31 Despite these endeavors, the study in this field is still rather limited and it is highly desirable to develop more exquisite plasmonic PEC bioanalysis. With these motivations, on the basis of the Au NPs-TiO2 nanotubes (NTs) electrode, this work reported an advanced plasmonic PEC study that is capable of label-free yet amplified immunoassay (experimental details were included in the Supporting Information). Specifically, as shown in Scheme 1, Au NPs were coupled with TiO2 NTs for constructing the Received: March 31, 2016 Accepted: May 6, 2016

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DOI: 10.1021/acs.analchem.6b01261 Anal. Chem. XXXX, XXX, XXX−XXX

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Upon the subsequent formation of immunocomplex without further labeling steps, the plasmonic charge separation from Au NPs to the conduction band (CB) of TiO2 NTs could be influenced greatly and hence reduce the plasmonic photocurrent response. The degree of signal suppression is directly associated with the target concentration. As such, a novel plasmonic label-free and amplified PEC immunoassay could be developed for the sensitive and specific detection of the model protein p53 and to the best of our knowledge has never been reported. It is worth noting that the operating principle of this study could be extended as a general protocol for other targets of interest.

Scheme 1. Proposed Plasmonic PEC Immunoassay Using an Au NPs-TiO2 NTs Electrode

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RESULTS AND DISCUSSION TiO2 nanostructures have long been used for various bioanalytical applications due to their good biocompatible properties and chemical/thermal stabilities.19 Here, the hierarchically ordered TiO2 NTs were first fabricated with a two-step electrochemical anodic oxidation technique, and the fabrication process was illustrated in Scheme S1 and revealed by scanning electron microscopy (SEM) as shown in Figure S1A−H. Figure 1A presents a typical top morphology of the TiO2 NTs microscopic structure with the magnified SEM image as shown in the Figure 1A inset, and the detailed description was included in the Supporting Information. On the other hand, Au NPs were synthesized using the well-established method and then characterized with UV−vis absorption spectra and transmission electron microscopy (TEM) imaging, with the results recorded in Figure 1B and inset, respectively. As shown, the Au NPs have the maximum plasmon absorption peak at ca. 520 nm and appeared as quasi-spherical particles with the sizes corresponding to ca. 20 nm. The photoelectrode was then

photoresponsive transducer due to its excellent plasmonic photoelectrochemistry. Then, a protein G molecular membrane was used as matrix for immobilization of alkaline phosphatase (ALP) conjugated anti-p53 (ALP-a-p53) molecules since it not only specifically binds to the Fc fragment of IgG but also strongly adsorbs to the Au NPs-TiO2 NTs electrode surface. The bound ALP-a-p53 on protein G layer can retain both its enzyme catalytic activity to the hydrolysis of ascorbic acid 2phosphate (AAP) to in situ generate ascorbic acid (AA) for efficient electron donating and its immunoactivity toward binding with p53 protein, an important transcription factor that regulates the cell cycle and functions as a tumor suppressor.

Figure 1. (A) SEM image and the magnified image (inset) of the as-prepared TiO2 NTs. (B) UV−vis spectra of Au NPs. Inset: TEM image of Au NPs. (C) SEM image and the magnified image (inset) of Au NPs-TiO2 NTs. (D) Photocurrent action spectra generated by TiO2 NTs (black) and Au NPs-TiO2 NTs (red). Inset: Transient photocurrent responses of TiO2 NTs (black) and Au NPs-TiO2 NTs (red) under visible light irradiation (λ > 410 nm). The tests were performed in 0.1 M PBS solution containing 0.1 M AA with a constant potential of 0.0 V. B

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Figure 2. AFM images of (A) Au NPs-TiO2 NTs electrode; (B) after protein G immobilization; (C) after the Ab immobilization. (D) Photocurrent response of the developed immunosystem in (a) the 0.10 M Tris-HCl without both AAP and p53, (b) 0.10 M Tris-HCl containing 0.01 M AAP without p53, and (c) 0.10 M Tris-HCl containing 0.01 M AAP with p53 corresponding to 10 ng mL−1. All experiments were performed under visible light irradiation (λ > 410 nm) with 0.0 V applied potential.

prepared by anchoring the Au NPs onto the TiO2 NTs through a convenient poly(diallyldimethylammonium chloride) (PDDA)-mediated adsorption technique. We verified the successful decoration of Au NPs by SEM imaging and performed the layer optimization by PEC measurements, with the results recorded in Figures S2 and S3, respectively. Typically, as shown in Figure 1C, the Au NPs were thickly distributed on the entire TiO2 NTs scaffold, and the magnified image in Figure 1C inset demonstrated that Au NPs located evenly on the walls and in the tubes of TiO2 NTs with good conformability. Since the Au NPs could facilitate the protein adsorption due to their superior biocompatibility, such uniform distribution would be advantageous to following biomolecular assay development. To obtain more enriched information on the light-harvesting properties of the nanostructures, the PEC performances of the TiO2 NTs and Au NPs-TiO2 NTs were then disclosed by photocurrent action spectra under the irradiation from 300 to 800 nm. Figure 1D demonstrates the substantial plasmonic change of TiO2 NTs prior to and after Au NPs loading. As shown, bare TiO2 NTs only had an obvious response in the UV region, whereas the Au NPs-TiO2 NTs electrode had a remarkable response in the broad visible spectrum, implying the crucial role of Au NPs to photocurrent generation under visible light illumination through the LSPR enhanced effect and also their high suitability for use as a photoelectrode for the PEC bioassay. The underlying reason could be ascribed to a unique plasmonic charge separation and transfer process as included in Scheme 1. Upon visible irradiation (>410 nm in the present work), photoexcitation of the Au NPs would result in the collective oscillations of hot electrons and hence the plasmonic charge separation at the Au

NPs surface. When in intimate contact with TiO2 NTs, these electrons can be injected spatially from Au NPs to the CB of TiO2 NTs, leaving the oxidized Au NPs restored by the ascorbic acid (AA) as electron donor. The PEC properties were further probed by transient photocurrent responses, and the Figure 1D inset records the measured chronoamperometric I−t curves upon illumination of visible light with wavelength >410 nm. With the light turned on and off, a considerable photocurrent enhancement was produced by the Au NPsTiO2 NTs electrode, further indicating the excellent photoactivities of the hybrid Au NPs-TiO2 NTs. With the onset of illumination, the rapid rise of the photocurrent signal implied the prompt charge separation and transfer in the Au NPs, the excellent electrical contact between the Au NPs and the vertical TiO2 NTs substrate, and also the efficient collection of the injected electrons by the Ti foil as photocurrent signal. The Au NPs-TiO2 NTs were further applied for the proposed bioassay development. The changes of surface topographies along with the development process were recorded by atomic force microscopy (AFM) imaging as shown in Figures 2A−C and S4, and the detailed discussion about these figures was included in the Supporting Information due to the space limit. The proposed assay was then tested by the stepwise transient photocurrent responses upon the intermittent light irradiation. Figure 2D shows the response of the system to the ALP-catalyzed reaction and further to p53 at a level of 10 ng mL−1 in the presence and absence of 0.01 M AAP at room temperature. As shown in curves a and b, the ALP-a-p53 modified electrode exhibited a low photocurrent response in the absence of AAP, whereas a much enhanced signal was observed upon the addition of AAP. Specifically, the C

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Figure 3. (A) Plot of the photocurrent vs p53 concentration. Inset: the derived calibration curve. (B) Selectivity of the proposed immunoassay to p53 by comparing it to the interfering proteins at the 100 ng mL−1 level: glucose oxidase (GOD), prostate specific antigen (PSA), lysozyme (LZM), and thrombin. ΔI was the photocurrent decrement corresponding to variable p53 concentration. Data were recorded in 0.10 M Tris-HCl containing 0.01 M AAP with 0.0 V applied potential and visible light irradiation (λ > 410 nm).

serum samples is about 0.55 ng mL−1,39 these above results indicated that this assay may be applied for real sample testing.

ALP enzymatic reaction in the presence of AAP could in situ cause the generation of AA for scavenging the holes localized on Au NPs and thereby contribute to the obvious signal growth. Subsequently, as shown in curve c, the immunocomplexing with 10 ng mL−1 p53 would obviously decrease the photocurrent signal. In this process, such photocurrent reduction should be attributed to the following possible reasons: (1) the active site of tagged ALP can be shielded upon the immunobinding and the access to its substrate would be partially or completely blocked, inhibiting the enzymatic catalysis and thus the in situ AA generation;32−34 (2) the steric hindrance of the formed immunocomponent could further impede the interfacial mass transfer, retarding the interfacial electron communication;6,21 (3) direct capturing of p53 proteins close to the Au NPs-TiO2 NTs interface may change the ambient dielectric permittivity26 and influence the capability of attenuation of energy coupling between Au NPs and TiO2 NTs.19 These results proved the feasibility of the proposed protocol. Besides, some experimental optimizations were further performed as included in Figure S5. Since the signal decrease extent associates closely with the target concentration, a novel PEC p53 assay can be accomplished by recording the photocurrent variation. Figure 3A manifests the final photocurrent intensities after immunorecognition with variable target concentrations, and the Figure 3A inset includes the derived calibration curve. As shown, the signal declined with the increased p53 concentration up to 100 ng mL−1, proving the analyte-induced signal depression. The dropoff of the signal decrease at higher p53 concentrations was attributed to the saturation of target capture on the electrode surface. Under the current experimental conditions, the target could be detected at levels down to 0.05 ng mL−1, which is comparable to those of previous techniques of ELISA,35 quartz crystal microbalance,36 surface plasmon resonance,37 and fieldeffect transistor.38 Reproducibility of this assay was assessed by an interassay relative standard deviation (RSD) through testing 10 ng mL−1 samples with five electrodes, and RSD of 8.5% was calculated. As shown in Figure 3B, the selectivity of this work was further evaluated by using the interfering proteins including glucose oxidase (GOD), prostate-specific antigen (PSA), lysozyme (LZM), and thrombin. These results demonstrated that these interfering proteins could not cause obvious signal variation and thereby the good selectivity of this assay. In light of the fact that the average level of p53 in lung cancer patient

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CONCLUSIONS In summary, we have developed a plasmonic PEC immunoassay, in which Au NPs were loaded on the hierarchically ordered TiO2 NTs as efficient light-harvesting photoelectrode, followed by protein G assisted immobilization of ALP-a-p53 for capturing model protein p53. Due to the SPR-enhanced photoelectrochemistry, the well-defined Au NPs-TiO2 NTs nanostructure possessed much improved PEC property as compared to the bare TiO2 NTs. In the immunocomplexing, the binding of the target to the probe could cause an obvious photocurrent reduction that originated from ternary synergistic effects, leading to an advanced label-free-based PEC immunoassay with signal amplification. It is believed that our work could offer a new perspective for the rational implementation of various SPR nanostructures for novel PEC sensors, and the mechanism proposed here could also serve as a general basis for future immunoassay development.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b01261. Scheme S1, Figures S1−S5, materials and apparatus, synthesis of Au NPs and TiO2 NTs, fabrication of Au NPs modified TiO2 NTs electrode, the immunoassay development, and experimental optimizations (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone/Fax: +86-25-89684862. *E-mail: [email protected]. Phone/Fax: +86-25-89687294. Author Contributions †

Y.-C.Z. and N.Z. contributed to this work equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from the 973 Program (Grant 2012CB932600), the National Natural Science Foundation of China (Grant Nos. 21327902, 21135003, and 21305063), and D

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(31) Yan, Z. Y.; Wang, Z. H.; Miao, Z.; Liu, Y. Anal. Chem. 2016, 88, 922. (32) Zhang, X. T.; Wu, Y. F.; Tu, Y. F.; Liu, S. Q. Analyst 2008, 133, 485. (33) Su, B. L.; Tang, J.; Huang, J. X.; Yang, H. H.; Qiu, B.; Chen, G. N.; Tang, D. P. Electroanalysis 2010, 22, 2720. (34) Kheiri, F.; Sabzi, R. E.; Jannatdoust, E.; Shojaeefar, E.; Sedghi, H. Biosens. Bioelectron. 2011, 26, 4457. (35) Jagelská, E.; Brázda, V.; Pospisilová, S.; Vojtesek, B.; Palecek, E. J. Immunol. Methods 2002, 267, 227. (36) Altintas, Z.; Tothill, I. E. Sens. Actuators, B 2012, 169, 188. (37) Jiang, T.; Minunni, M.; Wilson, P.; Zhang, J.; Turner, A. P. F.; Mascini, M. Biosens. Bioelectron. 2005, 20, 1939. (38) Han, S. H.; Kim, S. K.; Park, K.; Yi, S. Y.; Park, H. J.; Lyu, H. K.; Kim, M.; Chung, B. H. Anal. Chim. Acta 2010, 665, 79. (39) Luo, J. C.; Zehab, R.; Anttila, S.; Ridanpaa, M.; HusgafvelPursiainen, K.; Vainio, H.; Carney, W.; Devivo, I.; Milling, C.; BrandtRauf, P. W. J. Occup. Environ. Med. 1994, 36, 155−160.

the Natural Science Funds of Jiangsu Province (Grant BK20130553) is appreciated. This work was also supported by a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.

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