Article pubs.acs.org/ac
Unraveling the Role of Hydrogen Peroxide in α‑Synuclein Aggregation Using an Ultrasensitive Nanoplasmonic Probe Yan Xu,† Kun Li,† Weiwei Qin,† Bing Zhu,† Ziang Zhou,‡ Jiye Shi,†,§ Kun Wang,† Jun Hu,† Chunhai Fan,† and Di Li*,† †
Division of Physical Biology and Bioimaging Centre, Shanghai Synchrotron Radiation Facility, CAS Key Laboratory of Interfacial Physics and Technology, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, China ‡ The Johns Hopkins University, Baltimore, Maryland 21211, United States § UCB Pharm, Slough SL1 3WE, Berks U.K. S Supporting Information *
ABSTRACT: Aggregation of α-Synuclein (α-Syn) in Lewy bodies is largely responsible for the demise and death of dopamine neurons. Oxidative stress associated with the aggregation-induced oxidative damage is considered as a possible origin of the toxicity. However, the cellular mechanism of H2O2 in the aggregation of α-Syn remains a debate, i.e., whether the aggregation is caused by endogenously secreted or exogenous H2O2 from upstream. Here, we report on the development of an ultrasensitive plasmonic assay with a designed nanoplasmonic probe to unravel the role of H2O2 in the aggregation of α-Syn. The nanoplasmonic probe is composed of a Au nanoparticle with surface-attached double-stranded DNA and horseradish peroxidase (HRP). In the presence of H2O2, HRP initiates the polymerization of aniline, which in turn results in the in situ formation of a layer of conducting polymer on the nanoplasmonic probe. By monitoring the associated plasmonic response, we can sensitively detect H2O2 with a remarkably low detection limit of 8 nM. With this ultrasensitive plasmonic assay, we find that exogenous H2O2 plays a dominant role for the aggregation of α-Syn in vitro, whereas the contribution from endogenously secreted H2O2 is negligible. sensors is at the level of 10−6 to 10−7 M (listed in Table S1 of the Supporting Information (SI)), which is inadequate for monitoring the extremely low amount of H2O2 secreted in cellular activities. Meanwhile, conventional electrochemical sensors can hardly provide spatial information in cells.20 In addition, many fluorescent molecules that are sensitive to H2O2 have been developed as probes for intracellular and extracellular fluorescent assays.21−25 However, since fluorophores are easily photobleachable, it is usually difficult to dynamically trace the fate of H2O2 under physiological conditions for a long period. Herein, we report a conceptually new nanoplasmonic assay of H2O2 to clarify its role in the aggregation of α-Syn. Localized surface plasmon resonance (LSPR), the collective oscillations of free electrons on noble metal nanoparticles (NPs), is highly sensitive to the refractive index of surrounding environments.26−29 Thus, LSPR response of plasmonic NPs could be finely tuned by surface-deposited active coatings including metals, biomolecules, and polymers.30 Recently, Wang et al. demonstrated that the plasmonic response of Au nanorods could be modulated by surfactant-assisted polymerization of aniline on its surface, suggesting polyaniline (PANI) is an effective candidate to regulate
Alpha-Synuclein (α-Syn) is an abundant presynaptic protein associated with several important biological activities.1,2 α-Syn is an intrinsically unfolded protein, and accumulated evidence suggests that α-Syn aggregates with abnormally high quantities in Lewy bodies are largely responsible for the demise and death of dopamine neurons, which has been regarded as a cardinal hallmark lesion of Parkinson’s disease (PD) pathology.3−6 However, the toxicity origin of aggregated α-Syn remains unclear. One possible explanation is that the toxicity arises from the oxidative stress associated with the aggregation that leads to oxidative damage.7 However, it leads to another controversy: it is still not clear whether oxidative stress caused by reactive oxygen species (ROS) is a causative factor8 or a result of α-Syn aggregation.9,10 The key reason for this controversy is the lack of an ultrasensitive ROS sensor. Current studies still rely on electron paramagnetic resonance (EPR)8−10 for analyzing hydroxyl radicals in the aggregation process, which obviously lacks spatial information and suffers from low sensitivity. H2O2, the “signaling face” of ROS, plays a critical role in the regulation of various physiological processes11 and has been recently regarded as a “necessary evil” because of its dual characters in living systems, host defense and cellular signal transduction.12,13 Considerable efforts have been devoted to develop various sensors for H2O2 analysis. Among them, electrochemical methods are direct and have real-time convenience.14−19 However, so far the limit of detection (LOD) of most reported electrochemical H2O2 © 2015 American Chemical Society
Received: November 24, 2014 Accepted: January 15, 2015 Published: January 15, 2015 1968
DOI: 10.1021/ac5043895 Anal. Chem. 2015, 87, 1968−1973
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Analytical Chemistry LSPR responses.31 In addition, enzymatic polymerization of aniline with horseradish peroxidase (HRP) has been proven to be a simple and environmentally benign alternative.32 A negatively charged polyelectrolyte is, however, needed as a template in the enzymeinitiated polymerization to minimize the parasitic branching and promote a more para-directed polymerization.33−36 In the present work, we integrated the nanoplasmonic properties of Au NPs with HRP-initiated polymerization of aniline on DNA templates, based on which we designed an ultrasensitive plasmonic assay for H2O2. With this ultrasensitive nanoplasmonic probe, we found that exogenous H2O2 plays a dominant role for the aggregation of α-Syn in vitro, whereas the contribution from endogenously secreted H2O2 is negligible.
Hek 293 cells were cultured in a DMEM medium containing 10% fetal bovine serum (FBS) in a humidified 5% CO2 atmosphere at 37 °C. The Hek 293 cells were seeded in a 12-well plate (2 × 105 cells per well) after 24 h for experiments. Prior to nanoplasmonic detection, the media were removed and 1 mL of PBS containing 20 mM glucose was added for the real sample measurements. Then 100 μM of 1-methyl-4-phenylpyridinium (MPP+) was added to the solution to stimulate cells to release H2O2. In a control group, 4 μM catalase was added with MPP+ to scavenge the released H2O2. The collected supernatant containing released H2O2 was then cast on the Au plasmonic probes modified glass slide and incubated with reaction buffer (PBS, pH 4.3) containing 2 mM aniline. PRRS spectra and the corresponding true color images of single Au NPs were collected by DFM. Analysis of H2O2 Released in the Aggregation of α-Syn. α-Syn was diluted to 50 μM with 10 mM PBS (pH 7.4) and then incubated at 37 °C in a parafilm-sealed 1.5-mL tube under gentle shaking. In control groups, equal concentrations of bovine serum albumin (BSA), ovalbumin (OVA), and lysozyme were also incubated under the same condition of α-Syn. After incubation for 7 days, the samples were taken from the tube, and the secreted H2O2 was test by DFM in a procedure as described before. Thioflavin T (ThT) Assay. A 100 mM aqueous solution of Thioflavin T was prepared and filtered through a 0.22-mm polyether sulfone filter for further use. α-Syn (50 μM) was incubated at 37 °C with gentle agitation. At various time points, 3-μL aliquots of the a-Syn incubations were added to 100 μL of 10 μM Thio T (50 mM glycine-NaOH, pH 8.5). Fluorescence measurements were performed on an Edinburgh FS920 fluorimeter at 490 nm with excitation at 450 nm. DFM Imaging and Scattering Spectroscopy Measurements. The dark-field measurements were carried out on an inverted microscope (Olympus IX71, Japan) equipped with a dark-field condenser (0.8 < NA < 0.95) and a 60× objective lens (NA = 0.8). The sample slides were immobilized on a platform, and a 100-W halogen lamp provided white light source to excite the Au NPs to generate plasmon resonance scattering light. The scattered light was collected by a true-color digital camera (Olympus DP70, Japan) to generate the dark-field color images, and was also split by a monochromator (Acton SP2300i, Princeton Instruments, USA) which was equipped with a grating (grating density 300 lines/mm; blazed wavelength 500 nm) and recorded by a spectrograph CCD (CASCADE 512B, Roper Scientific, Princeton Instruments, USA) to obtain the scattering spectra. The scattering spectra were integrated as 10 s. The spectra of an individual nanoparticle were corrected by subtracting the background spectra taken from the adjacent regions without the Au NPs and dividing with the calibrated response curve of the entire optical system. AFM Measurement. AFM was used to monitor the structural changes of α-Syn upon incubating with iron and H2O2. α-Syn (50 μM) in 10 mM PBS (pH 7.4) was incubated with 1 μM Fe (III) or 50 μM H2O2. The mixture was incubated at 37 °C in a parafilm-sealed 1.5-mL tube with gentle agitation. At given time points, 2 μL of the mixture was taken out for AFM measurement. The sample was scanned under tapping mode S3 using a J scanner of a Multimode Nanoscope IIIa AFM (Vecco/Digital Instruments) with a silicon nitride cantilever with sharpened pyramidal tip (OMCL-TR400PSA, Olympus). SEM Measurement. The Au nanoplasmonic probes were drop-cast on ITO glass slides for 5 min followed by thoroughly washing with PBS and then drying with N2. Then, 100 μL of
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EXPERIMENTAL SECTION Materials. HAuCl4·3H2O, H2O2, and aniline were purchased from Sigma-Aldrich. The concentration of H2O2 was determined by titration with cerium(IV) to a ferroin end-point.37 Streptavidin-HRP (SA-HRP) was purchased from New England Biolabs. α-Synuclein was purchased from Sino Biological Inc. and used as received. Thioflavin T was purchased from Sigma-Aldrich Co. Nucleic acids were synthesized by TaKaRa Biotechnology Co. (Dalian, China) and purified with HPLC. The sequences of nucleic acids employed in this study were as follows: thiolatedDNA, 5′-HS-TTTTTATAACAGCTGCTGCAGCTCG-3′; biotinylated -DNA, 5′-botin-TTTTTCGAGCTGCAGCAGCTGTTAT-3′. Preparation of Au Nanoplasmonic Probes. Au NPs with average diameter of 50 nm were synthesized according to a seedmediated growth method.38 Modification of the as-prepared Au NPs with ss-DNA was carried out according to our previous work. Briefly, 5 μL of thiolated ss-DNA (100 μM) was added to 400 μL of Au NPs solution. The solution was slowly brought up to a final salt concentration of 0.1 M NaCl and 10 mM phosphate (pH 7) and allowed to stand for 40 h. The mixtures were then centrifuged (1500 rcf, 5 min, 4 °C), and the supernatant was discarded. The centrifuge procedure was repeated three times to remove excess of unbound ss-DNA. The resulting ss-DNA modified Au NPs were redispersed with equal volume of 10 mM phosphate buffer saline (PBS, pH 7.2). A 5-μL portion of biotinmodified ss-DNA with complementary sequence (100 μM) was then added to the mixture, and it was incubated at 25 °C for 40 h to form a stable duplex. The mixtures were washed three times by centrifuge (1500 rcf, 5 min, 4 °C). The ds-DNA modified Au NPs were redispersed with equal volume of 10 mM PBS (pH 7.2). Then, 4 μL of SA-HRP was added to the mixture and incubated at 25 °C for 30 min. The resulting mixture was washed three times by centrifuge (1500 rcf, 5 min, 4 °C) to remove excess SA-HRP. The HRPs-bound AuNPs were redispersed with equal volume of 10 mM PBS (pH 7.2) and stored at 4 °C for further use. The numbers of ss- and ds-DNA on Au NPs were determined using a fluorescence-based method proposed by Mirkin et al. (SI Figure S1).39 Sensitivity Test with Exogenously Added H2O2 and Released H2O2 from Hek 293 Cell. The HRP-bound Au plasmonic probes were drop-cast on silanized glass slides for 5 min followed by thoroughly washing with PBS and then drying with N2. Next, 100 μL of reaction buffer (PBS, pH 4.3) containing 2 mM aniline and different concentrations of H2O2 (0−2 mM) was cast on the modified glass slides. PRRS spectra and the corresponding true color images of single Au NPs were collected by DFM every 10 min for 30 min. Experiments were performed in triplicate. 1969
DOI: 10.1021/ac5043895 Anal. Chem. 2015, 87, 1968−1973
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Figure 1. (A) Principle of the plasmonic assay for H2O2. Au NPs of 50 nm modified with ds-DNA and HRP are designed as nanoplasmonic probes. In the presence of H2O2, HRP initiates the polymerization of aniline on ds-DNA templates, leading to true color changes and plasmon band shifts. (B) Dark-field images of (a) bare Au NPs, (b) ds-DNA-loaded Au NPs, and (c) ds-DNA-HRP-loaded Au NPs. (C) PRRS spectra of Au NPs during sequential modifications.
reaction buffer (PBS, pH 4.3) containing 2 mM aniline and 2 mM H2O2 was brought up to the glass slide. After reaction for 30 min, the reaction buffer was discarded and the glass slide were thoroughly washed with PBS and dried with N2. The morphology changes of Au NPs were observed through scanning electron microscope (Zeiss, LEO 1530VP, Germany) and field emission scanning electron microscope (FEI, Magellan 400, USA) with a TLD detector in SE mode.
enlarged the cross section of scattering light, leading to a further redshift of λmax to 559 nm. We then carried out HRP-initiated polymerization on the prepared nanoplasmonic probes. Because HRP is present only on the gold surface, a given concentration of H2O2 will result in a certain depth of PANI coating with a certain polymerization time interval, leading to a change of dielectric constant. Thereby, the change of LSPR spectrum is H2O2 concentration- and timedependent. Figure 2A shows the dark-field images of the nanoplasmonic probes upon interaction with different concentrations of H2O2 for 30 min. With the increase of H2O2, we observed a green-to-red color change. In addition, we examined the timedependent LSPR changes upon incubating with different concentrations of H2O2 for different polymerization times. Figure 2B depicts the PRRS spectra of a single nanoplasmonic probe upon incubating with different concentrations of H2O2 (from 0 to 2 mM) with a time interval of 10 min. With the increase of H2O2 concentrations, we observed a rapid red shift in 10 min and then a plateau in 30 min. Even for the lowest concentration of H2O2 (10−8 M), the redshift of PRRS peak is also plateaued in 30 min with λmax red-shifted to 567 nm. Therefore, we chose 30 min as the fixed time-interval for the growth of PANI in further quantitative analysis. It should be noted that changing the medium from pure water to aniline (2 mM) changes the dielectric surroundings of the nanoplasmonic probes even in the absence of H2O2, which contributes to a 5-nm red-shift of λmax. Therefore, 5 nm is considered as background and will be subtracted in further quantitative analysis. Meanwhile, LSPR response of the HRP-loading nanoplasmonic probes is highly selective to H2O2. We performed a control experiment to interrogate the nanoplasmonic probes with aniline, H2O2, and catalase (a selective scavenger of H2O2) and observed only a red-shift of 5 nm, corresponding to medium change-induced background (Figure 2C). The enzymatic coating of plasmonic Au NPs with PANI was also confirmed by field emission scanning electron microscopy (FESEM) (Figure 2D); a shell structure on Au NPs was clearly observed. Next, we challenged the nanoplasmonic probes with exogenously added and stimuli-released H2O2 from living cells.
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RESULTS AND DISCUSSION The detailed principle of plasmonic assay is illustrated in Figure 1A. Au NPs of 50 nm were used as building blocks for surface attachment of double stranded (ds-) DNA and HRP. Briefly, a thiolated (ss-) DNA was first attached on Au NPs via Au−S chemistry. A biotinylated ss-DNA with complementary sequence was then hybridized with the surface-attached ss-DNA to form duplex with overhang biotin groups to act as templates for PANI. Next, Streptavidin-HRP (SA-HRP) was brought up to the surface of Au NPs via biotin−avidin interactions. The ds-DNA-HRP loaded Au NPs were used as nanoplasmonic probes. HRP initiates the polymerization of aniline on ds-DNA templates with the aid of H2O2, leading to true color changes and plasmon band shifts, which lay the foundation for plasmonic detection of H2O2. The coverage of ds-DNA and HRP on the surface of Au NPs were determined to be ca. 250 and 60, which were balanced between steric hindrance and catalytic activity (SI Figures S1 and S2). We first compared the true color (Figure 1B) and plasmon band shifts of Au NPs during sequential modifications with DNA and HRP (Figure 1C). In this work, LSPR change of the nanoplasmonic probes was monitored and recorded at real time by dark-field microscopy (DFM) combined with plasmonic resonance Rayleigh scattering (PRRS) spectroscopy.40−43 Bare Au NPs of 50 nm deposited on silanized glass slides exhibited green light with the maximum plasmon band (λmax) at 550 nm. Modification with ds-DNA led to slight changes of plasmon band with λmax red-shifted to 553 nm. Further modification with HRP 1970
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Figure 2. (A) Dark-field images of the nanoplasmonic probes after polymerization for 30 min in the presence of different concentration of H2O2: (a) 0 nM, (b) 10 nM, (c) 1 μM, and (d) 1 mM. (B) Time- and concentration-dependent wavelength shift (Δλmax) of the nanoplasmonic probes upon interrogating with different concentrations of H2O2 with a time interval of 10 min. H2O2 concentrations are 0 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM, 1 mM, and 2 mM, respectively. (C) Selectivity of the proposed Au nanoplasmonic probe toward H2O2. Control: 0 mM H2O2; 1 mM: 1 mM H2O2; 1 mM + catalase: 1 mM H2O2 + 4 μM catalase. In all experiments, the growth of PANI was performed in 10 mM PBS buffer (0.1 M NaCl and 10 mM phosphate, pH 4.3) containing 2 mM aniline. (D) FESEM images of (a) Au nanoplasmonic probes and (b) Au nanoplasmonic probes with a coating layer of PANI.
Figure 3. (A) PRRS spectra upon challenging the nanoplasmonic probes with different concentrations of H2O2. H2O2 concentrations are 0 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM, and 1 mM, respectively. The inset images are the true color images of light scattered from a representative plasmonic probe. (B) The derived calibration curve. (C) Plasmon band shift upon detection of H2O2 released from MPP+-stimulated Hek 293 cells. Control: Hek 293 cells without incubation with MPP+. MPP+: Hek 293 cells upon stimulation by 100 μM MPP+. MPP+ + catalase: Hek 293 cell upon stimulation by 100 μM MPP+ and then with 4 μM catalase to scavenge the released H2O2. (D) Plasmon band shift upon detection of H2O2 that released from different quantities of MPP+-stimulated cells. Error bars represent standard deviations for measurements taken from three independent experiments.
from 0 M to 1 mM) for 30 min. Similarly, as the concentration of H2O2 was increased, the corresponding λmax was gradually redshifted with the true color changed from green to red. Figure 3B
Figure 3A shows the end-point PRRS spectra and the corresponding true color images of a typical Au plasmonic probe after incubating with different concentrations of H2O2 (ranging 1971
DOI: 10.1021/ac5043895 Anal. Chem. 2015, 87, 1968−1973
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Figure 4. (A) AFM images showing the structural changes of α-Syn induced by exogenous added H2O2 (50 μM) upon incubation at 37 °C for (a) 0 day, (b) 1 day, (c) 3 days, and (d) 6 days. (B) AFM images showing the structural changes of α-Syn without incubating with H2O2 for (a) 0 day, (b) 1 day, (c) 3 days, and (d) 6 days. C) ThT fluorescence assay of α-Syn with (□) and without (○) incubation with H2O2. D) Plasmon band shifts upon the detection of H2O2 released in the aggregation of α-Syn. Control: freshly prepared α-Syn (50 μM). Syn: 50 μM of α-Syn after incubation with equal concentration of H2O2 for 7 days at 37 °C. Syn+catalase: 50 μM α-Syn after incubation with equal concentration of H2O2 for 7 days at 37 °C and then with 4 μM catalase to scavenge released H2O2.
plots the Δλmax (Δλmax = λmax (H2O2) − 5 nm) with H2O2 concentration, and the LOD was calculated as 8.0 nM (>3σ). In addition to exogenously added H2O2, the constructed plasmonic assay was also used for tracking stimuli-released H2O2 from living human embryonic kidney (Hek) 293 cells. 1-Methyl-4-phenylpyridinium (MPP+), a neurotoxin and an oxidant, was used to stimulate the cells for 3 h to release ROS (Figure 3C). The MPP+ stimuli-released H2O2 from 200 cells could be easily distinguished (Figure 3D), and the rate of H2O2 releasing by MPP+ stimuli was calculated to be 67 ± 11 nmol/105 cells/h. Having established the plasmonic assay for H2O2, we further employed DFM and atomic force microscopy (AFM) to clarify its role in the aggregation of α-Syn. The cellular mechanism of H2O2 in the aggregation of α-Syn is still in debate, whether the aggregation is caused by endogenously secreted or exogenous H2O2 from upstream. That is, it is important to clarify H2O2 whether is a cause factor or a result of α-Syn aggregation. We employed AFM to monitor the structural changes of α-Syn to establish the relationship between aggregation of α-Syn with H2O2. AFM images show the structural changes of α-Syn in vitro upon incubation in the presence (50 μM, Figure 4A) and absence of exogenously added H2O2 (Figure 4B). Clearly, exogenously added H2O2 significantly accelerated the aggregation of α-Syn. We further adopted thioflavin T (ThT) assay to give a quantitative comparison of the growth rate of α-Syn with and without the aid of H2O2 (Figure 4C). We found that the growth rate of α-Syn increased ca. 2-fold in the presence of H2O2. To test the secreted H2O2 from the aggregated α-Syn, we examined the sample of α-Syn without incubating with H2O2 using the established plasmonic assay (Figure 4D). We observed a Δλmax of 2.8 nm, corresponding to 10 nM H2O2, and upon the addition of catalase this 2.8-nm redshift was diminished. To further confirm
that this 2.8 nm of redshift is a result of secreted H2O2-induced coating of PANI, we also performed another control experiment by interrogating the nanoplasmonic probes with three other proteins (BSA, OVA, and lysozyme) incubated under the same conditions. ThT assay suggested that these three proteins did not aggregate after incubation (SI Figure S3A). Meanwhile, the proposed plasmonic assay indeed detected 70 nM H2O2 for BSA and 18 nM H2O2 for OVA, respectively, while it did not detect H2O2 for lysozyme (SI Figure S3B), a result consistent with previous work with a fluorescent probe.44 From the abovedescribed extracellular assays, we conclude that (1) exogenous H2O2 from upstream is the main driving force for the in vitro aggregation of α-Syn, (2) the aggregation of α-Syn indeed secretes 10 nM H2O2, and (3) the secreted H2O2 causes negligible toxicity. However, it should be noted that our extracellular α-Syn aggregation studies do not directly reflect intracellular or in vivo settings, which should have further efforts undertaken to explore.
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CONCLUSIONS In summary, we have developed a plasmonic assay for H2O2 with designed nanoplasmonic probes. HRP-initiated polymerization of aniline with the aid of H2O2 leads to coatings of the nanoplasmonic probes with a conducting polymer that changes the LSPR response. The plasmonic assay is highly sensitive to H2O2, with a detection limit reaching as low as 8 nM. The proposed plasmonic assay was employed to clarify the role of H2O2 in the aggregation of α-Syn in vitro. We found that exogenous H2O2 plays a dominant role for the aggregation of α-Syn, whereas the contribution from endogenously secreted H2O2 is negligible. Hence, the nanoplasmonic probe has proven to identify the role of H2O2 in signal transduction with spatial and temporal information through in situ imaging and monitoring its concentration level. 1972
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ASSOCIATED CONTENT
S Supporting Information *
Figures S1 and S2, and Table S1 as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the National Basic Research Program of China (973 program, 2013CB933800, 2012CB825800), the National Natural Science Foundation of China ((21222508, 21390414 and 21329501), the Shanghai Municipal Commission for Science and Technology (13QH1402), and the Chinese Academy of Sciences.
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DOI: 10.1021/ac5043895 Anal. Chem. 2015, 87, 1968−1973
