Efficient Modulation of Î²-Amyloid Peptide Fibrillation with Polymer

Subscriber access provided by UNIV OF SOUTHERN INDIANA

Article

Efficient Modulation of #-Amyloid Peptide Fibrillation with Polymer Nanoparticles Revealed by Super-Resolution Optical Microscopy Zhongju Ye, Lin Wei, Yiliang Li, and Lehui Xiao Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b01877 • Publication Date (Web): 31 May 2019 Downloaded from http://pubs.acs.org on June 1, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Efficient Modulation of β-Amyloid Peptide Fibrillation with

Polymer

Nanoparticles

Revealed

by

Super-

Resolution Optical Microscopy Zhongju Ye,† Lin Wei,‡ Yiliang Li,§ and Lehui Xiao*,† †

State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and

Molecular Recognition, College of Chemistry, Nankai University, Tianjin, 300071, China ‡

Key Laboratory of Phytochemical R&D of Hunan Province, College of Chemistry and Chemical

Engineering, Hunan Normal University, Changsha, 410081, China § Department

of Rehabilitation Medicine, The Affiliated Baoan Hospital of Southern Medical University,

The Second Affiliated Hospital of Shenzhen University, The People's Hospital of Baoan Shenzhen, Shenzhen, 510530, China

ABSTRACT: β-Amyloid peptide (Aβ) aggregation is the essential hallmark of neurodegenerative disorders such as Alzheimer’s disease. Efficient inhibitors are highly desired for the prevention of Aβ assembly that has been considered as the primary therapeutic strategy for neurodegenerative diseases. Apart from this, visualization of the aggregates and morphology at high spatial resolution is widely considered of crucial significance on biological treatment. In this work, we have developed small-sized (with diameter of ~4.7 nm) and positively charged fluorescent conjugated polymer nanoparticles (CPNPs) with strong inhibition effect on Aβ1-40 peptides fibrillation. Interestingly, the 1

ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

CPNPs also possess excellent photo-physical properties, including high photon counts, robust blinking and repetitive fluorescence switching, that are especially suitable for localization-based super-resolution imaging. Spatial resolution of ~20 nm for these blinking CPNPs is readily achieved. According to the optical microscopic results, it was found that binding of CPNPs to the terminal of seed fibrils can effectively inhibit the fibrillation process. Owing to these attractive biological and unique photo-physical properties, the small-sized CPNPs show high potential in a variety of super-resolution based biological applications.

2


Page 2 of 30

Page 3 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


INTRODUCTION Amyloid fibrils have been regarded as the primary source of many neurodegenerative disorders, such as Alzheimer’s disease (AD), Parkinson’s (PD), and Huntington’s diseases (HD).1-2 AD, the most common form of neurodegenerative disease on account of the aggregation of monomeric β-amyloid peptides (Aβ), always lead to progressive memory loss, cognitive deprivation, and death ultimately to the patients that has become an urgent medical problem to the society.3-4 Aβ is predominantly consisted of 40 or 42 residues in length (i.e. Aβ1-40 and Aβ1-42), which is formed from the cleavage of amyloid precursor protein. Nucleation and elongation phases are the main processes in Aβ assembly which are associated with the conformational transition from random coil to cross-β sheet structure. Both of these aggregated soluble seeds and mature fibrils from Aβ are of great damage to neuron system and brain cells by disturbing synaptic plasticity, deregulation of Ca2+ homeostasis.1-2 Efficient fibrillation inhibitors that can suppress the formation of oligomeric and fibrillar conformations is of great demand for the treatment of AD.5 Recently, numerous inhibitors have been developed to handle this issue. Peptides or peptide mimetics, small organic molecules and nanometer-sized materials have been used to accomplish this goal.5-13 For instance, peptide or protein analogues with the capability of association with Aβ peptide exhibit noticeable inhibitory effect on Aβ fibrillation.7 Polyphenols and antioxidants have been used to regulate Aβ fibrillation successfully and even applied to clinical trials.14 Recently, Aβ aggregation has been efficiently disturbed by inorganic nanoparticles such as gold nanoparticles and semiconducting quantum dots (Qdots).8, 11, 15-16 In addition, polymeric nanoparticles of various sizes and hydrophobicities have been applied to adsorb the Aβ peptides onto the particle surface to control their nucleation and fibrillation kinetics.17-18 3



Driven by the demand for biomedical development, visualization of the structure and morphology of the Aβ fibrils at higher spatial resolution is urgently desired, which may largely facilitate the understanding of Aβ pathogenesis and benefit the development of therapeutic agent. To solve this problem, several methods have been developed to resolve the fibril structure in situ.19-26 Among these methods, super-resolution imaging far surpassed the optical diffraction limit has become a powerful tool on this aspect.19, 23-24 Recently, super-resolution imaging techniques have undergone rapid development including single-molecule localization microscopy (SLM), photoactivated localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM) and direct STORM (dSTORM), enable imaging of cellular components and structures in an unprecedented spatial resolution.27-31 These techniques are typically achieved by modulating the fluorophores on an “on” (fluorescent) or “off” (dark) state. In other words, only a few parts of fluorophores are switched “on” at a moment. The fluorescent state should last for at least several camera frames but not too long to enable detection of sufficient photons and a certain flicker for accurate localization and image reconstruction, and then switched “off” or bleached when residual fluorophores blinked during data acquisition. The repetitive cycling of this process allows the reconstruction of fluorescence images from numerous individual molecular localization.32-39 On this regard, the development of new functional nanomaterials with high spatial resolution imaging capability and excellent inhibitory effect on the amyloid fibrillation process should be of great interest, which will afford insightful information for the comprehending of the self-assembly mechanisms and shed new light on the rational design of AD drug.19-21, 23, 26 In this work, we designed small-sized fluorescent conjugated polymer nanoparticles (CPNPs) for the controllable regulation of Aβ1-40 peptide fibrillation with unprecedented efficiency. The CPNPs were made by the one-pot co-precipitation of fluorescent conjugated polymer (i.e. poly [(9,9-dioctylfluorenyl4


Page 4 of 30

Page 5 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2,7-diyl)-alt-co-(1,4-benzo-(2,1',3)-thiadiazole)], PFBT) and zwitterionic lipid molecules (i.e., propylene glycol amine cationic lipid) together from anhydrous tetrahydrofuran (THF) solution to water, Figure 1a. According to the microscopic and spectroscopic characterizations, the CPNPs doped with the lipid molecules exhibit small size dimension (with diameter of 4.7±0.5 nm for CPNPs20, TEM), superior fluorescent brightness for single-particle localization imaging. The real-time fluorescence microscopic imaging results demonstrate that the CPNPs display strong interaction with the amyloid fibrils. The fibrillation process of Aβ1-40 peptides can be effectively inhibited by regulating the peptide folding process in phosphate buffer (PB) as well as binding CPNPs on the seed fibrils (i.e., blocking the growth points on the seeds). More interestingly, according to the time-dependent single-particle fluorescence characterizations, the small-sized CPNPs show a character of burst-like blinking behavior analogous to Qdots, which is well suited for localization-based super-resolution imaging.32,

39

By sequentially

recording the particle position, a spatial resolution of 20 nm was readily achieved, which was further applied for the super-resolution imaging of the Aβ1-40 fibril structure after staining the small-sized CPNPs on the backbone. As a consequence of these attractive biological and photo-physical properties, the small-sized CPNPs demonstrated herein will find promising applications in various fields in the future. EXPERIMENTAL SECTION Chemicals and Materials. NaH2PO4∙2H2O, Na2HPO4∙12H2O and NH3∙H2O were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Rhodamine B was purchased from Aladdin (Shanghai, China). GdSe/ZnS Qdots (FL 524 nm) was purchased from Suzhou Xingshuo Nanotech Co. Ltd. (Suzhou, China). Fluorescent beads (green sphere, 505/515 nm) were obtained from Invitrogen Ltd. (Paisley, UK). Poly [(9,9-dioctylfluorenyl-2,7-diyl)-alt-co-(1,4-benzo-(2,1',3)-thiadiazole)] (PFBT) was purchased from American Dye Source, Inc. (Quebec, Canada). Propylene glycol amine cationic lipids 5



were gifted from Prof. Dr. Youlin Zeng (Hunan Normal University, China, Chinese patent: #201110444269.5).40 The chemical structure of propylene glycol amine cationic lipid is shown in Figure 1a. β-amyloid (1-40) was purchased from AnaSpec, Inc. (Fremont, USA). Dimethyl sulfoxide (DMSO), anhydrous tetrahydrofuran (THF) and other chemicals not mentioned were purchased from SigmaAldrich (St. Louis, Mo, USA). Fabrication of Small-Sized CPNPs and Characterizations. The small-sized CPNPs used in this experiment were synthesized based on the nanoprecipitation method which is similar to the procedure described before with minor modifications.41 In order to gain the optimum blinking candidate, we synthesized four types of CPNPs by adjusting the mass ratio between propylene glycol amine cationic lipid and PFBT from 0-20, which are noted as CPNPs0, CPNPs5, CPNPs10 and CPNPs20 respectively. Taking the CPNPs20 as an example, the particles were fabricated as below, 5 μL of PFBT (1 mg/mL in THF) and 10 μL of propylene glycol amine cationic lipid (10 mg/mL in THF) were added to 1 mL of THF solution in a vial. The mixture was then rapidly injected into 6 mL of deionized (DI) water. THF was then removed by blowing the sample with nitrogen at 85 °C for 2 h. After the reaction was completed, the solution was gradually cooled down to room temperature and then concentrated to 1.5 mL by centrifugation. The solution was stored at 4 °C prior to use. Ultraviolet-visible (UV-vis) absorption spectra were collected using a Shimadzu UV-2450 spectrophotometer. Fluorescence spectra were recorded on the Hitachi F-7000 spectrometer. The size and morphology characterizations of these CPNPs were performed on a transmission electron microscopy (TEM, JEM2100, JEOL, Japan) and dynamic light scattering (DLS, malvern, Nano-ZS90). Preparation of β-Amyloid Fibrils and Modulation of the Fibrillation Process with Small-Sized CPNPs. Monomeric β-Amyloid (Aβ1-40) peptide stock solution was prepared by dissolving 1 mg of Aβ1-40 6


Page 6 of 30

Page 7 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


powder in 400 μL of ice-cold 0.02% ammonia solution without any purification and stored at -20 °C before use. Sample preparation of amyloid fibrils was described as the follow: 5 µL of stock monomeric Aβ1-40 was diluted to 45 µL of PB (25 mM, pH 7.4), and then incubated at 37 °C for 12 h. Prior to use, the Aβ1-40 fibrils were stored at 4 °C. The morphology of these resultant Aβ1-40 fibrils were visualized by labelling them with thioflavin T (ThT, a dye that is commonly adopted to stain the amyloid fibrils, λex = 450 nm, λem = 482 nm) under home-built objective type total internal reflection fluorescence microscopic (TIRFM) imaging system. Time dependent ThT fluorescence intensity measurements from the Aβ1-40 growth solution in the presence and absence of CPNPs20 were recorded by the Hitachi F-7000 spectrometer. The final concentrations of CPNPs20 is 5.8 µg/mL. TEM was used to further characterize the size and morphology of these fibrils. To investigate the inhibition effect of the CPNPs, the Aβ1-40 monomer solution was co-cultured with CPNPs with different concentrations (with final mass concentrations from 2.9 to 12 µg/mL) in 25 mM PB. The mixture was incubated at 37 °C for 12 h. The resulted Aβ1-40 aggregates or fibrils were imaged and captured with the TIRFM imaging system. The length of the fibrils was analyzed by the free-domain software ImageJ (http://rsbweb.nih.gov/ij/). RESULTS AND DISCUSSION Fabrication and Characterization of the Fluorescent CPNPs. In this work, nano-precipitation of the conjugated polymer from THF to water was adopted to synthesize the fluorescent CPNPs.41-43 An amphiphilic lipid molecule, propylene glycol amine cationic lipid, was utilized as the ligand molecule to transfer the hydrophobic conjugated polymer (PFBT) into a compact water soluble and fluorescent nanoparticle, Figure 1a-b. Without addition of phase transfer molecules, the conjugated polymer could still form aggregated polymer nanoparticle in water with poor colloidal stability. The fluorescence intensity of those nanoparticles determined by the spectrometer is typically weaker than that after the 7



addition of amphiphilic molecules (Figure 1c). This might be due to the compact - stacking from the intra-band fluorophores, resulting in the reduced energy band gap and then accelerate the energy quenching process.44-45 Interestingly, by gradually changing the mass ratio between cationic lipid molecule and PFBT from 0-20 (noted as CPNPs0, CPNPs5, CPNPs10 and CPNPs20, respectively), the fluorescence intensity of the CPNPs was also progressively increased. Around five-fold augment was observed when the ratio was 20 as illustrated in Figure 1c. From the DLS measurement, the hydrodynamic size of the as-synthesized CPNPs gradually decreases from CPNPs0 to CPNPs20, Figure 1d and S1. Only 5.6 nm was found for CPNPs20, which is further corroborated by the TEM measurement, Figure 1d. In the TEM image, the CPNPs20 exhibit homogeneous and small size distribution with diameter around 4.7±0.5 nm. It is worth to point out that the addition of the amphiphilic lipid molecule does not change the absorption spectrum of the resulted conjugated polymer nanoparticle, Figure 1e. Therefore, basically, the color of the synthesized CPNPs can be readily modulated by changing the composition of the polymer core. ξ potential measurements further confirmed that these CPNPs doped with cationic lipids are positively charged in water solution (26.9, 31.9 and 36 mV for CPNPs5, CPNPs10 and CPNPs20, respectively), which is essentially ascribed to the protonation of the amine group from the lipid molecule. However, without the introduction of the cationic lipid, the ξ potential of the resulted particle is -14.7 mV. Modulation of the Aβ1-40 Peptide Fibrillation with Positively Charged CPNPs. According to the earlier explorations, particles with strong interaction (e.g. hydrophobic association, hydrogen bonding and so on) with the Aβ peptide might disturb the fibrillation process.5-6, 8, 13, 17 On this account, we explored the effect of CPNPs on the Aβ1-40 peptide fibrillation process because these as-synthesized particles are positively charged and also encompass hydrophobic carbon tail as well as aromatic structure 8


Page 8 of 30

Page 9 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


from the conjugated polymer chain, where strong association might exist with Aβ1-40 peptide. To explore the effect of CPNPs on the peptide fibrillation process, firstly, the native fibrillation kinetics of Aβ1-40 peptide in PB was examined. Through staining the sample with ThT, the aggregated fibrils can be readily observed by fluorescence microscopy. As illustrated in Figure 2a, initially (0-4 h), only some optical diffraction-limited bright spots appeared in the fluorescence image. No preformed fibrils exist in the peptide stock solution. As time goes on, 4 h later at 37 oC, some short fibrils gradually appeared with length less than 3 µm. On further lengthening the incubation time (12 h, 37 oC), many well-defined fibrils exist in the solution with length around 3.5±0.7 µm according to the fluorescence image. Based on the aforementioned characterizations, since CPNPs20 exhibit the highest positive charge, we firstly explored the effect of CPNPs20 on Aβ1-40 fibrillation process. Freshly synthesized CPNPs20 (with a final concentration of 12 µg/mL) were co-incubated with the Aβ1-40 growth solution under the same reaction condition. At 0 h, Figure 2b, analogous to the sample without CPNPs20, only diffraction-limited bright spots displayed in the fluorescence image. However, distinct from the control, no elongated fibril was observed 4 h later, indicative of the broken seed formation process. On further extending the incubation time to 12 h, the result is still comparable to the observation at 0 h, demonstrating the excellent inhibition effect of CPNPs20 on the Aβ1-40 fibrillation process. Gradually reducing the dosage of CPNPs20 in the solution, a dosage dependent inhibition effect was observed, Figure 2c-d. No acceleration effect was found under dilute concentration. Besides the CPNPs20, under the same dosage (5.8 µg/mL) and reaction condition, other positively charged CPNPs also exhibited inhibition effect on the Aβ1-40 fibrillation process in contrast to the control, where comparable interaction mechanism might be involved in the modulation process, Figure S2. It is worth to point out that, among these CPNPs, CPNPs20 display the best inhibition efficiency as shown in Figure S2d. Apart from the microscopic characterizations, the 9



inhibition effect from CPNPs20 is also confirmed by the spectroscopic measurements, Figure S3. Without the introduction of CPNPs20, the fluorescence intensity of the sample stained with ThT grew up as a function of time. A plateau was achieved at around 9 h. Provided CPNPs20 was introduced, no observable fluorescence increase was observed even after 12 h, agreeing well with the microscopic results. Encouraged by the good inhibition efficiency, we further investigated the cell viability (PC12 cells) based on the MTT assay (Figure S4). The cells were co-incubated with the Aβ1-40 fibril growth solution for 24 h and analyzed by MTT assay. Evidently, nearly 35% of cells were dead in contrast to the control without the addition of Aβ1-40 fibrils. Negligible toxic effect was noted from CPNPs20 under the dosage of 5.8 µg/mL (more than 95% of cells were survived). Through co-incubation of CPNPs20 and Aβ1-40 fibril growth solution together, evidently reduced toxic effect was found. Only 10% of cells were dead in this case, indicating excellent cell protection effect from the CPNPs20. Since the CPNPs20 synthesized herein is positively charged and contains hydrophobic components, the electrostatic interaction between the positively charged CPNPs20 and negatively charged Aβ1-40 peptide (the isoelectric point of the peptide is ~5.2) should play an important inhibition role in the peptide fibrillation process. As illustrated in the TEM image, where ordered fibril structure was observed, Figure S5a. Interestingly, after being co-incubated with the CPNPs20 for 12 h, only very short seeds exist, Figure S5b. According to the earlier explorations, two interaction modes play significant roles in controlling Aβ peptides fibrillation.46-51 One is the π-π interaction between aromatic amino acid residues. The other is the hydrophobic interaction between hydrophobic amino acids. On this account, the strategy to disturb or break the β-sheet structure of those monomers or oligomers should be an efficient route to inhibit the Aβ fibrillation process. For instance, the conformation change of Aβ after adsorption onto the surface of nanoparticles or the anchoring of functional molecules (or nanoparticles) onto the growth points of the 10


Page 10 of 30

Page 11 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


seeds may influence their biological nature, and subsequently disturb their aggregation behavior accordingly.8, 13 To be a more definitive confirmation of the strong interaction of CPNPs20 with the Aβ1-40 fibrils, we mixed the preformed fibrils with the CPNPs20 together and then imaged with fluorescence microscopy. As shown in Figure S5c, without the CPNPs20, the preformed Aβ1-40 fibrils exhibited welldefined fibrillar structure when stained by ThT (the left panel). On mixing the CPNPs20 with the fibril solution (without ThT), comparable fluorescence image was obtained. Those fibrils in the solution were specifically stained by CPNPs20 (the right panel), further confirming the decent affinity of CPNPs20 toward the Aβ1-40 fibrils. As a consequence, the high-efficiency binding of CPNPs20 on the fibrils also plays an essential role in the inhibition of the Aβ1-40 fibrillation process. Due to the limited spatial resolution of the conventional fluorescence microscopy, it is a grand challenge to disclose the detailed binding sites of the particles on the preformed Aβ1-40 fibrils. These messages are fundamentally important for disclosing the fibrillation inhibition mechanism. For example, what is the discrepancy after the particles binding onto the backbone or the terminals of the fibrils? Interestingly, on the further inspection of the fluorescence image with higher tempo-resolution (50 ms) after stained with CPNPs20, stochastic blinking effect (“on” and “off” transitions comparable to Qdots) was noted on the backbone of the fibrils, which are basically resulted from individual CPNPs20. Recently, several breakthrough optical imaging methods have been developed to overcome the optical diffraction limit. Generally, these methods can be grouped into two major systems, based on fluorescent inhibition properties (e.g., stimulated emission depletion (STED)) or on stochastic methods (e.g., PALM, STORM, points accumulation for imaging in nanoscale topography (PAINT) and so on).28-30 In the latter case, the fluorophores are separated in time and measured as diffraction-limited spots on a CCD camera.

11



The coordinates of each fluorophores are determined with sub-pixel resolution by fitting each emission pattern with the point spread function (PSF) of the microscope. Evaluation of the Blinking Properties of CPNPs by Single-Particle Fluorescence Imaging. To localize the accurate position of the particles under a densely labeling condition, photoactivation, photobleaching, and stochastic adsorption and desorption are the commonly adopted strategies. Most of these techniques require complicated optical setup or image analysis software for the discrimination of individual objects in a temporally resolved manner.28 Since the “on”/“off” transition from the blinking particles is a randomly distributed process, without artificial modulation, the particles can be randomly light up separately. Charge trapping and redistribution on the conjugated polymer chain can trigger the transitions between emissive and dark states.45 Introducing the amphiphilic molecule into the CPNPs plays two essential roles in facilitating the blinking effect. One is the efficient reducing of the particle size, which effectively avoids the formation of large aggregates from hydrophobic conjugated polymers. The aggregation of multi-polymer chains into single particle usually results in photo-stable nanoparticles, which is not suitable for localization imaging under densely labeling condition as described above. The other is that the intercalating of the carbon tails into the polymer core can reduce the inter- and intrachain π-π stacking, resulting in greatly improved quantum yield as well as the efficient separation of the charge along the chain. On this basis, to optimize the blinking effect of CPNPs for the super-resolution imaging, the optical properties of the particles with different ligand/polymer ratios (w/w) were explored (i.e., CPNPs5, CPNPs10, and CPNPs20, respectively). Without introducing ligand molecules, the resulted CPNPs exhibited no blinking/bleaching process as illustrated in Figure 3a-c and S6, which is more suitable for biological labeling applications. After addition of the ligand molecules, noticeable blinking effect was observed even the mass ratio is only 5, 12


Page 12 of 30

Page 13 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


CPNPs5. Since the Qdots have been extensively adopted as the labels for super-resolution imaging,32 by using semiconductor Qdots as the control, it was found that, most of the Qdots display long-time “on” state, Figure 3d, while the majority of the CPNPs intercalated with the ligand molecules exhibit relative long-time “off” state. Normally for the single-particle localization orientated applications under densely labeling condition, the shorter the “on” time the better for the localization analysis. In this case, only few particles can be light up at once. As a more quantitative understanding of the blinking character of the CPNPs, the duty cycle of “on” time and the frequency of “on”/“off” switching were statistically analyzed from dozens of particles. As can be seen in Figure 3e, CPNPs doped with different mass ratio of ligand molecules exhibit comparable lower duty cycle close to ~0.23, while the Qdots show apparently larger value of ~0.55. Therefore, the probability to simultaneously observe two or more Qdots beyond the diffraction limit of optical microscopy should be greatly increased in contrast to CPNPs. This makes densely packed particles difficult to be resolved by successively recording their positions. Moreover, from the distribution of the lifetime of the fluorescent state (τon), Figure 3f-h, the majority of CPNPs hold the value closed to 0.2-0.4 s that would be appropriate to allow the detection of sufficient photons for the high precision single- particle localization. Sufficient repeats of “on”/“off” transition from a sequence can also increase the accuracy of localization. As shown in Figure 3j-l, obviously, CPNPs20 exhibit a relative narrower frequency distribution with the highest averaged value close to 60 under the observation of 250 s. According to these results, we chose the CPNPs20 for the following localization imaging experiments. Particle Localization and Super-Resolution Imaging with Small-Sized CPNPs. To get the localization information of individual particles in the image beyond the optical diffraction limit, the commonly adopted strategy is to localize the centroid position of the nanoparticle with high precision, 13



Figure S7a. A molecule or nanoparticle with size below the optical diffraction limit yields a photon emission pattern on the detector that is distributed according to the PSF of the microscope. This can be modeled and fitted with a spatial photon distribution function, typically using a Gaussian function for computational convenience. By using a fluorescent micro-bead as the reference, the standard deviation of the mechanic drift is estimated to be 𝜎𝑥𝑦 = 7.6 nm. The uncertainty in this work is determined to be ~8.5 nm, which corresponds to a spatial resolution of about 20 nm (2.35𝜎𝑐𝑜𝑟𝑟), see the supporting information. To demonstrate the super-resolution imaging capability, individual CPNPs20 were randomly adsorbed on the glass slide surface. An image stack with more than 5000 frames was then acquired. Through localize the centroid of each particle with the 2D Gaussian fitting, the exact position of the particle on the cover glass was then reconstructed as illustrated in Figure S7a. Without image processing, some of the particles in the image plane are overlapped together and hardly to resolve due to the limited resolution of epi-fluorescence mode. Representative particle pair with gap distance close to the optical diffraction limit is illustrated in the enlarged image, Figure S7f-g. From the intensity profile through the center of the particle pair, the PSF from those particles are partly overlapped. However, from the reconstructed image, Figure S7h-i, two evident bright spots can be observed with center-to-center distance around ~660 nm. Then, the CPNPs20 was adopted for the super-resolution imaging of the preformed Aβ1-40 fibrils. The morphology of preformed Aβ1-40 fibrils was firstly characterized by ThT (Figure S5a). Characteristic fibril structure was observed which is further confirmed by the TEM results, Figure S5a. For the superresolution imaging experiment, the preformed Aβ1-40 fibrils without dye labeling were injected into a flow channel made of two coverslips that were functionalized by amino groups. Excessive fibrils were 14


Page 14 of 30

Page 15 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


washed away by DI water. CPNPs20 were added to the flow channel for five minutes to label the fibrils. Low illumination power (

Efficient Modulation of Î²-Amyloid Peptide Fibrillation with Polymer

Recommend Documents