Detecting the Formation and Transformation of Oligomers during

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Detecting the formation and transformation of oligomers during insulin fibrillation by a dendrimer conjugated with aggregation-induced emission molecule Qin Huang, Jing Xie, Yanpeng Liu, Anna Zhou, and Jianshu Li Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00665 • Publication Date (Web): 23 Jan 2017 Downloaded from http://pubs.acs.org on January 25, 2017

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Bioconjugate Chemistry

Detecting the formation and transformation of oligomers during insulin fibrillation by a dendrimer conjugated with aggregation-induced emission molecule Qin Huang,& Jing Xie,& Yanpeng Liu, Anna Zhou, and Jianshu Li* & These authors contributed equally to this work and should be considered as co-first authors. Department of Biomedical Polymers and Artificial Organs, College of Polymer Science and Engineering, Sichuan University, Chengdu 610065, China KEYWORDS Insulin fibrillation • Oligomers • Fluorescence probe • Aggression-induced emission • Dendrimer

ABSTRACT: The fibrillation of protein is harmful and impedes the use of protein drugs. It also relates to various debilitating diseases such as Alzheimer’s diseases. Thus investigating the protein fibrillation process is necessary. In this study, poly(amido amine) dendrimer (PAMAM) of generation 3 (G3) and generation 4 (G4) were synthesized and conjugated with 4aminobiphenyl, an aggregation-induced emission (AIE) moiety, at varied grafted ratios. Among them, one fluorescence probe named G3-biph-3 that was grafted average 3.25 4-aminobiphenyl

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to the G3, can detect the transformations both from native insulin to oligomers and from oligomers to fibrils. The size difference of native insulin, oligomers and fibrils was proposed to be the main factor leading to the detection of above transformations. Different molecular weights of sodium polyacrylate (PAAS) were also applied as a model to interact with G3-biph-3 to further reveal the mechanism. The results indicated that PAMAM with a certain generation and grafted with appropriate AIE groups can detect the oligomers formation and transformation during the insulin fibrillation process.

INTRODUCTION Many proteins can form amyloid-like fibrils at denaturing conditions.1-4 These amyloid fibrils from non-homologous proteins present a similar β-sheets rich structure and deposits, which may induce debilitating diseases4 such as Alzheimer’s disease, spongiform encephalopathies, hereditary cerebral haemorrhage with amyloidosis and Creutzfeldt-Jakob disease. Revealing the mechanism of the protein misfolding is necessary for scientists to prevent the age-related neurodegeneration and also promote the use of protein drugs. For example, insulin is used to treat diabetes, but it can form amyloid deposits at the site of repeated injection.5 The insulin fibrils spine assumes a steric zipper structure,6 and are also full of β-sheets structures.7 The fibrillation of insulin under many conditions8 including low pH,9 different salt concentration,9 denaturants,9 exposure to air10, 11 and hydrophobic interfaces10, 11 at physiologic condition were investigated, and the B-chain segment LVEALYL has been proven to be the main factor of insulin fibrillation.12 Some researches show that the secondary structure of the peptides adsorbed on the hydrophobic interface can promote or inhibit the insulin fibrillation.13Such as the peptides (LK)nL at substoichiometric concentration can promote insulin fibrillation due to the β-strand secondary structure after adsorbing on the hydrophobic interface. However, at high concentration


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with much more (LK)nL peptides in solution, the fibrillation was supressed.14 The esterification and acylation of insulin shows that the amino groups rather than carboxyl groups can disrupt insulin stability at low pH.15 It was also reported that removing the B26-B30 or B23-B30 of Bchain C-terminal can promote the insulin fibrillation. It suggests that the hydrophobic interfaces which are necessary to form fibril are buried under the B-chain C-terminal.8, 16 The formation of insulin partial unfolded monomers is also an inevitable step in fibrillation process.17, 18 Generally, protein fibrillation can be classified into three phases: nucleation, elongation and equilibrium. In particular, soluble protein molecules form seeds, and other soluble protein molecules bind to the seed and change their conformation to form fibrils.15, 19-21 To monitor the insulin fibrillation, the proportion of different secondary structure can be investigated by fourier transform infrared spectroscopy (FTIR), circular dichroism (CD), and the morphology change can be attained by scanning electron microscopy (SEM) and transmission electron microscopy (TEM).22-27 During the fibrillation process, insulin disassociates to monomers. Then monomers convert into molten globule state and bind to each other to form oligomers. These oligomers act both as seeds and elongating species to form fibrils.17, 18, 28, 29 Since oligomers are more toxic than mature fibrils,30 it is important to investigate the structure and the forming conditions of these intermediates. Many characterization techniques including phase diagram method31 are competent to detect the intermediate state during the fibrillation. Fluorescence technology is widely used to investigate the fibrillation. For example, the fluorescence of insulin itself can be an indicator of fibrillation.32 And it was reported that the β-sheet structures have interesting photoluminescence ability and can be used to investigate fibrillation.33, 34 Moreover, 1-anilinonaphthalene-8-sulfonate (ANS) is a fluorescent molecule that is sensitive to the hydrophobic region. It can be used to probe the molten globule state of protein because of the blue shift


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caused by the access to the exposed hydrophobic pockets of the molten globules.29, 35 ThioflavinT (ThT) is another fluorescent molecule that can specifically bind to the grooves of β-sheets and change the maximum emission wavelength, so it can be used to detect the formation of βsheets.36-38 Some types of luminescent conjugated oligothiophenes were reported to present different emission wavelength shift while binding to fibrils from different proteins or different amyloid morphologies from the same protein. Meanwhile, some of these conjugated oligothiophenes can bind to pre-fibrils.39-42 Among various fluorescent molecules, aggregationinduced emission (AIE) molecules are non-emissive at isolated state while emitting fluorescence at aggregated state. The AIE activity is mainly due to the restriction of intramolecular rotation (RIR) and the restriction of intramolecular vibration.43-45 Different from the traditional fluorescence molecules, AIE molecules can give strong fluorescence signal at high concentration, even in solid state and avoid the influence of aggregation-caused quenching (ACQ).44 Since it was first discovered by Tang,46 AIE molecules became powerful biological probes. The AIE molecules can be utilized to detect macromolecules such as heparin47-49, DNA50-53 and proteins. 54-56 Furthermore, the AIE molecules can also detect more complex things like the protein unfolding,57, 58 the existence and the activity of enzymes59, 60 and the binding between protein and peptide.61 An interesting water soluble tetraphenylethyene derivative (an AIE molecule) has also been developed to monitor the fibrillation of insulin and exhibited a strong signal when the fibrils formed.62 Inspired by the property and the reported applications of AIE molecules, we synthesized a series of 4-aminobiphenyl (an AIE molecule) grafted polyamidoamine dendrimer (PAMAM), of generation 3 and 4, to monitor the insulin fibrillation. It was reported that some dendrimers can bind to proteins63 through electrostatic interaction64, 65 or metal-ligand interaction66, and during


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the electrostatic interaction binding, the binding ability varies with the size changes of protein and dendrimers,67 so the fluorescence intensity of the AIE-grafted dendrimers can reflect their different binding abilities to proteins of different sizes. Thus the oligomers which have different binding ability from the native insulin are expected to be identified by the fluorescence change. The result showed that one grafted dendrimer named G3-biph-3 can detect the formation and transformation of intermediate, i.e., oligomers, with the change of fluorescence intensity through static interaction with the sample taken from the incubation solution during the insulin fibrillation process under acidic and high temperature condition (25 mM HCl and 60 oC). It is presumed that the oligomers pile around the grafted PAMAM, forming a looser scaffold-like aggregates than the packing of grafted PAMAM with native insulin, due to its larger size than the native insulin.28 The looser scaffold-like aggregates led to a significant fluorescence difference between oligomers and native insulin. This assumption was also tested by using sodium polyacrylate (PAAS) of different molecular weights as a substitution model. In addition, PAMAM grafted with higher ratio of 4-aminobiphenyl was also prepared to monitor the insulin fibrillation through hydrophobic interaction.

RESULTS AND DISCUSSION Synthesis of 4-biphenylamide succinic acid grafted PAMAM of generation 3 and 4. Dendrimer G3 and G4 were synthesized through a step-by-step route,68-70 and their structure were confirmed by 1HNMR (Supporting Information Figure S1)71 and MALDI-TOF MS (Supporting Information Figure S2).72 4-biphenylamide succinic acid (Compound 1) was synthesized from 4-aminobiphenyl and succinic anhydride. The 1H NMR spectra indicated the forming of Compound 1 (Supporting Information Figure S3). The protons Ha and Hb resonated


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at 2.5 ppm merged with solvent peaks at 2.5 ppm. The Hc of arylamide resonated at 10 ppm. The Hd of biphenyl resonated between 7.25 and 7.75 ppm. This demonstrated the success synthesis of Compound 1. Then it was grafted on the G3 and G4 at different ratios through 1hydroxybenzotriazole (HOBT), 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI) and 4Methylmorpholine (NMM) in DMSO (Scheme 1).

Scheme 1. The synthesis process of compound 1, G3-biph-1 to 4, and G4-biph-1 to 4.

Table 1. The relationship between the real ratio of grafted compound 1 and the theoretical grafted ratio deduced from the total amount of compound 1 added during the grafting reaction. The molecular weight of G3 and G4 are 3256 g/mol and 6909 g/mol respectively and the molecular weight of Compound 1 is 269 g/mol G3biph-1

The feeding molar ratioa

6.25%

G3G3G3biph-2 biph-3 biph-4

12.50 %

25.00 %

50.00 %

G4biph-1

G4biph-2

G4biph-3

G4biph-4

6.25%

12.50 %

25.00 %

50.00 %


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Real ratio of grafted Compound 1b

11.11 %

14.08 %

20.31 %

50.09 %

5.06%

10.10 %

19.47 %

39.59 %

Molecular weightc (g/mol)

3699

3818

4069

5266

7316

7721

8475

10093

a

The ratio is the mole of feeding Compound 1 to the mole of primary amino groups in PAMAM b

The ratios were acquired from the MALDI-TOF (Supporting Information Figure S4 and S5) c

The molecular weight were acquired from the MALDI-TOF (Supporting Information Figure S4 and S5)

Then, Compound 1 was attached to G3 and G4 through an amide bone formed by the carboxyl groups of Compound 1 and the peripheral amino groups of G3 and G4 to synthesize 4biphenylamide succinic acid grafted polyamidoamines of generation 3 (AIE-grafted G3) and generation 4 (AIE-grafted G4). The molar ratio of feeding Compound 1 to primary amino groups in reaction were 6.25%, 12.50%, 25.00% and 50.00%, respectively, forming G3-biph-1/G4-biph1, G3-biph-2/G4-biph-2, G3-biph-3/G3-biph-3 and G3-biph-4/G3-biph-4 (Table 1). To verify whether Compound 1 was successful grafted on G3 and G4, the 1H NMR spectra of grafted G3 and G4 were investigated (Figure 1). The chemical shift is accordant with the report.73 From G3biph-1 to G3-biph-4 and from G4-biph-1 to G4-biph-4, when the molar ratio of Compound 1 in reaction increased, the signal of He ranging from 7.25 ppm to 7.75 ppm, which represented the hydrogen of biphenyl in grafted Compound 1, also increased. And the broad peaks Hi at about 3.2 ppm corresponding to the remaining amino groups in modified G3 and G4 gradually weakened. This was due to the formation of amide bone to transform the amino groups to amidegroups. The broad peaks Hd at 3.09 ppm represented the hydrogens of methylene directly


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connected the nitrogen of amide groups. Both from G3-biph-1 to G3-biph-4 and from G4-biph-1 to G4-biph-4, these peaks became stronger and stronger respectively, reflecting more and more amide bonds were formed with the rising molar ratio of Compound 1. Meanwhile, the peak of Hh was merged with the solvent peaks at 2.5 ppm, the rise of the peak with the increasing molar ratio of Compound 1 can not be identified. All of this suggested Compound 1 was successfully grafted on the G3 and G4. As MALDI-TOF MS can be used to investigate the average number of chromophores grafted around dendrimer,74 the average graft ratios of peripheral amino groups were 11.11%, 14.08%, 20.31%, 50.09% for G3-biph-1 to 4, and 5.06%, 10.10%, 19.47%, 39.59% for G4-biph-1 to 4, respectively (Table 1) according to MALDI-TOF MS (Figure S4 and Figure S5). The higher real graft ratio of G3-biph-1 and G3-biph-2 than the theoretical one was probably due to the influence of purification and the deviation of the average molecular weight acquired from mass spectrum.


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Figure 1. (a) The chemical structure of AIE-grafted PAMAM. (b) The 1H NMR of G3-biph-1 to 4. (c) The 1H NMR of G4-biph-1 to 4.


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Figure 2. The relationship between fluorescence of AIE-grafted G3 and insulin/fibrils concentration. The concentration of AIE-grafted G3 was kept at 10 µΜ. The fluorescence measurement was carried out in 10 mM PBS pH =7.4 with 0 M NaCl (a), 0.1 M NaCl (b), 0.2 M NaCl and 0.4 M NaCl (c). Sifting the proper AIE-grafted PAMAM among the candidates (G3-biph-1 to 4 and G4biph-1 to 4). By comparing with signal difference between the binding to native insulin and the binding to insulin fibrils among the eight AIE-grafted PAMAM, some of the candidates were chosen to monitor the insulin fibrillation in situ. As insulin exhibits negative charge above pH 5.3, it is presumed that insulin molecules should be adsorbed around the positively charged AIEgrafted PAMAM in the PBS (without NaCl, pH = 7.4), restricting the rotation of the peripheral biphenyl groups (AIE groups) and resulting in fluorescence signals. When insulin fibrils were added to the PBS solution of AIE-grafted PAMAM (without NaCl, pH = 7.4), the fluorescence


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signals were supposed to be weaker than that of native insulin, due to the smaller total surface area and larger size, which meant less binding sites and more difficulties in moving. Furthermore, the NaCl concentration, which can shield the static interaction between the AIEgrafted PAMAM and the insulin, was also taken into consideration. After being excited at wavelength of 220 nm, the insulin fluorescence intensity reaches maximum at 304 nm,75 the fluorescence climax of AIE-grafted G3 and AIE-grafted G4 is at 367 nm. As shown by the complete fluorescence spectra of AIE-grafted G3 and AIE-grafted G4 induced by insulin ranging between 250 nm and 400 nm (Figure S6 and S7), the interference from the fluorescence of insulin itself was negligible. Figure 2 showed the fluorescence intensity which was caused by the constant 10 μM AIE-grafted G3 binding to native insulin and insulin fibrils separately. The concentration of native insulin and insulin fibrils both ranged from 0 to 25 μM. The binding was carried out in the 10 mM PBS pH = 7.4, and the NaCl concentration was tested at 0 M (Figure 2a), 0.1 M (Figure 2b), 0.2 M and 0.4 M (Figure 2c). Comparing with fluorescence intensity caused by adding native insulin into G3-biph-1 (Figure 2a black triangle), G3-biph-2 (Figure 2a red triangle), G3-biph-3 (Figure 2a blue triangle) and G3-biph-4 (Figure 2a green triangle), it suggested that without NaCl, higher grafting ratio of Compound 1 to the G3 led to stronger fluorescence when AIE-grafted G3 was binding to the native insulin. It is because that when more Compound 1 was grafted to G3, one native insulin molecule can contact with more grafted biphenyl groups thus more biphenyl groups can emit fluorescence according to the AIE mechanism.43-45 Differing from G3-biph-1 ~ G3-biph-3 which almost presented approximately linear relationship between the fluorescence intensity and native insulin concentration, the fluorescence intensity of G3-biph-4 increased fast from 0 μM to 4 μM, then it increased slower, and formed a plateau at last. This is due to the higher grafting ratio of G3-biph-4 which meant


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that a lot of amino groups were consumed, so the positive charge in the G3-biph-4 was weaker and can be saturated by less amount of native insulin. The fluorescence caused by adding insulin fibrils into G3-biph-1 (Figure 2a black diamond), G3-biph-2 (Figure 2a red diamond), G3-biph-3 (Figure 2a blue diamond) and G3-biph-4 (Figure 2a green diamond) were investigated. The fluorescence intensity difference among G3-biph-1, G3-biph-2, and G3-biph-3 was negligible. However the fluorescence intensity of G3-biph-4 was relatively higher, probably the hydrophobic interaction between the grafted Compound 1 and the exposed hydrophobic region of insulin fibrils contributed to the fluorescence emitting. The fluorescence intensity differences between native insulin and insulin fibrils, which corresponded to G3-biph-1 (Figure 2a black triangle and black diamond), G3-biph-2 (Figure 2a red triangle and red diamond), G3-biph-3 (Figure 2a blue triangle and blue diamond) and G3-biph-4 (Figure 2a green triangle and green diamond), were investigated. The fluorescence of G3-biph-3 and G3-biph-4 caused by native insulin was significantly stronger than the corresponding fluorescence caused by the fibrils at the same concentration, which was in accordance with the presumption. In addition, it was noticeable that the signal difference between the binding to native insulin and binding to insulin fibrils of G3-biph-3 was the most significant. To probe the mechanism by which AIE-grafted G3 bound to native insulin and insulin fibrils, the binding was carried out in 10 mM PBS pH = 7.4 with 0.1 M NaCl (Figure 2b). The fluorescence caused by adding native insulin to G3-biph-1 (Figure 2b black triangle), G3-biph-2 (Figure 2b red triangle), G3-biph-3 (Figure 2b blue triangle) and G3-biph-4 (Figure 2b green triangle) was investigated. The fluorescence intensity of G3-biph-1, G3-biph-2, G3-biph-3 ranged from 0 to 90 a.u. and the difference among them was negligible, while in PBS without NaCl the difference was significant (black, red and blue triangles in Figure 2a). Furthermore


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there was a shape decline of G3-biph-3 fluorescence caused by native insulin with 0.1 M NaCl (Figure 2b blue triangle) comparing with that measured in PBS without NaCl (Figure 2a blue triangle). This decline indicated that the amount of native insulin adsorbed around G3-biph-3 decreased, i.e. the interaction between native insulin and G3-biph-3 was weakened. Given the fact that NaCl can shield the static interaction, it can be inferred that the interaction between native insulin and G3-biph-3 was mainly due to the static interaction. As an exception, the fluorescence of G3-biph-4 was almost unchanged, and this may be attributed to the high grafting ratio presented considerable fluorescence intensity. The fluorescence of G3-biph-1 (Figure 2b black diamond), G3-biph-2 (Figure 2b red diamond), G3-biph-3 (Figure 2b blue diamond), and G3-biph-4 (Figure 2b green diamond) which were induced by the addition of insulin fibrils in 0.1 M NaCl PBS were also investigated. These fluorescence curves from G3-biph-1, G3-biph-2 and G3-biph-3 lay low in Figure 2b and the fluorescence curve of G3-biph-4 was relatively higher. These four fluorescence curves were similar to their counterparts in Figure 2a. To G3-biph-1, G3-biph-2 and G3-biph-3, it can be inferred that hydrophobic interaction between them and the insulin fibril may have an effect. Another reason was that their fluorescence generated through bindings to the insulin fibrils were so weak that its reduction can not be detected. The higher fluorescence of G3-biph-4 (Figure 2b green diamond) clearly suggested there might be hydrophobic interaction which was not influenced by NaCl between G3-biph-4 and insulin fibrils. Further, in order to verify the static interaction between G3-biph-4 and native insulin, together with the hydrophobic interactions between G3-biph-4 and insulin fibrils, higher concentration of NaCl was added to the PBS (Figure 2c). Fluorescence comparison by mixing G3-biph-4 and native insulin in the 0.2 M NaCl PBS (Figure 2c black triangle) and 0.4 M NaCl


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PBS (Figure 2c red triangle) was carried out. The fluorescence of G3-biph-4 with native insulin in 0.4 M NaCl PBS declined significantly, which indicated that the interaction between G3-biph4 and native insulin were static interaction and the high fluorescence of G3-biph-4 with native insulin in 0.1 NaCl PBS (Figure 2b green triangle) and 0.2 NaCl PBS (Figure 2c black triangle) were just due to weak shielding effect of not enough NaCl concentration. The negligible fluorescence reduction from G3-biph-4 with insulin fibrils in 0.2 M NaCl PBS (Figure 2c black diamond) to G3-biph-4 with insulin fibrils in 0.4 M NaCl PBS (Figure 2c red diamond) indicated that even high concentration of NaCl can not block the interaction between G3-biph-4 and insulin fibrils, leading to the conclusion that the interaction between G3-biph-4 and insulin fibrils was hydrophobic interaction which can not be affected by the concentration of NaCl. Comparing the fluorescence of G3-biph-4 induced by adding native insulin (Figure 2c red triangle) with that by adding insulin fibrils in 0.4 NaCl PBS (Figure 2c red diamond), the higher fluorescence intensity of the former (Figure 2c red diamond) indicated a feasible way to use the G3-biph-4 as a probe to monitor the insulin fibrillation process in situ through the detection the exposed hydrophobic region. In conclusion, G3-biph-1 and G3-biph-2 emitted weak fluorescence both with native insulin and with insulin fibrils. In PBS without NaCl, G3-biph-3 presented the most significant signal difference among the four candidates, and the speculation that the binding between G3-biph-3 and native insulin is the static interaction was proved by adding NaCl to shield the static interaction. Through the adjusting of NaCl concentration, the binding between G3-biph-4 and native insulin was also proved as a result of static interaction. Besides, the binding between G3biph-4 and insulin fibrils which caused higher fluorescence signal than that caused by adding insulin fibrils into G3-biph-1, G3-biph-2 and G3-biph-3 in PBS without NaCl, was mainly due to


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the stronger hydrophobic interaction between the abundant grafted AIE groups of G3-biph-4 and the hydrophobic region of insulin fibrils. The fluorescence behavior of AIE-grafted G4 (Figure 3) was investigated through the process similar to what was used on AIE-grafted G3. The results indicated that in PBS without NaCl G4-biph-3 fluorescence (Figure 3a blue downtriangle and blue solid circle) showed the most significant difference between the addition of native insulin and the addition of insulin fibrils, and static interaction was also the main reason inducing the binding of G4-biph-3 and G4-biph-4 to native insulin. Just like G3-biph-4, the maintenance of the higher fluorescence signal of G4-biph-4 with insulin fibrils in 0.4 M NaCl indicated that hydrophobic interaction was mainly attributed to the binding between G4-biph-4 and insulin fibrils. Considering the fluorescence changes in all conditions, G3-biph-3 was chosen as a probe to monitor the insulin fibrils process via static interaction in PBS without NaCl, and G3-biph-4 was chosen as a probe to monitor insulin fibrils process via hydrophobic interaction in PBS containing 0.4 M NaCl. For the same reason, G4-biph-3 and G4-biph-4 were chosen to monitor the insulin fibrillation in PBS without NaCl and 0.4 M NaCl PBS, respectively.


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Figure 3. The relationship between fluorescence of AIE-grafted G4 and insulin/fibrils concentration. The concentration of AIE-grafted G4 was kept at 10 µΜ. The fluorescence measurement was carried out in 10 mM PBS pH = 7.4 with 0 M NaCl (a), 0.1 M NaCl (b), 0.2 M NaCl and 0.4 M NaCl (c). The mixture of native insulin and insulin fibrils were prepared with constant total concentration but various proportions, to further investigate the fluorescence signals of G3-biph3, G3-biph-4, G4-biph-3 and G4-biph-4 (Figure 4). It shows that with the increase of the fraction of fibrils, the fluorescence of G3-biph-3 decreased, and the fluorescence of G3-biph-4 and G4biph-4 increased throughout the measurement. As for G4-biph-3, its fluorescence declined significantly when the fibrils fraction was below 80%. Thus, the fluorescence change of G4-


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biph-3 can indicate the fibrillation under 80% conversion, while all other three fluorescence probes (G3-biph-3, G3-biph-4 and G4-biph-4) can detect the whole fibrillation process.

Figure 4. The fluorescence intensity of G3-biph-3 (a), G3-biph-4 (b), G4-biph-3 (c) and G4biph-4 (d) in the mixture of native insulin and insulin fibrils. The abscissa is the ratio of insulin fibrils. The fluorescence measurement of G3-biph-3 and G4-biph-3 were carried out in 10 mM PBS pH = 7.4 without NaCl. For G3-biph-4 and G4-biph-4 the measurement were carried out in 10 mM PBS pH = 7.4 with 0.4 M NaCl.

As the fluorescence intensity of the four AIE-grafted PAMAM was sensitive to the fraction of insulin fibrils in the mixture of native insulin and insulin fibrils, they were applied to monitor the insulin fibrillation process. First, the G3-biph-4 and G4-biph-4 hydrophobic binding to the insulin exposed hydrophobic region were exploited to investigate the process of fibrillation


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(Figure 5). Just like traditional fluorescence molecule, ANS (red dot) in Figure 6, the G3-biph-4 and G4-biph-4 fluorescence intensity increased with the increase of incubation time. Both fluorescence curves were sigmoidal, reflecting the nucleation, elongation and equilibration stages of fibrillation process.20, 32

Figure 5. Monitoring the insulin fibrillation by G3-biph-4 (a) and G4-biph-4 (b).

While G3-biph-3 and G4-biph-3 were utilized to monitor the insulin fibrillation, more interesting phenomena were observed. The 300 µM insulin in 25 mM HCl solution was heated to 60 oC and stirred at 100 rpm to form fibrils. The insulin transformed to molten-globe state then formed oligomers and finally became mature fibrils.29 ANS was used to investigate the fibrillation as a comparison as it can detect the molten-globe state and the exposure of hydrophobic region. With the solvent accessible hydrophobic region appearing, the fluorescence intensity of ANS enhanced and its wavelength shifted from about 510 nm to about 460 nm.35, 76, 77

Aliquots were taken from the incubation solution at suitable intervals to PBS, then G3-biph-3,

G4-biph-3 and ANS were added separately. Figure 6a shows that the fluorescence intensity of G3-biph-3 decreased while incubation time increased (black square), indicating the fibrillation of insulin. Different from traditional small fluorescence molecules like ANS which showed the three distinct phases of fibrillation including nucleation, elongation and equilibration,20, 29, 32 G3-


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biph-3 showed a nucleation stage at first, then the fluorescence intensity dramatically lowered to a platform at about 165 a.u. (marked with purple circle), after another 30 min the fluorescence decreased again to the lowest plateau indicating the formation of mature fibrils. To certify the middle platform is not accidental, another two batches of incubation solution were monitored with G3-biph-3, and the middle platform appeared again. Supporting Information Figure S8 showed the G3-biph-3 fluorescence changes in all the three replication of monitor and all the middle platform were marked with purple circles. The fluorescence middle platform of G3-biph3 indicated an intermediate during the insulin fibrillation. To classify the intermediate indicated by G3-biph-3, the ANS MAX emission wavelength was investigated. The ANS MAX emission wavelength shifted towards shorter wavelength at the initiation of the incubation (blue triangle), suggesting that the hydrophobic region appeared earlier than the decrease of G3-biph-3 fluorescence intensity. The molten-globe protein have some hydrophobic region to adsorb ANS molecules,35, 76 so the blue shift at early stage should be attributed to the molten-globe insulin, and the subsequent blue shift was due to the bond of ANS to oligomers and fibrils. However, the G3-biph-3 fluorescence did not weaken at the beginning, indicating that the molten-globe state did not weaken the G3-biph-3 fluorescence. Because insulin can transform from molten-globe state to oligomers during fibrillation, the middle platform of G3-biph-3 fluorescence should be classified into the oligomers of insulin. Comparing with the ANS fluorescence intensity which was measured collaterally (red dot), the G3-biph-3 fluorescence decrease significantly to the middle platform, while the ANS fluorescence did not show a notable rise, presenting G3-biph-3 a higher sensitivity to oligomers than ANS. The G4-biph-3 (Figure 6b), however, did not show a significant middle platform as that of G3-biph-3. The blue shift of ANS emission MAX wavelength appeared early than the decrease of G4-biph-3 fluorescence intensity, just as the case


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of G3-biph-3, indicating G4-biph-3 fluorescence decrease was not caused by the exposure of hydrophobic region, but by the formation of oligomers and mature fibrils.

Figure 6. The monitoring of insulin fibrillation by G3-biph-3 (a), G4-biph-3 (b) and ANS. The middle platform is marked with purple circle which is supposed to be oligomers.

The difference between G3-biph-3 and G4-biph-3 were probably attributed to their difference sizes in different generations. It is reported that PAMAM have an optimal generation to bind to a certain protein through static interaction.64, 67 For the insulin oligomers of a certain size,28 it can be regarded as an aggregate which has a bigger size than insulin. As the oligomers have bigger surface, the PAMAM with larger generation bond to it more tightly.67 Because during the measurement the weight percentage of G3-biph-3 to insulin in the buffer equalled that of G4biph-3 to insulin, the different binding abilities caused by the sizes of G4-biph-3 and G3-biph-3 can be compared with each other.78 G4-biph-3 has larger contact interface to the insulin oligomers than that of G3-biph-3, so the interface reduction of G4-biph-3 from native insulin to oligomers was less than that of G3-biph-3. Because more interface area indicated stronger fluorescence, the G4-biph-3 fluorescence difference between insulin and oligomers was smaller


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than the G3-biph-3. So G4-biph-3 fluorescence did not show a platform. The mechanism of G3biph-3 to differentiate insulin oligomers will be discussed later. The mechanism AIE-grafted PAMAM to identify the fibrillation intermediates. The mechanism of G3-biph-3 and G4-biph-3 to monitor the fibrillation is different from the traditional fluorescence molecule like ANS, ThT (Thioflavin-T) and Congo red which binds to the exposed pockets or the grooves of β-sheets.35-38, 77 The native insulin molecules (small size) piled around G3-biph-3 and G4-biph-3 tightly, restricting the grafted biphenyl rotation, and the fluorescence intensity went up (Scheme 2 the left part of (a) and (b)). Then several insulin molecules integrated into one oligomer (larger size).28 When G3-biph-3 and G4-biph-3 attracted oligomers through static interaction, the oligomers piled around G3-biph-3 and G4-biph-3, and will repel against each other because of their large volume and charge. As a result the oligomers formed scaffold-like structures, and both the G3-biph-3 and G4-biph-3 were located at the holes in the structures (Scheme 2 the middle part of (a) and (b)). The hole was large enough that when some space was occupied by G3-biph-3 or G4-biph-3, the remaining space partially maintained the rotation of grafted biphenyl, while the native insulin inhibited the biphenyl rotation more effectively. It can be inferred that the fluorescence generated from the oligomers was weaker than the fluorescence generated from native insulin. The big difference here between G4-biph-3 and G3-biph-3 is that G4-biph-3 had larger volume than G3-biph-3, when they are located in the hole of piled oligomers. Thus the remaining space of G4-biph-3 was smaller than that of G3biph-3, and the biphenyl rotation of G4-biph-3 was more difficult to maintain, so the fluorescence decrease was relatively less significant than G3-biph-3 fluorescence decrease when oligomers gradually appeared. For this reason, G4-biph-3 fluorescence can not form a middle platform and can not identify the oligomers. On the contrary, G3-biph-3 can maintain more


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biphenyl rotation due to its smaller volume, leading to relatively significant fluorescence decrease when oligomers gradually appeared. As a result, G3-biph-3 formed a middle platform that can identify the formation of oligomers as shown in Figure 6a and Supporting Information Figure S8.

Scheme 2. Schematic illustration of (a) G3-biph-3 and (b) G4-biph-3 interacted with native insulin, oligomers and fibrils, respectively. The fluorescence of G3-biph-3 and G4-biph-3 declined from the left to the right.

After oligomers transformed into fibrils (the largest size), the large bulky volume of fibrils make it difficult to piled around G3-biph-3 and G4-biph-3 (Scheme 2 the right part of (a) and (b)). Thus the inhibition of biphenyl rotation of G3-biph-3 and G4-biph-3 was negligible, leading to the fluorescence decrease of them to the bottom.


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The morphology of insulin at nucleation, oligomers and fibrils were investigated by scanning electron microscopy (SEM) (Figure 7). At nucleation stage, aliquot of incubation solution was added to the PBS. The morphology of pure insulin in the PBS was showed in Figure 7a, and no evident aggregation was observed. After adding G3-biph-3 to the sample, aggregates of different size were formed (Figure 7b). At oligomers stage, the pure oligomers in the PBS could not be detected due to the small size28 (Figure 7c). After adding G3-biph-3 to the sample, obvious aggregation formed. The oligomers aggregation induced by G3-biph-3 (Figure 7d) was less obvious than the nucleation insulin aggregation induced by G3-biph-3, corresponding to the weaker G3-biph-3 fluorescence with oligomers than with native insulin (Figure 6a). The insulin fibrils in the PBS almost maintained its morphology after addition of G3-biph-3. Large aggregates could not be detected both without and with G3-biph-3 (Figure 7e and f). All of these results could certify that the fluorescence decrease (Figure 6) was due to the reduced aggregation induced by G3-biph-3 (Figure 7).


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Figure 7. SEM images of Insulin at nucleation state without G3-biph-3 (a) and with 10 µM G3biph-3 (b) in PBS; insulin at oligomers state without G3-biph-3 (c) and with 10 µM G3-biph-3 (d) in PBS; insulin at fibrils state without G3-biph-3 (e) and with 10 µM G3-biph-3 (f) in PBS. All the insulin concentration was kept at 10 µM. The CD spectra (Figure 8) showed the secondary structure of insulin changed during the incubation. In the beginning, the secondary structure was dominated by α-helices corresponding to the two negative bands at 209 nm and 222 nm. After being incubated 4 hours, the CD spectra changed to a single negative band at 219 nm,79 corresponding to the β-sheets which dominated in the fibrils.24


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Figure 8. The CD spectra of insulin during the incubation. In order to verify whether the different quantity of electric charge of the native insulin, oligomers and insulin fibrils can influence the G3-biph-3 fluorescence intensity, their zeta potential before and after addition of G3-biph-3 in 0 M NaCl PBS were investigated. As G3biph-3 adsorbed on native insulin, oligomers and fibrils through static interaction, the zeta potential can reflect the intensity of the interaction. Table 2 showed that from the native insulin to the fibrils, the zeta potential maintain negative and the absolute value became higher. After adding the G3-biph-3 probes, the zeta-potential reversed to positive, and the data are 3.48, 3.78 and 5.34 for the three samples, respectively. It can be deduced that G3-biph-3 can attract oligomers more intensively than the native insulin, and the attraction to fibrils should be the strongest. However, the decreasing G3-biph-3 fluorescence along with the increase of the incubation time (Figure 6a) indicated that the interface area between G3-biph-3 and native insulin was bigger than G3-biph-3 and oligomers, and the interface area between G3-biph-3 and fibrils is the smallest. Hence, besides static interaction between G3-biph-3 and insulin at


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different state other factors which can influence the interface area should be considered. One reason was the size effect as mentioned before; another reason was the reduction of insulin total surface area due to the self-assembly during the fibrillation, which decrease the total potential binding sites for G3-biph-3.

Table 2. The zeta potential of native insulin, oligomers and fibrils with and without G3-biph-3. Samplea Native Insulin Oligomers Fibrils Zeta without G3-biph-3 -7.05 -15.00 -20.9 potential/mV with G3-biph-3b 3.48 3.78 5.34 a

All the sample were measured in 10 mM PBS without NaCl at 10 µM.

b

The concentration of G3-biph-3 was 10 µM.

To further demonstrate that the size effect is important in monitoring the fibrillation process, sodium polyacrylate (PAAS) with different molecular weight was used as a model to interact with G3-biph-3 and G4-biph-3. The PAAS with high molecular weight (PAAS-HM) can be regarded as several PAAS with low molecular weight (PAAS-LM) linked together through chemical bonds. This linkage caused negligible surface reduction, as compared with that of the insulin which assembled though the overlap of hydrophobic surfaces. As shown in Figure 9a, when the PAAS-HM concentration increased, the fluorescence of G3-biph-3 rose to a maximum then declined and the curve of PAAS-LM presented the similar tendency. It is because at low concentration only a few PAAS molecules were attracted by G3-biph-3, the mutual repulsion from the attracted PAAS molecules was weak and some PAAS molecules can stay closer to G3biph-3 molecule and raised the fluorescence intensity. However, when the concentration increased further, more PAAS molecules were attracted by one G3-biph-3 molecule. Many PAAS competed to pile around a G3-biph-3 molecule and repelled each other. Consequently none of them can stay close enough to the G3-biph-3 molecule, so there was some space between


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G3-biph-3 and the attracted PAAS, maintaining the rotation of biphenyl groups grafted to G3biph-3 and leading to the G3-biph-3 fluorescence reduction at high PAAS concentration. This fluorescence decline was a result of size effect caused by the high PAAS concentration.

Figure 9. (a) The fluorescence of 10 µM G3-biph-3 with varied concentration of PAAS-HM and PAAS-LM. (b) The fluorescence of 10 µM G4-biph-3 with varied concentration of PAAS-HM and PAAS-LM. Figure 9a also showed that at low mass concentration, the PAAS-LM fluorescence was weaker than the PAAS-HM fluorescence at the same concentration (Stage 1). Since there were more carboxyl groups on one molecule of PAAS-HM than that of PAAS-LM, G3-biph-3 can bind to PAAS-HM more easily (full extended chain because of the low concentration throughout the measurement). So the fluorescence of PAAS-HM increased more quickly than PAAS-LM at low concentration range and had a larger fluorescence maximum. On the other hand, because PAASHM had larger volume and more negative charges than PAAS-LM, the repulsion of the attracted PAAS-HM molecules were stronger than that of PAAS-LM. This strong repulsion led to the faster and earlier fluorescence decrease of G3-biph-3 fluorescence than the fluorescence decrease induced by the addition of PAAS-LM when more PAAS-HM was added. So above a certain


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concentration (Stage 2), the fluorescence corresponding to PAAS-LM was higher than that corresponding to PAAS-HM. As the total surface area of PAAS-HM was almost same to the total surface area of PAAS-LM, the surface reduction effect was negligible. The stronger fluorescence at high concentration of PAAS-LM than PAAS-HM should be ascribed to the larger size of PAAS-HM than PAAS-LM, i.e. the size effect. The G4-biph-3 represents the similar tendency to G3-biph-3. Because G4-biph-3 had more positive charges and larger volume, the attraction between G4-biph-3 and PAAS was stronger than that of G3-biph-3, and the more intensive repulsion among the attracted PAAS is necessary to weaken the fluorescence. The G4biph-3 fluorescence rose faster and declined at higher concentration than G3-biph-3 (Figure 9b). In this case of PAAS, its fluorescence detection by AIE-grafted PAMAM also demonstrated the size effect and sensitivity to the volume changes at molecule level. It helps to explain the detection of oligomers formation and transformation during insulin fibrillation process by AIEgrafted PAMAM.

Conclusions In this work, we synthesized a series of biphenyl groups grafted PAMAM, and these AIE probes are applied to investigate insulin fibrillation by mainly probing the size changes. The G3biph-3 fluorescence probe, with certain generation of PAMAM and appropriate graft ratio, can detect the change from native insulin to oligomers and the transformation from oligomers to fibrils. The mechanism how G3-biph-3 works was preliminary investigated, and one of the main factors, size effect, was proposed. Through the size effect, these dendrimer-based fluorescence probes may provide more details of the dynamic process of protein fibrillation, guiding a way to demonstrate the intermediate state in the protein fibrillation.


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Experimental Section Materials: Monopeak porcine insulin was purchased from Xuzhou Wanbang Biological Pharmaceutical Enterprise (Jiangsu, China). Two sodium polyacrylate (PAAS) solution with Mw = 1200 g/mol (PAAS-LM) and Mw = 15000 g/mol (PAAS-HM) were purchased from Aldrich. Ethylenediamine (EDA), acetone, methyl acrylate (MA), dichloromethane, toluene, methanol, dimethyl sulfoxide (DMSO), trifluoroacetic acid (TFA), ethyl acetate, alkaline aluminium oxide between 200 mesh and 300 mesh, were purchased from Tianjin Bodi Chemical Holding Company, N-methylmorpholine (NMM) and hydroxylbenotriazole (HOBT) were purchased from Aladdin. D-(+)-fucose, 2,5-dihydroxybenzoic acid (DHB), 4-aminobiphenyl and 1-Ethyl-3(3-dimethylaminopropyl)carbodiimide (EDCI) were purchased from J&K. Toluene , methanol and TFA were at HPLC degree. Others were at analytical degree. Sodium polyacrylate solution were precipitated in the acetone and lyophilized, then dissolved in the ultrapure water at 1 mg/mL to prepare the stock solution. 1 L EDA was shaked with CaO 50 g and KOH 15 g for 12 h. The supernatant was distilled under vacumm and stored under argon. MA passed through alkaline aluminium oxide column to remove the hydroquinone. Other reagents were used as received. Preparation of 4-biphenylamide succinic acid (compound 1): Succinic anhydride (10 mmol) was dissolved in dichloromethane (25 mL) in the 50 mL flask. 4-aminobiphenyl (10 mmol) was dissolved in dichloromethane (6 mL) and dropped into the flask, reacting for 7 hours. The precipitation was filtered and washed with dichloromethane for two times, then crystalized in the mixture solvent (DMSO: ethyl acetate=1:5). Yield: 43%.


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Preparations of G3-biph-1, G3-biph-2, G3-biph-3, G3-biph-4, G4-biph-1, G4-biph-2, G4biph-3, G4-biph-4: PAMAM dendrimers were synthesized through a step-by-step method reported by Tomalia.68-70 Their 1HNMR71 were showed in Supporting Information Figure S1. G3 (176 mg) was dissolved in DMSO (8 mL). Compound 1 (n mmol), EDCI (1.2n mmol), HOBT (1.2n mmol) and NMM (4n mmol) were added, respectively. The molar ratio of Compound 1 to the G3 was 1, 2, 4 and 8 from G3-biph-1 to G3-biph-4. Reaction was carried out under argon for 24 hours. The solution was precipitated with ethyl acetate at 5 times volume. After centrifugation, the precipitant was dissolved in DMSO and precipitated with ethyl acetate once again to further remove the residual Compound 1. Then the precipitation was dialysed with ultrapure water for 24 hours. The solution was filtered through 0.45 µm membrane and lyophilized. The syntheses of G4-biph-1 to 4 are similar to that of G3-biph-1 to 4, except the molar ratio of compound 1 to G4 was 2, 4, 8, 16 from G4-biph-1 to G4-biph-4, respectively. All the AIE-grafted PAMAM samples were dissolved in 25 mM HCl solution to prepare the stock solution (400 µM), and reserved under 4 oC. 1

H NMR spectroscopy: 1H NMR spectra of G3 and G4 in D2O solution and the AIE-grafted

G3 and G4 in [D6]DMSO were recorded with a Bruker Avance spectrometer (AVII-400; Bruker, Karlsruche, Germany) at 400 MHz. MALDI-TOF MS: the simple were dissolved in 0.1% THF aqueous solution at 1 mg/mL. Matrix was dissolved in solvent prepared by mix equal volume of ultrapure water and acetonitrile at 10 mg/mL. The matrixe used for PAMAM G3 and G4 was the mixture of D-(+)fucose and DHB (1:1 mass ratio).

72

The matrixe used for AIE-grafted G3 and AIE-grafted G4


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was DHB. MLDI-TOF MS in positive reflector mode was used for PAMAM G3. The other nine samples were investigated in positive linear mode. Fluorescence measurement: The fluorescence of AIE-grafted G3 and G4 were measured by Shimadzu RF-5301PC luminescence spectrometer at RT (Japan), at the excitation 220 nm with slit width 5 and emission ranges from 250 to 400 nm with slit width 3. The fluorescence intensity was the average of three scans. Preparation of the insulin fibrils: Porcine insulin was dissolved in 25 mM HCl, filtered with 0.45 µm membrane. The concentration was determined by absorption at 280 nm (ε = 5.53 mM-1 cm-1).14 The insulin concentration was kept at 300 µM. Tube vial (20 mL) which was made from borosilicate glass with a 1 cm long stirrer in it was washed with surfactant water solution, ultrapure water, 0.1 M HCl solution, ethyl alcohol sequentially in order and dried. Adding insulin solution 12.5 mL to the vial and sealed with rubber plug. The vial was incubated at 60 oC and stirred at 100 rpm for 48 hours. Monitoring the insulin fibrillation process: The insulin solution was incubated as the former part described. Aliquot of the incubation solution was added to the PBS buffer at a desired interval, then AIE-grafted G3, grafted G4 and ANS were added individually. The final sample concentration in the buffer was 10 µΜ, 3 µΜ, 20 µΜ, 4 µΜ and 10 µΜ corresponding to the respective addition of 10 µΜ G3-biph-3, 10 µΜ G3-biph-4, 10 µΜ G4-biph-3, 10 µΜ G4-biph-4 and 250 µΜ ANS to the buffer. Because in these range the corresponding grafted G3 and G4 fluorescence intensity demonstrated linear dependence on the pure native insulin concentration or pure insulin fibrils concentration (Figure 2 and Figure 3) and the fluorescence signal difference is significant enough.The PBS buffer used for G3-biph-3, G4-biph-3 and ANS was 10


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mM at 7.4 pH without NaCl. The PBS buffer used for G3-biph-4, G4-biph-4 contains 0.4 M NaCl. Measurement of the fluorescence caused by PAAS and AIE-grafted PAMAM: Different concentration of PAAS-LM and PAAS-HM were added to the 10 mM PBS without NaCl. Then 10 µM G3-biph-3 and G4-biph-3 were added to the buffer separately. Then the fluorescence of the buffer was investigated. Measurement of the zeta potential of native insulin, oligomers and fibrils before and after the addition of G3-biph-3: The incubation solution at native insulin state, oligomers state and fibrils state were taken to the 10 mM PBS without NaCl at 10 µM. Before and after the addition of 10 µM G3-biph-3, their zeta potentials were recorded by zeta potential instrument (Zetasizer Nano ZS 90, Malvern, UK). Scanning electron microscopy: The solutions containing 10 µM native insulin, oligomers or fibrils separately in 10 mM PBS without NaCl were dropped on the silica slide and dried at RT. The same three batches of solution containing additional 10 µM G-biph-3 was also dropped on the slide and dried. The microscopy was carried out at 15 kV with a field emission-scanning electron microscope (Inspect F, FEI, USA). Circular dichroism spectra: The incubation solutions were taken at 0 min, 240 min 360 min individually, and their circular dichroism spectra were recorded from 195 nm to 300 nm in the 1 mm cuvette with the instrument (J-1500-150ST).

ASSOCIATED CONTENT Supporting information This material is available free of charge on the ACS Publication website.


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The 1HNMR spectra of G3 and G4; The MALDI-TOF MS of G3 and G4; The 1HNMR spectra of Compound 1; The MALDI-TOF MS of AIE-grafted G3; The MALDI-TOF MS of AIE-grafted G4; The complete fluorescent spectra of AIE-grafted G3; The complete fluorescent spectra of AIE-grafted G4; The replication of the monitoring insulin fibrillation with G3-biph-3.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT Financial support from the National Natural Science Foundation of China (21534008 and 51322303) are gratefully acknowledged.

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Table of content

Fluorescence probe: A positive charged dendrimer conjugated with aggregation-induced emission (AIE) molecule was synthesized. This probe can detect the transformations both from native insulin to oligomers and from oligomers to fibrils during insulin fibrillation. The mechanism was investigated and the probe may help to discover intermediates in the fibrillation process of proteins.


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Detecting the Formation and Transformation of Oligomers during

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