Research Article www.acsami.org
Detection and Monitoring of Amyloid Fibrillation Using a Fluorescence “Switch-On” Probe Nibedita Pradhan,† Debabrata Jana,‡ Binay K. Ghorai,*,‡ and Nikhil R. Jana*,† †
Centre for Advanced Materials, Indian Association for the Cultivation of Science, Kolkata 700 032, India Department of Chemistry, Indian Institute of Engineering Science and Technology, Shibpur, Howrah 711 103, India
‡
S Supporting Information *
ABSTRACT: Amyloid protein fibrillation is associated with a variety of neurodegenerative and other diseases, and their efficient detection and monitoring can greatly advance early diagnosis and therapy. Herein, we report a fluorescent “switchon” probe for the reliable detection and monitoring of amyloid fibrils. The probe consists of a peptide component for binding with amyloid structure and a color component with an aggregation-induced green emission property. This probe is nonfluorescent in the presence of amyloid forming monomer protein/peptide, but fluorescence “switch-on” occurs after binding with amyloid fibrils. Compared to conventionally used thioflavin T, this probe offers a high signal-to-noise ratio, which is unaffected by the quencher ion/nanoparticle. The proposed new probe has been used for the detection and monitoring of amyloid fibrils produced by a wide variety of amyloid protein/peptides and can be extended for in vitro diagnostic applications. KEYWORDS: amyloid fibril, fluorescence probe, Alzheimer’s, aggregation-induced emission, tetraphenylethene, amyloid detection
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INTRODUCTION Amyloid fibrils are threadlike protein aggregates with crossed βsheet secondary structures that are associated with a diverse range of neurodegenerative and other diseases that include Alzheimers, Parkinsons, and diabetes type II.1−4 A variety of peptides/proteins have been identified that fold abnormally in the formation of pathological amyloid deposits/aggregates.1−3 Although amyloid related diseases are at the center of intense research efforts, currently, no definite diagnostic tool exists for this type of disease.4 Thus, reliable detection of amyloid fibrils and plaques and the mechanistic understanding of amyloid fibrillation are currently the focus of extensive research efforts.4 Several spectroscopic and microscopic methods have been developed for studying amyloid fibrillation, including fluorescence spectroscopy,5−11 scanning electron microscopy (SEM),12 atomic force microscopy (AFM),13 transmission electron microscopy (TEM),14 circular dichroism (CD),15 and NMR.16 Among them, the fluorescence-based approach is most widely used because of its simplicity. Environmentally sensitive fluorescent dyes are commonly used for such probe development, such as thioflavin T,5 a naphthalene derivative,6 a conjugated oligothiophene,7 a curcumin derivative,8 and other dyes.9−11 These molecular probes are sensitive to a hydrophobic environment, and their emission becomes intense after binding with the hydrophobic β-sheet region of an amyloid fibril. However, accumulation of multiple fluorophores in hydrophobic sites can lead to quenched emission and poor signal reproducibility due to the staking of aromatic rings.17 © XXXX American Chemical Society
Other common drawbacks include low specificity, small Stokes shift, poor sensitivity, false-positive response, and incapability in detecting oligomeric intermediates. Despite all of these limitations, they have been routinely been used for amyloid assays over the last 50 years. Recently, a new class of molecules has been discovered that is nonfluorescent under a molecularly dissolved state but becomes highly fluorescent after forming aggregates.18−20 The origin of aggregation-induced emission (AIE) lies with aggregationinduced restriction of intramolecular rotations21 and the restricted transition from a local excited state to the intramolecular charge transfer state accompanying twisting.22 Examples include silole,18 arylbenzene,19 arylethene,19 derivatives of aminobenzoic acid,19 and some metal complexes of platinum23 and iridium.24 AIE-based probes are expected to resolve the fluorescence quenching issue of conventional fluorescent probes, which arises at high concentration or under an aggregation state. This is because AIE-based molecules have the natural tendency to “switch-on” their fluorescence under aggregation stage with the advantage of signal enhancement rather than signal loss under high concentration or aggregation condition.18−20 This advantage of AIE-molecules has been utilized in the development of fluorescent probe for cell apoptosis,25 integrin receptors,26 Received: August 20, 2015 Accepted: November 5, 2015
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DOI: 10.1021/acsami.5b07751 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
Scheme 1. (a) Synthetic Steps for the Tetraphenylethene (TPE) Derivative Used in This Worka and (b) Structure of the Fluorescent Probe Showing the Aggregation-Induced Emission (AIE) and Amyloid Binding Peptide Components
a Reagents and conditions: (i) Zn (powder), TiCl4, THF, reflux, 5 h, 35%; (ii) n-BuLi, THF, −78 °C then DMF, rt, 12 h, 76%; (iii) BBr3, CH2Cl2, rt, 24 h, 65%.
mitochondria,27 cell membranes,28 nucleic acids,29 and insulin fibrils.30 Herein, we report an AIE-based probe for specific and reliable detection of amyloid fibrillation. The probe consists of a tetraphenylethene (TPE)-based color component and an RGKLVFFGR-based peptide component. The TPE component offers an AIE property,31 and the peptide component offers selective binding with the amyloid structure.32,33 Compared to conventionally used thioflavin T and reported AIE-based probes for insulin fibril,30 the presented probe is more specific and sensitive in detecting Aβ fibril,3 insulin fibril,34 and lysozyme fibril,35 and detection is unaffected by commonly occurring fluorescence quenchers.
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Instrumentation. Fluorescence spectra were measured with a BioTek Synergy MX microplate reader. All UV−visible spectra were recorded on a Shimadzu UV-2550 spectrophotometer from sample solutions in a quartz cell of 1-cm path length. High resolution mass spectra (HRMS) were recorded on a Waters QTOF Micro YA263 spectrometer. Mass spectral measurements of the TPE-peptide were carried out on a Bruker ultraflextreme MALDI-Mass spectrometer equipped with a 337 nm nitrogen laser and α-cyano-4-hydroxycinnamic acid as matrix. The TPE-peptide solution was mixed with the matrix solution (1:1, v/v), and ∼1 μL of the sample was placed on a metal sample plate and allowed to air-dry at ambient temperature. The mass spectrum was then acquired in positive linear mode with an acceleration voltage of 25 kV. HRTEM was performed with an FEI Tecnai G2 F20 microscope with a field-emission gun operating at 200 kV. FESEM was performed with a Supra 40 (Carl Zeiss) microscope. Secondary structure of amyloid fibril was determined using a JASCO J815 CD spectrometer (model J-815-1508). Synthesis of Aldehyde-Functionalized Tetraphenylethene (TPE) Derivatives. The detailed synthetic approach for TPE-aldehyde is outlined in Scheme 1. Conventional titanium(0)-catalyzed crossMcMurry reaction between 4,4′-dimethoxybenzophenone (1) and 4bromobenzophenone (2) was used for the synthesis of bromosubstituted TPE (3) with 35% yield.31 Lithiation of 3 with 1.2 equiv of n-BuLi at −78 °C, followed by the addition of dimethylformamide, afforded 4-[2,2-bis(4-methoxyphenyl)-1-phenylvinyl]benzaldehyde (4) with 76% yield. Demethylation of compound 4 with boron tribromide in dichloromethane at room temperature yielded 4-[2,2bis(4-hydroxyphenyl)-1-phenylvinyl]benzaldehyde (5) with 65% yield. The structures of synthesized compounds 3−5 were confirmed by their NMR and mass spectral analyses. The presence of hydroxyl groups in 5 is confirmed by the 1H NMR spectral analysis utilizing D2O shaking. The hydroxyl proton signal of 5 appeared at δ 9.52 ppm
EXPERIMENTAL SECTION
Materials. L-Aspartic acid, anhydrous AuCl3, Igepal, didodecyldimethylammonium bromide (DDAB), L-histidine monohydrochloride hydrate, 2,4,6-tris(bromomethyl)mesitylene, tetrabutylammoniumborohydride (TBAB), gold(III) chloride, 3-marcaptopropyltrimethoxysilane (MPS), 2-aminoethyl-aminopropyltrimetoxysilane (AEAPS), sodium borohydride, didodecyldimethylammonium bromide (DDAB), 3-(mercaptopropyl)-trimethoxysilane (MPS), tetrabutylammonium borohydride (TBAB), sodium chloride, and thioflavin T were purchased from Sigma-Aldrich. Amyloid β-protein fragment 1−40 (>90%) was purchased from Sigma-Aldrich. Amyloid β-protein fragment 1−42 (>90%) and peptide with sequence RGKLVFFGR (95%) were purchased from Genpro Biotech. All chemicals were used as received. Insulin powder from bovine pancreas (MW 5733) and lysozyme powder from chicken egg white (MW 14.3 Kda) were purchased from Sigma-Aldrich. B
DOI: 10.1021/acsami.5b07751 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces (s, 1H) and δ 9.50 ppm (s, 1H) in CDCl3, and it is exchangeable with D2O. Details of the synthesis and characterization are described in Figures S1−S6. Synthesis of Peptide-Functionalized TPE (TPE-Peptide). TPE was covalently conjugated with the N-terminus of the RGKLVFFGR peptide. Typically, 0.4 mg of TPE was dissolved in 150 μL of methanol. In a separate vial, 1.07 mg of RGKLVFFGR peptide was dissolved in 150 μL of dimethylformamide. The two solutions were mixed, and then a 50 μL solution of triethylamine in DMF was added. The mixture was kept at 4 °C with stirring, and after 30 min, 20 μL of sodium borohydride solution (0.37 mg dissolved in 20 μL H2O) was added. The reaction was continued overnight at 4 °C, and the formation of the TPE-peptide was confirmed by mass spectroscopy (Figures S7 and S8). In one case, the reaction mixture was directly used, and it was purified in another case. The HRMS results show similar results for both cases. In a typical purification step, yellow supernatant was isolated after centrifugation at 14,000 rpm for 5 min. Then, acetone was added to precipitate the sample. The precipitate was isolated by centrifugation, dried, and solubilized in anhydrous DMF. Next, excess absolute alcohol was added and centrifuged at 14,000 rpm for 5 min, and sample precipitate was dried in air and dissolved in dry DMSO. Synthesis of L-Aspartic Acid-Functionalized TPE (TPEAspartic Acid). Typically, a 500 μL methanol solution of TPE (10 mM) was mixed with 25 μL of a borate buffer solution (pH 9.0) of Laspartic acid (200 mM) followed by the addition of 100 μL of a triethylamine-methanol mixture (1:10 v:v). After 30 min of stirring at room temperature, a 20 μL aqueous solution of sodium borohydride (10 mM) was added. The reaction was continued overnight with stirring at room temperature. The yellow colored supernatant was isolated after centrifugation at 14,000 rpm for 5 min and used for further experimentation. The formation of TPE-aspartic acid was confirmed by mass spectroscopic analysis (Figure S9) Nanoparticle Synthesis. Gold, silver, and iron oxide (γ-Fe2O3) nanoparticles have been used in this study. Monodispersed hydrophobic nanoparticles of
