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A smart self-assembled organic nanoprobe for protein-specific detection: design, synthesis, application and mechanism studies Tang Gao, Shuqi Yang, Xiaozheng Cao, Jie Dong, Ning Zhao, Peng Ge, Wenbin Zeng, and Zhen Cheng Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02923 • Publication Date (Web): 22 Aug 2017 Downloaded from http://pubs.acs.org on August 23, 2017

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Analytical Chemistry

A smart self-assembled organic nanoprobe for protein-specific detection: design, synthesis, application and mechanism studies Tang Gao1, Shuqi Yang1, Xiaozheng Cao1, Jie Dong1, Ning Zhao2, Peng Ge1, Wenbin Zeng1,*, Zhen Cheng2,* 1

Xiangya School of Pharmaceutical Sciences, Central South University,

Changsha 410013, China. 2

Molecular Imaging Program at Stanford (MIPS), Canary Center at

Stanford for Cancer Early Detection, Department of Radiology and Bio-X Program, School of Medicine, Stanford University, California, USA. * Corresponding author: Tel/Fax: 0086-731-82650459, E-mail address: [email protected]; Tel/Fax: 001-650-736-7925, E-mail address: [email protected] (Z. Cheng)

ACS Paragon Plus Environment


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Abstract Specific detection or imaging protein has high potential to contribute greatly to medical diagnosis, biological research and therapeutic applications. The level of Human Serum Albumin (HSA) in blood is related to a variety of diseases and thus serves as an important biomarker for fast clinical diagnosis. Here we report the use of aggregation-induced emission (AIE) based supramolecular assembly to design biomolecular responsive smart organic nanomaterials for detection protein HSA. The designed nanoprobes were aggregates of small molecules and silent in fluorescence, but in the presence of HSA they disassembled and produced a clear turn-on fluorescent signal. Of a small library of nanoprobes constructed for HSA detection, structure-optical signaling and screening studies revealed that nanoprobe 7 is the most efficient one. Mechanism studies showed that nanoprobe 7 was bonded with Site I of HSA through the multiple noncovalent interactions. The resultant restriction of intramolecular rotation of nanoprobe 7 in the hydrophobic cavity of HSA induced fluorescent emission, which was validated by competitive binding assays and molecular docking. More importantly, nanoprobe 7 was successfully applied to recognize and quantify HSA in human serum samples. This study demonstrates nanoprobe 7 is a promising tool for clinical real and fast detection of HSA and thus may find many applications, and the molecular assembly based on AIE also opens a new avenue for designing smart nanomaterials for the sensitive and selective detection for varied analytes.


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Introduction Protein detection is crucial in biomedical diagnosis and treatment. For example, human serum albumin (HSA) is a major and versatile protein in human blood, and it plays a key role in maintaining plasma colloid osmotic pressure and transporting endogenous and exogenous molecules.

1, 2

As a reliable health indicator, the normal

level of serum albumin in serum is approximately 35–55 gL-1 (527-828 µmolL-1).3 Low level of HSA in the blood plasma is associated with liver cirrhosis, failure and chronic hepatitis.4 On the contrary, the presence of excess HSA in urine can result in microalbuminuria, which has been recognized in diabetes mellitus and hypertension as an indicator for patients with kidney disease and cardiovascular disease.5,6 Therefore, accurate detection of HSA with high selectivity and sensitivity in body fluids, especially in blood serum, has great importance in clinical diagnosis. Up to now, some methods, such as fluorescence-based assays, antibody based methods, LC-MS/MS and so on, have been developed for HSA quantitative detection in blood samples.7,

8

Among them, fluorescence assays have been widespread applied to

quantify HSA in multiple samples samples, due to their favorable perpertied for the rapid, convenient, sensitive and non-destructive detecton for bilogical samples.9-13 However, the reported fluorescent bioprobes for detection of HSA possess a few limitations. For example, some bioprobes are insoluble in aqueous media,14-20 which makes it difficult for assays in biological systems; a few bioprobes selectively recognize protein by covalent reactions with the thiol or amino moiety of the protein, however, this irreversible binding may lead to denaturation of proteins.21, 22 What is



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more, some bioprobes for protein detection comprising conventional organic dye molecules tend to aggregate when dispersed in physiological buffer, which usually quenches fluorescence attributed to the aggregates‘ non-radiative relaxation.23-26 Limitations of probes for protein detection listed above has forced researchers to employ single molecules in extreme dilute solutions as bioprobes. However, the dilute solution may lead to a weak emission and the poor sensitivity in fluorescent bioassays. Additionally, the aggregation-caused quenching (ACQ) effect is unavoidable even in dilute solutions， because in a bioanalysis, fluorescent probe may accumulate on the surface or in the hydrophobic cavity of biomacromolecules.27-29 Therefore, it is critical to design a probe with high water solubility and overcome the effect of ACQ to detect HSA levels in biological samples. Supramolecular

chemistry

refers

to

molecular

assemblies

built

upon

intermolecular noncovalent interactions including hydrogen bonding, electrostatic interaction, charge-transfer interactions, π–π interaction, hydrophobic effect and etc. 30,31

Compared with individual molecular building blocks, the supramolecular

self-assemblies possess some distinctive physical and chemical properties, which make them appealing as powerful tools for biomedicine.32-35 More importantly, small-molecules

self-assembled

nanoparticles

present

good

photo-stability,

biocompatibility, great diversity, flexibility in molecular design and tunability in optical properties and functionalities.36-38 However, not much such nanoparticles have been employed in protein sensing applications until now 39.


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Having taken the value of protein detection in clinical diagnosis and the limitations of reported probes, we have devoted in design and synthesis of self-assembled nanoprobes with high water solubility and excellent fluorescence characteristics for detection protein, particularly HSA as an example. Inspired by the fact that imidazole-cored molecular rotors often emit weak fluorescence in dispersed state due to intramolecular rotations and turn on its fluorescence in aggregate state or restrict the intramolecular rotations,40 we prepared a series of bola-type small molecules containing substituted imidazole as the fluorophore, an alkyl chain as the hydrophobic chain and a pyridinium salt group as the hydrophilic terminated group (Scheme 1). We hypothesize that the graft of aggregation-induced emission (AIE) effect and bola amphiphilic will bring about stable nanoprobes with good water-solubility and biocompatibility. The self-assembled nanoparticles are formed when probes are dissolved in aqueous solution. After combining the hydrophobic cavity of HSA, the nanostructures are disassembled. The results of trapping probes into HSA lead to the restriction of intramolecular rotation and induce the significant fluorescence emission (Scheme 1).



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Scheme 1. Chemical structures of probe molecules, the self-assembling turn on fluorescent nanoprobe for HSA. Experimental Section Materials

and

1,2-dibromoethane(98%),

apparatus.

Benzil

(98%),

1,4-dibromobutane(98%),

Aniline(99.5%),

1,6-dibromohexane(98%),

1,8-dibromooctane(98%), 1,10-dibromodecane(98%), 1,12-dibromododecane(98%) and 1,4-bis(bromomethyl)benzene (98%) was purchased from Energy Chemical Co., Ltd (China). 4-Pyridine carboxaldehyde(98%) and pyridine was purchased from Sinopharm Chemical Reagent Co., Ltd (China). All proteins were purchased from Sigma-Aldrich. Ultrapure water was obtained by using a Millipore water purification system. Other chemical reagents and solvents were used without further puriﬁcation. UV-vis absorption spectra were performed on a UV-2550 scanning spectrophotometer (Shimadzu). Fluorescent spectra were recorded on a Shimadzu RF-5301 equipped with a 1 cm quartz cell. Dynamic light scattering measurements were performed at 25


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o

C on Zestier Nano ZS (Malvern Instruments Ltd, Uk). The morphology of nanoprobe

7 was characterized by double beam electron microscope (Helios Nanolab 600i) and JEM-2100 transmission electron microscope with an accelerating voltage of 200 kV. 1

H NMR and 13C NMR chemical shifts were measured on a Bruker AVII-400 MHz or

500 MHz spectromete in DMSO or CDCl3(TMS as internal standard). HRMS was conducted on an Orbitrap Velos Pro LC-MS spectrometer (Thermo Scientific). Preparation of nanoprobe 7. A stock solution of 7 (3 × 10−3 M) was prepared from dimethyl sulfoxide. Thirty microliter of this solution was injected into phosphate buffer (3 mL) maintained at pH 7.4, and the solution (30 µM) was kept under room temperature. Nanoparticle formation was confirmed by DLS，SEM and TEM analyses of the solution by drop casting on freshly cleaved mica surface or carbon-coated copper grid (400 mesh), respectively, after drying in vacuum. Protein sensing experiments. The stock solutions of the required proteins were prepared by dissolving in phosphate buffer (10 mM, pH = 7.4). Concentrations of these stock solutions were calculated from the absorbance at a particular wavelength and molar extinction coefficient values. Protein (0 − 50 µL) from the stock solution was added to a stirring solution of nanoprobe 7 (30 µM, phosphate buffer at pH = 7.4) in a quartz cell with a path length of 1 cm at room temperature (25 °C). The solution was kept for 1 min, and the fluorescence intensity at 480 nm was measured after exciting at 380 nm. For protein selectivity studies, different proteins (50 µL) and small molecules from the stock solution were added slowly to a stirring solution of nanoprobe 7 (30 µM) and kept for 5 min at room temperature. The change of



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fluorescence intensity at 480 nm was measured at an excitation wavelength of 380 nm. Denaturing experiments. The stock solution of guanidinium chloride (GdnHCl) was dissolved in phosphate buffer solution. The mixture of HSA and nanoprobe 7 (10 µM) was incubated under different concentrations of GdnHCl at 37oC for 10 h, respectively. The measure of fluorescence emissions were conducted under excitation at 380 nm. Competitive assay. The competitive solvents warfarin and ibuprofen were dissolved in DMSO, respectively. In the assay, nanoprobe 7 was treated with HSA and the fluorescence emission spectrum was recorded. Then, the mixed solution was added with a various concentration of warfarin or ibuprofen. The fluorescence emission spectrum of the resultant solvents was measured by fluorescence spectrophotometer. Molecular docking. Protein−ligand docking studies were carried out using MOE (Molecular Operating Environment, 2014.10). Step 1: Using structure preparation to prepare complex of proteins and ligands. Use the default parameters to correct all possible error messages. The crystal structure of HSA compounded with phenylbutazone (PDB code 2BXP) was obtained from the Protein Data Bank (www.pdb.org). Step 2: Use the protein ligand as template to find the docking pocket (ligand molecule as the center radius of 4.5 Å). Step 3: 30 optimal structure of nanoprobe 7 was searched by the default parameter. Make use of these optimal structures and the pocket for docking (Placement: Triangle Matcher; Rescoring 1:


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London dG; Retain: 30; Refinement: Force field; Rescoring 2: GBVI/WSA dG; Retain: 30). Step 4: The docking results including interaction relations and spatial conformation maps were collected from the function of ligand interactions. Cell viability assay. The cell viability effect of nanoprobe 7 was determined by CCK-8 assay using a cell counting kit-8 (Beyotime Biotechnology, China) according to the manufacture’s protocols. Briefly, Hela cells were seeded in a 96-well plate (5 x 103 cells/well) and treated with different concentrations of nanoprobe 7 (2.5 µM, 5 µM, 10 µM, 20 µM, 50 µM) for 24 h. Then 10 µl of CKK-8 was added to the each well for 4 h and the absorbance at 450 nm was examined with a microplate reader (Synergy HT, Biotech). The optical density (OD) values were determined to reflect the viable cell population from each well. Detection of HSA in human blood serum. Blood samples (5 mL each) were collected from healthy donors into a blood collecting tube using sterilized syringe and needle. Then, the blood samples were centrifuged at 5000 rpm for 10 min to separate the serum from the red blood cells. The supernatant is then pipetted out into another centrifuge tube which was used for the analysis. The HSA content in blood serum was detected with nanoprobe 7 by using standard addition method. Fluorescence response of nanoprobe 7 (30 µM) in diluted plasma sample (1000 fold dilution) upon addition of different concentration of HAS (0-22 mgL-1). A calibration plot was prepared by measuring the emission maximum at 480 nm (I480). The unknown concentration of HSA protein in the blood serum was calculated from the calibration curve by diluting the serum sample appropriately within the linear range.



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Results and discussion Synthesis of probes. The bola-type probes (3, 5, 7, 9, 11, 13 and 15) were synthesized as shown in Scheme S1. The tetraaryl imidazole (compound 1) was achieved by a one-pot two-step multicomponent reaction of isonicotinaldehyde, primary aromatic amine, benzyl, and ammonium acetate. Compound 1 was reacted with 1,2-dibromoethane, 1,4-dibromobutane, 1,6-dibromohexane, 1,8-dibromooctane, 1,10-dibromodecane, 1,12-dibromododecane and 1,4-bis(bromomethyl)-benzene, to yield the intermediate 2, 4, 6, 8, 10, 12 and 14, respectively. Reaction of the related intermediate with pyridine afforded the probes. All probes were characterized by NMR. Optical properties of 7. The absorption spectra of probe 7 in different solvents were shown in Figure S1a. In CH3CN, DMF, or DMSO, the absorption maxima in 380 nm was observed. In EtOH, the absorption went through a small redshift to 400 nm due to the solvatochromic effect. Interestingly, in H2O, the absorption spectra broadened with a leveled-off tail known as the Mie effect, which indicated the aggregation of 7 in aqueous. This phenomenon can be interpreted as 7 disperse well in solvents, the intramolecular rotation blocks the radiative excitons and populates the nonradiative relaxation channel (Figure S1b).41 To check the AIE characteristics, we used water and THF as solvent and non-solvent. As shown in Figure 1a, in aqueous solution almost no fluorescence was observed. While the THF ƒw >80 (fraction by volume %), the emission of 7 dramatically increased and the fluorescent quantum efficiency (φF) of 7 in THF solution was determined as 0.13. Since probe 7 formed


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the aggregations and blocked the non-radiative relaxation pathways of the excited state, which indicated the probe 7 has the significant AIE effect.

Figure 1. (a) Fluorescent spectra of 7 in THF-water mixtures with different water fractions (ƒw); (b) Plots of fluorescent intensity vs. THF fractions of 7. Determine the critical aggregation concentration (CAC). To investigate the critical aggregation concentration, the conductivity experiment with probe 7 was carried out and the related results were plotted against its concentration (Figure S2b). It was observed that the conductivity increased linearly as the concentration increased up to 60 µM. However, at a concentration higher than 60 µM, the plot was found to be linear with a lower slope. Such break between the two slopes indicated that new micelles were formed, which is similar to the reported literature.42 When the fluorescence intensity at 480 nm was plotted with the corresponding concentration, there are two linear segments in the curve, and the slope decreases abruptly, which means that the CAC is about 60 µM. It was noteworthy that 7 showed very weak fluorescence in water in various concentrations ranging from 5 µM to 100 µM (Figure S3). Self-assembly behavior of probe 7. With the assistance of dynamic light scattering (DLS), scanning electronic microscopy (SEM) and transmission electron



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microscope (TEM), we further explored the self-assembled behavior of 7. The observed DLS studies indicated the aggregates of 7 had an average diameter of 180 nm (Figure S4a). Furthermore, the SEM (Figure S4b) and TEM (Figure S4c) images discovered a spherical structure of the aggregates. The aggregates with non-emission states may be attributed to the loosely packed characteristics and still has enough free volume to consume the radiative energy by the intramolecular rotation, leading to nearly no florescence. 28 Fluorescent sensing to HSA. The self-assembled nanoparticles of 7 with nonemissive were used as the nanoprobe. The UV-Vis absorption and fluorescence spectra of nanoprobe 7 in the absence and presence of HSA were investigated, respectively. As shown in Figure S5, upon addition of HSA, the broad absorption of 7 changed and the absorption peak at 380 nm increased, suggesting that the nanoaggregates of 7 in the presence of HSA were changed. The fluorescence spectra of 7 excited at 380 nm showed non-fluorescence in aqueous, but upon addition of HSA the fluorescence intensity at 480 nm increased dramatically (Figure 2a), which may owe to the bonding with the protein and restricting the intramolecular rotation. As shown in Figure S6, no fluorescence change was observed when common ions were added. Furthermore, other proteins and biomolecules cannot interface the fluorescence of the nanoprobe (Figure 2b). This observation revealed that 7 displayed a high selectivity for HSA. Thus, the nanoprobe would provide potential applications for biological detection. As shown in Figure 2c, the fluorescence intensity gradually enhanced with the


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increasing HSA concentrations. Moreover, the fluorescence intensity at 480 nm exhibited an excellent linearity relationship with HSA concentrations (0 to 15 µM) (R2 = 0.997) (Figure 2d). The detection limit (3σ/slope) of nanoprobe for HSA in PBS was determined to be 0.14 µM (9.3 mgL-1), which is below the regular concentration (35–55 gL-1) of HSA in blood. This result demonstrated that the nanoprobe 7 can be a sensitive fluorescent nanoprobe for quantitative detection of HSA in an aqueous environment. The time-dependence of fluorescence intensity to HSA was evaluated. As shown in Figure S7, the emission intensity was immediately enhanced after the addition of HSA and maintained constant over an extended period. The response time is about 15 s. Therefore, nanoprobe 7 could be a great choice for rapid detection of HSA in clinical samples.

Figure 2. (a) Fluorescence response of nanoprobe 7 (30 µM) to HSA (15 µM), λex = 380 nm. Inset: Fluorescence changes of nanoprobe 7 upon addition of HSA under



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excitation with UV light (365 nm). (b)

Fluorescence spectra of nanoprobe 7 (30

µM) toward different proteins and amino acids, 15 µM HSA and 50 µM other proteins and amino acids in PBS buffer, pH = 7.4, λex = 380 nm. (c) Fluorescence emission spectra of nanoprobe 7 (30 µM) with increasing concentrations of HSA (0–21.0 µM) in PBS buffer (pH = 7.4, 10 mM). (d) The linear relationship between the fluorescence intensity at 480 nm of nanoprobe 7 (30 µM) and the concentrations of HSA (0–15.0 µM), Y =3.2012 + 6.1946 X, R2 = 0.997. Conditions: PBS buffer (pH = 7.4, 10 mM), λex = 380 nm. Investigation of the detection mechanism of HSA. To investigate the influence of molecular structures, particularly the length of the alkyl chain and the rigidity, we designed and synthesized a series of molecular probes. Interesting effects of different alky chains and rigidity were discovered. The alky chain with length of less than 6-C atoms (compound 3 and 5) induced fluorescence emission with less intensity toward HSA, while probes with the chain length of more than 6-C atoms (compound 9, 11 and 13) were observed to be silent toward HSA detection (Figure S8) These results indicated that it is necessary to induce fluorescence enhancement an optimum hydrophobicity, and possibly, 7 possesses a suitable hydrophobicity to interact with the protein. For compounds with longer chain length, the steric factor predominates over hydrophobicity and hinders their interactions with the hydrophobic cleft of the protein. Furthermore, when the flexible alkyl chain was replaced by the rigid benzene ring, compound 15 also couldn’t induce strong interaction with the protein leading to silence optical signal.


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These experimental observations suggest that the flexibility and appropriate length of the connecting arm are important for the probe to interact with the protein. We further explored the detection mechanism. Since molecular aggregation is the major reason to affect the luminescence of AIE fluorophores, DLS analysis was applied to check whether larger nanoaggregates were formed. Surprisingly, after the addition of HSA, the corresponding peak at 178 nm of nanoprobe 7 disappeared. Also, a new peak at 10 nm showed up (Figure 3c), which corresponded to the size of HSA in solution (Figure 3a).17, 19 To illustrate the change of the nanoprobe, TEM characterization was further conducted. As shown in Figure 3e, the average diameter of nanoprobe is about 150 nm in agreement with DLS (Figure 3b). In presence of HSA led to the disaggregation of nanoprobe and formation some smaller nanoparticles (Figure 3f). These results revealed that the binding between HSA and nanoprobe led to the disassembly of the latter for which the hydrophobic interaction may be the primary driving force.

Figure 3. (a) DLS and (d) TEM analyses of HSA(15 µM). (b) DLS and (e) TEM analyses of nanoprobe 7 (30 µM) spherical assemblies obtained from phosphate



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buffer (pH 7.4, 10 mM ). (c) DLS and (f ) TEM analysis of nanoprobe 7 (30 µM) in the presence of HSA (15 µM). To testify the interaction of 7 with the hydrophobic pockets of HSA, the unfolding process of HSA induced by GdnHCl denaturant was investigated. As shown in Figure S9, the binding of nanoprobe to HSA showed bright green fluorescence. In the presence of GdnHCl, we found three transition steps during the denaturation process (Figure S9b). At low concentrations of GdnHCl (

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