Specific and Quantitative Detection of Albumin in Biological Fluids by

Subscriber access provided by BUFFALO STATE

Biological and Medical Applications of Materials and Interfaces

Specific and quantitative detection of albumin in biological fluids by tetrazolate-functionalized water-soluble AIEgens Yujie Tu, Yeqing Yu, Zhibiao Zhou, Sheng Xie, Bicheng Yao, Shujuan Guan, Bo Situ, Yong Liu, Ryan T. K. Kwok, Jacky W. Y. Lam, Sijie Chen, Xuhui Huang, Zebing Zeng, and Ben Zhong Tang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b10359 • Publication Date (Web): 25 Jul 2019 Downloaded from pubs.acs.org on July 26, 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 35 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

ACS Applied Materials & Interfaces

Specific and Quantitative Detection of Albumin in Biological Fluids by Tetrazolate-Functionalized Water-Soluble AIEgens Yujie Tu†‡§, Yeqing Yu‡, Zhibiao Zhou†, Sheng Xie*†‡, Bicheng Yao‡§, Shujuan Guan⊥, Bo Situ⊥, Yong Liu‡§, Ryan T. K. Kwok‡§, Jacky W. Y. Lam‡§, Sijie Chen∇, Xuhui Huang‡, Zebing Zeng†, Ben Zhong Tang*†‡§ † State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China. ‡ Department of Chemistry, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China; §Hong Kong Branch of Chinese National Engineering Research Center for Tissue Restoration and Reconstruction, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China; ⊥Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, 510515 China ∇Ming Wai Lau Center for Reparative Medicine, Karolinska Institutet, Hong Kong, China.

ACS Paragon Plus Environment

1

ACS Applied Materials & Interfaces 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

Page 2 of 35

KEYWORDS: albumin, aggregation-induced emission, water-soluble fluorescent probe, tetrazolate-lysine interaction, diagnostic detection.

ABSTRACT: The analysis of albumin has clinical significance in diagnostic tests and obvious value to researches on the albumin-mediated drug delivery and therapeutics. The present immunoassay, instrumental techniques, and colorimetric methods for albumin detection are either expensive, troublesome or insensitive. Herein, a class of water-soluble tetrazolatefunctionalized derivatives with aggregation-induced emission (AIE) characteristics are introduced as novel fluorescent probes for albumin detection. They can be selectively lighted up by site-specific binding with albumin. The resulting albumin fluorescent assay exhibits a low detection limit (0.21 nM), high robustness in aqueous buffer (pH = 6~9), and a broad tunable linear dynamic range (0.02~3000 mg/L) for quantification. The tetrazolate functionality endows the probes with a superior water solubility (> 0.01 M) and a high binding affinity to albumin (KD = 0.25 µM). To explore the detection mechanism, three unique polar binding sites on albumin are computationally identified, where the multivalent tetrazolate-lysine interactions contribute to the tight binding and restriction of molecular motion of the AIE probes. The key role of lysine residues is verified by the detection of poly-L-lysine. Moreover, we applied the fluorogenic method to quantify urinary albumin in clinical samples and found it a feasible and practical strategy for albumin analysis in complex biological fluids.

INTRODUCTION Human serum albumin (HSA) plays important physiological roles such as maintenance of intravascular osmotic pressure, substrate transportation, buffering capacity, free radical


2

Page 3 of 35 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


scavenging, coagulation and wound healing.1 The albumin level is delicately controlled in plasma, intercellular and interstitial spaces of organs, thereby an abnormal albumin level in biological fluid becomes a marker of diseases. For example, low concentration of albumin in blood, known as hypoalbuminemia (< 35 mg/L), indicates an unhealthy nutrition status, disorder of the digestive system, or even liver failure.2-4 Presence of albumin in urine, known as albuminuria(> 30 mg/L), is a kidney disease signal which can be associated with diabetes, high blood pressure, urinary tract infection, etc.5-8 Thereby the regular monitoring of albumin in body fluids is of obvious values for disease warning, early diagnosis, and timely treatment. Meanwhile, as a nonimmunogenic, nontoxic, and biodegradable carrier, albumin is also widely applied in pharmaceutical formulations;9-12 for example, the remarkable paclitaxelalbumin-stabilized nanoparticles (Abraxane®), and many newly emerging albumin-based diagnostic and therapeutic platforms.13-20 In addition, albumin solutions have been approved in the market for protein therapies since the 1940s.2 Therefore, quantitative analysis of albumin is necessary for the quality assurance of albumin-based pharmaceutical products. Classic colorimetric methods, such as sulfosalicylic acid test,21 Bradford test,22 Lowry assays,23 Biuret reagent test24 are not accurate and not selective to albumin protein in mixtures. Bromocresol purple (BCP) assays are better in selectivity but not good enough in sensitivity. 25 Instrumental techniques such as capillary electrophoresis and immunoassays. are more accurate but often expensive, troublesome and time-consuming.8 Fluorescence-based analytical methods enjoy advantages in terms of selectivity, sensitivity, simplicity, cost efficiency, and the in-time and on-site monitoring of the target.26-28 Organic fluorescent probes have been developed based on different working mechanisms, mainly including 1) aromatic amides/esters which can undergo enzymatic hydrolysis in the presence of


3


Page 4 of 35

albumins due to albumin’s pseudo-esterase activity (Fig. S1A, in supporting information),29-31 2) push-pull probes by modulating the twisted intramolecular charge transfer (TICT) process between the non-polar albumin binding sites and polar aqueous environment (Fig. S1B), 32-37 3) caged dyes or dark aggregates which can disassemble, disperse into albumin cavities and turn on the emission (Fig. S1C).38-47 So far, many probes are not validated and applied in complex biological fluids except for the commercialized Albumin Blue Fluorescent Assays.48-50 Nevertheless, these kits suffer from short shelf-lives due to their poor stability and a specific fluorescence reader is additionally required due to the small Stokes shifts. Aggregation-induced emission (AIE) has emerged as a reliable, effective and straightforward design strategy to obtain fluorescent probes for bio-macromolecules including proteins.51-53 When molecularly dissolved, AIE luminogens (AIEgens) are weakly or nonemissive due to their flexible molecular motion which favors the non-radiative decay. By binding with the protein target, their fluorescence can be switched on due to the restriction of intramolecular motions (RIM).52 For the albumin detection, the modular design of site-specific fluorescent probes was utilized (Fig. S2): an AIE-active core with periphery charged groups such as anionic sulfonate54-56 and carboxylate57 groups or cationic trialkyl-ammonium58,

59

and

pyridinium60 groups.53 When applied in biological fluids, the anionic ones usually displayed better performance than the cationic ones in terms of selectivity and robustness, due to the rich presence of anionic interferents such as lipopolysaccharides. Unfortunately, these anionic probes have not been optimized for clinical applications, because their binding affinities to albumin is usually low so that interferents such as fatty acids and bilirubin would compete for the same binding sites, leading to insensitive and unstable responses.


4

Page 5 of 35 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


Figure 1. Tetrazolate-tagged TPE probes for the albumin protein. (A) Structures of TPE-4TA, (Z)-TPE-2TA and (E)-TPE-2TA. (B) Schematic illustration of the fluorescence switch in aqueous solutions and when binding to albumin. (C) Comparison of acid bioisosteres in terms of the acidity (pKa) and the hydrophilicity (by the cLogP value). 61

In this work, tetrazolate-tagged tetraphenylethylene derivatives (TPE-TAs, Fig. 1A) are introduced as novel AIE-active anionic fluorescent albumin probes (Fig. 1B). As an isostere of the carboxylate and sulfonate group, the rarely explored tetrazolate group has a similar pKa but much-enhanced lipophilicity (Fig. 1C).62 The replacement leads to strengthened binding affinity towards albumin, which is similar to the pharmacological substitution effect of the tetrazole in medicinal chemistry. Moreover, the tetrazolate group is biocompatible and metabolism-resistant, making the probes suitable and durable in biological mixtures.63 This article concludes the synthesis, characterization of the probes’ performance in albumin detection, decipherment of detection mechanism, and the feasibility in the clinical application.


5


Page 6 of 35

EXPERIMENTAL General information. The probe TPE-4TA was donated by AIEgen Biotech Co. Ltd. Chemicals for the synthesis of other probes were purchased from Thermo-fisher and J&K Chemical Ltd. and used as received. Fatty acid-free HSA was purchased from Sigma-Aldrich in the form of lyophilized powder. Urine samples were collected from Southern Medical University with numeric identification due to patients’ personal data safety. UV-vis absorption spectra were recorded on a Varian Cary 50 UV-Visible spectrophotometer. DLS results were measured on a NanoBrook ZetaPALS Potential Analyzer. Fluorescence spectra were measured on a Fluorolog®-3 spectrofluorometer. Isothermal titration calorimetry experiments were performed on MicroCal VP-ITC instrument. Fluorescence intensities of urine samples in albumin assays were recorded by a TECAN infinite200 Pro microplate reader.

Figure 2. Preparation of (Z/E)-TPE-2TA

Preparation of (Z/E)-TPE-2TA (Fig. 2): (1) Synthesis of (Z/E)-TPE-2Br. n-butyllithium in hexanes (2.2 M, 10 mL) and compound 1 (3.36 g, 20 mmol) in anhydrous THF (50 mL) was added into a round-bottomed flask at 0 °C under Ar. After stirring for 1 h, a solution of compound 2 (5.40 g, 17 mmol) in THF was added


6

Page 7 of 35 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


and the mixture was warmed to room temperature and stirred for additional 10 h. The reaction was quenched with the addition of 10% sodium bicarbonate solution. The mixture was then extracted with dichloromethane (50 mL × 3) and the organic layers were dried with anhydrous magnesium sulfate. The solvent was evaporated to get the crude. The crude was next dissolved in 80 mL toluene. A catalytic amount of p-toluenesulfonic acid (0.68 g) was added and the mixture was refluxed for 12 h. After the mixture was cooled down, the toluene layer was washed with 10% aqueous sodium bicarbonate solution (25 mL × 2) and dried over anhydrous magnesium sulfate and evaporated to afford the crude. The crude was purified by flash column chromatography to give the (Z/E)-TPE-2Br as white solids (3.6 g, 46%). 1H NMR (400 MHz, CDCl3): δ 7.19-7.24 (m, 2H), 7.08-7.13 (m, 8H), 6.98-7.0 (m, 4H), 6.85-6.89 (m, 4H). 13C NMR (100 MHz, CDCl3): δ 142. 9, 142.3, 140.2, 132. 9, 131.2, 128.0, 127.8, 126.9, 120.7. (2) Synthesis of (Z/E)-TPE-2CN. The (Z/E)-TPE-2Br mixture (975 mg, 0.2 mmol), CuCN (560 mg), and DMF (10 mL) were added into a two-necked round bottom flask. The mixture was heated at reflux for 60 h under N2 and then suspended into 300 mL water. After ethylenediamine (10 mL) was added, the mixture was stirred at 100 °C for 1 h and was then filtered. The precipitated solid was extracted with dichloromethane (150 mL × 3). The combined organic phase was dried with anhydrous magnesium sulfate. After filtration and solvent evaporation, the residue was purified by silica gel column chromatography use hexane/dichloromethane (1/1, v/v) as eluent to give (E)-TPE-2CN (Rf = 0.27) in 38% yield and (Z)-TPE-2CN (Rf = 0.18) in 15% yield. For (E)-TPE-2CN, 1H NMR (400 MHz, CDCl3): δ 7.39 (dm, 4H, J=4.0 Hz), 7.18 (m, 6H), 7.10 (dm, 4H), 6.96 (dm, 4H). 13C NMR (100 MHz, CDCl3): δ 148.0, 141.7, 141.6, 131.9, 131.8, 128.5, 127.9, 118.9, 110.6. HRMS (MALDI-TOF), m/z calcd. for C28H18N2: 382.1470; found 382.1467. For (Z)-TPE-2CN, 1H NMR (400 MHz, CDCl3): δ 7.43 (d, 4H, J = 4.0 Hz), 7.15 (m,


7


Page 8 of 35

6H), 7.11 (d, 4H, J = 4 Hz), 6.96 (dm, 4H). 13C NMR (100 MHz, CDCl3): δ 147.1, 141.1, 140.7, 131.23, 131.19, 130.4, 127.5, 126.9, 118.0, 110.1. HRMS (MALDI-TOF), m/z calcd. for C28H18N2: 382.1470; found 382.1456. (3) Synthesis of (Z/E)-TPE-2TAH. Into a 25 mL flask were added sodium azide (100 mg), zinc bromide (180 mg) and water (0.5 mL). (Z)-TPE-2CN (100 mg) in 4.5 mL of Nmethylpyrrolidone was injected into the above solution. The mixture was stirred overnight at 150 °C. The mixture was acidified to pH~1 with aqueous HCl solution (3 M) and was stirred vigorously for 30 minutes. The organic mixture was extracted with ethyl acetate (20 mL × 2), washed with 3 M HCl (50 mL × 2) and concentrated to yield the crude solid. This crude was added into NaOH solution (0.25 M, 15 mL) and was then stirred vigorously for 1 h. Afterwards, the resulting suspension was filtered to remove the solid. The filtrate was washed with ethyl acetate (10 mL × 2) and acidified to pH 1 with 3 M HCl. The tetrazole product precipitated out upon stirring, which was again extracted into 20 mL ethyl acetate and the organic layer was separated. The aqueous layer was washed with ethyl acetate (20 mL × 2). The organic layers were combined, concentrated and dried under vacuum to yield the corresponding (E)-TPE-2TAH as a pale white solid (100 mg, 82%). 1H NMR (400 MHz, DMSO-d6): δ 7.82 (d, 4H, J=4.4 Hz), 7.2 (m, 10H), 7.06 (d, 4H, J=4.4 Hz).

13

C NMR (100 MHz, DMSO-d6): δ 145.7, 142.0, 140.6,

131.4, 130.6, 128.0, 127.0, 126.4. HRMS (MALDI-TOF), m/z calcd. for C28H20N8: 468.1811; found 468.1824. The (Z)-TPE-2TAH was prepared similarly to give a pale solid in 75% yield. 1H NMR (400 MHz, DMSO-d6): δ 7.84 (d, 4H, J = 4.0 Hz), 7.24 (d, 4H, J = 4.0 Hz), 7.17 (m, 6H), 7.03 (dm, 4H, J = 4.4 Hz). 13C NMR (100 MHz, DMSO-d6): δ 145.6, 142.1, 140.6, 131.5, 130.5, 127.8, 126.9, 126.5. HRMS (MALDI-TOF), m/z calcd. for C28H20N8: 468.1811; found 468.1798.


8

Page 9 of 35 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


Isothermal titration calorimetry (ITC) measurements. ITC experiments were conducted with a MicroCal VP-ITC instrument at 25℃. HSA solutions and the probe solutions were prepared in pH = 7.4 PBS. For a typical titration, a series of 10 µL aliquots of the probe solution was injected into the HSA solution within a duration of 20 s. The spacing time between two continuous injections was two minutes. The heat for each injection was determined by the integration of the peak area in the thermogram with respect to the reaction time. The binding constant values were derived by fitting the isotherm curves with the built-in Origin 7.0 software of the instrument. Molecular docking and MD simulation. The crystal structure of HSA was retrieved from the Protein Data Bank (PDB 2vue). The pdb files of the three ligands were generated in ChemBioDraw 3D. Both the macromolecule and the three ligands were imported in Autodock Tools.64 Polar hydrogen atoms were added and Gasteiger charges were assigned. All the ringstructures in the ligands were set rotatable to have full torsional freedom during modeling. To consider all possible sites for docking, Autodock Vina software was used to find 100 binding structures on the entire protein. We computed the root mean square deviation (RMSD) value between any two of the 100 docked protein-ligand conformations and cluster them with RMSD cut-off of 2.0 Å. The docked conformations were visualized by the VMD software.65 The binding energy and nearest contact residues sampling of HSA and ligand system were obtained by all-atom MD simulation conducted by Gromacs 5.1.3.66 The force field of ligands was built by AmbeTools18, the force field parameters of the ligands were fitted with the GAUSSIAN09 package67 in B3LYP/6-31g*, where the ESP charges68 are used all ligands molecules.

The protein system was dissolved in 25,874 TIP3P water

molecules69 and the system is placed in the center of a cubic box with a minimum distance of 1.2


9


Page 10 of 35

nm between the protein surface and box edges. 18 Na+ ions were added to make the whole system neutral, and the final system contained 86,824 atoms. The applied force field of protein is Amber03.70 We chose a cut-off of 1.0 nm for Lennard-Jones potential and short-range electrostatic interactions. Long-range electrostatic interactions were treated with the ParticleMesh Ewald (PME) method.71, 72 Chemical bonds were constrained by the LINCS algorithm. The energy minimization was conducted using the steepest descent minimization method, followed by 50 ps MD simulation with positions of protein heavy atoms restrained. The NVT equilibrium 100 ps MD simulation were collected at 300 K using velocity rescaling thermostat with time constant 0.1 ps.73 The NPT equilibrium 100 ps MD simulation were collected at 300 K used the velocity rescaling thermostat with time constant 0.1 ps and pressure coupling used ParrinelloRahman pressure bath with time constant 2.0 ps.74 Finally, we did 50 ns MD simulation for each complex at 300 K used the velocity rescaling thermostat with time constant 0.1 ps and pressure coupling used Parrinello-Rahman pressure bath with time constant 2.0 ps. The binding free energy was calculated by Molecular mechanics Poisson-Boltzmann surface area (MMPBSA) method using g_mmpbsa module.75 Binding energy contains van der Waal energy, electrostatic energy, polar solvation energy, and non-polar solvation energy. These terms were calculated using the structures from HSA-ligand complex MD simulations. Polar solvation energy was calculated by Linear Poisson-Boltzmann solver. The box for polar solvation energy calculation was created by using the extreme coordinates of the complex in each dimension, then expand it by 1.5-fold at each direction (cfac = 1.5). Ionic concentration of the system is 0.15 M (pconc = 0.15, nconc = 0.15) with 0.95 Å (prad = 0.95) and 1.81 Å (nrad = 1.81) radius of positive and negative ions, respectively. Vacuum, solute, and solvent dielectric constants were set as 1, 2, and 80, respectively. Non-polar solvation energy was calculated using solvent


10

Page 11 of 35 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


accessible surface area method, with probe radius = 1.4 Å, γ = 0.022678 kJ mol-1Å-2, and offset = 3.84982 kJ mol-1Å-2. The distribution of the nearest neighbor residues

was collected by

extracting 5000 frames at intervals of 10 ps. Albumin assay for urine samples. After collection, urine samples were stored in -20 °C refrigerator. The probe stock solution (50 μM) was prepared in PBS and was stored at 4°C in the dark. In a test, 150 μL of the probe stock solution was mixed with 25 μL of urine sample in a well of a 96-well plate. 150 μL of PBS and 25 μL of urine sample were mixed as the blank for deduction of urine auto-fluorescence. The TECAN infinite200 Pro microplate reader was used to measure the fluorescence intensities with excitation at 370 nm and emission at 490 nm with 10 seconds shaking before the first scan.

RESULTS AND DISCUSSION Fluorogenic detection of albumin. The probe TPE-4TA was prepared by the reported protocol.76 The probes (Z)-TPE-2TA and (E)-TPE-2TA were prepared efficiently by the synthetic routes (Fig. 2). These probes were initially obtained in the neutral form with the (1H)tetrazole groups. These probes can be deprotonated and fully dissolved at 100 mM concentration in pH = 7.4 PBS with a fluorescence quantum yield < 0.1%, in accord with the AIE characteristics. Of these probes, TPE-4TA showed the best solubility (> 0.01 M) in neutral-tobasic buffers. In PBS solutions of the three probes (5 µM), the addition of HSA induced a strong turn-on emission (Fig. 3). The emission maximums of TPE-4TA, (Z)-TPE-2TA, (E)-TPE-2TA with HSA were 490 nm, 476 nm, 476 nm, respectively, and no obvious red- or blue-shifts of absorption and emission could be observed (Fig. 3A/C/E, S3). DLS analysis showed the size of HSA particles in the absence and presence of probes did not change (Fig. S4), indicating the


11


Page 12 of 35

sensing process was a molecularly protein-ligand complexation which would not cause physical changes of target samples.

Figure 3. Fluorogenic detection of the HSA in pH 7.4 PBS. The fluorescence response and the corresponding peak intensity at λem against [HSA] (A/B, TPE-4TA; C/D, (Z)-TPE-2TA; E/F, (E)-TPE-2TA). [Probe] = 5 µM, λex = 370 nm. I0 equals the intensity of [HSA] = 0 mg/L.


12

Page 13 of 35 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


The responses of the three probes were examined by plotting the fluorescence intensity at the corresponding maximums versus the HSA gradient (Fig. 3B/D/F). Their limits of detection (LOD) were estimated in the range of 0.21~0.31 nM, which are among the best values in the reported fluorogenic albumin probes so far (Table S1). This sensitivity is more than enough for practical uses in clinical urine and blood samples. When the probes were used at 5 μM, the linear dynamic ranges (LDR, defined as R2 > 0.98) of the three probes extended to 230 mg/L, 80 mg/L, 130 mg/L, respectively. Thanks to the intriguing light-up AIE sensing characteristics and the good solubility of TPE-TA, the LDR for albumin quantification can be modulated to cover the range of albumins in biological samples by adjusting the concentration of the probe. For example, by setting up [TPE-4TA] = 125 μM, the LDR can be tuned as wide as 3000 mg/L (Fig. S5B). Above all, the three AIEgens are highly sensitive towards albumin with good linear responses and low enough detection limits, in which TPE-4TA exhibits the best performance (LOD = 0.21 nM, LDR = 0~230 mg/L) thus chosen as the representative in further studies (Table 1).

Table 1. Photophysical properties of the probes in the detection of HSAa

Probes

λex/ λem

LDRb

LODc

(nm)

(mg/L)

(nM)

TPE-4TA

360/490

0-230

0.21

(Z)-TPE-2TA

335/476

0-80

0.26

(E)-TPE-2TA

335/476

0-130

0.31

High-affinity site by ITC measurementsd KD (µM)

ΔH (kcal/mol)

0.25 ± 0.08 -26.2 ± 0.2 -e

-

0.29 ± 0.03 -24.7 ± 0.6

-TΔS (kcal/mol)

ΔG (kcal/mol)

1.4

-24.8

-

-

1.3

-23.4

a

pH 7.4 PBS, [Probe] = 5 µM, λex = 370 nm. bLDR: Linear dynamic range with R2 > 0.98. cLOD: Limit of detection. dEstimated by fitting with the sequential binding site model. KD: dissociation constant, calculated by 1/ (associate constant KA). eNot consistent results.


13


Page 14 of 35

Figure 4. (A) ITC calorimetric curves during the titration of HSA with serial injections of TPE4TA. [TPE-4TA] = 0.07 mM, [HSA] = 0.001 mM, at 25°C. (B) The fitting curve of integration of the peak area of each injection with sequential binding sites model. (C) The Job plot for determination of the binding stoichiometry of TPE-4TA and HSA, [TPE-4TA] + [HSA] kept constant as 2mM in pH 7.4 PBS. (D) The three probable binding positions of three probes in HSA. (E) Summary of the top 100 docked conformations in HSA for TPE-4TA (a), (Z)-TPE2TA (b), (E)-TPE-2TA (c) clustered at the three positions. For each cluster, the horizontal and vertical axes represent the average binding energy and the number of docked conformations, respectively. Blue bar = Cleft 1, red bar = Cleft 2, black bar = DIII.


14

Page 15 of 35 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


Detection mechanism.

To explore the detection mechanism, we investigated the

thermodynamic process of the binding between these probes and HSA (Fig. 4). Isothermal titration calorimetry (ITC) is a gold standard for characterization of biomolecular recognition and has been used to study the albumin-ligand binding. In the experiments, the ITC assays measure the raw heat release by progressively titrating the probes into the HSA solution (Fig. 4A, S7). For the albumin protein with multiple binding sites, it is difficult to interpret the ITC results because of the undetermined binding stoichiometry and fitting model.77 Thereby we performed the Job plot analysis (Fig. 4C), which suggested a binding stoichiometry of [Probe]/[HSA] = 2:1~3:1. We also carried out molecular docking analysis to evaluate the number of favorable binding sites. In the top dynamically favorable conformations of the protein-probe complex, the probe ligands are mainly distributed in three clusters of albumin protein, corresponding to three most stable binding positions (Fig. 4D). This agreed well with the Job plot results. Considering the general cooperativity between different binding sites of HSA,78, 79 the integrated heat release curves could be well fitted to the sequential isothermal binding model. For TPE-4TA, the curve can be successfully fitted with site number N = 3 (Fig. 4B). The docking results of (Z)-TPE-2TA and (E)-TPE-2TA also showed three clusters at the same site positions (Fig. 4E). However, the fitting qualities were comparably poorer when fitting with N = 3, which may be attributed to the allosteric effect of albumin since the protein structure was assumed unchanged in the computational process. As a result, N = 2 was applied in the sequential binding model (Fig. S6). The binding constants for the high-affinity sites (KD) were estimated to be 0.25 µM for TPE-4TA and 0.29 µM for (E)-TPE- 2TA (Table 1). These binding affinities are high enough among the reported albumin fluorescent probes (Table. S1). In the case of (Z)-TPE-2TA, ITC results were not consistent in repeated measurements, which was likely attributed to its relatively poor


15


Page 16 of 35

aqueous solubility. In these probe-HSA binding systems, (i) ΔH has a negative value and ΔS has a positive value, (ii) the absolute value of ΔH (-26.2 and -24.7 kcal/mol) is larger than TΔS (1.4 and 1.3 kcal/mol, respectively). That means the probe-albumin binding is an enthalpy-driven process. Since the enthalpically-favored interactions tend to be polar interactions such as hydrogen bonds, Coulomb forces, and van der Waals interactions, it agrees well with the property of the ionic tetrazolate group as both hydrogen-bonding donor and acceptor. To gain insights into the binding sites and to understand the favored binding interactions, we conducted the docking and all-atom MD simulation analysis on the probe-HSA complexes. The binding positions of the ligands labeled by cleft 1, cleft 2 and DIII are visualized in figure 4D with the case of TPE-4TA as an example. It reveals that the most favorable site (cleft 1 region) locates in the intersection of Domain I and Domain III. The cleft 2 region locates in the cleft of Domain I, Domain II and Domain III. The DIII region locates in Domain III. In general, the two clefts 1/2 sites have a less rigid environment, enabling more adapted conformations of the ligands (Fig. 4E). In comparison, the DIII site allows the ligands smaller degree of freedom. The statistical distribution of the nearest neighbor residues around the ligand was identified (Fig. 5), thereby the possible intermolecular interactions could be evaluated. In the cleft 1 region with the highest binding affinity, the dominated contacted residues, such as Asn and Lys (Fig. 5B), are polar amino acids, suggesting the driving force is dominated by polar interactions such as Coulomb forces and hydrogen bonds (Fig. 5A). This observation agrees well with the ITC study. For example, Arg186 serves as the proton donor to form hydrogen bonds with the nitrogen atoms on a tetrazolate ring. Arg114 provides electrostatic interactions with the tetrazolate anion. A similar binding mode is found in the cleft 2 (Fig. 5C), where polar contacting amino acids residues are


16

Page 17 of 35 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


dominant (Lys and Asn, Fig. 5D). Specifically, Lys444 interacts with a tetrazolate nitrogen by hydrogen bonds while Lys195, Lys436, and His440 contact other tetrazolate anions along with different directions via Coulomb forces. The docking situation of DIII resembles a key and lock model (Fig. 5E). Half of the ligand points out to the proteinsolvent boundary and the other half is buried in a polar cavity, where the dominated contacted residues are cationic Lys and His (Fig. 5F).


17


Page 18 of 35

Figure 5. Details of the best binding conformations with the lowest binding free energies in cleft 1 (A, B), cleft 2 (C, D) and DIII (E, F) sites. In each case, the TPE-4TA (Hydrogen atoms are omitted for clarity) and surrounding amino acid residues are shown in stick representation with a color scheme of green for carbon, blue for nitrogen, red for oxygen, and yellow for sulfur. Red dashed lines and orange double-dashed lines represent hydrogen bonds and Coulomb forces, respectively. Distribution of nearest neighbor residues of MD trajectories initiated from cleft 1 (B), cleft 2 (D) and DIII (F) sites, respectively. Since lysine residues (Lys) are dominant contacting units in all the three binding sites, we further explored the potential role of lysine in the fluorogenic detection. As a cationic amino acid, lysine has a four-carbon flexible alkyl chain and a protonatable amine head. Therefore, it is assumed to interact with and constrain the AIE-active ligand by whatever hydrogen bonding, electrostatic or hydrophobic interactions. The fluorescence of TPE-4TA could not be turned on by monomeric lysine molecules, whereas could be efficiently lighted up by a poly-L-lysine aqueous solution (MW. = 150,000-300,000) at the same condition (Fig. 6A). In this respect, the turn-on fluorescence should be largely attributed to the multivalent interactions between the tetrazolate and lysine residue as well as the rigidification effect of macromolecules, which restricted the molecular motion of the AIEgen and thereby deactivated the non-radiative decay (Fig. 6B). Since these kinds of lysine-rich polymers do not naturally exist in biological fluids, there is no worry about their interferences in practical applications.


18

Page 19 of 35 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


Figure 6. (A) Sensing response of TPE-4TA towards poly-L-lysine (MW = 150,000-300,000) and L-lysine. [Targets] = 1 mg/L, [TPE-4TA] = 10 μM in PBS. (B) The multivalent interactions of lysine-tetrazolate non-covalent interactions in the fluorogenic detection. Taking together, the tetrazolate-tagged AIE probes single-molecularly and non-covalently bind with albumin at specific sites. The driving forces mainly include polar interactions such as hydrogen-bonding and electrostatic interactions. The binding sites and the turn-on mechanism are quite different from many other fluorescent albumin probes, where hydrophobic interactions are commonly responsible for the fluorescence response.12-14,18 Thus these probes may provide an alternative platform to study the conformation transitions of albumin in processes such as folding /unfolding, denaturing and aggregation, in proteomic and pharmaceutical researches.53 Bioapplication. Since we aimed to apply the analysis in biomedical applications, the detection performance at different conditions was evaluated, including at varied pH values and in the presence of interference components of body fluids (Fig. 7). We selected TPE-4TA as the representative primarily because of its good water solubility and sensitivity. Firstly, we carried out the fluorogenic sensing of albumin in pH = 3~11 PBS solutions. At pH ≤ 4, the tetrazole group of a pKa 4~5 was mainly in the neutral form (TPE-4TAH), thereby the solubility decreased distinctly, leading to the aggregate formation and aggregation-induced emission (Fig.


19


Page 20 of 35

7A). At pH ≥ 5, the weakly acidic probe completely dissolved and became non-emissive. As a result, the fluorescence turn-on effect became manifest after binding with albumin. At pH > 9, a decline of fluorescence enhancement was observed which is likely due to the unfolding of albumin at high basic conditions.80 To be noticed, at 6≤ pH ≤9, the maximum fluorescence turnon response kept stable (Fig. 7B, red line), indicating the detection is robust in the physiological pH region. The potential interference from biomolecules was also evaluated. Figure 7C indicates that the fluorescence of TPE-4TA dramatically increases when binding with HSA and BSA, but no obvious response to proteins with isoelectric points ranging 1~10 (e.g. Hemoglobin, Pepsin, Lysozyme). The assay sensitivity is also not affected by the presence of the major chemical components in urines such as urea, uric acid, and creatine (Fig. 7D).


20

Page 21 of 35 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


Figure 7. Evaluation of the specific albumin sensing performance by TPE-4TA at different conditions. (A) Fluorescence of TPE-4TA in a pH gradient showing the probe remains in dark at pH>5 PBS, [TPE-4TA] = 5 µM. (B) Fluorescence intensity change of TPE-4TA (black line) and the corresponding HSA-TPE-4TA mixture at 490 nm in a pH gradient. [TPE-4TA] = 5 µM, [HSA] = 0.5 µM. (C) Fluorescence response of the TPE-4TA probe towards biomolecules in PBS. [TPE-4TA] = 5 µM, [Biomolecules] = 1 mg/mL. I0: intensity of the blank probe solution at 490 nm. (D) Interference of common components in urines on the fluorescence intensity of AIEgen-HSA complex at 490 nm. [TPE-4TA] = 5 µM, [HSA] = 5 µM, [urine component] = 10 mg/mL, in PBS buffer. I0: the intensity of the blank TPE-4TA solution.

To examine the feasibility of the fluorogenic albumin assay in body fluids, we collected patients’ urine samples whose albumin concentrations were determined by the turbidimetric inhibition immunoassay method and used as the references. The same urine samples were analyzed using TPE-4TA. Since some urine samples displayed autofluorescence at 440 nm (Fig. S7) which overlapped partially with the sensing signal at 490 nm, a blank for each urine sample group (without the addition of TPE-4TA) was adopted to correct the sample differences. In the data analysis, the fluorescence intensity of TPE-4TA/urine mixture at 490 nm (I) was firstly subtracted by the intensity of the corresponding urine blank group (I0), and the corrected values (I-I0) of all samples were then plotted against their known albumin concentrations (Fig. 8). The linear correlation coefficient of all the 15 samples showed to be 0.99, indicative of a good performance in the quantification of urinary albumin even within the microalbuminuria range. Therefore, the fluorescence method was proved to be viable.


21


Page 22 of 35

Figure 8. Plot of albumin concentration in clinical urines samples by a turbidimetric inhibition immunoassay method (X-axis) versus the corresponding fluorescence intensity at 490 nm by the fluorescent assay using TPE-4TA (Y-axis). I0 equals to the fluorescence intensity at 490 nm of urine blanks in the absence of the probe. Five repeats for each sample.

CONCLUSION In summary, we reported a general design of fluorescent probes for the specific and quantitative analysis of albumins. These probes consist of an AIE-active scaffold and multiple tetrazolate moieties on the periphery. They specifically bind with the albumin protein and induce a sensitive turn-on fluorescence. Mechanistic investigations by ITC, molecular docking, and MD simulation indicate that the main driving forces to trap the probes in the albumin are enthalpically-favored polar interactions including hydrogen bonds and Columbic forces. Particularly, we highlighted the multivalent interactions between cationic amino acid (e.g. lysine) and the tetrazolate groups. In comparison with the commonly used anionic groups such as sulfonates and carboxylates, the tetrazolate functionality exhibits a higher binding affinity with


22

Page 23 of 35 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


albumin, higher resistance to biodegradation, making the current fluorogenic albumin sensing an attractive alternative strategy. As an example, the TPE-4TA was proved to be a sensitive, selective light-up probe with high binding affinities (KD = 0.25 µM), a good linear dynamic range (0.02~2500 mg/L) covering the biological albumin concentration and a LOD down to 0.21 nM. The feasibility of the protein assay was furthermore verified using patients’ urine samples for clinic diagnosis, resulting in a satisfactory accuracy and linearity. In addition, since the binding sites of these probes are unique, leaving the major hydrophobic sites in the albumin unoccupied, they are promising to be applied in the pharmacokinetics analysis of drug-albumin complexes, and in the development of new drug-albumin formulations for improved drug delivery. Studies in this direction are currently being investigated in our lab.

ASSOCIATED CONTENT Supporting Information. UV-vis characterizations; Autofluorescence in some urine samples; Fluorescence lifetime measurements; TEM images of the HSA-probe complexes; and supplementary computational results can be found in the Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org AUTHOR INFORMATION Corresponding Author *[email protected] *[email protected]


23


Page 24 of 35

Notes The authors declare no competing financial interest. ACKNOWLEDGMENT We are grateful for financial support from the National Science Foundation of China (21788102, 21490570, 21490574, and 81501591), the Research Grants of Council of Hong Kong (AHKUST605/16 and C6009-17G), the Innovation of Technology Commission (ITCCNERC14SC01 and ITS/254/17), the National Key Research and Development Program of China (2018YFE0190200), and the AIEgen Biotech Co. Ltd. REFERENCES (1) Fanali, G.; di Masi, A.; Trezza, V.; Marino, M.; Fasano, M.; Ascenzi, P. Human Serum Albumin: From Bench to Bedside. Mol. Aspects Med. 2012, 33, 209-290. (2) Farrugia, A. Albumin Usage in Clinical Medicine: Tradition or Therapeutic? Transfus. Med. Rev. 2010, 24, 53-63. (3) Gatta, A.; Verardo, A.; Bolognesi, M. Hypoalbuminemia. Intern. Emerg. Med. 2012, 7 Suppl 3, S193-199. (4) Akirov, A.; Masri-Iraqi, H.; Atamna, A.; Shimon, I. Low Albumin Levels Are Associated with Mortality Risk in Hospitalized Patients. Am. J. Med. 2017, 130(12),1465-e11. (5) Clavant, S. P.; Osicka, T. M.; Comper, W. D. Albuminuria: Its Importance in Disease Detection. Lab. Med. 2007, 38, 35-38. (6) Farrugia, A. Albumin Usage in Clinical Medicine: Tradition or Therapeutic? Transfus. Med. Rev. 2010, 24, 53-63.


24

Page 25 of 35 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


(7) Levey, A. S.; Becker, C.; Inker, L. A. Glomerular Filtration Rate and Albuminuria for Detection and Staging of Acute and Chronic Kidney Disease in Adults: A Systematic Review. JAMA. 2015, 313, 837-846. (8) Kumar, D.; Banerjee, D. Methods of Albumin Estimation in Clinical Biochemistry: Past, Present, and Future. Clin. Chim. Acta. 2017, 469, 150-160. (9) Larsen, M. T.; Kuhlmann, M.; Hvam, M. L.; Howard, K. A. Albumin-Based Drug Delivery: Harnessing Nature to Cure Disease. Mol Cell Ther. 2016, 4, 3. (10) An, F. F.; Zhang, X. H. Strategies for Preparing Albumin-Based Nanoparticles for Multifunctional Bioimaging and Drug Delivery. Theranostics. 2017, 7, 3667-3689. (11) Hoogenboezem, E. N.; Duvall, C. L. Harnessing Albumin as a Carrier for Cancer Therapies. Adv Drug Deliv Rev. 2018, 130, 73-89. (12) Tan, Y. L.; Ho, H. K. Navigating Albumin-Based Nanoparticles through Various Drug Delivery Routes. Drug Discov. Today. 2018, 23, 1108-1114. (13) Xu, J.; Wang, J.; Luft, J. C.; Tian, S.; Owens, G., Jr.; Pandya, A. A.; Berglund, P.; Pohlhaus, P.; Maynor, B. W.; Smith, J.; Hubby, B.; Napier, M. E.; DeSimone, J. M. Rendering Protein-Based Particles Transiently Insoluble for Therapeutic Applications. J. Am. Chem. Soc. 2012, 134, 8774-8777. (14) Bergonzi, M. C.; Guccione, C.; Grossi, C.; Piazzini, V.; Torracchi, A.; Luccarini, I.; Casamenti, F.; Bilia, A. R. Albumin Nanoparticles for Brain Delivery: A Comparison of Chemical Versus Thermal Methods and in Vivo Behavior. Chem. Med. Chem. 2016, 11, 18401849.


25


Page 26 of 35

(15) Lacroix, A.; Edwardson, T. G. W.; Hancock, M. A.; Dore, M. D.; Sleiman, H. F. Development of DNA Nanostructures for High-Affinity Binding to Human Serum Albumin. J. Am. Chem. Soc. 2017, 139, 7355-7362. (16) Zhu, G.; Lynn, G. M.; Jacobson, O.; Chen, K.; Liu, Y.; Zhang, H.; Ma, Y.; Zhang, F.; Tian, R.; Ni, Q.; Cheng, S.; Wang, Z.; Lu, N.; Yung, B. C.; Wang, Z.; Lang, L.; Fu, X.; Jin, A.; Weiss, I. D.; Vishwasrao, H.; Niu, G.; Shroff, H.; Klinman, D. M.; Seder, R. A.; Chen, X. Albumin/Vaccine Nanocomplexes That Assemble in Vivo for Combination Cancer Immunotherapy. Nat. Commun. 2017, 8, 1954. (17) Jin, C.; Zhang, H.; Zou, J.; Liu, Y.; Zhang, L.; Li, F.; Wang, R.; Xuan, W.; Ye, M.; Tan, W. Floxuridine Homomeric Oligonucleotides "Hitchhike" with Albumin in Situ for Cancer Chemotherapy. Angew. Chem. Int. Ed. Engl. 2018, 57, 8994-8997. (18) Popova, M.; Soboleva, T.; Ayad, S.; Benninghoff, A. D.; Berreau, L. M. Visible-LightActivated Quinolone Carbon-Monoxide-Releasing Molecule: Prodrug and Albumin-Assisted Delivery Enables Anticancer and Potent Anti-Inflammatory Effects. J. Am. Chem. Soc. 2018, 140, 9721-9729. (19) Gao, S.; Wei, G.; Zhang, S.; Zheng, B.; Xu, J.; Chen, G.; Li, M.; Song, S.; Fu, W.; Xiao, Z.; Lu, W. Albumin Tailoring Fluorescence and Photothermal Conversion Effect of nearInfrared-Ii Fluorophore with Aggregation-Induced Emission Characteristics. Nat. Commun. 2019, 10, 2206.


26

Page 27 of 35 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


(20) Wang, S.; Hu, F.; Pan, Y.; Ng, L. G.; Liu, B. Bright Aiegen – Protein Hybrid Nanocomposite for Deep and High‐Resolution in Vivo Two‐Photon Brain Imaging. Adv. Funct. Mater. 2019, 1902717. (21) Grauer, G. F. Proteinuria: Measurement and Interpretation. Top. Companion Anim. Med. 2011, 26, 121-127. (22) Bradford, M. M. A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Anal. Biochem. 1976, 72, 248-254. (23) Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. Protein Measurement with the Folin Phenol Reagent. J. Biol. Chem. 1951, 193, 265-275. (24) Eckfeldt, J. H.; Kershaw, M. J.; Dahl, I. I. Direct Analysis for Urinary Protein with Biuret Reagent, with Use of Urine Ultrafiltrate Blanking - Comparison with a Manual Biuret Method Involving Trichloroacetic-Acid Precipitation. Clin. Chem. 1984, 30, 443-446. (25) Affonso, A.; Lasky, F. D. Bromocresol Purple Dye-Binding Method for the Estimation of Serum-Albumin Adapted to the Sma-12/60. Clin. Biochem. 1985, 18, 285-289. (26) Li, X.; Gao, X.; Shi, W.; Ma, H. Design Strategies for Water-Soluble Small Molecular Chromogenic and Fluorogenic Probes. Chem. Rev. 2014, 114, 590-659. (27) Klymchenko, A. S. Solvatochromic and Fluorogenic Dyes as Environment-Sensitive Probes: Design and Biological Applications. Acc. Chem. Res. 2017, 50, 366-375.


27


Page 28 of 35

(28) Zhang, J.; Chai, X.; He, X. P.; Kim, H. J.; Yoon, J.; Tian, H. Fluorogenic Probes for Disease-Relevant Enzymes. Chem. Soc. Rev. 2019, 48, 683-722. (29) Zeng, X.; Zhang, X.; Zhu, B.; Jia, H.; Li, Y.; Xue, J. A Colorimetric and Ratiometric Fluorescent Probe for Quantification of Bovine Serum Albumin. Analyst. 2011, 136, 4008-4012. (30) Ge, G. B.; Feng, L.; Jin, Q.; Wang, Y. R.; Liu, Z. M.; Zhu, X. Y.; Wang, P.; Hou, J.; Cui, J. N.; Yang, L. A Novel Substrate-Inspired Fluorescent Probe to Monitor Native Albumin in Human Plasma and Living Cells. Anal. Chim. Acta. 2017, 989, 71-79. (31) Wang, Y.-R.; Feng, L.; Xu, L.; Hou, J.; Jin, Q.; Zhou, N.; Lin, Y.; Cui, J.-N.; Ge, G.-B. An Ultrasensitive and Conformation Sensitive Fluorescent Probe for Sensing Human Albumin in Complex Biological Samples. Sensors Actuators B: Chem. 2017, 245, 923-931. (32) Li, H.; Yao, Q.; Fan, J.; Du, J.; Wang, J.; Peng, X. An Nir Fluorescent Probe of Uric Hsa for Renal Diseases Warning. Dyes and Pigments. 2016, 133, 79-85. (33) Reja, S. I.; Khan, I. A.; Bhalla, V.; Kumar, M. A Tict Based Nir-Fluorescent Probe for Human Serum Albumin: A Pre-Clinical Diagnosis in Blood Serum. Chem. Commun. (Camb.). 2016, 52, 1182-1185. (34) Liu, C.; Yang, W.; Shen, P.; Gao, Q.; Du, J.; Yang, C. A Dual-Modal Red-Emitting Fluorescence Probe for Proteins Based on Modulation of Aie or Tict State. Heteroat. Chem. 2017, 28, e21371. (35) Li, P.; Wang, Y.; Zhang, S.; Xu, L.; Wang, G.; Cui, J. An Ultrasensitive Rapid-Response Fluorescent Probe for Highly Selective Detection of Hsa. Tetrahedron Lett. 2018, 59, 1390-1393.


28

Page 29 of 35 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


(36) Luo, Z.; Liu, B.; Zhu, K.; Huang, Y.; Pan, C.; Wang, B.; Wang, L. An EnvironmentSensitive Fluorescent Probe for Quantification of Human Serum Albumin: Design, Sensing Mechanism, and Its Application in Clinical Diagnosis of Hypoalbuminemia. Dyes and Pigments. 2018, 152, 60-66. (37) Samanta, S.; Halder, S.; Das, G. Twisted-Intramolecular-Charge-Transfer-Based Turn-on Fluorogenic Nanoprobe for Real-Time Detection of Serum Albumin in Physiological Conditions. Anal. Chem. 2018, 90, 7561-7568. (38) Jisha, V. S.; Arun, K. T.; Hariharan, M.; Ramaiah, D. Site-Selective Binding and Dual Mode Recognition of Serum Albumin by a Squaraine Dye. J. Am. Chem. Soc. 2006, 128, 60246025. (39) Suzuki, Y.; Yokoyama, K. A Protein-Responsive Chromophore Based on Squaraine and Its Application to Visual Protein Detection on a Gel for Sds-Page. Angew. Chem. Int. Ed. Engl. 2007, 46, 4097-4099. (40) Xu, Y.; Li, Z.; Malkovskiy, A.; Sun, S.; Pang, Y. Aggregation Control of Squaraines and Their Use as near-Infrared Fluorescent Sensors for Protein. J. Phys. Chem. B. 2010, 114, 85748580. (41) Pisoni, D. d. S.; de Abreu, M. P.; Petzhold, C. L.; Rodembusch, F. S.; Campo, L. F. Synthesis, Photophysical Study and Bsa Association of Water-Insoluble Squaraine Dyes. J. Photochem. Photobiol. A: Chem. 2013, 252, 77-83.


29


Page 30 of 35

(42) Anees, P.; Sreejith, S.; Ajayaghosh, A. Self-Assembled near-Infrared Dye Nanoparticles as a Selective Protein Sensor by Activation of a Dormant Fluorophore. J. Am. Chem. Soc. 2014, 136, 13233-13239. (43) Gao, F. P.; Lin, Y. X.; Li, L. L.; Liu, Y.; Mayerhoffer, U.; Spenst, P.; Su, J. G.; Li, J. Y.; Wurthner, F.; Wang, H. Supramolecular Adducts of Squaraine and Protein for Noninvasive Tumor Imaging and Photothermal Therapy in Vivo. Biomaterials. 2014, 35, 1004-1014. (44) Sun, W.; Guo, S.; Hu, C.; Fan, J.; Peng, X. Recent Development of Chemosensors Based on Cyanine Platforms. Chem. Rev. 2016, 116, 7768-7817. (45) Yu, Y.; Huang, Y.; Hu, F.; Jin, Y.; Zhang, G.; Zhang, D.; Zhao, R. Self-Assembled Nanostructures Based on Activatable Red Fluorescent Dye for Site-Specific Protein Probing and Conformational Transition Detection. Anal. Chem. 2016, 88, 6374-6381. (46) Gao, T.; Yang, S.; Cao, X.; Dong, J.; Zhao, N.; Ge, P.; Zeng, W.; Cheng, Z. Smart SelfAssembled Organic Nanoprobe for Protein-Specific Detection: Design, Synthesis, Application, and Mechanism Studies. Anal. Chem. 2017, 89, 10085-10093. (47) Guo, Y.; Chen, Y.; Zhu, X.; Pan, Z.; Zhang, X.; Wang, J.; Fu, N. Self-Assembled Nanosensor Based on Squaraine Dye for Specific Recognition and Detection of Human Serum Albumin. Sensors Actuators B: Chem. 2018, 255, 977-985. (48) Kessler, M. A.; Hubmann, M. R.; Dremel, B. A.; Wolfbeis, O. S. Nonimmunological Assay of Urinary Albumin Based on Laser-Induced Fluorescence. Clin. Chem. 1992, 38, 20892092.


30

Page 31 of 35 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


(49) Kessler, M. A.; Meinitzer, A.; Petek, W.; Wolfbeis, O. S. Microalbuminuria and Borderline-Increase Albumin Excretion Determined with a Centrifugal Analyzer and the Albumin Blue 580 Fluorescence Assay. Clin. Chem. 1997, 43, 996-1002. (50) Kessler, M. A.; Meinitzer, A.; Wolfbeis, O. S. Albumin Blue 580 Fluorescence Assay for Albumin. Anal. Biochem. 1997, 248, 180-182. (51) Kwok, R. T. K.; Leung, C. W. T.; Lam, J. W. Y.; Tang, B. Z. Biosensing by Luminogens with Aggregation-Induced Emission Characteristics. Chem. Soc. Rev. 2015, 44, 4228-4238. (52) Mei, J.; Leung, N. L. C.; Kwok, R. T. K.; Lam, J. W. Y.; Tang, B. Z. AggregationInduced Emission: Together We Shine, United We Soar! Chem. Rev. 2015, 115, 11718-11940. (53) Xie, S.; Wong, A. Y. H.; Chen, S.; Tang, B. Z. Fluorogenic Detection and Characterization of Proteins by Aggregation-Induced Emission Methods. Chemistry (Easton). 2019, 25, 5824-5847. (54) Hong, Y. N.; Feng, C.; Yu, Y.; Liu, J. Z.; Lam, J. W. Y.; Luo, K. Q.; Tang, B. Z. Quantitation, Visualization, and Monitoring of Conformational Transitions of Human Serum Albumin by a Tetraphenylethene Derivative with Aggregation-Induced Emission Characteristics. Anal. Chem. 2010, 82, 7035-7043. (55) Wang, Z.; Ma, K.; Xu, B.; Li, X.; Tian, W. A Highly Sensitive “Turn-on” Fluorescent Probe for Bovine Serum Albumin Protein Detection and Quantification Based on Aie-Active Distyrylanthracene Derivative. Sci. China. Chem. 2013, 56, 1234-1238.


31


Page 32 of 35

(56) Li, J.; Wu, J.; Cui, F.; Zhao, X.; Li, Y.; Lin, Y.; Li, Y.; Hu, J.; Ju, Y. A Dual Functional Fluorescent Sensor for Human Serum Albumin and Chitosan. Sensors Actuators B: Chem. 2017, 243, 831-837. (57) Li, W.; Chen, D.; Wang, H.; Luo, S.; Dong, L.; Zhang, Y.; Shi, J.; Tong, B.; Dong, Y. Quantitation of Albumin in Serum Using "Turn-on" Fluorescent Probe with AggregationEnhanced Emission Characteristics. ACS Appl. Mater. Interfaces. 2015, 7, 26094-26100. (58) Tong, H.; Hong, Y.; Dong, Y.; Haussler, M.; Lam, J. W.; Li, Z.; Guo, Z.; Tang, B. Z. Fluorescent "Light-up" Bioprobes Based on Tetraphenylethylene Derivatives with AggregationInduced Emission Characteristics. Chem. Commun. (Camb.). 2006, 35, 3705-3707. (59) Xu, X.; Huang, J.; Li, J.; Yan, J.; Qin, J.; Li, Z. A Graphene Oxide-Based Aie Biosensor with High Selectivity toward Bovine Serum Albumin. Chem. Commun. (Camb.). 2011, 47, 12385-7. (60) Tong, J.; Hu, T.; Qin, A.; Sun, J. Z.; Tang, B. Z. Deciphering the Binding Behaviours of Bsa Using Ionic Aie-Active Fluorescent Probes. Faraday Discuss. 2017, 196, 285-303. (61)

Cambridge

MedChem

Consulting.

https://www.cambridgemedchemconsulting.com/resources/bioisoteres/acid_bioisosteres.html. (62) Patani, G. A.; LaVoie, E. J. Bioisosterism: A Rational Approach in Drug Design. Chem. Rev. 1996, 96, 3147-3176. (63) Malik, M. A.; Wani, M. Y.; Al-Thabaiti, S. A.; Shiekh, R. A. Tetrazoles as Carboxylic Acid Isosteres: Chemistry and Biology. J. Incl. Phenom. Macrocycl. Chem. 2013, 78, 15-37.


32

Page 33 of 35 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


(64) Trott, O.; Olson, A. J. Software News and Update Autodock Vina: Improving the Speed and Accuracy of Docking with a New Scoring Function, Efficient Optimization, and Multithreading. J. Comput. Chem. 2010, 31, 455-461. (65) Humphrey, W.; Dalke, A.; Schulten, K. Vmd: Visual Molecular Dynamics. J. Mol. Graph. Model. 1996, 14, 33-38. (66) Abraham, M. J.; Murtola, T.; Schulz, R.; Páll, S.; Smith, J. C.; Hess, B.; Lindahl, E. Gromacs: High Performance Molecular Simulations through Multi-Level Parallelism from Laptops to Supercomputers. SoftwareX. 2015, 1-2, 19-25. (67) Gaussian 09 (Gaussian, Inc., Wallingford CT, 2009). http://gaussian.com/glossary/g09/. (68) Singh, U. C.; Kollman, P. A. An Approach to Computing Electrostatic Charges for Molecules. J. Comput. Chem. 1984, 5, 129-145. (69) Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R. W.; Klein, M. L. Comparison of Simple Potential Functions for Simulating Liquid Water. J. Chem. Phys. 1983, 79, 926-935. (70) Sanborn, N. E.; Hayre, N. R.; Singh, R. R.; Cox, D. L. All-Atom Molecular Dynamics Simulations of the Projection Domain of the Intrinsically Disordered Htau40 Protein. arXiv preprint arXiv:1610.09696. 2016. (71) Darden, T.; York, D.; Pedersen, L. Particle Mesh Ewald - an N.Log(N) Method for Ewald Sums in Large Systems. J. Chem. Phys. 1993, 98, 10089-10092. (72) Essmann, U.; Perera, L.; Berkowitz, M. L.; Darden, T.; Lee, H.; Pedersen, L. G. A Smooth Particle Mesh Ewald Method. J. Chem. Phys. 1995, 103, 8577-8593.


33


Page 34 of 35

(73) Bussi, G.; Donadio, D.; Parrinello, M. Canonical Sampling through Velocity Rescaling. J. Chem. Phys. 2007, 126, 014101. (74) Berendsen, H. J. C.; Postma, J. P. M.; Vangunsteren, W. F.; Dinola, A.; Haak, J. R. Molecular-Dynamics with Coupling to an External Bath. J. Chem. Phys. 1984, 81, 3684-3690. (75) Kumari, R.; Kumar, R.; Lynn, A.; Consort, O. S. D. D. G_Mmpbsa-a Gromacs Tool for High-Throughput Mm-Pbsa Calculations. J. Chem. Inf. Model. 2014, 54, 1951-1962. (76) Xie, S.; Wong, A. Y. H.; Kwok, R. T. K.; Li, Y.; Su, H. F.; Lam, J. W. Y.; Chen, S. J.; Tang, B. Z. Fluorogenic Ag+-Tetrazolate Aggregation Enables Efficient Fluorescent Biological Silver Staining. Angewandte Chemie-International Edition. 2018, 57, 5750-5753. (77) Ghai, R.; Falconer, R. J.; Collins, B. M. Applications of Isothermal Titration Calorimetry in Pure and Applied Research--Survey of the Literature from 2010. J. Mol. Recognit. 2012, 25, 32-52. (78) Ascenzi, P.; Bocedi, A.; Notari, S.; Fanali, G.; Fesce, R.; Fasano, M. Allosteric Modulation of Drug Binding to Human Serum Albumin. Mini Rev. Med. Chem. 2006, 6, 483-489. (79) Ascenzi, P.; Fasano, M. Allostery in a Monomeric Protein: The Case of Human Serum Albumin. Biophys. Chem. 2010, 148, 16-22. (80) Varga, N.; Hornok, V.; Sebok, D.; Dekany, I. Comprehensive Study on the Structure of the Bsa from Extended-to Aged Form in Wide (2-12) Ph Range. Int. J. Biol. Macromol. 2016, 88, 51-58.


34

Page 35 of 35 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


TOC:


35

Specific and Quantitative Detection of Albumin in Biological Fluids by

Recommend Documents