Emissive H-Aggregates of an Ultrafast Molecular Rotor: A Promising

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Emissive H-aggregates of an Ultrafast Molecular Rotor: A Promising Platform for Sensing Heparin Niyati H. Mudliar, and Prabhat K. Singh ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b12729 • Publication Date (Web): 10 Nov 2016 Downloaded from http://pubs.acs.org on November 14, 2016

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ACS Applied Materials & Interfaces

Emissive H-aggregates of an Ultrafast Molecular Rotor: A Promising Platform for Sensing Heparin Niyati H. Mudliar and Prabhat K. Singh* Radiation & Photochemistry Division, Bhabha Atomic Research Centre, Mumbai 400 085, INDIA. ABSTRACT: Constructing "turn on" fluorescent probes for Heparin, a most widely used anticoagulant in clinics, from commercially available materials is of great importance, but remains challenging. Here, we report the formation of a rarely observed emissive H-aggregate of an ultrafast molecular rotor dye, Thioflavin-T, in the presence of Heparin, which provides an excellent platform for simple, economic and rapid fluorescence turn-on sensing of Heparin. Generally, Haggregates are considered as serious problem in the field of bio-molecular sensing, owing to their poorly emissive nature resulting from excitonic interaction. To the best of our knowledge, this is the first report, where contrastingly, the turn-on emission from the H-aggregates has been utilized in the bio-molecule sensing scheme, and enables a very efficient and selective detection of a vital bio-molecule and a drug with its extensive medical applications, i.e., Heparin. Our sensor system offers several advantages including, emission in the biologically advantageous red-region, dual sensing i.e., both by fluorimetry and colorimetry, and most importantly constructed from in-expensive commercially available dye molecule, which is expected to impart a large impact on the sensing field of Heparin. Our system displays good performance in complex biological media of serum samples. The novel Thioflavin-T aggregate emission could be also used to probe the interaction of Heparin with its only clinically approved antidote, Protamine. Keywords: Thioflavin-T, Ultrafast molecular rotor, Heparin sensor, H-aggregates, Protamine, Fluorescence

Heparin belongs to the family of glycosaminoglycan with a very high degree of sulfation, and carries the highest negative charge density known for any bio1 macromolecule. Heparin is the second most widely used natural drug, and clinically, Heparin is widely used as an anticoagulant during surgery to prevent thrombosis, and in the treatment of thrombotic diseases. However, Heparin overdose induces certain complications such as hemorrhages, Heparin-induced 2 3 thrombocytopenia, hyperkalemia, and osteoporosis. Thus, it is very essential to monitor the level of Heparin during and after surgery, and control the amount of Heparin for anticoagulant therapy to avoid complications induced by Heparin-overdose. Hence, intensive efforts have been dedicated to develop sensitive and simple assays for detecting Heparin. Various methods have been developed for detecting 4-9 Heparin, based on techniques such as fluorimetry,

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colorimetry, capillary electrophoresis, ion- ex13 change chromatography, and electrochemical meth14 ods. Among them, fluorometric methods have attracted significant attention due to its high sensitivity and simple read-out, and its extensive usage in medical 4, 6, 9, 15-17 applications. Recently, small molecular probes, 1, 7-8 conjugated polymers, fluorophore labeled biomole18 19 cules, and quantum dots have been utilized for Heparin detection using fluorimetry. However, most of these assays operate through fluorescence quenching (turn-off) upon interaction with Heparin, which is often unfavorable for detection of Heparin, owing to large environment effects and low sensitivity. For example, a phenylboronic acid and ammonium group based synthetic receptor was reported to display a high affinity and selectivity for Heparin, but the receptor showed fluorescence quenching upon interaction with 9 Heparin. On the other hand, a peptide based synthetic sensor was reported to display an increase in fluorescence intensity (turn-on) on interaction with Hepa20 rin. However, the turn-on assay was limited by the detection window (0–3.4 µM), which is out of the clinical range. Despite the development of few turn-on sensing approaches for Heparin, one of the major factors limiting the widespread use of many Heparin sensors is the complicated and time consuming multistep synthesis and purification associated with them. Thus, for Heparin detection, much attention still focuses on commercially available molecules such as methylene 21 22 blue, azure A, which exhibits only a colorimetric response to the Heparin, although fluorescence is a more sensitive technique. Therefore, a Heparin sensor from a commercially available molecule exhibiting response through turn-on emission is highly desirable 1 but remained challenging. Herein, we report the formation of a rarely observed emissive H-aggregate of an ultrafast molecular rotor dye, Thioflavin-T (ThT) in the presence of Heparin, which provides an excellent platform for sensing Heparin. It is important to note that H-aggregates are considered as a serious problem in the field of biomolecule sensing, owing to their weakly emissive nature resulting from excitonic interaction. To the best of our knowledge, this is probably the first report, where

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contrastingly, the turn-on emission from the Haggregates of the dye has been utilized in the biomolecule sensing scheme, and enables very efficient and selective detection of a vital bio-molecule and a drug with extensive medical applications, i.e., Heparin. The reported sensor has the advantages of being label free and most importantly, involves the use of a widely available commercial dye, and still displays comparable sensitivity and very good selectivity, as compared to many of the synthetically demanding Heparin sensing systems reported. Figure 1A presents the emission spectra of ThT in the presence of Heparin. Upon addition of Heparin to an aqueous solution of ThT, a largely red-shifted emission band appears at ~560 nm, which is in sharp contrast to a very weak emission band for ThT at 490 nm (Figure 1A). Further addition of Heparin causes a gradual increase in the emission intensity of the red-shifted emission band and leads to a large emission enhancement of ~45 times at the saturation condition. ThT is virtually non-fluorescent in aqueous solution owing to 23 its ultrafast molecular rotor property. Ultrafast Molecular rotors are characterized by their ability to twist around a single bond (Central C-C bond in ThT, 23-24 Scheme S1†) This twisting motion constitutes a 12 -1 very efficient non-radiative process (knr ~10 S ) for ThT, which leads to a quick dissipation of excitation 23 energy, and leads to a very low emission yield. However, this twisting motion is strongly affected by the rigidity of its micro-environment, which in turn immensely influences its emission yield. Owing to the immense sensitivity of the emission yield of ThT towards the rigidity in its surrounding environment, ThT is employed as a sensor for micro-viscosity in several chemical and biological environments, including amy23, 25 loid fibrils.

Figure 1. (A) Steady-state fluorescence spectrum (λex = 400 nm) of ThT (24 μM) at varying concentration of Heparin. The emission spectrum of ThT in water is represented by the dashed line. Inset: Variation in the emission intensity of ThT with the increasing concentration of Heparin (B) Groundstate absorption spectra of ThT (24 μM) at varying concentration of Heparin. The dashed line represents the absorption spectrum of ThT in water. Inset: Variation in the absorbance ratio (OD393/OD413) of ThT with the increasing concentration of Heparin.

Thus, although it can be argued that the increase in ThT emission, in the present case, is due to confine-

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ment of its monomeric form upon binding to Heparin, but according to the previous literature, the monomeric ThT emits at 490 nm. Thus, the presence of a strikingly large red-shifted emission band at 560 nm, in the present case, suggests that the ThT molecules associated with Heparin are not the usual monomer form, but rather it is present in a different molecular form. Very recently, a similar red-shifted emission band has been observed for ThT in the presence of highly negatively charged sulphated β-CD, and has been attributed to H26 aggregates of ThT. Since Heparin is known to bear the highest negative charge density for any known biological macromolecule, so it is very likely that Heparin will induce the formation of ThT aggregates. The proposition that the red-shifted emission originates from H-aggregates, is further confirmed by excitation spectra (see discussion later). In the aggregated state, 26 the torsional relaxation of ThT is strongly hindered which leads to a turn-on emission in the presence of Heparin. The emission intensity increases in a linear fashion with the increasing concentrations of Heparin in a concentration range of 0 – 15µM, and the linear 2 regression is I560= 58+ 184.8 x [Heparin/μM] (R = 0.996). The detection limit (LOD) of Heparin based on 3.3σ/s was calculated to be 18 nM, where σ represents the standard deviation of 10 blank measurements (ThT in aqueous solution), and s represents the slope of the fluorescence intensity (at 560 nm) with Heparin concentration (Figure 1A, inset). Importantly, Haggregates of ThT formed on Heparin template displays emission in long-wavelength region, which offers the advantage of being free from the intrinsic fluorescence by other biological contaminants such as DNA, RNA and proteins. To further confirm about the molecular form of species responsible for the origin of this red-shifted emission, and to explore the possibility of detection of Heparin through colorimetry measurements, absorption spectra of ThT, in the presence of Heparin, were recorded. As displayed in Figure 1B, the addition of Heparin to an aqueous solution of ThT leads to a blueshifted absorption band at 393 nm with a gradual decrease in absorbance. However, in aqueous solution, ThT displays an absorption maximum at 413 nm. The blue shift in the absorption spectra of ThT in the presence of Heparin is in sharp contrast to the association of monomeric ThT with the other negatively charged 25 surfaces such as DNA, SDS micelles, etc, where a red shift in the absorption spectra have been observed. This contrasting absorption spectral features of ThT in Heparin again indicates the association of ThT with Heparin, in a form other than monomeric form. Recently, a blue shift in the absorption spectra of ThT in the presence of negatively charged sulfated β-CD has 26 been ascribed to H-aggregates of ThT. Thus, the blue-shifted band presenting an absorption maximum


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at 393 nm for ThT in the presence of Heparin can be attributed to H-aggregation between ThT molecules on the highly negatively charged surface of Heparin. This blue shift can be understood in terms of molecular exciton theory. According to the molecular exciton 26-27 theory, when chromophores adopt a head-to-head cofacial arrangement (H-aggregate), the transition from ground state to the higher energy exciton state is allowed, which results in the blue shift of the absorption spectra. The excitonic interaction between the ThT molecules in the aggregated form is further confirmed by electronic circular dichroism (CD) measurements, which displays a clear bi-signate CD signal in the absorption region of ThT chromophores (Figure 2A). The bi-signate feature is characteristic of the ag26 gregates, and can be assigned to excitonic coupling 26, 28 between transition dipole moments of ThT. With an aim to establish that the enhanced fluorescence of ThT, in presence of Heparin, arises from ThT Haggregates, the excitation spectrum was measured for the red-shifted emission band of 560 nm. The excitation spectra (Figure S1†) displays a maximum at 393 nm which matches quite nicely with the absorption maximum (~393 nm) collected for ThT H-aggregates, clearly suggesting that the increased fluorescence band at 560 nm correspond to H-aggregate. The existence of such ThT-Heparin aggregates was also confirmed by DLS analysis displaying particles with an average size of ~98 nm (Figure S2†). Since the absorption maxima of monomer and Haggregates of ThT are reasonably well separated (~20 nm), so we attempted to analyze our absorption titration data in terms of variation of absorbance at two different wavelengths, i.e., at 393 nm, corresponding to ThT H-aggregate, and at 413 nm, corresponding to the ThT monomer band. The ratio of the absorbance at these two wavelengths was found to increase linearly (Figure 1B, Inset) with the increasing concentration of Heparin in a concentration range of 0- 15 μM, and the linear regression was OD393/OD413= 0.784 + 0.039 x 2 [Heparin/μM] (R = 0.995). The calculated LOD of Heparin based on 3.3σ/s was 26 nM. Thus, the present system provides a unique advantage of dual read out of signal for Heparin, both in terms of colorimetry and fluorimetry. Time-resolved emission measurements are crucial to gain insights into the excited state relaxation of dye molecules, which provides important clues for understanding the mechanism of enhanced emission of molecular rotors. In the aqueous solution, the monomer form of ThT decays very fast, which is within the limit of our equipment (the limit is 0.12 ns). It is reported to 23 be ~ 1 ps in water, which is assigned to the ultrafast twisting dynamics of ThT around the central C-C single bond in the excited state which quickly dissipate the excitation energy. On the contrary, the transient

decay for ThT aggregates, in the presence of Heparin, (monitored at 560 nm) extends upto few nanoseconds (Figure 2B), with a multi-exponential decay kinetics.

Figure 2.(A)Circular dichroism spectrum of ThT in aqueous solution (dashed red line) and ThT in Heparin (blue solid line). The green dotted line represents Heparin in water without ThT(B) Excited-state decay trace for ThT in (i) aqueous solution and (ii) Heparin (λex = 406 nm, λem = 560 nm). The solid black line represents instrument response function (IRF).

The average excited-state lifetime (equation 2†) for ThT in the aggregated state is calculated to be ~ 1.4 ns. The long lifetime indicates the presence of new aggregated species, and is also in conformity with the signif8 icantly reduced non-radiative processes (knr ~ 7.4 x 10 -1 12 -1 S in Heparin as compared to knr ~ 10 S for ThT in water) of ThT aggregates in the presence of Heparin, leading to observed emission enhancement for Haggregates of ThT. A similar long lifetime for the ThT aggregates has been also observed in presence of sulfated β-CD and its multi-exponential decay nature has been attributed to the different conformational struc26 ture of the ThT in the excitonic or aggregated states. The aggregates of ThT on Heparin surface was further demonstrated by temperature-dependent emission, absorption, and lifetime-measurements. As the self-assembled systems often integrate multiple weak interactions, such as van der Waals, London dispersion forces, and hydrophobic interactions, so the present system is expected to be responsive to temperature. Upon gradual increase in temperature from 25°C to 70°C, the emission intensity gradually decreases (Figure 3A). The increase in temperature weakens the noncovalent forces involved in the formation of the fluorescent H-aggregate assembly, and leads to gradual conversion of aggregate to monomer, which will weaken the emission capability of the system. The transient emission measurements further support this observation, where transient decay traces progressively became faster with increase in temperature (Figure 3B), implying the gradual conversion of the self-assembled fluorescent aggregates of ThT from the Heparin surface towards monomeric ThT. This is also supported by absorption measurements (Figure S3†).



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Figure 4. (A) Variation in emission intensity (λex = 400 nm, λem = 560 nm).of ThT at increasing concentrations of Hp, ChS and HA. (B) Selectivity analysis with various saccharides and anions.

Figure 3. (A) Steady-state emission spectrum of ThT in Heparin at various temperatures. Inset: Change in emission intensity at 560nm with increasing temperature. (B) Excitedstate decay trace for ThT in Heparin (λex = 406 nm, λem = 560 nm) at different temperatures. The solid black line represents the instrument response function (IRF). Inset: Decrease in average lifetime with temperature.

Selectivity is a very important parameter to evaluate the performance of a sensor. The structurally similar two glucosoaminogylcan analogues, Hyaluronic acid (HA) and Chondroitin sulfate (ChS) are often the main contaminants of clinical Heparin, thus, they were selected to evaluate the selectivity performance of ThT. The most intense fluorescence enhancement was observed for ThT in the presence of Heparin, while for Chs and HA, the fluorescence enhancement of ThT was found to be insignificant (Figure 4A). Thus, these results suggest that ThT displays exceptional selectivity for Heparin over its two structurally similar common contaminants. This selectivity for Heparin may be due to lower charge density and disadvantageous spatial orientation as well as more distant spatial distribution of anions on ChS and HA as compared to those of Heparin. On average, Heparin possesses three sulfate groups and one carboxylate group per disaccharide unit, among which one sulfate and carboxylate group reside at the same side of one saccharide ring (Scheme S2†). ChS carries one carboxylate and one sulfate group per disaccharide unit, whereas HA carries only one carboxyl group per disaccharide unit. The predominance of electrostatic interaction between ThT and Heparin is supported by the effect of ionic strength on the ThT-heparin interaction (Figure S4 & S5†). The selectivity experiments for ThT were also extended to some mono- and disaccharides and various anions. None of these anions and molecules produce distinguished fluorescence enhancement for ThT (Figure 4B).

These results demonstrate that our sensor system displays high selectivity towards Heparin. It should be noted that ThT has been also projected as a sensor for 23 29 amyloid fibrils and G-quadruplex structures, based on its emission enhancement, however, in those cases, ThT operates through its monomeric emission, centered at 490 nm, whereas in the case of Heparin, the enhanced emission is centered at a large red-shifted wavelength at 560 nm, characteristic of ThT aggregates, thus, constituting a unique case for Heparin. To evaluate the practical usefulness of our sensor system for Heparin in real biological samples, we attempted to detect the amount of Heparin in Fetal Bovine Serum (FBS) using ThT. In diluted FBS containing ThT, the fluorescence intensity of ThT increased linearly with the increased concentration of Heparin, (Figure S6†). The linear regression is I560 = 367 + 45 [Hepa2 rin/μM], (Linear range = 0-13 μM, R = 0.983). The LOD was 34 nM, which is quite comparable to other fluores4, 6-7, 30 cence based synthetic Heparin sensors. This detection range is suitable for Heparin monitoring during 30 postoperative and long-term care (1.7-10 μM). We also analyzed the colorimetric response of ThT towards Heparin spiked FBS samples. The absorbance ratio at two wavelengths (OD393/OD413) increases linearly with the increasing concentrations of Heparin in a concentration range of 0-14 μM (Figure S7†), and the linear regression was OD393/OD413 = 0.796 + 0.013 [Heparin/μM] (R2 = 0.997). The calculated detection limit of Heparin was 52 nM. The method was also tested for Human serum-Heparin mixtures (Figure S8 & S9†), and the calculated LOD was ~90 nM. Thus, these results suggest that the developed ThT H-aggregate based detection method can be used to detect Heparin without significant matrix interference. To reduce the risk of medical complications, in case 1, 19 of Heparin overdose, Protamine, the only clinically approved antidote for Heparin, is administered to re1 verse the anticoagulant effect of Heparin. Here, we anticipated if we can monitor interaction of Protamine with Heparin, using the same system of ThT aggregates. Figure 5A shows that the fluorescence intensity of the ThT-Heparin complex decreases drastically with the gradual addition of Protamine. This decrease in emission intensity can be attributed to the disassembly of the ThT H-aggregates from Heparin surface, as a consequence of stronger electrostatic interaction of cationic Protamine with anionic Heparin. Since the free ThT is weakly emissive in nature, so the disassembly of the H-aggregates of ThT from the Heparin surface towards the monomeric form leads to a decrease


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in emission intensity. This dissociation of ThT aggregates was further supported by time-resolved emission measurements, where decay trace for ThT-Heparin complex reaches a situation similar to that of bulk water, upon addition of Protamine (Figure 5B). These results were also supported by ground-state absorption and circular dichroism measurements (Figure S10 & S11†).

field of Heparin. The assay shows reasonably good heparin quantification range with very low LOD, even in human serum samples. Further, our system also represents one of the very few which can operate both through fluorimetry and colorimetry, providing great versatility to the sensing system. ThT can be also suitably used as a probe to explore the interaction of Heparin with its antidote, Protamine. Thus, our assay is expected to facilitate the Heparin related biochemical and biomedical research.

ASSOCIATED CONTENT Supporting Information† “The supporting information containing Material and Methods, and Figures (S1-S11) is available free of charge at http://pubs.acs.org.” Figure 5. (A) Steady-state emission spectrum (λex = 400 nm) of ThT in Heparin at varying concentration of Protamine (PrS). ThT in only water is represented by the black dotted line. Inset: Variation in the emission intensity at 560 nm with increasing concentration of PrS (B) Transient decay trace for ThT in Heparin and in Heparin with 3.7 μM PrS. The dotted red line represents the decay in water.

Thus, these data suggests that H-aggregates of ThT formed on the Heparin surface can be used for investigating the interaction of Heparin with its only medically approved antidote Protamine (Scheme-1).

Scheme 1. Schematic representation of Heparin induced ThT aggregates and its dissociation upon Heparin-Protamine interaction.

In Summary, we have developed a turn-on sensor system for quick, economic and convenient detection of Heparin. This utilizes emission from “H-aggregates”, which are otherwise considered as non-emissive and rather problematic in designing a biosensor. Hence our system provides a unique case for sensing a vital biomolecule and a drug with extensive medical applications. Compared with the use of sophisticated fluorescent sensors, obtained through complicated and timeconsuming synthetic steps, for identifying Heparin, the self-assembly induced, rather unusual H-aggregate emission reported here, exploits non-covalent interaction. Our sensor system offers several advantages including, label free approach, high sensitivity and selectivity, emission in the biologically advantageous redregion, and most importantly constructed from inexpensive commercially available dye molecule, which is expected to impart a large impact on the sensing

AUTHOR INFORMATION Corresponding Author Email: [email protected]; [email protected]

Notes The authors declare no competing financial interests.

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Emissive H-Aggregates of an Ultrafast Molecular Rotor: A Promising

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